U.S. patent application number 11/415722 was filed with the patent office on 2006-11-16 for macrocyclic isoquinoline peptide inhibitors of hepatitis c virus.
Invention is credited to Wenying Li.
Application Number | 20060257980 11/415722 |
Document ID | / |
Family ID | 33310775 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060257980 |
Kind Code |
A1 |
Li; Wenying |
November 16, 2006 |
Macrocyclic isoquinoline peptide inhibitors of hepatitis C
virus
Abstract
Macrocyclic isoquinoline peptides are disclosed having the
general formula: A compound of formula I: ##STR1## wherein R.sub.1
to R.sub.9, Q and W are described in the description. Compositions
comprising the compounds and methods for using the compounds to
inhibit HCV are also disclosed.
Inventors: |
Li; Wenying; (Pennington,
NJ) |
Correspondence
Address: |
LOUIS J. WILLE;BRISTOL-MYERS SQUIBB COMPANY
PATENT DEPARTMENT
P O BOX 4000
PRINCETON
NJ
08543-4000
US
|
Family ID: |
33310775 |
Appl. No.: |
11/415722 |
Filed: |
May 2, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10825693 |
Apr 16, 2004 |
|
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11415722 |
May 2, 2006 |
|
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60463423 |
Apr 16, 2003 |
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Current U.S.
Class: |
435/106 |
Current CPC
Class: |
A61P 1/16 20180101; C12P
13/04 20130101; A61K 38/00 20130101; C07K 5/0804 20130101; C12P
41/005 20130101; C12P 7/62 20130101; A61P 31/22 20180101; A61P
31/14 20180101; A61P 43/00 20180101 |
Class at
Publication: |
435/106 |
International
Class: |
C12P 13/04 20060101
C12P013/04 |
Claims
1. A process for resolving a mixture of alkyl ester enantiomers
comprising contacting the mixture with an enzyme effective to
preferentially promote the hydrolysis of one of the enantiomers;
characterized in that the contacting is conducted in the presence
of a buffer.
2. The process of claim 1 wherein the alkyl ester has the following
formula: ##STR3016## wherein: R.sub.25 is an amino protecting
group; and R.sub.26 is selected from the group consisting of
C.sub.1-10 alkyl, C.sub.6-14 aryl, C.sub.7-16 alkylaryl, C.sub.3-7
cycloalkyl or C.sub.3-10 alkyl cycloalkyl.
3. The process of claim 1 wherein the buffer is selected from the
group consisting of phosphates, borates and carbonates.
4. The process of claim 1 wherein the enzyme is a protease.
5. The process of claim 1 wherein the enzyme is selected from the
group consisting of Bacillus globigii, Bacillus licheniformis,
Bacillus halodurans, Bacillus clausii, Aspergillus oryzase and
mixtures thereof.
6. The process of claim 5 wherein the enzyme is selected from the
group consisting of Alcalase.RTM. (subtilisin protease),
Savinase.RTM. (subtilisin protease), Esperase.RTM. (subtilisn
protease), Flavourzyme.TM. (fungal protease) and mixtures
thereof.
7. The process of claim 1 wherein the contacting is conducted at pH
of from about 7.0 to 11.
8. The process of claim 1 wherein the contacting is conducted at a
temperature of from about 30 to 60.degree. C.
9. The process of claim 1 wherein the contacting is conducted for a
time of less than about seven days.
10. The process of claim 9 wherein the contacting is conducted for
a time of from about two hours to three days.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisonal of U.S. application Ser. No.
10/825,693, filed on Apr. 16, 2004, which claims priority to U.S.
Provisional Application Ser. No. 60/463,423 filed Apr. 16,
2003.
FIELD OF THE INVENTION
[0002] The present invention is generally directed to antiviral
compounds, and more specifically directed to compounds which
inhibit the functioning of the NS3 protease (also referred to
herein as "serine protease") encoded by Hepatitis C virus (HCV),
compositions comprising such compounds, and methods for inhibiting
the functioning of the NS3 protease.
BACKGROUND OF THE INVENTION
[0003] HCV is a major human pathogen, infecting an estimated 170
million persons worldwide--roughly five times the number infected
by human immunodeficiency virus type 1. A substantial fraction of
these HCV infected individuals develop serious progressive liver
disease, including cirrhosis and hepatocellular carcinoma. (Lauer,
G. M.; Walker, B. D. N. Engl. J. Med. (2001), 345, 41-52).
[0004] Presently, the most effective HCV therapy employs a
combination of alpha-interferon and ribavirin, leading to sustained
efficacy in 40% of patients. (Poynard, T. et al. Lancet (1998),
352, 1426-1432). Recent clinical results demonstrate that pegylated
alpha-interferon is superior to unmodified alpha-interferon as
monotherapy (Zeuzem, S. et al. N. Engl. J. Med. (2000), 343,
1666-1672). However, even with experimental therapeutic regimens
involving combinations of pegylated alpha-interferon and ribavirin,
a substantial fraction of patients do not have a sustained
reduction in viral load. Thus, there is a clear and unmet medical
need to develop effective therapeutics for treatment of HCV
infection.
[0005] HCV is a positive-stranded RNA virus. Based on a comparison
of the deduced amino acid sequence and the extensive similarity in
the 5' untranslated region, HCV has been classified as a separate
genus in the Flaviviridae family. All members of the Flaviviridae
family have enveloped virions that contain a positive stranded RNA
genome encoding all known virus-specific proteins via translation
of a single, uninterrupted, open reading frame.
[0006] Considerable heterogeneity is found within the nucleotide
and encoded amino acid sequence throughout the HCV genome. At least
six major genotypes have been characterized, and more than 50
subtypes have been described. The major genotypes of HCV differ in
their distribution worldwide, and the clinical significance of the
genetic heterogeneity of HCV remains elusive despite numerous
studies of the possible effect of genotypes on pathogenesis and
therapy.
[0007] The single strand HCV RNA genome is approximately 9500
nucleotides in length and has a single open reading frame (ORF)
encoding a single large polyprotein of about 3000 amino acids. In
infected cells, this polyprotein is cleaved at multiple sites by
cellular and viral proteases to produce the structural and
non-structural (NS) proteins. In the case of HCV, the generation of
mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and
NS5B) is effected by two viral proteases. The first one cleaves at
the NS2-NS3 junction; the second one is a serine protease contained
within the N-terminal region of NS3 and mediates all the subsequent
cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage
site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A,
NS5A-NS5B sites. The NS4A protein appears to serve multiple
functions, acting as a cofactor for the NS3 protease and possibly
assisting in the membrane localization of NS3 and other viral
replicase components. Complex formation between the NS3 protease
domain and its cofactor, NS4A, is essential for efficient
proteolytic cleavage of the four respective sites. The NS3 protein
also exhibits nucleoside triphosphatase and RNA helicase
activities. NS5B is a RNA-dependent RNA polymerase that is involved
in the replication of HCV.
[0008] Among the compounds that have demonstrated efficacy in
inhibiting HCV replication, as selective HCV serine protease
inhibitors, are the macrocyclic peptide compounds disclosed in
International Application PCT/CA00/00353 (Publication No. WO
00/59929).
SUMMARY OF THE INVENTION
[0009] The present invention provides macrocyclic isoquinoline
compounds of the following formula: A compound of formula I:
##STR2## [0010] wherein: [0011] (a) R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 are each independently H; C.sub.1-6
alkyl; C.sub.3-7 cycloalkyl; C.sub.1-6 alkoxy; C.sub.3-7
cycloalkoxy; halo-C.sub.1-6 alkoxy; halo-C.sub.1-6 alkyl; cyano;
halo; hydroxyl; C.sub.1-6 alkanoyl; nitro; amino; mono or
di-(C.sub.1-6)alkyl amine; mono or di-(C.sub.3-7)cycloalkyl amine;
mono or di-C.sub.1-6 alkylamide; mono or di-(C.sub.3-7)cycloalkyl
amide; carboxyl; (C.sub.1-6)carboxyester; thiol; C.sub.1-6
thioalkyl; C.sub.1-6alkylsulfoxide; C.sub.1-6alkylsulfone;
C.sub.1-6alkylsulfonamide; C.sub.6-10 aryl optionally substituted
with Het; C.sub.7-14 alkylaryl; C.sub.6-10 aryloxy; C.sub.7-14
alkylaryloxy; 5-7 membered monocyclic heteroaryloxy; or Het; said
R.sub.1 to R.sub.6 optionally attached to the isoquinoline group by
a C.sub.1-6 alkyl linking group; [0012] (b) R.sub.7 is NH.sub.2 or
--NR.sub.10R.sub.11; wherein R.sub.10 is C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C(O)--NR.sub.12R.sub.13, C(O)--OR.sub.14,
C(O)--SR.sub.15, or --C(O)--R.sub.16; R.sub.11 is H, C.sub.1-6
alkyl or C.sub.1-6 haloalkyl, provided that if either R.sub.12 or
R.sub.13 is H then R.sub.11 is H; [0013] R.sub.12 and R.sub.13 are
each independently H; C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl or
C.sub.4-10 alkylcycloalkyl, each optionally substituted with halo,
C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy, C.sub.1-3 alkyl or
C.sub.1-3 haloalkyl; or aryl; and wherein R.sub.12 and R.sub.13
together with the nitrogen to which they are bonded can form a 4-7
membered heterocycle; R.sub.14 and R.sub.15 are each independently
C.sub.1-6 alkyl, C.sub.3-7cycloalkyl or C.sub.4-10 alkylcycloalkyl,
each optionally substituted with halo, C.sub.1-3 alkoxy, C.sub.1-3
haloalkoxy, C.sub.1-3 alkyl or C.sub.1-3 haloalkyl; aryl or Het;
R.sub.16 is H; C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl or C.sub.4-10
alkylcycloalkyl, each optionally substituted with halo, C.sub.1-3
alkoxy, C.sub.1-3 haloalkoxy, C.sub.1-3 alkyl or C.sub.1-3
haloalkyl; aryl or Het; (c) R.sub.8 and R.sub.9 are each
independently H or C.sub.1-3 alkyl optionally substituted with
halo, or C.sub.1-3 alkoxy, or C.sub.1-3 haloalkoxy; (d) Q is a
C.sub.3-9 saturated or unsaturated chain optionally containing one
to three heteroatoms independently selected from O, S(O).sub.m;
wherein m is 0, 1 or 2, or NR.sub.17, wherein R.sub.17 is H;
C.sub.1-6 alkyl or C.sub.1-6 cycloalkyl, each optionally
substituted with halo, C.sub.1-6 alkoxy, cyano or C.sub.1-6
haloalkoxy; --C(O)--R.sub.18, C(O)--OR.sub.19,
C(O)--NR.sub.2OR.sub.21 or --SO.sub.2R.sub.22; R.sub.18, R.sub.20,
and R.sub.21 are each independently H; C.sub.1-6 alkyl or C.sub.1-6
cycloalkyl, each optionally substituted with halo, C.sub.1-6
alkoxy, cyano or C.sub.1-6 haloalkoxy; R.sub.19 is C.sub.1-6 alkyl
or C.sub.1-6 cycloalkyl, each optionally substituted with halo,
C.sub.1-6 alkoxy, cyano or C.sub.1-6 haloalkoxy; R.sub.22 is aryl,
C.sub.1-6 alkyl or C.sub.1-6 cycloalkyl, each optionally
substituted with halo, C.sub.1-6 alkoxy, cyano or C.sub.1-6
haloalkoxy; and (e) W is OH, --NH--SO.sub.n--R.sub.23, or
NH--SO.sub.n--R.sub.24; wherein n is 1 or 2, R.sub.23 is C.sub.1-8
alkyl, C.sub.4-10 alkylcycloalkyl, unsubstituted C.sub.3-7
cycloalkyl, or cyclopropyl or cyclobutyl optionally substituted
with C.sub.7-9 alkylaryl or C.sub.1-4 alkyl optionally substituted
with halo, C.sub.1-3 alkoxy, cyano, amine, mono or di-C.sub.1-6
alkylamine, mono or di-C.sub.1-6 alkylamide or carboxylate; and
R.sub.24 is C.sub.6-10 aryl or Het; or a pharmaceutically
acceptable enantiomer, diastereomer, salt, solvate or prodrug
thereof.
[0014] The present invention also provides compositions comprising
the compounds or pharmaceutically acceptable salts, solvates or
prodrugs thereof and a pharmaceutically acceptable carrier. In
particular, the present invention provides pharmaceutical
compositions useful for inhibiting HCV NS3 protease comprising a
therapeutically effective amount of a compound of the present
invention, or a pharmaceutically acceptable salt, solvate or
prodrug thereof, and a pharmaceutically acceptable carrier.
[0015] The present invention further provides methods for treating
patients infected with HCV, comprising administering to the patient
a therapeutically effective amount of a compound of the present
invention, or a pharmaceutically acceptable salt, solvate or
prodrug thereof. Additionally, the present invention provides
methods of inhibiting HCV NS3 protease by contacting the NS3
protease with a compound of the present invention.
[0016] By virtue of the present invention, it is now possible to
provide improved drugs comprising the compounds of the invention
which can be effective in the treatment of patients infected with
HCV. Specifically, the present invention provides peptide compounds
that can inhibit the functioning of the NS3 protease, e.g., in
combination with the NS4A protease. Further, the present invention
makes it possible to administer combination therapy to a patient
whereby a compound in accordance with the present invention, which
is effective to inhibit the HCV NS3 protease, can be administered
with another compound having anti-HCV activity, e.g., a compound
which is effective to inhibit the function of a target selected
from the group consisting of HCV metalloprotease, HCV serine
protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV
entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH and a
nucleoside analog for the treatment of an HCV infection.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Stereochemical definitions and conventions used herein
generally follow McGraw-Hill Dictionary of Chemical Terms, S. P.
Parker, Ed., McGraw-Hill Book Company, New York (1984) and
Stereochemistry of Organic Compounds, Eliel, E. and Wilen, S., John
Wiley & Sons, Inc., New York (1994). Many organic compounds
exist in optically active forms, i.e., they have the ability to
rotate the plane of plane-polarized light. In describing an
optically active compound, the prefixes D and L or R and S are used
to denote the absolute configuration of the molecule about its
chiral center(s). The prefixes d and l or (+) and (-) are employed
to designate the sign of rotation of plane-polarized light by the
compound, with (-) or l meaning that the compound is levorotatory
and (+) or d, meaning the compound, is dextrorotatory. For a given
chemical structure, these compounds, called stereoisomers, are
identical except that they are mirror images of one another. A
specific stereoisomer of a mirror image pair may also be referred
to as an enantiomer, and a mixture of such isomers is often called
an enantiomeric mixture. With reference to the instances where (R)
or (S) is used, it is to designate the absolute configuration of a
substituent in context to the whole compound and not in context to
the substituent alone.
[0018] Unless otherwise specifically noted herein, the terms set
forth below will have the following definitions.
[0019] The terms "racemic mixture" and "racemate" refer to an
equimolar mixture of two enantiomeric species, devoid of optical
activity.
[0020] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0021] The term "stereoisomers" refers to compounds which have
identical chemical composition, but differ with regard to the
arrangement of the atoms or groups in space.
[0022] The term "diastereomer" refers to a stereoisomer which is
not an enantiomer, e.g., a stereoisomer with two or more centers of
chirality and whose molecules are not mirror images of one another.
Diastereomers have different physical properties, e.g. melting
points, boiling points, spectral properties, and reactivities.
Mixtures of diastereomers may separate under high resolution
analytical procedures such as electrophoresis and
chromatography.
[0023] The term "enantiomers" refers to two stereoisomers of a
compound which are non-superimposable mirror images of one
another.
[0024] The term "therapeutically effective amount" means the total
amount of each active component that is sufficient to show a
meaningful patient benefit, e.g., a sustained reduction in viral
load. When applied to an individual active ingredient, administered
alone, the term refers to that ingredient alone. When applied to a
combination, the term refers to combined amounts of the active
ingredients that result in the therapeutic effect, whether
administered in combination, serially or simultaneously.
[0025] The term "compounds of the invention", and equivalent
expressions, are meant to embrace compounds of formula I, and
pharmaceutically acceptable enantiomer, diastereomer salts, and
solvates, e.g. hydrates and prodrugs. Similarly, references to
intermediates, are meant to embrace their salts, and solvates,
where the context so permits. References to the compound of the
invention also include the preferred compounds, e.g. formula II and
A-M.
[0026] The term "derivative" means a chemically modified compound
wherein the modification is considered routine by the ordinary
skilled chemist, such as an ester or an amide of an acid,
protecting groups, such as a benzyl group for an alcohol or thiol,
and tert-butoxycarbonyl group for an amine.
[0027] The term "solvate" means a physical association of a
compound of this invention with one or more solvent molecules,
whether organic or inorganic. This physical association includes
hydrogen bonding. In certain instances the solvate will be capable
of isolation, for example when one or more solvent molecules are
incorporated in the crystal lattice of the crystalline solid.
"Solvate" encompasses both solution-phase and isolable solvates.
Exemplary solvates include hydrates, ethanolates, methanolates,
isopropanolates and the like.
[0028] The term "prodrug" as used herein means derivatives of the
compounds of the invention which have chemically or metabolically
cleavable groups and become, by solvolysis or under physiological
conditions, the compounds of the invention which are
pharmaceutically active in vivo. A prodrug of a compound may be
formed in a conventional manner with a functional group of the
compounds such as with an amino, hydroxy or carboxy group when
present. The prodrug derivative form often offers advantages of
solubility, tissue compatibility, or delayed release in a mammalian
organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24,
Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well
known to practitioners of the art, such as, for example, esters
prepared by reaction of the parent acidic compound with a suitable
alcohol, or amides prepared by reaction of the parent acid compound
with a suitable amine.
[0029] The term "patient" includes both human and other
mammals.
[0030] The term "pharmaceutical composition" means a composition
comprising a compound of the invention in combination with at least
one additional pharmaceutical carrier, i.e., adjuvant, excipient or
vehicle, such as diluents, preserving agents, fillers, flow
regulating agents, disintegrating agents, wetting agents,
emulsifying agents, suspending agents, sweetening agents, flavoring
agents, perfuming agents, antibacterial agents, antifungal agents,
lubricating agents and dispensing agents, depending on the nature
of the mode of administration and dosage forms. Ingredients listed
in Remington's Pharmaceutical Sciences, 18.sup.th ed., Mack
Publishing Company, Easton, Pa. (1999) for example, may be
used.
[0031] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, e.g., the
compounds and their enantiomers, diastereomers, salts, solvates or
prodrugs, and/or dosage forms which are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of
patients for treatment without excessive toxicity, irritation,
allergic response, or other problem or complication commensurate
with a reasonable risk/benefit ratio.
[0032] The term "treating" refers to: (i) preventing a disease,
disorder or condition from occurring in a patient which may be
predisposed to the disease, disorder and/or condition but has not
yet been diagnosed as having it; (ii) inhibiting the disease,
disorder or condition, i.e., arresting its development; and (iii)
relieving the disease, disorder or condition, i.e., causing
regression of the disease, disorder and/or condition.
[0033] The term "substituted" as used herein includes substitution
at from one to the maximum number of possible binding sites on the
core, e.g., organic radical, to which the subsitutent is bonded,
e.g., mono-, di-, tri- or tetra-substituted, unless otherwise
specifically stated.
[0034] The nomenclature used to describe organic radicals, e.g.,
hydrocarbons and substituted hydrocarbons, generally follows
standard nomenclature known in the art, unless otherwise
specifically defined. Combinations of groups, e.g.,
alkylalkoxyamine or arylalkyl, include all possible stable
configurations, unless otherwise specifically stated. Thus, an
alkylaryl substituent can be bonded to a core segment by either the
alkyl portion or the aryl portion of the alkylaryl substituent,
provided the resulting compound is stable. Certain radicals and
combinations are defined below for purposes of illustration.
[0035] The term "halo" as used herein means a halogen substituent
selected from bromo, chloro, fluoro or iodo. The term "haloalkyl"
means an alkyl group that in substituted with one or more halo
substituents.
[0036] The term "alkyl" as used herein means acyclic, straight or
branched chain hydrocarbon substituents having the specified number
of carbon atoms and includes, for example, methyl, ethyl, propyl,
butyl, tert-butyl, hexyl, 1-methylethyl, 1-methylpropyl,
2-methypropyl, 1,1-dimethylethyl. Thus, C.sub.1-6 alkyl refers to
an alkyl group having from one to six carbon atoms. The term "lower
alkyl" means an alkyl group having from one to six, preferably from
one to four carbon atoms. The term "alkylester" means an alkyl
group additionally containing on ester group. Generally, a stated
carbon number range, e.g., C.sub.2-6 alkylester, includes all of
the carbon atoms in the radical.
[0037] The term "alkenyl" as used herein means an alkyl radical
containing at least F one double bond, e.g., ethenyl(vinyl) and
alkyl.
[0038] The term "alkoxy" as used herein means an alkyl group with
the indicated number of carbon atoms attached to an oxygen atom.
Alkoxy includes, for example, methoxy, ethoxy, propoxy,
1-methylethoxy, butoxy and 1,1-dimethylethoxy. The latter radical
is referred to in the art as tert-butoxy. The term "alkoxycarbonyl"
means an alkoxy group additionally containing a carbonyl group.
[0039] The term "cycloalkyl" as used herein means a cyclic
hydrocarbon substituent containing the indicated number of carbon
atoms and includes, for example, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl and spiro cyclic groups such
as spirocyclopropyl and spirocyclobutyl. The term "cycloalkoxy" as
used herein means a cycloalkyl group linked to an oxygen atom, such
as, for example, cyclobutyloxy or cyclopropyloxy. The term
"alkylcycloalkyl" means a cycloalkyl group linked to an alkyl
group. The stated carbon number range includes the total number of
carbons in the radical, unless otherwise specfically stated. Thus a
C.sub.4-10 alkylcycloalkyl may contain from 1-7 carbon atoms in the
alkyl group and from 3-9 carbon atoms in the ring, e.g.,
cyclopropylmethyl or cyclohexylethyl.
[0040] The term "aryl" as used herein means an aromatic moiety
containing the indicated number of carbon atoms, such as, but not
limited to phenyl, indanyl or naphthyl. For example, C.sub.6-10
aryl refers to an aromatic moiety having from six to ten carbon
atoms which may be in the form of a monocyclic or bicyclic
structure. The term "haloaryl" as used herein refers to an aryl
mono, di or tri substituted with one or more halogen atoms. The
terms "alkylaryl", "arylalkyl" and "aralkyl" mean an aryl group
substituted with one or more alkyl groups. Unless the carbon range
of each group is specified, the stated range applies to the entire
substituent. Thus, a C.sub.7-14 alkylaryl group many have from 1-8
carbon atoms in the alkyl group for a monocyclic aromatic and from
1-4 carbon atoms in the alkyl group for a fused aromatic. The
attachment of the group to bonding site on the molecule can be
either at the aryl group or the alkyl group. Unless a specific aryl
radical is specified, e.g., fluoro-phenyl, or the radical is stated
to be unsubstituted, the aryl radicals, e.g., in an aryl
substituent or an alkylaryl substituent, include those substituted
with typical substituents known to those skilled in the art, e.g.,
halogen, hydroxy, carboxy, carbonyl, nitro, sulfo, amino, cyano,
dialkylamino haloalkyl, CF.sub.3, haloalkoxy, thioalkyl, alkanoyl,
SH, alkylamino, alkylamide, dialkylamide, carboxyester,
alkylsulfone, alkylsulfonamide and alkyl(alkoxy)amine. Examples of
alkylaryl groups include benzyl, butylphenyl and
1-naphthylmethyl.
[0041] The term "alkanoyl" as used herein means straight or
branched 1-oxoalkyl radicals containing the indicated number of
carbon atoms and includes, for example, formyl, acetyl,
1-oxopropyl(propionyl), 2-methyl-1-oxopropyl, 1-oxohexyl and the
like.
[0042] The term "alkylamide" as used herein means an amide
mono-substituted with an alkyl, such as ##STR3##
[0043] The term "heterocycle", also referred to as "Het", as used
herein means 7-12 membered bicyclic heterocycles and 4-7 membered
monocyclic heterocycles.
[0044] Preferred bicyclic heterocycles are 7-12 membered fused
bicyclic ring systems (both rings share an adjacent pair of atoms)
containing from one to four heteroatoms selected from nitrogen,
oxygen and sulfur, wherein both rings of the heterocycle are fully
unsaturated. The nitrogen and sulfur heteroatoms atoms may be
optionally oxidized. The bicyclic heterocycle may contain the
heteroatoms in one or both rings. Unless a specific heterocycle is
specified, e.g., a fluorinated 7-12 membered bicyclic heterocycle,
or the heterocycle is stated to be unsubstituted, the heterocycles
include those substituted with typical substituents known to those
skilled in the art. For example, the bicyclic heterocycle may also
contain substituents on any of the ring carbon atoms, e.g., one to
three substituents. Examples of suitable substituents include
C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, C.sub.1-6 alkoxy, C.sub.3-7
cycloalkoxy, halo-C.sub.1-6 alkyl, CF.sub.3, mono- or
di-halo-C.sub.1-6 alkoxy, cyano, halo, thioalkyl, hydroxy,
alkanoyl, NO.sub.2, SH, amino, C.sub.1-6 alkylamino,
di(C.sub.1-6)alkylamino, di(C.sub.1-6)alkylamide, carboxyl,
(C.sub.1-6)carboxyester, C.sub.1-6 alkylsulfone, C.sub.1-6
alkylsulfonamide, C.sub.1-6 alkylsulfoxide,
di(C.sub.1-6)alkyl(alkoxy)amine, C.sub.6-10 aryl, C.sub.7-14
alkylaryl, and a 4-7 membered monocyclic heterocycle. When two
substituents are attached to vicinal carbon atoms of the bicyclic
heterocycle, they can join to form a ring, e.g., a five, six or
seven membered ring system containing up to two heteroatoms
selecting from oxygen and nitrogen. The bicyclic heterocycle may be
attached to the molecule, e.g. R.sub.1 in formula I, at any atom in
the ring and preferably carbon.
[0045] Examples of bicyclic heterocycles include, but are not
limited to, the following ring systems: ##STR4##
[0046] Preferred monocyclic heterocycles are 4-7 membered
saturated, partially saturated or fully unsaturated ring system
(this latter subset also herein referred to as unsaturated
heteroaromatic) containing in the ring from one to four heteroatoms
selected from nitrogen, oxygen and sulfur, wherein the sulfur and
nitrogen heteroatoms may be optionally oxidized. Unless a specific
heterocycle is specified, e.g., a C.sub.1-6 alkoxy substituted 4-7
membered monocyclic heterocycle, or the heterocycle is stated to be
unsubstituted, the heterocycles include those substituted with
typical substituents known to those skilled in the art. For
example, the monocyclic heterocycle may also contain substituents
on any of the ring atoms, e.g., one to three substituents. Examples
of suitable substituents include C.sub.1-6 alkyl, C.sub.3-7
cycloalkyl, C.sub.1-6 alkoxy, C.sub.3-7 cycloalkoxy, halo-C.sub.1-6
alkyl, CF.sub.3, mono- or di-halo-C.sub.1-6 alkoxy, cyano, halo,
thioalkyl, hydroxy, alkanoyl, NO.sub.2, SH, amino, C.sub.1-6
alkylamino, di(C.sub.1-6)alkylamino, di(C.sub.1-6)alkylamide,
carboxyl, (C.sub.1-6)carboxyester, C.sub.1-6 alkylsulfone,
C.sub.1-6 alkylsulfoxide, C.sub.1-6 alkylsulfonamide,
di(C.sub.1-6)alkyl(alkoxy)amine, C.sub.6-10 aryl, C.sub.7-14
alkylaryl and an additional 4-7 membered monocyclic heterocycle.
The monocyclic heterocycle may be attached to the molecule, e.g.
R.sub.1 in formula I, at any atom in the ring.
[0047] Examples of monocyclic heterocycles include, but are not
limited to, the following (and their tautomers): ##STR5##
[0048] Those skilled in the art will recognize that the
heterocycles used in the compounds of the present invention should
be stable. Generally, stable compounds are those which can be
synthesized, isolated and formulated using techniques known the
those skilled in the art without degradation of the compound.
[0049] The term "substituent" with reference to an amino acid or
amino acid derivative means a radical derived from the
corresponding .alpha.-amino acid. For instance, the substituents
methyl, iso-propyl, and phenyl represent the amino acids alanine,
valine, and phenyl glycine, respectively.
[0050] Where used in naming compounds of the present invention, the
designations "P1', P1, P2, P3 and P4", as used herein, map the
relative positions of the amino acid residues of a protease
inhibitor binding relative to the binding of the natural peptide
cleavage substrate. Cleavage occurs in the natural substrate
between P1 and P1' where the nonprime positions designate amino
acids starting from the C-terminus end of the peptide natural
cleavage site extending towards the N-terminus; whereas, the prime
positions emanate from the N-terminus end of the cleavage site
designation and extend towards the C-terminus. For example, P1'
refers to the first position away from the right hand end of the
C-terminus of the cleavage site (ie. N-terminus first position);
whereas P1 starts the numbering from the left hand side of the
C-terminus cleavage site, P2: second position from the C-terminus,
etc.)[see Berger A. & Schechter I., Transactions of the Royal
Society London series (1970), B257, 249-264].
[0051] In accordance with the present invention, there are provided
compounds of formula I: ##STR6## [0052] wherein: [0053] (a)
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are each
independently H; C.sub.1-6 alkyl; C.sub.3-7 cycloalkyl;
C.sub.1-6alkoxy; C.sub.3-7 cycloalkoxy; halo-C.sub.1-6 alkoxy;
halo-C.sub.1-6 alkyl; cyano; halo; hydroxyl; C.sub.1-6 alkanoyl;
nitro; amino; mono or di-(C.sub.1-6)alkyl amine; mono or
di-(C.sub.3-7)cycloalkyl amine; mono or di-C.sub.1-6 alkylamide;
mono or di-(C.sub.3-7)cycloalkyl amide; carboxyl;
(C.sub.1-6)carboxyester; thiol; C.sub.1-6 thioalkyl; C.sub.1-6
alkylsulfoxide; C.sub.1-6 alkylsulfone; C.sub.1-6 alkylsulfonamide;
C.sub.6-10 aryl optionally substituted with Het; C.sub.7-14
alkylaryl; C.sub.6-10 aryloxy; C.sub.7-14 alkylaryloxy; 4-7
membered monocyclic heteroaryloxy; or Het; said R.sub.1 to R.sub.6
optionally attached to the isoquinoline group by a C.sub.1-6 alkyl
linking group; [0054] (b) R.sub.7 is NH.sub.2 or
--NR.sub.10R.sub.11; wherein R.sub.10 is C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C(O)--NR.sub.12R.sub.13, C(O)--OR.sub.14,
C(O)--SR.sub.15, or --C(O)--R.sub.16; R.sub.11 is H, C.sub.1-6
alkyl or C.sub.1-6 haloalkyl, provided that if either R.sub.12 or
R.sub.13 is H then R.sub.11 is H; R.sub.12 and R.sub.13 are each
independently H; C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl or
C.sub.4-10 alkylcycloalkyl, each optionally substituted with halo,
C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy, C.sub.1-3 alkyl or
C.sub.1-3 haloalkyl; or aryl; and wherein R.sub.12 and R.sub.13
together with the nitrogen to which they are bonded can form a 4-7
membered heterocycle; R.sub.14 and R.sub.15 are each independently
C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl or C.sub.4-10
alkylcycloalkyl, each optionally substituted with halo, C.sub.1-3
alkoxy, C.sub.1-3 haloalkoxy, C.sub.1-3 alkyl or C.sub.1-3
haloalkyl; aryl or Het; R.sub.16 is H; C.sub.1-6 alkyl, C.sub.3-7
cycloalkyl or C.sub.4-10 alkylcycloalkyl, each optionally
substituted with halo, C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy,
C.sub.1-3 alkyl or C.sub.1-3 haloalkyl; aryl or Het; [0055] (c)
R.sub.8 and R.sub.9 are each independently H or C.sub.1-3 alkyl
optionally substituted with halo, or C.sub.1-3 alkoxy, or C.sub.1-3
haloalkoxy; [0056] (d) Q is a C.sub.3-9 saturated or unsaturated
chain optionally containing one to three heteroatoms independently
selected from O, S(O).sub.m; wherein m is 0, 1 or 2, or NR.sub.17,
wherein R.sub.17 is H; C.sub.1-6 alkyl or C.sub.1-6 cycloalkyl,
each optionally substituted with halo, C.sub.1-6 alkoxy, cyano or
C.sub.1-6 haloalkoxy; --C(O)--R.sub.18, C(O)--OR.sub.19,
C(O)--NR.sub.2OR.sub.21 or --SO.sub.2R.sub.22; R.sub.18, R.sub.20,
and R.sub.21 are each independently H; C.sub.1-6 alkyl or C.sub.1-6
cycloalkyl, each optionally substituted with halo, C.sub.1-6
alkoxy, cyano or C.sub.1-6 haloalkoxy; R.sub.19 is C.sub.1-6 alkyl
or C.sub.1-6 cycloalkyl, each optionally substituted with halo,
C.sub.1-6 alkoxy, cyano or C.sub.1-6 haloalkoxy; R.sub.22 is aryl,
C.sub.1-6 alkyl or C.sub.1-6 cycloalkyl, each optionally
substituted with halo, C.sub.1-6 alkoxy, cyano or C.sub.1-6
haloalkoxy; and [0057] (e) W is OH, --NH--SO, --R.sub.23, or
NH--SO.sub.n--R.sub.24; wherein n is 1 or 2, R.sub.23 is C.sub.1-8
alkyl, C.sub.4-10 alkylcycloalkyl, unsubstituted C.sub.3-7
cycloalkyl, or cyclopropyl or cyclobutyl optionally substituted
with C.sub.7-9 alkylaryl or C.sub.1-4 alkyl optionally substituted
with halo, C.sub.1-3 alkoxy, cyano, amine, mono or di-C.sub.1-6
alkylamine, mono or di-C.sub.1-6 alkylamide or carboxylate; and
R.sub.24 is C.sub.6-10 aryl or Het; or a pharmaceutically
acceptable enantiomer, diastereomer, salt, solvate or prodrug
thereof.
[0058] Preferably, R.sub.1 is bonded to the C.sub.3 position and is
selected from H; C.sub.1-6 alkyl; C.sub.3-7 cycloalkyl; C.sub.1-6
alkoxy; C.sub.3-7 cycloalkoxy; halo-C.sub.1-6 alkoxy;
halo-C.sub.1-6 alkyl; cyano; halo; C.sub.1-6 alkanoyl; mono or
di-(C.sub.1-6)alkyl amine; mono or di-C.sub.1-6 alkylamide;
carboxyl; C.sub.6-10 aryl optionally substituted with Het;
C.sub.7-14 alkylaryl; C.sub.6-10 aryloxy or Het. Preferably,
R.sub.2, R.sub.3, and R.sub.4 are bonded to the C.sub.4, C.sub.5
and C.sub.6 positions, respectively, and are each independently
selected from H; C.sub.1-6 alkyl; C.sub.3-7 cycloalkyl; C.sub.1-6
alkoxy; C.sub.3-7 cycloalkoxy; halo-C.sub.1-6 alkoxy;
halo-C.sub.1-6 alkyl; cyano; halo; hydroxyl; C.sub.1-6 alkanoyl;
mono or di-(C.sub.1-6)alkyl amine; mono or di-(C.sub.3-7)cycloalkyl
amine; mono or di-C.sub.1-6 alkylamide; mono or
di-(C.sub.3-7)cycloalkyl amide; carboxyl; C.sub.6-10 aryl
optionally substituted with Het; C.sub.7-14 alkylaryl; C.sub.6-10
aryloxy; or Het. Preferably, R.sub.5 and R.sub.6 are bonded to the
C.sub.7 and C.sub.8 positions, respectively, and are each
independently selected from H; C.sub.1-3 alkyl; C.sub.3-4
cycloalkyl; C.sub.1-3 alkoxy; C.sub.3-4 cycloalkoxy; halo-C.sub.1-3
alkoxy; halo-C.sub.1-3 alkyl; cyano; halo; hydroxyl; C.sub.1-3
alkanoyl; mono or di-(C.sub.1-3)alkyl amine; mono or
di-(C.sub.3-4)cycloalkyl amine; mono or di-C.sub.1-3 alkylamide;
mono or di-(C.sub.3-4)cycloalkyl amide; or carboxyl. If desired,
one or more of the R.sub.1 R.sub.2, R.sub.3, R.sub.4, R.sub.5 and
R.sub.6 substituents can be attached to the isoquinoline group by a
C.sub.1-6 alkyl linking group, e.g., a methylene group.
[0059] Preferably, Q is a C.sub.3-9 saturated or unsaturated chain
optionally containing one to three heteroatoms independently
selected from O, S(O).sub.m; wherein m is 0, 1 or 2, or NR.sub.17,
wherein R.sub.17 is H; C.sub.1-6 alkyl, C.sub.1-6 cycloalkyl,
--C(O)--R.sub.18, C(O)--OR.sub.19, C(O)--NR.sub.20R.sub.21 or
--SO.sub.2R.sub.22. Preferably, R.sub.18, R.sub.20, and R.sub.21
are each independently H; C.sub.1-6 alkyl or C.sub.1-6 cycloalkyl;
R.sub.19 is C.sub.1-6 alkyl or C.sub.1-6 cycloalkyl; and R.sub.22
is aryl, C.sub.1-6 alkyl or C.sub.1-6 cycloalkyl, each optionally
substituted with halo.
[0060] Preferably, W is OH, --NH--SO.sub.n--R.sub.23, or
NH--SO.sub.n--R.sub.24 wherein n is 1 or 2, R.sub.23 is
unsubstituted C.sub.3-7 cycloalkyl, cyclopropyl, or cyclobutyl
optionally substituted with C.sub.7-9 alkylaryl or C.sub.1-4 alkyl;
and R.sub.24 is C.sub.6-10 aryl or Het.
[0061] In accordance with one aspect of the present invention,
there are provided compounds of formula II: ##STR7## [0062]
wherein: [0063] (a) R.sub.1 is H; C.sub.1-6 alkyl; C.sub.3-7
cycloalkyl; C.sub.1-6 alkoxy; C.sub.3-7 cycloalkoxy; halo-C.sub.1-6
alkoxy; halo-C.sub.1-6 alkyl; cyano; halo; C.sub.1-6 alkanoyl; mono
or di-(C.sub.1-6)alkyl amine; mono or di-C.sub.1-6 alkylamide;
carboxyl; C.sub.6-10 aryl optionally substituted with Het;
C.sub.7-14 alkylaryl; C.sub.6-10 aryloxy or Het; said R.sub.1
optionally attached to the isoquinoline group by a C.sub.1-6 alkyl
linking group; R.sub.2, R.sub.3, and R.sub.4 are each independently
H; C.sub.1-6 alkyl; C.sub.3-7 cycloalkyl; C.sub.1-6 alkoxy;
C.sub.3-7 cycloalkoxy; halo-C.sub.1-6 alkoxy; halo-C.sub.1-6 alkyl;
cyano; halo; hydroxyl; C.sub.1-6 alkanoyl; mono or
di-(C.sub.1-6)alkyl amine; mono or di-(C.sub.3-7)cycloalkyl amine;
mono or di-C.sub.1-6alkylamide; mono or di-(C.sub.3-7)cycloalkyl
amide; carboxyl; C.sub.6-10 aryl optionally substituted with Het;
C.sub.7-14 alkylaryl; C.sub.6-10 aryloxy; or Het; said R.sub.2 to
R.sub.4 optionally attached to the isoquinoline group by a
C.sub.1-3 alkyl linking group; R.sub.5 and R.sub.6 are each
independently H; C.sub.1-3 alkyl; C.sub.3-4 cycloalkyl; C.sub.1-3
alkoxy; C.sub.3-4 cycloalkoxy; halo-C.sub.1-3 alkoxy;
halo-C.sub.1-3 alkyl; cyano; halo; hydroxyl; C.sub.1-3 alkanoyl;
mono or di-(C.sub.1-3)alkyl amine; mono or di-(C.sub.3-4)
cycloalkyl amine; mono or di-C.sub.1-3 alkylamide; mono or
di-(C.sub.3-4)cycloalkyl amide; or carboxyl; [0064] (b) R.sub.7 is
NH.sub.2 or --NR.sub.10R.sub.11; wherein R.sub.10 is C.sub.1-6
alkyl, C.sub.1-6 haloalkyl, C(O)--NR.sub.12R.sub.13,
C(O)--OR.sub.14, or --C(O)--R.sub.16; R.sub.11 is H, C.sub.1-6
alkyl or C.sub.1-6 haloalkyl, provided that if either R.sub.12 or
R.sub.13 is H then R.sub.11 is H; [0065] R.sub.12 and R.sub.13 are
each independently H; C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl or
C.sub.4-10 alkylcycloalkyl, each optionally substituted with halo,
C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy, C.sub.1-3 alkyl or
C.sub.1-3 haloalkyl; and wherein R.sub.12 and R.sub.13 together
with the nitrogen to which they are bonded can form a 4-7 membered
heterocycle; R.sub.14 and R.sub.15 are each independently C.sub.1-6
alkyl, C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, each
optionally substituted with halo, C.sub.1-3 alkoxy, C.sub.1-3
haloalkoxy, C.sub.1-3 alkyl or C.sub.1-3 haloalkyl; R.sub.16 is H;
C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl or C.sub.4-10
alkylcycloalkyl, each optionally substituted with halo, C.sub.1-3
alkoxy, C.sub.1-3 haloalkoxy, C.sub.1-3 alkyl or C.sub.1-3
haloalkyl; aryl or Het; [0066] (c) R.sub.8 and R.sub.9 are each
independently H or C.sub.1-3 alkyl optionally substituted with
halo, or C.sub.1-3 alkoxy, or C.sub.1-3 haloalkoxy; [0067] (d) Q is
a C.sub.3-9 saturated or unsaturated chain optionally containing
one to three heteroatoms independently selected from O, S(O).sub.m;
wherein m is 0, 1 or 2, or NR.sub.17, wherein R.sub.17 is H;
C.sub.1-6 alkyl, C.sub.1-6 cycloalkyl, --C(O)--R.sub.18,
C(O)--OR.sub.19, C(O)--NR.sub.2OR.sub.21 or --SO.sub.2R.sub.22;
R.sub.18, R.sub.20, and R.sub.21 are each independently H;
C.sub.1-6 alkyl or C.sub.1-6 cycloalkyl; R.sub.19 is C.sub.1-6
alkyl or C.sub.1-6 cycloalkyl; R.sub.22 is aryl, C.sub.1-6 alkyl or
C.sub.1-6 cycloalkyl, each optionally substituted with halo; and
[0068] (e) W is OH, --NH--SO.sub.n--R.sub.23 or
NH--SO.sub.n--R.sub.24, wherein n is 1 or 2, R.sub.23 is
unsubstituted C.sub.3-7 cycloalkyl, or cyclopropyl or cyclobutyl
optionally substituted with C.sub.7-9 alkylaryl or C.sub.1-4 alkyl;
and R.sub.24 is C.sub.6-10 aryl or Het; or a pharmaceutically
acceptable enantiomer, diastereomer, salt, solvate or prodrug
thereof.
[0069] Preferably, R.sub.1 is H; C.sub.1-3 alkoxy; mono or
di-(C.sub.1-6)alkyl amine; a 5 or 6 membered monocyclic
heterocycle; or C.sub.6-10 aryl optionally substituted with a 5 or
6 membered monocyclic heterocycle. Preferably, R.sub.2, R.sub.3,
R.sub.4 and R.sub.5 are each independently H; C.sub.1-6 alkoxy;
halo-C.sub.1-6 alkoxy; hydroxyl; or mono or di-(C.sub.1-6)alkyl
amine.
[0070] Preferably, R.sub.7 is NH.sub.2 or --NHR.sub.10; wherein
R.sub.10 is C(O)--NR.sub.12R.sub.13, or C(O)--OR.sub.14; and
R.sub.12 and R.sub.13 are C.sub.1-6 alkyl optionally substituted
with halo; and R.sub.14 is C.sub.1-6 alkyl or C.sub.3-7 cycloalkyl
optionally substituted with halo.
[0071] Preferably, Q is a C.sub.5-7 membered chain having one
double bond optionally containing one heteroatom independently
selected from O, S(O).sub.m; wherein m is 0, 1 or 2, or NR.sub.17,
wherein R.sub.17 is H; C.sub.1-6 alkyl or C.sub.1-6 cycloalkyl. In
a preferred aspect of the invention, Q has the following structure:
##STR8## wherein P is a C.sub.3 saturated chain optionally
containing one heteroatom independently selected from O,
S(O).sub.m; wherein m is 0, 1 or 2, or NR.sub.17 (as defined in the
compound of Formula II).
[0072] In one aspect of the invention, W is preferably
--NH--SO.sub.n--R.sub.23, wherein n is 1 or 2 and R.sub.23 is
unsubstituted C.sub.3-7 cycloalkyl, or cyclopropyl or cyclobutyl
optionally substituted with C.sub.7-9 alkylaryl or C.sub.1-4 alkyl.
In another aspect of the invention, W is NH--SO.sub.n--R.sub.24,
wherein n is 1 or 2 and R.sub.24 is Het.
[0073] Preferably, when R.sub.24 is Het, the Het is selected from
the group consisting of: ##STR9##
[0074] In accordance with another aspect of the present invention,
there are provided compounds of formula III: ##STR10## [0075]
wherein: [0076] (a) R.sub.1 is H; C.sub.1-3 alkoxy;
di-(C.sub.1-6)alkyl amine; a 5 or 6 membered monocyclic
heterocycle; or C.sub.6-10 aryl optionally substituted with a 5 or
6 membered monocyclic heterocycle; [0077] R.sub.2, R.sub.3, R.sub.4
and R.sub.5 are each independently H; C.sub.1-3 alkoxy; halo; or
di-(C.sub.1-6)alkyl amine; [0078] (b) R.sub.7 is --NHR.sub.10;
wherein R.sub.10 is C(O)--NHR.sub.13, or C(O)--OR.sub.14; R.sub.13
and R.sub.14 are C.sub.1-6 alkyl; [0079] (c) Q is a C.sub.5-7
membered chain having one double bond optionally containing one
heteroatom independently selected from O, S(O).sub.m; wherein m is
0, 1 or 2, or NR.sub.17, wherein R.sub.17 is H; C.sub.1-6 alkyl or
C.sub.1-6 cycloalkyl; and [0080] (d) R.sub.23 is unsubstituted
C.sub.3-7 cycloalkyl, or cyclopropyl or cyclobutyl optionally
substituted with C.sub.7-9 alkylaryl or C.sub.1-4 alkyl; or a
pharmaceutically acceptable enantiomer, diastereomer, salt, solvate
or prodrug thereof.
[0081] Preferably, R.sub.1 is selected from the group consisting of
pyridine, pyrrolidine, morpholine, piperazine, oxazole, isoxazole,
thiazole, imidazole, pyrrole and pyrazole. In one preferred aspect,
R.sub.1 is phenyl optionally substituted with one or more members
selected from the group consisting of selected from the group
consisting of C.sub.1-3 alkoxy, halo, carboxyl, di-(C.sub.1-3)alkyl
amine, C.sub.1-3 haloalkyl, trifluoromethyl, trifluoromethoxy and
hydroxy. In another preferred aspect, R.sub.1 is
di-(C.sub.1-3)alkyl amine. In another preferred aspect, R.sub.1 is
piperazine substituted with one or more members selected from the
group consisting of C.sub.1-3 alkyl, C.sub.5-7 cycloalkyl or
pyridine. Preferably, R.sub.2 is chloro or fluoro. In one preferred
aspect, R.sub.2 is di-(C.sub.1-3)alkyl amine or methoxy.
[0082] In one preferred aspect, Q has a structure selected from the
following: ##STR11##
[0083] In another preferred aspect, Q has a structure selected from
the following: ##STR12##
[0084] Examples of preferred compounds in accordance with the
invention are selected from the group consisting of ##STR13##
##STR14## ##STR15## ##STR16## ##STR17## ##STR18## ##STR19##
##STR20## ##STR21## ##STR22## ##STR23## ##STR24## ##STR25##
##STR26## ##STR27## ##STR28## ##STR29## ##STR30##
[0085] The compounds of the present invention, which contain a
basic moiety, can form salts by the addition of a pharmaceutically
acceptable acid. The acid addition salts are formed from a compound
of Formula I and a pharmaceutically acceptable inorganic acid,
including but not limited to hydrochloric, hydrobromic, hydroiodic,
sulfuric, phosphoric, or organic acid such as p-toluenesulfonic,
methanesulfonic, acetic, benzoic, citric, malonic, fumaric, maleic,
oxalic, succinic, sulfamic, or tartaric. Thus, examples of such
pharmaceutically acceptable salts include chloride, bromide,
iodide, sulfate, phosphate, methanesulfonate, citrate, acetate,
malonate, fumarate, sulfamate, and tartrate.
[0086] Salts of an amine group may also comprise quaternary
ammonium salts in which the amino nitrogen carries a suitable
organic group such as an alkyl, alkenyl, alkynyl or aralkyl
moiety.
[0087] Compounds of the present invention, which are substituted
with an acidic group, may exist as salts formed through base
addition. Such base addition salts include those derived from
inorganic bases which include, for example, alkali metal salts
(e.g. sodium and potassium), alkaline earth metal salts (e.g.
calcium and magnesium), aluminum salts and ammonium salts. In
addition, suitable base addition salts include salts of
physiologically acceptable organic bases such as trimethylamine,
triethylamine, morpholine, pyridine, piperidine, picoline,
dicyclohexylamine, N,N'-dibenzylethylenediamine,
2-hydroxyethylamine, bis-(2-hydroxyethyl)amine,
tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine,
N-benzyl-.beta.-phenethylamine, dehydroabietylamine,
N,N'-bishydroabietylamine, glucamine, N-methylglucamine, collidine,
quinine, quinoline, ethylenediamine, ornithine, choline,
N,N'-benzylphenethylamine, chloroprocaine, diethanolamine,
diethylamine, piperazine, tris(hydroxymethyl)aminomethane and
tetramethylammonium hydroxide and basic amino acids such as lysine,
arginine and N-methylglutamine. These salts may be prepared by
methods known to those skilled in the art.
[0088] Lists of suitable salts are found, for example, in
Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing
Company, Easton, Pa., 1990, p. 1445.
[0089] Certain compounds of the present invention, and their salts,
may also exist in the form of solvates with water, for example
hydrates, or with organic solvents such as methanol, ethanol or
acetonitrile to form, respectively, a methanolate, ethanolate or
acetonitrilate. The present invention includes each solvate and
mixtures thereof.
[0090] In addition, compounds of the present invention, or a salt
or solvate thereof, may exhibit polymorphism. The present invention
also encompasses any such polymorphic form.
[0091] Compounds of the present invention (Formula I, II or III)
also contain two or more chiral centers and exist in different
optically active forms. For example, compounds of Formula I may
include a cyclopropyl group as represented in the P1 fragment
below: ##STR31## wherein C.sub.1 and C.sub.2 each represent an
asymmetric carbon atom at positions 1 and 2 of the cyclopropyl
ring. Notwithstanding other possible asymmetric centers at other
segments of the compounds of the invention, the presence of these
two asymetric centers means that the compounds can exist as
mixtures of diastereomers, such as the diastereomers of compounds
of Formula III wherein Q is configured either syn to the amide or
syn to the carbonyl as shown below. ##STR32##
[0092] The present invention includes both enantiomers and mixtures
of enantiomers of the compunds, such as racemic mixtures.
[0093] The enantiomers may be resolved by methods known to those
skilled in the art, for example, by formation of diastereoisomeric
salts which may be separated by crystallization, gas-liquid or
liquid chromatography, selective reaction of one enantiomer with an
enantiomer-specific reagent. It will be appreciated that where the
desired enantiomer is converted into another chemical entity by a
separation technique, then an additional step is required to form
the desired enantiomeric form. Alternatively, specific enantiomers
may be synthesized by asymmetric synthesis using optically active
reagents, substrates, catalysts or solvents, or by converting one
enantiomer into the other by asymmetric transformation.
[0094] Certain compounds of the present invention may also exist in
different stable conformational forms which may be separable.
Torsional asymmetry due to restricted rotation about an asymmetric
single bond, for example because of steric hindrance or ring
strain, may permit separation of different conformers. The present
invention includes each conformational isomer of these compounds
and mixtures thereof.
[0095] Certain compounds of the present invention may exist in
zwitterionic form and the present invention includes each
zwitterionic form of these compounds and mixtures thereof.
[0096] The starting materials useful to synthesize the compounds of
the present invention are known to those skilled in the art and can
be readily manufactured or are commercially available.
[0097] The compounds of the present invention can be manufactured
by methods known to those skilled in the art. The following methods
set forth below are provided for illustrative purposes and are not
intended to limit the scope of the claimed invention. For example,
compounds of the present invention having the structure of Formula
I, II or III can be synthesized, as shown in the following scheme,
from compounds of Formula IV, Formula VIIA or VIIB. It will be
recognized that it may be preferred or necessary to prepare such a
compound in which a functional group is protected using a
conventional protecting group then to remove the protecting group
to provide a compound of the present invention. The details
concerning the use of protecting groups in accordance with the
present invention are known to those skilled in the art. ##STR33##
##STR34##
[0098] The .alpha.-carboxylic acid of a compound of Formula VIIA or
VIIB is coupled with R.sub.23SO.sub.2NH.sub.2 (R.sub.23 as defied
above) in the presence of peptide coupling agent, such as CDI or
EDAC, and in the presence of a base, such as
4-dimethylaminopyridine (4-DMAP) and/or
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), to form a compound of
Formula I, II or III. In the construction of compounds of Formula
I, the P1' terminus is incorporated into the molecules using one of
the general processes outlined above and described in more detail
below. In some examples the P1' elements, that is the
cycloalkylsulfonamides, alkyl sulfonamides or
heteroarylsulfonamides are commercially available or can be
prepared from the corresponding alkyl- or cycloalkyl-sulfonyl
chloride by treating said sulfonyl chloride with ammonia.
