U.S. patent application number 14/194520 was filed with the patent office on 2014-10-02 for inhibitors of human immunodeficiency virus replication.
This patent application is currently assigned to Gilead Sciences, Inc.. The applicant listed for this patent is Gilead Sciences, Inc.. Invention is credited to Murray D. Bailey, Francois Bilodeau, Rebekah J. Carson, Rene Coulombe, Lee Fader, Teddy Halmos, Stephen Kawai, Serge R. Landry, Steven LaPlante, Sebastien Morin, Mathieu Parisien, Marc-Andre Poupart, Bruno Simoneau, Youla S. Tsantrizos.
Application Number | 20140296228 14/194520 |
Document ID | / |
Family ID | 40638275 |
Filed Date | 2014-10-02 |
United States Patent
Application |
20140296228 |
Kind Code |
A1 |
Tsantrizos; Youla S. ; et
al. |
October 2, 2014 |
INHIBITORS OF HUMAN IMMUNODEFICIENCY VIRUS REPLICATION
Abstract
Compounds of formula I: ##STR00001## wherein R.sup.4, R.sup.6
and R.sup.7 are defined herein, are useful as inhibitors of HIV
replication.
Inventors: |
Tsantrizos; Youla S.;
(Laval, CA) ; Bailey; Murray D.; (Laval, CA)
; Bilodeau; Francois; (Laval, CA) ; Coulombe;
Rene; (Laval, CA) ; Halmos; Teddy; (Laval,
CA) ; Kawai; Stephen; (Laval, CA) ; Landry;
Serge R.; (Laval, CA) ; LaPlante; Steven;
(Laval, CA) ; Morin; Sebastien; (Laval, CA)
; Poupart; Marc-Andre; (Laval, CA) ; Simoneau;
Bruno; (Laval, CA) ; Fader; Lee; (Laval,
CA) ; Carson; Rebekah J.; (Laval, CA) ;
Parisien; Mathieu; (Laval, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gilead Sciences, Inc. |
Foster City |
CA |
US |
|
|
Assignee: |
Gilead Sciences, Inc.
Foster City
CA
|
Family ID: |
40638275 |
Appl. No.: |
14/194520 |
Filed: |
February 28, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13683761 |
Nov 21, 2012 |
8710230 |
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14194520 |
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12742997 |
Aug 19, 2010 |
8354429 |
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PCT/CA2008/001611 |
Sep 11, 2008 |
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13683761 |
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60988686 |
Nov 16, 2007 |
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Current U.S.
Class: |
514/230.2 ;
514/230.5; 514/291; 514/296; 514/311; 514/314 |
Current CPC
Class: |
C07D 215/14 20130101;
A61K 31/47 20130101; C07D 498/06 20130101; A61K 31/4741 20130101;
C07D 221/16 20130101; A61K 31/5383 20130101; A61P 43/00 20180101;
A61P 31/12 20180101; A61P 31/18 20180101; C07D 417/04 20130101;
C07D 491/06 20130101; A61K 31/538 20130101; C07D 215/18 20130101;
C07D 409/04 20130101; C07D 413/04 20130101; C07D 405/04 20130101;
C07D 401/04 20130101; A61K 31/473 20130101; A61K 31/4709
20130101 |
Class at
Publication: |
514/230.2 ;
514/311; 514/314; 514/230.5; 514/291; 514/296 |
International
Class: |
C07D 215/14 20060101
C07D215/14; C07D 405/04 20060101 C07D405/04; A61K 31/4709 20060101
A61K031/4709; C07D 409/04 20060101 C07D409/04; C07D 413/04 20060101
C07D413/04; A61K 31/538 20060101 A61K031/538; C07D 401/04 20060101
C07D401/04; C07D 491/06 20060101 C07D491/06; A61K 31/4741 20060101
A61K031/4741; C07D 417/04 20060101 C07D417/04; C07D 221/16 20060101
C07D221/16; A61K 31/473 20060101 A61K031/473; C07D 498/06 20060101
C07D498/06; A61K 31/5383 20060101 A61K031/5383; A61K 31/47 20060101
A61K031/47 |
Claims
1-38. (canceled)
39. A method of treating an HIV infection in a mammal having or at
risk of having the infection, the method comprising administering
to the mammal a therapeutically effective amount of a combination
of a compound of formula (I): ##STR00252## or a pharmaceutically
acceptable salt thereof, wherein: R.sup.4 is Het, wherein Het is
optionally substituted with 1 to 3 substituents each independently
selected from halo, (C.sub.1-6)alkyl, (C.sub.2-6)alkenyl,
(C.sub.1-6)haloalkyl, (C.sub.3-7)cycloalkyl, --OH,
--O(C.sub.1-6)alkyl, --SH, --S(C.sub.1-6)alkyl, --NH.sub.2,
--NH(C.sub.1-6)alkyl and --N((C.sub.1-6)alkyl).sub.2, wherein
(C.sub.1-6)alkyl is optionally substituted with hydroxy, cyano or
oxo; R.sup.6 and R.sup.7 are each independently selected from H,
halo, (C.sub.1-6)alkyl and (C.sub.1-6)haloalkyl; and Het is a 4- to
7-membered saturated, unsaturated or aromatic heterocycle having 1
to 4 heteroatoms each independently selected from O, N and S, or a
7- to 14-membered saturated, unsaturated or aromatic
heteropolycycle having wherever possible 1 to 5 heteroatoms, each
independently selected from O, N and S; or a salt thereof; and at
least one other antiviral agent.
40. The method according to claim 39 wherein R.sup.4 is Het
optionally substituted with 1 to 2 substituents each independently
selected from halo, (C.sub.1-3)alkyl and --O(C.sub.1-3)alkyl.
41. The method according to claim 40 wherein R.sup.4 is Het
optionally substituted with 1 to 2 substituents each independently
selected from Cl, F, CH.sub.3 and CH.sub.2CH.sub.3 wherein Het is a
7- to 14-membered saturated, unsaturated or aromatic
heteropolycycle having wherever possible 1 to 2 heteroatoms, each
independently selected from O, N and S.
42. The method according to claim 39 wherein R.sup.4 is selected
from: ##STR00253## optionally substituted with 1 to 3 substituents
each independently selected from halo, (C.sub.1-3)alkyl and
--O(C.sub.1-3)alkyl.
43. The method according to claim 42 wherein R.sup.4 is selected
from: ##STR00254## optionally substituted with 1 to 2 substituents
each independently selected from halo, (C.sub.1-3)alkyl and
--O(C.sub.1-3)alkyl.
44. The method according to claim 39 wherein R.sup.6 is H, F, Cl or
(C.sub.1-2)alkyl.
45. The method according to claim 44 wherein R.sup.6 is H or
CH.sub.3.
46. The method according to claim 39 wherein R.sup.7 is H, F, Cl or
CH.sub.3.
47. The method according to claim 46 wherein R.sup.7 is H or
CH.sub.3.
48. The method according to claim 39 wherein the compound of
formula (I) has following formula: ##STR00255## wherein R.sup.4,
R.sup.6 and R.sup.7 are defined as: TABLE-US-00006 R.sup.4 R.sup.6
R.sup.7 ##STR00256## H H.
49. The method according to claim 39 wherein the at least one other
antiviral agent comprises at least one NNRTI.
50. The method according to claim 39 wherein the at least one other
antiviral agent comprises at least one NRTI.
51. The method according to claim 39 wherein the at least one other
antiviral agent comprises at least one protease inhibitor.
52. The method according to claim 39 wherein the at least one other
antiviral agent comprises at least one entry inhibitor.
53. The method according to claim 39 wherein the at least one other
antiviral agent comprises at least one integrase inhibitor.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/683,761, filed Nov. 21, 2012; which is a
continuation of U.S. patent application Ser. No. 12/742,997, filed
Aug. 19, 2010 (now U.S. Pat. No. 8,354,429); which is a 371 of
PCT/CA2008/001611, filed Sep. 11, 2008; which claims the benefit of
U.S. Application No. 60/988,686, filed Nov. 16, 2007. The foregoing
applications are incorporated herein by reference in their
entireties.
SUBMISSION OF THE SEQUENCE LISTING ON ASClI TEXT FILE
[0002] The content of the following submission on ASClI text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
GILE-032.sub.--03 USSeqList.txt, date recorded: Feb. 28, 2014,
size: 3,691 bytes).
FIELD OF THE INVENTION
[0003] The present invention relates to compounds, compositions and
methods for the treatment of human immunodeficiency virus (HIV)
infection. In particular, the present invention provides novel
inhibitors of HIV replication, pharmaceutical compositions
containing such compounds and methods for using these compounds in
the treatment of HIV infection. More specifically, the present
invention provides novel inhibitors of the HIV integrase enzyme,
pharmaceutical compositions containing such compounds and methods
for using these compounds to reduce HIV replication and in the
treatment of HIV infection.
BACKGROUND OF THE INVENTION
[0004] Acquired immune deficiency syndrome (AIDS) is caused by the
human immunodeficiency virus (HIV), particularly the HIV-1 strain.
Most currently approved therapies for HIV infection target the
viral reverse transcriptase and protease enzymes. There is
additionally one approved drug targeting gp41 to inhibit viral
entry and one approved drug targeting the integrase enzyme. Within
the reverse transcriptase inhibitor and protease inhibitor classes,
resistance of HIV to existing drugs is a problem. Therefore, it is
important to discover and develop new antiretroviral compounds.
[0005] The inherent genetic variation within HIV has led to the
identification of many HIV mutants, commonly referred to as
variants, which exhibit altered drug susceptibility. On the
integrase enzyme, residues 124 and 125 are recognized as highly
variable across the HIV-1 virus from infected patients found in
major market and developing countries. The approximate prevalence
of these integrase variants are Thr124/Thr125 (44%), Ala124/Thr125
(17%), Ala124/Ala125 (16%), Thr124/Ala125 (10%), Asn124/Thr125
(6%), and Asn124/Ala125 (1%) for viruses sequenced from major
market countries reported in the Los Alamos database
(http://www.hiv.lanl.gov/content/hiv-db). These integrase variants
may be generated using known methods in the art and publicly
available polypeptide sequences for the integrase enzyme, for
example from the NL4.3 strain of HIV-1 integrase (SEQ ID NO:
1).
SUMMARY OF THE INVENTION
[0006] The present invention provides a novel series of compounds
having inhibitory activity against HIV replication. The compounds
of the present invention have an affinity for the HIV integrase
enzyme. Therefore, the compounds of the invention may be used to
inhibit the activity of HIV integrase and may be used to reduce HIV
replication. The compounds of the invention exhibit at least one of
the following surprising advantages: [0007] unexpectedly good
activity in a cell-based HIV-1 replication assay in four of the
major integrase variants at residues 124/125 (Thr124/Thr125,
Ala124/Thr125, Ala124/Ala125 and Thr124/Ala125); and/or [0008]
unexpectedly good activity in a cell-based HIV-1 replication assay
in all six of the major integrase variants at residues 124/125
(Thr124/Thr125, Ala124/Thr125, Ala124/Ala125, Thr124/Ala125,
Asn124/Thr125, and Asn124/Ala125); and/or [0009] unexpectedly good
pharmacological properties.
[0010] The compounds of the invention exhibit unexpectedly good
potency against four of major integrase variants at the 124/125
residues (.about.>85% natural abundance) and/or all six of the
major integrase variants at the 124/125 residues. The implication
of this unexpectedly good potency observed across the
aforementioned 124/125 variable residues is that some HIV infected
patients, who carry a virus with 124/125 variant residues of
integrase and who have a pre-existing anti-viral resistance against
drugs of this class, may be expected to respond to the compounds of
the invention.
[0011] Further objects of this invention arise for the one skilled
in the art from the following description and the examples.
[0012] One aspect of the invention provides an isomer, racemate,
enantiomer or diasteriomer of compounds of formula (I):
##STR00002##
wherein [0013] R.sup.4 is aryl or Het, wherein each of the aryl and
Het is optionally substituted with 1 to 3 substituents each
independently selected from halo, (C.sub.1-6)alkyl,
(C.sub.2-6)alkenyl, (C.sub.1-6)haloalkyl, (C.sub.3-7)cycloalkyl,
--OH, --O(C.sub.1-6)alkyl, --SH, --S(C.sub.1-6)alkyl, --NH.sub.2,
--NH(C.sub.1-6)alkyl and --N((C.sub.1-6)alkyl).sub.2; wherein the
(C.sub.1-6)alkyl is optionally substituted with hydroxy, cyano or
oxo; [0014] R.sup.6 and R.sup.7 are each independently selected
from H, halo, (C.sub.1-6)alkyl and (C.sub.1-6)haloalkyl; [0015]
wherein Het is a 4- to 7-membered saturated, unsaturated or
aromatic heterocycle having 1 to 4 heteroatoms each independently
selected from O, N and S, or a 7- to 14-membered saturated,
unsaturated or aromatic heteropolycycle having wherever possible 1
to 5 heteroatoms, each independently selected from O, N and S; or a
salt or an ester thereof.
[0016] Another aspect of the invention provides an isomer,
racemate, enantiomer or diasteriomer of compounds of formula
(I):
##STR00003##
wherein [0017] R.sup.4 is aryl or Het, wherein each of the aryl and
Het is optionally substituted with 1 to 3 substituents each
independently selected from halo, (C.sub.1-6)alkyl,
(C.sub.2-6)alkenyl, (C.sub.1-6)haloalkyl, (C.sub.3-7)cycloalkyl,
--OH, --O(C.sub.1-6)alkyl, --SH, --S(C.sub.1-6)alkyl, --NH.sub.2,
--NH(C.sub.1-6)alkyl and --N((C.sub.1-6)alkyl).sub.2; wherein the
(C.sub.1-6)alkyl is optionally substituted with hydroxy, cyano or
oxo; and wherein the aryl is not monosubstituted at the para
position; [0018] R.sup.6 and R.sup.7 are each independently
selected from H, halo, (C.sub.1-6)alkyl and (C.sub.1-6)haloalkyl;
[0019] wherein Het is a 4- to 7-membered saturated, unsaturated or
aromatic heterocycle having 1 to 4 heteroatoms each independently
selected from O, N and S, or a 7- to 14-membered saturated,
unsaturated or aromatic heteropolycycle having wherever possible 1
to 5 heteroatoms, each independently selected from O, N and S; or a
salt or an ester thereof.
[0020] Another aspect of this invention provides a compound of
formula (I), or a pharmaceutically acceptable salt or ester
thereof, exhibiting at least one of the following surprising
advantages: [0021] unexpectedly good activity in a cell-based HIV-1
replication assay in four of the major integrase variants at
residues 124/125 (Thr124/Thr125, Ala124/Thr125, Ala124/Ala125 and
Thr124/Ala125); and/or [0022] unexpectedly good activity in a
cell-based HIV-1 replication assay in all six of the major
integrase variants at residues 124/125 (Thr124/Thr125,
Ala124/Thr125, Ala124/Ala125, Thr124/Ala125, Asn124/Thr125, and
Asn124/Ala125); and/or [0023] unexpectedly good pharmacological
properties.
[0024] Another aspect of this invention provides a compound of
formula (I) or a pharmaceutically acceptable salt or ester thereof,
as a medicament.
[0025] Still another aspect of this invention provides a
pharmaceutical composition comprising a therapeutically effective
amount of a compound of formula (I) or a pharmaceutically
acceptable salt or ester thereof; and one or more pharmaceutically
acceptable carriers.
[0026] According to an embodiment of this aspect, the
pharmaceutical composition according to this invention additionally
comprises at least one other antiviral agent.
[0027] The invention also provides the use of a pharmaceutical
composition as described hereinabove for the treatment of an HIV
infection in a mammal having or at risk of having the
infection.
[0028] A further aspect of the invention involves a method of
treating an HIV infection in a mammal having or at risk of having
the infection, the method comprising administering to the mammal a
therapeutically effective amount of a compound of formula (I), a
pharmaceutically acceptable salt or ester thereof, or a composition
thereof as described hereinabove.
[0029] Another aspect of the invention involves a method of
treating an HIV infection in a mammal having or at risk of having
the infection, the method comprising administering to the mammal a
therapeutically effective amount of a combination of a compound of
formula (I) or a pharmaceutically acceptable salt or ester thereof,
and at least one other antiviral agent; or a composition
thereof.
[0030] Also within the scope of this invention is the use of a
compound of formula (I) as described herein, or a pharmaceutically
acceptable salt or ester thereof, for the treatment of an HIV
infection in a mammal having or at risk of having the
infection.
[0031] Another aspect of this invention provides the use of a
compound of formula (I) as described herein, or a pharmaceutically
acceptable salt or ester thereof, for the manufacture of a
medicament for the treatment of an HIV infection in a mammal having
or at risk of having the infection.
[0032] An additional aspect of this invention refers to an article
of manufacture comprising a composition effective to treat an HIV
infection; and packaging material comprising a label which
indicates that the composition can be used to treat infection by
HIV; wherein the composition comprises a compound of formula (I)
according to this invention or a pharmaceutically acceptable salt
or ester thereof.
[0033] Still another aspect of this invention relates to a method
of inhibiting the replication of HIV comprising exposing the virus
to an effective amount of the compound of formula (I), or a salt or
ester thereof, under conditions where replication of HIV is
inhibited.
[0034] Further included in the scope of the invention is the use of
a compound of formula (I) to inhibit the activity of the HIV
integrase enzyme.
[0035] Further included in the scope of the invention is the use of
a compound of formula (I), or a salt or ester thereof, to inhibit
the replication of HIV.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0036] As used herein, the following definitions apply unless
otherwise noted:
[0037] The term "substituent", as used herein and unless specified
otherwise, is intended to mean an atom, radical or group which may
be bonded to a carbon atom, a heteroatom or any other atom which
may form part of a molecule or fragment thereof, which would
otherwise be bonded to at least one hydrogen atom. Substituents
contemplated in the context of a specific molecule or fragment
thereof are those which give rise to chemically stable compounds,
such as are recognized by those skilled in the art.
[0038] The term "(C.sub.1-n)alkyl" as used herein, wherein n is an
integer, either alone or in combination with another radical, is
intended to mean acyclic, straight or branched chain alkyl radicals
containing from 1 to n carbon atoms. "(C.sub.1-6)alkyl" includes,
but is not limited to, methyl, ethyl, propyl (n-propyl), butyl
(n-butyl), 1-methylethyl (iso-propyl), 1-methylpropyl (sec-butyl),
2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), pentyl
and hexyl. The abbreviation Me denotes a methyl group; Et denotes
an ethyl group, Pr denotes a propyl group, iPr denotes a
1-methylethyl group, Bu denotes a butyl group and tBu denotes a
1,1-dimethylethyl group.
[0039] The term "(C.sub.2-n)alkenyl", as used herein, wherein n is
an integer, either alone or in combination with another radical, is
intended to mean an unsaturated, acyclic straight or branched chain
radical containing two to n carbon atoms, at least two of which are
bonded to each other by a double bond. Examples of such radicals
include, but are not limited to, ethenyl (vinyl), 1-propenyl,
2-propenyl, and 1-butenyl. Unless specified otherwise, the term
"(C.sub.2-n)alkenyl" is understood to encompass individual
stereoisomers where possible, including but not limited to (E) and
(Z) isomers, and mixtures thereof. When a (C.sub.2-n)alkenyl group
is substituted, it is understood to be substituted on any carbon
atom thereof which would otherwise bear a hydrogen atom, unless
specified otherwise, such that the substitution would give rise to
a chemically stable compound, such as are recognized by those
skilled in the art.
[0040] The term "(C.sub.3-m)cycloalkyl" as used herein, wherein m
is an integer, either alone or in combination with another radical,
is intended to mean a cycloalkyl substituent containing from 3 to m
carbon atoms and includes, but is not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
[0041] The term "aryl" as used herein, either alone or in
combination with another radical, is intended to mean a carbocyclic
aromatic monocyclic group containing 6 carbon atoms which may be
further fused to a second 5- or 6-membered carbocyclic group which
may be aromatic, saturated or unsaturated. Aryl includes, but is
not limited to, phenyl, indanyl, indenyl, 1-naphthyl, 2-naphthyl,
tetrahydronaphthyl and dihydronaphthyl.
[0042] The term "carbocycle" as used herein, either alone or in
combination with another radical, is intended to mean a cyclic
compound, either aromatic or non-aromatic, saturated or
unsaturated, in which all of the ring members are carbon atoms. The
carbocycle group may be containing 5 or 6 carbon atom and may be
further fused to a second 5- or 6-membered carbocyclic group which
may be aromatic, saturated or unsaturated. The carbocycle may be
substituted. When the carbocycle is substituted, it is understood
that substituents may be attached to any carbon atom which would
otherwise bear a hydrogen atom, unless specified otherwise, such
that the substitution would give rise to a chemically stable
compound, such as are recognized by those skilled in the art.
[0043] The term "Het" as used herein, either alone or in
combination with another radical, is intended to mean a 4- to
7-membered saturated, unsaturated or aromatic heterocycle having 1
to 4 heteroatoms each independently selected from O, N and S, or a
7- to 14-membered saturated, unsaturated or aromatic
heteropolycycle having wherever possible 1 to 5 heteroatoms, each
independently selected from 0, N and S, unless specified otherwise.
When a Het group is substituted, it is understood that substituents
may be attached to any carbon atom or heteroatom thereof which
would otherwise bear a hydrogen atom, unless specified otherwise,
such that the substitution would give rise to a chemically stable
compound, such as are recognized by those skilled in the art.
[0044] The term "heteroatom" as used herein is intended to mean O,
S or N.
[0045] The term "heterocycle" as used herein and unless specified
otherwise, either alone or in combination with another radical, is
intended to mean a 3- to 7-membered saturated, unsaturated or
aromatic heterocycle containing from 1 to 4 heteroatoms each
independently selected from O, N and S; or a monovalent radical
derived by removal of a hydrogen atom therefrom. Examples of such
heterocycles include, but are not limited to, azetidine,
pyrrolidine, tetrahydrofuran, tetrahydrothiophene, thiazolidine,
oxazolidine, pyrrole, thiophene, furan, pyrazole, imidazole,
isoxazole, oxazole, isothiazole, thiazole, triazole, tetrazole,
piperidine, piperazine, azepine, diazepine, pyran, 1,4-dioxane,
4-morpholine, 4-thiomorpholine, pyridine, pyridine-N-oxide,
pyridazine, pyrazine and pyrimidine, and saturated, unsaturated and
aromatic derivatives thereof.
[0046] The term "heteropolycycle" as used herein and unless
specified otherwise, either alone or in combination with another
radical, is intended to mean a heterocycle as defined above fused
to one or more other cycle, including a carbocycle, a heterocycle
or any other cycle; or a monovalent radical derived by removal of a
hydrogen atom therefrom. Examples of such heteropolycycles include,
but are not limited to, indole, isoindole, benzimidazole,
benzothiophene, benzofuran, benzopyran, benzodioxole, benzodioxane,
benzothiazole, quinoline, isoquinoline, and naphthyridine, and
saturated, unsaturated and aromatic derivatives thereof.
[0047] The term "halo" as used herein is intended to mean a halogen
substituent selected from fluoro, chloro, bromo or iodo.
[0048] The term "(C.sub.1-n)haloalkyl" as used herein, wherein n is
an integer, either alone or in combination with another radical, is
intended to mean an alkyl radical having 1 to n carbon atoms as
defined above wherein one or more hydrogen atoms are each replaced
by a halo substituent. Examples of (C.sub.1-n)haloalkyl include but
are not limited to chloromethyl, chloroethyl, dichloroethyl,
bromomethyl, bromoethyl, dibromoethyl, fluoromethyl,
difluoromethyl, trifluoromethyl, fluoroethyl and difluoroethyl.
[0049] The terms "--O--(C.sub.1-n)alkyl" or "(C.sub.1-n)alkoxy" as
used herein interchangeably, wherein n is an integer, either alone
or in combination with another radical, is intended to mean an
oxygen atom further bonded to an alkyl radical having 1 to n carbon
atoms as defined above. Examples of --O--(C.sub.1-n)alkyl include
but are not limited to methoxy (CH.sub.3O--), ethoxy
(CH.sub.3CH.sub.2O--), propoxy (CH.sub.3CH.sub.2CH.sub.2O--),
1-methylethoxy (iso-propoxy; (CH.sub.3).sub.2CH--O--) and
1,1-dimethylethoxy (tert-butoxy; (CH.sub.3).sub.3C--O--). When an
--O--(C.sub.1-n)alkyl radical is substituted, it is understood to
be substituted on the (C.sub.1-n)alkyl portion thereof, such that
the substitution would give rise to a chemically stable compound,
such as are recognized by those skilled in the art.
[0050] The terms "--S--(C.sub.1-n)alkyl" or "(C.sub.1-n)alkylthio"
as used herein interchangeably, wherein n is an integer, either
alone or in combination with another radical, is intended to mean
an sulfur atom further bonded to an alkyl radical having 1 to n
carbon atoms as defined above. Examples of --S--(C.sub.1-n)alkyl
include but are not limited to methylthio (CH.sub.3S--), ethylthio
(CH.sub.3CH.sub.2S--), propylthio (CH.sub.3CH.sub.2CH.sub.2S--),
1-methylethylthio (isopropylthio; (CH.sub.3).sub.2CH--S--) and
1,1-dimethylethylthio (tert-butylthio; (CH.sub.3).sub.3C--S--).
When --S--(C.sub.1-n)alkyl radical, or an oxidized derivative
thereof, such as an --SO--(C.sub.1-n)alkyl radical or an
--SO.sub.2--(C.sub.1-n)alkyl radical, is substituted, each is
understood to be substituted on the (C.sub.1-n)alkyl portion
thereof, such that the substitution would give rise to a chemically
stable compound, such as are recognized by those skilled in the
art.
[0051] The term "oxo" as used herein is intended to mean an oxygen
atom attached to a carbon atom as a substituent by a double bond
(.dbd.O).
[0052] The term "cyano" as used herein is intended to mean an
carbon atom attached to a nitrogen atom as a substituent by a
triple bond.
[0053] The term "functional group equivalent" as used herein is
intended to mean an atom or group that may replace another atom or
group which has similar electronic, hybridization or bonding
properties.
[0054] The term "protecting group" as used herein is intended to
mean protecting groups that can be used during synthetic
transformation, including but not limited to examples which are
listed in Greene, "Protective Groups in Organic Chemistry", John
Wiley & Sons, New York (1981), and more recent editions
thereof, herein incorporated by reference.
[0055] The following designation is used in sub-formulas to
indicate the bond which is connected to the rest of the molecule as
defined.
[0056] The term "salt thereof" as used herein is intended to mean
any acid and/or base addition salt of a compound according to the
invention, including but not limited to a pharmaceutically
acceptable salt thereof.
[0057] The term "pharmaceutically acceptable salt" as used herein
is intended to mean a salt of a compound according to the invention
which is, within the scope of sound medical judgment, suitable for
use in contact with the tissues of humans and lower animals without
undue toxicity, irritation, allergic response, and the like,
commensurate with a reasonable benefit/risk ratio, generally water
or oil-soluble or dispersible, and effective for their intended
use. The term includes pharmaceutically-acceptable acid addition
salts and pharmaceutically-acceptable base addition salts. Lists of
suitable salts are found in, for example, S. M. Birge et al., J.
Pharm. Sci., 1977, 66, pp. 1-19, herein incorporated by
reference.
[0058] The term "pharmaceutically-acceptable acid addition salt" as
used herein is intended to mean those salts which retain the
biological effectiveness and properties of the free bases and which
are not biologically or otherwise undesirable, formed with
inorganic acids including but not limited to hydrochloric acid,
hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid,
phosphoric acid and the like, and organic acids including but not
limited to acetic acid, trifluoroacetic acid, adipic acid, ascorbic
acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric
acid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric
acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic
acid, glycerophosphoric acid, hemisulfic acid, hexanoic acid,
formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (isethionic
acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid,
mandelic acid, mesitylenesulfonic acid, methanesulfonic acid,
naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic
acid, oxalic acid, pamoic acid, pectinic acid, phenylacetic acid,
3-phenylpropionic acid, pivalic acid, propionic acid, pyruvic acid,
salicylic acid, stearic acid, succinic acid, sulfanilic acid,
tartaric acid, p-toluenesulfonic acid, undecanoic acid and the
like.
[0059] The term "pharmaceutically-acceptable base addition salt" as
used herein is intended to mean those salts which retain the
biological effectiveness and properties of the free acids and which
are not biologically or otherwise undesirable, formed with
inorganic bases including but not limited to ammonia or the
hydroxide, carbonate, or bicarbonate of ammonium or a metal cation
such as sodium, potassium, lithium, calcium, magnesium, iron, zinc,
copper, manganese, aluminum and the like. Particularly preferred
are the ammonium, potassium, sodium, calcium, and magnesium salts.
Salts derived from pharmaceutically-acceptable organic nontoxic
bases include but are not limited to salts of primary, secondary,
and tertiary amines, quaternary amine compounds, substituted amines
including naturally occurring substituted amines, cyclic amines and
basic ion-exchange resins, such as methylamine, dimethylamine,
trimethylamine, ethylamine, diethylamine, triethylamine,
isopropylamine, tripropylamine, tributylamine, ethanolamine,
diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,
dicyclohexylamine, lysine, arginine, histidine, caffeine,
hydrabamine, choline, betaine, ethylenediamine, glucosamine,
methylglucamine, theobromine, purines, piperazine, piperidine,
N-ethylpiperidine, tetramethylammonium compounds,
tetraethylammonium compounds, pyridine, N,N-dimethylaniline,
N-methylpiperidine, N-methylmorpholine, dicyclohexylamine,
dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine,
N,N'-dibenzylethylenediamine, polyamine resins and the like.
Particularly preferred organic nontoxic bases are isopropylamine,
diethylamine, ethanolamine, trimethylamine, dicyclohexylamine,
choline, and caffeine.
[0060] The term "ester thereof" as used herein is intended to mean
any ester of a compound according to the invention in which any of
the --COOH substituents of the molecule is replaced by a --COOR
substituent, in which the R moiety of the ester is any
carbon-containing group which forms a stable ester moiety,
including but not limited to alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl,
each of which being optionally further substituted. The term "ester
thereof" includes but is not limited to pharmaceutically acceptable
esters thereof.
[0061] The term "pharmaceutically acceptable ester" as used herein
is intended to mean esters of the compound according to the
invention in which any of the COOH substituents of the molecule are
replaced by a --COOR substituent, in which the R moiety of the
ester is selected from alkyl (including, but not limited to,
methyl, ethyl, propyl, 1-methylethyl, 1,1-dimethylethyl, butyl);
alkoxyalkyl (including, but not limited to methoxymethyl);
acyloxyalkyl (including, but not limited to acetoxymethyl);
arylalkyl (including, but not limited to, benzyl); aryloxyalkyl
(including, but not limited to, phenoxymethyl); and aryl
(including, but not limited to phenyl) optionally substituted with
halogen, (C.sub.1-4)alkyl or (C.sub.1-4)alkoxy. Other suitable
esters can be found in Design of Prodrugs, Bundgaard, H. Ed.
Elsevier (1985), herein incorporated by reference. Such
pharmaceutically acceptable esters are usually hydrolyzed in vivo
when injected into a mammal and transformed into the acid form of
the compound according to the invention. With regard to the esters
described above, unless otherwise specified, any alkyl moiety
present preferably contains 1 to 16 carbon atoms, more preferably 1
to 6 carbon atoms. Any aryl moiety present in such esters
preferably comprises a phenyl group. In particular the esters may
be a (C.sub.1-16) alkyl ester, an unsubstituted benzyl ester or a
benzyl ester substituted with at least one halogen,
(C.sub.1-6)alkyl, (C.sub.1-6)alkoxy, nitro or trifluoromethyl.
