Source: http://www.google.com/patents/US6544951?dq=5,758,352
Timestamp: 2014-03-13 11:00:10
Document Index: 583216869

Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 623', 'Application No. 623', 'Application No. 618', 'Application No. 533', 'Application No. 528', 'Application No. 618']

Patent US6544951 - C-terminal modified oxamyl dipeptides as inhibitors of the ICE/ced-3 family ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThis invention is directed to novel oxamyl dipeptide ICE/ced-3 family inhibitor compounds. The invention is also directed to pharmaceutical compositions containing these compounds, as well as to the use of such compositions in the treatment of patients suffering inflammatory, autoimmune and neurodegenerative...http://www.google.com/patents/US6544951?utm_source=gb-gplus-sharePatent US6544951 - C-terminal modified oxamyl dipeptides as inhibitors of the ICE/ced-3 family of cysteine proteasesAdvanced Patent SearchPublication numberUS6544951 B2Publication typeGrantApplication numberUS 09/745,204Publication dateApr 8, 2003Filing dateDec 19, 2000Priority dateJul 2, 1998Fee statusPaidAlso published asUS20020028774Publication number09745204, 745204, US 6544951 B2, US 6544951B2, US-B2-6544951, US6544951 B2, US6544951B2InventorsDonald S. Karanewsky, Robert J. TernanskyOriginal AssigneeIdun Pharmaceuticals, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (7), Non-Patent Citations (8), Referenced by (3), Classifications (55), Legal Events (4) External Links: USPTO, USPTO Assignment, EspacenetC-terminal modified oxamyl dipeptides as inhibitors of the ICE/ced-3 family of cysteine proteasesUS 6544951 B2Abstract This invention is directed to novel oxamyl dipeptide ICE/ced-3 family inhibitor compounds. The invention is also directed to pharmaceutical compositions containing these compounds, as well as to the use of such compositions in the treatment of patients suffering inflammatory, autoimmune and neurodegenerative diseases, for the prevention of ischemic injury, and for the preservation of organs that are to undergo a transplantation procedure.
Images(38) Claims(104)
We claim: 1. A compound of the following Formula I: wherein:
A is has the following Formula IIa: B is hydrogen, halomethyl, CH2ZR15 wherein Z is oxygen, or CH2OPOR16R17; R1 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, naphthyl, substituted naphthyl, (1 or 2 naphthyl)alkyl, heteroaryl, substituted heteroaryl, (heteroaryl)alkyl, substituted (heteroaryl)alkyl or R1a(R1b)N; R1a and R1b are phenyl; R2 is hydrogen; R3 is C1-6 lower alkyl; R3a is hydrogen; R15 is substituted phenyl; and R16 and R17 are independently lower alkyl or phenyl; or a pharmaceutically acceptable salt thereof. 2. The compound of claim 1 wherein R1 is phenyl.
