U.S. patent application number 12/557638 was filed with the patent office on 2010-01-07 for hcv ns-3 serine protease inhibitors.
This patent application is currently assigned to Medivir AB. Invention is credited to Susana Ayesa, Bjorn Classon, Per-Ola Johansson, Ingemar Kvarnstrom, Laszlo Rakos, Asa Rosenquist, Bertil Samuelsson, Fredrik Thorstensson.
Application Number | 20100003216 12/557638 |
Document ID | / |
Family ID | 34831013 |
Filed Date | 2010-01-07 |
United States Patent
Application |
20100003216 |
Kind Code |
A1 |
Rosenquist; Asa ; et
al. |
January 7, 2010 |
HCV NS-3 Serine Protease Inhibitors
Abstract
Compounds of the formula ##STR00001## where the variables are as
defined in the specification inhibit the NS3 protease of flavivirus
sych as hepatitis C virus (HCV). The compounds comprise a novel
linkage between a heterocyclic P2 unit and those portions of the
inhibitor more distal to the nominal cleavage site of the native
substrate, which linkage reverses the orientation of peptidic bonds
on the distal side relative to those proximal to the cleavage
site.
Inventors: |
Rosenquist; Asa; (Huddinge,
SE) ; Thorstensson; Fredrik; (Linkoping, SE) ;
Johansson; Per-Ola; (Linkoping, SE) ; Kvarnstrom;
Ingemar; (Linkoping, SE) ; Ayesa; Susana;
(Huddinge, SE) ; Classon; Bjorn; (Huddinge,
SE) ; Rakos; Laszlo; (Huddinge, SE) ;
Samuelsson; Bertil; (Huddinge, SE) |
Correspondence
Address: |
BUTZEL LONG;IP DOCKETING DEPT
350 SOUTH MAIN STREET, SUITE 300
ANN ARBOR
MI
48104
US
|
Assignee: |
Medivir AB
Huddinge
SE
|
Family ID: |
34831013 |
Appl. No.: |
12/557638 |
Filed: |
September 11, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10572418 |
Jan 22, 2007 |
7608590 |
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PCT/SE05/00096 |
Jan 28, 2005 |
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12557638 |
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Current U.S.
Class: |
424/85.4 ;
514/1.1; 514/312 |
Current CPC
Class: |
C07D 401/12 20130101;
C07C 271/22 20130101; C07D 215/233 20130101; C07D 417/14 20130101;
C07K 5/06034 20130101; C07C 309/73 20130101; C07D 245/04 20130101;
C07C 2601/02 20170501; A61P 31/00 20180101; A61P 31/14 20180101;
C07C 235/40 20130101; C07C 2601/08 20170501; A61P 31/12 20180101;
C07D 413/14 20130101; C07K 5/06052 20130101; C07C 237/04 20130101;
C07D 207/16 20130101; A61K 31/47 20130101; C07D 417/04 20130101;
C07D 487/04 20130101; C07C 2601/10 20170501; A61P 43/00 20180101;
C07K 5/0205 20130101; C07C 311/51 20130101; C07C 281/02 20130101;
C07D 405/14 20130101; C07D 215/20 20130101; C07D 409/14 20130101;
C07C 237/10 20130101; C07C 247/04 20130101 |
Class at
Publication: |
424/85.4 ;
514/312; 514/18 |
International
Class: |
A61K 38/21 20060101
A61K038/21; A61K 31/47 20060101 A61K031/47; A61K 38/06 20060101
A61K038/06; A61P 31/12 20060101 A61P031/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 30, 2004 |
SE |
0400199-6 |
May 19, 2004 |
SE |
0401288-6 |
Oct 22, 2004 |
SE |
0402562-3 |
Claims
1-56. (canceled)
57. A method for treatment or prophylaxis of flavivirus infection
comprising administering to an individual afflicted with or at risk
of flavivirus infection an effective amount of a compound of
formula I': ##STR00213## wherein A is C(.dbd.O)OR.sup.1, or
C(.dbd.O)NHSO.sub.2R.sup.2, wherein; R.sup.1 is hydrogen,
C.sub.1-C.sub.6alkyl, C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl; R.sup.2 is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl;
wherein R.sup.2 is optionally substituted with 1 to 3 substituents
independently selected from the group consisting of halo, oxo,
nitrile, azido, nitro, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl,
NH.sub.2CO--, Y--NRaRb, Y--O--R.sub.b, Y--C(.dbd.O)Rb,
Y--(C.dbd.O)NRaRb, Y--NRaC(.dbd.O)Rb, Y--NHSO.sub.pRb,
Y--S(.dbd.O).sub.pRb, Y--S(.dbd.O).sub.pNRaRb, Y--C(.dbd.O)ORb,
Y--NRaC(.dbd.O)ORb; Y is independently a bond or
C.sub.1-C.sub.3alkyl; Ra is independently H or
C.sub.1-C.sub.3alkyl; Rb is independently H, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl or
C.sub.0-C.sub.3alkylheterocyclyl; p is independently 1 or 2; M is
CR.sup.7R.sup.7'; R.sup.7 taken together with R.sup.7' forms a
C.sub.3-C.sub.6cycloalkyl ring substituted with J; q is 1 and k is
1; W is --CH.sub.2--, --O--, --OC(.dbd.O)NH, --OC(.dbd.O), --S--,
--NH--, --NRa, --NHSO.sub.2--, --NHC(.dbd.O)NH-- or
--NHC(.dbd.O)--, --NHC(.dbd.S)NH-- or a bond; R.sup.8 is a ring
system containing 1 or 2 saturated, partially saturated or
unsaturated rings each of which has 4-7 ring atoms and each of
which has 0 to 4 hereto atoms independently selected from S, O and
N, the ring system being optionally spaced from W by a
C.sub.1-C.sub.3 alkylene group; any of which R.sup.8 groups can be
optionally mono-, di-, or tri-substituted with R.sup.9, wherein
R.sup.9 is independently selected from the group consisting of
halo, oxo, nitrile, azido, nitro, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl,
NH.sub.2C(.dbd.O)--, Y--NRaRb, Y--O--Rb, Y--C(.dbd.O)Rb,
Y--(C.dbd.O)NRaRb, Y--NRaC(.dbd.O)Rb, Y--NHSO.sub.pRb,
Y--S(.dbd.O).sub.pRb, Y--S(.dbd.O).sub.pNRaRb, Y--C(.dbd.O)ORb,
Y--NRaC(.dbd.O)ORb; wherein said carbocyclyl or heterocyclyl is
optionally substituted with R.sup.10; wherein R.sup.10 is
C.sub.1-C.sub.6alkyl, C.sub.3-C.sub.7cycloalkyl,
C.sub.1-C.sub.6alkoxy, amino, amido, sulfonyl,
(C.sub.1-C.sub.3alkyl)sulfonyl, NO.sub.2, OH, SH, halo, haloalkyl,
carboxyl; E is --C(.dbd.O)--, --C(.dbd.S)--,
--S(.dbd.O).sub.2.sup.-, --S(.dbd.O)--, --C(.dbd.N--Rf)-; Rf is H,
--CN, --C(.dbd.O)NRaRb; --C(.dbd.O)C.sub.1-C.sub.3alkyl; J is a
single 3 to 10-membered saturated or partially unsaturated alkylene
chain that extends from the R.sup.7/R.sup.7' cycloalkyl- to G and
forms a macrocycle, which chain is optionally interrupted by one to
three heteroatoms independently selected from: --O--, --S-- or
--NR.sup.12--, and wherein 0 to 3 carbon atoms in the chain are
optionally substituted with R.sup.14; wherein; R.sup.12 is H,
C.sub.1-C.sub.6alkyl, C.sub.3-C.sub.6cycloalkyl, or
C(.dbd.O)R.sup.13; R.sup.13 is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl;
R.sup.14 is independently selected from the group consisting of H,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy, hydroxyl, halo, amino, oxo, thio and
C.sub.1-C.sub.6thioalkyl; m is 0; n is 0; Ry is J; R.sup.16 is H;
or C.sub.1-C.sub.6alkyl, C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl, any of which can be substituted
with halo, oxo, nitrile, azido, nitro, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl,
NH.sub.2CO--, Y--NRaRb, Y--O--Rb, Y--C(.dbd.O)Rb,
Y--(C.dbd.O)NRaRb, Y--NRaC(.dbd.O)Rb, Y--NHSO.sub.pRb,
Y--S(.dbd.O).sub.pRb, Y--S(.dbd.O).sub.pNRaRb, Y--C(.dbd.O)ORb,
Y--NRac(.dbd.O)ORb; or a pharmaceutically acceptable salt
thereof.
58. The method according to claim 57, wherein said compound has the
partial structure Ia', Ib' or Iaa': ##STR00214## where e is 1 or
2.
59. The method according to claim 57, wherein E is
--C(.dbd.O)--.
60. The method according to claim 57, wherein R.sup.16 is H,
C.sub.1-C.sub.3 alkyl or C.sub.3-C.sub.6 cycloalkyl.
61. The method according to claim 57, wherein W is --OC(.dbd.O)--,
--NRa--, NHS(O).sub.2--; --NHC(.dbd.O)--; --OC(.dbd.O)NH--; --S--,
a bond or --O--.
62. The method according to claim 61 wherein R.sup.8 is optionally
substituted C.sub.0-C.sub.3alkylcarbocyclyl or optionally
substituted C.sub.0-C.sub.3-alkylheterocyclyl.
63. The method according to claim 62, wherein the C.sub.0-C.sub.3
alkyl moiety is methylene or a bond.
64. The method according to claim 63, wherein R.sup.8 is
1-naphthylmethyl, 2-naphthylmethyl, benzyl, 1-naphthyl, 2-naphthyl,
or quinolinyl any of which is unsubstituted, mono, or distributed
with R.sup.9.
65. The method according to claim 64, wherein R.sup.8 is
1-naphthylmethyl, or quinolinyl any of which is unsubstituted,
mono, or distributed with R.sup.9.
66. The method according to claim 65 wherein R.sup.8 is
##STR00215## wherein R.sup.9a is C.sub.1-C.sub.6alkyl;
C.sub.1-C.sub.6alkoxy; thioC.sub.1-C.sub.3alkyl; amino optionally
substituted with C.sub.1-C.sub.6alkyl; C.sub.0-C.sub.3alkylaryl; or
C.sub.0-C.sub.3alkylheteroaryl, C.sub.0-C.sub.3alkylheterocyclyl,
said aryl, heteroaryl or heterocycle being optionally substituted
with R.sup.10 wherein R.sup.10 is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylC.sub.3-C.sub.7cycloalkyl,
C.sub.1-C.sub.6alkoxy, amino optionally mono- or di-substituted
with C1-C6alkyl, amido, C1-C3 amide; and R.sup.9b is
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6-alkoxy, amino,
di(C.sub.1-C.sub.3alkyl)amino, (C.sub.1-C.sub.3alkyl)amide,
NO.sub.2, OH, halo, trifluoromethyl, carboxyl.
67. The method according to claim 66, wherein R.sup.9a is aryl or
heteroaryl, either of which is optionally substituted with
R.sup.10.
68. The method according to claim 67, wherein R.sup.9a is selected
from the group consisted of: ##STR00216## wherein R.sup.10 is H,
C.sub.1-C.sub.6alkyl, or C.sub.0-C.sub.3alkylcycloalkyl, amino
optionally mono- or di-substituted with C.sub.1-C.sub.6alkyl,
amido, (C.sub.1-C.sub.3alkyl)amide.
69. The method according to claim 67, wherein R.sup.9a is phenyl,
optionally substituted with C.sub.1-C.sub.6alkyl;
C.sub.1-C.sub.6alkoxy; or halo.
70. The method according to claim 66, wherein R.sup.8 is:
##STR00217## wherein R.sup.10a is H, C.sub.1-C.sub.6alkyl, or
C.sub.0-C.sub.3alkylcarbocyclyl, amino optionally mono- or
di-substituted with C.sub.1-C.sub.6alkyl, amido, heteroaryl or
heterocyclyl; and R.sup.9b is C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6-alkoxy, amino, di(C.sub.1-C.sub.3 alkyl)amino,
amide, NO.sub.2, OH, halo, trifluoromethyl, or carboxyl.
71. The method according to claim 66, wherein R.sup.9b is
C.sub.1-C.sub.6-alkoxy.
72. The method according to claim 57, wherein A is
C(.dbd.O)NHSO.sub.2R.sup.2.
73. The method according to claim 72, wherein R.sup.2 is optionally
substituted C.sub.1-C.sub.6 alkyl.
74. The method according to claim 72, wherein R.sup.2 is optionally
substituted C.sub.3-C.sub.7cycloalkyl.
75. The method according to claim 72, wherein R.sup.2 is optionally
substituted C.sub.0-C.sub.6alkylary.
76. The method according to claim 57, wherein A is
C(.dbd.O)OR.sup.1.
77. The method according to claim 76, wherein R.sup.1 is H or
C.sub.1-C.sub.6 alkyl.
78. The method according to claim 57, wherein R.sup.7 and R7'
together define a spiro-cyclopropyl.
79. The method according to claim 57, wherein J is a 3 to
8-membered saturated or unsaturated alkylene chain optionally
containing one to two heteroatoms independently selected from:
--O--, --S-- or --NR.sup.12--, wherein R.sup.12 is H,
C.sub.1-C.sub.6 alkyl, or --C(.dbd.O)C.sub.1-C.sub.6 alkyl.
80. The method according to claim 79, wherein J is a 4 to
7-membered saturated or unsaturated, all carbon alkylene chain.
81. The method according to claim 79, wherein J is saturated or
mono-unsaturated.
82. The method according to claim 79, wherein J is dimensioned to
provide a macrocycle of 14 or 15 ring atoms.
83. The method according to claim 57 wherein said compound has
formula Ihe': ##STR00218## or a pharmaceutically acceptable salt
thereof wherein R.sup.16 is H, or C.sub.1-C.sub.6alkyl; J is a
single 3 to 10-membered saturated or partially unsaturated alkylene
chain; q is 1 and k is 1; A is C(.dbd.O)OR.sup.1, or
C(.dbd.O)NHSO.sub.2R.sup.2, wherein R.sup.1 is hydrogen or
C.sub.1-C.sub.6alkyl; R.sup.2 is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl;
W is --O-- or --OC(.dbd.O)NH--; R.sup.8 is C.sub.0-C.sub.3alkylaryl
or C.sub.0-C.sub.3alkylheteroaryl, either of which is optionally
mono, di, or tri substituted with R.sup.9, wherein; R.sup.9 is
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, NO.sub.2, OH, halo,
trifluoromethyl, amino or amido optionally mono- or di-substituted
with C.sub.1-C.sub.6alkyl, C.sub.0-C.sub.3alkylaryl,
C.sub.0-C.sub.3alkylheteroaryl, carboxyl, said aryl or heteroaryl
being optionally substituted with R.sup.10; wherein R.sup.10 is
C.sub.1-C.sub.6alkyl, C.sub.3-C.sub.7cycloalkyl,
C.sub.1-C.sub.6alkoxy, amino optionally mono- or di-substituted
with C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.3 alkyl amide,
sulfonylC.sub.1-C.sub.3alkyl, NO.sub.2, OH, halo, trifluoromethyl,
carboxyl or heteroaryl.
84. The method according to claim 83, wherein J is a single 5-8
membered saturated or partially unsaturated alkylene chain.
85. The method according to claims 83, wherein J is
monounsaturated.
86. The method according to claim 85, wherein J has one double bond
spaced one carbon atom from the cyclopropyl group depicted in the
formula Ihe'.
87. The method according to claim 83, wherein R.sup.8 is the group
##STR00219## wherein R.sup.9a is C.sub.0-C.sub.3alkylaryl,
C.sub.0-C.sub.3alkylheteroaryl, or
C.sub.0-C.sub.3alkylheterocyclyl; said aryl, heteroaryl or
heterocyclyl being optionally substituted with R.sup.10 wherein
R.sup.10 is C.sub.1-C.sub.6alkyl, amino, amino mono- or
disubstituted with C.sub.1-C.sub.6alkyl or
NHC(.dbd.O)C.sub.1-C.sub.6alkyl; and R.sup.9b is
C.sub.1-C.sub.6alkoxy; or R.sup.8 is C.sub.0-C.sub.3alkylaryl
wherein the aryl group is optionally substituted with 1-2
substituents selected from C.sub.0-C.sub.3alkylheterocyclyl and
trifluoC.sub.1-C.sub.6alkyl; and wherein the
C.sub.0-C.sub.3alkylheterocyclyl is optionally substituted with
R.sup.10.
88. The method according to claim 87, wherein R.sup.9a is phenyl,
##STR00220## wherein R.sup.10 is H, C.sub.1-C.sub.6alkyl, amino,
amino mono or disubstituted with C.sub.1-C.sub.3alkyl.
89. The method according to claim 83, wherein A is
C(.dbd.O)NHS(.dbd.O).sub.2R.sup.2.
90. The method according to claim 89, wherein R.sup.2 is optionally
substituted cycloalkyl.
91. The method according to claim 90 wherein R.sup.2 is optionally
substituted cyclopropyl.
92. The method according to claim 60, wherein R.sup.16 is
methyl.
93. The method according to claim 57 wherein the flavivirus
infection is HCV infection.
94. The method according to claim 57 further comprising sequential
or simultaneous administration of an additional HCV antiviral
selected from nucleoside analogue polymerase inhibitors, protease
inhibitors, ribavirin, and interferon.
95. The method according to claim 57 wherein said administering
further comprises administering said compound in a pharmaceutical
composition comprising said compound and a pharmaceutically
acceptable carrier.
96. The method of claim 95, wherein said pharmaceutical composition
further comprises an additional HCV antiviral selected from
nucleoside analogue polymerase inhibitors, protease inhibitors,
ribavirin, and interferon.
97. A method for treatment or prophylaxis of flavivirus infection
comprising administering to an individual afflicted with or at risk
of flavivirus infection an effective amount of a compound of
formula I': ##STR00221## wherein A is C(.dbd.O)OR.sup.1, or
C(.dbd.O)NHSO.sub.2R.sup.2, wherein; R.sup.1 is hydrogen,
C.sub.1-C.sub.6alkyl, C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl; R.sup.2 is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl;
wherein R.sup.2 is optionally substituted with 1 to 3 substituents
independently selected from the group consisting of halo, oxo,
nitrile, azido, nitro, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl,
NH.sub.2CO--, Y--NRaRb, Y--O--R.sub.b, Y--C(.dbd.O)Rb,
Y--(C.dbd.O)NRaRb, Y--NRaC(.dbd.O)Rb, Y--NHSO.sub.pRb,
Y--S(.dbd.O).sub.pRb, Y--S(.dbd.O).sub.pNRaRb, Y--C(.dbd.O)ORb,
Y--NRaC(.dbd.O)ORb; Y is independently a bond or
C.sub.1-C.sub.3alkyl; Ra is independently H or
C.sub.1-C.sub.3alkyl; Rb is independently H, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl or
C.sub.0-C.sub.3alkylheterocyclyl; p is independently 1 or 2; M is
CR.sup.7R.sup.7'; R.sup.7 taken together with R.sup.7' forms a
C.sub.3-C.sub.6cycloalkyl ring substituted with J; q is 1 and k is
1; W is --O--, --OC(.dbd.O)NH, --OC(.dbd.O), --S--, --NRa,
--NHSO.sub.2--, --NHC(.dbd.O)--, or a bond; R.sup.8 is
C.sub.0-C.sub.3alkylaryl, or C.sub.0-C.sub.3alkylheteroaryl, either
of which is optionally mono, di, or tri substituted with R.sup.9,
wherein; R.sup.9 is C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6alkoxy,
NO.sub.2, OH, halo, trifluoromethyl, amino, amido optionally mono-
or di-substituted with C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylaryl, C.sub.0-C.sub.3alkylheteroaryl, carboxyl,
aryl or heteroaryl being optionally substituted with R.sup.10;
wherein R.sup.10 is C.sub.1-C.sub.6alkyl,
C.sub.3-C.sub.7cycloalkyl, C.sub.1-C.sub.6alkoxy, amino optionally
mono- or di-substituted with C.sub.1-C.sub.6alkyl, amido,
sulfonylC.sub.1-C.sub.6alkyl, NO.sub.2, OH, halo, trifluoromethyl,
carboxyl, or heteroaryl; E is --C(.dbd.O)--, --C(.dbd.S)--,
--S(.dbd.O).sub.2--, --S(.dbd.O)--, --C(.dbd.N--Rf)-; Rf is H,
--CN, --C(.dbd.O)NRaRb; --C(.dbd.O)C.sub.1-C.sub.3alkyl; J is a
single 3 to 10-membered saturated or partially unsaturated alkylene
chain that extends from the R.sup.7/R.sup.7' cycloalkyl to G and
forms a macrocycle, which chain is optionally interrupted by one to
three heteroatoms independently selected from: --O--, --S-- or
--NR.sup.12--, and wherein 0 to 3 carbon atoms in the chain are
optionally substituted with R.sup.14; wherein; R.sup.12 is H,
C.sub.1-C.sub.6alkyl, C.sub.3-C.sub.6cycloalkyl, or
C(.dbd.O)R.sup.13; R.sup.13 is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl;
R.sup.14 is independently selected from the group consisting of H,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy, hydroxyl, halo, amino, oxo, thio and
C.sub.1-C.sub.6thioalkyl; m is 0; n is 0; G is --NRy-; Ry is J;
R.sup.16 is H; or C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl,
any of which can be substituted with halo, oxo, nitrile, azido,
nitro, C.sub.1-C.sub.6alkyl, C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl, NH.sub.2CO--, Y--NRaRb, Y--O--Rb,
Y--C(.dbd.O)Rb, Y--(C.dbd.O)NRaRb, Y--NRaC(.dbd.O)Rb,
Y--NHSO.sub.pRb, Y--S(.dbd.O).sub.pRb, Y--S(.dbd.O).sub.pNRaRb,
Y--C(.dbd.O)ORb, Y--NRac(.dbd.O)ORb; or a pharmaceutically
acceptable salt thereof.
98. The method according to claim 97 wherein R.sup.9 is
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6alkoxy, amino,
di-(C.sub.1-C.sub.3 alkyl)amino, C.sub.1-C.sub.3alkylamide, aryl or
heteroaryl, the aryl or heteroaryl being optionally substituted
with R.sup.10; wherein R.sup.10 is C.sub.1-C.sub.6alkyl,
C.sub.3-C.sub.7cycloalkyl, C.sub.1-C.sub.6alkoxy, amino, mono- or
di-C.sub.1-C.sub.3alkylamino, amido, halo, trifluoromethyl, or
heteroaryl.
99. The method according to claim 98, wherein R.sup.10 is
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, amino optionally mono-
or di substituted with C.sub.1-C.sub.3 alkyl, amido,
C.sub.1-C.sub.3-alkylamide, halo, or heteroaryl.
100. The method according to claim 99 wherein R.sup.10 is methyl,
ethyl, isopropyl, tert-butyl, methoxy, chloro, amino optionally
mono- or di substituted with C.sub.1-C.sub.3 alkyl, amido, or
C.sub.1-C.sub.3alkyl thiazolyl.
101. The method according to claim 97, wherein W is --O--.
102. The method according to claim 97 wherein the flavivirus
infection is HCV infection.
103. The method according to claim 97 further comprising sequential
or simultaneous administration of an additional HCV antiviral
selected from nucleoside analogue polymerase inhibitors, protease
inhibitors, ribavirin, and interferon.
104. The method according to claim 97 wherein said administering
further comprises administering said compound in a pharmaceutical
composition comprising said compound and a pharmaceutically
acceptable carrier.
105. The method of claim 104, wherein said pharmaceutical
composition further comprises an additional HCV antiviral selected
from nucleoside analogue polymerase inhibitors, protease
inhibitors, ribavirin, and interferon.
106. A method for treatment or prophylaxis of flavivirus infection
comprising administering to an individual afflicted with or at risk
of flavivirus infection an effective amount of a compound selected
from the group consisting of:
19-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-2,16-dioxo-3,15,17-triaza-tricyc-
lo[15.3.0.0*4,6*]icos-7-ene-4,14-dicarboxylic acid 4-ethyl ester
14-methyl ester;
19-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-2,16-dioxo-3,15,17-triaza-
-tricyclo[15.3.0.0*4,6*]icos-7-ene-3,14-dicarboxylic acid 3-ethyl
ester;
14-[(Cyclohexyl-methylcarbamoyl-methyl)-19-(7-methoxy-2-phenyl-quinolin-4-
-yloxy)-2,16-dioxo-3,15,17-triaza-tricyclo[15.3.0.0*4,6*]icos-7-ene-4-carb-
oxylic acid 3-ethyl ester;
14-[(Cyclohexyl-methylcarbamoyl-methyl)-19-(7-methoxy-2-phenyl-quinolin-4-
-yloxy)-2,16-dioxo-3,15,17-triaza-tricyclo[15.3.0.0*4,6*]icos-7-ene-4-carb-
oxylic acid;
[14-Cyclopropanesulfonylaminocarbonyl-17(7-methoxy-2-phenyl-quinolin-4-yl-
oxy)-2,14-dioxo-3,13,15-triaza-tricyclo[13.3.0.0*4,6*]octadec-7-en-13-yl]--
carbamic acid ter.butyl ester;
17-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-2,14-dioxo-3,13,15-triaza-tricyc-
lo[13.3.0.0*4,6*]octadec-7-ene-4-carboxylic acid ethyl ester;
17-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-2,14-dioxo-3,13,15-triaza-tricyc-
lo[13.3.0.0*4,6*]octadec-7-ene-4-carboxylic acid; or a
pharmaceutically acceptable salt thereof.
107. The method according to claim 106 wherein the flavivirus
infection is HCV infection.
108. The method according to claim 106 further comprising
sequential or simultaneous administration of an additional HCV
antiviral selected from nucleoside analogue polymerase inhibitors,
protease inhibitors, ribavirin, and interferon.
109. The method according to claim 106 wherein said administering
further comprises administering said compound in a pharmaceutical
composition comprising said compound and a pharmaceutically
acceptable carrier.
110. The method of claim 109, wherein said pharmaceutical
composition further comprises an additional HCV antiviral selected
from nucleoside analogue polymerase inhibitors, protease
inhibitors, ribavirin, and interferon.
Description
TECHNICAL FIELD
[0001] This invention relates to novel inhibitors of the NS3 serine
protease of the flavivirus HCV and to methods for their use in the
treatment or prophylaxis of HCV.
BACKGROUND ART
[0002] The NS3 serine protease of HCV is a multifunctional protein
which contains a serine protease domain and a RNA helicase domain.
The protease cofactor NS4A, which is a relatively small protein, is
absolutely required for enhanced serine protease activity. The NS3
serine protease is essential in the viral lifecycle. From analysis
of the substrate binding site as revealed by X-ray crystal
structure, it has been shown that the binding site of the NS3
protease is remarkably shallow and solvent exposed making small
molecule inhibitor design a challenge.
[0003] It is believed that two HCV protease inhibitors have entered
clinical trials, namely Boehringer Ingelheim's BILN-2061 disclosed
in WO 0059929 and Vertex' VX-950 disclosed in WO 0387092. A number
of similarly peptidomimetic HCV protease inhibitors have also been
proposed in the academic and patent literature. Common for the vast
majority of such prior art peptidomimetics is the presence of an
L-proline derivative at the P2 position of the inhibitor and
interacting with the S2 subsite of the HCV protease enzyme. In the
case of BILN-2061, the L-proline is 4-substituted with a quinoline
ether, whereas VX-950 has a carboyclic ring fused to the L-proline
ring. Most peptidomimetics additionally comprise additional L-amino
acid derivatives peptide bonded at the P3 position, with many
proposed inhibitors also including additional L-amino acid
derivatives extending into P4, P5 and P6.
[0004] It has already become apparent that the sustained
administration of BILN-2061 or VX-950 selects HCV mutants which are
resistant to the respective drug, so called drug escape mutants.
These drug escape mutants have characteristic mutations in the HCV
protease genome, notably D168V, D168Y and/or A165S. Treatment
paradigms for HCV will thus have to resemble HIV treatment, where
drug escape mutations also arise readily. Accordingly, additional
drugs with different resistance patterns will consistently be
required to provide failing patients with treatment options, and
combination therapy with multiple drugs is likely to be the norm in
the future, even for first line treatment.
[0005] Experience with HIV drugs, and HIV protease inhibitors in
particular, has further emphasized that sub-optimal
pharmacokinetics and complex dosage regimes quickly result in
inadvertent compliance failures. This in turn means that the 24
hour trough concentration (minimum plasma concentration) for the
respective drugs in an HIV regime frequently falls below the
IC.sub.90 or ED.sub.90 threshold for large parts of the day. It is
considered that a 24 hour trough level of at least the IC.sub.50,
and more realistically, the IC.sub.90 or ED.sub.90 is essential to
slow down the development of drug escape mutants and achieving the
necessary pharmacokinetics and drug metabolism to allow such trough
levels provides a stringent challenge to drug design. The strongly
peptidomimetic nature of prior art HCV protease inhibitors, with
multiple peptide bonds in native configurations poses
pharmacokinetic hurdles to effective dosage regimes.
BRIEF DESCRIPTION OF THE INVENTION
[0006] In accordance with a first aspect of the invention, there
are provided compounds of the formula I:
##STR00002##
[0007] wherein
[0008] A is C(.dbd.O)OR.sup.1, C(.dbd.O)NHSO.sub.2R.sup.2,
C(.dbd.O)NHR.sup.3, or CR.sup.4R.sup.4' wherein;
[0009] R.sup.1 is hydrogen, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl;
[0010] R.sup.2 is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl;
[0011] R.sup.3 is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl,
--OC.sub.1-C.sub.6alkyl, --OC.sub.0-C.sub.3alkylcarbocyclyl,
--OC.sub.0-C.sub.3alkylheterocyclyl;
[0012] R.sup.4 is halo, amino, or OH; or R.sup.4 and R.sup.4'n
together are .dbd.O;
[0013] R.sup.4' is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl;
[0014] wherein R.sup.2, R.sup.3, and R.sup.4' are each optionally
substituted with 1 to 3 substituents independently selected from
the group consisting of halo, oxo, nitrile, azido, nitro,
C.sub.1-C.sub.6alkyl, C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl, NH.sub.2CO--, Y--NRaRb,
Y--O--R.sub.b, Y--C(.dbd.O)Rb, Y--(C.dbd.O)NRaRb,
Y--NRaC(.dbd.O)Rb, Y--NHSO.sub.pRb, Y--S(.dbd.O).sub.pRb,
Y--S(.dbd.O).sub.pNRaRb, Y--C(.dbd.O)Orb and
Y--NRaC(.dbd.O)ORb;
[0015] Y is independently a bond or C.sub.1-C.sub.3alkylene;
[0016] Ra is independently H or C.sub.1-C.sub.3alkyl;
[0017] Rb is independently H, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl or
C.sub.0-C.sub.3alkylheterocyclyl;
[0018] p is independently 1 or 2;
[0019] M is CR.sup.7R.sup.7' or NRu;
[0020] R.sup.7 is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylC.sub.3-C.sub.7cycloalkyl, or
C.sub.2-C.sub.6alkenyl, any of which is optionally substituted with
1-3 halo atoms, or an amino, --SH or C.sub.0-C.sub.3alkylcycloalkyl
group; or
[0021] R.sup.7 is J;
[0022] R.sup.7' is H or taken together with R.sup.7 forms a
C.sub.3-C.sub.6cycloalkyl ring optionally substituted with
R.sup.7'a wherein; [0023] R.sup.7'a is C.sub.1-C.sub.6alkyl,
C.sub.3-C.sub.5cycloalkyl, C.sub.2-C.sub.6alkenyl any of which may
be optionally substituted with halo; or R.sup.7'a can be J;
[0024] q is 0 to 3 and k is 0to 3; where q+k.gtoreq.1;
[0025] W is --CH.sub.2--, --O--, --OC(.dbd.O)H--, --OC(.dbd.O)--,
--S--, --NH--, --NRa, --NHSO.sub.2--, --NHC(.dbd.O)NH--or
--NHC(.dbd.O)--, --NHC(.dbd.S)NH-- or a bond;
[0026] R.sup.8 is a ring system containing 1 or 2 saturated,
partially unsaturated or unsaturated rings each of which has 4-7
ring atoms and each of which has 0 to 4 hetero atoms independently
selected from S, O and N, the ring system being optionally spaced
from W by a C.sub.1-C.sub.3 alkyl group; or R.sup.8 is
C.sub.1-C.sub.6 alkyl; any of which R.sup.8 groups can be
optionally mono, di, or tri substituted with R.sup.9, wherein
[0027] R.sup.9 is independently selected from the group consisting
of halo, oxo, nitrile, azido, nitro, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl,
NH.sub.2C(.dbd.O)--, Y--NRaRb, Y--O--Rb, Y--C(.dbd.O)Rb,
Y--(C.dbd.O)NRaRb, Y--NRaC(.dbd.O)Rb, Y--NHSO.sub.pRb,
Y--S(.dbd.O).sub.pRb, Y--S(.dbd.O).sub.pNRaRb, Y--C(.dbd.O)ORb and
Y--NRaC(.dbd.O)ORb; wherein said carbocyclyl or heterocyclyl moiety
is optionally substituted with R.sup.10; wherein [0028] R.sup.10 is
C.sub.1-C.sub.6alkyl, C.sub.3-C.sub.7cycloalkyl,
C.sub.1-C.sub.6alkoxy, amino, sulfonyl, (C.sub.1-C.sub.3
alkyl)sulfonyl, NO.sub.2, OH, SH, halo, haloalkyl, carboxyl,
amido;
[0029] E is --C(.dbd.O)--, --C(.dbd.S)--, --S(.dbd.O).sub.2--,
--S(.dbd.O)--, --C(.dbd.N--Rf)--;
[0030] Rf is H, --CN, --C(.dbd.O)NRaRb;
--C(.dbd.O)C.sub.1-C.sub.3alkyl;
[0031] X is --NRx- where Rx is H, C.sub.1-C.sub.5alkyl or J; or in
the case where E is --C(.dbd.O), X can also be --O-- or
--NRjNRj-;
[0032] wherein one of Rj is H and the other is H, C.sub.1-C.sub.5
alkyl or J;
[0033] R.sup.11 is H, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl,
any of which can be substituted with halo, oxo, nitrile, azido,
nitro, C.sub.1-C.sub.6alkyl, C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl, NH.sub.2CO--, Y--NRaRb, Y--O--Rb,
Y--C(.dbd.O)Rb, Y--(C.dbd.O)NRaRb, Y--NRaC(.dbd.O)Rb,
Y--NHSO.sub.pRb, Y--S(.dbd.O).sub.pRb, Y--S(.dbd.O).sub.pNRaRb,
Y--C(.dbd.O)ORb, Y--NRaC(.dbd.O)ORb; or R.sup.11 is J;
[0034] J, if present, is a single 3 to 10-membered saturated or
partially unsaturated alkylene chain extending from the
R.sup.7/R.sup.7' cycloalkyl or from the carbon atom to which
R.sup.7 is attached to one of Rj, Rx, Ry or R.sup.11 to form a
macrocycle, which chain is optionally interrupted by one to three
heteroatoms independently selected from: --O--, --S-- or
--NR.sup.12--, and wherein 0 to 3 carbon atoms in the chain are
optionally substituted with R.sup.14; [0035] wherein; [0036]
R.sup.12 is H, C.sub.1-C.sub.6alkyl, C.sub.3-C.sub.6cycloalkyl, or
C(.dbd.O)R.sup.13; [0037] R.sup.13 is C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl;
[0038] R.sup.14 is independently selected from the group consisting
of H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy, hydroxy, halo, amino, oxo, thio and
C.sub.1-C.sub.6thioalkyl;
[0039] Ru is independently H or C.sub.1-C.sub.3alkyl;
[0040] m is 0 or 1;n is 0 or 1;
[0041] U is .dbd.O or is absent;
[0042] R.sup.15 is H, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl,
any of which can be substituted with halo, oxo, nitrile, azido,
nitro, C.sub.1-C.sub.6 alkyl, C.sub.0-C.sub.3alkylheterocyclyl,
C.sub.0-C.sub.3alkylcarbocyclyl, NH.sub.2CO--, Y--NRaRb, Y--O--Rb,
Y--C(.dbd.O)Rb, Y--(C.dbd.O)NRaRb, Y--NRaC(.dbd.O)Rb,
Y--NHS(.dbd.O).sub.pRb, Y--S(.dbd.O).sub.pRb,
Y--S(.dbd.O).sub.pNRaRb, Y--C(.dbd.O)ORb, Y--NRaC(.dbd.O)ORb;
[0043] G is --O--, --NRy-, --NRjNRj-: where one Rj is H and the
other is H, C.sub.1-C.sub.5 alkyl or J;
[0044] Ry is H, C.sub.1-C.sub.3 alkyl; or Ry is J;
[0045] R.sup.16 is H; or C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl,
any of which can be substituted with halo, oxo, nitrile, azido,
nitro, C.sub.1-C.sub.6alkyl, C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl, NH.sub.2CO--, Y--NRaRb, Y--O--Rb,
Y--C(.dbd.O)Rb, Y--(C.dbd.O)NRaRb, Y--NRaC(.dbd.O)Rb,
Y--NHSO.sub.pRb, Y--S(.dbd.O).sub.pRb, Y--S(.dbd.O).sub.pNRaRb,
Y--C(.dbd.O)ORb, Y--NRaC(.dbd.O)ORb;
[0046] with the proviso that when m=n=0 and G is 0 then R.sup.16 is
not tert.butyl or phenyl;
[0047] or a pharmaceutically acceptable salt or prodrug
thereof.