[0062] The term "mammal" as used herein is intended to encompass
humans, as well as non-human mammals which are susceptible to
infection by HIV. Non-human mammals include but are not limited to
domestic animals, such as cows, pigs, horses, dogs, cats, rabbits,
rats and mice, and non-domestic animals.
[0063] The term "treatment" as used herein is intended to mean the
administration of a compound or composition according to the
present invention to alleviate or eliminate symptoms of HIV
infection and/or to reduce viral load in a patient. The term
"treatment" also encompasses the administration of a compound or
composition according to the present invention post-exposure of the
individual to the virus but before the appearance of symptoms of
the disease, and/or prior to the detection of the virus in the
blood, to prevent the appearance of symptoms of the disease and/or
to prevent the virus from reaching detectible levels in the blood,
and the administration of a compound or composition according to
the present invention to prevent perinatal transmission of HIV from
mother to baby, by administration to the mother before giving birth
and to the child within the first days of life.
[0064] The term "antiviral agent" as used herein is intended to
mean an agent that is effective to inhibit the formation and/or
replication of a virus in a mammal, including but not limited to
agents that interfere with either host or viral mechanisms
necessary for the formation and/or replication of a virus in a
mammal.
[0065] The term "inhibitor of HIV replication" as used herein is
intended to mean an agent capable of reducing or eliminating the
ability of HIV to replicate in a host cell, whether in vitro, ex
vivo or in vivo.
[0066] The term "HIV integrase" or "integrase", used herein
interchangeably, means the integrase enzyme encoded by the human
immunodeficiency virus type 1.
[0067] The term "therapeutically effective amount" means an amount
of a compound according to the invention, which when administered
to a patient in need thereof, is sufficient to effect treatment for
disease-states, conditions, or disorders for which the compounds
have utility. Such an amount would be sufficient to elicit the
biological or medical response of a tissue system, or patient that
is sought by a researcher or clinician. The amount of a compound
according to the invention which constitutes a therapeutically
effective amount will vary depending on such factors as the
compound and its biological activity, the composition used for
administration, the time of administration, the route of
administration, the rate of excretion of the compound, the duration
of the treatment, the type of disease-state or disorder being
treated and its severity, drugs used in combination with or
coincidentally with the compounds of the invention, and the age,
body weight, general health, sex and diet of the patient. Such a
therapeutically effective amount can be determined routinely by one
of ordinary skill in the art having regard to their own knowledge,
the state of the art, and this disclosure.
PREFERRED EMBODIMENTS
[0068] In the following preferred embodiments, groups and
substituents of the compounds of formula (I):
##STR00004##
according to this invention are described in detail.
R.sup.4:
[0069] R.sup.4-A: In one embodiment, R.sup.4 is aryl or Het
optionally substituted with 1 to 3 substituents each independently
selected from halo, (C.sub.1-6)alkyl, (C.sub.2-6)alkenyl,
(C.sub.1-6)haloalkyl, (C.sub.3-7)cycloalkyl, --OH,
--O(C.sub.1-6)alkyl, --SH, --S(C.sub.1-6)alkyl, --NH.sub.2,
--NH(C.sub.1-6)alkyl and --N((C.sub.1-6)alkyl).sub.2; wherein the
(C.sub.1-6)alkyl is optionally substituted with hydroxy, cyano or
oxo. [0070] R.sup.4-B: In another embodiment, R.sup.4 is Het
optionally substituted with 1 to 3 substituents each independently
selected from halo, (C.sub.1-6)alkyl, (C.sub.2-6)alkenyl,
(C.sub.1-6)haloalkyl, (C.sub.3-7)cycloalkyl, --OH,
--O(C.sub.1-6)alkyl, --SH, --S(C.sub.1-6)alkyl, --NH.sub.2,
--NH(C.sub.1-6)alkyl and --N((C.sub.1-6)alkyl).sub.2. [0071]
R.sup.4-C: In another embodiment, R.sup.4 is naphthyl or phenyl,
optionally substituted with 1 to 3 substituents each independently
selected from halo, (C.sub.1-4)alkyl, (C.sub.1-4)haloalkyl,
--O(C.sub.1-4)alkyl, --NH.sub.2, --NH(C.sub.1-6)alkyl and
--N((C.sub.1-6)alkyl).sub.2. [0072] R.sup.4-D: In another
embodiment, R.sup.4 is phenyl optionally substituted with 1 to 3
substituents each independently selected from halo,
(C.sub.1-4)alkyl, (C.sub.1-4)haloalkyl, --O(C.sub.1-4)alkyl,
--NH.sub.2, --NH(C.sub.1-6)alkyl and --N((C.sub.1-6)alkyl).sub.2.
[0073] R.sup.4-E: In one embodiment, R.sup.4 is Het optionally
substituted with 1 to 2 substituents each independently selected
from halo, (C.sub.1-3)alkyl and O--(C.sub.1-3)alkyl. [0074]
R.sup.4-F: In one embodiment, R.sup.4 is Het optionally substituted
with 1 to 2 substituents each independently selected from Cl, F,
CH.sub.3 and CH.sub.2CH.sub.3 wherein said Het is defined as a 7-
to 14-membered saturated, unsaturated or aromatic heteropolycycle
having wherever possible 1 to 2 heteroatoms, each independently
selected from O, N and S. [0075] R.sup.4-G: In one embodiment,
R.sup.4 is Het optionally substituted with 1 to 2 substituents each
independently selected from halo, (C.sub.1-3)alkyl and
O--(C.sub.1-3)alkyl, wherein said Het is defined as a 9- or
10-membered saturated, unsaturated or aromatic heteropolycycle
having wherever possible 1 to 2 heteroatoms, each independently
selected from O, N and S. [0076] R.sup.4-H: In another embodiment,
R.sup.4 is phenyl optionally substituted with 1 to 3 substituents
each independently selected from halo, (C.sub.1-4)alkyl,
(C.sub.1-4)haloalkyl, --O(C.sub.1-4)alkyl, --NH.sub.2,
--NH(C.sub.1-6)alkyl and --N((C.sub.1-6)alkyl).sub.2 or R.sup.4 is
Het optionally substituted with 1 to 3 substituents each
independently selected from halo, (C.sub.1-4)alkyl and
O--(C.sub.1-4)alkyl, wherein said Het is defined as a 7- to
14-membered saturated, unsaturated or aromatic heteropolycycle
having wherever possible 1 to 2 heteroatoms, each independently
selected from O, N and S. [0077] R.sup.4-I: In another embodiment,
R.sup.4 is phenyl optionally substituted with 1 to 3 substituents
each independently selected from F, Cl, Br, --CH.sub.3,
--CH(CH.sub.3).sub.2, CH.sub.2F, --CH.sub.2CH.sub.2F, --OCH.sub.3
and --NH.sub.2 or R.sup.4 is Het optionally substituted with 1 to 3
substituents each independently selected from Cl, F, CH.sub.3,
CH.sub.2CH.sub.3 and OCH.sub.3, wherein said Het is defined as a 7-
or 14-membered saturated, unsaturated or aromatic heteropolycycle
having wherever possible 1 to 3 heteroatoms, each independently
selected from O, N and S. [0078] R.sup.4-J: In another embodiment,
R.sup.4 is selected from:
[0078] ##STR00005## [0079] being optionally substituted 1 to 3
times with halo, (C.sub.1-3)alkyl and O--(C.sub.1-3)alkyl. [0080]
R.sup.4-K: In another embodiment, R.sup.4 is selected from:
[0080] ##STR00006## [0081] being optionally substituted 1 to 2
times with halo, (C.sub.1-3)alkyl and O--(C.sub.1-3)alkyl. [0082]
R.sup.4-L: In another embodiment, R.sup.4 is selected from:
[0082] ##STR00007## [0083] being optionally substituted 1 to 2
times with halo, (C.sub.1-3)alkyl and O--(C.sub.1-3)alkyl. [0084]
R.sup.4-M: In another embodiment, R.sup.4 is selected from:
[0084] ##STR00008## [0085] being optionally substituted 1 to 2
times with halo, (C.sub.1-3)alkyl and O--(C.sub.1-3)alkyl. [0086]
R.sup.4-N: In another embodiment, R.sup.4 is selected from:
[0086] ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## [0087] R.sup.4-O: In another embodiment,
R.sup.4 is selected from:
##STR00015## ##STR00016## ##STR00017##
[0088] One skilled in the art will recognize that when the R.sup.4
substituent is not symmetrically substituted about the axis of
rotation of the bond attaching R.sup.4 to Core, rotational isomers
or atropisomers are possible. Compounds of the invention in which
the R.sup.4 substituent is not symmetrically substituted about the
axis of rotation of the bond attaching R.sup.4 to Core and in which
the carbon atom bonded to the --COOH and R.sup.3 substituents is
chiral, as described above, will have two chiral centers, a chiral
carbon atom and a rotational axis of asymmetry, and thus the
atropisomers will exist as diastereomers. However, individual
diastereomeric atropisomers may or may not be detectable and/or
separable, depending upon the relative amounts of each atropisomer
formed during synthesis, present at equilibrium, and the degree of
steric hindrance to rotation about the C-4 chiral axis, and
therefore, the rate at which interconversion between these
atropoisomers occurs. Once separated, individual atropoisomers may
be very stable or interconvert, rapidly or slowly, with each other
to form an equilibrium mixture of atropoisomers. [0089] R.sup.4-P:
In another embodiment, R.sup.4 is selected from:
[0089] ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022## ##STR00023## [0090] R.sup.4-Q: in another embodiment,
R.sup.4 is selected from:
[0090] ##STR00024## ##STR00025## ##STR00026## [0091] R.sup.4-R: in
another embodiment, R.sup.4 is aryl or Het, wherein each of the
aryl and Het is optionally substituted With 1 to 3 substituents
each independently selected from halo, (C.sub.1-6)alkyl,
(C.sub.2-6)alkenyl, (C.sub.1-6)haloalkyl, (C.sub.3-7)cycloalkyl,
--OH, --O(C.sub.1-6)alkyl, --SH, --S(C.sub.1-6)alkyl, --NH.sub.2,
--NH(C.sub.1-6)alkyl and --N(C.sub.1-6)alkyl).sub.2; wherein the
(C.sub.1-6)alkyl is optionally substituted with hydroxy, cyano or
oxo; and wherein the aryl is not monosubstituted at the para
position;
[0092] Any and each individual definition of R.sup.4 as set out
herein may be combined with any and each individual definition of
R.sup.6 and R.sup.7 as set out herein.
R.sup.b:
[0093] R.sup.6-A: In one embodiment, R.sup.6 is H, halo,
(C.sub.1-6)alkyl or (C.sub.1-6)haloalkyl, [0094] R.sup.6-B: In
another embodiment, R.sup.6 is H, halo or (C.sub.1-3)alkyl. [0095]
R.sup.6-C: In another embodiment, R.sup.6 is H, F, Cl or
(C.sub.1-2)alkyl. [0096] R.sup.6-D: In another embodiment, R.sup.6
is H, F, Cl or CH.sub.3. [0097] R.sup.6-E: In another embodiment,
R.sup.6 is H, CH.sub.3 or CH.sub.2CH.sub.3. [0098] R.sup.6-F: In
another embodiment, R.sup.6 is H or CH.sub.3. [0099] R.sup.6-G: In
another embodiment, R.sup.6 is H.
[0100] Any and each individual definition of R.sup.6 as set out
herein may be combined with any and each individual definition of
R.sup.4 and R.sup.7 as set out herein.
R.sup.7:
[0101] R.sup.7-A: In one embodiment, R.sup.7 is H, halo,
(C.sub.1-6)alkyl or (C.sub.1-6)haloalkyl. [0102] R.sup.7-B: In
another embodiment, R.sup.7 is H, halo or (C.sub.1-3)alkyl. [0103]
R.sup.7-C: In another embodiment, R.sup.7 is H, F, Cl or
(C.sub.1-2)alkyl. [0104] R.sup.7-D: In another embodiment, R.sup.7
is H, F, Cl or CH.sub.3. [0105] R.sup.7-E: In one embodiment,
R.sup.7 is H, F or CH.sub.3. [0106] R.sup.7-F: In one embodiment,
R.sup.7 is H or CH.sub.3. [0107] R.sup.7-G: In another embodiment,
R.sup.7 is H.
[0108] Any and each individual definition of R.sup.7 as set out
herein may be combined with any and each individual definition of
R.sup.4 and R.sup.6 as set out herein.
[0109] Examples of preferred subgeneric embodiments of the present
invention are set forth in the following table, wherein each
substituent group of each embodiment is defined according to the
definitions set forth above:
TABLE-US-00001 Embodiment R.sup.4 R.sup.6 R.sup.7 E-1 R.sup.4-A
R.sup.6-B R.sup.7-C E-2 R.sup.4-A R.sup.6-D R.sup.7-C E-3 R.sup.4-A
R.sup.6-E R.sup.7-F E-4 R.sup.4-A R.sup.6-F R.sup.7-A E-5 R.sup.4-A
R.sup.6-E R.sup.7-E E-6 R.sup.4-B R.sup.6-E R.sup.7-E E-7 R.sup.4-B
R.sup.6-B R.sup.7-F E-8 R.sup.4-B R.sup.6-F R.sup.7-A E-9 R.sup.4-C
R.sup.6-A R.sup.7-A E-10 R.sup.4-C R.sup.6-D R.sup.7-A E-11
R.sup.4-C R.sup.6-F R.sup.7-D E-12 R.sup.4-D R.sup.6-E R.sup.7-F
E-13 R.sup.4-D R.sup.6-D R.sup.7-F E-14 R.sup.4-D R.sup.6-G
R.sup.7-F E-15 R.sup.4-D R.sup.6-E R.sup.7-G E-16 R.sup.4-D
R.sup.6-D R.sup.7-C E-17 R.sup.4-D R.sup.6-E R.sup.7-C E-18
R.sup.4-E R.sup.6-C R.sup.7-A E-19 R.sup.4-E R.sup.6-B R.sup.7-C
E-20 R.sup.4-E R.sup.6-B R.sup.7-B E-21 R.sup.4-E R.sup.6-B
R.sup.7-E E-22 R.sup.4-E R.sup.6-E R.sup.7-C E-23 R.sup.4-F
R.sup.6-D R.sup.7-A E-24 R.sup.4-F R.sup.6-E R.sup.7-C E-25
R.sup.4-F R.sup.6-C R.sup.7-B E-26 R.sup.4-F R.sup.6-B R.sup.7-E
E-27 R.sup.4-G R.sup.6-A R.sup.7-D E-28 R.sup.4-G R.sup.6-C
R.sup.7-B E-29 R.sup.4-G R.sup.6-B R.sup.7-A E-30 R.sup.4-G
R.sup.6-B R.sup.7-G E-31 R.sup.4-G R.sup.6-A R.sup.7-F E-32
R.sup.4-G R.sup.6-A R.sup.7-E E-33 R.sup.4-G R.sup.6-C R.sup.7-D
E-34 R.sup.4-H R.sup.6-C R.sup.7-E E-35 R.sup.4-H R.sup.6-C
R.sup.7-F E-36 R.sup.4-H R.sup.6-D R.sup.7-E E-37 R.sup.4-H
R.sup.6-D R.sup.7-F E-38 R.sup.4-H R.sup.6-E R.sup.7-E E-39
R.sup.4-H R.sup.6-E R.sup.7-F E-40 R.sup.4-H R.sup.6-G R.sup.7-B
E-41 R.sup.4-H R.sup.6-C R.sup.7-F E-42 R.sup.4-H R.sup.6-F
R.sup.7-C E-43 R.sup.4-H R.sup.6-G R.sup.7-E E-44 R.sup.4-H
R.sup.6-D R.sup.7-E E-45 R.sup.4-H R.sup.6-D R.sup.7-A E-46
R.sup.4-H R.sup.6-A R.sup.7-B E-47 R.sup.4-I R.sup.6-C R.sup.7-E
E-48 R.sup.4-I R.sup.6-C R.sup.7-F E-49 R.sup.4-I R.sup.6-D
R.sup.7-E E-50 R.sup.4-I R.sup.6-D R.sup.7-F E-51 R.sup.4-I
R.sup.6-E R.sup.7-E E-52 R.sup.4-I R.sup.6-E R.sup.7-F E-53
R.sup.4-I R.sup.6-B R.sup.7-C E-54 R.sup.4-I R.sup.6-A R.sup.7-G
E-55 R.sup.4-I R.sup.6-B R.sup.7-C E-56 R.sup.4-J R.sup.6-C
R.sup.7-E E-57 R.sup.4-J R.sup.6-C R.sup.7-F E-58 R.sup.4-J
R.sup.6-D R.sup.7-E E-59 R.sup.4-J R.sup.6-D R.sup.7-F E-60
R.sup.4-J R.sup.6-E R.sup.7-E E-61 R.sup.4-J R.sup.6-E R.sup.7-F
E-62 R.sup.4-J R.sup.6-F R.sup.7-D E-63 R.sup.4-J R.sup.6-A
R.sup.7-A E-64 R.sup.4-J R.sup.6-F R.sup.7-G E-65 R.sup.4-J
R.sup.6-G R.sup.7-F E-66 R.sup.4-J R.sup.6-C R.sup.7-F E-67
R.sup.4-J R.sup.6-D R.sup.7-G E-68 R.sup.4-J R.sup.6-G R.sup.7-E
E-69 R.sup.4-K R.sup.6-C R.sup.7-E E-70 R.sup.4-K R.sup.6-C
R.sup.7-F E-71 R.sup.4-K R.sup.6-D R.sup.7-E E-72 R.sup.4-K
R.sup.6-D R.sup.7-F E-73 R.sup.4-K R.sup.6-E R.sup.7-E E-74
R.sup.4-K R.sup.6-E R.sup.7-F E-75 R.sup.4-K R.sup.6-G R.sup.7-A
E-76 R.sup.4-K R.sup.6-B R.sup.7-C E-77 R.sup.4-K R.sup.6-G
R.sup.7-E E-78 R.sup.4-L R.sup.6-C R.sup.7-F E-79 R.sup.4-L
R.sup.6-D R.sup.7-E E-80 R.sup.4-L R.sup.6-D R.sup.7-F E-81
R.sup.4-L R.sup.6-E R.sup.7-E E-82 R.sup.4-L R.sup.6-E R.sup.7-F
E-83 R.sup.4-L R.sup.6-G R.sup.7-A E-84 R.sup.4-L R.sup.6-B
R.sup.7-C E-85 R.sup.4-L R.sup.6-G R.sup.7-E E-86 R.sup.4-L
R.sup.6-C R.sup.7-F E-87 R.sup.4-L R.sup.6-F R.sup.7-F E-88
R.sup.4-L R.sup.6-F R.sup.7-G E-89 R.sup.4-L R.sup.6-A R.sup.7-C
E-90 R.sup.4-L R.sup.6-D R.sup.7-A E-91 R.sup.4-M R.sup.6-C
R.sup.7-F E-92 R.sup.4-M R.sup.6-D R.sup.7-E E-93 R.sup.4-M
R.sup.6-D R.sup.7-F E-94 R.sup.4-M R.sup.6-E R.sup.7-E E-95
R.sup.4-M R.sup.6-E R.sup.7-F E-96 R.sup.4-M R.sup.6-G R.sup.7-A
E-97 R.sup.4-M R.sup.6-B R.sup.7-C E-98 R.sup.4-M R.sup.6-G
R.sup.7-E E-99 R.sup.4-M R.sup.6-C R.sup.7-F E-100 R.sup.4-M
R.sup.6-F R.sup.7-F E-101 R.sup.4-M R.sup.6-F R.sup.7-G E-102
R.sup.4-M R.sup.6-A R.sup.7-D E-103 R.sup.4-M R.sup.6-C R.sup.7-B
E-104 R.sup.4-N R.sup.6-C R.sup.7-E E-105 R.sup.4-N R.sup.6-C
R.sup.7-F E-106 R.sup.4-N R.sup.6-D R.sup.7-E E-107 R.sup.4-N
R.sup.6-D R.sup.7-F E-108 R.sup.4-N R.sup.6-E R.sup.7-E E-109
R.sup.4-N R.sup.6-E R.sup.7-F E-110 R.sup.4-N R.sup.6-F R.sup.7-G
E-111 R.sup.4-N R.sup.6-B R.sup.7-B E-112 R.sup.4-N R.sup.6-G
R.sup.7-A E-113 R.sup.4-N R.sup.6-A R.sup.7-G E-114 R.sup.4-O
R.sup.6-C R.sup.7-E E-115 R.sup.4-O R.sup.6-C R.sup.7-F E-116
R.sup.4-O R.sup.6-D R.sup.7-E E-117 R.sup.4-O R.sup.6-D R.sup.7-F
E-118 R.sup.4-O R.sup.6-E R.sup.7-E E-119 R.sup.4-O R.sup.6-E
R.sup.7-F E-120 R.sup.4-O R.sup.6-B R.sup.7-F E-121 R.sup.4-O
R.sup.6-G R.sup.7-B E-122 R.sup.4-O R.sup.6-F R.sup.7-A E-123
R.sup.4-P R.sup.6-B R.sup.7-A E-124 R.sup.4-P R.sup.6-C R.sup.7-B
E-125 R.sup.4-P R.sup.6-D R.sup.7-C E-126 R.sup.4-P R.sup.6-E
R.sup.7-D E-127 R.sup.4-P R.sup.6-F R.sup.7-E E-128 R.sup.4-P
R.sup.6-G R.sup.7-F E-129 R.sup.4-P R.sup.6-A R.sup.7-G E-130
R.sup.4-P R.sup.6-A R.sup.7-A E-131 R.sup.4-P R.sup.6-B R.sup.7-E
E-132 R.sup.4-P R.sup.6-C R.sup.7-F E-133 R.sup.4-P R.sup.6-D
R.sup.7-G E-134 R.sup.4-P R.sup.6-E R.sup.7-E E-135 R.sup.4-P
R.sup.6-F R.sup.7-F E-136 R.sup.4-P R.sup.6-G R.sup.7-G E-137
R.sup.4-Q R.sup.6-B R.sup.7-A E-138 R.sup.4-Q R.sup.6-C R.sup.7-B
E-139 R.sup.4-Q R.sup.6-D R.sup.7-C E-140 R.sup.4-Q R.sup.6-E
R.sup.7-D E-141 R.sup.4-Q R.sup.6-F R.sup.7-E E-142 R.sup.4-Q
R.sup.6-G R.sup.7-F E-143 R.sup.4-Q R.sup.6-A R.sup.7-G E-144
R.sup.4-Q R.sup.6-A R.sup.7-A E-145 R.sup.4-Q R.sup.6-B R.sup.7-E
E-146 R.sup.4-Q R.sup.6-C R.sup.7-F E-147 R.sup.4-Q R.sup.6-D
R.sup.7-G E-148 R.sup.4-Q R.sup.6-E R.sup.7-E E-149 R.sup.4-Q
R.sup.6-F R.sup.7-F E-150 R.sup.4-Q R.sup.6-G R.sup.7-G
[0110] Examples of most preferred compounds according to this
invention are each single compound listed in the following Table
1.
[0111] In general, all tautomeric and isomeric forms and mixtures
thereof, for example, individual tautomers, geometric isomers,
stereoisomers, atropisomers, enantiomers, diastereomers, racemates,
racemic or non-racemic mixtures of stereoisomers, mixtures of
diastereomers, or mixtures of any of the foregoing forms of a
chemical structure or compound is intended, unless the specific
stereochemistry or isomeric form is specifically indicated in the
compound name or structure.
[0112] It is well-known in the art that the biological and
pharmacological activity of a compound is sensitive to the
stereochemistry of the compound. Thus, for example, enantiomers
often exhibit strikingly different biological activity including
differences in pharmacokinetic properties, including metabolism,
protein binding, and the like, and pharmacological properties,
including the type of activity displayed, the degree of activity,
toxicity, and the like. Thus, one skilled in the art will
appreciate that one enantiomer may be more active or may exhibit
beneficial effects when enriched relative to the other enantiomer
or when separated from the other enantiomer. Additionally, one
skilled in the art would know how to separate, enrich, or
selectively prepare the enantiomers of the compounds of the present
invention from this disclosure and the knowledge in the art.
[0113] Preparation of pure stereoisomers, e.g. enantiomers and
diastereomers, or mixtures of desired enantiomeric excess (ee) or
enantiomeric purity, are accomplished by one or more of the many
methods of (a) separation or resolution of enantiomers, or (b)
enantioselective synthesis known to those of skill in the art, or a
combination thereof. These resolution methods generally rely on
chiral recognition and include, for example, chromatography using
chiral stationary phases, enantioselective host-guest complexation,
resolution or synthesis using chiral auxiliaries, enantioselective
synthesis, enzymatic and nonenzymatic kinetic resolution, or
spontaneous enantioselective crystallization. Such methods are
disclosed generally in Chiral Separation Techniques: A Practical
Approach (2nd Ed.), G. Subramanian (ed.), Wiley-VCH, 2000; T. E.
Beesley and R. P. W. Scott, Chiral Chromatography, John Wiley &
Sons, 1999; and Satinder Ahuja, Chiral Separations by
Chromatography, Am. Chem. Soc., 2000, herein incorporated by
reference. Furthermore, there are equally well-known methods for
the quantitation of enantiomeric excess or purity, for example, GC,
HPLC, CE, or NMR, and assignment of absolute configuration and
conformation, for example, CD, ORD, X-ray crystallography, or
NMR.
Pharmaceutical Composition
[0114] Compounds of the present invention may be administered to a
mammal in need of treatment for HIV infection as a pharmaceutical
composition comprising a therapeutically effective amount of a
compound according to the invention or a pharmaceutically
acceptable salt or ester thereof; and one or more conventional
non-toxic pharmaceutically-acceptable carriers, adjuvants or
vehicles. The specific formulation of the composition is determined
by the solubility and chemical nature of the compound, the chosen
route of administration and standard pharmaceutical practice. The
pharmaceutical composition according to the present invention may
be administered orally or systemically.
[0115] When one enantiomer of a chiral active ingredient has a
different biological activity than the other, it is contemplated
that the pharmaceutical composition according to the invention may
comprise a racemic mixture of the active ingredient, a mixture
enriched in one enantiomer of the active ingredient or a pure
enantiomer of the active ingredient. The mixture enriched in one
enantiomer of the active ingredient is contemplated to contain from
more than 50% to about 100% of one enantiomer of the active
ingredient and from about 0% to less than 50% of the other
enantiomer of the active ingredient. Preferably, when the
composition comprises a mixture enriched in one enantiomer of the
active ingredient or a pure enantiomer of the active ingredient,
the composition comprises from more than 50% to about 100% of, or
only, the more physiologically active enantiomer and/or the less
toxic enantiomer. It is well known that one enantiomer of an active
ingredient may be the more physiologically active for one
therapeutic indication while the other enantiomer of the active
ingredient may be the more physiologically active for a different
therapeutic indication; therefore the preferred enantiomeric makeup
of the pharmaceutical composition may differ for use of the
composition in treating different therapeutic indications.
[0116] For oral administration, the compound, or a pharmaceutically
acceptable salt or ester thereof, can be formulated in any orally
acceptable dosage form including but not limited to aqueous
suspensions and solutions, capsules, powders, syrups, elixirs or
tablets. For systemic administration, including but not limited to
administration by subcutaneous, intracutaneous, intravenous,
intramuscular, intra-articular, intrasynovial, intrasternal,
intrathecal, and intralesional injection or infusion techniques, it
is preferred to use a solution of the compound, or a
pharmaceutically acceptable salt or ester thereof, in a
pharmaceutically acceptable sterile aqueous vehicle.
[0117] Pharmaceutically acceptable carriers, adjuvants, vehicles,
diluents, excipients and additives as well as methods of
formulating pharmaceutical compositions for various modes of
administration are well-known to those of skill in the art and are
described in pharmaceutical texts such as Remington: The Science
and Practice of Pharmacy, 21st Edition, Lippincott Williams &
Wilkins, 2005; and L. V. Allen, N. G. Popovish and H. C. Ansel,
Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th ed.,
Lippincott Williams & Wilkins, 2004, herein incorporated by
reference.
[0118] The dosage administered will vary depending upon known
factors, including but not limited to the activity and
pharmacodynamic characteristics of the specific compound employed
and its mode, time and route of administration; the age, diet,
gender, body weight and general health status of the recipient; the
nature and extent of the symptoms; the severity and course of the
infection; the kind of concurrent treatment; the frequency of
treatment; the effect desired; and the judgment of the treating
physician. In general, the compound is most desirably administered
at a dosage level that will generally afford antivirally effective
results without causing any harmful or deleterious side
effects.
[0119] A daily dosage of active ingredient can be expected to be
about 0.001 to about 100 milligrams per kilogram of body weight,
with the preferred dose being about 0.01 to about 50 mg/kg.
Typically, the pharmaceutical composition of this invention will be
administered from about 1 to about 5 times per day or
alternatively, as a continuous infusion. Such administration can be
used as a chronic or acute therapy. The amount of active ingredient
that may be combined with the carrier materials to produce a single
dosage form will vary depending upon the host treated and the
particular mode of administration. A typical preparation will
contain from about 5% to about 95% active compound (w/w).
Preferably, such preparations contain from about 20% to about 80%
active compound.
[0120] Therefore, according to one embodiment, the pharmaceutical
composition according to the invention comprises a racemic mixture
of the compound of formula (I), or a pharmaceutically acceptable
salt or ester thereof.
[0121] An alternative embodiment provides a pharmaceutical
composition comprising a mixture enriched in one enantiomer of the
compound of formula (I), or a pharmaceutically acceptable salt or
ester thereof.
[0122] A further embodiment provides a pharmaceutical composition
comprising a pure enantiomer of the compound of formula (I), or a
pharmaceutically acceptable salt or ester thereof.
Combination Therapy
[0123] Combination therapy is contemplated wherein a compound
according to the invention, or a pharmaceutically acceptable salt
or ester thereof, is co-administered with at least one additional
antiviral agent. The additional agents may be combined with
compounds of this invention to create a single dosage form.
Alternatively these additional agents may be separately
administered, concurrently or sequentially, as part of a multiple
dosage form.
[0124] When the pharmaceutical composition of this invention
comprises a combination of a compound according to the invention,
or a pharmaceutically acceptable salt or ester thereof, and one or
more additional antiviral agent, both the compound and the
additional agent should be present at dosage levels of between
about 10 to 100%, and more preferably between about 10 and 80% of
the dosage normally administered in a monotherapy regimen. In the
case of a synergistic interaction between the compound of the
invention and the additional antiviral agent or agents, the dosage
of any or all of the active agents in the combination may be
reduced compared to the dosage normally administered in a
monotherapy regimen.
[0125] Antiviral agents contemplated for use in such combination
therapy include agents (compounds or biologicals) that are
effective to inhibit the formation and/or replication of a virus in
a mammal, including but not limited to agents that interfere with
either host or viral mechanisms necessary for the formation and/or
replication of a virus in a mammal. Such agents can be selected
from: [0126] NRTIs (nucleoside or nucleotide reverse transcriptase
inhibitors) including but not limited to zidovudine (AZT),
didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine
(3TC), emtricitabine, abacavir succinate, elvucitabine, adefovir
dipivoxil, lobucavir (BMS-180194) lodenosine (FddA) and tenofovir
including tenofovir disoproxil and tenofovir disoproxil fumarate
salt, COMBIVIR.TM. (contains 3TC and AZT), TRIZIVIR.TM. (contains
abacavir, 3TC and AZT), TRUVADA.TM. (contains tenofovir and
emtricitabine), EPZICOM.TM. (contains abacavir and 3TC); [0127]
NNRTIs (non-nucleoside reverse transcriptase inhibitors) including
but not limited to nevirapine, delaviradine, efavirenz, etravirine
and rilpivirine; [0128] protease inhibitors including but not
limited to ritonavir, tipranavir, saquinavir, nelfinavir,
indinavir, amprenavir, fosamprenavir, atazanavir, lopinavir,
darunavir (TMC-114), lasinavir and brecanavir (VX-385); [0129]
entry inhibitors including but not limited to [0130] CCR5
antagonists (including but not limited to maraviroc, vicriviroc,
INCB9471 and TAK-652), [0131] CXCR4 antagonists (including but not
limited to AMD-11070), [0132] fusion inhibitors (including but not
limited to enfuvirtide (T-20), TR1-1144 and TR1-999) and [0133]
others (including but not limited to BMS-488043); [0134] integrase
inhibitors (including but not limited to raltegravir (MK-0518),
BMS-707035 and elvitegravir (GS 9137)); [0135] TAT inhibitors;
[0136] maturation inhibitors (including but not limited to
berivimat (PA-457)); [0137] immunomodulating agents (including but
not limited to levamisole); and [0138] other antiviral agents
including hydroxyurea, ribavirin, IL-2, IL-12 and pensafuside.