3. The compound of claim 1 wherein R1 is substituted phenyl.
4. The compound of claim 3 wherein R1 is 2-halophenyl.
5. The compound of claim 4 wherein R1 is 2-fluorophenyl.
6. The compound of claim 4 wherein R1 is 2-chlorophenyl.
7. The compound of claim 4 wherein R1 is 2-bromophenyl.
8. The compound of claim 4 wherein R1 is 2-iodophenyl.
9. The compound of claim 3 wherein R1 is 4-halophenyl.
10. The compound of claim 9 wherein R1 is 4-fluorophenyl.
11. The compound of claim 9 wherein R1 is 4-chlorophenyl.
12. The compound of claim 9 wherein R1 is 4-bromophenyl.
13. The compound of claim 9 wherein R1 is 4-iodophenyl.
14. The compound of claim 3 wherein R1 is 2,6-difluorophenyl.
15. The compound of claim 3 wherein R1 is 2,6-dichlorophenyl.
16. The compound of claim 3 wherein R1 is 2,4-difluorobenzyl.
17. The compound of claim 3 wherein R1 is 2,3,5,6-tetrachlorophenyl.
18. The compound of claim 3 wherein R1 is 2-trifluoromethylphenyl.
19. The compound of claim 3 wherein R1 is 3-trifluoromethylphenyl.
20. The compound of claim 3 wherein R1 is 2-methoxyphenyl.
21. The compound of claim 3 wherein R1 is 4-methoxyphenyl.
22. The compound of claim 3 wherein R1 is 3,4-dimethoxybenzyl.
23. The compound of claim 3 wherein R1 is 3,4,5-trimethoxyphenyl.
24. The compound of claim 3 wherein R1 is 2-phenyiphenyl.
25. The compound of claim 3 wherein R1 is 2-benzylphenyl.
26. The compound of claim 3 wherein R1 is 2-tert-butylphenyl.
27. The compound of claim 3 wherein R1 is 2,5-di-tert-butylphenyl.
28. The compound of claim 3 wherein R1 is 2-tert-butyl-5-acetaminophenyl.
29. The compound of claim 3 wherein R1 is 2-(2-methylphenyl)phenyl.
30. The compound of claim 3 wherein R1 is 2-(2-methoxyphenyl)phenyl.
31. The compound of claim 3 wherein R1 is 3-(3-methoxyphenyl)phenyl.
32. The compound of claim 3 wherein R1 is 3-(4-methoxyphenyl)phenyl.
33. The compound of claim 3 wherein R1 is 2-(3-methylphenyl)phenyl.
34. The compound of claim 3 wherein R1 is 2-(4-methylphenyl)phenyl.
35. The compound of claim 3 wherein R1 is 2-phenoxyphenyl.
36. The compound of claim 3 wherein R1 is 4-(n-heptyl)phenyl.
37. The compound of claim 3 wherein R1 is 2-(1-naphthyl)phenyl.
38. The compound of claim 1 wherein R1 is naphthyl.
39. The compound of claim 38 wherein R1 is 1-naphthyl.
40. The compound of claim 38 wherein R1 is 2-naphthyl.
41. The compound of claim 1 wherein R1 is substituted naphthyl.
42. The compound of claim 41 wherein R1 is 1-(4-chloro)naphthyl.
43. The compound of claim 1 wherein R1 is phenylalkyl.
44. The compound of claim 43 wherein R1 is benzyl.
45. The compound of claim 43 wherein R1 is �(CH2)2phenyl.
46. The compound of claim 1 wherein R1 is substituted phenylalky.
47. The compound of claim 46 wherein R1 is 2-tert-butylbenzyl.
48. The compound of claim 46 wherein R1 is 3,4,5-trimethoxybenzyl.
49. The compound of claim 46 wherein R1 is �CH2CH2(2-fluorophenyl).
50. The compound of claim 1 wherein R1 is cycloalkyl.
51. The compound of claim 50 wherein cycloalkyl is a bicyclic ring.
52. The compound of claim 51 wherein the bicyclic ring is partially unsaturated.
53. The compound of claim 52 wherein the partially unsaturated bicyclic ring is 1-(5,6,7,8-tetrahydro)naphthalene.
54. The compound of claim 50 wherein cycloalkyl is a tricyclic ring.
55. The compound of claim 54 wherein the tricyclic ring is 1-adamantanyl.
56. The compound of claim 54 wherein the tricyclic ring is partially unsaturated.
57. The compound of claim 1 wherein R1 is (1 or 2naphthyl)alkyl.
58. The compound of claim 57 wherein R1 is �CH2(1-naphthyl).
59. The compound of claim 57 wherein R1 is �CH2(2-naphthyl).
60. The compound of claim 1 wherein R1 is heteroaryl.
61. The compound of claim 60 wherein R1 is 4-pyridyl.
62. The compound of claim 60 wherein R1 is 2-pyrazinyl.
63. The compound of claim 1 wherein R1 is R1a(R1b)N.
64. The compound of claim 1 wherein B is hydrogen.
65. The compound of claim 1 wherein B is halomethyl.
66. The compound of claim 65 wherein B is �CH2F.
67. The compound of claim 1 wherein B is �CH2ZR15.
68. The compound of claim 67 wherein R15 is phenyl substituted with one or more halogen atoms.
69. The compound of claim 68 wherein R15 is 2,6-dihalophenyl.
70. The compound of claim 68 wherein R15 is 2,4,6-trihalophenyl.
71. The compound of claim 68 wherein R15 is 2,3,5,6-tetrahalophenyl.
72. The compound of claim 67 wherein R15 is phenyl substituted with one or more fluorine atoms.
73. The compound of claim 72 wherein R15 is 2,6-difluorophenyl.
74. The compound of claim 72 wherein R15 is 2,4,6-trifluorophenyl.
75. The compound of claim 72 wherein R15 is 2,3,5,6-tetrafluorophenyl.
76. The compound of claim 1 wherein B is CH2OPOR16R17.
77. The compound of claim 76 wherein R16 is methyl.
78. The compound of claim 76 wherein R16 is phenyl.
79. The compound of claim 76 wherein R17 is phenyl.
80. The compound of claim 1 wherein R3 methyl.
81. The compound of claim 1 wherein R3 is ethyl.
82. The compound of claim 1 wherein R3 is isopropyl.
83. The compound of claim 1 wherein R3 is isobutyl.
84. The compound of claim 1 wherein R3 is tert-butyl.
85. A composition comprising a compound of claim 1 in combination with a pharmaceutically acceptable carrier.
86. The compound of claim 1 wherein the compound is:
(3S)-3-[N-(N′-(2-Benzylphenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(Benzyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Phenoxyphenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthylmethyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(4-Chloro-1-Naphthyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Anthryl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(Phenethyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(Phenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Phenylphenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(4-n-Heptylphenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(5,6,7,8-Tetrahydro-1-Naphthyl Oxamyl)-Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Adamantanyl)Oxamyl)-Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(4-Fluorophenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Naphthyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Methoxyphenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(N″-N″′-Diphenylamino)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(4-Pyridinyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Pyrazinyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1,2,3,4-Tetrahydro-1-Naphthyl)Oxamyl)-Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(3,4,5-Trimethoxybenzyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(Benzhydryl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(3-Phenoxylphenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-tert-Butylphenyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Pyridinyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2,3,5,6-Tetrafluoro-4-Pyridinyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Iodophenyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2,6-Difluorophenyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2,5-Di-tert-Butylphenyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(5-Indanyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(Methyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(n-Heptyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(tert-Octyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(Cyclohexyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(5-Phenyl-3-Pyrazolyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2,3,4,5-Tetrafluorophenyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2,3,4,6-Tetrafluorophenyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2,3,5,6-Tetrachlorophenyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2,3,4,5,6-Pentafluorophenyl)OxamylValinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Benzimidazolyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-(2′,6′-Difluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-(2′,4′,6′-Trifluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-(Diphenylphosphinyloxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(I-Naphthyl)Oxamyl)Valinyl]Amino-5-(Methylphenylphosphinyloxy)-4-Oxopentanoic Acid; or (3RS)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-Fluoro-4-Oxopentanoic Acid. 87. The compound of claim 1 wherein the compound is:
(3S)-3-[N-(N′-(Phenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(5,6,7,8-Tetrahydro-1-Naphthyl)Oxamyl)-Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Phenylphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Benzylphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthylmethyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Phenoxyphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(3-Phenoxylphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(4-Phenylphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(Benzhydryl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Phenylphenyl)Oxamyl)Alaninyl]Amino-5-(2′-Fluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-tert-Butylphenyl)Oxamyl)Alaninyl]Amino-5-(2′-Fluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(2-Phenyphenyl)Oxamyl)Alaninyl]Amino-5-(Diphenylphosphinyloxy)-4-Oxopentanoic Acid; or (3S)-3-[N-(N′-(1-Naphthylmethyl)Oxamyl)Alaninyl]Amino-5-(Diphenylphosphinyloxy)-4-Oxopentanoic Acid. 88. The compound of claim 1 wherein the compound is:
(3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-(2′,4′,6′-Trifluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)tert-Butyl)Glycinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Norleucinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)(tert-Butyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-(Methylphenylphosphinyloxy)-4-Oxopentanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-Fluoro-4-Oxopentanoic Acid; or (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-Diphenylphosphinyloxy)-4-Oxopentanoic Acid. 89. The compound of claim 1 wherein the compound is:
(3S)-3-[N-(N′-(2-(1H-Tetrazol-5-yl)Phenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(1-Adamantanyl)Phenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(Phenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(Benzyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(Phenethyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(2-Phenoxyphenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(2-Naphthyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(4-Chloro-1-Naphthyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(5,6,7,8-Tetrahydro-1-Naphthyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(1,2,3,4-Tetrahydro-1-Naphthyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; (3S)-3-[N-(N′-(1-Napthylmethyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid; or (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-4-Oxobutanoic Acid. 90. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-Fluoro-4-Iodophenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
91. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-Chlorophenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
92. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-Bromophenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
93. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-Fluorophenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
94. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-Trifluoromethylphenyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
95. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(1-Anthryl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
96. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-tert-Butylphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
97. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-Trifluoromethylphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
98. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2,6-Difluorophenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
99. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
100. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(4-Methoxyphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid.
101. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-Trifluoromethylphenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid.
102. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-tert-Butylmethylphenyl)Oxamyl)Valinyl]Amino-4--Oxobutanoic Acid.
103. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-Benzylphenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid.
104. The compound of claim 1 wherein the compound is (3S)-3-[N-(N′-(2-Phenylphenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid.
CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of PCT Application No. PCT/US99/15074 filed Jul. 1, 1999, and is a continuation of U.S. application Ser. No. 09/177,549 filed Oct. 22, 1998 and now issued as U.S. Pat. No. 6,197,750, which claims the benefit of U.S. Provisional Application No. 60/091,689, filed Jul. 2, 1998.
TECHNICAL FIELD The present invention relates to novel classes of compounds which are inhibitors of interleukin-1β converting enzyme and related proteases (�ICE/ced-3 family of cysteine proteases�), as well as pharmaceutical compositions comprising these compounds and to methods of using such pharmaceutical compositions.
BACKGROUND OF THE INVENTION Interleukin 1 (�IL-1�) is a major pro-inflammatory and immunoregulatory protein that stimulates fibroblast differentiation and proliferation, the production of prostaglandins, collagenase and phospholipase by synovial cells and chondrocytes, basophil and eosinophil degranulation and neutrophil activation. Oppenheim, J. H. et al., Immunology Today, 7:45-56 (1986). As such, it is involved in the pathogenesis of chronic and acute inflammatory and autoimmune diseases. IL-1 is predominantly produced by peripheral blood monocytes as part of the inflammatory response. Mosely, B. S. et al., Proc. Nat. Acad. Sci., 84:4572-4576 (1987); Lonnemann, G. et al., Eur. J. Immunol., 19:1531-1536 (1989).