Alternatively, these sulfonamides can be synthesized using the
general process outline in the following Scheme. Therein
commercially available 3-chloro-propylsulfonyl chloride (1) is
converted to a suitable protected sulfonamide as for example by
treatment with tert-butyl amine. The sulfonamide obtained (2) is
then converted to the corresponding cycloalkylsulfonamide by
treatment with two equivalents of a base such as butyl lithium in a
solvent such as THF at low temperature. The resulting
cycloalkylsulfonamide can be deprotected by treatment with an acid
to provide the desired unprotected cycloalkylsulfoamide. Said P1'
fragment can be incorporated into compounds of Formula I.
##STR35##
[0099] Compounds having the structure of Formula IV, VI, VIIA or
VIIB can be prepared, for example, as described herein, and in
International Application Number PCT/US01/45145, Publication No. WO
02/060926, published Aug. 8, 2002; International Application Number
PCT/CA00/00353, Publication No. WO 00/59929, published Oct. 12,
2000; and U.S. Pat. No. 6,323,180 granted Nov. 27, 2001.
[0100] Compounds having the structure of Formula IV can also be
prepared by coupling chemical precursor A with chemical precursor B
as shown below. ##STR36##
[0101] Chemical precursor B, which is also shown above, may for
example, be synthesized as follows. ##STR37##
[0102] Treatment of commercially available or readily synthesized
imine (B1) with 1,4-dihalobutene (B2) in presence of a base
provides the imine (B3). Acid hydrolysis of B3 then provides B,
which has a vinyl substituent syn to the carboxyl group. It is
preferred that for compounds B3 and B that the vinyl group is syn
to the ester. The amine moiety of B can protected using a Boc group
to provide the fully protected amino acid B4. This intermediate is
a racemate which can be resolved by an enzymatic process wherein
the ester moiety of B4 is cleaved to provide the corresponding
carboxylic acid. Without being bound to any particular theory, it
is believed that this reaction is selective in that one of the
enantiomers B4 (1S, 2R) undergoes the reaction at a much greater
rate than its mirror image B4 (1R, 2S) providing for a kinetic
resolution of the intermediate racemate. In the example cited
above, the more preferred stereoisomer for integration into the
tripeptdes is B4(1R, 2S). In the presence of the enzyme, this
enantiomer does not undergo ester cleavage and thereby this
enantiomer B5 is recovered from the reaction mixture. However, the
less preferred enantiomer B4(1S, 2R) undergoes ester cleavage,
i.e., enzyme catalyzed hydrolysis, to provide the free acid B6. The
ester (B5) can be separated from the acid product (B6) by rountine
methods such as, for example, aqueous extraction methods or
chromatography. Compounds of Formula 1 can also be converted into
other compounds of Formula I as described herein. An example of
such a process is the Scheme below, wherein a compound of Formula I
(1) which bears a Boc group at the P4 position is converted into a
compound of Formula I (3) wherein said compound bears a urea group
at the P4 position. The conversion of (1) to (3) can be carried out
in a two step process the first of which is the conversion of (1)
to amine (2) by treatment of (1) with an acid such as TFA in a
solvent such as methylene chloride. The resulting amine TFA salt
can be treated with an isocyanate in the presence of one equivalent
of base to provide a compound of Formula I (3) wherein the P3
moiety is capped with a urea. As previously noted one skilled in
the art will recognize that intermediate (2) can be used as
starting materials for the preparation of compounds of Formula I
wherein the P3 group is capped with an amide or a carbamate. The
construction of said compounds of Formula I can be achieved using
standard conditions for the formation of said P4 functionalities
from amines. ##STR38## Procedures for making P2 intermediates and
compounds of Formula I are shown in the Schemes below. It should be
noted that in many cases reactions are depicted for only one
position of an intermediate. However, it is to be understood that
such reactions could be used to impart modifications to other
positions within this intermediate. Moreover, said intermediates,
reaction conditions and methods given in the specific examples are
broadly applicable to compounds with other substitution patterns.
The general Schemes outlined below are followed with examples
herein. Both general and specific examples are non-limiting.
##STR39## The above Scheme shows the coupling of an N-protected
C4-hydroxyproline moiety with an isoquinoline heterocycle to form
intermediate (4) and the subsequent modification of said
intermediate (4) to a compound of Formula I by the process of
peptide elongation and a ring-closing olefin metathesis reaction as
described herein. It should be noted that in the first step, that
is the coupling of the C4-hydroxy proline group with the
isoquinoline element, a base is employed. One skilled in the art
would recognize that this coupling can be done using bases such as
potassium tert-butoxide, or sodium hydride, in solvent such as DMF
or DMSO or THF. This coupling to the isoquinoline ring system
occurs at the C1 position (numbering for isoquinoline ring system
shown in intermediate 2) and is directed by the chloro group which
is displaced in this process. It should be noted that alternative
leaving groups can be utilized at this position such as a fluoro as
shown in the Scheme. Said fluoro intermediates (3) are available
from the corresponding chloro compound using literature procedures
described herein. In a subset of examples herein, isoquinolines are
incorporated into the final compounds and specifically into the P2
region of said compounds. One skilled in the art would recognize
that a number of general methods are available for the synthesis of
isoquinolines. Moreoever, said isoquinolines generated by these
methods can be readily incorporated into final compounds of Formula
I using the processes described herein. One general methodology for
the synthesis of isoquinolines is shown in the scheme below,
wherein cinnamic acid derivatives, shown in general form as
structure (2) are ##STR40## converted to 1-chloroisoquinolines in a
four step process. Said chloroisoquinolines can be subsequently
used in coupling reactions to C4-hydroxyproline derivatives as
described herein. The conversion of cinnamic acids to
chloroquinolines begins with the treatment of cinnamic acid with an
alkylcholorformate in the presence of a base. The resulting
anhydride is then treated with sodium azide which results in the
formation of an acylazide (3) as shown in the Scheme. Alternate
methods are available for the formation of acylazides from
carboxylic acids as for example said carboxylic acid can be treated
with diphenylphosphorylazide (DPPA) in an aprotic solvent such as
methylene chloride in the presence of a base. In the next step of
the reaction sequence the acylazide (3) is coverted to the
corresponding isoquinolone (4) as shown in the Scheme. In the event
the acylazide is heated to a temperature of approximately 190
degress celcius in a high boiling solvent such a diphenylmethane.
This reaction is general and provides moderate to good yields of
substituted isoquinolone from the corresponding cinnamic acid
derivatives. It should be noted that said cinnamic acid derivatives
are available commercially or can be obtained from the
corresponding benzaldehyde (1) derivative by direct condensation
with malonic acid or derivatives thereof and also by employing a
Wittig reaction. The intermediate isoquinolones (4) can be
converted to the corresponding 1-chloroisoquinoline by treatment
with phosphorous oxychloride. This reaction is general and can be
applied to any of the isoquinolones shown herein to covert a
hydroxy substituent to the corresponding chloro compound. An
alternative method for the synthesis of the isoquinoline ring
system is the Pomeranz-Fritsh procedure. This general method is
outlined below. The process begins with the conversion of a
benzaldehyde derivative (1) to a functionalized imine (2). Said
imine is then converted to the isoquinoline ring system by
treatment with acid at elevated ##STR41## temperature. This
isoquinoline synthesis as outlined above is general, and it should
be noted that this process is particularly useful in procuring
isoquinoline intermediates that are substituted at the C8 position
(note: in intermediate (3) of the above Scheme the C8 position of
the isoquinoline ring is substituted with substutuent R.sub.6). The
intermediate isoquinolines (3) can be converted to the
corresponding 1-chloroquinolines (5) in a two step process as
shown. The first step in this sequence is the formation of the
isoquinoline N-oxide (4) by treatment of isoquinoline (3) with
meta-chloroperbenzoic acid in an aprotic solvent such as
dichloromethane. Intermediate (4) can be converted to the
corresponding 1-chloroisoquinoline by treatment with phosphorous
oxychloride in refluxing chloroform. Another method for the
synthesis of the isoquinoline ring system is shown below. In this
process an ortho-alkylbenzamide derivative (1) is treated with a
strong ##STR42## base such as tert-butyl lithium in a solvent such
as THF at low temperature. To this reaction mixture is then added a
nitrile derivative, which undergoes an addition reaction with the
anion derived from deprotonation of (1), resulting in the formation
of (2). This reaction is general and can be used for the formation
of substituted isoquinolines. Intermediate (2) of the above scheme
can be converted to the corresponding 1-chloroisoquinoline by the
methods described herein. An additional method for the synthesis of
isoquinolines is shown below. The deprotonation of intermediate (1)
using tert-butyl lithium is described above. In the present method
however, said intermediate anion is trapped by an ester, resulting
in the formation of intermediate (2) as shown below. In a
subsequent reaction ketone (2) is condensed with ammonium acetate
at elevated temperature providing for the formation of quinolone
(3). This reaction is general and can be applied for the
construction of substituted isoquinolones which can then be
converted to the corresponding 1-chloroisoquinolines as described
herein. ##STR43## Yet an additional method for the construction of
isoquinolines is found in the scheme below. In the first step of
this process an ortho-alkylarylimine derivative such as (1) is
subjected to deprotonation conditions (sec-butyl lithium, THF) and
the resulting anion is quenched by ##STR44## the addition of an
activated carboxylic acid derivative such as a Weinreb amide. The
resulting keto imine (2) can be converted to the corresponding
isoquinoline by condensation with ammonium acetate at elevated
temperatures. This method is general and can be used for the
synthesis of substituted isoquinolines. Said isoquinolines can be
converted to the corresponding 1-chloroisoquinoline by the methods
described herein. The construction of functionalized isoquinoline
ring systems is also possible employing [4+2] cycloaddition
reactions. For example as shown below the use of vinyl isocyantes
(1) in cycloaddition reactions with benzyne precusors (2) provides
functionalized isoquinolones (3). Said isoquinolines can be
incorporated into compounds of Formula I using the methods
described herein. ##STR45## The isoquinolines described herein, and
which are incorporated into the compounds of Formula I can be
further functionalized. It is obvious to one skilled in the art
that additional functionalization of said heterocycles can be done
either before or after incorporation of these functionalities into
compounds of Formula I. The following schemes illustrate this
point. For example the scheme below shows the conversion of a
1-chloro- ##STR46## 6-fluoro-isoquinoline to the corresponding
1-chloro-6-alkoxy-isoquinoline species, by treatment of (1) of (eq.
1) with a sodium or potassium alkoxide species in the alcohol
solvent from which the alkoxide is derived at room temperature. In
some cases it may be necessary to heat the reaction to drive it to
completion. Intermediate (2) of equation 1 can be incorporated into
compounds of Formula I by the teachings herein. As shown in
equation 2 the analogous reaction can also be conducted on a
substrate wherein the 6-fluoro-isoquinoline element has been
incorporated into the macrocycle. Therein treatment of intermediate
2 of equation 2 with a sodium or potassium alkoxide species in the
alcohol solvent from which the alkoxide is derived can provide
compounds of Formula 1. The scheme below provides a general example
for the modification of isoquinolines as defined herein by
employing palladium mediated coupling reactions. Said couplings can
be employed to functionalize an isoquinoline at each position of
the ring system providing said ring is suitably activated or
functionalized, as for example with a chloride as shown in the
scheme. This sequence begins with 1-chloroisoquinoline (1) which
upon treatment with metachloroperbenzoic acid can be converted to
the corresponding N-oxide (2). Said intermediate (2) can be
converted to the corresponding 1,3-dichloroisoquinoline (3) by
treatment with phosphorous oxychloride in refluxing chloroform.
Intermediate (3) can be coupled with N-Boc-4-hydroxyproline by the
methods described herein to provide intermediate (5) as shown in
the Scheme. Intermediate (5) can undergo a Suzuki coupling with an
aryl boronic acid, in the presence of a palladium reagent and base,
and in a solvent such as THF or toluene or DMF to provide the
C3-arylisoquinoline intermediate (6). Heteroarylboronic acids can
also be employed in this Pd mediated coupling process to provide
C3-heteroarylisoquinolines. Intermediate (6) can be converted into
final compounds of Formula I by the methods described herein.
##STR47## Palladium mediated couplings of heteroaryl systems with
aryl or heteroaryl elements can also be employed at a later
synthetic stage in the construction of compounds of Formula I as
shown below. Therein macrocycle tripeptide acylsulfonamide
intermediate (1) is coupled to a 1-chloro-3-bromoisoquinoline (2)
using the previously described process of alkoxide displacement of
an heteroarylhalo moiety to provide intermediate (3). The coupling
of (1) and (2) can be executed in the presence of a catalyst such
as lanthanum chloride. The isoquinoline ring system of intermediate
(3) can be further functionalized by employing either Suzuki
couplings (Process 1: subjecting (3) to heteroaryl or aryl boronic
acids in the presence of a palladium catalyst such as palladium
tetrakis(triphenylphosphine) and a base such as cesium carbonate in
solvents such as DMF) or Stille couplings (Process 2: subjecting
(3) to heteraryl or aryl tin dervatives in the presence of
palladium catalyst such as palladium tetrakis(triphenylphosphine in
solvents such as toluene). ##STR48##
[0103] The present invention also provides compositions comprising
a compound of the present invention, or a pharmaceutically
acceptable enantiomer, diastereomer, salt, solvate or prodrug
thereof, and a pharmaceutically acceptable carrier. Pharmaceutical
compositions of the present invention comprise a therapeutically
effective amount of a compound of the invention, or a
pharmaceutically acceptable enantiomer, diastereomer, salt, solvate
or prodrug thereof, and a pharmaceutically acceptable carrier, with
a pharmaceutically acceptable carrier, e.g., excipient, or vehicle
diluent.
[0104] The active ingredient, i.e., compound, in such compositions
typically comprises from 0.1 weight percent to 99.9 percent by
weight of the composition, and often comprises from about 5 to 95
weight percent.
[0105] Thus, in one aspect of the invention, there is provided a
composition comprising the compound of formula 1 and a
pharmaceutically acceptable carrier. Preferably, the composition
further comprises a compound having anti-HCV activity. As used
herein, the term "anti-HCV activity" means the compound is
effective to inhibit the function of a target selected from the
group consisting of HCV metalloprotease, HCV serine protease, HCV
polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV
assembly, HCV egress, HCV NS5A protein, IMPDH and a nucleoside
analog for the treatment of an HCV infection. Often, the other
compound having anti-HCV activity is effective to inhibit the
function of target in the HCV life cycle other than the HCV NS3
protease protein.
[0106] In one preferred aspect, the compound having anti-HCV
activity is an interferon. Preferably, the interferon is selected
from the group consisting of interferon alpha 2B, pegylated
interferon alpha, consensus interferon, interferon alpha 2A,
lymphoblastiod interferon tau.
[0107] In another aspect of the invention, the compound having
anti-HCV activity is selected from the group consisting of
interleukin 2, interleukin 6, interleukin 12, a compound that
enhances the development of a type 1 helper T cell response,
interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine
5'-monophospate dehydrogenase inhibitor, amantadine, and
rimantadine.
[0108] In one preferred aspect of the invention, the composition
comprises a compound of the invention, an interferon and
ribavirin.
[0109] In another preferred aspect of the invention, the compound
having anti-HCV activity is a small molecule compound. As used
herein, the term "small molecule compound" means a compound having
a molecular weight of less than 1,500 daltons, preferably less than
1000 daltons. Preferably, the small molecule compound is effective
to inhibit the function of a target selected from the group
consisting of HCV metalloprotease, HCV serine protease, HCV
polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV
assembly, HCV egress, HCV NS5A protein, inosine monophophate
dehydrogenase ("IMPDH") and a nucleoside analog for the treatment
of an HCV infection.
[0110] Certain illustrative HCV inhibitor compounds which can be
administered with the compounds of the present invention include
those disclosed in the following publications: WO 02/04425 A2
published Jan. 17, 2002, WO 03/007945 A1 published Jan. 30, 2003,
WO 03/010141 A2 published Feb. 6, 2003, WO 03/010142 A2 published
Feb. 6, 2003, WO 03/010143 A1 published Feb. 6, 2003, WO 03/000254
A1 published Jan. 3, 2003, WO 01/32153 A2 published May 10, 2001,
WO 00/06529 published Feb. 10, 2000, WO 00/18231 published Apr. 6,
2000, WO 00/10573 published Mar. 2, 2000, WO 00/13708 published
Mar. 16, 2000, WO 01/85172 A1 published Nov. 15, 2001, WO 03/037893
A1 published May 8, 2003, WO 03/037894 A1 published May 8, 2003, WO
03/037895 A1 published May 8, 2003, WO 02/100851 A2 published Dec.
19, 2002, WO 02/100846 A1 published Dec. 19, 2002, EP 1256628 A2
published Nov. 13, 2002, WO 99/01582 published Jan. 14, 1999, WO
00/09543 published Feb. 24, 2000.
[0111] Table 1 below lists some illustrative examples of compounds
that can be administered with the compounds of this invention. The
compounds of the invention can be administered with other anti-HCV
activity compounds in combination therapy, either jointly or
separately, or by combining the compounds into a composition.
TABLE-US-00001 TABLE 1 Type of Inhibitor or Brand Name Target
Source Company Omega IFN IFN-.omega. BioMedicines Inc., Emeryville,
CA BILN-2061 serine protease inhibitor Boehringer Ingelheim Pharma
KG, Ingelheim, Germany Summetrel antiviral Endo Pharmaceuticals
Holdings Inc., Chadds Ford, PA Roferon A IFN-.alpha.2a F.
Hoffmann-La Roche LTD, Basel, Switzerland Pegasys PEGylated
IFN-.alpha.2a F. Hoffmann-La Roche LTD, Basel, Switzerland Pegasys
and Ribavirin PEGylated IFN-.alpha.2a/ribavirin F. Hoffmann-La
Roche LTD, Basel, Switzerland CellCept HCV IgG F. Hoffmann-La Roche
LTD, immunosuppressant Basel, Switzerland Wellferon lymphoblastoid
IFN-.alpha.n1 GlaxoSmithKline plc, Uxbridge, UK Albuferon-.alpha.
albumin IFN-.alpha.2b Human Genome Sciences Inc., Rockville, MD
Levovirin ribavirin ICN Pharmaceuticals, Costa Mesa, CA IDN-6556
caspase inhibitor Idun Pharmaceuticals Inc., San Diego, CA IP-501
antifibrotic Indevus Pharmaceuticals Inc., Lexington, MA Actimmune
INF-.gamma. InterMune Inc., Brisbane, CA Infergen A IFN alfacon-1
InterMune Pharmaceuticals Inc., Brisbane, CA ISIS 14803 antisense
ISIS Pharmaceuticals Inc, Carlsbad, CA/Elan Phamaceuticals Inc.,
New York, NY JTK-003 RdRp inhibitor Japan Tobacco Inc., Tokyo,
Japan Pegasys and Ceplene PEGylated IFN-.alpha.2a/ Maxim
Pharmaceuticals Inc., immune modulator San Diego, CA Ceplene immune
modulator Maxim Pharmaceuticals Inc., San Diego, CA Civacir HCV IgG
Nabi Biopharmaceuticals immunosuppressant Inc., Boca Raton, FL
Intron A and Zadaxin IFN-.alpha.2b/.alpha.1-thymosin RegeneRx
Biopharmiceuticals Inc., Bethesda, MD/ SciClone Pharmaceuticals
Inc, San Mateo, CA Levovirin IMPDH inhibitor Ribapharm Inc., Costa
Mesa, CA Viramidine IMPDH inhibitor Ribapharm Inc., Costa Mesa, CA
Heptazyme ribozyme Ribozyme Pharmaceuticals Inc., Boulder, CO
Intron A IFN-.alpha.2b Schering-Plough Corporation, Kenilworth, NJ
PEG-Intron PEGylated IFN-.alpha.2b Schering-Plough Corporation,
Kenilworth, NJ Rebetron IFN-.alpha.2b/ribavirin Schering-Plough
Corporation, Kenilworth, NJ Ribavirin ribavirin Schering-Plough
Corporation, Kenilworth, NJ PEG-Intron/Ribavirin PEGylated
IFN-.alpha.2b/ribavirin Schering-Plough Corporation, Kenilworth, NJ
Zadazim immune modulator SciClone Pharmaceuticals Inc., San Mateo,
CA Rebif IFN-.beta.1a Serono, Geneva, Switzerland IFN-.beta. and
EMZ701 IFN-.beta. and EMZ701 Transition Therapeutics Inc., Ontario,
Canada T67 .beta.-tubulin inhibitor Tularik Inc., South San
Francisco, CA VX-497 IMPDH inhibitor Vertex Pharmaceuticals Inc.,
Cambridge, MA VX-950/LY-570310 serine protease inhibitor Vertex
Pharmaceuticals Inc., Cambridge, MA/Eli Lilly and Co. Inc.,
Indianapolis, IN Omniferon natural IFN-.alpha. Viragen Inc.,
Plantation, FL XTL-002 monoclonal antibody XTL Biopharmaceuticals
Ltd., Rehovot, Isreal
[0112] The pharmaceutical compositions of this invention may be
administered orally, parenterally or via an implanted reservoir.
Oral administration or administration by injection are preferred.
In some cases, the pH of the formulation may be adjusted with
pharmaceutically acceptable acids, bases or buffers to enhance the
stability of the formulated compound or its delivery form. The term
parenteral as used herein includes subcutaneous, intracutaneous,
intravenous, intramuscular, intra-articular, intrasynovial,
intrasternal, intrathecal, and intralesional injection or infusion
techniques.
[0113] When orally administered, the pharmaceutical compositions of
this invention may be administered in any orally acceptable dosage
form including, but not limited to, capsules, tablets, and aqueous
suspensions and solutions. In the case of tablets for oral use,
carriers which are commonly used include lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically
added. For oral administration in a capsule form, useful diluents
include lactose and dried corn starch. When aqueous suspensions are
administered orally, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening
and/or flavoring and/or coloring agents may be added. Other
suitable carriers for the above noted compositions can be found in
standard pharmaceutical texts, e.g. in "Remington's Pharmaceutical
Sciences", 19th ed., Mack Publishing Company, Easton, Pa.,
1995.
[0114] The pharmaceutical compositions can be prepared by known
procedures using well-known and readily available ingredients. The
compositions of this invention may be formulated so as to provide
quick, sustained or delayed release of the active ingredient after
administration to the patient by employing procedures well known in
the art. In making the compositions of the present invention, the
active ingredient will usually be admixed with a carrier, or
diluted by a carrier, or enclosed within a carrier which may be in
the form of a capsule, sachet, paper or other container. When the
carrier serves as a diluent, it may be a solid, semi-solid or
liquid material which acts as a vehicle, excipient or medium for
the active ingredient. Thus, the compositions can be in the form of
tablets, pills, powders, beadlets, lozenges, sachets, elixirs,
suspensions, emulsions, solutions, syrups, aerosols, (as a solid or
in a liquid medium), soft and hard gelatin capsules, suppositories,
sterile injectable solutions, sterile packaged powders and the
like. Further details concerning the design and preparation of
suitable delivery forms of the pharmaceutical compositions of the
invention are known to those skilled in the art.
[0115] Dosage levels of between about 0.01 and about 1000 milligram
per kilogram ("mg/kg") body weight per day, preferably between
about 0.5 and about 250 mg/kg body weight per day of the compounds
of the invention are typical in a monotherapy for the prevention
and treatment of HCV mediated disease. Typically, the
pharmaceutical compositions 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.
[0116] As the skilled artisan will appreciate, lower or higher
doses than those recited above may be required. Specific dosage and
treatment regimens for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health status, sex, diet,
time of administration, rate of excretion, drug combination, the
severity and course of the infection, the patient's disposition to
the infection and the judgment of the treating physician.
Generally, treatment is initiated with small dosages substantially
less than the optimum dose of the peptide. Thereafter, the dosage
is increased by small increments until the optimum effect under the
circumstances is reached. In general, the compound is most
desirably administered at a concentration level that will generally
afford antivirally effective results without causing any harmful or
deleterious side effects.
[0117] When the compositions of this invention comprise a
combination of a compound of the invention and one or more
additional therapeutic or prophylactic agent, both the compound and
the additional agent are usually 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.
[0118] When these compounds or their pharmaceutically acceptable
enantiomers, diastereomers, salts, solvates or prodrugs are
formulated together with a pharmaceutically acceptable carrier, the
resulting composition may be administered in vivo to mammals, such
as man, to inhibit HCV NS3 protease or to treat or prevent HCV
virus infection.
[0119] Accordingly, another aspect of this invention provides
methods of inhibiting HCV NS3 protease activity in patients by
administering a compound of the present invention or a
pharmaceutically acceptable enantiomer, diastereomer, salt or
solvate thereof.
[0120] In one aspect of the invention, there is provided a method
of treating an HCV infection in a patient, comprising administering
to the patient a therapeutically effective amount of the compound
of the invention, or a pharmaceutically acceptable enantiomer,
diastereomer, solvate, prodrug or salt thereof.
[0121] Preferably, the method of administering the compound is
effective to inhibit the function of the HCV NS3 protease protein.
In a preferred aspect, the method further comprises administering
another compound having anti-HCV activity (as described above)
prior to, after or concurrently with a compound of the
invention.
[0122] The compounds of the invention may also be used as
laboratory reagents. Compounds may be instrumental in providing
research tools for designing of viral replication assays,
validation of animal assay systems and structural biology studies
to further enhance knowledge of the HCV disease mechanisms.
Further, the compounds of the present invention are useful in
establishing or determining the binding site of other antiviral
compounds, for example, by competitive inhibition.
[0123] The compounds of this invention may also 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 or transfusion apparatuses and
materials.
[0124] Further, the compounds and compositions of the invention can
be used for the manufacture of a medicament for treating HCV
infection in a patient.
[0125] The present invention also provides processes for resolving
a mixture of alkyl ester enantiomers comprising contacting the
mixture with an enzyme effective to preferentially promote the
hydrolysis of one of the enantiomers; characterized in that the
contacting is conducted in the presence of a buffer. Such a mixture
of alkyl ester enantiomers can result from the preparation of
chemical precursor B, described above.
[0126] Preferably, the alkyl ester has the following Formula
##STR49## wherein: R.sub.25 is an amino protecting group, such as,
for example carbamates, e.g., BOC imines, or amides; and R.sub.26
is selected from the group consisting of C.sub.1-10 alkyl,
C.sub.6-14 aryl, C.sub.7-16 alkylaryl, C.sub.3-7 cycloalkyl or
C.sub.3-10 alkyl cycloalkyl.
[0127] The particular buffer used in the processes of the present
invention is not critical. Typically, the buffer will have a pKa of
.+-.1 of the pH at which the hydrolysis is conducted, e.g., pH from
about 7.0 to 11. Thus, if the hydrolysis is conducted, e.g., at a
pH of 8.0, a buffer with a pKa of from about 7.0 to 9.0 can be
used. Preferred buffers are phosphates, borates and carbonates.
Examples of suitable buffers include:
2-[(2-amino-2-oxoethyl)amino]ethanesulphonic acid,
N-(2-acetamido)-2-iminodiacetic acid,
n,n-bis(2-hydroxyethyl)glycine,
1,3-bis[tris(hydroxymethyl)methylamino]propane, o-Boric acid,
carbonic acid, 2-(cyclohexylamino)ethanesulphonic acid,
Diethylmalonic acid, Glycylglycine,
N-2-hydroxyethylpiperazine-N'-2-ethane-sulphonic acid (HEPES),
N-2-hydroxyethylpiperazine-N'-3-propane-sulphonic acid, imidazole,
3-(N-morpholino)propanesulphonic acid, o-phosphoric acid,
piperazine-N-N'-bis(2-ethanesulphonic acid),
piperazine-1,4-bis(2-hydroxypropanesulfonic acid),
3-[tris(hydroxymethyl)methyl]amino propanesulphonic acid,
2-[tris(hydroxymothyl)methyl]amino ethanesulphonic acid,
N-[tris(hydroxymethyl)methyl]glycine, and
tris(hydroxylmethyl)aminomethane.
[0128] Any enzyme effective to preferentially hydrolyze the desired
alkyl ester enantiomer can be used in accordance with the present
invention. Proteases, lipases and esterases are often used for
hydrolysis of carboxylic ester bonds. The enzymes suitable for use
in accordance with the present invention, regardless of origin or
purity, may be employed in the free state, either liquid or dry
forms, or immobilized on a support such as, for example, by
physical adsorption or entrapment. Examples of lipases include
lipases from Candida rugosa, Wheat Germ, Porcine Pancreas, Rhizopus
arrhizus, Candida antarctica, Mucor miehei, Aspergillus niger,
Pseudomonas species, Candida lipolytica, Humicola langinosa and
Mucor javanicus. Examples of esterase include pig liver esterase
(PLE), horse liver esterase (HLE), acetylcholine esterase (ACE),
cholesterol esterase, esterase from Bacillus subtilis (carboxyl
esterase NP). Proteases are generally defined as hydrolases acting
on peptide bonds as their natural substrates, but they are capable
of cleaving a carboxylic ester bond since an ester bond is much
weaker than a peptide (amide) bond. Examples of proteases include
.alpha.-chymotrypsin, thermolysin, papain, proteases from
Aspergillus sp., Bacillus sp., Rhizopus sp., Penicillum sp.,
Streptomyces griseus.
[0129] In the ester of Formula VIII, the chiral center in the acid
moiety is fully substituted. This represents a difficult target for
hydralitic enzymes since their natural substrates (such as natural
amino acids) usually contain at least one .alpha.-hydrogen. Quite
surprisingly, it was found in accordance with the present invention
that certain proteases were highly effective for the
enantioselective hydrolysis of ester VIII. Preferred proteases
include proteases from strains selected from the group consisting
of Bacillus globigii, Bacillus licheniformis, Bacillus halodurans,
Bacillus clausii and Aspergillus oryzae. Examples of suitable
enzymes include those selected from the group consisting of
Alcalase.RTM. (subtilisin, alkaline protease, Novozymes North
America Inc., Franklington, N.C.), Savinase.RTM. (substilisin,
alkaline protease, Novozymes), ChiroCLEC.TM.-BL (subtilisin,
alkaline protease, Altus Biologics, Inc.,), Protease N (subtilisin,
alkaline protease, Amano) and Flavourzyme.TM. (fungal
protease/peptidase, Novozymes). Further details of such enzymes are
known in the art. Such enzymes are commercially available. These
enzymes can be employed alone or used as mixtures thereof.
[0130] The ester hydrolysis leads to the release of an alcohol and
a carboxylic acid, which can lower the pH significantly if no
buffer is present. In accordance with the present invention, it has
been found that including buffer in the reaction mixture can
stabilize the pH in the course of hydrolysis, thus contributing to
higher enzyme activity and stability.
[0131] Futhermore, by virtue of the present invention, it is
possible to conduct the hydrolysis at elevated temperatures,
preferably about 30 to 60.degree. C., and for short time periods,
preferably less than about seven days, more preferably from about
two hours to three days.
EXAMPLES
[0132] The specific examples that follow illustrate the syntheses
of the compounds of the present invention, and are not to be
construed as limiting the scope of the claims which follow. The
methods may be adapted to variations in order to produce compounds
embraced by this invention but not specifically disclosed. Further,
variations of the methods to produce the same compounds in somewhat
different manner will also be evident to one skilled in the
art.
[0133] Chemical abbreviations commonly used to identify chemical
compounds disclosed herein include Bn: benzyl; Boc:
tert-butyloxycarbonyl {Me.sub.3COC(O)}; BSA: bovine serum albumin;
CDI: carbonyldiimidazole; DBU: 1,8-diazabicyclo[5.4.0]-undec-7-ene;
CH.sub.2Cl.sub.2=DCM: methylene chloride; TBME: tert-butyl methyl
ether; DEAD: diethylazodicarboxylate; DIAD:
diisopropylazodicarboxylate; DIEA: diisopropylethylamine; DIPEA:
diisopropylethylamine; 4-DMAP: 4-dimethylaminopyridine; DCC:
1,3-dicyclohexylcarbodiimide; DMF: dimethylformamide; DMSO:
dimethylsulfoxide; DPPA: diphenylphosphoryl azide; Et: ethyl; EtOH:
ethanol; EtOAc: ethyl acetate; Et.sub.2O: diethyl ether; Grubb's
Catalyst: bis(tricyclohexylphosphine)benzylidene ruthenium (IV)
dichloride; Grubb's 2.sup.nd Generation Catalyst:
tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-
-2-ylidene][benzylidene]ruthenium (IV) dichloride; HATU:
[O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate; HBTU:
[O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate; HOBT, 1-hydroxybenzotriazole; HOAT,
1-hydroxy-7-azabenzotriazole; HPLC: high performance liquid
chromatography; MS: mass spectrometry; Me: methyl; MeOH: methanol;
NMM: N-methylmorpholine; NMP: N-methylpyrrolidine; Pr: propyl; PPA:
polyphosphoric acid; TBAF: tetra-n-butylammonium fluoride; 1,2-DCE
or DCE: 1,2-dichloroethane; TFA: trifluoroacetic acid; THF:
tetrahydrofuran.
[0134] Solution percentages express a weight to volume
relationship, and solution ratios express a volume to volume
relationship, unless stated otherwise. Nuclear magnetic resonance
(NMR) spectra were recorded either on a Bruker 300, 400 or 500
megahertz (MHz) spectrometer; the chemical shifts (.delta.) are
reported in parts per million. Flash chromatography was carried out
on silica gel (SiO.sub.2) evident to one skilled in the art. All
Liquid Chromatography (LC) data were recorded on a Shimadzu LC-10AS
liquid chromatograph using a SPD-10AV UV-Vis detector and Mass
Spectrometry (MS) data were determined with a Micromass Platform
for LC in electrospray mode (ES+).
[0135] Unless otherwise noted, each compound was analyzed, by
LC/MS, using one of three methodologies, having the following
conditions. [0136] Columns: Method A)--YMC Xterra ODS S7 micrometer
(".mu.m") 3.0.times.50 millimeter ("mm") i.e., 3.0 mm diameter by
50 mm length. [0137] Gradient: 100% Solvent A/0% Solvent B to
[0138] 0% Solvent A/100% Solvent B [0139] Gradient time: 4 min (A)
[0140] Hold time: 1 min (A); 2 min [0141] Flow rate: 4 mL/min
[0142] Detector Wavelength: 220 nanometer ("nm") [0143] Solvent A:
10% MeOH/90% H.sub.2O/0.1% TFA [0144] Solvent B: 10% H.sub.2O/90%
MeOH/0.1% TFA. [0145] Columns: Method B)--YMC Xterra ODS S7
3.0.times.50 mm Gradient: 100% Solvent A/0% Solvent B to [0146] 0%
Solvent A/100% Solvent B [0147] Gradient time: 2 min [0148] Hold
time: 1 min [0149] Flow rate: 5 mL/min [0150] Detector Wavelength:
220 nm [0151] Solvent A: 10% MeOH/90% H.sub.2O/0.1% TFA [0152]
Solvent B: 10% H.sub.2O/90% MeOH/0.1% TFA. [0153] Columns: Method
D)--YMC Xterra C18 5 .mu.M 3.0.times.50 mm Gradient: 100% Solvent
A/0% Solvent B to 0% Solvent A/100% Solvent B [0154] Gradient time:
3 min [0155] Hold time: 1 min [0156] Flow rate: 5 mL/min [0157]
Detector Wavelength: 220 nm [0158] Solvent A: 10% MeOH/90%
H.sub.2O/0.1% TFA [0159] Solvent B: 10% H.sub.2O/90% MeOH/0.1% TFA.
Unless otherwise noted, preparative HPLC was carried out under the
following conditions. [0160] Gradient time: 10 min [0161] Hold
time: 2 min [0162] Flow rate: 25 mL/min [0163] Detector wavelength:
220 nM [0164] Solvent A: 10% MeOH/90% H.sub.2O/0.1% TFA [0165]
Solvent B: 10% H.sub.2O/90% MeOH/0.1% TFA The pure fractions were
combined and 0.1N NaOH was used to bring the pH up to 7. The
solution was concentrated in vacuo, and then the pH was carefully
lowered to pH 3-4 using 0.1N HCl. The mixture was quickly extracted
with ethyl acetate (3.times.). The combined extracts were dried
(MgSO.sub.4) and concentrated in vacuo to give the purified
products.
[0166] The compounds and chemical intermediates of the present
invention, described in the following examples, were prepared
according to the following methods.
Example 1
Preparation of Representative Intermediates
Preparation of P2 Isoquinoline Intermediates for Incorporation into
Compounds of Formula 1
[0167] Method A ##STR50## Step 1:
[0168] To a solution of 3-methoxy cinnamic acid (11.04 g, 62 mmol)
and triethylamine (12.52 g, 124 mmol) in acetone (80 mL) was added
ethyl chloroformate (approximately 1.5 equivalents) dropwise at
0.degree. C. After stirring at this temperature for 1 h, aqueous
NaN.sub.3 (6.40 g, 100 mmol in 35 mL H.sub.2O; appropriate
precautions must be taken when using sodium azide) was added
dropwise and the reaction mixture was stirred for 16 h at the
ambient temperature. Water (100 mL) was added to the mixture and
the volatile was removed in vacuo. The resulting slurry was
extracted with toluene (3.times.50 mL) and the combined organic
layers were dried over MgSO.sub.4. This dried solution was added
dropwise to a heated solution of diphenylmethane (50 mL) and
tributylamine (30 mL) at 190.degree. C. The toluene was distilled
off as added. After complete addition, the reaction temperature was
raised to 210.degree. C. for 2 h. After cooling, the precipitated
product was collected by filtration, washed with hexane (2.times.50
mL), and dried to yield the desired product as a white solid (5.53
g, 51%) (Nicolas Briet at el, Tetrahedron, 2002, 5761-5766).
[0169] LC-MS (retention time: 0.82 min, method B), MS m/z 176
(M.sup.++H).
[0170] An altenative procedure to the above employs
diphenylphosphoryl azide for the conversion of the carboxylic acid
to the corresponding acylazide. In a one pot procedure then the
acid is converted to the corresponding quinolone. The process is
described below for the prepapration of
4-methyl-2H-isoquinolin-1-one from 3-phenyl-but-2-enoic acid:
[0171] A solution of 3-phenyl-but-2-enoic acid (16.2 g),
diphenylphosphoryl azide (27.5 g), and triethylamine (10.1 g) in
benzene (100 mL) was stirred for 1 h. After filtration through a
silica gel plug washing with benzene and concentration, the residue
was dissolved in diphenylmethane (80 mL) and refluxed for 3 h.
After cooling to rt, solids were collected through a plug washing
with benzene and dried to give 10 g (63%) of the desired
4-methyl-2H-isoquinolin-1-one as a solid. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. ppm 2.30 (s, 3H), 7.00 (s, 1H), 7.54 (m, 1H),
7.77 (m, 2H), 8.33 (d, J=7.34 Hz, 1H).
Step 2:
[0172] 6-Methoxy-2H-isoquinolin-1-one (5.0 g, 28.4 mmol) in
POCl.sub.3 (10 mL) was heated to gentle reflux for 3 h the
evaporated in vacuo (Nicolas Briet at el, Tetrahedron, 2002,
5761-5766). The residue was poured into iced water (20 mL) and
neutralized to pH 10 with 10 M NaOH. Extracted with CHCl.sub.3. The
organic layer was washed with brine, dried over MgSO.sub.4,
filtered, evaporated. The residue was purified by flash
chromatography (1:1 hexane-EtOAc) to afford 4.41 g (80%) of the
desired product as a white solid.
[0173] .sup.1H NMR (CD.sub.3OD) .delta. 3.98 (s, 3H), 7.34-7.38 (m,
2H), 7.69 (d, J=5.5 Hz, 1H), 8.10 (d, J=6.0 Hz, 1H), 8.23 (d, J=9.5
Hz, 1H);
[0174] LC-MS (retention time: 1.42 min, method B), MS m/z 194
(M.sup.++H).
Step 3:
[0175] To a solution of N-BOC-3-(R)-hydroxy-L-proline (892 mg, 3.89
mmol) in DMSO (40 mL) at the ambient temperature was added
potassium tert-butoxide (1.34 g, 12.0 mmol) in one portion. The
formed suspension was stirred at this temperature for 30 min before
being cooled to 10.degree. C. 1-chloro-6-methoxy-isoquinoline
(example 11, Step 2) (785 mg, 4.05 mmol) was added as solid in one
portion and the final mixture was stirred at the ambient
temperature for 12 h. Quenched with iced 5% citric acid (aq),
extracted with EtOAC (100 mL). The aqueous phase was extracted with
EtOAC again. The combined organic layers were washed with 5% citric
acid (aq) and brine respectively, dried over MgSO.sub.4, filtered.
The filtrate was evaporated in vacuo to dryness to yield 1.49 g
(99%) of the desired product as an off-white foam. This material
was used in the next step reaction as crude without further
purification. .sup.1H NMR (CD.sub.3OD) .delta. 1.42, 1.44
(rotamers, 9H), 2.38-2.43 (m, 1H), 2.66-2.72 (m, 1H), 3.80-3.87 (m,
2H), 3.92 (s, 3H), 4.44-4.52 (m, 1H), 5.73 (b, 1H), 7.16-7.18 (m,
2H), 7.24-7.25 (m, 1H), 7.87-7.88 (m, 1H), 8.07 (d, J=8.5 Hz,
1H);
[0176] LC-MS (retention time: 1.62 min, method B), MS m/z 389
(M.sup.++H). The following intermediates were prepared as described
herein and can be incorporated into compounds of Formula 1
##STR51## Step 1: Modifications: 15 g 3-methoxy-3-phenyl-acrylic
acid used, 250 mg product obtained (2% yield). Product:
##STR52##
[0177] .sup.1H NMR (400 MHz, CD.sub.3COCD.sub.3) .delta. ppm 3.85
(s, 3H), 6.96 (s, 1H), 7.54 (m, 1H), 7.71 (m, 1H), 7.86 (d, J=8.07
Hz, 1H), 8.31 (d, J=8.07 Hz, 1H).
Step 2:
Modifications: 200 mg 4-methoxy-2H-isoquinolin-1-one used, 150 mg
product obtained (68% yield).
[0178] Product: ##STR53##
[0179] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 4.05 (s, 2H),
7.71 (m, 1H), 7.72 (m, 2H), 7.80 (s, 1H), 8.23 (dd, J=18.71, 7.70
Hz, 2H).
Step 3:
Modifications: 122 mg 1-chloro-4-methoxy-isoquinoline used, 218 mg
product obtained (89% yield).
[0180] Product: ##STR54##
[0181] MS: (M+Na).sup.+ 411.
The following intermediates were prepared as described herein and
can be incorporated into compounds of Formula 1
[0182] 1 ##STR55## Step 1: Modifications: 20 g 2-methylcinnamic
acid used, 14.3 g product obtained (72% yield) Product:
##STR56##
[0183] Data: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 2.54 (s,
1H), 6.69 (d, J=7.3 Hz, 1H), 7.23 (d, J=7.3 Hz, 1H), 7.39 (t, J=7.8
Hz, 1H), 7.50 (d, J=7.1 Hz, 1H), 8.30 (d, J=8.1 Hz, 1H), 11.62 (s,
1H); MS: (M+H).sup.+ 160.
Step 2:
Modifications: 14.4 g 5-methyl-2H-isoquinolin-1-one used, 10.6 g
product obtained (66% yield).
[0184] Product: ##STR57##
[0185] Data: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 2.67 (s,
3H), 7.55 (m, 2H), 7.70 (dd, J=5.9, 1.0 Hz, 1H), 8.19 (m, 1H), 8.28
(d, J=5.9 Hz, 1H); MS: (M+H).sup.+ 178.
Step 3:
Modifications: 533 mg 1-chloro-5-methyl-isoquinoline used, 1116 mg
product obtained (100% yield).
[0186] Product: ##STR58##
[0187] Data: MS: (M+H).sup.+ 373. The following intermediates were
prepared as described herein and can be incorporated into compounds
of Formula 1 ##STR59## Step 1: Modifications: 10 g 2-methoxy
cinnamic acid used, 5.3 g product obtained (53% yield). Product:
##STR60##
[0188] Data: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 3.95 (s,
3H), 6.94 (d, J=7.3 Hz, 1H), 7.08 (d, J=8.1 Hz, 1H), 7.14 (d, J=7.3
Hz, 1H), 7.43 (t, J=8.1 Hz, 1H), 7.99 (d, J=8.1 Hz, 1H), 10.92 (s,
1H); MS: (M+H).sup.+ 176.
Step 2:
Modifications: 5.3 g 5-methoxy-2H-isoquinolin-1-one used, 5.38 g
product obtained (92% yield).
[0189] Product: ##STR61##
[0190] Data: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 4.01 (s,
3H), 7.04 (d, J=7.8 Hz, 1H), 7.57 (t, J=8.1 Hz, 1H), 7.88 (d, J=8.6
Hz, 1H), 7.97 (d, J=5.9 Hz, 1H), 8.25 (d, J=5.9 Hz, 1H); MS:
(M+H).sup.+ 194.
Step 3:
Modifications: 581 mg 1-chloro-5-methoxy-isoquinoline used, 1163 mg
product obtained (100% yield).
[0191] Product: ##STR62##
[0192] Data: MS: (M+H).sup.+ 389. The following intermediates were
prepared as described herein and can be incorporated into compounds
of Formula 1 ##STR63## Step 1: Modifications: 25 g 2-chlorocinnamic
acid used, 14.6 g product obtained (59% yield). Product:
##STR64##
[0193] Data: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 7.22 (d,
J=7.3 Hz, 1H), 7.42 (t, J=7.8 Hz, 1H), 7.73 (d, J=7.8 Hz, 1H), 8.34
(d, J=8.1 Hz, 1H), 10.61 (s, 1H); MS: (M+H).sup.+ 180.
Step 2:
Modifications: 14.2 g 5-chloro-2H-isoquinolin-1-one used, 8.28 g
product obtained (53% yield).
[0194] Product: ##STR65##
[0195] Data: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.60
(dd, J=8.6, 7.6 Hz, 1H), 7.83 (m, 1H), 8.00 (d, J=5.9 Hz, 1H), 8.29
(dt, J=8.9, 1.0 Hz, 1H), 8.38 (d, J=5.9 Hz, 1H); MS: (M+H)+198.
Step 3:
Modifications: 594 mg 1,5-dichloro-isoquinoline used, 1174 mg
product obtained (100% yield).
[0196] Product: ##STR66##
[0197] Data: MS: (M+H).sup.+ 393. The following intermediates were
prepared as described herein and can be incorporated into compounds
of Formula 1 ##STR67## Step 1: Modifications: 16.6
g2-fluorocinnamic acid used, 8.55 g product obtained (51% yield).
Product: ##STR68##
[0198] Data: .sup.1H NMR (400 MHz, CD.sub.3COCD.sub.3) .delta. ppm
6.62 (d, J=7.3 Hz, 1H), 7.32 (d, J=7.3 Hz, 1H), 7.47 (m, 2H), 8.09
(m, 1H).
Step 2:
Modifications: 8.4 g 5-fluoro-2H-isoquinolin-1-one used, 7.5 g
product obtained (80% yield).
[0199] Product: ##STR69##
[0200] Data: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.43
(ddd, J=9.7, 7.8, 0.9 Hz, 1H), 7.62 (td, J=8.2, 5.4 Hz, 1H), 7.84
(d, J=5.6 Hz, 1H), 8.14 (d, J=8.6 Hz, 1H), 8.33 (d, J=5.9 Hz, 1H);
MS: (M+H).sup.+ 182.
Step 3:
Modifications: 203 mg 1-chloro-5-fluoro-isoquinoline used, 384 mg
product obtained (90% yield).
[0201] Product: ##STR70##
[0202] Data: .sup.1H NMR (400 MHz, CD.sub.3SOCD.sub.3) .delta. ppm
1.34, 1.36 (2s, 9H, rotamers), 2.35 (m, 1H), 2.61 (m, 1H), 3.65 (d,
J=12.23 Hz, 1H), 3.80 (m, 1H), 4.35 (m, 1H), 5.70 (s, 1H), 7.48 (d,
J=6.11 Hz, 1H), 7.63 (m, 2H), 7.99 (m, 1H), 8.10 (d, J=5.87 Hz,
1H); MS: (M+Na).sup.+ 399. The following intermediates were
prepared as described herein and can be incorporated into compounds
of Formula 1 ##STR71## Step 1: Modifications: 16.6 g
4-fluorocinnamic acid used, 8.2 g product obtained (49% yield).
Product: ##STR72##
[0203] Data: .sup.1H NMR (400 MHz, CD.sub.3COCD.sub.3) .delta. ppm
6.57 (d, J=7.09 Hz, 1H), 7.21 (d, J=7.09 Hz, 1H), 7.50 (m, 1H),
7.72 (dd, J=8.68, 5.26 Hz, 1H), 7.90 (dd, J=9.54, 2.93 Hz, 1H).
Step 2:
Modifications: 8.15 g 7-fluoro-2H-isoquinolin-1-one used, 7.6 g
product obtained (84% yield).
[0204] Product: ##STR73##
[0205] Data: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.52
(td, J=8.6, 2.6 Hz, 1H), 7.59 (d, J=5.6 Hz, 1H), 7.86 (dd, J=9.1,
5.4 Hz, 1H), 7.95 (dd, J=9.5, 2.5 Hz, 1H), 8.26 (d, J=5.6 Hz, 1H);
MS: (M+H)+182.
Step 3:
Modifications: 191 mg 1-chloro-7-fluoro-isoquinoline used, 350 mg
product obtained (93% yield).
[0206] Product: ##STR74##
[0207] Data: MS: (M+Na).sup.+ 399. The following intermediates were
prepared as described herein and can be incorporated into compounds
of Formula 1 ##STR75## Step 1: Modifications: 9.13 g
4-chlorocinnamic acid used, 4 g product obtained (44% yield).
Product: ##STR76##
[0208] Data: .sup.1H NMR (400 MHz, CD.sub.3SOCD.sub.3) .delta. ppm
6.58 (d, J=7.1 Hz, 1H), 7.20 (dd, J=7.1, 5.9 Hz, 1H), 7.72 (m, 2H),
8.10 (m, 1H).
Step 2:
Modifications: 3.5 g 7-chloro-2H-isoquinolin-1-one used, 2.8 g
product obtained (72% yield).
[0209] Product: ##STR77##
[0210] Data: .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm 7.59 (d,
J=5.5 Hz, 1H), 7.69 (dd, J=8.9, 2.1 Hz, 1H), 7.80 (d, J=8.6 Hz,
1H), 8.29 (d, J=5.5 Hz, 1H), 8.34 (s, 1H); MS: (M+H).sup.+ 198.
Step 3:
Modifications: 208 mg 1,7-dichloro-isoquinoline used, 350 mg
product obtained (89% yield).
[0211] Product: ##STR78##
[0212] Data: MS: (M+Na).sup.+ 415. The following intermediates were
prepared as described herein and can be incorporated into compounds
of Formula 1 ##STR79## Step 1: Modifications: 25 g 4-methylcinnamic
acid used, 15.3 g product obtained (62% yield). Product:
##STR80##
[0213] Data: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 2.50 (s,
3H), 6.54 (d, J=7.1 Hz, 1H), 7.13 (d, J=7.1 Hz, 1H), 7.49 (m, 2H),
8.22 (s, 1H), 11.49 (s, 1H); MS: (M+H).sup.+ 160.
Step 2:
Modifications: 15.3 g 7-methyl-2H-isoquinolin-1-one used, 5.15 g
product obtained (30% yield).
[0214] Product: ##STR81##
[0215] Data: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 2.58 (s,
3H), 7.56 (m, 2H), 7.73 (d, J=8.3 Hz, 1H), 8.09 (s, 1H), 8.20 (d,
J=5.6 Hz, 1H); MS: (M+H).sup.+ 178.
Step 3:
Modifications: 205 mg 1-chloro-7-methyl-isoquinoline used, 350 mg
product obtained (89% yield).
[0216] Product: ##STR82##
[0217] Data: MS: (M+H).sup.+ 373. The following intermediates were
prepared as described herein and can be incorporated into compounds
of Formula 1 ##STR83## Step 1: Modifications: 33 g using
4-methoxycinnamic acid used, 7 g product obtained (33% yield).
Product: ##STR84##
[0218] Data: .sup.1H NMR (500 MHz, CD.sub.3COCD.sub.3) .delta. ppm
3.90 (s, 3H), 6.49 (d, J=7.0 Hz, 1H), 7.10 (d, J=7.3 Hz, 1H), 7.28
(dd, J=8.6, 2.8 Hz, 1H), 7.57 (d, J=8.9 Hz, 1H), 7.71 (d, J=2.8 Hz,
1H).
Step 2:
Modifications: 4 g 7-methoxy-2H-isoquinolin-1-one used, 3 g product
obtained (68% yield).
[0219] Product: ##STR85##
[0220] Data: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 3.98 (s,
3H), 7.38 (dd, J=8.9, 2.6 Hz, 1H), 7.52 (m, 2H), 7.73 (d, J=8.8 Hz,
1H), 8.16 (d, J=5.4 Hz, 1H).
Step 3:
Modifications: 533 mg 1-chloro-7-methoxy-isoquinoline used, 1115 mg
product obtained (100% yield).
