U.S. patent application number 10/554287 was filed with the patent office on 2007-08-16 for method and compositions for identifying anti-hiv therapeutic compounds.
Invention is credited to Gabriel Birkus, James M. Chen, Xiaowu Chen, Tomas Cihlar, Eugene J. Eisenberg, Marcos Hatada, Gong-Xin He, Choung U. Kim, William A. Lee, Martin J. McDermott, Sundaramoorthi Swaminathan.
Application Number | 20070190523 10/554287 |
Document ID | / |
Family ID | 29273807 |
Filed Date | 2007-08-16 |
United States Patent
Application |
20070190523 |
Kind Code |
A1 |
Birkus; Gabriel ; et
al. |
August 16, 2007 |
Method and compositions for identifying anti-hiv therapeutic
compounds
Abstract
The invention relates to methods and compositions for
identifying compounds having therapeutic activity against human
immunodeficiency virus (HIV).
Inventors: |
Birkus; Gabriel; (San
Francisco, CA) ; Chen; James M.; (San Ramon, CA)
; Chen; Xiaowu; (San Mateo, CA) ; Cihlar;
Tomas; (Foster City, CA) ; Eisenberg; Eugene J.;
(San Carlos, CA) ; Hatada; Marcos; (Fremont,
CA) ; He; Gong-Xin; (Cupertino, CA) ; Kim;
Choung U.; (San Carlos, CA) ; Lee; William A.;
(Los Altos, CA) ; McDermott; Martin J.; (Boulder,
CO) ; Swaminathan; Sundaramoorthi; (Burlingame,
CA) |
Correspondence
Address: |
Gilead Sciences Inc
333 Lakeside Drive
Foster City
CA
94404
US
|
Family ID: |
29273807 |
Appl. No.: |
10/554287 |
Filed: |
November 6, 2003 |
PCT Filed: |
November 6, 2003 |
PCT NO: |
PCT/EP03/12423 |
371 Date: |
February 12, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60375622 |
Apr 26, 2002 |
|
|
|
60375779 |
Apr 26, 2002 |
|
|
|
60375834 |
Apr 26, 2002 |
|
|
|
60375665 |
Apr 26, 2002 |
|
|
|
Current U.S.
Class: |
435/5 ; 435/7.1;
544/224; 548/112 |
Current CPC
Class: |
A61K 31/66 20130101;
C07F 9/65583 20130101; A61K 31/675 20130101; G01N 33/5038 20130101;
C07D 233/42 20130101; C07D 307/68 20130101; C07F 9/6506 20130101;
C07F 9/65515 20130101; C07F 9/65335 20130101; C07D 403/06 20130101;
A61P 43/00 20180101; C07D 231/20 20130101; C07D 213/85 20130101;
C07F 9/650947 20130101; C07D 239/49 20130101; C40B 40/04 20130101;
C07F 9/645 20130101; C12N 9/16 20130101; C07D 265/18 20130101; C07D
491/04 20130101; C12Q 1/44 20130101; C07F 9/650905 20130101; C07F
9/58 20130101; C07F 9/6512 20130101; C07D 243/04 20130101; C07D
401/06 20130101; C07D 239/80 20130101; C07F 9/6561 20130101; C07D
471/14 20130101; C12Q 1/37 20130101; C07D 213/75 20130101; G01N
2500/04 20130101; A61P 31/18 20180101; C07F 9/65128 20130101; C07F
9/4006 20130101; C12Q 1/18 20130101; C07D 231/12 20130101; G01N
2333/16 20130101 |
Class at
Publication: |
435/005 ;
435/007.1; 548/112; 544/224 |
International
Class: |
C40B 30/06 20060101
C40B030/06; C40B 40/04 20060101 C40B040/04; C12Q 1/70 20060101
C12Q001/70 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 25, 2003 |
US |
US03/12901 |
Apr 25, 2003 |
US |
US03/12943 |
Apr 25, 2003 |
US |
US03/12926 |
Claims
1. A method comprising (a) identifying a non-nucleotide prototype
compound; (b) substituting the prototype compound with a
phosphonate-containing group to produce a candidate compound; and
(c) determining the anti-HIV activity of the candidate
compound.
2-250. (canceled)
Description
[0001] This non-provisional application claims the benefit of
Provisional Application No. 60/375,622, filed Apr. 26, 2002,
Provisional Application No. 60/375,779 filed Apr. 26, 2002,
Provisional Application No. 60/375,834 filed Apr. 26, 2002 and
Provisional Application No. 60/375,665 filed Apr. 26, 2002, which
are incorporated herein by reference. Additionally,
FIELD OF THE INVENTION
[0002] The invention relates generally to methods and compositions
for identifying compounds having therapeutic activity against human
immunodeficiency virus (HIV).
BACKGROUND OF THE INVENTION
[0003] Anti-HIV compounds are well established and have achieved
significant therapeutic benefit. However, existing therapeutics
remain less than optimal. Conspiring to reduce patient compliance
and therapeutic efficacy are toxicity, resistant HIV, poor
bioavailability, low potency, and frequent and inconvenient dosing
schedules, among other failings. The need to administer very large
tablets and requirements for frequent dosing characterize a number
of important anti-HIV therapeutics, most particularly the HIV
protease inhibitors. While significant advances have been made in
preparing improved nucleotide analogue anti-HIV therapeutics (see
WO 02/08241, EP 820,461 and WO 95/07920, all of which are hereby
incorporated by reference), other anti-HIV therapeutic drug classes
remain encumbered with severe deficiencies.
SUMMARY OF THE INVENTION
[0004] The present invention provides methods and compositions for
identifying therapeutic anti-HIV compounds having improved
pharmacological and therapeutic properties. In particular, this
invention provides for novel candidate therapeutic anti-HIV
compounds and methods for screening them to identify compounds
having such beneficial properties.
[0005] In accordance with this invention, a method is provided that
comprises [0006] (a) identifying a non-nucleotide prototype
compound; [0007] (b) substituting the prototype compound with an
esterified carboxyl or esterified phosphonate-containing group to
produce a candidate compound; and [0008] (c) determining the
anti-HIV activity of the candidate compound.
[0009] In another embodiment, a method is provided that comprises
[0010] (a) selecting a non-nucleotide candidate compound containing
at least one esterified carboxyl or esterified
phosphonate-containing group and [0011] (b) determining the
intracellular persistence of the candidate compound or a
esterolytic metabolite of the esterified carboxyl or
phosphonate-containing group thereof.
[0012] In a further embodiment, determining the anti-HIV activity
of the candidate compound comprises determining the anti-HIV
activity of a carboxylic acid or phosphonic acid-containing
metabolite of the candidate compound, which carboxyl acid or
phosphonic acid-containing metabolite is produced by esterolytic
metabolic cleavage of the esterified carboxyl or
phosphonate-containing group. In another embodiment determining
anti-HIV activity comprises determining the tissue selectivity
and/or the intracellular residence time of at least one of said
intracellular carboxylic acid or phosphonic acid-containing
metabolites.
[0013] In another embodiment of this invention, a library of
anti-HIV candidate compounds is provided that comprises at least
one non-nucleotide prototype compound substituted by an esterified
carboxyl or phosphonate group. Such libraries facilitate
large-scale screening of candidate compounds.
[0014] This invention is an improvement in the conventional methods
for identifying therapeutic anti-HIV compounds. Thus, in a method
for identifying an anti-HIV therapeutic compound, the improvement
comprises substituting a prototype compound with an esterified
carboxyl or phosphonate and assaying the resulting candidate
compound for its anti-HIV activity.
[0015] Adding the esterified carboxyl or phosphonate group to the
prototype molecule produces significant advantages in the
pharmacologic properties of the prototype. Without being held to
any particular method of operation of the invention, it is believed
that the ester(s) mask the charge of the carboxyl or phosphonate
and permit the candidate to enter HIV infected cells, in particular
peripheral blood mononuclear cells (PBMCs). Once the candidate has
entered the cells it is processed by biological mechanisms (most
notably, it is believed, by a newly discovered PBMC enzyme which we
designate GS-7340 Ester Hydrolase) to produce at least one
metabolite containing a free carboxylic acid and/or phosphonic
acid. This metabolite is antivirally active against HIV. These
charged metabolic depot forms are exceptionally persistent in the
cells, thereby permitting substantial reductions in the frequency
of dosing compared to the parental prototype, among other
advantages. In addition, the esterified carboxyl or phosphonate
substituent may direct the selective distribution of the prototype
to tissues (most particularly lymphoid tissues such as PBMCs) which
are noted sites of HIV infection, thereby potentially reducing
systemic dose and toxicity.
[0016] In further embodiments, assaying for anti-HIV activity
optionally comprises screening the candidate compounds for their
susceptibility to esterolytic cleavage by isolated GS-7340 Ester
Hydrolase. The isolated Hydrolase is a further embodiment of this
invention.
[0017] Since GS-7340 Ester Hydrolase may interact with other
compounds than the anti-HIV candidates, it will be of pharmacologic
utility to determine if the enzyme is cleaving such other
compounds. Thus, another embodiment of this invention is a method
comprising obtaining a substantially pure organic molecule,
optionally contacting the organic molecule with another molecule to
produce a composition, contacting GS-7340 Ester Hydrolase with said
organic molecule or composition, and optionally determining whether
the organic molecule has been cleaved by the Hydrolase.
[0018] In another embodiment, a method is provided comprising
contacting GS-7340 Ester Hydrolase with an organic compound in a
cell-free environment.
[0019] In a further embodiment, a method is provided comprising
contacting GS-7340 Ester Hydrolase with an organic compound in an
in vitro or cell culture environment.
[0020] In another embodiment, a composition is provided comprising
a substantially pure organic compound and isolated GS-7340 Ester
Hydrolase.
[0021] In another embodiment, a composition is provided comprising
an organic compound and GS-7340 Ester Hydrolase in an in vitro or
cell culture environment.
[0022] These and other embodiments of this invention are more fully
described in the following disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The following disclosure contains detailed embodiments of
the practice of the invention. These are provided to more fully
describe the invention, but the invention is not limited to these
embodiments.
[0024] "Anti-HIV activity" of candidates is determined by any
method for assaying the HIV inhibitory activity of a substance.
Many such methods are well known, and range from in vitro enzyme
assays (e.g., HIV reverse transcriptase or integrase assays) to
animal studies (e.g., SIV in chimps) and human clinical trials.
Included with this term are any assays bearing on the therapeutic
anti-HIV efficacy of a substance, e.g., HIV resistance
determinations, biodistribution, and intracellular persistence.
[0025] "Candidate compound" is an organic compound containing an
esterified carboxylate or phosphonate. Optionally, candidate
compounds excluded compounds heretofore known to have anti-HIV
activity. With respect to the United States, the candidate
compounds herein exclude compounds that are anticipated under 35
USC .sctn. 102 or obvious under 35 USC .sctn. 103 over the prior
art. In other jurisdictions using the novelty and inventive step
criteria, the candidate compounds exclude compounds not novel or
which lack inventive step over the prior art. However, libraries
containing candidate compounds optionally comprise known compounds.
These may be, for example, reference compounds having known
anti-HIV activity.
[0026] "Non-nucleotide" means any compound that has all of the
following characteristics: It does not already contain an
esterified carboxyl or phosphonate, it is not a phosphonate or
phosphate-containing compound disclosed in WO 02/08241, EP 820,461
or WO 95/07920 and it does not already contain a phosphonate group.
GS-7340 is an example of a nucleotide anti-HIV compound. Many other
examples of such compounds are known. These compounds are excluded
from the scope of prototype compounds and are not employed in the
candidate compound screening method or candidate compound
compositions of this invention. For the most part, the nucleotide
analogues comprise the substructure --OC(H).sub.2P(O).dbd. coupled
(usually at the 9 position of purine bases or the 1 position of
pyrimidine bases) via a sugar or cyclic or acyclic sugar analogue
(aglycon) to a nucleotide base or an analogue thereof. The base
analogues typically are substituted, usually at extracyclic N
atoms, or are the aza or deaza analogues of the naturally occurring
base scaffolds. They are fully set forth in the above described art
and are well known in the field. See for example U.S. Pat. No.
5,641,763 and related patents and publications by Antonin Holy.
[0027] Optionally excluded from the scope of the libraries of this
invention are any phosphonates disclosed by WO99/33815, WO99/33792,
WO99/33793, WO00/76961 and their related, progeny and parental
filings, all of which are hereby incorporated by reference.
However, unless expressly excluded by the claims herein, such
compounds shall be considered candidate compounds. Further, the act
of making and screening the phosphonates of such filings to
determine their intracellular persistence (whether by preclinical
assays such as that using GS-7340 Ester Hydrolase, or by clinical
studies) falls within the scope hereof, as does obtaining
regulatory approval to market one of them and selling the selected
phosphonate.
[0028] "Non-nucleoside" means any compound that is not a nucleotide
base linked to a sugar or aglycon (cyclic or acyclic) and
terminating at the 5' position (or the analogous position in
nucleosides containing sugar analogues) by hydroxyl or a group
which is metabolized in vivo to hydroxyl. The nucleosides are
distinguishable from the nucleotides in not containing a phosphate
or, in the case of relevant nucleotide analogues, a
phosphonate.
[0029] "Phosphonate-containing group" is a group comprising a
phosphorus atom singly bonded to carbon, double bonded to oxygen
and singly bonded to two other groups through oxygen, sulfur, or
nitrogen. In general, the carbon bond is to a carbon atom of the
prototype or a liking group to the prototype and the single bonds
to oxygen, nitrogen or sulfur are bonds to oxy or thioesters or are
amino acid amidates in which the terminal carboxyl group(s) are
esterified.
[0030] "Carboxyl-containing groups" are any group having a free
carboxyl serving as the site for esterification. An "organic acid"
is any compound containing carboxyl and at least one additional
carbon atom.
[0031] The "esterified carboxyl or esterified phosphonate group" is
any group capable of intracellular processing to yield a free
carboxyl and/or free phosphonic acid. The structure of these groups
is not important other than that the free acid be produced
intracellularly. Preferably, systemic or digestive esterolysis is
minimized in preference to intracellular hydrolysis. This permits
maximum migration of the candidate into target cells and maximum
intracellular retention of the acid metabolites.
[0032] Suitable exemplary esterified carboxyl or phosphonate groups
are described herein. Others are identified by screening for
esterolysis in vivo, in PBMCs or using GS-7340 Ester Hydrolase.
These groups have the structure A.sup.3, wherein A.sup.3 is a group
of the formula ##STR1## in which:
[0033] Y.sup.1 is independently O, S, --N(R.sup.x),
--N(O)(R.sup.x), --N(OR.sup.x), --N(O)(OR.sup.x), or
N(N(R.sup.x)(R.sup.x));
[0034] Y.sup.2 is independently a bond, O, --N(R.sup.x),
--N(O)(R.sup.x), --N(OR.sup.x), --N(O)(OR.sup.x),
--N(N(R.sup.x)(R.sup.x)), --S(O).sub.M2--, or
--S(O).sub.M2--S(O).sub.M2--;
[0035] R.sup.x is independently H, W.sup.3, a protecting group, or
a group of the formula: ##STR2##
[0036] R.sup.y is independently H, W.sup.3, R.sup.2 or a protecting
group;
[0037] R.sup.1 is independently H or alkyl of 1 to 18 carbon
atoms;
[0038] R.sup.2 is independently H, R.sup.3 or R.sup.4 wherein each
R.sup.4 is independently substituted with 0 to 3 R.sup.3
groups;
[0039] R.sup.3 is R.sup.3a, R.sup.3b, R.sup.3c or R.sup.3d,
provided that when R.sup.3 is bound to a heteroatom, then R.sup.3
is R.sup.3c or R.sup.3d;
[0040] R.sup.3a is F, Cl, Br, I, --CN, N.sub.3 or --NO.sub.2;
[0041] R.sup.3b is Y.sup.1;
[0042] R.sup.3c is --R.sup.x, --N(R.sup.x)(R.sup.x), --SR.sup.x,
--S(O)R.sup.x, --S(O).sub.2R.sup.x, --S(O)(OR.sup.x),
--S(O).sub.2(OR.sup.x), --OC(Y.sup.1)R.sup.x,
--OC(Y.sup.1)OR.sup.x, --OC(Y.sup.1)(N(R.sup.x)(R.sup.x)),
--SC(Y.sup.1)R.sup.x, --SC(Y.sup.1)OR.sup.x,
--SC(Y.sup.1)(N(R.sup.x)(R.sup.x)), --N(R.sup.x)C(Y.sup.1)R.sup.x,
--N(R.sup.x)C(Y.sup.1)OR.sup.x, or
--N(R.sup.x)C(Y.sup.1)(N(R.sup.x)(R.sup.x));
[0043] R.sup.3d is --C(Y.sup.1)R.sup.x, --C(Y.sup.1)OR.sup.x or
--C(Y.sup.1)(N(R.sup.x)(R.sup.x));
[0044] R.sup.4 is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to
18 carbon atoms, or alkynyl of 2 to 18 carbon atoms;
[0045] R.sup.5 is R.sup.4 wherein each R.sup.4 is substituted with
0 to 3 R.sup.3 groups;
[0046] R.sup.5a is independently alkylene of 1 to 18 carbon atoms,
alkenylene of 2 to 18 carbon atoms, or alkynylene of 2-18 carbon
atoms any one of which alkylene, alkenylene or alkynylene is
substituted with 0-3 R.sup.3 groups;
[0047] W.sup.3 is W.sup.4 or W.sup.5;
[0048] W.sup.4 is R.sup.5, --C(Y.sup.1)R.sup.5,
--C(Y.sup.1)W.sup.5, --SO.sub.2R.sup.5, or --SO.sub.2W.sup.5;
[0049] W.sup.5 is carbocycle or heterocycle wherein W.sup.5 is
independently substituted with 0 to 3 R.sup.2 groups;
[0050] M2 is 0, 1 or 2;
[0051] M12a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
[0052] M12b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
[0053] M1a, M1c, and M1d are independently 0 or 1; and
[0054] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.
[0055] The esterified group is attached to the prototype through a
bond or via intermediary linking groups such as the A.sup.1
subgroup
--[Y.sup.2--(C(R.sup.2).sub.2).sub.m12a].sub.m12bY.sup.2W.sup.6--
defined below.
[0056] Candidates optionally are substituted with a single
substituent which contains both an esterified carboxyl and an
esterified phosphonate. In addition, or as an alternative, the
candidate contains separate substituents bearing esterified
carboxyl and/or phosphonate groups. An example of a combined group
would a phosphonate in which a free valence of the phosphorus atom
is bonded to the hydroxy of an hydroxyorganic acid or to the amino
group of an amino acid wherein the carboxyl groups of the organic
acid or amino acid are esterified.
[0057] "Esterified" means that the phosphonate or carboxyl is
bonded to a carbon atom-containing group through oxygen or sulfur,
as in --P(O)(OR)-- or --COOR for example, where R is a carbon
containing group such as alkyl or aryl.
[0058] "Protecting group" is a group covalently bonded to a labile
site on the candidate compound, which site is expected to be labile
under the conditions to be encountered by the candidate, for
example during synthetic procedures, during exposure to ambient
conditions, and the conditions found in in vivo environments. The
protecting group serves to prevent degradation or otherwise
undesired conversions at the labile site. Extensive disclosure of
various exemplary protecting groups is found infra.
[0059] "Intracellular depot metabolite" is an esterolytic
metabolite of the esterified carboxyl or phosphonate whereby a
charged carboxyl or phosphonic acid is revealed. An example is
Metabolite X, further described in the examples.
[0060] "Tissue selectivity" of candidate compounds is determined by
procedures set forth in WO02/08241. The object of this
determination is to find whether or not the candidate (and by
extension its depot forms) are enriched in one tissue or another.
It is expected that compounds containing the carboxyl or
phosphonate groups as described herein will be preferentially
enriched in lymphoid tissue such as PBMCs.
[0061] "Intracellular residence time," "intracellular persistence,"
"intracellular half life" and the like refers to a measure of the
time that a candidate molecule or its anti-HIV active metabolite is
found within a given cell after introduction of the esterified
candidate into the cell. Any technique is suitable that
demonstrates how long a candidate or its anti-HIV active
metabolite(s) remain in a cell. Further description of suitable
assay procedures are set forth infra. Ideally, the method for
measuring residence time will measure the retention time of the
metabolite at a concentration adequate to inhibit HIV.
[0062] A "prototype compound" is any organic compound. In general,
in the method of this invention one will select prototype compounds
having known structures and synthesis routes in order to reduce the
synthetic burden and development costs. Typically, the prototype
compound will be one that has, or at least is suspected, to have
anti-HIV activity. However, since the prototype compound is serving
only as a starting point for preparing candidate compounds to be
screened, it is not essential that it have, or be known or
suspected to have, preexisting anti-HIV activity. The prototype
compound need not be published or known generally to the public. In
fact, the method of this invention is advantageously practiced in
on-going proprietary research programs where anti-HIV compounds are
continually identified and optimized. It also should be understood
that identification or selection of the prototype compound need not
be temporally related to that of the candidate compound. This means
that the prototype might be identified after one or more related
candidate compounds are made, or the prototype might be an early
version of a compound class that has advanced further into
development before the candidate based on the early prototype is
actually synthesized. The prototype compound also may be entirely
conceptual or may be in various phases of development. No actual
prototype need to have been made, nor tested for activity or any
other properties. This is often the case with candidates that are
the product of truncating an existing compound and then inserting a
linker group in place of all or a part of the omitted portion. In
addition, it is not necessary that the prototype compound be
conceived independently of the esterified substituent, i.e., it is
not necessary to have the prototype in mind before designing the
esterified substitution. The conception of the candidate compound
optionally is a single act. Of course, the candidate compound may
be based on a prototype which is in fact a previously made
candidate compound and the subsequent candidate is multiply
substituted with the carboxyl or phosphonate ester. Also, it will
be understood that a candidate or group of candidates compounds
optionally are based on an original prototype even though
intervening candidates or libraries of candidates have been
made.
[0063] The prototypes generally serve as the starting point for
designing and identifying candidate compounds. Generally a
prototype will not contain a phosphonate or carboxyl group, but it
may do so if the phosphonate or carboxyl are not esterified (since
candidates contain esterified phosphonate or carboxyl groups). It
is most efficient to start with prototypes already known to have
anti-HIV activity (preferably compounds active against anti-HIV
protease, HIV integrase or HIV polymerase), but it is not essential
to do so. For example, a prototype optionally is a subsegment or
fragment of a compound known to possess anti-HIV activity, even
though the fragment need not be active against HIV in its own
right. In this instance, the phosphonate or carboxyl group restores
anti-HIV activity to the candidate.
[0064] "Linker" or "link" is a bond or an assembly of atoms binding
the prototype to the esterified phosphonate or carboxyl-containing
group. The nature of the linker is not critical. The linker need
not be involved in the interactions of the esterified carboxyl or
phosphonate group with GS-7340 Ester Hydrolase or other processing
enzymes, nor need it be involved in the therapeutic interaction of
the prototype with its target protein. This is not to say that
these functions could not be enhanced or influenced by the linker,
but it is not necessary that the linker perform or contribute to
such functions. Thus, it is a straight-forward matter of elemental
organic chemistry to devise suitable linkergroups and methods for
joining the esterified groups.
[0065] Some general principles are useful in selecting suitable
linkergroups, despite their lack of criticality. First, they will
not be so bulky as to interfere with the interaction of the
remainder of the prototype with its target protein, e.g., HIV
protease inhibitor, nor will they bear reactive or unstable groups
once the linkage has been accomplished. Such chemically reactive
groups will be well known to the artisan, and the parameters of
bulky linkers can be evaluated by molecular modeling. Resources are
available to model proteins involved in a number of diseases and
disorders of lymphoid tissues, in particular HIV protease. In
general, the linker will be relatively small, on the order of about
16-500 MW, typically about 16-250, ordinarily about 16-200,
although as noted the linker can be as small as a bond. It
generally will be substantially linear, containing less than about
40% of the total MW of the linkeratoms being found in branching
groups, typically less than 30% and ordinarily less than about
20%.
[0066] The backbone of such linkergroups ideally will not contain
any atom that is known to be labile to cleavage by biological
processes or otherwise subject to hydrolysis in biological fluids.
Typical suspect groups would be esters or amides in the backbone of
the linker. The object is for the carboxyl or phosphonate to
survive intracellular processing, with only the ester(s) being
hydrolyzed, and the presence of labile groups in the backbone would
jeopardize this function. However, if enzymatic access to labile
atoms or groups is sterically hindered, e.g., by a cycloalkyl group
or branched alkyl group, then labile sites optionally may be used
in the linker. Labile groups also optionally are can be found in
locations other than backbone positions, e.g. on branching groups
or cyclic substituents, where their potential cleavage would not
result in the loss of the free acid functionality. Backbone alkyls,
alkyl ethers (S or O), or alkyl containing N in any oxidation state
are usually satisfactory. Generally the linker backbone is linear
rather than branched or cyclic (although it may be desired to use
branching or cyclic backbones when multiple esterified groups are
substituted onto the prototype). The linker generally is chosen to
permit substantial rotational freedom to the esterified group, and
for this reason backbone double or triple bonds are not favored
unless it is expected that they would be metabolized to less
rotationally confined structures in vivo (e.g., oxidized to
hydroxyl substituents). If it is desired to avoid interactions with
the target protein then the linker optimally will have neither
highly charged nor strongly hydrophobic character, although as
noted such properties can have advantages in enhancing anti-HIV
activity.
[0067] The typical linker to phosphonate will comprise at least the
group --OCH.sub.2-- (wherein the carbon is linked to the
phosphorous atom), but many others will be apparent to the artisan
or are described elsewhere herein.
[0068] Synthetic ease optionally will play a role in selection of
the linker. For this reason, many linkers will contain a backbone
or chain heteroatom such as 1 to 3 S, N or O. However, occasionally
the prototype compound will contain a convenient site for insertion
of the linker, e.g., a pendant hydroxyl, thus enabling a small
linkergroup because the phosphorous atom can be linked directly, or
virtually directly, to the prototype. Synthetic routes also can be
devised readily that permit direct linkage of the phosphorous atom
to the prototype, in which case the linker is merely a bond.
[0069] The linker optionally is grafted onto the prototype, or the
prototype compound is optionally is modified to remove group(s)
which then are replaced with linker(s). This may facilitate the
synthesis of the candidate compound or, in some instances, may
fortuitously improve the properties of the candidate. This may or
may not be more efficient that simply grafting A.sup.3 onto the
prototype.
[0070] Typically, the starting point in devising a facile synthetic
route for a candidate compound is to analyze the synthons employed
in known methods for preparing the remainder of the prototype
compound, concentrating on synthons which could contribute at least
a part of the esterified group. Such synthons optionally are
modified to contain the esterified group or a portion thereof
(e.g., the acid, which is then esterified in a later step). They
are then introduced into the remainder of the candidate in
substantially the same fashion as the prototype or antecedent
compound. Alternatively, a reactive group is introduced into the
synthon before it is assembled into the precursor, and it is this
group that is reacted with an intermediate for the carboxyl or
phosphonate group. If necessary, suitable protecting groups are
employed to facilitate the synthesis.
[0071] The site for insertion of the esterified carboxyl or
phosphonate group on the prototype will vary widely. The esterified
group preferably is substituted at any location on the prototype
that does not bind substantially with the target protein or affect
the functioning of a group that does interact with the target
protein. These sites are identified by molecular modeling, by
consulting systematic SAR studies or by preparing pilot candidate
compounds. However, it is also within the scope of this invention
to insert the esterified groups at a site which is involved in
binding the prototype to the target protein. Such sites optionally
are used if (a) the linker reasonably replicates the function of
the group on the prototype that it is displacing, e.g., it
possesses a side chain containing the group, (b) if the loss in
binding affinity is not critical to the functioning of the
prototype or (c) if other substitutents are introduced into the
prototype that compensate for any loss in activity caused by the
insertion of the linker.
[0072] The linker generally will contain at least two free valences
(1 for the prototype and 1-3 for the esterified groups).
Multivalent linkergroups can be employed to form a cyclic
structure, being joined at 2 or more sites on the prototype and
forming a bridge, the bridge in turn being substituted with one or
more esterified carboxyl or phosphonate groups or including at
least one atom encompassed within such groups. In addition, the
linker does not need to be bound to the esterified group and/or the
remainder of the prototype by a covalent bond, nor need it consist
solely of covalently bonded atoms. Any bond meeting the basic
criteria herein will be satisfactory, as for example linkage by
chelation or other stable non-covalent attachment systems are
included within the scope of the term "bond" as used herein.
[0073] Linkers also include polymers, e.g., those containing
repeating units of alkyloxy (e.g. polyethylenoxy, PEG,
polymethyleneoxy) and/or alkylamino (e.g. polyethyleneamino,
Jeffamine.TM.). Other linker groups include diacid ester and amides
including succinate, succinamide, diglycolate, malonate, and
caproamide.
[0074] Suitable linker groups optionally are prescreened by testing
model candidates in the same fashion set forth herein for disclosed
candidate compounds, e.g., screening using the Ester Hydrolase
described herein, or by studying the effect of a model
linker-containing candidate compound in PBMCs.
[0075] Typical linkers have the A.sup.1 substructure
--[Y.sup.2--(C(R.sup.2).sub.2).sub.m12a].sub.m12bY.sup.2W.sup.6--
wherein Y.sup.2, R.sup.2, m12a and m12b are defined elsewhere
herein, W.sup.6 is W.sup.3 having from 1 to 3 free valences and the
prototype is bound to the Y.sup.2 with free valence. However, many
other structures would be apparent to the ordinary artisan and can
be prepared by conventional means using the guidance herein.
Defined Chemical Terms
[0076] "Alkyl" is C.sub.1-C.sub.18 hydrocarbon containing normal
secondary, tertiary or cyclic carbon atoms. Examples are methyl
(Me, --CH.sub.3), ethyl (Et, --CH.sub.2CH.sub.3), 1-propyl (n-Pr,
n-propyl, --CH.sub.2CH.sub.2CH.sub.3), 2-propyl (i-Pr, i-propyl,
--CH(CH.sub.3).sub.2), 1-butyl (n-Bu, n-butyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-methyl-1-propyl (i-Bu,
i-butyl, --CH.sub.2CH(CH.sub.3).sub.2), 2-butyl (s-Bu, s-butyl,
--CH(CH.sub.3)CH.sub.2CH.sub.3), 2-methyl-2-propyl (t-Bu, t-butyl,
--C(CH.sub.3).sub.3), 1-pentyl (n-pentyl
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-pentyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.3), 3-pentyl
(--CH(CH.sub.2CH.sub.3).sub.2), 2-methyl-2-butyl
(--C(CH.sub.3).sub.2CH.sub.2CH.sub.3), 3-methyl-2-butyl
(--CH(CH.sub.3)CH(CH.sub.3).sub.2), 3-methyl-1-butyl
(--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2), 2-methyl-1-butyl
(--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.3), 1-hexyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-hexyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 3-hexyl
(--CH(CH.sub.2CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3)),
2-methyl-2-pentyl (--C(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3),
3-methyl-2-pentyl (--CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH.sub.3),
4-methyl-2-pentyl (--CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2),
3-methyl-3-pentyl (--C(CH.sub.3)(CH.sub.2CH.sub.3).sub.2),
2-methyl-3-pentyl (--CH(CH.sub.2CH.sub.3)CH(CH.sub.3).sub.2),
2,3-dimethyl-2-butyl (--C(CH.sub.3).sub.2CH(CH.sub.3).sub.2),
3,3-dimethyl-2-butyl (--CH(CH.sub.3)C(CH.sub.3).sub.3.
[0077] "Alkenyl" is C.sub.2-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms with at least one site
of unsaturation, i.e. a carbon-carbon, sp.sup.2 double bond.
Examples include, but are not limited to: ethylene or vinyl
(--CH.dbd.CH.sub.2), allyl (--CH.sub.2CH.dbd.CH.sub.2),
cyclopentenyl (--C.sub.5H.sub.7), and 5-hexenyl (--CH.sub.2
CH.sub.2CH.sub.2CH.sub.2CH.dbd.CH.sub.2),
[0078] "Alkynyl" is C.sub.2-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms with at least one site
of unsaturation, i.e. a carbon-carbon, sp triple bond. Examples
include, but are not limited to: acetylenic (--C.ident.CH) and
propargyl (--CH.sub.2C.ident.CH),
[0079] "Alkylene" refers to a saturated, branched or straight chain
or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two
monovalent radical centers derived by the removal of two hydrogen
atoms from the same or two different carbon atoms of a parent
alkane. Typical alkylene radicals include, but are not limited to:
methylene (--CH.sub.2--) 1,2-ethyl (--CH.sub.2CH.sub.2--),
1,3-propyl (--CH.sub.2CH.sub.2CH.sub.2--), 1,4-butyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and the like.
[0080] "Alkenylene" refers to an unsaturated, branched or straight
chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and
having two monovalent radical centers derived by the removal of two
hydrogen atoms from the same or two different carbon atoms of a
parent alkene. Typical alkenylene radicals include, but are not
limited to: 1,2-ethylene (--CH.dbd.CH--).
[0081] "Alkynylene" refers to an unsaturated, branched or straight
chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and
having two monovalent radical centers derived by the removal of two
hydrogen atoms from the same or two different carbon atoms of a
parent alkyne. Typical alkynylene radicals include, but are not
limited to: acetylene (--C.ident.C--), propargyl
(--CH.sub.2C.ident.C--), and 4-pentynyl
(--CH.sub.2CH.sub.2CH.sub.2C.ident.CH--).
[0082] "Aryl" means a monovalent aromatic hydrocarbon radical of
6-20 carbon atoms derived by the removal of one hydrogen atom from
a single carbon atom of a parent aromatic ring system. Typical aryl
groups include, but are not limited to, radicals derived from
benzene, substituted benzene, naphthalene, anthracene, biphenyl,
and the like.
[0083] "Arylalkyl" refers to an acyclic alkyl radical in which one
of the hydrogen atoms bonded to a carbon atom, typically a terminal
or sp.sup.3 carbon atom, is replaced with an aryl radical. Typical
arylalkyl groups include, but are not limited to, benzyl,
2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,
2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,
2-naphthophenylethan-1-yl and the like. The arylalkyl group
comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, including
alkanyl, alkenyl or alkynyl groups, of the arylalkyl group is 1 to
6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.
[0084] "Substituted alkyl", "substituted aryl", and "substituted
arylalkyl" mean alkyl aryl and arylalkyl respectively, in which one
or more hydrogen atoms are each independently replaced with a
substituent. Typical substituents include, but are not limited to,
--X, --R, --O.sup.-, --OR, --SR, --S--, --NR.sub.2, --NR.sub.3,
.dbd.NR, --CX.sub.3, --CN, --OCN, --SCN, --N.dbd.C.dbd.O, --NCS,
--NO, --NO.sub.2, .dbd.N.sub.2, --N.sub.3, NC(.dbd.O)R,
--C(.dbd.O)R, --C(.dbd.O)NRR --S(.dbd.O).sub.2O--,
--S(.dbd.O).sub.2OH, --S(.dbd.O).sub.2R, --OS(.dbd.O).sub.2OR,
--S(.dbd.O).sub.2NR, --S(.dbd.O)R, --OP(.dbd.O)O.sub.2RR,
--P(.dbd.O)O.sub.2RR --P(.dbd.O)(O.sup.-).sub.2,
--P(.dbd.O)(OH).sub.2, --C(.dbd.O)R, --C(.dbd.O)X, --C(S)R,
--C(O)OR, --C(O)O, --C(S)OR, --C(O)SR, --C(S)SR, --C(O)NRR,
--C(S)NRR, --C(NR)NRR, where each X is independently a halogen: F,
Cl, Br, or I; and each R is independently --H, alkyl aryl,
heterocycle, protecting group or prodrug moiety. Alkylene,
alkenylene, and alkynylene groups may also be similarly
substituted.
[0085] "Heterocycle" as used herein includes by way of example and
not limitation these heterocycles described in Paquette, Leo A.;
"Principles of Modern Heterocyclic Chemistry" (W. A. Benjamin, New
York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The
Chemistry of Heterocyclic Compounds, A series of Monographs" (John
Wiley & Sons, New York, 1950 to present), in particular Volumes
13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.
[0086] Examples of heterocycles include by way of example and not
limitation pyridyl dihydroypyridyl tetrahydropyridyl (piperidyl),
thiazolyl, tetrahydrothiophenyl, sulfur oxidized
tetrahydrothiophenyl pyrimidinyl, furanyl, thienyl, pyrrolyl
pyrazolyl, imidazolyl tetrazolyl benzofuranyl, thianaphthalenyl,
indolyl, indolenyl, quinolinyl isoquinolinyl benzimidazolyl,
piperidinyl, 4-piperidonyl, pyrrolidinyl 2-pyrrolidonyl,
pyrrolinyl, tetrahydrofuranyl tetrahydroquinolinyl
tetrahydroisoquinolinyl, decahydroquinolinyl,
octahydroisoquinolinyl, azocinyl, triazinyl 6H-1,2,5-thiadiazinyl,
2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl pyranyl
isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,
isothiazolyl isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, 1H-indazoly, purinyl 4H-quinolizinyl
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,
cinnolinyl pteridinyl, 4aH-carbazolyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl acridinyl, pyrimidinyl,
phenanthrolinyl phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl
isochromanyl, chromanyl, imidazolidinyl imidazolinyl pyrazolidinyl
pyrazolinyl, piperazinyl indolinyl isoindolinyl quinuclidinyl,
morpholinyl oxazolidinyl, benzotriazolyl, benzisoxazolyl,
oxindolyl, benzoxazolinyl, and isatinoyl.
[0087] By way of example and not limitation, carbon bonded
heterocycles are bonded at position 2, 3, 4, 5, or 6 of a pyridine,
position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a
pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4,
or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or
tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or
thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or
isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4
of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or
position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more
typically, carbon bonded heterocycles include 2-pyridyl, 3-pyridyl,
4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,
5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,
5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,
5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or
5-thiazolyl.
[0088] By way of example and not limitation, nitrogen bonded
heterocycles are bonded at position 1 of an aziridine, azetidine,
pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,
imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,
2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole,
indoline, 1H-indazole, position 2 of a isoindole, or isoindoline,
position 4 of a morpholine, and position 9 of a carbazole, or
.beta.-carboline. Still more typically, nitrogen bonded
heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,
1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
[0089] "Carbocycle" means a saturated, unsaturated or aromatic ring
having 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms
as a bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, still
more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12
ring atoms, e.g. arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6]
system, or 9 or 10 ring atoms arranged as a bicyclo [5,6] or [6,6]
system. Examples of monocyclic carbocycles include cyclopropyl,
cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,
1-cyclohex-2-enyl, 1-cyclohex-3-enyl, phenyl spiryl and
naphthyl.
[0090] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0091] The term "stereoisomers" refers to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space.
[0092] "Diastereomer" refers to a stereoisomer with two or more
centers of chirality and whose molecules are not mirror images of
one another. Diastereomers have different physical properties, e.g.
melting points, boiling points, spectral properties, and
reactivities. Mixtures of diastereomers may separate under high
resolution analytical procedures such as electrophoresis and
chromatography.
[0093] "Enantiomers" refer to two stereoisomers of a compound which
are non-superimposable mirror images of one another.
[0094] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of
Chemical Terms (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds
(1994) John Wiley & Sons, Inc., New York. Many organic
compounds exist in optically active forms, i.e., they have the
ability to rotate the plane of plane-polarized light. In describing
an optically active compound, the prefixes D and the linkeror R and
S are used to denote the absolute configuration of the molecule
about its chiral center(s). The prefixes d and the linkeror (+) and
(-) are employed to designate the sign of rotation of
plane-polarized light by the compound, with (-) or 1 meaning that
the compound is levorotatory. A compound prefixed with (+) or d is
dextrorotatory. For a given chemical structure, these stereoisomers
are identical except that they are mirror images of one another. A
specific stereoisomer may also be referred to as an enantiomer, and
a mixture of such isomers is often called an enantiomeric mixture.
A 50:50 mixture of enantiomers is referred to as a racemic mixture
or a racemate, which may occur where there has been no
stereoselection or stereospecificity in a chemical reaction or
process. The terms "racemic mixture" and "racemate" refer to an
equimolar mixture of two enantiomeric species, devoid of optical
activity.
[0095] Whenever a compound described herein is substituted with
more than one of the same designated group, e.g., "R.sup.1" or
"R.sup.6a", then it will be understood that the groups may be the
same or different, i.e., each group is independently selected.
[0096] Candidate compounds contain at least one A.sup.1 (which in
turn contains 1-3 A.sup.3 groups) but also may contain at least one
A.sup.2 group.
[0097] A.sup.1 is: ##STR3##
[0098] A.sup.2 is: ##STR4##
[0099] A.sup.3 is: ##STR5##
[0100] Y.sup.1 is independently O, S, N(R.sup.x), --N(O)(R.sup.x),
--N(OR.sup.x), --N(O)(OR.sup.x), or N(N(R.sup.x)(R.sup.x));
[0101] Y.sup.2 is independently a bond, O, N(R.sup.x),
--N(O)(R.sup.x), --N(OR.sup.x), --N(O)(OR.sup.x),
--N(N(R.sup.x)(R.sup.x)), --S(O).sub.M2--, or
--S(O).sub.M2--S(O).sub.M2--;
[0102] R.sup.x is independently H, R.sup.1, W.sup.3, a protecting
group, or the formula: ##STR6##
[0103] R.sup.y is independently H, W.sup.3, R.sup.2 or a protecting
group;
[0104] R.sup.1 is independently H or an alkyl of 1 to 18 carbon
atoms;
[0105] R.sup.2 is independently H, R.sup.1, R.sup.3 or R.sup.4
wherein each R.sup.4 is independently substituted with 0 to 3
R.sup.3 groups;
[0106] R.sup.3 is R.sup.3a, R.sup.3b, R.sup.3c or R.sup.3d,
provided that when R.sup.3 is bound to a heteroatom, then R.sup.3
is R.sup.3c or R.sup.3d;
[0107] R.sup.3a is F, Cl, Br, I, --CN, N.sub.3 or --NO.sub.2;
[0108] R.sup.3b is Y.sup.1;
[0109] R.sup.3 is --R.sup.x, --N(R.sup.x)(R.sup.x), --SR.sup.x,
--S(O)R.sup.x, --S(O)R.sup.x, --S(O)(OR.sup.x),
--S(O).sub.2(OR.sup.x), --OC(Y.sup.1)R.sup.x,
--OC(Y.sup.1)OR.sup.x, --OC(Y.sup.1)(N(R.sup.x)(R.sup.x)),
--SC(Y.sup.1)R.sup.x, --SC(Y.sup.1)OR.sup.x,
--SC(Y.sup.1)(N(R.sup.x)(R.sup.x)), --N(R.sup.x)C(Y.sup.1)R.sup.x,
--N(R.sup.x)C(Y.sup.1)OR.sup.x, or
--N(R.sup.x)C(Y.sup.1)(N(R.sup.x)(R.sup.x));
[0110] R.sup.3d is --C(Y.sup.1)R.sup.x, --C(Y.sup.1)OR.sup.x or
--C(Y.sup.1)(N(R.sup.x)(R.sup.x));
[0111] R.sup.4 is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to
18 carbon atoms, or alkynyl of 2 to 18 carbon atoms;
[0112] R.sup.5 is R.sup.4 wherein each R.sup.4 is substituted with
0 to 3 R.sup.3 groups;
[0113] W.sup.3 is W.sup.4 or W.sup.5;
[0114] W.sup.4 is R.sup.5, --C(Y.sup.1)R.sup.5,
--C(Y.sup.1)W.sup.5, --SO.sub.2R.sup.5, or --SO.sub.2W.sup.5;
[0115] W.sup.5 is carbocycle or heterocycle wherein W.sup.5 is
independently substituted with 0 to 3 R.sup.2 groups;
[0116] W.sup.6 is W.sup.3 independently substituted with 1, 2, or 3
A.sup.3 groups;
[0117] W.sup.7 is a heterocycle bonded through a nitrogen atom of
said heterocycle and independently substituted with 0, 1 or 2
A.sup.0 groups;
[0118] M2 is 0, 1 or 2;
[0119] M12a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
[0120] M12b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
[0121] M1a, M1c, and M1d are independently 0 or 1; and
[0122] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.
[0123] W.sup.5 carbocycles and W.sup.5 heterocycles may be
independently substituted with 0 to 3 R.sup.2 groups. W.sup.5 may
be a saturated, unsaturated or aromatic ring comprising a mono- or
bicyclic carbocycle or heterocycle. W.sup.5 may have 3 to 10 ring
atoms, e.g., 3 to 7 ring atoms. The W.sup.5 rings are saturated
when containing 3 ring atoms, saturated or mono-unsaturated when
containing 4 ring atoms, saturated, or mono- or di-unsaturated when
containing 5 ring atoms, and saturated, mono- or di-unsaturated, or
aromatic when containing 6 ring atoms.
[0124] A W.sup.5 heterocycle may be a monocycle having 3 to 7 ring
members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from
N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9
carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S).
W.sup.5 heterocyclic monocycles may have 3 to 6 ring atoms (2 to 5
carbon atoms and 1 to 2 heteroatoms selected from N, O, and S); or
5 or 6 ring atoms (3 to 5 carbon atoms and 1 to 2 heteroatoms
selected from N and S). W.sup.5 heterocyclic bicycles have 7 to 10
ring atoms (6 to 9 carbon atoms and 1 to 2 heteroatoms selected
from N, O, and S) arranged as a bicyclo [4,5], [5,5], [5,6], or
[6,6] system; or 9 to 10 ring atoms (8 to 9 carbon atoms and 1 to 2
hetero atoms selected from N and S) arranged as a bicyclo [5,6] or
[6,6] system. The W.sup.5 heterocycle may be bonded to Y.sup.2
through a carbon, nitrogen, sulfur or other atom by a stable
covalent bond.
[0125] W.sup.5 heterocycles include for example, pyridyl,
dihydropyridyl isomers, piperidine, pyridazinyl, pyrimidinyl
pyrazinyl, s-triazinyl oxazolyl, imidazolyl thiazolyl isoxazolyl,
pyrazolyl isothiazolyl, furanyl thiofuranyl, thienyl, and pyrrolyl.
W.sup.5 also includes, but is not limited to, examples such as:
##STR7##
[0126] W.sup.5 carbocycles and heterocycles may be independently
substituted with 0 to 3 R.sup.2 groups, as defined above. For
example, substituted W.sup.5 carbocycles include: ##STR8##
[0127] Examples of substituted phenyl carbocycles include:
##STR9##
Embodiments
[0128] The following embodiments represent preferred choices for
various substituents found on the candidate compounds of this
invention. Each embodiment is to be construed as representing the
enumerated substituent (or assembly of substituents) in combination
with each and every other substituent that is not enumerated in the
embodiment. For example, if W.sup.3 is specified in an embodiment,
then W.sup.3 is locked but the remaining substituents can be set in
any combination possible within the definition of A.sup.3.
[0129] In an embodiment A.sup.1 is ##STR10##
[0130] In an embodiment A.sup.1 is ##STR11##
[0131] An embodiment of A.sup.3 includes where M2 is 0, such as:
##STR12## and where M12b is 1, Y.sup.1 is oxygen, and Y.sup.2b is
oxygen (O) or nitrogen (N(R.sup.x)) such as: ##STR13##
[0132] Another embodiment of A.sup.3 is: ##STR14## where W.sup.5 is
a carbocycle such as phenyl or substituted phenyl. Such embodiments
include: ##STR15## where Y.sup.2b is O or N(R.sup.x); M12d is 1, 2,
3, 4, 5, 6, 7 or 8; and the phenyl carbocycle is substituted with 0
to 3 R.sup.2 groups. Such embodiments of A.sup.3 include phenyl
phosphonamidate-alanate esters and phenyl phosphonate-lactate
esters: ##STR16##
[0133] Embodiments of R.sup.x include esters, carbamates,
carbonates, thioesters, amides, thioamides, and urea groups:
##STR17##
[0134] Embodiments of A.sup.2 include where W.sup.3 is W.sup.5,
such as: ##STR18## Alternatively, A.sup.2 is phenyl, substituted
phenyl, benzyl, substituted benzyl, pyridyl or substituted
pyridyl.
[0135] In other embodiments W.sup.4 may be R.sup.4, W.sup.5a is a
carbocycle or heterocycle and W.sup.5a is optionally and
independently substituted with 1, 2, or 3 R.sup.2 groups. For
example, W.sup.5a may be 3,5-dichlorophenyl.
[0136] An embodiment of A.sup.1 is: ##STR19## n is an integer from
1 to 18;
[0137] An embodiment of A.sup.3 optionally is of the formula:
##STR20## and Y.sup.2c is O, --N(R.sup.y) or S. For example,
R.sup.1 may be H and n may be 1.
[0138] An embodiment of A.sup.1 optionally comprises a phosphonate
group attached to an imidazole nitrogen through a heterocycle
linker, such as: ##STR21## where Y.sup.2b is O or N(R.sup.2); and
M12d is 1, 2, 3, 4, 5, 6, 7 or 8. The A.sup.3 unit may be attached
at any of the W.sup.5 carbocycle or heterocycle ring atoms, e.g.
ortho, meta, or para on a disubstituted W.sup.5.
[0139] A.sup.1 optionally is
--(X.sub.2--(C(R.sub.2)(R.sub.2)).sub.m1--X.sub.3).sub.m1--W.sup.3,
and W.sup.3 is substituted with 1 to 3 A.sub.3 groups.
[0140] A.sub.2 optionally is
--(X.sub.2--(C(R.sub.2)(R.sub.2)).sub.m1--X.sub.3).sub.m1--W.sup.3.
[0141] A.sub.3 optionally is
--(X.sub.2--(C(R.sub.2)(R.sub.2)).sub.m1--X.sub.3).sub.m1--P(Y.sub.1)(Y.s-
ub.1R.sub.6a)(Y.sub.1R.sub.6a).
[0142] X.sub.2 and X.sub.3 optionally are independently a bond,
--O--, --N(R.sub.2)--, --N(OR.sub.2)--, --N(N(R.sub.2)(R.sub.2))--,
--S--, --SO--, or --SO2-.
[0143] Each Y.sub.1 optionally is independently O, --N(R.sub.2),
--N(OR.sub.2), or N(N(R.sub.2)(R.sub.2)), wherein each Y.sub.1 is
bound by two single bonds or one dot
[0144] R.sub.1 optionally is independently H or alkyl of 1 to 12
carbon atoms.
[0145] R.sub.2 optionally is independently H, R.sub.3 or R.sub.4
wherein each R.sub.4 is independently substituted with 0 to 3
R.sub.3 groups.
[0146] R.sub.3 optionally is independently F, Cl, Br, I, --CN,
N.sub.3, --NO.sub.2, --OR.sub.6a, --OR.sub.1, --N(R.sub.1).sub.2,
--N(R.sub.1)(R.sub.6b), --N(R.sub.6b).sub.2, --SR.sub.1,
--SR.sub.6a, --S(O)R.sub.1, --S(O).sub.2R.sub.1, --S(O)OR.sub.1,
--S(O)OR.sub.6a, --S(O).sub.2OR.sub.1, --S(O).sub.2OR.sub.6a,
--C(O)OR.sub.1, --C(O)R.sub.6c, --C(O)OR.sub.6a, --OC(O)R.sub.1,
--N(R.sub.1)(C(O)R.sub.1), --N(R.sub.6b)(C(O)R.sub.1),
--N(R.sub.1)(C(O)OR.sub.1), --N(R.sub.6b)(C(O)OR.sub.1),
--C(O)N(R.sub.1).sub.2, --C(O)N(R.sub.6b)(R.sub.1),
--C(O)N(R.sub.6b).sub.2, --C(NR.sub.1)(N(R.sub.1).sub.2),
--C(N(R.sub.6b))(N(R.sub.1).sub.2),
--C(N(R.sub.1))(N(R.sub.1)(R.sub.6b)),
--C(N(R.sub.6b))(N(R.sub.1)(R.sub.6b)),
--C(N(R.sub.1))(N(R.sub.6b).sub.2),
--C(N(R.sub.6b))(N(R.sub.6b).sub.2),
--N(R.sub.1)C(N(R.sub.1))(N(R.sub.1).sub.2),
--N(R.sub.1)C(N(R.sub.1))(N(R.sub.1)(R.sub.6b)),
--N(R.sub.1)C(N(R.sub.6b))(N(R.sub.1).sub.2),
--N(R.sub.6b)C(N(R.sub.1))(N(R.sub.1).sub.2),
--N(R.sub.6b)C(N(R.sub.6b))(N(R.sub.1).sub.2),
--N(R.sub.6b)C(N(R.sub.1))(N(R.sub.1)(R.sub.6b)),
--N(R.sub.1)C(N(R.sub.6b))(N(R.sub.1)(R.sub.6b)),
--N(R.sub.1)C(N(R.sub.1))(N(R.sub.6b).sub.2),
--N(R.sub.6b)C(N(R.sub.6b))(N(R.sub.1)(R.sub.6b)),
--N(R.sub.6b)C(N(R.sub.1))(N(R.sub.6b).sub.2),
--N(R.sub.1)C(N(R.sub.6b))(N(R.sub.6b).sub.2),
--N(R.sub.6b)C(N(R.sub.6b))(N(R.sub.6b).sub.2), .dbd.O, .dbd.S,
.dbd.N(R.sub.1), .dbd.N(R.sub.6b) or W.sub.5.
[0147] R.sub.4 optionally is independently alkyl of 1 to 12 carbon
atoms, alkenyl of 2 to 12 carbon atoms, or alkynyl of 2 to 12
carbon atoms.
[0148] R.sub.5 optionally is independently R.sub.4 wherein each
R.sub.4 is substituted with 0 to 3 R.sub.3 groups; or R.sub.5 is
independently alkylene of 1 to 12 carbon atoms, alkenylene of 2 to
12 carbon atoms, or alkynylene of 2-12 carbon atoms any one of
which alkylene, alkenylene or alkynylene is substituted with 0-3
R.sub.3 groups.
[0149] R.sub.6a is independently H or an ether- or ester-forming
group.
[0150] R.sub.6b is independently H, a protecting group for amino or
the residue of a carboxyl-containing compound.
[0151] R.sub.6c is independently H or the residue of an
amino-containing compound.
[0152] W.sub.4 is R.sub.5, --C(Y.sub.1)R.sub.5,
--C(Y.sub.1)W.sub.5, --SO.sub.2R.sub.5, or --SO.sub.2W.sup.5.
[0153] W.sub.5 is carbocycle or heterocycle wherein W.sub.5 is
independently substituted with 0 to 3 R.sub.2 groups.
[0154] m1 is independently an integer from 0 to 12, wherein the sum
of all m1's within each individual embodiment of A.sub.1, A.sub.2
or A.sub.3 is 12 or less.
[0155] m2 is independently an integer from 0 to 2.
[0156] In another embodiment A.sub.1 is
--(C(R.sub.2)(R.sub.2)).sub.m1--W.sup.3, wherein W.sub.3 is
substituted with 1 A.sub.3 group, A.sub.2 is
--(C(R.sub.2)(R.sub.2)).sub.m1--W.sub.3, and A.sub.3 is
--(C(R.sub.2)(R.sub.2)).sub.m1P(Y.sub.1)(Y.sub.1R.sub.6a)(Y.sub.1R.sub.6a-
).
[0157] In an embodiment A.sup.1 is of the formula: ##STR22##
[0158] In an embodiment A.sup.1 is of the formula: ##STR23##
[0159] In an embodiment A.sup.1 is of the formula: ##STR24##
[0160] In an embodiment A.sup.1 is of the formula: ##STR25##
[0161] and W.sup.5a is a carbocycle or a heterocycle where W.sup.5a
is independently substituted with 0 or 1 R.sup.2 groups.
[0162] In an embodiment M12a is 1.
[0163] In an embodiment A.sup.3 is of the formula: ##STR26##
[0164] In an embodiment A.sup.3 is of the formula: ##STR27##
[0165] In an embodiment A.sup.3 is of the formula: ##STR28##
[0166] Y.sup.1a is O or S; and
[0167] Y.sup.2a is O, --N(R.sup.x) or S.
[0168] In an embodiment A.sup.3 is of the formula: ##STR29##
[0169] and Y.sup.2b is O or N(R.sup.x).
[0170] In an embodiment A.sup.3 is of the formula: ##STR30##
[0171] Y.sup.2b is O or N(R.sup.x); and
[0172] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0173] In an embodiment A.sup.3 is of the formula: ##STR31##
[0174] Y.sup.2b is O or N(R.sup.x); and
[0175] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0176] In an embodiment M12d is 1.
[0177] In an embodiment A.sup.3 is of the formula: ##STR32##
[0178] In an embodiment A.sup.3 is of the formula: ##STR33##
[0179] In an embodiment W.sup.5 is a carbocycle.
[0180] In an embodiment A.sup.3 is of the formula: ##STR34##
[0181] In an embodiment W.sup.5 is phenyl.
[0182] In an embodiment M12b is 1.
[0183] In an embodiment A.sup.3 is of the formula: ##STR35##
[0184] Y.sup.1a is O or S; and
[0185] Y.sup.2a is O, N(R.sup.x) or S.
[0186] In an embodiment A.sup.3 is of the formula: ##STR36##
[0187] and Y.sup.2b is O or N(R.sup.x).
[0188] In an embodiment A.sup.3 is of the formula: ##STR37##
[0189] Y.sup.2b is O or N(R.sup.x); and
[0190] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0191] In an embodiment R.sup.1 is H.
[0192] In an embodiment M12d is 1.
[0193] In an embodiment A.sup.3 is of the formula: ##STR38##
[0194] wherein the phenyl carbocycle is substituted with 0 to 3
R.sup.2 groups.
[0195] In an embodiment A.sup.3 is of the formula: ##STR39##
[0196] In an embodiment A.sup.3 is of the formula: ##STR40##
[0197] In an embodiment A.sup.3 is of the formula: ##STR41##
[0198] In an embodiment R.sup.x is of the formula: ##STR42##
[0199] In an embodiment R.sup.x is of the formula: ##STR43##
[0200] Y.sup.1a is O or S; and
[0201] Y.sup.2c is O, --N(R.sup.y) or S.
[0202] In an embodiment R.sup.x is of the formula: ##STR44##
[0203] Y.sup.1a is O or S; and
[0204] Y.sup.2d is O or N(R.sup.y).
[0205] In an embodiment R.sup.x is of the formula: ##STR45##
[0206] In an embodiment R.sup.x is of the formula: ##STR46##
[0207] In an embodiment R.sup.x is of the formula: ##STR47##
[0208] In an embodiment A.sup.3 is of the formula: ##STR48##
[0209] In an embodiment A.sup.3 is of the formula: ##STR49##
[0210] R.sup.x is of the formula: ##STR50##
[0211] In an embodiment A.sup.3 is of the formula: ##STR51##
[0212] Y.sup.1a is O or S; and
[0213] Y.sup.2a O, --N(R.sup.2) or S.
[0214] In an embodiment A.sup.3 is of the formula: ##STR52##
[0215] Y.sup.1a is O or S;
[0216] Y.sup.2b is O or N(R.sup.2); and
[0217] Y.sup.2c is O, --N(R.sup.y) or S.
[0218] In an embodiment A.sup.3 is of the formula: ##STR53##
[0219] Y.sup.1a is O or S;
[0220] Y.sup.2b is O or N(R.sup.2);
[0221] Y.sup.2d is O or N(R.sup.y); and
[0222] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0223] In an embodiment A.sup.3 is of the formula: ##STR54##
[0224] Y.sup.2b is O or N(R.sup.2); and
[0225] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0226] In an embodiment A.sup.3 is of the formula: ##STR55##
[0227] and Y.sup.2b is O or N(R.sup.2).
[0228] In an embodiment A.sup.3 is of the formula: ##STR56##
[0229] In an embodiment is of the formula: ##STR57##
[0230] In an embodiment A.sup.3 is of the formula: ##STR58##
##STR59##
[0231] Y.sup.1a is O or S; and
[0232] Y.sup.2a is O, --N(R.sup.2) or S.
[0233] In an embodiment A.sup.3 is of the formula: ##STR60##
[0234] Y.sup.1a is O or S;
[0235] Y.sup.2b is O or N(R.sup.2); and
[0236] Y.sup.2c is O, --N(R.sup.y) or S.
[0237] In an embodiment A.sup.3 is of the formula: ##STR61##
[0238] Y.sup.1a is O or S;
[0239] Y.sup.2b is O or N(R.sup.2);
[0240] Y.sup.2d is O or N(R.sup.y); and
[0241] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0242] In an embodiment A.sup.3 is of the formula: ##STR62##
[0243] Y.sup.2b is O or N(R.sup.2); and
[0244] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0245] In an embodiment A.sup.3 is of the formula: ##STR63##
[0246] and Y.sup.2b is O or N(R.sup.2).
[0247] In an embodiment A.sup.1 is of the formula: ##STR64##
[0248] A.sup.3 is of the formula: ##STR65##
[0249] In an embodiment A.sup.1 is of the formula: ##STR66##
[0250] A.sup.3 is of the formula: ##STR67##
[0251] R.sup.x is of the formula: ##STR68##
[0252] In an embodiment A.sup.1 is of the formula: ##STR69##
[0253] A.sup.3 is of the formula: ##STR70##
[0254] Y.sup.1a is O or S; and
[0255] Y.sup.2a is O, --N(R.sup.2) or S.
[0256] In an embodiment A.sup.1 is of the formula: ##STR71##
[0257] W.sup.5a is a carbocycle independently substituted with 0 or
1 R.sup.2 groups;
[0258] A.sup.3 is of the formula: ##STR72##
[0259] Y.sup.1a is O or S;
[0260] Y.sup.2b is O or N(R.sup.2); and
[0261] Y.sup.2c is O, --N(R.sup.y) or S.
[0262] In an embodiment A.sup.1 is of the formula: ##STR73##
[0263] W.sup.5a is a carbocycle independently substituted with 0 or
1 R.sup.2 groups;
[0264] A.sup.3 is of the formula: ##STR74##
[0265] Y.sup.1a is O or S;
[0266] Y.sup.2b is O or N(R.sup.2);
[0267] Y.sup.2d is O or N(R.sup.y); and
[0268] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0269] In an embodiment A.sup.1 is of the formula: ##STR75##
[0270] Y.sup.2b is O or N(R.sup.2); and
[0271] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0272] In an embodiment A.sup.1 is of the formula: ##STR76##
[0273] A.sup.3 is of the formula: ##STR77##
[0274] In an embodiment A.sup.1 is of the formula: ##STR78##
[0275] A.sup.3 is of the formula: ##STR79##
[0276] R.sup.x is of the formula: ##STR80##
[0277] In an embodiment A.sup.1 is of the formula: ##STR81##
[0278] A.sup.3 is of the formula: ##STR82##
[0279] Y.sup.1a is O or S; and
[0280] Y.sup.2a is O, N(R.sup.2) or S.
[0281] In an embodiment A.sup.1 is of the formula: ##STR83##
[0282] W.sup.5a is a carbocycle independently substituted with 0 or
1 R.sup.2 groups;
[0283] A.sup.3 is of the formula: ##STR84##
[0284] Y.sup.1a is O or S;
[0285] Y.sup.2b is O or N(R.sup.2); and
[0286] Y.sup.2c is O, --N(R.sup.y) or S.
[0287] In an embodiment A.sup.3 is of the formula: ##STR85##
[0288] wherein the phenyl carbocycle is substituted with 0 to 3
R.sup.2 groups.
[0289] In an embodiment A.sup.1 is of the formula: ##STR86##
[0290] W.sup.5a is a carbocycle or heterocycle where W.sup.5a is
independently substituted with 0 or 1 R.sup.2 groups;
[0291] A.sup.3 is of the formula: ##STR87##
[0292] Y.sup.1a is O or S;
[0293] Y.sup.2b is O or N(R.sup.2);
[0294] Y.sup.2d is O or N(R.sup.y); and
[0295] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0296] In an embodiment A.sup.1 is of the formula: ##STR88##
[0297] Y.sup.2b is O or N(R.sup.2); and
[0298] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0299] In an embodiment A.sup.2 is of the formula: ##STR89##
[0300] In an embodiment A.sup.2 is of the formula: ##STR90##
[0301] In an embodiment M12b is 1.
[0302] In an embodiment M12b is 0, Y.sup.2 is a bond and W.sup.5 is
a carbocycle or heterocycle where W.sup.5 is optionally and
independently substituted with 1, 2, or 3 R.sup.2 groups.
[0303] In an embodiment A.sup.2 is of the formula: ##STR91##
[0304] and W.sup.5a is a carbocycle or heterocycle where W.sup.5a
is optionally and independently substituted with 1, 2, or 3 R.sup.2
groups.
[0305] In an embodiment M12a is 1.
[0306] In an embodiment A.sup.2 is selected from phenyl,
substituted phenyl, benzyl, substituted benzyl, pyridyl and
substituted pyridyl.
[0307] In an embodiment A.sup.2 is of the formula: ##STR92##
[0308] In an embodiment A.sup.2 is of the formula: ##STR93##
[0309] In an embodiment M12b is 1.
[0310] In an embodiment A.sup.1 is of the formula: ##STR94##
[0311] A.sup.3 is of the formula: ##STR95##
[0312] In an embodiment A.sup.3 is of the formula: ##STR96##
[0313] In an embodiment R.sup.x is of the formula: ##STR97##
[0314] In an embodiment A.sup.3 is of the formula: ##STR98##
[0315] In an embodiment R.sup.x is of the formula: ##STR99##
[0316] In an embodiment A.sup.3 is of the formula: ##STR100##
[0317] In an embodiment R.sup.4 is isopropyl.
[0318] In an embodiment A.sup.1 is of the formula: ##STR101##
[0319] A.sup.3 is of the formula: ##STR102##
[0320] and Y.sup.1a is O or S.
[0321] In an embodiment A.sup.3 is of the formula: ##STR103##
[0322] and Y.sup.2a is O, --N(R.sup.2) or S.
[0323] In an embodiment A.sup.3 is of the formula: ##STR104##
[0324] Y.sup.2b is O or N(R.sup.2); and
[0325] Y.sup.2c is O, --N(R.sup.y) or S.
[0326] In an embodiment A.sup.3 is of the formula: ##STR105##
[0327] Y.sup.1a is O or S;
[0328] Y.sup.2b is O or N(R.sup.2);
[0329] Y.sup.2d is O or N(R.sup.y); and
[0330] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0331] In an embodiment A.sup.1 is of the formula: ##STR106##
[0332] Y.sup.2b is O or N(R.sup.2); and
[0333] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0334] In an embodiment A.sup.1 is of the formula: ##STR107##
[0335] and Y.sup.2b is O or N(R.sup.2); and
[0336] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0337] In an embodiment A.sup.1 is of the formula: ##STR108##
[0338] n is an integer from 1 to 18; A.sup.3 is of the formula:
##STR109##
[0339] and Y.sup.2c is O, --N(R.sup.y) or S.
[0340] In an embodiment R.sup.1 is H and n is 1.
[0341] In an embodiment A.sup.1 is of the formula: ##STR110##
[0342] A.sup.3 is of the formula: ##STR111##
[0343] In an embodiment A.sup.3 is of the formula: ##STR112##
[0344] In an embodiment R.sup.x is of the formula: ##STR113##
[0345] In an embodiment A.sup.3 is of the formula: ##STR114##
[0346] In an embodiment R.sup.x is of the formula: ##STR115##
[0347] In an embodiment A.sup.3 is of the formula: ##STR116##
[0348] In an embodiment A.sup.2 is selected from: ##STR117##
[0349] where W.sup.5 is a carbocycle or a heterocycle and where
W.sup.5 is independently substituted with 0 to 3 R.sup.2
groups.
[0350] In an embodiment A.sup.3 is of the formula: ##STR118##
[0351] and Y.sup.2a is O, --N(R.sup.2) or S.
[0352] In an embodiment A.sup.3 is of the formula: ##STR119##
[0353] and Y.sup.2c is O, --N(R.sup.y) or S.
[0354] In an embodiment A.sup.1 is of the formula: ##STR120##
[0355] A.sup.3 is of the formula: ##STR121##
[0356] W.sup.5a is a carbocycle or a heterocycle where the
carbocycle or heterocycle is independently substituted with 0 to 3
R.sup.2 groups;
[0357] Y.sup.2b is O or N(R.sup.2); and
[0358] Y.sup.2c is O, --N(R.sup.y) or S.
[0359] In an embodiment A.sup.1 is of the formula: ##STR122##
[0360] A.sup.3 is of the formula: ##STR123##
[0361] Y.sup.1a is O or S;
[0362] Y.sup.2b is O or N(R.sup.2);
[0363] Y.sup.2d is O or N(R.sup.y); and
[0364] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0365] In an embodiment A.sup.1 is of the formula: ##STR124##
[0366] Y.sup.2b is O or N(R.sup.2); and
[0367] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0368] In an embodiment A.sup.1 is of the formula: ##STR125##
[0369] and Y.sup.2b is O or N(R.sup.2); and
[0370] M12d is 1, 2, 3, 4, 5, 6, 7 or 8.
[0371] In an embodiment A.sup.2 is a phenyl substituted with 0 to 3
R.sup.2 groups.
[0372] In an embodiment W.sup.4 is of the formula: ##STR126##
[0373] wherein n is an integer from 1 to 18; and Y.sup.2b is O or
N(R.sup.2).
[0374] In an embodiment
[0375] A.sub.1 is
--(X.sub.2--(C(R.sub.2)(R.sub.2)).sub.m1--X.sub.3).sub.m1--W.sub.3,
wherein W.sub.3 is substituted with 1 to 3 A.sub.3 groups;
[0376] A.sub.2 is
--(X.sub.2--(C(R.sub.2)(R.sub.2)).sub.m1--X.sub.3).sub.m1--W.sup.3;
[0377] A.sub.3 is
--(X.sub.2--(C(R.sub.2)(R.sub.2)).sub.m1--X.sub.3).sub.m1--P(Y.sub.1)(Y.s-
ub.1R.sub.6a)(Y.sub.1R.sub.6a);
[0378] X.sub.2 and X.sub.3 are independently a bond, --O--,
--N(R.sub.2)--, --N(OR.sub.2)--, --N(N(R.sub.2)(R.sub.2))--, --S--,
--SO--, or --SO.sub.2--;
[0379] each Y.sub.1 is independently O, --N(R.sub.2),
--N(OR.sub.2), or N(N(R.sub.2)(R.sub.2)), wherein each Y.sub.1 is
bound by two single bonds or one double bond;
[0380] R.sub.1 is independently H or alkyl of 1 to 12 carbon
atoms;
[0381] R.sub.2 is independently H, R.sub.3 or R.sub.4 wherein each
R.sub.4 is independently substituted with 0 to 3 R.sub.3
groups;
[0382] R.sub.3 is independently F, Cl, Br, I, --CN, N.sub.3,
--NO.sub.2, --OR.sub.6a, --OR.sub.1, --N(R.sub.1).sub.2,
--N(R.sub.1)(R.sub.6b), --N(R.sub.6b).sub.2, --SR.sub.1,
--SR.sub.6a, --S(O)R.sub.1, --S(O).sub.2R.sub.1, --S(O)OR.sub.1,
--S(O)OR.sub.6a, --S(O).sub.2OR.sub.1, --S(O).sub.2OR.sub.6a,
--C(O)OR.sub.1, --C(O)R.sub.6c, --C(O)OR.sub.6a, --OC(O)R.sub.1,
--N(R.sub.1)(C(O)R.sub.1), --N(R.sub.6b)(C(O)R.sub.1),
--N(R.sub.1)(C(O)OR.sub.1), --N(R.sub.6b)(C(O)OR.sub.1),
--C(O)N(R.sub.1).sub.2, --C(O)N(R.sub.6b)(R.sub.1),
--C(O)N(R.sub.6b).sub.2, --C(NR.sub.1)(N(R.sub.1).sub.2),
--C(N(R.sub.6b))(N(R.sub.1).sub.2),
--C(N(R.sub.1))(N(R.sub.1)(R.sub.6b)),
--C(N(R.sub.6b))(N(R.sub.1)(R.sub.6b)),
--C(N(R.sub.1))(N(R.sub.6b).sub.2),
--C(N(R.sub.6b))(N(R.sub.6b).sub.2),
--N(R.sub.1)C(N(R.sub.1))(N(R.sub.1).sub.2),
--N(R.sub.1)C(N(R.sub.1))(N(R.sub.1)(R.sub.6b)),
--N(R.sub.1)C(N(R.sub.6b))(N(R.sub.1).sub.2),
--N(R.sub.6b)C(N(R.sub.1))(N(R.sub.1).sub.2),
--N(R.sub.6b)C(N(R.sub.6b))(N(R.sub.1).sub.2),
--N(R.sub.6b)C(N(R.sub.1))(N(R.sub.1)(R.sub.6b)),
--N(R.sub.1)C(N(R.sub.6b))(N(R.sub.1)(R.sub.6b)),
--N(R.sub.1)C(N(R.sub.1))(N(R.sub.6b).sub.2),
--N(R.sub.6b)C(N(R.sub.6b))(N(R.sub.1)(R.sub.6b)),
--N(R.sub.6b)C(N(R.sub.1))(N(R.sub.6b).sub.2),
--N(R.sub.1)C(N(R.sub.6b))(R.sub.6b).sub.2),
--N(R.sub.6b)C(N(R.sub.6b))(N(R.sub.6b).sub.2), .dbd.O, .dbd.S,
.dbd.N(R.sub.1), .dbd.N(R.sub.6b) or W.sub.5;
[0383] R.sub.4 is independently alkyl of 1 to 12 carbon atoms,
alkenyl of 2 to 12 carbon atoms, or alkynyl of 2 to 12 carbon
atoms;
[0384] R.sub.5 is independently R.sub.4 wherein each R.sub.4 is
substituted with 0 to 3 R.sub.3 groups;
[0385] R.sub.5a is independently alkylene of 1 to 12 carbon atoms,
alkenylene of 2 to 12 carbon atoms, or alkynylene of 2-12 carbon
atoms any one of which alkylene, alkenylene or alkynylene is
substituted with 0-3 R.sub.3 groups;
[0386] R.sub.6a is independently H or an ether- or ester-forming
group;
[0387] R.sub.6b is independently H, a protecting group for amino or
the residue of a carboxyl-containing compound;
[0388] R.sub.6c is independently H or the residue of an
amino-containing compound;
[0389] W.sub.3 is W.sub.4 or W.sub.5;
[0390] W.sub.4 is R.sub.5, --C(Y.sub.1)R.sub.5,
--C(Y.sub.1)W.sub.5, --SO.sub.2R.sub.5, or --SO.sub.2W.sup.5;
[0391] W.sub.5 is carbocycle or heterocycle wherein W.sub.5 is
independently substituted with 0 to 3 R.sub.2 groups;
[0392] m1 is independently an integer from 0 to 12, wherein the sum
of all m1's within each individual embodiment of A.sub.1, A.sub.2
or A.sub.3 is 12 or less; and
[0393] m2 is independently an integer from 0 to 2.
[0394] In an embodiment
[0395] A.sub.1 is --(C(R.sub.2)(R.sub.2)).sub.m1--W.sub.3, wherein
W.sub.3 is substituted with 1 A.sub.3 group;
[0396] A.sub.2 is --(C(R.sub.2)(R.sub.2)).sub.m1--W.sub.3; and
[0397] A.sub.3 is
--(C(R.sub.2)(R.sub.2)).sub.m1--P(Y.sub.1)(Y.sub.1R.sub.6a)(Y.sub.1R.sub.-
6a).
Protecting Groups
[0398] The chemical substructure of a protecting group varies
widely. One function of a protecting group is to serve as
intermediates in the synthesis of the parental drug substance.
Chemical protecting groups and strategies for
protection/deprotection are well known in the art. See: "Protective
Groups in Organic Chemistry", Theodora W. Greene (John Wiley &
Sons, Inc., New York, 1991. Protecting groups are often utilized to
mask the reactivity of certain functional groups, to assist in the
efficiency of desired chemical reactions, e.g. making and breaking
chemical bonds in an ordered and planned fashion. Protection of
functional groups of nal group, such as the polarity, lipophilicity
(hydrophobicity), and other properties which can be measured by
common analytical tools. Chemically protected intermediates may
themselves be biologically active or inactive. Protected compounds
may also exhibit altered, and in some cases, optimized properties
in vitro and in vivo, such as passage through cellular membranes
and resistance to enzymatic degradation or sequestration. In this
role, protected compounds may in themselves exhibit therapeutic
activity and need not be limited to the role of chemical
intermediates or precursors. The protecting group need not be
physiologically acceptable upon deprotection, although in general
it is more desirable if such products are pharmacologically
innocuous a compound alters other physical properties besides the
reactivity of the protected function.
[0399] In the context of the present invention, embodiments of
protecting groups include prodrug moieties and chemical protecting
groups.
[0400] Protecting groups are available, commonly known and used,
and are optionally used to prevent side reactions with the
protected group during synthetic procedures, i.e. routes or methods
to prepare the compounds of the invention. For the most part the
decision as to which groups to protect, when to do so, and the
nature of the chemical protecting group "PRT" will be dependent
upon the chemistry of the reaction to be protected against (e.g.,
acidic, basic, oxidative, reductive or other conditions) and the
intended direction of the synthesis. The PRT groups do not need to
be, and generally are not, the same if the compound is substituted
with multiple PRT. In general, PRT will be used to protect
functional groups such as carboxyl hydroxyl or amino groups and to
thus prevent side reactions or to otherwise facilitate the
synthetic efficiency. The order of deprotection to yield free,
deprotected groups is dependent upon the intended direction of the
synthesis and the reaction conditions to be encountered, and may
occur in any order as determined by the artisan.
[0401] Various functional groups of the compounds of the invention
may be protection. For example, protecting groups for --OH groups
(whether hydroxyl, carboxylic acid, phosphonic acid, or other
functions) are embodiments of "ether- or ester-forming groups".
Ether- or ester-forming groups are capable of functioning as
chemical protecting groups in the synthetic schemes set forth
herein. However, some hydroxyl and thio protecting groups are
neither ether- nor ester-forming groups, as will be understood by
those skilled in the art, and are included with amides, discussed
below.
[0402] A very large number of hydroxyl protecting groups and
amide-forming groups and corresponding chemical cleavage reactions
are described in "Protective Groups in Organic Chemistry", Theodora
W. Greene (John Wiley & Sons, Inc., New York, 1991, ISBN
0-471-62301-6) ("Greene"). See also Kocienski Philip J.;
"Protecting Groups" (Georg Thieme Verlag Stuttgart, N.Y., 1994),
which is incorporated by reference in its entirety herein. In
particular Chapter 1, Protecting Groups: An Overview, pages 1-20,
Chapter 2, Hydroxyl Protecting Groups, pages 21-94, Chapter 3, Diol
Protecting Groups, pages 95-117, Chapter 4, Carboxyl Protecting
Groups, pages 118-154, Chapter 5, Carbonyl Protecting Groups, pages
155-184. For protecting groups for carboxylic acid, phosphonic
acid, phosphonate, sulfonic acid and other protecting groups for
acids see Greene as set forth below. Such groups include by way of
example and not limitation, esters, amides, hydrazides, and the
like.
Ether- and Ester-Forming Protecting Groups
[0403] Ester-forming groups include: (1) phosphonate ester-forming
groups, such as phosphonamidate esters, phosphorothioate esters,
phosphonate esters, and phosphon-bis-amidates; (2) carboxyl
ester-forming groups, and (3) sulphur ester-forming groups, such as
sulphonate, sulfate, and sulfinate.
[0404] The phosphonate moieties of the compounds of the invention
may or may not be prodrug moieties, i.e. they may or may be
susceptible to hydrolytic or enzymatic cleavage or modification.
Certain phosphonate moieties are stable under most or nearly all
metabolic conditions. For example, a dialkylphosphonate, where the
alkyl groups are two or more carbons, may have appreciable
stability in vivo due to a slow rate of hydrolysis.
[0405] Within the context of phosphonate prodrug moieties, a large
number of structurally-diverse prodrugs have been described for
phosphonic acids (Freeman and Ross in Progress in Medicinal
Chemistry 34: 112-147 (1997) and are included within the scope of
the present invention. An exemplary embodiment of a phosphonate
ester-forming group is the phenyl carbocycle in substructure
A.sub.3 having the formula: ##STR127##
[0406] wherein m1 is 1, 2, 3, 4, 5, 6, 7 or 8, and the phenyl
carbocycle is substituted with 0 to 3 R.sub.2 groups. Also, in this
embodiment, where Y.sub.1 is O, a lactate ester is formed.
Alternatively, where Y.sub.1 is N(R.sub.2), --N(OR.sub.2) or
N(N(R.sub.2).sub.2, then phosphonamidate esters result. R.sub.1 may
be H or C.sub.1-C.sub.12 alkyl.
[0407] In its ester-forming role, a protecting group typically is
bound to any acidic group such as, by way of example and not
limitation, a --CO.sub.2H or --C(S)OH group, thereby resulting in
--CO.sub.2R.sup.x where R.sup.x is defined herein. Also, R.sup.x
for example includes the enumerated ester groups of WO
95/07920.
[0408] Examples of protecting groups include:
[0409] C.sub.3-C.sub.12 heterocycle (described above) or aryl.
These aromatic groups optionally are polycyclic or monocyclic.
Examples include phenyl, spiryl, 2- and 3-pyrrolyl, 2- and
3-thienyl, 2- and 4-imidazolyl, 2-, 4- and 5-oxazolyl, 3- and
4-isoxazolyl, 2-, 4- and 5-thiazolyl, 3-, 4- and 5-isothiazolyl, 3-
and 4-pyrazolyl 1-, 2-, 3- and 4-pyridinyl, and 1-, 2-, 4- and
5-pyrimidinyl, C.sub.3-C.sub.12 heterocycle or aryl substituted
with halo, R.sup.1, R.sup.1--O--C.sub.1-C.sub.12 alkylene,
C.sub.1-C.sub.12 alkoxy, CN, NO.sub.2, OH, carboxy, carboxyester,
thiol, thioester, C.sub.1-C.sub.12 haloalkyl (1-6 halogen atoms),
C.sub.2-C.sub.12 alkenyl or C.sub.2-C.sub.12 alkynyl. Such groups
include 2-, 3- and 4-alkoxyphenyl (C.sub.1-C.sub.12 alkyl), 2-, 3-
and 4-methoxyphenyl, 2-, 3- and 4-ethoxyphenyl, 2,3-, 2,4-, 2,5-,
2,6-, 3,4- and 3,5-diethoxyphenyl, 2- and
3-carboethoxy-4-hydroxyphenyl, 2- and 3-ethoxy-4-hydroxyphenyl, 2-
and 3-ethoxy-5-hydroxyphenyl, 2- and 3-ethoxy-6-hydroxyphenyl, 2-,
3- and 4-O-acetylphenyl, 2-, 3- and 4-dimethylaminophenyl, 2-, 3-
and 4-methylmercaptophenyl 2-, 3- and 4-halophenyl (including 2-,
3- and 4-fluorophenyl and 2-, 3- and 4-chlorophenyl), 2,3-, 2,4-,
2,5-, 2,6-, 3,4- and 3,5-dimethylphenyl, 2,3-, 2,4-, 2,5-, 2,6-,
3,4- and 3,5-biscarboxyethylphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4-
and 3,5-dimethoxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and
3,5-dihalophenyl (including 2,4-difluorophenyl and
3,5-difluorophenyl), 2-, 3- and 4-haloalkylphenyl (1 to 5 halogen
atoms, C.sub.1-C.sub.12 alkyl including 4-trifluoromethylphenyl),
2-, 3- and 4-cyanophenyl, 2-, 3- and 4-nitrophenyl, 2-, 3- and
4-haloalkylbenzyl (1 to 5 halogen atoms, C.sub.1-C.sub.12 alkyl
including 4-trifluoromethylbenzyl and 2-, 3- and
4-trichloromethylphenyl and 2-, 3- and 4-trichloromethylphenyl),
4-N-methylpiperidinyl, 3-N-methylpiperidinyl, 1-ethylpiperazinyl,
benzyl, alkylsalicylphenyl (C.sub.1-C.sub.4 alkyl including 2-, 3-
and 4-ethylsalicylphenyl), 2-, 3- and 4-acetylphenyl,
1,8-dihydroxynaphthyl (--C.sub.10H.sub.6--OH) and aryloxy ethyl
[C.sub.6-C.sub.9 aryl (including phenoxy ethyl)],
2,2'-dihydroxybiphenyl, 2-, 3- and 4-N,N-dialkylaminophenol,
--C.sub.6H.sub.4CH.sub.2--N(CH.sub.3).sub.2, trimethoxybenzyl
triethoxybenzyl, 2-alkyl pyridinyl (C.sub.1-4 alkyl); ##STR128##
C.sub.4-C.sub.8 esters of 2-carboxyphenyl; and C.sub.1-C.sub.4
alkylene-C.sub.3-C.sub.6 aryl (including benzyl,
--CH.sub.2-pyrrolyl, --CH.sub.2-thienyl, --CH.sub.2-imidazolyl,
--CH.sub.2-oxazolyl, --CH.sub.2-isoxazolyl, --CH.sub.2-thiazolyl,
--CH.sub.2-isothiazolyl, --CH.sub.2-pyrazolyl, --CH.sub.2-pyridinyl
and --CH.sub.2-pyrimidinyl) substituted in the aryl moiety by 3 to
5 halogen atoms or 1 to 2 atoms or groups selected from halogen,
C.sub.1-C.sub.12 alkoxy (including methoxy and ethoxy), cyano,
nitro, OH, C.sub.1-C.sub.12 haloalkyl (1 to 6 halogen atoms;
including --CH.sub.2CCl.sub.3), C.sub.1-C.sub.12 alkyl (including
methyl and ethyl), C.sub.2-C.sub.12 alkenyl or C.sub.2-C.sub.12
alkynyl; alkoxy ethyl [C.sub.1-C.sub.6 alkyl including
--CH.sub.2--CH.sub.2--O--CH.sub.3 (methoxy ethyl)]; alkyl
substituted by any of the groups set forth above for aryl, in
particular OH or by 1 to 3 halo atoms (including --CH.sub.3,
--CH(CH.sub.3).sub.2, --C(CH.sub.3).sub.3, --CH.sub.2CH.sub.3,
--(CH.sub.2).sub.2CH.sub.3, --(CH.sub.2).sub.3CH.sub.3,
--(CH.sub.2).sub.4CH.sub.3, --(CH.sub.2).sub.5CH.sub.3,
--CH.sub.2CH.sub.2F, --CH.sub.2CH.sub.2Cl, --CH.sub.2CF.sub.3, and
--CH.sub.2CCl.sub.3); ##STR129## --N-2-propylmorpholino,
2,3-dihydro-6-hydroxyindene, sesamol catechol monoester,
--CH.sub.2--C(O)--N(R.sup.1).sub.2, --CH.sub.2--S(O)(R.sup.1),
--CH.sub.2--S(O).sub.2(R.sup.1),
--CH.sub.2--CH(OC(O)CH.sub.2R.sup.1)--CH.sub.2(OC(O)CH.sub.2R.sup.1),
cholesteryl, enolpyruvate (HOOC--C(.dbd.CH.sub.2)--), glycerol;
[0410] a 5 or 6 carbon monosaccharide, disaccharide or
oligosaccharide (3 to 9 monosaccharide residues);
[0411] triglycerides such as .alpha.-D-.beta.-diglycerides (wherein
the fatty acids composing glyceride lipids generally are naturally
occurring saturated or unsaturated C.sub.6-26, C.sub.6-18 or
C.sub.6-10 fatty acids such as linoleic, lauric, myristic,
palmitic, stearic, oleic, palmitoleic, linolenic and the like fatty
acids) linked to acyl of the parental compounds herein through a
glyceryl oxygen of the triglyceride;
[0412] phospholipids linked to the carboxyl group through the
phosphate of the phospholipid;
[0413] phthalidyl (shown in FIG. 1 of Clayton et al., Antimicrob.
Agents Chemo. (1974) 5(6):670-671;
[0414] cyclic carbonates such as
(5-R.sub.d-2-oxo-1,3-dioxolen-4-yl) methyl esters (Sakamoto et al.,
Chem. Pharm. Bull. (1984) 32(6)2241-2248) where R.sub.d is R.sub.1,
R.sub.4 or aryl; and ##STR130##
[0415] The hydroxyl groups of the compounds of this invention
optionally are substituted with one of groups III, IV or V
disclosed in WO 94/21604, or with isopropyl.
[0416] As further embodiments, Table A lists examples of protecting
group ester moieties that for example can be bonded via oxygen to
--C(O)O-- and --P(O)(O--).sub.2 groups. Several amidates also are
shown, which are bound directly to --C(O)-- or --P(O).sub.2. Esters
of structures 1-5, 8-10 and 16, 17, 19-22 are synthesized by
reacting the compound herein having a free hydroxyl with the
corresponding halide (chloride or acyl chloride and the like) and
N,N-dicyclohexyl-N-morpholine carboxamidine (or another base such
as DBU, triethylamine, CsCO.sub.3, N,N-dimethylaniline and the
like) in DMF (or other solvent such as acetonitrile or
N-methylpyrrolidone). When the compound to be protected is a
phosphonate, the esters of structures 5-7, 11, 12, 21, and 23-26
are synthesized by reaction of the alcohol or alkoxide salt (or the
corresponding amines in the case of compounds such as 13, 14 and
15) with the monochlorophosphonate or dichlorophosphonate (or
another activated phosphonate). TABLE-US-00001 TABLE A 1.
--CH.sub.2--C(O)--N(R.sub.1).sub.2* 2. --CH.sub.2--S(O)(R.sub.1) 3.
--CH.sub.2--S(O).sub.2(R.sub.1) 4.
--CH.sub.2--O--C(O)--CH.sub.2--C.sub.6H.sub.5 5. 3-cholesteryl 6.
3-pyridyl 7. N-ethylmorpholino 8.
--CH.sub.2--O--C(O)--C.sub.6H.sub.5 9.
--CH.sub.2--O--C(O)--CH.sub.2CH.sub.3 10.
--CH.sub.2--O--C(O)--C(CH.sub.3).sub.3 11. --CH.sub.2--CCl.sub.3
12. --C.sub.6H.sub.5 13. --NH--CH.sub.2--C(O)O--CH.sub.2CH.sub.3
14. --N(CH.sub.3)--CH.sub.2--C(O)O--CH.sub.2CH.sub.3 15.
--NHR.sub.1 16. --CH.sub.2--O--C(O)--C.sub.10H.sub.15 17.
--CH.sub.2--O--C(O)--CH(CH.sub.3).sub.2 18.
--CH.sub.2--C#H(OC(O)CH.sub.2R.sub.1)--CH.sub.2--(OC(O)CH.sub.2R.sub.-
1)* 19. ##STR131## 20. ##STR132## 21. ##STR133## 22. ##STR134## 23.
##STR135## 24. ##STR136## 25. ##STR137## 26. ##STR138## #--chiral
center is (R), (S) or racemate.
[0417] Other esters that are suitable for use herein are described
in EP 632048.
[0418] Protecting groups also includes "double ester" forming
profunctionalities such as --CH.sub.2OC(O)OCH.sub.3, ##STR139##
--CH.sub.2SCOCH.sub.3, --CH.sub.2OCON(CH.sub.3).sub.2, or alkyl- or
aryl-acyloxyakyl groups of the structure --CH(R.sup.1 or
W.sup.5)O((CO)R.sup.37) or --CH(R.sup.1 or W.sup.5)((CO)OR.sup.38)
(linked to oxygen of the acidic group) wherein R.sup.37 and
R.sup.38 are alkyl, aryl, or alkylaryl groups (see U.S. Pat. No.
4,968,788). Frequently R.sup.37 and R.sup.38 are bulky groups such
as branched alkyl ortho-substituted aryl, meta-substituted aryl, or
combinations thereof, including normal, secondary, iso- and
tertiary alkyls of 1-6 carbon atoms. An example is the
pivaloyloxymethyl group. These are of particular use with prodrugs
for oral administration. Examples of such useful protecting groups
are alkylacyloxymethyl esters and their derivatives, including
--CH(CH.sub.2CH.sub.2OCH.sub.3)OC(O)C(CH.sub.3).sub.3, ##STR140##
CH.sub.2OC(P)C.sub.10H.sub.15, --CH.sub.2OC(O)C(CH.sub.3).sub.3,
--CH(CH.sub.2OCH.sub.3)OC(O)C(CH.sub.3).sub.3,
--CH(CH(CH.sub.3).sub.2)OC(O)C(CH.sub.3).sub.3,
--CH.sub.2OC(O)CH.sub.2CH(CH.sub.3).sub.2,
--CH.sub.2OC(O)C.sub.6H.sub.11, --CH.sub.2OC(P)C.sub.6H.sub.5,
--CH.sub.2OC(O)C.sub.10H.sub.15, --CH.sub.2OC(O)CH.sub.2CH.sub.3,
--CH.sub.2OC(O)CH(CH.sub.3).sub.2, --CH.sub.2OC(O)C(CH.sub.3).sub.3
and --CH.sub.2OC(O)CH.sub.2C.sub.6H.sub.5.
[0419] For prodrug purposes, the ester typically chosen is one
heretofore used for antibiotic drugs, in particular the cyclic
carbonates, double esters, or the phthalidyl, aryl or alkyl
esters.
[0420] In some embodiments the protected acidic group is an ester
of the acidic group and is the residue of a hydroxyl-containing
functionality. In other embodiments, an amino compound is used to
protect the acid functionality. The residues of suitable hydroxyl
or amino-containing functionalities are set forth above or are
found in WO 95/07920. Of particular interest are the residues of
amino acids, amino acid esters, polypeptides, or aryl alcohols.
Typical amino acid, polypeptide and carboxyl-esterified amino acid
residues are described on pages 11-18 and related text of WO
95/07920 as groups L1 or L2. WO 95/07920 expressly teaches the
amidates of phosphonic acids, but it will be understood that such
amidates are formed with any of the acid groups set forth herein
and the amino acid residues set forth in WO 95/07920.
[0421] Typical esters for protecting acidic functionalities are
also described in WO 95/07920, again understanding that the same
esters can be formed with the acidic groups herein as with the
phosphonate of the '920 publication. Typical ester groups are
defined at least on WO 95/07920 pages 89-93 (under R.sup.31 or
R.sup.35), the table on page 105, and pages 21-23 (as R). Of
particular interest are esters of unsubstituted aryl such as phenyl
or arylalkyl such benzyl, or hydroxy-, halo-, alkoxy-, carboxy-
and/or alkylestercarboxy-substituted aryl or alkylaryl, especially
phenyl ortho-ethoxyphenyl, or C.sub.1-C.sub.4
alkylestercarboxyphenyl (salicylate C.sub.1-C.sub.12
alkylesters).
[0422] The protected acidic groups, particularly when using the
esters or amides of WO 95/07920, are useful as prodrugs for oral
administration. However, it is not essential that the acidic group
be protected in order for the compounds of this invention to be
effectively administered by the oral route. When the compounds of
the invention having protected groups, in particular amino acid
amidates or substituted and unsubstituted aryl esters are
administered systemically or orally they are capable of hydrolytic
cleavage in vivo to yield the free acid.
[0423] One or more of the acidic hydroxyls are protected. If more
than one acidic hydroxyl is protected then the same or a different
protecting group is employed, e.g., the esters may be different or
the same, or a mixed amidate and ester may be used.
[0424] Typical hydroxy protecting groups described in Greene (pages
14-118) include substituted methyl and alkyl ethers, substituted
benzyl ethers, silyl ethers, esters including sulfonic acid esters,
and carbonates. For example: [0425] Ethers (methyl, t-butyl,
allyl); [0426] Substituted Methyl Ethers (Methoxymethyl,
Methylthiomethyl, t-Butylthiomethyl,
(Phenyldimethylsilyl)methoxymethyl, Benzyloxymethyl,
p-Methoxybenzyloxymethyl, (4-Methoxyphenoxy)methyl, Guaiacolmethyl,
t-Butoxymethyl, 4-Pentenyloxymethyl, Siloxymethyl,
2-Methoxyethoxymethyl, 2,2,2-Trichloroethoxymethyl,
Bis(2-chloroethoxy)methyl, 2-(Trimethylsilyl)ethoxymethyl,
Tetrahydropyranyl, 3-Bromotetrahydropyranyl,
Tetrahydropthiopyranyl, 1-Methoxycyclohexyl,
4-Methoxytetrahydropyranyl, 4-Methoxytetrahydrothiopyranyl,
4-Methoxytetrahydropthiopyranyl S,S-Dioxido,
1-[(2-Chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl,
1,4-Dioxan-2-yl, Tetrahydrofuranyl, Tetrahydrothiofuranyl,
2,3,3a,4,5,6,7,7a-Octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl));
[0427] Substituted Ethyl Ethers (1-Ethoxyethyl,
1-(2-Chloroethoxy)ethyl, 1-Methyl-1-methoxyethyl,
1-Methyl-1-benzyloxyethyl, 1-Methyl-1-benzyloxy-2-fluoroethyl,
2,2,2-Trichloroethyl, 2-Trimethylsilylethyl,
2-(Phenylselenyl)ethyl, [0428] p-Chlorophenyl, p-Methoxyphenyl,
2,4-Dinitrophenyl, Benzyl); [0429] Substituted Benzyl Ethers
(p-Methoxybenzyl, 3,4-Dimethoxybenzyl, o-Nitrobenzyl,
p-Nitrobenzyl, p-Halobenzyl, 2,6-Dichlorobenzyl, p-Cyanobenzyl,
p-Phenylbenzyl, 2- and 4-Picolyl, 3-Methyl-2-picolyl N-Oxido,
Diphenylmethyl, p',p'-Dinitrobenzhydryl, 5-Dibenzosuberyl,
Triphenylmethyl, .alpha.-Naphthyldiphenylmethyl,
p-methoxyphenyldiphenylmethyl, Di(p-methoxyphenyl)phenylmethyl,
Tri(p-methoxyphenyl)methyl,
4-(4'-Bromophenacyloxy)phenyldiphenylmethyl,
4,4',4''-Tris(4,5-dichlorophthalimidophenyl)methyl,
4,4',4''-Tris(levulinoyloxyphenyl)methyl,
4,4',4''-Tris(benzoyloxyphenyl)methyl,
3-(Imidazol-1-ylmethyl)bis(4',4''-dimethoxyphenyl)methyl,
1,1-Bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-Anthryl,
9-(9-Phenyl)xanthenyl, 9-(9-Phenyl-10-oxo)anthryl,
1,3-Benzodithiolan-2-yl, Benzisothiazolyl S,S-Dioxido); [0430]
Silyl Ethers (Trimethylsilyl, Triethylsilyl, Triisopropylsilyl,
Dimethylisopropylsilyl, Diethylisopropylsilyl, Dimethylthexylsilyl,
t-Butyldimethylsilyl, t-Butyldiphenylsilyl, Tribenzylsilyl,
Tri-p-xylylsilyl, Triphenylsilyl, Diphenylmethylsilyl,
t-Butylmethoxyphenylsilyl); [0431] Esters (Formate, Benzoylformate,
Acetate, Choroacetate, Dichloroacetate, Trichloroacetate,
Trifluoroacetate, Methoxyacetate, Triphenylmethoxyacetate,
Phenoxyacetate, p-Chlorophenoxyacetate, p-poly-Phenylacetate,
3-Phenylpropionate, 4-Oxopentanoate (Levulinate),
4,4-(Ethylenedithio)pentanoate, Pivaloate, Adamantoate, Crotonate,
4-Methoxycrotonate, Benzoate, p-Phenylbenzoate,
2,4,6-Trimethylbenzoate (Mesitoate)); [0432] Carbonates (Methyl,
9-Fluorenylmethyl, Ethyl, 2,2,2-Trichloroethyl,
2-(Trimethylsilyl)ethyl, 2-(Phenylsulfonyl)ethyl,
2-(Triphenylphosphonio)ethyl, Isobutyl, Vinyl, Allyl,
p-Nitrophenyl, Benzyl, p-Methoxybenzyl, 3,4-Dimethoxybenzyl,
o-Nitrobenzyl, p-Nitrobenzyl, S-Benzyl Thiocarbonate,
4-Ethoxy-1-naphthyl, Methyl Dithiocarbonate); [0433] Groups With
Assisted Cleavage (2-Iodobenzoate, 4-Azidobutyrate,
4-Nitro-4-methylpentanoate, o-(Dibromomethyl)benzoate,
2-Formylbenzenesulfonate, 2-(Methylthiomethoxy)ethyl Carbonate,
4-(Methylthiomethoxy)butyrate,
2-(Methylthiomethoxymethyl)benzoate); Miscellaneous Esters
(2,6-Dichloro-4-methylphenoxyacetate, 2,6-Dichloro-4-(1,1,3,3
tetramethylbutyl)phenoxyacetate,
2,4-Bis(1,1-dimethylpropyl)phenoxyacetate, Chlorodiphenylacetate,
Isobutyrate, Monosuccinate, (E)-2-Methyl-2-butenoate (Tigloate),
o-(Methoxycarbonyl)benzoate, p-poly-Benzoate, .alpha.-Naphthoate,
Nitrate, Alkyl N,N,N',N'-Tetramethylphosphorodiamidate,
N-Phenylcarbamate, Borate, Dimethylphosphinothioyl,
2,4-Dinitrophenylsulfenate); and [0434] Sulfonates (Sulfate,
Methanesulfonate (Mesylate), Benzylsulfonate, Tosylate). [0435]
Typical 1,2-diol protecting groups (thus, generally where two OH
groups are taken together with the protecting functionality) are
described in Greene at pages 118-142 and include Cyclic Acetals and
Ketals (Methylene, Ethylidene, 1-t-Butylethylidene,
1-Phenylethylidene, (4-Methoxyphenyl)ethylidene,
2,2,2-Trichloroethylidene, Acetonide (Isopropylidene),
Cyclopentylidene, Cyclohexylidene, Cycloheptylidene, Benzylidene,
p-Methoxybenzylidene, 2,4-Dimethoxybenzylidene,
3,4-Dimethoxybenzylidene, 2-Nitrobenzylidene); Cyclic Ortho Esters
(Methoxymethylene, Ethoxymethylene, Dimethoxymethylene,
1-Methoxyethylidene, 1-Ethoxyethylidine, 1,2-Dimethoxyethylidene,
.alpha.-Methoxybenzylidene, 1-(N,N-Dimethylamino)ethylidene
Derivative, .alpha.-(N,N-Dimethylamino)benzylidene Derivative,
2-Oxacyclopentylidene); Silyl Derivatives (Di-t-butylsilylene
Group, 1,3-(1,1,3,3-Tetraisopropyldisiloxanylidene), and
Tetra-t-butoxydisiloxane-1,3-diylidene), Cyclic Carbonates, Cyclic
Boronates, Ethyl Boronate and Phenyl Boronate.
[0436] More typically, 1,2-diol protecting groups include those
shown in Table B, still more typically, epoxides, acetonides,
cyclic ketals and aryl acetals. TABLE-US-00002 TABLE B ##STR141##
##STR142## ##STR143## ##STR144## ##STR145## ##STR146## ##STR147##
##STR148## ##STR149## ##STR150## ##STR151## wherein R.sup.9 is
C.sub.1-C.sub.6 alkyl.
Amino Protecting Groups
[0437] Another set of protecting groups include any of the typical
amino protecting groups described by Greene at pages 315-385. They
include: [0438] Carbamates: (methyl and ethyl, 9-fluorenylmethyl,
9(2-sulfo)fluorenylmethyl, 9-(2,7-dibromo)fluorenylmethyl
2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl
4-methoxyphenacyl); [0439] Substituted Ethyl:
(2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-phenylethyl,
1-(1-adamantyl)-1-methylethyl, 1,1-dimethyl-2-haloethyl,
1,1-dimethyl-2,2-dibromoethyl, 1,1-dimethyl-2,2,2-trichloroethyl,
1-methyl-1-(4-biphenylyl)ethyl,
1-(3,5-di-t-butylphenyl)-1-methylethyl 2-(2'- and 4-pyridyl)ethyl,
2-(N,N-dicyclohexylcarboxamido)ethyl, t-butyl, 1-adamantyl, vinyl,
allyl, 1-isopropylallyl, cinnamyl, 4-nitrocinnamyl, 8-quinolyl,
N-hydroxypiperidinyl alkyldithio, benzyl, p-methoxybenzyl,
p-nitrobenzyl, p-bromobenzyl, p-chlorobenzyl 2,4-dichlorobenzyl,
4-methylsulfinylbenzyl, 9-anthrylmethyl, diphenylmethyl); [0440]
Groups With Assisted Cleavage: (2-methylthioethyl,
2-methylsulfonylethyl, 2-(p-toluenesulfonyl)ethyl,
[2-(1,3-dithianyl)]methyl, 4-methylthiophenyl,
2,4-dimethylthiophenyl, 2-phosphonioethyl,
2-triphenylphosphonioisopropyl, 1,1-dimethyl-2-cyanoethyl,
m-choro-p-acyloxybenzyl, p-(dihydroxyboryl)benzyl,
5-benzisoxazolylmethyl, 2-(trifluoromethyl)-6-chromonylmethyl);
[0441] Groups Capable of Photolytic Cleavage: (m-nitrophenyl,
3,5-dimethoxybenzyl, o-nitrobenzyl, 3,4-dimethoxy-6-nitrobenzyl,
phenyl(o-nitrophenyl)methyl); Urea-Type Derivatives
(phenothiazinyl-(10)-carbonyl, N'-p-toluenesulfonylaminocarbonyl,
N'phenylaminothiocarbonyl); [0442] Miscellaneous Carbamates:
(t-amyl S-benzyl thiocarbamate, p-cyanobenzyl, cyclobutyl
cyclohexyl, cyclopentyl, cyclopropylmethyl p-decyloxybenzyl
diisopropylmethyl, 2,2-dimethoxycarbonylvinyl
o-(N,N-dimethylcarboxamido)benzyl,
1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl,
1,1-dimethylpropynyl, di(2-pyridyl)methyl, 2-furanylmethyl,
2-Iodoethyl, Isobornyl, Isobutyl, Isonicotinyl,
p-(p'-Methoxyphenylazo)benzyl, 1-methylcyclobutyl
1-methylcyclohexyl, 1-methyl-1-cyclopropylmethyl,
1-methyl-1-(3,5-dimethoxyphenyl)ethyl,
1-methyl-1-(p-phenylazophenyl)ethyl, 1-methyl-1-phenylethyl,
1-methyl-1-(4-pyridyl)ethyl, phenyl, p-(phenylazo)benzyl,
2,4,6-tri-t-butylphenyl, 4-(trimethylammonium)benzyl,
2,4,6-trimethylbenzyl); [0443] Amides: (N-formyl, N-acetyl,
N-choroacetyl, N-trichoroacetyl, N-trifluoroacetyl, N-phenylacetyl,
N-3-phenylpropionyl, N-picolinoyl, N-3-pyridylcarboxamide,
N-benzoylphenylalanyl, N-benzoyl, N-p-phenylbenzoyl); [0444] Amides
With Assisted Cleavage: (N-o-nitrophenylacetyl,
N-o-nitrophenoxyacetyl, N-acetoacetyl,
(N'-dithiobenzyloxycarbonylamino)acetyl,
N-3-(p-hydroxyphenyl)propionyl, N-3-(o-nitrophenyl)propionyl,
N-2-methyl-2-(o-nitrophenoxy)propionyl,
N-2-methyl-2-(o-phenylazophenoxy)propionyl, N-4-chlorobutyryl,
N-3-methyl-3-nitrobutyryl, N-o-nitrocinnamoyl, N-acetylmethionine,
N-o-nitrobenzoyl, N-o-(benzoyloxymethyl)benzoyl,
4,5-diphenyl-3-oxazolin-2-one); [0445] Cyclic Imide Derivatives:
(N-phthalimide, N-dithiasuccinoyl, N-2,3-diphenylmaleoyl,
N-2,5-dimethylpyrrolyl, N-1,1,4,4-tetramethyldisilylazacyclopentane
adduct, 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one,
5-substituted 1,3-dibenzyl-1,3-5-triazacyclohexan-2-one,
1-substituted 3,5-dinitro-4-pyridonyl); [0446] N-Alkyl and N-Aryl
Amines: (N-methyl, N-allyl, N-[2-(trimethylsilyl)ethoxy]methyl,
N-3-acetoxypropyl N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl),
Quaternary Ammonium Salts, N-benzyl, N-di(4-methoxyphenyl)methyl,
N-5-dibenzosuberyl, N-triphenylmethyl,
N-(4-methoxyphenyl)diphenylmethyl, N-9-phenylfluorenyl,
N-2,7-dichloro-9-fluorenylmethylene, N-ferrocenylmethyl
N-2-picolylamine N'-oxide); [0447] Imine Derivatives:
(N-1,1-dimethylthiomethylene, N-benzylidene, N-p-methoxybenylidene,
N-diphenylmethylene, N-[(2-pyridyl)mesityl]methylene,
N,(N',N'-dimethylaminomethylene, N,N'-isopropylidene,
N-p-nitrobenzylidene, N-salicylidene, N-5-chlorosalicylidene,
N-(5-chloro-2-hydroxyphenyl)phenylmethylene, N-cyclohexylidene);
[0448] Enamine Derivatives:
(N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)); [0449] N-Metal Derivatives
(N-borane derivatives, N-diphenylborinic acid derivatives,
N-[phenyl(pentacarbonylchromium- or -tungsten)]carbenyl, N-copper
or N-zinc chelate); [0450] N--N Derivatives: (N-nitro, N-nitroso,
N-oxide); [0451] N--P Derivatives: (N-diphenylphosphinyl,
N-dimethylthiophosphinyl, N-diphenylthiophosphinyl, N-dialkyl
phosphoryl, N-dibenzyl phosphoryl N-diphenyl phosphoryl); [0452]
N--Si Derivatives, N--S Derivatives, and N-Sulfenyl Derivatives:
(N-benzenesulfenyl, N-o-nitrobenzenesulfenyl,
N-2,4-dinitrobenzenesulfenyl, N-pentachlorobenzenesulfenyl,
N-2-nitro-4-methoxybenzenesulfenyl, N-triphenylmethylsulfenyl,
N-3-nitropyridinesulfenyl); and N-sulfonyl Derivatives
(N-p-toluenesulfonyl, N-benzenesulfonyl,
N-2,3,6-trimethyl-4-methoxybenzenesulfonyl,
N-2,4,6-trimethoxybenzenesulfonyl,
N-2,6-dimethyl-4-methoxybenzenesulfonyl,
N-pentamethylbenzenesulfonyl,
N-2,3,5,6,-tetramethyl-4-methoxybenzenesulfonyl,
N-4-methoxybenzenesulfonyl, N-2,4,6-trimethylbenzenesulfonyl,
N-2,6-dimethoxy-4-methylbenzenesulfonyl,
N-2,2,5,7,8-pentamethylchroman-6-sulfonyl, N-methanesulfonyl,
N-.beta.-trimethylsilyethanesulfonyl, N-9-anthracenesulfonyl,
N-4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonyl,
N-benzylsulfonyl, N-trifluoromethylsulfonyl,
N-phenacylsulfonyl).
[0453] More typically, protected amino groups include carbamates
and amides, still more typically, --NHC(O)R.sup.1 or
--N.dbd.CR.sup.1N(R.sup.1).sub.2. Another protecting group, also
useful as a prodrug for amino or --NH(R.sup.5), is: ##STR152## See
for example Alexander, J. etal (1996) J. Med. Chem. 39:480-486.
Amino Acid and Polypeptide Protecting Group and Conjugates
[0454] An amino acid or polypeptide protecting group of a compound
of the invention has the structure R.sup.15NHCH(R.sup.16)C(O)--,
where R.sup.15 is H, an amino acid or polypeptide residue, or
R.sup.5, and R.sup.16 is defined below.
[0455] R.sub.16 is lower alkyl or lower alkyl (C.sub.1-C.sub.6)
substituted with amino, carboxyl, amide, carboxyl ester, hydroxyl,
C.sub.6-C.sub.7 aryl, guanidinyl, imidazolyl, indolyl, sulfhydryl,
sulfoxide, and/or alkylphosphate. R.sup.10 also is taken together
with the amino acid .alpha. N to form a proline residue
(R.sup.10=--CH.sub.2).sub.3--). However, R.sup.10 is generally the
side group of a naturally-occurring amino acid such as H,
--CH.sub.3, --CH(CH.sub.3).sub.2, --CH.sub.2--CH(CH.sub.3).sub.2,
--CHCH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2--C.sub.6H.sub.5,
--CH.sub.2CH.sub.2--S--CH.sub.3, --CH.sub.2OH, --CH(OH)--CH.sub.3,
--CH.sub.2--SH, --CH.sub.2--C.sub.6H.sub.4OH,
--CH.sub.2--CO--NH.sub.2, --CH.sub.2--CH.sub.2--CO--NH.sub.2,
--CH.sub.2--COOH, --CH.sub.2--CH.sub.2--COOH,
--(CH.sub.2).sub.4--NH.sub.2 and
--(CH.sub.2).sub.3--NH--C(NH.sub.2)--NH.sub.2. R.sub.10 also
includes 1-guanidinoprop-3-yl, benzyl 4-hydroxybenzyl
imidazol-4-yl, indol-3-yl, methoxyphenyl and ethoxyphenyl.
[0456] Another set of protecting groups include the residue of an
amino-containing compound, in particular an amino acid, a
polypeptide, a protecting group, --NHSO.sub.2R, NHC(O)R,
--N(R).sub.2, NH.sub.2 or --NH(R)(H), whereby for example a
carboxylic acid is reacted, i.e. coupled, with the amine to form an
amide, as in C(O)NR.sub.2. A phosphonic acid may be reacted with
the amine to form a phosphonamidate, as in
--P(O)(OR)(NR.sub.2).
[0457] In general, amino acids have the structure
R.sup.17C(O)CH(R.sup.16)NH--, where R.sup.7 is --OH, --OR, an amino
acid or a polypeptide residue. Amino acids are low molecular weight
compounds, on the order of less than about 1000 MW and which
contain at least one amino or imino group and at least one carboxyl
group. Generally the amino acids will be found in nature, i.e., can
be detected in biological material such as bacteria or other
microbes, plants, animals or man. Suitable amino acids typically
are alpha amino acids, i.e. compounds characterized by one amino or
imino nitrogen atom separated from the carbon atom of one carboxyl
group by a single substituted or unsubstituted alpha carbon atom.
Of particular interest are hydrophobic residues such as mono- or
di-alkyl or aryl amino acids, cycloalkylamino acids and the like.
These residues contribute to cell permeability by increasing the
partition coefficient of the parental drug. Typically, the residue
does not contain a sulfhydryl or guanidino substituent.
[0458] Naturally-occurring amino acid residues are those residues
found naturally in plants, animals or microbes, especially proteins
thereof. Polypeptides most typically will be substantially composed
of such naturally-occurring amino acid residues. These amino acids
are glycine, alanine, valine, leucine, isoleucine, serine,
threonine, cysteine, methionine, glutamic acid, aspartic acid,
lysine, hydroxylysine, arginine, histidine, phenylalanine,
tyrosine, tryptophan, proline, asparagine, glutamine and
hydroxyproline. Additionally, unnatural amino acids, for example,
valanine, phenylglycine and homoarginine are also included.
Commonly encountered amino acids that are not gene-encoded may also
be used in the present invention. All of the amino acids used in
the present invention may be either the D- or L-optical isomer. In
addition, other peptidomimetics are also useful in the present
invention. For a general review, see Spatola, A. F., in Chemistry
and Biochemistry of Amino Acids, Peptides and Proteins, B.
Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).
[0459] When protecting groups are single amino acid residues or
polypeptides they optionally are substituted at R.sup.3 of
substituents A.sup.1, A.sup.2 or A.sup.3, or substituted at R.sub.3
of substituents A.sub.1, A.sub.2 or A.sub.3. These conjugates are
produced by forming an amide bond between a carboxyl group of the
amino acid (or C-terminal amino acid of a polypeptide for example).
Similarly, conjugates are formed between R.sup.3 or R.sub.3 and an
amino group of an amino acid or polypeptide. Generally, only one of
any site in the parental molecule is amidated with an amino acid as
described herein, although it is within the scope of this invention
to introduce amino acids at more than one permitted site. Usually,
a carboxyl group of R.sup.3 is amidated with an amino acid. In
general the .alpha.-amino or .alpha.-carboxyl group of the amino
acid or the terminal amino or carboxyl group of a polypeptide are
bonded to the parental functionalities, i.e., carboxyl or amino
groups in the amino acid side chains generally are not used to form
the amide bonds with the parental compound (although these groups
may need to be protected during synthesis of the conjugates as
described further below).
[0460] With respect to the carboxyl-containing side chains of amino
acids or polypeptides it will be understood that the carboxyl group
optionally will be blocked, e.g. by R.sup.1, esterified with
R.sup.5 or amidated. Similarly, the amino side chains R.sup.16
optionally will be blocked with R.sup.1 or substituted with
R.sup.5.
[0461] Such ester or amide bonds with side chain amino or carboxyl
groups, like the esters or amides with the parental molecule,
optionally are hydrolyzable in vivo or in vitro under acidic
(pH<3) or basic (pH>10) conditions. Alternatively, they are
substantially stable in the gastrointestinal tract of humans but
are hydrolyzed enzymatically in blood or in intracellular
environments. The esters or amino acid or polypeptide amidates also
are useful as intermediates for the preparation of the parental
molecule containing free amino or carboxyl groups. The free acid or
base of the parental compound, for example, is readily formed from
the esters or amino acid or polypeptide conjugates of this
invention by conventional hydrolysis procedures.
[0462] When an amino acid residue contains one or more chiral
centers, any of the D, L, meso, threo or erythro (as appropriate)
racemates, scalemates or mixtures thereof may be used. In general,
if the intermediates are to be hydrolyzed non-enzymatically (as
would be the case where the amides are used as chemical
intermediates for the free acids or free amines), D isomers are
useful On the other hand, the linkerisomers are more versatile
since they can be susceptible to both non-enzymatic and enzymatic
hydrolysis, and are more efficiently transported by amino acid or
dipeptidyl transport systems in the gastrointestinal tract.
[0463] Examples of suitable amino acids whose residues are
represented by R.sup.x or R.sup.y include the following:
[0464] Glycine;
[0465] Aminopolycarboxylic acids, e.g., aspartic acid,
.beta.-hydroxyaspartic acid, glutamic acid, .beta.-hydroxyglutamic
acid, .beta.-methylaspartic acid, .beta.-methylglutamic acid,
.beta.,.beta.-dimethylaspartic acid, .gamma.-hydroxyglutamic acid,
.beta.,.gamma.-dihydroxyglutamic acid, .beta.-phenylglutamic acid,
.gamma.-methyleneglutamic acid, 3-aminoadipic acid, 2-aminopimelic
acid, 2-aminosuberic acid and 2-aminosebacic acid;
[0466] Amino acid amides such as glutamine and asparagine;
[0467] Polyamino- or polybasic-monocarboxylic acids such as
arginine, lysine, .beta.-aminoalanine, .gamma.-aminobutyrine,
ornithine, citruline, homoarginine, homocitrulline, hydroxylysine,
allohydroxylsine and diaminobutyric acid;
[0468] Other basic amino acid residues such as histidine;
[0469] Diaminodicarboxylic acids such as
.alpha.,.alpha.'-diaminosuccinic acid,
.alpha.,.alpha.'-diaminoglutaric acid,
.alpha.,.alpha.'-diaminoadipic acid,
.alpha.,.alpha.'-diaminopimelic acid,
.alpha.,.alpha.'-diamino-O-hydroxypimelic acid,
.alpha.,.alpha.'-diaminosuberic acid,
.alpha.,.alpha.'-diaminoazelaic acid, and
.alpha.,.alpha.'-diaminosebacic acid;
[0470] Imino acids such as proline, hydroxyproline,
allohydroxyproline, .gamma.-methylproline, pipecolic acid,
5-hydroxypipecolic acid, and azetidine-2-carboxylic acid;
[0471] A mono- or di-alkyl (typically C.sub.1-C.sub.8 branched or
normal) amino acid such as alanine, valine, leucine, allylglycine,
butyrine, norvaline, norleucine, heptyline, .alpha.-methylserine,
.alpha.-amino-.alpha.-methyl-.gamma.-hydroxyvaleric acid,
.alpha.-amino-.alpha.-methyl-.delta.-hydroxyvaleric acid,
.alpha.-amino-.alpha.-methyl-.epsilon.-hydroxycaproic acid,
isovaline, .alpha.-methylglutamic acid, .alpha.-aminoisobutyric
acid, .alpha.-aminodiethylacetic acid,
.alpha.-aminodiisopropylacetic acid, .alpha.-aminodi-n-propylacetic
acid, .alpha.-aminodiisobutylacetic acid,
.alpha.-aminodi-n-butylacetic acid,
.alpha.-aminoethylisopropylacetic acid,
.alpha.-amino-n-propylacetic acid, .alpha.-aminodiisoamyacetic
acid, .alpha.-methylaspartic acid, .alpha.-methylglutamic acid,
1-aminocyclopropane-1-carboxylic acid, isoleucine, alloisoleucine,
tert-leucine, .beta.-methyltryptophan and
.alpha.-amino-.beta.-ethyl-.beta.-phenylpropionic acid;
[0472] .beta.-phenylserinyl;
[0473] Aliphatic .alpha.-amino-.beta.-hydroxy acids such as serine,
.beta.-hydroxyleucine, .beta.-hydroxynorleucine,
.beta.-hydroxynorvaline, and .alpha.-amino-.beta.-hydroxystearic
acid;
[0474] .alpha.-Amino, .alpha.-, .gamma.-, .delta.- or
.epsilon.-hydroxy acids such as homoserine,
.delta.-hydroxynorvaline, .gamma.-hydroxynorvaline and
.epsilon.-hydroxynorleucine residues; canavine and canaline;
.gamma.-hydroxyornithine;
[0475] 2-hexosaminic acids such as D-glucosaminic acid or
D-galactosaminic acid;
[0476] .alpha.-Amino-.beta.-thiols such as penicillamine,
.beta.-thiolnorvaline or .beta.-thiolbutyrine;
[0477] Other sulfur containing amino acid residues including
cysteine; homocystine, .beta.-phenylmethionine, methionine,
S-allyl-L-cysteine sulfoxide, 2-thiolhistidine, cystathionine, and
thiol ethers of cysteine or homocysteine;
[0478] Phenylalanine, tryptophan and ring-substituted .alpha.-amino
acids such as the phenyl- or cyclohexylamino acids
.alpha.-aminophenylacetic acid, .alpha.-aminocyclohexylacetic acid
and .alpha.-amino-.beta.-cyclohexylpropionic acid; phenylalanine
analogues and derivatives comprising aryl, lower alkyl, hydroxy,
guanidino, oxyalkylether, nitro, sulfur or halo-substituted phenyl
(e.g., tyrosine, methyltyrosine and o-chloro-, p-chloro-,
3,4-dichloro, o-, m- or p-methyl-, 2,4,6-trimethyl-,
2-ethoxy-5-nitro-, 2-hydroxy-5-nitro- and p-nitro-phenylalanine);
furyl-, thienyl-, pyridyl-, pyrimidinyl-, purinyl- or
naphthyl-alanines; and tryptophan analogues and derivatives
including kynurenine, 3-hydroxykynurenine, 2-hydroxytryptophan and
4-carboxytryptophan;
[0479] .alpha.-Amino substituted amino acids including sarcosine
(N-methylglycine), N-benzylglycine, N-methylalanine,
N-benzylalanine, N-methylphenylalanine, N-benzylphenylalanine,
N-methylvaline and N-benzylvaline; and
[0480] .alpha.-Hydroxy and substituted .alpha.-hydroxy amino acids
including serine, threonine, allothreonine, phosphoserine and
phosphothreonine.
[0481] Polypeptides are polymers of amino acids in which a carboxyl
group of one amino acid monomer is bonded to an amino or imino
group of the next amino acid monomer by an amide bond. Polypeptides
include dipeptides, low molecular weight polypeptides (about
1500-5000 MW) and proteins. Proteins optionally contain 3, 5, 10,
50, 75, 100 or more residues, and suitably are substantially
sequence-homologous with human, animal, plant or microbial
proteins. They include enzymes (e.g., hydrogen peroxidase) as well
as immunogens such as KLH, or antibodies or proteins of any type
against which one wishes to raise an immune response. The nature
and identity of the polypeptide may vary widely.
[0482] The polypeptide amidates are useful as immunogens in raising
antibodies against either the polypeptide (if it is not immunogenic
in the animal to which it is administered) or against the epitopes
on the remainder of the compound of this invention.
[0483] Antibodies capable of binding to the parental non-peptidyl
compound are used to separate the parental compound from mixtures,
for example in diagnosis or manufacturing of the parental compound.
The conjugates of parental compound and polypeptide generally are
more immunogenic than the polypeptides in closely homologous
animals, and therefore make the polypeptide more immunogenic for
facilitating raising antibodies against it. Accordingly, the
polypeptide or protein may not need to be immunogenic in an animal
typically used to raise antibodies, e.g., rabbit, mouse, horse, or
rat, but the final product conjugate should be immunogenic in at
least one of such animals. The polypeptide optionally contains a
peptidolytic enzyme cleavage site at the peptide bond between the
first and second residues adjacent to the acidic heteroatom. Such
cleavage sites are flanked by enzymatic recognition structures,
e.g. a particular sequence of residues recognized by a peptidolytic
enzyme.
[0484] Peptidolytic enzymes for cleaving the polypeptide conjugates
of this invention are well known, and in particular include
carboxypeptidases. Carboxypeptidases digest polypeptides by
removing C-terminal residues, and are specific in many instances
for particular C-terminal sequences. Such enzymes and their
substrate requirements in general are well known. For example, a
dipeptide (having a given pair of residues and a free carboxyl
terminus) is covalently bonded through its .alpha.-amino group to
the phosphorus or carbon atoms of the compounds herein. In
embodiments where W.sub.1 is phosphonate it is expected that this
peptide will be cleaved by the appropriate peptidolytic enzyme,
leaving the carboxyl of the proximal amino acid residue to
autocatalytically cleave the phosphonoamidate bond.
[0485] Suitable dipeptidyl groups (designated by their single
letter code) are AA, AR, AN, AD, AC, AE, AQ, AG, AH, AI, AL, AK,
AM, AF, AP, AS, AT, AW, AY, AV, RA, RR, RN, RD, RC, RE, RQ, RG, RH,
RI, RL, RK, RM, RF, RP, RS, RT, RW, RY, RV, NA, NR, NN, ND, NC, NE,
NQ, NG, NH, NI, NL, NK, NM, NF, NP, NS, NT, NW, NY, NV, DA, DR, DN,
DD, DC, DE, DQ, DG, DH, DI, DL, DK, DM, DF, DP, DS, DT, DW, DY, DV,
CA, CR, CN, CD, CC, CE, CQ, CG, CH, CI, CL, CK, CM, CF, CP, CS, CT,
CW, CY, CV, EA, ER, EN, ED, EC, EE, EQ, EG, EH, EI, EL, EK, EM, EF,
EP, ES, ET, EW, EY, BV, QA, QR, QN, QD, QC, QE, QQ, QG, QH, QI, QL,
QK, QM, QF, QP, QS, QT, QW, QY, QV, GA, GR, GN, GD, GC, GE, GQ, GG,
GH, GI, GL, GK, GM, GF, GP, GS, GT, GW, GY, GV, HA, HR, HN, HD, HC,
HE, HQ, HG, HH, HI, HL, HK, HM, HF, HP, HS, HT, HW, HY, HV, IA, IR,
IN, ID, IC, IE, IQ, IG, IH, II, IL, IK, IM, IF, IP, IS, IT, IW, IY,
IV, LA, LR, LN, LD, LC, LE, LQ, LG, LH, LI, LL, LK, LM, LF, LP, LS,
LT, LW, LY, LV, KA, KR, KN, KD, KC, KE, KQ, KG, KH, KI, KL, KK, KM,
KF, KP, KS, KT, KW, KY, KV, MA, MR, MN, MD, MC, ME, MQ, MG, MH, MI,
ML, MK, MM, MF, MP, MS, MT, MW, MY, MV, FA, FR, FN, FD, FC, FE, FQ,
FG, FH, FI, FL, FK, FM, FF, FP, FS, FT, FW, FY, FV, PA, PR, PN, PD,
PC, PE, PQ, PG, PH, PI, PL, PK, PM, PF, PP, PS, PT, PW, PY, PV, SA,
SR, SN, SD, SC, SE, SQ, SG, SH, SI, SL, SK, SM, SF, SP, SS, ST, SW,
SY, SV, TA, TR, TN, TD, TC, TE, TQ, TG, TH, TI, TL, TK, TM, TF, TP,
TS, TT, TW, TY, TV, WA, WR, WN, WD, WC, WE, WQ, WG, WH, WI, WL, WK,
WM, WF, WP, WS, WT, WW, WY, WV, YA, YR, YN, YD, YC, YE, YQ, YG, YH,
YI, YL, YK, YM, YF, YP, YS, YT, YW, YY, YV, VA, VR, VN, VD, VC, VE,
VQ, VG, VH, VI, VL, VK, VM, VF, VP, VS, VT, VW, VY and VV.
[0486] Tripeptide residues are also useful as protecting groups.
When a phosphonate is to be protected, the sequence
--X.sup.4-pro-X.sup.5-- (where X.sup.4 is any amino acid residue
and X.sup.5 is an amino acid residue, a carboxyl ester of proline,
or hydrogen) will be cleaved by luminal carboxypeptidase to yield
X.sup.4 with a free carboxyl, which in turn is expected to
autocatalytically cleave the phosphonoamidate bond. The carboxy
group of X.sup.5 optionally is esterified with benzyl.
[0487] Dipeptide or tripeptide species can be selected on the basis
of known transport properties and/or susceptibility to peptidases
that can affect transport to intestinal mucosal or other cell
types. Dipeptides and tripeptides lacking an .alpha.-amino group
are transport substrates for the peptide transporter found in brush
border membrane of intestinal mucosal cells (Bai, J. P. F., (1992)
Pharm Res. 9:969-978. Transport competent peptides can thus be used
to enhance bioavailability of the amidate compounds. Di- or
tripeptides having one or more amino acids in the D configuration
are also compatible with peptide transport and can be utilized in
the amidate compounds of this invention. Amino acids in the D
configuration can be used to reduce the susceptibility of a di- or
tripeptide to hydrolysis by proteases common to the brush border
such as aminopeptidase N. In addition, di- or tripeptides
alternatively are selected on the basis of their relative
resistance to hydrolysis by proteases found in the lumen of the
intestine. For example, tripeptides or polypeptides lacking asp
and/or glu are poor substrates for aminopeptidase A, di- or
tripeptides lacking amino acid residues on the N-terminal side of
hydrophobic amino acids (leu, tyr, phe, val, trp) are poor
substrates for endopeptidase, and peptides lacking a pro residue at
the penultimate position at a free carboxyl terminus are poor
substrates for carboxypeptidase P. Similar considerations can also
be applied to the selection of peptides that are either relatively
resistant or relatively susceptible to hydrolysis by cytosolic,
renal, hepatic, serum or other peptidases. Such poorly cleaved
polypeptide amidates are immunogens or are useful for bonding to
proteins in order to prepare immunogens.
[0488] Prototype compounds contain at least one functional group
capable of bonding to the phosphorus atom in the phosphonate
moiety. The phosphonate candidate compounds are cleaved
intracellularly after they have reached the desired site of action,
e.g., inside a lymphoid cell. The mechanism by which this occurs is
further described below in the examples. As noted, the free acid of
the phosphonate is phosphorylated in the cell.
[0489] From the foregoing, it will be apparent that many different
prototypes can be derivatized in accord with the present invention.
Numerous such prototypes are specifically mentioned herein.
However, it should be understood that the discussion of anti-HIV
drug families and their specific members for derivatization
according to this invention is not intended to be exhaustive, but
merely illustrative.
[0490] When the prototype compound contains multiple reactive
hydroxyl functions, a mixture of intermediates and final products
may be obtained. In the unusual case in which all hydroxy groups
are approximately equally reactive, there is not expected to be a
single, predominant product, as each mono-substituted product will
be obtained in approximately equal amounts, while a lesser amount
of multiple-substituted candidate compound will also result.
Generally speaking, however, one of the hydroxyl groups will be
more susceptible to substitution than the other(s), e.g. a primary
hydroxyl will be more reactive than a secondary hydroxyl, an
unhindered hydroxyl will be more reactive than a hindered one.
Consequently, the major product will be a mono-substituted one in
which the most reactive hydroxyl has been derivatized while other
mono-substituted and multiply-substituted products may be obtained
as minor products.
Stereoisomers
[0491] The candidate compounds may have chiral centers, e.g. chiral
carbon or phosphorus atoms. The compounds thus include racemic
mixtures of all stereoisomers, including enantiomers,
diastereomers, and atropisomers. In addition, the compounds include
enriched or resolved optical isomers at any or all asymmetric,
chiral atoms. In other words, the chiral centers apparent from the
depictions are provided as the chiral isomers or racemic mixtures.
Both racemic and diastereomeric mixtures, as well as the individual
optical isomers isolated or synthesized, substantially free of
their enantiomeric or diastereomeric partners, are all suitable for
use as candidate compounds. The racemic mixtures are separated into
their individual substantially optically pure isomers through
well-known techniques such as, for example, the separation of
diastereomeric salts formed with optically active adjuncts, e.g.,
acids or bases followed by conversion back to the optically active
substances. In most instances, the desired optical isomer is
synthesized by means of stereospecific reactions, beginning with
the appropriate stereoisomer of the desired starting material.
[0492] The compounds can also exist as tautomeric isomers in
certain cases. All though only one delocalized resonance structure
may be depicted, all such forms are contemplated within the scope
of the invention. For example, ene-amine tautomers can exist for
purine, pyrimidine, imidazole, guanidine, amidine, and tetrazole
systems and all their possible tautomeric forms are within the
scope of the invention.
[0493] The optimal absolute configuration at the phosphorus atom
for use in candidate compounds is that of GS-7340, depicted in the
examples.
Salts and Hydrates
[0494] Any reference to any of the compounds of the invention also
includes a reference to a physiologically acceptable salt thereof.
Examples of physiologically acceptable salts of the compounds of
the invention include salts derived from an appropriate base, such
as an alkali metal (for example, sodium), an alkaline earth (for
example, magnesium), ammonium and NX.sub.4.sup.+ (wherein X is
C.sub.1-C.sub.4 alkyl). Physiologically acceptable salts of a
hydrogen atom or an amino group include salts of organic carboxylic
acids such as acetic, benzoic, lactic, fumaric, tartaric, maleic,
malonic, malic, isethionic, lactobionic and succinic acids; organic
sulfonic acids, such as methanesulfonic, ethanesulfonic,
benzenesulfonic and p-toluenesulfonic acids; and inorganic acids,
such as hydrochloric, sulfuric, phosphoric and sulfamic acids.
[0495] Physiologically acceptable salts of a compound of an hydroxy
group include the anion of said compound in combination with a
suitable cation such as Na.sup.+ and NX.sub.4.sup.+ (wherein X is
independently selected from H or a C.sub.1-C.sub.4 alkyl
group).
[0496] For therapeutic use, salts of active ingredients of the
candidate compounds will be physiologically acceptable, i.e. they
will be salts derived from a physiologically acceptable acid find
use, for example, in the preparation or purification of a
physiologically acceptable compound. All salts, whether or not
derived form a physiologically acceptable acid or base, are within
the scope of the present invention.
[0497] Pharmaceutically acceptable non-toxic salts of candidate
compounds containing, for example, Na.sup.+, Li.sup.+, K.sup.+,
Ca.sup.+2 and Mg.sup.+2, fall within the scope herein. Such salts
may include those derived by combination of appropriate cations
such as alkali and alkaline earth metal ions or ammonium and
quaternary amino ions with an acid anion moiety, typically a
carboxylic acid. Monovalent salts are preferred if a water soluble
salt is desired.
[0498] Metal salts typically are prepared by reacting the metal
hydroxide with a compound of this invention. Examples of metal
salts which are prepared in this way are salts containing Li.sup.+,
Na.sup.+, and K.sup.+. A less soluble metal salt can be
precipitated from the solution of a more soluble salt by addition
of the suitable metal compound.
[0499] In addition, salts may be formed from acid addition of
certain organic and inorganic acids, e.g., HCl, HBr,
H.sub.2SO.sub.4, H.sub.3PO.sub.4 or organic sulfonic acids, to
basic centers, typically amines, or to acidic groups. Finally, it
is to be understood that the compositions herein comprise compounds
of the invention in their un-ionized, as well as zwitterionic form,
and combinations with stoichiometric amounts of water as in
hydrates.
[0500] Salts of the candidate compounds with amino acids also fall
within the scope of this invention. Any of the amino acids
described above are suitable, especially the naturally-occurring
amino acids found as protein components, although the amino acid
typically is one bearing a side chain with a basic or acidic group,
e.g., lysine, arginine or glutamic acid, or a neutral group such as
glycine, serine, threonine, alanine, isoleucine, or leucine.
Methods for Assay of Anti-HIV Activity
[0501] The anti-HIV activity of a candidate compound is assayed by
any method heretofore known for determining inhibition of growth,
replication, or other characteristic of HIV infection, including
direct and indirect methods of detecting HIV activity.
Quantitative, qualitative, and semiquantitative methods of
determining HIV activity are all contemplated. Typically any one of
the in vitro or cell culture screening methods known to the art are
employed, as are clinical trials in humans, studies in animal
models (SIV), and the like. In screening candidate compounds it
should be kept in mind that the results of enzyme assays may not
correlate with cell culture assays. Thus, a cell based assay is
often the primary screening tool. Candidate compounds having an in
vitro Ki (inhibitory constant) of less then about 5.times.10.sup.-6
M, typically less than about 1.times.10.sup.-7 M and preferably
less than about 5.times.10.sup.-8 M are preferred for in vivo
development, but the analytical point of selection of a candidate
compound for further development is essentially a matter of
choice.
Pharmaceutical Formulations
[0502] Candidate compounds selected for further development in vivo
are formulated with conventional carriers and excipients, which
will be selected in accord with ordinary practice. Tablets will
contain excipients, glidants, fillers, binders and the like.
Aqueous formulations are prepared in sterile form, and when
intended for delivery by other than oral administration generally
will be isotonic. All formulations will optionally contain
excipients such as those set forth in the "Handbook of
Pharmaceutical Excipients" (1986). Excipients include ascorbic acid
and other antioxidants, chelating agents such as EDTA,
carbohydrates such as dextrin, hydroxyalkylcellulose,
hydroxyalkylmethylcellulose, stearic acid and the like. The pH of
the formulations ranges from about 3 to about 11, but is ordinarily
about 7 to 10.
[0503] While it is possible for the active ingredients to be
administered alone it may be preferable to present them as
pharmaceutical formulations. The formulations, both for veterinary
and for human use, of the invention comprise at least one active
ingredient, as above defined, together with one or more acceptable
carriers therefor and optionally other therapeutic ingredients. The
carrier(s) must be "acceptable" in the sense of being compatible
with the other ingredients of the formulation and physiologically
innocuous to the recipient thereof.
[0504] The formulations include those suitable for the foregoing
administration routes. The formulations may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well known in the art of pharmacy. Techniques and
formulations generally are found in Remington's Pharmaceutical
Sciences (Mack Publishing Co., Easton, Pa.). Such methods include
the step of bringing into association the active ingredient with
the carrier which constitutes one or more accessory ingredients. In
general the formulations are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers or finely divided solid carriers or both, and then, if
necessary, shaping the product.
[0505] Formulations of candidate compounds suitable for oral
administration may be presented as discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the
active ingredient; as a powder or granules; as a solution or a
suspension in an aqueous or non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The
active ingredient may also be administered as a bolus, electuary or
paste.
[0506] A tablet is made by compression or molding, optionally with
one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with a binder, lubricant, inert diluent, preservative,
surface active or dispersing agent. Molded tablets may be made by
molding in a suitable machine a mixture of the powdered active
ingredient moistened with an inert liquid diluent. The tablets may
optionally be coated or scored and optionally are formulated so as
to provide slow or controlled release of the active ingredient
therefrom.
[0507] For infections of the eye or other external tissues e.g.
mouth and skin, the formulations are preferably applied as a
topical ointment or cream containing the active ingredient(s) in an
amount of, for example, 0.075 to 20% w/w (including active
ingredient(s) in a range between 0.1% and 20% in increments of 0.1%
w/w such as 0.6% w/w, 0.7% w/w, etc.), preferably 0.2 to 15% w/w
and most preferably 0.5 to 10% w/w. When formulated in an ointment,
the active ingredients may be employed with either a paraffinic or
a water-miscible ointment base. Alternatively, the active
ingredients may be formulated in a cream with an oil-in-water cream
base.
[0508] If desired, the aqueous phase of the cream base may include,
for example, at least 30% w/w of a polyhydric alcohol, i.e. an
alcohol having two or more hydroxyl groups such as propylene glycol
butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene
glycol (including PEG 400) and mixtures thereof. The topical
formulations may desirably include a compound which enhances
absorption or penetration of the active ingredient through the skin
or other affected areas. Examples of such dermal penetration
enhancers include dimethyl sulphoxide and related analogs.
[0509] The oily phase of the emulsions of this invention may be
constituted from known ingredients in a known manner. While the
phase may comprise merely an emulsifier (otherwise known as an
emulgent), it desirably comprises a mixture of at least one
emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a hydrophilic emulsifier is included together with a
lipophilic emulsifier which acts as a stabilizer. It is also
preferred to include both an oil and a fat. Together, the
emulsifier(s) with or without stabilizer(s) make up the so-called
emulsifying wax, and the wax together with the oil and fat make up
the so-called emulsifying ointment base which forms the oily
dispersed phase of the cream formulations.
[0510] Emulgents and emulsion stabilizers suitable for use in the
formulation of the invention include Tween.RTM. 60, Span.RTM. 80,
cetostearyl alcohol benzyl alcohol myristyl alcohol, glyceryl
mono-stearate and sodium lauryl sulfate.
[0511] The choice of suitable oils or fats for the formulation is
based on achieving the desired cosmetic properties. The cream
should preferably be a non-greasy, non-staining and washable
product with suitable consistency to avoid leakage from tubes or
other containers. Straight or branched chain, mono- or dibasic
alkyl esters such as di-isoadipate, isocetyl stearate, propylene
glycol diester of coconut fatty acids, isopropyl myristate, decyl
oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate
or a blend of branched chain esters known as Crodamol CAP may be
used, the last three being preferred esters. These may be used
alone or in combination depending on the properties required.
Alternatively, high melting point lipids such as white soft
paraffin and/or liquid paraffin or other mineral oils are used.
[0512] Pharmaceutical formulations according to the present
invention comprise a combination according to the invention
together with one or more pharmaceutically acceptable carriers or
excipients and optionally other therapeutic agents. Pharmaceutical
formulations containing the active ingredient may be in any form
suitable for the intended method of administration. When used for
oral use for example, tablets, troches, lozenges, aqueous or oil
suspensions, dispersible powders or granules, emulsions, hard or
soft capsules, syrups or elixirs may be prepared. Compositions
intended for oral use may be prepared according to any method known
to the art for the manufacture of pharmaceutical compositions and
such compositions may contain one or more agents including
sweetening agents, flavoring agents, coloring agents and preserving
agents, in order to provide a palatable preparation. Tablets
containing the active ingredient in admixture with non-toxic
pharmaceutically acceptable excipient which are suitable for
manufacture of tablets are acceptable. These excipients may be, for
example, inert diluents, such as calcium or sodium carbonate,
lactose, calcium or sodium phosphate; granulating and
disintegrating agents, such as maize starch, or alginic acid;
binding agents, such as starch, gelatin or acacia; and lubricating
agents, such as magnesium stearate, stearic acid or talc. Tablets
may be uncoated or may be coated by known techniques including
microencapsulation to delay disintegration and adsorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate alone or with a wax
may be employed.
[0513] Formulations for oral use may be also presented as hard
gelatin capsules where the active ingredient is mixed with an inert
solid diluent, for example calcium phosphate or kaolin, or as soft
gelatin capsules wherein the active ingredient is mixed with water
or an oil medium, such as peanut oil, liquid paraffin or olive
oil.
[0514] Aqueous suspensions of the invention contain the active
materials in admixture with excipients suitable for the manufacture
of aqueous suspensions. Such excipients include a suspending agent,
such as sodium carboxymethylcellulose, methylcellulose,
hydroxypropyl methylcelluose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous
suspension may also contain one or more preservatives such as ethyl
or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or
more flavoring agents and one or more sweetening agents, such as
sucrose or saccharin.
[0515] Oil suspensions may be formulated by suspending the active
ingredient in a vegetable oil, such as arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oral suspensions may contain a thickening agent, such
as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such
as those set forth above, and flavoring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an antioxidant such as ascorbic
acid.
[0516] Dispersible powders and granules of the invention suitable
for preparation of an aqueous suspension by the addition of water
provide the active ingredient in admixture with a dispersing or
wetting agent, a suspending agent, and one or more preservatives.
Suitable dispersing or wetting agents and suspending agents are
exemplified by those disclosed above. Additional excipients, for
example sweetening, flavoring and coloring agents, may also be
present.
[0517] The pharmaceutical compositions of the candidate compounds
may also be in the form of oil-in-water emulsions. The oily phase
may be a vegetable oil, such as olive oil or arachis oil, a mineral
oil, such as liquid paraffin, or a mixture of these. Suitable
emulsifying agents include naturally-occurring gums, such as gum
acacia and gum tragacanth, naturally occurring phosphatides, such
as soybean lecithin, esters or partial esters derived from fatty
acids and hexitol anhydrides, such as sorbitan monooleate, and
condensation products of these partial esters with ethylene oxide,
such as polyoxyethylene sorbitan monooleate. The emulsion may also
contain sweetening and flavoring agents. Syrups and elixirs may be
formulated with sweetening agents, such as glycerol sorbitol or
sucrose. Such formulations may also contain a demulcent, a
preservative, a flavoring, or a coloring agent.
[0518] The pharmaceutical compositions of the candidate compounds
may be in the form of a sterile injectable preparation, such as a
sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents which
have been mentioned above. The sterile injectable preparation may
also be a sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent, such as a solution in
1,3-butane-diol or prepared as a lyophilized powder. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile fixed oils may conventionally be employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid may likewise be used in
the preparation of injectables.
[0519] The amount of active ingredient that may be combined with
the carrier material to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. For example, a time-release formulation intended
for oral administration to humans may contain approximately 1 to
1000 mg of active material compounded with an appropriate and
convenient amount of carrier material which may vary from about 5
to about 95% of the total compositions (weight:weight). The
pharmaceutical composition can be prepared to provide easily
measurable amounts for administration. For example, an aqueous
solution intended for intravenous infusion may contain from about 3
to 500 .mu.g of the active ingredient per milliliter of solution in
order that infusion of a suitable volume at a rate of about 30
mL/hr can occur.
[0520] Formulations suitable for topical administration to the eye
also include eye drops wherein the active ingredient is dissolved
or suspended in a suitable carrier, especially an aqueous solvent
for the active ingredient. The active ingredient is preferably
present in such formulations in a concentration of 0.5 to 20%,
advantageously 0.5 to 10% particularly about 1.5% w/w.
[0521] Formulations suitable for topical administration in the
mouth include lozenges comprising the active ingredient in a
flavored basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0522] Formulations for rectal administration may be presented as a
suppository with a suitable base comprising for example cocoa
butter or a salicylate.
[0523] Formulations suitable for intrapulmonary or nasal
administration have a particle size for example in the range of 0.1
to 500 microns (including particle sizes in a range between 0.1 and
500 microns in increments microns such as 0.5, 1, 30 microns, 35
microns, etc.), which is administered by rapid inhalation through
the nasal passage or by inhalation through the mouth so as to reach
the alveolar sacs. Suitable formulations include aqueous or oily
solutions of the active ingredient. Formulations suitable for
aerosol or dry powder administration may be prepared according to
conventional methods and may be delivered with other therapeutic
agents such as compounds heretofore used in the treatment or
prophylaxis of HIV infections as described below.
[0524] Formulations suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing in addition to the active ingredient
such carriers as are known in the art to be appropriate.
[0525] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents.
[0526] The formulations are presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water for
injection, immediately prior to use. Extemporaneous injection
solutions and suspensions are prepared from sterile powders,
granules and tablets of the kind previously described. Preferred
unit dosage formulations are those containing a daily dose or unit
daily sub-dose, as herein above recited, or an appropriate fraction
thereof, of the active ingredient.
[0527] It should be understood that in addition to the ingredients
particularly mentioned above the formulations of candidate
compounds may include other agents conventional in the art having
regard to the type of formulation in question, for example those
suitable for oral administration may include flavoring agents.
[0528] The invention further provides veterinary compositions
comprising at least one active ingredient as above defined together
with a veterinary carrier therefor.
[0529] Veterinary carriers are materials useful for the purpose of
administering the composition and may be solid, liquid or gaseous
materials which are otherwise inert or acceptable in the veterinary
art and are compatible with the active ingredient. These veterinary
compositions may be administered orally, parenterally or by any
other desired route.
[0530] Compounds of the invention are used to provide controlled
release pharmaceutical formulations containing as active ingredient
one or more compounds of the invention ("controlled release
formulations") in which the release of the active ingredient are
controlled and regulated to allow less frequency dosing or to
improve the pharmacokinetic or toxicity profile of a given active
ingredient.
[0531] An effective dose of candidate compound depends at least on
the nature of the condition being treated, toxicity, whether the
compound is being used prophylactically (lower doses) or against an
active HIV infection, the method of delivery, and the
pharmaceutical formulation, and will be determined by the clinician
using conventional dose escalation studies. It can be expected to
be from about 0.0001 to about 100 mg/kg body weight per day.
Typically, from about 0.01 to about 10 mg/kg body weight per day.
More typically, from about 0.01 to about 5 mg/kg body weight per
day. More typically, from about 0.05 to about 0.5 mg/kg body weight
per day. For example, the daily candidate dose for an adult human
of approximately 70 kg body weight will range from 1 mg to 1000 mg,
preferably between 5 mg and 500 mg, and may take the form of single
or multiple doses.
Routes of Administration
[0532] One or more candidate compounds (herein referred to as the
active ingredients) are administered by any route appropriate to
the condition to be treated. Suitable routes include oral, rectal,
nasal, topical (including buccal and sublingual), vaginal and
parenteral (including subcutaneous, intramuscular, intravenous,
intradermal, intrathecal and epidural), and the like. It will be
appreciated that the preferred route may vary with for example the
condition of the recipient. An advantage of the compounds of this
invention is that they are orally bioavailable and can be dosed
orally.
Combination Therapy
[0533] Candidate compound are also used in combination with other
active ingredients. Such combinations are selected based on the
condition to be treated, cross-reactivities of ingredients and
pharmaco-compounds. Other active ingredients include adefovir
dipivoxil and/or any other product currently marketed for therapy
of HIV infection properties. It is also possible to combine any
compound of the invention with one or more other active ingredients
in a unitary dosage form for simultaneous or sequential
administration to an HIV infected patient. The combination therapy
may be administered as a simultaneous or sequential regimen. When
administered sequentially, the combination may be administered in
two or more administrations. Second and third active ingredients in
the combination may have anti-HIV activity and include HIV.
[0534] The combination therapy may be synergistic, i.e. the effect
achieved when the active ingredients used together is greater than
the sum of the effects that results from using the compounds
separately. A synergistic effect may be attained when the active
ingredients are: (1) co-formulated and administered or delivered
simultaneously in a combined formulation; (2) delivered by
alternation or in parallel as separate formulations; or (3) by some
other regimen. When delivered in alternation therapy, a synergistic
effect may be attained when the compounds are administered or
delivered sequentially, e.g. in separate tablets, pills or
capsules, or by different injections in separate syringes. In
general, during alternation therapy, an effective dosage of each
active ingredient is administered sequentially, i.e. serially,
whereas in combination therapy, effective dosages of two or more
active ingredients are administered together. A synergistic
anti-viral effect denotes an antiviral effect which is greater than
the predicted purely additive effects of the individual compounds
of the combination.
Metabolites of the Candidate Compounds
[0535] The candidate compounds are metabolized in vivo. In
particular, the group R.sup.x is hydrolytically cleaved to produce
a charged metabolite, and in some cases the substituents on the
phosphonate such as
--Y.sup.2[P((.dbd.Y.sup.1)(Y.sup.2)).sub.m2R.sup.x].sub.2 are
hydrolyzed as well. An example showing exemplary metabolites is
found in the examples herein. While this example is concerned with
the metabolites of GS-7340, a nucleotide analogue, the metabolic
changes to be found with candidate compounds are believed to be
substantially the same at the phosphonate substituent. This charged
metabolite functions as an intracellular depot form of the
candidate. However, other changes may result for example from the
oxidation, reduction, hydrolysis, amidation, esterification and the
like of the administered compound, primarily due to enzymatic
processes. Accordingly, candidate compounds include metabolites of
candidate compounds produced by a process comprising contacting a
compound of this invention with a mammal for a period of time
sufficient to yield a metabolic product thereof. Such products
typically are identified by preparing a radiolabelled (e.g.
C.sup.14 or H.sup.3) compound of the invention, administering it
parenterally in a detectable dose (e.g. greater than about 0.5
mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to
man, allowing sufficient time for metabolism to occur (typically
about 30 seconds to 30 hours) and isolating its conversion products
from the urine, blood or other biological samples. These products
are easily isolated since they are labeled (others are isolated by
the use of antibodies capable of binding epitopes surviving in the
metabolite). The metabolite structures are determined in
conventional fashion, e.g. by MS or NMR analysis. In general,
analysis of metabolites is done in the same way as conventional
drug metabolism studies well-known to those skilled in the art. The
conversion products, so long as they are not otherwise found in
vivo, are useful in diagnostic assays for therapeutic dosing of the
candidate compounds even if they possess no HIV inhibitory activity
of their own.
[0536] Recipes and methods for determining stability of compounds
in surrogate gastrointestinal secretions are known. Compounds are
defined herein as stable in the gastrointestinal tract where less
than about 50 mole percent of the protected groups are deprotected
in surrogate intestinal or gastric juice upon incubation for 1 hour
at 37.degree. C. Simply because the compounds are stable to the
gastrointestinal tract does not mean that they cannot be hydrolyzed
in vivo. The phosphonate prodrugs of the invention typically will
be stable in the digestive system but are substantially hydrolyzed
to the parental drug in the digestive lumen, liver or other
metabolic organ, or within cells in general.
Exemplary Methods of Making Candidate Compounds
[0537] The candidate compounds are prepared by any of the
applicable techniques of organic synthesis. Many such techniques
are well known in the art. However, many of the known techniques
are elaborated in "Compendium of Organic Synthetic Methods" (John
Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen
Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974;
Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G.
Wade, jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6,
Michael B. Smith; as well as March, J., "Advanced Organic
Chemistry, Third Edition", (John Wiley & Sons, New York, 1985),
"Comprehensive Organic Synthesis. Selectivity, Strategy &
Efficiency in Modern Organic Chemistry. In 9 Volumes", Barry M.
Trost, Editor-in-Chief (Pergamon Press, New York, 1993
printing).
[0538] Dialkyl phosphonates may be prepared according to the
methods of: Quast etal (1974) Synthesis 490; Stowell etal (1990)
Tetrahedron Lett. 3261; U.S. Pat. No. 5,663,159.
[0539] In general synthesis of phosphonate esters is achieved by
coupling a nucleophile amine or alcohol with the corresponding
activated phosphonate electrophilic precursor. For example,
chlorophosphonate addition on to 5'-hydroxy of nucleoside is a well
known method for preparation of nucleoside phosphate monoesters.
The activated precursor can be prepared by several well known
methods. Chlorophosphonates useful for synthesis of the prodrugs
are prepared from the substituted-1,3-propanediol (Wissner, et al,
(1992) J. Med. Chem. 35:1650). Chlorophosphonates are made by
oxidation of the corresponding chlorophospholanes (Anderson, et al,
(1984) J. Org. Chem. 49:1304) which are obtained by reaction of the
substituted diol with phosphorus trichloride. Alternatively, the
chlorophosphonate agent is made by treating substituted-1,3-diols
with phosphorusoxychloride (Patois, etal (1990) J. Chem. Soc.