ICE is a cysteine protease localized primarily in monocytes. In addition to promoting the pro-inflammatory and immunoregulatory properties of IL-1β, ICE, and particularly its homologues, also appear to be involved in the regulation of cell death or apoptosis. Yuan, J. et al., Cell, 75:641-652 (1993); Miura, M. et al., Cell, 75:653-660 (1993); Nett-Giordalisi, M. A. et al., J. Cell Biochem., 17B: 117 (1993). In particular, ICE or ICE/ced-3 homologues are thought to be associated with the regulation of apoptosis in neurogenerative diseases, such as Alzheimer's and Parkinson's disease. Marx, J. and M. Baringa, Science 259:760-762 (1993); Gagliardini, V. et al., Science, 263:826-828 (1994).
Thus, disease states in which inhibitors of the ICE/ced-3 family of cysteine proteases may be useful as therapeutic agents include: infectious diseases, such as meningitis and salpingitis; septic shock, respiratory diseases; inflammatory conditions, such as arthritis, cholangitis, colitis, encephalitis, endocerolitis, hepatitis, pancreatitis and reperfusion injury, ischemic diseases such as the myocardial infarction, stroke and ischemic kidney disease: immune-based diseases, such as hypersensitivity; auto-immune diseases, such as multiple sclerosis; bone diseases; and certain neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Such inhibitors are also useful for the repopulation of hematopoietic cells following chemo- and radiation therapy and for prolonging organ viability for use in transplantation.
SUMMARY OF THE INVENTION In general, the compounds of this invention incorporate a (N-substituted)oxamyl group as a dipeptide mimetic. The resulting compounds exhibit improved properties relative to their peptidic counterparts, for example, such as improved cell penetration or improved absorption and metabolic stability resulting in enhanced bioavailability. This application claims priority from U.S. Provisional Application No. 60/091,689, filed Jul. 2, 1998, and U.S. Application Ser. No. 09/177,549, filed Oct. 22, 1998 (both of which are hereby incorporated by reference in their entirety).
One aspect of the instant invention is the compounds of the Formula I: wherein A, B R1 and R2 are as defined below, as well as pharmaceutically acceptable salts thereof.
A further aspect of the instant invention is a pharmaceutical composition comprising a compound of the above Formula I and a pharmaceutically-acceptable carrier therefor.
Yet another aspect of the instant invention is a method for treating an inflammatory disease comprising administering an effective amount of a pharmaceutical composition discussed above to a patient in need of such treatment.
A further aspect of the instant invention is a method for treating a neurodegenerative disease comprising administering an effective amount of a pharmaceutical composition discussed above to a patient in need of such treatment.
A further aspect of the instant invention is a method for expanding of hematopoietic cell populations and/or enhancing their survival by contacting the cells with an effective amount of the pharmaceutical composition discussed above. Cell populations included in the method of the invention include (but are not limited to) granulocytes, monocytes, erthrocytes, lymphocytes and platelets for use in cell transfusions.
An alternate aspect of the instant invention is a method of prolonging the viability of an organ that has been removed from the donor for the purpose of a future transplantation procedure, which comprises applying an effective amount of the pharmaceutical composition discussed above to the organ, thereby prolonging the viability of the organ as compared to an untreated organ. The organ may be an intact organ, or isolated cells derived from an organ (e.g., isolated pancreatic islet cells, isolated dopaminergic neurons, blood or hematopoietic cells).
DETAILED DESCRIPTION OF THE INVENTION As mentioned above, one aspect of the instant invention is the compounds of the Formula I: wherein:
A is a natural or unnatural amino acid of Formula IIa-i: B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, substituted naphthyl, 2-benzoxazolyl, substituted 2-oxazolyl, (CH2)ncycloalkyl, (CH2)nphenyl, (CH2)n(substituted phenyl), (CH2)n(1 or 2-naphthyl), (CH2)n(substituted 1 or 2-naphthyl), (CH2)n(heteroaryl), (CH2)n(substituted heteroaryl), halomethyl, CO2R12, CONR13R14, CH2ZR15, CH2OCO(aryl), CH2OCO(heteroaryl), or CH2OPO(R16)R17, where Z is an oxygen or a sulfur atom, or B is a group of the Formula IIIa-c: R1 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, naphthyl, substituted naphthyl, (1 or 2 naphthyl)alkyl, substituted (1 or 2 naphthyl)alkyl, heteroaryl, substituted heteroaryl, (heteroaryl)alkyl, substituted (heteroaryl)alkyl, R1a(R1b)N, or R1cO; and
R2 is hydrogen, lower alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, naphthyl, substituted naphthyl, (1 or 2 naphthyl)alkyl, or substituted 1 or 2 naphthyl)alkyl;
R3 is C1-6 lower alkyl, cycloalkyl, phenyl, substituted phenyl, (CH2)nNH2, (CH2)nNHCOR9, (CH2)nN(C═NH)NH2, (CH2)mCO2R2, (CH2)mOR10, (CH2)mSR11, (CH2)ncycloalkyl, (CH2)nphenyl, (CH2)n(substituted phenyl), (CH2)n(1 or 2-naphthyl) or (CH2)n(heteroaryl), wherein heteroaryl includes pyridyl, thienyl, furyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, pyrazinyl, pyrimidyl, triazinyl, tetrazolyl, and indolyl;
R3a is hydrogen or methyl, or R3 and R3a taken together are �(CH2)d� where d is an interger from 2 to 6;
R6 is hydrogen, fluorine, oxo, lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH2)ncycloalkyl, (CH2)nphenyl, (CH2)n(substituted phenyl), (CH2)n(1 or 2-naphthyl), OR10, SR11 or NHCOR9;
R7 is hydrogen, oxo (i.e., ═O), lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH2)ncycloalkyl, (CH2)nphenyl, (CH2)n(substituted phenyl), or (CH2)n(1 or 2-naphthyl);
or R13 and R14 taken together form a five to seven membered carbocyclic or heterocyclic ring, such as morpholine, or N-substituted piperazine;
R16 and R17 are independently lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, phenylalkyl, substituted phenylalkyl, or (cycloalkyl)alkyl;
R18 and R19 are independently hydrogen, alkyl, phenyl, substituted phenyl, (CH2)nphenyl, (CH2)n(substituted phenyl), or R18 and R19 taken together are �(CH═CH)2�;
Y2 is CH2, O or NR23;
As used herein, the term �alkyl� means a straight or branched C1 to C10 carbon chain, such as methyl, ethyl, tert-butyl, iso-propyl, n-octyl, and the like. The term �lower alkyl� means a straight chain or branched C1 to C6 carbon chain, such as methyl, ethyl, iso-propyl, and the like.
The term �cycloalkyl� means a mono-, bi-, or tricyclic ring that is either fully saturated or partially unsaturated. Examples of such a ring include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin, bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl, 1,4-cyclooctadienyl, and the like.
The term �substituted phenyl� specifies a phenyl group substituted with one or more substituents chosen from halogen, hydroxy, protected hydroxy, cyano, nitro, trifluoromethyl, alkyl, alkoxy, acyl, acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(lower alkyl)carboxamide, protected N-(lower alkyl)carboxamide, N,N-di(lower alkyl)carboxamide, N-((lower alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or by a substituted or unsubstituted phenyl group, such that in the latter case a biphenyl or naphthyl group results, or wherein two adjacent alkyl substituents on the substituted phenyl ring taken together form a cycloalkyl to yield, for example, tetrahydronaphthyl or indanyl.