[0221] Product: ##STR86## The following intermediates were prepared
as described herein and can be incorporated into compounds of
Formula 1 ##STR87## Step 1: Modifications: 19.6 g
4-fluoro-3-methoxycinnamic acid used, 9.5 g product obtained (48%
yield). Product: ##STR88##
[0222] Data: .sup.1H NMR (400 MHz, CD.sub.3COCD.sub.3) .delta. ppm
4.00 (s, 1H), 6.49 (d, J=7.34 Hz, 1H), 7.19 (d, J=7.09 Hz, 1H),
7.29 (d, J=8.07 Hz, 1H), 7.86 (d, J=11.74 Hz, 1H).
Step 2:
Modifications: 9 g 7-fluoro-6-methoxy-2H-isoquinolin-1-one used, 7
g product obtained (70% yield).
[0223] Product: ##STR89## Data: .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. ppm 4.04 (s, 3H), 7.17 (d, J=8.07 Hz, 1H), 7.48 (d, J=5.62
Hz, 1H), 7.94 (d, J=11.49 Hz, 1H), 8.20 (d, J=5.62 Hz, 1H). Step 3:
Modifications: 222 mg 1-chloro-7-fluoro-6-methoxy-isoquinoline
used, 406 mg products obtained. Products: ##STR90## The following
intermediates were prepared as described herein and can be
incorporated into compounds of Formula 1 ##STR91## Step 1:
Modifications: 3.8 g 3-(2,3-dihydro-benzofuran-7-yl)-acrylic acid
used, 2 g product obtained (53% yield). Product: ##STR92##
[0224] Data: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 3.37 (t,
J=9.05 Hz, 1H), 4.73 (t, J=9.05 Hz, 2H), 6.67 (d, J=7.09 Hz, 1H),
7.10 (d, J=7.09 Hz, 1H), 7.37 (d, J=8.07 Hz, 1H), 7.81 (d, J=8.07
Hz, 1H); MS: (M+H).sup.+ 188.
Step 2:
Modifications: 1.87 g 2,3-dihydro-7H-furo[2,3-f]isoquinolin-6-one
used, 1.84 g product obtained (90% yield).
[0225] Product: ##STR93##
[0226] Data: .sup.1H NMR (400 Hz, CDCl.sub.3) .delta. ppm 3.43 (t,
J=9.05 Hz, 2H), 4.82 (t, J=9.05 Hz, 2H), 7.52 (d, J=8.56 Hz, 1H),
7.66 (d, J=5.62 Hz, 1H), 7.84 (d, J=8.31 Hz, 1H), 8.19 (d, J=5.62
Hz, 1H); MS (M+H).sup.+ 206.
Step 3:
Modifications: 206 mg 6-chloro-2,3-dihydro-furo[2,3-f]isoquinoline
used, 300 mg products mixture obtained.
[0227] Products: ##STR94## The following intermediates were
prepared as described herein and can be incorporated into compounds
of Formula 1 ##STR95## Step 1: Modifications: 1.14 g
3-(2,3-dihydro-benzofuran-4-yl)-acrylic acid used, 600 mg product
obtained (52% yield). Product: ##STR96##
[0228] Data: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 3.35 (t,
J=8.93 Hz, 2H), 4.74 (t, J=8.93 Hz, 2H), 6.49 (d, J=7.09 Hz, 1H),
6.95 (d, J=8.56 Hz, 1H), 7.25 (d, J=7.09 Hz, 1H), 8.13 (d, J=8.80
Hz, 1H); MS (M+H).sup.+ 188.
Step 2:
Modifications: 560 mg 1,7-dihydro-2H-furo[3,2-f]isoquinolin-6-one
used, 380 mg product obtained (48% yield).
[0229] Product: ##STR97##
[0230] Data: .sup.1H NMR (400 Hz, CDCl.sub.3) .delta. ppm 3.47 (t,
J=9.05 Hz, 2H), 4.84 (t, J=9.05 Hz, 2H), 7.24 (d, J=8.56 Hz, 1H),
7.33 (d, J=5.87 Hz, 1H), 8.20 (m, 2H); MS (M+H).sup.+ 206.
Step 3:
Modifications: 105 mg 6-chloro-1,2-dihydro-furo[3,2-f]isoquinoline
used, 390 mg products mixture obtained.
[0231] Products: ##STR98## The following intermediates were
prepared as described herein and can be incorporated into compounds
of Formula 1 ##STR99## Step 1: Modifications: A mixture of
6-methoxy-2H-isoquinolin-1-one (700 mg) and NCS (532 mg) in MeCN
(10 mL) was refluxed for 3 h. Filtration gave 600 mg (72%) of the
desired product as a solid. Product: ##STR100##
[0232] Data: .sup.1H NMR(400 MHz, CD.sub.3OD) .delta. ppm 3.96 (s,
1H), 7.19 (dd, J=8.80, 2.45 Hz, 1H), 7.28 (d, J=2.45 Hz, 1H), 7.34
(s, 1H), 8.25 (d, J=9.05 Hz, 1H); MS: (M+H).sup.+ 210.
Step 2:
Modifications: 500 mg 4-chloro-6-methoxy-2H-isoquinolin-1-one used,
400 mg product obtained.
[0233] Product: ##STR101##
[0234] Data: .sup.1H NMR (400 Hz, CDCl.sub.3) .delta. ppm 4.01 (s,
3H), 7.35 (d, J=2.45 Hz, 1H), 7.41 (d, J=2.45 Hz, 1H), 8.24 (d,
J=9.29 Hz, 1H), 8.27 (s, 1H); MS: (M+H).sup.+ 229. Method B
##STR102## Step 1:
[0235] A mixture of 4-methoxy-2-methyl-benzoic acid (5.00 g, 30.1
mmol) and thionyl chloride (20.0 g, 0.17 mol) was heated to reflux
for 30 min. Removed the volatile in vacuo. After pumping overnight,
the viscous oily acid chloride was used as crude for the next
reaction without any purification.
[0236] To a solution of 4-methoxy-2-methyl-benzoyl chloride in
CH.sub.2Cl.sub.2 (60 mL) at 0.degree. C. was added diethylamine
dropwise. The formed mixture was allowed to warm up to the ambient
temperature for 2 h with stirring. Removed the volatiles in vacuo.
The residue was triturated with EtOAc (100 mL) and filtered. The
filtrate was washed with 1M HCl, 1M NaOH and brine, dried over
MgSO.sub.4. Evaporation of the solvent yielded 6.51 g (98%) of the
desired product as a viscous oil.
[0237] LC-MS (retention time: 1.20 min, method B), MS m/z 222
(M.sup.++H).
Step 2:
[0238] To a solution of N,N-diethyl-4-methoxy-2-methyl-benzamide
(221 mg, 1.0 mmol) in THF (2 mL) at -78.degree. C. was added n-BuLi
(0.84 mL of 2.5 M in hexane, 2.10 mmol) dropwise. The formed orange
solution was kept at this temperature for additional 30 min before
dropwise addition of benzonitrile (103 mg, 1.0 mmol). The final
solution was allowed to warm up to the ambient temperature over
night with stirring. Quenched with iced 5% citric acid. Filtered,
washed with water, dried. Trituration with 2:1 hexane-EtOAc (5 mL)
yielded 205 mg (82%) of the desired product as a white solid.
[0239] .sup.1H NMR (d.sub.6-DMSO) .delta. 3.89 (s, 3H), 6.84 (s,
1H), 7.05-7.07 (m, 1H), 7.18 (d, J=2.5 Hz, 1H), 7.44-7.51 (m, 3H),
7.78 (d, J=7.0 Hz, 1H), 8.11 (d, J=9.0 Hz, 1H);
[0240] LC-MS (retention time: 1.20 min, method B), MS m/z 252
(M.sup.++H).
Step 3:
[0241] This product, 1-chloro-6-methoxy-3-phenyl-isoquinoline, was
prepared by the same method as described above except using
6-methoxy-3-phenyl-2H-isoquinolin-1-one instead.
[0242] .sup.1H NMR (CDCl.sub.3) .delta. 3.97 (s, 3H), 7.12 (d,
J=2.5 Hz, 1H), 7.23-7.26 (m, 1H), 7.40-7.42 (m, 1H), 7.46-7.50 (m,
2H), 7.89 (s, 1H), 8.08 (d, J=7.0 Hz, 2H), 8.21 (d, J=9.0 Hz, 1H);
LC-MS (retention time: 1.90 min, method B), MS m/z 270, 271
(M.sup.++H). The following intermediates were prepared as described
herein and can be incorporated into compounds of Formula 1
##STR103## To a solution of
N,N-Diethyl-4-methoxy-2-methyl-benzamide (332 mg, 1.5 mmol) in THF
(15 mL) at -78.degree. C., t-BuLi (1.7 M solution in pentane, 1.3
mL, 2.25 mmol) was added. The resulting red solution was stirred at
-78.degree. C. for 10 min, then 2-cyanopyridine (156 mg, 1.5 mmol)
was added. The reaction mixture was then warmed to rt and stirred
for overnight. The reaction was quenched with saturated NH.sub.4Cl
solution and extracted with ethyl acetate twice. The combined
organic layers were dried (MgSO.sub.4) and concentrated. The crude
product was purified by Prep. HPLC to give yellowish solid as TFA
salt. (85 mg, 15% yield)
[0243] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 3.91 (m, 3H), 7.09
(dd, J=9.05, 2.45 Hz, 1H), 7.17 (d, J=2.45 Hz, 1H), 7.37 (s, 1H),
7.42 (m, 1H), 7.92 (m, 1H), 8.08 (d, J=8.07 Hz, 1H), 8.18 (d,
J=9.05 Hz, 1H), 8.65 (d, J=4.89 Hz, 1H).
[0244] LC-MS (retention time: 2.14 min.), MS m/z 253 (MH.sup.+).
Step 2 (Scheme 3, Step 1): ##STR104##
6-Methoxy-3-pyridin-2-yl-2H-isoquinolin-1-one TFA salt (85 mg,
0.232 mmol) was heated under reflux with POCl.sub.3 (3.0 mL) for 2
days. Then POCl.sub.3 was distilled off and the residue was
quenched with ice. It was then neutralized with 10 N NaOH solution
and the brown solid was collected as pure product. (62 mg, 99%
yield)
[0245] LC-MS (retention time: 2.063 min.), MS m/z 271 (MH.sup.+).
The following intermediates were prepared as described herein and
can be incorporated into compounds of Formula 1 ##STR105## To a
solution of N,N-Diethyl-4-methoxy-2-methyl-benzamide (332 mg, 1.5
mmol) in THF (15 mL) at -78.degree. C., t-BuLi (1.7 M solution in
pentane, 1.3 mL, 2.25 mmol) was added. The resulting red solution
was stirred at -78.degree. C. for 10 min, then 4-cyanopyridine (164
mg, 1.575 mmol) was added. The reaction mixture was then warmed to
rt and stirred for overnight. The reaction was quenched with
saturated NH.sub.4Cl solution and the yellow precipitate was
collected as pure product. (145 mg, 38% yield)
[0246] .sup.1H NMR(CD.sub.3OD, 400 MHz) .delta. 3.91 (s, 3H), 7.18
(dd, J=8.8 Hz, 2.8 Hz, 1H), 7.26 (m, 2H), 8.06 (d, J=6.0 Hz, 2H),
8.16 (d, J=8.8 Hz, 1H), 8.84 (d, J=6.0 Hz, 2H).
[0247] LC-MS (retention time: 1.300 min.), MS m/z 253 (MH.sup.+).
Step 2 (Scheme 3, step 1): ##STR106##
6-Methoxy-3-pyridin-4-yl-2H-isoquinolin-1-one (134 mg, 0.531 mmol)
was heated under reflux with POCl.sub.3 (6.0 mL) for 5 days. Then
POCl.sub.3 was distilled off and the residue was quenched with ice.
It was then neutralized with saturated NaHCO.sub.3 solution and the
brown solid was collected as pure product. (125 mg, 87% yield)
[0248] .sup.1H NMR(DMSO-d.sup.6, 400 MHz) .delta. 3.99 (s, 3H),
7.53 (dd, J=9.04 Hz, 2.44 Hz, 1H), 7.59 (d, J=2.69 Hz, 1H), 8.26
(d, J=9.05 Hz, 1H), 8.30 (d, J=5.38 Hz, 2H), 8.73 (s, 1H), 8.85 (d,
J=6.36 Hz, 2H).
[0249] LC-MS (retention time: 2.027 min.), MS m/z 271 (MH.sup.+).
The following intermediates were prepared as described herein and
can be incorporated into compounds of Formula 1 ##STR107## To a
solution of N,N-Diethyl-4-methoxy-2-methyl-benzamide (332 mg, 1.5
mmol) in THF (15 mL) at -78.degree. C., t-BuLi (1.7 M solution in
pentane, 1.3 mL, 2.25 mmol) was added. The resulting red solution
was stirred at -78.degree. C. for 10 min, then 4-dimethylamino
benzonitrile (219 mg, 1.5 mmol) was added. The reaction mixture was
then warmed to rt and stirred for overnight. The reaction was
quenched with saturated NH.sub.4Cl solution and the yellow
precipitate was collected and triturated with ether to give an
off-white solid as pure product. (247 mg, 56% yield)
[0250] .sup.1H NMR(DMSO-d.sup.6, 400 MHz) .delta. 2.97 (s, 6H),
3.87 (s, 3H), 6.72 (s, 1H), 6.78 (d, J=8.80 Hz, 2H), 6.97 (dd,
J=8.80, 2.45 Hz, 1H), 7.10 (d, J=2.45 Hz, 1H), 7.65 (d, J=8.80 Hz,
2H), 8.05 (d, J=8.80 Hz, 1H), 11.11 (s, 1H).
[0251] LC-MS (retention time: 2.023 min.), MS m/z 295 (MH.sup.+).
##STR108##
3-(4-Dimethylamino-phenyl)-6-methoxy-2H-isoquinolin-1-one (245 mg,
0.83 mmol) was heated under reflux with POCl.sub.3 (10.0 mL) for 2
days. Then POCl.sub.3 was distilled off and the residue was
quenched with ice. It was then neutralized with 10 N NaOH solution
and extracted with ethyl acetate twice. The organic layers were
combined and dried (MgSO4). Evaporation of solvent gave an orange
solid as product (215 mg, 83% yield)
[0252] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 3.01 (s, 6H), 3.96
(s, 3H), 6.88 (d, J=9.05 Hz, 2H), 7.20 (dd, J=9.17, 2.57 Hz, 1H),
7.28 (d, J=2.45 Hz, 1H), 7.94 (s, 1H), 7.96 (d, J=9.05 Hz, 2H),
8.13 (d, J=9.29 Hz, 1H).
[0253] LC-MS (retention time: 2.543 min.), MS m/z 313 (MH.sup.+).
##STR109## A mixture of
[4-(1-Chloro-6-methoxy-isoquinolin-3-yl)-phenyl]-dimethyl-amine
(110 mg, 0.35 mmol) and tetrabutyl phosphonium hydrogen difluoride
(0.5 g) was heated at 140.degree. C. in Smith microwave reactor for
20 min. Then it was added water and extracted with ethyl acetate.
The organic layer was separated, washed with water and dried
(MgSO.sub.4). Evaporation of solvent gave a brownish solid as
product. (85 mg, 82% yield)
[0254] LC-MS (retention time: 2.320 min.), MS m/z 297 (MH.sup.+).
Method C ##STR110## Step 1:
[0255] To a solution of N-BOC-3-(R)-hydroxy-L-proline (6.22 g, 26.9
mmol) in DMF (250 mL) at 0.degree. C. was added NaH (60%, 3.23 g,
80.8 mmol) by several portions. The formed suspension was stirred
at this temperature for 30 min. 1,3-dichloroisoquinoline (5.33 g,
26.9 mmol) was added as solid in one portion and the final mixture
was stirred at the ambient temperature for 12 h. Quenched with iced
5% citric acid (aq), extracted with EtOAC (300 mL). The aqueous
phase was extracted with EtOAC again. The combined organic layers
were washed with 5% citric acid (aq) and brine respectively, dried
over MgSO.sub.4, filtered. The filtrate was evaporated in vacuo to
dryness to yield 10.53 g (99.8%) of
4-(6-methoxy-isoquinolin-1-yloxy)-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester as an off-white foam. This material was used in
the next step reaction as crude without further purification.
[0256] .sup.1H NMR (CD.sub.3OD) .delta. 1.43, 1.44 (rotamers, 9H),
2.39-2.44 (m, 1H), 2.68-2.72 (m, 1H), 3.80-3.90 (m, 2H), 4.44-4.52
(m, 1H), 5.77 (b, 1H), 7.39 (s, 1H), 7.58 (t, J=7.3 Hz, 1H),
7.71-7.78 (m, 2H), 8.16 (d, J=7.5 Hz, 1H);
[0257] LC-MS (retention time: 1.80 min, method B), MS m/z 392
(M.sup.++H).
Step 2:
[0258] A mixture of of
4-(6-methoxy-isoquinolin-1-yloxy)-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester (39 mg, 0.10 mmol), phenylboronic acid (14.6 mg,
0.12 mmol), sodium tert-butoxide (38 mg, 0.40 mmol) and
((t-Bu).sub.2POH).sub.2PdCl.sub.2 (POPd) (5 mg, 0.01 mmol) in THF
(2 mL) was heated to reflux for 4 h. After cooling down, the formed
mixture was quenched with 5% citric acid (aq) and extracted with
EtOAc (20 mL). The organic layer was washed with brine, dried over
MgSO.sub.4, filtered, evaporated. The residue was purified by
prep-HPLC to yield 36 mg (83%) of the desired product as an
off-white foam.
[0259] .sup.1H NMR (CD.sub.3OD) .delta. 1.43, 1.45 (rotamers, 9H),
2.51-2.56 (m, 1H), 2.74-2.82 (m, 1H), 3.88-3.92 (m, 1H), 3.98-4.01
(m, 1H), 4.50-4.57 (m, 1H), 5.95 (b, 1H), 7.36-7.39 (m, 1H),
7.45-7.48 (m, 2H), 7.55 (t, J=7.3 Hz, 1H), 7.70 (t, J=7.5 Hz, 1H),
7.84-7.89 (m, 2H), 8.14-8.17 (m, 3H), 9.05 (b, 1H);
[0260] LC-MS (retention time: 1.97 min, method B), MS m/z 435
(M.sup.++H). The following intermediates were prepared as described
herein and can be incorporated into compounds of Formula 1
##STR111## Prepared using 4-methoxyphenylboronic acid
[0261] .sup.1H NMR (CD.sub.3OD) .delta. 1.40, 1.45 (rotamers, 9H),
2.50-2.55 (m, 1H), 2.73-2.81 (m, 1H), 3.81-3.89 (m, 4H), 3.98-4.01
(m, 1H), 4.50-4.57 (m, 1H), 5.93 (b, 1H), 7.02 (d, J=9.0 Hz, 2H),
7.50 (t, J=7.3 Hz, 1H), 7.67 (t, J=7.5 Hz, 1H), 7.73 (s, 1H), 7.83
(d, J=8.5 Hz, 1H), 8.09 (d, J=8.5 Hz, 2H), 8.15 (d, J=8.0 Hz,
1H);
[0262] LC-MS (retention time: 2.00 min, method B), MS m/z 465
(M.sup.++H). The following intermediate was prepared as described
above: ##STR112## Prepared using 4-pyridylboronic acid
[0263] .sup.1H NMR (CD.sub.3OD) .delta. 1.43, 1.46 (rotamers, 9H),
2.53-2.56 (m, 1H), 2.80-2.89 (m, 1H), 3.90-3.93 (m, 1H), 4.00-4.05
(m, 1H), 4.50-4.57 (m, 1H), 6.00, 6.05(rotamers, 1H), 7.80 (t,
J=7.3 Hz, 1H), 7.87 (t, J=7.5 Hz, 1H), 8.08 (d, J=8.5 Hz, 1H), 8.32
(d, J=8.0 Hz, 1H), 8.49 (s, 1H), 8.84 (d, J=6.0 Hz, 2H), 8.84 (d,
J=6.5 Hz, 2H);
[0264] LC-MS (retention time: 1.39 min, method B), MS m/z 436
(M.sup.++H). The following intermediates were prepared as described
herein and can be incorporated into compounds of Formula 1
##STR113## Prepared using 4-N,N-dimethylamino-phenylboronic
acid
[0265] LC-MS (retention time: 1.64 min, method B), MS m/z 478
(M.sup.++H). Method D ##STR114## Step 1:
[0266] To a solution of N,N-diethyl-4-methoxy-2-methyl-benzamide
(633 mg, 2.9 mmol) in THF (15 mL) at -78.degree. C. was added
n-BuLi (2.3 mL of 2.5 M in hexane, 5.74 mmol) dropwise. The formed
red solution was kept at this temperature for additional 30 min
before being cannulated to a solution of thiazole-2-carboxylic acid
ethyl ester (A. Medici et al, Tetrahedron Lett. 1983, p2901) (450
mg, 2.9 mmol) in THF (5 mL) at -78.degree. C. The final dark green
solution was kept to this temperature for 2 h with stirring.
Quenched with sat. NH.sub.4Cl (aq) and extracted with EtOAc (50
mL). The organic layer was washed with sat. NH.sub.4Cl (aq) and
brine, dried, purified by flash column chromatography, eluting with
2:1 EtOAc:hexane to provide 405 mg (45%) of the desired product as
an off-white viscous oil.
[0267] .sup.1H NMR (CDCl.sub.3) .delta. 1.08 (t, J=7.0 Hz, 6H),
3.22 (b, 2H), 3.44 (b, 2H), 3.79 (s, 3H), 4.59 (s, 2H), 6.79-6.81
(m, 1H), 6.86 (d, J=2.5 Hz, 1H), 7.16 (d, J=8.5 Hz, 1H), 7.66 (d,
J=3.0 Hz, 1H), 8.00 (d, J=3.0 Hz, 1H);
[0268] LC-MS (retention time: 1.30 min, method B), MS m/z 333
(M.sup.++H).
Step 2:
[0269] A mixture of
N,N-diethyl-4-methoxy-2-(2-oxo-2-thiazol-2-yl-ethyl)-benzamide (405
mg, 1.22 mmol) and NH.sub.4OAc (3.0 g, 38.9 mmol) was heated to
140.degree. C. in a sealed tube for 1 h. The melted solution was
poured into iced water, filtered, washed the cake thoroughly with
water. The dried brownish solid (240 mg, 76%) was used as crude for
the next reaction without further purification.
[0270] LC-MS (retention time: 1.24 min, method B), MS m/z 259
(M.sup.++H).
Step 3:
[0271] This product,
1-chloro-6-methoxy-3-thiazol-2-yl-isoquinoline, was prepared as
described above except using
6-methoxy-3-thiazol-2-yl-2H-isoquinolin-1-one instead.
[0272] .sup.1H NMR (CDCl.sub.3) .delta. 3.97 (s, 3H), 7.16 (d,
J=4.0 Hz, 1H), 7.27-7.31 (m, 1H), 7.46 (d, J=5.0 Hz, 1H), 7.93 (d,
J=5.5 Hz, 1H), 8.22 (d, J=15.5 Hz, 1H), 8.39 (s, 1H);
[0273] LC-MS (retention time: 1.66 min, method B), MS m/z 277
(M.sup.++H).
Step 4:
[0274] This product was prepared by the same method as described
above except using 1-chloro-6-methoxy-3-thiazol-2-yl-isoquinoline
instead.
[0275] .sup.1H NMR (CD.sub.3OD) .delta. 0.97-1.09 (m, 12H),
1.24-1.29 (m, 10H), 1.44-1.46 (m, 1H), 1.87-1.90 (m, 1H), 2.20-2.26
(m, 1H), 2.30-2.36 (m. 1H), 2.65-2.71 (m, 1H), 2.93-2.96 (m, 1H),
3.96 (s, 3H), 4.12-4.27 (m, 2H), 4.38-4.52 (m, 2H), 5.12 (d, J=10.5
Hz, 1H), 5.29 (d, J=17.5 Hz, 1H), 5.69-5.74 (m, 1H), 5.99 (b, 1H),
7.14 (d, J=9.0 Hz, 1H), 7.33 (s, 1H), 7.66 (d, J=3.5 Hz, 1H), 7.93
(d, J=3.0 Hz, 1H), 8.05 (s, 1H), 8.11 (d, J=9.0 Hz, 1H), 9.14 (b,
1H);
[0276] LC-MS (retention time: 1.89 min, method B), MS m/z 797
(M.sup.++H). The following intermediates were prepared as described
herein and can be incorporated into compounds of Formula 1
##STR115##
6-methoxy-3-(3-methoxy-isoxazol-5-yl)-2H-isoquinolin-1-one was
prepared using
N,N-diethyl-4-methoxy-2-[2-(3-methoxy-isoxazol-5-yl)-2-oxo-ethyl]-b-
enzamide.
[0277] .sup.1H NMR (DMSO-d.sub.6) .delta. 3.89 (s, 3H), 3.97 (s,
3H), 7.01 (s, 1H), 7.14-7.16 (m, 2H), 7.43 (s, 1H), 8.13 (d, J=8.5
Hz, 1H);
[0278] LC-MS (retention time: 1.31 min, method B), MS m/z 273
(M.sup.++H). ##STR116##
1-chloro-6-methoxy-3-(3-methoxy-isoxazol-5-yl)-isoquinoline was
prepared using
6-methoxy-3-(3-methoxy-isoxazol-5-yl)-2H-isoquinolin-1-one
[0279] .sup.1H NMR (CDCl.sub.3) .delta. 3.97 (s, 3H), 4.04 (s, 3H),
6.60 (s, 1H), 7.17 (d, J=2.5 Hz, 1H), 7.31-7.33 (m, 1H), 8.02 (s,
1H), 8.23 (d, J=9.0 Hz, 1H);
[0280] LC-MS (retention time: 1.73 min, method B), MS m/z 291, 293
(M.sup.++H) The following intermediates were prepared as described
herein and can be incorporated into compounds of Formula 1
##STR117##
N,N-diethyl-4-methoxy-2-[2-(5-methoxy-oxazol-2-yl)-2-oxo-ethyl]-benzamide-
, was prepared using 5-methoxy-oxazole-2-carboxylic acid ethyl
ester
[0281] LC-MS (retention time: 1.24 min, method B), MS m/z 347
(M.sup.++H). ##STR118##
6-methoxy-3-(5-methoxy-oxazol-2-yl)-2H-isoquinolin-1-one, was
prepared using
N,N-diethyl-4-methoxy-2-[2-(5-methoxy-oxazol-2-yl)-2-oxo-ethyl]-ben-
zamide.
[0282] .sup.1H NMR (DMSO-d.sub.6) .delta. 3.94 (s, 3H), 4.01 (s,
3H), 6.34 (s, 1H), 6.99 (d, J=2.0 Hz, 1H), 7.12-7.14 (m, 1H), 7.25
(s, 1H), 8.32 (d, J=9.0 Hz, 1H);
[0283] LC-MS (retention time: 1.22 min, method B), MS m/z 274
(M.sup.++H). ##STR119##
1-chloro-6-methoxy-3-(5-methoxy-oxazol-2-yl)-isoquinoline, was
prepared using
6-methoxy-3-(5-methoxy-oxazole-2-yl)-2H-isoquinolin-1-one.
[0284] .sup.1H NMR (CDCl.sub.3) .delta. 3.96 (s, 3H), 4.00 (s, 3H),
6.34 (s, 1H), 7.12 (d, J=2.5 Hz, 1H), 7.28-7.31 (m, 1H), 8.13 (s,
1H), 8.23 (d, J=9.0 Hz, 1H);
[0285] LC-MS (retention time: 1.58 min, method B), MS m/z 291, 293
(M.sup.++H). The following intermediates were prepared as described
herein and can be incorporated into compounds of Formula 1
##STR120## To a solution of
N,N-Diethyl-4-methoxy-2-methyl-benzamide (332 mg, 1.5 mmol) in THF
(15 mL) at -78.degree. C., t-BuLi (1.7 M solution in pentane, 2.12
mL, 3.6 mmol) was added. The resulting red solution was stirred at
-78.degree. C. for 10 min, then methyl nicotinate (206 mg, 1.5
mmol) was added. The reaction mixture was stirred at -78.degree. C.
for 2 h. Then the reaction was quenched with saturated NH.sub.4Cl
solution and extracted with ethyl acetate twice. The combined
organic layers were dried (MgSO.sub.4) and concentrated. The crude
product was purified by Prep. HPLC to give yellowish thick oil as
TFA salt. (124 mg, 19% yield)
[0286] LC-MS (retention time: 1.740 min.), MS m/z 349 (M+Na.sup.+).
##STR121##
N,N-Diethyl-4-methoxy-2-(2-oxo-2-pyridin-3-yl-ethyl)-benzamide (120
mg, 0.272 mmol) was heated with ammonium acetate (1 g) for 3 hr.
Then it was cooled down and added water. Extracted with ethyl
acetate and the organic layer was separated. It was then dried
(MgSO.sub.4) and concentrated to give a brownish solid as product.
(65 mg, 95% yield)
[0287] .sup.1H NMR (400 MHz, DMSO-d.sup.6) .delta. 3.89 (s, 3H),
6.93 (s, 1H), 7.10 (dd, J=8.80, 2.45 Hz, 1H), 7.19 (d, J=2.45 Hz,
1H), 7.52 (dd, J=7.46, 4.77 Hz, 1H), 8.15 (m, 2H), 8.64 (dd,
J=4.89, 1.47 Hz, 1H), 8.96 (d, J=1.71 Hz, 1H), 11.51 (s, 1H).
[0288] LC-MS (retention time: 1.377 min.), MS m/z 253 (MH.sup.+).
##STR122## 6-Methoxy-3-pyridin-3-yl-2H-isoquinolin-1-one (65 mg,
0.258 mmol) was heated under reflux with POCl.sub.3 (2.5 mL) for 7
days. Then POCl.sub.3 was distilled off and the residue was
quenched with ice. It was then neutralized with 10 N NaOH solution
and extracted with ethyl acetate twice. The combined organic layers
were dried (MgSO.sub.4) and concentrated to give yellow solid as
product. (27 mg, 39% yield)
[0289] LC-MS (retention time: 2.090 min.), MS m/z 271 (MH.sup.+).
The following intermediates were prepared as described herein and
can be incorporated into compounds of Formula 1 ##STR123## To a
solution of N,N-Diethyl-4-methoxy-2-methyl-benzamide (332 mg, 1.5
mmol) in THF (15 mL) at -78.degree. C., t-BuLi (1.7 M solution in
pentane, 2.2 mL, 3.75 mmol) was added. The resulting red solution
was stirred at -78.degree. C. for 10 min, then
N,N-dimethylanthranilic acid methyl ester (269 mg, 1.5 mmol) was
added. The reaction mixture was stirred at -78.degree. C. for 2 h.
Then the reaction was quenched with saturated NH.sub.4Cl solution
and extracted with ethyl acetate twice. The combined organic layers
were dried (MgSO.sub.4) and concentrated. The crude product was
purified by Prep. HPLC to give yellowish thick oil as product. (256
mg, 46% yield)
[0290] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.99-1.13 (m, 6H),
3.23-3.31 (m, 8H), 3.39 (m, 2H), 3.82 (s, 3H), 4.35 (s, 2H), 6.91
(dd, J=8.44, 2.57 Hz, 1H), 6.99 (d, J=2.45 Hz, 1H), 7.22 (d, J=8.56
Hz, 1H), 7.69 (t, J=7.70 Hz, 1H), 7.84 (m, 1H), 7.96 (d, J=8.31 Hz,
1H), 8.18 (d, J=7.83 Hz, 1H).
[0291] LC-MS (retention time: 1.557 min.), MS m/z 369(MH.sup.+).
##STR124##
2-[2-(2-Dimethylamino-phenyl)-2-oxo-ethyl]-N,N-diethyl-4-methoxy-benzamid-
e (250 mg, 0.678 mmol) was heated with ammonium acetate (1.5 g) for
2 hr. Then it was cooled down and added water. Extracted with ethyl
acetate and the organic layer was separated. It was then dried
(MgSO.sub.4) and concentrated to give a yellowish solid as product.
(125 mg, 63% yield)
[0292] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 2.95 (s, 6H), 3.92
(s, 3H), 6.92 (s, 1H), 7.12 (dd, J=8.80, 2.45 Hz, 1H), 7.16 (d,
J=2.45 Hz, 1H), 7.35 (m, 1H), 7.55 (m, 2H), 7.63 (d, J=7.83 Hz,
1H), 8.20 (d, J=9.05 Hz, 1H).
[0293] LC-MS (retention time: 2.097 min.), MS m/z 295 (MH.sup.+).
##STR125##
3-(2-Dimethylamino-phenyl)-6-methoxy-2H-isoquinolin-1-one (125 mg,
0.425 mmol) was heated under reflux with POCl.sub.3 (4.0 mL) for
one day. Then POCl.sub.3 was distilled off and the residue was
quenched with ice. It was then neutralized with 10 N NaOH solution
and extracted with ethyl acetate twice. The organic layers were
combined and dried (MgSO.sub.4). Evaporation of solvent gave a
brownish solid as product (82 mg, 62% yield)
[0294] LC-MS (retention time: 2.040 min.), MS m/z 313 (MH.sup.+).
##STR126## A mixture of
[2-(1-Chloro-6-methoxy-isoquinolin-3-yl)-phenyl]-dimethyl-amine (82
mg, 0.262 mmol) and tetrabutyl phosphonium hydrogen difluoride (1.0
g) was heated at 140.degree. C. in Smith microwave reactor for 20
min. Then it was added water and extracted with ethyl acetate. The
organic layer was separated, washed with water and dried
(MgSO.sub.4). Evaporation of solvent gave the crude product which
was purified by Prep. HPLC to afford a yellowish oil as product.
(85 mg)
[0295] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 3.41 (s, 6H), 4.00
(s, 3H), 7.42 (dd, J=9.05, 2.45 Hz, 1H), 7.53 (s, 1H), 7.71 (m,
2H), 7.99 (m, 1H), 8.16 (m, 2H), 8.31 (s, 1H).
[0296] LC-MS (retention time: 1.873 min.), MS m/z 297 (MH.sup.+).
The following intermediates were prepared as described herein and
can be incorporated into compounds of Formula 1 ##STR127## To a
solution of N,N-Diethyl-4-methoxy-2-methyl-benzamide (332 mg, 1.5
mmol) in THF (15 mL) at -78.degree. C., t-BuLi (1.7 M solution in
pentane, 2.2 mL, 3.75 mmol) was added. The resulting red solution
was stirred at -78.degree. C. for 10 min, then
(3-dimethylamino)benzoic acid methyl ester (269 mg, 1.5 mmol) was
added. The reaction mixture was stirred at -78.degree. C. for 2 h.
Then the reaction was quenched with saturated NH.sub.4Cl solution
and extracted with ethyl acetate twice. The combined organic layers
were dried (MgSO.sub.4) and concentrated. The crude product was
purified by Prep. HPLC to give yellowish thick oil as TFA salt.
(245 mg, 33% yield)
[0297] 1H NMR (400 MHz, CD.sub.3OD) .delta. 1.01 (t, J=6.85 Hz,
3H), 1.09 (m, 3H), 3.11 (s, 6H), 3.21 (m, 2H), 3.40 (m, 2H), 3.79
(s, 3H), 4.39 (s, 2H), 6.84-6.91 (m, 2H), 7.19 (d, J=8.32 Hz, 1H),
7.35 (m, 1H), 7.49 (t, J=8.07 Hz, 1H), 7.66-7.71 (m, 2H).
[0298] LC-MS (retention time: 1.930 min.), MS m/z 369(MH.sup.+).
##STR128##
2-[2-(3-Dimethylamino-phenyl)-2-oxo-ethyl]-N,N-diethyl-4-methoxy-benzamid-
e (240 mg, 0.497 mmol) was heated with ammonium acetate (2.0 g) for
2.5 hr. Then it was cooled down and added water. A brownish solid
was collected as pure product. (95 mg, 65% yield)
[0299] 1H NMR (400 MHz, CD.sub.3OD) .delta. 2.98 (s, 6H), 3.88 (s,
3H), 6.74-6.87 (m, 2H), 7.01-7.07 (m, 3H), 7.18 (d, J=2.44 Hz, 1H),
7.28 (t, J=7.82 Hz, 1H), 8.10 (d, J=8.80 Hz, 1H).
[0300] LC-MS (retention time: 1.773 min.), MS m/z 295 (MH.sup.+).
##STR129##
3-(3-Dimethylamino-phenyl)-6-methoxy-2H-isoquinolin-1-one (92 mg,
0.312 mmol) was heated under reflux with POCl.sub.3 (3.0 mL) for 2
days. Then POCl.sub.3 was distilled off and the residue was
quenched with ice. It was then neutralized with saturated
NaHCO.sub.3 solution and extracted with ethyl acetate twice. The
organic layers were combined and dried (MgSO.sub.4). Evaporation of
solvent gave a brownish thick oil as product. (72 mg, 74%
yield)
[0301] LC-MS (retention time: 2.297 min.), MS m/z 313 (MH.sup.+).
##STR130## A mixture of
[3-(1-Chloro-6-methoxy-isoquinolin-3-yl)-phenyl]-dimethylamine (72
mg, 0.23 mmol) and tetrabutyl phosphonium hydrogen difluoride (0.5
g) was heated at 140.degree. C. in Smith microwave reactor for 20
min. Then it was added water and extracted with ethyl acetate. The
organic layer was separated, washed with water and dried
(MgSO.sub.4). Evaporation of solvent gave a brownish oil as
product. (58 mg, 85% yield)
[0302] LC-MS (retention time: 2.193 min.), MS m/z 297 (MH.sup.+).
The following intermediates were prepared as described herein and
can be incorporated into compounds of Formula 1 ##STR131##
Condensation of ethyl bromopyruvate with ethyl thiourea in
refluxing dioxane afforded the monoalkylamino thiazole as HBr salt
in quantitative yield. Alkylation of
2-ethylamino-thiazole-4-carboxylic acid ethyl ester with EtI in DMF
provided 2-diethylamino-thiazole-4-carboxylic acid ethyl ester.
LC/MS m/z 229 (MH).sup.+ Method E ##STR132## Step 1: A suspension
of 2-cyanomethyl-4-methoxy-benzoic acid methyl ester (1.9 g and
TsOH. H.sub.2O (0.15 g, mmol) in morpholine 5 mL) was refluxed for
4 h and removed the solvent in vavuo. The residue was recrystalyzed
from EtOAc/hexanes with drops of MeOH to provide the product (0.43
g, 17%): LC-MS retention time: 1.07 method H), MS m/z 266
(M.sup.++1). Step 2: A mixture of
6-methoxy-3-morpholin-4-yl-isoquinolin-1-ol (0.298 g, 1.15 mmol) in
POCl.sub.3 (20 mL) was refluxed for 2 h, removed the solvent in
vacuo and cold water was added. The pH was adjustde to >1 by
addition of 1.0 N NaOH. The aqueous layer was extracted with EtOAc.
The extract was dried (MgSO.sub.4), removed the solvent in vacuo to
provide the produt (0.299 g, 94%): LC-MS retention time: 1.68
method H), MS m/z 279 (M.sup.++1). Step 3: A mixture of
1-Chloro-6-methoxy-3-morpholin-4-yl-isoquinoline (0.050 g, 0.18
mmol) and tetrabutyl phosphorium hydrgen difloride (0.8 g, 2.8
mmol) [Synlett 1992, (4), 345-6] was heated at 140.degree. C. in
microwave for 10 min. the reaction mixture was diluted with EtOAc
and filtered through an ISCO 25 g precolumn with a layer of silicon
gel on the top, removed the solvent to provide the product (0.037
mg, 77%): .sup.1H NMR (CHLOROFORM-D) .delta. ppm 3.48 (m, 4H), 3.84
(m, 4H), 3.89 (s, 3H), 6.46 (d, J=1.22 Hz, 1H), 6.85 (s, 1H), 6.90
(dd, J=9.16, 2.44 Hz, 1H), 7.82 (d, J=8.85 Hz, 1H). LC-MS retention
time: 1.56 method H), MS m/z 263 (M.sup.++1). Method F 6-fluoro and
6-alkyl isoquinolines used in the preparation of compounds of
Formula 1 were prepared via a Pomeranz-Fritsch synthesis (Typical
procedure: Preparation of optically active 8,8-disubstituted
1,1-biisoquinoline, K. Hirao, R. Tsuchiya, Y. Yano, H. Tsue,
Heterocycles 42(1) 1996, 415-422) as outlined below. The products
were converted into the 1-chloro derivatives via N-oxide
intermediates. ##STR133##
[0303] Reagents and reaction conditions: (a) reflux in benzene,
azeotropic removal of water; (b) first step: ethyl chloroformate,
trimethyl phosphite in THF, second step: titanium tetrachloride in
chloroform; (c) MCPBA in CH.sub.2Cl.sub.2; (d) POCl.sub.3 in
benzene TABLE-US-00002 R Isoquinoline, Yield 1-Chloride, combined
yield F 20 43 Et 76 65 i-Pr 14 18 t-Bu 47 55
Preparation of 6-isopropoxyl and 6-tert-butoxyl isoquinoline
intermediates: Some 6-alkoxy-1-chloro isoquinolines were prepared
by a direct, ipso displacement of the 6-fluoro-1-chloroisoquinoline
with the corresponding alkoxide metal ions such as potassium
tert-butoxide (53%) and sodium isopropoxide (54%). ##STR134## The
6-fluoro-1-chloroisoquinoline was subjected to an aromatic
nucleophilic displacement with sodium isopropoxide and potassium
tert-butoxide in DMF to give the corresponding 6-isopropoxyl (54%):
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.43 (d, J=6.11 Hz,
6H) 4.76 (m, J=6.11 Hz, 1H) 7.08 (d, J=2.45 Hz, 1H) 7.29 (dd,
J=9.29, 2.45 Hz, 1H) 7.50 (d, J=5.62 Hz, 1H) 8.18 (d, J=5.87 Hz,
1H) 8.24 (d, J=9.29 Hz, 1H) and 6-tert-butoxyl-1-chloro
isoquinolines (55%): .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.
ppm 1.48 (s, 9H) 7.31 (m, 2H) 7.47 (d, J=5.62 Hz, 1H) 8.18 (d,
J=5.62 Hz, 1H) 8.21 (d, J=9.78 Hz, 1H) as the major product
respectively. These 6-alkoxyl-1-chloro isoquinolines were
incorporated into compounds of Formula 1 as described herein.
Method G ##STR135## This synthesis made use of the technologies
described, in part, in the following references: [0304] (1) Hojo,
Masaru; Masuda, Ryoichi; Sakaguchi, Syuhei; Takagawa, Makoto,
Synthesis (1986), (12), 1016-17 [0305] (2) Rigby, James H.;
Holsworth, Daniel D.; James, Kelly. Vinyl Isocyanates In Synthesis.
[4+2] Cycloaddition Reactions With Benzyne Addends. Journal Of
Organic Chemistry (1989), 54(17), 4019-20 [0306] (3) Uchibori, Y.;
Umeno, M.; Yoshiokai, H.; Heterocycles, 1992, 34 (8), 1507-1510
Step 1d: Preparation of racemic
(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid ethyl
ester hydrochloride (Method A and Method B) and chiral resolution
of this racemate for the preparation of
N-(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid ethyl ester
hydrochloride (Method C)
[0307] ##STR136##
[0308] The named compound was made (Step d) racemic by each of the
following methods A and B. This racemate could also be resolved to
afford chiral Boc-(1R,2S)-1-amino-2-vinylcyclopropyl carboxylic
acid ester which was deprotected under acid conditions to afford
(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid ester
hydrochloride (Method C).
Method A
A.1 Preparation of N-Benzyl Imine of Glycine Ethyl Ester
[0309] ##STR137##
[0310] Glycine ethyl ester hydrochloride (303.8 g, 2.16 mole) was
suspended in tert-butylmethyl ether (1.6 L). Benzaldehyde (231 g,
2.16 mole) and anhydrous sodium sulfate (154.6 g, 1.09 mole) were
added and the mixture cooled to 0.degree. C. using an ice-water
bath. Triethylamine (455 mL, 3.26 mole) was added dropwise over 30
min and the mixture stirred for 48 h at rt. The reaction was then
quenched by addition of ice-cold water (1 L) and the organic layer
was separated. The aqueous phase was extracted with
tert-butylmethyl ether (0.5 L) and the combined organic phases
washed with a mixture of saturated aqueous NaHCO.sub.3 (1 L) and
brine (1 L). The solution was dried over MgSO.sub.4, concentrated
in vacuo to afford 392.4 g of the N-benzyl imine product as a thick
yellow oil that was used directly in the next step. .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. 1.32 (t, J=7.1 Hz, 3H), 4.24 (q,
J=7.1 Hz, 2H), 4.41 (d, J=1.1 Hz, 2H), 7.39-7.47 (m, 3H), 7.78-7.81
(m, 2H), 8.31 (s, 1H).
A.2 Preparation of racemic
N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester
[0311] ##STR138##
[0312] To a suspension of lithium tert-butoxide (84.06 g, 1.05 mol)
in dry toluene (1.2 L), was added dropwise a mixture of the
N-benzyl imine of glycine ethyl ester (100.4 g, 0.526 mol) and
trans-1,4-dibromo-2-butene (107.0 g, 0.500 mol) in dry toluene (0.6
L) over 60 min. After completion of the addition, the deep red
mixture was quenched by addition of water (1 L) and
tert-butylmethyl ether (TBME, 1 L). The aqueous phase was separated
and extracted a second time with TBME (1 L). The organic phases
were combined, 1 N HCl (1 L) was added and the mixture stirred at
room temperature for 2 h. The organic phase was separated and
extracted with water (0.8 L). The aqueous phases were then
combined, saturated with salt (700 g), TBME (1 L) was added and the
mixture cooled to 0.degree. C. The stirred mixture was then
basified to pH 14 by the dropwise addition of 10 N NaOH, the
organic layer separated, and the aqueous phase extracted with TBME
(2.times.500 mL). The combined organic extracts were dried
(MgSO.sub.4) and concentrated to a volume of 1 L. To this solution
of free amine, was added BOC.sub.2O or di-tert-butyldicarbonate
(131.0 g, 0.6 mol) and the mixture stirred 4 days at rt. Additional
di-tert-butyldicarbonate (50 g, 0.23 mol) was added to the
reaction, the mixture refluxed for 3 h, and was then allowed cool
to room temperature overnight. The reaction mixture was dried over
MgSO.sub.4 and concentrated in vacuo to afford 80 g of crude
material. This residue was purified by flash chromatography (2.5 Kg
of SiO.sub.2, eluted with 1% to 2% MeOH/CH.sub.2Cl.sub.2) to afford
57 g (53%) of racemic
N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester as a yellow oil which solidified while sitting in the
refrigerator: .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 1.26 (t,
J=7.1 Hz, 3H), 1.46 (s, 9H), 1.43-1.49 (m, 1H), 1.76-1.82 (br m,
1H), 2.14 (q, J=8.6 Hz, 1H), 4.18 (q, J=7.2 Hz, 2H), 5.12 (dd
J=10.3, 1.7 Hz, 1H), 5.25 (br s, 1H), 5.29 (dd, J=17.6, 1.7 Hz,
1H), 5.77 (ddd, J=17.6, 10.3, 8.9 Hz, 1H); MS m/z 254.16 (M-1)
A.3 Preparation of Racemic (1R,2S)/(1S,2R)
1-amino-2-vinylcyclopropane carboxylic acid ethyl ester
hydrochloride
[0313] ##STR139##
[0314] N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic
acid ethyl ester (9.39 g, 36.8 mmol) was dissolved in 4 N
HCl/dioxane (90 ml, 360 mmol) and was stirred for 2 h at rt. The
reaction mixture was concentrated to supply
(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid ethyl
ester hydrochloride in quanitative yield (7 g, 100%). .sup.1H NMR
(methanol-d.sub.4) .delta. 1.32 (t, J=7.1, 3H), 1.72 (dd, J=10.2,
6.6 Hz, 1H), 1.81 (dd, J=8.3, 6.6 Hz, 1H), 2.38 (q, J=8.3 Hz, 1H),
4.26-4.34 (m, 2H), 5.24 (dd, 10.3, 1.3 Hz, 1H) 5.40 (d, J=17.2,
1H), 5.69-5.81 (m, 1H).
B.1 Preparation of Racemic N-Boc-1-amino-2-vinylcyclopropane
carboxylic acid ethyl ester hydrochloride
Method B
[0315] ##STR140##
[0316] To a solution of potassium tert-butoxide (11.55 g, 102.9
mmol) in THF (450 mL) at -78.degree. C. was added the commercially
available N,N-dibenzyl imine of glycine ethyl ester (25.0 g, 93.53
mmol) in THF (112 mL). The reaction mixture was warmed to 0.degree.
C., stirred for 40 min, and was then cooled back to -78.degree. C.
To this solution was added trans-1,4-dibromo-2-butene (20.0 g,
93.50 mmol), the mixture stirred for 1 h at 0.degree. C. and was
cooled back to -78.degree. C. Potassium tert-butoxide (11.55 g,
102.9 mmol) was added, the mixture immediately warmed to 0.degree.
C., and was stirred one more hour before concentrating in vacuo.
The crude product was taken up in Et.sub.2O (530 mL), 1N aq. HCl
solution (106 mL, 106 mmol) added and the resulting biphasic
mixture stirred for 3.5 h at rt. The layers were separated and the
aqueous layer was washed with Et.sub.2O (2.times.) and basified
with a saturated aq. NaHCO.sub.3 solution. The desired amine was
extracted with Et.sub.2O (3.times.) and the combined organic
extract was washed with brine, dried (MgSO.sub.4), and concentrated
in vacuo to obtain the free amine. This material was treated with a
4N HCl solution in dioxane (100 mL, 400 mmol) and concentrated to
afford (1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester hydrochloride as a brown semisolid (5.3 g, 34% yield)
identical to the material obtained from procedure A, except for the
presence of a small unidentified aromatic impurity (8%).
Method C
C.1 Resolution of N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane
carboxylic acid ethyl ester
[0317] ##STR141## Resolution A
[0318] To an aqueous solution of sodium phosphate buffer (0.1 M,
4.25 liter ("L"), pH 8) housed in a 12 Liter jacked reactor,
maintained at 39.degree. C., and stirred at 300 rpm was added 511
grams of Alcalase 2.4 L (about 425 mL) (Novozymes North America
Inc.). When the temperature of the mixture reached 39.degree. C.,
the pH was adjusted to 8.0 by the addition of a 50% NaOH in water.
A solution of the racemic
N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester (85 g) in 850 mL of DMSO was then added over a period
of 40 min. The reaction temperature was then maintained at
40.degree. C. for 24.5 h during which time the pH of the mixture
was adjusted to 8.0 at the 1.5 h and 19.5 h time points using 50%
NaOH in water. After 24.5 h, the enantio-excess of the ester was
determined to be 97.2%, and the reaction was cooled to room
temperature (26.degree. C.) and stirred overnight (16 h) after
which the enantio-excess of the ester was determined to be 100%.
The pH of the reaction mixture was then adjusted to 8.5 with 50%
NaOH and the resulting mixture was extracted with MTBE (2.times.2
L). The combined MTBE extract was then washed with 5% NaHCO.sub.3
(3.times.100 mL), water (3.times.100 mL), and evaporated in vacuo
to give the enantiomerically pure
N-Boc-(1R,2S)/-1-amino-2-vinylcyclopropane carboxylic acid ethyl
ester as light yellow solid (42.55 g; purity: 97% @ 210 nm,
containing no acid; 100% enantiomeric excess ("ee").
[0319] The aqueous layer from the extraction process was then
acidified to pH 2 with 50% H.sub.2SO.sub.4 and extracted with MTBE
(2.times.2 L). The MTBE extract was washed with water (3.times.100
mL) and evaporated to give the acid as light yellow solid (42.74 g;
purity: 99% @ 210 nm, containing no ester). TABLE-US-00003
##STR142## ##STR143## ester acid High (+) ESI, C13H22NO4, (-) ESI,
C11H16NO4, Resolution [M + H].sup.+, [M - H].sup.-, Mass Spec cal.
256.1549, cal. 226.1079, found 256.1542 found 226.1089 NMR observed
chemical shift Solvent: CDCl.sub.3 (proton .delta. 7.24 ppm, C-13
.delta. 77.0 ppm) Bruker DRX-500C: proton 500.032 MHz, carbon
125.746 MHz Proton Proton (pattern) C-13 (pattern) C-13 Position
ppm ppm ppm ppm 1 -- 40.9 -- 40.7 2 2.10(q, J=9.0Hz) 34.1 2.17(q,
J=9.0Hz) 35.0 .sup. 3a 1.76(br) 23.2 1.79(br) 23.4 3b 1.46(br)
1.51,(br) 4 -- 170.8 -- 175.8 5 5.74(ddd, J=9.0, 10.0, 133.7
5.75(m) 133.4 17.0Hz) .sup. 6a 5.25(d, J=17.0Hz) 117.6 5.28(d,
J=17.0Hz) 118.1 6b 5.08(dd, J=10.0, 1.5Hz) 5.12(d, J=10.5Hz) 7 --
155.8 -- 156.2 8 -- 80.0 80.6 9 1.43(s) 28.3 1.43(s) 28.3 10
4.16(m) 61.3 -- -- 11 1.23(t, J=7.5Hz) 14.2 -- --
Resolution B
[0320] To 0.5 mL 100 mM Heps.Na buffer (pH 8.5) in a well of a 24
well plate (capacity: 10 ml/well), 0.1 mL of Savinase 16.0 L
(protease from Bacillus clausii) (Novozymes North America Inc.) and
a solution of the racemic
N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester (10 mg) in 0.1 mL of DMSO were added. The plate was
sealed and incubated at 250 rpm at 40.degree. C. After 18 h,
enantio-excess of the ester was determined to be 44.3% as
following: 0.1 mL of the reaction mixture was removed and mixed
well with 1 mL ethanol; after centrifugation, 10 microliter
(".mu.l") of the supernatant was analyzed with the chiral HPLC. To
the remaining reaction mixture, 0.1 mL of DMSO was added, and the
plate was incubated for additional 3 days at 250 rpm at 40.degree.