Perkin Trans. I, 1577). Chlorophosphonate species may also be
generated in situ from corresponding cyclic phosphites (Silverburg,
et al., (1996) Tetrahedron lett., 37:771-774), which in turn can be
either made from chlorophospholane or phosphoramidate intermediate.
The phosphoroflouridate intermediate prepared either from
pyrophosphate or phosphoric acid may also act as precursor in
preparation of cyclic prodrugs (Watanabe et al., (1988) Tetrahedron
lett., 29:5763-66).
[0540] Candidate compounds comprising a prodrug functionality may
also be prepared from the free acid by Mitsunobu reactions
(Mitsunobu, (1981) Synthesis, 1; Campbell, (1992) J. Org. Chem.,
52:6331), and other acid coupling reagents including, but not
limited to, carbodiimides (Alexander, et al, (1994) Collect. Czech.
Chem. Commun. 59:1853; Casara, et al, (1992) Bioorg. Med. Chem.
Lett., 2:145; Ohashi, et al, (1988) Tetrahedron Lea., 29:1189), and
benzotriazolyloxytris-(dimethylamino)phosphonium salts (Campagne,
et al, (1993) Tetrahedron Lett., 34:6743).
[0541] Aryl halides undergo Ni.sup.+2 catalyzed reaction with
phosphite derivatives to give aryl phosphonate containing compounds
(Balthazar, etal (1980) J. Org. Chem. 45:5425). Phosphonates may
also be prepared from the chlorophosphonate in the presence of a
palladium catalyst using aromatic triflates (Petrakis, etal (1987)
J. Am. Chem. Soc. 109:2831; Lu, et al, (1987) Synthesis, 726). In
another method, aryl phosphonate esters are prepared from aryl
phosphates under anionic rearrangement conditions (Melvin (1981)
Tetrahedron Lett. 22:3375; Casteel, etal (1991) Synthesis, 691).
N-Alkoxy aryl salts with alkali metal derivatives of cyclic alkyl
phosphonate provide general synthesis for heteroaryl-2-phosphonate
linkers (Redmore (1970) J. Org. Chem. 35:4114). These above
mentioned methods can also be extended to compounds where the
W.sup.5 group is a heterocycle. Cyclic-1,3-propanyl prodrugs of
phosphonates are also synthesized from phosphonic diacids and
substituted propane-1,3-diols using a coupling reagent such as
1,3-dicyclohexylcarbodiimide (DCC) in presence of a base (e.g.,
pyridine). Other carbodiimide based coupling agents like
1,3-disopropylcarbodiimide or water soluble reagent,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
can also be utilized for the synthesis of cyclic phosphonate
prodrugs.
[0542] The carbamoyl group may be formed by reaction of a hydroxy
group according to the methods known in the art, including the
teachings of Ellis, U.S. 2002/0103378 A1 and Hajima, U.S. Pat. No.
6,018,049.
[0543] A number of exemplary methods for the preparation of the
candidate compounds are provided below. These methods are intended
to illustrate the nature of such preparations and do not limit the
scope of this invention. Many of the compounds set forth below have
been screened and demonstrated to have anti-HIV activity. In view
of this these compounds are no longer candidate compounds for use
in the screening method of this invention. However, they are
illustrative of the manner in which the artisan can substitute
prototype compounds with A.sup.3 in various ways. In addition,
taken cumulatively, they are illustrative of the typical component
candidate compounds to be found in a screening library.
[0544] Generally; the reaction conditions such as temperature,
reaction time, solvents, work-up procedures, and the like, will be
those common in the art for the particular reaction to be
performed. The cited reference material together with material
cited therein, contains detailed descriptions of such conditions.
Typically the temperatures will be -100.degree. C. to 200.degree.
C., solvents will be aprotic or protic, and reaction times will be
10 seconds to 10 days. Work-up typically consists of quenching any
unreacted reagents followed by partition between a water/organic
layer system (extraction) and separating the layer containing the
product.
[0545] Oxidation and reduction reactions are typically carried out
at temperatures near room temperature (about 20.degree. C.),
although for metal hydride reductions frequently the temperature is
reduced to 0.degree. C. to -100.degree. C., solvents are typically
aprotic for reductions and may be either protic or aprotic for
oxidations. Reaction times are adjusted to achieve desired
conversions.
[0546] Condensation reactions are typically carried out at
temperatures near room temperature, although for non-equilibrating,
kinetically controlled condensations reduced temperatures
(0.degree. C. to -100.degree. C.) are also common. Solvents can be
either protic (common in equilibrating reactions) or aprotic
(common in kinetically controlled reactions).
[0547] Standard synthetic techniques such as azeotropic removal of
reaction by-products and use of anhydrous reaction conditions (e.g.
inert gas environments) are common in the art and will be applied
when applicable.
Schemes
[0548] General aspects of these exemplary methods are described
below and in the Examples. Each of the products of the following
processes are optionally separated, isolated, and/or purified prior
to its use in subsequent processes.
[0549] The terms "treated", "treating", "treatment", and the like,
mean contacting, mixing, reacting, allowing to react, bringing into
contact, and other terms common in the art for indicating that one
or more chemical entities is treated in such a manner as to convert
it to one or more other chemical entities. This means that
"treating compound one with compound two" is synonymous with
"allowing compound one to react with compound two", "contacting
compound one with compound two", "reacting compound one with
compound two", and other expressions common in the art of organic
synthesis for reasonably indicating that compound one was
"treated", "reacted", "allowed to react", etc., with compound
two.
[0550] "Treating" indicates the reasonable and usual manner in
which organic chemicals are allowed to react. Normal concentrations
(0.01M to 10M, typically 0.1M to 1M), temperatures (-100.degree. C.
to 250.degree. C., typically -78.degree. C. to 150.degree. C., more
typically -78.degree. C. to 100.degree. C., still more typically
0.degree. C. to 100.degree. C.), reaction vessels (typically glass,
plastic, metal), solvents, pressures, atmospheres (typically air
for oxygen and water insensitive reactions or nitrogen or argon for
oxygen or water sensitive), etc., are intended unless otherwise
indicated. The knowledge of similar reactions known in the art of
organic synthesis are used in selecting the conditions and
apparatus for "treating" in a given process. In particular, one of
ordinary skill in the art of organic synthesis selects conditions
and apparatus reasonably expected to successfully carry out the
chemical reactions of the described processes based on the
knowledge in the art.
[0551] Modifications of each of the exemplary schemes above and in
the examples (hereafter "exemplary schemes") leads to various
analogs of the candidate compounds. The above cited citations
describing suitable methods of organic synthesis are applicable to
such modifications.
[0552] In each of the exemplary schemes it may be advantageous to
separate reaction products from one another and/or from starting
materials. The desired products of each step or series of steps is
separated and/or purified (hereinafter separated) to the desired
degree of homogeneity by the techniques common in the art.
Typically such separations involve multiphase extraction,
crystallization from a solvent or solvent mixture, distillation,
sublimation, or chromatography. Chromatography can involve any
number of methods including, for example: reverse-phase and normal
phase; size exclusion; ion exchange; high, medium, and low pressure
liquid chromatography methods and apparatus; small scale
analytical; simulated moving bed (SMB) and preparative thin or
thick layer chromatography, as well as techniques of small scale
thin layer and flash chromatography.
[0553] Another class of separation methods involves treatment of a
mixture with a reagent selected to bind to or render otherwise
separable a desired product, unreacted starting material, reaction
by product, or the like. Such reagents include adsorbents such as
activated carbon, molecular sieves, ion exchange media, or the
like. Alternatively, the reagents can be acids in the case of a
basic material, bases in the case of an acidic material, binding
reagents such as antibodies, binding proteins, selective chelators
such as crown ethers, liquid/liquid ion extraction reagents (LIX),
or the like.
[0554] Selection of appropriate methods of separation depends on
the nature of the materials involved. These include boiling point
and molecular weight in distillation and sublimation, presence or
absence of polar functional groups in chromatography, stability of
materials in acidic and basic media in multiphase extraction, and
the like. One skilled in the art will apply techniques most likely
to achieve the desired separation.
[0555] A single stereoisomer, e.g. an enantiomer, substantially
free of its stereoisomer may be obtained by resolution of the
racemic mixture using a method such as formation of diastereomers
using optically active resolving agents ("Stereochemistry of Carbon
Compounds," (1962) by E. L. Eliel, McGraw Hill; Lochmuller, C. H.,
(1975) J. Chromatogr., 113:(3) 283-302). Racemic mixtures of chiral
compounds of the invention can be separated and isolated by any
suitable method, including: (1) formation of ionic, diastereomeric
salts with chiral compounds and separation by fractional
crystallization or other methods, (2) formation of diastereomeric
compounds with chiral derivatizing reagents, separation of the
diastereomers, and conversion to the pure stereoisomers, and (3)
separation of the substantially pure or enriched stereoisomers
directly under chiral conditions.
[0556] Under method (1), diastereomeric salts can be formed by
reaction of enantiomerically pure chiral bases such as brucine,
quinine, ephedrine, strychnine,
.alpha.-methyl-.beta.-phenylethylamine (amphetamine), and the like
with asymmetric compounds bearing acidic functionality, such as
carboxylic acid and sulfonic acid. The diastereomeric salts may be
induced to separate by fractional crystallization or ionic
chromatography. For separation of the optical isomers of amino
compounds, addition of chiral carboxylic or sulfonic acids, such as
camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid
can result in formation of the diastereomeric salts.
[0557] Alternatively, by method (2), the substrate to be resolved
is reacted with one enantiomer of a chiral compound to form a
diastereomeric pair (Eliel. E. and Wilen, S. (1994) Stereochemistry
of Organic Compounds, John Wiley & Sons, Inc., p. 322).
Diastereomeric compounds can be formed by reacting asymmetric
compounds with enantiomerically pure chiral derivatizing reagents,
such as menthyl derivatives, followed by separation of the
diastereomers and hydrolysis to yield the free, enantiomerically
enriched xanthene. A method of determining optical purity involves
making chiral esters, such as a menthyl ester, e.g. (-) menthyl
chloroformate in the presence of base, or Mosher ester,
.alpha.-methoxy-.alpha.-(trifluoromethyl)phenyl acetate (Jacob III.
(1982) J. Org. Chem. 47:4165), of the racemic mixture, and
analyzing the NMR spectrum for the presence of the two
atropisomeric diastereomers. Stable diastereomers of atropisomeric
compounds can be separated and isolated by normal- and
reverse-phase chromatography following methods for separation of
atropisomeric naphthyl-isoquinolines (Hoye, T., WO 96/15111). By
method (3), a racemic mixture of two enantiomers can be separated
by chromatography using a chiral stationary phase ("Chiral Liquid
Chromatography" (1989) W. J. Lough, Ed. Chapman and Hall, New York;
Okamoto, (1990) J. of Chromatogr. 513:375-378). Enriched or
purified enantiomers can be distinguished by methods used to
distinguish other chiral molecules with asymmetric carbon atoms,
such as optical rotation and circular dichroism.
[0558] The articles "and" and "or" shall be construed as meaning
"and/or" unless otherwise required by context or usage. Use of
"and/or" herein shall not be construed as foreclosing "and/or" when
only "and" or "or" are employed in other circumstances.
[0559] This invention includes all novel and unobvious compounds
disclosed herein, whether or not such compounds are described in
the context of methods or other disclosure and whether or not such
compounds are claimed upon filing or are set forth in the summary
of invention.
[0560] The invention has been described in detail sufficient to
allow one of ordinary skill in the art to make and use the subject
matter of the following examples. It is apparent that certain
modifications of the methods and compositions of the following
examples can be made within the scope and spirit of the
invention.
Examples General Section
[0561] Some Examples have been performed multiple times. In
repeated Examples, reaction conditions such as time, temperature,
concentration and the like, and yields were within normal
experimental ranges. In repeated Examples where significant
modifications were made, these have been noted where the results
varied significantly from those described. In Examples where
different starting materials were used, these are noted. When the
repeated Examples refer to a "corresponding" analog of a compound,
such as a "corresponding ethyl ester", this intends that an
otherwise present group, in this case typically a methyl ester, is
taken to be the same group modified as indicated.
Exemplary Methods of Making the Compounds of the Invention.
[0562] The invention provides many methods of making the
compositions of the invention. The compositions are prepared by any
of the applicable techniques of organic synthesis. Many such
techniques are well known in the art. Such as those elaborated in
"Compendium of Organic Synthetic Methods" (John Wiley & Sons,
New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol.
2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S.
Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, jr., 1980,
Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6, Michael B. Smith; as
well as March, J., "Advanced Organic Chemistry, Third Edition",
(John Wiley & Sons, New York, 1985), "Comprehensive Organic
Synthesis. Selectivity, Strategy & Efficiency in Modern Organic
Chemistry. In 9 Volumes", Barry M. Trost, Editor-in-Chief (Pergamon
Press, New York, 1993 printing).
[0563] Dialkyl phosphonates may be prepared according to the
methods of: Quast et al (1974) Synthesis 490; Stowell et al (1990)
Tetrahedron Lett. 3261; U.S. Pat. No. 5,663,159.
[0564] In general, synthesis of phosphonate esters is achieved by
coupling a nucleophile amine or alcohol with the corresponding
activated phosphonate electrophilic precursor for example,
Chlorophosphonate addition on to 5'-hydroxy of nucleoside is a well
known method for preparation of nucleoside phosphate monoesters.
The activated precursor can be prepared by several well known
methods. Chlorophosphonates useful for synthesis of the prodrugs
are prepared from the substituted-1,3-propanediol (Wissner, et al,
(1992) J. Med. Chem. 35:1650). Chlorophosphonates are made by
oxidation of the corresponding chlorophospholanes (Anderson, et al,
(1984) J. Org. Chem. 49:1304) which are obtained by reaction of the
substituted diol with phosphorus trichloride. Alternatively, the
chlorophosphonate agent is made by treating substituted-1,3-diols
with phosphorusoxychloride (Patois, et al, (1990) J. Chem. Soc.
Perkin Trans. I, 1577). Chlorophosphonate species may also be
generated in situ from corresponding cyclic phosphites (Silverburg,
et al., (1996) Tetrahedron lett., 37:771-774), which in turn can be
either made from chlorophospholane or phosphoramidate intermediate.
Phosphoroflouridate intermediate prepared either from pyrophosphate
or phosphoric acid may also act as precursor in preparation of
cyclic prodrugs (Watanabe et al., (1988) Tetrahedron lett.,
29:5763-66). Caution: fluorophosphonate compounds may be highly
toxic!
SCHEMES AND EXAMPLES
[0565] General aspects of these exemplary methods are described
below and in the Examples. Each of the products of the following
processes is optionally separated, isolated, and/or purified prior
to its use in subsequent processes.
[0566] A number of exemplary methods for the preparation of the
compositions of the invention are provided below. These methods are
intended to illustrate the nature of such preparations are not
intended to limit the scope of applicable methods.
[0567] The terms "treated", "treating", "treatment", and the like,
mean contacting, mixing, reacting, allowing to react, bringing into
contact, and other terms common in the art for indicating that one
or more chemical entities is treated in such a manner as to convert
it to one or more other chemical entities. This means that
"treating compound one with compound two" is synonymous with
"allowing compound one to react with compound two", "contacting
compound one with compound two", "reacting compound one with
compound two", and other expressions common in the art of organic
synthesis for reasonably indicating that compound one was
"treated", "reacted", "allowed to react", etc., with compound
two.
[0568] "Treating" indicates the reasonable and usual manner in
which organic chemicals are allowed to react. Normal concentrations
(0.01M to 10M, typically 0.1M to 1M), temperatures (-100.degree. C.
to 250.degree. C., typically -78.degree. C. to 150.degree. C., more
typically -78.degree. C. to 100.degree. C., still more typically
0.degree. C. to 100.degree. C.), reaction vessels (typically glass,
plastic, metal), solvents, pressures, atmospheres (typically air
for oxygen and water insensitive reactions or nitrogen or argon for
oxygen or water sensitive), etc., are intended unless otherwise
indicated. The knowledge of similar reactions known in the art of
organic synthesis are used in selecting the conditions and
apparatus for "treating" in a given process. In particular, one of
ordinary skill in the art of organic synthesis selects conditions
and apparatus reasonably expected to successfully carry out the
chemical reactions of the described processes based on the
knowledge in the art.
[0569] Modifications of each of the exemplary schemes above and in
the examples (hereafter "exemplary schemes") leads to various
analogs of the specific exemplary materials produce. The above
cited citations describing suitable methods of organic synthesis
are applicable to such modifications.
[0570] In each of the exemplary schemes it may be advantageous to
separate reaction products from one another and/or from starting
materials. The desired products of each step or series of steps is
separated and/or purified (hereinafter separated) to the desired
degree of homogeneity by the techniques common in the art.
Typically such separations involve multiphase extraction,
crystallization from a solvent or solvent mixture, distillation,
sublimation, or chromatography. Chromatography can involve any
number of methods including, for example: reverse-phase and normal
phase; size exclusion; ion exchange; high, medium, and low pressure
liquid chromatography methods and apparatus; small scale
analytical; simulated moving bed (SMB) and preparative thin or
thick layer chromatography, as well as techniques of small scale
thin layer and flash chromatography.
[0571] Another class of separation methods involves treatment of a
mixture with a reagent selected to bind to or render otherwise
separable a desired product, unreacted starting material, reaction
by product, or the like. Such reagents include adsorbents or
absorbents such as activated carbon, molecular sieves, ion exchange
media, or the like. Alternatively, the reagents can be acids in the
case of a basic material bases in the case of an acidic material
binding reagents such as antibodies, binding proteins, selective
chelators such as crown ethers, liquid/liquid ion extraction
reagents (LIX), or the like.
[0572] Selection of appropriate methods of separation depends on
the nature of the materials involved. For example, boiling point,
and molecular weight in distillation and sublimation, presence or
absence of polar functional groups in chromatography, stability of
materials in acidic and basic media in multiphase extraction, and
the like. One skilled in the art will apply techniques most likely
to achieve the desired separation.
[0573] A single stereoisomer, e.g. an enantiomer, substantially
free of its stereoisomer may be obtained by resolution of the
racemic mixture using a method such as formation of diastereomers
using optically active resolving agents ("Stereochemistry of Carbon
Compounds," (1962) by E. L. Eliel, McGraw Hill; Lochmuller, C. H.,
(1975) J. Chromatogr., 113:(3) 283-302). Racemic mixtures of chiral
compounds of the invention can be separated and isolated by any
suitable method, including: (1) formation of ionic, diastereomeric
salts with chiral compounds and separation by fractional
crystallization or other methods, (2) formation of diastereomeric
compounds with chiral derivatizing reagents, separation of the
diastereomers, and conversion to the pure stereoisomers, and (3)
separation of the substantially pure or enriched stereoisomers
directly under chiral conditions.
[0574] Under method (1), diastereomeric salts can be formed by
reaction of enantiomerically pure chiral bases such as brucine,
quinine, ephedrine, strychnine,
.alpha.-methyl-.beta.-phenylethylamine (amphetamine), and the like
with asymmetric compounds bearing acidic functionality, such as
carboxylic acid and sulfonic acid. The diastereomeric salts may be
induced to separate by fractional crystallization or ionic
chromatography. For separation of the optical isomers of amino
compounds, addition of chiral carboxylic or sulfonic acids, such as
camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid
can result in formation of the diastereomeric salts.
[0575] Alternatively, by method (2), the substrate to be resolved
is reacted with one enantiomer of a chiral compound to form a
diastereomeric pair (Eliel, E. and Wilen, S. (1994) Stereochemistry
of Organic Compounds, John Wiley & Sons, Inc., p. 322).
Diastereomeric compounds can be formed by reacting asymmetric
compounds with enantiomerically pure chiral derivatizing reagents,
such as menthyl derivatives, followed by separation of the
diastereomers and hydrolysis to yield the free, enantiomerically
enriched xanthene. A method of determining optical purity involves
making chiral esters, such as a menthyl ester, e.g. (-) menthyl
chloroformate in the presence of base, or Mosher ester,
.alpha.-methoxy-.alpha.-(trifluoromethyl)phenyl acetate (Jacob III.
(1982) J. Org. Chem. 47:4165), of the racemic mixture, and
analyzing the NMR spectrum for the presence of the two
atropisomeric diastereomers. Stable diastereomers of atropisomeric
compounds can be separated and isolated by normal- and
reverse-phase chromatography following methods for separation of
atropisomeric naphthyl-isoquinolines (Hoye, T., WO 96/15111). By
method (3), a racemic mixture of two enantiomers can be separated
by chromatography using a chiral stationary phase ("Chiral Liquid
Chromatography" (1989) W. J. Lough, Ed. Chapman and Hall, New York;
Okamoto, (1990) J. of Chromatogr. 513:375-378). Enriched or
purified enantiomers can be distinguished by methods used to
distinguish other chiral molecules with asymmetric carbon atoms,
such as optical rotation and circular dichroism.
[0576] All literature and patent citations above are hereby
expressly incorporated by reference at the locations of their
citation. Specifically cited sections or pages of the above cited
works are incorporated by reference with specificity. The invention
has been described in detail sufficient to allow one of ordinary
skill in the art to make and use the subject matter of the
following Embodiments. It is apparent that certain modifications of
the methods and compositions of the following Embodiments can be
made within the scope and spirit of the invention. ##STR153##
[0577] Scheme A shows the general interconversions of certain
phosphonate compounds: acids --P(O)(OH).sub.2; mono-esters
--P(O)(OR.sub.1)(OH); and diesters --P(O)(OR.sub.1).sub.2 in which
the R.sup.1 groups are independently selected, and defined herein
before, and the phosphorus is attached through a carbon moiety
(link, i.e. linker), which is attached to the rest of the molecule,
e.g. drug or drug intermediate (R). The R.sup.1 groups attached to
the phosphonate esters in Scheme 1 may be changed using established
chemical transformations. The interconversions may be carried out
in the precursor compounds or the final products using the methods
described below. The methods employed for a given phosphonate
transformation depend on the nature of the substituent R.sup.1. The
preparation and hydrolysis of phosphonate esters is described in
Organic Phosphorus Compounds, G. M. Kosolapoff, L. Maeir, eds,
Wiley, 1976, p. 9ff.
[0578] The conversion of a phosphonate diester 27.1 into the
corresponding phosphonate monoester 27.2 (Scheme A, Reaction 1) can
be accomplished by a number of methods. For example, the ester 27.1
in which R.sup.1 is an arylalkyl group such as benzyl, can be
converted into the monoester compound 27.2 by reaction with a
tertiary organic base such as diazabicyclooctane (DABCO) or
quinuclidine, as described in J. Org. Chem., 1995, 60:2946. The
reaction is performed in an inert hydrocarbon solvent such as
toluene or xylene, at about 110.degree. C. The conversion of the
diester 27.1 in which R.sup.1 is an aryl group such as phenyl or an
alkenyl group such as allyl, into the monoester 27.2 can be
effected by treatment of the ester 27.1 with a base such as aqueous
sodium hydroxide in acetonitrile or lithium hydroxide in aqueous
tetrahydrofuran. Phosphonate diesters 27.2 in which one of the
groups R.sup.1 is arylalkyl, such as benzyl and the other is alkyl,
can be converted into the monoesters 27.2 in which R.sup.1 is
alkyl, by hydrogenation, for example using a palladium on carbon
catalyst. Phosphonate diesters in which both of the groups R.sup.1
are alkenyl, such as allyl, can be converted into the monoester
27.2 in which R.sup.1 is alkenyl, by treatment with
chlorotris(triphenylphosphine)rhodium (Wilkinson's catalyst) in
aqueous ethanol at reflux, optionally in the presence of
diazabicyclooctane, for example by using the procedure described in
J. Org. Chem., 38:3224 1973 for the cleavage of allyl
carboxylates.
[0579] The conversion of a phosphonate diester 27.1 or a
phosphonate monoester 27.2 into the corresponding phosphonic acid
27.3 (Scheme A, Reactions 2 and 3) can effected by reaction of the
diester or the monoester with trimethylsilyl bromide, as described
in J. Chem. Soc., Chem. Comm., 739, 1979. The reaction is conducted
in an inert solvent such as, for example, dichloromethane,
optionally in the presence of a silylating agent such as
bis(trimethylsilyl)trifluoroacetamide, at ambient temperature. A
phosphonate monoester 27.2 in which R.sup.1 is arylalkyl such as
benzyl, can be converted into the corresponding phosphonic acid
27.3 by hydrogenation over a palladium catalyst, or by treatment
with hydrogen chloride in an ethereal solvent such as dioxane. A
phosphonate monoester 27.2 in which R.sup.1 is alkenyl such as, for
example, allyl, can be converted into the phosphonic acid 27.3 by
reaction with Wilkinson's catalyst in an aqueous organic solvent,
for example in 15% aqueous acetonitrile, or in aqueous ethanol, for
example using the procedure described in Helv. Chim. Acta., 68:618,
1985. Palladium catalyzed hydrogenolysis of phosphonate esters 27.1
in which R.sup.1 is benzyl is described in J. Org. Chem., 24:434,
1959. Platinum-catalyzed hydrogenolysis of phosphonate esters 27.1
in which R.sup.1 is phenyl is described in J. Amer. Chem. Soc.,
78:2336, 1956.
[0580] The conversion of a phosphonate monoester 27.2 into a
phosphonate diester 27.1 (Scheme A, Reaction 4) in which the newly
introduced R.sub.1 group is alkyl, arylalkyl, or haloalkyl such as
chloroethyl, can be effected by a number of reactions in which the
substrate 27.2 is reacted with a hydroxy compound R.sup.1OH, in the
presence of a coupling agent. Suitable coupling agents are those
employed for the preparation of carboxylate esters, and include a
carbodiimide such as dicyclohexylcarbodiimide, in which case the
reaction is preferably conducted in a basic organic solvent such as
pyridine, or (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PYBOP, Sigma), in which case the reaction is
performed in a polar solvent such as dimethylformamide, in the
presence of a tertiary organic base such as diisopropylethylamine,
or Aldrithiol-2 (Aldrich) in which case the reaction is conducted
in a basic solvent such as pyridine, in the presence of a triaryl
phosphine such as triphenylphosphine. Alternatively, the conversion
of the phosphonate monoester 27.1 to the diester 27.1 can be
effected by the use of the Mitsunobu reaction. The substrate is
reacted with the hydroxy compound R.sup.1OH, in the presence of
diethyl azodicarboxylate and a triarylphosphine such as triphenyl
phosphine. Alternatively, the phosphonate monoester 27.2 can be
transformed into the phosphonate diester 27.1, in which the
introduced R.sup.1 group is alkenyl or arylalkyl, by reaction of
the monoester with the halide R.sup.1Br, in which R.sup.1 is as
alkenyl or arylalkyl. The alkylation reaction is conducted in a
polar organic solvent such as dimethylformamide or acetonitrile, in
the presence of a base such as cesium carbonate. Alternatively, the
phosphonate monoester can be transformed into the phosphonate
diester in a two step procedure. In the first step, the phosphonate
monoester 27.2 is transformed into the chloro analog
--P(O)(OR.sup.1)Cl by reaction with thionyl chloride or oxalyl
chloride and the like, as described in Organic Phosphorus
Compounds, G. M. Kosolapoff, L. Maeir, eds, Wiley, 1976, p. 17, and
the thus-obtained product --P(O)(OR.sup.1)Cl is then reacted with
the hydroxy compound R.sup.1OH, in the presence of a base such as
triethylamine, to afford the phosphonate diester 27.1.
[0581] A phosphonic acid --P(O)(OH).sub.2 can be transformed into a
phosphonate monoester --P(O)(OR.sup.1)(OH) (Scheme A, Reaction 5)
by means of the methods described above of for the preparation of
the phosphonate diester --P(O)(OR.sup.1).sub.2 27.1, except that
only one molar proportion of the component R.sup.1OH or R.sup.1Br
is employed.
[0582] A phosphonic acid --P(O)(OH).sub.2 27.3 can be transformed
into a phosphonate diester --P(O)(OR.sup.1).sub.2 27.1 (Scheme A,
Reaction 6) by a coupling reaction with the hydroxy compound
R.sup.1OH, in the presence of a coupling agent such as Aldrithiol-2
(Aldrich) and triphenylphosphine. The reaction is conducted in a
basic solvent such as pyridine. Alternatively, phosphonic acids
27.3 can be transformed into phosphonic esters 27.1 in which
R.sup.1 is aryl, such as phenyl by means of a coupling reaction
employing, for example, phenol and dicyclohexylcarbodiimide in
pyridine at about 70.degree. C. Alternatively, phosphonic acids
27.3 can be transformed into phosphonic esters 27.1 in which
R.sup.1 is alkenyl, by means of an alkylation reaction. The
phosphonic acid is reacted with the alkenyl bromide R.sup.1Br in a
polar organic solvent such as acetonitrile solution at reflux
temperature, in the presence of a base such as cesium carbonate, to
afford the phosphonic ester 27.1.
[0583] Phosphonate prodrugs of the present invention may also be
prepared from the precursor free acid by Mitsunobu reactions
(Mitsunobu, (1981) Synthesis, 1; Campbell, (1992) J. Org. Chem.,
52:6331), and other acid coupling reagents including, but not
limited to, carbodiimides (Alexander, et al, (1994) Collect. Czech.
Chem. Commun. 59:1853; Casara, et al, (1992) Bioorg. Med. Chem.
Lett., 2:145; Ohashi, et al, (1988) Tetrahedron Lett., 29:1189),
and benzotriazolyloxytris-(dimethylamino)phosphonium salts
(Campagne, et al, (1993) Tetrahedron Lett., 34:6743).
Preparation of Carboalkoxy-Substituted Phosphonate Bisamidates,
Monoamidates, Diesters and Monoesters.
[0584] A number of methods are available for the conversion of
phosphonic acids into amidates and esters. In one group of methods,
the phosphonic acid is either converted into an isolated activated
intermediate such as a phosphoryl chloride, or the phosphonic acid
is activated in situ for reaction with an amine or a hydroxy
compound.
[0585] The conversion of phosphonic acids into phosphoryl chlorides
is accomplished by reaction with thionyl chloride, for example as
described in J. Gen. Chem. USSR, 1983, 53, 480, Zh. Obschei Khim.,
1958, 28, 1063, or J. Org. Chem., 1994, 59, 6144, or by reaction
with oxalyl chloride, as described in J. Am. Chem. Soc., 1994, 116,
3251, or J. Org. Chem., 1994, 59, 6144, or by reaction with
phosphorus pentachloride, as described in J. Org. Chem., 2001, 66,
329, or in J. Med. Chem., 1995, 38, 1372. The resultant phosphoryl
chlorides are then reacted with amines or hydroxy compounds in the
presence of a base to afford the amidate or ester products.
[0586] Phosphonic acids are converted into activated imidazolyl
derivatives by reaction with carbonyl diimidazole, as described in
J. Chem. Soc., Chem. Comm., 1991, 312, or Nucleosides Nucleotides
2000, 19, 1885. Activated sulfonyloxy derivatives are obtained by
the reaction of phosphonic acids with trichloromethylsulfonyl
chloride, as described in J. Med. Chem. 1995, 38, 4958, or with
triisopropylbenzenesulfonyl chloride, as described in Tet. Lett.,
1996, 7857, or Bioorg. Med. Chem. Lett., 1998, 8, 663. The
activated sulfonyloxy derivatives are then reacted with amines or
hydroxy compounds to afford amidates or esters.
[0587] Alternatively, the phosphonic acid and the amine or hydroxy
reactant are combined in the presence of a diimide coupling agent.
The preparation of phosphonic amidates and esters by means of
coupling reactions in the presence of dicyclohexyl carbodiimide is
described, for example, in J. Chem. Soc., Chem. Comm., 1991, 312,
or J. Med. Chem., 1980, 23, 1299 or Coll. Czech. Chem. Comm., 1987,
52, 2792. The use of ethyl dimethylaminopropyl carbodiimide for
activation and coupling of phosphonic acids is described in Tet.
Lett., 2001, 42, 8841, or Nucleosides Nucleotides, 2000, 19,
1885.
[0588] A number of additional coupling reagents have been described
for the preparation of amidates and esters from phosphonic acids.
The agents include Aldrithiol-2, and PYBOP and BOP, as described in
J. Org. Chem., 1995, 60, 5214, and J. Med. Chem., 1997, 40, 3842,
mesitylene-2-sulfonyl-3-nitro-1,2,4-triazole (MSNT), as described
in J. Med. Chem., 1996, 39, 4958, diphenylphosphoryl azide, as
described in J. Org. Chem., 1984, 49, 1158,
1-(2,4,6-triisopropylbenzenesulfonyl-3-nitro-1,2,4-triazole (TPSNT)
as described in Bioorg. Med. Chem. Lett., 1998, 8, 1013,
bromotris(dimethylamino)phosphonium hexafluorophosphate (BroP), as
described in Tet. Lett., 1996, 37, 3997,
2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane, as described in
Nucleosides Nucleotides 1995, 14, 871, and diphenyl
chlorophosphate, as described in J. Med. Chem., 1988, 31, 1305.
[0589] Phosphonic acids are converted into amidates and esters by
means of the Mitsonobu reaction, in which the phosphonic acid and
the amine or hydroxy reactant are combined in the presence of a
triaryl phosphine and a dialkyl azodicarboxylate. The procedure is
described in Org. Lett., 2001, 3, 643, or J. Med. Chem., 1997, 40,
3842.
[0590] Phosphonic esters are also obtained by the reaction between
phosphonic acids and halo compounds, in the presence of a suitable
base. The method is described, for example, in Anal. Chem., 1987,
59, 1056, or J. Chem. Soc. Perkin Trans., I, 1993, 19, 2303, or J.
Med. Chem., 1995, 38, 1372, or Tet. Lett., 2002, 43, 1161.
[0591] Schemes 1-4 illustrate the conversion of phosphonate esters
and phosphonic acids into carboalkoxy-substituted
phosphorobisamidates (Scheme 1), phosphoroamidates (Scheme 2),
phosphonate monoesters (Scheme 3) and phosphonate diesters, (Scheme
4).
[0592] Scheme 1 illustrates various methods for the conversion of
phosphonate diesters 1.1 into phosphorobisamidates 1.5. The diester
1.1, prepared as described previously, is hydrolyzed, either to the
monoester 1.2 or to the phosphonic acid 1.6. The methods employed
for these transformations are described above. The monoester 1.2 is
converted into the monoamidate 1.3 by reaction with an aminoester
1.9, in which the group R.sup.2 is H or alkyl the group R.sup.4 is
an alkylene moiety such as, for example, CHCH.sub.3, CHPr.sup.I,
CH(CH.sub.2Ph), CH.sub.2CH(CH.sub.3) and the like, or a group
present in natural or modified aminoacids, and the group R.sup.5 is
alkyl. The reactants are combined in the presence of a coupling
agent such as a carbodiimide, for example dicyclohexyl
carbodiimide, as described in J. Am. Chem. Soc., 1957, 79, 3575,
optionally in the presence of an activating agent such as
hydroxybenzotriazole, to yield the amidate product 1.3. The
amidate-forming reaction is also effected in the presence of
coupling agents such as BOP, as described in J. Org. Chem., 1995,
60, 5214, Aldrithiol, PYBOP and similar coupling agents used for
the preparation of amides and esters. Alternatively, the reactants
1.2 and 1.9 are transformed into the monoamidate 1.3 by means of a
Mitsonobu reaction. The preparation of amidates by means of the
Mitsonobu reaction is described in J. Med. Chem., 1995, 38, 2742.
Equimolar amounts of the reactants are combined in an inert solvent
such as tetrahydrofuran in the presence of a triaryl phosphine and
a dialkyl azodicarboxylate. The thus-obtained monoamidate ester 1.3
is then transformed into amidate phosphonic acid 1.4. The
conditions used for the hydrolysis reaction depend on the nature of
the R.sup.1 group, as described previously. The phosphonic acid
amidate 1.4 is then reacted with an aminoester 1.9, as described
above, to yield the bisamidate product 1.5, in which the amino
substituents are the same or different.
[0593] An example of this procedure is shown in Scheme 1, Example
1. In this procedure, a dibenzyl phosphonate 1.14 is reacted with
diazabicyclooctane (DABCO) in toluene at reflux, as described in J.
Org. Chem., 1995, 60, 2946, to afford the monobenzyl phosphonate
1.15. The product is then reacted with equimolar amounts of ethyl
alaninate 1.16 and dicyclohexyl carbodiimide in pyridine, to yield
the amidate product 1.17. The benzyl group is then removed, for
example by hydrogenolysis over a palladium catalyst, to give the
monoacid product 1.18. This compound is then reacted in a Mitsonobu
reaction with ethyl leucinate 1.19, triphenyl phosphine and
diethylazodicarboxylate, as described in J. Med. Chem., 1995, 38,
2742, to produce the bisamidate product 1.20.
[0594] Using the above procedures, but employing, in place of ethyl
leucinate 1.19 or ethyl alaninate 1.16, different aminoesters 1.9,
the corresponding products 1.5 are obtained.
[0595] Alternatively, the phosphonic acid 1.6 is converted into the
bisamidate 1.5 by use of the coupling reactions described above.
The reaction is performed in one step, in which case the
nitrogen-related substituents present in the product 1.5 are the
same, or in two steps, in which case the nitrogen-related
substituents can be different.
[0596] An example of the method is shown in Scheme 1, Example 2. In
this procedure, a phosphonic acid 1.6 is reacted in pyridine
solution with excess ethyl phenylalaninate 1.21 and
dicyclohexylcarbodiimide, for example as described in J. Chem.
Soc., Chem. Comm., 1991, 1063, to give the bisamidate product
1.22.
[0597] Using the above procedures, but employing, in place of ethyl
phenylalaninate, different aminoesters 1.9, the corresponding
products 1.5 are obtained.
[0598] As a further alternative, the phosphonic acid 1.6 is
converted into the mono or bis-activated derivative 1.7, in which
Lv is a leaving group such as chloro, imidazolyl,
triisopropylbenzenesulfonyloxy etc. The conversion of phosphonic
acids into chlorides 1.7 (Lv=Cl) is effected by reaction with
thionyl chloride or oxalyl chloride and the like, as described in
Organic Phosphorus Compounds, G. M. Kosolapoff, L. Maeir, eds,
Wiley, 1976, p. 17. The conversion of phosphonic acids into
monoimidazolides 1.7 (Lv=irridazolyl) is described in J. Med.
Chem., 2002, 45, 1284 and in J. Chem. Soc. Chem. Comm, 1991, 312.
Alternatively, the phosphonic acid is activated by reaction with
triisopropylbenzenesulfonyl chloride, as described in Nucleosides
and Nucleotides, 2000, 10, 1885. The activated product is then
reacted with the aminoester 1.9, in the presence of a base, to give
the bisamidate 1.5. The reaction is performed in one step, in which
case the nitrogen substituents present in the product 1.5 are the
same, or in two steps, via the intermediate 1.11, in which case the
nitrogen substituents can be different.
[0599] Examples of these methods are shown in Scheme 1, Examples 3
and 5. In the procedure illustrated in Scheme 1, Example 3, a
phosphonic acid 1.6 is reacted with ten molar equivalents of
thionyl chloride, as described in Zh. Obschei Khim, 1958, 28, 1063,
to give the dichloro compound 1.23. The product is then reacted at
reflux temperature in a polar aprotic solvent such as acetonitrile,
and in the presence of a base such as triethylamine, with butyl
serinate 1.24 to afford the bisamidate product 1.25.
[0600] Using the above procedures, but employing, in place of butyl
serinate 1.24, different aminoesters 1.9, the corresponding
products 1.5 are obtained.
[0601] In the procedure illustrated in Scheme 1, Example 5, the
phosphonic acid 1.6 is reacted, as described in J. Chem. Soc. Chem.
Comm., 1991, 312, with carbonyl diimidazole to give the imidazolide
1.32. The product is then reacted in acetonitrile solution at
ambient temperature, with one molar equivalent of ethyl alaninate
1.33 to yield the monodisplacement product 1.34. The latter
compound is then reacted with carbonyl diimidazole to produce the
activated intermediate 1.35, and the product is then reacted, under
the same conditions, with ethyl N-methylalaninate 1.33a to give the
bisamidate product 1.36.
[0602] Using the above procedures, but employing, in place of ethyl
alaninate 1.33 or ethyl N-methylalaninate 1.33a, different
aminoesters 1.9, the corresponding products 1.5 are obtained.
[0603] The intermediate monoamidate 1.3 is also prepared from the
monoester 1.2 by first converting the monoester into the activated
derivative 1.8 in which Lv is a leaving group such as halo,
imidazolyl etc, using the procedures described above. The product
1.8 is then reacted with an aminoester 1.9 in the presence of a
base such as pyridine, to give an intermediate monoamidate product
1.3. The latter compound is then converted, by removal of the
R.sup.1 group and coupling of the product with the aminoester 1.9,
as described above, into the bisamidate 1.5.
[0604] An example of this procedure, in which the phosphonic acid
is activated by conversion to the chloro derivative 1.26, is shown
in Scheme 1, Example 4. In this procedure, the phosphonic
monobenzyl ester 1.15 is reacted, in dichloromethane, with thionyl
chloride, as described in Tet. Let., 1994, 35, 4097, to afford the
phosphoryl chloride 1.26. The product is then reacted in
acetonitrile solution at ambient temperature with one molar
equivalent of ethyl 3-amino-2-methylpropionate 1.27 to yield the
monoamidate product 1.28. The latter compound is hydrogenated in
ethyl acetate over a 5% palladium on carbon catalyst to produce the
monoacid product 1.29. The product is subjected to a Mitsonobu
coupling procedure, with equimolar amounts of butyl alaninate 1.30,
triphenyl phosphine, diethylazodicarboxylate and triethylamine in
tetrahydrofuran, to give the bisamidate product 1.31.
[0605] Using the above procedures, but employing, in place of ethyl
3-amino-2-methylpropionate 1.27 or butyl alaninate 1.30, different
aminoesters 1.9, the corresponding products 1.5 are obtained.
[0606] The activated phosphonic acid derivative 1.7 is also
converted into the bisamidate 1.5 via the diamino compound 1.10.
The conversion of activated phosphonic acid derivatives such as
phosphoryl chlorides into the corresponding amino analogs 1.10, by
reaction with ammonia, is described in Organic Phosphorus
Compounds, G. M. Kosolapoff, L. Maeir, eds, Wiley, 1976. The
diamino compound 1.10 is then reacted at elevated temperature with
a haloester 1.12, in a polar organic solvent such as
dimethylformamide, in the presence of a base such as
dimethylaminopyridine or potassium carbonate, to yield the
bisamidate 1.5. An example of this procedure is shown in Scheme 1,
Example 6. In this method, a dichlorophosphonate 1.23 is reacted
with ammonia to afford the diamide 1.37. The reaction is performed
in aqueous, aqueous alcoholic or alcoholic solution, at reflux
temperature. The resulting diamino compound is then reacted with
two molar equivalents of ethyl 2-bromo-3-methylbutyrate 1.38, in a
polar organic solvent such as N-methylpyrrolidinone at ca.
150.degree. C., in the presence of a base such as potassium
carbonate, and optionally in the presence of a catalytic amount of
potassium iodide, to afford the bisamidate product 1.39.
[0607] Using the above procedures, but employing, in place of ethyl
2-bromo-3-methylbutyrate 1.38, different haloesters 1.12 the
corresponding products 1.5 are obtained.
[0608] The procedures shown in Scheme 1 are also applicable to the
preparation of bisamidates in which the aminoester moiety
incorporates different functional groups. Scheme 1, Example 7
illustrates the preparation of bisamidates derived from tyrosine.
In this procedure, the monoimidazolide 1.32 is reacted with propyl
tyrosinate 1.40, as described in Example 5, to yield the
monoamidate 1.41. The product is reacted with carbonyl diimidazole
to give the imidazolide 1.42, and this material is reacted with a
further molar equivalent of propyl tyrosinate to produce the
bisamidate product 1.43.
[0609] Using the above procedures, but employing, in place of
propyl tyrosinate 1.40, different aminoesters 1.9, the
corresponding products 1.5 are obtained. The aminoesters employed
in the two stages of the above procedure can be the same or
different, so that bisamidates with the same or different amino
substituents are prepared.
[0610] Scheme 2 illustrates methods for the preparation of
phosphonate monoamidates. In one procedure, a phosphonate monoester
1.1 is converted, as described in Scheme 1, into the activated
derivative 1.8. This compound is then reacted, as described above,
with an aminoester 1.9, in the presence of a base, to afford the
monoamidate product 2.1. The procedure is illustrated in Scheme 2,
Example 1. In this method, a monophenyl phosphonate 2.7 is reacted
with, for example, thionyl chloride, as described in J. Gen. Chem.
USSR., 1983, 32, 367, to give the chloro product 2.8. The product
is then reacted, as described in Scheme 1, with ethyl alaninate
2.9, to yield the amidate 2.10.
[0611] Using the above procedures, but employing, in place of ethyl
alaninate 2.9, different aminoesters 1.9, the corresponding
products 2.1 are obtained.
[0612] Alternatively, the phosphonate monoester 1.1 is coupled, as
described in Scheme 1, with an aminoester 1.9 to produce the
amidate 2.1. If necessary, the R.sup.1 substituent is then altered,
by initial cleavage to afford the phosphonic acid 2.2. The
procedures for this transformation depend on the nature of the
R.sup.1 group, and are described above. The phosphonic acid is then
transformed into the ester amidate product 2.3, by reaction with
the hydroxy compound R.sup.3OH, in which the group R.sup.3 is aryl
heteroaryl, alkyl cycloalkyl, haloalkyl etc, using the same
coupling procedures (carbodiide, Aldrithiol-2, PYBOP, Mitsonobu
reaction etc) described in Scheme 1 for the coupling of amines and
phosphonic acids. ##STR154## ##STR155## ##STR156##
[0613] Examples of this method are shown in Scheme 2, Examples and
2 and 3. In the sequence shown in Example 2, a monobenzyl
phosphonate 2.11 is transformed by reaction with ethyl alaninate,
using one of the methods described above, into the monoamidate
2.12. The benzyl group is then removed by catalytic hydrogenation
in ethyl acetate solution over a 5% palladium on carbon catalyst,
to afford the phosphonic acid amidate 2.13. The product is then
reacted in dichloromethane solution at ambient temperature with
equimolar amounts of 1-(dimethylaminopropyl)-3-ethylcarbodiimide
and trifluoroethanol 2.14, for example as described in Tet. Lett.,
2001, 42, 8841, to yield the amidate ester 2.15.
[0614] In the sequence shown in Scheme 2, Example 3, the
monoamidate 2.13 is coupled, in tetrahydrofuran solution at ambient
temperature, with equimolar amounts of dicyclohexyl carbodiimide
and 4-hydroxy-N-methylpiperidine 2.16, to produce the amidate ester
product 2.17.
[0615] Using the above procedures, but employing, in place of the
ethyl alaninate product 2.12 different monoacids 2.2, and in place
of trifluoroethanol 2.14 or 4-hydroxy-N-methylpiperidine 2.16,
different hydroxy compounds R.sup.3OH, the corresponding products
2.3 are obtained.
[0616] Alternatively, the activated phosphonate ester 1.8 is
reacted with ammonia to yield the amidate 2.4. The product is then
reacted, as described in Scheme 1, with a haloester 2.5, in the
presence of a base, to produce the amidate product 2.6. If
appropriate, the nature of the R.sup.1 group is changed, using the
procedures described above, to give the product 2.3. The method is
illustrated in Scheme 2, Example 4. In this sequence, the
monophenyl phosphoryl chloride 2.18 is reacted, as described in
Scheme 1, with ammonia, to yield the amino product 2.19.
[0617] This material is then reacted in N-methylpyrrolidinone
solution at 170.degree. C. with butyl 2-bromo-3-phenylpropionate
2.20 and potassium carbonate, to afford the amidate product 2.21.
Using these procedures, but employing, in place of butyl
2-bromo-3-phenylpropionate 2.20, different haloesters 2.5, the
corresponding products 2.6 are obtained.
[0618] The monoamidate products 2.3 are also prepared from the
doubly activated phosphonate derivatives 1.7. In this procedure,
examples of which are described in Synlett., 1998, 1, 73, the
intermediate 1.7 is reacted with a limited amount of the aminoester
1.9 to give the mono-displacement product 1.11. The latter compound
is then reacted with the hydroxy compound R.sup.3OH in a polar
organic solvent such as dimethylformamide, in the presence of a
base such as diisopropylethylamine, to yield the monoamidate ester
2.3.
[0619] The method is illustrated in Scheme 2, Example 5. In this
method, the phosphoryl dichloride 2.22 is reacted in
dichloromethane solution with one molar equivalent of ethyl
N-methyl tyrosinate 2.23 and dimethylaminopyridine, to generate the
monoamidate 2.24. The product is then reacted with phenol 2.25 in
dimethylformamide containing potassium carbonate, to yield the
ester amidate product 2.26.
[0620] Using these procedures, but employing, in place of ethyl
N-methyl tyrosinate 2.23 or phenol 2.25, the aminoesters 1.9 and/or
the hydroxy compounds R.sup.3OH, the corresponding products 2.3 are
obtained. ##STR157## ##STR158##
[0621] Scheme 3 illustrates methods for the preparation of
carboalkoxy-substituted phosphonate diesters in which one of the
ester groups incorporates a carboalkoxy substituent. In one
procedure, a phosphonate monoester 1.1, prepared as described
above, is coupled, using one of the methods described above, with a
hydroxyester 3.1, in which the groups R.sup.4 and R.sup.5 are as
described in Scheme 1. For example, equimolar amounts of the
reactants are coupled in the presence of a carbodiimide such as
dicyclohexyl carbodiimide, as described in Aust. J. Chem., 1963,
609, optionally in the presence of dimethylaminopyridine, as
described in Tet., 1999, 55, 12997. The reaction is conducted in an
inert solvent at ambient temperature.
[0622] The procedure is illustrated in Scheme 3, Example 1. In this
method, a monophenyl phosphonate 3.9 is coupled, in dichloromethane
solution in the presence of dicyclohexyl carbodiimide, with ethyl
3-hydroxy-2-methylpropionate 3.10 to yield the phosphonate mixed
diester 3.11.
[0623] Using this procedure, but employing, in place of ethyl
3-hydroxy-2-methylpropionate 3.10, different hydroxyesters 3.1, the
corresponding products 3.2 are obtained.
[0624] The conversion of a phosphonate monoester 1.1 into a mixed
diester 3.2 is also accomplished by means of a Mitsonobu coupling
reaction with the hydroxyester 3.1, as described in Org. Lett.,
2001, 643. In this method, the reactants 1.1 and 3.1 are combined
in a polar solvent such as tetrahydrofaran, in the presence of a
triarylphosphine and a dialkyl azodicarboxylate, to give the mixed
diester 3.2. The R.sup.1 substituent is varied by cleavage, using
the methods described previously, to afford the monoacid product
3.3. The product is then coupled, for example using methods
described above, with the hydroxy compound R.sup.3OH, to give the
diester product 3.4.
[0625] The procedure is illustrated in Scheme 3, Example 2. In this
method, a monoallyl phosphonate 3.12 is coupled in tetrahydrofuran
solution, in the presence of triphenylphosphine and
diethylazodicarboxylate, with ethyl lactate 3.13 to give the mixed
diester 3.14. The product is reacted with tris(triphenylphosphine)
rhodium chloride (Wilkinson catalyst) in acetonitrile, as described
previously, to remove the allyl group and produce the monoacid
product 3.15. The latter compound is then coupled, in pyridine
solution at ambient temperature, in the presence of dicyclohexyl
carbodiimide, with one molar equivalent of 3-hydroxypyridine 3.16
to yield the mixed diester 3.17.
[0626] Using the above procedures, but employing, in place of the
ethyl lactate 3.13 or 3-hydroxypyridine, a different hydroxyester
3.1 and/or a different hydroxy compound R.sup.3OH, the
corresponding products 3.4 are obtained.
[0627] The mixed diesters 3.2 are also obtained from the monoesters
1.1 via the intermediacy of the activated monoesters 3.5. In this
procedure, the monoester 1.1 is converted into the activated
compound 3.5 by reaction with, for example, phosphorus
pentachloride, as described in J. Org. Chem., 2001, 66, 329, or
with thionyl chloride or oxalyl chloride (Lv=Cl), or with
triisopropylbenzenesulfonyl chloride in pyridine, as described in
Nucleosides and Nucleotides, 2000, 19, 1.885, or with carbonyl
diimidazole, as described in J. Med. Chem., 2002, 45, 1284. The
resultant activated monoester is then reacted with the hydroxyester
3.1, as described above, to yield the mixed diester 3.2.
[0628] The procedure is illustrated in Scheme 3, Example 3. In this
sequence, a monophenyl phosphonate 3.9 is reacted, in acetonitrile
solution at 70.degree. C., with ten equivalents of thionyl
chloride, so as to produce the phosphoryl chloride 3.19. The
product is then reacted with ethyl 4-carbamoyl-2-hydroxybutyrate
3.20 in dichloromethane containing triethylamine, to give the mixed
diester 3.21.
[0629] Using the above procedures, but employing, in place of ethyl
4-carbamoyl-2-hydroxybutyrate 3.20, different hydroxyesters 3.1,
the corresponding products 3.2 are obtained.
[0630] The mixed phosphonate diesters are also obtained by an
alternative route for incorporation of the R.sup.3O group into
intermediates 3.3 in which the hydroxyester moiety is already
incorporated. In this procedure, the monoacid intermediate 3.3 is
converted into the activated derivative 3.6 in which Lv is a
leaving group such as chloro, imidazole, and the like, as
previously described. The activated intermediate is then reacted
with the hydroxy compound R.sup.3OH, in the presence of a base, to
yield the mixed diester product 3.4.
[0631] The method is illustrated in Scheme 3, Example 4. In this
sequence, the phosphonate monoacid 3.22 is reacted with
trichloromethanesulfonyl chloride in tetrahydrofuran containing
collidine, as described in J. Med. Chem., 1995, 38, 4648, to
produce the trichloromethanesulfonyloxy product 3.23. This compound
is reacted with 3-(morpholinomethyl)phenol 3.24 in dichloromethane
containing triethylamine, to yield the mixed diester product
3.25.
[0632] Using the above procedures, but employing, in place of with
3-(morpholinomethyl)phenol 3.24, different carbinols R.sup.3OH, the
corresponding products 3.4 are obtained.
[0633] The phosphonate esters 3.4 are also obtained by means of
alkylation reactions performed on the monoesters 1.1. The reaction
between the monoacid 1.1 and the haloester 3.7 is performed in a
polar solvent in the presence of a base such as
diisopropylethylamine, as described in Anal. Chem., 1987, 59, 1056,
or triethylamine, as described in J. Med. Chem., 1995, 38, 1372, or
in a non-polar solvent such as benzene, in the presence of
18-crown-6, as described in Syn. Comm., 1995, 25, 3565.
[0634] The method is illustrated in Scheme 3, Example 5. In this
procedure, the monoacid 3.26 is reacted with ethyl
2-bromo-3-phenylpropionate 3.27 and diisopropylethylamine in
dimethylformamide at 80.degree. C. to afford the mixed diester
product 3.28.
[0635] Using the above procedure, but employing, in place of ethyl
2-bromo-3-phenylpropionate 3.27, different haloesters 3.7, the
corresponding products 3.4 are obtained. ##STR159## ##STR160##
[0636] Scheme 4 illustrates methods for the preparation of
phosphonate diesters in which both the ester substituents
incorporate carboalkoxy groups.
[0637] The compounds are prepared directly or indirectly from the
phosphonic acids 1.6. In one alternative, the phosphonic acid is
coupled with the hydroxyester 4.2, using the conditions described
previously in Schemes 1-3, such as coupling reactions using
dicyclohexyl carbodiimide or similar reagents, or under the
conditions of the Mitsonobu reaction, to afford the diester product
4.3 in which the ester substituents are identical.
[0638] This method is illustrated in Scheme 4, Example 1. In this
procedure, the phosphonic acid 1.6 is reacted with three molar
equivalents of butyl lactate 4.5 in the presence of Aldrithiol-2
and triphenyl phosphine in pyridine at ca. 70.degree. C., to afford
the diester 4.6.
[0639] Using the above procedure, but employing, in place of butyl
lactate 4.5, different hydroxyesters 4.2, the corresponding
products 4.3 are obtained.
[0640] Alternatively, the diesters 4.3 are obtained by alkylation
of the phosphonic acid 1.6 with a haloester 4.1. The alkylation
reaction is performed as described in Scheme 3 for the preparation
of the esters 3.4.
[0641] This method is illustrated in Scheme 4, Example 2. In this
procedure, the phosphonic acid 1.6 is reacted with excess ethyl
3-bromo-2-methylpropionate 4.7 and diisopropylethylamine in
dimethylformamide at ca. 80.degree. C., as described in Anal.
Chem., 1987, 59, 1056, to produce the diester 4.8.
[0642] Using the above procedure, but employing, in place of ethyl
3-bromo-2-methylpropionate 4.7, different haloesters 4.1, the
corresponding products 4.3 are obtained.
[0643] The diesters 4.3 are also obtained by displacement reactions
of activated derivatives 1.7 of the phosphonic acid with the
hydroxyesters 4.2. The displacement reaction is performed in a
polar solvent in the presence of a suitable base, as described in
Scheme 3. The displacement reaction is performed in the presence of
an excess of the hydroxyester, to afford the diester product 4.3 in
which the ester substituents are identical, or sequentially with
limited amounts of different hydroxyesters, to prepare diesters 4.3
in which the ester substituents are different. The methods are
illustrated in Scheme 4, Examples 3 and 4. As shown in Example 3,
the phosphoryl dichloride 2.22 is reacted with three molar
equivalents of ethyl 3-hydroxy-2-(hydroxymethyl)propionate 4.9 in
tetrahydrofuran containing potassium carbonate, to obtain the
diester product 4.10.
[0644] Using the above procedure, but employing, in place of ethyl
3-hydroxy-2-(hydroxymethyl)propionate 4.9, different hydroxyesters
4.2, the corresponding products 4.3 are obtained.
[0645] Scheme 4, Example 4 depicts the displacement reaction
between equimolar amounts of the phosphoryl dichloride 2.22 and
ethyl 2-methyl-3-hydroxypropionate 4.11, to yield the monoester
product 4.12. The reaction is conducted in acetonitrile at
70.degree. C. in the presence of diisopropylethylamine. The product
4.12 is then reacted, under the same conditions, with one molar
equivalent of ethyl lactate 4.13, to give the diester product
4.14.
[0646] Using the above procedures, but employing, in place of ethyl
2-methyl-3-hydroxypropionate 4.11 and ethyl lactate 4.13,
sequential reactions with different hydroxyesters 4.2, the
corresponding products 4.3 are obtained. ##STR161## ##STR162##
[0647] Aryl halides undergo Ni.sup.+2 catalyzed reaction with
phosphite derivatives to give aryl phosphonate containing compounds
(Balthazar, et al (1980) J. Org. Chem. 45:5425). Phosphonates may
also be prepared from the chlorophosphonate in the presence of a
palladium catalyst using aromatic triflates (Petrakis, et al.
(1987) J. Am. Chem. Soc. 109:2831; Lu, et al, (1987) Synthesis,
726). In another method, aryl phosphonate esters are prepared from
aryl phosphates under anionic rearrangement conditions (Melvin
(1981) Tetrahedron Lett. 22:3375; Casteel, et al, (1991) Synthesis,
691). N-Alkoxy aryl salts with alkali metal derivatives of cyclic
alkyl phosphonate provide general synthesis for
heteroaryl-2-phosphonate linkers (Redmore (1970) J. Org. Chem.
35:4114). These above mentioned methods can also be extended to
compounds where the W.sup.5 group is a heterocycle.
Cyclic-1,3-propanyl prodrugs of phosphonates are also synthesized
from phosphonic diacids and substituted propane-1,3-diols using a
coupling reagent such as 1,3-dicyclohexylcarbodiimide (DCC) in
presence of a base (e.g., pyridine). Other carbodiimide based
coupling agents like 1,3-disopropylcarbodiimide or water soluble
reagent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCI) can also be utilized for the synthesis of
cyclic phosphonate prodrugs.
[0648] The carbamoyl group may be formed by reaction of a hydroxy
group according to the methods known in the art, including the
teachings of Ellis, US 2002/0103378 A1 and Hajima, U.S. Pat. No.
6,018,049.
[0649] Generally, the reaction conditions such as temperature,
reaction time, solvents, work-up procedures, and the like, will be
those common in the art for the particular reaction to be
performed. The cited reference material, together with material
cited therein, contains detailed descriptions of such conditions.
Typically the temperatures will be -100.degree. C. to 200.degree.
C., solvents will be aprotic or protic, and reaction times will be
10 seconds to 10 days. Work-up typically consists of quenching any
unreacted reagents followed by partition between a water/organic
layer system (extraction) and separating the layer containing the
product.
[0650] Oxidation and reduction reactions are typically carried out
at temperatures near room temperature (about 20.degree. C.),
although for metal hydride reductions frequently the temperature is
reduced to 0.degree. C. to -100.degree. C., solvents are typically
aprotic for reductions and may be either protic or aprotic for
oxidations. Reaction times are adjusted to achieve desired
conversions.
[0651] Condensation reactions are typically carried out at
temperatures near room temperature, although for non-equilibrating,
kinetically controlled condensations reduced temperatures
(0.degree. C. to -100.degree. C.) are also common. Solvents can be
either protic (common in equilibrating reactions) or aprotic
(common in kinetically controlled reactions).
[0652] Standard synthetic techniques such as azeotropic removal of
reaction by-products and use of anhydrous reaction conditions (e.g.
inert gas environments) are common in the art and will be applied
when applicable.
[0653] General synthetic routes to substituted imidazoles are well
established. See Ogata M (1988) Annals of the New York Academy of
Sciences 544:12-31; Takahashi et al (1985) Heterocycles 23:6,
1483-1492; Ogata et al (1980) CHEM IND LONDON 2:5-8.6; Yanagisawa
et al U.S. Pat. No. 5,646,171; Rachwal et al US 2002/0115693 A1;
Carlson et al U.S. Pat. Nos. 3,790,593; 3,761,491 and 3773781; Aono
et al U.S. Pat. No. 6,054,591; Hajima et al U.S. Pat. No.
6,057,448; Sugimoto et al EP 00552060 and U.S. Pat. No.
5,326,780.
[0654] Amino alkyl phosphonate compounds 809: ##STR163## are a
generic representative of compounds 811, 813, 814, 816 and 818
(Scheme 2). The alkylene chain may be any length from 1 to 18
methylene groups (n=1-18). Commercial amino phosphonic acid 810 was
protected as carbamate 811. The phosphonic acid 811 was converted
to phosphonate 812 upon treatment with ROH in the presence of DCC
or other conventional coupling reagents. Coupling of phosphonic
acid 811 with esters of amino acid 820 provided bisamidate 817.
Conversion of acid 811 to bisphenyl phosphonate followed by
hydrolysis gave mono-phosphonic acid 814
(Cbz=C.sub.6H.sub.5CH.sub.2C(O)--), which was then transformed to
mono-phosphonic amidate 815. Carbamates 813, 816 and 818 were
converted to their corresponding amines upon hydrogenation.
Compounds 811, 813, 814, 816 and 818 are useful intermediates to
form the phosphonate compounds of the invention. ##STR164##
[0655] Following the similar procedures, replacement of amino acid
esters 820 with lactates 821 (Scheme 3) provides mono-phosphonic
lactates 823. Lactates 823 are useful intermediates to form the
phosphonate compounds of the invention. ##STR165##
Examples General Section
[0656] The following Examples refer to the Schemes. Some Examples
have been performed multiple times. In repeated Examples, reaction
conditions such as time, temperature, concentration and the like,
and yields were within normal experimental ranges. In repeated
Examples where significant modifications were made, these have been
noted where the results varied significantly from those described.
In Examples where different starting materials were used, these are
noted. When the repeated Examples refer to a "corresponding" analog
of a compound, such as a "corresponding ethyl ester", this intends
that an otherwise present group, in this case typically a methyl
ester, is taken to be the same group modified as indicated.
Example 1
[0657] To a solution of 2-aminoethylphosphonic acid (810 where n=2,
1.26 g, 10.1 mmol) in 2N NaOH (10.1 mL, 20.2 mmol) was added benzyl
chloroformate (1.7 mL, 12.1 mmol). See Scheme 5. After the reaction
mixture was stirred for 2 d at room temperature, the mixture was
partitioned between Et.sub.2O and water. The aqueous phase was
acidified with 6N HCl until pH=2. The resulting colorless solid was
dissolved in MeOH (75 mL) and treated with Dowex 50 W.times.8-200
(7 g). After the mixture was stirred for 30 minutes, it was
filtered and evaporated under reduced pressure to give carbamate 28
(2.37 g, 91%) as a colorless solid.
[0658] To a solution of carbamate 28 (2.35 g, 9.1 mmol) in pyridine
(40 mL) was added phenol (8.53 g, 90.6 mmol) and
1,3-dicyclohexylcarbodiimide (7.47 g, 36.2 mmol). After the
reaction mixture was warmed to 70.degree. C. and stirred for 5 h,
the mixture was diluted with CH.sub.3CN and filtered. The filtrate
was concentrated under reduced pressure and diluted with EtOAc. The
organic phase was washed with sat. NH.sub.4Cl, sat. NaHCO.sub.3,
and brine, then dried over Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was
chromatographed on silica gel twice (eluting 40-60% EtOAc/hexane)
to give phosphonate 29 (2.13 g, 57%) as a colorless solid.
[0659] To a solution of phosphonate 29 (262 mg, 0.637 mmol) in
iPrOH (5 mL) was added TFA (0.05 mL, 0.637 mmol) and 10% Pd/C (26
mg). After the reaction mixture was stirred under H.sub.2
atmosphere (balloon) for 1 h, the mixture was filtered through
Celite. The filtrate was evaporated under reduced pressure to give
amine 30 (249 mg, 100%) as a colorless oil (Scheme 5).
[0660] Following the similar procedures, replacement of amino acid
esters with lactates (Scheme 6) provided mono-phosphonic lactates,
e.g. 823. ##STR166##
[0661] Treatment of alcohol 801 (prepared according to literature)
with MsCl and TEA afforded chloride 802 (Scheme 7). Chloride 802
was converted to compound 803 by reacting with 809, which
preparation is detailed in Schemes 3 and 4, in the presence of
base. When mesylate 802 was treated with NaCN, imidazole nitrile
804 was provided. Reduction of 804 with DIBAL followed by
NaBH.sub.4 yielded imidazole alcohol 806. Repeating the same
procedure several times furnished alcohol 807 with the desired
length. Hydrolysis of imidazole nitrile 804 provided acid 805.
Coupling of acid 805 in the presence of conventional reagents
afforded the amide 808. Phosphorus compound 807' was produced by
transforming alcohol 807 to its corresponding mesylate followed by
treating with amine 809. ##STR167##
[0662] Alcohol 825 was converted to bromide 826 by first
transformed to its mesylate and then treated with NaBr, this
conversion was also realized by reacting alcohol 825 with Ph.sub.3P
and CBr.sub.4 (Scheme 8). Upon treating with P(OR).sub.3,
phosphonate 827 was produced. Esters was then removed to form acid,
and following the similar procedure described in Scheme 2 and 3,
desired phosphonate, bisphosphoamidate, mono-phosphoamidate, and
monophospholactate were produced. ##STR168##
[0663] In Scheme 9, alcohol 830 was converted to carbonate 831 by
reacting with either p-nitrophenyl chloroformate or p-nitrophenyl
carboxy anhyride. Treatment of carbonate 831 with amine 809 in the
presence of suitable base afforded desired phosphonate compounds
832. ##STR169##
[0664] Phosphorus compound 838 was produced according to the
procedures described in Scheme 10. Replacement of chloride group in
compound 833 with azide followed by reduction with
triphenylphosphine provided amine 834. Replacement of chloride
group in compound 833 with cyanide, e.g. sodium cyanide, provided
amine 835. Reduction of nitrile 835 furnished amine 836. Reaction
of amines, e.g. 834 or 836, with triflate 841 in the presence of a
base afforded phosphonate 837. Removal of benzyl group of 837 gave
its corresponding phosphonic acid, e.g. 838 where R.sub.1.dbd.H,
which was converted to various phosphorus compounds according to
the procedure described in the previous Schemes. ##STR170##
[0665] Phosphorus compound 840 was produced in a similar way as
described in Scheme 10 except by replacing amines with alcohols
801, or generally, 807 (Scheme 11). ##STR171##
[0666] Phosphorus compound 848 was synthesized according to
procedures described in Scheme 12. Iodoimidazole 842 was converted
to imidazole phenyl thioether 843 by reacting with LiH and
substituted phenyl disulfide (Scheme 12). Treatment of imidazole
with NaH and 4-picolyl chloride gave imidazole 844. Benzyl and
methyl groups were removed by treating with strong acid to provide
alcohol 845. Conversion of phenol 845 to phosphonate 846 was
accomplished by reacting phenol 845 with triflate 841 in the
presence of base. Alcohol 846 was reacting with trichloroacetyl
isocyanate followed by treatment of alumina afforded carbamate 847.
Phosphonate 847 was transformed to all kinds of phosphorus compound
848 followed the procedure described for 838 in Scheme 10.
##STR172##
[0667] Phosphorus compound 854 was prepared as shown in Scheme 13.
Imidazole 849 (prepared according to U.S. Pat. Nos. 5,910,506 and
6,057,448) was converted to 850 by reacting with chloride in the
presence of base. Benzyl and methyl groups were removed by treating
ether 850 with strong protonic or Lewis acid to furnish phenol 851.
Treatment of phenol 851 with base followed by triflate 841 gave
phosphonate 852. Following similar procedures described in Scheme
12 transforming alcohol 846 to phosphorus compound 848, alcohol 852
was converted to phosphorus compound 854. ##STR173##
[0668] Preparation of phosphorus compound 861 is shown in Scheme
14. Imidazole 855 was synthesized by treating compound 842 with NaH
followed by allyl bromide. Hydroboration followed by oxidative work
up gave alcohol 856. Ozonolysis followed by reduction of the
resulting aldehyde afforded alcohol 857. Alcohol 858, which has
variation of length, was obtained by following the same
transformation of alcohol 806 to 807 as exhibited in Scheme 7.
Mitsunobu reaction of alcohol 859 with substituted phenols gave
imidazole 860. Phenol ether 860 was converted to phosphonate 861 by
following same procedure of transforming compound 850 to 854 as
described in Scheme 13. ##STR174##
[0669] In Scheme 15, preparation of phosphorus compounds 864 is
shown. Alcohol 858 was converted to mesylate 862 by reacting with
MsCl. Removal of benzyl group, followed by conversion of the
resultant alcohol to the corresponding carbamate (described in
previous Schemes) furnished compound 863. Substitution of mesylate
with amine 809 generated phosphorus compound 864. ##STR175##
[0670] Synthesis of phosphorus compound 866 is described in Scheme
16. Protection of alcohol 858 to its acetate 865, followed by the
conversion of the benzyl, --OBn group to the corresponding
carbamate as described for transforming compound 862 to 863 in
Scheme 15, gave compound 865. Hydrolysis of acetate, and treatment
of the resultant alcohol with triflate 841 in the presence of base
afforded phosphonate 866. ##STR176##
[0671] Scheme 17 describes synthesis of phosphorus compound 672.
Mesylate 862 was transformed to bromide 867 by reacting with NaBr.
Arbusov reaction gave phosphonate 868. Both benzyl and ethyl groups
were cleaved when treated with TMSBr to yield compound 869.
Coupling of phosphonic acid 869 with PhOH provided bisphenyl
phosphonate 670. Compound 670 was converted to various phosphorus
compounds 671 according to the procedures described in Schemes 1, 2
and 3. Phosphorus compound 672 was obtained by repeating the
procedures shown before. ##STR177## ##STR178## ##STR179##
Example 10
[0672] ##STR180##
[0673] To a solution of alcohol 15 (42 mg, 0.10 mmol) in
CH.sub.2Cl.sub.2 (5 mL) was added triethylamine (24 .mu.L, 0.17
mmol) and bis(4-nitrophenyl) carbonate (46 mg, 0.15 mmol). See
Scheme 18. After the reaction mixture was stirred for 4 h at room
temperature, the mixture was partitioned between CH.sub.2Cl.sub.2
and water. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was chromatographed on silica gel (eluting 60-70% EtOAc/hexane) to
give carbonic acid
5-(3,5-dichloro-phenylsulfanyl)-4-isopropyl-1-pyridin-4-ylmethyl-1H-imida-
zol-2-ylmethyl ester 4-nitro-phenyl ester 16 (47 mg, 82%) as a
colorless oil.
Example 11A
[0674] ##STR181##
[0675] To a solution of carbonate 16 (14 mg, 0.024 mmol) in
CH.sub.3CN (2 mL) was added diethyl(aminomethyl)phosphonate (10 mg,
0.037 mmol) and diisopropylethylamine (8 .mu.L, 0.048 mmol). See
Scheme 18. After the reaction mixture was stirred for 16 h at room
temperature, the mixture was concentrated under reduced pressure.
The residue was purified by preparative thin layer chromatography
(eluting 5% MeOH/CH.sub.2Cl.sub.2) to give
{[5-(3,5-dichloro-phenylsulfanyl)-4-isopropyl-1-pyridin-4-ylmethy-
l-1H-imidazol-2-ylmethoxycarbonylamino]-methyl}-phosphonic acid
diethyl ester 17 (13 mg, 90%) as a pale yellow oil. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 8.44 (d, 2H), 7.04 (t, 1H), 6.78 (d,
2H), 6.68 (d, 2H), 5.25 (s, 2H), 5.19 (s, 2H), 4.98 (bt, 1H), 4.11
(dq, 4H), 3.49 (ABq, 2H), 3.17 (dq, 1H), 1.30 (m, 12H). .sup.31P
NMR (300 MHz, CDCl.sub.3) .delta. 21.9.
Example 11B
[0676] ##STR182##
[0677] To a solution of carbonate 16 (82 mg, 0.143 mmol) in
CH.sub.3CN (5 mL) was added diethyl(aminoethyl)phosphonate (58 mg,
0.214 mmol) and diisopropylethylamine (0.05 mL, 0.286 mmol). See
Scheme 20. After the reaction mixture was stirred for 16 h at room
temperature, the mixture was concentrated under reduced pressure.
The residue was chromatographed on silica gel (eluting 5-7.5%
MeOH/CH.sub.2Cl.sub.2) to give
{2-[5-(3,5-Dichloro-phenylsulfanyl)-4-isopropyl-1-pyridin-4-ylmethyl-1H-i-
midazol-2-ylmethoxycarbonylamino]-ethyl}-phosphonic acid diethyl
ester 18 (79 mg, 90%) as a pale yellow oil. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 8.43 (d, 2H), 7.02 (s, 1H), 6.77 (d, 2H), 6.67
(s, 2H), 5.32 (t, 1H), 5.24 (s, 2H), 5.16 (s, 2H), 4.08 (m, 4H),
3.35 (m, 2H), 3.15 (m, 1H), 1.86 (m, 2H), 1.30 (m, 6H), 1.29 (s,
6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 31.5.
##STR183##
Example 11C
[0678] ##STR184##
[0679] To a solution of 3-aminopropylphosphonic acid 19 (500 g,
3.59 mmol) in 2N NaOH (3.6 mL, 7.19 mmol) was added benzyl
chloroformate (0.62 mL, 4.31 mmol) according to Scheme 19. After
the reaction mixture was stirred for 16 hours at room temperature,
the mixture was partitioned between Et.sub.2O and water. The
aqueous phase was acidified with 6N HCl until pH=2. The resulting
colorless solid was dissolved in MeOH (75 mL) and treated with
Dowex 50 W.times.8-200 (2.5 g). After the mixture was stirred for
30 minutes, it was filtered and evaporated under reduced pressure
to give carbamate 20 (880 mg, 90%) as a colorless solid.
[0680] To a solution of carbamate 20 (246 mg, 0.90 mmol) in benzene
(5 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene phenol (0.27
mL, 1.8 mmol) and iodoethane (0.22 mL, 2.7 mmol). After the
reaction mixture was warmed to 60.degree. C. and stirred for 16 h,
the mixture was concentrated under reduced pressure and partitioned
between EtOAc and sat. NH.sub.4Cl. The crude product was
chromatographed on silica gel (eluting 3-4% MeOH/CH.sub.2Cl.sub.2)
to give phosphonate 21 (56 mg, 19%) as a colorless oil.
[0681] To a solution of phosphonate 21 (56 mg, 0.17 mmol) in EtOH
(3 mL) was added TFA (13 .mu.L, 0.17 mmol) and 10% Pd/C (11 mg).
After the reaction mixture was stirred under H.sub.2 atmosphere
(balloon) for 1 h, the mixture was filtered through Celite. The
filtrate was evaporated under reduced pressure to give amine 22 (52
mg, 99%) as a colorless oil.
[0682] To a solution of carbonate 16 (15 mg, 0.026 mmol) in
CH.sub.3CN (2 mL) was added diethyl(aminopropyl)phosphonate (16 mg,
0.052 mmol) and diisopropylethylamine (11 .mu.L, 0.065 mmol). After
the reaction mixture was stirred for 16 h at room temperature, the
mixture was concentrated under reduced pressure. The residue was
purified by preparative thin layer chromatography (eluting 5%
MeOH/CH.sub.2Cl.sub.2) to give
{3-[5-(3,5-dichloro-phenylsulfanyl)-4-isopropyl-1-pyridin-4-ylmethyl-1H-i-
midazol-2-ylmethoxycarbonylamino]-propyl}-phosphonic acid diethyl
ester 23 (13 mg, 79%) as a pale yellow oil. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 8.44 (d, 2H), 7.04 (t, 1H), 6.80 (d, 2H), 6.68
(d, 2H), 5.26 (s, 2H), 5.18 (s, 2H), 5.08 (bt, 1H), 4.08 (m, 4H),
3.15 (m, 3H), 1.72 (m, 4H), 1.31 (m, 12H). .sup.31P NMR (300 MHz,
CDCl.sub.3) .delta. 31.5. ##STR185##
Example 12A
[0683] ##STR186##
[0684] To a solution of aminomethylphosphonic acid (8 mg, 0.073
mmol) in water (1 mL) was added 1N NaOH (0.15 mL, 0.15 mmol) and
carbonate 16 (21 mg, 0.037 mmol) in dioxane (1 mL). See Scheme 20.
After the reaction mixture was stirred for 6 h at room temperature,
the mixture was concentrated under reduced pressure. The residue
was purified by HPLC on C18 reverse phase chromatography (eluting
30% CH.sub.3CN/water) to give a mixture of phosphonic acid 24 and
alcohol 15. The mixture was further purified by preparative thin
layer chromatography (eluting 7.5% MeOH/CH.sub.2Cl.sub.2) to give
{[5-(3,5-dichloro-phenylsulfanyl)-4-isopropyl-1-pyridin-4-ylmethyl-1H-imi-
dazol-2-ylmethoxycarbonyl amino]-methyl}-phosphonic acid 24 (8 mg,
40%) as a colorless solid. .sup.1H NMR (300 MHz, CD.sub.3OD)
.delta. 8.33 (bs, 2H), 7.10 (t, 1H), 7.04 (bs, (2H), 6.72 (d, 2H),
5.44 (s, 2H), 5.25 (s, 2H), 3.24 (m, 2H), 3.17 (m, 1H), 1.28 (d,
6H).
Example 12B
[0685] ##STR187##
[0686] To a solution of 2-aminoethylphosphonic acid (12 mg, 0.098
mmol) in water (1 mL) was added 1N NaOH (0.2 mL, 0.20 mmol) and
carbonate 16 (28 mg, 0.049 mmol) in dioxane (1 mL). See Scheme 20.
After the reaction mixture was stirred for 6 h at room temperature,
the mixture was concentrated under reduced pressure. The residue
was purified by HPLC on C18 reverse phase chromatography (eluting
30% CH.sub.3CN/water) to give a mixture of phosphonic acid 25 and
alcohol 15. The mixture was further purified by preparative thin
layer chromatography (eluting 7.5% MeOH/CH.sub.2Cl.sub.2) to give
{2-[5-(3,5-dichloro-phenylsulfanyl)-4-isopropyl-1-pyridin-4-ylmethyl-1H-i-
midazol-2-ylmethoxycarbonylamino]-ethyl}-phosphonic acid 25 (13 mg,
47%) as a colorless solid. .sup.1H NMR (300 MHz, CD.sub.3OD)
.delta. 8.32 (d, 2H), 7.11 (s, 1H), 7.02 (d, 2H), 6.72 (s, 2H),
5.42 (s, 2H), 5.23 (s, 2H), 3.30 (m, 2H), 3.17 (m, 1H), 1.71 (m,
2H), 1.28 (d, 6H). .sup.31P NMR (300 MHz, CD.sub.3OD) .delta.
20.1.
Example 12C
[0687] ##STR188##
[0688] To a solution of 3-aminopropylphosphonic acid (12 mg, 0.084
mmol) in water (1 mL) was added 1N NaOH (0.17 mL, 0.17 mmol) and
carbonate 16 (24 mg, 0.042 mmol) in dioxane (1 mL). See Scheme 20.
After the reaction mixture was stirred for 6 h at room temperature,
the mixture was concentrated under reduced pressure. The residue
was purified by HPLC on C18 reverse phase chromatography (eluting
30% CH.sub.3CN/water) to give a mixture of phosphonic acid 26 and
alcohol 15. The mixture was further purified by preparative thin
layer chromatography (eluting 7.5% MeOH/CH.sub.2Cl.sub.2) to give
{3-[5-(3,5-dichloro-phenylsulfanyl)-4-isopropyl-1-pyridin-4-ylmethyl-1H-i-
midazol-2-ylmethoxycarbonylamino]-propyl}-phosphonic acid 26 (11
mg, 46%) as a colorless solid. .sup.1H NMR (300 MHz, CD.sub.3OD)
.delta. 8.34 (bs, 2H), 7.11 (s, 1H), 7.02 (bs, 2H), 6.73 (d, 2H),
5.43 (s, 2H), 5.23 (s, 2H), 3.32 (m, 1H), 3.06 (bs, 2H), 1.69 (bs,
2H), 1.50 (bs, 2H), 1.28 (d, 6H). ##STR189##
Example 13
[0689] ##STR190##
[0690] To a solution of 2-aminoethylphosphonic acid (1.26 g, 10.1
mmol) in 2N NaOH (10.1 mL, 20.2 mmol) was added benzyl
chloroformate (1.7 mL, 12.1 mmol). See Scheme 21. After the
reaction mixture was stirred for 2 d at room temperature, the
mixture was partitioned between Et.sub.2O and water. The aqueous
phase was acidified with 6N HCl until pH=2. The resulting colorless
solid was dissolved in MeOH (75 mL) and treated with Dowex 50
W.times.8-200 (7 g). After the mixture was stirred for 30 minutes,
it was filtered and evaporated under reduced pressure to give
carbamate 28 (2.37 g, 91%) as a colorless solid.
[0691] To a solution of carbamate 28 (2.35 g, 9.1 mmol) in pyridine
(40 mL) was added phenol (8.53 g, 90.6 mmol) and
1,3-dicyclohexylcarbodiimide (7.47 g, 36.2 mmol). After the
reaction mixture was warmed to 70.degree. C. and stirred for 5 h,
the mixture was diluted with CH.sub.3CN and filtered. The filtrate
was concentrated under reduced pressure and diluted with EtOAc. The
organic phase was washed with sat. NH.sub.4Cl, sat. NaHCO.sub.3,
and brine, then dried over Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was
chromatographed on silica gel twice (eluting 40-60% EtOAc/hexane)
to give phosphonate 29 (2.13 g, 57%) as a colorless solid.
[0692] To a solution of phosphonate 29 (262 mg, 0.637 mmol) in
isopropanol (iPrOH) (5 mL) was added TFA (0.05 mL, 0.637 mmol) and
10% Pd/C (26 mg). After the reaction mixture was stirred under
H.sub.2 atmosphere (balloon) for 1 h, the mixture was filtered
through Celite. The filtrate was evaporated under reduced pressure
to give amine 30 (249 mg, 100%) as a colorless oil.
[0693] To a solution of carbonate 16 (40 mg, 0.070 mmol) and amine
30 (82 mg, 0.21 mmol) in CH.sub.3CN (5 mL) was added
diisopropylethylamine (0.05 mL, 0.28 mmol). After the reaction
mixture was stirred for 2 h at room temperature, the mixture was
concentrated under reduced pressure. The residue was
chromatographed on silica gel (eluting 3-4% MeOH/CH.sub.2Cl.sub.2)
to give
{2-[5-(3,5-dichloro-phenylsulfanyl)-4-isopropyl-1-pyridin-4-ylmethyl-1H-i-
midazol-2-ylmethoxycarbonylamino]-ethyl}-phosphonic acid diphenyl
ester 31 (36 mg, 72%) as a colorless oil. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 8.37 (d, 2H), 7.22 (m, 4H), 7.14 (m, 2H), 7.10
(m, 2H), 6.99 (t, 1H), 6.72 (d, 2H), 6.62 (d, 2H), 5.30 (bt, 1H),
5.18 (s, 2H), 5.13 (s, 2H), 3.50 (m, 2H), 3.12 (m, 1H), 2.21 (m,
2H), 1.26 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta.
22.4.
Example 14
[0694] ##STR191##
[0695] To a solution of phosphonate 31 (11 mg, 0.015 mmol) in
CH.sub.3CN (0.5 mL) was added 1N LiOH (46 .mu.L, 0.046 mmol) at
0.degree. C. See Scheme 21. After the reaction mixture was stirred
for 2 h at 0.degree. C., Dowex 50 W.times.8-200 (26 mg) was added
and stirring was continued for an additional 30 min. The reaction
mixture was filtered, rinsed with CH.sub.3CN, and concentrated
under reduced pressure to give
{2-[5-(3,5-dichloro-phenylsulfanyl)-4-isopropyl-1-pyridin-4-ylmethyl-1H-i-
midazol-2-ylmethoxycarbonylamino]-ethyl}-phosphonic acid monophenyl
ester 32 (10 mg, 100%) as a colorless oil. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 8.52 (d, 2H), 7.28 (m, 6H), 6.79 (m, 4H), 5.60
(s, 2H), 5.29 (s, 2H), 3.29 (m, 3H), 1.83 (m, 2H), 1.31 (d, 6H).
.sup.31P NMR (300 MHz, CD.sub.3OD) .delta. 20.2. ##STR192##
##STR193##
Example 15
[0696] ##STR194##
[0697] To a solution of 3-methoxybenzenethiol (0.88 mL, 7.13 mmol)
in CH.sub.3CN (15 mL) was added sodium iodide (214 mg, 1.43 mmol)
and ferric chloride (232 mg, 1.43 mmol). See Scheme 22. After the
reaction mixture was warmed to 60.degree. C. and stirred for 3 d,
the mixture was concentrated under reduced pressure and partitioned
between CH.sub.2Cl.sub.2 and water. The organic phase was dried
over Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was chromatographed on silica gel
(eluting 5-6% EtOAc/hexane) to give disulfide 34 (851 mg, 86%) as a
yellow oil. To a solution of disulfide 34 (850 mg, 3.05 mmol) in
DMSO (10 mL) was added iodide 35, also denoted previously as
compound 842, (1.21 g, 3.39 mmol) and lithium hydride (32 mg, 4.07
mmol). After the reaction mixture was warmed to 60.degree. C. and
stirred for 16 h, the mixture was partitioned between EtOAc and
water. The organic phase was washed with brine, dried over
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was chromatographed on silica gel (eluting 30-50%
EtOAc/hexane) to give
2-benzyloxymethyl-4-isopropyl-5-(3-methoxy-phenylsulfanyl)-1H-imidazole
36 (247 mg, 22%) as a yellow oil.
Example 16
[0698] ##STR195##
[0699] To a solution of sulfide 36 (247 mg, 0.67 mmol) in THF (10
mL) was added 4-picolylchloride (220 mg, 1.34 mmol), powder NaOH
(59 mg, 1.47 mmol), lithium iodide (44 mg, 0.33 mmol), and
tetrabutylammonium bromide (22 mg, 0.067 mmol). See Scheme 22.
After the reaction mixture was stirred for 2 d at room temperature,
the mixture was partitioned between EtOAc and sat. NH.sub.4Cl. The
organic phase was dried over Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was
chromatographed on silica gel (eluting 60-100% EtOAc/hexane) to
give
4-[2-benzyloxymethyl-4-isopropyl-5-(3-methoxy-phenylsulfanyl)-imidazol-1--
ylmethyl]-pyridine 37 (201 mg, 65%) as a yellow oil.
Example 17
[0700] ##STR196##
[0701] To a solution of amine 37 (101 mg, 0.220 mmol) in EtOH (5
mL) was added conc. HCl (5 mL). See Scheme 22. After the reaction
mixture was warmed to 80.degree. C. and stirred for 16 h, the
mixture was concentrated under reduced pressure and partitioned
between EtOAc and sat. NaHCO.sub.3. The organic phase was dried
over Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was chromatographed on silica gel
(eluting 5-7% MeOH/CH.sub.2Cl.sub.2) to give [4
isopropyl-5-(3-methoxy-phenylsulfanyl)-1-pyridinylmethyl-1H-imida-
zol-2-yl]-methanol 38 (71 mg, 87%) as a pale yellow oil.
Example 18
[0702] ##STR197##
[0703] To a solution of alcohol 38 (56 mg, 0.15 mmol) in
CH.sub.2Cl.sub.2 (2 mL) was added 1M BBr.sub.3 in CH.sub.2Cl.sub.2
at 0.degree. C. See Scheme 22. After the reaction mixture was
stirred for 1 h at 0.degree. C., the mixture was partitioned
between CH.sub.2Cl.sub.2 and sat. NaHCO.sub.3. The aqueous phase
was neutralized with solid NaHCO.sub.3 and extracted with
CH.sub.2Cl.sub.2 and EtOAc. The organic phase was dried over
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was chromatographed on silica gel (eluting 5-10%
MeOH/CH.sub.2Cl.sub.2) to give
3-(2-hydroxymethyl-5-isopropyl-3-pyridin-4-ylmethyl-3H-imidazol-4-ylsulfa-
nyl)-phenol 39 (43 mg, 81%) as a colorless solid.
Example 19
[0704] ##STR198##
[0705] To a solution of phenol 39 (25 mg, 0.070 mmol) and triflate
(33 mg, 0.11 mmol) in THF (2 mL) and CH.sub.3CN (2 mL) was added
Cs.sub.2CO.sub.3 (46 mg, 0.14 mmol). See Scheme 22. After the
reaction mixture was stirred for 1 h at room temperature, the
mixture was partitioned between EtOAc and water. The organic phase
was dried over Na.sub.2SO.sub.4, filtered, and evaporated under
reduced pressure. The crude product was purified by preparative
thin layer chromatography (eluting 10% MeOH/CH.sub.2Cl.sub.2) to
give
[3-(2-Hydroxymethyl-5-isopropyl-3-pyridin-4-ylmethyl-3H-imidazol-4-ylsulf-
anyl)-phenoxymethyl]-phosphonic acid diethyl ester 40 (10 mg, 28%)
as a colorless oil.
Example 20
[0706] ##STR199##
[0707] To a solution of diethylphosphonate 40 (10 mg, 0.020 mmol)
in THF (2 mL) was added trichloroacetyl isocyanate (7 .mu.L, 0.059
mmol). See Scheme 22. After the reaction mixture was stirred for 30
min at room temperature, the mixture was evaporated under reduced
pressure. To a solution of the concentrated residue in MeOH (2 mL)
was added 1M K.sub.2CO.sub.3 (0.2 mL, 0.20 mmol) at 0.degree. C.
After the reaction mixture was warmed to room temperature and
stirred for 3 h, the mixture was partitioned between EtOAc and sat.
NH.sub.4Cl. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was purified by preparative thin layer chromatography (eluting 10%
MeOH/CH.sub.2Cl.sub.2) to give
[3-(2-hydroxymethyl-5-isopropyl-3-pyridin-4-ylmethyl-3H-imidazol-4-ylsulf-
anyl)-phenoxymethyl]-phosphonic acid diethyl ester 41 (10 mg, 91%)
as a colorless oil. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.50
(d, 2H), 7.16 (m, 1H), 6.85 (m, 1H), 6.75 (m, 1H), 6.73 (m, 1H),
6.17 (s, 1H), 5.31 (s, 2H), 5.02 (s, 2H), 4.23 (m, 4H), 4.16 (d,
2H), 3.23 (m, 1H), 1.37 (t, 6H), 1.29 (d, 6H). .sup.31P NMR (300
MHz, CDCl.sub.3) .delta. 19.6. ##STR200##
Example 21
[0708] ##STR201##
[0709] To a solution of phenol 39 (20 mg, 0.056 mmol) in THF (1 mL)
and CH.sub.3CN (1 mL) was added sodium hydride (60%, 5 mg, 0.112
mmol) at 0.degree. C. See Scheme 23. After the reaction mixture was
stirred for 30 min at 0.degree. C., dibenzylphosphonyl
methyltriflate (21 mg, 0.050 mmol) in THF (1 mL) was added. After
the reaction mixture was stirred for 1 h at 0.degree. C., the
mixture was evaporated under reduced pressure and partitioned
between EtOAc and sat. NH.sub.4Cl. The organic phase was dried over
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by preparative thin layer
chromatography (eluting 10% MeOH/CH.sub.2Cl.sub.2) to give
dibenzylphosphonate 42 (5 mg, 16%) as a pale yellow oil.
Example 22
[0710] ##STR202##
[0711] To a solution of dibenzylphosphonate 42 (5 mg, 0.0079 mmol)
in CH.sub.2Cl.sub.2 (1 mL) was added trichloroacetyl isocyanate (5
.mu.L, 0.049 mmol). See Scheme 23. After the reaction mixture was
stirred for 15 min at room temperature, the mixture was transferred
on to a 2-inch column of neutral Al.sub.2O.sub.3. After the
reaction mixture was soaked for 30 min, the mixture was rinsed off
the column with 10% MeOH/CH.sub.2Cl.sub.2 and evaporated under
reduced pressure. The crude product was purified by preparative
thin layer chromatography (eluting 10% MeOH/CH.sub.2Cl.sub.2) to
give carbamate 43 (3 mg, 56%) as a pale yellow oil. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 8.48 (d, 2H), 7.35 (m, 10H), 7.12 (t,
1H), 6.88 (m, 2H), 6.70 (d, 1H), 6.66 (dd, 1H), 6.10 (t, 1H), 5.29
(s, 2H), 5.13 (dd, 6H), 5.05 (s, 2H), 4.14 (d, 2H), 3.24 (m, 1H),
1.30 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 20.3.
[0712] Preparation of phosphorus compound 874 was displayed in
Scheme 24. Starting with imidazole 842, Ar1 and Ar2 were introduced
following the procedure described in U.S. Pat. No. 5,326,780.
Benzyl group was then removed and converted to phosphorus analog
874 using the procedure described previously. ##STR203##
[0713] Scheme 25 describes preparation of compound 880. Compound
875 was synthesized from compound 842 using the procedures
described in U.S. Pat. No. 5,326,780. Treatment of 875 with HCl
removed the benzyl group to give alcohol 876, which was then
introduced phenyl group with substitution of Y. Y is a function
which can be converted to alcohol, aldehyde or amine, for example
--NO.sub.2, --COOMe, N.sub.3, and etc. Conversion of Y to the amine
or alcohol gave compound 878 and/or 879, which were then used as
attachment site of phosphorus to afford phosphorus compound 880.
Hydroxyl group in compound 880 was then converted to the desired
side chain including but not limit to carbamate 881, urea 882,
substituted amine 883. ##STR204##
[0714] Preparation of phosphorus compound 887 is shown in Scheme
26. Compound 877 was converted to amine 884 and/or aldehyde 885,
which then reacted with aldehyde and/or amine respectively to
provide phosphorus compound 886. Treatment of compound 886 with
Cl.sub.3CCONCO provide the carbamate 887. ##STR205##
Example 22
[0715] ##STR206##
[0716] Compound 44 was prepared following the sequence of steps
described in Example 13, by substituting compound 20 for compound
28. Purification of the crude product on silica gel eluted with 34%
MeOH/CH.sub.2Cl.sub.2 provided 37 mg of 48, the title compound.
.sup.1H NMR (500 MHz, CDCl.sub.3) (1.3:1 diastereomeric ratio)
.delta. 8.50 (bs, 2H), 7.35 (t, 2H), 7.20 (m, 3H), 7.06 (s, 1H),
6.90 (bs, 2H), 6.70 (s, 2H), 5.26 (bs, 2H), 5.21 (s, 2H), 4.97 (m,
1H), 4.22 (q, 2H), 3.24 (m, 2H), 3.19 (m, 1H), 2.05 (m, 2H), 1.92
(m, 2H), 1.37 (d, 3H), 1.33 (d, 6H), 1.28 (t, 3H). .sup.31P NMR
(300 MHz, CDCl.sub.3) .delta. 30.0.
Example 23
[0717] ##STR207##
[0718] The title compound 49 was prepared following the sequence of
steps described in Example 22, except for using scalmeric mixture
46 (around 13:1 ratio). Purification of the crude final product on
silica gel eluted with 3-4% MeOH/CH.sub.2Cl.sub.2 provided 40 mg of
the title compound. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.44
(bd, 2H), 7.32 (m, 2H), 7.19 (m, 3H), 7.04 (d, 1H), 6.80 (bs, 2H),
6.68 (m, 2H), 5.27 (d, 2H), 5.19 (d, 2H), 4.96 (m, 1H), 4.15 (m,
2H), 3.18 (m, 3H), 1.93 (m, 4H), 1.55 (d, 1.5H), 1.34 (d, 1.5H),
1.31 (d, 6H), 1.21 (m, 3H). .sup.31P NMR (300 MHz, CDCl.sub.3)
830.0, 28.3.
Example 24
[0719] ##STR208##
[0720] Amidate 49: A solution of phosphonic acid 45 (66 mg, 0.19
mmol) in CH.sub.3CN (5 mL) was treated with thionyl chloride-(42
.mu.L, 0.57 mmol). After the reaction mixture was warmed to
70.degree. C. and stirred for 2 h, the mixture was concentrated
under reduced pressure. The residue was dissolved in
CH.sub.2Cl.sub.2 (5 mL) and cooled to 0.degree. C. Triethylamine
(0.11 mL, 0.76 mmol) and L-alanine n-butyl ester (104 mg, 0.57
mmol) were added. After stirring for 1 h at 0.degree. C. and 1 h at
room temperature, the reaction mixture was neutralized with sat.
NH.sub.4Cl and extracted with CH.sub.2Cl.sub.2 and EtOAc. The
organic phase was dried over Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was purified
on silica gel (eluting 60-80% EtOAc/hexane) to give amidate 49 (35
mg, 39%) as a colorless oil.
[0721] Amine 50: A mixture of benzyl carbamate 49 (35 mg, 0.073
mmol), trifluoroacetic acid (8 .mu.L, 0.11 mmol) and 10% Pd/C (7
mg) in isopropyl alcohol (2 mL) was stirred under H.sub.2
atmosphere (balloon) for 1 h. The mixture was then filtered through
Celite. The filtrate was evaporated under reduced pressure to give
amine 50 (33 mg, 99%) as a colorless oil.
[0722] Title compound 51: A solution of 4-nitrophenylcarbonate 16
(35 mg, 0.061 mmol) in CH.sub.3CN (2 mL) was treated with amine 50
(33 mg, 0.072 mmol) and iPr.sub.2NEt (21 .mu.L, 0.122 mmol). After
the reaction mixture was stirred for 1 h at room temperature, the
mixture was concentrated under reduced pressure. The residue was
purified on silica gel (eluting 4-5% MeOH/CH.sub.2Cl.sub.2) to give
the title compound 51 (43 mg, 91%) as a pale yellow oil. .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. 8.46 (bs, 2H), 7.31 (m, 2H), 7.20
(d, 2H), 7.14 (m, 1H), 7.05 (s, 1H), 6.81 (bd, 2H), 6.71 (d, 2H),
5.27 (bs, 2H), 5.19 (bs, 2H), 4.07 (m, 2H), 3.98 (m, 1H), 3.63 (m,
1H), 3.18 (m, 3H), 1.83 (m, 2H), 1.80 (m, 2H), 1.58 (m, 2H), 1.35
(m, 2H), 1.32 (d, 6H), 1.30 (d, 1.5H), 1.24 (d, 1.5H), 0.93 (t,
3H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 31.6, 31.3.
Example 25
[0723] ##STR209##
[0724] The title compound was prepared following the sequence of
steps described in Example 24, except for substituting alanine
ethyl ester for alanine n-butyl ester. Purification of the crude
final product on a preparative TLC plate (5%
CH.sub.3OH/CH.sub.2Cl.sub.2) provided 5 mg (75%) of the title
compound. .sup.1H NMR(CDCl.sub.3, 500 MHz): .delta. 8.46 (d, 2H),
7.32 (d, 2H), 7.20 (d, 2H), 7.15 (s, 1H), 7.05 (s, 1H), 6.82 (d,
2H), 6.70 (s, 2H), 5.27 (s, 2H), 5.19 (s, 2H), 4.12 (m, 2H), 3.70
(t, 2H), 3.19 (m, 2H), 3.12 (t, 2H), 1.48 (m, 3H), 1.47 (t, 3H),
1.25 (d,6H).
Example 26
[0725] ##STR210##
[0726] Imidazole 54: A solution of imidazole 53 (267 mg, 0.655
mmol) in THF (10 mL) was treated with 4-methoxybenzyl chloride
(0.18 mL, 1.31 mmol), powder NaOH (105 mg, 2.62 mmol), lithium
iodide (88 mg, 0.655 mmol), and tetrabutylammonium bromide (105 mg,
0.327 mmol). After stirring for 4 days at room temperature, the
resulting mixture was partitioned between EtOAc and sat.
NH.sub.4Cl. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was purified on silica gel (eluting 20-40% EtOAc/hexane) to give
imidazole 54 (289 mg, 84%) as a colorless oil.
[0727] Phenol 55: A solution of benzyl ether 54 (151 mg, 0.286
mmol) in EtOH (5 mL) was treated with conc. HCl (5 mL). After the
reaction mixture was warmed to 80.degree. C. and stirred for 2 d,
the mixture was concentrated under reduced pressure and partitioned
between EtOAc and sat. aqueous NaHCO.sub.3. The organic phase was
dried over Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was purified on silica gel (eluting
60-70% EtOAc/hexane) to give the alcohol (99 mg, 79%) as a
colorless solid. A solution of the alcohol (77 mg, 0.18 mmol) in
CH.sub.2Cl.sub.2 (3 mL) was added 1M BBr.sub.3 in CH.sub.2Cl.sub.2
(0.90 mL, 0.90 mmol) at 0.degree. C. After the reaction mixture was
stirred for 1 h at 0.degree. C., the mixture was neutralized with
sat. NaHCO.sub.3 and extracted with CH.sub.2Cl.sub.2 and EtOAc. The
organic phase was dried over Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was
chromatographed on silica gel (eluting 4-5% MeOH/CH.sub.2Cl.sub.2)
to give phenol 55 (68 mg, 89%) as a colorless solid.
[0728] Diethylphosphonate 56: To a solution of phenol 55 (21 mg,
0.050 mmol) in CH.sub.3CN (1 mL) and THF (1 mL) was added
trifluoro-methanesulfonic acid diethoxy-phosphorylmethyl ester (18
mg, 0.060 mmol) in CH.sub.3CN (1 mL). After the addition of
Cs.sub.2CO.sub.3 (20 mg, 0.060 mmol), the reaction mixture was
stirred for 2 h at room temperature. Additional triflate (18 mg,
0.060 mmol) and Cs.sub.2CO.sub.3 (20 mg, 0.060 mmol) were
introduced. After the reaction mixture was stirred for another 2 h
at room temperature, the mixture was concentrated under reduced
pressure. The residue was partitioned between EtOAc and sat.
NH.sub.4Cl. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was purified by preparative thin layer chromatography (eluting 5%
MeOH/CH.sub.2Cl.sub.2) to give diethylphosphonate 56 (26 mg, 91%)
as a pale yellow oil.
[0729] Title compound carbamate 57: A solution of
diethylphosphonate 56 (26 mg, 0.045 mmol) in CH.sub.2Cl.sub.2 (2
mL) was treated with trichloroacetyl isocyanate (27 .mu.L, 0.23
mmol). After the reaction mixture was stirred for 10 min at room
temperature, the mixture was concentrated under reduced pressure.
The residue was transferred to an Al.sub.2O.sub.3 column in 10%
MeOH/CH.sub.2Cl.sub.2. After soaking on the column for 30 min, the
crude product was flushed out with 10% MeOH(CH.sub.2Cl.sub.2 and
concentrated under reduced pressure. The crude product was purified
by preparative thin layer chromatography eluted with 5%
MeOH/CH.sub.2Cl.sub.2 to give title compound carbamate 57 (22 mg,
79%) as a pale yellow oil. .sup.1H NMR (500 MHz, CDCl.sub.3), 7.00
(s, 1H), 6.88 (d, 2H), 6.76 (d, 2H), 6.62 (s, 2H), 5.24 (s, 2H),
5.18 (s, 2H), 4.26 (q, 4H), 4.21 (d, 2H), 3.15 (m, 1H), 1.38 (t,
6H), 1.29 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta.
19.1.
Example 27
[0730] ##STR211##
[0731] The title compound 58 was prepared following the sequence of
steps described in Example 27 with substitution of
trifluoro-methanesulfonic acid bis-benzyloxy-phosphorylmethyl ester
for trifluoro-methanesulfonic acid diethoxy-phosphorylmethyl ester.
Purification of the crude final product on silica gel eluted with
3-4% MeOH/CH.sub.2Cl.sub.2 provided 33 mg of the title compound.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.37 (m, 10H), 6.96 (s,
1H), 6.85 (d, 2H), 6.70 (d, 2H), 6.62 (s, 2H), 5.23 (s, 2H), 5.17
(s, 2H), 5.13 (m, 4H), 4.18 (d, 2H), 3.16 (m, 1H), 1.30 (d, 6H).
.sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 20.1.
Example 28
[0732] ##STR212##
[0733] A solution of dibenzylphosphonate 58 (15 mg, 0.020 mmol) was
treated 4M HCl in dioxane (1 mL). After the reaction mixture was
stirred for 18 h at room temperature, the mixture was concentrated
under reduced pressure. The crude product was purified on a C-18
column (eluting 30-40% CH.sub.3CN/H.sub.2O) to give phosphonic acid
59 (8 mg, 71%) as a colorless oil. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 7.19 (s, 1H), 7.08 (d, 2H), 6.81 (d, 2H), 6.69
(s, 2H), 5.48 (s, 2H), 5.44 (s, 2H), 4.12 (d, 2H), 3.32 (m, 1H),
1.33 (d, 6H). .sup.31P NMR (300 MHz, CD.sub.3OD) .delta. 17.1.
Example 29
[0734] ##STR213##
[0735] The title compound 60 was prepared following the sequence of
steps described in Example 25, except for substituting 3-methoxy
benzyl chloride for 4-methoxyl benzyl chloride. Purification of the
crude final product on preparative thin layer chromatography eluted
with 5% MeOH/CH.sub.2Cl.sub.2 provided 28 mg of the title compound.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.12 (t, 1H), 7.03 (s,
1H), 6.75 (d, 1H), 6.66 (s, 2H), 6.60 (d, 1H), 6.55 (s, 1H), 5.24
(s, 2H), 5.19 (s, 2H), 4.22 (q, 4H), 4.20 (d, 2H), 3.17 (m, 1H),
1.37 (t, 6H), 1.31 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3)
819.2.
Example 30
[0736] ##STR214##
[0737] The title compound 61 was prepared following the sequence of
steps described in Example 26, except for substituting 3-methoxy
benzyl chloride for 4-methoxyl benzyl chloride. Purification of the
crude final product on silica gel eluted with 3-4%
MeOH/CH.sub.2Cl.sub.2 provided 36 mg of the title compound. .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. 7.36 (m, 10H), 7.10 (t, 1H), 7.00
(s, 1H), 6.68 (d, 1H), 6.64 (s, 2H), 6.59 (d, 1H), 6.53 (s, 1H),
5.23 (s, 2H), 5.17 (s, 2H), 5.11 (m, 4H), 4.18 (d, 2H), 3.16 (m,
1H), 1.31 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta.
20.2.
Example 31
[0738] ##STR215##
[0739] The title compound 62 was prepared following the sequence of
steps described in Example 29, except for substituting compound 61
for compound 58. Purification of the crude final product with HPLC
(eluting 30-40% CH.sub.3CN/H.sub.2O) provided 7 mg of the title
compound. .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 7.18 (s, 1H),
7.13 (t, 1H), 6.81 (d, 1H), 6.77 (s, 2H), 6.72 (s, 1H), 6.68 (d,
1H), 5.49 (s, 2H), 5.37 (s, 2H), 4.12 (d, 2H), 3.33 (m, 1H), 1.34
(d, 6H). .sup.31P NMR (300 MHz, CD.sub.3OD) .delta. 17.0.
Example 32
[0740] ##STR216## ##STR217##
[0741] Alcohol 64: A solution of methyl 6-methoxynicotinate 63 (2.0
g, 12 mmol) in Et.sub.2O (50 mL) was treated with 1.5M DIBAL-H in
toluene (16.8 mL, 25.1 mmol) at 0.degree. C. After the reaction
mixture was stirred for 1 h at 0.degree. C., the mixture was
quenched with 1M sodium potassium tartrate and stirred for an
additional 2 h. The aqueous phase was extracted with Et.sub.2O and
concentrated to give alcohol 64 (1.54 g, 92%) as a pale yellow
oil.
[0742] Bromide 65: A solution of alcohol 64 (700 mg, 5.0 mmol) in
CH.sub.2Cl.sub.2 (50 mL) was treated with carbon tetrabromide (2.49
g, 7.5 mmol) and triphenylphosphine (1.44 g, 5.5 mmol) at 0.degree.
C. After the reaction mixture was stirred for 30 min at room
temperature, the mixture was partitioned between CH.sub.2Cl.sub.2
and sat. aqueous NaHCO.sub.3. The organic phase was dried over
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified on silica gel (eluting 5-10%
MeOH/CH.sub.2Cl.sub.2) to give bromide 65 (754 mg, 75%) as
colorless crystals.
[0743] Imidazole 66: A solution of imidazole 53 (760 mg, 1.86 mmol)
and bromide 65 (752 mg, 3.72 mmol) in THF (10 mL) was treated with
powder NaOH (298 mg, 7.44 mmol), lithium iodide (249 mg, 1.86
mmol), and tetrabutylammonium bromide (300 mg, 0.93 mmol). After
stirring for 14 h at room temperature, the mixture was partitioned
between EtOAc and sat. NH.sub.4Cl. The organic phase was dried over
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified on silica gel (eluting 20-30%
EtOAc/hexane) to give imidazole 66 (818 mg, 83%) as a pale yellow
oil.
[0744] Diol 67: A solution of benzyl ether 66 (348 mg, 0.658 mmol)
in EtOH (3 mL) was treated with conc. HCl (3 mL). After the
reaction mixture was warmed to 80.degree. C. and stirred for 18 h,
the mixture was concentrated under reduced pressure. The crude
product was chromatographed on silica gel (eluting 5-10%
MeOH/CH.sub.2Cl.sub.2) to give diol 67 (275 mg, 98%) as a colorless
solid.
[0745] Title compound diethylphosphonate 68: A solution of diol 67
(40 mg, 0.094 mmol) in THF (1 mL) was treated with
trifluoro-methanesulfonic acid diethoxy-phosphorylmethyl ester (114
mg, 0.38 mmol) in THF (1 mL). After the addition of
Ag.sub.2CO.sub.3 (52 mg, 0.19 mmol), the reaction mixture was
stirred for 5 d at room temperature. The mixture was quenched with
sat. NaHCO.sub.3 and sat. NaCl, and extracted with EtOAc. The
organic phase was dried over Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was
chromatographed by silica gel (eluting 3-4% MeOH/CH.sub.2Cl.sub.2)
and by preparative thin layer chromatography (eluting 4%
MeOH/CH.sub.2Cl.sub.2) to give the title compound
diethylphosphonate 68 (23 mg, 43%) as a colorless oil. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 7.92 (s, 1H), 7.39 (d, 1H), 7.00 (s,
1H), 6.65 (d, 1H), 6.55 (d, 2H), 5.20 (s, 2H), 4.81 (s, 2H), 4.55
(d, 2H), 4.21 (m, 4H), 3.08 (m, 1H), 1.35 (t, 6H), 1.20 (d, 6H).
.sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 20.7.
Example 33
[0746] ##STR218##
[0747] A solution of diethylphosphonate 68 (13 mg, 0.023 mmol) in
CH.sub.2Cl.sub.2 (0.5 mL) was treated with trichloroacetyl
isocyanate (13 .mu.L, 0.11 mmol). After the reaction mixture was
stirred for 10 min at room temperature, the mixture was
concentrated under reduced pressure. The residue was transferred to
an Al.sub.2O.sub.3 column in 10% MeOH/CH.sub.2Cl.sub.2. After
soaking on the column for 30 min, the crude product was flushed out
with 10% MeOH/CH.sub.2Cl.sub.2 and concentrated under reduced
pressure. The crude product was purified by preparative thin layer
chromatography (eluting 5% MeOH/CH.sub.2Cl.sub.2) to give carbamate
69 (13 mg, 92%) as a pale yellow oil. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 7.78 (d, 1H), 7.20 (dd, 1H), 7.03 (t, 1H), 6.65
(d, 1H), 6.62 (d, 2H), 5.24 (s, 2H), 5.16 (s, 2H), 4.74 (bs, 2H),
4.58 (d, 2H), 4.20 (m, 4H), 3.13 (m, 1H), 1.35 (t, 6H), 1.27 (d,
6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 20.7.
Example 34
[0748] ##STR219##
[0749] The title compound 70 was prepared following the sequence of
steps described in Example 32, except for substituting
trifluoro-methanesulfonic acid bis-benzyloxy-phosphorylmethyl ester
for trifluoro-methanesulfonic acid diethoxy-phosphorylmethyl ester.
Purification of the crude final product on silica gel eluted with
50-60% CH.sub.3CN/H.sub.2O provided 12 mg of the title compound.
.sup.1H NMR (300 MHz; CDCl.sub.3) .delta. 7.78 (s, 1H), 7.34 (m,
10H), 7.19 (dd, 1H), 7.02 (t, 1H), 6.63 (s, 1H), 6.61 (d, 2H), 5.38
(s, 2H), 5.25 (s, 2H), 5.11 (m, 4H), 4.62 (d, 2H), 3.24 (m, 1H),
1.33 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 21.4.
Example 35
[0750] ##STR220##
[0751] The title compound 71 was prepared following the sequence of
steps described in Example 29, except for substituting compound 70
for compound 28. Purification of the crude final product with HPLC
provided 2 mg of the title compound. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 7.90 (s, 1H), 7.44 (d, 1H), 7.13 (t, 1H), 6.72
(m, 3H), 5.39 (s, 2H), 5.34 (s, 2H), 4.39 (d, 2H), 3.30 (m, 1H),
1.28 (d, 6H).
Example 36
[0752] ##STR221##
[0753] To a solution of phosphonic acid 72 (33 mg, 0.058 mmol) in
DMF (2 mL) was added benzotriazol-1-yloxytripyrrolidino-phosphonium
hexafluorophosphate (91 mg, 0.175 mmol), iPr.sub.2NEt (30 .mu.L
0.175 mmol), and MeOH (0.24 mL, 5.83 mmol). After the reaction
mixture was stirred for 2 d at room temperature, the mixture was
partitioned between EtOAc and sat. NH.sub.4Cl. The organic phase
was dried over Na.sub.2SO.sub.4, filtered, and evaporated under
reduced pressure. Purification of the crude final product on silica
gel eluted with 3-5% MeOH/CH.sub.2Cl.sub.2 and by preparative thin
layer chromatography (eluting 5% MeOH/CH.sub.2Cl.sub.2) provided 6
mg of the title compound as a colorless solid. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 7.79 (d, 1H), 7.21 (dd, 1H), 7.04 (s, 1H),
6.66 (d, 1H), 6.62 (d, 2H), 5.25 (s, 2H), 5.17 (s, 2H), 4.70 (bs,
2H), 4.63 (d, 2H), 3.84 (d, 6H), 3.14 (m, 1H), 1.28 (d, 6H).
.sup.31P NMR (300 MHz, CDCl.sub.3) 323.2.
Example 37
[0754] ##STR222##
[0755] A solution of diol 67 (50 mg, 0.118 mmol) in
CH.sub.2Cl.sub.2 (5 mL) was treated with diethyl
(2-bromoethyl)-phosphonate (64 .mu.L, 0.354 mmol) and
Ag.sub.2CO.sub.3 (65 mg, 0.236 mmol). After the reaction mixture
was stirred for 3 d at 40.degree. C., additional phosphonate (64
.mu.L, 0.354 mmol), Ag.sub.2CO.sub.3 (65 mg, 0.236 mmol), and
benzene (5 mL) were introduced. After the reaction mixture was
stirred for another 4 days at 70.degree. C., the mixture was
filtered through a medium-fritted funnel. The crude product was
chromatographed by silica gel (eluting 4-5% MeOH(CH.sub.2Cl.sub.2)
to give diethylphosphonate 74 (8 mg, 12%) as a colorless oil.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.81 (bs, 1H), 7.17 (dd,
1H), 7.03 (t, 1H), 6.60 (d, 2H), 6.52 (d, 2H), 5.25 (s, 2H), 5.15
(s, 2H), 4.71 (bs, 2H), 4.47 (m, 2H), 4.14 (m, 4H), 3.12 (m, 1H),
2.27 (m, 2H), 1.34 (t, 6H), 1.27 (d, 6H). .sup.31P NMR (300 MHz,
CDCl.sub.3) .delta. 28.0.
Example 38
[0756] ##STR223##
[0757] The title compound 74 was prepared following the sequence of
steps described in Example 33, except for substituting
6-bromomethyl-3-methoxy pyridine for 5-bromomethyl-2-methoxy
pyridine 65. Purification of the crude final product on silica gel
with 4-5% MeOH/CH.sub.2Cl.sub.2 provided 66 mg of the title
compound. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.17 (d, 1H),
7.01 (d, 1H), 6.93 (m, 2H), 6.41 (d, 2H), 5.26 (s, 2H), 4.94 (s,
2H), 4.22 (q, 4H), 4.12 (m, 2H), 3.08 (m, 1H), 1.38 (t, 6H), 1.25
(d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 17.7.
Example 39
[0758] ##STR224##
[0759] The title compound 75 was prepared following the sequence of
steps described in Example 34, except for substituting compound 74
for compound 33. Purification of the crude final product on
preparative thin layer chromatography eluted with 5%
MeOH/CH.sub.2Cl.sub.2 provided 15 mg the title compound. .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. 8.18 (d, 1H), 6.98 (m, 1H), 6.96
(m, 1H), 6.79 (d, 1H), 6.58 (d, 2H), 5.35 (s, 2H), 5.32 (s, 2H),
4.83 (bs, 2H), 4.25 (q, 4H), 4.24 (m, 2H), 3.14 (m, 1H), 1.39 (t,
6H), 1.28 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta.
18.1.
Example 40
[0760] ##STR225##
[0761] The title compound 76 was prepared following the sequence of
steps described in Example 39, except for substituting
trifluoro-methanesulfonic acid bis-benzyloxy-phosphorylmethyl ester
for trifluoro-methanesulfonic acid diethoxy-phosphorylmethyl ester.
Purification of the crude final product on silica gel eluted with
4% MeOH/CH.sub.2Cl.sub.2 provided 67 mg of the title compound.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.05 (d, 1H), 7.36 (m,
10H), 6.95 (d, 1H), 6.81 (m, 2H), 6.37 (d, 2H), 5.22 (s, 2H), 5.13
(m, 4H), 4.91 (s, 2H), 4.11 (d, 2H), 3.05 (m, 1H), 1.22 (d, 6H).
.sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 18.8.
Example 41
[0762] ##STR226##
[0763] The title compound 77 was prepared following the sequence of
steps described in Example 34, except for substituting compound 76
for compound 33. Purification of the crude final product on silica
gel eluted with 4-5% MeOH/CH.sub.2Cl.sub.2 provided 35 mg of the
title compound. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.07 (d,
1H), 7.36 (m, 10H), 6.85 (m, 2H), 6.72 (d, 1H), 6.55 (d, 2H), 5.35
(s, 2H), 5.29 (s, 2H), 5.13 (m, 4H), 4.74 (bs, 2H), 4.15 (d, 2H),
3.13 (m, 1H), 1.28 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3)
.delta. 19.2.
Example 42
[0764] ##STR227##
[0765] The title compound 78 was prepared following the sequence of
steps described in Example 29, except for substituting compound 77
for compound 28. Purification of the crude final product on a C-18
column eluted with 30% CH.sub.3CN/H.sub.2O provided 6 mg of the
title compound. .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 8.16 (bs,
1H), 7.21 (bs, 2H), 7.18 (bs, 1H), 6.70 (d, 2H), 5.64 (s, 2H), 5.49
(s, 2H), 4.21 (d, 2H), 3.34 (m, 1H), 1.34 (d, 6H). .sup.31P NMR
(300 MHz, CD.sub.3OD) .delta. 16.0. ##STR228##
[0766] Diphenylphosphonate 79: A solution of phosphonic acid 59
(389 mg, 0.694 mmol) in pyridine (5 mL) was treated with phenol
(653 mg, 6.94 mmol) and 1,3-dicyclohexylcarbodiimide (573 mg, 2.78
mmol). After stirring at 70.degree. C. for 2 h, the mixture was
diluted with CH.sub.3CN and filtered through a fritted funnel. The
filtrate was partitioned between EtOAc and sat. NH.sub.4Cl, and
extracted with EtOAc. The organic phase was dried over
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified on silica gel (eluting 60-80%
EtOAc/hexane) to give diphenylphosphonate 79 (278 mg, 56%) as a
colorless oil.
[0767] Phosphonic acid 80: A solution of diphenylphosphonate 79
(258 mg, 0.362 mmol) in CH.sub.3CN (20 mL) was treated with 1N NaOH
(0.72 mL, 0.724 mmol) at 0.degree. C. After the reaction mixture
was stirred for 3 h at 0.degree. C., the mixture was filtered
through Dowex 50 W.times.8-400 acidic resin (380 mg), rinsed with
MeOH, and concentrated under reduced pressure to give phosphonic
acid 80 (157 mg, 68%) as a colorless solid.
[0768] Title compound 81: A solution of phosphonic acid 80 (35 mg,
0.055 mmol) in CH.sub.3CN (1 mL) and THF (1 mL) was treated with
thionyl chloride (12 .mu.L, 0.16 mmol). After the reaction mixture
was warmed to 70.degree. C. and stirred for 2 h, the mixture was
concentrated under reduced pressure. The residue was then dissolved
in CH.sub.2Cl.sub.2 (2 mL) and cooled to 0.degree. C. Triethylamine
(31 .mu.L, 0.22 mmol) and ethyl S-(-)-lactate (19 .mu.L, 0.16 mmol)
were added. After stirring for 1 h at 0.degree. C. and 1 h at room
temperature, the reaction mixture was neutralized with sat.
NH.sub.4Cl and extracted with CH.sub.2Cl.sub.2 and EtOAc. The
organic phase was dried over Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was purified
by preparative thin layer chromatography (eluting 70% EtOAc/hexane)
to give ethyl lactate 81 (7 mg, 17%) as a colorless solid. .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 7.30 (m, 5H), 6.99 (d, 1H), 6.82
(m, 4H), 6.63 (d, 2H), 5.23 (s, 2H), 5.18 (s, 2H), 5.14 (m, 1H),
4.67 (bs, 2H), 4.51 (d, 2H), 4.20 (m, 2H), 3.16 (m, 1H), 1.61 (d,
1.5H), 1.50 (d, 1.5H), 1.30 (d, 6H), 1.24 (m, 3H). .sup.31P NMR
(300 MHz, CDCl.sub.3) .delta. 17.0, 15.0.
Example 44
[0769] ##STR229##
[0770] The title compound 82 was prepared following the sequence of
steps described in Example 44, except for reacting monophosphonic
acid 80 with isopropyl lactate. Purification of the crude final
product on silica gel eluted with 70-90% EtOAc/hexane provided 5.4
mg of the title compound. .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.
7.35 (m, 3H), 7.25 (m, 3H), 7.0 (s, 0.5H), 6.98 (s, 0.5H), 6.86 (m,
2H), 6.79 (m, 2H), 6.64 (s, 1H), 6.61 (s, 1H), 5.22 (s, 2H), 5.17
(s, 2H), 5.06 (b, 1H), 4.62 (b, 2H), 4.53 (m, 2H), 4.38 (q, 1H),
3.15 (m, 1H), 1.60 (d, 1.5H), 1.48 (d, 1.5H), 1.30 (d, 3H), 1.28
(d, 3H), 1.20 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3) &
17.04, 14.94 (1:1 diastereomeric ratio).
Example 45
[0771] ##STR230##
[0772] The title compound 83 was prepared following the sequence of
steps described in Example 44, except for reacting monophosphonic
acid 80 with methyl lactate. Purification of the crude final
product on silica gel eluted with 70-90% EtOAc/hexane provided 2.7
mg of the title compound. .sup.1H NMR (300 MHz, CD.sub.3CN) .delta.
7.40 (m, 2H), 7.25 (m, 3H), 7.08 (s, 1H), 6.98 (d, 2H), 6.77 (d,
2H), 6.64 (s, 2H), 5.20 (s, 2H), 5.16 (s, 2), 5.13 (b, 1H), 4.47
(m, 2H), 3.72 (s, 2H), 3.67 (s, 1H), 3.09 (m, 1H), 1.56 (d, 1H),
1.51 (d, 2H), 1.20 (d, 6H). .sup.31P NMR (300 MHz, CD.sub.3CN)
.delta. 16.86, 15.80 (2.37:1 diastereomeric ratio).
Example 46
[0773] ##STR231##
[0774] A solution of mono-lactate phosphonate compound 83 (131 mg,
0.18 mmol) in DMSO/MeCN (1 mL/2 mL) and PBS buffer (10 mL) was
treated with esterase (400 .mu.L). After the reaction mixture was
warmed to 40.degree. C. and stirred for 7 days, the mixture was
filtered and concentrated under reduced pressure. Purification of
the crude product on C.sub.18 column eluted with MeCN/H.sub.2O
provided 17.3 mg (15%) of the title compound 84. .sup.1H NMR (300
MHz, CD.sub.3OD) .delta. 7.20 (s, 1H), 7.02 (d, 2H), 6.79 (d, 2H),
6.71 (s, 2H), 5.40 (s, 2H), 5.35 (s, 2H), 5.34 (b, 1H) 4.10 (bd,
2H), 3.26 (m, 1H), 1.50 (d, 3H), 1.30 (d, 6H). .sup.31P NMR (300
MHz, CD.sub.3OD) .delta. 14.2.
Example 47
[0775] ##STR232##
[0776] The title compound 85 was prepared following the sequence of
steps described in Example 44, except for reacting monophosphonic
acid 80 with L-alanine ethyl ester. Purification of the crude final
product on preparative thin layer chromatography eluted with 80%
EtOAc/hexane provided 7 mg of the title compound. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 7.26 (m, 5H), 6.98 (d, 1H), 6.87 (d, 2H),
6.73 (t, 2H), 6.62 (s, 2H), 5.21 (s, 2H), 5.17 (s, 2H), 4.28 (bs,
2H), 4.25 (m, 2H), 4.10 (m, 2H), 4.02 (m, 1H), 3.66 (m, 1H), 3.14
(m, 1H), 1.28 (d, 6H), 1.24 (m, 6H). .sup.31P NMR (300 MHz,
CDCl.sub.3) .delta. 20.2, 19.1.
Example 48
[0777] ##STR233##
[0778] The title compound 86 was prepared following the sequence of
steps described in Example 44, except for reacting monophosphonic
acid 80 with L-alanine methyl ester. Purification of the crude
final product on preparative thin layer chromatography eluted with
80% EtOAc/hexane provided 8 mg of the title compound. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 7.25 (m, 5H), 6.98 (d, 1H), 6.88 (d,
2H), 6.73 (t, 2H), 6.61 (bs, 2H), 5.21 (d, 2H), 5.17 (s, 2H), 4.66
(bs, 2H), 4.25 (m, 3H), 3.66 (s, 1.5H), 3.64 (m, 1H), 3.59 (m,
1.5H), 3.14 (m, 1H), 1.36 (t, 6H), 1.28 (d, 6H). .sup.31P NMR (300
MHz, CDCl.sub.3) .delta. 20.2, 19.0.
Example 49
[0779] ##STR234##
[0780] The title compound 87 was prepared following the sequence of
steps described in Example 44, except for reacting monophosphonic
acid 80 with L-alanine isopropyl ester. Purification of the crude
final product on preparative thin layer chromatography eluted with
80% EtOAc/hexane provided 7 mg of the title compound. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. =b 7.25 (m, 5H), 6.98 (m, 1H), 6.87
(d, 2H), 6.74 (m, 2H), 6.61 (bs, 2H), 5.22 (d, 2H), 5.18 (s, 2H),
4.93 (m, 1H), 4.68 (bs, 2H), 4.25 (m, 3H), 3.66 (s, 1H), 3.15 (m,
1H), 1.34 (m, 3H), 1.29 (d, 6H), 1.17 (m, 6H). .sup.31P NMR (300
MHz, CDCl.sub.3) .delta. 20.1, 19.1.
Example 50
[0781] ##STR235##
[0782] The title compound 88 was prepared following the sequence of
steps described in Example 44, except for reacting monophosphonic
acid 80 with L-alanine n-butyl ester. Purification of the crude
final product on preparative thin layer chromatography eluted with
80% EtOAc/hexane provided 6 mg of the title compound. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 7.25 (m, 5H), 6.98 (bd, 1H), 6.88 (d,
2H), 6.73 (t, 2H), 6.61 (d, 2H), 5.22 (d, 2H), 5.17 (s, 2H), 4.63
(bs, 2H), 4.25 (m, 3H), 4.06 (m, 2H), 3.65 (m, 1H), 3.14 (m, 1H),
1.58 (m, 4H), 1.36 (m, 3H), 1.28 (d, 6H), 0.90 (t, 3H). .sup.31P
NMR (300 MHz, CDCl.sub.3) .delta. 20.2, 19.1.
Example 51
[0783] ##STR236##
[0784] The title compound 89 was prepared following the sequence of
steps described in Example 44, except for reacting monophosphonic
acid 80 with L-alanine n-butyl ester. Purification of the crude
final product on preparative thin layer chromatography eluted with
80% EtOAc/hexane provided 4 mg of the title compound. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 7.24 (m, 5H), 6.98 (m, 1H), 6.87 (d,
2H), 6.74 (t, 2H), 6.62 (d, 2H), 5.21 (d, 2H), 5.17 (s, 2H), 4.64
(bs, 2H), 4.24 (m, 2H), 4.11 (m, 3H), 3.58 (m, 1H), 3.15 (m, 1H),
1.28 (d, 6H), 1.19 (m, 5H), 0.84 (m, 3H). .sup.31P NMR (300 MHz,
CDCl.sub.3) .delta. 20.4, 19.4.
Example 52
[0785] ##STR237##
[0786] To a solution of phosphonic acid 59 (61 mg, 0.11 mmol) in
DMF (1 mL) was added benzotriazol-1-yloxytripyrrolidino-phosphonium
hexafluorophosphate (169 mg, 0.32 mmol), L-alanine ethyl ester (50
mg, 0.32 mmol), and DIEA (151 .mu.L, 0.87 mmol). The reaction
mixture was stirred for 5 hours at room temperature. Then the
mixture was concentrated under reduced pressure. The residue was
dissolved in EtOAc, washed with HCl (5% aq), and extracted with
EtOAc (3.times.). The organic phase was washed with sat.
NaHCO.sub.3, dried over Na.sub.2SO.sub.4, and evaporated under
reduced pressure. The crude product was purified on silica gel
eluted with 5-8% MeOH/CH.sub.2Cl.sub.2 to give 5.5 mg of compound
bis-amidate 90 as white solid. .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.06 (s, 1H), 6.88 (d, 2H), 6.73 (d, 2H), 6.62 (s, 2H),
5.23 (s, 2H), 5.17 (s, 2H), 4.70 (bs, 2H), 4.25 (bm, 8H), 3.40 (q,
2H), 3.16 (m, 1H), 1.44 (t, 6H), 1.24 (d, 6H). .sup.31P NMR (300
MHz, CDCl.sub.3) .delta. 19.41.
Example 53
[0787] ##STR238##
[0788] The title compound 91 was prepared following the sequence of
steps described in Example 52, except for substituting ethyl amine
for L-alanine ethyl ester. Purification of the crude final product
on silica gel eluted with 4-10% MeOH/CH.sub.2Cl.sub.2 provided 14.8
mg of the title compound. .sup.1H NMR (300 MHz, CD.sub.3OD) .delta.
7.07 (s, 1H), 6.99 (d, 2H), 6.77 (d, 2H), 6.60 (s, 2H), 5.27 (s,
2H), 5.22 (s, 2H), 4.07 (d, 2H), 3.09 (m, 1H), 3.01 (bm, 4H), 1.24
(d, 6H), 1.16 (t, 6H). .sup.31P NMR (300 MHz, CD.sub.3OD) .delta.
24.66.
Example 54
[0789] ##STR239##
[0790] Diethylphosphonate 93: A solution of alcohol 92 (200 mg,
0.609 mmol) in THF (5 mL) was treated with 60% NaH in mineral oil
(37 mg, 0.914 mmol) at 0.degree. C. After the reaction mixture was
stirred for 5 min at 0.degree. C., trifluoro-methanesulfonic acid
diethoxy-phosphorylmethyl ester (219 mg, 0.731 mmol) was added in
THF (3 mL). After the reaction mixture was stirred for an
additional 30 min, the mixture was quenched with sat. NH.sub.4Cl
and extracted with EtOAc. The organic phase was dried over
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure
to give crude diethylphosphonate 93 as a colorless oil.
[0791] Alcohol 94: A solution of diethylphosphonate 93 (291 mg,
0.609 mmol) in CH.sub.2Cl.sub.2 (5 mL) was treated with
trifluoroacetic acid (0.5 mL). After the reaction mixture was
stirred for 30 min at room temperature, the mixture was
concentrated under reduced pressure. The crude product was purified
on silica gel (eluting 4-5% MeOH/CH.sub.2Cl.sub.2) to give alcohol
94 (135 mg, 94% over 2 steps) as a colorless oil.
[0792] Bromide 95: A solution of alcohol 94 (134 mg, 0.567 mmol) in
CH.sub.2Cl.sub.2 (5 mL) was treated with carbon tetrabromide (282
mg, 0.851 mmol) and triphenylphosphine (164 mg, 0.624 mmol). After
stirring at room temperature for 1 h, the mixture was partitioned
between CH.sub.2Cl.sub.2 and sat. NaHCO.sub.3. The organic phase
was dried over Na.sub.2SO.sub.4, filtered, and evaporated under
reduced pressure. The crude product was purified twice on silica
gel (eluting 60-100% EtOAc/hexane, followed by eluting 0-2%
MeOH/CH.sub.2Cl.sub.2) to give bromide 95 (80 mg, 47%) as a
colorless oil.
[0793] Imidazole 96: A solution of benzyl ether 53 (2.58 g, 6.34
mmol) in EtOH (60 mL) was treated with conc. HCl (60 mL). After the
reaction mixture was warmed to 100.degree. C. and stirred for 18 h,
the mixture was concentrated under reduced pressure. The residue
was partitioned between EtOAc and sat. NaHCO.sub.3. The organic
phase was dried over Na.sub.2SO.sub.4, filtered, and evaporated
under reduced pressure. The crude product was chromatographed on
silica gel (eluting 8-9% MeOH/CH.sub.2Cl.sub.2) to give imidazole
96 (1.86 g, 93%) as a colorless solid.
[0794] Title compound 97: A solution of imidazole 96 (54 mg, 0.170
mmol) and bromide 95 (56 mg, 0.187 mmol) in THF (3 mL) was treated
with powder NaOH (14 mg, 0.340 mmol), lithium iodide (23 mg, 0.170
mmol), and tetrabutylammonium bromide (27 mg, 0.085 mmol) were then
added. After stirring at room temperature for 2 h, the mixture was
partitioned between EtOAc and sat. NH.sub.4Cl. The organic phase
was dried over Na.sub.2SO.sub.4, filtered, and evaporated under
reduced pressure. The crude product was purified on silica gel
(eluting 3-4% MeOH/CH.sub.2Cl.sub.2) and by preparative thin layer
chromatography (eluting 5% MeOH/CH.sub.2Cl.sub.2) to give alcohol
97 (42 mg, 46%) as a pale yellow oil. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 7.13 (bs, 1H), 6.86 (d, 2H), 492 (s, 2H), 4.87
(s, 2H), 4.16 (m, 6H), 3.73 (d, 2H), 3.10 (m, 1H), 1.34 (t, 6H),
1.21 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 20.8.
Example 55
[0795] ##STR240##
[0796] The title compound 97a was prepared following the sequence
of steps described in Example 32 by substituting compound 97a for
compound 68. Purification of the crude final product on-silica gel
eluted with 3-4% MeOH(CH.sub.2Cl.sub.2 provided 13 mg of the title
compound. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.13 (t, 1H),
6.87 (d, 2H), 5.29 (s, 2H), 4.87 (s, 2H), 4.14 (m, 6H), 3.72 (d,
2H), 3.13 (m, 1H), 1.33 (t, 6H), 1.26 (d, 6H). .sup.31P NMR (300
MHz, CDCl.sub.3) .delta. 21.2.
Example 56
[0797] ##STR241## ##STR242##
[0798] Monophenol Allylphosphonate 99c: To a solution of
allylphosphonic dichloride 99a (4 g, 25.4 mmol) and phenol (5.2 g,
55.3 mmol) in CH.sub.2Cl.sub.2 (40 mL) at 0.degree. C. was added
TEA (8.4 mL, 60 mmol). After stirred at room temperature for 1.5 h,
the mixture was diluted with hexane-ethyl acetate and washed with
HCl (0.3 N) and water. The organic phase was dried over MgSO.sub.4,
filtered and concentrated under reduced pressure. The residue was
filtered through a pad of silica gel (eluted with 2:1 hexane-ethyl
acetate) to afford crude product diphenol allylphosphonate 99b (7.8
g, containing the excessive phenol) as an oil which was used
directly without any further purification. The crude material was
dissolved in CH.sub.3CN (60 mL), and NaOH (4.4N, 15 mL) was added
at 0.degree. C. The resulted mixture was stirred at room
temperature for 3 h, then neutralized with acetic acid to pH=8 and
concentrated under reduced pressure to remove most of the
acetonitrile. The residue was dissolved in water (50 mL) and washed
with CH.sub.2Cl.sub.2 (3.times.25 mL). The aqueous phase was
acidified with concentrated HCl at 0.degree. C. and extracted with
ethyl acetate. The organic phase was dried over MgSO.sub.4,
filtered, evaporated and co-evaporated with toluene under reduced
pressure to yield desired monophenol allylphosphonate 99c (4.75 g.
95%) as an oil.
[0799] Monolactate Allylphosphonate 99e: A solution of monophenol
allylphosphonate 99c (4.75 g, 24 mmol) in toluene (30 mL) was
treated with SOCl.sub.2 (5 mL, 68 mmol) and DMF (0.05 mL). After
stirred at 65.degree. C. for 4 h, the reaction was completed as
shown by .sup.31P NMR. The reaction mixture was evaporated and
co-evaporated with toluene under reduced pressure to give mono
chloride 99d (5.5 g) as an oil. A solution of chloride 99d in
CH.sub.2Cl.sub.2 (25 mL) at 0.degree. C. was added ethyl
(s)-lactate (3.3 mL, 28.8 mmol), followed by TEA. The mixture was
stirred at 0.degree. C. for 5 min then at room temperature for 1 h,
and concentrated under reduced pressure. The residue was
partitioned between ethyl acetate and HCl (0.2N), the organic phase
was washed with water, dried over MgSO.sub.4, filtered and
concentrated under reduced pressure. The residue was purified by
chromatography on silica gel to afford desired monolactate 99e
(5.75 g, 80%) as an oil (2:1 mixture of two isomers).
[0800] Aldehyde 99f: A solution of allylphosphonate 99e (2.5 g,
8.38 mmol) in CH.sub.2Cl.sub.2 (30 mL) was bubbled with ozone air
at -78.degree. C. until the solution became blue, then bubbled with
nitrogen until the blue color disappeared. Methyl sulfide (3 mL)
was added at -78.degree. C. The mixture was warmed up to room
temperature, stirred for 16 h and concentrated under reduced
pressure to give desired aldehyde 99f (3.2 g, as a 1:1 mixture of
DMSO).
[0801] Compound 98 was prepared from compound 29 following the
sequence of steps described in Example 22. Compound 99 was prepared
from compound 96 following the sequence of steps described in
Example 54 and 55, except for substituting 4-nitro benzyl bromide
for compound 95.
[0802] Aniline 100: To a solution of compound 99 (100 mg, 0.202
mmol) in EtOH (2 mL) was added acetic acid (2 mL) and zinc dust (40
mg, 0.606 mmol). After the reaction mixture was stirred for 30 min
at room temperature, the mixture was concentrated under reduced
pressure. The crude product was purified on silica gel (eluting
5-6% MeOH/CH.sub.2Cl.sub.2) to give aniline 100 (43 mg, 41%) as a
yellow oil.
[0803] Title compound phosphonate 101: To a solution of aniline 100
(22 mg, 0.042 mmol) and aldehyde 99f (17 mg, 0.046 mmol) in MeOH (2
mL) was added acetic acid (10 .mu.L, 0.17 mmol) and 4 .ANG.
molecular sieves (10 mg). After the reaction mixture was stirred
for 2 h at room temperature, NaCNBH.sub.3 (5 mg, 0.084 mmol) was
added. After the reaction mixture was stirred for an additional 4 h
at room temperature, the mixture was concentrated under reduced
pressure. The residue was partitioned between EtOAc and sat.
NaHCO.sub.3. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was purified on silica gel (eluting 5-6% MeOH/CH.sub.2Cl.sub.2) to
give title compound phosphonate 101 (25 mg, 79%) as a colorless
oil. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.34 (dd, 2H), 7.21
(m, 3H), 7.02 (bs, 1H), 6.79 (d, 2H), 6.64 (t, 2H), 6.42 (dd, 2H),
5.21 (s, 2H), 5.10 (s, 2H), 5.02 (m, 1H), 4.75 (bs, 2H), 4.20 (m,
2H), 3.53 (m, 2H), 3.13 (m, 1H), 2.31 (m, 2H), 1.58 (d, 1.5H), 1.38
(d, 1.5H), 1.28 (d, 6H), 1.25 (t, 3H). .sup.31P NMR (300 MHz,
CDCl.sub.3) .delta. 28.4, 26.5.
Example 57
[0804] ##STR243##
[0805] Compound 102 was prepared from compound 96 following the
sequence of steps described in Example 54, except for substituting
methyl 4-bromomethyl benzoate for compound 95.
[0806] Amide 103: A solution of ester 102 (262 mg, 0.563 mmol) in
THF (5 mL) and CH.sub.3CN (2 mL) was treated with 1N NaOH (1.13 mL,
1.13 mmol). After the reaction mixture was stirred for 2 h at
60.degree. C., the mixture was concentrated under reduced pressure.
The residue was partitioned between EtOAc and 1N HCl. The organic
phase was dried over Na.sub.2SO.sub.4, filtered, and evaporated
under reduced pressure. The crude product was chromatographed on
silica gel (eluting 5-10% MeOH/CH.sub.2Cl.sub.2) to give the
carboxylic acid (120 mg, 47%) as a colorless oil. A solution of the
above carboxylic acid (120 mg, 0.266 mmol) and
N,O-dimethylhydroxylamine (29 mg, 0.293 mmol) in DMF (3 mL) was
treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodimide
hydrochloride (61 mg, 0.319 mmol), 1-hydroxybenzotriazole hydrate
(43 mg, 0.319 mmol), and triethylamine (55 .mu.L, 0.399 mmol).
After the reaction mixture was stirred for 18 h at room
temperature, the mixture was partitioned between EtOAc and
H.sub.2O. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was chromatographed on silica gel (eluting 3-4%
MeOH/CH.sub.2Cl.sub.2) to give the amide 103 (107 mg, 81%) as a
colorless oil.
[0807] Aldehyde 104: A solution of amide 103 (106 mg, 0.214 mmol)
in THF (5 mL) was treated with 1.5M DIBAL-H in toluene (0.43 mL,
0.642 mmol) at 0.degree. C. After the reaction mixture was stirred
for 1 h at 0.degree. C., the mixture was quenched with 1M sodium
potassium tartrate and stirred for an additional 3 d. The aqueous
phase was extracted with EtOAc, and the organic phase was dried
over Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure to give crude aldehyde 104 as a colorless oil.
[0808] Title compound 105: To a solution of aldehyde 104 (91 mg,
0.21 mmol) in MeOH (5 mL) was added diethyl(aminoethyl) phosphonate
(63 mg, 0.231 mmol), acetic acid (48 .mu.L, 0.231 mmol) and 4 .ANG.
molecular sieves (10 mg). After the reaction mixture was stirred
for 2 h at room temperature, NaCNBH.sub.3 (26 mg, 0.42 mmol) was
added. After the reaction mixture was stirred for an additional 18
h at room temperature, the mixture was concentrated under reduced
pressure. The residue was partitioned between EtOAc and sat.
NaHCO.sub.3. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was chromatographed on silica gel (eluting 5-10%
MeOH/CH.sub.2Cl.sub.2) to give phosphonate 105 (10 mg, 8% over 2
steps) as a colorless oil. .sup.1H NMR (300 MHz, CD.sub.3OD)
.delta. 7.15; (d, 2H), 7.10 (t, 1H), 7.06 (d, 2H), 6.65 (t, 2H),
5.34 (s, 2H), 4.73 (s, 2H), 4.09 (m, 4H), 3.68 (s, 2H), 3.12 (m,
1H), 2.83 (m, 2H), 2.04 (m, 2H), 1.30 (t, 6H), 1.24 (d, 6H).
.sup.31P NMR (300 MHz, CD.sub.3OD) .delta. 30.6.
Example 58
[0809] ##STR244##
[0810] The title compound 106 was prepared following the sequence
of steps described in Example 34, except for substituting compound
105 for compound 68. Purification of the crude final product on
preparative thin layer chromatography eluted with 7%
MeOH(CH.sub.2Cl.sub.2 provided 6 mg of the title compound. .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 7.15 (d, 2H), 7.02 (bs, 1H), 6.88
(d, 2H), 6.67 (t, 2H), 5.21 (s, 2H), 5.17 (s, 2H), 4.76 (bs, 2H),
4.08 (m, 4H), 3.70 (s, 2 Hz, 3.15 (m, 1H), 2.86 (m, 2H), 1.97 (m,
2H), 1.31 (t, 6H), 1.29 (d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3)
.delta. 30.6.
Example 59
[0811] ##STR245##
[0812] Compound 107 was prepared following the sequence of steps
described in Example 34, except for substituting compound 104 for
compound 68. The title compound was prepared following the sequence
of steps described in Example 58, except for substituting compound
98 for aminoethyl phosphonic acid diethyl ester. Purification of
the crude final product on preparative thin layer chromatography
eluted with 7% MeOH/CH.sub.2Cl.sub.2 provided 24 mg of the title
compound 108. .sup.1H NMR (300 MHz, CDCl.sub.3) (5:1 diastereomeric
ratio) .delta. 7.34 (t, 2H), 7.17 (m, 5H), 7.01 (t, 1H), 6.86 (d,
2H), 6.66 (t, 2H), 5.20 (bs, 4H), 4.96 (m, 1H), 4.63 (bs, 2H), 4.19
(m, 2H), 3.73 (s, 2H), 3.15 (m, 1H), 3.02 (m, 2H), 2.27 (m, 2H),
1.36 (d, 3H), 1.29 (d, 6H) 1.27 (r, 3H). .sup.31P NMR (300 MHz,
CDCl.sub.3) .delta. 29.1, 27.4.
Example 60
[0813] ##STR246##
[0814] Compound 109 was prepared from compound 29 following the
sequence of steps described in Example 22. The title compound was
prepared following the sequence of steps described in Example 58,
except for substituting compound 109 for aminoethyl phosphonic acid
diethyl ester. Purification of the crude final product on silica
gel eluted with 5-6% MeOH/CH.sub.2Cl.sub.2 provided 8 mg of the
title compound. .sup.1H NMR (300 MHz, CDCl.sub.3) (1.8:1
diastereomeric ratio) .delta. 7.31 (m, 2H), 7.16 (m, 5H), 7.01 (bs,
1H), 6.88 (d, 2H), 6.66 (bs, 2H), 5.21 (s, 2H), 5.20 (s, 2H), 4.69
(bd, 2H), 4.27 (bt, 1H), 4.12 (m, 3H), 3.75 (m, 2H), 3.16 (m, 1H),
2.99 (m, 2H), 2.11 (m, 2H), 1.30 (d, 6H), 1.22 (m, 6H). .sup.31P
NMR (300 MHz, CDCl.sub.3) .delta. 31.3, 30.8.
Example 61
[0815] ##STR247##
[0816] Compound 112: A solution of methyl 4-hydroxybenzoate 111
(0.977 g, 6.42 mmol) and trifluoro-methanesulfonic acid
diethoxy-phosphorylmethyl ester (2.12 g, 7.06 mmol) in THF (50 mL)
was treated with Cs.sub.2CO.sub.3 (4.18 g, 12.84 mmol). The
resulting reaction mixture was stirred for 1 h at room temperature
before it was partitioned between EtOAc and sat. aqueous NH.sub.4Cl
and extracted with EtOAc (3.times.). The organic phase was washed
with brine, dried over Na.sub.2SO.sub.4, and evaporated under
reduced pressure. Purification of the crude product on silica gel
(eluted with 60-90% EtOAc/hexane) provided 1.94 g (quantitative) of
methyl phosphonobenzoate compound 112 as a clear oil.
[0817] Alcohol 112a: A solution of 112 (1.94 g, 6.42 mmol) in
Et.sub.2O (40 mL) was treated with LiBH.sub.4 (0.699 g, 32.1 mmol)
and THF (10 mL). After the reaction mixture was stirred for 12 h at
room temperature, the mixture was quenched with water and extracted
with EtOAc (3.times.). The organic phase was dried over
Na.sub.2SO.sub.4 and evaporated under reduced pressure. The crude
product was purified on silica gel (eluted with 2-5%
MeOH/CH.sub.2Cl.sub.2) to give 1.48 g (84%) of alcohol compound
112a as a colorless oil.
[0818] Chloride 112b: A solution of 112a (315 mg, 1.15 mmol) in
MeCN (6 mL) was treated with methanesulfonyl chloride (97.6 .mu.L,
1.26 mmol), TEA (175 .mu.L, 1.26 mmol), LiCl (74.5 mg, 1.72 mmol).
After stirring at room temperature for 30 min., the mixture was
concentrated under reduced pressure, partitioned between EtOAc and
sat. NaHCO.sub.3, and extracted with EtOAc (3.times.). The organic
phase was dried over Na.sub.2SO.sub.4 and evaporated under reduced
pressure. Purification of the crude product on silica gel (eluted
with 2-4% MeOH/CH.sub.2Cl.sub.2) provided 287 mg (85%) of chloride
compound 112b as a clear pale yellow oil.
[0819] Alcohol compound 113: A solution of benzyl ether 36 (120 mg,
0.326 mmol) in EtOH (2 mL) was treated with conc. HCl (2 mL). After
the reaction mixture was refluxed at 100.degree. C. for 1 day, the
mixture was concentrated under reduced pressure, partitioned
between EtOAc and sat. NaHCO.sub.3, and extracted with EtOAc
(3.times.). The organic phase was dried over Na.sub.2SO.sub.4 and
evaporated under reduced pressure to provide the crude alcohol
compound 113 (90 mg, 99%) as a white solid.
[0820] Compound 114: A solution of alcohol compound 113 (16.8 mg,
0.060 mmol) and chloride compound 112b (21.1 mg, 0.072 mmol) in THF
(1.5 mL) was treated with powder NaOH (3.5 mg, 0.090 mmol), lithium
iodide (12.0 mg, 0.090 mmol), and tetrabutylammonium bromide (9.70
mg, 0.030 mmol). After the reaction mixture was stirred at room
temperature for 15 h, the mixture was partitioned between EtOAc and
sat. NH.sub.4Cl. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was purified on silica gel (eluted with 3-6% MeOH/CH.sub.2Cl.sub.2)
to give compound 114 (19.7 mg, 61%) as a colorless oil.
[0821] Title compound 115: A solution of 114 (19.7 mg, 0.037 mmol)
in CH.sub.2Cl.sub.2 (1 mL) was treated with trichloroacetyl
isocyanate (13.2 .mu.L, 0.111 mmol). After the reaction mixture was
stirred at room temperature for 20 min, 2 ml, of CH.sub.2Cl.sub.2
(saturated with NH.sub.3) was added to the mixture. After stirring
at room temperature for 1 h, the mixture was bubbled with N.sub.2
for 1 h. The mixture was then concentrated under reduced pressure
and purified on silica gel (eluted with 4-6% MeOH/CH.sub.2Cl.sub.2)
to give the titled compound 115 (18.5 mg, 87%) as a clear oil.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.09 (t, 1H), 6.90 (d,
2H), 6.78 (d, 2H), 6.63 (dd, 1H), 6.51 (dd, 1H), 6.40 (t, 1H), 5.15
(s, 2H), 5.11 (s, 2H), 4.70 (b, 2H), 4.21 (m, 6H), 3.70 (s, 3H),
3.22 (m, 1H), 1.36 (t, 6H), 1.29 (d, 6H). .sup.31P NMR (300 MHz,
CDCl.sub.3) .delta. 19.2.
Example 62
[0822] ##STR248##
[0823] A suspension of compound 116 (15 mg, 0.03 mmol) in acetone
d-6 was treated with trifluoro-methanesulfonic acid
diethoxy-phosphorylmethyl ester (12 mg, 0.04 mmol). The solution
was stirred overnight at ambient temperature. Concentration
afforded compound 117. Compound 117 (22 mg, 0.03 mmol) was
suspended in EtOH (2 mL) and an excess of sodium borohydride (15
mg, 0.39 mmol) was added. The solution was stirred at room
temperature. After 30 minutes, sodium borohydride (15 mg, 0.39
mmol) was added again. Acetic-acid (1 ml) in EtOH was added 2 hours
later followed by the addition of sodium borohydride (15 mg, 0.39
mmol). After 30 minutes, the solution was concentrated. The residue
was dissolved in saturated aqueous NaHCO.sub.3 and extracted with
EtOAc (.times.3). The organic layers were washed with brine and
dried over MgSO.sub.4. The solution was filtered, concentrated and
purified using a TLC plate (5% CH.sub.3OH/CH.sub.2Cl.sub.2) to give
14 mg (80%) of the desired product. .sup.1H NMR (CDCl.sub.3, 500
mHz): 7.13 (s, 1H), 6.83 (s, 2H), 5.16 (s, 2H), 5.01 (s, 1H), 4.51
(s, 2H), 4.14 (m, 4H), 3.15 (m, 1H), 3.00 (s, 2H), 2.80 (d, 2H),
2.68 (t, 2H), 1.97 (s, 2H), 1.33 (t, 6H), 1.29 (d, 6H).
Example 63
[0824] ##STR249##
[0825] Title compound 119 was prepared following the sequence of
steps described in Example 62 by substituting
trifluoro-methanesulfonic acid bis-benzyloxy-phosphorylmethyl ester
for trifluoro-methanesulfonic acid diethoxy-phosphorylmethyl ester.
Purification of the crude final product on silica gel eluted with
(2.5%-5% CH.sub.3OH/CH.sub.2Cl.sub.2) provided 71 mg (65%) of the
title compound. .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.35 (s, 10H),
7.11 (s,1H) 6.82 (s, 2H), 5.16 (s, 2H), 5.04 (d, 4H), 4.99 (s, 1H),
4.49 (s, 2H), 3.15 (m, 1H), 2.96 (s, 2H), 2.81 (d, 2H), 2.63 (t,
2H), 1.91 (s, 2H), 1.29 ppm(d, 6H).
