A racemate diastereoisomer and optical isomer of a compound of formula (I):
##STR00001##
wherein B is H, a C6 or C10 aryl, C7-16 aralkyl; Het or (lower alkyl)-Het, all of which may be optionally substituted with C1-6 alkyl; C1-6 alkoxy; C1-6 alkanoyl; hydroxy; hydroxyalkyl; halo; haloalkyl; nitro; cyano; cyanoalkyl; amino optionally substituted with C1-6 alkyl; amido; or (lower alkyl)amide; or
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1. A racemate, diastereoisomer or optical isomer of a compound of formula (I):
##STR00319##
wherein B is H, a C6 or C10 aryl, C7-16 aralkyl; Het or (lower alkyl)-Het, all of which optionally substituted with C1-6 alkyl; C1-6 alkoxy; C1-6 alkanoyl; hydroxy; hydroxyalkyl; halo; haloalkyl; nitro; cyano; cyanoalkyl; amino optionally substituted with C1-6 alkyl; amido; or (lower alkyl)amide;
or B is an acyl derivative of formula r4—C(O)—; a carboxyl derivative of formula r4—O—C(O)—; an amide derivative of formula r4—N(r5)—C(O)—; a thioamide derivative of formula r4—N(r5)—C(S)—; or a sulfonyl derivative of formula r4—SO2 wherein
r4 is (i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxyl, C1-6 alkoxy, amino optionally mono- or di-substituted with C1-6 alkyl, or amido, or (lower alkyl) amide ; and when B is r4—O—C(O)—; r4—N(r5)—C(O)—; r4—N(r5)—C(S)—; or r4—SO2, then r4 may additionally be selected from C1-10 alkyl substituted with (lower alkyl) amide;
(ii) C3-7 cycloalkyl, or C3-7 cycloalkoxy, or C4-10 alkylcycloalkyl, all optionally substituted with hydroxy, carboxyl, (C1-6 alkoxy)carbonyl, amino optionally mono- or di-substituted with C1-6 alkyl, amido, or (lower alkyl) amide; or r4 is C4-10 alkylcycloalkyl, optionally substituted with hydroxy, carboxyl, (C1-6 alkoxy)carbonyl, amino optionally mono- or di-substituted with C1-6 alkyl, or amido; and when B is r4—O—C(O)—; r4—N(r5)—C(O)—; r4—N(r5)—C(S)—; r4—SO2, then r4 may additionally be C4-10 alkylcycloalkyl substituted with (lower alkyl) amide;
(iii) amino optionally mono- or di-substituted with C1-6 alkyl; amido; or (lower alkyl)amide;
(iv) C6 or C10 aryl or C7-16 aralkyl, all optionally substituted with C1-6 alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C1-6 alkyl; or
(v) Het or (lower alkyl)-Het, both optionally substituted with C1-6 alkyl, hydroxy, amido, (lower alkyl) amide, or amino optionally mono- or di-substituted with C1-6 alkyl;
r5 is H or C1-6 alkyl; with the proviso that when B is a carboxyl derivative, an amide derivative or a thioamide derivative, r4 is not a cycloalkoxy;
Y is H or C1-6 alkyl;
r3 is C1-8 alkyl, C3-7 cycloalkyl, or C4-10 alkylcycloalkyl, all optionally substituted with hydroxy, C1-6 alkoxy, C1-6 thioalkyl, amido, (lower alkyl)amido, C6 or C10 aryl, or C7-16 aralkyl;
r2 is CH2—R20, NH—R20, O—R20 or S—R20, wherein r20 is pyridinyl, quinolyl, (lower alkyl)-pyridinyl or (lower alkyl)-quinolyl, each optionally mono-, di- or tri-substituted with r21,
wherein each r21 is independently C1-6 alkyl; C1-6 alkoxy; lower thioalkyl; sulfonyl; NO2; OH; SH; halo; haloalkyl; amino optionally mono- or di-substituted with C1-6 alkyl, C6 or C10 aryl, C7-14 aralkyl, Het or (lower alkyl)-Het; amido optionally mono-substituted with C1-6 alkyl, C6 or C1-10 aryl, C6 or C10 aryl, C7-14 aralkyl, Het or (lower alkyl)-Het; carboxyl; carboxy(lower alkyl); C6 or C10 aryl, C7-14 aralkyl or Het, said aryl, aralkyl or Het being optionally substituted with r22;
wherein r22 is C1-6 alkyl; C3-7 cycloalkyl; C1-6 alkoxy; amino optionally mono- or di-substituted with C1-6 alkyl; sulfonyl; (lower alkyl)sulfonyl; NO2; OH; SH; halo; haloalkyl; carboxyl; amide; (lower alkyl)amide; or Het optionally substituted with C1-6 alkyl;
r1 is H, C1-6 alkyl, C3-7 cycloalkyl, C2-6 alkenyl, or C2-6 alkynyl, all optionally substituted with halogen;
or a pharmaceutically acceptable salt or ester thereof; wherein “Het” is defined as a five-membered saturated or unsaturated, aromatic or non-aromatic, heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur, wherein said heterocycle is optionally fused to a benzene ring.
2. A compound of formula I according to
B is a C6 or C10 aryl or C7-16 aralkyl, all optionally substituted with C1-6 alkyl, C1-6 alkoxy, C1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substituted with C1-6 alkyl; or
B is Het or (lower alkyl)-Het, all optionally substituted with C1-6 alkyl, C1-6 alkoxy, C1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substituted with C1-6 alkyl.
3. A compound of formula I according to
4. A compound of formula I according to
(i) C1-10 alkyl optionally substituted with carboxyl, hydroxy or C1-6 alkoxy, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C1-6 alkyl;
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl, (C1-6 alkoxy) carbonyl, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C1-6 alkyl; or r4 is C4-10 alkylcycloalkyl optionally substituted with hydroxy, carboxyl, (C1-6 alkoxy)carbonyl, amido, or amino optionally mono- or di-substituted with C1-6 alkyl;
(iv) C6 or C10 aryl or C7-16 aralkyl, all optionally substituted with C1-6 alkyl, hydroxy, amido, (lower alkyl) amide, or amino optionally substituted with C1-6 alkyl;
(v) Het or (lower alkyl)-Het, both optionally substituted with C1-6 alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionally substituted with C1-6 alkyl.
5. A compound of formula I according to
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxy, C1-6 alkoxy, amino optionally mono- or di-substituted with C1-6 alkyl, amido or (lower alkyl)amide;
(ii) C3-7 cycloalkyl, or C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy)carbonyl, amino optionally mono- or di-substituted with C1-6 alkyl, amido or (lower alkyl)amide;
(iv) C6 or C10 aryl or C7-16 aralkyli, all optionally substituted with C1-6 alkyl, hydroxy, amido, (lower alkyl)amido, or amino optionally mono- or di-substituted with C1-6 alkyl; or
(v) Het or (lower alkyl)-Het, both optionally substituted with C1-6 alkyl, hydroxy, amino optionally mono- or di-substituted with C1-6 alkyl, amido or (lower alkyl) amido.
6. A compound of formula I according to
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxy, C1-6 alkoxy, amido, (lower alkyl) amido, or amino optionally mono- or di-substituted with C1-6 alkyl;
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy)carbonyl, amido, (lower alkyl)amido, or amino optionally mono- or di-substituted with C1-6 alkyl;
(iii) amino optionally mono- or di-substituted with C1-3 alkyl;
(iv) C6 or C10 aryl or C7-16 aralkyl, optionally substituted with C1-6 alkyl, hydroxy, amido, (lower alkyl) amide, or amino optionally substituted with C1-6 alkyl; or
(v) Het or (lower alkyl)-Het, both optionally substituted with C1-6 alkyl, hydroxy, amino optionally substituted with C1-6 alkyl, amido or (lower alkyl)amide; and
r5 is H or methyl.
7. A compound of formula I according to
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl or C1-6 alkoxy;
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy)carbonyl, amino or amido.