[0048] Without in any way wishing to be bound by theory, or the
ascription of tentative binding modes for specific variables, the
notional concepts P1, P2, P3 and P4 as used herein are provided for
convenience only and have substantially their conventional
meanings, as illustrated by Schechter & Berger, (1976) Biochem
Biophys Res Comm 27 157-162, and denote those portions of the
inhibitor believed to fill the S1, S2, S3 and S4 subsites
respectively of the enzyme, where S1 is adjacent the cleavage site
and S4 remote from the cleavage site. Regardless of binding mode,
the components defined by Formula I are intended to be within the
scope of the invention. For example it is expected that capping
group R.sup.16-G may interact with the S3 and S4 subsites
especially when m and/or n is 0.
[0049] Various embodiments of the present invention can be
notionally represented as R.sup.16-G-P4-P3-link-P2-P1, wherein P3
and/or P4 may be absent, and P1, P3 and P4 each represents a
building block constituted of a derivative of a natural or
unnatural amino acid, P2 is a heterocyclic residue and G-R.sup.16
is a capping group. The link is a carbonyl or other function as
defined for E. The P1 and P2 building blocks and the P3 and P4
building blocks are thus typically linked together by amide bonds
whereas the P2 and P3 building blocks are linked through the above
described link. The amide bonds are thereby typically reversed
relative to each other on each side of the link in the compounds of
the invention.
[0050] Additional aspects of the invention include a pharmaceutical
composition comprising a compound of the invention as defined above
and a pharmaceutically acceptable carrier or diluent therefor.
[0051] The compounds and compositions of the invention have utility
in methods of medical treatment or prophylaxis of HCV infections in
humans. Accordingly, a further aspect of the invention is the use
of a compound as defined above in therapy, such as in the
manufacture of a medicament for the prophylaxis or treatment of
flavivirus infections in humans or animals. Exemplary flavivirus
include BVDV, dengue and especially HCV.
[0052] The compounds of the invention have a non-peptidic linkage
at the bond between P2 and P3 building blocks, resulting in the
orientation of the P3 and P4 residues being reversed relative to a
native substrate. This non-peptidic link is also typically longer
than the corresponding peptide bond would have been and means that
the P3 and/or P4 groups (including the R16 cap to the extent this
interacts with S3 or S4) are displaced outwardly relative to a
native peptide substrate. Reversal and displacement in this fashion
would be expected to favour the non-natural D stereochemistries for
the pocket filling groups (eg side chains) of P3 and/or P4 and/or
R16. Indeed, such compounds are typically highly active and within
the scope of the invention. However, it has been surprisingly found
that even compounds of the invention bearing L-amino acid side
chains at P3 and/or P4 exhibit good activity, notwithstanding that
the respective side chain entity must approach the S3 or S4 pocket
from a different angle relative to a native peptide substrate.
Accordingly L-stereochemistry at R.sup.11 and/or R.sup.15 and/or
the corresponding configuration at R.sup.16 to mimic L
stereochemistry represents a favoured aspect of the invention.
[0053] The different angle of approach to the S3 and/or S4 pockets
also has implications for the ability of the compounds of the
invention to avoid resistance patterns exhibited by prior art HCV
protease inhibitors which hitherto have all had a conventional
peptide backbone of natural or non-natural L-amino acid residues.
As with the reverse transcriptase of HIV which is notorious for
quickly generating drug escape mutants under the selective pressure
of antiviral therapy, the RNA dependent RNA polymerase NS5A of HCV
has a very poor proof reading capacity. This in turn means that the
HCV polymerase is highly error prone and it is likely that
characteristic resistance patterns will arise when HCV antivirals
are administered over long periods. Even before launch, it is
apparent that BILN 2061 with a substantially peptidic backbone
(albeit macrocyclised) and Vertex' NS3 protease inhibitor VX-950
with a linear peptide backbone at P3 and P4 quickly give rise to
characteristic resistance mutations at positions 155, 156 or 168 of
the NS3 protease (Lin et al J Biol Chem 2004 279(17):1
7808-17).
[0054] A preferred group of compounds of the invention comprises
those wherein P1 represents a hydrazine derivative, that is M is
NRu where Ru is typically H or C.sub.1-C.sub.3alkyl. Compounds
wherein M is CR.sup.7R.sup.7' constitute a further preferred aspect
of the invention.
[0055] Preferred embodiments wherein M is CR.sup.7R.sup.7' in
formulae I include formulae IA:
##STR00003##
[0056] Preferred values for q and k in Formula I include 2:1, 2:2,
2:3, 3:2, 3:3, more preferably 1:2 and 1:0; and most preferably
1:1, in which case preferred compounds have the partial
structure:
##STR00004##
[0057] where e is 1 or 2.
[0058] It is currently preferred that E is --C(.dbd.O)-- or
--C.dbd.N--Rf, for example where Rf is --CN or
--C(.dbd.O)NH.sub.2.
[0059] Compounds of the invention may comprise both a P3 and a P4
function, viz m and n are each 1. Favoured embodiments within
formula I include formula Ida-Idd below:
##STR00005##
[0060] Alternative embodiments include the structures corresponding
to Ida, Idb, Idc and Idd wherein M is NRu.
[0061] Alternative configurations of the compounds of the invention
comprise a P3, but no P4 function, viz m is 1 and n is zero.
Preferred embodiments within Formula I include formulae Iea-Iee
below:
##STR00006##
[0062] Alternative embodiments include the structures corresponding
to Iea, Ieb, Iec, Ied and Iee wherein M is NRu.
[0063] Still further alternative configurations of the compounds of
the invention include those where m and n are zero and thus groups
R.sup.16-G abut P2, but as mentioned above, the capping group R16-G
may interact favorably with S3 and/or S4.
[0064] Favoured embodiments within Formula I include formulae
Ifa-Ife below:
##STR00007##
[0065] R.sup.16 in figure Ifb and elsewhere is typically H,
C.sub.1-C.sub.3alkyl, C.sub.5-C.sub.6alkyl,
C.sub.0-C.sub.3alkylheterocyclyl, C.sub.1-C.sub.3alkylcarbocyclyl
or C.sub.3-C.sub.7cycloalkyl, any of which being optionally
substituted, as described above. For example, R.sup.16 ca be phenyl
substituted as described above.
[0066] Alternative embodiments include the structures corresponding
to Ifa, Ifb, Ifc, Ife, and Ife wherein M is NRu.
[0067] The compounds of the invention may comprise linear
molecules, as depicted above. Alternatively, in embodiments wherein
R.sup.7 and R.sup.7' together define a spiro cycloalkyl group, such
as spiro-cyclopropyl, the compounds of the invention may be
configured as macrocycles, wherein a linking group J extends
between one of Rj, Rx, Ry or R.sup.11 of formula I. Alternatively
the macrocycle J may extend from the carbon adjacent to R.sup.7 to
one of Rj, Rx, Ry or R.sup.11.
[0068] Favoured embodiments of such macrocyclic structures within
formula I wherein m is 0 and n is 1 include those of Formulae
Iga-Igd below:
##STR00008##
[0069] The corresponding structures wherein the J chain bonds to
the carbon adjacent R.sup.7 are also favoured.
[0070] Additional favoured embodiments of such macrocyclic
structures within formula I wherein m is 0 and n is 1 include those
of Formulae Ige-Igf below:
##STR00009##
[0071] The corresponding structures wherein the J chain bonds to
the carbon adjacent R.sup.7 are also favoured
[0072] Favoured macrocyclic structures within Formula I, comprising
both a P3 and P4 function, ie wherein m and n are each 1, include
those of the formulae Iha-Ihd below.
##STR00010##
[0073] The corresponding structures wherein the J chain bonds to
the carbon adjacent R.sup.7 are also favoured.
[0074] Favoured macrocyclic structures within Formula I, wherein
both of the P3 and P4 functions are absent, i.e. wherein m and n
are each 0, include those of the formulae Ihe-Ihh below, especially
1he and 1hf.
##STR00011##
[0075] The corresponding structures wherein the J chain bonds to
the carbon adjacent R.sup.7 are also favoured, especially formula
Ihe and 1hf:
[0076] In general, in the optionally macrocyclic structures such as
those illustrated above, linker J is a 3 to 10 chain atom,
preferably 5 to 8 chain atom, such as 6 or 7 chain atom, saturated
alkylene chain or a partially unsaturated alkylene chain, that is
an alkylene chain bearing 1 to 3 unsaturated bonds between adjacent
carbons, typically one unsaturation. The length of the chain will,
of course, depend on whether J extends from Rd, Rj, Rx, Ry,
R.sup.11 or the carbon adjacent to R.sup.7. Suitable chains are
described in detail in WO 00/59929. Typically J will be dimensioned
to provide a macrocycle of 13 to 16 ring atoms (including those
atoms in the P1, P2 and if present P3 groups contributing to the
ring). Conveniently J is dimensioned to provide a macrocycle of 14
or 15 ring atoms.
[0077] Conveniently, the J chain contains one or two heteroatoms
selected from: O, S, NH, NC.sub.1-C.sub.6 alkyl or
N--C(.dbd.O)C.sub.1-C.sub.6alkyl. More preferably, the J chain
optionally contains one heteroatom selected from: NH, or
N--C(.dbd.O)C.sub.1-C.sub.6alkyl, most preferably N(Ac). Most
preferably, the chain containing a nitrogen atom is saturated. In
an alternative embodiment, J contains one heteroatom selected from
O or S. The chain may be substituted with R.sup.14, such as H or
methyl.
[0078] Typically, the J linker structure is saturated.
Alternatively, J contains 1 to 3, preferably 1 double bond,
typically spaced one carbon from the cycloalkyl R.sup.7 function,
if present. The double bond may be cis or trans.
[0079] Representative examples of J thus include pentylene,
hexylene, heptylene, any of which are substituted with
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy, hydroxyl, halo, amino, oxo, thio or
C.sub.1-C.sub.6 thioalkyl; penten-3-yl, hexen-4-yl, hepten-5-yl,
where 3, 4 or 5 refers to a double bond between carbon atoms 3 and
4, 4 and 5 etc.
[0080] Convenient R.sup.7 and R.sup.7' groups include those wherein
R.sup.7' is H and R.sup.7 is n-ethyl, n-propyl, cyclopropylmethyl,
cyclopropyl, cyclobutylmethyl, cyclobutyl, 2,2-difluoroethyl, or
mercaptomethyl. Preferred embodiments include those wherein R.sup.7
is n-propyl or 2,2-difluoroethyl.
[0081] Alternative favoured configurations for R.sup.7 and R.sup.7'
include those wherein R.sup.7' is H and R.sup.7 is C.sub.3-C.sub.7
cycloalkyl or C.sub.1-C.sub.3alkylC.sub.3-C.sub.7cycloalkyl.
[0082] Still further favoured configurations for R.sup.7 and
R.sup.7' include these wherein R.sup.7' is H and R.sup.7 is J.
[0083] Alternatively, R.sup.7 and R.sup.7' together define a
spiro-cycloalkyl function, such as a spiro-cyclobutyl ring, and
more preferably a spiro-cyclopropyl ring. "Spiro" in this context
simply means that the cycloalkyl ring shares a single carbon atom
with the peptidic backbone of the compound. The ring is substituted
or unsubstituted. Preferred substituents include mono or
di-substitutions with R.sup.7'a wherein R.sup.7'a is
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.5cycloalkyl, or
C.sub.2-C.sub.6 alkenyl, any of which is optionally substituted
with halo. Alternatively the substituent may be a J linker as
described above. Currently preferred stereochemistries for a
spiro-cyclopropyl ring are defined below.
[0084] Particularly preferred substituents include R.sup.7'a as
ethyl, vinyl, cyclopropyl (ie a spiro-cyclopropyl substituent to
the "spiro" cycloalkyl ring of R.sup.7/R.sup.7'), 1- or
2-bromoethyl, 1-or 2-fluoroethyl, 2-bromovinyl or 2-fluorethyl.
[0085] In one embodiment of the invention, A is --CR.sup.4R.sup.4'
as illustrated in detail in PCT/EP03/10595, the contents of which
are incorporated by reference.
[0086] Convenient R.sup.4' groups thus include
C.sub.1-C.sub.6alkyl, such as methyl, ethyl, propyl, ethenyl and
--CHCHCH.sub.3 Alternative preferred R.sup.4' groups include aryl
or heteroaryl such as optionally substituted phenyl, pyridyl,
thiazolyl or benzimidazolyl or C.sub.1-C.sub.3alkylaryl or
C.sub.1-C.sub.3alkylheteroaryl, where the alkyl moiety is methyl,
ethyl, propyl, ethenyl and --CH.dbd.CHCH.sub.3. Preferred aryl
moieties include optionally substituted: phenyl, benzothiazole and
benzimidazole.
[0087] Favoured R.sup.4 groups include --NH.sub.2, fluoro or
chloro. Alternative preferred R.sup.4 groups include --OH and
especially .dbd.O.
[0088] An alternative embodiment for A is C(.dbd.O)NHR.sup.3, where
R.sup.3 is optionally substituted C.sub.0-C.sub.3alkylaryl,
C.sub.0-C.sub.3alkylheteroaryl, OC.sub.0-C.sub.3alkylaryl or
OC.sub.0-C.sub.3alkylheteroaryl. Appropriate substituents appear in
the definitions section below.
[0089] A currently favoured configuration for A is
C(.dbd.O)OR.sup.1, especially where R.sup.1 is
C.sub.1-C.sub.6alkyl, such as methyl, ethyl, or tert-butyl and most
preferably hydrogen.
[0090] A particularly preferred configuration for A is
C(.dbd.O)NHSO.sub.2R.sup.2, especially where R.sup.2 is optionally
substituted C.sub.1-C.sub.6alkyl, preferably methyl, or optionally
substituted C.sub.3-C.sub.7cycloalkyl, preferably cyclopropyl, or
optionally substituted C.sub.0-C.sub.6alkylaryl, preferably
optionally substituted phenyl. Appropriate substituents appear in
the definitions section below.
[0091] Substituent --W--R8 on the cyclic P2 group can employ any of
the proline substituents which are extensively described in WO
00/59929, WO 00/09543, WO 00/09558, WO 99/07734, WO 99/07733, WO
02/60926, W003/35060, WO 03/53349, WO03/064416, W=03/66103,
WO03/064455, WO03/064456, WO03/62265, WO03/062228, WO03/87092, WO
03/99274, WO03/99316, WO03/99274, WO04/03670, WO04/032827,
WO04/037855, WO04/43339, WO04/92161, WO04/72243, 5WO04/93798.
WO04/93915, WO04/94452, WO04/101505, WO04/101602, WO04/103996,
WO04113365 and the like.
[0092] Favoured W functions include W as --OC(.dbd.O)NH--,
--OC(.dbd.O)--, --NH--, --NR.sup.8'--, --NHS(O).sub.2-- or
--NHC(.dbd.O)--, especially --OC(.dbd.O)NH-- or --NH--. Favoured
R.sup.8 groups for such W functions include optionally substituted
C.sub.0-C.sub.3alkylcarbocyclyl or
C.sub.0-C.sub.3alkyl-heterocyclyl, including those described in
WO0009543, WO0009558 and WO 00/174768. For example ester
substituents, --W--R.sup.8, on the cyclic P2 group, include those
disclosed in WO 01/74768 such as C.sub.1-C.sub.6alkanoyloxy,
C.sub.0-C.sub.3alkylaryloyloxy, particularly (optionally
substituted) benzoyloxy or C.sub.0-C.sub.3alkylheterocycloyloxy,
especially
##STR00012##
[0093] This publication also describes alternative --W--R.sup.8
possibilities for example C.sub.1-C.sub.6alkyl, such as ethyl,
isopropyl, C.sub.0-C.sub.3alkylcarbocyclyl such as cyclohexyl,
2,2-difluoroethyl, --C(.dbd.O)NRc, where Rc is C.sub.1-C.sub.6
alkyl, C.sub.0-C.sub.3alkylcyclopropyl, C.sub.0-C.sub.3alkylaryl or
C.sub.0-C.sub.3alkylheterocyclyl.
[0094] Currently preferred W functions include --S-- and especially
--O--. Convenient values for R.sup.8 in such embodiments include
C.sub.0-C.sub.3alkylaryl, or C.sub.0-C.sub.3alkylheteroaryl either
of which is optionally mono, di, or tri substituted with R.sup.9,
wherein; [0095] R.sup.9 is C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6alkoxy, NO.sub.2, OH, halo, trifluoromethyl, amino
or amido (for example amido or amino optionally mono- or
di-substituted with C.sub.1-C.sub.6alkyl),
C.sub.0-C.sub.3alkylaryl, C.sub.0-C.sub.3alkylheteroaryl, or
carboxyl, wherein the aryl or heteroaryl moiety is optionally
substituted with R.sup.10; wherein [0096] R.sup.10 is
C.sub.1-C.sub.6alkyl, C.sub.3-C.sub.7cycloalkyl,
C.sub.1-C.sub.6alkoxy, amino, amido, sulfonylC.sub.1-C.sub.3alkyl,
NO.sub.2, OH, halo, trifluoromethyl, carboxyl, or heteroaryl.
[0097] Typically, the C.sub.0-C.sub.3 alkyl component of R.sup.8 as
C.sub.0-C.sub.3alkylaryl, or C.sub.0-C.sub.3alkylheteroaryl is
methyl and especially absent, ie C.sub.0. The aryl or heteroaryl
component is as extensively illustrated in the definition section
below.
[0098] Preferred R.sup.9 include C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6alkoxy, amino, (such as
di-C.sub.1-C.sub.3alkylamino), amido (such as
--NHC(O)C.sub.1-C.sub.6alkyl or C(.dbd.O)NHC.sub.1-C.sub.6alkyl),
aryl or heteroaryl, the aryl or heteroaryl being optionally
substituted with R.sup.10; wherein [0099] R.sup.10 is
C.sub.1-C.sub.6alkyl, C.sub.3-C.sub.7cycloalkyl,
C.sub.1-C.sub.6alkoxy, amino,(such as mono- or di-C.sub.1-C.sub.3
alkylamino), amido (such as as --NHC(O)C.sub.1-C.sub.3alkyl or
C(.dbd.O)NHC.sub.1-C.sub.3alkyl), halo, trifluoromethyl, or
heteroaryl.
[0100] Preferred R.sup.10 include C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, amino, amido (such as as
--NHC(O)C.sub.1-C.sub.6alkyl or C(.dbd.O)NHC.sub.1-C.sub.6alkyl),
halo, or heteroaryl.
[0101] Particularly preferred R.sup.10 include methyl, ethyl,
isopropyl, tert-butyl, methoxy, chloro, amino, amido (such as as
--NHC(O)C.sub.1-C.sub.6alkyl, for example
--NC(.dbd.O)CHC(CH.sub.3).sub.3, or
C(.dbd.O)NHC.sub.1-C.sub.3alkyl) or C.sub.1-C.sub.3alkyl
thiazole.
[0102] Favoured embodiments of R.sup.8 include 1-naphthylmethyl,
2-naphthylmethyl, benzyl, 1-naphthyl, 2-naphthyl, or quinolinyl,
any of which is unsubstituted, mono, or disubstituted with R.sup.9
as defined, in particular 1-naphthylmethyl, or quinolinyl
unsubstituted, mono-, or disubstituted with R.sup.9 as defined.
[0103] A currently preferred R.sup.8 is:
##STR00013##
[0104] wherein R.sup.9a is C.sub.1-C.sub.6alkyl;
C.sub.1-C.sub.6alkoxy; thioC.sub.1-C.sub.3alkyl; amino optionally
substituted with C.sub.1-C.sub.6alkyl; C.sub.0-C.sub.3alkylaryl; or
C.sub.0-C.sub.3alkylheteroaryl, C.sub.0-C.sub.3alkylheterocyclyl,
said aryl, heteroaryl or heterocycle being optionally substituted
with R.sup.10 wherein [0105] R.sup.10 is C.sub.1-C.sub.6alkyl,
C.sub.3-C.sub.7cycloalkyl, C.sub.1-C.sub.6alkoxy, amino, amido,
heteroaryl or heterocyclyl; and
[0106] R.sup.9b is C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy,
amino, amido, NO.sub.2, OH, halo, trifluoromethyl, carboxyl.
[0107] Convenient R.sup.9a include aryl or heteroaryl, all
optionally substituted with R.sup.10 as defined, especially where
R.sup.9a is selected from the group consisted of:
##STR00014##
[0108] wherein R.sup.10 is H, C.sub.1-C.sub.6alkyl, or
C.sub.0-C.sub.3alkyl-C.sub.3-C.sub.6cycloalkyl, amino optionally
mono- or di-substituted with C.sub.1-C.sub.6alkyl, amido (such as
as --NHC(O)C.sub.1-C.sub.6alkyl or
C(.dbd.O)NHC.sub.1-C.sub.6alkyl), heteroaryl or heterocyclyl.
[0109] R.sup.9a is conveniently phenyl and thus R.sup.8 is:
##STR00015##
[0110] wherein R.sup.10a is H, C.sub.1-C.sub.6alkyl;
C.sub.1-C.sub.6alkoxy; or halo; and R.sup.9b is C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6-alkoxy, amino such as
di(C.sub.1-C.sub.3alkyl)amine, amido (such as as
--NHC(O)C.sub.1-C.sub.3alkyl or C(.dbd.O)NHC.sub.1-C.sub.3alkyl),
NO.sub.2, OH, halo, trifluoromethyl, carboxyl.
[0111] An alternative preferred R.sup.8 is:
##STR00016##
[0112] wherein R.sup.10a is H, C.sub.1-C.sub.6alkyl, or
C.sub.0-C.sub.3alkyl-C.sub.3-C.sub.6cycloalkyl, amine (such as
amine mono- or di-substituted with C.sub.1-C.sub.6alkyl), amido
(such as as --NHC(O)C.sub.1-C.sub.6alkyl or
C(.dbd.O)NHC.sub.1-C.sub.6alkyl), heteroaryl or heterocyclyl; and
R.sup.9b is C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6-alkoxy, amino
(such as di(C.sub.1-C.sub.3 alkyl)amino), amido (such as as
--NHC(O)C.sub.1-C.sub.3alkyl or C(.dbd.O)NHC.sub.1-C.sub.3alkyl),
NO.sub.2, OH, halo, trifluoromethyl, or carboxyl.
[0113] In the immediately above described embodiments R.sup.9b is
conveniently C.sub.1-C.sub.6-alkoxy, preferably methoxy.
[0114] A further convenient R.sup.8, for example when W is an
ether, has the formula
##STR00017##
[0115] where W' is N or CH, r is 0 or 1, Ra' is H, C.sub.1-C.sub.6
alkyl, C.sub.0-C.sub.3alkylcycloalkyl, C.sub.1-C.sub.6alkyloxy,
hydroxy or amine and Rb' is H, halo, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcycloalkyl, C.sub.1-C.sub.6alkyloxy,
C.sub.1-C.sub.6thioalkyl, cycloalkylC.sub.0-C.sub.3alkyloxy,
C.sub.1-C.sub.3alkyloxyC.sub.1-C.sub.3alkyl,
C.sub.0-C.sub.3alkylaryl or C.sub.0-C.sub.3alkylheterocyclyl. A
particularly preferred ether substituent is
7-methoxy-2-phenyl-quinolin-4-yl oxy.
[0116] When W is a bond then R.sup.8 is preferably a substituted or
unsubstituted heterocyclic ring system as described in
WO2004/072243 or WO2004/113665.
[0117] Representative examples of R.sup.8 when W is a bond include
the following aromatics which may optionally be substituted:
1H-pyrrole, 1H-imidazole, 1H-pyrazole, furan, thiophene, oxazole,
thiazole, isoxazole, isothiazole, pyridine, pyndazine, pyrimidine,
pyrazine, phthalazine, quinoxaline, quinazoline, quinoline,
cinnoline, 1H-pyrrolo[2,3]-b]pyridine, 1H-indole,
1H-benzoimidazole, 1H-indazole, 7H-purine, benzothiazole,
benzooxazole, 1H-imidazo[4,5-c]pyridine, 1H-imidazo[4,5-b]pyridine,
1, 3-dihydro-benzoimidazol-2-one,
1,3-dihydro-benzoimidazol-2-thione, 2,3-dihydro-1H-indole,
1,3-dihydro-indol-2-one, 1H-indole-2,3-dione,
1,3-dihydro-benzoimidazole-2-one, 1H, 1H-pyrrolo[2, 3-c]pyridine,
benzofuran, benzo[b]thiophene, benzo[d]isoxazole,
benzo[d]isothiazole, 1H-quinotin-2-one, 1H-quinolin-4-one,
1H-quinazolin-4-one, 9H-carbazole, 1H-quinazolin-2-one.
[0118] Additional representative examples of R.sup.8 when W is a
bond, include the following non-aromatics, which may be optionally
substituted: aziridine, azetidine, pyrrolidine,
4,5-dihydro-1H-pyrazole, pyrazolidine, imidazolidin-2-one,
imidazolidine-2-thione, pyrrolidin-2-one, pyrolidine-2,5-dione,
piperidine-2,6-dione, piperidin-2-one, piperazine-2,6-dione,
piperazin-2-one, piperazine, morpholine,
thiomorpholine-1,1-dioxide, pyrazolidin-3-one,
imidazolidine-2,4-dione, piperidine, tetrahydrofuran,
tetrahydropyran, [1,4]dioxane, 1,2,3,6-tetrahydropyridine.
[0119] Preferred values for R.sup.8 when W is a bond, include
tetrazole and derivatives thereof. The tetrazole moiety is linked
to the cyclic P2 scaffold and optionally substituted as shown
below:
##STR00018##
wherein Q* is selected from the group consisting of absent,
--CH.sub.2--, --O--, --NH--, --N(R.sup.1*), --S--,
--S(.dbd.O).sub.2-- and --(C.dbd.O)--; Q* is selected from the
group consisting of: absent, --CH.sub.2-- and --NH; Y* is selected
from the group consisting of: H, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3aryl, C.sub.0-C.sub.3heterocyclyl; R.sup.1* is
selected from the group consisting of: H, C.sub.1-C.sub.6alkyl,
carbocyclyl, C.sub.0-C.sub.3aryl, C.sub.0-C.sub.3heterocyclyl,
[0120] Representative examples of substituted tetrazoles are as
described in table 1 of WO2004/072243 and the structures following
immediately after, or WO2004/113665.
[0121] Further preferred values for R.sup.8 when W is a bond,
include triazole and derivatives thereof. The triazole moiety is
linked to the cyclic P2 scaffold and optionally substituted as
shown below:
##STR00019##
[0122] wherein X* and Y* are independently selected from the group
consisting of: H, halogen, C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3carbocyclyl, --CH.sub.2-amino, --CH.sub.2-arylamino,
--CH.sub.2-diarylamino, --(C.dbd.O)-amino, --(C.dbd.O)-arylamino,
--(C.dbd.O)-diarylamino, C.sub.0-C.sub.3aryl,
C.sub.0-C.sub.3heterocyclyl or alternatively, X* and Y* taken
together with the carbon atoms to which they are attached, form a
cyclic moiety selected from the group consisting of aryl and
heteroaryl.
[0123] Representative examples of substituted triazoles are as
described in table 2 of WO2004/072243 and the structures following
immediately after, and in the tables of WO20041113365.
[0124] Further preferred values for R.sup.8 when W is a bond,
include pyridazinone and derivatives thereof. The pyridazinone
moiety is linked to the cyclic P2 scaffold and optionally
substituted as shown below:
##STR00020##
[0125] wherein X*, Y* and Z* are independently selected from the
group consisting of: H, N.sub.3, halogen, C.sub.1-C.sub.6alkyl,
carbocyclyl, amino, C.sub.0-C.sub.3aryl, --S-aryl, --O-aryl,
--NH-aryl, diarylamino, diheteroarylamino,
C.sub.0-C.sub.3heterocyclyl, --S-heteroaryl, --O-heteroaryl,
NH-heteroaryl or, alternatively, X and Y or Y and Z taken together
with the carbon atoms to which they are attached, form an aryl or
heteroaryl cyclic moiety.
[0126] Representative examples of substituted pyridazinones are as
described in table 3 of WO2004/072243 and the structures following
immediately after, and in the tables of WO2004/113365.
[0127] Preferred P3 groups, i.e. when m is 1, resemble natural or
unnatural amino acids, especially aliphatic amino acids, such as
L-valyl, L-leucyl, L-isoleucyl or L-t-leucyl Further preferred P3
groups, as shown in WO 02/01898 include
C.sub.0-C.sub.3alkylcycloalkylalanine, especially
cyclohexylalanine, optionally substituted with CO.sub.2Rg, where Rg
is H, is C.sub.1-C.sub.6alkyl, C.sub.0-C.sub.3alkylaryl,
C.sub.0-C.sub.3alkylheterocyclyl, C.sub.0-C.sub.3alkylcycloalkyl or
amine; or N-acetylpiperidine or tetrahydropyran. Preferred R.sup.11
groups thus include C.sub.1-C.sub.6alkyl,
C.sub.0-C.sub.3alkylcarbocyclyl for example
C.sub.0-C.sub.3alkylC.sub.3-C.sub.7cycloalkylyl,
C.sub.0-C.sub.3alkylaryl or C.sub.0-C.sub.3alkylheteroaryl, any of
which is optionally substituted with hydroxy, halo, amino,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6thioalkyl,
C(.dbd.O)OR.sup.14, carboxyl, (C.sub.1-C.sub.6alkoxy)carbonyl,
aryl, heteroaryl or heterocyclyl, especially where the substituent
is hydroxy or C(.dbd.O)OR.sup.14.
[0128] Particularly preferred R.sup.11 include tert-butyl,
iso-butyl, cyclohexyl, phenylethyl, 2,2-dimethyl-propyl,
cyclohexylmethyl, phenylmethyl, 2-pyridylmethyl,
4-hydroxy-phenylmethyl, or carboxylpropyl. The most preferred
R.sup.11 values are currently tert-butyl, isobutyl, or
cyclohexyl.
[0129] An embodiment of the invention include compounds wherein P4
is absent (ie n is 0) and wherein the P3 function lacks a carbonyl,
ie U is absent. Representative substructures include those of
formula Ii below:
##STR00021##
[0130] wherein
[0131] Rx and Ry are as defined above, preferably H,
[0132] R.sup.11' is C.sub.1-C.sub.6 alkyl, preferably
C.sub.3-C.sub.5 branched alkyl such as the side chains of L-valyl,
L-leucyl, L-isoleucyl, L-t-leucyl; or
C.sub.0-C.sub.2alkylC.sub.3-C.sub.7 cycloalkyl such as cyclohexyl
or cyclohexylmethyl;
[0133] R.sup.16a is -Rba, --S(.dbd.O)pRba, --C(.dbd.O)Rba;
[0134] Rba is C.sub.1-C.sub.6 alkyl,
C.sub.0-C.sub.3alkylheterocyclyl,
C.sub.0-C.sub.3alkylcarbocyclyl.