[0139] Furthermore, a compound according to the invention can be
used with at least one other compound according to the invention or
with one or more antifungal or antibacterial agents (including but
not limited to fluconazole).
[0140] Therefore, according to one embodiment, the pharmaceutical
composition of this invention additionally comprises one or more
antiviral agents.
[0141] A further embodiment provides the pharmaceutical composition
of this invention wherein the one or more antiviral agent comprises
at least one NNRTI.
[0142] According to another embodiment of the pharmaceutical
composition of this invention, the one or more antiviral agent
comprises at least one NRTI.
[0143] According to yet another embodiment of the pharmaceutical
composition of this invention, the one or more antiviral agent
comprises at least one protease inhibitor.
[0144] According to still another embodiment of the pharmaceutical
composition of this invention, the one or more antiviral agent
comprises at least one entry inhibitor.
[0145] According to a further embodiment of the pharmaceutical
composition of this invention, the one or more antiviral agent
comprises at least one integrase inhibitor.
[0146] A compound according to the present invention may also be
used as a laboratory reagent or a research reagent. For example, a
compound of the present invention may be used as positive control
to validate assays, including but not limited to surrogate
cell-based assays and in vitro or in vivo viral replication
assays.
[0147] Furthermore, a compound according to the present invention
may be used to treat or prevent viral contamination of materials
and therefore reduce the risk of viral infection of laboratory or
medical personnel or patients who come in contact with such
materials (e.g. blood, tissue, surgical instruments and garments,
laboratory instruments and garments, and blood collection
apparatuses and materials).
Derivatives Comprising a Detectable Label
[0148] Another aspect of the invention provides a derivative of a
compound of formula (I), the derivative comprising a detectable
label. Such a label allows recognition either directly or
indirectly of the derivative such that it can be detected, measured
or quantified. The detectable label may itself be detectable,
measurable or quantifiable, or it may interact with one or more
other moities which themselves comprise one or more detectable
labels, so that the interaction therebetween allows the derivative
to be detected, measured or quantified.
[0149] Such derivatives may be used as probes to study HIV
replication, including but not limited to study of the mechanism of
action of viral and host proteins involved in HIV replication,
study of conformational changes undergone by such viral and host
proteins under various conditions and study of interactions with
entities which bind to or otherwise interact with these viral and
host proteins. Derivatives according to this aspect of the
invention may be used in assays to identify compounds which
interact with viral and host proteins, the assays including but not
limited to displacement assays which measure the extent to which
the derivative is displaced from interacting with the viral and
host proteins. A preferred used of derivivatives according to this
aspect of the invention is in displacement assays to identify HIV
integrase inhibitors. Such derivatives may also be used to form
covalent or non-covalent interactions with the viral and host
proteins or to identify residues of the viral and host proteins
which interact with the compounds of the invention.
[0150] Detectable labels contemplated for use with derivatives of
the compounds of the invention include, but are not limited to,
fluorescent labels, chemiluminescent labels, chromophores,
antibodies, enzymatic markers, radioactive isotopes, affinity tags
and photoreactive groups.
[0151] A fluorescent label is a label which fluoresces, emitting
light of one wavelength upon absorption of light of a different
wavelength. Fluorescent labels include but are not limited to
fluorescein; Texas Red; aminomethylcoumarin; rhodamine dyes,
including but not limited to tetramethylrhodamine (TAMRA); Alexa
dyes including but not limited to Alexa Fluor.RTM. 555; cyanine
dyes including but not limited to Cy3; europium or lanthanide
series based fluorescent molecules; and the like.
[0152] A chemiluminescent label is a label which can undergo a
chemical reaction which produces light. Chemiluminescent labels
include but are not limited to luminol, luciferin, lucigenin, and
the like.
[0153] A chromophore is a label which selectively absorbs certain
wavelengths of visible light while transmitting or reflecting
others, thereby causing the compounds which contain the chromophore
to appear colored. Chromophores include but are not limited to
natural and synthetic dyes.
[0154] An antibody is a protein produced by a mammalian immune
system in response to a specific antigen, which binds specifically
to that antigen. Antibodies contemplated for use as detectable
labels according to the invention include but are not limited to
antibodies against the following: polyhistidine tags,
glutathione-S-transferase (GST), hemagglutinin (HA), FLAG.RTM.
epitope tags, Myc tag, maltose binding protein (MBP), green
fluorescent protein (GFP) and the like.
[0155] An enzymatic marker is an enzyme whose presence may be
detected by means of an assay specific to the catalytic activity of
the enzyme. Enzymatic markers contemplated for use as detectable
labels according to the invention include but are not limited to
luciferase, horseradish peroxidase (HRP), .beta.-galactosidase and
the like.
[0156] A radioactive isotope is an isotope of an atom which
produces radiation upon radioactive decay. Radioactive isotopes
include but are not limited to .sup.14C, .sup.3H, .sup.31P,
.sup.121I, .sup.125I and the like.
[0157] An affinity tag is a label which has a strong affinity for
another moiety, designated herein as a binding partner. Such an
affinity tag can be used to form a complex with the binding partner
so that the complex may be selectively detected or separated from a
mixture. Affinity tags include but are not limited to biotin or a
derivative thereof, a histidine polypeptide, a polyarginine, an
amylose sugar moiety or a defined epitope recognizable by a
specific antibody; suitable epitopes include but are not limited to
glutathione-S-transferase (GST), hemagglutinin (HA), FLAG.RTM.
epitope tags, Myc tag, maltose binding protein (MBP), green
fluorescent protein (GFP) and the like.
[0158] Furthermore, compounds of the invention used as probes may
be labelled with a photoreactive group which is transformed, upon
activation by light, from an inert group to a reactive species,
such as a free radical. Such a group may be used to activate the
derivative so that it can form a covalent bond with one or more
residues of a viral or host protein. Photoreactive groups include
but are not limited to photoaffinity labels such as benzophenone
and azide groups.
Methodology and Synthesis
[0159] The synthesis of compounds of formula (I) according to this
invention is conveniently accomplished following the general
procedure outlined in the schemes below wherein R.sup.4, R.sup.6
and R.sup.7 are as defined herein. Further instruction is provided
to one skilled in the art by the specific examples set out herein
below.
##STR00027##
wherein R.sup.42, R.sup.43, R.sup.44, R.sup.45 and R.sup.46 may
either be substituents on the phenyl moiety or (R.sup.42 and
R.sup.43), (R.sup.43 and R.sup.44), (R.sup.44 and R.sup.45) or
(R.sup.45 and R.sup.46) may be linked so to as to form a carbocycle
or heterocycle, W is iodo, bromo, chloro or OTf, Y is B(OH).sub.2
or boronate esters such as B(OCH.sub.3).sub.2 and
B(OC(CH.sub.3).sub.2C(CH.sub.3).sub.2O), iodo, SnR.sub.3 wherein R
is (C.sub.1-6)alkyl, ZnX wherein X is halo, and P is a protecting
group, such as commonly used protecting groups for carboxylic
acids, including, but not limited to a methyl or ethyl ester.
[0160] Several coupling methods between the intermediate (I) (i.e.
quinoline scaffold) and the intermediate II (i.e. R.sup.4
substituent) can be contemplated by those skilled in the art. For
examples, but not limited to, Suzuki cross-coupling between the
boronic acid or boronate ester derivative of intermediate II and
the halo or triflate derivative of intermediate I, copper catalyzed
Ullmann cross-coupling between the iodo derivatives of
intermediates I and II, Negishi cross-coupling between the arylzinc
reagent of the intermediate II and the iodo or triflate derivative
of intermediate I, and Stille coupling between the arylltin reagent
of intermediate II and the bromo or iodo derivative of I as shown
above can lead, after saponification, to the compounds of formula
(I).
[0161] Alternatively, the same cross-coupling methods can be used
by interchanging the coupling partners as shown below. For
examples, Suzuki, Negishi, and Stille type cross-coupling between
boronic acid or boronate ester derivative, the arylzinc reagent or
the arylltin reagent of quinoline intermediate III and the required
iodo, bromo, chloro or triflate derivative of intermediate IV can
also lead, after saponification, to the compounds of the invention
of formula (I).
##STR00028##
wherein R.sup.42, R.sup.43, R.sup.44, R.sup.45 and, R.sup.46 and P
are as defined above and W is iodo, bromo, chloro or OTf, Y is
B(OH).sub.2 or boronate esters such as B(OCH.sub.3).sub.2 and
B(OC(CH.sub.3).sub.2C(CH.sub.3).sub.2O), SnR.sub.3 wherein R is
(C.sub.1-6)alkyl, and ZnX wherein X is halo.
[0162] Furthermore, downstream modifications to the product can be
contemplated, such as conversion of an aniline-type amine to a
chloro or bromo substituent via Sandmeyer reaction or alkylation,
or dehalogenation via reduction.
[0163] Additionally, intermediate III can be used for
decarboxylative biaryl cross-coupling reactions similar to those
described by Forgione, Bilodeau and coworkers, J. Am. Chem. Soc.
2006, 128, 11350-11351, herein incorporated by reference, as shown
below:
##STR00029##
wherein W is iodo, bromo, chloro or OTf, R may be a substituent on
the ring and P is as defined herein.
[0164] There are a number of transformations known to access
quinoline scaffolds. As shown in Scheme 1A, a Friedlander approach
can be followed in which appropriately substituted aniline is
condensed with a functionalized ketone under dehydration
conditions. This intermediate is then cyclized under thermal
conditions followed by halogenation of the resulting alcohol. The
acetic acid ester side chain can be oxidized and protected to
furnish the alpha t-butoxy acetic acid ester moiety as shown.
Separation of the enantiomers can be accomplished by formation of
diastereomers by addition of a chiral auxiliary such as an
oxazolidinone followed by conversion to the corresponding ester by
known means.
##STR00030##
[0165] Alternatively, a modification of this approach can also be
used to prepare the quinoline scaffold as shown in Scheme 2. In
this method a properly substituted anthranilic acid derivative can
be condensed under dehydration conditions with an appropriate
ketone and subsequently cyclized under DMAP/POCl.sub.3 conditions
to the 4-chloroquinoline. Further elaboration can then be performed
as outlined in Scheme 1A.
##STR00031##
[0166] Furthermore, in an alternative route the quinoline scaffold
can be accessed in an enantioselective manner as outlined in Scheme
3.
##STR00032## ##STR00033##
[0167] A quinoline precursor can be selectively brominated in the
3-position and subsequently elaborated into the chiral diol by
standard methods known in the literature. The chiral diol can be
differentially protected to the t-butyl ether followed by
liberation of the primary alcohol. This alcohol can then be
oxidized to the corresponding carboxylic acid and subsequently
protected as the methyl ester to furnish the key chiral
4-iodoquinoline intermediate.
##STR00034##
[0168] In an alternate route to compounds of general formula I, the
known aldehyde Via is transformed to terminal alkyne VIb. Those
skilled in the art will recognize that there are a number of
methods for accomplishing this transformation, such as, but not
limited to the Bestmann-Ohira reaction or the Corey-Fuchs reaction.
The R.sup.4 group is then attached to the alkyne using conditions
well-known to those skilled in the art, preferentially via a
Sonogashira coupling between the alkyne and the aryl iodide
derivative of the R.sup.4 group, to give the internal alkyne VIc.
Other methods may include the Castro-Stevens reaction, or the
silver mediated, palladium catalyzed coupling of alkyne VIb and the
boronic acid or ester derivative of the R.sup.4 fragment as
reported by Zou and coworkers (Tetrahedron Lett. 2003, 44,
8709-8711) to give the internal alkyne VIc. In this method a
properly substituted benzoylacetonitrile can be condensed in the
presence of sulfur with an appropriate ketone or aldehyde by
standard methods known in the literature. VIc then undergoes a
cyclocondensation with amide VId to give quinoline Vie. Those
skilled in the art will recognize this may involve activation of
amide VId to facilitate the overall condensation. This is
preferentially achieved by the action of triflic anhydride and in
the presence of 2-chloropyridine as described by Movassaghi (J. Am.
Chem. Soc., 129 (33), 10096-10097, 2007), but may also be achieved
in other ways. Amides VId are typically commercially available,
although those skilled in the art will recognize that they are also
easily obtained from commercially available aniline or nitro arene
precursors. The cyclic diketal is then hydrolyzed to give diol VIf
under acidic conditions. The terminal alcohol is then protected to
give VIg, where P can be a number of different protecting groups
including, but not limited to, a trimethylacetyl group. The
secondary alcohol is then derivatized with a tert-butyl group to
give compound VIh. Those skilled in the art will recognize that
this can be accomplished in more than one way, including an
SN.sub.1 reaction or acid catalyzed addition to isobutylene. The
protecting group is then removed to give primary alcohol VIj, which
in turn is oxidized to carboxylic acid VIk. It will be obvious that
the oxidation of VIj to VIk can be accomplished in one or two
synthetic steps. In the preferred method, Dess-Martin oxidation to
an intermediate aldehyde followed by Lindgren oxidation is
employed.
##STR00035##
[0169] In yet another route to compounds of general formula I,
synthesis of intermediate VIh may also be accomplished following a
path that begins with acid catalyzed hydrolysis of the cyclic
diketal of terminal alkyne VIb to give diol VIIa. The terminal
alcohol is then protected to give VIIb, where P can be a number of
different protecting groups including, but not limited to, a
trimethylacetyl group. The secondary alcohol is then derviatized
with the tert-butyl group to give compound VIIc. Those skilled in
the art will recognize that this can be accomplished in more than
one way, including an SN.sub.1 reaction or acid catalyzed addition
to isobutylene. The R.sup.4 group is then attached to the alkyne
using conditions well-known to those skilled in the art,
preferentially via a Sonogashira coupling between the alkyne and
the aryl iodide derivative of the R.sup.4 group, to give the
internal alkyne VIId. The internal alkyne VIId then undergoes a
cyclocondensation with amide VId to give quinoline VIh,
preferentially achieved by the action of triflic anhydride and in
the presence of 2-chloropyridine as described for step 3 of Scheme
4. From intermediate VIh, the synthesis compounds of general
formula I is then accomplished following steps 7 and 8 of Scheme
4.
EXAMPLES
[0170] Other features of the present invention will become apparent
from the following non-limiting examples which illustrate, by way
of example, the principles of the invention. It will be apparent to
a skilled person that the procedures exemplified below may be used,
with appropriate modifications, to prepare other compounds of the
invention as described herein.
[0171] As is well known to a person skilled in the art, reactions
are performed in an inert atmosphere (including but not limited to
nitrogen or argon) where necessary to protect reaction components
from air or moisture. Temperatures are given in degrees Celsius
(.degree. C.). Solution percentages and ratios express a volume to
volume relationship, unless stated otherwise. Flash chromatography
is carried out on silica gel (SiO.sub.2) according to the procedure
of W. C. Still et al., J. Org. Chem., (1978), 43, 2923. Mass
spectral analyses are recorded using electrospray mass
spectrometry. A number of intermediate and final products are
purified using CombiFlash.RTM. Companion apparatus, purchased from
Teledyne Isco Inc, employing pre-packed silica gel cartridges and
EtOAc and hexanes as solvents. These cartridges are available
either from Silicycle Inc (SiliaFlash, 40-63 microns silica) or
from Teledyne Isco (RediSep, 40-63 microns silica). Preparative
HPLC is carried out under standard conditions using a SunFire.TM.
Prep C18 OBD 5 .mu.M reverse phase column, 19.times.50 mm and a
linear gradient employing 0.1% TFA/acetonitrile and 0.1% TFA/water
as solvents. Compounds are isolated as TFA salts when
applicable.
[0172] Analytical HPLC is carried out under standard conditions
using a Combiscreen ODS-AQ C18 reverse phase column, YMC,
50.times.4.6 mm i.d., 5 .mu.M, 120 .ANG. at 220 nM, elution with a
linear gradient as described in the following table (Solvent A is
0.06% TFA in H.sub.2O; solvent B is 0.06% TFA in CH.sub.3CN):
TABLE-US-00002 Time Flow Solvent A Solvent B (min) (mL/min) (%) (%)
0 3.0 95 5 0.5 3.0 95 5 6.0 3.0 50 50 10.5 3.5 0 100
[0173] Abbreviations or symbols used herein include:
Ac: acetyl; AcOH: acetic acid; Ac.sub.2O: acetic anhydride;
Anti-his XL665: XL665 labeled anti-His antibody; BOC or Boc:
tert-butyloxycarbonyl; BSA: bovine serum albumin; Bu: butyl; CD:
circular dichroism DABCO: 1,4-diazabicyclo[2.2.2]octane Dba:
dibenzylidene acetone; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene;
DCE: dichloroethane; DEAD: diethyl azodicarboxylate; DCM:
dichloromethane; DIAD: diisopropyl azodicarboxylate; DIBAL:
diisobutyl aluminum hydride; DIPEA: diisopropylethylamine DMAP:
N,N-dimethyl-4-aminopyridine; DME: 1,2-dimethoxyethane;
DMF: N,N-dimethylformamide;
[0174] DMSO: dimethylsulfoxide;
Dppf: 1,1'-Bis(diphenylphosphino)ferrocene;
[0175] EC.sub.50: 50% effective concentration; Eq: equivalent; Et:
ethyl; Et.sub.3N: triethylamine; Et.sub.2O: diethyl ether; EtOAc:
ethyl acetate; EtOH: ethanol; HATU: O-(7-Azabenzotriazole-1-A-N,
N,N'N'-tetramethyluronium hexafluorophosphate: HBTU:
O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate;
HEPES: N-2-hydroxyethyl piperazine N-ethane sulfonic acid; HPLC:
high performance liquid chromatography; IC.sub.50: 50% inhibitory
concentration; ITC: Isothermal calorimetry; .sup.iPr or i-Pr:
1-methylethyl (iso-propyl); Kd.sub.app: apparent affinity constant;
KHMDS: potassium hexamethyl disilazane; LiHMDS: lithium
hexamethyldisilazide; Me: methyl; MeCN: acetonitrile; MeOH:
methanol; MOI: multiplicity of infection; MS: mass spectrometry
(ES: electrospray); n-BuONa: sodium n-butoxide n-BuOH: n-butanol;
n-BuLi: n-butyl lithium; NMR: nuclear magnetic resonance
spectroscopy; OD: optical density; ORD: optical rotary dispersion;
Ph: phenyl; PhMe: toluene; PG: protecting group; PPh.sub.3:
triphenylphosphine; Pr: propyl; RPMI: Roswell Park Memorial
Institute (cell culture medium); RT: room temperature
(approximately 18.degree. C. to 25.degree. C.); SM: starting
material; Strep-EuK: Streptavidin labeled with europium cryptate;
tert-butyl or t-butyl: 1,1-dimethylethyl; TCEP:
tris[2-carboxyethyl]phosphine; Tf: trifluoromethanesulfonyl;
Tf.sub.2O: trifluoromethanesulfonic anhydride; TFA: trifluoroacetic
acid; THF: tetrahydrofuran; and TLC: thin layer chromatography.
Example 1
Synthesis of Quinoline Scaffold 1i
##STR00036##
[0177] Step 1:
[0178] In a 4-neck 500 mL round bottom flask equipped with a
magnetic stir bar, condenser and Dean-Stark trap, diethyl
acetylsuccinate (6 g, 0.026 mol), aniline 1a (2.5 mL, 0.028 mol),
Amberlyst.RTM. 15 (0.08 g) and toluene (30 mL) are added. The
resulting mixture is heated at reflux temperature for approximately
3 days at which time TLC shows only traces of SM. The reaction
mixture is cooled to RT and the Amberlyst.RTM. 15 is removed by
filtration. The filtrate is concentrated in vacuo to give a
suspension of a solid in brown liquid. The filtrate is diluted with
diethyl ether and cooled. The solid is filtered and the filtrate is
concentrated in vacuo leaving a brown oil (.about.7.8 g), which
contains 1b and some cyclised intermediate. This crude intermediate
is used in the next step without further purification.
[0179] Step 2:
[0180] In a 3-neck 100 mL round bottom flask a mixture of the crude
intermediate 1 b (.about.7.8 g) and diphenyl ether (.about.50 mL)
are heated quickly in a pre-heated (250.degree. C.) heating mantle
for 6 min (internal temperature reaches .about.250.degree. C.) at
which time the flask is removed from the heating mantle and is
stirred until the internal temperature reaches below 100.degree. C.
The reaction mixture is then mixed with hexane (15 mL), at which
time a light brown solid is formed. The solid is filtered and
washed with hexane (3.times.10 mL) to give approximately 2.4 g of
the intermediate cyclised product. A portion of this sample (1.4 g,
5.87 mmol) is dissolved in phosphorus oxychloride (5 mL) and heated
at reflux for 2.5 h. The reaction mixture is cooled to RT and is
concentrated on vacuum. The residue is treated with sodium
bicarbonate powder then partitioned between EtOAc and water. The
combined organic layer is washed with brine, dried over anhydrous
Na.sub.2SO.sub.4, filtered, passed through a silica gel pad and
concentrated to give 1c as a crude light brown solid (.about.2.35
g).
[0181] Step 3:
[0182] Crude chloro quinoline 1c (1.36 g, 5.17 mmol) is dissolved
in THF (20 mL), and HCl in dioxane (4 M, 5.4 mL, 0.022 mol) is
added to this solution slowly. The resulting reaction mixture is
stirred at RT for 40 min. The solvent is then removed in vacuo and
the residue is dried on vacuum. The resulting solid and NaI (3.87
g) are suspended in MeCN (20 mL), and the resulting reaction
mixture is heated to reflux for 16 h. The reaction mixture is
cooled to RT and treated with a saturated aqueous solution of
NaHCO.sub.3 (20 mL). The aqueous layer is extracted with DCM, and
the combined organic layer is dried over anhydrous MgSO.sub.4,
filtered and concentrated to give a brown syrup. Purification by
silica gel chromatography (30% EtOAc/hexanes) provides iodo
quinoline 1d as an off-white solid (1.72 g, 94% yield).
[0183] Step 4:
[0184] To a solution of KHMDS (0.5 M in toluene, 3 mL, 1.5 mmol) in
THF (8 mL) at -78.degree. C. is added a solution of 1d (0.35 g,
0.99 mmol) in THF (8 mL). As the ester is added, the solution
becomes scarlet red. This is allowed to stir at -78.degree. C. for
30 min before being treated with the Davis reagent (0.39 g, 1.5
mmol). After addition of the oxidizing agent, the solution becomes
pale yellow and this is stirred for an additional 30 min at
-78.degree. C. The reaction is quenched with saturated NH.sub.4Cl
aqueous solution (8 mL) is warmed to RT and is diluted with EtOAc.
The mixture is washed with brine and the organic phase dried
(Na.sub.2SO.sub.4), filtered and concentrated to afford a solid.
Purification by silica gel chromatography (hexanes/EtOAc: 6/4)
provides 1e as a beige solid (0.50 g, >98% yield).
[0185] Step 5:
[0186] To a suspension of iodoalcohol 1e (0.53 g, 1.4 mmol) in
tert-butyl acetate (12 mL) at RT is added perchloric acid (0.66 mL,
4.6 mmol). The reaction is left to stir for 2 h at RT (suspension
turns into a clear solution). The reaction is quenched with water
(12 mL) and basified with solid NaHCO.sub.3 until pH .about.6. The
crude product is extracted with EtOAc (3.times.10 mL), washed with
brine (10 mL), dried over MgSO.sub.4, filtered and concentrated to
afford the crude product. Purification by silica gel chromatography
(hexanes/EtOAc: 85/15) affords 1f as a pale yellow oil (0.56 g, 91%
yield).
[0187] Step 6:
[0188] Intermediate 1f (0.59 g, 1.4 mmol) is dissolved in a 2 M
NaOH aqueous solution (7 mL, 0.014 mol) with ethanol (10 mL) and is
stirred for 4 h at RT. The ethanol is then removed in vacuo. The
resulting residue is diluted with water (3 mL) and acidified with 2
M HCl solution until pH .about.3-4. The residue is then extracted
with DCM (3.times.10 mL), dried over Na.sub.2SO.sub.4, filtered,
concentrated and dried under high vacuum to afford 1g as a foamy
solid (0.56 g, >98% yield).
[0189] Step 7:
[0190] To a solution of acid 1g (0.39 g, 0.97 mmol) and HBTU (0.48
g, 1.3 mmol) in anhydrous THF (5 mL) is added diisopropylethylamine
(0.5 mL, 2.9 mmol). The mixture is stirred for 5.5 h at
30-35.degree. C. (internal temperature) at which time the sodium
salt of R-(+)-benzyloxazolidinone (which is prepared by adding
sodium hydride (60% dispersion in mineral oil, 78 mg, 1.95 mmol) to
a solution of R-(+)-benzyloxazolidinone (0.35 g, 1.9 mmol) in
anhydrous THF (5 mL) is added. The resulting solution is then
stirred at RT for 16 h. The solvent is removed in vacuo and
partitioned between water and EtOAc. The aqueous phase is then
extracted with EtOAc, and the combined organic extracts are dried
over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated in
vacuo to afford a pale yellow solid. The crude product is purified
by silica gel chromatography (10->30% EtOAc:hexanes), yielding
the desired diastereomer 1h (190 mg, 35% yield, more polar product,
>99% ee by chiral column).
[0191] Step 8:
[0192] To a solution of oxazolidinone 1h (190 mg, 0.34 mmol) in
THF/H.sub.2O (2 mL/1 mL) at 0.degree. C. is added H.sub.2O.sub.2
(30%, 0.36 mL) followed by LiOH monohydrate (17 mg, 0.41 mmol)
dissolved in water (1 mL). The reaction mixture is stirred at
0.degree. C. for 30 min at which time 10% Na.sub.2SO.sub.3 (0.26
mL) is added. The resulting mixture is stirred for .about.10 min
and then acidified with 2 N HCl to pH .about.4-5. The product is
then extracted with DCM (3.times.10 mL). The combined organic
extracts are dried over sodium sulfate and concentrated in vacuo to
yield the crude acid intermediate as a white foam (0.13 g, 96%
yield), which is used in the next step without further
purification. The acid (130 mg) is suspended in diethyl ether (3
mL) and treated with diazomethane in diethyl ether until all of the
acid SM is consumed (as indicated by TLC). The reaction is quenched
with a very small amount of glacial AcOH and then concentrated in
vacuo to give an off-white solid. The crude ester product is
purified by silica gel chromatography (10-15% EtOAc/hexanes)
yielding the quinoline fragment 1i (120 mg, 89% yield) in high
enantiomeric purity (>99% ee by chiral HPLC).
Example 2
Synthesis of Fragment 2f
##STR00037##
[0194] Step 1:
[0195] Aldehyde 2a (5.85 g, 28.6 mmol, for preparation see: Michel,
P. and Ley, S. V. Synthesis 2003, 10, 1598-1602, herein
incorporated by reference), phoshonate 2b (6.6 g, 34 mmol) and
K.sub.2CO.sub.3 (8.8 g, 64 mmol) are combined in MeOH (125 mL) and
the reaction is stirred overnight at RT. The reaction is evaporated
nearly to dryness and the residue is partitioned between H.sub.2O
(250 mL) and EtOAc (500 mL). The water layer is washed with EtOAc
(2.times.250 mL) and the combined organic layers dried over
anhydrous Na.sub.2SO.sub.4 and concentrated to give alkyne 2c (5.55
g, 97% yield).
[0196] Step 2:
[0197] Alkyne 2c (5.0 g, 25 mmol) is dissolved in TFA (35 mL) and
water (3.6 mL) and the solution is stirred at RT. After 30 min, the
reaction is concentrated under reduced pressure and the residue is
purified by CombiFlash.RTM. Companion to give diol 2d (1.8 g, 84%
yield).
[0198] Step 3:
[0199] A solution of diol 2d (1.2 g, 14 mmol) and triethylamine
(1.7 mL, 12 mmol) in DCM (80 mL) is cooled to 0.degree. C. under
N.sub.2. Trimethylacetylchloride is added dropwise and the
resulting mixture is allowed to come to RT and stir overnight. The
reaction is then quenched with MeOH (100 mL) and stirring is
continued for 20 min. The mixture is then concentrated under
reduced pressure and the residue is purified by CombiFlash.RTM.
Companion to give the desired mono ester 2e (550 mg, 40% yield)
along with the undesired regioisomeric mono ester (378 mg, 27%
yield).
[0200] Step 4:
[0201] In a sealable reaction flask, a solution of the propargylic
alcohol 2e (375 mg, 2.20 mmol) and Amberlyst.RTM. H-15 resin (150
mg) in hexane (3 mL) is cooled to -78.degree. C. Isobutene is then
bubbled through the solution until the volume approximately
doubles. The tube is then sealed, brought to RT and is stirred
overnight. The tube is then cooled to -78.degree. C., is opened and
brought back to RT. The mixture is then filtered through a plug of
SiO.sub.2 (EtOAc wash) and concentrated under reduced pressure to
provide pure tert-butyl ether 2f (390 mg, 78% yield).
Example 3
Synthesis of Alkyne 3a
##STR00038##
[0203] Step 1:
[0204] Solid Pd(PPh.sub.3).sub.4 (444 mg, 0.385 mmol) and CuI (146
mg, 0.769 mmol) are successively added to a solution of
6-iodochroman (10 g, 34 mmol) and alkyne 2c (11 g, 55 mmol)
dissolved in DMF (23 mL) and diethylamine (115 mL). The reaction
mixture is stirred overnight at RT and then concentrated, diluted
with EtOAc (300 mL) and successively washed with brine, 1 N aqueous
HCl and water (300 mL each). The organic layer is dried over
Na.sub.2SO.sub.4 and the residue purified by CombiFlash.RTM.
Companion to give alkyne 3a (10.8 g, 84% yield).
Example 4
Synthesis of Boronate Fragment 4f (Used in Preparation of 1086)
##STR00039##
[0206] Step 1:
[0207] To a solution of 4a (6 g, 37 mmol) in nitrobenzene (12 mL),
chloroacetyl chloride (4.6 mL, 57.5 mmol) is added, followed by the
addition of AlCl.sub.3 (20.4 g, 152 mmol). As the AlCl.sub.3 is
added, the mixture becomes viscous and gas evolution is observed.
The resulting brown syrupy mixture is left to stir overnight at RT.
(Reference: Y. Takeuchi et. al., Chem. Pharm. Bull. 1997, 45(12),
2011-2015.) The thick reaction mixture is cooled and ice water is
added very carefully (Very exothermic!!) a few drops at a time.