Examples of the term �substituted phenyl� includes a mono-, di-, tri-, tetra- or penta(halo)phenyl group such as 2-, 3- or 4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 2-,3- or 4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2-, 3- or 4-fluorophenyl, 2,4,6-trifluorophenyl, 2,3,5,6-tetrafluorophenyl, 2,3,4,5-tetrafluorophenyl, 2,3,4,5,6-pentafluorophenyl, and the like; a mono or di(hydroxy)phenyl group such as 2-, 3-, or 4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivatives thereof and the like; a nitrophenyl group such as 2-, 3-, or 4-nitrophenyl; a cyanophenyl group, for example, 2-,3- or 4-cyanophenyl; a mono- or di(alkyl)phenyl group such as 2-, 3-, or 4-methylphenyl, 2,4-dimethylphenyl, 2-, 3- or 4-(iso-propyl)phenyl, 2-, 3-, or 4-ethylphenyl, 2-, 3- or 4-(n-propyl)phenyl and the like; a mono or di(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2-, 3- or 4-(iso-propoxy)phenyl, 2-, 3- or 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl and the like; 2-, 3- or 4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2-, 3- or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- or di(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2-, 3- or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; a mono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as 2-, 3- or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or a mono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or 4-(N-(methylsulfonylamino))phenyl. Also, the term �substituted phenyl� represents disubstituted phenyl groups wherein the substituents are different, for example, 3-methyl-4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy-4-chlorophenyl, and the like.
The term �phenylalkyl� means one of the above phenyl groups attached to one of the above-described alkyl groups, and the term �substituted phenylalkyl� means that either the phenyl or the alkyl, or both, are substituted with one or more of the above-defined substituents. Examples of such groups include 2-phenyl-1-chloroethyl, 2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl, 2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′-dimethylphenyl)n-propyl, 4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl), 5-(4′-aminomethylphenyl)-3-(aminomethyl)n-pentyl, 5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl, and the like.
The term �substituted naphthyl� means a naphthyl group sustituted with one or more of the above-identified subtituents, and the term �(1 or 2 naphthyl)alkyl� means a naphthyl (1 or 2) attached to one of the above-described alkyl groups.
The terms �halo� and �halogen� refer to the fluoro, chloro, bromo or iodo groups. These terms may also be used to describe one or more halogens, which are the same or different. Preferred halogens in the context of this invention are chloro and fluoro.
The term �heteroaryl� denotes optionally substituted aromatic five-membered or six-membered heterocyclic rings that have 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, in particular nitrogen, either alone or in conjunction with sulfur or oxygen ring atoms.
The following ring systems are representative examples of the heterocyclic radicals denoted by the term �heteroaryl� (whether substituted or unsubstituted): thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, as well as benzo-fused derivatives, for example, benzoxazolyl, benzothiazolyl, benzimidazolyl and indolyl.
Substituents for the above optionally substituted heteroaryl rings are from one to three halo, trihalomethyl, amino, protected amino, amino salts, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, carboxylate salts, hydroxy, protected hydroxy, salts of a hydroxy group, lower alkoxy, lower alkylthio, lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and substituted phenylalkyl groups.
Substituents for the heteroaryl group are as defined above, or as set forth below. As used in conjunction with the above substituents for heteroaryl rings, �trihalomethyl� can be trifluoromethyl, trichloromethyl, tribromomethyl or triiodomethyl, �lower alkoxy� means a C1 to C4 alkoxy group, similarly, �lower alkylthio� means a C1 to C4 alkylthio group. The term �substituted lower alkyl� means the above-defined lower alkyl group substituted from one to three times by a hydroxy, protected hydroxy, amino, protected amino, cyano, halo, trifluoromethyl, mono-substituted amino, di-substituted amino, lower alkoxy, lower alkylthio, carboxy, protected carboxy, or a carboxy, amino, and/or hydroxy salt.
As used in conjunction with the substituents for the heteroaryl rings, the terms �substituted (cycloalkyl)alkyl� and �substituted cycloalkyl� are as defined above substituted with the same groups as listed for a �substituted alkyl� group. The term �(monosubstituted)amino� refers to an amino group with one substituent chosen from the group consisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C1 to C7 acyl, C2 to C7 alkenyl, C2 to C7 substituted alkenyl, C2 to C7 alkynyl, C7 to C16 alkylaryl, C7 to C16 substituted alkylaryl and heteroaryl group. The (monosubstituted)amino can additionally have an amino-protecting group as encompassed by the term �protected (monosubstituted)amino.� The term �(disubstituted)amino� refers to amino groups with two substituents chosen from the group consisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C1 to C7 acyl, C2 to C7 alkenyl, C2 to C7 alkynyl, C7 to C16 alkylaryl, C7 to C16 substituted alkylaryl and heteroaryl. The two substituents can be the same or different. The term �heteroaryl(alkyl)� denotes an alkyl group as defined above, substituted at any position by a heteroaryl group, as above defined.
Furthermore, the above optionally substituted five-membered or six-membered heterocyclic rings, and the above cycloalky rings, can optionally be fused to a aromatic 5-membered or 6-membered aryl or heteroaryl ring system. For example, the rings can be optionally fused to an aromatic 5-membered or 6-membered ring system such as a pyridine or a triazole system, and preferably to a benzene ring.
The term �pharmaceutically-acceptable salt� encompasses those salts that form with the carboxylate anions and includes salts formed with the organic and inorganic cations such as those chosen from the alkali and alkaline earth metals, (for example, lithium, sodium, potassium, magnesium, barium and calcium); and ammonium ion; and the organic cations (for example, dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium, phenylethylbenzyl-ammonium, dibenzylethylenediammonium, and like cations.) Other cations encompassed by the above term include the protonated form of procaine, quinine and N-methylglucosamine, the protonated forms of basic amino acids such as glycine, ornithine, histidine, phenylglycine, lysine, and arginine. Furthermore, any zwitterionic form of the instant compounds formed by a carboxylic acid and an amino group is referred to by this term. A preferred cation for the carboxylate anion is the sodium cation. Furthermore, the term includes salts that form by standard acid-base reactions with basic groups (such as amino groups) and includes organic or inorganic acids. Such acids include hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicyclic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and the like acids.
The term �carboxy-protecting group� as used herein refers to one of the ester derivatives of the carboxylic acid group commonly employed to block or protect the carboxylic acid group while reactions are carried out on other functional groups on the compound. Examples of such carboxylic acid protecting groups include t-butyl, 4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl, 4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl, 2-phenylpropyl, trimethylsilyl, t-butyldimethylsilyl, phenacyl, 2,2,2-trichloroethyl, β-(trimethylsilyl)ethyl, β-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl, 4-nitrobenzylsulfonylethyl, allyl, cinnamyl, 1-(trimethylsilylmethyl)propenyl and like moieties. The species of carboxy-protecting group employed is not critical so long as the derivatized carboxylic acid is stable to the conditions of subsequent reaction(s) and can be removed at the appropriate point without disrupting the remainder of the molecule. Further examples of these groups are found in C. B. Reese and E. Haslani, �Protective Groups in Organic Chemistry,� J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, respectively, and T. W. Greene and P. G. M. Wuts, �Protective Groups in Organic Synthesis,� 2nd ed., John Wiley and Sons, New York, N.Y., 1991, Chapter 5, each of which is incorporated herein by reference. A related term is �protected carboxy,� which refers to a carboxy group substituted with one of the above carboxy-protecting groups.
