U.S. patent application number 10/614432 was filed with the patent office on 2004-04-22 for inhibitors of serine proteases, particularly hepatitis c virus ns3 protease.
Invention is credited to Bhisetti, Govinda Rao, Farmer, Luc J., Tung, Roger D..
Application Number | 20040077600 10/614432 |
Document ID | / |
Family ID | 22155006 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040077600 |
Kind Code |
A1 |
Tung, Roger D. ; et
al. |
April 22, 2004 |
Inhibitors of serine proteases, particularly hepatitis C virus NS3
protease
Abstract
The present invention relates to compounds, methods and
pharmaceutical compositions for inhibiting proteases, particularly
serine proteases, and more particularly HCV NS3 proteases. The
compounds, and the compositions and methods that utilize them, can
be used, either alone or in combination to inhibit viruses,
particularly HCV virus.
Inventors: |
Tung, Roger D.; (San Diego,
CA) ; Bhisetti, Govinda Rao; (Lexington, MA) ;
Farmer, Luc J.; (Foxboro, MA) |
Correspondence
Address: |
VERTEX PHARMACEUTICALS INC.
130 WAVERLY STREET
CAMBRIDGE
MA
02139-4242
US
|
Family ID: |
22155006 |
Appl. No.: |
10/614432 |
Filed: |
July 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10614432 |
Jul 7, 2003 |
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09677382 |
Sep 29, 2000 |
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6608067 |
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09677382 |
Sep 29, 2000 |
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PCT/US99/07149 |
Mar 31, 1999 |
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60080060 |
Mar 31, 1998 |
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Current U.S.
Class: |
514/64 ; 514/114;
514/20.3; 514/21.91; 514/4.3; 514/553; 514/561; 514/563; 514/616;
562/11; 562/30; 562/507; 562/7; 564/152 |
Current CPC
Class: |
A61P 31/14 20180101;
A61P 43/00 20180101; C07D 241/24 20130101; A61P 31/18 20180101;
A61P 31/12 20180101; C07C 237/42 20130101; C07D 213/82 20130101;
A61P 1/16 20180101 |
Class at
Publication: |
514/064 ;
514/114; 514/561; 514/563; 514/553; 514/616; 562/007; 562/011;
562/030; 562/507; 564/152; 514/019; 514/007 |
International
Class: |
A61K 038/04; C07F
005/02; C07F 009/22; C07K 005/04 |
Claims
What is claimed is:
1. A compound of the formula (I): 40wherein: W is: 41wherein: m is
0 or 1; each R.sup.1 is hydroxy, alkoxy, or aryloxy, or each
R.sup.1 is an oxygen atom and together with the boron, to which
they are each bound, form a 5-7 membered ring, wherein the ring
atoms are carbon, nitrogen, or oxygen; each R.sup.2 is
independently hydrogen, alkyl, alkenyl, aryl, aralkyl, aralkenyl,
cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl,
heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heteroaryl,
or heteroaralkyl, or two R.sup.2 groups, which are bound to the
same nitrogen atom, form together with that nitrogen atom, a 5-7
membered monocyclic heterocyclic ring system; wherein any R.sup.2
carbon atom is optionally substituted with J; J is alkyl, aryl,
aralkyl, alkoxy, aryloxy, aralkoxy, cycloalkyl, cycloalkoxy,
heterocyclyl, heterocyclyloxy, heterocyclylalkyl, keto, hydroxy,
amino, alkylamino, alkanoylamino, aroylamino, aralkanoylamino,
carboxy, carboxyalkyl, carboxamidoalkyl, halo, cyano, nitro,
formyl, acyl, sulfonyl, or sulfonamido and is optionally
substituted with 1-3 J.sup.1 groups; and J.sup.1 is alkyl, aryl,
aralkyl, alkoxy, aryloxy, heterocyclyl, heterocyclyloxy, keto,
hydroxy, amino, alkanoylamino, aroylamino, carboxy, carboxyalkyl,
carboxamidoalkyl, halo, cyano, nitro, formyl, sulfonyl, or
sulfonamido; L is alkyl, alkenyl, or alkynyl, wherein any hydrogen
is optionally substituted with halogen, and wherein any hydrogen or
halogen atom bound to any terminal carbon atom is optionally
substituted with sulfhydryl or hydroxy; each M is independently
alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,
heteroaryl, or heteroaralkyl, and is optionally substituted by 1 to
3 J groups, wherein any alkyl carbon atom may be replaced by a
heteroatom; R.sup.18 is a bond, --N(R.sup.11)--, or --C(O)--;
R.sup.11 is hydrogen or C.sub.1-C.sub.3 alkyl; each R.sup.19 is
independently H or R.sup.21-aryl, or 2 adjacent R.sup.19 may be
bound to one another to form a 5-7 membered aromatic ring; wherein
any R.sup.19 is optionally substituted with 1 to 4 independently
selected J' groups; each R.sup.21 is independently
C.sub.1-C.sub.3-straight or branched alkyl,
C.sub.2-C.sub.3-straight or branched alkenyl,
O--(C.sub.1-C.sub.3)-straight or branched alkyl, or
O--(C.sub.2-C.sub.3)-straight or branched alkenyl; n is 0 or 1; the
ring to which R.sup.18 and R.sup.19 are attached may be saturated,
partially saturated, aromatic or fully unsaturated; and 1 to 3
carbon atoms that make up the ring to which R.sup.18 and R.sup.19
are attached are optionally replaced with a heteroatom which is
independently selected from O, S, S(O), S(O).sub.2 or N(R.sup.11);
A.sup.2 is a bond or --N(R.sup.11)--R.sup.17(M)-R.sup.22--, wherein
R.sup.17 is --CH-- or --N--; and R.sup.22 is --C(O)-- or
--S(O).sub.2--; V is a bond, --CH(R.sup.11)--, --O--, --S--, or
--N(R.sup.11)--; K is a bond, --O--, --S--, --C(O)--, --S(O)--,
--S(O).sub.2--, or --S(O)NR.sup.11--; T is --R.sup.12,
-alkyl-R.sup.12, -alkenyl-R.sup.12, -alkynyl-R.sup.12, --OR.sup.12,
--N(R.sup.12).sub.2, --C(O)R.sup.12, --C (.dbd.NO-alkyl).sup.12, or
42wherein: each R.sup.12 is independently selected from hydrogen,
aryl, heteroaryl, cycloalkyl, heterocyclyl, cycloalkylidenyl, or
heterocycloalkylidenyl, and is optionally substituted with 1 to 3 J
groups; or a first R.sup.12 and a second R.sup.12, together with
the nitrogen to which they are bound, form a mono- or bicyclic ring
system optionally substituted by 1 to 3 J groups; R.sup.10 is
alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,
heteroaryl, heteroaralkyl, carboxyalkyl, or carboxamidoalkyl, and
is optionally substituted with 1 to 3 hydrogens J groups; R.sup.15
is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,
heterocyclylalkyl, heteroaryl, heteroaralkyl, carboxyalkyl, or
carboxamidoalkyl, and is optionally substituted with 1 to 3 J
groups; and R.sup.16 is hydrogen, alkyl, aryl, heteroaryl,
cycloalkyl, or heterocyclyl.
2. The compound according to claim 1, wherein W is selected from:
43
3. The compound according to claim 2, wherein W is --C(O)H.
4. The compound according to claim 1, wherein J is selected from
alkyl, alkoxy, aryloxy, aryl, aralkyl, aralkoxy, halo, heteroaryl,
cyano, amino, nitro, heterocyclyl, acyl, carboxy, carboxyalkyl,
alkylamino, hydroxy, heterocyclylalkyl, aralkanoylamino,
aroylamino, alkanoylamino, formyl or keto.
5. The compound according to claim 4, wherein J is selected from
t-butyl, methyl, trifluoromethyl, hydroxy, methoxy, ethoxy,
trifluoromethoxy, carboxy, phenyl, benzyl, phenoxy, benzyloxy,
fluoro, chloro, bromo, isoxazolyl, pyridinyl, piperidinyl,
carboxymethyl, carboxyethyl, dialkylamino, morpholinylmethyl,
phenylacetylamino, or acylamino.
6. The compound according to claim 1, wherein each J.sup.1 is
independently selected from alkoxy, alkyl, halo or aryl.
7. The compound according to claim 6, wherein each J.sup.1 is
independently selected from C.sub.1-3 alkoxy, chloro, C.sub.1-3
alkyl, or phenyl.
8. The compound according to claim 1, wherein L is selected from
trihalomethyl, sulfhydryl or alkyl substituted with trihalomethyl,
sulfhydryl, or hydroxy.
9. The compound according to claim 8, wherein L is
--CH.sub.2CH.sub.3 or --CH.sub.2CF.sub.3.
10. The compound according to claim 1, wherein R.sup.2 is selected
from H, fluorine, trifluoromethyl, alkyl, aryl, aralkyl,
heteroaralkyl, heterocyclyl, or heterocyclylalkyl.
11. The compound according to claim 10, wherein R.sup.2 is H.
12. The compound according to claim 1, wherein each M is
independently selected from isopropyl, propyl, methyl,
pyridylmethyl, benzyl, naphthylmethyl, phenyl, imidazolylmethyl,
thiophenylmethyl, cyclohexylmethyl, phenethyl, benzylthiomethyl, or
benzyloxyethyl.
13. The compound according to claim 12, wherein each M is
isopropyl.
14. The compound according to claim 1, wherein one R.sup.19 is
R.sup.21-aryl and the other two R.sup.19 are H, or two R.sup.19 are
bound together to form an aromatic ring and the other R.sup.19 is
H.
15. The compound according to claim 14, wherein one R.sup.19 is
--O--(C.sub.1-C.sub.3)-alkyl-aryl.
16. The compound according to claim 15, wherein 14, wherein one
R.sup.19 is --O-benzyl.
17. The compound according to claim 14, wherein the two R.sup.19
that are bound together form a 6-membered aromatic ring.
18. The compound according to claim 17, wherein the two R.sup.19
that are bound together form phenyl.
19. The compound according to claim 1, wherein R.sup.18 is
--N(R.sup.11)--.
20. The compound according to claim 19, wherein R.sup.18 is
--N(H)-- or --N(CH.sub.3)--.
21. The compound according to claim 1, wherein A.sup.2 is a bond or
--N(R.sup.11)--C(M)-C(O)--.
22. The compound according to claim 21, wherein A.sup.2 is a bond
or --N(H)--C(M)-C(O)--, wherein M is isopropyl.
23. The compound according to claim 1, wherein V is
--N(R.sup.11)--.
24. The compound according to claim 23, wherein V is --NH--.
25. The compound according to claim 1, wherein K is --C(O)-- or
--S(O).sub.2--.
26. The compound according to claim 25, wherein K is --C(O)--.
27. The compound according to claim 1, wherein T is selected from
--R.sup.12, -alkyl-R.sup.12, -alkenyl-R.sup.12, --OR.sup.12,
--N(R.sup.12).sub.2, --C(.dbd.NO-alkyl)-R.sup.12 or 44
28. The compound according to claim 27, wherein T is --R.sup.12 or
-alkyl-R.sup.12.
29. The compound according to claim 1, wherein R.sup.12 is aryl or
heteroaryl and is optionally substituted by 1-3 J groups.
30. The compound according to claim 29, wherein R.sup.12 is
naphthyl, pyrazinyl, or pyridyl, any of which is optionally
substituted with a hydroxy group.
31. The compound according to claim 1, wherein R.sup.10 is alkyl
substituted with carboxy.
32. The compound according to claim 1, wherein R.sup.15 is alkyl
substituted with carboxy.
33. The compound according to claim 1, wherein the ring to which
R.sup.18 and R.sup.19 are attached is aromatic.
34. A pharmaceutically acceptable composition comprising: a) a
compound according to any one of claims 1-33 in an amount effective
to inhibit HCV NS3 protease; and b) a pharmaceutically suitable
carrier.
35. The use of a compound according to any one of claims 1-33 or a
pharmaceutical composition according to claim 34 in the manufacture
of a medicament for inhibiting serine protease activity in a
patient.
36. The use according to claim 35, wherein the serine protease is
HCV NS3 protease.
37. The use of a compound according to any one of claims 1-33 or a
pharmaceutical composition according to claim 34 in the manufacture
of a medicament for treating or preventing a hepatitis C viral
infection in a patient.