C., after which four mL of ethanol was added to the well. After
centrifugation, 10 .mu.l of the supernatant was analyzed with the
chiral HPLC and enantio-excess of the ester was determined to be
100%.
Resolution C
[0321] To 0.5 ml 100 mM Heps.Na buffer (pH 8.5) in a well of a 24
well plate (capacity: 10 mL/well), 0.1 ml of Esperase 8.0 L,
(protease from Bacillus halodurans) (Novozymes North America Inc.)
and a solution of the racemic
N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester (10 mg) in 0.1 mL of DMSO were added. The plate was
sealed and incubated at 250 rpm at 40.degree. C. After 18 hour,
enantio-excess of the ester was determined to be 39.6% as
following: 0.1 mL of the reaction mixture was removed and mixed
well with 1 mL ethanol; after cenrifugation, 10 .mu.l of the
supernatant was analyzed with the chiral HPLC. To the remaining
reaction mixture, 0.1 mL of DMSO was added, and the plate was
incubated for additional 3 days at 250 rpm at 40.degree. C., after
which four mL of ethanol was added to the well. After
centrifugation, 10 .mu.l of the supernatant was analyzed with the
chiral HPLC and enantio-excess of the ester was determined to be
100%.
Samples analysis was carried out in the following manner:
1) Sample preparation: About 0.5 ml of the reaction mixture was
mixed well with 10 volume of EtOH. After centrifugation, 10 .mu.l
of the supernatant was injected onto HPLC column.
2) Conversion determination:
Column: YMC ODS A, 4.6.times.50 mm, S-5 .mu.m
Solvent: A, 1 mM HCl in water; B, MeCN
Gradient: 30% B for 1 min; 30% to 45% B over 0.5 min; 45% B for 1.5
min; 45% to 30% B over 0.5 min.
Flow rate: 2 ml/min
UV Detection: 210 nm
Retention time: acid, 1.2 min; ester, 2.8 min.
3) Enantio-excess determination for the ester:
Column: CHIRACEL OD-RH, 4.6.times.150 mm, S-5 .mu.m
Mobile phase: MeCN/50 mM HClO.sub.4 in water (67/33)
Flow rate: 0.75 ml/min.
UV Detection: 210 nm.
Retention time:
(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid 5.2 min;
Racemate (1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic
acid ethyl ester 18.5 min and 20.0 min;
(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid ethyl ester
18.5 min.
Resolution D
[0322] 5 L of 0.3 M sodium phosphate buffer (pH 8) was maintained
at 38.degree. C. in a 20 Liter jacked reactor, stirred at 130 rpm.
Four liters of Alcalase 2.4 L (Novozymes North America Inc.) and 1
liter of DI water were added to the reactor. When temperature of
the mixture closed to 38.degree. C., pH was adjusted to 7.8 with 10
N NaOH. A solution of the racemic
N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester (500 grams) in 5 liters DMSO was added to the reactor
over a period of 1 hour via an addition funel. The reaction
temperature was then adjusted to 48.degree. C. After 21 hours,
enantio-excess of the ester reached 99.3%. Heating was stopped at
24 hour and the reaction was slowly cooled down to room temperature
(about 25.degree. C.) and stirred overnight. pH of the reaction
mixture was adjusted to 8.5 with 10 N NaOH and the mixture was
extracted with MTBE (2.times.4 L). The combined MTBE extract was
washed with 5% NaHCO.sub.3 (3.times.400 ml) and water (3.times.400
ml), and evaporated to give enantiomerically pure
N-Boc-(1R,2S)/-1-amino-2-vinylcyclopropane carboxylic acid ethyl
ester as light yellow crystal (259 g; purity: 96.9% @ 210 nm,
containing no acid; 100% ee).
Resolution E
[0323] 10 L of 0.1 M sodium phosphate buffer (pH 8) was maintained
at 40.degree. C. in a 20 Liter jacked reactor, stirred at 360 rpm.
1.5 liters of Alcalase 2.4 L (Novozymes North America Inc.) was
added to the reactor. When temperature of the mixture closed to
38.degree. C., pH was adjusted to 8.0 with 10 N NaOH. A solution of
the racemic N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane
carboxylic acid ethyl ester (200 grams) in 2 liters DMSO was added
to the reactor over a period of 1 hour via an addition funel. The
reaction temperature was then adjusted to 40.degree. C. After 3
hours, pH was adjusted to 8.0 with 10 N NaOH. After 21 hours, the
reaction was cooled down to 25.degree. C. pH of the reaction
mixture was adjusted to 8.5 with 10 N NaOH and the mixture was
extracted with MTBE (2.times.5 L). The combined MTBE extract was
washed with 5% NaHCO.sub.3 (3.times.500 ml) and water (3.times.200
ml), and evaporated to give 110 gram of yellow oil. The oil was set
at room temperature under house vacuum and gave enantiomerically
pure N-Boc-(1R,2S)/-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester as colorless long rod crystal (101 g; purity: 97.9% @
210 nm, containing no acid; 100% ee).
[0324] The crystal structure enantiomerically pure
N-Boc-(1R,2S)/-1-amino-2-vinylcyclopropane carboxylic acid ethyl
ester has been characterized by single crystal analysis (X-ray NB#:
52795-093, refcode: 634592N1). The absolute configuration is not
established for lack of a known chiral center or heavier atom(s). A
chain structure along the crystallographic a-axis is formed via
intermolecular hydrogen bonding between the amide group and the
carbonyl oxygen atom (N . . . O 3.159 .ANG.).
Structure of N-Boc-(1R,2S)-1-amino-2-vinylcyclopropane carboxylic
acid ethyl ester
[0325] TABLE-US-00004 ##STR144## Crystal Data: Experimental:
Chemical formula: C13H21N1O4 Crystallization Crystal system:
Orthorhombic Crystal source: MTBE Space Group:
P2.sub.12.sub.12.sub.1 Crystal description: Colorless a = 5.2902(1)
.ANG. .alpha. = 90.degree. rod b = 13.8946(2) .ANG. .beta. =
90.degree. Crystal size (mm): c = 19.9768(3) .ANG. .gamma. =
90.degree. 0.12 .times. 0.26 .times. 0.30 V = 1468.40(4)
.ANG..sup.3 Data Collection Z = 4 d.sub.x = 1.155 g cm.sup.-3
Temperature (K.): 293 No. of reflections for lattice
.theta..sub.max (.degree.): 65.2 (Cu K.alpha.) parameters: 6817 No.
of reflections measured: .theta. range for lattice parameters
(.degree.): 7518 2.2-65.2 No. of independent reflections:
Absorption coefficient (mm.sup.-1): 2390 (R.sub.int = 0.0776) 0.700
No. of observed reflections (I .gtoreq. 2.sigma.): 2284 Absorption
correction (T.sub.min-T.sub.max): 0.688-1.000
Resolution F 5 L of 0.2 M sodium borate buffer (pH 9) was
maintained at 45.degree. C. in a 20 liter jacked reactor, stirred
at 400 rpm. Three liter of DI water and four liters of Savinase 16
L, type EX (Novozymes North America Inc.) were added to the
reactor. When temperature of the mixture closed to 45.degree. C.,
pH was adjusted to 8.5 with 10 N NaOH. A solution of the racemic
N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester (200 grams) in 2 liters DMSO was added to the reactor
over a period of 40 min, via an addition funel. The reaction
temperature was then adjusted to 48.degree. C. After 2 hours, pH
was adjusted to pH 9.0 with 10 N NaOH. At 18 hour, enantio-excess
of the ester reached 72%, pH was adjusted to 9.0 with 10 N NaOH. At
24 hour, temperature was lowered to 35.degree. C. At 42 hour,
temperature was raised to 48.degree. C. and pH was adjusted to 9.0
with 10 N NaOH. Heating was stopped at 48 hour and the reaction was
slowly cooled down to room temperature (about 25.degree. C.) and
stirred overnight. At 66 hour, pH of the reaction mixture was 8.6.
The mixture was extracted with MTBE (2.times.4 L). The combined
MTBE extract was washed with 5% NaHCO.sub.3 (6.times.300 ml) and
water (3.times.300 ml), and evaporated to give give
enantiomerically pure N-Boc-(1R,2S)/-1-amino-2-vinylcyclopropane
carboxylic acid ethyl ester as light yellow crystal (101 Ag;
purity: 95.9% @ 210 nm, containing no acid; 98.6% ee).
C.2 Preparation of chiral (1R,2S)-1-amino-2-vinylcyclopropane
carboxylic acid ethyl ester hydrochloride
[0326] ##STR145##
[0327] N-BOC-(1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid
ethyl ester (8.5 g, 33.3 mmol) was stirred under an N.sub.2
atmosphere with 200 mL of 4N HCl/dioxane (Aldrich) at rt for 3 h.
The solvent was removed under reduced pressure keeping the
temperature below 40 C. This gave 6.57 g (.about.100%) of
(1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester
hydrochloride as a light tan solid. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 1.31 (t, J=7.0 Hz, 3H), 1.69-1.82 (m, 2H), 2.38
(q, J=8.8 Hz, 1H), 4.29 (q, J=7.0 Hz, 2H), 5.22 (d, J=10.3 Hz, 1H),
5.40 (d, J=17.2 Hz, 1H), 5.69-5.81 (m, 1H). LC-MS (Method A,
retention time: 0.58 min), MS m/z 156 (M.sup.++1).
Example 2
Preparation of Compound
1,14-tert-Butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-di-
aza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic acid
[0328] ##STR146##
Example 2, Compound 1
Step 2A, Preparation of
2-(1-Ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl)-4-(isoquinolin-1-yloxy)-
-pyrrolidine-1-carboxylic acid tert-butyl ester
[0329] ##STR147##
[0330] A stirred slurry of
4-(isoquinolin-1-yloxy)-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester (5.69 g, 15.9 mmol) in 200 mL of methylene
chloride was treated sequentially with diisopropylethylamine (13.8
mL, 79.0 mmol), HBTU (7.10 g, 18.7 mmol), HOBT.H.sub.2O (2.86 g,
18.7 mmol), and 1R,2S-1-amino-2-vinylcyclopropane carboxylic acid
ethyl ester hydrochloride (3.19 g, 16.7 mmol). The gold homogeneous
solution was stirred at rt under N.sub.2 for 18 h, and then
concentrated in vacuo to give 10 g of a brown oil. This was
partitioned between ethyl acetate and sat. aq. NaHCO.sub.3. The
organic phase was washed with brine, dried (MgSO.sub.4), and
concentrated in vacuo. Flash chromatography (2-5% MeOH in methylene
chloride) gave 5.5 g (70%) of
2-(1-ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl)-4-(isoquinolin-1-yloxy)-
-pyrrolidine-1-carboxylic acid tert-butyl ester: LC-MS (Method A,
retention time: 3.42 min), MS m/z 496 (M.sup.++1).
Step 2B, Preparation of
1-{[4-(Isoquinolin-1-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclop-
ropanecarboxylic acid ethyl ester, bis hydrochloride
[0331] ##STR148##
[0332] A stirred slurry of
2-(1-ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl)-4-(isoquinolin-1-yloxy)-
-pyrrolidine-1-carboxylic acid tert-butyl ester (5.40 g, 10.9 mmol)
was treated with 2N HCl/ether (Aldrich)(250 mL) for 24 h. The
reaction mixture was concentrated in vacuo to give 5.3 g
(.about.100%) of
1-{[4-(isoquinolin-1-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclop-
ropanecarboxylic acid ethyl ester, bis hydrochloride as a white
solid: LC-MS (Method A, retention time: 2.40 min), MS m/z 396
(M.sup.++1).
Step 2C, Preparation of
1-{[1-(2-tert-Butoxycarbonylamino-non-8-enoyl)-4-(isoquinolin-1-yloxy)-py-
rrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
ethyl ester
[0333] ##STR149##
[0334] 2(S)-tert-butoxycarbonylamino-8-nonenoic acid (purchased
from RSP Amino Acids) (1.0 g, 3.68 mmol) dissolved in 100 mL of DMF
was treated sequentially with
1-{[4-(isoquinolin-1-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclop-
ropanecarboxylic acid ethyl ester, bis hydrochloride (1.46 g, 3.68
mmol), N-methyl morpholine (1.4 mL, 12.9 mmol), and HATU (PE
biosystems) (1.68 g, 4.42 mmol). The reaction mixture was stirred
at rt under N.sub.2 for 24 h, and then concentrated in vacuo. The
residue was partitioned between ethyl acetate and pH 4 buffer
(biphthalate). The organic phase was washed with sat. aq.
NaHCO.sub.3, dried (MgSO.sub.4), and concentrated in vacuo to give
2.2 g of the crude product. Flash chromatography (20-50% ethyl
acetate/hexane) gave 1.9 g (79%) of
1-{[1-(2-tert-butoxycarbonylamino-non-8-enoyl)-4-(isoquinolin-1-yloxy)-py-
rrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
ethyl ester as a glassy colorless solid: LC-MS (Method A, retention
time: 3.72 min), MS m/z 649 (M.sup.++1).
Step 2D, Preparation of
14-tert-Butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaz-
a-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic acid ethyl
ester
[0335] ##STR150##
[0336] A solution of
1-{[1-(2-tert-butoxycarbonylamino-non-8-enoyl)-4-(isoquinolin-1-yloxy)-py-
rrolidine-2-carbonyl]-amino}-2-vinylcyclopropanecarboxylic acid
ethyl ester (1.72 g, 2.65 mmol) and
tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-
-2-ylidene][benzylidene]ruthenium (IV) dichloride (Strem) (225 mg,
0.265 mmol) in 650 mL of methylene chloride was refluxed under
N.sub.2. The light orange homogeneous solution was refluxed for 24
h to give a dark orange solution. The reaction mixture was cooled
to rt and concentrated in vacuo to give 1.7 g of an orange oil.
Flash chromatography (20-40% ethyl acetate/hexane) gave 1.4 g (85%)
of
14-tert-butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaz-
a-tricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic acid ethyl
ester as a white solid: .sup.1H NMR (300 MHz, CD.sub.3Cl.sub.3)
.delta. 1.29 (t, J=7.3 Hz, 3H), 1.34 (s, 9H), 1.35-1.71 (m, 9H),
1.86-1.97 (m, 2H), 2.08-2.27 (m, 2H), 2.38-2.47 (m, 1H), 2.96-3.04
(m, 1H), 4.02-4.23 (m, 4H), 4.55 (m, 1H), 4.91 (dd, J=8.4 Hz, 4.0
Hz, 1H), 5.25 (t, J=9.5 Hz, 1H), 5.32 (d, J=8.8 Hz, 1H), 5.50 (m,
1H), 5.81 (m, 1H), 6.96 (s, 1H), 7.23 (m, 1H), 7.51 (t, J=7.3 Hz,
1H), 7.65 (t, J=8.4 Hz, 1H), 7.73 (d, J=8.4 Hz, 1H), 7.93 (d, J=5.9
Hz, 1H), 8.17 (d, J=8.4 Hz, 1H). LC-MS (Method A, retention time:
3.52 min), MS m/z 621 (M.sup.++1).
Step 2E, Preparation of Compound
1,14-tert-Butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-di-
aza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic acid
[0337] ##STR151##
[0338]
14-tert-butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,-
16-diaza-tricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic acid
ethyl ester (1.10 g, 1.77 mmol) was dissolved in the mixed solvent
system; THF (18 mL), methanol (8 mL), and water (2 mL). Powdered
lithium hydroxide hydrate (744 mg, 17.7 mmol) was added. The light
yellow slurry was stirred at rt under N2 for 24 h, and then
concentrated in vacuo. The residue was partitioned between ether
and water. The ether phase was discarded, and the aqueous phase was
treated with 4N HCl until the pH was 4. This acidic solution was
extracted with ether four times. The combined ether extracts were
dried (MgSO.sub.4) and concentrated in vacuo to give 0.95 g (90%)
of
14-tert-butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaz-
a-tricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic acid as a
white solid: .sup.1H NMR (300 MHz, CD.sub.3Cl.sub.3) .delta. 1.30
(s, 9H), 1.34-1.46 (m, 4H), 1.52-1.68 (m, 2H), 1.84 (m, 2H),
2.08-2.58 (m, 6H), 2.78-2.87 (m, 1H), 4.07 (m, 1H), 4.32 (d, J=1.3
Hz, 1H), 4.41 (m, 1H), 4.79 (t, J=7.3 Hz, 1H), 5.14 (t, J=9.5 Hz,
1H), 5.23 (d, J=7.3 Hz, 1H), 5.60 (q, J=8.8 Hz, 1H), 5.86 (s, 1H),
7.08 (s, 1H), 7.22 (d, J=6.2 Hz, 1H), 7.48 (t, J=7.7 Hz, 1H), 7.64
(t, J=8.0 Hz, 1H), 7.72 (d, J=8.1 Hz, 1H), 7.94 (d, J=5.9 Hz, 1H),
8.17 (s, J=8.4 Hz, 1H). LC-MS (Method A, retention time: 3.32 min),
MS m/z 593 (M.sup.++1)
Example 3a
Preparation of Cyclopropylsulfonamide
[0339] Method A: ##STR152##
[0340] To a solution of 100 mL of THF cooled to 0.degree. C. was
bubbled in gaseous ammonia until saturation was reached. To this
solution was added a solution of 5 g (28.45 mmol) of
cyclopropylsulfonyl chloride (purchased from Array Biopharma) in 50
mL of THF, the solution warmed to rt overnite and stirred one
additional day. The mixture was concentrated until 1-2 mL of
solvent remained, applied on to 30 g plug of SiO.sub.2 (eluted with
30% to 60% EtOAc/Hexanes) to afford 3.45 g (100%) of cyclopropyl
sulfonamide as a white solid. .sup.1H NMR (Methanol-d.sub.4)
.delta. 0.94-1.07 (m, 4H), 2.52-2.60 (m, 1H); .sup.13C NMR
(methanol-d.sub.4) .delta. 5.92, 33.01.
Method B:
Step 1: Preparation of N-tert-Butyl-(3-chloro)propylsulfonamide
[0341] ##STR153## tert-Butylamine (3.0 mol, 315.3 mL) was dissolved
in THF (2.5 L). The solution was cooled to -20.degree. C.
3-Chloropropanesulfonyl chloride (1.5 mol, 182.4 mL) was added
slowly. The reaction mixture was allowed to warm to rt and stirred
for 24 h. The mixture was filtered, and the filtrate was
concentrated in vacuo. The residue was dissolved in
CH.sub.2Cl.sub.2 (2.0 L). The resulting solution was washed with 1
N HCl (1.0 L), water (1.0 L), brine (1.0 L) and dried over
Na.sub.2SO.sub.4. It was filtered and concentrated in vacuo to give
a slightly yellow solid, which was crystallized from hexane to
afford the product as a white solid (316.0 g, 99%).
[0342] .sup.1H NMR (CDCl.sub.3) .delta. 1.38 (s, 9H), 2.30-2.27 (m,
2H), 3.22 (t, J=7.35 Hz, 2H), 3.68 (t, J=6.2 Hz, 2H), 4.35 (b,
1H).
Step 2: preparation of Cyclopropanesulfonic acid
tert-butylamide
[0343] ##STR154## To a solution of
N-tert-butyl-(3-chloro)propylsulfonamide (2.14 g, 10.0 mmol) in THF
(100 mL) was added n-BuLi (2.5 M in hexane, 8.0 mL, 20.0 mmol) at
-78.degree. C. The reation mixture was allowed to warm up to room
temperature over period of 1 h. The volatiles were removed in
vacuo. The residue was partitioned between EtOAC and water (200 mL,
200 mL). The separated organic phase was washed with brine, dried
over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The
residue was recrystallized from hexane to yield the desired product
as a white solid (1.0 g, 56%).
[0344] .sup.1H NMR (CDCl.sub.3) .delta. 0.98-1.00 (m, 2H),
1.18-1.19 (m, 2H), 1.39 (s, 9H), 2.48-2.51 (m, 1H), 4.19 (b,
1H).
Step 3: Preparation of Cyclopropylsulfonamide
[0345] ##STR155## A solution of cyclopropanesulfonic acid
tert-butylamide (110.0 g, 0.62 mol) in TFA (500 mL) was stirred at
room temperature for 16 h. The volatile was removed in vacuo. The
residue was recrystallized from EtOAC/hexane (60 mL/240 mL) to
yield the desired product as a white solid (68.5 g, 91%).
[0346] .sup.1H NMR (DMSO-d.sub.6) .delta. 0.84-0.88 (m, 2H),
0.95-0.98 (m, 2H), 2.41-2.58 (m, 1H), 6.56 (b, 2H).
Example 3b
Preparation of C1-Substituted Cyclopropylsulfonamides
Preparation of N-tert-butyl-(1-methyl)cyclopropyl-sulfonamide
[0347] ##STR156##
Step 1a Preparation of N-tert-butyl-(3-chloro)propylsulfonamide
[0348] ##STR157## As shown above.
Step 1b. Preparation of
N-tert-Butyl-(1-methyl)cyclopropyl-sulfonamide
[0349] ##STR158## A solution of
N-tert-butyl-(3-chloro)propylsulfonamide (4.3 g, 20 mmol) was
dissolved in dry THF (100 mL) and cooled to -78.degree. C. To this
solution was added n-BuLi (17.6 mL, 44 mmol, 2.5 M in hexane)
slowly. The dry ice bath was removed and the reaction mixture was
allowed to warm to rt over a period of 1.5 h. This mixture was then
cooled to -78.degree. C., and a solution of n-BuLi (20 mmol, 8 mL,
2.5 M in hexane) was added. The reaction mixture was warmed to rt,
recooled to -78.degree. C. over a period of 2 h and a neat solution
of methyl iodide (5.68 g, 40 mmol) added. The reaction mixture was
allowed to warm to rt overnight, quenched with saturated NH.sub.4Cl
(100 mL) at rt. It was extracted with EtOAc (100 mL). The organic
phase was washed with brine (100 mL), dried (MgSO.sub.4), and
concentrated in vacuo to give a yellow oil which was crystallized
from hexane to afford the product as a slightly yellow solid (3.1
g, 81%): .sup.1H NMR (CDCl.sub.3) .delta. 0.79 (m, 2H), 1.36 (s,
9H), 1.52 (m, 2H), 1.62 (s, 3H), 4.10 (bs, 1H).
Step 1c: Preparation of 1-methylcyclopropylsulfonamide
[0350] ##STR159## A solution of
N-tert-butyl-(1-methyl)cyclopropylsulfonamide (1.91 g, 10 mmol) was
dissolved in TFA (30 mL), and the reaction mixture stirred at rt
for 16 h. The solvent was removed in vacuo to give a yellow oil
which was crystallized from EtOAc/hexane (1:4, 40 mL) to yield
Example 3,1-methylcyclopropylsulfonamide, as a white solid (1.25 g,
96%): .sup.1H NMR (CDCl.sub.3) .delta. 0.84 (m, 2H), 1.41 (m, 2H),
1.58 (s, 3H), 4.65 (bs, 2H). Anal. Calcd. For
C.sub.4H.sub.9NO.sub.2S: C, 35.54; H, 6.71; N, 10.36. Found: C,
35.67; H, 6.80; N, 10.40.
Preparation of 1-Benzylcyclopropylsulfonamide
[0351] ##STR160##
Steps 1b: Preparation of
N-tert-Butyl-(1-benzyl)cyclopropyl-sulfonamide
[0352] ##STR161## This compound was obtained in 60% yield using the
procedure described for the synthesis of
N-tert-butyl-(1-methyl)cyclopropylsulfonamide except 1.05
equivalents of benzyl bromide were used, followed by trituration
with 10% EtOAc in hexane: .sup.1H NMR (CDCl.sub.3) .delta. 0.92 (m,
2H), 1.36 (m, 2H), 1.43 (s, 9H), 3.25 (s, 2H), 4.62 (bs, 1H),
7.29-7.36 (m, 5H).
Steps 1c: Preparation of 1-Benzylcyclo-propylsulfonamide
[0353] ##STR162## This compound 1-benzylcyclopropylsulfonamide, was
obtained in 66% yield from
N-tert-butyl(1-benzyl)cyclopropylsulfonamide using the procedure
described for the synthesis of 1-methylcyclopropylsulfonamide,
followed by recrystallization from the minimum amount of 10% EtOAc
in hexane: .sup.1H NMR (CDCl.sub.3) .delta. 0.90 (m, 2H), 1.42 (m,
2H), 3.25 (s, 2H), 4.05 (s, 2H), 7.29 (m, 3H), 7.34 (m, 2H);
.sup.13C NMR (CDCl.sub.3) .delta. 11.1, 36.8, 41.9, 127.4, 128.8,
129.9, 136.5.
Preparation of 1-Propylcyclopropylsulfonamide
[0354] ##STR163##
Steps 1b: Preparation of
N-tert-Butyl-(1-benzyl)cyclopropyl-sulfonamide
[0355] ##STR164## This compound was prepared using the process
desribed for the preparation of 1-methylcyclopropylsulfonamide
except propyl halide was utilized in place of methyl iodide in the
second step of this process.
Preparation of N-tert-Butyl-(1-allyl)cyclopropylsulfonamide
[0356] ##STR165## This compound,
N-tert-Butyl-(1-allyl)cyclopropylsulfonamide, was obtained in 97%
yield according to the procedure described in the synthesis of
N-tert-Butyl-(1-methyl)cyclopropylsul-fonamide except 1.25
equivalents of allyl bromide were used as electrophile. The
compound was taken directly into the next reaction without
purification: .sup.1H NMR (CDCl.sub.3) .delta. 0.83 (m, 2H), 1.34
(s, 9H), 1.37 (m, 2H), 2.64 (d, J=7.3 Hz, 2H), 4.25 (bs, 1H),
5.07-5.10 (m, 2H), 6.70-6.85 (m, 1H).
Preparation of 1-allylcyclopropylsulfonamide
[0357] ##STR166## This compound, 1-allylcyclopropylsulfonamide, was
obtained in 40% yield from
N-tert-butyl-(1-allyl)cyclopropylsulfonamide according to the
procedure described in the synthesis of
1-Methylcyclopropylsulfonamide. The compound was purified by column
chromotography over SiO.sub.2 using 2% MeOH in CH.sub.2Cl.sub.2 as
the eluent: .sup.1H NMR (CDCl.sub.3) .delta. 0.88 (m, 2H), 1.37 (m,
2H), 2.66 (d, J=7.0 Hz, 2H), 4.80 (s, 2H), 5.16 (m, 2H), 5.82 (m,
1H); .sup.13C NMR (CDCl.sub.3) .delta. 11.2, 35.6, 40.7, 119.0,
133.6.
Preparation of
N-tert-Butyl-[1-(1-hydroxy)cyclohexyl]-cyclopropylsulfonamide
[0358] ##STR167## This compound was obtained in 84% yield using to
the procedure described for the synthesis of
N-tert-Butyl-(1-methyl)cyclopropylsul-fonamide except 1.30
equivalents of cyclohexanone were used, followed by
recrystallization from the minimum amount of 20% EtOAc in hexane:
.sup.1H NMR (CDCl.sub.3) .delta. 1.05 (m, 4H), 1.26 (m, 2H), 1.37
(s, 9H), 1.57-1.59 (m, 6H), 1.97 (m, 2H), 2.87 (bs, 1H), 4.55 (bs,
1H).
Preparation of 1-(1-cyclohexenyl)cyclopropyl-sulfonamide
[0359] ##STR168## This compound,
1-(1-cyclohexenyl)-cyclopropylsulfonamide was obtained in 85% yield
from N-tert-butyl-[1-(1-hydroxy)cyclohexyl]-cyclopropylsulfonamide
using the procedure described for the synthesis of
1-methylcyclopropylsulfonamide, followed by recrystallization from
the minimum amount of EtOAc and hexane: .sup.1H NMR (DMSO-d.sub.6)
.delta. 0.82 (m, 2H), 1.28 (m, 2H), 1.51 (m, 2H), 1.55 (m, 2H),
2.01 (s, 2H), 2.16 (s, 2H), 5.89 (s, 1H), 6.46 (s, 2H); .sup.13C
NMR (DMSO-d.sub.6) .delta. 11.6, 21.5, 22.3, 25.0, 27.2, 46.9,
131.6, 132.2; LR-MS (ESI): 200 (M.sup.+-1).
Preparation of N-tert-Butyl-(1-benzoyl)cyclopropyl-sulfonamide
[0360] ##STR169## This compound was obtained in 66% yield using the
procedure described for the synthesis of
N-tert-Butyl-(1-methyl)cyclopropylsulfonamide except 1.2
equivalents of methyl benzoate was used as the electrophile. The
compound was purified by column chromatography over SiO.sub.2 using
30% to 100% CH.sub.2Cl.sub.2 in hexane: .sup.1H NMR (CDCl.sub.3)
.delta. 1.31 (s, 9H), 1.52 (m, 2H), 1.81 (m, 2H), 4.16 (bs, 1H),
7.46 (m, 2H), 7.57 (m, 1H), 8.05 (d, J=8.5 Hz, 2H).
Preparation of 1-benzoylcyclo-propylsulfonamide
[0361] ##STR170## This compound 1-benzoylcyclopropyl-sulfonamide,
was obtained in 87% yield from
N-tert-butyl(1-benzoyl)cyclopropylsul-fonamide using the procedure
described for the synthesis of 1-Methylcyclopropylsulfonamide,
followed by recrystallization from the minimum amount of EtOAc in
hexane: .sup.1H NMR (DMSO-d.sub.6) .delta. 1.39 (m, 2H), 1.61 (m,
2H), 7.22 (s, 2H), 7.53 (t, J=7.6 Hz, 2H), 7.65 (t, J=7.6 Hz, 1H),
8.06 (d, J=8.2 Hz, 2H); .sup.13C NMR (DMSO-d.sub.6) .delta. 12.3,
48.4, 128.1, 130.0, 133.4, 135.3, 192.0.
Preparation of
N-tert-Butyl-(1-phenylaminocarboxy)-cyclopropylsulfonamide
[0362] ##STR171##
[0363] This compound was obtained in 42% yield using the procedure
described for the synthesis of
N-tert-Butyl-(1-methyl)cyclopropylsulfonamide using 1 equivalent of
phenylisocyanate, followed by recrystallization from the minimum
amount of EtOAc in hexane .sup.1H NMR (CDCl.sub.3) .delta. 1.38 (s,
9H), 1.67-1.71 (m, 4H), 4.30 (bs, 1H), 7.10 (t, J=7.5 Hz, 1H), 7.34
(t, J=7.5 Hz, 2H), 7.53 (t, J=7.5 Hz, 2H).
Example 4
Preparation of Cycloalkylsulfonamides from Cyloalkylbromides
Preparation of Cyclobutylsulfonamide from Cylobutylbromide
[0364] ##STR172##
[0365] To a solution of 5.0 g (37.0 mmol) of cyclobutyl bromide in
30 mL of anhydrous diethyl ether (Et.sub.2O) cooled to -78.degree.
C. was added 44 mL (74.8 mmol) of 1.7M tert-butyl lithium in
pentanes and the solution slowly warmed to -35.degree. C. over 1.5
h. This mixture was cannulated slowly into a solution of 5.0 g
(37.0 mmol) of freshly distilled sulfuryl chloride in 100 mL of
hexanes cooled to -40.degree. C., warmed to 0.degree. C. over 1 h
and carefully concentrated in vacuo. This mixture was redissolved
in Et.sub.2O, washed once with some ice-cold water, dried
(MgSO.sub.4) and concentrated carefully. This mixture was
redissolved in 20 mL of THF, added dropwise to 500 mL of saturated
NH.sub.3 in THF and was allowed to stir overnite. The mixture was
concentrated in vacuo to a crude yellow solid and was
recrystallized from the minimum amount of CH.sub.2Cl.sub.2 in
hexanes with 1-2 drops of MeOH to afford 1.90 g (38%) of
cyclobutylsulfonamide as a white solid. .sup.1H NMR (CDCl.sub.3)
.delta. 1.95-2.06 (m, 2H), 2.30-2.54 (m, 4H), 3.86 (p, J=8 Hz, 1H),
4.75 (brs, 2H); .sup.13C NMR (CDCl.sub.3) .delta. 16.43, 23.93,
56.29. HRMS m/z (M-H).sup.- calcd for C.sub.4H.sub.8NSO.sub.2:
134.0276, found 134.0282.
Preparation of Cyclopentyl Sulfonamide
[0366] ##STR173## A solution of 18.5 mL (37.0 mmol) of 2M
cyclopentyl-magnesium chloride in ether was added dropwise to a
solution of 3.0 mL (37.0 mmol) freshly distilled sulfuryl chloride
(obtained from Aldrich) in 100 mL of hexanes cooled to -78.degree.
C. The mixture was warmed to 0.degree. C. over 1 h and was then
carefully concentrated in vacuo. This mixture was redissolved in
Et.sub.2O (200 mL), washed once with some ice-cold water (200 mL),
dried (MgSO.sub.4) and concentrated carefully. This mixture was
redissolved in 35 mL of THF, added dropwise to 500 mL of saturated
NH.sub.3 in THF and was allowed to stir overnite. The mixture was
concentrated in vacuo to a crude yellow solid, the residue filtered
through 50 g of silica gel using 70% EtOAc-hexanes as the eluent
and the solution was then concentrated. The residue was
recrystallized from the minimum amount of CH.sub.2Cl.sub.2 in
hexanes with 1-2 drops of MeOH to afford 2.49 g (41%) of
cyclopentylsulfonamide as a white solid. .sup.1H NMR (CDCl.sub.3)
.delta. 1.58-1.72 (m, 2H), 1.74-1.88 (m, 2H), 1.94-2.14 (m, 4H),
3.48-3.59 (m, 1H), 4.80 (bs, 2H); .sup.13C NMR (CDCl.sub.3) .delta.
25.90, 28.33, 63.54; MS m/e 148 (M-H).sup.-.
Preparation of Cyclohexyl Sulfonamide
[0367] ##STR174## A solution of 18.5 mL (37.0 mmol) of 2M
cyclohexylmagnesium chloride (TCI Americas) in ether was added
dropwise to a solution of 3.0 mL (37.0 mmol) freshly distilled
sulfuryl chloride in 100 mL of hexanes cooled to -78.degree. C. The
mixture was warmed to 0.degree. C. over 1 h and was then carefully
concentrated in vacuo. This mixture was redissolved in Et.sub.2O
(200 mL), washed once with some ice-cold water (200 mL), dried
(MgSO.sub.4) and concentrated carefully. This mixture was
redissolved in 35 mL of THF, added dropwise to 500 mL of saturated
NH.sub.3 in THF and was allowed to stir overnite. The mixture was
concentrated in vacuo to a crude yellow solid, the residue filtered
through 50 g of silica gel using 70% EtOAc-hexanes as the eluent
and was concentrated. The residue was recrystallized from the
minimum amount of CH.sub.2Cl.sub.2 in hexanes with 1-2 drops of
MeOH to afford 1.66 g (30%) of cyclohexyl-sulfonamide as a white
solid: .sup.1H NMR (CDCl.sub.3) .delta. 1.11-1.37 (m, 3H),
1.43-1.56 (m, 2H), 1.67-1.76 (m, 1H), 1.86-1.96 (m, 2H), 2.18-2.28
(m, 2H), 2.91 (tt, J=12, 3.5 Hz, 1H), 4.70 (bs, 2H); .sup.13CH NMR
(CDCl.sub.3) .delta. 25.04, 25.04, 26.56, 62.74; MS m/e 162
(M-1).sup.-.
Example 5
Preparation of Compound 2,
[18-(Isoquinolin-1-yloxy)-4-methanesulfonylaminocarbonyl-2,15-dioxo-3,16--
diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic acid
tert-butyl ester
[0368] ##STR175##
[0369] Prepared from
14-tert-butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaz-
a-tricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic acid (50
mg, 0.075 mmol) and methane sulfonamide (9.3 mg, 0.098 mmol) as
described in the general procedure above to give
[18-(isoquinolin-1-yloxy)-4-methanesulfonylaminocarbonyl-2,15-dioxo-3,16--
diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic acid
tert-butyl ester as a white powder: .sup.1H NMR (300 MHz,
CD.sub.3Cl.sub.3) .delta. 1.18 (s, 9H), 1.25-1.96 (m, 11H), 2.26
(m, 1H), 2.54 (m, 1H), 2.71 (m, 2H), 3.18 (s, 3H), 4.08 (m, 1H),
4.26 (m, 1H), 4.64 (m, 2H), 5.00 (m, 2H), 5.73 (m, 1H), 6.01 (s,
1H), 6.73 (s, 1H), 7.28 (d, J=5.5 Hz, 1H), 7.50 (t, J=7.3 Hz, 1H),
7.65-7.76 (m, 2H), 7.98 (d, J=5.9 Hz, 1H), 8.19 (d, J=7.7 Hz, 1H),
10.28 (s, 1H). LC-MS (Method A, retention time: 3.57 min), MS m/z
670 (M.sup.++1).
Example 6
Preparation of Compound 3,
[18-(Isoquinolin-1-yloxy)-2,15-dioxo-4-(propane-2-sulfonylaminocarbonyl)--
3,16-diaza-tricyclo-[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid tert-butyl ester
[0370] ##STR176##
14-tert-butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaz-
a-tricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic acid (67
mg, 0.10 mmol) was dissolved in 5 mL of THF and treated with CDI
(21 mg, 0.13 mmol). (Care was taken to avoid moisture by using oven
dried glassware and maintaining a dry N2 atmosphere.) After
refluxing the reaction mixture for one hour, it was cooled to rt
and treated sequentially with isopropylsulfonamide (16 mg, 0.13
mmol) and DBU (20 mg, 0.13 mmol). After stirring for 24 h at rt,
the THF was removed by rotary evaporation. The residue was
partitioned between ethyl acetate and pH 4 buffer. The organic
phase was dried (MgSO4) and concentrated in vacuo to give the crude
product. Flash chromatography (1-5% MeOH/methylene chloride) gave
50 mg (71%) of the desired product. Further purification by
preparative HPLC (YMC ODS-A, S5, 20.times.100 mm, gradient: 60% to
100% B) gave 30 mg (43%) of
[18-(isoquinolin-1-yloxy)-2,15-dioxo-4-(propane-2-sulfonylaminoc-
arbonyl)-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid tert-butyl ester as a white powder: .sup.1H NMR (300 MHz,
CD.sub.3Cl.sub.3) .delta. 1.22 (S, 9H), 1.31 (d. J=6.6 Hz, 3H),
1.41 (d, J=6.9 Hz, 3H), 1.20-1.96 (m, 11H), 2.30 (q, J=8.4 Hz, 1H),
2.56 (m, 1H), 2.69 (m, 2H), 3.69 (m, 1H), 4.03 (m, 1H), 4.29 (m,
1H), 4.62 (m, 2H), 4.97-5.06 (m, 2H), 5.68 (q, J=9.5 Hz, 1H), 5.91
(s, 1H), 6.76 (s, 1H), 7.23 (m, 1H), 7.45 (t, J=7.7 Hz, 1H), 7.63
(t, J=8.1 Hz, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.97 (d, J=5.9 Hz, 1H),
8.17 (d, J=8.1 Hz, 1H), 9.89 (s, 1H). LC-MS (Method A, retention
time: 3.79 min), MS m/z 670 (M.sup.++1).
Example 7
Preparation of Compound 4,
[4-Cyclopropanesulfonylaminocarbonyl-18-(isoquinolin-1-yloxy)-2,15-dioxo--
3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid tert-butyl ester
[0371] ##STR177##
[0372] Prepared from
14-tert-butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaz-
a-tricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic acid
etherate (100 mg, 0.15 mmol) and cyclopropylsulfonamide (24 mg,
0.20 mmol) as described in the general procedure above. Flash
chromatography (2% methanol/methylene chloride) gave 55 mg (53%) of
[4-cyclopropanesulfonylaminocarbonyl-18-(isoquinolin-1-yloxy)-2,15-dioxo--
3,16-diaza-tricyclo-[14.3.0.0.sup.4,6]-nonadec-7-en-14-yl]-carbamic
acid tert-butyl ester as a white powder: .sup.1H NMR (500 MHz,
CD.sub.3Cl.sub.3) .delta. 0.89-0.95 (m, 1H), 1.03-1.16 (m, 3H),
1.22 (s, 9H), 1.18-1.48 (m, 8H), 1.78 (m, 1H), 1.87 (m, 1H), 1.94
(m, 1H), 2.29 (q, J=9.0 Hz, 1H), 2.56 (m, 1H), 2.67 (dd, J=7.9 Hz,
2.7 Hz, 2H), 2.90 (m, 1H), 4.04 (dd, J=1.3 Hz, 4.0 Hz, 1H), 4.29
(m, 1H), 4.61 (m, 2H), 4.97-5.03 (m, 2H), 5.70 (m, 1H), 5.94 (s,
1H), 6.67 (s, 1H), 7.25 (m, 1H), 7.47 (t, J=7.3 Hz, 1H), 7.64 (t,
J=7.3 Hz, 1H), 7.72 (d, J=7.9 Hz, 1H), 7.97 (d, J=5.8 Hz, I H),
8.17 (d, J=8.5 Hz, 1H), 10.20 (s, 1H). LC-MS (Method A, retention
time: 3.67 min), MS m/z 696 (M.sup.++1).
Example 8
[4-(1-Benzyl-cyclopropanesulfonylaminocarbonyl)-18-(isoquinolin-1-yloxy)-2-
,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbami-
c acid tert-butyl ester
[0373] ##STR178##
[0374] Prepared from
14-tert-butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaz-
a-tricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic acid (50
mg, 0.075 mmol) and 1-benzyl-cyclopropanesulfonic acid amide (21
mg, 0.098 mmol, prepared as described above) to give
[4-(1-benzyl-cyclopropanesulfonylaminocarbonyl)-18-(isoquinolin-1-yloxy)--
2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester as a white powder: .sup.1H NMR (500 MHz,
CD.sub.3Cl.sub.3) .delta. 1.23 (s, 9H), 1.04-1.92 (m, 14H), 1.96
(m, 1H), 2.32 (dd, J=17.7 Hz, 9.5 Hz, 1H), 2.59 (m, 1H), 2.70 (m,
2H), 3.20 (d, J=13.4 Hz, 1H), 3.41 (d, J=13.7 Hz, 1H), 4.02 (m,
1H), 4.29(m, 1H), 4.61 (m, 2H), 5.01 (d, J=7.6 Hz, 1H), 5.15 (m,
1H), 5.78 (m, 1H), 5.92 (s, 1H), 6.65 (s, 1H), 7.08 (d, J=8.2 Hz,
2H), 7.26 (m, 4H), 7.46 (t, J=7.3 Hz, 1H), 7.64 (t, J=7.3 Hz, 1H),
7.72 (d, J=8.2 Hz, 1H), 7.97 (d, J=5.8 Hz, 1H), 8.17 (d, J=8.9 Hz,
1H), 10.05 (s, 1H). LC-MS (Method A, retention time: 3.87 min), MS
m/z 786 (M.sup.++1).
Example 9
Preparation of Compound 6,
[18-(Isoquinolin-1-yloxy)-2,15-dioxo-4-(1-propyl-cyclopropanesulfonylamin-
ocarbonyl)-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester
[0375] ##STR179##
[0376] Prepared from
14-tert-butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaz-
a-tricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic acid (50
mg, 0.075 mmol) and 1-propyl-cyclopropanesulfonic acid amide (16
mg, 0.098 mmol, prepared as described above) to give
[18-(isoquinolin-1-yloxy)-2,15-dioxo-4-(1-propyl-cyclopropanesulfonylamin-
ocarbonyl)-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester as a white powder: .sup.1H NMR (300 MHz,
CD.sub.3Cl.sub.3) .delta. 0.82-0.93 (m, 5H), 1.23 (s, 9H),
1.30-1.94 (m, 17H), 2.28 (m, 1H), 2.54 (m, 1H), 2.68 (dd, J=8.1 Hz,
3.2 Hz, 2H), 4.05 (m, 1H), 4.30 (m, 1H), 4.63-4.66 (m, 2H),
4.96-5.09 (m, 2H), 5.70 (m, 1H), 5.92 (s, 1H), 6.69 (s, 1H), 7.23
(m, 1H), 7.46 (t, J=8.1 Hz, 1H), 7.63 (t, J=8.1 Hz, 1H), 7.72 (d,
J=8.1 Hz, 1H), 7.97(d, J=5.9 Hz, 1H), 8.17 (d, J=8.4 Hz, 1H), 10.12
(s, 1H). LC-MS (Method A, retention time: 3.77 min), MS m/z 738
(M.sup.++1).
Example 10
Preparation of Compound 7,
[18-(Isoquinolin-1-yloxy)-2,15-dioxo-4-(1-propyl-cyclopropanesulfonylamin-
ocarbonyl)-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-14-yl]-carbamic
acid tert-butyl ester
[0377] ##STR180##
[0378] To a mixture of 73 mg (0.1 mmol) of
[18-(Isoquinolin-1-yloxy)-2,15-dioxo-4-(1-propylcyclopropanesulfonylamino-
-carbonyl)-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester in 3 mL of methanol was added 332 mg (2
mmol) of dipotassium azodicarboxylate. Glacial acetic acid (240 mg,
4 mmol) in 2 mL of methanol was added slowly by a syringe pump over
5 h. The mixture was stirred at rt. After 5 h, another 332 mg (2
mmol) of dipotassium azodicarboxylate and 240 mg (4 mmol) of
glacial acetic acid was added over the course of 5 h and the
stirring was continued overnight. This cycle was repeated twice.
LC-MS showed about 40% starting material still remaining. The
solvent was then removed on a rotary evaporator. Water was added to
the residue and the mixture was extracted three times with ethyl
acetate. It was then dried with magnesium sulfate, filtered and
concentrated in vacuo. The resulting oil was dissolved in methanol
and purified by preparative HPLC (YMC ODS-A, S5, 20.times.100 mm,
gradient: 80% B to 85% B, 15 min, hold 2 min, flow rate 25 mL/min)
to isolate the product as a white powder (25 mg, 34%).
[0379] .sup.1H NMR (500 MHz, CD.sub.3Cl.sub.3) .delta. 0.91 (m,
5H), 1.30 (s, 9H), 1.20-1.80 (m, 21H), 1.86-1.93 (m, 2H), 2.57 (m,
1H), 2.67 (m, 1H), 4.10 (m, 1H), 4.36 (d, J=12.2 Hz, 1H), 4.44 (t,
J=8.2 Hz, 1H), 4.61 (t, J=7.9 Hz, 1H), 5.16 (d, J=8.2 Hz, 1H), 5.92
(bs, 1H), 6.58 (bs, 1H), 7.25 (m, 1H), 7.49 (t, J=7.3 Hz, 1H), 7.65
(t, J=7.3 Hz, 1H), 7.73 (d, J=8.2 Hz, 1H), 7.97 (d, J=5.8 Hz, 1H),
8.15 (d, J=8.2 Hz, 1H), 10.09 (s, 1H). LC-MS (Method A, retention
time: 3.83 min), MS m/z 740(M.sup.++1).
Example 11
Preparation of Compound 8,
[18-(Isoquinolin-1-yloxy)-4-(1-methyl-cyclopropanesulfonylaminocarbonyl)--
2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester
[0380] ##STR181##
[0381] Prepared from
14-tert-butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaz-
a-tricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic acid
etherate (105 mg, 0.177 mmol) and 1-methyl-cyclopropanesulfonic
acid amide (31 mg, 0.23 mmol, prepared as described above). Flash
chromatography (2% methanol/methylene chloride) gave 73 mg (58%) of
[18-(isoquinolin-1-yloxy)-4-(1-methyl-cyclopropanesulfonylaminocarbonyl)--
2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester as a white powder: LC-MS (Method A,
retention time: 3.50 min), MS m/z 710 (M.sup.++1).
Example 12
Preparation of Compoun 9,
[4-Cyclobutanesulfonylaminocarbonyl-18-(isoquinolin-1-yloxy)-2,15-dioxo-3-
,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid tert-butyl ester
[0382] ##STR182##
[0383] Prepared from
14-tert-butoxycarbonylamino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaz-
a-tricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic acid
etherate (114 mg, 0.192 mmol) and cyclobutanesulfonic acid amide
(34 mg, 0.25 mmol) as described in the general procedure above.
Flash chromatography (2% methanol/methylene chloride) gave 73 mg
(58%) of
[18-(isoquinolin-1-yloxy)-4-(1-methyl-cyclopropanesulfonylaminocarbonyl)--
2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester as a white powder: LC-MS (Method A,
retention time: 3.55 min), MS m/z 710 (M.sup.++1).
Example 13
Preparation of Compound 10,
[4-Cyclopropanesulfonylaminocarbonyl-18-(isoquinolin-1-yloxy)-2,15-dioxo--
3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-14-yl]-carbamic acid
tert-butyl ester
[0384] ##STR183##
[0385] To a mixture of 140 mg (0.2 mmol) of
[4-cyclopropanesulfonylaminocarbonyl-18-(isoquinolin-1-yloxy)-2,15-dioxo--
3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid tert-butyl ester in 3 mL of methanol was added 656 mg (4 mmol)
of dipotassium azodicarboxylate. Glacial acetic acid (480 mg, 8
mmol) in 2 mL of methanol was added slowly by a syringe pump over 5
h. The mixture was stirred at rt. After 5 h, another 656 mg (4
mmol) of dipotassium azodicarboxylate and 480 mg (8 mmol) of
glacial acetic acid was added over the course of 5 h and the
stirring was continued overnight. This cycle was repeated twice.
LC-MS showed about 40% starting material still was remaining. The
solvent was then removed on a rotary evaporator. Water was added to
the residue and the mixture was extracted three times with ethyl
acetate. It was then dried with magnesium sulfate, filtered and
concentrated in vacuo. The resulting oil was dissolved in methanol
and purified by preparative HPLC (YMC XTERRA, S5, 30.times.50 mm,
gradient: 72% B to 78% B, 15 min, hold 2 min, flow rate 40 ml/min)
to isolate the product as a white powder (80 mg, 57%). LC-MS
(Method A, retention time: 3.59 min), MS m/z 698 (M.sup.++1).
Example 14
Preparation of Compound 11, Cyclopropanesulfonic acid
[14-amino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.-
0.sup.4,6]nonadec-7-ene-4-carbonyl]-amide bis hydrochloride
[0386] ##STR184##
[0387] A stirred slurry of
[4-cyclopropane-sulfonylaminocarbonyl-18-(isoquinolin
1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14--
yl]-carbamic acid tert-butyl ester (1.20 g, 1.7 mmol) was treated
with 4N HCl/dioxane (Aldrich)(10 mL) for 4 h. The reaction mixture
was concentrated in vacuo to give 1.10 g (92%) of
cyclopropanesulfonic acid
[14-amino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.-
0.sup.4,6]nonadec-7-ene-4-carbonyl]-amide bis hydrochloride as a
white solid: LC-MS (Method B, retention time: 1.39 min), MS m/z 596
(M.sup.++1-2 HCl).
Example 15
Preparation of Compound 12
[4-Cyclopropanesulfonylaminocarbonyl-18-(isoquinolin-1-yloxy)-2,15-dioxo--
3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid methyl ester
[0388] ##STR185##
[0389] To a mixture of cyclopropanesulfonic acid
[14-amino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.-
0.sup.4,6]nonadec-7-ene-4-carbonyl]-amide bis hydrochloride (50 mg,
0.075 mmol) in 2 mL of DCM was added 44 .mu.L (0.25 mmol) of DIPEA
and 9 mg (0.10 mmol) of methyl chloroformate. The mixture was
stirred at rt for 2 h. It was then diluted with EtOAc and washed
with pH 4 buffer (2.times.) and brine (1.times.). The organic phase
was dried (MgSO.sub.4) and concentrated in vacuo to give the crude
product. Flash chromatography (2% MeOH/DCM) gave 40 mg (82%) of the
desired product as a white solid: LC-MS (Method A, retention time:
3.05 min), MS m/z 654 (M.sup.++1).
Example 16
Preparation of Compound 13,
[4-Cyclopropanesulfonylaminocarbonyl-18-(isoquinolin-1-yloxy)-2,15-dioxo--
3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid 2,2-dimethyl-propyl ester
[0390] ##STR186##
[0391] To a mixture of cyclopropanesulfonic acid
[14-amino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.-
0.sup.4,6]nonadec-7-ene-4-carbonyl]-amide bis hydrochloride (50 mg,
0.075 mmol) in 2 mL of DCM was added 44 .mu.L (0.25 mmol) of DIPEA
and 15 mg (0.1 mmol) of neopentyl chloroformate. The mixture was
stirred at rt for 2 h. It was then diluted with EtOAc and washed
with pH 4 buffer (2.times.) and brine (1.times.). The organic phase
was dried (MgSO.sub.4) and concentrated in vacuo to give the crude
product. The resulting oil was dissolved in methanol and purified
by preparative HPLC (YMC XTERRA, S5, 30.times.50 mm, gradient: 65%
B to 100% B, 15 min, hold 2 min, flow rate 40 mL/min) to isolate
the product as a white powder (42 mg, 79%): LC-MS (Method A,
retention time: 3.58 min), MS m/z 710 (M.sup.++1).
