U.S. patent application number 10/934141 was filed with the patent office on 2005-03-17 for novel peptides as ns3-serine protease inhibitors of hepatitis c virus.
This patent application is currently assigned to Dendreon Corporation. Invention is credited to Arasappan, Ashok, Bennett, Frank, Bogen, Stephane L., Brunck, Terence K., Ganguly, Ashit K., Girijavallabhan, Viyyoor Moopil, Jao, Edwin, Kemp, Scott Jeffrey, Levy, Odile Esther, Lim-Wilby, Marguerita, Liu, Yi-Tsung, Lovey, Raymond G., McCormick, Jinping L., Njoroge, F. George, Parekh, Tejal, Pike, Russell E., Pinto, Patrick A., Saksena, Anil K., Wang, Haiyan.
Application Number | 20050059606 10/934141 |
Document ID | / |
Family ID | 22822101 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050059606 |
Kind Code |
A1 |
Saksena, Anil K. ; et
al. |
March 17, 2005 |
Novel peptides as NS3-serine protease inhibitors of hepatitis C
virus
Abstract
The present invention discloses novel peptide compounds which
have HCV protease inhibitory activity as well as methods for
preparing such compounds. In another embodiment, the invention
discloses pharmaceutical compositions comprising such peptides as
well as methods of using them to treat disorders associated with
the HCV protease.
Inventors: |
Saksena, Anil K.; (Upper
Montclair, NJ) ; Girijavallabhan, Viyyoor Moopil;
(Parsippany, NJ) ; Lovey, Raymond G.; (West
Caldwell, NJ) ; Jao, Edwin; (Warren, NJ) ;
Arasappan, Ashok; (Bridgewater, NJ) ; Parekh,
Tejal; (Mountain View, CA) ; Pinto, Patrick A.;
(Morris Plains, NJ) ; Bennett, Frank; (Cranford,
NJ) ; McCormick, Jinping L.; (Edison, NJ) ;
Wang, Haiyan; (Cranbury, NJ) ; Pike, Russell E.;
(Stanhope, NJ) ; Bogen, Stephane L.; (Somerset,
NJ) ; Liu, Yi-Tsung; (Morris Township, NJ) ;
Njoroge, F. George; (Warren, NJ) ; Ganguly, Ashit
K.; (Upper Montclair, NJ) ; Brunck, Terence K.;
(Santa Fe, NM) ; Kemp, Scott Jeffrey; (San Diego,
CA) ; Levy, Odile Esther; (San Diego, CA) ;
Lim-Wilby, Marguerita; (La Jolla, CA) |
Correspondence
Address: |
LERNER, DAVID, LITTENBERG,
KRUMHOLZ & MENTLIK
600 SOUTH AVENUE WEST
WESTFIELD
NJ
07090
US
|
Assignee: |
Dendreon Corporation
San Diego
CA
Schering Corporation
Kenilworth
NJ
|
Family ID: |
22822101 |
Appl. No.: |
10/934141 |
Filed: |
September 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10934141 |
Sep 3, 2004 |
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09909062 |
Jul 19, 2001 |
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6800434 |
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60220109 |
Jul 21, 2000 |
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Current U.S.
Class: |
435/5 ; 435/6.13;
514/20.3; 514/4.3; 530/329 |
Current CPC
Class: |
A61P 31/16 20180101;
A61P 35/00 20180101; A61P 31/14 20180101; C07K 7/06 20130101; A61P
31/12 20180101; A61P 43/00 20180101; A61P 1/16 20180101; A61K 38/00
20130101; C07K 7/02 20130101 |
Class at
Publication: |
514/017 ;
530/329 |
International
Class: |
A61K 038/08; C07K
007/06 |
Claims
What is claimed is:
1. A compound, including enantiomers, stereoisomers, rotomers and
tautomers of said compound, and pharmaceutically acceptable salts,
solvates or derivatives thereof, with said compound having the
general structure shown in Formula I: 288wherein: Z is O, NH or
NR.sup.12; X is alkylsulfonyl, heterocyclylsulfonyl,
heterocyclylalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl,
alkylcarbonyl, heterocyclylcarbonyl, heterocyclylalkylcarbonyl,
arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl,
heterocyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,
alkyaminocarbonyl, heterocyclylaminocarbonyl, arylaminocarbonyl, or
heteroarylaminocarbonyl moiety, with the proviso that X may be
additionally optionally substituted with R.sup.12 or R.sup.13;
X.sup.1 is H; C.sub.1-C.sub.4 straight chain alkyl; C.sub.1-C.sub.4
branched alkyl or; CH.sub.2-aryl (substituted or unsubstituted);
R.sup.12 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl,
heterocyclyl, heterocyclylalkyl, aryl, alkylaryl, arylalkyl,
heteroaryl, alkylheteroaryl, or heteroarylalkyl moiety, with the
proviso that R.sup.12 may be additionally optionally substituted
with R.sup.13. R.sup.13 is hydroxy, alkoxy, aryloxy, thio,
alkylthio, arylthio, amino, alkylamino, arylamino, alkylsulfonyl,
arylsulfonyl, alkylsulfonamido, arylsulfonamido, carboxy,
carbalkoxy, carboxamido, alkoxycarbonylamino, alkoxycarbonyloxy,
alkylureido, arylureido, halogen, cyano, or nitro moiety, with the
proviso that the alkyl, alkoxy, and aryl may be additionally
optionally substituted with moieties independently selected from
R.sup.13. P1a, P1b, P2, P3, P4, P5, and P6 are independently: H;
C.sub.1-C.sub.10 straight or branched chain alkyl; C2-C10 straight
or branched chain alkenyl; C3-C8 cycloalkyl, C3-C8 heterocyclic;
(cycloalkyl)alkyl or (heterocyclyl)alkyl, wherein said cycloalkyl
is made up of 3 to 8 carbon atoms, and zero to 6 oxygen, nitrogen,
sulfur, or phosphorus atoms, and said alkyl is of 1 to 6 carbon
atoms; aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein
said alkyl is of 1 to 6 carbon atoms; wherein said alkyl, alkenyl,
cycloalkyl, heterocyclyl; (cycloalkyl)alkyl and (heterocyclyl)alkyl
moieties may be optionally substituted with R.sup.13, and further
wherein said P1 a and P1 b may optionally be joined to each other
to form a spirocyclic or,.spiroheterocyclic ring, with said
spirocyclic or spiroheterocyclic ring containing zero to six
oxygen, nitrogen, sulfur, or phosphorus atoms, and may be
additionally optionally substituted with R.sup.13; and P1' is H,
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl,
heterocyclyl, heterocyclyl-alkyl, aryl, aryl-alkyl, heteroaryl, or
heteroaryl-alkyl; with the proviso that said P1' may be
additionally optionally substituted with R.sup.13.
2. The compound of claim 1, wherein X is selected from the group
consisting of: 289wherein Alkyl is a C1 to C4 straight or branched
chain, and Aryl is a phenyl or substituted phenyl.
3. The compound of claim 2, wherein X is --CO--CH.sub.3.
4. The compound of claim 2, wherein X is --CO-phenyl.
5. The compound of claim 1, wherein P5 and P6 are the same and are:
--(CH.sub.2).sub.n--C(O)--R.sup.1, where n=14 and R.sup.1 is OH,
O-t-Bu, OR.sup.3, NHR.sup.3, NH-phenyl or NH-trityl, with R.sup.3
being selected from H, C.sub.1-C.sub.4 straight or branched chain
alkyl.
6. The compound of claim 1, wherein P5 and P6 are different and
are: --(CH.sub.2).sub.n--C(O)--R.sup.1, where n=1-4 and R.sup.1 is
OH, O-t-Bu, OR.sup.3, NHR.sup.3, NH-phenyl or NH-trityl, with
R.sup.3 being selected from H, C.sub.1-C.sub.4 straight or branched
chain alkyl.
7. The compound of claim 5, wherein P5 and P6 are
--CH.sub.2--CH.sub.2--C(- O)--O--C(CH.sub.3).sub.3 or
--CH.sub.2--CH.sub.2--C(O)--OH.
8. The compound of claim 6, wherein P5 and P6 are independently
selected from --CH.sub.2--CH.sub.2--C(O)--O--C(CH.sub.3).sub.3 or
--CH.sub.2--CH.sub.2--C(O)--OH.
9. The compound of claim 1, wherein P3 and P4 are the same.
10. The compound of claim 1, wherein P3 and P4 are different.
11. The compound of claim 1, wherein P3 and P4 are independently
selected from the group consisting of: 290
12. The compound of claim 1, wherein P2 is selected from the group
consisting of: 291wherein n is 0, 1, 2 or 3.
13. The compound of claim 1, wherein P1a and P1b are independently
selected from the group consisting of: 292
14. The compound of claim 1, wherein P1' is selected from the group
consisting of: 293
15. The compound of claim 1, wherein Z is NH and X.sup.1 is H.
16. A compound, including enantiomers, stereoisomers, rotomers and
tautomers of said compound, and pharmaceutically acceptable salts
or solvates of said compound having the general structure shown in
Formula II: 294wherein: Z is O, NH or NR.sup.12; X is
alkylsulfonyl, heterocyclylsulfonyl, heterocyclylalkylsulfonyl,
arylsulfonyl, heteroarylsulfonyl, alkylcarbonyl,
heterocyclylcarbonyl, heterocyclylalkylcarbonyl, arylcarbonyl,
heteroarylcarbonyl, alkoxycarbonyl, heterocyclyloxycarbonyl,
aryloxycarbonyl, heteroaryloxycarbonyl, alkyaminocarbonyl,
heterocyclylaminocarbonyl, arylaminocarbonyl, or
heteroarylaminocarbonyl moiety, with the proviso that X may be
additionally optionally substituted with R.sup.12 or R.sup.13;
R.sup.12 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl,
heterocyclyl, heterocyclylalkyl, aryl, alkylaryl, arylalkyl,
heteroaryl, alkylheteroaryl, or heteroarylalkyl moiety, with the
proviso that R.sup.12 may be additionally optionally substituted
with R.sup.13; R.sup.13 is hydroxy, alkoxy, aryloxy, thio,
alkylthio, arylthio, amino, alkylamino, arylamino, alkylsulfonyl,
arylsulfonyl, alkylsulfonamido, arylsulfonamido, carboxy,
carbalkoxy, carboxamido, alkoxycarbonylamino, alkoxycarbonyloxy,
alkylureido, arylureido, halogen, cyano, or nitro moiety, with the
proviso that the alkyl, alkoxy, and aryl may be additionally
optionally substituted with moieties independently selected from
R.sup.13; P1a, P1b, P2, P3, P4, P5, and P6 are independently: H;
C1-C10 straight or branched chain alkyl; C2-C10 straight or
branched chain alkenyl; C3-C8 cycloalkyl, C3-C8 heterocyclic;
(cycloalkyl)alkyl or (heterocyclyl)alkyl, wherein said cycloalkyl
is made up of 3 to 8 carbon atoms, and zero to six oxygen,
nitrogen, sulfur, or phosphorus atoms, and said alkyl is of 1 to 6
carbon atoms; or aryl, heteroaryl, arylalkyl, or heteroarylalkyl,
wherein said alkyl is of 1 to 6 carbon atoms; wherein said alkyl,
alkenyl, cycloalkyl, heterocyclyi, (cycloalkyl)alkyl and
(heterocyclyl)alkyl moieties may be optionally substituted with
R.sup.13 and further wherein said P1 may optionally be a
spirocyclic or spiroheterocyclic ring, with said spirocyclic or
spiroheterocyclic ring containing zero to six oxygen, nitrogen,
sulfur, or phosphorus atoms, and may be additionally optionally
substituted with R.sup.13; and P1' is H, alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkyl-alkyl, heterocyclyl, heterocyclyl-alkyl,
aryl, aryl-alkyl, heteroaryl, or heteroaryl-alkyl; with the proviso
that said P1' may be additionally optionally substituted with
R.sup.13; and 295indicates a cyclic ring structure, with the
proviso that said cyclic ring structure does not contain a carbonyl
group as part of the cyclic ring.
17. The compound of claim 16, wherein said 296indicates a
five-membered ring.
18. The compound of claim 16, wherein said 297indicates a
six-membered ring.
19. The compound of claim 16, wherein X is selected from the group
consisting of: 298wherein Alkyl is a C1 to C4 straight or branched
chain, and Aryl is a phenyl or substituted phenyl.
20. The compound of claim 19, wherein X is --CO--CH.sub.3.
21. The compound of claim 19, wherein X is --CO-phenyl.
22. The compound of claim 16, wherein P5 and P6 are the same and
are: --(CH.sub.2).sub.n--C(O)--R.sup.1, where n=14 and R.sup.1 is
OH, O-t-Bu, OR.sup.3, NHR.sup.3, NH-phenyl or NH-trityl, with
R.sup.3 being selected from H, C.sub.1-C.sub.4 straight or branched
chain alkyl.
23. The compound of claim 16, wherein P5 and P6 are different and
are: --(CH.sub.2).sub.n--C(O)--R.sup.1, where n=14 and R.sup.1 is
OH, O-t-Bu, OR.sup.3, NHR.sup.3, NH-phenyl or NH-trityl, with
R.sup.3 being selected from H, C.sub.1-C.sub.4 straight or branched
chain alkyl.
24. The compound of claim 22, wherein P5 and P6 are
--CH.sub.2CH.sub.2--C(O)--O--C(CH.sub.3).sub.3 or
--CH.sub.2--CH.sub.2--C- (O)--OH.
25. The compound of claim 23, wherein P5 and P6 are independently
selected from --CH.sub.2--CH.sub.2--C(O)--O--C(CH.sub.3).sub.3 or
--CH.sub.2--CH.sub.2C(O)--OH.
26. The compound of claim 16, wherein P3 and P4 are the same.
27. The compound of claim 16, wherein P3 and P4 are different.
28. The compound of claim 16, wherein P3 and P4 are independently
selected from the group consisting of: 299
29. The compound of claim 16, wherein P2 is selected from the group
consisting of: 300wherein n=0, 1, 2, or 3; and R.sup.2=R.sup.3=H;
R.sup.2=C.sub.1 to C6 straight chainalkyl or cycloalkyl; R.sup.3=H
R.sup.4=COAlkyl (straight chain or cyclic, C.sub.1 to C.sub.6);
COAryl; COOAlkyl; COOAryl R.sup.5=H; R.sup.6=Alkyl (C.sub.1 to
C.sub.3); R.sup.6=H; R.sup.5=Alkyl (C.sub.1 to C3) R.sup.7=H;
R.sup.8=Alkyl (C.sub.1 to C.sub.3), CH.sub.2OH; R.sup.8=H;
R.sup.7=Alkyl (C.sub.1 to C.sub.3), CH.sub.2OH;
R.sup.7=R.sup.8=Alkyl (C.sub.1 to C.sub.3), CH.sub.2OH
R.sup.9=R.sup.10=Alkyl (C.sub.1 to C.sub.3); R.sup.9=H,
R.sup.10=Alkyl (C.sub.1 to C.sub.3), COOMe, COOH, CH.sub.2OH;
R.sup.10=H, R.sup.9=Alkyl (C.sub.1 to C.sub.3), COOMe,
COOH,CH.sub.2OH; R.sup.11=Alkyl (C.sub.1 to C.sub.6 straight chain,
branched or cyclic), CH.sub.2Aryl (may be substituted)
Z.sup.1=Z.sup.2=S, O; Z=S, Z.sup.2=O;Z
=O,Z.sup.2=S;Z.sup.1=CH.sub.2,Z.sup.2=O;Z.sup.1=O,Z.sup.2=CH.sub.2;
Z.sub.1=S, Z.sub.2=CH.sub.2; Z.sup.1=CH.sub.2,Z.sup.2=S
Z.sup.3=CH.sub.2, S, SO.sub.2, NH, NR.sup.4 Z.sup.4=Z.sup.5=S,
O
29. The compound of claim 16, wherein P1a and P1b are independently
selected from the group consisting of: 301
30. The compound of claim 16, wherein P1' is selected from the
group consisting of: 302
31. The compound of claim 16, wherein Z is NH.
32. A pharmaceutical composition comprising as an active ingredient
a compound of claim 1 or claim 16.
33. The pharmaceutical composition of claim 32 for use in treating
disorders associated with Hepatitis C virus.
34. The pharmaceutical composition of claim 32 additionally
comprising a pharmaceutically acceptable carrier.
35. A method of treating disorders associated with the HCV
protease, said method comprising administering to a patient in need
of such treatment a pharmaceutical composition which composition
comprises therapeutically effective amounts of a compound of claim
1 or claim 16.
36. The method of claim 35, wherein said administration is via
subcutaneous administration.
37. The use of a compound of claim 1 or claim 16 forthe manufacture
of a medicament to treat disorders associated with the HCV
protease.
38. A method of preparing a pharmaceutical composition for treating
disorders associated with the HCV protease, said method comprising
bringing into intimate contact a compound of claim 1 or claim 16
and a pharmaceutically acceptable carrier.
39. A compound exhibiting HCV protease inhibitory activity,
including enantiomers, stereoisomers, rotamers and tautomers of
said compound, and pharmaceutically acceptable salts or solvates of
said compound, said compound being selected from the group of
compounds with structures listed below:
303304305306307308309310311312313314315316317318319
40. A pharmaceutical composition for treating disorders associated
with the HCV protease, said composition comprising therapeutically
effective amount of one or more compounds in claim 39 and a
pharmaceutically acceptable carrier.
41. The pharmaceutical composition of claim 40, additionally
containing an antiviral agent.
42. The pharmaceutical composition of claim 40 or claim 41, still
additionally containing an interferon.
43. The pharmaceutical composition of claim 42, wherein said
antiviral agent is ribavirin and said interferon is
.alpha.-interferon.
44. A compound selected from the group consisting of:
320321322323324325326327328329330331332333334335336337338339340or
an enantiomer, sterioisomer, rotamer or tautomer thereof, or a
pharmaceutically acceptable salt or solvate thereof, wherein the
compound exhibits HCV inhibitory activity.
45. A pharmaceutical composition, comprising one or more compounds
of claim 44.
47. A method of treatment of an hepatitis C virus associated
disorder, comprising administering an effective amount of one or
more compounds of claim 44.
47. A method of modulating the activity of hepatitis C virus (HCV)
protease, comprising contacting HCV protease with one or more
compounds of claim 44.
48. A method of treating, preventing, or ameliorating one or more
symptoms of hepatitis C, comprising administering an effective
amount of one or more compounds of claim 44.
49. The method of claim 47, wherein the HCV protease is the
NS3/NS4a protease.
50. The method of claim 49, wherein the compound or compounds
inhibit HCV NS3/NS4a protease.
51. A method of modulating the processing of hepatitis C virus
(HCV) polypeptide, comprising contacting a composition containing
the HCV polypeptide under conditions in which the polypeptide is
processed with one or more compounds of claim 44.
52. The compound of claim 17, wherein P2 is selected from the group
consisting of: 341wherein: n is 0, 1, 2 or 3; R.sup.20 is
alkylene-COOH; R.sup.21 is C(O)alkyl, CO.sub.2alkyl, C(O)aryl,
CO.sub.2aryl, SO.sub.2alkyl, SO.sub.2aryl, CONHalkyl, or CONHaryl;
R.sup.22 is alkyl or alkylene-COOH; and R.sup.23 is alkyl.
53. The compound of claim 52, wherein: R.sup.20 is CH.sub.2COOH;
R.sup.21 is CO.sub.2Ph, COPh, CO.sub.2CH.sub.2-9-fluorenyl,
CO-(3-phenoxyphenyl), SO.sub.2Ph or CONHPh; R.sup.22 is methyl or
CH.sub.2COOH; and R.sup.23 is methyl.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel hepatitis C virus
("HGV") protease inhibitors, pharmaceutical compositions containing
one or more such inhibitors, methods of preparing such inhibitors
and methods of using such inhibitors to treat hepatitis C and
related disorders. This invention specifically discloses novel
peptide compounds as inhibitors of the HCV NS3/NS4a serine
protease.
BACKGROUND OF THE INVENTION
[0002] Hepatitis C virus (HCV) is a (+)-sense single-stranded RNA
virus that has been implicated as the major causative agent in
non-A, non-B hepatitis (NANBH), particularly in blood-associated
NANBH (BB-NANBH)(see, International Patent Application Publication
No. WO 89/04669 and European Patent Application Publication No. EP
381 216). NANBH is to be distinguished from other types of
viral-induced liver disease, such as hepatitis A virus (HAV),
hepatitis B virus (HBV), delta hepatitis virus (HDV),
cytomegalovirus (CMV) and Epstein-Barr virus (EBV), as well as from
other forms of liver disease such as alcoholism and primary biliar
cirrhosis.
[0003] Recently, an HCV protease necessary for polypeptide
processing and viral replication has been identified, cloned and
expressed; (see, e.g., U.S. Pat. No. 5,712,145). This approximately
3000 amino acid polyprotein contains, from the amino terminus to
the carboxy terminus, a nucleocapsid protein (C), envelope proteins
(E1 and E2) and several non-structural proteins (NS1, 2, 3, 4a, 5a
and 5b). NS3 is an approximately 68 kda protein, encoded by
approximately 1893 nucleotides of the HCV genome, and has two
distinct domains: (a) a serine protease domain consisting of
approximately 200 of the N-terminal amino acids; and (b) an
RNA-dependent ATPase domain at the C-terminus of the protein. The
NS3 protease is considered a member of the chymotrypsin family
because of similarities in protein sequence, overall
three-dimensional structure and mechanism of catalysis. Other
chymotrypsin-like enzymes are elastase, factor Xa, thrombin,
trypsin, plasmin, urokinase, tPA and PSA. The HCV NS3 serine
protease is responsible for proteolysis of the polypeptide
(polyprotein) at the NS3/NS4a, NS4a/NS4b, NS4b/NS5a and NS5a/NS5b
junctions and is thus responsible for generating four viral
proteins during viral replication. This has made the HCV NS3 serine
protease an attractive target for antiviral chemotherapy.
[0004] It has been determined that the NS4a protein, an
approximately 6 kda polypeptide, is a co-factor for the serine
protease activity of NS3. Autocleavage of the NS3/NS4a junction by
the NS3/NS4a serine protease occurs intramolecularly (i.e., cis)
while the other cleavage sites are processed intermolecularly
(i.e., trans).
[0005] Analysis of the natural cleavage sites for HCV protease
revealed the presence of cysteine at P1 and serine at P1' and that
these residues are strictly conserved in the NS4a/NS4b, NS4b/NS5a
and NS5a/NS5b junctions. The NS3/NS4a junction contains a threonine
at P1 and a serine at P1'. The Cys.fwdarw.Thr substitution at
NS3/NS4a is postulated to account for the requirement of cis rather
than trans processing at this junction. See, e.g., Pizzi et al.