Once gas evolution and bubbling is subsided, cold water is further
added followed by EtOAc. The mixture is stirred for 5 min and the
product extracted with EtOAc (3.times.). The combined organic
layers are washed with brine (1.times.), dried over
Na.sub.2SO.sub.4, filtered and concentrated to afford the
uncyclized chloroketone (24 g of crude; contaminated with some
nitrobenzene) as a pale yellow solid. This intermediate is then
taken up in EtOH (100 mL), NaOAc is added (20.4 g, 248 mmol) and
the reaction is brought to reflux for 40 min. The EtOH is
evaporated, the residue is taken up in EtOAc (300 mL) and washed
with 5% K.sub.2CO.sub.3 (2.times.200 mL) and the aqueous layer then
acidified with aqueous HCl (1 N; pH=.about.5). This acidic layer is
extracted with EtOAc (2.times.250 mL), washed with brine
(1.times.), dried over Na.sub.2SO.sub.4, filtered and concentrated
to afford the crude product. This material is purified by
CombiFlash.RTM. Companion (120 g) to afford intermediate 4b as a
yellow solid (4.7 g).
[0208] Step 2:
[0209] The ketone 4b (127 mg, 0.64 mmol) is dissolved in EtOH (2
mL) and treated with hydrazine hydrate (500 .mu.L, 16 mmol). The
mixture is heated to reflux for 45 min before allowing it to cool
to RT. The solvent is removed by evaporation and the residue is
dissolved in diethylene glycol (1 mL) before being treated with KOH
(108 mg, 1.92 mmol) and then heated to 110-120.degree. C. for 2.5
h. The reaction mixture is diluted with EtOAc and the pH is
adjusted with 1 N HCl to pH <4. The organic phase is separated,
washed with saturated brine, dried over anhydrous MgSO.sub.4,
filtered and concentrated. The crude material is purified by
CombiFlash.RTM. Companion (eluent: 0-50% EtOAc/hexanes) to give
intermediate 4c as a yellow oil (62 mg).
[0210] Step 3:
[0211] A solution of 4c (61 mg, 0.33 mmol) is cooled to -78.degree.
C. in DCM (2 mL) and then treated with BBr.sub.3 (1 M in DCM, 825
.mu.L, 0.82 mmol). After 15 min, the bath is removed and the
reaction is allowed to reach RT. The reaction is then stirred for
1.5 h. The reaction is cooled to 0.degree. C. before quenching by
the careful dropwise addition of water. The mixture is treated with
saturated NaHCO.sub.3 (to pH .about.8) and the phases separated.
The organic phase is washed with saturated brine, dried over
MgSO.sub.4, filtered and concentrated to dryness. The product is
purified by CombiFlash.RTM. Companion (0-50% EtOAc/hexanes) to give
intermediate 4d as colorless oil, which solidifies upon standing
(40 mg, 71% yield).
[0212] Step 4:
[0213] The phenol 4d (40 mg, 0.23 mmol) is dissolved in DCM (2 mL),
cooled to 0.degree. C. and treated with pyridine (95 .mu.L, 1.17
mmol), followed by Tf.sub.2O (44 .mu.L, 0.26 mmol). The reaction is
allowed to stir at this temperature for 10 min before warming to RT
over a period of 1 h. The reaction mixture is diluted with DCM and
the organic phase washed with 10% citric acid and then brine. The
organic phase is dried over anhydrous MgSO.sub.4, filtered,
concentrated and purified by CombiFlash.RTM. Companion (0-50%
EtOAc/hexanes) to give 4e as a yellow oil (67 mg, 94% yield).
[0214] Step 5:
[0215] To a solution of the triflate 4e (66 mg, 0.22 mmol) in DMF
(2 mL), bis-(pinacolato)diborone (72 mg, 0.28 mmol) and potassium
acetate (64 mg, 0.65 mmol) are added. This solution is de-gassed
(with bubbling Ar) for 10 min before adding
PdCl.sub.2(dppf)-CH.sub.2Cl.sub.2, (27 mg, 0.03 mmol). The mixture
is de-gassed a further 5 min before being heated to 90.degree. C.
for 16 h. The mixture is cooled to RT and diluted with EtOAc/water.
The organic phase is washed with saturated brine (3.times.), dried
over anhydrous MgSO.sub.4, filtered and concentrated. The crude
material is purified by CombiFlash.RTM. Companion (0-70% EtOAc in
hexanes) to afford the boronate 4f as a white solid (41 mg, 67%
yield).
Example 5
Synthesis of Boronate Fragment 5f (Used in Preparation of 1077,
1091, 1095, 1099, 1100, 1118)
##STR00040##
[0217] Step 1:
[0218] The nitrophenol 5a (5.23 g, 34.1 mmol) is dissolved in
acetic acid (20 mL) and the solution is cooled in an ice bath.
Bromine (1.75 mL, 34.15 mmol), dissolved in 5 mL acetic acid) is
added dropwise with stirring. The mixture is stirred for 1 h at
0.degree. C. before being poured into ice water (250 mL). The
mixture is extracted with EtOAc (2.times.100 mL) and then washed
with 5% NaHCO.sub.3 (2.times.50 mL) before being dried over
anhydrous MgSO.sub.4, filtered and concentrated to give the desired
crude product 5b as an orange solid (8.2 g, quantitative yield).
This material is used in the next step without further
purification.
[0219] Step 2:
[0220] To a well stirred ethanol solution (75 mL) of 5b (8.1 g,
34.9 mmol), SnCl.sub.2 (20 g, 105 mmol) is added. The reaction
mixture is stirred at reflux for 2.5 h. After that period, the
transformation is incomplete, therefore, more SnCl.sub.2 (2 g, 10
mmol) is added and the reaction mixture is heated at reflux for 1 h
before being cooled to RT. The mixture is poured onto 250 g of ice
and the pH adjusted to approximately 7.5 with aqueous 5%
NaHCO.sub.3. The product is extracted with EtOAc (3.times.100 mL)
before being washed with saturated brine (2.times.100 mL). The
organic phase is dried over anhydrous MgSO.sub.4, filtered and
concentrated to dryness to give the aniline intermediate 5c as a
gray solid (8.25 g, .about.100% yield; this material contained some
tin residues, nonetheless, it is used as such for the following
step).
[0221] Step 3:
[0222] To a stirring, ice cold, DMF (5 mL) suspension of potassium
carbonate (2.05 g, 14.8 mmol) and aniline 5c (750 mg, 3.71 mmol)
under nitrogen, chloroacetyl chloride (355 .mu.L, 4.45 mmol) is
added dropwise. The mixture is allowed to warm to RT over a period
of 15 min and then heated to -60.degree. C. for 1 h. The mixture is
allowed to cool to RT, is poured into a mixture of ice/water (250
mL) and is stirred for 15 min. The suspension is centrifuged, and
the supernatant is discarded. The solid material is left drying
under suction overnight to give intermediate 5d (280 mg, 31%
yield).
[0223] Step 4:
[0224] To an ice cold THF (6 mL) solution of the cyclic amide 5d
(280 mg, 1.16 mmol) under nitrogen, a borane-THF solution (1 M in
THF, 1.74 mL, 1.74 mmol) is added slowly. The reaction mixture is
slowly allowed to warm to RT, then is stirred at RT for
approximately 1.5 h and then gently heated to reflux for 1 h to
complete the conversion. The mixture is cooled in an ice bath and
is carefully quenched with aqueous 1 M NaOH (4 mL) over 10 min. The
reaction mixture is partitioned between EtOAc (150 mL) and water
(25 mL). The organic layer is washed with aqueous 1 N NaOH (20 mL),
saturated aqueous NaCl, and finally dried over anhydrous
MgSO.sub.4, filtered and concentrated to give the crude 5e as an
amber oil (212 mg, 81% yield). This product is used as such for
next transformation.
[0225] Step 5:
[0226] A well stirred DMF (15 mL) solution of the arylbromide 5e
(0.50 g, 2.19 mmol), potassium acetate (0.728 g, 7.67 mmol) and
bis(pinacolato)diborane (0.83 g, 3.3 mmol) is degassed by bubbling
Ar through the solution for 20 min. PdCl.sub.2(dppf)-DCM (320 mg,
0.44 mmol) is added and degassing is continued for 15 min. The
system is sealed (teflon screw cap vessel) under Ar and heated to
-90.degree. C. for 5 h. The reaction mixture is allowed to cool to
RT, dilute with EtOAc (150 mL), washed with brine (3.times.100 mL)
and water (2.times.100 mL), dried over anhydrous MgSO.sub.4,
filtered and concentrated to dryness. The residue is purified by
CombiFlash.RTM. Companion (EtOAc/hexanes) to give the desired
boronate 5f (389 mg, 65% yield) as a yellowish waxy solid.
Example 6
Synthesis of Boronate Fragment 6i (Used in Preparation of 1038,
1039, 1053, 1054, 1055, 1056, 1083)
##STR00041##
[0228] Step 1:
[0229] Sodium hydride (60%, 7.78 g, 194 mmol) is added to a well
stirred suspension of 6a (12.5 g, 97.2 mmol) in THF (100 mL). After
stirring the reaction mixture for 1 h, N,N-diethylcarbamoyl
chloride (24.64 mL, 194 mmol) is added at RT. After stirring the
reaction overnight, the reaction mixture is quenched with water
(100 mL), extracted with EtOAc (3.times.50 mL), is dried over
anhydrous MgSO.sub.4, filtered and evaporated under reduced
pressure to obtain 6b (33 g, 75% yield) in high purity.
[0230] Step 2:
[0231] Diisopropylamine (21.0 mL, 121 mmol) in THF (330 mL) is
treated with a solution of n-BuLi (2.5 M in hexanes, 48.2 mL, 121
mmol) at 0.degree. C. After 30 min at this temperature, the
solution is cooled to -78.degree. C. and carbamate 6b (33.29 g,
109.7 mmol, 75% pure) is added. The reaction is stirred at this
temperature for 30 min and then iodine (33.4 g, 132 mmol) is added.
The solution is stirred for 30 min at 0.degree. C. and is then
warmed to RT. After 2 h, the reaction mixture is quenched with
water (250 mL) and the volatile organic solvents are removed under
reduced pressure. The aqueous phase is then extracted with EtOAc
(3.times.100 mL), washed with 1 N HCl (200 mL), dry MgSO.sub.4,
filtered and evaporated under reduced pressure to obtain 6c (18.6
g, 39% yield).
[0232] Step 3:
[0233] The iodocarbamate 6c (10 g, 28 mmol), propargyl alcohol (3.3
mL, 56 mmol), Pd(PPh.sub.3).sub.4 (3.27 g, 2.83 mmol) and copper
iodide (1.08 g, 5.66 mmol) are combined in diisopropylamine (39 mL,
39 mmol) in a sealable tube under Ar and heated at 100.degree. C.
After 1 h, the reaction mixture is cooled to RT and poured into
EtOAc (100 mL) and this mixture is extracted with 10% HCl
(2.times.100 mL). The organic layer is dried over MgSO.sub.4 and
concentrated to dryness. The crude product is purified by
CombiFlash.RTM. Companion to obtain 6d (3.65 g, 46% yield).
[0234] Step 4:
[0235] 6d (3.63 g, 12.9 mmol) is dissolved in EtOAc (81 mL) and
treated with Rh--Al.sub.2O.sub.3 (5% w/w, 3.45 g, 1.68 mmol). The
flask is evacuated and charged with 1 atmosphere of H.sub.2
(balloon) and the reaction is stirred overnight at RT. The reaction
mixture is filtered through Celite.RTM. (EtOAc wash) and the
filtrate is concentrated under reduced pressure. The residue is
then purified by CombiFlash.RTM. Companion to obtain 6e (3.7 g, 71%
yield).
[0236] Step 5:
[0237] Solid NaOH (920 mg, 23 mmol) is added to a solution of 6e
(2.63 g, 9.20 mmoL) in EtOH (93 mL) and the mixture is heated to
reflux and is stirred overnight. The mixture is then cooled to RT
and the organic solvent removed under reduced pressure. Water is
added (100 mL) and the mixture extracted with Et.sub.2O
(3.times.100 mL), dried over MgSO.sub.4, filtered and evaporated
under reduced pressure to obtain phenol 6f (869 mg, 51% yield).
[0238] Step 6:
[0239] Diethyl azodicarboxylate (953 .mu.L, 6.05 mmol) is added
dropwise to a solution of phenol 6f (869 mg, 4.66 mmol) and
PPh.sub.3 (1.59 g, 6.053 mmol) in THF (65 mL) and the reaction is
stirred at RT. After 4 h, the reaction mixture is evaporated under
reduced pressure. The residue is then purified by CombiFlash.RTM.
Companion to obtain the chroman 6g (387 mg, 49% yield).
[0240] Step 7:
[0241] Iodine (583 mg, 2.295 mmol) is added to a solution of
chroman 6g (387 mg, 2.29 mmol) and AgNO.sub.3 (429 mg, 2.52 mmol)
in MeOH (23 mL). After 20 min, a 0.5 M solution of sodium
thiosulfate (10 mL) is added and the aqueous phase extracted with
EtOAc (3.times.25 mL). The combined organic phases are washed with
brine, then dried (MgSO.sub.4), filtered and evaporated to obtain
aryl iodide 6h (647 mg, 96% yield).
[0242] Step 8:
[0243] A solution of iodo intermediate 6h (647 mg, 2.20 mmol),
bis(pinocolato)diborane (0.725 g, 2.86 mmol) and potassium acetate
(0.626 g, 6.59 mmol) in DMF (17 mL) is degassed with Ar for 10 min.
PdCl.sub.2(dppf)-DCM complex (179 mg, 0.22 mmol) is then added and
the mixture is degassed with Ar for approximately another 5 min.
The reaction is then heated to 95.degree. C. in a sealable tube and
is stirred overnight. The reaction is cooled to RT and EtOAc (100
mL) is added. The solution is washed with brine (3.times.150 mL),
water (1.times.150 mL), dried over MgSO.sub.4, filtered and solvent
removed under reduced pressure. The residue is purified by
CombiFlash.RTM. Companion to afford boronate ester 6i (260 mg, 40%
yield).
Example 7
Synthesis of Boronate Fragment 7d (Used in Preparation of 1065,
1107)
##STR00042##
[0245] Step 1:
[0246] A solution of phenol 7a (0.91 g, 5.74 mmol) in dry DMF (1
mL) is added dropwise to a slurry of NaH (60% in oil, 0.60 g, 15
mmol) in dry DMF (1 mL) cooled to 10-15.degree. C. (cold water
bath) and the mixture is stirred for 20 min. This results in a
thick, frothy white mixture. A solution of 3-bromopropionic acid
(1.1 g, 6.9 mmol) in dry DMF (0.5 mL) is then added dropwise and
the reaction stirred at RT overnight. After 16 h, MeOH (1.2 mL) is
added to help break up the thick, pasty reaction mixture which is
then added to diluted HCl (.about.12 mL, 1 N HCl in 100 mL water)
and extracted with EtOAc (80 mL; the pH of the aqueous phase is
adjusted to pH <3). The organic layer is dried over anhydrous
Na.sub.2SO.sub.4 and evaporated to give 7b as a white solid
material, contaminated with some unreacted SM (1.29 g of crude
material). This material is used in the next step without
purification.
[0247] Step 2:
[0248] The crude compound 7b (1.53 g, 6.63 mmol) is combined with
polyphosphoric acid (approximately 7 g) and heated to 75.degree. C.
to give a cherry red colored solution. During the reaction time,
the reaction mixture becomes viscous and stirring becomes
difficult. After 4 h, ice and water are slowly added with rapid
stirring to give a thick suspension. This mixture is transferred to
a separatory funnel where the product is extracted with EtOAc (100
mL) and washed with water (100 mL), saturated NaHCO.sub.3
(2.times.100 mL) and brine (75 mL). The organic phase is dried over
anhydrous MgSO.sub.4 and evaporated to give 7c as a sticky violet
solid which is used as such (1.29 g).
[0249] Step 3:
[0250] Intermediate 7c is analogous to intermediate 4b in Example
4; those skilled in the art would recognize that the same synthetic
methodologies used to convert 4b to the boronate 4f can be applied
for the conversion of 7c to the corresponding boronate 7d.
Example 8
Synthesis of Boronate Fragment 8h (Used in Preparation of 1069)
##STR00043##
[0252] Step 1
[0253] 2-Amino-m-cresol 8a (5.7 g, 46.3 mmol) is dissolved in
H.sub.2O (30 mL) and 1,4-dioxan (15 mL). The mixture is heated to
reflux and then HBr (48%, 17 mL, 0.31 mol) is added dropwise over a
period of 20 min. The reflux is maintained for an additional 15 min
after the addition is complete. The reaction is cooled to 0.degree.
C., and NaNO.sub.2 in H.sub.2O (20 mL) is added over a period of 30
min. The stirring is continued for 15 min at 0.degree. C., the
mixture is then transferred in one shot to a stirring mixture of
Cu(I)Br (7.64 g, 53.2 mmol) in H.sub.2O (20 mL) and HBr (48%, 17
mL, 0.31 mol) at 0.degree. C. (protected from light). The reaction
is stirred for 15 min at 0.degree. C., warmed to 60.degree. C.,
stirred for an additional 15 min, cooled to RT and then stirred
overnight. The reaction mixture is then transferred to a separatory
funnel and extracted with EtOAc (3.times.). The organic layers are
combined, washed with brine, dried over anhydrous MgSO.sub.4,
filtered and concentrated over silica to afford a mixture that is
purified using the CombiFlash.RTM. Companion (20% EtOAc/hexanes) to
afford the desired bromide 8b (1.46 g, 17% yield) as a red-brown
oil.
[0254] Step 2:
[0255] To a solution of the bromide 8b (1.36 g; 7.27 mmol) and
(PPh.sub.3).sub.2PdCl.sub.2 (766 mg, 1.09 mmol, 15 mol %) in DMF
(12 mL), 1-ethoxyvinyl-tri-n-butyltin (2.7 mL, 8.0 mmol) is added.
The mixture is capped and heated in a microwave at 160.degree. C.
for 15 min. HPLC and LC-MS analysis indicate approximately 70%
conversion. More 1-ethoxyvinyl-tri-n-butyltin (2.7 mL; 8.0 mmol)
and catalyst (PPh.sub.3).sub.2PdCl.sub.2 (380 mg, 0.05 mol %) are
added and the solution is again subjected to the same microwave
conditions. The reaction is quenched with 6N HCl (1.5 mL) and
stirred at RT for 1 h to effect hydrolysis of the intermediate. The
mixture is poured into EtOAc (150 mL), washed with brine
(3.times.), dried over MgSO.sub.4, filtered and concentrated over
silica to afford the mixture that is purified using the
CombiFlash.RTM. Companion to afford the desired ketone 8c (947 mg,
87% yield) as an orange oil.
[0256] Step 3:
[0257] The methyl ketone 8c (1.02 g, 6.8 mmol) is dissolved in
EtOAc (15 mL) and CHCl.sub.3 (15 mL) before being treated with
Cu(II)Br.sub.2 (3.03 g, 13.6 mmol). The mixture is heated to reflux
for 16 h. The mixture is cooled to RT, the product filtered and
washed with EtOAc (1.times.). The solution is concentrated over
silica to afford the mixture that is purified using the
CombiFlash.RTM. Companion (10% EtOAc/hexanes) to afford the
.alpha.-bromoketone 8d (710 mg, 46% yield) as an orange oil.
[0258] Step 4:
[0259] To a solution of the bromoketone 8d (710 mg, 3.1 mmol) in
anhydrous DMF (12 mL), KF (400 mg, 6.95 mmol) is added. The
reaction is stirred at RT for 16 h. The mixture is taken up in
EtOAc (150 mL), washed with brine (3.times.), dried over anhydrous
MgSO.sub.4, filtered and concentrated over silica to afford the
mixture that is purified using the CombiFlash.RTM. Companion (20%
EtOAc/hexanes) to afford the cyclic ketone 8e (280 mg, 61% yield)
as a pale orange solid.
[0260] Step 5:
[0261] Zn Dust Pre-Activation Procedure:
[0262] Zinc dust (20 g, 350 mesh) is placed in a round bottom flask
and 1 N HCl (50 mL) is added. This suspension is sonicated for 1
min before decanting off the liquid. This procedure is repeated for
a second time after which the solid is washed with EtOH (2.times.),
Et.sub.2O (2.times.) and dried under high vacuum. To a solution of
the ketone 8e (280 mg, 1.89 mmol) in AcOH (10 mL) pre-activated Zn
dust (1.24 g, 18.9 mmol) is added. The reaction mixture is then
heated to 75.degree. C. for 2 h. The reaction mixture is filtered
(with EtOAc washing of the solids). The solvent is evaporated over
silica and the mixture is directly purified using the
CombiFlash.RTM. Companion (10% EtOAc/hexanes) to afford the desired
dihyrobenzofuran 8f (174 mg, 69% yield) as a colorless oil.
[0263] Step 6:
[0264] To a solution of the dihydrobenzofuran 8f (240 mg, 1.8 mmol)
in MeOH (5 mL), AgNO.sub.3 (304 mg, 1.79 mmol) is added followed by
iodine (453 mg, 1.79 mmol). The yellow mixture is stirred at RT for
1 h. To the reaction mixture is added a solution of 10%
Na.sub.2S.sub.2O.sub.3 and the mixture is stirred for 15 min at RT.
The mixture is diluted with EtOAc (100 mL), and the organic layer
is washed with brine (3.times.) and 10% Na.sub.2S.sub.2O.sub.3
(2.times.). The organic phase is dried over anhydrous MgSO.sub.4,
filtered and concentrated over silica to give a mixture. This
mixture is purified using the CombiFlash.RTM. Companion (10%
EtOAc/hexanes) to afford the iodo derivative 8g (400 mg, 86% yield)
as a white amorphous solid.
[0265] Step 7:
[0266] A mixture of the iodo derivative 8g (400 mg, 1.54 mmol),
bis(pinocolato)diborane (585 mg, 2.31 mmol), potassium acetate (511
mg, 5.4 mmol) in DMF (20 mL) is deoxygenated (Ar balloon and
sonication for 5 min); then the catalyst (PdCl.sub.2dppf, 188 mg,
0.23 mmol) is added with additional degassing (Ar balloon and
sonication for 2 min). The mixture is then heated to 95.degree. C.
for 4 h. The mixture is cooled, EtOAc (200 mL) is added, washed
with brine (3.times.), water (2.times.), dried over anhydrous
MgSO.sub.4, filtered and solvent evaporation over silica affords
the mixture that is purified using the CombiFlash.RTM. Companion
(10% EtOAc/hexanes) to afford the desired boronate 8h (315 mg, 79%
yield) as a yellow oil.
Example 9
Synthesis of Boronate Fragment 9b (Used in Example 43)
##STR00044##
[0268] Anhydrous DMF (60 mL) is added to a flask charged with
bromide 9a (5.00 g, 22.2 mmol), bis-(pinacolato)diborone (8.48 g,
33.4 mmol) and potassium acetate (6.35 g, 66.8 mmol) and the
resulting suspension is deoxygenated by bubbling a stream of
N.sub.2 gas through the mixture for 45 min.
1,1'-bis(diphenylphosphino)ferrocene (2.73 g, 3.34 mmol) is then
added and the mixture is deoxygenated for approximately a further 5
min and is then heated to 95.degree. C. After 16 h, the dark
reaction mixture is cooled, extracted with EtOAc (500 mL and 300
mL) and washed with 1:1 water/brine (600 mL) and brine (600 mL).
The combined extracts are dried over anhydrous MgSO.sub.4, filtered
and evaporated to a black syrup which is purified by flash column
chromatography (EtOAc/hexanes) to afford the boronate 9b as white
solid contaminated with <25% of the diboron reagent (4.24 g, 62%
yield).
Example 10
Synthesis of Boronate Fragment 10g (Used in Preparation of 1102,
1108, 1109, 1110, 1111, 1119, 1142)
##STR00045##
[0270] Step 1:
[0271] 2-Chloro-6-fluoronitrobenzene 10a (6.62 g, 37.7 mmol) and
LiOH monohydrate (6.33 g, 151 mmol) are dissolved in THF (45 mL)
and water (65 mL) and an aqueous solution of H.sub.2O.sub.2 (30%,
8.60 mL, 80.0 mmol) added. The resulting turbid solution is sealed
and is heated to 60.degree. C. with rapid stirring. After 3 days,
the dark orange mixture is cooled and is added to half-saturated
aqueous sodium thiosulfate (200 mL) and shaken vigorously in a
separatory funnel. The mixture is then acidified to pH <3 with 1
N HCl, extracted with EtOAc (400 mL+100 mL) and washed with brine
(400 mL). The combined extracts are dried over magnesium sulfate,
filtered and evaporated to a deep yellow oil (aminophenol 10b)
containing some solid particles (residual starting material) which
is used as such (6.37 g, 97% yield).
[0272] Step 2:
[0273] The crude aminophenol 10b (6.37 g, 36.7 mmol) is dissolved
in THF (100 mL) and tin powder (17.4 g, 147 mmol) is added followed
by 1 N HCl (220 mL, 220 mmol). The resulting mixture is stirred
vigorously at RT. After 16 h, the reaction is cooled to 0.degree.
C., the acid neutralized with 10 N NaOH (22 mL) and the resulting
milky suspension stirred vigorously for 15 min. The mixture is then
filtered through a pad of Celite.RTM. and the solids washed
thoroughly with EtOAc (4.times.200 mL). The filtrate is transferred
to a separatory funnel and the aqueous phase acidified with 1 N HCl
(4 mL), diluted with brine (400 mL) and the organic phase washed
with brine (400 mL). The extract is then dried over sodium sulfate,
filtered and evaporated to afford aminophenol 10c as a waxy, pale
brown solid (2.91 g, 55% yield).
[0274] Step 3:
[0275] Chloroacetyl chloride (1.94 mL, 24.3 mmol) is added to an
ice-cold mixture of aminophenol 10c (2.91 g, 20.3 mmol) and
potassium carbonate (8.40 g, 60.8 mmol) in anhydrous DMF (200 mL)
under a N.sub.2 atmosphere. After 5 min, the reaction is allowed to
warm to RT and, after a further 45 min, is heated to 50.degree. C.
After 15 h, the reaction is cooled and extracted with EtOAc (600
mL) and washed with water/brine (1 L), half-saturated sodium
bicarbonate (1 L) and brine (600 mL). The organic phase is then
dried over MgSO.sub.4, filtered and evaporated to afford lactam 10d
as a fibrous, pale-olive solid (3.15 g, 85% yield).
[0276] Step 4:
[0277] Bromine (1.8 mL; 35 mmol) is slowly added dropwise to a
stirred solution of lactam 10d (3.15 g; 17.1 mmol) in anhydrous DCM
(40 mL) at RT. After 3 h, the resulting suspension is slowly added
to saturated aqueous sodium thiosulfate (200 mL) and extracted with
DCM (4.times.100 mL). The combined extracts are then washed with
brine (200 mL), dried over magnesium sulfate, filtered and
evaporated to afford the bromide 10e as a pale beige powder (4.00
g, 89% yield).
[0278] Step 5:
[0279] A solution of borane in THF (1.0 M, 18.5 mL, 18.5 mmol) is
added dropwise to an ice-cold solution of lactam 10e (4.00 g, 15.2
mmol) in anhydrous THF (75 mL), and the reaction is allowed to warm
to RT. After 30 min, the solution is heated to gentle reflux under
a N.sub.2 atmosphere. After 2 h, the reaction is cooled to
0.degree. C. and carefully quenched with 1 N NaOH (19 mL) and
stirred for 15 min. The mixture is then diluted with water (30 mL)
and the THF is evaporated. The aqueous residue is then extracted
with EtOAc (400 mL+50 mL) and washed with water/brine (200 mL), 0.5
N NaOH (200 mL) and brine (100 mL). The combined extracts are dried
over magnesium sulfate, filtered and evaporated to afford the
morpholine derivative 10f as a yellow syrup (3.90 g, quantitative.
yield).
[0280] Step 6:
[0281] Anhydrous DMF (30 mL) is added to a flask charged with aryl
bromide 10f (1.84 g, 7.42 mmol), bis(pinacolato)diborane (2.83 g,
11.1 mmol) and potassium acetate (2.47 g, 26.0 mmol) and the
resulting suspension is then deoxygenated by bubbling a stream of
N.sub.2 gas through the mixture for 15 min.
1,1'-bis(diphenylphosphino)ferrocene (909 mg, 1.11 mmol) is then
added and the mixture is deoxygenated for a further 5 min and then
heated to 95.degree. C. After 16 h, the dark reaction mixture is
cooled, diluted with EtOAc (300 mL) and washed with 1:1 water/brine
(500 mL) and brine (200 mL). The extract is then dried over
MgSO.sub.4, filtered and evaporated to a brown syrup which is
chromatographed over silica gel (EtOAc/hexanes) to afford the
boronate 10g as a white solid contaminated with 0.8 eq of the
diboron reagent (1.52 g, 69% yield).
Example 11
Synthesis of Boronate Fragment 11d (Used in Preparation of 1006,
1021, 1022)
##STR00046##
[0283] Step 1:
[0284] Commercially available chromanone 11a (9.78 g, 66.0 mmol)
dissolved in AcOH (20 mL) is added to a suspension of zinc dust
(108 g, 1.65 mol) in AcOH (150 mL). The mixture is heated to
100.degree. C. and is stirred mechanically overnight. The mixture
is then filtered through Celite.RTM. (washed with EtOAc, 100 mL),
diluted with PhMe (300 mL) and the solution is evaporated to give
chroman intermediate 11 b (8.45 g, 95% yield).
[0285] Step 2:
[0286] AgNO.sub.3 (12.0 g, 70.6 mmol) and I.sub.2 (15.8 g, 62.3
mmol) are added sequentially to a solution of 11b (8.45 g, 63.0
mmol) dissolved in MeOH (225 mL). The reaction is allowed to stir
for 1 h, filtered on Celite.RTM. and the filtrate concentrated
under reduced pressure. The crude mixture is diluted with EtOAc
(250 mL) and washed with saturated sodium thiosulfate (250 mL). The
organic layer is washed with water (200 mL) and then dried over
Na.sub.2SO.sub.4, filtered and concentrated. The crude mixture is
further purified by CombiFlash.RTM. Companion to give 6-iodochroman
11c (12.1 g, 74% yield).
[0287] Step 3:
[0288] A solution of the 6-iodochroman 11c (1.0 g, 3.85 mmol),
bis[pinocolato]diborane (1.22 g, 4.81 mmol) and potassium acetate
(1.10 g, 11.5 mmol) in DMF (36 mL) is degassed with Ar for 5 min
followed by the addition of the PdCl.sub.2dppf-DCM complex (314 mg,
0.38 mmol). The reaction mixture is then degassed for an additional
5 min before being heated to 95.degree. C. for 5 h. The reaction is
then cooled to RT. The crude reaction mixture is diluted with water
and the product is extracted 3 times with EtOAc (3.times.100 mL).
The combined organics are washed with water (100 mL) and brine (100
mL). The organic phase is then dried over MgSO.sub.4 and filtered
and concentrated. The crude mixture is further purified by
CombiFlash.RTM. Companion using a gradient of EtOAc/hexanes to
afford the borane fragment 11d (840 mg, 84% yield).
Example 12
Synthesis of Boronate Fragment 12g (Used in Preparation of 1037,
1042)
##STR00047##
[0290] Step 1:
[0291] The phenol 12a (6.75 g, 47.3 mmol) is dissolved in DMF (270
mL) and is treated with allyl bromide (6.55 mL, 75.7 mmol). To this
solution, NaH (60%, 4 g, 99.4 mmol) is added portionwise and
stirring is continued overnight. The reaction mixture is diluted
with EtOAc (500 mL) and washed with H.sub.2O (3.times.500 mL). The
organic layer is dried over MgSO.sub.4, filtered and concentrated
to dryness to obtain the desired product 12b, which is used as such
in the next step.
[0292] Step 2:
[0293] The ether 12b (9.67 g) is placed in a microwave vial neat
with a stir bar and is heated to 240.degree. C. for 20 min at which
point the Claisen rearrangement reaction is complete. The crude
product 12c (9.3 g) is used in the following step without further
purification.