38. A process for preparing a compound of the formula (I):
45wherein: W is: 46wherein: m is 0 or 1; each R.sup.1 is hydroxy,
alkoxy, or aryloxy, or each R.sup.1 is an oxygen atom and together
with the boron, to which they are each bound, form a 5-7 membered
ring, wherein the ring atoms are carbon, nitrogen, or oxygen; each
R.sup.2 is independently hydrogen, alkyl, alkenyl, aryl, aralkyl,
aralkenyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl,
cycloalkenylalkyl, heterocyclyl, heterocyclylalkyl,
heterocyclylalkenyl, heteroaryl, or heteroaralkyl, or two R.sup.2
groups, which are bound to the same nitrogen atom, form together
with that nitrogen atom, a 5-7 membered monocyclic heterocyclic
ring system; wherein any R.sup.2 carbon atom is optionally
substituted with J; J is alkyl, aryl, aralkyl, alkoxy, aryloxy,
aralkoxy, cycloalkyl, cycloalkoxy, heterocyclyl, heterocyclyloxy,
heterocyclylalkyl, keto, hydroxy, amino, alkylamino, alkanoylamino,
aroylamino, aralkanoylamino, carboxy, carboxyalkyl,
carboxamidoalkyl, halo, cyano, nitro, formyl, acyl, sulfonyl, or
sulfonamido and is optionally substituted with 1-3 J.sup.1 groups;
and J.sup.1 is alkyl, aryl, aralkyl, alkoxy, aryloxy, heterocyclyl,
heterocyclyloxy, keto, hydroxy, amino, alkanoylamino, aroylamino,
carboxy, carboxyalkyl, carboxamidoalkyl, halo, cyano, nitro,
formyl, sulfonyl, or sulfonamido; L is alkyl, alkenyl, or alkynyl,
wherein any hydrogen is optionally substituted with halogen, and
wherein any hydrogen or halogen atom bound to any terminal carbon
atom is optionally substituted with sulfhydryl or hydroxy; each M
is independently alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,
heterocyclylalkyl, heteroaryl, or heteroaralkyl, and is optionally
substituted by 1 to 3 J groups, wherein any alkyl carbon atom may
be replaced by a heteroatom; R.sup.18 is a bond, --N(R.sup.11)--,
or --C(O)--; R.sup.11 is hydrogen or C.sub.1-C.sub.3 alkyl; each
R.sup.19 is independently H or R.sup.21-aryl, or 2 adjacent
R.sup.19 may be bound to one another to form a 5-7 membered
aromatic ring; wherein any R.sup.19 is optionally substituted with
1 to 4 independently selected J' groups; each R.sup.21 is
independently C.sub.1-C.sub.3-straight or branched alkyl,
C.sub.2-C.sub.3-straight or branched alkenyl,
O--(C.sub.1-C.sub.3)-straig- ht or branched alkyl, or
O--(C.sub.2-C.sub.3)-straight or branched alkenyl; n is 0 or 1; the
ring to which R.sup.18 and R.sup.19 are attached may be saturated,
partially saturated, aromatic or fully unsaturated; and 1 to 3
carbon atoms that make up the ring to which R.sup.18 and R.sup.19
are attached are optionally replaced with a heteroatom which is
independently selected from O, S, S(O), S(O).sub.2 or N(R.sup.11);
A.sup.2 is a bond or --N(R.sup.11)--R.sup.17(M)-R.sup.22--, wherein
R.sup.17 is --CH-- or --N--; and R.sup.22 is --C(O)-- or
--S(O).sub.2--; V is a bond, --CH(R.sup.11)--, --O--, --S--, or
--N(R.sup.11)--; K is a bond, --O--, --S--, --C(O)--, --S(O)--,
--S(O).sub.2--, or --S(O)NR.sup.11--; T is --R.sup.12,
-alkyl-R.sup.12, -alkenyl-R.sup.12, -alkynyl-R.sup.12, --OR.sup.12,
--N(R.sup.12).sub.2, --C(O)R.sup.12, --C (.dbd.NO-alkyl) R.sup.12,
or 47 wherein: each R.sup.12 is independently selected from
hydrogen, aryl, heteroaryl, cycloalkyl, heterocyclyl,
cycloalkylidenyl, or heterocycloalkylidenyl, and is optionally
substituted with 1 to 3 J groups; or a first R.sup.12 and a second
R.sup.12, together with the nitrogen to which they are bound, form
a mono- or bicyclic ring system optionally substituted by 1 to 3 J
groups; R.sup.10 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,
heterocyclylalkyl, heteroaryl, heteroaralkyl, carboxyalkyl, or
carboxamidoalkyl, and is optionally substituted with 1 to 3
hydrogens J groups; R.sup.15 is alkyl, cycloalkyl, aryl, aralkyl,
heterocyclyl, heterocyclylalkyl, heteroaryl, heteroaralkyl,
carboxyalkyl, or carboxamidoalkyl, and is optionally substituted
with 1 to 3 J groups; and R.sup.16 is hydrogen, alkyl, aryl,
heteroaryl, cycloalkyl, or heterocyclyl; comprising the step of:
reacting a compound of formula (II): 48, wherein LG is OH or an
appropriate leaving group and the other substituents are as defined
above; with a compound of formula (III): 49wherein the NH.sub.2
group is optionally protected and L and W are as defined above; in
the presence of a coupling reagent, provided that the compound of
formula (II) or the compound of formula (III) is optionally bound
to a resin.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to compounds that are useful
as protease inhibitors, particularly as serine protease inhibitors,
and more particularly as hepatitis C NS3 protease inhibitors. As
such, they act by interfering with the life cycle of the hepatitis
C virus and are also useful as antiviral agents.
[0002] This invention also relates to pharmaceutical compositions
comprising these compounds. The compounds and pharmaceutical
compositions of this invention are particularly well suited for
inhibiting HCV NS3 protease activity and consequently, may be
advantageously used as therapeutic agents against the hepatitis C
virus and other viruses that are dependent upon a serine protease
for proliferation. This invention also relates to methods for
inhibiting the activity of proteases, including hepatitis C virus
NS3 protease and other serine proteases, using the compounds of
this invention and related compounds.
BACKGROUND OF THE INVENTION
[0003] Infection by hepatitis C virus ("HCV") is a compelling human
medical problem. HCV is recognized as the causative agent for most
cases of non-A, non-B hepatitis, with an estimated human
seroprevalence of 1% globally [Purcell, R. H., "Hepatitis C virus:
Historical perspective and current concepts" FEMS Microbiology
Reviews 14, pp. 181-192 (1994); Van der Poel, C. L., "Hepatitis C
Virus. Epidemiology, Transmission and Prevention in Hepatitis C
Virus. Current Studies in Hematology and Blood Transfusion, H. W.
Reesink, Ed., (Basel: Karger), pp. 137-163 (1994)]. Four million
individuals may be infected in the United States alone [Alter, M.
J. and Mast, E. E., "The Epidemiology of Viral Hepatitis in the
United States, Gastroenterol. Clin. North Am. 23, pp. 437-455
(1994)].
[0004] Upon first exposure to HCV only about 20% of infected
individuals develop acute clinical hepatitis while others appear to
resolve the infection spontaneously. In most instances, however,
the virus establishes a chronic infection that persists for decades
[Iwarson, S. "The Natural Course of Chronic Hepatitis" FEMS
Microbiology Reviews 14, pp. 201-204 (1994)]. This usually results
in recurrent and progressively worsening liver inflammation, which
often leads to more severe disease states such as cirrhosis and
hepatocellular carcinoma [Kew, M. C., "Hepatitis C and
Hepatocellular Carcinoma", FEMS Microbiology Reviews, 14, pp.
211-220 (1994); Saito, I., et al. "Hepatitis C Virus Infection is
Associated with the Development of Hepatocellular Carcinoma" Proc.
Natl. Acad. Sci. USA 87, pp. 6547-6549 (1990)). Unfortunately,
there are no broadly effective treatments for the debilitating
progression of chronic HCV.
[0005] The HCV genome encodes a polyprotein of 30103033 amino acids
[Choo, Q.-L., et al. "Genetic organization and Diversity of the
Hepatitis C Virus", Proc. Natl. Acad. Sci. USA, 88, pp. 2451-2455
(1991); Kato, N. et al., Molecular Cloning of the Human Hepatitis C
Virus Genome From Japanese Patients with Non-A, Non-B Hepatitis",
Proc. Natl. Acad. Sci. USA, 87, pp. 9524-9528 (1990); Takamizawa,
A. et al., "Structure and Organization of the Hepatitis C Virus
Genome Isolated From Human Carriers", J. Virol., 65, pp. 1105-1113
(1991)]. The HCV nonstructural (NS) proteins are presumed to
provide the essential catalytic machinery for viral replication.
The NS proteins are derived by proteolytic cleavage of the
polyprotein [Bartenschlager, R. et al., "Nonstructural Protein 3 of
the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for
Cleavage at the NS3/4 and NS4/5 Junctions", J. Virol., 67, pp.
3835-3844 (1993); Grakoui, A. et al. "Characterization of the
Hepatitis C Virus-Encoded Serine Proteinase: Determination of
Proteinase-Dependent Polyprotein Cleavage Sites", J. Virol., 67,
pp. 2832-2843 (1993); Grakoui, A. et al., Expression and
Identification of Hepatitis C Virus Polyprotein Cleavage Products",
J. Virol., 67, pp. 1385-1395 (1993); Tomei, L. et al., "NS3 is a
serine protease required for processing of hepatitis C virus
polyprotein", J. Virol., 67, pp. 4017-4026 (1993)].
[0006] The HCV NS protein 3 (NS3) contains a serine protease
activity that helps process the majority of the viral enzymes, and
is thus considered essential for viral replication and infectivity.
It is known that mutations in the yellow fever virus NS3 protease
decreases viral infectivity [Chambers, T. J. et. al., "Evidence
that the N-terminal Domain of Nonstructural Protein NS3 From Yellow
Fever Virus is a Serine Protease Responsible for Site-Specific
Cleavages in the Viral Polyprotein", Proc. Natl. Acad. Sci. USA,
87, pp. 8898-8902 (1990)). The first 181 amino acids of NS3
(residues 1027-1207 of the viral polyprotein) have been shown to
contain the serine protease domain of NS3 that processes all four
downstream sites of the HCV polyprotein [C. Lin et al., "Hepatitis
C Virus NS3 Serine Proteinase: Trans-Cleavage Requirements and
Processing Kinetics", J. Virol., 68, pp. 8147-8157 (1994))]
[0007] The HCV NS3 serine protease and its associated cofactor,
NS4A, helps process all of the viral enzymes, and is thus
considered essential for viral replication. This processing appears
to be analogous to that carried out by the human immunodeficiency
virus aspartyl protease, which is also involved in viral enzyme
processing HIV protease inhibitors, which inhibit viral protein
processing are potent antiviral agents in man, indicating that
interrupting this stage of the viral life cycle results in
therapeutically active agents. Consequently it is an attractive
target for drug discovery. Unfortunately, there are no serine
protease inhibitors available currently as anti-HCV agents.
[0008] Furthermore, the current understanding of HCV has not led to
any other satisfactory anti-HCV agents or treatments. The only
established therapy for HCV disease is interferon treatment.
However, interferons have significant side effects (Janssen et al.,
1994; Renault and Hoofnagle, 1989) [Janssen, H. L. A., et al.
"Suicide Associated with Alfa-Interferon Therapy for Chronic Viral
Hepatitis" J. Hepatol., 21, pp. 241-243 (1994)]; Renault, P. F. and
Hoofnagle, J. H., "Side effects of alpha interferon. Seminars in
Liver Disease 9, 273-277. (1989)] and induce long term remission in
only a fraction (-25%) of cases [Weiland, O. "Interferon Therapy in
Chronic Hepatitis C Virus Infection", FEMS Microbiol. Rev., 14, PP.
279-288 (1994)]. Moreover, the prospects for effective anti-HCV
vaccines remain uncertain.
[0009] Thus, there is a need for more effective anti-HCV therapies.
Such inhibitors would have therapeutic potential as protease
inhibitors, particularly as serine protease inhibitors, and more
particularly as HCV NS3 protease inhibitors. Specifically, such
compounds may be useful as antiviral agents, particularly as
anti-HCV agents.
SUMMARY OF THE INVENTION
[0010] The present invention provides compounds, and
pharmaceutically acceptable derivatives thereof, that are useful as
protease inhibitors, particularly as serine protease inhibitors,
and more particularly as HCV NS3 protease inhibitors. These
compounds can be used alone or in combination with immunomodulatory
agents, such as .alpha.-, .beta.- or .gamma.-interferons; other
antiviral agents such as ribavirin and amantadine; other inhibitors
of hepatitis C protease; inhibitors of other targets in the HCV
life cycle including the helicase, polymerase, metalloprotease, or
internal ribosome entry; or combinations thereof.
[0011] The present invention also provides methods for inhibiting
proteases, particularly serine proteases, and more particularly HCV
NS3 protease.
[0012] The present invention also provides pharmaceutical
compositions comprising the compounds of this invention, as well as
multi-component compositions comprising additional immunomodulatory
agents, such as .alpha.-, .beta.- or .gamma.-interferons; other
antiviral agents such as ribavirin and amantadine; other inhibitors
of hepatitis C protease; inhibitors of other targets in the HCV
life cycle including the helicase, polymerase, metalloprotease, or
internal ribosome entry; or combinations thereof. The invention
also provides methods of using the compounds of this invention, as
well as other related compounds, for the inhibition of HCV.
DETAILED DESCRIPTION OF THE INVENTION
[0013] In order that the invention herein described may be more
fully understood, the following detailed description is set forth.
In the description, the following abbreviations are used:
1 Designation Reagent or Fragment Abu aminobutyric acid Ac acetyl
AcOH acetic acid Bn benzyl Boc tert-butyloxycarbonyl Bz benzoyl Cbz
carbobenzyloxy CDI carbonyldiimidazole DCE 1,2-dichloroethane DCM
dichioromethane DIEA diisopropylethylamine DMA dimethylacetamide
DMAP dimethylaminopyridine DMF dimethylformamide DPPA
diphenylphosphorylazide DMSO dimethylsulfoxide Et ethyl EtOAc ethyl
acetate FMOC 9-fluorenylmethoxycarbonyl HbtU
O-benzotriazolyl-N,N,N',N'- tetramethyluronium hexafluorophosphate
HOBt N-hydroxybenzotriazole HPLG high performance liquid
chromatography Me methyl MS mass spectrometry NMP N-methyl
pyrrolidinone ND not determined Pip piperidine Prz piperazine
PyBrop bromo-tris-pyrrolidinophosphonium hexafluorophosphate Pyr
pyridine THF tetrahydrofuran TFA trifluoroacetic acid TFE
trifluoroethanol Tol toluene
[0014] The following terms are used herein:
[0015] Unless expressly stated to the contrary, the terms
"--SO.sub.2--" and "--S(O).sub.2--" as used herein refer to a
sulfone or sulfone derivative (i.e., both appended groups linked to
the S), and not a sulfinate ester.