(1994) Proc. Natl. Acad. Sci (USA) 91:888-892, Failla et al. (1996)
Folding & Design 1:35-42. The NS3/NS4a cleavage site is also
more tolerant of mutagenesis than the other sites. See, e.g.,
Kollykhalov et al. (1994) J. Virol. 68:7525-7533. It has also been
found that acidic residues in the region upstream of the cleavage
site are required for efficient cleavage. See, e.g., Komoda et al.
(1994) J. Virol. 68:7351-7357.
[0006] Inhibitors of HCV protease that have been reported include
antioxidants (see, International Patent Application Publication No.
WO 98/14181), certain peptides and peptide analogs (see,
International Patent Application Publication No. WO 98/17679,
Landro et al. (1997) Biochem. 36:9340-9348, ingallinella et al.
(1998) Biochem. 37:8906-8914, Llins-Brunet et al. (1998) Bioorg.
Med. Chem. Lett. 8:1713.-1718), inhibitors based on the 70-amino
acid polypeptide eglin c (Martin et al. (1998) Biochem.
37:11459-11468, inhibitors affinity selected from human pancreatic
secretory trypsin inhibitor (hPSTI-C3) and minibody repertoires
(MBip) (Dimasi et al. (1997) J. Virol. 71:7461-7469), cV.sub.HE2 (a
"camelized" variable domain antibody fragment) (Martin et al.(1997)
Protein Eng. 10:607-614), and .alpha.1-antichymotrypsin (ACT)
(Elzouki et al.) (1997) J. Hepat. 27:42-28). A ribozyme designed to
selectively destroy hepatitis C virus RNA has recently been
disclosed (see, BioWorld Today 9(217): 4 (Nov. 10, 1998)).
[0007] Reference is also made to the PCT Publications, No. WO
98/17679, published Apr. 30, 1998 (Vertex Pharmaceuticals
Incorporated); WO 98/22496, published May 28, 1998 (F. Hoffmann-La
Roche AG); and WO 99/07734, published Feb. 18, 1999 (Boehringer
Ingelheim Canada Ltd.).
[0008] HCV has been implicated in cirrhosis of the liver and in
induction of hepatocellular carcinoma. The prognosis for patients
suffering from HCV infection is currently poor. HCV infection is
more difficult to treat than other forms of hepatitis due to the
lack of immunity or remission associated with HCV infection.
Current data indicates a less than 50% survival rate at four years
post cirrhosis diagnosis. Patients diagnosed with localized
resectable hepatocellular carcinoma have a five-year survival rate
of 10-30%, whereas those with localized unresectable hepatocellular
carcinoma have a five-year survival rate of less than 1%.
[0009] Reference is made to A. Marchetti et al, Synlett, S1,
1000-1002 (1999) describing the synthesis of bicylic analogs of an
inhibitor of HCV NS3 protease. A compound disclosed therein has the
formula: 1
[0010] Reference is also made to WO 00/09558 (Assignee: Boehringer
Ingelheim Limited; Published Feb. 24, 2000) which discloses peptide
derivatives of the formula: 2
[0011] where the various elements are defined therein. An
illustrative compound of that series is: 3
[0012] Reference is also made to WO 00/09543 (Assignee: Boehringer
Ingelheim Limited; Published Feb. 24, 2000) which discloses peptide
derivatives of the formula: 4
[0013] where the various elements are defined therein. An
illustrative compound of that series is: 5
[0014] Current therapies for hepatitis C include interferon-.alpha.
(INF.sub..alpha.) and combination therapy with ribavirin and
interferon. See, e.g., Beremguer et al. (1998) Proc. Assoc. Am.
Physicians 110(2):98-112. These therapies suffer from a low
sustained response rate and frequent side effects. See, eg.
Hoofnagle et al. (1997) N. Engl. J. Med. 336:347. Currently, no
vaccine is available for HCV infection.
[0015] Pending and copending U.S. patent applications, Ser. No.
60/220,110, filed Jul. 21, 2000, Ser. No. 60/220,107, filed Jul.
21, 2000, Ser. No. 60/220,108, filed Jul. 21, 2000, Ser. No.
60/220,101, filed Jul. 21, 2000, Ser. No. 60/254,869, filed Dec.
12, 2000, Ser. No. 60/194,607, filed Apr. 5, 2000, and Ser. No.
60/198,204, filed Apr. 19, 2000 disclose various types of peptides
as NS-3 serine protease inhibitors of hepatitis C virus.
[0016] There is a need for new treatments and therapies for HCV
infection. It is, therefore, an object of this invention to provide
compounds useful in the treatment or prevention or amelioration of
one or more symptoms of hepatitis C.
[0017] It is a further object herein to provide methods of
treatment or prevention or amelioration of one or more symptoms of
hepatitis C.
[0018] A still further object of the present invention is to
provide methods for modulating the activity of serine proteases,
particularly the HCV NS3/NS4a serine protease, using the compounds
provided herein.
[0019] Another object herein is to provide methods of modulating
the processing of the HCV polypeptide using the compounds provided
herein.
SUMMARY OF THE INVENTION
[0020] In its many embodiments, the present invention provides a
novel class of inhibitors of the HCV protease, pharmaceutical
compositions containing one or more of the compounds, methods of
preparing pharmaceutical formulations comprising one or more such
compounds, and methods of treatment, prevention or amelioration or
one or more of the symptoms of hepatitis C. Also provided are
methods of modulating the interaction of an HCV polypeptide with
HCV protease. Among the compounds provided herein, compounds that
inhibit HCV NS3/NS4a serine protease activity are preferred. The
presently disclosed compounds generally contain about four or more
amino acid residues and less than about twelve amino acid residues.
Specifically, the present application discloses peptide compounds,
defined further below.
[0021] In its first embodiment, the present invention provides a
compound of Formula I: 6
[0022] or a pharmaceutically acceptable salt, solvate or derivative
thereof, wherein:
[0023] Z is O, NH or NR.sup.12;
[0024] X is alkylsulfonyl, heterocyclylsulfonyl,
heterocyclylalkylsulfonyl- , arylsulfonyl, heteroarylsulfonyl,
alkylcarbonyl, heterocyclylcarbonyl, heterocyclylalkylcarbonyl,
arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl,
heterocyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,
alkyaminocarbonyl, heterocyclylaminocarbonyl, arylaminocarbonyl, or
heteroarylaminocarbonyl moiety, with the proviso that X may be
additionally optionally substituted with R.sup.12 or R.sup.13;
[0025] X.sup.1 is H; C.sub.1-C.sub.4 straight chain alkyl;
C.sub.1-C.sub.4 branched alkyl or; CH.sub.2-aryl (substituted or
unsubstituted);
[0026] R.sup.12 is alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkyl-alkyl, heterocyclyl, heterocyclylalkyl, aryl, alkylaryl,
arylalkyl, heteroaryl, alkylheteroaryl, or heteroarylalkyl moiety,
with the proviso that R.sup.12 may be additionally optionally
substituted with R.sup.13.
[0027] R.sup.13 is hydroxy, alkoxy, aryloxy, thio, alkylthio,
arylthio, amino, alkylamino, arylamino, alkylsulfonyl,
arylsulfonyl, alkylsulfonamido, arylsulfonamido, carboxy,
carbalkoxy, carboxamido, alkoxycarbonylamino, alkoxycarbonyloxy,
alkylureido, arylureido, halogen, cyano, or nitro moiety, with the
proviso that the alkyl, alkoxy, and aryl may be additionally
optionally substituted with moieties independently selected from
R.sup.13.
[0028] P1a, P1b, P2, P3, P4, P5, and P6 are independently:
[0029] H; C1-C10 straight or branched chain alkyl; C2-C10 straight
or branched chain alkenyl;
[0030] C3-C8 cycloalkyl, C3-C8 heterocyclic; (cycloalkyl)alkyl or
(heterocyclyl)alkyl, wherein said cycloalkyl is made up of 3 to 8
carbon atoms, and zero to 6 oxygen, nitrogen, sulfur, or phosphorus
atoms, and said alkyl is of 1 to 6 carbon atoms;
[0031] aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein
said alkyl is of 1 to 6 carbon atoms;
[0032] wherein all aforesaid alkyl, alkenyl, cycloalkyl,
heterocyclyl; (cycloalkyl)alkyl and (heterocyclyl)alkyl moieties
may be optionally substituted with R.sup.13. Additionally, the
atoms of P1a and P1b may be joined to each other in such a fashion
to form a spirocyclic or spiroheterocyclic ring, with said
spirocyclic or spiroheterocyclic ring containing zero to 6 oxygen,
nitrogen, sulfur, or phosphorus atoms, and may be optionally
substituted with R.sup.13.
[0033] P1' is H, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkyl-alkyl, heterocyclyl, heterocyclyl-alkyl, aryl,
aryl-alkyl, heteroaryl, or heteroaryl-alkyl; with the proviso that
said P1' may be additionally optionally substituted with
R.sup.13.
[0034] Among the above-stated definitions for X, R12, R13, P1a,
P1b, P2, P3, P4, P5, and P1', the preferred groups for the various
moieties are as follows: Preferred moieties for X are: 7
[0035] wherein Alkyl is C1 to C4 straight or branched chain, and
wherein Aryl is phenyl or substituted phenyl.
[0036] Preferred moieties for P6 are: 8
[0037] with n being 1-4.
[0038] Preferred moieties for P5 are: 9
[0039] with n being 1-4; additionally, P6 and P5 may be the same or
different. Preferred moieties for R.sup.1 are OH, O-t-Bu, OR.sup.3,
NHR.sup.3, NH-phenyl or NH-trityl.
[0040] Preferred moieties for R.sup.3 are H, Cl to C.sub.4 straight
or branched chain alkyl. P3 and P4 may be the same or different and
preferred moieties for P3 and P4 are: 10
[0041] Preferred moieties for P2 are: 11
[0042] wherein n=0, 1, 2 or 3;
[0043] Preferred moieties for P1a and P1b are: 12
[0044] Preferred moieties for P1' are: 13
[0045] In another embodiment, the present invention provides a
compound of Formula II: 14
[0046] wherein X, P6, P5, P4, P3, P2, P1a, P1b and P1' are as
defined above. Z is O, NH or NR.sup.12 where R.sup.12 has been
defined before. In Formula II, P2, when connected with the N atom
adjacent to C atom it is attached to, forms a cyclic ring, with the
proviso that said cyclic ring does not contain a carbonyl group as
part of the cyclic ring structure. The cyclic ring moiety comprised
of P2, the carbon atom to which P2 is attached, and the nitrogen
atom adjacent to that carbon atom is noted above as: 15
[0047] which may denote a five-membered ring or a six-membered ring
structure. Preferred representatives for that cyclic ring structure
are selected from the following: 16
[0048] wherein:
[0049] R.sup.2 and R.sup.3 may be the same or different and are
selected from H; C.sub.1-C.sub.6 straight chain alkyl;
C.sub.1-C.sub.6 branched alkyl or cycloalkyl;
[0050] R.sup.4is CO-Alkyl (alkyl being C1-C6 straight chain or
branched or cycloalkyl); CO-aryl; COO-alkyl or COO-aryl;
[0051] R.sup.5 and R.sup.6 may be the same or different and are
selected from H; C.sub.1-C.sub.3 straight chain alkyl; or
C.sub.1-C.sub.3 branched alkyl;
[0052] R.sup.7 and R.sup.8 may be the same or different and are
selected from H; C.sub.1-C.sub.3 straight chain alkyl;
C.sub.1-C.sub.3 branched alkyl or CH.sub.2OH;
[0053] R.sup.9 and R.sup.10 may be the same or different and are
selected from H; C.sub.1-C.sub.3 straight chain alkyl;
C.sub.1-C.sub.3 branched alkyl; COOMe; COOH or CH.sub.2OH;
[0054] R.sup.11 is C.sub.1-C.sub.6 straight chain alkyl;
C.sub.1-C.sub.6 branched alkyl; cyclocalkyl; or CH.sub.2-aryl
(substituted or unsubstituted);
[0055] Z.sup.1 and Z.sup.2 may be the same or different and are
selected from S; O; or CH.sub.2;
[0056] Z.sup.3 is CH.sub.2; S, SO.sub.2; NH or NR.sup.4;
[0057] Z.sup.4 and Z.sup.5 may be the same or different and are
selected from S, O or CH.sub.2.
[0058] In yet another embodiment, the present invention discloses
compounds of Formula III: 17
[0059] where the various elements are as defined above.
[0060] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. Thus, for
example, the term alkyl (including the alkyl portions of alkoxy)
refers to a monovalent group derived from a straight or branched
chain saturated hydrocarbon by the removal of a single atom having
from 1 to 8 carbon atoms, preferably from 1 to 6;
[0061] aryl--represents a carbocyclic group having from 6 to 14
carbon atoms and having at least one benzenoid ring, with all
available substitutable aromatic carbon atoms of the carbocyclic
group being intended as possible points of attachment. Preferred
aryl groups include phenyl, 1-naphthyl, 2-naphthyl and indanyl, and
especially phenyl and substituted phenyl;
[0062] aralkyl--represents a moiety containing an aryl group linked
vial a lower alkyl;
[0063] alkylaryl--represents a moiety containing a lower alkyl
linked via an aryl group;
[0064] cycloalkyl--represents a saturated carbocyclic ring having
from 3 to 8 carbon atoms, preferably 5 or 6, optionally
substituted.
[0065] heterocyclic--represents, in addition to the heteroaryl
groups defined below, saturated and unsaturated cyclic organic
groups having at least one O, S and/or N atom interrupting a
carbocyclic ring structure that consists of one ring or two fused
rings, wherein each ring is 5-, 6- or 7-membered and may or may not
have double bonds that lack delocalized pi electrons, which ring
structure has from 2 to 8, preferably from 3 to 6 carbon atoms,
e.g., 2- or 3-piperidinyl, 2- or 3-piperazinyl, 2- or
3-morpholinyl, or 2- or 3-thiomorpholinyl;
[0066] halogen--represents fluorine, chlorine, bromine and
iodine;
[0067] heteroaryl--represents a cyclic organic group having at
least one O, S and/or N atom interrupting a carbocyclic ring
structure and having a sufficient number of delocalized pi
electrons to provide aromatic character, with the aromatic
heterocyclyl group having from 2 to 14, preferably 4 or 5 carbon
atoms, e.g., 2-, 3- or 4-pyridyl, 2- or 3-furyl, 2- or 3-thienyl,
2-, 4- or 5-thiazolyl, 2- or 4-imidazolyl, 2-, 4- or 5-pyrimidinyl,
2-pyrazinyl, or 3- or 4-pyridazinyl, etc. Preferred heteroaryl
groups are 2-, 3- and 4-pyridyl; such heteroaryl groups may also be
optionally substituted.
[0068] Also included in the invention are tautomers, rotamers,
enantiomers and other optical isomers of compounds of Formula I,
Formula II and Formula II, as well as pharmaceutically acceptable
salts, solvates and derivatives thereof.
[0069] A further feature of the invention is pharmaceutical
compositions containing as active ingredient a compound of Formula
I, Formula II or Formula III (or its salt, solvate or isomers)
together with a pharmaceutically acceptable carrier or
excipient.
[0070] The invention also provides methods for preparing compounds
of Formulas I, II and III, as well as methods for treating diseases
such as, for example, HCV and related disorders. The methods for
treating comprise administering to a patient suffering from said
disease or diseases a therapeutically effective amount of a
compound of Formula I, II or III, or pharmaceutical compositions
comprising a compound of Formula I, II or III.
[0071] Also disclosed is the use of a compound of Formulas I, II or
III for the manufacture of a medicament for treating HCV and
related disorders.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0072] In one embodiment, the present invention discloses compounds
of Formula I as inhibitors of HCV protease, especially the HCV
NS3/NS4a serine protease, or a pharmaceutically acceptable
derivative thereof, where the various definitions are given
above.
[0073] In another embodiment, the present invention discloses
compounds of Formula II as inhibitors of HCV protease, especially
the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable
derivative thereof, where the various definitions are given
above.
[0074] In another embodiment, the present invention discloses
compounds of Formula III as inhibitors of HCV protease, especially
the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable
derivative thereof, where the various definitions are given
above:
[0075] Representative compounds of the invention which exhibit
excellent HCV protease inhibitory activity are listed below in
Table 1 along with their activity (ranges of K.sub.i* values in
nanomolar, nM).
1TABLE 1 Compounds and HCV protease continuous assay results
Compound from Ki* Example No. Range 1 b 2 b 3 c 4 b 5 b 6 c 7 c 8 b
9 b 10 c 11 c 12 b 13 c 14 b 15 b 16 c 17 c 18 c 19 c 20 c 21 c 22
c 23 c 24 c 25 a 26 b 27 a 28 c 29 c 30 c 31 b 32 b 33 b 34 c 35 c
36 a 37 b 38 a 39 a 40 a 41 c 42 a 43 a 44 a 45 a 46 c 47 c 48 b 49
a 50 c 51 b 52 b 53 b 54 b 55 a 56 b 57 b 58 c 59 b 60 b 61 a 62 a
63 a 64 b 65 b 66 b 67 b 68 c 69 c 70 c 71 b 72 b 73 c 74 c 75 c 76
c 77 c 78 b 79 a 80 a 81 a 82 a 83 a 84 a 85 a 86 a 87 a 88 b
[0076] HCV continous assay Ki* range:
[0077] Category a=1-100 nM; Category b=101-999 nM; Category
c=1000-10,000.
[0078] Some of the types of the inventive compounds and methods of
synthesizing the various types of the inventive compounds of both
Formula I and Formula II are listed below, then schematically
described, followed by the illustrative Examples. 18192021
[0079] Depending upon their structure, the compounds of the
invention may form pharmaceutically acceptable salts with organic
or inorganic acids, or organic or inorganic bases. Examples of
suitable acids for such salt formation are hydrochloric, sulfuric,
phosphoric, acetic, citric, malonic, salicylic, malic, fumaric,
succinic, ascorbic, maleic, methanesulfonic and other mineral and
carboxylic acids well known to those skilled in the art. For
formation of salts with bases, suitable bases are, for example,
NaOH, KOH, NH.sub.4OH, tetraalkylammonium hydroxide, and the
like.
[0080] In another embodiment, this invention provides
pharmaceutical compositions comprising the inventive peptides as an
active ingredient. The pharmaceutical compositions generally
additionally comprise a pharmaceutically acceptable carrier
diluent, excipient or carrier (collectively referred to herein as
carrier materials). Because of their HCV inhibitory activity, such
pharmaceutical compositions possess utility in treating hepatitis C
and related disorders.
[0081] In yet another embodiment, the present invention discloses
methods for preparing pharmaceutical compositions comprising the
inventive compounds as an active ingredient. In the pharmaceutical
compositions and methods of the present invention, the active
ingredients will typically be administered in admixture with
suitable carrier materials suitably selected with respect to the
intended form of administration, i.e. oral tablets, capsules
(either solid-filled, semi-solid filled or liquid filled), powders
for constitution, oral gels, elixirs, dispersible granules, syrups,
suspensions, and the like, and consistent with conventional
pharmaceutical practices. For example, for oral administration in
the form of tablets or capsules, the active drug component may be
combined with any oral non-toxic pharmaceutically acceptable inert
carrier, such as lactose, starch, sucrose, cellulose, magnesium
stearate, dicalcium phosphate, calcium sulfate, talc, mannitol,
ethyl alcohol (liquid forms) and the like. Moreover, when desired
or lo needed, suitable binders, lubricants, disintegrating agents
and coloring agents may also be incorporated in the mixture.
Powders and tablets may be comprised of from about 5 to about 95
percent inventive composition. Suitable binders include starch,
gelatin, natural sugars, corn sweeteners, natural and synthetic
gums such as acacia, sodium alginate, carboxymethylcellulose,
polyethylene is glycol and waxes. Among the lubricants there may be
mentioned for use in these dosage forms, boric acid, sodium
benzoate, sodium acetate, sodium chloride, and the like.
Disintegrants include starch, methylcellulose, guar gum and the
like.
[0082] Sweetening and flavoring agents and preservatives may also
be included where appropriate. Some of the terms noted above,
namely disintegrants, diluents, lubricants, binders and the like,
are discussed in more detail below.
[0083] Additionally, the compositions of the present invention may
be formulated in sustained release form to provide the rate
controlled release of any one or more of the components or active
ingredients to optimize the therapeutic effects, i.e.
[0084] HCV inhibitory activity and the like. Suitable dosage forms
for sustained release include layered tablets containing layers of
varying disintegration rates or controlled release polymeric
matrices impregnated with the active components and shaped in
tablet form or capsules containing such impregnated or encapsulated
porous polymeric matrices.
[0085] Liquid form preparations include solutions, suspensions and
emulsions. As an example may be mentioned water or water-propylene
glycol solutions for parenteral injections or addition of
sweeteners and pacifiers for oral solutions, suspensions and
emulsions. Liquid form preparations may also include solutions for
intranasal administration.
[0086] Aerosol preparations suitable for inhalation may include
solutions and solids in powder form, which may be in combination
with a pharmaceutically acceptable carrier such as inert compressed
gas, e.g. nitrogen.
[0087] For preparing suppositories, a low melting wax such as a
mixture of fatty acid glycerides such as cocoa butter is first
melted, and the active ingredient is dispersed homogeneously
therein by stirring or similar mixing. The molten homogeneous
mixture is then poured into convenient sized molds, allowed to cool
and thereby solidify.
[0088] Also included are solid form preparations which are intended
to be converted, shortly before use, to liquid form preparations
for either oral or parenteral administration. Such liquid forms
include solutions, suspensions and emulsions.
[0089] The compounds of the invention may also be deliverable
transdermally. The transdermal compositions may take the form of
creams, lotions, aerosols and/or emulsions and can be included in a
transdermal patch of the matrix or reservoir type as are
conventional in the art for this purpose.
[0090] Preferably the compound is administered orally,
intravenously or subcutaneously.
[0091] Preferably, the pharmaceutical preparation is in a unit
dosage form. In such form, the preparation is subdivided into
suitably sized unit doses containing appropriate quantities of the
active components, e.g., an effective amount to achieve the desired
purpose.
[0092] The quantity of the inventive active composition in a unit
dose of preparation may be generally varied or adjusted from about
1.0 milligram to about 1,000 milligrams, preferably from about 1.0
to about 950 milligrams, more preferably from about 1.0 to about
500 milligrams, and typically from about 1 to about 250 milligrams,
according to the particular application. The actual dosage employed
may be varied depending upon the patient's age, sex, weight and
severity of the condition being treated. Such techniques are well
known to those skilled in the art.
[0093] Generally, the human oral dosage form containing the active
ingredients can be administered 1 or 2 times per day. The amount
and frequency of the administration will be regulated according to
the judgment of the attending clinician. A generally recommended
daily dosage regimen for oral administration may range from about
1.0 milligram to about 1,000 milligrams per day, in single or
divided doses.
[0094] Some useful terms are described below:
[0095] Capsule--refers to a special container or enclosure made of
methyl cellulose, polyvinyl alcohols, or denatured gelatins or
starch for holding or containing compositions comprising the active
ingredients. Hard shell capsules are typically made of blends of
relatively high gel strength bone and pork skin gelatins. The
capsule itself may contain small amounts of dyes, opaquing agents,
plasticizers and preservatives.