Example 64
[0826] ##STR250##
[0827] Compound 119 was stirred in 4M HCl/dioxane overnight at
ambient temperature. The mixture was concentrated and purified
using HPLC (20% CH.sub.3CN/H.sub.2O) to provide 20 mg of the title
compound 120. .sup.1H NMR (CD.sub.3OD.sub.3, 500 MHz) 7.33 (s,1H)
7.00 (s, 2H), 5.22 (s, 2H), 5.12 (s, 1H), 4.79 (s, 2H), 3.80 (s,
2H), 3.49 (s, 2H), 3.23 (m, 2H), 3.21 (m, 1H), 2.40 (s, 2H), 1.28
(d, 6H).
Example 65
[0828] ##STR251##
[0829] Compound 121 was prepared following the sequence of steps
described in Example 62 by substituting trifluoro-methanesulfonic
acid dimethoxy-phosphorylethyl ester for trifluoro-methanesulfonic
acid diethoxy-phosphorylmethyl ester. Purification of the crude
final product on TLC plate eluted with (5%
CH.sub.3OH/CH.sub.2Cl.sub.2) provided 11 mg (65%) of the title
compound. .sup.1H NMR (CDCl.sub.3, 500 MHz): 7.34 (d, 2H). 7.20 (d,
2H), 7.19 (d,1H) 7.13 (s, 1H), 6.83 (s, 2H), 5.18 (s, 2H), 5.03 (s,
1H), 4.98 (m, 1H), 4.52 (s, 2H), 4.22 (m, 2H), 3.15 (m, 1H), 2.91
(s, 2H), 2.81 (s, 2H), 2.54 (s, 2H), 2.29 (m, 2H), 2.01 (d, 2H),
1.56 (d, 3H), 1.38 (d,3H), 1.28 (q, 3H), 1.28 (d, 6H).
Example 66
[0830] ##STR252##
[0831] A solution of 25 (33.2 mg, 0.081 mmol) in DMF (3 mL) under
N.sub.2 at 0.degree. C. was treated with NaH. After stirring at
0.degree. C. for 10 min, 95 (23 mg, 0.077 mmol) was added, and the
resulting mixture was slowly raised to room temperature and stirred
at room temperature for 8 h. The mixture was then poured into
water, and extracted with EtOAc. The combined organic layers were
washed with brine, dried (Na.sub.2SO.sub.4), filtered, and
evaporated under reduced pressure. The crude product was purified
on TLC plate (eluted with 3% MeOH/CH.sub.2Cl.sub.2) to provide 17.9
mg of the title compound 122. .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 8.45 (d, 2H), 7.04 (t, 1H), 6.88 (d, 2H), 6.67 (d, 2H),
5.24 (s, 2H), 4.67 (s, 2H), 5.02 (m, 1H), 4.27 (bs, 2H), 4.22 (bs,
2H), 4.19 (m, 4H), 3.82 (m, 2H), 3.16 (m, 1H), 1.35 (t, 6H), 1.30
(d, 6H). .sup.31P NMR (300 MHz, CDCl.sub.3) .delta. 20.8.
Example 67
Anti-HIV-1 Cell Culture Assay
[0832] The assay is based on quantification of the HIV-1-associated
cytopathic effect by a colorimetric detection of the viability of
virus-infected cells in the presence or absence of tested
inhibitors. The HIV-1-induced cell death is determined using a
metabolic substrate
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT) which is converted only by intact cells into a product with
specific absorption characteristics as described by Weislow O S,
Kiser R, Fine D L, Bader J, Shoemaker R H and Boyd M R (1989) J
Natl Cancer Inst 81, 577.
Assay Protocol for Determination of EC50:
[0833] 1. Maintain MT2 cells in RPMI-1640 medium supplemented with
5% fetal bovine serum and antibiotics. [0834] 2. Infect the cells
with the wild-type HIV-1 strain MB (Advanced Biotechnologies,
Columbia, Md.) for 3 hours at 37.degree. C. using the virus
inoculum corresponding to a multiplicity of infection equal to
0.01. [0835] 3. Distribute the infected cells into a 96-well plate
(20,000 cells in 100 .mu.L/well) and add various concentrations of
the tested inhibitor in triplicate (100 .mu.L/well in culture
media). Include untreated infected and untreated mock-infected
control cells. [0836] 4. Incubate the cells for 5 days at
37.degree. C. [0837] 5. Prepare XTT solution (6 ml per assay plate)
at a concentration of 2 mg/mL in a phosphate-buffered saline pH
7.4. Heat the solution in water-bath for 5 min at 55.degree. C. Add
50 .mu.L of N-methylphenazonium methasulfate (5 .mu.g/mL) per 6 ml,
of XTT solution. [0838] 6. Remove 100 .mu.L media from each well on
the assay plate. [0839] 7. Add 100 .mu.L of the XTT substrate
solution per well and incubate at 37.degree. C. for 45 to 60 min in
a CO.sub.2 incubator. [0840] 8. Add 20 .mu.L of 2% Triton X-100 per
well to inactivate the virus. [0841] 9. Read the absorbance at 450
nm with subtracting off the background absorbance at 650 [0842] 10.
Plot the percentage absorbance relative to untreated control and
estimate the EC50 value as drug concentration resulting in a 50%
protection of the infected cells.
Example 68
Cytotoxicity Cell Culture Assay (Determination of CC50)
[0843] The assay is based on the evaluation of cytotoxic effect of
tested compounds using a metabolic substrate
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT) as described by Weislow O S, Kiser R, Fine D L, Bader J,
Shoemaker R H and Boyd M R (1989) J Natl Cancer Ins 81, 577.
Assay Protocol for Determination of CC50:
[0844] 1. Maintain MT-2 cells in RPMI-1640 medium supplemented with
5% fetal bovine serum and antibiotics. [0845] 2. Distribute the
cells into a 96-well plate (20,000 cell in 100 .mu.l, media per
well) and add various concentrations of the tested compound in
triplicate (100 .mu.L/well). Include untreated control. [0846] 3.
Incubate the cells for 5 days at 37.degree. C. [0847] 4. Prepare
XTT solution (6 ml per assay plate) in dark at a concentration of 2
mg/mL in a phosphate-buffered saline pH 7.4. Heat the solution in a
water-bath at 55.degree. C. for 5 min. Add 50 .mu.L of
N-methylphenazonium methasulfate (5 .mu.g/mL) per 6 ml, of XTT
solution. [0848] 5. Remove 100 .mu.L media from each well on the
assay plate and add 100 .mu.L of the XTT substrate solution per
well. Incubate at 37.degree. C. for 45 to 60 min in a CO.sub.2
incubator. [0849] 6. Add 20 .mu.l, of 2% Triton X-100 per well to
stop the metabolic conversion of XTT. [0850] 7. Read the absorbance
at 450 nm with subtracting off the background at 650 nm. [0851] 8.
Plot the percentage absorbance relative to untreated control and
estimate the CC50 value as drug concentration resulting in a 50%
inhibition of the cell growth. Consider the absorbance being
directly proportional to the cell growth. PETT-Like Phosphonate
NNRTI Compounds
[0852] The PETT class of compound has demonstrated activity in
inhibiting HIV replication. The present invention provides novel
analogs of PETT class of compound. Such novel PETT analogs possess
all the utilities of PETT and optionally provide cellular
accumulation as set forth below. ##STR253##
[0853] The intermediate phosphonate esters required for conversion
into the prodrug phosphonate moieties bearing amino acid, or
lactate esters are shown in FIG. 2.
[0854] PETT 1 compounds, analogs of trovirdine, are obtained
following the procedures described in WO/9303022 and J. Med. Chem.
1995, 38, 4929-4936 and 1996, 39, 4261-4274. Preparation of
PETT-like phosphonate NNRTI compounds, e.g. phosphonate analog type
2 is outlined in Scheme 1. PETT analog 1a is obtained following the
above mentioned literature procedure. Alkyl group of 1a is then
removed using such as, for example BCl.sub.3 to give phenol 7, many
examples are described in Greene and Wuts, Protecting Groups in
Organic Synthesis, 3.sup.rd Edition, John Wiley and Sons Inc.
Conversion of 7 to the desired phosphonate analogs is realized by
treatment of 7 with the phosphonate reagent 6 under suitable
conditions.
[0855] For example (Example 1), PETT 1a is treated with BCl.sub.3
to give phenol 7. Treatment of 7 with phosphonate 6.1 in the
presence of base, for example, Cs.sub.2CO.sub.3, affords the
phosphonate 2a.1. Using the above procedure but employing a
different phosphonate reagent 5 in place of 6.1, corresponding
products 2 with different linking groups are obtained.
##STR254##
Example 1
[0856] ##STR255##
[0857] Scheme 2 shows the preparation of phosphonate type 3 in FIG.
2. PETT 1b is obtained as described in WO/9303022 and J. Med. Chem.
1995, 38, 4929-4936 and 1996, 39, 4261-4274. Alkyl group of 1b is
then removed using such as, for example BCl.sub.3 to give phenol 8,
many examples are described in Greene and Wuts, Protecting Groups
in Organic Synthesis, 3.sup.rd Edition, John Wiley and Sons Inc.
Conversion of 8 to the desired phosphonate analogs is realized by
treatment of S with the phosphonate reagent 6 under suitable
conditions.
[0858] For example (Example 1), PETT 1a is treated with BCl.sub.3
to give phenol 7. Treatment of 7 with triflate methyl phosphonic
acid diethyl ester 6.1 in the presence of base, for example,
Cs.sub.2CO.sub.3, affords the phosphonate 2a.1. Using the above
procedure but employing a different phosphonate reagent 6 in place
of 6.1, corresponding products 3 with different linking groups are
obtained. ##STR256##
Example 2
[0859] ##STR257##
[0860] Scheme 3 shows of the preparation of the phosphonate linkage
of type 4 and 5 to PETT. PETT 1c is first treated with a suitable
base to remove the thiourea proton, the product is then treated
with 1 equivalent of a phosphonate reagent 5 bearing a leaving
group such as, for example, bromine, mesyl tosyl etc to give the
alkylated product 4 and 5. The phosphonates 4 and 5 are separated
by chromatography. For example (Example 3), PETT 1, in DMF, is
treated with sodium hydride followed by one equivalent of
bromomethyl phosphonic acid dibenzyl ester 6.2 to give phosphonate
4a and 5a. Phosphonate product 4a and 5a are then separated by
chromatography to give pure 4a and 5a respectively. Using the above
procedure but employing a different phosphonate reagent 5 in place
of 6.2, corresponding products 4 and 5 with different linking
groups are obtained. ##STR258##
Example 3
[0861] ##STR259## Pyrazole-Like Phosphonate NNRTI Compounds
[0862] The present invention includes pyrazole-like phosphonate
NNRTI compounds and describes methods for their preparation.
Pyrazole-like phosphonate NNRTI compounds are potential anti-HIV
agents.
[0863] A link group includes a portion of the structure that links
two substructures, one of which is pyrazole class of HIV inhibiting
agents having the general formula shown above, the other is a
phosphonate group bearing the appropriate R and R.sub.5 groups. The
link has at least one uninterrupted chain of atoms other than
hydrogen.
[0864] Pyrazole class of compounds has shown to be inhibitors of
HIV RT. The present invention provides novel analogs of pyrazole
class of compound. Such novel pyrazole analogs possess all the
utilities of pyrazoles and optionally provide cellular accumulation
as set forth below.
[0865] The intermediate phosphonate esters required for conversion
into the prodrug phosphonate moieties bearing amino acid, or
lactate esters are shown in FIG. 2, where R.sub.1, R.sub.2,
R.sub.3, R.sub.4 and X are as described in WO02/04424.
[0866] Pyrazole 1 is obtained following the procedures described in
WO02/04424. Preparation of phosphonate analog type 2 is outlined in
Scheme 1. Pyrazole analog 1a, which R.sub.2 bears a function group
can be used as attaching site for phosphonate prodrug, is obtained
as described in the above mentioned literature. Conversion of 1a to
the desired phosphonate analogs is realized by treatment of 2a with
the phosphonate reagent 4 under suitable conditions.
[0867] For example (Example 1), treatment of pyrazole 1a.1 with
phosphonate 4.1 in the presence of base, for example, Mg(OtBu) 2,
affords the phosphonate 2a.1. Using the above procedure but
employing a different phosphonate reagent 4 in place of 4.1,
corresponding products 2a with different linking groups are
obtained. Alternatively, activation of the hydroxyl group with
bis(4-nitrophenyl) carbonate, following by treatment with amino
ethyl phosphonate 4.2 provides phosphonate 2a.2. Using different
phosphonate 4 in place of 4.2 and/or different methods for linking
them together affords 2 with different linker. ##STR260##
Example 1
[0868] ##STR261##
[0869] Scheme 2 shows the preparation of phosphonate type 3
conjugate to pyrazole in FIG. 2. Pyrazole 1b, bearing a functional
group at position R.sub.1 can be used as attaching site for
phosphonate prodrug, is obtained as described in WO02/04424.
Conversion of 1b to the desired phosphonate 3 analogs is realized
by treatment of 1b with the phosphonate reagent 4 under suitable
conditions. For example (Example 2), pyrazole 1b reacts with
phosphonate 4.3 in the presence of triphenyl phosphine and DEAD in
THF, affords the phosphonate 3a.1. Phosphonate 3a.2 is obtained by
first reducing the ester to alcohol, and then by treating the
resulting alcohol with trichloroacetyl isocyanate, and followed by
alumina. Using the above procedure but employing a different
phosphonate reagent 4 in place of 4.3, corresponding products 3
with different linking groups are obtained. ##STR262##
Example 2
[0870] ##STR263##
[0871] Alternatively, as shown in Example 3, reaction of pyrazolone
1b.1 with a moiety bearing a protected function group which can be
used to attach phosphonate, for example benzyl alcohol with a
protected hydroxyl or amino group, under Mitsunobu condition
affords compound 5. The protecting group of Z is then removed, and
the resulting product is reacted with phosphonate reagent yields
phosphonate 3b.1. Phosphonate 3b.1 is converted to phosphonate 3b.2
following the procedures described Example 2. Reaction of
pyrazolone 1b.1 with benzyl alcohol 6b with Ph.sub.3P/DEAD produces
5a. The protecting group MOM- is then removed with TFA to give
phenol 5b. Treatment of phenol with triflate methyl phosphonic acid
dibenzyl ester 4a to give phosphonate 3b.11, which is also
converted to 3b.2 type of compound.
Example 3
[0872] ##STR264## ##STR265## Urea-PETT-Like Phosphonate NNRTI
Compounds
[0873] The present invention include describes Urea-PETT-like
phosphonate NNRTI compounds and methods for their preparation.
Urea-PETT-like phosphonate NNRTI compounds are potential anti-HIV
agents.
[0874] A link group includes a portion of the structure that links
two substructures, one of which is urea-PETT class of HIV
inhibiting agents having the general formula shown above, the other
is a phosphonate group bearing the appropriate R and R1 groups. The
link has at least one uninterrupted chain of atoms other than
hydrogen.
[0875] Urea-PETT class of compound has demonstrated activity in
inhibiting HIV replication. The present invention provides novel
analogs of urea-PETT class of compound. Such novel urea-PETT
analogs possess all the utilities of urea-PETT and optionally
provide cellular accumulation as set forth below.
[0876] The intermediate phosphonate esters required for conversion
into the prodrug phosphonate moieties bearing amino acid, or
lactate esters are shown in FIG. 2.
[0877] Preparation of phosphonate analog type 2 is outlined in
Scheme 1. Urea-PETT 1 is described in U.S. Pat. No. 6,486,183 and
J. Med. Chem. 1999, 42, 4150-4160. Conversion of 1 to the desired
phosphonate analogs is realized by treatment of 1 with the
phosphonate reagent 5 under suitable conditions. For example
(Example 1), urea-PETT 1a is activated as it p-nitro-phenol
carbonate by reacting with bis(4-nitrophenyl)carbonate. Reaction of
the resulting carbonate with amino ethyl phosphonate 5.1 in the
presence of base, for example, Hunig's base, affords the
phosphonate 2.1. ##STR266##
Example 1
[0878] ##STR267##
[0879] Scheme 2 shows of the preparation of the phosphonate linkage
of type 2 and 3 to urea-PETT. The hydroxyl group of urea-PETT 1 is
protected with a suitable protecting group, for example, trityl,
silyl, benzyl or MOM- etc to give 6 as described in Greene and
Wuts, Protecting Groups in Organic Synthesis, 3.sup.rd Edition,
John Wiley and Sons Inc. The resulting protected urea-PETT 6 is
first treated with a suitable base to remove the urea proton, the
product is then treated with 1 equivalent of a phosphonate reagent
5 bearing a leaving group such as, for example, bromine, mesyl,
tosyl etc to give the alkylated product 7 and 8. The phosphonates 7
and 8 are separated by chromatography and independently deprotected
using conventional conditions described in Greene and Wuts,
Protecting Groups in Organic Synthesis, 3.sup.rd Edition, John
Wiley and Sons Inc. p 116-121. For example (Example 2), urea-PETR 1
is protected as t-butyl dimethyl silyl ether 6a by reacting with
TBSCl and imidazole. Compound 6a, in DMF, is treated with sodium
hydride followed by one equivalent of bromomethyl phosphonic acid
dibenzyl ester 5.2 to give phosphonate 7a and 8a respectively.
phosphonates 7a and 8a are separated by chromatography, and then
independently deprotected by treatment with TBAF in an aprotic
solvent such as THF or acetonitrile to give 3a and 4a respectively
in which the linkage is a methylene group. Using the above
procedure but employing a different phosphonate reagent 5 in place
of 5.2, corresponding products 3 and 4 with different linking
groups are obtained. ##STR268##
Example 2
[0880] ##STR269## Nevaripine-Like Phosphonate NNRTI Compounds
[0881] The present invention describes methods for the preparation
of phosphonate analogs of nevaripine class of HIV inhibiting agents
shown in FIG. 1 that are potential anti-HIV agents.
[0882] A link group includes a portion of the structure that links
two substructures, one of which is nevapine class of HIV inhibiting
agents having the general formula shown above, the other is a
phosphonate group bearing the appropriate R and R1 groups. The link
has at least one uninterrupted chain of atoms other than hydrogen.
Nevirapine-type compounds are inhibitors of HIV RT, and nevirapine
is currently used in clinical for treatment of HIV infection and
AIDs. The present invention provides novel analogs of nevirapine
class of compound. Such novel nevirapine analogs possess all the
utilities of nevirapine and optionally provide cellular
accumulation as set forth below.
[0883] The intermediate phosphonate esters required for conversion
into the prodrug phosphonate moieties bearing amino acid, or
lactate esters are shown in FIG. 2.
[0884] Compound 1 is synthesized as described in U.S. Pat. No.
5,366,972 and J. Med. Chem. 1991, 34, 2231. Preparation of
phosphonate analog 2 is outlined in Scheme 1 and 2. Amide 7 is
prepared as described in U.S. Pat. No. 5,366,972 and J. Med. Chem.
1998, 41, 2960-2971 and 2972-2984. Amide 7 is converted to
dipyridodizaepinone 10 following the procedures described in U.S.
Pat. No. 5,366,972 and J. Med. Chem. 1998, 41, 2960-2971 and
2972-2984. Namely, treatment of dipyridine amide 7 with base
provides the dipyridodizaepinone 8. Alkylation of the amide N-- is
achieved with base and alkyls bearing a leaving group, such as, for
example, bromide, iodide, mesylate etc. Displacement of chloride
with p-methoxybenzylamine, followed by removal of the
p-methoxybenzyl group affords amine 10. The amine group serves as
the attachment site for introduction of a phosphonate group.
Reaction of amine 10 with reagent 6 provides 2 with different
linker attached to amine.
[0885] Alternatively (Scheme 2), amine 10 is transformed to phenol
11 as described in J. Med. Chem. 1998, 41, 2972-2984, many examples
are also described in R. C. Larock, Comprehensive Organic
Transformation, John Wiley & Sons, 2.sup.nd Ed. the hydroxyl
group then serves as the linking site for a suitable phosphonate
group. Reaction of amine 11 with reagent 6 provides 2 with
different linker attached to hydroxyl group. For example (Example
1), amide 7a, obtained as described in J. Med. Chem. 1998, 41,
2960-2971 and 2972-2984, is treated with sodium
hexamethyldisilazane in pyridine to give diazepinone 9a. Amine 10a
is synthesized from 9a by displacement of the chloride with
p-methoxybenzylamine followed by removal of the protecting group of
amine. Diazotization of the amine 10a and subsequent in situ
conversion to hydroxy yields phenol 11a. Phosphonate with different
linker is then able to be attached at the phenol site. For example,
the phenol is activated as p-nitro-benzyl carbonate, subsequent
treatment with amino ethyl phosphonate 6.1 in the presence of
Hunig's base affords carbamate 2b.1. ##STR270## ##STR271##
Example 1
[0886] ##STR272##
[0887] Scheme 2 shows the preparation of phosphonate conjugates
compounds type 3 in FIG. 2. Diazapinone 13 is obtained from
dipyrido amide 7 following the procedure described in J. Med. Chem.
1998, 41, 2960-2971 and 2972-2984, which is then converted to
aldehyde 14 and phenol 14a following the procedures in the same
literature. Aldehyde 14 and phenol 14a are then converted to 3a and
3b respectively by reacting with suitable phosphonate reagents 6.
Amine 14b is obtained using the method described in J. Med. Chem.
1998, 41, 2960-2971, which is converted to phosphonate 3c.
[0888] For example (Example 2), amine 14b.1, obtained by using the
procedures described in J. Med. Chem. 1998, 41, 2960-2971, reacts
with phosphonic acid dibenzyl ester 6.2 under reductive amination
conditions to give phosphonate 3c.1. ##STR273##
Example 2
[0889] ##STR274##
[0890] Preparation of phosphonate analog type 4 in FIG. 2 is shown
in Scheme 3. nevirapine analog 1 is dissolved in suitable solvent
such as, for example, DMF or other protic solvent, and treated with
the phosphonate reagent 9, bearing a leaving group, such as, for
example, bromine, mesyl, tosyl, or triflate, in the presence of a
suitable organic or inorganic base, to give phosphonate 4. For
example, 1 was dissolved in DMF, is treated with sodium hydride and
1 equivalent of bromomethyl phosphonic acid dibenzyl ester 6.2 to
give phosphonate 4a in which the linkage is a methylene group.
##STR275##
Example 3
[0891] ##STR276##
[0892] Scheme 4 shows the preparation of phosphonate type 5 in FIG.
2. Amine 15 is prepared according to the procedures described in
U.S. Pat. No. 5,366,972 and J. Med. Chem. 1998, 41, 2960-2971 and
2972-2984. Substituted alkyl amines, which bearing a protected
amino or hydroxyl group, or a precursor of amino group, are used in
displacement of alkyls described in U.S. Pat. No. 5,366,972 and J.
Med. Chem. 1998, 41, 2960-2971 and 2972-2984, react with the
chloropyridine 15 in the presence of base to give amine 16. These
alkyl amines include but not limit to examples in Scheme 4. These
substituted alkyl amines are obtained from commercial sources by
protection of the amino or hydroxyl group with a suitable
protecting group, for example trityl, silyl, benzyl etc as
described in Greene and Wuts, Protecting Groups in Organic
Synthesis, 3.sup.rd Edition, John Wiley and Sons Inc. Formation of
the diazepinone ring in the presence of a suitable base produces
17. Removal of protecting group or conversion to amine group from a
precursor, such as a nitro group, followed by treatment with
reagent 6 yield 5a. For example (Example 4), the hydroxyl group of
2-hydroxy ethylamine is protected as its MOM-ether (19). Selective
displacement of 2'-chloro substituent of the pyridinecarboxamide
ring with substituted ethylamine 19 produce 16a. Formation of the
diazepinone ring in the presence of sodium hexamethyldisilazane
affords 17a. MOM- is then removed to provide alcohol 18a. The
hydroxyl group is then used for attaching the phosphonate group.
The alcohol is first converted to carbonate by reacting with
bis(4-nitrobenzyl)carbonate, subsequent treatment of the resulting
carbonate with aminoethyl phosphonate 6.2 provides phosphonate
5a.1. ##STR277##
Example 5
[0893] ##STR278## Quinazolinone-Like Phosphonate NNRTI
Compounds
[0894] The present invention describes methods for the preparation
of phosphonate analogs of quinazolinones shown in FIG. 1 that are
potential anti-HIV agents.
[0895] A link group includes a portion of the structure that links
two substructures, one of which is quinazolinones having the
general formula shown above, the other is a phosphonate group
bearing the appropriate R and R.sub.4 groups. The link has at least
one uninterrupted chain of atoms other than hydrogen.
[0896] Quinozolinone class of compound, act as NNRTI, has
demonstrated to inhibit HIV replication. DPC-083, one of
representative analogs of this class of compounds, is in clinical
phase II studies for treatment of HIV infection and AIDs. The
present invention provides novel analogs of quinozolinone class of
compound. Such novel quinozolinone analogs possess all the
utilities of quinozolinone and optionally provide cellular
accumulation as set forth below.
[0897] The intermediate phosphonate esters required for conversion
into the prodrug phosphonate moieties bearing amino acid, or
lactate esters are shown in FIG. 2.
[0898] Preparation of phosphonate 2 is outlined in Scheme 1.
Quinazolinone 1, synthesized as described in Patent EP0530994,
WO93/04047 and U.S. Pat. No. 6,423,718, is dissolved in suitable
solvent such as, for example, DMF or other protic solvent is first
treated with a suitable base to remove the urea proton, the product
is then treated with 1 equivalent of a phosphonate reagent 8
bearing a leaving group such as, for example, bromine, mesyl tosyl
etc to give the alkylated product 2 and 3. The phosphonates 2 and 3
are separated by chromatography. For example, 1 is dissolved in
DMF, is treated with sodium hydride and 1 equivalent of bromomethyl
phosphonic acid diethyl ester 8.1 prepared to give quinazolinone
phosphonate 2 in which the linkage is a methylene group. Using the
above procedure but employing different phosphonate reagents 8 in
place of 8.1, the corresponding products 2 and 3 are obtained
bearing different linking group. ##STR279##
Example 1
[0899] ##STR280##
[0900] Scheme 2 shows the preparation of phosphonate analogs type 2
and 3 attached with an alternative way. Quinazolinone 1, dissolved
in a suitable solvent such as, for example, DMF or other protic
solvents, is first treated with a suitable base to remove the urea
proton, the product is then treated with 1 equivalent of reagent B,
which bears a leaving group such as, for example, bromine, mesyl,
tosyl etc, to give the alkylated product 7a and 7b. Compound B
possesses a protected NH.sub.2 or OH group, or a precursor for
them. The alkylated product 7a and 7b are separated by
chromatography. Protecting group is then removed, and the resulting
alcohol or amine then reacts with reagent 8 to afford 2b and 3b
respectively.
[0901] Alternatively (Scheme 3), alkylation of 1 with bromoacetate
provides 9a and 9b, which are separated by chromatography. The
ester group of 9 is reduced to alcohol to give 10. The alcohol 11
is also transformed to amine 12 under conventional conditions, many
examples are described in R. C. Larock, Comprehensive Organic
Transformation, John Wiley & Sons, 2.sup.nd Ed. The hydroxyl
group of 10 and amino group of 12 then serve as the attachment site
for linking phosphonate to provide 2c. Similarly, ester 10a is
converted to phosphonate 3c following the procedures of
transformation of 10 to 2c. ##STR281## ##STR282##
[0902] Scheme 4 shows the preparation of quinazolinone-phosphonate
conjugates type 4 in FIG. 2. Substituted aniline 6 with a
functional group Z, which is bearing a protected alcohol or amino
group, or protected alcohol or amino alkyl, is converted to
trifluoromethyl phenyl ketone 13, which is subsequently converted
to quinozolinone 14a, following the procedure described in U.S.
Pat. No. 6,423,718. Deprotection of the protecting group, followed
by reacting with reagents 8 under suitable conditions give the
desired the phosphonate 4a. Quinazoline 14b, prepared according to
U.S. Pat. No. 6,423,718, is converted to phosphonate 4b by reacting
with phosphonate reagent 8 directly (R.sub.3.dbd.NH.sub.2), or
after deprotection (R.sub.3.dbd.OMe) under the condition such as
for example, BCl.sub.3, many examples are described in Greene and
Wuts, Protecting Groups in Organic Synthesis, 3r Edition, John
Wiley and Sons Inc. Synthesis of compound 6 is described in Scheme
5. ##STR283##
[0903] Scheme 5 shows compounds 6 are obtained through modification
of commercial available material 2-halo-5-nitroaniline, or
5-halo-2-nitroaniline (6.0a). The amino group of 6.0a is first
protected with a suitable protecting group, for example trityl Cbz,
or Boc etc as described in Greene and Wuts, Protecting Groups in
Organic Synthesis, 3r' Edition, John Wiley and Sons Inc. Reduction
of the nitro group of 6.1a with a reducing agent, many examples are
described in R. C. Larock, Comprehensive Organic Transformation,
John Wiley & Sons, 2.sup.nd Ed, gives 6.1b, which is then used
in the transformation described in Scheme 4.
[0904] The amino group of 6.0a is converted to hydroxyl group to
give 6.2a by established procedures, for example, diazotization
followed by treatment with H.sub.2O/H.sub.2SO.sub.4, many examples
are described in R. C. Larock, Comprehensive Organic
Transformation, John Wiley & Sons, 2.sup.nd Ed. The hydroxyl
group is then protected with a suitable protecting group, for
example trityl ethers, silyl ethers, methoxy methyl ethers etc as
described in Greene and Wuts, Protecting Groups in Organic
Synthesis, 3.sup.rd Edition, John Wiley and Sons Inc. The nitro
group of the resulting compound is then reduced with the above
mentioned methods to give 6.2b, which is then used in the
transformation described in Scheme 4.
[0905] The hydroxyl or amino alkyls are obtained using the
following methods. The amino group of 6.0a is converted to nitrile
6.3a with the known method, for example diazotization followed by
treatment with cuprous cyanide, many examples are described in R.
C. Larock, Comprehensive Organic Transformation, John Wiley &
Sons, 2.sup.nd Ed. The nitrile group is then selectively reduced
with a reducing agent, many examples are described in R. C. Larock,
Comprehensive Organic Transformation, John Wiley & Sons,
2.sup.nd Ed, to give amine 6.3b. With the mentioned methods above,
the amino group is protected and nitro group is reduced
respectively to give 6.3c. Alternatively, the nitrile 6.3a is
converted to acid 6.4a and the acid is subsequently reduced to
alcohol to give 6.4b using the examples described in R. C. Larock,
Comprehensive Organic Transformation, John Wiley & Sons, 2n Ed.
Similarly, protection of hydroxyl group followed by reduction of
nitro to amine gives 6.4c. Compound 6.3c and 6.4c are used in
Scheme 4 respectively.
[0906] The homologated hydroxyl or amino alkyls are obtained using
the following methods (Scheme 3). The acid 6.4a are extended to
acid 6.5a, which is transformed to nitrile 6.5b, these two
transformation are described in R. C. Larock, Comprehensive Organic
Transformation, John Wiley & Sons, 2.sup.nd Ed, Nitrile 6.5b is
converted to aniline 6.5c using the similar methods described
above. Alternatively, nitrile 6.5b is obtained by first convert
benzyl alcohol 6.4b to benzyl halide, then treated with CN--
nucleophile. Reduction of acid 6.5a provided alcohol 6.6b, which is
protected using the protecting groups described above to give the
required aniline 6.6c. Compound 6.5c and 6.6c are used in Scheme 4
respectively.
[0907] For example aniline 6.0a (Example 2) is treated with
NaNO.sub.2 in the presence of acid at 0.degree. C., then the
resulting mixture was heated in H.sub.2O to give phenol 6.2a. The
hydroxyl group is then protected as methoxyl methyl ether by
treating phenol 6.2a with MOMCl in the presence of Hunig's base to
yield 6.21b. Hydrogenation of nitrobenzene affords aniline 6a.
Aniline 6a is converted to phenyl trifluoromethyl ketone 13a.1,
which is subsequently transformed to quinazolinone analog 14a.1,
using the method described in U.S. Pat. No. 6,423,718. Deprotection
of the MOM-ether with trifluoroacidic acid provides phenol 15.
Treatment of 15, in acetonitrile, with triflate methyl phosphonic
acid dibenzyl ester 8.2 in the presence of Cs.sub.2CO.sub.3 gives
4a.1. Alternatively, reaction of phenol 15 with ethylenediol under
the Mitsunobu condition produces 16. Hydroxyl group of 16 as
activated as carbamate, subsequent treatment with amino methyl
phosphonate 8.3 affords phosphonate analog 4a.2.
[0908] Example 3 shows 2-chloro-5-nitro aniline 6.0b transformed to
nitrite 6.31a by reacting with NaNO.sub.2 and then CuCN
subsequently. Hydrolysis of nitrile 6.31a gives acid 6.41a.
Treatment of 6.41a with ClCOOEt in the presence of base at
0.degree. C. followed by CH.sub.2N.sub.2 provides diazoketone,
which is converted to methyl ester 6.51a upon treating with silver
perchlorate in methanol. The ester group is then reduced to give
alcohol, which is protected as MOM-ether to provide 6.61c. The
nitro group is then reduced to amine to afford 6b. Aniline 6b is
converted to quinazolinone analog 14 using the method described in
U.S. Pat. No. 6,423,718. Deprotection of the MOM-ether with
trifluoroacidic acid provide alcohol 16. The aldehyde 17 is
obtained by oxidation of alcohol. Reductive amination of 17 with
amino ethyl phosphonate 8.4 afford analog 4a.3. ##STR284##
Example 2
[0909] ##STR285##
Example 3
[0910] ##STR286## ##STR287##
[0911] Preparation of phosphonate analog type 5 from quinazolinone
1 is outlined in Scheme 6. Quinazolinone 1, which R.sub.1 contains
OH, or NH.sub.2 or NHR.sub.1' as the attachment site for connecting
phosphonate, reacts with reagent 8 under suitable conditions to
provide phosphonate analog 5. For example (Example 4),
Quinozalinone 1b.1, obtained as described in U.S. Pat. No.
6,423,718, is treated with phosphonate reagents 8.2 in the presence
of Cs.sub.2CO.sub.3, give phosphonate 5a. ##STR288##
Example 4
[0912] ##STR289## Efavirenz-Like Phosphonate NTT Compounds
[0913] The present invention includes efavirenz-like phosphonate
NNRTI compounds and methods for the preparation of efavirenz
phosphonate analogs shown in FIG. 1.
[0914] A link group includes a portion of the structure that links
two substructures, one of which is efavirenz having the general
formula shown above, the other is a phosphonate group bearing the
appropriate R and R.sub.1 groups. The link has at least one
uninterrupted chain of atoms other than hydrogen.
[0915] Efavirenz and its analogs have demonstrated therapeutic
activity against HIV replication, and efavirenz is currently used
in clinical for treatment of HIV infection and AIDS. The present
invention provides novel analogs of efavirenz. Such novel efavirenz
analogs possess all the utilities of efavirenz and optionally
provide cellular accumulation as set forth below.
[0916] The intermediate phosphonate esters required for conversion
into the prodrug phosphonate moieties bearing amino acid, or
lactate esters are shown in FIG. 2.
[0917] Compound 1 can be synthesized as described in U.S. Pat. No.
5,519,021. Preparation of compound 2 from efavirenz 1 is outlined
in Scheme 1. Efavirenz 1 is dissolved in suitable solvent such as,
for example, DMF or other protic solvent, and treated with the
phosphonate reagent 5 in the presence of a suitable organic or
inorganic base. For example, 1 is dissolved in DMF, is treated with
sodium hydride and 1 equivalent of triflate methyl phosphonic acid
dibenzyl ester 5.1 prepared to give EFV phosphonate 2 in which the
linkage is a methylene group. Using the above procedure but
employing different phosphonate reagents 5 in place of 5.1, the
corresponding products 2 are obtained bearing different linking
group. ##STR290##
Example 1
[0918] ##STR291##
[0919] Scheme 2 shows the preparation of EFV-phosphonate conjugates
compounds 3 in FIG. 2. p-Chloro aniline with functional group Z,
which bears a protected alcohol or amino group, or protected
alcohol or amino alkyl, is converted to compound 7 following the
procedure described in U.S. Pat. No. 5,519,021. Deprotection of the
protecting group, followed by reacting with reagent 5 in the above
mentioned conditions give the desired the compound 3. As shown in
Scheme 3, compounds 6 are obtained through modification of
commercial available material 2-chloro-5-nitroaniline, or
5-chloro-2-nitroaniline (6.0a). ##STR292##
[0920] The amino group of 6.0a is first protected with a suitable
protecting group (Scheme 3), for example trityl Cbz, or Boc etc as
described in Greene and Wuts, Protecting Groups in Organic
Synthesis, 3.sup.rd Edition, John Wiley and Sons Inc. Reduction of
the nitro group in 6.1a with a reducing agent, many examples are
described in R. C. Larock, Comprehensive Organic Transformation,
John Wiley & Sons, 2.sup.nd Ed, give 6.1b, which is then used
in the transformation described in Scheme 2.
[0921] Alternatively, the amino group of 6.0a is converted to
hydroxyl group to give 6.2a by established procedures, for example,
diazotization followed by treatment with H.sub.2O/H.sub.2SO.sub.4,
many examples are described in R. C. Larock, Comprehensive Organic
Transformation, John Wiley & Sons, 2.sup.nd Ed. The hydroxyl
group is then protected with a suitable protecting group, for
example trityl ethers, silyl ethers, methoxy methyl ethers etc as
described in Greene and Wuts, Protecting Groups in Organic
Synthesis, 3.sup.rd Edition, John Wiley and Sons Inc. The nitro
group of the resulting compound is then reduced with the above
mentioned methods to give 6.2b, which is then used in the
transformation described in Scheme 2.
[0922] The hydroxyl or amino alkyls are obtained using the
following methods. The amino group in 6.0a is converted to nitrile
6.3a with the known method, for example diazotization followed by
treatment with cuprous cyanide, many examples are described in R.
C. Larock, Comprehensive Organic Transformation, John Wiley &
Sons, 2.sup.nd Ed. The nitrile group is then selectively reduced
with a reducing agent, many examples are described in R. C. Larock,
Comprehensive Organic Transformation, John Wiley & Sons,
2.sup.nd Ed, to give amine 6.3b. With the mentioned methods above,
the amino group is protected and nitro group is reduced
respectively to give 6.3c. In addition, the nitrile 6.3a is
converted to acid 6.4a and the acid is subsequently reduced to
alcohol to give 6.4b, and the reduction of nitro to amine give
6.4c, using the methods described in R. C. Larock, Comprehensive
Organic Transformation, John Wiley & Sons, 2.sup.nd Ed. Both
6.3c and 6.4c used in the transformation described in Scheme 2. The
homologated hydroxyl or amino alkyls are obtained using the
following methods (Scheme 3). The acid 6.4a are extended to acid
6.5a, which is transformed to nitrile 6.5b, these two
transformation are described in R. C. Larock, Comprehensive Organic
Transformation, John Wiley & Sons, 2.sup.nd Ed, Nitrile 6.5b is
converted to aniline 6.5c using the similar methods described
above. Alternatively, nitrile 6.5b is obtained by first convert
benzyl alcohol 6.4b to benzyl halide, then treated with CN--
nucleophile. Reduction of acid 6.5a provided alcohol 6.6b, which is
protected using the protecting groups described above to give the
required aniline 6.6c. Both 6.5c and 6.6c used in the
transformation described in Scheme 2.
[0923] For example aniline 6.0a (Example 2) is treated with
NaNO.sub.2 in the presence of acid at 0.degree. C., then the
resulting mixture was heated in H.sub.2O to give phenol 6.2a. The
hydroxyl group is then protected as methoxyl methyl ether by
treating phenol 6.2a with MOMCl in the presence of Hunig's base to
yield 6.21b. Hydrogenation of nitrobenzene affords aniline 6.2a.
Aniline 6a is converted to efavirenz analog 7.1. Deprotection of
the MOM-ether with trifluoroacidic acid provides phenol 8.
Treatment of 8 in acetonitrile with
(trifluorosulfonylmethyl)-phosphonic acid dibenzyl ester 5.1 in the
presence of Cs.sub.2CO.sub.3 gives 3a.
[0924] In Example 3, 2-chloro-5-nitro aniline 6.0b is transformed
to nitrile 6.31a by reacting with NaNO.sub.2 and then CuCN
subsequently. Hydrolysis of nitrile 6.31a gives acid 6.41a.
Treatment of 6.41a with ClCOOEt in the presence of base at
0.degree. C. followed by CH.sub.2N.sub.2 provides diazoketone,
which is converted to methyl ester 6.51a upon treating with silver
perchlorate in methanol. The ester group is then reduced to give
alcohol, which is protected as MOM-ether to provide 6.61c. The
nitro group is then reduced to amine to afford 6b. Aniline 6a is
converted to efavirenz analog 7.1. Deprotection of the MOM-ether
with trifluoroacetic acid provides phenol 9. The aldehyde 10 is
obtained by oxidation of alcohol. Reductive amination of 10 with
agent 5.2 affords analog 3b. ##STR293##
Example 2
[0925] ##STR294##
Example 3
[0926] ##STR295## ##STR296##
[0927] Preparation of compound 2 from efavirenz 1 is outlined in
Scheme 4. Compound 12, obtained as described in U.S. Pat. No.
5,519,021, reacting with Grignard reagent, generated from protected
acetylene 11 following the procedure described in U.S. Pat. No.
5,519,021, gives compound 13a. The hydroxyl group in 11 is
protected as its silyl ether, trityl ether etc. Removal of the
protecting group of 13a yields alcohol 14a. Alkylation of 14a with
agent 5 affords phosphonate 4.1. Alternatively, compound 15,
obtained as described in U.S. Pat. No. 5,519,021, reacts with
aldehyde or ketone to give alcohol 14b, which is converted to
analog 4b using the conditions described above. Amine 14c is
obtained from alcohol 14b under the standard conditions. Amine 14c
is converted to phosphonate 4c either by reacting with agent or
reductive amination with a phosphonate reagents containing an
aldehyde group. For example, treatment of compound 14 with n-BuLi
followed by paraformaldehyde gives alcohol 14b.1. Treatment of
alcohol 14b.1 with Mg(OtBu).sub.2 followed by phosphonate provides
phosphonate 4.2b. ##STR297##
Example 4
[0928] ##STR298## Benzophenone-Like Phosphonate NNRTI Compounds
[0929] The present invention describes methods for the preparation
of phosphonate analogs of benzophenone class of HIV inhibiting
pyrimidines shown in FIG. 1 that are potential anti-HIV agents.
[0930] A link group includes a portion of the structure that links
two substructures, one of which is benzophenone class of HIV
inhibiting agents having the general formula shown above, the other
is a phosphonate group bearing the appropriate R and R.sub.3
groups. The link has at least one uninterrupted chain of atoms
other than hydrogen.
[0931] Benzophenone class of compounds has shown to be inhibitors
of HIV RT. The present invention provides novel analogs of
benzophenone class of compound. Such novel benzophenone analogs
possess all the utilities of benzophenone and optionally provide
cellular accumulation as set forth below.
[0932] The intermediate phosphonate esters required for conversion
into the prodrug phosphonate moieties bearing amino acid, or
lactate esters are shown in FIG. 2.
[0933] Preparation of phosphonate analog 4 is outlined in Scheme 1.
Benzophenone 8 is obtained from Freidel-Crafts reaction of
substituted benzoyl chloride 7 and 4-chloro-phenol methyl ether
which bearing a protected amine or hydroxyl group Z. Phenol ether
is obtained by selective protection of commercially available
4-chlorophenol substituted with amino- or hydroxyl group. Benzoyl
chloride is obtained either from commercial sources or prepared
from commercial available benzoic acid. Benzophenone 8 is also
obtained from oxidation of the corresponding alcohol, which in turn
is obtained from the reaction of benzaldehyde and anion. Removal of
methyl provides phenol 9. Alkylation of phenol with bromoacetate
such as ethyl bromoacetate affords ester 10. The ester is then
converted to acid. Formation of amide 12 from acid 11 and aniline
10 is achieved following the standard amide formation methods, many
examples are described in R. C. Larock, Comprehensive Organic
Transformation, John Wiley & Sons, 2.sup.nd Ed. Removal of the
protecting group of Z followed by reacting with reagent 6 affords
phosphonate analog 4a.
[0934] For example (Example 1), commercially available
3-cyanobenzoyl chloride is treated with trichloroaluminum followed
by 3,4-dimethoxy chlorobenzene to give benzophenone 8a. Treatment
of 8 with BCl.sub.3 removes the methyl to give diphenol, which is
selectively protected as its mono MOM-ether to give 9a. Alkylation
of phenol 9a with ethyl bromoacetate gives ester 10a. Hydrolysis of
the ester affords acid 1a. Coupling if the acid Ha with aniline
produces 12a. The MOM-group is then removed to yield phenol 12b.
Phenol is then activated as its 4-nitro-phenyl carbonate by
reacting with bis(4-nitro-phenyl)carbonate, which is subsequently
treated with aminoethyl phosphonate to give 4a.1.
[0935] Alternatively (Scheme 2), amine 10 is transformed to phenol
11 as described in, the hydroxyl group is then serves as the
linking site for a suitable phosphonate group. ##STR299##
Example 1
[0936] ##STR300##
[0937] Scheme 2 shows the preparation of phosphonate analog type 5.
Benzophenone 11b reacts with aniline 14, bearing a protect hydroxyl
or amino group, gives amide 13. Formation of amide 13 from acid 11
b and aniline 14 is achieved following the standard amide formation
methods, many examples are described in R. C. Larock, Comprehensive
Organic Transformation, John Wiley & Sons, 2.sup.nd Ed. Removal
of the protecting group of Z followed by reacting with reagent 6
affords phosphonate analog 5a. For example (Example 2), acid 11b
couples with aniline 14 provides amide 13a. The MOM-group is then
deprotected with TFA to afford phenol 13b, which is then coupled
with hydroxy ethyl phosphonic acid dibenzyl ester in the presence
of Ph3P/DEAD to give phosphonate 5a. Protected aniline 14a is
obtained by treating the commercially available 4-amino-m-cresol
with MOMCl in the presence of base, for example Hunig's base.
##STR301##
Example 2
[0938] ##STR302## Pyrimidine-Like Phosphonate NNRTI Compounds
[0939] The present invention includes Pyrimidine-like phosphonate
NNRTI compounds. The present invention also includes methods for
the preparation of phosphonate analogs of TMC-125 and TMC-120 class
of HIV inhibiting pyrimidines as shown in FIG. 1 which are
potential anti-HIV agents.
[0940] A link group includes a portion of the structure that links
two substructures, one of which is TMC-120 and TMC-125 class of
pyrimidines having the general formula shown above, the other is a
phosphonate group bearing the appropriate R and R1 groups. The link
has at least one uninterrupted chain of atoms other than
hydrogen.
[0941] TMC-125 and TMC-120 class of pyrimidines have demonstrated
to be potent in inhibition of HIV replication. Both TMC-125 and
TMC-120 are currently in clinical phase II studies for treatment of
HIV infection and AIDs. The present invention provides novel
analogs of TMC-120 and TMC-125 class of compound. Such novel
TMC-120 and TMC-125 class analogs possess all the utilities of
TMC-120 and TMC-125 class and optionally provide cellular
accumulation as set forth below.
[0942] The intermediate phosphonate esters required for conversion
into the prodrug phosphonate moieties bearing amino acid, or
lactate esters are shown in FIG. 2.
[0943] Compounds 1 and 2 can be synthesized as described in U.S.
Pat. No. 6,197,779 and WO 0027825. Preparation of phosphonate
analog 3 and 7 is outlined in Scheme 1. TMC-125 1 is dissolved in
suitable solvent such as, for example, DMF or other protic solvent,
and treated with the phosphonate reagent 9, bearing a leaving
group, such as, for example, bromine, mesyl tosyl; or
trifluoromethanesulfonyl in the presence of a suitable organic or
inorganic base, either 3a or 7a is obtained as the major product
depending on the base. For example, 1 was dissolved in DMF, is
treated with n-butyl lithium and 1 equivalent of triflate methyl
phosphonic acid dibenzyl ester 9.1 prepared to give phosphonate
3a.1 as the major product. Alternatively, treatment of 1 with 9.1
in acetonitrile in the presence of triethylamine provides 7a.1 as
the major product. The above procedure provides phosphonate analog
3 in which the linkage is a methylene group. Using the above
procedure but employing different phosphonate reagents 9 in place
of 9.1, the corresponding products 3 and 7 are obtained bearing
different linking group. ##STR303##
Example 1
[0944] ##STR304##
[0945] Scheme 2 shows the preparation of phosphonate conjugates
compounds type 3 and 8 in FIG. 2. TMC-120 2 is treated with base,
and subsequently treated with phosphonate reagent 9 bearing a
leaving group, such as, for example, bromine, mesyl, tosyl, or
trifluoromethanesulfonyl. The alkylated products are then separated
by chromatography. For example (Example 2), treatment of TMC-120 2
with NaH in DMF, followed by bromomethyl phosphonic acid dibenzyl
ester 9.2 gives phosphonate 3b.1 and 8a.1. The mixture of
phosphonates 3b.1 and 8a.1 is separated by chromatography to give
pure 3b.1 and 8a.1 respectively. ##STR305##
Example 2
[0946] ##STR306##
[0947] Preparation of phosphonate analogs type 4 in FIG. 2 is shown
in Scheme 3, 4 and 5. Nitration of commercially available
3,5-dimethyl phenol 10 gives 11, subsequent reduction of the
resulting nitrobenzene 11 provide 12, many examples are described
in R. C. Larock, Comprehensive Organic Transformation, John Wiley
& Sons, 2.sup.nd Ed. The hydroxyl group of phenol 12 is
protected with a suitable protecting group, for example trityl
silyl, benzyl or MOM- etc to give 13 as described in Greene and
Wuts, Protecting Groups in Organic Synthesis, 3.sup.rd Edition,
John Wiley and Sons Inc. Treatment of 14 with 13 following the
procedures described in U.S. Pat. No. 6,197,779 and WO 0027825 give
15. Removal of the protecting group gives phenol 16. Reaction of
phenol 16 with phosphonate reagent 9 in the presence of base in a
protic solvent provides 4a. Nitration (Scheme 4) of commercially
available 2,6-dimethyl phenol provides 18. Reduction of nitro group
to amine, followed by protection of the resultant amine with
protecting group, for example, such as trityl, Boc, Cbz etc as
described in Greene and Wuts, Protecting Groups in Organic
Synthesis, 3.sup.rd Edition, John Wiley and Sons Inc. Treatment of
14a with 19 following the procedures described in U.S. Pat. No.
6,197,779 and WO 0027825 give 20. Phenol 21 is obtained by treating
20 with NH.sub.3 using the procedure described in U.S. Pat. No.
6,197,779 and WO 0027825, followed by removal of the protecting
group. Reaction of phenol 21 with phosphonate reagent 9 provides
4b. As shown in Scheme 5, the commercially available
2,6-dimethyl-4-cyano-phenol 22 is reduced to benzyl amine, and the
resultant amine is protected as described above. Phenol 23 is
converted to phosphonate 4c following the procedure described above
for the transformation 19 to 4b, just replace 19 with 23. For
example (Example 3), nitration of 2,6-dimethyl phenol with
HNO.sub.3 in H.sub.2SO.sub.4 gives phenol 18. The nitro group is
reduced under catalytic hydrogenation condition, and subsequent
protection of the resulting amine with Boc-gives phenol 19a.
Treatment of phenol 18 with sodium hydride, followed by reacting
the resulting sodium phenoxide with 13 in dioxane provides 20a.
Removal of the Boc- with TFA followed by treatment of the resulting
product with NH.sub.3 in isopropyl alcohol according to U.S. Pat.
No. 6,197,779 and WO 0027825 replaces the Cl- with NH.sub.2 group
to give 21. The amine group in the phenyl ring is used as
attachment site for introduction of phosphonate. Reductive
amination of amine with aldehyde 9.3 provides 4b.1. Treatment of 21
with p-nitro-phenyl carbonate, followed by aminoethyl phosphonate
9.4 affords urea linker 4b.2. ##STR307## ##STR308## ##STR309##
Example 3
[0948] ##STR310##
[0949] Scheme 6 shows the preparation of phosphonate type 6 in FIG.
2. Substituted 4-amino-benzonitriles 24 or 27, which bearing a
protected amino or hydroxyl group, or a precursor of amino group,
are used in the replacement of 4-amino-benzonitrile for the
preparation of TMC-125 and TMC-120 class of analogs as described in
U.S. Pat. No. 6,197,779 and WO 0027825. TMC-120 and TMC-125 analogs
25 and 29 are thus obtained. Removal of protecting group or
conversion to amine group from a precursor, such as a nitro group,
provide 26 or 30 respectively. Treatment of 26 and/or 30 with
reagent 9 yield 6a and/or 6b respectively. For example (Example 4),
the hydroxyl group of 4-amino-2-hydroxy-benzonitrile 27a is
protected as its MOM-ether to give 28a. Following the procedure in
U.S. Pat. No. 6,197,779 and WO 0027825, 28a is converted to TMC-120
analog 29a. Removal of MOM-ether with TFA provides phenol 30a,
which is treated with trifluoromethylsulfonyl phosphonic acid
benzyl ester together with Cs.sub.2CO.sub.3 in acetonitrile affords
phosphonate analog 6b.1. ##STR311##
Example 4
[0950] ##STR312##
[0951] Preparation of phosphonate analog type 5 in FIG. 2 is shown
in Scheme 7. Substituted aniline, which bearing a protected amino
or hydroxyl group, is converted to TMC-120 or TMC-125 analogs
following the procedures described in U.S. Pat. No. 6,197,779 and
WO 0027825. Removal of the protecting group gives analog 34. The
amino or hydroxyl group in 33 serves as attachment site for
introduction of phosphonate. Reaction of 33 with reagent 9 provides
5a. For example (Example 5), commercially available
2-amino-2,4,6-trimethyl-aniline is selectively protected as
Boc-carbamate. Reaction of 32a with 13 provides 33a. Removal of Boc
with TFA affords aniline 34a. Reductive amination with reagent 9.2
yields phosphonate analog 5a.1. ##STR313##
Example 6
[0952] ##STR314## SJ3366-Like Phosphonate NNRTI Compounds
##STR315##
[0953] SJ3366 is described in U.S. Pat. No. 5,922,727. The present
invention provides novel phosphonate analogs of SJ3366 which
possess all the utilities of SJ3366 and optionally provide cellular
accumulation as set forth below.
[0954] The present invention also relates to the delivery of
SJ3366-like phosphonate compounds which are optionally targeted for
site-specific accumulation in cells, tissues or organs. More
particularly, this invention relates to analogs of SJ3366 which
comprise SJ3366 linked to a PO(R.sub.1)(R.sub.2) moiety.
[0955] SJ3366 may be covalently bonded directly or indirectly by a
link to the PO(R.sub.1)(R.sub.2) moiety. An R group of the
PO(R.sub.1)(R.sub.2) moiety can possibly be cleaved within the
desired delivery site, thereby forming an ionic species which does
not exit the cell easily. This may cause accumulation within the
cell and can optionally protect the SJ3366 analog from exposure to
metabolic enzymes which would metabolize the analog if not
protected within the cell. The cleavage may occur as a result of
normal displacement by cellular nucleophiles or enzymatic action,
but is preferably caused to occur selectively at a predetermined
release site. The advantage of this method is that the SJ3366
analog may optionally be delivered site-specifically, may
optionally accumulate within the cell and may optionally be
shielded from metabolic enzymes.
[0956] The following examples illustrate various aspects of the
present invention and are not to be construed to limit the types of
analogs that may employ this strategy of linking SJ3366 or an
SJ3366 analog to a PO(R.sub.1)(R.sub.2) moiety in any manner
whatsoever.
[0957] Preparation of compounds of type A require a link which can
react with SJ3366 or an intermediate or analog thereof, to result
in a covalent bond between the link and the drug-like compound. The
link is also attached to the phosphorous containing moiety as shown
in an example of type A, namely A1.
[0958] Examples of type A can be made by 1-alkylation of the
3-phenacyl derivatives 35 and 36 (synthesis described in J. Med.
Chem. 1995, 38, 1860-2865, and so numbered 35 and 36 therein) with
alkyl halide containing links followed by deprotection of the
3-phenacyl group. An example synthesis is as follows, and is shown
in Scheme 1.
6-Benzyl-5-isopropyl-3-(2-phenyl-allyl)-dihydro-pyrimidine-2,4-dione,
as prepared in J. Med. Chem. 1995, 38, 15, 2860-2865, is treated
analogously to the reference article authors' treatment in
preparing their compounds 37-40, but in the case of compound A1,
commercially available chloromethyldiethylphosphonate is used as
the alkylating agent. Alternatively the link is connected by
starting with the same drug-like compound and using a triflated
link. The triflated link is prepared, for example, by reaction of
allyl bromide with dibenzylphosphite and potassium carbonate in
acetonitrile at 65.degree. C. Ozonolysis of the double bond
followed by treatment with sodium borohydride would provide the
alcohol, which could then be reacted with triflic anhydride with
2,6 lutidine in dichloromethane to produce the triflate. The
triflated material could then be attached by stirring it with, for
example
6-Benzyl-5-isopropyl-3-(2-phenyl-allyl)-dihydro-pyrimidine-2,4-dione
with 2,6 lutidine or other base in an appropriate solvent such as
acetone. This procedure will provide examples A1 and A2. ##STR316##
##STR317##
[0959] Scheme 1 can be extended to include analogs with various
moieties at C6 in addition to substituted benzyl rings. For
example, the LDA treatment described in J. Med. Chem. 1995, 38, 15,
2860-2865 followed by disulfide addition provides intermediates
which can then be treated similarly to those in scheme 1 to install
the link PO(R.sub.1)(R.sub.2) at the 1 position ##STR318##
[0960] Scheme 3 also demonstrates a method to prepare analogs with
oxygen or nitrogen at Y.sub.2 attached to the 6 position. This
method is explained fully in J. Med. Chem. 1991, 34, 1, 349-357.
Using this method allows for aryl and alkyl groups to be attached
to the 6 position by either oxygen or nitrogen. A specific example
is shown in the bottom row of the boxes in Scheme 7 below.
##STR319##
[0961] Alternatively the 5 position may be functionalized after the
nucleophile is appended by the TFA/water deprotection and
alkylation strategy shown in Scheme 2. Analogs with methylene, a
secondary alcohol or a ketone at the 6 position are readily
prepared following the LDA procedure in Scheme 2, but using
substituted or unsubstituted PhCOCl in place of a disulfide, as is
done in J. Med. Chem. 1991, 34, 1 page 351. The resultant ketone
can be converted to an oxime ether (Scheme 4), an ether (Scheme 5)
or reduced to a methylene (Scheme 6). Scheme 6 can be extended with
the deprotection and alkylation steps described in Scheme 2. The
methylene, secondary alcohol and ether are all described in J. Med.
Chem. 1991, 34, 1 page 349-357, and the oxime ether can be prepared
as described below (Scheme 4). ##STR320## ##STR321##
[0962] Alternatively the ketone containing compound could undergo
deprotection at the 1 position and attachment of the link
PO(R.sub.1)(R.sub.2) as in Scheme 2 above. ##STR322##
##STR323##
[0963] The above shown compounds could also have a reactive group
at the aryl or alkyl substituent on the 5 or the 6 position that
would allow for attachment of the PO(R.sub.1)(R.sub.2) group. These
reactive groups are protected by a protecting group, or be present
in the form of a masked functionality, such as the manner in which
a nitro group would mask an amine. Scheme 7 shows some more
representative examples of the many ways an attachment of a
PO(R.sub.1)(R.sub.2) is made. The chemistry involved is explained
above, except for the BBr3 demethylation, which is a common
procedure (J. F. W. McOmie and D. E. West, Org. Synth. Collect.
Vol. V, 412, (1973) for demethylating methoxyaryl rings. The
compounds in box A are treated with hydrogen gas and stirred in a
solvent such as ethanol or methanol with a suspension of 10%
palladium on carbon. The anilines or alcohols are then treated with
a triflated PO(R.sub.1)(R.sub.2) containing group as described
above. ##STR324## ##STR325## Delavirdine-Like Phosphonate NNRTI
Compounds
[0964] Diaromatic compounds refer to any diaromatic substituted
compound, more specifically, bis(heteroaryl) piperazine (BHAP),
more specifically
1{5-methanesulfonamidoindolyl-2-carbonyl}-4-{3-(1-methylethylamino)-2-pyr-
idinyl}piperazine as found in U.S. Pat. No. 5,563,142 claim 8
column 90 line 49-51, and pharmaceutically acceptable salts
thereof. ##STR326##
[0965] Preparation of compounds of type A, B, and C require a link
which can react with a drug-like compound which is either 1
{5-methanesulfonamidoindolyl-2-carbonyl}-4-{3-(1-methylethylamino)-2-pyri-
dinyl}piperazine or an intermediate thereof, to result in a
covalent bond between the link and the drug-like compound. The link
is also attached to the phosphorous containing moiety shown in
examples of type A, B and C, namely A1, B1 and C1. ##STR327##
[0966] Examples of type A can be made by reacting the aminoindole
NH.sub.2 of the immediate precursor to delavirdine
(1-[5-amidoindolyl-2-carbonyl]-4-[3-(1-methylethylamino)-2-pyridinyl]pipe-
razine, such as example 101 in U.S. Pat. No. 5,563,142, synthesis
described therein, with the phosphorous containing moiety having an
aldehyde as the reactive part of the link. The aldehyde and
NH.sub.2 group react through a reductive amination reaction, which
can be performed by stirring both reagents in, for example
dichloroethane, for approximately two hours and then adding acetic
acid and sodium cyanoborohydride, or by other standard methods
known to most organic chemists. Commercially available
aldehyde-containing phosphonates such as that shown in the below
scheme 1 can be used to prepare example A1.
[0967] This method may be extended to synthesize molecules with the
link attached at other positions on the indole phenyl ring by
following the procedures described in U.S. Pat. No. 5,563,142 but
substituting starting materials as relevant to obtain the indole
with the desired substitution pattern.
[0968] Examples of type B can be prepared by reacting the indole NH
of delavirdine with, for example, a link which contains an alkyl
chloride in the presence of KOH in DMSO as described in J. Med.
Chem. 34, 3, 1991, 1099-1110. The alkyl chloride link is for
example commercially available chloromethyl diethoxyphosphonate,
giving example B1. ##STR328##
[0969] Examples of type C can be made by reacting the secondary
amine of delavirdine with the phosphorous containing moiety having
an aldehyde as the reactive part of the link. The aldehyde and NH
group react through a reductive amination reaction, which can be
performed by stirring both reagents in, for example dichloroethane,
for approximately two hours and then adding acetic acid and sodium
cyanoborohydride, or by other standard methods known to most
organic chemists. In this example the aldehyde containing
phosphonate is commercially available. This procedure will provide
example C1. ##STR329## ##STR330##
[0970] The present invention provides novel analogs of
1{5-methanesulfonamidoindolyl-2-carbonyl}-4-{3-(1-methylethylamino)-2-pyr-
idinyl}piperazine. Such novel
1{5-methanesulfonamidoindolyl-2-carbonyl}-4-{3-(1-methylethylamino)-2-pyr-
idinyl}piperazine analogs possess all the utilities of
1{5-methanesulfonamidoindolyl-2-carbonyl)}{3-(1-methylethylamino)-2-pyrid-
inyl}piperazine and optionally provide cellular accumulation as set
forth below. ##STR331##
[0971] The present invention provides novel phosphonate analogs of
Emivirine and pharmaceutically acceptable salts thereof. Emivirine
is described in U.S. Pat. No. 5,461,060. Such novel Emivirine
analogs possess all the utilities of Emivirine and optionally
provide cellular accumulation as set forth below.
[0972] The present invention also relates to the delivery of
Emivirine-like phosphonate compounds which are optionally targeted
for site-specific accumulation in cells, tissues or organs. More
particularly, this invention relates to analogs of Emivirine which
comprise Emivirine linked to a PO(R.sub.1)(R.sub.2) moiety.
[0973] Emivirine is covalently bonded directly or indirectly by a
link to the PO(R.sub.1)(R.sub.2) moiety. An R group of the
PO(R.sub.1)(R.sub.2) moiety can possibly be cleaved within the
desired delivery site, thereby forming an ionic species which does
not exit the cell easily. This may cause accumulation within the
cell and can optionally protect the Emivirine analog from exposure
to metabolic enzymes which would metabolize the analog if not
protected within the cell. The cleavage may occur as a result of
normal displacement by cellular nucleophiles or enzymatic action,
but is preferably caused to occur selectively at a predetermined
release site. The advantage of this method is that the Emivirine
analog may optionally be delivered site-specifically, may
optionally accumulate within the cell and may optionally be
shielded from metabolic enzymes.
[0974] Link: an atom or molecule which covalently binds together
two components. In the present invention, a link is intended to
include atoms and molecules which can be used to covalently bind
Emivirine or an analog thereof at one end of the link to the
PO(R.sub.1)(R.sub.2) at the other end of the link. The link must
not prevent the binding of the analog with its appropriate
receptor. Examples of suitable links include, but are not limited
to, polymethylene [--(CH.sub.2).sub.n, where n is 1-10]1, ester,
amine, carbonate, carbamate, ether, olefin, aromatic ring, acetal,
heteroatom containing ring, or any combination of two or more of
these units. The PO(R.sub.1)(R.sub.2) may also be directly
attached. A skilled artisan will readily recognize other links
which can be used in accordance with the present invention.
[0975] The preceding Schemes 1-7 for SJ3366-like phosphonate NNRTI
compounds illustrate various aspects of the present invention and
are not to be construed to limit the types of analogs that may
employ this strategy of linking Emivirine or an Emivirine analog to
a PO(R.sub.1)(R.sub.2) moiety in any manner whatsoever.
Loviride-Like Phosphonate NNRTI Compounds
[0976] The present invention relates to Loviride-like phosphonate
NNRTI compounds and their delivery to cells, tissue or organs which
are optionally targeted for site-specific accumulation. More
particularly, this invention relates to phosphonate analogs of
Loviride, and their pharmaceutically acceptable salts and
formulations, which comprise Loviride linked to a phosphonate, i.e.
PO(R.sub.1)(R.sub.2) moiety.
[0977] The groups R.sub.1-R.sub.10 are as described in U.S. Pat.
No. 5,556,886, and also can be link PO(R.sub.1)(R.sub.2). The
present invention provides novel phosphonate analogs of Loviride.
Such novel Loviride analogs possess all the utilities of NNRTI
properties as Loviride and optionally provide cellular accumulation
as set forth below. ##STR332##
[0978] Loviride may be covalently bonded directly or indirectly by
a link to the PO(R.sub.1)(R.sub.2) moiety. An R group of the
PO(R.sub.1)(R.sub.2) moiety can possibly be cleaved within the
desired delivery site, thereby forming an ionic species which does
not exit the cell easily. This may cause accumulation within the
cell and can optionally protect the Loviride analog from exposure
to metabolic enzymes which would metabolize the analog if not
charged or protected within the cell. The cleavage may occur as a
result of normal displacement by cellular nucleophiles or enzymatic
action, but is preferably caused to occur selectively at a
predetermined release site. The advantage of this method is that
the Loviride analog may optionally be delivered site-specifically,
may optionally accumulate within the cell and may optionally be
shielded from metabolic enzymes.
[0979] The following examples illustrate various aspects of the
present invention and are not to be construed to limit the types of
analogs that may employ this strategy of lining Loviride or an
Loviride analog to a PO(R.sub.1)(R.sub.2) moiety in any manner
whatsoever. UC781-Like Phosphonate NNRTI Compounds ##STR333##
[0980] The present invention includes UC781-like phosphonate
compounds and pharmaceutically acceptable salts thereof. UC781 is
described in U.S. Pat. No. 6,143,780.
[0981] A, X, Y, Q and R.sup.6 in the formula above are as defined
in U.S. Pat. No. 6,143,780. Z represents any substitution of the
heteroatom ring. Also the heteroatom ring may be six membered. The
present invention provides novel phosphonate analogs of UC781. Such
novel UC781 analogs possess all the utilities of Emivirine and
optionally provide cellular accumulation as set forth below. The
present invention also relates to the delivery of UC781-like
phosphonate compounds which are optionally targeted for
site-specific accumulation in cells, tissues or organs. More
particularly, this invention relates to analogs of UC781 which
comprise UC781 linked to a PO(R.sub.1)(R.sub.2) moiety.
[0982] UC781 is covalently bonded directly or indirectly by a link
to the PO(R.sub.1)(R.sub.2) moiety. An R group of the
PO(R.sub.1)(R.sub.2) moiety can possibly be cleaved within the
desired delivery site, thereby forming an ionic species which does
not exit the cell easily. This may cause accumulation within the
cell and can optionally protect the UC781e analog from exposure to
metabolic enzymes which would metabolize the analog if not
protected within the cell. The cleavage may occur as a result of
normal displacement by cellular nucleophiles or enzymatic action,
but is preferably caused to occur selectively at a predetermined
release site. The advantage of this method is that the UC781 analog
may optionally be delivered site-specifically, may optionally
accumulate within the cell and may optionally be shielded from
metabolic enzymes.
[0983] Link is any moiety which covalently binds together UC781 or
an analog of UC781 and a phosphonate group. In the present
invention, a link is intended to include atoms and molecules which
can be used to covalently bind UC781 or an analog thereof at one
end of the link to the PO(R.sub.1)(R.sub.2) at the other end of the
link. The link should not prevent the binding of the analog with
its appropriate receptor. Examples of suitable links include, but
are not limited to, polymethylene [--(CH.sub.2).sub.n, where n is
1-10], ester, amine, carbonate, carbamate, ether, olefin, aromatic
ring, acetal, heteroatom containing ring or any combination of two
or more of these units. Direct attachment of the
PO(R.sub.1)(R.sub.2) is also possible. A skilled artisan will
readily recognize other links which can be used in accordance with
the present invention.
[0984] The following examples illustrate various aspects of the
present invention and are not to be construed to limit the types of
analogs that may employ this strategy of linking UC781 or an UC781
analog to a PO(R.sub.1)(R.sub.2) moiety in any manner
whatsoever.
[0985] Preparation of compounds of type A may proceed via a link
which can react with UC781 or an analog or intermediate thereof, to
result in a covalent bond between the link and the drug-like
compound. The link is also attached to the phosphorous containing
moiety as shown in an example of type A, namely A1.
[0986] Preparation of
N-3-((2-chlorophenoxy)methyl)-4-chlorophenyl-2-methyl-3-furancarbothioami-
de, compound 12 in scheme 1 and intermediates 2, 4-11, as per U.S.
Pat. No. 6,143,780.
[0987] Step 1: Preparation of 2-chloro-5-nitrobenzoyl alcohol 30 g
of 2-chloro-5-nitrobenzaldehyde was dissolved in 500 ml, of
methanol and cooled to 0.degree. C. A solution of 10 g of sodium
borohydride in 100 ml, of water was then added dropwise over 90
minutes while maintaining the temperature below 10.degree. C. The
resultant reaction mixture was then stirred for one hour, then
acidified with 2N HCl and left stirring overnight. The solids were
then, washed with water and dried, to produce 27 g of
2-chloro-5-nitrobenzyl alcohol as a white solid.
[0988] Step 2: Preparation of 2-chloro-5-nitrobenzoyl acetate 27 g
of the 2-chloro-5-nitrobenzyl alcohol prepared above in Step 1, was
dissolved in 122 ml, of toluene. 22 mL of triethylamine was then
added. The resultant reaction mixture was cooled to 20.degree. C.
and then a solution of 10.2 ml, of acetyl chloride in 10 ml, of
toluene, was added dropwise, keeping the temperature below
20.degree. C. The reaction mixture was then stirred overnight. 2.1
ml, of triethylamine and 1.1 ml, of acetyl chloride/toluene
solution were then added and the reaction mixture was stirred for
one hour. 100 ml, of water was then added, followed by 50 mL of
ether. The resulting organic phase was separated, washed with 2N
HCl, aqueous sodium bicarbonate solution and water. The washed
organic phase was then dried over magnesium sulfate and the solvent
was evaporated, to produce 29.6 g of 2-chloro-5-nitrobenzoyl
acetate as a white solid.
[0989] Step 3: Preparation of 5-amino-2-chlorobenzoyl acetate 24 g
of iron powder was added to a solution of 1.6 ml, of concentrated
HCl, 16.8 ml, of water, and 70 ml, of ethanol. 29.6 g of the
2-chloro-5-nitrobenzoyl acetate prepared above in Step 2 dissolved
in 45 ml, of ethanol, was then added to the mixture in three equal
portions. The resultant reaction mixture was refluxed for 5 hours.
An additional 2.4 g of iron and 0.1 ml, of concentrated HCl was
then added to the reaction mixture. The reaction mixture was then
refluxed for an additional one hour, filtered through Celite and
evaporated. 100 ml, of water was then added to the evaporated
material and the resultant mixture was extracted with 100 ml, of
ether. The ether solution was washed with water, dried over
magnesium sulfate, and evaporated, to produce 22.9 g of
5-amino-2-chlorobenzoyl acetate as an oil.
[0990] Step 4: Preparation of
N-(3-acetoxymethylchlorophenyl)-2-methyl-3-furancarboxanilide. A
solution of 22.8 g of the 5-amino-2-chlorobenzoyl acetate from Step
3 above and 17.2 ml, of triethylamine in 118 ml, ether was prepared
and then added dropwise to a second solution of 16.6 g
2-methyl-3-thiophenecarboxylic acid chloride in 118 ml, ether at
0.degree. C. to 10.degree. C. and the resultant mixture was stirred
at room temperature overnight. 100 ml, of water and 100 ml, of
ethyl acetate were then added to the mixture, the organic phase
separated, washed with 2N hydrochloric acid and water, dried over
magnesium sulfate, and the solvents removed in vacuo, to produce
29.87 g of
N-(3-acetoxymethyl-4-chlorophenyl)-2-methyl-3-furancarboxamide as a
beige solid.
[0991] Step 5: Preparation of
N-(4-chloro-3-hydroxymethylphenyl)-2-methyl-3-furancarboxamide. A
solution of 29 g of the
N-(3-acetoxymethyl-4-chlorophenyl)-2-methyl-3-furancarboxamide
prepared in Step 4 above and 14.5 g potassium hydroxide in 110 ml,
water, was prepared. The solution was then heated at 70.degree. C.
for 16 hours and then acidified with 2N hydrochloric. The resulting
solid was collected, washed with water, and dried, producing 23.65
g of N-(4-chloro-3-hydroxymethylphenyl)-2-methyl-3-furancarboxamide
as a white solid.
[0992] Step 6: Preparation of
N-(3-bromomethyl-4-chlorophenyl)-2-methyl-3-furancarboxamide. 12 g
of the
N-(4-chloro-3-hydroxymethylphenyl)-2-methyl-3-furancarboxamide
prepared in Step 5 above, was dissolved in 180 ml, ethyl acetate.
1.8 ml, of phosphorus tribromide was then added. The resultant
mixture was stirred for 90 minutes at room temperature. 100 ml, of
water was then added to the mixture. The resultant organic phase
was separated, washed with water, aqueous sodium bicarbonate
solution and water, and then dried over magnesium sulfate. The
solvent was evaporated off to produce 12.97 g of
N-(3-bromomethyl-4-chlorophenyl)-2-methyl-3-furancarboxamide as a
solid.
[0993] Step 7: Preparation of
N-3-((2-chlorophenoxy)methyl)-4-chlorophenyl-2-methyl-3-furancarboxamide.
2 g of the
N-(3-bromomethyl-4-chlorophenyl)-2-methyl-3-furancarboxamide
produced in Step 6, was dissolved in 20 ml, of 2-butanone to
produce a solution. 0.84 g of potassium carbonate, 0.79 g of
2-chlorophenol and 0.2 g of tetrabutylammonium bromide were then
added to the solution. The resultant reaction mixture was stirred
at room temperature overnight, the solvents removed in vacuo, and
the residue extracted with ethyl acetate, to produce a second
solution. This second solution was washed with 2N aqueous sodium
hydroxide and water, and then dried over magnesium sulfate. The
solvent was removed to produce 2.7 g of a solid, which was purified
by dissolving in ethyl acetate:hexane (20:80) and running the
resultant solution through a plug of silica gel. Removal of solvent
produced 2.0 g of
N-3-((2-chlorophenoxy)methyl)-4-chlorophenyl-2-methyl-3-furancarboxamide
as a white solid. Step 8: Preparation of
N-3-((2-chlorophenoxy)methyl)-4-chlorophenyl-2-methyl-3-furancarbothioami-
de. 1.5 g of the
N-3-((2-chlorophenoxy)methyl)-4-chlorophenyl-2-methyl-3-furancarboxamide
prepared in Step 7 above, 0.8 g of Lawesson's reagent (0.8 g) and
1.6 g of sodium bicarbonate were added to 35 ml, of toluene, and
the resultant reaction mixture was refluxed for five hours. The
reaction mixture was then passed through a plug of neutral aluminum
oxide, eluted with 1:1 ether/hexane and purified by column
chromatography on silica gel, to produce 0.77 g of
N-3-((2-chlorophenoxy)methyl)-4-chlorophenyl-2-methyl-3-furancarbothioami-
de. ##STR334##
[0994] The above protocol can easily be modified to attach the
link-PO(R.sub.1)(R.sub.2). To prepare compounds of type A in FIG.
1, the following route is performed. Compound 8 above, when R.sup.6
is chloro, is transformed into a triflate by reacting it with
triflic anhydride and 2,6 lutidine in dichloromethane at
-40.degree. C. The addition of hydroxyethyldimethoxyphosphonate
will effect the attachment of the link PO(R.sub.1)(R.sub.2) group.
Treatment with Lawesson's reagent as above will provide compound
A2.
[0995] By replacing 2-chloro 5-nitrobenzaldehyde with other
nitrobenzaldehyes and following a similar procedure as that used to
make compound A2, the relative positions of attachment of the ether
and the amide is changed. Furthermore, the chloro substituent shown
as R.sup.6 above is switched to other positions, and other
substituents are used in combination with or without the chloro
atom or other substituents anywhere on the ring (shown as Q below).
This would allow for compounds of type B2 of FIG. 2 to be prepared.
As with all analogs that are amenable to such treatment, Lawesson's
reagent would then be used to convert to the corresponding
sulfamide.
[0996] Type B1 compounds would include Type B2 and are prepared
using the above steps with the center aryl ring being considered
part of the link. Prior to treatment with Lawesson's reagent the
amide proton is abstracted by treatment with base to allow for
attachment of the PO(R.sub.1)(R.sub.2) moiety. Lawesson's reagent
would then be used to convert to the corresponding sulfamide. This
would allow for compounds of the general form Type C shown in FIG.
3.
[0997] The furan ring of UC781 is switched to 5 or 6-membered
heterocycles easily by substituting different heterocyclic acid
chlorides for 2-methyl-3-thiophenecarboxylic acid chloride in step
4 in the above written synthesis of
N-3-((2-chlorophenoxy)methyl)-4-chlorophenyl-2-methyl-3-furancarbothioami-
de. This will afford Type D compounds as exemplified below. The
link PO(R.sub.1)(R.sub.2) moiety is attached directly to the
heterocycle by starting with for example the diester of the desired
heterocycle. Mono acid formation of the heterocycle by hydrolysis
of one ester would allow for attachment of the PO(R.sub.1)(R.sub.2)
group. This would be followed by hydrolysis of the remaining ester
by base, acid chloride formation as above and amide formation by
reaction with the desired amine. D1, a specific exemplification of
Type D compounds having in this case R.sub.1 and R.sub.2.dbd.OMe
and link=CH.sub.2CH.sub.2 is prepared as shown below in FIG. 4.
[0998] All amides shown can be converted to sulfamides by treatment
with Lawesson's reagent. ##STR335## ##STR336##
[0999] The details of the first two steps of Scheme 1 shown above
are thoroughly covered in U.S. Pat. No. 5,556,886. The synthesis
can be extended as shown to allow for the attachment of the link
PO(R.sub.1)(R.sub.2) at various sites on either aryl ring.
[1000] To attach on the ortho, meta or para positions of the ring
that starts out as the substituted aniline, a moiety must be
present that will allow for such an attachment of the
PO(R.sub.1)(R.sub.2) moiety. In this case a nitro group is used as
an amine precursor. The reduction of the nitro can be effected by
tin chloride and acetic acid in an appropriate solvent, or through
some other catalytic hydrogenation method. From there, compounds
such as compound 5 with a free anilino NH.sub.2 can be reacted
with, for example, a commercially available phosphonate such as
compound 6 above in a reductive amination reaction. This reductive
amination is performed using dichloroethane as solvent, and after
stirring under dry conditions, sodium cyanoborohydride and acetic
acid is added to complete the reaction giving compound 7. Using
commercially available meta and para nitroanilines leads to
compounds 8, 9 and 10. Other substitution patterns are also
possible. Also, other means of attachment are also possible to
attach the drug-like compound to the PO(R.sub.1)(R.sub.2) piece. By
varying the position of the nitro group, PO(R.sub.1)(R.sub.2) is
attached at any position on the anilino ring. FIG. 1 below contains
examples of nitroanilines that allow for the attachment at various
positions.
[1001] Alternatively, the nitroanilines is attached to the
PO(R.sub.1)(R.sub.2) moiety prior to coupling with the aldehyde.
The nitro is then reduced to form the aniline needed for coupling
with the aldehyde. Hydrolysis of the cyano group to the amide is
conducted as above, as illustrated in Scheme 2. ##STR337##
[1002] the ketone of Loviride or Loviride analogs also serves as a
point of attachment for the PO(R.sub.1)(R.sub.2) groups. The
synthesis of such an attachment is shown in Scheme 3.
##STR338##
[1003] By using a variation of the benzaldehyde shown as compound 1
in Scheme 1, further points of attachment are also attainable. By
using, for example, 2,6-dichloro (3, 4, or 5 nitro) benzaldehyde,
and following Scheme 1, the PO(R.sub.1)(R.sub.2) is attached at any
position of the ring which starts out as the benzaldehyde. Further
examples of compounds that can be made in this way are compounds
11, 12 and 13 below. ##STR339## Scheme General Section
[1004] General aspects of these exemplary methods are described
below and in the Example. Each of the products of the following
processes is optionally separated, isolated, and/or purified prior
to its use in subsequent processes.
[1005] The terms "treated", "treating", "treatment", and the like,
mean contacting, mixing, reacting, allowing to react, bringing into
contact, and other terms common in the art for indicating that one
or more chemical entities is treated in such a manner as to convert
it to one or more other chemical entities. This means that
"treating compound one with compound two" is synonymous with
"allowing compound one to react with compound two", "contacting
compound one with compound two", "reacting compound one with
compound two", and other expressions common in the art of organic
synthesis for reasonably indicating that compound one was
"treated", "reacted", "allowed to react", etc., with compound
two.
[1006] "Treating" indicates the reasonable and usual manner in
which organic chemicals are allowed to react. Normal concentrations
(0.01M to 10M, typically 0.1M to 1M), temperatures (-100.degree. C.
to 250.degree. C., typically -78.degree. C. to 150.degree. C., more
typically -78.degree. C. to 100.degree. C., still more typically
0.degree. C. to 100.degree. C.), reaction vessels (typically glass,
plastic, metal), solvents, pressures, atmospheres (typically air
for oxygen and water insensitive reactions or nitrogen or argon for
oxygen or water sensitive), etc., are intended unless otherwise
indicated. The knowledge of similar reactions known in the art of
organic synthesis is used in selecting the conditions and apparatus
for "treating" in a given process. In particular, one of ordinary
skill in the art of organic synthesis selects conditions and
apparatus reasonably expected to successfully carry out the
chemical reactions of the described processes based on the
knowledge in the art.
[1007] Modifications of each of the exemplary schemes above and in
the examples (hereafter "exemplary schemes") leads to various
analogs of the specific exemplary materials produce. The above
cited citations describing suitable methods of organic synthesis
are applicable to such modifications.
[1008] In each of the exemplary schemes it may be advantageous to
separate reaction products from one another and/or from starting
materials. The desired products of each step or series of steps is
separated and/or purified (hereinafter separated) to the desired
degree of homogeneity by the techniques common in the art.
Typically such separations involve multiphase extraction,
crystallization from a solvent or solvent mixture, distillation,
sublimation, or chromatography. Chromatography can involve any
number of methods including, for example, size exclusion or ion
exchange chromatography, high, medium, or low pressure liquid
chromatography, small scale and preparative thin or thick layer
chromatography, as well as techniques of small scale thin layer and
flash chromatography.
[1009] Another class of separation methods involves treatment of a
mixture with a reagent selected to bind to or render otherwise
separable a desired product, unreacted starting material, reaction
by product, or the like. Such reagents include adsorbents or
absorbents such as activated carbon, molecular sieves, ion exchange
media, or the like. Alternatively, the reagents can be acids in the
case of a basic material, bases in the case of an acidic material,
binding reagents such as antibodies, binding proteins, selective
chelators such as crown ethers, liquid/liquid ion extraction
reagents (LIX), or the like.
[1010] Selection of appropriate methods of separation depends on
the nature of the materials involved. For example, boiling point,
and molecular weight in distillation and sublimation, presence or
absence of polar functional groups in chromatography, stability of
materials in acidic and basic media in multiphase extraction, and
the like. One skilled in the art will apply techniques most likely
to achieve the desired separation.
[1011] All literature and patent citations above are hereby
expressly incorporated by reference at the locations of their
citation. Specifically cited sections or pages of the above cited
works are incorporated by reference with specificity. The invention
has been described in detail sufficient to allow one of ordinary
skill in the art to make and use the subject matter of the
following Embodiments. It is apparent that certain modifications of
the methods and compositions of the following Embodiments can be
made within the scope and spirit of the invention. ##STR340##
[1012] Scheme 1001 shows the interconversions of certain
phosphonate compounds: acids --P(O)(OH).sub.2; mono-esters
--P(O)(OR.sub.1)(OH); and diesters --P(O)(OR.sub.1).sub.2 in which
the R.sup.1 groups are independently selected, and defined herein
before, and the phosphorus is attached through a carbon moiety
(link, i.e. linker), which is attached to the rest of the molecule,
e.g. drug or drug intermediate (R). The R.sup.1 groups attached to
the phosphonate esters in Scheme 1001 may be changed using
established chemical transformations. The interconversions may be
carried out in the precursor compounds or the final products using
the methods described below. The methods employed for a given
phosphonate transformation depend on the nature of the substituent
R.sup.1. The preparation and hydrolysis of phosphonate esters is
described in Organic Phosphorus Compounds, G. M. Kosolapoff, L.
Maeir, eds, Wiley, 1976, p. 9ff.
[1013] The conversion of a phosphonate diester 27.1 into the
corresponding phosphonate monoester 27.2 (Scheme 1001, Reaction 1)
can be accomplished by a number of methods. For example, the ester
27.1 in which R.sup.1 is an arylalkyl group such as benzyl can be
converted into the monoester compound 27.2 by reaction with a
tertiary organic base such as diazabicyclooctane (DABCO) or
quinuclidine, as described in J. Org. Chem., 1995, 60:2946. The
reaction is performed in an inert hydrocarbon solvent such as
toluene or xylene, at about 110.degree. C. The conversion of the
diester 27.1 in which R.sup.1 is an aryl group such as phenyl or an
alkenyl group such as allyl, into the monoester 27.2 can be
effected by treatment of the ester 27.1 with a base such as aqueous
sodium hydroxide in acetonitrile or lithium hydroxide in aqueous
tetrahydrofuran. Phosphonate diesters 27.2 in which one of the
groups R.sup.1 is arylalkyl, such as benzyl, and the other is
alkyl, can be converted into the monoesters 27.2 in which R.sup.1
is alkyl, by hydrogenation, for example using a palladium on carbon
catalyst. Phosphonate diesters in which both of the groups R.sup.1
are alkenyl, such as allyl, can be converted into the monoester
27.2 in which R.sup.1 is alkenyl, by treatment with
chlorotris(triphenylphosphine)rhodium (Wilkinson's catalyst) in
aqueous ethanol at reflux, optionally in the presence of
diazabicyclooctane, for example by using the procedure described in
J. Org. Chem., 38:3224 1973 for the cleavage of allyl
carboxylates.
[1014] The conversion of a phosphonate diester 27.1 or a
phosphonate monoester 27.2 into the corresponding phosphonic acid
27.3 (Scheme 1001, Reactions 2 and 3) can be effected by reaction
of the diester or the monoester with trimethylsilyl bromide, as
described in J. Chem. Soc., Chem. Comm., 739, 1979. The reaction is
conducted in an inert solvent such as, for example,
dichloromethane, optionally in the presence of a silylating agent
such as bis(trimethylsilyl)trifluoroacetamide, at ambient
temperature. A phosphonate monoester 27.2 in which R.sup.1 is
arylalkyl such as benzyl, can be converted into the corresponding
phosphonic acid 27.3 by hydrogenation over a palladium catalyst, or
by treatment with hydrogen chloride in an ethereal solvent such as
dioxane. A phosphonate monoester 27.2 in which R.sup.1 is alkenyl
such as, for example, allyl, can be converted into the phosphonic
acid 27.3 by reaction with Wilkinson's catalyst in an aqueous
organic solvent, for example in 15% aqueous acetonitrile, or in
aqueous ethanol, for example using the procedure described in Helv.
Chim. Acta., 68:618, 1985. Palladium catalyzed hydrogenolysis of
phosphonate esters 27.1 in which R.sup.1 is benzyl is described in
J. Org. Chem., 24:434, 1959. Platinum-catalyzed hydrogenolysis of
phosphonate esters 27.1 in which R.sup.1 is phenyl is described in
J. Amer. Chem. Soc., 78:2336, 1956.
[1015] The conversion of a phosphonate monoester 27.2 into a
phosphonate diester 27.1 (Scheme 1001, Reaction 4) in which the
newly introduced R.sup.1 group is alkyl, arylalkyl, or haloalkyl
such as chloroethyl, can be effected by a number of reactions in
which the substrate 27.2 is reacted with a hydroxy compound
R.sup.1OH, in the presence of a coupling agent. Suitable coupling
agents are those employed for the preparation of carboxylate
esters, and include a carbodiimide such as
dicyclohexylcarbodiimide, in which case the reaction is preferably
conducted in a basic organic solvent such as pyridine, or
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PYBOP, Sigma), in which case the reaction is performed in a polar
solvent such as dimethylformamide, in the presence of a tertiary
organic base such as diisopropylethylamine, or Aldrithiol-2
(Aldrich) in which case the reaction is conducted in a basic
solvent such as pyridine, in the presence of a triaryl phosphine
such as triphenylphosphine. Alternatively, the conversion of the
phosphonate monoester 27.1 to the diester 27.1 can be effected by
the use of the Mitsunobu reaction. The substrate is reacted with
the hydroxy compound R.sup.1OH, in the presence of diethyl
azodicarboxylate and a triarylphosphine such as triphenyl
phosphine. Alternatively, the phosphonate monoester 27.2 can be
transformed into the phosphonate diester 27.1, in which the
introduced R.sup.1 group is alkenyl or arylalkyl, by reaction of
the monoester with the halide R.sup.1Br, in which R.sup.1 is as
alkenyl or arylalkyl. The alkylation reaction is conducted in a
polar organic solvent such as diethylformamide or acetonitrile, in
the presence of a base such as cesium carbonate. Alternatively, the
phosphonate monoester can be transformed into the phosphonate
diester in a two step procedure. In the first step, the phosphonate
monoester 27.2 is transformed into the chloro analog
--P(O)(OR.sup.1)Cl by reaction with thionyl chloride or oxalyl
chloride and the like, as described in Organic Phosphorus
Compounds, G. M. Kosolapoff, L. Maeir, eds, Wiley, 1976, p. 17, and
the thus-obtained product --P(O)(OR.sup.1)Cl is then reacted with
the hydroxy compound R.sup.1OH, in the presence of a base such as
triethylamine, to afford the phosphonate diester 27.1.
[1016] A phosphonic acid --P(O)(OH).sub.2 can be transformed into a
phosphonate monoester --P(O)(OR.sup.1)(OH) (Scheme 1001, Reaction
5) by means of the methods described above of for the preparation
of the phosphonate diester --P(O)(OR.sup.1).sub.2 27.1, except that
only one molar proportion of the component R.sup.1OH or R.sup.1Br
is employed.
[1017] A phosphonic acid --P(O)(OH).sub.2 27.3 can be transformed
into a phosphonate diester --P(O)(OR.sup.1).sub.2 27.1 (Scheme 1,
Reaction 6) by a coupling reaction with the hydroxy compound
R.sup.1OH, in the presence of a coupling agent such as Aldrithiol-2
(Aldrich) and triphenylphosphine. The reaction is conducted in a
basic solvent such as pyridine. Alternatively, phosphonic acids
27.3 can be transformed into phosphonic esters 27.1 in which
R.sup.1 is aryl, such as phenyl, by means of a coupling reaction
employing, for example, phenol and dicyclohexylcarbodiimide in
pyridine at about 70.degree. C. Alternatively, phosphonic acids
27.3 can be transformed into phosphonic esters 27.1 in which
R.sup.1 is alkenyl, by means of an alkylation reaction. The
phosphonic acid is reacted with the alkenyl bromide R.sup.1Br in a
polar organic solvent such as acetonitrile solution at reflux
temperature, in the presence of a base such as cesium carbonate, to
afford the phosphonic ester 27.1.
[1018] Amino alkyl phosphonate compounds 809: ##STR341## are a
generic representative of compounds 811, 813, 814, 816 and 818.
Some methods to prepare embodiments of 809 are shown in Scheme
1002. Commercial amino phosphonic acid 810 was protected as
carbamate 811. The phosphonic acid 811 was converted to phosphonate
812 upon treatment with ROH in the presence of DCC or other
conventional coupling reagents. Coupling of phosphonic acid 811
with esters of amino acid 820 provided bisamidate 817. Conversion
of acid 811 to bisphenyl phosphonate followed by hydrolysis gave
mono-phosphonic acid 814 (Cbz=C.sub.6H.sub.5CH.sub.2C(O)--), which
was then transformed to mono-phosphonic amidate 815. Carbamates
813, 816 and 818 were converted to their corresponding amines upon
hydrogenation. Compounds 811, 813, 814, 816 and 818 are useful
intermediates to form the phosphonate compounds of the invention.
Preparation of Carboalkoxy-Substituted Phosphonate Bisamidates,
Monoamidates, Diesters and Monoesters.
[1019] A number of methods are available for the conversion of
phosphonic acids into amidates and esters. In one group of methods,
the phosphonic acid is either converted into an isolated activated
intermediate such as a phosphoryl chloride, or the phosphonic acid
is activated in situ for reaction with an amine or a hydroxy
compound.
[1020] The conversion of phosphonic acids into phosphoryl chlorides
is accomplished by reaction with thionyl chloride, for example as
described in J. Gen. Chem. USSR, 1983, 53, 480, Zh. Obschei Khim.,
1958, 28, 1063, or J. Org. Chem., 1994, 59, 6144, or by reaction
with oxalyl chloride, as described in J. Am. Chem. Soc., 1994, 116,
3251, or J. Org. Chem., 1994, 59, 6144, or by reaction with
phosphorus pentachloride, as described in J. Org. Chem., 2001, 66,
329, or in J. Med. Chem., 1995, 38, 1372. The resultant phosphoryl
chlorides are then reacted with amines or hydroxy compounds in the
presence of a base to afford the amidate or ester products.
[1021] Phosphonic acids are converted into activated imidazolyl
derivatives by reaction with carbonyl diimidazole, as described in
J. Chem. Soc., Chem. Comm., 1991, 312, or Nucleosides Nucleotides
2000, 19, 1885. Activated sulfonyloxy derivatives are obtained by
the reaction of phosphonic acids with trichloromethylsulfonyl
chloride, as described in J. Med. Chem. 1995, 38, 4958, or with
triisopropylbenzenesulfonyl chloride, as described in Tet. Lett.,
1996, 7857, or Bioorg. Med. Chem. Lett., 1998, 8, 663. The
activated sulfonyloxy derivatives are then reacted with amines or
hydroxy compounds to afford amidates or esters. Alternatively, the
phosphonic acid and the amine or hydroxy reactant are combined in
the presence of a diimide coupling agent. The preparation of
phosphonic amidates and esters by means of coupling reactions in
the presence of dicyclohexyl carbodiimide is described, for
example, in J. Chem. Soc., Chem. Comm., 1991, 312, or J. Med.
Chem., 1980, 23, 1299 or Coil. Czech. Chem. Comm., 1987, 52, 2792.
The use of ethyl dimethylaminopropyl carbodiimide for activation
and coupling of phosphonic acids is described in Tet. Lett., 2001,
42, 8841, or Nucleosides Nucleotides, 2000, 19, 1885.
[1022] A number of additional coupling reagents have been described
for the preparation of amidates and esters from phosphonic acids.
The agents include Aldrithiol-2, and PYBOP and BOP, as described in
J. Org. Chem., 1995, 60, 5214, and J. Med. Chem., 1997, 40, 3842,
mesitylene-2-sulfonyl-3-nitro-1,2,4-triazole (MSNT), as described
in J. Med. Chem., 1996, 39, 4958, diphenylphosphoryl azide, as
described in J. Org. Chem., 1984, 49, 1158,
1-(2,4,6-triisopropylbenzenesulfonyl-3-nitro-1,2,4-triazole (TPSNT)
as described in Bioorg. Med. Chem. Lett., 1998, 8, 1013,
bromotris(dimethylamino)phosphonium hexafluorophosphate (BroP), as
described in Tet. Lett., 1996, 37, 3997,
2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane, as described in
Nucleosides Nucleotides 1995, 14, 871, and diphenyl
chlorophosphate, as described in J. Med. Chem., 1988, 31, 1305.
[1023] Phosphonic acids are converted into amidates and esters by
means of the Mitsonobu reaction, in which the phosphonic acid and
the amine or hydroxy reactant are combined in the presence of a
triaryl phosphine and a dialkyl azodicarboxylate. The procedure is
described in Org. Lett., 2001, 3, 643, or J. Med. Chem., 1997, 40,
3842.
[1024] Phosphonic esters are also obtained by the reaction between
phosphonic acids and halo compounds, in the presence of a suitable
base. The method is described, for example, in Anal. Chem., 1987,
59, 1056, or J. Chem. Soc. Perkin Trans., I, 1993, 19, 2303, or J.
Med. Chem., 1995, 38, 1372, or Tet. Lett., 2002, 43, 1161.
[1025] Schemes 1-4 illustrate the conversion of phosphonate esters
and phosphonic acids into carboalkoxy-substituted
phosphorobisamidates (Scheme 1), phosphoroamidates (Scheme 2),
phosphonate monoesters (Scheme 3) and phosphonate diesters, (Scheme
4).
[1026] Scheme 1 illustrates various methods for the conversion of
phosphonate diesters 1.1 into phosphorobisamidates 1.5. The diester
1.1, prepared as described previously, is hydrolyzed, either to the
monoester 1.2 or to the phosphonic acid 1.6. The methods employed
for these transformations are described above. The monoester 1.2 is
converted into the monoamidate 1.3 by reaction with an aminoester
1.9, in which the group R.sup.2 is H or alkyl, the group R.sup.4 is
an alkylene moiety such as, for example, CHCH.sub.3, CHPr.sup.I,
CH(CH.sub.2Ph), CH.sub.2CH(CH.sub.3) and the like, or a group
present in natural or modified aminoacids, and the group R.sup.5 is
alkyl. The reactants are combined in the presence of a coupling
agent such as a carbodiimide, for example dicyclohexyl
carbodiimide, as described in J. Am. Chem. Soc., 1957, 79, 3575,
optionally in the presence of an activating agent such as
hydroxybenzotriazole, to yield the amidate product 1.3. The
amidate-forming reaction is also effected in the presence of
coupling agents such as BOP, as described in J. Org. Chem., 1995,
60, 5214, Aldrithiol, PYBOP and similar coupling agents used for
the preparation of amides and esters. Alternatively, the reactants
1.2 and 1.9 are transformed into the monoamidate 1.3 by means of a
Mitsonobu reaction. The preparation of amidates by means of the
Mitsonobu reaction is described in J. Med. Chem., 1995, 38, 2742.
Equimolar amounts of the reactants are combined in an inert solvent
such as tetrahydrofuran in the presence of a triaryl phosphine and
a dialkyl azodicarboxylate. The thus-obtained monoamidate ester 1.3
is then transformed into amidate phosphonic acid 1.4. The
conditions used for the hydrolysis reaction depend on the nature of
the R.sup.1 group, as described previously. The phosphonic acid
amidate 1.4 is then reacted with an aminoester 1.9, as described
above, to yield the bisamidate product 1.5, in which the amino
substituents are the same or different.
[1027] An example of this procedure is shown in Scheme 1, Example
1. In this procedure, a dibenzyl phosphonate 1.14 is reacted with
diazabicyclooctane (DABCO) in toluene at reflux, as described in J.
Org. Chem., 1995, 60, 2946, to afford the monobenzyl phosphonate
1.15. The product is then reacted with equimolar amounts of ethyl
alaninate 1.16 and dicyclohexyl carbodiimide in pyridine, to yield
the amidate product 1.17. The benzyl group is then removed, for
example by hydrogenolysis over a palladium catalyst, to give the
monoacid product 1.18. This compound is then reacted in a Mitsonobu
reaction with ethyl leucinate 1.19, triphenyl phosphine and
diethylazodicarboxylate, as described in J. Med. Chem., 1995, 38,
2742, to produce the bisamidate product 1.20.
[1028] Using the above procedures, but employing, in place of ethyl
leucinate 1.19 or ethyl alaninate 1.16, different aminoesters 1.9,
the corresponding products 1.5 are obtained.
[1029] Alternatively, the phosphonic acid 1.6 is converted into the
bisamidate 1.5 by use of the coupling reactions described above.
The reaction is performed in one step, in which case the
nitrogen-related substituents present in the product 1.5 are the
same, or in two steps, in which case the nitrogen-related
substituents can be different.
[1030] An example of the method is shown in Scheme 1, Example 2. In
this procedure, a phosphonic acid 1.6 is reacted in pyridine
solution with excess ethyl phenylalaninate 1.21 and
dicyclohexylcarbodiimide, for example as described in J. Chem.
Soc., Chem. Comm., 1991, 1063, to give the bisamidate product
1.22.
[1031] Using the above procedures, but employing, in place of ethyl
phenylalaninate, different aminoesters 1.9, the corresponding
products 1.5 are obtained.
[1032] As a further alternative, the phosphonic acid 1.6 is
converted into the mono or bis-activated derivative 1.7, in which
Lv is a leaving group such as chloro, imidazolyl,
triisopropylbenzenesulfonyloxy etc. The conversion of phosphonic
acids into chlorides 1.7 (Lv=Cl) is effected by reaction with
thionyl chloride or oxalyl chloride and the like, as described in
Organic Phosphorus Compounds, G. M. Kosolapoff, L. Maeir, eds,
Wiley, 1976, p. 17. The conversion of phosphonic acids into
monoimidazolides 1.7 (Lv=imidazolyl) is described in J. Med. Chem.,
2002, 45, 1284 and in J. Chem. Soc. Chem. Comm., 1991, 312.
Alternatively, the phosphonic acid is activated by reaction with
triisopropylbenzenesulfonyl chloride, as described in Nucleosides
and Nucleotides, 2000, 10, 1885. The activated product is then
reacted with the aminoester 1.9, in the presence of a base, to give
the bisamidate 1.5. The reaction is performed in one step, in which
case the nitrogen substituents present in the product 1.5 are the
same, or in two steps, via the intermediate 1.11, in which case the
nitrogen substituents can be different.
[1033] Examples of these methods are shown in Scheme 1, Examples 3
and 5. In the procedure illustrated in Scheme 1, Example 3, a
phosphonic acid 1.6 is reacted with ten molar equivalents of
thionyl chloride, as described in Zh. Obschei Khim., 1958, 28,
1063, to give the dichloro compound 1.23. The product is then
reacted at reflux temperature in a polar aprotic solvent such as
acetonitrile, and in the presence of a base such as triethylamine,
with butyl serinate 1.24 to afford the bisamidate product 1.25.
[1034] Using the above procedures, but employing, in place of butyl
serinate 1.24, different aminoesters 1.9, the corresponding
products 1.5 are obtained.
[1035] In the procedure illustrated in Scheme 1, Example 5, the
phosphonic acid 1.6 is reacted, as described in J. Chem. Soc. Chem.
Comm, 1991, 312, with carbonyl diimidazole to give the imidazolide
1.32. The product is then reacted in acetonitrile solution at
ambient temperature, with one molar equivalent of ethyl alaninate
1.33 to yield the monodisplacement product 1.34. The latter
compound is then reacted with carbonyl diimidazole to produce the
activated intermediate 1.35, and the product is then reacted, under
the same conditions, with ethyl N-methylalaninate 1.33a to give the
bisamidate product 1.36.
[1036] Using the above procedures, but employing, in place of ethyl
alaninate 1.33 or ethyl N-methylalaninate 1.33a, different
aminoesters 1.9, the corresponding products 1.5 are obtained.
[1037] The intermediate monoamidate 1.3 is also prepared from the
monoester 1.2 by first converting the monoester into the activated
derivative 1.8 in which Lv is a leaving group such as halo,
imidazolyl etc, using the procedures described above. The product
1.8 is then reacted with an aminoester 1.9 in the presence of a
base such as pyridine, to give an intermediate monoamidate product
1.3. The latter compound is then converted, by removal of the
R.sup.1 group and coupling of the product with the aminoester 1.9,
as described above, into the bisamidate 1.5.
[1038] An example of this procedure, in which the phosphonic acid
is activated by conversion to the chloro derivative 1.26, is shown
in Scheme 1, Example 4. In this procedure, the phosphonic
monobenzyl ester 1.15 is reacted, in dichloromethane, with thionyl
chloride, as described in Tet. Let., 1994, 35, 4097, to afford the
phosphoryl chloride 1.26. The product is then reacted in
acetonitrile solution at ambient temperature with one molar
equivalent of ethyl 3-amino-2-methylpropionate 1.27 to yield the
monoamidate product 1.28. The latter compound is hydrogenated in
ethyl acetate over a 5% palladium on carbon catalyst to produce the
monoacid product 1.29. The product is subjected to a Mitsonobu
coupling procedure, with equimolar amounts of butyl alaninate 1.30,
triphenyl phosphine, diethylazodicarboxylate and triethylamine in
tetrahydrofuran, to give the bisamidate product 1.31.
[1039] Using the above procedures, but employing, in place of ethyl
3-amino-2-methylpropionate 1.27 or butyl alaninate 1.30, different
aminoesters 1.9, the corresponding products 1.5 are obtained.
[1040] The activated phosphonic acid derivative 1.7 is also
converted into the bisamidate 1.5 via the diamino compound 1.10.
The conversion of activated phosphonic acid derivatives such as
phosphoryl chlorides into the corresponding amino analogs 1.10, by
reaction with ammonia, is described in Organic Phosphorus
Compounds, G. M. Kosolapoff, L. Maeir, eds, Wiley, 1976. The
diamino compound 1.10 is then reacted at elevated temperature with
a haloester 1.12, in a polar organic solvent such as
dimethylformamide, in the presence of a base such as
dimethylaminopyridine or potassium carbonate, to yield the
bisamidate 1.5.
[1041] An example of this procedure is shown in Scheme 1, Example
6. In this method, a dichlorophosphonate 1.23 is reacted with
ammonia to afford the diamide 1.37. The reaction is performed in
aqueous, aqueous alcoholic or alcoholic solution, at reflux
temperature. The resulting diamino compound is then reacted with
two molar equivalents of ethyl 2-bromo-3-methylbutyrate 1.38, in a
polar organic solvent such as N-methylpyrrolidinone at ca
150.degree. C., in the presence of a base such as potassium
carbonate, and optionally in the presence of a catalytic amount of
potassium iodide, to afford the bisamidate product 1.39.
[1042] Using the above procedures, but employing, in place of ethyl
2-bromo-3-methylbutyrate 1.38, different haloesters 1.12 the
corresponding products 1.5 are obtained.
[1043] The procedures shown in Scheme 1 are also applicable to the
preparation of bisamidates in which the aminoester moiety
incorporates different functional groups. Scheme 1, Example 7
illustrates the preparation of bisamidates derived from tyrosine.
In this procedure, the monoimidazolide 1.32 is reacted with propyl
tyrosinate 1.40, as described in Example 5, to yield the
monoamidate 1.41. The product is reacted with carbonyl diimidazole
to give the imidazolide 1.42, and this material is reacted with a
further molar equivalent of propyl tyrosinate to produce the
bisamidate product 1.43.
[1044] Using the above procedures, but employing, in place of
propyl tyrosinate 1.40, different aminoesters 1.9, the
corresponding products 1.5 are obtained. The aminoesters employed
in the two stages of the above procedure can be the same or
different, so that bisamidates with the same or different amino
substituents are prepared.
[1045] Scheme 2 illustrates methods for the preparation of
phosphonate monoamidates. In one procedure, a phosphonate monoester
1.1 is converted, as described in Scheme 1, into the activated
derivative 1.8. This compound is then reacted, as described above,
with an aminoester 1.9, in the presence of a base, to afford the
monoamidate product 2.1. The procedure is illustrated in Scheme 2,
Example 1. In this method, a monophenyl phosphonate 2.7 is reacted
with, for example, thionyl chloride, as described in J. Gen. Chem.
USSR., 1983, 32, 367, to give the chloro product 2.8. The product
is then reacted, as described in Scheme 1, with ethyl alaninate
2.9, to yield the amidate 2.10.
[1046] Using the above procedures, but employing, in place of ethyl
alaninate 2.9, different aminoesters 1.9, the corresponding
products 2.1 are obtained.
[1047] Alternatively, the phosphonate monoester 1.1 is coupled, as
described in Scheme 1, with an aminoester 1.9 to produce the
amidate 2.1. If necessary, the R.sup.1 substituent is then altered,
by initial cleavage to afford the phosphonic acid 2.2. The
procedures for this transformation depend on the nature of the
R.sup.1 group, and are described above. The phosphonic acid is then
transformed into the ester amidate product 2.3, by reaction with
the hydroxy compound R.sup.3OH, in which the group R.sup.3 is aryl,
heteroaryl, alkyl, cycloalkyl, haloalkyl etc, using the same
coupling procedures (carbodiimide, Aldrithiol-2, PYBOP, Mitsonobu
reaction etc) described in Scheme 1 for the coupling of amines and
phosphonic acids. ##STR342## ##STR343## ##STR344##
[1048] Examples of this method are shown in Scheme 2, Examples and
2 and 3. In the sequence shown in Example 2, a monobenzyl
phosphonate 2.11 is transformed by reaction with ethyl alaninate,
using one of the methods described above, into the monoamidate
2.12. The benzyl group is then removed by catalytic hydrogenation
in ethyl acetate solution over a 5% palladium on carbon catalyst,
to afford the phosphonic acid amidate 2.13. The product is then
reacted in dichloromethane solution at ambient temperature with
equimolar amounts of 1-(dimethylaminopropyl)-3-ethylcarbodiimide
and trifluoroethanol 2.14, for example as described in Tet. Lett.,
2001, 42, 8841, to yield the amidate ester 2.15.
[1049] In the sequence shown in Scheme 2, Example 3, the
monoamidate 2.13 is coupled, in tetrahydrofuran solution at ambient
temperature, with equimolar amounts of dicyclohexyl carbodiimide
and 4-hydroxy-N-methylpiperidine 2.16, to produce the amidate ester
product 2.17.
[1050] Using the above procedures, but employing, in place of the
ethyl alaninate product 2.12 different monoacids 2.2, and in place
of trifluoroethanol 2.14 or 4-hydroxy-N-methylpiperidine 2.16,
different hydroxy compounds R.sup.3OH, the corresponding products
2.3 are obtained.
[1051] Alternatively, the activated phosphonate ester 1.8 is
reacted with ammonia to yield the amidate 2.4. The product is then
reacted, as described in Scheme 1, with a haloester 2.5, in the
presence of a base, to produce the amidate product 2.6. If
appropriate, the nature of the R.sup.1 group is changed, using the
procedures described above, to give the product 2.3. The method is
illustrated in Scheme 2, Example 4. In this sequence, the
monophenyl phosphoryl chloride 2.18 is reacted, as described in
Scheme 1, with ammonia, to yield the amino product 2.19. This
material is then reacted in N-methylpyrrolidinone solution at
170.degree. C. with butyl 2-bromo-3-phenylpropionate 2.20 and
potassium carbonate, to afford the amidate product 2.21. Using
these procedures, but employing, in place of butyl
2-bromo-3-phenylpropionate 2.20, different haloesters 2.5, the
corresponding products 2.6 are obtained.
[1052] The monoamidate products 2.3 are also prepared from the
doubly activated phosphonate derivatives 1.7. In this procedure,
examples of which are described in Synlett., 1998, 1, 73, the
intermediate 1.7 is reacted with a limited amount of the aminoester
1.9 to give the mono-displacement product 1.11. The latter compound
is then reacted with the hydroxy compound R.sup.3OH in a polar
organic solvent such as dimethylformamide, in the presence of a
base such as diisopropylethylamine, to yield the monoamidate ester
2.3.
[1053] The method is illustrated in Scheme 2, Example 5. In this
method, the phosphoryl dichloride 2.22 is reacted in
dichloromethane solution with one molar equivalent of ethyl
N-methyl tyrosinate 2.23 and dimethylaminopyridine, to generate the
monoamidate 2.24. The product is then reacted with phenol 2.25 in
dimethylformamide containing potassium carbonate, to yield the
ester amidate product 2.26.
[1054] Using these procedures, but employing, in place of ethyl
N-methyl tyrosinate 2.23 or phenol 2.25, the aminoesters 1.9 and/or
the hydroxy compounds R.sup.3OH, the corresponding products 2.3 are
obtained. ##STR345## ##STR346##
[1055] Scheme 3 illustrates methods for the preparation of
carboalkoxy-substituted phosphonate diesters in which one of the
ester groups incorporates a carboalkoxy substituent.
[1056] In one procedure, a phosphonate monoester 1.1, prepared as
described above, is coupled, using one of the methods described
above, with a hydroxyester 3.1, in which the groups R.sup.4 and
R.sup.5 are as described in Scheme 1. For example, equimolar
amounts of the reactants are coupled in the presence of a
carbodiimide such as dicyclohexyl carbodiimide, as described in
Aust. J. Chem., 1963, 609, optionally in the presence of
dimethylaminopyridine, as described in Tet., 1999, 55, 12997. The
reaction is conducted in an inert solvent at ambient
temperature.
[1057] The procedure is illustrated in Scheme 3, Example 1. In this
method, a monophenyl phosphonate 3.9 is coupled, in dichloromethane
solution in the presence of dicyclohexyl carbodiimide, with ethyl
3-hydroxy-2-methylpropionate 3.10 to yield the phosphonate mixed
diester 3.11.
[1058] Using this procedure, but employing, in place of ethyl
3-hydroxy-2-methylpropionate 3.10, different hydroxyesters 3.1, the
corresponding products 3.2 are obtained.
[1059] The conversion of a phosphonate monoester 1.1 into a mixed
diester 3.2 is also accomplished by means of a Mitsonobu coupling
reaction with the hydroxyester 3.1, as described in Org. Lett.,
2001, 643. In this method, the reactants 1.1 and 3.1 are combined
in a polar solvent such as tetrahydrofuran, in the presence of a
triarylphosphine and a dialkyl azodicarboxylate, to give the mixed
diester 3.2. The R.sup.1 substituent is varied by cleavage, using
the methods described previously, to afford the monoacid product
3.3. The product is then coupled, for example using methods
described above, with the hydroxy compound R.sup.3OH, to give the
diester product 3.4.
[1060] The procedure is illustrated in Scheme 3, Example 2. In this
method, a monoallyl phosphonate 3.12 is coupled in tetrahydrofuran
solution, in the presence of triphenylphosphine and
diethylazodicarboxylate, with ethyl lactate 3.13 to give the mixed
diester 3.14. The product is reacted with tris(triphenylphosphine)
rhodium chloride (Wilkinson catalyst) in acetonitrile, as described
previously, to remove the allyl group and produce the monoacid
product 3.15. The latter compound is then coupled, in pyridine
solution at ambient temperature, in the presence of dicyclohexyl
carbodiimide, with one molar equivalent of 3-hydroxypyridine 3.16
to yield the mixed diester 3.17.
[1061] Using the above procedures, but employing, in place of the
ethyl-lactate 3.13 or 3-hydroxypyridine, a different hydroxyester
3.1 and/or a different hydroxy compound R.sup.3OH, the
corresponding products 3.4 are obtained.
[1062] The mixed diesters 3.2 are also obtained from the monoesters
1.1 via the intermediacy of the activated monoesters 3.5. In this
procedure, the monoester 1.1 is converted into the activated
compound 3.5 by reaction with, for example, phosphorus
pentachloride, as described in J. Org. Chem., 2001, 66, 329, or
with thionyl chloride or oxalyl chloride (Lv=Cl), or with
triisopropylbenzenesulfonyl chloride in pyridine, as described in
Nucleosides and Nucleotides, 2000, 19, 1885, or with carbonyl
diimidazole, as described in J. Med. Chem., 2002, 45, 1284. The
resultant activated monoester is then reacted with the hydroxyester
3.1, as described above, to yield the mixed diester 3.2.
[1063] The procedure is illustrated in Scheme 3, Example 3. In this
sequence, a monophenyl phosphonate 3.9 is reacted, in acetonitrile
solution at 70.degree. C., with ten equivalents of thionyl
chloride, so as to produce the phosphoryl chloride 3.19. The
product is then reacted with ethyl 4-carbamoyl-2-hydroxybutyrate
3.20 in dichloromethane containing triethylamine, to give the mixed
diester 3.21.
[1064] Using the above procedures, but employing, in place of ethyl
4-carbamoyl-2-hydroxybutyrate 3.20, different hydroxyesters 3.1,
the corresponding products 3.2 are obtained.
[1065] The mixed phosphonate diesters are also obtained by an
alternative route for incorporation of the R.sup.3O group into
intermediates 3.3 in which the hydroxyester moiety is already
incorporated. In this procedure, the monoacid intermediate 3.3 is
converted into the activated derivative 3.6 in which Lv is a
leaving group such as chloro, imidazole, and the like, as
previously described. The activated intermediate is then reacted
with the hydroxy compound R.sup.3OH, in the presence of a base, to
yield the mixed diester product 3.4.
[1066] The method is illustrated in Scheme 3, Example 4. In this
sequence, the phosphonate monoacid 3.22 is reacted with
trichloromethanesulfonyl chloride in tetrahydrofuran containing
collidine, as described in J. Med. Chem., 1995, 38, 4648, to
produce the trichloromethanesulfonyloxy product 3.23. This compound
is reacted with 3-(morpholinomethyl)phenol 3.24 in dichloromethane
containing triethylamine, to yield the mixed diester product
3.25.
[1067] Using the above procedures, but employing, in place of with
3-(morpholinomethyl)phenol 3.24, different carbinols R.sup.3OH, the
corresponding products 3.4 are obtained.
[1068] The phosphonate esters 3.4 are also obtained by means of
alkylation reactions performed on the monoesters 1.1. The reaction
between the monoacid 1.1 and the haloester 3.7 is performed in a
polar solvent in the presence of a base such as
diisopropylethylamine, as described in Anal. Chem., 1987, 59, 1056,
or triethylamine, as described in J. Med. Chem., 1995, 38, 1372, or
in a non-polar solvent such as benzene, in the presence of
18-crown-6, as described in Syn. Comm., 1995, 25, 3565.
[1069] The method is illustrated in Scheme 3, Example 5. In this
procedure, the monoacid 3.26 is reacted with ethyl
2-bromo-3-phenylpropionate 3.27 and diisopropylethylamine in
dimethylformamide at 80.degree. C. to afford the mixed diester
product 3.28.
[1070] Using the above procedure, but employing, in place of ethyl
2-bromo-3-phenylpropionate 3.27, different haloesters 3.7, the
corresponding products 3.4 are obtained. ##STR347## ##STR348##
[1071] Scheme 4 illustrates methods for the preparation of
phosphonate diesters in which both the ester substituents
incorporate carboalkoxy groups.
[1072] The compounds are prepared directly or indirectly from the
phosphonic acids 1.6. In one alternative, the phosphonic acid is
coupled with the hydroxyester 4.2, using the conditions described
previously in Schemes 1-3, such as coupling reactions using
dicyclohexyl carbodiimide or similar reagents, or under the
conditions of the Mitsonobu reaction, to afford the diester product
4.3 in which the ester substituents are identical.
[1073] This method is illustrated in Scheme 4, Example 1. In this
procedure, the phosphonic acid 1.6 is reacted with three molar
equivalents of butyl lactate 4.5 in the presence of Aldrithiol-2
and triphenyl phosphine in pyridine at ca. 70.degree. C., to afford
the diester 4.6.
[1074] Using the above procedure, but employing, in place of butyl
lactate 4.5, different hydroxyesters 4.2, the corresponding
products 4.3 are obtained.
[1075] Alternatively, the diesters 4.3 are obtained by alkylation
of the phosphonic acid 1.6 with a haloester 4.1. The alkylation
reaction is performed as described in Scheme 3 for the preparation
of the esters 3.4.
[1076] This method is illustrated in Scheme 4, Example 2. In this
procedure, the phosphonic acid 1.6 is reacted with excess ethyl
3-bromo-2-methylpropionate 4.7 and diisopropylethylamine in
dimethylformamide at ca. 80.degree. C., as described in Anal.
Chem., 1987, 59, 1056, to produce the diester 4.8.
[1077] Using the above procedure, but employing, in place of ethyl
3-bromo-2-methylpropionate 4.7, different haloesters 4.1, the
corresponding products 4.3 are obtained.
[1078] The diesters 4.3 are also obtained by displacement reactions
of activated derivatives 1.7 of the phosphonic acid with the
hydroxyesters 4.2. The displacement reaction is performed in a
polar solvent in the presence of a suitable base, as described in
Scheme 3. The displacement reaction is performed in the presence of
an excess of the hydroxyester, to afford the diester product 4.3 in
which the ester substituents are identical, or sequentially with
limited amounts of different hydroxyesters, to prepare diesters 4.3
in which the ester substituents are different. The methods are
illustrated in Scheme 4, Examples 3 and 4. As shown in Example 3,
the phosphoryl dichloride 2.22 is reacted with three molar
equivalents of ethyl 3-hydroxy-2-(hydroxymethyl)propionate 4.9 in
tetrahydrofuran containing potassium carbonate, to obtain the
diester product 4.10.
[1079] Using the above procedure, but employing, in place of ethyl
3-hydroxy-2-(hydroxymethyl)propionate 4.9, different hydroxyesters
4.2, the corresponding products 4.3 are obtained.
[1080] Scheme 4, Example 4 depicts the displacement reaction
between equimolar amounts of the phosphoryl dichloride 2.22 and
ethyl 2-methyl-3-hydroxypropionate 4.11, to yield the monoester
product 4.12. The reaction is conducted in acetonitrile at
70.degree. C. in the presence of diisopropylethylamine. The product
4.12 is then reacted, under the same conditions, with one molar
equivalent of ethyl lactate 4.13, to give the diester product
4.14.
[1081] Using the above procedures, but employing, in place of ethyl
2-methyl-3-hydroxypropionate 4.11 and ethyl lactate 4.13,
sequential reactions with different hydroxyesters 4.2, the
corresponding products 4.3 are obtained. ##STR349## ##STR350##
##STR351##
[1082] Following the similar procedures, replacement of amino acid
esters 820 with lactates 821 (Scheme 1003) provides mono-phosphonic
lactates 823. Lactates 823 are useful intermediates to form the
phosphonate compounds of the invention. ##STR352## ##STR353##
##STR354##
Example 1
[1083] To a solution of 2-aminoethylphosphonic acid (1.26 g, 10.1
mmol) in 2N NaOH (10.1 mL, 20.2 mmol) was added benzyl
chloroformate (1.7 mL, 12.1 mmol). After the reaction mixture was
stirred for 2 d at room temperature, the mixture was partitioned
between Et.sub.2O and water. The aqueous phase was acidified with
6N HCl until pH=2. The resulting colorless solid was dissolved in
MeOH (75 mL) and treated with Dowex 50 W.times.8-200 (7 g). After
the mixture was stirred for 30 minutes, it was filtered and
evaporated under reduced pressure to give carbamate 28 (2.37 g,
91%) as a colorless solid (Scheme 1005).
[1084] To a solution of carbamate 28 (2.35 g, 9.1 mmol) in pyridine
(40 mL) was added phenol (8.53 g, 90.6 mmol) and
1,3-dicyclohexylcarbodiimide (7.47 g, 36.2 mmol). After the
reaction mixture was warmed to 70.degree. C. and stirred for 5 h,
the mixture was diluted with CH.sub.3CN and filtered. The filtrate
was concentrated under reduced pressure and diluted with EtOAc. The
organic phase was washed with sat. NH.sub.4Cl, sat. NaHCO.sub.3,
and brine, then dried over Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was
chromatographed on silica gel twice (eluting 40-60% EtOAc/hexane)
to give phosphonate 29 (2.13 g, 57%) as a colorless solid.
[1085] To a solution of phosphonate 29 (262 mg, 0.637 mmol) in
iPrOH (5 mL) was added TFA (0.05 mL, 0.637 mmol) and 10% Pd/C (26
mg). After the reaction mixture was stirred under H.sub.2
atmosphere (balloon) for 1 h, the mixture was filtered through
Celite. The filtrate was evaporated under reduced pressure to give
amine 30 (249 mg, 100%) as a colorless oil (Scheme 1005).
Scheme Section A
[1086] Exemplary methods of preparing the compounds of the
invention are shown in Schemes 1-7 below. A detailed description of
the methods is found in the Experimental section below. ##STR355##
##STR356## ##STR357## ##STR358## ##STR359## ##STR360## ##STR361##
##STR362## ##STR363## ##STR364## Scheme Section B
[1087] Alternative exemplary methods of preparing the compounds of
the invention are shown in Schemes 101-113 below. ##STR365##
[1088] Treatment of commercially available epoxide 1 with sodium
azide (Bioorg. & Med. Chem. Lett., 5, 459, 1995) furnishes the
azide intermediate 2. The free hydroxyl is converted to benzyl
ether 3 by treating it with benzyl bromide in the presence of base
such as potassium carbonate. Compound 4 is achieved by the
reduction of the azide group with triphenyl phosphine, as described
in the publication Bioorg. & Med. Chem. Lett., 7, 1847, 1997.
Conversion of the amino group to its sulfonamide derivative 5 is
achieved by treating the amine with stoichiometric amounts of
sulfonyl chloride. Regioselective alkylation is performed (as shown
in the article J. Med. Chem., 40, 2525, 1997) on the sulfonamide
nitrogen using the iodide 6 (J. Med. Chem., 35, 2958, 1992) to get
the compound 7. Upon TFA catalyzed deprotection of BOC group
followed by the reaction with bisfuranyl carbonate 8 (for a similar
coupling see, J. Med. Chem., 39, 3278, 1996) furnishes the compound
9. Final deprotection of the protecting groups by catalytic
hydrogenolysis result the compound 10. ##STR366##
[1089] The sulfonamide 11 is readily alkylated with the iodide 6
(J. Med. Chem., 35, 2958, 1992) to get the intermediate 12.
Regioselective epoxide opening (JP-9124630) of the epoxide 1 with
12 furnishes the intermediate 13. Deprotection of the BOC group
followed by the treatment of bisfuranyl carbonate 8 yields the
intermediate 14 which is subjected to hydrogenation to furnish the
compound 10. ##STR367##
[1090] The epoxide 1 is converted to the aminohydroxyl derivative
15 using the known procedure (J. Med. Chem., 37, 1758, 1994).
Sulfonylation of 15 using benzene sulfonylchloride affords the
compound 16. Installation of the side chain to get the intermediate
13 is achieved by alkylation of sulfonamide nitrogen with iodide 6.
The intermediate 13 is converted to the compound 10 using the same
sequence as shown in scheme 102. ##STR368##
[1091] Sulfonamide 5 is alkylated under basic conditions using the
allyl bromide 17 (Chem. Pharm. Bull., 30, 111, 1982) to get the
intermediate 18. Similar transformation is reported in literature
(J. Med. Chem., 40, 2525, 1997). Hydrolysis of BOC group with TFA
and acylation of the resulting amine 19 with bisfuranyl carbonate 8
yields the compound 20. Hydrogenation using Pd/C catalysis under
H.sub.2 atmosphere affords the phosphonic acid 21. ##STR369##
##STR370##
[1092] Sulfonamide 5 is converted to 22 via hydrolysis of BOC group
with TFA and acylation with bisfuranyl carbonate 8. The sulfonamide
22 is alkylated with the bromide 23 (J. Med. Chem., 40, 2525, 1997)
to get the compound 24, which upon hydrogenolysis gives the
catechol 25. Alkylation of the phenolic groups using
dibenzylhydroxymethyl phosphonate (J. Org. Chem., 53, 3457, 1988)
affords regioisomeric compounds 26 and 27. These compounds 26 and
27 are hydrogenated to get the phosphonic acids 28 and 29,
respectively. Individual cyclic phosphonic acids 30 and 31 are
obtained under basic (like NaH) conditions (U.S. Pat. No.
5,886,179) followed by hydrogenolysis of the dibenzyl ester
derivatives 26 and 27.
Scheme 106
[1093] In this route, compound 25 is obtained by conducting a
reaction between the epoxide 32 and the sulfonamide 33 using the
conditions described in the Japanese Patent No. 9124630.
##STR371##
[1094] Epoxide 32 and sulfonamide 33 are synthesized utilizing
similar methodology delineated in the same patent. ##STR372##
[1095] Compound 34 is obtained from 32 using similar sequence
depicted in J. Med. Chem., 37, 1758, 1994. Reductive amination (for
similar transformation see WO 00/47551) of compound 34 with
aldehyde 35 furnishes the intermediate 36 which is converted to the
compound 25 by sulfonylation followed by hydrogenation.
##STR373##
[1096] Treatment of epoxide 32 with sulfonamides 37 and/or 38 under
conditions described in Japanese Patent No. 9124630 furnishes 26
and 27.
Scheme 109
[1097] Reductive amination of aminohydroxyl intermediate 34 with
the aldehydes 39 and 40 as described in patent WO 00/47551, furnish
41 and 42 which undergoes smooth sulfonylation to give 26 and 27.
##STR374## ##STR375## Scheme 110
[1098] In an alternative approach, where epoxide 32 is opened with
benzyl amines 43 and 44 under conditions described above furnishes
41 and 42, respectively. Similar transformations were documented in
the Japanese Patent No. 9124630. ##STR376## ##STR377##
[1099] Reductive amination of the bromoaldehyde 45 (J. Organomet.
Chem., FR; 122, 123, 1976) with the amine 34 gives 46 which then
undergoes sulfonylation to furnish 47. The bromoderivative 47 is
converted to the phosphonate 48 under Michaelis-Arbuzov reaction
conditions (Bioorg. Med. Chem. Lett., 9, 3069, 1999). Final
hydrogenation of 48 delivers the phosphonic acid 49. ##STR378##
[1100] The intermediate 48 is also obtained as shown in scheme 112.
Reductive amination of the aldehyde 52 with the amine 34 offers the
phosphonate 52 and sulfonylation of this intermediate furnishes 48.
##STR379##
[1101] Alternatively, compound 52 is obtained from the epoxide 32
by a ring opening reaction with the aminophosphonate 53 (Scheme
113).
Scheme Section C
[1102] Scheme 9 is described in the Examples. ##STR380## Scheme
Section D
[1103] The following schemes are described in the Examples.
##STR381## ##STR382## ##STR383## ##STR384## Scheme Section E
[1104] Schemes 1-3 are described in the examples. ##STR385##
##STR386## ##STR387## Scheme Section F
[1105] Schemes 1-5 are described in the examples. ##STR388##
##STR389## ##STR390## ##STR391## ##STR392## Scheme Section G
[1106] Schemes 1 to 9 are described in the examples. ##STR393##
##STR394## ##STR395## ##STR396## ##STR397## ##STR398## ##STR399##
##STR400## ##STR401## ##STR402## ##STR403## ##STR404## ##STR405##
Scheme Section H
[1107] Schemes 1 to 14 are described in the examples. ##STR406##
##STR407## ##STR408## ##STR409## ##STR410## ##STR411## ##STR412##
##STR413## ##STR414## ##STR415## ##STR416## ##STR417## ##STR418##
##STR419## ##STR420## Scheme Section I
[1108] Schemes 1 to 3 are described in the examples. ##STR421##
##STR422## ##STR423## Scheme Section T
[1109] Schemes 14 are described in the examples. ##STR424##
##STR425## ##STR426## ##STR427## ##STR428## ##STR429## Scheme
Section K
[1110] Schemes 1 to 9 are described in the examples. ##STR430##
##STR431## ##STR432## ##STR433## ##STR434## ##STR435## ##STR436##
##STR437## ##STR438## Scheme Section L
[1111] Schemes 1-9 are described in the examples. ##STR439##
##STR440## ##STR441## ##STR442## TABLE-US-00003 Scheme 4 Synthesis
of Bisamidates ##STR443## ##STR444## Compound R.sub.1 R.sub.2 16a
Gly-Et Gly-Et 16b Gly-Bu Gly-Bu 16j Phe-Bu Phe-Bu 16k NHEt NHEt
[1112] TABLE-US-00004 Scheme 5 Synthesis of Monoamidates ##STR445##
##STR446## Compound R.sub.1 R.sub.2 30a OPh Ala-Me 30b OPh Ala-Et
30c OPh (D)-Ala-iPr 30d OPh Ala-Bu 30e OBn Ala-Et
[1113] ##STR447## TABLE-US-00005 Scheme 7 Synthesis of Lactates
##STR448## ##STR449## Compound R.sub.1 R.sub.2 31a OPh Lac-iPr 31b
OPh Lac-Et 31c OPh Lac-Bu 31d OPh (R)-Lac-Me 31e OPh (R)-Lac-Et
[1114] ##STR450## ##STR451##
Examples
[1115] The following Examples refer to the Schemes.
[1116] Some Examples have been performed multiple times. In
repeated Examples, reaction conditions such as time, temperature,
concentration and the like, and yields were within normal
experimental ranges. In repeated Examples where significant
modifications were made, these have been noted where the results
varied significantly from those described. In Examples where
different starting materials were used, these are noted. When the
repeated Examples refer to a "corresponding" analog of a compound,
such as a "corresponding ethyl ester", this intends that an
otherwise present group, in this case typically a methyl ester, is
taken to be the same group modified as indicated.
Example Section A
Example 1
[1117] Diazo Ketone 1: To a solution of
N-tert-Butoxycarbonyl-O-benzyl-L-tyrosine (11 g, 30 mmol, Fluka) in
dry THF (55 mL) at -25-30.degree. C. (external bath temperature)
was added isobutylchloroformate (3.9 mL, 30 mmol) followed by the
slow addition of N.methylmorpholine (3.3 mL, 30 mmol). The mixture
was stirred for 25 min, filtered while cold, and the filter cake
was rinsed with cold (0.degree. C.) THF (50 mL), The filtrate was
cooled to -25.degree. C. and diazomethane (.about.50 mmol,
generated from 15 g Diazald according to Aldrichimica Acta 1983,
16, 3) in ether (.about.150 nm) was poured into the mixed anhydride
solution. The reaction was stirred for 15 min and was then placed
in an icebath at 0.degree. C., allowing the bath to warm to room
temperature while stirring overnight for 15 h. The solvent was
evaporated under reduced pressure and the residue was dissolved in
EtOAc, washed with water, saturated NaHCO.sub.3, saturated NaCl,
dried (MgSO.sub.4), filtered and evaporated to a pale yellow solid.
The crude solid was slurried in hexane, filtered, and dried to
afford the diazo ketone (10.9 g, 92%) which was used directly in
the next step.
Example 2
[1118] Chloroketone 2: To a suspension of diazoketone 1 (10.8 g, 27
mmol) in ether (606 mL) at 0.degree. C. was added 4M HCl in dioxane
(7.5 mL, 30 mmol). The solution was removed from the cooling bath,
and allowed to warm to room temperature at which time the reaction
was stirred 1 h. The reaction solvent was evaporated under reduced
pressure to give a solid residue that was dissolved in ether and
passed through a short column of silica gel. The solvent was
evaporated to afford the chloroketone (10.7 g, 97%) as a solid.
Example 3
[1119] Chloroalcohol 3: To a solution of chloroketone 2 (10.6 g, 26
mmol) in THF (90 mL) was added water (10 mL) and the solution was
cooled to 3-4.degree. C. (internal temperature). A solution of
NaBH.sub.4 (1.5 g, 39 mmol) in water (5 mL) was added dropwise over
a period of 10 min. The mixture was stirred for 1 h at 0.degree. C.
and saturated KHSO.sub.4 was slowly added until the pH<4
followed by saturated NaCl. The organic phase was washed with
saturated NaCl, dried (MgSO.sub.4) filtered and evaporated under
reduced pressure. The crude product consisted of a 70:30 mixture of
diastereomers by HPLC analysis (mobile phase,
77:25-CH.sub.3CN:H.sub.2O; flow rate: 1 mL/min; detection: 254 nm;
sample volume: 20 .mu.L; column: 5.mu. C18, 4.6.times.250 mm,
Varian; retention times: major diastereomer 3, 5.4 min, minor
diastereomer 4, 6.1 min). The residue was recrystallized from
EtOAc/hexane twice to afford the chloro alcohol 3 (4.86 g, >99%
diastereomeric purity by HPLC analysis) as a white solid.
Example 4
[1120] Epoxide 5: A solution of chloroalcohol 3 (4.32 g, 10.6 mmol)
in EtOH (250 mL) and THF (100 mL) was treated with K.sub.2CO.sub.3
(4.4 g, 325 mesh, 31.9 mmol) and the mixture was stirred for at
room temperature for 20 h. The reaction mixture was filtered and
was evaporated under reduced pressure. The residue was partitioned
between EtOAc and water and the organic phase was washed with
saturated NaCl, dried (MgSO.sub.4), filtered, and evaporated under
reduced pressure. The crude product was chromatographed on silica
gel to afford the epoxide (3.68 g, 94%) as a white solid.
Example 5
[1121] Sulfonamide 6: To a suspension of epoxide 5 (2.08 g, 5.6
mmol) in 2-propanol (20 mL) was added isobutylamine (10.7 mL, 108
mmol) and the solution was refluxed for 30 min. The solution was
evaporated under reduced pressure and the crude solid was dissolved
in CH.sub.2Cl.sub.2 (20 mL) and cooled to 0.degree. C.
N,N'-diisopropylethylamine (1.96 mL, 11.3 mmol) was added followed
by the addition of 4-methoxybenzenesulfonyl chloride (1.45 g, 7
mmol) in CH.sub.2Cl.sub.2 (5 mL) and the solution was stirred for
40 min at 0.degree. C., warmed to room temperature and evaporated
under reduced pressure. The residue was partitioned between EtOAc
and saturated NaHCO.sub.3. The organic phase was washed with
saturated NaCl, dried (MgSO.sub.4), filtered and evaporated under
reduced pressure. The crude product was recrystallized from
EtOAc/hexane to give the sulfonamide (2.79 g, 81%) as a small white
needles: mp 122-124.degree. C. (uncorrected).
Example 6
[1122] Carbamate 7: A solution of sulfonamide 6 (500 mg, 0.82 mmol)
in CH.sub.2Cl.sub.2 (5 mL) at 0.degree. C. was treated with
trifluoroacetic acid (5 mL). The solution was stirred at 0.degree.
C. for 30 min and was removed from the cold bath stirring for an
additional 30 min. Volatiles were evaporated under reduced pressure
and the residue was partitioned between CH.sub.2Cl.sub.2 and
saturated NaHCO.sub.3. The aqueous phase was extracted twice with
CH.sub.2Cl.sub.2 and the combined organic extracts were washed with
saturated NaCl, dried (MgSO.sub.4), filtered, and evaporated under
reduced pressure. The residue was dissolved in CH.sub.3CN (5 mL)
and was treated with (3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl
4-nitrophenyl carbonate (263 mg, 0.89 mmol, prepared according to
Ghosh et al., J. Med. Chem., 1996, 39, 3278.) and
N,N-dimethylaminopyridine (197 mg, 1.62 mmol). After stirring for
1.5 h at room temperature, the reaction solvent was evaporated
under reduced pressure and the residue was partitioned between
EtOAc and 5% citric acid. The organic phase was washed twice with
1% K.sub.2CO.sub.3, and then was washed with saturated NaCl dried
(MgSO.sub.4), filtered, and evaporated under reduced pressure. The
crude product was purified by chromatography on silica gel
(1/1-EtOAc/hexane) affording the carbamate (454 mg, 83%) as a
solid: mp 128-129.degree. C. (MeOH, uncorrected).
Example 7
[1123] Phenol 8: A solution of carbamate 7 (1.15 g, 1.7 mmol) in
EtOH (50 mL) and EtOAc (20 mL) was treated with 10% Pd/C (115 mg)
and was stirred under H.sub.2 atmosphere (balloon) for 18 h. The
reaction solution was purged with N.sub.2, filtered through a 0.45
.mu.M filter and was evaporated under reduced pressure to afford
the phenol as a solid that contained residual solvent: mp
131-134.degree. C. (EtOAc/hexane, uncorrected).
Example 8
[1124] Dibenzylphosphonate 10: To a solution of
dibenzylhydroxymethyl phosphonate (527 mg, 1.8 mmol) in
CH.sub.2Cl.sub.2 (5 mL) was treated with 2,6-lutidine (300 .mu.L,
2.6 mmol) and the reaction flask was cooled to -50.degree. C.
(external temperature). Trifluoromethanesulfonic anhydride (360
.mu.L, 2.1 mmol) was added and the reaction mixture was stirred for
15 min and then the cooling bath was allowed to warm to 0.degree.
C. over 45 min. The reaction mixture was partitioned between ether
and ice-cold water. The organic phase was washed with cold 1M
H.sub.3PO.sub.4, saturated NaCl, dried (MgSO.sub.4), filtered and
evaporated under reduced pressure to afford triflate 9 (697 mg,
91%) as an oil which was used directly without any further
purification. To a solution of phenol 8 (775 mg, 1.3 mmol) in THF
(5 mL) was added Cs.sub.2CO.sub.3 (423 mg, 1.3 mmol) and triflate 9
(710 mg, 1.7 mmol) in THF (2 mL). After stirring the reaction
mixture for 30 min at room temperature additional Cs.sub.2CO.sub.3
(423 mg, 1.3 mmol and triflate (178 mg, 0.33 mmol) were added and
the mixture was stirred for 3.5 h. The reaction mixture was
evaporated under reduced pressure and the residue was partitioned
between EtOAc and saturated NaCl. The organic phase was dried
(MgSO.sub.4), filtered and evaporated under reduced pressure. The
crude product was chromatographed on silica gel eluting (5%
2-propanol/CH.sub.2Cl.sub.2) to give the dibenzylphosphonate as an
oil that solidified upon standing. The solid was dissolved in
EtOAc, ether was added, and the solid was precipitated at room
temperature overnight. After cooling to 0.degree. C., the solid was
filtered and washed with cold ether to afford the
dibenzylphosphonate (836 mg, 76%) as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 7.66 (d, 2H), 7.31 (s, 10H), 7.08 (d, 2H),
6.94 (d, 2H), 6.76 (d, 2H), 5.59 (d, 1H), 5.15-4.89 (m, 6H), 4.15
(d, 2H), 3.94-3.62 (m, 10H), 3.13-2.69 (m, 7H), 1.78 (m, 1H),
1.70-1.44 (m, 2H), 0.89-0.82 (2d, 6H); .sup.31P NMR (CDCl.sub.3) a
18.7; MS (ESI) 853 (M+H).
Example 9
[1125] Phosphonic acid 11: A solution of dibenzylphosphonate 10
(0.81 g) was dissolved in EtOH/EtOAc (30 mL/10 mL), treated with
10% Pd/C (80 mg) and was stirred under H.sub.2 atmosphere (balloon)
for 1.5 h. The reaction was purged with N.sub.2, and the catalyst
was removed by filtration through celite. The filtrate was
evaporated under reduced pressure and the residue was dissolved in
MeOH and filtered with a 0.45 .mu.M filter. After evaporation of
the filtrate, the residue was triturated with ether and the solid
was collected by filtration to afford the phosphonic acid (634 mg,
99%) as a white solid: .sup.1H NMR (CDCl.sub.3) .delta. 7.77 (d,
2H), 7.19 (d, 2H), 7.09 (d, 2H), 6.92 (d, 2H), 5.60 (d, 1H), 4.95
(m, 1H), 4.17 (d, 2H), 3.94 (m, 1H), 3.89 (s, 3H), 3.85-3.68 (m,
5H), 3.42 (dd, 1H), 3.16-3.06 (m, 2H), 2.96-2.84 (m, 3H), 2.50 (m,
1H), 2.02 (m, 1H), 1.58 (m, 1H), 1.40 (dd, 1H), 0.94 (d, 3H), 0.89
(d, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 16.2; MS (ESI) 671
(M-H).
Example 10
[1126] Diethylphosphonate 13: Triflate 12 was prepared from diethyl
hydroxymethylphosphonate (2 g, 11.9 mmol), 2,6-lutidine (2.1 mL,
17.9 mmol), and trifluoromethanesulfonic anhydride (2.5 mL, 14.9
mmol) as described for compound 9. To a solution of phenol 8 (60
mg, 0.10 mmol) in THF (2 mL) was added Cs.sub.2CO.sub.3 (65 mg,
0.20 mmol) and triflate 12 (45 mg, 0.15 mmol) in THF (0.25 mL). The
mixture was stirred at room temperature for 2 h and additional
triflate (0.15 mmol) in THF (0.25 mL) was added. After 2 h the
reaction mixture was partitioned between EtOAc and saturated NaCl.
The organic phase was dried (MgSO.sub.4), filtered, and evaporated
under reduced pressure. The crude product was chromatographed on
silica gel (EtOAc) to give a residue that was purified by
chromatography on silica gel (5% 2-propanol/CH.sub.2Cl.sub.2) to
afford the diethylphosphonate as a foam: .sup.1H NMR (CDCl.sub.3)
.delta. 7.66 (d, 2H), 7.10 (d, 2H), 6.94 (d, 2H), 6.82 (d, 2H),
5.60 (d, 1H), 4.97 (d, 2H), 4.23-4.13 (m, 6H), 3.93-3.62 (m, 10H),
3.12-2.68 (m, 7H), 1.84-1.44 (m, 3H), 1.31 (t, 6H), 0.88-0.82 (2d,
6H); .sup.31P NMR (CDCl.sub.3) .delta. 17.7; MS (ESI) 729 (M+M.
Example 11
[1127] Diphenylphosphonate 14: To a solution of 11 (100 mg, 0.15
mmol) and phenol (141 mg, 1.5 mmol) in pyridine (1.5 mL) was added
N,N-diisopropylcarbodiimide (50 .mu.L, 0.38 mmol). The solution was
stirred for 31 h at room temperature and for 20 h at 50.degree. C.
The solvent was evaporated under reduced pressure and the residue
was purified by chromatography on silica gel eluting (EtOAc) to
provide diphenylphosphonate 14 (16 mg) as a foam: .sup.31P NMR
(CDCl.sub.3) .delta. 10.9; MS (ESI) 847 (M+Na).
Example 12
[1128] Bis-Poc-phosphonate 15: To a solution of 11 (50 mg, 0.74
mmol) and isopropylchloromethyl carbonate (29 mg, 0.19 mmol) in DMF
(0.5 mL) was added triethylamine (26 .mu.L, 0.19 mmol) and the
solution was heated at 70.degree. C. (bath temperature) for 4.5 h.
The reaction was concentrated under reduced pressure and the
residue was purified by preparative layer chromatography (2%
2-propanol/CH.sub.2Cl.sub.2) to afford 15 (7 mg): .sup.1H NMR
(CDCl.sub.3) .delta. 7.71 (d, 2H), 7.15 (d, 2H); 7.01 (d, 2H), 6.93
(d, 2H), 5.80-5.71 (m, 4H), 5.67 (d, 1H), 5.07-4.87 (m7, 4H), 4.35
(d, 2H), 4.04-3.68 (m, 10H), 3.13 (dd, 1H), 3.04-2.90 (m, 5H), 2.79
(dd, 1H), 1.88-1.50 (m, 3H+H.sub.2O peak), 1.30 (m, 12H), 0.93 (d,
3H), 0.88 (d, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 19.6.
Example 13
[1129] Synthesis of Bisamidates 16a-j. Representative Procedure,
Bisamidate 16f: A solution of phosphonic acid 11 (100 mg, 0.15
mmol) and (S)-2-aminobutyric acid butyl ester hydrochloride (116
mg, 0.59 mmol) was dissolved in pyridine (5 mL) and the solvent was
distilled under reduced pressure at 40-60.degree. C. The residue
was treated with a solution of Ph.sub.3P (117 mg, 0.45 mmol) and
2,2'-dipyridyl disulfide (98 mg, 0.45 mmol) in pyridine (1 mL)
stirring for 20 h at room temperature. The solvent was evaporated
under reduced pressure and the residue was chromatographed on
silica gel (1% to 5% 2-propanol/CH.sub.2Cl.sub.2). The purified
product was suspended in ether and was evaporated under reduced
pressure to afford bisamidate 16f (106 mg, 75%) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, 2H), 7.15 (d, 2H), 7.01
(d, 2H), 6.87 (d, 2H), 5.67 (d, 1H), 5.05 (m, 1H), 4.96 (d, 1H),
4.19-3.71 (m overlapping s, 18H,), 3.42 (t, 1H), 3.30 (t, 1H), 3.20
(dd, 1H), 3.20-2.97 (m, 4H), 2.80 (dd, 2H), 1.87-1.54 (m, 19H),
1.42-1.35 (4H), 9.97-0.88 (m, 18H); .sup.31P NMR (CDCl.sub.3)
.delta. 20.3; MS (ESI) 955 (M+H), TABLE-US-00006 Compound R.sub.1
R.sub.2 Amino Acid 16a H Et Gly 16b H Bu Gly 16c Me Et Ala 16d Me
Bu Ala 16e Et Et Aba.sup.1 16f Et Bu Aba.sup.1 16g iBu Et Leu 16h
iBu Bu Leu 16i Bn Et Phe 16j Bn Bu Phe .sup.1Aba, 2-aminobutyric
acid
Example 14
[1130] Diazo ketone 17: To a solution of
N-tert-Butoxycarbonyl-p-bromo-L-phenylalanine (9.9 g, 28.8 mmol,
Synthetech) in dry THF (55 mL) at -25-30.degree. C. (external bath
temperature) was added isobutylchloroformate (3.74 mL, 28.8 mmol)
followed by the slow addition of N-methylmorpholine (3.16 mL, 28.8
mmol). The mixture was stirred for 25 min, filtered while cold, and
the filter cake was rinsed with cold (0.degree. C.) THF (50 mL).
The filtrate was cooled to -25.degree. C. and diazomethane
(.about.50 mmol, generated from 15 g diazald according to
Aldrichimica Acta 1983, 16, 3) in ether (.about.150 mL) was poured
into the mixed anhydride solution. The reaction was stirred for 15
min and was then placed in an icebath at 0.degree. C., allowing the
bath to warm to room temperature while stirring overnight for 15 h.
The solvent was evaporated under reduced pressure and the residue
was suspended in ether, washed with water, saturated NaHCO.sub.3,
saturated NaCl, dried (MgSO.sub.4), filtered and evaporated to a
pale yellow solid. The crude solid was slurried in hexane,
filtered, and dried to afford diazo ketone 17 (9.73 g, 90%) which
was used directly in the next step.
Example 15
[1131] Chloroketone 18: To a solution of diazoketone 17 (9.73 g, 26
mmol) in ether (500 mL) at 0.degree. C. was added 4M HCl in dioxane
(6.6 mL, 26 mmol). The solution was stirred for 1 h at 0.degree. C.
and 4M HCl in dioxane (1 mL) was added. After 1 h, the reaction
solvent was evaporated under reduced pressure to afford the
chloroketone 18 (9.79 g, 98%) as a white solid.
Example 16
[1132] Chloroalcohol 19: A solution of chloroketone 18 (9.79 g, 26
mmol) in TH-F (180 mL) and water (16 mL) was cooled to 0.degree. C.
(internal temperature). Solid NaBH.sub.4 (2.5 g, 66 mmol) was added
in several portions over a period of 15 min while maintaining the
internal temperature below 5.degree. C. The mixture was stirred for
45 min and saturated KHSO.sub.4 was slowly added until the pH<3.
The mixture was partitioned between EtOAc and water. The aqueous
phase was extracted with EtOAc and the combined organic extracts
were washed with brine, dried (MgSO.sub.4) filtered and evaporated
under reduced pressure. The residue was dissolved in EtOAc, and was
passed through a short column of silica gel, and the solvent was
evaporated. The solid residue was recrystallized from EtOAc/hexane
to afford the chloroalcohol 19 (3.84 g) as a white solid.
Example 17
[1133] Epoxide 21: A partial suspension of chloroalcohol 19 (1.16
g, 3.1 mmol) in EtOH (50 mL) was treated with K.sub.2CO.sub.3 (2 g,
14.5 mmol) and the mixture was stirred for 4 h at room temperature.
The reaction mixture was diluted with EtOAc, filtered, and the
solvents were evaporated under reduced pressure. The residue was
partitioned between EtOAc and saturated NaCl, and the organic phase
was dried (MgSO.sub.4), filtered, and evaporated under reduced
pressure to afford epoxide 21 (1.05 g, 92%) as a white crystalline
solid.
Example 18
[1134] Sulfonamide 22: To a solution of epoxide 21 (1.05 g, 3.1
mmol) in 2-propanol (40 mL) was added isobutylamine (6 mL, 61 mmol)
and the solution was refluxed for 30 min. The solution was
evaporated under reduced pressure and the crude solid was dissolved
in CH.sub.2Cl.sub.2 (20 mL) and cooled to 0.degree. C.
Triethylamine (642 .mu.L, 4.6 mmol) was added followed by the
addition of (634 mg, 3.4 mmol) in CH.sub.2Cl.sub.2 (5 mL) and the
solution was stirred for 2 h at 0.degree. C. at which time the
reaction solution was treated with additional triethylamine (1.5
mmol) and 4-methoxybenzenesulfonyl chloride (0.31 mmol). After 1.5
h, the reaction solution was evaporated under reduced pressure. The
residue was partitioned between EtOAc and cold 1M H.sub.3PO.sub.4.
The organic phase was washed with saturated NaHCO.sub.3, saturated
NaCl, dried (MgSO.sub.4), filtered and the solvent was evaporated
under reduced pressure. The crude product was purified on silica
gel (15/1-CH.sub.2Cl.sub.2/EtOAc) to afford 1.67 g of a solid which
was recrystallized from EtOAc/hexane to give sulfonamide 22 (1.54
g, 86%) as a white crystalline solid.
Example 19
[1135] Silyl ether 23: To a solution of the sulfonamide 22 (1.53 g,
2.6 mmol) in CH.sub.2Cl.sub.2 (12 mL) at 0.degree. C. was added
N,N-diisopropylethylamine (0.68 mL, 3.9 mmol) followed by
tert-butyldimethylsilyl trifluoromethanesulfonate (0.75 mL, 3.3
mmol). The reaction solution was stirred for 1 h at 0.degree. C.
and was warmed to room temperature, stirring for 17 h Additional
N,N-diisopropylethylamine (3.9 mmol) and tert-butyldimethylsilyl
trifluoromethanesulfonate (1.6 mmol) was added, stirred for 2.5 h,
then heated to reflux for 3 h and stirred at room temperature for
12 h. The reaction mixture was partitioned between EtOAc and cold
1M H.sub.3PO.sub.4. The organic phase was washed with saturated
NaHCO.sub.3, saturated NaCl, and was dried (MgSO.sub.4), filtered
and evaporated under reduced pressure. The crude product was
purified on silica gel (2/1-hexane/ether) to afford silyl ether 23
(780 mg, 43%) as an oil.