General Procedure for Preparation of Chloroformates
[0392] ##STR187##
[0393] This procedure was used for the preparation of
non-commercially available chloroformates. To a solution of 5.96 g
(67.6 mmol) of commercially available reagents
(S)-3-hydroxytetrahydrofuran and pyridine (5.8 mL; 72 mmol) in THF
(150 mL) cooled to 0.degree. C. was added a 1.93 M solution of
phosgene in toluene (48 mL, 92.6 mmol over 10 min under argon. The
resulting solution was allowed to warm to rt over 2 h, the
resulting solid filtered, and the mother liquor carefully
concentrated in vacuo at room temperature until theoretical mass
was obtained. The resulting residue was dissolved in 100 mL of THF
to prepare a 0.68M stock solution of 3(S)-oxo-tetrahydrofuran
chloroformate that could be stored in the freezer until use. In
analogous fashion, other commercially available alcohols could be
converted to 0.68M stock solutions of the corresponding
chloroformates.
Example 17
Preparation of Compound 14,
[4-Cyclopropanesulfonylaminocarbonyl-18-(isoquinolin-1-yloxy)-2,15-dioxo--
3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid tetrahydro-pyran-4-yl ester
[0394] ##STR188##
[0395] Prepared from cyclopropanesulfonic acid
[14-amino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.-
0.sup.4,6]nonadec-7-ene-4-carbonyl]-amide bis hydrochloride (50 mg,
0.075 mmol, 44 .mu.L (0.25 mmol) of DIPEA and 0.17 mL (0.1 mmol) of
tetrahydropyran chloroformate (prepared by treating
tetrahydro-pyran-4-ol with phosgene) in DCM as described in the
procedure above to give 28 mg (52%) of a white solid. Preparative
HPLC condition: YMC XTERRA, S5, 19.times.100 mm, gradient: 50% B to
100% B, 10 min, hold 2 min, flow rate 25 mL/min. LC-MS (Method A,
retention time: 3.13 min), MS m/z 724 (M.sup.++1).
Example 18
Preparation of Compound 15,
[4-Cyclopropanesulfonylaminocarbonyl-18-(isoquinolin-1-yloxy)-2,15-dioxo--
3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid tetrahydro-furan-3-yl ester
[0396] ##STR189##
[0397] Prepared from cyclopropanesulfonic acid
[14-amino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.-
0.sup.4,6]nonadec-7-ene-4-carbonyl]-amide bis hydrochloride (50 mg,
0.075 mmol), 44 .mu.L (0.25 mmol) of DIPEA and 0.10 mL (0.1 mmol)
of tetrahydro-furan-3-yl chloroformate (prepared by treating
tetrahydro-furan-3-ol with phosgene) in DCM as described in the
procedure above to give 28 mg (52%) of a white solid. Preparative
HPLC condition: YMC ODS-A, S5, 30.times.50 mm, gradient: 50% B to
85% B, 8 min, hold 2 min, flow rate 45 ml/min. LC-MS (Method A,
retention time: 3.05 min), MS m/z 710 (M.sup.++1).
Example 19
Preparation of Compound 16,
[4-Cyclopropanesulfonylaminocarbonyl-18-(isoquinolin-1-yloxy)-2,15-dioxo--
3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid isopropyl ester
[0398] ##STR190##
[0399] Prepared from cyclopropanesulfonic acid
[14-amino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.-
0.sup.4,6]nonadec-7-ene-4-carbonyl]-amide bis hydrochloride (50 mg,
0.075 mmol), 44 .mu.L (0.25 mmol) of DIPEA and 0.10 mL (0.1 mmol)
of 1 M isopropyl chloroformate in toluene (Aldrich) as described in
the procedure above to give 40 mg (78%) of a white solid.
Preparative HPLC condition: YMC XTERRA, S5, 30.times.50 mm,
gradient: 50% B to 100% B, 15 min, hold 2 min, flow rate 40 mL/min)
NMR (300 MHz, CD.sub.3OD) 8. LC-MS (Method A, retention time: 3.32
min), MS m/z 682 (M.sup.++1).
Example 20
Preparation of
(1S,4R,6S,14S,18R)-7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2,15-diox-
o-3,16-diazatricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid for use in the preparation of Compound 17 of Example 21
[0400] ##STR191##
Example 20
Step 20 A: Preparation of
1-(2(S)-tert-Butoxycarbonylamino-non-8-enoyl)-4(R)-hydroxy-pyrrolidine-2(-
S)-carboxylic acid methyl ester
[0401] ##STR192##
[0402] A solution of 2(S)-tert-butoxycarbonylamino-8-nonenoic acid
(purchased from RSP Amino Acids) (3.5 g, 12.9 mmol) in 200 mL of
DCM was treated sequentially with
4(R)-hydroxypyrrolidine-2(S)-carboxylic acid methyl ester
hydrochloride (2.15 g, 11.8 mmol), N-methyl morpholine (4.25 mL,
38.6 mmol), and HATU (5.37 g, 14.1 mmol). The reaction mixture was
stirred at rt under N.sub.2 for 3 days, and then concentrated in
vacuo. The residue was partitioned between ethyl acetate and pH 4
buffer (biphthalate). The organic phase was washed with sat. aq.
NaHCO.sub.3, dried (MgSO.sub.4), and concentrated in vacuo to give
the crude product. Flash chromatography (50% ethyl acetate/hexane
to 100% ethyl acetate) gave 4.7 g (100%) of
1-(2(S)-tert-Butoxycarbonylamino-non-8-enoyl)-4(R)-hydroxypyrrolidine-2(S-
)-carboxylic acid methyl ester as a colorless oil: .sup.1H NMR (500
MHz, CD.sub.3OD) .delta. 1.33-1.50(m, 8H), 1.46 (s, 9H), 1.57 (m,
1H), 1.72 (m, 1H) 2.08 (m, 2H), 2.28 (m, 1H), 3.72 (s, 3H,)
3.75-3.87 (m, 2H), 4.36 (m, 1H), 4.51 (bs, 1H), 4.57 (t, J=8.2 Hz,
1H), 4.95 (d, J=10.4 Hz, 1H), 5.01 (m, 1H), 5.83 (m, 1H). LC-MS
(Method A, retention time: 3.01 min), MS m/z 399 (M.sup.++1).
Step 20 B: Preparation of
1-{[1-(2(S)-tert-Butoxycarbonylamino-non-8-enoyl)-4(R)-hydroxy-pyrrolidin-
e-2(S)carbonyl]-(1R)-amino}-2(S)-vinyl-cyclopropanecarboxylic acid
ethyl ester
[0403] ##STR193##
[0404]
1-(2(S)-tert-Butoxycarbonylamino-non-8-enoyl)-4(R)-hydroxy-pyrroli-
dine-2(S)-carboxylic acid methyl ester (4.7 g, 11.8 mmol) was
dissolved in THF (80 mL), methanol (20 mL), and water (40 mL).
Powdered lithium hydroxide (5.6 g, 233 mmol) was added. The light
yellow slurry was stirred at rt under N.sub.2 for 16 h, and then
concentrated in vacuo. The residue was partioned between ether and
water. The ether phase was discarded, and the aqueous phase was
treated with 1N HCl until the pH was 4. This acidic solution was
extracted with EtOAc (3.times.). The combined EtOAc extracts were
dried (MgSO.sub.4) and concentrated in vacuo to give 4.36 g (96%)
of
1-(2(S)-tert-butoxycarbonylamino-8-nonenoyl)-4(R)-hydroxy-pyrrolidine-2(S-
)-carboxylic acid as a white solid. This acid was then dissolved in
150 mL of DMF and (1R,2S)-1-amino-2-vinylcyclopropane carboxylic
acid ethyl ester hydrochloride (2.61 g, 13.6 mmol), N-methyl
morpholine (2.5 mL, 22.6 mmol), and HATU (5.2 g, 13.7 mmol) was
added. The reaction mixture was stirred at rt under N.sub.2 for 16
h, and then concentrated in vacuo. The residue was partitioned
between ethyl acetate and pH 4 buffer (biphthalate). The organic
phase was washed with sat. aq. NaHCO.sub.3, dried (MgSO.sub.4), and
concentrated in vacuo to give the crude product. Flash
chromatography (60%-80% ethyl acetate/hexane) gave 6.0 g (98%) of
1-{[1-(2(S)-tert-Butoxycarbonylamino-non-8-enoyl)-4(R)-hydroxy-pyrrolidin-
e-2(S)carbonyl]-(1R)-amino}-2(S)-vinyl-cyclopropanecarboxylic acid
ethyl ester as a white solid: .sup.1H NMR (500 MHz, CD.sub.3OD)
.delta. 1.25 (t, J=7.2 Hz, 3H), 1.33-1.80 (m, 10H), 1.46 (s, 9H),
2.09 (m, 3H), 2.25 (m, 2H), 3.76 (m, 2H), 4.14 (m, 2H), 4.27 (dd,
J=8.5, 5.2 Hz, 1H), 4.50 (m, 2H), 4.94 (d, J=10.1 Hz, 1H), 5.01
(dd, J=17.1, 1.8 Hz, 1H), 5.11 (dd, J=10.4, 1.8 Hz, 1H), 5.30 (d,
J=15.6 Hz, 1H), 5.80 (m, 2H), 8.57 (s, 1H). LC-MS (Method A,
retention time: 3.21 min), MS m/z 522 (M.sup.++1).
Step 20C: Preparation of
(1S,4R,6S,14S,18R)-7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2,15-diox-
o-3,16-diazatricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid ehtyl ester
[0405] ##STR194##
[0406] A solution of
1-{[1-(2(S)-tert-Butoxycarbonyl-amino-non-8-enoyl)-4(R)-hydroxy-pyrrolidi-
ne-2(S)carbonyl]-(1R)-amino}-2(S)-vinylcyclopropane-carboxylic acid
ethyl ester (800 mg, 1.53 mmol) in 2 L of methylene chloride was
flushed with N.sub.2 for 0.5 h. Then
tricyclohexylphosphine[1,3-bis(2,4,6-trimethyl-phenyl)-4,5-dihydroimidazo-
l-2-ylidene][benzylidene]-ruthenium (IV) dichloride (Strem) (64 mg,
0.075 mmol) was added, and the mixture was flushed with N.sub.2 for
another 10 min. The light orange homogeneous solution was refluxed
for 2 h to give a dark orange solution. The reaction mixture was
cooled to rt and concentrated in vacuo to give an orange oil. Flash
chromatography (ethyl acetate) gave 460 mg (61%) of
(1S,4R,6S,14S,18R)-7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2,15-diox-
o-3,16-diazatricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic
acid ethyl ester as a gray solid. .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 1.19 (t, J=7.2 Hz, 3H), 1.42 (s, 9H), 1.22-1.8 (m, 8H),
1.87 (m, 2H), 2.03-2.22 (m, 4H), 2.63 (m, 1H), 3.65 (m, 1H), 4.09
(m, 3H), 4.45 (m, 1H), 4.56 (s, 1H), 4.82 (m, 1H), 5.23 (m, 1H),
5.51 (s, 1H), 7.16 (s, 1H). LC-MS (Method A, retention time: 2.97
min), MS m/z 494 (M.sup.++1).
Step 20 D:
(1S,4R,6S,14S,18R)-7-cis-14-tert-butoxycarbonylamino-18-hydroxy-
-2,15-dioxo-3,16-diazatricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxyli-
c acid
[0407] ##STR195##
[0408] To a solution of
(1S,4R,6S,14S,18R)-7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2,15-diox-
o-3,16-diazatricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic
acid ethyl ester (493 mg, 1.0 mmol) in THF (4 mL), methanol (1 mL),
and water (2 mL), was added powdered lithium hydroxide (480 mg, 20
mmol), and the light yellow slurry stirred at rt under N.sub.2 for
16 h. The mixture was then concentrated in vacuo and the residue
partioned between ether and water. The ether phase was discarded,
and the aqueous phase was treated with 1 N HCl until pH 4. This
acidic solution was extracted with EtOAc three times. The combined
EtOAc extracts were dried (MgSO.sub.4) and concentrated in vacuo to
give 460 mg (98%) of Example 26,
(1S,4R,6S,14S,18R)-7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2,15-diox-
o-3,16-diazatricyclo[14.3.0.0.sup.4,6]-nonadec-7-ene-4-carboxylic
acid as a gray solid. .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. ppm
1.26 (t, J=7.2 Hz, 3H), 1.35-1.52 (m, 15H), 1.57-1.68 (m, 3H), 1.79
(m, 1H), 2.04 (m, 1H), 2.16-2.41 (m, 3H), 3.80 (dd, J=10.7, 4.3 Hz,
1H), 3.88 (m, 1H), 4.38 (dd, J=8.9, 3.1 Hz, 1H), 4.55 (m, 2H), 5.39
(t, J=9.8 Hz, 1H), 5.58 (m, 1H). LC-MS (Method A, retention time:
2.64 min), MS m/z 466 (M.sup.++1).
Example 21
Preparation of Compound 17B,
[4-Cyclopropanesulfonylaminocarbonyl-18-(6-methoxy-isoquinolin-1-yloxy)-2-
,15-dioxo-3,16-diazatricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbamic
acid tert-butyl ester
[0409] ##STR196##
[0410] To a mixture of
4-tert-butoxycarbonylamino-18-hydroxy-2,15-dioxo-3,16-diaza-tricyclo[14.3-
.0.0.sup.4,6]nonadec-7-ene-4-carboxylic acid (215 mg, 0.46 mmol) in
DMSO (5 mL) was added t-BuOK (125 mg, 1.11 mmol) and
1-chloro-6-methoxy-isoquinoline (110 mg, 0.56 mmol). The reaction
was stirred for 16 h at rt. The reaction mixture then was
partitioned between ether (10 mL) and water (10 mL). The aqueous
phase was acidified to pH 4 using 1 N HCl. The resulting solution
was extracted with EtOAc (3.times.20 mL). The combined EtOAc
extracts were dried (MgSO.sub.4), filtered, and concentrated in
vacuo to give a white solid. Flash chromatography (2%
MeOH/CH.sub.2Cl.sub.2) gave 140 mg (49%) of the carboxylic acid
derivative as a white solid. LC-MS (Method B, retention time: 1.80
min), MS m/z 543 (M.sup.++1). The above solid (140 mg, 0.22 mmol)
was treated with cyclopropylsulfonamide (35 mg, 0.28 mmol) as
described in the general procedure above to give the crude product.
Flash chromatography (2% MeOH/DCM) gave 90 mg of the desired
product. Further purification by preparative HPLC (YMC Xterra, S5,
30.times.50 mm, 50% to 100% B, gradient 9 min, hold 1 min, flow
rate 40 mL/min) gave 30 mg (19%) of the product as a white powder:
LC-MS (Method B, retention time: 1.86 min), MS m/z 726
(M.sup.++1).
Example 22
Preparation of
1-{[4-(6-Methoxy-isoquinolin-1-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vi-
nyl-cyclopropanecarboxylic acid ethyl ester (as bis HCl salt) use
in Example 24
[0411] ##STR197##
Example 22
[0412] Prepared utilizing the same synthetic sequence employed for
the preparation of the product of Step 2B of Example 3 except
6-Methoxy-1-chloroquinoline (described in Example 1, eg. Step 1a
and Step 1b) was used in place of 1-chloroquinoline. Example 23,
Preparation of Example 23,
2(S)-tert-butoxycarbonylamino-3-pent-4-enylsulfanylpropionic acid
for use in Example 24. ##STR198##
[0413] Step 1: To a solution of N-Boc-cysteine methyl ester (3.36
g, 0.014 mol) in methanol (166 mL) at RT was added triethylamine
(10.8 mL) and 1-bromopent-4-ene (3.19 g, 21 mmol, 1.5 equivalents)
and the resulting solution was stirred at room temperature
overnight. The mixture was then concentrated in vacuo and the
resulting residual mixture was purified using flash chromatography
(hexane, ethyl acetate gradient) to provide 1.76 g (41%) of the
desired thioether. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.43
(s, 9H), 1.64 (m, 2H), 2.11 (m, 2H), 2.51 (m, 2H), 2.95 (m, 2H),
3.75 (s, 3H), 4.51 (m, 1H), 4.95-5.03 (m, 2H), 5.34 (m, 1H), 5.80
(1H, m). LC-MS (Method B, except gradient time was 3 min., and flow
rate was 4 ml/min, retention time: 2.29 min), MS m/z
304(M.sup.++1).
[0414] Step 2: The thioether product of step 1 (9.51 g, 31.4 mmol)
was added to a mixture of 1M LiOH in water (200 mL) and THF (200
mL) and the resulting mixture was stirred at room temperature
overnight. The reaction mixture was then acidified using 1N
hydrochloric acid and the resulting mixture was extracted several
times with ethyl acetate. The extracts were combined, dried over
magnesium sulfate, and concentrated in vacuo to provide the desired
acid which was used as is in the next reaction.
Example 24
Preparation of Compound 18B,
[4-Cyclopropanesulfonylaminocarbonyl-18-(6-methoxy-isoquinolin-1-yloxy)-2-
,15-dioxo-12-thia-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-
-carbamic acid tert-butyl ester
[0415] ##STR199##
Step 24 A, Preparation of
1-{[1-(2-tert-Butoxycarbonylamino-3-pent-4-enylsulfanyl-propionyl)-4-(6-m-
ethoxy-isoquinolin-1-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopr-
opanecarboxylic acid ethyl ester
[0416] ##STR200## To a solution of the carboxylic acid of Example
23 (289 mg, 1.00 mmol, 1 eq) in DMF (9 mL) was added sequentially
the dipeptide product of Example 22 (498 mg, 1.00 mmol, 1 eq), HATU
(456 mg, 1.20 mmol, 1.2 eq), and N-methyl morpholine (385 .mu.L,
3.50 mmol, 3.5 eq). The solution was allowed to stir at room
temperature overnight, at which time LC/MS indicated the formation
of product and disappearance of starting material 2. A pH 4 buffer
solution was added, and the mixture extracted three times with
ethyl acetate. The organic layers were washed with brine, dried
over magnesium sulfate, and concentrated in vacuo. Chromatography
was performed (Silica Gel, Ethyl acetate/Hexane gradient) to obtain
pure tripeptide 3 in quantitative yield.
[0417] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.03 (d, J=9.3 Hz,
1H), 7.89 (d, J=5.9 Hz, 1H), 7.56 (br s, 1H), 7.15-7.09 (m, 2H),
7.01 (d, J=2.5 Hz, 1H), 5.86-5.68 (m, 3H), 5.39-5.22 (m, 2H),
5.12-4.82 (m, 4H), 4.61 (app q, J=6.8 Hz, 1H), 4.24-4.04 (m, 3H),
3.91 (s, 3H), 3.02-2.90 (m, 1H), 2.84-2.69 (m, 2H), 2.66 (m, 3H),
2.24-2.08 (m, 2H), 1.88 (dd, J=5.5, 8.0 Hz, 1H), 1.68 (app quint,
J=7.5 Hz, 2H), 1.56 (dd, J=5.4, 9.5 Hz, 1H), 1.42 (m, 1H), 1.31 (s,
9H), 1.27-1.20 (m, 4H).[m/z]+H 697, ERRA 3.0.times.50 mm S7
retention time=1.937 min, HPLC method 2 min gradient 0% B to 100%
B, then 1 min at 100% B (3 min total run time)
Step 24 B, Preparation of
14-tert-Butoxycarbonylamino-18-(6-methoxy-isoquinolin-1-yloxy)-2,15-dioxo-
-12-thia-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid ethyl ester
[0418] ##STR201## To a solution of tripeptide prodict of Step 24A
(384.6 mg, 553 .mu.mol, 1 eq) in dichloroethane (159 mL) was added
tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-
-2-ylidene[benzylidine]ruthenium(IV)dichloride (48.5 mg, 57
.mu.mol, 0.10 eq) and refluxed for 16 h. The reaction was
concentrated in vacuo. Chromatography was performed (Silica Gel,
Ethyl acetate/Hexane gradient) to obtain pure macrocycle, in 73%
yield.
[0419] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.04 (d, J=9.2 Hz,
1H), 7.86 (d, J=5.8 Hz, 1H), 7.59 (br s, 1H), 7.16-7.09 (m, 2H),
7.00 (d, J=2.4 Hz, 1H), 5.78-5.71 (m, 1H), 5.64 (d, J=7.6 Hz, 1H),
5.58-5.50 (m, 1H), 5.36 (m, 1H), 5.03 (dd, J=4.2, 8.3 Hz, 1H),
4.81-4.75 (m, 1H), 4.27-4.14 (m, 2H), 3.98-3.93 (m, 1H), 3.91 (s,
3H), 3.13-3.02 (m, 2H), 3.00-2.94 (m, 1H), 2.69 (dt, J=3.9, 11.2
Hz, 1H), 2.52-2.42 (m, 2H), 2.36-2.26 (m, 2H), 2.16-2.08 (m, 1H),
1.96 (dd, J=5.5, 8.2 Hz, 1H), 1.86-1.76 (m, 1H), 1.68-1.58 (m, 2H),
1.37 (s, 9H), 1.29 (t, J=7.2 Hz, 1H), 1.27-1.21 (m, 1H).
[0420] [m/z]+H 669
XTERRA 3.0.times.50 mm S7 retention time=1.797, HPLC method 2 min
gradient 0% B to 100% B, then 1 min at 100% B (3 min total run
time)
Step 24 C, Preparation of 18A,
14-tert-Butoxycarbonylamino-18-(6-methoxy-isoquinolin-1-yloxy)-2,15-dioxo-
-12-thia-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid
[0421] ##STR202## To a solution of the macrocycle ester product of
Step 24B (271 mg, 406 .mu.mol, 1 eq) in THF (4.1 mL) was added LiOH
(97 mg, 4.04 mmol, 10 eq), water (0.45 mL), and MeOH (1.9 mL). The
reaction was allowed to stir for 16 h, at which time LC/MS
indicated the hydrolysis was complete. A 1 M solution of HCl in
water was added, and the resulting mixture was extracted 3 times
into ethyl acetate. The organic extracts were washed with brine,
dried over magnesium sulfate, and concentrated in vacuo to provide
pure carboxylic acid in quantitative yield.
[0422] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 12.50 (br s,
1H), 8.59 (br s, 1H), 8.22 (d, J=7.8 Hz, 1H), 8.14-8.07 (m, 1H),
7.46 (br s, 2H), 7.33-7.25 (m, 2H), 5.95 (br s, 1H), 5.79-5.56 (m,
2H), 4.66-4.57 (m, 1H), 4.50-4.37 (m, 2H), 4.24-4.11 (m, 2H), 4.05
(s, 3H), 3.60-3.40 (m, 2H), 3.09-2.97 (m, 1H), 2.91-2.81 (m, 1H),
2.65-2.58 (m, 1H), 2.56-2.44 (m, 2H), 2.44-2.33 (m, 1H), 1.84-1.73
(m, 1H), 1.72-1.57 (m, 2H), 1.34 (s, 9H), 1.08-0.95 (m, 1H).
[0423] [m/z]+H 641
XTERRA 3.0.times.50 mm S7 retention time=1.673, HPLC method 2 min
gradient 0% B to 100% B, then 1 min at 100% B (3 min total run
time)
Step 24D, Preparation of Compound 18B,
[4-Cyclopropanesulfonylaminocarbonyl-18-(6-methoxy-isoquinolin-1-yloxy)-2-
,15-dioxo-12-thia-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-
-carbamic acid tert-butyl ester
[0424] ##STR203## To a solution of the carboxylic acid product of
Step 24 C (236.3 mg, 369 .mu.mol, 1 eq) in THF (5.2 mL) was added
carbonyl diimidazole (143 mg, 738 .mu.mol, 2 eq) and refluxed for
2.5 h. The solution was cooled to room temperature, at which time
cyclopropyl sulfonamide (158 mg, 1.1 mmol, 3 eq) and DBU (127
.mu.L, 849 .mu.mol, 2.3 eq) were added. The reaction was allowed to
stir at room temperature for 16 h. A 1M solution of HCl in water
was added, and the product was extracted three times into methylene
chloride. The organic layers were washed with brine, dried over
magnesium sulfate, and concentrated in vacuo. Chromatography was
performed (Silica Gel, Ethyl acetate/Hexane gradient) to obtain
pure sulfonamide Compound 18, in 78% yield.
[0425] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.10 (d, J=9.3 Hz,
1H), 7.89-7.83 (m, 2H), 7.21 (d, J=5.9 Hz, 1H), 7.13 (br s, 1H),
7.05 (d, J=9.1 Hz, 1H), 5.81 (br s, 1H), 5.68 (app q, J=8.9 Hz,
1H), 5.16 (app t, J=9.7 Hz, 1H), 4.59-4.52 (m, 2H), 4.40-4.32 (m,
1H), 4.12-4.04 (m, 1H), 3.89 (s, 3H), 2.98-2.78 (m, 3H), 2.73-2.41
(m, 6H), 2.02-1.98 (m, 1H), 1.75 (dd, J=5.5, 8.2 Hz, 1H), 1.71-1.53
(m, 3H), 1.32-1.20 (m, 1H), 1.16 (s, 9H), 1.14-0.95 (m, 2H).
[0426] [m/z]+H 744
XTERRA 3.0.times.50 mm, S7 retention time=1.700, HPLC method 2 min
gradient 0% B to 100% B, then 1 min at 100% B (3 min total run
time)
Example 25
Preparation of Compound 19
4-Cyclopropanesulfonylaminocarbonyl-18-(6-methoxy-isoquinolin-1-yloxy)-2,-
12,12,15-tetraoxo-12-thia-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-e-
n-14-yl]-carbamic acid tert-butyl ester
[0427] ##STR204## To a solution of Compound 18 (84.7 mg, 114
.mu.mol) in MeOH (1.4 mL) and water (460 .mu.L) at 0.degree. C. was
added oxone (210.6 mg, 343 .mu.mol, 3.0 eq). The mixture was
stirred for 20 min at 0.degree. C., then allowed to stir for 3 h at
ambient temperature. Water and methylene chloride were then added,
and the layers separated. The aqueous layer was extracted twice
more with methylene chloride. The combined organic layers were
dried over magnesium sulfate and concentrated in vacuo. The product
was purified by prep TLC (ethyl acetate) to afford the sulfone
(37.2 mg, 42% yield).
[0428] .sup.1H NMR (CD.sub.3OD) .delta. 8.11 (d, J=9.5 Hz, 1H),
7.88 (d, J=6.1 Hz, 1H), 7.23 (d, J=5.8 Hz, 1H), 7.20-7.15 (m, 1H),
7.11 (dd, J=1.8, 8.9 Hz, 1H), 5.87-5.81 (m, 1H), 5.61-5.47 (m, 2H),
4.96-4.90 (m, 1H), 4.67 (t, J=7.8 Hz, 1H), 4.46-4.39 (m, 1H),
4.27-4.20 (m, 1H), 3.92 (s, 3H), 3.84-3.72 (m, 1H), 2.94-2.84 (m,
2H), 2.67-2.58 (m, 2H), 2.47-2.37 (m, 1H), 2.37-2.25 (m, 2H),
1.85-1.73 (m, 2H), 1.72-1.64 (m, 1H), 1.49-1.38 (m, 1H), 1.20 (s,
9H), 1.16-1.02 (m, 3H), 1.00-0.82 (m, 3H).
Example 26
Preparation of Compound 27 and Compound 28
[0429] ##STR205##
Step 26A: Preparation of Isopropyl
pyrrolidin-5-one-2(S)-carboxylate
[0430] ##STR206## A solution of L-pyroglutamic acid (Aldrich, 25.0
g, 195 mmol) and paratoluenesulfonic acid mono hydrate (3.71 g,
19.5 mmol) was refluxed in isopropanol (40 mL) under nitrogen for 6
hours using a Dean-Stark trap variation (condensate returned
through a Soxhlet extractor filled with 4 .ANG. molecular sieves).
After cooling to room temperature, the reaction was diluted with
ether, washed with saturated aqueous sodium bicarbonate and then
saturated aqueous NaCl, dried (MgSO.sub.4) and evaporated to give a
colorless syrup. It crystallized upon setting. Triturating the
crystalline residue in hexane provided 31.9 g (96%) of isopropyl
pyrrolidin-5-one-2(S)-carboxylate as white prisms: .sup.1H NMR (300
MHz, Chloroform-D) .delta. 6.35 (br s, 1H), 5.04 (sept. 1H, J=6.2
Hz), 4.18 (dd, 1H, J=8.4, 5.3 Hz), 2.51-2.28 (m, 3H), 2.27-2.12 (m,
1H), 1.24 (d, 6H, J=6.2 Hz). LCMS m/z 172 (M+H).sup.+.
Step 26B: Preparation of Isopropyl
1-(tert-butoxycarbonyl)-pyrrolidin-5-one-2(S)-carboxylate
[0431] ##STR207##
[0432] A solution of isopropyl pyrrolidin-5-one-2(S)-carboxylate
(product of step 26A, 31.9 g, 188 mmol), di-tert-butyl dicarbonate
(48.6 g, 225 mmol) and DMAP (2.30 g, 8.8 mmol) in acetonitrile (300
mL) was stirred at room temperature under N.sub.2 for 30 minutes.
The reaction was evaporated to about 100 mL, diluted with ether,
washed with 1N HCl then saturated aqueous NaCl, dried (MgSO.sub.4)
and evaporated to give isopropyl
1-(tert-butoxycarbonyl)pyrrolidin-5-one-2(S) carboxylate as a light
yellow oil, 50.1 g (99%): .sup.1H NMR (300 MHz, Chloroform-D)
.delta. 5.06 (sept. 1H, J=6.2 Hz), 4.53 (dd, 1H, J=9.5, 2.9 Hz),
2.66-2.40 (m, 2H), 2.36-2.22 (m, 1H), 2.03-1.93 (m, 1H), 1.47 (s,
9H), 1.26 (d, 3H, J=6.2 Hz), 1.24 (d, 3H, J=6.2 Hz). LCMS m/z 272
(M+H).sup.+.
Step 26C: Preparation of Isopropyl
2(S)-(tert-butoxycarbonylamino)-5-hydroxypentanoate
[0433] ##STR208##
[0434] To a solution of isopropyl
1-(tert-butoxycarbonyl)pyrrolidin-5-one-2(S)-carboxylate (product
of step 26B, 49.5 g, 183 mmol) in methanol (300 mL) was added
sodium borohydride (10.0 g, 263 mmol) in .about.1 g portions over
1.5 hours. The reaction was stirred under nitrogen for another 10
minutes. It was diluted with water, extracted with ether, combined
organic fractions washed with saturated aqueous NaCl, dried
(MgSO.sub.4) and evaporated to give a light yellow oil. Flash
chromatography (silica gel, 20-30% ethyl acetate/hexane) gave 31.8
g (64%) of isopropyl
2(S)-(tert-butoxycarbonylamino)-5-hydroxypentanoate as a colorless
syrup:
[0435] .sup.1H NMR (300 MHz, Chloroform-D) .delta. 5.16 (br d, 1H,
J=7.3 Hz), 5.03 (sept., 1H, J=6.2 Hz), 4.28 (br d, 1H, J=6.2 Hz),
3.67 (br dd, J=10.2, 5.5 Hz), 1.94-1.79 (m, 2H), 1.76-1.67 (m, 1H),
1.66-1.56 (m, 2H), 1.43 (s, 9H), 1.25 (d, 3H, J=6.2 Hz), 1.23 (d,
3H, J=6.2 Hz). LCMS m/z 276 (M+H).sup.+.
Step 26D: Preparation of
Isopropyl-5-allyloxy-2(S)-(tert-butoxycarbonylamino)pentanoate
[0436] ##STR209##
[0437] A degassed mixture of isopropyl
2(S)-(tert-butoxycarbonylamino)-5-hydroxypentanoate (product of
step 26C, 17.6 g, 63.9 mmol), allyl methyl carbonate (24.0 ml, 213
mmol), Pd.sub.2(dba).sub.3 (1.62 g, 1.78 mmol) and BINAP (4.42 g,
7.10 mmol) in THF (150 mL) was refluxed under nitrogen for 3 hours.
After cooling to room temperature, the reaction was diluted with
ether, filtered through celite and evaporated giving a dark brown
syrup. Flash chromatography of the residue (silica gel, 30%
ether/hexane) gave isopropyl
5-allyloxy-2(S)-(tert-butoxycarbonylamino)pentanoate as a viscous
colorless oil, 16.3 g (81%): .sup.1H NMR (300 MHz, Chloroform-D)
.delta. 5.88 (ddt, 1H, 17.4, 10.4, 5.5), 5.28 (m, 1H), 5.22-5.11
(m, 1H), 5.02 (sept., 1H, J=6.2 Hz), 4.21 (br t, 1H, J=6.7 Hz),
3.94 (dt, 2H, J=5.9, 1.5 Hz), 3.42 (t, 2H, J=5.9 Hz), 1.90-1.82 (m,
1H), 1.75-1.57 (m, 3H), 1.42 (s, 9H), 1.21 (d, 3H, J=6.2 Hz), 1.19
(d, 3H, J=6.2 Hz). LCMS m/z 316 (M+H).sup.+.
Step 26E: Preparation of
5-Allyloxy-2(S)-(tert-butoxycarbonylamino)pentanoic acid
[0438] ##STR210##
[0439] A mixture of isopropyl
5-allyloxy-2(S)-(tert-butoxycarbonylamino)pentanoate (product of
step 26D, 16.1 g, 51.1 mmol) and lithium hydroxide hydrate (4.19 g,
102 mmol) in THF/water (100 mL/20 mL) was stirred at room
temperature under nitrogen for 16 hours. The reaction was diluted
with water, washed with ether, pH of aqueous fraction adjusted to
4, extracted with ether, combined organic fractions washed with
saturated NaCl, dried (MgSO.sub.4) and evaporated giving
5-allyloxy-2(S)-(tert-butoxycarbonylamino)pentanoic acid as a light
yellow syrup: .sup.1H NMR (300 MHz, Chloroform-D) .delta. 5.89
(ddt, 1H, J=17.4, 10.4, 5.5), 5.25 (dd, 1H, J=17.4, 1.6 Hz), 5.17
(dd, 1H, J=10.4, 1.6 Hz), 4.30 (br d, 1H, J=6.2), 3.96 (dt, 2H,
J=5.9, 1.5 Hz), 3.46 (t, 2H, J=5.9 Hz), 1.96-1.86 (m, 1H),
1.85-1.77 (m, 1H), 1.75-1.64 (m, 2H), 1.43 (s, 9H). LCMS m/z 274
(M+H).sup.+.
Step 26F Preparation of
1-{[1-(5-Allyloxy-2-tert-butoxycarbonylamino-pentanoyl)-4-(6-methoxy-isoq-
uinolin-1-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinylcyclopropanecarboxy-
lic acid ethyl ester
[0440] ##STR211##
[0441] A solution of
1-{[4-(6-Methoxy-isoquinolin-1-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vi-
nyl-cyclopropanecarboxylic acid ethyl ester dihydrochloride
(Example 22) (3.60 g, 7.25 mmol) and
5-allyloxy-2-tert-butoxycarbonylamino-pentanoic acid (the product
of step 26E, 1.98 g, 7.25 mmol) in 75 ml of DMF was treated with
HATU (3.31 g, 8.7 mmol) and N-methylmorpholine (2.79 ml, 25.4
mmol). The reaction mixture was stirred at rt under N.sub.2 for 4
h, and then quenched with pH 4 buffer (biphthalate). The resulting
mixture was extracted with EtOAc. The organic phase was washed with
sat. aq. NaCl, dried (MgSO.sub.4), and concentrated in vacuo to
give the crude product. Flash chromatography (silica gel, 10-60%
ethyl acetate/hexane) gave 3.91 g (-79%) of
1-{[1-(5-Allyloxy-2-tert-butoxycarbonylamino-pentanoyl)-4-(6-methoxy-isoq-
uinolin-1-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarbox-
ylic acid ethyl ester as a yellow collapsed foam: .sup.1H NMR (500
MHz, METHANOL-D4) .delta. 1.05-1.16 (m, 1H), 1.32 (s, 9H),
1.37-1.50 (m, 3H), 1.51-1.67 (m, 3H), 1.68-1.84 (m, 2H), 2.17-2.30
(m, 1H), 2.32-2.50 (m, 1H), 2.58-2.73 (m, 1H), 2.80 (s, 1H),
3.33-3.52 (m, 2H), 3.86-3.95 (m, 5H), 4.00-4.06 (m, 1H), 4.07-4.22
(m, 3H), 4.23-4.38 (m, 1H), 4.58-4.67 (m, 1H), 5.04-5.17 (m, 2H),
5.18-5.34 (m, 2H), 5.65-5.94 (m, 3H), 7.07-7.28 (m, 3H), 7.82-7.92
(m, 1H), 7.96-8.13 (m, 1H). LC-MS (Method B, retention time: 1.75
min), MS m/z 681 (M.sup.++1).
Step 26G: Preparation of
14-tert-Butoxycarbonylamino-18-(6-methoxy-isoquinolin-1-yloxy)-2,15-dioxo-
-10-oxa-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid ethyl ester
[0442] ##STR212##
[0443] A solution of
1-{[1-(5-Allyloxy-2-tert-butoxycarbonylamino-pentanoyl)-4-(6-methoxy-isoq-
uinolin-1-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinylcyclopropanecarboxy-
lic acid ethyl ester (the product of step 26F, 2.0 g, 2.94 mmol) in
800 ml of benzene was treated with Grubb's second generation
catalyst (375 mg, 0.44 mmol). The reaction mixture was heated to
50.degree. C., stirred under N.sub.2 for 2 h, and then concentrated
in vacuo. Flash chromatography (silica gel, 25-100% ethyl
acetate/hexane) and treatment with activated carbon gave 677.6 mg
(.about.35%) of
14-tert-butoxycarbonylamino-18-(6-methoxy-isoquinolin-1-yloxy)-2,15-dioxo-
-10-oxa-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid ethyl ester as a brown collapsed foam: .sup.1H NMR (500 MHz,
METHANOL-D4) .delta. 1.06-1.30 (m, 12H), 1.51-2.03 (m, 6H),
2.41-2.70 (m, 3H), 3.39-3.50 (m, 1H), 3.51-3.61 (m, 1H), 3.77-3.87
(m, 1H), 3.92 (s, 3H), 4.02-4.18 (m, 3H), 4.24 (d, J=6.41 Hz, 1H),
4.36-4.48 (m, 1H), 4.55 (d, J=1.29 Hz, 1H), 4.65 (t, J=8.39 Hz,
1H), 5.55-5.72 (m, 2H), 5.85 (s, 1H), 7.08 (d, J=8.85 Hz, 1H), 7.17
(s, 1H), 7.24 (d, J=5.80 Hz, 1H), 7.89 (d, J=5.80 Hz, 1H), 8.16 (d,
J=9.16 Hz, 1H). LC-MS (Method B, retention time: 1.58 min), MS m/z
653 (M.sup.++1).
Step 26H, Preparation of Compound 27,
14-tert-Butoxycarbonylamino-18-(6-methoxy-isoquinolin-1-yloxy)-2,15-dioxo-
-10-oxa-3,16-diazatricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid
[0444] ##STR213##
[0445] To a solution of
14-tert-butoxycarbonylamino-18-(6-methoxy-isoquinolin-1-yloxy)-2,15-dioxo-
-10-oxa-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid ethyl ester (the product of step 26G, 667 mg, 1.02 mmol) in
25.7 ml of THF was added 2.9 ml of water, 11.4 ml of methanol, and
powdered lithium hydroxide (489 mg, 20.4 mmol). The reaction
mixture was stirred at rt for 6 h, and then concentrated in vacuo.
The residue was partitioned between ethyl acetate and 20.4 ml of 1
N aq. HCl. To the resulting solution was added pH 4 buffer
(biphthalate). The organic phase was washed with water, sat. aq.
NaCl, dried (MgSO.sub.4), and concentrated in vacuo to give the
crude product. Flash chromatography (silica gel, 0-10%
methanol/chloroform) gave 446 mg (.about.70%) of
14-tert-butoxycarbonylamino-18-(6-methoxy-isoquinolin-1-yloxy)-2,15-dioxo-
-10-oxa-3,16-diazatricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid as a slightly pink collapsed foam: .sup.1H NMR (500 MHz,
METHANOL-D4) .delta. 0.99-1.16 (m, 1H), 1.22 (s, 9H), 1.50-2.04 (m,
6H), 2.40-2.73 (m, 3H), 3.41-3.50 (m, 1H), 3.51-3.62 (m, 1H),
3.73-3.86 (m, 1H), 3.90 (s, 3H), 4.03 (d, J=10.52 Hz, 1H), 4.21 (d,
J=8.31 Hz, 1H), 4.42-4.72 (m, 3H), 5.65 (s, 2H), 5.82 (s, 1H), 7.06
(d, J=9.05 Hz, 1H), 7.15 (s, 1H), 7.22 (d, J=5.87 Hz, 1H), 7.87 (m,
J=9.29 Hz, 2H), 8.14 (d, J=8.56 Hz, 1H). LC-MS (Method B, retention
time: 1.46 min), MS m/z 625 (M.sup.++1).
Step 26I, Preparation of Compound 28,
[4-Cyclopropanesulfonylaminocarbonyl-18-(6-methoxy-isoquinolin-1-yloxy)-2-
,15-dioxo-10-oxa-3,16-diazatricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-c-
arbamic acid tert-butyl ester
[0446] ##STR214##
[0447] A solution of
14-tert-butoxycarbonylamino-18-(6-methoxy-isoquinolin-1-yloxy)-2,15-dioxo-
-10-oxa-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid (the product of step 26H, 146.5 mg, 0.235 mmol) and
carbonyldiimidazole (76 mg, 0.47 mmol) in 5 ml of THF was stirred
at reflux under N.sub.2 for 2 h, and then allowed to come to rt. To
the reaction mixture was added cyclopropanesulfonic acid amide (86
mg, 0.71 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (81 .mu.l,
0.54 mmol). The reaction mixture was stirred at rt under N.sub.2
for 4 h, and then quenched with pH 4 buffer (biphthalate). The
resulting mixture was extracted with EtOAc. The organic phase was
washed with sat. aq. NaCl, dried (MgSO.sub.4), and concentrated in
vacuo to give the crude product. Flash chromatography (silica gel,
0-8% methanol/chloroform) gave 111.7 mg (.about.65%) of
[4-Cyclopropanesulfonylaminocarbonyl-18-(6-methoxy-isoquinolin-1-yloxy)-2-
,15-dioxo-10-oxa-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]--
carbamic acid tert-butyl ester (compound 27) as a white powder:
.sup.1H NMR (500 MHz, METHANOL-D4) .delta. 0.96-1.17 (m, 6H), 1.20
(s, 9H), 1.24-1.32 (m, 1H), 1.49-1.64 (m, 2H), 1.65-1.79 (m, 2H),
1.79-2.06 (m, 2H), 2.45-2.60 (m, 1H), 2.62-2.76 (m, 2H), 2.86-2.98
(m, 1H), 3.47-3.61 (m, 2H), 3.62-3.73 (m, 1H), 3.92 (s, 3H), 4.00
(d, J=10.38 Hz, 1H), 4.18 (d, J=10.99 Hz, 1H), 4.55-4.72 (m, 3H),
5.44 (t, J=9.92 Hz, 1H), 5.69-5.78 (m, 1H), 5.86 (s, 1H), 7.07 (d,
J=7.02 Hz, 1H), 7.17 (s, 1H), 7.24 (d, J=5.80 Hz, 1H), 7.85-7.95
(m, 2H), 8.18 (d, J=8.85 Hz, 1H). LC-MS (Method B, retention time:
1.52 min), MS m/z 728 (M.sup.++1).
Preparation of Compound 61
[0448] ##STR215## ##STR216## The preparation of Compound 61 was
carried-out as shown in the above scheme and using procedures
outlined below:
Preparation of Example 28
[0449] Example 28 was prepared by adding a DMF solution of N-trityl
protected threonine to a DMF solution of sodium hydride cooled to
-15 C. The reaction mixture was stirred for 30 minutes at -15 C
after which 5-bromo-1-pentene was added and the resulting mixture
was warmed to -5 C. The reaction mixture was maintained at -5 C for
3 days after which time the reaction was quenched by the addition
of 1N aqueous HCl and worked up using standard extraction
procedures as described above. Example 28 was obtained in pure form
by standard chromatography procedures.
Preparation of Example 29
Example 29 was prepared by coupling Example 28 with Example 22 as
shown in the scheme using the general procedure described above for
the preparation of structurally related Compounds and Examples.
Preparation of Example 30
Example 30 was prepared from Example 29 as shown in the scheme
using procedures described above for the preparation of
structurally related Compounds and Examples.
Preparation of Example 31
[0450] Example 31 was prepared from Example 30 as shown in the
scheme. In the event 100 mg of Example 30 was dissolved in 2 ml of
DCM and the resulting solution was treated with 82 microliters of a
4:1 solution of water and trifluoroacetic acid. The resulting
mixture was stirred at room temperature for several hours after
which the reaction mixture was concentrated and the resulting crude
product mixture was purified by chromatography to provide Example
31.
Preparation of Compound 61
Compound 61 was prepared from Example 31 as shown in the scheme and
using procedures described above for the preparation of related
Compounds and Examples.
Preparation of Compound 62, Compound 66, and Compound 70
[0451] ##STR217## Compound 62 was prepared from Compound 61 as
shown in the scheme, and by using the procedures decribed above for
the preparation of related compounds and Examples.
Preparation of Compound 63, 64, 65, Compound 67, 68, 69, Compound
71, 72, 73
Preparation of Compound 63, Compound 67, Compound 71
[0452] ##STR218##
Preparation of Compound 64, Compound 68, Compound 72
[0453] ##STR219##
Preparation of Compound 65, Compound 69, Compound 73
[0454] ##STR220## Compounds 63, 64, 65, 67, 68, 69, 71, 72 and 73
were prepared as shown in the above synthetic scheme, and by using
procedures described herein for the synthesis of related Examples
and Compounds.
Example 36
Preparation of Compound 79 and Compound 80
[0455] ##STR221##
Step 36A: Preparation of
N-t-Butoxycarbonyl-3-(4-pentenylthio)-L-valine, methyl ester
[0456] ##STR222##
[0457] To a solution of 7.12 g (48 mmol, 1.0 eq) of L-penicillamine
in 100 mL of 1,4-dioxane and 25 mL of water at room temperature was
added 9.60 mL (96 mmol, 2.0 eq) of 10N aqueous sodium hydroxide
solution, followed by the dropwise addition of 12.00 mL (101 mmol,
2.1 eq) of 5-bromo-1-pentene over several minutes. The resulting
mixture was stirred at room temperature for 68 hours. At this point
12.50 g (57 mmol, 1.2 eq) of di-tert-butyl dicarbonate was added,
and the mixture was stirred at room temperature for another 6
hours. The mixture was concentrated under vacuum, and the residue
was dissolved in water. The aqueous mixture was washed with diethyl
ether, adjusted to pH 3 employing 1N hydrochloric acid, and then
extracted with ethyl acetate. The combined extracts were washed
with brine, dried over anhydrous magnesium sulfate, filtered, and
concentrated under vacuum.
[0458] The crude product (12.20 g) was dissolved in 120 mL of
anhydrous dimethylsulfoxide. To this solution was added 10.50 g (76
mmol) of potassium carbonate and 4.70 mL (76 mmol) of iodomethane,
and the resulting mixture was stirred at room temperature for 24
hours. The reaction mixture was diluted with water and extracted
with ethyl acetate. The combined extracts were washed with water
(2.times.) and brine, dried over anhydrous sodium sulfate,
filtered, and concentrated under vacuum. Column chromatography on
silica gel (elution: 2-10% ethyl acetate/hexane) provided 8.54 g of
N-tert-butoxycarbonyl-3-(4-pentenylthio)-L-valine, methyl ester as
a colorless oil. NMR (300 MHz, CDCl.sub.3): .delta. 5.76 (d of d of
t, 1H, J=17.2, 10.3, 6.6 Hz), 5.35 (br d, 1H, J=9.0 Hz), 5.05-4.94
(m, 2H), 4.27 (br d, 1H, J=9.0 Hz), 3.73 (s, 3H), 2.52 (m, 2H),
2.13 (quart., 2H, J=7.3 Hz), 1.61 (quint., 2H, J=7.3 Hz), 1.43 (s,
9H), 1.35 (s, 3H), 1.33 (s, 3H).
Step 36B: Preparation of
N-tert-Butoxycarbonyl-3-(4-pentenylthio)-L-valine
[0459] ##STR223##
[0460] To a solution of 8.52 g (25.7 mmol) of
N-tert-butoxycarbonyl-3-(4-pentenylthio)-L-valine, methyl ester in
200 mL of tetrahydrofuran at room temperature was added a solution
of 1.10 g (26.2 mmol) of lithium hydroxide monohydrate in 50 mL of
water. The resulting mixture was stirred at room temperature for 65
hours. To the reaction mixture then was added 28 mL of 1.00N
hydrochloric acid. The mixture was diluted with diethyl ether,
washed with water (3.times.) and brine, dried over anhydrous sodium
sulfate, filtered, and concentrated under vacuum to afford 8.10 g
of N-tert-butoxycarbonyl-3-(4-pentenylthio)-L-valine as a colorless
oil. NMR (300 MHz, CDCl.sub.3): .delta. 5.75 (d of d of t, 1H,
J=17.2, 10.3, 6.6 Hz), 5.40 (br s, 1H), 5.05-4.94 (m, 2H), 4.28 (br
s, 1H), 2.56 (m, 2H), 2.13 (quart., 2H, J=7.3 Hz), 1.63 (quint.,
2H, J=7.3 Hz), 1.44 (s, 9H), 1.39 (s, 3H), 1.37 (s, 3H).
Step 36C: Preparation of
(1S,4R,6S,14R,18R)-7-cis-14-tert-Butoxycarbonylamino-13,13-dimethyl-18-(6-
-methoxy-isoquinolin-1-yloxy)-2,15-dioxo-12-thia-3,16-diazatricyclo[14.3.0-
.0.sup.4,6]nonadec-7-ene-4-carboxylic acid (Compound 79)
[0461] ##STR224##
[0462] This compound was prepared from
N-tert-butoxycarbonyl-3-(4-pentenylthio)-L-valine employing the
procedures described in example 24. ESI-mass spectrum: m/e: 669
(M+H).sup.+, 667 (M-H).sup.-; high resolution mass spectrum: calc'd
for C.sub.34H.sub.45N.sub.4O.sub.8S: 669.2958, found: 669.2975; NMR
(500 MHz, CD.sub.3OD) .delta. 8.06 (d, 1H, J=8.8 Hz), 7.90 (d, 1H,
J=5.8 Hz), 7.24 (d, 1H, J=5.8 Hz), 7.16 (s, 1H), 7.09 (d, 1H, J=8.8
Hz), 6.69 (d, 1H, J=8.5 Hz), 5.80 (s, 1H), 5.72 (quart., 1H, J=8.5
Hz), 5.46 (t, 1H, J=8.5 Hz), 4.71 (t, 1H, J=8.5 Hz), 4.64 (d, 1H,
J=9.1 Hz), 4.45 (d, 1H, J=11.6 Hz), 4.22 (m, 1H), 3.91 (s, 3H),
2.78 (m, 1H), 2.74-2.68 (m, 2H), 2.56 (m, 1H), 2.36-2.28 (m, 2H),
2.21 (m, 1H), 1.75 (m, 1H), 1.72-1.64 (m, 2H), 1.60 (m, 1H), 1.45
(s, 3H), 1.39 (s, 3H), 1.21 (s, 9H).
Step 36D: Preparation of
(1S,4R,6S,14R,18R)-[7-cis-4-Cyclopropanesulfonylaminocarbonyl-13,13-dimet-
hyl-18-(6-methoxyisoquinolin-1-yloxy)-2,15-dioxo-12-thia-3,16-diaza-tricyc-
lo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]carbamic acid, tert-butyl
ester (Compound 80)
[0463] ##STR225##
[0464] This compound was prepared from compound 79 employing the
procedure described in example 24D. ESI-mass spectrum: m/e: 772
(M+H).sup.+, 770 (M-H).sup.-; high resolution mass spectrum: calc'd
for C.sub.37H.sub.50N.sub.5O.sub.9S.sub.2: 772.3050, found:
772.3060; NMR (500 MHz, CD.sub.3OD) .delta. 8.06 (d, 1H, J=8.8 Hz),
7.90 (d, 1H, J=5.8 Hz), 7.24 (d, 1H, J=5.8 Hz), 7.17 (s 1H), 7.08
(d, 1H, J=8.8 Hz), 6.62 (d, 1H, J=8.5 Hz), 5.83 (s, 1H), 5.74
(quart., 1H, J=8.5 Hz), 5.26 (t, 1H, J=8.5 Hz), 4.67 (m, 1H), 4.46
(m, 2H), 4.19 (m, 1H), 3.92 (s, 3H), 2.93 (m, 1H), 2.74-2.67 (m,
3H), 2.58-2.48 (m, 3H), 2.11 (m, 1H), 1.82-1.75 (m, 2H), 1.61 (m,
1H), 1.54 (m, 1H), 1.45 (s, 3H), 1.37 (s, 3H), 1.27 (m, 1H), 1.18
(s, 9H), 1.09 (m, 1H), 1.07-0.99 (m, 2H).
Example 37
Preparation of Compound 84 and Compound 85
[0465] ##STR226##
Step 37A: Preparation of
N-tert-Butoxycarbonyl-O-(4-pentenyl)-L-serine methyl ester
[0466] ##STR227##
[0467] To a solution of 10.26 g (50 mmol, 1.0 eq) of
N-tert-butoxycarbonyl-L-serine in 500 mL of anhydrous
dimethylsulfoxide at room temperature was added 2.00 g (50 mmol,
1.0 eq) of 60% sodium hydride in mineral oil. This mixture was
stirred at room temperature for 0.5 hour until the evolution of gas
had ceased. To the resulting solution was added 6.00 mL (50 mmol,
1.0 eq) of 5-bromo-1-pentene followed immediately by another 2.00 g
(50 mmol, 1.0 eq) of 60% sodium hydride in mineral oil. The
reaction mixture then was stirred at room temperature for 16 hours.