Examples of such amino-protecting groups include the formyl (�For�) group, the trityl group, the phthalimido group, the trichloroacetyl group, the trifluoroacetyl group, the chloroacetyl, bromoacetyl, and iodoacetyl groups, urethane-type protecting groups, such as t-butoxycarbonyl (�Boc�), 2-(4-biphenylyl)propyl-2-oxycarbonyl (�Bpoc�), 2-phenylpropyl-2-oxycarbonyl (�Poc�), 2-(4-xenyl)isopropoxycarbonyl, 1,1-diphenylethyl-1-oxycarbonyl, 1,1-diphenylpropyl-1-oxycarbonyl, 2-(3,5-dimethoxyphenyl)propyl-2-oxycarbonyl (�Ddz�), 2-(p-toluyl)propyl-2-oxycarbonyl, cyclopentanyloxycarbonyl, 1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl, 1-methyl-cyclohexanyloxycarbonyl, 2-methylcyclohexanyl-oxycarbonyl, 2-(4-toluylsulfonyl)ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl, 2-(triphenylphosphino)-ethoxycarbonyl, 9-fluorenylmethoxycarbonyl (�Fmoc�), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, 1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl, 5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyl-oxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl, cyclopropylmethoxycarbonyl, isobornyloxycarbonyl, 1-piperidyloxycarbonyl, benzyloxycarbonyl (�Cbz�), 4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl, α-2,4,5,-tetramethylbenzyl-oxycarbonyl (�Tmz�), 4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, 4-(decyloxy)benzyloxycarbonyl and the like; the benzoylmethylsulfonyl group, the 2,2,5,7,8-pentamethylchroman-6-sulfonyl group (�PMC�), the dithiasuccinoyl (�Dts�) group, the 2-(nitro)phenyl-sulfenyl group (�Nps�), the diphenylphosphine oxide group, and like amino-protecting groups. The species of amino-protecting group employed is not critical so long as the derivatized amino group is stable to the conditions of the subsequent reaction(s) and can be removed at the appropriate point without disrupting the remainder of the molecule. Preferred amino-protecting groups are Boc, Cbz and Fmoc. Further examples of amino-protecting groups embraced by the above term are well known in organic synthesis and the peptide art and are described by, for example, T. W. Greene and P. G. M. Wuts, �Protective Groups in Organic Synthesis,� 2nd ed., John Wiley and Sons, New York, N.Y., 1991, Chapter 7, M. Bodanzsky, �Principles of Peptide Synthesis,� 1st and 2nd revised Ed., Springer-Verlag, New York, N.Y., 1984 and 1993, and J. M. Stewart and J. D. Young, �Solid Phase Peptide Synthesis,� 2nd Ed., Pierce Chemical Co., Rockford, Ill., 1984, E. Atherton and R. C. Shephard, �Solid Phase Peptide Synthesis�A Practical Approach� IRL Press, Oxford, England (1989), each of which is incorporated herein by reference. The related term �protected amino� defines an amino group substituted with an amino-protecting group discussed above.
The terms �natural and unnatural amino acid� refers to both the naturally occurring amino acids and other non-proteinogenic α-amino acids commonly utilized by those in the peptide chemistry arts when preparing synthetic analogues of naturally occurring peptides, including D and L forms. The naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, omithine and lysine. Examples of unnatural alpha-amino acids include hydroxylysine, citrulline, kynurenine, (4-aminophenyl)alanine, 3-(2′-naphthyl)alanine, 3-(1′-naphthyl)alanine, methionine sulfone, (t-butyl)alanine, (t-butyl)glycine, 4-hydroxyphenyl-glycine, aminoalanine, phenylglycine, vinylalanine, propargyl-gylcine, 1,2,4-triazolo-3-alanine, thyronine, 6-hydroxytryptophan, 5-hydroxytryptophan, 3-hydroxykynurenine, 3-aminotyrosine, trifluoromethylalanine, 2-thienylalanine, (2-(4-pyridyl)ethyl)cysteine, 3,4-dimethoxy-phenylalanine, 3-(2′-thiazolyl)alanine, ibotenic acid, 1-amino-1-cyclopentane-carboxylic acid, 1-amino-1-cyclohexanecarboxylic acid, quisqualic acid, 3-(trifluoromethylphenyl)alanine, (cyclohexyl)glycine, thiohistidine, 3-methoxytyrosine, norleucine, norvaline, alloisoleucine, homoarginine, thioproline, dehydroproline, hydroxyproline, homoproline, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 1,2,3,4-tetrahydroquinoline-2-carboxylic acid, α-amino-n-butyric acid, cyclohexylalanine, 2-amino-3-phenylbutyric acid, phenylalanine substituted at the ortho, meta, or para position of the phenyl moiety with one or two of the following groups: a (C1 to C4)alkyl, a (C1 to C4)alkoxy, a halogen or a nitro group, or substituted once with a methylenedioxy group; β-2- and 3-thienylalanine; β-2- and 3-furanylalanine; β-2-, 3- and 4-pyridylalanine; β-(benzothienyl-2- and 3-yl)alanine; β-(1- and 2-naphthyl)alanine; O-alkylated derivatives of serine, threonine or tyrosine; S-alkylated cysteine, S-alkylated homocysteine, the O-sulfate, O-phosphate and O-carboxylate esters of tyrosine; 3-(sulfo)tyrosine, 3-(carboxy)tyrosine, 3-(phospho)tyrosine, the 4-methane-sulfonic acid ester of tyrosine, 4-methanephosphonic acid ester of tyrosine, 3,5-diiodotyrosine, 3-nitrotyrosine, ε-alkyllysine, and delta-alkyl ornithine. Any of these (X-amino acids may be substituted with a methyl group at the alpha position, a halogen at any position of the aromatic residue on the α-amino side chain, or an appropriate protective group at the O, N, or S atoms of the side chain residues. Appropriate protective groups are discussed above.
Depending on the choice of solvent and other conditions known to the practitioner skilled in the art, compounds of this invention may also take the ketal or acetal form, which forms are included in the instant invention. In particular, compounds of Formula I in which R2 is a hydrogen atom (i.e., Formula Ia) may exist in the cyclic ketal or acetal form Formula Ia′ shown below: In addition, it should be understood that the equilibrium forms of the compounds of this invention may include tautomeric forms. All such forms of these compounds are expressly included in the present invention.
The compounds of this invention may be modified by appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of exertion. In addition, the compounds may be altered to pro-drug form such that the desired compound is created in the body of the patient as the result of the action of metabolic or other biochemical processes on the pro-drug. Some examples of pro-drug forms include ketal, acetal, oxime, and hydrazone forms of compounds which contain ketone or aldehyde groups, especially where they occur in the group donated as �A� in Formula I or the modified aspartic acid residue attached to the group denoted as �A�.
Compounds of this invention with respect to the group �R1� in Formula I, include those wherein:
R1 is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, naphthyl, substituted naphthyl, (1 or 2 naphthyl)alkyl, heteroaryl, or (heteroaryl)alkyl.
More typically, the compounds of this invention with respect to the group �R1� include those wherein:
R1 is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, naphthyl, substituted naphthyl, or (1 or 2 naphthyl)alkyl.
Compounds of this invention with respect to the group �A� in Formula I, include those of Formula IIa wherein:
R3 is lower alkyl, cycloalkyl, phenyl, substituted phenyl, (CH2)1NH2, (CH2)mOR10, (CH2)mSR11, (CH2)ncycloalkyl,(CH2)nphenyl, (CH2)n(substituted phenyl), or (CH2)n(1 or 2-naphthyl);
R11 is lower alky, cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH2)ncycloalkyl, (CH2)nphenyl, (CH2)n(substituted phenyl), or (CH2)n(1 or 2-naphthyl); and
n=1-4 and m=1 or 2.