Example 20
[1136] Phosphonate 24: A solution of 23 (260 mg, 0.37 mmol),
triethylamine (0.52 mL, 3.7 mmol), and diethylphosphite (0.24 mmol,
1.85 mmol) in toluene (2 mL) was purged with argon and to the
solution was added (Ph.sub.3P).sub.4Pd (43 mg, 10 mol %). The
reaction mixture, was heated at 110.degree. C. (bath temperature)
for 6 h, and was then allowed to stir at room temperature for 12 h.
The solvent was evaporated under reduced pressure and the residue
was partitioned between ether and water. The aqueous phase was
extracted with ether and the combined organic extracts were washed
with saturated NaCl, dried (MgSO.sub.4), filtered, and the solvent
was evaporated under reduced pressure. The residue was purified by
chromatography on silica gel (2/1-ethyl acetate/hexane) to afford
diethylphosphonate 24 (153 mg, 55%).
Example 21
[1137] Phosphonic acid 26: To a solution of 24 (143 mg) in MeOH (5
mL) was added 4N HCl (2 mL). The solution was stirred at room
temperature for 9 h and was evaporated under reduced pressure. The
residue was triturated with ether and the solid was collected by
filtration to provide hydrochloride salt 25 (100 mg, 92%) as a
white powder. To a solution of X (47 mg, 0.87 mmol) in CH.sub.3CN
(1 mL) at 0.degree. C. was added TMSBr (130 .mu.L, 0.97 mmol). The
reaction was warmed to room temperature and stirred for 6.5 h at
which time TMSBr (0.87 mmol) was added and stirring was continued
for 16 h. The solution was cooled to 0.degree. C. and was quenched
with several drops of ice-cold water. The solvents were evaporated
under reduced pressure and the residue was dissolved in several
milliliters of MeOH and treated with propylene oxide (2 mL). The
mixture was heated to gentle boiling and evaporated. The residue
was triturated with acetone and the solid was collected by
filtration to give phosphonic acid 26 (32 mg, 76%) as a white
solid.
Example 22
[1138] Phosphonate 27: To a suspension of 26 (32 mg, 0.66 mmol) in
CH.sub.3CN (1 mL) was added bis(trimethylsilyl)acetamide (100
.mu.L, 0.40 mmol) and the solution was stirred for 30 min at room
temperature. The solvent was evaporated under reduced pressure and
the residue was dissolved in CH.sub.3CN (1 mL). To this solution
was added (3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl 4-nitrophenyl
carbonate (20 mg, 0.069 mmol, prepared according to Ghosh et al. J.
Med. Chem. 1996, 39, 3278.), N,N-diisopropylethylamine (35 .mu.L,
0.20 mmol), and N,N-dimethylaminopyridine (catalytic amount). The
solution was stirred for 22 h at room temperature, diluted with
water (0.5 mL) and was stirred with IR 120 ion exchange resin (325
mg, H.sup.+ form) until the pH was <2. The resin was removed by
filtration, washed with methanol and the filtrate was concentrated
under reduced pressure. The residue was dissolved water, treated
with solid NaHCO.sub.3 until pH=8 and was evaporated to dryness.
The residue was dissolved in water and was purified on C18 reverse
phase chromatography eluting with water followed by 5%, 10% and 20%
MeOH in water to give the disodium salt 27 (24 mg) as a pale yellow
solid: .sup.1H NMR (D.sub.2O) .delta. 7.72 (d, 2H), 7.52 (dd, 2H),
7.13 (dd, 2H), 7.05 (d, 2H), 5.58 (d, 1H), 4.87 (m, 1H), 3.86-3.53
(m overlapping s, 10H), 3.22 (dd, 1H), 3.12-2.85 (6H), 2.44 (m,
1H), 1.83 (m, 1H), 1.61 (m, 1H) 1.12 (dd, 1H), 0.77 (m, 6H);
.sup.31P NMR (D.sub.2O) .delta. 11.23; MS (ESI) 641 (M-H).
Example 23
[1139] Diethylphosphonate 28: To a solution of 25 (16 mg, 0.028
mmol) in CH.sub.3CN (0.5 mL) was added
(3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl 4-nitrophenyl carbonate
(9 mg, 0.031 mmol), N,N-diisopropylethylamine (20 .mu.L, 0.11
mmol), and N,N-dimethylaminopyridine (catalytic amount). The
solution was stirred at room temperature for 48 h and was then
concentrated under reduced pressure. The residue was partitioned
between EtOAc and saturated NaHCO.sub.3. The organic phase was
washed with saturated NaHCO.sub.3, saturated NaCl, and was dried
(MgSO.sub.4), filtered, and concentrated under reduced pressure.
The residue was purified by silica gel chromatography (2.5-5%
2-propanol/CH.sub.2Cl.sub.2). The residue obtained was further
purified by preparative layer chromatography (5%
MeOH/CH.sub.2Cl.sub.2) followed by column chromatography on silica
gel (10% 2-propanol/CH.sub.2Cl.sub.2) to afford diethylphosphonate
28 (7 mg) as a foam: .sup.1H NMR (CDCl.sub.3) .delta. 7.72-7.66 (m,
4H), 7.32-7.28 (2H), 6.96 (d, 2H), 5.60 (d, 1H), 4.97 (m, 2H),
4.18-4.01 (m, 4H), 3.94-3.60 (m overlapping s, 10H), 3.15-2.72 (m,
7H), 1.78 (m, 1H), 1.61 (m+H.sub.2O, .about.3H), 1.28 (t; 6H), 0.86
(m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 18.6; MS (ESI) 699
(M+H).
Prospective Example 24
[1140] Diphenyl phosphonate 14 is treated with aqueous sodium
hydroxide to provide monophenyl phosphonate 29 according to the
method found in J. Med. Chem. 1994, 37, 1857. Monophenyl
phosphonate 29 is then converted to the monoamidate 30 by reaction
with an amino acid ester in the presence of Ph.sub.3 and
2,2'-dipyridyl disulfide as described in the synthesis of
bisamidate 16f. Alternatively, monoamidate 30 is prepared by
treating 29 with an amino acid ester and DCC. Coupling conditions
of this type are found in Bull. Chem. Soc. Jpn. 1988, 61, 4491.
Example 25
[1141] Diazo ketone 1: To a solution of
N-tert-Butoxycarbonyl-O-benzyl-L-tyrosine (25 g, 67 mmol, Fluka) in
dry THF (150 mL) at -25-30.degree. C. (external bath temperature)
was added isobutylchloroformate (8.9 mL, 69 mmol) followed by the
slow addition of N.methylmorpholine (37.5 mL, 69 mmol). The mixture
was stirred for 40 min, and diazomethane (170 mmol, generated from
25 g 1-methyl-3-nitro-1-nitroso-guanidine according to Aldrichimica
Acta 1983, 16, 3) in ether (400 mL) was poured into the mixed
anhydride solution. The reaction was stirred for 15 min allowing
the bath to warm to room temperature while stirring overnight for 4
h. The mixture was bubbled with N.sub.2 for 30 min., washed with
water, saturated NaHCO.sub.3, saturated NaCl, dried (MgSO.sub.4),
filtered and evaporated to a pale yellow solid. The crude solid was
slurried in hexane, filtered, and dried to afford the diazo ketone
(26.8 g, 99%) which was used directly in the next step.
Example 26
[1142] Chloroketone 2: To a suspension of diazoketone 1 (26.8 g, 67
mmol) in ether/THF (750 mL, 3/2) at 0.degree. C. was added 4M HCl
in dioxane (16.9 mL, 67 mmol). The solution was stirred at
0.degree. C. for 2 hr. The reaction solvent was evaporated under
reduced pressure to give the chloroketone (27.7 g, 97%) as a
solid.
Example 27
[1143] Chloroalcohol 3: To a solution of chloroketone 2 (127.1 g,
67 mmol) in THF (350 mL) was added water (40 mL) and the solution
was cooled to 3-4.degree. C. (internal temperature). NaBH.sub.4
(6.3 g, 168 mmol) was added in portions. The mixture was stirred
for 1 h at 0.degree. C. and the solvents were removed. The mixture
was diluted with ethyl acetate and saturated KHSO.sub.4 was slowly
added until the pH<4 followed by saturated NaCl. The organic
phase was washed with saturated NaCl, dried (MgSO.sub.4) filtered
and evaporated under reduced pressure. The crude product consisted
of a 70:30 mixture of diastereomers by HPLC analysis (mobile phase,
77:25-CH.sub.3CN:H.sub.2O; flow rate: 1 mL/min; detection: 254 nm;
sample volume: 20 .mu.L; column: 5.mu., C18, 4.6.times.250 mm,
Varian; retention times: major diastereomer 3, 5.4 min, minor
diastereomer 4, 6.1 min). The residue was recrystallized from
EtOAc/hexane twice to afford the chloro alcohol 3 (12.2 g, >96%
diastereomeric purity by HPLC analysis) as a white solid.
Example 28
[1144] Epoxide 5: To a solution of chloroalcohol 3 (12.17 g, 130
mmol) in EtOH (300 mL) was added KOH/EtOH solution (0.71N, 51 mL,
36 mmol). The mixture was stirred for at room temperature for 1.5
h. The reaction mixture was evaporated under reduced pressure. The
residue was partitioned between EtOAc and water and the organic
phase was washed with saturated NH.sub.4Cl, dried (MgSO.sub.4),
filtered, and evaporated under reduced pressure to afford the
epoxide (10.8 g, 97%) as a white solid.
Example 29
[1145] Sulfonamide 6: To a suspension of epoxide 5 (10.8 g, 30
mmol) in 2-propanol (100 mL) was added isobutylamine (129.8 mL, 300
mmol) and the solution was refluxed for 1 hr. The solution was
evaporated under reduced pressure to give a crude solid. The solid
(42 mmol) was dissolved in CH.sub.2Cl.sub.2 (200 mL) and cooled to
0.degree. C. Triethylamine (11.7 mL, 84 mmol) was added followed by
the addition of 4-methoxybenzenesulfonyl chloride (8.68 g, 42 mmol)
and the solution was stirred for 40 min at 0.degree. C., warmed to
room temperature and evaporated under reduced pressure. The residue
was partitioned between EtOAc and saturated NaHCO.sub.3. The
organic phase was washed with saturated NaCl, dried (MgSO.sub.4),
filtered and evaporated under reduced pressure. The crude product
was recrystallized from EtOAc/hexane to give the sulfonamide (23.4
g, 91%) as a small white needles: mp 122-124.degree. C.
(uncorrected).
Example 30
[1146] Carbamate 7: A solution of sulfonamide 6 (6.29 mg, 10.1
mmol) in CH.sub.2Cl.sub.2 (20 mL) was treated with trifluoroacetic
acid (10 mL). The solution was stirred for 3 hr. Volatiles were
evaporated under reduced pressure and the residue was partitioned
between EtOAc and 0.5 N NaOH. The organic phase were washed with
0.5 N NaOH (2.times.), water (2.times.) and saturated NaCl, dried
(MgSO.sub.4), filtered, and evaporated under reduced pressure. The
residue was dissolved in CH.sub.3CN (60 mL), cooled to 0.degree. C.
and was treated with (3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl
4-nitrophenyl carbonate (298.5 g, 10 mmol, prepared according to
Ghosh et al. J. Med. Chem. 1996, 39, 3278.) and
N,N-dimethylaminopyridine (2.4 g, 20 mmol). After stirring for 1 h
at 0.degree. C., the reaction solvent was evaporated under reduced
pressure and the residue was partitioned between EtOAc and 5%
citric acid. The organic phase was washed twice with 1%
K.sub.2CO.sub.3, and then was washed with saturated NaCl, dried
(MgSO.sub.4), filtered, and evaporated under reduced pressure. The
crude product was purified by chromatography on silica gel
(1/1-EtOAc/hexane) affording the carbamate (5.4 g, 83%) as a solid:
mp 128-129.degree. C. (MeOH, uncorrected).
Example 31
[1147] Phenol 8: A solution of carbamate 7 (5.4 g, 8.0 mmol) in
EtOH (260 mL) and EtOAc (130 mL) was treated with 10% Pd/C (540 mg)
and was stirred under H.sub.2 atmosphere (balloon) for 3 h. The
reaction solution stirred with celite for 10 min, and passed
through a pad of celite. The filtrate was evaporated under reduced
pressure to afford the phenol as a solid (4.9 g) that contained
residual solvent: mp 131-134.degree. C. (EtOAc/hexane,
uncorrected).
Example 32
[1148] Dibenzylphosphonate 10: To a solution of
dibenzylhydroxymethyl phosphonate (3.1 g, 10.6 mmol) in
CH.sub.2Cl.sub.2 (30 mL) was treated with 2,6-lutidine (1.8 mL,
15.6 mmol) and the reaction flask was cooled to -50.degree. C.
(external temperature). Trifluoromethanesulfonic anhydride (2.11
mL, 12.6 mmol) was added and the reaction mixture was stirred for
15 min and then the cooling bath was allowed to warm to 0.degree.
C. over 45 min. The reaction mixture was partitioned between ether
and ice-cold water. The organic phase was washed with cold 1M
H.sub.3PO.sub.4, saturated NaCl, dried (MgSO.sub.4), filtered and
evaporated under reduced pressure to afford triflate 9 (3.6 g, 80%)
as an oil which was used directly without any further purification.
To a solution of phenol 8 (3.61 g, 6.3 mmol) in THF (90 mL) was
added Cs.sub.2CO.sub.3 (4.1 g, 12.6 mmol) and triflate 9 (4.1 g,
9.5 mmol) in THF (10 mL). After stirring the reaction mixture for
30 min at room temperature additional Cs.sub.2CO.sub.3 (6.96 g, 3
mmol) and triflate (1.26 g, 3 mmol) were added and the mixture was
stirred for 3.5 h. The reaction mixture was evaporated under
reduced pressure and the residue was partitioned between EtOAc and
saturated NaCl. The organic phase was dried (MgSO.sub.4), filtered
and evaporated under reduced pressure. The crude product was
chromatographed on silica gel eluting (5%
2-propanol/CH.sub.2Cl.sub.2) to give the dibenzylphosphonate as an
oil that solidified upon standing. The solid was dissolved in
EtOAc, ether was added, and the solid was precipitated at room
temperature overnight. After cooling to 0.degree. C. the solid was
filtered and washed with cold ether to afford the
dibenzylphosphonate (3.43 g, 64%) as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 7.66 (d, 2H), 7.31 (s, 10H), 7.08 (d, 2H),
6.94 (d, 2H), 6.76 (d, 2H), 5.59 (d, 1H), 5.15-4.89 (m, 6H), 4.15
(d, 2H), 3.94-3.62 (m, 10H), 3.13-2.69 (m, 7H), 1.78 (m, 1H),
1.70-1.44 (m, 2H), 0.89-0.82 (2d, 6H); .sup.31P NMR (CDCl.sub.3)
.delta. 18.7; MS (ESI) 853 (M+H).
Example 33
[1149] Phosphonic acid 11: A solution of dibenzylphosphonate 10
(3.43 g) was dissolved in EtOH/EtOAc (150 mL/50 mL), treated with
10% Pd/C (350 mg) and was stirred under H.sub.2 atmosphere
(balloon) for 3 h. The reaction mixture was stirred with celite,
and the catalyst was removed by filtration through celite. The
filtrate was evaporated under reduced pressure and the residue was
dissolved in MeOH and filtered with a 0.45 .mu.M filter. After
evaporation of the filtrate, the residue was triturated with ether
and the solid was collected by filtration to afford the phosphonic
acid (2.6 g, 94%) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 7.77 (d, 2H), 7.19 (d, 2H), 7.09 (d, 2H), 6.92 (d, 2H),
5.60 (d, 1H), 4.95 (m, 1H), 4.17 (d, 2H), 3.94 (m, 1H), 3.89 (s,
3H), 3.85-3.68 (m, 5H), 3.42 (dd, 1H), 3.16-3.06 (m, 2H), 2.96-2.84
(m, 3H), 2.50 (m, 1H), 2.02 (m, 1H), 1.58 (m, 1H), 1.40 (dd, 1H),
0.94 (d, 3H), 0.89 (d, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 16.2;
MS (ESI) 671 (M-H).
Example Section B
[1150] There is no Section B in this application.
Example Section C
Example 1
[1151] Diphenyl phosphonate 31: To a solution of phosphonic acid 30
(11 g, 16.4 mmol) and phenol (11 g, 117 mmol) in pyridine (100 mL)
was added 1,3-dicyclohexylcarbodiimide (13.5 g, 65.5 mmol). The
solution was stirred at room temperature for 5 min and then at
70.degree. C. for 2 h. The reaction mixture was cooled to room
temperature, diluted with ethyl acetate (100 mL) and filtered. The
filtrate was evaporated under reduced pressure to remove pyridine.
The residue was dissolved in ethyl acetate (250 mL) and acidified
to pH=4 by addition of HCl (0.5 N) at 0.degree. C. The mixture was
stirred at 0.degree. C. for 0.5 h, filtered and the organic phase
was separated and washed with brine, dried over MgSO.sub.4,
filtered and concentrated under reduced pressure. The residue was
purified on silica gel to give diphenyl phosphonate 31 (9 g, 67%)
as a solid. .sup.31P NMR (CDCl.sub.3) d 12.5.
Example 2
[1152] Monophenyl phosphonate 32: To a solution of
diphenylphosphonate 31 (9.0 g, 10.9 mmol) in acetonitrile (400 mL)
was added NaOH (1N, 27 mL) at 0.degree. C. The reaction mixture was
stirred at 0.degree. C. for 1 h, and then treated with Dowex (50
W.times.8-200, 12 g). The mixture was stirred for 0.5 h at
0.degree. C., and then filtered. The filtrate was concentrated
under reduced pressure and co-evaporated with toluene. The residue
was dissolved in ethyl acetate and hexane was added to precipitate
out the monophenyl phosphonate 32 (8.1 g, 100%). .sup.31P NMR
(CDCl.sub.3) d 18.3.
Example 3
[1153] Monoamidate 33a (R.sub.1=Me, R.sub.2=n-Bu): To a flask
charged with monophenyl phosphonate 32 (4.0 g, 5.35 mmol), was
added L-alanine n-butyl ester hydrochloride (4.0 g, 22 mmol),
1,3-dicyclohexylcarbodiimide (6.6 g, 32 mmol), and finally pyridine
(30 mL) under nitrogen. The resultant mixture was stirred at
60-70.degree. C. for 1 h, then cooled to room temperature and
diluted with ethyl acetate. The mixture was filtered and the
filtrate was concentrated under reduced pressure. The residue was
partitioned between ethyl acetate and HCl (0.2 N) and the organic
layer was separated. The ethyl acetate phase was washed with water,
saturated NaHCO.sub.3, dried over MgSO.sub.4, filtered and
concentrated under reduced pressure. The residue was purified on
silica gel (pre-treated with 10% MeOH/CH.sub.3CO.sub.2Et, eluting
with 40% CH.sub.2Cl.sub.2/CH.sub.3CO.sub.2Et and
CH.sub.3CO.sub.2Et) to give two isomers of 33a in a total yield of
51%. Isomer A (1.1 g): .sup.1H NMR (CDCl.sub.3) d 0.88 (m, 9H), 1.3
(m, 2H), 1.35 (d, J=7 Hz, 3H), 1.55 (m, 2H), 1.55-1.7 (m, 2H), 1.8
(m, 1H), 2.7-3.2 (m, 7H), 3.65-4.1 (m, 9H), 3.85 (s, 3H), 4.2 (m,
1H), 4.3 (d, J=9.6 Hz, 2H), 5.0 (m, 2H), 5.65 (d, J=5.4 Hz, 1H),
6.85 (d, J=8.7 Hz, 2H), 7.0 (d, J=8.7 Hz, 2H), 7.1-7.3 (m, 7H), 7.7
(d, J=8.7 Hz, 2H); .sup.31P NMR (CDCl.sub.3) d 20.5. Isomer B (1.3
g) .sup.1H NMR (CDCl.sub.3) d 0.88 (m, 9H), 1.3 (m, 2H), 1.35 (d,
J=7 Hz, 3H), 1.55 (m, 2H), 1.55-1.7 (m, 2H), 1.8 (m, 1H), 2.7-3.2
(m, 7H), 3.65-4.1 (m, 9H), 3.85 (s, 3H), 4.2-4.35 (m, 3H), 5.0 (m,
2H), 5.65 (d, J=5.4 Hz, 1H), 6.85 (d, J=8.7 Hz, 2H), 7.0 (d, J=8.7
Hz, 2H), 7.1-7.3 (m, 7H), 7.7 (d, J=8.7 Hz, 2H); .sup.31P NMR
(CDCl.sub.3) d 19.4.
Example 4
[1154] Monoamidate 33b (R.sub.1=Me, R.sub.2=i-Pr) was synthesized
in the same manner as 33a in 77% yield. Isomer A: .sup.1H NMR
(CDCl3) d 0.9 (2d, J=6.3 Hz, 6H), 1.2 (d, J=7 Hz, 6H), 1.38 (d, J=7
Hz, 3H), 1.55-1.9 (m, 3H), 2.7-3.2 (m, 7H), 3.65-4.1 (m, 8H), 3.85
(s, 3H), 4.2 (m, 1H), 4.3 (d, J=9.6 Hz, 2H), 5.0 (m, 2H), 5.65 (d,
J=5.4 Hz, 1H), 6.85 (d, J=8.7 Hz, 2H), 7.0 (d, J=8.7 Hz, 2H),
7.1-7.3 (m, 7H), 7.7 (d, J=8.7 Hz, 2H); .sup.31P NMR (CDCl.sub.3) d
20.4. Isomer B: .sup.1H NMR (CDCl3) d 0.9 (2d, J=6.3 Hz, 6H), 1.2
(d, J=7 Hz, 6H), 1.38 (d, J=7 Hz, 3H), 1.55-1.9 (m, 3H), 2.7-3.2
(m, 7H), 3.65-4.1 (m, 8H), 3.85 (s, 3H), 4.2 (m, 1H), 4.3 (d, J=9.6
Hz, 2H), 5.0 (, 2H), 5.65 (d, J=5.4 Hz, 1H), 6.85 (d, J=8.7 Hz,
2H), 7.0 (d, J=8.7 Hz, 2H), 7.1-7.3 (m, 7H), 7.7 (d, J=8.7 Hz, 2H);
.sup.31P NMR (CDCl.sub.3) d 19.5.
Example Section D
Example 1
[1155] Cyclic Anhydride 1 (6.57 g, 51.3 mmol) was treated according
to the procedure of Brown et al., J. Amer. Chem. Soc. 1955, 77,
1089-1091 to afford amino alcohol 3 (2.00 g, 33%). for intermediate
2: .sup.1H NMR (CD.sub.3OD) .delta. 2.40 (S, 2H), 1.20 (s, 6H).
Example 2
[1156] Amino alcohol 3 (2.0 g, 17 mmol) was stirred in 30 ml, 1:1
THF:water. Sodium Bicarbonate (7.2 g, 86 mmol) was added, followed
by Boc Anhydride (4.1 g, 19 mmol). The reaction was stirred for 1
hour, at which time TLC in 5% methanol/DCM with ninhydrin stain
showed completion. The reaction was partitioned between water and
ethyl acetate. The organic layer was dried and concentrated, and
the resulting mixture was chromatographed on silica in 1:1
hexane:ethyl acetate to afford two fractions, "upper" and "lower"
each having the correct mass. By NMR the correct product 4 was
"lower" (0.56 g, 14%) .sup.1H NMR (CDCl.sub.3) .delta. 3.7 (t, 2H),
3.0 (d,2H), 1.45 (t, 2H) 1.4 (s, 9H), 0.85 (s, 6H), MS (ESI): 240
(M+23).
Example 3
[1157] Sodium Hydride (60% emulsion in oil) was added to a solution
of the alcohol 4 (1.1 g, 5.2 mmol) in dry DMF in a 3-neck flask
under dry nitrogen. Shortly afterward triflate 35 (2.4 g, 5.7 mmol)
was added with stirring for 1.5 hrs. Mass spectrometry showed the
presence of the starting material (240, M+23), thus 100 mg more 60%
sodium hydride emulsion as well as .about.1 g more triflate were
added with an additional hour of stirring. The reaction was
quenched by the addition of saturated NaHCO.sub.3 then partitioned
between ethyl acetate and water. The organic layer was dried with
brine and MgSO.sub.4 and eluted on silica with 1:1 hexane:ethyl
acetate to afford 5 (0.445 g, 15%). NMR showed some contamination
with alcohol 4 starting material .sup.1H NMR (CDCl.sub.3): .delta.
7.28 (s, 10H), 5.00 (m, 4H), 3.70 (t, 2H), 2.94, (d, 2H), 1.44 (t,
2H), 1.40 (s, 9H), 0.83 (s, 6H) MS (ESI): 514 (M+23).
Example 4
[1158] Phosphonate ester 5 (0.445 g, 0.906 mmol) was stirred with
20% TFA in DCM. (5 mL) TLC showed completion in 1 hr time. The
reaction was azeotroped with toluene then run on a silica gel
column with 10% methanol in DCM. Subsequently, the product was
dissolved in ethyl acetate and shaken with saturated sodium
bicarbonate:water (1:1), dried with brine and magnesium sulfate to
afford the free amine 6 (30 mg, 8.5%). .sup.1H NMR (CDCl.sub.3):
.delta. 7.30 (s, 10H), 5.00 (m, 4H), 3.67 (d, 2H), 3.47, (t, 2H),
2.4-2.6 (brs) 1.45 (t, 2H), 0.82 (s, 6H), MS (ESI): 393 (M+1).
Example 5
[1159] Amine 6 (30 mg, 0.08 mmol) and epoxide 7 (21 mg, 0.08 mmol)
were dissolved in 2 mL IprOH and heated to reflux for 1 hr then
monitored by TLC in 10% MeOH/DCM. Added .about.20 mg more epoxide 7
and continued reflux for 1 hr. Cool to room temperature, dilute
with ethyl acetate, shake with water and brine, dry with magnesium
sulfate. Silica gel chromatography using first 5% then 10% MeOH in
EtOAc yielded amine 8 (18 mg, 36%). .sup.1H NMR (CDCl.sub.3):
.delta. 7.30 (s, 10H), 7.20-7-14 (m, 5H), 5.25-4.91 (m, 4H), 3.83,
(m, 1H), 3.71 (d, 2H) 3.64 (m, 1H), 3.54 (t, 2H), 3.02-2.61 (m,
5H), 2.65-2.36 (dd, 2H) (t, 2H), 1.30 (s, 9H) 0.93 (s, 9H) 0.83 (t,
2H) MS (ESI) 655 (M+1).
Example 6
[1160] Amine 8 (18 mg, 0.027 mmol) was dissolved in 1 mL DCM then
acid chloride 9 (6 mg, 0.2 mmol) followed by triethylamine (0.004
mL, 0.029 mmol). The reaction was monitored by TLC. Upon completion
the reaction was diluted with DCM shaken with 5% citric acid,
saturated sodium bicarbonate, brine, and dried with MgSO.sub.4.
Purification on silica (1:1 Hexane:EtOAc) afforded sulfonamide 10
(10.5 mg, 46%). .sup.1H NMR (CDCl.sub.3): .delta. 7.69 (d, 2H),
7.30 (s, 10H), 7.24-7-18 (m, 5H), 5.00 (m, 4H), 4.73, (d, 1H), 4.19
(s, 1H) 3.81 (m, 1H), 3.80 (s, 3H), 3.71 (d,2H), 3.57 (t, 2H),
3.11-2.95 (m, 5H) 2.75 (m,1H) 1.25 (s, 1H), 0.90 (s, 6H) MS (ESI)
847 (M+Na.sup.+).
Example 7
[1161] Sulfonamide 10 (10.5 mg, 0.013 mmol) was stirred at room
temperature in 20% TFA/DCM. Once Boc deprotection was complete by
TLC (1:1 Hexane:EtOAc) and MS, the reaction was azeotroped with
toluene. The TFA salt of the amine was dissolved in acetonitrile
(0.5 mg) and to this were added carbonate 11 (4.3 mg, 0.014 mmol)
followed by DMAP (4.6 mg, 0.038 mg). Stir at room temp until TLC
(1:1 Hexane:EtOAc) shows completion. Solvent was evaporated and the
residue was redissolved in EtOAc then shaken with saturated
NaHCO.sub.3. The organic layer was washed with water and brine,
then dried with MgSO.sub.4 Purification on silica with Hexane:EtOAc
afforded compound 12 (7.1 mg, 50%). .sup.1H NMR (CDCl.sub.3):
.delta. 7.75 (d, 2H) 7.24-7.35 (15H) 6.98 (d, 2H), 5.62 (d, 1H)
5.04 (m, 4H) 4.98 (m, 1H) 4.03 (m, 1H), 3.85 (s, 3H), 3.61-3.91
(9H), 3.23-3.04 (5H) 2.85 (m, 1H), 2.74 (m,1H) 1.61 (d, 2H), 1.55
(m, 1H) 1.36 (m, 1H) 0.96 (d, 6H) MS (ESI): 903 (M+23).
Example 8
[1162] Compound 12 (6.1 mg, 0.007 mmol) was dissolved in 1 mL, 3:1
EtOH:EtoAc. Palladium catalyst (10% on C, 1 mg) was added and the
mixture was purged three times to vacuum with 1 atmosphere hydrogen
gas using a balloon. The reaction was stirred for 2 hrs, when MS
and TLC showed completion. The reaction was filtered through Celite
with EtOH washing and all solvent to was evaporated to afford final
compound 13 (5 mg, 100%). .sup.1H NMR (CD.sub.3OD): .delta. 87.79
(d, 2H) 7.16-7.24 (5H) 7.09 (d, 2H) 5.58 (d, 1H) 4.92 (m, 1H) 3.97
(m, 1H), 3.92 (dd,1H) 3.89 (s, 3H) 3.66-3.78 (8H) 3.40 (d,1H), 3.37
(dd, 1H), 3.15 (m, 1H) 3.12 (dd,1H) 2.96 (d, 1H), 2.87 (m, 1H),
2.74 (m,1H) 2.53 (m, 1H) 1.70 (m, 2H), 1.53 (m, 1H) 1.32 (m, 1H)
1.04 (d, 6H) MS (ESI): 723 (M+23).
Example 9
[1163] Amino Alcohol 14 (2.67 g, 25.9 mmol) was dissolved in THF
with stirring and Boc Anhydride (6.78 g, 31.1 mmol) was added. Heat
and gas evolution ensued. TEA (3.97 mL, 28.5 mmol) was added and
the reaction was stirred overnight. In the morning, the reaction
was quenched by the addition of saturated NaHCO.sub.3. The organic
layer was separated out and shaken with water, dried with brine and
MgSO.sub.4 to afford 15 which was used without further
purification. (100% yield) (some contamination): .sup.1H NMR
(CDCl.sub.3): .delta. 3.76 (t,1H) 3.20, (d,2H), 2.97 (d, 2H), 1.44
(s, 9H), 0.85 (s, 6H).
Example 10
[1164] A solution of the alcohol 15 (500 mg, 2.45 mmol) in dry THF
was cooled under dry N.sub.2 with stirring. To this was added
n-butyl lithium (1.29 mL, 2.71 mmol) as a solution in hexane in a
manner similar to that described in Tetrahedron. 1995, 51 #35,
9737-9746. Triflate 35 (1.15 g, 2.71 mmol) was added neat with a
tared syringe. The reaction was stirred for four hours, then
quenched with saturated NaHCO.sub.3. The mixture was then
partitioned between water and EtOAc. The organic layer was dried
with brine and MgSO.sub.4, then chromatographed on silica in 1:1
Hexane:EtOAc to afford phosphonate 16 (445 mg, 38%) .sup.1H NMR
(CDCl.sub.3): .delta. 7.37 (m, 10H), 5.09 (m, 4H), 3.73-3.75 (m,
2H), 3.24 (s,2H), 3.02 (d, 2H), 1.43 (s, 9H), 0.86 (s, 6H).
Example 11
[1165] Phosphonate 16 (249 mg, 0.522 mmol) was stirred in 20%
TFA/DCM for 1 hr. The reaction was then azeotroped with toluene.
The residue was re-dissolved in EtOAc, then shaken with water:
saturated NaHCO.sub.3 (1:1). The organic layer was dried with brine
and MgSO.sub.4 and solvent was removed to afford amine 17 (143 mg,
73%) .sup.1H NMR (CDCl.sub.3): .delta. 7.30 (s, 10H), 5.05-4.99 (m,
4H), 3.73 (d, 2H), 3.23 (s, 2H), 2.46 (brs, 2H), 0.80 (s, 6H)
.sup.31P NMR (CDCl.sub.3): .delta. 23.77 (s).
Example 12
[1166] Amine 17 (143 mg, 0.379 mmol) and epoxide 7 (95 mg, 0.360
mmol) were dissolved in 3 mL IprOH and heated to 85.degree. C. for
1 hr. The reaction was cooled to room temperature overnight then
heated to 85.degree. C. for 1 hr more in the morning. The reaction
was then diluted with EtOAc, shaken with water, dried with brine
MgSO.sub.4 and concentrated. The residue was eluted on silica in a
gradient from 5% to 10% MeOH in DCM to afford compound 18 (33 mg,
14%).
Example 13
[1167] Mix compound 18 (33 mg, 0.051 mmol) and chlorosulfonyl
compound 9 (11 mg, 0.054 mmol) in 2 mL DCM then add TEA (0.0075 mL,
0.054 mmol), stir for 5 hrs. TLC in 1:1 EtOAc:hexane shows reaction
not complete. Place in freezer overnight. In the morning, take out
of freezer, stir for 2 hrs, TLC shows completion. Workup done with
5% citric acid, saturated NaHCO.sub.3, then dry with brine and
MgSO.sub.4. The reaction mixture was concentrated and
chromatographed on a Monster Pipette column in 1:1 hexane:EtOAc
then 7:3 hexane:EtOAc to avail compound 19 (28 mg, 67%) .sup.1H NMR
(CDCl.sub.3): .delta. 7.37 (d, 2H), 7.20 (m, 15H), 6.90 (d, 2H),
5.07-4.93 (m, 4H), 4.16 (brs, 1H), 3.80 (s, 3H), 3.75-3.37 (m, 4H),
3.36 (d, 1H), 3.20-2.93 (m, 6H), 2.80-2.75 (dd, 1H).
Example 14
[1168] Compound 19 (28 mg, 0.35 mmol) was stirred in 4 mL DCM with
addition of 1 mL TFA. Stir for 45 minutes, at which time complete
deprotection was noted by TLC as well as MS. Azeotrope with
toluene. The residue was dissolved in 1 mL CH.sub.3CN, cooled to
0.degree. C. Bis-Furan para-Nitro phenol carbonate 11 (12 mg, 0.038
mmol), dimethyl amino pyridine (1 mg, 0.008 mmol) and
diisopropylethylamine (0.018 mL, 0.103 mmol) were added. The
mixture was stirred and allowed to come to room temperature and
stirred until TLC in 1:1 hexane:EtOAc showed completion. The
reaction mixture was concentrated and the residue was partitioned
between saturated NaHCO.sub.3 and EtOAc. The organic layer was
dried with brine and MgSO.sub.4, then chromatographed on silica
with hexane:EtOAc to afford compound 20 (20 mg, 67%). .sup.1NMR
(CDCl.sub.3): .delta. 7.76 (d, 2H), 7.34-7.16 (m, 15H), 7.07 (d,
2H), 5.56 (d, 1H), 5.09 (m, 4H), 4.87 (m, 1H), 4.01 (m, 1H), 3.91
(m, 2H), 3.87 (s, 3H), 3.86 (m, 1H), 3.69 (m, 1H), 3.67 (m, 1H)
3.60 (d, 2H) 3.28 (m, 1H) 3.25 (d, 2H), 3.32 (d, 1H), 3.13 (m, 1H),
3.02 (m, 1H) 2.85 (d, 1H), 2.83 (m, 1H) 2.52 (m, 1H) 1.47 (m, 1H),
1.31 (m, 1H) 0.98 (s, 3H), 0.95 (s,3H).
Example 15
[1169] Compound 20 (7 mg, 0.008 mmol) was treated in a manner
identical to example 8 to afford compound 21 (5 mg, 90%) .sup.1H
NMR (CDCl.sub.3): .delta. 7.80 (d, 2H), 7.25-7.16 (m, 5H), 7.09 (d,
2H), 5.58 (d, 1H), 4.92 (m, 1H), 3.99 (m, 1H), 3.92 (m, 1H), 3.88
(s, 3H), 3.86 (m, 1H), 3.77 (m, 1H), 3.75 (m, 1H), 3.73 (m, 1H),
3.71 (m, 1H) 3.71 (m, 1H), 3.68 (m, 1H), 3.57 (d,1H), 3.41 (d, 1H),
3.36 (m, 1H), 3.29 (d, 1H), 3.25 (d, 2H), 3.18 (m, 1H), 3.12 (m,
1H), 3.01 (d, 1H) 2.86 (m, 1H), 2.53 (m, 1H) 1.50 (m, 1H), 1.33 (m,
1H), 1.02 (s, 3H), 0.99 (s, 3H).
Example 16
[1170] Compound 15 (1.86 g, 9.20 mmol) was treated with triflate 22
in a manner identical to example 10 to afford compound 23 (0.71 g,
21.8%) .sup.1H NMR (CDCl.sub.3): .delta. 5.21 (brs, 1H) 4.16-4.07
(m, 4H), 3.71-3.69 (d, 2H), 3.24 (s, 2H), 1.43 (s, 9H), 1.34-1.28
(m, 6H) 0.86 (s, 6H).
Example 17
[1171] Compound 23 (151 mg, 0.427 mmol) was dissolved in 10 mL DCM
and 1.0 mL TFA was added. The reaction was stirred until
completion. The reaction was azeotroped with toluene and the
residue was then dissolved in THF and treated with basic Dowex
resin beads. Afterwards, the beads were filtered away and solvent
was removed to avail compound 24 (100 mg, 92%) .sup.1H NMR
(CDCl.sub.3): .delta. 4.15-4.05 (m, 4H), 3.72-3.69 (d, 2H), 3.27
(s, 2H), 1.30-1.26 (m, 6H) 0.81 (s, 6H).
Example 18
[1172] Compound 24 (100 mg, 0.395 mmol) was treated in a manner
identical to example 12 to avail compound 25 (123 mg, 60%). .sup.1H
NMR (CDCl.sub.3): .delta. 7.26-7.13 (m, 5H), 4.48-4.83 (d, 1H)
4.17-4.06 (m, 4H), 3.75 (d, 2H) 3.56 (brs, 1H), 3.33 (s, 2H),
2.93-2.69 (ma, 4H), 2.44-2.55 (dd, 2H) 1.32 (m, 6H), 0.916 (s,
6H).
Example 19
[1173] Compound 25 (88 mg, 0.171 mmol) was treated in a manner
identical to example 13 to afford compound 26 (65 mg, 55%) .sup.1H
NMR (CDCl.sub.3): .delta. 7.26-7.13 (m, 5H), 4.48-4.83 (d, 1H)
4.17-4.06 (m, 4H), 3.75 (d, 2H) 3.56 (brs, 1H), 3.33 (s, 2H),
2.93-2.69 (m, 4H), 2.44-2.55 (dd, 2H) 1.32 (m, 6H), 0.916 (s,
6H).
Example 20
[1174] Compound 26 (65 mg, 0.171 mmol) was treated in a manner
identical to example 14 to afford compound 27 (49 mg, 70%) .sup.1H
NMR: (CDCl.sub.3): .delta. 7.75 (d, 2H), 7.25-7.24 (m,4H), 7.18 (m,
1H) 6.99 (d, 2H), 5.63 (d, 1H), 5.01 (m, 1H), 4.16 (m, 4H), 3.94
(m, 1H), 3.88 (m, 1H), 3.88 (s, 3H), 3.84 (m, 1H), 3.81 (m, 1H),
3.74 (m, 2H),), 3.70 (m, 1H), 3.69 (m, 1H) 3.43 (m, 1H), 3.24 (m,
1H), 3.22 (m, 2H) 3.21 (m, 2H) 3.12 (m, 1H), 3.02 (m, 1H) 2.86 (m,
1H), 2.72 (m, 1H), 1.54 (m, 1H), 1.38 (m, 1H) 1.35 (m, 6H) 1.00 (s,
3H), 0.96 (s,3H).
Example 21
[1175] Boc protected amine 28 (103 mg, 0.153 mmol) was dissolved in
DCM (5 mL). The stirred solution was cooled to 0.degree. C.
BBr.sub.3 as a 1.0 M solution in DCM (0.92 mL, 0.92 mmol) was added
dropwise over 10 min, and the reaction was allowed to continue
stirring at 0.degree. C. for 20 min. The reaction was warmed to
room temperature and stirring was continued for 2 hours. The
reaction was then cooled to 0.degree. C. and quenched by dropwise
addition of MeOH (1 mL). The reaction mixture was evaporated and
the residue suspended in methanol which was removed under reduced
pressure. The procedure was repeated for EtOAc and finally toluene
to afford free amine HBr salt 29 (107 mg, >100%) which was used
without further purification.
Example 22
[1176] Amine HBr salt 29 (50 mg, 0.102 mmol) was suspended in 2 mL
CH.sub.3CN with stirring then cooled to 0.degree. C. DMAP (25 mg,
0.205 mmol) was added, followed by Carbonate 11. The reaction was
stirred at 0.degree. C. for 1.5 hrs then allowed to warm to room
temperature. The reaction was stirred overnight. A few drops Acetic
acid were added to the reaction mixture, which was concentrated and
re-diluted with ethyl acetate, shaken with 10% citric acid then
saturated NaHCO.sub.3. The organic layer was dried with brine and
MgSO.sub.4 and eluted on silica to afford di-phenol 30 (16 mg, 28%)
.sup.1H NMR (CD.sub.3OD): .delta. 7.61, (d, 2H), 7.01 (d, 2H), 6.87
(d, 2H), 6.62 (d, 2H), 5.55 (d, 1H), 4.93 (m, 1H), 3.92 (m, 2H),
3.79 (m, 5H), 3.35 (m, 1H), 3.07 (m, 2H), 2.88 (m, 3H), 2.41 (m,
1H), 2.00 (m, 1H), 1.54 (m, 1H), 1.31 (dd, 1H) 0.89-0.82 (dd,
6H).
Example 23
[1177] A solution of di-phenol 30 (100 mg, 0.177 mmol) was made in
CH.sub.3CN that had been dried over K.sub.2CO.sub.3. To this, the
triflate (0.084 mL, 0.23 mmol) was added, followed by
Cs.sub.2CO.sub.3 (173 mg, 0.531 mmol). The reaction was stirred for
1 hr. TLC (5% IprOH/DCM) showed 2 spots with no starting materials
left. Solvent was evaporated and the residue was partitioned
between EtOAc and water. The organic layer was washed with
saturated NaHCO.sub.3, then dried with brine and MgSO.sub.4. The
mixture was separated by column chromatography on silica with 3%
IprOH in DCM. The upper spot 31 (90 mg, 46%) was confirmed to be
the bis alkylation product. The lower spot required further
purification on silica gel plates to afford a single mono
alkylation product 32 (37 mg, 26%). The other possible alkylation
product was not observed. NMR: .sup.1H NMR (CDCl.sub.3): for 31:
.delta. 7.57 (d, 2H), 7.37 (m, 10H) 7.03 (d, 2H), 6.99 (d, 2H),
6.73 (d, 2H), 5.69 (d, 1H), 5.15-5.09 (m, 4H), 5.10 (m, 1H), 4.32
(d, 2H), 4.02 (d, 1H), 3.82 (m, 1H) 3.81 (m, 1H), 3.93-3.81 (m,
2H), 3.74 (d, 1H), 3.06 (m, 1H), 3.00 (m, 1H), 2.96 (m, 1H), 2.91
(m, 1H) 2.77 (m, 1H) 2.64 (m, 1H) 2.47 (m, 1H) 1.82 (m, 2H) 1.79
(m, 1H), 0.94-0.86 (dd, 6H) for 32: .delta. 7.68 (d, 2H), 7.33-7.35
(m, 20H), 7.11 (d, 2H), 6.96 (d, 2H), 6.80 (d, 2H), 5.26 (d, 1H),
5.11(m, 8H), 5.00 (m, 1H) 4.23 (d, 2H), 4.19 (d, 2H), 3.93 (m, 1H),
3.82-3.83 (m, 3H), 3.68-3.69 (m, 2H) 3.12-2.75 (m, 7H), 1.82 (m,
1H), 1.62-1.52 (d, 2H), 0.89-0.86 (dd, 6H).
Example 24
[1178] Ref: J. Med. Chem. 1992, 35 10, 1681-1701
[1179] To a solution of phosphonate 32 (100 mg, 0.119 mmol) in dry
dioxane was added Cs.sub.2CO.sub.3 (233 mg, 0.715 mmol), followed
by 2-(dimethylamino) ethyl chloride hydrochloride salt (69 mg, 0.48
mmol). The reaction was stirred at room temperature and monitored
by TLC. When it was determined that starting material remained,
additional Cs.sub.2CO.sub.3 (233 mg, 0.715 mmol) as well as amine
salt (69 mg, 0.48 mmol) were added and the reaction was stirred
overnight at 60.degree. C. In the morning when TLC showed
completion the reaction was cooled to room temperature, filtered,
and concentrated. The product amine 33 (40 mg, 37%) was purified on
silica. Decomposition was noted as lower spots were seen to emerge
with time using 15% MeOH in DCM on silica.
Example 25
[1180] Amine 33 (19 mg, 0.021 mmol) was dissolved in 1.5 mL DCM.
This solution was stirred in an icebath. Methane sulfonic acid
(0.0015 mL, 0.023 mmol) was added and the reaction was stirred for
20 minutes. The reaction was warmed to room temperature and stirred
for 1 hour. The product, amine mesylate salt 34 (20 mg, 95%) was
precipitated-out by addition of hexane. .sup.1H NMR (CD.sub.3OD):
.delta. 7.69 (d, 2H), 7.35 (m, 10H), 7.15 (m, 4H) 6.85 (m, 2H),
5.49 (d, 1H), 5.10 (m, 4H), 4.83 (ma, 1H), 4.62 (d, 2H), 4.22 (m,
2H), 3.82 (m, 1H), 3.56 (m, 1H), 3.48 (m, 2H), 3.35 (m, 1H), 2.99
(m, 1H), 2.95 (m, 1H), 2.84 (s, 6H), 2.78 (m, 1H), 2.75 (m, 1H),
2.70 (m, 1H), 2.40 (m, 1H) 1.94 (m, 1H), 1.43 (m, 1H), 1.27 (m,
1H), 0.77 (dd, 6H).
Example Section E
[1181] ##STR452## ##STR453##
Example 1
[1182] To a solution of phenol 3 (336 mg, 0.68 mmol) in THF (10 mL)
was added Cs.sub.2CO.sub.3 (717 mg, 2.2 mmol) and triflate (636 mg,
1.5 mmol) in THF (3 mL). After the reaction mixture was stirred for
30 min at room temperature, the mixture was partitioned between
EtOAc and water. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was chromatographed on silica gel (eluting 40-50% EtOAc/hexane) to
give dibenzylphosphonate 4 (420 mg, 80%) as a colorless oil.
Example 2
[1183] ##STR454##
[1184] To a solution of dibenzylphosphonate 4 (420 mg, 0.548 mmol)
in CH.sub.2Cl.sub.2 (10 mL) was added TFA (0.21 mL, 2.74 mmol).
After the reaction mixture was stirred for 2 h at room temperature,
additional TFA (0.84 mL, 11 mmol) was added and the mixture was
stirred for 3 h. The reaction mixture was evaporated under reduced
pressure and the residue was partitioned between EtOAc and 1M
NaHCO.sub.3. The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure to give amine 5
(325 mg, 89%).
Example 3
[1185] ##STR455##
[1186] To a solution of carbonate (79 mg, 0.27 mmol), amine 5 (178
mg, 0.27 mmol), and CH.sub.3CN (10 mL) was added DMAP (66 mg, 0.54
mmol) at 0.degree. C. After the reaction mixture was warmed to room
temperature and stirred for 16 hours, the mixture was concentrated
under reduced pressure. The residue was chromatographed on silica
gel (eluting 60-90% EtOAc/hexane) to give a mixture of carbamate 6
and starting carbonate. The mixture was further purified by HPLC on
C18 reverse phase chromatography (eluting 60% CH.sub.3CN/water) to
give carbamate 6 (49 mg, 22%) as a colorless oil. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 7.68 (d, 2H), 7.22 (m, 15H), 6.95 (d, 2H),
5.62 (d, 1H), 5.15 (dt, 4H), 5.00 (m, 2H), 4.21 (d, 2H), 3.88 (m,
4H), 3.67 (m, 3H), 3.15 (r, 2H), 2.98 (m, 3H), 2.80 (m, 2H), 1.82
(m, 1H), 1.61 (m, 1H), 0.93 (d, 3H), 0.88 (d, 3H).
Example 4
[1187] ##STR456##
[1188] To a solution of carbamate 6 (21 mg, 0.026 mmol) in
EtOH/EtOAc (2 mL/1 mL) was added 10% Pd/C (11 mg). After the
reaction mixture was stirred under H.sub.2 atmosphere (balloon) for
2 hours, the mixture was filtered through Celite. The filtrate was
evaporated under reduced pressure to give phosphonic acid 7 (17 mg,
100%) as a colorless solid. .sup.1H NMR (300 MHz, CD.sub.3OD)
.delta. 7.73 (d, 2H), 7.19 (m, 5H), 7.13 (d, 2H), 5.53 (d, 1H),
4.26 (d, 2H), 3.86 (m, 1H), 3.64 (m, 5H), 3.38 (d, 1H), 3.13 (d,
1H), 3.03 (dd, 1H), 2.86 (m, 3H), 2.48 (m, 1H), 1.97 (m, 1H), 1.47
(m, 1H), 1.28 (m, 2H), 1.13 (t, 1H), 0.88 (d, 3H), 0.83 (d, 3H).
##STR457##
Example 5
[1189] ##STR458##
[1190] To a solution of phenol 8 (20 mg, 0.036 mmol) and triflate
(22 mg, 0.073 mmol) in THF (2 mL) was added Cs.sub.2CO.sub.3 (29
mg, 0.090 mmol). After the reaction mixture was stirred for 30 min
at room temperature, the mixture was partitioned between EtOAc and
water. The organic phase was dried over Na.sub.2SO.sub.4, filtered,
and evaporated under reduced pressure. The crude product was
purified by preparative thin layer chromatography (eluting 80%
EtOAc/hexane) to give diethylphosphonate 9 (21 mg, 83%) as a
colorless oil. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.73 (d,
2H), 7.25 (m, 5H), 7.07 (d, 2H), 5.64 (d, 1H), 5.01 (m, 2H), 4.25
(m, 6H), 3.88 (m, 4H), 3.70 (m, 3H), 2.97 (m, 6H), 1.70 (m, 4H),
1.38 (t, 6H), 0.92 (d, 3H), 0.88 (d, 3H). .sup.31P NMR (300 MHz,
CDCl.sub.3) .delta. 18.1. ##STR459##
Example 6
[1191] ##STR460##
[1192] To a solution of phosphonic acid 10 (520 mg, 2.57 mmol) in
CH.sub.3CN (5 mL) was added thionyl chloride (0.75 mL, 10.3 mmol)
and heated to 70.degree. C. in an oil bath. After the reaction
mixture was stirred for 2 h at 70.degree. C., the mixture was
concentrated and azeotroped with toluene. To a solution of the
crude chloridate in toluene (5 mL) was added tetrazole (18 mg, 0.26
mmol) at 0.degree. C. To this mixture was added phenol (121 mg,
1.28 mmol) and triethylamine (0.18 mL, 1.28 mmol) in toluene (3 mL)
at 0.degree. C. After the reaction mixture was warmed to room
temperature and stirred for 2 h, ethyl lactate (0.29 mL, 2.57 mmol)
and triethylamine (0.36 mL, 2.57 mmol) in toluene (2.5 mL) were
added. The reaction mixture was stirred for 16 hours at room
temperature, at which time the mixture was partitioned between
EtOAc and sat. NH.sub.4Cl. The organic phase was washed with sat.
NH.sub.4Cl, 1M NaHCO.sub.3, and brine, then dried over
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was chromatographed on silica gel (eluting 20-40%
EtOAc/hexane) to give two diastereomers of phosphonate 11 (66 mg,
109 mg, 18% total) as colorless oils.
Example 7A
[1193] ##STR461##
[1194] To a solution of phosphonate 11 isomer A (66 mg, 0.174 mmol)
in EtOH (2 mL) was added 10% Pd/C (13 mg). After the reaction
mixture was stirred under H.sub.2 atmosphere (balloon) for 6 h, the
mixture was filtered through Celite. The filtrate was evaporated
under reduced pressure to give alcohol 12 isomer A (49 mg, 98%) as
a colorless oil.
Example 7B
[1195] To a solution of phosphonate 11 isomer B (110 mg, 0.291
mmol) in EtOH (3 mL) was added 10% Pd/C (22 mg). After the reaction
mixture was stirred under H.sub.2 atmosphere (balloon) for 6 h, it
was filtered through Celite. The filtrate was evaporated under
reduced pressure to give alcohol 12 isomer B (80 mg, 95%) as a
colorless oil.
Example 8A
[1196] ##STR462##
[1197] To a solution of alcohol 12 isomer A (48 mg, 0.167 mmol) in
CH.sub.2Cl.sub.2 (2 mL) was added 2,6-lutidine (0.03 mL, 0.250
mmol) and trifluoromethanesulfonic anhydride (0.04 mL, 0.217 mmol)
at -40.degree. C. (dry ice-CH.sub.3CN bath). After the reaction
mixture was stirred for 15 min at -40.degree. C., the mixture was
warmed to 0.degree. C. and partitioned between Et.sub.2O and 1M
H.sub.3PO.sub.4. The organic phase was washed with 1M
H.sub.3PO.sub.4 (3 times), dried over Na.sub.2SO.sub.4, filtered,
and evaporated under reduced pressure to give triflate 13 isomer A
(70 mg, 100%) as a pale yellow oil.
Example 8B
[1198] To a solution of alcohol 12 isomer B (80 mg, 0.278 mmol) in
CH.sub.2Cl.sub.2 (3 mL) was added 2,6-lutidine (0.05 mL, 0.417
mmol) and trifluoromethanesulfonic anhydride (0.06 mL, 0.361 mmol)
at -40.degree. C. (dry ice-CH.sub.3CN bath). After the reaction
mixture was stirred for 15 min at -40.degree. C., the mixture was
warmed to 0.degree. C. and partitioned between Et.sub.2O and 1M
H.sub.3PO.sub.4. The organic phase was washed with 1M
H.sub.3PO.sub.4 (3 times), dried over Na.sub.2SO.sub.4, filtered,
and evaporated under reduced pressure to give triflate 13 isomer B
(115 mg, 98%) as a pale yellow oil.
Example 9A
[1199] ##STR463##
[1200] To a solution of phenol (64 mg, 0.111 mmol): ##STR464## and
triflate 13 isomer A (70 mg, 0.167 mmol) in THF (2 mL) was added
Cs.sub.2CO.sub.3 (72 mg, 0.222 mmol). After the reaction mixture
was stirred for 30 min at room temperature, the mixture was
partitioned between EtOAc and water. The organic phase was dried
over Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was chromatographed on silica gel
(eluting 60-80% EtOAc/hexane) to give a mixture. The mixture was
further purified by HPLC on C18 reverse phase chromatography
(eluting 55% CH.sub.3CN/water) to give phosphonate 14 isomer A (30
mg, 32%) as a colorless solid. .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.71 (d, 2H), 7.26 (m, 6H), 7.00 (m, 5H), 5.65 (d, 1H),
5.14 (m, 1H), 5.00 (m, 2H), 4.54 (dd, 1H), 4.44 (dd, 1H), 4.17 (m,
2H), 3.96 (dd, 1H), 3.86 (m, 5H), 3.72 (m, 3H), 3.14 (m, 1H), 2.97
(m, 4H), 2.79 (m, 2H), 1.83 (m, 1H), 1.62 (m, 3H), 1.50 (d, 3H),
1.25 (m, 3H), 0.93 (d, 3H), 0.88 (d, 3H). .sup.31P NMR (300 MHz,
CDCl.sub.3) .delta. 17.4.
Example 9B
[1201] To a solution of phenol (106 mg, 0.183 mmol): ##STR465## and
triflate 13 isomer B (115 mg, 0.274 mmol) in THF (2 mL) was added
Cs.sub.2CO.sub.3 (119 mg, 0.366 mmol). After the reaction mixture
was stirred for 30 min at room temperature, the mixture was
partitioned between EtOAc and water. The organic phase was dried
over Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was chromatographed on silica gel
(eluting 60-80% EtOAc/hexane) to give a mixture. The mixture was
further purified by HPLC on C18 reverse phase chromatography
(eluting 55% CH.sub.3CN/water) to give phosphonate 14 isomer B (28
mg, 18%) as a colorless solid. .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.71 (d, 2H), 7.26 (m, 6H), 6.94 (m, 5H), 5.66 (d, 1H),
5.17 (m, 1H), 4.99 (m, 2H), 4.55 (m, 1H), 4.42 (m, 1H), 4.16 (m,
2H), 3.97 (m, 1H), 3.85 (m, 5H), 3.72 (m, 3H), 3.13 (m, 1H), 2.97
(m, 4H), 2.80 (m, 2H), 1.83 (m, 1H), 1.60 (m, 6H), 1.22 (m, 3H),
0.93 (d, 3H), 0.88 (d, 3H). .sup.31P NMR (300 MHz, CDCl.sub.3)
.delta. 15.3. Resolution of Compound 14 Diastereomers
[1202] Analysis was performed on an analytical Alltech Econosil
column, conditions described below, with a total of about 0.5 mg 14
injected onto the column. This lot was a mixture of major and minor
diastereomers where the lactate ester carbon is a mix of R and S
configurations (FIG. 1). Up to 2 mg could be resolved on the
analytical column. Larger scale injections (up to 50 mg 14) were
performed on an Alltech Econosil semi-preparative column (FIG. 2),
conditions described below.
[1203] The isolated diastereomer fractions were stripped to dryness
on a rotary evaporator under house vacuum, followed by a final high
vacuum strip on a vacuum pump. The chromatographic solvents were
displaced by two portions of dichloromethane before the final high
vacuum strip to aid in removal of trace solvents, and to yield a
friable foam.
[1204] The bulk of the diastereomer resolution was performed with
n-heptane substituted for hexanes for safety considerations.
[1205] Sample Dissolution: While a fairly polar solvent mixture is
described below, the sample may be dissolved in mobile phase with a
minimal quantity of ethyl alcohol added to dissolve the sample.
Analytical Column, 0.45 mg Injection, Hexanes--IPA (90:10) (FIG.
1)
[1206] HPLC Conditions [1207] Column: Alltech Econosil, 5 .mu.m,
4.6.times.250 mm [1208] Mobile Phase: Hexanes-Isopropyl Alcohol
(90:10) [1209] Flow Rate: 1.5 mL/min [1210] Run Time: 50 min [1211]
Detection: UV at 242 nm [1212] Temperature: Ambient [1213]
Injection Size: 100 .mu.L [1214] Sample Prep.: .about.5 mg/mL,
dissolved in hexanes-ethyl alcohol (75:25) [1215] Retention Times:
14.about.22 min [1216] : 14.about.29 min [1217] : Less Polar
Impurity .about.19 min Semi-Preparative Column, 50 mg Injection,
n-Heptane-IPA (84:16) (FIG. 2)
[1218] HPLC Conditions [1219] Column: Alltech Econosil, 10 .mu.m,
22.times.250 mm [1220] Mobile Phase: n-Heptane-Isopropyl Alcohol
(84:16) [1221] Flow Rate: 10 mL/min [1222] Run Time: 65 min [1223]
Detection: UV at 257 nm [1224] Temperature: Ambient [1225]
Injection Size: .about.50 mg [1226] Dissolution: 2 ml, mobile phase
plus .about.0.75 ml, ethyl alcohol [1227] Retention Times:
14.about.41 min [1228] : 14.about.54 min [1229] : Less Polar
Impurity.about.Not resolved
Example Section F
Example 1
[1230] Phosphonic acid 2: To a solution of compound 1 (A. Flohr et
al, J. Med. Chem., 42, 12, 1999; 2633-2640) (4.45 g, 17 mmol) in
CH.sub.2Cl.sub.2 (50 mL) at room temperature was added
bromotrimethylsilane (1.16 mL, 98.6 mmol). The solution was stirred
for 19 h. The volatiles were evaporated under reduced pressure to
give the oily phosphonic acid 2 (3.44 g, 100%). .sup.1H NMR
(CDCl.sub.3) .delta. 7.30 (m, 5H), 4.61 (s, 2H), 3.69 (d, 2H).
Example 2
[1231] Compound 3: To a solution of phosphonic acid 2 (0.67 g, 3.3
mmol) in CH.sub.3CN (5 mL) was added thionyl chloride (1 mL, 13.7
mmol) and the solution was heated at 70.degree. C. for 2.5 h. The
volatiles were evaporated under reduced pressure and dried in vacuo
to afford an oily phophonyl dichloride. The crude chloride
intermediate was dissolved in CH.sub.2Cl.sub.2 (20 mL) and cooled
in an ice/water bath. Ethyl lactate (1.5 mL, 13.2 mmol) and
triethyl amine (1.8 mL, 13.2 mmol) were added dropwise. The mixture
was stirred for 4 h at room temperature and diluted with more
CH.sub.2Cl.sub.2 (100 mL). The organic solution was washed with
0.1N HCl, saturated aqueous NaHCO.sub.3, and brine, dried
(MgSO.sub.4) filtered and evaporated under reduced pressure. The
crude product was chromatographed on silica gel to afford oily
compound 3 (0.548 g, 41%). .sup.1H NMR (CDCl.sub.3) .delta. 7.30
(m, 5H), 5.00-5.20 (m, 2H), 4.65 (m, 2H), 4.20 (m, 4H), 3.90 (d,
2H), 1.52 (t, 6H), 1.20 (t, 6H).
Example 3
[1232] Alcohol 4: A solution of compound 3 (0.54 g, 1.34 mmol) in
EtOH (15 mL) was treated with 10% Pd/C (0.1 g) under H.sub.2 (100
psi) for 4 h. The mixture was filtered and the filtrate was treated
with fresh 10% PD/C (0.1 g) under H.sub.2 (1 atmosphere) for 18 h.
The mixture was filtered and the filtrate was evaporated to afford
alcohol 4 (0.395 g, 94%) as an oil. .sup.1H NMR (CDCl.sub.3)
.delta. 4.90-5.17 (m, 2H), 4.65 (q, 2H), 4.22 (m, 4H), 4.01 (m,
2H), 1.55 (t, 6H), 1.21 (t, 6H); .sup.31P NMR (CDCl.sub.3) .delta.
22.8.
Example 4
[1233] Triflate 5: To absolution of alcohol 4 (122.8 mg, 0.393
mmol) in CH.sub.2Cl.sub.2 (5 mL) at -40.degree. C. were added
2,6-lutidine (0.069 mL, 0.59 mmol) and trifluoromethansulfonic
anhydride (0.086 mL, 0.51 mmol). Stirring was continued at
0.degree. C. for 2 h. and the mixture partitioned in
CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3. The organic layer was
washed with 0.1N HCl, saturated NaCl, dried (MgSO.sub.4), filtered
and evaporated under reduced pressure. The crude product 5 (150 mg,
87%) was used for the next step without further purification.
.sup.1H NMR (CDCl.sub.3) .delta. 5.0-5.20 (m, 2H), 4.93 (d, 2H),
4.22 (m, 4H), 1.59 (m, 6H), 1.29 (t, 611.
Example 5
[1234] Phosphonate 6: A solution of phenol 8 (see Scheme Section A,
Scheme 1 and 2) (32 mg, 0.055 mmol) and triflate 5 (50 mg, 0.11
mmol) in THF (1.5 mL) at room temperature was treated with
Cs.sub.2CO.sub.3 (45.6 mg, 0.14 mmol). The mixture was stirred for
2.5 h and partitioned in EtOAc and saturated NaHCO.sub.3. The
organic layer was washed with 0.1N HCl, saturated NaCl, dried
(MgSO.sub.4), filtered and evaporated under reduced pressure. The
crude product was purified by chromatography on silica gel (30-70%
EtOAc/hexane) affording the phosphonate 6 (41 mg, 84%) as a solid.
.sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, 2H), 7.13 (d, 2H), 7.00
(d, 2H), 6.90 (d, 2H), 5.65 (d, 1H), 4.90-5.22 (m, 3H), 4.40 (m,
2H), 4.20 (m, 4H), 3.90 (s, 3H), 3.65-4.00 (m, 5H), 2.70-3.20 (m,
6H), 1.52-1.87 (m, 12H), 1.25 (m, 6H), 0.85-0.90 (m, 6H); .sup.31P
NMR (CDCl.sub.3) .delta. 20.0.
Example 6
[1235] Compound 7: To a solution of phosphonic acid 2 (0.48 g, 2.37
mmol) in CH.sub.3CN (4 mL) was added thionyl chloride (0.65 mL,
9.48 mmol) and the solution was heated at 70.degree. C. for 2.5 h.
The volatiles were evaporated under reduced pressure and dried in
vacuo to afford an oily phophonyl dichloride. The crude chloride
intermediate was dissolved in CH.sub.2Cl.sub.2 (5 mL) and cooled in
an ice/water bath. Ethyl glycolate (0.9 mL, 9.5 mmol) and triethyl
amine (1.3 mL, 9.5 mmol) were added dropwise. The mixture was
stirred for 2 h at room temperature and diluted with more
CH.sub.2Cl.sub.2 (100 mL). The organic solution was washed with
0.1N HCl, saturated aqueous NaHCO.sub.3, and saturated NaCl, dried
(MgSO.sub.4) filtered and concentrated under reduced pressure. The
crude product was chromatographed on silica gel to afford oily
compound 7 (0.223 g, 27%). .sup.1H NMR (CDCl.sub.3) .delta. 7.30
(m, 5H), 4.65 (m, 6H), 4.25 (q, 4H), 3.96 (d, 2H), 1.27 (t, 6H);
.sup.31P NMR (CDCl.sub.3) .delta. 24.0.
Example 7
[1236] Alcohol 8: A solution of compound 7 (0.22 g, 0.65 mmol) in
EtOH (8 mL) was treated with 10% Pd/C (0.04 g) under H.sub.2 (1
atmosphere) for 4 h. The mixture was filtered and the filtrate was
evaporated to afford alcohol 8 (0.156 g, 96%) as an oil. .sup.1H
NMR (CDCl.sub.3) .delta. 4.66 (m, 4H), 4.23 (q, 4H), 4.06 (d, 2H),
1.55 (t, 6H), 1.26 (t, 6H); .sup.31P NMR (CDCl.sub.3) .delta.
26.8.
Example 8
[1237] Triflate 9: To a solution of alcohol 8 (156 mg, 0.62 mmol)
in CH.sub.2Cl.sub.2 (5 mL) at -40.degree. C. were added
2,6-lutidine (0.11 mL, 0.93 mmol) and trifluoromethansulfonic
anhydride (0.136 mL, 0.8 mmol). Stirring was continued at 0.degree.
C. for 2 h. and the mixture partitioned in CH.sub.2Cl.sub.2 and
saturated NaHCO.sub.3. The organic layer was washed with 0.1N HCl,
saturated NaCl, dried (MgSO.sub.4), filtered and evaporated under
reduced pressure. The crude product 9 (210 mg, 88%) was used for
the next step without further purification. .sup.1H NMR
(CDCl.sub.3) .delta. 4.90 (d, 2H), 4.76 (d, 4H), 4.27 (q, 4H), 1.30
(t, 6H).
Example 9
[1238] Phosphonate 10: A solution of phenol 8 (30 mg, 0.052 mmol)
and triflate 9 (30 mg, 0.078 mmol) in THF (1.5 mL) at room
temperature was treated with Cs.sub.2CO.sub.3 (34 mg, 0.1 mmol).
The mixture was stirred for 2.5 h and partitioned in EtOAc and
saturated NaHCO.sub.3. The organic layer was washed with 0.1N HCl,
saturated NaCl, dried (MgSO.sub.4), filtered and evaporated under
reduced pressure. The crude product was purified by chromatography
on silica gel (30-70% EtOAc/hexane) affording the unreacted phenol
(xx) (12 mg, 40%) and the phosphonate 10 (16.6 mg, 38%) as a solid.
.sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, 2H), 7.13 (d, 2H), 7.00
(d, 2H), 6.90 (d, 2H), 5.65 (d, 1H), 5.00 (m, 2H), 4.75 (m, 4H),
4.48 (d, 2H), 4.23 (q, 4H), 3.90 (s, 3H), 3.65-4.00 (m, 5H),
2.70-3.20 (m, 6H), 2.23 (b.s., 2H), 1.52-1.87 (m, 4H), 1.25 (t,
6H), 0.85-0.90 (m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 22.0.
Example 10
[1239] Compound 11: To a solution of phosphonic acid 2 (0.512 g,
2.533 mmol) in CH.sub.3CN (5 mL) was added thionyl chloride (0.74
mL, 10 mmol) and the solution was heated at 70.degree. C. for 2.5
h. The volatiles were evaporated under reduced pressure and dried
in vacuo to afford an oily phophonyl dichloride. The crude chloride
intermediate was dissolved in toluene (8 mL) and cooled in an
ice/water bath. A catalytic amount of tetrazol (16 mg, 0.21 mmol)
was added followed by the addition of a solution of triethylamine
(0.35 mL, 2.53 mmol) and phenol (238 mg, 2.53 mmol) in toluene (5
mL). The mixture was stirred at room temperature for 3 h. A
solution of ethyl glycolate (0.36 mL, 3.8 mmol) and triethyl amine
(0.53 mL, 3.8 mmol) in toluent (3 mL) was added dropwise. The
mixture was stirred for 18 h at room temperature and partitioned in
EtOAc and 0.1N HCl. The organic solution was washed with saturated
aqueous NaHCO.sub.3, and saturated NaCl, dried (MgSO.sub.4)
filtered and concentrated under reduced pressure. The crude product
was chromatographed on silica gel to afford diphenyl phosphonate as
a by product (130 mg) and compound 11 (0.16 g, 18%). .sup.1H NMR
(CDCl.sub.3) .delta. 7.15-7.40 (m, 10H), 4.58-4.83 (m, 4H), 4.22
(q, 2H), 4.04 (dd, 2H), 1.24 (t, 3H).
Example 11
[1240] Alcohol 12: A solution of compound 11 (0.16 g, 0.44 mmol) in
EtOH (5 mL) was treated with 10% Pd/C (0.036 g) under H.sub.2 (1
atmosphere) for 22 h. The mixture was filtered and the filtrate was
evaporated to afford alcohol 12 (0.112 g, 93%) as an oil. .sup.1H
NMR (CDCl.sub.3) .delta. 7.15-7.36 (m, 5H), 4.81 (dd, 1H), 4.55
(dd, 1H), 4.22 (q, 2H), 4.12 (m, 2H), 3.78 (b.s., 1H), 1.26 (t,
6H); .sup.31P NMR (CDCl.sub.3) .delta. 22.9
Example 12
[1241] Triflate 13: To a solution of alcohol 12 (112 mg, 0.41 mmol)
in CH.sub.2Cl.sub.2 (5 mL) at -40.degree. C. were added
2,6-lutidine (0.072 mL, 0.62 mmol) and trifluoromethansulfonic
anhydride (0.09 mL, 0.53 mmol). Stirring was continued at 0.degree.
C. for 3 h. and the mixture partitioned in CH.sub.2Cl.sub.2 and
saturated NaHCO.sub.3. The organic layer was washed with 0.1N HCl,
saturated NaCl, dried (MgSO.sub.4), filtered and evaporated under
reduced pressure. The crude product was purified by chromatography
on silica gel (30% EtOAc/hexane) affording triflate 13 (106 mg,
64%). .sup.1H NMR (CDCl.sub.3) .delta. 7.36 (m, 2H), 7.25 (m, 3H),
4.80-5.10 (m, 3H), 4.60 (dd, 1H), 4.27 (q, 2H), 1.28 (t, 3H);
.sup.31P NMR (CDCl.sub.3) .delta. 11.1
Example 13
[1242] Phosphonate 14: A solution of phenol 8 (32 mg, 0.052 mmol)
and triflate 13 (32 mg, 0.079 mmol) in CH.sub.3CN (1.5 mL) at room
temperature was treated with Cs.sub.2CO.sub.3 (34 mg, 0.1 mmol).
The mixture was stirred for 1 h and partitioned in EtOAc and
saturated NaHCO.sub.3. The organic layer was washed with saturated
NaCl, dried (MgSO.sub.4), filtered and evaporated under reduced
pressure. The crude product was purified by chromatography on
silica gel (70% EtOAc/hexane) affording phosphonate 14 (18 mg,
40%). .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, 2H), 6.75-7.35 (m,
11H, 5.65 (d, 1H), 5.00 (m, 2H), 4.50-4.88 (m, 3H), 4.20 (q, 2H),
3.84 (s, 3H), 3.65-4.00 (m, 5H), 2.70-3.20 (m, 6H), 1.52-1.87 (m,
6H), 1.25 (t, 3H), 0.85-0.90 (m, 6H); .sup.31P NMR (CDCl.sub.3)
.delta. 17.9, 17.7.
Example 14
[1243] Piperidine 16: A solution of compound 15 (3.1 g, 3.673 mmol)
in MeOH (100 mL) was treated with 10% Pd/C (0.35 g) under H.sub.2
(1 atmosphere) for 18 h. The mixture was filtered and the filtrate
was evaporated to afford phenol 16 (2 g, 88%). .sup.1H NMR
(CD.sub.3OD) .delta. 7.76 (d, 2H), 7.08 (d, 2H), 7.04 (d, 2H), 6.65
(d, 2H), 5.59 (d, 1H), 4.95 (m, 1H), 3.98 (s, 3H), 3.65-4.00 (m,
5H), 3.30-3.50 (m, 3H), 2.80-3.26 (m, 5H), 2.40-2.70 (m, 3H),
1.35-2.00 (m, 7H), 1.16 (m, 2H); MS (ESI) 620 (M+H).
Example 15
[1244] Formamide 17: Piperidine 16 obtained above (193 mg, 0.3118
mmol) in DMF (4 mL) was treated with formic acid (0.035 mL, 0.936
mmol), triethylamine (0.173 mL, 1.25 mmol) and EDCI (179 mg, 0.936
mmol) at room temperature. The mixture was stirred for 18 h and
partitioned in EtOAc and saturated NaHCO.sub.3. The organic layer
was washed with saturated NaCl, dried (MgSO.sub.4), filtered and
evaporated under reduced pressure. The crude product was purified
by chromatography on silica gel (EtOAC/hexane) affording formamide
17 (162 mg, 80%). .sup.1H NMR (CDCl.sub.3) .delta. 7.96 (s, 1H),
7.68 (d, 2H), 7.04 (d, 2H), 6.97 (d, 2H), 6.76 (d, 2H), 5.63 (d,
1H), 5.37 (bs, 1H), 5.04 (m, 1H), 4.36 (m, 1H), 3.93 (s, 3H),
3.52-3.95 (m, 7H), 2.70-3.20 (m, 8H), 1.48-2.00 (m, 7H), 1.02 (m,
2H).
Example 16
[1245] Dibenzyl phosphonate 18: A solution of phenol 17 (123 mg,
0.19 mmol) and dibenzyl trifluoromethansulfonyloxymethanphosphonate
YY (120 mg, 0.28 mmol) in CH.sub.3CN (1.5 mL) at room temperature
was treated Cs.sub.2CO.sub.3 (124 mg, 0.38 mmol). The mixture was
stirred for 3 h and partitioned in CH.sub.2Cl.sub.2 and saturated
NaHCO.sub.3. The organic layer was washed with 0.1N HCl, saturated
NaCl, dried (MgSO.sub.4), filtered and evaporated under reduced
pressure. The crude product was purified by chromatography on
silica gel (10% MeOH/CH.sub.2Cl.sub.2) affording phosphonate 18
(154 mg, 88%). .sup.1H NMR (CDCl.sub.3) .delta. 7.96 (s, 1H), 7.68
(d, 2H), 7.35 (m, 10H), 7.10 (d, 2H), 6.97 (d, 2H), 6.80 (d, 2H),
5.63 (d, 1H), 4.96-5.24 (m, 6H), 4.37 (m, 1H), 4.20 (d, 2H), 3.84
(s, 3H), 3.52-3.95 (m, 7H), 2.55-3.20 (m, 8H), 1.48-2.00 (m, 7H),
1.02 (m, 2H). .sup.31P NMR (CDCl.sub.3) .delta. 20.3.
Example 17
[1246] Phosphonic acid 19: A solution of phosphonate 18 (24 mg,
0.026 mmol) in MeOH (3 mL) was treated with 10% Pd/C (5 mg) under
H.sub.2 (1 atmosphere) for 4 h. The mixture was filtered and the
filtrate was evaporated to afford phosphonic acid 19 as a solid (18
mg, 93%). .sup.1H NMR (CD.sub.3OD) .delta. 8.00 (s, 1H), 7.67 (d,
2H), 7.18 (d, 2H), 7.09 (d, 2H), 6.90 (d, 2H), 5.60 (d, 1H), 4.30
(m, 1H), 4.16 (d, 2H), 3.88 (s, 3H), 3.60-4.00 (m, 7H), 3.04-3.58
(m, 5H), 2.44-2.92 (m, 5H), 1.28-2.15 (m, 5H), 1.08 (m, 2H).
.sup.31P NMR (CDCl.sub.3) .delta. 16.3.
Example 18
[1247] Diethyl phosphonate 20: A solution of phenol 17 (66 mg, 0.1
mmol) and diethyl trifluoromethansulfonyloxymethanphosphonate XY
(46 mg, 0.15 mmol) in CH.sub.3CN (1.5 mL) at room temperature was
treated Cs.sub.2CO.sub.3 (66 mg, 0.2 mmol). The mixture was stirred
for 3 h and partitioned in CH.sub.2Cl.sub.2 and saturated
NaHCO.sub.3. The organic layer was washed with 0.1N HCl, saturated
NaCl, dried (MgSO.sub.4), filtered and evaporated under reduced
pressure. The crude product was purified by chromatography on
silica gel (10% MeOH/CH.sub.2Cl.sub.2) affording the unreacted 17
(17 mg, 26%) and diethyl phosphonate 20 (24.5 mg, 41%). .sup.1H NMR
(CDCl.sub.3) .delta. 8.00 (s, 1H), 7.70 (d, 2H), 7.16 (d, 2H),
7.00(d, 2H), 6.88 (d, 2H), 5.66 (d, 1H), 4.98-5.10 (m, 2H), 4.39
(m, 1H), 4.24 (m, 5H), 3.89 (s, 3H), 3.602-3.98 (m, 7H), 2.55-3.16
(m, 8H), 1.50-2.00 (m, 7H), 1.36 (t, 6H), 1.08 (m, 2H). .sup.31P
NMR (CDCl.sub.3) .delta. 19.2
Example 19
[1248] N-methyl piperidine diethyl phosphonate 21: A solution of
compound 20 (22.2 mg, 0.0278 mmol) in THF (1.5 mL) at 0.degree. C.
was treated with a solution of borane in THF (1M, 0.083 mL). The
mixture was stirred for 2 h at room temperature and the starting
material was consumed completely as monitored by TLC. The reaction
mixture was cooled in an ice/water bath and excess methanol (1 mL)
was added to quench the reaction. The solution was concentrated in
vacuo and the crude product was chromatographed on silica gel with
MeOH/EtOAc to afford compound 21 (7 mg, 32%). .sup.1H NMR
(CDCl.sub.3) .delta. 7.70 (d, 2H), 7.16 (d, 2H), 7.00(d, 2H), 6.88
(d, 2H), 5.66 (d, 1H), 4.98-5.10 (m, 2H), 4.24 (m, 4H), 3.89 (s,
3H), 3.602-3.98 (m, 7H), 2.62-3.15 (m, 9H), 2.26 (s, 3H), 1.52-2.15
(m, 10H), 1.36 (t, 6H). .sup.31P NMR (CDCl.sub.3) .delta. 19.3
Example Section G
Example 1
[1249] Compound 1: To a solution of 4-nitrobenzyl bromide (21.6 g,
100 mmol) in toluene (100 mL) was added triethyl phosphite (17.15
mL, 100 mL). The mixture was heated at 120.degree. C. for 14 hrs.
The evaporation under reduced pressure gave a brown oil, which was
purified by flash column chromatography (hexane/EtOAc=2/1 to 100%
EtOAc) to afford compound 1.
Example 2
[1250] Compound 2: To a solution of compound 1 (1.0 g) in ethanol
(60 mL) was added 10% Pd--C (300 mg). The mixture was hydrogenated
for 14 hrs. Celite was added and the mixture was stirred for 5
mins. The mixture was filtered through a pad of celite, and washed
with ethanol. Concentration gave compound 2.
Example 3
[1251] Compound 3: To a solution of compound 3 (292 mg, 1.2 mmol)
and aldehyde (111 mg, 0.2 mmol) in methanol (3 mL) was added acetic
acid (48 .mu.L, 0.8 mmol). The mixture was stirred for 5 mins, and
sodium cyanoborohydride (25 mg, 0.4 mmol) was added. The mixture
was stirred for 14 hrs, and methanol was removed under reduced
pressure. Water was added, and was extracted with EtOAc. The
organic phase was washed 0.5 N NaOH solution (1.times.), water
(2.times.), and brine (1.times.), and was dried over MgSO.sub.4.
Purification by flash column chromatography
(CH.sub.2Cl.sub.2/MeOH=100/3) gave compound 3.
Example 4
[1252] Compound 4: To a solution of compound 3 (79 mg, 0.1 mmol) in
CH.sub.2Cl.sub.2 (5 mL) was added trifluoroacetic acid (1 mL). The
mixture was stirred for 2 hrs, and solvents were evaporated under
reduced pressure. Coevaporation with EtOAc and CH.sub.2Cl.sub.2
gave an oil. The oil was dissolved in THF (1 mL) and
tetrabutylammonium fluoride (0.9 mL, 0.9 mmol) was added. The
mixture was stirred for 1 hr, and solvent was removed. Purification
by flash column chromatography (CH.sub.2Cl.sub.2/MeOH=100/7) gave
compound 4.
Example 5
[1253] Compound 5: To a solution of compound 4 (0.1 mmol) in
acetonitrile (1 mL) at 0.degree. C. was added DMAP (22 mg, 0.18
mmol), followed by bisfurancarbonate (27 mg, 0.09 mmol). The
mixture was stirred for 3 hrs at 0.degree. C., and diluted with
EtOAc. The organic phase was washed with 0.5 N NaOH solution
(2.times.), water (2.times.), and brine (1.times.), and dried over
MgSO.sub.4. Purification by flash column chromotography
(CH.sub.2Cl.sub.2/MeOH=100/3 to 100/5) afford compound 5 (50 mg):
.sup.1H NMR (CDCl.sub.3) .delta. 7.70 (2H, d, J=8.9 Hz), 7.11 (2H,
d, J=8.5 Hz), 6.98 (2H, d, J=8.9 Hz), 6.61 (2H, d, J=8.5 Hz), 5.71
(1H, d, J=5.2 Hz), 5.45 (1H, m), 5.13 (1H, m), 4.0 (6H, m),
3.98-3.70 (4H, m), 3.86 (3H, s), 3.38 (2H, m), 3.22 (1H, m), 3.02
(5H, m), 2.8 (1H, m), 2.0-1.8 (3H, m), 1.26 (6H, t, J=7.0 Hz), 0.95
(3H, d, J=6.7 Hz), 0.89 (3H, d, J=6.7 Hz).
Example 6
[1254] Compound 6: To a solution of compound 5 (30 mg, 0.04 mmol)
in MeOH (0.8 mL) was added 37% fomaldehyde (30 .mu.L, 0.4 mmol),
followed by acetic acid (23 .mu.L, 0.4 mmol). The mixture was
stirred for 5 mins, and sodium cyanoborohydride (25 mg, 0.4 mmol)
was added. The reaction mixture was stirred for 14 hrs, and diluted
with EtOAc. The organic phase was washed 0.5 N NaOH solution
(2.times.), water (2.times.), and brine, and dried over MgSO.sub.4.
Purification by flash column chromatography
(CH.sub.2Cl.sub.2/MeOH=100/3) gave compound 6 (11 mg): .sup.1H NMR
(CDCl.sub.3) .delta. 7.60 (2H, d, J=8.9 Hz), 7.17 (2H, m), 6.95
(2H, d, J=8.9 Hz), 6.77 (2H, d, J=8.5 Hz), 5.68 (1H, d, J=5.2 Hz),
5.21 (1H, m), 5.09 (1H, m), 4.01 (6H, m), 3.87 (3H, s), 3.8-3.3
(4H, m), 3.1-2.6 (7H, m), 2.90 (3H, s), 1.8 (3H, m), 1.25 (6H, m),
0.91 (6H, m).
Example 7
[1255] Compound 7: To a solution of compound 1 (24.6 g, 89.8 mmol)
in acetonitrile (500 mL) was added TMSBr (36 mL, 269 mmol). The
reaction mixture was stirred for 14 hrs, and evaporated under
reduced pressure. The mixture was coevaporated with MeOH
(2.times.), toluene (2.times.), EtOAc (2.times.), and
CH.sub.2Cl.sub.2 to give a yellow solid (20 g). To the suspension
of above yellow solid (15.8 g, 72.5 mmol) in toluene (140 mL) was
added DMF (1.9 mL), followed by thionyl chloride (53 mL, 725 mmol).
The reaction mixture was heated at 60.degree. C. for 5 hrs, and
evaporated under reduced pressure. The mixture was coevaporated
with toluene (2.times.), EtOAc, and CH.sub.2Cl.sub.2 (2.times.) to
afford a brown solid. To the solution of the brown solid in
CH.sub.2Cl.sub.2 at 0.degree. C. was added benzyl alcohol (29 mL,
290 mmol), followed by slow addition of pyridine (35 mL, 435 mmol).
The reaction mixture was allowed to warm to 25.degree. C. and
stirred for 14 hrs. Solvents were removed under reduced pressure.
The mixture was diluted with EtOAc, and washed with water
(3.times.) and brine (1.times.), and dried over MgSO.sub.4.
Concentration gave a dark oil, which was purified by flash column
chromatography (hexanes/EtOAc=2/1 to 1/1) to afford compound 7.
Example 8
[1256] Compound 8: To a solution of compound 7 (15.3 g) in acetic
acid (190 mL) was added Zinc dust (20 g). The mixture was stirred
for 14 hrs, and celite was added. The suspension was filtered
through a pad of celite, and washed with EtOAc. The solution was
concentrated under reduced pressure to dryness. The mixture was
diluted with EtOAc, and was washed with 2N NaOH (2.times.), water
(2.times.), and brine (1.times.), and dried over MgSO.sub.4.
Concentration under reduced pressure gave compound 8 as an oil (15
g).
Example 9
[1257] Compound 9: To a solution of compound 8 (13.5 g, 36.8 mmol)
and aldehyde (3.9 g, 7.0 mmol) in methanol (105 mL) was added
acetic acid (1.68 mL, 28 mmol). The mixture was stirred for 5 mins,
and sodium cyanoborohydride (882 mg, 14 mmol) was added. The
mixture was stirred for 14 hrs, and methanol was removed under
reduced pressure. Water was added, and was extracted with EtOAc.
The organic phase was washed 0.5 N NaOH solution (1.times.), water
(2.times.), and brine (1.times.), and was dried over MgSO.sub.4.
Purification by flash column chromatography
(CH.sub.2Cl.sub.2/MeOH=100/3) gave compound 9 (6.0 g).
Example 10
[1258] Compound 10: To a solution of compound 9 (6.2 g, 6.8 mmol)
in CH.sub.2Cl.sub.2 (100 mL) was added trifluoroacetic acid (20
mL). The mixture was stirred for 2 hrs, and solvents were
evaporated under reduced pressure. Coevaporation with EtOAc and
CH.sub.2Cl.sub.2 gave an oil. The oil was dissolved in THF (1 mL)
and tetrabutylammonium fluoride (0.9 mL, 0.9 mmol) was added. The
mixture was stirred for 1 hr, and solvent was removed. Purification
by flash column chromatography (CH.sub.2Cl.sub.2/MeOH=100/7) gave
compound 10.
Example 11
[1259] Compound 11: To a solution of compound 10 (5.6 mmol) in
acetonitrile (60 mL) at 0.degree. C. was added DMAP (1.36 g, 11.1
mmol), followed by bisfurancarbonate (1.65 g, 5.6 mmol). The
mixture was stirred for 3 hrs at 0.degree. C., and diluted with
EtOAc. The organic phase was washed with 0.5 N NaOH solution
(2.times.), water (2.times.), and brine (1.times.), and dried over
MgSO.sub.4. Purification by flash column chromotography
(CH.sub.2Cl.sub.2/MeOH=100/3 to 100/5) afford compound 11 (3.6 g):
.sup.1H NMR (CDCl.sub.3) .delta. 7.70 (2H, d, J=8.9 Hz), 7.30 (10H,
m), 7.07 (2H, m), 6.97 (2H, d, J=8.9 Hz), 6.58 (2H, d, J=8.2 Hz),
5.70 (1H, d, J=5.2 Hz), 5.42 (1H, m), 5.12 (1H, m), 4.91 (4H, m),
4.0-3.7 (6H, m), 3.85 (3H, s), 3.4 (2H, m), 3.25 (1H, m), 3.06 (2H,
d, J=21 Hz), 3.0 (3H, m), 2.8 (1H, m), 1.95 (1H, m), 1.82 (2H, m),
0.91 (6H, m).
Example 12
[1260] Compound 12: To a solution of compound 11 (3.6 g) in ethanol
(175 mL) was added 10% Pd--C (1.5 g). The reaction mixture was
hydrogenated for 14 hrs. The mixture was stirred with celite for 5
mins, and filtered through a pad of celite. Concentration under
reduced pressure gave compound 12 as a white solid (2.8 g): .sup.1H
NMR (DMSO-d.sub.6) .delta. 7.68 (2H, m), 7.08 (2H, m), 6.93 (2H,
m), 6.48 (2H, m), 5.95 (1H, m), 5.0 (2H, m), 3.9-3.6 (6H, m), 3.82
(3H, s), 3.25 (3H, m), 3.05 (4H, m), 2.72 (2H, d, J=20.1 Hz),
2.0-1.6 (3H, m), 0.81 (6H, m).
Example 13
[1261] Compound 13: Compound 12 (2.6 g, 3.9 mmol) and L-alanine
ethyl ester hydrochloride (3.575 g, 23 mmol) were coevaporated with
pyridine (2.times.). The mixture was dissolved in, pyridine (20 mL)
and diisopropylethylamine (4.1 mL, 23 mmol) was added. To above
mixture was added a solution of Aldrithiol (3.46 g, 15.6 mmol) and
triphenylphosphine (4.08 g, 15.6 g) in pyridine (20 mL). The
reaction mixture was stirred for 20 hrs, and solvents were
evaporated under reduced pressure. The mixture was diluted with
ethyl acetate, and was washed with 0.5 N NaOH solution (2.times.),
water (2.times.), and brine, and dried over MgSO.sub.4.
Concentration under reduced pressure gave a yellow oil, which was
purified by flash column chromatography
(CH.sub.2Cl.sub.2/MeOH=100/5 to 100/10) to afford compound 13 (750
mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (2H, d, J=8.8 Hz), 7.13
(2H, m), 6.98 (2H, d, J=8.8 Hz), 6.61 (2H, d, J=8.0 Hz), 5.71 (1H,
d, J=5.2 Hz), 5.54 (1H, m), 5.16 (1H, m), 4.15 (6H, m), 4.1-3.6
(6H, m), 3.86 (3H, s), 3.4-3.2 (3H, m), 3.1-2.8 (8H, m), 2.0 (1H,
m), 1.82 (2H, m), 1.3 (12H, m), 0.92 (6H, m).
Example 14
[1262] Compound 14: To a solution of 4-hydroxypiperidine (19.5 g,
193 mmol) in THF at 0.degree. C. was added sodium hydroxide
solution (160 mL, 8.10 g, 203 mmol), followed by di-tert-butyl
dicarbonate (42.1 g, 193 mmol). The mixture was warmed to
25.degree. C., and stirred for 12 hours. THF was removed under
reduced pressure, and the aqueous phase was extracted with EtOAc
(2.times.). The combined organic layer was washed with water
(2.times.) and brine, and dried over MgSO.sub.4. Concentration gave
a compound 14 as a white solid (35 g).
Example 15
[1263] Compound 15: To a solution of alcohol 14 (5.25 g, 25 mmol)
in THF (100 mL) was added sodium hydride (1.2 g, 30 mmol, 60%). The
suspension was stirred for 30 mins, and chloromethyl methyl sulfide
(2.3 mL, 27.5 mmol) was added. Starting material alcohol 14 still
existed after 12 hrs. Dimethyl sulfoxide (50 mL) and additional
chloromethyl methyl sulfide (2.3 mL, 27.5 mmol) were added. The
mixture was stirred for additional 3 hrs, and THF was removed under
reduced pressure. The reaction was quenched with water, and
extracted with ethyl acetate. The organic phase was washed with
water and brine, and was dried over MgSO.sub.4. Purification by
flash column chromatography (hexanes/EtOAc=8/1) gave compound 15
(1.24 g).
Example 16
[1264] Compound 16: To a solution of compound 15 (693 mg, 2.7 mmol)
in CH.sub.2Cl.sub.2 (50 mL) at -78.degree. C. was added a solution
of sulfuryl chloride (214 .mu.L, 2.7 mmol) in CH.sub.2Cl.sub.2 (5
mL). The reaction mixture was kept at -78.degree. C. for 3 hrs, and
solvents were removed to give a white solid. The white solid was
dissolved in toluene (7 mL), and triethyl phosphite (4.5 mL, 26.6
mmol) was added. The reaction mixture was heated at 120.degree. C.
for 12 hrs. Solvent and excess reagent was removed under reduced
pressure to give compound 16.
Example 17
[1265] Compound 17: To a solution of compound 17 (600 mg) in
CH.sub.2Cl.sub.2 (10 mL) was added trifluoroacetic acid (2 mL). The
mixture was stirred for 2 hrs, and was concentrated under reduced
pressure to give an oil. The oil was diluted with methylene
chloride and base resin was added. The suspension was filtered and
the organic phase was concentrated to give compound 17.
Example 18
[1266] Compound 18: To a solution of compound 17 (350 mg, 1.4 mmol)
and aldehyde (100 mg, 0.2 mmol) in methanol (4 mL) was added acetic
acid (156 .mu.L, 2.6 mmol). The mixture was stirred for 5 mins, and
sodium cyanoborohydride (164 mg, 2.6 mmol) was added. The mixture
was stirred for 14 hrs, and methanol was removed under reduced
pressure. Water was added, and was extracted with EtOAc. The
organic phase was washed 0.5 N NaOH solution (1.times.), water
(2.times.), and brine (1.times.), and was dried over MgSO.sub.4.
Purification by flash column chromatography
(CH.sub.2Cl.sub.2/MeOH=100/3) gave compound 18 (62 mg).
Example 19
[1267] Compound 19: To a solution of compound 18 (62 mg, 0.08 mmol)
in THF (3 mL) were added acetic acid (9 .mu.L, 0.15 mmol) and
tetrabutylammonium fluoride (0.45 mL, 1.0 N, 0.45 mmol). The
mixture was stirred for 3 hr, and solvent was removed. Purification
by flash column chromotography (CH.sub.2Cl.sub.2/MeOH=100/5) gave
an oil. To a solution of above oil in CH.sub.2Cl.sub.2 (2 mL) was
added trifluoroacetic acid (2 mL). The mixture was stirred for 1
hrs, and was concentrated under reduced pressure. Coevaporation
with EtOAc and CH.sub.2Cl.sub.2 gave compound 19.
Example 20
[1268] Compound 20: To a solution of compound 19 (55 mg 0.08 mmol)
in acetonitrile (1 mL) at 0.degree. C. was added DMAP (20 mg, 0.16
mmol), followed by bisfurancarbonate (24 mg, 0.08 mmol). The
mixture was stirred for 3 hrs at 0.degree. C., and diluted with
EtOAc. The organic phase was washed with 0.5 N NaOH solution
(2.times.), water (2.times.), and brine (1.times.), and dried over
MgSO.sub.4. Purification by flash column chromotography
(CH.sub.2Cl.sub.2/MeOH=100/3 to 100/5) afford compound 20 (46 mg):
.sup.1H NMR (CDCl.sub.3) .delta. 7.70 (2H, d, J=8.9 Hz), 7.01 (2H,
d, J=8.9 Hz), 5.73 (1H, d, J=5.1 Hz), 5.51 (1H, m), 5.14 (1H, m),
4.16 (1H, m), 4.06 (1H, m), 3.94 (3H, m), 3.86 (3H, s), 3.80 (1H,
m), 3.75 (2H, d, J=9.1 Hz), 3.58 (1H, m), 3.47 (1H, m), 3.30 (1H,
m), 3.1-2.6 (8H, m), 2.3 (2H, m), 2.1-1.8 (5H, m), 1.40 (2H, m),
1.36 (6H, t, J=7.0 Hz), 0.93 (3H, d, J=6.7 Hz), 0.86 (3 h, d, J=6.7
Hz).
Example 21
[1269] Compound 21: Compound 21 was made from
Boc-4-Nitro-L-Phenylalanine (Fluka) following the procedure for
Compound 2 in Scheme Section A, Scheme 1.
Example 22
[1270] Compound 22: To a solution of chloroketone 21 (2.76 g, 8
mmol) in THF (50 mL) and water (6 mL) at 0.degree. C. (internal
temperature) was added solid NaBH.sub.4 (766 mg, 20 mmol) in
several portions over a period of 15 min while maintaining the
internal temperature below 5.degree. C. The mixture was stirred for
1.5 hrs at 0.degree. C. and solvent was removed under reduced
pressure. The mixture was quenched with saturated KHSO.sub.3 and
extracted with EtOAc. The organic phase was washed with waster and
brine, and dried over MgSO.sub.4. Concentration gave a solid, which
was recrystallized from EtOAc/hexane (1/1) to afford the
chloroalcohol 22 (1.72 g).
Example 23
[1271] Compound 23: To a suspension of chloroalcohol 22 (1.8 g, 5.2
mmol) in EtOH (50 mL) was added a solution of KOH in ethanol (8.8
mL, 0.71 N, 6.2 mmol). The mixture was stirred for 2 h at room
temperature and ethanol was removed under reduced pressure. The
reaction mixture was diluted with EtOAc, and washed with water
(2.times.), saturated NH.sub.4Cl (2.times.), water, and brine, and
dried over MgSO.sub.4. Concentration under reduced pressure
afforded epoxide 23 (1.57 g) as a white crystalline solid.
Example 24
[1272] Compound 24: To a solution of epoxide 23 (20 g, 65 mmol) in
2-propanol (250 mL) was added isobutylamine (65 mL) and the
solution was refluxed for 90 min. The reaction mixture was
concentrated under reduced pressure and was coevaporated with MeOH,
CH.sub.3CN, and CH.sub.2Cl.sub.2 to give a white solid. To a
solution of the white solid in CH.sub.2Cl.sub.2 (300 mL) at
0.degree. C. was added triethylamine (19 mL, 136 mmol), followed by
the addition of 4-methoxybenzenesulfonyl chloride (14.1 g, 65 mmol)
in CH.sub.2Cl.sub.2 (50 mL). The reaction mixture was stirred at
0.degree. C. for 30 min, and warmed to room temperature and stirred
for additional 2 hrs. The reaction solution was concentrated under
reduced pressure and was diluted with EtOAc. The organic phase was
washed with saturated NaHCO.sub.3, water and brine, and dried over
MgSO.sub.4. Concentration under reduced pressure gave compound 24
as a white solid (37.5 g).
Example 25
[1273] Compound 25: To a solution of compound 24 (37.5 g, 68 mmol)
in CH.sub.2Cl.sub.2 (100 mL) at 0.degree. C. was added a solution
of tribromoborane in CH.sub.2Cl.sub.2 (340 mL, 1.0 N, 340 mmol).
The reaction mixture was kept at 0.degree. C. for 1 hr, and warmed
to room temperature and stirred for additional 3 hrs. The mixture
was cooled to 0.degree. C., and methanol (200 mL) was added slowly.
The mixture was stirred for 1 hr and solvents were removed under
reduced pressure to give a brown oil. The brown oil was
coevaporated with EtOAc and toluene to afford compound 25 as a
brown solid, which was dried under vacuum for 48 hrs.
Example 26
[1274] Compound 26: To a solution of compound 25 in THF (80 mL) was
added a saturated sodium bicarbonate solution (25 mL), followed by
a solution of Boc2O (982 mg, 4.5 mmol) in THF (20 mL). The reaction
mixture was stirred for 5 hrs. THF was removed under reduced
pressure, and aqueous phase was extracted with EtOAc. The organic
phase was washed with water (2.times.) and Brine (1.times.), and
dried over MgSO.sub.4. Purification by flash column chromatography
(hexanes/EtOAc=1/1) gave compound 26 (467 mg).
Example 27
[1275] Compound 27: To a solution of compound 26 (300 mg, 0.56
mmol) in THF (6 mL) was added Cs.sub.2CO.sub.3 (546 mg, 1.68 mmol),
followed by a solution of triflate (420 mg, 1.39 mmol) in THF (2
mL). The reaction mixture was stirred for 1.5 hrs. The mixture was
diluted with EtOAc, and washed with water (3.times.) and brine
(1.times.), and dried over MgSO.sub.4. Purification by flash column
chromatography (hexanes/EtOAc=1/1 to 1/3) gave compound 27 (300
mg).
Example 28
[1276] Compound 28: To a solution of compound 27 (300 mg, 0.38
mmol) in CH.sub.2Cl.sub.2 (2 mL) was added trifluoroacetic acid (2
mL). The mixture was stirred for 2.5 hrs, and was concentrated
under reduced pressure. The mixture was diluted with EtOAc and was
washed with 0.5 N NaOH solution (3.times.), water (2.times.), and
brine (1.times.), and dried over MgSO.sub.4. Concentration gave a
white solid. To the solution of above white solid in acetonitrile
(3 mL) at 0.degree. C. was added DMAP (93 mg, 0.76 mmol), followed
by bisfurancarbonate (112 mg, 0.38 mmol). The mixture was stirred
for 3 hrs at 0.degree. C., and diluted with EtOAc. The organic
phase was washed with 0.5 N NaOH solution (2.times.), water
(2.times.), and brine (1.times.), and dried over MgSO.sub.4.
Purification by flash column chromotography
(CH.sub.2Cl.sub.2/MeOH=100/3 to 100/5) afford compound 28 (230 mg):
.sup.1H NMR (CDCl.sub.3) .delta. 8.16 (2H, d, J=8.5 Hz), 7.73 (2H,
d, J=9.2 Hz), 7.42 (2H, d, J=8.5 Hz), 7.10 (2H, d, J=9.2 Hz), 5.65
(1H, d, J=4.8 Hz), 5.0 (2H, m), 4.34 (2H, d, J=10 Hz), 4.25 (4H,
m), 4.0-3.6 (6H, m), 3.2-2.8 (7H, m), 1.82 (1H, m), 1.6 (2H, m),
1.39 (6H, t, J=7.0 Hz), 0.95 (6H, m).
Example 29
[1277] Compound 29: To a solution of compound 28 (50 mg) in ethanol
(5 mL) was added 10% Pd--C (20 mg). The mixture was hydrogenated
for 5 hrs. Celite was added, and the mixture was stirred for 5
mins. The reaction mixture was filtered through a pad of celite.
Concentration under reduced pressure gave compound 29 (50 mg):
.sup.1H NMR (CDCl.sub.3) .delta. 7.72 (2H, d, J=8.8 Hz), 7.07 (2H,
2H, d, J=8.8 Hz), 7.00 (2H, d, J=8.5 Hz), 6.61 (2H, d, J=8.5 Hz),
5.67 (1H, d, J=5.2 Hz), 5.05 (1H, m), 4.90 (1H, m), 4.34 (2H, d,
J=10.3 Hz), 4.26 (2H, m), 4.0-3.7 (6H, m), 3.17 (1H, m), 2.95 (4H,
m), 2.75 (2H, m), 1.82 (1H, m), 1.65 (2H, m), 1.39 (6H, t, J=7.0
Hz), 0.93 (3 h, d, J=6.4 Hz), 0.87 (3 h, d, J=6.4 Hz).
Example 30
[1278] Compound 30: To a solution of compound 29 (50 mg, 0.07 mmol)
and formaldehyde (52 .mu.L, 37%, 0.7 mmol) in methanol (1 mL) was
added acetic acid (40 .mu.L, 0.7 mmol). The mixture was stirred for
5 mins, and sodium cyanoborohydride (44 mg, 0.7 mmol) was added.
The mixture was stirred for 14 hrs, and methanol was removed under
reduced pressure. Water was added, and was extracted with EtOAc.
The organic phase was washed 0.5 N NaOH solution (1.times.), water
(2.times.), and brine (1.times.), and was dried over MgSO.sub.4.
Purification by flash column chromatography
(CH.sub.2Cl.sub.2/MeOH=100/3) gave compound 30 (40 mg): .sup.1H NMR
(CDCl.sub.3) .delta. 7.73 (2H, d, J=8.9 Hz), 7.10 (4H, m), 6.66
(2H, d, J=8.2 Hz), 5.66 (1H, d, J=5.2 Hz), 5.02 (1H, m), 4.88 (1H,
m), 4.32 (2H, d, J=10.1 Hz), 4.26 (4H, m), 3.98 (1H, m), 3.85 (3H,
m), 3.75 (2H, m), 3.19 (1H, m), 2.98 (4H, m), 2.93 (6H, s), 2.80
(2H, m), 1.82 (1H, m), 1.62 (2H, m), 1.39 (6H, t, J=7.0 Hz), 0.90
(6H, m).
Example 31
[1279] Compound 31: To a suspension of compound 25 (2.55 g, 5 mmol)
in CH.sub.2Cl.sub.2 (20 mL) at 0.degree. C. was added triethylamine
(2.8 mL, 20 mmol), followed by TMSCl (1.26 mL, 10 mmol). The
mixture was stirred at 0.degree. C. for 30 mins, and warmed to
25.degree. C. and stirred for additional 1 hr. Concentration gave a
yellow solid. The yellow solid was dissolved in acetonitrile (30
mL) and cooled to 0.degree. C. To this solution was added DMAP
(1.22 g, 10 mmol) and Bisfurancarbonate (1.48 g, 5 mmol). The
reaction mixture was stirred at 0.degree. C. for 2 hrs and for
additional 1 hr at 25.degree. C. Acetonitrile was removed under
reduced pressure. The mixture was diluted with EtOAc, and washed
with 5% citric acid (2.times.), water (2.times.), and brine
(1.times.), and dried over MgSO.sub.4. Concentration gave a yellow
solid. The yellow solid was dissolved in THF (40 mL), and acetic
acid (1.3 mL, 20 mmol) and tetrabutylammonium fluoride (8 mL, 1.0
N, 8 mmol) were added. The mixture was stirred for 20 mins, and THF
was removed under reduced pressure. Purification by flash column
chromatography (hexenes/EtOAc=1/1) gave compound 31 (1.5 g).
Example 32
[1280] Compound 32: To a solution of compound 31 (3.04 g, 5.1 mmol)
in THF (75 mL) was added Cs.sub.2CO.sub.3 (3.31 g, 10.2 mmol),
followed by a solution of triflate (3.24 g, 7.65 mmol) in THF (2
mL). The reaction mixture was stirred for 1.5 hrs, and THF was
removed under reduced pressure. The mixture was diluted with EtOAc,
and washed with water (3.times.) and brine (1.times.), and dried
over MgSO.sub.4. Purification by flash column chromatography
(hexanes/EtOAc=1/1 to 1/3) gave compound 32 (2.4 g): .sup.1H NMR
(CDCl.sub.3) .delta. 8.17 (2H, d, J=8.5 Hz), 7.70 (2H, J=9.2 Hz),
7.43 (2H, d, J=8.5 Hz), 7.37 (10H, m), 6.99 (2H, d, J=9.2 Hz), 5.66
(1H, d, J 5.2 Hz), 5.15 (4H, m), 5.05 (2H, m), 4.26 (2H, d, J=10.2
Hz), 3.9-3.8 (4H, m), 3.75 (2H, m), 3.2-2.8 (7H, m), 1.82 (1H, m),
1.62 (2H, m), 0.92 (6H, m).
Example 33
[1281] Compound 33: To a solution of compound 32 (45 mg) in acetic
acid (3 mL) was added zinc (200 mg). The mixture was stirred for 5
hrs. Celite was added, and the mixture was filtered and washed with
EtOAc. The solution was concentrated to dryness and diluted with
EtOAc. The organic phase was washed with 0.5 N NaOH solution,
water, and brine, and dried over MgSO.sub.4. Purification by flash
column chromatography (CH.sub.2Cl.sub.2/isoproanol=100/5) gave
compound 33 (25 mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.67 (2H, d,
J=8.8 Hz), 7.36 (10H, m), 6.98 (4H, m), 6.60 (2H, d, J=8.0 Hz),
5.67 (1H, d, J=4.9 Hz), 5.12 (4H, m), 5.05 (1H, m), 4.90 (1H, m),
4.24 (2H, d, J=10.4 Hz), 4.0-3.6 (6H, m), 3.12 (1H, m), 3.95 (4H,
m), 2.75 (2H, m), 1.80 (1H, m), 1.2 (2H, m), 0.9 (6H, m).
Example 34
[1282] Compound 34: To a solution of compound 32 (2.4 g) in ethanol
(140 mL) was added 10% Pd--C (1.0 g). The mixture was hydrogenated
for 14 hrs. Celite was added, and the mixture was stirred for 5
mins. The slurry was filtered through a pad of celite, and washed
with pyridine. Concentration under reduced pressure gave compound
34: .sup.1H NMR (DMSO-d.sub.6) .delta. 7.67 (2H, d, J=8.9 Hz), 7.14
(2H, d, J=8.9 Hz), 6.83 (2H, d, J=8.0 Hz), 6.41 (2H, d, J=8.0 Hz),
5.51 (1H, d, J=5.2 Hz), 5.0-4.8 (2H, m), 4.15 (2H, d, J=10.0 Hz),
3.9-3.2 (8H, m), 3.0 (2H, m), 2.8 (4H, m), 2.25 (1H, m), 1.4 (2H,
m), 0.8 (6H, m).
Example 35
[1283] Compound 35: Compound 34 (1.62 g, 2.47 mmol) and L-alanine
butyl ester hydrochloride (2.69 g, 14.8 mmol) were coevaporated
with pyridine (2.times.). The mixture was dissolved in pyridine (12
mL) and diisopropylethylamine (2.6 mL, 14.8 mmol) was added. To
above mixture was added a solution of Aldrithiol (3.29 g, 14.8
mmol) and triphenylphosphine (3.88 g, 14.8 g) in pyridine (12 mL).
The reaction mixture was stirred for 20 hrs, and solvents were
evaporated under reduced pressure. The mixture was diluted with
ethyl acetate, and was washed with 0.5 N NaOH solution (2.times.),
water (2.times.), and brine, and dried over MgSO.sub.4.
Concentration under reduced pressure gave a yellow oil, which was
purified by flash column chromatography
(CH.sub.2Cl.sub.2/MeOH=100/5 to 100/15) to afford compound 35 (1.17
g): .sup.1H NMR (CDCl.sub.3) .delta. 7.70 (2H, d, J=8.6 Hz), 7.05
(2H, d, J=8.6 Hz), 6.99 (2H, d, J=8.0 Hz), 6.61 (2H, d, J=8.0 Hz),
5.67 (1H, d, J=5.2 Hz), 5.05 (1H, m), 4.96 (1H, m), 4.28 (2H, m),
4.10 (6H, m), 4.0-3.6 (6H, m), 3.12 (2H, m), 2.92 (3H, m), 2.72
(2H, m), 1.82 (1H, m), 1.75-1.65 (2H, m), 1.60 (4H, m), 1.43 (6H,
m), 1.35 (4H, m), 0.91 (12H, m).
Example 36
[1284] Compound 37: Compound 36 (100 mg, 0.15 mmol) and L-alanine
butyl ester hydrochloride (109 mg, 0.60 mmol) were coevaporated
with pyridine (2.times.). The mixture was dissolved in pyridine (1
mL) and diisopropylethylamine (105 .mu.L, 0.6 mmol) was added. To
above mixture was added a solution of Aldrithiol (100 mg, 0.45
mmol) and triphenylphosphine (118 mg, 0.45 mmol) in pyridine (1
mL). The reaction mixture was stirred for 20 hrs, and solvents were
evaporated under reduced pressure. The mixture was diluted with
ethyl acetate, and was washed with water (2.times.), and brine, and
dried over MgSO.sub.4. Concentration under reduced pressure gave an
oil, which was purified by flash column chromatography
(CH.sub.2Cl.sub.21MeOH=100/5 to 100/15) to afford compound 37 (21
mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (2H, d, J=8.8 Hz), 7.15
(2H, d, J=8.2 Hz), 7.01 (2H, d, J=8.8 Hz), 6.87 (2H, d, J=8.2 Hz),
5.66 (1H, d, J=5.2 Hz), 5.03 (1H, m), 4.95 (1H, m), 4.2-4.0 (8H,
m), 3.98 (1H, m), 3.89 (3H, s), 3.88-3.65 (5H, m), 3.15 (1H, m),
2.98 (4H, m), 2.82 (2H, m), 1.83 (1H, m), 1.63 (4H, m), 1.42 (6H,
m), 1.35 (4H, m), 0.95 (12H, m).
Example 37
[1285] Compound 38: Compound 36 (100 mg, 0.15 mmol) and L-leucine
ethyl ester hydrochloride (117 mg, 0.60 mmol) were coevaporated
with pyridine (2.times.). The mixture was dissolved in pyridine (1
mL) and diisopropylethylamine (105 .mu.L, 0.6 mmol) was added. To
above mixture was added a solution of Aldrithiol (100 mg, 0.45
mmol) and triphenylphosphine (118 mg, 0.45 mmol) in pyridine (1
mL). The reaction mixture was stirred for 20 hrs, and solvents were
evaporated under reduced pressure. The mixture was diluted with
ethyl acetate, and was washed with water (2.times.), and brine, and
dried over MgSO.sub.4. Concentration under reduced pressure gave an
oil, which was purified by flash column chromatography
(CH.sub.2Cl.sub.2/MeOH=100/5 to 100/15) to afford compound 38 (12
mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (2H, d, J=8.5 Hz), 7.14
(2H, d, J=8.0 Hz), 7.00 (2H, d, J=8.5 Hz), 6.86 (2H, d, J=8.0 Hz),
5.66 (1H, d, J=5.2 Hz), 5.05 (1H, m), 4.95 (1H, m), 4.2-4.0 (8H,
m), 4.0-3.68 (6H, m), 3.88 (3H, s), 3.2-2.9 (5H, m), 2.80 (2H, m),
1.80 (1H, m), 1.65 (4H, m), 1.65-1.50 (4H, m), 1.24 (6H, m), 0.94
(18H, m).
Example 38
[1286] Compound 39: Compound 36 (100 mg, 0.15 mmol) and L-leucine
butyl ester hydrochloride (117 mg, 0.60 mmol) were coevaporated
with pyridine (2.times.). The mixture was dissolved in pyridine (1
mL) and diisopropylethylamine (105 .mu.L, 0.6 mmol) was added. To
above mixture was added a solution of Aldrithiol (100 mg, 0.45
mmol) and triphenylphosphine (118 mg, 0.45 mmol) in pyridine (1
mL). The reaction mixture was stirred for 20 hrs, and solvents were
evaporated under reduced pressure. The mixture was diluted with
ethyl acetate, and was washed with water (2.times.), and brine, and
dried over MgSO.sub.4. Concentration under reduced pressure gave an
oil, which was purified by flash column chromatography
(CH.sub.2Cl.sub.2/MeOH 100/5 to 100/15) to afford compound 39 (32
mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (2H, d, J=8.8 Hz), 7.15
(2H, d, J=8.0 Hz), 7.0 (2H, d, J=8.8 Hz), 6.89 (2H, d, J=8.0 Hz),
5.66 (1H, d, J=4.3 Hz), 5.07 (1H, m), 4.94 (1H, m), 4.2-4.0 (8H,
m), 3.89 (3H, s), 4.0-3.6 (6H, m), 3.2-2.9 (5H, m), 2.8 (2H, m),
1.81 (1H, m), 1.78-1.44 (10H, m), 1.35 (4H, m), 0.95 (24H, m).
Example 39
[1287] Compound 41: Compound 40 (82 mg, 0.1 mmol) and L-alanine
isopropyl ester hydrochloride (92 mg, 0.53 mmol) were coevaporated
with pyridine (2.times.). The mixture was dissolved in pyridine (1
mL) and diisopropylethylamine (136 .mu.L, 0.78 mmol) was added. To
above mixture was added a solution of Aldrithiol (72 mg, 0.33 mmol)
and triphenylphosphine (87 mg, 0.33 mmol) in pyridine (1 mL). The
reaction mixture was stirred at 75.degree. C. for 20 hrs, and
solvents were evaporated under reduced pressure. The mixture was
diluted with ethyl acetate, and was washed with water (2.times.),
and brine, and dried over MgSO.sub.4. Concentration under reduced
pressure gave an oil, which was purified by flash column
chromatography (CH.sub.2Cl.sub.2/MeOH=100/1 to 100/3) to afford
compound 41 (19 mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (2H, d,
J=8.9 Hz), 7.2-7.35 (5H, m), 7.15 (2H, m), 7.01 (2H, d, J=8.9 Hz),
6.87 (2H, m), 5.65 (1H, d, J=5.4 Hz), 5.05-4.93 (2H, m), 4.3 (2H,
m), 4.19 (1H, m), 3.98 (1H, m), 3.88 (3H, s), 3.80 (2H, m), 3.70
(3H, m), 3.18 (1H, m), 2.95 (4H, m), 2.78 (2H, m), 1.82 (1H, m),
1.62 (2H, m), 1.35 (3H, m), 1.25-1.17 (6H, m), 0.93 (3H, d, J=6.4
Hz), 0.88 (3H, d, J=6.4 Hz).
Example 40
[1288] Compound 42: Compound 40 (100 mg, 0.13 mmol) and L-glycine
butyl ester hydrochloride (88 mg, 0.53 mmol) were coevaporated with
pyridine (2.times.). The mixture was dissolved in pyridine (1 mL)
and diisopropylethylamine (136 .mu.L, 0.78 mmol) was added. To
above mixture was added a solution of Aldrithiol (72 mg, 0.33 mmol)
and triphenylphosphine (87 mg, 0.33 mmol) in pyridine (1 mL). The
reaction mixture was stirred at 75.degree. C. for 20 hrs, and
solvents were evaporated under reduced pressure. The mixture was
diluted with ethyl acetate, and was washed with water (2.times.),
and brine, and dried over MgSO.sub.4. Concentration under reduced
pressure gave an oil, which was purified by flash column
chromatography (CH.sub.2Cl.sub.2/MeOH=100/1 to 100/3) to afford
compound 42 (18 mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (2H, d,
J=9.2 Hz), 7.35-7.24 (5H, m), 7.14 (2H, m), 7.00 (2H, d, J=8.8 Hz),
6.87 (2H, m), 5.65 (1H, d, J=5.2 Hz), 5.04 (1H, m), 4.92 (1H, m),
4.36 (2H, m), 4.08 (2H, m), 3.95 (3H, m), 3.88 (3H, s), 3.80 (2H,
m), 3.76 (3H, m), 3.54 (1H, m), 3.15 (1H, m), 2.97 (4H, m), 2.80
(2H, m), 1.82 (1H, m), 1.62 (4H, m), 1.35 (2H, m), 0.9 (9H, m).
Example Section H
Example 1
[1289] Sulfonamide 1: To a suspension of epoxide (20 g, 54.13 mmol)
in 2-propanol (250 mL) was added isobutylamine (54 mL, 541 mmol)
and the solution was refluxed for 30 min. The solution was
evaporated under reduced pressure and the crude solid was dissolved
in CH.sub.2Cl.sub.2 (250 mL) and cooled to 0.degree. C.
Triethylamine (15.1 mL, 108.26 mmol) was added followed by the
addition of 4-nitrobenzenesulfonyl chloride (12 g, 54.13 mmol) and
the solution was stirred for 40 min at 0.degree. C., warmed to room
temperature for 2 h, and evaporated under reduced pressure. The
residue was partitioned between EtOAc and saturated NaHCO.sub.3.
The organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was recrystallized from EtOAc/hexane to give the
sulfonamide (30.59 g, 90%) as an off-white solid.
Example 2
[1290] Phenol 2: A solution of sulfonamide 1 (15.58 g, 24.82 mmol)
in EtOH (450 mL) and CH.sub.2Cl.sub.2 (60 mL) was treated with 10%
Pd/C (6 g). The suspension was stirred under H.sub.2 atmosphere
(balloon) at room temperature for 24 h. The reaction mixture was
filtered through a plug of celite and concentrated under reduced
pressure. The crude product was purified by column chromatography
on silica gel (6% MeOH/CH.sub.2Cl.sub.2) to give the phenol (11.34
g, 90%) as a white solid.
Example 3
[1291] Dibenzylphosphonate 3: To a solution of phenol 2 (18.25 g,
35.95 mmol) in CH.sub.3CN (200 mL) was added Cs.sub.2CO.sub.3
(23.43 g, 71.90 mmol) and triflate (19.83 g, 46.74 mmol). The
reaction mixture was stirred at room temperature for 1 h and the
solvent was evaporated under reduced pressure. The residue was
partitioned between EtOAc and saturated NaCl. The organic phase was
dried with Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was purified by column chromatography
on silica gel (2/1-EtOAc/hexane) to give the dibenzylphosphonate
(16.87 g, 60%) as a white solid.
Example 4
[1292] Amine 4: A solution of dibenzylphosphonate (16.87 g, 21.56
mmol) in CH.sub.2Cl.sub.2 (60 mL) at 0.degree. C. was treated with
trifluoroacetic acid (30 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. Volatiles were evaporated under reduced pressure
and the residue was partitioned between EtOAc and 0.5 N NaOH. The
organic phase was washed with 0.5 N NaOH (2.times.), water
(2.times.), saturated NaCl, dried with Na.sub.2SO.sub.4, filtered,
and evaporated under reduced pressure to give the amine (12.94 g,
88%) as a white solid.
Example 5
[1293] Carbonate 5: To a solution of
(S)-(+)-3-hydroxytetrahydrofuran (5.00 g, 56.75 mmol) in
CH.sub.2Cl.sub.2 (80 mL) was added triethylamine (11.86 mL, 85.12
mmol) and bis(4-nitrophenyl)carbonate (25.90 g, 85.12 mmol). The
reaction mixture was stirred at room temperature for 24 h and
partitioned between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3. The
CH.sub.2Cl.sub.2 layer was dried with Na.sub.2SO.sub.4, filtered,
and concentrated. The crude product was purified by column
chromatography on silica gel (2/1-EtOAc/hexane) to give the
carbonate (8.62 g, 60%) as a pale yellow oil which solidified upon
refrigerating.
Example 6
[1294] Carbamate 6: Two methods have been used.
[1295] Method 1: To a solution of 4 (6.8 g, 9.97 mmol) and 5 (2.65
g, 10.47 mmol) in CH.sub.3CN (70 mL) at 0.degree. C. was added
4-(dimethylamino)pyridine (2.44 g, 19.95 mmol). The reaction
mixture was stirred at 0.degree. C. for 3 h and concentrated. The
residue was dissolved in EtOAc and washed with 0.5 N NaOH,
saturated NaHCO.sub.3, H.sub.2O, dried with Na.sub.2SO.sub.4,
filtered, and concentrated. The crude product was purified by
column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the carbamate (3.97 g, 50%) as
a pale yellow solid.
[1296] Method 2: To a solution of 4 (6.0 g, 8.80 mmol) and 5 (2.34
g, 9.24 mmol) in CH.sub.3CN (60 mL) at 0.degree. C. was added
4-(dimethylamino)pyridine (0.22 g, 1.76 mmol) and
N,N-diisopropylethylamine (3.07 mL, 17.60 mmol). The reaction
mixture was stirred at 0.degree. C. for 1 h and warmed to room
temperature overnight. The solvent was evaporated under reduced
pressure. The crude product was dissolved in EtOAc and washed with
0.5 N NaOH, saturated NaHCO.sub.3, H.sub.2O, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the carbamate (3.85 g, 55%) as
a pale yellow solid.
Example 7
[1297] Phosphonic Acid 7: To a solution of 6 (7.52 g, 9.45 mmol) in
MeOH (350 mL) was added 10% Pd/C (3 g). The suspension was stirred
under H.sub.2 atmosphere (balloon) at room temperature for 48 h.
The reaction mixture was filtered through a plug of celite. The
filtrate was concentrated and dried under vacuum to give the
phosphonic acid (5.24 g, 90%) as a white solid.
Example 8
[1298] Cbz Amide 8: To a solution of 7 (5.23 g, 8.50 mmol) in
CH.sub.3CN (50 mL) was added N,O-bis(trimethylsilyl)acetamide
(16.54 mL, 68 mmol) and then heated to 70.degree. C. for 3 h. The
reaction mixture was cooled to room temperature and concentrated.
The residue was co-evaporated with toluene and dried under vacuum
to afford the silylated intermediate which was used directly
without any further purification. To a solution of the silylated
intermediate in CH.sub.2Cl.sub.2 (40 mL) at 0.degree. C. was added
pyridine (1.72 mL, 21.25 mmol) and benzyl chloroformate (1.33 mL,
9.35 mmol). The reaction mixture was stirred at 0.degree. C. for 1
h and warmed to room temperature overnight. A solution of MeOH (50
mL) and 1% aqueous HCl (150 mL) was added at 0.degree. C. and
stirred for 30 min. CH.sub.2Cl.sub.2 was added and two layers were
separated. The organic layer was dried with Na.sub.2SO.sub.4,
filtered, concentrated, co-evaporated with toluene, and dried under
vacuum to give the Cbz amide (4.46 g, 70%) as an off-white
solid.
Example 9
[1299] Diphenylphosphonate 9: A solution of 8 (4.454 g, 5.94 mmol)
and phenol (5.591 g, 59.4 mmol) in pyridine (40 mL) was heated to
70.degree. C. and 1,3-dicyclohexylcarbodiimide (4.903 g, 23.76
mmol) was added. The reaction mixture was stirred at 70.degree. C.
for 4 h and cooled to room temperature. EtOAc was added and the
side product 1,3-dicyclohexyl urea was filtered off. The filtrate
was concentrated and dissolved in CH.sub.3CN (20 mL) at 0.degree.
C. The mixture was treated with DOWEX 50 W.times.8-400 ion-exchange
resin and stirred for 30 min at 0.degree. C. The resin was filtered
off and the filtrate was concentrated. The crude product was
purified by column chromatography on silica gel (4%
2-propanol/CH.sub.2Cl.sub.2) to give the diphenylphosphonate (2.947
g, 55%) as a white solid.
Example 10
[1300] Monophosphonic Acid 10: To a solution of 9 (2.945 g, 3.27
mmol) in CH.sub.3CN (25 mL) at 0.degree. C. was added 1N NaOH (8.2
mL, 8.2 mmol). The reaction mixture was stirred at 0.degree. C. for
1 h. DOWEX 50 W.times.8-400 ion-exchange resin was added and the
reaction mixture was stirred for 30 min at 0.degree. C. The resin
was filtered off and the filtrate was concentrated and
co-evaporated with toluene. The crude product was triturated with
EtOAc/hexane (1/2) to give the monophosphonic acid (2.427 g, 90%)
as a white solid.
Example 11
[1301] Cbz Protected Monophosphoamidate 11: A solution of 10 (2.421
g, 2.93 mmol) and L-alanine isopropyl ester hydrochloride (1.969 g,
11.73 mmol) in pyridine (20 mL) was heated to 70.degree. C. and
1,3-dicyclohexylcarbodiimide (3.629 g, 17.58 mmol) was added. The
reaction mixture was stirred at 70.degree. C. for 2 h and cooled to
room temperature. The solvent was evaporated under reduced pressure
and the residue was partitioned between EtOAc and 0.2 N HCl. The
EtOAc layer was washed with 0.2 N HCl, H.sub.2O, saturated
NaHCO.sub.3, dried with Na.sub.2SO.sub.4, filtered, and
concentrated. The crude product was purified by column
chromatography on silica gel (4% 2-propanol/CH.sub.2Cl.sub.2) to
give the monoamidate (1.569 g, 57%) as a white solid.
Example 12
[1302] Monophosphoamidate 12: To a solution of 11 (1.569 g, 1.67
mmol) in EtOAc (80 mL) was added 10% Pd/C (0.47 g). The suspension
was stirred under H.sub.2 atmosphere (balloon) at room temperature
overnight. The reaction mixture was filtered through a plug of
celite. The filtrate was concentrated and the crude product was
purified by column chromatography on silica gel (CH.sub.2Cl.sub.2
to 1-8% 2-propanol/CH.sub.2Cl.sub.2) to give the monophosphoamidate
12a (1.12 g, 83%, GS 108577, 1:1 diastereomeric mixture A/B) as a
white solid: .sup.1H NMR (CDCl.sub.3) .delta. 7.45 (dd, 2H),
7.41-7.17 (m, 7H), 6.88 (dd, 2H), 6.67 (d, J=8.4 Hz, 2H), 5.16
(broad s, 1H), 4.95 (m, 1H), 4.37-4.22 (m, 5H), 3.82-3.67 (m, 7H),
2.99-2.70 (m, 6H), 2.11-1.69 (m, 3H), 1.38 (m, 3H), 1.19 (m, 6H),
0.92 (d, J=6.3 Hz, 3H), 0.86 (d, J=6.3 Hz, 3H); .sup.31P NMR
(CDCl.sub.3) .delta. 20.5, 19.6. 12b (29 mg, 2%, GS108578,
diastereomer A) as a white solid: .sup.1H NMR (CDCl.sub.3) .delta.
7.43 (d, J=7.8 Hz, 2H), 7.35-7.17 (m, 7H), 6.89 (d, J=8.4 Hz, 2H),
6.67 (d, J=8.4 Hz, 2H), 5.16 (broad s, 1H), 4.96 (m, 1H), 4.38-4.32
(m, 4H), 4.20 (m, 1H), 3.82-3.69 (m, 7H), 2.99-2.61 (m, 6H), 2.10
(m, 1H), 1.98 (m, 1H), 1.80 (m, 1H), 1.38 (d, J=7.2 Hz, 3H), 1.20
(d, J=6.3 Hz, 6H), 0.92 (d, J=6.3 Hz, 3H), 0.86 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CDCl.sub.3) .delta. 20.5. 12c (22 mg, 1.6%, GS
108579, diastereomer B) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 7.45 (d, J=8.1 Hz, 2H), 7.36-7.20 (m, 7H), 6.87 (d, J=8.7
Hz, 2H), 6.67 (d, J=8.4 Hz, 2H), 5.15 (broad s, 1H), 4.95 (m, 1H),
4.34-4.22 (m, 5H), 3.83-3.67 (m, 7H), 2.99-2.64 (m, 6H), 2.11-1.68
(m, 3H), 1.33 (d, J=6.9 Hz, 3H), 1.20 (d, J=6.0 Hz, 6H), 0.92 (d,
J=6.3 Hz, 3H), 0.86 (d, J=6.3 Hz, 3H); .sup.31P NMR (CDCl.sub.3)
.delta. 19.6.
Example 13
[1303] Sulfonamide 13: To a suspension of epoxide (1.67 g, 4.52
mmol) in 2-propanol (25 mL) was added isobutylamine (4.5 mL, 45.2
mmol) and the solution was refluxed for 30 min. The solution was
evaporated under reduced pressure and the crude solid was dissolved
in CH.sub.2Cl.sub.2 (20 mL) and cooled to 0.degree. C.
Triethylamine (1.26 mL, 9.04 mmol) was added followed by the
treatment of 3-nitrobenzenesulfonyl chloride (1.00 g, 4.52 mmol).
The solution was stirred for 40 min at 0.degree. C., warmed to room
temperature for 2 h, and evaporated under reduced pressure. The
residue was partitioned between EtOAc and saturated NaHCO.sub.3.
The organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (1/1-EtOAc/hexane) to give the sulfonamide (1.99 g, 70%) as a
white solid.
Example 14
[1304] Phenol 14: Sulfonamide 13 (1.50 g, 2.39 mmol) was suspended
in HOAc (40 mL) and concentrated HCl (20 mL) and heated to reflux
for 3 h. The reaction mixture was cooled to room temperature and
concentrated under reduced pressure. The crude product was
partitioned between 10% MeOH/CH.sub.2Cl.sub.2 and saturated
NaHCO.sub.3. The organic layers were washed with NaHCO.sub.3,
H.sub.2O, dried with Na.sub.2SO.sub.4, filtered, and concentrated
to give a yellow solid. The crude product was dissolved in
CHCl.sub.3 (20 mL) and treated with triethylamine (0.9 mL, 6.45
mmol) followed by the addition of Boc.sub.2O (0.61 g, 2.79 mmol).
The reaction mixture was stirred at room temperature for 6 h. The
product was partitioned between CHCl.sub.3 and H.sub.2O. The
CHCl.sub.3 layer was washed with NaHCO.sub.3, H.sub.2O, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (1-5%
MeOH/CH.sub.2Cl.sub.2) to give the phenol (0.52 g, 45%) as a pale
yellow solid.
Example 15
[1305] Dibenzylphosphonate 15: To a solution of phenol 14 (0.51 g,
0.95 mmol) in CH.sub.3CN (8 mL) was added Cs.sub.2CO.sub.3 (0.77 g,
2.37 mmol) and triflate (0.8 g, 1.90 mmol). The reaction mixture
was stirred at room temperature for 1.5 h and the solvent was
evaporated under reduced pressure. The residue was partitioned
between EtOAc and saturated NaCl. The organic phase was dried
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (3% MeOH/CH.sub.2Cl.sub.2) to give the dibenzylphosphonate
(0.62 g, 80%) as a white solid.
Example 16
[1306] Amine 16: A solution of dibenzylphosphonate 15 (0.61 g, 0.75
mmol) in CH.sub.2Cl.sub.2 (8 mL) at 0.degree. C. was treated with
trifluoroacetic acid (2 mL). The solution was stirred for 30 min at
0.degree. C. and then warmed to room temperature for an additional
30 min. Volatiles were evaporated under reduced pressure and the
residue was partitioned between EtOAc and 0.5 N NaOH. The organic
phase was washed with 0.5 N NaOH (2.times.), water (2.times.),
saturated NaCl, dried Na.sub.2SO.sub.4), filtered, and evaporated
under reduced pressure to give the amine (0.48 g, 90%) which was
used directly without any further purification.
Example 17
[1307] Carbamate 17: To a solution of amine 16 (0.48 g, 0.67 mmol)
in CH.sub.3CN (8 mL) at 0.degree. C. was treated with
(3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl 4-nitrophenyl carbonate
(0.2 g, 0.67 mmol, prepared according to Ghosh et al. J. Med. Chem.
1996, 39, 3278.) and 4-(dimethylamino)pyridine (0.17 g, 1.34 mmol).
After stirring for 2 h at 0.degree. C., the reaction solvent was
evaporated under reduced pressure and the residue was partitioned
between EtOAc and 0.5 N NaOH. The organic phase was washed with
0.5N NaOH (2.times.), 5% citric acid (2.times.), saturated
NaHCO.sub.3, dried with Na.sub.2SO.sub.4, filtered, and evaporated
under reduced pressure. The crude product was purified by column
chromatography on silica gel (3% 2-propanol/CH.sub.2Cl.sub.2) to
give the carbamate (0.234 g, 40%) as a white solid.
Example 18
[1308] Analine 18: To a solution of carbamate 17 (78 mg, 0.09 mmol)
in 2 mL HOAc was added zinc powder. The reaction mixture was
stirred at room temperature for 1.5 h and filtered through a small
plug of celite. The filtrate was concentrated and co-evaporated
with toluene. The crude product was purified by column
chromatography on silica gel (5% 2-propanol/CH.sub.2Cl.sub.2) to
give the analine (50 mg, 66%) as a white solid.
Example 19
[1309] Phosphonic Acid 19: To a solution of analine (28 mg, 0.033
mmol) in MeOH (1 mL) and HOAc (0.5 mL) was added 10% Pd/C (14 mg).
The suspension was stirred under H.sub.2 atmosphere (balloon) at
room temperature for 6 h. The reaction mixture was filtered through
a small plug of celite. The filtrate was concentrated,
co-evaporated with toluene, and dried under vacuum to give the
phosphonic acid (15 mg, 68%, GS 17424) as a white solid: .sup.1H
NMR (DMSO-d.sub.6) .delta. 7.16-6.82 (m, 8H), 5.50 (d, 1H), 4.84
(m, 1H), 3.86-3.37 (m, 9H), 2.95-2.40 (m, 6H), 1.98 (m, 1H),
1.42-1.23 (m, 2H), 0.84 (d, J=6.3 Hz, 3H), 0.79 (d, J=6.3 Hz, 3H).
MS (ESI) 657 (M-H).
Example 20
[1310] Phenol 21: A suspension of aminohydrobromide salt 20 (22.75
g, 44 mmol) in CH.sub.2Cl.sub.2 (200 mL) at 0.degree. C. was
treated with triethylamine (24.6 mL, 176 mmol) followed by slow
addition of chlorotrimethylsilane (11.1 mL, 88 mmol). The reaction
mixture was stirred at 0.degree. C. for 30 min and warmed to room
temperature for 1 h. The solvent was removed under reduced pressure
to give a yellow solid. The crude product was dissolved in
CH.sub.2Cl.sub.2 (300 mL) and treated with triethylamine (18.4 mL,
132 mmol) and Boc.sub.2O (12 g, 55 mmol). The reaction mixture was
stirred at room temperature overnight. The product was partitioned
between CH.sub.2Cl.sub.2 and H.sub.2O. The CH.sub.2Cl.sub.2 layer
was washed with NaHCO.sub.3, H.sub.2O, dried with Na.sub.2SO.sub.4,
filtered, and concentrated. The crude product was dissolved in THF
(200 mL) and treated with 1.0 M TBAF (102 mL, 102 mmol) and HOAc
(13 mL). The reaction mixture was stirred at room temperature for 1
h and concentrated under reduced pressure. The residue was
partitioned between CH.sub.2Cl.sub.2 and H.sub.2O, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (1-3%
2-propanol/CH.sub.2Cl.sub.2) to give the phenol (13.75 g, 58%) as a
white solid.
Example 21
[1311] Dibenzylphosphonate 22: To a solution of phenol 21 (13.70 g,
25.48 mmol) in THF (200 mL) was added Cs.sub.2CO.sub.3 (16.61 g,
56.96 mmol) and triflate (16.22 g, 38.22 mmol). The reaction
mixture was stirred at room temperature for 1 h and the solvent was
evaporated under reduced pressure. The residue was partitioned
between EtOAc and saturated NaCl. The organic phase was dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (3% MeOH/CH.sub.2Cl.sub.2) to give the dibenzylphosphonate
(17.59 g, 85%) as a white solid.
Example 22
[1312] Amine 23: A solution of dibenzylphosphonate 22 (17.58 g,
21.65 mmol) in CH.sub.2Cl.sub.2 (60 mL) at 0.degree. C. was treated
with trifluoroacetic acid (30 mL). The solution was stirred for 30
min at 0.degree. C. and then warmed to room temperature for an
additional 1.5 h. Volatiles were evaporated under reduced pressure
and the residue was partitioned between EtOAc and 0.5 N NaOH. The
organic phase was washed with 0.5 N NaOH (2.times.), water
(2.times.), saturated NaCl, dried with Na.sub.2SO.sub.4, filtered,
and evaporated under reduced pressure to give the amine (14.64 g,
95%) which was used directly without any further purification.
Example 23
[1313] Carbamate 24: To a solution of amine 23 (14.64 g, 20.57
mmol) in CH.sub.3CN (200 mL) at 0.degree. C. was treated with
(3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl 4-nitrophenyl carbonate
(6.07 g, 20.57 mmol, prepared according to Ghosh et al., J. Med.
Chem. 1996, 39, 3278.) and 4-(dimethylamino)pyridine (5.03 g, 41.14
mmol). After stirring for 2 h at 0.degree. C., the reaction solvent
was evaporated under reduced pressure and the residue was
partitioned between EtOAc and 0.5 N NaOH. The organic phase was
washed with 0.5N NaOH (2.times.), 5% citric acid (2.times.),
saturated NaHCO.sub.3, dried with Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was purified
by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the carbamate (10 g, 56%) as a
white solid.
Example 24
[1314] Phosphonic Acid 25: To a solution of carbamate 24 (8 g, 9.22
mmol) in EtOH (500 ml, was added 10% Pd/C (4 g). The suspension was
stirred under H.sub.2 atmosphere (balloon) at room temperature for
30 h. The reaction mixture was filtered through a plug of celite.
The celite paste was suspended in pyridine and stirred for 30 min
and filtered. This process was repeated twice. The combined
solution was concentrated under reduced pressure to give the
phosphonic acid (5.46 g, 90%) as an off-white solid.
Example 25
[1315] Cbz Amide 26: To a solution of 25 (5.26 g, 7.99 mmol) in
CH.sub.3CN (50 mL) was added N,O-bis(trimethylsilyl)acetamide (15.6
mL, 63.92 mmol) and then heated to 70.degree. C. for 3 h. The
reaction mixture was cooled to room temperature and concentrated.
The residue was co-evaporated with toluene and dried under vacuum
to afford the silylated intermediate which was used directly
without any further purification. To a solution of the silylated
intermediate in CH.sub.2Cl.sub.2 (40 mL) at 0.degree. C. was added
pyridine (1.49 mL, 18.38 mmol) and benzyl chloro-formate (1.25 mL,
8.79 mmol). The reaction mixture was stirred at 0.degree. C. for 1
h and warmed to room temperature overnight. A solution of MeOH (50
mL) and 1% aqueous HCl (150 mL) was added at 0.degree. C. and
stirred for 30 min. CH.sub.2Cl.sub.2 was added and two layers were
separated. The organic layer was dried with Na.sub.2SO.sub.4,
filtered, concentrated, co-evaporated with toluene, and dried under
vacuum to give the Cbz amide (4.43 g, 70%) as an off-white
solid.
Example 26
[1316] Diphenylphosphonate 27: A solution of 26 (4.43 g, 5.59 mmol)
and phenol (4.21 g, 44.72 mmol) in pyridine (40 mL) was heated to
70.degree. C. and 1,3-dicyclohexylcarbodiimide (4.62 g, 22.36 mmol)
was added. The reaction mixture was stirred at 70.degree. C. for 36
h and cooled to room temperature. EtOAc was added and the side
product 1,3-dicyclohexyl urea was filtered off. The filtrate was
concentrated and dissolved in CH.sub.3CN (20 mL) at 0.degree. C.
The mixture was treated with DOWEX 50 W.times.8-400 ion-exchange
resin and stirred for 30 min at 0.degree. C. The resin was filtered
off and the filtrate was concentrated. The crude product was
purified by column chromatography on silica gel (2/1-EtOAc/hexane
to EtOAc) to give the diphenylphosphonate (2.11 g, 40%) as a pale
yellow solid.
Example 27
[1317] Monophosphonic Acid 28: To a solution of 27 (2.11 g, 2.24
mmol) in CH.sub.3CN (15 mL) at 0.degree. C. was added 1N NaOH (5.59
mL, 5.59 mmol). The reaction mixture was stirred at 0.degree. C.
for 1 h. DOWEX 50 W.times.8400 ion-exchange resin was added and the
reaction mixture was stirred for 30 min at 0.degree. C. The resin
was filtered off and the filtrate was concentrated and
co-evaporated with toluene. The crude product was triturated with
EtOAc/hexane (1/2) to give the monophosphonic acid (1.75 g, 90%) as
a white solid.
Example 28
[1318] Cbz Protected Monophosphoamidate 29: A solution of 28 (1.54
g, 1.77 mmol) and L-alanine isopropyl ester hydrochloride (2.38 g,
14.16 mmol) in pyridine (15 mL) was heated to 70.degree. C. and
1,3-dicyclohexylcarbodiimide (2.20 g, 10.62 mmol) was added. The
reaction mixture was stirred at 70.degree. C. overnight and cooled
to room temperature. The solvent was removed under reduced pressure
and the residue was partitioned between EtOAc and 0.2 N HCl. The
EtOAc layer was washed with 0.2 N HCl, H.sub.2O, saturated
NaHCO.sub.3, dried with Na.sub.2SO.sub.4, filtered, and
concentrated. The crude product was purified by column
chromatography on silica gel (3% MeOH/CH.sub.2Cl.sub.2) to give the
monophosphoamidate (0.70 g, 40%) as an off-white solid.
Example 29
[1319] Monophosphoamidate 30a-b: To a solution of 29 (0.70 g, 0.71
mmol) in EtOH (10 mL) was added 10% Pd/C (0.3 g). The suspension
was stirred under H.sub.2 atmosphere (balloon) at room temperature
for 6 h. The reaction mixture was filtered through a small plug of
celite. The filtrate was concentrated and the crude products were
purified by column chromatography on silica gel (7-10%
MeOH/CH.sub.2Cl.sub.2) to give the monoamidates 30a (0.106 g, 18%,
GS 77369, 1/1 diastereomeric mixture) as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 7.71 (d, J=8.7 Hz, 2H), 7.73-7.16 (m, 5H),
7.10-6.98 9m, 4H), 6.61 (d, J=8.1 Hz, 2H), 5.67 (d, J=4.8 Hz, 1H),
5.31-4.91 (m, 2H), 4.44 (m, 2H), 4.20 (m, 1H), 4.00-3.61 (m, 6H),
3.18-2.74 (m, 7H), 1.86-1.64 (m, 3H), 1.38 (m, 3H), 1.20 (m, 6H),
0.93 (d, J=6.6 Hz, 3H), 0.87 (d, J=6.6 Hz, 3H); .sup.31P NMR
(CDCl.sub.3) .quadrature. 19.1, 18; MS(ESI) 869 (M+Na). 30b (0.200
g, 33%, GS 77425, 1/1 diastereomeric mixture) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.73 (dd, J=8.7 Hz, J=1.5 Hz, 2H),
7.36-7.16 (m, 5H), 7.09-7.00 (m, 4H), 6.53 (d, J=8.7 Hz, 2H), 5.66
(d, J=5.4 Hz, 1H), 5.06-4.91 (m, 2H), 4.40 (m, 2H), 4.20 (m, 1H),
4.00-3.60 (m, 6H), 3.14 (m, 3H), 3.00-2.65 (m, 6H), 1.86-1.60 (m,
3H), 1.35 (m, 3H), 1.20 (m, 9H), 0.92 (d, J=6.6 Hz, 3H), 0.87 (d,
J=6.6 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .quadrature. 19.0, 17.9.
MS (ESI) 897 (M+Na).
Example 30
[1320] Synthesis of Bisamidates 32: A solution of phosphonic acid
31 (100 mg, 0.15 mmol) and L-valine ethyl ester hydrochloride (108
mg, 0.60 mmol) was dissolved in pyridine (5 mL) and the solvent was
distilled under reduced pressure at 40-60.degree. C. The residue
was treated with a solution of Ph.sub.3P (117 mg, 0.45 mmol) and
2,2'-dipyridyl disulfide (98 mg, 0.45 mmol) in pyridine (1 mL)
followed by addition of N,N-diisopropylethylamine (0.1 mL, 0.60
mmol). The reaction mixture was stirred at room temperature for two
days. The solvent was evaporated under reduced pressure and the
residue was purified by column chromatography on silica gel to give
the bisamidate (73 mg, 53%, GS 17389) as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H), 7.15 (d, J=8.1 Hz,
2H), 7.00 (d, J=8.7 Hz, 2H), 6.86 (d, J=8.1 Hz, 2H), 5.66 (d, J=4.8
Hz, 1H), 5.05 (m, 1H), 4.95 (d, J=8.7 Hz, 1H), 4.23-4.00 (m,4H,),
3.97-3.68 (m, 11H), 3.39-2.77 (m, 9H), 2.16 (m, 2H), 1.82-1.60 (m,
3H), 1.31-1.18 (m, 6H), 1.01-0.87 (m, 18H); .sup.31P NMR
(CDCl.sub.3) .delta. 21.3; MS (ESI) 950 (M+Na).
Example 31
[1321] Triflate 34: To a solution of phenol 33 (2.00 g, 3.46 mmol)
in THF (15 mL) and CH.sub.2Cl.sub.2 (5 mL) was added
N-phenyltrifluoromethanesulfonimide (1.40 g, 3.92 mmol) and cesium
carbonate (1.40 g, 3.92 mmol). The reaction mixture was stirred at
room temperature overnight and concentrated. The crude product was
partitioned between CH.sub.2Cl.sub.2 and saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (3%
MeOH/CH.sub.2Cl.sub.2) to give the triflate (2.09 g, 85%) as a
white solid.
Example 32
[1322] Aldehyde 35: To a suspension of triflate 34 (1.45 g, 2.05
mmol), palladium (II) acetate (46 mg, 0.20 mmol) and
1,3-bis(diphenylphosphino)propane (84 mg, 0.2 mmol) in DMF (8 mL)
under CO atmosphere (balloon) was slowly added triethylamine (1.65
mL, 11.87 mmol) and triethylsilane (1.90 mL, 11.87 mmol). The
reaction mixture was heated to 70.degree. C. under CO atmosphere
(balloon) and stirred overnight. The solvent was concentrated under
reduced pressure and partitioned between CH.sub.2Cl.sub.2 and
H.sub.2O. The organic phase was dried with Na.sub.2SO.sub.4,
filtered, and concentrated. The crude product was purified by
column chromatography on silica gel (4%
2-propanol/CH.sub.2Cl.sub.2) to give the aldehyde (0.80 g, 66%) as
a white solid.
Example 33
[1323] Substituted Benzyl Alcohol 36: To a solution of aldehyde 35
(0.80 g, 1.35 mmol) in THF (9 mL) and H.sub.2O (1 mL) at
-10.degree. C. was added NaBH.sub.4 (0.13 g, 3.39 mmol). The
reaction mixture was stirred for 1 h at -10.degree. C. and the
solvent was evaporated under reduced pressure. The residue was
dissolved in CH.sub.2Cl.sub.2 and washed with NaHSO.sub.4,
H.sub.2O, dried with Na.sub.2SO.sub.4, filtered, and concentrated.
The crude product was purified by column chromatography on silica
gel (6% 2-propanol/CH.sub.2Cl.sub.2) to give the alcohol (0.56 g,
70%) as a white solid.
Example 34
[1324] Substituted Benzyl Bromide 37: To a solution of alcohol 36
(77 mg, 0.13 mmol) in THF (1 mL) and CH.sub.2Cl.sub.2 (1 mL) at
0.degree. C. was added triethylamine (0.027 mL, 0.20 mmol) and
methanesulfonyl chloride (0.011 mL, 0.14 mmol). The reaction
mixture was stirred at 0.degree. C. for 30 min and warmed to room
temperature for 3 h. Lithium bromide (60 mg, 0.69 mmol) was added
and stirred for 45 min. The reaction mixture was concentrated and
the residue was partitioned between CH.sub.2Cl.sub.2 and H.sub.2O,
dried with Na.sub.2SO.sub.4, filtered, and concentrated. The crude
product was purified by column chromatography on silica gel (2%
MeOH/CH.sub.2Cl.sub.2) to give the bromide (60 mg, 70%).
Example 35
[1325] Diethylphosphonate 38: A solution of bromide 37 (49 mg,
0.075 mmol) and triethylphosphite (0.13 mL, 0.75 mmol) in toluene
(1.5 mL) was heated to 120.degree. C. and stirred overnight. The
reaction mixture was cooled to room temperature and concentrated
under reduced pressure. The crude product was purified by column
chromatography on silica gel (6% MeOH/CH.sub.2Cl.sub.2) to give the
diethylphosphonate (35 mg, 66%, GS 191338) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H), 7.27-7.16
(m, 4H), 7.00 (d, J=8.7 Hz, 2H), 5.66 (d, J=5.1 Hz, 1H), 5.00 (m,
2H), 4.04-3.73 (m, 13H), 3.13-2.80 (m, 9H), 1.82-1.64 (m, 3H), 1.25
(t, J=6.9 Hz, 6H), 0.92 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CDCl.sub.3) .quadrature. 26.4; MS (ESI) 735
(M+Na).
Example 36
[1326] N-tert-Butoxycarbonyl-O-benzyl-L-serine 39: To a solution of
Boc-L-serine (15 g, 73.09 mmol) in DMF (300 mL) at 0.degree. C. was
added NaH (6.43 g, 160.80 mmol, 60% in mineral oil) and stirred for
1.5 h at 0.degree. C. After the addition of benzyl bromide (13.75
g, 80.40 mmol), the reaction mixture was warmed to room temperature
and stirred overnight. The solvent was evaporated under reduced
pressure and the residue was dissolved in H.sub.2O. The crude
product was partitioned between H.sub.2O and Et.sub.2O. The aqueous
phase was acidified to pH<4 with 3 N HCl and extracted with
EtOAc three times. The combined EtOAc solution was washed with
H.sub.2O, dried with Na.sub.2SO.sub.4, filtered, and concentrated
to give the N-tert-butoxycarbonyl-O-benzyl-L-serine (17.27 g,
80%).
Example 37
[1327] Diazo Ketone 40: To a solution of
N-tert-Butoxycarbonyl-O-benzyl-L-serine 39 (10 g, 33.86 mmol) in
dry THF (120 mL) at -15.degree. C. was added 4-methylmorpholine
(3.8 mL, 34.54 mmol) followed by the slow addition of
isobutylchloroformate (4.40 mL, 33.86 mmol). The reaction mixture
was stirred for 30 min and diazomethane (.about.50 mmol, generated
from 15 g Diazald according to Aldrichimica Acta 1983, 16, 3) in
ether (.about.150 mL) was poured into the mixed anhydride solution.
The reaction was stirred for 15 min and was then placed in an ice
bath at 0.degree. C. and stirred for 1 h. The reaction was allowed
to warm to room temperature and stirred overnight. The solvent was
evaporated under reduced pressure and the residue was dissolved in
EtOAc, washed with water, saturated NaHCO.sub.3, saturated NaCl,
dried with Na.sub.2SO.sub.4, filtered and evaporated. The crude
product was purified by column chromatography (EtOAc/hexane) to
afford the diazo ketone (7.50 g, 69%) as a yellow oil.
Example 38
[1328] Chloroketone 41: To a suspension of diazoketone 40 (7.50 g,
23.48 mmol) in ether (160 mL) at 0.degree. C. was added 4N HCl in
dioxane (5.87 mL, 23.48 mmol). The reaction mixture was stirred at
0.degree. C. for 1 h. The reaction solvent was evaporated under
reduced pressure to give the chloroketone which was used directly
without any further purification.
Example 39
[1329] Chloroalcohol 42: To a solution of chloroketone 41 (7.70 g,
23.48 mmol) in THF (90 mL) was added water (10 mL) and the solution
was cooled to 0.degree. C. A solution of NaBH.sub.4 (2.67 g, 70.45
mmol) in water (4 mL) was added dropwise over a period of 10 min.
The mixture was stirred for 1 h at 0.degree. C. and saturated
KHSO.sub.4 was slowly added until the pH<4 followed by saturated
NaCl. The organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (1/4 EtOAc/hexane) to give the chloroalcohol (6.20 g, 80%) as a
diastereomeric mixture.
Example 40
[1330] Epoxide 43: A solution of chloroalcohol 42 (6.20 g, 18.79
mmol) in EtOH (150 mL) was treated with 0.71 M KOH (1.27 g, 22.55
mmol) and the mixture was stirred at room temperature for 1 h. The
reaction mixture was evaporated under reduced pressure and the
residue was partitioned between EtOAc and water. The organic phase
was washed with saturated NaCl, dried with Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was purified by column chromatography on silica gel (1/6
EtOAc/hexane) to afford the desired epoxide 43 (2.79 g, 45%) and a
mixture of diastereomers 44 (1.43 g, 23%).
Example 41
[1331] Sulfonamide 45: To a suspension of epoxide 43 (2.79 g, 8.46
mmol) in 2-propanol (30 mL) was added isobutylamine (8.40 mL, 84.60
mmol) and the solution was refluxed for 1 h. The solution was
evaporated under reduced pressure and the crude solid was dissolved
in CH.sub.2Cl.sub.2 (40 mL) and cooled to 0.degree. C.