[0294] Step 3:
[0295] To a solution of the allyl intermediate 12c (9.3 g, 45.8
mmol) in anhydrous THF (300 mL) at 0.degree. C., borane (1 M in
THF, 96 mL, 96 mmol) is added. The solution is allowed to warm to
RT and then is stirred for 2.5 h. The solution is then cooled to
0.degree. C. and treated with 10 N NaOH dropwise, followed by slow
addition of 30% H.sub.2O.sub.2 (104 ml, 916 mmol, 20 eq). The
resulting mixture is allowed to warm to RT and then is stirred at
RT for 1 h. The reaction mixture is diluted with HCl (10%, 100 mL)
and extracted with EtOAc (3.times.200 mL). The combined organic
phases are dried over MgSO.sub.4 and concentrated. The crude
product is purified by CombiFlash.RTM. Companion to give 12d (7.1
g, 77% yield).
[0296] Step 4:
[0297] To a solution of the diol 12d (7.1 g, 35.3 mmol) in THF (500
mL), PPh.sub.3 (12 g, 45.9 mmol), followed by DEAD (7.2 mL, 45.9
mmol) are added. The solution is stirred at RT for 4 h. The
reaction mixture is evaporated under reduced pressure and purified
by CombiFlash.RTM. Companion to obtain the desired product 12e
(5.26 g, 82% yield).
[0298] Step 5:
[0299] The chroman derivative 12e (5.26 g, 28.8 mmol) is dissolved
in AcOH (70 mL) and is then treated with Br.sub.2 in AcOH (40 mL).
The reaction is stirred at RT for 15 min, then diluted with toluene
and concentrated to dryness. The residue is taken up in EtOAc (25
mL) and washed with saturated Na.sub.2S.sub.2O.sub.3 (25 mL) and
saturated NaHCO.sub.3 (25 mL). The organic layer is dried over
MgSO.sub.4, concentrated and purified by CombiFlash.RTM. Companion
to obtain the desired product 12f (2.7 g, 36% yield).
[0300] Step 6:
[0301] The bromide 12f (2.71 g, 10.4 mmol) is dissolved in DMF (120
mL) and treated with bispinocolatoborane (4 g, 15.5 mmol) and
potassium acetate (3.45 g, 36.3 mmol). The mixture is degassed
(using an Ar balloon) before the introduction of the catalyst
(PdCl.sub.2dppf, 845 mg, 1.04 mmol). The mixture is then degassed
again (using an Ar balloon) and heated to 95.degree. C. for 16 h.
The mixture is cooled to RT, diluted with H.sub.2O (300 mL) and
extracted with EtOAc (2.times.300 mL). The combined organic layers
are washed with water (3.times.300 mL) dried over MgSO.sub.4,
filtered and concentrated. The product is then purified by
CombiFlash.RTM. Companion. The semi-purified product is then
triturated with hexanes (3.times.50 mL) in order to remove the
excess disborane and obtain clean compound 12g (1.74 g, 54%
yield).
Example 13
Synthesis of Boronate Fragment 13a (Used in Preparation of 1043,
1044, 1090, 1092, 1096, 1122)
##STR00048##
[0303] Step 1:
[0304] Palladium on activated charcoal (10% Pd by weight, 0.63 mg,
0.59 mmol) is added to a solution of aryl chloride 12g (0.91 g,
2.95 mmol) and ammonium formate (1.92 g, 30.4 mmol) dissolved in
MeOH and the mixture is heated to reflux. After 15 min, the
reaction is cooled to RT and filtered through Celite.RTM. (MeOH
rinse). The filtrate is evaporated to dryness and the residue
partitioned between water and EtOAc (10 mL each). The organic layer
is dried over anhydrous MgSO.sub.4 and concentrated to obtain
boronic ester 13a (0.78 g, 97% yield).
Example 14
Synthesis of Boronate Fragment 14g (Used in Preparation of
1017)
##STR00049##
[0306] Step 1:
[0307] Allyl bromide (9.3 mL, 110 mmol) followed by potassium
carbonate (20 g, 150 mmol) are added to a solution of 14a (10 g, 73
mmol) dissolved in DMF (110 mL). The reaction is allowed to stir
under Ar at RT overnight. The reaction is diluted with water (400
mL) and extracted with EtOAc (400 mL). The organic layer is washed
with water (2.times.400 mL), dried over Na.sub.2SO.sub.4 and
concentrated. The product is then purified by CombiFlash.RTM.
Companion in two batches (120 g column) to provide allyl ether 14b
(12 g, 92% yield).
[0308] Step 2:
[0309] A solution of n-BuLi in hexanes (2.5 M, 6.4 mL, 16 mmol) is
added dropwise to a precooled (-78.degree. C.) suspension of
methyltriphenylphosphonium bromide (6.6 g, 19 mmol) in THF (90 mL).
The resulting bright yellow mixture is stirred for 5 min at
-78.degree. C., warmed to RT over approximately 5 min and then
recooled to -78.degree. C. Aldehyde 14b (2.4 g, 14 mmol) dissolved
in THF (10 mL) is added dropwise and the reaction is allowed to
proceed for 10 min at -78.degree. C. before being allowed to warm
to RT and stir overnight. The reaction is quenched with brine (100
mL), diluted with water (100 mL) and extracted with EtOAc (100 mL).
The organic layer is then washed with water (2.times.100 mL), dried
over Na.sub.2SO.sub.4 and concentrated. The crude yellow liquid is
then taken up in EtOAc (1 mL) and diluted with hexanes (20 mL),
after which Ph.sub.3PO precipitates as a white solid. The solid is
removed by filtration, washed with 1:9 EtOAc/hexanes (50 mL) and
the filtrates are evaporated to dryness. The product is purified by
CombiFlash.RTM. Companion to give diene 14c (1.3 g, 54% yield).
[0310] Step 3:
[0311] Grubb's second generation catalyst (50 mg, 0.075 mmol) is
added to a degassed solution of diene 14c (1.3 g, 7.5 mmol). After
stirring under Ar for 2.5 h, the reaction is concentrated onto
SiO.sub.2 (2 g) and the product purified by CombiFlash.RTM.
Companion to give benzopyran 14d (940 mg, 86% yield) as a clear
oil.
[0312] Step 4:
[0313] Solid Pd--C(10% w/w, 680 mg, 0.64 mmol) is added to a
solution of benzopyran 14d (940 mg, 6.4 mmol) in EtOH (8.5 mL) and
the flask is evacuated and backfilled with H.sub.2 gas (balloon).
After stirring the reaction at RT for 2.5 h, the mixture is
filtered through Celite.RTM. (EtOAc washing) and then the filtrate
is concentrated to dryness. The product is purified by
CombiFlash.RTM. Companion to provide chroman 14e (800 mg, 84%
yield).
[0314] Step 5:
[0315] Neat Br.sub.2 (275 .mu.L, 5.4 mmol) is added dropwise to a
solution of chroman 14e (800 mg, 5.4 mmol) dissolved in AcOH (25
mL). The reaction is then diluted with water (50 mL) and EtOAc (50
mL). The organic layer is washed with water (2.times.50 mL) and
saturated NaHCO.sub.3 (2.times.50 mL). The organic layer is dried
over Na.sub.2SO.sub.4 and concentrated to dryness. The product is
purified by CombiFlash.RTM. Companion to give bromide 14f as a
mixture with the dibromide (1.3 g, 68% by mass 14f, 51% yield).
[0316] Step 6:
[0317] A solution of the bromide 14f (950 mg, 2.8 mmol),
bis[pinocolato]diborane (840 mg, 3.3 mmol) and potassium acetate
(920 g, 9.6 mmol) in DMF (30 mL) is degassed with Ar for 5 min
followed by the addition of the PdCl.sub.2dppf-DCM complex (290 mg,
0.36 mmol). The reaction mixture is then degassed for an additional
5 min before being heated to 95.degree. C. for 3 h. The reaction is
then cooled to RT. The crude reaction mixture is diluted with water
and the product is extracted with EtOAc (3.times.20 mL). The
combined organics are washed with water (2.times.20 mL). The
organic phase is then dried over Na.sub.2SO.sub.4, filtered and
concentrated. The crude mixture is further purified by
CombiFlash.RTM. Companion to afford boronic ester 14g (403 mg, 53%
yield) as a pale yellow solid.
Example 15
Synthesis of Boronate Fragment 151 (Used in Preparation of
1098)
##STR00050##
[0319] Step 1:
[0320] An ethereal solution of diazomethane (0.7 M, 100 mL) is
added to a solution of 15a (5.0 g, 30 mmol) in ether (20 mL). After
consumption of the SM (TLC monitoring), the reaction is
concentrated onto SiO.sub.2 (10 g) and the product purified by
CombiFlash.RTM. Companion to yield ester 15b (5.2 g, 95%
yield).
[0321] Step 2:
[0322] A solution of NaNO.sub.2 (2.1 g, 30 mmol) in water (10 mL)
is slowly added to a solution of aniline 15b (5.0 g, 28 mmol)
dissolved in AcOH (50 mL) and 2 M HCl (75 mL) at 0.degree. C. The
resulting mixture is stirred at this temperature for 1 h. Solid
CuCl (8.4 g, 85 mmol) is added portionwise (over 2 min). The
reaction is allowed to come to RT, is stirred for 30 min and then
is warmed to 60.degree. C. for 40 min. The mixture is poured into
water (200 mL) and extracted with EtOAc (2.times.200 mL). The
organic layer is dried with MgSO.sub.4, filtered and evaporated to
dryness. The product is purified by CombiFlash.RTM. Companion to
afford aryl chloride 15c (3.8 g, 68% yield).
[0323] Step 3:
[0324] A solution of DIBAL in DCM (1 M, 42 mL, 42 mmol) is added
dropwise over a period of 25 min to a precooled (-78.degree. C.)
solution of ester 15c (3.8 g, 19 mmol) in dry CH.sub.2Cl.sub.2 (100
mL). The reaction is allowed to stir for 2 h at -78.degree. C. The
reaction is quenched at -78.degree. C. by the dropwise addition of
1 N HCl (8 mL). The reaction is allowed to warm to RT and the
organic phase washed with a 5% solution of Rochelle's salt (100
mL), dried over MgSO.sub.4, filtered and concentrated under reduced
pressure to give crude benzyl alcohol 15d (3.2 g, 99% yield), which
is used in the next step without any further purification.
[0325] Step 4:
[0326] Solid Dess Martin reagent (8.7 g, 20 mmol) is added to a
precooled (0.degree. C.) solution of alcohol 15d in dry
CH.sub.2Cl.sub.2 (100 mL). The reaction is allowed to stir for 2 h
while slowly warming to RT. At this time, another 0.5 g of Dess
Martin Periodinane is added and the reaction continues for another
1 h. A 1:1 mixture of saturated NaHCO.sub.3 and 0.5 M
Na.sub.2S.sub.2O.sub.3 (100 mL) is added and this mixture is
stirred vigorously until the phases become clear (approximately 30
min). The organic phase is separated and the aqueous phase is
extracted with DCM (100 mL) and washed with saturated
NaHCO.sub.3(100 mL). The combined organic phases are then dried
over MgSO.sub.4 and evaporated. The product is purified by
CombiFlash.RTM. Companion to give aldehyde 15e (2.9 g, 90%
yield).
[0327] Step 5:
[0328] A solution of methyl ether 15e (720 mg, 4.2 mmol) in
anhydrous CH.sub.2Cl.sub.2 (20 mL) is added slowly to a precooled
(-30.degree. C.) solution of BBr.sub.3 (1 M, 8.4 mL, 8.4 mmol). The
solution is warmed to 0.degree. C. and is stirred for 3 h. The
reaction is quenched carefully with methanol (1 mL) and washed with
saturated NaHCO.sub.3 and then brine (25 mL each). The organic
layer is dried over MgSO.sub.4, filtered and concentrated and the
product is purified by CombiFlash.RTM. Companion to give phenol 15f
(530 mg, 80% yield).
[0329] Step 6:
[0330] A mixture of the aldehyde 15f (1.1 g, 7.2 mmol),
acrylonitrile (2.4 mL, 36 mmol) and DABCO (190 mg, 1.7 mmol) are
refluxed for 5 h. The reaction mixture is cooled to RT, diluted
with EtOAc (50 mL) and washed with 1 N NaOH (20 mL) and then with 1
N HCl (20 mL). The organic phase is dried over MgSO.sub.4 and
concentrated to dryness. The product is purified by CombiFlash.RTM.
Companion to afford the nitrile 15g (650 mg, 47% yield).
[0331] Step 7:
[0332] A mixture of nitrile 15g (650 mg, 3.4 mmol), 10% NaOH (10
mL, 25 mmol) and EtOH (95%, 0.5 mL) is heated to reflux for 5 days.
The reaction is then cooled to RT and 1 N HCl is then added until
pH .about.4. The precipitate is then collected by filtration,
washed with water and dried in vacuo to give acid 15h (740 mg,
>99% yield).
[0333] Step 8:
[0334] Triethylamine (0.56 mL, 4.0 mmol) and diphenylphosphoryl
azide (0.75 mL, 3.5 mmol) are added successively to a solution of
acid 15h (714 mg, 3.4 mmol) in dry toluene (40 mL). This mixture is
heated to 85.degree. C. for 2 h and then cooled to RT and treated
with 6 N HCl (6 mL). The mixture is brought to reflux and is
stirred at this temperature for 2 h. The reaction is then cooled to
RT, diluted with EtOAc (100 mL) and washed with saturated
NaHCO.sub.3 (2.times.100 mL), water (2.times.100 mL) and brine (100
mL). The organic layer is dried over MgSO.sub.4, filtered and
evaporated to dryness. The product is then purified by
CombiFlash.RTM. Companion to give ketone 15i (269 mg, 44%
yield).
[0335] Step 9:
[0336] Deoxofluor.RTM. (0.54 mL, 2.9 mmol) is added to a solution
of ketone 15i (270 mg, 1.5 mmol) in CH.sub.2Cl.sub.2 (0.6 mL) and
EtOH (17 .mu.L) in a sealed tube. The sealed tube is heated to
40.degree. C. for 24 h. The tube is then unsealed, cooled to
0.degree. C. and the reaction quenched by the slow (Caution!
Exothermic!) addition of saturated NaHCO.sub.3 (1 mL). The crude
reaction mixture is diluted with water (20 mL) and extracted with
DCM (3.times.20 mL). The combined organics are washed with water
(20 mL) and the organic phase is dried over MgSO.sub.4, filtered
and concentrated. The product is purified by CombiFlash.RTM.
Companion to provide difluorochroman 15j (225 mg, 71% yield).
[0337] Step 10:
[0338] Solid silver nitrate (187 mg, 1.1 mmol) and iodine (279 mg,
1.1 mmol) are added successively to a solution of difluorochroman
15j (225 mg, 1.1 mmol) dissolved in MeOH (7.8 mL). The reaction is
stirred at RT for 90 min and then filtered through a pad of
Celite.RTM.. The filtrate is treated with a drop of 0.5 N
Na.sub.2S.sub.2O.sub.3 (orange color dissipated) then concentrated
under reduced pressure. The residue is partitioned between
H.sub.2O, 0.5N Na.sub.2S.sub.2O.sub.3 and EtOAc (20 mL each). The
water layer is extracted with EtOAc (3.times.20 mL) and the
combined organics are washed with brine (20 mL), dried over
MgSO.sub.4, filtered and concentrated. The product is purified by
CombiFlash.RTM. Companion to give aryl iodide 15k (158 mg, 44%
yield).
[0339] Step 11:
[0340] A solution of the aryl iodide 15k (150 mg, 0.45 mmol),
bis[pinocolato]diborane (150 mg, 0.59 mmol) and potassium acetate
(130 mg, 1.4 mmol) in DMF (5 mL) is degassed with Ar for 5 min
followed by the addition of the PdCl.sub.2dppf-DCM complex (44 mg,
0.054 mmol). The reaction mixture is then degassed for an
additional 5 min before being heated to 85.degree. C. for
approximately 9 h. The reaction is then cooled to RT. The crude
reaction mixture is diluted with water and the product is extracted
with EtOAc (3.times.10 mL). The combined organics are washed with
water (10 mL) and brine (10 mL). The organic phase is then dried
over MgSO.sub.4 and filtered and concentrated. The crude mixture is
further purified by CombiFlash.RTM. Companion to afford boronic
ester 151 (123 mg, 70% pure by NMR, 57% yield).
Example 16
Synthesis of Boronate Fragment 16c (Used in Preparation of
1088)
##STR00051##
[0342] Step 1:
[0343] Solid NaBH.sub.4 (342 mg, 9.0 mmol) is added to a solution
of ketone 4b (1.5 g, 7.5 mmol) dissolved in MeOH (10 mL) and THF
(25 mL) at 0.degree. C. is then added. The reaction is warmed to RT
and is allowed to stir for 1 h. The reaction is quenched with
aqueous HCl (1 N, 5 mL), the MeOH is removed by concentration and
the product extracted with EtOAc (2.times.50 mL). The organic layer
is washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered
and concentrated to afford alcohol 16a (1.52 g>99% yield). This
material is used as is in the next step.
[0344] Step 2:
[0345] TFA (2.9 mL) is added dropwise to a solution of crude
alcohol 16a (1.5 g; 7.47 mmol) in CH.sub.2Cl.sub.2 (28 mL) at
0.degree. C. The solution is stirred for 30 min, and then
concentrated to dryness. The residue is taken up in EtOAc, washed
with NaHCO.sub.3 (saturated), brine, dried over Na.sub.2SO.sub.4,
filtered and concentrated to a pale yellow gum. The product is
purified by CombiFlash.RTM. Companion to afford benzofuran 16b
(0.30 g, 22% yield) as a white solid.
[0346] Step 3:
[0347] Compound 16c is prepared from 16b following a synthetic
sequence identical to steps 3 to 5 of Example 4.
Example 17
Synthesis of Boronate Fragment 17g (Used in Preparation of 1047,
1048, 1049)
##STR00052##
[0349] Step 1:
[0350] Zn dust (7.89 g, 121 mmol) is added to a solution of 17a
(5.0 g, 24 mmol) in AcOH (100 mL). The reaction mixture is then
heated to 100.degree. C. and is stirred overnight. The reaction is
cooled to RT and the mixture is filtered (EtOAc washing), the
solvent is evaporated and the residue purified by CombiFlash.RTM.
Companion to afford aniline 17b (3.06 g, 72% yield) as a yellow
solid.
[0351] Step 2:
[0352] A solution of NaNO.sub.2 (640 mg, 9.3 mmol) in water (3 mL)
is slowly added to a solution of aniline 17b (1.5 g, 8.5 mmol)
dissolved in AcOH (12 mL) and 2 M HCl (25 mL) at 0.degree. C. The
resulting mixture is stirred at this temperature for 1 h. Solid
CuCl (2.6 g, 26 mmol) is added portionwise (over 2 min) and the
reaction is allowed to come to RT, is then stirred for 30 min and
then is warmed to 60.degree. C. for 40 min. The mixture is poured
into water (100 mL) and extracted with EtOAc (2.times.100 mL). The
organic layer is dried with MgSO.sub.4, filtered and evaporated to
dryness. The product is purified by CombiFlash.RTM. Companion to
afford aryl chloride 17c (1.11 g, 99% yield) as a pale yellow
solid.
[0353] Step 3:
[0354] Solid pre-activated Zn dust is added to a solution of ketone
17c in AcOH. The reaction mixture is then heated to 100.degree. C.
and stirred at that temperature for 4 h. The reaction mixture is
filtered (EtOAc washing), the filtrate is evaporated to dryness and
the product purified by CombiFlash.RTM. Companion to afford indane
17d (902 mg, 88% yield) as a white crystalline solid.
[0355] Step 4:
[0356] A solution of BBr.sub.3 in DCM (1 M, 9.9 mL, 9.9 mmol) is
added dropwise to a precooled (-78.degree. C.) solution of methyl
ether 17d (902 mg, 4.9 mmol) dissolved in DCM (20 mL). The reaction
solution is stirred at this temperature for 10 min and allowed to
warm to RT. After stirring for 1.5 h, water (50 mL) is added
(caution! Exothermic!) and the mixture is extracted with DCM
(3.times.50 mL). The combined organic layers are dried over
MgSO.sub.4, filtered and evaporated to dryness. The product is
purified by CombiFlash.RTM. Companion to afford phenol 17e (700 mg,
84% yield) as an off-white solid.
[0357] Step 5:
[0358] Tf.sub.2O (1.05 mL, 12 mmol) is added to a precooled
(0.degree. C.) solution of phenol 17e (700 mg, 4.1 mmol) and
Et.sub.3N (1.7 mL, 12 mmol) in DCM (20 mL). The resulting dark
solution is allowed to warm to RT. After 25 min, the reaction is
quenched with saturated NaHCO.sub.3 (10 mL), diluted with DCM, and
the organic layer washed with water, brine, dried over MgSO.sub.4
and evaporated to dryness. The product is purified by
CombiFlash.RTM. Companion to afford triflate 17f (1.21 g, 97%
yield) as a yellow oil.
[0359] Step 6:
[0360] A solution of triflate 17f (1.2 g, 4.0 mmol),
bis[pinocolato]diborane (1.5 g, 6.0 mmol) and potassium acetate
(1.3 g, 14 mmol) in DMF (20 mL) is degassed with Ar for 5 min
followed by the addition of the PdCl.sub.2dppf-DCM complex (490 mg,
0.60 mmol). The reaction mixture is then degassed for an additional
5 min before being heated to 95.degree. C. for 5 h. The reaction is
then cooled to RT. The crude reaction mixture is diluted with water
and the product is extracted with EtOAc (3.times.100 mL). The
combined organics are washed with water (100 mL) and brine (100
mL). The organic phase is then dried over MgSO.sub.4 and filtered
and concentrated. The crude mixture is further purified by
CombiFlash.RTM. Companion to afford boronic ester 17g (593 mg, 53%
yield) as a pale yellow solid.
Example 18
Synthesis of Boronate Fragment 18d (Used in Preparation of
1067)
##STR00053##
[0362] Step 1:
[0363] Neat Tf.sub.2O (0.83 mL, 4.9 mmol) is added dropwise to a
cooled (0.degree. C.) solution of phenol 18a (0.50 g, 3.1 mmol) and
pyridine (1.3 mL, 17 mmol) in DCM (15 mL). The reaction is allowed
to warm to RT and stir overnight. The reaction is quenched by the
addition of a 10% citric acid solution (50 mL) and the mixture is
extracted with DCM (3.times.50 mL). The combined organics are
washed with water (50 mL), dried over MgSO.sub.4, filtered and
concentrated. The product is purified by CombiFlash.RTM. Companion
to give triflate 18b (500 mg, 94% yield).
[0364] Step 2:
[0365] Deoxyfluor.RTM. (0.83 mL, 4.2 mmol) followed by EtOH (10 uL,
0.2 mmol) are added to neat triflate 18b (500 mg, 1.7 mmol) in a
sealable tube. The tube is sealed and the reaction is heated in an
oil bath at 85.degree. C. and is stirred overnight. The reaction is
then cooled to 0.degree. C. and quenched by the slow addition of
NaHCO.sub.3 (100 .mu.L, CAUTION! Exothermic!). The mixture is
diluted with water (50 mL) and extracted with DCM (3.times.50 mL).
The combined organic layers are washed with water (50 mL) and brine
(50 mL). The organic phase is then dried over MgSO.sub.4, filtered
and concentrated. The crude product is purified by CombiFlash.RTM.
Companion to provide the difluorotetrahydronaphtyl triflate 18c
(175 mg, 33% yield).
[0366] Step 3:
[0367] Step three is performed exactly as in step 6 of Example 17
to provide boronic ester 18d.
Example 19
Synthesis of Boronate Fragment 19d (Used in Preparation of 1070,
1078)
##STR00054##
[0369] Step 1:
[0370] Solid N-chlorosuccinimide (2.2 g, 16 mmol) is added in
portions over 5 min to a solution of naphthylamine 19a (2.3 g, 16
mmol) dissolved in CCl.sub.4 (150 mL). The reaction is then heated
to 50.degree. C. and is stirred for 40 min. The reaction is then
cooled to RT, solids are removed by filtration and the filtrate is
washed with water (100 mL), dried over MgSO.sub.4 and evaporated to
dryness to provide chloroaniline 19b (2.8 g, 96% yield).
[0371] Step 2:
[0372] A solution of NaNO.sub.2 (1.2 g, 17 mmol) in water (5 mL) is
slowly added to a precooled (0.degree. C.) suspension of aniline
19b (2.8 g, 15 mmol) in 12 N HCl (7 mL) and ice (9.7 g), so as to
maintain the temperature below 5.degree. C. The mixture is stirred
for 15 min and then is transferred to a solution of KI (8.7 g, 52
mmol) in water (30 mL) and the resulting mixture is stirred for 2
h. The mixture is extracted with Et.sub.2O (3.times.100 mL) and the
combined organic layers washed successively with 3 N NaOH
(2.times.50 mL), 5% NaHSO.sub.3 (50 mL) and brine (100 mL). The
organic phase is dried over MgSO.sub.4, filtered and concentrated
to dryness. The crude product is purified by flash chromatography
(EtOAc/hexanes) to provide aryl iodide 19c (2.4 g, 54% yield).
[0373] Step 3:
[0374] Step three is carried out exactly as described in step 11 of
Example 15 to provide boronic ester 19d.
Example 20
Synthesis of Boronate Fragment 20d (Used in Preparation of
1064)
##STR00055##
[0376] Step 1:
[0377] Allyl bromide (2.1 mL, 25 mmol) followed by potassium
carbonate (7.2 g, 52 mmol) are added to a solution of
6-chlororesorcinol 20a (10 g, 69 mmol) dissolved in DMF (120 mL).
The reaction is stirred overnight, diluted with EtOAc (500 mL) and
washed with water (3.times.500 mL). The organic layer is dried over
MgSO.sub.4 and concentrated to dryness. The crude product is
purified by CombiFlash.RTM. Companion to obtain allyl ether 20b
(1.8 g, 40% yield).
[0378] Step 2:
[0379] Methyl iodide (1.2 mL, 20 mmol) followed by potassium
carbonate (3.8 g, 27 mmol) are added to a solution of phenol 20b
(1.8 g, 9.8 mmol) dissolved in DMF (12 mL). The reaction is stirred
for 2 h, diluted with EtOAc (50 mL) and washed with water
(3.times.50 mL). The organic layer is dried over MgSO.sub.4 and
concentrated to dryness. The crude product is purified by
CombiFlash.RTM. Companion to obtain methyl ether 20c (1.8 g, 40%
yield).
[0380] Step 3:
[0381] Step 3 is comprised of a sequence of steps identical to
steps 2 through 6 of Example 12, followed by step 1 of Example 13
to provide boronic ester 20d.
Example 21
Synthesis of Boronate Fragment 21g (Used in Preparation of
1071)
##STR00056##
[0383] Step 1:
[0384] Solid CuBr.sub.2 (7.9 g; 35 mmol) is added to a solution of
21a (4.0 g, 23 mmol) dissolved in EtOAc (32 mL) and CHCl.sub.3 (32
mL). The mixture is heated to reflux and is stirred for 8 h.
CuBr.sub.2 (3.9 g, mmol) is then added and the mixture continues to
stir at reflux for an additional 15 h. The mixture is cooled to RT,
the solids removed by filtration (EtOAc washing). The filtrate is
concentrated to afford the crude bromoketone 21 b (6.3 g), which is
used directly in the next step.
[0385] Step 2:
[0386] Solid KF (2.5 g, 43 mmol) is added to a solution of crude
bromoketone 21b (6.3 g, 23 mmol) dissolved in DMF (21 mL). The
reaction is stirred at RT for 3 h and then taken up in ether (300
mL), washed with brine (3.times.100 mL), dried over MgSO.sub.4,
filtered and concentrated to dryness. The crude product is purified
by CombiFlash.RTM. Companion to afford ether 21c (2.1 g, 49% yield
over two steps).
[0387] Step 3:
[0388] Solid NaBH.sub.4 (270 mg, 7.1 mmol) is added to a precooled
(0.degree. C.) solution of ketone 21c (1.0 g, 5.9 mmol) dissolved
in MeOH (20 mL). The reaction is allowed to stir for 1 h and then
quenched with aqueous HCl (1 N, 1 mL). The volatiles are removed in
vacuo and the product extracted with EtOAc (20 mL). The organic
layer is washed with brine (20 mL), dried (Na.sub.2SO.sub.4),
filtered and concentrated to afford the crude alcohol 21d (1.0 g),
which is used directly in the next step.
[0389] Step 4:
[0390] Solid AgNO.sub.3 (1.0 g, 6.1 mmol) followed by I.sub.2(1.6
g, 6.2 mmol) are added to a solution of alcohol 21d (1.0 g, 6.2
mmol) dissolved in MeOH (58 mL). The mixture is stirred at RT for 1
h and then a solution of Na.sub.2S.sub.2O.sub.4(0.5 M, 10 mL) is
added and the mixture is stirred for 30 min. The MeOH is removed in
vacuo and the residue taken up in EtOAc (50 mL), washed with water
(50 mL), brine (50 mL), dried (Na.sub.2SO.sub.4), filtered and
concentrated to afford aryl iodide 21e (1.6 g), which is used
directly in the next step.
[0391] Step 5:
[0392] Crude alcohol 21e (1.6 g; .about.5 mmol) is dissolved in a
mixture of DCM (20 mL) and TFA (2.2 mL). The reaction is stirred
for 45 min and then concentrated to dryness. The residue is taken
up in EtOAc (50 mL), washed with saturated NaHCO.sub.3 (50 mL) and
brine (50 mL). The organic layer is dried over Na.sub.2SO.sub.4,
filtered and concentrated to dryness. The crude product is purified
by CombiFlash.RTM. Companion to provide benzofuran 21f (978 mg, 65%
yield over 3 steps).
[0393] Step 6:
[0394] Step 6 is carried out exactly as described for step 11 of
Example 15 to provide boronic ester 21g.
Example 22
Synthesis of Boronate Fragment 22d (Used in Preparation of
1068)
##STR00057##
[0396] Step 1:
[0397] Neat 3-bromo-2-methylpropene (1.7 mL, 16 mmol) is added to a
suspension of phenol 22a (3.0 g, 14 mmol) and potassium carbonate
(5.6 g, 41 mmol) in DMF (35 mL). The reaction is stirred for 2 h
and then quenched with water (100 mL) and extracted with hexanes
(2.times.100 mL). The organic phase is washed with brine
(2.times.100 mL) and concentrated to give ether 22b (3.3 g, 87%
yield).
[0398] Step 2:
[0399] Neat tributyltin hydride (2.3 mL, 8.8 mmol) is added to a
solution of aryliodide 22b (2.0 g, 7.3 mmol) and AIBN (120 mg, 0.73
mmol) in PhMe (40 mL) and the reaction is then stirred at reflux
under N.sub.2. After 1 h, the reaction is concentrated to dryness
and the crude product purified by CombiFlash.RTM. Companion to
provide dihydrobenzofuran 22c (785 mg, 73% yield).
[0400] Step 3:
[0401] Step 3 is comprised of a sequence of synthetic steps
identical to steps 10 and 11 of Example 15 to provide boronic ester
22d.
Example 23
Synthesis of Boronate Fragment 23c (Used in Preparation of 1040,
1041, 1057)
##STR00058##
[0403] Step 1:
[0404] Neat Tf.sub.2O (056 mL, 3.3 mmol) is added dropwise to a
cooled (0.degree. C.) solution of phenol 23a (350 mg, 2.1 mmol;
prepared according to Doi et al Bull. Chem. Soc. Jpn. 2004 77,
2257-2263) and pyridine (0.91 mL, 11 mmol) in DCM (10 mL) under an
Ar atmosphere. The reaction is allowed to warm to RT and then is
stirred for abut 2 h. The reaction is quenched by the addition of a
10% citric acid solution (20 mL) and extracted with DCM (3.times.20
mL). The combined organic layers are washed with water (20 mL),
dried over MgSO.sub.4, filtered and concentrated to dryness. The
crude product is purified by CombiFlash.RTM. Companion to provide
triflate 23b (512 mg, 82% yield).