Compounds of this invention with respect to the group �A� in Formula I, also include those of Formula IIb wherein:
R4 is phenyl, substituted phenyl, (CH2)mphenyl, (CH2)m(substituted phenyl), cycloalkyl, or 2-indanyl; and
Another group of compounds with respect to the group �A� in Formula I, include those of Formula IId wherein:
R6 is hydrogen, fluorine, cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH2)ncycloalkyl, (CH2)nphenyl, (CH2)n(substituted phenyl), (CH2)n(1 or 2-naphthyl), OR10, or SR11;
R10 and R11 are independently cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH2)ncycloalkyl, (CH2)nphenyl, (CH2)n(substituted phenyl), or (CH2)n(1 or 2-naphthyl); and
A fourth group of compounds with respect to the group �A� in Formula I, include those of Formula IIe wherein:
R7 is hydrogen, oxo, cycloalkyl, phenyl, substituted phenyl, or naphthyl; and
X═CH2, (CH2)2, (CH2)3, or S.
Another group of compounds with respect to the group �A� in Formula I, include those of Formula IIh wherein:
a=0 and b=or 2.
Compounds of this invention with respect to the group �B� in Formula I, include those wherein:
B is hydrogen, 2-benzoxazolyl, substituted 2-oxazolyl, CH2ZR15, CH2OCO(aryl), or CH2OPO(R16)R17, where Z is an oxygen or a sulfur atom;
R16 and R17 are independently alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, phenylalkyl, substituted phenylalkyl, or (cycloalkyl)alkyl.
Another group of compounds with respect to the group �B� in Formula I, include those of Formula IIIa-c wherein:
R18 and R19 are independently hydrogen, alkyl, or phenyl, or R18 and R19 taken together are �(CH═CH)2�;
R20 is hydrogen, alkyl, phenyl, substituted phenyl, (CH2)nphenyl, or (CH2)n(substituted phenyl);
R21, R22 and R23 are independently hydrogen or alkyl.
The compounds of Formula I may be synthesized using conventional techniques as discussed below. Advantageously, these compounds are conveniently synthesized from readily available starting materials.
One synthetic route for synthesizing the instant compounds is set forth in the following Scheme 1: In the above Scheme 1, Formula (V), that is H2N�C, is a modified aspartic acid residue of Formulas Va through Vd: In the above Scheme 1, �PG� stands for an amino protecting group and �A� stands for a natural or unnatural amino acid of formula Ia through IIi, as discussed above. In Formula Vb through Vd, R2′ is a carboxyl protecting group as described in the definition of R2 in Formula I with the exception that R2′ cannot be a hydrogen atom.
The modified aspartic acids of Formula Va-d can be prepared by methods well known in the art. See, for example, European Patent Application 519,748; PCT Patent Application No. PCT/EP92/02472; PCT Patent Application No. PCT/US91/06595; PCT Patent Application No. PCT/US91/02339; European Patent Application No. 623,592; World Patent Application No. WO93/09135; PCT Patent Application No. PCT/US94/08868; European Patent Application No. 623,606; European Patent Application No. 618,223; European Patent Application No. 533,226; European Patent Application No. 528,487; European Patent Application No. 618,233; PCT Patent Application No. PCT/EP92/02472; World Patent Application No. WO93/09135; PCT Patent Application No. PCT/US93/03589; and PCT Patent Application No. PCT/US93/00481, all of which are herein incorporated by reference.
The coupling reactions carried out under Step A are performed in the presence of a standard peptide coupling agent such as the combination of the combination of dicyclohexylcarbodiimide(DCC) and 1-hydroxy-benzotriazole(HOBt), as well as the BOP (benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate) reagent, pyBOP (benzotriazolyloxy-tris(N-pyrolidinyl)phosphoniumhexafluorophosphate), HBTU (O-benzotriazolyly-tetramethylisouronium-hexafluorophosphate), and EEDQ (1-ethyloxycarbonyl-2-ethyloxy-1,2-dihydroquinoline) reagents, the combination of 1-ethyl(3,3′-dimethyl-1′-aminopropyl)carbodimide (EDAC) and HOBt, and the like, as discussed in J. Jones, �Amino Acid and Peptide Synthesis,� Steven G. Davis ed., Oxford University Press, Oxford, pp. 25-41 (1992); M. Bodanzky, �Principles of Peptide Synthesis,� Haffer et al. ed., Springer-Verlag, Berlin Heidelberg, pp. 9-52 and pp. 202-251 (1984); M:. Bodanzky, �Peptide Chemistry, A Practical Textbook,� Springer-Verlag, Berlin Heidelberg, pp. 55-73 and pp. 129-180; and Stewart and Young, �Solid Phase Peptide Synthesis,� Pierce Chemical Company, (1984), all of which are herein incorporated by reference. The amino protecting group is then removed and the resulting amine is coupled to the (N-substituted) oxamic acid of Formula VII (Step B). Again, this coupling reaction uses the standard peptide coupling reactions mentioned above.
Alternatively, the (N-substituted)oxamic acid of Formula VII can be coupled to an amino ester of Formula IX (Step D). Again, this coupling reaction uses the standard peptide coupling reactions mentioned above. In Formula IX, the group R is a carboxil protecting group such as methyl, allyl, benzyl or tert-butyl. After removal of the carboxyl protecting group under standard conditions well known in the art, the resulting carboxylic acid is coupled to amine V using the standard peptide coupling methods described above (Step E).
In the case where the coupling reaction depicted by either Step A or Step E was carried out with the amino alcohol of Formula Vc, the alcohol moiety must be oxidized to the corresponding carbonyl compound prior to removal of the protecting groups. Preferred methods for the oxidation reaction include Swern oxidation (oxalyl chloride-dimethyl sulfoxide, methylene chloride at −78� C. followed by triethylamine); and Dess-Martin oxidation (Dess-Martin periodinane, t-butanol, and methylene chloride.) The protecting groups contained in substructures of the Formula Va-d and A are removed by methods well known in the art. These reactions and removal of some or all of the protecting groups are involved in Step C in the above Scheme 1.
An alternative synthetic route for synthesizing the instant compounds is set forth in the following Scheme 2: In the above Scheme 2, �PG� stands for an amino protecting group and �A� stands for a natural or unnatural amino acid of formula IIa through IIi, as discussed above. The group R is a carboxyl protecting group such as trimethylsilyl, methyl, allyl, benzyl or tert-butyl.
The coupling reactions carried out under Step F and Step G are performed in the presence of a standard peptide coupling agent as discussed above. In Step G, the amino protecting group must be removed prior to the coupling step. In Step H the alpha-carboxy protecting group R of the compound of Formula XIII is selectively removed and the resulting mono-carboxylic acid treated sequentially with diazomethane and hydrobromic acid to give the alpha-bromoketone of Formula XIV.
In Step I, the bromoketone of Formula XIV is treated with either R15Z-H, (aryl)-CO2H, (heteroaryl)-CO2H, or R16(R17)PO2H in the presence of an inorganic base such as potassium carbonate or potassium fluoride in an inert solvent such as dimethyl formamide to give the corresponding compound of Formula I in which B is CH2ZR15, CH2OCO(aryl), CH2OCO(heteroaryl), or CH2OPO(R16)R17, respectively. Compounds of Formula I in which B is a fragment of Formula III may also be prepared in a similar fashion. The protecting groups contained in substructures of the Formula XI and A are removed by methods well known in the art. These reactions and removal of some or all of the protecting groups are involved in Step I in the above Scheme 2.