Triethylamine (2.36 mL, 16.92 mmol) was added followed by the
addition of 4-methoxybenzenesulfonyl chloride (1.75 g, 8.46 mmol).
The solution was stirred for 40 min at 0.degree. C., warmed to room
temperature, and evaporated under reduced pressure. The residue was
partitioned between EtOAc and saturated NaHCO.sub.3. The organic
phase was washed with saturated NaCl, dried with Na.sub.2SO.sub.4,
filtered, and evaporated under reduced pressure. The crude product
was directly used without any further purification.
Example 42
[1332] Silyl Ether 46: A solution of sulfonamide 45 (5.10 g, 8.46
mmol) in CH.sub.2Cl.sub.2 (50 mL) was treated with triethylamine
(4.7 mL, 33.82 mmol) and TMSOTf (3.88 mL, 16.91 mmol). The reaction
mixture was stirred at room temperature for 1 h and partitioned
between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3. The aqueous
phase was extracted twice with CH.sub.2Cl.sub.2 and the combined
organic extracts were washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (1/6 EtOAc/hexane) to give the silyl ether (4.50 g, 84%) as a
thick oil.
Example 43
[1333] Alcohol 47: To a solution of silyl ether 46 (4.5 g, 7.14
mmol) in MeOH (50 mL) was added 10% Pd/C (0.5 g). The suspension
was stirred under H.sub.2 atmosphere (balloon) at room temperature
for 2 h. The reaction mixture was filtered through a plug of celite
and concentrated under reduced pressure. The crude product was
purified by column chromatography on silica gel (3%
MeOH/CH.sub.2Cl.sub.2) to give the alcohol (3.40 g, 85%) as a white
solid.
Example 44
[1334] Aldehyde 48: To a solution of alcohol 47 (0.60 g, 1.07 mmol)
in CH.sub.2Cl.sub.2 (6 mL) at 0.degree. C. was added Dess Martin
reagent (0.77 g, 1.82 mmol). The reaction mixture was stirred at
0.degree. C. for 3 h and partitioned between CH.sub.2Cl.sub.2 and
NaHCO.sub.3. The organic phase was washed with H.sub.2O, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (1/4 EtOAc/hexane)
to give the aldehyde (0.45 g, 75%) as a pale yellow solid.
Example 45
[1335] Sulfonamide 50: To a suspension of epoxide (2.00 g, 5.41
mmol) in 2-propanol (20 mL) was added amine 49 (4.03 g, 16.23 mmol)
(prepared in 3 steps starting from 4-(aminomethyl)piperidine
according to Bioorg. Med. Chem. Lett., 2001, 11, 1261.). The
reaction mixture was heated to 80.degree. C. and stirred for 1 h.
The solution was evaporated under reduced pressure and the crude
solid was dissolved in CH.sub.2Cl.sub.2 (20 mL) and cooled to
0.degree. C. Triethylamine (4.53 mL, 32.46 mmol) was added followed
by the addition of 4-methoxybenzenesulfonyl chloride (3.36 g, 16.23
mmol). The solution was stirred for 40 min at 0.degree. C., warmed
to room temperature for 1.5 h, and evaporated under reduced
pressure. The residue was partitioned between EtOAc and saturated
NaHCO.sub.3. The organic phase was washed with saturated NaCl,
dried with Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was purified by column chromatography
on silica gel (3% 2-propanol/CH.sub.2Cl.sub.2) to give the
sulfonamide (2.50 g, 59%).
Example 46
[1336] Amine 51: A solution of sulfonamide 50 (2.50 g, 3.17 mmol)
in CH.sub.2Cl.sub.2 (6 mL) at 0.degree. C. was treated with
trifluoroacetic acid (3 mL). The solution was stirred for 30 min at
0.degree. C. and then warmed to room temperature for an additional
1.5 h. Volatiles were evaporated under reduced pressure and the
residue was partitioned between EtOAc and 0.5 N NaOH. The organic
phase was washed with 0.5 N NaOH (2.times.), water (2.times.) and
saturated NaCl, dried with Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure to give the amine (1.96 g, 90%)
which was used directly without any further purification.
Example 47
[1337] Carbamate 52: To a solution of amine 51 (1.96 g, 2.85 mmol)
in CH.sub.3CN (15 mL) at 0.degree. C. was treated with
(3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl 4-nitrophenyl carbonate
(0.84 g, 2.85 mmol, prepared according to Ghosh et al., J. Med.
Chem. 1996, 39, 3278.) and 4-(dimethylamino)pyridine (0.70 g, 5.70
mmol). After stirring for 2 h at 0.degree. C., the reaction solvent
was evaporated under reduced pressure and the residue was
partitioned between EtOAc and 0.5 N NaOH. The organic phase was
washed with 0.5N NaOH (2.times.), 5% citric acid (2.times.),
saturated NaHCO.sub.3, dried with Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was purified
by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the carbamate (1.44 g, 60%) as
a white solid.
Example Section I
Example 1
[1338] Carbonate 2: To a solution of
(R)-(+)-3-hydroxytetrahydrofuran (1.23 g, 14 mmol) in
CH.sub.2Cl.sub.2 (50 mL) was added triethylamine (2.9 mL, 21 mmol)
and bis(4-nitrophenyl)carbonate (4.7 g, 15.4 mmol). The reaction
mixture was stirred at room temperature for 24 h and partitioned
between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3. The
CH.sub.2Cl.sub.2 layer was dried with Na.sub.2SO.sub.4, filtered,
and concentrated. The crude product was purified by column
chromatography on silica gel (2/1-EtOAc/hexane) to give the
carbonate (2.3 g, 65%) as a pale yellow oil which solidified upon
standing.
Example 2
[1339] Carbamate 3: To a solution of 1 (0.385 g, 0.75 mmol) and 2
(0.210 g, 0.83 mmol) in CH.sub.3CN (7 mL) at room temperature was
added N,N-diisopropylethylamine (0.16 mL, 0.90 mmol). The reaction
mixture was stirred at room temperature for 44 h. The solvent was
evaporated under reduced pressure. The crude product was dissolved
in EtOAc and washed with saturated NaHCO.sub.3, brine, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (1/1-EtOAc/hexane)
to give the carbamate (0.322 g, 69%) as a white solid: mp
98-100.degree. C. (uncorrected).
Example 3
[1340] Phenol 4: To a solution of 3 (0.31 g, 0.49 mmol) in EtOH (10
mL) and EtOAc (5 mL) was added 10% Pd/C (30 mg). The suspension was
stirred under H.sub.2 atmosphere (balloon) at room temperature for
15 h. The reaction mixture was filtered through a plug of celite.
The filtrate was concentrated and dried under vacuum to give the
phenol (0.265 g) in quantitative yield.
Example 4
[1341] Diethylphosphonate 5: To a solution of phenol 4 (100 mg,
0.19 mmol) in THF (3 mL) was added Cs.sub.2CO.sub.3 (124 mg, 0.38
mmol) and triflate (85 mg, 0.29 mmol). The reaction mixture was
stirred at room temperature for 4 h and the solvent was evaporated
under reduced pressure. The residue was partitioned between EtOAc
and saturated NaCl. The organic phase was dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (5% 2-propanol/CH.sub.2Cl.sub.2) to give the diethylphosphonate
(63 mg, 49%, GS 16573) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 7.65 (d, J=8.7 Hz, 2H), 7.21 (d, J=8.7 Hz, 2H), 6.95 (d,
J=9 Hz, 2H), 6.84 (d, J=8.4 Hz, 2H), 5.06 (broad, s, 1H), 4.80 (d,
J=7.5 Hz, 1H), 4.19 (m, 6H), 3.83 (s, 3H), 3.80-3.70 (m, 6H),
3.09-2.72 (m, 6H), 2.00 (m, 1H), 1.79 (m, 2H), 1.32 (t, J=7.5 Hz,
6H), 0.86 (d, J=6.6 Hz, 3H), 0.83 (d, J=6.6 Hz, 3H); .sup.31P NMR
.delta. 17.8.
Example 5
[1342] Dibenzylphosphonate 6: To a solution of phenol 4 (100 mg,
0.19 mmol) in THF (3 mL) was added Cs.sub.2CO.sub.3 (137 mg, 0.42
mmol) and triflate (165 mg, 0.39 mmol). The reaction mixture was
stirred at room temperature for 6 h and the solvent was evaporated
under reduced pressure. The residue was partitioned between EtOAc
and saturated NaCl. The organic phase was dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (5% 2-propanol/CH.sub.2Cl.sub.2) to give the
dibenzylphosphonate (130 mg, 84%, GS 16574) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.65 (d, J=9 Hz, 2H), 7.30 (m,
10H), 7.08 (d, J=8.4 Hz, 2H), 6.94 (d, J=9 Hz, 2H), 6.77 (d, J=8.7
Hz, 2H), 5.16-5.04 (m, 5H), 4.80 (d, J=8.1 Hz, 1H), 4.16 (d, J=10.2
Hz, 2H), 3.82 (s, 3H), 3.75-3.71 (m, 6H), 3.10-2.72 (m, 6H), 2.00
(m, 1H), 1.79 (m, 2H), 0.86 (d, J=6.6 Hz, 3H), 0.83 (d, J=6.6 Hz,
3H); .sup.31P NMR (CDCl.sub.3) .delta. 18.8.
Example 6
[1343] Phosphonic Acid 7: To a solution of 6 (66 mg, 0.08 mmol) in
EtOH (3 mL) was added 10% Pd/C (12 mg). The suspension was stirred
under H.sub.2 atmosphere (balloon) at room temperature for 15 h.
The reaction mixture was filtered through a plug of celite. The
filtrate was concentrated under reduced pressure and triturated
with EtOAc to give the phosphonic acid (40 mg, 78%, GS 16575) as a
white solid.
Example 7
[1344] Carbonate 8: To a solution of
(S)-(+)-3-hydroxytetrahydrofuran (2 g, 22.7 mmol) in CH.sub.3CN (50
mL) was added triethylamine (6.75 mL, 48.4 mmol) and
N,N'-disuccinimidyl carbonate (6.4 g, 25 mmol). The reaction
mixture was stirred at room temperature for 5 h and concentrated
under reduced pressure. The residue was partitioned between EtOAc
and H.sub.2O. The organic phase was dried with Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. The crude
product was purified by column chromatography on silica gel (EtOAc
as eluant) followed by recrystallization (EtOAc/hexane) to give the
carbonate (2.3 g, 44%) as a white solid.
Example 8
[1345] Carbamate 9: To a solution of 1 (0.218 g, 0.42 mmol) and 8
(0.12 g, 0.53 mmol) in CH.sub.3CN (3 mL) at room temperature was
added N,N-diisopropylethylamine (0.11 mL, 0.63 mmol). The reaction
mixture was stirred at room temperature for 2 h. The solvent was
evaporated and the residue was partitioned between EtOAc and
saturated NaHCO.sub.3. The organic phase was washed with brine,
dried with Na.sub.2SO.sub.4, filtered, and concentrated. The crude
product was purified by column chromatography on silica gel
(1/1-EtOAc/hexane) to give the carbamate (0.176 g, 66%) as a white
solid.
Example 9
[1346] Phenol 10: To a solution of 9 (0.176 g, 0.28 mmol) in EtOH
(10 mL) was added 10% Pd/C (20 mg). The suspension was stirred
under H.sub.2 atmosphere (balloon) at room temperature for 4 h. The
reaction mixture was filtered through a plug of celite. The
filtrate was concentrated and dried under vacuum to give the phenol
(0.151 g, GS 10) in quantitative yield.
Example 10
[1347] Diethylphosphonate 11: To a solution of phenol 10 (60 mg,
0.11 mmol) in THF (3 mL) was added Cs.sub.2CO.sub.3 (72 mg, 0.22
mmol) and triflate (66 mg, 0.22 mmol). The reaction mixture was
stirred at room temperature for 4 h and the solvent was evaporated
under reduced pressure. The residue was partitioned between EtOAc
and saturated NaCl. The organic phase was dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (5% 2-propanol/CH.sub.2Cl.sub.2) to give the diethylphosphonate
(38 mg, 49%, GS 11) as a white solid.
Example Section J
Example 1
[1348] Triflate 1: To a solution of A (4 g, 6.9 mmol) in THF (30
mL) and CH.sub.2Cl.sub.2 (10 mL) was added Cs.sub.2CO.sub.3 (2.7 g,
8 mmol) and N-phenyltrifluoromethanesulfonimide (2.8 g, 8.0 mmol)
and stirred at room temperature for 16 h. The reaction mixture was
concentrated under reduced pressure. The residue was partitioned
between CH.sub.2Cl.sub.2 and saturated brine twice. The organic
phase was dried over sodium sulfate and used for next reaction
without further purification.
Example 2
[1349] Aldehyde 2: A solution of crude above triflate 1 (.about.6.9
mmol) in DMF (20 mL) was degassed (high vacuum for 5 min, argon
purge, repeat 3 times). To this solution were quickly added
Pd(OAc).sub.2 (120 mg, 266 .mu.mol) and
bis(diphenylphosphino-propane (dppp, 220 mg, 266 .mu.mol), and
heated to 70.degree. C. To this reaction mixture was rapidly
introduced carbon monoxide, and stirred at room temperature under
an atmospheric pressure of carbon monoxide, followed by slow
addition of TEA (5.4 mL, 38 mmol) and triethylsilane (3 mL, 18
mmol). The resultant mixture was stirred at 70.degree. C. for 16 h,
then cooled to room temperature, concentrated under reduced
pressure, partitioned between CH.sub.2Cl.sub.2 and saturated brine.
The organic phase was concentrated under reduced pressure and
purified on silica gel column to afford aldehyde 2 (2.1 g, 51%) as
white solid.
Example 3
[1350] Compounds 3a-3e: Representative Procedure, 3c: A solution of
aldehyde 2 (0.35 g, 0.59 mmol), L-alanine isopropyl ester
hydrochloride (0.2 g, 1.18 mmol), glacial acetic acid (0.21 g, 3.5
mmol) in 1,2-dichloroethane (10 mL) was stirred at room temperature
for 16 h, followed by addition of sodium cyanoborohydride (0.22 g,
3.5 mmol) and methanol (0.5 mL). The resulting solution was stirred
at room temperature for one h. The reaction mixture was washed with
sodium bicarbonate solution, saturated brine, and chromatographed
on silica gel to afford 3c (0.17 g, 40%). .sup.1H NMR (CDCl.sub.3):
.delta. 7.72 (d, 2H), 7.26 (d, 2H), 7.20 (d, 2H), 7.0 (d, 2H), 5.65
(d, 1H), 4.90-5.30 (m, 3H), 3.53-4.0 (m overlapping s, 13H), 3.31
(q, 1H), 2.70-3.20 (m, 7H), 1.50-1.85 (m, 3H), 1.25-1.31 (m, 9H),
0.92 (d, 3H), 0.88 (d, 3H). MS: 706 (M+1). TABLE-US-00007 Compound
R.sub.1 R.sub.2 Amino Acid 3a Me Me Ala 3b Me Et Ala 3c Me iPr Ala
3d Me Bn Ala 3e iPr Et Val
Example 4
[1351] Sulfonamide 1: To a solution of crude amine A (1 g, 3 mmol)
in CH.sub.2Cl.sub.2 was added TEA (0.6 g, 5.9 mmol) and
3-methoxybenzenesulfonyl chloride (0.6 g, 3 mmol). The resulting
solution was stirred at room temperature for 5 h, and evaporated
under reduced pressure. The residue was chromatographed on silica
gel to afford sulfonamide 1 (1.0 g, 67%).
Example 5
[1352] Amine 2: To a 0.degree. C. cold solution of sulfonamide 1
(0.85 g, 1.6 mmol) in CH.sub.2Cl.sub.2 (40 mL) was treated with
BBr.sub.3 in CH.sub.2Cl.sub.2 (10 ml, of 1 M solution, 10 mmol).
The solution was stirred at 0.degree. C. 10 min and then warmed to
room temperature and stirred for 1.5 h. The reaction mixture was
quenched with CH.sub.3OH, concentrated under reduced pressure,
azeotroped with CH.sub.3CN three times. The crude amine 2 was used
for next reaction without further purification.
Example 6
[1353] Carbamate 3: A solution of crude amine 2 (0.83 mmol) in
CH.sub.3CN (20 mL) and was treated with
(3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl 4-nitrophenyl carbonate
(245 mg, 0.83 mmol, prepared according to Ghosh et al., J. Med.
Chem. 1996, 39, 3278.) and N,N-dimethylaminopyridine (202 mg, 1.7
mmol). After stirring for 16 h at room temperature, the reaction
solvent was evaporated under reduced pressure and the residue was
partitioned between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3
three times. The organic phase was evaporated under reduced
pressure. The residue was purified by chromatography on silica gel
affording the carbamate 3 (150 mg, 33%) as a solid.
Example 7
[1354] Diethylphosphonate 4: To a solution of carbamate 3 (30 mg,
54 .mu.mol) in THF (5 mL) was added Cs.sub.2CO.sub.3 (54 mg, 164
.mu.mol) and triflate # (33 mg, 109 .mu.mol). After stirring the
reaction mixture for 30 min at room temperature, additional
Cs.sub.2CO.sub.3 (20 mg, 61 .mu.mol) and triflate (15 mg, 50
.mu.mol) were added and the mixture was stirred for 1 more hour.
The reaction mixture was evaporated under reduced pressure and the
residue was partitioned between CH.sub.2Cl.sub.2 and water. The
organic phase was dried (Na.sub.2SO.sub.4), filtered and evaporated
under reduced pressure. The crude product was chromatographed on
silica gel and repurified by HPLC (50% CH.sub.3CN-50% H.sub.2O on
C18 column) to give the diethylphosphonate 4 (15 mg, 39%). .sup.1H
NMR (CDCl.sub.3): .delta. 7.45 (m, 3H), 7.17-7.30 (m, 6H), 5.64 (d,
1H), 5.10 (d, 1H), 5.02 (q, 1H), 4.36 (d, 2H), 4.18-4.29 (2 q
overlap, 4H), 3.60-3.98 (m, 7H), 2.70-3.10 (m, 7H), 1.80-1.90 (m,
1H), 1.44-1.70 (m, 2H+H2O), 1.38 (t, 6H), 0.94 (d, 3H), 0.90 (d,
3H). .sup.31P NMR (CDCl.sub.3): 18.7 ppm; MS (ESI) 699 (M+H).
Example 8
[1355] Dibenzylphosphonate 5: To a solution of carbamate 3 (100 mg,
182 .mu.mol) in THF (10 mL) was added Cs.sub.2CO.sub.3 (180 mg, 550
.mu.mol) and dibenzylhydroxymethyl phosphonate triflate, Section A,
Scheme 2, Compound 9, (150 mg, 360 .mu.mol). After stirring the
reaction mixture for 1 h at room temperature, the reaction mixture
was evaporated under reduced pressure and the residue was
partitioned between CH.sub.2Cl.sub.2 and water. The organic phase
was dried (Na.sub.2SO.sub.4), filtered and evaporated under reduced
pressure. The residue was purified by HPLC (50% CH.sub.3CN-50%
H.sub.2O on C18 column) to give the dibenzylphosphonate 5 (110 mg,
72%). .sup.1H NMR (CDCl.sub.3): .delta. 7.41 (d, 2H), 7.35 (s,
10H), 7.17-7.30 (m, 6H), 7.09-7.11 (m, 1H), 5.64 (d, 1H), 4.90-5.15
(m, 6H), 4.26 (d, 2H), 3.81-3.95 (m, 4H), 3.64-3.70 (m, 2H),
2.85-3.25 (m, 7H), 1.80-1.95 (m, 1H), 1.35-1.50 (m, 1H), 0.94 (d,
3H), 0.91 (d, 3H). .sup.31P NMR (CDCl.sub.3) .delta. 19.4 ppm; MS
(ESI): 845 (M+Na), 1666 (2M+Na).
Example 9
[1356] Phosphonic acid 6: A solution of dibenzylphosphonate 5 (85
mg, 0.1 mmol) was dissolved in MeOH (10 mL) treated with 10% Pd/C
(40 mg) and stirred under H.sub.2 atmosphere (balloon) overnight.
The reaction was purged with N.sub.2, and the catalyst was removed
by filtration through celite. The filtrate was evaporated under
reduced pressure to afford phosphonic acid 6 (67 mg,
quantitatively). .sup.1H NMR (CD.sub.3OD): .delta. 7.40-7.55 (m,
3H), 7.10-7.35 (m, 6H), 5.57 (d, 1H), 4.32 (d, 2H), 3.90-3.95 (m,
1H), 3.64-3.78 (m, 5H), 3.47 (m, 1H), 2.85-3.31 (m, 5H), 2.50-2.60
(m, 1H), 2.00-2.06 (m, 1H), 1.46-1.60 (m, 1H), 1.30-1.34 (m, 1H),
0.9 (d, 3H), 0.90 (d, 3H). .sup.31P NMR (CD.sub.3OD): 16.60 ppm; MS
(ESI): 641 (M-H).
Example 10
[1357] Sulfonamide 1: To a solution of crude amine A (0.67 g, 2
mmol) in CH.sub.2Cl.sub.2 (50 mL) was added TEA (0.24 g, 24 mmol)
and crude 3-acetoxy-4-methoxybenzenesulfonyl chloride (0.58 g, 2.1
mmol, was prepared according to Kratzl et al., Monatsh. Chem. 1952,
83, 1042-1043), and the solution was stirred at room temperature
for 4 h, and evaporated under reduced pressure. The residue was
chromatographed on silica gel to afford sulfonamide 1 (0.64 g,
54%). MS: 587 (M+Na), 1150 (2M+Na)
[1358] Phenol 2: Sulfonamide 1 (0.64 g, 1.1 mmol) was treated with
saturated NH.sub.3 in MeOH (15 mL) at room temperature for 15 min.,
then evaporated under reduced pressure. The residue was purified on
silica gel column to afford phenol 2 (0.57 g, 96%).
Example 11
[1359] Dibenzylphosphonate 3a: To a solution of phenol 2 (0.3 g,
0.57 mmol) in THF (8 mL) was added Cs.sub.2CO.sub.3 (0.55 g, 1.7
mmol)) and dibenzylhydroxymethyl phosphonate triflate (0.5 g, 1.1
mmol). After stirring the reaction mixture for 1 h at room
temperature, the reaction mixture was quenched with water and
partitioned between CH.sub.2Cl.sub.2 and saturated ammonium
chloride aqueous solution. The organic phase was dried
(Na.sub.2SO.sub.4), filtered and evaporated under reduced pressure.
The residue was chromatographed on silica gel (40% EtOAc/60%
hexane) to give the dibenzylphosphonate 3a (0.36 g, 82%). .sup.1H
NMR (CDCl.sub.3): .delta. 7.20-7.40 (m, 17H), 6.91 (d, 1H),
5.10-5.25 (2 q(ab) overlap, 4H), 4.58-4.70 (m, 1H), 4.34 (d, 2H),
3.66-3.87 (m+s, 5H), 2.85-3.25 (m, 6H), 1.80-1.95 (m, 1H), 1.58 (s,
9H), 0.86-0.92 (2d, 6H).
Example 12
[1360] Diethylphosphonate 3b: To a solution of phenol 2 (0.15 g,
0.28 mmol) in THF (4 mL) was added Cs.sub.2CO.sub.3 (0.3 g, 0.92
mmol)) and diethylhydroxymethyl phosphonate triflate (0.4 g, 1.3
mmol). After stirring the reaction mixture for 1 h at room
temperature, the reaction mixture was quenched with water and
partitioned between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3
aqueous solution. The organic phase was dried (Na.sub.2SO.sub.4),
filtered and evaporated under reduced pressure. The residue was
chromatographed on silica gel (1% CH.sub.3OH--CH.sub.2Cl.sub.2) to
give the diethylphosphonate 3b (0.14 g, 73%).
Example 13
[1361] Amine 4a: To a solution of 3a (0.35 g, 0.44 mmol) in
CH.sub.2Cl.sub.2 (10 mL) was treated with TFA (0.75 g, 6.6 mmol) at
room temperature for 2 h. The reaction was evaporated under reduced
pressure, azeotroped with CH.sub.3CN twice, dried to afford crude
amine 4a. This crude 4a was used for next reaction without further
purification.
Example 14
[1362] Amine 4b: To a solution of 3b (60 mg, 89 .mu.mol) in
CH.sub.2Cl.sub.2 (1 mL) was treated with TFA (0.1 mL, 1.2 mmol) at
room temperature for 2 h. The reaction was evaporated under reduced
pressure, azeotroped with CH.sub.3CN twice, dried to afford crude
amine 4b (68 mg). This crude 4b was used for next reaction without
further purification.
Example 15
[1363] Carbamate 5a: An ice-cold solution of crude amine 4a (0.44
mmol) in CH.sub.3CN (10 mL) and was treated with
(3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl 4-nitrophenyl carbonate
(120 mg, 0.4 mmol) and N,N-dimethylaminopyridine (DMAP, 110 mg,
0.88 mmol). After 4 h, more DMAP (0.55 g, 4.4 mmol) was added to
the reaction mixture. After stirring for 1.5 h at room temperature,
the reaction solvent was evaporated under reduced pressure and the
residue was partitioned between CH.sub.2Cl.sub.2 and saturated
NaHCO.sub.3. The organic phase was evaporated under reduced
pressure. The residue was purified by chromatography on silica gel
affording the crude carbamate 5a (220 mg) containing some
p-nitrophenol. The crude 5a was repurified by HPLC (50%
CH.sub.3CN/50% H.sub.2O) to afford pure carbamate 5a (176 mg, 46%,
2 steps). .sup.1H NMR (CDCl.sub.3): .delta. 7.20-7.36 (m, 1H), 6.94
(d, 1H), 5.64 (d, 1H), 5.10-5.25 (2 q(ab) overlap, 4H), 4.90-5.10
(m, 1H), 4.90 (d, 1H), 4.34 (d, 2H), 3.82-3.91 (m+s, 6H), 3.63-3.70
(m, 3H), 2.79-3.30 (m, 7H), 1.80-1.90 (m, 1H), 1.40-1.50 (m, 1H),
0.94 (d, 3H), 0.89 (d, 3H). .sup.31P NMR (CDCl.sub.3): 17.2
ppm.
Example 16
[1364] Carbamate 5b: An ice-cold solution of crude amine 4b (89
.mu.mol)) in CH.sub.3CN (5 mL) and was treated with
(3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl 4-nitrophenyl carbonate
(26 mg, 89 .mu.mol) and N,N-dimethylaminopyridine (DMAP, 22 mg,
0.17 mmol). After 1 h at 0.degree. C., more DMAP (10 mg. 82
.mu.mol) was added to the reaction mixture. After stirring for 2 h
at room temperature, the reaction solvent was evaporated under
reduced pressure and the residue was partitioned between
CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3. The organic phase was
evaporated under reduced pressure. The residue was purified by HPLC
(C18 column, 45% CH.sub.3CN/55% H.sub.2O) to afford pure carbamate
5b (18.8 mg, 29%, 3 steps). .sup.1H NMR (CDCl.sub.3): .delta. 7.38
(d, 2H), 7.20-7.36 (m, 6H), 7.0 (d, 1H), 5.64 (d, 1H), 4.96-5.03
(m, 2H), 4.39 (d, 2H), 4.20-4.31 (2q overlap, 4H) 3.80-4.00 ((s
overlap with m, 7H), 3.60-3.73 (m, 2H), 3.64-3.70 (m, 2H),
2.85-3.30 (m, 7H), 1.80-1.95 (m, 1H), 1.55-1.75 (m, 1H), 1.35-1.50
(s overlap with m, 7H), 0.94 (d, 3H), 0.88 (d, 3H). .sup.31P NMR
(CDCl.sub.3): 18.1 ppm.
Example 17
[1365] Phosphonic acid 6: A solution of dibenzylphosphonate 5a (50
mg, 58 .mu.mol) was dissolved in MeOH (5 mL) and EtOAc (3 mL) and
treated with 10% Pd/C (25 mg) and was stirred at room temperature
under H.sub.2 atmosphere (balloon) for 8 h. The catalyst was
filtered off. The filtrate was concentrated and redissolved in MeOH
(5 mL), treated with 10% Pd/C (25 mg) and was stirred at room
temperature under H.sub.2 atmosphere (balloon) overnight. The
catalyst was filtered off. The filtrate was evaporated under
reduced pressure to afford phosphonic acid 6 (38 mg,
quantitatively). .sup.1H NMR (CD.sub.3OD): .delta. 7.42 (m, 1H),
7.36 (s, 1H), 7.10-7.25 (m, 6H), 5.58 7 (d, 1H), 4.32 (d, 2H), 3.90
(s, 3H), 3.60-3.80 (m, 6H), 3.38 (d, 1H), 2.85-3.25 (m, 5H),
2.50-2.60 (m, 1H), 1.95-2.06 (m, 1H), 1.46-1.60 (m, 1H), 1.30-1.40
(m, 1H), 0.93(d, 3H), 0.89 (d, 3H). .sup.31P NMR (CD.sub.3OD): 14.8
ppm; MS (ESI): 671 (M-H).
Example 18
[1366] Amine 7: To a 0.degree. C. cold solution of
diethylphosphonate 3b (80 mg, 0.118 mmol) in CH.sub.2Cl.sub.2 was
treated with BBr.sub.3 in CH.sub.2Cl.sub.2 (0.1 ml, of 1 M
solution, 1 mmol). The solution was stirred at 0.degree. C. 10 min
and then warmed to room temperature and stirred for 3 h. The
reaction mixture was concentrated under reduced pressure. The
residue was redissolved in CH.sub.2Cl.sub.2 (containing some
CH.sub.3OH, concentrated, azeotroped with CH.sub.3CN three times.
The crude amine 7 was used for next reaction without further
purification.
Example 19
[1367] Carbamate 8: An ice-cold solution of crude amine 7 (0.118
mmol) in CH.sub.3CN (5 mL) and was treated with
(3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl 4-nitrophenyl carbonate
(35 mg, 0.118 mmol) and N,N-dimethylaminopyridine (29 mg, 0.24
mmol), warmed to room temperature. After stirring for 1 h at room
temperature, more DMAP (20 mg, 0.16 mmol) was added to reaction
mixture. After 2 h stirred at room temperature, the reaction
solvent was evaporated under reduced pressure and the residue was
partitioned between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3. The
organic phase was evaporated under reduced pressure. The residue
was purified by HPLC on C18 (CH.sub.3CN-55% H.sub.2O) to afford the
desired carbamate 8 (11.4 mg, 13.4%) as an off-white solid. .sup.1H
NMR (CDCl.sub.3): .delta. 7.20-7.40 (m, 7H), 7.00 (d, 1H), 5.64 (d,
1H), 5.00-5.31 (m, 2H), 4.35 (d, 2H), 4.19-4.30 (2q overlap, 4H),
3.80-4.00 (m, 4H), 3.68-3.74 (m, 2H), 3.08-3.20 (m, 3H), 2.75-3.00
(m, 4H), 1.80-1.90 (m, 1H), 1.55-1.75 (m, 1H), 1.38 (t, 6H), 0.91
(2d overlap, 6H). .sup.31P NMR (CD.sub.3OD): 619.5 ppm.
Example Section K
Example 1
[1368] Monophenyl-monolactate 3: A mixture of monoacid 1 (0.500 g,
0.7 mmol), alcohol 2 (0.276 g, 2.09 mmol) and
dicyclohexylcarbodiimide (0.431 g, 2.09 mmol) in dry pyridine (4
mL) was placed into a 70.degree. C. oil bath and heated for two
hours. The reaction was monitored by TLC assay (SiO.sub.2, 70%
ethyl acetate in hexanes as eluent, product R.sub.f=0.68,
visualization by UV). The reaction contents were cooled to ambient
temperature with the aid of a cool bath and diluted with
dichloromethane (25 mL). TLC assay may show presence of starting
material. The diluted reaction mixture was filtered to remove
solids. The filtrate was then cooled to 0.degree. C. and charged
with 0.1 N HCl (10 mL). The pH 4 mixture was stirred for 10 minutes
and poured into separatory funnel to allow the layers to separate.
The lower organic layer was collected and dried over sodium
sulfate. The drying agent was filtered off and the filtrate
concentrated to an oil via rotary evaporator (<30.degree. C.
warm bath). The crude product oil was purified on pretreated silica
gel (deactivated using 10% methanol in dichloromethane followed by
rinse with 60% ethyl acetate in dichloromethane). The product was
eluted with 60% ethyl acetate in dichloromethane to afford the
product monophenyl-monolactate 3 as a white foam (0.497 g, 86%
yield). .sup.1H NMR (CDCl.sub.3) .delta. 7.75 (d, 2H), 7.40-7.00
(m, 4H), 5.65 (d, 1H), 5.20-4.90 (m, 4H), 4.70 (d, 1H), 4.55-4.50
(m, 1H), 4.00-3.80 (m, 4H), 3.80-3.60 (m, 3H), 3.25-2.75 (m, 7H),
1.50 (d, 3H), 1.30-1.20 (m, 7H), 0.95 (d, 3H), 0.85 (d, 3H).
.sup.31P NMR (CDCl.sub.3) .delta. 16.2, 13.9.
Example 2
[1369] Monophenyl-monoamidate 5: A mixture of monoacid 1 (0.500 g,
0.70 mmol), amine hydrochloride 4 (0.467 g, 2.78 mmol) and
dicyclohexylcarbodiimide (0.862 g, 4.18 mmol) in dry pyridine (8
mL) was placed into a 60.degree. C. oil bath, and heated for one
hour (at this temperature, product degrades if heating continues
beyond this point). The reaction was monitored by TLC assay
(SiO.sub.2, 70% ethyl acetate in hexanes as eluent, product
R.sub.f=0.39, visualization by UV). The contents were cooled to
ambient temperature and diluted with ethyl acetate (15 mL) to
precipitate a white solid. The mixture was filtered to remove
solids and the filtrate was concentrated via rotary evaporator to
an oil. The oil was diluted with dichloromethane (20 mL) and washed
with 0.1 N HCl (2.times.20 mL), water (1.times.20 mL) and dilute
sodium bicarbonate (1.times.20 mL). The organic layer was dried
over sodium sulfate, filtered, and concentrated to an oil via
rotary evaporator. The crude product oil was dissolved in
dichloromethane (10 mL). Hexane was slowly charged to the stirring
solution until cloudiness persisted. The cloudy mixture was stirred
for a few minutes until TLC assay showed that the
dichloromethane/hexane layer contained no product. The
dichloromethane/hexanes layer was decanted and the solid was
further purified on silica gel first pretreated with 10% methanol
in ethyl acetate and rinsed with 50% ethyl acetate in hexanes. The
product 5 was eluted with 50% ethyl acetate in hexanes to afford a
white foam (0.255 g, 44% yield) upon removal of solvents. .sup.1H
NMR (CDCl.sub.3) .delta. 7.75 (d, 2H), 7.40-7.15 (m, 10H),
7.15-7.00 (t, 2H), 5.65 (d, 1H), 5.10-4.90 (m, 3H), 4.50-4.35 (m,
2H), 4.25-4.10 (m, 1H), 4.00-3.60 (m, 8H), 3.20-2.75 (m, 7H),
1.40-1.20 (m, 1H), 0.95 (d, 3H), 0.85 (d, 3H). .sup.31P NMR
(CDCl.sub.3) .delta. 19.1, 18.0.
Example 3
[1370] Bisamidate 8: A solution of triphenylphosphine (1.71 g, 6.54
mmol) and aldrithiol (1.44 g, 6.54 mmol) in dry pyridine (5 mL),
stirred for at least 20 minutes at room temperature, was charged
into a solution of diacid 6 (1.20 g, 1.87 mmol) and amine
hydrochloride 7 (1.30 g, 7.47 mmol) in dry pyridine (10 mL).
Diisopropylethylamine (0.97 g, 7.48 mmol) was then added to this
combined solution and the contents were stirred at room temperature
for 20 hours. The reaction was monitored by TLC assay (SiO.sub.2,
5:5:1 ethyl acetate/hexanes/methanol as eluent, product
R.sub.f=0.29, visualization by UV). The reaction mixture was
concentrated via rotary evaporator and dissolved in dichloromethane
(50 mL). Brine (25 mL) was charged to wash the organic layer. The
aqueous layer was back extracted with dichloromethane (1.times.50
mL). The combined organic layers were dried over sodium sulfate,
filtered, and concentrated via rotary evaporator to afford an oil.
The crude product oil was purified on silica gel using 4%
isopropanol in dichloromethane as eluent. The combined fractions
containing the product may have residual amine contamination. If
so, the fractions were concentrated via rotary evaporator and
further purified by silica gel chromatography using a gradient of
1:1 ethyl acetate/hexanes to 5:5:1 ethyl acetate/hexanes/methanol
solution as eluent to afford the product 8 as a foam (0.500 g, 30%
yield).
Example 4
[1371] Diacid 6: A solution of dibenzylphosphonate 9 (8.0 g, 9.72
mmol) in ethanol (160 mL) and ethyl acetate (65 mL) under a
nitrogen atmosphere and at room temperature was charged 10% Pd/C
(1.60 g, 20 wt %). The mixture was stirred and evacuated by vacuum
and purged with hydrogen several times. The contents were then
placed under atmospheric pressure of hydrogen via a balloon. The
reaction was monitored by TLC assay (SiO.sub.2, 7:2.5:0.5
dichloromethane/methanol/ammonium hydroxide as eluent, product
R.sub.f=0.05, visualization by UV) and was judged complete in 4 to
5 hours. The reaction mixture was filtered through a pad of celite
to remove Pd/C and the filter cake rinsed with ethanol/ethyl
acetate mixture (50 mL). The filtrate was concentrated via rotary
evaporation followed by several co-evaporations using ethyl acetate
(3.times.50 mL) to remove ethanol. The semi-solid diacid 6, free of
ethanol, was carried forward to the next step without
purification.
Example 5
[1372] Diphenylphosphonate 10: To a solution of diacid 6 (5.6 g,
8.71 mmol) in pyridine (58 mL) at room temperature was charged
phenol (5.95 g, 63.1 mmol). To this mixture, while stirring, was
charged dicyclohexylcarbodiimide (7.45 g, 36.0 mmol). The resulting
cloudy, yellow mixture was placed in a 70-80.degree. C. oil bath.
The reaction was monitored by TLC assay (SiO.sub.2, 7:2.5:0.5
dichloromethane/methanol/ammonium hydroxide as eluent, diacid
R.sub.f=0.05, visualization by UV for the disappearance of starting
material. SiO.sub.2, 60% ethyl acetate in hexanes as eluent,
diphenyl R.sub.f=0.40, visualization by UV) and was judged complete
in 2 hours. To the reaction mixture was charged isopropyl acetate
(60 mL) to produce a white precipitation. The slurry was filtered
through a pad of celite to remove the white precipitate and the
filter cake rinsed with isopropyl acetate (25 mL). The filtrate was
concentrated via rotary evaporator. To the resulting yellow oil was
charged a premixed solution of water (58 mL) and 1N HCl (55 mL)
followed by isopropyl acetate (145 mL). The mixture was stirred for
one hour in an ice bath. After separating the layers, the aqueous
layer was back extracted with ethyl acetate (2.times.50 mL). The
combined organic layers were dried over sodium sulfate, filtered,
and concentrated via rotary evaporator. The crude product oil was
purified by silica gel column chromatography using 50% ethyl
acetate in hexanes as eluent to afford the product 10 as a white
foam (3.52 g, 51% yield). .sup.1H NMR (CDCl.sub.3) .delta. 7.75
(d,2H), 7.40-7.20 (m, 15H), 7.10 (d, 2H), 5.65 (d, 1H), 5.10-4.90
(m, 2H), 4.65 (d, 2H), 4.00-3.80 (m, 4H), 3.75-3.65 (m, 3H),
3.25-2.75 (m, 7H), 1.90-1.75 (m, 1H), 1.70-1.60 (m, 1H), 1.50-1.40
(m, H), 0.90 (d, 3H), 0.85 (d, 3H). .sup.31P NMR (CDCl.sub.3)
.delta. 10.9.
Example 6
[1373] Monophenyl 1: To a solution of diphenyl 10 (3.40 g, 4.28
mmol) in acetonitrile (170 mL) at 0.degree..degree. C. was charged
1N sodium hydroxide (4.28 mL). The reaction was monitored by TLC
assay (SiO.sub.2, 7:2.5:0.5 dichloromethane/methanol/ammonium
hydroxide as eluent, diphenyl R.sub.f=0.65, visualization by UV for
the disappearance of starting material. Product monophenyl
R.sub.f=0.80, visualization by UV). Additional 1N NaOH was added
(if necessary) until the reaction was judged complete. To the
reaction contents at 0.degree. C. was charged Dowex H.sup.+ (Dowex
50 W.times.8-200) (4.42 g) and stirred for 30 minutes at which time
the pH of the mixture reached pH 1 (monitored by pH paper). The
mixture was filtered to remove the Dowex resin and the filtrate was
concentrated via rotary evaporation (water bath<40.degree. C.).
The resulting solution was co-evaporated with toluene to remove
water (3.times.50 mL). The white foam was dissolved in ethyl
acetate (8 mL) followed by slow addition of hexanes (16 mL) over 30
minutes to induce precipitation. A premixed solution of 2:1
hexanes/ethyl acetate solution (39 mL) was charged to the
precipitated material and stirred. The product 1 was filtered and
rinsed with premixed solution of 2:1 hexanes/ethyl acetate solution
(75 mL) and dried under vacuum to afford a white powder (2.84 g,
92% yield). .sup.1H NMR (CD.sub.3OD) .delta. 7.80 (d, 2H),
7.40-7.30 (m, 2H), 7.20-7.15 (m, 1H), 5.55 (d, 1H), 4.50 (d, 2H),
3.95-3.85 (m, 1H), 3.80-3.60 (m, 5H), 3.45 (bd, 1H), 3.25-3.15 (m,
2H), 3.00-2.80 (m, 3H), 2.60-2.45 (m, 1H), 2.10-1.95 (m, 2H),
1.85-1.60 (m, 2H), 1.50-1.40 (m, 1H), 1.40-1.30 (m, 1H), 0.95 (d,
3H), 0.85 (d, 3H). .sup.31P NMR (CDCl.sub.3) .delta. 13.8. The
monophenyl product 1 is sensitive to silica gel. On contact with
silica gel 1 converts to an unknown compound possessing .sup.31P
NMR chemical shift of 8 ppm. However, the desired monophenyl
product 1 can be regenerated by treatment of the unknown compound
with 2.5 M NaOH in acetonitrile at 0.degree. C. for one hour
followed by Dowex H.sup.+ treatment as described above.
Example 7
[1374] Dibenzylphosphonate 9: To a solution of phenol 11 (6.45 g,
11.8 mmol) in tetrahydrofuran (161 mL) at room temperature was
charged triflate reagent 12 (6.48 g, 15.3 mmol). Cesium carbonate
(1.5 g, 35.3 mmol) was added and the mixture was stirred and
monitored by TLC assay (SiO.sub.2, 5% methanol in dichloromethane
as eluent, dibenzyl product R.sub.f=0.26, visualization by UV or
ninhydrin stain and heat). Additional Cs.sub.2CO.sub.3 was added
until the reaction was judged complete. To the reaction contents
was charged water (160 mL) and the mixture extracted with ethyl
acetate (2.times.160 mL). The combined organic layer was dried over
sodium sulfate, filtered, and concentrated via rotary evaporator to
afford a viscous oil. The crude oil was purified by silica gel
column chromatography using a gradient of 100% dichloromethane to
1% methanol in dichloromethane to afford product 9 as a white foam
(8.68 g, 90% yield). .sup.1H NMR (CDCl.sub.3) .delta. 7.75 (d, 2H),
7.40-7.20 (m, 16H), 6.95 (d, 2H), 5.65 (d, 1H), 5.20-4.90 (m, 6H),
4.25 (d, 2H), 4.00-3.80 (m, 4H), 3.75-3.65 (m, 3H), 3.20-2.75 (m,
7H), 1.90-1.75 (m, 1H), 1.30-1.20 (m, 1H), 0.90 (d, 3H), 0.85 (d,
3H). .sup.31P NMR (CDCl.sub.3) .delta. 19.1.
Example 7a
[1375] Hydroxyphenylsulfonamide 14: To a solution of
methoxyphenylsulfonamide 13 (35.9 g, 70.8 mmol) in dichloromethane
(3.5 L) at 0.degree. C. was charged boron tribromide (1M in DCM,
40.1 mL, 425 mmol). The reaction content was allowed to warm to
room temperature, stirred over two hours, and monitored by TLC
assay (SiO.sub.2, 10% methanol in dichloromethane as eluent,
dibenzyl product R.sub.f=0.16, visualization by UV). To the
contents at 0.degree. C. was slowly charged propylene oxide (82 g,
1.42 mmol). Methanol (200 mL) was added and the reaction mixture
was concentrated via rotary evaporator to afford a viscous oil. The
crude product mixture was purified by silica gel column
chromatography using 10% methanol in dichloromethane to afford the
product 14 as a foam (22 g, 80% yield). .sup.1H NMR (DMSO) .delta.
7.60 (d, 2H), 7.30-7.20 (m, 5H), 6.95 (d, 2H), 3.90-3.75 (m, 1H),
3.45-3.20 (m, 5H), 3.00-2.55 (m, 5H), 2.50-2.40 (m, 1H), 1.95-1.85
(m, 1H), 0.85 (d, 3H), 0.80 (d, 3H).
Example 8
[1376] Cisfuran carbamate 16: To a solution of amine 14 (20.4 g,
52.0 mmol) in acetonitrile (600 mL) at room temperature was charged
dimethylaminopyridine (13.4 g, 109 mmol) followed by cisfuran
p-nitrophenylcarbonate reagent 15 (14.6 g, 49.5 mmol). The
resulting solution was stirred at room temperature for at least 48
hours and monitored by TLC assay (SiO.sub.2, 10% methanol in
dichloromethane as eluent, cisfuran product R.sub.f=0.34,
visualization by UV). The reaction mixture was concentrated via
rotary evaporator. The crude product mixture was purified by silica
gel column chromatography using a gradient of 60% ethyl acetate in
hexanes to 70% ethyl acetate in hexanes to afford the product 16 as
a solid (18.2 g, 64% yield). .sup.1H NMR (DMSO) .delta. 10.4 (bs,
1H), 7.60 (d, 2H), 7.30-7.10 (m, 6H), 6.95 (d, 2H), 5.50 (d, 1H),
4.85 (m, 1H), 3.85 (m, 1H), 3.70 (m, 1H), 3.65-3.50 (m, 4H), 3.30
(d, 1H), 3.05-2.95 (m, 2H), 2.80-2.65 (m, 3H), 2.50-2.40 (m, 1H),
2.00-1.90 (m, 1H), 1.45-1.20 (m, 2H), 0.85 (d, 3H), 0.80 (d,
3H).
Example Section L
Example 1
[1377] Monobenzyl phosphonate 2 A solution of dibenzylphosphonate 1
(150 mg, 0.175 mmol) was dissolved in toluene (1 mL), treated with
DABCO (20 mg, 0.178 mmol) and was refluxed under N2 atmosphere
(balloon) for 3 h. The solvent was removed and the residual was
dissolved in aqueous HCl (5%). The aqueous layer was extracted with
ethyl acetate and the organic layer was dried over sodium sulfate.
After evaporation to yield the monobenzyl phosphonate 2 (107 mg,
80%) as a white powder. .sup.1H NMR (CD.sub.3OD) .delta. 7.75 (d,
J=5.4 Hz, 2H), 7.42-7.31 (m, 5H) 7.16 (d, J=5.4 Hz, 2H), 7.01 (d,
J=5.4 Hz, 2H), 6.86 (d, J=5.4 Hz, 2H), 5.55 (d, J=3.3 Hz, 1H), 5.14
(d, J=5.1 Hz, 2H), 4.91 (m, 1H), 4.24-3.66 (m overlapping s, 11H),
3.45 (m, 2H), 3.14-2.82 (m, 6H), 2.49 (m, 1H), 2.01 (m, 1H),
1.51-1.34 (m, 2H), 0.92 (d, J=3.9 Hz, 3H), 0.87 (d, J=3.9 Hz, 3H);
.sup.31P NMR (CD.sub.3OD) .delta. 20.5; MS (ESI) 761 (M-H).
Example 2
[1378] Monobenzyl, ethyl phosphonate 3 To a solution of monobenzyl
phosphonate 2 (100 mg, 0.13 mmol) in dry THF (5 mL) at room
temperature under N.sub.2 was added Ph.sub.3P (136 mg, 0.52 mmol)
and ethanol (30 .mu.L, 0.52 mmol). After cooled to 0.degree. C.,
DEAD (78 .mu.L, 0.52 mmol) was added. The mixture was stirred for
20 h at room temperature. The solvent was evaporated under reduced
pressure and the residue was purified by using chromatograph on
silica gel (10% to 30% ethyl acetate/hexane) to afford the
monobenzyl, ethyl phosphonate 3 (66 mg, 64%) as white solid.
.sup.1H NMR (CDCl.sub.3) 7.70 (d, J=8.7 Hz, 2H), 7.43-7.34 (m, 5H)
7.14 (d, J=8.4 Hz, 2H), 7.01 (d, J=8.7 Hz, 2H), 6.84 (d, J=8.4 Hz,
2H), 5.56 (d, J=5.4 Hz, 1H), 5.19 (d, J=8.7 Hz, 2H), 5.00 (m, 2H),
4.22-3.67 (m overlapping s, 13H), 3.18-2.76 (m, 7H), 1.82-1.54 (m,
3H), 1.33 (t, J=7.0 Hz, 3H), 0.92 (d, J=6.6 Hz, 3H), 0.88 (d, J=6.6
Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 19.8; MS (ESI) 813
(M+Na).
Example 3
[1379] Monoethyl phosphonate 4 A solution of monobenzyl, ethyl
phosphonate 3 (60 mg) was dissolved in EtOAc (2 mL), treated with
10% Pd/C (6 mg) and was stirred under H.sub.2 atmosphere (balloon)
for 2 h. The catalyst was removed by filtration through celite. The
filtered was evaporated under reduced pressure, the residue was
triturated with ether and the solid was collected by filtration to
afford the monoethyl phosphonate 4 (50 mg, 94%) as white solid.
.sup.1H NMR (CD.sub.3OD) 7.76 (d, J=8.7 Hz, 2H), 7.18 (d, J=8.4 Hz,
2H), 7.01 (d, J=8.7 Hz, 2H), 6.89 (d, J=8.4 Hz, 2H), 5.58 (d, J=5.4
Hz, 1H), 5.90 (m, 1H), 4.22-3.67 (m overlapping s, 13H), 3.18-2.50
(m, 7H), 1.98(m, 1H), 1.56 (m, 2H), 1.33 (t, J=6.9 Hz, 3H), 0.92
(d, J=6.6 Hz, 3H), 0.87 (d, J=6.6 Hz, 3H); .sup.31P NMR
(CD.sub.3OD) .delta. 18.7; MS (ESI) 700 (M-H).
Example 4
[1380] Monophenyl, ethyl phosphonate 5 To a solution of phosphonic
acid 11 (800 mg, 1.19 mmol) and phenol (1.12 g, 11.9 mmol) in
pyridine (8 mL) was added ethanol (69 .mu.L, 1.19 mmol) and
1,3-dicyclohexylcarbodiimide (1 g, 4.8 mmol). The solution was
stirred at 70.degree. C. for 2 h. The reaction mixture was cooled
to room temperature, then diluted with ethyl acetate (10 mL) and
filtered. The filtrate was evaporated under reduced pressure to
remove pyridine. The residue was dissolved in ethyl acetate and the
organic phase was separated and washed with brine, dried over
MgSO.sub.4, filtered and concentrated. The residue was purified by
chromatography on silica gel to give monophenyl, ethyl phosphonate
5 (600 mg, 65%) as white solid. .sup.1H NMR (CDCl.sub.3) 7.72 (d,
J=9 Hz, 2H), 7.36-7.18 (m, 5H), 7.15 (d, J=8.7 Hz, 2H), 6.98 (d,
J=9 Hz, 2H), 6.87 (d, J=8.7 Hz, 2H), 5.64 (d, J=5.4 Hz, 1H), 5.00
(m, 2H), 4.34 (m, 4H), 3.94-3.67 (m overlapping s, 9H), 3.18-2.77
(m, 7H), 1.82-1.54 (m, 3H), 1.36 (t, J=7.2 Hz, 3H), 0.92 (d, J=6.6
Hz, 3H), 0.87 (d, J=6.6 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta.
16.1; MS (ESI) 799 (M+Na).
Example 5
[1381] Sulfonamide 6 To a suspension of epoxide 5 (3 g, 8.12 mmol)
in 2-propanol (30 mL) was added isobutylamine (8 mL, 81.2 mmol) and
the solution was stirred at 80.degree. C. for 1 h. The solution was
evaporated under reduced pressure and the crude solid was dissolved
in CH.sub.2Cl.sub.2 (40 mL) and cooled to 0.degree. C. TEA (2.3 mL,
16.3 mmol) was added followed by the addition of
4-nitrobenzenesulfonyl chloride (1.8 g, 8.13 mmol) in
CH.sub.2Cl.sub.2 (5 mL) and the solution was stirred for 30 min at
0.degree. C., warmed to room temperature and evaporated under
reduced pressure. The residue was partitioned between EtOAc and
saturated NaHCO.sub.3. The organic phase was washed with saturated
NaCl, dried over Na.sub.2SO.sub.4, filtered and evaporated under
reduced pressure. The crude product was recrystallized from
EtOAc/hexane to give the sulfonamide 6 (4.6 g, 91%) as an off-white
solid. MS (ESI) 650 (M+Na).
Example 6
[1382] Phenol 7 A solution of sulfonamide 6 (4.5 g, 7.1 mmol) in
CH.sub.2Cl.sub.2 (50 mL) at 0.degree. C. was treated with BBr.sub.3
(1M in CH.sub.2Cl.sub.2, 50 mL). The solution was stirred at
0.degree. C. to room temperature for 48 h. CH.sub.3OH (10 mL) was
carefully added. The solvent was evaporated under reduced pressure
and the residue was partitioned between EtOAc and saturated
NaHCO.sub.3. The organic phase washed with saturated NaCl, dried
over Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was purified by chromatography on
silica gel (10%--MeOH/CH.sub.2Cl.sub.2) to give the phenol 7 (2.5
g, 80%) as an off-white solid. MS (ESI) 528 (M+H).
Example 7
[1383] Carbamate 8 A solution of sulfonamide 7 (2.5 g, 5.7 mmol) in
CH.sub.3CN (100 mL) and was treated with proton-sponge (3 g, 14
mmol) and followed by (3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl
4-nitrophenyl carbonate (1.7 g, 5.7 mmol) at 0.degree. C. After
stirring for 48 h at room temperature, the reaction solvent was
evaporated under reduced pressure and the residue was partitioned
between EtOAc and 10% HCl. The organic phase was washed with
saturated NaCl, dried over Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was purified
by chromatography on silica gel (10% MeOH/CH.sub.2Cl.sub.2)
affording the carbamate 8 (2.1 g, 62%) as a white solid. MS (ESI)
616 (M+Na).
Example 8
[1384] Diethylphosphonate 9 To a solution of carbamate 8 (2.1 g,
3.5 mmol) in CH.sub.3CN (50 mL) was added Cs.sub.2CO.sub.3 (3.2 g,
9.8 mmol) and diethyltriflate (1.6 g, 5.3 mmol). The mixture was
stirred at room temperature for 1 h. After removed the solvent, the
residue was partitioned between EtOAc and saturated NaCl. The
organic phase was dried over Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was
chromatographed on silica gel (1% to 5% MeOH/CH.sub.2Cl.sub.2) to
afford the diethylphosphonate 9 as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 8.35 (d, J=9 Hz, 2H), 7.96 (d, J=9 Hz, 2H),
7.13 (d, J=8.4 Hz, 2H), 6.85 (d, J=8.4 Hz, 2H), 5.63 (d, J=5.1 Hz,
1H), 5.18-5.01 (m, 2H), 4.27-4.17 (m, 6H), 3.94-3.67 (m, 7H),
3.20-2.73 (m, 7H), 1.92-1.51 (m, 3H), 1.35 (t, J=7.2 Hz, 6H),
0.88-0.85 (m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 19.2; MS (ESI)
756 (M+Na).
Example 9
[1385] Amine 10 A solution of diethylphosphonate 9 (1 g) was
dissolved in EtOH (100 mL), treated with 10% Pd/C (300 mg) and was
stirred under H.sub.2 atmosphere (balloon) for 3 h. The reaction
was purged with N.sub.2, and the catalyst was removed by filtration
through celite. After evaporation of the filtrate, the residue was
triturated with ether and the solid was collected by filtration to
afford the amine 10 (920 mg, 96%) as a white solid. .sup.1H NMR
(CDCl.sub.3) .sup.1H NMR (CDCl.sub.3) .delta. 7.41 (d, J=8.4 Hz,
2H), 7.17 (d, J=8.4 Hz, 2H), 6.88 (d, J=8.4 Hz, 2H), 6.68 (d, J=8.4
Hz, 2H), 5.67 (d, J=5.1 Hz, 1H), 5.13-5.05 (m, 2H), 4.42 (s, 2H),
4.29-4.20 (m, 6H), 4.00-3.69 (m, 7H), 3.00-2.66 (m, 7H), 1.80-1.69
(m, 3H), 1.38 (m, 6H), 0.94 (d, J=6.4 Hz, 3H), 0.86 (d, J=6.4 Hz,
6H); .sup.31P NMR (CDCl.sub.3) .delta. 19.4; MS (ESI) 736 (M+Na).
TABLE-US-00008 Compound R.sub.1 R.sub.2 16a Gly-Et Gly-Et 16b
Gly-Bu Gly-Bu 16j Phe-Bu Phe-Bu 16k NHEt NHEt
Example 10
[1386] Synthesis of Bisamidates 16a. A solution of phosphonic acid
11 (100 mg, 0.15 mmol) L-alanine ethyl ester hydrochloride (84 mg,
0.6 mmol) was dissolved in pyridine (5 mL) and the solvent was
distilled under reduced pressure at 40-60.degree. C. The residue
was treated with a solution of Ph.sub.3P (118 mg, 0.45 mmol) and
2,2'-dipyridyl disulfide (99 mg, 0.45 mmol) in pyridine (1 mL)
stirring for 20 h at room temperature. The solvent was evaporated
under reduced pressure and the residue was chromatographed on
silica gel (1% to 5% 2-propanol/CH.sub.2Cl.sub.2). The purified
product was suspended in ether and was evaporated under reduced
pressure to afford bisamidate 16a (90 mg, 72%) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H), 7.15 (d,
J=8.7 Hz, 2H), 7.01 (d, J=8.7 Hz, 2H), 6.87 (d, J=8.7 Hz, 2H), 5.68
(d, J=5.1 Hz, 1H), 5.05 (m, 1H), 4.25 (d, J=9.9 Hz, 2H), 4.19 (q,
4H), 3.99-3.65 (m overlapping s, 13H,), 3.41 (m, 1H), 3.20-2.81 (m,
7H), 1.85-1.60 (m, 3H), 1.27 (t, J=7.2 Hz, 6H), 0.93 (d, J=6.3 Hz,
3H), 0.89 (d, J=6.3 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta.
21.8; MS (ESI) 843 (M+H).
Example 11
[1387] Synthesis of Bisamidates 16b. A solution of phosphonic acid
11 (100 mg, 0.15 mmol) L-alanine n-butyl ester hydrochloride (101
mg, 0.6 mmol) was dissolved in pyridine (5 mL) and the solvent was
distilled under reduced pressure at 40-60.degree. C. The residue
was treated with a solution of Ph.sub.3P (118 mg, 0.45 mmol) and
2,2'-dipyridyl disulfide (99 mg, 0.45 mmol) in pyridine (1 mL)
stirring for 20 h at room temperature. The solvent was evaporated
under reduced pressure and the residue was chromatographed on
silica gel (1% to 5% 2-propanol/CH.sub.2Cl.sub.2). The purified
product was suspended in ether and was evaporated under reduced
pressure to afford bisamidate 16b (100 mg, 74%) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=9 Hz, 2H), 7.15 (d, J=9
Hz, 2H), 7.01 (d, J=9 Hz, 2H), 6.87 (d, J=9 Hz, 2H), 5.67 (d, J=5.4
Hz, 1H), 5.05 (m, 1H), 4.96 (m, 1H), 4.25 (d, J=9.9 Hz, 2H), 4.11
(t, J=6.9 Hz, 4H), 3.99-3.71 (m overlapping s, 13H,), 3.41 (m, 1H),
3.20-2.80 (m, 7H), 1.87-1.60 (m, 7H), 1.42 (m, 4H), 0.96-0.88 (m,
12H); .sup.31P NMR (CDCl.sub.3) .delta. 21.8; MS (ESI) 890
(M+H).
Example 12
[1388] Synthesis of Bisamidates 16j. A solution of phosphonic acid
11 (100 mg, 0.15 mmol) L-phenylalanine n-butyl ester hydrochloride
(155 mg, 0.6 mmol) was dissolved in pyridine (5 mL) and the solvent
was distilled under reduced pressure at 40-60.degree. C. The
residue was treated with a solution of Ph.sub.3P (118 mg, 0.45
mmol) and 2,2'-dipyridyl disulfide (99 mg, 0.45 mmol) in pyridine
(1 mL) stirring for 36 h at room temperature. The solvent was
evaporated under reduced pressure and the residue was
chromatographed on silica gel (1% to 5%
2-propanol/CH.sub.2Cl.sub.2). The purified product was suspended in
ether and was evaporated under reduced pressure to afford
bisamidate 16j (106 mg, 66%) as a white solid. .sup.1H NMR
(CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H), 7.31-7.10 (m, 12H),
7.01 (d, J=9 Hz, 2H), 6.72 (d, J=8.7 Hz, 2H), 5.67 (d, J=5.1 Hz,
1H), 5.05 (m, 1H), 4.96 (m, 1H), 4.35-3.98 (m, 7H), 3.90-3.61 (m
overlapping s, 10H,), 3.19-2.78 (m, 11H), 1.87-1.25 (m, 11H),
0.96-0.88 (m, 12H); .sup.31P NMR (CDCl.sub.3) .delta. 19.3; MS
(ESI) 1080 (M+H).
Example 13
[1389] Synthesis of Bisamidates 16k. A solution of phosphonic acid
11 (80 mg, 0.12 mmol), ethylamine (0.3 mL, 2M in TF, 0.6 mmol) was
dissolved in pyridine (5 mL) and the solvent was distilled under
reduced pressure at 40-60.degree. C. The residue was treated with a
solution of Ph.sub.3P (109 mg, 0.42 mmol) and 2,2'-dipyridyl
disulfide (93 mg, 0.42 mmol) in pyridine (1 mL) stirring for 48 h
at room temperature. The solvent was evaporated under reduced
pressure and the residue was chromatographed on silica gel (1% to
5% 2-propanol/CH.sub.2Cl.sub.2). The purified product was suspended
in ether and was evaporated under reduced pressure to afford
bisamidate 16k (60 mg, 70%) as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H), 7.15 (d, J=8.7 Hz,
2H), 7.01 (d, J=8.7 Hz, 2H), 6.87 (d, J=8.7 Hz, 2H), 5.67 (d, J=5.1
Hz, 1H), 5.05-4.95 (m, 2H), 4.15 (d, J=9.6 Hz, 2H), 3.99-3.72 (m
overlapping s, 9H,), 3.18-2.81 (m, 1H), 2.55 (br, 1H), 1.85-1.65
(m,3H), 1.18 (t, J=7.2 Hz, 6H), 0.93 (d, J=6.3 Hz, 3H), 0.89 (d,
J=6.3 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 21.6; MS (ESI) 749
(M+Na). TABLE-US-00009 Compound R.sub.1 R.sub.2 30a OPh Ala-Me 30b
OPh Ala-Et 30c OPh (D)-Ala-iPr 30d OPh Ala-Bu 30e OBn Ala-Et
Example 14
[1390] Monoamidate 30a (R1=OPh, R2=Ala-Me) To a flask was charged
with monophenyl phosphonate 29 (75 mg, 0.1 mmol), L-alanine methyl
ester hydrochloride (4.0 g, 22 mmol) and
1,3-dicyclohexylcarbodiimide (84 mg, 0.6 mmol), then pyridine (1
mL) was added under N2. The resulted mixture was stirred at
60-70.degree. C. for 2 h, then cooled to room temperature and
diluted with ethyl acetate. The mixture was filtered and the
filtrate was evaporated. The residue was partitioned between ethyl
acetate and HCl (0.2 N), the ethyl acetate phase was washed with
water and NaHCO.sub.3, dried over Na.sub.2SO.sub.4 filtered and
concentrated. The residue was purified by chromatography on silica
gel (ethyl acetate/hexane 1:5) to give 30a (25 mg, 30%) as a white
solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H),
7.73-7.24 (m, 5H) 7.19-7.15 (m, 2H), 7.01 (d, J=8.7 Hz, 2H),
6.90-6.83 (m, 2H), 5.65 (d, J=5.1 Hz, 1H), 5.01 (m, 2H), 4.30 (m,
2H), 3.97-3.51 (m overlapping s, 12H), 3.20-2.77 (m, 7H), 1.81 (m,
1H), 1.58 (m, 3H), 0.92 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CDCl.sub.3) .delta. 20.4 and 19.3; MS (ESI) 856
(M+Na).
Example 15
[1391] Monoamidate 30b (R1=OPh, R2=Ala-Et) was synthesized in the
same manner in 35% yield. .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d,
J=8.7 Hz, 2H), 7.73-7.24 (m, 5H) 7.19-7.15 (m, 2H), 7.01 (d, J=8.7
Hz, 2H), 6.90-6.83 (m, 2H), 5.65 (d, J=5.4 Hz, 1H), 5.01 (m, 3H),
4.30-3.67 (m overlapping s, 14H), 3.18-2.77 (m, 7H), 1.81-1.35 (m,
6H), 1.22 (m, 3H), 0.92 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CDCl.sub.3) .delta. 20.4 and 19.3; MS (ESI) 870
(M+Na).
Example 16
[1392] Monoamidate 30c (R1=OPh, R2=(D)-Ala-iPr) was synthesized in
the same manner in 52% yield. Isomer A .sup.1H NMR (CDCl.sub.3)
.delta. 7.72 (d, J=8.7 Hz, 2H), 7.73-7.24 (m, 5H) 7.19-7.15 (m,
2H), 7.01 (d, J=8.7 Hz, 2H), 6.90-6.83 (m, 2H), 5.66 (m, 1H), 5.01
(m, 3H), 4.30-3.67 (m overlapping s, 14H), 3.18-2.77 (m, 7H),
1.81-1.35 (m, 6H), 1.23 (m, 6H), 0.92 (d, J=6.3 Hz, 3H), 0.88 (d,
J=6.3 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 20.4; MS (ESI) 884
(M+Na). Isomer B .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7
Hz, 2H), 7.73-7.24 (m, 5H) 7.19-7.15 (m, 2H), 7.01 (d, J=8.7 Hz,
2H), 6.90-6.83 (m, 2H), 5.66 (m, 1H), 5.01 (m, 3H), 4.30-3.67 (m
overlapping s, 14H), 3.18-2.77 (m, 7H), 1.81-1.35 (m, 6H), 1.23 (m,
6H), 0.92 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H); .sup.31P NMR
(CDCl.sub.3) .delta. 19.3; MS (ESI) 884 (M+Na).
Example 17
[1393] Monoamidate 30d (R1=OPh, R2=Ala-Bu) was synthesized in the
same manner in 25% yield. .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d,
J=8.7 Hz, 2H), 7.73-7.24 (m, 5H) 7.19-7.15 (m, 2H), 7.01 (d, J=8.7
Hz, 2H), 6.90-6.83 (m, 2H), 5.65 (d, J=5.4 Hz, 1H), 5.01 (m, 3H),
4.30-3.67 (m overlapping s, 16H), 3.18-2.77 (m, 7H), 1.81-1.35 (m,
8H), 1.22 (m, 3H), 0.92 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CDCl.sub.3) .delta. 20.4 and 19.4; MS (ESI) 898
(M+Na).
Example 18
[1394] Monoamidate 30e (R1=OBn, R2=Ala-Et) To a flask was charged
with monobenzyl phosphonate 2 (76 mg, 0.1 mmol), L-alanine methyl
ester hydrochloride (4.0 g, 22 mmol) and
1,3-dicyclohexylcarbodiimide (84 mg, 0.6 mmol), then pyridine (1
mL) was added under N2. The resulted mixture was stirred at
60-70.degree. C. for 2 h, then cooled to room temperature and
diluted with ethyl acetate. The mixture was filtered and the
filtrate was evaporated. The residue was partitioned between ethyl
acetate and HCl (0.2 N), the ethyl acetate phase was washed with
water and NaHCO.sub.3, dried over Na.sub.2SO.sub.4 filtered and
concentrated. The residue was purified by chromatography on silica
gel (ethyl acetate/hexane 1:5) to give 30a (25 mg, 30%) as a white
solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H),
7.38-7.34 (m, 5H), 7.13 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz, 2H),
6.86-6.80 (m, 2H), 5.65 (d, J=5.4 Hz, 1H), 5.15-5.01 (m, 5H),
4.30-3.67 (m overlapping s, 14H), 3.18-2.77 (m, 7H), 1.81-1.35 (m,
6H), 1.22 (m, 3H), 0.92 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CDCl.sub.3) .delta. 23.3 and 22.4; MS (ESI) 884
(M+Na). TABLE-US-00010 Compound R.sub.1 R.sub.2 31a OPh Lac-iPr 31b
OPh Lac-Et 31c OPh Lac-Bu 31d OPh (R)-Lac-Me 31e OPh (R)-Lac-Et
Example 19
[1395] Monolactate 31a (R1=OPh, R2=Lac-iPr): To a flask was charged
with monophenyl phosphonate 29 (1.5 g, 2 mmol),
isopropyl-(s)-lactate (0.88 mL, 6.6 mmol) and
1,3-dicyclohexylcarbodiimide (1.36 g, 6.6 mmol), then pyridine (15
mL) was added under N.sub.2. The resulted mixture was stirred at
60-70.degree. C. for 2 h, then cooled to room temperature and
diluted with ethyl acetate. The mixture was filtered and the
filtrate was evaporated. The residue was washed with ethyl acetate
and the combined organic phase was washed with NH.sub.4Cl, brine
and water, dried over Na.sub.2SO.sub.4, filtered and concentrated.
The residue was purified by chromatography on silica gel (ethyl
acetate/CH.sub.2Cl.sub.2 1:5) to give 31a (1.39 g, 81%) as a white
solid. Isomer A .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz,
2H), 7.73-7.19 (m, 5H), 7.15 (d, J=8.4 Hz, 2H), 7.00 (d, J=8.7 Hz,
2H), 6.92 (d, J=8.4 Hz, 2H), 5.65 (d, J=5.4 Hz, 1H), 5.15-5.00 (m,
4H), 4.56-4.44 (m, 2H), 3.96-3.68 (m overlapping s, 9H), 3.13-2.78
(m, 7H), 1.81-1.23 (m, 6H), 1.22 (m, 6H), 0.92 (d, J=6.6 Hz, 3H),
0.88 (d, J=6.6 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.4; MS
(ESI) 885 (M+Na). Isomer B .sup.1H NMR (CDCl.sub.3) .delta. 7.72
(d, J=8.7 Hz, 2H), 7.73-7.19 (m, 5H), 7.14 (d, J=8.4 Hz, 2H), 7.00
(d, J=8.7 Hz, 2H), 6.88 (d, J=8.4 Hz, 2H), 5.64 (d, J=5.4 Hz, 1H),
5.15-5.00 (m, 4H), 4.53-4.41 (m, 2H), 3.96-3.68 (m overlapping s,
9H), 3.13-2.78 (m, 7H), 1.81-1.23 (m, 6H), 1.22 (m, 6H), 0.92 (d,
J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H); .sup.31P NMR (CDCl.sub.3)
.delta. 15.3; MS (ESI) 885 (M+Na).
Example 20
[1396] Monolactate 31b (R1=OPh, R2=Lac-Et) was synthesized in the
same manner in 75% yield. .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d,
J=8.7 Hz, 2H), 7.73-7.14 (m, 7H), 6.99 (d, J=8.7 Hz, 2H), 6.88 (d,
J=8.7 Hz, 2H), 5.63 (m, 1H), 5.19-4.95 (m, 3H), 4.44-4.40 (m, 2H),
4.17-4.12 (m, 2H), 3.95-3.67 (m overlapping s, 9H), 3.15-2.77 (m,
7H), 1.81-1.58 (m, 6H), 1.23 (m, 3H), 0.91 (d, J=6.6 Hz, 3H), 0.87
(d, J=6.6 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.5 and 15.4;
MS (ESI) 872 (M+Na).
Example 21
[1397] Monolactate 31c (R1=OPh, R2=Lac-Bu) was synthesized in the
same manner in 58% yield. Isomer A .sup.1H NMR (CDCl.sub.3) .delta.
7.72 (d, J=8.7 Hz, 2H), 7.73-7.19 (m, 5H), 7.14 (d, J=8.4 Hz, 2H),
7.00 (d, J=8.7 Hz, 2H), 6.90 (d, J=8.4 Hz, 2H), 5.63 (d, J=5.4 Hz,
1H), 5.15-5.00 (m, 3H), 4.56-4.51 (m, 2H), 4.17-4.10 (m, 2H),
3.95-3.67 (m overlapping s, 9H), 3.10-2.77 (m, 7H), 1.81-1.23 (m,
101H), 1.23 (m, 6H), 0.91 (d, J=6.6 Hz, 3H), 0.87 (d, J=6.6 Hz,
3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.3; MS (ESI) 899 (M+Na).
Isomer B .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H),
7.73-7.19 (m, 5H), 7.14 (d, J=8.4 Hz, 2H), 7.00 (d, J=8.7 Hz, 2H),
6.90 (d, J=8.4 Hz, 2H), 5.64 (d, J=5.4 Hz, 1H), 5.15-5.00 (m, 3H),
4.44-4.39 (m, 2H), 4.17-4.10 (m, 2H), 3.95-3.67 (m overlapping s,
9H), 3.10-2.77 (m, 7H), 1.81-1.23 (m, 101H), 1.23 (m, 6H), 0.91 (d,
J=6.6 Hz, 3H), 0.87 (d, J=6.6 Hz, 3H); .sup.31P NMR (CDCl.sub.3)
.delta. 15.3; MS (ESI) 899 (M+Na).
Example 22
[1398] Monolactate 31d (R1=OPh, R2=(R)-Lac-Me): To a stirred
solution of monophenyl phosphonate 29 (100 mg, 0.13 mmol) in 10 ml,
of THF at room temperature under N.sub.2 was added
methyl-(S)-lactate (54 mg, 0.52 mmol) and Ph.sub.3P (136 mg g, 0.52
mmol), followed by DEAD (82 .mu.L, 0.52 mmol). After 2 h, the
solvent was removed under reduced pressure, and the resulting crude
mixture was purified by chromatography on silica gel (ethyl
acetate/hexane 1:1) to give 31d (33 mg, 30%) as a white solid.
.sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H), 7.73-7.14
(m, 7H), 6.99 (d, J=8.7 Hz, 2H), 6.88 (d, J=8.7 Hz, 2H), 5.63 (m,
1H), 5.19-4.95 (m, 3H), 4.44-4.40 (m, 2H), 3.95-3.64 (m overlapping
s, 12H), 3.15-2.77 (m, 7H), 1.81-1.55 (m, 4H), 0.91 (d, J=6.6 Hz,
3H), 0.87 (d, J=6.6 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.4
and 15.3; MS (ESI) 857 (M+Na).
Example 23
[1399] Monolactate 31e (R1=OPh, R2=(R)-Lac-Et): To a stirred
solution of monophenyl phosphonate 29 (50 mg, 0.065 mmol) in 2.5
ml, of THF at room temperature under N.sub.2 was added
ethyl-(s)-lactate (31 mg, 0.52 mmol) and Ph.sub.3P (68 mg g, 0.26
mmol), followed by DEAD (41 .mu.L, 0.52 mmol). After 2 h, the
solvent was removed under reduced pressure, and the resulting crude
mixture was purified by chromatography on silica gel (ethyl
acetate/hexane 1:1) to give 31e (28 mg, 50%) as a white solid.
.sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H), 7.73-7.14
(m, 7H), 6.99 (d, J=8.7 Hz, 2H), 6.85(m, 2H), 5.63 (m, 1H),
5.19-4.95 (m, 3H), 4.44-4.40 (m, 2H), 4.17-4.12 (m, 2H), 3.95-3.67
(m overlapping s, 9H), 3.15-2.77 (m, 7H), 1.81-1.58 (m, 6H), 1.23
(m, 3H), 0.91 (d, J=6.6 Hz, 3H), 0.87 (d, J=6.6 Hz, 3H); .sup.31P
NMR (CDCl.sub.3) .delta. 17.5 and 15.4; MS (ESI) 872 (M+Na).
Example 24
[1400] Monolactate 32 (R1=OBn, R2=(S)-Lac-Bn): To a stirred
solution of monobenzyl phosphonate 2 (76 mg, 0.1 mmol) in 0.5 ml,
of DMF at room temperature under N.sub.2 was added
benzyl-(s)-lactate (27 mg, 0.15 mmol) and PyBOP (78 mg, 0.15 mmol),
followed by DIEA (70 .mu.L, 0.4 mmol). After 3 h, the solvent was
removed under reduced pressure, and the resulting crude mixture was
purified by chromatography on silica gel (ethyl acetate/hexane 1:1)
to give 32 (46 mg, 50%) as a white solid. .sup.1H NMR (CDCl.sub.3)
.delta. 7.72 (d, J=8.7 Hz, 2H), 7.38-7.44 (m, 10H), 7.13 (d, J=8.4
Hz, 2H), 6.99 (d, J=8.7 Hz, 2H), 6.81(m, 2H), 5.63 (d, J=5.1 Hz,
1H), 5.23-4.92 (m, 7H), 4.44-22 (m, 2H), 3.96-3.67 (m overlapping
s, 9H), 3.15-2.77 (m, 7H), 1.81-1.58 (m, 6H), 0.93 (d, J=6.3 Hz,
3H), 0.88 (d, J=6.3 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 20.8
and 19.6; MS (ESI) 947 (M+Na).
Example 25
[1401] Monolactate 33 (R1=OBn, R2=(R)-Lac-Bn): To a stirred
solution of monobenzyl phosphonate 2 (76 mg, 0.1 mmol) in 5 ml, of
THF at room temperature under N.sub.2 was added benzyl-(s)-lactate
(72 mg, 0.4 mmol) and Ph.sub.3P (105 mg g, 0.4 mmol), followed by
DEAD (60 .mu.L, 0.4 mmol). After 20 h, the solvent was removed
under reduced pressure, and the resulting crude mixture was
purified by chromatography on silica gel (ethyl acetate/hexane 1:1)
to give 33 (44 mg, 45%) as a white solid. .sup.1H NMR (CDCl.sub.3)
.delta. 7.72 (d, J=8.7 Hz, 2H), 7.38-7.44 (m, 101H), 7.13 (m, 2H),
6.99 (d, J=8.7 Hz, 2H), 6.81(m, 2H), 5.63 (m, 1H), 5.23-4.92 (m,
7H), 4.44-22 (m, 2H), 3.96-3.67 (m overlapping s, 9H), 3.15-2.77
(m, 7H), 1.81-1.58 (m, 6H), 0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3
Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 20.8 and 19.6; MS (EST)
947 (M+Na).
Example 26
[1402] Monophosphonic acid 34: A solution of monobenzyllactate 32
(20 mg) was dissolved in EtOH/EtOAc (3 mL/1 mL), treated with 10%
Pd/C (4 mg) and was stirred under H2 atmosphere (balloon) for 1.5
h. The catalyst was removed by filtration through celite. The
filtered was evaporated under reduced pressure, the residue was
triturated with ether and the solid was collected by filtration to
afford the monophosphonic acid 33 (15 mg, 94%) as a white solid.
.sup.1H NMR (CD.sub.3OD) .delta. 7.76 (d, J=8.7 Hz, 2H), 7.18 (d,
J=8.7 Hz, 2H), 7.08 (d, J=8.7 Hz, 2H), 6.90 (d, J=8.7 Hz, 2H), 5.69
(d, J=5.7 Hz, 1H), 5.03-4.95 (m, 2H), 4.20 (m, 2H), 3.90-3.65 (m
overlapping s, 9H), 3.41 (m, 2H), 3.18-2.78 (m, 5H), 2.44 (m, 1H),
2.00 (m, 1H), 1.61-1.38 (m, 5H), 0.93 (d, J=6.3 Hz, 3H), 0.88 (d,
J=6.3 Hz, 3H); .sup.31P NMR (CD.sub.3OD) .delta. 18.0; MS (ESI) 767
(M+Na).
Example 27
[1403] Monophosphonic acid 35: A solution of monobenzyllactate 33
(20 mg) was dissolved in EtOH (3 mL), treated with 10% Pd/C (4 mg)
and was stirred under H2 atmosphere (balloon) for 1 h. The catalyst
was removed by filtration through celite. The filtered was
evaporated under reduced pressure, the residue was triturated with
ether and the solid was collected by filtration to afford the
monophosphonic acid 35 (15 mg, 94%) as a white solid. .sup.1H NMR
(CD.sub.3OD) .delta. 7.76 (d, J=8.7 Hz, 2H), 7.18 (d, J=8.7 Hz,
2H), 7.08 (d, J=8.7 Hz, 2H), 6.90 (d, J=8.7 Hz, 2H), 5.69 (d, J=5.7
Hz, 1H), 5.03-4.95 (m, 2H), 4.20 (m, 2H), 3.90-3.65 (m overlapping
s, 9H), 3.41 (m, 2H), 3.18-2.78 (m, 5H), 2.44 (m, 1H), 2.00 (m,
1H), 1.61-1.38 (m, 5H), 0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz,
3H); .sup.31P NMR (CD.sub.3OD) .delta. 18.0; MS (ESI) 767
(M+Na).
Example 28
[1404] Synthesis of Bislactate 36: A solution of phosphonic acid 11
(100 mg, 0.15 mmol) isopropyl-(S)-lactate (79 mg, 0.66 mmol) was
dissolved in pyridine (1 mL) and the solvent was distilled under
reduced pressure at 40-60.degree. C. The residue was treated with a
solution of Ph.sub.3P (137 mg, 0.53 mmol) and 2,2'-dipyridyl
disulfide (116 mg, 0.53 mmol) in pyridine (1 mL) stirring for 20 h
at room temperature. The solvent was evaporated under reduced
pressure and the residue was chromatographed on silica gel (1% to
5% 2-propanol/CH.sub.2Cl.sub.2). The purified product was suspended
in ether and was evaporated under reduced pressure to afford
bislactate 36 (42 mg, 32%) as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz, 2H), 7.14 (d, J=8.7 Hz,
2H), 7.01 (d, J=8.7 Hz, 2H), 6.89 (d, J=8.7 Hz, 2H), 5.66 (d, J=5.1
Hz, 1H), 5.05 (m, 3H), 4.25 (d, J=9.9 Hz, 2H), 4.19 (q, 4H),
3.99-3.65 (m overlapping s, 9H,), 3.41 (m, 1H), 3.20-2.81 (m, 7H),
1.85-1.60 (m, 3H), 1.58 (m, 6H), 1.26 (m, 12H), 0.93 (d, J=6.3 Hz,
3H), 0.89 (d, J=6.3 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta.
21.1; MS (ESI) 923 (M+Na).
Example 29
[1405] Triflate derivative 1: A THF--CH.sub.2Cl.sub.2 solution (30
mL-10 mL) of 8 (4 g, 6.9 mmol), cesium carbonate (2.7 g, 8 mmol),
and N-phenyltrifluoromethane sulfonimide (2.8 g, 8 mmol) was
reacted overnight. The reaction mixture was worked up, and
concentrated to dryness to give crude triflate derivative 1.
[1406] Aldehyde 2: Crude triflate 1 (4.5 g, 6.9 mmole) was
dissolved in DMF (20 mL), and the solution was degassed (high
vacuum for 2 min, Ar purge, repeat 3 times). Pd(OAc).sub.2 (0.12 g,
0.27 mmol), and bis(diphenylphosphino)propane (dppp, 0.22 g, 0.27
mmol) were added and the solution was heated to 70.degree. C.
Carbon monoxide was rapidly bubbled through the solution, then
under 1 atmosphere of carbon monoxide. To this solution were slowly
added TEA (5.4 mL, 38 mmol), and triethylsilane (3 mL, 18 mmol).
The resulting solution was stirred overnight at room temperature.
The reaction mixture was worked up, and purified on silica gel
column chromatograph to afford aldehyde 2 (2.1 g, 51%). (Hostetler,
et al. J. Org. Chem., 1999. 64, 178-185).
[1407] Lactate prodrug 4: Compound 4 is prepared as described above
procedure for 3a-e by the reductive amination between 2 and 3 with
NaBH.sub.3CN in 1,2-dichloroethane in the presence of HOAc.
##STR466##
Example 30
[1408] Preparation of compound 3 Diethyl (cyano(dimethyl)methyl)
phosphonate 5: A THF solution (30 mL) of NaH (3.4 g of 60% oil
dispersion, 85 mmole) was cooled to -10.degree. C., followed by the
addition of diethyl (cyanomethyl)phosphonate (5 g, 28.2 mmol) and
iodomethane (17 g, 112 mmol). The resulting solution was stirred at
-10.degree. C. for 2 hr. then 0.degree. C. for 1 hr, was worked up,
and purified to give dimethyl derivative 5 (5 g, 86%). Diethyl
(2-amino-1,1-dimethyl-ethyl)phosphonate 6: Compound 5 was reduced
to amine derivative 6 by the described procedure (J. Med. Chem.
1999, 42, 5010-5019). A ethanol (150 mL) and 1N HCl aqueous
solution (22 mL) of 5 (2.2 g, 10.7 mmol) was hydrogenated at 1
atmosphere in the presence of PtO.sub.2 (1.25 g) at room
temperature overnight. The catalyst was filtered through a celite
pad. The filtrate was concentrated to dryness, to give crude 6 (2.5
g, as HCl salt).
[1409] 2-Amino-1,1-dimethyl-ethyl phosphonic acid 7: A CH.sub.3CN
(30 mL) of crude 6 (2.5 g) was cooled to 0.degree. C., and treated
with TMSBr (8 g, 52 mmol) for 5 hr. The reaction mixture was
stirred with methanol for 1.5 hr at room temperature, concentrated,
recharged with methanol, concentrated to dryness to give crude 7
which was used for next reaction without further purification.
[1410] Lactate phenyl (2-amino-1,1-dimethyl-ethyl)phosphonate 3:
Compound 3 is synthesized according to the procedures described in
a previous scheme for the preparation of a lactate phenyl
2-aminoethyl phosphonate. Compound 7 is protected with CBZ,
followed by the reaction with thionyl chloride at 70.degree. C. The
CBZ protected dichlorodate is reacted phenol in the presence of
DIPEA. Removal of one phenol, follow by coupling with ethyl
L-lactate leads N--CBZ-2-amino-1,1-dimethyl-ethyl phosphonated
derivative. Hydrogenation of N--CBZ derivative at 1 atmosphere in
the presence of 10% Pd/C and 1 equivalent of TFA affords compound 3
as TFA salt. ##STR467##
Example Section M
[1411] ##STR468## ##STR469## ##STR470## ##STR471## ##STR472##
Example 1
[1412] Cbz Amide 1: To a suspension of epoxide (34 g, 92.03 mmol)
in 2-propanol (300 mL) was added isobutylamine (91.5 mL, 920 mmol)
and the solution was refluxed for 1 h. The solution was evaporated
under reduced pressure and the crude solid was dried under vacuum
to give the amine (38.7 g, 95%) which was dissolved in
CH.sub.2Cl.sub.2 (300 mL) and cooled to 0.degree. C. Triethylamine
(18.3 mL, 131 mmol) was added followed by the addition of benzyl
chloroformate (13.7 mL, 96.14 mmol) and the solution was stirred
for 30 min at 0.degree. C., warmed to room temperature overnight,
and evaporated under reduced pressure. The residue was partitioned
between EtOAc and 0.5 M H.sub.3PO.sub.4. The organic phase was
washed with saturated NaHCO.sub.3, brine, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (1/2-EtOAc/hexane) to give the Cbz amide (45.37 g, 90%) as a
white solid.
Example 2
[1413] Amine 2: A solution of Cbz amide 1 (45.37 g, 78.67 mmol) in
CH.sub.2Cl.sub.2 (160 mL) at 0.degree. C. was treated with
trifluoroacetic acid (80 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. Volatiles were evaporated under reduced pressure
and the residue was partitioned between EtOAc and 0.5 N NaOH. The
organic phase was washed with 0.5 N NaOH (2.times.), water
(2.times.), saturated NaCl, dried with Na.sub.2SO.sub.4, filtered,
and evaporated under reduced pressure to give the amine (35.62 g,
95%) as a white solid.
Example 3
[1414] Carbamate 3: To a solution of amine 2 (20.99 g, 44.03 mmol)
in CH.sub.3CN (250 mL) at 0.degree. C. was treated with
(3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl 4-nitrophenyl carbonate
(13.00 g, 44.03 mmol, prepared according to Ghosh et al. J. Med.
Chem. 1996, 39, 3278.), N,N-diisopropylethylamine (15.50 mL, 88.06
mmol) and 4-dimethylaminopyridine (1.08 g, 8.81 mmol). The reaction
mixture was stirred at 0.degree. C. for 30 min and then warmed to
room temperature overnight. The reaction solvent was evaporated
under reduced pressure and the residue was partitioned between
EtOAc and 0.5 N NaOH. The organic phase was washed with 0.5 N NaOH
(2.times.), 5% citric acid (2.times.), saturated NaHCO.sub.3, dried
with Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was purified by column chromatography
on silica gel (3% 2-propanol/CH.sub.2Cl.sub.2) to give the
carbamate (23.00 g, 83%) as a white solid.
Example 4
[1415] Amine 4: To a solution of 3 (23.00 g, 36.35 mmol) in EtOH
(200 mL) and EtOAc (50 mL) was added 20% Pd(OH).sub.2/C (2.30 g).
The suspension was stirred under H.sub.2 atmosphere (balloon) at
room temperature for 3 h. The reaction mixture was filtered through
a plug of celite. The filtrate was concentrated and dried under
vacuum to give the amine (14.00 g, 94%) as a white solid.
Example 5
[1416] Phenol 5: To a solution of amine 4 (14.00 g, 34.27 mmol) in
H.sub.2O (80 mL) and 1,4-dioxane (80 mL) at 0.degree. C. was added
Na.sub.2CO.sub.3 (5.09 g, 47.98 mmol) and di-tert-butyl dicarbonate
(8.98 g, 41.13 mmol). The reaction mixture was stirred at 0.degree.
C. for 2 h and then warmed to room temperature for 30 min. The
residue was partitioned between EtOAc and H.sub.2O. The organic
layer was dried with Na.sub.2SO.sub.4, filtered, and concentrated.
The crude product was purified by column chromatography on silica
gel (3% MeOH/CH.sub.2Cl.sub.2) to give the phenol (15.69 g, 90%) as
a white solid.
Example 6
[1417] Dibenzylphosphonate 6: To a solution of phenol 5 (15.68 g,
30.83 mmol) in CH.sub.3CN (200 mL) was added Cs.sub.2CO.sub.3
(15.07 g, 46.24 mmol) and triflate (17.00 g, 40.08 mmol). The
reaction mixture was stirred at room temperature for 1 h, the salt
was filtered off, and the solvent was evaporated under reduced
pressure. The residue was partitioned between EtOAc and saturated
NaCl. The organic phase was dried with Na.sub.2SO.sub.4, filtered,
and evaporated under reduced pressure. The crude product was
purified by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the dibenzylphosphonate (15.37
g, 73%) as a white solid.
Example 7
[1418] Sulfonamide 7: A solution of dibenzylphosphonate 6 (0.21 g,
0.26 mmol) in CH.sub.2Cl.sub.2 (0.5 mL) at 0.degree. C. was treated
with trifluoroacetic acid (0.25 mL). The solution was stirred for
30 min at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (3 mL) and cooled to 0.degree. C.
Triethylamine (0.15 mL, 1.04 mmol) was added followed by the
treatment of benzenesulfonyl chloride (47 mg, 0.26 mmol). The
solution was stirred for 1 h at 0.degree. C. and the product was
partitioned between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3. The
organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (3% 2-propanol/CH.sub.2Cl.sub.2) to give the sulfonamide 7
(0.12 g, 55%, GS 191477) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 7.79 (dd, 2H), 7.61-7.56 (m, 3H), 7.38-7.36 (m, 10H), 7.13
(d, J=8.4 Hz, 2H), 6.81 (d, J=8.4 Hz, 2H), 5.65 (d, J=5.4 Hz, 1H),
5.18 (m, 4H), 5.05 (m, 1H), 4.93 (d, J=8.7 Hz, 1H), 4.20 (d, J=10.2
Hz, 2H), 4.0-3.67 (m, 7H), 3.15-2.8 (m, 7H), 1.84 (m, 1H),
1.65-1.59 (m, 2H), 0.93 (d, J=6.6 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CDCl.sub.3) .delta. 20.36.
Example 8
[1419] Phosphonic Acid 8: To a solution of 7 (70 mg, 0.09 mmol) in
MeOH (4 mL) was added 10% Pd/C (20 mg). The suspension was stirred
under H.sub.2 atmosphere (balloon) at room temperature overnight.
The reaction mixture was filtered through a plug of celite. The
filtrate was concentrated and dried under vacuum to give the
phosphonic acid (49 mg, 90% GS 191478) as a white solid: .sup.1H
NMR (CD.sub.3OD) .delta. 7.83 (dd, 2H), 7.65-7.56 (m, 3H), 7.18 (d,
J=8.4 Hz, 2H), 6.91 (d, J=7.8 Hz, 2H), 5.59 (d, J=5.4 Hz, 1H), 4.96
(m, 1H), 4.15 (d, J=9.9 Hz, 2H), 3.95-3.68 (m, 6H), 3.44 (dd, 2H),
3.16 (m, 2H), 2.99-2.84 (m, 4H), 2.48 (m, 1H), 2.02 (m, 1H), 1.6
(m, 1H), 1.37 (m, 1H), 0.93 (d, J=6.3 Hz, 3H), 0.87 (d, J=6.3 Hz,
3H); .sup.31P NMR (CD.sub.3OD) .delta. 17.45.
Example 9
[1420] Sulfonamide 9: A solution of dibenzylphosphonate 6 (0.24 g,
0.31 mmol) in CH.sub.2Cl.sub.2 (0.5 mL) at 0.degree. C. was treated
with trifluoroacetic acid (0.25 mL). The solution was stirred for
30 min at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (3 mL) and cooled to 0.degree. C.
Triethylamine (0.17 mL, 1.20 mmol) was added followed by the
treatment of 4-cyanobenzenesulfonyl chloride (61.4 mg, 0.30 mmol).
The solution was stirred for 1 h at 0.degree. C. and the product
was partitioned between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3.
The organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (3% 2-propanol/CH.sub.2Cl.sub.2) to give the sulfonamide 9
(0.20 g, 77%, GS 191717) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 7.90 (d, J=8.4 Hz, 2H), 7.83 (d, J=7.8 Hz, 2H), 7.36 (m,
10H), 7.11 (d, J=8.4 Hz, 2H), 6.82 (d, J=8.7 Hz, 2H), 5.65 (d,
J=5.4 Hz, 1H), 5.2-4.9 (m, 5H), 4.8 (d, 1H), 4.2 (d, J=9.9 Hz, 2H),
3.99 (m 1H), 3.94 (m, 3H), 3.7 (m, 2H), 3.48 (broad, s, 1H),
3.18-2.78 (m, 7H), 1.87 (m, 1H), 1.66-1.47 (m, 2H), 0.91 (d, J=6.3
Hz, 3H), 0.87 (d, J=6.3 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta.
20.3.
Example 10
[1421] Sulfonamide 10: A solution of dibenzylphosphonate 6 (0.23 g,
0.29 mmol) in CH.sub.2Cl.sub.2 (0.5 mL) at 0.degree. C. was treated
with trifluoroacetic acid (0.25 mL). The solution was stirred for
30 min at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (3 mL) and cooled to 0.degree. C.
Triethylamine (0.16 mL, 1.17 mmol) was added followed by the
treatment of 4-trifluoromethyl benzenesulfonyl chloride (72 mg,
0.29 mmol). The solution was stirred for 1 h at 0.degree. C. and
the product was partitioned between CH.sub.2Cl.sub.2 and saturated
NaHCO.sub.3. The organic phase was washed with saturated NaCl,
dried with Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was purified by column chromatography
on silica gel (3% 2-propanol/CH.sub.2Cl.sub.2) to give the
sulfonamide (0.13 g, 50%, GS 191479) as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 7.92 (d, J=8.1 Hz, 2H), 7.81 (d, J=8.1 Hz,
2H), 7.36 (m, 10H), 7.12 (d, J=8.4 Hz, 2H), 6.81 (d, J=8.4 Hz, 2H),
5.65 (d, J=5.1 Hz, 1H), 5.20-4.89 (m, 6H), 4.20 (d, J=9.9 Hz, 2H),
3.95 (m, 1H), 3.86 (m, 3H), 3.71 (m, 2H), 3.19-2.78 (m, 7H), 1.86
(m, 1H), 1.65 (m, 2H), 0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz,
3H); .sup.31P NMR (CDCl.sub.3) .delta. 20.3.
Example 11
[1422] Phosphonic Acid 11: To a solution of 10 (70 mg, 0.079 mmol)
in MeOH (4 mL) was added 10% Pd/C (20 mg). The suspension was
stirred under H.sub.2 atmosphere (balloon) at room temperature
overnight. The reaction mixture was filtered through a plug of
celite. The filtrate was concentrated and dried under vacuum to
give the phosphonic acid (50 mg, 90%, GS 191480) as a white solid:
.sup.1H NMR (CD.sub.3OD) .delta. 8.03 (dd, 2H), 7.90 (dd, 2H), 7.17
(d, J=8.1 Hz, 2H), 6.91 (d, J=7.8 Hz, 2H), 5.59 (d, J=5.7 Hz, 1H),
4.94 (m, 1H), 4.15 (d, J=10.2 Hz, 2H), 3.94-3.72 (m, 6H), 3.48 (m,
1H), 3.2-3.1 (m, 3H), 3.0-2.9 (m, 2H), 2.47 (m, 1H), 2.06 (m, 1H),
1.56 (m, 1H), 1.37 (m, 1H), 0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3
Hz, 3H); .sup.31P NMR (CD.sub.3OD) .delta. 17.5.
Example 12
[1423] Sulfonamide 12: A solution of dibenzylphosphonate 6 (0.23 g,
0.29 mmol) in CH.sub.2Cl.sub.2 (0.5 mL) at 0.degree. C. was treated
with trifluoroacetic acid (0.25 mL). The solution was stirred for
30 min at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (3 mL) and cooled to 0.degree. C.
Triethylamine (0.16 mL, 1.17 mmol) was added followed by the
treatment of 4-fluorobenzenesulfonyl chloride (57 mg, 0.29 mmol).
The solution was stirred for 1 h at 0.degree. C. and the product
was partitioned between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3.
The organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (3% 2-propanol/CH.sub.2Cl.sub.2) to give the sulfonamide (0.13
g, 55%, GS 191482) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 7.81 (m, 2H), 7.38 (m, 10H), 7.24 (m, 2H), 7.12 (d, J=8.1
Hz, 2H), 6.82 (d, J=8.4 Hz, 2H), 5.65 (d, J=5.4 Hz, 1H), 5.17 (m,
4H), 5.0 (m, 1H), 4.90 (d, 1H), 4.20 (d, J=9.9 Hz, 2H), 3.97 (m,
1H), 3.86 (m, 3H), 3.73 (m, 2H), 3.6 (broad, s, 1H), 3.13 (m, 1H),
3.03-2.79 (m, 6H), 1.86 (m, 1H), 1.66-1.58 (m, 2H), 0.92 (d, J=6.6
Hz, 3H), 0.88 (d, J=6.6 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta.
20.3.
Example 13
[1424] Phosphonic Acid 13: To a solution of 12 (70 mg, 0.083 mmol)
in MeOH (4 mL) was added 10% Pd/C (20 mg). The suspension was
stiffed under H.sub.2 atmosphere (balloon) at room temperature
overnight. The reaction mixture was filtered through a plug of
celite. The filtrate was concentrated and dried under vacuum to
give the phosphonic acid (49 mg, 90%, GS 191483) as a white solid:
.sup.1H NMR (CD.sub.3OD) .delta. 7.89 (m, 2H), 7.32 (m, 2H), 7.18
(d, J=8.4 Hz, 2H), 6.9 (d, J=8.1 Hz, 2H), 5.59 (d, J=5.1 Hz, 1H),
4.94 (m, 1H), 4.16 (d, J=9.9 Hz, 2H), 3.94 (m, 1H), 3.85-3.7 (m,
5H), 3.43 (dd, 1H), 3.15-2.87 (m, 5H), 2.48 (m, 1H), 2.03 (m, 1H),
1.59-1.36 (m, 2H), 0.93 (d, J=6.3 Hz, 3H), 0.87 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CD.sub.3OD) .delta. 17.5.
Example 14
[1425] Sulfonamide 14: A solution of dibenzylphosphonate 6 (0.21 g,
0.26 mmol) in CH.sub.2Cl.sub.2 (0.5 mL) at 0.degree. C. was treated
with trifluoroacetic acid (0.25 mL). The solution was stirred for
30 min at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (3 mL) and cooled to 0.degree. C.
Triethylamine (0.15 mL, 1.04 mmol) was added followed by the
treatment of 4-trifluoromethoxybenzenesulfonyl chloride (69 mg,
0.26 mmol). The solution was stirred for 1 h at 0.degree. C. and
the product was partitioned between CH.sub.2Cl.sub.2 and saturated
NaHCO.sub.3. The organic phase was washed with saturated NaCl,
dried with Na.sub.2SO.sub.4, filtered, and evaporated under reduced
pressure. The crude product was purified by column chromatography
on silica gel (3% 2-propanol/CH.sub.2Cl.sub.2) to give the
sulfonamide (0.17 g, 70%, GS 191508) as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 7.84 (d, J=9 Hz, 2H), 7.36 (m, 12H), 7.12 (d,
J=8.7 Hz, 2H), 6.81 (d, J=8.7 Hz, 2H), 5.65 (d, J=5.4 Hz, 1H), 5.16
(m, 4H), 5.03 (m, 1H), 4.89 (d, 1H), 4.2 (d, J=9.9 Hz, 2H), 3.97
(m, 1H), 3.85 (m, 3H), 3.7 (m, 2H), 3.59 (broad, s, 1H), 3.18 (m,
1H), 3.1-3.0 (m, 3H), 2.96-2.78 (m, 3H), 1.86 (m, 1H), 1.66-1.5 (m,
2H), 0.93 (d, J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H); .sup.1P NMR
(CDCl.sub.3) .delta. 20.3.
Example 15
[1426] Phosphonic Acid 15: To a solution of 14 (70 mg, 0.083 mmol)
in MeOH (4 mL) was added 10% Pd/C (20 mg). The suspension was
stirred under H.sub.2 atmosphere (balloon) at room temperature
overnight. The reaction mixture was filtered through a plug of
celite. The filtrate was concentrated and dried under vacuum to
give the phosphonic acid (50 mg, 90%, GS 192041) as a white solid:
.sup.1H NMR (CD.sub.3OD) .delta. 7.95 (dd, 2H), 7.49 (dd, 2H), 7.17
(dd, 2H), 6.92 (dd, 2H), 5.58 (d, J=5.4 Hz, 1H), 4.89 (m, 1H), 4.17
(d, J=9 Hz, 2H), 3.9 (m, 1H), 3.82-3.7 (m, 5H), 3.44 (m, 1H),
3.19-2.9 (m, 5H), 2.48 (m, 1H), 2.0 (m, 1H), 1.6 (m, 1H), 1.35 (m,
1H), 0.93 (d, J=6.0 Hz, 3H), 0.88 (d, J=6.0 Hz, 3H); .sup.31P NMR
(CD.sub.3OD) .delta. 17.4.
Example 16
[1427] Sulfonamide 16: A solution of dibenzylphosphonate 6 (0.59 g,
0.76 mmol) in CH.sub.2Cl.sub.2 (2.0 mL) at 0.degree. C. was treated
with trifluoroacetic acid (1.0 mL). The solution was stirred for 30
min at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (3 mL) and cooled to 0.degree. C.
Triethylamine (0.53 mL, 3.80 mmol) was added followed by the
treatment of hydrogen chloride salt of 3-pyridinylsulfonyl chloride
(0.17 g, 0.80 mmol, prepared according to Karaman, R. et al. J. Am.
Chem. Soc. 1992, 114, 4889). The solution was stirred for 30 min at
0.degree. C. and warmed to room temperature for 30 min. The product
was partitioned between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3.
The organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (4% 2-propanol/CH.sub.2Cl.sub.2) to give the sulfonamide (0.50
g, 80%, GS 273805) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 9.0 (d, J=1.5 Hz, 1H), 8.8 (dd, 1H), 8.05 (d, J=8.7 Hz,
1H), 7.48 (m, 1H), 7.36 (m, 10H), 7.12 (d, J=8.4 Hz, 2H), 6.82 (d,
J=9.0 Hz, 2H), 5.65 (d, J=5.1 Hz, 1H), 5.18 (m, 4H), 5.06 (m, 1H),
4.93 (d, 1H), 4.21 (d, J=8.4 Hz, 2H), 3.97 (m, 1H), 3.86 (m, 3H),
3.74 (m, 2H), 3.2 (m, 1H), 3.1-2.83 (m, 5H), 2.76 (m, 1H), 1.88 (m,
1H), 1.62 (m, 2H), 0.92 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CDCl.sub.3) .delta. 20.3.
Example 17
[1428] Phosphonic Acid 17: To a solution of 16 (40 mg, 0.049 mmol)
in MeOH (3 mL) and AcOH (1 mL) was added 10% Pd/C (10 mg). The
suspension was stirred under H.sub.2 atmosphere (balloon) at room
temperature overnight. The reaction mixture was filtered through a
plug of celite. The filtrate was concentrated and dried under
vacuum to give the phosphonic acid (28 mg, 90%, GS 273845) as a
white solid: .sup.1H NMR (CD.sub.3OD) .delta. 8.98 (s, 1H), 8.77
(broad, s, 1H), 8.25 (dd, 1H), 7.6 (m, 1H), 7.15 (m, 2H), 6.90 (m,
2H), 5.6 (d, J=5.4 Hz, 1H), 4.98 (m, 1H), 4.15 (d, 2H), 3.97-3.7
(m, 6H), 3.45-2.89 (m, 6H), 2.50 (m, 1H), 2.0 (m, 1H), 1.6-1.35 (m,
2H), 0.9 (m, 6H).
Example 18
[1429] Sulfonamide 18: A solution of dibenzylphosphonate 6 (0.15 g,
0.19 mmol) in CH.sub.2Cl.sub.2 (0.60 mL) at 0.degree. C. was
treated with trifluoroacetic acid (0.30 mL). The solution was
stirred for 30 min at 0.degree. C. and then warmed to room
temperature for an additional 30 min. The reaction mixture was
diluted with toluene and concentrated under reduced pressure. The
residue was co-evaporated with toluene (2.times.), chloroform
(2.times.), and dried under vacuum to give the ammonium triflate
salt which was dissolved in CH.sub.2Cl.sub.2 (2 mL) and cooled to
0.degree. C. Triethylamine (0.11 mL, 0.76 mmol) was added followed
by the treatment of 4-formylbenzenesulfonyl chloride (43 mg, 0.21
mmol). The solution was stirred for 30 min at 0.degree..degree. C.
and warmed to room temperature for 30 min. The product was
partitioned between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3. The
organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (3% 2-propanol/CH.sub.2Cl.sub.2) to give the sulfonamide (0.13
g, 80%, GS 278114) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 10.1 (s, 1H), 8.04 (d, J=8.1 Hz, 2H), 7.94 (d, J=8.1 Hz,
2H), 7.35 (m, 10H), 7.13 (m, J=8.1 Hz, 2H), 6.82 (d, J=8.1 Hz, 2H),
5.65 (d, J=5.4 Hz, 1H), 5.17 (m, 4H), 5.06 (m, 1H), 4.93 (m, 1H),
4.2 (d, J=9.9 Hz, 2H), 3.94 (m, 1H), 3.85 (m, 3H), 3.7 (m, 2H),
3.18-2.87 (m, 5H), 2.78 (m, 1H), 1.86 (m, 1H), 1.67-1.58 (m, 2H),
0.93 (d, J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H); .sup.31P NMR
(CDCl.sub.3) .delta. 20.3.
Example 19
[1430] Phosphonic Acid 19: To a solution of 18 (0.12 g, 0.15 mmol)
in EtOAc (4 mL) was added 10% Pd/C (20 mg). The suspension was
stirred under H.sub.2 atmosphere (balloon) at room temperature for
6 h. The reaction mixture was filtered through a plug of celite.
The filtrate was concentrated and dried under vacuum to give the
phosphonic acid (93 mg, 95%) as a white solid.
Example 20
[1431] Phosphonic Acids 20 and 21: Compound 19 (93 mg, 0.14 mmol)
was dissolved in CH.sub.3CN (2 mL).
N,O-Bis(trimethylsilyl)acetamide (BSA, 0.28 g, 1.4 mmol) was added.
The reaction mixture was heated to reflux for 1 h, cooled to room
temperature and concentrated. The residue was co-evaporated with
toluene and chloroform and dried under vacuum to give a semi-solid
which was dissolved in EtOAc (2 mL). Morpholine (60 .mu.L, 0.9
mmol), AcOH (32 .mu.L, 0.56 mmol), and NaBH.sub.3CN (17 mg, 0.28
mmol) were added and the reaction mixture was stirred at room
temperature overnight. The reaction was quenched with H.sub.2O,
stirred for 2 h, filtered, and concentrated. The crude product was
purified by HPLC to give the phosphonic acid 20 (10 mg, GS 278118)
as a white solid: .sup.1H NMR (CD.sub.3OD) .delta. 7.80 (d, J=7.8
Hz, 2H), 7.56 (d, J=7.5 Hz, 2H), 7.17 (d, J=7.8 Hz, 2), 6.91 (d,
J=7.5 Hz, 2H), 5.59 (d, J=5.1 Hz, 1H), 5.06 (m, 1H), 4.7 (s, 2H),
4.15 (d, J=10.2 Hz, 2H), 3.92 (m, 1H), 3.82-3.7 (m, 5H), 3.43 (dd,
1H), 3.11-2.89 (m, 6H), 2.50 (m, 1H), 2.0 (m, 1H), 1.6-1.35 (m,
2H), 0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H); .sup.31P NMR
(CD.sub.3OD) .delta. 17.3. Phosphonic acid 21 (15 mg, GS 278117) as
a white solid: .sup.1H NMR (CD.sub.3OD) .delta. 7.8-7.7 (m, 4H),
7.20 (d, J=8.4 Hz, 2H), 6.95 (d, J=8.4 Hz, 2H), 5.62 (d, J=5.1 Hz,
1H), 5.00 (m, 1H), 4.42 (s, 2H), 4.20 (dd, 2H), 3.98-3.68 (m, 9H),
3.3-2.92 (m, 11H), 2.6 (m, 1H), 2.0 (m, 1H), 1.6 (m, 2H), 0.92 (d,
J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H); .sup.31P NMR (CD.sub.3OD)
.delta. 16.2. ##STR473## ##STR474## ##STR475## ##STR476##
##STR477## ##STR478##
Example 21
[1432] Phosphonic Acid 22: To a solution of dibenzylphosphonate 6
(5.00 g, 6.39 mmol) in EtOH (100 mL) was added 10% Pd/C (1.4 g).
The suspension was stirred under H.sub.2 atmosphere (balloon) at
room temperature overnight. The reaction mixture was filtered
through a plug of celite. The filtrate was concentrated and dried
under vacuum to give the phosphonic acid (3.66 g, 95%) as a white
solid.
Example 22
[1433] Diphenylphosphonate 23: A solution of 22 (3.65 g, 6.06 mmol)
and phenol (5.70 g, 60.6 mmol) in pyridine (30 mL) was heated to
70.degree. C. and 1,3-dicyclohexylcarbodiimide (5.00 g, 24.24 mmol)
was added. The reaction mixture was stirred at 70.degree. C. for 2
h and cooled to room temperature. EtOAc was added and the side
product 1,3-dicyclohexyl urea was filtered off. The filtrate was
concentrated and dissolved in CH.sub.3CN (20 mL) at 0.degree. C.
The mixture was treated with DOWEX 50 W.times.8-400 ion-exchange
resin and stirred for 30 min at 0.degree. C. The resin was filtered
off and the filtrate was concentrated. The crude product was
purified by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the diphenylphosphonate (2.74
g, 60%) as a white solid.
Example 23
[1434] Monophosphonic Acid 24: To a solution of 23 (2.74 g, 3.63
mmol) in CH.sub.3CN (40 mL) at 0.degree. C. was added 1 N NaOH
(9.07 mL, 9.07 mmol). The reaction mixture was stirred at 0.degree.
C. for 1 h. DOWEX 50 W.times.8400 ion-exchange resin was added and
the reaction mixture was stirred for 30 min at 0.degree. C. The
resin was filtered off and the filtrate was concentrated and
co-evaporated with toluene. The crude product was triturated with
EtOAc/hexane (1/2) to give the monophosphonic acid (2.34 g, 95%) as
a white solid.
Example 24
[1435] Monophospholactate 25: A solution of 24 (2.00 g, 2.95 mmol)
and ethyl-(S)-(-)-lactate (1.34 mL, 11.80 mmol) in pyridine (20 mL)
was heated to 70.degree. C. and 1,3-dicyclohexylcarbodiimide (2.43
g, 11.80 mmol) was added. The reaction mixture was stirred at
70.degree. C. for 2 h and cooled to room temperature. The solvent
was removed under reduced pressure. The residue was suspended in
EtOAc and 1,3-dicyclohexyl urea was filtered off. The product was
partitioned between EtOAc and 0.2 N HCl. The EtOAc layer was washed
with 0.2 N HCl, H.sub.2O, saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (1.38
g, 60%) as a white solid.
Example 25
[1436] Monophospholactate 26: A solution of 25 (0.37 g, 0.48 mmol)
in CH.sub.2Cl.sub.2 (0.80 mL) at 0.degree. C. was treated with
trifluoroacetic acid (0.40 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (3 mL) and cooled to 0.degree. C.
Triethylamine (0.27 mL, 1.92 mmol) was added followed by the
treatment of benzenesulfonyl chloride (84 mg, 0.48 mmol). The
solution was stirred for 30 min at 0.degree. C. and then warmed to
room temperature for 30 min. The product was partitioned between
CH.sub.2Cl.sub.2 and 0.2 N HCl. The organic phase was washed with
saturated NaCl, dried with Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was purified
by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (0.33
g, 85%, GS 192779, 1:1 diastereomeric mixture) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.78 (dd, 2H), 7.59 (m, 3H),
7.38-7.18 (m, 7H), 6.93 (dd, 2H), 5.66 (m, 1H), 5.18-4.93 (m, 3H),
4.56-4.4 (m, 2H), 4.2 (m, 2H), 4.1-3.7 (m, 6H), 3.17 (m, 1H),
3.02-2.8 (m, 6H), 1.84 (m, 1H), 1.82-1.5 (m, 5H), 1.27 (m, 3H),
0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H); .sup.31P NMR
(CDCl.sub.3) .delta. 17.4, 15.3.
Example 26
[1437] Monophospholactate 27: A solution of 25 (0.50 g, 0.64 mmol)
in CH.sub.2Cl.sub.2 (1.0 mL) at 0.degree. C. was treated with
trifluoroacetic acid (0.5 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (4 mL) and cooled to 0.degree. C.
Triethylamine (0.36 mL, 2.56 mmol) was added followed by the
treatment of 4-fluorobenzenesulfonyl chloride (0.13 g, 0.64 mmol).
The solution was stirred for 30 min at 0.degree. C. and then warmed
to room temperature for 30 min. The product was partitioned between
CH.sub.2Cl.sub.2 and 0.2 N HCl. The organic phase was washed with
saturated NaCl, dried with Na.sub.2SO.sub.4, filtered, and
evaporated under reduced pressure. The crude product was purified
by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (0.44
g, 81%, GS 192776, 3/2 diastereomeric mixture) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.80 (m, 2H), 7.38-7.15 (m, 9H),
6.92 (m, 2H), 5.66 (m, 1H), 5.2-4.9 (m, 3H), 4.57-4.4 (m, 2H), 4.2
(m, 2H), 4.1-3.7 (m, 6H), 3.6 (broad, s, 1H), 3.17 (m, 1H),
3.02-2.75 (m, 6H), 1.85 (m, 1H), 1.7-1.5 (m, 5H), 1.26 (m, 3H),
0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H); .sup.31P NMR
(CDCl.sub.3) .delta. 17.3, 15.2.
Example 27
[1438] Monophospholactate 28: A solution of 25 (0.50 g, 0.64 mmol)
in CH.sub.2Cl.sub.2 (1.0 mL) at 0.degree. C. was treated with
trifluoroacetic acid (0.5 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (3 mL) and cooled to 0.degree. C.
Triethylamine (0.45 mL, 3.20 mmol) was added followed by the
treatment of hydrogen chloride salt of 3-pyridinylsulfonyl chloride
(0.14 g, 0.65 mmol). The solution was stirred for 30 min at
0.degree. C. and then warmed to room temperature for 30 min. The
product was partitioned between CH.sub.2Cl.sub.2 and H.sub.2O. The
organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (4% 2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate
(0.41 g, 79%, GS 273806, 1:1 diastereomeric mixture) as a white
solid: .sup.1H NMR (CDCl.sub.3) .delta. 9.0 (s, 1H), 8.83 (dd, 1H),
8.06 (d, J=7.8 Hz, 1H), 7.5 (m, 1H), 7.38-7.15 (m, 7H), 6.92 (m,
2H), 5.66 (m, 1H), 5.18-4.95 (m, 3H), 4.6-4.41 (m, 2H), 4.2 (m,
2H), 4.0 (m, 1H), 3.95-3.76 (m, 6H), 3.23-2.8 (m, 7H), 1.88 (m,
1H), 1.7-1.5 (m, 5H), 1.26 (m, 3H), 0.93 (d, J=6.6 Hz, 3H), 0.83
(d, J=6.6 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.3,
15.3.
Example 28
[1439] Monophospholactate 29: A solution of compound 28 (0.82 g,
1.00 mmol) in CH.sub.2Cl.sub.2 (8 mL) at 0.degree. C. was treated
with mCPBA (1.25 eq). The solution was stirred for 1 h at 0.degree.
C. and then warmed to room temperature for an additional 6 h. The
reaction mixture was partitioned between CH.sub.2Cl.sub.2 and
saturated NaHCO.sub.3. The organic phase was washed with saturated
NaCl, dried with Na.sub.2SO.sub.4, filtered, and evaporated under
reduced pressure. The crude product was purified by column
chromatography on silica gel (10% 2-propanol/CH.sub.2Cl.sub.2) to
give the monophospholactate (0.59 g, 70%, GS 273851, 1:1
diastereomeric mixture) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 8.63 (dd, 1H), 8.3 (dd, 1H), 7.57 (m, 1H), 7.44 (m, 1H),
7.38-7.13 (m, 7H), 6.92 (m, 2H), 5.66 (m, 1H), 5.2-5.05 (m, 2H),
4.57-4.4 (m, 2H), 4.2 (m, 2H), 4.0-3.73 (m, 6H), 3.2 (m, 2H), 3.0
(m, 4H), 2.77 (m, 1H), 1.92 (m, 1H), 1.7-1.49 (m, 5H), 1.26 (m,
3H), 0.91 (m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 17.3,
15.3.
Example 29
[1440] Monophospholactate 30: A solution of compound 28 (71 mg,
0.087 mmol) in CHCl.sub.3 (1 mL) was treated with MeOTf (18 mg,
0.11 mmol). The solution was stirred at room temperature for 1 h.
The reaction mixture was concentrated and co-evaporated with
toluene (2.times.), CHCl.sub.3 (2.times.) and dried under vacuum to
give the monophospholactate (81 mg, 95%, GS 273813, 1:1
diastereomeric mixture) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 9.0 (dd, 1H), 8.76 (m, 2H), 8.1 (m, 1H), 7.35-7.1 (m, 7H),
6.89 (m, 2H), 5.64 (m, 1H), 5.25-5.0 (m, 3H), 4.6-4.41 (m, 5H), 4.2
(m, 2H), 3.92-3.72 (m, 6H), 3.28 (m, 2H), 3.04-2.85 (m, 3H), 2.62
(m, 1H), 1.97 (m, 1H), 1.62-1.5 (m, 5H), 1.25 (m, 3H), 0.97 (m,
6H); .sup.31P NMR (CDCl.sub.3) .delta. 17.4, 15.4.
Example 30
[1441] Dibenzylphosphonate 31: A solution of compound 16 (0.15 g,
0.18 mmol) in CHCl.sub.3 (2 mL) was treated with MeOTf (37 mg, 0.23
mmol). The solution was stirred at room temperature for 2 h. The
reaction mixture was concentrated and co-evaporated with toluene
(2.times.), CHCl.sub.3 (2.times.) and dried under vacuum to give
the dibenzylphosphonate (0.17 g, 95%, GS 273812) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 9.0 (dd, 1H), 8.73 (m, 2H), 8.09
(m, 1H), 7.35 (m, 101H), 7.09 (d, J=8.4 Hz, 2H), 6.79 (d, J=8.1 Hz,
2H), 5.61 (d, J=4.2 Hz, 1H), 5.2-4.96 (m, 6H), 4.54 (s, 3H), 4.2
(dd, 2H), 3.92-3.69 (m, 6H), 3.3 (m, 2H), 3.04-2.6 (m, 5H), 1.97
(m, 1H), 1.6 (m, 2H), 0.98 (m, 6H); .sup.31P NMR (CDCl.sub.3)
.delta. 20.4.
Example 31
[1442] Dibenzylphosphonate 32: A solution of compound 16 (0.15 g,
0.18 mmol) in CH.sub.2Cl.sub.2 (3 mL) at 0.degree. C. was treated
with mCPBA (1.25 eq). The solution was stirred for 1 h at 0.degree.
C. and then warmed to room temperature overnight. The reaction
mixture was partitioned between 10% 2-propanol/CH.sub.2Cl.sub.2 and
saturated NaHCO.sub.3. The organic phase was washed with saturated
NaCl, dried with Na.sub.2SO.sub.4, filtered, and evaporated under
reduced pressure. The crude product was purified by column
chromatography on silica gel (10% 2-propanol/CH.sub.2Cl.sub.2) to
give the dibenzylphosphonate (0.11 g, 70%, GS 277774) as a white
solid: .sup.1H NMR (CDCl.sub.3) .delta. 8.64 (m, 1H), 8.27 (d,
J=6.9 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.36 (m, 1H), 7.10 (d, J=8.4
Hz, 2H), 6.81 (d, J=8.7 Hz, 2H), 5.65 (d, J=5.4 Hz, 1H), 5.22-5.02
(m, 6H), 4.21 (dd, 2H), 3.99-3.65 (m, 6H), 3.2 (m, 2H), 3.03-2.73
(m, 5H), 1.90 (m, 1H), 1.66-1.56 (m, 2H), 0.91 (m, 6H); .sup.31P
NMR (CDCl.sub.3) .delta. 20.3.
Example 32
[1443] Phosphonic Acid 33: To a solution of dibenzylphosphonate 32
(0.1 g, 0.12 mmol) in MeOH (4 mL) was added 10% Pd/C (20 mg). The
suspension was stirred under H.sub.2 atmosphere (balloon) at room
temperature for 1 h. The reaction mixture was filtered through a
plug of celite. The filtrate was concentrated and purified by HPLC
to give the phosphonic acid (17 mg, GS 277775) as a white solid:
.sup.1H NMR (CD.sub.3OD) .delta. 8.68 (s, 1H), 8.47 (d, J=6.0 Hz,
1H), 7.92 (d, J=7.8 Hz, 1H), 7.68 (m, 1H), 7.14 (m, 2H), 6.90 (d,
J=7.8 Hz, 2H), 5.58 (d, J=5.4 Hz, 1H), 5.00 (m, 1H), 4.08 (d, J=9.9
Hz, 2H), 3.93-3.69 (m, 6H), 3.4-2.9 (m, 7H), 2.5 (m, 1H), 2.04 (m,
1H), 1.6-1.35 (m, 2H), 0.92 (m, 6H); .sup.31P NMR (CD.sub.3OD)
.delta. 15.8.
Example 33
[1444] Monophospholactate 34: A solution of 25 (2.50 g, 3.21 mmol)
in CH.sub.2Cl.sub.2 (5.0 mL) at 0.degree. C. was treated with
trifluoroacetic acid (2.5 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (30 mL) and cooled to 0.degree. C.
Triethylamine (1.79 mL, 12.84 mmol) was added followed by the
treatment of 4-formylbenzenesulfonyl chloride (0.72 g, 3.53 mmol)
and the solution was stirred at 0.degree. C. for 1 h. The product
was partitioned between CH.sub.2Cl.sub.2 and 5% HCl. The organic
phase was washed with H.sub.2O, saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (3% 2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate
(2.11 g, 77%, GS 278052, 1:1 diastereomeric mixture) as a white
solid: .sup.1H NMR (CDCl.sub.3) .delta. 10.12 (s, 1H), 8.05 (d,
J=8.7 Hz, 2H), 7.95 (d, J=7.5 Hz, 2H), 7.38-7.15 (m, 7H), 6.94 (m,
2H), 5.67 (m, 1H), 5.18-4.91 (m, 3H), 4.57-4.4 (m, 2H), 4.2 (m,
2H), 4.0-3.69 (m, 6H), 3.57 (broad, s, 1H), 3.19-2.8 (m, 7H), 1.87
(m, 1H), 1.69-1.48 (m, 5H), 1.25 (m, 3H), 0.93 (d, J=6.3 Hz, 3H),
0.88 (d, J=6.3 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.3,
15.2.
Example 34
[1445] Monophospholactate 35: A solution of 34 (0.60 g, 0.71 mmol)
and morpholine (0.31 mL, 3.54 mmol) in EtOAc (8 mL) was treated
with HOAc (0.16 mL, 2.83 mmol) and NaBH.sub.3CN (89 mg, 1.42 mmol).
The reaction mixture was stirred at room temperature for 4 h. The
product was partitioned between EtOAc and H.sub.2O. The organic
phase was washed with brine, dried with Na.sub.2SO.sub.4, filtered,
and concentrated. The crude product was purified by column
chromatography on silica gel (6% 2-propanol/CH.sub.2Cl.sub.2) to
give the monophospholactate (0.46 g, 70%, GS 278115, 1:1
diastereomeric mixture) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 7.74 (d, J=8.4 Hz, 2H), 7.52 (d, J=8.4 Hz, 2H), 7.38-7.15
(m, 7H), 6.92 (m, 2H), 5.66 (m, 1H), 5.2-5.0 (m, 2H), 4.57-4.4 (m,
2H), 4.2 (m, 2H), 3.97-3.57 (m, 12H), 3.2-2.78 (m, 7H), 2.46
(broad, s, 4H), 1.87 (m, 1H), 1.64-1.5 (m, 5H), 1.25 (m, 3H), 0.93
(d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H); .sup.31P NMR
(CDCl.sub.3) .delta. 17.3, 15.3.
Example 35
[1446] Monophospholactate 37: A solution of 25 (0.50 g, 0.64 mmol)
in CH.sub.2Cl.sub.2 (2.0 mL) at 0.degree. C. was treated with
trifluoroacetic acid (1 mL). The solution was stirred for 30 min at
0.degree. C. and then warmed to room temperature for an additional
30 min. The reaction mixture was diluted with toluene and
concentrated under reduced pressure. The residue was co-evaporated
with toluene (2.times.), chloroform (2.times.), and dried under
vacuum to give the ammonium triflate salt which was dissolved in
CH.sub.2Cl.sub.2 (3 mL) and cooled to 0.degree. C. Triethylamine
(0.45 mL, 3.20 mmol) was added followed by the treatment of
4-benzyloxybenzenesulfonyl chloride (0.18 g, 0.64 mmol, prepared
according to Toja, E. et al. Eur. J. Med. Chem. 1991, 26, 403). The
solution was stirred for 30 min at 0.degree. C. and then warmed to
room temperature for 30 min. The product was partitioned between
CH.sub.2Cl.sub.2 and 0.1 N HCl. The organic phase was washed with
saturated NaCl, dried with Na.sub.2SO.sub.4, filtered, and
concentrated. The crude product was purified by column
chromatography on silica gel (4% 2-propanol/CH.sub.2Cl.sub.2) to
give the monophospholactate (0.51 g, 85%) as a white solid.
Example 36
[1447] Monophospholactate 38: To a solution of 37 (0.48 g, 0.52
mmol) in EtOH (15 mL) was added 10% Pd/C (0.10 g). The suspension
was stirred under H.sub.2 atmosphere (balloon) at room temperature
overnight. The reaction mixture was filtered through a plug of
celite. The filtrate was concentrated and the crude product was
purified by column chromatography on silica gel (5%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (0.38
g, 88%, GS 273838, 1:1 diastereomeric mixture) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 8.86 (dd, 1H), 7.42-7.25 (m, 9H),
6.91 (m, 4H), 5.73 (d, J=5.1 Hz, 1H), 5.42 (m, 1H), 5.18 (m, 2H),
4.76-4.31 (m, 2H), 4.22 (m, 2H), 4.12-3.75 (m, 6H), 3.63 (broad, s,
1H), 3.13 (m, 3H), 2.87 (m, 1H), 2.63 (m, 1H), 2.4 (m, 1H), 2.05
(m, 2H), 1.9 (m, 1H), 1.8(m, 1H), 1.6 (m, 3H), 1.25 (m, 3H), 0.95
(d, J=6.6 Hz, 3H), 0.85 (d, J=6.6 Hz, 3H); .sup.31P NMR
(CDCl.sub.3) .delta. 17.1, 15.7.
Example 37
[1448] Monophospholactate 40: A solution of 25 (0.75 g, 0.96 mmol)
in CH.sub.2Cl.sub.2 (2.0 mL) at 0.degree. C. was treated with
trifluoroacetic acid (1 mL). The solution was stirred for 30 min at
0.degree. C. and then warmed to room temperature for an additional
30 min. The reaction mixture was diluted with toluene and
concentrated under reduced pressure. The residue was co-evaporated
with toluene (2.times.), chloroform (2.times.), and dried under
vacuum to give the ammonium triflate salt which was dissolved in
CH.sub.2Cl.sub.2 (4 mL) and cooled to 0.degree. C. Triethylamine
(0.67 mL, 4.80 mmol) was added followed by the treatment of
4-(4'-benzyloxycarbonyl piperazinyl)benzenesulfonyl chloride (0.48
g, 1.22 mmol, prepared according to Toja, E. et al. Arzneim.
Forsch. 1994, 44, 501). The solution was stirred at 0.degree. C.
for 1 h and then warmed to room temperature for 30 min. The product
was partitioned between 10% 2-propanol/CH.sub.2Cl.sub.2 and 0.1 N
HCl. The organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (0.63
g, 60%) as a white solid.
Example 38
[1449] Monophospholactate 41: To a solution of 40 (0.62 g, 0.60
mmol) in MeOH (8 mL) and EtOAc (2 mL) was added 10% Pd/C (0.20 g).
The suspension was stirred under H.sub.2 atmosphere (balloon) at
room temperature overnight. The reaction mixture was filtered
through a plug of celite. The filtrate was treated with 1.2
equivalent of TFA, co-evaporated with CHCl.sub.3 and dried under
vacuum to give the monophospholactate (0.55 g, 90%) as a white
solid.
Example 39
[1450] Monophospholactate 42: A solution of 41 (0.54 g, 0.53 mmol)
and formaldehyde (0.16 mL, 5.30 mmol) in EtOAc (10 mL) was treated
with HOAc (0.30 mL, 5.30 mmol) and NaBH.sub.3CN (0.33 g, 5.30
mmol). The reaction mixture was stirred at room temperature
overnight. The product was partitioned between EtOAc and H.sub.2O.
The organic phase was washed with brine, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (6%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (97.2
mg, 20%, GS 277937, 1:1 diastereomeric mixture) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.64 (d, J=9.0 Hz, 2H), 7.38-7.17
(m, 7H), 6.95-6.88 (m, 4H), 5.67 (m, 1H), 5.2-4.96 (m, 2H),
4.57-4.4 (m, 2H), 4.2 (m, 2H), 3.97-3.64 (m, 8H), 3.49-3.37 (m,
4H), 3.05-2.78 (m, 12H), 1.88-1.62 (m, 3H), 1.58 (m, 3H), 1.25 (m,
3H), 0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H); .sup.31P NMR
(CDCl.sub.3) .delta. 17.3, 15.3.
Example 40
[1451] Monophospholactate 45: A solution of 43 (0.12 g, 0.16 mmol)
and lactate 44 (0.22 g, 1.02 mmol) in pyridine (1 mL) was heated to
70.degree. C. and 1,3-dicyclohexylcarbodiimide (0.17 g, 0.83 mmol)
was added. The reaction mixture was stirred at 70.degree. C. for 4
h and cooled to room temperature. The solvent was removed under
reduced pressure. The residue was suspended in EtOAc and
1,3-dicyclohexyl urea was filtered off. The product was partitioned
between EtOAc and 0.2 N HCl. The EtOAc layer was washed with 0.2 N
HCl, H.sub.2O, saturated NaCl, dried with Na.sub.2SO.sub.4,
filtered, and concentrated. The crude product was purified by
column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (45 mg,
26%) as a white solid.
Example 41
[1452] Alcohol 46: To a solution of 45 (40 mg, 0.042 mmol) in EtOAc
(2 mL) was added 20% Pd(OH).sub.2/C (10 mg). The suspension was
stirred under H2 atmosphere (balloon) at room temperature for 3 h.
The reaction mixture was filtered through a plug of celite. The
filtrate was concentrated and the product was dried under vacuum to
give the alcohol (33 mg, 90%, GS 278809, 3/2 diastereomeric
mixture) as a white solid: .sup.1H NMR (CDCl.sub.3) .delta. 7.72
(d, J=8.7 Hz, 2H), 7.39-7.15 (m, 7H), 7.02-6.88 (m, 4H), 5.66 (d,
J=4.5 Hz, 1H), 5.13-5.02 (m, 2H), 4.54-4.10 (m, 4H), 4.00-3.69 (m,
11H), 3.14 (m, 1H), 3.02-2.77 (m, 6H), 1.85-1.6 (m, 6H), 0.94 (d,
J=6.3 Hz, 3H), 0.89 (d, J=6.3 Hz, 3H); .sup.31P NMR (CDCl.sub.3)
.delta. 17.4, 15.9. ##STR479## ##STR480## ##STR481## ##STR482##
Example 42
[1453] Monobenzylphosphonate 47: A solution of 6 (2.00 g, 2.55
mmol) and DABCO (0.29 g, 2.55 mmol) in toluene (10 mL) was heated
to reflux for 2 h. The solvent was evaporated under reduced
pressure. The residue was partitioned between EtOAc and 0.2 N HCl.
The EtOAc layer was washed with H.sub.2O, saturated NaCl, dried
with Na.sub.2SO.sub.4, filtered, and concentrated. The crude
product was dried under vacuum to give the monobenzylphosphonate
(1.68 g, 95%) as a white solid.
Example 43
[1454] Monophospholactate 48: To a solution of 47 (2.5 g, 3.61
mmol) and benzyl-(S)-(-)-lactate (0.87 mL, 5.42 mmol) in DMF (12
mL) was added PyBop (2.82 g, 5.42 mmol) and
N,N-diisopropylethylamine (2.51 mL, 14.44 mmol). The reaction
mixture was stirred at room temperature for 3 h and concentrated.
The residue was partitioned between EtOAc and 0.2 N HCl. The EtOAc
layer was washed with H.sub.2O, saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (1.58
g, 51%) as a white solid.
Example 44
[1455] Monophospholactate 49: A solution of 48 (0.30 g, 0.35 mmol)
in CH.sub.2Cl.sub.2 (0.6 mL) at 0.degree. C. was treated with
trifluoroacetic acid (0.3 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (2 mL) and cooled to 0.degree. C.
Triethylamine (0.20 mL, 1.40 mmol) was added followed by the
treatment of benzenesulfonyl chloride (62 mg, 0.35 mmol). The
solution was stirred at 0.degree. C. for 30 min and then warmed to
room temperature for 30 min. The product was partitioned between
CH.sub.2Cl.sub.2 and 0.1 N HCl. The organic phase was washed with
saturated NaCl, dried with Na.sub.2SO.sub.4, filtered, and
concentrated. The crude product was purified by column
chromatography on silica gel (3% 2-propanol/CH.sub.2Cl.sub.2) to
give the monophospholactate (0.17 g, 53%) as a white solid.
Example 45
[1456] Metabolite X 50: To a solution of 49 (80 mg, 0.09 mmol) in
EtOH (6 mL) and EtOAc (2 mL) was added 10% Pd/C (20 mg). The
suspension was stirred under H.sub.2 atmosphere (balloon) at room
temperature for 8 h. The reaction mixture was filtered through a
plug of celite. The filtrate was concentrated, co-evaporated with
CHCl.sub.3 and dried under vacuum to give the metabolite X (61 mg,
95%, GS 224342) as a white solid: .sup.1H NMR (CD.sub.3OD) .delta.
7.83 (d, J=6.9 Hz, 2H), 7.65-7.58 (m, 3H), 7.18 (d, J=7.8 Hz, 2H),
6.90 (d, J=7.8 Hz, 2H), 5.59 (d, J=4.8 Hz, 1H), 5.0 (m, 1H), 4.27
(d, J=10.2 Hz, 2H), 3.95-3.68 (m, 6H), 3.45 (dd, 1H), 3.18-2.84 (m,
6H), 2.50 (m, 1H), 2.02 (m, 1H), 1.6-1.38 (m, 5H), 0.93 (d, J=6.3
Hz, 3H), 0.88 (d, J=6.3 Hz, 3H); .sup.31P NMR (CD.sub.3OD), .delta.
18.0.
Example 46
[1457] Monophospholactate 51: A solution of 48 (0.28 g, 0.33 mmol)
in CH.sub.2Cl.sub.2 (0.6 mL) at 0.degree. C. was treated with
trifluoroacetic acid (0.3 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (2 mL) and cooled to 0.degree. C.
Triethylamine (0.18 mL, 1.32 mmol) was added followed by the
treatment of 4-fluorobenzenesulfonyl chloride (64 mg, 0.33 mmol).
The solution was stirred at 0.degree. C. for 30 min and then warmed
to room temperature for 30 min. The product was partitioned between
CH.sub.2Cl.sub.2 and 0.1 N HCl. The organic phase was washed with
saturated NaCl, dried with Na.sub.2SO.sub.4, filtered, and
concentrated. The crude product was purified by column
chromatography on silica gel (3% 2-propanol/CH.sub.2Cl.sub.2) to
give the monophospholactate (0.16 g, 52%) as a white solid.
Example 47
[1458] Metabolite X 52: To a solution of 51 (80 mg, 0.09 mmol) in
EtOH (6 mL) and EtOAc (2 mL) was added 10% Pd/C (20 mg). The
suspension was stirred under H.sub.2 atmosphere (balloon) at room
temperature for 8 h. The reaction mixture was filtered through a
plug of celite. The filtrate was concentrated, co-evaporated with
CHCl.sub.3 and dried under vacuum to give the metabolite X (61 mg,
95%, GS 224343) as a white solid: .sup.1H NMR (CD.sub.3OD) .delta.
7.9 (dd, 2H), 7.32 (m, 2H), 7.18 (dd, 2H), 6.90 (dd, 2H), 5.59 (d,
J=5.4 Hz, 1H), 5.0 (m, 1H), 4.28 (d, J=10.2 Hz, 2H), 3.95-3.72 (m,
6H), 3.44 (dd, 1H), 3.15-2.85 (m, 6H), 2.5 (m, 1H), 2.02 (m, 1H),
1.55-1.38 (m, 5H), 0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H).
.sup.31P NMR (CD.sub.3OD) .delta. 18.2.
Example 48
[1459] Monophospholactate 53: A solution of 48 (0.20 g, 0.24 mmol)
in CH.sub.2Cl.sub.2 (0.6 mL) at 0.degree. C. was treated with
trifluoroacetic acid (0.3 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (2 mL) and cooled to 0.degree. C.
Triethylamine (0.16 mL, 1.20 mmol) was added followed by the
treatment of hydrogen chloride salt of 3-pyridinysulfonyl chloride
(50 mg, 0.24 mmol). The solution was stirred at 0.degree. C. for 30
min and then warmed to room temperature for 30 min. The product was
partitioned between CH.sub.2Cl.sub.2 and H.sub.2O. The organic
phase was washed with saturated NaCl, dried with Na.sub.2SO.sub.4,
filtered, and concentrated. The crude product was purified by
column chromatography on silica gel (4%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (0.11
g, 53%) as a white solid.
Example 49
[1460] Metabolite X 54: To a solution of 53 (70 mg, 0.09 mmol) in
EtOH (5 mL) was added 10% Pd/C (20 mg). The suspension was stirred
under H.sub.2 atmosphere (balloon) at room temperature for 5 h. The
reaction mixture was filtered through a plug of celite. The
filtrate was concentrated, co-evaporated with CHCl.sub.3 and dried
under vacuum to give the metabolite X (53 mg, 95%, GS 273834) as a
white solid: .sup.1H NMR (CD.sub.3OD) .delta. 8.99 (s, 1H), 8.79
(d, J=4.2 Hz, 1H), 8.29 (d, J=7.5 Hz, 1H), 7.7 (m, 1H), 7.15 (d,
J=8.4 Hz, 2H), 6.9 (d, J=7.8 Hz, 2H), 5.59 (d, J=5.4 Hz, 1H), 5.0
(m, 1H), 4.28 (d, J=9.9 Hz, 2H), 3.97-3.70 (m, 6H), 3.44 (dd, 1H),
3.17-2.85 (m, 6H), 2.5 (m, 1H), 2.03 (m, 1H), 1.65-1.38 (m, 5H),
0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H). .sup.31P NMR
(CD.sub.3OD) .delta. 17.8.
Example 50
[1461] Monophospholactate 55: A solution of 48 (0.15 g, 0.18 mmol)
in CH.sub.2Cl.sub.2 (1 mL) at 0.degree. C. was treated with
trifluoroacetic acid (0.5 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (2 mL) and cooled to 0.degree. C.
Triethylamine (0.12 mL, 0.88 mmol) was added followed by the
treatment of 4-benzyloxybenzenesulfonyl chloride (50 mg, 0.18
mmol). The solution was stirred at 0.degree. C. for 30 min and then
warmed to room temperature for 30 min. The product was partitioned
between CH.sub.2Cl.sub.2 and 0.1 N HCl. The organic phase was
washed with saturated NaCl, dried with Na.sub.2SO.sub.4, filtered,
and concentrated. The crude product was purified by column
chromatography on silica gel (3% 2-propanol/CH.sub.2Cl.sub.2) to
give the monophospholactate (0.11 g, 63%) as a white solid.
Example 51
[1462] Metabolite X 56: To a solution of 55 (70 mg, 0.07 mmol) in
EtOH (4 mL) was added 10% Pd/C (20 mg). The suspension was stirred
under H.sub.2 atmosphere (balloon) at room temperature for 4 h. The
reaction mixture was filtered through a plug of celite. The
filtrate was concentrated, co-evaporated with CHCl.sub.3 and dried
under vacuum to give the metabolite X (46 mg, 90%, GS 273847) as a
white solid: .sup.1H NMR (CD.sub.3OD), .delta. 7.91 (s, 1H), 7.65
(d, J=8.4 Hz, 2H), 7.17 (d, J=8.1 Hz, 2H), 6.91 (m, 4H), 5.59 (d,
J=5.1 Hz, 1H), 5.0 (m, 1H), 4.27 (d, J=10.2 Hz, 2H), 3.97-3.74 (m,
6H), 3.4 (dd, 1H), 3.17-2.8 (m, 6H), 2.5 (m, 1H), 2.0 (m, 1H),
1.6-1.38 (m, 5H), 0.93 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CD.sub.3OD) .delta. 17.9.
Example 52
[1463] Metabolite X 57: To a suspension of 29 (40 mg, 0.05 mmol) in
CH.sub.3CN (1 mL), DMSO (0.5 mL), and 1.0 M PBS buffer (5 mL) was
added esterase (200 .mu.L). The suspension was heated to 40.degree.
C. for 48 h. The reaction mixture was concentrated, suspended in
MeOH and filtered. The filtrate was concentrated and purified by
HPLC to give the metabolite X (20 mg, 57%, GS 277777) as a white
solid: .sup.1H NMR (CD.sub.3OD) .delta. 8.68 (s, 1H), 8.47 (d,
J=6.0 Hz, 1H), 7.93 (d, J=7.8 Hz, 1H), 7.68 (m, 1H), 7.15 (d, J=8.4
Hz, 2H), 6.9 (d, J=8.4 Hz, 2H), 5.59 (d, J=5.4 Hz, 1H), 5.0 (m,
1H), 4.23 (d, J=10.5 Hz, 2H), 3.97-3.68 (m, 6H), 3.45 (dd, 1H),
3.15-2.87 (m, 6H), 2.46 (m, 1H), 2.0 (m, 1H), 1.6-1.38 (m, 5H),
0.95 (d, J=6.6 Hz, 3H), 0.92 (d, J=6.6 Hz, 3H); .sup.31P NMR
(CD.sub.3OD) .delta. 17.2.
Example 53
[1464] Metabolite X 58: To a suspension of 35 (60 mg, 0.07 mmol) in
CH.sub.3CN (1 mL), DMSO (0.5 mL), and 1.0 M PBS buffer (5 mL) was
added esterase (400 .mu.L). The suspension was heated to 40.degree.
C. for 3 days. The reaction mixture was concentrated, suspended in
MeOH and filtered. The filtrate was concentrated and purified by
HPLC to give the metabolite X (20 mg, 38%, GS 278116) as a white
solid: .sup.1H NMR (CD.sub.3OD) .delta. 7.74 (d, J=6.9 Hz, 2H),
7.63 (d, J=7.5 Hz, 2H), 7.21 (d, J=8.4 Hz, 2H), 6.95 (d, J=8.1 Hz,
2H), 5.64 (d, J=5.1 Hz, 1H), 5.0 (m, 2H), 4.41 (m, 2H), 4.22 (m,
2H), 3.97-3.65 (m, 12H), 3.15-2.9 (m, 8H), 2.75 (m, 1H), 2.0 (m,
1H), 1.8 (m, 2H), 1.53 (d, J=6.9 Hz, 3H), 0.88 (m, 6H).
Example 54
[1465] Monophospholactate 59: A solution of 34 (2.10 g, 2.48 mmol)
in THF (72 mL) and H.sub.2O (8 mL) at -15.degree. C. was treated
with NaBH.sub.4 (0.24 g, 6.20 mmol). The reaction mixture was
stirred for 10 min at -15.degree. C. The reaction was quenched with
5% aqueous NaHSO.sub.3 and extracted with CH.sub.2Cl.sub.2
(3.times.). The combined organic layers were washed with H.sub.2O,
dried with Na.sub.2SO.sub.4, filtered, and concentrated. The crude
product was purified by column chromatography on silica gel (5%
2-propanol/CH.sub.2Cl.sub.2) to give monophospholactate (1.89 g,
90%, GS 278053, 1:1 diastereomeric mixture) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.64 (m, 2H), 7.51(m, 2H),
7.38-7.19 (m, 7H), 6.92 (m, 2H), 5.69 (d, J=4.8 Hz, 1H), 5.15 (m,
2H), 4.76 (s, 2H), 4.54 (d, J=10.5 Hz, 1H), 4.44 (m, 1H), 4.2 (m,
2H), 4.04-3.68 (m, 6H), 3.06-2.62 (m, 7H), 1.8 (m, 3H), 1.62-1.5
(dd, 3H), 1.25 (m, 3H), 0.94 (d, J=6.3 Hz, 3H), 0.87 (d, J=6.3 Hz,
3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.4, 15.4.
Example 55
[1466] Metabolite X 60: To a suspension of 59 (70 mg, 0.08 mmol) in
CH.sub.3CN (1 mL), DMSO (0.5 mL), and 1.0 M PBS buffer (5 mL) was
added esterase (600 .mu.L). The suspension was heated to 40.degree.
C. for 36 h. The reaction mixture was concentrated, suspended in
MeOH and filtered. The filtrate was concentrated and purified by
HPLC to give the metabolite X (22 mg, 36%, GS 278764) as a white
solid: .sup.1H NMR (CD.sub.3OD) .delta. 7.78 (dd, 2H), 7.54 (dd,
2H), 7.15 (m, 2H), 6.9 (m, 2H), 5.57 (d, 1H), 5.0 (m, 2H), 4.65 (m,
4H), 4.2 (m, 2H), 3.9-3.53 (m, 6H), 3.06-2.82 (m, 6H), 2.5 (m, 1H),
2.0 (m, 2H), 1.62-1.35 (m, 3H), 0.94 (m, 6H). ##STR483## ##STR484##
##STR485##
Example 56
[1467] Phosphonic Acid 63: Compound 62 (0.30 g, 1.12 mmol) was
dissolved in CH.sub.3CN (5 mL). N,O-Bis(trimethylsilyl)acetamide
(BSA, 2.2 mL, 8.96 mmol) was added. The reaction mixture was heated
to reflux for 2 h, cooled to room temperature, and concentrated.
The residue was co-evaporated with toluene and chloroform and dried
under vacuum to give a thick oil which was dissolved in EtOAc (4
mL) and cooled to 0.degree. C. Aldehyde 61 (0.20 g, 0.33 mmol),
AcOH (0.18 mL, 3.30 mmol), and NaBH.sub.3CN (0.20 g, 3.30 mmol)
were added. The reaction mixture was warmed to room temperature and
stirred overnight. The reaction was quenched with H.sub.2O, stirred
for 30 min, filtered, and concentrated. The crude product was
dissolved in CH.sub.3CN (13 mL) and 48% aqueous HF (0.5 mL) was
added. The reaction mixture was stirred at room temperature for 2 h
and concentrated. The crude product was purified by HPLC to give
the phosphonic acid (70 mg, 32%, GS 277929) as a white solid:
.sup.1H NMR (CD.sub.3OD) .delta. 7.92 (dd, 2H), 7.73 (d, J=8.7 Hz,
2H), 7.63 (dd, 2H), 7.12 (d, J=8.7 Hz, 2H), 5.68 (d, J=5.1 Hz, 1H),
5.13 (m, 1H), 4.4 (m, 2H), 4.05-3.89 (m, 8H), 3.75 (m, 1H), 3.5 (m,
1H), 3.37 (m, 1H), 3.23-3.0 (m, 3H), 2.88-2.7 (m, 2H), 2.2 (m, 1H),
1.8 (m, 2H), 0.92 (d, J=6.3 Hz, 3H), 0.85 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CD.sub.3OD) .delta. 14.5.
Example 57
[1468] Phosphonic Acid 64: A solution of 63 (50 mg, 0.07 mmol) and
formaldehyde (60 mg, 0.70 mmol) in EtOAc (2 mL) was treated with
HOAc (43 .mu.L, 0.70 mmol) and NaBH.sub.3CN (47 mg, 0.7 mmol). The
reaction mixture was stirred at room temperature for 26 h. The
reaction was quenched with H.sub.2O, stirred for 20 min, and
concentrated. The crude product was purified by HPLC to give the
phosphonic acid (15 mg, 29%, GS 277935) as a white solid: .sup.1H
NMR (CD.sub.3OD) .delta. 7.93 (m, 2H), 7.75 (m, 2H), 7.62 (m, 2H),
7.11 (m, 2H), 5.66 (m, 1H), 5.13 (m, 1H), 4.4 (m, 2H), 4.05-3.89
(m, 8H), 3.75 (m, 2H), 3.09-2.71 (m, 6H), 2.2 (m, 1H), 1.9 (m, 5H),
0.92 (d, J=6.3 Hz, 3H), 0.85 (d, J=6.3 Hz, 3H); .sup.31P NMR
(CD.sub.3OD) .delta. 14.0.
Example 58
[1469] Phosphonic Acid 66: 2-Aminoethylphosphonic acid (2.60 g,
21.66 mmol) was dissolved in CH.sub.3CN (40 mL).
N,O-Bis(trimethylsilyl)acetamide (BSA, 40 mL) was added. The
reaction mixture was heated to reflux for 2 h and cooled to room
temperature and concentrated. The residue was co-evaporated with
toluene and chloroform and dried under vacuum to give a thick oil
which was dissolved in EtOAc (40 mL). Aldehyde 65 (1.33 g, 2.25
mmol), AcOH (1.30 mL, 22.5 mmol) and NaBH.sub.3CN (1.42 g, 22.5
mmol) were added. The reaction mixture was stirred at room
temperature overnight. The reaction was quenched with H.sub.2O,
stirred for 1 h, filtered, and concentrated. The residue was
dissolved in MeOH and filtered. The crude product was purified by
HPLC to give the phosphonic acid (1.00 g, 63%) as a white
solid.
Example 59
[1470] Phosphonic Acid 67: Phosphonic acid 66 (0.13 g, 0.19 mmol)
was dissolved in CH.sub.3CN (4 mL).
N,O-Bis(trimethylsilyl)acetamide (BSA, 0.45 mL, 1.90 mmol) was
added. The reaction mixture was heated to reflux for 2 h, cooled to
room temperature, and concentrated. The residue was co-evaporated
with toluene and chloroform and dried under vacuum to give a thick
oil which was dissolved in EtOAc (3 mL). Formaldehyde (0.15 mL,
1.90 mmol), AcOH (0.11 mL, 1.90 mmol) and NaBH.sub.3CN (63 mg, 1.90
mmol) were added. The reaction mixture was stirred at room
temperature overnight. The reaction was quenched with H.sub.2O,
stirred for 6 h, filtered, and concentrated. The residue was
dissolved in MeOH and filtered. The crude product was purified by
HPLC to give the phosphonic acid (40 mg, 30%, GS 277957) as a white
solid: .sup.1H NMR (CD.sub.3OD) .delta. 7.78 (d, J=8.4 Hz, 2H), 7.4
(m, 4H), 7.09 (d, J=8.4 Hz, 2H), 5.6 (d, J=5.1 Hz, 1H), 4.33 (m,
2H), 3.95-3.65 (m, 9H), 3.5-3.05 (m, 6H), 2.91-2.6 (m, 7H), 2.0 (m,
3H), 1.5 (m, 2H), 0.93 (d, J=6.3 Hz, 3H), 0.87 (d, J=6.3 Hz, 3H);
.sup.31P NMR (CD.sub.3OD) .delta. 19.7.
Example 60
[1471] Metabolite X 69: Monophospholactate 68 (1.4 g, 1.60 mmol)
was dissolved in CH.sub.3CN (20 mL) and H.sub.2O (20 mL). 1.0 N
NaOH (3.20 mL, 3.20 mmol) was added. The reaction mixture was
stirred at room temperature for 1.5 h and cooled to 0.degree. C.