[0405] Step 2:
[0406] A solution of the triflate 23b (510 mg, 1.7 mmol),
bis[pinocolato]diborane (560 mg, 2.2 mmol) and potassium acetate
(500 mg, 5.1 mmol) in DMF (18 mL) is degassed with Ar for 5 min
followed by the addition of the PdCl.sub.2dppf-DCM complex (140 mg,
0.17 mmol). The reaction mixture is then degassed for an additional
5 min before being heated to 100.degree. C. by microwave
irradiation for 10 min. The reaction is then cooled to RT. The
crude reaction mixture is diluted with EtOAc (60 mL) and washed
with brine (3.times.60 mL). The organic layer is dried over
MgSO.sub.4, filtered and concentrated. The crude mixture is further
purified by CombiFlash.RTM. Companion to afford boronic ester 23c
(200 mg, 42% yield).
Example 24
Synthesis of Boronate Fragment 24b (Used in Preparation of
1061)
##STR00059##
[0408] Step 1:
[0409] Compound 24b is prepared from 24a following a synthetic
sequence identical to steps 1 to 6 of Example 12.
Example 25
Synthesis of Boronate Fragment 25b (Used in Preparation of
1059)
##STR00060##
[0411] Step 1:
[0412] Compound 25b is prepared from 25a following a synthetic
sequence identical to steps 1 to 6 of Example 12.
Example 26
Synthesis of Boronate Fragment 26b (Used in Preparation of 1105,
1106)
##STR00061##
[0414] Step 1:
[0415] Compound 26b is prepared from 26a following a synthetic
sequence identical to steps 1 to 6 of Example 12.
Example 27
Synthesis of Boronate Fragment 27b (Used in Preparation of
1033)
##STR00062##
[0417] Step 1:
[0418] Compound 27b is prepared from 27a following a synthetic
sequence identical to steps 1 to 6 of Example 14.
Example 28
Synthesis of Boronate Fragment 28b (Used in Preparation of
1060)
##STR00063##
[0420] Step 1:
[0421] Compound 28b is prepared from 28a following a synthetic
sequence identical to steps 1 to 8 of Example 6.
Example 29
Synthesis of Boronate Fragment 29b (Used in Preparation of
1052)
##STR00064##
[0423] Step 1:
[0424] Compound 29b is prepared from 29a following a synthetic
sequence identical to steps 1 to 6 of Example 14.
Example 30
Synthesis of Boronate Fragment 30b (Used in Preparation of
1080)
##STR00065##
[0426] Step 1:
[0427] Compound 30b is prepared from 30a following a synthetic
sequence identical to steps 2 and 3 of Example 18.
Example 31
Synthesis of Boronate Fragment 31b (Used in Preparation of
1013)
##STR00066##
[0429] Step 1:
[0430] Compound 31b is prepared from 31a following a synthetic
sequence identical to steps 9 to 11 of Example 15.
Example 32
Synthesis of Boronate Fragment 32b (Used in Preparation of
1005)
##STR00067##
[0432] Step 1:
[0433] Compound 32b is prepared from 32a following a synthetic
sequence identical to steps 5 to 6 of Example 17.
Example 33
Synthesis of Boronate Fragment 33b (Used in Preparation of 1023,
1034)
##STR00068##
[0435] Step 1:
[0436] Compound 33b is prepared from 33a following a synthetic
sequence identical to steps 1 and 3 of Example 11.
Example 34
Synthesis of Boronate Fragment 34f (Used in Preparation of
1094)
##STR00069##
[0438] Step 1:
[0439] Benzyl bromide (25 mL, 210 mmol) followed by potassium
carbonate (44 g, 320 mmol) are added to a solution of
2-methylresorcinol 34a (38 g, 310 mmol) dissolved in DMF (1 L). The
reaction is stirred overnight, diluted with EtOAc (2 L) and washed
with water (3.times.2 L). The organic layer is dried over
Na.sub.2SO.sub.4and concentrated to dryness. The crude product is
purified by CombiFlash.RTM. Companion to obtain benzyl ether 34b
(18.6 g, 39% yield).
[0440] Step 2:
[0441] Allyl bromide (3.0 mL, 35 mmol) followed by potassium
carbonate (6.5 g, 47 mmol) are added to a solution of phenol 34b (5
g, 23 mmol) dissolved in DMF (100 mL). The reaction is stirred
overnight, diluted with EtOAc (500 mL) and washed with water
(3.times.500 mL). The organic layer is dried over Na.sub.2SO.sub.4
and concentrated to dryness. The crude product is purified by
CombiFlash.RTM. Companion to obtain benzyl ether 34c (4.4 g, 75%
yield).
[0442] Step 3:
[0443] Compound 34d is prepared from 34c following a synthetic
sequence identical to steps 2 to 4 of Example 12.
[0444] Step 4:
[0445] Benzyl ether 34d and Pd--C(10% w/w, 100 mg, 0.094 mmol) are
combined in EtOAc (5 mL) and the flask is evacuated and backfilled
with a H.sub.2 atmosphere (balloon). After stirring for 3 h, the
reaction is filtered through Celite.RTM. (EtOAc washing) and the
filtrated concentrated to give phenol 34e (145 mg, 95% yield).
[0446] Step 5:
[0447] Compound 34f is prepared from 34e following a synthetic
sequence identical to steps 5 to 6 of Example 17.
Example 35
Synthesis of Boronate Fragment 35e (Used in Preparation of
1047)
##STR00070##
[0449] Steps 1 through 4 are done in analogy to steps 3 through 6
from Example 17.
Example 36
Synthesis of Boronate Fragment 36d (Used in Preparation of 1075,
1076)
##STR00071##
[0451] Step 1:
[0452] 4-bromo-3-nitrotoluene 36a (5.0 g, 22.9 mmol) is dissolved
in 50 mL ethyl acetate and solid tin(II) chloride dihydrate (20.0
g, 86.9 mmol) is added. The mixture is heated under nitrogen
atmosphere at 70.degree. C. for 2 h (note: temporary overheating to
100.degree. C. is observed! Caution should be exercised!). The
mixture is cooled down and is poured into 200 mL of ice-water. 50
mL of 5% aqueous NaHCO.sub.3 solution is added (rapid foaming!),
followed by 10 N aqueous NaOH to bring the pH .about.7-8. Large
volume of gelatinous yellowish precipitate is formed. This
heterogeneous mixture is shaken with EtOAc (200 mL) and the mixture
is centrifuged in 50 mL portions, resulting in good separation of a
yellowish solid. The clear supernatant is decanted and is extracted
with EtOAc. Combined organic phase is washed with brine, dried over
sodium sulphate, filtered and concentrated under vacuum to give an
orange oily residue. This residue is re-dissolved in 100 mL of
ether and the solution is washed with 10% Na.sub.2CO.sub.3 (20 mL)
followed by 2.5 M aqueous NaOH (20 mL). The dark brown organic
solution is then stirred with MgSO.sub.4 and active charcoal and
filtered to give a light yellow solution, which darkened rapidly on
standing in open flask. The solvent is removed under vacuum to give
the desired compound 36b as a brown-red oil which is used in the
next step without further purification (3.31 g, 78% yield).
[0453] Step 2:
[0454] A mixture of compound 36b (3.3 g, 17.7 mmol), glycerin (3.3
g, 35.5 mmol), nitrobenzene (2.2 g, 17.7 mmol) and 75% aqueous
sulfuric acid (10 mL, 138 mmol) is stirred at 150.degree. C. for 3
h (mixture turns black and viscous). The reaction mixture is cooled
down, poured into ice-water (200 mL) and 10 N aqueous NaOH is added
(30 mL, 300 mmol). The black mixture is then shaken with EtOAc (100
mL) and is centrifuged in 50 mL portions. The upper EtOAc layers
are combined and the bottom aqueous layers containing the black tar
are shaken with EtOAc and re-centrifuged. All EtOAc extracts are
combined, washed with brine, dried over Na.sub.2SO.sub.4, filtered
and concentrated under vacuum to give 4.8 g of a brown-red oil.
This material is chromatographed on 80 g silica gel column
(CombiFlash.RTM. Companion apparatus, hexanes-EtOAc gradient). The
fractions containing the compound are concentrated under vacuum to
afford compound 36c as a white solid (3.26 g, 83% yield).
[0455] Step 3:
[0456] To a cooled (-78.degree. C.) solution of compound 36c (500
mg, 2.25 mmole) in anhydrous Et.sub.2O (20 mL), is added over 5 min
under an Ar atmosphere a 1.6 M solution of n-BuLi in hexane (3.5
mL, 5.60 mmol). The mixture is stirred at -78.degree. C. for 50
min, triisopropylborate (2.00 mL, 8.55 mmol) is then added dropwise
and the mixture is stirred for 2 h at that temperature. The mixture
is slowly allowed to reach RT over a 2 h period and it is poured
into 1 M aqueous HCl (30 mL). The mixture is transferred into a
separatory funnel, the organic layer is separated and the aqueous
layer is washed with Et.sub.2O. The aqueous layer is then
transferred into a 500 mL Erlenmeyer flask and the pH of the
solution is adjusted to approximately 6.3 (measured with a pH
meter) by slowly adding a saturated solution of NaHCO.sub.3 in
water (.about.25 mL, careful: foaming!). The suspension is filtered
off and the separated light-beige solid is washed with water and
dried under high vacuum. This crude product (383 mg) is triturated
with Et.sub.2O/hexanes to give a first crop of the desired compound
36d as a free base (120 mg, 28% yield). The mother liquors are
concentrated under vacuum and are purified by reversed-phase HPLC
using a CH.sub.3CN/H.sub.2O gradient containing 0.06% TFA (ODS-AQ,
C-18 column, 75.times.30 mm, 5-.mu.m particle size). After
lyophilization, a second crop of compound 36d is obtained as a TFA
salt (102 mg, 15% yield), (total yield: 43%).
Example 37
Synthesis of Boronate Fragment 37d (Used in Preparation of
1084)
##STR00072##
[0458] Step 1:
[0459] 1-bromo-4-chloro-2-nitrobenzene 37a is transformed to
compound 37b using the procedure of example 36b, except for the
fact that Et.sub.2O is used for the extractions instead of
EtOAc.
[0460] Step 2:
[0461] Compound 37b (4.2 g, 20.3 mmol) is melted at 50.degree. C.
in a 100 mL round-bottomed flask containing a stirring bar and
immersed in an oil bath. A solution of zinc chloride (700 mg, 5.03
mmol) and ferric chloride (540 mg, 3.25 mmol) in water(3.3 mL) is
added in one portion followed by absolute EtOH (20 mL). The flask
is stoppered with a rubber septa and a needle is inserted to avoid
any pressure build-up. The mixture is warmed to 80.degree. C. and
acrolein (1.68 mL, 24.4 mmol) is added via a syringe pump over a 2
h period. After the addition, the mixture is stirred at 80.degree.
C. for 1 h and an additional amount of solid ferric chloride is
added (4.1 g, 25.3 mmol). The mixture is stirred at 80.degree. C.
for an extra 24 h and then concentrated under vacuum to give a
semi-solid residue. Water (200 mL) is added followed by a 10 N
aqueous solution of NaOH (20 mL) and CH.sub.2Cl.sub.2 (200 mL).
After shaking the mixture for a few min, the solid is filtered over
a pad of Celite.RTM. and the filtrate is transferred into a
separatory funnel. The organic layer is separated and the aqueous
layer is extracted with CH.sub.2Cl.sub.2. The combined organic
extracts are washed with brine, dried (Na.sub.2SO.sub.4), filtered
and concentrated under vacuum to give 3.69 g of a brown solid. This
solid is triturated in hot CH.sub.3CN and filtered. The solid is
discarded and the filtrate is concentrated under vacuum to give 2.3
g of a brown semi-solid. This material is purified on a
CombiFlash.RTM. Companion apparatus on 40 g silica gel column
eluted with EtOAc/hexanes gradient. After evaporation of the
solvent under vacuum, the desired compound 37c is isolated as a
yellow solid (390 mg, 8% yield).
[0462] Step 3:
[0463] Compound 37c is transformed to compound 37d using the
procedure of example 36d.
Example 38
Synthesis of Boronate Fragment 38c (Used in Preparation of
1085)
##STR00073##
[0465] Step 1:
[0466] 2-bromoaniline 38a is transformed to compound 38b using the
procedure of example 37c except that methyl vinyl ketone is used
instead of acrolein.
[0467] Step 2:
[0468] Compound 38b is transformed to compound 38c using the
procedure of example 36d.
Example 39
Synthesis of Boronate Fragment 39k (Used in Preparation of 1131 and
in Example 46)
##STR00074## ##STR00075##
[0469] REFERENCE
[0470] Feliu, L.; Ajana, W.; Alvarez, M.; Joule, J. A. Tetrahedron
1997, 53, 4511.
[0471] Step 1:
[0472] Meldrum's acid 39b (47.04 g, 326 mmol) is taken in trimethyl
orthoformate (360 mL) and refluxed for 2 h. Then 2,5-dimethoxy
aniline 39a (50 g, 326 mmol) is added and the mixture is refluxed
for an extra 5 h. The reaction mixture is cooled down to RT and the
solid which forms upon cooling is collected by filtration. It is
further crystallized from MeOH to afford compound 39c as a yellow
solid (63 g, 63% yield).
[0473] Step 2:
[0474] Compound 39c (62.00 g, 202 mmol) is dissolved in diphenyl
ether (310 mL) and refluxed at 240.degree. C. for 30 min. The
mixture is then cooled down to RT and n-hexane is added, which
causes a brown precipitate to form. This solid is separated by
filtration and is washed with n-pentane and n-hexane to remove
non-polar impurities and the remaining dark brown solid (compound
39d) is used as is in the next step (27 g, 65% yield).
[0475] Step 3:
[0476] A mixture of compound 39d (30.0 g, 146 mmol), DMAP (3.75 g,
30.7 mmol) and 2,6-lutidine (24.4 mL; 208 mmol) in DCM (1.4 L) is
cooled to 0.degree. C. and Tf.sub.2O (29.6 mL, 175 mmol) is added
slowly at 0.degree. C. The resulting mixture is stirred at
0.degree. C. for 2 h and at RT for 1 h. It is then diluted with
DCM, washed with H.sub.2O and brine and dried (Na.sub.2SO.sub.4).
The solvent is removed under reduced pressure and the residue is
purified by flash chromatography on silica gel (20% EtOAc/petroleum
ether). The desired compound 39e is isolated as a yellow solid (35
g, 71% yield).
[0477] Step 4:
[0478] A mixture of diisopropylethyl amine (46.5 mL, 267 mmol) in
dry DMF (250 mL) is degassed with argon for 30 min and is added to
a mixture of compound 39e (30.0 g, 88.5 mmol), triphenylphosphine
(7.70 g, 29.4 mmol),
tris(dibenzylideneacetone)di-palladium(0)-chloroform adduct (9.21
g, 8.9 mmol). The resulting mixture is stirred for 5 min at
0.degree. C. and TMS acetylene (13.4 g, 136 mmol) is added
dropwise. The temperature is raised to RT and the mixture is
stirred for 4 h. Diethyl ether and water is added, the aqueous
layer is separated and washed with diethyl ether. The combined
organic layers are washed with H.sub.2O and brine. After drying on
Na.sub.2SO.sub.4, the solvent is removed under reduced pressure and
the residue is purified by flash chromatography on silica gel (30%
EtOAc/petroleum ether). Compound 39f is isolated as a yellow solid
(18 g, 70% yield).
[0479] Step 5:
[0480] A solution of ceric ammonium nitrate (42.3 g, 77.2 mmol) in
H.sub.2O (47 mL) is added under argon atmosphere to a solution of
compound 39f (11.0 g, 38.3 mmol) in acetonitrile (366 mL). The
reaction mixture is degassed with argon for 10 min and the mixture
is stirred at RT for 20 min. Water is then added and the solution
is extracted with CH.sub.2Cl.sub.2. The organic extracts are
combined, washed with H.sub.2O, brine and dried (Na.sub.2SO.sub.4).
The solvent is removed under reduced pressure and the residue is
purified by flash chromatography on silica gel (40% EtOAc/petroleum
ether). The desired compound 39g is isolated as a yellow solid (5.0
g, 52% yield).
[0481] Step 6:
[0482] Compound 39g (1.80 g, 7.1 mmol) is taken in distilled acetic
acid (72 mL) under argon atmosphere. Ammonium chloride (7.55 g, 141
mmol) is added and the reaction is refluxed for 45 min. The
reaction mixture is cooled to RT, H.sub.2O is added and the
solution is washed with EtOAc. The aqueous layer is neutralized
with a saturated aqueous solution of NaHCO.sub.3 and is extracted
with EtOAc. The combined organic extracts are washed with H.sub.2O,
brine and dried (Na.sub.2SO.sub.4). The solvent is removed under
reduced pressure to afford compound 39h as a brown solid (250 mg,
19% yield).
[0483] Step 7:
[0484] Compound 39h (230 mg, 1.24 mmol) is dissolved in absolute
EtOH (11 mL) and 10% palladium on carbon is added (10% w/w, 23 mg)
under nitrogen atmosphere. The mixture is stirred for 15 h under
one atmosphere of hydrogen. The reaction is degassed with nitrogen,
filtered through Celite.RTM., and the Celite.RTM. bed is washed
with an EtOH--CHCl.sub.3 mixture. The solvent is removed under
reduced pressure to give compound 39i as a brown sticky solid (200
mg, 86% yield).
[0485] Step 8:
[0486] Compound 39i (600 mg, 3.21 mmol) is taken in dry
CH.sub.2Cl.sub.2 (30 mL) under nitrogen atmosphere. The solution is
cooled to 0.degree. C. and triethylamine (0.89 mL, 6.42 mmol) is
added dropwise followed by Tf.sub.2O (0.65 mL, 3.87 mmol). The
temperature is raised to RT and the reaction mixture is stirred for
2 h. The mixture is diluted with CH.sub.2Cl.sub.2 and is washed
with H.sub.2O, brine and dried (Na.sub.2SO.sub.4). The solvent is
removed under reduced pressure to afford a residue which is
purified by flash chromatography (10% EtOAc/hexanes). Compound 39j
is isolated as a brown solid (630 mg, 61% yield).
[0487] Step 9:
[0488] In a dry (oven-dried for 30 min) 5-mL glass microwave vessel
containing a magnetic stirring bar, are added compounds 39j (250
mg, 0.78 mmol), bis(pinacolato)diboron (250 mg, 0.94 mmol),
anhydrous potassium acetate (150 mg, 1.51 mmol),
Pd(PCy.sub.3).sub.2 (62.0 mg, 0.091 mmol) and anhydrous,
deoxygenated (argon bubbling for 30 min) 1,4-dioxane (4 mL). The
vial is capped tightly with a septum-cap and the vessel is flushed
with argon. The mixture is stirred at 95.degree. C. (oil bath
temperature) under an atmosphere of argon for 16 h. The reaction
mixture is then concentrated under vacuum, the brown oily residue
is dissolved in 7 mL of glacial AcOH and is filtered via 45 .mu.m
membrane filter. The dark brown solution is divided into
5.times.1.5 mL portions and is injected on an automatic preparative
reversed-phase HPLC-MS apparatus (CH.sub.3CN/H.sub.2O gradient
containing 0.06% TFA, ODS-AQ, C-18 column, 50.times.19 mm, 5-.mu.m
particle size). The collected fractions are lyophylized to give the
desired compound 39k as a yellow amorphous solid (115 mg, 45% yield
for the TFA salt).
Example 40
Synthesis of Triflate Fragment 40e (Used in Example 47)
##STR00076##
[0490] Step 1:
[0491] A solution of fuming HNO.sub.3 (6.0 mL, 142 mmol) and
concentrated H.sub.2SO.sub.4 (0.2 mL, 3.8 mmol) in chloroform (150
mL) is added to a solution of 2-fluoro 4-methyl phenol 40a (20.0 g,
159 mmol) in chloroform (100 mL). The resulting mixture is stirred
for 2 h at RT and is transferred in a separatory funnel. The
solution is washed with H.sub.2O, brine, the organic layer is dried
(Na.sub.2SO.sub.4) and the solvent is evaporated under reduced
pressure. The reddish crude solid is crystallized from aqueous
ethanol to give compound 40b as a yellowish solid (17 g, 62%
yield).
[0492] Step 2:
[0493] Compound 40b is transformed to compound 40c using the
procedure of example 36b.
[0494] Step 3:
[0495] A mixture of concentrated sulfuric acid (12.5 mL, 235 mmol),
water (10 mL), glycerol (10.0 mL, 137 mmol) and sodium
3-nitrobenzenesulfonate (9.57 g, 42.5 mmol) is heated gently until
everything dissolved. Compound 40c (5.0 g, 35.5 mmol) is then added
slowly to the warm (60.degree. C.) solution and the mixture is
heated at reflux for 2 h (bath temperature: 140.degree. C.). The
reaction mixture is then cooled to room temperature and poured into
ice-water. The solution is brought to pH 6-7 with an aqueous
ammonia solution. A brown precipitate of compound 40d formed, it is
collected by filtration and dried under high vacuum (5.0 g, 79%
yield). This compound is used in the next step without further
purification.
[0496] Step 4:
[0497] To a solution of compound 40d (5.0 g, 28.0 mmol) and triflic
anhydride (5.25 mL, 31.0 mmol) in CH.sub.2Cl.sub.2 (150 mL) is
added dropwise at 0.degree. C. Et.sub.3N (4.7 mL, 33.6 mmol). The
resulting mixture is warmed to RT and stirred for 3 h. The reaction
mixture is diluted with CH.sub.2Cl.sub.2 (50 mL), the solution is
washed with 1N HCl, water, brine and it is dried
(Na.sub.2SO.sub.4). The solvent is removed under vacuum to give a
dark solid, which is purified by flash chromatography (10%
EtOAc/hexanes) to afford the desired compound 40e as an off-white
solid (6.0 g, 68% yield).
Example 41
Synthesis of Compound 1006
##STR00077##
[0499] Step 1:
[0500] Solid Pd(PPh.sub.3).sub.4 (9 mg, 0.008 mmol) and CuI (3 mg,
0.015 mmol) are successively added to a solution of 11c (200 mg,
0.75 mmol) and alkyne 2f (190 mg, 1.1 mmol) dissolved in DMF (0.46
mL) and diethylamine (2.3 mL). The reaction mixture is stirred
overnight at RT and then concentrated, diluted with EtOAc (10 mL)
and successively washed with brine, 1 N aqueous HCl and water (10
mL each). The organic layer is dried over Na.sub.2SO.sub.4,
concentrated under reduced pressure and the residue purified by
CombiFlash.RTM. Companion to give alkyne 41a (126 mg, 46%
yield)
[0501] Step 2:
[0502] Tf.sub.2O (96 .mu.L, 0.57 mmol) is added via syringe over
the period of 1 min to a stirred mixture of acetanilide (77 mg,
0.57 mmol) and 2-chloropyridine (67 .mu.L 0.71 mmol) in DCM (1.0
mL) at -78.degree. C. After 5 min, the reaction flask is placed in
an ice-water bath and is warmed to 0.degree. C. Alkyne 41a (102 mg,
0.29 mmol) in DCM (1 mL) is added via syringe. The resulting
solution is allowed to warm to RT. After stirring for 30 min,
Et.sub.3N (1 mL) is added and the mixture is partitioned between
DCM (50 mL) and brine (50 mL). The organic layer is washed with
brine (50 mL), is dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The residue is then purified by CombiFlash.RTM.
Companion giving quinoline 41b (81 mg, 60% yield).
[0503] Step 3:
[0504] LiBH.sub.4 in THF (2 M, 255 .mu.L, 0.51 mmol) is added to a
solution of ester 41 b (81 mg, 0.17 mmol) dissolved in THF (900
.mu.L) and the reaction is stirred overnight at RT. Excess reagent
is quenched with HCl (three drops, lots of effervescence) and the
mixture neutralized with saturated NaHCO.sub.3 (10 mL) and
extracted with EtOAc (3.times.10 mL). The combined organic layers
are dried over anhydrous Na.sub.2SO.sub.4 and concentrated to give
alcohol 41c (38 mg, 57% yield).
[0505] Step 4:
[0506] Dess-Martin periodinane (46 mg, 0.11 mmol) is added to a
solution of alcohol 41c (33 mg, 0.084 mmol) dissolved in DCM (1
mL). After 2 h, the reaction is applied to a pad of SiO.sub.2
(1.5.times.1 cm) and the product is eluted with 1:1 hexanes/EtOAc
(20 mL). The filtrate is evaporated to give the crude aldehyde. The
aldehyde is then dissolved in 2:2:1 THF/H.sub.2O/t-butanol (2.5 mL)
and one drop of 2,3-dimethyl-2-butene (0.8 mL, 1 M in THF) is
added. NaClO.sub.2 (62 mg, 0.68 mmol) and NaH.sub.2PO.sub.4 (51 mg,
0.42 mmol) are added as solids to the solution and the reaction is
stirred at RT. After 30 min, the reaction is diluted with H.sub.2O
(5 mL) and extracted with EtOAc (3.times.10 mL). The organic layer
is dried over anhydrous MgSO.sub.4 and concentrated. The residue is
purified by preparative HPLC to give compound 1006 (12 mg, 27%
yield).
[0507] It would be apparent to those skilled in the art that the
above synthetic protocols can also be used in the synthesis of
other inhibitors where either 11c is replaced by another aromatic
halide in Step 1 and/or the acetanilide is replaced with another
aryl-NH--CO--R.sup.2, or heteroaryl-NH--CO--R.sup.2
(R.sup.2=CH.sub.3) in Step 2.
Example 42
Synthesis of Compound 1017
##STR00078##
[0509] Step 1:
[0510] Quinoline 1i (260 mg, 0.62 mmol), boronic ester 14g (350 mg,
1.3 mmol) and Pd[P(t-Bu).sub.3].sub.2 (50 mg, 0.098 mmol) are
dissolved in DMF (4.3 mL) in a microwave vial and a solution of
Na.sub.2CO.sub.3 (1.25 mL, 2 M, 2.5 mmol) is added. The solution is
degassed (Ar balloon) and the mixture is then submitted to
microwave heating at 120.degree. C. for 10 min. The crude reaction
mixture is diluted with water (15 mL) and the product is extracted
with EtOAc (15 mL). The organic layer is washed with water
(2.times.15 mL) and dried over anhydrous Na.sub.2SO.sub.4, filtered
and concentrated. The crude product is then purified by
CombiFlash.RTM. Companion (gradient of EtOAc in hexanes) to give
quinoline 42a as a mixture of atropisomers (175 mg, 65% yield).
[0511] Step 2:
[0512] An aqueous solution of LiOH (4 mL, 1 N, 4 mmol) is added to
a solution of esters 42a in THF (25 mL), MeOH (6 mL) and water (12
mL) at RT and the reaction is heated to 50.degree. C. After 4 h,
the reaction is evaporated to a white slurry under reduced
pressure, diluted with 1 N NaOH (5 mL) and extracted with EtOAc
(2.times.25 mL). The water layer is then acidified to pH .about.3
with 10% HCl and extracted with DCM (2.times.100 mL) and EtOAc (100
mL). The combined organic layers are dried over anhydrous
Na.sub.2SO.sub.4 and concentrated. The desired product is isolated
after purification of this crude by preparative HPLC to give the
desired pure atropisomer (diastereomer) 1017 (7.5 mg, 4.4%
yield).
Example 43
Synthesis of Compound 1125
##STR00079##
[0514] Step 1:
[0515] In two separate batches, DMF (15 mL) and distilled water
(3.0 mL) are added to two microwave vials each charged with
boronate 9b (560 mg, 2.06 mmol), iodoquinoline 1i 600 mg, 1.45
mmol), potassium carbonate (602 mg, 4.35 mmol) and
Pd(PPh.sub.3).sub.4 (252 mg, 0.218 mmol). The vials are then sealed
and heated in a microwave reactor (7 min, 140'C). The resulting
mixtures are cooled, pooled and extracted with EtOAc (200 mL) and
washed with half-saturated aqueous NaHCO.sub.3 (200 mL) and brine
(200 mL). The extract is dried over MgSO.sub.4, filtered and
evaporated to a red syrup which is chromatographed over silica gel
(EtOAc/hexanes) to afford the pure atropisomers 43a (160 mg, 13%
yield) and 43b (175 mg, 14% yield) as pale yellow amorphous solids,
as well as a sample consisting of a mixture of atropisomers which
is set aside for later separation (275 mg, 22% yield).
[0516] Step 2:
[0517] A solution of anilines 43a/43b (mixture of atropisomers; 50
mg; 0.116 mmol) in anhydrous acetonitrile (0.4 mL) is added to a
stirred mixture of copper (II) bromide (32 mg; 0.145 mmol) and
tert-butyl nitrite (22 .mu.L; 0.19 mmol) in anhydrous acetonitrile
(0.6 mL) at RT under an argon atmosphere. After 1 h, the reaction
is quenched with 1.0 N HCl and extracted with EtOAc (20 mL) and
washed with water (20 mL) and brine (20 mL). The extract is dried
over MgSO.sub.4, filtered and evaporated to afford a mixture of
aryl bromides 43c/43d as a green solid which is used as such (51
mg; 89% yield).
[0518] Step 3:
[0519] Sodium hydroxide (1.0 N, 1.00 mL; 1.00 mmol) is added to a
stirred solution of the ester mixture 43c and 43d (51 mg; 0.103
mmol) in MeOH (1.5 mL) and THF (3 mL) and the reaction heated to
50.degree. C. After 16 h, the solution is acidified with 1.0 N HCl
to a pH of -4 and extracted with DCM (20 mL). The extract is dried
over MgSO.sub.4, filtered and evaporated to solid which is diluted
with acetic acid and acetonitrile (to a volume of 2 mL) and
purified by preparative HPLC (0.1% TFA water/acetonitrile). The
relevant fractions are pooled and lyophilized to yield the TFA
salts of inhibitors 43e (13 mg; 27% yield) and 1125 (18 mg; 37%
yield) as white powders.
[0520] It would be obvious to those skilled in the art that
intermediate 43b could also be used, employing the same methods, to
prepare other inhibitors such as the para-chloro analogue 1112. The
bromo derivative 43d could also be transformed via Suzuki coupling
to para-alkyl derivatives such as 1127.
Example 44
Synthesis of Compound 1103
##STR00080##
[0522] Step 1:
[0523] To a solution of 3-bromo-2-methylaniline 44a (0.77 g, 4.15
mmol) in MeCN (20 mL) is added .beta.-propiolactone (435 .mu.L, 6.2
mmol). The reaction mixture is heated at reflux for 16 h. The
reaction is found to be incomplete so an equivalent amount of the
lactone is added again and the reaction mixture is heated at reflux
for 24 h. The solvent is evaporated before the residue is taken up
into EtOAc and washed with 1 N HCl (aq) and brine before being
dried (MgSO.sub.4), filtered and concentrated. Purification by
CombiFlash.RTM. Companion (hexanes/EtOAc) gives intermediate 44b as
a white solid (606 mg, 57% yield).
[0524] Step 2:
[0525] Compound 44b (1.1 g, 4.3 mmol) is combined with
polyphosphoric acid (40 g) and heated at 100.degree. C. for 22 h.
The cooled mixture is diluted with EtOAc and ice before being
basified to pH .about.8 with 10 N NaOH. The phases are separated
and the aqueous phase again extracted with EtOAc (3.times.). The
combined organic phases are dried (MgSO.sub.4) filtered and
concentrated. Purification by CombiFlash.RTM. Companion
(hexanes/EtOAc) gives the desired ketone 44c as a yellow solid (535
mg, 52% yield).