An alternative method for the prepartion of compounds of the instant invention of Formula I in which R2 and B are both hydrogen (i.e., Formula Ib) is set forth below in Scheme 3: In Scheme 3, Fmoc is the amino protecting group 9-fluorenylmethoxycarbonyl and the shaded circle labeled �PS� represents polystryene resin.
The coupling of the acid of Formula XV to a primary amine on solid support, preferably aminomethyl polystyrene, is carried out using standard peptide coupling agents, preferably using benzotriazolyloxy-triso-pyrolidinyl)phosphoniumhexafluorophosphate (pyBOP) in a inert solvent such as dimethylformamide or N-methyl pyrrolidone (Step J). After removal of the Fmoc protecting group of XVI by treatment with pyrrolidine-dimethylformamide, the resulting amine is coupled to Fmoc-amino acid of Formula IVa using standard peptide coupling conditions as discussed above (Step K).
In Step L the Fmoc protecting group of the compound of Formula XVII is removed again by treatment with pyrrolidine-dimethylformamide and the resulting amine coupled to the (N-substituted)oxamic acid of Formula VII again using standard peptide coupling conditions as discussed above. The tert-butyl ester of the compound of Formula XVIII is removed by treatment with trfluoroacetic acid-methylene chloride in the presence of a trapping agent such as anisole and the resulting acid cleaved from the solid support by treatment with 37% aqueous formaldehyde/acetic acid/tetrahydrofuran/ trifluoroacetic acid, preferably in a ratio of 1/1/5/0.025, to give the aspartyl aldehyde of Formula Ib (Step M).
Pharmaceutical compositions of this invention comprise any of the compounds of the present invention, and pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle (hereinafter collectively referred to as �pharmaceutically-acceptable carriers�). Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin; buffer substances such as the various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids; water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts; colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat, and the like.
The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or by an implanted reservoir. Oral and parenteral administration are preferred. The term �parenteral� as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articulaor, intrasynovial, intrastemal, intrathecal, intralesional and intracranial injection or infusion techniques.
When the compounds of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to the patient. Alternatively, pharmaceutical compositions according to this invention may be comprised of a combination of a compound of Formula I and another therapeutic or prophylactic agent mentioned above.
Inflammatory disease which may be treated or prevented include, for example, septic shock, septicemia, and adult respiratory distress syndrome. Target autoimmune diseases include, for example, rheumatoid, arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves′ disease, autoimmune gastritis, insulin-dependent diabetes mellitus, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, myasthenia gravis and multiple sclerosis. Target neurodegenerative diseases include, for example, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and primary lateral sclerosis. The pharmaceutical compositions of this invention may also be used to promote wound healing. Target diseases associated with harmful, apoptosis, in other words, those associated with ischemic injury, includes myocardial infarction, stroke, and ischemic kidney disease. The pharmaceutical compositions of this invention may also be used to treat infectious diseases, especially those involved with viral infections.
The amount of the compounds of Formula I that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 0.5 milligrams to 5 grams of a compound of Formula I combined with an appropriate and convenient amount of a pharmaceutically-acceptable carrier which may vary from about 5 to about 95 percent of the total composition. Dosage unit forms will generally contain between from about 1 milligram to about 500 milligrams of an active compound of Formula I.
PREPARATION 1 Preparation of (3S)-Amino-4-Oxobutanoic Acid (tert)-Butyl Ester Semicarbazone, p-Toluenesulfonate Salt Part A: N-(Benzyloxycarbonyl)-L-(N′-Methyl-N′-Methoxy)aspartamide D-(tert-Butyl) Ester
To a solution of N-(benzyloxycarbonyl)-L-aspartic acid-β-(tert-butyl) ester (14.65 g, 45.3 mmol, Bachem) in CH2Cl2 (150 mL) at 0� C. (ice bath) under a nitrogen atmosphere was added 1-hydroxybenzotriazole hydrate (7.29 g, 47.6 mmol, Aldrich) followed by 1-ethyl-3-(3′,3′-dimethyl-1′-aminopropyl)carbodiimide hydrochloride (9.55 g, 49.8 mmol, Sigma). After stirring at 0� C. for 15 min., N,O-dimethylhydroxylamine hydrochloride (5.10 g, 52.3 mmol, Aldrich) and N-methylmorpholine (5.8 mL, 53 mmol, Aldrich) were added. The mixture was allowed to warm to room temperature over 3 hours then stirred at room temperature for 16 hours. The solution was concentrated under vacuum and the residue partitioned between ethyl acetate-5% KHSO4 (200 mL each). The organic phase was washed in turn with 5% KHSO4, saturated sodium bicarbonate and saturated sodium chloride solutions; dried over anhydrous sodium sulfate and evaporated to an oil. The oil was crystallized from hexane to give the title product (16.10 g, 97% yield) as a fluffy white crystalline solid. TLC (ethyl acetate), single spot (TV and PMA): Rf=0.37.
A similar procedure to the one above, starting with 29.3 g of N-(benzyloxycarbonyl)-L-aspartic acid-β-(tert-butyl)ester (2-fold scale up) gave 31.18 g (94% yield) of the title product.
Part B: (3S)-(Benzyloxycarbonyl)Amino-4-Oxobutanoic Acid (tert)-Butyl Ester Semicarbazone.
To a solution of N-(benzyloxycarbonyl)-L-(N′-methyl-N′-methoxy)aspartamide-β-(tert-butyl) ester (15.50 g, 42.3 mmol) in anhydrous ether (400 mL) at 0� C. (ice bath) under a nitrogen atmosphere was added dropwise to a 1.0 M solution of LiAlH4 in ether (22.0 mL, 22.0 mmol, Aldrich) at such a rate as to keep the reaction solution temperature between 0-5� C. (addition time 15-20 min). After the addition of the lithium aluminum hydride reagent was complete, the mixture was stirred at 0-5� C. for 1 hr, then quenched by the dropwise addition of 0.3 N KHSO4 solution (100 mL). The resultant mixture was transferred to a separatory funnel adding sufficient 5% KHSO4 solution (75 mL) to dissolve the solids. The organic phase was separated and the combined aqueous washes back-extracted with ether (100 mL). The combined ether extracts were washed with saturated NaCl solution, dried over anhydrous sodium sulfate and concentrated in vacuo with minimal heating. TLC (ethyl acetate): streaky spot (UV and PMA) Rf=0.48. TLC (methanol/methylene chloride, 1:9) major spot (UV and PMA): Rf=0.75.
The crude aldehyde was immediately taken up in aqueous ethanol (45 mL water/105 mL alcohol), placed in an ice bath and treated with sodium acetate (3.82 g, 46.65 mmol) and semicarbazide hydrochloride (5.20 g, 46.6 mmol, Aldrich). The mixture was stirred at 0� C. (ice bath) under a nitrogen atmosphere for 3 hrs, allowed to warm to room temperature, and stirred overnight (16 hrs). Most of the ethanol was removed under vacuum and the residue partitioned between ethyl acetate and water (100 mL each). The organic phase was washed sequentially with 5% KHSO4, saturated sodium bicarbonate and saturated sodium chloride solutions; dried over anhydrous sodium sulfate and evaporated to dryness. The crude product of this reaction was combined with that of two similar procedures starting with 15.40 g arid 4.625 g of N-(benzyloxycarbonyl)-L-(N′-methyl-N′-methoxy)aspartamide-p-(tert-butyl ester) (total: 35.525 g, 97 mmol) and these combined products were purified by flash chromotagraphy on silica gel eluting with acetone/methylene chloride (3:7) then methanol-acetone-methylene chloride (0.5:3:7) to give pure title product (27.73 g, 78.5%) as a colorless foam. TLC (MeOH�CH2Cl2, 1:9): single spot (UV and PMA), Rf=0.51.