[0016] The term "substituted" refers to the replacement of one or
more hydrogen radicals in a given structure with a radical selected
from a specified group. When more than one hydrogen radical may be
replaced with a substituent selected from the same specified group,
the substituents may be either the same or different at every
position.
[0017] As used herein, the term "amino" refers to a trivalent
nitrogen which may be primary or which may be substituted with 1-2
alkyl groups.
[0018] The term "alkyl" or "alkane", alone or in combination with
any other term, refers to a straight-chain or branched-chain
saturated aliphatic hydrocarbon radical containing the specified
number of carbon atoms, or where no number is specified, preferably
from 1-10 and more preferably from 1-5 carbon atoms. Examples of
alkyl radicals include, but are not limited to, methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,
pentyl, isoamyl, n-hexyl and the like.
[0019] The term "alkenyl" or "alkene", alone or in combination with
any other term, refers to a straight-chain or branched-chain mono-
or poly-unsaturated aliphatic hydrocarbon radical containing the
specified number of carbon atoms, or where no number is specified,
preferably from 2-10 carbon atoms and more preferably, from 2-5
carbon atoms. Examples of alkenyl radicals include, but are not
limited to, ethenyl, E- and Z-propenyl, E- and Z-isobutenyl, E- and
Z-pentenyl, E- and Z-hexenyl, E,E-, E,Z-, Z,E-, and Z-Z-hexadienyl
and the like.
[0020] The term "alkynyl" or "alkyne", alone or in combination with
any other term, refers to a straight-chain or branched-chain mono
or poly-unsaturated aliphatic hydrocarbon radical containing the
specified number of carbon atoms, or where no number is specified,
preferably from 2-10 carbon atoms and more preferably, from 2-5
carbon atoms, wherein at least one of the unsaturated aliphatic
hydrocarbon radicals comprises a triple bond. Examples of alkynyl
radicals include, but are not limited to, ethynyl, propynyl,
isobutynyl, pentynyl, hexynyl, hexenynyl, and the like.
[0021] The term "aryl", alone or in combination with any other
term, refers to a carbocyclic aromatic radical containing the
specified number of carbon atoms, and which may be optionally
fused, for example benzofused, with one to three cycloalkyl,
aromatic, heterocyclic or heteroaromatic rings. Preferred aryl
groups have from 6-14 carbon atoms, and more preferred groups from
6-10 carbon atoms. Examples of aryl radicals include, but are not
limited to, phenyl, naphthyl, anthracenyl and the like.
[0022] The term "carbocycle", alone or in combination with any
other term, refers to a stable non-aromatic 3- to 8-membered carbon
ring radical which may be saturated, mono-unsaturated or
poly-unsaturated, and which may be optionally fused, for example
benzofused, with one to three cycloalkyl, aromatic, heterocyclic or
heteroaromatic rings. The carbocycle may be attached at any
endocyclic carbon atom which results in a stable structure.
[0023] The terms "cycloalkyl" or "cycloalkane", alone or in
combination with any other term, refers to a stable non-aromatic 3-
to 8-membered carbon ring radical which is saturated and which may
be optionally fused, for example benzofused, with one to three
cycloalkyl, aromatic, heterocyclic or heteroaromatic rings. The
cycloalkyl may be attached at any endocyclic carbon atom which
results in a stable structure. Preferred carbocycles have 5 to 6
carbons. Examples of carbocycle radicals include, but are not
limited to, cyclopropyl, cyclbutyl, cyclpentyl, cyclohexyl,
cycloheptyl, cyclopentenyl, cyclohexenyl, indane,
tetrahydronaphthalene and the like.
[0024] The term "cycloalkenyl" or "cycloalkene" alone or in
combination with any other term, refers to a stable cyclic
hydrocarbon ring radical containing at least one endocyclic
carbon-carbon double bond. The carbocycle may be attached at any
cyclic carbon atom which results in a stable structure. Where no
number of carbon atoms is specified, a cycloalkenyl radical
preferably has from 5-7 carbon atoms. Examples of cycloalkenyl
radicals include, but are not limited to, cyclopentenyl,
cyclohexenyl, cyclopentadienyl, indenyl and the like.
[0025] The term "cycloalkylidenyl", alone or in combination with
any other term, refers to a stable cyclic hydrocarbon ring radical
containing at least one exocyclic carbon-carbon double bond,
wherein the cyclic hydrocarbon ring may be optionally fused, for
example benzofused, with one to three cycloalkyl, aromatic,
heterocyclic or heteroaromatic rings. The carbocycle may be
attached at any cyclic carbon atom, which results in a stable
structure. Where no number of carbon atoms is specified, a
cycloalkylidenyl radical preferably has from 5-7 carbon atoms.
Examples of cycloalkylidenyl radicals include, but are not limited
to, cyclopentylidenyl, cyclohexylidenyl, cyclopentenylidenyl and
the like.
[0026] The skilled practitioner would realize that certain groups
could be classified either as cycloalkanes or as aryl groups.
Examples of such groups include indanyl and tetrahydro naphthyl
groups.
[0027] The term "monocycle" or "monocyclic" alone or in combination
with any other term, unless otherwise defined herein, refers to a
5-7 membered ring system.
[0028] The term "bicycle" or "bicyclic" alone or in combination
with any other term, unless otherwise defined herein, refers to a
6-11 membered ring system.
[0029] The term "tricycle" or "tricyclic" alone or in combination
with any other term, unless otherwise defined herein, refers to a
11-15 membered ring system.
[0030] The terms "heterocyclyl" and "heterocycle", alone or in
combination with any other term, unless otherwise defined herein,
refers to a stable 5- to 15-membered mono-, bi-, or tricyclic,
heterocyclic ring which is either saturated or partially
unsaturated, but not aromatic, and which may be optionally fused,
for example benzofused, with one to three cycloalkyl, aromatic,
heterocyclic or heteroaromatic rings. Each heterocycle consists of
one or more carbon atoms and from one to four heteroatoms selected
from the group consisting of nitrogen, oxygen and sulfur. As used
herein, the terms "nitrogen and sulfur heteroatoms" include any
oxidized form of nitrogen and sulfur, and the quaternized form of
any basic nitrogen. A heterocycle may be attached at any endocyclic
carbon or heteroatom which results in the creation of a stable
structure.
[0031] Preferred heterocycles defined above include, for example,
imidazolidinyl, indazolinolyl, perhydropyridazyl, pyrrolinyl,
pyrrolidinyl, piperidinyl, pyrazolinyl, piperazinyl, morpholinyl,
thiamorpholinyl, .beta.-carbolinyl, thiazolidinyl, thiamorpholinyl
sulfone, oxopiperidinyl, oxopyrroldinyl, oxoazepinyl, azepinyl,
furazanyl, tetrahydropyranyl, tetrahydrofuranyl, oxathiolyl,
dithiolyl, tetrahydrothiophenyl, dioxanyl, dioxolanyl,
tetrahydrofurotetrahydrofuran- yl,
tetrahydropyranotetrahydrofuranyl, tetrahydrofurodihydrofuranyl,
tetrahydropyranodihydrofuranyl, dihydropyranyl, dihydrofuranyl,
dihydrofurotetrahydrofuranyl, dihydropyranotetrahydrofuranyl,
sulfolanyl and the like.
[0032] The terms "heteroaryl" and "heteroaromatic" alone or in
combination with any other term, unless otherwise defined herein,
refers to a stable 3- to 7-membered monocyclic heterocyclic ring
which is aromatic, and which may be optionally fused, for example,
benzofused, with one to three cycloalkyl, aromatic, heterocyclic or
heteroaromatic rings. Each heteroaromatic ring consists of one or
more carbon atoms and from one to four heteroatoms selected from
the group consisting of nitrogen, oxygen and sulfur. As used
herein, the terms "nitrogen and sulfur heteroatoms" include any
oxidized form of nitrogen and sulfur, and the quaternized form of
any basic nitrogen. A heteroaromatic ring may be attached at any
endocyclic carbon or heteroatom which results in the creation of a
stable, aromatic structure.
[0033] Preferred heteroaromatics defined above include, for
example, benzimidazolyl, imidazolyl, quinolyl, isoquinolyl,
indolyl, indazolyl, pyridazyl, pyridyl, pyrrolyl, pyrazolyl,
pyrazinyl, quinoxolyl, pyranyl, pyrimidinyl, pyridazinyl, furyl,
thienyl, triazolyl, thiazolyl, tetrazolyl, benzofuranyl, oxazolyl,
benzoxazolyl, isoxazolyl, isothiazolyl, thiadiazolyl, thiophenyl,
oxadiazolyl, oxatriazolyl, thiatriazolyl, dithiazolyl, dioxazolyl,
oxathiazolyl, triazinyl and the like.
[0034] The term "halo" refers to a radical of fluorine, chlorine,
bromine or iodine. Preferred halogen radicals include fluorine and
chlorine.
[0035] In chemical formulas, parentheses are used herein to
indicate 1) the presence of more than one atom or group bonded to
the same atom or group; or 2) a branching point in a chain (i.e.,
the group or atom immediately before the open parenthesis is bonded
directly to the group or atom immediately after the closed
parenthesis). An example of the first use is "N(R.sup.1).sub.2"
denoting two R.sup.1 groups bound to the nitrogen atom. An example
of the second use is "--C(O)R.sup.1" denoting an oxygen atom and a
R.sup.1 bound to the carbon atom, as in the following structure:
1
[0036] As used herein, "B" indicates a boron atom.
[0037] Those of skill in the art will realize that certain
combinations of moiety choices for variables in the generic
structures set forth throughout this application will produce
chemically unstable or unfeasible compounds. Such compounds are not
intended to be part of the present invention.
[0038] The present invention provides compounds that are useful as
protease inhibitors, particularly as serine protease inhibitors,
and more particularly as HCV NS3 protease inhibitors. As such, they
act by interfering with the life cycle of the HCV virus and other
viruses that are dependent upon a serine protease for
proliferation. Therefore, these compounds are useful as antiviral
agents.
[0039] According to one embodiment, the present invention provides
a compound of the formula (I): 2
[0040] In these compounds W is selected from: 3
[0041] wherein m is 0 or 1.
[0042] Each R.sup.1 is hydroxy, alkoxy, or aryloxy, or each R.sup.1
is an oxygen atom and together with the boron, to which they are
each bound, form a 5-7 membered ring, wherein the ring atoms are
carbon, nitrogen, or oxygen.
[0043] Each R.sup.2 is independently hydrogen, halo, alkyl,
alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl,
cycloalkenyl, cycloalkenylalkyl, heterocyclyl, heterocyclylalkyl,
heterocyclylalkenyl, heteroaryl, or heteroaralkyl, or two R.sup.2
groups, which are bound to the same nitrogen atom, form together
with that nitrogen atom, a 5-7 membered monocyclic heterocyclic
ring system; wherein any R.sup.2 carbon atom is optionally
substituted with J.
[0044] Each J is independently alkyl, aryl, aralkyl, alkoxy,
aryloxy, aralkoxy, cycloalkyl, cycloalkoxy, heterocyclyl,
heterocyclyloxy, heterocyclylalkyl, keto, hydroxy, amino,
alkylamino, alkanoylamino, aroylamino, aralkanoylamino, carboxy,
carboxyalkyl, carboxamidoalkyl, halo, cyano, nitro, formyl, acyl,
sulfonyl, or sulfonamido and is optionally substituted with 1 to 3
J.sup.1 groups.
[0045] Each J.sup.1 is independently selected from alkyl, aryl,
aralkyl, alkoxy, aryloxy, heterocyclyl, heterocyclyloxy, keto,
hydroxy, amino, alkanoylamino, aroylamino, carboxy, carboxyalkyl,
carboxamidoalkyl, halo, cyano, nitro, formyl, sulfonyl, or
sulfonamido.
[0046] L is alkyl, alkenyl, or alkynyl, wherein any hydrogen bound
to a carbon atoms is optionally substituted with halogen, and
wherein any hydrogen or halogen atom bound to any terminal carbon
atom is optionally substituted with sulfhydryl or hydroxy.
[0047] Each M is independently alkyl, cycloalkyl, aryl, aralkyl,
heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroaralkyl, and
is optionally substituted by 1 to 3 J groups, wherein any alkyl
carbon atom may be replaced by a heteroatom.
[0048] R.sup.18 is a bond, --N(R.sup.11)--, or --C(O)--.
[0049] R.sup.11 is hydrogen or C.sub.1-C.sub.3 alkyl.
[0050] Each R.sup.19 is independently H or R.sup.21-aryl, or 2
adjacent R.sup.19 may be bound to one another to form a 5-7
membered aromatic ring, wherein any R.sup.19 is optionally
substituted with 1 to 4 independently selected J' groups. When 2
adjacent R.sup.19 are bound to one another to form a 5-7 membered
aromatic ring a bicyclic ring system is formed consisting of the
aromatic ring shown in formula I and the aromatic ring formed by
two adjacent R.sup.19 groups. When R.sup.19 is R.sup.21-aryl, this
optional substitution may occur on one or more carbon atoms of
R.sup.21 and/or on one or more ring atoms of said aryl. When 2
adjacent R.sup.19 are bound to one another to form a 5-7 membered
aromatic ring, the optional substitution may occur on one or more
atoms of the resulting aromatic ring.