[0526] Step 3:
[0527] To a solution of ketone 44c (489 mg, 2.04 mmol) in DCE (20
mL) is added zinc iodide (975 mg, 3.06 mmol) and sodium
cyanoborohydride (960 mg, 15.3 mmol). The mixture is heated at
85.degree. C. for 1.5 h. The mixture is cooled to RT and diluted
with EtOAc and a saturated solution of NH.sub.4Cl (containing 10%
by volume of 6 N HCl). The mixture is stirred for 30 min before the
phases are separated. The organic phase is washed with saturated
brine and then dried (MgSO.sub.4) filtered and concentrated. Pure
intermediate 44d (232 mg, 50% yield) is isolated after purification
of the crude by CombiFlash.RTM. Companion (hexanes/EtOAc).
[0528] Step 4:
[0529] To compound 44d (260 mg, 1.15 mmol) in anhydrous DMF (10 mL)
is added bis(pinacolato)borane (380 mg, 1.5 mmol) followed by
potassium acetate (339 mg, 3.45 mmol). The mixture is degassed with
Ar for 10 min before the catalyst PdCl.sub.2(dPPf)-CH.sub.2Cl.sub.2
complex (141 mg, 0.17 mmol) is added. The mixture is heated at
95.degree. C. for 20 h before being cooled and diluted with EtOAc
and water. The organic phase is washed with saturated brine
(3.times.) before being dried (MgSO.sub.4), filtered and
concentrated. The crude material is purified by CombiFlash.RTM.
Companion (hexanes/EtOAc) to give the boronate 44e as a yellow
solid (252 mg, 80% yield).
[0530] Step 5:
[0531] In a vessel suitable for microwave heating, quinoline 1i (62
mg, 0.15 mmol), boronate 44e (50 mg, 0.18 mmol), potassium
carbonate (62 mg, 0.45 mmol) and Pd[(PPh.sub.3)].sub.4 (26 mg,
0.023 mmol) in DMF (2.5 mL) and water (0.25 mL) are added. The
mixture is irradiated at 110.degree. C. for 15 min in a microwave
before being cooled and diluted with EtOAc. The organic phase is
washed with brine (3.times.) before being dried (MgSO.sub.4),
filtered, concentrated and purified by combiflash (hexanes/EtOAc)
to obtain a mixture of atropisomers (diastereomers) as a yellow
oil. This material (68 mg, 0.16 mmol) is dissolved in THF (1.5 mL)
and MeOH (0.5 mL) before being treated with 5 N NaOH (0.32 mL, 1.57
mmol, 10 eq). The mixture is heated at 50.degree. C. for 18 h
before being cooled. The pH is adjusted to -5 with aqueous 1 N HCl
and the mixture is extracted with EtOAc. The organic phase is
washed with saturated brine before being dried (MgSO.sub.4),
filtered and concentrated. The atropisomers (diastereomers) are
separated by preparative HPLC to give the desired compound 1103 as
a light orange solid (16.8 mg, 20% yield over two steps).
Example 45
Synthesis of a Compound 1074 Via Decarboxylative Biaryl Cross
Coupling Reactions
[0532] The decarboxylative cross coupling reactions are used to
prepare a variety of inhibitors; the details of the synthetic
methodology can be found in the literature reference: J. Am. Chem.
Soc. 2006, 128, 11350-11351. An example is shown below:
##STR00081##
[0533] In a vial suitable for microwave reactions is added
5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxylic acid (73 mg,
0.44 mmol), 4-iodoquinoline 1i (100 mg, 0.24 mmol),
tetrabutylammonium chloride hydrate (67 mg, 0.24 mmol), cesium
carbonate (118 mg, 0.36 mmol), and the catalyst
Pd[(PtBu).sub.3].sub.2 (12.4 mg, 0.02 mmol) in DMF (3 mL). The vial
is then capped and submitted directly to the microwave conditions:
170.degree. C. for 8 min. After cooling, the reaction is diluted
with EtOAc (100 mL) and the mixture is washed with brine
(3.times.), water (1.times.), before being dried (MgSO.sub.4),
filtered and concentrated. The residue is purified by
CombiFlash.RTM. Companion (hexanes/EtOAc) to afford the methyl
ester of the desired product (79 mg, 80% yield) as a foamy solid.
The final compound 1074 is obtained after a saponification step
followed by HPLC purification.
Example 46
Synthesis of Compound 1131
##STR00082##
[0535] In a 5-mL glass microwave vessel containing a magnetic
stirring bar, are added compound 46a (100 mg, 0.234 mmol), compound
39k (90 mg, 0.273 mmol), anhydrous potassium carbonate (150 mg,
1.08 mmol), Pd(PPh.sub.3).sub.4 (40.0 mg; 0.035 mmol), anhydrous,
deoxygenated (Argon bubbling for 30 min) dimethylacetamide (3 mL)
and deoxygenated H.sub.2O (0.35 mL). The vial is capped and is
heated in a microwave at 100.degree. C. for 25 min (Biotage
Initiator apparatus). The mixture is cooled and THF (3 mL),
H.sub.2O (1 mL), and MeOH (3 mL) are added followed by a 10 N
aqueous solution of NaOH (0.50 mL, 5.0 mmol). The reaction mixture
is heated to 60.degree. C. 1 h. The reaction is cooled and the
volatiles are removed under vacuum to give a brown oily residue
which is diluted with 7 mL of acetic acid, filtered on a 45 .mu.m
membrane filter and injected in 1.5-mL batches into a preparative
reversed-phase HPLC-MS for purification (CH.sub.3CN/H.sub.2O
gradient containing 0.06% TFA, ODS-AQ, C-18 column, 50.times.19 mm,
5-.mu.m particle size). The desired atropisomer is re-purified
under the same conditions as described above. 1131 as a white
amorphous solid (52.0 mg, 32% yield, bis-TFA salt).
Example 47
Synthesis of Compound 1101
##STR00083##
[0537] In a dry pressure tube, compound 1i (100 mg, 0.24 mmol) is
dissolved in anhydrous, deoxygenated THF (2.5 mL, Ar bubbling for
30 min) and the mixture is cooled to -40.degree. C. under Ar
atmosphere. A freshly titrated solution of i-PrMgCl--LiCl complex
in THF (the titration is done following the protocol in Lin, H. S.;
Paquette, L. A. Synth. Commun. 1994, 24, 2503; 0.83 M solution,
0.400 mL, 0.328 mmol) is added and is stirred at -40.degree. C. for
30 min.
[0538] In a separate flask, a 0.65M solution of zinc chloride in
THF is prepared in the following manner: 115 mg (0.84 mmole) of
anhydrous zinc chloride is placed in an oven-dried 2 mL glass
microwave vessel and is dried at 180.degree. C. (oil bath) under
high vacuum overnight. The vessel is cooled to room temperature and
1.3 mL of anhydrous, argon-degassed THF is added. The mixture is
sonicated until all the zinc chloride dissolved.
[0539] The 0.65M solution of zinc chloride in THF (0.50 mL, 0.30
mmol), is added to the reaction mixture at -40.degree. C. It is
stirred at this temperature for 5 min and is warmed to -5.degree.
C. and kept at this temperature for 1 h and finally at RT for an
extra hour. Pd.sub.2(dba).sub.3 (24.0 mg, 0 0262 mmol) and RuPhos
(24.0 mg, 0.051 mmol, Strem Chemicals) are added under an Ar
atmosphere and the mixture is stirred at RT for 5 min. Compound 40e
(80.0 mg, 0.259 mmol) is then added, the reaction vessel is purged
with Ar, sealed and heated on an oil bath set at 80.degree. C. for
40 h. The reaction mixture is cooled down and H.sub.2O (0.30 mL),
MeOH (0.30 mL) and 10 N aqueous NaOH (0.30 mL, 3.0 mmol) are added
sequentially and the mixture is heated to 60.degree. C. for 2 h.
Acetic acid is then added (2 mL), the mixture is filtered over a 45
.mu.m membrane filter and the compound is purified in two portions
through direct injection into a semi-preparative reversed-phase
HPLC-MS (CH.sub.3CN/H.sub.2O gradient containing 0.06% TFA, ODS-AQ,
C-18 column, 75.times.30 mm, 5-.mu.m particle size). A mixture of
two atropisomers is isolated (20 mg, 13% yield for bis-TFA salt).
Both atropisomers are separated using silica-gel chromatography
(CombiFlash.RTM. Companion apparatus, 4 g column,
MeOH--CH.sub.2Cl.sub.2 gradient) to give compound 1101 (5.5 mg, 5%
yield) as a pure atropisomer.
Example 48
Synthesis of Boronate Fragment 48b (Used for the Preparation of
1136)
##STR00084##
[0541] Step 1:
[0542] A stirred DMF (5 mL) solution of the arylbromide 48a (0.152
g, 0.71 mmol), potassium acetate (0.209 g, 2.1 mmol) and
bis(pinacolato)diborane (0.234 g, 0.92 mmol) is degassed by
bubbling Ar through the solution for 20 min. PdCl.sub.2(dppf)-DCM
(87 mg, 0.11 mmol) is added and degassing is continued for 15 min.
The system is sealed (Teflon screw cap vessel) under Ar and heated
to 90.degree. C. for 16 h. The reaction mixture is allowed to cool
to RT, dilute with EtOAc (150 mL), washed with brine (3.times.100
mL) and water (2.times.100 mL), dried over anhydrous MgSO.sub.4,
filtered and concentrated to dryness. The residue is purified by
CombiFlash.RTM. Companion (EtOAc/hexanes) to give the desired
boronate 48b (144 mg, 77% yield) as a yellowish solid.
Example 49
Alternative Synthesis of Boronate Fragment 39K (Used for the
Preparation of 1143, 1144, 1150, 1151, 1152, 1153)
##STR00085##
[0544] Step 1:
[0545] 1,3-acetonedicarboxylic acid 49a (30 g, 205.3 mmol) is added
in portions to acetic anhydride (55 g, 587.7 mmol) and the mixture
is stirred at 35.degree. C. for 23 h. The mixture is filtered and
the filtrate is diluted with benzene (200 mL) and the solution
stored at 5.degree. C. for 3 h. The precipitate that formed is
filtered and dried under vacuum to give compound 49b as a pale
yellow solid (26.9 g, 70% yield).
[0546] Step 2:
[0547] To a stirred solution of aniline 49c (7.5 g, 44 mmol) in
AcOH (50 mL) is added 49b (8.0 g, 40 mmol) portionwise. Following
addition, the reaction mixture is warmed to 35.degree. C. After 2
h, the reaction mixture is cooled to room temperature and poured in
ice/water (600 mL). The resulting precipitate is isolated by
filtration, rinsed with water (100 mL) and dried under vacuum to
give 49d (9.1 g, 61% yield).
[0548] Step 3:
[0549] Compound 49d (5.7 g, 15.4 mmol) is added portionwise to
concentrated sulfuric acid (20 mL) at RT, temperature of the
reaction mixture is kept below 30.degree. C. during addition. The
mixture is stirred at RT for 30 min and then poured in ice/water
(400 mL). The resulting precipitate is isolated by filtration,
rinsed with water and dried under vacuum to give 49e (3.5 g, 72%
yield) as a white solid.
[0550] Step 4:
[0551] The borane solution (1.0 M in THF, 10.5 ml, 10.5 mmol) is
added dropwise to an ice cold solution of quinolone 49e (1.5 g, 4.8
mmol) in dry THF (40 mL) under a N.sub.2 atmosphere. After the
addition, the reaction is allowed to warm to RT and stirred for 22
h (reaction not completed by HPLC, 15% starting material). An extra
equivalent of BH.sub.3 is added at 0.degree. C. and the reaction
mixture is heated to 45.degree. C. for 2 h. The reaction mixture is
carefully quenched with 1.0 N NaOH (10 mL) and THF is removed under
vacuum. The mixture is poured in EtOAc (100 mL) and the desired
compound crashed out of the solution under these conditions. The
solid 49f is filtered and dried under vacuum (1.1 g, 79% yield) as
grey solid.
[0552] Step 5:
[0553] To a solution of 49f (1.1 g, 3.8 mmol) in DCM (60 mL) at
-78.degree. C. is added dropwise a 1.0 M BBr.sub.3 solution (23 mL,
23 mmol). The cooling bath is removed after 1 h and the mixture is
stirred at RT for 16 h (by HPLC, .about.30% cyclized product 49h is
formed). The mixture is poured in ice/water (100 mL) and the white
precipitate that formed is filtered and dried under vacuum to give
49g (773 mg, 71% yield).
[0554] Step 6:
[0555] To a solution of compound 49g (773 mg, 2.27 mmol) in THF (30
mL) is added PPh.sub.3 (928 mg, 3.5 mmol) followed by DIAD (0.69
ml, 3.5 mmol) (dropwise) and the solution is stirred at RT for 2 h.
The reaction mixture is concentrated under vacuum and the crude
product is directly added portionwise to POCl.sub.3 (2 mL) at RT.
The reaction mixture is stirred at 100.degree. C. for 45 min and
then cooled to RT. The mixture is concentrated under vacuum (to
remove POCl.sub.3) and the crude product is diluted with DCM. The
organic phase is washed with 1.0 N NaOH, water, and brine, dried
(MgSO.sub.4), filtered and concentrated under vacuum. The crude
product is purified in two batches by combi-flash (330 column
hexanes/EtOAc 9/1 to 1/1) to give 49h as a pale yellow solid (445
mg, 91% yield).
[0556] Step 7:
[0557] To a solution of chloroquinoline 49h (30 mg, 0.1 mmol) in
TFA (1 mL) is added zinc (34 mg, 0.5 mmol). The reaction mixture is
stirred at RT for 16 h. The mixture is filtered, concentrated under
vacuum, then diluted with 1.0 N NaOH (5 mL) and extracted with DCM
(3.times.). The combined organic extracts are washed with water and
brine, dried (MgSO.sub.4), filtered and concentrated under vacuum.
The crude product was purified by combi flash (hexanes/EtOAc 6/4 to
4/6) to give 49i as a pale yellow solid (26 mg, quantitative
yield).
[0558] Step 8:
[0559] The reaction is done following a procedure similar to the
one in step 9 of example 39 using Pd(PPh.sub.3).sub.4 as catalyst
and starting with 49i to give 39k as a white solid.
Example 50
Synthesis of Boronate Fragment 50d (Used for the Preparation of
1018, 1020)
##STR00086##
[0561] Step 1:
[0562] Solid NaBH.sub.4 (603 mg, 15.9 mmol) is added to a solution
of ketone 50a (4.11 g, 19.92 mmol) dissolved in MeOH (62 mL) at
0.degree. C. The reaction is warmed to RT and is allowed to stir
for 2 h. The reaction is quenched with aqueous HCl (1 N, 20 mL),
the MeOH is removed by concentration and the product extracted with
EtOAc (2.times.50 mL). The organic layer is washed with brine (50
mL), dried over MgSO.sub.4, filtered and concentrated to afford
alcohol 50b (4.1 g, 97% yield). This material is used as is in the
next step.
[0563] Step 2:
[0564] To a cold solution (0.degree. C.) of 50b (3.96 g, 19.31
mmol) in DCM (12 mL) is added diethylamino sulfur trifluoride (2.78
mL, 21.25 mmol). The reaction is warmed to RT and is allowed to
stir for 2 h. The reaction is quenched with aqueous NaHCO.sub.3 and
extracted with DCM. The organic layer is dried with MgSO.sub.4,
filtered and evaporated to dryness. The product is purified by
CombiFlash.RTM. Companion to afford 50c (2.1 g, 52% yield) as a
colorless oil.
[0565] Step 3:
[0566] Step 3 is carried out exactly as in step 1 of example 48 to
provide boronic ester 50d.
Example 51
Synthesis of Boronate Fragment 51a (Used for the Preparation of
1115)
##STR00087##
[0568] Step 1:
[0569] To a cooled solution (0.degree. C.) of boronate 5f (400 mg,
1.45 mmol) in anhydrous DMF (8 mL) is added NaH (87.4 mg, 2.18
mmol, 60% dispersion in oil). The mixture is stirred for 30 min
before being treated with iodoethane (233 .mu.L, 2.9 mmol). The
resultant mixture is stirred for 18 h before being quenched with
water and extracted with EtOAc. The organic phase is washed with
brine and dried over MgSO.sub.4, filtered and concentrated. The
residue is purified by CombiFlash.RTM. Companion (EtOAc/hexanes) to
give 51a as colourless oil (317 mg, 72%).
Example 52
Synthesis of Boronate Fragment 52g (Used for the Preparation of
1149)
##STR00088##
[0571] Step 1:
[0572] To a solution of 4-bromo-1-methoxy-2-nitro-benzene 52a (6 g,
25.9 mmol) in dry THF (250 mL) at -40.degree. C. is added dropwise
the vinylmagnesium bromide solution (1 M in THF, 90.5 mL, 90.5
mmol). The reaction mixture is stirred at -40.degree. C. for 4 h
then poured into saturated NH.sub.4Cl solution. The reaction
mixture is extracted with Et.sub.2O (2.times.); the combined
organic layers are washed with brine, dried over Na.sub.2SO.sub.4,
filtered and concentrated in vacuo. The crude material was purified
by flash chromatography eluting EtOAc/hexanes (10% to 40%)
affording 52b (620 mg, 11% yield).
[0573] Step 2:
[0574] A solution of indole 52b (619 mg, 2.7 mmol) in DMF (5 mL) at
23.degree. C. is treated with NaH (60% in oil dispersion, 125 mg,
5.2 mmol) and stirred at 23.degree. C. for 5 min. Ethyl
bromoacetate (637 .mu.L, 5.75 mmol) is added and the solution is
stirred at 23.degree. C. for 24 h; UPLC/MS analysis indicated 62%
conversion. To this mixture, additional amounts of NaH (60% in oil
dispersion, 77 mg, 1.9 mmol) and ethyl bromoacetate (244 .mu.L, 2.2
mmol) are added. After 10 min, the UPLC analysis indicated >90%
conversion. The reaction is diluted with EtOAc, washed with
saturated NH.sub.4Cl solution and brine (4.times.), dried over
MgSO.sub.4, filtrated, concentrated and purified by flash
chromatography (5-20%; EtOAc/hexanes) to give 52c (541 mg, 63%
yield) as a yellow oil.
[0575] Step 3:
[0576] To a solution of 52c (385 mg, 1.2 mmol) in THF (12.3 mL) is
added LiBH.sub.4 solution (2 M in THF, 1.54 mL, 3.1 mmol). The
mixture is stirred at RT for 16 h, then cooled to 0.degree. C. and
neutralized with NH.sub.4Cl (sat). The resulting solution is
extracted with ethyl acetate (2.times.) and combined organic layers
are washed with water, brine, dried over Na.sub.2SO.sub.4, filtered
and concentrated in vacuo providing the compound 52d (317 mg, 95%
yield).
[0577] Step 4:
[0578] To a solution of 52d (284 mg, 1.05 mmol) in DCM (9.7 mL) at
RT is added the AlCl.sub.3 (561 mg, 4.2 mmol). This mixture is
stirred at RT for 16h, then cooled to 0.degree. C. and methanol is
added. The resulting solution is concentrated in vacuo. The residue
is diluted with DCM containing 5% MeOH and a mixture of brine/water
50:50. The resulting solution is extracted with DCM until no more
products remained in the aqueous layer. The organic layer is dried
over Na.sub.2SO.sub.4, filtered and concentrated in vacuo.
Purification by flash chromatography (2-10%, methanol/DCM) gives
the compound 52e (200 mg, 74% yield).
[0579] Step 5:
[0580] To a solution of 52e (170 mg, 0.66 mmol) in DCM (10 mL) at
0.degree. C. is added the triethylamine (204 .mu.L, 1.46 mmol).
Methanesulfonyl chloride (62 .mu.L, 0.8 mmol) is added and the
mixture is stirred at 0.degree. C. for 10 min. The reaction is not
completed, additional methanesulfonyl chloride (30 .mu.L, 0.4 mmol)
is added and the mixture is stirred at 0.degree. C. for 10 min.
Poured into ice water the extracted with CH.sub.3Cl (3.times.).
Combined organic layers are washed with saturated NH.sub.4Cl,
NaHCO.sub.3, brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. The crude intermediate (261 mg, 0.78 mmol)
is diluted in DMF (5.3 mL) and NaH (60% in oil dispersion, 52.9 mg,
1.3 mmol) is added at 0.degree. C. The mixture is stirred at RT for
16 h. Et.sub.2O and brine are added, layers separated and the
organic layer is washed with brine (2.times.), dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The product
is then purified by flash chromatography eluting EtOAc/hexanes
(10-25%) to give 52f (170 mg, 91% yield).
[0581] Step 6:
[0582] Step 6 is carried out exactly as in step 1 of example 48 to
provide boronic ester 52g.
Example 53
Synthesis of Boronate Fragment 53i (Used for the Preparation of
1141, 1148)
##STR00089##
[0584] Step 1:
[0585] To a solution of 53a (10.0 g, 48.3 mmol) in acetic acid (150
mL) at RT is added iron (10.8 g, 193 mmol) and the reaction mixture
is stirred at 70.degree. C. for 2 h. The cooled reaction mixture is
filtered and the filtate concentrated under vacuum. The residue
diluted with EtOAc (300 mL) is washed with water (100 mL), brine
(100 mL), dried over MgSO.sub.4, filtered and concentrated under
vacuum to give 53b as a yellow solid (8.8 g, 100% yield). This
material is used in the next step without further purification.
[0586] Step 2:
[0587] To a solution of aniline 53b (8.8 g, 49.7 mmol) and
diethylphosphonoacetic acid (8.8 mL, 54.6 mmol) in DCM (300 mL) is
added HATU (22.7 g, 59.6 mmol) followed by DIPEA (21.6 mL, 124
mmol). The reaction mixture is stirred at RT for 3 h. After that
period, the transformation is incomplete, therefore, more
diethylphosphonoacetic acid (4.0 mL, 24.9 mmol), HATU (9.4 g, 24.9
mmol) and DIPEA (4.3 mL, 24.9 mmol) are added. The mixture is
stirred for another 2 h. The mixture is diluted with DCM (300 mL),
washed with aqueous 0.2N HCl (3.times.100 mL), aqueous 0.2N NaOH
(3.times.100 mL), water (100 mL) and brine (100 mL). The organic
phase is dried over MgSO.sub.4, filtered and concentrated under
vacuum. The residue is dried under vacuum overnight to give the
desired product 53c as a pale orange solid (16.0 g, 60% yield).
This material is used in the next step without further
purification.
[0588] Step 3:
[0589] To a solution of 53c (16.0 g, 26.9 mmol) in THF (180 mL) at
50.degree. C. is carefully added NaH (60% in oil, 1.2 g, 29.6 mmol)
and the reaction mixture is stirred for 2 h. The cooled reaction
mixture is quenched with MeOH (20 mL) and silica gel (50 g) is
added. The mixture is concentrated under vacuum and purified by
CombiFlash.RTM. Companion (DCM/MeOH) to give the desired
intermediate 53d (1.8 g, 27% yield).
[0590] Step 4:
[0591] A solution of 53d (1.8 g, 7.1 mmol) in POCl.sub.3 (30 mL,
322 mmol) is stirred at 110.degree. C. for 45 min. The cooled
reaction mixture is concentrated under vacuum. The residue is
diluted with DCM (100 mL), washed with aqueous 1.0N NaOH (50 mL),
water (50 mL), and brine (50 mL), dried over MgSO.sub.4, filtered
and concentrated under vacuum. The crude product is purified by
CombiFlash.RTM. Companion (DCM/MeOH) to give the desired
chloroquinoline 53e (1.3 g, 81% yield).
[0592] Step 5:
[0593] A solution of 53e (1.3 g, 5.8 mmol) in EtOH (100 mL) is
degassed by bubbling argon for 45 min. Palladium (10 wt. % on
activated carbon, 1.0 g) is added to the solution and the reaction
mixture is stirred under an atmospheric pressure of hydrogen for 6
h. The reaction mixture is filtered and the filtrate concentrated
under vacuum to give 53f (1.1 g, 100% yield). This material is used
in the next step without further purification.
[0594] Step 6:
[0595] To a solution of 53f (1.1 g, 5.8 mmol) in DCM (40 mL) at
0.degree. C. is added a BBr.sub.3 solution (1M in heptane, 12.7 mL,
12.7 mmol). The reaction mixture is stirred for 30 min at 0.degree.
C. and then slowly allowed to warm to Rt and stirred at this
temperature for 24 h. The reaction mixture is quenched with MeOH
(10 mL) and neutralized with aqueous 1.0 N NaOH. The yellow
precipitate that formed is filtered and dried under vacuum to give
the desired product 53g (984 mg, 100% yield). This material is used
in the next step without further purification.
[0596] Step 7:
[0597] To a solution of 53g (984 mg, 5.7 mmol) and Et.sub.3N (4.0
mL, 28.7 mmol) in DCM (40 mL) cooled to -78.degree. C. is added
Tf.sub.2O) (2.1 mL, 12.6 mmol). The resulting dark solution is
stirred for 15 min at -78.degree. C. and then slowly allowed to
warm to RT and then stirred at this temperature for 3 h. The
reaction mixture is diluted with DCM (50 mL), washed with aqueous
0.2 N HCl (25 mL), aqueous saturated NaHCO.sub.3 (25 mL), water (25
mL), brine (25 mL), dried over MgSO.sub.4, filtered and
concentrated under vacuum. The residue is purified by
CombiFlash.RTM. Companion (DCM/MeOH) to give triflate 53h (755 mg,
43% yield).
[0598] Step 8:
[0599] A well stirred DMF (7 mL) solution of triflate 53h (555 mg,
1.8 mmol), potassium acetate (608 mg, 6.4 mmol) and
bis(pinacolato)diborane (697 mg, 2.7 mmol) is degassed by bubbling
argon through the solution for 20 min. PdCl.sub.2(dppf)-DCM (224
mg, 0.27 mmol) is added and degassing is continued for 15 min. The
system is sealed (Teflon screw cap vessel) under argon and heated
to 95.degree. C. for 7 h. The mixture is poured in aqueous 1N HCl
(30 mL) and diluted with EtOAc (15 mL). The layers are separated
and the aqueous layer is neutralized to pH 7 with aqueous 1.0 N
NaOH. This neutral aqueous layer is extracted with EtOAc
(3.times.25 mL). The combined organic extracts are washed with
brine (25 mL), dried over MgSO.sub.4, filtered and concentrated
under vacuum. The boronic acid 53i (290 mg, 80% yield) is used as
such for the following step.
Example 54
Synthesis of Boronate Fragment 54b (Used for the Preparation of
1145)
##STR00090##
[0601] Step 1:
[0602] Compound 54b is prepared from 54a following a synthetic
sequence identical to steps 1 to 8 of Example 53.
Example 55
Synthesis of Boronate Fragment 55g (Used for the Preparation of
1147)
##STR00091##
[0604] Step 1:
[0605] To a 1 L flask equipped with a mechanical stirrer is added
55a (30 g, 129 mmol) in acetic acid (300 mL). To this mixture is
added iron powder (14.4 g, 258 mmol) slowly in aliquots. The
mixture is heated at 50.degree. C. for 2 h before an additional 7.2
g (129 mmol) of iron powder is slowly added. After 1.5 h at
50.degree. C., the conversion to the aniline is complete. The
solution is cooled to RT and diluted with 500 mL of EtOAc before
being filtered through Celite. The filtrate is concentrated and the
crude product is partitioned between EtOAc (10 and 200 mL of water.
The mixture is vigorously shaken and the organic phase is washed
with 200 mL of brine, dried over MgSO.sub.4, filtered and
concentrated to give aniline 55b (23.7 g, 91%) as a brown oil which
is used directly in the next step.
[0606] Step 2:
[0607] Aniline 55b (600 mg, 3 mmol) is treated with 6N HCl (8 mL)
and sonicated until a white suspension appears (HCl salt). This
mixture is heated to 100.degree. C. before being treated with the
vinyl ketone 55c (1.2 g, 5.9 mmol) which is prepared similarly to
that reported (Ref: Bull. Korean Chem. Soc. 24 (2003) 1, 13-14) but
via the Weinreb amide. The reaction mixture is heated at reflux for
6 h, then stirred at RT for 16 h. The mixture is diluted with EtOAc
and basified with 10N NaOH. The organic phase is separated and the
aqueous phase re-extracted with EtOAc. The combined organic layers
are washed with brine, dried (MgSO.sub.4), filtered and
concentrated. The material is purified by Combiflash.RTM. Companion
(EtOAc/hexanes) to afford the desired alcohol 55d (330 mg, 37.5%)
as a pale brown solid.
[0608] Step 3:
[0609] BBr.sub.3 (3.34 mL, 3.34 mmol, 1M solution in DCM) is added
to a solution of alcohol 55d (330 mg, 1.12 mmol) in DCM (10 mL) at
RT. The reaction mixture is stirred at RT for 16 h, then is
quenched with MeOH and concentrated to dryness. The residue is
taken up into DCM and washed with water and brine before being
dried (MgSO.sub.4) filtered and concentrated to afford the
bromophenol 55e (384 mg, 100%). This material is used in the next
step.
[0610] Step 4:
[0611] a mixture of bromophenol bromophenol 55e (345 mg, 1.14 mmol)
and K.sub.2CO.sub.3 (315 mg, 2.28 mmol) in MeCN (20 mL) is treated
at RT for 2 h. The mixture is then concentrated to dryness and the
residue dissolved in EtOAc before being washed with water and
brine. The organic phase is dried (MgSO.sub.4), filtered and
concentrated before the residue is purified by Combiflash.RTM.
Companion (EtOAc/hexanes) to afford the cyclic ether 55f (301 mg,
60%) as an amber solid.
[0612] Step 5:
[0613] In a screw-cap pressure tube is added the cyclic ether 55f
(180 mg, 0.68 mmol), bispinocolatoborane (260 mg, 1.0 mmol),
potassium acetate (226 mg, 2.4 mmol) and Pd(dppf)Cl.sub.2.DCM
complex (83 mg, 0.10 mmol) in dry DMF (4 mL). The resulting mixture
is de-gased with Argon (5 min). The tube is sealed and stirred at
95.degree. C. for 2.5 h. The mixture is treated with 1N HCl (10 mL)
before being diluted with EtOAc. After separation of the layers,
the aqueous phase was neutralized with 1N NaOH (using a pH meter)
to pH 7.0 and is extracted with EtOAc (3.times.). The combined
organic layers are washed with brine, dried (MgSO.sub.4), filtered
and concentrated to afford the boronic acid 55g (124 mg, 79%). This
material is used as is in the final cross coupling reaction.
Biological Data
[0614] The compounds of the invention have valuable pharmacological
properties. Compounds from this class strongly associate with the
integrase target as demonstrated by an integrase displacement
assay, are particularly effective at inhibiting HIV integrase and
additionally show unexpected potency across at least four or all
six major HIV-1 virus HIV variants at residues 124, 125.
Integrase Displacement Assay
[0615] A displacement assay is used to evaluate the relative
affinity of compounds of the invention to bind reversibly with HIV
integrase. The displacement assay measures the extent to which
Probe I (consisting of a HIV integrase inhibitor connected by a
flexible linker to a biotin molecule), in complex with a His-tagged
HIV integrase, is displaced by compounds of the invention. The
integrase-Probe I complex is formed in the presence or absence of
inhibitor compounds and the interaction is monitored by a
homogeneous time resolved fluorescence (hTRF) assay system.
Example 56
Production of Probe I
##STR00092##
[0617] Step 1:
[0618] Dimethyl carbonate (22 mL, 269 mmol) and NaH (60% in oil,
10.8 g, 270 mmol) are combined in toluene (80 mL) and heated to
90.degree. C. for 20 min, then 4-chloroacetophenone P1 (14 mL, 109
mmol) is added dropwise over ca. 15 min. The mixture is stirred at
90.degree. C. for 30 min, then cooled and carefully treated with 5%
HCl (aq) (100 mL) and EtOAc (100 mL). The organic phase is washed
with brine, dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue is purified by flash chromatography (15%
EtOAc/hexanes) to give compound P2.