8. A compound of formula I according to
9. A compound of formula I according to
10. A compound of formula I according to
(i) C1-10 alkyl optionally substituted with carboxyl, hydroxy or C1-6 alkoxy; or
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl, (C1-6 alkoxy) carbonyl, or
(iv) C6 or C10 aryl or C7-16 aralkyl, all optionally substituted with C1-6 alkyl, hydroxy, or (v) Het optionally substituted with C1-6 alkyl, hydroxy, amido or amino.
11. A compound of formula I according to
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxy, C1-6 alkoxy or amido, (lower alkyl) amide, amino optionally mono- or di-substituted with C1-6 alkyl;
(ii) C3-7 cycloalkyl, C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C1-6 alkyl, or
(iv) C6 or C10 aryl or C7-16 aralkyl, optionally substituted with C1-6 alkyl, hydroxy, amino optionally substituted with C1-6 alkyl; or
(v) Het or (lower alkyl)-Het, both optionally substituted with C1-6 alkyl, hydroxy, amido, or amino optionally mono-substituted with C1-6 alkyl.
12. A compound of formula I according to
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxy, C1-6 alkoxy, amido, (lower alkyl) amide, amino optionally mono- or di-substituted with C1-6 alkyl;
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C1-6 alkyl;
(iii) amino optionally mono- or di-substituted with C1-3 alkyl, or
(iv) C6 or C10 aryl or C7-16 aralkyl, optionally substituted with C1-6 alkyl, hydroxy, amino or amido optionally substituted with C1-6 alkyl; or
(v) Het optionally substituted with C1-6 alkyl, hydroxy, amino or amido, and r5 is H.
13. A compound of formula I according to
14. A compound of formula I according to
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxy, C1-6 alkoxy amido, (lower alkyl) amide, amino optionally mono- or di-substituted with C1-6 alkyl;
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C1-6 alkyl;
(iv) C6 or C10 aryl or C7-16 aralkyl optionally substituted with C1-6 alkyl, hydroxy, amino or amido.
18. A compound of formula I according to
19. A compound of formula I according to
21. A compound of formula I according to
r21 is C1-6 alkyl; C1-6 alkoxy; lower thioalkyl; amino or amido optionally mono- or di-substituted with C1-6 alkyl, C6 or C10 aryl, C7-16 aralkyl, Het or (lower alkyl)-Het; NO2; OH; halo; trifluoromethyl; carboxyl; C6 or C10 aryl, C7-16 aralkyl, or Het, said aryl, aralkyl or Het being optionally substituted with r22, wherein r22 is C1-6 alkyl; C3-7 cycloalkyl; C1-6 alkoxy; amino; mono- or di-(lower alkyl)amino; (lower alkyl)amide; sulfonylalkyl; NO2; OH; halo; trifluoromethyl; carboxyl or Het.
22. A compound of formula I according to
23. A compound of formula I according to
24. A compound of formula I according to
26. A compound of formula I according to
27. A compound of formula I according to
28. A compound of formula I according to
wherein r21A is C1-6 alkyl; C1-6 alkoxy; lower thioalkyl; halo; amino optionally mono-substituted with C1-6 alkyl; or C6, C10 aryl, C7-16 aralkyl, or Het, said aryl, aralkyl or Het optionally substituted with r22 wherein r22 is C1-6 alkyl, C1-6 alkoxy, amido, (lower alkyl) amide, amino optionally mono- or di-substituted with C1-6 alkyl, or Het; and
r21B is C1-6 alkyl, C1-6 alkoxy, amino, di(lower alkyl) amino, (lower alkyl)amide, NO2, OH, halo, trifluoromethyl, or carboxyl.
29. A compound of formula I according to
30. A compound of formula I according to
31. A compound of formula I according to
wherein r22A is C1-6 alkyl; C1-6 alkoxy; or halo; and r21B is C1-6 alkyl, C1-6 alkoxy, amino, di(lower alkyl)amino, (lower alkyl)amide, NO2, OH, halo, trifluoromethyl, or carboxyl.
wherein r22B is C1-6 alkyl, amino optionally mono-substituted with C1-6 alkyl, amido, or (lower alkyl)amide and r21B is C1-6 alkyl, C1-6 alkoxy, amino, di(lower alkyl) amino, (lower alkyl)amide, NO2, OH, halo, trifluoromethyl, or carboxyl.
33. A compound of formula I according to
35. A compound of formula I according to
36. A compound of formula I according to
and r1 is ethyl, vinyl, cyclopropyl, 1 or 2-bromoethyl or 1 or 2-bromovinyl.
38. A compound of formula I according to
39. A compound of formula I according to
40. A compound of formula I according to
41. An optical isomer of a compound of formula I according to
42. A compound of formula I according to
43. A compound of formula I according to
44. A compound of formula I according to
B is a C6 or C10 aryl or C7-16 aralkyl, all optionally substituted with C1-6 alkyl, C1-6 alkoxy, C1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substituted with C1-6 alkyl; or Het or (lower alkyl)-Het, all optionally substituted with C1-6 alkyl, C1-6 alkoxy, C1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substituted with C1-6 alkyl, or
B is r4—SO2 wherein r4 is preferably amido; (lower alkyl)amide; C6 or C10 aryl, C7-14 aralkyl or Het, all optionally substituted with C1-6 alkyl, or
B is an acyl derivative of formula r4—C(O)— wherein r4 is
(i) C1-10 alkyl optionally substituted with carboxyl, hydroxy or C1-6 alkoxy, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C1-6 alkyl;
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl, (C1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C1-6 alkyl;
(iv) C6 or C10 aryl or C7-16 aralkyl, all optionally substituted with C1-6 alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionally substituted with C1-6 alkyl;
(v) Het or (lower alkyl)-Het, both optionally substituted with C1-6 alkyl, hydroxy, amino optionally substituted with C1-6 alkyl, amido, (lower alkyl)amide, or amino optionally substituted with C1-6 alkyl, or
B is a carboxyl derivative of formula r4—O—C(O)—, wherein r4 is
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxy, C1-6 alkoxy, amino optionally mono- or di-substituted with C1-6 alkyl, amido or (lower alkyl)amide;
(ii) C3-7 cycloalkyl, C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy) carbonyl, amino optionally mono- or di-substituted with C1-6 alkyl, amido or (lower alkyl)amide;
(iv) C6 or C10 aryl or C7-16 aralkyl optionally substituted with C1-6 alkyl, hydroxy, amido, (lower alkyl) amido, or amino optionally mono- or di-substituted with C1-6 alkyl; or
(v) Het or (lower alkyl)-Het, both optionally substituted with C1-6 alkyl, hydroxy, amino optionally mono- or di-substituted with C1-6 alkyl, amido or (lower alkyl) amido, or
B is an amide derivative of formula r4—N(r5)—C(O)— wherein r4 is
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxy, C1-6 alkoxy, amido, (lower alkyl)amido, or amino optionally mono- or di-substituted with C1-6 alkyl;
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy) carbonyl, amido, (lower alkyl)amido, or amino optionally mono- or di-substituted with C1-6 alkyl;
(iii) amino optionally mono- or di-substituted with C1-3 alkyl;
(iv) C6 or C10 aryl or C7-16 aralkyl, all optionally substituted with C1-6 alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionally substituted with C1-6 alkyl; or
(v) Het or (lower alkyl)-Het, both optionally substituted with C1-6 alkyl, hydroxy, amino optionally substituted with C1-6 alkyl, amido, (lower alkyl)amide; and
r5 is preferably H or methyl, or
B is thioamide derivative of formula r4—NH—C(S)—; wherein r4 is
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl or C1-6 alkoxy;
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy) carbonyl, amino or amido;
Y is H or methyl;
r3 is C1-6 alkyl, C3-7 cycloalkyl, or C4-10 alkylcycloalkyl, all optionally substituted with hydroxy, C1-6 alkoxy, C1-6 thioalkyl, acetamido, C6 or C10 aryl, or C7-16 aralkyl;
r2 is S—R20 or O—R20 wherein r20 is pyridinyl, quinolyl, —CH2-pyridinyl or —CH2-quinolyl, all optionally mono-, di- or tri-substituted with r21, wherein
r21 is C1-6 alkyl; C1-6 alkoxy; lower thioalkyl; amino or amido optionally mono- or di-substituted with C1-6 alkyl, C6 or C10 aryl, C7-16 aralkyl, Het or (lower alkyl)-Het; NO2; OH; halo; trifluoromethyl; carboxyl; C6 or C10 aryl, C7-16 aralkyl, or Het, said aryl, aralkyl or Het being optionally substituted with r22, wherein
r22 is C1-6 alkyl; C3-7 cycloalkyl; C1-6 alkoxy; amino; mono- or di-(lower alkyl)amino; (lower alkyl)amide; sulfonylalkyl; NO2; OH; halo; trifluoromethyl; carboxyl or Het;
or r2 is quinolinoxy unsubstituted, mono- or di-substituted with r21 as defined above; and
P1 is:
##STR00333##
wherein r1 is H, C1-3 alkyl, C3-5 cycloalkyl, or C2-4 alkenyl optionally substituted with halo, and said r1 at carbon 2 is orientated syn to the carbonyl at position 1, represented by the radical:
##STR00334##
or a pharmaceutically acceptable salt or ester thereof.