[0135] Alternatively, compounds of partial structure Ii may be
macrocyclised between an appropriate value of R.sup.7 and one of
Rx, Ry or R.sup.11'.
[0136] Representative embodiments of P3 groups which lack a carboxy
function (ie variable U is absent) include those of formula Iia-Iid
below:
##STR00022##
[0137] where Ar is carbocyclyl or heterocyclyl, especially aryl or
heteroaryl, any of which is optionally substituted with R.sup.9.
Although the partial structures of Formulae Iia-Iid have been
illustrated in the context of a compound within Formula I, it will
be apparent that such configurations of Formula Ii apply also to
other values of q and k. Similarly, although the partial structures
of formulae Iic and Iid show an R.sup.11 group corresponding to
leucine, it will be apparent that these configurations will be
applicable to other R.sup.11 groups, especially those resembling
the side chains of natural or unnatural L-amino acids, for example
t-butyl alanine/t-leucine.
[0138] R.sup.15 in those compounds of the invention wherein n is 1,
is preferably optionally substituted C.sub.1-C.sub.6alkyl or
C.sub.0-C.sub.3alkylcarbocyclyl for example
C.sub.0-C.sub.3alkylC.sub.3-C.sub.7cycloalkyl, any of which may be
optionally substituted. Preferred P4 groups are typically analogues
of natural or unnatural amino acids, especially aliphatic amino
acids such as L-valyl, L-leucyl, L-isoleucyl, L-t-leucyl or
L-cyclohexylalanine and thus favoured R.sup.15 groups include
cyclohexyl, cyclohexylmethyl, tert-butyl, iso-propyl, or
iso-butyl.
[0139] Preferred G values include --NRy-, especially wherein Ry is
methyl or preferably H, or hydrazine.
[0140] A further preferred G value is O thereby defining an ester
with the carbonyl of P4 (if present) or the carbonyl of P3 (if
present) or an ether in the case of variants wherein group U is
absent. Conventional pharmaceutically acceptable ethers or esters
capping groups for R.sup.16 include C.sub.1-C.sub.6alkyl
(especially methyl or t-butyl), C.sub.0-C.sub.3alkylheterocyclyl
(especially pyridyl, benzimidazolyl, piperidyl, morpholinyl,
piperazinyl) or C.sub.0-C.sub.3alkylcarbocyclyl (especially phenyl,
benzyl, indanyl) any of which is optionally substituted with
hydroxy, halo, amino, or C.sub.1-C.sub.6alkoxy.
[0141] It will be apparent that for compounds of formula 1, when
m=n=0, then R.sup.16G- is not a BOC or CBz protecting group, but
this restriction does not apply to other permutations of m and n.
The Boc or CBz protected-4-substituted proline synthetic
intermediates described for example in WO 0059929 are thus outside
the scope of the invention.
[0142] Favoured compounds of the invention can comprise a hydrazine
functionality, for example where X is --NHNH-- and m is 1; with n
being zero or 1. Alternatively, especially where m is zero, G can
be --NRjNRj- such as --NHNH--. Compounds will generally not
comprise a hydrazine at both G and X. Typical hydrazines within
Formula I, wherein m and n are zero include compounds of the
partial structures Ija-Ijb below:
##STR00023##
[0143] R.sup.16' in formulae Ija and Ijb can be regarded as an
alkyl (or C.sub.1-C.sub.3-alkylheterocyclyl or C.sub.1-C.sub.3alkyl
carbocyclyl) wherein the first alkyl carbon is substituted with an
oxo group to define the keto function and R.sup.16' is the
remainder of the alkyl, alkylheterocyclyl or alkylcarbocyclyl
moiety. Formula Ijb depicts a variant where R.sup.16 is a methylene
group whose carbon is substituted with an oxo substituent and also
--ORb, where Rb is as defined above, typically, C.sub.1-C.sub.6
alkyl, such as t-butyl, C.sub.0-C.sub.3alkylheterocyclyl such as
pyridyl, or C.sub.0-C.sub.3alkylcarbocyclyl, such as benzyl or
phenyl, any of which is optionally substituted as defined above.
Compounds of partial structures Ija and Ijb can be linear molecules
as shown (both Rj are H), or preferably one of the depicted Rj
groups can be macrocyclised via J to an appropriate R.sup.7
group.
[0144] Alternative hydrazines of Formula I where m is 1 include
those of partial structures Ijc and Ijd below:
##STR00024##
[0145] where R.sup.16, G, R.sup.11, R.sup.15, Rj and Ru are as
defined for formula I above. Compounds of partial structures Ijc
and Ijd can be linear molecules as shown (both Rj are H), or
preferably one of the depicted Rj groups, or the R.sup.11 group can
be macrocyclised via J to an appropriate R.sup.7 group
[0146] Although formulae Ija-Ijd are depicted with a proline
analogue as P2, it will be apparent that this aspect of the
invention is equally adapted to other configurations of q and
k.
[0147] Alternative hydrazine-like configuration are found when G is
amino, and m and n are 0, and R.sup.16 is an N-linked unsaturated
heterocycle as defined below, for example pyridyl or pyrimidyl or a
saturated heterocycle as defined below, such as piperazinyl,
piperidinyl and especially morpholinyl. Examples of such
embodiments include those of the formulae Ije:
##STR00025##
[0148] Compounds of partial structures Ije can be linear molecules
as shown or preferably Rx can be macrocyclised via J to an
appropriate R.sup.7 group. Although these partial structures are
depicted with a five membered ring for P2, it will be readily
apparent that this configuration extends to other values of q and
k. Similarly these configurations will be applicable to other
N-linked heterocycles as R.sup.16.
[0149] Returning now to Formulae I in general, favoured R.sup.16
groups for the compounds of the invention include 2-indanol,
indanyl, 2-hydroxy-1-phenyl-ethyl, 2-thiophenemethyl,
cyclohexylmethyl, 2,3-methylenedioxybenzyl, cyclohexyl, phenyl,
benzyl, 2-pyridylmethyl, cyclobutyl, iso-butyl, n-propyl, methyl,
or 4-methoxyphenylethyl.
[0150] Currently preferred R.sup.16 groups include 2-indanol,
indan, 2-hydroxy-1-phenyl-ethyl, 2-thiophenemethyl,
2,3-methylenedioxybenzyl, or cyclohexylmethyl.
[0151] Unnatural amino acids include L-amino acids wherein the side
chain is not one of the 20 naturally occurring amino acids.
Examples of non-natural amino acids include
L-beta-methylsulfonylmethylalanine, L-cyclohexylalanine,
L-tertiary-leucine, L-norleucine, L-norvaline, L-ornithine,
L-sarcosine, L-citurline, L-homophenylalanine, L-homoserine,
L-beta-(1-napthyl)alanine, L-beta-(2-napthyl)alanine etc. Non
natural amino acids also include the D-amino acids corresponding to
the 20 natural amino acids and D-amino acids bearing other side
chains, such as those listed above.
[0152] `C.sub.1-C.sub.6alkyl` (also abbreviated as
C.sub.1-C.sub.6alk, or used in compound expressions such as
C.sub.1-C.sub.6alkyloxy etc) as applied herein is meant to include
straight and branched chain aliphatic carbon chains such as methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl,
isopentyl, hexyl, heptyl and any simple isomers thereof. The alkyl
group may have an unsaturated bond. Additionally, any C atom in
C.sub.1-C.sub.6alkyl may optionally be substituted by one, two or
where valency permits three halogens and/or substituted or the
alkyl chain interrupted by a heteroatom S, O, NH. If the heteroatom
is located at a chain terminus then it is appropriately substituted
with one or 2 hydrogen atoms. C.sub.1-C.sub.4alkyl and
C.sub.1-C.sub.5alkyl have the corresponding meaning to
C.sub.1-C.sub.6alkyl adjusted as necessary for the carbon
number.
[0153] `C.sub.1-C.sub.3alkyl` as applied herein includes methyl,
ethyl, propyl, isopropyl, cyclopropyl, any of which may be
optionally substituted or heteroatom interrupted as described in
the paragraph above or in the case of C.sub.2 or C.sub.3, bear an
unsaturated bond such as CH.sub.2.dbd.CH.
[0154] "C.sub.1-C.sub.3alkylene" as applied herein describes a
divalent C.sub.1-C.sub.3alkyldiyl moiety, including propylene,
ethylene and especially methylene. The typically longer alkylene
chains for J may comprise 1 to 3 unsaturations and/or interruptions
with heteroatoms as defined above.
[0155] `Amino` includes NH.sub.2, NHC.sub.1-C.sub.6alkyl or
N(C.sub.1-C.sub.6-alkyl).sub.2, especially C.sub.1-C.sub.3 alkyl
variants
[0156] `Amido` includes C(.dbd.O)NH.sub.2, and alkylamido, such as
C(.dbd.O)NHC.sub.1-C.sub.6alkyl,
C(.dbd.O)N(C.sub.1-C.sub.6alkyl).sub.2 especially
C(.dbd.O)NHC.sub.1-C.sub.3alkyl,
C(.dbd.O)N(C.sub.1-C.sub.3alkyl).sub.2 or
--NH(C.dbd.O)C.sub.1-C.sub.6alkyl, for example
--NHC(.dbd.O)CHC(CH.sub.3).sub.3, including
--NH(C.dbd.O)C.sub.1-C.sub.3alkyl.
[0157] `Halo` or halogen as applied herein is meant to include F,
Cl, Br, I, particularly chloro and preferably fluoro.
[0158] `C.sub.0-C.sub.3alkylaryl` as applied herein is meant to
include an aryl moiety such as a phenyl, naphthyl or phenyl fused
to a C.sub.3-C.sub.7cycloalkyl (for example indanyl), which aryl is
directly bonded (i.e. C.sub.0) or through an intermediate methyl,
ethyl, or propyl group as defined for C.sub.1-C.sub.3alkylene
above. Unless otherwise indicated the aryl and/or its fused
cycloalkyl moiety is optionally substituted with 1-3 substituents
selected from halo, hydroxy, nitro, cyano, carboxy,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6alkoxyC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkanoyl,
amino, azido, oxo, mercapto, nitro C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl. "Aryl" has the corresponding
meaning, i.e. where the C.sub.0-C.sub.3alkyl linkage is absent.
[0159] `C.sub.0-C.sub.3alkylC.sub.3C.sub.7cycloalkyl` as applied
herein is meant to include a C.sub.3-C.sub.7cycloalkyl group such
as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl,
which cycloalkyl is directly bonded (i.e. C.sub.0alkyl) or through
an intermediate methyl, ethyl, proyl or isopropyl group as defined
for C.sub.1-C.sub.3alkylene above. The cycloalkyl group may contain
an unsaturated bond. Unless otherwise indicated the cycloalkyl
moiety is optionally substituted with 1-3 substituents selected
from halo, hydroxy, nitro, cyano, carboxy, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6alkoxyC.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkanoyl, amino, azido, oxo, mercapto, nitro
C.sub.0-C.sub.3alkylcarbocyclyl,
C.sub.0-C.sub.3alkylheterocyclyl.
[0160] `C.sub.0-C.sub.3alkylcarbocyclyl` as applied herein is meant
to include C.sub.0-C.sub.3alkylaryl and
C.sub.0-C.sub.3alkylC.sub.3-C.sub.7cycloalkyl. Unless otherwise
indicated the aryl or cycloalkyl group is optionally substituted
with 1-3 substituents selected from halo, hydroxy, nitro, cyano,
carboxy, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6alkoxyC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkanoyl,
amino, azido, oxo, mercapto, nitro, C.sub.0-C.sub.3alkylcarbocyclyl
and/or C.sub.0-C.sub.3alkylheterocyclyl. "Carbocyclyl" has the
corresponding meaning, i.e. where the C.sub.0-C.sub.3alkyl linkage
is absent
[0161] `C.sub.0-C.sub.3alkylheterocycylyl` as applied herein is
meant to include a monocyclic, saturated or unsaturated,
heteroatom-containing ring such as piperidinyl, morpholinyl,
piperazinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl,
thiazinolyl, isothiazinolyl, thiazolyl, oxadiazolyl,
1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, furanyl, thienyl,
pyridyl, pyrimidyl, pyridazinyl, pyrazolyl, or any of such groups
fused to a phenyl ring, such as quinolinyl, benzimidazolyl,
benzoxazolyl, benzisoxazolyl, benzothiazinolyl, benzisothiazinolyl,
benzothiazolyl, benzoxadiazolyl, benzo-1,2,3-triazolyl,
benzo-1,2,4-triazolyl, benzotetrazolyl, benzofuranyl, benzothienyl,
benzopyridyl, benzopyrimidyl, benzopyridazinyl, benzopyrazolyl etc,
which ring is bonded directly i.e. (C.sub.0), or through an
intermediate methyl, ethyl, propyl, or isopropyl group as defined
for C.sub.1-C.sub.3alkylene above. Any such non-saturated rings
having an aromatic character may be referred to as heteroaryl
herein. Unless otherwise indicated the hetero ring and/or its fused
phenyl moiety is optionally substituted with 1-3 substituents
selected from halo, hydroxy, nitro, cyano, carboxy,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6alkoxyC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkanoyl,
amino, azido, oxo, mercapto, nitro,
C.sub.0-C.sub.3alkylcarbocyclyl, C.sub.0-C.sub.3alkylheterocyclyl.
"Heterocyclyl" and "Heteroaryl" have the corresponding meaning,
i.e. where the C.sub.0-C.sub.3alkyl linkage is absent.
[0162] Typically heterocycyl and carbocyclyl moieties within the
scope of the above definitions are thus a monocyclic ring with 5 or
especially 6 ring atoms, or a bicyclic ring structure comprising a
6 membered ring fused to a 4, 5 or 6 membered ring.
[0163] Typical such groups include C.sub.3-C.sub.8cycloalkyl,
phenyl, benzyl, tetrahydronaphthyl, indenyl, indanyl, heterocyclyl
such as from azepanyl, azocanyl, pyrrolidinyl, piperidinyl,
morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl,
tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl,
tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl,
thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, tetrazolyl, pyrazolyl, indolyl, benzofuranyl,
benzothienyl, benzimidazolyl, benzthiazolyl, benzoxazolyl,
benzisoxazolyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl,
tetrahydroisoquinolinyl, quinazolinyl, tetrahydroquinazolinyl and
quinoxalinyl, any of which may be optionally substituted as defined
herein.
[0164] The saturated heterocycle moiety thus includes radicals such
as pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,
piperidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl,
piperazinyl, indolinyl, azetidinyl, tetrahydropyranyl,
tetrahydrothiopyranyl, tetrahydrofuranyl, hexahydropyrimidinyl,
hexahydropyridazinyl, 1,4,5,6-tetrahydropyrimidinylamine,
dihydro-oxazolyl, 1,2-thiazinanyl-1,1-dioxide,
1,2,6-thiadiazinanyl-1,1-dioxide, isothiazolidinyl-1,1-dioxide and
imidazolidinyl-2,4-dione, whereas the unsaturated heterocycle
include radicals with an aromatic character such as furanyl,
thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,
isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl,
thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl,
indolizinyl, indolyl, isoindolyl. In each case the heterocycle may
be condensed with a phenyl ring to form a bicyclic ring system.
[0165] Synthesis
[0166] Synthesis of the compounds of the present invention can be
performed by different chemical strategies in solution or solid
phase or a combination of both. The suitably protected individual
building blocks can first be prepared and subsequently coupled
together i.e. P2+P1.fwdarw.P2-P1. Alternatively, precursors of the
building blocks can be coupled together and modified at a later
stage of the synthesis of the inhibitor sequence. Further building
blocks, precursors of building blocks or prefabricated bigger
fragments of the desired structure, can then be coupled to the
growing chain, e.g.
R.sup.16-G-P3+E-P2-P1.fwdarw.R.sup.16-G-P3-P2-P1 or
R.sup.16-G-P4-P3+E-P2-P1.fwdarw.R.sup.16-G-P4-P3-E-P2-P1.
[0167] Coupling between two amino acids, an amino acid and a
peptide, or two peptide fragments can be carried out using standard
coupling procedures such as the azide method, mixed
carbonic-carboxylic acid anhydride (isobutyl chloroformate) method,
carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or
water-soluble carbodiimide) method, active ester (pnitrophenyl
ester, N-hydroxysuccinic imido ester) method, Woodward reagent
K-method, carbonyidiimidazole method, phosphorus reagents or
oxidation-reduction methods. Some of these methods (especially the
carbodiimide method) can be enhanced by adding
1-hydroxybenzotriazole or 4-DMAP. These coupling reactions can be
performed in either solution (liquid phase) or solid phase.
[0168] More explicitly, the coupling step involves the dehydrative
coupling of a free carboxyl of one reactant with the free amino
group of the other reactant in the present of a coupling agent to
form a linking amide bond. Descriptions of such coupling agents are
found in general textbooks on peptide chemistry, for example, M.
Bodanszky, "Peptide Chemistry", 2nd rev ed., Springer-Verlag,
Berlin, Germany, (1993) hereafter simply referred to as Bodanszky,
the contents of which are hereby incorporated by reference.
Examples of suitable coupling agents are
N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole in the
presence of N,N'-dicyclohexylcarbodiimide or
N-ethyl-N'-[(3dimethylamino)propyl]carbodiimide. A practical and
useful coupling agent is the commercially available
(benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium
hexafluorophosphate, either by itself or in the present of
1-hydroxybenzotriazole or 4-DMAP. Another practical and useful
coupling agent is commercially available
2-(IH-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate. Still another practical and useful coupling
agent is commercially available 0-(7-azabenzotrizol-1-yl)-N
N,N',N'-tetramethyluronium hexafluorophosphate.
[0169] The coupling reaction is conducted in an inert solvent, e.
g. dichloromethane, acetonitrile or dimethylformamide. An excess of
a tertiary amine, e. g. diisopropylethylamine, N-methylmorpholine,
N-methylpyrrolidine or 4-DMAP is added to maintain the reaction
mixture at a pH of about 8. The reaction temperature usually ranges
between 0.degree. C. and 50.degree. C. and the reaction time
usually ranges between 15 min and 24 h.
[0170] The functional groups of the constituent amino acids
generally must be protected during the coupling reactions to avoid
formation of undesired bonds. The protecting groups that can be
used are listed in Greene, "Protective Groups in Organic
Chemistry", John Wiley & Sons, New York (1981) and "The
Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press,
New York (1981), hereafter referred to simply as Greene, the
disclosures of which are hereby incorporated by reference.
[0171] The .alpha.-carboxyl group of the C-terminal residue is
usually protected as an ester that can be cleaved to give the
carboxylic acid. Protecting groups that can be used include 1)
alkyl esters such as methyl, trimethylsilyl and t.butyl, 2) aralkyl
esters such as benzyl and substituted benzyl, or 3) esters that can
be cleaved by mild base or mild reductive means such as
trichloroethyl and phenacyl esters.
[0172] The .alpha.-amino group of each amino acid to be coupled is
typically protected. Any protecting group known in the art can be
used. Examples of such groups include: 1) acyl groups such as
formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2)
aromatic carbamate groups such as benzyloxycarbonyl (Cbz or Z) and
substituted bensyloxycarbonyls, and 9-fluorenylmethyloxycarbonyl
(Fmoc); 3) aliphatic carbamate groups such as tertbutyloxycarbonyl
(Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and
allyloxycarbonyl; 4) cyclic alkyl carbamate groups such as
cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl groups
such as triphenylmethyl and benzyl; 6) trialkylsilyl such as
trimethylsilyl; and 7) thiol containing groups such
asphenylthiocarbonyl anddithiasuccinoyl. The preferred
.alpha.-amino protecting group is either Boc or Fmoc. Many amino
acid derivatives suitably protected for peptide synthesis are
commercially available.
[0173] The .alpha.-amino protecting group is cleaved prior to the
next coupling step. When the Boc group is used, the methods of
choice are trifluoroacetic acid, neat or in dichloromethane, or HCl
in dioxane or in ethyl acetate. The resulting ammonium salt is then
neutralized either prior to the coupling or in situ with basic
solutions such as aqueous buffers, or tertiary amines in
dichloromethane or acetonitrile or dimethylformamide. When the Fmoc
group is used, the reagents of choice are piperidine or substituted
piperidine in dimethylformamide, but any secondary amine can be
used. The deprotection is carried out at a temperature between
0.degree. C. and room temperature usually 20-22.degree. C.
[0174] Any of the natural or non-natural amino acids having side
chain functionalities will typically be protected during the
preparation of the peptide using any of the above described groups.
Those skilled in the art will appreciate that the selection and use
of appropriate protecting groups for these side chain
functionalities depend upon the amino acid and presence of other
protecting groups in the peptide. In the selection of such
protecting groups it is desirable that the group is not removed
during the deprotection and coupling of the .alpha.-amino
group.
[0175] For example, when Boc is used as the .alpha.-amino
protecting group, the following side chain protecting groups are
suitable: p-toluenesulfonyl (tosyl) moieties can be used to protect
the amino side chain of amino acids such as Lys and Arg;
acetamidomethyl, benzyl (Bn), or tert-butylsulfonyl moieties can be
used to protect the sulfide containing side chain of cysteine;
benzyl (Bn) ethers can be used to protect the hydroxy containing
side chains of serine, threonine or hydroxyproline; and benzyl
esters can be used to protect the carboxy containing side chains of
aspartic acid and glutamic acid.
[0176] When Fmoc is chosen for the .alpha.-amine protection,
usually tert. butyl based protecting groups are acceptable. For
instance, Boc can be used for lysine and arginine, tert.butyl ether
for serine, threonine and hydroxyproline, and tert-butyl ester for
aspartic acid and glutamic acid. Triphenylmethyl (Trityl) moiety
can be used to protect the sulfide containing side chain of
cysteine.
[0177] Once the inhibitor sequence is completed any protecting
groups are removed in whatever manner is dictated by the choice of
protecting groups. These procedures are well known to those skilled
in the art.
[0178] In compounds of Formula I, the P2 unit comprises a
nitrogen-containing ring residue which is substituted with the W
and R8 moieties.
[0179] Synthesis of Heterocyclic P2 Building Blocks
[0180] The R.sup.8 group can be coupled to the P2 scaffold at any
convenient stage of the synthesis of compounds according to the
present invention. One approach is to first couple the R8 group to
the P2 scaffold and subsequently add the other desired building
blocks, i.e. P1 and optionally P3 and P4. Another approach is to
couple the P1 and, if present P3 and P4 using an unsubstituted P2
scaffold and add the R.sup.8 group afterwards.
[0181] Compounds wherein W is O and R.sup.8 is alkyl,
C.sub.0-C.sub.3alkylcarbocycylyl, C.sub.0-C.sub.3alkylheterocycylyl
can be prepared according to the procedure described by E. M. Smith
et al. (J. Med. Chem. (1988), 31, 875-885), as depicted in Scheme
1, which illustrates the technique in a moiety wherein q and k are
1.
##STR00026##
[0182] Commercially available Boc-4-(R)-hydroxyproline, or any
suitable hydroxy substituted proline analogue, such as an
hydroxypiperidoic acid is treated with a base such as sodium
hydride or potassium t.butoxide in a solvent like dimethylformamide
and the resulting alkoxide is reacted with an alkylating agent,
R.sup.8--X, wherein X is a suitable leaving group such as a
halide,mesylate, triflate or tosylate or the like, providing the
desired substituted proline derivative.
[0183] Alternatively, when W is O or S and R.sup.8 is carbocyclyl
such as phenyl or heterocyclylyl such as heteroaryl, the P2
building blocks can also be prepared via a Mitsunobu reaction
(Mitsunobu, 1981, Synthesis, January, 1-28; Rano et al.,
Tetrahedron Lett., 1995, 36, 22, 3779-3792; Krchnak et al.,
Tetrahedron Lett., 1995, 36, 5, 6193-6196; Richter et al.,
Tetrahedron Lett., 1994, 35, 27, 4705-4706) as shown in Scheme 2,
which illustrates the technique in a moiety wherein q and k are
1.
##STR00027##
[0184] Treatment of the appropriate hydroxy substituted proline
analogue, such as a hydroxypiperidoic acid, here shown as
commercially available Boc-4-hydroxyproline methyl ester, with the
desired alcohol or thiol (R.sup.8--WH) in the presence of
triphenylphosphine and an activating agent like diethyl
azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD) or the
like, provides the ester compound (2b). Hydrolysation of the ester
to the acid by standard procedures provides the P2 building block
(2c).
[0185] Alcohol (2a) can alternatively be treated with phosgene thus
providing the corresponding chloroformate which upon reaction with
an amine, R.sup.8NH.sub.2, in the presence of a base like sodium
hydrogen carbonate or triethylamine, provides carbamates i.e. W is
--OC(.dbd.O)NH--, whereas reaction of alcohol (2a) with an
acylating agent, R8-CO--X, like an acid anhydride or acid halide
for instance the acid chloride, to provide esters, i.e. W is
--OC(.dbd.O)--.
[0186] Various alcohols R.sup.8--OH, and alkylating agents
R.sup.8--X are described in WO 00/09543 and WO00/59929. An example
of the synthesis wherein R.sup.8 is a substituted quinoline
derivative is shown in Scheme 3.
##STR00028##
[0187] Friedel-Craft acylation of a suitable substituted aniline
(3a), available either commercially or in the literature, using an
acylating agent like acetyl chloride or the like in the presence of
boron trichloride and aluminum trichloride in a solvent like
dichloromethane provides (3b). Coupling of (3b) to a heterocyclic
carboxylic acid (3c) under basic conditions, such as in pyridine,
in the presence of an activating agent for the carboxylate group,
for instance POCl.sub.3, followed by ring closure and dehydration
under basic conditions like potassium tert-butoxide in tert-butanol
provides quinoline derivative (3e). Quinoline derivative (3e) can
be coupled in a Mitsunobu reaction to an alcohol as described
above, or the hydroxy group can be displaced by a suitable leaving
group such as a halide like chloride, bromide or iodide, by
treatment of quinoline (3e) with an appropriate halogenating agent
for example phosphoryl chloride or the like.
[0188] A variety of carboxylic acids with the general structure
(3c) can be used in Scheme 3. These acids are available either
commercially or in the literature. An example of the preparation of
2-(substituted)-amino-carboxy-aminothiazole derivatives, following
the procedure by Berdikhina et al. Chem. Heterocycl. Compd. (Engl.
Transl.) (1991), 427-433, is shown below.
##STR00029##
[0189] Thiourea (4c) with different alkyl substituents R' can be
formed by reaction of the appropriate amine (4a) with
tert-butylisothiocyanate in the presence of a base like
diisopropylethylamine in a solvent like dichloromethane followed by
removal of the tert-butyl group under acidic conditions. Subsequent
condensation of thiourea derivative (4c) with 3-bromopyruvic acid
provides the acid (4d).
[0190] P2 building blocks wherein the R.sup.8 substituent is
attached via an amine, amide, urea or sulphonamide, can be prepared
from aminoproline analogues achieved either from a suitable
commercially available aminoproline, etc derivative or by
transforming the hydroxy group of the corresponding hydroxy
derivative into an azide group for example by transforming the
hydroxy group into a suitable leaving group such as a mesylate or
halogen like chloride, followed by substitution of the leaving
group with azide or by the use of an azide transfer agent like
diphenylphosphoryl azide (DPPA). Reduction of the azide by
catalytic hydrogenation or any other suitable reduction method
provides the amine. The amino derivative can be reacted in a
displacement reaction with an alkylating agent of the general
formula R.sup.8--X wherein R.sup.8 and X are as described for
scheme 1, to form P2 building blocks for use in the preparation of
compounds of general formula I, wherein W is --NH--. Reaction of
the aminoproline analogue with an acid of the general formula
R.sup.8--COOH under standard amide coupling conditions provides
compounds wherein the R8 substituent is linked via an amide bond,
whereas reaction of the aminoproline analogue with an appropriate
derivative of sulphonic acid, R.sup.8--S(O).sub.2--X where X is a
leaving group for example chloride, in the presence of a base,
provides sulphonamides. Compounds wherein the linkage between the
cyclic scaffold and the R8 substituent is constituted of a urea
group can for example be achieved by treatment of amino proline
analogue with phosgene to afford the corresponding chlorocarbamate
followed by reaction with the desired amine. Alternatively, the
amino proline analogue can be reacted with the carbamoyl chloride
or isocyanate of the desired R8 substituent for the formation of
the urea linkage. It will be apparent that corresponding reactions
will be available for P2 groups with other ring sizes and
substitution pattern.
[0191] 4-Substituted heterocyclyl derivatives such as 4-substituted
proline for use as P2 building blocks where W is --CH.sub.2-- can
be prepared as shown in Scheme 5, which illustrates the technique
on a moiety where q and k is 1, according to the procedures
described by J. Ezquerra et al., Tetrahedron, 1993, 38, 8665-8678
and C. Pedregal et al. Tetrahedron Lett., 1994, 35, 2053-2056.
##STR00030##
[0192] Treatment of suitably acid protected pyrrolidone or
piperidinone such as commercially available Boc-pyroglutamic acid
(5a) with a strong base such as lithium diisopropylamide in a
solvent like tetrahydrofuran followed by addition of an alkylating
agent R.sup.8--CH.sub.2--X where X is a suitable leaving group such
as a halide like chloride or bromide, followed by reduction of the
amide and deprotection of the ester gives the desired compound
(5d).
[0193] Compounds of the present invention wherein a heterocyclic
R.sup.8 group is attached directly to the cyclic P2 scaffold, i.e.
W is a bond in general formula 1, can be prepared for example by
using a replacement reaction wherein a suitable leaving group on
the P2 scaffold is replaced by the desired R.sup.8 group such as a
heterocyclic group.
[0194] Alternatively the R8 group can be introduced by way of a
Mitsunobu reaction wherein the hydroxy group of the P2 scaffold is
reacted with a nitrogen atom in the heterocyclic R.sup.8 group.
##STR00031##
[0195] Compounds wherein a tetrazole derivative is attached through
a carbon atom of the heterocyclic ring are conveniently prepared by
building up the tetrazole moiety directly on the P2 precursor. This
can be achieved for instance by transforming the hydroxy group of
the P2 precursor into a cyano group followed by reaction with an
azide reagent like sodium azide. Triazole derivatives can also be
built up directly on the P2 precursor for example by transforming
the hydroxy group of the P2 precursor into an azide group followed
by a 3+2 cycloaddition reaction of the afforded azide and a
suitable alkyne derivative.
[0196] Structurally diverse tetrazoles for use in the above
described substitution or Mitsunobu reactions can be prepared by
reacting commercially available nitrile compounds with sodium
azide. Triazole derivatives can be prepared by reaction of an
alkyne compound and trimethylsilyl azide. Useful alkyne compounds
are available either commercially or they can be prepared for
instance according to the Sonogashira reaction i.e. reaction of a
primary alkyne, an aryl halide and triethylamine in the presence of
PdCl.sub.2(PPh).sub.3 and CuI as described for example in A.
Elangovan, Y.-H. Wang, T.-I. Ho, Org. Lett., 2003, 5, 1841-1844.
The heterocyclic substituent can also be modified when attached to
the P2 building block either before or after coupling of the P2
building block to the other building blocks.
[0197] These methods and further alternatives for the preparation
of compounds wherein W is a bond and R8 is an optionally
substituted heterocycle are extensively described in
WO2004/072243.
[0198] Compounds with alternative ring size and/or position of the
W--R.sup.8 substituent of the proline derivatives in scheme 1, 2
and 5 may also be used in the preparation of compounds according to
the present invention. For example, alkylation of commercially
available 3-hydroxyproline provides compounds of the general
formula (I) wherein k is O and q is 2. Correspondingly, alkylation
of 5-hydroxyproline, prepared for example as described by Hallberg
et al., J. Med. Chem. (1999), 4524-4537, provides compounds of the
general formula (I) wherein k is 2 and q is 0.
[0199] Various methods for the preparation of hydroxylated
2-piperidine carboxylic acids are described in the literature se
for instance Celestini et al., Org. Lett., (2002), 1367-1370,
Hoarau et al., Tetrahedron: Asymmetry, (1996), 2585-2594, Zhu et
al., Tetrahedron Lett., 41, (2000), 7033-7036. For example, the
corresponding pyridine carboxylic acids can be reduced to provide
hydroxylated 2-piperidine carboxylic acids. Enzymatical methods can
also be used for the preparation of hydroxylated proline analogues.
For example, a 3-hydroxy substituent can be introduced on
commercially available 4, 5, and 6 membered heterocyclic acids by
the use of proline 3-hydroxylase as described by Ozaki et al., Tet.
Letters, 40, (1999), 5227-5230.
[0200] Synthesis and Introduction of P1 Building Blocks.
[0201] The amino acids used in the preparation of P1 fragments are
available either commercially or in the literature, see for example
WO 00/09543 and WO00/59929 from Boehringer-Ingelheim or
US2004/0048802 from BMS.
[0202] Scheme 6 shows an example of the preparation of a
sulphonamide derivative to be used as a P1 fragment, and the
subsequent coupling to a Boc protected P2 building block.