[0051] Each R.sup.21" is independently C.sub.1-C.sub.3-straight or
branched alkyl, C.sub.2-C.sub.3-straight or branched alkenyl,
O--(C.sub.1-C.sub.3)-straight or branched alkyl, or
O--(C.sub.2-C.sub.3)-straight or branched alkenyl.
[0052] n is 0 or 1.
[0053] The ring to which R.sup.18 and R.sup.19 are attached may be
saturated, partially saturated, aromatic or fully unsaturated. Up
to 3 carbon atoms that make up the ring to which R.sup.18 and
R.sup.19 are attached are optionally replaced with a heteroatom
which is independently selected from O, S, S(O), S(O).sub.2 or
N(R.sup.11).
[0054] A.sup.2 is a bond or --N(R.sup.11)--R.sup.17(M)-R.sup.22--,
wherein R.sup.17 is --CH-- or --N--; and R.sup.22 is --C(O)-- or
--S(O).sub.2--.
[0055] V is a bond, --CH(R.sup.11)--, --O--, --S--, or
--N(R.sup.11)--.
[0056] K is a bond, --O--, --S--, --C(O)--, --S(O)--,
--S(O).sub.2--, or --S(O)NR.sup.11--.
[0057] T is --R.sup.12, -alkyl-R.sup.12, -alkenyl-R.sup.12,
-alkynyl-R.sup.12, --OR.sup.12]-N(R.sup.12).sub.21--C(O)R.sup.12,
--C(.dbd.NO-alkyl)R.sup.12, or 4
[0058] Each R.sup.12 is independently selected from hydrogen, aryl,
heteroaryl, cycloalkyl, heterocyclyl, cycloalkylidenyl, or
heterocycloalkylidenyl, and is optionally substituted with 1 to 3 J
groups; or a first R.sup.12 and a second R.sup.12, together with
the nitrogen to which they are bound, form a mono- or bicyclic ring
system optionally substituted by 1 to 3 J groups.
[0059] R.sup.10 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,
heterocyclylalkyl, heteroaryl, heteroaralkyl, carboxyalkyl, or
carboxamidoalkyl, and is optionally substituted with 1 to 3 J
groups.
[0060] R.sup.15 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,
heterocyclylalkyl, heteroaryl, heteroaralkyl, carboxyalkyl, or
carboxamidoalkyl, and is optionally substituted with 1 to 3 J
groups.
[0061] R.sup.16 is hydrogen, alkyl, aryl, heteroaryl, cycloalkyl,
or heterocyclyl.
[0062] Preferably, W is 5
[0063] Most preferably, W is --C(O)H.
[0064] Preferably, J is alkyl, alkoxy, aryloxy, aryl, aralkyl,
aralkoxy, halo, heteroaryl, cyano, amino, nitro, heterocyclyl,
acyl, carboxy, carboxyalkyl, alkylamino, hydroxy,
heterocyclylalkyl, aralkanoylamino, aroylamino, alkanoylamino,
formyl or keto.
[0065] More preferably, J is t-butyl, methyl, trifluoromethyl,
hydroxy, methoxy, ethoxy, trifluoromethoxy, carboxy, phenyl,
benzyl, phenoxy, benzyloxy, fluoro, chloro, bromo, isoxazolyl,
pyridinyl, piperidinyl, carboxymethyl, carboxyethyl, dialkylamino,
morpholinylmethyl, phenylacetylamino, or acylamino.
[0066] Preferably, J.sup.1 is alkoxy, alkyl, halo or aryl.
[0067] More preferably, J.sup.1 is C.sub.1-3 alkoxy, chloro,
C.sub.1-3 alkyl, or phenyl.
[0068] Preferably, L is alkyl, alkenyl, allyl, or propargyl.
[0069] More preferably, L is trihalomethyl, sulfhydryl or alkyl
substituted with trihalomethyl, sulfhydryl, or hydroxy. Most
preferably, L is --CH.sub.2CH.sub.3 or --CH.sub.2CF.sub.3.
[0070] Preferably, R.sup.2 is H, fluorine, trifluoromethyl, alkyl,
aryl, aralkyl, heteroaralkyl, heterocyclyl, or heterocyclylalkyl.
More preferred is when R.sup.2 is H.
[0071] Preferably, M is alkyl, heteroaralkyl, aryl,
cycloalkylalkyl, aralkyl, or aralkyl, wherein one of the alkyl
carbon atoms is replaced by O or S.
[0072] More preferably M is isopropyl, propyl, methyl,
pyridylmethyl, benzyl, naphthylmethyl, phenyl, imidazolylmethyl,
thiophenylmethyl, cyclohexylmethyl, phenethyl, benzylthiomethyl, or
benzyloxyethyl. Most preferably, M is isopropyl.
[0073] Preferably, one R.sup.19 is R.sup.21-aryl and the other two
R.sup.19 are H or two R.sup.19 are bound together to form an
aromatic ring and the other R.sup.19 is H. More preferred is when
one R.sup.19 is --O--(C.sub.1-C.sub.3)-alkyl-aryl or the two
R.sup.19 bound together form a 6-membered aromatic ring. Most
preferred is when one R.sup.19 is --O-benzyl or the two R.sup.19
bound together form phenyl.
[0074] Preferably, R.sup.18 is --N(R.sup.11)--. More preferably,
R.sup.18 is --N(H)-- or --N(CH.sub.3)--.
[0075] Preferably, the ring to which R.sup.18 and R.sup.19 are
attached is aromatic.
[0076] It is preferred that A.sup.2 be a bond or
--N(R.sup.11)--C(M)-C(O)-- -. More preferred is when A.sup.2 is a
bond or --N(H)--C(M)-C(O)--, wherein M is isopropyl.
[0077] Preferably, V is --N(R.sup.11)--. More preferably, V is
--NH--.
[0078] Preferably, K is --C(O)-- or --S(O).sub.2--. More
preferably, K is --C(O)--
[0079] Preferably, T is --R.sup.12, -alkyl-R.sup.12,
-alkenyl-R.sup.12--OR.sup.12, --N(R.sup.12).sub.2,
--C(.dbd.NO-alkyl)-R.sup.12, or 6
[0080] More preferably, T is --R.sup.12 or -alkyl-R.sup.12.
[0081] Preferably, R.sup.12 is aryl or heteroaryl and is optionally
substituted by 1-3 J groups. More preferably, R.sup.12 is naphthyl,
pyrazinyl, or pyridyl, any of which is optionally substituted with
a hydroxy group
[0082] Preferably, R.sup.10 is alkyl substituted with carboxy. More
preferably, R.sup.10 is C.sub.1-3 alkyl substituted with
carboxy.
[0083] Preferably, R.sup.15 is alkyl substituted with carboxy. More
preferably, R.sup.15 is C.sub.1-3 alkyl substituted with
carboxy.
[0084] The most preferred compounds of this invention are listed in
Table 1, below.
2TABLE 1 Preferred Compounds of the formula: 7 Compound T A.sup.2
R.sup.18 R.sup.19A R.sup.19B L 101 8 9 --N(H)-- H O-benzyl
--CH.sub.2CH.sub.3 102 10 11 --N(H)-- H O-benzyl --CH.sub.2CF.sub.3
103 12 13 --N(CH.sub.3)-- H O-benzyl --CH.sub.2CH.sub.3 104 14 bond
--N(H)-- H O-benzyl --CH.sub.2CH.sub.3 105 15 bond --N(H)-- H
O-benzyl --CH.sub.2CH.sub.3 106 16 bond --N(H)-- H O-benzyl
--CH.sub.2CH.sub.3 107 17 18 --N(H)-- H O-3,4- dichloro benzyl
--CH.sub.2CH.sub.3 108 19 20 --N(H)-- H H --CH.sub.2CH.sub.3 109 21
22 --N(H)-- bound to one another to form a benzofused ring
--CH.sub.2CH.sub.3
[0085] This invention anticipates that many active-site directed
inhibitors of the NS3 protease may be peptidomimetic in nature and
thus may be designed from the natural substrate. Therefore,
preferred substituents in peptidomimetic inhibitors of this
invention include those which correspond to the backbone or side
chains of naturally occurring substrates or synthetic substrates
with high affinity for the enzyme (low K.sub.m).
[0086] The skilled practitioner would realize that some certain
groups could be classified either as heterocycles or
heteroaromatics, depending on the point of attachment.
[0087] The compounds of this invention may contain one or more
asymmetric carbon atoms and thus may occur as racemates and racemic
mixtures, single enantiomers, diastereomeric mixtures and
individual diastereomers. All such isomeric forms of these
compounds are expressly included in the present invention. Each
stereogenic carbon may be of the R or S configuration. Combinations
of substituents and variables envisioned by this invention are only
those that result in the formation of stable compounds. The term
"stable", as used herein, refers to compounds which possess
stability sufficient to allow manufacture and which maintains the
integrity of the compound for a sufficient period of time to be
useful for the purposes detailed herein (e.g., therapeutic or
prophylactic administration to a mammal or for use in affinity
chromatography applications). Typically, such compounds are stable
at a temperature of 40.degree. C. or less, in the absence of
moisture or other chemically reactive conditions, for at least a
week.
[0088] The compounds of this invention may be synthesized using
conventional techniques. Advantageously, these compounds are
conveniently synthesized from readily available starting
materials.
[0089] As used herein, the compounds of this invention are defined
to include pharmaceutically acceptable derivatives or prodrugs
thereof. A "pharmaceutically acceptable derivative or prodrug"
means any pharmaceutically acceptable salt, ester, salt of an
ester, or other derivative of a compound of this invention which,
upon administration to a recipient, is capable of providing
(directly or indirectly) a compound of this invention.
[0090] Accordingly, this invention also provides prodrugs of the
compounds of this invention, which are derivatives that are
designed to enhance biological properties such as oral absorption,
clearance, metabolism or compartmental distribution. Such
derivations are well known in the art.
[0091] As the skilled practitioner realizes, the compounds of this
invention may be modified by appending appropriate functionalities
to enhance selective biological properties. Such modifications are
known in the art and include those which increase biological
penetration into a given biological compartment (e.g., blood,
lymphatic system, central nervous system), increase oral
availability, increase solubility to allow administration by
injection, alter metabolism and alter rate of excretion.
[0092] The term "protected" refers to when the designated
functional group is attached to a suitable chemical group
(protecting group). Examples of suitable amino protecting groups
and protecting groups are described in T. W. Greene and P. G. M.
Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley
and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's
Reagents for Organic Synthesis, John Wiley and Sons (1994); L.
Paquette, ed. Encyclopedia of Reagents for Organic Synthesis, John
Wiley and Sons (1995) and are exemplified in certain of the
specific compounds used in this invention.
[0093] Particularly favored derivatives and prodrugs are those that
increase the bioavailability of the compounds of this invention
when such compounds are administered to a mammal (e.g., by allowing
an orally administered compound to be more readily absorbed into
the blood), have more favorable clearance rates or metabolic
profiles, or which enhance delivery of the parent compound to a
biological compartment (e.g., the brain or lymphatic system)
relative to the parent species. Preferred prodrugs include
derivatives where a group which enhances aqueous solubility or
active transport through the gut membrane is appended to the
structure of formula (I).
[0094] Pharmaceutically acceptable salts of the compounds of this
invention include those derived from pharmaceutically acceptable
inorganic and organic acids and bases. Examples of suitable acid
salts include acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptanoate, glycerophosphate, glycolate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxyethanesulfonate, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, salicylate, succinate,
sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other
acids, such as oxalic, while not in themselves pharmaceutically
acceptable, may be employed in the preparation of salts useful as
intermediates in obtaining the compounds of the invention and their
pharmaceutically acceptable acid addition salts.
[0095] Salts derived from appropriate bases include alkali metal
(e.g., sodium and potassium), alkaline earth metal (e.g., calcium
and magnesium), salts with organic bases, such as dicyclohexylamine
salts, N-methyl-D-glucamine, salts with amino acids such as
arginine and lysine, ammonium and N--(C.sub.1-4 alkyl).sub.4.sup.+
salts.
[0096] This invention also envisions the quaternization of any
basic nitrogen-containing groups of the compounds disclosed herein
with such agents as lower alkyl halides, such as methyl, ethyl,
propyl, and butyl chloride, bromides and iodides; dialkyl sulfates,
such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain
halides such as decyl, lauryl, myristyl and stearyl chlorides,
bromides and iodides, aralkyl halides, such as benzyl and phenethyl
bromides and others. Water or oil-soluble or dispersible products
may be obtained by such quaternization.
[0097] In general, compounds of formula (I) are obtained via
methods illustrated in the Examples. As can be appreciated by the
skilled artisan however the synthetic schemes set forth herein are
not intended to comprise a comprehensive list of all means by which
the compounds described and claimed in this application may be
synthesized. Further methods will be evident to those of ordinary
skill in the art. Additionally, the various synthetic steps
described above may be performed in an alternate sequence or order
to give the desired compounds.
[0098] Without being bound by theory, we believe that the compounds
of this invention interact either covalently or noncovalently with
the active site of the HCV NS3 protease and other serine proteases,
inhibiting the ability of such an enzyme to cleave natural or
synthetic substrates. Noncovalent interactions are advantageous in
that they impart relatively greater specificity of inhibition and
will not inhibit other undesirable targets, e.g. cysteine
proteases. These compounds will therefore have a greater
therapeutic index when administered to mammals than covalent
protease inhibitors, which can interact with a wide range of
proteases and cause undesirable toxic effects. In contrast,
covalent interactions are advantageous in that they impart greater
inhibitory potency allowing lower doses may be administered and
thus ameliorating any lack of specificity problems.