[0619] Step 2:
[0620] A mixture of compound P2 (7.1 g, 33.4 mmol) and
4-chloroaniline (5.9 g, 46.3 mmol) in DMF/xylene (7 mL/40 mL) is
heated at 140.degree. C. for 10 h. The cooled mixture is
partitioned between 1M HCl (40 mL) and EtOAc (150 mL). The organic
layer is washed with 1M HCl, water and brine, dried over
MgSO.sub.4, filtered and concentrated to dryness. The residue is
purified by flash chromatography (SiO.sub.2, 15% to 20%
EtOAc/hexanes) to give compound P3.
[0621] Step 3:
[0622] To a mixture of compound P3 (4.79 g; 15.5 mmol), KOtBu (2.0
g; 18.57 mmol) and DMF (23 mL) is added ethyl-2-bromovalerate (3.2
mL, 18.25 mmol). The mixture is allowed to stir at RT for 16 h,
then is poured over ice into a solution of 1N HCl (100 mL) and the
mixture is extracted with EtOAc (2.times.100 mL). The combined
organic extracts are washed with brine (4.times.), dried
(Na.sub.2SO.sub.4), filtered and concentrated to afford, after
purification by chromaotography (EtOAc/hexanes) compound P4 as a
mixture of diastereoisomers.
[0623] Step 4:
[0624] A mixture of compound P4 (in separate portions of 1.26 g,
0.77 g and 1.09 g; 7.15 mmol total) and H.sub.2SO.sub.4 (in
separate portions of 24 mL, 15 mL and 19 mL) is allowed to react at
150.degree. C. for 20 minutes. The combined reaction mixture is
allowed to cool slightly and added dropwise to ice-water. The
mixture is extracted with EtOAc (3.times.), washed with brine
(1.times.), dried (Na.sub.2SO.sub.4), filtered and concentrated in
vacuo. The residue (0.69 g, 1.76 mmol) is dissolved in EtOH (25 mL)
and to this solution is added POCl.sub.3 (2.4 mL; 27 mmol). The
reaction is heated at reflux for 1 h, then poured into ice-water
and extracted with CH.sub.2Cl.sub.2 (3.times.). The organic phase
is washed with brine, dried (MgSO.sub.4), filtered and concentrated
and the residue is purified by chromatography to afford compound
P5.
[0625] Step 5:
[0626] A solution of (Boc).sub.2O (1 M in THF, 2.47 mL, 2.47 mmol)
is added to a solution of H.sub.2NCH.sub.2CH.sub.2Br--HBr (506 mg,
2.47 mmol) and Et.sub.3N (860 .mu.L, 6.175 mmol) in THF (10 mL).
The reaction mixture is stirred at RT for 18 h and is partitioned
between EtOAc (100 mL) and saturated aqueous NaHCO.sub.3 (25 mL).
The organic phase is washed with brine, dried over anhydrous
MgSO.sub.4 and concentrated. The residue is purified by
chromatography (5% to 20% EtOAc/hexanes) to give
BocNHCH.sub.2CH.sub.2Br. To a cooled solution (0.degree. C.) of
compound P5 (200 mg, 0.495 mmol) in DMF (3 mL) is added KOtBu (67
mg, 0.598 mmol). The mixture is stirred for 15 min, then a solution
of BocNHCH.sub.2CH.sub.2Br (160 mg, 0.717 mmol) in DMF (2 mL) is
added. The reaction mixture is stirred at RT for 18 h. Water (1 mL)
is added, the mixture was diluted with EtOAc (100 mL) and the
organic phase is washed with saturated aqueous NaHCO.sub.3 (25 mL)
and brine, dried over anhydrous MgSO.sub.4 and concentrated. The
residue is purified by chromatography (10% to 30% EtOAc/hexanes) to
give compound P6.
[0627] Step 6:
[0628] To a solution of compound P6 (96 mg, 0.175 mmol) in DMSO
(2.5 mL) is added 5N NaOH (175 .mu.L, 0.875 mmol). The mixture is
stirred for 30 min and purified by semi preparative HPLC to afford
the Boc-deprotected carboxylic acid. The compound is treated with
Boc.sub.2O in the presence of NaOH to provide compound P7 as a
racemic mixture. Separation by chiral HPLC, using a ChiralCel OD-R
column (20.times.250 mm from Chiral Technologies Inc) and an
isocratic solvent system of 20% H.sub.2O (containing 0.06% TFA) and
80% of a solvent mixture composed of 75% MeCN in H.sub.2O
(containing 0.06% TFA) provides the (S)-enantiomer P7.
[0629] Step 7:
[0630] To a mixture of compound P7 (9.2 mg, 0.017 mmol) and
CH.sub.2Cl.sub.2 (1.5 mL) is added TFA (750 .mu.L). The mixture is
stirred at RT for 1 h and concentrated. The residue is dissolved in
CH.sub.2Cl.sub.2 (1.0 mL) and to this mixture is added Et.sub.3N (7
.mu.L, 0.051 mmol), followed by EZ-Link.TM. TFP-PEO-biotin (Pierce;
17.2 mg, 0.025 mmol). The reaction mixture is stirred at RT for 18
h, the solvent is evaporated and the residue is purified by
semi-preparative HPLC to afford biotinylated Probe I.
[0631] Probe I is determined to have a dissociation constant (Kd)
of 1 .mu.M, as measured by Isothermal Titration calorimetry (ITC)
according to known methods, for example as described in Shaw-Reid
et al. J Biol Chem. 278(5):2777-80 (2003).
[0632] His-tapped HIV inteqrase: His-tagged integrase is cloned and
expressed in a similar manner as outlined in Barsov et al., J.
Virol., Vol 70, No. 7, 4484-4494, (1996). Briefly, the integrase
gene is PCR amplified from a plasmid containing the HXB2 provirus
using forward and reverse primers that span the first and final
codons of integrase, respectively. Primers contain a 5' NdeI
(forward primer) and 3' XhoI site (reverse primer) which allows for
cloning of the NdeI/XhoI fragment into the Pet28a bacterial
expression vector (Novagen). DH5.mu.E. coli cells are used to
generate and propagate the DNA vectors while BL21 pLysS E. coli
cells are used for expression of the His-tagged protein.
[0633] His-tagged HIV-1 integrase is expressed in BL21 pLysS cells
(Stratagene) to an O.D. of 1.4 in a 30 L fermentor at 37.degree. C.
and induced with 0.5 mM IPTG for 3 hours at 37.degree. C. The
bacterial pellet is re-suspended in freshly prepared extraction
buffer (20 mM NaPi pH 5.8, 1 M NaCl, 1 M urea, 1 mM TCEP, 20 mM
imidazole, 1 mM PMSF, and 1.5 mL Sigma protease inhibitor cocktail
for 35 mg pellet) and sonicated. The cell lysate is subjected to
centrifugation at 100,000 G for 30 min and the supernatant is
loaded onto a HiTrap Ni.sup.2+ column. The column is washed with
the extraction buffer containing 100 mM imidazole and eluted in a
15 min linear gradient to 1 M imidazole. The His-Integrase
fractions are pooled and diluted 1 to 8 in fresh S column buffer
(20 mM Na Pi pH 5.8, 1 mM TCEP, 1 mM EDTA, 1 M urea, 10% glycerol)
and loaded onto a HiTrap S column. The column is washed with S
column buffer then eluted in an 80 min linear gradient from 0 to 1
M NaCl. Protein fractions are run on an SDS-PAGE gel and the most
concentrated samples without contaminant bands are pooled and
precipitated with 60% ammonium sulfate. The precipitate is then
re-suspended in storage buffer (20 mM Hepes pH 7.5, 500 mM NaCl,
10% glycerol, 0.5 mM TCEP) then loaded onto a SD200 gel filtration
column. The His-tagged Integrase containing fractions are pooled,
dosed for protein concentration and stored at -80.degree. C. in
aliquots. The His-tagged integrase samples are thawed on ice and
diluted to the desired concentration in assay buffer for use in the
integrase displacement assay.
Homogenous Time Resolved Fluorescence (HTRF) Assay System
[0634] The relative binding affinity of compounds of the invention
to the HIV integrase target is assessed using a HTRF system based
on fluorescence resonance energy transfer (FRET) between europium
cryptate (EuK) as energy donor and cross linked allophycocyanin
(XL665) as acceptor. EuK labeled streptavidin (CysBio) binding to
Probe I and XL665 labeled anti-His antibody (CysBio) binding to
His-integrase are used in the system. Interaction between integrase
and Probe I is monitored by energy transfer between the EuK and
XL665 in the integrase-probe complex. Displacement of the
biotinylated probe from integrase by a compound of the invention
results in a loss of fluorescence from the XL665 labeled
antibody.
[0635] Assay solutions are prepared using an assay buffer
consisting of 50 mM HEPES (pH 7.5); 50 mM NaCl; 150 mM KF; 1 mg/ml
BSA, 1.0 mM TCEP, 0.05% Tween 20. Assay solution A is prepared with
Probe I (30 nM) and His-integrase (300 nM) in assay buffer with
4.5% DMSO. Assay solutions B is prepared with a compound of the
invention diluted to 120 .mu.M in assay buffer containing 4.5%
DMSO, then serial diluted 11 times in 2-fold steps of the same
buffers. Finally, assay solution C is prepared with
Streptavidin-EuK and Anti-his XL665 premixed at concentrations of 6
nM and 150 nM respectively in assay buffer.
[0636] The reaction mixture was prepared by adding 5 .mu.L of each
assay solution (solutions A and C with one of the serial dilutions
of compound in solution B for each reaction mixture) to a 384-well
black round bottom, low volume NBS plates (Corning catalog #3676)
giving a final concentration of 100 nM His-integrase, 10 nM probe,
compound of invention (40 .mu.M down to 37.5 nM), 50 nM anti-his
XL665, 2 nM Strep-EuK and 3% DMSO with a total volume of 15 .mu.L.
The reaction mixture was incubated at room temperature for one
hour. The fluorescence was read on a Victor 1420 Multilable HTS
reader at 615 nm and 665 nm.
[0637] The compounds of the invention have greater affinity for the
HIV integrase target than that of Probe I. However, the increased
avidity of the biotinylated probe due to tetramerization with
Strep-EuK allows the evaluation of compounds of the invention that
have significantly stronger association with HIV integrase.
Example 57
C8166 HIV-1 Luciferase Assay (EC.sub.50)
[0638] C8166 cells are derived from a human T-lymphotrophic virus
type 1 immortalized but nonexpressing line of cord blood
lymphocytes (NIH AIDS reagent 404) and are highly permissive to
HIV-1 infection. The pGL3 Basic LTR/TAR plasmid is made by
introducing the HIV-1 HxB2 LTR sequence from nucleotide -138 to +80
(Sca1-HindIII) upstream of the luciferase gene in the pGL3 Basic
Vector (a promoterless luciferase expression vector from Promega
catalogue #E1751) with the gene for blasticidine resistance cloned
in. The reporter cells are made by electroporating C8166 cells with
pGL3 Basic LTR/TAR and selecting positive clones with blasticidine.
Clone C8166-LTRluc #A8-F5-G7 was selected by 3 consecutive rounds
of limiting dilution under blasticidine selection. Cultures are
maintained in complete media (consisting of: Roswell Park Memorial
Institute medium (RPMI) 1640+10% FBS+10.sup.-5 M
.beta.-mercaptoethanol+10 .mu.g/mL gentamycin) with 5 .mu.g/mL
blasticidine, however, blasticidine selection is removed from the
cells before performing the viral replication assay.
Luciferase Assay Protocol
Preparation of Compounds
[0639] Serial dilutions of HIV-1 inhibitor compounds are prepared
in complete media from 10 mM DMSO stock solutions. Eleven serial
dilutions of 2.5.times. are made at 8.times. desired final
concentration in a 1 ml deep well titer plate (96 wells). The
12.sup.th well contains complete media with no inhibitor and serves
as the positive control. All samples contain the same concentration
of DMSO (.ltoreq.0.1% DMSO). A 25 .mu.L aliquot of inhibitor is
added, to triplicate wells, of a 96 well tissue culture treated
clear view black microtiter plate (Corning Costar catalogue #3904).
The total volume per well is 200 .mu.L of media containing cells
and inhibitor. The last row is reserved for uninfected C8166 LTRluc
cells to serve as the background blank control and the first row is
media alone.
Infection of Cells
[0640] C8166 LTRluc cells are counted and placed in a minimal
volume of complete RPMI 1640 in a tissue culture flask (ex.
30.times.10.sup.6 cells in 10 mL media/25 cm.sup.2 flask). Cells
are infected with HIV-1 or virus with variant integrase generated
as described below at a molecules of infection (moi) of 0.005.
Cells are incubated for 1.5 h at 37.degree. C. on a rotating rack
in a 5% CO.sub.2 incubator and re-suspended in complete RPMI to
give a final concentration of 25,000-cells/175 .mu.L. 175 .mu.L of
cell mix is added to wells of a 96 well microtiter plate containing
25 .mu.L 8.times. inhibitors. 25,000 uninfected C8166-LTRluc
cells/well in 200 .mu.L complete RPMI are added to the last row for
background control. Cells are incubated at 37.degree. C. in 5%
CO.sub.2 incubator for 3 days.
Luciferase Assay
[0641] 50 .mu.L Steady Glo (luciferase substrate T.sub.1/2=5 hours
Promega catalogue # E2520) is added to each well of the 96 well
plate. The relative light units (RLU) of luciferase is determined
using the LUMIstar Galaxy luminometer (BMG LabTechnologies). Plates
are read from the bottom for 2 seconds per well with a gain of
240.
[0642] The level of inhibition (% inhibition) of each well
containing inhibitor is calculated as follows:
% inhibition = ( 1 - [ RLU well - RLU blank RLU control - RLU blank
] ) * 100 ##EQU00001##
[0643] The calculated % inhibition values are used to determine
EC.sub.50, slope factor (n) and maximum inhibition (I.sub.max) by
the non-linear regression routine NLIN procedure of SAS using the
following equation:
% inhibition = I max .times. [ inhibitor ] n [ inhibitor ] n + IC
50 n ##EQU00002##
[0644] Compounds of the invention tested in the cellular assay
described above are particularly effective at inhibiting HIV
integrase. Compounds of Table 1 were found to have EC.sub.50 values
of 300 nM or less. Furthermore, compounds of the invention show
unexpected potency across major HIV-1 virus HIV variants. Compounds
of Table 1 show unexpected potency in at least four or in all six
of the known variants at residues 124 and 125 of HIV-1 virus from
infected patients, namely Thr124/Thr125, Ala124/Thr125,
Ala124/Ala125, Thr124/Ala125, Asn124/Thr125 and Asn124/Ala125. The
results of representative compounds are shown in Table 2.
TABLE-US-00003 TABLE 2 EC.sub.50 (nM) EC.sub.50 (nM) EC.sub.50 (nM)
EC.sub.50 (nM) Compound A124/T125 T124/T125 T124/A125 A124/A125
1008 11 59 49 55 1010 12 41 76 49 1014 19 87 76 68 1018 35 55 93
110 1023 12 29 31 37 1038 3 9 15 8 1052 28 120 59 64 1059 3 14 11 9
1136 91 200 140 170 2001 71 110 120 67 2002 9.9 19 24 13
Generation of Viruses with 124/125 Variant Residues of
Integrase
[0645] The 2.12 virus with NL4.3 strain of HIV-1 integrase (SEQ ID
NO: 1) is used for the Thr124/Thr125 variant integrase residues and
HXB2 integrase is introduced into the 2.12 virus for the
Ala124/Thr125 variant integrase residues. The remaining variants
viruses are generated by site directed mutagenesis of NL4.3
integrase to introduce the Ala124/Ala125, Thr124/Ala125,
Asn124/Thr125, or Asn124/Ala125 variants in the 2.12 virus.
Molecular biology and generation of virus are performed in a
similar manner to Doyon et al. J Virol. 70(6):3763-9 (1996).
Briefly, site directed mutagenesis is used to introduce silent
mutations in the integrase gene of the 2.12 virus. The mutations in
NL4.3 integrase introduced unique Cla I and Xba I restriction sites
in the 2.12 provirus (using the primers 5'-TTT AGA TGG AAT CGA TAA
GGC CCA AGA AG-3' (SEQ ID NO:2) and 5'-CTT CTT GGG CCT TAT CGA TTC
CAT CTA AA-3' (SEQ ID NO:3) to introduce Cla I site and 5'-GGT TTA
TTA CAG GGA CTC TAG AGA TCC AGT TTG GA-3' (SEQ ID NO:4) and 5'-TCC
AAA CTG GAT CTC TAG AGT CCC TGT AAT AAA CC-3' (SEQ ID NO:5) to
introduce XbaI site). The 2.12 virus is generated with the Cla I
and Xba I restriction sites in integrase replicates and responds to
compounds of the invention in a similar manner to the original 2.12
virus. Point mutations at residues 124 and 125 are introduced into
NL4.3 integrase in a Pet 28a vector (Novagen) by using the
QuikChange site-directed mutagenesis kit (Stratagene). After
sequencing to ensure that the desired 124 and/or 125 residue
mutations are obtained, the Cla I/Xba I fragment is PCR
amplification with Pfu Ultra (Stratagene) and cloned into the
unique Cla I/Xba I sites in the 2.12 virus.
TABLES OF COMPOUNDS
[0646] The following tables list compounds of the invention.
Compounds of the invention tested in the cellular assay described
above in Example 57 are particularly effective at inhibiting HIV
integrase. Compounds of Table 1 were found to have EC.sub.K values
of 300 nM or less in at least four or in all six of the known
variants at residues 124 and 125 of HIV-1 virus from infected
patients, namely Thr124/Thr125, Ala124/Thr125, Ala124/Ala125,
Thr124/Ala125, Asn124/Thr125 and Asn124/Ala125.
[0647] Retention times (t.sub.R) for each compound are measured
using the standard analytical HPLC conditions described in the
Examples. As is well known to one skilled in the art, retention
time values are sensitive to the specific measurement conditions.
Therefore, even if identical conditions of solvent, flow rate,
linear gradient, and the like are used, the retention time values
may vary when measured, for example, on different HPLC instruments.
Even when measured on the same instrument, the values may vary when
measured, for example, using different individual HPLC columns, or,
when measured on the same instrument and the same individual
column, the values may vary, for example, between individual
measurements taken on different occasions.
TABLE-US-00004 TABLE 1 ##STR00093## t.sub.R MS Cpd R.sup.4 R.sup.6
R.sup.7 (min) (M + H).sup.+ 1001 ##STR00094## CH.sub.3 H 4.7 398.1/
400.1 1002 ##STR00095## H CH.sub.3 4.6 398.1/ 400.1 1003
##STR00096## H F 4.5 402.2/ 404.1 1004 ##STR00097## H H 3.9 396.2
1005 ##STR00098## H H 5.1 404.2 1006 ##STR00099## H H 4.3 406.2
1007 ##STR00100## H H 4.5 364.2 1008 ##STR00101## H H 4.8 378.2
1009 ##STR00102## H H 4.6 382.1 1010 ##STR00103## H H 4.8 402.1/
404.1 1011 ##STR00104## H H 4.7 406.2 1012 ##STR00105## H H 4.7
428.0/ 430.0 1013 ##STR00106## H H 3.9 442.1 1014 ##STR00107## H H
3.7 392.1 1015 ##STR00108## H H 5.0 398.1/ 400.1 1016 ##STR00109##
H H 5.6 418.2 1017 ##STR00110## H H 5.0 420.2 1018 ##STR00111## H H
3.7 400.1 1019 ##STR00112## H H 3.7 382.1 1020 ##STR00113## H
CH.sub.3 4.0 413.2 1021 ##STR00114## H CH.sub.3 4.3 420.1 1022
##STR00115## F H 4.9 424.2 1023 ##STR00116## H H 4.4 390.1 1024
##STR00117## H H 5.2 420.1/ 422.1 1025 ##STR00118## H CH.sub.3 4.4
364.2 1026 ##STR00119## H CH.sub.3 5.3 432.1/ 434.1/ 436.1 1027
##STR00120## H CH.sub.3 5.5 406.2 1028 ##STR00121## H CH.sub.3 3.6
415.2 1029 ##STR00122## H CH.sub.3 5.1 460.1/ 462.1 1030
##STR00123## H CH.sub.3 4.4 416.1/ 418.2 1031 ##STR00124## H
CH.sub.3 4.8 396.2 1032 ##STR00125## H CH.sub.3 4.8 430.2 1033
##STR00126## H CH.sub.3 5.3 454.1/ 456.1 1034 ##STR00127## H
CH.sub.3 4.6 404.2 1035 ##STR00128## H H 4.9 398.1/ 400.1 1036
##STR00129## H CH.sub.3 4.8 410.2 1037 ##STR00130## H H 5.3 454.2/
456.2 1038 ##STR00131## H H 4.9 440.2/ 442.2 1039 ##STR00132## H H
5.1 440.2/ 442.2 1040 ##STR00133## H H 4.1 424.1 1041 ##STR00134##
H CH.sub.3 4.3 438.2 1042 ##STR00135## H CH.sub.3 5.6 468.1/ 470.1
1043 ##STR00136## CH.sub.3 H 5.2 434.2 1044 ##STR00137## H H 4.9
420.2 1045 ##STR00138## CH.sub.3 H 4.4 416.1/ 418.1 1046
##STR00139## H H 4.8 398.1/ 400.1 1047 ##STR00140## H H 4.7 424.1
1048 ##STR00141## H H 4.5 424.1/ 426.1 1049 ##STR00142## H H 4.8
424.1/ 426.1 1050 ##STR00143## H H 3.9 390.1 1051 ##STR00144## H
CH.sub.3 4.3 416.1/ 418.1 1052 ##STR00145## H H 4.1 420.2 1053
##STR00146## CH.sub.3 H 5.1 454.1/ 456.1 1054 ##STR00147## CH.sub.3
H 5.4 454.1/ 456.1 1055 ##STR00148## H CH.sub.3 5.2 454.1/ 456.1
1056 ##STR00149## H CH.sub.3 5.4 454.1/ 456.1 1057 ##STR00150##
CH.sub.3 H 5.1 438.3 1058 ##STR00151## CH.sub.2CH.sub.3 H 5.5
412.2/ 414.2 1059 ##STR00152## H H 4.9 438.2 1060 ##STR00153## H H
3.7 424.4 1061 ##STR00154## H H 3.9 442.2 1062 ##STR00155## H H 4.5
426.2/ 428.2 1063 ##STR00156## H H 3.7 406.2 1064 ##STR00157## H H
4.8 436.3 1065 ##STR00158## H H 3.8 440.2/ 442.1 1066 ##STR00159##
H H 4.6 406.2 1067 ##STR00160## H H 4.1 440.2 1068 ##STR00161## H H
4.9 420.2 1069 ##STR00162## H H 4.6 406.2 1070 ##STR00163## H H 4.4
434.1/ 436.1 1071 ##STR00164## H H 5.0 424.1/ 426.1 1072
##STR00165## H H 4.7 424.1/ 426.1 1073 ##STR00166## H H 4.9 424.1/
426.1 1074 ##STR00167## H H 5.0 396.2 1075 ##STR00168## H H 3.6
415.3 1076 ##STR00169## H CH.sub.3 3.9 429.2 1077 ##STR00170## H H
3.8 421.2 1078 ##STR00171## H CH.sub.3 5.7 448.1/ 450.1 1079
##STR00172## H H 5.2 442.2 1080 ##STR00173## H H 5.4 440.1 1081
##STR00174## H H 4.6 398.2 1082 ##STR00175## H CH.sub.3 4.9 403.2
1083 ##STR00176## F H 5.6 458.1/ 460.1 1084 ##STR00177## H CH.sub.3
4.5 449.2/ 451.2 1085 ##STR00178## H CH.sub.3 3.4 429.3 1086
##STR00179## H H 5.0 426.2/ 428.2 1087 ##STR00180## H H 4.8 428.2/
430.2 1088 ##STR00181## H H 4.8 424.1/ 426.1 1089 ##STR00182## H H
4.9 416.2/ 418.2 1090 ##STR00183## F H 5.5 438.2 1091 ##STR00184##
Cl H 4.8 455.2/ 457.2 1092 ##STR00185## Cl H 5.4 454.2/ 456.2 1093
##STR00186## H H 4.1 434.2 1094 ##STR00187## H H 5.5 420.2 1095
##STR00188## H CH.sub.3 4.6 435.2 1096 ##STR00189## CH.sub.3
CH.sub.3 5.3 448.3 1097 ##STR00190## H H 4.3 455.2/ 457.2 1098
##STR00191## H H 5.5 476.1/ 478.1 1099 ##STR00192## F H 4.4 439.2
1100 ##STR00193## CH.sub.3 H 4.5 435.2 1101 ##STR00194## H H 3.9
432.2 1102 ##STR00195## H H 4.4 441.2/ 443.1 1103 ##STR00196## H H
4.1 419.3 1104 ##STR00197## H H 4.5 440.2/ 442.1 1105 ##STR00198##
H H 5.1 458.2/ 460.2 1106 ##STR00199## Cl H 5.8 492.1/ 494.1/ 496.1
1107 ##STR00200## Cl H 5.6 474.2/ 476.2 1108 ##STR00201## F H 4.2
459.2/ 461.1 1109 ##STR00202## Cl H 4.7 475.1/ 477.1/ 479.1 1110
##STR00203## CH.sub.3 H 4.6 455.2/ 457.2 1111 ##STR00204## H
CH.sub.3 4.5 455.2/ 457.2 1112 ##STR00205## H H 5.0 436.1/ 438.1/
440.1 1113 ##STR00206## H H 5.4 432.1/ 434.1/ 438.1 1114
##STR00207## H H 4.4 384.1/ 386.1 1115 ##STR00208## H H 4.3 449.3
1116 ##STR00209## H H 4.4 469.2/ 471.2 1117 ##STR00210## H H 4.5
402.1/ 404.1 1118 ##STR00211## F CH.sub.3 4.4 453.2 1119
##STR00212## F CH.sub.3 5.4 473.2/ 475.2 1120 ##STR00213## H H 4.6
402.1/ 404.1 1121 ##STR00214## H H 4.4 382.2 1122 ##STR00215## F
CH.sub.3 5.5 452.3
1123 ##STR00216## H CH.sub.3 5.3 494.0/ 496.0/ 498.0 1124
##STR00217## H H 4.5 402.2/ 404.2 1125 ##STR00218## H H 5.1 480.1/
482.1/ 484.1 1126 ##STR00219## Cl H 5.9 514.0/ 516.0/ 518.0 1127
##STR00220## H H 4.9 416.2/ 418.2 1128 ##STR00221## F H 5.4 498.0/
500.0/ 502.0 1129 ##STR00222## H H 5.3 442.2/ 444.2 1130
##STR00223## H H 4.9 460.1/ 462.1 1131 ##STR00224## H --CH.sub.3
3.6 457.3 1132 ##STR00225## H CH.sub.3 5.2 450.1/ 452.1/ 454.1 1133
##STR00226## F H 5.4 454.1/ 456.1/ 458.1 1134 ##STR00227## F H 5.2
434.2/ 436.2 1135 ##STR00228## Cl H 5.6 450.1/ 452.1/ 454.1 1136
##STR00229## H H 3.0 407.1 1137 ##STR00230## H Me 3.7 471.3 1138
##STR00231## H Me 5.0 463.2/ 465.2 1139 ##STR00232## H Me 4.8
468.2/ 469.2 1140 ##STR00233## H Me 4.4 447.3 1141 ##STR00234## H
Me 3.1 441.2 1142 ##STR00235## H Cl 5.4 475.1/ 477.1/ 479.1 1143
##STR00236## H Cl 3.1 477.2/ 479.2 1144 ##STR00237## H H 3.7 443.2
1145 ##STR00238## H H 3.2 441.3 1146 ##STR00239## H H 4.1 433.3
1147 ##STR00240## H H 3.8 457.2 1148 ##STR00241## H H 2.8 472.2
1149 ##STR00242## H H 4.5 430.0 1150 ##STR00243## Me H 3.7 457.2
1151 ##STR00244## Cl H 3.0 477.3/ 479.3 1152 ##STR00245## F H 2.8
461.3 1153 ##STR00246## F Me 2.9 475.1
TABLE-US-00005 TABLE 2 ##STR00247## MS t.sub.R (M + Cpd R.sup.4
R.sup.3 (min) H).sup.+ 2001 ##STR00248## ##STR00249## 3.0 429.2
2002 ##STR00250## ##STR00251## 2.9 457.3
[0648] Each of the references including all patents, patent
applications and publications cited in the present application is
incorporated herein by reference in its entirety, as if each of
them is individually incorporated. Further, it would be appreciated
that, in the above teaching of invention, the skilled in the art
could make certain changes or modifications to the invention, and
these equivalents would still be within the scope of the invention
defined by the appended claims of the application.
Sequence CWU 1
1
51288PRTHuman immunodeficiency virus type 1 1Phe Leu Asp Gly Ile
Asp Lys Ala Gln Glu Glu His Glu Lys Tyr His 1 5 10 15 Ser Asn Trp
Arg Ala Met Ala Ser Asp Phe Asn Leu Pro Pro Val Val 20 25 30 Ala
Lys Glu Ile Val Ala Ser Cys Asp Lys Cys Gln Leu Lys Gly Glu 35 40
45 Ala Met His Gly Gln Val Asp Cys Ser Pro Gly Ile Trp Gln Leu Asp
50 55 60 Cys Thr His Leu Glu Gly Lys Val Ile Leu Val Ala Val His
Val Ala 65 70 75 80 Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro Ala Glu
Thr Gly Gln Glu 85 90 95 Thr Ala Tyr Phe Leu Leu Lys Leu Ala Gly
Arg Trp Pro Val Lys Thr 100 105 110 Val His Thr Asp Asn Gly Ser Asn
Phe Thr Ser Thr Thr Val Lys Ala 115 120 125 Ala Cys Trp Trp Ala Gly
Ile Lys Gln Glu Phe Gly Ile Pro Tyr Asn 130 135 140 Pro Gln Ser Gln
Gly Val Ile Glu Ser Met Asn Lys Glu Leu Lys Lys 145 150 155 160 Ile
Ile Gly Gln Val Arg Asp Gln Ala Glu His Leu Lys Thr Ala Val 165 170
175 Gln Met Ala Val Phe Ile His Asn Phe Lys Arg Lys Gly Gly Ile Gly
180 185 190 Gly Tyr Ser Ala Gly Glu Arg Ile Val Asp Ile Ile Ala Thr
Asp Ile 195 200 205 Gln Thr Lys Glu Leu Gln Lys Gln Ile Thr Lys Ile
Gln Asn Phe Arg 210 215 220 Val Tyr Tyr Arg Asp Ser Arg Asp Pro Val
Trp Lys Gly Pro Ala Lys 225 230 235 240 Leu Leu Trp Lys Gly Glu Gly
Ala Val Val Ile Gln Asp Asn Ser Asp 245 250 255 Ile Lys Val Val Pro
Arg Arg Lys Ala Lys Ile Ile Arg Asp Tyr Gly 260 265 270 Lys Gln Met
Ala Gly Asp Asp Cys Val Ala Ser Arg Gln Asp Glu Asp 275 280 285
229DNAArtificial SequenceSynthetic primer 2tttagatgga atcgataagg
cccaagaag 29329DNAArtificial SequenceSynthetic primer 3cttcttgggc
cttatcgatt ccatctaaa 29435DNAArtificial SequenceSynthetic primer
4ggtttattac agggactcta gagatccagt ttgga 35535DNAArtificial
SequenceSynthetic primer 5tccaaactgg atctctagag tccctgtaat aaacc
35
* * * * *
References