##STR00032##
[0203] The sulphonamide group can be introduced on a suitably
protected amino acid (6a) by treatment of the amino acid with a
coupling agent, for example N,N'-carbonyldiimidazole (CDI) or the
like, in a solvent like THF followed by reaction with the desired
sulphonamide (6b) in the presence of a strong base such as
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Alternatively the amino
acid can be treated with the desired sulphonamide (6b) in the
presence of a base like diisopropyl ethylamine followed by
treatment with a coupling agent like PyBOP.RTM. to effect the
introduction of the sulphonamide group. Removal of the amino
protecting group by standard methods and subsequent coupling to a
P2 building block, prepared as described above, using standard
methods for amide bond formation, like with a coupling agent as
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) in the presence of a base such as
diisopropylamine in a solvent like dimethylformamide, gives Boc
protected P2-P1 compound (6e). Alternatively, the sulphonamide
group can be introduced at a later stage of the synthesis, for
example as the last step. In this case an amino acid with the
reversed protection pattern, i.e. having an unprotected amino
function and a protected acid function, is coupled to the acid
function of the P2 building block using standard peptide coupling
conditions for example as described above. Removal of the acid
protection group, using the appropriate conditions for the
protection group used, followed by coupling of the sulphonamide as
described above, yields compound 6e
[0204] P1 building blocks for the preparation of compounds
according to general formula I wherein A is an ester or an amide
can be prepared by reacting amino acid (6a) with the appropriate
amine or alcohol respectively under standard conditions for amide
or ester formation. Compounds according to general formula I
wherein A is CR.sup.4R.sup.4' can be prepared by coupling of the
appropriate PI building block to the P2 building block as described
in Oscarsson et al Bioorg Med Chem 2003 11(13) 2955-2963 and
PCT/EP03/10595 filed Sep. 23, 2003, the contents of which are
incorporated by reference.
[0205] Compounds comprising an azapeptide P1 residue, i.e. Q is NRu
in general formula I can be prepared by using a suitable P1
aza-amino acyl moiety in the coupling to the P2 fragment. The
preparation of aza-amino acyl moieties is described by M. D. Bailey
et al. in J. Med. Chem., 47, (2004), 3788-3799, and an example is
shown in scheme 6A.
##STR00033##
[0206] Incorporation of the appropriate N-linked side chain, Ru, on
commercially available tert-butylhydrazine can be performed for
example by a reductive amination reaction with the appropriate
aldehyde or ketone as described in scheme 19 below which produces
the N-alkylated carbazate (6Aa). Condensation of 6Aa with a desired
chloroformate in the presence of a base like triethylamine or
diisopropylethylamine in a solvent like THF provides 6Ab. The R1'
moiety can then optionally be removed using the appropriate
conditions depending on the specific R1', such as catalytic
hydrogenation for R1' being benzyl, which gives the corresponding
acids. Subsequent reaction of the afforded acid with a desired
sulphonamide derivative as described in scheme 6 yields
sulphonamide capped building blocks. Alternatively, reaction of
carbazate 6Aa with an isocyanate, R3-N.dbd.C.dbd.O, provides
building blocks for the preparation of compounds according to
general formula I, wherein M is NRu and A is CONHR.sup.3.
[0207] The P2 and P3 moieties may be linked together prior to or
after the introduction of the P1 building block.
[0208] Synthesis of Capped P3 and P3-P4 Building Blocks
[0209] The building blocks R.sup.16-G-P3 and R.sup.16-G-P4-P3 can
be prepared as generally depicted in scheme 7.
##STR00034##
[0210] A suitable N-protected amino acid (7a) can be coupled with
an amino capping group (R.sup.16--NHRy) using standard peptide
coupling conditions like with coupling agents such as HATU, DCC,
HOBt or the like in the presence of a base such as DIEA or DMAP in
a solvent like dichloromethane, chloroform or dimethylformamide or
a mixture thereof and ester formation conditions like providing
amides i.e. G is NHRy (7b). Alternatively, reaction of amino acid
(7a) with a compound of general formula R.sup.16--X where R.sup.16
is as defined above and X is a leaving group such as a halide, in
the presence of a base like cesium carbonate or silver (I) oxide
provides esters, i.e. G is O (7b). On the other hand, amino acid
(7a) can be coupled to a second, suitably O-protected, amino acid
(7d) using standard peptide coupling conditions as described above,
providing (7e). Displacement of the ester group with a suitable
capping group (7b) provides fragment (7f) useful for the
preparation of compounds according to the present invention wherein
m and n are 1.
[0211] When G is N-Ry, the capped P3 or P2 building block can also
be prepared on solid support as exemplified in Scheme 8.
##STR00035##
[0212] An appropriate N-protected, for example Boc protected, amino
acid (8a) can be immobilized on a solid support, here exemplified
by Agronaut resin PS-TFP, by reacting the amino acid with the
desired solid support in the presence of coupling reagent like
N,N'-diisopropylcarbodiimide and a base like DMAP in a solvent like
dichloromethane and dimethylformamide. The immobilized amino acid
can then be cleaved from the support with a suitable capping group
(8c) thus giving fragments useful for the preparation of compounds
according to the present invention wherein m or n is 1. Optionally
the amino protecting group can be removed followed by coupling of
an appropriate amino acid using standard methods thus providing
fragments useful for the preparation of compounds according to the
present invention wherein m and n are 1.
[0213] Coupling of a Capping Group or a Capped Building Block to
the P2-P1 Construct
[0214] The R.sup.16-G, R.sup.16-G-P3 or R.sup.16-G-P4-P3 building
block linked via a urea functionality to the P2-P1 construct, can
be introduced as depicted in scheme 9, which illustrates the
technique with a variant in which the P2 scaffold is a 5-membered
ring.
##STR00036##
[0215] A chlorocarbamate group can be formed onto the ring amine of
the P2-P1 construct (9a) by removal of the amine protection group
by standard procedures, like acidic treatment with for example TFA
in dichloromethane or the like when the Boc group is used, followed
by reaction of the free amine with phosgene in toluene in the
presence of a base such as sodium hydrogen carbonate or
triethylamine in a solvent like tetrahydrofuran. Subsequent
reaction of the formed electrophilic center with the amino group of
a R.sup.16--NH.sub.2, R.sup.16--NH--NH.sub.2, R.sup.16-G-P3 or
R.sup.16-G-P4-P3 building block (9c) in a solvent like
dichloromethane in the presence of a base like sodium hydrogen
carbonate provides (9d). Compounds of general formula (I) wherein E
is C.dbd.S, S(.dbd.O) or S(.dbd.O).sub.2 can be prepared according
to the above procedure but with the use of reagents like
thiocarbonyl diimidazole, thionyl chloride or sulphuryl chloride
respectively instead of phosgene.
[0216] Compounds containing a hydrazine group linked to the P2
unit, i.e. X is --NRjNRj- in general formula I, or when the P3 and
P4 units are absent and G is NRjNRj, can be prepared as depicted
below. Scheme 10 shows the introduction of a hydrazine derivative
to a 5-membered P2 building block.
##STR00037##
[0217] Reaction of tert-butyl carbazate (10a), optionally alkyl
substituted on one or both nitrgogens, with p-nitrophenyl
chloroformate in the presence of a base like sodium hydrogen
carbonat followed by addition of the P2 building block (10b)
provides the urea derivative 10c. The phosgene method described in
scheme 9 can alternatively be used to effect the linkage of the
fragments 10a and 10b. Optional removal of the boc group by
standard procedures like acidic treatment with for example TFA in a
suitable solvent such as dichloromethane, provides the hydrazine
containing derivative (10d). Alternatively, any appropriate
hydrazine derivative, such as morpholin-1-ylamine,
piperidin-1-ylamine or the like can be linked to 9Ab instead of the
tert-butyl carbazate derivative.
[0218] The achieved compound can then be further extended by
coupling of a P3 or P4-P3 building block to the primary amine of
compound 9Ad for example as shown in scheme 11.
##STR00038##
[0219] Treatment of the .alpha.-amino compound (11a) with sodium
nitrite, potassium bromide and sulphuric acid (Yang et al. J. Org.
Chem. (2001), 66, 7303-7312) provides the corresponding
.alpha.-bromo compound (11b) which upon reaction with the above
described derivative (10d) provides the hydrazine containing
derivative (11c).
[0220] The linkage between the P2 and P3 building blocks may also
be constituted of a carbamate group and a general route to such
compounds is depicted in Scheme 12, which illustrates the technique
with a variant in which P2 is a proline derivative.
##STR00039##
[0221] The desired, optionally protected, amino capping group (12a)
is coupled to a hydroxy acid (10b) using standard peptide coupling
techniques followed by reaction with the electrophilic P2 building
block (12d) described above and optional deprotection provides
construct (12e).
[0222] Compounds lacking a carboxy group in the P3 unit can be
prepared as illustrated in Scheme 13, which illustrates the
technique as applied to a compound of Formula I
##STR00040##
[0223] Chlorocarbamoyl derivative (13a) can be reacted in a
displacement reaction with an azide derivative (13b), prepared by
methods known from the literature, in the presence of a base like
sodium hydrogen carbonate to give (13c). X is as described for
general formula (I). Reduction of the azide function for example by
polymer bound triphenyl phosphine in a solvent like methanol or any
other suitable reduction method provides intermediate (13d) which
subsequently can be reacted with an acid under peptide coupling
conditions or with an amine in a reductive amination reaction
providing amides and secondary amines respectively.
[0224] Scheme 14 shows an alternative route towards compounds
lacking a carboxy group in the P3 unit.
##STR00041##
[0225] Instead of using the azide derivative (13b) in scheme 13 the
corresponding, optionally protected, hydroxy derivative (14b) can
be used in the displacement reaction with the chlorocarbamate (14a)
and thus introducing a primary alcohol. The alcohol (14c) can then,
after optional deprotection, be oxidized with a suitable oxidizing
agent like for example Dess-Martin periodinane to form the
corresponding aldehyde. Reaction of the aldehyde with a desired
amine in a reductive amination reaction using a reagent like for
example polystyrene bound cyanoborohydride in a solvent like THF
provides amine derivatives (14e).
[0226] Alternatively alcohol (14c) can be reacted with a suitable
acylating or alkylating agent under the appropriate conditions to
provide ester and ether compounds respectively, i.e. G is O in
general formula (I).
[0227] Subsequent reaction of the formed alcohol with a suitable
acylating or alkylating agent using the appropriate conditions
provides the ester and ether compounds respectively, i.e. G is O in
general formula (I).
[0228] Alternatively the linkage between the P2 and P3 building
blocks can be via a guanidine group and a general route to such
compounds is depicted in Scheme 15.
##STR00042##
[0229] Treatment of the P2-building block (15a) with thiocarbonyl
diimidazole or the like in a solvent like dimethylformamide
followed by condensation with sodium cyanamide in a solvent like
ethanol affords the thiolate intermediate (15b). Reaction of
intermediate (15b) with the desired building block, here shown as a
capped P3 building block (12c) provides the cyanoguanidine
derivative (15d). Other building blocks, R.sup.16-G or
R.sup.16-G-P4-P3, can alternatively be coupled to the intermediate
(15b). Hydrolysis of the cyano group by treatment of (15d) with
diluted hydrochloric acid gives the guanylurea derivative
(15e).
[0230] When R7, R7' and A' contains functional groups, these are
suitably protected by methods recognized by persons skilled in the
art, see for example Bodanzky or Greene cited above.
[0231] Formation of Macrocyclic Compounds
[0232] Compounds according to the present invention wherein an
alkylene chain extending from the R.sup.7/R.sup.7' cycloalkyl to Rx
or R.sup.11 thus forming a macrocycle, can be prepared as described
below. Suitable P1, P2 and P3 building blocks, or precursors
thereof, are coupled together using the strategies described above,
followed by a ring-closing reaction (macrocyclization). The
substituent W--R.sup.8 of the P2 building block can be incorporated
via a Mitsunobu reaction as described above, before or after
formation of the macrocycle or the desired building blocks can be
coupled together using the appropriately substituted P2-building
block. For macrocyclic structures extending from the
R.sup.7/R.sup.7' cycloalkyl to R.sup.11, P3 amino acids containing
the appropriate side chain can be prepared as described in
WO00/59929.
[0233] A typical route to macrocyclic compounds is shown in Scheme
18 which illustrates the technique applied to a compound having a
5-membered P2 scaffold and a spiro-cyclopropyl group in the P1
moiety, where the macrocycle extends from the P3 side chain.
##STR00043##
[0234] Coupling of proline derivative (16a) with the appropriate,
acid protected, amino acid (16b) using e.g. the phosgene conditions
described above provides (16c). Formation of the macrocycle can
then be carried out via an olefin metathesis reaction using a
Ru-based catalyst such as the one reported by Miller, S. J.,
Blackwell, H. E.; Grubbs, R. H. J. Am. Chem. Soc. 118, (1996),
9606-9614, Kingsbury, J. S., Harrity, J. P. A., Bonitatebus, P. J.,
Hoveyda, A. H., J. Am. Chem. Soc. 121, (1999), 791-799 and Huang et
al., J. Am. Chem. Soc. 121, (1999), 2674-2678. It will also be
recognized that catalysts containing other transition metals such
as Mo can be used for this reaction. Optionally the double bond is
reduced and/or the ethyl ester is hydrolysed by standard
hydrogenation and/or hydrolysation methods respectively well known
in the art. Alternatively the methyl ester can be selectively
hydrolysed followed by coupling of a R.sup.16-G-P4 building block
by standard peptide coupling conditions. The macrocyclisation step
described in Scheme 16 can also be applied to the corresponding
carbocyclic analogues described above. When the linker contains a
nitrogen atom the ring closure can be carried out by reductive
amination as described in WO00/59929.
[0235] Macrocyclic compounds without the cyclopropyl moiety in the
P1 part, i.e. the macrocyclic ring extends directly from the
peptidic backbone at the carbon adjacent R.sup.7, can be prepared
using the methods described herein. An example wherein a proline
derivative is used as the cyclic P2 scaffold is shown in scheme
17.
##STR00044##
[0236] Coupling of a suitable allylglycine derivative (17a), to the
acid function of the P2 building block (17b) using standard peptide
coupling conditions yields the amide derivative (17c). Removal of
the Boc protection group by acidic treatment followed by formation
of a chlorocarbamate by treatment with phosgene in the presence of
sodium hydrogencarbonate and subsequent reaction with the olefin
substituted amino acid (17d) provides the urea compound (17e). A
ring closing metathesis reaction is then effected by using for
example Hoveyda-Grubbs catalyst which gives the macrocyclic
compound (17f).
[0237] Even though scheme 17 shows the synthetic sequence using a
P2 building block wherein the R8 substituent is attached to the
scaffold, it will be apparent that an unsubstituted P2 scaffold
could be used and the R.sup.8 group introduced at any suitable
stage of the synthesis, using any of the methods described
herein.
[0238] Building blocks to be used in the preparation of compounds
wherein the macrocycle extends from the nitrogen in the linkage
between the P2 and P3 fragments i.e. X is NRx in general formula I,
or in the preparation of compounds wherein the P3 and P4 fragments
are absent, i.e. m and n are 0 and G is NRj in general formula I,
can typically be prepared as outlined in scheme 18B.
##STR00045##
[0239] Carbamate 18a, which is commercially available or is readily
prepared for instance by reaction of the desired alkyl amine with
di-tert-butyl dicarbonate, can be reacted with an appropriate
.omega.-unsaturated alcohol under Mitsunobu conditions to provide
the alkylated carbamate (18b). Subjection of 18b to acidic
conditions like for example treatment with trifluoroacetic acid in
a solvent like dichloromethane gives the free amine (18c) which can
be linked to a P2 fragment using any of the previously described
strategies.
[0240] Macrocyclic structures containing a hydrazine group i.e. X
is NRjNRj or m and n are 0 and G is NRjNRj, in general formula I,
can be prepared by linking a suitably N-alkylated carbazate
derivative to the P2 fragment. Alkylated carbazate derivatives can
be prepared, for example, as described in Scheme 19.
##STR00046##
[0241] Oxidation of the appropriate alcohol (19a) effected by a
suitable oxidation method like for example with N-methyl morpholine
oxide and tetrapropylammonium perruthenate in a solvent like
dichloromethane provides aldehyde (19b). Reductive alkylation of
tert-butyl carbazate with the afforded aldehyde gives the desired
N-alkylated building block (19c). Alternatively, any desired
hydrazine derivative such as morpholin-1-ylamine,
piperidin-1-ylamine or the like can be used instead of tert-butyl
carbazate in the reaction with aldehyde 19b.
[0242] Scheme 20 illustrates synthetic sequences to building blocks
suitable for the preparation of compounds wherein the "outer"
nitrogen of the hydrazine group is alkylated, either with an
.omega.-unsaturated alkyl chain appropriate for subsequent
macrocycle formation or with any other suitable alkyl group.
##STR00047##
[0243] Reaction of a suitably protected hydrazine derivative, for
example (1,3-dioxo-1,3-dihydro-isonidol-2-yl)-carbamic acid
tert-butyl ester (20a), which can easily be prepared by a person
skilled in the art, with a desired alcohol, R--OH, under Mitsunobu
conditions provides N-alkylated hydrazine compound (20b). Removal
of the phtalimido group effected by treatment with hydrazine or a
derivative thereof like hydrazine hydrate or hydrazine acetate
provides the carbazate (20c). The afforded primary amine can then
either be be coupled to any desired P2 fragment using any of the
methods previously described to give the urea derivative (20d) or
alternatively it can be further alkylated using for example the
reductive amination method described in scheme 19 followed by
coupling to a P2 fragment as previously described to give 20e.
[0244] Scheme 21 exemplifies the coupling of a hydrazine containing
P3 building block to a cyclopentane scaffold followed by
macrocyclisation.
##STR00048##
[0245] Coupling of the carbazate derivative (21b) to the P2-P1
building block (21a) using standard peptide coupling conditions
provides intermediate (21c). Ring closure of (21c) by an olefin
metathesis reaction as described in scheme 18 gives the macrocyclic
compound (21d).
[0246] The term "N-protecting group" or "N-protected" as used
herein refers to those groups intended to protect the N-terminus of
an amino acid or peptide or to protect an amino group against
undesirable reactions during synthetic procedures. Commonly used
N-protecting groups are disclosed in Greene, "Protective Groups in
Organic Synthesis" (John Wiley & Sons, New York, 1981), which
is hereby incorporated by reference. N-protecting groups include
acyl groups such as formyl, acetyl, propionyl, pivaloyl,
t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoracetyl,
trichloroacetyl, phthalyl, o-nitrophenoxyacetyl,
.alpha.-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl,
4-nitrobenzoyl, and the like; sulfonyl groups such as
benzenesulfonyl, p-toluenesulfonyl, and the like, carbamate forming
groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,
p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
2-nitro-4,5-dimethoxybenzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl,
1-(p-biphenylyl)-1-methylethoxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
benzhydryloxycarbonyl, t-butoxycarbonyl,
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,
phenoxycarbonyl, 4-nitrophenoxycarbonyl,
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl,
and the like; alkyl groups such as benzyl, triphenylmethyl,
benzyloxymethyl and the like; and silyl groups such as
trimethylsilyl and the like. Favoured N-protecting groups include
formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl,
benzyl, t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).
[0247] Hydroxy protecting group as used herein refers to a
substituent which protects hydroxyl groups against undesirable
reactions during synthetic procedures such as those O-protecting
groups disclosed in Greene, "Protective Groups In Organic
Synthesis," (John Wiley & Sons, New York (1981)). Hydroxy
protecting groups comprise substituted methyl ethers, for example,
methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl,
2-(trimethylsilyl)ethoxymethyl, t-butyl and other lower alkyl
ethers, such as isopropyl, ethyl and especially methyl, benzyl and
triphenylmethyl; tetrahydropyranyl ethers; substituted ethyl
ethers, for example, 2,2,2-trichloroethyl; silyl ethers, for
example, trimethylsilyl, t-butyidimethylsilyl and
t-butyldiphenylsilyl; and esters prepared by reacting the hydroxyl
group with a carboxylic acid, for example, acetate, propionate,
benzoate and the like.
[0248] In treating conditions caused by flavivirus such as HCV, the
compounds of formula I are typically administered in an amount to
achieve a plasma level of around 100 to 5000 nM, such as 300 to
2000 nM. This corresponds to a dosage rate, depending on the
bioavailability of the formulation, of the order 0.01 to 10
mg/kg/day, preferably 0.1 to 2 mg/kg/day. A typical dosage rate for
a normal adult will be around 0.05 to 5 g per day, preferably 0.1
to 2 g such as 500-750 mg, in one to four dosage units per day. As
with all pharmaceuticals, dosage rates will vary with the size and
metabolic condition of the patient as well as the severity of the
infection and may need to be adjusted for concomitant
medications.
[0249] As is good prescribing practice with antiviral therapy, the
compounds of formula I are typically coadministered with other HCV
therapies to avoid the generation of drug escape mutants. Examples
of such additional HCV antiviral therapies include ribavirin,
interferons, including pegylated interferons. Additionally a number
of nucleoside analogues and protease inhibitors are in clinical or
preclinical development and will be amenable to co-administration
with the compounds of the invention.
[0250] Accordingly a further aspect of the invention provides a
composition comprising a compound of formula I and at least one
further HCV antiviral in a common dosage unit, such as any of the
dosage forms described below, but especially an orally administered
tablet, or capsule or a liquid suspension or solution for oral or
injection use. A further aspect of the invention provides a method
for the treatment or prophylaxis of flavivirus infection, such as
HCV, comprising the sequential or simultaneous administration of a
compound of formula I and at least one further HCV antiviral. A
related aspect of the invention provides a patient pack comprising
a first pharmaceutical composition, preferably in unit dosage form,
of the compound of formula I and a second pharmaceutical
composition, typically also in unit dosage form and generally in a
separate container within the patient pack, of a second HCV
antiviral. A patient pack will conveniently also be provided with
instructions printed on the package or a container therein, or on a
package insert, for the simultaneous or sequential administration
of the respective pharmaceutical compositions.
[0251] Many HCV patients are co-infected, or prone to
superinfection, with other infectious diseases. Accordingly, a
further aspect of the invention provides combination therapies
comprising the compound of the invention co-formulated in the same
dosage unit or co-packaged with at least one further anti-infective
pharmaceutical. The compound of the invention and the at least one
further antinfective are administered simultaneously or
sequentially, typically at doses corresponding to the monotherapy
dose for the agent concerned. However, certain antifectives can
induce a synergistic response, allowing one or both of the active
ingredients to be administered at a lower dose that the
corresponding monotherapy. For example in drugs prone to rapid
metabolism by Cyp3A4, co-dosing with the HIV protease inhibitor
ritonavir can allow lower dosage regimes to be administered.
[0252] Typical coinfections or superinfections with HCV include
hepatitis B virus or HIV. Accordingly the compound of the invention
is advantageously co-administered (either in the same dosage unit,
co-packaged or separately prescribed dosage unit) with at least one
HIV antiviral and/or at least one HBV antiviral.
[0253] Representative HIV antivirals include NRTI such as alovudine
(FLT), zudovudine (AZT, ZDV), stavudine (d4T, Zerit), zalcitabine
(ddC), didanosine (ddl, Videx), abacavir, (ABC, Ziagen), lamivudine
(3TC, Epivir), emtricitabine (FTC, Emtriva), racevir (racemic FTC),
adefovir (ADV), entacavir (BMS 200475), alovudine (FLT), tenofovir
disoproxil fumarate (TNF, Viread), amdoxavir (DAPD), D-d4FC
(DPC-817), -dOTC (Shire SPD754), elvucitabine (Achillion
ACH-126443), BCH 10681 (Shire) SPD-756, racivir, D-FDOC, GS7340,
INK-20 (thioether phospholipid AZT, Kucera),
2'3'-dideoxy-3'-fluoroguanosine (FLG) & its prodrgus such as
MIV-210, reverset (RVT, D-D4FC, Pharmasset DPC-817).
[0254] Representative NNRTI include delavirdine (Rescriptor),
efavirenz (DMP-266, Sustiva), nevirapine (BIRG-587, Viramune),
(+)calanolide A and B (Advanced Life Sciences), capravirine
(AG1549f S-1153; Pfizer), GW-695634 (GW-8248; GSK), MIV-150
(Medivir), MV026048 (R-1495; Medivir AB/Roche), NV-05 2 2 (Idenix
Pharm.), R-278474 (Johnson & Johnson), RS-1588 (Idenix Pharm.),
TMC-120/125 (Johnson & Johnson), TMC-125 (R-165335; Johnson
& Johnson), UC-781 (Biosyn Inc.) and YM215389 (Yamanoushi).
[0255] Representative HIV protease inhibitors include PA-457
(Panacos), KPC-2 (Kucera Pharm.), 5 HGTV-43 (Enzo Biochem),
amprenavir (VX-478, Agenerase), atazanavir (Reyataz), indinavir
sulfate (MK-639, Crixivan), Lexiva (fosamprenavir calcium,
GW-433908 or 908, VX-175), ritonavir (Norvir), lopinavir+ritonavir
(ABT-378, Kaletra), tipranavir, nelfinavir mesylate (Viracept),
saquinavir (Invirase, Fortovase), AG 1776 (JE-2147, KNI-764; Nippon
Mining Holdings), AG-1859 (Pfizer), DPC-681/684 (BMS), GS224338;
Gilead Sciences), KNI-272 (Nippon Mining Holdings), Nar-DG-35
(Narhex), P(PL)-100 (P-1946; Procyon Biopharma), P-1946 (Procyon
Biopharma), R-944 (Hoffmann-LaRoche), RO-0334649
(Hoffmann-LaRoche), TMC-114 (Johnson & Johnson), VX-385
(GW640385; GSK/Vertex), VX-478 (Vertex/GSK).
[0256] Other HIV antivirals include entry inhibitors, including
fusion inhibitors, inhibitors of the CD4 receptor, inhibitors of
the CCR5 co-receptor and inhibitors of the CXCR4 coreceptor, or a
pharmaceutically acceptable salt or prodrug thereof. Examples of
entry inhibitors are AMD-070 (AMD11070; AnorMed), BlockAide/CR
(ADVENTRX Pharm.), BMS 806 (BMS-378806; BMS), Enfurvirtide (T-20,
R698, Fuzeon), KRH1636 (Kureha Pharmaceuticals), ONO-4128
(GW-873140, AK-602, E-913; ONO Pharmaceuticals), Pro-140 (Progenics
Pharm), PRO542 (Progenics Pharm.), SCH-D (SCH-417690;
Schering-Plough), T-1249 (R724; Roche/Trimeris), TAK-220 (Takeda
Chem. Ind.), TNX-355 (Tanox) and UK-427,857 (Pfizer). Examples of
integrase inhibitors are L-870810 (Merck & Co.), c-2507 (Merck
& Co.) and S(RSC)-1838 (shionogi/GSK).
[0257] Examples of HBV antivirals include adefovir dipivoxil
(Hepsera), and especially lamivudine and
2'3'-dideoxy-3'-fluoroguanosine (FLG) & its prodrugs such as
MIV-210, the 5'-O-valyl-L-lactyl prodrug of FLG. These latter HBV
antivirals are particularly convenient as they are also active
against HIV.
[0258] While it is possible for the active agent to be administered
alone, it is preferable to present it as part of a pharmaceutical
formulation. Such a formulation will comprise the above defined
active agent together with one or more acceptable carriers or
excipients and optionally other therapeutic ingredients. The
carrier(s) must be acceptable in the sense of being compatible with
the other ingredients of the formulation and not deleterious to the
recipient.
[0259] The formulations include those suitable for rectal, nasal,
topical (including buccal and sublingual), vaginal or parenteral
(including subcutaneous, intramuscular, intravenous and
intradermal) administration, but preferably the formulation is an
orally administered formulation. The formulations may conveniently
be presented in unit dosage form, e.g. tablets and sustained
release capsules, and may be prepared by any methods well known in
the art of pharmacy.
[0260] Such methods include the step of bringing into association
the above defined active agent with the carrier. In general, the
formulations are prepared by uniformly and intimately bringing into
association the active agent with liquid carriers or finely divided
solid carriers or both, and then if necessary shaping the product.
The invention extends to methods for preparing a pharmaceutical
composition comprising bringing a compound of Formula I or its
pharmaceutically acceptable salt in conjunction or association with
a pharmaceutically acceptable carrier or vehicle. If the
manufacture of pharmaceutical formulations involves intimate mixing
of pharmaceutical excipients and the active ingredient in salt
form, then it is often preferred to use excipients which are
non-basic in nature, i.e. either acidic or neutral. Formulations
for oral administration in the present invention may be presented
as discrete units such as capsules, cachets or tablets each
containing a predetermined amount of the active agent; as a powder
or granules; as a solution or a suspension of the active agent in
an aqueous liquid or a non-aqueous liquid; or as an oil-in-water
liquid emulsion or a water in oil liquid emulsion and as a bolus
etc.
[0261] With regard to compositions for oral administration (e.g.
tablets and capsules), the term suitable carrier includes vehicles
such as common excipients e.g. binding agents, for example syrup,
acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone
(Povidone), methylcellulose, ethylcellulose, sodium
carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and
starch; fillers and carriers, for example corn starch, gelatin,
lactose, sucrose, microcrystalline cellulose, kaolin, mannitol,
dicalcium phosphate, sodium chloride and alginic acid; and
lubricants such as magnesium stearate, sodium stearate and other
metallic stearates, stearic acid, glycerol stearate, silicone
fluid, talc waxes, oils and colloidal silica. Flavouring agents
such as peppermint, oil of wintergreen, cherry flavouring or the
like can also be used. It may be desirable to add a colouring agent
to make the dosage form readily identifiable. Tablets may also be
coated by methods well known in the art.
[0262] A tablet may be made by compression or moulding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active agent in a
free flowing form such as a powder or granules, optionally mixed
with a binder, lubricant, inert diluent, preservative,
surface-active or dispersing agent. Moulded tablets may be made by
moulding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets may be
optionally be coated or scored and may be formulated so as to
provide slow or controlled release of the active agent.
[0263] Other formulations suitable for oral administration include
lozenges comprising the active agent in a flavoured base, usually
sucrose and acacia or tragacanth; pastilles comprising the active
agent in an inert base such as gelatin and glycerin, or sucrose and
acacia; and mouthwashes comprising the active agent in a suitable
liquid carrier.
[0264] The compounds of formula I can form salts which form an
additional aspect of the invention. Appropriate pharmaceutically
acceptable salts of the compounds of formula I include salts of
organic acids, especially carboxylic acids, including but not
limited to acetate, trifluoroacetate, lactate, gluconate, citrate,
tartrate, maleate, malate, pantothenate, isethionate, adipate,
alginate, aspartate, benzoate, butyrate, digluconate,
cyclopentanate, glucoheptanate, glycerophosphate, oxalate,
heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate,
3-phenylpropionate, picrate, pivalate, proprionate, tartrate,
lactobionate, pivolate, camphorate, undecanoate and succinate,
organic sulphonic acids such as methanesulphonate,
ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate,
2-napthalenesulphonate, benzenesulphonate,
p-chlorobenzenesulphonate and p-toluenesulphonate; and inorganic
acids such as hydrochloride, hydrobromide, hydroiodide, sulphate,
bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and
sulphonic acids. The invention further extends to salts of the
compounds of formula I which may or may not be pharmaceutically
acceptable, but which are useful as synthetic intermediates, the
salt moiety being displaced or replaced as necessary.
[0265] The invention includes prodrugs of the compounds of formula
I. Prodrugs of the compounds of formula I are those compounds which
following administration to a patient release a compound of the
formula I in vivo generally following hydrolysis in the gut, liver
or plasma. Typical prodrugs are pharmaceutically acceptable ethers
and especially esters (including phosphate esters) of hydroxy
functions, pharmaceutically acceptable amides or carbamates of
amine functions or pharmaceutically acceptable esters of carboxy
functions. Preferred pharmaceutically acceptable esters include
alkyl esters, including acetyl, ethanoyl, butyryl, t-butyryl,
stearyl and pivaloyl, phosphate esters and sulphonic esters (ie
those derived from RSO.sub.2OH, where R is lower alkyl or aryl).
Pharmaceutically acceptable esters include lower alkyl ethers and
the ethers disclosed in WO00/47561, especially methoxyaminoacyl and
ethoxyaminoacyl.
[0266] The compounds of the invention have various steric centres
and the invention extends to racemates and enantiomers at each of
these steric centres.
[0267] Typically, the stereochemistry of the groups corresponding
to the P3 and P4 side chains (ie R.sup.15 and/or R.sup.11) will
correspond to an L-amino acid configuration, although the invention
also extends to D-isomers at one or both of these centres. It is
noteworthy that the L configuration is active nothwithstanding that
the nature of the E moiety means that P3 and P4 are typically
translated one atom relative to a conventional polypeptide and the
fact that the reversal of a peptide residue, as envisaged for P3
and P4 then pitches the amine acid side chain to the opposite side
compared to a conventional peptide substrate.
[0268] The stereochemistry of the backbone component of the cyclic
P2 group (i.e. spanning the carbonyl of the P1 amide bond and the
carbonyl or E extending of P3 will typically correspond to
L-proline. The stereochemistry of the P2 ring atom to which W is
bonded is typically as shown
##STR00049##
[0269] In compounds of the invention wherein R.sup.7 and R.sup.7'
together define a spiroalkyl group, such a spiro-cycloalkyl will
typically comprise an R.sup.7'a substituent on the
spiro-cyclopropyl ring which is is orientated syn to A:
##STR00050##
[0270] or anti to A:
##STR00051##
[0271] Conveniently, the Spiro carbon of such a spiro-cyclopropyl
ring has the R configuration:
##STR00052##
[0272] Conveniently an R.sup.7'a substituent on a spiro-cyclopropyl
ring adjacent to A is in a syn orientation in the following
absolute configuration:
##STR00053##
[0273] Particularly preferred variants have R.sup.7'a include
ethyl, hence the asymmetric carbon atoms at position 1 and 2 have
the R, R configuration. Alternative preferred R.sup.7'a include
vinyl, hence the asymmetric carbon atoms at position 1 and 2 have
the R, S configuration.
[0274] Where the compound of the invention is a macrocycle
comprising a J group, J is preferably a diastereomer represented by
partial structures (i) or (ii):
##STR00054##
[0275] especially where J is syn to A.
Detailed Description of the Embodiments
[0276] Various embodiments of the invention will now be described
by way of illustration only with reference to the following
non-limiting examples.
Example 1
##STR00055##
[0277] 7-Methoxy-2-phenyl-quinolin-4-ol (1)
[0278] To a stirred round bottled flask with toluene (100 mL) ethyl
benzoyl acetate (18.7 g, 97 mmol) and m-anisidine (12 g, 97 mmol)
was added. 4 M HCl in dioxane (0.5 mL) was added and the reaction
mixture was refluxed for 6 h (140.degree. C.). The mixture was
co-evaporated with toluene. To the crude mixture diphenyl ether (50
mL) was added and the mixture was heated to 280.degree. C. for 2 h.