[0099] The novel compounds of the present invention are excellent
inhibitors of proteases, particularly serine proteases, and more
particularly HCV NS3 protease inhibitors. Accordingly, these
compounds are capable of targeting and inhibiting proteases,
particularly serine proteases, and more particularly HCV NS3
proteases. As such, these compounds interfere with the life cycle
of viruses, including HCV and are thus useful as antiviral agents.
Inhibition can be measured by various methods such as the methods
of Example 3.
[0100] The term "antiviral agent" refers to a compound or drug
which possesses viral inhibitory activity. Such agents include
reverse transcriptase inhibitors (including nucleoside and
non-nucleoside analogs) and protease inhibitors. Preferably the
protease inhibitor is a HCV protease inhibitor.
[0101] The term "treating" as used herein refers to the alleviation
of symptoms of a particular disorder in a patient or the
improvement of an ascertainable measurement associated with a
particular disorder. As used herein, the term "patient" refers to a
mammal, including a human.
[0102] Thus, according to another embodiment this invention
provides pharmaceutical compositions comprising a compound of
formula (I) or a pharmaceutically acceptable salt thereof; an
additional agent selected from, but not including, an
immunomodulatory agent, such as .alpha.-, .beta.-, or
.gamma.-interferon; other antiviral agents, such as ribavarin or
amantadine; other inhibitors of HCV protease; inhibitors of other
targets in the HCV life cycle such as helicase, polymerase, or
metalloprotease; or combinations thereof and any pharmaceutically
acceptable carrier, adjuvant or vehicle. An alternate embodiment
provides compositions comprising a compound of formula (I) or a
pharmaceutically acceptable salt thereof; and a pharmaceutically
acceptable carrier, adjuvant or vehicle. Such composition may
optionally comprise an additional agent selected from an
immunomodulatory agent, such as .alpha.-, .beta.-, or
.gamma.-interferon; other antiviral agents, such as ribavarin;
other inhibitors of HCV protease; inhibitors of the HCV helicase;
or combinations thereof.
[0103] The term "pharmaceutically acceptable carrier or adjuvant"
refers to a carrier or adjuvant that may be administered to a
patient, together with a compound of this invention, and which does
not destroy the pharmacological activity thereof and is nontoxic
when administered in doses sufficient to deliver a therapeutic
amount of the compound.
[0104] Pharmaceutically acceptable carriers, adjuvants and vehicles
that may be used in the pharmaceutical compositions of this
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, self-emulsifying drug delivery systems
(SEDDS) such as d.alpha.-tocopherol, polyethyleneglycol 1000
succinate, or TPGS, surfactants used in pharmaceutical dosage forms
such as Tweens or other similar polymeric delivery matrices, serum
proteins, such as human serum albumin, gelatin, buffer substances
such as phosphates, glycine, sorbic acid, potassium sorbate,
partial glyceride mixtures of saturated vegetable fatty acids,
water, salts or electrolytes, such as protamine sulfate, disodium
hydrogen phosphate, potassium hydrogen phosphate, sodium chloride,
zinc salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, polylacetic acid, ployacetic polyglycollic acid,
citric acid, cellulose-based substances, such as HPC and HPMC,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol, wool fat. Cyclodextrins such as .alpha.-, .beta.-, and
.gamma.-cyclodextrin, or chemically modified derivatives such as
hydroxyalkylcyclodextrins, including 2- and
3-hydroxypropyl-.beta.-cyclodextrins, or other solubilized
derivatives may also be advantageously used to enhance delivery of
compounds of formula (I).
[0105] The pharmaceutical compositions of this invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. We prefer oral administration or administration by
injection. The pharmaceutical compositions of this invention may
contain any conventional non-toxic pharmaceutically-acceptable
carriers, adjuvants or vehicles. In some cases, the pH of the
formulation may be adjusted with pharmaceutically acceptable acids,
bases or buffers to enhance the stability of the formulated
compound or its delivery form. The term parenteral as used herein
includes subcutaneous, intracutaneous, intravenous, intramuscular,
intra-articular, intrasynovial, intrasternal, intrathecal,
intralesional and intracranial injection or infusion
techniques.
[0106] The pharmaceutical compositions may be in the form of a
sterile injectable preparation, for example, as a sterile
injectable aqueous or oleaginous suspension. This suspension may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents (such as, for example, Tween 80) and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are mannitol, water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil may be
employed including synthetic mono- or diglycerides. Fatty acids,
such as oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant such as those described in Pharmacopeia Helvetica (Ph.
Helv.) or a similar alcohol, or carboxymethyl cellulose or similar
dispersing agents which are commonly used in the formulation of
pharmaceutically acceptable dosage forms such as emulsions and/or
suspensions. Other commonly used surfactants such as Tweens or
Spans and/or other similar emulsifying agents or bioavailability
enhancers which are commonly used in the manufacture of
pharmaceutically acceptable solid, liquid, or other dosage forms
may also be used for the purposes of formulation.
[0107] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, and aqueous suspensions and
solutions. In the case of tablets for oral use, carriers which are
commonly used include lactose, corn starch, dicalcium phosphate and
microcrystalline cellulose (Avicel). Lubricating agents, such as
magnesium stearate and talc, are also typically added. For oral
administration in a capsule form, useful diluents include lactose,
dried corn starch and TPGS, as well as the other diluents used in
tablets. For oral administration in a soft gelatin capsule form
(filled with either a suspension or a solution of a compound of
this invention), useful diluents include PEG400, TPGS, propylene
glycol, Labrasol, Gelucire, Transcutol, PVP and potassium acetate.
When aqueous suspensions are administered orally, the active
ingredient is combined with emulsifying and suspending agents, such
as sodium CMC, methyl cellulose, pectin and gelatin. If desired,
certain sweetening and/or flavoring and/or coloring agents may be
added.
[0108] The pharmaceutical compositions of this invention may also
be administered in the form of suppositories for rectal
administration. These compositions can be prepared by mixing a
compound of this invention with a suitable non-irritating excipient
which is solid at room temperature but liquid at the rectal
temperature and therefore will melt in the rectum to release the
active components. Such materials include, but are not limited to,
cocoa butter, beeswax, gelatin, glycerin and polyethylene
glycols.
[0109] Topical administration of the pharmaceutical compositions of
this invention is especially useful when the desired treatment
involves areas or organs readily accessible by topical application.
For application topically to the skin, the pharmaceutical
composition should be formulated with a suitable ointment
containing the active components suspended or dissolved in a
carrier. Carriers for topical administration of the compounds of
this invention include, but are not limited to, mineral oil, liquid
petroleum, white petroleum, propylene glycol, polyoxyethylene
polyoxypropylene compound, emulsifying wax, stearic acid, cetyl
stearate, cetyl alcohol, lanolin, magnesium hydroxide, kaolin and
water. Alternatively, the pharmaceutical composition can be
formulated with a suitable lotion or cream containing the active
compound suspended or dissolved in a carrier. Suitable carriers
include, but are not limited to, mineral oil, sorbitan
monostearate, polysorbate 60, cetyl esters, wax, cetyl alcohol,
2-octyldodecanol, benzyl alcohol and water. The pharmaceutical
compositions of this invention may also be topically applied to the
lower intestinal tract by rectal suppository formulation or in a
suitable enema formulation. Topically-transdermal patches are also
included in this invention.
[0110] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with our without a preservative
such as benzylalkonium chloride. Alternatively, for ophthalmic
uses, the pharmaceutical compositions may be formulated in an
ointment such as petrolatum.
[0111] The pharmaceutical compositions of this invention may be
administered by nasal aerosol or inhalation. Such compositions are
prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other solubilizing or dispersing agents known in the
art.
[0112] Dosage levels of between about 0.01 and about 100 mg/kg body
weight per day, preferably between about 0.5 and about 75 mg/kg
body weight per day of the protease inhibitor compounds described
herein are useful in a monotherapy for the prevention and treatment
of antiviral, particularly anti-HCV mediated disease. Typically,
the pharmaceutical compositions of this invention will be
administered from about 1 to about 5 times per day or
alternatively, as a continuous infusion. Such administration can be
used as a chronic or acute therapy. The amount of active ingredient
that may be combined with the carrier materials to produce a single
dosage form will vary depending upon the host treated and the
particular mode of administration. A typical preparation will
contain from about 5% to about 95% active compound (w/w).
Preferably, such preparations contain from about 20% to about 80%
active compound.
[0113] When the compositions of this invention comprise a
combination of a compound of formula (I) and one or more additional
therapeutic or prophylactic agents, both the compound and the
additional agent should be present at dosage levels of between
about 10 to 100%, and more preferably between about 10 to 80% of
the dosage normally administered in a monotherapy regimen.
[0114] According to one embodiment, the pharmaceutical compositions
of this invention comprise an additional immunomodulatory agent.
Examples of additional immunomodulatory agents include, but are not
limited to, .alpha.-, .beta.-, and .gamma.-interferons.
[0115] According to an alternate embodiment, the pharmaceutical
compositions of this invention may additionally comprise an
anti-viral agent. Examples of anti-viral agents include, ribavirin
and amantadine.
[0116] According to another alternate embodiment, the
pharmaceutical compositions of this invention may additionally
comprise other inhibitors of HCV protease.
[0117] According to yet another alternate embodiment, the
pharmaceutical compositions of this invention may additionally
comprise an inhibitor of other targets in the HCV life cycle, such
as helicase, polymerase, or metalloprotease.
[0118] Upon improvement of a patient's condition, a maintenance
dose of a compound, composition or combination of this invention
may be administered, if necessary. Subsequently, the dosage or
frequency of administration, or both, may be reduced, as a function
of the symptoms, to a level at which the improved condition is
retained when the symptoms have been alleviated to the desired
level, treatment should cease. Patients may, however, require
intermittent treatment on a long-term basis upon any recurrence of
disease symptoms.
[0119] As the skilled artisan will appreciate, lower or higher
doses than those recited above may be required. Specific dosage and
treatment regimens for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health status, sex, diet,
time of administration, rate of excretion, drug combination, the
severity and course of the infection, the patient's disposition to
the infection and the judgment of the treating physician.
[0120] When these compounds or their pharmaceutically acceptable
salts are formulated together with a pharmaceutically acceptable
carrier, the resulting composition may be administered in vivo to
mammals, such as man, to inhibit serine proteases, particularly HCV
NS3 protease or to treat or prevent viral infection, particularly
HCV virus infection. Such treatment may also be achieved using the
compounds of this invention in combination with agents which
include, but are not limited to: immunomodulatory agents, such as
.alpha.-, .beta.-, or .gamma.-interferons; other antiviral agents
such as ribavirin, amantadine; other inhibitors of HCV NS3
protease; inhibitors of other targets in the HCV life cycle such as
helicase, polymerase, metalloprotease, or internal ribosome entry;
or combinations thereof. These additional agents may be combined
with the compounds of this invention to create a single dosage
form. Alternatively these additional agents may be separately
administered to a mammal as part of a multiple dosage form.
[0121] Accordingly, another embodiment of this invention provides
methods of inhibiting serine protease activity in mammals by
administering a compound of the formula (I), wherein the
substituents are as defined above. Preferably, the serine protease
is HCV NS3.
[0122] In an alternate embodiment, the invention provides methods
of inhibiting HCV or HCV NS3 activity in a mammal comprising the
step of administering to said mammal, a compound of formula (I),
wherein the substituents are as defined above.
[0123] In an alternate embodiment, this invention provides methods
of decreasing serine protease activity in a mammal comprising the
step of administrating to said mammal any of the pharmaceutical
compositions and combinations described above. If the
pharmaceutical composition comprises only a compound of this
invention as the active component, such methods may additionally
comprise the step of administering to said mammal an agent selected
from an immunomodulatory agent, an antiviral agent, a HCV protease
inhibitor, or an inhibitor of other targets in the HCV life cycle.
Such additional agent may be administered to the mammal prior to,
concurrently with, or following the administration of the HCV
inhibitor composition.
[0124] In a preferred embodiment, these methods are useful in
decreasing HCV NS3 protease activity in a mammal. If the
pharmaceutical composition comprises only a compound of this
invention as the active component, such methods may additionally
comprise the step of administering to said mammal an agent selected
from an immunomodulatory agent, an antiviral agent, a HCV protease
inhibitor, or an inhibitor of other targets in the HCV life cycle
such as helicase, polymerase, or metalloprotease. Such additional
agent may be administered to the mammal prior to, concurrently
with, or following the administration of the compositions of this
invention.
[0125] In an alternate preferred embodiment, these methods are
useful for inhibiting viral replication in a mammal. Such methods
are useful in treating or preventing, for example, viral diseases,
such as HCV. If the pharmaceutical composition comprises only a
compound of this invention as the active component, such methods
may additionally comprise the step of administering to said mammal
an agent selected from an immunomodulatory agent, an antiviral
agent, a HCV protease inhibitor, or an inhibitor of other targets
in the HCV life cycle. Such additional agent may be administered to
the mammal prior to, concurrently with, or following the
administration of the composition according to this invention.
[0126] The compounds set forth herein may also be used as
laboratory reagents. The compounds of this invention may also be
used to treat or prevent viral contamination of materials and
therefore reduce the risk of viral infection of laboratory or
medical personnel or patients who come in contact with such
materials. These materials include, but are not limited to,
biological materials, such as blood, tissue, etc; surgical
instruments and garments; laboratory instruments and garments; and
blood collection apparatuses and materials.