45. A compound of formula I according to
B is r4—SO2 wherein r4 is C6 or C10 aryl, a C7-14 aralkyl or Het all optionally substituted with C1-6 alkyl; amido, (lower alkyl)amide; B is an acyl derivative of formula r4—C(O)— wherein r4 is
(i) C1-10 alkyl optionally substituted with carboxyl, hydroxy or C1-6 alkoxy; or
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl, (C1-6 alkoxy)carbonyl; or
(iv) C6 or C10 aryl or C7-16 aralkyl, all optionally substituted with C1-6 alkyl, hydroxy; or
(v) Het optionally substituted with C1-6 alkyl, hydroxy, amido or amino;
or B is a carboxyl derivative of formula r4—O—C(O)—, wherein r4 is
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxy, C1-6 alkoxy or amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C1-6 alkyl;
(ii) C3-7 cycloalkyl, C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C1-6 alkyl; or
(iv) C6 or C10 aryl or C7-16 aralkyl, all optionally substituted with C1-6 alkyl, hydroxy, amino optionally substituted with C1-6 alkyl; or
(v) Het or (lower alkyl)-Het, both optionally substituted with C1-6 alkyl, hydroxy, amido, or amino optionally mono-substituted with C1-6 alkyl;
or B is an amide derivative of formula r4—N(r5)—C(O)— wherein r4 is
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxy, C1-6 alkoxy, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C1-6 alkyl;
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy) carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C1-6 alkyl; and r5 is H or methyl; or
r4 is (iii) amino optionally mono- or di-substituted with C1-3 alkyl; or
(iv) C6 or C10 aryl or C7-16 aralkyl, all optionally substituted with C1-6 alkyl, hydroxy, amino or amido optionally substituted with C1-6 alkyl; or
(v) Het optionally substituted with C1-6 alkyl, hydroxy, amino or amido; or
B is a thioamide derivative of formula r4—NH—C(S)—; wherein RR4 is
(i) C1-10 alkyl; or (ii) C3-7 cycloalkyl; or
Y is H;
r3 is the side chain or Tbg, Ile, Val, Chg or:
##STR00335##
r2 is quinolinoxy unsubstituted, mono- or di-substituted with r21 as defined above, or r2 is:
##STR00336##
wherein r21 is C1-6 alkyl; C1-6 alkoxy; C6, C10 aryl or Het; lower thioalkyl; halo; amino optionally mono-substituted with C1-6 alkyl; or C6, C10 aryl, C7-16 aralkyl or Het, optionally substituted with r22 wherein r22 is C1-6 alkyl, C1-6 alkoxy, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C1-6 alkyl, or Het; and r21B is C1-6 alkyl, C1-6 alkoxy, amino, di(lower alkyl)amino, (lower alkyl)amide, NO2, OH, halo, trifluoromethyl, or carboxyl;
P1 is:
##STR00337##
r1 is ethyl, vinyl, cyclopropyl, 1 or 2-bromoethyl or 1 or 2-bromovinyl.
46. A compound of formula I according to claims claim 45, wherein
B is an amide derivative of formula r4—NH—C(O)— wherein r4 is
(i) C1-10 alkyl optionally substituted with carboxyl, C1-6 alkanoyl, hydroxy, C1-6 alkoxy, amido, (lower alkyl) amide, amino optionally mono- or di-substituted with C1-6 alkyl;
(ii) C3-7 cycloalkyl or C4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C1-6 alkoxy) carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C1-6 alkyl;
(iv) C6 or C10 aryl or C7-16 aralkyl optionally substituted with C1-6 alkyl, hydroxy, amino or amido;
r3 is the side chain of Tbg, Chg or Val;
r2 is:
##STR00338##
wherein r22A is C1-6 alkyl, C1-6 alkoxy; r22B is C1-6 alkyl, amino optionally mono-substituted with C1-6 alkyl, or (lower alkyl)amide; and r21B is C1-6 alkyl, C1-6 alkoxy, amino, di(lower alkyl)amino, (lower alkyl) amide, NO2, OH, halo, trifluoromethyl, or carboxyl;
and P1 is:
##STR00339##
47. A compound according to
wherein B, r3, r2 are as defined below:
!
49. A compound according to
wherein B, r3, r2, R1 are defined below:
!
51. A compound according to
wherein B, r3, r2, r1 are as defined below:
!
52. A compound according to
53. A compound according to
wherein B, r3, r2 and r1 are as defined below:
!
54. A compound according to
55. A compound according to
!
wherein r3 is as defined below:
##STR00392##
56. A compound according to
57. A compound according to
wherein r3, R21A, r21B are as defined below:
!
58. A compound according to
59. A compound according to
wherein r3 and r21A are as defined below:
!
60. A compound according to
61. A compound according to
wherein B, r3, and r22 are as defined below:
!
62. A compound according to
63. A compound according to
wherein B is as defined below:
!
64. A compound according to
wherein B—N(Y)—, r3, and r2 are as defined below:
!
65. A pharmaceutical composition comprising an anti-hepatitis C virally effective amount of a compound of formula I according to
66. A method of treating a hepatitis C viral infection in a mammal comprises administering to the mammal an anti-hepatitis C virally effective amount of the compound of formula I according to
67. A method of treating a hepatitis C viral infection in a mammal comprises administering to the mammal an anti-hepatitis C virally effective amount of the composition according to
68. A method of inhibiting the replication of hepatitis C virus comprises exposing the virus to a hepatitis C viral NS3 protease inhibiting amount of the compound of formula I according to
69. A method of treating a hepatitis C viral infection in a mammal comprises administering thereto an anti-hepatitis C virally effective amount of a combination of the compound of formula I according to
70. A method according to
71. A method according to
72. A process for the preparation of a peptide analog of formula (I) according to
coupling a peptide selected from the group consisting of: APG-P3-P2; or APG-P2; with a P1 intermediate of formula:
##STR00511##
wherein r1 is C1-6 alkyl, cycloalkyl or C2-6 alkenyl, all optionally substituted with halogen,
CPG is a carboxyl protecting group and APG is an amino protecting group and P3 and P2 are as defined above.
73. A process for the preparation of: a peptide analog of formula (I) according to
coupling a suitably protected amino acid, peptide or peptide fragment with a P1 intermediate of formula:
##STR00512##
wherein r1 is C1-6 alkyl, cycloalkyl or C2-6 alkenyl, all optionally substituted with halogen, and CPG is a carboxyl protecting group.