[0096] Tablet--refers to a compressed or molded solid dosage form
containing the active ingredients with suitable diluents. The
tablet can be prepared by compression of mixtures or granulations
obtained by wet granulation, dry granulation or by compaction.
[0097] Oral gel--refers to the active ingredients dispersed or
solubilized in a hydrophillic semi-solid matrix.
[0098] Powder for constitution refers to powder blends containing
the active ingredients and suitable diluents which can be suspended
in water or juices.
[0099] Diluent--refers to substances that usually make up the major
portion of the composition or dosage form. Suitable diluents
include sugars such as lactose, sucrose, mannitol and sorbitol;
starches derived from wheat, corn, rice and potato; and celluloses
such as microcrystalline cellulose. The amount of diluent in the
composition can range from about 10 to about 90% by weight of the
total composition, preferably from about 25 to about 75%, more
preferably from about 30 to about 60% by weight, even more
preferably from about 12 to about 60%.
[0100] Disintegrant--refers to materials added to the composition
to help it break apart (disintegrate) and release the medicaments.
Suitable disintegrants include starches; "cold water soluble"
modified starches such as sodium carboxymethyl starch; natural and
synthetic gums such as locust bean, karaya, guar, tragacanth and
agar; cellulose derivatives such as methylcellulose and sodium
carboxymethylcellulose; microcrystalline celluloses and
cross-linked microcrystalline celluloses such as sodium
croscarmellose; alginates such as alginic acid and sodium alginate;
clays such as bentonites; and effervescent mixtures. The amount of
disintegrant in the composition can range from about 2 to about 15%
by weight of the composition, more preferably from about 4 to about
10% by weight.
[0101] Binder--refers to substances that bind or "glue" powders
together and make them cohesive by forming granules, thus serving
as the "adhesive" in the formulation. Binders add cohesive strength
already available in the diluent or bulking agent. Suitable binders
include sugars such as sucrose; starches derived from wheat, corn
rice and potato; natural gums such as acacia, gelatin and
tragacanth; derivatives of seaweed such as alginic acid, sodium
alginate and ammonium calcium alginate; cellulosic materials such
as methylcellulose and sodium carboxymethylcellulose and
hydroxypropylmethylcellulose; polyvinylpyrrolidone; and inorganics
such as magnesium aluminum silicate. The amount of binder in the
composition can range from about 2 to about 20% by weight of the
composition, more preferably from about 3 to about 10% by weight,
even more preferably from about 3 to about 6% by weight.
[0102] Lubricant--refers to a substance added to the dosage form to
enable the tablet, granules, etc. after it has been compressed, to
release from the mold or die by reducing friction or wear. Suitable
lubricants include metallic stearates such as magnesium stearate,
calcium stearate or potassium stearate; stearic acid; high melting
point waxes; and water soluble lubricants such as sodium chloride,
sodium benzoate, sodium acetate, sodium oleate, polyethylene
glycols and d'l-leucine. Lubricants are usually added at the very
last step before compression, since they must be present on the
surfaces of the granules and in between them and the parts of the
tablet press. The amount of lubricant in the composition can range
from about 0.2 to about 5% by weight of the composition, preferably
from about 0.5 to about 2%, more preferably from about 0.3 to about
1.5% by weight.
[0103] Glident--material that prevents caking and improve the flow
characteristics of granulations, so that flow is smooth and
uniform. Suitable glidents include silicon dioxide and talc. The
amount of glident in the composition can range from about 0.1% to
about 5% by weight of the total composition, preferably from about
0.5 to about 2% by weight.
[0104] Coloring agents--excipients that provide coloration to the
composition or the dosage form. Such excipients can include food
grade dyes and food grade dyes adsorbed onto a suitable adsorbent
such as clay or aluminum oxide. The amount of the coloring agent
can vary from about 0.1 to about 5% by weight of the composition,
preferably from about 0.1 to about 1%.
[0105] Bioavailability--refers to the rate and extent to which the
active drug ingredient or therapeutic moiety is absorbed into the
systemic circulation from an administered dosage form as compared
to a standard or control.
[0106] Conventional methods for preparing tablets are known. Such
methods include dry methods such as direct compression and
compression of granulation produced by compaction, or wet methods
or other special procedures. Conventional methods for making other
forms for administration such as, for example, capsules,
suppositories and the like are also well known.
[0107] Another embodiment of the invention discloses the use of the
pharmaceutical compositions disclosed above for treatment of
diseases such as, for example, hepatitis C and the like. The method
comprises administering a therapeutically effective amount of the
inventive pharmaceutical composition to a patient having such a
disease or diseases and in need of such a treatment.
[0108] In yet another embodiment, the compounds of the invention
may be used for the treatment of HCV in humans in monotherapy mode
or in a combination therapy mode such as, for example, in
combination with antiviral agents such as, for example, ribavirin
and/or interferon such as, for example, .alpha.-interferon and the
like.
[0109] As stated earlier, the invention includes tautomers,
rotamers, enantiomers and other stereoisomers of the compounds
also. Thus, as one skilled in the art appreciates, some of the
inventive compounds may exist in suitable isomeric forms. Such
variations are contemplated to be within the scope of the
invention.
[0110] Another embodiment of the invention discloses a method of
making the compounds disclosed herein. The compounds may be
prepared by several techniques known in the art. Representative
illustrative procedures are outlined in the following reaction
schemes. It is to be understood that while the following
illustrative schemes describe the preparation of a few
representative inventive compounds, suitable substitution of any of
both the natural and unnatural amino acids will result in the
formation of the desired compounds based on such substitution. Such
variations are contemplated to be within the scope of the
invention.
[0111] Abbreviations which are used in the descriptions of the
schemes, preparations and the examples that follow are:
[0112] THF: Tetrahydrofuran
[0113] DMF: N,N-Dimethylformamide
[0114] EtOAc: Ethyl acetate
[0115] AcOH: Acetic acid
[0116] HOOBt: 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one
[0117] EDCI:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
[0118] NMM: N-Methylmorpholine
[0119] ADDP: 1,1'-(Azodicarbobyl)dipiperidine
[0120] DEAD: Diethylazodicarboxylate
[0121] DCC: Dicyclohexylcarbodiimide
[0122] HOBt: Hydroxybezotriazole
[0123] HATU:
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
[0124] hexafluorophosphate
[0125] TEMPO: 2,2,6,6-tetramethyl-1-piperidinyloxy free radical
[0126] TBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium
tetrafluoroborate
[0127] PAM: 4-Hydroxymethylphenylacetamidomethyl
[0128] DTT: Dithiothreitol
[0129] Hunigs base (DIPEA or DIEA): Diisopropylethyl amine
[0130] DCM: Dichloromethane
[0131] MeOH: Methanol
[0132] EtOH: Ethanol
[0133] Et2O: Diethyl ether
[0134] PyBrOP: Bromo-tris-pyrrolidinophosphonium
hexafluorophosphate
[0135] Trt: Trityl
[0136] PMB: Para-methoxybenzyl
[0137] Bzl=Bn: Benzyl
[0138] Boc: tert-Butyloxycarbonyl
[0139] Cbz: Benzyloxycarbonyl
[0140] Cp: Cylcopentyldienyl
[0141] Ts: p-toluenesulfonyl
[0142] Me: Methyl
[0143] General Preparative Schemes:
[0144] The following schemes describe the methods of synthesis of
intermediate building blocks: 22 23 24 2526
[0145] Preparation of Intermediates:
[0146] The procedures to modify an amino acid with N-Boc, N-Cbz,
COOBzl, COOBu.sup.t, Obzl, Obu.sup.t, COOMe, both putting them on
or taking them off in the presence of each other in various
combinations, are generally well known to those skilled in the art.
Any modifications from the known procedures are, noted herein.
[0147] Commercially Available Intermediates:
[0148] The following amino acids, used as P2 units in the
preparation of the various inventive compounds, are commercially
available, and were converted to their N-Boc derivatives with
di-tert-butyldicarbonate, using known procedures. 27
[0149] The following N-Boc-amino acids, used as P2 units, are
commercially available. 28
[0150] The following N-Boc-amino acid, used as P2 unit, is
commercially available. After coupling the carboxylic acid, the
Fmoc is removed by known treatment with piperidine before
subsequent coupling. 29
[0151] Certain intermediates which were not commercially available
were synthesized, as needed, by following the procedures given
below: 30
[0152] A. Mesylate:
[0153] A mixture of triphenylphosphine (8.7 g), toluene (200 mL),
and methanesulfonic acid (2.07 mL) was stirred at 15.degree. C.
while slowly adding diethylazidodicarboxylate (7.18 g) to maintain
the temperature below 35.degree. C. The mixture was cooled to
20.degree. C., and the N-Boc amino acid (7.4 g, Bachem Biosciences,
Inc.), and Et.sub.3N (1.45 mL) were added, and then the mixture was
stirred at 70.degree. C. for 5 hr. The mixture was cooled to
5.degree. C., the organic supernate decanted, and solvent was
removed from it in vacuo. The residue was stirred with Et.sub.2O
(200 mL) until a precipitate deposits, the mixture was filtered,
and the ethereal solution was chromatographed on silica gel (5:95
to 20:80 EtOAc-Et.sub.2O) to obtain the product (9.3 g), which was
carried into the next step.
[0154] B. Azide
[0155] Sodium azide (1.98 g) was added to a solution of the product
of the step above (9.3 g) in DMF (100 mL), and the mixture stirred
at 70.degree. C. for 8 hr. The mixture was cooled, and poured into
5% aqueous NaHCO.sub.3, and extracted with EtOAc. The organic layer
was washed with brine, then dried over anhydrous MgSO.sub.4. The
mixture was filtered, and the filtrate evaporated in vacuo, to
obtain the product (6.2 g), which was carried into the next
step.
[0156] C. N-Cbz(4-N-Boc)-PrO--OMe
[0157] A solution of the product of the step above (0.6 g) in
dioxane (40 mL) was treated with di-tert-butyldicarbonate (0.8 g),
10% Pd-C (0.03 g), and hydrogen at one atmosphere for 18 hr. The
mixture was filtered, the filtrate evaporated in vacuo, and the
residue chromatographed on silica gel (1:1 to 2:1 Et.sub.2O-hexane)
to obtain the product.
[0158] D. N-Cbz(4-N-Boc)-PrO-OH was prepared using known ester
hydrolysis using LiOH. 31
[0159] These were prepared by following the procedure of U.
Larsson, et al., Acta Chem. Scan., (1994), 48(6), 517-525. A
solution of oxone.RTM. (20.2 g, from Aldrich Chemical Co.) in water
(110 mL) was added slowly to a 0.degree. C. solution of the sulfide
(7.2 g, from Bachem Biosciences, Inc.) in MeOH (100 mL). The cold
bath was removed and the mixture stirred for 4 hr. The mixture was
concentrated to 1/2 volume on a rotary evaporator, cold water (100
mL) added, extracted with EtOAc, the extract washed with brine, and
then it was dried over anhydrous MgSO4. The mixture was filtered,
and the filtrate evaporated in vacuo, to obtain the product as a
white solid (7.7 g). A portion was crystallized from (i-Pr).sub.2O
to obtain an analytical sample, [.alpha.].sub.D+8.6 (c 0.8,
CHCl.sub.3). Using the same procedure, the other sulfides shown
were oxidized to sulfones to lead to the subject targets.
PREPARATIVE EXAMPLE 1
Preparation of Ac-Glu(OBut)Glu(OBut)-Val-Val-OH(1.6) (Scheme 1)
[0160] Step A Compound (1.1).
[0161] To a mixture of Ac-Glu(OBzl)-OH (2.0 g), H-Glu(OBzl)-OMe.HCl
(2.0 g), HOOBt (1.35 g), N-methylmorpholine (0.87 mL), and
dimethylformamide (70 mL) at -20.degree. C. was added EDCl (2.50 g)
and stirred for 48 hr. The reaction mixture was poured into 5%
aqueous KH.sub.2PO.sub.4 (500 mL) and extracted with ethyl acetate
(2.times.150 mL). The combined organic layer was washed with cold
5% aqueous K.sub.2CO.sub.3, then 5% aqueous KH.sub.2PO.sub.4, then
brine, and dried over anhydrous MgSO.sub.4. The mixture was
filtered and evaporated under vacuum. The residue was triturated
with hexane (200 mL), and filtered to leave the title compound (3.4
g, 96% yield), TLC (EtOAc) Rf=0.7.
[0162] Step B Compound (1.2)
[0163] A solution of Compound (1.1) from Step A (3.4 g), MeOH (125
mL), and 1 M aqueous LiOH (7.25 mL) was stirred at room temperature
for 18 hr. The mixture was concentrated, treated with cold 0.5 N
HCl (250 mL), and extracted with ethyl acetate (2.times.150 mL).
The combined organic layer was washed with cold water, then brine.
The mixture was evaporated under vacuum, triturated with
Et.sub.2O-hexane, and filtered to leave the title compound (2.8 g,
85% yield).
[0164] Step C Compound (1.3)
[0165] In essentially the same manner as in Step A, but
substituting proportional amounts of compound (1.2) the product of
Step B for Ac-Glu(OBzl)-OH, and H-Val-OMe-HCl for H-Glu(OBzl)-Ome,
the title compound was prepared (80% yield).
[0166] Step D Compound (1.4)
[0167] In essentially the same manner as in Step B, but
substituting a proportional amount of compound (1.3) the product of
Step C for compound (1.1) the product of Step A, the title compound
was prepared (41% yield); C.sub.25H.sub.43N.sub.3O.sub.9 (529.64),
mass spec. (FAB) M+1=530.3; [a].sub.D-24.6 (c=0.7, MeOH).
[0168] Step E Compound (1.5)
[0169] In essentially the same manner as in Step A, but
substituting proportional amounts of compound (1.4), the product of
Step D for Ac-Glu(OBzl)-OH, and H-Val-OBzl.HCl for H-Glu(OBzl)-Ome,
the title compound was prepared (86% yield),
C.sub.37H.sub.58N.sub.4O.sub.10 (718.90) mass spec. (FAB)
M+1=719.3.
[0170] Step F Compound (1.6)
[0171] A mixture of compound (1.5) the product of Step E (2.6 g),
10% Pd/C (0.2 g), and EtOH-dioxane (1:1, 240 mL) was stirred under
1 atm. H.sub.2 for 18 hr. The mixture was filtered and evaporated
to dryness under vacuum to afford the title compound (2.1 g, 93%
yield), C.sub.30H.sub.52N.sub.4O.sub.10 (628.77) mass spec. (FAB)
M+1=629.5.
PREPARATIVE EXAMPLE 2
[0172] Step A Compound (2.2) 32
[0173] In a pot were combined N-Cbz-hydroxyproline methyl ester
(available from Bachem Biosciences, Incorporated, King of Prussia,
Pa.), compound (2.1) (3.0 g), toluene (30 mL), and ethyl acetate
(30 mL). The mixture was stirred vigorously, and then a solution of
NaBr/water (1.28 g/5 mL) was added. To this was added
2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO, 17 mg,
from Aldrich Chemicals, Milwaukee, Wis.). The stirred mixture was
cooled to 5.degree. C. and then was added a prepared solution of
oxidant [commercially available bleach, Clorox.RTM. (18 mL), NaHCO3
(2.75 g) and water to make up 40 mL] dropwise over 0.5 hr. To this
was added 2-propanol (0.2 mL). The organic layer was separated, and
the aqueous layer extracted with ethyl acetate. The organic
extracts were combined, washed with 2% sodium thiosulfate, then
saturated brine. The organic solution was dried over anhydrous
MgSO.sub.4, filtered, and evaporated the filtrate under vacuum to
leave a pale yellow gum suitable for subsequent reactions (2.9 g,
97% yield), C.sub.14H.sub.15NO.sub.5 (277.28), mass spec. (FAB)
M+1=278.1.
[0174] Step B Compound (2.3). 33
[0175] Compound (2.2) from Step A above (7.8 g) was dissolved in
dichloromethane (100 mL), and cooled to 15.degree. C. To this
mixture was first added 1,3-propanedithiol (3.1 mL), followed by
freshly distilled boron trifluoride etherate (3.7 mL). The mixture
was stirred at room temperature for 18 h. While stirring
vigorously, a solution of K.sub.2CO.sub.3/water (2 g/30 mL)was
carefully added, followed by saturated NaHCO.sub.3 (10 mL). The
organic layer was separated from the aqueous layer (pH -7.4),
washed with water (10 mL), then brine. The organic solution was
dried over anhydrous MgSO.sub.4, filtered, and evaporated under
vacuum. The residue was chromatographed on silica gel, eluting with
toluene, then a with a gradient of hexane-Et.sub.2O (2:3 to 0:1) to
afford a brown oil (7.0 g, 68% yield), C.sub.17H.sub.21NO.sub.4S.-
sub.2 (367.48), mass spec. (FAB) M+1=368.1.
[0176] Steg C Compound (2.4) 34
[0177] A solution of compound (2.3) from Step B above (45 g) in
acetonitrile (800 mL) at 20.degree. C. was treated with freshly
distilled iodotrimethylsilane (53 mL) at once. The reaction was
stirred for 30 min., then poured into a freshly prepared solution
of di-t-butyldicarbonate (107 g), ethyl ether (150 mL), and
diisopropylethylamine (66.5 mL). The mixture stirred for 30 min.
more then was washed with hexane (2.times.500 mL). Ethyl acetate
(1000 mL) was added to the lower acetonitrile layer, and then the
layer was washed with 10% aqueous KH.sub.2PO.sub.4 (2.times.700
mL), and brine. The filtrate was evaporated under vacuum in a
25.degree. C. water bath, taken up in fresh ethyl acetate (1000
mL), and washed successively with 0.1 N HCl, 0.1 N NaOH, 10%
aqueous KH.sub.2PO.sub.4, and brine. The organic solution was dried
over anhydrous MgSO.sub.4, filtered, and evaporated under vacuum.
The residue (66 g) was chromatographed on silica gel (2 kg),
eluting with hexane (2 L), then Et.sub.2O/hexane (55:45, 2 L), then
Et.sub.2O (2 L) to afford an orange gum which slowly crystallized
on standing (28 g, 69% yield), C.sub.14H.sub.23NO.sub.4S.sub.2
(333.46), mass spec. (FAB) M+1=334.1.
[0178] Step D Compound (2.5) 35
[0179] A solution of compound (2.4) from Step C above (11 g) in
dioxane (150 mL) at 20.degree. C. was treated with 1 N aqueous LiOH
(47 mL) and stirred for 30 h. The mixture was concentrated under
vacuum in a 30.degree. C. water bath to half volume. The remainder
was diluted with water (300 mL), extracted with Et.sub.2O
(2.times.200 mL). The aqueous layer was acidified to pH .about.4
with 12 N HCl (34 mL), extracted with ethyl acetate, and washed
with brine. The organic solution was dried over anhydrous
MgSO.sub.4, filtered, and evaporated under vacuum to leave the
title compound (8.1 g, 78%), C.sub.13H.sub.21NO.sub.4S.sub.2
(319.44), mass spec. (FAB) M+1=320.1.
[0180] Step E Compound (2.6). 36
[0181] To a solution of compound (2.4) from Step C above (1 g) in
dioxane (5 mL), was added 4 N HCl-dioxane solution (50 mL). The
mixture was stirred vigorously for 1 hr. The mixture was evaporated
under vacuum in a 25.degree. C. water bath. The residue was
triturated with Et.sub.2O, and filtered to leave the title compound
(0.76 g, 93% yield), C.sub.9H.sub.15NO.sub.2S.sub.2.HCl (269.81),
mass spec. (FAB) M+1=234.0.
[0182] Step F Compound (2.7). 37
[0183] A mixture of compound (2.6) from Step E above (1.12 g),
N-Boc-cyclohexylglycine (1.0 g, from Sigma Chemicals, St. Louis,
Mo.), dimethylformamide (20 mL), and PyBrOP coupling reagent (2.1
g) was cooled to 5.degree. C. To this was added
diisopropylethylamine (DIEA or DIPEA, 2.8 mL). The mixture was
stirred cold for 1 min., then stirred at room temperature for 6 hr.
The reaction mixture was poured into cold 5% aqueous
H.sub.3PO.sub.4 (150 mL) and extracted with ethyl acetate
(2.times.150 mL). The combined organic layer was washed with cold
5% aqueous K.sub.2CO.sub.3, then 5% aqueous KH.sub.2PO.sub.4, then
brine. The organic solution was dried over anhydrous MgSO.sub.4,
filtered, and evaporated under vacuum. The residue was
chromatographed on silica gel, eluting with EtOAc-CH.sub.2Cl.sub.2
to afford a white solid (0.8 g, 50% yield),
C.sub.22H.sub.36N.sub.2O.sub.5S.sub.2 (472.66), mass spec. (FAB)
M+1=473.2.
[0184] Step G Compound (2.8). 38
[0185] Following essentially the same procedure of Step D above,
but substituting compound (2.7) (0.8 g) as starting material,
compound (2.8) (0.7 g) was obtained
C.sub.21H.sub.34N.sub.2O.sub.5S.sub.2 (458.64), mass spec. (FAB)
M+1=459.2.
[0186] Step H Compound (2.9). 39
[0187] Following essentially the same procedure of Step E above,
but substituting compound (2.8) as starting material, compound
(2.9) was obtained. C.sub.17H.sub.28N.sub.2O.sub.3S.sub.2.HCl
(409.01), mass spec. (FAB) M+1=373.2.
PREPARATIVE EXAMPLE 3
[0188] Step A Compound (3.1) 40
[0189] Following essentially the same procedure of Preparative
Example 2, Step B, substituting ethane dithiol for propane dithiol,
compound (3.1) was obtained.
[0190] Step B Compound (3.2). 41
[0191] Following essentially the same procedure of Preparative
Example 2, Step C, substituting compound (3.1) for compound (2.3),
the product compound (3.2) was obtained.
[0192] Step C Compound (3.3) 42
[0193] Following essentially the same procedure of Preparative
Example 2, Step D, substituting compound (3.2) for compound (2.4)
the product compound (3.3) was obtained.
[0194] Step D Compound (3.4) 43
[0195] Following essentially the same procedure of Preparative
Example 2, Step E, substituting compound (3.2) for compound (2.4)
the product compound (3.4) was obtained.
[0196] Step E Compound (3.5) 44
[0197] Following essentially the same procedure of Preparative
Example 2, Step F, substituting compound (3.4) for compound (2.6)
the product compound (3.5) was obtained.
[0198] Step F Compound (3.6) 45
[0199] Following essentially the same procedure of Preparative
Example 2, Step G, substituting compound (3.5) for compound (2.7)
the product compound (3.6) was obtained.
[0200] Step G Compound (3.7) 46
[0201] Following essentially the same procedure of Preparative
Example 2, Step H, substituting compound (3.5) for compound (2.7)
the product compound (3.7) was obtained.