[0127] In order that this invention be more fully understood, the
following examples are set forth. These examples are for the
purpose of illustration only and are not to be construed as
limiting the scope of the invention in any way.
[0128] General Materials and Methods
[0129] Compounds 101 through 109 were prepared using the generic
synthesis scheme 1, depicted below. Synthesis of specific compounds
are described in the following examples.
[0130] The correct (M+H).sup.+ and/or (M+Na).sup.+ molecular ions
for all compounds as set forth in Table 2 were obtained by either
matrix-assisted laser desorption mass spectrometry (Kratos MALDI I)
or by electro spray mass spectrometry (MICROMASS Quatro II).
[0131] Numerous amino acids for use in the synthesis of peptidyl
and peptidomimetic compounds of this invention may be purchased
commercially from, for instance, Sigma Chemical Company or Bachem
Feinchemikalien AG (Switzerland). Amino acids that are not
commercially available can be made by known synthetic routes
("Kinetic Resolution of Unnatural and Rarely Occurring Amino Acids:
Enantioselective Hydrolysis of N-Acyl Amino Acids Catalyzed by
Acylase I", Chenault, H. K. et. al., J. Am. Chem. Soc. 111,
6354-6364 (1989) and references cited therein; "Synthesis of
.beta.-.gamma.-Unsaturated Amino Acids by the Strecker Reaction,
Greenlee, W. J., J. Org. Chem. 49, 2632-2634 (1984); "Recent
Stereoselective Synthetic Approaches to Beta-amino Acids", Cole, D.
Tetrahedron 50: 9517 (1994); "The Chemistry of Cyclic Alpha Imino
Acids", Mauger, A. B; Volume 4 of "Chemistry and Biochemistry of
Amino Acids, Peptides, and Proteins", Weinstein, B. editor, Marcel
Dekker (1977); "Recent Progress in the Synthesis and Reactions of
Substituted Piperidines", Org. Prep. Procedure Int. 24, 585-621
(1992), all of which are incorporated herein by reference).
[0132] Certain compounds of formula (I) may be synthesized from
amino acids by procedures which are well known in the art of
peptide and organic chemical synthesis. Examples of such syntheses
are generally set forth in Bodanszky and Bodanszky, "The Practice
of Peptide Synthesis", Springer-Verlag, Berlin, Germany (1984),
"The Peptides", Gross and Meinhofer, eds.; Academic Press, 1979,
Vols. I-III, and Stewart, J. M. and Young, J. D., "Solid Phase
Peptide Synthesis, Second Edition", Pierce Chemical Company,
Rockford, Ill. (1984); and "Recent Advances in the Generation of
Molecular Diversity", Moos, W. H., Green, G. D. and Pavia, M. R. in
"Annual Reports in Medicinal Chemistry, Vol. 28" pp. 315-324;
Bristol, J. A., ed.; Academic Press, San Diego, Calif. (1993), all
of which are incorporated herein by reference.
[0133] Typically, for solution phase synthesis of peptides, the
.alpha.-amine of the amino acid to be coupled is protected by a
urethane such as Boc, Cbz, Fmoc or Alloc while the free carboxyl is
activated by reaction with a carbodiimide such as DCC, EDC, or DIC,
optionally in the presence of a catalyst such as HOBT, HOAt, HOSu,
or DMAP. Other methods, which proceed through the intermediacy of
activated esters, acid halides, enzyme-activated amino acids and
anhydrides including phosphonium reagents such as BOP, Py-BOP,
N-carboxy-anhydrides, symmetrical anhydrides, mixed carbonic
anhydrides, carbonic-phosphinic and carbonic-phosphoric anhydrides,
are also suitable. After the peptide has been formed, protecting
groups may be removed by methods described in the references listed
above, such as by hydrogenation in the presence of a palladium,
platinum or rhodium catalyst, treatment with sodium in liquid
ammonia, hydrochloric, hydrofluoric, hydrobromic, formic,
trifluoromethanesulfonic, or trifluoroacetic acid, secondary
amines, fluoride ion, trimethylsilyl halides including bromide and
iodide, or alkali. Automation of the synthetic process, using
techniques such as those set forth above, can be accomplished by
use of commercially available instrumentation, including but not
limited to the Advanced Chemtech 357 FBS and 496 MOS; Tecan
CombiTec, and Applied Biosystems 433A among others. Specific
application of these methods and their equivalents, depending upon
the target compound, will be apparent to those skilled in the art.
Modifications of chemical processes and choice of instrumentation
is within the skill of the ordinary practitioner.
[0134] Although some of the schemes depicted below indicate
particular stereochemistry for certain groups, it should be
apparent to those of skill in the art that the synthesis schemes
may be modified to allow for the use of those certain groups having
the opposite stereochemistry. Therefore, the indication of
stereochemistry in these scheme is not intended to limit the
depicted synthesis to any particular stereochemistry of any
intermediate or final product. 2324
EXAMPLE 1
Synthesis of Compound 101
[0135] Synthesis of 2. To a solution of Boc-5-hydroxyanthranilic
acid (1) (1.5 g, 5.8 mmol) in 45 mL of anhydrous THF at room
temperature was added portionwise, sodium hydride (581 mg, 14.5
mmol). The mixture was stirred and heated to reflux for 2 hours and
cooled to room temperature. Benzyl bromide (691 mL, 5.8 mmol) was
then added and the reaction mixture was heated to reflux for 1.5
hours, cooled to room temperature and stirred overnight. The
reaction was quenched with ice-water (40 mL) and carefully
acidified to pH 3 with HCl 2N. Extraction with ethyl acetate (100
mL) followed by a water (50 mL) and a brine (40 mL) wash gave,
after drying over Na.sub.2SO.sub.4 and concentration in vacuo, a
solid residue. Flash chromatography of the crude mixture (10%
methanol-90% dichloromethane) provided 1.42 g (70%) of the desired
product 2.
[0136] Synthesis of 4. 4-Methyl Benzhydrylamine resin (1.05 mmol/g,
20.0 g) was placed in a sintered glass funnel and washed with
dimethylformamide (3.times.75 mL), 10% (v/v) diisopropylethylamine
(DIEA) in dimethylformamide (2.times.75 mL) and finally with
dimethylformamide (4.times.75 mL). Sufficient dimethylformamide was
added to the resin to obtain a slurry followed by: 25
[0137] (8.0 g, 20.8 mmol, prepared from (2S)
2-(t-Butyloxycarbonylamino)-b- utyraldehyde according to A. M.
Murphy et. al. J. Am. Chem. Soc., 114, 3156-3157 (1992)),
1-hydroxybenzotriazole hydrate (HOBT.H.sub.2O; 3.22 g, 21.0 mmol),
O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HBTU; 8.0 g, 21.0 mmol), and DIEA (11.0 mL, 63 mmol). The reaction
mixture was agitated overnight at room temperature using a wrist
arm shaker. The resin was isolated on a sintered glass funnel by
suction filtration and washed with dimethylformamide (3.times.75
mL). Unreacted amine groups were then capped by reacting the resin
with 20% (v/v) acetic anhydride/dimethylformamide (2.times.50 mL)
directly in the funnel (10 min/wash). The resin was washed with
dimethylformamide (3.times.75 mL) and dichloromethane (3.times.75
mL) prior to drying overnight in vacuo to yield an intermediate
resin (26.3 g, 81% yield).
[0138] The t-Boc protecting group was removed from the intermediate
resin using the Advanced ChemTech 396 Multiple Peptide synthesizer
by the following procedure. The intermediate resin (0.05 mmol) was
swelled by washing with dichloromethane (3.times.1 mL) followed by
cleavage of the t-Boc protecting group with 50% (v/v)
TFA/dichloromethane (1.0 mL) for 10 minutes (with shaking) followed
by fresh reagent (1 mL) for 30 minutes. The resin was then washed
with dichloromethane (3.times.1 ml), followed by DMF (3.times.1
mL), then 10% DIEA/dimethylformamide (v/v) (2.times.1 mL), and
finally with N-methypyrrolidone (3.times.1 mL) to yield resin
4.
[0139] Synthesis of 5. Resin 4 (554 mg, 0.36 mmol) was placed in a
Nalgene syringe equipped with a filter at the bottom and coupled to
2 (251 mg, 0.72 mmol) with HOBt (110 mg, 0.72 mmol), HBTU (273 mg,
0.72 mmol) and DIEA (376 mL, 2.16 mmol) in 4 mL of NMP (with
shaking) for 72 hours. The solvent was removed by suction and the
resin washed sequentially with NMP (3.times.4 mL) and
dichloromethane (3.times.4 mL) to provide resin 5.
[0140] Synthesis of 6. The t-Boc protecting group was removed from
the resin 5 (in the Nalgene syringe) with 50% (v/v)
TFA/dichloromethane (6 mL) for 20 min. (with shaking). The resin
was washed with dichloromethane (3.times.5 mL), followed by 10%
DIEA/dichloromethane (3.times.5 mL) and finally with
dichloromethane (3.times.5 mL). To the resulting resin was added
Fmoc-valine-N-carboxy anhydride (526 mg, 1.44 mmol) and 6 mL of
NMP. The reaction mixture was agitated for 72 hours at room
temperature and the solvent was removed by suction. The resin 6 was
washed sequentially with NMP (3.times.5 mL) and dichloromethane
(3.times.5 mL) and used directly for the next step.
[0141] Synthesis of 8. This compound was prepared from resin 6 (0.1
mmol) using an Applied Biosystems Model 433A Peptide synthesizer.
N.sup..alpha.-Fmoc-protected amino acid and pyrazine-2-carboxylic
acid were added sequentially to resin 6 with standard coupling
cycles using HBTU with HOBt as coupling agents in NMP to yield
resin 8.
[0142] Syntheses of Compound 101. The aldehyde was cleaved from the
resin 8 by treatment with 10 mL of a solution of THF/30%
formalin/AcOH/0.1N HCl 5:1:1:1 (v:v:v:v) for 1 hour at room
temperature. After washing the resin with cleavage reagent (5 mL),
the combined filtrates were diluted with water (15 mL) and 5 mL of
that mixture was purified by RP-HPLC with a Waters DeltaPak 300 A
C18 column (15 m, 30.times.300 mm) eluting with a linear
acetonitrile gradient containing 0.1% TFA (v/v) over 45 min. at 20
mL/min. Fractions containing the desired product were pooled and
lyophilized to provide 101. M+H=617.3.
EXAMPLE 2
Synthesis of Compound 102
[0143] Intermediates 1 through 4 were prepared as in Example 1.
[0144] Synthesis of 5. Resin 4 (W.dbd.CF.sub.3) (1.2 g, 0.384 mmol)
was placed in a Nalgene syringe equipped with a filter at the
bottom and coupled to 2 (267 mg, 0.768 mmol) with HOBt (118 mg,
0.768 mmol), HBTU (291 mg, 0.768 mmol) and DIEA (401 mL, 2.3 mmol)
in 4 mL of NMP (with shaking) for 72 hours. The solvent was removed
by suction and the resin washed sequentially with NMP (3.times.4
mL) and dichloromethane (3.times.4 mL) to provide resin 5.
[0145] Synthesis of 6. The t-Boc protecting group was removed from
the resin 5 (in the Nalgene syringe) with 50% (v/v)
TFA/dichloromethane (6 mL) for 20 minutes (with shaking). The resin
was washed with dichloromethane (3.times.5 mL), followed by 10%
DIEA/dichloromethane (3.times.5 mL) and finally with
dichloromethane (3.times.5 mL). To the resulting resin was added
Fmoc-valine-N-carboxy anhydride (562 mg, 1.54 mmol) and 8 mL of
NMP. The reaction mixture was agitated for 72 hours at room
temperature and the solvent was removed by suction. The resin 6 was
washed sequentially with NMP (3.times.6 mL) and dichloromethane
(3.times.6 mL) and used directly for the next step.
[0146] Synthesis of 8. This compound was prepared from resin 6 (0.1
mmol) using an Applied Biosystems Model 433A Peptide synthesizer.
N.sub.a-Fmoc-protected amino acid and pyrazine-2-carboxylic acid
were added sequentially to resin 6 with standard coupling cycles
using HBTU with HOBt as coupling agents in NMP to yield resin
8.
[0147] Syntheses of 102. The aldehyde was cleaved from the resin 8
by treatment with 10 mL of a solution of THF/30% formalin/AcOH/0.1N
HCl 5:1:1:1 (v:v:v:v) for 1 hour at room temperature. After washing
the resin with cleavage reagent (5 mL), the combined filtrates were
diluted with water (15 ml) and 5 mL of that mixture was purified by
RP-HPLC with a Waters DeltaPak 300 A C18 column (15 m, 30.times.300
mm) eluting with a linear acetonitrile gradient containing 0.1% TFA
(v/v) over 45 min. at 20 mL/min. Fraction containing the desired
product were pooled and lyophilized to provide 102. M+H 671.2
EXAMPLE 3
Synthesis of Compound 103
[0148] Synthesis of 3 (R.sub.2.dbd.Me). To a solution of 2 (350 mg,
0.001 mol) in 10 mL of anhydrous DMF at room temperature was added,
portionwise, NaH (100 mg, 0.0025 mol). The mixture was stirred for
40 minutes, then methyl iodide (0.175 mL, 0.0028 mol) was added and
the reaction mixture was stirred overnight. The reaction was
quenched with water and extracted with ethyl acetate. The organic
phase was dried (Na.sub.2SO.sub.4) and concentrated in vacuo to
provide 335 mg (89%) of an oil that was used directly for the next
step.