The mixture was diluted with 2000 mL of water, adjusted to pH 3-4
by the addition of 50 mL of 1.00N hydrochloric acid, and extracted
with ethyl acetate. The organic phase was washed with water
(2.times.) and brine, dried over anhydrous sodium sulfate,
filtered, and concentrated under vacuum. To remove the residual
mineral oil the resulting material was dissolved in a dilute
aqueous sodium hydroxide solution. This aqueous solution was washed
with hexane and then adjusted to pH 4 employing hydrochloric acid,
and extracted with ethyl acetate. The extract was washed with water
(2.times.) and brine, dried over anhydrous sodium sulfate,
filtered, and concentrated under vacuum.
[0468] The crude product (7.70 g) was dissolved in 100 mL of
anhydrous dimethylsulfoxide. To this solution was added 7.80 g (56
mmol) of potassium carbonate and 3.50 mL (56 mmol) of iodomethane,
and the resulting mixture was stirred at room temperature for 24
hours. The reaction mixture was diluted with water and extracted
with ethyl acetate. The combined extracts were washed with water
(2.times.) and brine, dried over anhydrous sodium sulfate,
filtered, and concentrated under vacuum. Column chromatography on
silica gel (elution: 2-10% ethyl acetate/hexane) provided 6.70 g of
N-tert-butoxycarbonyl-O-(4-pentenyl)-L-serine, methyl ester as a
colorless oil. NMR (300 MHz, CDCl.sub.3): .delta. 5.78 (d of d of
t, 1H, J=17.2, 10.2, 6.6 Hz), 5.34 (br d, 1H, J=8.0 Hz), 5.03-4.92
(m, 2H), 4.40 (m, 1H), 3.81 (d of d, 1H, J=9.5, 2.9 Hz), 3.74 (s,
3H), 3.61 (d of d, 1H, J=9.5, 3.5 Hz), 3.42 (m, 2H), 2.06 (quart.,
2H, J=7.3 Hz), 1.61 (quint., 2H, J=7.3 Hz), 1.44 (s, 9H).
Step 37B: Preparation of
N-tert-Butoxycarbonyl-O-(4-pentenyl)-L-serine
[0469] ##STR228##
[0470] To a solution of 6.65 g (23 mmol) of
N-tert-butoxycarbonyl-O-(4-pentenyl)-L-serine, methyl ester in 500
mL of tetrahydrofuran at room temperature was added a solution of
1.95 g (46 mmol) of lithium hydroxide monohydrate in 100 mL of
water. The resulting mixture was stirred at room temperature for 40
hours. To the reaction mixture then was added 46 mL of 1.00N
hydrochloric acid. The mixture was diluted with ethyl acetate,
washed with water (3.times.) and brine, dried over anhydrous sodium
sulfate, filtered, and concentrated under vacuum to afford 6.30 g
of N-tert-butoxycarbonyl-O-(4-pentenyl)-L-serine as a colorless
oil. NMR (300 MHz, CDCl.sub.3): .delta. 5.77 (d of d of t, 1H,
J=17.2, 10.2, 6.6 Hz), 5.37 (br d, 1H, J=8.0 Hz), 5.03-4.92 (m,
2H), 4.42 (m, 1H), 3.87 (d of d, 1H, J=9.5, 2.6 Hz), 3.63 (d of d,
1H, J=9.5, 4.0 Hz), 3.45 (t, 2H, J=6.6 Hz), 2.07 (quart., 2H, J=7.3
Hz), 1.64 (quint., 2H, J=7.3 Hz), 1.44 (s, 9H).
Step 37C: Preparation of
(1S,4R,6S,14S,18R)-7-cis-14-tert-Butoxycarbonylamino-18-(6-methoxyisoquin-
olin-1-yloxy)-2,15-dioxo-12-oxa-3,16-diazatricyclo[14.3.0.0.sup.4,6]-nonad-
ec-7-ene-4-carboxylic acid (Compound 84)
[0471] ##STR229##
[0472] This compound was prepared from
N-tert-butoxycarbonyl-3-(4-pentenylthio)-L-valine employing the
procedures described in example 26. ESI-mass spectrum: m/e: 625
(M+H).sup.+, 623 (M-H).sup.-; high resolution mass spectrum: calc'd
for C.sub.32H.sub.41N.sub.4O.sub.9: 625.2874, found: 625.2871; NMR
(500 MHz, CD.sub.3OD) .delta. 8.12 (d, 1H, J=9.0 Hz), 7.89 (d, 1H,
J=5.8 Hz), 7.24 (d, 1H, J=5.8 Hz), 7.17 (s, 1H), 7.12 (d, 1H, J=9.0
Hz), 5.83 (s, 1H), 5.63 (quart., 1H, J=8.8 Hz), 5.45 (t, 1H, J=8.8
Hz), 4.74 (m, 1H), 4.60 (brs, 1H), 4.36 (d, 1H, J=11.0 Hz), 4.16
(d, 1H, J=8.0 Hz), 3.91 (s, 3H), 3.74 (m, 2H), 3.62 (m, 1H), 3.49
(m, 1H), 2.66 (m, 1H), 2.57 (m, 1H), 2.47-2.33 (m, 2H), 2.19 (m,
1H), 1.78 (m, 2H), 1.69 (m, 1H), 1.48 (m, 1H), 1.30 (s, 9H).
Step 37D: Preparation of
(1S,4R,6S,14S,18R)-[7-cis-4-Cyclopropanesulfonylaminocarbonyl-18-(6-metho-
xy-isoquinolin-1-yloxy)-2,15-dioxo-12-oxa-3,16-diazatricyclo[14.3.0.0.sup.-
4,6]nonadec-7-en-14-yl]-carbamic acid, tert-butyl ester (Compound
85)
[0473] ##STR230##
[0474] This compound was prepared from compound 84 employing the
procedure described in example 261. ESI-mass spectrum: m/e: 728
(M+H).sup.+, 726 (M-H).sup.-; high resolution mass spectrum: calc'd
for C.sub.35H.sub.46N.sub.5O.sub.10S: 728.2966, found: 728.2972;
NMR (500 MHz, CD.sub.3OD) .delta. 8.12 (m, 1H), 7.89 (d, 1H, J=5.8
Hz), 7.25 (d, 1H, J=5.8 Hz), 7.18 (s, 1H), 7.12 (d, 1H, J=8.0 Hz),
5.89 (s, 1H), 5.66 (quart., 1H, J=8.8 Hz), 5.19 (t, 1H, J=8.8 Hz),
4.71 (m, 1H), 4.52 (br s, 1H), 4.36 (d, 1H, J=10.0 Hz), 4.12 (m,
1H), 3.92 (s, 3H), 3.65 (m, 2H), 3.47 (m, 2H), 2.92 (m, 1H), 2.64
(m, 1H), 2.56 (m, 1H), 2.45 (m, 2H), 2.19 (m, 1H), 1.77 (m, 1H),
1.70-1.60 (m, 2H), 1.54 (m, 1H), 1.28 (s, 9H), 1.15-0.98 (m,
4H).
Example 38
Preparation of Compound 58
[0475] ##STR231## ##STR232##
Step, 38a: Preparation of
1-{[1-(2-tert-Butoxycarbonylamino-non-8-enoyl)-4-(tert-butyl-dimethyl-sil-
anyloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinylcyclopropanecarboxylic
acid ethyl ester
[0476] ##STR233## To a mixture of
1-{[1-(2(S)-tert-Butoxycarbonylamino-non-8-enoyl)-4(R)-hydroxy-pyrrolidin-
e-2(S)carbonyl]-(1R)-amino}-2(S)-vinyl-cyclopropanecarboxylic acid
ethyl ester (1.5 g, 2.87 mmoL) in 10 mL of DMF was added imidazole
(0.25 g, 3.67 mmoL) and tert-butyl-dimethylsilyl chloride (516 mg,
3.44 mmoL). The mixture was stirred at rt for two days. The
reaction mixture was then concentrated in vacuo, and the residue
was dissolved in ethyl acetate. This solution was washed with
water, dried over magnesium sulfate, and concentrated in vacuo to
obtain a crude solid. Purification by flash chromatography (eluting
with 20% ethyl acetate in hexane) gave 1.43 g (78%) of
1-{[1-(2-tert-butoxycarbonylamino-non-8-enoyl)-4-(tert-butyldime-
thyl-silanyloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinylcyclopropanecarboxy-
lic acid ethyl ester as a white solid.
[0477] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.10 (s, 6H), 0.89
(s, 9H), 1.22 (m, 3H), 1.31-1.48 (m, 16H), 1.50-1.75 (m, 3H), 2.06
(m, 3H), 2.11-2.33 (m, 2H), 3.70 (m, 2H), 4.03-4.19 (m, 2H), 4.21
(m, 1H), 4.45 (t, J=7.87 Hz, 1H), 4.59 (m, 1H), 4.91 (d, J=9.15 Hz,
1H), 4.98 (d, 117.20 Hz, 1H), 5.08 (dd, 110.25, 1.83 Hz, 1H), 5.27
(dd, J=17.38, 1.65 Hz, 1H), 5.65-5.87 (m, 2H). LC-MS (Method A,
retention time: 4.00 min), MS m/z 636 (M.sup.++1).
Step 38b: Preparation of
14-tert-Butoxycarbonylamino-18-(tert-butyl-dimethylsilanyloxy)-2,15-dioxo-
-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid, ethyl ester
[0478] ##STR234## To a solution of
1-{[1-(2-tert-butoxycarbonylamino-non-8-enoyl)-4-(tert-butyldimethyl-sila-
nyloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinylcyclopropanecarboxylic
acid ethyl ester (1.63 g, 2.56 mmoL) in 640 mL of methylene
chloride was added 215 mg (0.26 mmoL) of
tricyclohexylphosphine[1,3-bis(2,4,6-tri[benzylidene]ruthenium(IV)dichlor-
ide. The mixture was heated at reflux for 15 min. The residue was
concentrated in vacuo, and then purified by flash chromatography
eluting with 30% ethyl acetate/hexane. To further decolorize the
sample, the crude product was chromatographed a second time eluting
with 50% ether in hexane to give 1.5 g (96%) of
14-tert-butoxycarbonylamino-18-(tert-butyl-dimethyl-silanyloxy)-2,15-diox-
o-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid ethyl ester as a white solid. .sup.1H NMR (500 MHz,
CD.sub.3Cl) .delta. 0.06 (s, 3H), 0.07 (s, 3H), 0.86 (s, 9H),
1.18-1.24 (m, 6H), 1.34-1.64 (m, 14H), 1.86-1.96 (m, 3H), 2.02-2.09
(m, 1H), 2.11-2.17 (m, 1H), 2.19-2.28 (m, 1H), 2.57-2.63 (m, 1H),
3.50-3.54 (m, 1H), 3.71 (dd, J=10.22, 6.26 Hz, 1H), 4.06-4.17 (m,
2H), 4.52-4.58 (m, 2H), 4.75 (d, J=8.55 Hz, 1H), 5.21 (t, J=9.92
Hz, 1H), 5.35 (d, J=7.63 Hz, 1H), 5.45-5.50 (m, 1H), 6.94 (s, 1H).
LC-MS (Method A, retention time: 3.88 min), MS m/z 608
(M.sup.++1).
Step 38c: Preparation of
14-tert-butoxycarbonylamino-18-(tert-butyl-dimethylsilanyloxy)-2,15-dioxo-
-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid
[0479] ##STR235## To a solution of
14-tert-butoxycarbonylamino-18-(tert-butyl-dimethyl-silanyloxy)-2,15-diox-
o-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid ethyl ester (1.5 g, 2.47 mmoL) in a mixed solvent system of
THF (4 mL), methanol (1 mL), and water (2 mL), was added powdered
lithium hydroxide monohydrate (1.0 g, 50 mmoL). The light yellow
slurry was stirred at rt under N.sub.2 for 4 h. The mixture was
then concentrated in vacuo, and the residue partioned between ether
and water. The ether phase was discarded, and the aqueous phase was
treated with 1 N HCl until reaching pH 4. This acidic solution was
extracted with EtOAc (3.times.). The combined EtOAc extracts were
dried (MgSO.sub.4), and concentrated in vacuo to give 1.2 g (84%)
of
14-tert-butoxycarbonylamino-18-(tert-butyl-dimethyl-silanyloxy)-2,15-diox-
o-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid as an off-white solid.
[0480] .sup.1H NMR (300 MHz, CD.sub.3OD) 0.12 (s, 6H), 0.89 (s,
9H), 1.23-1.64 (m, 17H), 1.70-1.87 (m, 1H), 1.90-2.49 (m, 6H),
3.70-3.80 (m, 1H), 3.83-3.90 (m, 1H), 4.28-4.36 (m, 1H), 4.47-4.55
(m, 1H), 4.65 (s, 1H), 5.30-5.39 (m, 1H), 5.53-5.62 (m, 1H). LC-MS
(Method A, retention time: 3.69 min), MS m/z 580 (M.sup.++1).
Step 38d: Preparation of
[18-(tert-butyl-dimethyl-silanyloxy)-4-cyclopropanesulfonylaminocarbonyl--
2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester
[0481] ##STR236##
14-tert-Butoxycarbonylamino-18-(tert-butyl-dimethyl-silanyloxy)-2,15-diox-
o-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid (500 mg, 0.86 mmoL) was dissolved in 25 mL of THF and treated
with CDI (180 mg, 1.12 mmoL). (Care was taken to avoid moisture by
using oven dried glassware and maintaining a dry N2 atmosphere).
After refluxing the reaction mixture for 2 h, it was cooled to rt
and treated sequentially with cyclopropylsulfonamide (135 mg, 1.12
mmoL) and DBU (170 mg, 1.12 mmoL). The reaction mixture was stirred
for 4 h at rt, and the THF was removed by rotary evaporation. The
residue was partitioned between ethyl acetate and pH 4 buffer. The
organic phase was dried (MgSO4) and concentrated in vacuo to give
the crude product. It was then purified by flash chromatography
(eluting with 33% ethyl acetate in hexane) to give 300 mg (51%) of
[18-(tert-butyl-dimethyl-silanyloxy)-4-cyclopropanesulfonylaminocarbonyl--
2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester as a white solid. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 1H 0.07 (s, 3H), 0.08 (s, 3H), 0.85 (s, 9H),
0.87-1.49 (m, 21H), 1.73-1.95 (m, 3H), 2.08-2.16 (m, 1H), 2.25-2.36
(m, 2H), 2.42-2.56 (m, 1H), 2.85-2.93 (m, 1H), 3.65-3.74(dd,
J=10.61, 3.66 Hz, 1H), 3.89 (d, J=10.25 Hz, 1H), 4.34 (m, J=9.70,
9.70 Hz, 1H), 4.43 (t, J=7.87 Hz, 1H), 4.57 (s, 1H), 4.94-5.01 (m,
1H), 5.10 (d, J=8.78 Hz, 1H), 5.66-5.75 (m, 1H), 6.55 (s, 1 H),
10.13 (s, 1H). LC-MS (Method A, retention time: 3.81 min), MS m/z
683 (M.sup.++1).
Step 38e:
(4-Cyclopropanesulfonylaminocarbonyl-18-hydroxy-2,15-dioxo-3,16--
diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl)-carbamic acid
tert-butyl ester
[0482] ##STR237## To a mixture of
[18-(tert-butyl-dimethylsilanyloxy)-4-cyclopropanesulfonylaminocarbonyl-2-
,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbami-
c acid tert-butyl ester (330 mg, 0.48 mmoL) in 25 mL of THF was
added tetrabutylammonium floride (150 mg, 0.54 mmoL). The reaction
mixture was stirred at rt for 18 h, and then the THF was removed by
rotary evaporation. The residue was partitioned between ethyl
acetate and water. The organic phase was dried (MgSO.sub.4) and
concentrated in vacuo to give the crude product. It was then
purified by triturating with hexane to yield 200 mg (73%) of
(4-cyclopropanesulfonylaminocarbonyl-18-hydroxy-2,15-dioxo-3,16-diaza-tri-
cyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl)-carbamic acid tert-butyl
ester as a white solid. .sup.1H NMR (500 MHz, CD.sub.3Cl) .delta.
1.87-1.64 (m, 21H), 1.70-1.98 (m, 3H), 2.15-2.56 (m, 5H), 2.85-2.94
(m, 1H), 3.71 (d, J=13.91 Hz, 1H), 4.10-4.26 (m, 2H), 4.51 (t,
J=7.87 Hz, 1H), 4.62 (s, 1H), 4.98 (m, 1H), 5.06 (d, J=8.78 Hz,
1H), 5.64-5.71 (m, 1H), 6.72 (s, 1H), 10.24 (s, 1H). LC-MS (Method
A, retention time: 2.85 min), MS m/z 569 (M.sup.++1).
Step 38f: Preparation of compound 58:
[(Z)-(1S,4R,14S,18R)-4-cyclopropanesulfonylaminocarbonyl-2,15-dioxo-18-(3-
-phenyl-isoquinolin-1-yloxy)-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec--
7-en-14-yl]-carbamic acid tert-butyl ester
[0483] ##STR238## To a mixture of
(4-cyclopropanesulfonylaminocarbonyl-18-hydroxy-2,15-dioxo-3,16-diaza-tri-
cyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl)-carbamic acid tert-butyl
ester (20 mg, 0.035 mmoL) in 1 mL of DMF was added potassium
t-butoxide (22 mg, 0.196 mmoL). The mixture was stirred at rt for 5
min, and then 1-chloro-3-phenylisoquinoline (15 mg, 0.062 mmoL) was
added. The reaction mixture was stirred at rt for 15 h, and then
concentrated in vacuo. This crude product was triturated with
ether. The residue was dissolved in MeOH, and then purified by
preparative HPLC.(YMC XTERRA, S5, 19.times.100 mm, gradient: 60% B
to 100% B, 15 min, hold 2 min, flow rate 25 mL/min) to give 10 mg
(39%) of compound 58,
[(Z)-(1S,4R,14S,18R)-4-cyclopropanesulfonylaminocarbonyl-2,15-dioxo-18-(3-
-phenyl-isoquinolin-1-yloxy)-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec--
7-en-14-yl]-carbamic acid tert-butyl ester, as a white solid.
.sup.1H NMR (500 MHz, CD.sub.3Cl) .delta. 0.90-1.64 (m, 21H),
1.76-1.97 (m, 3H), 2.26-2.31 (m, 1H), 2.52-2.63 (br s, 1H),
2.69-2.81 (m, 2H), 2.88-2.93 (m, 1H), 4.11 (d, J=11.60 Hz, 1H),
4.33 (m, 1H), 4.61 (d, J=7.94 Hz, 2H), 4.99 (t, J=9.31 Hz, 1H),
5.05 (d, J=7.93 Hz, 1H), 5.69-5.74 (m, 1H), 6.08 (s, 1H), 6.60 (s,
1H), 7.38-7.47(m, 2H), 7.50 (t, J=7.63 Hz, 2H), 7.63 (t, J=7.32 Hz,
1H), 7.71 (s, 1H), 7.77 (d, J=8.24 Hz, 1H), 8.10 (d, J=7.32 Hz,
2H), 8.18 (d, J=7.93 Hz, 1H), 10.27 (s, 1H). LC-MS (Method A,
retention time: 3.72 min), MS m/z 772 (M.sup.++1).
Example 39
Preparation of compounds 144 and 145
[0484] ##STR239##
Step 39a: Preparation of
(S)-2-(tert-butoxycarbonyl)-3-(2-nitro-N-(pent-4-enyl)phenylsulfonamido)p-
ropanoic acid v
[0485] ##STR240##
Preparation of (S)-methyl 3-amino-2-(tert-butoxycarbonyl)propanoate
ii
[0486] To a mixture of i (Boc-DAP-OH) (3.0 g 14.7 mmol) in 50 mL of
methylene chloride was added 5 mL of methanol. To this solution was
slowly added (trimethylsilyl)diazomethane (2 M in ether, 7.9 mL,
15.8 mmoL). The mixture was stirred at rt for 2 h until all of the
solid dissolved and the solution turned light yellow. It was then
concentrated to yield 3.2 g (99%) of (S)-methyl
3-amino-2-(tert-butoxycarbonyl)propanoate ii as a colorless
oil.
[0487] .sup.1H NMR (CD.sub.3OD, 300 MHz) .delta. 1.46 (s, 9H),
2.82-3.00 (m, 2H), 3.71 (s, 3H), 4.14 (brs, 1H).
Preparation of (S)-methyl
2-(tert-butoxycarbonyl)-3-(2-nitrophenylsulfonamido)propanoate
iii
[0488] To a mixture of (S)-methyl
3-amino-2-(tert-butoxycarbonyl)propanoate ii (1.6 g, 7.3 mmoL) in
DCM (50 mL) was added DIPEA (1.64 mL, 9.4 mmoL) and 2-nitrobenzene
sulfonyl chloride (1.62 g, 7.3 mmoL). The mixture was stirred at rt
for 2 h. It was then concentrated, dissolved in ethyl acetate,
which was then washed with sat. sodium bicarbonate, brine and dried
over magnesium sulfate. It was then filtered, concentrated to yield
2.9 g (98%) of (S)-methyl
2-(tert-butoxycarbonyl)-3-(2-nitrophenylsulfonamido)propanoate iii
as a yellow foam. .sup.1H NMR (CD.sub.3OD, 300 MHz) .delta. 1.41
(s, 9H), 3.36-3.51 (m, 2H), 3.71 (s, 3H), 4.22 (m, 1H), 7.80-7.90
(m, 3H), 8.07-8.10 (m, 1H).
Preparation of (S)-methyl
2-(tert-butoxycarbonyl)-3-(2-nitro-N-(pent-4-enyl)phenylsulfonamido)propa-
noate iv
[0489] To a mixture of (S)-methyl
2-(tert-butoxycarbonyl)-3-(2-nitrophenylsulfonamido)-propanoate iii
(150 mg, 0.37 mmol) in 3 mL of DMF was added potassium carbonate
(102 mg, 0.74 mmoL). This mixture was stirred at rt for 20 min
followed by the addition of 5-bromo-1-pentene (65 .mu.L, 0.55
mmoL). The reaction mixture was stirred at rt for 2 days. It was
then filtered, concentrated and purified by silica gel
chromatography (eluting with 25% ethyl acetate in hexane) to give
75 mg (43%) of (S)-methyl
2-(tert-butoxycarbonyl)-3-(2-nitro-N-(pent-4-enyl)phenylsulfonamido)propa-
noate iv as a yellow solid. .sup.1H NMR (CD.sub.3OD, 300 MHz)
.delta. 1.42 (s, 9H), 1.54-1.64 (m, 2H), 1.97 (q, J=7.20 Hz, 2H),
3.37 (m, 2H), 3.57-3.80 (m, 2H), 3.72 (s, 3H), 4.42 (dd, J=8.60,
5.31 Hz, 1H), 4.91-5.01 (m, 2H), 5.69-5.79 (m, 1H), 7.75-7.85 (m,
3H), 8.04 (m, 1H). LC-MS (Method D, retention time: 2.68 min), MS
m/z 372 (M.sup.++1-Boc).
Preparation of
(S)-2-(tert-butoxycarbonyl)-3-(2-nitro-N-(pent-4-enyl)phenylsulfonamido)p-
ropanoic acid v
[0490] (S)-methyl
2-(tert-butoxycarbonyl)-3-(2-nitro-N-(pent-4-enyl)phenylsulfonamido)-prop-
anoate iv (500 mg, 1.06 mmol) was dissolved in the mixed solvent
system: THF (4 mL), methanol (1 mL), and water (2 mL). Powdered
lithium hydroxide (250 mg, 10.4 mmol) was added. The light yellow
slurry was stirred at rt for 15 h, and then concentrated in vacuo.
The residue was partitioned between ether and water. The ether
phase was discarded, and the aqueous phase was treated with 1 N HCl
until the pH was 4. This acidic solution was extracted with ethyl
acetate four times. The combined ethyl acetate extracts were dried
(MgSO.sub.4) and concentrated in vacuo to give 430 mg (89%) of
(S)-2-(tert-butoxycarbonyl)-3-(2-nitro-N-(pent-4-enyl)phenylsulf-
onamido)propanoic acid v as a yellow oil. .sup.1H NMR (CD.sub.3OD,
300 MHz) .delta. 1.38 (s, 9H), 1.51-1.60 (m, 2H), 1.89-1.98 (m,
2H), 3.28-3.32 (m, 2H), 3.59-3.64 (dd, J=14.95, 9.46 Hz, 1H),
3.71-3.74 (m, 1H), 4.33 (dd, J=9.61, 4.43 Hz, 1H), 4.87-4.94 (m,
2H), 5.63-5.72 (m, 1H), 7.71-7.77 (m, 3H), 8.01 (dd, J=7.48, 1.37
Hz, 1H). LC-MS (Method D, retention time: 2.04 min), MS m/z 358
(M.sup.++1-Boc).
Step 39b: Preparation of (1R,2S)-ethyl
1-((3R,5S)-1-((S)-2-(tert-butoxycarbonyl)-3-(2-nitro-N-(pent-4-enyl)pheny-
lsulfonamido)propanoyl)-3-(isoquinolin-1-yloxy)pyrrolidine-5-carboxamido)--
2-vinylcyclopropanecarboxylate
[0491] ##STR241##
(S)-2-(tert-butoxycarbonyl)-3-(2-nitro-N-(pent-4-enyl)phenylsulfonamido)p-
ropanoic acid (1.10 g, 2.40 mmol) dissolved in 20 mL of DCM was
treated sequentially with
1-{[4-(isoquinolin-1-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclop-
ropanecarboxylic acid ethyl ester, bis hydrochloride (1.27 g, 2.72
mmol), N-methyl morpholine (0.92 mL, 8.34 mmol), and HATU (PE
biosystems)(1.28 g, 3.36 mmol). The reaction mixture was stirred at
rt under N.sub.2 for 15 h, and then concentrated in vacuo. The
residue was partitioned between ethyl acetate and pH 4 buffer
(biphthalate). The organic phase was washed with sat. aq.
NaHCO.sub.3, dried (MgSO.sub.4), and concentrated in vacuo to give
2.0 g of the crude product. Flash chromatography (30% hexane/ethyl
acetate) gave 1.4 g (70%) of (1R,2S)-ethyl
1-((3R,5S)-1-((S)-2-(tert-butoxycarbonyl)-3-(2-nitro-N-(pent-4-enyl)pheny-
lsulfonamido)propanoyl)-3-(isoquinolin-1-yloxy)pyrrolidine-5-carboxamido)--
2-vinylcyclopropanecarboxylate as a purple solid. .sup.1H NMR
(CD.sub.3OD, 500 MHz) .delta. 1.23-1.31 (m, 9H), 1.46-1.52 (m, 4H),
1.59 (br s, 1H), 1.66 (br s, 1H), 1.74 (dd, J=7.93, 5.19 Hz, 1H),
2.00 (br s, 2H), 2.29 (q, J=8.95 Hz, 1H), 2.47 (m, 1H), 2.72 (m,
1H), 3.38-3.51 (m, 2H), 3.68 (m, 2H), 4.09-4.26 (m, 3H), 4.37 (d,
J=11.29 Hz, 1H), 4.64 (m, 1H), 4.72 (m, 1H), 4.95 (d, J=10.99 Hz,
1H), 4.99 (dd, J=18.92, 1.53 Hz, 1H), 5.12 (d, J=10.68 Hz, 1H),
5.32 (d, J=17.40 Hz, 1H), 5.73-5.86 (m, 2H), 5.92 (s, 1H), 7.36 (m,
1H), 7.58 (m, 1H), 7.73 (m, 1H), 7.80-7.88 (m, 4H), 7.99 (m, 1H),
8.11 (m, 1H), 8.24 (d, J=8.24 Hz, 1H). LC-MS (Method D, retention
time: 3.15 min), MS m/z 835 (M.sup.++1). Step 39c: ##STR242## A
solution of (1R,2S)-ethyl
1-((3R,5S)-1-((S)-2-(tert-butoxycarbonyl)-3-(2-nitro-N-(pent-4-enyl)pheny-
lsulfonamido)propanoyl)-3-(isoquinolin-1-yloxy)pyrrolidine-5-carboxamido)--
2-vinylcyclopropanecarboxylate (1.40 g, 1.67 mmol) and
tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-
-2-ylidene][benzylidene]ruthenium (IV) dichloride (Aldrich), (204
mg, 0.24 mmol) in 1500 mL of methylene chloride was refluxed under
N.sub.2. Shortly after reaching reflux temperature the reaction
mixture was homogeneous and light orange in color. After refluxing
for 2 h, the dark orange reaction mixture was cooled to rt and
concentrated in vacuo to give 1.7 g of an orange oil. Flash
chromatography (30% hexane/ethyl acetate) gave 1.1 g (82%) of the
ester shown above as a purple solid. .sup.1H NMR (CD.sub.3OD, 300
MHz) .delta. 1.11-1.31 (m, 12H), 1.51-1.80 (m, 4H), 1.97-2.15 (m,
2H), 2.34-2.50 (m, 2H), 2.62-2.69 (m, 1H), 3.17-3.33 (m, 2H),
3.44-3.60 (m, 2H), 4.01-4.05 (m, 1H), 4.12 (q, J=7.32 Hz, 2H), 4.37
(d, J=13.17 Hz, 1H), 4.58 (t, J=8.42 Hz, 1H), 4.71 (m, 1H), 5.61
(m, 2H), 5.88 (s, 1H), 6.91 (d, J=9.15 Hz, 1H), 7.30 (d, J=5.49 Hz,
1H), 7.52 (t, J=7.50 Hz, 1H), 7.66-7.73 (m, 1H), 7.76-7.80 (m, 4H),
7.95 (d, J=5.86 Hz, 1H), 8.11 (d, J=7.32 Hz, 1H), 8.17 (d, J=8.05
Hz, 1H), 8.84 (s, 1H). LC-MS (Method D, retention time: 2.91 min),
MS m/z 807 (M.sup.++1).
Step 39d: Preparation of Compound 144
[0492] ##STR243## The ethyl ester prepared in step 39c (1.1 g, 1.3
mmol) was dissolved in the mixed solvent system: THF (16 mL),
methanol (4 mL), and water (8 mL). Powdered lithium hydroxide
hydrate (540 mg, 13.2 mmol) was added. The light yellow slurry was
stirred at rt for 15 h, and then concentrated in vacuo. The residue
was partitioned between ether and water. The ether phase was
discarded, and the aqueous phase was treated with 1 N HCl until the
pH was 4. This acidic solution was extracted with ethyl acetate
four times. The combined ethyl acetate extracts were dried
(MgSO.sub.4) and concentrated in vacuo to give 878 mg (85%) of
compound 144 as a purple solid. .sup.1H NMR (CD.sub.3OD, 300 MHz)
.delta. 1.11, 1.31 (s, 9H), 1.56-1.68 (m, 4H), 2.04-2.16 (m, 2H),
2.42-2.66 (m, 2H), 2.68-2.75 (m, 1H), 3.22-3.30 (m, 2H), 3.48-3.63
(m, 2H), 4.02-4.14 (m, 1H), 4.46 (d, J=12.44 Hz, 1H), 4.62 (t,
J=8.42 Hz, 1H), 4.70-4.73 (m, 1H), 5.64 (m, 2H), 5.90 (s, 1H), 7.36
(d, J=5.86 Hz, 1H), 7.57 (t, J=7.68 Hz, 1H), 7.71-7.87 (m, 5H),
7.98 (d, J=5.86 Hz, 1H), 8.15 (m, 1H), 8.22 (d, J=8.05 Hz, 1H).
LC-MS (Method D, retention time: 2.78 min), MS m/z 779
(M.sup.++1).
Step 39e: Preparation of Compound 145
[0493] ##STR244## Compound 144 (0.878 g, 1.1 mmol) was dissolved in
15 mL of THF and treated with CDI (255 mg, 1.58 mmol). (Care was
taken to avoid moisture by using oven dried glassware and
maintaining a dry N.sub.2 atmosphere.) After refluxing the reaction
mixture for one hour, it was cooled to rt and treated sequentially
with cyclopropylsulfonamide (194 mg, 1.6 mmol) and DBU (245 mg, 1.6
mmol). After stirring for 24 h at rt, the THF was removed by rotary
evaporation. The residue was partitioned between ethyl acetate and
pH 4 buffer. The organic phase was dried (MgSO.sub.4) and
concentrated in vacuo to give the crude product. Flash
chromatography (3% MeOH/methylene chloride) gave 0.68 g (70%) of
compound 145 as a white solid. .sup.1H NMR (CD.sub.3OD, 300 MHz)
.delta. 1.07-1.37 (m, 13H), 1.11 (s, 9H), 1.60-1.78 (m, 4H),
1.97-2.02 (m, 1H), 2.28-2.35 (m, 1H), 2.44-2.51 (m, 1H), 2.75-2.68
(m, 1H), 2.75-2.80 (m, 1H), 2.94-3.00 (m, 1H), 3.20-3.25 (m, 1H),
3.28-3.47 (m, 2H), 3.57-3.62 (m, 1H), 4.05-4.08 (m, 1H), 4.51 (d,
J=12.21 Hz, 1H), 4.65-4.68 (m, 1H), 4.84 (m, 1H), 5.15-5.23 (m,
1H), 5.70-5.75 (m, 1H), 5.94 (s, 1H), 7.35 (d, J=6.10 Hz, 1H), 7.57
(t, J=7.32 Hz, 1H), 7.74 (t, J=7.78 Hz, 1H), 7.78-7.89 (m, 4H),
8.00 (d, J=5.80 Hz, 1H), 8.20-8.25 (m, 2H). LC-MS (Method D,
retention time: 2.89 min), MS m/z 882 (M.sup.++1).
Example 40
Preparation of Compound 151
[0494] ##STR245## To a mixture of compound 145 (680 mg, 0.77 mmol)
in acetonitrile (20 mL) was added potassium carbonate (319 mg, 2.31
mmol) and benzenethiol (186 mg, 1.69 mmol). The mixture was stirred
at rt overnight and then concentrated in vacuo. The residue was
diluted with water, and this aqueous solution was adjusted to pH 5
with 1 N HCl. A white solid precipitated formed in the solution.
This heterogeneous mixture was extracted with ethyl acetate
(3.times.). The combined extracts were dried (MgSO.sub.4) and
concentrated to yield a light yellow solid. This crude solid was
then triturated with hexane several times to yield 510 mg (95%) of
compound 151 as an off-white solid.
[0495] .sup.1H NMR (CD.sub.3OD, 300 MHz) .delta. 1.06-1.88 (m,
13H), 1.23, 1.31 (s, 9H), 1.65 (dd, J=9.70, 5.67 Hz, 1H), 1.80 (dd,
J=8.23, 5.67 Hz, 2H), 2.03 (brs, 1H), 2.18 (br s, 1H), 2.37-2.52
(m, 3H), 2.85-2.99 (m, 2H), 3.09-3.15 (m, 1H), 3.27 (m, 1H), 3.44
(m, 2H), 4.15-4.20 (m, 1H), 4.46 (m, 1H), 4.74-4.85 (m, 2H), 5.32
(t, J=10.25 Hz, 1H), 5.71 (m, 1H), 5.98(br s, 1H), 7.37 (d, J=5.86
Hz, 1H), 7.58 (t, J=8.23 Hz, 1H), 7.74 (t, J=7.68 Hz, 1H), 7.85 (d,
J=8.05 Hz, 1H), 8.00 (d, J=5.86 Hz, 1H), 8.22 (d, J=8.05 Hz, 1H).
LC-MS (Method D, retention time: 2.01 min), MS m/z 697
(M.sup.++1).
Example 41
Preparation of Compound 146
[0496] ##STR246## To a mixture of compound 151 (20 mg, 0.029 mmol)
in 2 mL of DCM was added triethylamine (14 mg, 0.138 mmol) and
acetic anhydride (8 mg, 0.078 mmol). This reaction mixture was
stirred at rt for 2 h, and then concentrated in vacuo. The residue
was dissolved in methanol and purified by preparative HPLC (YMC
XTERRA, S5, 30.times.50 mm, gradient: 45% B to 85% B, 15 min, hold
2 min, flow rate 25 mL/min) to give 10 mg (47%) of compound 146 as
a white solid. .sup.1H NMR (CD.sub.3OD, 300 MHz, 60.degree. C.)
.delta. 1.08-1.22 (m, 13H), 1.55-1.74 (m, 4H), 2.09-2.21(m, 2H),
2.13 (s, 3H), 2.33-2.48 (m, 2H), 2.66-2.75 (m, 1H), 2.90-2.99 (m,
1H), 3.12-3.25 (m, 2H), 3.45-3.60 (m, 2H), 3.75-3.84 (m, 1H),
4.98-4.05 (m, 1H), 4.70 (t, J=7.68 Hz, 1H), 4.89-4.97 (m, 1H), 5.26
(t, J=10.43 Hz, 1H), 5.2-5.75 (m, 1H), 5.92 (s, 1H), 7.29 (d,
J=5.86 Hz, 1H), 7.50 (t, J=7.50 Hz, 1H), 7.64-7.69 (m, 1H), 7.77
(m, 1H), 7.95 (d, J=5.86 Hz, 1H), 8.18 (d, J=8.78 Hz, 1H). LC-MS
(Method D, retention time: 2.64 min), MS m/z 739 (M.sup.++1).
Example 42
Preparation of Compound 147
[0497] ##STR247## To a mixture of compound 151 (100 mg, 0.144 mmol)
in 5 mL of DMF was added potassium carbonate (40 mg, 0.290 mmol)
and methyl iodide (13 .mu.L, 0.21 mmol). This reaction mixture was
stirred at rt for 2 h. It was then concentrated in vacuo. The
residue was dissolved in methanol and purified by preparative HPLC
(YMC XTERRA, S5, 30.times.50 mm, gradient: 45% B to 70% B, 15 min,
hold 2 min, flow rate 25 mL/min) to give 75 mg (73%) of compound
147 as a white solid. .sup.1H NMR (CD.sub.3OD, 300 MHz, 60.degree.
C.) .delta. 1.02-1.28 (m, 13H), 1.64 (dd, J=9.70, 5.67 Hz, 1H),
1.72-1.81(m, 2H), 1.91-2.10 (m, 1H), 2.13-2.30 (m, 1H), 2.36-2.50
(m, 2H), 2.54-2.68 (m, 1H), 2.76-2.99 (m, 2H), 3.05 (s, 3H),
3.11-3.46 (m, 4H), 3.79-3.96 (m, 1H), 4.17 (m, 1H), 4.76 (m, 2H),
5.31 (t, J=10.06 Hz, 1H), 5.71 (m, 1H), 5.99 (s, 1H), 7.33 (d,
J=5.86 Hz, 1H), 7.55 (t, J=7.50 Hz, 1H), 7.70 (t, J=7.68 Hz, 1H),
7.81 (m, 1H), 7.96 (d, J=5.86 Hz, 1H), 8.17 (d, J=8.42 Hz, 1H).
LC-MS (Method D, retention time: 1.98 min), MS m/z 711
(M.sup.++1).
Example 43
Preparation of Compound 148
[0498] ##STR248## To a mixture of compound 151 (20 mg, 0.029 mmol)
in 2 mL of DCM was added diisopropylamine (15 .mu.L, 0.086 mmol)
and methyl chloroformate (8 mg, 0.085 mmol). This reaction mixture
was stirred at rt for 2 h, and then concentrated in vacuo. The
residue was dissolved in methanol and purified by preparative HPLC
(YMC XTERRA, S5, 30.times.50 mm, gradient: 50% B to 90% B, 15 min,
hold 2 min, flow rate 25 mL/min) to give 15 mg (69%) of compound
148 as a white solid. .sup.1H NMR (CD.sub.3OD, 300 MHz) .delta.
1.09 (s, 9H), 1.07-1.45 (m, 13H), 1.56-1.61 (m, 1H), 1.70-1.76 (m,
3H), 2.95-2.10 (m, 1H), 2.13-2.26 (m, 1H), 2.33-2.55 (m, 2H),
2.71-2.78 (m, 1H), 2.93-3.02 (m, 1H), 3.33 (m, 2H), 3.48-3.57 (m,
2H), 3.75 (s, 3H), 4.04-4.12 (m, 1H), 4.64 (t, J=8.42 Hz, 1H), 4.90
(m, 2H), 5.19 (t, J=10.25 Hz, 1H), 5.69-5.77 (m, 1H), 5.93 (s, 1H),
7.34 (d, J=5.86 Hz, 1H), 7.55 (t, J=6.95 Hz, 1H), 7.72 (t, J=7.32
Hz, 1H), 7.83 (d, J=7.32 Hz, 1H), 7.98 (d, J=5.86 Hz, 1H), 8.20 (d,
J=8.42 Hz, 1H), 9.21 (s, 1H). LC-MS (Method D, retention time: 2.75
min), MS m/z 755 (M.sup.++1).
Example 44
Preparation of Compound 150
[0499] ##STR249## To a mixture of compound 151 (20 mg, 0.029 mmol)
in 2 mL of DCM was added diisopropylamine (15 .mu.L, 0.086 mmol)
and tert-butyl isocyanate (8 .mu.L, 0.058 mmol). The reaction
mixture was stirred at rt for 2 h, and then concentrated in vacuo.
The residue was dissolved in methanol and purified by preparative
HPLC (YMC XTERRA, S5, 30.times.50 mm, gradient: 55% B to 90% B, 15
min, hold 2 min, flow rate 25 mL/min) to give 15 mg (65%) of
compound 150 as a white solid (15 mg, 65%). .sup.1H NMR
(CD.sub.3OD, 300 MHz) .delta. 1.06-1.34 (m, 13H), 1.21 (s, 9H),
1.40 (s, 9H), 1.58-1.75 (m, 3H), 1.97-2.09 (m, 1H), 2.11-2.23 (m,
1H), 2.37-2.50 (m, 2H), 2.69-2.82 (m, 1H), 2.94-3.04 (m, 1H), 3.22
(m, 1H), 0.36-3.46 (m, 2H), 4.12 (d, J=12.44 Hz, 1H), 4.39 (d,
J=11.34 Hz, 1H), 4.50-4.57 (m, 1H), 4.69 (t, J=8.23 Hz, 1H),
5.20-5.27 (m, 1H), 5.69-5.78 (m, 1H), 5.90 (s, 1H), 7.34 (d, J=5.86
Hz, 1H), 7.57 (t, J=7.14 Hz, 1H), 7.73 (m, 1H), 7.83 (m, 1H), 7.98
(d, J=5.86 Hz, 1H), 8.24 (d, J=8.05 Hz, 1H). LC-MS (Method D,
retention time: 3.04 min), MS m/z 796 (M.sup.++1).
Example 45
Preparation of Compound 153
[0500] ##STR250## To a mixture of compound 151 (20 mg, 0.029 mmol)
in 2 mL of DCM was added diisopropylamine (15 .mu.L, 0.086 mmol)
and methanesulfonyl chloride (5 .mu.L 0.065 mmol). The reaction
mixture was stirred at rt for 2 h, and then concentrated in vacuo.
The residue was dissolved in methanol and purified by preparative
HPLC (YMC XTERRA, S5, 30.times.50 mm, gradient: 50% B to 90% B, 15
min, hold 2 min, flow rate 25 mL/min) to give 17 mg (76%) of
compound 153 as a white solid. .sup.1H NMR (CD.sub.3OD, 300 MHz)
.delta. 1.02-1.49(m, 13H), 1.59 (m, 1H), 1.72-1.86 (m, 3H),
1.96-2.09 (m, 1H), 2.37-2.50 (m, 2H), 2.63-2.80 (m, 2H), 2.91-3.08
(m, 5H), 2.97 (s, 3H), 3.22-3.45 (m, 3H), 4.19 (dd, J=1.89, 3.48
Hz, 1H), 4.52 (d, J=1.71 Hz, 1H), 4.62 (dd, J=9.88, 7.32 Hz, 1H),
4.83-4.96 (m, 1H), 5.17 (t, J=10.06 Hz, 1H), 5.75 (m, 1H), 5.94 (s,
1H), 7.33 (d, J=5.86 Hz, 1H), 7.57 (d, J=8.05 Hz, 1H), 7.72 (t,
J=7.50 Hz, 1H), 7.82 (m, 1H), 7.99 (d, J=6.22 Hz, 1H), 8.20 (d,
J=8.42 Hz, 1H). LC-MS (Method D, retention time: 2.59 min), MS m/z
775 (M.sup.++1).
Example 46
Preparation of compound 141:
[(Z)-(1S,4R,13S,17R)-4-cyclopropanesulfonylaminocarbonyl-17-(6-methoxy-is-
oquinolin-1-yloxy)-2,14-dioxo-10-oxa-3,15-diaza-tricyclo[13.3.0.0.sup.4,6]-
octadec-7-en-13-yl]-carbamic acid tert-butyl ester
[0501] ##STR251##
Step 46a: Preparation of
(S)-4-allyloxy-2-(tert-butoxycarbonylamino)butyric acid
[0502] ##STR252## To a mixture of sodium hydride (913 mg, 22.8
mmoL) in DMF at 0.degree. C. was added N-t-Boc-L-homoserine (2 g,
9.13 mmoL). This reaction mixture was stirred at 0.degree. C. for
15 min, and then allyl bromide (1.38 g, 11.4 mmoL) was added. The
mixture was warmed up to rt, and stirred for 2 h. It was then
concentrated in vacuo. The residue was diluted with water, and
sequentially washed with hexane and ether. The organic layers were
discarded, and the aqueous layer was carefully adjusted to pH 3
with 1 N HCl. This acidic aqueous solution was extracted with ethyl
acetate. The organic phase was dried (MgSO.sub.4), and concentrated
in vacuo to yield 2.2 g (93%) of
(S)-4-allyloxy-2-(tert-butoxycarbonylamino)butyric acid as a
colorless oil. .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 1.42 (s,
9H), 1.80-1.90 (m, 1H), 2.04-2.16 (m, 1H), 3.50-3.54 (m, 2H), 3.97
(d, J=4.39 Hz, 2H), 4.23 (dd, J=8.78, 4.39 Hz, 1H), 5.15 (d,
J=10.25 Hz, 1H), 5.26 (dd, J=17.38, 1.65 Hz, 1H), 5.84-5.97 (m,
1H). This starting material was employed in the synthesis of
(1R,2S)-ethyl
1-((3R,5S)-1-((S)-4-(allyloxy)-2-(tert-butoxycarbonyl)butanoyl)-3-(6-meth-
oxyisoquinolin-1-yloxy)pyrrolidine-5-carboxamido)-2-vinylcyclopropane-carb-
oxylate as described in example 2.
Step 46b: Preparation of
(Z)-(1S,4R,13S,17R)-13-tert-butoxycarbonylamino-17-(6-methoxy-isoquinolin-
-1-yloxy)-2,14-dioxo-10-oxa-3,15-diaza-tricyclo[13.3.0.0.sup.4,6]octadec-7-
-ene-4-carboxylic acid ethyl ester
[0503] ##STR253## A solution of (1R,2S)-ethyl
1-((3R,5S)-1-((S)-4-(allyloxy)-2-(tert-butoxycarbonyl)butanoyl)-3-(6-meth-
oxyisoquinolin-1-yloxy)pyrrolidine-5-carboxamido)-2-vinylcyclopropanecarbo-
xylate (220 mg, 0.33 mmol) in 84 mL of methylene chloride was added
tricyclohexylphosphine[1,3-bis(2,4,6-trimethyl-phenyl)-4,5-dihydroimidazo-
l-2-ylidene][benzylidene]-ruthenium (IV) dichloride (27 mg, 0.032
mmol). The light orange homogeneous solution was refluxed for 3 h
to give a dark orange solution. At this time another portion of the
ruthenium catalyst (13 mg, 0.016 mmoL) was added, and refluxing was
continued for another 2 h. The reaction mixture was cooled to rt,
and concentrated in vacuo to give an orange oil. Flash
chromatography (ethyl acetate then 10% MeOH in ethyl acetate) gave
174 mg (80%) of
(Z)-(1S,4R,13S,17R)-13-tert-butoxycarbonylamino-17-(6-methoxy-isoquinolin-
-1-yloxy)-2,14-dioxo-10-oxa-3,15-diaza-tricyclo[13.3.0.0.sup.4,6]octadec-7-
-ene-4-carboxylic acid ethyl ester as a white solid. LC-MS (Method
A, retention time: 3.74 min), MS m/z 639(M.sup.++1).
Step 46c: Preparation of Compounds 140 and 141
[0504] ##STR254##
(Z)-(1S,4R,13S,17R)-13-tert-butoxycarbonylamino-17-(6-methoxy-isoquinolin-
-1-yloxy)-2,14-dioxo-10-oxa-3,15-diaza-tricyclo[13.3.0.0.sup.4,6]octadec-7-
-ene-4-carboxylic acid ethyl ester was hydrolyzed according to
Example 2 (step 2E) to give compound 140. Compound 140 was
converted to compound 141,
[(Z)-(1S,4R,13S,17R)-4-cyclopropanesulfonylaminocarbonyl-17-(6-metho-
xy-isoquinolin-1-yloxy)-2,14-dioxo-10-oxa-3,15-diaza-tricyclo[13.3.0.0.sup-
.4,6]octadec-7-en-13-yl]-carbamic acid tert-butyl ester, according
to Example 7. LC-MS (Method A, retention time: 3.51 min), MS m/z
714(M.sup.++1).
Example 47
General Procedure for Preparing P4-Carbamates from Tertiary
Alcohols
Preparation of compound 56:
[(Z)-(1S,4R,14S,18R)-4-cyclopropanesulfonylaminocarbonyl-18-(isoquinolin--
1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-y-
l]-carbamic acid 1-methyl-cyclobutyl ester
[0505] ##STR255##
Step 47a: Preparation of 1-methylcyclobutanol
[0506] ##STR256## To a solution of 6 mL of methylmagnesium iodide
(3M in ether) at -10.degree. C. was added cyclobutanone (1 g, 14.3
mmol) dropwise. The mixture was stirred for 2.5 h (-10.degree. C.
to rt). Dilute HCl was added to the reaction mixture until all of
the precipitate was dissolved. The homogeneous reaction mixture was
extracted with ether (2.times.). The combined ether extracts were
dried (MgSO.sub.4), filtered, and concentrated in vacuo to give 0.9
g (73%) of 1-methylcyclobutanol as a yellow oil.
Step 47b. Preparation of
[(Z)-(1S,4R,14S,18R)-4-cyclopropanesulfonylaminocarbonyl-18-(isoquinolin--
1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-y-
l]-carbamic acid 1-methyl-cyclobutyl ester
[0507] To a slurry of KH (400 mg, 35% in oil, 3.5 mmol) was added 5
mL of hexane. It was stirred for 5 min, and then the hexane was
removed. This process was repeated three times. THF (5 mL) was
added to this prewashed KH, and then 1-methylcyclobutanol (200 mg,
2.3 mmol) was added at 0.degree. C. The mixture was stirred
(0.degree. C. to rt) for 1 h, and then carbonic acid dipyridin-2-yl
ester (753 mg, 3.5 mmol) was added. The mixture was stirred at rt
for 4 h, and the reaction was diluted with sat. aq. NH.sub.4Cl. The
mixture was then extracted with EtOAc, and the solvent was removed
in vacuo to obtain 200 mg of the crude carbonic acid
1-methyl-cyclobutyl ester pyridin-2-yl ester as an orange solid. It
was used directly in the next step without further
purification.
[0508] To a suspension of 50 mg (0.073 mmol) of compound 11
[1-(2-Amino-3,3-dimethyl-butyryl)-4-(6-methoxy-isoquinolin-1-yloxy)-pyrro-
lidine-2-carboxylic acid
(1-cyclopropanesulfonylaminocarbonyl-2-vinyl-cyclopropyl)-amide bis
hydrochloride salt] in CH.sub.2Cl.sub.2 was added
diisopropylethylamine (29 mg, 0.22 mmoL) and 70 mg (0.33 mmol) of
the reagent above (carbonic acid 1-methylcyclobutyl ester
pyridin-2-yl ester). This reaction mixture was stirred at rt
overnight, and then concentrated in vacuo. The residue was purified
by preparative HPLC to give 32 mg (62%) of compound 56 as a white
solid. Preparative HPLC condition: YMC XTERRA, S5, 19.times.100 mm,
gradient: 50% B to 90% B, 15 min, hold 2 min, flow rate 25 mL/min.
LC-MS (Method A, retention time: 3.47 min), MS m/z 708
(M.sup.++1).
[0509] Compound 59 was prepared by the general method used for
compound 56 (example 47): 1-methylcyclopropanol was converted to
the reagent carbonic acid 1-methyl-cyclopropyl ester pyridin-2-yl
ester was prepared according to the method above (example 47).
1-Methyl-1-cyclopropanol was prepared by a slight modification of
the procedure described by Kulinkovich, O. G.; Sviridov, S. V.; and
Vasilevski, D. A. Synthesis, 1991, 234: To a stirred solution of
methyl acetate (1.85 g, 25 mmol) and Ti(OPr-i).sub.4 (744 .mu.L,
2.5 mmol) in diethyl ether (80 mL) is added ethylmagnesium bromide
(3M solution in diethyl ether) (18 mL, 53 mmol) in diethyl ether
(60 mL) slowly over a period of 1 h while maintaining the reaction
temperature at 18-20.degree. C. Once the addition is complete,
stirring is continued for 10 min. The mixture is poured into cooled
(5.degree. C.) 10% aq. H.sub.2SO.sub.4 (250 mL) and the product is
extracted with diethyl ether (3.times.50 mL). The combined ether
extracts are washed with water (50 mL), dried (MgSO.sub.4), and
carefully concentrated by rotory evaporation (-20 torr, rt) to give
1.7 g of 1-methyl-1-cyclopropanol (purity-80%). .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 0.42-0.45 (m, 2H), 0.73-0.77 (m, 2H), 1.44
(s, 3H), 1.64 (br s, 1H).