PREPARATIVE EXAMPLE 4
[0202] Step A Compound (4.1) 47
[0203] To a stirred solution of compound (4.01) (3.00 g, 12.0 mmol)
prepared according to S. L. Harbeson et al., J.Med.Chem. 37 (18),
2918-2929 (1994), in DMF (15 mL) and CH.sub.2Cl.sub.2 (15 mL) at
-20.degree. C. was added HOOBt (1.97 g, 12.0 mmol), N-methyl
morpholine (4.0 mL, 36.0 mmol) and EDCl (2.79 g, 14.5 mmol). The
reaction mixture stirred for 10 minutes, followed by the addition
of HCl.H.sub.2N-Gly-Oallyl (2.56 g, 13.0 mmol). The resulting
solution was stirred at -20.degree. C. for 2 hrs, then kept in the
refrigerator overnight. The solution was concentrated to dryness,
then diluted with EtOAc (150 mL). The EtOAc solution was then
washed twice with saturated NaHCO3, H2O, 5% H.sub.3PO.sub.4, and
brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to
dryness to give the product (4.1). LRMS m/z MH.sup.+=345.2.
[0204] Step B Compound (4.2) 48
[0205] Following essentially the same procedure of Preparative
Example 2, Step E, but substituting compound (4.1) from Step A
above, as the starting material, compound (4.2) was obtained
C.sub.11H.sub.20N.sub.2O.s- ub.4.HCl (280.75).
[0206] Step C Compound (4.3) 49
[0207] Following essentially the same procedure of Preparative
Example 1, Step A, reacting compound (4.2) from Step B above, with
compound (3.3) from Preparative example 3, Step C, compound (4.3)
was obtained. C.sub.28H.sub.37N.sub.3O.sub.7S.sub.2 (531.69).
[0208] Step D Compound (4.4). 50
[0209] Following essentially the same procedure of Preparative
Example 2, Step E, but substituting compound (4.3) from Step C
above, as starting material, compound (4.4) was obtained.
C.sub.18H.sub.30N.sub.4O.sub.5S.su- b.2.HCl (483.05).
[0210] Step E Compound (4.5) 51
[0211] Following essentially the same procedure of Preparative
Example 1, Step A, reacting compound (4.4) from Step D above, with
N-Boc-cyclohexylglycine, compound (4.5) was obtained.
C.sub.31H.sub.50N.sub.4O.sub.8S.sub.2 (670.88).
[0212] Step F Compound (4.6). 52
[0213] Following essentially the same procedure of Preparative
Example 2, Step E, but substituting compound (4.5) from Step E
above, as starting material, compound (4.6) was obtained.
C.sub.26H.sub.42N.sub.4O.sub.6S.su- b.2.HCl (607.23).
[0214] Step G Compound (4.7). 53
[0215] Following essentially the same procedure of Preparative
Example 1, Step A, reacting compound (4.6) from Step F above, with
compound (1.4) from Preparative example 1, Step D, compound (4.7)
was obtained. C.sub.51H.sub.83N.sub.7O.sub.14S.sub.2 (1082.38).
[0216] Step H Compound (4.8). 54
[0217] A mixture of compound (4.7) from Step G above (0.47 g),
dichloromethane (20 mL), methyl sulfoxide (0.9 mL), and
2,2-dichloroacetic acid (0.142 mL) was stirred at 5.degree. C. To
this was added a solution of 1 M dicyclohexylcarbodiimide in
CH.sub.2Cl.sub.2 (9.6 mL), and the resulting mixture was stirred
cold for 5 min., at room temperature for 3 h. A solution of oxalic
acid (0.35 g) in methanol (3 mL) was added to destroy excess
oxidant, stirred for 15 min., and then filtered to remove the
precipitated urea. The filtrate was diluted with excess ethyl
acetate, and washed with cold 0.1 N NaOH, then cold 0.2 N
H.sub.3PO.sub.4, then brine, The organic solution was dried over
anhydrous MgSO.sub.4, filtered, and evaporated under vacuum. The
residue was chromatographed on silica gel, eluting with a gradient
of MeOH--CH.sub.2Cl.sub.2 (1:99 to 5:95) to obtain the title
compound (4.8) (0.25 g, 53% yield)
C.sub.51H.sub.81N.sub.7O.sub.14S.sub.2 (1080.36), mass spec. (FAB)
M+1=1080.6.
[0218] Step I Compound (4.9). 55
[0219] Following essentially the same procedure of Preparative
Example 2, Step D, but substituting compound (4.8) from Step H
above, as the starting material, compound (4.9) was obtained
C.sub.48H.sub.73N.sub.7O.s- ub.14S.sub.2 (1036.26), mass spec.
(FAB) M+1=1036.6.
[0220] Step J Compound (86). 56
[0221] Compound (4.9) from Step I above (0.10 g) was treated with a
solution of anhydrous trifluoroacetic acid-dichloromethane (1:1, 10
mL) for 2 h. The solution was diluted with xylene (50 mL) and
evaporated under vacuum. The residue was triturated with Et.sub.2O,
and filtered to leave the title compound (86) (0.04 g),
C.sub.40H.sub.61N7O.sub.14S.sub.2 (928.08), mass spec. (FAB)
M+1=928.4.
EXAMPLES
[0222] Using the procedures of Preparative Example 1, Step A, and
Preparative Example 2, Step F, for couplings; Preparative Example
1, Step B, Preparative Example 1, Step F, Preparative Example 2,
Step D, and Preparative Example 4, Step J for ester deprotection;
Preparative Example 2, Step E, and Preparative Example 4, Step J,
for amine deprotection; and Preparative Example 4, Step H, for
oxidation of hydroxyamides to ketoamides--together with the
.alpha.-amino acids of the above examples or those commercially
available or those described in the literature, in the necessary
various combinations, the following compounds in Table 2 were
prepared:
2TABLE 2 Compound from Molecular Example No. STRUCTURE Weight 1 57
950.149 2 58 837.932 3 59 849.944 4 60 779.895 5 61 892.112 6 62
836.948 7 63 949.164 8 64 781.911 9 65 894.128 10 66 793.922 11 67
1065.28 12 68 777.879 13 69 890.096 14 70 948.133 15 71 835.916 16
72 825.921 17 73 853.975 18 74 966.192 19 75 938.138 20 76 915.166
21 77 889.408 22 78 1099.30 23 79 931.062 24 80 1043.28 25 81
952.165 26 82 811.894 27 83 839.948 28 84 827.894 29 85 841.921 30
86 1010.25 31 87 996.219 32 88 878.085 33 89 765.868 34 90 1025.22
35 91 932.177 36 92 876.069 37 93 1043.19 38 94 819.961 39 95
987.083 40 96 1022.26 41 97 1080.41 42 98 853.932 43 99 982.278 44
100 926.170 45 101 870.062 46 102 896.057 47 103 823.905 48 104
906.139 49 105 793.922 50 106 793.922 51 107 910.149 52 108 797.932
53 109 829.931 54 110 990.214 55 111 877.998 56 112 910.084 57 113
797.867 58 114 1044.31 59 115 1004.24 60 116 892.025 61 117 932.09
62 118 914.031 63 119 1026.25 64 120 954.097 65 121 906.139 66 122
793.922 67 123 807.950 68 124 920.166 69 125 900.004 70 126 1012.22
71 127 940.069 72 128 851.960 73 129 964.176 74 130 1004.24 75 131
871.950 76 132 966.192 77 133 1006.26 78 134 894.041 79 135 851.960
80 136 964.176 81 137 934.106 82 138 894.04 83 139 956.112 84 140
1026.25 85 141 914.031 86 142 928.099 87 143 1060.27 88 144
889.984
[0223] Additional compounds were prepared by the following
procedures and examples. The compounds are listed in Tables
immediately following the examples. The structures of many of the
so-prepared compounds and their activity are given in the attached
Table 3.
[0224] General Procedure for Preparation of the Compounds of Table
3:
[0225] Solid-phase synthesis is useful for the production of small
amounts of certain compounds of the present invention. As with the
conventional solid-phase synthesis of peptides, reactors for the
solid-phase synthesis of peptidyl argininals are comprised of a
reactor vessel with at least one surface permeable to solvent and
dissolved reagents, but not permeable to synthesis resin of the
selected mesh size. Such reactors include glass solid phase
reaction vessels with a sintered glass frit, polypropylene tubes or
columns with frits, or reactor Kans.TM. made by Irori Inc., San
Diego Calif. The type of reactor chosen depends on volume of
solid-phase resin needed, and different reactor types might be used
at different stages of a synthesis.
General Procedure for the Synthesis of 4-alkylprolines
(Intermediates Used in the Synthesis (Steps 14 Below):
[0226] Step 1
tert-Butyl N-.alpha.-t-butoxycarbonyl-L-pyroglutamate
[0227] 145
[0228] To a solution of L-pyroglutamic acid (100 g, 775 mmol) in
tert-butyl acetate (1.3 L) was added 70% aqueous perchloric acid
(25 mL). After stirring for 18 hours in a 3-liter round bottom
flask sealed with a rubber septum, the reaction mixture was poured
carefully into saturated aqueous sodium bicarbonate (800 mL) and
extracted with ethyl acetate (1 L, then 300 mL). The combined
organic layers were dried (sodium sulfate), filtered and
concentrated to provide tert-Butyl L-pyroglutamate (100.5 g, 70%
mass recovery). The tert-butyl L-pyroglutamate was dissolved in
acetonitrile (1.5 L) with 4-dimethylaminopyridine (6.0 g, 49.1
mmol) and cooled to 0.degree. C. A solution of
di-tert-butyl-dicarbonate (154 g, 705 mmol) in acetonitrile (150
mL) was added over 30 min and after an additional 30 min the
cooling bath was removed. After stirring at room temperature for 48
hours, the reaction mixture was concentrated. The residue was
dissolved in ether (1 L) and hexanes (1 L), then washed with
saturated aqueous sodium bicarbonate (2.times.100 mL) and saturated
aqueous sodium chloride. The solution was dried (sodium sulfate),
filtered and concentrated to give a yellow oil (140 g). The residue
was purified by recrystallization from hexanes (800 ml) and ethyl
acetate (25 mL), and the mother liquor was recrystallized from
hexanes (300 mL) and ethyl acetate (10 mL) to give two crops of the
title compound (122 g, 428 mmol, 55%) as a white solid. NMR data
was consistent with previously reported material: R. A. August et
al, J. Chem. Soc., Perkin Trans. 1 (1996) 507-514.
[0229] Step 2
tert-Butyl N-.alpha.-t-butoxycarbonyl-4-alkyl-L-pyroglutamate
[0230] 146
[0231] To a solution of the compound of Step 1 (1.15 g, 4.0 mmol)
in tetrahydrofuran (20 mL) stirring at -78 .degree. C., was added a
1M solution of lithium hexamethyldisilazide in tetrahydrofuran (4.4
mL, 4.4 mmol) dropwise over 5 min. After 40 min, alkylbromide (4.8
mmol) in tetrahydrofuran (5 mL) was added. After 2 h at -78.degree.
C., the cooling bath was removed and saturated aqueous ammonium
chloride (20 mL) was added. The solution was stirred for 20
minutes, then extracted with 50% ether/ethyl acetate (3.times.70
mL). The combined organic layers were washed with brine (30 mL),
dried (sodium sulfate), filtered, and concentrated. The resulting
residues were purified by flash chromatography and/or
recrystallization to afford the title compounds.
3 Compound Alkylbromide Yield (%) R.dbd.Bn benzyl bromide 63
R.dbd.PMB p-methoxybenzyl bromide 57 R.dbd.allyl allyl bromide 22
R.dbd.CH2CO2Bn benzyl a-bromoacetate 51
[0232] Step 3
4-alkyl-proline
[0233] 147
[0234] (Modification of known procedure: C. Pedregal et al,
Tetrahedron Letters 35 (13) (1994) 35(13) 2053-2056.) To a solution
of tert-butyl N-tert-butoxycarbonyl-4-alkylpyroglutamate (2.0 mmol)
in tetrahydrofuran (5 mL) stirring at -78 .degree. C., was added a
1 M solution of lithium triethylborohydride in tetrahydrofuran (2.4
mL, 2.4 mmol) dropwise over 5 min. After 30 min, the cooling bath
was removed and saturated aqueous sodium bicarbonate (5 mL) was
added. The reaction mixture was immersed in an ice/water bath and
30% aqueous hydrogen peroxide (10 drops) was added. The solution
was stirred for 20 minutes at 0.degree. C., then the reaction
mixture was concentrated in vacuo to remove the tetrahydrofuran.
The aqueous solution was diluted with water (10 mL) and extracted
with dichloromethane (3.times.40 mL). The organic layers were dried
(sodium sulfate), filtered and concentrated. The residue was
dissolved in dichloromethane (20 mL) and triethylsilane (310 .mu.L,
2.0 mmol), then cooled to -78.degree. C. and boron trifluoride
etherate (270 .mu.L, 2.13 mmol) was added dropwise. Stirring was
continued for 30 min, at which time additional triethylsilane (310
.mu.L, 2.0 mmol) and boron trifluoride etherate (270 .mu.L, 2.13
mmol) were added. After stirring at -78.degree. C. for an
additional 2 h, the cooling bath was removed and saturated aqueous
sodium bicarbonate (4 mL) was added. After 5 min the mixture was
extracted with dichloromethane (3.times.40 mL). The organic layers
were dried (sodium sulfate), filtered and concentrated. The residue
was dissolved in dichloromethane (5 mL) and trifluoroacetic (5 mL)
and stirred at ambient temperature for 5 h. The solution was
concentrated and then dried under high vacuum to afford the title
compound.
4 Compound Yield (%) R.dbd.Bn 72 R.dbd.PMB 76 R.dbd.allyl 65
R.dbd.CH2CO2Bn 34
[0235] Step 4
N-.alpha.-t-butoxycarbonyl-4-alkyl-proline
[0236] 148
[0237] To a solution of 4-alkylproline trifluoroacetate salt (1.5
mmol) in dioxane (7 mL), acetonitrile (12 mL) and
diisopropylethylamine (700 .mu.L, 4 mmol) was added
di-tert-butyl-dicarbonate (475 mg, 2.18 mmol) in acetonitrile (5
mL). After stirring for 12 h at room temperature the solution was
concentrated in vacuo, dissolved in saturated aqueous sodium
bicarbonate(50 mL) and washed with ether (3.times.40 mL). The
aqueous layer was acidified to pH 3 with citric acid, then
extracted with dichloromethane (3.times.40 mL). The combined
organic layers were dried (sodium sulfate), filtered and
concentrated to afford the title compounds, which where pure enough
to be used without further purification.
5 Compound Yield (%) R.dbd.Bn 59 R.dbd.PMB 69 R.dbd.allyl 58
R.dbd.CH2CO2Bn 67
Synthesis of Other Intermediates:
4-methylsulfinyl-2S-fluorenylmethyloxycarboaminobutyric Acid
(Fmoc-Met(O2)-OH
[0238] 149
[0239] To a solution of
N-.alpha.-fluorenylmethyloxycarbonyl-methionine (3.72 g, 10 mmol)
in chloroform (225 mL) at 0.degree. C., was added 56-87%
m-chloroperbenzoic acid (20-31mmol) in portions over 10 min. The
cold bath was removed and the reaction was stirred for 18 h. The
solid (4.09 g) was collected by filtration (filtrate was
discarded), dissolved in hot methanol (90 mL), allowed to cool,
then filtered and concentrated. The crude product was
recrystallized from ethyl acetate and hexanes to yield the title
compound (2.577 g, 6.39 mmol, 64 %).
N-.alpha.-fluorenylmethyloxycarbonyl-3-methylsulfinylalanine
(Fmoc-Cvs(O2,Me)--OH
[0240] 150
[0241] To a solution of
N-.alpha.-fluorenylmethyloxycarbonyl-S-methylcyste- ine (893 mg,
2.5 mmol) in chloroform (50 mL) at 0.degree. C., was added 56-87%
m-chloroperbenzoic acid (1.555 g, 5.0-7.8 mmol, >2 eq) in
portions over 10 min. The cold bath was removed and the reaction
was stirred for 18 h. The solid was collected by filtration
(filtrate was discarded), dried in vacuo, dissolved in hot ethyl
acetate (150 mL), filtered and concentrated. The solid was
dissolved in a minimal amount of hot methanol, then crystallized by
the addition of isopropyl ether, followed by 10% ethyl acetate in
hexanes. The solid (658.6 mg) was collected and recrystallized
again to give the title compound (520 mg, 1.335 mmol, 53%).
Synthesis of
N-tert-butoxycarbonyl-trans-4-(N-fluorenylmethyloxycarbonyl
amino)-L-proline (Boc-Pro(4t-NHFmoc): (Steps 1-3 Below)
[0242] Step 1
N-tert-butoxycarbonyl-cis-4-chloro-L-proline benzyl ester
[0243] 151
[0244] A mixture of N- -t-butoxycarbonyl-trans-4-hydroxy-proline
(8.79 g, 38 mmol), potassium carbonate (13.0 g, 94 mmol), benzyl
bromide (4.5 ml, 38 mmol) and dimethylformamide (150 mL) was
stirred for 18 h. Addition of ethyl acetate (100 mL) was followed
by filtration. The white cloudy filtrate was clarified by the
addition of 1M HCl (100 mL). The layers were separated and the
aqueous layer was extracted with additional ethyl acetate
(2.times.100 mL). The combined organic layers were washed with
water (2.times.50 mL), dried (sodium sulfate), filtered and
concentrated. Toluene was added to the crude benzyl ester, and the
solution was filtered and reconcentrated. Dichloromethane (70 mL)
and carbon tetrachloride (70 mL) was added, followed by
triphenylphosphine (21.11 g, 80 mmol). The reaction mixture was
stirred for 10 h, quenched with ethanol (7 mL) and stirred for 5
more h. The solution was concentrated to approx. 100 ml, then
dichloromethane (40 mL) was added, followed by the addition of
ether (200 mL) while stirring. The solution was cooled for 4 h,
filtered and concentrated to give a yellow-brown oil which was
purified by flash chromatography using ether/hexane/dichloromethane
2:2:1 to give the title compound (9.13 g, 26.9 mmol, 71 %) as a
white solid.
[0245] Step 2
N-.alpha.-t-butoxycarbonyl-trans-4-azido-L-proline benzyl ester
[0246] 152
[0247] A solution of the compound of step 1 above (9.0 g, 26.5
mmol) and sodium azide (7.36 g, 113 mmol) in dimethylformamide (270
mL) was heated at 75.degree. C. for 2 days. Water (100 mL) was
added and the reaction mixture was extracted with ethyl acetate
(3.times.100 mL). The combined organic layers were washed with
water (3.times.50 mL), dried (sodium sulfate), filtered and
concentrated. The oil was purified by flash chromatography using
ethyl acetate/hexane 1:1 to give the title compound (8.59 g, 24.8
mmol, 94%).
[0248] Step 3
N-tert-butoxycarbonyl-trans-4-(N-fluorenylmethyloxycarbonyl_amino)-L-proli-
ne
[0249] 153
[0250] A mixture of the compound of step 2 above (8.59 g, 24.8
mmol) and 10% palladium on carbon (900 mg) in ethanol (500 mL) was
hydrogenated at 50 psi for 14 h using a Parr hydrogenation
apparatus. The mixture was filtered, concentrated, dissolved in
methanol (60 mL), refiltered and concentrated to give a colorless
oil. The oil was dissolved in water (53 mL) containing sodium
carbonate (5.31 g, 50.1 mmol) and a solution of fluorenylmethyl
succinyl carbonate (8.37 g, 29.8 mmol) in dioxane (60 mL) was added
over 40 min. The reaction mixture was stirred at room temperature
for 17 h, then concentrated to remove the dioxane and diluted with
water (200 mL). The solution was washed with ether (3.times.100
mL). The pH of the aqueous solution was adjusted to 2 by the
addition of citric acid (caution! foaming!) and water (100 mL). The
mixture was extracted with dichloromethane (400 mL, 100 mL, 100 mL)
and the combined organic layers were dried (sodium sulfate),
filtered and concentrated to give the title compound.
N-.alpha.-t-butoxycarbonyl-4-trans-(N-fluorenylmethyloxycarbonyl
aminomethyl)-L-proline (Boc-Pro(4t-MeNHFmoc)-OH) (Steps 1-4
Below)
[0251] Step 1
N-.alpha.-t-butoxycarbonyl cis-4-hydroxy-L-proline benzyl ester
[0252] 154
[0253] To a mixture of cis-hydroxy-L-proline (5 g, 38.1 mmol) in
benzene (45 mL) and benzyl alcohol (45 mL) was added
p-toluenesulfonic acid monohydrate (7.6 g, 40.0 mmol). The reaction
mixture was heated at 125.degree. C. for 20 h while water (2 ml)
was removed using a Dean-Stark trap. The solution was filtered
while still hot, and then ether (150 ml) was added. The solution
was allowed to cool for three h at room temperature, then three h
at 4.degree. C. The resulting solid was collected, washed with
ether (100 mL) and dried in vacuo for 1 h to give 13.5 grams of
white solid. The solid was dissolved in dioxane (40 mL) and
diisopropylethylamine (7.6 mL), and then di-tert-butyl-dicarbonate
(10 g, 45.8 mmol) was added over 5 min while using an ice bath to
maintain a constant reaction temperature. After 10 h at room
temperature the reaction mixture was poured into cold water (200
mL) and extracted with ethyl acetate (3.times.200 mL). The combined
organic layers were washed with water (3.times.100 mL) and
saturated aqueous sodium chloride (50 mL), dried (sodium sulfate),
filtered and concentrated. The crude product was purified by flash
chromatography using 40-60% ethyl acetate in hexanes to give the
title compound (10.04 g, 31.24 mmol, 82%).
[0254] Step 2
N-.alpha.-t-butoxycarbonyl cis-4-mesyloxy-L-proline benzyl
ester
[0255] 155
[0256] To a solution of the compound of step 2 above (8.45 g, 26.3
mmol) in pyridine (65 mL) at 0.degree. C., was added
methanesulfonyl chloride (3.4 mL, 44 mmol) dropwise over 7 min. The
reaction mixture was allowed to warm to room temperature over 2 h,
then stirred overnight. A solution of 10% water in pyridine (20 mL)
was added over 15 min and the reaction mixture was concentrated.
The residue was dissolved in water and extracted with ethyl acetate
(2.times.200 mL). The combined organic layers were washed with
water (2.times.50 mL) saturated aqueous sodium bicarbonate (50 mL)
and saturated aqueous sodium chloride (50 mL), dried (sodium
sulfate), filtered and concentrated. The resulting residue was
dissolved in toluene (100 mL) and concentrated to remove traces of
pyridine. The residue was dried in vacuo for 30 min to afford the
title compound (10.7 g, 102%), then used in the next step without
purification.