When the theoretical amount ethanol (6 mL) was collected in a Dean
Stark trap the heating was stopped and the mixture was cooled to
rt. The crude mixture was dissolved in CH.sub.2Cl.sub.2 (100 mL)
and stirred for 30 min. The formed precipitate was filtered off and
dried which gave 1 (4.12 g, 16.4 mmol, 17 %): pale yellow
powder.
[0279] .sup.1H (300 MHz, DMSO-D.sub.6): .delta. 3.8 (s, 3H), 6.24
(s, 1H), 6.88-6.96 (dd, 1H, J=9.07 Hz, J=2.47 Hz), 7.19 (d, 1H,
J=2.19 Hz), 7.56 (t, 3H, J=2.19 Hz), 7.8 (dd, 2H, J=7.14 Hz, J=2.19
Hz), 8.0 (d, 1H, J=9.06 Hz); .sup.13C (75.5 MHz, DMSO-D.sub.6):
.delta. 55.3, 99.6, 106.9, 113.1, 119.1, 126.4, 127.5, 128.8,
130.2, 134.1, 142.2, 149.4, 161.8, 176.4.
Example 2
##STR00056##
[0280] Boc-L-tert-leucine-OH (2)
[0281] Triethylamine (890 .mu.L, 6.40 mmol) was added dropwise to a
stirred solution of L-tert-leucine (300 mg, 2.29 mmol) and
di-tert-butyl dicarbonate (599 mg, 2.74 mmol) in dioxane/water 1:1
(8 mL) and the solution was stirred overnight. The mixture was
extracted with petroleum ether (2.times.) and the aqueous phase was
cooled to 0.degree. C. and carefully acidified to pH 3 by slow
addition of 4M NaHSO.sub.4.H.sub.2O. The acidified water phase was
extracted with EtOAc (3.times.) and the combined organic phases
were washed with brine (2.times.) and was then dried, filtered and
concentrated to give the title compound (522 mg, 99%) as a
colorless powder. No further purification was needed.
[0282] .sup.1H-NMR (300 MHz, CD.sub.3OD) .delta. 0.99 (s, 9H), 1.44
(s, 9H), 3.96 (s, 1H); .sup.13C-NMR (75.5 MHz, CD.sub.3OD) .delta.
27.1, 28.7, 34.9, 68.0, 80.5, 157.8, 174.7.
Example 3
##STR00057##
[0283] ((S)-Cyclohexyl-methylcarbamoyl-methyl)-carbamic acid
tert-butyl ester (3)
[0284] Boc-Chg-OH (387 mg, 1.50 mmol) was coupled to methylamine
hydrochloride (111 mg, 1.65 mmol) using the same HATU coupling
conditions as in the synthesis of compound 7. The crude product was
extracted with EtOAc, washed with brine and concentrated.
Purification by flash column chromatography (EtOAc) provided the
title compound (307 mg, 76%) as a colorless solid.
[0285] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 0.91-1.13 (m, 2H),
1.14-1.31 (m, 3H), 1.44 (s, 9H), 1.61-1.80 (m, 6H), 2.80 (d, J=4.7
Hz, 3H), 3.91 (dd, J=7.1, 9.1 Hz, 1H), 5.23 (b, 1H), 6.52 (bs, 1H);
.sup.13C-NMR (75.5 MHz, CDCl.sub.3) .delta. 25.9, 26.0, 26.1, 28.3,
28.5, 29.6, 40.5, 59.5, 79.7, 155.9, 172.4.
Example 4
##STR00058##
[0286]
{(S)-1-[((S)-Cyclohexyl-methylcarbamoyl-methyl)-carbamoyl]-2,2-dime-
thyl-propyl)-carbamic acid tert-butyl ester (4)
[0287] To a solution of compound 3 (98 mg, 0.362 mmol) in methylene
chloride (3 mL) were added triethylsilane (115 mL, 0.742 mmol) and
TFA (3 mL). The mixture was stirred for 2 h at room temperature and
was then evaporated and coevaporated with toluene.
[0288] The deprotected amine was dissolved in DMF (5 mL) and
coupled to compound 2 (84 mg, 0.363 mmol) using the same HATU
coupling conditions as in the synthesis of 7. The crude product was
extracted with EtOAc, washed with brine, dried, filtered and
concentrated. Purification by flash column chromatography
(toluene/EtOAc 1:1) provided the title compound (128 mg, 92%) as a
colorless solid.
[0289] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 0.99 (s, 9H),
1.02-1.30 (m, 5H), 1.44 (s, 9H), 1.58-1.77 (m, 4H), 1.78-1.89 (m,
2H), 2.79 (d, J=4.7 Hz, 3H), 4.11 (d, J=9.3 Hz, 1H), 4.33 (app. t,
J=8.5 Hz, 1H), 5.65 (b, 1H), 7.25 (b, 1H), 7.39 (b, 1H);
.sup.13C-NMR (75.5 MHz, CDCl.sub.3) .delta. 25.9, 25.9, 26.0, 26.2,
26.8, 28.4, 29.0, 29.7, 34.5, 39.7, 58.4, 62.4, 79.4, 156.0, 171.4,
171.8.
Example 5
##STR00059##
[0290] Hept-6-enal (5)
[0291] To a solution of hept-6-en-1-ol (1 mL, 7.44 mmol) and
N-methylmorpholine N-oxide (1.308 g, 11.17 mmol) in DCM (17 mL) was
added ground molecular sieves (3.5 g, 4 .ANG.). The mixture was
stirred for 10 min at room temperature under nitrogen atmosphere
before tetrapropylammonium perruthenate (TPAP) (131 mg, 0.37 mmol)
was added. After stirring for additional 2.5 h the solution was
filtered through celite. The solvent was then carefully evaporated
and the remaining liquid was purified by flash column
chromatography (DCM) to give the volatile aldehyde 5 (620 mg, 74%)
as an oil.
Example 6
##STR00060##
[0292] N'-Hept-6-en-(E)-ylidene-hydrazinecarboxylic acid tert-butyl
ester (6)
[0293] To a solution of 5 (68 mg, 0.610 mmol) and tert-butyl
carbazate (81 mg, 0.613 mmol) in MeOH (5 mL) was added ground
molecular sieves (115 mg, 3 .ANG.). The mixture was stirred for 3 h
after which it was filtered through celite and evaporated. The
residue was dissolved in dry THF (3 mL) and AcOH (3 mL).
NaBH.sub.3CN (95 mg, 1.51 mmol) was added and the solution was
stirred over night. The reaction mixture was diluted with saturated
NaHCO.sub.3 solution (6 mL) and EtOAc (6 mL). The organic phase was
washed with brine, saturated NaHCO.sub.3, brine, dried over
MgSO.sub.4 and evaporated. The cyanoborane adduct was hydrolyzed by
treatment with MeOH (3 mL) and 2 M NaOH (1.9 mL). The mixture was
stirred for 2 h and the MeOH was evaporated. H.sub.2O (5 mL) and
DCM (5 mL) were added and the water phase was extracted three times
with DCM. The combined organic phases were dried and evaporated.
Purification by flash column chromatography (toluene/ethyl acetate
9:1 with 1% triethylamine and toluene/ethyl acetate 6:1 with 1%
triethylamine) provided the title compound (85 mg, 61%) as an
oil.
Example 7
##STR00061##
[0294] ((S)-1-Cyclopentylcarbamoyl-2,2-dimethyl-propyl)-carbamic
acid tert-butyl ester (7).
[0295] To a cold solution of 2 (133 mg, 0.575 mmol),
cyclopentylamine (64 .mu.L, 0.648 mmol) and DIEA (301 .mu.L, 1.73
mmol) in DMF (3 mL) was added the coupling reagent HATU (240 mg,
0.631 mmol). The mixture was stirred for half an hour and for
additional two hours at room temperature. The solvent was removed
by heating the reaction flask in a water bath under diminished
pressure and the residue was dissolved in ethyl acetate, after
which the organic phase was washed three times with brine, dried,
filtered and evaporated. Purification by flash column
chromatography (toluene/ethyl acetate 4:1) provided the title
compound (140 mg, 82%) as colorless crystals.
[0296] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 0.95 (s, 9H),
1.28-1.48 (m, overlapped, 2H), 1.40 (s, 9H), 1.49-1.71 (m, 4H),
1.86-2.01 (m, 2H), 3.76 (b, 1H), 4.09-4.23 (m, 1H), 5.32 (b, 1H),
5.91 (b, 1H); .sup.13C-NMR (75.5 MHz, CDCl.sub.3): .delta. 23.6,
23.7, 26.5, 28.3, 32.6, 33.1, 34.5, 51.0, 62.2, 79.4, 155.9,
170.3.
Example 8
##STR00062##
[0297] (S)-tert-Butoxycarbonylamino-cyclohexyl-acetic acid methyl
ester (8)
[0298] To a solution of Boc-Chg-OH (53 mg, 0.206 mmol) in acetone
(3 mL) were added methyl iodide (195 .mu.L, 3.1 mmol) and silver
(I) oxide (53 mg, 0.229 mmol). The mixture was allowed to stir over
night in a reaction flask that was covered with aluminum foil.
Thereafter the solution was filtered through celite and evaporated.
Purification by flash column chromatography (toluene/ethyl acetate
15:1) provided methyl ester 8 (56 mg, 100%) as a colorless oil.
[0299] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 1.00-1.34 (m,
5H), 1.44 (s, 9H), 1.54-1.82 (m, 6H), 3.73 (s, 3H), 4.20 (dd,
J=2.8, 5.0 Hz, 1H), 5.05 (bs, 1H); .sup.13C-NMR (75.5 MHz,
CDCl.sub.3): .delta. 26.0, 28.2, 28.3, 29.5, 41.1, 52.0, 58.3,
79.7, 155.6, 172.9.
Example 9
##STR00063##
[0300]
(S)-((S)-2-Benzyloxycarbonylamino-3-methyl-butyrylamino)-cyclohexyl-
-acetic acid methyl ester (9)
[0301] Compound 8 (93 mg, 0.343 mmol) was deprotected and coupled
to Z-Val-OH (95 mg, 0.378 mmol) according to the method for the
preparation of 39. Flash column chromatography (toluene/ethyl
acetate 4:1) gave the title compound (131 mg, 94%) as a colorless
solid.
[0302] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 0.92-1.30 (m,
11H), 1.54-1.88 (m, 6H), 2.02-2.18 (m, 1H), 3.72 (s, 3H), 4.05-4.18
(m, 1H), 4.52 (dd, J=3.0, 5.5 Hz, 1H), 5.12 (s, 2H), 5.49 (bs, 1H),
6.52 (bs, 1H), 7.34 (s, 5H); .sup.13C-NMR (75.5 MHz, CDCl.sub.3):
.delta. 17.8, 19.0, 25.8, 28.2, 29.3, 31.2, 40.5, 51.9, 56.8, 60.0,
66.8, 127.7, 127.9, 128.1, 128.3, 136.2, 156.3, 171.3, 172.2.
Example 10
##STR00064##
[0303] N-Boc-4R-(2-phenyl-7-methoxyquinoilne-4-oxo)proline (10)
[0304] To a stirred solution of N-Boc-trans-4-hydroxy-L-proline
(3.9 g, 16.9 mmol) in DMSO (90 mL) was added potassium
tert.butoxide (4.5 g, 40.1 mmol). After 1 hrs
4-chloro-2-phenyl-7-methoxy quinoline (4.5 g, 16.7 mmol) was added
and stirred at RT for 12 hrs. The mixture was diluted with water
(180 mL), washed with ethyl acetate (1.times.30 mL) and neutralized
with 1N HCl. The solid was filtered, washed with water and dried
giving (4.65 g, 10 mmol) of product. >95% purity by HPLC.
M+H.sup.+ 464.2.
Example 11
##STR00065##
[0305]
2-(1-Ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl)-4-(7-methoxy-2-ph-
enyl-quinoline-4-yloxy)-pyrrolidine-1-carboxylic acid tert.butyl
ester (11)
[0306] To a solution of 1-amino-2-vinyl-cyclopropanecarboxylic acid
ethyl ester (41 mg, 0.26 mmol), 10 (11 mg, 0.22 mmol), HATU (204
mg, 0.54 mmol) in DMF (4 mL) was added diisopropyehtylamine (187
.mu.L, 1.08 mmol). After stirring at RT for 1 hrs, dichloromethane
(4 mL) was added. The solution was washed with aqueous NaHCO.sub.3
(sat) and with two portions of water. The organic layer was dried
and concentrated. The product was pure enough (>95% by HPLC) to
be used in the next step. M+H.sup.+ 602.2.
Example 12
##STR00066##
[0307]
1-{[4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-
-amino}-2-vinyl-cyclopropanecarboxylic acid ethyl ester (12)
[0308] Compound 11 was kept in TFA-DCM 1:2 (3 mL) at RT for 60 min.
Toluene (3 mL) was added. The sample was co-evaporated to dryness.
Purity by HPLC>95%. M+H.sup.+ 502.4.
Example 13
##STR00067##
[0309]
1-{[1-[1-(2-Hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamo-
yl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-
-2-vinyl-cyclopropanecarboxylic acid ethyl ester (13)
[0310] To a solution of compound 12 (0.13 mmol) in THF (2 mL), was
added a large excess of NaHCO.sub.3 (s) and a solution of phosgene
in toluene (1.6 M, 600 .mu.L). After 10 min of agitation the slurry
was filtered and concentrated to dryness. The solid was redissolved
in dichloromethane and a large excess of NaHCO.sub.3 (s) and
2-Amino-N-(2-hydroxy-indan-1-yl)-3,3-dimethyl-butyramide (0.65
mmol) was added. The slurry was agitated for 24-40 hrs at RT. The
slurry was filtered, concentrated and subjected to silica column
chromatography (gradient elution from 100% DCM to MeOH/DCM 2:98) to
give the title compound (89.6 mg, 0.11 mmol). Purity by
HPLC>95%. M+H.sup.+ 790.3.
Example 14
##STR00068##
[0311]
1-[1-[1-(2-Hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propyl]-4-(6-m-
ethoxy-3-phenyl-naphthalen-1-yloxy)-pyrrolidin-2-yl]-2-vinyl-cyclopropanec-
arboxylic acid (14)
[0312] To a solution of 13 (76.7 mg, 0.097 mmol) in THF-MeOH 2:3 (2
mL) was added 1M LiOH 5 equiv. The solution was kept at 60.degree.
C. for 60 min. After cooling to RT, HOAc 15-30 eq. was added
followed by toluene (2 mL) and then concentrated to dryness. The
residue was taken up in DCM and washed with water. The organic
layer was dried and concentrated to give the title compound (72 mg,
0.094 mmol). Purity >95% by HPLC M+H.sup.+ 762.2.
Example 15
##STR00069##
[0313]
N-(2-Hydroxy-indan-1-yl)-2-[4-(6-methoxy-3-phenyl-naphthalen-1-ylox-
y)-2-(1-phenylmethanesulfonylaminocarbonyl-2-vinyl-cyclopropyl)-pyrrolidin-
-1-yl]-3,3-dimethyl-butyramide (15)
[0314] To solution of 14 (25 mg, 0.033 mmol) in chloroform (1 mL)
was added benzenesulfonamide (10.5 mg, 0.066 mmol) followed by
diisopropylethylamine (34 .mu.L, 0.197 mmol). The solution was
stirred at RT for 10 min and then at -20.degree. C. for 30 min.
PyBOP (76 mg, 0.13 mmol) was then added as a solid. The solution
was kept at -20.degree. C. for 48 hours. The solution was then
poured into aqueous NaHCO.sub.3 (sat.) and washed with water. The
organic layer was dried, concentrated and subjected to purification
by HPLC, affording the title compound as a white solid.
Example 16
##STR00070##
[0315] Resin bound 2-tert.butoxycarbonylamino-3,3-dimetylbutyric
acid (16)
[0316] To Argonaut resin PS-TFP (1.38 mmol/g, 10 g) and
2-tert-butoxycarbonylamino-3,3-imethyl-butyric acid (4.5 g, 20.7
mmol) was added dichloromethane (40 mL) and DMF (10 mL). To this
mixture was added DMAP (1 g, 8.28 mmol) and then DIC (9.5 mL, 60.7
mmol). After 3 hrs of agitation at RT the resin was filtered and
washed successively with DMF, THF, DCM, THF, DCM and ether and then
dried in a vacuum.
Example 17
##STR00071##
[0317]
[1-(2-Hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propyl]-carbamic
acid tert.butyl ester (17)
[0318] To a portion of 16 (200 mg) in DCM aminoindanol (0.14 mmol)
was added. The mixture was agitated for 2 hrs. The liquid was
filtered of and the resin washed with 2.times.DCM. The combined
liquids were combined and concentrated to dryness to afford the
title compound (20.5 mg, 0.055 mmol) Purity >95% by HPLC.
M+H.sup.+ 363.15.
[0319] .sup.13C NMR .delta..sub.C (100 MHz; CDCl.sub.3; Me.sub.4Si)
27.0, 28.5, 34.2, 39.8, 50.8, 57.9, 68.2, 73.7, 124.8, 125.6,
127.4, 128.5, 140.4, 171.6. .sup.1H NMR .delta..sub.H (400 MHz;
CDCl.sub.3; Me.sub.4Si) 1.07 (9H, s, CCH.sub.3), 1.44 (9H, s,
OCCH.sub.3), 2.93 (1H, dd, J.sub.gem 16.4 Hz, J.sub.3,2 2.3 Hz,
CH.sub.2), 3.15 (1H, dd, J.sub.gem 16.4 Hz, J.sub.3,2 5.2 Hz,
CH.sub.2),
Example 18
##STR00072##
[0320] 2-Amino-N-(2-hydroxy-indan-1-yl)-3,3-dimethyl butyramide
(18)
[0321] Compound 17 was kept in DCM-TFA 2:1 (2 mL) for 60 min at RT.
The solution was co-evaporated with toluene to dryness.
Example 19
##STR00073##
[0322]
(2-tert-Butoxycarbonylamino-3,3-dimethyl-butyrylamino)-cyclohexyl-a-
cetic acid methyl ester (19)
[0323] To a solution of 2-tert.butoxycarbonylamino-3,3-dimethyl
butyric acid (500 mg, 2.16 mmol), Amino-cyclohexyl-acetic acid
methyl ester (444 mg, 2.59 mmol) and HATU (2 g, 5.40 mmol) in DMF
(20 mL) was added diisopropylethylamine (1.88 mL, 10.8 mmol). The
solution was stirred for 1 hrs at r.t. and diluted with
dichloromethane (40 mL). This solution was washed with aqueous.
NaHCO.sub.3 (sat.) and water (.times.2), dried and concentrated.
The product was >95% pure. M+H.sup.+ 385.4.
Example 20
##STR00074##
[0324]
{1-[(Cyclohexyl-methylcarbamoyl-methyl)-carbamoyl]-2,2-dimethyl-pro-
pyl}-carbamic acid tert-butyl ester (20)
[0325] To compound 19 in EtOH-THF 1:2 was added a large excess of
methylamine (30% in water) and left at rt. for 2 weeks. The
solution was concentrated to dryness and the residue subjected to a
short silica gel column eluted with 2% MeOH in dichloromethane to
give a pure (>95%) product M+H.sup.+ 384.5.
Example 21
##STR00075##
[0326]
2-Amino-N-(cyclohexyl-methylcarbamoyl-methyl)-3,3-dimethyl-butyrami-
de (71)
[0327] Compound 20 was kept in dichloromethane-trifuoroacetic acid
2:1 for 1 h at rt and concentrated to dryness. The residue was
dried in a vacuum for 16 hrs. Reversed phase C18 HPLC showed
>95% purity M+H.sup.+ 283.1.
Example 22
##STR00076##
[0328]
(1R,2S)-1-{[(2S,4R)-1-((1S,2R)-2-Hydroxy-indan-1-ylcarbamoyl)-4-(7--
methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl--
cyclopropanecarboxylic acid (22)
[0329] Compound 12 was treated as described for the preparation of
13 but with the use of (1S,2R)-cis-1-amino-2-indanol instead of
2-amino-N-(2-hydroxyindan-1-yl)-3,3-dimethyl butyramide followed by
ester hydrolysis as described for the preparation of compound 14
which gave the title compound. Purity by HPLC>95%. M+H.sup.+
649.1.
Example 23
##STR00077##
[0330]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-(Cyclohexylmethyl-carbamoyl)-2-methyl-
-propylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-ca-
rbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (23)
[0331] N-(tert-butoxycarbonyl)-L-valine was attached to the resin
as described for the preparation of compound 16 followed by
reaction with cyclohexylamine as described for the preparation of
17 and removal of the Boc group as described for 18. The afforded
compound was then reacted with the chlorocarbamate achieved from 12
as described for the preparation of 13 which gave the title
compound. Purity by HPLC>95%. M+H.sup.+ 712.3.
Example 24
##STR00078##
[0332]
(1R,2S)-1-{[(2S,4R)-1-((1R)-2-Hydroxy-1-phenyl-ethylcarbamoyl)-4-(7-
-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-
-cyclopropanecarboxylic acid (24)
[0333] Compound 12 was treated as described for the preparation of
13 but with the use of (R)-2-phenylglycinol instead of
2-amino-N-(2-hydroxyindan-1-yl)-3,3-dimethyl butyramide instead of
2-amino-N-(2-hydroxy-indan-1-yl)-3,3-dimethyl-butyramide followed
by ester hydrolysis as described for the preparation of compound 14
which gave the title compound. Purity by HPLC>95%. M+H.sup.+
637.1.
Example 25
##STR00079##
[0334]
(1R,2S)-1-{[(2S,4R)-1-{[(1S)-Cyclohexyl-(cyclohexylmethyl-carbamoyl-
)-methyl]-carbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-
-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (25)
[0335] N-(tert-butoxycarbonyl)-L-cyclohexylglycine was attached to
the resin as described for the preparation of compound 16 followed
by reaction with cyclohexanemethylamine as described for the
preparation of 17 and removal of the Boc group as described for 18.
The afforded compound was then reacted with the chlorocarbamate
achieved from 12 as described for the preparation of 13 which gave
the title compound. Purity by HPLC>95%. M+H.sup.+ 752.4.
Example 26
##STR00080##
[0336]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-2-Cyclohexyl-1-(cyclohexylmethyl-carbam-
oyl)-ethylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-
-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (26)
[0337] N-(tert-butoxycarbonyl)-L-cyclohexylalanine was attached to
the resin as described for the preparation of compound 16 followed
by reaction with cyclohexanemethylamine as described for the
preparation of 17 and removal of the Boc group as described for 18.
The afforded compound was then reacted with the chlorocarbamate
achieved from 12 as described for the preparation of 13 which gave
the title compound. Purity by HPLC>95%. M+H.sup.+ 766.4.
Example 27
##STR00081##
[0338]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-(Cyclohexylmethyl-carbamoyl)-2,2-dime-
thyl-propylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine--
2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (27)
[0339] N-(tert-butoxycarbonyl)-L-tert-butyglycine was attached to
the resin as described for the preparation of compound 16 followed
by reaction with cyclohexanemethylamine as described for the
preparation of 17 and removal of the Boc group as described for 18.
The afforded compound was then reacted with the chlorocarbamate
achieved from 12 as described for the preparation of 13 which gave
the title compound. Purity by HPLC>95%. M+H.sup.+ 726.3.
Example 28
##STR00082##
[0340]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-(Cyclohexylmethyl-carbamoyl)-2-phenyl-
-ethylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-car-
bonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (28)
[0341] N-(tert-butoxycarbonyl)-L-phenylalanine was attached to the
resin as described for the preparation of compound 16 followed by
reaction with cyclohexanemethylamine as described for the
preparation of 17 and removal of the Boc group as described for 18.
The afforded compound was then reacted with the chlorocarbamate
achieved from 12 as described for the preparation of 13 which gave
the title compound. Purity by HPLC>95%. M+H.sup.+ 760.4.
Example 29
##STR00083##
[0342]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1S,2R)-2-Hydroxy-indan-1-ylcarbamoy-
l)-3-phenyl-propylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrro-
lidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(29)
[0343] N-(tert.butoxycarbonyl)-L-phenethylglycine was attached to
the resin as described for the preparation of compound 16 followed
by reaction with (1S,2R)-cis-1-amino-2-indanol as described for the
preparation of 17 and removal of the Boc group as described for 18.
The afforded compound was then reacted with the chlorocarbamate
achieved from 12 as described for the preparation of 13 which gave
the title compound. Purity by HPLC>95%. M+H.sup.+ 810.4.
Example 30
##STR00084##
[0344]
(1R,2S)-1-{[(2S,4R)-1-((1S)-1-Benzylcarbamoyl-2-methyl-propylcarbam-
oyl)-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino-
}-2-vinyl-cyclopropanecarboxylic acid (30)
[0345] N-(tert-butoxycarbonyl)-L-valine was attached to the resin
as described for the preparation of compound 16 followed by
reaction with benzylamine as described for the preparation of 17
and removal of the Boc group as described for 18. The afforded
compound was then reacted with the chlorocarbamate achieved from 12
as described for the preparation of 13 which gave the title
compound. Purity by HPLC>95%. M+H.sup.+ 706.2.
Example 31
##STR00085##
[0346]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1R)-2-Hydroxy-1-phenyl-ethylcarbamo-
yl)-2,2-dimethyl-propylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)--
pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(31)
[0347] N-(tert-butoxycarbonyl)-L-tert-butyglycine was attached to
the resin as described for the preparation of compound 16 followed
by reaction with (R)-2-phenylglycinol as described for the
preparation of 17 and removal of the Boc group as described for
18.
[0348] The afforded compound was then reacted with the
chlorocarbamate achieved from 12 as described for the preparation
of 13 which gave the title compound. Purity by HPLC>95%.
M+H.sup.+ 750.3.
Example 32
##STR00086##
[0349]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1R)-lndan-1-ylcarbamoyl)-2-methyl-p-
ropylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carb-
onyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (32)
[0350] (2S)-tert-butoxycarbonylamino-3-methylbutyric acid was
attached to the resin as described for the preparation of compound
16 followed by reaction with (1R)-1-aminoindane as described for
the preparation of 17 and removal of the Boc group as described for
18. The afforded compound was then reacted with the chlorocarbamate
achieved from 12 as described for the preparation of 13 which,
after purification by HPLC, gave the title compound (12.5 mg, 28%
yield), Purity by HPLC>90%. M+H.sup.+ 732.2.
Example 33
##STR00087##
[0351]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1S)-lndan-1-ylcarbamoyl)-2-methyl-p-
ropylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carb-
onyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (33)
[0352] (2S)-tert-butoxycarbonylamino-3-methylbutyric acid was
attached to the resin as described for the preparation of compound
16 followed by reaction with (1S)-1-aminoindane as described for
the preparation of 17 and removal of the Boc group as described for
18. The afforded compound was then reacted with the chlorocarbamate
achieved from 12 as described for the preparation of 13 which,
after purification by HPLC, gave the title compound (22 mg, 49%
yield), Purity by HPLC>90% M+H.sup.+ 732.2.
Example 34
##STR00088##
[0353]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-(2-hydroxyethylcarbamoyl)-2-methyl-pr-
opylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbo-
nyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (34)
[0354] (2S)-tert-butoxycarbonylamino-3-methylbutyric acid was
attached to the resin as described for the preparation of compound
16 followed by reaction with 2-aminoethanol as described for the
preparation of 17 and removal of the Boc group as described for 18.
The afforded compound was then reacted with the chlorocarbamate
achieved from 12 as described for the preparation of 13 which,
after purification by HPLC, gave the title compound (3 mg, 8%
yield), Purity by HPLC>90% M+H.sup.+ 660.2.
Example 35
##STR00089##
[0355]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1S,2R)-2-Hydroxy-indan-1-ylcarbamoy-
l)-2-methyl-propylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrro-
lidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(35)
[0356] (2S)-tert-butoxycarbonylamino-3-methylbutyric acid was
attached to the resin as described for the preparation of compound
16 followed by reaction with (1S,2R)-1-amino-2-indanol as described
for the preparation of 17 and removal of the Boc group as described
for 18. The afforded compound was then reacted with the
chlorocarbamate achieved from 12 as described for the preparation
of 13 which, after purification by HPLC, gave the title compound
(10 mg, 22% yield), Purity by HPLC>90% M+H.sup.+ 748.2.
Example 36
##STR00090##
[0357]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1R,2S)-2-Hydroxy-indan-1-ylcarbamoy-
l)-2-methyl-propylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrro-
lidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(36)
[0358] (2S)-tert-butoxycarbonylamino-3-methylbutyric acid was
attached to the resin as described for the preparation of compound
16 followed by reaction with (1R,2S)-1-amino-2-indanol as described
for the preparation of 17 and removal of the Boc group as described
for 18. The afforded compound was then reacted with the
chlorocarbamate achieved from 12 as described for the preparation
of 13 which, after purification by HPLC, gave the title compound
(11 mg, 24% yield), Purity by HPLC>75% M+H.sup.+ 748.
Example 37
##STR00091##
[0359]
(1R,2S)-1-{[(2S,4R)-1-{[Cyclohexyl-(S)-((1S,2R)-2-hydroxy-indan-1-y-
lcarbamoyl)-methyl]-carbamoyl)-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyr-
rolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(37)
[0360] (2S)-tert.butoxycarbonylamino-cyclohexylacetic acid was
attached to the resin as described for the preparation of compound
16 followed by reaction with (1S,2R)-1-amino-2-indanol as described
for the preparation of 17 and removal of the Boc group as described
for 18. The afforded compound was then reacted with the
chlorocarbamate achieved from 12 as described for the preparation
of 13 which, after purification by HPLC, gave the title compound
(7.5 mg, 16% yield), Purity by HPLC>95% M+H.sup.+ 788.3.
Example 38
##STR00092##
[0361]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1S,2R)-2-Hydroxy-indan-1-ylcarbamoy-
l)-2.2-dimethyl-propylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-p-
yrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(38)
[0362] (2S)-tert-butoxycarbonylamino-3,3-dimethylbutyric acid was
attached to the resin as described for the preparation of compound
16 followed by reaction with (1S,2R)-1-amino-2-indanol as described
for the preparation of 17 and removal of the Boc group as described
for 18. The afforded compound was then reacted with the
chlorocarbamate achieved from 12 as described for the preparation
of 13 which, after purification by HPLC, gave the title compound
(12 mg, 26% yield), Purity by HPLC>95% M+H.sup.+ 762.3.
Example 39
##STR00093##
[0363]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1S,2R)-2-Hydroxy-indan-1-ylcarbamoy-
l)-3,3-dimethyl-butylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-py-
rrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(39)
[0364] (2S)-tert-butoxycarbonylamino-4,4-dimethylpentanoic acid was
attached to the resin as described for the preparation of compound
16 followed by reaction with (1S,2R)-1-amino-2-indanol as described
for the preparation of 17 and removal of the Boc group as described
for 18. The afforded compound was then reacted with the
chlorocarbamate achieved from 12 as described for the preparation
of 13 which, after purification by HPLC, gave the title compound
(14.2 mg, 30% yield), Purity by HPLC>95% M+H.sup.+ 776.3.
Example 40
##STR00094##
[0365]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1S,2R)-2-Hydroxy-indan-1-ylcarbamoy-
l)-2-phenyl-etylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrroli-
dine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(40)
[0366] (2S)-tert-butoxycarbonylamino-3-phenylpropanoic acid was
attached to the resin as described for the preparation of compound
16 followed by reaction with (1S,2R)-1-amino-2-indanol as described
for the preparation of 17 and removal of the Boc group as described
for 18. The afforded compound was then reacted with the
chlorocarbamate achieved from 12 as described for the preparation
of 13 which, after purification by HPLC, gave the title compound
(2.4 mg, 5% yield), Purity by HPLC>95% M+H.sup.+ 796.2.
Example 41
##STR00095##
[0367]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-2-Cyclohexyl-1-((1S,2R)-2-hydroxy-indan-
-1-ylcarbamoyl)-ethylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-py-
rrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(41)
[0368] (2S)-tert-Butoxycarbonylamino-3-cyclohexylpropanoic acid was
attached to the resin as described for the preparation of compound
16 followed by reaction with (1S,2R)-1-amino-2-indanol as described
for the preparation of 17 and removal of the Boc group as described
for 18. The afforded compound was then reacted with the
chlorocarbamate achieved from 12 as described for the preparation
of 13 which, after purification by HPLC, gave the title compound
(12.3 mg, 25% yield), Purity by HPLC>95% M+H.sup.+ 802.3.
Example 42
##STR00096##
[0369]
(1R,2S)-1-{[(2S,4R)-1-{(1S)-1-[(S)-(Cyclohexyl-methylcarbamoyl-meth-
yl)-carbamoyl]-2,2-dimethyl-propylcarbamoyl}-4-(7-methoxy-2-phenyl-quinoli-
n-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic
acid (42)
[0370] Compound 12 was treated as described for the preparation of
13 but with the use of 21 instead of
2-amino-N-(2-hydroxy-indan-1-yl)-3,3-dimethyl-butyramide followed
by ester hydrolysis as described for the preparation of compound 14
which, after purification by HPLC, gave the title compound(8.6 mg,
18% yield). Purity by HPLC>95%. M+H.sup.+ 783.3.
Example 43
##STR00097##
[0371] 1-(2-Amino-4-methoxyphenyl)ethanone (43)
[0372] m-Anisidine (10.0 g, 82 mmol) was dissolved in
CH.sub.2Cl.sub.2 (50 mL), and the solution was cooled to
-50.degree. C. BCl.sub.3 (1 M in CH.sub.2Cl.sub.2, 82 mL, 82 mmol)
was added slowly during 20 min, after which the mixture was stirred
at -50.degree. C. for 30 min, followed by sequential addition of
AcCl (6.0 mL, 84 mmol) and AlCl.sub.3 (11 g, 82 mmol). The mixture
was stirred at -50.degree. C. for 1 h and was then allowed to
assume rt. After stirring at rt overnight, the solution was heated
at 40.degree. C. for 4 h, after which the mixture was poured over
ice. The aqueous mixture was made alkaline with 10% NaOH (w/v) and
extracted with EtOAc (4.times.200 mL). The combined organic phases
were washed with brine, dried (MgSO.sub.4), and evaporated to give
a black solid, which was purified by flash column chromatography
(ether/CH.sub.2Cl.sub.2 20:80). The resulting solid was
recrystallized from ether/hexane to give compound 93 as shiny tan
leaflets (5.6 g, 42%).