The reaction mixture was acidified to pH=1-2 with 2 N HCl (1.6 mL,
3.20 mmoL). The solvent was evaporated under reduced pressure. The
crude product was purified by HPLC to give the metabolite X (0.60
g, 49%, GS 273842) as a white solid: .sup.1H NMR (DMSO-d.sub.6)
.delta. 7.72 (d, J=8.7 Hz, 2H), 7.33 (m, 4H), 7.09 (d, J=9.0 Hz,
2H), 5.52 (d, J=5.7 Hz, 1H), 5.1 (broad, s, 1H), 4.85 (m, 1H), 4.63
(m, 1H), 4.13 (m, 2H), 3.8 (m, 5H), 3.6 (m, 4H), 3.36 (m, 1H), 3.03
(m, 4H), 2.79 (m, 3H), 2.5 (m, 1H), 2.0 (m, 3H), 1.5-1.3 (m, 5H),
0.85 (d, J=6.6 Hz, 3H), 0.79 (d, J=6.6 Hz, 3H); .sup.31P NMR
(DMSO-d.sub.6) .delta. 21.9. ##STR486## ##STR487## ##STR488##
Example 61
[1472] Monophospholactate 70: A solution of 59 (1.48 g, 1.74 mmol)
and Boc-L-valine (0.38 g, 1.74 mmol) in CH.sub.2Cl.sub.2 (30 mL) at
0.degree. C. was treated with 1,3-dicyclohexylcarbodiimide (0.45 g,
2.18 mmol) and 4-dimethylaminopyridine (26 mg, 0.21 mmol). The
reaction mixture was stirred at 0.degree. C. for 1 h and then
warmed to room temperature for 2 h. The product was partitioned
between CH.sub.2Cl.sub.2 and 0.2 N HCl. The organic layer was
washed with H.sub.2O, dried with Na.sub.2SO.sub.4, filtered, and
concentrated. The crude product was purified by column
chromatography on silica gel (4% 2-propanol/CH.sub.2Cl.sub.2) to
give the monophospholactate (1.65 g, 90%) as a white solid.
Example 62
[1473] Monophospholactate 71: A solution of 70 (1.65 g, 1.57 mmol)
in CH.sub.2Cl.sub.2 (8 mL) at 0.degree. C. was treated with
trifluoroacetic acid (4 mL). The solution was stirred for 30 min at
0.degree. C. and then warmed to room temperature for an additional
30 min. The reaction mixture was diluted with toluene and
concentrated under reduced pressure. The crude product was purified
by column chromatography on silica gel (10%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (1.42
g, 85%, GS 278635, 2/3 diastereomeric mixture) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.73 (m, 2H), 7.49 (d, J=7.2 Hz,
2H), 7.4-7.1 (m, 7H), 6.89 (m, 2H), 5.64 (m, 1H), 5.47 (m, 1H),
5.33-5.06 (m, 4H), 4.57-4.41 (m, 2H), 4.2 (m, 2H), 3.96-3.7 (m,
7H), 3.15-2.73 (m, 7H), 2.38 (m, 1H), 1.9 (m, 1H), 1.7 (m, 1H),
1.63-1.5 (m, 4H), 1.24 (m, 3H), 1.19 (m, 6H), 0.91 (d, 3H), 0.88
(d, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.3, 15.4.
Example 63
[1474] Monophospholactate 73: A solution of 72 (0.43 g, 0.50 mmol)
and Boc-L-valine (0.11 g, 0.50 mmol) in CH.sub.2Cl.sub.2 (6 mL) was
treated with 1,3-dicyclohexylcarbodiimide (0.13 g, 0.63 mmol) and
4-dimethylaminopyridine (62 mg, 0.5 mmol). The reaction mixture was
stirred at room temperature overnight. The product was partitioned
between CH.sub.2Cl.sub.2 and 0.2 N HCl. The organic layer was
washed with H.sub.2O, dried with Na.sub.2SO.sub.4, filtered, and
concentrated. The crude product was purified by column
chromatography on silica gel (2% 2-propanol/CH.sub.2Cl.sub.2) to
give the monophospholactate (0.45 g, 85%) as a white solid.
Example 64
[1475] Monophospholactate 74: A solution of 73 (0.44 g, 0.42 mmol)
in CH.sub.2Cl.sub.2 (1 mL) at 0.degree. C. was treated with
trifluoroacetic acid (0.5 mL). The solution was stirred for 30 min
at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The crude product was
purified by column chromatography on silica get (10%
2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (0.40
g, 90%, GS 278785, 1:1 diastereomeric mixture) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.69 (d, J=8.4 Hz, 2H), 7.34-7.2
(m, 7H), 6.98 (d, J=8.4 Hz, 2H), 6.88 (m, 2H), 6.16 (m, 1H), 5.64
(m, 1H), 5.46 (m, 1H), 5.2-5.0 (m, 2H), 4.5 (m, 2H), 4.2 (m, 3H),
4.0-3.4 (m, 9H), 3.3 (m, 1H), 3.0-2.8 (m, 5H), 2.5 (m, 1H), 1.83
(m, 1H), 1.6-1.5 (m, 5H), 125 (m, 3H), 1.15 (m, 6H), 0.82 (d, J=6.0
Hz, 3H), 0.76 (d, J=6.0 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta.
17.3, 15.5.
Example 65
[1476] Cbz Amide 76: Compound 75 (0.35 g, 0.69 mmol) was dissolved
in CH.sub.3CN (6 mL). N,O-Bis(trimethylsilyl)acetamide (BSA, 0.67
mL, 2.76 mmol) was added. The reaction mixture was heated to reflux
for 1 h, cooled to room temperature, and concentrated. The residue
was co-evaporated with toluene and chloroform and dried under
vacuum to give a thick oil which was dissolved in CH.sub.2Cl.sub.2
(3 mL) and cooled to 0.degree. C. Pyridine (0.17 mL, 2.07 mmol) and
benzyl chloroformate (0.12 mL, 0.83 mmol) were added. The reaction
mixture was stirred at 0.degree. C. for 1 h and then warmed to room
temperature overnight. The reaction was quenched with MeOH (5 mL)
and 10% HCl (20 mL) at 0.degree. C. and stirred for 1 h. The
product was extracted with CH.sub.2Cl.sub.2, washed with brine,
dried with Na.sub.2SO.sub.4, filtered, and concentrated. The crude
product was purified by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the CBz amide (0.40 g, 90%) as
a white solid.
Example 66
[1477] Dibenzylphosphonate 77: A solution of 76 (0.39 g, 0.61 mmol)
and 1H-tetrazole (54 mg, 0.92 mmol) in CH.sub.2Cl.sub.2 (8 mL) was
treated with dibenzyldiisopropylphosphoramidite (0.32 g, 0.92 mmol)
and stirred at room temperature overnight. The solution was cooled
to 0.degree. C., treated with mCPBA, stirred for 1 h at 0.degree.
C. and then warmed to room temperature for 1 h. The reaction
mixture was poured into a mixture of aqueous Na.sub.2SO.sub.3 and
NaHCO.sub.3 and extracted with CH.sub.2Cl.sub.2. The organic layer
was washed with H.sub.2O, dried with Na.sub.2SO.sub.4, filtered,
and concentrated. The crude product was purified by column
chromatography on silica gel (3% 2-propanol/CH.sub.2Cl.sub.2) to
give the dibenzylphosphonate (0.42 g, 76%) as a white solid.
Example 67
[1478] Disodium Salt of Phosphonic Acid 78: To a solution of 77
(0.18 g, 0.20 mmol) in EtOH (20 mL) and EtOAc (4 mL) was added 10%
Pd/C (40 mg). The suspension was stirred under H.sub.2 atmosphere
(balloon) at room temperature for 4 h. The reaction mixture was
filtered through a plug of celite. The filtrate was concentrated
and dried under vacuum to give the phosphonic acid (0.11 g, 95%)
which was dissolved in H.sub.2O (4 mL) and treated with NaHCO.sub.3
(32 mg, 0.38 mmol). The reaction mixture was stirred at room
temperature for 1 h and lyopholyzed overnight to give the disodium
salt of phosphonic acid (0.12 g, 99%, GS 277962) as a white solid:
.sup.1H NMR (D.sub.2O) .delta. 7.55 (dd, 2H), 7.2 (m, 5H), 7.77
(dd, 2H), 4.65 (m, 1H), 4.24 (m, 1H), 4.07 (m, 1H), 3.78-2.6 (m,
12H), 1.88-1.6 (m, 3H), 0.75 (m, 6H). ##STR489## ##STR490##
Example 1
[1479] Compound 1 was prepared by methods from Examples herein.
Example 2
[1480] Compound 2: To a solution of compound 1 (47.3 g) in
EtOH/EtOAc (1000 mL/500 mL) was added 10% Pd--C (5 g). The mixture
was hydrogenated for 19 hours. Celite was added and the mixture was
stirred for 10 minutes. The mixture was filtered through a pad of
celite and was washed with ethyl acetate. Concentration gave
compound 2 (42.1 g).
Example 3
[1481] Compound 3: To a solution of compound 2 (42.3 g, 81 mmol) in
CH.sub.2Cl.sub.2 (833 mL) was added
N-phenyltrifluoromethanesulfonimide (31.8 g, 89 mmol), followed by
cesium carbonate (28.9 g, 89 mmol). The mixture was stirred for 24
hours. The solvent was removed under reduced pressure, and ethyl
acetate was added. The reaction mixture was washed with water
(3.times.) and brine (1.times.), and was dried over MgSO.sub.4.
Purification by flash column chromatography
(CH.sub.2Cl.sub.2/EtOAc=13/1) gave compound 3 (49.5 g) as a white
powder.
Example 4
[1482] Compound 4: To a solution of compound 3 (25.2, 38.5 mmol) in
DMF (240 mL) was added lithium chloride (11.45 g, 270 mmol),
followed by dichlorobis(triphenylphosphine) palladium(II) (540 mg,
0.77 mmol). The mixture was stirred for 3 minutes under high vacuum
and recharged with nitrogen. To the above solution was added
tributylvinyltin (11.25 mL). The reaction mixture was heated at
90.degree. C. for 6 hours and cooled to 25.degree. C. Water was
added to the reaction, and the mixture was extracted with ethyl
acetate (3.times.). The combined organic layer was washed with
water (6.times.) and brine, and dried over MgSO.sub.4.
Concentration gave an oil. The oil was diluted with dichloromethane
(40 mL), water (0.693 mL, 38.5 mmol) and DBU (5.76 mL, 38.5 mmol)
were added. The mixture was stirred for 5 minutes, and subjected to
flash column chromatography (hexanes/EtOAc=2.5/1).
[1483] Compound 4 was obtained as white solid (18.4 g).
Example 5
[1484] Compound 5: To a solution of compound 4 (18.4 g, 34.5 mmol)
in CH.sub.2Cl.sub.2 (70 mL) at 0.degree. C. was added
trifluoroacetic acid (35 mL). The mixture was stirred at 0.degree.
C. for 2 hrs, and solvents were evaporated under reduced pressure.
The reaction mixture was quenched with saturated sodium carbonate
solution, and was extracted with ethyl acetate (3.times.). The
combined organic layer was washed with saturated sodium carbonate
solution (1.times.), water (2.times.), and brine (1.times.), and
dried over MgSO.sub.4. Concentration gave a solid. To a solution of
the above solid in acetonitrile (220 mL) at 0.degree. C. was added
bisfurancarbonate (10.09 g, 34.2 mmol), followed by
di-isopropylethylamine (12.0 mL, 69.1 mmol) and DMAP (843 mg, 6.9
mmol). The mixture was warmed to 25.degree. C. and stirred for 12
hours. Solvents were removed under reduced pressure. The mixture
was diluted with ethyl acetate, and was washed with water
(2.times.), 5% hydrochloric acid (2.times.), water (2.times.), 1N
sodium hydroxide (2.times.), water (2.times.), and brine
(1.times.), and dried over MgSO.sub.4. Purification by flash column
chromatography (hexanes/EtOAc=1/1)) gave compound 5 (13.5 g).
Example 6
[1485] Compound 6: To a solution of compound 5 (13.5 g, 23 mmol) in
ethyl acetate (135 mL) was added water (135 mL), followed by 2.5%
osmium tetraoxide/tert-butanol (17 mL). Sodium periodate (11.5 g)
was added in portions over 2 minutes period. The mixture was
stirred for 90 minutes, and was diluted with ethyl acetate. The
organic layer was separated and washed with water (3.times.) and
brine (1.times.), and dried over MgSO.sub.4. Purification by flash
column chromatography (hexanes/EtOAc=1/2) gave compound 6 as white
powder (12 g): .sup.1H NMR (CDCl.sub.3) .delta. 91.98 (1H, s), 7.82
(2H, m), 7.75 (2H, m), 7.43 (2H, m), 6.99 (2H, m), 5.64 (1H, m),
5.02 (2H, m), 4.0-3.8 (9H, m), 3.2-2.7 (7H, m), 1.9-1.4 (3H, m),
0.94 (6H, m). ##STR491## ##STR492## ##STR493##
Example 8
[1486] Compound 8: To the suspension of compound 7 (15.8 g, 72.5
mmol) in toluene (140 mL) was added DMF (1.9 mL), followed by
thionyl chloride (53 mL, 725 mmol). The reaction mixture was heated
at 60.degree. C. for 5 hrs, and evaporated under reduced pressure.
The mixture was coevaporated with toluene (2.times.), EtOAc, and
CH.sub.2Cl.sub.2 (2.times.) to afford a brown solid. To the
solution of the brown solid in CH.sub.2Cl.sub.2 at 0.degree. C. was
added phenol (27.2 g, 290 mmol), followed by slow addition of
pyridine (35 mL, 435 mmol). The reaction mixture was allowed to
warm to 25.degree. C. and stirred for 14 hrs. Solvents were removed
under reduced pressure. The mixture was diluted with EtOAc, and
washed with water (3.times.) and brine (1.times.), and dried over
MgSO.sub.4. Concentration gave a dark oil, which was purified by
flash column chromatography (hexanes/EtOAc=411 to 1/1) to afford
compound 8 (12.5 g).
Example 9
[1487] Compound 9: To a solution of compound 8 (2.21 g, 6 mmol) in
THF (30 mL) was added 12 ml, of 1.0 N NaOH solution. The mixture
was stirred at 25.degree. C. for 2 hours, and THF was removed under
reduced pressure. The mixture was diluted with water, and acetic
acid (343 mL, 6 mmol) was added. The aqueous phase was washed with
EtOAc (3.times.), and then acidified with concentrated HCl until
pH=1. The aqueous was extracted with EtOAc (3.times.). The combined
organic layer was washed with water (1.times.) and brine
(1.times.), and dried over MgSO.sub.4. Concentration under reduced
pressure gave compound 9 as a solid (1.1 g).
Example 10
[1488] Compound 10: To a suspension of compound 9 (380 mg, 1.3
mmol) in toluene (2.5 mL) was added thionyl chloride (1 mL, 13
mmol), followed by DMF (1 drop). The mixture was heated at
60.degree. C. for 2 hours. The solvent and reagent were removed
under reduced pressure. The mixture was coevaporated with toluene
(2.times.) and CH.sub.2Cl.sub.2 to give a white solid. To the
solution of the above solid in CH.sub.2Cl.sub.2 (5 ml) at
-20.degree. C. was added ethyl lactate (294 .mu.L, 2.6 mmol),
followed by pyridine (420 .mu.L, 5.2 mmol). The mixture was warmed
to 25.degree. C. and stirred for 12 hours. The reaction mixture was
concentrated under reduced pressure to give a yellow solid, which
was purified by flash column chromatography to generate compound 10
(427 mg).
Example 11
[1489] Compound 11: To a solution of compound 10 (480 mg) in EtOAc
(20 mL) was added 10% Pd--C (80 mg). The reaction mixture was
hydrogenated for 6 hrs. The mixture was stirred with celite for 5
mins, and filtered through a pad of celite. Concentration under
reduced pressure gave compound 11 (460 mg).
Example 12
[1490] Compound 12 was prepared by the methods of the Examples
herein
Example 13
[1491] Compound 13: To a solution of compound 12 (536 mg, 1.0 mmol)
in CH.sub.2Cl.sub.2 (10 mL) was added trifluoroacetic acid (2 mL).
The mixture was stirred for 2 hrs, and was concentrated under
reduced pressure. The liquid was coevaporated with CH.sub.2Cl.sub.2
(3.times.) and EtOAc (3.times.) to give a brown solid. To the
solution of above brown solid in acetonitrile (6.5 mL) at 0.degree.
C. was added bisfurancarbonate (295 mg, 1.0 mmol), followed by
diisopropylethylamine (350 .mu.L, 2.0 mmol) and DMAP (24 mg). The
mixture was warmed to 25.degree. C., and was stirred for 12 hrs.
The mixture was diluted with EtOAc, and was washed sequentially
with water (2.times.), 0.5 N HCl (2.times.), water (2.times.), 0.5
N NaOH solution (2.times.), water (2.times.), and brine (1.times.),
and dried over MgSO.sub.4. Purification by flash column
chromatography (hexanes/EtOAc=1/1) afford compound 13 (540 mg).
Example 14
[1492] Compound 14: To a solution of compound 13 (400 mg, 0.67
mmol) in DMF (3 mL) was added imidazole (143 mg, 2.10 mmol),
followed by triethylchlorosilane (224 .mu.L, 1.34 mmol). The
mixture was stirred for 12 hours. The mixture was diluted with
EtOAc, and was washed with water (5.times.) and brine, and dried
over MgSO.sub.4. Purification by flash column chromatography
(hexanes/EtOAc=2/1) gave a white solid (427 mg). To the solution of
above solid in isopropanol (18 mL) was added 20% palladium(II)
hydroxide on carbon (120 mg). The mixture was hydrogenated for 12
hours. The mixture was stirred with celite for 5 mins, and filtered
through a pad of celite. Concentration under reduced pressure gave
compound 14 (360 mg).
Example 15
[1493] Compound 15: To a solution of compound 14 (101 mg, 0.18
mmol) in CH.sub.2Cl.sub.2 (5 mL) was added Dess-Martin periodiane
(136 mg, 0.36 mmol). The mixture was stirred for 1 hour.
Purification by flash column chromatography (hexanes/EtOAc=2/1)
gave compound 15 (98 mg).
Example 16
[1494] Compound 16: To a solution of compound 15 (50 mg, 0.08 mmol)
in EtOAc (0.5 mL) was added compound 11 (150 mg, 0.41 mmol). The
mixture was cooled to 0.degree. C., acetic acid (19 .mu.L, 0.32
mmol) was added, followed by sodium cyanoborohydride (10 mg, 0.16
mmol). The mixture was warmed to 25.degree. C., and was stirred for
14 hrs. The mixture was diluted with EtOAc, and was washed with
water (3.times.) and brine, and was dried over MgSO.sub.4.
Concentration gave a oil. To the solution of above oil in
acetonitrile (2.5 mL) was added 48% HF/CH.sub.3CN (0.1 mL). The
mixture was stirred for 30 minutes, and was diluted with EtOAc. The
organic phase was washed with water (3.times.) and brine
(1.times.), and was dried over MgSO.sub.4. Purification by flash
column chromatography (CH.sub.2Cl.sub.2/iPrOH=100/3) gave compound
16 (50 mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (2H, d, J=8.9
Hz), 7.15-7.05 (7H, m), 7.30 (2H, d, J=8.9 Hz), 6.64 (2H, m), 5.73
(1H, m), 5.45 (1H, m), 5.13 (1H, m), 4.93 (1H, m), 4.22-3.75 (1H,
m), 3.4 (4H, m), 3.35-2.80 (5H, m), 2.1-1.8 (3H, m), 1.40-1.25 (6H,
m), 0.94 (6H, m).
Example 17
[1495] Compound 17: To a solution of compound 16 (30 mg, 0.04 mmol)
in EtOAc (0.8 mL) was added 37% formaldehyde (26 .mu.L, 0.4 mmol).
The mixture was cooled to 0.degree. C., acetic acid (20 .mu.L, 0.4
mmol) was added, followed by sodium cyanoborohydride (22 mg, 0.4
mmol). The mixture was warmed to 25.degree. C., and was stirred for
14 hrs. The mixture was diluted with EtOAc, and was washed with
water (3.times.) and brine, and was dried over MgSO.sub.4.
Purification by flash column chromatography
(CH.sub.2Cl.sub.2/iPrOH=100/3) gave compound 17 (22 mg): .sup.1H
NMR (CDCl.sub.3) .delta. 7.63 (2H, m), 7.3-6.9 (9H, m), 6.79 (2H,
m), 5.68 (1H, m), 5.2 (1H, m), 5.10 (1H, m), 4.95 (1H, m), 4.22
(2H, m), 4.2-3.7 (21H, m), 2.0-1.7 (3H, m), 1.4-1.2 (6H, m), 0.93
(6H, m). ##STR494## ##STR495##
Example 18
[1496] Compound 18: Compound 18 was purchased from Aldrich.
Example 19
[1497] Compound 19: To compound 18 (12.25 g, 81.1 mmol) was added
37% formaldehyde (6.15 mL, 82.7 mmol) slowly. The mixture was
heated at 100.degree. C. for 1 hour. The mixture was cooled to
25.degree. C., and was diluted with benzene, and was washed with
water (2.times.). Concentration under reduced pressure gave a
yellow oil. To above oil was added 20% HCl (16 mL), and the mixture
was heated at 100.degree. C. for 12 hours. The mixture was basified
with 40% KOH solution at 0.degree. C., and was extracted with EtOAc
(3.times.). The combined organic layer was washed with water and
brine, and was dried over MgSO.sub.4. Concentration gave a oil. To
the oil was added 48% HBr (320 mL), and the mixture was heated at
120.degree. C. for 3 hours. Water was removed at 100.degree. C.
under reduced pressure to give a brown solid. To the solution of
above solid in water/dioxane (200 mL/200 mL) at 0.degree. C. was
added sodium carbonate (25.7 g, 243 mmol) slowly, followed by
di-tert-butyl dicarbonate (19.4 g, 89 mmol). The mixture was warmed
to 25.degree. C. and stirred for 12 hours. Dioxane was removed
under reduced pressure, and the remaining was extracted with EtOAc
(3.times.). The combined organic phase was washed with water
(3.times.) and brine, and was dried over MgSO.sub.4. Purification
by flash column chromatography (hexanes/EtOAc=4/1 to 3/1) gave
compound 19 as white solid (13.6 g).
Example 20
[1498] Compound 20: To a solution of compound 19 (2.49 g, 10 mmol)
in CH.sub.2Cl.sub.2 (100 mL) was added
N-phenyltrifluoromethanesulfonimide (3.93 g, 11 mmol), followed by
cesium carbonate (3.58 g, 11 mmol). The mixture was stirred for 48
hours. The solvent was removed under reduced pressure, and ethyl
acetate was added. The reaction mixture was washed with water
(3.times.) and brine (1.times.), and was dried over MgSO.sub.4.
Purification by flash column chromatography (hexanes/EtOAc=6/1)
gave a white solid (3.3 g). To the solution of above solid (2.7 g,
7.1 mmol) in DMF (40 mL) was added lithium chloride (2.11 g, 49.7
mmol), followed by dichlorobis(triphenylphosphine) palladium(II)
(100 mg, 0.14 mmol). The mixture was stirred for 3 minutes under
high vacuum and recharged with nitrogen. To the above solution was
added tributylvinyltin (2.07 mL, 7.1 mmol). The reaction mixture
was heated at 90.degree. C. for 3 hours and cooled to 25.degree. C.
Water was added to the reaction, and the mixture was extracted with
ethyl acetate (3.times.). The combined organic layer was washed
with water (6.times.) and brine, and dried over MgSO.sub.4.
Concentration gave an oil. The oil was diluted with
CH.sub.2Cl.sub.2 (5 mL), water (128 .mu.L, 7.1 mmol) and DBU (1 mL,
7.1 mmol) were added. The mixture was stirred for 5 minutes, and
was subjected to flash column chromatography (hexanes/EtOAc=9/1).
Compound 20 was obtained as white solid (1.43 g).
Example 21
[1499] Compound 21: To a solution of compound 20 (1.36 g, 5.25
mmol) in ethyl acetate (16 mL) was added water (16 mL), followed by
2.5% osmium tetraoxide/tert-butanol (2.63 mL). Sodium periodate
(2.44 g) was added in portions over 2 minutes period. The mixture
was stirred for 45 minutes, and was diluted with ethyl acetate. The
organic layer was separated and washed with water (3.times.) and
brine (1.times.), and dried over MgSO.sub.4. Concentration gave a
brown solid. To the solution of above solid in methanol (100 mL) at
0.degree. C. was added sodium borohydride. The mixture was stirred
for 1 hour at 0.degree. C., and was quenched with saturated
NH.sub.4Cl (40 mL). Methanol was removed under reduced pressure,
and the remaining was extracted with EtOAc (3.times.). The combined
organic layer was washed with water and brine, and was dried over
MgSO.sub.4. Purification by flash column chromatography
(hexanes/EtOAc 2/1) gave compound 21 (1.0 g).
Example 22
[1500] Compound 22: To a solution of compound 21 (657 mg, 2.57
mmol) in CH.sub.2Cl.sub.2 (2 mL) was added a solution of
tetrabromocarbon (1.276 g, 3.86 mmol) in CH.sub.2Cl.sub.2 (2 mL).
To the above mixture was added a solution of triphenylphosphine
(673 mg, 2.57 mmol) in CH.sub.2Cl.sub.2 (2 mL) over 30 minutes
period. The mixture was stirred for 2 hours, and was concentrated
under reduced pressure. Purification by flash column chromatography
(hexanes/EtOAc=9/1) gave the bromide intermediate (549 mg). To the
solution of above bromide (548 mg, 1.69 mmol) in acetonitrile (4.8
mL) was added dibenzyl phosphite (0.48 mL, 2.19 mmol), followed by
cesium carbonate (828 mg, 2.54 mmol). The mixture was stirred for
48 hours, and was diluted with EtOAc. The mixture was washed with
water (3.times.) and brine, and was dried over MgSO.sub.4.
Purification by flash column chromatography (hexanes/EtOAc=3/1 to
100% EtOAc) gave compound 22 (863 mg).
Example 23
[1501] Compound 23: To a solution of compound 22 (840 mg) in
ethanol (80 mL) was added 10% palladium on carbon (200 mg). The
mixture was hydrogenated for 2 hours. The mixture was stirred with
celite for 5 mins, and was filtered through a pad of celite.
Concentration under reduced pressure gave compound 23 (504 mg).
Example 24
[1502] Compound 24: To a solution of compound 23 (504 mg, 1.54
mmol) in pyridine (10.5 mL) was added phenol (1.45 g, 15.4 mmol),
followed by DCC (1.28 g, 6.2 mmol). The mixture was heated at
65.degree. C. for 3 hours, and pyridine was removed under reduced
pressure. The mixture was diluted with EtOAc (5 ml), and was
filtered and washed with EtOAc (2.times.5 mL). Concentration gave a
oil, which was purified by flash column chromatography
(CH.sub.2Cl.sub.2/isopropanol=100/3) to give diphenylphosphonate
intermediate (340 mg). To a solution of above compound (341 mg,
0.71 mmol) in THF (1 mL) was added 0.85 ml, of 1.0 N NaOH solution.
The mixture was stirred at 25.degree. C. for 3 hours, and THF was
removed under reduced pressure. The mixture was diluted with water,
and was washed with EtOAc (3.times.), and then acidified with
concentrated HCl until pH=1. The aqueous was extracted with EtOAc
(3.times.). The combined organic layer was washed with water
(1.times.) and brine (1.times.), and dried over MgSO.sub.4.
Concentration under reduced pressure gave compound 24 as a solid
(270 mg).
Example 25
[1503] Compound 25: To a solution of compound 24 (230 mg, 0.57
mmol) in DMF (2 mL) was added ethyl (s)-lactate (130 mL, 1.14
mmol), followed by diisopropylethylamine (400 .mu.L, 2.28 mmol) and
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (504 mg, 1.14 mmol). The mixture was stirred
for 14 hours, was diluted with EtOAc. The organic phase was washed
with water (5.times.) and brine (1.times.), and was dried over
MgSO.sub.4. Purification by flash column chromatography
(CH.sub.2Cl.sub.2/isopropanol=100/3) gave compound 25 (220 mg).
Example 26
[1504] Compound 26: To a solution of compound 25 (220 mg) in
CH.sub.2Cl.sub.2 (2 mL) was added trifluoroacetic acid (1 mL). The
mixture was stirred for 2 hrs, and was concentrated under reduced
pressure. The mixture was diluted with EtOAc, and was washed with
saturated sodium carbonate solution, water, and brine, and was
dried over MgSO.sub.4. Concentration gave compound 26 (170 mg).
Example 27
[1505] Compound 27: To a solution of compound 15 (258 mg, 0.42
mmol) in EtOAc (2.6 mL) was added compound 26 (170 mg, 0.42 mmol),
followed by acetic acid (75 .mu.L, 1.26 mmol). The mixture was
stirred for 5 minutes, and sodium cyanoborohydride (53 mg, 0.84
mmol) was added. The mixture was stirred for 14 hrs. The mixture
was diluted with EtOAc, and was washed with saturated sodium
bicarbonate solution, water (3.times.) and brine, and was dried
over MgSO.sub.4. Purification by flash column chromatography
(CH.sub.2Cl.sub.2/iPrOH=100/4 to 100/6) gave the intermediate (440
mg). To the solution of above compound (440 mg) in acetonitrile (10
mL) was added 48% HF/CH.sub.3CN (0.4 mL). The mixture was stirred
for 2 hours, and acetonitrile was removed under reduced pressure.
The remaining was diluted with EtOAc, and was washed with water
(3.times.) and brine (1.times.), and was dried over MgSO.sub.4.
Purification by flash column chromatography
(CH.sub.2Cl.sub.2/iPrOH=100/5) gave compound 27 (120 mg): .sup.1H
NMR (CDCl.sub.3) .delta. 7.70 (2H, m), 7.27 (2H, m), 7.15 (5H, m),
6.95 (3H, m), 5.73 (1H, m), 5.6-5.4 (1H, m), 5.16 (1H, m), 4.96
(1H, m), 4.22-3.60 (13H, m), 3.42 (2H, m), 3.4-2.6 (11H, m),
2.1-3.8 (3H, m), 1.39 (3H, m), 1.24 (3H, m), 0.84 (6H, m).
##STR496## ##STR497##
Example 28
[1506] Compound 28: To a solution of compound 19 (7.5 g, 30 mmol)
in acetonitrile (420 mL) was added dibenzyl triflate (17.8 g, 42
mmol), followed by cesium carbonate (29.4 g, 90 mmol). The mixture
was stirred for 2.5 hours, and was filtered. Acetonitrile was
removed under reduced pressure, and the remaining was diluted with
EtOAc. The mixture was washed with water (3.times.) and brine, and
was dried over MgSO.sub.4. Purification by flash column
chromatography (hexanes/EtOAc=2/1 to 1/1) gave compound 28 (14.3
g).
Example 29
[1507] Compound 29: To a solution of compound 28 (14.3 g) in
ethanol (500 mL) was added 10% palladium on carbon (1.45 g). The
mixture was hydrogenated for 2 hours. The mixture was stirred with
celite for 5 mins, and was filtered through a pad of celite.
Concentration under reduced pressure gave compound 29 (9.1 g).
Example 30
[1508] Compound 30: To a solution of compound 29 (9.1 g) in
CH.sub.2Cl.sub.2 (60 mL) was added trifluoroacetic acid (30 mL).
The mixture was stirred for 4 hrs, and was concentrated under
reduced pressure. The mixture was coevaporated with
CH.sub.2Cl.sub.2 (3.times.) and toluene, and was dried under high
vacuum to give a white solid. The white solid was dissolved in 2.0
N NaOH solution (45 mL, 90 mmol), and was cooled to 0.degree. C. To
the above solution was added slowly a solution of benzyl
chloroformate (6.4 mL, 45 mmol) in toluene (7 mL). The mixture was
warmed to 25.degree. C., and was stirred for 6 hours. 2.0 N sodium
hydroxide was added to above solution until pH=11. The aqueous was
extracted with ethyl ether (3.times.), and was cooled to 0.degree.
C. To the above aqueous phase at 0.degree. C. was added
concentrated HCl until pH=1. The aqueous was extracted with EtOAc
(3.times.). The combine organic layers were washed with brine, and
were dried over MgSO.sub.4. Concentration gave compound 30 (11.3 g)
as a white solid.
Example 31
[1509] Compound 31: To the suspension of compound 30 (11.3 g, 30
mmol) in toluene (150 mL) was added thionyl chloride (13 mL, 180
mmol), followed by DMF (a few drops). The reaction mixture was
heated at 65.degree. C. for 4.5 hrs, and evaporated under reduced
pressure. The mixture was coevaporated with toluene (2.times.) to
afford a brown solid. To the solution of the brown solid in
CH.sub.2Cl.sub.2 (120 ml) at 0.degree. C. was added phenol (11.28
g, 120 mmol), followed by slow addition of pyridine (14.6 mL, 180
mmol). The reaction mixture was allowed to warm to 25.degree. C.
and stirred for 14 hrs. Solvents were removed under reduced
pressure. The mixture was diluted with EtOAc, and washed with water
(3.times.) and brine (1.times.), and dried over MgSO.sub.4.
Concentration gave a dark oil, which was purified by flash column
chromatography (hexanes/EtOAc=3/1 to 1/1) to afford compound 31
(9.8 g).
Example 32
[1510] Compound 32: To a solution of compound 31 (9.8 g, 18.5 mmol)
in THF (26 mL) was added 20.3 ml, of 1.0 N NaOH solution. The
mixture was stirred at 25.degree. C. for 2.5 hours, and THF was
removed under reduced pressure. The mixture was diluted with water,
and was washed with EtOAc (3.times.). The aqueous phase was cooled
to 0.degree. C., and was acidified with concentrated HCl until
pH=1. The aqueous was extracted with EtOAc (3.times.). The combined
organic layer was washed with water (1.times.) and brine
(1.times.), and dried over MgSO.sub.4. Concentration under reduced
pressure gave a solid (8.2 g). To a suspension of above solid (4.5
g, 10 mmol) in toluene (50 mL) was added thionyl chloride (4.4 mL,
60 mmol), followed by DMF (0.2 mL). The mixture was heated at
70.degree. C. for 3.5 hours. The solvent and reagent were removed
under reduced pressure. The mixture was coevaporated with toluene
(2.times.) to give a white solid. To the solution of the above
solid in CH.sub.2Cl.sub.2 (40 mL) at 0.degree. C. was added ethyl
(s)-lactate (2.3 mL, 20 mmol), followed by pyridine (3.2 mL, 40
mmol). The mixture was warmed to 25.degree. C. and stirred for 12
hours. The reaction mixture was concentrated under reduced
pressure, and was diluted with EtOAc. The organic phase was washed
with 1 N HCl, water, and brine, and was dried over MgSO.sub.4.
Purification by flash column chromatography (hexanes/EtOAc 2/1 to
1/1) gave compound 32 (4.1 g).
Example 33
[1511] Compound 33: To a solution of compound 32 (3.8 g, 6.9 mmol)
in EtOAc/EtOH (30 mL/30 mL) was added 10% palladium on carbon (380
mg), followed by acetic acid (400 .mu.L, 6.9 mmol). The mixture was
hydrogenated for 3 hours. The mixture was stirred with celite for 5
mins, and was filtered through a pad of celite. Concentration under
reduced pressure gave compound 33 (3.5 g).
Example 34
[1512] Compound 34: To a solution of compound 15 (1.70 g, 2.76
mmol) in EtOAc (17 mL) was added compound 33 (3.50 g, 6.9 mmol).
The mixture was stirred for 5 minutes, and was cooled to 0.degree.
C., and sodium cyanoborohydride (347 mg, 5.52 mmol) was added. The
mixture was stirred for 6 hrs. The mixture was diluted with EtOAc,
and was washed with saturated sodium bicarbonate solution, water
(3.times.) and brine, and was dried over MgSO.sub.4. Purification
by flash column chromatography (CH.sub.2Cl.sub.2/iPrOH=100/6) gave
the intermediate (3.4 g). To the solution of above compound (3.4 g)
in acetonitrile (100 mL) was added 48% HF/CH.sub.3CN (4 mL). The
mixture was stirred for 2 hours, and acetonitrile was removed under
reduced pressure. The remaining was diluted with EtOAc, and was
washed with saturated sodium carbonate, water (3.times.), and brine
(1.times.), and was dried over MgSO.sub.4. Purification by flash
column chromatography (CH.sub.2Cl.sub.2/iPrOH=100/5) gave compound
34 (920 mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (2H, m),
7.38-7.19 (5H, m), 6.92 (3H, m), 6.75 (2H, m), 5.73 (1H, m),
5.57-5.35 (1H, m), 5.16 (2H, m), 4.5 (2H, m), 4.2-3.6 (13H, m),
3.25-2.50 (11H, m), 2.0-1.8 (3H, m), 1.5 (3H, m), 1.23 (3H, m),
0.89 (6H, m).
Example 35
[1513] Compound 35: To a solution of compound 34 (40 mg) in
CH.sub.3CN/DMSO (1 mL/0.5 mL) was added 1.0 M PBS buffer (5 mL),
followed by esterase (200 .mu.L). The mixture was heated at
40.degree. C. for 48 hours. The mixture was purified by reverse
phase HPLC to give compound 35 (11 mg). ##STR498##
Example 36
[1514] Compound 36: Compound 36 was purchased from Aldrich.
Example 37
[1515] Compound 37: To a solution of compound 36 (5.0 g, 40 mmol)
in chloroform (50 mL) was added thionyl chloride (12 mL) slowly.
The mixture was heated at 60.degree. C. for 2.5 hours. The mixture
was concentrated under reduced pressure to give a yellow solid. To
the suspension of above solid (5.2 g, 37 mmol) in toluene (250 mL)
was added triethyl phosphite (19 mL, 370 mmol). The mixture was
heated at 120.degree. C. for 4 hours, and was concentrated under
reduced pressure to give a brown solid. The solid was dissolved in
EtOAc, and was basified with 1.0 N NaOH. The organic phase was
separated and was washed with water (2.times.) and brine, and was
dried over MgSO.sub.4. Purification by flash column chromatography
(CH.sub.2Cl.sub.2/iPrOH=9/1) gave compound 37 (4.8 g).
Example 38
[1516] Compound 38: To a solution of compound 14 (100 mg, 0.16
mmol) and compound 37 (232 mg, 0.74 mmol) in CH.sub.2Cl.sub.2 (1
mL) at -40.degree. C. was added triflic anhydride (40 .mu.L, 0.24
mmol) slowly. The mixture was warmed to 25.degree. C. slowly, and
was stirred for 12 hours. The mixture was concentrated, and was
diluted with EtOH/EtOAc (2 mL/0.4 mL). To the above solution at
0.degree. C. was added sodium borohydride (91 mg) in portions. The
mixture was stirred at 0.degree. C. for 3 hours, and was diluted
with EtOAc. The mixture was washed with saturated sodium
bicarbonate, water, and brine, and was dried over MgSO.sub.4.
Purification by flash column chromatograph
(CH.sub.2Cl.sub.2/iPrOH=10015 to 100/10) gave the intermediate (33
mg). To the solution of above intermediate in acetonitrile (2.5 mL)
was added 48% HF/CH.sub.3CN (0.1 mL). The mixture was stirred for
30 minutes, and was diluted with EtOAc. The organic solution was
washed with 0.5 N sodium hydroxide, water, and brine, was dried
over MgSO.sub.4. Purification by reverse HPLC gave compound 38 (12
mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (2H, d, J=8.9 Hz), 7.02
(2H, d, J=8.9 Hz), 5.70 (1H, m), 5.45 (1H, m), 5.05 (1H, m),
4.2-3.4 (19H, m), 3.4-2.8 (5H, m), 2.45-2.20 (4H, m), 2.15-1.81
(5H, m), 1.33 (6H, m), 0.89 (6H, m). ##STR499##
Example 39
[1517] Compound 39 was prepared by the methods of the previous
Examples.
Example 40
[1518] Compound 40: To the suspension of compound 39 (4.25 g, 16.4
mmol) in toluene (60 mL) was added thionyl chloride (7.2 mL, 99
mmol), followed by DMF (a few drops). The reaction mixture was
heated at 65.degree. C. for 5 hrs, and evaporated under reduced
pressure. The mixture was coevaporated with toluene (2.times.) to
afford a brown solid. To the solution of the brown solid in
CH.sub.2Cl.sub.2 (60 ml) at 0.degree. C. was added
2,6-dimethylphenol (8.1 g, 66 mmol), followed by slow addition of
pyridine (8 mL, 99 mmol). The reaction mixture was allowed to warm
to 25.degree. C. and stirred for 14 hrs. Solvents were removed
under reduced pressure. The mixture was diluted with EtOAc, and
washed with water (3.times.) and brine (1.times.), and dried over
MgSO.sub.4. Purification by flash column chromatography
(hexanes/EtOAc=3/1 to 1/1) afforded compound 40 (1.38 g).
Example 41
[1519] Compound 41: To a solution of compound 40 (1.38 g, 1.96
mmol) in THF (6 mL) was added 3.55 ml, of 1.0 N NaOH solution. The
mixture was stirred at 25.degree. C. for 24 hours, and THF was
removed under reduced pressure. The mixture was diluted with water,
and was washed with EtOAc (3.times.). The aqueous phase was cooled
to 0.degree. C., and was acidified with concentrated HCl until
pH=1. The aqueous was extracted with EtOAc (3.times.). The combined
organic layer was washed with water (1.times.) and brine
(1.times.), and dried over MgSO.sub.4. Concentration under reduced
pressure gave compound 41 as a white solid (860 mg).
Example 42
[1520] Compound 42: To a suspension of compound 41 (1.00 g, 2.75
mmol) in toluene (15 mL) was added thionyl chloride (1.20 mL, 16.5
mmol), followed by DMF (3 drops). The mixture was heated at
65.degree. C. for 5 hours. The solvent and reagent were removed
under reduced pressure. The mixture was coevaporated with toluene
(2.times.) to give a brown solid. To the solution of the above
solid in CH.sub.2Cl.sub.2 (11 mL) at 0.degree. C. was added ethyl
(s)-lactate (1.25, 11 mmol), followed by pyridine (1.33 mL, 16.6
mmol). The mixture was warmed to 25.degree. C. and stirred for 12
hours. The reaction mixture was concentrated under reduced
pressure, and was diluted with EtOAc. The organic phase was washed
with 1 N HCl, water, and brine, and was dried over MgSO.sub.4.
Purification by flash column chromatography (hexanes/EtOAc=1.5/1 to
1/1) gave compound 42 (470 mg).
Example 43
[1521] Compound 43: To a solution of compound 42 (470 mg) in EtOH
(10 mL) was added 10% palladium on carbon (90 mg), followed by
acetic acid (150 .mu.L). The mixture was hydrogenated for 6 hours.
The mixture was stirred with celite for 5 mins, and was filtered
through a pad of celite. Concentration under reduced pressure gave
compound 43 (400 mg).
Example 44
[1522] Compound 44: To a solution of compound 6 (551 mg, 0.93 mmol)
in 1,2-dichloroethane (4 mL) was added compound 43 (400 mg, 1.0
mmol), followed by MgSO.sub.4 (1 g). The mixture was stiffed for 3
hours, and acetic acid (148 .mu.L) and sodium cyanoborohydride (117
mg, 1.86 mmol) were added sequentially. The mixture was stirred for
1 hour. The mixture was diluted with EtOAc, and was washed with
saturated sodium bicarbonate solution, water (3.times.) and brine,
and was dried over MgSO.sub.4. Purification by flash column
chromatography (EtOAc to EtOAc/EtOH=9/1) gave compound 44. Compound
44 was dissolved in CH.sub.2Cl.sub.2 (25 mL), and trifluoroacetic
acid (100 mL) was added. The mixture was concentrated to give
compound 44 as a TFA salt (560 mg): .sup.1H NMR (CDCl.sub.3)
.delta. 7.74 (2H, m), 7.39 (2H, m), 7.20 (2H, m), 7.03 (5H, m),
5.68 (1H, m), 5.43 (1H, m), 5.01 (1H, m), 4.79 (1H, m), 4.35-4.20
(4H, m), 4.18-3.4 (1H, m), 3.2-2.6 (9H, m), 2.30 (6H, m), 1.82 (1H,
m), 1.70 (2H, m), 1.40-1.18 (6H, m), 0.91 (6H, m). ##STR500##
Example 45
[1523] Compound 45: To a suspension of compound 41 (863 mg, 2.4
mmol) in toluene (13 mL) was added thionyl chloride (1.0 mL, 14.3
mmol), followed by DMF (3 drops). The mixture was heated at
65.degree. C. for 5 hours. The solvent and reagent were removed
under reduced pressure. The mixture was coevaporated with toluene
(2.times.) to give a brown solid. To the solution of the above
solid in CH.sub.2Cl.sub.2 (10 mL) at 0.degree. C. was added propyl
(s)-lactate (1.2 mL, 9.6 mmol), followed by triethylamine (2.0 mL,
14.4 mmol). The mixture was warmed to 25.degree. C. and stirred for
12 hours. The reaction mixture was concentrated under reduced
pressure, and was diluted with EtOAc. The organic phase was washed
with water and brine, and was dried over MgSO.sub.4. Purification
by flash column chromatography (hexanes/EtOAc=1.5/1 to 1/1) gave
compound 45 (800 mg).
Example 46
[1524] Compound 46: To a solution of compound 45 (785 mg) in EtOH
(17 mL) was added 10% palladium on carbon (150 mg), followed by
acetic acid (250 .mu.L). The mixture was hydrogenated for 16 hours.
The mixture was stirred with celite for 5 mins, and was filtered
through a pad of celite. Concentration under reduced pressure gave
compound 46 (700 mg).
Example 47
[1525] Compound 47: To a solution of compound 6 (550 mg, 0.93 mmol)
in 1,2-dichloroethane (4 mL) was added compound 43 (404 mg, 1.0
mmol), followed by MgSO.sub.4 (1 g). The mixture was stirred for 3
hours, and acetic acid (148 .mu.L) and sodium cyanoborohydride (117
mg, 1.86 mmol) were added sequentially. The mixture was stirred for
1 hour. The mixture was diluted with EtOAc, and was washed with
saturated sodium bicarbonate solution, water (3.times.) and brine,
and was dried over MgSO.sub.4. Purification by flash column
chromatography (EtOAc to EtOAc/EtOH=9/1) gave compound 47. Compound
47 was dissolved in CH.sub.2Cl.sub.2 (25 mL), and trifluoroacetic
acid (100 .mu.L) was added. The mixture was concentrated to give
compound 47 as a TFA salt (650 mg): .sup.1H NMR (CDCl.sub.3)
.delta. 7.74 (2H, m), 7.41 (2H, m), 7.25-7.1 (2H, m), 7.02 (5H, m),
5.65 (1H, m), 5.50 (1H, m), 5.0-4.75 (2H, m), 4.25-4.05 (4H, m),
4.0-3.4 (11H, m), 3.2-2.6 (9H, m), 2.31 (6H, m), 1.82-1.51 (3H, m),
1.45-1.2 (5H, m), 0.93 (9H, m). ##STR501##
Example 48
[1526] Compound 48 was made by the methods of the previous
Examples.
Example 49
[1527] Compound 49: To a solution of compound 48 (100 mg, 0.13
mmol) in pyridine (0.75 mL) was added L-alanine methyl ester
hydrochloride (73 mg, 0.52 mmol), followed by DCC (161 mg, 0.78
mmol). The mixture was heated at 60.degree. C. for 1 hour. The
mixture was diluted with EtOAc, and was washed with 0.2 N HCl,
water, 5% sodium bicarbonate, and brine, and was dried over
MgSO.sub.4. Purification by flash column chromatography
(CH.sub.2Cl.sub.2/iPrOH=100/5) gave compound 49 (46 mg): .sup.1H
NMR (CDCl.sub.3) .delta. 7.73 (2H, m), 7.38-7.18 (7H, m), 7.03 (2H,
m), 6.89 (2H, m), 5.68 (1H, m), 5.05 (1H, m), 4.95 (1H, m), 4.30
(3H, m), 4.0-3.6 (12H, m), 3.2-2.8 (7H, m), 1.84-1.60 (3H, m), 1.38
(3H, m), 0.93 (6H, m).
Example 50
[1528] Compound 50: To a solution of compound 48 (100 mg, 0.13
mmol) in pyridine (0.75 mL) was added methyl (s)-lactate (41 mg,
0.39 mmol), followed by DCC (81 mg, 0.39 mmol). The mixture was
heated at 60.degree. C. for 2 hours, and pyridine was removed under
reduced pressure. The mixture was diluted with EtOAc (5 mL), and
was filtered. Purification by flash column chromatography
(CH.sub.2Cl.sub.2/iPrOH=100/5) gave compound 50 (83 mg): .sup.1H
NMR (CDCl.sub.3) .delta. 7.74 (2H, m), 7.38-7.14 (7H, m), 7.02 (2H,
m), 6.93 (2H, m), 5.67 (1H, m), 5.18 (1H, m), 5.04 (1H, m), 4.92
(1H, m), 4.5 (2H, m), 4.0-3.68 (12H, m), 3.2-2.75 (7H, m), 1.82
(1H, m), 1.75-1.50 (5H, m), 0.93 (6H, m). ##STR502##
Example 51
[1529] Compound 51: To a solution of benzyl (s)-lactate (4.0 g, 20
mmol) in DMF (40 mL) was added imidazole (2.7 g, 20 mmol), followed
by tert-butyldimethylsilyl chloride (3.3 g, 22 mmol). The mixture
was stirred for 14 hours, and diluted with EtOAc. The organic phase
was washed with 1.0 N HCl solution (2.times.), water (2.times.),
and brine (1.times.), and dried over MgSO.sub.4. Concentration gave
the lactate intermediate (6.0 g). To the solution of the above
intermediate in EtOAc (200 mL) was added 10% Palladium on carbon
(700 mg). The mixture was hydrogenated for 2 hours. The mixture was
stirred with celite for 5 minutes, and was filtered through a pad
of celite. Concentration gave compound 51 (3.8 g).
Example 52
[1530] Compound 52: To a solution of compound 51 (1.55 g, 7.6 mmol)
in CH.sub.2Cl.sub.2 (20 mL) was added
4-benzyloxycarbonylpiperidineethanol (2.00 g, 7.6 mmol), followed
by benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate (4.74 g, 9.1 mmol) and diisopropylethylamine
(1.58 mL, 9.1 mmol). The mixture was stirred for 14 hours, and
dichloromethane was removed. The mixture was diluted with EtOAc,
and was washed with brine, and dried with MgSO.sub.4. Purification
by flash column chromatography (hexanes/EtOAc=10/1) gave compound
52 (1.50 g).
Example 53
[1531] Compound 53: To a solution of compound 52 (1.50 g) in
CH.sub.3CN was added 58% FF/CH.sub.3CN (5 mL). The mixture was
stirred for 30 minutes, and acetonitrile was removed under reduced
pressure. The mixture was diluted with EtOAc, and was washed with
water and brine, and was dried over MgSO.sub.4. Purification by
flash column chromatography (hexanes/EtOAc=1/1) gave compound 53
(1.00 g).
Example 54
[1532] Compound 54: To a solution of compound 48 (769 mg, 1.0 mmol)
in pyridine (6.0 mL) was added compound 53 (1.0 g, 3.0 mmol),
followed by DCC (618 mg, 3.0 mmol). The mixture was heated at
60.degree. C. for 2 hours, and pyridine was removed under reduced
pressure. The mixture was diluted with EtOAc (5 mL), and was
filtered. Purification by flash column chromatography
(CH.sub.2Cl.sub.2/iPrOH=100/4) gave compound 54 (630 mg).
Example 55
[1533] Compound 55: To a solution of compound 54 (630 mg, 0.58
mmol) in EtOAc (30 mL) was added 10% Palladium on carbon (63 mg),
followed by acetic acid (80 .mu.L). The mixture was hydrogenated
for 2 hours. The mixture was stirred with celite for 5 minutes, and
was filtered through a pad of celite. Concentration gave the
intermediate. To the solution of the above intermediate in EtOAc
(10 mL) was added 37% formaldehyde (88 .mu.L, 1.18 mmol), followed
by acetic acid (101 .mu.L, 1.77 mmol). The mixture was cooled to
0.degree. C., and sodium cyanoborohydride (74 mg, 1.18 mmol) was
added. The mixture was stirred at 25.degree. C. for 80 minutes, and
was diluted with EtOAc. The mixture was washed with water and
brine, and was dried over MgSO.sub.4. Concentration gave compound
55 as a white solid (530 mg): .sup.1H NMR (CDCl.sub.3) .delta. 7.74
(2H, m), 7.40-7.15 (7H, m), 7.03 (2H, m), 6.92 (2H, m), 5.66 (1H,
m), 5.20-5.00 (3H, m), 4.58-4.41 (2H, m), 4.16 (2H, m), 4.0-3.7
(9H, m), 3.4-2.6 (14H, m), 1.90-1.50 (13H, m), 0.92 (6H, m).
##STR503##
Example 56
[1534] Compound 56 was made by the methods of the previous
Examples.
Example 57
[1535] Compound 57: To a solution of compound 56 (100 mg, 0.12
mmol) in pyridine (0.6 mL) was added N-hydroxymorpholine (50 mg,
0.48 mmol), followed by DCC (99 mg, 0.48 mmol). The mixture was
stirred for 14 hours, and pyridine was removed under reduced
pressure. The mixture was diluted with EtOAc, and was filtered.
Purification by flash column chromatography
(CH.sub.2Cl.sub.2/iPrOH=100/5) gave compound 57 (53 mg): .sup.1H
NMR (CDCl.sub.3) .delta. 7.71 (2H, d, J=8.6 Hz), 7.15 (2H, d, J=7.6
Hz), 6.99 (2H, d, J=8.8 Hz), 6.90 (2H, m), 5.67 (1H, m), 5.18 (1H,
m), 5.05 (1H, m), 4.95 (1H, m), 4.58-4.38 (2H, m), 4.21 (2H, m),
4.02-3.80 (13H, m), 3.55-3.38 (2H, m), 3.2-2.78 (9H, m), 1.9-1.8
(1H, m), 1.8-0.95 (5H, m), 1.29 (3H, m), 0.93 (6H, m).
Example 58
[1536] Compound 58: To a solution of compound 56 (100 mg, 0.12
mmol) in pyridine (0.6 mL) was added N,N-dimethylhydroxylamine
hydrochloride (47 mg, 0.48 mmol), followed by DCC (99 mg, 0.48
mmol). The mixture was stirred for 6 hours, and pyridine was
removed under reduced pressure. The mixture was diluted with EtOAc,
and was filtered. Purification by flash column chromatography
(CH.sub.2Cl.sub.2/iPrOH=100/5) gave compound 58 (35 mg). .sup.1H
NMR (CDCl.sub.3) .delta. 7.71 (2H, d, J=8.9 Hz), 7.15 (2H, d, J=8.2
Hz), 6.99 (2H, d, J=8.4 Hz), 6.89 (2H, m), 5.65 (1H, d, J=5.2 Hz),
5.15 (1H, m), 4.98 (2H, m), 4.42 (2H, m), 4.18 (2H, m), 4.0-3.6
(9H, m), 3.2-2.7 (13H, m), 1.92-1.45 (6H, m), 1.25 (3H, m), 0.90
(6H, m). ##STR504##
[1537] Aminomethylphosphonic acid 59 is protected as benzyl
carbamate. The phosphonic acid is treated with thionyl chloride to
generate dichloridate, which reacts with phenol or
2,6-dimethylphenol to give compound 60. Compound 60 is hydrolyzed
with sodium hydroxide, followed by acidification to afford monoacid
61. Monoacid 61 is treated with thionyl chloride to generate
monochloridate, which reacts with different alkyl (s)-lactates to
form compound 62. Compound 62 is hydrogenated with 10% Pd--C in the
presence of acetic acid to give compound 63. Compound 63 reacts
with aldehyde 6 in the presence of MgSO.sub.4 to form imine, which
is reduced with sodium cyanoborohydride to generate compound 64.
##STR505##
[1538] I.a. n-BuLi; b. compound 15; 11. H.sub.2/10% Pd--C/HOAc; IV.
PPh.sub.3/DEAD
[1539] Compound 65 is prepared from 2-hydroxy-5-bromopyridine by
alkylation. J. Med. Chem. 1992, 35, 3525. Compound 65 is treated
with n-Butyl lithium to generate aryl lithium, which reacts with
aldehyde 15 to form compound 66. J. Med. Chem. 1994, 37, 3492.
Compound 66 is hydrogenated with 10% Pd--C in the presence of
acetic acid to give compound 67. J. Med. Chem. 2000, 43, 721.
Compound 68 is prepared from compound 67 with corresponding alcohol
under Mitsunobu reaction conditions. Bioorg. Med. Chem. Lett. 1999,
9, 2747. ##STR506##
Example 1
[1540] Methyl
2-(S)-(dimethylethoxycarbonylamino)-3-(4-pyridyl)propanoate (2): A
solution of N-tert-Butoxycarbonyl-4-pyridylalanine (1, 9.854 g, 37
mmol, Peptech), 4-dimethylaminopyridine (4.52 g, 37 mmol, Aldrich),
and dicyclohexylcarbodiimide (15.30 g, 74.2 mmol, Aldrich) in
methanol (300 mL) was stirred at 0.degree. C. for 2 h and at room
temperature for 12 h. After the solids were removed by filtration,
the filtrate was concentrated under reduced pressure. More
dicyclohexylurea was removed by repeated trituration of the
concentrated residue in EtOAc followed by filtration. The residue
was chromatographed on silica gel to afford the methyl ester 2
(9.088 g, 88%): .sup.1H NMR (CDCl.sub.3) .delta. 8.53 (d, 2H, J=5.7
Hz), 7.09 (d, 2H, J=5.7 Hz), 5.04 (br, 1H), 4.64 (br, 1H), 3.74 (s,
3H), 3.16 (dd, 1H, J=13.5 and 5.7 Hz), 3.02 (dd, 1H, J=13.5 and 6.3
Hz), 1.42 (s, 9H); MS (ESI) 281 (M+H).
Example 2
[1541]
1-Chloro-3-(S)-(dimethylethoxycarbonylamino)-4-(4-pyridyl)-2-(S)-b-
utanol (3): A solution of diisopropylamine (37.3 mL, 266 mmol,
Aldrich) in THF (135 mL) was stirred at -78.degree. C. as a
solution of n-butyllithium (102 ml, of 2.3 M solution and 18 ml, of
1.4 M solution 260 mmol, Aldrich) in hexane was added. After 10
min, the cold bath was removed and stirred the solution for 10 min
at the ambient temperature. The solution was cooled at -78.degree.
C. again and stirred as a solution of chloroacetic acid (12.255 g,
130 mmol, Aldrich) in THF (50 mL) was added over 20 min. After the
solution was stirred for 15 min, this dianion solution was
transferred to a stirred solution of the methyl ester 2 (9.087 g,
32.4 mmol) in THF (100 mL) at 0.degree. C. over 15 min. The
resulting yellow slurry was stirred at 0.degree. C. for 10 min and
cooled at -78.degree. C. A solution of acetic acid (29 mL, 507
mmol, Aldrich) in THF (29 mL) was added quickly to the slurry and
the resulting slurry was stirred at -78.degree. C. for 30 min, at
0.degree. C. for 30 min, and at room temperature for 15 min. The
resulting slurry was dissolved in saturated NaHCO.sub.3 solution
(750 mL) and EtOAc (500 mL). The separated aqueous layer was
extracted with EtOAc (300 mL.times.2) and the combined organic
fractions were washed with water (750 mL.times.2) and saturated
NaCl solution (250 mL). The resulting solution was dried
(MgSO.sub.4) and evaporated under reduced pressure.
[1542] A solution of the residue in THF (170 mL) and water (19 mL)
was stirred at 0.degree. C. as NaBH.sub.4 (3.375 g, 89.2 mmol,
Aldrich) was added. After 30 min, the solution was evaporated under
reduced pressure and the residue was dissolved in EtOAc, acidified
with aqueous NaHSO.sub.4, and then neutralized by adding saturated
aqueous NaHCO.sub.3 solution. The separated aqueous fraction was
extracted with EtOAc (100 mL) and the combined organic fractions
were washed with water (500 mL) and saturated NaCl solution (100
mL). The solution was dried (MgSO.sub.4) and evaporated under
reduced pressure. The residue was chromatographed on silica gel to
afford the chlorohydrin 3 and 4 (4.587 g, 47%) as a mixture of two
diastereomers (3.about.4:1). The obtained mixture was
recrystallized from EtOAc-hexane twice to obtain pure desired
diastereomer 3 (2.444 g, 25%) as yellow crystals: .sup.1H NMR
(CDCl.sub.3) .delta. 8.53 (d, 2H, J=5.7 Hz), 7.18 (d, 2H, J=5.7
Hz), 4.58 (br, 1H), 3.94 (m, 1H), 3.87 (br, 1H), 3.75-3.54 (m, 2H),
3.05 (dd, 1H, J=13.8 and 3.9 Hz), 2.90 (dd, 1H, J=13.8 and 8.4 Hz),
1.36 (s, 9H); MS (ESI) 301 (M+H).
Example 3
[1543] The epoxide 5: A solution of the chlorohydrin 3 (1.171 g,
3.89 mmol) in ethanol (39 mL) was stirred at room temperature as
0.71 M KOH in ethanol (6.6 mL) was added. After 1.5 h, the mixture
was concentrated under reduced pressure and the residue was
dissolved in EtOAc (60 mL) and water (60 mL). The separated aqueous
fraction was extracted with EtOAc (60 mL) and the combined organic
fractions were washed with saturated NaCl solution, dried
(MgSO.sub.4), and concentrated under reduced pressure to obtain the
epoxide (1.058 g, quantitative): .sup.1H NMR (CDCl.sub.3) .delta.
8.52 (d, 2H, J=6.0 Hz), 7.16 (d, 2H, J=6.0 Hz), 4.57 (d, 1H, J=7.8
Hz), 3.76 (br, 1H), 3.02-2.92 (m, 2H), 2.85-2.79 (m, 2H), 2.78-2.73
(m, 1H), 1.37 (s, 9H); MS (ESI) 265 (M+H).
Example 4
[1544] The hydroxy-amine 6: A solution of the epoxide 5 obtained
above and i-BuNH.sub.2 (3.9 mL, 39.2 mmol, Aldrich) in 58 ml, of
i-PrOH was stirred at 65.degree. C. for 2 h and the solution was
concentrated under reduced pressure. The residual i-PrOH was
removed by dissolving the residue in toluene and concentration of
the solution twice: .sup.1H NMR (CDCl.sub.3) .delta. 8.51 (d, 2H,
J=6.0 Hz), 7.18 (d, 2H, J=6.0 Hz), 4.70 (d, 1H, J=9.6 Hz), 3.86
(br, 1H), 3.46 (q, 1H, J=5.8 Hz), 3.06 (dd, 1H, J=14.1 and 3.9 Hz),
2.79 (dd, 1H, J=14.1 and 9.0 Hz), 2.76-2.63 (m, 3H), 2.43 (m, 2H,
J=6.9 Hz), 1.73 (m, 1H, J=6.6 Hz), 1.36 (s, 9H), 0.93 (d, 3H, J=6.6
Hz), 0.92 (d, 3H, J=6.6 Hz); MS (ESI) 338 (M+H).
Example 5
[1545] The sulfoamide 7: A solution of the crude 6 and
p-methoxybenzene sulfonyl chloride (890 mg, 4.31 mmol, Aldrich) in
CH.sub.2Cl.sub.2 (24 mL) was stirred at 0.degree. C. for 2 h and at
room temperature for 13 h. The solution was washed with saturated
NaHCO.sub.3 solution and the aqueous washing was extracted with
CH.sub.2Cl.sub.2 (60 mL). After the combined organic fractions were
dried (MgSO.sub.4) and concentrated under reduced pressure, the
residue was purified by chromatography on silica gel to obtain the
sulfoamide 7 (1.484 g, 75%): .sup.1H NMR (CDCl.sub.3) .delta. 8.51
(d, 2H, J=5.7 Hz), 7.73 (d, 2H, J=8.7 Hz), 7.21 (d, 2H, J=5.7 Hz),
7.00 (d, 2H, J=8.7 Hz), 4.68 (d, 1H, J=8.1 Hz), 4.08 (br, 1H), 3.88
(s, 3H), 3.83 (br, 2H), 3.09 (d, 2H, J=5.1 Hz), 3.06-2.80 (m, 4H),
1.85 (m, 1H, J=7.0 Hz), 1.34 (s, 9H), 0.92 (d, 3H, J=6.3 Hz), 0.89
(d, 3H, J=6.6 Hz); MS (ESI) 508 (M+H).
Example 6
[1546] The bisfurancarbamate 9: A solution of the sulfoamide 7
(1.484 g, 2.92 mmol) and trifluoroacetic acid (6.8 mL, 88.3 mmol,
Aldrich) in CH.sub.2Cl.sub.2 (18 mL) was stirred at room
temperature for 2 h. After the solution was evaporated under
reduced pressure, the residue was dissolved in acetonitrile (10 mL)
and toluene (10 mL), and evaporated to dryness twice to result
crude amine as TFA salt. A solution of the crude amine,
dimethylaminopyridine (72 mg, 0.59 mmol, Aldrich),
diisopropylethylamine (2.55 mL, 14.6 mmol, Aldrich) in acetonitrile
was stirred at 0.degree. C. as the bisfurancarbonate 8 (907 mg,
3.07 mmol, obtained from Azar) was added in portion. The solution
was stirred at 0.degree. C. for 1 h and at room temperature for 19
h, and concentrated under reduced pressure. The residue was
dissolved in EtOAc (60 mL) and washed with saturated NaHCO.sub.3
solution (60 mL). After the aqueous washing was extracted with
EtOAc (60 mL), the combined organic fractions were washed with
saturated NaHCO.sub.3 (60 mL) and saturated NaCl solution (60 mL),
dried (MgSO.sub.4), and concentrated under reduced pressure. The
residue was purified by chromatography on silica gel to obtain the
carbamate 9 (1.452 g, 88%): .sup.1H NMR (CDCl.sub.3) .delta. 8.50
(d, 2H, J=5.7 Hz), 7.72 (d, 2H, J=8.7 Hz), 7.19 (d, 2H, J=5.7 Hz),
7.01 (d, 2H, J=8.7 Hz), 5.65 (d, 1H, J=5.1 Hz), 5.12 (d, 1H, J=9.3
Hz), 5.02 (q, 1H, J=6.7 Hz), 4.01-3.77 (m, 4H), 3.88 (s, 3H),
3.76-3.63 (m, 2H), 3.18-2.76 (m, 7H), 1.95-1.77 (m, 1H), 1.77-1.56
(m, 2H), 1.56-1.41 (m, 1H), 0.94 (d, 3H, J=6.6 Hz), 0.90 (d, 3H,
J=6.9 Hz); MS (ESI) 564 (M+H). ##STR507##
Example 7
[1547] The tetrahydropyridine-diethyl phosphonate 11: A solution of
the pyridine 9 (10.4 mg, 0.018 mmol) and the triflate 10 (8.1 mg,
0.027 mmol, in acetone-4 (0.75 mL) was stored at room temperature
for 9 h and the solution was concentrated under reduced pressure:
.sup.31P NMR (acetone-d.sub.3) .delta. 14.7; MS (ESI) 714
(M.sup.+). The concentrated crude pyridinium salt was dissolved in
ethanol (2 mL) and stirred at room temperature as NaBH.sub.4
(.about.10 mg, Aldrich) was added occasionally over 4 h. To the
mixture was added a solution of acetic acid (0.6 mL, Aldrich) in
ethanol (3 mL) until the pH of the mixture became 3-4. More
NaBH.sub.4 and acetic acid were added until the reaction was
completed. The mixture was carefully concentrated under reduced
pressure and the residue was dissolved in saturated NaHCO.sub.3
solution (10 mL). The product was extracted using EtOAc (10
mL.times.3) and washed with saturated NaCl solution, dried
(MgSO.sub.4), and concentrated under reduced pressure. The residue
was purified by chromatography on silica gel to obtain the product
11 (8.5 mg, 64%): .sup.1H NMR (CDCl.sub.3) .delta. 7.73 (d, 2H,
J=8.7 Hz), 7.00 (d, 2H, J=8.7 Hz), 5.71 (d, 1H, J=5.1 Hz), 5.41
(br, 1H), 5.15-5.08 (m, 1H), 5.00 (br, 1H), 4.14 (dq, 4H, J=7.2
Hz), 4.06-3.94 (m, 2H), 3.88 (s, 3H), 3.92-3.80 (m, 2H), 3.75 (dd,
1H, J=9.6 and 6.6 Hz), 3.79-3.61 (m, 1H), 3.24-2.94 (m, 6H), 2.85
(d, 2H, J=11.7 Hz), 2.88-2.76 (m, 2H), 2.75-2.63 (m, 1H), 2.38-2.29
(m, 1H), 2.24-2.2.12 (m, 2H), 2.12-1.78 (m, 4H), 1.30 (t, 6H, J=7.1
Hz), 0.94 (d, 3H, J=6.6 Hz), 0.91 (d, 3H, J=6.3 Hz); .sup.31P NMR
(CDCl.sub.3) .delta. 24.6; MS (ESI) 740 (M+Na). ##STR508##
Example 8
[1548] The tetrahydropyridine-dibenzyl phosphonate 13: The compound
13 was obtained by the same procedure as described for compound 11
using the pyridine 9 (10.0 mg, 0.018 mmol) and the triflate 12 (9.4
mg, 0.022 mmol). The product 13 was purified by preparative TLC to
afford the dibenzyl phosphonate 13 (8.8 mg, 59%): .sup.1H NMR
(CDCl.sub.3) .delta. 7.73 (d, 2H, J=8.7 Hz), 7.35 (s, 10H), 7.00
(d, 2H, J=8.7 Hz), 5.65 (d, 1H2H, J=5.1 Hz), 5.39 (br, 1H),
5.15-4.92 (m, 6H), 4.03-3.77 (m, 6H), 3.77-3.62 (m, 2H), 3.56 (br,
1H), 3.24-2.62 (m, 9H), 2.32 (d, 1H, J=13.5 Hz), 2.24-1.75 (m, 6H),
0.94 (d, 3H, J=6.6 Hz), 0.89 (d, 3H, J=6.3 Hz); .sup.31P NMR
(CDCl.sub.3) .delta. 25.5; MS (ESI) 842 (M+H).
Example 9
[1549] The phosphonic acid 14: A mixture of the dibenzyl
phosphonate 13 (8.8 mg, 0.011 mmol) and 10% Pd/C in EtOAc (2 mL)
and EtOH (0.5 mL) was stirred under H.sub.2 atmosphere for 10 h at
room temperature. After the mixture was filtered through celite,
the filtrate was concentrated to dryness to afford the product 14
(6.7 mg, quantitative): .sup.1H NMR (CD.sub.3OD) .delta. 7.76 (d,
2H, J=9.0 Hz), 7.10 (d, 2H, J=9.0 Hz), 5.68 (d, 1H, J=5.1 Hz), 5.49
(br, 1H), 5.11 (m, 1H), 3.90 (s, 3H), 4.04-3.38 (m, 10H), 3.22 (d,
2H, J=12.9 Hz), 3.18-3.00 (m, 2H), 2.89-2.75 (m, 2H), 2.68-2.30 (m,
3H), 2.21-1.80 (m, 4H), 0.92 (d, 3H, J=6.3 Hz), 0.85 (d, 3H, J=6.3
Hz); .sup.31P NMR (CD.sub.3OD) .delta. 6.29; MS (ESI) 662 (M+H).
##STR509##
Example 10
[1550] Diphenyl benzyloxymethylphosphonate 15: To a solution of
diphenylphosphite (46.8 g, 200 mmol, Aldrich) in acetonitrile (400
mL) (at ambient temperature) was added potassium carbonate (55.2 g,
400 mmol) followed by the slow addition of benzyl chloromethyl
ether (42 mL, 300 mmol, about 60%, Fluka). The mixture was stirred
overnight, and was concentrated under reduced pressure. The residue
was dissolved in EtOAc, washed with water, saturated NaCl, dried
(Na.sub.2SO.sub.4), filtered and evaporated. The crude product was
chromatographed on silica gel to afford the benzylether (6.8 g,
9.6%) as a colorless liquid.
Example 111
[1551] Monoacid 16: To a solution of diphenyl
benzyloxymethylphosphonate 15 (6.8 g, 19.1 mmol) in THF (100 mL) at
room temperature was added 1N NaOH in water (21 mL, 21 mmol). The
solution was stirred 3 h. The THF was evaporated under reduced
pressure and water (100 mL) was added. The aqueous solution was
cooled to 0.degree. C., neutralized to pH 7 with 3N HCl and washed
with EtOAc. The aqueous solution was again cooled to 0.degree. C.,
acidified with 3N HCl to pH 1, saturated with sodium chloride, and
extracted with EtOAc. The organic layer was washed with brine and
dried (Na.sub.2SO.sub.4), filtered and evaporated, then
co-evaporated with toluene to yield the monoacid (4.0 g, 75%) as a
colorless liquid. .sup.1H NMR (CDCl.sub.3) .delta. 7.28-7.09 (m,
10H), 4.61 (s, 2H), 3.81 (d, 2H); .sup.31P NMR (CDCl.sub.3) B
20.8.
Example 12
[1552] Ethyl lactate phosphonate 18: To a solution of monoacid 16
(2.18 g, 7.86 mmol) in anhydrous acetonitrile (50 mL) under a
nitrogen atmosphere was slowly added thionyl chloride (5.7 mL, 78
mmol). The solution was stirred in a 70.degree. C. oil bath for
three hours, cooled to room temperature and concentrated. The
residue was dissolved in anhydrous dichloromethane (50 mL), and
this solution cooled to 0.degree. C. and stirred under a nitrogen
atmosphere. To the stirring solution was added ethyl
(S)-(-)-lactate (2.66 mL, 23.5 mmol) and triethylamine (4.28 mL,
31.4 mmol). The solution was warmed to room temperature and allowed
to stir for one hour. The solution was diluted with ethyl acetate,
washed with water, brine, citric acid and brine again, dried
(MgSO.sub.4), filtered through Celite, concentrated under reduced
pressure and chromatographed on silica gel using 30% ethylacetate
in hexane. The two diastereomers were pooled together. .sup.1H NMR
(CDCl.sub.3) .delta. 7.40-7.16 (m, 20H), 5.18-5.13 (m, 2H), 4.73
(s, 2H), 4.66 (d, 2H), 4.28-4.11 (m, 5H), 4.05 (d, 2H), 3.95 (d,
2H), 1.62 (d, 3H), 1.46 (d, 3H), 1.30-1.18 (m, 6H); .sup.31P NMR
(CDCl.sub.3) .delta. 19.6, 17.7.
Example 13
[1553] Ethyl lactate phosphonate with free alcohol 19: Ethyl
lactate phosphonate 18 was dissolved in EtOH (50 mL) and under a
nitrogen atmosphere 10% Pd--C (approximately 20 wt %) was added.
The nitrogen atmosphere was replaced with hydrogen (1 atm) and the
suspension stirred for two hours. 10% Pd--C was again added (20 wt
%) and the suspension stirred five hours longer. Celite was added,
the reaction mixture was filtered through Celite and the filtrate
was concentrated to afford 1.61 g (71% from monoacid 16) of the
alcohol as a colorless liquid. .sup.1H NMR (CDCl.sub.3) .delta.
7.40-7.16 (m, 10H), 5.16-5.03 (m, 2H), 4.36-4.00 (m, 8H), 1.62 (d,
3H), 1.46 (d, 3H), 1.30-1.22 (m, 6H); .sup.31P NMR (CDCl.sub.3)
.delta. 22.3, 20.0.
Example 14
[1554] Triflate 20: To a solution of ethyl lactate phosphonate with
free alcohol 19 (800 mg, 2.79 mmol) in anhydrous dichloromethane
(45 mL) chilled to -40.degree. C. under a nitrogen atmosphere was
added triflic anhydride (0.516 mL, 3.07 mmol) and 2-6 lutidine
(0.390 mL, 3.34 mmol). The solution was stirred for 3 hr, then
warmed to -20.degree. C. and stirred one hour longer. 0.1
equivalents of triflic anhydride and 2-6 lutidine were then added
and stirring was resumed for 90 minutes more. The reaction mixture
was diluted with ice-cold dichloromethane, washed with ice-cold
water, washed with ice-cold brine and the organic layer was dried
(MgSO.sub.4) and filtered. The filtrate was concentrated and
chromatographed on silica gel using 30% EtOAc in hexane as eluent
to afford 602 mg (51%) of the triflate diastereomers as a slightly
pink, transparent liquid. .sup.1H NMR (CDCl.sub.3) .delta.
7.45-7.31 (m, 4H), 7.31-7.19 (m, 6H), 5.15-4.75 (m, 6H), 4.32-4.10
(4H), 1.62 (d, 3H), 1.50 (d, 3H), 1.30-1.22 (m, 6H); .sup.31P NMR
(CDCl.sub.3) .delta. 10.3, 8.3.
Example 15
[1555] The tetrahydropyridine-prodrug 21: A solution of the
pyridine 9 (11.1 mg, 0.020 mmol) and the triflate 20 (11.4 mg,
0.027 mmol) in acetone-d.sub.6 (0.67 mL, Aldrich) was stored at
room temperature for 7 h and the solution was concentrated under
reduced pressure: .sup.31P NMR (acetone-d.sub.6) .delta. 11.7,
10.9; MS (ESI) 838 (M+H). The concentrated crude pyridinium salt
was dissolved in ethanol (1 mL) and added 2-3 drops of a solution
of acetic acid (0.6 mL, Aldrich) in ethanol (3 mL). The solution
was stirred at 0.degree. C. as NaBH.sub.4 (7-8 mg, Aldrich) was
added. More acetic acid solution was added to adjust pH 3-4 of the
reaction mixture. Additions of NaBH.sub.4 and the acetic acid
solution were repeated until the reaction was completed. The
mixture was carefully concentrated under reduced pressure and the
residue was purified by chromatography on C18 reverse phase column
material followed by preparative TLC using C18 reverse phase plate
to obtain the prodrug 21 (13.6 mg, 70%) as a 2:3 mixture of two
diastereomers: .sup.1H NMR (CD.sub.3CN) .delta. 7.78 (d, 2H, J=9.0
Hz), 7.48-7.42 (m, 2H), 7.35-7.27 (m, 3H), 7.10 (d, 2H, J=9.0 Hz),
5.86 (m, 1H), 5.60 (m, 1H), 5.48 (br, 1H), 5.14-5.03 (m, 2H),
4.29-4.13 (m, 2H), 3.89 (s, 3H), 3.97-3.32 (m, 12H), 3.29 (br,
0.4H), 3.24 (br, 0.6H), 3.02-2.82 (m, 4H), 2.64-2.26 (m, 3H),
2.26-2.08 (m, 1H), 1.94-1.76 (m, 3H), 1.57 (d, 1.8H, J=6.9 Hz),
1.46 (d, 1.2H, J=6.9 Hz), 1.28 (d, 1.2H, J=6.9 Hz), 1.21 (d, 1.8H,
J=7.2 Hz), 0.92-0.88 (m, 6H); .sup.31P NMR (CD.sub.3CN) .delta.
14.4 (0.4P), 13.7 (0.6P); MS (ESI) 838 (M+H).
Example 16
[1556] Metabolite 22: To a solution of the prodrug 21 (10.3 mg,
0.011 mmol) in DMSO (0.1 mL) and acetonitrile (0.2 mL) was added
0.1 M PBS buffer (3 mL) mixed thoroughly to result a suspension. To
the suspension was added porcine liver esterase suspension (0.05
mL, EC3.1.1.1, Sigma). After the suspension was stored in
37.degree. C. for 1.5 h, the mixture was centrifuged and the
supernatant was taken. The product was purified by HPLC and the
collected fraction was lyophilized to result the product 22 as
trifluoroacetic acid salt (7.9 mg, 86%): .sup.1H NMR (D.sub.2O)
.delta. 7.70 (d, 1H), 7.05 (d, 2H), 5.66 (d, 1H), 5.40 (br, 1H),
5.02 (r, 1H), 4.70 (br, 1H), 3.99-3.89 (m, 2H), 3.81 (s, 3H),
3.83-3.50 (m, 8H), 3.34-2.80 (m, 7H), 2.50-2.18 (m, 3H), 2.03 (m,
1H), 1.92-1.70 (m, 3H), 1.39 (d, 3H), 0.94 (d, 3H), 0.93 (d, 3H);
.sup.31P NMR (020) .delta. 9.0, 8.8; MS (ESI) 734 (M+H).
##STR510##
Example 17
[1557] Triflate 24: Triflate 24 was prepared analogously to
triflate 20, except that dimethylhydroxyethylphosphonate 23
(Aldrich) was substituted for ethyl lactate phosphonate with free
alcohol 19.
Example 18
[1558] Tetrahydropyridine 25: Tetrahydropyridine 25 was prepared
analogously to tetrahydropyridine 30, except that triflate 24 was
substituted for triflate 29. .sup.1H NMR (CDCl.sub.3) .delta. 7.71
(d, 2H), 7.01 (d, 2H), 5.71 (d, 2H), 5.43 (bs, 1H), 5.07-4.87 (m,
1H), 4.16-3.46 (m, 13H), 3.34-3.18 (m, 3H), 3.16-2.80 (m, 5H),
2.52-1.80 (m, 12H), 1.28-1.04 (m, 3H+H.sub.2O peak), 0.98-0.68 (m,
6H). ##STR511##
Example 19
[1559] Dibenzyl phosphonate with double bond 27: To a stirring
solution of allyl bromide (4.15 g, 34 mmol, Aldrich) and
dibenzylphosphite (6 g, 23 mmol, Aldrich) in acetonitrile (25 mL)
was added potassium carbonate (6.3 g, 46 mmol, powder 325 mesh
Aldrich) to create a suspension, which was heated to 65.degree. C.
and stirred for 72 hours. The suspension was cooled to room
temperature, diluted with ethyl acetate, filtered, and the filtrate
was washed with water, then brine, dried (MgSO.sub.4), concentrated
and used directly in the next step.
Example 20
[1560] Dibenzylhydroxyethylphosphonate 28: Dibenzyl phosphonate
with double bond 27 was dissolved in methanol (50 mL), chilled to
-78.degree. C., stirred, and subjected to ozone by bubbling ozone
into the solution for three hours until the solution turned pale
blue. The ozone flow was stopped and oxygen bubbling was done for
15 minutes until the solution became colorless. Sodium borohydride
(5 g, excess) was added slowly portionwise. After the evolution of
gas subsided the solution was allowed to warm to room temperature,
concentrated, diluted with ethyl acetate, made acidic with acetic
acid and water and partitioned. The ethyl acetate layer was washed
with water, then brine and dried (MgSO.sub.4), filtered,
concentrated and chromatographed on silica gel eluting with a
gradient of eluent from 50% ethyl acetate in hexane to 100% ethyl
acetate, affording 2.76 g of the desired product. .sup.1H NMR
(CDCl.sub.3) .delta. 7.36 (m, 10H), 5.16-4.95 (m, 4H), 3.94-3.80
(dt, 2H), 2.13-2.01 (dt, 2H); .sup.31P NMR (CDCl.sub.3) .delta.
31.6.
Example 21
[1561] Dibenzyl phosphonate 30: A solution of the alcohol 28 (53.3
mg, 0.174 mmol) and 2,6-lutidine (0.025 mL, 0.215 mmol, Aldrich) in
CH.sub.2Cl.sub.2 (1 mL) was stirred at -45.degree. C. as
trifluoromethanesulfonic anhydride (0.029 mL, 0.172 mmol, Aldrich)
was added. The solution was stirred for 1 h at -45.degree. C. and
evaporated under reduced pressure to obtain the crude triflate
29.
[1562] A solution of the crude triflate 29, 2,6-lutidine (0.025 mL,
0.215 mmol, Aldrich), and the pyridine 9 in acetone-d.sub.6 (1.5
mL, Aldrich) was stored at room temperature for 2 h. The solution
was concentrated under reduced pressure to obtain crude pyridinium
product: .sup.31P NMR (acetone-d.sub.6) .delta. 25.8; MS (ESI) 852
(MW).
[1563] To a solution of the crude pyridinium salt in ethanol (2 mL)
was added 7-8 drops of a solution of acetic acid (0.4 mL, Aldrich)
in ethanol (2 mL). The solution was stirred at 0.degree. C. as
NaBH.sub.4 (7-8 mg) was added. The solution was maintained to be pH
3-4 by adding the acetic acid solution. More NaBH.sub.4 and the
acetic acid were added until the reduction was completed. After 4
h, the mixture was concentrated and the remaining residue was
dissolved in saturated NaHCO.sub.3 (10 mL). The product was
extracted with EtOAc (10 mL.times.3), dried (MgSO.sub.4), and
concentrated under reduced pressure. The residue was purified by
repeated chromatography on silica gel followed by HPLC
purification. Lyophilization of the collected fraction resulted the
product 30 (13.5 mg, 26%) as trifluoroacetic acid salt: .sup.1H NMR
(CDCl.sub.3) .delta. 7.72 (d, 2H, J=8.7 Hz), 7.36 (br, 10H), 7.00
(d, 2H, J=8.7 Hz), 5.69 (d, 1H, J=5.1 Hz), 5.41 (br, 1H), 5.13-4.93
(m, 6H), 4.05-2.5 (m, 19H), 3.88 (s, 3H), 2.5-1.9 (m, 5H),
1.90-1.74 (m, 2H), 0.88 (d, 6H, J=6.1 Hz); .sup.31P NMR
(CDCl.sub.3) .delta. 25.8; MS (ESI) 856 (M+H).
Example 22
[1564] Phosphonic acid 31: A mixture of the dibenzyl phosphonate 30
(9.0 mg, 0.009 mmol) and 10% Pd/C (5.2 mg, Aldrich) in EtOAc (2 mL)
and ethanol (0.5 mL) was stirred under H.sub.2 atmosphere for 3 h
at room temperature. After the mixture was filtered through celite,
a drop of trifluoroacetic acid (Aldrich) was added to the filtrate
and the filtrate was concentrated to dryness to afford the product
31 (6.3 mg, 86%): .sup.1H NMR (CD.sub.3OD) .delta. 7.76 (d, 2H,
J=9.0 Hz), 7.11 (d, 2H, J=9.0 Hz), 5.69 (d, 1H, J=5.1 Hz), 5.54
(br, 1H), 5.09 (br, 1H), 4.05-3.84 (m, 4H), 3.89 (s, 3H), 3.84-3.38
(m, 9H), 3.07 (dd, 2H, J=13.5 and 8.4 Hz), 2.9-2.31 (m, 5H),
2.31-1.83 (m, 6H), 0.92 (d, 3H, J=6.3 Hz), 0.85 (d, 3H, J=6.9 Hz);
.sup.31P NMR (CD.sub.3OD) .delta. 21.6; MS (ESI) 676 (M+H).
##STR512##
Example 23
[1565] Benzylether 32: A solution of dimethyl
hydroxyethylphosphonate (5.0 g, 32.5 mmol, Across) and benzyl
2,2,2-trichloroacetimidate (97.24 mL, 39.0 mmol, Aldrich) in
CH.sub.2Cl.sub.2 (100 mL) at 0.degree. C. under a nitrogen
atmosphere was treated with trifluoromethanesulfonic acid (0.40
mL). Stirring was performed for three hours at 0.degree. C. and the
reaction was then allowed to warm to room temperature while
stirring continued. The reaction continued for 15 hours, and the
reaction mixture was then diluted with dichloromethane, washed with
saturated sodium bicarbonate, washed with brine, dried
(MgSO.sub.4), concentrated under reduced pressure and
chromatographed on silica gel eluting with a gradient of eluent
from 60% EtOAc in hexane to 100% EtOAc to afford 4.5 g, (57%) of
the benzyl ether as a colorless liquid. .sup.31P NMR (CDCl.sub.3)
.delta. 31.5.
Example 24
[1566] Diacid 33: A solution of benzylether 32 (4.5 g, 18.4 mmol)
was dissolved in anhydrous acetonitrile (100 mL), chilled to
0.degree. C. under a nitrogen atmosphere and treated with TMS
bromide (9.73 mL, 74 mmol). The reaction mixture was warmed to room
temperature and after 15 hours of stirring was concentrated
repeatedly with MeOH/water to afford the diacid, which was used
directly in the next step. .sup.31P NMR (CDCl.sub.3) .delta.
31.9.
Example 25
[1567] Diphenylphosphonate 34: Diacid 33 (6.0 g, 27 mmol) was
dissolved in toluene and concentrated under reduced pressure three
times, dissolved in anhydrous acetonitrile, stirred under a
nitrogen atmosphere, and treated with thionyl chloride (20 mL, 270
mmol) by slow addition. The solution was heated to 70.degree. C.
for two hours, then cooled to room temperature, concentrated and
dissolved in anhydrous dichloromethane, chilled to -78.degree. C.
and treated with phenol (15 g, 162 mmol) and triethylamine (37 mL,
270 mmol). The reaction mixture was warmed to room temperature and
stirred for 15 hours, and was then diluted with ice cold
dichloromethane, washed with ice cold 1 N. NaOH, washed with ice
cold water, dried (MgSO.sub.4), and concentrated under reduced
pressure. The resulting residue was used directly in the next step.
.sup.1H NMR (CDCl.sub.3) .delta. 7.40-7.16 (d, 15H), 4.55 (s, 2H),
3.98-3.84 (m, 2H), 2.55-2.41 (m, 2H); .sup.31P NMR (CDCl.sub.3)
.delta. 22.1.
Example 26
[1568] Mono acid 35: Monoacid 35 was prepared using conditions
analogous to those used to prepare monoacid 16, except that
diphenylphosphonate 34 was substituted for benzylether 15. .sup.1H
NMR (CDCl.sub.3) .delta. 7.38-7.16 (d, 10H), 4.55 (s, 2H),
3.82-3.60 (m, 3H), 2.33-2.21 (m, 2H); .sup.31P NMR (CDCl.sub.3)
.delta. 29.0.
Example 27
[1569] Ethyl lactate phosphonate 36: Ethyl lactate phosphonate 36
was prepared analogously to ethyl lactate phosphonate 18 except
monoacid 35 was substituted for monoacid 16. .sup.31P NMR
(CDCl.sub.3) .delta. 27.0, 25.6.
Example 28
[1570] Ethyl lactate phosphonate with free alcohol 37: Ethyl
lactate phosphonate with free alcohol 37 was prepared analogously
to ethyl lactate phosphonate with free alcohol 19 except that ethyl
lactate phosphonate 36 was substituted for ethyl lactate
phosphonate 18. .sup.31P NMR (CDCl.sub.3) .delta. 28.9, 26.8.
Example 29
[1571] Triflate 38: A solution of the alcohol 37 (663 mg, 2.19
mmol) and 2,6-lutidine (0.385 mL, 3.31 mmol, Aldrich) in
CH.sub.2Cl.sub.2 (5 mL) was stirred at -45.degree. C. as
trifluoromethanesulfonic anhydride (0.48 mL, 2.85 mmol, Aldrich)
was added. The solution was stirred for 1.5 h at -45.degree. C.,
diluted with ice-cold water (50 mL), and extracted with EtOAc (30
mL.times.2). The combined extracts were washed with ice cold water
(50 mL), dried (MgSO.sub.4), and concentrated under reduced
pressure to obtain a crude mixture of two diastereomers (910 mg,
96%, 1:3 ratio): .sup.1H NMR (acetone-d.sub.6) .delta. 7.48-7.37
(m, 2H), 7.37-7.18 (m, 3H), 5.2-4.95 (m, 3H), 4.3-4.02 (m, 2H),
3.38-3.0 (m, 1H), 3.0-2.7 (m, 2H), 2.1-1.9 (m, 1H), 1.52 (d, 1H),
1.4 (d, 2H), 1.4-1.1) m, 3H); .sup.31P NMR (acetone-d.sub.6)
.delta. 21.8 (0.75P), 20.5 (0.25P).
Example 30
[1572] The prodrug 39: A solution of the crude triflate 38 (499 mg,
1.15 mmol) and the pyridine 9 (494 mg, 0.877 mmol) in acetone (5
mL) was stirred at room temperature for 16.5 h. The solution was
concentrated under reduced pressure to obtain the crude pyridinium
salt. To a solution of the crude pyridinium salt in ethanol (10 mL)
was added 5 drops of a solution of acetic acid (1 mL) in ethanol (5
mL). The solution was stirred at 0.degree. C. as NaBH.sub.4
(.about.10 mg, Aldrich) was added. The solution was maintained to
be pH 3-4 by adding the acetic acid solution. More NaBH.sub.4 and
the acetic acid were added until the reduction was completed. After
5.5 h, the mixture was concentrated under reduced pressure and the
remaining residue was dissolved in ice-cold saturated NaHCO.sub.3
(50 mL). The product was extracted with ice-cold EtOAc (30
mL.times.2) and the combined extracts were washed with 50%
saturated NaHCO.sub.3 (50 mL), dried (MgSO.sub.4), and concentrated
under reduced pressure. The residue was purified by a
chromatography on silica gel followed by a chromatography on C18
reverse phase column material. Lyophilization of the collected
fraction resulted the product 39 mixture (376 mg, 50%, 2.5:1 ratio)
as trifluoroacetic acid salt: .sup.1H NMR (CD.sub.3CN+TFA) .delta.
7.78 (d, 2H, J=8.7 Hz), 7.52-7.42 (m, 2H); 7.37-7.22 (m 3H), 7.10
(d, 2H, J=8.7 Hz), 5.78 (d, 1H, J=9.0 Hz), 5.64 (m, 1H), 5.50 (br,
1H), 5.08 (m, 2H), 4.31-4.12 (m, 2H), 4.04-3.42 (m, 11H), 3.90 (s,
3H), 3.29 (m, 2H), 3.23-3.16 (m, 1H), 3.08-2.78 (m, 6H), 2.76-2.27
(m, 5H), 2.23-2.11 (m, 1H), 2.08-1.77 (m, 3H), 1.58 (d, 0.9H, J=7.2
Hz), 1.45 (d, 2.1H, J=6.6 Hz), 1.32-1.20 (m, 3H), 0.95-0.84 (m,
6H); .sup.31P NMR (CD.sub.3CN+TFA) .delta. 24.1 and 23.8, 22.2 and
22.1; MS (ESI) 852 (M+H).
Example 31
[1573] Metabolite 40: To a solution of the prodrug 39 (35.4 mg,
0.037 mmol) in DMSO (0.35 mL) and acetonitrile (0.70 mL) was added
0.1 M PBS buffer (10.5 mL) mixed thoroughly to result a suspension.
To the suspension was added porcine liver esterase suspension
(0.175 mL, EC3.1.1.1, Sigma). After the suspension was stored in
37.degree. C. for 6.5 h, the mixture was filtered through 0.45 um
membrane filter and the filtrate was purified by HPLC. The
collected fraction was lyophilized to result the product 40 as
trifluoroacetic acid salt (28.8 mg, 90%): .sup.1H NMR (D.sub.2O)
.delta. 7.96 (d, 2H, J=8.7 Hz), 7.32 (d, 2H, J=8.7 Hz), 5.89 (d,
1H, J=5.1 Hz), 5.66 (br, 1H), 5.27 (m, 1H), 4.97 (m, 1H), 4.23-4.12
(m, 2H), 4.08 (s, 3H), 4.06-3.10 (m, 14H), 3.03 (dd, 1H, J=14.1 and
6.6 Hz), 2.78-1.97 (m, 9H), 1.66 (d, 3H, J=6.9 Hz), 1.03 (d, 3H,
J=7.5 Hz), 1.01 (d, 3H, J=6.9 Hz); .sup.31P NMR (CD.sub.3CN+TFA)
.delta. 20.0, 19.8; MS (ESI) 748 (M+H). ##STR513## ##STR514##
Example 32
[1574] Compound 42: The dibenzyl phosphonate 41 (947 mg, 1.21 mmol)
was treated with DABCO (140.9 mg, 1.26 mmol, Aldrich) in 4.5 ml,
toluene to obtain the monoacid (890 mg, 106%). The crude monoacid
(890 mg) was dried by evaporation with toluene twice and dissolved
in DMF (5.3 mL) with ethyl (S)-lactate (0.3 mL, 2.65 mmol, Aldrich)
and pyBOP (945 mg, 1.82 mmol, Aldrich) at room temperature. After
diisopropylethylamine (0.85 mL, 4.88 mmol, Aldrich) was added, the
solution was stirred at room temperature for 4 h and concentrated
under reduced pressure to a half volume. The resulting solution was
diluted with 5% aqueous HCl (30 mL) and the product was extracted
with EtOAc (30 mL.times.3). After the combined extracts were dried
(MgSO.sub.4) and concentrated, the residue was chromatographed on
silica gel to afford the compound 42 (686 mg, 72%) as a mixture of
two diastereomers (2:3 ratio): .sup.1H NMR (CDCl.sub.3) .delta.
7.46-7.32 (m, 5H), 7.13 (d, 2H, J=8.1 Hz), 6.85 (t, 2H, J=8.1 Hz),
5.65 (m, 1H), 5.35-4.98 (m, 4H), 4.39 (d, 0.8H, J=10.2H), 4.30-4.14
(m, 3.2H), 3.98 (dd, 1H, J=9.3 and 6.0 Hz), 3.92-3.78 (m, 3H),
3.78-3.55 (m, 3H), 3.16-2.68 (m, 6H), 1.85 (m, 1H), 1.74-1.55 (m,
2H), 1.56 (d, 1.8H, J=7.2 Hz), 1.49 (d, 1.2H), 1.48 (s, 9H),
1.30-1.23 (m, 3H), 0.88 (d, 3H, J=6.3 Hz), 0.87 (d, 3H, J=6.3 Hz);
.sup.31P NMR (CDCl.sub.3) .delta. 20.8 (0.4P), 19.5 (0.6P); MS
(ESI) 793 (M+H).
Example 33
[1575] Compound 45: A solution of compound 42 (101 mg, 0.127 mmol)
and trifluoroacetic acid (0.27 mL, 3.5 mmol, Aldrich) in
CH.sub.2Cl.sub.2 (0.6 mL) was stirred at 0.degree. C. for 3.5 h and
concentrated under reduced pressure. The resulting residue was
dried in vacuum to result the crude amine as TFA salt.
[1576] A solution of the crude amine salt and triethylamine (0.072
mL, 0.52 mmol, Aldrich) in CH.sub.2Cl.sub.2 (1 mL) was stirred at
0.degree. C. as the sulfonyl chloride 42 (37 mg, 0.14 mmol) was
added. After the solution was stirred at 0.degree. C. for 4 h and
0.5 h at room temperature, the reaction mixture was diluted with
saturated NaHCO.sub.3 (20 mL) and extracted with EtOAc (20
mL.times.1; 15 mL.times.2). The combined organic fractions were
washed with saturated NaCl solution, dried (MgSO.sub.4), and
concentrated under reduced pressure. Purification by chromatography
on silica gel provided the sulfonamide 45 (85 mg, 72%) as a mixture
of two diastereomers (.about.1:2 ratio): .sup.1H NMR (CDCl.sub.3)
.delta. 7.45-7.31 (m, 7H), 7.19 (d, 1H, J=8.4 Hz), 7.12 (d, 2H,
J=7.8 Hz), 6.85 (m, 2H), 5.65 (d, 1H, J=5.4 Hz), 5.34-5.16 (m, 2H),
5.13-4.97 (m, 2H), 4.97-4.86 (m, 1H), 4.38 (d, 0.7H, J=10.8 Hz),
4.29-4.12 (m, 3.3H), 3.96 (dd, 1H, J=9.3 and 6.3 Hz), 3.89 (s, 3H),
3.92-3.76 (m, 3H), 3.76-3.64 (m, 2H), 3.64-3.56 (br, 1H), 3.34-3.13
(m, 1H), 3.11-2.70 (m, 6H), 2.34 (s, 3H), 1.86 (m, 1H, J=7.0 Hz),
1.75-1.58 (m, 2H), 1.56 (d, 2H, J=7.2 Hz), 1.49 (d, 1H, J=7.2 Hz),
1.29-1.22 (m, 3H), 0.94 (d, 3H, J=6.6 Hz), 0.90 (d, 3H, J=6.9 Hz);
.sup.31P NMR (CDCl.sub.3) .delta. 20.7 (0.3P), 19.5 (0.7P); MS
(ESI) 921 (M+H).
Example 34
[1577] Compound 46: Compound 45 (257 mg, 0.279 mmol) was stirred in
a saturated solution of ammonia in ethanol (5 mL) at 0.degree. C.
for 15 min and the solution was concentrated under reduced
pressure. Purification of the residue by chromatography on silica
gel provided compound 46 (2.6 mg, 84%): .sup.1H NMR (CDCl.sub.3)
.delta. 7.48-7.34 (m, 4H), 7.22-7.05 (m, 5H), 7.01 (d, 1H, J=8.1
Hz), 6.87-6.80 (m, 2H), 5.68 (d, 1H, J=4.8 Hz), 5.32 (dd, 1.3H,
J=8.7 and 1.8 Hz), 5.22 (d, 0.7H, J=9.0 Hz), 5.11-5.00 (m, 3H),
4.47-4.14 (m, 4H), 4.00 (dd, 1H, J=9.9 and 6.6 Hz), 3.93 (s, 3H),
3.95-3.63 (m, 5H), 3.07-2.90 (m, 4H), 2.85-2.75 (m, 1H), 2.75-2.63
(m, 2H), 1.88-1.67 (m, 3H), 1.65-1.55 (m, 2H), 1.57 (d, 2H, J=6.9
Hz), 1.50 (d, 1H, J=7.2 Hz), 1.31-1.20 (m, 3H), 0.95 (d, 3H, J=6.6
Hz), 0.88 (d, 3H, J=6.3 Hz); .sup.31P NMR (CDCl.sub.3) .delta. 20.7
(0.3P), 19.6 (0.7P); MS (ESI) 879 (M+H).
Example 35
[1578] Compound 47: A mixture of compound 46 (176 mg, 0.200 mmol)
and 10% Pd/C (9.8 mg, Aldrich) in EtOAc (4 mL) and ethanol (1 mL)
was stirred under H.sub.2 atmosphere for 3 h at room temperature.
After the mixture was filtered through celite, the filtrate was
concentrated to dryness to afford compound 47 (158 mg, 100%) as
white powder: .sup.1H NMR (CDCl.sub.3) .delta. 7.30-7.16 (m, 2H),
7.12 (d, 2H, J=7.5 Hz), 7.01 (d, 1H, J=7.8 Hz), 6.84 (d, 2H, J=7.5
Hz), 5.66 (d, 1H, J=4.5 Hz), 5.13-4.97 (m, 2H), 4.38-4.10 (m, 4H),
3.93 (s, 3H), 4.02-3.66 (m, 6H), 3.13-2.69 (m, 7H), 1.96-1.50 (m,
3H), 1.57 (d, 3H, J=6.6 Hz), 1.26 (t, 3H, J=7.2 Hz), 0.93 (d, 3H,
J=6.0 Hz), 0.88 (d, 3H, J==6.0 Hz); .sup.31P NMR (CDCl.sub.3)
.delta. 20.1; MS (ESI) 789 (4+H).
Example 36
[1579] Compound 48A and 48B: A solution of pyBOP (191 mg, 0.368
mmol, Aldrich) and diisopropylethylamine (0.1 mL, 0.574 mmol,
Aldrich) in DMF (35 mL) was stirred at room temperature as a
solution of compound 47 (29 mg, 0.036 mmol) in DMF (5.5 mL) was
added over 16 h. After addition, the solution was stirred at room
temperature for 3 h and concentrated under reduced pressure. The
residue was dissolved in ice-cold water and extracted with EtOAc
(20 mL.times.1; 10 mL.times.2). The combined extracts were dried
(MgSO.sub.4) and concentrated under reduced pressure. The residue
was purified by chromatography on silica gel followed by
preparative TLC gave two isomers of structure 48 (1.0 mg, 3.6% and
3.6 mg, 13%). Isomer 48A: .sup.1H NMR (CDCl.sub.3) .delta. 7.39 (m,
1H), 7.12 (br, 1H), 7.01 (d, 2H, J=8.1 Hz), 6.98 (br, 1H), 6.60 (d,
2H, J=8.1 Hz), 5.75 (d, 1H, J=5.1 Hz), 5.37-5.28 (m, 2H), 5.18 (q,
1H, J=8.7 Hz), 4.71 (dd, 1H, J=14.1 and 7.5 Hz), 4.29 (m, 3H),
4.15-4.06 (m, 1H), 3.99 (s, 3H), 4.05-3.6 (m, 5H), 3.35 (m, 1H),
3.09 (br, 1H), 2.90-2.78 (m, 3H), 2.2-2.0 (m, 3H), 1.71 (d, 3H,
J=6.6 Hz), 1.34 (t, 3H, J=6.9 Hz), 1.01 (d, 3H, J=6.3 Hz), 0.95 (d,
3H, J=6.3 Hz); .sup.31P NMR (CDCl.sub.3) .delta. 17.8; MS (ESI) 793
(M+Na); isomer 48B: .sup.1H NMR (CDCl.sub.3) .delta. 7.46 (d, 1H,
J=9.3 Hz), 7.24 (br, 1H), 7.00 (d, 2H, J=8.7 Hz), 6.91 (d, 1H,
J=8.7 Hz), 6.53 (d, 2H, J=8.7 Hz), 5.74 (d, 1H, J=5.1 Hz), 5.44 (m,
1H), 5.35 (d, 1H, J=9.0 Hz), 5.18 (q, 1H, J=7.2 Hz), 4.68 (dd, 1H,
J=14.4 and 6.3 Hz), 4.23 (m, 3H), 4.10 (m, 1H), 4.04 (s, 3H),
3.77-4.04 (m, 6H), 3.46 (dd, 1H, J=12.9 and 11.4 Hz), 3.08 (br,
1H), 2.85 (m, 2H), 2.76 (dd, 1H, J=12.9 and 4.8 Hz), 1.79-2.11 (m,
3H), 1.75 (d, 3H, J=6.6 Hz), 1.70 (m, 2H), 1.27 (t, 3H, J=6.9 Hz),
1.01 (d, 3H, J=6.6 Hz), 0.93 (d, 3H, J=6.6 Hz); .sup.31P NMR
(CDCl.sub.3) .delta. 15.4; MS (ESI) 793 (M+Na).
Example 1
[1580] ##STR515##
Example 1A
[1581] Dimethylphosphonic ester 2 (R.dbd.CH.sub.3): To a flask was
charged with phosphonic acid 1 (67 mg, 0.1 mmol), methanol (0.1 mL,
2.5 mmol) and 1,3-dicyclohexylcarbodiimide (83 mg, 0.4 mmol), then
pyridine (1 mL) was added under N.sub.2. The resulted mixture was
stirred at 60-70.degree. C. for 2 h, then cooled to room
temperature and diluted with ethyl acetate. The mixture was
filtered and the filtrate was evaporated. The residue was diluted
with ethyl acetate and the combined organic phase was washed with
NH.sub.4Cl, brine and water, dried over Na.sub.2SO.sub.4, filtered
and concentrated. The residue was purified by chromatography on
silica gel (isopropanol/CH.sub.2Cl.sub.2, 1% to 7%) to give 2 (39
mg, 56%) as a white solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.71(d,
J=8.7 Hz, 2H), 7.15 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz, 2H), 6.87
(d, J=8.7 Hz, 2H), 5.65 (d, J=5.1 Hz, 1H), 5.10-4.92 (m, 4H), 4.26
(d, J=9.9 Hz, 2H), 3.96-3.65 (m overlapping s, 15H), 3.14-2.76 (m,
7H), 1.81-1.55 (m, 3H), 0.91 (d, J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz,
3H); .sup.31P NMR (CDCl.sub.3) .delta. 21.7; MS (ESI) 723
(M+Na).
Example 1B
[1582] Diisopropylphosphonic ester 3 (R.dbd.CH(CH.sub.3).sub.2) was
synthesized in the same manner in 60% yield. .sup.1H NMR
(CDCl.sub.3) .delta. 7.71 (d, J=8.7 Hz, 2H), 7.15 (d, J=8.7 Hz,
2H), 7.15 (d, J=8.7 Hz, 2H), 6.99 (d, J=8.7 Hz, 2H), 6.87 (d, J=8.7
Hz, 2H), 5.66 (d, J=5.1 Hz, 1H), 5.08-4.92 (m, 3H), 4.16 (d, J=10.5
Hz, 2H), 3.98-3.68 (m overlapping s, 9H), 3.16-2.78 (m, 7H),
1.82-1.56 (m, 3H), 1.37 (t, J=6.3 Hz, 6H), 0.93 (d, J=6.6 Hz, 3H),
0.88 (d, J=6.6 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.3; MS
(ESI) 779 (M+Na).
Example 2
[1583] TABLE-US-00011 ##STR516## ##STR517## Compound R.sub.1
R.sub.2 5a OPh mix-Hba-Et 5b OPh (S)-Hba-Et 5c OPh (S)-Hba-tBu 5d
OPh (S)-Hba-EtMor 5e OPh (R)-Hba-Et
Example 2A
[1584] Monolactate 5a (R1=OPh, R2=Hba-Et): To a flask was charged
with monophenyl phosphonate 4 (250 mg, 0.33 mmol),
2-hydroxy-n-butyric acid ethyl ester (145 mg, 1.1 mmol) and
1,3-dicyclohexylcarbodiimide (226 mg, 1.1 mmol), then pyridine (2.5
mL) was added under N.sub.2. The resulted mixture was stirred at
60-70.degree. C. for 2 h, then cooled to room temperature and
diluted with ethyl acetate. The mixture was filtered and the
filtrate was evaporated. The residue was diluted with ethyl acetate
and the combined organic phase was washed with NH.sub.4Cl, brine
and water, dried over Na.sub.2SO.sub.4, filtered and concentrated.
The residue was purified by chromatography on silica gel
(EtOAc/CH.sub.2Cl.sub.2, 1:1) to give 5a (150 mg, 52%) as a white
solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.70 (d, J=8.7 Hz, 2H),
7.37-7.19 (m, 5H), 7.14 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz, 2H),
6.91 (d, J=8.7 Hz, 1H), 6.86 (d, J=8.7 Hz, 1H), 5.65 (m, 1H),
5.10-4.95 (m, 3H), 4.57-4.39 (m, 2H), 4.26 (m, 2H), 3.96-3.68 (m
overlapping s, 9H), 3.15-2.77 (m, 7H), 1.81-1.55 (m, 5H), 1.21 (m,
3H), 1.04-0.86 (m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 17.5 and
15.1; MS (ESI) 885 (M+Na).
Example 2B
[1585] Monolactate 5b (R1=OPh, R2=(S)-Hba-Et): To a flask was
charged with monophenyl phosphonate 4 (600 mg, 0.8 mmol),
(s)-2-hydroxy-n-butyric acid ethyl ester (317 mg, 2.4 mmol) and
1,3-dicyclohexylcarbodiimide (495 mg, 2.4 mmol), then pyridine (6
mL) was added under N.sub.2. The resulted mixture was stirred at
60-70.degree. C. for 2 h, then cooled to room temperature and
diluted with ethyl acetate. The mixture was filtered and the
filtrate was evaporated. The residue was diluted with ethyl acetate
and the combined organic phase was washed with NH.sub.4Cl, brine
and water, dried over Na.sub.2SO.sub.4, filtered and concentrated.
The residue was purified by chromatography on silica gel
(EtOAc/CH.sub.2Cl.sub.2, 1:1) to give 5b (360 mg, 52%) as a white
solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, J=8.7 Hz, 2H),
7.37-7.19 (m, 5H), 7.15 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz, 2H),
6.92 (d, J=8.7 Hz, 1H), 6.86 (d, J=8.7 Hz, 1H), 5.65 (m, 1H),
5.10-4.95 (m, 3H), 4.57-4.39 (m, 2H), 4.26 (m, 2H), 3.96-3.68 (m
overlapping s, 9H), 3.15-2.77 (m, 7H), 1.81-1.55 (m, 5H), 1.23 (m,
3H), 1.04-0.86 (m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 17.5 and
15.2; MS (ESI) 885 (M+Na).
Example 2C
[1586] Monolactate 5c (R1=OPh, R2=(S)-Hba-tBu): To a flask was
charged with monophenyl phosphonate 4 (120 mg, 0.16 mmol),
tert-butyl (S)-2-hydroxybutyrate (77 mg, 0.48 mmol) and
1,3-dicyclohexylcarbodiimide (99 mg, 0.48 mmol), then pyridine (1
mL) was added under N.sub.2. The resulted mixture was stirred at
60-70.degree. C. for 2 h, then cooled to room temperature and
diluted with ethyl acetate. The mixture was filtered and the
filtrate was evaporated. The residue was diluted with ethyl acetate
and the combined organic phase was washed with NH.sub.4Cl, brine
and water, dried over Na.sub.2SO.sub.4, filtered and concentrated.
The residue was purified by chromatography on silica gel
(EtOAc/CH.sub.2Cl.sub.2, 1:1) to give 5c (68 mg, 48%) as a white
solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, J=8.7 Hz, 2H),
7.37-7.19 (m, 5H), 7.14 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz, 2H),
6.93 (d, J=8.7 Hz, 1H), 6.86 (d, J=8.7 Hz, 1H), 5.64 (m, 1H),
5.10-4.95 (m, 3H), 4.57-4.39 (m, 2H), 4.26 (m, 2H), 3.96-3.68 (m
overlapping s, 9H), 3.15-2.77 (m, 7H), 1.81-1.55 (m, 5H), 1.44 (d,
J=11 Hz, 9H), 1.04-0.86 (m, 9H); .sup.31P NMR (CDCl.sub.3) .delta.
17.5 and 15.2; MS (ESI) 913 (M+Na).
Example 2D
[1587] Monolactate 5d (R1=OPh, R2=(S)-Lac-EtMor): To a flask was
charged with monophenyl phosphonate 4 (188 mg, 0.25 mmol),
(S)-lactate ethylmorpholine ester (152 mg, 0.75 mmol) and
1,3-dicyclohexylcarbodiimide (155 mg, 0.75 mmol), then pyridine (2
mL) was added under N.sub.2. The resulted mixture was stirred at
60-70.degree. C. for 2 h, then cooled to room temperature and
diluted with ethyl acetate. The mixture was filtered and the
filtrate was evaporated. The residue was washed with ethyl acetate
and the combined organic phase was washed with NH.sub.4Cl, brine
and water, dried over Na.sub.2SO.sub.4, filtered and concentrated.
The residue was purified by chromatography on silica gel
(isopropanol/CH.sub.2Cl.sub.2, 1:9) to give 5d (98 mg, 42%) as a
white solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz,
2H), 7.34-7.20 (m, 5H), 7.15 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz,
2H), 6.92 (d, J=8.7 Hz, 1H), 6.87 (d, J=8.7 Hz, 1H), 5.65 (m, 1H),
5.21-4.99 (m, 3H), 4.57-4.20 (m, 4H), 3.97-3.63 (m overlapping s,
13H), 3.01-2.44 (m, 13H), 1.85-1.50 (m, 6H), 0.92 (d, J=6.5 Hz,
3H), 0.88 (d, J=6.5, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.4
and 15.3; MS (ESI) 934(M).
Example 2E
[1588] Monolactate 5e (R1=OPh, R2=(R)-Hba-Et): To a flask was
charged with monophenyl phosphonate 4 (600 mg, 0.8 mmol),
(R)-2-hydroxy-n-butyric acid ethyl ester (317 mg, 2.4 mmol) and
1,3-dicyclohexylcarbodiimide (495 mg, 2.4 mmol), then pyridine (6
mL) was added under N.sub.2. The resulted mixture was stirred at
60-70.degree. C. for 2 h, then cooled to room temperature and
diluted with ethyl acetate. The mixture was filtered and the
filtrate was evaporated. The residue was diluted with ethyl acetate
and the combined organic phase was washed with NH.sub.4Cl, brine
and water, dried over Na.sub.2SO.sub.4, filtered and concentrated.
The residue was purified by chromatography on silica gel
(EtOAc/CH.sub.2Cl.sub.2, 1:1) to give 5e (345 mg, 50%) as a white
solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.70 (d, J=8.7 Hz, 2H),
7.37-7.19 (m, 5H), 7.15 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz, 2H),
6.92 (d, J=8.7 Hz, 1H), 6.86 (d, J=8.7 Hz, 1H), 5.65 (m, 1H),
5.10-4.95 (m, 3H), 4.57-4.39 (m, 2H), 4.26 (m, 2H), 3.96-3.68 (m
overlapping s, 9H), 3.15-2.77 (m, 7H), 1.81-1.55 (m, 5H), 1.23 (m,
3H), 1.04-0.86 (m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 17.5 and
15.1; MS (ESI) 885 (M+Na). ##STR518##
Example 3
[1589] Monoamidate 6: To a flask was charged with monophenyl
phosphonate 4 (120 mg, 0.16 mmol), L-alanine butyric acid ethyl
ester hydrochloride (160 mg, 0.94 mmol) and
1,3-dicyclohexylcarbodiimide (132 mg, 0.64 mmol), then pyridine (1
mL) was added under N.sub.2. The resulted mixture was stirred at
60-70.degree. C. for 2 h, then cooled to room temperature and
diluted with ethyl acetate. The mixture was filtered and the
filtrate was evaporated. The residue was diluted with ethyl acetate
and the combined organic phase was washed with NH.sub.4Cl, brine
and water, dried over Na.sub.2SO.sub.4, filtered and concentrated.
The residue was purified by chromatography on silica gel
(isopropanol/CH.sub.2Cl.sub.2, 1:9) to give 6 (55 mg, 40%) as a
white solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.72 (d, J=8.7 Hz,
2H), 7.37-7.23 (m, 5H), 7.16 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz,
2H), 6.90-6.83 (m, 2H), 5.65 (d, J=5.1 Hz, 1H), 5.10-4.92 (m, 3H),
4.28 (m, 2H), 3.96-3.68 (m overlapping s, 9H), 3.15-2.77 (m, 7H),
1.81-1.55 (m, 5H), 1.23 (m, 3H), 1.04-0.86 (m, 6H); .sup.31P NMR
(CDCl.sub.3) .delta. 20.7 and 19.6; MS (ESI) 884(M+Na).
##STR519##
Example 4A
[1590] Compound 8: To a stirred solution of monobenzyl phosphonate
7 (195 mg, 0.26 mmol) in 1 ml, of DMF at room temperature under
N.sub.2 was added benzyl-(s)-lactate (76 mg, 0.39 mmol) and PyBOP
(203 mg, 0.39 mmol), followed by DIEA (181 .mu.L, 1 mmol). After 3
h, the solvent was removed under reduced pressure, and the
resulting crude mixture was purified by chromatography on silica
gel (ethyl acetate/hexane 1:1) to give 8 (120 mg, 50%) as a white
solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, J=8.7 Hz, 2H),
7.38-7.34 (m, 5H), 7.12 (d, J=8.7 Hz, 2H), 6.99 (d, J=8.7 Hz, 2H),
6.81(d, J=8.7 Hz, 2H), 5.64 (d, J=5.4 Hz, 1H), 5.24-4.92 (m, 7H),
4.28 (m, 2H), 3.96-3.67 (m overlapping s, 9H), 3.16-2.76 (m, 7H),
1.95-1.62 (m, 5H), 0.99-0.87 (m, 9H); .sup.31P NMR (CDCl.sub.3)
.delta. 21.0 and 19.7; MS (ESI) 962 (M+Na).
Example 4B
[1591] Compound 9: A solution of compound 8 (100 mg) was dissolved
in EtOH/EtOAc (9 mL/3 mL), treated with 10% Pd/C (10 mg) and was
stirred under H.sub.2 atmosphere (balloon) for 1.5 h. The catalyst
was removed by filtration through celite. The filtered was
evaporated under reduced pressure, the residue was triturated with
ether and the solid was collected by filtration to afford the
compound 9 (76 mg, 94%) as a white solid. .sup.1H NMR (CD.sub.3OD)
.delta. 7.76 (d, J=8.7 Hz, 2H), 7.18 (d, J=8.7 Hz, 2H), 7.08 (d,
J=8.7 Hz, 2H), 6.90 (d, J=8.7 Hz, 2H), 5.59 (d, J=5.4 Hz, 1H),
5.03-4.95 (m, 2H), 4.28 (m, 2H), 3.90-3.65 (m overlapping s, 9H),
3.41 (m, 2H), 3.18-2.78 (m, 5H), 2.44 (m, 1H), 1.96 (m, 3H), 1.61
(m, 2H), 1.18 (m, 3H), 0.93 (d, J=6.3 Hz, 3H), 0.87 (d, J=6.3 Hz,
3H); .sup.31P NMR (CD.sub.3OD) .delta. 18.3; MS (ESI) 782 (M+Na).
##STR520##
Example 5A
[1592] Compound 11: To a stirred solution of compound 10 (1 g, 1.3
mmol) in 6 ml, of DMF at room temperature under N.sub.2 was added
3-hydroxybenzaldehyde (292 mg, 2.6 mmol) and PyBOP (1 g, 1.95
mmol), followed by DIEA (0.9 mL, 5.2 mmol). After 5 h, the solvent
was removed under reduced pressure, and the resulting crude mixture
was purified by chromatography on silica gel (ethyl acetate/hexane
1:1) to give 11 (800 mg, 70%) as a white solid. .sup.1H NMR
(CDCl.sub.3) .delta. 9.98 (s, 1H), 7.79-6.88 (m, 12H), 5.65 (m,
1H), 5.21-4.99 (m, 3H), 4.62-4.16 (m, 4H), 3.99-3.61 (m overlapping
s, 9H), 3.11-2.79 (m, 5H), 1.85-1.53 (m, 6H), 1.25 (m, 3H), 0.90
(m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 17.9 and 15.9; MS (ESI)
899 (M+Na).
Example 5B
[1593] Compound 12: To a stirred solution of compound 11 (920 mg,
1.05 mmol) in 10 ml, of ethyl acetate at room temperature under
N.sub.2 was added morpholine (460 mg, 5.25 mmol) and acidic acid
(0.25 mL, 4.2 mmol), followed by sodium cyanoborohydride (132 mg,
2.1 mmol). After 20 h, the solvent was removed under reduced
pressure, and the residue was diluted with ethyl acetate and the
combined organic phase was washed with NH.sub.4Cl, brine and water,
dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue
was purified by chromatography on silica gel
(isopropanol/CH.sub.2Cl.sub.2, 6%) to give 12 (600 mg, 60%) as a
white solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, J=8.7 Hz,
2H), 7.27 (m, 4H), 7.15 (d, J=8.7 Hz, 2H), 6.95 (d, J=8.7 Hz, 2H),
6.89 (m, 2H), 5.65 (m, 1H), 5.21-5.02 (m, 3H), 4.58-4.38 (m, 2H),
4.21-4.16 (m, 2H), 3.99-3.63 (m overlapping s, 15H), 3.47 (s, 2H),
3.18-2.77 (m, 7H), 2.41 (s, 4H), 1.85-1.53 (m, 6H), 1.25 (m, 3H),
0.90 (m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 17.4 and 15.2; MS
(ESI) 971 (M+Na). ##STR521##
Example 6A
[1594] Compound 14: To a stirred solution of compound 13 (1 g, 3
mmol) in 30 ml, of acetonitrile at room temperature under N.sub.2
was added thionyl chloride (0.67 mL, 9 mmol). The resulted mixture
was stirred at 60-70.degree. C. for 0.5 h. After cooled to room
temperature, the solvent was removed under reduced pressure, and
the residue was added 30 ml, of DCM, followed by DIEA (1.7 mL, 10
mmol), L-alanine butyric acid ethyl ester hydrochloride (1.7 g, 10
mmol) and TEA (1.7 mL, 12 mmol). After 4 h at room temperature, the
solvent was removed under reduced pressure, and the residue was
diluted with DCM and washed with brine and water, dried over
Na.sub.2SO.sub.4, filtered and concentrated. The residue was
purified by chromatography on silica gel (Hexane/EtOAc 1:1) to give
14 (670 mg, 50%) as a yellow oil. .sup.1H NMR (CDCl.sub.3) .delta.
7.33-7.11 (m, 10H), 5.70 (m, 1H), 5.10 (s, 2H), 4.13-3.53 (m, 5H),
2.20-2.10 (m, 2H), 1.76-1.55 (m, 2H), 1.25-1.19 (m, 3H), 0.85-0.71
(m, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 30.2 and 29.9; MS (ESI)
471 (M+Na).
Example 6B
[1595] Compound 15: A solution of compound 14 (450 mg) was
dissolved in 9 ml, of EtOH, then 0.15 ml, of acetic acid and 10%
Pd/C (90 mg) was added. The resulted mixture was stirred under H2
atmosphere (balloon) for 4 h. After filtration through celite, the
filtered was evaporated under reduced pressure to afford the
compound 15 (300 mg, 95%) as a colorless oil. .sup.1H NMR
(CDCl.sub.3) .delta. 7.29-7.12 (m, 5H), 4.13-3.53 (m, 5H),
2.20-2.10 (m, 2H), 1.70-1.55 (m, 2H), 1.24-1.19 (m, 3H),
0.84-0.73(m, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 29.1 and 28.5;
MS (ESI) 315 (M+1).
Example 6C
[1596] Monoamdidate 17: To a stirred solution of compound 16 (532
mg, 0.9 mmol) in 4 ml, of 1,2-dichloroethane was added compound 15
(300 mg, 0.96 mmol) and MgSO.sub.4 (50 mg), the resulted mixture
was stirred at room temperature under argon for 3 h, then acetic
acid (1.3 mL, 23 mmol) and sodium cyanoborohydride (1.13 g, 18
mmol) were added. The reaction mixture was stirred at room
temperature for 1 h under argon. Then aqueous NaHCO.sub.3 (50 mL)
was added, and the mixture was extracted with ethyl acetate, and
the combined organic layers were washed with brine and water, dried
over Na.sub.2SO.sub.4, filtered and concentrated. The residue was
purified by chromatography on silica gel (EtOH/EtOAc, 1/9) to give
17 (600 mg, 60%) as a white solid. .sup.1H NMR (CDCl.sub.3) .delta.
7.73 (d, J=8.7 Hz, 2H), 7.33-7.13 (m, 9H), 7.00 (d, J=8.7 Hz, 2H),
5.65 (d, J=5.4 Hz, 1H), 5.11-4.98 (m, 2H), 4.22-3.68 (m overlapping
s, 15H), 3.20-2.75 (m, 9H), 2.21-2.10 (m, 2H), 1.88-1.55(m, 5H),
1.29-1.19 (m, 3H), 0.94-0.70 (m, 9H); .sup.31P NMR (CDCl.sub.3)
.delta. 31.8 and 31.0; MS (ESI) 889 (M).
Example 7
[1597] ##STR522##
Example 7A
[1598] Compound 19: To a stirred solution of compound 18 (3.7 g,
14.3 mmol) in 70 ml, of acetonitrile at room temperature under
N.sub.2 was added thionyl chloride (6.3 mL, 86 mmol). The resulted
mixture was stirred at 60-70.degree. C. for 2 h. After cooled to
room temperature, the solvent was removed under reduced pressure,
and the residue was added 150 ml, of DCM, followed by TEA (12 mL,
86 mmol) and 2-ethoxyphenol (7.2 mL, 57.2 mmol). After 20 h at room
temperature, the solvent was removed under reduced pressure, and
the residue was diluted with ethyl acetate and washed with brine
and water, dried over Na.sub.2SO.sub.4, filtered and concentrated.
The residue was purified by chromatography on silica gel (DCM/EtOAc
9:1) to give 19 (4.2 g, 60%) as a yellow oil. .sup.1H NMR
(CDCl.sub.3) .delta. 7.32-6.83 (m, 13H), 5.22 (m, 1H), 5.12 (s,
2H), 4.12-3.73 (m, 6H), 2.52-2.42 (m, 2H), 1.41-1.37 (m, 6H);
.sup.31P NMR (CDCl.sub.3) .delta. 25.4; MS (ESI) 522 (M+Na).
Example 7B
[1599] Compound 20: A solution of compound 19 (3 g, 6 mmol) was
dissolved in 70 ml, of acetonitrile at 0.degree. C., then 2N NaOH
(12 mL, 24 mmol) was added dropwisely. The reaction mixture was
stirred at room temperature for 1.5 h. Then the solvent was removed
under reduced pressure, and the residue diluted with water and
extracted with ethyl acetate. The aqueous layer was acidified with
conc. HCl to PH=1, then extracted with ethyl acetate, combined the
organic layer and dried over Na.sub.2SO.sub.4, filtered and
concentrated to give compound 20 (2 g, 88%) as a off-white solid.
.sup.1H NMR (CDCl.sub.3) .delta. 7.33-6.79 (m, 9H), 5.10 (s, 2H),
4.12-3.51 (m, 6H), 2.15-2.05 (m, 2H), 1.47-1.33 (m, 3H); .sup.31P
NMR (CDCl.sub.3) .delta. 30.5; MS (ESI) 380 (M+1).
Example 7C
[1600] Compound 21: To a stirred solution of compound 20 (1 g, 2.6
mmol) in 20 ml, of acetonitrile at room temperature under N.sub.2
was added thionyl chloride (1.1 mL, 15.6 mmol). The resulted
mixture was stirred at 60-70.degree. C. for 45 min. After cooled to
room temperature, the solvent was removed under reduced pressure,
and the residue was added 25 ml, of DCM, followed by TEA (1.5 mL,
10.4 mmol) and (S) lactate ethyl ester (0.9 mL, 7.8 mmol). After 20
h at room temperature, the solvent was removed under reduced
pressure, and the residue was diluted with DCM and washed with
brine and water, dried over Na.sub.2SO.sub.4, filtered and
concentrated. The residue was purified by chromatography on silica
gel (DCM/EtOAc 3:1) to give 21 (370 mg, 30%) as a yellow oil.
.sup.1H NMR (CDCl.sub.3) .delta. 7.33-6.84 (m, 9H), 6.17-6.01 (m,
1H), 5.70 (m, 1H), 5.18-5.01 (m, 3H), 4.25-4.04 (m, 4H), 3.78-3.57
(m, 2H), 2.38-2.27 (m, 2H), 1.5-1.23 (m, 9H); .sup.31P NMR
(CDCl.sub.3) .delta. 29.2 and 27.3; MS (ESI) 502 (M+Na).
Example 7D
[1601] Compound 22: A solution of compound 21 (370 mg) was
dissolved in 8 ml, of EtOH, then 0.12 ml, of acetic acid and 10%
Pd/C (72 mg) was added. The resulted mixture was stirred under
H.sub.2 atmosphere (balloon) for 4 h. After filtration through
celite, the filtered was evaporated under reduced pressure to
afford the compound 22 (320 mg, 96%) as a colorless oil. .sup.1H
NMR (CDCl.sub.3) 7.27-6.86 (m, 4H), 5.98 (s, 2H), 5.18-5.02 (m,
1H), 4.25-4.06 (m, 4H), 3.34-3.24 (m, 2H), 2.44-2.30 (m, 2H),
1.62-1.24 (m, 9H); .sup.31P NMR (CDCl.sub.3) .delta. 28.3 and 26.8;
MS (ESI) 346 (M+1). ##STR523##
Example 8A
[1602] Compound 24: Compound 23 was purified using a Dynamax SD-200
HPLC system. The mobile phase consisted of acetonitrile:water
(65:35, v/v) at a flow rate of 70 mL/min. The injection volume was
4 mL. The detection was by fluorescence at 245 nm and peak area
ratios were used for quantitations. Retention time was 8.2 min for
compound 24 as yellow oil. .sup.1H NMR (CDCl.sub.3) .delta.
7.36-7.19 (m, 10H), 5.88 (m, 1H), 5.12 (s, 2H), 4.90-4.86 (m, 1H),
4.26-4.12 (m, 2H), 3.72-3.61(m, 2H), 2.36-2.29 (m, 2H), 1.79-1.74
(m, 2H); 1.27 (t, J=7.2 Hz, 3H), 0.82 (t, J=7.2 Hz, 3H); .sup.31P
NMR (CDCl.sub.3) .delta. 28.3; MS (ESI) 472 (M+Na).
Example 8B
[1603] Compound 25 was purified in the same manner and retention
time was 7.9 min for compound 25 as yellow oil. .sup.1H NMR
(CDCl.sub.3) .delta. 7.34-7.14 (m, 101H), 5.75 (m, 1H), 5.10 (s,
2H), 4.96-4.91 (m, 1H), 4.18-4.12 (m, 2H), 3.66-3.55(m, 2H),
2.29-2.19 (m, 2H), 1.97-1.89 (m, 2H); 1.21 (t, J=7.2 Hz, 3H), 0.97
(t, J=7.2 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 26.2; MS (ESI)
472 (M+Na).
Example 8C
[1604] Compound 26: A solution of compound 24 (1 g) was dissolved
in 20 ml, of EtOH, then 0.3 ml, of acetic acid and 10% Pd/C (200
mg) was added. The resulted mixture was stirred under H2 atmosphere
(balloon) for 4 h. After filtration through celite, the filtered
was evaporated under reduced pressure to afford the compound 26
(830 mg, 99%) as a colorless oil. .sup.1H NMR (CDCl.sub.3) .delta.
7.46-7.19 (m, 5H), 4.92-4.81 (m, 1H), 4.24-4.21 (m, 2H), 3.41-3.28
(m, 2H), 2.54-2.38 (m, 2H), 1.79-1.74 (m, 2H), 1.27 (t, J=7.2 Hz,
3H), 0.80 (t, J=7.2 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta.
26.9; MS (ESI) 316 (M+1).
Example 8D
[1605] Compound 27: A solution of compound 25 (700 g) was dissolved
in 14 ml, of EtOH, then 0.21 ml, of acetic acid and 10% Pd/C (140
mg) was added. The resulted mixture was stirred under H2 atmosphere
(balloon) for 4 h. After filtration through celite, the filtered
was evaporated under reduced pressure to afford the compound 27
(510 mg, 98%) as a colorless oil. .sup.1H NMR (CDCl.sub.3) .delta.
7.39-7.18 (m, 5H), 4.98-4.85 (m, 1H), 4.25-4.22 (m, 2H), 3.43-3.28
(m, 2H), 2.59-2.41 (m, 2H), 1.99-1.85 (m, 2H), 1.28 (t, J=7.2 Hz,
3H), 1.02 (t, J=7.2 Hz, 3H); .sup.31P NMR (CDCl.sub.3) .delta.
24.2; MS (ESI) 316 (M+1).
Example 8E
[1606] Compound 28: To a stirred solution of compound 16 (1.18 g, 2
mmol) in 9 ml, of 1,2-dichloroethane was added compound 26 (830 mg,
2.2 mmol) and MgSO.sub.4 (80 mg), the resulted mixture was stirred
at room temperature under argon for 3 h, then acetic acid (0.34 mL,
6 mmol) and sodium cyanoborohydride (251 mg, 4 mmol) were added.
The reaction mixture was stirred at room temperature for 2 h under
argon. Then aqueous NaHCO.sub.3 (50 mL) was added, and the mixture
was extracted with ethyl acetate, and the combined organic layers
were washed with brine and water, dried over Na.sub.2SO.sub.4,
filtered and concentrated. The residue was purified by
chromatography on silica gel (EtOH/EtOAc, 1/9) to give 28 (880 mg,
50%) as a white solid. .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d,
J=8.7 Hz, 2H), 7.35-7.16 (m, 9H), 6.99 (d, J=8.7 Hz, 2H), 5.64 (d,
J=5.4 Hz, 1H), 5.03-4.85 (m, 3H), 4.24-3.67 (m overlapping s, 15H),
3.14-2.70 (m, 9H), 2.39-2.28 (m, 2H), 1.85-1.51 (m, 5H), 1.29-1.25
(m, 3H), 0.93-0.78 (m, 9H); .sup.31P NMR (CDCl.sub.3) .delta. 29.2;
MS (ESI) 912 (M+Na).
Example 8F
[1607] Compound 29: To a stirred solution of compound 16 (857 g,
1.45 mmol) in 7 ml, of 1,2-dichloroethane was added compound 27
(600 mg, 1.6 mmol) and MgSO.sub.4 (60 mg), the resulted mixture was
stirred at room temperature under argon for 3 h, then acetic acid
(0.23 mL, 3 mmol) and sodium cyanoborohydride (183 mg, 2.9 mmol)
were added. The reaction mixture was stirred at room temperature
for 2 h under argon. Then aqueous NaHCO.sub.3 (50 mL) was added,
and the mixture was extracted with ethyl acetate, and the combined
organic layers were washed with brine and water, dried over
Na.sub.2SO.sub.4, filtered and concentrated. The residue was
purified by chromatography on silica gel (EtOH/EtOAc, 1/9) to give
29 (650 mg, 50%) as a white solid. .sup.1H NMR (CDCl.sub.3) .delta.
7.72 (d, J=8.7 Hz, 2H), 7.35-7.16 (m, 9H), 7.00 (d, J=8.7 Hz, 2H),
5.64 (d, J=5.4 Hz, 1H), 5.03-4.90 (m, 3H), 4.17-3.67 (m overlapping
s, 15H), 3.16-2.77 (m, 9H), 2.26-2.19 (m, 2H), 1.94-1.53 (m, 5H),
1.26-1.18 (m, 3H), 1.00-0.87 (m, 9H); .sup.31P NMR (CDCl.sub.3)
.delta. 27.4; MS (ESI) 912 (M+Na). ##STR524##
Example 9A
[1608] Compound 31: To a stirred solution of compound 30 (20 g, 60
mmol) in 320 ml, of toluene at room temperature under N.sub.2 was
added thionyl chloride (17.5 mL, 240 mmol) and a few drops of DMF.
The resulted mixture was stirred at 60-70.degree. C. for 3 h. After
cooled to room temperature, the solvent was removed under reduced
pressure, and the residue was added 280 ml, of DCM, followed by TEA
(50 mL, 360 mmol) and (S) lactate ethyl ester (17 mL, 150 mmol).
After 20 h at room temperature, the solvent was removed under
reduced pressure, and the residue was diluted with DCM and washed
with brine and water, dried over Na.sub.2SO.sub.4, filtered and
concentrated. The residue was purified by chromatography on silica
gel (DCM/EtOAc, 1:1) to give 31 (24 g, 92%) as a yellow oil.
.sup.1H NMR (CDCl.sub.3) .delta. 7.33-7.18 (m, 10H), 5.94-6.63 (m,
1H), 5.70 (m, 1H), 5.12-4.95 (m, 3H), 4.24-4.14 (m, 2H),
3.72-3.59(m, 2H), 2.35-2.20 (m, 2H), 1.58-1.19 (m, 6H); .sup.31P
NMR (CDCl.sub.3) .delta. 28.2 and 26.2; MS (ESI) 458 (M+Na).
Example 9B
[1609] Compound 32: Compound 31 was purified using a Dynamax SD-200
HPLC system. The mobile phase consisted of acetonitrile:water
(60:40, v/v) at a flow rate of 70 mL/min. The injection volume was
3 mL. The detection was by fluorescence at 245 nm and peak area
ratios were used for quantitations. Retention time was 8.1 min for
compound 32 as yellow oil. .sup.1H NMR (CDCl.sub.3) .delta.
7.33-7.18 (m, 10H), 5.94-6.63 (m, 1H), 5.70 (m, 1H), 5.12-4.95 (m,
3H), 4.24-4.14 (m, 2H), 3.72-3.59(m, 2H), 2.35-2.20 (m, 2H),
1.58-1.19 (m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 28.2; MS (ESI)
458 (M+Na).
Example 9C
[1610] Compound 33 was purified in the same manner and retention
time was 7.9 min for compound 33 as yellow oil. .sup.1H NMR
(CDCl.sub.3) .delta. 7.33-7.18 (m, 10H), 5.94-6.63 (m, 1H), 5.70
(m, 1H), 5.12-4.95 (m, 3H), 4.24-4.14 (m, 2H), 3.72-3.59(m, 2H),
2.35-2.20 (m, 2H), 1.58-1.19 (m, 6H); .sup.31P NMR (CDCl.sub.3)
.delta. 26.2; MS (ESI) 458 (M+Na).
Example 9D
[1611] Compound 34: A solution of compound 33 (3.2 g) was dissolved
in 60 ml, of EtOH, then 0.9 ml, of acetic acid and 10% Pd/C (640
mg) was added. The resulted mixture was stirred under H.sub.2
atmosphere (balloon) for 4 h. After filtration through celite, the
filtered was evaporated under reduced pressure to afford the
compound 34 (2.7 g, 99%) as a colorless oil. .sup.1H NMR
(CDCl.sub.3) .delta. 7.42-7.18 (m, 5H), 6.10 (s, 1H), 5.15-5.02
(m,1H), 4.24-4.05 (m, 2H), 3.25-3.16 (m, 2H), 2.36-2.21 (m, 2H),
1.61-1.58 (m, 3H), 1.35-1.18, m, 3H); .sup.31P NMR .delta.
(CDCl.sub.3) .delta. 26.1; MS (ESI) 302 (M+1).
Example 9E
[1612] Compound 35: To a stirred solution of compound 16 (8.9 g, 15
mmol) in 70 ml, of 1,2-dichloroethane was added compound 34 (8.3 g,
23 mmol) and MgSO.sub.4 (80 mg), the resulted mixture was stirred
at room temperature under argon for 2.5 h, then acetic acid (3 mL,
52.5 mmol) and sodium cyanoborohydride (1.9 g, 30 mmol) were added.
The reaction mixture was stirred at room temperature for 1.5 h
under argon. Then aqueous NaHCO.sub.3 (100 mL) was added, and the
mixture was extracted with ethyl acetate, and the combined organic
layers were washed with brine and water, dried over
Na.sub.2SO.sub.4, filtered and concentrated. The residue was
purified by chromatography on silica gel (EtOH/EtOAc, 1/9) to give
35 (8.4 g, 64%) as a white solid. .sup.1H NMR (CDCl.sub.3) .delta.
7.73 (d, J=8.7 Hz, 2H), 7.36-7.17(m, 9H), 7.00 (d, J=8.7 Hz, 2H),
5.64 (d, J=5.1 Hz, 1H), 5.07-4.97 (m, 3H), 4.19-3.67 (m overlapping
s, 13H), 3.15-2.78 (m, 9H), 2.25-2.19 (m, 2H), 1.91-1.54 (m, 6H),
1.24-1.20 (m, 3H), 0.94-0.87 (m, 6H); .sup.31P NMR (CDCl.sub.3)
.delta. 27.4; MS (ESI) 876 (M+1).
Resolution of Compound 35 Diastereomers
[1613] Analysis was performed on an analytical Daicel Chiralcel OD
column (FIG. 3, 4), conditions described below, with a total of
about 3.5 mg compound 35 free base injected onto the column. This
lot was about a 3:1 mixture of major to minor diastereomers where
the lactate ester carbon is a 3:1 mix of R and S
configurations.
[1614] Two injections of 3.8 and 3.5 mg each were made using the
conditions described below. The isolated major diastereomer
fractions were evaporated to dryness on a rotary evaporator under
house vacuum. The chromatographic solvents were displaced by two
portions of ethyl acetate followed by a single portion of ethyl
acetate-trifluoroacetic acid (about 95:5) and a final high vacuum
strip to aid in removal of trace solvents. This yielded the major
diastereomer trifluoroacetate salt as a gummy solid.
[1615] The resolved minor diastereomer was isolated for biological
evaluation by an 11 mg injection, performed on an analytical Daicel
Chiralcel OD column, using the conditions described in below. The
minor diastereomer of 35 was isolated as the trifluoroacetate salt
by the conditions described above.
[1616] Larger scale injections (.about.300 mg 35 per injection)
were later performed on a Daicel Chiralcel OD column
semi-preparative column with a guard column (FIG. 5), conditions
described below. A minimal quantity of isopropyl alcohol was added
to heptane to dissolve the 3:1 diastereomeric mix of 35 and the
resolved diastereomers sample, and the isolated fractions were
refrigerated until the eluted mobile phase was stripped.
Analytical Column, .about.4 mg Injection, Heptane-EtOH (20:80)
Initial (FIG. 3)
[1617] HPLC Conditions [1618] Column: Chiralcel OD, 10 .mu.m,
4.6.times.250 mm [1619] Mobile Phase: Heptane-Ethyl Alcohol (20:80
initial) [1620] : 100% Ethyl Alcohol (final) [1621] Note: Final
began after first peak eluted [1622] Flow Rate: 1.0 mL/min [1623]
Run Time: As needed [1624] Detection: UV at 250 nm [1625]
Temperature: Ambient [1626] Injection: .about.4 mg on Column [1627]
Sample Prep.: Dissolved in .about.1 ml, heptane-ethyl alcohol
(50:50) [1628] Retention Times: 35 Minor .about.14 min [1629] 35
Majors .about.25 min Analytical Column, .about.6 mg Injection,
Heptane-EtOH (65:35) Initial (FIG. 4)
[1630] HPLC Conditions [1631] Column: Chiralcel OD, 10 .mu.m,
4.6.times.250 mm [1632] : Mobile Phase: Heptane-Ethyl Alcohol
(65:35 initial) [1633] : Heptane-Ethyl Alcohol (57.5:42.5
intermediate) [1634] Note: Intermediate began after impurity peaks
eluted [1635] : Heptane-Ethyl Alcohol (20:80 final) [1636] Note:
Final mobile phase began after minor diastereomer eluted [1637]
Flow Rate: 1.0 mL/min [1638] Run Time: As needed [1639] Detection:
V at 250 nm [1640] Temperature: Ambient [1641] Injection: .about.4
mg on Column [1642] Sample Prep.: Dissolved in .about.1 ml,
heptane-ethyl alcohol (50:50) [1643] Retention Times: 35 Minor
.about.14 min [1644] : 35 Major .about.40 min Semi-Preparative
Column, .about.300 mg Injection, Heptane-EtOH (65:35) Initial (FIG.
5)
[1645] HPLC Conditions [1646] Columns: Chiralcel OD, 20 .mu.m,
21.times.50 mm (guard) [1647] : Chiralcel OD, 20 .mu.m,
21.times.250 mm [1648] Mobile Phase: Heptane-Ethyl Alcohol (65:35
initial) [1649] : Heptane-Ethyl Alcohol (50:50 intermediate) [1650]
Note: Intermediate began after minor diastereomer peak eluted
[1651] : Heptane-Ethyl Alcohol (20:80 final) [1652] Note: Final
mobile phase began after major diastereomer began to elute [1653]
Flow Rate: 10.0 mL/min [1654] Run Time: As needed [1655] Detection:
UV at 260 nm [1656] Temperature: Ambient [1657] Injection:
.about.300 mg on Column [1658] Sample Prep.: Dissolved in
.about.3.5 ml, hetpane-ethyl alcohol (70:30) [1659] Retention
Times: 35 Minor .about.14 min [1660] : 35 Major .about.40 min
##STR525## ##STR526##
Example 29
[1661] Triflate derivative 1: A THF--CH.sub.2Cl.sub.2 solution (30
mL-10 mL) of 8 (4 g, 6.9 mmol), cesium carbonate (2.7 g, 8 mmol),
and N-phenyltrifluoromethane sulfonimide (2.8 g, 8 mmol) was
reacted overnight. The reaction mixture was worked up, and
concentrated to dryness to give crude triflate derivative 1.
[1662] Aldehyde 2: Crude triflate 1 (4.5 g, 6.9 mmol) was dissolved
in DMF (20 mL), and the solution was degassed (high vacuum for 2
min, Ar purge, repeat 3 times). Pd(OAc).sub.2 (0.12 g, 0.27 mmol),
and bis(diphenylphosphino)propane (dppp, 0.22 g, 0.27 mmol) were
added, the solution was heated to 70.degree. C. Carbon monoxide was
rapidly bubbled through the solution, then under 1 atmosphere of
carbon monoxide. To this solution were slowly added TEA (5.4 mL, 38
mmol), and triethylsilane (3 ml), 18 mmol). The resulting solution
was stirred overnight at room temperature. The reaction mixture was
worked up, and purified on silica gel column chromatograph to
afford aldehyde 2 (2.1 g, 51%). (Hostetler, et al J. Org. Chem.,
1999. 64, 178-185).
[1663] Lactate prodrug 4: Compound 4 is prepared as described above
procedure for Example 9E, Compound 35 by the reductive amination
between 2 and 3 with NaBH.sub.3CN in 1,2-dichloroethane in the
presence of HOAc.
Example 30 Preparation of Compound 3
[1664] Diethyl (cyano(dimethyl)methyl) phosphonate 5: A THF
solution (30 mL) of NaH (3.4 g of 60% oil dispersion, 85 mmol) was
cooled to -10.degree. C., followed by the addition of diethyl
(cyanomethyl)phosphonate (5 g, 28.2 mmol) and iodomethane (17 g,
112 mmol). The resulting solution was stirred at -10.degree. C. for
2 hr, then 0.degree. C. for 1 hr, was worked up, and purified to
give dimethyl derivative 5 (5 g, 86%).
[1665] Diethyl (2-amino-1 .mu.l-dimethyl-ethyl)phosphonate 6:
Compound 5 was reduced to amine derivative 6 by the described
procedure (J. Med. Chem. 1999, 42, 5010-5019).
[1666] A solution of ethanol (150 mL) and 1N HCl aqueous solution
(22 mL) of 5 (2.2 g, 10.7 mmol) was hydrogenated at 1 atmosphere in
the presence of PtO.sub.2 (1.25 g) at room temperature overnight.
The catalyst was filtered through a celite pad. The filtrate was
concentrated to dryness, to give crude 6 (2.5 g, as HCl salt).
[1667] 2-Amino-1,1-dimethyl-ethyl phosphonic acid 7: A solution of
CH.sub.3CN (30 mL) of crude 6 (2.5 g) was cooled to 0.degree. C.,
and treated with TMSBr (8 g, 52 mmol) for 5 hr. The reaction
mixture was stirred with methanol for 1.5 hr at room temperature,
concentrated, recharged with methanol, concentrated to dryness to
give crude 7 which was used for next reaction without further
purification.
[1668] Lactate phenyl (2-amino-1,1-dimethyl-ethyl)phosphonate 3:
Compound 3 is synthesized according to the procedures described in
Example 9D, Compound 34 for the preparation of lactate phenyl
2-aminoethyl phosphonate 34. Compound 7 is protected with CBZ,
followed by the reaction with thionyl chloride at 70.degree. C. The
CBZ protected dichlorodate is reacted phenol in the presence of
DIPEA. Removal of one phenol, follow by coupling with ethyl
L-lactate leads N--CBZ-2-amino-1,1-dimethyl-ethyl phosphonate
derivative. Hydrogenation of N--CBZ derivative at 1 atmosphere in
the presence of 10% Pd/C and 1 eq. of TFA affords compound 3 as TFA
salt. ##STR527##
Example 1
[1669] Monophenol Allylphosphonate 2: To a solution of
allylphosphonic dichloride (4 g, 25.4 mmol) and phenol (5.2 g, 55.3
mmol) in CH.sub.2Cl.sub.2 (40 mL) at 0.degree. C. was added TEA
(8.4 mL, 60 mmol). After stirred at room temperature for 1.5 h, the
mixture was diluted with hexane-ethyl acetate and washed with HCl
(0.3 N) and water. The organic phase was dried over MgSO.sub.4,
filtered and concentrated under reduced pressure. The residue was
filtered through a pad of silica gel (eluted with 2:1 hexane-ethyl
acetate) to afford crude product diphenol allylphosphonate 1 (7.8
g, containing the excessive phenol) as an oil which was used
directly without any further purification. The crude material was
dissolved in CH.sub.3CN (60 mL), and NaOH (4.4N, 15 mL) was added
at 0.degree. C. The resulted mixture was stirred at room
temperature for 3 h, then neutralized with acetic acid to pH=8 and
concentrated under reduced pressure to remove most of the
acetonitrile. The residue was dissolved in water (50 mL) and washed
with CH.sub.2Cl.sub.2 (3.times.25 mL). The aqueous phase was
acidified with concentrated HCl at 0.degree. C. and extracted with
ethyl acetate. The organic phase was dried over MgSO.sub.4,
filtered, evaporated and co-evaporated with toluene under reduced
pressure to yield desired monophenol allylphosphonate 2 (4.75 g.
95%) as an oil.
Example 2
[1670] Monolactate Allylphosphonate 4: To a solution of monophenol
allylphosphonate 2 (4.75 g, 24 mmol) in toluene (30 mL) was added
SOCl.sub.2 (5 mL, 68 mmol) and DMF (0.05 mL). After stirred at
65.degree. C. for 4 h, the reaction was completed as shown by
.sup.31P NMR. The reaction mixture was evaporated and co-evaporated
with toluene under reduced pressure to give mono chloride 3 (5.5 g)
as an oil. To a solution of chloride 3 in CH.sub.2Cl.sub.2 (25 mL)
at 0.degree. C. was added ethyl (s)-lactate (3.3 mL, 28.8 mmol),
followed by TEA. The mixture was stirred at 0.degree. C. for 5 min
then at room temperature for 1 h, and concentrated under reduced
pressure. The residue was partitioned between ethyl acetate and HCl
(0.2N), the organic phase was washed with water, dried over
MgSO.sub.4, filtered and concentrated under reduced pressure. The
residue was purified by chromatography on silica gel to afford
desired monolactate 4 (5.75 g, 80%) as an oil (2:1 mixture of two
isomers): .sup.1H NMR (CDCl.sub.3) .delta. 7.1-7.4 (m, 5H), 5.9 (m,
1H), 5.3 (m, 2H), 5.0 (m, 1H), 4.2 (m, 2H), 2.9 (m, 2H), 1.6; 1.4
(d, 3H), 1.25 (m, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 25.4,
23.9.
Example 3
[1671] Aldehyde 5: A solution of allylphosphonate 4 (2.5 g, 8.38
mmol) in CH.sub.2Cl.sub.2 (30 mL) was bubbled with ozone air at
-78.degree. C. until the solution became blue, then bubbled with
nitrogen until the blue color disappeared. Methyl sulfide (3 mL)
was added at -78.degree. C. The mixture was warmed up to room
temperature, stirred for 16 h and concentrated under reduced
pressure to give desired aldehyde 5 (3.2 g, as a 1:1 mixture of
DMSO): .sup.1H NMR (CDCl.sub.3) .delta. 9.8 (m, 1H), 7.1-7.4 (m,
5H), 5.0 (m, 1H), 4.2 (m, 2H), 3.4 (m, 2H), 1.6; 1.4 (d, 3H), 1.25
(m, 3H); .sup.31P NMR (CDCl.sub.3) .delta. 17.7, 15.4.
Example 4
[1672] Compound 7: To a solution of aniline 6 (reported before)
(1.62 g, 2.81 mmol) in THF (40 mL) was added acetic acid (0.8 mL,
14 mmol), followed by aldehyde 5 (1.3 g, 80%, 3.46 mmol) and
MgSO.sub.4 (3 g). The mixture was stirred at room temperature for
0.5 h, then NaBH.sub.3CN (0.4 g, 6.37 mmol) was added. After
stirred for 1 h, the reaction mixture was filtered. The filtrate
was diluted with ethyl acetate and washed with NaHCO.sub.3, dried
over MgSO.sub.4, filtered and concentrated under reduced pressure.
The residue was purified by chromatography on silica gel to give
compound 6 (1.1 g, 45%) as a 3:2 mixture of two isomers, which were
separated by HPLC (mobile phase, 70% CH.sub.3CN/H.sub.2O; flow
rate: 70 mL/min; detection: 254 mm; column: 8.mu. C18, 41.times.250
mm, Varian). Isomer A (0.39 g): .sup.1H NMR (CDCl.sub.3) .delta.
7.75 (d, 2H), 7.1-7.4 (m, 5H), 7.0 (m, 4H), 6.6 (d, 2H), 5.65 (d,
1H), 5.05 (m, 2H), 4.9 (d, 1H), 4.3 (brs, 1H), 4.2 (q, 2H), 3.5-4.0
(m, 6H), 3.9 (s, 3H), 2.6-3.2 (m, 9H), 2.3 (m, 2), 1.6-1.9 (m, 5H),
1.25 (t, 3H), 0.9 (2d, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 26.5;
MS (ESI): 862 (M+H). Isomer B (0.59 g): .sup.1H NMR (CDCl.sub.3)
.delta. 7.75 (d, 2H), 7.1-7.4 (m, 5H), 7.0 (m, 4H), 6.6 (d, 2H),
5.65 (d, 1H), 5.05 (m, 2H), 4.9 (d, 1H), 4.5 (brs, 1H), 4.2 (q,
2H), 3.5-4.0 (m, 6H), 3.9 (s, 3H), 2.7-3.2 (m, 9H), 2.4 (m, 2),
1.6-1.9 (m, 2H), 1.4 (d, 3H), 1.25 (t, 3H), 0.9 (2d, 6H); .sup.31P
NMR (CDCl.sub.3) .delta. 28.4; MS (ESI): 862 (M+H). ##STR528##
Example 5
[1673] Acid 8: To a solution of compound 7 (25 mg, 0.029 mmol) in
acetonitrile (1 mL) at 0.degree. C. was added NaOH (1N, 0.125 mL).