74. A process for the preparation of: a peptide analog of formula (I) according to
coupling a suitably protected amino acid, peptide or peptide fragment with a P1 intermediate of formula:
##STR00513##
wherein CPG is a carboxyl protecting group.
75. A process according to
alkyl esters, aralkyl esters, and esters being cleavable by mild base treatment or mild reductive means.
76. A Method of preparing a composition for treating a hepatitis C viral infection in a mammal comprising combining an anti-hepatitis C virally effective amount of the compound of formula I according to
77. Method of preparing a composition for inhibiting the replication of hepatitis C virus comprising combining a hepatitis C viral NS3 protease inhibiting amount of the compound of formula I according to
78. Method of preparing a composition for treating a hepatitis C viral infection in a mammal comprising combining an anti-hepatitis C virally effective amount of a combination of the compound of formula I according to
79. A compound of formula (I) according to
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The present application claims benefit to U.S. Provisional Applications No. 60/095,931, filed on Aug. 10, 1998, and Ser. No. 60/132,386, filed on May 4, 1999.
The present invention relates to compounds, process for their synthesis, compositions and methods for the treatment of hepatitis C virus (HCV) infection. In particular, the present invention provides novel peptide analogs, pharmaceutical compositions containing such analogs and methods for using these analogs in the treatment of HCV infection. The present invention also provides processes ana intermediates for the synthesis of these peptide analogs.
Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 15 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so-called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system. In addition, the roles of cellular and humoral immune responses in protection against HCV infection and disease have yet to be established. Immunoglobulins have been reported for prophylaxis of transfusion-associated viral hepatitis, however, the Center for Disease Control does not presently recommend immunoglobulins treatment for this purpose. The lack of an effective protective immune response is hampering the development of a vaccine or adequate post-exposure prophylaxis measures, so in the near-term, hopes are firmly pinned on antiviral interventions.
Various clinical studies have been conducted with the goal of identifying pharmaceutical agents capable of effectively treating HCV infection in patients afflicted with chronic hepatitis C. These studies have involved the use of interferon-alpha, alone an in combination with other anti-viral agents. Such studies have shown that a substantial number of the participants do not respond to these therapies, and of those that do respond favorably, a large proportion were found to relapse after termination of treatment.
Until recently, interferon (IFN) was the only available therapy of proven benefit approved in the clinic for patients with chronic hepatitis C. However the sustained response rate is low, and interferon treatment also induces severe side-effects (i.e. retinopathy, thyroiditis, acute pancreatitis, depression) that diminish the quality of life of treated patients. Recently, interferon in combination with ribavirin has been approved for patients non-responsive to IFN alone. However, the side effects caused by IFN are not alleviated with this combination therapy.
Therefore, a need exists for the development of effective antiviral agents for treatment of HCV infection that overcomes the limitations of existing pharmaceutical therapies.
HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. In the case of HCV, the generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one, as yet poorly characterized, cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (henceforth referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation of the NS3 protein with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
A general strategy for the development of antiviral agents is to inactivate virally encoded enzymes that are essential for the replication of the virus. In this vein, patent application WO 97/06804 describes the (−) enantiomer of the nucleoside analogue cytosine-1,3-oxathiolane (also known as 3TC) as active against HCV. This compound, although reported as safe in previous clinical trials against HIV and HBV, has yet to be clinically proven active against HCV and its mechanism of action against the virus has yet to be reported. Intense efforts to discover compounds which inhibit the NS3 protease or RNA helicase of HCV have led to the following disclosures:
Several studies have reported compounds inhibitory to other serine proteases, such as human leukocyte elastase. One family of these compounds is reported in WO 95/33764 (Hoechst Marion Roussel, 1995). The peptides disclosed in this application are morpholinylcarbonyl-benzoyl-peptide analogues that are structurally different from the peptides of the present invention.
Steinkühler et al. and Ingallinella et al. have published on NS4A-4B product inhibition (Biochemistry (1998), 37, 8899-8905 and 8906-8914). However, the peptides and peptide analogues presented do not include nor do they lead to the design of the peptides of the present invention.
One advantage of the present invention is that it provides tripeptides that are inhibitory to the NS3 protease of the hepatitis C virus.
A further advantage of one aspect of the present invention resides in the fact that these peptides specifically inhibit the NS3 protease and do not show significant inhibitory activity at concentrations up to 300 μM against other serine proteases such as human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), or bovine pancreatic chymotrypsin, or cysteine proteases such as human liver cathepsin B (Cat B).
A further advantage of the present invention is that it provides small peptides of low molecular weight that may be capable of penetrating cell membranes and may be active in cell culture and in vivo with good pharmacokinetic profile.
Included in the scope of the invention are racemates, diastereoisomers and optical isomers of a compound of formula (I): ##STR00002##
Included within the scope of this invention is a pharmaceutical composition comprising an anti-hepatitis C virally effective amount of a compound of formula 1, or a therapeutically acceptable salt or ester thereof, in admixture with a pharmaceutically acceptable carrier medium or auxiliary agent.
An important aspect of the invention involves a method of treating a hepatitis C viral infection in a mammal by administering to the mammal an anti-hepatitis C virally effective amount of the compound of formula 1, or a therapeutically acceptable salt or ester thereof or a composition as described above.
Another important aspect involves a method of inhibiting the replication of hepatitis C virus by exposing the virus to a hepatitis C viral NS3 protease inhibiting amount of the compound of formula 1, or a therapeutically acceptable salt or ester thereof or a composition as described above.
Still another aspect involves a method of treating a hepatitis C viral infection in a mammal by administering thereto an anti-hepatitis C virally effective amount of a combination of the compound of formula 1, or a therapeutically acceptable salt or ester thereof. According to one embodiment, the pharmaceutical compositions of this invention comprise an additional immunomodulatory agent. Examples of additional immunomodulatory agents include but are not limited to, α-, β-, and δ-interferons.
Definitions
As used herein, the following definitions apply unless otherwise noted: With reference to the instances where (R) or (S) is used to designate the configuration of a substituent, e.g. R1 of the compound of formula 1, the designation is done in the context of the compound and not in the context of the substituent alone.
The natural amino acids, with exception of glycine, contain a chiral carbon atom. Unless otherwise specifically indicated, the compounds containing natural amino acids with the L-configuration are preferred. However, applicants contemplate that when specified, some amino acids of the formula I can be of either D- or L-configuration or can be mixtures of D- and L-isomers, including racemic mixtures. The designation “P1, P2 and P3” as used herein refer to the position of the amino acid residues starting from the C-terminus end of the peptide analogues and extending towards the N-terminus [i.e. P1 refers to position 1 from the C-terminus, P2: second position from the C-terminus, etc.) (see Berger A. & Schechter I., Transactions of the Royal Society London series (1970), B257, 249-264].
The abbreviations for the α-amino acids used in this application are set forth in
TABLE A
Amino Acid
Symbol
1-aminocyclopropyl-carboxylic acid
Acca
Alanine
Ala
Aspartic acid
Asp
Cysteine
Cys
Cyclohexylglycine
Chg
(also named: 2-amino-2-cyclohexylacetic acid)
Glutamic acid
Glu
Isoleucine
Ile
Leucine
Leu
Phenylalanine
Phe
Proline
Pro
Valine
Val
tert-Butylglycine
Tbg
As used herein the term “1-aminocyclopropyl-carboxylic acid” (Acca) refers to a compound of formula: ##STR00003##
As used herein the term “tert-butylglycine” (Acca) refers to a compound of formula: ##STR00004##
The term “residue” with reference to an amino acid or amino acid derivative means a radical derived from the corresponding α-amino acid by eliminating the hydroxyl of the carboxy group and one hydrogen of the α-amino group. For instance, the terms Gin, Ala, Gly, lie, Arg, Asp, Phe, Ser, Leu, Cys, Asn, Sar and Tyr represent the “residues” of L-glutamine, L-alanine, glycine, L-isoleucine, L-arginine, L-aspartic acid, L-phenylalanine, L-serine, L-leucine, L-cysteine, L-asparagine, sarcosine and L-tyrosine, respectively.