Example 44
N-(tert-Butyl)-N'-isopropylthiourea (44)
[0373] To a solution of tert-butylisothiocyanate (5.0 mL, 39 mmol)
in CH.sub.2Cl.sub.2 (200 mL) were added isopropylamine (4.0 mL, 47
mmol) and diisopropylethylamine (DIEA) (6.8 mL, 39 mmol), and the
mixture was stirred at rt for 2 h. The reaction mixture was diluted
with EtOAc, washed with 10% citric acid (2.times.), saturated
NaHCO.sub.3 (2.times.), H.sub.2O (2.times.), and brine (1.times.).
The organic layer was dried (MgSO.sub.4) and evaporated to yield
compound 94 (3.3 g, 52%) as a white solid which was used without
further purification.
Example 45
N-Isopropylthiourea (45)
[0374] Compound 44 (3.3 g, 20 mmol) was dissolved in conc. HCl (45
mL) and the solution was refluxed for 40 min. The mixture was
allowed to cool to rt and then cooled in an ice bath and basified
to pH 9.5 with solid and saturated NaHCO.sub.3, after which the
product was extracted into EtOAc (3.times.). The combined organic
phases were washed with H.sub.2O (2.times.) and brine (1.times.),
dried (MgSO.sub.4), and evaporated to yield crude compound 95 (2.1
g, 90%) which was used without further purification.
Example 46
##STR00098##
[0375] 2-(Isopropylamino)-1,3-thiazole-4-carboxylic acid
hydrobromide (46)
[0376] A suspension of compound 45 (2.1 g, 18 mmol) and
3-bromopyruvic acid (3.0 g, 18 mmol) in dioxane (180 mL) was heated
to 80.degree. C. Upon reaching 80.degree. C. the mixture became
clear, and soon thereafter the product started to precipitate as a
white solid. After 2 h of heating, the reaction mixture was cooled
to rt and the precipitate was filtered off and collected. This
yielded pure compound 46 (4.4 g, 94%).
Example 47
##STR00099##
[0377]
N-(2-Acetyl-5-methoxyphenyl)-2-(isopropylamino)-1,3-thiazole-4-carb-
oxamide (47)
[0378] A mixture of compound 46 (4.4 g, 16.5 mmol) and the aniline
derivative 93 (2.75 g, 16.5 mmol) in pyridine (140 mL) was cooled
to -30.degree. C. (upon cooling, the clear solution became
partially a suspension). POCl.sub.3 (3.3 mL, 35 mmol) was added
slowly over a 5 min period. The mixture was stirred at -30.degree.
C. for 1 h, and was then allowed to assume rt. After stirring at rt
for 1.5 h the reaction mixture was poured over ice, and the pH was
adjusted to about 9-10 using solid and saturated NaHCO.sub.3. The
crude product was extracted into CH.sub.2Cl.sub.2 (3.times.) and
the combined organic phases were dried (MgSO.sub.4) and evaporated.
The crude dark-beige solid was purified by flash column
chromatography (hexane/EtOAc 55:45) to give compound 47 (5.6 g,
76%) as a pale yellow solid.
Example 48
##STR00100##
[0379]
2-[2-(Isopropylamino)-1,3-thiazol-4-yl]-7-methoxyquinolin-4-ol
(48)
[0380] A solution of t.BuOK (2.42 g, 21 mmol) in anhydrous t.BuOH
(40 mL) was heated to reflux. Compound 47 (1.8 g, 5.4 mmol) was
added portion-wise over a 5 min period, and the dark red solution
formed was stirred at reflux for an additional 20 min. The mixture
was cooled to rt, and HCl (4 M in dioxane, 8.0 mL, 32 mmol) was
added, after which the reaction mixture was concentrated under
vacuum. In order to assure that all of the HCl and dioxane were
removed, the crude product was re-dissolved in CH.sub.2Cl.sub.2
twice and thoroughly evaporated to obtain the slightly impure HCl
salt of compound 98 (1.62 g) as a brown solid. The product was
dissolved in CH.sub.2Cl.sub.2 and washed with saturated
NaHCO.sub.3, after which the aqueous phase was extracted several
times with CH.sub.2Cl.sub.2. The combined organic phases were dried
(MgSO.sub.4) and evaporated to give the title compound (1.38 g,
81%) as a light brown solid (>95% pure according to HPLC tests).
.sup.1H-NMR (MeOH-d.sub.4, 400 MHz): .delta. 1.30 (d, J=6.0 Hz,
6H), 3.93 (s, 3H), 3.95-4.07 (m, 1H), 6.73 (s, 1H), 6.99 (dd,
J=2.4, 9.2 Hz, 1H), 7.26 (d, J=2.4 Hz, 1H), 7.37 (s, 1H), 8.10 (d,
J=9.2 Hz, 1H).
Example 49
##STR00101##
[0381]
(1S)-1-{[(2S,4R)-2-(1-Methoxycarbonyl-butylcarbamoyl)-4-(7-methoxy--
2-phenyl-quinolin-4-yloxy)]-pyrrolidine}-carboxylic acid tert-butyl
ester (49)
[0382] Reaction of 10 with Nva-OMe hydrochloride according to the
method described in example 11 provided the title compound. Purity
>95% by HPLC, M+H.sup.+ 578.24.
Example 50
##STR00102##
[0383]
(1S)-1-{[(2S,4R)-2-[4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrroli-
dine-2-carbonyl]-amino}-pentanoic acid methyl ester (50)
[0384] Compound 49 was kept in TFA-DCM 1:2 (3 mL) at RT for 60 min.
Toluene (3 mL) was added. The sample was co-evaporated to dryness.
Purity by HPLC>95%. M+H.sup.+ 478.21.
Example 51
##STR00103##
[0385]
(1S)-2-{[(2S,4R)-1-[(1S)-1-(Cyclohexylmethyl-carbamoyl)-2-methyl-pr-
opylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbo-
nyl]-amino}-pentanoic acid methyl ester (51)
[0386] To a solution of 50 (0.1 mmol) in THF (4 mL), cooled to
0.degree. C., was added a large excess of NaHCO.sub.3 (s) and a
solution of phosgene in toluene (0.2 mmol, 21 .mu.L). After 10 min
of agitation the slurry was filtered and concentrated to dryness.
The solid was redissolved in dichloromethane and a large excess of
NaHCO.sub.3 (s) and 2-amino-N-cyclohexylmethyl-3-methyl-butyramide,
described in example 23, (0.15 mmol) was added. The slurry was
agitated 30 hrs at RT. The slurry was filtered, concentrated and
subjected to silica column chromatography (gradient elution from
100% DCM to MeOH/DCM 2:98) to give the title compound (30 mg, 0.042
mmol). Purity by HPLC>95%. M+H.sup.+ 716.40.
Example 52
##STR00104##
[0387]
(1S)-2-{[(2S,4R)-1-[(1S)-1-(Cyclohexylmethyl-carbamoyl)-2-methyl-pr-
opylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbo-
nyl]-amino}-pentanoic acid (52)
[0388] To a solution of 51 (26 mg, 0.036 mmol) in THF-MeOH 2:3 (2
mL) was added 1M LiOH 1.5 equiv. The solution was kept at
60.degree. C. for 60 min. After cooling to RT, HOAc was added
followed by toluene (2 mL) and then concentrated to dryness to give
the title compound (25 mg, 0.035 mmol). Purity >95% by HPLC
M+H.sup.+ 702.34.
Example 53
##STR00105##
[0389]
(1R,2S)-1-{[(2S,4R)-1-[2-(2-Methoxy-phenoxy)-ethylcarbamoyl]-4-(7-m-
ethoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-c-
yclopropanecarboxylic acid ethyl ester (53)
[0390] To a solution of 12 (0.06 mmol) in THF (2 mL), was added a
large excess of NaHCO3 (s) and a solution of phosgene in toluene
(0.078 mmol). After 10 min of agitation the slurry was filtered and
concentrated to dryness. The solid was redissolved in
dichloromethane and a large excess of NaHCO.sub.3 (s) and
2-(2-methoxy-phenoxy)-ethylamine (15 mg, 0.09 mmol) was added. The
slurry was agitated for 30 hrs at RT. The slurry was filtered,
concentrated to dryness, redissolved in MeOH and subjected HPLC
purification to give the title compound (10.6 mg, 0.015 mmol).
Purity by HPLC>95%. M+H.sup.+ 695.17.
Example 54
##STR00106##
[0391]
(1R,2S)-1-{[(2S,4R)-1-[2-(2-Methoxy-phenoxy)-ethylcarbamoyl]-4-(7-m-
ethoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-c-
yclopropanecarboxylic acid (54)
[0392] To a solution of 53 (10.6 mg, 0.0153 mmol) in THF-MeOH 2:3
(2 mL) was added 1M LiOH 10 equiv. The solution was kept at
50.degree. C. for 60 min. After cooling to RT, HOAc 25 equiv. was
added followed by toluene (2 mL) and then concentrated to dryness.
The residue was taken up in ethyl acetate, filtered and
concentrated to dryness to give the title compound (9.4 mg, 0.014
mmol). Purity >95% by HPLC M+H.sup.+ 667.14.
Example 55
##STR00107##
[0393]
(1R,2S)-1-{[(2S,4R)-1-((1S,2R)-5-Hydroxy-4,5,6,7-tetrahydro-benzo[b-
]thiophen-4-yl-carbamoyl))-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrroli-
dine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(55)
[0394] The procedure described in example 53 was followed but with
the use of 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-5-ol instead
of 2-(2-methoxy-phenoxy)-ethylamine, followed by hydrolysis of the
ethyl ester as described in example 54 which gave the title
compound (7.5 mg, 0.011 mmol). Purity >95% by HPLC M+H.sup.+
669.
Example 56
##STR00108##
[0395]
(1R,2S)-1-{[(2S,4R)-1-[(3R)-3-Hydroxy-pyrrolidine-1-carbonyl)]-4-(7-
-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-
-cyclopropanecarboxylic acid (56)
[0396] The procedure described in example 53 was followed but with
the use of (R)-3-pyrrolidinol instead of
2-(2-methoxy-phenoxy)-ethylamine, followed by hydrolysis of the
ethyl ester as described in example 54 which gave the title
compound (4 mg, 0.007 mmol). Purity >95% by HPLC M+H.sup.+
587.1.
Example 57
##STR00109##
[0397]
(1R,2S)-1-{[(2S,4R)-4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-1-[(thi-
ophen-2-yl-methyl)-carbamoyl]-pyrrolidine-2-carbonyl}-amino)-2-vinyl-cyclo-
propanecarboxylic acid (57)
[0398] The procedure described in example 53 was followed but with
the use of thiophene-2-methylamine instead of
2-(2-methoxy-phenoxy)-ethylamine, followed by hydrolysis of the
ethyl ester as described in example 54 which gave the title
compound (8 mg, 0.013 mmol). Purity >95% by HPLC M+H.sup.+
613.08.
Example 58
##STR00110##
[0399]
(1R,2S)-1-{[(2S,4R)-1[(1,1-Dioxo-tetrahydro-1-.lamda..sup.6-thiophe-
n-3-yl-carbamoyl)-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-ca-
rbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (58)
[0400] The procedure described in example 53 was followed but with
the use of 3-aminotetrahydro-1H-1.lamda..sup.6-thiophene-1,1-dione
instead of 2-(2-methoxy-phenoxy)-ethylamine, followed by hydrolysis
of the ethyl ester as described in example 54 which gave the title
compound (13 mg, 0.02 mmol). Purity >95% by HPLC M+H.sup.+
635.05.
Example 59
##STR00111##
[0401] 2-Amino-3,3-dimethyl-N-thiophen-2-yl-methyl-butyramide
(59)
[0402] The title compound was prepared as described in example 17
but with the use of thiophene-2-methylamine instead of
aminoindanole followed by removal of the Boc group as described in
example 18.
Example 60
##STR00112##
[0403]
2-Amino-N-(6-hydroxy-4,5,6,7-tetrahydro-benzo[b]thiophen-5-yl)-3,3--
dimethyl-butyramide (60)
[0404] The title compound was prepared as described in example 17
but with the use of
2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-5-ol instead of
aminoindanole followed by removal of the Boc group as described in
example 18.
Example 61
##STR00113##
[0405] 2-Amino-N-(2-diethylamino-ethyl)-3,3-dimethyl-butyramide
(61)
[0406] The title compound was prepared as described in example 17
but with the use of N,N-diethylethylenediamine instead of
aminoindanole followed by removal of the Boc group as described in
example 18.
Example 62
##STR00114##
[0407]
2-Amino-N-[2-(2-methoxy-phenoxy)-ethyl]-3,3-dimethyl-butyramide
(62)
[0408] The title compound was prepared as described in example 17
but with the use of 2-methoxyphenoxyethylamine instead of
aminoindanole followed by removal of the Boc group as described in
example 18.
Example 63
##STR00115##
[0409]
2-Amino-1-(3-hydroxy-pyrrolidin-1-yl)-3,3-dimethyl-butan-1-one
(63)
[0410] The title compound was prepared as described in example 17
but with the use of (R)-3-pyrrolidinone instead of aminoindanole
followed by removal of the Boc group as described in example
18.
Example 64
##STR00116##
[0411]
2-Amino-N-(1,1-dioxo-tetrahydro-1-.lamda..sup.6-thiophen-3-yl)-3,3--
dimethyl-butyramide (64)
[0412] The title compound was prepared as described in example 17
but with the use of 2-methoxyphenoxyethylamine instead of
aminoindanole followed by removal of the Boc group as described in
example 18.
Example 65
##STR00117##
[0413]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-(2,2-Dimethyl-1-[(thiophen-2-yl-methy-
l)-carbamoyl]-propylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyr-
rolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
ethyl ester (65)
[0414] To a solution of 12 (0.06 mmol) in THF (2 mL), was added a
large excess of NaHCO.sub.3 (s) and a solution of phosgene in
toluene (0.078 mmol). After 10 min of agitation the slurry was
filtered and concentrated to dryness. The solid was redissolved in
dichloromethane and a large excess of NaHCO.sub.3 (s) and 59 (0.09
mmol) was added. The slurry was agitated for 30 hrs at RT. The
slurry was filtered, concentrated to dryness, re-dissolved in MeOH
and subjected HPLC purification to give the title compound (15.5
mg, 0.02 mmol). Purity by HPLC>95%. M+H.sup.+ 754.2.
Example 66
##STR00118##
[0415]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-(2,2-Dimethyl-1-[(thiophen-2-ylmethyl-
)-carbmoyl]-propylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrro-
lidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(66)
[0416] To a solution of 65 (14 mg, 0.017 mmol) in THF-MeOH 2:3 (2
mL) was added 1M LiOH 10 equiv. The solution was kept at 50.degree.
C. for 60 min. After cooling to RT, HOAc 20 equiv. was added
followed by toluene (2 mL) and then concentrated to dryness. The
residue was taken up in ethyl acetate, filtered and concentrated to
dryness to give the title compound (13 mg, 0.017 mmol). Purity
>95% by HPLC M+H.sup.+ 748.13.
Example 67
##STR00119##
[0417]
(1R,2S)-1-{[(2S,4R)-(1S)-1-[(1S,2R)-1-[1-(5-Hydroxy-4,5,6,7-tetrahy-
dro-benzo[b]thiophen-4-yl-carbamoyl)-2,2-dimethyl-propylcarbamoyl]-4-(7-me-
thoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cy-
clopropanecarboxylic acid (67)
[0418] The procedure described in example 65 was followed but with
the use of 60 instead of 59, followed by hydrolysis of the ethyl
ester as described in example 66 which gave the title compound (4
mg, 0.005 mmol). Purity >95% by HPLC M+H.sup.+ 782.16.
Example 68
##STR00120##
[0419]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-(2-Diethylamino-ethylcarbamoyl)-2,2-d-
imethyl-propylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidi-
ne-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (68)
[0420] The procedure described in example 65 was followed but with
the use of 61 instead of 59, followed by hydrolysis of the ethyl
ester as described in example 66 which gave the title compound (6
mg, 0.008 mmol). Purity >95% by HPLC M+H.sup.+ 729.24.
Example 69
##STR00121##
[0421]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-[2-(2-Methoxy-phenoxy)-ethylcarbamoyl-
]-2,2-dimethyl-propylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-py-
rrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(69)
[0422] The procedure described in example 65 was followed but with
the use of 62 instead of 59, followed by hydrolysis of the ethyl
ester as described in example 66 which gave the title compound (3
mg, 0.004 mmol). Purity >95% by HPLC M+H.sup.+ 780.19.
Example 70
##STR00122##
[0423]
(1R,2S)-1-{[(2S,4R)-(1S)-1-[(3R)-1-(3-Hydroxy-pyrrolidine-1-carbony-
l)-2,2-dimethyl-propylcarbamoyl]-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-p-
yrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(70)
[0424] The procedure described in example 65 was followed but with
the use of 63 instead of 59, followed by hydrolysis of the ethyl
ester as described in example 66 which gave the title compound
(12.4 mg, 0.02 mmol). Purity >95% by HPLC M+H.sup.+ 700.16.
Example 71
##STR00123##
[0425]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-(1,1-Dioxo-tetrahydro-1-.lamda..sup.6-
-thiophen-3-yl-carbamoyl)-2,2-dimethyl-propylcarbamoyl]-4-(7-methoxy-2-phe-
nyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanec-
arboxylic acid (71)
[0426] The procedure described in example 65 was followed but with
the use of 64 instead of 59, followed by hydrolysis of the ethyl
ester as described in example 66 which gave the title compound (13
mg, 0.014 mmol). Purity >95% by HPLC M+H.sup.+ 748.13.
Example 72
##STR00124##
[0427]
(4R)-1-(tert-butoxycarbonyl)-4-[(7-methoxy-2-phenylquinolin-4-yl)ox-
y]-L-prolyl-N.sup.1-(phenylsulfonyl)-L-norvalinamide (72)
[0428] To a solution of 10 (60 mg, 0.13 mmol) in DMF, HATU (124 mg,
0.325 mmol), diisopropylethylamine (114 .mu.L, 0.65 mmol) was added
and agitated for 30 min at RT. A solution of 75 (0.157 mmol) in DMF
was added. The slurry was agitated for 16 hrs at RT followed by
concentration to dryness. The residue was taken up in DCM and
washed with NaHCO.sub.3 (sat.), and water. The organic layer was
dried, concentrated and subjected to silica column chromatography
(gradient elution from 100% DCM to 2% MeOH/DCM) to give the title
compound (61 mg, 0.087 mmol). Purity >90% by HPLC. M+H.sup.+
703.23.
Example 73
##STR00125##
[0429]
(4R)-4-[(7-methoxy-2-phenylquinolin-4-yl)oxy]-L-prolyl-N.sup.1-(phe-
nylsulfonyl)-L-norvalinamide (73)
[0430] Compound 72 was kept in DCM-TFA 2:1 (2 mL) for 2.5 hr at RT.
The solution was co-evaporated with toluene to dryness. Yield 100%.
M+H 603.12
Example 74
##STR00126##
[0431] Carbamic acid,
[(1S)-1-[[(phenylsulfonyl)amino]carbonyl]butyl]-, phenylmethyl
ester (74)
[0432] To a stirred solution of Z-Nva-OH (150 mg, 0.59 mmol) in THF
(6 mL), CDI (400 mg, 2.4 mmol) was added. The slurry was agitated
for 30 min at RT followed by the addition of DBU (200 .mu.L, 1.3
mmol) and a solution of benzenesulfonamide (250 mg, 1.59 mmol) in
THF (2 mL). The mixture was stirred at 60.degree. C. for 48 hrs
followed by concentration to dryness. The residue was dissolved in
MeOH and subjected to HPLC purification to give the title compound
(118.5 mg, 0.304 mmol). Purity >95% by HPLC. M-H.sup.+ 389.0,
+Na 412.96.
Example 75
##STR00127##
[0433] (2S)-2-Amino-N-(phenylsulphonyl)pentanamide (75)
[0434] Compound 74 was dissolved in MeOH (5 mL) followed by the
addition of Pd/C and subjected to hydrogenation for 2 hrs. The
slurry was filtered through celite, washed with MeOH and
concentrated to dryness to give the title compound. Yield 100%.
[0435] M+H.sup.+ 257.3.
Example 76
##STR00128##
[0436]
4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-1,2-dicarboxyli-
c acid
1-({1-[(cyclohexyl-methylcarbamoyl-methyl)-carbamoyl]-2,2-dimethyl--
propyl}-amide)-2-[(1-phenylmethanesulfonylaminocarbonyl-2-vinyl-cyclopropy-
l)-amide] (76)
[0437] To solution of 42 (8.7 mg, 0.011 mmol) in chloroform (1 ml)
was added .alpha.-toluenesulfonamide (7 mg, 0.04 mmol) followed by
diisopropylethylamine (21 .mu.L, 0.12 mmol). The solution was
stirred at RT for 10 min and then at -20.degree. C. for 30 min.
PyBOP (46.5 mg, 0.08 mmol) was then added as a solid. The solution
was kept at -20.degree. C. for 48 hours. The solution was then
poured into aqueous NaHCO.sub.3 (sat.) and washed with water. The
organic layer was dried, concentrated and subjected to purification
by HPLC, affording the title compound as a white solid (2.8 mg,
0.0049 mmol), Purity by HPLC>95%, M+H.sup.+ 936.26.
Example 77
##STR00129##
[0438]
N-(2-Hydroxy-indan-1-yl)-2-[4-(6-methoxy-3-phenyl-naphthalen-1-ylox-
y)-2-(1-methanesulfonylaminocarbonyl-2-vinyl-cyclopropyl)-pyrrolidin-1-yl]-
-3,3-dimethyl-butyramide (77)
[0439] The title compound was prepared as described in example 76,
using 14 as carboxylic acid starting material and
methanesulfonamide instead of .alpha.-toluenesulfonamide.
[0440] Yield 13%, Purity by HPLC>95%, M+H.sup.+ 839.16.
Example 78
##STR00130##
[0441]
4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-1,2-dicarboxyli-
c acid
1-{[1-(cyclohexylmethyl-carbamoyl)-2-methyl-propyl]-amide}2-[(1-phe-
nylmethanesulfonylaminocarbonyl-2-vinyl-cyclopropyl)-amide]
(78)
[0442] The title compound was prepared as described in example 76,
using 23 as carboxylic acid starting material. Yield 2%. Purity
>95% by HPLC. M+H.sup.+ 865.28.
Example 79
##STR00131##
[0443]
4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-1,2-dicarboxyli-
c acid
1-{[1-(cyclohexylmethyl-carbamoyl)-2-methyl-propyl]-amide}2-[(1-phe-
nylmethanesulfonylaminocarbonyl-butyl)-amide] (79)
[0444] The title compound was prepared as described in example 76,
using 52 as carboxylic acid starting material. Yield 8%. Purity
>95% by HPLC. M+H.sup.+ 855.28.
Example 80
##STR00132##
[0445]
4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-1,2-dicarboxyli-
c acid
2-[(1-benzenesulfonylaminocarbonyl-butyl)-amide]1-{[1-(cyclohexylme-
thyl-carbamoyl)-2-methyl-propyl]-amide} (80)
[0446] The title compound was prepared as described in example 76,
using 52 as carboxylic acid starting material and
benzensulpnonamide instead of .alpha.-toluenesulfonamide.
[0447] Yield 21.5%. Purity >95% by HPLC. M+H.sup.+ 841.28.
Example 81
##STR00133##
[0448]
4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-1,2-dicarboxyli-
c acid
2-[(1-benzenesulfonylaminocarbonyl-2-vinyl-cyclopropyl)-amide]1-({1-
-[(cyclohexyl-methylcarbamoyl-methyl)-carbamoyl]-2,2-dimethyl-propyl}-amid-
e) (81)
[0449] The title compound was prepared as described in example 76,
using benzenesulfonamide instead of .alpha.-toluenesulfonamide.
Yield 26%. Purity by HPLC>95%, M+H.sup.+ 922.23.
Example 82
##STR00134##
[0450]
4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-1,2-dicarboxyli-
c acid
2-[(1-benzenesulfonylaminocarbonyl-butyl)-amide]1-{[1-(2-hydroxy-in-
dan-1-ylcarbamoyl)-2-methyl-propyl]-amide} (82)
[0451] To a solution of 73 (24.1 mg, 0.04 mmol) in DCM (2 ml), was
added a large excess of NaHCO.sub.3 (s) and a solution of phosgene
in toluene (50 .mu.L, 0.096 mmol). After 10 min of agitation the
slurry was filtered and concentrated to dryness. The solid was
redissolved in DCM and a large excess of NaHCO.sub.3 (s) and
2-amino-N-(2-hydroxy-indan-1-yl)-3-methyl-butyramide, described in
example 35, (0.1 mmol) was added. The slurry was agitated for 40
hrs at RT. The slurry was filtered, concentrated and subjected to
HPLC purification, to give the title compound (1.6 mg, 0.0018
mmol).
[0452] Purity >95% by HPLC. M+H.sup.+ 877.21.
Example 83
##STR00135##
[0453]
4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-1,2-dicarboxyli-
c acid
2-[(1-benzenesulfonylaminocarbonyl-butyl)-amide]1-({1-[(cyclohexyl--
methylcarbamoyl-methyl)-carbamoyl]-2,2-dimethyl-propyl}-amide)
(83)
[0454] The title compound was prepared as described in example 82
but using 21 instead of
2-amino-N-(2-hydroxy-indan-1-yl)-3-methyl-butyramide. Yield 2%.
Purity >95% by HPLC. M+H.sup.+ 912.25.
Example 84
##STR00136##
[0455]
(1R,2S)-1-{[(4R,2S)1-(1-(1S)-Hydroxymethyl-2,2-dimethyl-propylcarba-
moyl)-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amin-
o}-2-vinyl-cyclopropanecarboxylic acid ethyl ester (84)
[0456] Treatment of compound 12 as described for the preparation 13
but with the use of (S)-tert-leucinol instead of
2-amino-N-(2-hydoxy-indan-1-yl) 3,3-dimethyl-butyramide provided
the title product. M+H.sup.+ 645.2.
Example 85
##STR00137##
[0457]
(1R,2S)-1-{[(4R,2S)1-(1-(1S)-Formyl-2,2-dimethyl-propylcarbamoyl)-4-
-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vi-
nyl-cyclopropanecarboxylic acid ethyl ester (85)
[0458] To a stirred solution of compound 84 (64 mg) in
dichloromethane Dess-Martin periodinane (80 mg) was added at
ambient temperature. After 4 hrs the slurry was filtered through
basic alumina and concentrated to dryness. M+H.sup.+ 643.2.
Example 86
##STR00138##
[0459]
(1R,2S)-1-{[(4R,2S)1-{1-[((1S,2R)-2-Hydroxy-indan-1-ylamino)-propyl-
carbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-
-amino}-2-vinyl-cyclopropanecarboxylic acid ethyl ester (86)
[0460] To a solution of compound 85 in THF (2 ml) and HOAc (0.5 mL)
polystyrene bound cyanoborohydride (2.36 mmol/g, 100 mg) and
(1S,2R)-1-aminoindan-2-ol (18 mg) was added and agitated for 4 hrs.
The mixture was filtered, concentrated and purified on a prep.
HPLC. Purity by HPLC>90%. M+H.sup.+ 776.5
Example 87
##STR00139##
[0461] (1R,2S)-1-{[(4R,2S)1-{1-[((1S,2R)-2-Hydroxy-indan-1-yl
amino)-methyl]-2,2-dimethyl-propylcarbamoyl}-4-(7-methoxy-2-phenyl-quinol-
in-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic
acid (87)
[0462] To a solution of compound 86 in THF (2 mL) and MeOH (1 mL)
1N LiOH (0.2 mL) was added and the solution was kept at 60.degree.
C. for 1.5 hrs. The slurry was neutralized with 1N HCl to pH 7,
concentrated and purified on a prep. HPLC giving pure product by
HPLC>95%. M+H.sup.+ 748.4.
Example 88
##STR00140##
[0463]
(1R,2S)-1-{[(4R,2S)1-(1-{[(1S)-(Cyclohexyl-methylcarbamoyl-methyl)--
amino]-methyl}-2,2-dimethyl-propylcarbamoyl)-4-(7-methoxy-2-phenyl-quinoli-
n-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic
acid (B8)
[0464] Treatment of compound 85 as described for the preparation of
86 but with the use of 2-amino-2-cyclohexyl-N-metyl-acetamide (17
mg) instead of (1S,2R)-1-aminoindan-2-ol followed by hydrolysis of
the ethyl ester as described in example 87 provided the title
product. Purity by HPLC>95%. M+H.sup.+ 769.5
Example 89
##STR00141##
[0465] Acetic acid
(1S,2R)-1-((2S)-2-amino-3,3-dimethyl-butyrylamino)-indan-2-yl ester
(89)
[0466] A solution of compound 17 (4g) was kept in pyridine-acetic
anhydride 2:1 for 30 min. DCM was added and the solution was washed
with citric acid (aq) and NaHCO.sub.3 (aq). The organic layer was
concentrated to dryness which gave the acetylated product >90%
pure by HPLC. The afforded compound was then kept in a solution of
30% TFA in DCM for 1.5 hrs and then concentrated to dryness.
Co-evaporation twice from toluene gave the title product >90%
pure by HPLC.
Example 90
##STR00142##
[0467]
(2S,4R)-2-((1S,2R)1-Ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl)-4--
hydroxy-pyrrolidine-1-carboxylic acid tert.butyl ester (90)
[0468] A solution of HATU (6 g), diisopropylethylamine (6.8 mL),
(1R,2S)-1-amino-2-vinyl-cyclopropanecarboxylic acid ethyl ester
(1.5 g) and BOC-L-hydroxyproline (1.6 g) in dichloromethane was
stirred for 1 hrs. The mixture was extracted with DCM-NaHCO.sub.3
(aq) dried and concentrated. HPLC purity ca 90% M+H.sup.+
369.1.
Example 91
##STR00143##
[0469]
(1S,2R)-1-[(2S,4R)-(4-Hydroxy-pyrrolidine-2-carbonyl)-amino]-2-viny-
l-cyclopropanecarboxylic acid ethyl ester (91)
[0470] Compound 90 was kept in 30% trifluoroacetic acid in
dichloromethane and 1% MeOH for 2 hrs before it was concentrated to
dryness. The residue was re-dissolved in dichloromethane and during
stirring 1N NaOH was added to pH 10-11. The organic layer was
separated and concentrated which gave 1.6 g of the title product.
HPLC purity ca. 90% M+H.sup.+ 269.1.
Example 92
##STR00144##
[0471]
(1R,2S)-1-({(2S,4R)-1-[(1S)-1-((1S,2R)-2-Acetoxy-indan-1-ylcarbamoy-
l)-2,2-dimethyl-propylcarbamoyl]-4-hydroxy-pyrrolidine-2-carbonyl}-amino)--
2-vinyl-cyclopropanecarboxylic acid ethyl ester (92)
[0472] To a stirred solution of compound 89 (1.81 g) in
acetonitrile at 0.degree. C. solid NaHCO.sub.3 (800 mg) and
p-nitrophenychlorocarbonate (1.2 g) was added. The slurry was taken
up to ambient temperature and stirred for another 30 min. To this
slurry a solution of compound 91 (1.6 g) in acetonitrile (5 mL)
diisopropylethylamine (1 mL) was added. After 10 min the resulting
mixture was concentrated, re-dissolved in ethyl acetate and washed
with K.sub.2CO.sub.3 (aq) and then with 0.5 N HCl. Dried and
concentrated which gave a >80% pure product by HPLC M+H.sup.+
599.6
Example 93
##STR00145##
[0473]
(1R,2S)-1-({(2S,4R)-1-[(1)-1-((1S,2R)-2-Acetoxy-indan-1-ylcarbamoyl-
)-2,2-dimethyl-propylcarbamoyl]-4-phenylcarbamoyloxy-pyrrolidine-2-carbony-
l}-amino)-2-vinyl-cyclopropanecarboxylic acid ethyl ester (93)
[0474] To a stirred solution of compound 92 (20mg) in DCM and solid
K.sub.2CO.sub.3 (200 mg) 20% phosgene in toluene (1 mL) was added.
After 6 hrs the slurry was filtered and concentrated to dryness. To
this residue a mixture of aniline (30 mg) DCM (3 mL) and solid
NaHCO.sub.3 (50 mg) was added and agitated for 10 hrs. The mixture
was filtered, concentrated and purified on a prep. HPLC which gave
the title product, >95% pure M+H.sup.+ 718.6.
Example 94
##STR00146##
[0475]
(1R,2S)-1-({(2S,4R)-1-[1-((1S,2R)-2-Hydroxy-indan-1-ylcarbamoyl)-2,-
2-dimethyl-propylcarbamoyl]-4-phenylcarbamoyloxy-pyrrolidine-2-carbonyl}-a-
mino)-2-vinyl-cyclopropanecarboxylic acid (94)
[0476] To a solution of compound 93 in THF-MeOH 2:1 (3 mL) was
added 1N LiOH (0.2 mL). The solution was heated to 60.degree. C.
for 2 hrs. After cooling to ambient temperature acetic acid (0.5
mL) was added and the solution was concentrated to dryness. The
remaining residue was purified on a prep. HPLC which gave the title
product >95% pure M+H+ 648.5.
Example 95
##STR00147##
[0477] (5S,3R)-3,4-Dihydro-1H-isoquinoline-2-carboxylic acid
5-((1R,2S)-1-carboxy-2-vinyl-cyclopropylcarbamoyl)-1-[1-((1S,2R)-2-hydrox-
y-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamoyl]-pyrrolidin-3-yl
ester (95)
[0478] Treatment of compound 92 as described for the preparation of
93 but with the use of 12,3,4-tetrahydro-isoquinoline instead of
aniline followed by hydrolysis of the ethyl ester as described in
example 94 gave the title compound. Purity >90%. M+H.sup.+
688.6.