[0257] Step 3
N-.alpha.-t-butoxycarbonyl-trans-4-(R)-cyano-L-proline benzyl
ester
[0258] 156
[0259] A solution of the compound of Step 3 above (10.7 g, 26.3
mmol) and tetrabutylammonium cyanide (15.0 g, 56 mmol) in
dimethylformamide (100 mL) was heated in an oil bath at 55.degree.
C. for 28 h. After cooling, water (150 mL) was added and the
mixture was extracted with ethyl acetate (3.times.200 mL). The
combined organic layers were washed with water (3.times.100 mL) and
saturated aqueous sodium chloride (100 mL), dried (sodium sulfate),
filtered and concentrated. The resulting residue was purified by
flash chromatography (1:1 ether/hexanes) and then recrystallized
from ethyl acetate/hexanes to provide the title compound (2.40 g,
7.26 mmol, 28%).
[0260] Step 4
N-.alpha.-t-butoxycarbonyl-4-trans-(N-fluorenyimethyloxycarbonyl
aminomethyl)-L-proline
[0261] 157
[0262] A mixture the com pound of Step 3 above (2.31 g, 7 mmol),
water (10 mL), methanol (85 mL) and 10% palladium on carbon (700
mg) was hydrogenated at 50 psi for 11 h using a Parr hydrogenation
apparatus. The mixture was filtered and concentrated. Water (15 mL)
and sodium carbonate (1.5 g, 14.2 mmol) was added to the residue. A
solution of fluorenylmethyl succinyl carbonate (2.36 g, 7.0 mmol)
in dioxane (17 mL) was added over 5 min and stirring was continued
for 28 h at room temperature. The reaction was concentrated in
vacuo to a 15 mL volume, and water (100 mL) was added. The solution
was washed with ether (3.times.75 mL). The pH of the aqueous
solution was adjusted to 2 by the addition of citric acid (approx.
20 g, caution! foaming!) and water (100 mL). The mixture was
extracted with dichloromethane (4.times.100 mL), and the combined
organic layers were dried (sodium sulfate), filtered and
concentrated. The crude product contained a major impurity which
necessitated a three step purification. The crude product was
dissolved in dichloromethane (50 mL) and trifluoroacetic acid (50
mL) and stirred for 5 h before being concentrated. The residue was
purified by preparatory reverse-phase HPLC. The pure
4-(N-fluorenylmethyloxycarbonyl aminomethyl)proline
trifluoroacetate salt (1.887 g, 3.93 mmol) was dissolved in dioxane
(10 mL), acetonitrile (20 mL) and diisopropylethylamine (1.4 mL, 8
mmol). To the reaction mixture was added a solution of
di-tert-butyldicarbonate (1.1g, 5 mmol) in dioxane (5 mL). After
stirring for 18 h, the pH of the solution was adjusted to 2 by the
addition of citric acid (caution: foaming!) and water (100 mL). The
mixture was extracted with ethyl acetate (3.times.150 mL) and the
combined organic layers were washed with saturated aqueous sodium
chloride (100 mL), dried (sodium sulfate), filtered and
concentrated. The crude product was dissolved in saturated aqueous
sodium bicarbonate(100 mL) and washed with ether (3.times.75 mL).
The pH of the aqueous layer was adjusted to 3 by the addition of
citric acid, then extracted with dichloromethane (4.times.100 mL).
The combined organic layers were dried (sodium sulfate), filtered
and concentrated to the title compound (1.373 g, 2.94 mmol,
42%).
[0263] Procedure to Synthesize Inventive Compounds:
[0264] Procedure A: Coupling reaction: To the resin suspended in
DMF (10-15 mL/gram resin) was added Fmoc-amino acid (1 eq), HOBt (1
eq), TBTU (1 eq) and DIEA (1 eq). The mixture was let to react for
448 hours. The reactants were drained and the resin was washed
successively with dimethylformamide, dichloromethane, methanol,
dichloromethane and diethylether (use 10-15 mL solvent/gram resin).
The resin was then dried in vacuo.
[0265] Procedure B: Coupling reaction: To the resin suspended in
N-methylpyrrolidine (NMP) (10-15 mL/gram resin) was added
Fmoc-amino acid (2 eq), HOAt (2 eq), HATU (2 eq) and
diisopropylethylamine (4 eq). The mixture was let to react for 4-48
hours. The reactants were drained and the resin was washed
successively with dimethylformamide, dichloromethane, methanol,
dichloromethane and diethylether (use 10-15 mL solvent/gram resin).
The resin was then dried in vacuo.
[0266] Procedure C: Fmoc deprotection: The Fmoc-protected resin was
treated with 20% piperidine in dimethylformamide (10 mL reagent/g
resin) for 30 minutes. The reagents were drained and the resin was
washed successively with dimethylformamide, dichloromethane,
methanol, dichloromethane and diethyl ether (10 mL solvent/gram
resin).
[0267] Procedure D: Boc deprotection: The Boc-protected resin was
treated with a 1:1 mixture of dichloromethane and trifluoroacetic
acid for 20-60 minutes (10 mL solvent/gram resin). The reagents
were drained and the resin was washed successively with
dichloromethane, dimethylformamide, 5% diisopropylethylamine in
dimethylformamide, dimethylformamide, dichloromethane and
dimethylformamide (10 mL solvent/gram resin).
[0268] Procedure E: Acetylation with acetic anhydride: The resin
was suspended in dimethylformamide. The acetylating reagent,
prepared by adding 5 mmol (0.47 mL) acetic anhydride and 5 mmol
(0.70 mL) triethylamine to 15 mL Dimethylformamide, was added to
the resin and the resin was agitated for 30 minutes. The resin was
washed successively with dimethylformamide, dichloromethane,
methanol, dichloromethane and diethyl ether (10 mL solvent/gram
resin).
[0269] Procedure F: Semicarbazone hydrolysis: The resin was
suspended in the cleavage cocktail (10 mL/g resin) consisting of
trifluoroacetic acid: pyruvic acid: dichloromethane: water 9:2:2:1
for 2 hours. The reactants were drained and the procedure was
repeated three more times. The resin was washed successively with
dichloromethane, water and dichloromethane and dried under
vacuum.
[0270] Procedure G: HF cleavage: The dried peptide-nVal(CO)-G-O-PAM
resin (50 mg) was placed in an HF vessel containing a small stir
bar. Anisole (10% of total volume) was added as a scavenger. In the
presence of glutamic acid and cysteine amino acids, thioanisole
(10%) and 1,2-ethanedithiol (0.2%) were also added. The HF vessel
was then hooked up to the HF apparatus (from Immuno Dynamics,
Incorporated) and the system was flushed with nitrogen for five
minutes. It was then cooled down to -70.degree. C. with a dry
ice/isopropanol bath. After 20 minutes, HF was distilled to the
desired volume (10 mL HF/g resin). The reaction was let to proceed
for one and a half hour at 0.degree. C. Work up consisted of
removing all the HF using nitrogen. Dichloromethane was then added
to the resin and the mixture was stirred for five minutes. This was
followed by the addition of 20% acetic acid in water (4 mL). After
stirring for 20 minutes, the resin was filtered using a fritted
funnel and the dichloromethane was removed under reduced pressure.
Hexane was added to the remaining residue and the mixture was
agitated, and the layers separated (this was repeated twice to
remove scavengers). Meanwhile, the resin was soaked in 1 mL
methanol. The aqueous layer (20% HOAC) was added back to the resin
and the mixture was agitated for five minutes and then filtered.
The methanol was removed under reduced pressure and the aqueous
layer was lyophilized. The peptide was then dissolved in 10-25%
methanol (containing 0.1% trifluoroacetic acid) and purified by
reverse phase HPLC.
[0271] Procedure H: HF pyridine cleavage: To dried
peptide-nVal(CO)-G-O-PA- M resin (50 mg) in a HDPE 20 mL
scintillation vial containing a 1/2 inch football stir bar was
added thioanisole (100 .mu.L) using a pipetman. A commercially
available solution of 70% HF/Pyridine (1 mL) was added. The vial
was immediately sealed tight with a polypropylene lined cap and
placed in an ice bath with gentle stirring. After stirring in a
0.degree. C. bath for one hour, the bath was removed and the
mixture was stirred at room temperature for one hour. During the
two hour cleavage process, the vial was inspected periodically and
if necessary the resin which built up on the sides of the flask was
dispersed back into the HF solution. The vial was cooled back to
0.degree. C. and its cap was carefully removed. The vial were then
cooled to -15.degree. C. using a salt/ice bath. A 10 mL B-D syringe
body equipped with a 23 gauge needle was held above the vial. To
this syringe body was added methoxytrimethylsilane (2 mL) using a
repetitive autopipette. After the syringe body dripped empty,
additional portions of methoxytrimethylsilane (2.times.2 mL, 3 mL,
(9 mL total)) were added until the HF was neutralized. The vial was
stirred for 5 minutes and then removed from the bath and stirred
for 20 min. The vial was loaded in the speed vac and concentrated.
Methylene chloride (5 mL) was added and the resin was stirred for
10 minutes and filtered into a 16.times.100 mm test tube using a 5
mL disposable column. The methylene chloride filtrate was
concentrated. The resin was additionally washed with 20% AcOH in
water (2.times.3 mL) and diethyl ether (2 mL) into the before
mentioned test tube. The mixture was agitated to dissolve the oil.
The ether layer was then removed and the aqueous layer was
lyophilized. The peptide was then dissolved in 10-25% methanol
(containing 0.1% trifluoroacetic acid) and purified by reverse
phase HPLC.
Example I
Solid Phase Synthesis of Ac-EEWP-nV(CO)-G-OH
[0272] 158
[0273] Step I. Synthesis of H-nVal(dpsc)-Gly-PAM Resin 159
a) Preparation of N-.alpha.-t-butoxycarbonyl-norvalinol
[0274] 160
[0275] To a solution of N-.alpha.-t-butoxycarbonyl-norvaline (25.0
g, 0.115 mol) in tetrahydrofuran (461 mL), cooled to 0.degree. C.,
was added borane/tetrahydrofuran complex (461 mL of a 1.0M solution
in THF) dropwise. After 1 h at 0.degree. C., the solution was
warmed to room temperature over a period of 1.5 h. TLC indicated
that the reaction was complete. Methanol was added to quench the
reaction. The solution was concentrated to yield the title compound
(22.56 g, 96%) as a foamy syrup. TLC of the products indicated
satisfactory purity. R.sub.f=0.34 (40% ethyl acetate/hexanes).
b) Preparation of N-.alpha.-t-butoxycarbonyl-norvaline hydroxy
t-butyl amide
[0276] 161
[0277] Part I: To a solution of the compound of step la (7.77 g, 38
mmol), in anhydrous dimethylsulfoxide (153 mL) and toluene (153 mL)
was added EDC (73.32 g, 382 mmol). After the solution was cooled to
0.degree. C., dichloroacetic acid (15.8 mL, 191 mmol) was added
dropwise. After addition was complete, the reaction was stirred for
15 min. The solution was allowed to warm to room temperature over a
period of 2 h. The reaction mixture was concentrated to remove the
toluene, then dissolved in ethyl acetate. The solution was washed
successively with 1N sodium bisulfate, saturated sodium bicarbonate
and brine, dried over sodium sulfate, then concentrated to afford
crude N-.alpha.-t-butoxycarbonyl-nor- valinal which was used
directly in the next step. R.sub.f=0.84 (40% ethyl
acetate/hexanes).
[0278] Part II: To a solution of the crude N-
-t-butoxycarbonyl-norvalinal in dichloromethane (153 mL) was added
t-butyl isocyanide (5.19 mL, 46 mmol) and 2,4,6-collidine (20.2mL,
153 mmol). After the solution was cooled to 0.degree. C.,
trifluoroacetic acid (7.64 mL, 76 mmol) was added dropwise. After
stirring for 1 h, the solution was stirred at room temperature for
3 days while allowing the solvent from the reaction mixture in an
uncovered vessel to evaporate under ambient conditions. The
reaction mixture was concentrated to remove the toluene, then
dissolved in ethyl acetate. The solution was washed successively
with 1N sodium bisulfate, saturated sodium bicarbonate and brine,
dried over sodium sulfate, then concentrated. The residue was
purified by flash silica gel chromatography eluting with 10-40%
ethyl acetate/hexanes to afford 8.12 g of the title compound (70%
yield) as a yellow syrup. R.sub.f=0.44 (40% ethyl
acetate/hexanes).
c) Preparation of Ethyl
3-(N-t-butoxycarbonyl)amino-2-hydroxy-hexanoate
[0279] 162
[0280] The compound of step Ib above (22.7 g, 75 mmol) was refluxed
in 6N HCl (480 mL) for 4 h. The reaction mixture was allowed to
cool to room temperature, then washed with dichloromethane
(3.times.100 mL). The aqueous layer was concentrated to dryness.
The residue was triturated with toluene/n-heptane (3.times.), dried
in vacuo to give crude 3-amino-2-hydroxy-hexanoic acid. The crude
3-amino-2-hydroxy-hexanoic acid was dissolved in hydrogen chloride
saturated ethanol (40 mL). After 4 h, the solution was concentrated
under reduced pressure. The residue was triturated with n-heptane
(3.times.), then dried in vacuo to give crude ethyl
3-amino-2-hydroxy-hexanoate. To the crude ethyl
3-amino-2-hydroxy-hexanoate in water (150 mL) was added potassium
carbonate (37.32 g, 270 mmol). After dissolution was complete,
dioxane was added (150 mL), followed by di-t-butyldicarbonate (17.7
g, 81.0 mmol). The reaction mixture was allowed to stir overnight,
then concentrated under reduced pressure to remove the dioxane. The
solution was extracted with ether (3.times.), then acidified to pH
2-3 with 1N sodium bisulfate. The solution was extracted with ethyl
acetate (3.times.200 mL), dried over sodium sulfate, and the
solvent was removed in vacuo to give 13.21 g of the title compound
in 71 % yield.
d) Preparation of Ethyl N-.alpha.-t-butoxycarbonyl-norvalyl
carboxylate diphenylmethylsemicarbazone (Part I-Part III Below)
[0281] 163
Part I: Synthesis of 1-t-Butoxycarbonyl-semicarbazid-4-yl
diphenylmethane
[0282] 164
[0283] A solution of carbonyidiimidazole (16.2 g, 0.10 mole) in 225
mL of dimethylformamide was prepared at room temperature and
allowed to stir under nitrogen. A solution of t-butyl carbazate
(13.2 g, 0.100 mol) in 225 mL DMF was then added dropwise over a 30
min. period. Diphenylmethylamine (18.3 9, 0.10 mol) was added next
over a 30min. period. The reaction was allowed to stir at room
temperature under nitrogen for one hour. Water (10 mL) was added
and the mixture was concentrated to about 150 mL under reduced
pressure. This solution was poured into 500 mL water and extracted
with 400 mL of ethyl acetate. The ethylacetate phase was extracted
two times each with 75 mL 1 N HCl, H.sub.2O, saturated sodium
bicarbonate solution and sodium chloride, and dried with magnesium
sulfate. The mixture was filtered and the solution was concentrated
to give 29.5 g (85% yield) of a white foam. This material could be
purified by recrystallization from ethyl acetate/hexane, but was
pure enough to use directly in the next step: mp 142-143.degree. C.
.sup.1H NMR (CDCl.sub.3) d 1.45 (s, 9H), 6.10 (dd, 2H), 6.42 (s,
1H), 6.67 (bs, 1H), 7.21-7.31 (m, 1OH). Anal: Calcd. for
C.sub.19H.sub.23N.sub.3O.sub.3: C, 66.84; H, 6.79; N, 12.31. Found:
C, 66.46; H, 6.75; N; 12.90.
Part II): Synthesis of diphenylmethyl semicarbazide (dpsc)
trifluoroacetate salt
[0284] 165
[0285] A solution of the product obtained in part I (above) (3.43
9,10 mmol) in 12.5 mL of dichloromethane was treated with 12.5 mL
of trifluoroacetic acid at room temperature and allowed to stir for
30 min. The solution was added dropwise to 75 mL of ether and the
resulting precipitate (2.7 g, 80%) was filtered on a glass funnel.
mp 182-184.degree. C. .sup.1H NMR (CD.sub.3OD) d 6.05 (s, 1H),
7.21-7.35 (m, 10H). .sup.13C NMR (CD.sub.3OD) d 57.6, 118.3 (q,
CF.sub.3), 126.7, 127.9, 141.6, 156.9, 160.9 (q,
CF.sub.3CO.sub.2H).
[0286] Part III): Preparation of Ethyl
N-.alpha.-t-butoxycarbonyl-norvalyl carboxylate
diphenylmethylsemicarbazone
[0287] To a cooled (0.degree. C.) solution of the compound of step
Ic above (13.21 g, 48 mmol) and EDC (92.1 g, 0.48 mol) in
dimethylsulfoxide (20 mL) and toluene (20 mL), was added
dichloroacetic acid (20.6 mL, 0.24 mol) dropwise. The reaction
mixture was stirred for 15 min, then allowed to warm to room
temperature over a 2 h period. The reaction mixture was
concentrated under reduced pressure, then diluted with ethyl
acetate, and washed successively with 1N sodium bisulfate,
saturated sodium bicarbonate and brine. The organic layer was dried
over sodium sulfate, and the solvent was removed in vacuo to afford
crude ethyl N-.alpha.-t-butoxycarbonyl-norvalyl carboxylate. Ethyl
N-.alpha.-t-butoxycarbonyl-norvalyl carboxylate was dissolved in
ethanol (180 mL), and water (60 mL), diphenylmethylsemicarbazide
(obtained in part 11 above)(34.12 g, 96 mmol) and sodium acetate
(4.73 g, 57.6 mmol) was added. The reaction mixture was refluxed
overnight, then allowed to cool to room temperature. The reaction
mixture was concentrated under reduced solvent, then diluted with
ethyl acetate. The organic layer was washed successively with 1N
sodium bisulfate, saturated sodium bicarbonate and brine, dried
over sodium sulfate, and the solvent was removed in vacuo. The
residue was chromatographed over flash silica gel using 20-30%
ethyl acetate/hexanes as eluent to afford 4.02g of the title
compound in 17% yield as a white powder. R.sub.f=0.60 (40% ethyl
acetate/hexanes).
e) Preparation of N-.alpha.-t-butoxycarbonyl-norvalyl carboxylic
acid diphenylmethylsemicarbazone (Boc-nVal(dpsc)-OH)
[0288] 166
[0289] To the compound of step Id above (4.02 g, 8.1 mmol) in
ethanol (40.5 mL) was slowly added 1N lithium hydroxide (64.8 mL,
64.8 mmol). After 3 h, Dowex 50WX8400 ion exchange resin was added
until the pH of the solution reached 4, and the mixture was stirred
for 5 min. The mixture was filtered, washed with methanol, and
concentrated. The solution was diluted with water, washed with
ether. The aqueous solution was concentrated in vacuo. The
resulting solid was triturated with toluene, then dried in vacuo to
yield 1.44 g of the title compound (38%) as a white solid.
[0290] HPLC: R.sub.t=16.2 and 17.7 minutes in a 30% to 90%
acetonitrile gradient in 0.1% aqueous TFA buffer on a 4.6.times.250
mm, 5 mM particle, 100 .ANG. pore, C18 column at a 1.0 mL/min flow
rate.
f) Synthesis of H-nVal(dpsc)-Gly-PAM Resin
[0291] 167
[0292] The commercially available Boc-Gly-PAM resin (5 g, 3.35
mmol) was deprotected according to Procedure D in a 250 mL fritted
solid phase reaction vessel equipped with a nitrogen inlet. It was
then coupled to Boc-nVal(dpsc)-OH (step Ie above) (2.81 g, 6
mmol)according to Procedure A. The resin was then subjected to Boc
deprotection according to procedure D.
[0293] Step II. Synthesis of Fmoc-Pro-nVal(dpsc)-Gly-PAM resin
[0294] The resin obtained in step 1 (3.5 9, 1.80 mmol) was reacted
with Fmoc-Pro-OH (4.5 mmol, 1.52 g) according to Procedure A. After
18 hours, qualitative ninhydrin analysis showed colorless beads and
solution indicating a high yield of coupling.
[0295] Step III. Synthesis of Fmoc-Val-Pro-nVal(dpsc)-Gly-PAM
Resin
[0296] The compound of step II above (100 mg) was transferred to a
fritted polypropylene tube and was deprotected according to
Procedure C. A ninhydrin assay on a small aliquot gave dark blue
resin and solution, indicating a high yield for the deprotection.
The resin was resuspended in DMF (1 mL) and coupled to Fmoc-Val-OH
(51 mg, 0.15 mmol) according to Procedure A. A small aliquot was
taken for qualitative ninhydrin analysis which showed colorless
beads and a dark red solution indicating a high yield of
coupling.
[0297] Step IV. Synthesis of Fmoc-Val-Val-Pro-nVal(dpsc)-Gly-PAM
Resin
[0298] The compound of the previous step (100 mg) was deprotected
according to Procedure C. A ninhydrin assay on a small aliquot gave
dark blue resin and solution showing a high yield for the
deprotection. The resin was resuspended in DMF (1 mL) and was
coupled to Fmoc-Val-OH (51 mg, 0.15 mmol), according to Procedure A
for 20 hours. A small aliquot was taken for qualitative ninhydrin
analysis which showed colorless beads and solution indicating a
high yield of coupling.
[0299] Step V. Synthesis of
Fmoc-Glu(OtBu)-Val-Val-Pro-nVal(dpsc)-Gly-PAM Resin
[0300] The compound of the previous step (100 mg) was deprotected
according to Procedure C. A ninhydrin assay on a small aliquot gave
dark blue resin and solution showing a high yield for the
deprotection. The resin was resuspended in DMF (1 mL) and was
coupled to Fmoc-Glu(OtBu)-OH (64 mg, 0.15 mmol), according to
Procedure A for 5 hours. A small aliquot was taken for qualitative
ninhydrin analysis which showed colorless beads and solution
indicating a high yield of coupling.
[0301] Step VI. Synthesis of
Fmoc-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(dps- c)-Gly-PAM Resin
[0302] The compound of the previous step (100 mg) was deprotected
according to Procedure C. A ninhydrin assay on a small aliquot gave
dark blue resin and solution showing a high yield for the
deprotection. The resin was resuspended in DMF (1 ml) and was
coupled to Fmoc-Glu(OtBu)-OH (64 mg, 0.15 mmol), according to
Procedure A for 5 hours. A small aliquot was taken for qualitative
ninhydrin analysis which showed colorless beads and solution
indicating a high yield of coupling.
[0303] Step VII. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(dpsc- )-Gly-PAM Resin
[0304] The compound of the previous step (100 mg) was deprotected
according to Procedure C and acylated according to Procedure E. The
resin was vacuum dried and a small aliquot was taken for
qualitative ninhydrin analysis which showed colorless beads and
solution indicating a high yield of coupling.
[0305] Step VIII. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal-(CO- )-Gly-PAM Resin
[0306] The compound of the previous step (100 mg) was subjected to
semicarbazone hydrolysis Procedure F.