[0149] To a solution of the above oil (335 mg, 0.0089 mol) in 8 mL
of methanol room temperature was added 2.2 mL of 2N NaOH. The
reaction was stirred overnight at room temperature after which the
methanol was removed under vacuum. The resulting aqueous phase was
diluted with water then slowly acidified to pH 3 with 1N HCl. The
aqueous phase was extracted with ethyl acetate and the organic
phase dried (Na.sub.2SO.sub.4) and concentrated in vacuo to give
300 mg (93%) of 3 that was used directly for the next step.
[0150] Synthesis of 6. The t-Boc protecting group was removed from
the resin 5 (in the Nalgene syringe) with 50% (v/v)
TFA/dichloromethane (6 mL) for 20 min. (with shaking). The resin
was washed with dichloromethane (3.times.5 mL), followed by 10%
DIEA/dichloromethane (3.times.5 mL) and finally with
dichloromethane (3.times.5 mL). To the resulting resin was added
Fmoc-valine-N-carboxy anhydride (606 mg, 1.66 mmol) and 10 mL of
NMP. The reaction mixture was agitated for 72 hours at room
temperature and the solvent was removed by suction. The resin 6 was
washed sequentially with NMP (3.times.6 mL) and dichloromethane
(3.times.6 mL) and used directly for the next step.
[0151] Synthesis of 8. This compound was prepared from resin 6 (0.1
mmol) using an Applied Biosystems Model 433A Peptide synthesizer.
N.sub.a-Fmoc-protected amino acid and pyrazine-2-carboxylic acid
were added sequentially to resin 6 with standard coupling cycles
using HBTU with HOBt as coupling agents in NMP to yield resin
8.
[0152] Syntheses of 103. The aldehyde was cleaved from the resin 8
by treatment with 10 mL of a solution of THF/30% formalin/AcOH/0.1N
HCl 5:1:1:1 (v:v:v:v) for 1 hour at room temperature. After washing
the resin with cleavage reagent (5 mL), the combined filtrates were
diluted with water (15 mL) and 5 mL of that mixture was purified by
RP-HPLC with a Waters DeltaPak 300 A C18 column (15 m, 30.times.300
mm) eluting with a linear acetonitrile gradient containing 0.1% TFA
(v/v) over 45 min. at 20 mL/min. Fraction containing the desired
product were pooled and lyophilized to provide 103.
[0153] M+H 631.3.
EXAMPLE 4
Synthesis of Compound 104
[0154] Intermediates 1 through 6 were prepared as in Example 1.
[0155] Synthesis of 7. This compound was prepared from resin 6 (0.1
mmol) using an Applied Biosystems Model 433A Peptide synthesizer.
2-Hydroxynaphthoic acid was added to resin 6 with standard coupling
cycles using HBTU with HOBt as coupling agents in NMP to yield
resin 7.
[0156] Syntheses of 104. The aldehyde was cleaved from the resin 7
by treatment with 3 mL of a solution of THF/30% formalin/AcOH/0.1N
HCl 5:1:1:1 (v:v:v:v) for 1 hour at room temperature. After washing
the resin with cleavage reagent (1.5 mL), the combined filtrates
were diluted with water (4.5 mL) and 5 mL of that mixture was
purified by RP-HPLC with a Waters DeltaPak 300 A C18 column (15 m,
30.times.300 mm) eluting with a linear acetonitrile gradient
containing 0.1% TFA (v/v) over 45 min. at 20 mL/min. Fraction
containing the desired product were pooled and lyophilized to
provide 104.
[0157] M+H=582.2.
EXAMPLE 5
Synthesis of Compound 105
[0158] Intermediates 1 through 6 were prepared as in Example 1.
[0159] Synthesis of 7. This compound was prepared from resin 6 (0.1
mmol) using an Applied Biosystems Model 433A Peptide synthesizer.
2-Hydroxy-4-nicotinic acid was added to resin 6 with standard
coupling cycles using HBTU with HOBt as coupling agents in NMP to
yield resin 7.
[0160] Syntheses of 105. The aldehyde was cleaved from the resin 8
by treatment with 3 mL of a solution of THF/30% formalin/AcOH/0.1N
HCl 5:1:1:1 (v:v:v:v) for 1 hour at room temperature. After washing
the resin with cleavage reagent (1.5 mL), the combined filtrates
were diluted with water (4.5 mL) and 5 mL of that mixture was
purified by RP-HPLC with a Waters DeltaPak 300 A C18 column (15 m,
30.times.300 mm) eluting with a linear acetonitrile gradient
containing 0.1% TFA (v/v) over 45 min. at 20 mL/min. Fraction
containing the desired product were pooled and lyophilized to
provide 105.
[0161] M+H=533.2
EXAMPLE 6
Synthesis of Compound 106
[0162] Intermediates 1 through 6 were prepared as in Example 1.
[0163] Synthesis of 7. This compound was prepared from resin 6 (0.1
mmol) using an Applied Biosystems Model 433A Peptide synthesizer.
6-Hydroxynaphthoic acid was added to resin 6 with standard coupling
cycles using HBTU with HOBt as coupling agents in NMP to yield
resin 7.
[0164] Syntheses of 106. The aldehyde was cleaved from the resin 8
by treatment with 3 mL of a solution of THF/30% formalin/AcOH/0.1N
HCl 5:1:1:1 (v:v:v:v) for 1 hour at room temperature. After washing
the resin with cleavage reagent (1.5 mL), the combined filtrates
were diluted with water (4.5 mL) and 5 mL of that mixture was
purified by RP-HPLC with a Waters DeltaPak 300 A C18 column (15 m,
30.times.300 mm) eluting with a linear acetonitrile gradient
containing 0.1% TFA (v/v) over 45 min. at 20 mL/min. Fraction
containing the desired product were pooled and lyophilized to
provide 106.
[0165] M+H=582.2.
EXAMPLE 7
Synthesis of Compound 107
[0166] Synthesis of 2. To a solution of Boc-5-hydroxyanthranilic
acid (1) (1.5 g, 5.8 mmol) in 45 mL of anhydrous THF at room
temperature was added portionwise, sodium hydride (581 mg, 14.5
mmol). The mixture was stirred and heated to reflux for 2 hours and
cooled to room temperature. 3,4-dichloro benzyl bromide (1.39 g,
5.8 mmol) was then added and the reaction mixture was heated to
reflux for 1.5 hours, cooled to room temperature and stirred
overnight. The reaction was quenched with ice-water (40 mL) and
carefully acidified to pH 3 with 2N HCl. Extraction with ethyl
acetate (100 mL) followed by a water (50 mL) and a brine (40 mL)
wash gave, after drying (Na.sub.2SO.sub.4) and concentration in
vacuo, a solid residue. Subjection of the crude mixture to flash
chromatography (10% methanol-90% dichloromethane) provided 1.37 g
(57%) of the desired product 2.
[0167] Synthesis of 5. Resin 4 (1.0 g, 0.65 mmol) (from Example 1)
was placed in a Nalgene syringe equipped with a filter at the
bottom and coupled to 2 (542 mg, 1.3 mmol) with HOBt (199 mg, 1.3
mmol), HBTU (493 mg, 1.3 mmol) and DIEA (679 mL, 3.9 mmol) in 8 mL
of NMP (with shaking) for 72 hours. The solvent was removed by
suction and the resin washed sequentially with NMP (3.times.8 mL)
and dichloromethane (3.times.8 mL) to provide resin 5.
[0168] Synthesis of 6. The t-Boc protecting group was removed from
the resin 5 (in the Nalgene syringe) with 25% (v/v)
TFA/dichloromethane (10 mL) for 30 minutes (with shaking). The
resin was washed with dichloromethane (3.times.7 mL), followed by
10% DIEA/dichloromethane (3.times.7 mL) and finally with
dichloromethane (3.times.7 mL). To the resulting resin was added
Fmoc-valine-N-carboxy anhydride (950 mg, 2.6 mmol) and 8 mL of NMP.
The reaction mixture was agitated for 72 hours at room temperature
and the solvent was removed by suction. The resin 6 was washed
sequentially with NMP (3.times.7 mL) and dichloromethane (3.times.7
mL) and used directly for the next step.
[0169] Synthesis of 8. This compound was prepared from resin 6 (0.1
mmol) using an Applied Biosystems Model 433A Peptide synthesizer.
N.sup..alpha.-Fmoc-protected amino acid and pyrazine-2-carboxylic
acid were added sequentially to resin 6 with standard coupling
cycles using HBTU with HOBt as coupling agents in NMP to yield
resin 8.
[0170] Syntheses of 107. The aldehyde was cleaved from the resin 8
by treatment with 10 mL of a solution of THF/30% formalin/AcOH/0.1N
HCl 5:1:1:1 (v:v:v:v) for 1 hour at room temperature. After washing
the resin with cleavage reagent (5 mL), the combined filtrates were
diluted with water (15 mL) and 5 mL of that mixture was purified by
RP-HPLC with a Waters DeltaPak 300 A C18 column (15 m, 30.times.300
mm) eluting with a linear acetonitrile gradient containing 0.1% TFA
(v/v) over 45 minutes at 20 mL/minute. Fraction containing the
desired product were pooled and lyophilized to provide 107.
[0171] M+H=685.1.
EXAMPLE 8
Synthesis of Compound 108
[0172] Synthesis of 5. Resin 4 (640 mg, 0.414 mmol) (from Example
1) was placed in a Nalgene syringe equipped with a filter at the
bottom and coupled to 1 (wherein X, Y and Z are hydrogen; 200 mg,
0.828 mmol) with HOBt (126 mg, 0.828 mmol), HBTU (314 mg, 0.828
mmol) and DIEA (433 mL, 3.9 mmol) in 6 mL of NMP (with shaking) for
24 h. The solvent was removed by suction and the resin washed
sequentially with NMP (3.times.5 mL) and dichloromethane (3.times.5
mL) to provide resin 5.
[0173] Synthesis of 6. The t-Boc protecting group was removed from
the resin 5 (in the Nalgene syringe) with 50% (v/v)
TFA/dichloromethane (6 mL) for 20 minutes (with shaking). The resin
was washed with dichloromethane (3.times.6 mL), followed by 10%
DIEA/dichloromethane (3.times.6 mL) and finally with
dichloromethane (3.times.6 mL). To the resulting resin was added
Fmoc-valine-N-carboxy anhydride (605 mg, 1.66 mmol) and 10 mL of
NMP. The reaction mixture was agitated for 72 hours at room
temperature and the solvent was removed by suction. The resin 6 was
washed sequentially with NMP (3.times.7 mL) and dichloromethane
(3.times.7 mL) and used directly for the next step.
[0174] Synthesis of 8. This compound was prepared from resin 6 (0.1
mmol) using an Applied Biosystems Model 433A Peptide synthesizer.
N.sup..alpha.-Fmoc-protected amino acid and pyrazine-2-carboxylic
acid were added sequentially to resin 6 with standard coupling
cycles using HBTU with HOBt as coupling agents in NMP to yield
resin 8.
[0175] Syntheses of 108. The aldehyde was cleaved from the resin 8
by treatment with 10 mL of a solution of THF/30% formalin/AcOH/0.1N
HCl 5:1:1:1 (v:v:v:v) for 1 hour at room temperature. After washing
the resin with cleavage reagent (5 mL), the combined filtrates were
diluted with water (15 mL) and 5 mL of that mixture was purified by
RP-HPLC with a Waters DeltaPak 300 A C18 column (15 m, 30.times.300
mm) eluting with a linear acetonitrile gradient containing 0.1% TFA
(v/v) over 45 minutes at 20 mL/minute. Fraction containing the
desired product were pooled and lyophilized to provide 108.
[0176] M+H=511.3.
EXAMPLE 9
Synthesis of Compound 109
[0177] Synthesis of 5. Resin 4 (640 mg, 0.414 mmol) (from Example
1) was placed in a Nalgene syringe equipped with a filter at the
bottom and coupled to 1 (238 mg, 0.828 mmol) with HOBt (126 mg,
0.828 mmol), HBTU (314 mg, 0.828 mmol) and DIEA (433 mL, 3.9 mmol)
in 6 mL of NMP (with shaking) for 24 hours. The solvent was removed
by suction and the resin washed sequentially with NMP (3.times.5
mL) and dichloromethane (3.times.5 mL) to provide resin 5.
[0178] Synthesis of 6. The t-Boc protecting group was removed from
the resin 5 (in the Nalgene syringe) with 50% (v/v)
TFA/dichloromethane (6 mL) for 20 minutes (with shaking). The resin
was washed with dichloromethane (3.times.6 mL), followed by 10%
DIEA/dichloromethane (3.times.6 mL) and finally with
dichloromethane (3.times.6 mL). To the resulting resin was added
Fmoc-valine-N-carboxy anhydride (605 mg, 1.66 mmol) and 10 mL of
NMP. The reaction mixture was agitated for 72 hours at room
temperature and the solvent was removed by suction. The resin 6 was
washed sequentially with NMP (3.times.7 mL) and dichloromethane
(3.times.7 mL) and used directly for the next step.
[0179] Synthesis of 8. This compound was prepared from resin 6 (0.1
mmol) using an Applied Biosystems Model 433A Peptide synthesizer.
N.sup..alpha.-Fmoc-protected amino acid and pyrazine-2-carboxylic
acid were added sequentially to resin 6 with standard coupling
cycles using HBTU with HOBt as coupling agents in NMP to yield
resin 8.