The mixture was stirred at 0.degree. C. for 0.5 h and at room
temperature for 1 h. The reaction was quenched with acetic acid and
purified by HPLC to give acid 8 (10 mg, 45%). .sup.1H NMR
(CD.sub.3OD) .delta. 7.8 (d, 2H), 7.5 (d, 2H), 7.4 (d, 2H), 7.1 (d,
2H), 5.6 (d, 1H), 4.9 (m, 3H), 3.2-4.0 (m, 6H), 3.9 (s, 3H),
2.6-3.2 (m, 9H), 2.05 (m, 2), 1.4-1.7 (m, 2H), 1.5 (d, 3H), 0.9
(2d, 6H); .sup.31P NMR (CD.sub.3OD) .delta. 20.6; MS (ESI): 758
(M+H).
Example 6
[1674] Diacid 10: To a solution of triflate 9 (94 mg, 0.214 mmol)
in CH.sub.2Cl.sub.2 (2 mL) was added a solution of aniline 6 (100
mg, 0.173 mmol) in CH.sub.2Cl.sub.2 (2 mL) at -40.degree. C.,
followed by 2,6-lutidine (0.026 mL). The mixture was warmed up to
room temperature and stirred for 1 h. Cesium carbonate (60 mg) was
added and the reaction mixture was stirred for additional 1 h. The
mixture was diluted with ethyl acetate, washed with HCl (0.2N),
dried over MgSO.sub.4, filtered and concentrated under reduced
pressure. The residue was purified by HPLC to afford dibenzyl
phosphonate (40 mg). To a solution of this dibenzyl phosphonate in
ethanol (3 mL) and ethyl acetate (1 mL) was added 10% Pd/C (40 mg).
The mixture was stirred under hydrogen atmosphere (balloon) for 4
h. The reaction mixture was diluted with methanol, filtered and
concentrated under reduced pressure. The residue was washed with
ethyl acetate and dried to give desired product diacid 10 (20 mg).
.sup.1H NMR (CD.sub.3OD) .delta. 7.8 (d, 2H), 7.3 (d, 2H), 7.1 (2d,
4H), 5.6 (d, 1H), 4.9 (m, 2H), 3.4-4.0 (m, 6H), 3.9 (s, 3H),
2.5-3.2 (m, 9H), 2.0 (m, 2), 1.4-1.7 (m, 2H), 0.9 (2d, 6H);
.sup.31P NMR (CD.sub.3OD) .delta. 22.1; MS (ESI): 686 (M+H).
##STR529##
[1675] The synthesis of compound 19 is outlined in Scheme 3.
Condensation of 2-methyl-2-propanesulfinamide with acetone give
sulfinyl imine 11 (J. Org. Chem. 1999, 64, 12). Addition of
dimethyl methylphosphonate lithium to 11 afford 12. Acidic
methanolysis of 12 provide amine 13. Protection of amine with Cbz
group and removal of methyl groups yield phosphonic acid 14, which
can be converted to desired 15 using methods reported earlier on.
An alternative synthesis of compound 14 is also shown in Scheme 3.
Commercially available 2-amino-2-methyl-1-propanol is converted to
aziridines 16 according to literature methods (J. Org. Chem. 1992,
57, 5813; and Syn. Lett. 1997, 8, 893). Aziridine opening with
phosphite give 17 (Tetrahedron Lett. 1980, 21, 1623). Deprotection
(and, if necessary, reprotection) of 17 afford 14. Reductive
amination of amine 15 and aldehyde 18 provides compound 19.
##STR530##
Example 1
[1676]
2-{[2-(4-{2-(Hexahydro-furo[2,3-b]furan-3-yloxycarbonylamino)-3-hy-
droxy-4-[isobutyl-(4-methoxy-benzenesulfonyl)-amino]-butyl}-benzylamino)-e-
thyl]-phenoxy-phosphinoyloxy}-propionic acid ethyl ester 2
(Compound 35, previous Example 9E).
[1677] A solution of 1 (2.07 g, 3.51 mmol) and 4 (1.33 g, 3.68 mmol
of a 4:1 mixture of two diastereomers at the phosphorous center)
were dissolved in 14 ml, of (CH.sub.2Cl).sub.2 to provide a clear
solution. Addition of MgSO.sub.4 (100 mg) to the solution resulted
in a white cloudy mixture. The solution was stirred at ambient
temperature for 3 hours when acetic acid (0.80 mL, 14.0 mmol) and
sodium cyanoborohydride (441 mg, 7.01 mmol) were added. Following
the reaction progress by TLC showed complete consumption of the
aldehyde starting materials in 1 hour. The reaction mixture was
worked up by addition of 200 ml, of saturated aqueous NaHCO.sub.3
and 400 ml, of CH.sub.2Cl.sub.2. The aqueous layer was extracted
with CH.sub.2Cl.sub.2 two more times (2.times.300 mL). The combined
organic extracts were dried in vacuo and purified by column
chromatography (EtOAc-10% MeOH:EtOAc) to provide the desired
product as a foam. The early eluting compound from the column was
collected and characterized as alcohol 3 (810 mg, 39%). Addition of
TFA (3.times.1 mL) generated the TFA salt which was lyopholized
from 50 ml, of a 1:1 CH.sub.3CN:H.sub.2O to provide 1.63 g (47%) of
the product 2 as a white powder. .sup.1H NMR (CD.sub.3CN) .delta.
8.23 (br s, 2H), 7.79 (d, J=8.4 Hz, 2H), 7.45-7.13 (m, 9H), 7.09
(d, J=8.4 Hz, 2H), 5.86 (d, J=9.0 Hz, 1H), 5.55 (d, J=4.8 Hz, 1H),
5.05-4.96 (m, 1H), 4.96-4.88 (m, 1H), 4.30-4.15 (m, 4H), 3.89 (s,
3H), 3.86-3.76 (m, 4H), 3.70-3.59 (m, 4H), 3.56-3.40 (m, 2H), 3.34
(d, J=15 Hz, 1H), 3.13 (d, J=13.5 Hz, 1H), 3.06-2.93 (m, 2H),
2.92-2.80 (m, 2H), 2.69-2.43 (m, 3H), 2.03-1.86 (m, 1H), 1.64-1.48
(m, 1H), 1.53 and 1.40 (d, J=6.3 Hz, J=6.6 Hz, 3H), 1.45-1.35 (m,
1H), 1.27 and 1.23 (t, J=6.9 Hz, J=7.2 Hz, 3H), 0.90 (t, J=6.9 Hz,
6H). .sup.31P NMR (CD.sub.3CN) .delta. 24.47, 22.86. ESI
(M+H).sup.+ 876.4.
Example 2
[1678] ##STR531##
[1679]
2-{[2-(4-{2-(Hexahydro-furo[2,3-b]furan-3-yloxycarbonylamino)-3-hy-
droxy-4-[isobutyl-(4-methoxy-benzenesulfonyl)-amino]-butyl}-benzylamino)-e-
thyl]-phenoxy-phosphinoyloxy}-propionic acid ethyl ester
(MF-1912-68):
[1680] A solution of MF-1912-67 (0.466 g, 0.789 mmol) and
ZY-1751-125 (0.320 g, 0.789 mmol of a 1:1 mixture of two
diastereomers at the phosphorous center) were dissolved in 3.1 ml,
of (CH.sub.2Cl.sub.2).sub.2 to provide a clear solution. Addition
of MgSO.sub.4 (20 mg) to the solution resulted in a white cloudy
mixture. The solution was stirred at ambient temperature for 3
hours when acetic acid (0.181 mL, 3.16 mmol) and sodium
cyanoborohydride (99 mg, 1.58 mmol) were added. Following the
reaction progress by TLC showed complete consumption of the
aldehyde starting materials in 1.5 hour. The reaction mixture was
worked up by addition of 50 ml, of saturated aqueous NaHCO.sub.3
and 200 ml, of CH.sub.2Cl.sub.2. The aqueous layer was extracted
with CH.sub.2Cl.sub.2 two more times (2.times.200 mL). The combined
organic extracts were dried in vacuo and purified by column
chromatography (EtOAc-10% MeOH:EtOAc) to provide the desired
product as a foam. The early eluting compound from the column was
collected and characterized to be MF-1912-48b alcohol (190 mg,
41%). Addition of TFA (3.times.1 mL) generated the TFA salt which
was lyopholized from 50 ml, of a 1:1 CH.sub.3CN:H.sub.2O to provide
0.389 g (48%) of the product as a white powder. .sup.1H NMR (CD3CN)
.delta. 8.39 (br s, 2H), 7.79 (d, J=8.7 Hz, 2H), 7.40 (d, J=7.5 Hz,
2H), 7.34 (d, J=8.1 Hz, 2H), 7.26-7.16 (m, 2H), 7.10 (d, J=9 Hz,
3H), 7.01-6.92 (m, 1H), 5.78 (d, J=9.0 Hz, 1H), 5.55 (d, J=5.1 Hz,
1H), 5.25-5.03 (m, 1H), 4.95-4.88 (m, 1H), 4.30-4.17 (m, 4H),
4.16-4.07 (m, 2H), 3.90 (s, 3H), 3.88-3.73 (m, 4H), 3.72-3.60 (m,
2H), 3.57-3.38 (m, 2H), 3.32 (br d, J=15.3 Hz, 1H), 3.13 (br d,
J=14.7 Hz, 1H), 3.05-2.92 (m, 2H), 2.92-2.78 (m, 2H), 2.68-2.48 (m,
3H), 2.03-1.90 (m, 1H), 1.62-1.51 (m, 1H), 1.57 and 1.46 (d, J=6.9
Hz, J=6.9 Hz, 3H), 1.36-1.50 (m, 1H), 1.43-1.35 (m, 4H), 1.33-1.22
(m, 3H), 0.91 (t, J=6.6 Hz, 6H). .sup.31P NMR (CD.sub.3CN) .delta.
25.27, 23.56. ESI (M+H).sup.+ 920.5. ##STR532## ##STR533##
Example 1
[1681] Mono-Ethyl mono-lactate 3: To a solution of 1 (96 mg, 0.137
mmol) and ethyl lactate 2 (0.31 mL, 2.7 mmol) in pyridine (2 mL)
was added N,N-dicyclohexylcarbodiimide (170 mg, 0.822 mmol). The
solution was stirred for 18 h at 70.degree. C. The mixture was
cooled to room temperature and diluted with dichloromethane. The
solid was removed by filtration and the filtrate was concentrated.
The residue was suspended in diethyl ether/dichloromethane and
filtered again. The filtrate was concentrated and mixture was
chromatographed on silica gel eluting with EtOAc/hexane to provide
compound 3 (43 mg, 40%) as a foam: .sup.1H NMR (CDCl.sub.3) .delta.
7.71 (d, 2H), 7.00 (d, 2H); 7.00 (d, 2H), 6.88 (d, 2H), 5.67 (d,
1H), 4.93-5.07 (m, 2H), 4.15-4.39 (m, 6H), 3.70-3.99 (m, 10H),
2.76-3.13 (m, 7H), 1.55-1.85 (m, 9H), 1.23-1.41 (m, 6H), 0.90 (dd,
6H); .sup.31P NMR (CDCl.sub.3) .delta. 19.1, 20.2; MS (ESI) 823
(M+Na).
Example 2
[1682] Bis-2,2,2-trifluoroethyl phosphonate 6: To a solution of 4
(154 mg, 0.228 mmol) and 222,-trifluoroethanol 5 (1 mL, 13.7 mmol)
in pyridine (3 mL) was added N,N-dicyclohexylcarbodiimide (283 mg,
1.37 mmol). The solution was stirred for 6.5 h at 70.degree. C. The
mixture was cooled to room temperature and diluted with
dichloromethane. The solid was removed by filtration and the
filtrate was concentrated. The residue was suspended in
dichloromethane and filtered again. The filtrate was concentrated
and mixture was chromatographed on silica gel eluting with
EtOAc/hexane to provide compound 6 (133 mg, 70%) as a foam: .sup.1H
NMR (CDCl.sub.3) .delta. 7.71 (d, 2H), 7.21 (d, 2H); 7.00 (d, 2H),
6.88 (dd, 2H), 5.66 (d, 1H), 4.94-5.10 (m, 3H), 4.39-4.56 (m, 6H),
3.71-4.00 (m, 10H), 2.77-3.18 (m, 7H), 1.67-1.83(m, 2H), 0.91 (dd,
4H); .sup.31P NMR (CDCl.sub.3) .delta. 22.2; MS (ESI) 859
(M+Na).
Example 3
[1683] Mono-2,2,2-trifluoroethyl phosphonate 7: To a solution of 6
(930 mg, 1.11 mmol) in THF (14 mL) and water (10 mL) was added an
aqueous solution of NaOH in water (1N, 2.2 mL). The solution was
stirred for 1 h at 0.degree. C. An excess amount of Dowex resin
(H.sup.+) was added to until pH=1. The mixture was filtered and the
filtrate was concentrated under reduced pressure. The concentrated
solution was azeotroped with EtOAc/toluene three times and the
white powder was dried in vacuo provide compound 7 (830 mg, 100%).
.sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, 2H), 7.11 (d, 2H); 6.99
(d, 2H), 6.85 (d, 2H), 5.63 (d, 1H), 5.26 (m, 1H), 5.02 (m, 1H),
4.40 (m, 1H), 4.14 (m, 4H), 3.60-3.95 (m, 12H), 2.62-3.15 (m, 15H),
1.45-1.84 (m, 3H), 1.29 (m, 4H), 0.89 (d, 6H); .sup.31P NMR
(CDCl.sub.3) .delta. 19.9; MS (ESI) 723 (M+Na).
Example 4
[1684] Mono-2,2,2-trifluoroethyl mono-lactate 8: To a solution of 7
(754 mg, 1 mmol) and N,N-dicyclohexylcarbodiimide (1.237 g, 6 mmol)
in pyridine (10 mL) was added ethyl lactate (2.26 mL, 20 mmol). The
solution was stirred for 4.5 h at 70.degree. C. The mixture was
concentrated and the residue was suspended in diethyl ether (5 mL)
and dichloromethane (5 mL) and filtered. The solid was washed a few
times with diethyl ether. The combined filtrate was concentrated
and the crude product was chromatographed on silica gel, eluting
with EtOAc and hexane to provide compound 8 (610 mg, 71%) as a
foam. .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, 2H), 7.16 (d, 2H);
6.99 (d, 2H), 6.88 (dd, 2H), 5.66 (d, 1H), 4.95-5.09 (m, 2H),
4.19-4.65 (m, 6H), 3.71-4.00 (m, 9H), 2.76-3.13 (m, 6H), 1.57-1.85
(m, 7H), 1.24-1.34 (m, 4H), 0.91 (dd, 6H); .sup.31P NMR
(CDCl.sub.3) .delta. 20.29, 21.58; MS (ESI) 855 (M+1).
Example 1
[1685] Boc-protected hydroxylamine 1: A solution of diethyl
hydroxymethyl phosphonate triflate (0.582 g, 1.94 mmol) in
dichloromethane (19.4 mL) was treated with triethylamine (0.541 mL,
3.88 mmol). Tert-butyl N-hydroxy-carbamate (0.284 g, 2.13 mmol) was
added and the reaction mixture was stirred at room temperature
overnight. The mixture was partitioned between dichloromethane and
water. The organic phase was washed with saturated NaCl, dried
(MgSO.sub.4) and evaporated under reduced pressure. The crude
product was purified by chromatography on silica gel (1/1-ethyl
acetate/hexane) affording the BOC-protected hydroxylamine 1 (0.41
g, 75%) as an oil: .sup.1H NMR (CDCl.sub.3) .delta. 7.83 (s, 1H),
4.21 (d, 2H), 4.18 (q, 4H), 1.47 (s, 9H), 1.36 (t, 6H); .sup.31P
NMR (CDCl.sub.3) .delta. 19.3.
Example 2
[1686] Hydroxylamine 2: A solution of BOC-protected hydroxylamine 1
(0.305 g, 1.08 mmol) in dichloromethane (2.40 mL) was treated with
trifluoroacetic acid (0.829 mL, 10.8 mmol). The reaction was
stirred for 1.5 hours at room temperature and then the volatiles
were evaporated under reduced pressure with toluene to afford the
hydroxylamine 2 (0.318 g, 100%) as the TFA salt which was used
directly without any further purification: .sup.1H NMR (CDCl.sub.3)
.delta. 10.87 (s, 2H), 4.45 (d, 2H), 4.24 (q, 4H), 1.38 (t, 6H);
.sup.31P NMR (CDCl.sub.3) .delta. 16.9; MS (ESI) 184 (M+H).
Example 3
[1687] Oxime 4: To a solution of aldehyde 3 (96 mg, 0.163 mmol) in
1,2-dichloroethane (0.65 mL) was added hydroxylamine 2 (72.5 mg,
0.244 mmol), triethylamine (22.7 .mu.L, 0.163 mmol) and MgSO.sub.4
(10 mg). The reaction mixture was stirred at room temperature for 2
hours then the mixture was partitioned between dichloromethane and
water. The organic phase was washed with saturated NaCl, dried
(MgSO.sub.4) and evaporated under reduced pressure. The crude
product was purified by chromatography on silica gel (90/10-ethyl
acetate/hexane) affording, GS-277771, oxime 4 (0.104 g, 85%) as a
solid: .sup.1H NMR (CDCl.sub.3) .delta. 8.13 (s, 1H), 7.72 (d, 2H),
7.51 (d, 2H), 7.27 (d, 2H), 7.00 (d, 2H), 5.67 (d, 1H), 5.02 (m,
2H), 4.54 (d, 2H), 4.21 (m, 4H), 3.92 (m, 1H), 3.89 (s, 3H), 3.88
(m, 1H), 3.97-3.71 (m, 2H), 3.85-3.70 (m, 2H), 3.16-2.99 (m, 2H),
3.16-2.81 (m, 7H), 1.84 (m, 1H), 1.64-1.48 (m, 2H), 1.37 (t, 6H),
0.94-0.90 (dd, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 20.0; MS
(ESI) 756 (M+H). ##STR534## ##STR535## ##STR536##
Example 1
[1688] Compound 1 was prepared according to methods from previous
Schemes
Example 2
[1689] Compound 2: To a solution of compound 1 (5.50 g, 7.30 mmol),
Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
(5.70 g, 10.95 mmol), and Ethyl(S)-(-)lactate (1.30 g, 10.95 mmol)
in DMF (50 mL) was added Diisopropylethylamine (5.08 mL, 29.2
mmol). The mixture was stirred for 7 hours after which was diluted
in EtOAc. The organic phase was washed with H.sub.2O (5.times.),
brine, dried over MgSO.sub.4 and concentrated in vacuo. The residue
was purified by silica gel chromatography
(CH.sub.2Cl.sub.2/Isopropanol=100/4) to give 3.45 g of compound
2.
Example 3
[1690] Compound 3: To the mixture of compound 2 (3.45 g) in
EtOH/EtOAc (300 mL/100 mL) was added 20% Pd/C (0.700 g). The
mixture was hydrogenated for 1 hour. Celite was added and the
mixture was stirred for 10 minutes. The mixture was filtered
through a pad of celite and washed with ethanol. Concentration gave
2.61 g of compound 3.
Example 4
[1691] Compound 4: To a solution of compound 3 (1.00 g, 1.29 mmol)
in dry dimethylformamide (5 mL) was added 3-Hydroxy-benzoic acid
benzyl ester (0.589 g, 2.58 mmol),
Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
(1.34 g, 2.58 mmol), followed by addition of Diisopropylethylamine
(900 .mu.L, 5.16 mmol). The mixture was stirred for 14 hours, the
resulting residue was diluted in EtOAc, washed with brine
(3.times.) and dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography (CH.sub.2Cl.sub.2/Isopropanol=100/3) to
provide 67.3 mg of compound 4: .sup.1H NMR (CDCl.sub.3) .delta.
7.91 (2H, d, J=8.9 Hz), 7.75 (2H, m), 7.73-7.3 (13H, m), 7.25 (2H,
m), 7.21-6.7(6H, m), 5.87(1H, m), 5.4-4.8(6H, m), 4.78-4.21 (4H,
m), 3.98 (3H,s), 2.1-1.75 (8H, m), 1.55 (3H, m), 1.28(3H, m),
0.99(6H, m).
Example 5
[1692] Compound 5: To a solution of compound 3 (1.40 g, 1.81 mmol)
in dry dimethylformamide (5 mL) was added
(4-Hydroxy-benzyl)-carbamic acid tert-butyl ester (0.80 g, 3.62
mmol), Benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate (1.74 g, 3.62 mmol), followed by addition of
Diisopropylethylamine (1.17 ml, 7.24 mmol). The mixture was stirred
for 14 hours, the resulting residue was diluted in EtOAc, washed
with brine (3.times.) and dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography (CH.sub.2Cl.sub.2/Isopropanol=100/3.5) to
provide 770 mg of compound 5: .sup.1H NMR (CDCl.sub.3) .delta.
7.8(2H, d, J=8.9 Hz), 7.4 (2H, m), 7.3-6.8 (8H, m), 5.75 (1H, m),
5.3-5.1(2H, m), 4.6-4.23 (4H, m), 3.98 (3H, s), 3.7-2.6 (15H, m),
2.2-1.8 (12H, m), 1.72 (3H, s), 1.58(3H, m), 1.25 (3H, m), 0.95
(6H, m).
Example 6
[1693] Compound 6: To a solution of compound 3 (1.00 g, 1.29 mmol)
in dry dimethylformamide (6 mL) was added 3-Hydroxybenzaldehyde
(0.320 g, 2.60 mmol), Benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate (1.35 g, 2.60 mmol), followed by addition of
Diisopropylethylamine (901 .mu.L, 5.16 mmol). The mixture was
stirred for 14 hours, the resulting residue was diluted in EtOAc,
washed with brine (3.times.) and dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The residue was
purified by silica gel chromatography
(CH.sub.2Cl.sub.2/Isopropanol=100/5) to provide 880 mg of compound
6.
Example 7
[1694] Compound 7: To a solution of compound 3 (150 mg, 0.190 mmol)
in dry dimethylformamide (1 mL) was added 2-Ethoxy-phenol (48.0
.mu.L, 0.380 mmol), Benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate (198 mg, 0.380 mmol), followed by addition of
Diisopropylethylamine (132 .mu.L, 0.760 mmol). The mixture was
stirred for 14 hours, the resulting residue was diluted in EtOAc,
washed with brine (3.times.) and dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The residue was
purified by silica gel chromatography
(CH.sub.2Cl.sub.2/Isopropanol=100/4) to provide 84.7 mg of compound
7: .sup.1H NMR (CDCl.sub.3) .delta. 7.73 (2H, d, J=8.9 Hz), 7.15
(2H, m), 7.01-6.9 (8H, m), 5.66 (1H, m), 5.22-5.04 (2H, m),
4.56-4.2 (6H, m), 4.08 (2H, m), 3.89 (3H, m), 3.85-3.69 (6H, m),
3.17-2.98 (7H, m), 2.80(3H, m) 1.86 (1H, m), 1.65(2H, m), 1.62-1.22
(6H, m), 0.92(6H, m).
Example 8
[1695] Compound 8: To a solution of compound 3 (50.0 mg, 0.0650
mmol) in dry dimethylformamide (1 mL) was added 2-(1-methylbutyl)
phenol (21.2 mg, 0.130 mmol),
Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
(67.1 mg, 0.130 mmol), followed by addition of
Diisopropylethylamine (45.0 .mu.L, 0.260 mmol). The mixture was
stirred for 14 hours, the resulting residue was diluted in EtOAc,
washed with brine (3.times.) and dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The residue was
purified by reversed phase HPLC to provide 8.20 mg of compound 8:
.sup.1H NMR (CDCl.sub.3) .delta. 7.73 (2H, d, J=8.9 Hz), 7.25 (2H,
m), 7.21-6.89 (8H, m), 5.7(1H, m), 5.29-4.9 (2H, m), 4.56-4.2 (6H,
m), 3.89 (3H, m), 3.85-3.69 (6H, m), 3.17-2.89 (8H, m), 2.85(3H,
m), 2.3-1.65(4H, m), 1.55-1.35 (6H, m), 0.92(6H, m).
Example 9
[1696] Compound 9: To a solution of compound 3 (50.0 mg, 0.0650
mmol) in dry dimethylformamide (1 mL) was added) 4-N-Butylphenol
(19.4 mg, 0.130 mmol),
Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
(67.1 mg, 0.130 mmol), followed by addition (45.0 .mu.L, 0.260
mmol) of Diisopropylethylamine. The mixture was stirred for 14
hours, the resulting residue was diluted in EtOAc, washed with
brine (3.times.) and dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified by
reversed phase HPLC to provide 9.61 mg of compound 9: .sup.1H NMR
(CDCl.sub.3) .delta. 7.8(2H, d, J=8.9 Hz), 7.4 (2H, m), 7.3-6.8
(8H, m), 5.75 (1H, m), 5.3-4.5 (4H, m), 4.3-3.4.1 (4H, m), 3.9 (3H,
m), 3.3-2.59 (1H, m), 2.25 (2H, m), 1.85-1.5 (5H, m), 1.4-1.1(10H,
m), 0.95(9H, m).
Example 10
[1697] Compound 10: To a solution of compound 3 (50.0 mg, 0.0650
mmol) in dry dimethylformamide (1 mL) was added 4-Octylphenol (26.6
mg, 0.130 mmol), Benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate (67.1 mg, 0.130 mmol), followed by addition of
Diisopropylethylamine (45.0 .mu.L, 0.260 mmol). The mixture was
stirred for 14 hours, the resulting residue was diluted in EtOAc,
washed with brine (3.times.) and dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The residue was
purified by reversed phase HPLC to provide 7.70 mg of compound 10:
.sup.1H NMR (CDCl.sub.3) .delta. 7.75 (2H, d, J=8.9 Hz), 7.3 (2H,
m), 7.2-6.8 (8H, m), 5.70 (1H, m), 5.3-4.9 (4H, m), 4.6-3.9 (4H,
m), 3.89 (3H, m), 3.85-2.59 (12H, m), 2.18-1.75 (10H, m), 1.69-1.50
(8H, m), 1.4-1.27(6H, m), 0.95(9H, m).
Example 11
[1698] Compound 11: To a solution of compound 3 (100 mg, 0.120
mmol) in dry dimethylformamide (1 mL) was added Isopropanol (20.0
.mu.L, 0.240 mmol), Benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate (135 mg, 0.240 mmol), followed by addition of
Diisopropylethylamine (83.0 .mu.L, 0.480 mmol). The mixture was
stirred for 14 hours, the resulting residue was diluted in EtOAc,
washed with brine (3.times.) and dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The residue was
purified by silica gel chromatography
(CH.sub.2Cl.sub.2/Isopropanol=100/4) to provide 12.2 mg of compound
11: .sup.1H NMR (CDCl.sub.3) .delta. 7.71 (2H, d, J=8.9 Hz), 7.15
(2H, m), 7.0 (2H, m), 6.89 (2H, m), 5.65 (1H, m), 5.03-4.86(4H, m),
4.34-4.19 (3H, m), 3.89 (3H, s), 3.88 (1H, m), 3.82 (2H, m), 3.65
(4H, m), 3.2-2.9 (1H, m), 2.80(3H, m) 1.65(2H, m), 1.86 (1H, m),
1.6(3H, m), 1.30(3H, m), 0.92(6H, m).
Example 12
[1699] Compound 12: To a solution of compound 3 (100 mg, 0.120
mmol) in dry dimethylformamide (1 mL) was added
4-Hydroxy-1-methylpiperidine (30.0 mg, 0.240 mmol),
Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
(135 mg, 0.240 mmol), followed by addition of Diisopropylethylamine
(83.0 .mu.L, 0.480 mmol). The mixture was stirred for 14 hours, the
resulting residue was diluted in EtOAc, washed with brine
(3.times.) and dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified by
reversed phase HPLC to provide 50.1 mg of compound 12: .sup.1H NMR
(CDCl.sub.3) .delta. 7.73 (2H, d, J=8.9 Hz), 7.18 (2H, m), 7.0 (2H,
m), 6.9 (2H, m), 5.67 (1H, m), 5.2-4.9 (4H, m), 4.30-4.11 (4H, m),
3.98 (1H, m), 3.89 (3H, s), 3.87 (1H, m), 3.75 (2H, m), 3.5-3.3
(4H, m), 3.2-2.9 (14H, m), 2.80(3H, m) 1.65(2H, m), 1.86 (1H, m),
1.6(3H, m), 1.30(3H, m), 0.92(6H, m). ##STR537## ##STR538##
##STR539##
Example 13
[1700] Compound 13: To a solution of compound 4 (4.9 g)) in EtOAc
(150 ml) was added 20% Pd/C (0.90 g), the reaction mixture was
hydrogenated for 1 hour. Celite was added and the mixture was
stirred for 10 minutes. The mixture was filtered through a pad of
celite and washed with ethanol. Concentration gave 4.1 g of
compound 13: .sup.1H NMR (CDCl.sub.3) .delta. 7.91 (2H, d, J=8.9
Hz), 7.75 (2H, m), 7.73-7.3 (8H, m), 7.25 (2H, m), 7.21-6.7(6H, m),
5.4-4.8(6H, m), 4.78-4.21 (4H, m), 3.98 (3H,s), 2.1-1.75 (8H, m),
1.55 (3H, m), 1.28(3H, m), 0.99(6H, m).
Example 14
[1701] Compound 14: To a solution of compound 5 (0.770 g, 0.790
mmol) in dichloromethane (10 mL), under ice-cooling, was added
trifluoroacetic acid (5 mL), the resulting mixture was stirred at
25.degree. C. for two hours. The reaction mixture was concentrated
under reduced pressure and the residue was co-evaporated with EtOAc
to provide an yellow oil. To a solution of the above oil in (10 mL)
of EtOAc, under ice-cooling and stirring was added formaldehyde
(210 .mu.L, 2.86 mmol), acetic acid (252 .mu.L, 4.30 mmol),
followed by sodium cyanoborohydride (178 mg, 2.86 mmol). The
mixture was further stirred at 25.degree. C. for 2 hours. The above
mixture was concentrated and diluted with EtOAc and washed with
H.sub.2O (3.times.), brine, dried over sodium sulfate, filtered,
and concentrated under reduced pressure. The residue was purified
using reversed-phase HPLC to provide 420 mg of compound 14: .sup.1H
NMR (CDCl.sub.3) .delta. 7.8(2H, d, J=8.9 Hz), 7.4 (2H, m), 7.3-6.8
(8H, m), 5.75 (1H, m), 5.3-5.1(2H, m), 4.6-4.23 (4H, m), 3.98 (3H,
s), 3.7-2.6 (15H, m), 2.2-1.8 (8H, m), 1.72 (3H, s), 1.58(3H, m),
1.25 (3H, m), 0.95 (6H, m).
Example 15
[1702] Compound 15: To a solution of compound 6 (100 mg, 0.114
mmol) in EtOAc (1 mL) was added 1-Methyl-piperazine (63.2 mg, 0.570
mmol), acetic acid (34.0 .mu.l, 0.570 mmol) followed by Sodium
Cyanoborohydride (14.3 mg, 0.228 mmol). The mixture was stirred at
25.degree. C. for 14 hours. The reaction mixture was concentrated
and diluted with EtOAc and washed with H.sub.2O (5.times.), brine
(2.times.), dried over sodium sulfate, filtered, and concentrated
under reduced pressure. The residue was purified using silica gel
chromatography (CH.sub.2Cl.sub.2/Isopropanol=100/6.5) to give 5.22
mg of compound 15: .sup.1H NMR (CDCl.sub.3) .delta. 7.73 (2H, d,
J=8.9 Hz), 7.4-7.18(8H, m), 7.1-6.89 (2H, m), 5.67 (1H, m), 5.2-4.9
(4H, m), 4.30-4.11 (4H, m), 3.98 (1H, m), 3.89 (3H, s), 3.87 (1H,
m), 3.75 (2H, m), 3.5-3.3 (4H, m), 3.2-2.9 (10H, m), 2.80-2.25 (8H,
m) 1.65(2H, m), 1.86 (1H, m), 1.6(3H, m), 1.30(3H, m), 0.92(6H, m).
##STR540## ##STR541##
Example 16
[1703] Compound 16: To a solution of compound 3 (100 mg, 0.120
mmol) in Pyridine (600 .mu.L) was added Piperidin-1-ol (48.5 mg,
0.480 mmol), followed by N,N-Dicyclohexylcarbodiimide (99.0 mg,
0.480 mmol). The mixture was stirred for 6 hours, the solvent was
concentrated under reduced pressure. The resulting residue was
purified by silica gel chromatography
(CH.sub.2Cl.sub.2/Methanol=100/5) to provide 17 mg of compound 16:
.sup.1H NMR (CDCl.sub.3) .delta. 7.73 (2H, d, J=8.9 Hz), 7.16 (2H,
m), 7.0 (2H, m), 6.9 (2H, m), 5.68 (1H, m), 5.17 (1H, m), 5.04 (1H,
m), 4.5-4.2 (4H, m), 3.90 (3H, s), 3.75 (2H, m), 3.5-3.3 (4H, m),
3.2-2.9 (10H, m), 2.80(3H, m) 1.65(2H, m), 1.86 (1H, m), 1.6(3H,
m), 1.5-1.27 (9H, m), 0.92(6H, m).
Example 17
[1704] Compound 18: To a solution of compound 17 (148 mg, 0.240
mmol) in 4 ml, of Methanol was added
(1,2,3,4-Tetrahydro-isoquinolin-6-ylmethyl)-phosphonic acid diethyl
ester (70.0 mg, 0.240 mmol), acetic acid (43.0 .mu.L, 0.720 mmol).
The reaction mixture was stirred for 3 minutes, followed by
addition of Sodium Cyanoborohydride (75.3 mg, 1.20 mmol). The
reaction mixture was stirred at 25.degree. C. for 14 hours. The
reaction mixture was diluted with EtOAc and washed with H.sub.2O
(3.times., brine, dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified using
silica gel chromatography (CH.sub.2Cl.sub.2/Isopropanol=100/5) to
give 59 mg of TES protected intermediate. 83 .mu.L of 48% HF
solution was added to acetonitrile (4 mL) to prepare the 2% HF
solution. The above 2% HF solution was added to TES protected
intermediate (47 mg, 0.053 mmol) and the reaction mixture was
stirred for 2 hours. The solvent was concentrated and the residue
was diluted with EtOAc, dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified using
silica gel chromatography (CH.sub.2Cl.sub.2/Methanol=100/10) to
give 35.2 mg of compound 18: .sup.1H NMR (CDCl.sub.3) .delta. 7.73
(2H, d, J=8.9 Hz), 7.05 (2H, m), 6.89 (2H, m), 6.76 (1H, m), 5.75
(1H, m), 5.67 (1H, m), 5.3 (2H, m), 4.2-3.6 (12H, m), 3.4-2.4 (11H,
m), 2.1-1.8 (6H, m), 1.4-1.28 (8H, m), 0.92(6H, m). ##STR542##
[1705] Compound 19 is prepared following the procedure for compound
2 by using monoacid 1.
[1706] Compound 20 is made following a hydrogenation of compound
19. Mono acid 20 reacts with corresponding amino esters in the
presence of Aldrithiol-2 and triphenylphosphine to form compound
21. ##STR543##
[1707] Monoacid 22 is treated with thionyl chloride at 60.degree.
C. to form monochloridate, which reacts with corresponding alkyl
(s)lactate to generate monolactate 23. Monolactate 23 is
hydrogenated with 10% Pd--C in the presence of acetic acid to form
amine 24. Aldehyde 25 reacts with amine 24 in the presence of
MgSO.sub.4 to form the intermediate imine, which is reduced with
sodium cyanoborohydride to afford compound 26. ##STR544##
##STR545##
Example 1
[1708] Compound 2: A 3 L, 3-neck flask was equipped with a
mechanical stirrer and addition funnel and charged with
2-aminoethyl phosphonic acid (60.0 g, 480 mmol). 2N Sodium
hydroxide (480 mL, 960 mmol) was added and flask cooled to
0.degree. C. Benzyl chloroformate (102.4 g, 600 mmol) in toluene
(160 mL) was added dropwise with vigorous stirring. The reaction
mixture was stirred at 0.degree. C. for 30 minutes, then at room
temperature for 4 h. 2N sodium hydroxide (240 mL, 480 mmol) was
added, followed by benzyl chloroformate (20.5 g, 120 mmol) and the
reaction mixture was vigorously stirred for 12 h. The reaction
mixture was washed with diethyl ether (3.times.). The aqueous layer
was acidified to pH 2 with concentrated HCl to give a white
precipitate. Ethyl acetate was added to the mixture and
concentrated HCl (80 mL, 960 mmol) was added. The aqueous layer was
extracted with ethyl acetate and combined organic layer was dried
(MgSO.sub.4) and concentrated to give a waxy, white solid (124 g,
479 mmol, 100%). .sup.1H NMR (300 MHz, CD.sub.3OD): .delta.
7.45-7.30 (m, 5H, Ar), 5.06 (d, J=14.7 Hz, 2H, CH.sub.2Ph),
3.44-3.31 (m, 2H, NCH.sub.2CH.sub.2), 2.03-1.91 (m, 2H,
CH.sub.2CH.sub.2P); .sup.31P NMR (121 MHz, CD.sub.3OD): .delta.
26.3.
Example 2
[1709] Compound 3: To a mixture of compound 2 (50.0 g, 193 mmol) in
toluene (1.0 L) was added DMF (1.0 mL) followed by thionyl chloride
(56 mL, 768 mmol). The reaction mixture was heated at 65.degree. C.
for 3-4 h under a stream of argon. The reaction mixture was cooled
to room temperature and concentrated. Residual solvent was removed
under high vacuum for 1 h. The residue was dissolved in
CH.sub.2Cl.sub.2 (1.0 L) and cooled to 0.degree. C. Triethylamine
(161 mL, 1158 mmol) was added, followed by phenol (54.5 g, 579
mmol). The reaction mixture was warmed to room temperature
overnight, then washed with 1.0N HCl, saturated NaHCO.sub.3
solution, brine and dried (MgSO.sub.4). Concentrated and purified
(silica gel, 1:1 EtOAc/Hex) to give a pale yellow solid (56 g, 136
mmol, 71%). .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.40-7.10
(m, 15H, Ar), 5.53 (br s, 1H, NH), 5.11 (br s, 2H, CH.sub.2Ph),
3.72-3.60 (m, 2H, NCH.sub.2CH.sub.2), 2.49-2.30 (m, 2H,
CH.sub.2CH.sub.2P); .sup.31P NMR (121 MHz, CDCl.sub.3): .delta.
22.9.
Example 3
[1710] Compound 4: To a solution of compound 3 (64 g, 155.6 mmol)
in acetonitrile (500 mL) at 0.degree. C. was added 2.0M sodium
hydroxide. The reaction mixture was stirred at 0.degree. C. for 30
min, then at room temperature for 2.5 h. The reaction mixture was
concentrated to 100 ml, and diluted with H.sub.2O (500 mL). The
aqueous solution was washed with EtOAc (3.times.300 mL). The
aqueous layer was acidified to pH 1 with concentrated HCl,
producing a white precipitated. The mixture was extracted with
EtOAc (4.times.300 mL) and combined organic layer was washed with
brine and dried (MgSO.sub.4). Concentration gave a solid, which was
recrystallized from hot EtOAc (450 mL) to give a white solid (41.04
g, 122 mmol, 79%). .sup.1H NMR (300 MHz, CD.sub.3OD): .delta.
7.45-7.10 (m, 10H, Ar), 5.09 (s, 2H, CH.sub.2Ph), 3.53-3.30 (m, 2H,
NCH.sub.2CH.sub.2), 2.25-2.10 (m, 2H, CH.sub.2CH.sub.2P); .sup.31P
NMR (121 MHz, CD.sub.3OD): .delta. 24.5.
Example 4
[1711] Compound 5: To a mixture of compound 4 (28 g, 83 mmol) in
toluene (500 mL) was added DMF (1.0 mL), followed by thionyl
chloride (36.4 mL, 499 mmol). The mixture was heated at 65.degree.
C. for 2 h providing a pale yellow solution. The reaction mixture
was concentrated and dried for 45 min under high vacuum. The
residue was dissolved in anhydrous CH.sub.2Cl.sub.2 (350 mL) and
cooled to 0.degree. C. Triethylamine (45.3 mL, 332 mmol) was added
slowly, followed by the dropwise addition of ethyl lactate (18.8
mL, 166 mmol). The reaction mixture was stirred at 0.degree. C. for
30 min, then warmed to room temperature overnight. The reaction
mixture was diluted with CH.sub.2Cl.sub.2 and washed with 1 N HCl,
saturated NaHCO.sub.3 solution, brine and dried (MgSO.sub.4).
Concentration and purification (silica gel, 1:5 to 1:0 EtOAc/Hex)
gave a pale yellow oil (30.7 g, 71 mmol, 85%) as a mixture of
diastereomers which were separated by HPLC (Dynamax reverse phase
C-18 column, 60% acetonitrile/H.sub.2O). More polar diastereomer:
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.40-7.10 (m, 10H, Ar),
5.65 (s, 1H, NB), 5.12 (s, 2H, CH.sub.2Ph), 5.10-5.00 (m,1H, OCHC)
4.17 (q, J=6.9 Hz, 2H, OCH.sub.2CH.sub.3), 3.62 (dt, J=20.4 Hz,
J.sub.2=6.0 Hz, 2H, NCH.sub.2CH.sub.2), 2.25 (dt, J=18.0 Hz,
J.sub.2=6.0 Hz, 2H, CH.sub.2CH.sub.2P), 1.60 (dd,
J.sub.1=J.sub.2=6.9 Hz, 3H, CHCH.sub.3), 1.23 (t, J=6.9 Hz, 3H,
OCH.sub.2CH.sub.3); .sup.31P NMR (121 MHz, CDCl.sub.3): .delta.
26.2. Less polar diastereomer: .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 7.40-7.10 (m, 10H, Ar), 5.87 (s, 1H, NB), 5.13 (s, 2H,
CH.sub.2Ph), 5.10-5.00 (dq, J.sub.1=J.sub.2=6.9 Hz, 1H, OCHC) 4.22
(q, J=7.2 Hz, 2H, OCH.sub.2CH.sub.3), 3.68 (dt, J=21.6 Hz,
J.sub.2=6.9 Hz, 2H, NCH.sub.2CH.sub.2), 2.40-2.20 (m, 2H,
CH.sub.2CH.sub.2P), 1.49 (dd, J=70.2 Hz, J.sub.2=6.9 Hz, 3H,
CHCH.sub.3), 1.28 (t, J=6.9 Hz, 3H, OCH.sub.2CH.sub.3); .sup.31P
NMR (121 MHz, CDCl.sub.3): .delta. 28.3.
Example 5
[1712] Compound 6: 2-Hydroxy-butyric acid ethyl ester was prepared
as follows: To a solution of L-2-aminobutyric acid (100 g, 970
mmol) in 1.0 N H.sub.2SO.sub.4 (2 L) at 0.degree. C. was added
NaNO.sub.2 (111 g, 1610 mmol) in H.sub.2O (400 mL) over 2 h. The
reaction mixture was stirred at room temperature for 18 h. Reaction
mixture was extracted with EtOAc (4.times.) and combined organic
layer was dried (MgSO.sub.4) and concentrated to give a yellow
solid (41.5 g). This solid was dissolved in absolute ethanol (500
mL) and concentrated HCl (3.27 mL, 39.9 mmol) was added. Reaction
mixture was heated to 80.degree. C. After 24 h, concentrated HCl (3
mL) was added and reaction continued for 24 h. Reaction mixture was
concentrated and product was distilled to give a colorless oil (31
g, 235 mmol, 59%).
[1713] To a mixture of compound 4 (0.22 g, 0.63 mmol) in anhydrous
acetonitrile (3.0 mL) was added thionyl chloride (0.184 mL, 2.52
mmol). The mixture was heated at 65.degree. C. for 1.5 h providing
a pale yellow solution. The reaction mixture was concentrated and
dried for 45 min under high vacuum. The residue was dissolved in
anhydrous CH.sub.2Cl.sub.2 (3.3 mL) and cooled to 0.degree. C.
Triethylamine (0.26 mL, 1.89 mmol) was added slowly, followed by
the dropwise addition of 2-hydroxy-butyric acid ethyl ester (0.167
mL, 1.26 mmol). The reaction mixture was stirred at 0.degree. C.
for 5 min, then warmed to room temperature overnight. The reaction
mixture was concentrated, dissolved in EtOAc and washed with 1.0 N
HCl, saturated NaHCO.sub.3 solution, brine and dried (MgSO.sub.4).
Concentration and purification (silica gel, 3:2 EtOAc/Hex) gave a
pale yellow oil (0.21 g, 0.47 mmol, 75%). For major diastereomer,
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.35-7.10 (m, 10H, Ar),
5.91 (s,1H, NB)), 5.12 (s, 2H, CH.sub.2Ph), 4.94-4.83 (m, 1H,
OCHC), 4.27-4.12 (m, 2H, OCH.sub.2CH.sub.3), 3.80-3.50 (m, 2H,
NCH.sub.2CH.sub.2), 2.39-2.19 (m, 2H, CH.sub.2CH.sub.2P), 1.82-1.71
(m, 2H, CHCH.sub.2CH.sub.3), 1.30-1.195 (m, 3H, OCH.sub.2CH.sub.3),
0.81 (t, J=7.5 Hz, 3H, CHCH.sub.2CH.sub.3); .sup.31P NMR (120 MHz,
CDCl.sub.3): 628.3. For minor diastereomer, .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 7.35-7.10 (m, 10H, Ar), 5.74 (s, 1H, NH)),
5.11 (s, 2H, CH.sub.2Ph), 4.98-4.94 (m,1H, OCHC), 4.27-4.12 (m, 2H,
OCH.sub.2CH.sub.3), 3.80-3.50 (m, 2H, NCH.sub.2CH.sub.2), 2.39-2.19
(m, 2H, CH.sub.2CH.sub.2P), 1.98-1.82 (m, 2H, CHCH.sub.2CH.sub.3),
1.30-1.195 (m, 3H, OCH.sub.2CH.sub.3), 1.00 (t, J=7.5 Hz, 3H,
CHCH.sub.2CH.sub.3); .sup.31P NMR (121 MHz, CDCl.sub.3): .delta.
26.2.
Example 6
[1714] Compound 7: A mixture of compound 6, (0.53 g, 1.18 mmol)
acetic acid (0.135 mL, 2.36 mmol) and 10% palladium on activated
carbon (0.08 g) in absolute ethanol (12 mL) was stirred under a
hydrogen atmosphere (1 atm) for 3 h. Reaction mixture was filtered
through Celite, concentrated, and resubjected to identical reaction
conditions. After 2 h, Celite was added to the reaction mixture and
mixture was stirred for 2 min, then filtered through a pad of
Celite and concentrated. Dried under high vacuum to give the
diasteromeric acetate salt as a oil (0.42 g, 1.11 mmol, 94%).
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.40-7.10 (m, 5H, Ar),
5.00-4.80 (m, 1H, OCHC), 4.28-4.10 (m, 2H, OCH.sub.2CH.sub.2),
3.32-3.14 (m, 2H, NCH.sub.2CH.sub.2), 2.45-2.22 (m, 2H,
CH.sub.2CH.sub.2P), 1.97 (s, 3H, Ac), 1.97-1.70 (m, 2H,
CHCH.sub.2CH.sub.3), 1.30-1.18 (m, 3H, OCH.sub.2CH.sub.3), 1.00 (t,
J=7.5 Hz, 1H, CHCH.sub.2CH.sub.3), 0.80 (t, J=7.5 Hz, 2H,
CHCH.sub.2CH.sub.3); .sup.31P NMR (121 MHz, CDCl.sub.3): .delta.
27.6 (major, 1.85), 26.0 (minor, 1.01).
Example 7
[1715] Compound 9: A solution of aldehyde 8 (0.596 g, 1.01 mmol)
and compound 7 (0.42 g, 1.11 mmol) were stirred together in
1,2-dichloroethane (4.0 mL) in the presence of MgSO.sub.4 for 3 h.
Acetic acid (0.231 mL, 4.04 mmol) and sodium cyanoborohydride
(0.127 g, 2.02 mmol) were added and reaction mixture was stirred
for 50 min at room temperature. Reaction mixture was quenched with
saturated NaHCO.sub.3 solution, diluted with EtOAc, and vigorously
stirred for 5 min. Brine was added and extracted with EtOAc
(2.times.). Combined organic layer was dried (MgSO.sub.4)
concentrated and purified (silica gel, EtOAc, then 10% EtOH/EtOAc)
to give a colorless foam. Acetonitrile (4 mL) and trifluoroacetic
acid (0.06 mL) were added and concentrated to a volume of 1 mL.
H.sub.2O (10 mL) was added and lyophilized to give the TFA salt as
a white powder (0.51 g, 0.508 mmol, 50%). .sup.1H NMR (300 MHz,
CD.sub.3CN): .delta. 7.79 (d, J=8.4 Hz, 2H, (SO.sub.2C(CH).sub.2),
7.43-7.20 (m, 9H, Ar), 7.10 (d, J=8.4 Hz, 2H,
(CH).sub.2COCH.sub.3), 5.85 (d, J=8.4 Hz, 1H, NB), 5.55 (d, J=4.5
Hz, 1H, OCHO), 5.00-4.75 (m, 2H, CH.sub.2CHOC(O), POCHC), 4.39-4.05
(m, 2H, PhCH.sub.2N, OCH.sub.2CH.sub.3), 3.89 (s, 3H, OCH.sub.3),
3.88-3.30 (m, 9H), 3.15-2.84 (m, 5H), 2.65-2.42 (m, 3H), 2.10-1.68
(m, 5H), 1.65-1.15 (m, 5H), 1.05-0.79 (m, 9H); .sup.31P NMR (121
MHz, CD.sub.3CN): .delta. 24.8 (major, 1.85), 23.1 (minor,
1.01).
Example 8
[1716] Compound 10: Compound 9 (0.041 g, 0.041 mmol) was dissolved
in DMSO (1.9 mL) and to this solution was added phosphate buffered
saline, pH 7.4 (10 mL) and pig liver esterase (Sigma, 0.2 mL).
Reaction mixture was stirred for 24 h at 40.degree. C. After 24 h,
additional esterase (0.2 mL) was added and reaction was continued
for 24 h. Reaction mixture was concentrated in methanol and
filtered. Filtrate was concentrated and purified by reverse phase
chromatography to give a white powder after lyophilization (8 mg,
0.010 mmol, 25%). .sup.1H NMR (500 MHz, CD.sub.3OD): .delta. 7.78
(d, J=8.9 Hz, 2H, (SO.sub.2C(CH).sub.2), 7.43-7.35 (m, 4H, Ar),
7.11 (d, J=8.9 Hz, 2H, (CH).sub.2COCH.sub.3), 5.62 (d, J=5.2 Hz,
1H, OCHO), 4.96-4.77 (m, 2H, CH.sub.2CHOC(O), POCHC), 4.21 (br s,
2H, PhCH.sub.2N), 3.97-3.70 (m, 6H), 3.90 (s, 3H, OCH.sub.3),
3.50-3.30 (m, 3H), 3.26-3.02 (m, 2H), 2.94-2.58 (m, 4H), 2.09-1.78
(m, 5H), 1.63-1.52 (m, 2H), 1.05-0.97 (m, 3H); 0.94 (d, J=6.7 Hz,
3H), 0.88 (d, J=6.7 Hz, 3H), .sup.31P NMR (121 MHz, CD.sub.3OD):
.delta. 20.8. ##STR546## ##STR547##
Example 9
[1717] Compound 12: To a solution of compound 11 (4.10 g, 9.66
mmol) and anhydrous ethylene glycol (5.39 mL, 96.6 mmol) in
anhydrous DMF (30 mL) at 0.degree. C. was added powdered magnesium
tert-butoxide (2.05 g, 12.02 mmol). The reaction mixture was
stirred at 0.degree. C. for 1.5 h, then concentrated. The residue
was partitioned between EtOAc and H.sub.2O and washed with 1 N HCl,
saturated NaHCO.sub.3 solution, and brine. Organic layer dried
(MgSO.sub.4), concentrated and purified (silica gel, 4%
MeOH/CH.sub.2Cl.sub.2) to give a colorless oil (1.55 g, 48%).
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.37 (s, 10H, Ar),
5.40-5.05 (m, 4H, CH.sub.2Ph), 3.84 (d, J=8.1 Hz, 2H, PCH.sub.2O),
3.70-3.60 (m, 4H, OCH.sub.2CH.sub.2O, OCH.sub.2CH.sub.2O); .sup.31P
NMR (121 MHz, CDCl.sub.3): .delta. 22.7.
Example 10
[1718] Compound 14: To a solution of compound 12 (0.75 g, 2.23
mmol) and 2,6-lutidine (0.78 mL, 6.69 mmol) in CH.sub.2Cl.sub.2 (20
mL) at -78.degree. C. was added trifluoromethanesulfonic anhydride
(0.45 mL, 2.68 mmol). The reaction mixture was stirred at
-78.degree. C. for 40 min, then diluted with CH.sub.2Cl.sub.2 and
washed with 1 N HCl, saturated NaHCO.sub.3 and dried (MgSO.sub.4).
Concentration gave a yellow oil that was dissolved in anhydrous
acetonitrile (20 mL). Phenol 13 (1.00 g, 1.73 mmol) was added to
the solution, which was cooled to 0.degree. C. Cesium carbonate
(0.619 g, 1.90 mmol) was added and reaction mixture was stirred at
0.degree. C. for 2 h, then at room temperature for 1.5 h Additional
cesium carbonate (0.200 g, 0.61 mmol) was added and reaction was
continued for 1.5 h, then filtered. Concentration of the filtrate
and purification (silica gel, 3% MeOH/CH.sub.2Cl.sub.2) gave a
yellow gum (1.005 g, 65%). .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 7.71 (d, J=8.7 Hz, 2H, SO.sub.2C(CH).sub.2), 7.34 (s, 10H,
PhCH.sub.2O), 7.11 (d, J=8.1 Hz, 2H,
CH.sub.2C(CH).sub.2(CH).sub.2), 6.98 (d, J=8.7 Hz, 2H,
(CH).sub.2COCH.sub.3), 6.78 (d, J=8.7 Hz, 2H,
(CH).sub.2COCH.sub.2), 5.62 (d, J=5.4 Hz, 1H, OCHO), 5.16-4.97 (m,
6H), 4.05-3.65 (m, 12H), 3.86 (s, 3H, OCH.sub.3), 3.19-2.66 (m,
7H), 1.95-1.46 (m, 3H), 0.92 (d, J=6.6 Hz, 3H, CH(CH.sub.3).sub.2),
0.88 (d, J=6.6 Hz, 3H, CH(CH.sub.3).sub.2); .sup.31P NMR (121 MHz,
CDCl.sub.3): 821.9.
Example 11
[1719] Compound 15: A mixture of compound 14 (0.410 g, 0.457 mmol)
and 10% palladium on carbon (0.066 g) in ethanol (5.0 mL) was
stirred under a hydrogen atmosphere (1 atm) for 16 h. Celite was
added and the mixture was stirred for 5 min, then filtered through
Celite and concentrated to give a foam (0.350 g, 107%). .sup.1H NMR
(300 MHz, CD.sub.3OD): .delta. 7.76 (d, J=8.7 Hz, 2H,
SO.sub.2C(CH).sub.2), 7.15 (d, J=8.4 Hz, 2H,
CH.sub.2C(CH).sub.2(CH).sub.2), 7.08 (d, J=8.4 Hz, 2H,
(CH).sub.2COCH.sub.3), 6.82 (d, J=8.4 Hz, 2H,
(CH).sub.2COCH.sub.2), 5.59 (d, J=5.4 Hz, 1H, OCHO), 5.16-4.97
(masked by CD.sub.3OH, 1H), 4.09-4.02 (m, 2H), 3.99-3.82 (m, 10H),
3.88 (s, 3H, OCH.sub.3), 3.52-3.32 (m, 1H), 3.21-2.75 (m, 5H),
2.55-2.40 (m, 1H), 2.10-1.95 (m, 1H), 1.75-1.25 (m, 2H), 0.93 (d,
J=6.3 Hz, 3H, CH(CH.sub.3).sub.2), 0.88 (d, J=6.6 Hz, 3H,
CH(CH.sub.3).sub.2); .sup.31P NMR (121 MHz, CD.sub.3OD): .delta.
19.5.
Example 12
[1720] Compound 16: Compound 15 (0.350 g, 0.488 mmol) was
coevaporated with anhydrous pyridine (3.times.10 mL), each time
filling with N.sub.2. Residue was dissolved in anhydrous pyridine
(2.5 mL) and phenol (0.459 g, 4.88 mmol) was added. This solution
was heated to 70.degree. C., then 1,3-dicyclohexylcarbodiimide
(0.403 g, 1.93 mmol) was added and reaction mixture was heated at
70.degree. C. for 7 h. Reaction mixture was concentrated,
coevaporated with toluene and residue obtained was diluted with
EtOAc, precipitating 1,3-dicyclohexylurea. The mixture was filtered
and filtrate concentrated and residue obtained was purified (silica
gel, 2% MeOH/CH.sub.2Cl.sub.2, then another column 75% EtOAc/Hex)
to give a clear oil (0.1324 g, 31%). .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 7.71 (d, J=8.7 Hz, 2H, SO.sub.2C(CH).sub.2),
7.41-7.18 (m, 10H, Ar), 7.14 (d, J=8.4 Hz, 2H,
CH.sub.2C(CH).sub.2(CH).sub.2), 6.99 (d, J=9.0 Hz, 2H,
(CH).sub.2COCH.sub.3), 6.83 (d, J=8.4 Hz, 2H,
(CH).sub.2COCH.sub.2), 5.64 (d, J=5.1 Hz, 1H, OCHO), 5.16-4.92 (m,
2H), 4.32-3.62 (m, 12H), 3.87 (s, 3H, OCH.sub.3), 3.22-2.73 (m,
7H), 1.95-1.75 (m, 3H), 0.93 (d, J=6.6 Hz, 3H, CH(CH.sub.3).sub.2),
0.88 (d, J=6.6 Hz, 3H, CH(CH.sub.3).sub.2); .sup.31P NMR (121 MHz,
CDCl.sub.3): .delta. 14.3.
Example 13
[1721] Compound 17: To a solution of compound 16 (0.132 g, 0.152
mmol) in acetonitrile (1.5 mL) at 0.degree. C. was added 1.0 M NaOH
(0.38 mL, 0.381 mmol). Reaction mixture was stirred for 2 h at
0.degree. C., then Dowex 50 (H+) resin was added until pH=1. The
resin was removed by filtration and the filtrate was concentrated
and washed with EtOAc/Hex (1:2, 25 mL), then dried under high
vacuum to give a clear film (0.103 g, 85%). This film was
coevaporated with anhydrous pyridine (3.times.5 mL), filling with
N.sub.2. The residue was dissolved in anhydrous pyridine (1 mL) and
ethyl lactate (0.15 mL, 1.30 mmol) was added and reaction mixture
was heated at 70.degree. C. After 5 min,
1,3-dicyclohexylcarbodiimide (0.107 g, 0.520 mmol) was added and
reaction mixture was stirred at 70.degree. C. for 2.5 h. Additional
1,3-dicyclohexylcarbodiimide (0.055 g, 0.270 mmol) was added and
reaction continued for another 1.5 h. Reaction mixture was
concentrated and coevaporated with toluene and diluted with EtOAc,
precipitating 1,3-dicyclohexylurea. The mixture was filtered and
filtrate concentrated and residue obtained was purified (silica
gel, 80 to 100% EtOAc/Hex) to give a white foam (0.0607 g, 52%).
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.71 (d, J=8.7 Hz, 2H,
SO.sub.2C(CH).sub.2), 7.39-7.16 (m, 5H, Ar), 7.13 (d, J=8.1 Hz, 2H,
CH.sub.2C(CH).sub.2(CH).sub.2), 6.99 (d, J=9.0 Hz, 2H,
(CH).sub.2COCH.sub.3), 6.82 (d, J=8.4 Hz, 2H,
(CH).sub.2COCH.sub.2), 5.64 (d, J=5.1 Hz, 1H, OCHO), 5.16-4.92 (m,
3H), 4.35-3.65 (m, 14H), 3.87 (s, 3H, OCH.sub.3), 3.22-2.73 (m,
7H), 1.95-1.80 (m, 3H), 1.59 (d, J=6.9 Hz, 1.5H, CCHCH.sub.3), 1.47
(d, J=7.2 Hz, 1.5H, CCHCH.sub.3), 1.37-1.18 (m, 3H), 0.92 (d, J=6.6
Hz, 3H, CH(CH.sub.3).sub.2), 0.88 (d, J=6.6 Hz, 3H,
CH(CH.sub.3).sub.2); .sup.31P NMR (121 MHz, CDCl.sub.3): .delta.
19.2, 17.2.
Example 14
[1722] Compound 18: Compound 17 (11.5 mg, 0.013 mmol) was dissolved
in DMSO (0.14 mL) and acetonitrile (0.29 mL). PBS (pH 7.4, 1.43 mL)
was added slowly with stirring. Porcine liver esterase (Sigma, 0.1
mL) was added and reaction mixture was gently stirred at 38.degree.
C. After 24 h, additional porcine liver esterase (0.1 mL) and DMSO
(0.14 mL) were added and reaction mixture stirred for 48 h at
38.degree. C. Reaction mixture concentrated and methanol was added
to precipitate the enzyme. The mixture was filtered, concentrated
and purified by reverse phase chromatography to give a white powder
after lyophilization (7.1 mg, 69%). .sup.1H NMR (300 MHz,
CD.sub.3OD): .delta. 7.76 (d, J=8.7 Hz, 2H, SO.sub.2C(CH).sub.2),
7.15 (d, J=8.4 Hz, 2H, CH.sub.2C(CH).sub.2(CH).sub.2), 7.08 (d,
J=9.0 Hz, 2H, (CH).sub.2COCH.sub.3), 6.83 (d, J=8.7 Hz, 2H,
(CH).sub.2COCH.sub.2), 5.59 (d, J=5.1 Hz, 1H, OCHO), 5.16-4.90
(masked by CD.sub.3OH, 2H), 4.19-3.65 (m, 12H), 3.88 (s, 3H,
OCH.sub.3), 3.50-3.27 (m, 1H), 3.20-2.78 (m, 5H), 2.55-2.40 (m,
1H), 2.05-1.90 (m, 1H), 1.75-1.30 (m, 2H), 1.53 (d, J=6.6 Hz, 3H,
CCHCH.sub.3), 0.93 (d, J=6.6 Hz, 3H, CH(CH.sub.3).sub.2), 0.88 (d,
J=6.6 Hz, 3H, CH(CH.sub.3).sub.2); .sup.31P NMR (121 MHz,
CD.sub.3OD): .delta. 16.7.
[1723] Alternatively, compound 17 was prepared as described below
(Scheme 3). ##STR548##
Example 15
[1724] Compound 19: To a solution of compound 14 (0.945 g, 1.05
mmol) in anhydrous toluene (10.0 mL) was added
1,4-diazobicyclo[2.2.2]octane (0.130 g, 1.16 mmol) and reaction
mixture was refluxed for 2 h. After cooling to room temperature,
reaction mixture was diluted with EtOAc and washed with 1.0 N HCl
and dried (MgSO.sub.4). Concentration gave a white foam (0.785 g,
93%). Residue was dissolved in anhydrous DMF (10.0 mL) and to this
solution was added ethyl (S)-lactate (0.23 mL, 2.00 mmol) and
diisopropylethylamine (0.70 mL, 4.00 mmol), followed by
benzotriazol-1-yloxytripyrroldinophosphonium hexafluorophosphate
(1.041 g, 2.00 mmol). Reaction mixture was stirred for 20 h, then
concentrated and residue was dissolved in EtOAc and washed with 1.0
N HCl, saturated NaHCO.sub.3, brine and dried (MgSO.sub.4).
Concentration and purification (silica gel, 2%
MeOH/CH.sub.2Cl.sub.2) gave an off-white foam (0.520 g, 59%).
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.72 (d, J=7.5 Hz, 2H,
SO.sub.2C(CH).sub.2), 7.50-7.27 (m, 4H, Ar), 7.12 (d, J=8.1 Hz, 2H,
CH.sub.2C(CH).sub.2(CH).sub.2), 7.00 (d, J=6.6 Hz, 2H,
(CH).sub.2COCH.sub.3), 6.81 (d, J=8.4 Hz, 2H,
(CH).sub.2COCH.sub.2), 5.64 (d, J=5.1 Hz, 1H, OCHO), 5.37-4.90 (m,
5H), 4.35-3.65 (m, 14H), 3.88 (s, 3H, OCH.sub.3), 3.24-2.70 (m,
7H), 1.90-1.70 (m, 3H), 1.54 (d, J=6.9 Hz, 1.5H, CCHCH.sub.3), 1.47
(d, J=6.9 Hz, 1.5H, CCHCH.sub.3), 1.37-1.22 (m, 3H), 0.93 (d, J=6.3
Hz, 3H, CH(CH.sub.3).sub.2), 0.89 (d, J=6.0 Hz, 3H,
CH(CH.sub.3).sub.2); .sup.31P NMR (121 MHz, CDCl.sub.3): .delta.
22.3, 21.2.
Example 16
[1725] Compound 17: A mixture of compound 19 (0.520 g, 0.573 mmol)
and 10% palladium on carbon (0.055 g) in ethanol (10 mL) was
stirred under a hydrogen atmosphere (1 atm) for 2 h. Celite was
added to the reaction mixture and stirred for 5 min, then mixture
was filtered through Celite and concentrated to give a white foam
(0.4649 g, 99%). Residue was dissolved in anhydrous DMF (5.0 mL)
and to this solution was added phenol (0.097 g, 1.03 mmol),
diisopropylethylamine (0.36 mL, 2.06 mmol) followed by
benzotriazol-1-yloxytripyrroldinophosphonium hexafluorophosphate
(0.536 g, 1.03 mmol). Reaction mixture was stirred for 20 h, then
concentrated and residue was dissolved in EtOAc and washed with 1 N
HCl, H.sub.2O, sat. NaHCO.sub.3, brine and dried (MgSO.sub.4).
Concentration and purification (silica gel, 2%
MeOH/CH.sub.2Cl.sub.2) gave a white foam (0.180 g, 35%).
##STR549##
Example 17
[1726] Compound 21: Compound 20 (11.5 g, 48.1 mmol) in 48% HBr (150
mL) was heated at 120.degree. C. for 4 h, then cooled to room
temperature and diluted with EtOAc. Mixture was neutralized with
saturated NaHCO.sub.3 solution and solid NaHCO.sub.3 and extracted
with EtOAc containing MeOH. Organic layer dried (MgSO.sub.4),
concentrated, and purified (silica gel, 1:2 EtOAc/Hex with 1% MeOH)
to give a brown solid (7.0 g, 65%). The resulting compound (7.0 g,
31.1 mmol) and 10% palladium hydroxide (2.1 g) in EtOH (310 mL) was
stirred under a hydrogen atmosphere for 1 d, then filtered through
Celite and concentrated to give an off-white solid (4.42 g, 100%).
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.01 (d, J=7.8 Hz, 1H,
Ar), 6.64 (s, 1H, Ar), 6.61 (d, J=8.1 Hz, 2H, Ar), 4.07 (s, 2H,
ArCH.sub.2N), 4.05 (s, 2H, ArCH.sub.2N).
Example 18
[1727] Compound 22: To a solution of compound 21 (4.42 g, 32.7
mmol) in 1.0 M NaOH (98 mL, 98.25 mmol) at 0.degree. C. was added
dropwise benzyl chloroformate (7.00 mL, 49.13 mmol) in toluene (7
mL). After addition was complete, reaction mixture was stirred
overnight at room temperature. Reaction mixture was diluted with
EtOAc and extracted with EtOAc (3.times.). Combined organic layer
was dried (MgSO.sub.4), concentrated and purified (silica gel, 2%
MeOH/CH.sub.2Cl.sub.2) to give a white solid (3.786 g, 43%). The
resulting compound (0.6546 g, 2.43 mmol) was dissolved in anhydrous
acetonitrile (10 mL), and compound 23 (0.782 g, 2.92 mmol) was
added, followed by cesium carbonate (1.583 g, 4.86 mmol). Reaction
mixture was stirred for 2 h at room temperature, then filtered,
concentrated, and purified (3% MeOH/CH.sub.2Cl.sub.2) to give a
brownish oil (1.01 g, 99%).
Example 19
[1728] Compound 25: To a solution of compound 22 (0.100 g, 0.238
mmol) in EtOAc/EtOH (2 mL, 1:1) was added acetic acid (14 .mu.L,
0.238 mmol) and 10% palladium on carbon (0.020 g) and the mixture
was stirred under a hydrogen atmosphere for 2 h. Celite was added
to the reaction mixture and stirred for 5 min, then filtered
through Celite. Concentration and drying under high vacuum gave a
reddish film (0.0777 g, 95%). The resulting amine (0.0777 g, 0.225
mmol) and aldehyde 24 (0.126 g, 0.205 mmol) in 1,2-dichloroethane
(1.2 mL) were stirred for 5 min at 0.degree. C., then sodium
triacetoxyborohydride (0.0608 g, 0.287 mmol) was added. Reaction
mixture was stirred for 1 h at 0.degree. C., then quenched with
saturated NaHCO.sub.3 solution and brine. Extracted with EtOAc, the
organic layer was dried (MgSO.sub.4), concentrated and purified
(silica gel, 2% MeOH/CH.sub.2Cl.sub.2) to give a brown foam (38.7
mg, 21%). .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.74 (d, J=8.7
Hz, 2H, Ar), 7.09 (d, J=8.7 Hz, 1H, Ar), 7.05-6.72 (m, 4H, Ar),
5.71 (d, J=5.1 Hz, 1H), 5.22-5.07 (m, 2H), 4.22-4.17 (m, 7H),
4.16-3.69 (m, 9H), 3.82 (s, 3H), 3.25-2.51 (m, 7H), 2.22-1.70 (m,
3H), 1.37 (t, J=6.9 Hz, 6H), 1.10-0.58 (m, 21H); .sup.31P NMR (121
MHz, CDCl.sub.3): .delta. 19.5.
Example 20
[1729] Compound 26: To a solution of compound 25 (38.7 mg, 0.0438
mmol) in acetonitrile (0.5 mL) at 0.degree. C. was added 48% HF
(0.02 mL). The reaction mixture was stirred at room temperature for
2 h, then quenched with saturated NaHCO.sub.3 solution and
extracted with EtOAc. Organic layer was separated, dried
(MgSO.sub.4), concentrated and purified (silica gel, 3 to 5%
MeOH/CH.sub.2Cl.sub.2) to give a red film (21.2 mg, 62%). .sup.1H
NMR (300 MHz, CDCl.sub.3): .delta. 7.73 (d, J=8.7 Hz, 2H, Ar), 7.10
(d, J=8.7 Hz, 1H, Ar), 6.97 (d, J=8.70 Hz, 2H), 6.90-6.76 (m, 2H),
5.72 (d, J=5.1 Hz, 1H), 5.41 (d, J=9.0 Hz, 1H), 5.15 (q, J=6.6 Hz,
1H), 4.38-4.17 (m, 7H), 4.16-3.65 (m, 9H), 3.87 (s, 3H), 3.20-2.82
(m, 7H), 2.75-1.79 (m, 3H), 1.37 (t, J=6.9 Hz, 6H), 0.90 (d, J=6.6
Hz, 3H), 0.88 (d, J=6.6 Hz, 3H); .sup.31P NMR (121 MHz,
CDCl.sub.3): .delta. 19.3. ##STR550## ##STR551##
Example 21
[1730] Compound 28: To a mixture of 4-bromobenzylamine
hydrochloride (15.23 g, 68.4 mmol) in H.sub.2O (300 mL) was added
sodium hydroxide (8.21 g, 205.2 mmol), followed by di-tert-butyl
dicarbonate (16.45 g, 75.3 mmol). Reaction mixture was vigorously
stirred for 18 h, then diluted with EtOAc (500 mL). Organic layer
separated and aqueous layer extracted with EtOAc (200 mL). Combined
organic layer was dried (MgSO.sub.4), concentrated and dried under
high vacuum to give a white solid (18.7 g, 96%). .sup.1H NMR (300
MHz, CDCl.sub.3): .delta. 7.41 (d, J=8.4 Hz, 2H), 7.12 (d, J=8.3
Hz, 2H), 4.82 (s, 1H, NH), 4.22 (d, J=6.1 Hz, 2H), 1.41 (s,
9H).
Example 22
[1731] Compound 29: Compound 28 (5.00 g, 17.47 mmol) was
coevaporated with toluene. Diethyl phosphite (11.3 mL, 87.36 mmol)
was added and mixture was coevaporated with toluent (2.times.).
Triethylamine (24.0 mL, 174.7 mmol) was added and mixture was
purged with argon for 10 min, then tetrakis(triphenylphosphine)
palladium(0) (4.00 g, 3.49 mmol) was added. Reaction mixture was
refluxed for 18 h, cooled, concentrated and diluted with EtOAc.
Washed with 0.5 N HCl, 0.5 M NaOH, H.sub.2O, brine and dried
(MgSO.sub.4). Concentrated and purification (silica gel, 70%
EtOAc/Hex) gave an impure reaction product as a yellow oil (6.0 g).
This material (6.0 g) was dissolved in anhydrous acetonitrile (30
mL) and cooled to 0.degree. C. Bromotrimethylsilane (11.5 mL, 87.4
mmol) was added and reaction mixture was warmed to room temperature
over 15 h. Reaction mixture was concentrated, dissolved in MeOH (50
mL) and stirred for 1.5 h. H.sub.2O (1 mL) was added and mixture
stirred for 2 h. Concentrated to dryness and dried under high
vacuum, then triturated with Et.sub.2O containing 2% MeOH to give a
white solid (3.06 g, 65%). .sup.1H NMR (300 MHz, D.sub.2O): .delta.
7.67 (dd, J=12.9, 7.6 Hz, 2H), 7.45-7.35 (m, 2H), 4.10 (s, 2H);
.sup.31P NMR (121 MHz, D.sub.2O): .delta. 12.1.
Example 23
[1732] Compound 30: Compound 29 (4.78 g, 17.84 mmol) was dissolved
in H.sub.2O (95 mL) containing sodium hydroxide (3.57 g, 89.20
mmol). Di-tert-butyl dicarbonate (7.63 g, 34.94 mmol) was added,
followed by THF (25 mL). The clear reaction mixture was stirred
overnight at room temperature then concentrated to 100 mL. Washed
with EtOAc and acidified to pH 1 with 1 N HCl and extracted with
EtOAc (7.times.). Combined organic layer was dried (MgSO.sub.4),
concentrated and dried under high vacuum. Trituration with
Et.sub.2O gave a white powder (4.56 g, 89%). .sup.1H NMR (300 MHz,
CD.sub.3OD): .delta. 7.85-7.71 (m, 2H), 7.39-7.30 (m, 2H), 4.26 (s,
2H), 1.46 (s, 9H); .sup.31P NMR (121 MHz, CD.sub.3OD): .delta.
16.3.
Example 24
[1733] Compound 31: Compound 30 (2.96 g, 10.32 mmol) was
coevaporated with anhydrous pyridine (3.times.10 mL). To this
residue was added phenol (9.71 g, 103.2 mmol) and mixture was
coevaporated with anhydrous pyridine (2.times.10 mL). Pyridine (50
mL) was added and solution heated to 70.degree. C. After 5 min,
1,3-dicyclohexylcarbodiimide (8.51 g, 41.26 mmol) was added and
resulting mixture was stirred for 8 h at 70.degree. C. Reaction
mixture was cooled and concentrated and coevaporated with toluene.
Residue obtained was diluted with EtOAc and the resulting
precipitate was removed by filtration. The filtrate was
concentrated and purified (silica gel, 20 to 40% EtOAc/Hex, another
column 30 to 40% EtOAc/Hex) to give a white solid (3.20 g, 71%).
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.90 (dd, J=13.8, 8.2
Hz, 2H), 7.41-7.10 (m, 14H), 5.17 (br s, 1H, NH), 4.35 (d, J=5.2
Hz, 2H), 1.46 (s, 9H); .sup.31P NMR (121 MHz, CDCl.sub.3): .delta.
11.8.
Example 25
[1734] Compound 32: To a solution of compound 31 (3.73 g, 8.49
mmol) in acetonitrile (85 mL) at 0.degree. C. was added 1 M NaOH
(21.2 mL, 21.21 mmol). Reaction mixture was stirred at 0.degree. C.
for 30 min, then warmed to room temperature over 4 h. Reaction
mixture cooled to 0.degree. C. and Dowex (H+) residue was added to
pH 2. Mixture was filtered, concentrated and residue obtained was
triturated with EtOAc/Hex (1:2) to give a white powder (2.889 g,
94%). This compound (2.00 g, 5.50 mmol) was coevaporated with
anhydrous pyridine (3.times.10 mL). The residue was dissolved in
anhydrous pyridine (30 mL) and ethyl (S)-lactate (6.24 mL, 55 mmol)
and reaction mixture was heated to 70.degree. C. After 5 min,
1,3-dicyclocarbodiiimide (4.54 g, 22.0 mmol) was added. Reaction
mixture was stirred at 70.degree. C. for 5 h, then cooled and
concentrated. Residue was dissolved in EtOAc and precipitate was
removed by filtration. The filtrate was concentrated and purified
(25 to 35% EtOAc/Hex, another column 40% EtOAc/Hex) to give a
colorless oil (2.02 g, 80%). .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 7.96-7.85 (m, 2H), 7.42-7.35 (m, 2H), 7.35-7.08 (m, 4H),
5.16-5.00 (m, 1H), 4.93 (s, 1H, 1H), 4.37 (d, J=5.5 Hz, 1H), 4.21
(q, J=7.3 Hz, 1H), 4.11 (dq, J=5.7, 2.2 Hz, 1H), 1.62-1.47 (m, 3H),
1.47 (s, 9H), 1.27 (t, J=7.3 Hz, 1.5H), 1.17 (t, J=7.3 Hz, 1.5H);
.sup.31P NMR (121 MHz, CDCl.sub.3): .delta. 16.1, 15.0.
Example 26
[1735] Compound 33: Compound 32 (2.02 g, 4.36 mmol) was dissolved
in CH.sub.2Cl.sub.2 (41 mL) and cooled to 0.degree. C. To this
solution was added trifluoroacetic acid (3.5 mL) and reaction
mixture was stirred at 0.degree. C. for 1 h, then at room
temperature for 3 h. Reaction mixture was concentrated,
coevaporated with EtOAc and diluted with H.sub.2O (400 mL). Mixture
was neutralized with Amberlite IRA-67 weakly basic resin, then
filtered and concentrated. Coevaporation with MeOH and dried under
high vacuum to give the TFA amine salt as a semi-solid (1.48 g,
94%). To a solution of the amine (1.48 g, 4.07 mmol) in absolute
ethanol (20 mL) at 0.degree. C. was added aldehyde 24 (1.39 g, 2.26
mmol), followed by acetic acid (0.14 mL, 2.49 mmol). After stirring
for 5 min, sodium cyanoborohydride (0.284 g, 4.52 mmol) was added
and reaction mixture stirred for 30 min at 0.degree. C. Reaction
was quenched with saturated NaHCO.sub.3 solution and diluted with
EtOAc and H.sub.2O. Aqueous layer was extracted with EtOAc
(3.times.) and combined organic layer was dried (MgSO.sub.4),
concentrated and purified (silica gel, 2 to 4%
MeOH/CH.sub.2Cl.sub.2) to give white foam (0.727 g, 33%). .sup.1H
NMR (300 MHz, CDCl.sub.3): .delta. 7.98-7.86 (m, 2H), 7.71 (d,
J=8.6 Hz, 2H), 7.49 (br s, 2H), 7.38-7.05 (m, 5H), 6.98 (d, J=8.8
Hz, 2H), 5.72 (d, J=5.1 Hz, 1H), 5.28-5.00 (m, 2H), 4.30-3.72 (m,
12H), 3.42-3.58 (m, 1H), 3.20-2.68 (m, 7H), 2.25-1.42 (m, 6H), 1.26
(t, J=7.2 Hz, 1.5H), 1.17 (t, J=7.2 Hz, 1.5H), 1.08-0.50 (m, 21H);
.sup.31P NMR (121 MHz, CDCl.sub.3): .delta. 16.1, 15.1.
Example 27
[1736] Compound 34: To a solution of compound 33 (0.727 g, 0.756
mmol) in acetonitrile (7.6 mL) at 0.degree. C. was added 48%
hydrofluoric acid (0.152 mL) and reaction mixture was stirred for
40 min at 0.degree. C., then diluted with EtOAc and H.sub.2O.
Saturated NaHCO.sub.3 was added and aqueous layer was extracted
with EtOAc (2.times.). Combined organic layer was dried
(MgSO.sub.4), concentrated and purified (silica gel, 4 to 5%
MeOH/CH.sub.2Cl.sub.2) to give a colorless foam (0.5655 g, 88%).
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.95-7.82 (m, 2H), 7.67
(d, J=8.1 Hz, 2H), 7.41 (br s, 2H), 7.38-7.05 (m, 5H), 6.95 (d,
J=7.2 Hz, 2H), 5.76 (d, J=7.9 Hz, 1H), 5.67 (d, J=5.0 Hz, 1H),
5.32-4.98 (m, 2H), 4.25-3.75 (m, 13H), 3.25-2.70 (m, 7H), 2.15-1.76
(m, 3H), 1.53-1.41 (m, 3H), 1.25-1.08 (m, 3H), 0.87 (d, J=4.2 Hz,
6H); .sup.31P NMR (121 MHz, CDCl.sub.3): .delta. 16.1, 15.0.
Example 28
[1737] Compound 35: To a solution of compound 33 (0.560 g, 0.660
mmol) in absolute ethanol (13 mL) at 0.degree. C. was added 37%
formaldehyde (0.54 mL, 6.60 mmol), followed by acetic acid (0.378
mL, 6.60 mmol). The reaction mixture was stirred at 0.degree. C.
for 5 min, then sodium cyanoborohydride (0.415 g, 6.60 mmol) was
added. Reaction mixture was warmed to room temperature over 2 h,
then quenched with saturated NaHCO.sub.3 solution. EtOAc was added
and mixture was washed with brine. Aqueous layer was extracted with
EtOAc (2.times.) and combined organic layer was dried (MgSO.sub.4),
concentrated and purified (silica gel, 3% MeOH/CH.sub.2Cl.sub.2) to
give a white foam (0.384 g, 67%). .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 7.95-7.82 (m, 2H), 7.71 (d, J=8.4 Hz, 2H),
7.38 (br s, 2H), 7.34-7.10 (m, 5H), 6.98 (d, J=8.8 Hz, 2H), 5.72
(d, J=5.0 Hz, 1H), 5.50 (br s, 1H), 5.19-5.01 (m, 2H), 4.29-3.75
(m, 10H), 3.85 (s, 3H), 3.35-2.70 (m, 7H), 2.23 (s, 3H), 2.17-1.79
(m, 3H), 1.54 (d, J=6.9 Hz, 1.5H), 1.48 (d, J=6.8 Hz, 1.5H), 1.25
(t, J=7.2 Hz, 1.5H), 1.16 (t, J=7.2 Hz, 1.5H), 0.92 (d, J=6.6 Hz,
3H), 0.87 (d, J=6.6 Hz, 3H). .sup.31P NMR (121 MHz, CDCl.sub.3):
.delta. 16.0, 14.8.
Example 29
[1738] Compound 36: To a solution of compound 35 (44 mg, 0.045
mmol) in acetonitrile (1.0 mL) and DMSO (0.5 mL) was added
phosphate buffered saline (pH 7.4, 5.0 mL) to give a cloudy white
suspension. Porcine liver esterase (200 .mu.L) was added and
reaction mixture was stirred for 48 h at 38.degree. C. Additional
esterase (600 .mu.L) was added and reaction was continued for 4 d.
Reaction mixture was concentrated, diluted with MeOH and the
resulting precipitate removed by filtration. Filtrate was
concentrated and purified by reverse phase HPLC to give a white
powder after lyophilization (7.2 mg, 21%). .sup.1H NMR (300 MHz,
CD.sub.3OD): .delta. 7.95 (br s, 2H), 7.76 (d, J=8.4 Hz, 2H), 7.64
(br s, 2H), 7.13 (d, J=8.7 Hz, 2H), 5.68 (d, J=5.1 Hz, 1H), 5.14
(br s, 1H), 4.77 (br s, 1H), 4.35-3.59 (m, 8H), 3.89 (s, 3H),
3.45-2.62 (m, 10H), 2.36-1.86 (m, 3H), 1.44 (d, J=6.3 Hz, 3H), 0.92
(d, J=6.6 Hz, 3H), 0.84 (d, J=6.6 Hz, 3H); .sup.31P NMR (121 MHz,
CD.sub.3OD): .delta. 13.8. ##STR552## ##STR553## ##STR554##
##STR555## ##STR556##
Example 1
[1739] Monophospholactate 2: A solution of 1 (0.11 g, 0.15 mmol)
and .alpha.-hydroxyisovaleric acid ethyl-(S)-ester (71 mg, 0.49
mmol) in pyridine (2 mL) was heated to 70.degree. C. and
1,3-dicyclohexylcarbodiimide (0.10 g, 0.49 mmol) was added. The
reaction mixture was stirred at 70.degree. C. for 2 h and cooled to
room temperature. The solvent was removed under reduced pressure.
The residue was suspended in EtOAc and 1,3-dicyclohexyl urea was
filtered off. The product was partitioned between EtOAc and 0.2 N
HCl. The EtOAc layer was washed with 0.2 N HCl, H.sub.2O, saturated
NaCl, dried with Na.sub.2SO.sub.4, filtered, and concentrated. The
crude product was purified by column chromatography on silica gel
(3% 2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (35
mg, 28%, GS 192771, 1/1 diastereomeric mixture) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, J=8.7 Hz, 2H), 7.36-7.14
(m, 7H), 6.99 (d, J=8.7 Hz, 2H), 6.94-6.84 (dd, 2H), 5.65 (d, J=5.4
Hz, 1H), 5.00-4.85 (m, 3H), 4.55 (dd, 1H), 4.41 (dd, 1H), 4.22-4.07
(m, 2H), 3.96-3.68 (m, 9H), 3.12-2.74 (m, 7H), 2.29 (m, 1H),
1.85-1.57 (m, 3H), 1.24 (m, 3H), 1.05 (d, J=6.6 Hz, 3H), 0.98 (d,
J=6.6 Hz, 3H), 0.9 (m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 17.7,
15.1.
Example 2
[1740] Monophospholactate 3: A solution of 1 (0.11 g, 0.15 mmol)
and .alpha.-hydroxyisovaleric acid ethyl-(R)-ester (71 mg, 0.49
mmol) in pyridine (2 mL) was heated to 70.degree. C. and
1,3-dicyclohexylcarbodiimide (0.10 g, 0.49 mmol) was added. The
reaction mixture was stirred at 70.degree. C. for 2 h and cooled to
room temperature. The solvent was removed under reduced pressure.
The residue was suspended in EtOAc and 1,3-dicyclohexyl urea was
filtered off. The product was partitioned between EtOAc and 0.2 N
HCl. The EtOAc layer was washed with 0.2 N HCl, H.sub.2O, saturated
NaCl, dried with Na.sub.2SO.sub.4, filtered, and concentrated. The
crude product was purified by column chromatography on silica gel
(3% 2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (35
mg, 28%, GS 192772, 1/1 diastereomeric mixture) as a white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 7.71 (d, J=8.7 Hz, 2H), 7.35-7.13
(m, 7H), 6.98 (d, J=8.7 Hz, 2H), 6.93-6.83 (dd, 2H), 5.64 (d, J=5.4
Hz, 1H), 5.04-4.85 (m, 3H), 4.54 (dd, 1H), 4.39 (dd, 1H), 4.21-4.06
(m, 2H), 3.97-3.67 (m, 9H), 3.12-2.75 (m, 7H), 2.27 (m, 1H),
1.83-1.57 (m, 3H), 1.26 (m, 3H), 1.05 (d, J=6.6 Hz, 3H), 0.98 (d,
J=6.6 Hz, 3H), 0.9 (m, 6H); .sup.31P NMR (CDCl.sub.3) .delta. 17.7,
15.1.
Example 3
[1741] Monophospholactate 4: A solution of 1 (0.10 g, 0.13 mmol)
and methyl-2,2-dimethyl-3-hydroxypropionate (56 .mu.L, 0.44 mmol)
in pyridine (1 mL) was heated to 70.degree. C. and
1,3-dicyclohexylcarbodiimide (91 mg, 0.44 mmol) was added. The
reaction mixture was stirred at 70.degree. C. for 2 h and cooled to
room temperature. The solvent was removed under reduced pressure.
The residue was suspended in EtOAc and 1,3-dicyclohexyl urea was
filtered off. The product was partitioned between EtOAc and 0.2 N
HCl. The EtOAc layer was washed with 0.2 N HCl, H.sub.2O, saturated
NaCl, dried with Na.sub.2SO.sub.4, filtered, and concentrated. The
crude product was purified by column chromatography on silica gel
(3% 2-propanol/CH.sub.2Cl.sub.2) to give the monophospholactate (72
mg, 62%, GS 191484) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 7.71 (d, J=8.7 Hz, 2H), 7.34 (m, 2H), 7.25-7.14 (m, 5H),
7.00 (d, J=9.0 Hz, 2H), 6.87 (d, J=8.7 Hz, 2H), 5.65 (d, J=5.4 Hz,
1H), 5.05 (m, 2H), 4.38 (d, J=9.6 Hz, 2H), 4.32-4.20 (m, 2H), 4.00
(m, 2H), 3.87-3.63 (m, 12H), 3.12-2.78 (m, 7H), 1.85-1.67 (m, 3H),
1.20 (m, 6H), 0.91 (d, J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H);
.sup.31P NMR (CDCl.sub.3) .delta. 16.0.
Example 4
[1742] Lactate 5: To a suspension of lactic acid sodium salt (5 g,
44.6 mmol) in 2-propanol (60 mL) was added
4-(3-chloropropyl)morpholine hydrochloride (8.30 g, 44.6 mmol). The
reaction mixture was heated to reflux for 18 h and cooled to room
temperature. The solid was filtered and the filtrate was
recrystallized from EtOAc/hexane to give the lactate (1.2 g,
12%).
Example 5
[1743] Monophospholactate 6: A solution of 1 (0.10 g, 0.13 mmol)
and lactate 5 (0.10 g, 0.48 mmol) in pyridine (2 mL) was heated to
70.degree. C. and 1,3-dicyclohexylcarbodiimide (0.10 g, 0.49 mmol)
was added. The reaction mixture was stirred at 70.degree. C. for 2
h and cooled to room temperature. The solvent was removed under
reduced pressure. The residue was suspended in EtOAc and
1,3-dicyclohexyl urea was filtered off. The product was partitioned
between EtOAc and H.sub.2O. The EtOAc layer was washed with
saturated NaCl, dried with Na.sub.2SO.sub.4, filtered, and
concentrated. The crude product was purified by column
chromatography on silica gel (4% 2-propanol/CH.sub.2Cl.sub.2) to
give the monophospholactate (30 mg, 24%, GS 192781, 1/1
diastereomeric mixture) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 7.71 (d, J=8.7 Hz, 2H), 7.38-7.15 (m, 7H), 7.00 (d, J=8.7
Hz, 2H), 6.91 (m, 2H), 5.65 (d, J=3.3 Hz, 1H), 5.18-4.98 (m, 3H),
4.54 (dd, 1H), 4.42 (dd, 1H), 4.2 (m, 2H), 4.00-3.67 (m, 16H),
3.13-2.77 (m, 7H), 2.4 (m, 5H), 1.85-1.5 (m, 5H), 1.25 (m, 2H),
0.93 (d, J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H); .sup.31P NMR
(CDCl.sub.3) .delta. 17.4, 15.4.
Example 6
[1744] Sulfonamide 8: A solution of dibenzylphosphonate 7 (0.1 g,
0.13 mmol) in CH.sub.2Cl.sub.2 (0.5 mL) at 0.degree. C. was treated
with trifluoroacetic acid (0.25 mL). The solution was stirred for
30 min at 0.degree. C. and then warmed to room temperature for an
additional 30 min. The reaction mixture was diluted with toluene
and concentrated under reduced pressure. The residue was
co-evaporated with toluene (2.times.), chloroform (2.times.), and
dried under vacuum to give the ammonium triflate salt which was
dissolved in CH.sub.2Cl.sub.2 (1 mL) and cooled to 0.degree. C.
Triethylamine (72 .mu.L, 0.52 mmol) was added followed by the
treatment of 4-methylpiperazinylsulfonyl chloride (25 mg, 0.13
mmol). The solution was stirred for 1 h at 0.degree. C. and the
product was partitioned between CH.sub.2Cl.sub.2 and H.sub.2O. The
organic phase was washed with saturated NaCl, dried with
Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel (5% 2-propanol/CH.sub.2Cl.sub.2) to give the sulfonamide 8 (32
mg, 30%, GS 273835) as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 7.35 (m, 10H), 7.11 (d, J=8.7 Hz, 2H), 6.81 (d, J=8.7 Hz,
2H), 5.65 (d, J=5.4 Hz, 1H), 5.2-4.91 (m, 4H), 4.2 (d, J=10.2 Hz,
2H), 4.0-3.69 (m, 6H), 3.4-3.19 (m, 5H), 3.07-2.75 (m, 5H), 2.45
(m, 4H), 2.3 (s, 3H), 1.89-1.44 (m, 7H), 0.93 (m, 6H); .sup.31P NMR
(CDCl.sub.3) .delta. 20.3.
Example 7
[1745] Phosphonic Acid 9: To a solution of 8 (20 mg, 0.02 mmol) in
EtOAc (2 mL) and 2-propanol (0.2 mL) was added 10% Pd/C (5 mg). The
suspension was stirred under H.sub.2 atmosphere (balloon) at room
temperature overnight. The reaction mixture was filtered through a
plug of celite. The filtrate was concentrated and dried under
vacuum to give the phosphonic acid (10 mg, 64%) as a white
solid.
Example 8
[1746] Dibenzylphosphonate 11: A solution of 10 (85 mg, 0.15 mmol)
and 1H-tetrazole (14 mg, 0.20 mmol) in CH.sub.2Cl.sub.2 (2 mL) was
treated with Dibenzyldiisopropylphosphoramidite (60 .mu.L, 0.20
mmol) and stirred at room temperature overnight. The product was
partitioned between CH.sub.2Cl.sub.2 and H.sub.2O, dried with
Na.sub.2SO.sub.4, filtered and concentrated. The crude product was
purified by column chromatography to give the intermediate
dibenzylphosphite (85 mg, 0.11 mmol) which was dissolved in
CH.sub.3CN (2 mL) and treated with iodobenzenediacetate (51 mg,
0.16 mmol). The reaction mixture was stirred at room temperature
for 3 h and concentrated. The residue was partitioned between EtOAc
and NaHCO.sub.3. The organic layer was washed with H.sub.2O, dried
with Na.sub.2SO.sub.4, filtered, and concentrated. The crude
product was purified by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the dibenzylphosphonate (45
mg, 52%) as a white solid.
Example 9
[1747] Disodium Salt of Phosphonic Acid 12: To a solution of 11 (25
mg, 0.03 mmol) in EtOAc (2 mL) was added 10% Pd/C (10 mg). The
suspension was stirred under H.sub.2 atmosphere (balloon) at room
temperature for 4 h. The reaction mixture was filtered through a
plug of celite. The filtrate was concentrated and dried under
vacuum to give the phosphonic acid which was dissolved in H.sub.2O
(1 mL) and treated with NaHCO.sub.3 (2.53 mg, 0.06 mmol). The
reaction mixture was stirred at room temperature for 1 h and
lyophilized overnight to give the disodium salt of phosphonic acid
(19.77 mg, 95%, GS 273777) as a white solid: .sup.1H NMR
(CD.sub.3OD) .delta. 7.81 (d, J=9.0 Hz, 2H), 7.35 (d, J=8.1 Hz,
2H), 7.27-7.09 (m, 5H), 5.57 (d, J=5.1 Hz, 1H), 5.07 (m, 1H),
4.87-4.40 (m, 3H), 3.93-3.62 (m, 6H), 3.45-2.6 (m, 6H), 2.0 (m,
2H), 1.55 (m, 1H), 0.95-0.84 (m, 6H).
Example 10
[1748] Dibenzylphosphonate 14: A solution of 13 (0.80 g, 0.93 mmol)
and 1H-tetrazole (98 mg, 1.39 mmol) in CH.sub.2Cl.sub.2 (15 mL) was
treated with dibenzyldiisopropylphosphoramidite (0.43 mL, 1.39
mmol) and stirred at room temperature overnight. The product was
partitioned between CH.sub.2Cl.sub.2 and H.sub.2O, dried with
Na.sub.2SO.sub.4, filtered and concentrated. The crude product was
purified by column chromatography to give the intermediate
dibenzylphosphite (0.68 g, 67%). To a solution of the
dibenzylphosphite (0.39 g, 0.35 mmol) in CH.sub.3CN (5 mL) was
added iodobenzenediacetate (0.17 g, 0.53 mmol). The reaction
mixture was stirred at room temperature for 2 h and concentrated.
The residue was partitioned between EtOAc and NaHCO.sub.3. The
organic layer was washed with H.sub.2O, dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (3%
2-propanol/CH.sub.2Cl.sub.2) to give the dibenzylphosphonate (0.35
g, 88%) as a white solid.
Example 11
[1749] Disodium Salt of Phosphonic Acid 15: To a solution of 14
(0.39 g, 0.35 mmol) in EtOAc (30 mL) was added 10% Pd/C (0.10 g).
The suspension was stirred under H2 atmosphere (balloon) at room
temperature for 4 h. The reaction mixture was filtered through a
plug of celite. The filtrate was concentrated and dried under
vacuum to give the phosphonic acid, which was dissolved in H.sub.2O
(3 mL) and treated with NaHCO.sub.3 (58 mg, 0.70 mmol). The
reaction mixture was stirred at room temperature for 1 h and
lyophilized overnight to give the disodium salt of phosphonic acid
(0.31 g, 90%, GS 273811) as a white solid: .sup.1H NMR (CD.sub.3OD)
.delta. 7.81 (d, J=9.0 Hz, 2H), 7.43-7.2 (m, 7H), 7.13 (d, J=9.0
Hz, 2H), 6.9 (m, 2H), 5.55 (d, J=4.8 Hz, 1H), 5.07 (m, 2H), 4.87(m,
1H), 4.64-4.4 (m, 4H), 3.93-3.62 (m, 9H), 3.33-2.63 (m, 5H), 2.11
(m, 1H), 1.6-1.42 (m, 4H), 1.38-1.25 (m, 7H), 0.95 (d, J=6.3 Hz,
3H), 0.84 (d, J=6.3 Hz, 3H).
[1750] Examples for the Preparation of Cyclic Carbonyl-Like
Phosphonate Protease Inhibitors (CCPPI) TABLE-US-00012
Phosphonamidate Prodrugs ##STR557## Scheme 1-2 Scaffold Synthesis
Scheme 3-10 P2'-Benzyl ether phosphonates Scheme 11-13 P2'-Alkyl
ether phosphonates Scheme 14-17 P2'-Benzyl Amide phosphonates
Scheme 18-25 P1-Phosphonates Scheme 50 Reagents
[1751] ##STR558##
[1752] The conversion of 1 to 1.1 is described in J. Org Chem.
1996, 61, p 444-450 ##STR559## ##STR560##
2-Benzyloxycarbonylamino-3-(4-tert-butoxy-phenyl)-propionic acid
methyl ester (2.3)
[1753] H-D-Tyr-O-me hydrochloride 2.1 (25 g, 107.7 mmol) is
dissolved in methylene chloride (150 mL) and aqueous sodium
bicarbonate (22 g in 150 ml, water), and then cooled to 0.degree.
C. To this resulting solution benzyl chloroformate (20 g, 118 mmol)
is slowly added. After complete addition, the resulting solution is
warmed to room temperature, and is then stirred for 2 h. The
organic phase is separated, dried over Na.sub.2SO.sub.4, and
concentrated under reduced pressure, to give the crude carbamate
2.2 (35 g). The crude CBZ-Tyr-OMe product is dissolved in methylene
chloride (300 mL) containing concentrated H.sub.2SO.sub.4.
Isobutene is bubbled though the solution for 6 h. The reaction is
then cooled to 0.degree. C., and neutralized with saturated
NaHCO.sub.3 aqueous solution. The organic phase is separated,
dried, concentrated under reduced pressure, and purified by silica
gel column chromatography to afford the tert-butyl ether 2.3 (25.7
g, 62%).
[2-(4-tert-Butoxy-phenyl)-1-formyl-ethyl]-carbamic acid benzyl
ester (2.4) (Reference J. O. C. 1997, 62, 3884)
[1754] To a stirred -78.degree. C. methylene chloride solution (60
mL) of 2.3, DIBAL (82 ml, of 1.5 M in toluene, 123 mmol) was added
over 15 min. The resultant solution was stirred at -78.degree. C.
for 30 min. Subsequently, a solution of EtOH/36% HCl (9/1; 15 mL)
is added slowly. The solution is added to a vigorously stirred
aqueous HCl solution (600 mL, 1N) at 0.degree. C. The layers are
then separated, and the aqueous phase is extracted with cold
methylene chloride. The combined organic phases are washed with
cold 1N HCl aqueous solution, water, dried over Na.sub.2SO.sub.4,
and then concentrated under reduced pressure to give the crude
aldehyde 2.4 (20 g, 91%).
[4-Benzyloycarbonylamino-1-(4-tert-butoxy-benzyl)-5-(4-tert-butoxy-phenyl)-
-2,3-dihydroxy-pentyl]-carbamic acid benzyl ester (2.5)
[1755] To a slurry of VCl.sub.3(THF).sub.3 in methylene chloride
(150 mL) at room temperature is added Zinc powder (2.9 g, 44 mmol),
and the resulting solution is then stirred at room temperature for
1 hour. A solution of aldehyde 2.4 (20 g, 56 mmol) in methylene
chloride (100 mL) is then added over 10 min. The resulting solution
is then stirred at room temperature overnight, poured into an
ice-cold H.sub.2SO.sub.4 aqueous solution (8 ml, in 200 mL), and
stirred at 0.degree. C. for 30 min. The methylene chloride solution
is separated, washed with 1N HCl until the washing solution is
light blue. The organic solution is then concentrated under reduced
pressure (solids are formed during concentration), and diluted with
hexane. The precipitate is collected and washed thoroughly with a
hexane/methylene chloride mixture to give the diol product 2.5. The
filtrate is concentrated under reduced pressure and subjected to
silica gel chromatography to afford a further 1.5 g of 2.5.
(Total=13 g, 65%).
[1-{5-[1-Benzyloxycarbonylamino-2-(4-tert-butoxy-phenyl)-ethyl]-2,2-dimeth-
yl-[1,3]dioxolan-4-yl}-2-(4-tert-butoxy-phenyl)-ethyl]-carbamic
acid benzyl ester (2.6)
[1756] Diol 2.5 (5 g, 7 mmol) is dissolved in acetone (120 mL),
2,2-dimethoxypropane (20 mL), and pyridinium p-toluenesulfonate
(120 mg, 0.5 mmol). The resulting solution is refluxed for 30 min.,
and then concentrated under reduced pressure to almost dryness. The
resulting mixture is partitioned between methylene chloride and
saturated NaHCO.sub.3 aqueous solution, dried, concentrated under
reduced pressure, and purified by silica gel column chromatography
to afford isopropylidene protected diol 2.6 (4.8 g, 92%).
4,8-Bis-(4-tert-butoxy-benzyl)-2,2-dimethyl-hexahydro-1,3-dioxa-5,7-diaza--
azulen-6-one (2.8)
[1757] The diol 2.6 is dissolved in EtOAc/EtOH (10 mL/2 mL) in the
presence of 10% Pd/C and hydrogenated at atmospheric pressure to
afford the diamino compound 2.7. To a solution of crude 2.7 in
1,1,2,2-tetrachloroethane is added 1,1-carboxydiimidazole (1.05 g,
6.5 mmol) at room temperature. The mixture is stirred for 10 min,
and the resulting solution is then added dropwise to a refluxing
1,1',2,2'-tetrachloroethane solution (150 mL). After 30 min., the
reaction mixture is cooled to room temperature, and washed with 5%
citric acid aqueous solution, dried over Na.sub.2SO.sub.4,
concentrated under reduced pressure, and purified by silica gel
column chromatography to afford the cyclourea derivative 2.8 (1.92
g, 60% over 2 steps).
5,6-Dihydroxy-4,7-bis-(4-hydroxy-benzyl)-[1,3]diazepan-2-one
(2.9)
[1758] Cyclic Urea 2.8 (0.4 g, 0.78 mmol) was dissolved in
dichloromethane (3 mL) and treated with TFA (1 mL). The mixture was
stirred at room temperature for 2 h upon which time a white solid
precipitated. 2 drops of water and methanol (2 mL) were added and
the homogeneous solution was stirred for 1 h and concentrated under
reduced pressure. The crude solid, 2.9, was dried overnight and
then used without further purification.
4,8-Bis-(4-hydroxy-benzyl)-2,2-dimethyl-hexahydro-1,3-dioxa-5,7-diaza-azul-
en-6-one (2.10)
[1759] Diol 2.9 (1.8 g, 5.03 mmol) was dissolved in DMF (6 mL) and
2,2-dimethoxypropane (12 mL). P-TsOH (95 mg) was added and the
mixture stirred at 65.degree. C. for 3 h. A vacuum was applied to
remove water and then the mixture was stirred at 65.degree. C. for
a further 1 h. The excess dimethoxypropane was then distilled and
the remaining DMF solution was then allowed to cool. The solution
of acetonide 2.10 can then used without further purification in
future reactions. ##STR561## ##STR562##
[1760] 3-Cyano-4-fluorobenzyl urea 3.1: A solution of urea 1.1 (1.6
g, 4.3 mmol) in THF was treated with sodium hydride (0.5 g of 60%
oil dispersion, 13 mmol). The mixture was stirred at room
temperature for 30 min and then treated with 3-cyano-4-fluorobenzyl
bromide 3.9 (1.0 g, 4.8 mmol). The resultant solution was stirred
at room temperature for 3 h, concentrated under reduced pressure,
and then partitioned between CH.sub.2Cl.sub.2 and saturated brine
solution containing 1% citric acid. The organic phase was
separated, dried over sodium sulfate, filtered and concentrated
under reduced pressure. The residue was purified by silica gel
eluting with 15-25% ethyl acetate in hexanes to yield urea 3.1 (1.5
g, 69%) as a white form.
[1761] Benzyl ether 3.2: A solution of 3.1 (0.56 g, 1.1 mmol) in
DMF (5 mL) was treated with sodium hydride (90 mg of 60% oil
dispersion, 2.2 mmol) and the resultant mixture stirred at room
temperature for 30 min. 4-Benzyloxy benzyl chloride 3.10 (0.31 g,
1.3 mmol) was added and the resultant solution stirred at room
temperature for 3 h. The mixture was concentrated under reduced
pressure and then partitioned between CH.sub.2Cl.sub.2 and
saturated brine solution. The organic phase was separated, dried
over sodium sulfate, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel eluting with 1-10%
ethyl acetate in hexanes to yield compound 3.2 (0.52 g, 67%) as
white form.
[1762] Indazole 3.3: Benzyl ether 3.2 (0.51 g, 0.73 mmol) was
dissolved in n-butanol (10 mL) and treated with hydrazine hydrate
(1 g, 20 mmol). The mixture was refluxed for 4 h and then allowed
to cool to room temperature. The mixture was concentrated under
reduced pressure and the residue was then partitioned between
CH.sub.2Cl.sub.2 and 10% citric acid solution. The organic phase
was separated, concentrated under reduced pressure, and then
purified by silica gel column eluting with 5% methanol in
CH.sub.2Cl.sub.2 to afford indazole 3.3 (0.42 g, 82%) as white
solid.
[1763] Boc-indazole 3.4: A solution of indazole 3.3 (0.4 g, 0.59
mmol) in CH.sub.2Cl.sub.2 (10 mL) was treated with
diisopropylethylamine (0.19 g, 1.5 mmol), DMAP (0.18 g, 1.4 mmol),
and di-tert-butyl dicarbonate (0.4 g, 2 mmol). The mixture was
stirred at room temperature for 3 h and then partitioned between
CH.sub.2Cl.sub.2 and 5% citric acid solution. The organic phase was
separated, dried over sodium sulfate, filtered and concentrated
under reduced pressure. The residue was purified by silica gel
eluting with 2% methanol in CH.sub.2Cl.sub.2 to afford 3.4 (0.42 g,
71%).
[1764] Phenol 3.5: A solution of 3.4 (300 mg, 0.3 mmol) in ethyl
acetate (10 mL) and methanol (10 mL) was treated with 10% Pd/C (40
mg) and stirred under a hydrogen atmosphere (balloon) for 16 h. The
catalyst was removed by filtration and the filtrate was
concentrated under reduced pressure to yield 3.5 as a white powder.
This was used without further purification.
[1765] Dibenzyl ester 3.6: A solution of 3.5 (0.1 mmol) in THF (5
mL) was treated with dibenzyl triflate 3.11 (90 mg, 0.2 mmol), and
cesium carbonate (0.19 g, 0.3 mmol). The mixture was stirred at
room temperature for 4 h and then concentrated under reduced
pressure. The residue was partitioned between CH.sub.2Cl.sub.2 and
saturated brine. The organic phase was separated, dried over sodium
sulfate, filtered and concentrated under reduced pressure. The
residue was purified by silica gel eluting with 20-40% ethyl
acetate in hexanes to afford 3.6 (70 mg, 59%). .sup.1H NMR
(CDCl.sub.3): .delta. 8.07 (d, 1H), 7.20-7.43 (m, 16H), 7.02-7.15
(m, 8H), 6.80 (d, 2H), 5.07-5.18 (m, 4H), 5.03 (d, 1H), 4.90 (d,
1H), 4.20 (d, 2H), 3.74-3.78 (m, 4H), 3.20 (d, 1H), 3.05 (d, 1H)
2.80-2.97 (m, 4H), 1.79 (s, 9H), 1.40 (s, 18H), 1.26 (s, 6H);
.sup.31P NMR (CDCl.sub.3): 20.5 ppm.
[1766] Phosphonic acid 3.7: A solution of dibenzylphosphonate 3.6
(30 mg) in EtOAc (10 mL) was treated with 10% Pd/C (10 mg) and the
mixture was stirred under a hydrogen atmosphere (balloon) for 3 h.
The catalyst was removed by filtration and the filtrate was
concentrated under reduced pressure to afford phosphonic acid 3.7.
This was used without further purification.
[1767] Phosphonic acid 3.8: The crude phosphonic acid 3.7 was
dissolved in CH.sub.2Cl.sub.2 (2 mL) and treated with
trifluoroacetic acid (0.4 mL). The resultant mixture was stirred at
room temperature for 4 h. The mixture was concentrated under
reduced pressure and then purified by preparative HPLC (35%
CH.sub.3CN/65% H.sub.2O) to afford the phosphonic acid 3.8 (9.4 mg,
55%). .sup.1H NMR (CD.sub.3OD): .delta. 7.71 (s, 1H), 7.60 (d, 1H),
6.95-7.40 (m, 15H), 4.65 (d, 2H), 4.17 (d, 2H), 3.50-3.70 (m, 3H),
3.42 (d, 1H), 2.03-3.14 (m, 6H); .sup.31P NMR (CDCl.sub.3): 17.30
##STR563##
[1768] Dibenzylphosphonate 4.1: A solution of 3.6 (30 mg, 25
.mu.mol) in CH.sub.2Cl.sub.2 (2 mL) was treated with TFA (0.4 mL)
and the resultant mixture was stirred at room temperature for 4 h.
The mixture was concentrated under reduced pressure and the residue
was purified by silica gel eluting with 50% ethyl acetate in
hexanes to afford 4.1 (5 mg, 24%). .sup.1H NMR (CDCl.sub.3):
.delta. 6.96-7.32 (m, 25H), 6.95 (d, 2H), 5.07-5.18 (m, 4H), 4.86
(d, 1H), 4.75 (d, 1H), 4.18 (d, 2H), 3.40-3.62 (m, 4H), 3.25 (d,
1H), 2.80-3.15 (m, 6H); .sup.31P NMR (CDCl.sub.3) 20.5 ppm; MS: 852
(M+H), 874 (M+Na). ##STR564##
[1769] Diethylphosphonate 5.1: A solution of phenol 3.5 (48 mg, 52
.mu.mol) in THF (5 mL) was treated with triflate 5.3 (50 mg, 165
.mu.mol), and cesium carbonate (22 mg, 0.2 mmol). The resultant
mixture was stirred at room temperature for 5 h and then
concentrated under reduced pressure. The residue was partitioned
between CH.sub.2Cl.sub.2 and saturated brine. The organic phase was
separated, dried over sodium sulfate, filtered and concentrated
under reduced pressure. The residue was purified by silica gel
eluting with 7% methanol in CH.sub.2Cl.sub.2 to afford 5.1 (28 mg,
50%). .sup.1H NMR (CDCl.sub.3): .delta. 8.06 (d, 1H), 7.30-7.43 (m,
7H), 7.02-7.30 (m, 7H), 6.88 (d, 2H), 5.03 (d, 1H), 4.90 (d, 1H),
4.10-4.25 (m, 6H), 3.64-3.80 (m, 4H), 3.20 (d, 1H), 3.05 (d, 1H)
2.80-2.97 (m, 4H), 1.79 (s, 9H), 1.20-1.50 (m, 30H); .sup.31P NMR
(CDCl.sub.3): 18.5 ppm; MS: 1068 (M+H), 1090 (M+Na).
[1770] Diethylphosphonate 5.2: A solution of 5.1 (28 mg, 26
.mu.mol) in CH.sub.2Cl.sub.2 (2 mL) was treated with TFA (0.4 mL)
and the resultant mixture was stirred at room temperature for 4
hrs. The mixture was concentrated under reduced pressure and the
residue was purified by silica gel to afford 5.2 (11 mg, 55%).
.sup.1H NMR (CDCl.sub.3+10% CD.sub.3OD): .delta. 6.96-7.35 (m,
15H), 6.82 (d, 2H), 4.86(d, 1H), 4.75 (d, 1H), 4.10-4.23 (M, 6H),
3.40-3.62 (m, 4H), 2.80-3.20 (m), 1.31 (t, 6H); .sup.31P NMR
(CDCl.sub.3+10% CD.sub.3OD): 19.80 ppm; MS: 728 (M+H). ##STR565##
##STR566##
[1771] 3-Benzyloxybenzyl urea 6.1: The urea 3.1 (0.87 g, 1.7 mmol)
was dissolved in DMF and treated with sodium hydride (60%
dispersion, 239 mg, 6.0 mmol) followed by m-benzyloxybenzylbromide
6.9 (0.60 g, 2.15 mmol). The mixture was stirred for 5 h and then
diluted with ethyl acetate. The solution was washed with water,
brine, dried over magnesium sulfate, filtered and concentrated
under reduced pressure. The residue was purified by silica gel
eluting with 25% ethyl acetate in hexanes to afford urea 6.1 (0.9
g, 75%).
[1772] Indazole 6.2: The urea 6.1 (41 mg, 59 .mu.mol) was dissolved
in n-butanol (1.5 mL) and treated with hydrazine hydrate (100
.mu.L, 100 mmol). The mixture was refluxed for 2 h and then allowed
to cool. The mixture was diluted with ethyl acetate, washed with
10% citric acid solution, brine, saturated NaHCO.sub.3, and finally
brine again. The organic phase was dried over sodium sulfate,
filtered and concentrated under reduced pressure to give the crude
product 6.2 (35 mg, 83%). (Chem. Biol. 1998, 5, 597-608).
[1773] Boc-indazole 6.3: The indazole 6.2 (1.04 g, 1.47 mmol) was
dissolved in CH.sub.2Cl.sub.2 (20 mL) and treated with di-t-butyl
dicarbonate (1.28 g, 5.9 mmol), DMAP (0.18 g, 1.9 mmol) and DIPEA
(1.02 ml, 9.9 mmol). The mixture was stirred for 3 h and then
diluted with ethyl acetate. The solution was washed with 5% citric
acid solution, NaHCO.sub.3, brine, dried over magnesium sulfate,
filtered and concentrated under reduced pressure. The residue was
purified by silica gel eluting with 50% ethyl acetate in hexanes to
give 6.3 (0.71 g, 49%).
[1774] Phenol 6.4: Compound 6.3 (20 mg, 0.021 mmol) was dissolved
in MeOH (1 mL) and EtOAc (1 mL) and treated with 10% Pd/C catalyst
(5 mg). The mixture was stirred under a hydrogen atmosphere
(balloon) until completion. The catalyst was removed by filtration
and the filtrate concentrated under reduced pressure to afford
compound 6.4 (19 mg, 100%).
[1775] Dibenzyl phosphonate 6.5: A solution of compound 6.4 (0.34
g, 0.37 mmol) in acetonitrile (5 mL) was treated with
Cs.sub.2CO.sub.3 (0.36 g, 1.1 mmol) and triflate 3.11 (0.18 mL,
0.52 mmol). The reaction mixture was stirred for 1 h. The reaction
mixture was filtered and the filtrate was then concentrated under
reduced pressure. The residue was re-dissolved in EtOAc, washed
with water, saturated NaHCO.sub.3, and finally brine, dried over
MgSO.sub.4, filtered and concentrated under reduced pressure. The
residue was purified by silica gel eluting with hexane:EtOAc (1:1)
to afford compound 6.5 (0.32 g, 73%).
[1776] Phosphonic acid 6.6: Compound 6.5 (208 mg, 0.174 mmol) was
treated in the same manner as benzyl phosphonate 3.6 in the
preparation of phosphonate diacid 3.7, except MeOH was used as the
solvent, to afford compound 6.6 (166 mg, 94%).
[1777] Phosphonic acid 6.7: Compound 6.6 (89 mg, 0.088 mmol) was
treated according to the conditions described in Scheme 3 for the
conversion of 3.7 into 3.8. The residue was purified by preparative
HPLC eluting with a gradient of 90% methanol in 100 mM TEA
bicarbonate buffer and 100% TEA bicarbonate buffer to afford
phosphonic acid 6.7 (16 mg, 27%)
[1778] Bisamidate 6.8: Triphenylphosphine (112 mg, 0.43 mmol) and
aldrithiol-2 (95 mg, 0.43 mmol) were mixed in dry pyridine (0.5
mL). In an adjacent flask the diacid 6.7 (48 mg, 0.71 mmol) was
suspended in dry pyridine (0.5 mL) and treated with DIPEA (0.075
ml, 0.43 mmol) and L-AlaButyl ester hydrochloride (78 mg, 0.43
mmol) and finally the triphenylphosphine, aldrithiol-2 mixture. The
reaction mixture was stirred under nitrogen for 24 h then
concentrated under reduced pressure. The residue was purified by
preparative HPLC eluting with a gradient of 5% to 95% acetonitrile
in water. The product obtained was then further purified by silica
gel eluting with CH.sub.2Cl.sub.2:MeOH (9:1) to give compound 6.8
(9 mg, 14%). ##STR567##
[1779] Diethyl phosphonate 7.1: Compound 6.4 (164 mg, 0.179 mmol)
was treated according to the procedure used to generate compound
6.5 except triflate 5.3 was used in place of triflate 3.11 to
afford compound 7.1 (142 mg, 74%).
[1780] Diethylphosphonate 7.2: Compound 7.1 (57 mg, 0.053 mmol) was
treated according to the conditions used to form 6.7 from 6.6. The
residue formed was purified by silica gel eluting with
CH.sub.2Cl.sub.2:MeOH (9:1) to afford compound 7.2 (13 mg, 33%).
##STR568##
[1781] Diphenylphosphonate 8.1: A solution of 6.6 (0.67 g, 0.66
mmol) in pyridine (10 mL) was treated with phenol (0.62 g, 6.6
mmol) and DCC (0.82 mg, 3.9 mmol). The resultant mixture was
stirred at room temperature for 5 min and then the solution was
heated at 70.degree. C. for 3 h. The mixture was allowed to cool to
room temperature and then diluted with EtOAc and water (2 mL). The
resultant mixture was stirred at room temperature for 30 min and
then concentrated under reduced pressure. The residue was
triturated with CH.sub.2Cl.sub.2, and the white solid that formed
was removed by filtration. The filtrate was concentrated under
reduced pressure and the resultant residue was purified by silica
gel eluting with 30% ethyl acetate in hexanes to yield 8.1 (0.5 g,
65%). .sup.1H NMR (CDCl.sub.3): .delta. 8.08 (d, 1H), 7.41 (d, 1H),
7.05-7.35 (m, 22H), 6.85 (d, 2H), 6.70 (s, 1H). 5.19 (d, 1H), 5.10
(d, 1H), 4.70 (d, 2H), 3.70-3.90 (m, 4H), 3.20 (d, 1H), 3.11 (d,
1H), 2.80-2.97 (m, 4H), 1.79 (s, 9H), 1.40 (s, 18H), 1.30 (s, 6H);
.sup.31P NMR (CDCl.sub.3): 12.43 ppm
[1782] Diphenylphosphonate 8.2: A solution of 8.1 (0.5 g, 0.42
mmol) in CH.sub.2Cl.sub.2 (4 mL) was treated with TFA (1 mL) and
the resultant mixture was stirred at room temperature for 4 h. The
reaction mixture was concentrated under reduced pressure and
azeotroped twice with CH.sub.3CN. The residue was purified by
silica gel eluting with 5% methanol in CH.sub.2Cl.sub.2 to afford
diphenylphosphonate 8.2 (0.25 g, 71%). .sup.1H NMR (CDCl.sub.3):
.delta. 7.03-7.40 (m, 21H), 6.81-6.90 (m, 3H), 4.96 (d, 1H), 4.90
(d, 1H) 4.60-4.70 (m, 2H), 3.43-3.57 (m, 4H), 3.20 (d, 1H),
2.80-2.97 (m, 5H); .sup.31P NMR (CDCl.sub.3): 12.13 ppm; MS: 824
(M+H).
[1783] Monophenol 8.3: The monophenol 8.3 (124 mg, 68%) was
prepared from the diphenol 8.2 by treating with 1N NaOH in
acetonitrile at 0.degree. C.
[1784] Monoamidate 8.4: To a pyridine solution (0.5 mL) of 8.3 (40
mg, 53 .mu.mol), n-butyl amidate HCl salt (116 mg, 640 .mu.mol),
and DIPEA (83 mg, 640 .mu.mol) was added a pyridine solution (0.5
mL) of triphenyl phosphine (140 mg, 640 .mu.mol), and aldrithiol-2
(120 mg, 640 .mu.mol). The resulting solution was stirred at
65.degree. C. overnight, worked up, and purified by preparative TLC
twice to give 8.4 (1.8 mg). .delta. 4.96 (d, 1H), 4.90 (d, 1H)
4.30-4.6 (m, 2H), 3.9-4.2 (m, 2H), 3.6-3.70 (m, 4H), 3.2-3.3 (d,
1H), 2.80-3.1 (m, 4H); MS: 875 (M+H) & 897 (M+Na)
##STR569##
[1785] Monolactate 9.1: The monolactate 9.1 is prepared from 8.3
using the conditions described above for the preparation of the
monoamidate 8.4 except n-butyl lactate was used in place of n-butyl
amidate HCl salt. ##STR570##
[1786] Dibenzylphosphonate 10.1: Compound 6.5 (16 mg, 0.014 mmol)
was dissolved in CH.sub.2Cl.sub.2 (2 mL) and cooled to 0.degree. C.
TFA (1 mL) was added and the reaction mixture was stirred for 0.5
h. The mixture was then allowed to warm to room temperature for 2
h. The reaction mixture was concentrated under reduced pressure and
azeotroped with toluene. The residue was purified by silica gel
eluting with CH.sub.2Cl.sub.2:MeOH (9:1) to afford compound 10.1 (4
mg, 32%).
[1787] Isopropylamino indazole 10.2: Compound 10.1 (30 mg, 0.35
mmol) was treated with acetone according to the method of Henke et
al (J. Med Chem. 40 17 (1997) 2706-2725) to yield 10.2 as a crude
residue. The residue was purified by silica gel eluting with
CH.sub.2Cl.sub.2:MeOH (93:7) to afford compound 10.2 (3.4 mg, 10%).
##STR571## ##STR572##
[1788] Benzyl ether 11.1: A DMF solution (5 mL) of 3.1 (0.98 g,
1.96 mmol) was treated with NaH (0.24 g of 60% oil dispersion, 6
mmol) for 30 min, followed by the addition of sodium iodide (0.3 g,
2 mmol), and benzoxypropyl bromide (0.55 g, 2.4 mmol). After the
reaction for 3 h at room temperature, the reaction mixture was
partitioned between methylene chloride and saturated NaCl, dried,
and purified to give 11.1 (0.62 g, 49%).
[1789] Aminoindazole 11.2: A n-butanol solution (10 mL) of 11.1
(0.6 g, 0.92 mmol) and hydrazine hydrate (0.93 g, 15.5 mmol) was
heated at reflux for 4 h. The reaction mixture was concentrated
under reduced pressure to give crude 11.2 (.about.0.6 g).
[1790] Tri-BOC-Aminoindazole 11.3: A methylene chloride solution
(10 mL) of crude 11.2, DIPEA (0.36 g, 2.8 mmol), (BOC).sub.2O (0.73
g, 3.3 mmol), and DMAP (0.34 g, 2.8 mmol) was stirred for 5 h at
room temperature, partitioned between methylene chloride and 5%
citric acid solution, dried, purified by silica gel column
chromatography to give 11.3 (0.51 g, 58%, 2 steps).
[1791] 3-Hydroxypropyl cyclic urea 11.4: An ethyl acetate/ethanol
solution (30 mL/5 mL) of 11.3 (0.5 g, 0.52 mmol) was hydrogenated
at 1 atm in the presence of 10% Pd/C (0.2 g) for 4 h. The catalyst
was removed by filtration. The filtrate was then concentrated under
reduced pressure to afford crude 11.4 (0.44 g, 98%).
[1792] Dibenzyl phosphonate 11.5: A THF solution (3 mL) of 11.4
(0.5 g, 0.57 mmol) and triflate dibenzyl phosphonate 3.11 (0.37 g,
0.86 mmol) was cooled to -3.degree. C., followed by addition of
n-BuLi (0.7 ml, of 2.5 M hexane solution, 1.7 mmol). After 2 h
reaction, the reaction mixture was partitioned between methylene
chloride and saturated NaCl solution, concentrated under reduced
pressure. The residue was redissolved in methylene chloride (10
mL), and reacted with (BOC).sub.2O (0.15 g, 0.7 mmol) in the
presence of DMAP (0.18 g, 0.57 mmol), DIPEA (0.18 g, 1.38 mmol) for
2 h at room temperature. The reaction mixture was worked up, and
purified by silica gel chromatography to give 11.5 (0.25 g,
43%).
[1793] Phosphonic diacid 11.7: An ethyl acetate solution (2 mL) of
11.5A (11 mg, 10.5 .mu.mol) was hydrogenated at 1 atm in the
presence of 10% Pd/C (10 mg) for 6 h. The catalyst was removed by
filtration, and the filtrate was concentrated under reduced
pressure to give crude 11.6. The crude 11.6 was redissolved in
methylene chloride (1 mL) and treated with TFA (0.2 mL) for 4 h at
room temperature. The reaction mixture was concentrated under
reduced pressure and purified by HPLC to give 11.7 (2 mg, 30%).
[1794] NMR (CD.sub.3OD): .delta. 7.1-7.3 (m, 11H), 7.0-7.1 (d, 2H),
4.95 (d, 1H), 3.95-4.1 (d, 1H), 2.9-3.3 (m, 4H), 2.3-2.45 (m, 1H),
1.6-1.8 (m, 2H). P NMR (CD.sub.3OD): 15.5 ppm. MS: 624 (M+1).
[1795] Diphenyl phosphonate 11.8: A pyridine solution (1 mL) of
11.6 (0.23 g, 0.23 mmol), phenol (0.27 g, 2.8 mmol), and DCC (0.3
g, 1.4 mmol) was stirred for 5 min. at room temperature, then
reacted at 70.degree. C. for 3 h. The reaction mixture was cooled
to room temperature, concentrated under reduced pressure, and
purified by silica gel column chromatograph to afford 11.8 (0.11 g,
41%).
[1796] Monophenyl phosphonate 11.9: An acetonitrile solution (2 mL)
of 11.8 (0.12 g, 0.107 mmol) at 0.degree. C. was treated with 1N
sodium hydroxide aqueous solution (0.2 mL) for 1.5 h., then
acidified with Dowex (50wx8-200, 120 mg). The Dowex was removed by
filtration, and the filtrate was concentrated under reduced
pressure. The residue was triturated with 10% EtOAc/90% hexane
twice to afford 11.9 (90 mg, 76%) as a white solid.
[1797] Mono-ethyl lactate phosphonate 11.10: A pyridine solution
(0.3 mL) of 11.9 (33 mg, 30 .mu.mol), ethyl lactate (41 mg, 340
.mu.mol), and DCC (31 mg, 146 .mu.mol) was stirred at room
temperature for 5 min, then reacted at 70.degree. C. for 1.5 h. The
reaction mixture was concentrated under reduced pressure,
partitioned between methylene chloride and saturated NaCl solution,
and purified by silica gel chromatography to give 11.10 (18 mg,
50%).
[1798] Ethyl lactate phosphonate 11.11: A methylene chloride
solution (0.8 mL) of 11.10 (18 mg, 15.8 .mu.mol) was treated with
TFA (0.2 mL) for 4 h, and then concentrated under reduced pressure.
The residue was purified by preparative TLC to give 11.11 (6 mg,
50%). NMR (CDCl.sub.3+.about.10% CD.sub.3OD): .delta. 7.0-7.3 (m,
16H), 6.8-7.0 (m, 2H), 4.9-5.0 (m, 1H), 4.75 (d, 1H), 4.1-4.2 (m,
2H). 3.5-4.0 (m, 10H), 2.18-2.3 (m, 1H), 1.6-1.7 (m, 1H), 1.47
& 1.41 (2d, 3H), 1.22 (t, 3H). P NMR (CDCl.sub.3+.about.10%
CD.sub.3OD): 19.72 & 17.86 ppm.
[1799] Diethyl phosphonate 11.13: Compound 11.13 (6 mg) was
prepared as described above in Scheme 5 from 11.4 (30 mg, 34
.mu.mol) and triflate phosphonate 5.3 (52 mg, 172 .mu.mol),
followed by TFA treatment. NMR (CDCl.sub.3+.about.10% CD.sub.3OD):
.delta. 7.1-7.32 (m, 11H), 6.9-7.0 (d, 2H), 4.75 (d, 1H), 4.1-4.2
(2q, 4H), 3.84-3.9 (m, 1H), 3.4-3.8 (m, 8H), 2.7-3.1 (m, 4H),
2.1-2.5 (m, 1H), 1.5-1.7 (m, 2H), 1.25-1.35 (2t, 6H). P NMR
(CDCl.sub.3+.about.10% CD.sub.3OD): 21.63 ppm. MS: 680 (M+1).
##STR573##
[1800] Butyl lactate phosphonate 12.2: A pyridine solution (0.3 mL)
of 11.9 (27 mg, 22 .mu.mol), butyl lactate (31 mg, 265 .mu.mol),
and DCC (28 mg, 132 .mu.mol) was stirred at room temperature for 5
min, then reacted at 70.degree. C. for 1.5 h. The reaction mixture
was concentrated under reduced pressure, partitioned between
methylene chloride and saturated NaCl solution, and purified by
preparative TLC to give 12.1 (12 mg). A methylene chloride solution
(0.8 mL) of 12.1 (12 mg) was treated with TFA (0.2 mL) for 4 h,
concentrate. The residue was purified by preparative TLC to give
12.2 (3 mg, 16%). NMR (CDCl.sub.3+.about.10% CD.sub.3OD): .delta.
6.8-7.4 (m, 18H), 6.4-6.6 (m), 4.9-5.05 (m, 1H), 4.75 (d, 1H),
4.1-4.2 (m, 2H). 3.5-4.0 (m, 10H), 3.1-3.25 (m, 2H), 2.2-2.35 (m,
1H), 1.8-1.9 (m, 1H), 1.4 & 1.8 (m, 7H), 1.22 (t, 3H). P NMR
(CDCl.sub.3+.about.10% CD.sub.3OD): 19.69 & 17.86 ppm.
##STR574##
[1801] Benzyl ether 13.1: A DMF solution (5 mL) of 3.1 (1 g, 2
mmol) was treated with NaH (0.24 g of 60% oil dispersion, 6 mmol)
for 30 min, followed by the addition of sodium iodide (0.3 g, 2
mmol), and benzoxybutyl bromide (0.58 g, 2.4 mmol). After the
reaction for 5 h at room temperature, the reaction mixture was
partitioned between methylene chloride and saturated NaCl, dried,
and purified to give 13.1 (0.58 g, 44%).
[1802] Aminoindazole 13.2: A n-butanol solution (10 mL) of 11.1
(0.58 g, 0.87 mmol) and hydrazine hydrate (0.88 g, 17.5 mmol) was
heated at reflux for 4 h. The reaction mixture was concentrated
under reduced pressure to give crude 13.2 (0.56 g).
[1803] Tri-BOC-aminoindazole 13.3: A methylene chloride solution
(10 mL) of 132 (0.55 g, 0.82 mmol), DIPEA (0.42 g, 3.2 mmol),
(BOC).sub.2O (0.71 g, 3.2 mmol), and DMAP (0.3 g, 2.4 mmol) was
stirred for 4 h at room temperature, partitioned between methylene
chloride and 5% citric acid solution, dried, purified by silica gel
chromatography to give 13.3 (0.56 g, 71%, 2 steps).
[1804] 3-Hydroxybutyl cyclic urea 13.4: An ethyl acetate/methanol
solution (30 mL/5 mL) of 11.3 (0.55 g, 0.56 mmol) was hydrogenated
at 1 atm in the presence of 10% Pd/C (0.2 g) for 3 h. The catalyst
was removed by filtration. The filtrate was concentrated under
reduced pressure to afford crude 13.4 (0.5 g, 98%).
[1805] Diethyl phosphonate 13.6: A THF solution (1 mL) of 13.4 (5
mg, 56 .mu.mol) and triflate diethyl phosphonate 5.3 (30 mg, 100
.mu.mol) was cooled to -3.degree. C., followed by addition of
n-BuLi (80 .mu.l of 2.5 M hexane solution, 200 .mu.mol). After 2 h
reaction, the reaction mixture was partitioned between methylene
chloride and saturated NaCl solution, concentrated under reduced
pressure to give crude 13.5. The residue was dissolved in methylene
chloride (0.8 mL) and treated with TFA (0.2 mL) for 4 h.
concentrated under reduced pressure, and purified by HPLC to give
13.6 (8 mg, 21%). NMR (CDCl.sub.3): .delta. 7.1-7.4 (m, 11H),
7.0-7.1 (m, 2H) 4.81 (d, 1H), 4.1-4.25 (m, 4H). 3.85-3.95 (m, 1H),
3.4-3.8 (m, 7H), 3.3-3.4 (m, 1H), 2.8-3.25 (m, 5H), 2.0-2.15 (m,
1H), 1.3-1.85 (m, 10H). P NMR (CDCl.sub.3): 21.45 ppm.
##STR575##
[1806] Phosphonic diacid 13.8: Compound 13.8 (4.5 mg) was prepared
from 13.4 as described above for the preparation of 11.7 from 11.4
(Scheme 11). NMR (CD.sub.3OD): .delta. 7.41 (s, 1H), 7.1-7.4 (m,
10H), 6.9-7.0 (m, 2H) 4.75 (d, 1H), 3.8-4.0 (m, 1H). 3.4-3.8 (m,
8H), 2.8-3.25 (m, 5H), 2.1-2.25 (m, 1H), 1.6-1.85 (m, 4H). MS: 638
(M+1). ##STR576##
[1807] t-Butyl ester 14.1: A DMF solution (3 mL) of 3.1 (0.5 g, 1
mmol) was treated with NaH (80 mg of 60% oil dispersion, 2 mmol)
for 10 min, followed by the addition of 14.5 (0.25 g, 1.1 mmol).
After the reaction for 1 h at room temperature, the reaction
mixture was partitioned between methylene chloride and saturated
NaCl, dried, and purified to give 14.1 (0.4 g, 59%).
[1808] Aminoindazole derivative 14.3: A methylene chloride solution
(5 mL) of 14.1 (0.4 g, 0.58 mmol) was treated with TFA (1 mL) at
room temperature for 1.5 h, and then concentrated under reduced
pressure to give crude 14.2. The crude 14.2 was dissolved in n-BuOH
(5 mL) and reacted with hydrazine hydrate (0.58 g, 11.6 mmol) at
reflux for 5 h. The reaction mixture was concentrated under reduced
pressure and purified by silica gel chromatography to give the
desired product 14.3 (0.37 g, quantitative yield).
[1809] Diethylphosphonate ester 14.4: A methylene chloride solution
(3 mL) of 14.3 (23 mg, 38 mmol) was reacted with
aminopropyl-diethylphosphonate 14.6 (58 mg, 190 mmol), DIPEA (50
mg, 380 mmol), and ByBOP (21 mg, 48 .mu.mol) at room temperature
for 2 h, and then concentrated under reduced pressure. The residue
was triturated with methylene chloride/hexane. The solid was
purified by preparative TLC to give 14.4 (9 mg, 34%). NMR
(CDCl.sub.3+.about.10% CD.sub.3O): .delta. 7.87 (t, 1H), 7.61 (b,
1H), 7.51 (s, 1H), 7.14-7.2 (m, 10H), 6.93-7.0 (m, 4H), 4.79 (d,
2H), 3.99-4.04 (m, 4H), 3.38-3.65 (m, 6H), 2.60-3.2 (m, 6H),
1.70-1.87 (m, 4H), 1.25 (t, 6H). P NMR (CDCl.sub.3+.about.10%
CD.sub.3OD): 32.7 ppm.
[1810] Diethylphosphonate ester 14.5: A methylene chloride solution
(2 mL) of 14.3 (13 mg, 21 mmol) was reacted with
aminoethyl-diethylphosphonate oxalate 14.7 (23 mg, 85 .mu.mol),
DIPEA (22 mg, 170 mmol), and ByBOP (12 mg, 25 .mu.mol) at room
temperature for 2 h, and then concentrated under reduced pressure.
The residue was triturated with methylene chloride/hexane. The
solid was purified by preparative TLC to give 14.5 (5 mg, 30%). Ms:
783 (M+1). NMR (CDCl.sub.3+.about.10% CD.sub.3O): .delta. 7.88 (b,
1H), 7.58 (b, 1H), 7.49 (s, 1H), 7.14-7.2 (m, 10H), 6.90-7.0 (m,
4H), 4.75 (d, 2H), 3.90-4.04 (m, 4H), 2.50-3.3 (m, 6H), 1.97-2.08
(m, 2H). P NMR (CDCl.sub.3+.about.10% CD.sub.3OD): 30.12 ppm.
##STR577##
[1811] Monophenol-ethyl lactate phosphonate prodrug 15.1: A
methylene chloride/DMF solution (2 mL/0.5 mL) of 14.3 (30 mg, 49
.mu.mol) was reacted with aminopropyl-phenol-ethyl lactate
phosphonate 15.5 (100 mg, 233 .mu.mol), DIPEA (64 mg, 495 .mu.mol),
and BOP reagent (45 mg, 100 .mu.mol) at room temperature for 2 h,
and then concentrated under reduced pressure. The residue was
triturated with methylene chloride/hexane. The solid was purified
by silica gel chromatography to give 15.1 (28 mg, 64%). NMR
(CDCl.sub.3+.about.10% CD.sub.3O): .delta. 7.83 (b, 1H), 7.59 (b,
1H), 7.51 (s, 1H), 7.14-7.2 (m, 11H), 6.90-7.0 (m, 4H), 4.75-4.87
(d+q, 3H), 4.10 (q, 2H), 3.3-3.61 (m, 6H), 2.60-3.2 (m, 6H),
1.92-2.12 (m, 4H), 1.30 (d, 3H), 1.18 (t, 3H). P NMR
(CDCl.sub.3+.about.10% CD.sub.3OD): 30.71 ppm. MS: 903 (M+1).
[1812] Phenol-ethyl alanine phosphonate prodrug 15.2: A methylene
chloride/DMF solution (2 mL/0.5 mL) of 14.3 (30 mg, 49 .mu.mol) was
reacted with aminopropyl-phenol-ethyl alanine phosphonate 15.6 (80
mg TFA salt, 186 .mu.mol), DIPEA (64 mg, 500 .mu.mol), and BOP
reagent (45 mg, 100 .mu.mol) at room temperature for 2 h, and then
concentrated under reduced pressure. The residue was triturated
with methylene chloride/hexane. The solid was purified by
preparative TLC to give 15.2 (12 mg, 27%). NMR
(CDCl.sub.3+.about.10% CD.sub.3O): .delta. 7.91 (b, 1H), 7.61 (b,
1H), 7.52 (s, 1H), 7.14-7.2 (m, 11H), 6.90-7.0 (m, 4H), 4.75 (d,
2H), 3.82-4.1 (2q, 3H), 3.4-3.65 (m, 6H), 2.60-3.15 (m, 6H),
1.8-2.0 (m, 4H), 1.3 (d, 3H). P NMR (CDCl.sub.3+.about.10%
CD.sub.3OD): 32.98 & 33.38 ppm. MS: 902 (M+1).
[1813] Dibenzyl phosphonate 15.3: A methylene chloride/DMF solution
(2 mL/0.5 mL) of 14.3 (30 mg, 49 .mu.mol) was reacted with
aminopropyl dibenzyl phosphonate 15.7 (86 mg TFA salt, 200
.mu.mol), DIPEA (64 mg, 500 .mu.mol), and BOP reagent (45 mg, 100
.mu.mol) at room temperature for 2 h, and then concentrated under
reduced pressure. The residue was triturated with methylene
chloride/hexane. The solid was purified by preparative TLC to give
15.3 (20 mg, 44%). NMR (CDCl.sub.3+.about.5% CD.sub.3O): .delta.
7.50-7.58 (m, 2H), 7.14-7.3 (m, 21H), 6.90-7.0 (m, 4H), 4.7-5.1 (m,
6H), 3.6-3.8 (m, 4H), 3.3-3.55 (m, 2H), 2.60-3.15 (m, 6H), 1.8-2.0
(m, 4H). P NMR (CDCl.sub.3+.about.5% CD.sub.3OD): 33.7 ppm. MS: 907
(M+1).
[1814] Phosphonic diacid 15.4: An ethanol solution (5 mL) of 15.3
(17 mg, 18.7 .mu.mol) was hydrogenated at 1 atm in the presence of
10% Pd/C for 4 h. The catalyst was removed by filtration, and the
filtrate was concentrated under reduced pressure to give the
desired product 15.4 (12 mg, 85%). NMR (CD.sub.3O+20% CDCl.sub.3):
.delta. 7.88 (b, 1H), 7.59 (b, 1H), 7.6 (s, 1H), 7.1-7.25 (m, 10H),
6.90-7.1 (m, 4H), 4.8 (d, 2H+water peak), 3.6-3.8 (m, 4H), 3.4-3.5
(m, 2H), 1.85-2.0 (m, 4H). ##STR578##
[1815] Monobenzyl derivative 16.1: A DMF solution (4 mL) of 1.1
(0.8 g, 2.2 mmol) was treated with NaH (0.18 g of 60% oil
dispersion, 4.4 mmol) for 10 min at room temperature followed by
the addition of 14.5 (0.5 g, 2.2 mmol). The resulting solution was
reacted at room temperature for 2 h, worked up, and then purified
to afford 16.1 (0.48 g, 40%).
[1816] 3-Nitrobenzyl cyclic urea derivative 16.2: A DME solution
(0.5 mL) of 16.1 (65 mg, 117 .mu.mol) was treated with NaH (15 mg
of 60% oil dispersion, 375 .mu.mol) for 10 min at room temperature,
followed by the addition of 3-nitrobenzyl bromide (33 mg, 152
.mu.mol). The resulting solution was reacted at room temperature
for 1 h, worked up, and purified by preparative TLC to afford 16.2
(66 mg, 82%).
[1817] Diol 16.3: A methylene chloride solution (2 mL) of 16.2 (46
mg, 61 .mu.mol) was treated with TFA (0.4 mL) for 2 h at room
temperature, and then concentrated under reduced pressure to afford
16.3. This material was used without further purification.
[1818] 3-Aminobenzyl cyclic urea 16.4: An ethyl acetate/ethanol (5
mL/1 mL) solution of 16.3 (crude) was hydrogenated at 1 atm in the
presence of 10% Pd/C for 2 h. The catalyst was removed by
filtration. The filtrate was concentrated under reduced pressure,
and purified by preparative TLC to afford 16.4 (26 mg, 70%, 2
steps).
[1819] Diethyl phosphonate 16.5: A methylene chloride/DMF solution
(2 mL/0.5 mL) of 16.4 (24 mg, 42 .mu.mol) was reacted with
aminopropyl-diethylphosphonate ester TFA salt 14.6 (39 mg, 127
.mu.mol), DIPEA (27 mg, 210 .mu.mol), and BOP reagent (28 mg, 63
.mu.mol) at room temperature for 2 h, and then concentrated under
reduced pressure. The residue was purified by preparative TLC to
give 16.5 (20.7 mg, 63%). NMR (CDCl.sub.3+.about.10% CD.sub.3O):
.delta. 7.62 (b, 1H), 7.51 (s, 1H), 7.0-7.35 (m, 12H), 6.95 (d,
2H), 6.85 (d, 2H), 4.6-4.71 (2d, 2H), 3.95-4.1 (m, 4H). 3.3-3.55
(m, 3H), 2.60-2.8 (m, 2H), 2.95-3.15 (m, 4H), 1.85-2.0 (m, 4H),
1.25 (t, 6H). P NMR (CDCl.sub.3+.about.10% CD.sub.3OD): 32.65 ppm.
##STR579##
[1820] p-Benzoxybenzyl cyclic urea derivative 17.1: A DMF solution
(0.5 mL) of 16.1 (65 mg, 117 .mu.mol) was treated with NaH (15 mg
of 60% oil dispersion, 375 .mu.mol) for 10 min at room temperature,
followed by the addition of 4-benzoxy benzyl chloride 3.10 (35 mg,
.mu.mol). The resulting solution was stirred for 2 h at room
temperature. The reaction mixture was concentrated under reduced
pressure, purified by preparative TLC to generate 17.1 (62 mg,
70%).
[1821] Diethyl phosphonate 17.3: A methylene chloride solution (2
mL) of 17.1 (46 mg, 61 .mu.mol) was treated with TFA (0.4 mL) for 2
h at room temperature, and then concentrated under reduced pressure
to give crude 17.2. An ethyl acetate/ethanol solution (3 mL/2 mL)
of the crude 17.2 was then hydrogenated at 1 atm in the presence of
10% Pd/C (10 mg) for 5 h at room temperature. The catalyst was
removed by filtration. The filtrate was concentrated under reduced
pressure to afford 17.3 (crude).
[1822] Diethyl phosphonate cyclic urea 17.4: A methylene
chloride/DMF solution (2 mL/0.5 mL) of 17.3 (25 mg, 42 .mu.mol) was
reacted with aminopropyl-diethylphosphonate ester TFA salt 14.6 (40
mg, 127 .mu.mol), DIPEA (27 mg, 210 .mu.mol), and BOP reagent (28
mg, 63 .mu.mol) at room temperature for 2 h, and then concentrated
under reduced pressure. The residue was purified by preparative TLC
to give 17.4 (14.6 mg, 44%). NMR (CDCl.sub.3+.about.10% CD.sub.3O):
.delta. 7.82 (t), 7.62 (d, 1H), 7.51 (s, 1H), 7.05-7.35 (m, 10H),
6.8-6.95 (2d, 4H), 6.85 (d, 2H), 4.8 (d, 1H), 4.65 (d, 1H),
3.95-4.1 (m, 4H). 3.4-3.75 (m, 6H), 2.60-3.2 (m), 1.85-2.0 (m, 4H),
1.25 (t, 6H). P NMR (CDCl.sub.3+.about.10% CD.sub.3OD): 32.72 ppm.
##STR580## ##STR581##
[1823] Dibenzyl derivative 18.1: A DMF solution (3 mL) of compound
2.8 (0.4 g, 0.78 mmol) was reacted with 60% NaH (0.13 g, 1.96
mmol), 4-benzoxy benzylchloride 3.10 (0.46 g, 1.96 mmol) and sodium
iodide (60 mg, 0.39 mmol) at room temperature for 4 h. The reaction
mixture was partitioned between methylene chloride and saturated
NaHCO.sub.3 solution. The organic phase was isolated, dried over
Na.sub.2SO.sub.4, concentrated under reduced pressure, and purified
by silica gel chromatography to give the desired product 18.1 (0.57
g, 81%).
[1824] Diol derivative 18.2 and diphenol derivative 20.1: A
methylene chloride solution (4 mL) of 18.1 (0.57 g, 0.63 mmol) was
treated with TFA (1 mL) at room temperature for 20 min,
concentrated under reduced pressure, and purified by silica gel
chromatography to give diol derivative 18.2 (133 mg, 28%) and
diphenol derivative 20.1 (288 mg. 57.6%).
[1825] Monophosphonate derivative 18.3: A THF solution (10 mL) of
18.2 (130 mg, 0.17 mmol) was stirred with cesium carbonate (70 mg,
0.21 mmol) and diethylphosphonate triflate 5.3 (52 mg, 0.17 mmol)
at room temperature for 4 h. The reaction mixture was concentrated
under reduced pressure and purified to give 18.3 (64 mg, 41%), and
recovered 18.2 (25 mg, 19%).
[1826] Methoxy derivative 18.4: A THF solution (2 mL) of 18.3 (28
mg, 25 .mu.mol) was treated with cesium carbonate (25 mg, 76
.mu.mol) and iodomethane (10 eq. Excess) at room temperature for 5
h. The reaction mixture was concentrated under reduced pressure and
partitioned between methylene chloride and saturated NaHCO.sub.3.
The organic phase was separated, concentrated under reduced
pressure and the residue purified by preparative TLC to afford 18.4
(22 mg, 78%).
[1827] Diethylphosphonate 18.5: An ethyl acetate/ethanol (2 mL/2
mL) solution of 18.4 (22 mg, 24 .mu.mol) was hydrogenated at 1 atm
in the presence of 10% Pd/C for 3 h. The catalyst was removed by
filtration, the filtrate was concentrated under reduced pressure to
give the desired product 18.5 (18 mg, quantitative). NMR
(CDCl.sub.3+.about.10% CD.sub.3O): .delta. 6.7-7.0 (m, 12H),
6.62-6.69 (m, 4H), 4.65 (d, 1H), 4.50 (d, 1H), 4.18-4.3 (m, 6H).
3.75 (s, 3H), 3.3-3.4 (m, 4H), 2.8-3.0 (m, 6H), 1.30 (t, 6H). P NMR
(CDCl.sub.3+.about.10% CD.sub.3OD): 20.16 ppm. ##STR582##
[1828] Diethyl phosphonate 19.1: An ethyl acetate/ethanol (2 mL/1
mL) solution of 18.3 (14 mg, 15.5 .mu.mol) was hydrogenated at 1
atm in the presence of 10% Pd/C (5 mg) for 3 h. The catalyst was
then removed by filtration, and the filtrate was concentrated under
reduced pressure to give the desired product 19.1 (10 mg, 90%). NMR
(CDCl.sub.3+.about.15% CD.sub.3O): .delta. 6.6-7.0 (m, 16H),
4.5-4.65 (2d, 2H), 4.1-4.3 (m, 6H). 2.7-3.0 (m, 6H), 1.29 (t, 6H).
P NMR (CDCl.sub.3+15% CD.sub.3OD): 20.12 ppm. ##STR583##
##STR584##
[1829] Monophosphonate 20.2: A THF solution (8 mL) of 20.1 (280 mg,
0.36 mmol) was stirred with cesium carbonate (140 mg, 0.43 mmol)
and diethylphosphonate triflate 5.3 (110 mg, 0.36 mmol) at room
temperature for 4 h. The reaction mixture was concentrated under
reduced pressure and purified to give 20.2 (130 mg, 39%), and
recovered 20.1 (76 mg, 27%).
[1830] Triflate derivative 20.3: A THF solution (6 mL) of 20.2 (130
mg, 0.13 mmol) was stirred with cesium carbonate (67 mg, 0.21 mmol)
and N-phenyltrifluoromethane-sulfonimide (60 mg, 0.17 mmol) at room
temperature for 4 h. The reaction mixture was concentrated under
reduced pressure and purified to give 20.3 (125 mg, 84%).
[1831] Benzyl ether 20.4: To a DMF solution (2 mL) of Pd(OAc).sub.2
(60 mg, 267 .mu.mol), and dppp (105 mg. 254 .mu.mol) was added 20.3
(120 mg, 111 .mu.mol) under nitrogen, followed by the addition of
triethylsilane (0.3 mL). The resulting solution was stirred at room
temperature for 4 h, then concentrated under reduced pressure. The
residue was purified by silica gel chromatography to afford 20.4
(94 mg, 92%).
[1832] Diethyl phosphonate 20.6: An ethyl acetate/ethanol (2 mL/2
mL) solution of 20.4 (28 mg, 30 .mu.mol) was hydrogenated at 1 atm
in the presence of 10% Pd/C (5 mg) for 3 h. The catalyst was
removed by filtration, and the filtrate was concentrated under
reduced pressure to give the desired product 20.5. The crude
product 20. 5 was redissolved in methylene chloride (2 mL) and
treated with TFA (0.4 mL) and a drop of water. After 1 h stirring
at room temperature, the reaction mixture was concentrated under
reduced pressure, and purified by preparative TLC plate to give
20.6 (18 mg, 85%, 2 steps). .delta. 6.6-7.3 (m, 17H), 4.65 (d, 1H),
4.58 (d, 1H), 4.18-4.3 (m, 6H), 3.3-3.5 (m, 4H), 2.8-3.1 (m), 1.34
(t, 6H). P NMR (CDCl.sub.3+.about.10% CD.sub.3OD): 20.16 ppm. MS:
705 (M+1). ##STR585## ##STR586##
[1833] Bis-(3-nitrobenzyl) derivative 21.1: A DMF solution (2 mL)
of compound 2.8 (0.3 g, 0.59 mmol) was reacted with 60% NaH (0.07
g, 1.76 mmol), 3-nitrobenzyl bromide (0.38 g, 1.76 mmol) and sodium
iodide (60 mg, 0.39 mmol) at room temperature for 3 h. The reaction
mixture was partitioned between methylene chloride and saturated
NaHCO.sub.3 solution. The organic phase was isolated, dried over
Na.sub.2SO.sub.4, concentrated under reduced pressure, and purified
by silica gel chromatography to give the desired product 21.1 (0.37
g, 82%).
[1834] Diphenol derivative 21.2: A methylene chloride solution (4
mL) of 21.1 (0.37 g, 0.47 mmol) was treated with TFA (1 mL) at room
temperature for 3 h, and then concentrated under reduced pressure,
and azeotroped with CH.sub.3CN twice to give diphenol derivative
21.2 (0.3 g, quantitative).
[1835] Monophosphonate derivative 21.3: A THF solution (8 mL) of
18.2 (0.28 g, 0.44 mmol) was stirred with cesium carbonate (0.17 g,
0.53 mmol) and diethylphosphonate triflate 5.3 (0.14 g, 0.44 mmol)
at room temperature for 4 h. The reaction mixture was concentrated
under reduced pressure and purified to give 21.3 (120 mg, 35%), and
recovered 21.2 (150 mg, 53%).
[1836] Methoxy derivative 21.4: A THF solution (2 mL) of 21.3 (9
mg, 11 .mu.mol) was treated with cesium carbonate (15 mg, 46
.mu.mol) and iodomethane (10 eq. Excess) at room temperature for 6
h. The reaction mixture was concentrated under reduced pressure and
partitioned between methylene chloride and saturated NaHCO.sub.3.