[0307] Step IX. Synthesis of
Ac-Glu-Glu-Val-Val-Pro-nVal-(CO)Gly-OH
[0308] The resin of the previous step (100 mg) was subjected to HF
cleavage condition (Procedure G) to yield the desired crude
product. The material was purified by HPLC using a 2.2.times.25 cm
reverse phase column, containing a C-18 resin comprised of 10
micron size gel particles with a 300 angstrom pore size, eluting
with a gradient using 5-25% acetonitrile in water. Analytical HPLC
using a 4.6.times.250 mm reverse phase column, containing a C-18
resin comprised of 5 micron size gel particles with a 300 angstrom
pore size, eluting with 5-25% acetonitrile (containing 0.1%
trifluoroacetic acid) showed one peak at 17.5 minutes. Low
resolution mass spectrum confirmed the desired mass (MH.sup.+
798.5).
Table of Compounds Synthesized According to Example I
[0309]
6 COMPOUND NAME SYNTHESIS Ac-EEVVP-nV-(CO)-G-OH example I
Ac-EEVV-Sar-nV-(CO)-G-OH step II: used Fmoc-Sar-OH
Ac-EEVV-Aze-nV-(CO)-G-OH step II: used Fmoc-azetidine-OH
Ac-EEV-G(Chx)-P-nV-(CO)-G-OH step III: used Fmoc- Gly(CHx)--OH
Ac-EEVFP-nV-(CO)-G-OH step III: used Fmoc-Phe-OH
Ac-EEVIP-nV-(CO)-G-OH step III: used Fmoc-Ile-OH
Ac-EEVV-dlPip-nV-(CO)-G-OH step II: used Boc-d,I-pipecolic acid
Ac-EEVV-Tiq-nV-(CO)-G-OH step II: used Fmoc-Tiq-OH
Ac-EEVV-C(Me)-nV-(CO)-G-OH step II: used Fmoc- Cys(Me)--OH
Ac-EEVV-C(O2,Me)-nV-(CO)-G-OH step II: used Fmoc- Cys(O2,Me)--OH
Ac-EEVV-C(2-AcOH)-nV-(CO)-G- step II: used Fmoc- OH
Cys(2-AcOtBu)--OH Ac-EEVV-M(O2)-nV-(CO)-G-OH step II: used Fmoc-
Met(O2)--OH Ac-EEVV-P(4t-Bn)-nV-(CO)-G-OH step II: used
Boc-Pro(4t-Bn)-OH Ac-EEVV-P(4t-Bn(4-OMe))-nV- step II: used Boc-
(CO)-G-OH Pro(4t-Bn(4-OMe))--OH Ac-EEVV-P(4t-allyl)-nV-(CO)-G- -OH
step II: used Boc- Pro(4t-allyl)-OH Ac-EEVVD-nV-(CO)-G-OH step II:
used Fmoc- Asp(OtBu)--OH Ac-EEVVE-nV-(CO)-G-OH step II: used Boc-
Glu(OtBu)--OH Ac-EEVVF-nV-(CO)-G-OH step II: used Fmoc-Phe-OH
Ac-EEVV-P(4t-AcOH)-nV-(CO)-G- step II: used Boc- OH
Pro(4t-AcOBn)-OH Ac-EESVP-nV-(CO)-G-OH step IV: used Fmoc-
Ser(tBu)--OH Ac-EAVVP-nV-(CO)-G-OH Step V: used Fmoc-Ala-OH
Ac-EEHVP-nV-(CO)-G-OH step IV: used Fmoc-His(Trt)-OH
Ac-EENVP-nV-(CO)-G-OH step IV: used Fmoc- Asn(Trt)-OH
Ac-EEVV-P(4t-Ph)-nV-(CO)-G-OH step II: used Boc- Pro(4t-Ph)--OH
Ac-EEVV-P(3t-Me)-nV-(CO)-G-OH step II: used Boc- Pro(3t-Me)--OH
Ac-EE-Orn-VP-nV-(CO)-G-OH step IV: used Fmoc- Orn(Boc)-OH
Ac-EdEVVP-nV-(CO)-G-OH step V: used Fmoc- dGlu(OtBu)--OH
Ac-EE-(s,s)alloT-VP-nV-(CO)-G- step IV: used Fmoc-(s,s)allo- OH
Thr-OH Ac-EE-Dif-VP-nV-(CO)-G-OH step III: used Fmoc-Dif-OH
Ac-EE-daba-VP-nV-(CO)-G-OH step III: used Fmoc- *Daba(Boc)-OH
Ac-EEDVP-nV-(CO)-G-OH step IV: used Fmoc- Asp(OtBu)--OH
Ac-EEEVP-nV-(CO)-G-OH step IV: used Fmoc- Glu(OtBu)--OH
Ac-EETVP-nV-(CO)-G-OH step IV: used Fmoc- Thr(tBu)--OH
Ac-AEVVP-nV-(CO)-G-OH step VI: used Fmoc-Ala-OH
Ac-EELVP-nV-(CO)-G-OH step IV: used Fmoc-Leu-OH *Note: Daba denotes
diaminobutyric acid
Example II
Solution Phase Synthesis of Ac-EEWP-nV-(CO)-G-allylAm
[0310] 168
[0311] Step I. Synthesis of Boc-nVal(CHOH)-Gly-OEt 169
[0312] Trifluoroacetic acid (4.15 mL, 41.54 mmol) was added
dropwise to a cooled solution (0.degree. C.) of Boc-nVal-aldehyde
(4.18 9, 20.77 mmol) (obtained in example I, step Ib(part I)),
ethylisocyanoacetate (2.72 mL, 24.93 mmol), and pyridine (6.72 mL,
83.09 mmol) in dichloromethane (83 mL). After two hours, the
reaction was brought to room temperature and stirred uncapped for
48 hours. The reaction mixture was concentrated and dissolved in
ethylacetate (80 mL). It was then extracted three times each with
10 mL portions of 1N sodium bisulfate, saturated sodium bicarbonate
and brine. The organic layer was dried and concentrated and the
remaining residue was subjected to purification using flash column
chromatography in 1:4 ethylacetate: hexane followed by 2:3
ethylacetate: hexane. The desired fractions were pulled and
concentrated to a yellowish flake (24.5 g, 78.6%). Thin layer
chromatography in 2:3 ethylacetate: hexane showed one spot with an
Rf of 0.16. Low resolution mass spectrum confirmed the desired mass
(M+Na.sup.+ 489).
[0313] Step II. Synthesis of HCl.H-nVal(CHOH)-Gly-OEt
[0314] The product obtained in step I above (1.12 g, 3.38 mmol) was
treated for one hour with 10 mL saturated solution of anhydrous HCl
in ethanol. It was then concentrated and triturated with n-heptane
to a solid (0.9 g, 99.2%).
[0315] Step III. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH
(Steps a-f Below)
[0316] a) Synthesis of Fmoc-Val-Pro-2ClTrt Resin
[0317] In a 1 L solid phase reaction vessel equipped with a
nitrogen inlet, 25 g of Pro-2ClTrt resin (200-400 mesh, 0.64 mmol/g
substitution) was suspended in dimethylformamide (213 mL).
Fmoc-Val-OH (1.5 g, 32 mmol) was coupled for four hours according
to Procedure A. A small aliquot was taken for colorimetric
ninhydrin analysis which showed a 99.5% coupling efficiency in the
production of the title compound.
[0318] b) Synthesis of Fmoc-Val-Val-Pro-2ClTrt Resin
[0319] The resin from the previous step (0.53 mmol/g) was
deprotected according to Procedure C. It was then coupled to
Fmoc-Val-OH (10.85 g, 32 mmol) according to Procedure A with 99.5%
efficiency.
[0320] c) Synthesis of Fmoc-Glu(OtBu)-Val-Val-Pro-2ClTrt Resin
[0321] The resin from the previous step (0.504 mmol/g) was
deprotected according to Procedure C. It was then coupled to
Fmoc-Glu(OtBu)-OH (13.63 g, 32 mmol) according to Procedure A with
99.4% efficiency.
[0322] d) Synthesis of Fmoc-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-2ClTrt
Resin
[0323] The resin from the previous step (0.461 mmol/g) was
deprotected according to Procedure C. It was then coupled to
Fmoc-Glu(OtBu)-OH (13.63 g, 32 mmol) according to procedure A with
99.2% efficiency to yield the titled compound.
[0324] e) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-2ClTrt
Resin
[0325] The resin from the previous step (0.42 mmol/g) was
deprotected according to procedure C. The N-terminus was then
capped according to Procedure D to yield the desired compound in
99.7% efficiency.
[0326] f) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH
[0327] The resin from the previous step was transferred to a 1 L
plastic bottle and cleaved in the presence of 525 ml solution of
acetic acid: trifluoroethanol: dichloromethane (1:1:3) with
vigorous shaking for two hours. The resin was filtered using a
fritted funnel and washed 3.times.50 mL with dichloromethane. The
brownish red filtrate was concentrated to an oil which was then
treated three times with 50 ml of a 1:1 mixture of dichloromethane:
n-heptane. The crude off-white powder (13 g) was then dissolved in
minimum amount of methanol and purified by HPLC using a
2.2.times.25 cm reverse phase column, containing a C-18 resin
comprised of 10 micron size gel particles with a 300 angstrom pore
size, eluting with a gradient ranging from 15-55% acetonitrile in
water. The pure fractions were pulled and concentrated to a fluffy,
white product (7.5 g, 65%). Analytical HPLC using a 4.6.times.250
mm reverse phase column, containing a C-18 resin comprised of 5
micron size gel particles with a 300 angstrom pore size ran at
5-50% acetonitrile (containing 0.1 % trifluoroacetic acid) showed
one peak with the retention time of 20.5 min. Low resolution mass
spectrum confirmed the desired mass (MH.sup.+ 726.5).
[0328] Step IV. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH)- -Gly-OEt
[0329] The compound of step III above
(Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-- OH) (0.72 g, 1 mmol) was
coupled to the compound of step II above (HCl.H-nVal(CHOH)-Gly-OEt)
(0.27 g, 1 mmol) using HOAt (0.204 g, 1.5 mmol), HATU (0.418 g, 1.1
mmol) and diisopropylethylamine (0.87 mL, 5 mmol) in DMF at room
temperature. After 18 hours, the reaction mixture was concentrated.
The remaining residue was picked up in ethylacetate and washed
three times each with 10 mL portions of 1N sodium bisulfate,
saturated sodium bicarbonate and brine. It was then dried over
sodium sulfate and concentrated to a crusty yellowish product which
was taken to the next step without further purification (0.98 g).
Analytical HPLC using a 4.6.times.250 mm reverse phase column,
containing a C-18 resin comprised of 5 micron size gel particles
with a 300 angstrom pore size, eluting with 5-50% acetonitrile
(containing 0.1% trifluoroacetic acid) showed a 2:1 ratio of
diastereomers with retention times of 21 minutes and 21.5 minutes,
respectively.
[0330] Step V. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH)-- Gly-OH
[0331] To the compound obtained in step IV above (0.94 g, 1 mmol)
in ethanol (15 mL) was added 1N lithium hydroxide (4 mL, 4 mmol)
and the reaction was stirred at room temperature for two hours. The
reaction was stopped by the addition of enough Dowex ion exchange
resin (50.times.8-400) to obtain an acidic solution, pH .about.3.
After stirring for 15 minutes, the reaction mixture was filtered
and concentrated. The crude product was subjected to HPLC
purification using a 5.5.times.30 cm reverse phase column,
containing a C-18 resin comprised of 5 micron size gel particles
with a 300 angstrom pore size, eluting with a 30 minute gradient
using 5-30% acetonitrile in water. The desired fractions were
pulled and concentrated to a white solid (238 mg, 26%).
[0332] Step VI. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH)- -Gly-allylamide
[0333] The compound of step V above (129 mg, 0.14 mmol) was coupled
to allylamine (13 l, 0.17 mmol) in the presence of HOBt (58.5 mg,
0.38 mmol), EDC (54.3 mg, 0.28 mmol) and diisopropylethylamine (124
l, 0.71 mmol) in dimethylformamide (10 ml). After 18 hours, the
reaction mixture was concentrated and the remaining residue was
picked-up in ethylacetate and washed three times each with 5 mL
portions of 1N sodium bisulfate, saturated sodium bicarbonate and
brine. After drying over sodium sulfate, the organic layer was
concentrated to give a white precipitate which was taken to the
next step without further purification (115 mg, 85%). Analytical
HPLC using a 4.6.times.250 mm reverse phase column, containing a
C-18 resin comprised of 5 micron size gel particles with a 300
angstrom pore size, eluting with 5-50% acetonitrile (containing
0.1% trifluoroacetic acid) showed two diastereomeric peaks with
retention times of 15.9 and 16.5 minutes, respectively. Low
resolution mass spectrum confirmed the desired mass (M+Na.sup.+
973.5).
[0334] Step VII. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CO)-- YGly-allylamide
[0335] Under a stream of nitrogen gas, the product of step VI above
(115.2 mg, 0.12 mmol) was dissolved in dimethylsulfoxide (5 mL) and
toluene (5 mL). Water soluble carbodiimide (EDC, 232.2 mg, 1.21
mmol) was then added in one batch. The reaction mixture was cooled
to 0.degree. C. and dichloroacetic acid (52 l, 0.60 mmol) was added
dropwise. Stirring at 0.degree. C. continued for 15 minutes. The
ice bath was removed and the reaction continued for two hours at
room temperature. The toluene was removed under reduced pressure.
The remaining solution was diluted with ethylacetate and washed
three times each with 5 mL portions of 1N sodium bisulfate,
saturated sodium bicarbonate and brine. It was then concentrated to
a yellowish foam (85.5 mg, 74.4%).
[0336] Step VIII. Synthesis of
Ac-Glu-Glu-Val-Val-Pro-nVal(CO)-Gly-allylam- ide
[0337] The product of step VII above (0.86 g, 0.91 mmol) was
treated with a 1:1 mixture of dichloromethane: trifluoroacetic acid
(20 ml) for one hour. The reaction mixture was then concentrated
and the remaining residue was purified using a 2.2.times.25 cm
reverse phase HPLC column, containing a C-18 resin comprised of 10
micron size gel particles with a 300 angstrom pore size, eluting
with a 30 minutes gradient using 10-25% acetonitrile in water. The
purified fractions were pulled and lyophilized to a white powder
(21.5 mg, 28.5%). Analytical HPLC using a 4.6.times.250 mm reverse
phase column, containing a C-18 resin comprised of 5 micron size
gel particles with a 300 angstrom pore size, eluting with 5-75%
acetonitrile (containing 0.1 % trifluoroacetic acid) showed one
peak at 9.5 minutes. Low resolution mass spectrum confirmed the
desired mass (MH.sup.+ 837.5).
Table of Compounds Synthesized According to Example II
[0338]
7 COMPOUND NAME SYNTHESIS Ac-EEVVP-nV-(CO)-G-allylAm example II
Ac-EEVVP-nV-(CO)-G-2PhEtAm step VI: used phenethylamine
Ac-EEVVP-nV-(CO)-G-PropAm step VI: used propylamine
Ac-EEVVP-nV-(CO)-G-propynylAm step VI: used propynylamine
Ac-EEVVP-nV-(CO)-G-iPrAm step VI: used isopropylamine
Example III
Solution Phase Synthesis of Ac-EEWP-nV-(CO)-G(Oallyl)
[0339] 170
[0340] Step I. Synthesis of Fmoc-nVal-Gly-Oallyl (Steps a-c Below)
171
[0341] a) Synthesis of allyl isocyanoacetate
[0342] a1) Ethyl isocyanoacetate (96.6 mL, 0.88 mol) was added
dropwise to a chilled solution of ethanol (1.5 L) and potassium
hydroxide (59.52 g, 1.0 mol). The reaction was slowly warmed to
room temperature. After two hours, the precipitated product was
filtered on a glass funnel and washed with several portions of
chilled ethanol. The potassium salt of isocyanoacetic acid thus
obtained was dried in vacuo to a golden-brown solid (99.92 g,
91.8%).
[0343] a2) To the product of step al (99.92 g, 0.81 mol) dissolved
in acetonitrile (810 mL) was added allyl bromide (92 mL, 1.05 mol).
After refluxing for four hours, a dark brown solution was obtained.
The reaction mixture was concentrated and the remaining residue was
picked-up in ether (1.5 L) and washed three times with water (500
ml). The organic layer was dried and concentrated to a dark brown
syrup. The crude was purified by vacuum distillation at 7 mm Hg
(98.degree. C.) to a clear oil (78.92 g, 77.7%). NMR .delta. ppm
(CDCI3): 5.9 (m, 1 H), 5.3 (m, 2H), 4.7 (d, 2H), 4.25 (s, 2H).
[0344] b) Synthesis of 9-fluorenylmethoxycarbonyl-norvalinal (Steps
b1-b3 Below)
[0345] b1) Synthesis of 9-fluorenylmethoxycarbonyl-norvaline methyl
ester:
[0346] To a chilled solution of Fmoc-norvaline (25 g, 73.75 mmol)
in anhydrous methanol (469 mL) was added thionyl chloride (53.76
mL, 0.74 mol) over one hour. Thin layer chromatography in
ethylacetate taken an hour later confirmed the completion of the
reaction (R.sub.f=0.85). The reaction mixture was concentrated and
the remaining residue was picked-up in ethylacetate. The organic
layer was washed with three 200 ml portions of saturated sodium
bicarbonate followed by brine. The organic layer was dried and
concentrated to afford the title product as a white solid (26.03 g)
in quantitative yield. NMR .delta. ppm (CD.sub.3OD): 7.7 (m, 2H),
7.6 (m, 2H), 7.4 (m, 2H), 7.3 (m, 2H), 4.3 (m, 2H), 4.1 (m, 2H),
3.7 (s, 3H), 1.7 (m, 1H), 1.6 (m,1H), 1.4 (m, 2H), 0.95 (t,
3H).
[0347] b2) Synthesis of 9-fluorenylmethoxycarbonyl-norvalinol:
[0348] To the product of step b1 (26.03 g, 73.75 mmol) in
tetrahydrofuran (123 mL) and methanol (246 mL) was added calcium
chloride (16.37 g, 147.49 mmol). The reaction mixture was cooled to
0.degree. C. and sodium borohydride (11.16 g, 0.3 mol) was added in
several batches. Methanol (500 mL) was added to the thick paste
obtained and the reaction was stirred at room temperature for 90
minutes. Thin layer chromatography in 2:3 ethylacetate: hexane
confirmed the completion of the reaction (R.sub.f=0.25). The
reaction was quenched with the slow addition of 100 mL water at
0.degree. C. The methanol was removed under reduced pressure and
the remaining aqueous phase was diluted with ethylacetate (500 mL).
The organic layer was washed three times each with 500 ml portions
of water, saturated sodium bicarbonate and brine. The organic layer
was dried over sodium sulfate and concentrated to a white solid
(21.70 g, 90.5%). NMR .delta. ppm (CD.sub.3OD): 7.8 (m, 2H), 7.7
(m, 2H), 7.4 (m, 2H), 7.3 (m, 2H), 4.3-4.5 (m, 2H), 4.2 (m, 1H),
3.6 (s, 1H), 3.5 (s, 2H), 1.5 (m, 1H), 1.3-1.4 (m, 3H), 0.99 (m,
3H).
[0349] b3) Synthesis of 9-fluorenylmethoxycarbonyl-norvalinal:
[0350] To the product of step b2 (21.70 g, 66.77 mmol) in
dichloromethane (668 mL) was added triethylamine (37.23 mL, 267.08
mmol) and the solution was cooled to 0.degree. C. A suspension of
pyridine sulfur trioxide complex (42.51 g, 267.08 mmol) in
dimethylsulfoxide (96 mL) was added to the chilled solution. After
one hour, thin layer chromatography in 2:3 ethylacetate: hexane
confirmed the completion of the reaction. The dichloromethane was
removed under reduced pressure and the remaining residue was
picked-up in ethylacetate and washed with several 50 mL portions of
water,1N saturated sodium bisulfate, saturated sodium bicarbonate
and brine. The organic layer was concentrated to yield a white
solid. Theoretical yield (21.57 g) was assumed and the reaction was
taken to the next step without further purification.
[0351] c) Synthesis of Fmoc-nVal(CHOH)-Gly-Oallyl 172
[0352] To a solution of 9-fluorenylmethoxycarbonyl-norvalinal
obtained from step b3 (5.47 g, 16.90 mmol) in dichloromethane (170
mL) was added allyl isocyanoacetate (step I a2 above) (2.46 mL,
20.28 mmol) and pyridine (5.47 mL, 67.61 mmol). The reaction
mixture was cooled to 0.degree. C. and trifluoroacetic acid (3.38
mL, 33.80 mmol) was added dropwise. The reaction was stirred at 0 C
for 1 h, and then at room temperature for 48 hours. Thin layer
chromatography taken in ethylacetate confirmed the completion of
the reaction. The reaction mixture was concentrated and subjected
to flash column chromatography using a gradient composed of 20:80
ethylacetate: hexane to 70:30 ethylacetate: hexane. Fractions
containing the desired product were pooled and concentrated to a
white foam (6.88 g, 87.3%). TLC in-50:50 ethylacetate showed one
spot (R.sub.f=0.37). NMR .delta. ppm (CD.sub.3OD): 7.8 (m, 2H),
7.65 (m, 2H), 7.4 (m, 2H), 7.3 (m, 2H), 5.9 (m,1H), 5.1-5.4 (m,
2H), 4.55-4.65 (m, 2H), 4.3-4.4 (m, 2H), 4.15-4.25 (m, 1H), 4.01
(s, 1H), 3.9-4.0 (m, 3H),1.5-1.6 (m, 2H), 1.35-1.45 (m, 3H), 0.9
(m, 3H).
[0353] Step II. Synthesis of H-nVal(CHOH)-Gly-Oallyl
[0354] To Fmoc-nVal(CHOH)-Gly-Oallyl (300 mg, 0.64 mmol) (obtained
in step Ic above) dissolved in tetrahydrofuran (5.8 mL) was added
diethylamine (0.64 mL) and the resulting solution was stirred at
room temperature for two hours. The reaction mixture was
concentrated and the remaining solid was triturated with a 1:4
ether: hexane mixture. The product was collected on a glass funnel
and washed several times with a 1:1 ether: hexane mixture (93.7 mg,
57%). Low resolution mass spectrum confirmed the desired mass
(MH.sup.+ 245.0).
[0355] Step III. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH- )-Gly-Oallyl
[0356] The product obtained from step II above (28.2 mg, 0.11 mmol)
was coupled to Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH (example II,
step III) (84 mg, 0.11 mmol) in the presence of HOAt (23.6 mg, 0.17
mmol), HATU (48.4 mg, 0.13 mmol), diisopropylethylamine (100.8
.mu.l, 0.58 mmol) in dimethylformamide (20 mL) for 4 hours at room
temperature. The DMF was removed under reduced pressure and the
remaining residue was picked up in ethylacetate and washed with 1N
sodium bisulfate, saturated sodium bicarbonate and brine. After
drying over sodium sulfate, the organic layer was concentrated to
give a white solid (100.3 mg, 91 %) which was taken to the next
step without further purification. Low resolution mass spectrum
confirmed the desired mass (M+Na.sup.+ 974.5).