Example 96
##STR00148##
[0479] (5S,3R)-3,4-Dihydro-2H-quinoline-1-carboxylic acid
5-((1R,2S)-1-carboxy-2-vinyl-cyclopropylcarbamoyl)-1-[1-((1S,2R)-2-hydrox-
y-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamoyl]-pyrrolidin-3-yl
ester (96)
[0480] Treatment of compound 92 as described for the preparation of
93 but with the use of 1,2,3,4-tetrahydro-quinoline instead of
aniline followed by hydrolysis of the ethyl ester as described in
example 94 gave the title compound. Purity >90%. M+H.sup.+
688.6.
Example 97
##STR00149##
[0481]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1S,2R)-2-Hydroxy-indan-1-ylcarbamoy-
l)-2,2-dimethyl-propylcarbamoyl]-4-(pyridin-3-ylmethylcarbamoyloxy)-pyrrol-
idine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(97)
[0482] Treatment of compound 92 as described for the preparation of
93 but with the use of 2-pyridine-3-yl-ethylamine instead of
aniline followed by hydrolysis of the ethyl ester as described in
example 94 gave the title compound. Purity >90%. M+H.sup.+
663.5.
Example 98
##STR00150##
[0483]
(1R,2S)-1-{[(2S,4R)-1-[(1S)-1-((1S,2R)-2-Hydroxy-indan-1-ylcarbamoy-
l)-2,2-dimethyl-propylcarbamoyl]-4-(methyl-phenethyl-carbamoyloxy)-pyrroli-
dine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(98)
[0484] Treatment of compound 92 as described for the preparation of
93 but with the use of N-methylphenethylamine instead of aniline
followed by hydrolysis of the ethyl ester as described in example
94 gave the title compound. Purity >90%. M+H.sup.+ 690.6.
Example 99
##STR00151##
[0485]
(1R,2S)-1-({(2S,4R)-4-Benzylcarbamoyloxy-1-[(1S)-1-((1S,2R)-2-hydro-
xy-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamoyl]-pyrrolidine-2-carbon-
yl}-amino)-2-vinyl-cyclopropanecarboxylic acid (99)
[0486] Treatment of compound 92 as described for the preparation of
93 but with the use of benzylamine instead of aniline followed by
hydrolysis of the ethyl aster as described in example 94 gave the
title compound. Purity >90%. M+H.sup.+ 662.4.
Example 100
##STR00152##
[0487]
(1R,2S)-1-({(2S,4R)-1-[(1S)-1-((1S,2R)-2-Hydroxy-indan-1-ylcarbamoy-
l)-2,2-dimethyl-propylcarbamoyl]-4-phenethylcarbamoyloxy-pyrrolidine-2-car-
bonyl}-amino)-2-vinyl-cyclopropanecarboxylic acid (100)
[0488] Treatment of compound 92 as described for the preparation of
93 but with the use of phenthylamine instead of aniline followed by
hydrolysis of the ethyl aster as described in example 94 gave the
title compound. Purity >90%. M+H.sup.+ 676.5.
Example 101
##STR00153##
[0489]
(1R,2S)-1-({(4R)-1-{[2-(tert-butoxycarbonyl)hydrazino]carbonyl}-4-[-
(7-methoxy-2-phenylquinolin-4-yl)oxy]-L-prolyl}amino)-2-vinylcyclopropanec-
arboxylic acid ethyl ester (101)
[0490] To a solution of tert-butyl carbazate (0.3 mmol) and p-nitro
phenyl chloroformate (0.3 mmol) in acetonitrile (6 ml) was added
sodium hydrogen carbonate (0.48 mmol) as solid. The solution was
stirred at RT for 5 hrs and then cooled down to 0.degree. C.
Compound 62 (0.3 mmol) dissolved in acetonitrile (10 mL) was mixed
together with diisopropylethylamine (0.75 mmol) at 0.degree. C.,
and then added to the previous solution. The mixture was stirred at
RT overnight and then concentrated to dryness. The residue was
dissolved in DCM and then washed with citric acid pH 4, followed by
NaHCO.sub.3 (aq) and water, dried over anhydrous sodium sulphate,
filtrated and concentrated to dryness. The crude was dissolved in
DCM and purified by column chromatography eluted with 0.1 to 0.2%
MeOH/DCM to yield the title compound (101 mg). Purity >95% by
HPLC, M+H.sup.+ 660.1.
Example 102
##STR00154##
[0491]
(1R,2S)-1-({(4R)-1-{[2-(tert-butoxycarbonyl)hydrazino]carbonyl}-4-[-
(7-methoxy-2-phenylquinolin-4-yl)oxy]-L-prolyl}amino)-2-vinylcyclopropanec-
arboxylic acid (159)
[0492] Method A: To a solution of compound 101 (0.0115 mmol) in
THF-MeOH 2:3 (2 ml) was added 1M LiOH (10 equiv) The solution was
kept at 50.degree. C. for 60 min. After cooling to RT, HOAc (20
equiv) was added followed by toluene (2 ml) and then concentrated
to dryness. The residue was taken up in MeOH and then purified by
Prep LCMS which gave the title compound (0.7 mg). Purity >95% by
HPLC M+H.sup.+ 732.2.
[0493] Method B: To a solution of tert-butyl carbazate (0.07 mmol)
and p-nitrophenyl chloroformate (0.07 mmol) in acetonitrile (3 ml)
was added sodium hydrogen carbonate (0.112 mmol) as a solid. The
solution was stirred at RT for 2.5 hrs and then cooled to 0.degree.
C. Compound 103 (described below) (0.07 mmol) dissolved in
acetonitrile (10 ml) was mixed together with diisopropylethylamine
(0.175 mmol) at 0.degree. C., and then added to the previous
solution. The mixture was stirred at RT overnight and then
concentrated to dryness. The crude material was dissolved in MeOH
and purified by Prep LCMS which gave the title compound (4.8 mg).
Purity >95% by HPLC M+H.sup.+ 632.2
Example 103
##STR00155##
[0494]
(1R,2S)-1-{[(2S,4R)-4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrroli-
dine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(103)
[0495] To a solution of compound 12 (0.067 mmol) in THF-MeOH 2:3 (2
ml) was added 1M LiOH 10 equiv. The solution was kept at 50.degree.
C. for 2.5 hrs. After cooling to RT, HOAc 20 equiv. was added
followed by toluene (2 ml) and then concentrated to dryness. The
residue was taken up in DCM and filtered form the salts which gave
the title compound (0.07 mmol). Purity >95% by HPLC M+H.sup.+
474.
Example 104
##STR00156##
[0496]
(1R,2S)-1-({(4R)-1-(hydrazinocarbonyl)-4-[(7-methoxy-2-phenylquinol-
in-4-yl)oxy]-L-prolyl}amino)-2-vinylcyclopropanecarboxylic acid
(104)
[0497] Compound (102) was kept in TFA-DCM 1:2 (3 ml) at RT for 60
min. Toluene (1 ml) was added. The sample was co-evaporated to
dryness which gave the title compound (10.5 mg) as the
trifluoracetic acid salt. Purity by HPLC>95%. M+H.sup.+ 532.
Example 105
##STR00157##
[0498]
(1R,2S)-1-({(4R)-1-(hydrazinocarbonyl)-4-[(7-methoxy-2-phenylquinol-
in-4-yl)oxy]-L-prolyl}amino)-2-vinylcyclopropanecarboxylic acid
ethyl ester (105)
[0499] Compound 101 (50 mg) was kept in TFA-DCM 1:2 (3 ml) at RT
for 60 min. Toluene (1 ml) was added. The sample was co-evaporated
to dryness and then taken up in DCM and washed with
K.sub.2CO.sub.3, dried over anhydrous sodium sulphate and
concentrated to dryness which gave the title compound (41.8 mg).
Purity by HPLC>95%. M+H.sup.+ 560.
Example 106
##STR00158##
[0500]
(1R,2S)-1-({(4R)-1-[(2-Benzylhydrazino)carbonyl]-4-[(7-methoxy-2-ph-
enylquinolin-4-yl)oxy]-L-prolyl}amino)-2-vinylcyclopropanecarboxylic
acid etyl ester (106)
[0501] To a solution of compound 105 (0.037 mmol) in MeOH:THF (4:1)
was added benzaldehyde (0.0448 mmol). The solution was stirred at
RT for 18 hrs. Borane-pyridine complex (0.37 mmol) was added
followed by HCl (37%, 400 .mu.l). The solution was stirred for 1.5
hrs and then filtrated and concentrated to dryness. The crude
material was dissolved in MeOH and purified by Prep LCMS which gave
the title compound (0.01 mmol). Purity by HPLC>95%. M+H.sup.+
650.
Example 107
##STR00159##
[0502]
(1R,2S)-1-({(4R)-1-[(2-benzylhydrazino)carbonyl]-4-[(7-methoxy-2-ph-
enylquinolin-4-yl)oxy]-L-prolyl}amino)-2-vinylcyclopropanecarboxylic
acid (164107
[0503] To a solution compound 106 (0.0101 mmol) in THF-MeOH 2:3 (3
ml) was added 1M LiOH 10 equiv. The solution was kept at 50.degree.
C. for 18 hrs. After cooling to RT the sample was neutralized with
HCl and concentrated to dryness. The crude material was dissolved
in DCM (2 ml) and a solution of TFA:TES 1:1 (1 ml) was added. The
mixture was stirred for 3 hrs at RT and then concentrated to
dryness. The crude material was dissolved in MeOH and purified by
Prep LCMS which gave the title compound (0.6 mg). Purity by
HPLC>95%. M+H.sup.+ 622.
Example 108
##STR00160##
[0504]
(1R,2S)-2-{[(2S,4R)-1-((1S)-1-Azidomethyl-3-methyl-butylcarbamoyl)--
4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-v-
inyl-cyclopropanecarboxylic acid ethyl ester (108)
##STR00161##
[0505] i) (2S)-Methanesulphonic acid
2-tert.butoxycarbonylamino-4-methyl-pentyl ester
[0506] To a solution of
((1S)-1-hydroxymethyl-3-methyl-butyl)-carbamic acid tert-butyl
ester (25 g, 115 mmol) in dichloromethane (500 ml) cooled by an
ice-water bath was successively added diisopropylethylamine (35.7
g, 276 mmol) and methanesulphonyl chloride (15.81 g, 138 mmol). The
resulting solution was stirred over night during which time the
mixture was allowed to gradually warm up to ambient temperature.
The mixture was washed successively with water, 10% citric acid
(aq), water and saturated NaHCO.sub.3 (aq), then dried with
Na.sub.2SO.sub.4 and concentrated to a brown solid (32.6 g, 96%)
which was used in the next reaction without further
purification.
##STR00162##
ii) ((1S)-1-Azidomethyl-3-methyl-butyl)-carbamic acid tert.butyl
ester
[0507] The mesylate from step i (32.6 g, 110 mmol) was treated with
sodium azide (21.45 g, 330 mmol) in DMF at 80.degree. C. for 24
hrs. The solvent was evaporated, the residue was taken up in DCM,
filtered and washed with saturated NaHCO.sub.3 (aq). The solution
was dried with Na.sub.2SO.sub.4 and concentrated to a brown oil
which was purified by flash chromatography using a gradient of
ethyl acetate and hexane to afford the title compound as a white
solid (19.55 g, 73%).
##STR00163##
iii) (1S)-1-Azidomethyl-3-methyl-butylamine
[0508] ((1S)-1-Azidomethyl-3-methyl-butyl)-carbamic acid tert-butyl
ester (9.64 g, 39.78 mmol) was treated with TFA (30 ml) in DCM (150
ml) for 3 hrs, the mixture was evaporated under reduced pressure
and the residue was dissolved in ethyl acetate and washed with
aqueous 1 M K.sub.2CO.sub.3, dried with Na.sub.2SO.sub.4 and
concentrated to a yellow liquid (4.55 g, 80%).
[0509] Compound 12 was treated with phosgene as described in
example 13 which gave the corresponding chlorocarbamate compound.
The afforded chlorocarbamate (568 mg, 1.13 mmol) was dissolved in a
solution of DCM-THF (1:1,10 ml) and
(1S)-1-azidomethyl-3-methyl-butylamine (401 mg, 2.82 mmol) and a
large excess of NaHCO.sub.3 (s) was added. The resulting mixture
was stirred for 18 hrs, filtered and washed with dilute citric acid
(aq, pH 5). The organic layer was dried with Na.sub.2SO.sub.4 and
evaporated to afford the desired product as a light yellow oil (837
mg, 99%) sufficiently pure to be used in the next step.
[0510] M+H.sup.+ 670.1.
Example 109
##STR00164##
[0511]
(1R,2S)-1-{[(2S,4R)-1-((1S)-1-Aminomethyl-3-methyl-butylcarbamoyl)--
4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-v-
inyl-cyclopropanecarboxylic acid ethyl ester (109)
[0512] A solution of 108 (717 mg, 1.07 mmol) in THF (25 ml) was
shaken together with PS-triphenylphosphine resin (diphenylphosphino
polystyrene) (3.24 g, 1.65 mmol PPh.sub.3/g) and methanol (2.5 ml)
for 78 hrs. The mixture was filtered and the polymer was washed
repeatedly with DCM and methanol. The combined filtrates were
evaporated to yield the title compound as a light beige solid foam
(685 mg, 99%) with more than 95% purity as determined by reversed
phase HPLC. M+H.sup.+ 644.1.
[0513] General Procedure 1A for the Preparation of Compounds
110-116
[0514] To a solution of the acyl chloride (0.075 mmol) in DCM (0.5
ml) was added NaHCO.sub.3 (s) (60 mg, 07 mmol) and a solution of
the amine 109 (25 mg, 0.037 mmol) in THF (1 ml). The resulting
mixture was stirred at room temperature over night, filtered and
then shaken in the presence of PS-trisamine resin
(tris-(2-aminoethyl)aminomethyl polystyrene) (3.91 mmol/g, 50 mg,
0.2 mmol) for 5 hrs. The mixture was filtered and evaporated. The
resulting solid residue was dissolved in MeOH-THF (2:1, 1.5 ml) and
treated with 1 M LiOH (aq) (170 .mu.l) at 50.degree. C. between 2
and 16 hrs. The reaction was monitored by HPLC-MS. The mixture was
acidified with acetic acid and evaporated to dryness. The residue
was dissolved in methanol and purified by reversed phase HPLC.
[0515] General Procedure 1B for the Preparation of Compounds
110-116
[0516] To the acid (0.039 mmol) was successively added a solution
of HATU (14.7 mg, 0.039 mmol) in DMF (0.5 ml), a solution of the
amine 109 (20 mg, 0.031 mmol) in DMF (0.5 ml) and
diisopropylethylamine (30 .mu.l, 0.155 mmol). The resulting mixture
was stirred for 16 hrs then the solvent was evaporated and the
residue was dissolved in DCM and washed with water and aqueous
saturated NaHCO.sub.3. The solvent was evaporated and the residue
was dissolved in methanol-THF (2:1, 1.5 ml). To this was added 1 M
LiOH (aq) (155 .mu.l) and the mixture was stirred at 60.degree. C.
for 3-5 hrs. Glacial acetic acid (50 .mu.l) was added and the
mixture was concentrated, dissolved in methanol and purified by
reversed phase HPLC.
Example 110
##STR00165##
[0517]
(1R,2S)-1-{[(2S,4R)-1-{(1S)-1-[(3-Fluoro-benzoylamino)-methyl]-3-me-
thylbutylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2--
carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid (110)
[0518] General procedure 1A was followed using 3-fluorobenzoyl
chloride (12 mg) as acyl chloride which gave the title compound as
a solid (13.6 mg, 50%). M+H.sup.+ 738.1.
Example 111
##STR00166##
[0519]
(1R,2S)-1-{[(2S,4R)-4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-1-((1S)-
-3-methyl-1-{[(pyridine-3-carbonyl)-amino]-methyl}-butylcarbamoyl)-pyrroli-
dine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(111)
[0520] General procedure 1A was followed using nicotinoyl chloride
(10.5 mg) as acyl chloride which gave the title compound as a solid
(10 mg, 37%). M+H.sup.+ 721.1.
Example 112
##STR00167##
[0521]
(1R,2S)-1-{[(2S,4R)-4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-1-((1S)-
-3-methyl-1-{[(pyrazine-2-carbonyl)-amino]-methyl}-butylcarbamoyl)-pyrroli-
dine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(112)
[0522] General procedure 1B was followed using
pyrazine-2-carboxylic acid (5 mg) as acid which gave the title
compound as a solid (5.7 mg, 25%). M+H.sup.+ 722.1.
Example 113
##STR00168##
[0523]
(1R,2S)-1-{[(2S,4R)-4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-1-((1S)-
-3-methyl-1-{[(thiophene-3-carbonyl)-amino]-methyl}-butylcarbamoyl)-pyrrol-
idine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(113)
[0524] General procedure 1A was followed using thiophene-3-carbonyl
chloride (11 mg) which gave the title compound as a solid (4.3 mg,
16%). M+H.sup.+ 726.1.
Example 114
##STR00169##
[0525]
(1R,2S)-1-{[(2S,4R)-4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-1-((1S)-
-3-methyl-1-{[(tetrahydro-furan-2-carbonyl)-amino]-methyl}-butylcarbamoyl)-
-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid
(114)
[0526] General procedure 1B was followed using
tetrahydrofurane-2-carboxylic acid (4.5 mg) as acid which gave the
title compound as a solid (7.9 mg, 36%). M+H.sup.+ 714.1.
Example 115
##STR00170##
[0527]
(1R,2S)-1-{[(2S,4R)-4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-1-((1S)-
-3-methyl-1-{[(5-phenyl-oxazole-4-carbonyl)-amino]-methyl}-butylcarbamoyl)-
-pyrrolidine-2-carbonyl]-amino)-2-vinyl-cyclopropanecarboxylic acid
(115)
[0528] General procedure 1B was followed using
5-phenyl-oxazole-4-carboxylic acid (7.5 mg) as acid which gave the
title compound as a solid (7.5 mg, 31%). M+H.sup.+ 787.1.
Example 116
##STR00171##
[0529]
(1R,2S)-1-{[(2S,4R)-1-((1S)-1-{[(Benzofuran-2-carbonyl)-amino]-meth-
yl}-3-methyl-amino}-2-vinyl-cyclopropanecarboxylic acid (116)
[0530] General procedure 1B was followed using
benzofuran-2-carboxylic acid (6.5 mg) as acid which gave the title
compound as a solid (5.4 mg, 23%). M+H.sup.+ 760.1.
[0531] General Procedure 2 for the Preparation of compounds
117-119
[0532] To a solution of the sulphonyl chloride (0.075 mmol) in DCM
(0.5 ml) was added NaHCO.sub.3 (s) (60 mg) and a solution of the
amine 109 (25 mg, 0.037 mmol) in THF (1 ml). The resulting mixture
was stirred at room temperature for 18 hrs, filtered and then
shaken with PS-trisamine (tris-(2-aminoethyl)aminomethyl
polystyrene, 3.91 mmol/g, .about.50 mg) for 5 hrs.
[0533] The mixture was filtered and the polymer was washed
successively with DCM, THF and methanol. The solid residue
resulting from evaporation of the combined filtrates was dissolved
in MeOH-THF (2:1, 1.5 ml) and treated with 1 M LiOH (aq) (170
.mu.l) at 50.degree. C. for reaction times varying from 18 hrs to
one week depending on the actual structure. The reaction was
monitored by HPLC-MS. The mixture was acidified with acetic acid
and evaporated to dryness. The residue was dissolved in methanol
and purified by reversed phase HPLC.
Example 117
##STR00172##
[0534]
(1R,2S)-1-({(2S,4R)-4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-1-[(1S)-
-3-methyl-1-(phenylmethanesulphonylamino-methyl)-butylcarbamoyl]-pyrrolidi-
ne-2-carbonyl}-amino)-2-vinyl-cyclopropanecarboxylic acid (117)
[0535] General procedure 2 was followed using
.alpha.-toluenesulphonyl chloride (14 mg) as sulphonyl chloride
which gave the title compound as a white solid (4.9 mg, 17%).
[0536] M+H.sup.+ 770.1.
Example 118
##STR00173##
[0537]
(1R,2S)-1-[((2S,4R)-4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-1-{(1S)-
-3-methyl-1-[(5-methyl-isoxazole-4-sulphonylamino)-methyl]-butylcarbamoyl}-
-pyrrolidine-2-carbonyl)-amino]-2-vinyl-cyclopropanecarboxylic acid
(118)
[0538] General procedure 2 was followed using
5-methyl-isoxazole-4-sulphonyl chloride (14 mg) as sulphonyl
chloride which gave the title compound as a white solid (1.6 mg,
6%). M+H.sup.+ 761.0.
Example 119
##STR00174##
[0539]
(1R,2S)-1-{[(2S,4R)-1-{(1S)-1-[(5-Isoxazol-3-yl-thiophene-2-sulphon-
ylamino-methyl]-3-methyl-butylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4--
yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic
acid (119)
[0540] General procedure 2 was followed using
5-isoxazol-3-yl-thiophene-2-sulphonyl chloride (19 mg) as sulphonyl
chloride which gave the title compound as a white solid (3.0 mg,
10%). M+H.sup.+ 828.98.
Example 120
##STR00175##
[0541] 1-{[1-(N'-tert.
Butoxycarbonyl-N-hept-6-enyl-hydrazinocarbonyl)-4-(7-methoxy-2-phenyl-qui-
nolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxyl-
ic acid ethyl ester (120)
[0542] Compound 12 (200 mg, 0.4 mmol) was dissolved in
tetrahydrofuran (10 ml). A tea-spoon of sodium hydrogencarbonate
was added, followed by phosgene (1.8 .mu.l, 1.9 M in toluene). The
reaction mixture was stirred for 30 min and filtrated. The solvent
was evaporated and the crude chloride was re-dissolved in
dichloromethane (10 ml). Sodium hydrogencarbonate (1 tea-spoon) and
N'-hept-6-enyl-hydrazinecarboxylic acid tert.butyl ester (182 mg,
0.8 mmol). The reaction mixture was stirred at room temp. for 40 h.
and then filtrated and purified by silica chromatography (1%
methanol in ether.fwdarw.2% methanol in ether) to give pure title
product (240 mg, 79%).
Example 121
##STR00176##
[0543]
14-tert.Butoxycarbonylamino-18-(7-methoxy-2-phenyl-quinolin-4-yloxy-
)-2,15-dioxo-3,14,16-triaza-tricyclo[14.3.0.0*4,6*]nonadec-7-ene-4-carboxy-
lic acid ethyl ester (121)
[0544] Compound 120 (200 mg, 0.26 mmol) was dissolved in degassed
dichloromethane (30 ml). Hoveyda-Grubbs catalyst II generation (16
mg, 0.026 mmol) was then added and the mixture was refluxed under
argon atmosphere overnight. The solvent was then evaporated and the
crude product was purified by silica chromatography (1% methanol in
ether) which gave 39 mg (20%) of the title product. MS (M+H.sup.+)
728.2
Example 122
##STR00177##
[0545]
14-tert.Butoxycarbonylamino-18-(7-methoxy-2-phenyl-quinolin-4-yloxy-
)-2,15-dioxo-3,14,16-triaza-tricyclo[14.3.0.0*4,6*]nonadec-7-ene-4-carboxy-
lic Acid (122)
[0546] Compound 121 (39 mg, 0.054 mmol) was dissolved in
tetrahydrofuran (3.5 ml), water (1.75 ml) and methanol (1.75 ml).
Lithium hydroxide (430 .mu.l, 1 M in water) was then added and the
reaction was stirred at room temperature for 24 h. The volume was
reduced to half and water (10 ml) was added. Acidification (pH=5)
followed by extraction with chloroform gave 34 mg (90%) of the pure
acid 179. MS (M+H.sup.+) 700.2
Example 123
##STR00178##
[0547]
1-{[1-(N'-tert.Butoxycarbonyl-N-hex-5-enyl-hydrazinocarbonyl)-4-(7--
methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl--
cyclopropanecarboxylic Acid ethyl ester (123)
[0548] The title compound was prepared from compound 12 (800 mg,
1.6 mmol) and N-hex-5-enyl-hydrazinecarboxylic acid tert.butyl
ester (620 mg, 2.9 mmol) according to the procedure described in
Example 120 which gave 1 g (85%). MS (M+H.sup.+) 742.37
Example 124
##STR00179##
[0549]
13-tert.Butoxycarbonylamino-17-(7-methoxy-2-phenyl-quinolin-4-yloxy-
)-2,14-dioxo-3,13,15-triaza-tricyclo[13.3.0.0*4,6*]octadec-7-ene-4-carboxy-
lic Acid ethyl ester (124)
[0550] Treatment of compound 123 (400 mg, 0.54 mmol) according to
the procedure described in example 121 gave a crude product.
Purification by silica gel chromatography (1% methanol in ether)
gave the title product (67 mg, 17%). MS (M+H.sup.+) 714.29
Example 125
##STR00180##
[0551]
13-tert.Butoxycarbonylamino-17-(7-methoxy-2-phenyl-quinolin-4-yloxy-
)-2,14-dioxo-3,13,15-triaza-tricyclo[13.3.0.0*4,6*]octadec-7-ene-4-carboxy-
lic Acid (125)
[0552] The title compound was prepared from compound 124 (67 mg,
0.09 mmol) by the same procedure as described for 122 which gave 46
mg (71%) of the pure acid. Chloroform was replaced by
1,2-dichloroethane in the extraction step for the preparation of
this compound. MS (M+H.sup.+) 686.33
Example 126
##STR00181##
[0553]
13-tert.Amino-17-(7-methoxy-2-phenyl-quinolin-4-yloxy)-2,14-dioxo-3-
,13,15-triaza-tricyclo[13.3.0.0*4,6*]octadec-7-ene-4-carboxylic
Acid (126)
[0554] Compound 125 (10 mg) was dissolved in dichloromethane (4
ml). Trifluoromethanesulphonic acid (4 ml) was added and the
mixture was left at 50.degree. C. for 6 hours. The solvent was
removed and the residue was washed with acetonitrile which gave 3
mg of the pure title product (35%). MS (M+H.sup.+) 586.25
Example 127
##STR00182##
[0555]
1-{[1-(1-Methoxycarbonyl-oct-7-enylcarbamoyl)-4-(7-methoxy-2-phenyl-
-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarb-
oxylic Acid ethyl ester (127)
[0556] The title compound was prepared from compound 12 (380 mg,
0.758 mmol) and 2-aminononan-8-enyl-carboxylic acid methyl ester
(250 mg, 1.89 mmol) using the conditions described in Example 120
which gave the pure product (405 mg, 75%).
Example 128
##STR00183##
[0557]
19-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-2,16-dioxo-3,15,17-triaza--
tricyclo[15.3.0.0*4,6*]icos-7-ene-4,14-dicarboxylic Acid 4-ethyl
ester 14 methyl ester (128)
[0558] Compound 127 (170 mg, 0.2385 mmol) was dissolved in
dichloromethane (40 ml) and degassed by bubbling nitrogen for 20
min. Hoveyda-Grubbs catalyst II generation (10 mg, 0.016 mmol, 6.7
mol %) was then added and the mixture was refluxed under nitrogen
atmosphere overnight. The solvent was then evaporated, catalyst and
salts were removed by flash chromatography (5% methanol in
chloroform) and the crude product (120 mg, 73% yield, 85-90%
purity) was used in next step MS (M+H.sup.+) 685
Example 129
##STR00184##
[0559]
19-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-2,16-dioxo-3,15,17-triaza--
tricyclo[15.3.0.0*4,6*]icos-7-ene-3,14-dicarboxylic Acid 3-ethyl
ester (129)
[0560] Compound 128 (120 mg, 0.175 mmol) was dissolved in dioxane
(9 ml) and water (6 ml). Lithium hydroxide (12 mg, 0.526 mmol) was
added and the reaction was stirred at room temperature for 3.5 h.
The mixture was acidified with acetic acid to pH=5, and
co-evaporated with toluene. The crude product was used in the next
step. MS (M+H.sup.+) 671
Example 130
##STR00185##
[0561]
14-[(Cyclohexyl-methylcarbamoyl-methyl)-19-(7-methoxy-2-phenyl-quin-
olin-4-yloxy)-2,16-dioxo-3,15,17-triaza-tricyclo[15.3.0.0*4,6*]icos-7-ene--
4-carboxylic Acid 3-ethyl ester (130)
[0562] Compound 129 (crude, 100 mg), indanolamine (33 mg, 0.209
mmol) and Hunig's base (DIEA) (0.2 ml) were dissolved in DMF (14
ml). After stirring at 0.degree. C. for 10 min HATU was added. The
reaction was monitored by LC-MS. After 5 h conversion was 100%. DMF
and DIEA were removed in vacuo. The residue was partitioned between
ethyl acetate and water. The organic layer was washed with brine,
dried and concentrated in vacuo. The crude yield was 120 mg, the
purification by prep. HPLC gave 21 mg (25%) of title product. MS
(M+H.sup.+) 802
Example 131
##STR00186##
[0563]
14-[(Cyclohexyl-methylcarbamoyl-methyl)-19-(7-methoxy-2-phenyl-quin-
olin-4-yloxy)-2,16-dioxo-3,15,17-triaza-tricyclo[15.3.0.0*4,6*]icos-7-ene--
4-carboxylic Acid (131)
[0564] To a solution of the ester 130 (19 mg, 0.024 mmol) in the
mixture of THF (0.2 ml) and methanol (0.3 ml) was added solution of
LiOH (6 mg, 0.237 mmol) in 0.15 ml of water. The resulting mixture
was stirred at 60.degree. C. for 3.5 h. After cooling to room
temperature, acetic acid was added (30 eq). The mixture was
co-evaporated with toluene. The residue was distributed between
chloroform and water phases, the water one was extracted with
chloroform and ethyl acetate, organic phases were combined, dried
over sodium sulphate, evaporated to give 15 mg of pure product.
[0565] MS (M+H.sup.+) 774
Example 132
##STR00187##
[0566]
[14-Cyclopropanesulfonylaminocarbonyl-17-(7-methoxy-2-phenyl-quinol-
in-4-yloxy)-2,14-dioxo-3,13,15-triaza-tricyclo[13.3.0.0*4,6*]octadec-7-en--
13-yl]-carbamic Acid ter.butyl ester (132)
[0567] To the acid 125 (19 mg, 0.028 mmol) in 0.5 ml of DMF was
added 5.5 mg (0.044 mmol) of DMAP and 10.7 mg (0.056 mmol) of EDC.
After 6.5 h of stirring 20 mg of cyclopropylsulphone amide and 0.04
ml of DBU were added. The mixture was stirred overnight, acidified
with 5% citric acid (in water) and extracted with ethyl acetate.
Dried, evaporated, purified by 5% to 10% methanol in chloroform (or
prep LC-MS) which gave 8 mg of the title compound (37%) MS
(M+H.sup.+) 783
Example 133
##STR00188##
[0568]
4-[2-(2-Isopropylamino-thiazol-4-yl)-7-methoxy-quinolin-4-yloxy]-py-
rrolidine-1,2-dicarboxylic Acid 1-tert.butyl ester (133)
[0569] To a stirred solution of N-Boc-trans-4-hydroxy-L-proline
(221 mg, 0.96 mmol) in DMSO was added potassium tert.butoxide (320
mg, 2,9 mmol). After 1 h
2-[2-isopropylamino)-1,3-thiazol-4-yl]-7-methoxyquinolin-4-ol (319
mg, 0.96 mmol) was added and the mixture was stirred at 70.degree.
C. for 72 hours. The mixture was diluted with water and extracted
with ethyl acetate. The product was used without further
purification. Yield 429 mg, 85%.
Example 134
##STR00189##
[0570]
2-(1-Ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl)-4-[2-(2-isopropyl-
amino-thiazol-4-yl)-7-methoxy-quinolin-4-yloxy]-pyrrolidine-1-carboxylic
Acid tert.butyl ester (134)
[0571] Compound 133 (300 mg, 0.56 mmol) was reacted with
1-amino-2-vinyl-cyclopropanecarboxylic acid ethyl ester (130 mg,
0.84 mmol) as described in Example 11 which gave the title compound
(302 mg, 80%).
Example 135
##STR00190##
[0572]
1-({4-[2-(2-Isopropylamino-thiazol-4-yl)-7-methoxy-quinolin-4-yloxy-
]-pyrrolidine-2-carbonyl}-amino)-2-vinyl-cyclopropanecarboxylic
Acid ethyl ester (135)
[0573] Compound 134 (302 mg, 0.45 mmol) was treated as described in
Example 12 which gave the title compound (195 mg, 76%).
Example 136
##STR00191##
[0574]
1-({1-[1-(2-Hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamo-
yl]-4-[2-(2-isopropylamino-thiazol-4-yl)-7-methoxy-quinolin-4-yloxy]-pyrro-
lidine-2-carbonyl}-amino-2-vinyl-cyclopropanecarboxylic Acid ethyl
ester (136)
[0575] Compound 135 (80 mg, 0.14 mmol) was treated as described in
Example 13 which gave the title product (87 mg, 72%).
Example 137
##STR00192##
[0576]
1-({1-[1-(2-Hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamo-
yl]-4-[2-(2-isopropylamino-thiazol-4-yl)-7-methoxy-quinolin-4-yloxy]-pyrro-
lidine-2-carbonyl}-amino-2-vinyl-cyclopropanecarboxylic Acid
(137)
[0577] The ethyl ester of compound 136 (80 mg, 0.09 mmol) was
hydrolyzed following the procedure described in Example 14 which
gave the title product Yield after preparative LC-MS (7.5 mg,
10%).
Example 138
##STR00193##
[0578]
1-{[1-Ethylcarbamoyl-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrol-
idine-2-carbonyl]amino}-2-vinyl-cyclopropanecarboxylic Acid ethyl
ester (138)
[0579] Reaction of compound 12 (330 mg, 0.66 mmol), phosgene (1.6
ml, 1.9 M in toluene, 3 mmol) and hex-5-enylamine hydrochloride
(500 mg, 3.68 mmol) following the procedure described in Example
120 gave the pure title product (328 mg, 80%), MS (M+H.sup.+)
627.