The term “side chain” with reference to an amino acid or amino acid residue means a group attached to the α-carbon atom of the α-amino acid. For example, the R-group side chain for glycine is hydrogen, for alanine it is methyl, for valine it is isopropyl. For the specific R-groups or side chains of the α-amino acids reference is made to A. L. Lehninger's text on Biochemistry (see chapter 4).
The term “halo” as used herein means a halogen substituent selected from bromo, chloro, fluoro or iodo.
The term “C1-6 alkyl” or “(lower)alkyl” as used herein, either alone or in combination with another substituent, means acyclic, straight or branched chain alkyl substituents containing from 1 to six carbon atoms and includes, for example, methyl, ethyl, propyl, butyl, tert-butyl, hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl.
The term “C3-7 cycloalkyl” as used herein, either alone or in combination with another substituent, means a cycloalkyl substituent containing from three to seven carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. This term also includes “spiro”-cyclic group such as spiro-cyclopropyl or spiro-cyclobutyl: ##STR00005##
The term “unsaturated cycloalkyl” includes, for example, cyclohexenyl: ##STR00006##
The term “C4-10 (alkylcycloalkyl) as used herein means a cycloalkyl radical containing from three to seven carbon atoms linked to an alkyl radical, the linked radicals containing up to ten carbon atoms; for example, cyclopropylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl or cycloheptylethyl.
The term “C2-10 alkenyl” as used herein, either alone or in combination with another radical, means an alkyl radical as defined above containing from 2 to 10 carbon atoms, and further containing at least one double bond. For example alkenyl includes allyl and vinyl.
The term “C1-6 alkanoyl” as used herein, either alone or in combination with another radical, means straight or branched 1-oxoalkyl radicals containing one to six carbon atoms and includes formyl, acetyl, 1-oxopropyl(propionyl), 2-methyl-1-oxopropyl, 1-oxohexyl and the like.
The term “C3-7 alkoxy” as used herein, either alone or in combination with another radical, means the radical —O(C1-6 alkyl) wherein alkyl is as defined above containing up to six carbon atoms. Alkoxy includes methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. The latter radical is known commonly as tert-butoxy.
The term “C3-7 cycloalkoxy” as used herein, either alone or in combination with another radical, means a C3-7 cycloalkyl group linked to an oxygen atom, such as, for example: ##STR00007##
The term “C6 or C10 aryl” as used herein, either alone or in combination with another radical, means either an aromatic monocyclic group containing 6 carbon atoms or an aromatic bicyclic group containing 10 carbon atoms. For example, aryl includes phenyl, 1-naphthyl or 2-naphthyl.
The term “C7-16 aralkyl” as used herein, either alone or in combination with another radical, means a C6 or C10 aryl as defined above linked to an alkyl group, wherein alkyl is as defined above containing from 1 to 6 carbon atoms. C7-16 aralkyl includes for example benzyl, butylphenyl, and 1-naphthylmethyl.
The term “amino aralkyl” as used herein, either alone or in combination with another radical, means an amino group substituted with a C7-16 aralkyl group, such as, for example, the amino aralkyl: ##STR00008##
The term “(lower alkyl)amide” as used herein, either alone or in combination with another radical, means an amide mono-substituted with a C1-6 alkyl, such as: ##STR00009##
The term “carboxy(lower)alkyl” as used herein, either alone or in combination with another radical, means a carboxyl group (COOH) linked through a (lower)alkyl group as defined above and includes for example butyric acid.
The term “heterocycle” or “Het” as used herein, either alone or in combination with another radical, means a monovalent radical derived by removal of a hydrogen from a five-, six-, or seven-membered saturated or unsaturated (including aromatic) heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur. Furthermore, “Het” as used herein, means a heterocycle as defined above fused to one or more other cycle, be it a heterocycle or any other cycle. Examples of suitable heterocycles include: pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, thiophene, diazepine, 1H-imidazole, isoxazole, thiazole, tetrazole, piperidine, 1,4-dioxane, 4-morpholine, pyridine, pyrimidine, thiazolo[4,5-b]-pyridine, quinoline, or indole, or the following heterocycles: ##STR00010##
The term “(lower alkyl)-Het” as used herein, means a heterocyclic radical as defined above linked through a chain or branched alkyl group, wherein alkyl is as defined above containing from 1 to 6 carbon atoms. Examples of (lower alkyl)-Het include: ##STR00011##
The term “pharmaceutically acceptable ester” as used herein, either alone or in combination with another substituent, means esters of the compound of formula I in which any of the carboxyl functions of the molecule, but preferably the carboxy terminus, is replaced by an alkoxycarbonyl function:
##STR00012##
in which the R moiety of the ester is selected from alkyl (e.g. methyl, ethyl, n-propyl, t-butyl, n-butyl); alkoxyalkyl (e.g. methoxymethyl); alkoxyacyl (e.g. acetoxymethyl); aralkyl (e.g. benzyl); aryloxyalkyl (e.g. phenoxymethyl); aryl (e.g. phenyl), optionally substituted with halogen, C1-4 alkyl or C1-4 alkoxy. Other suitable prodrug esters can be found in Design of prodrugs, Bundgaard, H. Ed. Elsevier (1985) incorporated herewith by reference. Such pharmaceutically acceptable esters are usually hydrolyzed in vivo when injected in a mammal and transformed into the acid form of the compound of formula I.
With regard to the esters described above, unless otherwise specified, any alkyl moiety present advantageously contains 1 to 16 carbon atoms, particularly 1 to 6 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group.
In particular the esters may be a C1-6 alkyl ester, an unsubstituted benzyl ester or a benzyl ester substituted with at least one halogen, C1-6 alkyl, C1-6 alkoxy, nitro or trifluoromethyl.
The term “pharmaceutically acceptable salt” as used herein includes those derived from pharmaceutically acceptable bases. Examples of suitable bases include choline, ethanolamine and ethylenediamine. Na+, K+, and Ca++ salts are also contemplated to be within the scope of the invention (also see Pharmaceutical salts, Birge, S. M. Et al., J. Pharm. Sci., (1977), 66, 1-19, incorporated herein by reference).
Preferred Embodiments
Included within the scope of this invention are compounds of formula I wherein
Alternatively, B is preferably R4—SO2 wherein R4 is preferably C1-6 alkyl; amido; (lower alkyl)amide; C6 or C10 aryl, C7-14 aralkyl or Het, all optionally substituted with C1-6 alkyl.
Alternatively, B is preferably an acyl derivative of formula R4—C(O)— wherein R4 is preferably
Alternatively, B is preferably a carboxyl of formula R4—O—C(O)—, wherein R4 is preferably
Alternatively, B is preferably an amide of formula R4—N (R5)—C(O)— wherein R4 is preferably
Alternatively, B is a preferably thioamide of formula R4—NH—C(S)—; wherein R4 is preferably
More preferably, B is a C6 or C10 aryl optionally substituted with C1-6 alkyl, C1-6 alkoxy, C1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C1-6 alkyl, such that B is for example:
##STR00013##
or B is more preferably Het optionally substituted with C1-6 alkyl, C1-6 alkoxy, C1-6 alkanoyl, hydroxy, halo, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C1-6 alkyl, such that B is for example:
##STR00014##
Alternatively, B is more preferably R4—SO2 wherein R4 is preferably C6 or C10 aryl, a C7-14 aralkyl or Het all optionally substituted with C1-6 alkyl; amido, (lower alkyl) amide, such that B is, for example: ##STR00015##
Alternatively, B is more preferably an acyl derivative of formula R4—C(O)— wherein R4 is preferably
Alternatively, B is more preferably a carboxyl of formula R4—O—C(O)—, wherein R4 is preferably
A non-linear curve fit with the Hill model was applied to the inhibition-concentration data, and the 50% effective concentration (IC50) was calculated by the use of SAS software (Statistical Software System; SAS Institute, Inc. Cary, N.C.).