[0510] The following compounds were also prepared using the general
procedure above: compound 50 was prepared from
1-methylcyclopentanol; compounds 39 and 51 were prepared from
1,1,1-trifluoro-2-methylpropan-2-ol, compound 55 was prepared from
1,1-difluoro-2-methylpropan-2-ol (1,1-difluoro-2-methylpropan-2-ol
was prepared according to the procedure described by Dickey, J. B.
and Towne, E. B. U.S. Pat. No. 2,700,686), compounds 40 and 52 were
prepared from 1-chloro-2-methylpropan-2-ol, compound 124 was
prepared from 1,1,1-trichloro-2-methylpropan-2-ol, compound 123 was
prepared from 3-hydroxy-3-methylbutan-2-one, and compound 139 was
prepared from S-tert-butyl O-4-nitrophenyl carbonothioate (shown
below) (This reagent was prepared as discribed by E. M. Gordon, J.
C. Barrish, G. S. Bisacchi, C-Q. Sun, J. A. Tino, G. D. Vite, and
R. Zahler in US005559256A.). ##STR257##
Example 48
Preparation of Compound 126
[0511] ##STR258##
[0512] To a mixture of compound 11 {cyclopropanesulfonic acid
[14-amino-18-(isoquinolin-1-yloxy)-2,15-dioxo-3,16-diaza-tricyclo[14.3.0.-
0.sup.4,6]nonadec-7-ene-4-carbonyl]-amide bis hydrochloride} (20
mg, 0.029 mmol) in 2 mL of DCM was added 18 .mu.L (0.10 mmol) of
DIPEA and 4 mg (0.04 mmol) of t-butyl isocyanate. The mixture was
stirred at rt overnight. It was then diluted with EtOA,c and washed
with pH 4 buffer (2.times.) and brine (1.times.). The organic phase
was dried (MgSO.sub.4) and concentrated in vacuo to give the crude
product. The resulting oil was dissolved in methanol and purified
by preparative HPLC (YMC XTERRA, S5, 19.times.100 mm, gradient: 50%
B to 100% B, 15 min, hold 2 min, flow rate 25 mL/min) to isolate
compound 126 as a white powder (17 mg, 82%): LC-MS (Method D,
retention time: 3.24 min), MS m/z 695 (M.sup.++1). .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 0.86-0.96 (m, 2H), 1.12 (s, 9H), 1.01-1.49
(m, 18H), 1.52-1.66 (m, 1H), 1.70-1.78 (m, 1H), 1.83-1.95(m, 2H),
2.14-2.25 (m, 1H), 2.49 (brs, 1H), 2.71 (m, 2H), 2.86-2.92 (m, 1H),
4.07 (dd, J=11.71, 4.03 Hz, 1H), 4.40-4.44 (m, 1H), 4.62-4.69 (m,
2H), 4.94-5.00 (m, 1H), 5.65-5.74 (m, 1H), 5.97 (br s, 1H), 7.03
(s, 1H), 7.32 (d, J=6.22 Hz, 1H), 7.50-7.52 (m, 1H), 7.68-7.78 (m,
2H), 8.00 (d, J=5.86 Hz, 1H), 8.22 (d, J=7.68 Hz, 1H), 10.15 (s,
1H).
Example 49
Preparation of Compound 58
[0513] ##STR259## ##STR260##
Step 49a: Preparation of
1-{[1-(2-tert-Butoxycarbonylamino-non-8-enoyl)-4-(tert-butyl-dimethylsila-
nyloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinylcyclopropanecarboxylic
acid ethyl ester
[0514] ##STR261## To a mixture of
1-{[1-(2(S)-tert-Butoxycarbonylamino-non-8-enoyl)-4(R)-hydroxy-pyrrolidin-
e-2(S)carbonyl]-(1R)-amino}-2(S)-vinyl-cyclopropanecarboxylic acid
ethyl ester (1.5 g, 2.87 mmoL) in 10 mL of DMF was added imidazole
(0.25 g, 3.67 mmoL) and tert-butyl-dimethylsilyl chloride (516 mg,
3.44 mmoL). The mixture was stirred at rt for two days. The
reaction mixture was then concentrated in vacuo, and the residue
was dissolved in ethyl acetate. This solution was washed with
water, dried over magnesium sulfate, and concentrated in vacuo to
obtain a crude solid. Purification by flash chromatography (eluting
with 20% ethyl acetate in hexane) gave 1.43 g (78%) of
1-{[1-(2-tert-butoxycarbonylamino-non-8-enoyl)-4-(tert-butyldime-
thylsilanyloxy)pyrrolidine-2-carbonyl]-amino}-2-vinylcyclopropanecarboxyli-
c acid ethyl ester as a white solid.
[0515] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.10 (s,6H), 0.89
(s, 9H), 1.22 (m, 3H), 1.31-1.48 (m, 16H), 1.50-1.75 (m, 3H), 2.06
(m, 3H), 2.11-2.33 (m, 2H), 3.70 (m, 2H), 4.03-4.19 (m, 2H), 4.21
(m, 1H), 4.45 (t, J=7.87 Hz, 1H), 4.59 (m, 1H), 4.91 (d, J=9.15 Hz,
1H), 4.98 (d, J=17.20 Hz, 1H), 5.08 (dd, J=10.25, 1.83 Hz, 1H),
5.27 (dd, J=17.38, 1.65 Hz, 1H), 5.65-5.87 (m, 2H). LC-MS (Method
A, retention time: 4.00 min), MS m/z 636 (M.sup.++1).
Step 49b: Preparation of
14-tert-butoxycarbonylamino-18-(tert-butyldimethyl-silanyloxy)-2,15-dioxo-
-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid ethyl ester
[0516] ##STR262## To a solution of
1-{[1-(2-tert-butoxycarbonylamino-non-8-enoyl)-4-(tert-butyl-dimethyl-sil-
anyloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic
acid ethyl ester (1.63 g, 2.56 mmoL) in 640 mL of methylene
chloride was added 215 mg (0.26 mmoL) of
tricyclohexylphosphine[1,3-bis(2,4,6-tri[benzylidene]ruthenium(IV)dichlor-
ide. The mixture was heated at reflux for 15 min. The residue was
concentrated in vacuo, and then purified by flash chromatography
eluting with 30% ethyl acetate/hexane. To further decolorize the
sample, the crude product was chromatographed a second time eluting
with 50% ether in hexane to give 1.5 g (96%) of
14-tert-butoxycarbonylamino-18-(tert-butyl-dimethyl-silanyloxy)-2,15-diox-
o-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid ethyl ester as a white solid. .sup.1H NMR (500 MHz,
CD.sub.3Cl) .delta. 0.06 (s, 3H), 0.07 (s, 3H), 0.86 (s, 9H),
1.18-1.24 (m, 6H), 1.34-1.64 (m, 14H), 1.86-1.96 (m, 3H), 2.02-2.09
(m, 1H), 2.11-2.17 (m, 1H), 2.19-2.28 (m, 1H), 2.57-2.63 (m, 1H),
3.50-3.54 (m, 1H), 3.71 (dd, J=10.22, 6.26 Hz, 1H), 4.06-4.17 (m,
2H), 4.52-4.58 (m, 2H), 4.75 (d, J=8.55 Hz, 1H), 5.21 (t, J=9.92
Hz, 1H), 5.35 (d, J=7.63 Hz, 1H), 5.45-5.50 (m, 1H), 6.94 (s,1H).
LC-MS (Method A, retention time: 3.88 min), MS m/z 608
(M.sup.++1).
Step 49c: Preparation of
14-tert-butoxycarbonylamino-18-(tert-butyl-dimethyl-silanyloxy)-2,15-diox-
o-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid
[0517] ##STR263## To a solution of
14-tert-butoxycarbonylamino-18-(tert-butyldimethylsilanyloxy)-2,15-dioxo--
3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid ethyl ester (1.5 g, 2.47 mmoL) in a mixed solvent system of
THF (4 mL), methanol (1 mL), and water (2 mL), was added powdered
lithium hydroxide monohydrate (1.0 g, 50 mmoL). The light yellow
slurry was stirred at rt under N.sub.2 for 4 h. The mixture was
then concentrated in vacuo, and the residue partioned between ether
and water. The ether phase was discarded, and the aqueous phase was
treated with 1 N HCl until reaching pH 4. This acidic solution was
extracted with EtOAc (3.times.). The combined EtOAc extracts were
dried (MgSO.sub.4), and concentrated in vacuo to give 1.2 g (84%)
of
14-tert-butoxycarbonylamino-18-(tert-butyl-dimethyl-silanyloxy)-2,15-diox-
o-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid as an off-white solid.
[0518] .sup.1H NMR (300 MHz, CD.sub.3OD) 0.12 (s, 6H), 0.89 (s,
9H), 1.23-1.64 (m, 17H), 1.70-1.87 (m, 1H), 1.90-2.49 (m, 6H),
3.70-3.80 (m, 1H), 3.83-3.90 (m, 1H), 4.28-4.36 (m, 1H), 4.47-4.55
(m, 1H), 4.65 (s, 1H), 5.30-5.39 (m, 1H), 5.53-5.62 (m, 1H). LC-MS
(Method A, retention time: 3.69 min), MS m/z 580 (M.sup.++1).
Step 49d: Preparation of
[18-(tert-butyl-dimethyl-silanyloxy)-4-cyclopropanesulfonylaminocarbonyl--
2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester
[0519] ##STR264##
14-tert-Butoxycarbonylamino-18-(tert-butyldimethylsilanyloxy)-2,15-dioxo--
3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-ene-4-carboxylic
acid (500 mg, 0.86 mmoL) was dissolved in 25 mL of THF and treated
with CDI (180 mg, 1.12 mmoL). (Care was taken to avoid moisture by
using oven dried glassware and maintaining a dry N2 atmosphere).
After refluxing the reaction mixture for 2 h, it was cooled to rt
and treated sequentially with cyclopropylsulfonamide (135 mg, 1.12
mmoL) and DBU (170 mg, 1.12 mmoL). The reaction mixture was stirred
for 4 h at rt, and the THF was removed by rotary evaporation. The
residue was partitioned between ethyl acetate and pH 4 buffer. The
organic phase was dried (MgSO4) and concentrated in vacuo to give
the crude product. It was then purified by flash chromatography
(eluting with 33% ethyl acetate in hexane) to give 300 mg (51%) of
[18-(tert-butyl-dimethyl-silanyloxy)-4-cyclopropanesulfonylaminocarbonyl--
2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester as a white solid. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 1H 0.07 (s, 3H), 0.08 (s, 3H), 0.85 (s, 9H),
0.87-1.49 (m, 21H), 1.73-1.95 (m, 3H), 2.08-2.16 (m, 1H), 2.25-2.36
(m, 2H), 2.42-2.56 (m, 1H), 2.85-2.93 (m, 1H), 3.65-3.74(dd,
J=10.61, 3.66 Hz, 1H), 3.89 (d, J=10.25 Hz, 1H), 4.34 (m, J=9.70,
9.70 Hz, 1H), 4.43 (t, J=7.87 Hz, 1H), 4.57 (s, 1H), 4.94-5.01 (m,
1H), 5.10 (d, J=8.78 Hz, 1H), 5.66-5.75 (m, 1H), 6.55 (s, 1H),
10.13 (s, 1H). LC-MS (Method A, retention time: 3.81 min), MS m/z
683 (M.sup.++1).
Step 49e:
(4-Cyclopropanesulfonylaminocarbonyl-18-hydroxy-2,15-dioxo-3,16--
diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl)-carbamic acid
tert-butyl ester
[0520] ##STR265## To a mixture of
[18-(tert-butyl-dimethylsilanyloxy)-4-cyclopropanesulfonyl-aminocarbonyl--
2,15-dioxo-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl]-carbam-
ic acid tert-butyl ester (330 mg, 0.48 mmoL) in 25 mL of THF was
added tetrabutylammonium fluoride (150 mg, 0.54 mmoL). The reaction
mixture was stirred at rt for 18 h, and then the THF was removed by
rotary evaporation. The residue was partitioned between ethyl
acetate and water. The organic phase was dried (MgSO.sub.4) and
concentrated in vacuo to give the crude product. It was then
purified by triturating with hexane to yield 200 mg (73%) of
(4-cyclopropanesulfonylaminocarbonyl-18-hydroxy-2,15-dioxo-3,16-diaza-tri-
cyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl)-carbamic acid tert-butyl
ester as a white solid. .sup.1H NMR (500 MHz, CD.sub.3Cl) .delta.
1.87-1.64 (m, 21H), 1.70-1.98 (m, 3H), 2.15-2.56 (m, 5H), 2.85-2.94
(m, 1H), 3.71 (d, J=13.91 Hz, 1H), 4.10-4.26 (m, 2H), 4.51 (t,
J=7.87 Hz, 1H), 4.62 (s, 1H), 4.98 (m, 1H), 5.06 (d, J=8.78 Hz,
1H), 5.64-5.71 (m, 1H), 6.72 (s, 1H), 10.24 (s, 1H). LC-MS (Method
A, retention time: 2.85 min), MS m/z 569 (M.sup.++1).
Step 49f: Preparation of compound 58:
[(Z)-(1S,4R,14S,18R)-4-cyclopropanesulfonylaminocarbonyl-2,15-dioxo-18-(3-
-phenyl-isoquinolin-1-yloxy)-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec--
7-en-14-yl]-carbamic acid tert-butyl ester
[0521] ##STR266## To a mixture of
(4-cyclopropanesulfonylaminocarbonyl-18-hydroxy-2,15-dioxo-3,16-diaza-tri-
cyclo[14.3.0.0.sup.4,6]nonadec-7-en-14-yl)-carbamic acid tert-butyl
ester (20 mg, 0.035 mmoL) in 1 mL of DMF was added potassium
t-butoxide (22 mg, 0.196 mmoL). The mixture was stirred at rt for 5
min, and then 1-chloro-3-phenylisoquinoline (15 mg, 0.062 mmoL) was
added. The reaction mixture was stirred at rt for 15 h, and then
concentrated in vacuo. This crude product was triturated with
ether. The residue was dissolved in MeOH, and then purified by
preparative HPLC.(YMC XTERRA, S5, 19.times.100 mm, gradient: 60% B
to 100% B, 15 min, hold 2 min, flow rate 25 mL/min) to give 10 mg
(39%) of compound 58,
[(Z)-(1S,4R,14S,18R)-4-cyclopropanesulfonylaminocarbonyl-2,15-dioxo-18-(3-
-phenyl-isoquinolin-1-yloxy)-3,16-diaza-tricyclo[14.3.0.0.sup.4,6]nonadec--
7-en-14-yl]-carbamic acid tert-butyl ester, as a white solid.
.sup.1H NMR (500 MHz, CD.sub.3Cl) .delta. 0.90-1.64 (m, 21H),
1.76-1.97 (m, 3H), 2.26-2.31 (m, 1H), 2.52-2.63 (br s, 1H),
2.69-2.81 (m, 2H), 2.88-2.93 (m, 1H), 4.11 (d, J=11.60 Hz, 1H),
4.33 (m, 1H), 4.61 (d, J=7.94 Hz, 2H), 4.99 (t, J=9.31 Hz, 1H),
5.05 (d, J=7.93 Hz, 1H), 5.69-5.74 (m, 1H), 6.08 (s, 1H), 6.60 (s,
1H), 7.38-7.47(m, 2H), 7.50 (t, J=7.63 Hz, 2H), 7.63 (t, J=7.32 Hz,
I H), 7.71 (s, 1H), 7.77 (d, J=8.24 Hz, 1H), 8.10 (d, J=7.32 Hz,
2H), 8.18 (d, J=7.93 Hz, 1H), 10.27 (s, 1H). LC-MS (Method A,
retention time: 3.72 min), MS m/z 772 (M.sup.++1).
[0522] The compounds of tables 2 and 3 were prepared employing the
procedures described in the above examples: TABLE-US-00005 TABLE 2
##STR267## Mass Spec. Cmpd R.sub.1 R.sub.3 R.sub.7 R.sub.8 R.sub.9
Q (M + H).sup.+ 1 ##STR268## ##STR269## ##STR270## ##STR271##
##STR272## ##STR273## 593 17A ##STR274## ##STR275## ##STR276##
##STR277## ##STR278## ##STR279## 623 18A ##STR280## ##STR281##
##STR282## ##STR283## ##STR284## ##STR285## 641 27 ##STR286##
##STR287## ##STR288## ##STR289## ##STR290## ##STR291## 625 61
##STR292## ##STR293## ##STR294## ##STR295## ##STR296## ##STR297##
639 79 ##STR298## ##STR299## ##STR300## ##STR301## ##STR302##
##STR303## 669 84 ##STR304## ##STR305## ##STR306## ##STR307##
##STR308## ##STR309## 625 140 ##STR310## ##STR311## ##STR312##
##STR313## ##STR314## ##STR315## 611 143 ##STR316## ##STR317##
##STR318## ##STR319## ##STR320## ##STR321## 609 144 ##STR322##
##STR323## ##STR324## ##STR325## ##STR326## ##STR327## 779
[0523] TABLE-US-00006 TABLE 3 ##STR328## Cmpd R.sub.23 R.sub.1
R.sub.3, R.sub.4 R.sub.8 R.sub.9 2 ##STR329## ##STR330## H, H
##STR331## ##STR332## 3 ##STR333## ##STR334## H, H ##STR335##
##STR336## 4 ##STR337## ##STR338## H, H ##STR339## ##STR340## 5
##STR341## ##STR342## H, H ##STR343## ##STR344## 6 ##STR345##
##STR346## H, H ##STR347## ##STR348## 7 ##STR349## ##STR350## H, H
##STR351## ##STR352## 8 ##STR353## ##STR354## H, H ##STR355##
##STR356## 9 ##STR357## ##STR358## H, H ##STR359## ##STR360## 10
##STR361## ##STR362## H, H ##STR363## ##STR364## 11 ##STR365##
##STR366## H, H ##STR367## ##STR368## 12 ##STR369## ##STR370## H, H
##STR371## ##STR372## 13 ##STR373## ##STR374## H, H ##STR375##
##STR376## 14 ##STR377## ##STR378## H, H ##STR379## ##STR380## 15
##STR381## ##STR382## H, H ##STR383## ##STR384## 16 ##STR385##
##STR386## H, H ##STR387## ##STR388## 17B ##STR389## ##STR390##
6-OMe, H ##STR391## ##STR392## 18B ##STR393## ##STR394## 6-OMe, H
##STR395## ##STR396## 19 ##STR397## ##STR398## 6-OMe, H ##STR399##
##STR400## 20 ##STR401## ##STR402## H, H ##STR403## ##STR404## 21
##STR405## ##STR406## H, H ##STR407## ##STR408## 22 ##STR409##
##STR410## H, H ##STR411## ##STR412## 23 ##STR413## ##STR414## H, H
##STR415## ##STR416## 24 ##STR417## ##STR418## H, H ##STR419##
##STR420## 25 ##STR421## ##STR422## H, H ##STR423## ##STR424## 26
##STR425## ##STR426## H, H ##STR427## ##STR428## 28 ##STR429##
##STR430## 6-OMe, H ##STR431## ##STR432## 29 ##STR433## ##STR434##
6-OMe, H ##STR435## ##STR436## 30 ##STR437## ##STR438## 6-OMe, H
##STR439## ##STR440## 31 ##STR441## ##STR442## 6-OMe, H ##STR443##
##STR444## 32 ##STR445## ##STR446## 6-OMe, H ##STR447## ##STR448##
33 ##STR449## ##STR450## 6-OMe, H ##STR451## ##STR452## 34
##STR453## ##STR454## 6-OMe, H ##STR455## ##STR456## 35 ##STR457##
##STR458## 6-OMe, H ##STR459## ##STR460## 36 ##STR461## ##STR462##
6-OMe, H ##STR463## ##STR464## 37 ##STR465## ##STR466## 6-OMe, H
##STR467## ##STR468## 38 ##STR469## ##STR470## 6-OMe, H ##STR471##
##STR472## 39 ##STR473## ##STR474## 6-OMe, H ##STR475## ##STR476##
40 ##STR477## ##STR478## 6-OMe, H ##STR479## ##STR480## 41
##STR481## ##STR482## 6-OMe, H ##STR483## ##STR484## 42 ##STR485##
##STR486## 6-OMe, H ##STR487## ##STR488## 43 ##STR489## ##STR490##
6-OMe, H ##STR491## ##STR492## 44 ##STR493## ##STR494## 6-OMe, H
##STR495## ##STR496## 45 ##STR497## ##STR498## 6-OMe, H ##STR499##
##STR500## 46 ##STR501## ##STR502## 6-OMe, H ##STR503## ##STR504##
47 ##STR505## ##STR506## 6-OMe, H ##STR507## ##STR508## 48
##STR509## ##STR510## 6-OMe, H ##STR511## ##STR512## 49 ##STR513##
##STR514## 6-OMe, H ##STR515## ##STR516## 50 ##STR517## ##STR518##
H, H ##STR519## ##STR520## 51 ##STR521## ##STR522## H, H ##STR523##
##STR524## 52 ##STR525## ##STR526## H, H ##STR527## ##STR528## 53
##STR529## ##STR530## 6-OMe, H ##STR531## ##STR532## 54 ##STR533##
##STR534## 6-OMe, H ##STR535## ##STR536## 55 ##STR537## ##STR538##
H, H ##STR539## ##STR540## 56 ##STR541## ##STR542## H, H ##STR543##
##STR544## 57 ##STR545## ##STR546## H, H ##STR547## ##STR548## 58
##STR549## ##STR550## H, H ##STR551## ##STR552## 59 ##STR553##
##STR554## H, H ##STR555## ##STR556## 60 ##STR557## ##STR558##
6-OMe, H ##STR559## ##STR560## 62 ##STR561## ##STR562## 6-OMe, H
##STR563## ##STR564## 63 ##STR565## ##STR566## 6-OMe, H ##STR567##
##STR568## 64 ##STR569## ##STR570## 6-OMe, H ##STR571## ##STR572##
65 ##STR573## ##STR574## 6-OMe, H ##STR575## ##STR576## 66
##STR577## ##STR578## 6-OMe, H ##STR579## ##STR580## 67 ##STR581##
##STR582## 6-OMe, H ##STR583## ##STR584## 68 ##STR585## ##STR586##
6-OMe, H ##STR587## ##STR588## 69 ##STR589## ##STR590## 6-OMe, H
##STR591## ##STR592## 70 ##STR593## ##STR594## 6-OMe, H ##STR595##
##STR596## 71 ##STR597## ##STR598## 6-OMe, H ##STR599## ##STR600##
72 ##STR601## ##STR602## 6-OMe, H ##STR603## ##STR604## 73
##STR605## ##STR606## 6-OMe, H ##STR607## ##STR608## 74 ##STR609##
##STR610## 6-OMe, H ##STR611## ##STR612## 75 ##STR613## ##STR614##
6-OMe, H ##STR615## ##STR616## 76 ##STR617## ##STR618## 6-OMe, 7-Cl
##STR619## ##STR620## 77 ##STR621## ##STR622## 5-OMe, H ##STR623##
##STR624## 78 ##STR625## ##STR626## 5-Cl, H ##STR627## ##STR628##
80 ##STR629## ##STR630## 6-OMe, H ##STR631## ##STR632## 81
##STR633## ##STR634## 6-OMe, H ##STR635## ##STR636## 82 ##STR637##
##STR638## 6-OMe, H ##STR639## ##STR640## 83 ##STR641## ##STR642##
6-OMe, H ##STR643## ##STR644## 85 ##STR645## ##STR646## 6-OMe, H
##STR647## ##STR648## 86 ##STR649## ##STR650## 6-OMe, H ##STR651##
##STR652## 87 ##STR653## ##STR654## 6-OMe, H ##STR655## ##STR656##
88 ##STR657## ##STR658## 6-OMe, H ##STR659## ##STR660## 89
##STR661## ##STR662## 6-OMe, H ##STR663## ##STR664## 90 ##STR665##
##STR666## 6-OMe, H ##STR667## ##STR668## 91 ##STR669## ##STR670##
6-OMe, H ##STR671## ##STR672## 92 ##STR673## ##STR674## 6-OMe, H
##STR675## ##STR676## 93 ##STR677## ##STR678## 6-OMe, H ##STR679##
##STR680## 94 ##STR681## ##STR682## 6-OMe, H ##STR683## ##STR684##
95 ##STR685## ##STR686## 6-OMe, H ##STR687## ##STR688## 96
##STR689## ##STR690## 6-OMe, H ##STR691## ##STR692## 97 ##STR693##
##STR694## 6-OMe, H ##STR695## ##STR696## 98 ##STR697## ##STR698##
6-OMe, H ##STR699## ##STR700## 99 ##STR701## ##STR702## 6-OMe, H
##STR703## ##STR704## 100 ##STR705## ##STR706## 6-OMe, H ##STR707##
##STR708## 101 ##STR709## ##STR710## 6-OMe, H ##STR711## ##STR712##
102 ##STR713## ##STR714## 6-OMe, H ##STR715## ##STR716## 103
##STR717## ##STR718## 6-OMe, H ##STR719## ##STR720## 104 ##STR721##
##STR722## 6-OMe, H ##STR723## ##STR724## 105 ##STR725## ##STR726##
6-OMe, H ##STR727## ##STR728## 106 ##STR729## ##STR730## 6-OMe, H
##STR731## ##STR732## 107 ##STR733## ##STR734## 6-OMe, H ##STR735##
##STR736## 108 ##STR737## ##STR738## 6-OMe, H ##STR739## ##STR740##
109 ##STR741## ##STR742## 6-OMe, H ##STR743## ##STR744## 110
##STR745## ##STR746## 6-OMe, H ##STR747## ##STR748## 111 ##STR749##
##STR750## 6-OMe, H ##STR751## ##STR752## 112 ##STR753## ##STR754##
6-OMe, H ##STR755## ##STR756## 113 ##STR757## ##STR758## 6-OMe, H
##STR759## ##STR760## 114 ##STR761## ##STR762## 6-OMe, H ##STR763##
##STR764## 115 ##STR765## ##STR766## 6-OMe, H ##STR767## ##STR768##
116 ##STR769## ##STR770## H, H ##STR771## ##STR772## 117 ##STR773##
##STR774## 6-OMe, H ##STR775## ##STR776## 118 ##STR777## ##STR778##
H, H ##STR779## ##STR780## 119 ##STR781## ##STR782## H, H
##STR783## ##STR784## 120 ##STR785## ##STR786## H, H ##STR787##
##STR788## 121 ##STR789## ##STR790## 6-OMe, H ##STR791## ##STR792##
122 ##STR793## ##STR794## H, H ##STR795## ##STR796## 123 ##STR797##
##STR798## H, H ##STR799## ##STR800## 124 ##STR801## ##STR802## H,
H ##STR803## ##STR804## 125 ##STR805## ##STR806## H, H ##STR807##
##STR808## 126 ##STR809## ##STR810## H, H ##STR811## ##STR812##
127 ##STR813## ##STR814## H, H ##STR815## ##STR816## 128 ##STR817##
##STR818## H, H ##STR819## ##STR820## 129 ##STR821## ##STR822## H,
H ##STR823## ##STR824## 130 ##STR825## ##STR826## 5-Cl, H
##STR827## ##STR828## 131 ##STR829## ##STR830## 6-OMe, 7-Cl
##STR831## ##STR832## 132 ##STR833## ##STR834## 5-F, 6-OMe
##STR835## ##STR836## 133 ##STR837## ##STR838## 5-OMe, H ##STR839##
##STR840## 134 ##STR841## ##STR842## 5,6-dimethoxy ##STR843##
##STR844## 135 ##STR845## ##STR846## 6-OMe, H ##STR847## ##STR848##
136 ##STR849## ##STR850## 6-OMe, H ##STR851## ##STR852## 137
##STR853## ##STR854## 6-OMe, H ##STR855## ##STR856## 138 ##STR857##
##STR858## H, H ##STR859## ##STR860## 139 ##STR861## ##STR862## H,
H ##STR863## ##STR864## 141 ##STR865## ##STR866## 6-OMe, H
##STR867## ##STR868## 142 ##STR869## ##STR870## 6-OMe, H ##STR871##
##STR872## 145 ##STR873## ##STR874## H, H ##STR875## ##STR876## 146
##STR877## ##STR878## H, H ##STR879## ##STR880## 147 ##STR881##
##STR882## H, H ##STR883## ##STR884## 148 ##STR885## ##STR886## H,
H ##STR887## ##STR888## 149 ##STR889## ##STR890## H, H ##STR891##
##STR892## 150 ##STR893## ##STR894## H, H ##STR895## ##STR896## 151
##STR897## ##STR898## H, H ##STR899## ##STR900## 152 ##STR901##
##STR902## H, H ##STR903## ##STR904## 153 ##STR905## ##STR906## H,
H ##STR907## ##STR908## Mass Spec. Cmpd R.sub.7 Q (M + H).sup.+ 2
##STR909## ##STR910## 670 3 ##STR911## ##STR912## 698 4 ##STR913##
##STR914## 696 5 ##STR915## ##STR916## 786 6 ##STR917## ##STR918##
738 7 ##STR919## ##STR920## 740 8 ##STR921## ##STR922## 710 9
##STR923## ##STR924## 710 10 ##STR925## ##STR926## 698 11
##STR927## ##STR928## 596 12 ##STR929## ##STR930## 654 13
##STR931## ##STR932## 710 14 ##STR933## ##STR934## 724 15
##STR935## ##STR936## 710 16 ##STR937## ##STR938## 682 17B
##STR939## ##STR940## 726 18B ##STR941## ##STR942## 744 19
##STR943## ##STR944## 776 20 ##STR945## ##STR946## 708 21
##STR947## ##STR948## 706 22 ##STR949## ##STR950## 678 23
##STR951## ##STR952## 681 24 ##STR953## ##STR954## 668 25
##STR955## ##STR956## 674 26 ##STR957## ##STR958## 638 28
##STR959## ##STR960## 728 29 ##STR961## ##STR962## 742 30
##STR963## ##STR964## 756 31 ##STR965## ##STR966## 714 32
##STR967## ##STR968## 742 33 ##STR969## ##STR970## 739 34
##STR971## ##STR972## 727 35 ##STR973## ##STR974## 728 36
##STR975## ##STR976## 753 37 ##STR977## ##STR978## 742 38
##STR979## ##STR980## 767 39 ##STR981## ##STR982## 782 40
##STR983## ##STR984## 762, 764 41 ##STR985## ##STR986## 744 42
##STR987## ##STR988## 772 43 ##STR989## ##STR990## 769 44
##STR991## ##STR992## 758 45 ##STR993## ##STR994## 786 46
##STR995## ##STR996## 783 47 ##STR997## ##STR998## 788 48
##STR999## ##STR1000## 804 49 ##STR1001## ##STR1002## 790 50
##STR1003## ##STR1004## 722 51 ##STR1005## ##STR1006## 750 52
##STR1007## ##STR1008## 730 53 ##STR1009## ##STR1010## 740 54
##STR1011## ##STR1012## 754 55 ##STR1013## ##STR1014## 732 56
##STR1015## ##STR1016## 708 57 ##STR1017## ##STR1018## 730 58
##STR1019## ##STR1020## 772 59 ##STR1021## ##STR1022## 694 60
##STR1023## ##STR1024## 723 62 ##STR1025## ##STR1026## 742 63
##STR1027## ##STR1028## 756 64 ##STR1029## ##STR1030## 728 65
##STR1031## ##STR1032## 753 66 ##STR1033## ##STR1034## 756 67
##STR1035## ##STR1036## 770 68 ##STR1037## ##STR1038## 742 69
##STR1039## ##STR1040## 767 70 ##STR1041## ##STR1042## 770 71
##STR1043## ##STR1044## 784 72 ##STR1045## ##STR1046## 756 73
##STR1047## ##STR1048## 781 74 ##STR1049## ##STR1050## 730 75
##STR1051## ##STR1052## 758 76 ##STR1053## ##STR1054## 778, 780 77
##STR1055## ##STR1056## 774 78 ##STR1057## ##STR1058## 748, 750 80
##STR1059## ##STR1060## 772 81 ##STR1061## ##STR1062## 771 82
##STR1063## ##STR1064## 783 83 ##STR1065## ##STR1066## 672 85
##STR1067## ##STR1068## 728 86 ##STR1069## ##STR1070## 727 87
##STR1071## ##STR1072## 739 88 ##STR1073## ##STR1074## 742 89
##STR1075## ##STR1076## 714 90 ##STR1077## ##STR1078## 740 91
##STR1079## ##STR1080## 804 92 ##STR1081## ##STR1082## 803 93
##STR1083## ##STR1084## 801 94 ##STR1085## ##STR1086## 816 95
##STR1087## ##STR1088## 818 96 ##STR1089## ##STR1090## 790 97
##STR1091## ##STR1092## 804 98 ##STR1093## ##STR1094## 790 99
##STR1095## ##STR1096## 816 100 ##STR1097## ##STR1098## 801 101
##STR1099## ##STR1100## 815 102 ##STR1101## ##STR1102## 803 103
##STR1103## ##STR1104## 818 104 ##STR1105## ##STR1106## 803 105
##STR1107## ##STR1108## 848
106 ##STR1109## ##STR1110## 816 107 ##STR1111## ##STR1112## 847 108
##STR1113## ##STR1114## 846 109 ##STR1115## ##STR1116## 833 110
##STR1117## ##STR1118## 859 111 ##STR1119## ##STR1120## 844 112
##STR1121## ##STR1122## 847 113 ##STR1123## ##STR1124## 833 114
##STR1125## ##STR1126## 861 115 ##STR1127## ##STR1128## 758 116
##STR1129## ##STR1130## 724 117 ##STR1131## ##STR1132## 644 118
##STR1133## ##STR1134## 684 119 ##STR1135## ##STR1136## 624 120
##STR1137## ##STR1138## 664 121 ##STR1139## ##STR1140## 772 122
##STR1141## ##STR1142## 708 123 ##STR1143## ##STR1144## 724 124
##STR1145## ##STR1146## 798, 800 125 ##STR1147## ##STR1148## 710
126 ##STR1149## ##STR1150## 695 127 ##STR1151## ##STR1152## 707 128
##STR1153## ##STR1154## 681 129 ##STR1155## ##STR1156## 751 130
##STR1157## ##STR1158## 730 131 ##STR1159## ##STR1160## 760 132
##STR1161## ##STR1162## 744 133 ##STR1163## ##STR1164## 726 134
##STR1165## ##STR1166## 756 135 ##STR1167## ##STR1168## 725 136
##STR1169## ##STR1170## 737 137 ##STR1171## ##STR1172## 802 138
##STR1173## ##STR1174## 772 139 ##STR1175## ##STR1176## 742 141
##STR1177## ##STR1178## 714 142 ##STR1179## ##STR1180## 845 145
##STR1181## ##STR1182## 882 146 ##STR1183## ##STR1184## 739 147
##STR1185## ##STR1186## 711 148 ##STR1187## ##STR1188## 755 149
##STR1189## ##STR1190## 811 150 ##STR1191## ##STR1192## 796 151
##STR1193## ##STR1194## 697 152 ##STR1195## ##STR1196## 765 153
##STR1197## ##STR1198## 775
[0524] The compounds of tables 4 and 5 could be prepared employing
the procedures described in the above examples: TABLE-US-00007
TABLE 4 ##STR1199## Cmpd. R.sub.23 R.sub.1 R.sub.3, R.sub.4 R.sub.8
R.sub.9 R.sub.7 Q 201 ##STR1200## ##STR1201## 6-OMe, H ##STR1202##
##STR1203## ##STR1204## ##STR1205## 202 ##STR1206## ##STR1207##
6-OMe, H ##STR1208## ##STR1209## ##STR1210## ##STR1211## 203
##STR1212## ##STR1213## 6-OMe, H ##STR1214## ##STR1215##
##STR1216## ##STR1217## 204 ##STR1218## ##STR1219## 6-OMe, H
##STR1220## ##STR1221## ##STR1222## ##STR1223## 205 ##STR1224##
##STR1225## H, H ##STR1226## ##STR1227## ##STR1228## ##STR1229##
206 ##STR1230## ##STR1231## H, H ##STR1232## ##STR1233##
##STR1234## ##STR1235## 207 ##STR1236## ##STR1237## 6-OMe, H
##STR1238## ##STR1239## ##STR1240## ##STR1241## 208 ##STR1242##
##STR1243## 6-OMe, H ##STR1244## ##STR1245## ##STR1246##
##STR1247## 209 ##STR1248## ##STR1249## 6-OMe, H ##STR1250##
##STR1251## ##STR1252## ##STR1253## 210 ##STR1254## ##STR1255##
6-OMe, H ##STR1256## ##STR1257## ##STR1258## ##STR1259## 211
##STR1260## ##STR1261## 6-OMe, H ##STR1262## ##STR1263##
##STR1264## ##STR1265## 212 ##STR1266## ##STR1267## H, H
##STR1268## ##STR1269## ##STR1270## ##STR1271## 213 ##STR1272##
##STR1273## 6-OMe, H ##STR1274## ##STR1275## ##STR1276##
##STR1277## 214 ##STR1278## ##STR1279## 6-OMe, H ##STR1280##
##STR1281## ##STR1282## ##STR1283## 215 ##STR1284## ##STR1285##
6-OMe, H ##STR1286## ##STR1287## ##STR1288## ##STR1289## 216
##STR1290## ##STR1291## 6-OMe, H ##STR1292## ##STR1293##
##STR1294## ##STR1295## 217 ##STR1296## ##STR1297## 6-OMe, H
##STR1298## ##STR1299## ##STR1300## ##STR1301## 218 ##STR1302##
##STR1303## H, H ##STR1304## ##STR1305## ##STR1306## ##STR1307##
219 ##STR1308## ##STR1309## 6-OMe, H ##STR1310## ##STR1311##
##STR1312## ##STR1313## 220 ##STR1314## ##STR1315## 6-OMe, H
##STR1316## ##STR1317## ##STR1318## ##STR1319## 221 ##STR1320##
##STR1321## 6-OMe, H ##STR1322## ##STR1323## ##STR1324##
##STR1325## 222 ##STR1326## ##STR1327## 6-OMe, H ##STR1328##
##STR1329## ##STR1330## ##STR1331## 223 ##STR1332## ##STR1333##
6-OMe, H ##STR1334## ##STR1335## ##STR1336## ##STR1337## 224
##STR1338## ##STR1339## 6-OMe, H ##STR1340## ##STR1341##
##STR1342## ##STR1343## 225 ##STR1344## ##STR1345## 6-OMe, H
##STR1346## ##STR1347## ##STR1348## ##STR1349## 226 ##STR1350##
##STR1351## 6-OMe, H ##STR1352## ##STR1353## ##STR1354##
##STR1355## 227 ##STR1356## ##STR1357## 6-OMe, H ##STR1358##
##STR1359## ##STR1360## ##STR1361## 228 ##STR1362## ##STR1363##
6-OMe, H ##STR1364## ##STR1365## ##STR1366## ##STR1367## 229
##STR1368## ##STR1369## H, H ##STR1370## ##STR1371## ##STR1372##
##STR1373## 230 ##STR1374## ##STR1375## 6-OMe, H ##STR1376##
##STR1377## ##STR1378## ##STR1379## 231 ##STR1380## ##STR1381##
6-OMe, H ##STR1382## ##STR1383## ##STR1384## ##STR1385## 232
##STR1386## ##STR1387## 6-OMe, H ##STR1388## ##STR1389##
##STR1390## ##STR1391## 233 ##STR1392## ##STR1393## 6-OMe, H
##STR1394## ##STR1395## ##STR1396## ##STR1397## 234 ##STR1398##
##STR1399## 6-OMe, H ##STR1400## ##STR1401## ##STR1402##
##STR1403## 235 ##STR1404## ##STR1405## H, H ##STR1406##
##STR1407## ##STR1408## ##STR1409## 236 ##STR1410## ##STR1411##
6-OMe, H ##STR1412## ##STR1413## ##STR1414## ##STR1415## 237
##STR1416## ##STR1417## 6-OMe, H ##STR1418## ##STR1419##
##STR1420## ##STR1421## 238 ##STR1422## ##STR1423## 6-OMe, H
##STR1424## ##STR1425## ##STR1426## ##STR1427## 239 ##STR1428##
##STR1429## 6-OMe, H ##STR1430## ##STR1431## ##STR1432##
##STR1433## 240 ##STR1434## ##STR1435## H, H ##STR1436##
##STR1437## ##STR1438## ##STR1439## 241 ##STR1440## ##STR1441##
6-OMe, H ##STR1442## ##STR1443## ##STR1444## ##STR1445## 242
##STR1446## ##STR1447## 6-OMe, H ##STR1448## ##STR1449##
##STR1450## ##STR1451## 243 ##STR1452## ##STR1453## 6-OMe, H
##STR1454## ##STR1455## ##STR1456## ##STR1457## 244 ##STR1458##
##STR1459## 6-OMe, H ##STR1460## ##STR1461## ##STR1462##
##STR1463## 245 ##STR1464## ##STR1465## 6-OMe, H ##STR1466##
##STR1467## ##STR1468## ##STR1469## 246 ##STR1470## ##STR1471##
6-OMe, H ##STR1472## ##STR1473## ##STR1474## ##STR1475## 247
##STR1476## ##STR1477## 6-OMe, H ##STR1478## ##STR1479##
##STR1480## ##STR1481## 248 ##STR1482## ##STR1483## 6-OMe, H
##STR1484## ##STR1485## ##STR1486## ##STR1487## 249 ##STR1488##
##STR1489## H, H ##STR1490## ##STR1491## ##STR1492## ##STR1493##
250 ##STR1494## ##STR1495## H, H ##STR1496## ##STR1497##
##STR1498## ##STR1499## 251 ##STR1500## ##STR1501## 6-OMe, H
##STR1502## ##STR1503## ##STR1504## ##STR1505## 252 ##STR1506##
##STR1507## 6-OMe, H ##STR1508## ##STR1509## ##STR1510##
##STR1511## 253 ##STR1512## ##STR1513## 6-OMe, H ##STR1514##
##STR1515## ##STR1516## ##STR1517## 254 ##STR1518## ##STR1519##
6-OMe, H ##STR1520## ##STR1521## ##STR1522## ##STR1523## 255
##STR1524## ##STR1525## 6-OMe, H ##STR1526## ##STR1527##
##STR1528## ##STR1529## 256 ##STR1530## ##STR1531## H, H
##STR1532## ##STR1533## ##STR1534## ##STR1535## 257 ##STR1536##
##STR1537## 6-OMe, H ##STR1538## ##STR1539## ##STR1540##
##STR1541## 258 ##STR1542## ##STR1543## 6-OMe, H ##STR1544##
##STR1545## ##STR1546## ##STR1547## 259 ##STR1548## ##STR1549##
6-OMe, H ##STR1550## ##STR1551## ##STR1552## ##STR1553## 260
##STR1554## ##STR1555## 6-OMe, H ##STR1556## ##STR1557##
##STR1558## ##STR1559##
[0525] TABLE-US-00008 TABLE 4 (cont'd.) ##STR1560## Cmpd R.sub.23
R.sub.1 R.sub.3, R.sub.4 R.sub.8 R.sub.9 R.sub.7 Q 261 ##STR1561##
##STR1562## 6-OMe, H ##STR1563## ##STR1564## ##STR1565##
##STR1566## 262 ##STR1567## ##STR1568## H, H ##STR1569##
##STR1570## ##STR1571## ##STR1572## 263 ##STR1573## ##STR1574##
6-OMe, H ##STR1575## ##STR1576## ##STR1577## ##STR1578## 264
##STR1579## ##STR1580## 6-OMe, H ##STR1581## ##STR1582##
##STR1583## ##STR1584## 265 ##STR1585## ##STR1586## 6-OMe, H
##STR1587## ##STR1588## ##STR1589## ##STR1590## 266 ##STR1591##
##STR1592## 6-OMe, H ##STR1593## ##STR1594## ##STR1595##
##STR1596## 267 ##STR1597## ##STR1598## H, H ##STR1599##
##STR1600## ##STR1601## ##STR1602## 268 ##STR1603## ##STR1604##
6-OMe, H ##STR1605## ##STR1606## ##STR1607## ##STR1608## 269
##STR1609## ##STR1610## 6-OMe, H ##STR1611## ##STR1612##
##STR1613## ##STR1614## 270 ##STR1615## ##STR1616## 6-OMe, H
##STR1617## ##STR1618## ##STR1619## ##STR1620## 271 ##STR1621##
##STR1622## 6-OMe, H ##STR1623## ##STR1624## ##STR1625##
##STR1626## 272 ##STR1627## ##STR1628## 6-OMe, H ##STR1629##
##STR1630## ##STR1631## ##STR1632## 273 ##STR1633## ##STR1634## H,
H ##STR1635## ##STR1636## ##STR1637## ##STR1638## 274 ##STR1639##
##STR1640## 6-OMe, H ##STR1641## ##STR1642## ##STR1643##
##STR1644## 275 ##STR1645## ##STR1646## 6-OMe, H ##STR1647##
##STR1648## ##STR1649## ##STR1650## 276 ##STR1651## ##STR1652##
6-OMe, H ##STR1653## ##STR1654## ##STR1655## ##STR1656## 277
##STR1657## ##STR1658## 6-OMe, H ##STR1659## ##STR1660##
##STR1661## ##STR1662## 278 ##STR1663## ##STR1664## 6-OMe, H
##STR1665## ##STR1666## ##STR1667## ##STR1668## 279 ##STR1669##
##STR1670## H, H ##STR1671## ##STR1672## ##STR1673## ##STR1674##
280 ##STR1675## ##STR1676## 6-OMe, H ##STR1677## ##STR1678##
##STR1679## ##STR1680## 281 ##STR1681## ##STR1682## 6-OMe, H
##STR1683## ##STR1684## ##STR1685## ##STR1686## 282 ##STR1687##
##STR1688## 6-OMe, H ##STR1689## ##STR1690## ##STR1691##
##STR1692## 283 ##STR1693## ##STR1694## 6-OMe, H ##STR1695##
##STR1696## ##STR1697## ##STR1698## 284 ##STR1699## ##STR1700##
6-OMe, H ##STR1701## ##STR1702## ##STR1703## ##STR1704## 285
##STR1705## ##STR1706## H, H ##STR1707## ##STR1708## ##STR1709##
##STR1710## 286 ##STR1711## ##STR1712## 6-OMe, H ##STR1713##
##STR1714## ##STR1715## ##STR1716## 287 ##STR1717## ##STR1718##
6-OMe, H ##STR1719## ##STR1720## ##STR1721## ##STR1722## 288
##STR1723## ##STR1724## 6-OMe, H ##STR1725## ##STR1726##
##STR1727## ##STR1728## 289 ##STR1729## ##STR1730## 6-OMe, H
##STR1731## ##STR1732## ##STR1733## ##STR1734## 290 ##STR1735##
##STR1736## 6-OMe, H ##STR1737## ##STR1738## ##STR1739##
##STR1740## 291 ##STR1741## ##STR1742## H, H ##STR1743##
##STR1744## ##STR1745## ##STR1746## 292 ##STR1747## ##STR1748##
6-OMe, H ##STR1749## ##STR1750## ##STR1751## ##STR1752## 293
##STR1753## ##STR1754## 6-OMe, H ##STR1755## ##STR1756##
##STR1757## ##STR1758## 294 ##STR1759## ##STR1760## 6-OMe, H
##STR1761## ##STR1762## ##STR1763## ##STR1764## 295 ##STR1765##
##STR1766## 6-OMe, H ##STR1767## ##STR1768## ##STR1769##
##STR1770## 296 ##STR1771## ##STR1772## 6-OMe, H ##STR1773##
##STR1774## ##STR1775## ##STR1776## 297 ##STR1777## ##STR1778##
6-OMe, H ##STR1779## ##STR1780## ##STR1781## ##STR1782## 298
##STR1783## ##STR1784## 6-OMe, H ##STR1785## ##STR1786##
##STR1787## ##STR1788## 299 ##STR1789## ##STR1790## 6-OMe, H
##STR1791## ##STR1792## ##STR1793## ##STR1794## 300 ##STR1795##
##STR1796## 6-OMe, H ##STR1797## ##STR1798## ##STR1799##
##STR1800## 301 ##STR1801## ##STR1802## 6-OMe, H ##STR1803##
##STR1804## ##STR1805## ##STR1806## 302 ##STR1807## ##STR1808##
6-OMe, H ##STR1809## ##STR1810## ##STR1811## ##STR1812## 303
##STR1813## ##STR1814## 6-OMe, H ##STR1815## ##STR1816##
##STR1817## ##STR1818## 304 ##STR1819## ##STR1820## 6-OMe, H
##STR1821## ##STR1822## ##STR1823## ##STR1824## 305 ##STR1825##
##STR1826## 6-OMe, H ##STR1827## ##STR1828## ##STR1829##
##STR1830## 306 ##STR1831## ##STR1832## 6-OMe, H ##STR1833##
##STR1834## ##STR1835## ##STR1836## 307 ##STR1837## ##STR1838##
6-OMe, H ##STR1839## ##STR1840## ##STR1841## ##STR1842## 308
##STR1843## ##STR1844## 6-OMe, H ##STR1845## ##STR1846##
##STR1847## ##STR1848## 309 ##STR1849## ##STR1850## 6-OMe, H
##STR1851## ##STR1852## ##STR1853## ##STR1854## 310 ##STR1855##
##STR1856## 6-OMe, H ##STR1857## ##STR1858## ##STR1859##
##STR1860## 311 ##STR1861## ##STR1862## 6-OMe, H ##STR1863##
##STR1864## ##STR1865## ##STR1866## 312 ##STR1867## ##STR1868##
6-OMe, H ##STR1869## ##STR1870## ##STR1871## ##STR1872## 313
##STR1873## ##STR1874## 6-OMe, H ##STR1875## ##STR1876##
##STR1877## ##STR1878## 314 ##STR1879## ##STR1880## 6-OMe, H
##STR1881## ##STR1882## ##STR1883## ##STR1884## 315 ##STR1885##
##STR1886## 6-OMe, H ##STR1887## ##STR1888## ##STR1889##
##STR1890## 316 ##STR1891## ##STR1892## 6-OMe, H ##STR1893##
##STR1894## ##STR1895## ##STR1896## 317 ##STR1897## ##STR1898##
6-OMe, H ##STR1899## ##STR1900## ##STR1901## ##STR1902## 318
##STR1903## ##STR1904## 6-OMe, H ##STR1905## ##STR1906##
##STR1907## ##STR1908## 319 ##STR1909## ##STR1910## 6-OMe, H
##STR1911## ##STR1912## ##STR1913## ##STR1914## 320 ##STR1915##
##STR1916## 6-OMe, H ##STR1917## ##STR1918## ##STR1919##
##STR1920## 321 ##STR1921## ##STR1922## 6-OMe, H ##STR1923##
##STR1924## ##STR1925## ##STR1926## 322 ##STR1927## ##STR1928##
6-OMe, H ##STR1929## ##STR1930## ##STR1931## ##STR1932## 323
##STR1933## ##STR1934## 6-OMe, H ##STR1935## ##STR1936##
##STR1937## ##STR1938## 324 ##STR1939## ##STR1940## 6-OMe, H
##STR1941## ##STR1942## ##STR1943## ##STR1944## 325 ##STR1945##
##STR1946## 6-OMe, H ##STR1947## ##STR1948## ##STR1949##
##STR1950## 326 ##STR1951## ##STR1952## 6-OMe, H ##STR1953##
##STR1954## ##STR1955## ##STR1956## 327 ##STR1957## ##STR1958##
6-OMe, H ##STR1959## ##STR1960## ##STR1961## ##STR1962## 328
##STR1963## ##STR1964## 6-OMe, H ##STR1965## ##STR1966##
##STR1967## ##STR1968## 329 ##STR1969## ##STR1970## 6-OMe, H
##STR1971## ##STR1972## ##STR1973## ##STR1974## 330 ##STR1975##
##STR1976## 6-OMe, H ##STR1977## ##STR1978## ##STR1979##
##STR1980## 331 ##STR1981## ##STR1982## 6-OMe, H ##STR1983##
##STR1984## ##STR1985## ##STR1986##
[0526] TABLE-US-00009 TABLE 5 ##STR1987## Cmpd R.sub.1 R.sub.2
R.sub.3 401 H H H 402 H H H 403 H H Me 404 H H --CF.sub.3 405 H H
--CHF.sub.2 406 H H H 407 H H H 408 H H --OCH.sub.3 409 H H H 410 H
--OCH.sub.3 H 411 H Me H 412 Cl --OCH.sub.3 H 413 H H H 414 H H H
415 H H H 416 H H H 417 H H F 418 H H --SCH.sub.3 419 H H H 420 H H
##STR1988## 421 H H H 422 H H ##STR1989## 423 ##STR1990## H H 424
##STR1991## H H 425 ##STR1992## H H 426 ##STR1993## H H 427
##STR1994## H H 428 ##STR1995## H H 429 ##STR1996## H H 430
##STR1997## H H 431 ##STR1998## H H 432 ##STR1999## --OCH.sub.3 H
433 ##STR2000## --OCH.sub.3 H 434 ##STR2001## --OCH.sub.3 H 435
##STR2002## H H 436 ##STR2003## H H 437 ##STR2004## H H 438
##STR2005## H --OCH.sub.3 439 ##STR2006## H --OCH.sub.3 440
##STR2007## H H 441 H ##STR2008## H 442 ##STR2009## H --OCH.sub.3
443 ##STR2010## H H 444 ##STR2011## H H 445 ##STR2012## --OH H 446
##STR2013## --OH H 447 ##STR2014## --OH H 448 ##STR2015## --OH H
449 ##STR2016## H H 450 ##STR2017## H H 451 ##STR2018## H H 452
##STR2019## H H 453 ##STR2020## H H 454 ##STR2021## H H 455
##STR2022## H --OCH.sub.3 456 ##STR2023## H H Cmpd R.sub.4 R.sub.5
R.sub.6 Q 401 H Me H ##STR2024## 402 H H Me ##STR2025## 403 H H H
##STR2026## 404 H H H ##STR2027## 405 H H H ##STR2028## 406
--OCF.sub.3 H H ##STR2029## 407 --OH H H ##STR2030## 408
--OCH.sub.3 H H ##STR2031## 409 H H --OCH.sub.3 ##STR2032## 410 H H
H ##STR2033## 411 H H H ##STR2034## 412 H H H ##STR2035## 413 Et H
H ##STR2036## 414 --OEt H H ##STR2037## 415 Br H H ##STR2038## 416
--OCH.sub.3 F H ##STR2039## 417 --OCH.sub.3 H H ##STR2040## 418
--OCH.sub.3 H H ##STR2041## 419 ##STR2042## H H ##STR2043## 420
--OCH.sub.3 H H ##STR2044## 421 ##STR2045## H H ##STR2046## 422 H H
H ##STR2047## 423 --OCH.sub.3 H H ##STR2048## 424 --OCH.sub.3 H H
##STR2049## 425 --OCH.sub.3 H H ##STR2050## 426 --OCH.sub.3 H H
##STR2051## 427 --OCH.sub.3 H H ##STR2052## 428 --OCH.sub.3 H H
##STR2053## 429 --OCH.sub.3 H H ##STR2054## 430 --OCH.sub.3 H H
##STR2055## 431 --OCH.sub.3 H H ##STR2056## 432 H H H ##STR2057##
433 H H H ##STR2058## 434 H H H ##STR2059## 435 H H H ##STR2060##
436 H H H ##STR2061## 437 --OCH.sub.3 H H ##STR2062## 438 H H H
##STR2063## 439 H H H ##STR2064## 440 --OCH.sub.3 H H ##STR2065##
441 H H H ##STR2066## 442 H H H ##STR2067## 443 --OCH.sub.3 H H
##STR2068## 444 --OCH.sub.3 H H ##STR2069## 445 H H H ##STR2070##
446 H H H ##STR2071## 447 H H H ##STR2072## 448 H H H ##STR2073##
449 H H H ##STR2074## 450 --OCH.sub.3 H H ##STR2075## 451
--OCH.sub.3 H H ##STR2076## 452 --OCH.sub.3 H H ##STR2077## 453
--OCH.sub.3 H H ##STR2078## 454 --OCH.sub.3 H H ##STR2079## 455 H H
H ##STR2080## 456 --OCH.sub.3 H H ##STR2081##
Example 50
Biological Studies
[0527] Recombinant HCV NS3/4A Protease Complex FRET Peptide
Assay
[0528] The purpose of this in vitro assay was to measure the
inhibition of HCV NS3 protease complexes, derived from the BMS
strain, H.sub.77C strain or J416S strain, as described below, by
compounds of the present invention. This assay provides an
indication of how effective compounds of the present invention
would be in inhibiting HCV proteolytic activity.