[0357] Step IV. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CO)-G- ly-Oallyl
[0358] Under a stream of nitrogen gas, the product of the previous
step (51.5 mg, 0.054 mmol) was dissolved in dimethylsulfoxide (1.2
mL) and toluene (1.2 mL).
[0359] Water soluble carbodiimide (EDC, 103.8 mg, 0.54 mmol) was
then added in one batch. The reaction mixture was cooled to
0.degree. C. and dichloroacetic acid (22.3 l, 0.27 mmol) was added
dropwise. Stirring at 0.degree. C. continued for 15 minutes. The
ice bath was removed and the reaction was slowly brought to room
temperature. The reaction was stopped after 90 minutes. The toluene
was removed under reduced pressure. The reaction was diluted with
ethylacetate and washed with 1N sodium bisulfate, saturated sodium
bicarbonate and brine. It was then concentrated to a yellowish foam
(40.4 mg, 79%) and taken to the next step without further
purification.
[0360] Step V. Synthesis of
Ac-Glu-Glu-Val-Val-Pro-nVal(CO)-Gly-Oallyl
[0361] The product of the previous step (40.4 mg, 0.042 mmol) was
treated with a 1:1 mixture of dichloromethane: trifluoroacetic acid
(4 mL) for two hours. The reaction mixture was then concentrated
and purified on a 1.times.25 cm reverse phase HPLC column,
containing a C-18 resin comprised of 10 micron size gel particles
with a 300 angstrom pore size, eluting with a 30 minute gradient
using 10-30% acetonitrile in water. The desired fractions were
pulled and concentrated to a white powder (8.5 mg, 24%). Analytical
HPLC using a 4.6.times.250 mm reverse phase column, containing a
C-18 resin comprised of 5 micron size gel particles with a 300
angstrom pore size, ran at 5-50% acetonitrile (containing 0.1%
trifluoroacetic acid) showed one peak at 15 minutes. Low resolution
mass spectrum confirmed the desired mass (MH.sup.+ 838.0).
Table: Compounds Synthesized According to Example III
[0362]
8 COMPOUND NAME SYNTHESIS Ac-EEVVPnV-(CO)-G-Oallyl example III
Ac-EEVVP-nL-(CO)-G-Oallyl step I(b1): used Fmoc-nLeu-OH
Ac-EEVVP-V-(CO)-G-Oallyl step I(b1): used Fmoc-Val-OH
Ac-EEVVPL-(CO)-G-Oallyl step I(b1): used Fmoc-Leu-OH
Ac-EEVVP-G(propynyl)-(CO)-G- step I(b1): used Fmoc- Oallyl
Gly(propynyl)-OH Ac-EEVVPnV-(CO)-G-OEt step Ic: used ethyl
isocyanoacetate Ac-EEVVP-G(allyl)-(CO)-G-Oallyl step I(b1): used
Fmoc- Gly(allyl)-OH Ac-EEVVG-L-(CO)-G-Oallyl step I(b1): used
Fmoc-Leu-OH, example II, step IIIa: used Gly- 2ClTrt-resin
Ac-EEVVPnV-(CO)-G-OtBu step Ic: used t-butyl isocyanoacetate
Ac-EEVVP-G(allyl)-(CO)-G-OEt step Ic: used ethyl isocyanoacetate,
step I(b1): used Fmoc- Gly(allyl)-OH Ac-EEVVP-C(Me)-(CO)-G-OMe step
Ic: used methyl isocyanoacetate, step I(b1): used
Boc-Cys(Me)--OH
Example IV
Solid Phase Synthesis of
Ac-EEW-G(N-Bu(4NH2,4-CO2H))-nV-(CO)-G-OH
[0363] 173
[0364] Step I. Synthesis of bromoacetyl-nVal(dpsc)-Gly-PAM Resin
174
[0365] In a solid phase vessel, bromoacetic acid (1.29 g, 9.27
mmol) was coupled to the resin obtained in example I (step If) (1.5
g, 0.77 mmol) in the presence of diisopropylcarbodiimide (1.27 g,
10.04 mmol) and dimethylformamide (10 mL) for five hours. The
reagents were filtered and the reaction was repeated once more for
18 hours, at which time qualitative ninhydrin test showed colorless
beads and solution indicating that the reaction had gone to
completion. All reagents were drained and the resin was washed
thoroughly with 15 mL portions of dimethylformamide, methanol and
dichloromethane.
[0366] Step II. Synthesis of
Gly(N-Bu(4NH-Boc,4-COOtBu)-nVal(dpsc)-Gly-PAM Resin 175
[0367] The product of the previous step (0.12 g, 0.06 mmol) was
treated with Boc-Orn-OtBu.AcOH (0.86 g, 2.47 mmol) in the presence
of diisopropylethylamine (0.05 mL, 0.31 mmol) and dimethylsulfoxide
(1.2 mL) for 18 hours. The reagents were drained and the procedure
was repeated for 5 hours using fresh reagents. All reagents were
drained and the resin was washed thoroughly with 2 mL portions of
dimethylformamide, methanol and dichloromethane.
[0368] Step III. Synthesis of
Ac-Glu-Glu-Val-Val-Gly(N-Bu(4NH2,4-COOH)-nVa- l(CO)-Gly-OH
[0369] The following steps were carried out sequentially:
[0370] a) Fmoc-Val-OH (0.04 g, 0.10 mmol) was double coupled to the
product of step II above (0.12 g, 0.05 mmol) according to Procedure
B.
[0371] b) The resin was deprotected according to Procedure C and
coupled to Fmoc-Val-OH (0.04 g, 0.10 mmol) according to Procedure
B.
[0372] c) The resin was deprotected according to Procedure C and
coupled to Fmoc-Glu(OtBu)-OH (0.04 g, 0.10 mmol) according to
Procedure B.
[0373] d) The resin was deprotected according to Procedure C and
coupled to Fmoc-Glu(OtBu)-OH (0.04 g, 0.10 mmol) according to
Procedure B.
[0374] e) The resin was deprotected according to Procedure C and
acylated at the N-terminus according to Procedure E.
[0375] f) The semicarbazone group of the product obtained in step e
was hydrolyzed according to Procedure F, and the product was
subjected to HF cleavage according to Procedure G. The crude
material was subjected to HPLC purification using a 1.times.25 cm
reverse phase column, containing a C-18 resin comprised of 10
micron size gel particles with a 300 angstrom pore size, eluting
with a 30 minute gradient using 1040% acetonitrile in water.
Analytical HPLC using a 4.6.times.250 mm reverse phase column,
containing a C-18 resin comprised of 5 micron size gel particles
with a 300 angstrom pore size, eluting with 5-75% acetonitrile
(containing 0.1% trifluoroacetic acid) showed one peak at 8
minutes. Low resolution mass spectrum confirmed the presence of the
desired product (MH.sup.+ 873.5).
Table of Compounds Synthesized According to Example IV
[0376]
9 COMPOUND NAME SYNTHESIS Ac-EEVV-G(N-Bu(4NH2,4-CO2H))-nV- example
IV (CO)-G-OH Ac-EEVV-G(N-Et(CO2H))-nV-(CO)-G- step II: used
-Ala(OtBu).HCl OH Ac-EEVV-G(N-EtPh(3,4diOMe))-nV- step II: used
3,4- (CO)-G-OH dimethoxyphenethylamine
Ac-EEVV-G(N-Pe(5-NH2,5-CO2H))-nV- step II: used CBz- (CO)-G-OH
Lys(OBzl).benzene sulfonate
Example V
Solid Phase Synthesis of Ac-EEW-G(N-Et(NHBz))-nV-(CO)-G-OH
[0377] 176
[0378] Step I. Synthesis of Gly(N-Et(NH-Boc))-nVal(dpsc)-Gly-PAM
Resin 177
[0379] The resin obtained in example IV (step I) (0.24 g, 0.12
mmol) was treated with t-butyl N-(2-aminoethyl)-carbamate (0.78 mL,
4.94 mmol) in the presence of diisopropylethylamine (0.11 mL, 0.62
mmol) and dimethylsulfoxide (2.4 mL) for 18 hours. The reagents
were drained and the procedure was repeated for 5 hours using fresh
reagents. All reagents were drained and the resin was washed
thoroughly with 2 mL portions of dimethylformamide, methanol and
dichloromethane.
[0380] Step II. Synthesis of
Fmoc-Val-Gly(N-Et(NH-Boc))-nVal(dpsc)Gly-PAM Resin 178
[0381] Fmoc-Val-OH (0.07 g, 0.21 mmol) was double coupled to the
product of step I above (0.24 g, 0.10 mmol) according to Procedure
B.
[0382] Step III. Synthesis of
Fmoc-Val-Gly(N-Et(NHBz))-nVal(dpsc)-Gly-PAM Resin 179
[0383] The product obtained from step II above (0.12 g, 0.06 mmol)
was treated with a 1:1 mixture of trifluoroacetic acid:
dichloromethane according to Procedure D. The free amine generated
was then capped with the addition of benzoyl chloride (0.02 mL,
0.19 mmol) in the presence of diisopropylethylamine (0.06 mL, 0.37
mmol) in N-methylpyrrolidine (1.24 mL).
[0384] Step IV. Synthesis of
Ac-Glu-Glu-Val-Val-Gly(N-Et(NHBz))-nVal(CO)-G- ly-OH
[0385] The following steps were carried out sequentially:
[0386] a) The resin obtained in step III above was deprotected
according to Procedure C and coupled to Fmoc-Val-OH (0.04 g, 0.10
mmol) according to Procedure B.
[0387] b) The resin obtained in the previous step was deprotected
according to Procedure C and coupled to Fmoc-Glu(OtBu)-OH (0.04 g,
0.10 mmol) according to Procedure B.
[0388] c) The resin obtained in the previous step was deprotected
according to Procedure C and coupled to Fmoc-Glu(OtBu)-OH (0.04 g,
0.10 mmol) according to Procedure B.
[0389] d) The resin obtained in the previous step (0.12 g) was
deprotected according to Procedure C and acylated at the N-terminus
according to Procedure E.
[0390] e) The semicarbazone group of the product obtained in step d
was hydrolyzed according to procedure F, and the product was
subjected to HF cleavage according to procedure G. Analytical HPLC
using a 4.6.times.250 mm reverse phase column, containing a C-18
resin comprised of 5 micron size gel particles with a 300 angstrom
pore size, ran at 5-50% acetonitrile (containing 0.1%
trifluoroacetic acid) showed one peak at 14 minutes. Low resolution
mass spectrum confirmed the presence of the desired product
(MH.sup.+ 905.5).
Table of Compounds Synthesized According to Example V
[0391]
10 COMPOUND NAME SYNTHESIS Ac-EEVV-G(N-Et(NHBz))-nV-(CO)-G- example
V OH Ac-EEVV-G(N-Et(NHBzl(3-OPh)))-nV- step III: used 3-phenoxy-
(CO)-G-OH benzoic acid as capping group Ac-EEVV-G(N-Prop(NHBz))-nV-
-(CO)-G- step I: used t-butyl N-(2- OH aminopropyl)-carbamate
Example VI
[0392] Solid Phase Synthesis of Ac-EEWP-nV(CO)-Am 180
[0393] Step I. Formation of HOBt Ammonium Salt
[0394] Ammonium hydroxide (0.5 mL) was added dropwise to a slurry
of HOBt (2 g, 13.07 mmol) in water (5 mL). The mixture was stirred
at room temperature until a clear solution was obtained. The
product was precipitated by the slow addition of acetone (50 mL).
It was then filtered on a glass funnel and washed thoroughly with
cold acetone (white powder, 2.23 g, 78%; mp 177-181.degree.
C.).
[0395] Step II. Synthesis of HCl.H-nVal-(CHOH)-CONH2 181
[0396] Boc-nVal-(CHOH)--COOH (295 mg, 1.19 mmol) (example V, step
II) was reacted with the product of the previous step (362 mg, 2.38
mmol) in the presence of EDC (342 mg, 1.78 mmol) in
dimethylformamide (10 mL) at room temperature for 18 hours. The
reaction mixture was concentrated and the remaining residue was
picked up in ethylacetate (5 mL) and washed three times each with 5
mL portions of 1N sodium bisulfate, saturated sodium bicarbonate
and brine. The organic layer was dried over sodium sulfate and
concentrated to a white solid (170 mg, 58%). After drying in vacuo,
the product was treated with SN anhydrous HCl in ethylacetate (5
mL) for 1 hour and concentrated (120 mg, 94%).
[0397] Step III. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal-(CHO- H)-CONH2
[0398] The product obtained from step II above (19 mg, 0.103 mmol)
was coupled to Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH (example II,
step IIIf) (50 mg, 0.069 mmol) in the presence of HOAt (14.1 mg,
0.103 mmol), HATu (28.8 mg, 0.076 mmol), diisopropylethylamine (60
.mu.l, 0.345 mmol) in dimethylformamide (10 mL) for 4 hours at room
temperature. The DMF was removed under reduced pressure and the
remaining residue was picked up in ethylacetate and washed with 1N
sodium bisulfate, saturated sodium bicarbonate and brine. After
drying over sodium sulfate it was concentrated to give a white
solid (40 mg, 68%) which was taken to the next step without further
purification. Low resolution mass spectrum confirmed the desired
mass (M+Na.sup.+ 876.5).
[0399] Step IV. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal-(CO)-- CONH2
[0400] Under a stream of nitrogen gas, the product of the previous
step (40 mg, 0.047 mmol) was dissolved in dimethylsulfoxide (4 mL)
and toluene (4 mL). Water soluble carbodiimide (EDC, 89.8 mg, 0.47
mmol) was then added in one batch. The reaction mixture was cooled
to 0.degree. C. and dichloroacetic acid (20 l, 0.23 mmol) was added
dropwise. Stirring at 0.degree. C. continued for 15 minutes. The
ice bath was removed and the reaction was slowly brought to room
temperature. The reaction was stopped after 90 minutes. The toluene
was removed under reduced pressure. The reaction was diluted with
ethylacetate and washed with 1N sodium bisulfate, saturated sodium
bicarbonate and brine. It was then concentrated to a yellowish foam
(40 mg, 53%) and taken to the next step without further
purification. Low resolution mass spectrum confirmed the desired
mass (M+Na.sup.+ 852.5).
[0401] Step V. Synthesis of
Ac-Glu-Glu-Val-Val-Pro-nVal-(CO)-CONH2
[0402] The product of the previous step (39.9 mg, 0.047 mmol) was
treated with a 1:1 mixture of dichloromethane: trifluoroacetic acid
(10 mL) for two hours. The reaction mixture was concentrated and
the remaining residue was subjected to HPLC purification using a
1.times.25 cm reverse phase column, containing a C-18 resin
comprised of 10 micron size gel particles with a 300 angstrom pore
size, eluting with a 30 minute gradient using 5-25% acetonitrile in
water. The purified fractions were pulled and lyophilized to a
white powder (3.6 mg, 10%). Low resolution mass spectrum confirmed
the desired mass (MH.sup.+ 740.0).
Table of Compounds Synthesized According to Example VI
[0403]
11 COMPOUND NAME SYNTHESIS Ac-EEVVP-nV-(CO)-Am example VI
Example VII
Solid Phase Synthesis of
Ac-EEW-P(4t-MeNHBzl(3-OPh))-nV-(CO)-G-OH
[0404] 182
[0405] Step I. Synthesis of H-Pro(4t-MeNHFmoc)-nVal-(dpsc)-Gly-PAM
Resin 183
[0406] The resin obtained from example I (step I) (0.70 g, 0.36
mmol) was coupled with Boc-Pro(4t-MeNHFmoc)-OH according to
procedure B for 18 hours, with 99.98% efficiency. The resin was
then subjected to deprotection according to procedure D to obtain
the title product.
[0407] Step II. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val
-Pro(4t-MeNHFmoc)-nVal-(dpsc)-Gly-PAM Resin
[0408] The resin obtained from the previous step (0.7 g, 0.29 mmoJ)
was double coupled to Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-OH (0.45 g,
0.72 mmol) (obtained similar to example II (step II) starting from
H-Val-2ClTrt-resin) according to procedure B.
[0409] Step III. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro(4t-MeNH2)- -nVal-(dpsc)-Gly-PAM
Resin 184
[0410] The Fmoc side chain protecting group of the product obtained
from the previous step was removed according to procedure C to
afford the title compound.
[0411] Step IV. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro(4t-MeNHBzl-
(3-OPh))-nVal-(dpsc)-Gly-PAM Resin
[0412] The resin obtained from the previous step (0.15 g, 0.05
mmol) was coupled to 3-phenoxy benzoic acid (0.02 g, 0.10 mmol)
according to procedure B with 99.97% efficiency.
[0413] Step V. Synthesis of
Ac-Glu-Glu-Val-Val-Pro(4t-MeNHBzl(3-OPh))-nVal- -(CO)-Gly-OH
[0414] The resin obtained from the previous step was subjected to
semicarbazone hydrolysis according to procedure F, followed by HF
cleavage according to procedure H to yield the crude product. The
material was subjected to HPLC purification using a 1.times.25 cm
reverse phase column, containing a C-18 resin comprised of 10
micron size gel particles with a 300 angstrom pore size, eluting
with a 30 minute gradient ranging from 10-35% acetonitrile in
water. Analytical HPLC using a 4.6.times.250 mm reverse phase
column, containing a C-18 resin comprised of 5 micron size gel
particles with a 300 angstrom pore size, eluting with 5-75%
acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak
at 14.5 minutes. Low resolution mass spectrum confirmed the
presence of the desired product (MH.sup.+ 1049.5).
Table of Compounds Synthesized According to Example VII
[0415]
12 COMPOUND NAME SYNTHESIS Ac-EEVV-P(4t-MeNHBzl(3-OPh))- example
VII nV-(CO)-G-OH Ac-EEVV-P(4t-MeNHCO2Ph)-nV- step IV: used phenyl
(CO)-G-OH chloroformate, DIEA, NMP Ac-EEVV-P(4t-MeNHCOPh)-nV- step
IV: used benzoyl chloride, (CO)-G-OH DIEA, NMP
Ac-EEVV-P(4t-MeNH-Fmoc)-nV- omitted steps III and IV (CO)-G-OH
Ac-EEVV-P(4t-MeNHSO2Ph)-nV- step IV: used benzenesulfonyl (CO)-G-OH
chloride, 2,4,6-collidine, NMP Ac-EEVV-P(4t-MeUreaPh)-nV- - step
IV: used phenyl isocyanate, (CO)-G-OH DIEA, NMP
Ac-EEVV-P(4t-NH-Fmoc)-nV- step I: used Boc-Pro(4t-NH-Fmoc)-
(CO)-G-OH OH
Example VIII
Solid Phase Synthesis of Ac-EEW-P(4t-NHBZI)-nV-(CO)-G-OH
[0416] 185
[0417] Step I. Synthesis of Boc-Pro(4t-NH2)-nVal-(dpsc)-Gly-PAM
Resin 186
[0418] The resin obtained from example I (step I) (1.3 g, 0.67
mmol) was coupled with Boc-Pro(4t-NHFmoc)-QH (0.61 g, 1.34 mmol)
according to procedure B for 18 hours, at which time qualitative
ninhydrin test confirmed completion of the reaction. The resin was
then subjected to deprotection according to Procedure C to obtain
the title product.
[0419] Step II. Synthesis of Boc-Pro(4t-NHBzl)-nVal-(dpsc)-Gly-PAM
Resin 187
[0420] The resin obtained from the previous step (0.13 g, 0.05
mmol) was transferred to a fritted polypropylene tube and was
suspended in N-methylpyrrolidine (1.5 mL). It was then capped with
benzoyl chloride (0.02 mL, 0.16 mmol) in the presence of
diisopropylethylamine (0.05 mL, 0.32 mmol) for four hours to yield
the title product.
[0421] Step III. Synthesis of
Fmoc-Val-Pro(4t-NHBzl)-nVal-(dpsc)-Gly-PAM resin
[0422] The compound of the previous step (100 mg, 0.05 mmol) was
deprotected according to Procedure D. A ninhydrin assay on a small
aliquot gave dark blue resin and solution showing a high yield for
the deprotection. The resin was resuspended in N-methylpyrrolidine
(1.47 mL) and coupled to Fmoc-Val-OH (0.03 g, 0.10 mmol) according
to Procedure B. A small aliquot was taken for qualitative ninhydrin
analysis which showed colorless beads and a dark red solution
indicating a high yield of coupling.
[0423] Step IV. Synthesis of
Fmoc-Val-Val-Pro(4t-NHBzl)-nVal-(dpsc)-Gly-PA- M resin
[0424] The compound of the previous step (100 mg) was deprotected
according to Procedure D. A ninhydrin assay on a small aliquot gave
dark blue resin and solution showing a high yield for the
deprotection. The resin was resuspended in N-methylpyrrolidine
(1.47 mL) and was coupled to Fmoc-Val-OH (0.03, 0.10 mmol) as in
step III.
[0425] Step V. Synthesis of
Fmoc-Glu(OtBu)-Val-Val-Pro(4t-NHBzl)-nVal-(dps- c)-Gly-PAM
Resin
[0426] The compound of the previous step (100 mg, 0.03 mmol) was
deprotected according to Procedure D. A ninhydrin assay on a small
aliquot gave dark blue resin and solution showing a high yield for
the deprotection. The resin was resuspended in N-methylpyrrolidine
(1.47 mL) and was coupled to Fmoc-Glu(OtBu)-OH (0.04 g, 0.10 mmol),
according to Procedure B for 5 hours. A small aliquot was taken for
qualitative ninhydrin analysis which showed colorless beads and
solution indicating a high yield of coupling.
[0427] Step VI. Synthesis of
Fmoc-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro(4t-NHBzl-
)-nVal-(dpsc)-Gly-PAM Resin
[0428] The compound of the previous step (100 mg) was deprotected
according to Procedure D and coupled to Fmoc-Glu(OtBu)-OH (0.04 g,
0.10 mmol) in the same manner.
[0429] Step VII. Synthesis of
Ac-Glu-Glu-Val-Val-Pro(4t-NHBzl)-nVal-(CO)-G- ly-OH
[0430] The compound of previous step (100 mg) was deprotected
according to Procedure C and acylated according to Procedure E. The
resin was vacuum dried and a small aliquot was taken for
qualitative ninhydrin analysis which showed colorless beads and
solution indicating a high yield of coupling. The resin was then
subjected to semicarbazone hydrolysis followed by HF cleavage
reactions according to Procedures F and H, respectively. The crude
product was subjected to HPLC purification using a 1.times.25 cm
reverse phase column, containing a C-18 resin comprised of 10
micron size gel particles with a 300 angstrom pore size, eluting
with a 30 minute gradient using 10-40% acetonitrile in water.