Full-length NS3-NS4A Heterodimer Protein Assay
The NS2-NS5B-3′ non coding region was cloned by RT-PCR into the pCR®3 vector (Invitrogen) using RNA extracted from the serum of an HCV genotype 1b infected individual (provided by Dr. Bernard Willems, Hôpital St-Luc, Montréal, Québec, Canada). The NS3-NS4A DNA region was then subcloned by PCR into the pFastBac™ HTa baculovirus expression vector (Gibco/BRL). The vector sequence includes a region encoding a 28-residue N-terminal sequence which contains a hexahistidine tag. The Bac-to-Bac™ baculovirus expression system (Gibco/BRL) was used to produce the recombinant baculovirus. The full length mature NS3 and NS4A heterodimer protein (His-NS3-NS4AFL) was expressed by infecting 106 Sf21 cells/mL with the recombinant baculovirus at a multiplicity of infection of 0.1-0.2 at 27° C. The infected culture was harvested 48 to 64 h later by centrifugation at 4° C. The cell pellet was homogenized in 50 mM NaPO4, pH 7.5, 40% glycerol (w/v), 2 mM β-mercaptoethanol, in presence of a cocktail of protease inhibitors. His-NS3-NS4AFL was then extracted from the cell lysate with 1.5% NP-40, 0.5% Triton X-100, 0.5M NaCl, and a DNase treatment. After ultracentrifugation, the soluble extract was diluted 4-fold and bound on a Pharmacia Hi-Trap Ni-chelating column. The His-NS3-NS4AFL was eluted in a >90% pure form (as judged by SDS-PAGE), using a 50 to 400 mM imidazole gradient. The His-NS3-NS4AFL was stored at −80° C. in 50 mM sodium phosphate, pH 7.5, 10% (w/v) glycerol, 0.5 M NaCl, 0.25 M imidazole, 0.1% NP-40. It was thawed on ice and diluted just prior to use. The protease activity of His-NS3-NS4AFL was assayed in 50 mM Tris-HCl, pH 8.0, 0.25 M sodium citrate, 0.01% (w/v) n-dodecyl-β-D-maltoside, 1 mM TCEP. Five (5) μM of the internally quenched substrate anthranilyl-DDIVPAbu[C(O)—O]-AMY(3-NO2)TW-OH in presence of various concentrations of inhibitor were incubated with 1.5 nM of His-NS3-NS4AFL for 45 min at 23° C. The final DMSO concentration did not exceed 5.25%. The reaction was terminated with the addition of 1M MES, pH 5.8. Fluorescence of the N-terminal product was monitored on a Perkin-Elmer LS-50B fluorometer equipped with a 96-well plate reader (excitation wavelength: 325 nm; emission wavelength: 423 nm).
The % inhibition was calculated with the following equation:
100−[(countsinh−countsblank)/(countsctl−countsblank)×100]
A non-linear curve fit with the Hill model was applied to the inhibition-concentration data, and the 50% effective concentration (IC50) was calculated by the use of SAS software (Statistical Software System; SAS Institute, Inc. Cary, N.C.).
NS3 Protease Cell-based Assay
This assay was done with Huh-7 cells, a human cell line derived from a hepatoma, co-transfected with 2 DNA constructs:
The polyprotein must be cleaved by the NS3 protease for the mature proteins to be released. Upon release of the mature proteins, it is believed that the viral proteins will form a complex at the membrane of the endoplasmic reticulum while tTA will migrate to the nucleus and transactivate the SEAP gene. Therefore, reduction of NS3 proteolytic activity should lead to reduction of mature tTA levels and concomitant decrease in SEAP activity.
To control for other effects of the compounds, a parallel transfection was done where a construct expressing tTA alone (called tTA) was co-transfected with the SEAP construct such that SEAP activity is independent of NS3 proteolytic activity. Protocol of the assay: Huh-7 cells, grown in CHO-SFMII+10% FCS (fetal calf serum), were co-transfected with either NS3 and SEAP or tTA and SEAP, using the FuGene protocol (Boehringer Mannheim). After 5 h at 37°, the cells were washed, trypsinized and plated (at 80,000 cells/well) in 96-well plates containing a range of concentrations of the compounds to be tested. After a 24-h incubation period, an aliquot of the medium was drawn and the SEAP activity in this aliquot was measured with the Phospha-Light kit (Tropix).
Analysis of the percent inhibition of SEAP activity with respect to compound concentration was performed with the SAS software to obtain the EC50.
The toxicity of the compound (TC50) was then assessed using the MTT assay as follows:
The TC50 was calculated in the same way as the EC50.
Specificity Assays
The specificity of the compounds was determined against a variety of serine proteases: human leukocyte elastase, porcine pancreatic elastase and bovine pancreatic α-chymotrypsin and one cysteine protease: human liver cathepsin B. In all cases a 96-well plate format protocol using a colorimetric p-nitroaniline (pNA) substrate specific for each enzyme was used. Each assay included a 1 h enzyme-inhibitor pre-incubation at 30° C. followed by addition of substrate and hydrolysis to ≈30% conversion as measured on a UV Thermomax® microplate reader. Substrate concentrations were kept as low as possible compared to KM to reduce substrate competition. Compound concentrations varied from 300 to 0.06 μM depending on their potency.
The final conditions for each assay were as follows:
A representative example is summarized below for porcine pancreatic elastase:
In a polystyrene flat-bottom 96-well plate were added using a Biomek liquid handler (Beckman):
After 60 min pre-incubation at 30° C., 20 μL of substrate solution (50 mM Tris-HCl, pH 8, 0.5 M Na2SO4, 50 mM NaCl, 0.1 mM EDTA, 665 μM Succ-AAA-pNA) were added to each well and the reaction was further incubated at 30° C. for 60 min after which time the absorbance was read on the UV Thermomax® plate reader. Rows of wells were allocated for controls (no inhibitor) and for blanks (no inhibitor and no enzyme).
The sequential 2-fold dilutions of the inhibitor solution were performed on a separate plate by the liquid handler using 50 mM Tris-HCl pH 8, 50 mM NaCl, 0.1 mM EDTA, 0.02% Tween-20, 15% DMSO. All other specificity assays were performed in a similar fashion.
The percentage of inhibition was calculated using the formula:
[1-((UVinh−UVblank)/(UVctl−UVblank))]×100
A non-linear curve fit with the Hill model was applied to the inhibition-concentration data, and the 50% effective concentration (IC50) was calculated by the use of SAS software (Statistical Software System; SAS Institute, Inc., Cary, N.C.).
The following tables list compounds representative of the invention. Compounds of the invention were assayed either in one or both of the assays of Examples 37 and 38 and were found to be active with IC50 below 50 μM.
Activity in Cells and Specificity:
Representative compounds of the invention were also tested in the surrogate cell-based assay of Example 39, and in one or several assays of Example 40. For example, compound 601 from Table 6 was found to have an IC50 of 50 nM in the assay of Example 37 and 30 nM in the assay of Example 38. The EC50 as determined by the assay of Example 39 is 8.2 μM. In the specificity assays of Example 40, the same compound was found to have the following activity: HLE>75 μM; PPE>75 μM; α-Chym.>75 μM; Cat. B>75 μM. These results indicate that this family of compounds is highly specific for the NS3 protease and at least certain members of this family are active in a surrogate cell-based assay.
The following abbreviations are used within the present tables: MS: Mass spectrometric data; Ac: acetyl; Bn: benzyl; Boc: tert-butyloxycarbonyl; cHex: cyclohexyl; Chg: cyclohexylglycine (2-amino-2-cyclohexyl-acetic acid); iPr: isopropyl; O-Bn: benzyloxy; Ph: phenyl: t-Bu: tert-butyl; Tbg: tert-butylglycine; 1- or 2-Np: 1- or 2-naphthyl; 1- or 2-NpCH2O: 1, or 2-naphthylmethoxy.