Example 139
##STR00194##
[0580]
17-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-2,14-dioxo-3,13,15-triaza--
tricyclo[13.3.0.0*4,6*]octadec-7-ene-4-carboxylic Acid ethyl ester
(139)
[0581] Compound 138 (200 mg, mol) was dissolved in degassed dry
dichloromethane (200 ml), bubbled with nitrogen. Then
Hoveyda-Grubbs (second generation) catalyst (5 mg, 2 mol %) was
added and the reaction mixture was refluxed for 20 h under
nitrogen. The resulting mixture was cooled down to room temperature
and concentrated by rotary evaporation. The resulting oil was
purified by column chromatography on YMC silica (ethyl
acetate-toluene 1:1 to 9:1) to give 55 mg of the title compound as
a beige solid. Yield 29%. MS (M+H.sup.+) 599.
Example 140
##STR00195##
[0582]
17-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-2,14-dioxo-3,13,15-triaza--
tricyclo[13.3.0.0*4,6*]octadec-7-ene-4-carboxylic Acid (140)
[0583] Compound 139 (55 mg, mol) was dissolved in 2 ml of methanol
and mixed with 3 eq. of aqueous NaOH and heated for 2 h at
60.degree. C. in a closed vial. The reaction mixture was then
extracted into ethyl acetate. The water solution was collected and
acidified with 1N HCl solution to pH 2. The resulting solution was
concentrated by rotary evaporation, dissolved in methanol and
purified by preparative HPLC (acetonitrile-water) to give 34 mg of
the title product. Yield 65%. MS (M+H.sup.+) 571.
Example 141
##STR00196##
[0584]
1-{[1-{1-[(Cyclohexyl-methoxycarbonyl-methyl)-carbamoyl]-2,2-dimeth-
yl-propylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2--
carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic Acid (141)
[0585] Compound 103 was dissolved in dichloromethane (3 ml) and
solid sodium bicarbonate (100 mg) and phosgene 20% in toluene (0.1
ml) was added. After 30 min at room temperature the mixture was
concentrated to dryness.
(S)-(2S-2-Amino-3,3-dimethyl-butyrylamino)-cyclohexyl-acetic acid
methyl ester (12 mg in dichloromethane 2 ml) was added. After 3
days of agitation at room temperature, the reaction mixture was
filtered, concentrated to dryness and purified on preparative
HPLC-MS which gave the title product (4.4 mg). M+H.sup.+ 784.7.
Example 142
##STR00197##
[0586]
1-{[1-(1-Aminomethyl-2,2-dimethyl-propylcarbamoyl-4-(7-methoxy-2-ph-
enyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropane-
carboxylic Acid ethyl ester (142)
[0587] The title compound was prepared from compound 12 (1.22 g,
2.43 mmol) by following the procedure described for the preparation
for compound 108 but using methanesulphonic acid
2-tert.butoxycarbonylamino-3,3-dimethyl-butyl ester instead of
methanesulphonic acid 2-tert.butoxycarbonylamino-4-methyl-pentyl
ester, in Example 165 step i). Reduction of the azide as described
in Example 109 gave the title compound (1.49 g, 95%). Purity
according to HPLC>95%, M+H.sup.+ 644.2.
Example 143
##STR00198##
[0588]
1-{[1-(2,2-Dimethyl-1-{[thiophene-3-carbonyl)-amino]-methyl}-propyl-
carbamoyl)-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-
-amino}-2-vinyl-cyclopropanecarboxylic Acid (143)
[0589] Compound 142 (100 mg, 0.155 mmol) was reacted according to
the general procedure IA for the preparation of compounds 110-116,
using thiophene-3-carbonyl chloride (28.5 mg, 0.194 mmol) as acyl
chloride which gave the title compound as a white solid (45 mg,
40%). Purity according to HPLC>95%, M+H.sup.+ 726.
Example 144
##STR00199##
[0590]
1-{[1-{1-[(5-Isoxazol-3-yl-thiophene-2-sulphonylamino)-methyl]-2,2--
dimethyl-propylcarbamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolid-
ine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic Acid
(144)
[0591] Compound 142 (25 mg, 0.039 mmol) was reacted according to
the general procedure 1A for the preparation of compounds 110-116,
using 5-isoxazole-3-yl-thiophene-2-sulphonyl chloride (14.5 mg,
0.058 mmol) as acyl chloride which gave the title compound as a
white solid (1.8 mg, 6%). Purity according to HPLC was >94%,
M+H.sup.+ 829.
Example 145
##STR00200##
[0592]
1-{[1-(3-Fluoro-benzoylamino)-methyl]-propylcarbamoyl}-4-(7-methoxy-
-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopr-
opanecarboxylic Acid (145)
[0593] Compound 142 (25 mg, 0.039 mmol) was reacted according to
the general procedure 1A for the preparation of compounds 110-116,
using 3-fluorobenzoyl chloride (12.3 mg, 0.078 mmol) as acyl
chloride which gave the title compound as a white solid (4.1 mg,
14%). Purity according to HPLC was >94%, M+H.sup.+ 738.
Example 146
##STR00201##
[0594]
1-{[1-(1{[(-Furan-3bcarbonyl)-amino]-methyl]-2,2-dimethyl-propylcar-
bamoyl}-4-(7-methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-2-carbonyl]-am-
ino}-2-vinyl-cyclopropanecarboxylic Acid (146)
[0595] Compound 142 (25 mg, 0.039 mmol) was reacted according to
the general procedure 1B for the preparation of compounds 110-116,
using 3-furanoic acid (5.5 mg, 0.049 mmol) as acyl chloride which
gave the title compound as a white solid (4.1 mg, 14%). Purity
according to HPLC was >99%, M+H.sup.+ 710.
Example 147
##STR00202##
[0596]
4-(7-Methoxy-2-phenyl-quinolin-4-yloxy)-pyrrolidine-1,2-dicarboxyli-
c acid
2-[(1-cyclopropanesulphonylaminocarbonyl-2-vinyl-cyclopropyl)-amide-
]1-[(2,2-dimethyl-1-{[(thiophene-3-carbonyl)amino]-methyl}-propyl)-amide
(147)
[0597] To solution of compound 143 (42.2 mg, 0.058 mmol) in
chloroform (3 ml) was added cyclopropylsulphonamide (14 mg, 0.116
mmol) followed by diisopropylethylamine (60.5 .mu.l, 0.17 mmol).
The solution was stirred at RT for 10 min and then at -20.degree.
C. for 30 min. PyBOP (121 mg, 0.116 mmol) was then added as a
solid. The solution was kept at -20.degree. C. for 10 days. The
solution was then poured into aqueous NaHCO.sub.3 (sat.) and washed
with water. The organic layer was dried, concentrated and subjected
to purification by HPLC, affording the title compound as a white
solid (2.3 mg, 0.0028 mmol), Purity by HPLC>95%, M+H.sup.+
830.
Example 148
##STR00203##
[0598]
Fmoc-4-amino-2-(1-ethoxycarbonyl-2-vinyl-cyclopropylcarbamoyl)-pyrr-
olidine-1-carbocyclic Acid tert.butyl ester (148)
[0599] (2S,4R) Fmoc-4-amino-1-Boc-pyrrolidine-2-carboxylic acid
(5.3 g, 11.8 mmol) was dissolved in DCM (100 ml), HATU (4.94 g,
12.99 mmol), DIEA (4.63 ml, 26.57 mmol) and vinylcyclopropylglycine
ethyl ester (2.26 g, 11.81 mmol) were added successively. The
mixture was stirred for 16 h at room temperature, and was then
diluted with DCM (50 ml), washed with citric acid (10% aq), water,
NaHCO.sub.3 (sat.aq) and water. The organic phase was dried over
Na.sub.2SO.sub.4 and concentrated to afford a beige solid foam
(8.11 g) which was subjected to silica gel column chromatography to
afford the title compound (7.14 g, 12.11 mmol).
Example 149
##STR00204##
[0600]
1-[(Fmoc-4-amino-pyrrolidine-2-carbonyl)-amino]-2-vinyl-cyclopropan-
ecarboxylic Acid ethyl ester (149)
[0601] Compound 148 (3.65 g, 6.04 mmol) was treated with a solution
of TFA/DCM (10 ml TFA, 50 ml DCM) for 2.5 h and then concentrated
to afford the titled compound (2.99 g, 6.12 mmol).
Example 150
##STR00205##
[0602]
1-({Fmoc-4-amino-1-[1-(2-hydroxy-indan-1-ylcarbamoyl-2,2-dimethyl-p-
ropylcarbamoyl]-pyrrolidine-2-carbonyl}-amino)-2-vinyl-cyclopropanecarboxy-
lic Acid ethyl ester (150)
[0603] The aminoproline derivative 149 (2.96 g, 6.04 mmol) was
stirred together with phosgene (1.93 M in toluene, 4 ml, 7.55 mmol)
for 10 min. The solvents and excess of phosgene were evaporated.
The residue was dissolved in DCM (30 ml) and t-Bug-aminoindanol
(1.9 g, 7.24 mmol) was added as a solution in DCM (30 ml), followed
by NaHCO.sub.3 (2 g). The mixture was stirred for 48 h, then
diluted with DCM, washed with water, 10% citric acid and
NaHCO.sub.3 (sat, aq), dried over Na.sub.2SO.sub.4, and evaporated
to dryness. The residue was subjected to column chromatography
purification, EtOAc-hexane 0-30% to afford the title compound (1 g,
1.3 mmol).
Example 151
##STR00206##
[0604]
1-({4-Amino-1-[1-(2-hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propy-
lcarbamoyl]-pyrrolidine-2-carbonyl}-amino)-2-vinyl-cyclopropanecarboxylic
Acid ethyl ester (151)
[0605] Compound 150 (595 mg, 0.765 mmol) was dissolve din DMF (20
ml) and treated with Si-piperazine (0.08 mmol/g, 4.78 g, 3.82 mmol)
for 48 h. The silica was filtered and washed once with DMF and then
with several portions of DCM. The solvents were evaporated and the
residue subjected to column chromatography to afford the title
compound (170 mg, 0.3 mmol).
Example 152
##STR00207##
[0606]
1-({1-[1-(2-Hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamo-
yl]-4-[(pyridine-3-carbonyl)-amino]-pyrrolidine-2-carbonyl}-amino)-2-vinyl-
-cyclopropanecarboxylic Acid (152)
[0607] To a stirred solution of compound 151 (35 mg, 0.064 mmol) in
DCM (1 ml), was added DIEA (0.12 mmol, 19 .mu.l) and nicotinoyl
chloride hydrochloride (0.12 mmol, 17 mg).The solution was stirred
at RT for 18 h, PS-trisamine was added then stirred at RT for 4 h.
After filtration, the solution was washed with citric acid (10% aq)
and NaHCO.sub.3 (sat, aq), the organic phase was dried over
Na.sub.2SO.sub.4 and concentrated. The residue was dissolved in
THF:MeOH (2:1, 1.5 ml). LiOH (1N aq, 3.2 mmol, 320 .mu.l) was
added. The solution was stirred at 60.degree. C. for 24 h. Acetic
acid was added and then concentrated. The residue was dissolved in
MeOH and subjected to purification by HPLC, affording the title
compound (19.5 mg, 0.03 mmol). Purity by HPLC>98%, M+H.sup.+
633.1.
Example 153
##STR00208##
[0608]
1-({1-[1-(2-Hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamo-
yl]-4-phenylacetamino-pyrrolidine-2-carbonyl}-amino)-2-vinyl-cyclopropanec-
arboxylic Acid (153)
[0609] The procedure described in Example 152 but using phenyl
acetyl chloride instead of nicotinoyl chloride hydrochloride, was
followed which gave the title compound (12.7 mg, 0.019 mmol).
Purity by HPLC>90%, M+H.sup.+ 646.1.
Example 154
##STR00209##
[0610]
1-({1-[1-(2-Hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamo-
yl]-4-[(5-methyl-3-phenyl-isoxazole-4-carbonyl)-amino]-pyrrolidine-2-carbo-
nyl}-amino)-2-vinyl-cyclopropanecarboxylic Acid (154)
[0611] The procedure described in Example 152 but using
5-methyl-3-phenyl-isoxazole-4-carbonyl chloride instead of
nicotinoyl chloride hydrochloride, was followed which gave the
title compound (3.6 mg, 00055 mmol). Purity by HPLC>98%,
M+H.sup.+ 713.1.
Example 155
##STR00210##
[0612]
1-{[1-[1-(2-Hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamo-
yl]-4-(3-phenyl-ureido)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropan-
ecarboxylic Acid (155)
[0613] To a stirred solution of compound 151 (30 mg, 0.054 mmol) in
acetonitrile:dichloromethane (2:1, 3 ml), triethylamine (0.0648
mmol, 9 .mu.l) and phenylisocyanate (0.0648 mmol, 7 .mu.l) was
added. The solution was stirred at room temperature for 3 h,
methanol was added (1 ml) and then it was concentrated. The residue
was dissolved in methanol and subjected to purification by HPLC,
affording the ester compound as a white solid (32.7 mg, 0.047
mmol), Purity by HPLC>95%, M+H.sup.+ 675.31. LiOH 1N aq. (0.47
mmol, 475 .mu.l) was added to the ester dissolved in THF:MeOH
(2:1). The reaction was stirred at 50.degree. C. for 15 min and
then at 8.degree. C. for 12 h followed by addition of acetic acid
(0.98 mmol, 53 .mu.l) before concentration. The residue was
dissolved in MeOH and subjected to purification by HPLC, affording
the title compound as a white solid (3.8 mg, 0.006 mmol), Purity by
HPLC>98%, M+H.sup.+ 675.31.
Example 156
##STR00211##
[0614]
1-({4-Benzenesulphonylamino-1-[1-(2-hydroxy-indan-1-ylcarbamoyl)-2,-
2-dimethyl-propylcarbamoyl]-pyrrolidine-2-carbonyl)-amino)-2-vinyl-cyclopr-
opanecarboxylic Acid (156)
[0615] To a stirred solution of compound 151 (30 mg, 0.054 mmol) in
DCM (2 ml), DIEA (0.0648 mmol, 11.5 .mu.l) and
phenysulfonylchloride (0.0648 mmol, 11.5 .mu.l) were successively
added The solution was stirred at RT for 3 h, and then it was
concentrated. The residue was dissolved in MeOH and subjected to
purification by HPLC, affording the ester compound as a white solid
(17.9 mg, 0.0257 mmol), Purity by HPLC>95%, M+H.sup.+ 696.24.
LiOH 1N aq, (0.25 mmol, 257 .mu.l) was added to the ester dissolved
in THF:MeOH (2:1). The reaction was stirred at 50.degree. C. for
1.5 h prior to the addition of acetic acid (0.98 mmol, 53 .mu.l).
The solution was concentrated. The residue was dissolved in DCM and
washed with water; the aqueous phase was acidified to pH 5 and then
extracted with dichloromethane and ethyl acetate. The combined
organic phases were dried over Na.sub.2SO.sub.4 and concentrated,
affording the title compound as a white solid (7.1 mg, 0.01 mmol),
Purity by HPLC>98%, M+H.sup.+ 668.19.
Example 157
##STR00212##
[0616]
1-{[1-[1-(2-Hydroxy-indan-1-ylcarbamoyl)-2,2-dimethyl-propylcarbamo-
yl]-4-(3-phenyl-thioureido)-pyrrolidine-2-carbonyl}-amino)-2-vinyl-cyclopr-
opanecarboxylic Acid (157)
[0617] To a stirred solution of compound 151 (30 mg, 0.054 mmol) in
acetonitril (3 ml), TEA (0.0648 mmol, 9 .mu.l) and
phenylthioisocyanate (0.0648 mmol, 7.8 .mu.l) were successively
added. The solution was stirred at RT for 16 h, and then it was
concentrated. The residue was dissolved in MeOH and subjected to
purification by HPLC, affording the ester compound as a white solid
(22.7 mg, 0.0328 mmol), Purity by HPLC>95%, M+H.sup.+ 691.2.
LiOH 1N aq, (0.33 mmol, 328 .mu.l) was added to the ester dissolved
in THF:MeOH (2:1). The reaction was stirred at 50.degree. C. for
2.5 h prior to the addition of acetic acid (0.98 mmol, 53 .mu.l).
The solution was concentration. The residue was dissolved in
dichloromethane and washed with water, the aqueous phase was
extracted with EtOAc. The combined organic phases were dried over
Na.sub.2SO.sub.4 and concentrated, affording the title compound as
a white solid (7.2 mg, 0.01 mmol), Purity by HPLC>98%, M+H.sup.+
663.26.
[0618] Assays
[0619] The compounds of the invention are conveniently assayed for
activity against the NS3 protease of flavivirus such as HCV using
conventional in vitro (enzyme) assays or cell culture assays.
[0620] A useful assay is the Bartenshlager replicon assay disclosed
in EP 1043399. An alternative replicon assay is described in WO
03064416.
[0621] A convenient enzyme assay involving the inhibition of
full-length hepatitis C NS3 is essentially as described in
Poliakov, 2002 Prot Expression & Purification 25 363 371.
Briefly, the hydrolysis of a depsipeptide substrate,
Ac-DED(Edans)EEAbu.psi.[COO]ASK(Dabcyl)-NH.sub.2 (AnaSpec, San
Jose, USA), is measured spectrofluorometrically in the presence of
a peptide cofactor, KKGSVVIVGRIVLSGK, as described by Landro, 1997
Biochem 36 9340-9348. The enzyme (1 nM) is incubated in a buffer
such as 50 mM HEPES, pH 7.5, 10 mM DTT, 40% glycerol, 0.1%
n-octyl-.beta.-D-glucoside, with 25 .mu.M cofactor and inhibitor at
say 30.degree. C. for 10 min, whereupon the reaction is initiated
by addition of substrate, typically 0.5 .mu.M substrate. Inhibitors
are typically dissolved in DMSO, sonicated for 30 s and vortexed.
The solutions are generally stored at -20.degree. C. between
measurements.
[0622] An alternative enzyme assay is described in WO 0399316 and
employs an HCV NS3/4A protease complex FRET peptide assay. The
purpose of this in vitro assay is to measure the inhibition of HCV
NS3 protease complexes, derived from the BMS, H77C or J416S
strains, 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.
[0623] Serum is taken from an HCV-infected patient. An engineered
full-length cDNA template of the HCV genome (BMS strain) was
constructed from DNA fragments obtained by reverse
transcription-PCR (RT-PCR) of serum RNA and using primers selected
on the basis of homology between other genotype Ia strains. From
the determination of the entire genome sequence, a genotype I a 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 Ia (H77C) and 87% identical to genotype lb (J4L6S).
The infectious clones, H77C (I a genotype) and J4L6S (I b genotype)
can be obtained from R. Purcell (NIH) and the sequences are
published in Genbank (AAB67036, see Yanagi, M., Purcell, R. H.,
Emerson, S. U. and Bukh. 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 Bukhj, Virology 244 (1),161
(1998)).
[0624] The BMS, H77C and J4L6S strains are conventional for 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):562032,
(1999)). Briefly, a three-lysine solubilizing tail was added at the
3'-end of the 3 0 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 can be
introduced by PCR at amino acid position 1454 to prevent the
autolytic cleavage in the NS3 helicase domain. The variant DNA
fragment can be cloned in the pET21b bacterial expression vector
(Novagen) and the NS3/4A complex can be 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 can be induced with 0.5 mM Isopropyl beta-D
thiogalactopyranoside (IPTG) for 22 hr at 20.degree. C. A typical
fermentation (I10 l) yields approximately 80 g of wet cell paste.
The cells are resuspended in lysis buffer (IO mL/g) consisting of
25 mM N-(2Hydroxyethyl)Piperazine-N'-(2-Ethane Sulfonic acid)
(HEPES), pH7.5, 20% glycerol, 5OO mM Sodium Chloride (NaCl), 0.5%
Triton-X100, I ug/mL lysozyme, 5 mM Magnesium Chloride (MgCl2), I
ug/mL Dnasel, 5 mM beta-Mercaptoethanol (BME), Protease
inhibitor--Ethylenediamine Tetraacetic acid (EDTA) free (Roche),
homogenized and incubated for 20 mins at VC. The homogenate is
sonicated and clarified by ultra-centrifugation at 235000 g for 1
hr at 4.degree. C.
[0625] Imidazole is added to the supernatant to a final
concentration of 15 mM and the pH adjusted to 8. The crude protein
extract is loaded on a Nickel Nitrilotriacetic acid (Ni-NTA) column
pre-equilibrated with buffer B (25 n-tM 2 0 HEPES, pH8 20%
glycerol, 5OO mM NaCl, 0.5% Triton-XIOO, 15 mM imidazole, 5 mM
BME). The sample is loaded at a flow rate of I mL/min. The column
is washed with 15 column volumes of buffer C (same as buffer B
except with 0.2% Triton-X100). The protein is eluted with 5 column
volumes of buffer D (same as buffer C except with 200 mM
imidazole).
[0626] NS3/4A protease complex-containing fractions are pooled and
loaded on a desalting column Superdex-S200 pre-equilibrated with
buffer D (25 MM HEPES, pH7.5, 20% glycerol, 3OO mM NaCl, 0.2%
Triton-XIOO, IO mM BME). Sample is loaded at a flow rate of ImUmin.
NS3/4A protease complex3 0 containing fractions are 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,
are typically judged to be greater than 90% by SDS-PAGE and mass
spectrometry analyses.
[0627] The enzyme is generally 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, is conveniently RET S 1 (Resonance
Energy Transfer Depsipeptide Substrate; AnaSpec, Inc. cat
#22991)(FRET peptide), described by Taliani et al. in Anal.
Biochem. 240(2):6067 (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 is 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. Useful reagents are as follow: HEPES and Glycerol (Ultrapure)
can be obtained from GIBCO-BRL. Dimethyl Sulfoxide (DMSO) is
obtained from Sigma. Beta-Mercaptoethanol is obtained from Bio
Rad.
[0628] Assay buffer: 50 m.M HEPES, pH7.5; 0.15M NaCl; 0. I %
Triton; 15% Glycerol; 10 mM BME. Substrate: 2 uM final
concentration (from a 2 mM stock 2 0 solution in DMSO stored at
-20.degree. C.). HCV NS3/4A type Ia (Ib), 2-3 nM final
concentration (from a 5 uM stock solution in 25 mM HEPES, pH7.5,
20% glycerol, 300 m.M NaCl, 0.2% Triton-X100, 10 mM BME). For
compounds with potencies approaching the assay limit, the assay can
be made more sensitive by adding 50 ug/mL BSA to the assay buffer
and/or reducing the end protease concentration to 300 pM.
[0629] The assay is conveniently performed in a 96-well polystyrene
black plate from Falcon. Each well contains 25 ul NS3/4A protease
complex in assay buffer, 50 ul of a compound of the present
invention in 10% DMSO/assay buffer and 25 ul substrate in assay
buffer. A control (no compound) is also prepared on the same assay
plate. The enzyme complex is mixed with compound or control
solution, typically for 1 min before initiating the enzymatic
reaction by the addition of substrate. The assay plate is generally
read immediately using a spectrophotometer such as a Cytofluor
Series 4000 (Perspective Biosystems). The instrument is
conveniently set to read an emission of 340 nm and excitation of
490 nm at 25.degree. C. Reactions are generally followed for
approximately 15 minutes.
[0630] The percent inhibition can be calculated with the following
equation.
100.-[(dF.sub.inh/dF.sub.con)XI00]
[0631] where dF is the change in fluorescence over the linear range
of the curve. A nonlinear curve fit is applied to the
inhibition-concentration data, and the 50% effective concentration
(IC.sub.50) is calculated by the use software such as Excel XI-fit
software using the equation:
y=A+((B-A)/(1+((C/x) D))).
[0632] Enzyme assays conveniently utilize a fluorescence resonance
energy transfer (FRET) principle to generate a spectroscopic
response to an HCV NS3 serine protease catalyzed NS4A/4B cleavage
event. The activity is typically measured in a continuous
fluorometric assay using an excitation wavelength of 355 nm and
emission wavelength of 500 nm. The initial velocity may be
determined from 10 minutes continuous reading of increased
fluorescence intensities as a result of the NS3 protease catalyzed
cleavage event.
[0633] An alternative enzyme assay can be carried out as
follows:
[0634] Materials
[0635] Recombinant HCV NS3 full length enzyme can be prepared as
shown in Poliakov et al Protein Expression & purification 25
(2002) 363-371.
[0636] The NS4A cofactor conveniently has an amino acid sequence of
KKGSVVIVGRIVLSGK (commercially available), generally prepared as a
10 mM stock solution in DMSO.
[0637] The FRET-substrate
(Ac-Asp-Glu-Asp(EDANS)-Glu-Glu-Abu-.psi.-[COO)Ala-Ser-Lys(DABCYL)-NH2,
MW1548.60 can be purchased from AnaSpec RET S1, CA. USA) and is
typically prepared as a 1.61 mM stock solution in DMSO. Aliquots
(50 .mu.l/tube) should be wrapped with aluminum foil to protect
from direct light and stored in -20.degree. C.
[0638] Reference compound-1, N-1725 with a sequence of
AcAsp-D-Gla-Leu-Ile-Cha-Cys, MW 830.95 may be purchased from
BACHEM, Switzerland and is generally prepare as a 2 mM stock
solution in DMSO and stored in aliquots in -20.degree. C.
[0639] 1M HEPES buffer may be purchased from Invitrogen
Corporation, storage at 20.degree. C. Glycerol may be purchased
from Sigma, 99% purity.
[0640] CHAPS,
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate: may be
purchased from Research Organics, Cleveland, Ohio 44125, USA.
MW614.90 DTT, DL-Dithiothreitol (Cleland Reagent: DL-DTT) 99%
purity, MW.154.2 Storage: +4.degree. C.
[0641] DMSO may be purchased from SDS, 13124 Peypin, France. 99.5%
purity.
[0642] TRIS, ultra pure (TRIS-(hydroxymethylaminomethane), may be
purchased from ICN Biomedicals Inc.
[0643] N-dodecyl-.beta.-D-maltoside, minimum 98%, may be purchased
from Sigma, storage -20.degree. C.
[0644] Equipment
[0645] Microtiter plates (white cliniplate, ThermoLab Systems cat
no. 9502890)
[0646] Eppendorf pipettes.
[0647] Biohit pipette, multi dosing.
[0648] Ascent fluorimeter, filterpair ex 355nm, em500 nm.
[0649] Method
[0650] Experimental Procedure:
[0651] 10 mM stock solutions of the compounds are made in DMSO. The
stock solutions are stored in room temperature while testing and
placed in -20.degree. C. at long-time storage.
[0652] Assay Buffer A: [0653] 50 mM HEPES buffer, pH=7.5, 40%
Glycerol, 0.1% CHAPS [0654] Storage: room temperature [0655] 10 mM
DTT (stored in aliquots at -20.degree. C. and added fresh at each
experiment)
[0656] Assay Buffer B: [0657] 25 mM TRIS pH7.5, 0.15 M NaCl, 10%
glycerol, 0.05% n-dodecyl-.beta.-D-maltoside [0658] 5 mM DTT
(stored in aliquots at -20.degree. C. and added fresh at each
experiment)
[0659] Experiment Sequence:
[0660] Preparation of Reaction Buffer (for One Plate, 100
Reactions)(Buffer A) [0661] 1. Prepare 9500 .mu.l assay buffer
(HEPES, pH=7.5, 40% glycerol and 0.1% CHAPS in de ionized water.
Add DTT giving a final concentration of 10 mM (freshly prepared for
every run). [0662] 2. Thaw rapidly the NS3 protease [0663] 3. Add
13.6 .mu.l NS3 protease and 13.6 .mu.l NS4A peptide and mix
properly. Leave the mixture for 15 minutes in room temperature.
[0664] 4. Place the enzyme stock solution back into liquid nitrogen
or -80.degree. C. as soon as possible.
[0665] Preparation of Reaction Buffer (for One Plate, 100
Reactions)(Buffer B) [0666] 5. Prepare 9500 .mu.l assay buffer
(TRIS, pH=7.5, 0.15 M NaCl, 0.5 mM EDTA, 10% glycerol and 0.05%
n-dodecyl .beta.-D-maltoside in de ionized water. Add DTT giving a
final concentration of 5 mM (freshly prepared for every run).
[0667] 6. Thaw the NS3 protease rapidly. [0668] 7. Add 27.2 .mu.l
NS3 protease and 13.6 .mu.l NS4A peptide and mix properly. Leave
the mixture for 15 minutes in room temperature. [0669] 8. Place the
enzyme stock solution back into liquid nitrogen or -80.degree. C.
as soon as possible.
[0670] Preparation of Inhibitor/Reference Compound
[0671] Make a dilution series of the inhibitors in DMSO to
100.times. the final concentrations 10, 1, 0.1, 0.01 and 0.001
.mu.M. The final DMSO concentration in 100 .mu.l total reaction
volume is 1%.
[0672] Make a dilution series of the reference compound, N-1725 in
DMSO to 100.times. the final concentrations 120, 60, 30,15, 7.5 and
3.75 nM.
[0673] Eight enzyme control wells are needed for every run.
[0674] Blank wells contain 95 .mu.L buffer (without NS3 PR), 1
.mu.L DMSO and 5 .mu.L substrate.
[0675] Preparation of FRET Substrate
[0676] Dilute the substrate stock solution (1.61 mM) with assay
buffer to 40 .mu.M working solution. Avoid exposure to light.
[0677] Assay Sequence
[0678] Use 96-well cliniplate, the total assay volume per well is
100 .mu.l. [0679] 1. Add 95 .mu.L of assay buffer to each well
[0680] 2. Add 1 .mu.l inhibitor/reference compound [0681] 3. Pre
incubate for 30 minutes at room temperature [0682] 4. Start the
reaction by adding 5 .mu.L 40 .mu.M substrate solution (final
concentration 2 .mu.M) [0683] 5. Read continuously for 20 minutes
at ex=355 nm and em=500 nm, monitoring the increased fluorescence
per minute. [0684] 6. Plot the progression curve (within linear
range, 8-10 time points) and determine the slope as an initial
velocity with respect to each individual inhibitor concentration.
[0685] 7. Calculate % inhibition with respect to enzyme
control.
[0686] Treatment of Results
[0687] The result is expressed as % inhibition at a certain
concentration (screen) or as a Ki value in nM or .mu.M.
[0688] Calculation of % inhibition.
[0689] The initial velocity is determined from 10 minutes
continuous reading of increased fluorescence intensities as a
result of the NS3 protease catalyzed cleavage event.
[0690] The change in slope for the inhibitor compared to the enzyme
control gives the % inhibition at a certain concentration.
[0691] Calculation of Ki.
[0692] All inhibitors are treated as if they follow the rules of
competitive inhibition.
[0693] The IC.sub.50 value is calculated from the inhibition values
of a series of inhibitor concentrations. The calculated value is
used in the following equation:
K.sub.i=IC.sub.50/(1+S/Km)
[0694] Plotting of the graph is done by help of two calculation
programs: Grafit and Graphpad
[0695] Various compounds of the invention exemplified above
displayed IC.sub.50s in the range 1 nM to 6.9 micromolar and
ED.sub.50s in the sub-micromolar to micromolar range.
[0696] Drug Escape Resistance Pattern and Rate
[0697] Replicon cultures in microtitre plates can be used to
determine resistance development rates and to select out drug
escape mutants. The compounds being tested are added at
concentrations around their ED.sub.50 using, say, 8 duplicates per
concentration. After the appropriate replicon incubation period the
protease activity in the supernatant or lysed cells is
measured.
[0698] The following procedure is followed at subsequent passages
of the cultures. Virus produced at the concentration of test
compound showing >50% of the protease activity of untreated
infected cells (SIC, Starting Inhibitory Concentration) are
passaged to fresh replicon cultures. An aliquot, say, 15 .mu.l
supernatant from each of the eight duplicates are transferred to
replicon cells without the test compound (control) and to cells
with test compound at the same concentration, and additionally two
respectively fivefold higher concentrations. (See the table below
below)
[0699] When the viral component of replicon propagation (for
example as measured by HCV protease activity) is permitted at the
highest non-toxic concentration (5-40 .mu.M), 2-4 parallel wells
are collected and expanded to give material for sequence analysis
and cross-wise resistance.
[0700] Key:
TABLE-US-00001 Viral growth permitted Virus production inhibited
125 x SIC 125 x SIC 25 x SIC .fwdarw. 25 x SIC 5 x SIC 25 x SIC 5 x
SIC .fwdarw. No compound 25 x SIC 5 x SIC .fwdarw. No compound 5 x
SIC SIC SIC .fwdarw. No compound SIC .fwdarw. No compound Pass 1
Pass 2 Pass 3 Pass 4 Pass 5
[0701] Alternative methods for assessing activity on drug escape
mutants include the preparation of mutant enzyme bearing the
distinctive mutation for use in standard Ki determinations as shown
above. For example WO 04/039970 describes constructions allowing
access to HCV proteases bearing the 155, 156 and/or 168 drug escape
mutants arising from the selective pressure of BILN-2061 and
VX-950. Susch constructs can then be engineered into replicon
vectors in place of the wild type protease, thereby allowing ready
assessment in a cellular assay, of whether a given compound is
active against a give drug escape mutant.
[0702] P450 Metabolism
[0703] The metabolism of compounds of the invention through the
main isoforms of the human cytochrome system P450 are conveniently
determined in baculovirus infected insect cells transfected with
human cytochrome P450 cDNA (supersomes) Gentest Corp. Woburn
USA.
[0704] The test compounds at concentrations 0.5, 5 and 50 .mu.M are
incubated in duplicate in the presence of supersomes overexpressing
various cytochrome P450 isoforms, including CYP1A2+P450 reductase,
CYP2A6+P450 reductase, CYP2C9-Arg 144+P450 reductase, CYP2C19+P450
reductase, CYP2D6-Val 374+P450 reductase and CYP3A4+P 450
reductase. Incubates contain a fixed concentration of cytochrome
P450 (eg 50 pmoles) and are conducted over 1 hour. The involvement
of a given isoform in the metabolism of the test compound is
determined by UV HPLC chromatographically measuring the
disappearance of parent compound.
* * * * *