[0529] Serum from an HCV-infected patient was obtained from Dr. T.
Wright, San Francisco Hospital. An engineered full-length cDNA
(compliment deoxyribonucleic acid) template of the HCV genome (BMS
strain) was constructed from DNA fragments obtained by reverse
transcription-PCR (RT-PCR) of serum RNA (ribonucleic acid) and
using primers selected on the basis of homology between other
genotype 1a strains. From the determination of the entire genome
sequence, a genotype 1a was assigned to the HCV isolate according
to the classification of Simmonds et al. (See P Simmonds, K A Rose,
S Graham, S W Chan, F McOmish, B C Dow, E A Follett, P L Yap and H
Marsden, J. Clin. Microbiol., 31(6), 1493-1503 (1993)). The amino
acid sequence of the nonstructural region, NS2-5B, was shown to be
>97% identical to HCV genotype 1a (H77C) and 87% identical to
genotype 1b (J4L6S). The infectious clones, H77C (1a genotype) and
J4L6S (1b genotype) were obtained from R. Purcell (NIH) and the
sequences are published in Genbank (AAB67036, see Yanagi, M.,
Purcell, R. H., Emerson, S. U. and Bukh, J. Proc. Natl. Acad. Sci.
U.S.A. 94(16), 8738-8743 (1997); AF054247, see Yanagi, M., St
Claire, M., Shapiro, M., Emerson, S. U., Purcell, R. H. and Bukh,
J, Virology 244 (1), 161-172. (1998)).
[0530] The H77C and J4L6S strains were used for production of
recombinant NS3/4A protease complexes. DNA encoding the recombinant
HCV NS3/4A protease complex (amino acids 1027 to 1711) for these
strains were manipulated as described by P. Gallinari et al. (see
Gallinari P, Paolini C, Brennan D, Nardi C, Steinkuhler C, De
Francesco R. Biochemistry. 38(17):5620-32, (1999)). Briefly, a
three-lysine solubilizing tail was added at the 3'-end of the NS4A
coding region. The cysteine in the P1 position of the NS4A-NS4B
cleavage site (amino acid 1711) was changed to a glycine to avoid
the proteolytic cleavage of the lysine tag. Furthermore, a cysteine
to serine mutation was introduced by PCR at amino acid position
1454 to prevent the autolytic cleavage in the NS3 helicase domain.
The variant DNA fragment was cloned in the pET21b bacterial
expression vector (Novagen) and the NS3/4A complex was expressed in
Escherichia coli strain BL21 (DE3) (Invitrogen) following the
protocol described by P. Gallinari et al. (see Gallinari P, Brennan
D, Nardi C, Brunetti M, Tomei L, Steinkuhler C, De Francesco R., J.
Virol. 72(8):6758-69 (1998)) with modifications. Briefly, NS3/4A
expression was induced with 0.5 mM Isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG) for 22 hr at 20.degree. C.
A typical fermentation (10 L) yielded approximately 80 g of wet
cell paste. The cells were resuspended in lysis buffer (10 mL/g)
consisting of 25 mM N-(2-Hydroxyethyl)piperazine-N'-(2-Ethane
Sulfonic acid) (HEPES), pH7.5, 20% glycerol, 500 mM Sodium Chloride
(NaCl), 0.5% Triton-X100, 1 microgram/milliliter (".mu.g/mL")
lysozyme, 5 mM Magnesium Chloride (MgCl.sub.2), 1 ug/ml DnaseI, 5
mM .beta.-Mercaptoethanol (.beta.ME), Protease
inhibitor-Ethylenediamine Tetraacetic acid (EDTA) free (Roche),
homogenized and incubated for 20 mins at 4.degree. C. The
homogenate was sonicated and clarified by ultra-centrifugation at
235000 g for 1 hr at 4.degree. C. Imidazole was added to the
supernatant to a final concentration of 15 mM and the pH adjusted
to 8.0. The crude protein extract was loaded on a
Nickel-Nitrilotriacetic acid (Ni-NTA) column pre-equilibrated with
buffer B (25 mM HEPES, pH8.0, 20% glycerol, 500 mM NaCl, 0.5%
Triton-X100, 15 mM imidazole, 5 mM .beta.ME). The sample was loaded
at a flow rate of 1 mL/min. The column was washed with 15 column
volumes of buffer C (same as buffer B except with 0.2%
Triton-X100). The protein was eluted with 5 column volumes of
buffer D (same as buffer C except with 200 mM Imidazole).
[0531] NS3/4A protease complex-containing fractions were pooled and
loaded on a desalting column Superdex-S200 pre-equilibrated with
buffer D (25 mM HEPES, pH7.5, 20% glycerol, 300 mM NaCl, 0.2%
Triton-X100, 10 mM PME). Sample was loaded at a flow rate of 1
mL/min. NS3/4A protease complex-containing fractions were pooled
and concentrated to approximately 0.5 mg/ml. The purity of the
NS3/4A protease complexes, derived from the BMS, H77C and J4L6S
strains, were judged to be greater than 90% by SDS-PAGE and mass
spectrometry analyses.
[0532] The enzyme was stored at -80.degree. C., thawed on ice and
diluted prior to use in assay buffer. The substrate used for the
NS3/4A protease assay was RET S I (Resonance Energy Transfer
Depsipeptide Substrate; AnaSpec, Inc. cat # 22991) (FRET peptide),
described by Taliani et al. in Anal. Biochem. 240(2):60-67 (1996).
The sequence of this peptide is loosely based on the NS4A/NS4B
natural cleavage site except there is an ester linkage rather than
an amide bond at the cleavage site. The peptide substrate was
incubated with one of the three recombinant NS3/4A complexes, in
the absence or presence of a compound of the present invention, and
the formation of fluorescent reaction product was followed in real
time using a Cytofluor Series 4000.
[0533] The reagents were as follow: HEPES and Glycerol (Ultrapure)
were obtained from GIBCO-BRL. Dimethyl Sulfoxide (DMSO) was
obtained from Sigma. .beta.-Mercaptoethanol was obtained from Bio
Rad.
[0534] Assay buffer: 50 mM HEPES, pH7.5; 0.15M NaCl; 0.1% Triton;
15% Glycerol; 10 mM 13ME. Substrate: 2 .mu.M final concentration
(from a 2 mM stock solution in DMSO stored at -20.degree. C.). HCV
NS3/4A type 1a (1b), 2-3 nM final concentration (from a 5 .mu.M
stock solution in 25 mM HEPES, pH7.5, 20% glycerol, 300 mM NaCl,
0.2% Triton-X100, 10 mM .beta.ME). For compounds with potencies
approaching the assay limit, the assay was made more sensitive by
adding 50 .mu.g/ml Bovine Serum Albumin (Sigma) to the assay buffer
and reducing the end protease concentration to 300 pM.
[0535] The assay was performed in a 96-well polystyrene black plate
from Falcon. Each well contained 25 microliter (".mu.l") NS3/4A
protease complex in assay buffer, 50 .mu.l of a compound of the
present invention in 10% DMSO/assay buffer and 25 .mu.l substrate
in assay buffer. A control (no compound) was also prepared on the
same assay plate. The enzyme complex was mixed with compound or
control solution for 1 min before initiating the enzymatic reaction
by the addition of substrate. The assay plate was read immediately
using the Cytofluor Series 4000 (Perspective Biosystems). The
instrument was set to read an emission of 340 nm and excitation of
490 nm at 25.degree. C. Reactions were generally followed for
approximately 15 minutes.
[0536] The percent inhibition was calculated with the following
equation: 100-[(.delta.F.sub.inh/.delta.F.sub.con).times.100] where
.delta.F is the change in fluorescence over the linear range of the
curve. A non-linear curve fit was applied to the
inhibition-concentration data, and the 50% effective concentration
(IC.sub.50) was calculated by the use of Excel Xl-fit software
using the equation, y=A+((B-A)/(1+((C/x) D))).
[0537] All of the compounds tested were found to have IC50s of 1.2
.mu.M or less. Further, compounds of the present invention, which
were tested against more than one type of NS3/4A complex, were
found to have similar inhibitory properties though the compounds
uniformly demonstrated greater potency against the 1b strains as
compared to the 1a strains.
Specificity Assays
[0538] The specificity assays were performed to demonstrate the
selectivity of the compounds of the present invention in inhibiting
HCV NS3/4A protease as compared to other serine or cysteine
proteases.
[0539] The specificities of compounds of the present invention were
determined against a variety of serine proteases: human neutrophil
elastase (HNE), porcine pancreatic elastase (PPE) and human
pancreatic chymotrypsin and one cysteine protease: human liver
cathepsin B. In all cases a 96-well plate format protocol using
colorimetric p-nitroaniline (pNA) substrate specific for each
enzyme was used as described previously (PCT Patent Application No.
WO 00/09543) with some modifications to the serine protease assays.
All enzymes were purchased from Sigma while the substrates were
from Bachem.
[0540] Each assay included a 2 hr enzyme-inhibitor pre-incubation
at room temperature followed by addition of substrate and
hydrolysis to .about.30% conversion as measured on a Spectramax Pro
microplate reader. Compound concentrations varied from 100 to 0.4
micromolar (".mu.M") depending on their potency.
[0541] The final conditions for each assay were as follows: [0542]
50 millimolar ("mM") Tris(hydroxymethyl)aminomethane hydrochloride
(Tris-HCl) pH8, 0.5M Sodium Sulfate (Na.sub.2SO.sub.4), 50 mM NaCl,
0.1 mM EDTA, 3% DMSO, 0.01% Tween-20 with: [0543] 133 .mu.M
succ-AAA-pNA and 20 nM HNE or 8 nM PPE; 100 .mu.M succ-AAPF-pNA and
250 pM Chymotrypsin. [0544] 100 mM NaHPO.sub.4 (Sodium Hydrogen
Phosphate) pH 6, 0.1 mM EDTA, 3% DMSO, 1 mM TCEP
(Tris(2-carboxyethyl)phosphine hydrochloride), 0.01% Tween-20, 30
.mu.M Z-FR-pNA and 5 nM Cathepsin B (enzyme stock activated in
buffer containing 20 mM TCEP before use).
[0545] The percentage of inhibition was calculated using the
formula:
[1-((UV.sub.inh-UV.sub.blank)/(UV.sub.ctl-UV.sub.blank))].times.100
[0546] A non-linear curve fit was applied to the
inhibition-concentration data, and the 50% effective concentration
(IC.sub.50) was calculated by the use of Excel Xl-fit software.
[0547] HCV Replion assays were utilized in the present invention,
and were prepared, conducted and validated as follows:
Generation of HCV Replicon
[0548] An HCV replicon whole cell system was established as
described by Lohmann V, Korner F, Koch J, Herian U, Theilmann L,
Bartenschlager R., Science 285(5424):110-3 (1999). This system
enabled us to evaluate the effects of our HCV Protease compounds on
HCV RNA replication. Briefly, using the HCV strain 1B sequence
described in the Lohmann paper (Assession number: AJ238799), an HCV
cDNA was synthesized by Operon Technologies, Inc. (Alameda,
Calif.), and the full-length replicon was then assembled in plasmid
pGem9zf(+) (Promega, Madison, Wis.) using standard molecular
biology techniques. The replicon consists of (i) the HCV 5' UTR
fused to the first 12 amino acids of the capsid protein, (ii) the
neomycin phosphotransferase gene (neo), (iii) the IRES from
encephalomyocarditis virus (EMCV), and (iv) HCV NS3 to NS5B genes
and the HCV 3' UTR. Plasmid DNAs were linearized with ScaI and RNA
transcripts were synthesized in vitro using the T7 MegaScript
transcription kit (Ambion, Austin, Tex.) according to
manufacturer's directions. To generate cell lines, 4.times.10.sup.6
Huh-7 cells (kindly provided by R. Bartenschlager and available
from Health Science Research Resources Bank, Japan Health Sciences
Foundation) were electroporated (GenePulser System, Bio-Rad) with
10 microgram (".mu.g") of RNA transcript and plated into 100-mm
dishes. After 24 h, selective media containing 1.0
milligrams/milliliter ("mg/ml") G418 was added and media was
changed every 3 to 5 days. Approximately 4 weeks after
electroporation, small colonies were visible which were isolated
and expanded for further analysis. These cell lines were maintained
at 37.degree. C., 5% CO.sub.2, 100% relative humidity in DMEM (Cat#
11965-084) Gibco-BRL, Rockville, Md., with 10% heat inactivated
calf serum (Sigma), 10 ml of 100.times. penicillin/streptomycin
(Cat# 15140-122) Gibco-BRL, Rockville, Md., Geneticin (Cat#
10131-027) Gibco-BRL, Rockville, Md. at 1 mg/ml. One of the cell
lines (deposited as ATCC Accession No. PTA-4583 in the American
Type Culture Collection) which had approximately 3,000 copies of
HCV replicon RNA/cell was used for development of the assay (HCV
1b-377-neo replicon cells).
FRET Assay
[0549] Huh7 cells, constitutively expressing the HCV replicon, were
grown in Dulbecco's Modified Eagle Media (DMEM) containing 10%
Fetal calf serum (FCS) and 1 mg/ml G418 (Gibco-BRL). Cells were
seeded the night before (1.5.times.10.sup.4 cells/well) in 96-well
tissue-culture sterile plates. Compound and no compound controls
were prepared in DMEM containing 4% FCS, 1:100
Penicillin/Streptomysin, 1:100 L-glutamine and 5% DMSO in the
dilution plate (0.5% DMSO final concentration in the assay).
Compound/DMSO mixes were added to the cells and incubated for 4
days at 37.degree. C. After 4 days, cells were first assessed for
cytotoxicity using alamar Blue (Trek Diagnotstic Systems) for a
CC.sub.50 reading. The toxicity of compound (CC.sub.50) was
determined by adding 1/10.sup.th volume of alamar Blue to the media
incubating the cells. After 4 hr, the fluorescence signal from each
well was read, with an excitation wavelength at 530 nm and an
emission wavelength of 580 nm, using the Cytofluor Series 4000
(Perspective Biosystems). Plates were then rinsed thoroughly with
Phosphate-Buffered Saline (PBS) (3 times 150 .mu.l). The cells were
lysed with 25 .mu.l of a lysis assay reagent containing an HCV
protease substrate (5.times. cell Luciferase cell culture lysis
reagent (Promega #E153A) diluted to 1.times. with distilled water,
NaCl added to 150 mM final, the FRET peptide diluted to 10 .mu.M
final from a 2 mM stock in 100% DMSO). The HCV protease substrate
(FRET peptide; AnaSpec, Inc. cat # 22991, described by Taliani et
al. in Anal. Biochem. 240(2):60-67 (1996)) contains a fluorescence
donor, EDANS, near one end of the peptide and an acceptor, DABCYL,
near the other end. The fluorescence of the peptide is quenched by
intermolecular resonance energy transfer (RET) between the donor
and the acceptor, but as the NS3 protease cleaves the peptide the
products are released from RET quenching and the fluorescence of
the donor becomes apparent. The plate was then placed into the
Cytofluor 4000 instrument which had been set to 340 nm
excitation/490 nm emission, automatic mode for 21 cycles and the
plate read in a kinetic mode at 25.degree. C. Reactions were
generally followed for approximately 15 minutes.
[0550] The percent inhibition for both efficacy and cytotoxicity
were calculated with the following equation:
100-[(.delta.F.sub.inh/.delta.F.sub.con).times.100]
[0551] where .delta.F is the change in fluorescence over the linear
range of the curve. A non-linear curve fit was applied to the
inhibition-concentration data, and the 50% effective concentration
(EC.sub.50 and CC.sub.50) was calculated by the use of Excel Xl-fit
software using the equation, y=A+((B-A)/(1+((C/x) D))).
Luciferase Assay
[0552] As a secondary assay, EC.sub.50 determinations from the
replicon FRET assay were confirmed in a luciferase reporter assay.
Utilization of a replicon luciferase reporter assay was first
described by Krieger et al (Krieger N, Lohmann V, and
Bartenschlager R, J. Virol. 75(10):4614-4624 (2001)). The replicon
construct described for our FRET assay was modified by replacing
the resistance gene neomycin with a cassette containing Renilla
Luciferase gene fused to a sequence representing an NS3 cleavage
site (NS4A/B site), followed by the Blasicidin resistance gene
(restriction sites Asc1/Pme1 used for the subcloning). The adaptive
mutation at position 1179 (serine to isoleucine) was also
introduced (Blight K J, Kolykhalov, A A, Rice, C M, Science
290(5498):1972-1974). A cell line containing the replicon was
selected and maintained in 1.25 .mu.g/ml Blasticidin. The
luciferase reporter assay was set up by seeding the replicon cells
the night before at a density of 7000 cells/well in 96-well plates.
One day later, media was changed to fresh DMEM containing 4% FBS
and compound dilutions were prepared and added to a final
concentration of 0.5% DMSO (final media volume of 150 .mu.l).
Following an additional 4-day incubation in a 37.degree. C./5%
CO.sub.2 incubator, cells were analyzed for Renilla luciferase
activity using the Promega Dual-Glo Luciferase Assay System. Media
(100 .mu.l) was removed from each well. To the remaining 50 .mu.l
of media, 50 .mu.l of Dual-Glo Luciferase Reagent was added, and
plates rocked for 10 minutes to 2 hours at room temperature.
Dual-Glo Stop & Glo Reagent (50 .mu.l) was then added to each
well, and plates were rocked again for an additional 10 minutes to
2 hours at room temperature. Plates were read on a Packard TopCount
NXT using a luminescence program.
[0553] The percentage inhibition was calculated using the formula
below: % .times. .times. control = average .times. .times.
luciferase .times. .times. signal .times. .times. in .times.
experimental .times. .times. wells .times. .times. ( + compound )
average .times. .times. luciferase .times. .times. signal .times.
.times. in DMSO .times. .times. control .times. .times. wells
.times. .times. ( - compound ) ##EQU1##
[0554] The values were graphed and analyzed using XLFit to obtain
the EC.sub.50 value.
[0555] Representative compounds of the invention were assessed in
the HCV NS3/4A protease recombinant enzyme assay, the HCV replicon
cell-based assay and/or in several of the outlined specificity
assays. For example, Compound 2 was found to have an IC.sub.50 of
26 nM against the BMS strain NS3/4A protease in the enzyme assay.
Similar potency values were obtained with the published H77C
(IC.sub.50 of 4.2 nM) and J4L6S (IC.sub.50 of 1.9 nM) strains. The
EC.sub.50 value in the replicon assay was 146 nM.
[0556] In the specificity assays, the same compound was found to
have the following activity: HNE>100 .mu.M; PPE>100 .mu.M;
Chymotrypsin>100 .mu.M; Cathepsin B>100 .mu.M. These results
indicate this family of compounds are highly specific for the NS3
protease and many of these members inhibit HCV replicon
replication.
[0557] The compounds tested were found to have activities in the
ranges as follow: IC50 Activity Ranges (NS3/4A BMS Strain): A is
<50 .mu.M; B is <5 .mu.M; C is <0.5 .mu.M;
D is <0.05 .mu.M
EC50 Activity Range (for compounds tested): A is <50 .mu.M; B is
<5 .mu.M; C is <0.5 .mu.M;
D is <0.05 .mu.M
Tables 6 & 7: Compound Activity Tables
[0558] TABLE-US-00010 TABLE 6 ##STR2082## IC50 EC50 Cmpd R.sub.1
R.sub.3 R.sub.7 R.sub.8 R.sub.9 Q (nM) (nM) 1 ##STR2083##
##STR2084## ##STR2085## ##STR2086## ##STR2087## ##STR2088## C B 17A
##STR2089## ##STR2090## ##STR2091## ##STR2092## ##STR2093##
##STR2094## D C 18A ##STR2095## ##STR2096## ##STR2097## ##STR2098##
##STR2099## ##STR2100## C B 27 ##STR2101## ##STR2102## ##STR2103##
##STR2104## ##STR2105## ##STR2106## C B 61 ##STR2107## ##STR2108##
##STR2109## ##STR2110## ##STR2111## ##STR2112## B A 79 ##STR2113##
##STR2114## ##STR2115## ##STR2116## ##STR2117## ##STR2118## A A 84
##STR2119## ##STR2120## ##STR2121## ##STR2122## ##STR2123##
##STR2124## C B 140 ##STR2125## ##STR2126## ##STR2127## ##STR2128##
##STR2129## ##STR2130## A NT 143 ##STR2131## ##STR2132##
##STR2133## ##STR2134## ##STR2135## ##STR2136## C B 144 ##STR2137##
##STR2138## ##STR2139## ##STR2140## ##STR2141## ##STR2142## B A
[0559] TABLE-US-00011 TABLE 7 ##STR2143## Cmpd R.sub.23 R.sub.1
R.sub.3, R.sub.4 R.sub.8 R.sub.9 2 ##STR2144## ##STR2145## H, H
##STR2146## ##STR2147## 3 ##STR2148## ##STR2149## H, H ##STR2150##
##STR2151## 4 ##STR2152## ##STR2153## H, H ##STR2154## ##STR2155##
5 ##STR2156## ##STR2157## H, H ##STR2158## ##STR2159## 6
##STR2160## ##STR2161## H, H ##STR2162## ##STR2163## 7 ##STR2164##
##STR2165## H, H ##STR2166## ##STR2167## 8 ##STR2168## ##STR2169##
H, H ##STR2170## ##STR2171## 9 ##STR2172## ##STR2173## H, H
##STR2174## ##STR2175## 10 ##STR2176## ##STR2177## H, H ##STR2178##
##STR2179## 11 ##STR2180## ##STR2181## H, H ##STR2182## ##STR2183##
12 ##STR2184## ##STR2185## H, H ##STR2186## ##STR2187## 13
##STR2188## ##STR2189## H, H ##STR2190## ##STR2191## 14 ##STR2192##
##STR2193## H, H ##STR2194## ##STR2195## 15 ##STR2196## ##STR2197##
H, H ##STR2198## ##STR2199## 16 ##STR2200## ##STR2201## H, H
##STR2202## ##STR2203## 17B ##STR2204## ##STR2205## 6-OMe, H
##STR2206## ##STR2207## 18B ##STR2208## ##STR2209## 6-OMe, H
##STR2210## ##STR2211## 19 ##STR2212## ##STR2213## 6-OMe, H
##STR2214## ##STR2215## 20 ##STR2216## ##STR2217## H, H ##STR2218##
##STR2219## 21 ##STR2220## ##STR2221## H, H ##STR2222## ##STR2223##
22 ##STR2224## ##STR2225## H, H ##STR2226## ##STR2227## 23
##STR2228## ##STR2229## H, H ##STR2230## ##STR2231## 24 ##STR2232##
##STR2233## H, H ##STR2234## ##STR2235## 25 ##STR2236## ##STR2237##
H, H ##STR2238## ##STR2239## 26 ##STR2240## ##STR2241## H, H
##STR2242## ##STR2243## 28 ##STR2244## ##STR2245## 6-OMe, H
##STR2246## ##STR2247## 29 ##STR2248## ##STR2249## 6-OMe, H
##STR2250## ##STR2251## 30 ##STR2252## ##STR2253## 6-OMe, H
##STR2254## ##STR2255## 31 ##STR2256## ##STR2257## 6-OMe, H
##STR2258## ##STR2259## 32 ##STR2260## ##STR2261## 6-OMe, H
##STR2262## ##STR2263## 33 ##STR2264## ##STR2265## 6-OMe, H
##STR2266## ##STR2267## 34 ##STR2268## ##STR2269## 6-OMe, H
##STR2270## ##STR2271## 35 ##STR2272## ##STR2273## 6-OMe, H
##STR2274## ##STR2275## 36 ##STR2276## ##STR2277## 6-OMe, H
##STR2278## ##STR2279## 37 ##STR2280## ##STR2281## 6-OMe, H
##STR2282## ##STR2283## 38 ##STR2284## ##STR2285## 6-OMe, H
##STR2286## ##STR2287## 39 ##STR2288## ##STR2289## 6-OMe, H
##STR2290## ##STR2291## 40 ##STR2292## ##STR2293## 6-OMe, H
##STR2294## ##STR2295## 41 ##STR2296## ##STR2297## 6-OMe, H
##STR2298## ##STR2299## 42 ##STR2300## ##STR2301## 6-OMe, H
##STR2302## ##STR2303## 43 ##STR2304## ##STR2305## 6-OMe, H
##STR2306## ##STR2307## 44 ##STR2308## ##STR2309## 6-OMe, H
##STR2310## ##STR2311## 45 ##STR2312## ##STR2313## 6-OMe, H
##STR2314## ##STR2315## 46 ##STR2316## ##STR2317## 6-OMe, H
##STR2318## ##STR2319## 47 ##STR2320## ##STR2321## 6-OMe, H
##STR2322## ##STR2323## 48 ##STR2324## ##STR2325## 6-OMe, H
##STR2326## ##STR2327## 49 ##STR2328## ##STR2329## 6-OMe, H
##STR2330## ##STR2331## 50 ##STR2332## ##STR2333## H, H ##STR2334##
##STR2335## 51 ##STR2336## ##STR2337## H, H ##STR2338## ##STR2339##
52 ##STR2340## ##STR2341## H, H ##STR2342## ##STR2343## 53
##STR2344## ##STR2345## 6-OMe, H ##STR2346## ##STR2347## 54
##STR2348## ##STR2349## 6-OMe, H ##STR2350## ##STR2351## 55
##STR2352## ##STR2353## H, H ##STR2354## ##STR2355## 56 ##STR2356##
##STR2357## H, H ##STR2358## ##STR2359## 57 ##STR2360## ##STR2361##
H, H ##STR2362## ##STR2363## 58 ##STR2364## ##STR2365## H, H
##STR2366## ##STR2367## 59 ##STR2368## ##STR2369## H, H ##STR2370##
##STR2371## 60 ##STR2372## ##STR2373## 6-OMe, H ##STR2374##
##STR2375## 62 ##STR2376## ##STR2377## 6-OMe, H ##STR2378##
##STR2379## 63 ##STR2380## ##STR2381## 6-OMe, H ##STR2382##
##STR2383## 64 ##STR2384## ##STR2385## 6-OMe, H ##STR2386##
##STR2387## 65 ##STR2388## ##STR2389## 6-OMe, H ##STR2390##
##STR2391## 66 ##STR2392## ##STR2393## 6-OMe, H ##STR2394##
##STR2395## 67 ##STR2396## ##STR2397## 6-OMe, H ##STR2398##
##STR2399## 68 ##STR2400## ##STR2401## 6-OMe, H ##STR2402##
##STR2403## 69 ##STR2404## ##STR2405## 6-OMe, H ##STR2406##
##STR2407## 70 ##STR2408## ##STR2409## 6-OMe, H ##STR2410##
##STR2411## 71 ##STR2412## ##STR2413## 6-OMe, H ##STR2414##
##STR2415## 72 ##STR2416## ##STR2417## 6-OMe, H ##STR2418##
##STR2419## 73 ##STR2420## ##STR2421## 6-OMe, H ##STR2422##
##STR2423## 74 ##STR2424## ##STR2425## 6-OMe, H ##STR2426##
##STR2427## 75 ##STR2428## ##STR2429## 6-OMe, H ##STR2430##
##STR2431## 76 ##STR2432## ##STR2433## 6-OMe, 7-Cl ##STR2434##
##STR2435## 77 ##STR2436## ##STR2437## 5-OMe, H ##STR2438##
##STR2439## 78 ##STR2440## ##STR2441## 5-Cl, H ##STR2442##
##STR2443## 80 ##STR2444## ##STR2445## 6-OMe, H ##STR2446##
##STR2447## 81 ##STR2448## ##STR2449## 6-OMe, H ##STR2450##
##STR2451## 82 ##STR2452## ##STR2453## 6-OMe, H ##STR2454##
##STR2455## 83 ##STR2456## ##STR2457## 6-OMe, H ##STR2458##
##STR2459## 85 ##STR2460## ##STR2461## 6-OMe, H ##STR2462##
##STR2463## 86 ##STR2464## ##STR2465## 6-OMe, H ##STR2466##
##STR2467## 87 ##STR2468## ##STR2469## 6-OMe, H ##STR2470##
##STR2471## 88 ##STR2472## ##STR2473## 6-OMe, H ##STR2474##
##STR2475## 89 ##STR2476## ##STR2477## 6-OMe, H ##STR2478##
##STR2479## 90 ##STR2480## ##STR2481## 6-OMe, H ##STR2482##
##STR2483## 91 ##STR2484## ##STR2485## 6-OMe, H ##STR2486##
##STR2487## 92 ##STR2488## ##STR2489## 6-OMe, H ##STR2490##
##STR2491## 93 ##STR2492## ##STR2493## 6-OMe, H ##STR2494##
##STR2495## 94 ##STR2496## ##STR2497## 6-OMe, H ##STR2498##
##STR2499## 95 ##STR2500## ##STR2501## 6-OMe, H ##STR2502##
##STR2503## 96 ##STR2504## ##STR2505## 6-OMe, H ##STR2506##
##STR2507## 97 ##STR2508## ##STR2509## 6-OMe, H ##STR2510##
##STR2511## 98 ##STR2512## ##STR2513## 6-OMe, H ##STR2514##
##STR2515## 99 ##STR2516## ##STR2517## 6-OMe, H ##STR2518##
##STR2519## 100 ##STR2520## ##STR2521## 6-OMe, H ##STR2522##
##STR2523## 101 ##STR2524## ##STR2525## 6-OMe, H ##STR2526##
##STR2527## 102 ##STR2528## ##STR2529## 6-OMe, H ##STR2530##
##STR2531## 103 ##STR2532## ##STR2533## 6-OMe, H ##STR2534##
##STR2535## 104 ##STR2536## ##STR2537## 6-OMe, H ##STR2538##
##STR2539## 105 ##STR2540## ##STR2541## 6-OMe, H ##STR2542##
##STR2543## 106 ##STR2544## ##STR2545## 6-OMe, H ##STR2546##
##STR2547## 107 ##STR2548## ##STR2549## 6-OMe, H ##STR2550##
##STR2551## 108 ##STR2552## ##STR2553## 6-OMe, H ##STR2554##
##STR2555## 109 ##STR2556## ##STR2557## 6-OMe, H ##STR2558##
##STR2559## 110 ##STR2560## ##STR2561## 6-OMe, H ##STR2562##
##STR2563## 111 ##STR2564## ##STR2565## 6-OMe, H ##STR2566##
##STR2567## 112 ##STR2568## ##STR2569## 6-OMe, H ##STR2570##
##STR2571## 113 ##STR2572## ##STR2573## 6-OMe, H ##STR2574##
##STR2575## 114 ##STR2576## ##STR2577## 6-OMe, H ##STR2578##
##STR2579## 115 ##STR2580## ##STR2581## 6-OMe, H ##STR2582##
##STR2583## 116 ##STR2584## ##STR2585## H, H ##STR2586##
##STR2587## 117 ##STR2588## ##STR2589## 6-OMe, H ##STR2590##
##STR2591## 118 ##STR2592## ##STR2593## H, H ##STR2594##
##STR2595## 119 ##STR2596## ##STR2597## H, H ##STR2598##
##STR2599## 120 ##STR2600## ##STR2601## H, H ##STR2602##
##STR2603## 121 ##STR2604## ##STR2605## 6-OMe, H ##STR2606##
##STR2607## 122 ##STR2608## ##STR2609## H, H ##STR2610##
##STR2611## 123 ##STR2612## ##STR2613## H, H ##STR2614##
##STR2615## 124 ##STR2616## ##STR2617## H, H ##STR2618##
##STR2619## 125 ##STR2620## ##STR2621## H, H ##STR2622##
##STR2623## 126 ##STR2624## ##STR2625## H, H ##STR2626##
##STR2627##
127 ##STR2628## ##STR2629## H, H ##STR2630## ##STR2631## 128
##STR2632## ##STR2633## H, H ##STR2634## ##STR2635## 129
##STR2636## ##STR2637## H, H ##STR2638## ##STR2639## 130
##STR2640## ##STR2641## 5-Cl, H ##STR2642## ##STR2643## 131
##STR2644## ##STR2645## 6-OMe, 7-Cl ##STR2646## ##STR2647## 132
##STR2648## ##STR2649## 5-F, 6-OMe ##STR2650## ##STR2651## 133
##STR2652## ##STR2653## 5-OMe, H ##STR2654## ##STR2655## 134
##STR2656## ##STR2657## 5,6-dimethoxy ##STR2658## ##STR2659## 135
##STR2660## ##STR2661## 6-OMe, H ##STR2662## ##STR2663## 136
##STR2664## ##STR2665## 6-OMe, H ##STR2666## ##STR2667## 137
##STR2668## ##STR2669## 6-OMe, H ##STR2670## ##STR2671## 138
##STR2672## ##STR2673## H, H ##STR2674## ##STR2675## 139
##STR2676## ##STR2677## H, H ##STR2678## ##STR2679## 141
##STR2680## ##STR2681## 6-OMe, H ##STR2682## ##STR2683## 142
##STR2684## ##STR2685## 6-OMe, H ##STR2686## ##STR2687## 145
##STR2688## ##STR2689## H, H ##STR2690## ##STR2691## 146
##STR2692## ##STR2693## H, H ##STR2694## ##STR2695## 147
##STR2696## ##STR2697## H, H ##STR2698## ##STR2699## 148
##STR2700## ##STR2701## H, H ##STR2702## ##STR2703## 149
##STR2704## ##STR2705## H, H ##STR2706## ##STR2707## 150
##STR2708## ##STR2709## H, H ##STR2710## ##STR2711## 151
##STR2712## ##STR2713## H, H ##STR2714## ##STR2715## 152
##STR2716## ##STR2717## H, H ##STR2718## ##STR2719## 153
##STR2720## ##STR2721## H, H ##STR2722## ##STR2723## IC50 EC50
Cmpd. R.sub.7 Q (nM) (nM) 2 ##STR2724## ##STR2725## D C 3
##STR2726## ##STR2727## D C 4 ##STR2728## ##STR2729## D D 5
##STR2730## ##STR2731## D C 6 ##STR2732## ##STR2733## D D 7
##STR2734## ##STR2735## D D 8 ##STR2736## ##STR2737## D D 9
##STR2738## ##STR2739## D D 10 ##STR2740## ##STR2741## D D 11
##STR2742## ##STR2743## D C 12 ##STR2744## ##STR2745## D D 13
##STR2746## ##STR2747## D D 14 ##STR2748## ##STR2749## D D 15
##STR2750## ##STR2751## D D 16 ##STR2752## ##STR2753## D D 17B
##STR2754## ##STR2755## D D 18B ##STR2756## ##STR2757## D D 19
##STR2758## ##STR2759## D C 20 ##STR2760## ##STR2761## D D 21
##STR2762## ##STR2763## D C 22 ##STR2764## ##STR2765## D D 23
##STR2766## ##STR2767## D D 24 ##STR2768## ##STR2769## D C 25
##STR2770## ##STR2771## D B 26 ##STR2772## ##STR2773## D C 28
##STR2774## ##STR2775## D D 29 ##STR2776## ##STR2777## D D 30
##STR2778## ##STR2779## D D 31 ##STR2780## ##STR2781## D D 32
##STR2782## ##STR2783## D D 33 ##STR2784## ##STR2785## D D 34
##STR2786## ##STR2787## D D 35 ##STR2788## ##STR2789## D D 36
##STR2790## ##STR2791## D D 37 ##STR2792## ##STR2793## D D 38
##STR2794## ##STR2795## D D 39 ##STR2796## ##STR2797## D D 40
##STR2798## ##STR2799## D C 41 ##STR2800## ##STR2801## D D 42
##STR2802## ##STR2803## D D 43 ##STR2804## ##STR2805## D D 44
##STR2806## ##STR2807## D D 45 ##STR2808## ##STR2809## D D 46
##STR2810## ##STR2811## D D 47 ##STR2812## ##STR2813## D D 48
##STR2814## ##STR2815## D C 49 ##STR2816## ##STR2817## C B 50
##STR2818## ##STR2819## D D 51 ##STR2820## ##STR2821## D D 52
##STR2822## ##STR2823## D D 53 ##STR2824## ##STR2825## D D 54
##STR2826## ##STR2827## D D 55 ##STR2828## ##STR2829## D D 56
##STR2830## ##STR2831## D D 57 ##STR2832## ##STR2833## D D 58
##STR2834## ##STR2835## D D 59 ##STR2836## ##STR2837## D D 60
##STR2838## ##STR2839## D D 62 ##STR2840## ##STR2841## D C 63
##STR2842## ##STR2843## D C 64 ##STR2844## ##STR2845## D C 65
##STR2846## ##STR2847## D D 66 ##STR2848## ##STR2849## D C 67
##STR2850## ##STR2851## D C 68 ##STR2852## ##STR2853## D C 69
##STR2854## ##STR2855## D D 70 ##STR2856## ##STR2857## D C 71
##STR2858## ##STR2859## D C 72 ##STR2860## ##STR2861## D C 73
##STR2862## ##STR2863## D C 74 ##STR2864## ##STR2865## D D 75
##STR2866## ##STR2867## D D 76 ##STR2868## ##STR2869## D D 77
##STR2870## ##STR2871## D D 78 ##STR2872## ##STR2873## D D 80
##STR2874## ##STR2875## D C 81 ##STR2876## ##STR2877## D C 82
##STR2878## ##STR2879## D C 83 ##STR2880## ##STR2881## B A 85
##STR2882## ##STR2883## D D 86 ##STR2884## ##STR2885## D D 87
##STR2886## ##STR2887## D D 88 ##STR2888## ##STR2889## D D 89
##STR2890## ##STR2891## D D 90 ##STR2892## ##STR2893## D D 91
##STR2894## ##STR2895## D D 92 ##STR2896## ##STR2897## D D 93
##STR2898## ##STR2899## D D 94 ##STR2900## ##STR2901## D D 95
##STR2902## ##STR2903## D D 96 ##STR2904## ##STR2905## D D 97
##STR2906## ##STR2907## D D 98 ##STR2908## ##STR2909## D D 99
##STR2910## ##STR2911## D D 100 ##STR2912## ##STR2913## D D 101
##STR2914## ##STR2915## D D 102 ##STR2916## ##STR2917## D D 103
##STR2918## ##STR2919## D D 104 ##STR2920## ##STR2921## D C 105
##STR2922## ##STR2923## D C
106 ##STR2924## ##STR2925## D B 107 ##STR2926## ##STR2927## D D 108
##STR2928## ##STR2929## D D 109 ##STR2930## ##STR2931## D D 110
##STR2932## ##STR2933## D D 111 ##STR2934## ##STR2935## D D 112
##STR2936## ##STR2937## D D 113 ##STR2938## ##STR2939## D D 114
##STR2940## ##STR2941## D D 115 ##STR2942## ##STR2943## D D 116
##STR2944## ##STR2945## D D 117 ##STR2946## ##STR2947## C C 118
##STR2948## ##STR2949## D D 119 ##STR2950## ##STR2951## D C 120
##STR2952## ##STR2953## D B 121 ##STR2954## ##STR2955## D D 122
##STR2956## ##STR2957## D D 123 ##STR2958## ##STR2959## D C 124
##STR2960## ##STR2961## D D 125 ##STR2962## ##STR2963## D D 126
##STR2964## ##STR2965## D D 127 ##STR2966## ##STR2967## D D 128
##STR2968## ##STR2969## D D 129 ##STR2970## ##STR2971## D D 130
##STR2972## ##STR2973## D D 131 ##STR2974## ##STR2975## D D 132
##STR2976## ##STR2977## D D 133 ##STR2978## ##STR2979## D D 134
##STR2980## ##STR2981## D D 135 ##STR2982## ##STR2983## D D 136
##STR2984## ##STR2985## D D 137 ##STR2986## ##STR2987## D D 138
##STR2988## ##STR2989## D D 139 ##STR2990## ##STR2991## D D 141
##STR2992## ##STR2993## C B 142 ##STR2994## ##STR2995## D D 145
##STR2996## ##STR2997## D C 146 ##STR2998## ##STR2999## D B 147
##STR3000## ##STR3001## D D 148 ##STR3002## ##STR3003## D C 149
##STR3004## ##STR3005## D C 150 ##STR3006## ##STR3007## D C 151
##STR3008## ##STR3009## D C 152 ##STR3010## ##STR3011## D C 153
##STR3012## ##STR3013## D C
Combination Studies
[0560] Since clinical drug resistance often develops in viral
infections following single-agent therapies, there is a need to
assess the additive, antagonistic, or synergistic properties of
combination therapies. We used the HCV replicon system, as
described above, to assess the potential use of our NS3 protease
macrocyclic inhibitor in combination therapies with Intron A and
inhibitors targeting other HCV proteins. Three HCV antivirals,
Intron A (recombinant Interferon alfa-2b) an HCV NS5A inhibitor
having the following structure: ##STR3014## MS(ESI) m/z=691.2
(MH.sup.+); HPLC rt 1.21 min; Purity (99%), and an NS5B replicase
inhibitor (compound 2006; WO 03/010141), were tested in 2-drug
combinations with the macrocyclic HCV NS3 protease inhibitor
compound 28. In addition, compound 28 was also tested in 3-drug
combinations with the NS5A and NS5B inhibitors.
[0561] For the experiments shown in Tables 8 and 9, inhibitors were
tested at eleven concentrations each. 200.times. stock solutions of
each inhibitor concentration were prepared by 2- or 3-fold dilution
in DMSO prior to addition to cells/media. The drugs were tested as
monotherapies and in combination with the NS3 protease inhibitor at
various concentration ratios. Cells were exposed to compounds for 4
days and the amount of HCV inhibition was then determined using the
FRET assay. The potential cytotoxicities of these combined agents
were also analyzed in parallel by Alamar Blue staining. The
CC.sub.50 values for the compounds presented were greater than the
highest tested inhibitor concentration. The degree of antagonism,
additivity, or synergy was determined over a range of drug
concentrations, and combination response curves were fit to assess
the antiviral effects of the drug treatment combinations. The
concentration ratios were analyzed using the method of Chou (Chou,
Ting-Chao, and Rideout (Editors), (1991) Synergism and Antagonism
in Chemotherapy, P. 61-101, Academic Press, New York). Tables 8 and
9 report the estimated EC.sub.50 values for the compounds tested,
as well as the combination indices (CI). All estimates were
computed using SAS Proc NLIN, and a two-parameter logistic. All
combination indices were tested for departure from additivity using
isobologram methods. Asymptotic confidence intervals were also
calculated for each of the combination indices. These intervals are
used to test for departure from additivity by comparing the bounds
to one--a lower bound of the interval greater than 1 indicates
antagonism, an upper bound of less than 1 indicates synergism, and
a value of 1 contained in the interval indicates additivity.
[0562] In addition to the combination index approach, above, the
Universal Response Surface Approach (URSA) was also used to assess
the antiviral effects of combinations of NS5A or NS5B inhibitors
with the NS3 protease inhibitor. The experiments were conducted in
a matrixed format with 8 concentrations of NS3 protease inhibitor
crossed against 10 concentrations of NS5A or NS5B inhibitor on each
of three plates. 200.times. stock solutions of each inhibitor
concentration were prepared by 3-fold dilution in DMSO. Effects on
replication were assessed in the HCV replicon system as described
above. The data was analyzed using the URSA as described in Greco,
Park and Rustum (Greco, Park, Sook, and Rustum (1990) Application
of a New Approach for the Quantitation of Drug Synergism to the
Combination of cis-Diamminedichloroplatinum and
1-.beta.-D-Arabinofuranosylcytosine, Cancer Research, 50,
5318-5327). The interaction of the two drugs was assessed by using
non-linear regression with a bisection algorithm. Seven parameters
were estimated. These include the minimal response or response in
the absence of drug, the maximal response or response in the
presence of infinite drug, EC.sub.50's for the two drugs, slope
parameters for the two drugs, and the drug interaction parameter
.alpha. which provides the assessment of synergism, antagonism or
additivity. Tables 10 and 11 present key estimated parameters and
standard errors. Additivity is implied when .alpha. is equal to
zero, synergy if the interaction parameter is larger than zero, and
antagonism when the interaction parameter is less than zero. The
confidence interval for the interaction parameter is also
presented. This interval is used to test for departure from
additivity by comparing the bounds to zero--a lower bound of the
interval greater than zero indicates synergy, an upper bound of
less than zero indicates antagonism, and a value of zero contained
in the interval indicates additivity. ##STR3015##
[0563] Table 8 summarizes data from combinations of compound 28
with Intron A. EC.sub.50 values for each monotherapy are also
presented. In two experiments, combination of compound 28 with
Intron A yielded either synergistic or additivity effects at the
75%, 90, and 95% effective doses.
[0564] The effects of compound 28 in combination with the HCV NS5A
inhibitor are summarized in Table 9. Synergy or additivity was
observed in two experiments at the 75, 90, and 95% effective doses
of compound 28. Importantly, no drug antagonism was observed at the
75, 90, or 95% effective doses when compound 28 was combined with
the HCV NS5A inhibitor, or Interferon alfa-2b. Follow-up
experiments using these same inhibitor combinations in the matrix
format (Table 10) yielded results similar to the combination index
experiments; additivity for the combination of the NS5A inhibitor
with compound 28.
[0565] The matrix format was also used to examine the activity of
combinations of the HCV NS5B inhibitor, with compound 28 (Table
11). In two experiments, overall additivity was observed.
[0566] Compound 28 was also tested in 3-drug combination
experiments with the NS5A inhibitor and the NS5B inhibitor (Table
12). The starting concentrations for the mono-therapies consisted
of a 0.667 .mu.M solution of the NS5A inhibitor, a 0.3 .mu.M
solution of compound 28, and a 2.5 .mu.M solution of the NS5B
inhibitor. The starting concentration for the triple combination
contained a mixture of 1/3 of each of the above concentrations.
Dose response curves for all three mono-therapies and the triple
combination therapy were obtained using 3-fold dilutions, and data
analysis was carried out as described above for combination index
experiments. Synergistic effects were observed using the triple
combination. No drug antagonism was observed at the 75, 90, or 95%
effective doses with the 3-drug combination.
[0567] These results demonstrate that combination treatment of
replicon cells with the HCV NS3 protease inhibitor, compound 28 and
Intron A, and/or inhibitors targeting HCV NS5A and/or NS5B, yield
additive to synergistic antiviral effects. The ability to use these
NS3 protease inhibitors in combination therapy can provide major
advantages over single drug therapy for the treatment of HCV.
TABLE-US-00012 TABLE 8 Two Drug Combinations with Interferon
alfa-2b Molar ratio, NS3 protease Combination Indices NS3 protease
Interferon inhibitor to (confidence interval) inhibitor alfa-2b
EC50, Interferon 75% 90% 95% Overall Expt EC50, .mu.M units per ml
alfa-2b effective effective effective Result 1 0.008 20 2:5 0.67
0.59 0.56 Synergy (0.53, 0.8) (0.4, 0.77) (0.33, 0.79) 1:1 0.57 0.5
0.46 Synergy (0.48, 0.66) (0.38, 0.62) (0.32, 0.61) 4:25 1.02 0.64
0.5 Additivity (0.88, 1.16) (0.5, 0.78) (0.36, 0.64) at 75%;
Synergy at 90, 95% 2 0.01 51 2:5 0.59 0.38 0.28 Synergy (0.43,
0.74) (0.22, 0.54) (0.13, 0.43) 1:1 0.50 0.33 0.25 Synergy (0.44,
0.56) (0.27, 0.39) (0.19, 0.31) 4:25 1.03 0.65 0.48 Additivity
(0.87, 1.19) (0.50, 0.81) (0.33, 0.63) at 75%; Synergy at 90,
95%
[0568] TABLE-US-00013 TABLE 9 Two Drug Combinations with an NS5A
inhibitor Molar ratio, NS3 protease Combination Indices NS3
protease NS5A inhibitor to (confidence interval) inhibitor
inhibitor NS5A 75% 90% 95% Overall Expt EC50, .mu.M EC50, .mu.M
inhibitor effective effective effective Result 1 0.009 0.043 3:100
0.66 0.61 0.59 Synergy (0.47, 0.85) (0.35, 0.88) (0.27, 0.91) 3:250
0.57 0.62 0.66 Additivity (0.41, 0.73) (0.36, 0.89) (0.3, 1.02) at
95%; Synergy at 75, 90% 3:40 0.73 0.64 0.59 Synergy (0.55, 0.9)
(0.43, 0.85) (0.34, 0.83) 2 0.011 0.005 1:5 1.1 1.15 1.18
Additivity (0.91, 1.3) (0.82, 1.47) (0.75, 1.61) 2:25 1.24 1.19
1.16 Additivity (1.0, 1.49) (0.81, 1.58) (0.67, 1.65) 1:2 1.0 0.83
0.73 Additivity (0.88, 1.14) (0.67, 1.0) (0.54, 0.92) at 75%;
Synergy at 90, 95%
[0569] TABLE-US-00014 TABLE 10 Two Drug Combinations with an NS5A
inhibitor: matrix studies Standard Error Overall Result Compound 28
EC50, .mu.M 0.009 0.52 NS5A inhibitor EC50, 0.008 0.46 .mu.M
Interaction parameter 0.63 0.37 Additivity alpha (-0.09, 1.36)
(confidence interval)
[0570] TABLE-US-00015 TABLE 11 Two Drug Combinations with an NS5B
inhibitor: matrix studies Standard Overall Standard Overall Expt 1
Error Result Expt 2 Error Result Compound 28 EC50, .mu.M 0.015 0.53
0.020 0.87 NS5B inhibitor EC50, 0.059 2.63 0.050 2.39 .mu.M
Interaction parameter 0.47 0.27 Additivity 0.31 0.27 Additivity
alpha (-0.05, 1.0) (-0.22, 0.83) (confidence interval)
[0571] TABLE-US-00016 TABLE 12 Three Drug Combination Studies Molar
ratio, NS3 protease inhibitor to NS3 Pr NS5A NS5B NS5A NS5B
Combination Indices inhibitor inhibitor inhibitor inhibitor to
inhibitor (confidence interval) EC50, EC50, EC50, NS5B EC50, 75%
90% 95% Overall Expt .mu.M .mu.M .mu.M inhibitor .mu.M effective
effective effective Result 1 0.008 0.005 0.052 9:20:75 0.052 0.78
0.7 0.65 Synergy (0.62, 0.94) (0.47, 0.93) (0.37, 0.93) 2 0.009
0.009 0.064 9:20:75 0.064 0.71 0.58 0.52 Synergy (0.6, 0.81) (0.44,
0.72) (0.36, 0.67)
[0572] Those skilled in the art will recognize that although the
invention has been described above with respect to specific
aspects, other aspects are intended to be within the scope of the
claims which follow. All documents referenced herein are hereby
incorporated by reference as if set out in full.
* * * * *