TABLE 1
##STR00121##
Tab 1
IC50
Cpd #
B
R3
R2
MS
(μM)
101
Boc
cHex
—O—CH2-1-naphthyl
594
43
102
##STR00122##
cHex
—O—CH2-1-naphthyl
632
45
103
##STR00123##
cHex
—O—CH2-1-naphthyl
642
42
104
##STR00124##
cHex
—O—CH2-1-naphthyl
728
29.5
105
##STR00125##
cHex
—O—CH2-1-naphthyl
619
47
106
Boc
cHex
##STR00126##
702
2.8
107
##STR00127##
cHex
—O—CH2-1-naphthyl
720 M + Na+
34
108
Boc
iPr
##STR00128##
662
8.9
109
acetyl
cHex
##STR00129##
644
6.3
110
Boc
i-Pr
##STR00130##
575.1
9.7
111
Boc
t-Bu
##STR00131##
661.3
0.475
TABLE 2
##STR00132##
Table 2
R1
IC50
Cpd #
B
R3
R2
anti to carboxy
MS
(μM)
201
Boc
cyclohexyl
—O—CH2-1-naphthyl
ethyl
622
15
(one isomer)
202
Boc
cyclohexyl
—O—CH2-1-naphthyl
ethyl
622
40
(one isomer)
203
Boc
t-Bu
##STR00133##
vinyl 1R, 2R
687.5
0.082
TABLE 3
##STR00134##
R1
Table 3
syn to
IC50
Cpd #
B
R3
R2
carboxyl
MS
(μM)
301
Boc
cHex
—O—CH2-1-naphthyl
ethyl
622
7.7
302
##STR00135##
iPr
—O—CH2-1-naphthyl
ethyl
582
12.5
303
##STR00136##
cHex
—O—CH2-1-naphthyl
ethyl
622
11
304
Boc
cHex
##STR00137##
ethyl
623
32
305
Boc
cHex
—O—CH2-1-naphthyl
vinyl
620
3.2
306
Boc
cHex
##STR00138##
vinyl
607
0.8
307
Boc
cHex
##STR00139##
vinyl
728
0.27
308
Boc
cHex
##STR00140##
vinyl
606
1.6
309
Boc
cHex
##STR00141##
vinyl
606
5
310
Boc
cHex
##STR00142##
vinyl
607
2.5
311
Boc
cHex
##STR00143##
vinyl
641
0.56
312
Boc
cHex
##STR00144##
vinyl
607
8.5
313
Boc
cHex
##STR00145##
vinyl
621
2.5
314
Boc
cHex
##STR00146##
vinyl
683
0.14
315
Boc
cHex
##STR00147##
vinyl
698
0.66
316
Acetyl
cHex
##STR00148##
vinyl
625
1.9
317
Boc
cHex
##STR00149##
vinyl
740
0.32
318
CF3—C(O)—
i-Pr
##STR00150##
vinyl
639.3
0.88
319
##STR00151##
cHex
##STR00152##
vinyl
732.3
1.2
320
##STR00153##
cHex
##STR00154##
vinyl
704.3
0.65
321
Boc
t-Bu
##STR00155##
vinyl
658.7
0.19
322
Boc
t-Bu
##STR00156##
vinyl
717.6
1.95
323
Boc
t-Bu
##STR00157##
##STR00158##
672.4
0.64
324
Boc
t-Bu
##STR00159##
vinyl
727.5
0.05
325
Boc
t-Bu
##STR00160##
##STR00161##
701.4
0.153
326
Boc
t-Bu
##STR00162##
vinyl
708.3
0.32
327
##STR00163##
t-Bu
##STR00164##
vinyl
610.3
0.045
328
Boc
t-Bu
##STR00165##
vinyl
615.3
3.2
329
Boc
t-Bu
##STR00166##
vinyl
685.3
0.36
330
Boc
t-Bu
##STR00167##
vinyl
627.5
6
331
##STR00168##
t-Bu
##STR00169##
vinyl
656.5
0.071
332
Boc
t-Bu
##STR00170##
ethyl
689.3
0.13
333
##STR00171##
t-Bu
##STR00172##
vinyl
778.3
0.003
334
##STR00173##
t-Bu
##STR00174##
vinyl
764.4
0.007
TABLE 4
##STR00175##
Table 4
IC50
Cpd #
B
R3
R2
R1
MS
(μM)
401
Boc
i-Pr
##STR00176##
H
589.1
5.8
402
Boc
t-Bu
##STR00177##
H
603.6
7.9
403
Boc
t-Bu
##STR00178##
H
675.4
0.132
404
Boc
t-Bu
##STR00179##
3-(═CH2)
687.1
0.6
405
Boc
t-Bu
##STR00180##
2-vinyl
702.3
0.220
406
Boc
t-Bu
##STR00181##
2-Et
703.3
0.4
TABLE 5
##STR00182##
Table 5
IC50
Cpd #
R3
MS
(μM)
501
t-Bu
581.3
0.4
502
H
539.2
6.2
503
##STR00183##
625.3
0.79
504
##STR00184##
582.6
2.6
505
##STR00185##
583.2
0.79
506
##STR00186##
659.2
1.3
507
##STR00187##
670.2
0.98
508
##STR00188##
703.3
3.1
509
##STR00189##
581.3
0.377
510
##STR00190##
581.2
0.255
511
##STR00191##
637.2
2.1
TABLE 6
##STR00192##
Table
6
Cpd
IC50
#
R3
R21A
R21B
MS
(μM)
601
i-Pr
Ph
7-OMe
673.3
0.05
602
t-Bu
Ph
8-OMe,
717.2
0.041
7-OMe
603
i-Pr
Ph
7-ethyl
671.2
0.195
604
t-Bu
—
7-OMe
611.2
0.073
605
t-Bu
Ph
7-O-iPr
715.3
0.195
606
t-Bu
—
7-Cl
615.2
0.48
607
iPr
—
7-Cl
601.2
0.45
608
CH2-iPr
—
7-Cl
615.3
1.45
609
t-Bu
##STR00193##
—
680.2
1.7
610
t-Bu
Cl
—
613.3
0.25
611
t-Bu
Ph
7-N(Me)2
700.5
0.035
612
t-Bu
##STR00194##
—
666.4
0.278
613
t-Bu
##STR00195##
—
650.4
1.0
614
t-Bu
##STR00196##
—
664.5
2.2
615
t-Bu
—
7-N(Me)2
624.5
0.16
616
t-Bu
##STR00197##
—
678.4 (M − H)+
0.087
617
t-Bu
##STR00198##
—
664.5
0.345
618
t-Bu
##STR00199##
—
638.5
2.3
619
t-Bu
##STR00200##
—
700.5
3.0
620
t-Bu
##STR00201##
—
679.5
0.72
621
t-Bu
##STR00202##
—
678.3
0.058
622
t-Bu
##STR00203##
—
625.4
0.16
623
t-Bu
MeO—
—
611.3
0.20
624
t-Bu
(Me)2N—
—
624.4
1.30
625
t-Bu
Ph
7-S(Me)
703.4
0.16
626
t-Bu
Ph
7-Br
737.3
0.24
627
t-Bu
Ph
7-F
675.3
0.33
628
t-Bu
##STR00204##
7-N(Me)2
764.2
0.011
629
t-Bu
##STR00205##
7-N(Me)2
764.3
0.02
630
t-Bu
##STR00206##
7-N(Et)2
792.3
0.043
TABLE 7
##STR00207##
Table 7
IC50
Cpd #
R3
R21A
MS
(μM)
701
t-Bu
##STR00208##
691.3
0.028
702
t-Bu
##STR00209##
713.4
0.10
703
t-Bu
##STR00210##
655.3
0.047
704
t-Bu
##STR00211##
728.4
0.24
705
t-Bu
##STR00212##
696.4
0.13
706
t-Bu
##STR00213##
693.3
0.032
707
t-Bu
##STR00214##
694.3
0.023
708
t-Bu
Ph—N(Me)—
716.4
0.15
709
t-Bu
##STR00215##
709.2
0.021
710
t-Bu