[0180] Syntheses of 109. The aldehyde was cleaved from the resin 8
by treatment with 10 mL of a solution of THF/30% formalin/AcOH/0.1N
HCl 5:1:1:1 (v:v:v:v) for 1 hour at room temperature. After washing
the resin with cleavage reagent (5 mL), the combined filtrates were
diluted with water (15 mL) and 5 mL of that mixture was purified by
RP-HPLC with a Waters DeltaPak 300 A C18 column (15 m, 30.times.300
mm) eluting with a linear acetonitrile gradient containing 0.1% TFA
(v/v) over 45 minutes at 20 mL/minute. Fraction containing the
desired product were pooled and lyophilized to provide 109.
[0181] M+H=561.3.
EXAMPLE 10
Synthesis of Para-Hydroxyanthranilic Acid Derivatives
[0182] The synthesis of compounds of the formulae: 26
[0183] is achieved using the steps set forth in Scheme 1 to produce
9 or 10 and intermediate 1 containing a hydroxy group at Y and
hydrogens at X and Z.
[0184] Synthesis of 1. This compound is prepared in three steps
from 2-nitro-4-aminobenzoic acid. Diazotization of
2-Nitro-4-aminobenzoic with, for example, sodium nitrite in 20%
sulfuric acid provides 2-nitro-4-hydroxybenzoic acid. Reduction of
2-nitro-4-hydroxybenzoic acid with, for example, tin in
concentrated hydrochloric acid provide the desired
p-hydroxyanthranilic acid. The amino group is then protected, for
example, by treatment with, for example Boc anhydride in
dichloromethane with diisopropylamine to give 1.
EXAMPLE 11
[0185] Scheme 2, set forth below, depicts the synthetic route to
obtain alkyl-substituted anthranilic acid derivative compounds of
this invention. 2728
[0186] Synthesis of 12. This compound is prepared in three steps
from the coupling of 11 (wherein X is Br; Y and Z are H) with
styrene in a Heck reaction using palladium acetate and
triethylamine. After deprotection and hydrogenation of the double
bond with palladium on carbon followed by protection of the amino
group as a carbamate with Boc anhydride in dichloromethane and DIEA
compound 12 is obtained.
[0187] Para-alkyl-substituted anthranilic acid derivatives are
similarly prepared using 11 wherein Y is Br and X and Z are H,
similar to the method described in this example.
EXAMPLE 12
[0188] Carbazate-containing compounds of this invention (compounds
27 and 28) are produced according to Scheme 3, depicted below:
2930
[0189] Synthesis of 22. This compound could be prepared for
example, in two steps from 4-hydroxyphthaloic acid (20) by
alkylation with, for example, an excess of benzyl bromide with a
base such as potassium carbonate in dimethylformamide. After
saponification of the diester 21 with, for example, potassium
hydroxide in ethanol provide compound 22.
[0190] Synthesis of 23 and 24. These compounds could be prepared by
coupling 22 with pyrazine-Val-isopropylcarbazate or with
arylcarboxylic acid (Cap) isopropylcarbazate with, for example,
HOBt, HBTU and DIEA in N-methylpyrrolidinone as previously
described in Example 1 to provide 23 or 24.
[0191] Synthesis of 25 and 26. These resins could be prepared from
resin 4 according to Scheme 3 and experimentals previously
described in Example 1.
[0192] Syntheses of 27 and 28. These compounds could be prepared
according to Scheme 2 and experimental previously described in
Examples 1 and 2.
EXAMPLE 13
[0193] 1,4-diketone-containing compounds of this invention
(compounds 32 and 33) are produced according to Scheme 4, depicted
below: 31
[0194] Synthesis of 30. This compound is prepared from 29 by
deprotonation/halogen-metal exchange with a base, such as butyl
lithium in tetrahydrofuran, followed by addition of
Boc-Val-C(O)N(OMe)Me (Weinreb amide).
[0195] Syntheses of 32 and 33. These compounds are prepared
according to Scheme 4 and experimentals previously described in
Examples 1 and 2.
EXAMPLE 14
[0196] 1,2-diketone-containing compounds of this invention
(compounds 39 and 40) are produced according to Scheme 5, depicted
below: 3233
[0197] Synthesis of 35. This compound is prepared from phosphonium
bromide 34 by treatment with a base such as potassium
hexamethyldisilazide in tetrahydrofuran, followed by addition of
Boc-Val-H.
[0198] Synthesis of 36. This compound is prepared from olefin 35 by
an osmylation reaction with, for example, osmium tetraoxide and
oxidation of the resulting diol with, for example, Dess-Martin
periodinane. This step is followed by a saponification reaction
with, for example, sodium hydroxide in methanol.
[0199] Syntheses of 39 and 40. These compounds are prepared
according to Scheme 5 and experimentals previously described in
Examples 1 and 2.
EXAMPLE 15
[0200] Sulfone-containing compounds of this invention (compounds 47
and 48) are prepared according to Scheme 6, below: 3435
[0201] Synthesis of 42. This compound is prepared from bromoester
41 by treatment with magnesium in ether followed by addition of
sulfur dioxide to the mixture.
[0202] Synthesis of 43. This compound is prepared from a mixture of
sulfinate salt 42 and benzylcarbamate in water by treatment with
isobutyraldehyde in methanol. This step is followed by addition of
formic acid with heating.
[0203] Synthesis of 43a. This compound is prepared by deprotection
of the amino group of compound 43 with hydrogen over palladium on
carbon. This is followed by saponification of the ester with 5% TFA
in dichloromethane, followed by treatment with Fmoc-OSU in
dioxane-water.
[0204] Syntheses of 47 and 48. These compounds are prepared
according to Scheme 6 and experimentals previously described in
Examples 1 and 2.
EXAMPLE 16
[0205] Sulfonyl-carbazate-containing compound of this invention
(compounds 54 and 55) are prepared according to Scheme 7, depicted
below. 3637
[0206] Synthesis of 49. This compound is prepared from bromoester
41 by treatment with magnesium in ether, followed by addition of
sulfur dioxide to the mixture and subsequent treatment with
sulfuryl chloride in dichloromethane at 0.degree. C.
[0207] Synthesis of 50. This compound is prepared by coupling
sulfinate 49 and Fmoc-isopropylcarbazate in the presence of DIEA in
dichloromethane, followed by treatment with 5% TFA in
dichloromethane.
[0208] Syntheses of 54 and 55. These compounds are prepared
according to Scheme 7 and experimentals previously described in
Examples 1 and 2.
EXAMPLE 17
[0209] Sulfonamide-containing compounds of this invention
(compounds 62 and 63) are prepared according to Scheme 8, depicted
below. 3839
[0210] Synthesis of 56a. This compound is prepared from ester 56 by
treatment with lithium diisopropylamide in THF at -78.degree. C.
followed by addition of sulfur dioxide to the mixture and
subsequent treatment with sulfuryl chloride in dichloromethane at
0.degree. C.
[0211] Synthesis of 57a. This compound is prepared by coupling
sulfonyl chloride 56a and anthranilate 57 in the presence of DIEA
in dichloromethane.
[0212] Synthesis of 58. This compound is prepared by saponification
of 57a with potassium hydroxide in ethanol followed by a Curtius
rearrangement using, for example, oxalyl chloride with sodium azide
in acetonitrile. Protection of the amino functional group with
Fmoc-OSU in dioxane-water, followed by hydrolysis of the t-butyl
ester with 5% TFA in dichloromethane provide compound 58.
[0213] Syntheses of 62 and 63. These compounds could be prepared
according to Scheme 8 and experimentals previously described in
Examples 1 and 2.
[0214] It will be readily apparent to those of skill in the art
that other compounds of this invention may be synthesized using
variations of Schemes 1 through 8 and/or the appropriate variants
of the reagents depicted in those schemes and set forth in Examples
1 through 17.
EXAMPLE 18
Inhibition of HCV NS3 Serine Protease
[0215] Insofar as compounds of formula (I) are able to inhibit NS3
serine protease, they are of evident clinical utility for the
treatment of viral diseases, including HCV. These tests are
predictive of the compounds ability to inhibit HCV in vivo.
[0216] Peptides and Assays.
[0217] Peptides EDVVabuCSMSY (Abu designates--aminobutyric acid),
DEMEECSQHLPYI, ECTTPCSGSWLRD and EDVV AbuC-p-nitroanilide was
purchased from AnaSpec Inc. (San Jose, Calif.).
[0218] Peptide content of purified, lyophilized peptides and
in-house peptides was determined by quantitative nitrogen analysis
and the appropriate values were used in preparing stock peptide
solutions (Galbreath). pKa determinations were determined by
Robertson Microlit Laboratories, Inc. (Madison, N.J.).
[0219] HPLC cleavage assays were performed using 25 nM to 3.0 .mu.M
enzyme in 100 .mu.L volumes at 30 C containing 50 mM HEPES-KOH (pH
7.8), 100 mM NaCl, 20% glycerol, 5 mM DTT and the appropriate
amount of substrate (in DMSO), with or without NS4A peptide, such
that the final concentration of DMSO did not exceed 4%. Separate
control experiments verified that this percentage of DMSO did not
effect enzymatic activity. Cleavage reactions were quenched by the
addition of an equal volume of a mixture of 10% TFA: acetonitrile
(1:1) and activity was assessed on a reversed phase HPLC column
(Rainin C18 Microsorb-MV, 5 mm, 4.6.times.250 mm; 0-50%
acetonitrile, 0.1% TFA @ 3.33% min) using a Hewlett Packard 1050
instrument with auto-injection and diode array detection at 210 nm
and 280 nm (where appropriate). Peptide elution fragments were
collected and identified by mass spectrometry and N-terminal
sequence analysis. Fragment identity and concentration was further
verified by authentic, synthesized products. Initial rates of
cleavage were determined at <20% substrate conversion and
catalytic parameters were determined assuming Michaelis-Menten
kinetics using the MultiFit program (Day Computing, Cambridge,
Mass.).
[0220] Spectrophotometric assays were run in a 96-well microtiter
plate at 30 C, using a SpectraMax 250 reader (Molecular Devices,
Sunnyvale, Calif.) with kinetic capability. Cleavage of EDVV
AbuC-p-nitroanilide (5A-pNA) substrate was performed with or
without NS4A in the same buffer used for HPLC assays at 30 C, and
pNA release was monitored at 405 nm. The extinction coefficient of
p-nitroaniline is independent of pH at values of 5.5. and above
[Tuppy, H., et al., Hoppe-Seyler's Z. Physiol. Chem., 329, pp.
278-288 (1962)]; Raybuck and Luong, unpublished observations). The
percentage of DMSO did not exceed 4% in these assays.
[0221] Determination of the pH dependence of Vmax, K.sub.m and
V.sub.max/K.sub.m was performed using a series of constant ionic
strength buffers containing 50 mM MES, 25 nM Tris, 25 mM
ethanolamine and 0.1 M NaCl [Morrison, J. F. and Stone, R. F.,
Biochemistry, 27, pp. 5499-5506 (1988)]. The inflection point for
log V data was calculated by nonlinear least squares fit of the
data to the equation.
log v=log[Vmax/(1+H/K.sub.a)]
[0222] [Dixon, M. and Webb, E. C. Enzymes; Academic Press: New
York; Vol., pp. 138-164 (1979)]. The inflection points for log
(V/K) data were calculated by nonlinear least squares fit of the
data to the equation:
log v=log[Vmax/(1+H/K.sub.a+K.sub.b/H)]
[0223] [Dixon, M. and Webb, E. C. Enzymes; Academic Press: New
York; Vol., pp. 138-164 (1979)]. The program KineTic (BioKin Ltd)
was used in both cases.
[0224] Kinetic constants for the rapid equilibrium ordered
bisubstrate reaction were determined from rate vs. [4A], [EDVV
AbuC-pNA] data by non-linear least squares fitting to equation 1
[Morrison, J. F. Biochim. Biophys. Acta., 185, pp. 269-286 (1969)]
as described in the text. K.sub.ii and K.sub.is values for peptidyl
inhibitors were determined from rate vs. [inhibitor], [substrate]
data and fitting to the equation for mixed inhibition:
rate=Vmax[S]/{Km(1+[I]/Kis)+[S](1+[I]/Kii)}
[0225] The commercial program KinetAsyst (State College, Pa.) was
used for both procedures. Ki values were calculated from rate vs.
[inhibitor] plots by a nonlinear least squares fit of the data to
the equation of Morrison for tight binding competitive inhibition
[Morrison, J. F. Biochim. Biophys. Acta., 185, pp. 269-286 (1969)].
The KineTic program (BioKin Ltd) was used for this procedure.
[0226] The results are shown in Table 2. K.sub.i values are
expressed in .mu.M. Category "A" indicates <1 .mu.M inhibition;
category "B" indicates 1-100 .mu.M inhibition; category "C"
indicates >100 .mu.M. The designation "ND" indicates that the
compound was not tested.
3TABLE 2 Enzyme inhibition data for compounds 1-9. Compound K.sub.i
Compound K.sub.i Compound K.sub.i 101 B 104 B 107 B 102 B 105 B 108
ND 103 B 106 B 109 ND
[0227] While we have hereinbefore presented a number of embodiments
of this invention, it is apparent that my basic construction can be
altered to provide other embodiments which utilize the methods of
this invention. Therefore, it will be appreciated that the scope of
this invention is to be defined by the claims appended hereto
rather than the specific embodiments which have been presented
hereinbefore by way of example.
* * * * *