The organic phase was separated, dried over sodium sulfate,
filtered and concentrated under reduced pressure. The residue was
purified by preparative TLC to afford 21.4 (9 mg)
[1837] Diethylphosphonate 21.5: A ethyl acetate/ethanol (2 mL/0.5
mL) solution of 21.4 (9 mg, 11 .mu.mol) was hydrogenated at 1 atm
in the presence of 10% Pd/C for 4 h. The catalyst was removed by
filtration, and the filtrate was concentrated under reduced
pressure to give the desired product 21.5 (4.3 mg, 49%, 2 steps).
NMR (CDCl.sub.3+.about.10% CD.sub.3O): .delta. 7.0-7.10 (m, 6H),
6.8-6.95 (m, 4H), 6.5-6.6 (m, 4H), 6.4-6.45 (m, 2H), 4.72 (d, 2H),
4.18-4.3 (m, 6H). 3.72 (s, 3H), 3.4-3.5 (m, 4H), 2.8-3.0 (m, 6H),
1.34 (t, 6H). P NMR (CDCl.sub.3+.about.10% CD.sub.3OD): 19.93
ppm.
[1838] Triflate 21.6: A THF solution (6 mL) of 21.3 (0.1 g, 0.14
mmol), cesium carbonate (0.07 g, 0.21 mmol), and
N-phenyltrifluoromethane-sulfonimide (60 mg, 0.17 mmol) was stirred
at room temperature for 4 h, and then concentrated under reduced
pressure, and worked up. The residue was purified by silica gel
chromatography to give 21.6 (116 mg, 90%).
[1839] Diamine 21.7: A DMF solution (2 mL) of 21.6 (116 mg, 127
.mu.mol), dppp (60 mg, 145 .mu.mol), and Pd(OAc).sub.2 (30 mg, 134
.mu.mol) was stirred under nitrogen, followed by addition of
triethylsilane (0.3 mL), and reacted for 4 h at room temperature.
The reaction mixture was worked up and purified to give 21.7 (50
mg).
[1840] Diethyl phosphonate 21.8: An acetonitrile solution (1 mL) of
crude 21.7 (50 mg) was treated with 48% HF (0.1 mL) for 4 h. The
reaction mixture was concentrated under reduced pressure, and
purified to give 21.8 (10 mg, 11% (2 steps). NMR
(CDCl.sub.3+.about.10% CD.sub.3O): .delta. 7.05-7.30 (m, 9H),
6.8-6.95 (d, 2H), 6.4-6.6 (m, 6H), 4.72 (d, 2H), 4.18-4.3 (m, 6H).
3.4-3.5 (m, 4H), 2.8-3.0 (m, 6H), 1.34 (t, 6H). P NMR
(CDCl.sub.3+.about.10% CD.sub.3OD): 19.83 ppm. ##STR587##
##STR588##
[1841] Acetonide 22.1: An acetone/2,2-diemethoxypropane solution
(15 mL/5 mL) of compound 21.2 (240 mg, 0.38 mmol) and pyridinium
toluenesulfonate (10 mg) was heated at reflux for 30 min. After
cooled to room temperature, the reaction mixture was concentrated
under reduced pressure. The residue was partitioned between
methylene chloride and saturated NaHCO.sub.3 aqueous solution,
dried, concentrated under reduced pressure and purified to afford
22.1 (225 mg, 88%).
[1842] Monomethoxy derivative 22.2: A THF solution (10 mL) of 22.1
(225 mg, 0.33 mmol) was treated with cesium carbonate (160 mg, 0.5
mmol) and iodomethane (52 mg. 0.37 mmol) at room temperature
overnight. The reaction mixture was concentrated under reduced
pressure, and purified by preparative silica gel column
chromatography to afford 22.2 (66 mg, 29%) and recovered starting
material 22.1 (25 mg, 11%).
[1843] Diethyl phosphonate 22.3: A methylene chloride solution (2
mL) of 22.2 (22 mg, 32 .mu.mol), DIPEA (9 mg, 66 .mu.mol), and
p-nitrophenyl chloroformate (8 mg, 40 .mu.mol) was stirred at room
temperature for 30 min. The resulting reaction mixture was reacted
with DIPEA (10 mg, 77 .mu.mol), and aminoethyl diethylphosphonate
14.7 (12 mg. 45 .mu.mol) at room temperature overnight. The
reaction mixture was washed with 5% citric acid solution, saturated
NaHCO.sub.3, dried, and purified by preparative TLC to afford 22.3
(12 mg, 43%).
[1844] Bis(3-aminobenzyl)-diethylphosphonate ester 22.5: An ethyl
acetate/t-BuOH (4 mL/2 mL) solution of 22.3 (12 mg, 13 .mu.mol) was
hydrogenated at 1 atm in the presence of 10% Pd/C 95 mg) at room
temperature for 5 h. The catalyst was removed by filtration. The
filtrate was concentrated under reduced pressure, and purified by
preparative TLC to give 22.4 (8 mg, 72%). A methylene chloride
solution (0.5 mL) of 22.4 (8 mg) was treated with TFA (0.1 mL) at
room temperature for 1 h., concentrated under reduced pressure, and
then azeotroped with CH.sub.3CN twice to afford 22.5 (8.1 mg, 81%).
NMR (CDCl.sub.3+.about.10% CD.sub.3OD): .delta. 7.2 (d, 1H),
6.95-7.15 (m, 6H), 6.75-6.9 (m, 5H), 4.66 (d, 1H), 4.46 (d, 1H),
4.06-4.15 (m, 4H). 3.75 (s, 3H), 3.6-3.7 (m, 4H), 2.6-3.1 (m, 6H),
2.0-2.1 (m, 2H), 1.30 (t, 6H). P NMR (CDCl.sub.3+10% CD.sub.3OD):
29.53 ppm. MS: 790 (M+1).
[1845] Bis(3-aminobenzyl) diethylphosphonate ester 22.7: Compound
22.7 was prepared from 22.2 (22 mg, 32 .mu.mol) and aminomethyl
diethylphosphonate 22.8 as shown above for the preparation of 22.5
from 22.2. NMR (CDCl.sub.3+.about.10% CD.sub.3OD): .delta. 7.24 (d,
1H), 6.8-7.12 (m, 1H), 4.66 (d, 1H), 4.45 (d, 1H), 4.06-4.15 (m,
4H). 3.75 (s, 3H), 2.6-3.1 (m, 6H), 1.30 (t, 6H). P NMR
(CDCl.sub.3+.about.10% CD.sub.3OD): 22.75 ppm. MS: 776 (M+1).
##STR589## ##STR590## ##STR591##
[1846] Diol 23.1: To a solution of compound 2.8 (2.98 g, 5.84 mmol)
in methylene chloride (14 mL) was added TFA (6 mL). The resulted
mixture was stirred at room temperature for 2 h. Methanol (5 mL)
and additional TFA (5 mL) were added. The reaction mixture was
stirred for additional 4 h and then concentrated under reduced
pressure. The residue was washed with hexane/ethyl acetate (1:1)
and dried to afford compound 23.1 (1.8 g, 86%) as an off-white
solid.
[1847] Benzyl ether 23.3: To a solution of compound 23.1 (1.8 g,
5.03 mmol) in DMF (6 mL) and 2,2-dimethoxyl propane (12 mL) was
added p-toluenesulfonic acid monohydrate (0.095 g, 0.5 mmol). The
resultant mixture was stirred at 65.degree. C. for 3 h. The excess
2,2-dimethoxyl propane was slowly distilled. The reaction mixture
was cooled to room temperature and charged with THF (50 mL), benzyl
bromide (0.8 mL, 6.73 mmol) and cesium carbonate (2.0 g, 6.13
mmol). The resulted mixture was stirred at 65.degree. C. for 16 h.
The reaction was quenched with acetic acid aqueous solution (4%,
100 mL) at 0.degree. C., and extracted with ethyl acetate. The
organic phase was dried over magnesium sulfate and concentrated
under reduced pressure. The residue was purified by chromatography
on silica gel to afford desired mono protected compound 23.3 (1.21
g, 49%).
[1848] Benzyl ether 23.5: To a solution of compound 23.3 (0.65 g,
1.33 mmol) and N-phenyltrifuoromethanesulfonimide (0.715 g, 2 mmol)
in THF (12 mL) was added cesium carbonate (0.65 g, 2 mmol). The
mixture was stirred at room temperature for 3 h. The reaction
mixture was filtered through a pad of silica gel and concentrated
under reduced pressure. The residue was purified on silica gel
chromatography to give triflate 23.4 (0.85 g). To a solution of
1,3-bis(diphenylphosphino)propane (0.275 g, 0.66 mmol) in DMF (10
mL) was added palladium(II) acetate (0.15 g, 0.66 mmol) under
argon. This mixture was stirred for 2 min. and then added to
triflate 23.4. After stirring for 2 min., triethylsilane was added
and the resulted mixture was stirred for 1.5 h. The solvent was
removed under reduced pressure and the residue was purified by
chromatography on silica gel to afford compound 23.5 (0.56 g,
89%).
[1849] Phenol 23.6: A solution of 23.5 (0.28 g, 0.593 mmol) in
ethyl acetate (5 mL) and isopropyl alcohol (5 mL) was treated with
10% Pd/C (0.05 g) and stirred under a hydrogen atmosphere (balloon)
for 16 h. The catalyst was removed by filtration and the filtrate
was concentrated under reduced pressure to yield 23.6 (0.22 g, 97%)
as a white solid.
[1850] Dibenzyl phosphonate 23.7: To a solution of compound 23.6
(0.215 g, 0.563 mmol) in THF (10 mL) was added dibenzyl triflate
3.11 (0.315 g, 0.74 mmol) and cesium carbonate (0.325 g, 1 mmol).
The mixture was stirred at room temperature for 2 h, then diluted
with ethyl acetate and washed with water. The organic phase was
dried over magnesium sulfate, filtered and concentrated under
reduced pressure. The residue was purified by chromatography on
silica gel to afford compound 23.7 (0.31 g, 84%).
[1851] Diphenyl ester 23.8: A solution of compound 23.7 (0.3 g,
0.457 mmol) and benzyl bromide (0.165 mL, 1.39 mmol) in THF (10 mL)
was treated with potassium tert-butoxide (1M/THF, 1.2 mL) for 0.5
h. The mixture was diluted with ethyl acetate and washed with HCl
(0.2N). The organic phase was dried over magnesium sulfate,
filtered and concentrated under reduced pressure. The residue was
dissolved in ethyl acetate and treated with 10% Pd/C (0.05 g) under
hydrogen atmosphere (balloon) for 16 h. The catalyst was removed by
filtration and the filtrate was concentrated under reduced
pressure. The residue was treated with TFA (1 mL) in methanol (5
mL) for 1 h, and then concentrated under reduced pressure. The
residue was dissolved in pyridine (1 mL) and mixed with phenol
(0.45 g, 4.8 mmol) and 1,3-dicyclohexylcarbodiimide (0.38 g, 1.85
mmol). The mixture was stirred at 70.degree. C. for 2 h, and then
concentrated under reduced pressure. The residue was partitioned
between ethyl acetate and HCl (0.2N). The organic phase was dried
over magnesium sulfate, filtered and concentrated. The residue was
purified by chromatography on silica gel to afford compound 23.8
(0.085 g, 24%).
[1852] Mono amidate 23.9: To a solution of 23.8 (0.085 g, 0.11
mmol) in acetonitrile (1 mL) was added sodium hydroxide (1N, 0.25
mL) at 0.degree. C. After stirred at 0.degree. C. for 1 h, the
mixture was acidified with Dowex resin to pH=3, and filtered. The
filtrate was concentrated under reduced pressure. The residue was
dissolved in pyridine (0.5 mL) and mixed with L-alanine ethyl ester
hydrochloride (0.062 g, 0.4 mmol) and 1,3-dicyclohexyl-carbodiimide
(0.125 g, 0.6 mmol). The mixture was stirred at 60.degree. C. for
0.5 h, and then concentrated under reduced pressure. The residue
was partitioned between ethyl acetate and HCl (0.2N). The organic
phase was dried over magnesium sulfate, filtered and concentrated.
The residue was purified by HPLC (C-18, 65% acetonitrile/water) to
afford compound 23.9 (0.02 g, 23%). .sup.1H NMR (CDCl.sub.3):
.delta. 1.2 (m, 3H), 1.4 (m, 3H), 1.8 (brs, 2H), 2.8-3.1 (m, 6H),
3.5-3.7 (m, 4H), 3.78 (m, 1H), 4.0-4.18 (m, 2H), 4.2-4.4 (m, 3H),
4.9 (m, 2H), 6.8-7.4 (m, 24H). .sup.31P NMR (CDCl.sub.3): d 20.9,
19.8. MS: 792 (M+1). ##STR592## ##STR593## ##STR594##
[1853] Di-tert butyl ether 24.1: To a solution of compound 2.8
(0.51 g, 1 mmol) and benzyl bromide (0.43 g, 2.5 mmol) in THF (6
mL) was added potassium tert-butoxide (1M/THF, 2.5 mL). The mixture
was stirred at room temperature for 0.5 h, then diluted with ethyl
acetate and washed with water. The organic phase was dried over
magnesium sulfate, filtered and concentrated under reduced
pressure. The residue was purified by chromatography on silica gel
to afford compound 24.1 (0.62 g, 90%).
[1854] Diol 24.2: To a solution of compound 24.1 (0.62 g, 0.9 mmol)
in methylene chloride (4 mL) was added TFA (1 mL) and water (0.1
mL). The mixture was stirred for 2 h, and then concentrated under
reduced pressure. The residue was purified by chromatography on
silica gel to afford compound 24.2 (0.443 g, 92%).
[1855] Benzyl ether 24.3: Compound 24.3 was prepared in 46% yield
according to the procedure described in Scheme 23 for the
preparation of 23.3.
[1856] Triflate 24.4: Compound 24.4 was prepared in 95% yield
according to the procedure described in Scheme 23 for the
preparation of 23.4.
[1857] Benzyl ether 24.5: Compound 24.5 was prepared in 93% yield
according to the procedure described in Scheme 23 for the
preparation of 23.5.
[1858] Phenol 24.6: Compound 24.6 was prepared in 96% yield
according to the procedure described in Scheme 23 for the
preparation of 23.6 from 23.5.
[1859] Dibenzyl phosphonate 24.7: Compound 24.7 was prepared in 82%
yield according to the procedure described in Scheme 23 for the
preparation of 23.7.
[1860] Diacid 24.8: A solution of 24.7 (0.16 g, 0.207 mmol) in
ethyl acetate (4 mL) and isopropyl alcohol (4 mL) was treated with
10% Pd/C (0.05 g) and stirred under a hydrogen atmosphere (balloon)
for 4 h. The catalyst was removed by filtration and the filtrate
was concentrated under reduced pressure to yield 24.8 (0.125 g,
98%) as a white solid.
[1861] Diphenyl ester 24.9: To a solution of compound 24.8 (0.12 g,
0.195 mmol) in pyridine (1 mL) was added phenol (0.19 g, 2 mmol)
and 1,3-dicyclohexylcarbodiimide (0.206 g, 1 mmol). The mixture was
stirred at 70.degree. C. for 2 h, and then concentrated under
reduced pressure. The residue was partitioned between ethyl acetate
and HCl (0.2N). The organic phase was dried over magnesium sulfate,
filtered and concentrated. The residue was purified by
chromatography on silica gel to afford compound 24.9 (0.038 g,
25%).
[1862] Mono lactate 24.11: Compound 24.9 was converted, via
compound 24.10, into compound 24.11 in 36% yield according to the
procedure described in Scheme 23 for the preparation of 23.9 except
utilizing the ethyl lactate ester in place of L-alanine ethyl
ester. .sup.1H NMR (CDCl3): .delta. 1.05 (t, J=8 Hz, 1.5H), 1.1 (t,
J=8 Hz, 1.5H), 1.45 (d, J=8 Hz, 1.5H), 1.55 (d, J=8 Hz, 1.5H), 2.6
(brs, 2H), 2.9-3.1 (m, 6H), 3.5-3.65 (m, 4H), 4.15-4.25 (m, 2H),
4.4-4.62 (m, 2H), 4.9 (m, 2H), 5.2 (m, 1H), 6.9-7.4 (m, 24H).
.sup.31P NMR (CDCl3): d 17.6, 15.5. MS: 793 (M+1). ##STR595##
[1863] Dibenzyl ether 25.1: The protection reaction of compound
2.10 with benzyl bromide was carried out in the same manner as
described in Scheme 23 to afford compound 25.1.
[1864] Bis indazole 25.2: The alkylation of compound 25.1 with
bromide 25.9 was carried out in the same manner as described in
Scheme 23 to afford compound 25.2 in 96% yield.
[1865] Diol 25.3: A solution of 25.2 (0.18 g, 0.178 mmol) in ethyl
acetate (5 mL)) and isopropyl alcohol (5 mL) was treated with 20%
Pd(OH).sub.2/C (0.09 g) and stirred under a hydrogen atmosphere
(balloon) for 24 h. The catalyst was removed by filtration and the
filtrate was concentrated under reduced pressure to afford 25.3 in
quantitative yield.
[1866] Diethyl phosphonate 25.4: To a solution of compound 25.3
(0.124 g, 0.15 mmol) in acetonitrile (8 mL) and DMF (1 mL) was
added potassium tert-butoxide (0.15 mL, 1M/THF). The mixture was
stirred for 10 min. to form a clear solution. Diethyl triflate 5.3
(0.045 g, 0.15 mmol) was added to the reaction mixture. After
stirred for 0.5 h, the reaction mixture was diluted with ethyl
acetate and washed with HCl (0.1N). The organic phase was dried
over magnesium sulfate, filtered and concentrated under reduced
pressure. The residue was purified by chromatography on silica gel
to afford compound 25.4 (0.039 g, 55% (based on recovered starting
material: 0.064 g, 52%).
[1867] Bisindazole 25.6: A mixture of compound 25.4 (0.027 g),
ethanol (1.5 mL), TFA (0.6 mL) and water (0.5 mL) was stirred at
60.degree. C. for 18 h. The mixture was concentrated under reduced
pressure, and the residue was purified by HPLC to afford compound
25.6 as a TFA salt (0.014 g, 51%). .sup.1H NMR (CD3OD): .delta. 1.4
(t, J=8 Hz, 6H), 2.9 (M, 4H), 3.2 (m, 2H), 3.58 (brs, 2H), 3.65 (m,
2H), 4.25 (m, 4H), 4.42 (d, J=10 Hz, 2H), 4.85 (m, 2H), 6.75 (d,
J=9 Hz, 2H), 6.9 (m, 4H), 7.0 (d, J=9 Hz, 2H), 7.4-7.6 (m, 6H), 8.1
(brs, 2H). .sup.31P NMR (CD3OD): .delta. 20.8. MS: 769 (M+1).
[1868] Diethyl phosphonate 25.7: Compound 25.4 was converted into
compound 25.7 in 76% yield according to the procedures described in
Scheme 23 for the conversion of 23.3 into 23.5.
[1869] Bis indazole 25.8: Compound 25.7 (0.029 g) was treated in
the same manner as compound 25.4 in the preparation of 25.6 to
afford compound 25.8 as a TFA salt (0.0175 g, 59%). .sup.1H NMR
(CD3OD): .delta. 1.4 (t, J=8 Hz, 6H), 3.0 (M, 4H), 3.15 (d, J=14
Hz, 1H), 3.25 (d, J=14 Hz, 1H), 3.58 (brs, 2H), 3.65 (m, 2H), 4.25
(m, 4H), 4.42 (d, J=10 Hz, 2H), 4.85 (m, 2H), 6.9 (d, J=9 Hz, 2H),
7.0 (d, J=9 Hz, 2H), 7.1 (d, J=7 Hz, 2H), 7.2-7.6 (m, 9H), 8.1
(brs, 2H). .sup.31P NMR (CD3OD): .delta. 20.8. MS: 753 (M+1).
Preparation of Alkylating and Phosphonate Reagents ##STR596##
##STR597##
[1870] 3-cyano-4-fluoro-benzylbromide 3.9: The commercially
available 2-fluoro-4-methylbenzonitrile 50.1 (10 g, 74 mmol) was
dissolved in carbon tetrachloride (50 mL) and then treated with NBS
(16 g, 90 mmol) followed by AIBN (0.6 g, 3.7 mmol). The mixture was
stirred at 85.degree. C. for 30 min and then allowed to cool to
room temperature. The mixture was filtered and the filtrate
concentrated under reduced pressure. The residue was purified by
silica gel eluting with 5-20% ethyl acetate in hexanes to give 3.9
(8.8 g, 56%).
[1871] 4-benzyloxy benzyl chloride 3.10 is purchased from
Aldrich
[1872] Dibenzyl triflate 3.11: To a solution of dibenzyl phosphite
50.2 (100 g, 381 mmol) and formaldehyde (37% in water, 65 mL, 860
mmol) in THF (200 mL) was added TEA (5 mL, 36 mmol). The resulted
mixture was stirred for 1 h, and then concentrated under reduced
pressure. The residue was dissolved in methylene chloride and
hexane (1:1, 300 mL), dried over sodium sulfate, filtered through a
pad of silica gel (600 g) and eluted with ethyl acetate and hexane
(1:1). The filtrate was concentrated under reduced pressure. The
residue 50.3 (95 g) was dissolved in methylene chloride (800 mL),
cooled to -78.degree. C. and then charged with pyridine (53 mL, 650
mmol). To this cooled solution was slowly added
trifluoromethanesulfonic anhydride (120 g, 423 mmol). The resulted
reaction mixture was stirred and gradually warmed up to -15.degree.
C. over 1.5 h period of time. The reaction mixture was cooled down
to about -50.degree. C., diluted with hexane-ethyl acetate (2:1,
500 mL) and quenched with aqueous phosphoric acid (1M, 100 mL) at
-10.degree. C. to 0.degree. C. The mixture diluted with
hexane-ethyl acetate (2:1, 1000 mL). The organic phase was washed
with water, dried over magnesium sulfate, filtered and concentrated
under reduced pressure. The residue was purified by chromatography
on silica gel to afford dibenzyl triflate 3.11 (66 g, 41%) as a
colorless oil.
[1873] Diethyl triflate 5.3 is prepared as described in Tet Lett.
1986, 27, p 1477-1480
[1874] 3-Benzyloxybenzylbromide 6.9: To a solution of triphenyl
phosphine (15.7 g, 60 mmol) in THF (150 mL) was added a solution of
carbon tetrabromide (20 g, 60 mmol) in THF (50 mL). A precipitation
was formed and stirred for 10 min. A solution of 3-benzyloxybenzyl
alcohol 50.4 (10 g, 46.7 mmol) was added. After stirred for 1.5 h,
the reaction mixture was filtered and concentrated under reduced
pressure. The majority of triphenyl phosphine oxide was removed by
precipitation from ethyl acetate-hexane. The crude product was
purified by chromatography on silica gel and precipitation from
hexane to give the desired product 3-Benzyloxybenzylbromide 6.9 (10
g, 77%) as a white solid.
[1875] t-Butyl-3-chloromethyl benzoate 14.5: A benzene solution (15
ml) of 3-chloromethylbenzoic acid 50.5 (1 g, 5.8 mmol) was heated
at reflux, followed by the slow addition of
N,N-dimethylforamide-di-t-butylacetal (5 m). The resulting solution
was refluxed for 4 h, concentrated under reduced pressure and
purified by silica gel column to afford 14.5 (0.8 g, 60%).
[1876] Aminopropyl-diethylphosphonate 14.6 is purchased from
Acros
[1877] Aminoethyl-diethylphosphonate oxalate 14.7 is purchased from
Acros
[1878] Aminopropyl-phenol-ethyl lactate phosphonate 15.5
[1879] N--CBZ-aminopropyl diphenylphosphonate 50.8: An aqueous
sodium hydroxide solution (50 ml, of 1 N solution, 50 mmol) of
3-aminopropyl phosphonic acid 50.6 (3 g, 1.5 mmol) was reacted with
CBZ-Cl (4.1 g, 24 mmol) at room temperature overnight. The reaction
mixture was washed with methylene chloride, acidified with Dowex
50wx8-200. The resin was filtered off. The filtrate was
concentrated to dryness. The crude N--CBZ-aminopropyl phosphonic
acid 50.7 (5.8 mmol) was suspended in CH.sub.3CN (40 mL), and
reacted with thionyl chloride (5.2 g, 44 mmol) at reflux for 4 hr,
concentrated, and azeotroped with CH.sub.3CN twice. The reaction
mixture was redissolved in methylene chloride (20 mL), followed by
the addition of phenol (3.2 g, 23 mmol), was cooled to 0.degree. C.
To this 0.degree. C. cold solution was added TEA (2.3 g, 23 mmol),
and stirred at room temperature overnight. The reaction mixture was
concentrated and purified on silica gel column chromatograph to
afford 50.8 (1.5 g, 62%).
[1880] Monophenol derivative 50.9: A CH.sub.3CN solution (5 mL) of
50.8 (0.8 g, 1.88 mmol) was cooled to 0.degree. C., and treated
with 1N NaOH aqueous solution (4 mL, 4 mmol) for 2 h. The reaction
was diluted with water, extracted with ethyl acetate, acidified
with Dowex 50wx8-200. The aqueous solution was concentrated to
dryness to afford 50.9 (0.56 g, 86%).
[1881] Monolactate derivative 50.10: A DMF solution (1 mL) of crude
50.9 (0.17 g, 0.48 mmol), BOP reagent (0.43 g, 0.97 mmol), ethyl
lactate (0.12 g, 1 mmol), and DIPEA (0.31 g, 2.4 mmol) was reacted
for 4 hr at room temperature. The reaction mixture was partitioned
between methylene chloride and 5% citric acid aqueous solution. The
organic solution was separated, concentrated, and purified on
preparative TLC to give 50.10 (0.14 g, 66%).
[1882] 3-Aminopropyl lactate phosphonate 15.5: An ethyl
acetate/ethanol solution (10 mL/2 mL) of 50.10 (0.14 g, 0.31 mmol)
was hydrogenated at 1 atm in the presence of 10% Pd/C (40 mg) for 3
hr. The catalyst was filtered off. The filtrate was concentrated to
dryness to afford 15.5 (0.14 g, quantitative). NMR (CDCl.sub.3):
.delta. 8.0-8.2 (b, 3H), 7.1-7.4 (m, 5H), 4.9-5.0 (m, 1H), 4.15-4.3
(m, 2H), 3.1-3.35 (m, 2H), 2.1-2.4 (m, 4H), 1.4 (d, 3H), 1.3 (t,
3H).
[1883] Aminopropyl-phenol-ethyl alanine phosphonate 15.6: Compound
15.6 (80 mg) was prepared from the reaction of 50.9 (160 mg, 0.45
mmol) and L-alanine ethyl ester hydrochloride salt (0.11 g, 0.68
mmol) in the presence of DIPEA and BOP reagent to give 50.11,
followed by the hydrogenation in the presence of 10% Pd/C and TFA
to yield 15.6. NMR (CDCl.sub.3+.about.10% CD.sub.3OD): .delta.
8.0-8.2 (b), 7.25-7.35 (t, 2H), 7.1-7.2 (m, 3H), 4.0-4.15 (m, 2H),
3.8-4.0 (m, 1H), 3.0-3.1 (m, 2H), 1.15-1.25 (m, 6H). P NMR
(CDCl.sub.3+.about.10% CD.sub.3OD): 32.1 & 32.4 ppm.
[1884] Aminopropyl dibenzyl phosphonate 15.7:
[1885] N--BOC-3-aminopropyl phosphonic acid 50.13: A THF-1N aqueous
solution (16 mL-16 mL) of 3-aminopropyl phosphonic acid 50.12 (1 g,
7.2 mmol) was reacted with (BOC).sub.2O (1.7 g, 7.9 mmol) overnight
at room temperature. The reaction mixture was concentrated, and
partitioned between methylene chloride and water. The aqueous
solution was acidified with Dowex 50wx8-200. The resin was filtered
off. The filtrate was concentrated to give 50.13 (2.2 g, 92%).
[1886] N--BOC-3-aminopropyl dibenzyl phosphonate 50.14: A
CH.sub.3CN solution (10 mL) of 50.13 (0.15 g, 0.63 mmol), cesium
carbonate (0.61 g, 1.88 mmol), and benzyl bromide (0.24 g, 1.57
mmol) was heated at reflux overnight. The reaction mixture was
cooled to room temperature, and diluted with methylene chloride.
The white solid was filtered off, washed thoroughly with methylene
chloride. The organic phase was concentrated, and purified on
preparative TLC to give 50.14 (0.18 g, 70%). MS: 442 (M+Na).
[1887] Aminopropyl dibenzyl phosphonate 15.7: A methylene chloride
solution (1.6 mL) of 50.14 (0.18 g) was treated with TFA (0.4 mL)
for 1 hr. The reaction mixture was concentrated to dryness, and
azeotroped with CH.sub.3CN twice to afford 15.7 (0.2 g, as TFA
salt). NMR (CDCl.sub.3): .delta. 8.6 (b, 2H), 7.9 (b, 2H), 7.2-7.4
(m, 10H), 4.71-5.0 (2 abq, 4H), 3.0 (b, 2H), 1.8-2 (m, 4H). 31P NMR
(CDCl.sub.3): 32.0 ppm. F NMR (CDCl.sub.3): -76.5 ppm.
[1888] Aminomethyl diethylphosphonate 22.8 is purchased from
Acros
[1889] Bromomethyl, tetrahydropyran indazole 25.9 is prepared
according to J. Org. Chem. 1997, 62, p 5627
Activity of the CCPPI Compounds
[1890] The enzyme inhibitory potency (Ki), antiviral activity
(EC50), and cytotoxicity (CC50) of the tested compounds were
measured and demonstrated.
Biological Assays Used for the Characterization of PI Prodrugs
HIV-1 Protease Enzyme Assay (Ki)
[1891] The assay is based on the fluorimetric detection of
synthetic hexapeptide substrate cleavage by HIV-1 protease in a
defined reaction buffer as initially described by M. V. Toth and G.
R. Marshall, Int. J. Peptide Protein Res. 36, 544 (1990)
Substrate: (2-aminobenzoyl)Thr-Ile-Nle-(p-nitro)Phe-Gln-Arg
Substrate supplied by Bachem California, Inc. (Torrance, Calif.;
Cat. no. H-2992)
Enzyme: recombinant HIV-1 protease expressed in E. Coli
Enzyme supplied by Bachem California, Inc. (Torrance, Calif.; Cat.
no. H-9040)
Reaction buffer: 100 mM ammonium acetate, pH 5.3
[1892] 1 M sodium chloride [1893] 1 mM ethylendiaminetetraacetic
acid [1894] 1 mM dithiothreitol [1895] 10% dimethylsulfoxide Assay
Protocol for the Determination of Inhibition Constant Ki: [1896] 1.
Prepare series of solutions containing identical amount of the
enzyme (1 to 2.5 nM) and a tested inhibitor at different
concentrations in the reaction buffer [1897] 2. Transfer the
solutions (190 uL each) into a white 96-well plate [1898] 3.
Preincubate for 15 min at 37.degree. C. [1899] 4. Solubilize the
substrate in 100% dimethylsulfoxide at a concentration of 800
.mu.M. Start the reaction by adding 10 .mu.L of 800 .mu.M substrate
into each well (final substrate concentration of 40 .mu.M [1900] 5.
Measure the real-time reaction kinetics at 37.degree. C. by using
Gemini 96-well plate fluorimeter (Molecular Devices, Sunnyvale,
Calif.) at .lamda.(Ex)=330 nm and .lamda.(Em)=420 nm [1901] 6.
Determine initial velocities of the reactions with different
inhibitor concentrations and calculate Ki (in picomolar
concentration units) value by using EnzFitter program (Biosoft,
Cambridge, U.K.) according to an algorithm for tight-binding
competitive inhibition described by Ermolieff J., Lin X., and Tang
J., Biochemistry 36, 12364 (1997) Anti-HIV-1 Cell Culture Assay
(EC.sub.50)
[1902] The assay is based on quantification of the HIV-1-associated
cytopathic effect by a calorimetric detection of the viability of
virus-infected cells in the presence or absence of tested
inhibitors. The HIV-1-induced cell death is determined using a
metabolic substrate
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT) which is converted only by intact cells into a product with
specific absorption characteristics as described by Weislow O S,
Kiser R, Fine D L, Bader J, Shoemaker R H and Boyd M R, J. Natl.
Cancer Inst. 81, 577 (1989).
Assay Protocol for Determination of EC.sub.50:
[1903] 1. Maintain MT2 cells in RPMI-1640 medium supplemented with
5% fetal bovine serum and antibiotics. [1904] 2. Infect the cells
with the wild-type HIV-1 strain IIIB (Advanced Biotechnologies,
Columbia, Md.) for 3 hours at 37.degree. C. using the virus
inoculum corresponding to a multiplicity of infection equal to
0.01. [1905] 3. Prepare a set of solutions containing various
concentrations of the tested inhibitor by making 5-fold serial
dilutions in 96-well plate (100 .mu.L/well). Distribute the
infected cells into the 96-well plate (20,000 cells in 100
.mu.L/well). Include samples with untreated infected and untreated
mock-infected control cells. [1906] 4. Incubate the cells for 5
days at 37.degree. C. [1907] 5. Prepare XTT solution (6 ml, per
assay plate) at a concentration of 2 mg/mL in a phosphate-buffered
saline pH 7.4. Heat the solution in water-bath for 5 min at
55.degree. C. Add 50 .mu.l, of N-methylphenazonium methasulfate (5
.mu.g/mL) per 6 ml, of XTT solution. [1908] 6. Remove 100 .mu.L
media from each well on the assay plate. [1909] 7. Add 100 .mu.L of
the XTT substrate solution per well and incubate at 37.degree. C.
for 45 to 60 min in a CO.sub.2 incubator. [1910] 8. Add 20 .mu.L,
of 2% Triton X-100 per well to inactivate the virus. [1911] 9. Read
the absorbance at 450 nm with subtracting off the background
absorbance at 650 nm. [1912] 10. Plot the percentage absorbance
relative to untreated control and estimate the EC.sub.50 value as
drug concentration resulting in a 50% protection of the infected
cells. Cytotoxicity Cell Culture Assay (CC):
[1913] The assay is based on the evaluation of cytotoxic effect of
tested compounds using a metabolic substrate
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT) as described by Weislow O S, Kiser R, Fine D L, Bader J,
Shoemaker R H and Boyd M R, J. Natl. Cancer Inst. 81, 577
(1989).
Assay Protocol for Determination of CC.sub.50:
[1914] 1. Maintain MT-2 cells in RPMI-1640 medium supplemented with
5% fetal bovine serum and antibiotics. [1915] 2. Prepare a set of
solutions containing various concentrations of the tested inhibitor
by making 5-fold serial dilutions in 96-well plate (100
.mu.L/well). Distribute cells into the 96-well plate (20,000 cells
in 100 .mu.L/well). Include samples with untreated cells as a
control. [1916] 3. Incubate the cells for 5 days at 37.degree. C.
[1917] 4. Prepare XTT solution (6 ml, per assay plate) in dark at a
concentration of 2 mg/mL in a phosphate-buffered saline pH 7.4.
Heat the solution in a water-bath at 55.degree. C. for 5 min. Add
50 .mu.L, of N-methylphenazonium methasulfate (5 .mu.g/mL) per 6
ml, of XTT solution. [1918] 5. Remove 100 .mu.L, media from each
well on the assay plate and add 100 .mu.L, of the XTT substrate
solution per well. Incubate at 37.degree. C. for 45 to 60 min in a
CO.sub.2 incubator. [1919] 6. Add 20 .mu.L, of 2% Triton X-100 per
well to stop the metabolic conversion of XTT. [1920] 7. Read the
absorbance at 450 nm with subtracting off the background at 650 nm.
[1921] 8. Plot the percentage absorbance relative to untreated
control and estimate the CC50 value as drug concentration resulting
in a 50% inhibition of the cell growth. Consider the absorbance
being directly proportional to the cell growth. Resistance
Evaluation (I50V and I84 V/L90M Fold Chance)
[1922] The assay is based on the determination of a difference in
the susceptibility to a particular HIV protease inhibitor between
the wild-type HIV-1 strain and a mutant HIV-1 strain containing
specific drug resistance-associated mutation(s) in the viral
protease gene. The absolute susceptibility of each virus
(EC.sub.50) to a particular tested compound is measured by using
the XTT-based cytopathic assay as described above. The degree of
resistance to a tested compound is calculated as fold difference in
EC.sub.50 between the wild type and a specific mutant virus. This
represents a standard approach for HIV drug resistance evaluation
as documented in various publications (e.g. Maguire et al.,
Antimicrob. Agents Chemother. 46: 731, 2002; Gong et al.,
Antimicrob. Agents Chemother. 44: 2319, 2000; Vandamme and De
Clercq, in Antiviral Therapy (Ed. E. De Clercq), pp. 243, ASM
Press, Washington, D.C., 2001).
HIV-1 Strains Used for the Resistance Evaluation:
[1923] Two strains of mutant viruses containing I50V mutation in
the protease gene have been used in the resistance assays: one with
M46F/I47V/I50V mutations (designated I50V #1) and the other with
L10I/M46I/I50V (designated I50V #2) mutations in the viral protease
gene. A third virus with I84V/L90M mutations was also employed in
the resistance assays. Mutants I50V #1 and I84V/L90M were
constructed by a homologous recombination between three overlapping
DNA fragments: 1. linearized plasmid containing wild-type HIV-1
proviral DNA (strain HXB2D) with the protease and reverse
transcriptase genes deleted, 2. DNA fragment generated by PCR
amplification containing reverse transcriptase gene from HXB2D
strain (wild-type), 3. DNA fragment of mutated viral protease gene
that has been generated by PCR amplification. An approach similar
to that described by Shi and Mellors in Antimicrob. Agents
Chemother. 41: 2781-85, 1997 was used for the construction of
mutant viruses from the generated DNA fragments. Mixture of DNA
fragments was delivered into Sup-T1 cells by using a standard
electroporation technique. The cells were cultured in RPMI-1640
medium supplemented with 10% fetal bovine serum and antibiotics
until the recombinant virus emerged (usually 10 to 15 days
following the electroporation). Cell culture supernatant containing
the recombinant virus was harvested and stored in aliquots. After
verification of protease gene sequence and determination of the
infectious virus titer, the viral stock was used for drug
resistance studies. Mutant I50V #2 is an amprenavir-resistant HIV-1
strain selected in vitro from the wild-type IIIB strain in the
presence of increasing concentration of amprenavir over a period of
>9 months using an approach similar to that described by
Partaledis et al., J. Virol. 69: 5228-5235, 1995. Virus capable of
growing in the presence of 5 .mu.M amprenavir was harvested from
the supernatant of infected cells and used for resistance assays
following the titration and protease gene sequencing.
Example 37
Activity of the Tested Compounds
[1924] The enzyme inhibitory potency (Ki), antiviral activity
(EC50), and cytotoxicity (CC50) of the tested compounds are
summarized in Table 1. TABLE-US-00013 TABLE 1 ##STR598## Enzyme
inhibition activity (Ki), antiviral cell culture activity (EC50),
and cytotoxicity (CC50) of the tested compounds. Substi- HIV-1
Anti-HIV-1 tution Phos- protease Cell Culture Cyto- of phonate
inhibition Activity toxicity (P1) Com- substi- Ki EC50 CC50 phenyl
pound tution [pM] [nM] [.mu.M] none Ampre- none 45.6 .+-. 18.2 16
.+-. 2.2 navir none 94-003 none 1.46 .+-. 0.58 1.4 .+-. 0.3 phos-
27 diacid 11.8 .+-. 6.0 >100,000 >100 phonyl 28 diethyl 1.2
.+-. 0.8 5.0 .+-. 2.8 70 phos- 11 diacid 2.1 .+-. 0.2 4,800 .+-.
1,800 >100 phonyl methoxy 13 diethyl 2.6 .+-. 1.5 3.0 .+-. 0 50
14 dibenzyl 12.7 .+-. 1.9 2.3 .+-. 0.4 35 .sup. 16c bis(Ala- 15.4
.+-. 0.85 105 .+-. 43 60 ethylester) 16d bis(Ala- 18.75 .+-. 3.04
6.0 .+-. 1.4 butylester) .sup. 16e bis(ABA- 8.8 .+-. 1.7 12.5 .+-.
3.5 ethylester) 16f bis(ABA- 3.5 .+-. 1.4 4.8 .+-. 1.8 butylester)
.sup. 16a bis(Gly- 29 .+-. 8.2 330 .+-. 230 ethylester) 16b
bis(Gly- 4.9 .+-. 1.8 17.5 .+-. 10.5 butylester) 16g bis(Leu- 29
.+-. 9 6.8 .+-. 0.4 ethylester) 16h bis(Leu- 31.7 .+-. 19.3 120
.+-. 42 butylester) 16i bis(Phe- 17 .+-. 12 ethylester) 16j
bis(Phe- 35 .+-. 7 butylester 15 bis(POC) 36 825 .+-. 106 11 Mono-
0.45 .+-. 0.15 700 .+-. 0 ethyl, monoacid
Cross-Resistance Profile Assay
[1925] The assay is based on the determination of a difference in
the susceptibility to a particular HIV protease inhibitor between
the wild-type HIV-1 strain and a recombinant HIV-1 strain
expressing specific drug resistance-associated mutation(s) in the
viral protease gene. The absolute susceptibility of each virus to a
particular tested compound is measured by using the XTT-based
cytopathic assay as described in Example B. The degree of
resistance to a tested compound is calculated as fold difference in
EC50 between the wild type and a specific mutant virus.
Recombinant HIV-1 Strains with Resistance Mutations in the Protease
Gene:
[1926] One mutant virus (82T/84V) was obtained from NIH AIDS
Research and Reference Reagent Program (Rockville, Md.). Majority
of the mutant HIV-1 strains were constructed by a homologous
recombination between three overlapping DNA fragments: 1.
linearized plasmid containing wild-type HIV-1 proviral DNA (strain
HXB2D) with the protease and reverse transcriptase genes deleted,
2. DNA fragment generated by PCR amplification containing reverse
transcriptase gene from HXB2D strain (wild-type), 3. DNA fragment
generated by RT-PCR amplification from patients plasma samples
containing viral protease gene with specific mutations selected
during antiretroviral therapy with various protease inhibitors.
Additional mutant HIV-1 strains were constructed by a modified
procedure relying on a homologous recombination of only two
overlapping DNA fragments: 1. linearized plasmid containing
wild-type HIV-1 proviral DNA (strain HXB2D) with only the protease
gene deleted, and 2. DNA fragment generated by RT-PCR amplification
from patients plasma samples containing viral protease gene with
specific mutations. In both cases, mixture of DNA fragments was
delivered into Sup-T1 cells by using a standard electroporation
technique. The cells were cultured in RPMI-1640 medium supplemented
with 10% fetal bovine serum and antibiotics until the recombinant
virus emerged (usually 10 to 15 days following the
electroporation). Cell culture supernatant containing the
recombinant virus was harvested and stored in aliquots. After
determination of the virus titer the virus stock was used for drug
resistance studies.
Example 39
Cross-Resistance Profile of the Tested Compounds
[1927] Cross-resistance profile of currently used HIV-1 protease
inhibitors was compared with that of the newly invented compounds
(Table 2). TABLE-US-00014 TABLE 2 Cross-resistance profile of HIV-1
protease inhibitors Fold Change in EC.sub.50 Relative to WT HIV-1
10F 10I 10I EC50 10I 10R 30N 46I 48V 48V 84V Total [nM] 8K.sup.a
48V 46I 46I 50S 54V 71V 71V 54V 71V No. of WT 46I 46I 54V 47V 82T
82I 71V 82T 82A 71V 73S Resistant Compound HIV-1 90M 84A 82A 50V
84V 88D 82S 90M 90M 82S 90M Viruses.sup.b Amprenavir 20 1.25 14 2
38 4 0.8 4 13 2.5 2 10 4 Nelfinavir 14 13 11 11.5 2 3 43 12 33 27
12 65 9 Indinavir 15 4 10 15 nd 7 1 10 13 28 23 43 8 Ritonavir 15
34 18 20 13 47 2 20 32 22 >50 42 10 Saquinavir 4 1 2.5 11 1 2.5
1 3 2.5 12 45 40 4 Lopinavir 8 nd 9 nd 19 11 nd nd 7.5 4.5 60 11 6
Tipranavir 80 nd 1 0.4 0.5 5 0.5 3.5 3 0.3 2 nd 1 94-003 0.5 nd 8
0.5 29 nd 0.4 3.5 nd nd nd 8 3 GS 16503 16 1.2 1 0.4 3.3 1 0.6 0.9
1 0.4 0.5 2 0 GS 16571 22 1.8 1 0.3 0.8 0.6 0.7 0.6 0.8 0.2 0.2 0.9
0 GS 16587 15 1.5 1 0.5 2 1 1 0.9 1 0.4 0.4 1 0
.sup.aResistance-associated mutations present in the viral
protease. The highlighted changes represent primary resistance
mutations. .sup.bResistance is considered as a 5-fold and higher
change in the EC50 value of the mutant virus relative to the
wild-type virus.
Example Section N
Plasma and PBMC Exposure Following Intravenous and Oral
Administration of Prodrug to Beagle Dogs
[1928] The pharmacokinetics of a phosphonate prodrug GS77366
(P1-monoLac-iPr), its active metabolite (metabolite X, or GS77568),
and GS8373 were studied in dogs following intravenous and oral
administration of the prodrug.
[1929] Dose Administration and Sample Collection. The in-life phase
of this study was conducted in accordance with the USDA Animal
Welfare Act and the Public Health Service Policy on Humane Care and
Use of Laboratory Animals, and followed the standards for animal
husbandry and care found in the Guide for the Care and Use of
Laboratory Animals, 7.sup.th Edition, Revised 1996. All animal
housing and study procedures involving live animals were carried
out at a facility which had been accredited by the Association for
Assessment and Accreditation of Laboratory Animal
Care--International (AAALAC).
[1930] Each animal in a group of 4 female beagle dogs was given a
bolus dose of GS77366 (P1-monoLac-iPr) intravenously at 1 mg/kg in
a formulation containing 40% PEG 300, 20% propylene glycol and 40%
of 5% dextrose. Another group of 4 female beagle dogs was dosed
with GS77366 via oral gavage at 20 mg/kg in a formulation
containing 60% Vitamin-E TPGS, 30% PEG 400 and 10% propylene
glycol.
[1931] Blood samples were collected pre-dose, and at 5 min, 15 min,
30 min, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr and 24 hr post-dose. Plasma
(0.5 to 1 mL) was prepared from each sample and kept at -70.degree.
C. until analysis. Blood samples (8 mL) were also collected from
each dog at 2, 8 and 24 hr post dose in Becton-Dickinson CPT
vacutainer tubes. PBMCs were isolated from the blood by
centrifugation for 15 minutes at 1500 to 1800 G. After
centrifugation, the fraction containing PBMCs was transferred to a
15 ml, conical centrifuge tube and the PBMCs were washed twice with
phosphate buffered saline (PBS) without Ca.sup.2+ and Mg.sup.2+.
The final wash of the cell pellet was kept at -70.degree. C. until
analysis.
[1932] Measurement of the prodrug, metabolite X and GS8373 in
plasma and PBMCs. For plasma sample analysis, the samples were
processed by a solid phase extraction (SPE) procedure outlined
below. Speedisk C18 solid phase extraction cartridges (1 mL, 20 mg,
10 .mu.M, from J. T. Baker) were conditioned with 200 .mu.L of
methanol followed by 200 .mu.L of water. An aliquot of 200 .mu.L of
plasma sample was applied to each cartridge, followed by two
washing steps each with 200 .mu.L of deionized water. The compounds
were eluted from the cartridges with a two-step process each with
125 .mu.L of methanol. Each well was added 50 .mu.L of water and
mixed. An aliquot of 25 .mu.L of the mixture was injected onto a
ThermoFinnigan TSQ Quantum LC/MS/MS system.
[1933] The column used in liquid chromatography was HyPURITY.RTM.
C18 (50.times.2.1 mm, 3.5 um) from Thermo-Hypersil. Mobile phase A
contained 10% acetonitrile in 10 mM ammonium formate, pH 3.0.
Mobile phase B contained 90% acetonitrile in 10 mM ammonium
formate, pH 4.6. The chromatography was carried out at a flow rate
of 250 .mu.L/min under an isocratic condition of 40% mobile phase A
and 60% mobile phase B. Selected reaction monitoring (SRM) were
used to measure GS77366, GS8373 and Metabolite X with the positive
ionization mode on the electrospray probe. The limit of
quantitation (LOQ) was 1 nM for GS77366, GS8373 and GS77568
(Metabolite X) in plasma.
[1934] For PBMC sample analysis, phosphate buffered saline (PBS)
was added to each PBMC pellet to bring the total sample volume to
500 .mu.L in each sample. An aliquot of 150 .mu.L from each PBMC
sample was mixed with an equal volume of methanol, followed by the
addition of 700 .mu.L of 1% formic acid in water. The resulting
mixture was applied to a Speedisk C18 solid phase extraction
cartridge (1 mL, 20 mg, 10 um, from J. T. Baker) which had been
conditioned as described above. The compounds were eluted with
methanol after washing the cartridge 3 times with 10% methanol. The
solvent was evaporated under a stream of N.sub.2, and the sample
was reconstituted in 150 .mu.L of 30% methanol. An aliquot of 75
.mu.L of the solution was injected for LC/MS/MS analysis. The limit
of quantitation was 0.1 ng/mL in the PBMC suspension.
[1935] Pharmacokinetic Calculations. The pharmacokinetic parameters
were calculated using Winonlin. Noncompartmental analysis was used
for all pharmacokinetic calculation. The intracellular
concentrations in PBMCs were calculated from the measured
concentrations in PBMC suspension on the basis of a reported volume
of 0.2 picoliter/cell (B. L. Robins, R. V. Srinivas, C. Kim, N.
Bischofberger, and A. Fridland, (1998) Antimicrob. Agents
Chemother. 42, 612).
Plasma and PBMC Concentration-Time Profiles.
[1936] The concentration-time profiles of GS77366, GS77568 and
GS8373 in plasma and PBMCs following intravenous dosing of GS77366
were compared at 1 mg/kg in dogs. The data demonstrate that the
prodrug can effectively deliver the active components (metabolite X
and GS8373) into cells that are primarily responsible for HIV
replication, and that the active components in these cells had much
longer half-life than in plasma.
[1937] The pharmacokinetic properties of GS77568 in PBMCs following
oral administration of GS77366 in dogs are compared with that of
nelfinavir and amprenavir, two marketed HIV protease inhibitors
(Table 3). These data show that the active component (GS77568) from
the phosphonate prodrug had sustained levels in PBMCs compared to
nelfinavir and amprenavir. TABLE-US-00015 TABLE 3 Comparison of
GS77568 with nelfinavir and amprenavir in PBMCs following oral
administration in beagle dogs. Compound Dose t.sub.1/2 (hr)
AUC.sub.(2-24 hr) Nelfinavir 17.5 mg/kg 3.0 hr 33,000 nM hr
Amprenavir 20 mg/kg 1.7 hr 102,000 nM hr GS77568 20 mg/kg of
GS77366 >20 hr 42,200 nM br
Example Section O
Intracellular Metabolism/In Vitro Stability
1. Uptake and Persistence in MT2 Cells, Quiescent and Stimulated
PBMC
[1938] The protease inhibitor (PI) phosphonate prodrugs undergo
rapid cell uptake and metabolism to produce acid metabolites
including the parent phosphonic acid. Due to the presence of
charges, the acid metabolites are significantly more persistent in
the cells than non-charged PI's. In order to estimate the relative
intracellular levels of the different PI prodrugs, three compounds
representative of three classes of phosphonate PI
prodrugs--bisamidate phosphonate, monoamidate phenoxy phosphonate
and monolactate phenoxy phosphonate (FIG. 1) were incubated at 10
.mu.M for 1 hr with MT-2 cells, stimulated and quiescent peripheral
blood mononuclear cells (PBMC) (pulse phase). After incubation, the
cells were washed, resuspended in the cell culture media and
incubated for 24 hr (chase phase). At specific time points, the
cells were washed, lysed and the lysates were analyzed by HPLC with
UV detection. Typically, the cell lysates were centrifuged and 100
uL of the supernatant were mixed with 200 .mu.L of 7.5 uM
amprenavir (Internal Standard) in 80% acetonitrile/20% water and
injected into an HPLC system (70 .mu.L).
HPLC Conditions:
Analytical Column: Prodigy ODS-3, 75.times.4.6, 3 u+C18 guard at
40.degree. C.
Gradient:
Mobile Phase A: 20 mM ammonium acetate in 10% ACN/90% H.sub.2O
Mobile Phase B: 20 mM ammonium acetate in 70% ACN/30% H.sub.2O
30-100% B in 4 min, 100% B for 2 min, 30% B for 2 min at 2.5
mL/min.
Run Time: 8 min
UV Detection at 245 nm
Concentrations of Intracellular metabolites were calculated based
on cell volume 0.2 .mu.L/mLn cells for PBMC and 0.338 .mu.L/mLn
(0.676 uL/mL) for MT-2 cells.
[1939] Chemical Structures of Selected Protease Inhibitor
Phosphonate Prodrugs and Intracellular Metabolites: TABLE-US-00016
TABLE 4 ##STR599## GS EC.sub.50 No. R1 R2 (nM) 8373 OH OH 4,800
.+-. 1,800 16503 HNCH(CH.sub.3)COOBu HNCH(CH.sub.3)COOBu 6.0 .+-.
1.4 16571 OPh HNCH(CH.sub.3)COOEt 15 .+-. 5 17394 OPh
OCH(CH.sub.3)COOEt 20 .+-. 7 16576 OPh HNCH(CH.sub.2CH.sub.3)COOEt
12.6 .+-. 4.8 Met X OH HNCH(CH.sub.3)COOH >10,000 Met LX OH
OCH(CH.sub.3)COOEt 1750 .+-. 354
[1940] A significant uptake and conversion of all 3 compounds in
all cell types was observed (Table 4). the uptake in the quiescent
PBMC was 2-3-fold greater than in the stimulated cells. GS-16503
and GS-16571 were metabolized to Metabolite X and GS-8373. GS-17394
metabolized to the Metabolite LX. Apparent intracellular half-lives
were similar for all metabolites in all cell types (7-12 hr). A
persistence of Total Acid Metabolites of Protease Inhibitor
Prodrugs in Stimulated (A), Quiescent PBMC (13) and MT-2 Cells (C)
(1 hr, 10 uM Pulse, 24 hr Chase) was observed.
2. Uptake and Persistence in Stimulated and Quiescent T-Cells
[1941] Since HIV mainly targets T-lymphocytes, it is important to
establish the uptake, metabolism and persistence of the metabolites
in the human T-cells. In order to estimate the relative
intracellular levels of the different PI prodrugs, GS-16503, 16571
and 17394 were incubated at 10 .mu.M for 1 hr with quiescent and
stimulated T-cells (pulse phase). The prodrugs were compared with a
non-prodrug PI, nelfinavir. After incubation, the cells were
washed, resuspended in the cell culture media and incubated for 4
hr (chase phase). At specific time points, the cells were washed,
lysed and the lysates were analyzed by HPLC with UV detection. The
sample preparation and analysis were similar to the ones described
for MT-2 cells, quiescent and stimulated PBMC.
[1942] Table 5 demonstrate the levels of total acid metabolites and
corresponding prodrugs in T-cells following pulse/chase and
continuous incubation. There was significant cell uptake/metabolism
in T-lymphocytes. There was no apparent difference in uptake
between stimulated and quiescent T-lymphocytes. There was
significantly higher uptake of phosphonate PI's than nelfinavir.
GS17394 demonstrates higher intracellular levels than GS16571 and
GS16503. The degree of conversion to acid metabolites varied
between different prodrugs. GS-17394 demonstrated the highest
degree of conversion, followed by GS-16503 and GS-16571. The
metabolites, generally, were an equal mixture of the
mono-phosphonic acid metabolite and GS-8373 except for GS-17394,
where Metabolite LX was stable, with no GS-8373 formed.
TABLE-US-00017 TABLE 5 Intracellular Levels of Metabolites and
Intact Prodrug Following Continuous and 1 hr Pulse/4 hr Chase
Incubation (10 .mu.M/0.7 mLn cells/1 mL) of 10 .mu.M PI Prodrugs
and Nelfinavir with Quiescent and Stimulated T-cell Continuous
Incubation 1 hr Pulse/4 hr Chase Quiescent Stimulated Quiescent
Stimulated T-cells T-cells T-cells T-cells Time Acid Met Prodrug
Acid Met Prodrug Acid Met Prodrug Acid Met Prodrug Compound (h)
(.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M)
16503 0 1180 42 2278 0 2989 40 1323 139 2 3170 88 1083 116 1867 4
1137 31 4 5262 0 3198 31 1054 119 1008 0 16571 0 388 1392 187 1417
1042 181 858 218 2 947 841 1895 807 1170 82 1006 35 4 3518 464 6147
474 1176 37 616 25 17394 0 948 1155 186 1194 4480 14 2818 10 2 7231
413 3748 471 2898 33 1083 51 4 10153 167 3867 228 1548 39 943 104
Nelfinavir 0 101 86 886 1239 2 856 846 725 770 4 992 1526 171
544
3. PBMC Uptake and Metabolism of Selected PI Prodrugs Following
1-hr Incubation in MT-2 Cells at 10, 5 and 1 .mu.M.
[1943] To were similar to the determine if the cell
uptake/metabolism is concentration dependent, selected PI's were
incubated with the 1 ml, of MT-2 cell suspension (2.74 mLn
cells/mL) for 1 hr at 37.degree. C. at 3 different concentrations:
10, 5 and 1 .mu.M. Following incubation, cells were washed twice
with the cell culture medium, lysed and assayed using HPLC with UV
detection. The sample preparation and analysis ones described for
MT-2 cells, quiescent and stimulated PBMC. Intracellular
concentrations were calculated based on cell count, a published
single cell volume of 0.338 pl for MT-2 cells, and concentrations
of analytes in cell lysates. Data are shown in Table 6.
[1944] Uptake of all three selected PI's in MT-2 cells appears to
be concentration-independent in the 1-10 .mu.M range. Metabolism
(conversion to acid metabolites) appeared to be
concentration-dependent for GS-16503 and GS-16577 (3-fold increase
at 1 .mu.M vs. 10 .mu.M) but independent for GS-17394
(monolactate). Conversion from a respective metabolite X to GS-8373
was concentration-independent for both GS-16503 and GS-16577 (no
conversion was observed for metabolite LX of GS-173394).
TABLE-US-00018 TABLE 6 Uptake and Metabolism of Selected PI
Prodrugs Following 1-hr Incubation in MT-2 Cells at 10, 5 and 1
.mu.M. Cell-Assosiated % Prodrug and Metabolites Conversion
Extracellular Concentration, .mu.M to acid Compound Concentration,
.mu.M Metabolite X GS8373 Prodrug Total metabolites GS-17394 10
1358 0 635 1993 68 5 916 0 449 1365 67 1 196 0 63 260 76 GS-16576
10 478 238 2519 3235 22 5 250 148 621 1043 40 1 65 36 61 168 64
GS-16503 10 120 86 1506 1712 12 5 58 60 579 697 17 1 12 18 74 104
29 * For GS16576, Metabolite X is mono-aminobutyric acid
4. PBMC Uptake and Metabolism of Selected PI Prodrugs Following
1-hr Incubation in Human Whole Blood at 10 .mu.M.
[1945] In order to estimate the relative intracellular levels of
the different PI prodrugs under conditions simulating the in vivo
environment, compounds representative of three classes of
phosphonate PI prodrugs--bisamidate phosphonate (GS-16503),
monoamidate phenoxy phosphonate (GS-16571) and monolactate phenoxy
phosphonate (GS-17394) were incubated at 10 .mu.M for 1 hr with
intact human whole blood at 37.degree. C. After incubation, PBMC
were isolated, then lysed and the lysates were analyzed by HPLC
with UV detection. The results of analysis are shown in Table 7.
There was significant cell uptake/metabolism following incubation
in whole blood. There was no apparent difference in uptake between
GS-16503 and GS-16571. GS-17394 demonstrated significantly higher
intracellular levels than GS-16571 and GS-16503.
[1946] The degree of conversion to acid metabolites varies between
different prodrugs after 1 hr incubation. GS-17394 demonstrated the
highest degree of conversion, followed by GS-16503 and GS-16571
(Table 7). The metabolites, generally, were an equimolar mixture of
the mono-phosphonic acid metabolite and GS-8373 (parent acid)
except for GS-17394, where Metabolite LX was stable with no GS-8373
formed. TABLE-US-00019 TABLE 7 PBMC Uptake and Metabolism of
Selected PI Prodrugs Following 1-hr Incubation in Human Whole Blood
at 10 .mu.M (Mean .+-. SD, N = 3). Intracellular Prodrug and Major
Metabolites Concentration, .mu.M Intracellular GS# Acid Metabolite
Prodrug, .mu.M Total, .mu.M Metabolites 16503 279 .+-. 47 61 .+-.
40 340 .+-. 35 X, GS-8373 16571 319 .+-. 112 137 .+-. 62 432 .+-.
208 X, GS-8373 17394 629 .+-. 303 69 .+-. 85 698 .+-. 301 LX * PBMC
Intracellular Volume = 0.2 .mu.L/mln
5. Distribution of PI Prodrugs in PBMC
[1947] In order to compare distribution and persistence of PI
phosphonate prodrugs with those of non-prodrug PI's, GS-16503,
GS-17394 and nelfinavir, were incubated at 10 .mu.M for 1 hr with
PBMC (pulse phase). After incubation, the cells were washed,
resuspended in the cell culture media and incubated for 20 more hr
(chase phase). At specific time points, the cells were washed and
lysed. The cell cytosol was separated from membranes by
centrifugation at 9000.times.g. Both cytosol and membranes were
extracted with acetonitrile and analyzed by HPLC with UV
detection.
[1948] Table 8 shows the levels of total acid metabolites and
corresponding prodrugs in the cytosol and membranes before and
after the 22 hr chase. Both prodrugs exhibited complete conversion
to the acid metabolites (GS-8373 and X for GS-16503 and LX for
GS-17394, respectively). The levels of the acid metabolites of the
PI phosphonate prodrugs in the cytosol fraction were 2-3-fold
greater than those in the membrane fraction after the 1 hr pulse
and 10-fold greater after the 22 hr chase. Nelfinavir was present
only in the membrane fractions. The uptake of GS-17394 was about
3-fold greater than that of GS-16503 and 30-fold greater than
nelfinavir. The metabolites were an equimolar mixture of metabolite
X and GS-8373 (parent acid) for GS-16503 and only metabolite LX for
GS-17394. TABLE-US-00020 TABLE 8 Uptake and Cell Distribution of
Metabolites and Intact Prodrugs Following Continuous and 1 hr
Pulse/22 hr Chase Incubation of 10 .mu.M PI Prodrugs and Nelfinavir
with Quiescent PBMC. Cell-Associated PI, pmol/mln cells 1 hr Pulse/
1 hr Pulse/ 0 hr Chase 22 hr Chase Acid Acid Cell Metabo- Metabo-
GS# Type Fraction lites Prodrug lites Prodrug GS-16503 PBMC
Membrane 228 0 9 0 GS-16503 PBMC Cytosol 390 0 130 0 GS-17394 PBMC
Membrane 335 0 26 0 GS-17394 PBMC Cytosol 894 0 249 0 Nelfinavir
PBMC Membrane 42 25 Nelfinavir PBMC Cytosol 0 0
[1949] Uptake and cell distribution of metabolites and intact
prodrugs following 1 hr pulse/22 hr chase incubation of 10 .mu.M PI
prodrugs and Nelfinavir with quiescent PBMC were measured.
6. PBMC Extract/Dog Plasma/Human Serum Stability of Selected PI
Prodrugs
[1950] The in vitro metabolism and stability of the PI phosphonate
prodrugs were determined in PBMC extract, dog plasma and human
serum (Table 9). Biological samples listed below (120 .mu.L) were
transferred into an 8-tube strip placed in the aluminum 37.degree.
C. heating block/holder and incubated at 37.degree. C. for 5 min.
Aliquots (2.5 .mu.L) of solution containing 1 mM of test compounds
in DMSO, were transferred to a clean 8-tube strip, placed in the
aluminum 37.degree. C. heating block/holder. 60 .mu.L aliquots of
80% acetonitrile/20% water containing 7.5 .mu.M of amprenavir as an
internal standard for HPLC analysis were placed into five 8-tube
strips and kept on ice/refrigerated prior to use. An enzymatic
reaction was started by adding 120 .mu.L aliquots of a biological
sample to the strip with the test compounds using a multichannel
pipet. The strip was immediately vortex-mixed and the reaction
mixture (20 .mu.L) was sampled and transferred to the Internal
Standard/ACN strip. The sample was considered the time-zero sample
(actual time was 1-2 min). Then, at specific time points, the
reaction mixture (20 .mu.L) was sampled and transferred to the
corresponding IS/ACN strip. Typical sampling times were 6, 20, 60
and 120 min. When all time points were sampled, an 80 .mu.L aliquot
of water was added to each tube and strips were centrifuged for 30
min at 3000.times.G. The supernatants were analyzed with HPLC under
the following conditions:
Column: Inertsil ODS-3, 75.times.4.6 mm, 3 .mu.m at 40.degree.
C.
Mobile Phase A: 20 mM ammonium acetate in 100% ACN/90% water
Mobile Phase B 20 mM ammonium acetate in 70% ACN/30% water
Gradient: 20% B to 100% B in 4 min, 2 min 100% B, 2 min 20% B
Flow Rate: 2 mL/min
Detection: UV at 243 nm
Run Time: 8 min
[1951] The biological samples evaluated were as follows:
[1952] PBMC cell extract was prepared from fresh cells using a
modified published procedure (A. Pompon, I. Lefebvre, J-L. Imbach,
S. Kahn, and D. Farquhar, Antiviral Chemistry & Chemotherapy,
5, 91-98 (1994)). Briefly, the extract was prepared as following:
The cells were separated from their culture medium by
centrifugation (1000 g, 15 min, ambient temperature). The residue
(about 100 .mu.L, 3.5.times.10.sup.8 cells) was resuspended in 4
ml, of a buffer (0.010 M HEPES, pH 7.4, 50 mM potassium chloride, 5
mM magnesium chloride and 5 mM dl-dithiothreitol) and sonicated.
The lysate was centrifuged (9000 g, 10 min, 4.degree. C.) to remove
membranes. The upper layer (0.5 mg protein/mL) was stored at
-70.degree. C. The reaction mixture contained the cell extract at
about 0.5 mg protein/mL.
[1953] Human serum (pooled normal human serum from George King
Biomedical Systems, Inc.). Protein concentration in the reaction
mixture was about 60 mg protein/mL.
[1954] Dog Plasma (pooled normal dog plasma (EDTA) from Pel Freez,
Inc.). Protein concentration in the reaction mixture was about 60
mg protein/mL. TABLE-US-00021 TABLE 9 PBMC Extract/Dog Plasma/Human
Serum Stability of Selected PI Prodrugs PBMC Dog Human
Extract.sup.1 Plasma Serum HIV EC.sub.50 GS# T.sub.1/2, min
T.sub.1/2, min T.sub.1/2, min (nM) 16503 2 368 >>400 6.0 .+-.
1.4 16571 49 126 110 15 .+-. 5 17394 15 144 49 20 .+-. 7
Example Section P
[1955] TABLE-US-00022 TABLE 10 Enzymatic and Cellular data Formula
II ALPPI activity ##STR600## ##STR601## Ki [pM] .ltoreq.10 +++
>10 to .ltoreq.100 ++ >100 to .ltoreq.1,000 + >1,000 -
EC.sub.50 [nM] .ltoreq.50 +++ >50 to .ltoreq.500 ++ >500 to
.ltoreq.5,000 + >5,000 - I50V and I84V/L90M fold change >30
+++ >10 to .ltoreq.30 ++ >3 to .ltoreq.10 + .ltoreq.3 -
CC.sub.50 [.mu.M] .ltoreq.5 ++ >5 to .ltoreq.50 + >50 - I50V
I50V (#1) (#2) I84V/L90M Ki EC.sub.50 fold fold fold CC.sub.50
Compound (pM) (nM) change change change (.mu.M) Saquinavir ++ +++ -
- +++ Nelfinavir + +++ - + +++ Indinavir + +++ - + +++ Ritonavir ++
+++ ++ ++ +++ Lopinavir ++ +++ ++ +++ ++ Amprenavir + +++ +++ +++
++ - Atazanavir ++ +++ - - +++ Tipranavir ++ ++ - - + 94-003 +++
+++ +++ +++ ++ + TMC114 +++ +++ ++ ++ -
[1956] TABLE-US-00023 P1-Phosphonic acid and esters ##STR602## I50V
(#1) I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1 R2 (pM) (nM)
change change (.mu.M) OH OH +++ + - - - OMe OMe ++ +++ OEt OEt +++
+++ - - + OCH.sub.2CF.sub.3 OCH.sub.2CF.sub.3 ++ - OiPr OiPr ++ +++
- - OPh OPh +++ OMe OPh ++ +++ OEt OPh +++ +++ OBn OBn ++ +++ - - +
OEt OBn ++ +++ ++ OPoc OPoc + OH OEt ++ OH OPh +++ - OH OBn + -
-
[1957] TABLE-US-00024 P1-Phosphonic acid and esters ##STR603## I50V
(#1) I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1 R2 (pM) (nM)
change change (.mu.M) OH OH +++ + Et Et +++ +++
[1958] TABLE-US-00025 P1-Direct phosphonic acid and esters
##STR604## I50V (#1) I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1
R2 (pM) (nM) change change (.mu.M) OH OH ++ - OEt OEt +++ +++ +
-
[1959] TABLE-US-00026 P1-CH.sub.2-phosphonic acid and esters
##STR605## I50V (#1) I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1
R2 (pM) (nM) change change (.mu.M) OE OE +++ +++ + +
[1960] TABLE-US-00027 P1-P-Bisamidates ##STR606## I50V (#1)
I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1 R2 (pM) (nM) change
change (.mu.M) NHEt NHEt +++ ++ - - Gly-Et Gly-Et ++ ++ Gly-Bu
Gly-Bu +++ +++ Ala-Et Ala-Et ++ ++ - - Ala-Bu Ala-Bu ++ +++ + -
Aba-Et Aba-Et +++ +++ Aba-Bu Aba-Bu +++ +++ ++ + Val-Et Val-Et +
+++ - - Leu-Et Leu-Et ++ +++ Leu-Bu Leu-Bu ++ ++ + + Phe-Et Phe-Et
+++ Phe-Bu Phe-Bu +++
[1961] TABLE-US-00028 P1-P-Bislactates ##STR607## I50V (#1)
I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1 R2 (pM) (nM) change
change (.mu.M) Glc-Et Glc-Et +++ + - - Lac-Et Lac-Et ++ ++ - -
Lac-iPr Lac-iPr ++ +++ -
[1962] TABLE-US-00029 P1-P-Monoamidates ##STR608## I50V (#1)
I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1 R2 (pM) (nM) change
change (.mu.M) OPh Gly-Bu ++ ++ - - OPh Ala-Me ++ +++ - OPh Ala-Et
+++ +++ - - OPh Ala-iPr ++ +++ - - OPh Ala-iPr +++ +++ OPh Ala-iPr
++ +++ OPh (D)Ala-iPr ++ +++ - OPh (D)Ala-iPr +++ +++ OPh
(D)Ala-iPr +++ +++ OPh Ala-Bu ++ +++ - - OPh Ala-Bu ++ +++ - OPh
Ala-Bu ++ +++ - OPh Aba-Et +++ OPh Aba-Et +++ - - OPh Aba-Et ++ OPh
Aba-Bu +++ + - OPh Aba-Bu ++ - - OBn Ala-Et +++ +++ - - OH Ala-OH
+++ - OH Ala-Bu -
[1963] TABLE-US-00030 P1-P-Monolactates (1) ##STR609## I50V I50V
(#1) (#2) I84V/L90M Ki EC.sub.50 fold fold fold CC.sub.50 R1 R2
(pM) (nM) change change change (.mu.M) OPh Glc-Et +++ +++ - - OPh
Lac-Me ++ - OPh Lac-Et +++ - + - + OPh Lac-Et +++ +++ - - OPh
Lac-Et ++ +++ - - OPh Lac-iPr ++ +++ - - OPh Lac-iPr +++ +++ OPh
Lac-iPr ++ +++ OPh Lac-Bu ++ ++ - OPh Lac-Bu ++ ++ OPh Lac-Bu ++ ++
OPh Lac-EtMor - OPh Lac-PrMor - OPh (R)Lac-Me +++ +++ OPh (R)Lac-Et
+++ +++ - - OEt Lac-Et ++ OCH.sub.2CF.sub.3 Lac-Et ++ OBn Lac-Bn ++
++ OBn (R)Lac-Bn OH Lac-OH +++ + - OH (R)Lac-OH ++ + -
[1964] TABLE-US-00031 P1-P-Monolactates (2) ##STR610## I50V (#1)
I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1 R2 (pM) (nM) change
change (.mu.M) OPh mix-Hba-Et ++ +++ + - OPh (S)Hba-Et + +++ OPh
(S)Hba-tBu +++ OH (S)Hba-OH ++ OPh (R)Hba-Et +++ OPh (S)MeBut-Et
+++ OPh (R)MeBut-Et +++ OPh DiMePro-Me ++ OPh (S)Lac-EtMor - OPh
(S)Lac-PrMor - OPh (S)Lac-EtPip ++ - -
[1965] TABLE-US-00032 P1-P-Monolactates (3) ##STR611## I50V I84V/
(#1) L90M Ki EC.sub.50 fold fold CC.sub.50 R1 R2 (pM) (nM) change
change (.mu.M) OPh-o-i-But (S)Lac-Et +++ OPh-p-n-Oct (S)Lac-Et ++
OPh-p-n-But (S)Lac-Et +++ OPh-m-COOBn (S)Lac-Et ++ OPh-m-COOH
(S)Lac-Et ++ OPh-m-CH.sub.2OH (S)Lac-Et ++ - -
OPh-m-CH.sub.2NH.sub.2 (S)Lac-Et ++ ++ OPh-m-CH.sub.2NMe.sub.2
(S)Lac-Et + OPh-m-CH.sub.2Mor (S)Lac-Et ++ - - OPh-m-CH.sub.2Pip
(S)Lac-Et ++ OPh-m- (S)Lac-Et ++ CH.sub.2NMeC2OM OPh-o-OEt
(S)Lac-Et +++ ONMe.sub.2 (S)Lac-Et ++ OPip (S)Lac-Et + OMor
(S)Lac-Et -
[1966] TABLE-US-00033 P1-C.sub.2H.sub.4--P-Monolactates ##STR612##
I50V (#1) I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1 R2 (pM)
(nM) change change (.mu.M) --OC.sub.2H.sub.4OBn +++ OEt OEt +++ - -
OPh Lac-Et ++ - - OH OH ++ OH Lac ++
[1967] TABLE-US-00034 P1-CH.sub.2N--P-diester and monolactate (1)
##STR613## I50V I50V (#1) (#2) I84V/L90M Ki EC.sub.50 fold fold
fold CC.sub.50 R1 R2 (pM) (nM) change change change (.mu.M) Et Et
++ +++ - H H ++ - + Ph Lac-Et ++ - ++ - Ph Lac-Et + + - - Ph Lac-Et
+ ++ - Ph Aba-Et + + - Ph-oEt Lac-Et ++ ++ - ++ - Ph-dM Lac-Et +++
+ + Ph-dM Lac-Pr +++ H Lac ++ Ph Hba-Et ++ ++ - Ph Hba-Et ++ ++ - +
Ph Hba-Et ++ ++ - H Hba +
[1968] TABLE-US-00035 P1-C.sub.2N--P-diester and monolactate (2)
##STR614## I50V (#1) I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1
R2 (pM) (nM) change change (.mu.M) Ph Lac-Et + ++ + + H H ++
[1969] TABLE-US-00036 P1-CH.sub.2N--P-diester and monolactate (3)
##STR615## I50V (#1) I84V/L90M Ki EC.sub.50 fold fold CC.sub.50 R1
R2 (pM) (nM) change change (.mu.M) Et Et ++ +++ -
[1970] TABLE-US-00037 P1-N-P1-Phosphonic acid and esters (1)
##STR616## I50V (#1) Ki EC.sub.50 fold I84V/L90M R1 (pM) (nM)
change fold change CC.sub.50 .mu.M ##STR617## - ++ ##STR618## - ++
##STR619## - ##STR620## ++ +++ + ##STR621## - ##STR622## -
##STR623## + ++ ##STR624## ++ +++ + ##STR625## - ##STR626## -
##STR627## - ##STR628## + +++ +
[1971] TABLE-US-00038 P1-N-P1-Phosphonic acid and esters (2)
##STR629## Ki EC.sub.50 I50V (#1) I84V/L90M R1 (pM) (nM) fold
change fold change CC.sub.50 .mu.M ##STR630## ++ + ##STR631## ++
+++ + ##STR632## ++ +++ ##STR633## ++ ++ - ##STR634## +++
##STR635## ++ +++ + ##STR636## +++ - ##STR637## - +++ ++ ##STR638##
- ##STR639## + +++ +++ - ##STR640## - ##STR641## +++ ++ +
##STR642## -
[1972] TABLE-US-00039 P1-N-P1-Phosphonic acid and esters (3)
##STR643## Ki EC.sub.50 I50V (#1) I84V/L90M R1 (pM) (nM) fold
change fold change CC.sub.50 .mu.M ##STR644## ++ +++ + + ##STR645##
+ ++ + + ##STR646## + ++ + + ##STR647## + ##STR648## ##STR649## -
-
[1973] TABLE-US-00040 P1-N-P1-Phosphonic acid and esters (4)
##STR650## Ki EC.sub.50 I50V (#1) I84V/L90M R1 (pM) (nM) fold
change fold change CC.sub.50 .mu.M ##STR651## +++ ##STR652## +++
+++ - - ##STR653## ++ +++ + - ##STR654## ++ +++ ##STR655## ++ ++
##STR656## +++ +++ ##STR657## +++ ++ - ##STR658## +++ ++ -
##STR659## ++ ##STR660## ++
[1974] TABLE-US-00041 P1-P-cyclic monolactate ##STR661## Ki
EC.sub.50 I50V (#1) I84V/L90M R.sub.1 R.sub.2 (pM) (nM) fold change
fold change CC.sub.50 .mu.M nd nd nd nd
[1975] TABLE-US-00042 P1'-N-P1-Phosphonic acid and esters
##STR662## Ki EC.sub.50 I50V (#1) I84V/L90M R1 R2 (pM) (nM) fold
change fold change CC.sub.50 .mu.M CH.sub.3 ##STR663## ++ +++ ++ +
OH ##STR664## +++ - - CH.sub.2OH ##STR665## +++ +++ - - OBn
##STR666## +++ +++ - - OH ##STR667## - ++ - - OBn ##STR668## - +++
- ##STR669## ##STR670## - - + + ##STR671## ##STR672## + ++ + + OH
##STR673## - - ##STR674## ##STR675## ++ - ##STR676## ##STR677## ++
- ##STR678## ##STR679## ++ ++ ##STR680## ##STR681## + -
[1976] TABLE-US-00043 P1'-Phosphonic acid and esters ##STR682## Ki
EC.sub.50 I50V (#1) I84V/L90M R1 (pM) (nM) fold change fold change
CC.sub.50 .mu.M ##STR683## ++ +++ +++ +++ ##STR684## +++ +++ +++
+++ ##STR685## ++ + +++ ##STR686## +++ +++ +++ ##STR687## +++ +++
++ ##STR688## ++ ++ ++ ++ ##STR689## ++ +++ +++ +++
[1977] TABLE-US-00044 P2-Monofuran-P1-phosphonic acid and esters
##STR690## I50V (#1) Ki EC.sub.50 fold I84V/L90M R1 R2 (pM) (nM)
change fold change CC.sub.50 .mu.M OMe OH - +++ +++ OMe OEt +++ +++
+++ ++ OMe OBn +++ ++ ++ OMe phenol +++ +++ +++ + OMe OEt ++ +++
+++ ++ NH.sub.2 phenol + ++ + - NH.sub.2 OH - + NH.sub.2 OBn ++ ++
+
[1978] TABLE-US-00045 P2-Monofuran-P1-P-monoamidates ##STR691##
I50V (#1) Ki EC.sub.50 fold I84V/L90M R1 R2 (pM) (nM) change fold
change CC.sub.50 .mu.M OPh Ala-iPr ++ ++ + OPh Ala-iPr ++ ++ OPh
Ala-iPr + ++
[1979] TABLE-US-00046 P2-Other modifications-P1-phosphonic acid and
esters ##STR692## Ki EC.sub.50 I50V (#1) I84V/L90M R1 R2 (pM) (nM)
fold change fold change CC.sub.50 .mu.M ##STR693## phenyl + +++ +++
++ ##STR694## phenol + ++ ++ + ##STR695## OH - - ++ - ##STR696##
OBn + ++ + - ##STR697## phenyl + ++ +++ + ##STR698## OH + - ++ +
##STR699## OBn + ++ +++ + ##STR700## phenyl - ++ ++ ##STR701##
phenol + + - ##STR702## OH + - - - ##STR703## OBn ++ ++ + -
[1980] TABLE-US-00047 P2'-Amino-P1-phosphonic acid and esters
##STR704## Ki EC.sub.50 I50V (#1) I84V/L90M R1 R2 (pM) (nM) fold
change fold change CC.sub.50 .mu.M OH p-NH.sub.2 ++ ++ - -
##STR705## p-NH.sub.2 ++ - + - ##STR706## p-NH.sub.2 ++ +++ -
##STR707## p-NO.sub.2 ++ +++ - ##STR708## p-NHEt ++ +++ -
##STR709## p-NH.sub.2 ++ +++ - - OH m-NH.sub.2 ++ ++ - ##STR710##
m-NH.sub.2 ++ + - ##STR711## m-NH.sub.2 ++ ++ - ##STR712##
m-NH.sub.2 ++ +++ - - ##STR713## m-NH.sub.2 + ++ - - ##STR714##
m-NH.sub.2 ++ ++ ##STR715## m-NH.sub.2 + ++
[1981] TABLE-US-00048 P2'-Substituted-P1-phosphonic acid and esters
(1) ##STR716## Ki EC.sub.50 I50V (#1) I84V/L90M R1 X (pM) (nM) fold
change fold change CC.sub.50 .mu.M ##STR717## p-OH +++ + ##STR718##
p-OH +++ +++ ##STR719## p-OH ++ ##STR720## p-OH +++ - ##STR721##
p-OBn ++ ##STR722## p-OBn - ##STR723## p-H ++ - ##STR724## p-H ++
+++ + ##STR725## p-H +++ + + ##STR726## p-H ++ ##STR727## p-H ++
##STR728## p-F ++ + ##STR729## p-F ++ +++ + ##STR730## p-F +++ + +
##STR731## p-F ++ + + ##STR732## p-F ++ ##STR733## p-CF.sub.3 +++ +
##STR734## p-CF.sub.3 ++ +++ - ##STR735## p-OCF.sub.3 ++ +
##STR736## p-OCF.sub.3 ++ +++ + ##STR737## p-CN ++ +++ - ##STR738##
p-Pip - - ##STR739## p-Pip-Me - -
[1982] TABLE-US-00049 P2'-Substituted-P1-phosphonic acid and esters
(2) ##STR740## Ki EC.sub.50 I50V (#1) I84V/L90M R1 X (pM) (nM) fold
change fold change CC.sub.50 .mu.M ##STR741## m-Py ++ +++
##STR742## m-Py ++ ##STR743## m-Py ++ ++ + - ##STR744## m-Py ++ ++
##STR745## m-Py ++ ##STR746## m-Py-Me.sup.+ + ##STR747##
m-Py-Me.sup.+ ++ ##STR748## m-Py-oxide ++ ##STR749## m-Py-oxide ++
##STR750## m-Py-oxide ++ ++ - ##STR751## m-Py-oxide + ##STR752##
m-Py-oxide - p-Py-oxide p-OMe ++ - ##STR753## p-CHO +++ ##STR754##
p-CHO +++ ##STR755## p-CH2 OH +++ - - ##STR756## p-CH2 OH ++
##STR757## p-CH2 OH ++ ##STR758## p-CH2 Mor ++ - - ##STR759## p-CH2
Mor - ##STR760## p-CH2 Mor -
[1983] TABLE-US-00050 P2'-Alkylsulfonyl-P1-phosphonic acid and
esters ##STR761## Ki EC.sub.50 I50V (#1) I84V/L90M CC.sub.50 R1 X
(pM) (nM) fold change fold change .mu.M ##STR762## ##STR763## - -
##STR764## ##STR765## + ++
[1984] TABLE-US-00051 P2'-Carbonyl-substituted-P1-phosphonic acid
and esters ##STR766## Ki EC.sub.50 I50V (#1) I84V/L90M R1 X (pM)
(nM) fold change fold change CC.sub.50 .mu.M ##STR767## ##STR768##
- ##STR769## ##STR770## - ++ ##STR771## ##STR772## +
[1985] TABLE-US-00052 P2'-Phosphonic acid and esters ##STR773## Ki
EC.sub.50 I50V (#1) I84V/L90M R (pM) (nM) fold change fold change
CC.sub.50 .mu.M ##STR774## +++ +++ - - ##STR775## +++ + - -
##STR776## ++ - ##STR777## ++ +++ ++ ++ ##STR778## + ++ +++ +++
##STR779## +++ +++ + + ##STR780## +++ +++ +++ ++ ##STR781## ++ ++
++ + ##STR782## +++ +++ +++ ++ ##STR783## ++ +++ ++ ++ ##STR784##
+++ +++ - - ##STR785## +++ ++ + - ##STR786## + ++ + + ##STR787## -
+ +++ ++ ##STR788## + ++ + -
[1986] TABLE-US-00053 P2'-P-Bisamidate, monoamidate, and
monolactate ##STR789## I50V I84V/ (#1) L90M Ki Ec.sub.50 fold fold
R.sub.1 R.sub.2 (pM) (nM) change change CC.sub.50 .mu.M Ala-Bu
Ala-Bu + ++ + + OPh Ala-iPr ++ ++ OPh Lac-iPr + + OH Ala-OH ++
[1987] TABLE-US-00054 P1-N-P2'-Phosphonic acid and esters
##STR790## Ki EC.sub.50 I50V (#1) I84V/L90M R.sub.1 R.sub.2 (pM)
(nM) fold change fold change CC.sub.50 .mu.M NO.sub.2 phenol +++ -
NH.sub.2 OH ++ - NH.sub.2 OEt + ++ ++ NH.sub.2 OBn + + + NMe.sub.2
OEt ++ +++ ++ OH OH ++ - OH OBn ++ ++ OC.sub.2H.sub.4NMe.sub.2 OH
+++ + OC.sub.2H.sub.4--NMe.sub.2 OBn ++ ++
[1988] TABLE-US-00055 P1-N-P2'-p-Bisamidate and monoamidate
##STR791## I50V I84V/ (#1) L90M Ki EC.sub.50 fold fold R.sub.1
R.sub.2 (pM) (nM) change change CC.sub.50 .mu.M Ala-Bu Ala-Bu + +
OPh Ala-iPr + - OPh Ala-iPr ++ -
[1989] TABLE-US-00056 P1-NEt-P2'-p-Bisamidate and monoamidate
##STR792## I50V I84V/ (#1) L90M Ki EC.sub.50 fold fold R.sub.1
R.sub.2 (pM) (nM) change change CC.sub.50 .mu.M OPh Ala-iPr + + OPh
Ala-iPr + + - -
[1990] TABLE-US-00057 Phosphate prodrug of ampenavir ##STR793## Ki
EC.sub.50 I50V (#1) I84V/L90M R.sub.1 R.sub.2 (pM) (nM) Fold change
fold change CC.sub.50 .mu.M ++
[1991] TABLE-US-00058 Phosphate prodrug of 94-003 ##STR794## Ki
EC.sub.50 I50V (#1) I84V/L90M R.sub.1 R.sub.2 (pM) (nM) fold change
fold change CC.sub.50 .mu.M +++
[1992] TABLE-US-00059 Phosphate prodrug of GS77366
(P1-mono(S)Lac-iPr) ##STR795## Ki EC.sub.50 I50V (#1) I84V/L90M
R.sub.1 R.sub.2 (pM) (nM) fold change fold change CC.sub.50 .mu.M
+++
[1993] TABLE-US-00060 Valine prodrug of(P1-mono(S)Lac-Et)
##STR796## Ki EC.sub.50 I50V (#1) I84V/L90M R.sub.1 R.sub.2 (pM)
(nM) fold change fold change CC.sub.50 .mu.M ++
[1994] TABLE-US-00061 Valine prodrug of GS278053
(P1-mono(S)Lac-Et,P2'-CH.sub.2OH) ##STR797## Ki EC.sub.50 I50V (#1)
I84V/L90M R.sub.1 R.sub.2 (pM) (nM) fold change fold change
CC.sub.50 .mu.M ++
[1995] TABLE-US-00062 TABLE 11 Enzymatic and Cellular Activity Data
Formula VIIIa CCLPPI activity ##STR798## ##STR799## Enzymatic assay
Cell-based assay (MT-4) EC.sub.50/nM 84V9 WT 0M 30N 48V5 48V5 48V8
K.sub.i IC.sub.50/ IC.sub.50/ 84V9 82I88 4V82 4V82 2A90 46I50
Structure, R (nM) nM nM WT 0M D A S M V H 0.033 3.0 9.1 165 819 82
82 73 45 88 (DMP-850) p-OH 0.029 3.0 12 149 143 79 32 39 19 55
p-OBn >5 353 781 2123 5312 1548 ND ND ND ND
p-OCH.sub.2PO.sub.3Bn.sub.2 >5 276 2042 2697 4963 2119 ND ND ND
ND p-OCH.sub.2PO.sub.3Et.sub.2 >5 627 1474 2480 >6000 1340 ND
ND ND ND p-OCH.sub.2PO.sub.3H.sub.2 >5 551 1657 >12000 ND ND
ND ND ND ND m-OH 0.128 1.6 12 151 475 249 84 104 m-OBn 0.253 6.9 27
218 2422 82 709 ND ND 601 m-OCH.sub.2PO.sub.3Bn.sub.2 1.54.sup.a 31
72 489 514 237 159 171 168 708 (N-iPr indazole)
m-OCH.sub.2PO.sub.3Bn.sub.2 0.177 18 43 898 >6000 705 2597 ND ND
3121 m-OCH.sub.2PO.sub.3Et.sub.2 1.93.sup.a 70 169 665 3005 93 513
ND ND 857 m-OCH.sub.2PO.sub.3H.sub.2 0.254 8.3 33 >12000 ND ND
ND ND ND ND m-OCH.sub.2PO.sub.3Ph.sub.2 0.543.sup.a 10 42 1349
>6000 1541 2183 ND ND 3380 m-OCH.sub.2PO.sub.3HPh 0.644 17 65
1745 >6000 ND ND ND ND ND m-mono-Ala-Bu 0.858.sup.a 6.6 39 1042
>6000 425 790 ND ND 797 m-mono-Ala-Et 35 68 1436 >6000 219
734 ND ND 1350 m-mono-Lac-Bu 15 34 2663 >6000 1089 ND ND ND ND
m-mono-Lac-Et 23 80 2609 >6000 516 5923 ND ND >6000
m-bis-Ala-Bu 1.279.sup.a 18 103 1079 >6000 2362 1854 ND ND 1536
m-bis-Ala-Et 1.987.sup.a 31 202 5620 >6000 1852 ND ND ND ND
[1996] TABLE-US-00063 ##STR800## Enzymatic assay 84V90 Cell-based
assay (MT-4) EC.sub.50/nM WT M 30N 48V5 48V5 48V8 K.sub.i
IC.sub.50/ IC.sub.50/ 84V9 82I88 4V82 4V82 2A90 46I50 Structure, R
(nM) nM nM WT 0M D A S M V H 0.033 3.0 9.1 165 819 82 82 73 45 88
(DMP-850) ##STR801## 0.091 3.4 27 1548 >600 >600 ND ND ND ND
##STR802## 0.354 3.3 25 168 909 750 277 489 ##STR803## 0.157 1.6 10
188 476 666 240 319 ##STR804## 0.044 5.0 27 491 387 234 238 192
##STR805## 0.362 7.3 70 5141 >600 4480 ND ND ND ND ##STR806##
0.112 1.4 6.4 603 1276 678 208 209 ##STR807## <0.03 1.3 7.5 625
708 899 301 398
[1997] TABLE-US-00064 ##STR808## Enzymatic assay 84V Cell-based
assay (MT-4) EC.sub.50/nM WT 90M 30N 48V 48V 48V K.sub.i IC.sub.50/
IC.sub.50/ 84V 82I8 54V 54V 82A 46I5 Structure, R1 Structure, R
(nM) nM nM WT 90M 8D 82A 82S 90M 0V CO.sub.2H ##STR809## 15 174
3055 >6000 887 ND ND ND ND CONH(CH.sub.2).sub.3PO.sub.3Et.sub.2
##STR810## 0.009 1.1 12 65 311 74 80 75 74 85 CO.sub.2H ##STR811##
18 299 2344 >6000 3360 ND ND ND ND
CONH(CH.sub.2).sub.3PO.sub.3Et.sub.2 ##STR812## <0.004 2.3 29
176 824 171 233 ND ND 195 CO.sub.2H ##STR813## 0.091 3.4 27 1548
>6000 >6000 ND ND ND ND CONH(CH.sub.2).sub.3PO.sub.3Et.sub.2
##STR814## 0.157 1.6 10 188 476 666 240 319
[1998] TABLE-US-00065 ##STR815## Enzymatic assay 84V9 Cell-based
assay (MT-4) EC.sub.50/nM WT 0M 30N 48V5 48V5 48V8 K.sub.i
IC.sub.50/ IC.sub.50/ 84V9 82I88 4V82 4V82 2A90 46I50 Structure, R
(nM) nM nM WT 0M D A S M V CH.sub.3 0.033 3.8 9.4 54 918 69 33 30
22 17 (DMP-851) OH 0.65.sup.a 6.1 77 356 2791 669 294 ND ND 683
OCH.sub.2PO.sub.3Et.sub.2 1.230.sup.a 23 157 356 >6000 145 175
ND ND 138 OCH.sub.2PO.sub.3H.sub.2 0.809 59 137 1074 >600 ND ND
ND ND ND O-mono-Lac-Et >2.0 93 553 >6000 >6000 ND ND ND ND
ND O-mono-Lac-Bu >2.0 25 249 >6000 >6000 ND ND ND ND ND
CH.sub.2OH 0.017 2.8 31 253 1106 486 413 ND ND 524
CH.sub.2OCH.sub.2PO.sub.3Et.sub.2 2.8 13 123 119 3295 267 430 ND ND
789 CH.sub.2OCH.sub.2PO.sub.3H.sub.2 42 20 5 1757 >4243 ND ND ND
ND ND
[1999] TABLE-US-00066 ##STR816## ##STR817## ##STR818## Enzymatic
assay 84V Cell-based assay (MT-4) EC.sub.50/nM WT 90M 30N 48V 48V
48V K.sub.i IC.sub.50/ IC.sub.50/ 84V 82I8 54V 54V 82A 46I5 R R1 R2
(nM) nM nM WT 90M 8D 82A 82S 90M 0V -- -- -- 0.03 3.0 9.1 165 819
82 82 73 45 88 -- -- -- 0.37 5.8 43.3 193 2312 281 705 ND ND 772 H
Ph H 34 631 2492 >600 3360 ND ND ND ND OH Ph OH 31 397 117 5609
756 2266 ND ND 928 OH Ph OCH.sub.2PO.sub.3 9 40 33 791 92 807 1103
1429 53 H Ph OCH.sub.2PO.sub.3 0.65 3.9 48 107 2456 293 1438 1899
3292 589 H Indazol H <0.0 2.5 13 11 22 <8 5.5 8 4 4.0 OH
Indazol OH 0.01 0.6 3.5 >600 2728 7224 ND ND ND ND OH Indazol
OCH.sub.2PO.sub.3 0.13 1.1 5.5 1698 1753 1998 ND ND ND ND H Indazol
OCH.sub.2PO.sub.3 0.02 1.4 6.2 57 40 68 28 26 32 27
[2000] TABLE-US-00067 ##STR819## Enzymatic assay Cell-based assay
(MT-4) EC.sub.50/nM 84V WT 90M 30N 48V 48V 48V K.sub.i IC.sub.50/
IC.sub.50/ 84V 82I8 54V 54V 82A 46I5 R R1 R2 (nM) nM nM WY 90M 8D
82A 82S 90M 0V -- -- -- 0.033 3.0 9.1 165 819 82 82 73 45 88 OH Ph
OCH.sub.2PO.sub.3Et.sub.2 9 40 33 791 92 807 1103 1429 53 H Ph
OCH.sub.2PO.sub.3Et.sub.2 0.656 3.9 48 107 2456 293 1438 1899 3292
589 OCH.sub.3 Ph OCH.sub.2PO.sub.3Et.sub.2 OH Ph-pOH
OCH.sub.2PO.sub.3Et.sub.2 <0.01 2.6 18 285 1912 211 986 ND ND
1107 H Ph-pOH OCH.sub.2PO.sub.3Et.sub.2 0.319 2.1 33 65 272 90 128
198 126 144 OCH.sub.3 Ph-pOH OCH.sub.2PO.sub.3Et.sub.2 0.043 1.8 17
29 146 23 67 106 48 68 OH Ph-mNH.sub.2/ OCH.sub.2PO.sub.3Et.sub.2
8.7 67 286 1902 562 789 1781 684 239 NHEt H Ph-mNH.sub.2
OCH.sub.2PO.sub.3Et.sub.2 0.126 3.4 39 65 328 16 168 146 74 46
OCH.sub.3 Ph-mNH.sub.2 OCH.sub.2PO.sub.3Et.sub.2 <0.01 3.6 56 63
535 18 202 117 102 36 OCH.sub.3 m- OCH.sub.2PO.sub.3Et.sub.2 115
765 106 1019 970 480 352 pyridine
[2001] TABLE-US-00068 ##STR820## Enzymatic assay Cell-based assay
(MT-4) EC.sub.50/nM 84V WT 90M 30N 48V5 48V5 38V8 K.sub.i
IC.sub.50/ IC.sub.50/ 84V9 82I88 4V82 4V82 2A90 46I50 R R1 R2 (nM)
nM nM WT 0M D A S M V -- -- -- 0.033 3.0 9.1 165 819 82 82 73 45 88
H Ph-mNH.sub.2 OCH.sub.2PO.sub.3Et.sub.2 0.126 3.4 39 65 328 16 168
146 74 46 OCH.sub.3 Ph-mNH.sub.2 OCH.sub.2PO.sub.3Et.sub.2 <0.01
3.6 56 63 535 18 202 117 102 36 OCH.sub.3 pH-mNH.sub.2
O(CH.sub.2).sub.2PO.sub.3Et.sub.2 OCH.sub.3 Ph-mNH.sub.2
OCONH(CH.sub.2).sub.2PO.sub.3Et.sub.2 1.3 116 74 2265 77 262 214
215 184 OCH.sub.3 Ph-mNH.sub.2 OCONH(CH.sub.2)PO.sub.3Et.sub.2 9.9
85 58 2151 68 223 203 185 104 H pH-pOH OCH.sub.2PO.sub.3Et.sub.2
0.319 2.1 33 65 272 90 128 222 146 144 OCH.sub.3 Ph-pOH
OCH.sub.2PO.sub.3Et.sub.2 0.045 1.8 17 30 148 25 70 129 54 90
OCH.sub.3 Ph-pOH OCONH(CH.sub.2).sub.2PO.sub.3Et.sub.2 6.6 49 33
495 31 74 51 55 223 -- -- -- 0.033 3.0 9.1 165 819 82 82 73 45 88 H
Ph OCH.sub.2PO.sub.3Et 0.656 3..9 48 107 2456 293 1438 1899 3292
589 H Ph OH 0.830 15 162 1261 >6000 2952 >6000 H Ph
OCH.sub.2PO.sub.3Bn.sub.2 0.125 7.4 158 1769 >6000 3135 >6000
H Ph PCH.sub.2PO.sub.3H.sub.2 0.586 9.7 210 >6000 >6000 ND ND
H Ph Mono-lac-Et 0.220 6.6 56 1726 >6000 2793 >6000 H Ph
Mono-Ala-Et 5 50 310 2943 238 2851 1948 2450 1250
[2002] TABLE-US-00069 Enzymatic assay Cell-based assay (MT-4)
EC.sub.50/nM 84V WY 90M 30N 48V5 48V5 48V8 X.sub.1 IC.sub.50/
IC.sub.50/ 84V9 82I88 4V82 4V82 2A90 46I50 R1 R2 (nM) nM nM WT 0M D
A S M V Phenyl ##STR821## 0.03 3.0 9.1 165 819 82 82 73 45 88
Phenyl ##STR822## 0.42 6.5 85 1226 >600 869 774 ND ND 937 Phenyl
##STR823## 0.37 5.8 43.3 193 2312 281 705 ND ND 772 Phenyl
##STR824## 109 >25 >600 ND ND ND ND ND ND Phenyl ##STR825##
Phenyl ##STR826## Phenyl ##STR827## ##STR828## ##STR829## 1.43 302
114 >600 >600 ND ND ND ND ND ##STR830## ##STR831## >5
>25 ND 5949 ND ND ND ND ND ND ##STR832## ##STR833## >5 130
348 2006 3121 ND ND ND ND ND
[2003] All publications and patent applications cited herein are
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
[2004] Although certain embodiments have been described in detail
above, those having ordinary skill in the art will clearly
understand that many modifications are possible in the embodiments
without departing from the teachings thereof. All such
modifications are intended to be encompassed within the claims of
the invention.
Example: Preliminary Study: Plasma and PBMC Exposure Following
Intravenous and Oral Administration of Candidate to Beagle Dogs
[2005] The pharmacokinetics of a phosphonate prodrug GS77366
(PI-monoLac-iPr, structure shown below), its active metabolite
(metabolite X, or GS77568), and GS8373 were studied in dogs
following intravenous and oral administration of the candidate.
[2006] Dose Administration and Sample Collection. The in-life phase
of this study was conducted in accordance with the USDA Animal
Welfare Act and the Public Health Service Policy on Humane Care and
Use of Laboratory Animals, and followed the standards for animal
husbandry and care found in the Guide for the Care and Use of
Laboratory Animals, 7.sup.th Edition, Revised 1996. All animal
housing and study procedures involving live animals were carried
out at a facility which had been accredited by the Association for
Assessment and Accreditation of Laboratory Animal
Care--International (AAALAC).
[2007] Each animal in a group of 4 female beagle dogs was given a
bolus dose of GS77366 (P1-monoLac-iPr) intravenously at 1 mg/kg in
a formulation containing 40% PEG 300, 20% propylene glycol and 40%
of 5% dextrose. Another group of 4 female beagle dogs was dosed
with GS77366 via oral gavage at 20 mg/kg in a formulation
containing 60% Vitamin-E TPGS, 30% PEG 400 and 10% propylene
glycol.
[2008] Blood samples were collected pre-dose, and at 5 min 15 min,
30 min, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr and 24 hr post-dose. Plasma
(0.5 to 1 mL) was prepared from each sample and kept at -70.degree.
C. until analysis. Blood samples (8 mL) were also collected from
each dog at 2, 8 and 24 hr post dose in Becton-Dickinson CPT
vacutainer tubes. PBMCs were isolated from the blood by
centrifugation for 15 minutes at 1500 to 1800 G. After
centrifugation, the fraction containing PBMCs was transferred to a
15 ml, conical centrifuge tube and the PBMCs were washed twice with
phosphate buffered saline (PBS) without Ca.sup.2+ and Mg.sup.2+.
The final wash of the cell pellet was kept at -70.degree. C. until
analysis.
[2009] Measurement of the candidate, metabolite X and GS8373 in
plasma and PBMCs. For plasma sample analysis, the samples were
processed by a solid phase extraction (SPE) procedure outlined
below. Speedisk C18 solid phase extraction cartridges (1 mL, 20 mg,
10 .mu.M, from J. T. Baker) were conditioned with 200 .mu.L of
methanol followed by 200 .mu.L of water. An aliquot of 200 .mu.L of
plasma sample was applied to each cartridge, followed by two
washing steps each with 200 .mu.L of deionized water. The compounds
were eluted from the cartridges with a two-step process each with
125 .mu.L of methanol. Each well was added 50 .mu.L of water and
mixed. An aliquot of 25 .mu.L of the mixture was injected onto a
ThermoFinnigan TSQ Quantum LC/MS/MS system.
[2010] The column used in liquid chromatography was HyPURITY.RTM.
C18 (50.times.2.1 mm, 3.5 .mu.m) from Thermo-Hypersil. Mobile phase
A contained 10% acetonitrile in 10 mM ammonium formate, pH 3.0.
Mobile phase B contained 90% acetonitrile in 10 mM ammonium
formate, pH 4.6. The chromatography was carried out at a flow rate
of 250 .mu.L/min under an isocratic condition of 40% mobile phase A
and 60% mobile phase B. Selected reaction monitoring (SRM) were
used to measure GS77366, GS8373 and Metabolite X with the positive
ionization mode on the electrospray probe. The limit of
quantitation (LOQ) was 1 nM for GS77366, GS8373 and GS77568
(Metabolite X) in plasma.
[2011] For PBMC sample analysis, phosphate buffered saline (PBS)
was added to each PBMC pellet to bring the total sample volume to
500 .mu.L in each sample. An aliquot of 150 .mu.L from each PBMC
sample was mixed with an equal volume of methanol, followed by the
addition of 700 .mu.L of 1% formic acid in water. The resulting
mixture was applied to a Speedisk C18 solid phase extraction
cartridge (1 mL, 20 mg, 10 um, from J. T. Baker) which had been
conditioned as described above. The compounds were eluted with
methanol after washing the cartridge 3 times with 10% methanol. The
solvent was evaporated under a stream of N.sub.2, and the sample
was reconstituted in 150 .mu.L of 30% methanol. An aliquot of 75 $I
of the solution was injected for LC/MS/MS analysis. The limit of
quantitation was 0.1 ng/mL in the PBMC suspension.
[2012] Pharmacokinetic Calculations. The pharmacokinetic parameters
were calculated using WinNonlin. Noncompartmental analysis was used
for all pharmacokinetic calculation. The intracellular
concentrations in PBMCs were calculated from the measured
concentrations in PBMC suspension on the basis of a reported volume
of 0.2 picoliter/cell (B. L. Robins, R. V. Srinivas, C. Kim, N.
Bischofberger, and A. Fridland, (1998) Antimicrob. Agents
Chemother. 42, 612).
[2013] Plasma and PBMC Concentration-time Profiles. The following
shows the concentration-time profiles of GS77366, GS77568 and
GS8373 in plasma and PBMCs following intravenous dosing of GS77366
at 1 mg/kg in dogs. The data demonstrate that the prodrug can
effectively deliver the active components (metabolite X and GS8373)
into cells that are primarily responsible for HIV replication, and
that the active components in these cells had much longer half-life
than in plasma.
[2014] Pharmacokinetic profiles of GS77366, GS77568 and GS8373 in
plasma and PBMCs following intravenous administration of GS77366 at
1 mg/kg in dogs are shown in FIG. 6.
[2015] The pharmacokinetic properties of GS77568 in PBMCs following
oral administration of GS77366 in dogs are compared with that of
nelfinavir and amprenavir, two marketed HIV protease inhibitors.
These data show that the active component (GS77568) from the
phosphonate prodrug had sustained levels in PBMCs compared to
nelfinavir and amprenavir.
[2016] Concentration-time profiles of GS77568, nelfinavir and
amprenavir in PBMCs following oral administration of GS77366 (20
mg/kg), nelfinavir (17.5 mg/kg) and amprenavir (20 mg/kg) in dogs
are shown in FIG. 7. TABLE-US-00070 TABLE 1a Comparison of GS77568
with nelfinavir and amprenavir in PBMCs following oral
administration in beagle dogs. Compound Dose t.sub.1/2 (hr)
AUC.sub.(2-24 hr) Nelfinavir 17.5 mg/kg 3.0 hr 33,000 nM hr
Amprenavir 20 mg/kg 1.7 hr 102,000 nM hr GS77568 20 mg/kg of
GS77366 >20 hr 42,200 nM hr
Intracellular Metabolism/In Vitro Stability 1. Uptake and
Persistence in MT2 Cells, Quiescent and Stimulated PBMC
[2017] The protease inhibitor (PI) phosphonate prodrugs undergo
rapid cell uptake and metabolism to produce acid metabolites
including the parent phosphonic acid. Due to the presence of
charges, the acid metabolites are significantly more persistent in
the cells than non-charged PI's. In order to estimate the relative
intracellular levels of the different PI prodrugs, three
phosphonate, monoamidate phenoxy phosphonate and monolactate
phenoxy phosphonate (FIG. 1) were incubated at 10 .mu.M for 1 hr
with MT-2 cells, stimulated and quiescent peripheral blood
mononuclear cells (PBMC) (pulse phase). After incubation, the cells
were washed, resuspended in the cell culture media and incubated
for 24 hr (chase phase). At specific time points, the cells were
washed, lysed and the lysates were analyzed by HPLC with UV
detection. Typically, the cell lysates were centrifuged and 100 uL
of the supernatant were mixed with 200 .mu.L of 7.5 uM amprenavir
(Internal Standard) in 80% acetonitrile/20% water and injected into
an HPLC system (70 A).
HPLC Conditions:
Analytical Column: Prodigy ODS-3, 75.times.4.6, 3 u+C18 guard at
40.degree. C.
Gradient:
Mobile Phase A: 20 mM ammonium acetate in 10% ACN/90% H.sub.2O
Mobile Phase B: 20 mM ammonium acetate in 70% ACN/30% H.sub.2O
30-100% B in 4 min, 100% B for 2 min, 30% B for 2 min at 2.5
mL/min.
Run Time: 8 min
UV Detection @ 245 nm
[2018] Concentrations of Intracellular metabolites were calculated
based on cell volume 0.2 mL/mln cells for PBMC and 0.338 .mu.L/mln
(0.676 uL/mL) for MT-2 cells.
[2019] Chemical Structures of Selected Protease Inhibitor
Phosphonate Prodrugs and Intracellular Metabolites. TABLE-US-00071
##STR834## GS EC.sub.50 No. R1 R2 (nM) 8373 OH OH 4,800 .+-. 1,800
16503 HNCH(CH.sub.3)COOBu HNCH(CH.sub.3)COOBu 6.0 .+-. 1.4 16571
OPh HNCH(CH.sub.3)COOEt 15 .+-. 5 17394 OPh OCH(CH.sub.3)COOEt 20
.+-. 7 16576 OPh HNCH(CH.sub.2CH.sub.3)COOEt 12.6 .+-. 4.8 Met X OH
HNCH(CH.sub.3)COOH >10,000 Met OH OCH(CH.sub.3)COOEt 1750 .+-.
354 LX
[2020] The foregoing data demonstrates that there was a significant
uptake and conversion of all 3 compounds in all cell types. The
uptake in the quiescent PBMC was 2-3-fold greater than in the
stimulated cells. GS-16503 and GS-16571 were metabolized to
Metabolite X and GS-8373. GS-17394 metabolized to the Metabolite
LX. Apparent intracellular half-lives were similar for all
metabolites in all cell types (7-12 hr).
[2021] Persistence of Total Acid Metabolites of Protease Inhibitor
Prodrugs in Stimulated (A), Quiescent PBMC (B) and MT-2 Cells (C)
(1 hr, 10 uM Pulse, 24 hr Chase) is shown in FIGS. 8 to 10.
2. Uptake and Persistence in Stimulated and Quiescent T-Cells
[2022] Since HIV mainly targets T-lymphocytes, it is important to
establish the uptake, metabolism and persistence of the metabolites
in the human T-cells. In order to estimate the relative
intracellular levels of the different PI prodrugs, GS-16503, 16571
and 17394 were incubated at 10 .mu.M for 1 hr with quiescent and
stimulated T-cells (pulse phase). The prodrugs were compared with a
non-prodrug PI, nelfinavir. After incubation, the cells were
washed, resuspended in the cell culture media and incubated for 4
hr (chase phase). At specific time points, the cells were washed,
lysed and the lysates were analyzed by HPLC with UV detection. The
sample preparation and analysis were similar to the ones described
for MT-2 cells, quiescent and stimulated PBMC.
[2023] Table 1b demonstrates the levels of total acid metabolites
and corresponding prodrugs in T-cells following pulse/chase and
continuous incubation. There was significant cell uptake/metabolism
in T-lymphocytes. There was no apparent difference in uptake
between stimulated and quiescent T-lymphocytes. There was
significantly higher uptake of phosphonate PI's than nelfinavir. GS
17394 demonstrates higher intracellular levels than GS 16571 and GS
16503. The degree of conversion to acid metabolites varied between
different prodrugs. GS-17394 demonstrated the highest degree of
conversion, followed by GS-16503 and GS-16571. The metabolites,
generally, were an equal mixture of the mono-phosphonic acid
metabolite and GS-8373 except for GS-17394, where Metabolite LX was
stable, with no GS-8373 formed. TABLE-US-00072 TABLE 1b
Intracellular Levels of Metabolites and Intact Prodrug Following
Continuous and 1 hr Pulse/4 hr Chase Incubation (10 .mu.M/0.7 mln
cells/1 mL) of 10 .mu.M PI Prodrugs and Nelfinavir with Quiescent
and Stimulated T-cells Continuous Incubation 1 hr Pulse/4 hr Chase
Quiescent Stimulated Quiescent Stimulated T-cells T-cells T-cells
T-cells Time Acid Met Prodrug Acid Met Prodrug Acid Met Prodrug
Acid Met Prodrug Compound (h) (.mu.M) (.mu.M) (.mu.M) (.mu.M)
(.mu.M) (.mu.M) (.mu.M) (.mu.M) 16503 0 1180 42 2278 0 2989 40 1323
139 2 3170 88 1083 116 1867 4 1137 31 4 5262 0 3198 31 1054 119
1008 0 16571 0 388 1392 187 1417 1042 181 858 218 2 947 841 1895
807 1170 82 1006 35 4 3518 464 6147 474 1176 37 616 25 17394 0 948
1155 186 1194 4480 14 2818 10 2 7231 413 3748 471 2898 33 1083 51 4
10153 167 3867 228 1548 39 943 104 Nelfinavir 0 101 86 886 1239 2
856 846 725 770 4 992 1526 171 544
3. PBMC Uptake and Metabolism of Selected PI Prodrugs Following
1-hr Incubation in MT- and 2 Cells at 10, 5 and 1 .mu.M.
[2024] To determine if the cell uptake/metabolism is concentration
dependent, selected PI's were incubated with the 1 ml, of MT-2 cell
suspension (2.74 mln cells/mL) for 1 hr at 37.degree. C. at 3
different concentrations: 10, 5 and 1 .mu.M. Following incubation,
cells were washed twice with the cell culture medium, lysed and
assayed using HPLC with UV detection. The sample preparation and
analysis were similar to the ones described for MT-2 cells,
quiescent and stimulated PBMC. Intracellular concentrations were
calculated based on cell count, a published single cell volume of
0.338 pl for MT-2 cells, and concentrations of analytes in cell
lysates. Data are shown in Table 2a.
[2025] Uptake of all three selected PI's in MT-2 cells appears to
be concentration-independent in the 1-10 uM range. Metabolism
(conversion to acid metabolites) appeared to be
concentration-dependent for GS-16503 and GS-16577 (3-fold increase
at 1 uM vs. 10 uM) but independent for GS-17394 (monolactate).
Conversion from a respective metabolite X to GS-8373 was
concentration-independent for both GS-16503 and GS-16577 (no
conversion was observed for metabolite LX of GS-17394).
TABLE-US-00073 TABLE 2a Uptake and Metabolism of Selected PI
Prodrugs Following 1-hr Incubation in MT-2 Cells at 10, 5 and 1
.mu.M. Cell-Assosiated Prodrug and % Metabolites Conversion
Extracellular Concentration, .mu.M to acid Compound Concentration,
.mu.M Metabolite X GS8373 Prodrug Total metabolites GS-17394 10
1358 0 635 1993 68 5 916 0 449 1365 67 1 196 0 63 260 76 GS-16576
10 478 238 2519 3235 22 5 250 148 621 1043 40 1 65 36 61 168 64
GS-16503 10 120 86 1506 1712 12 5 58 60 579 697 17 1 12 18 74 104
29 * For GS16576, Metabolite X is mono-aminobutyric acid
4. PBMC Uptake and Metabolism of Selected PI Candidates Following
1-hr Incubation in Human Whole Blood at 10 uM.
[2026] In order to estimate the relative intracellular levels of
the different PI prodrugs candidates under conditions simulating
the in vivo environment, compounds representative of three classes
of phosphonate PI prodrugs--bisamidate phosphonate (GS-16503),
monoamidate phenoxy phosphonate (GS-16571) and monolactate phenoxy
phosphonate (GS-17394) (FIG. 1) were incubated at 10 .mu.M for 1 hr
with intact human whole blood at 37.degree. C. After incubation,
PBMC were isolated, then lysed and the lysates were analyzed by
HPLC with UV detection.
[2027] The results of analysis are shown in Table 3. There was
significant cell uptake/metabolism following incubation in whole
blood. There was no apparent difference in uptake between GS-16503
and GS-16571. GS-17394 demonstrated significantly higher
intracellular levels than GS-16571 and GS-16503.
[2028] The degree of conversion to acid metabolites varies between
different prodrugs after 1 hr incubation. GS-17394 demonstrated the
highest degree of conversion, followed by GS-16503 and GS-16571.
The metabolites, generally, were an equimolar mixture of the
mono-phosphonic acid metabolite and GS-8373 (parent acid) except
for GS-17394, where Metabolite LX was stable with no GS-8373
formed. TABLE-US-00074 TABLE 3a PBMC Uptake and Metabolism of
Selected PI Prodrugs Following 1-hr Incubation in Human Whole Blood
at 10 uM (Mean .+-. SD, N = 3). Intracellular Prodrug and Major
Metabolites Concentration, uM Intracellular GS# Acid Metabolite
Prodrug, .mu.M Total, .mu.M Metabolites 16503 279 .+-. 47 61 .+-.
40 340 .+-. 35 X, GS-8373 16571 319 .+-. 112 137 .+-. 62 432 .+-.
208 X, GS-8373 17394 629 .+-. 303 69 .+-. 85 698 .+-. 301 LX * PBMC
Intracellular Volume = 0.2 .mu.L/mln
5. Distribution of PI Prodrug Candidates in PBMC
[2029] In order to compare distribution and persistence of PI
phosphonate prodrugs with those of non-prodrug PI's, GS-16503,
GS-17394 and nelfinavir, were incubated at 10 .mu.M for 1 hr with
PBMC (pulse phase). After incubation, the cells were washed,
resuspended in the cell culture media and incubated for 20 more hr
(chase phase). At specific time points, the cells were washed and
lysed. The cell cytosol was separated from membranes by
centrifugation at 9000.times.g. Both cytosol and membranes were
extracted with acetonitrile and analyzed by HPLC with UV
detection.
[2030] Table 4a and the accompanying bar graphs below show the
levels of total acid metabolites and corresponding prodrugs in the
cytosol and membranes before and after the 22 hr chase. Both
prodrugs exhibited complete conversion to the acid metabolites
(GS-8373 and X for GS-16503 and LX for GS-17394, respectively). The
levels of the acid metabolites of the PI phosphonate prodrugs in
the cytosol fraction were 2-3-fold greater than those in the
membrane fraction after the 1 hr pulse and 10-fold greater after
the 22 hr chase. Nelfinavir was present only in the membrane
fractions. The uptake of GS-17394 was about 3-fold greater than
that of GS-16503 and 30-fold greater than nelfinavir.
[2031] The metabolites were an equimolar mixture of metabolite X
and GS-8373 (parent acid) for GS-16503 and only metabolite LX for
GS-17394. TABLE-US-00075 TABLE 4a Uptake and Cell Distribution of
Metabolites and Intact Prodrugs Following Continuous and 1 hr
Pulse/22 hr Chase Incubation of 10 uM PI Prodrugs and Nelfinavir
with Quiescent PBMC. Cell-Associated PI, pmol/mln cells 1 hr Pulse/
1 hr Pulse/ 0 hr Chase 22 hr Chase Acid Acid Cell Metabo- Metabo-
GS# Type Fraction lites Prodrug lites Prodrug GS-16503 PBMC
Membrane 228 0 9 0 GS-16503 PBMC Cytosol 390 0 130 0 GS-17394 PBMC
Membrane 335 0 26 0 GS-17394 PBMC Cytosol 894 0 249 0 Nelfinavir
PBMC Membrane 42 25 Nelfinavir PBMC Cytosol 0 0
[2032] Uptake and Cell Distribution of Metabolites and Intact
Prodrugs Following 1 hr Pulse/22 hr Chase Incubation of 10 uM PI
Prodrugs and Nelfinavir with Quiescent PBMC is shown in FIG. 11 and
FIG. 12.
6. PBMC Extract/Dog Plasma/Human Serum Stability of Selected PI
Prodrug Candidates
[2033] The in vitro metabolism and stability of the PI phosphonate
prodrugs were determined in PBMC extract, dog plasma and human
serum. Biological samples listed below (120 .mu.l) were transferred
into an 8-tube strip placed in the aluminum 37.degree. C. heating
block/holder and incubated at 37.degree. C. for 5 min. Aliquots
(2.5 mL) of solution containing 1 mM of test compounds in DMSO,
were transferred to a clean 8-tube strip, placed in the aluminum
37.degree. C. heating block/holder. 60 .mu.L aliquots of 80%
acetonitrile/20% water containing 7.5 .mu.M of amprenavir as an
internal standard for HPLC analysis were placed into five 8-tube
strips and kept on ice/refrigerated prior to use. An enzymatic
reaction was started by adding 120 .mu.L aliquots of a biological
sample to the strip with the test compounds using a multichannel
pipet. The strip was immediately vortex-mixed and the reaction
mixture (20 .mu.L) was sampled and transferred to the Internal
Standard/ACN strip. The sample was considered the time-zero sample
(actual time was 1-2 min). Then, at specific time points, the
reaction mixture (20 .mu.L) was sampled and transferred to the
corresponding IS/ACN strip. sampling times were 6, 20, 60 and 120
min. When all time points were sampled, an 80 .mu.L aliquot of
water was added to each tube and strips were centrifuged for 30 min
at 3000.times.G. The supernatants were analyzed with HPLC under the
following conditions:
Column: Inertsil ODS-3, 75.times.4.6 mm, 3 .mu.m at 40.degree.
C.
Mobile Phase A: 20 mM ammonium acetate in 10% ACN/90% water
Mobile Phase B 20 mM ammonium acetate in 70% ACN/30% water
Gradient: 20% B to 100% B in 4 min, 2 min 100% B, 2 min 20% B
Flow Rate: 2 mL/min
Detection: UV at 243 nm
Run Time: 8 min
[2034] The biological samples evaluated were as follows:
[2035] PBMC cell extract was prepared from fresh cells using a
modified published procedure (A. Pompon, I. Lefebvre, J.-L. Imbach,
S. Kahn, and D. Farquhar, Antiviral Chemistry & Chemotherapy,
5, 91-98 (1994)). Briefly, the extract was prepared as following:
The cells were separated from their culture medium by
centrifugation (1000 g, 15 min, ambient temperature). The residue
(about 100 .mu.L, 3.5.times.10.sup.8 cells) was resuspended in 4
ml, of a buffer (0.010 M HEPES, pH 7.4, 50 mM potassium chloride, 5
mM magnesium chloride and 5 mM dl-dithiothreitol) and sonicated.
The lysate was centrifuged (9000 g, 10 min, 4.degree. C.) to remove
membranes. The upper layer (0.5 mg protein/mL) was stored at
-70.degree. C. The reaction mixture contained the cell extract at
about 0.5 mg protein/mL.
[2036] Human serum (pooled normal human serum from George King
Biomedical Systems, Inc.). Protein concentration in the reaction
mixture was about 60 mg protein/mL.
[2037] Dog Plasma (pooled normal dog plasma (EDTA) from Pel Freez,
Inc.). Protein concentration in the reaction mixture was about 60
mg protein/mL. TABLE-US-00076 TABLE 5a PBMC Extract/Dog
Plasma/Human Serum Stability of Selected PI Prodrugs PBMC Dog Human
Extract.sup.1 Plasma Serum HIV EC.sub.50 GS# T.sub.1/2, min
T.sub.1/2, min T.sub.1/2, min (nM) 16503 2 368 >>400 6.0 .+-.
1.4 16571 49 126 110 15 .+-. 5 17394 15 144 49 20 .+-. 7
Example: Pharmacokinetics in Plasma and PBMC Following Intravenous
or Oral Administration of Candidate Compounds to Beagle Dogs;
Method for Determining Intracellular Residence Time
[2038] The pharmacokinetics of several candidate compounds and
their active metabolites were studied in beagle dogs following
intravenous or oral administration of each candidate compound.
[2039] Dose Administration and Sample Collection. Each dosing group
consisted of 3 male beagle dogs that were fasted overnight before
dosing. For intravenous administration, each dog was dosed with the
candidate compound at 1 mg/kg via the cephalic vein as a slow bolus
injection over approximately 1 minute. Blood samples (1-2 mL) were
collected from the jugular vein pre-dose, and at 2 min, 15 min, 30
min, 1 hr, 2 hr, 4 hr, 8 hr and 24 hr post-dose into tubes
containing EDTA as the anticoagulant. For oral administration, each
dog was dosed with the candidate compound at 4 mg/kg through oral
gavage. Blood samples (1-2 mL) were collected pre-dose, and at 5
min, 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr and 12 hr post-dose
into tubes containing EDTA as the anticoagulant. The blood samples
were stored on ice and plasma samples were obtained by
centrifugation within 1 hour after blood collection. The plasma
samples were stored at approximately -70.degree. C. until analysis
for the concentrations of the candidate compound and its
metabolites in plasma.
[2040] Another set of blood samples was also collected from the
jugular vein for evaluation of the concentrations of candidate
compound and its metabolites in peripheral blood mononuclear cells
(PBMCs). Approximately 8 ml, of blood was collected either at 1 hr,
4 hr, 8 hr and 24 hr post-dose or at 2 hr, 8 hr and 24 hr post-dose
from the jugular vein into tubes containing EDTA as the
anticoagulant. An equal volume of sterile phosphate buffered saline
(PBS) was (Amersham Biosciences) in a 50 ml, conical tube. The tube
was centrifuged at approximately 500 g for 30 min at room
temperature. The upper layer containing plasma was drawn off and
discarded. The layer below the plasma layer is enriched with PBMCs.
This layer was collected with a clean pipette and transferred to a
15 ml, conical tube. The PBMC suspension was centrifuged at
approximately 500 g for 10 min at room temperature. The resulting
pellet was resuspended in 5 ml, of sterile PBS and then centrifuged
at approximately 500 g for 10 min at room temperature. The
supernatant was removed and 0.5 ml, of acetonitrile was added to
the pellet. The tube was vortexed, sealed and stored at -70.degree.
C. until analysis for concentrations of the candidate compound and
its metabolites.
[2041] Determination of the concentrations of the candidate
compound and its metabolites in plasma. The plasma concentrations
of the candidate compound and its metabolites were determined by an
LC/MS/MS assay. The plasma samples were processed with a solid
phase extraction (SPE) procedure outlined below. Speedisk C18 solid
phase extraction cartridges (1 mL, 20 mg, 10 um, from J. T. Baker)
in a 96-well plate were conditioned with 200 uL of methanol
followed by 200 uL of water. An aliquot of 200 uL of plasma sample
was applied to each cartridge, followed by two washing steps each
with 200 uL of deionized water. The analytes were eluted from the
cartridges by a two-step process each with 125 uL of methanol. Each
well was added 50 uL of water and mixed to reduce the organic
strength. An aliquot of 25 uL of the mixture was injected onto a
ThermoFinnigan TSQ Quantum LC/MS/MS system.
[2042] The column used in liquid chromatography (LC) was
HyPURITY.RTM. C18 (50.times.2.1 mm, 3.5 um) from Thermo-Hypersil.
Mobile phase A contained 10% acetonitrile in 10 mM ammonium
formate, 0.1% formic acid. Mobile phase B contained 90%
acetonitrile in 10 mM ammonium formate, 0.1% formic acid. The
chromatography was carried out at a flow rate of 250 .mu.L/min
under an isocratic condition of 40% mobile phase A and 60% mobile
phase B. Selected reaction monitoring (SRM) were used to measure
the candidate compound and its metabolites simultaneously with the
positive ionization mode on the electrospray probe. The limit of
quantitation (LOQ) was 1 nM for the candidate compound and its
metabolites in plasma.
Determination of the Concentrations of the Candidate Compound and
its Metabolites in PBMCs.
[2043] The concentrations of the candidate compound and its
metabolites in PBMCs were determined by an LC/MS/MS assay. The PBMC
samples were filtered through a Captiva.TM. filtration plate with
0.2 .mu.m pore size. An aliquot of 250 .mu.L of the filtrate was
evaporated under a stream of nitrogen. The samples were
reconstituted in 75 .mu.L of 20% acetonitrile in 0.1% formic acid.
An aliquot of 25 uL of the solution was injected onto a
ThermoFinnigan TSQ Quantum LC/MS/MS system.
[2044] The column used in liquid chromatography was HyPURITY.RTM.
C18 (50.times.2.1 mm, 3.5 um) from Thermo-Hypersil. Mobile phase A
(MPA) contained 10% acetonitrile in 10 mM ammonium formate, 0.1%
formic acid. Mobile phase B (MPB) contained 90% acetonitrile in 10
mM ammonium formate, 0.1% formic acid. The chromatography was
carried out at a flow rate of 300 .mu.L/min with a gradient elution
program: 5% MPB from 0 to 1.5 min; 5-95% MPB from 1.5 to 1.6 min;
95% MPB from 1.6 to 3.5 min; 95-5% MPB from 3.5 to 3.6 min; 5% MPB
till the end of the program (6 min). The first 2 min of the LC flow
was diverted to waste to alleviate salt buildup in the probe of the
mass spectrometer. Selected reaction monitoring was used to measure
the candidate compound and its metabolites simultaneously with the
positive ionization mode on the electrospray probe. The limit of
quantitation (LOQ) was 0.1 nM for the candidate compound and its
metabolites in PBMC suspension.
[2045] Pharmacokinetic Calculations. The pharmacokinetic parameters
were calculated using WinNonlin. Noncompartmental analysis was used
for all pharmacokinetic calculation. The intracellular
concentrations in PBMCs were extrapolated from the measured
concentrations in PBMC suspension on the basis of a reported volume
of 0.2 picoliter/cell (B. L. Robins, R. V. Srinivas, C. Kim, N.
Bischofberger, and A. Fridland, (1998) Antimicrob. Agents
Chemother. 42, 612).
[2046] Pharmacokinetic Profiles in Plasma and PBMC. Shown below are
the concentration-time profiles of three phosphonate candidate
compounds (GS-1, GS-2 and GS-3) and their metabolites in plasma and
PBMCs following intravenous administration of each candidate
compound at 1 mg/kg in dogs. The last profile shows the
concentration-time profiles of GS-3 and its metabolites in plasma
and PBMC following oral administration of GS-3 at 4 mg/kg in dogs.
The chemical structures of the candidate compounds and their
metabolites are shown in Table 1aa. The data demonstrate that the
candidate active components (metabolite X and diacid) into cells
that are primarily associated with HIV activity, and that the
half-lives of the active components in these cells are much longer
than in plasma TABLE-US-00077 TABLE 1aa Chemical Structures of
Candidate compounds and Their Metabolites. Metabolites Candidate
compound Metabolite X (MX) Diacid GS-1 ##STR835## ##STR836##
##STR837## GS-2 ##STR838## ##STR839## ##STR840## GS-3 ##STR841##
##STR842## ##STR843##
[2047] Pharmacokinetic profiles of GS-1 and its metabolites in
plasma and PBMCs following intravenous administration of GS-1 at 1
mg/kg in dogs are shown in FIG. 13.
[2048] Pharmacokinetic profiles of GS-2 and its metabolites in
plasma and PBMCs following intravenous administration of GS-2 at 1
mg/kg in dogs are shown in FIG. 14.
[2049] Pharmacokinetic profiles of GS-3 and its metabolites in
plasma and PBMCs following intravenous administration of GS-3 at 1
mg/kg in dogs are shown in FIG. 15.
[2050] Pharmacokinetic profiles of GS-3 and its metabolites in
plasma and PBMCs following oral administration of GS-3 at 4 mg/kg
in dogs are shown in FIG. 16. Example: Purification and Biochemical
Characterization of GS-7340 Ester Hydrolase: ##STR844## Metabolism
of GS-7340:
[2051] There is broad consensus that the bioactivation of
nucleotide amidate triesters follows a general scheme (FIG. 1)
(Valette, 1996; McGuigan, 1998a, 1998b; Saboulard, 1999; Siddiqui,
1999). Step A is the hydrolysis of the amino acid carboxylic ester.
A nucleophilic attack by the carboxylic acid of the phosphorous
(Step B) is believed to initiate the formation of the 5-membered
cyclic intermediate which in turn is quickly hydrolyzed to the
monoamidate diester (referred to as the amino acid nucleoside
monophosphate, AAM, or metabolite X, Step C). This compound is
considered an intracellular depot form of the antiviral nucleoside.
Various enzymes as well as non-enzymatic catalysis have been
implicated in Step D which is the hydrolysis of the amide bond
resulting in the formation of the nucleotide. The nucleotide is
activated by enzymatic phosphorylation to nucleotide di- and
tri-phosphates.
[2052] In the case of GS-7340, the efficient conversion of this
pro-drug to the amino acid nucleoside monophosphate (Metabolite X,
FIG. 2) is a necessary step for the observed accumulation of
Metabolite X is peripheral blood mononuclear cells (PBMC).
Purification of the Enzyme(s) responsible for the cleavage of
GS-7340 amino acid carboxylic ester resulting in the formation of
Metabolite X is the subject of this example.
Ester Hydrolase Assay:
[2053] The enzymatic production of metabolite X from GS-7340 was
monitored using the following Ester Hydrolase assay: Varying
amounts of peripheral blood mononuclear cell (PBMC) extracts,
column fractions or pools were incubated with [.sup.14C] GS-7340 at
37.degree. C. for 10-90 min. The production of [.sup.14C]
Metabolite X was monitored by measuring the amount of radioactivity
retained on an anion exchange resin (DE-81). HPLC and mass
spectrometry analysis of the reaction mixture and radioactivity
retained on the filter confirmed that only [.sup.14C]-Metabolite X
bound the DE-81 filter. Under the assay conditions, the more
hydrophobic [.sup.14C] GS-7340 is not retained on the DE-81
membrane. The final reaction conditions were: 25 mM
2-[N-morpholino]ethanesulfonic acid (MES), pH 6.5, 100 nM NaCl, 1
mM DTT, 30 .mu.M [.sup.14C] GS-7340, 0.1% NP40 and varying amounts
of enzyme in a final volume of 60 .mu.l. The reaction mixture was
incubated at 37.degree. C. and at 10, 30 and 90 minutes, 17 .mu.l
of the reaction mixture was spotted onto a DE-81 filter. The filter
was washed with 25 mM Tris, pH 7.5 100 mM NaCl, dried at room
temperature, placed in vials containing 5 ml of scintillation
fluid. [.sup.14C]-Metabolite X present on the filters was
determined using a scintillation counter (LS 6500, Beckman, _).
Activity was expressed as pmoles Metabolite X
produced/minute/volume enzyme sample. Ester Hydrolase Specific
Activity was expressed as pmoles Metabolite X produced/minute/.mu.g
protein.
Non-Specific Esterase Assay:
[2054] Non-specific ester hydrolase activity was monitored by
monitoring the enzymatic cleavage of alpha napthyl acetate (ANA)
(Mastropaolo, W and Yourno, J 1981). This substrate has been used
for both the measurement esterase enzyme activity and in situ
staining of esterases in tissue samples (Yourno, J and Mastropaolo,
W. 1981; Yourno, J et A11981; Yourno, J et al 1986). The method
described is a modification of the assay described by Mattes, P M
and Mattes, W B, 1992). Varying amounts of peripheral blood
mononuclear cell (PBMC) extracts column fractions or pools were
incubated with ANA at 37.degree. C. for 20 min. The final reaction
conditions were: 10 mM sodium phosphate, pH 6.5, 97 .mu.M ANA and
varying amounts of enzyme in a final volume of 150 .mu.l. The
reaction mixture was incubated at 37.degree. C. and at 20 minutes,
and the reaction was stopped by the addition of 20111 of 10 mM Blue
salt RR in 10% sodium dodecyl sulfate (SDS). The alpha napthyl-Blue
salt RR product was detected by reading absorbance at 405 nm.
Activity was expressed as pmoles product produced/minute/volume
enzyme sample.
Extraction of GS-7340 Ester Hydrolase from Human PBMCs:
[2055] Fresh human PBMC were obtained from patients undergoing
leukophoresis; cells were shipped in plasma and processed within 26
h of draw. PBMC cells were harvested by centrifugation at
1200.times.g for 5 minutes and washed three times by re-suspension
in RBC lysis buffer (155 mM NH.sub.4Cl, 1 mM EDTA, 10 mM
KHCO.sub.3). Washed cells (29.times.10.sup.9) were suspended in 150
ml of lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 20 mM
CaCl.sub.2, 1 mM DTT and 1% NP40) and incubated on ice for 20
minutes. The PBMC crude extract was centrifuged at 1000.times.g for
30 min to remove unlysed cells and the supernatant at 100,000 X g
for 1 h. The 100,000.times.g supernatant (PBMC Extract: PO) was
harvested (165 ml) and the pellets (1000.times.g and
100,000.times.g pellets) were resuspended in 10 mM Tris, pH 7.4,
150 mM NaCl, 20 mM CaCl.sub.2, 1 mM DTT and assayed for GS-GS-7340
ester hydrolase activity. Assays showed that <2% of the
GS-GS-7340 Ester Hydrolase enzymatic activity was present in the
pellets. The cell extract was snap frozen in liquid Nitrogen and
stored at -70.degree. C.
Anion Exchange Chromatography:
[2056] The PBMC Extract (15.times.10.sup.9 cells, 75-85 ml) was
diluted 1:10, (vol:vol) with 25 mM Tris, pH 7.5, 10% glycerol, 1 mM
DTT (Q15 Buffer A) and loaded onto an anion exchange column (2.5
cm.times.8.0 cm, Source Q15 (Amersham Biosciences)), previously
equilibrated with Q15 Buffer A. Bound protein was eluted with a
linear NaCl gradient (30 column volumes (CV)) to 0.5M NaCl. Eluting
protein was detected by monitoring Absorbance at 280 nm. Fractions
(12.0 ml) were collected and assayed for both GS-7340 Ester
Hydrolase and ANA Esterase activity. GS-7340 Ester Hydrolase
activity eluted as a single major peak at 50-75 mM NaCl (Table 1).
Recovery of Total GS-7340 Ester Hydrolase activity in the eluted
fractions was 50-65% of total activity loaded. Significant ANA
Esterase activity (30-40% of total activity loaded) was detected in
the column FT; however, 30% eluted in two peaks at 70-100 mM NaCl
(Table 1). Fractions containing GS-7340 Ester Hydrolase activity
(Q15 pool) were pooled, snap frozen in liquid nitrogen and stored
at -70.degree. C.
Hydrophobic Interaction (HIC) Chromatography:
[2057] The Q15 pool was defrosted and diluted 1:1, (vol:vol) with
25 mM Tris, pH 8.0, 0.5 M (NH.sub.4).sub.2SO.sub.4, 1 mM DTT, 10%
glycerol BS-HIC Buffer A). 1M (N).sub.2SO.sub.4 was added to yield
a final concentration of 0.5M (NH.sub.4).sub.2SO.sub.4 in the
sample. The sample (300 ml/10.times.10.sup.9 cells) was loaded onto
a Butyl Sepharose HIC column (5 ml HiTrap, Amersham Biosciences)
previously equilibrated with BS-HIC Buffer A. Bound protein was
eluted with a linear gradient (15 CV) decreasing to with 25 mM
Tris, pH 8.0, 1 mM DTT, 10% glycerol. Eluting protein was detected
by monitoring Absorbance at 280 nm. Fractions (4.0 ml) were
collected and assayed for both GS-7340 Ester Hydrolase and ANA
Esterase activity. GS-GS-7340 Ester Hydrolase activity eluted as a
single major peak at 200-75 mM (NH.sub.4).sub.2SO.sub.4 (Table 1).
Recovery of Total GS-7340 Ester Hydrolase activity in the eluted
fractions was 50-65% of total activity loaded (Table 1).
Significant ANA Esterase activity (85% of total activity loaded)
was detected in the column FT; however, .about.10-15% eluted in a
peak at 450-300 mM (NH.sub.4).sub.2SO.sub.4. Fractions containing
GS-7340 Ester Hydrolase activity (BS-HIC pool) were pooled, snap
frozen in liquid nitrogen and stored at -70.degree. C.
Hydroxyapatite (HAP) Chromatography:
[2058] The BS-HIC pool (40 ml/10.times.10.sup.9 cells) was
defrosted, concentrated to 2.0 ml using a 10 kDa molecular weight
cutoff concentrator (20 ml Vivaspin concentrator, Viva Science,
Carlsbad, Calif.), and diluted to 20 ml with 1 mM sodium phosphate,
pH 6.85, 10% glycerol, 1 mM DTT (HAP Buffer A). The sample
containing the GS-7340 Ester Hydrolase activity was loaded onto a
HAP column (0.75 ml, 5 mm.times.20 mm; ceramic hydroxyapatite,
BioRad, Hercules, Calif.), previously equilibrated with HAP Buffer
A. Bound protein was eluted with a 40 CV gradient to 500 mM sodium
phosphate, pH 6.85, 10% glycerol, 1 mM DTT. Eluting protein was
detected by monitoring Absorbance at 280 nm. Fractions (0.5 ml)
were collected and assayed for GS-7340 Ester Hydrolase. GS-7340
Ester Hydrolase activity eluted as a single major peak at 70-85 mM
sodium phosphate (Table 1a). Recovery of Total GS-7340 Ester
Hydrolase activity in the eluted fractions was 40-45% of total
activity loaded (Table 1a). Fractions containing GS-7340 Ester
Hydrolase activity (HAP pool) were pooled, snap frozen in liquid
nitrogen and stored at -70.degree. C.
High Resolution Gel Filtration Chromatography:
[2059] The BS-HIC pool (5 ml/1.25.times.10.sup.9 cells) was
defrosted, concentrated to 0.05 ml using a 5 kDa molecular weight
cutoff concentrator (20 ml Vivaspin concentrator, Viva Science,
Carlsbad, Calif.), and loaded onto a high resolution Gel Filtration
column (8 mm.times.300 mm, KW 802.5; Shodex, Thomas Instrument Co.,
Oceanside, Calif.), previously equilibrated with 25 mM Tris, pH
7.5, 150 mM NaCl, 10% glycerol, 20 mM CaCl.sub.2, 1 mM DTT (KW
802.5 column buffer). Eluting protein was detected by monitoring
Absorbance at 280 nm. Fractions (0.5 ml) were collected and assayed
for GS-7340 Ester Hydrolase. GS-7340 Ester Hydrolase activity
eluted as a single major peak at in fractions corresponding to an
apparent molecular weight of 70-100 kDa (Table 1a). Recovery of
Total GS-7340 Ester Hydrolase activity in the eluted fractions was
>75% of total activity loaded (Table 1a). Fractions containing
GS-7340 Ester Hydrolase activity (KW 802.5 pool) were pooled, snap
frozen in liquid nitrogen and stored at -70.degree. C.
Summary of GS-7340 Ester Hydrolase Purification:
[2060] The following table summarizes the purification of GS-7340
Ester Hydrolase achieved. Protein was measured by a Coomassie Blue
stain colorometric assay (Bradford Protein Assay, BioRad, Hercules,
Calif.). The Specific Activity (pmoles Metabolite X
produced/minute/.mu.g protein) of the partially purified GS-7340
Ester Hydrolase varied from 666 to 1500. This represents a 222-750
fold purification from the PBMC extracts. Overall Recovery of
GS-7340 Ester Hydrolase from PBMC extracts was approximately 10%.
TABLE-US-00078 TABLE 1c Purification Summary of GS-7340 Ester
Hydrolase: Protein Specific Sample concentration Volume Protein
Total Activity Activity % name PBMC (mg/ml) (ml) (mg) (pmol/min)
pmol/min/.mu.g Recovery P0 PBMC 30 .times. 10.sup.9 5.0 200 1000
2.0-3.0 .times. 10.sup.6 2.0-3.0 Q15 Pool 0.116-0.167 300 35-50
1.0-1.5 .times. 10.sup.6 20-42 .about.50 BS-HIC 0.02-0.035 100
2.0-3.5 0.5-0.75 .times. 10.sup.6 142-375 .about.50 Pool HAP Pool
0.02-0.03 10 0.2-0.3 0.2-0.3 .times. 10.sup.6 666-1500 .about.40 %
Total .about.10 Recovery
Biochemical Characterization of GS-7340 Ester Hydrolase:
Determination of the Isoelectric Point (pI) of GS-7340 Ester
Hydrolase:
[2061] The isoelectric point (pI) of a protein is defined as the pH
at which the protein has no net ionic charge. Chromatofocusing is a
chromatographic procedure in which a negatively charged protein is
bound to a hydrophilic column with a net positive ionic charge. The
protein is loaded at a pH 1 to 2 pH units higher that its estimated
pI, and the bound protein is eluted by generating a decreasing pH
gradient using a pH 3.0 to 4.0 buffer. The proteins will be eluted
at a pH corresponding to pI.
[2062] An aliquot of the BS HIC pool (20 ml, 5.times.10.sup.9
cells) was concentrated to 4.0 ml and prepared for chromatofocusing
chromatography by exchanging buffer using a desalting column. 1.0
ml aliquots of the concentrated BS HIC pool were loaded onto a 5.0
ml desalting column (5.0 ml HiTrap, Amersham Biosciences,
Piscataway, N.J.) previously equilibrated with 25 mM ethanolamine,
pH 7.8 (pH'd with iminodiacetic acid), 10% glycerol (Mono P Buffer
A). The desalted GS-7340 Ester Hydrolase activity was loaded onto a
chromatofocusing column (5 mm.times.5 mm HR Mono P, Amersham
Biosciences, Piscataway, N.J.) previously equilibrated with Mono P
Buffer A. Bound protein was eluted with a 20CV gradient to pH 3.6
with 10 ml/100 ml Polybuffer 74 (Amersham Biosciences) pH'd to 4.0
with iminodiacetic acid. This chromatofocusing protocol produces a
linear pH gradient from pH 7.8 to pH 3.6. Eluting protein was
detected by monitoring Absorbance at 280 nm. Fractions (0.5 ml)
were collected and assayed for GS-7340 Ester Hydrolase. GS-7340
Ester Hydrolase activity eluted as a single major peak at pH 5.5 to
4.5. Recovery of Total GS-7340 Ester Hydrolase activity in the
eluted fractions was 65-70% of total activity loaded. Fractions
containing GS-7340 Ester Hydrolase activity (KW 802.5 pool) were
pooled, snap frozen in liquid nitrogen and stored at -70.degree.
C.
Inhibition of GS-7340 Ester Hydrolases by Serine Hydrolase
Inhibitors:
[2063] Fluorophosphonate/fluorophosphate
(Diisopropylfluorophosphate (DFP)) derivatives, isocoumarins such
as 3,4 dichloroisocoumarin (3,4-DCI) and peptide carboxyl esters of
chloro- and fluoro-methyl ketones (AlaAlaProAla-CMK,
AlaAlaProVal-CMK, PheAla-FMK) are known effective inhibitors of
serine hydrolases (Powers and Harper 1986; Delbaere and Brayer,
1985; Bullock et al 1996; Yongsheng et al 1999; Kam et al 1993).
Inhibition of the enzymatic production of metabolite X from GS-7340
was monitored using the following Ester Hydrolase Inhibition assay:
Varying amounts of partially purified GS-7340 Ester Hydrolase and
control enzymes (human leukocyte elastase (huLE), porcine liver
carboxylesterase (PLCE)) were incubated with [.sup.14C] GS-7340 in
the presence and absence of varying amounts of known serine
hydrolase inhibitors at 37.degree. C. for 10-90 min. The production
of [.sup.14C] Metabolite X was monitored by measuring the amount of
radioactivity retained on an anion exchange resin (DE-81). The
final reaction conditions were: 25 mM
2-[N-morpholino]ethanesulfonic acid (MES), pH 6.5, 100 mM NaCl, 1
mM DTT, 30 .mu.M [.sup.14C] GS-7340, 0.1% NP40 varying amounts of
enzyme and inhibitors (1.0 .mu.M-1 mM) in a final volume of 60
.mu.l. The reaction mixture was incubated at 37.degree. C. and at
10, 30 and 90 minutes, 17 .mu.l of the reaction mixture was spotted
onto a DE-81 filter. The filter was processed and the amount of
[.sup.14C]-Metabolite X present was determined as described above.
Activity was expressed as pmoles Metabolite X
produced/minute/volume enzyme sample. Inhibition of Ester Hydrolase
and control hydrolases was expressed as percent activity present at
a given concentration of inhibitor compared to hydrolase activity
in the absence of the inhibitor. The results of the inhibition
experiments are shown in Table 2A/B. The serine hydrolase
inhibitors, 3,4-DCI and DFP inhibit GS-7340 Ester Hydrolase with
estimated IC50's of 4.0 and 30 .mu.M, respectively. The peptide
chloro- and fluoro-methyl ketones are less effective inhibitors
with estimated IC50's of 100-400 .mu.M (Table 2 A/B).
TABLE-US-00079 TABLE 2A Inhibition of GS-7340 Ester Hydrolase and
Control Enzymes by Serine Hydrolase Inhibitors IC50 (.mu.M) GS-7340
Ester Inhibitor Hydrolase PLCE huLE 3,4- 4.0 250 3.0
dichloroisocoumarin MeOSuC-Ala-Ala-Pro- 200-400 >1000 60 Ala-CMK
MeOSuc-Ala-Ala-Pro- 100 >1000 4.0 Val-CMK Biotin-Phe-Ala-FMK 100
>1000 100 DFP 30 0.05 --
[2064] TABLE-US-00080 TABLE 2B Inhibition of GS-7340 Ester
Hydrolase and Control Enzymes by Serine Hydrolase Inhibitors
Inhibitor Relative Activity IC50 (.mu.M) (%) (.mu.M) GS-7340 Ester
Hydrolase 3,4- 1.0 100 4.0 dichloroisocoumarin 10 25 100 5 1000
<2 DFP 1.0 100 30-40 10 90 100 35 1000 <2 Biotin-Phe-Ala-FMK
1.0 100 100 10 95 100 50 1000 <2 PLCE 3,4- 1.0 100 250
dichloroisocoumarin 10 100 100 90 1000 20 DFP 0.001 100 0.05 0.01
90 0.1 20 1.0 <2 Biotin-Phe-Ala-FMK 1.0 100 >1000 10 100 100
100 1000 80 huLE 3,4- 1.0 100 4.0 dichloroisocoumarin 10 25 100 5
1000 <2 Biotin-Phe-Ala-FMK 1.0 100 100 10 93 100 48 1000
<2
Summary of Biochemical Characterization of GS-7340 Ester
Hydrolase:
[2065] Summarizing, QS-7340 Ester Hydrolase is a novel enzyme
characterized by being capable of being recovered from human PBMCs
by a process comprising [2066] (a) lysing human PBMCs; [2067] (b)
extracting the lysed cells with detergent; [2068] (c) separating
the solids from supernatant and recovering the supernatant; [2069]
(d) contacting the supernatant with an anion exchange medium;
[2070] (e) eluting the Hydrolase from the anion exchange medium;
[2071] (f) contacting the eluate with a hydrophobic chromatographic
medium; and [2072] (g) eluting the Hydrolase from the hydrophobic
chromatographic medium.
[2073] GS-7340 Ester Hydrolase is useful in screening candidate
compounds to assess the likelihood that they can be processed to
form depot metabolites in lymphoid tissue. The candidates are
assayed in the same fashion as described herein for GS-7340, taking
into account differences in the nature of the suspected substrate
as will be apparent to the ordinary artisan.
[2074] GS-7340 Ester Hydrolase optionally is labelled with a
detectable group such as a radiolabel or covalently bound to an
insoluble matrix such as Sepharose using techniques heretofore
employed for other enzymes having similar properties, as will be
apparent to the ordinary artisan.
[2075] GS-7340 Ester Hydrolase has the following properties: [2076]
1) GS-7340 Ester Hydrolase can be partially purified from fresh
PBMC Extracts: SA=666-1500 pmoles MetX/min/ug protein. [2077] 2)
GS-7340 Ester Hydrolase can be separated from non-specific
Esterases capable of cleaving alpha-naphthyl acetate (ANA), a
non-specific substrate shown to be cleaved by many
carboxylesterases and hydrolases. [2078] 3) Multiple GS-7340 Ester
Hydrolase activity peaks are not eluted from columns during
purification. [2079] 4) The MW of GS-7340 Ester Hydrolase on Gel
Filtration is .about.70-100 kDa [2080] 5) The pI of GS-7340 Ester
Hydrolase is pH 4.5-5.5 [2081] 6) Evidence to date suggests that
the SA of isolated GS-7340 Ester Hydrolase is likely to be
>10,000. [2082] 7) The serine hydrolase inhibitors, 3,4-DCI and
DFP inhibit GS-7340 Ester Hydrolase with estimated IC50's of 4.0
and 30 .mu.M, respectively. The peptide chloro- and fluoro-methyl
ketones are less effective inhibitors with estimated IC50's of
100-400 .mu.M (Table 2 A/B).
REFERENCES
[2082] [2083] Bullock, T L et al 1996 J Mol Biol 255: 714-725.
[2084] Detbaere, L T and Brayer, G D 1985 J Mol Biol 183:89-103
[2085] Kam C et al 1993 Bioconjugate Chem. 4: 560-567 [2086]
Mastropaolo, W and Yourno, J 1981 Analytical Chemistry 115: 188-193
[2087] Mattes, P M, and Mattes, W B, 1992. Toxicol. Appl.
Pharmacol. 114:71-76 [2088] McGuigan, C P W et al 1998a Antiviral
Chem and Chemotherapy 9: 109-115 [2089] McGuigan, C P W et al 1998b
Antiviral Chem and Chemotherapy 9: 473-479 [2090] Powers, J C and
Harper, J W 1986 Inhibitors of serine proteinases. In Proteinase
Inhibitors (A J Barrett and G Salvesen, Eds.) Elsevier, Amsterdam,
N.Y., Oxford, pp 55-152) [2091] Saboulard, D L et al 1999 Molec
Pharmacol 56:693-704 [2092] Siddiqui, A Q C and McGuigan, C P W
1999 J Med Chem. 42:4122-4128 [2093] Valette, G A et al 1996 J Med
Chem. 39:1981-1990 [2094] Yongsheng, the linker et al 1999 Proc
Natl Acad Sci 96:14694-14699 [2095] Yourno, J and Mastropaolo, W.
1981 Blood, 58:939-945 [2096] Yourno, J et al 1981. Blood, 60:
24-29 [2097] Yourno, J et al 1986 J Histochem and Cytochem
34:727-33)
Example: Candidate Compounds
[2098] A large number of examples describing the preparation of
candidate compounds active against HIV protease, HIV integrase and
HIV polymerase (non-nucleotide reverse transcriptase inhibitors, or
NNRTIs) are found in copending applications and are set forth
below. These compounds are examples of candidate compounds that are
typical of those which are suitable for use in the method and
libraries of this invention.
Incorporation by Reference
[2099] All publications and patent applications cited herein are
incorporated by reference to the same extent as if the full text of
each individual publication or patent application was contained
herein. The incorporated text will be apparent from context if not
specifically set forth. Incorporated by reference are (a) U.S.
patent application 60/373,533 and 60/375,665 hereof based on such
applications and (b) U.S. patent application 60/375,622 (attorney
docket 260.P) and the section 111 (a) application filed of even
date hereof based on such application. Further, the content of
PCT/US 03/12901 and PCT/US 03/12926 and in particular all
embodiments thereof relating to the claims herein are incorporated
by reference in their entirety.
* * * * *