HOOC—
655.3
0.685
711
t-Bu
##STR00216##
708.2
0.016
712
t-Bu
(Me)2N—
654.3
0.10
713
t-Bu
##STR00217##
692.3 (M − H)−
0.026
714
t-Bu
##STR00218##
722.3
0.012
715
t-Bu
##STR00219##
688.3
0.031
716
t-Bu
##STR00220##
688.3
0.079
717
t-Bu
##STR00221##
723.3
0.028
718
t-Bu
NH2
626.3
0.16
719
t-Bu
##STR00222##
751.2
0.018
720
t-Bu
##STR00223##
733.4
0.03
721
t-Bu
##STR00224##
724.3
0.045
722
t-Bu
##STR00225##
737.3
0.048
723
t-Bu
##STR00226##
751.4
0.047
724
t-Bu
##STR00227##
708.4
0.075
725
t-Bu
##STR00228##
689.4
0.046
726
t-Bu
i-Pr
653.3
0.25
727
t-Bu
##STR00229##
688.3
0.07
728
t-Bu
##STR00230##
786.1
0.022
729
t-Bu
##STR00231##
689.3
0.2
730
t-Bu
##STR00232##
669.2
0.042
731
t-Bu
##STR00233##
669.2
0.031
732
t-Bu
##STR00234##
791.0
0.02
733
t-Bu
##STR00235##
765.3
0.028
734
t-Bu
##STR00236##
671.3
0.044
735
t-Bu
##STR00237##
683.3
0.058
736
t-Bu
t-Bu
667.4
0.25
737
t-Bu
CHex
693.4
0.11
TABLE 8
##STR00238##
Table 8
IC50
Cpd #
B
R3
R22
MS
(μM)
801
##STR00239##
t-Bu
—
686.7
0.006
802
##STR00240##
t-Bu
—
727.7
0.024
803
##STR00241##
t-Bu
—
685.7
0.12
804
##STR00242##
t-Bu
—
711.7
0.032
805
Ac
t-Bu
—
629.6
0.083
806
##STR00243##
t-Bu
—
725.7
0.036
807
##STR00244##
t-Bu
—
672.4
0.01
808
##STR00245##
t-Bu
—
712.4
0.008
809
##STR00246##
i-Pr
—
649.3
0.071
810
##STR00247##
t-Bu
—
749.3
0.45
811
Boc
t-Bu
4-Cl
721.3
0.04
812
##STR00248##
t-Bu
—
706.2
0.013
813
##STR00249##
t-Bu
—
702.2
0.02
814
Boc
t-Bu
2-Cl
721.3
0.13
815
Boc
t-Bu
3-Cl
721.3
0.16
816
##STR00250##
t-Bu
—
658.3
0.032
817
##STR00251##
t-Bu
—
720.2
0.017
818
##STR00252##
t-Bu
—
728.3
0.019
819
##STR00253##
i-Pr
—
762.3
0.32
820
##STR00254##
i-Pr
—
732.2
0.063
821
##STR00255##
i-Pr
—
679.1
0.12
822
##STR00256##
i-Pr
—
663.3
0.05
823
Boc
t-Bu
2-OMe
717.2
0.107
824
Boc
t-Bu
3-OMe
719.2
0.07
825
Boc
t-Bu
4-OMe
719.2
0.024
826
##STR00257##
i-Pr
—
663.3
0.78
827
##STR00258##
t-Bu
—
673.2
0.27
828
##STR00259##
i-Pr
—
691.3
0.10
829
##STR00260##
t-Bu
—
734.3
0.057
830
##STR00261##
t-Bu
—
645.3
0.111
831
##STR00262##
t-Bu
—
701.3
0.015
832
##STR00263##
t-Bu
—
801.3 701.3
0.11
833
##STR00264##
t-Bu
—
715.2
0.015
834
##STR00265##
i-Pr
—
663.3
0.074
835
##STR00266##
t-Bu
—
702.5
0.007
836
##STR00267##
i-Pr
—
694.4
0.13
837
##STR00268##
i-Pr
—
683.3
0.098
838
##STR00269##
i-Pr
—
679.1
0.094
839
##STR00270##
i-Pr
—
674.5
0.10
840
##STR00271##
i-Pr
—
667.4
0.085
841
Boc
t-Bu
2-Me
701.5
0.24
842
Boc
t-Bu
3-Me
701.5
0.073
843
Boc
t-Bu
4-Me
701.5
0.053
844
##STR00272##
t-Bu
4-OMe
716.6
0.006
845
##STR00273##
i-Pr
—
706.9
0.18
846
##STR00274##
i-Pr
—
693.4
0.104
847
Boc
cHex
—
713.4
0.037
848
Boc
##STR00275##
—
687.5
0.093
849
Boc
##STR00276##
—
701.5
0.110
850
Boc
##STR00277##
—
731.5
0.063
851
Boc
##STR00278##
—
689.5
0.12
852
Boc
##STR00279##
—
689.5
0.05
853
Boc
##STR00280##
—
765.5
0.17
854
##STR00281##
i-Pr
—
723.4 (M − H)+
0.37
855
##STR00282##
i-Pr
—
693.3
0.075
856
##STR00283##
i-Pr
—
688.3
0.11
857
##STR00284##
t-Bu
—
716.4
0.011
858
##STR00285##
t-Bu
—
700.4
0.205
859
##STR00286##
i-Pr
—
655.4
0.83
860
##STR00287##
i-Pr
—
759.3
0.24
861
##STR00288##
i-Pr
—
688.3
0.17
862
##STR00289##
i-Pr
—
685.3
0.23
863
##STR00290##
i-Pr
—
699.4
0.30
864
##STR00291##
i-Pr
—
667.3
0.45
865
##STR00292##
t-Bu
—
701.4
0.02
866
##STR00293##
t-Bu
—
702.4
0.20
867
##STR00294##
t-Bu
—
701.3
0.051
868
##STR00295##
t-Bu
—
713.3
0.03
869
##STR00296##
t-Bu
—
699.4
0.014
870
##STR00297##
t-Bu
—
700.4
0.009
871
##STR00298##
t-Bu
—
714.3
0.011
872
##STR00299##
t-Bu
—
714.4
0.005
873
##STR00300##
t-Bu
—
714.3
0.019
TABLE 9
##STR00301##
Table 9
IC50
Cpd #
B
MS
(μM)
901
Boc
685.3
0.025
902
##STR00302##
825.4
0.042
903
##STR00303##
769.3
0.005
904
##STR00304##
707.3
0.095
905
##STR00305##
685.2
0.029
906
##STR00306##
728.2
0.014
907
##STR00307##
717.2
0.025
908
##STR00308##
691.2
0.072
909
##STR00309##
727.2
0.036
910
##STR00310##
715.3
0.056
911
##STR00311##
721.3
0.039
912
##STR00312##
733.2
0.034
913
##STR00313##
713.3
0.030
914
##STR00314##
805.3
0.031
915
##STR00315##
692.2
0.026
916
##STR00316##
680.3
0.3
TABLE 10
##STR00317##
Table 10
IC50
Cpd #
B—X—
R3
Z
R21B
MS
(μM)
1001
Ph—N(Me)—
i-Pr
O
H
663.3
0.31
1002
Boc-NH—
t-Bu
S
OMe
703.4
0.32
1003
##STR00318##
i-Pr
O
—
663.3
0.31
Tsantrizos, Youla S., Bailey, Murray D., Llinas-Brunet, Montse, Ghiro, Elise, Cameron, Dale R., Faucher, Anne-Marie, Rancourt, Jean, Halmos, Teddy, Goudreau, Nathalie, Poupart, Marc-André , Wernic, Dominik M.
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