Analytical HPLC using a 4.6.times.250 mm reverse phase column,
containing a C-18 resin comprised of 5 micron size gel particles
with a 300 angstrom pore size, ran at 5-50% acetonitrile
(containing 0.1% trifluoroacetic acid) showed one peak at 13
minutes. Low resolution mass spectrum confirmed the presence of the
desired product (MH.sup.+ 917.5).
Table of Compounds Synthesized According to Example VIII
[0431]
13 COMPOUND NAME SYNTHESIS Ac-EEVV-P(4t-NHBzl)-nV-(CO)-G-OH example
VIII Ac-EEVV-P(4t-NHBzl(4-OMe))-nV- step II: used 4-methoxy-
(CO)-G-OH benzoyl chloride, DIEA, NMP
Ac-EEVV-P(4t-NHBzl(4-OPh))-nV- step II: used 4-phenoxy- (CO)-G-OH
benzoic acid Ac-EEVV-P(4t-NHBzl(3-OPh))-nV- step II: used
3-phenoxy- (CO)-G-OH benzoic acid Ac-EEVV-P(4t-NHBzl(3,4-OMeO))-nV-
step II: used piperonyloyl (CO)-G-OH chloride, DIEA, NMP
Ac-EEVV-P(4t-NHBzl(4F))-nV-(CO)-G- step II: used 4-fluoro- OH
benzoyl chloride, DIEA, NMP Ac-EEVV-P(4t-NHiBoc)-nV-(CO)-G-OH step
II: used isobutyl chloroformate, DIEA, NMP
Ac-EEVV-P(4t-NHSO2Ph)-nV-(CO)-G- step II: used benzene sulfonyl OH
chloride, 2,4,6-collidine, NMP Ac-EEVV-P(4t-NHSO2Ph(4-OMe)- )-nV-
step II: used 4-methoxy- (CO)-G-OH benzene sulfonyl chloride,
2,4,6-collidine, NMP Ac-EEVV-P(4t-UreaPh)-nV-(CO)-G-OH step II:
used phenyl isocyanate, DIEA, NMP Ac-EEVV-P(4t-UreaPh(4-OMe))-nV-
step II: used 4-methoxyphenyl (CO)-G-OH isocyanate, DIEA, NMP
Example IX: Solution Phase Synthesis of Ac-EEWP-nV-CO)--OH
[0432] 188
[0433] Step I. Synthesis of ethyl (R,S)-2-hydroxy-3-amino hexanoate
hydrochloride 189
[0434] The product obtained in example I(step Ib) (1.99 g, 6.59
mmol) was refluxed in 6N HCl (42 mL) for four hours. After cooling
to room temperature, the reaction mixture was extracted with
dichloromethane (3.times.30 mL) and the aqueous layer was
concentrated to dryness (1.65 g crude). Some of the resulting
product (0.88 g, 4.8 mmol) was stirred in 20 mL saturated solution
of anhydrous HCl in ethanol for 90 minutes. The mixture was
concentrated to a white solid (0.95 g, 93%).
[0435] Step II. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH)- -OEt
[0436] The product obtained from the previous step (88 mg, 0.41
mmol) was coupled to Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH (example
II, step IIIf) (200 mg, 0.26 mmol) in the presence of HOAt (56.3
mg, 0.41 mmol), HATu (115.3 mg, 0.30 mmol), diisopropylethylamine
(240 1, 1.37 mmol) in dimethylformamide (10 mL) for 18 hours at
room temperature. The DMF was removed under reduced pressure and
the remaining residue was subjected to HPLC purification using a
2.2.times.25 cm reverse phase column, containing a C-18 resin
comprised of 10 micron size gel particles with a 300 angstrom pore
size, eluting with a 30 minute gradient ranging from 15-50%
acetonitrile in water. The desired fractions were pulled and
concentrated to a solid (67 mg, 27.5%). Analytical HPLC using a
4.6.times.250 mm reverse phase column, containing a C-18 resin
comprised of 5 micron size gel particles with a 300 angstrom pore
size, eluting with 5-50% acetonitrile (containing 0.1 %
trifluoroacetic acid) showed one peak at 23.5 minutes. Low
resolution mass spectrum confirmed the desired mass (MH.sup.+
883.5).
[0437] Step III. Synthesis of
Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH- )-carboxylic acid
[0438] To the product obtained in the previous step (67 mg, 0.076
mmol) dissolved in ethanol (3.8 mL) was added 1N lithium hydroxide
(3041, 0.304 mmol) and the reaction was stirred at room temperature
for 90 minutes. The reaction was stopped by the addition of enough
Dowex ion exchange resin (50.times.8-400) to obtain an acidic
solution, pH .about.3. After stirring for 15 minutes, the reaction
mixture was filtered and concentrated to a white solid (53.4 mg,
82.2%).
[0439] Step IV. Synthesis of
Ac-Glu-Glu-Val-Val-Pro-nVal(CHOH)-carboxylic acid
[0440] The product obtained in the previous step (53.1 mg) stirred
in a 1:1 mixture of trifluoroacetic acid: dichloromethane (10 mL)
for 90 minutes. The reaction mixture was concentrated to a
yellowish solid (50 mg) which was taken to the next step without
further purification.
[0441] Step V. Synthesis of
Ac-Glu-Glu-Val-Val-Pro-nVal(CO)-carboxylic acid
[0442] The product obtained in the previous step (55.7 mg, 0.075
mmol) was dissolved in dichloromethane (8 mL) and dimethylsulfoxide
(2 mL). Triethylamine (125.5 l, 0.901 mmol) followed by pyridine
sulfur trioxide (143.4 mg, 0.901 mmol) were added and the reaction
was stirred at room temperature for two hours. Dichloromethane was
removed under reduced pressure and the remaining residue was
diluted with methanol (containing 0.1% TFA) and purified on a
reverse phase HPLC column (1.times.25 cm) containing a C-18 resin
comprised of 10 micron size gel particles with a 300 angstrom pore
size, eluting with a 30 minute gradient using 5-15% acetonitrile in
water. The desired fractions were pulled and concentrated to an oil
(15.2 mg, 27.4%). Analytical HPLC using a 4.6.times.250 mm reverse
phase column, containing a C-18 resin comprised of 5 micron size
gel particles with a 300 angstrom pore size, ran at 5-50%
acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak
at 11 minutes. Low resolution mass spectrum confirmed the desired
mass (MH.sup.+ 741.0).
Table of Compound Synthesized According to Example IX
[0443]
14 Ac-EEVVP-nV-(CO)-OH example IX
[0444] Assay for HCV Protease Inhibitory Activity:
[0445] Spectrophotometric Assay: Spectrophotometric assay for the
HCV serine protease was performed on the inventive compounds by
following the procedure described by R. Zhang et al, Analytical
Biochemistry, 270 (1999) 268-275, the disclosure of which is
incorporated herein by reference. The assay based on the
proteolysis of chromogenic ester substrates is suitable for the
continuous monitoring of HCV NS3 protease activity. The substrates
were derived from the P side of the NS5A-NS5B junction sequence
(Ac-DTEDWX(Nva), where X=A or P) whose C-terminal carboxyl groups
were esterified with one of four different chromophoric alcohols
(3- or 4-nitrophenol, 7-hydroxy4-methyl-coumarin, or
4-phenylazophenol). Presented below are the synthesis,
characterization and application of these novel spectrophotometric
ester substrates to high throughput screening and detailed kinetic
evaluation of HCV NS3 protease inhibitors.
[0446] Materials and Methods:
[0447] Materials: Chemical reagents for assay related buffers were
obtained from Sigma Chemical Company (St. Louis, Mo.). Reagents for
peptide synthesis were from Aldrich Chemicals, Novabiochem (San
Diego, Calif.), Applied Biosystems (Foster City, Calif.) and
Perseptive Biosystems (Framingham, Mass.). Peptides were
synthesized manually or on an automated ABI model 431A synthesizer
(from Applied Biosystems). UVNIS Spectrometer model LAMBDA 12 was
from Perkin Elmer (Norwalk, Conn.) and 96-well UV plates were
obtained from Coming (Coming, N.Y.). The prewarming block was from
USA Scientific (Ocala, Fla.) and the 96-well plate vortexer was
from Labline Instruments (Melrose Park, Ill.). A Spectramax Plus
microtiter plate reader with monochrometer was obtained from
Molecular Devices (Sunnyvale, Calif.).
[0448] Enzyme Preparation: Recombinant heterodimeric HCV NS3/NS4A
protease (strain 1a) was prepared by using the procedures published
previously (D. L. Sali et al, Biochemistry, 37 (1998) 3392-3401).
Protein concentrations were determined by the Biorad dye method
using recombinant HCV protease standards previously quantified by
amino acid analysis. Prior to assay initiation, the enzyme storage
buffer (50 mM sodium phosphate pH 8.0, 300 mM NaCl, 10% glycerol,
0.05% lauryl maltoside and 10 mM DTT) was exchanged for the assay
buffer (25 mM MOPS pH 6.5, 300 mM NaCl, 10% glycerol, 0.05% lauryl
maltoside, 5 .mu.M EDTA and 5 pM DTT) utilizing a Biorad Bio-Spin
P-6 prepacked column.
[0449] Substrate Synthesis and Purification: The synthesis of the
substrates was done as reported by R. Zhang et al, (ibid.) and was
initiated by anchoring Fmoc-Nva-OH to 2-chlorotrityl chloride resin
using a standard protocol (K. Barlos et al, Int. J. Pept. Protein
Res., 37 (1991), 513-520). The peptides were subsequently
assembled, using Fmoc chemistry, either manually or on an automatic
ABI model 431 peptide synthesizer. The N-acetylated and fully
protected peptide fragments were cleaved from the resin either by
10% acetic acid (HOAc) and 10% trifluoroethanol (TFE) in
dichloromethane (DCM) for 30 min, or by 2% trifluoroacetic acid
(TFA) in DCM for 10 min. The combined filtrate and DCM wash was
evaporated azeotropically (or repeatedly extracted by aqueous
Na.sub.2CO.sub.3 solution) to remove the acid used in cleavage. The
DCM phase was dried over Na.sub.2SO.sub.4 and evaporated.
[0450] The ester substrates were assembled using standard
acid-alcohol coupling procedures (K. Holmber et al, Acta Chem.
Scand., B33 (1979) 410-412). Peptide fragments were dissolved in
anhydrous pyridine (30-60 mg/ml) to which 10 molar equivalents of
chromophore and a catalytic amount (0.1 eq.) of
para-toluenesulfonic acid (pTSA) were added.
Dicyclohexylcarbodiimide (DCC, 3 eq.) was added to initiate the
coupling reactions. Product formation was monitored by HPLC and
found to be complete following 12-72 hour reaction at room
temperature. Pyridine solvent was evaporated under vacuum and
further removed by azeotropic evaporation with toluene. The peptide
ester was deprotected with 95% TFA in DCM for two hours and
extracted three times with anhydrous ethyl ether to remove excess
chromophore. The deprotected substrate was purified by reversed
phase HPLC on a C3 or C8 column with a 30% to 60% acetonitrile
gradient (using six column volumes). The overall yield following
HPLC purification was approximately 20-30%. The molecular mass was
confirmed by electrospray ionization mass spectroscopy. The
substrates were stored in dry powder form under desiccation.
[0451] Spectra of Substrates and Products: Spectra of substrates
and the corresponding chromophore products were obtained in the pH
6.5 assay buffer. Extinction coefficients were determined at the
optimal off-peak wavelength in 1-cm cuvettes (340 nm for 3-Np and
HMC, 370 nm for PAP and 400 nm for 4-Np) using multiple dilutions.
The optimal off-peak wavelength was defined as that wavelength
yielding the maximum fractional difference in absorbance between
substrate and product (product OD--substrate OD)/substrate OD).
[0452] Protease Assay: HCV protease assays were performed at
30.degree. C. using a 200 .mu.l reaction mix in a 96-well
microtiter plate. Assay buffer conditions (25 mM MOPS pH 6.5, 300
mM NaCl, 10% glycerol, 0.05% lauryl maltoside, 5 .mu.M EDTA and 5
pM DTT) were optimized for the NS3/NS4A heterodimer (D. L. Sali et
al, ibid.)).
[0453] Typically, 150 .mu.l mixtures of buffer, substrate and
inhibitor were placed in wells (final concentration of DMSO 4 %
v/v) and allowed to preincubate at 30.degree. C. for approximately
3 minutes. Fifty pis of prewarmed protease (12 nM, 30.degree. C.)
in assay buffer, was then used to initiate the reaction (final
volume 200 .mu.l).The plates were monitored over the length of the
assay (60 minutes) for change in absorbance at the appropriate
wavelength (340 nm for 3-Np and HMC, 370 nm for PAP, and 400 nm for
4-Np) using a Spectromax Plus microtiter plate reader equipped with
a monochrometer (acceptable results can be obtained with plate
readers that utilize cutoff filters). Proteolytic cleavage of the
ester linkage between the Nva and the chromophore was monitored at
the appropriate wavelength against a no enzyme blank as a control
for non-enzymatic hydrolysis. The evaluation of substrate kinetic
parameters was performed over a 30-fold substrate concentration
range (.about.6-200 .mu.M). Initial velocities were determined
using linear regression and kinetic constants were obtained by
fitting the data to the Michaelis-Menten equation using non-linear
regression analysis (Mac Curve Fit 1.1, K. Raner). Tumover numbers
(k.sub.cat) were calculated assuming the enzyme was fully
active.
[0454] Evaluation of Inhibitors and Inactivators: The inhibition
constants (K.sub.i) for the competitive inhibitors
Ac-D-(D-Gla)-L-I-(Cha)C-OH (27), Ac-DTEDWA(Nva)-OH and
Ac-DTEDWP(Nva)-OH were determined experimentally at fixed
concentrations of enzyme and substrate by plotting v.sub.o/v.sub.i
vs. inhibitor concentration ([I].sub.o) according to the rearranged
Michaelis-Menten equation for competitive inhibition kinetics:
v.sub.o/v.sub.i=1+[I].sub.o/(K.sub.i(1+[S].sub.o/K.sub.m)), where
v.sub.o is the uninhibited initial velocity, v.sub.i is the initial
velocity in the presence of inhibitor at any given inhibitor
concentration ([I].sub.o) and [S].sub.o is the substrate
concentration used. The resulting data were fitted using linear
regression and the resulting slope,
1/(K.sub.i(1+[S].sub.o/K.sub.m), was used to calculate the K.sub.i*
value.
[0455] The obtained K.sub.i* values for the various compounds of
the present invention are given in the Tables wherein the compounds
have been arranged in the order of ranges of K.sub.i* values. From
these test results, it would be apparent to the skilled artisan
that the compounds of the invention have excellent utility as
NS3-serine protease inhibitors.
[0456] While the present invention has been described with in
conjunction with the specific embodiments set forth above, many
alternatives, modifications and other variations thereof will be
apparent to those of ordinary skill in the art. All such
alternatives, modifications and variations are intended to fall
within the spirit and scope of the present invention.
15TABLE 3 Ki* STRUCTURE NAME Range 190 Ac-EEVVP-L- (CO)-G-Oallyl
191 Ac-EEVVP-nL- (CO)-G-Oallyl a 192 Ac-EEVVP-nV- (CO)-G-Oallyl a
193 Ac-EEVVG-L- (CO)-G-Oallyl b 194 Ac-EEVVP-nV- (CO)-G-OEt c 195
Ac-EEVVP-nV- (CO)-G- 2PhEtAm c 196 Ac-EEVVP-nV- (CO)-Am b 197
Ac-EEVVP-nV- (CO)-OH c 198 Ac-EEVVP-nV- (CO)-G-OH a 199
Ac-EEVVP-nV- (CO)-G-OtBu b 200 Ac-EEVVP-nV- (CO)-iPrAm c 201
Ac-EEVVP- G(allyl)-(CO)-G- Oallyl b 202 Ac-EEVVP- G(allyl)-(CO)-G-
OEt b 203 Ac-EEVVP-nV- (CO)-G-allylAm b 204 Ac-EEVVP-nV- (CO)-G-
propynylamide c 205 Ac-EEVVP-nV- (CO)-G-Am c 206 Ac-EEVVP-nV-
(CO)-G- Propylamide c 207 Ac-EEVVP- G(propynyl)- (CO)-G-Oallyl b
208 Ac-EEV-G(Ph)- P-nV-(CO)-G-OH b 209 Ac-EEVV-Sar- nV-(CO)-G-OH b
210 Ac-EEVV-Aze- nV-(CO)-G-OH a 211 Ac-EEVV-P(4t- O-2AcOH)-nV-
(CO)-G-OH a 212 Ac-EEV-G(Chx)- P-nV-(CO)-G-OH a 213 Ac-EEVFP-nV-
(CO)-G-OH b 214 Ac-EEVlP-nV- (CO)-G-OH a 215 Ac-EEVV-dlPip-
nV-(CO)-G-OH a 216 Ac-EEVV-Tiq- nV-(CO)-G-OH a 217 Ac-EEVV-thioP-
nV-(CO)-G-OH a 218 Ac-EEVV-C(Me) nV-(CO)-G-OH a 219 Ac-EEVV-
C(O2,Me)-nV- (CO)-G-OH a 220 Ac-EEVV-C(2- AcOH)-nV-(CO)- G-OH a 221
Ac-EEVV- M(O2)-nV-(CO)- G-OH b 222 Ac-EEVVP- C(Me)-(CO)-G- OMe c
223 Ac-EEVV-P(4t- MeNHCO2Ph)- nV-(CO)-G-OH a 224 Ac-EEVV-P(4t-
MeNHCOPh)- nV-(CO)-G-OH a 225 Ac-EEVV-P(4t- MeNH-Fmoc)-
nV-(CO)-G-OH a 226 Ac-EEVV-P(4t- MeNHBzl(3- OPh))-nV-(CO)- G-OH a
227 Ac-EEVV-P(4t- MeNHSO2Ph)- nV-(CO)-G-OH a 228 Ac-EEVV-P(4t-
NH-Fmoc)-nV- (CO)-G-OH a 229 Ac-EEVV-P(4t- MeUreaPh)-nV- (CO)-G-OH
a 230 Ac-EEVV-P(4t- NHBzl)-nV-(CO) G-OH a 231 Ac-EEVV-P(4t-
NHBzl(4-OMe))- nV-(CO)-G-OH a 232 Ac-EEVV-P(4t- NHBzl(4-OPh))-
nV-(CO)-G-OH a 233 Ac-EEVV-P(4t- NHBzl(3-OPh))- nV-(CO)-G-OH a 234
Ac-EEVV-P(4t- Bn)-nV-(CO)- G-OH a 235 Ac-EEVV-P(4t- Bn(4-OMe))-nV-
(CO)-G-OH a 236 Ac-EEVV-P(4t- allyl)-nV-(CO)- G-OH a 237
Ac-EEVV-P(4t- NHBzl(3,4- OMeO))-nV- (CO)-G-OH a 238 Ac-EEVV-P(4t-
NHBzl(4F))-nV- (CO)-G-OH a 239 Ac-EEVV-P(4t- NHiBoc)-nV- (CO)-G-OH
a 240 Ac-EEVV-P(4t- NHSO2Ph)-nV- (CO)-G-OH a 241 Ac-EEVV-P(4t-
NHSO2Ph(4- OMe))-nV-(CO)- G-OH a 242 Ac-EEVV-P(4t- UreaPh)-nV-
(CO)-G-OH a 243 Ac-EEVV-P(4t- UreaPh(4- OMe))-nV-(CO)- G-OH a 244
Ac-EEVVD-nV- (CO)-G-OH b 245 Ac-EEVVE-nV- (CO)-G-OH a 246
Ac-EEVVF-nV- (CO)-G-OH a 247 Ac-EEVV-P(4t- NH2)-nV-(CO)- G-OH b 248
Ac-EEVV-P(4t- AcOH)-nV-(CO)- G-OH a 249 Ac-EESVP-nV- (CO)-G-OH b
250 Ac-EAVVP-nV- (CO)-G-OH a 251 Ac-EEHVP-nV- (CO)-G-OH b 252
Ac-EENVP-nV- (CO)-G-OH b 253 Ac-EEVV-P(4t- Ph)-nV-(CO)-G- OH a 254
Ac-EEVV-P(3t- Me)-nV-(CO)-G- OH a 255 Ac-EEVV-G(N- Et(CO2H))-nV-
(CO)-G-OH a 256 Ac-EEVV-G(N- EtPh(3,4diOMe))- nV-(CO)-G-OH b 257
Ac-EEVV-G(N- Bu(4NH2,4- CO2H))-nV- (CO)-G-OH a 258 Ac-EE-Orn-VP-
nV-(CO)-G-OH b 259 Ac-EdEVVP-nV- (CO)-G-OH a 260 Ac-EE-(s,s)alloT
VP-nV-(CO)-G- OH a 261 Ac-EE-Dif-VP- nV-(CO)-G-OH a 262
Ac-EE-daba-VP- nV-(CO)-G-OH b 263 Ac-EEDVP-nV- (CO)-G-OH c 264
Ac-EEEVP-nV- (CO)-G-OH b 265 Ac-EETVP-nV- (CO)-G-OH b 266
Ac-AEVVP-nV- (CO)-G-OH b 267 Ac-EELVP-nV- (CO)-G-OH a 268
Ac-EEVV-G(N- Et(NHBz))-nV- (CO)-G-OH b 269 Ac-EEVV-G(N- Et(NHBzl(3-
OPh)))-nV-(CO)- G-OH a 270 Ac-EEVV-G(N- Prop(NHBz))-nV- (CO)-G-OH b
271 Ac-EEVV-G(N- Pe(5-NH2,5- CO2H))-nV- (CO)-G-OH a 272 Ac-EEA(1-
Np)VP-nV-(CO)- G-OH b 273 Ac-EEA(2- Np)VP-nV-(CO)- G-OH b 274
Ac-EEhSVP-nV- (CO)-G-OH c 275 Ac-EEF(alpha- Me)VP-nV-(CO)- G-OH c
276 Ac-EEVLP-nV- (CO)-G-OH b 277 Ac-EEVG(t- Bu)P-nV-(CO)- G-OH a
278 Ac-EEVSP-nV- (CO)-G-OH c 279 A-EEVTP-nV- (CO)-G-OH c 280
Ac-EEV-nL-P- nV-(CO)-G-OH b 281 Ac-EEVDifP-nV- (CO)-G-OH b 282
Ac-EEVS(Me)P- nV-(CO)-G-OH c 283 Ac-EEVNP-nV- (CO)-G-OH c 284
Ac-EEVQP-nV- (CO)-G-OH c 285 Ac-EEFVP-nV- (CO)-G-OH b 286
Ac-EEVMP-nV- (CO)-G-OH b 287 Ac-EEVCP-nV- (CO)-G-OH b
* * * * *