Part C: (3S)-Amino-4-Oxobutanoic Acid (tert)-Butyl Ester Semicarbazone, p-Toluenesulfonate Salt
To a solution of (3S)-(benzyloxycarbonyl)amino-4-oxobutanoic acid (tert)-butyl ester semicarbazone (13.84 g, 38.0 mmol) in absolute ethanol (250 mL) was added 10% Pd/C (1.50 g, Aldrich) and the resulting mixture stirred under an atmosphere of hydrogen (balloon) until TLC (methanol/methylene chloride, 1:9) indicated complete consumption of the starting material (60 min). Note: It is important to follow this reaction closely since the product can be over-reduced. The mixture was filtered though Celite and evaporated to an oil. The oil was chased with methylene chloride (2�75 mL) then with methvlene chloride/toluene (1:1, 75 mL) to give the crude amine as a white crystalline solid. TLC (EtOAc-pyridine-AcOH-H2O; 60:20:5:10) single spot (UV and PMA) Rf=0.24. Note: In this TLC system, any over-reduced product will show up immediately below the desired product, Rf=0.18 (PMA only).
The crude amine was taken up in CH3CN (60 mL) and treated with a solution of p-toluenesulfonic acid monohydrate (7.22 g, 38.0 mmol) in acetonitrile (60 mL). The crystalline precipitate was collected, washed with acetonitrile and ether, and air-dried to give the title compound (13.95 g, 92% yield) as a white, crystalline solid.
The optical purity of this material was checked by conversion to the corresponding Mosher amide [1.05 equiv (R)-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride, 2.1 equivalents of i-Pr2NEt in CH2Cl2, room temperature, 30 min]. The desired product has a doublet at 7.13 ppm (1H, d, J=2.4 Hz, CH═N) while the corresponding signal for its diastereomer is at 7.07 ppm. The optical purity of the title compound obtained from the above procedure is typically >95:5.
PREPARATION 2 Preparation of (3S)-(9-Fluorenylmethoxycabonyl)Amino-4-Oxobutanoic Acid (tert)-Butyl Ester Semicarbazonyl-4-[2′-(4-Ethyl-Phenoxyacetic Acid)] Part A: 4-[2′-(N-t-Butoxycarbonyl)Aminoethyl]Phenoxyacetic Acid, Methyl Ester
To a suspension 4-hydroxy-phenethylamine (7.00 g, 51.1 mmol, Aldrich) in dry dimethylformamide (50 mL) at room temperature under nitrogen was added di-tert-butyl dicarbonate (11.0 g, 50.5 mmol). After stirring at room temperature for 1 hr, the resulting clear solution was treated with methyl bromoacetate (7.5 mL, 79 mmol) and cesium carbonate (17.5 g, 53.7 mmol). After stirring at room temperature for 16 hrs, TLC (Et2O-toluene; 2:8) shows some unalkylated material remained (Rf=0.43) and a second portion of methyl bromoacetate (2.0 mL, 21 mmol) and cesium carbonate (4.5 g, 14 mmol) were added. After stirring for an additional 24 hrs, the mixture was partitioned between EtOAc-water (250 mnL each), organic phase washed succesively with water (3�), 5% potassium bisulfate and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. Trituration of the residue with hexane gave 15.87 g of a tan solid. Filtration of the crude product through a pad of silica gel eluting with EtOAc-hexane (2:8) and crystallization from hexane gave the title compound (14.75, 93%) as a white granular, crystalline solid. TLC (Et2O-toluene; 2:8) Rf=0.53.
Part B: 4-(2′-Aminoethyl)Phenoxyacetic Acid, Methyl Ester, Hydrochloride
To a solution 4-[2′-(N-t-butoxycarbonyl) aminoethyl]phenoxyacetic acid, methyl ester (18.31 g, 59.3 mmol) in dioxane (55 mL) at room temperature was added 4.0 N HCl in dioxane (55 mL). After stirring at room temperature for 16 hrs, the mixture was diluted with Et2O, the precipatate collected, washed throughly with Et2O and dried in vacuo to give the title compound (14.55 g, 94%) was a fluffy white, crystalline solid.
Part C: 1-tert-Butoxycarbonyl-Semicarbazidyl-4-[2′-(4-Ethyl-Phenoxyacetic Acid)]Methyl Ester.
A solution of t-butyl carbazate (6.60 g, 50 mmol) in dimethylfornamide (50 mL) was added dropwise to a solution carbonyldiimidazole (8.10 g, 50 mmol) in dimethylformamide (80 mL) over 40 min at room temperature under nitrogen. After stirring at room temperature for an additional 30 min, 4-(2′-aminoethyl)phenoxyacetic acid, methyl ester, hydrochloride (12.3 g, 50 mmol) was added as a solid in one portion followed by a triethylamine (8.0 mL, 58 mmol) added dropwise over 30 min. After stirring at room temperature for 18 hrs, the mixture was partitioned between EtOAc-water (300 mL each). The organic phase was washed succesively with water (3�), 5% potassium bisulfate, saturated sodium bicarbonate, and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. Crystallization of the residue from EtOAc-hexane gave the title compound (15.50, 84%) as an off-white crystalline solid. TLC (MeOH�CH2Cl2; 1:9) Rf=0.45.
Part D: 1-tert-Butoxycarbonyl-Semicarbazidyl-4-[2′-(4-Ethyl-Phenoxyacetic Acid)]
A solution of 1-tert-butoxycarbonyl-semicarbazidyl-4-[2′-(4-ethyl-phenoxyacetic acid)] methyl ester (14.68 g, 40 mmol) in dioxane (50 mL) at room temperature under nitrogen was added 1.0 N LiOH solution (50 mL). After stirring at room temperature for 1 hr, the mixture was acidified with conc. HCl and extracted with EtOAc (100 mL). The organic phase was washed with saturated NaCl solution, dried over anhydrous Na2SO4 and evaporated to a white solid. Recrystallization of the crude product from THF-EtOAc-hexane gave the title compound (13.44, 95%) as a white crystalline solid. TLC (AcOH�MeOH�CH2Cl2; 1:1:8) Rf=0.31.
Part E: Semicarbazidyl-4-[2′-(4-Ethyl-Phenoxyacetic Acid)] Hydrochloride
To a solution of 1-tert-butoxycarbonyl-semicarbazidyl-4-[2′-(4-ethyl-phenoxyacetic acid)] (13.43 g, 38.0 mmol) in dioxane (80 mL)-anisole (15 mL) at room temperature was added 4.0 N HCl in dioxane (35 mL). After stirring at room temperature for 18 hrs, additional 4.0 N HCl in dioxane (15 mL) was added. After an additional 6 hrs, the precipatate was collected, washed throughly with dioxane then Et2O and dried in vacuo to give the title compound (11.67 g, 100%) was a white, crystalline solid.
Part F: N-(9-Fluorenylmethoxycarbonyl)-L-(N′-Methyl-N′-Methoxy)aspartamide β-(tert-Butyl) Ester
To a solution of N-(9-fluorenylmethoxycarbonyl)-L-aspartic acid-β-(tert-butyl) ester (16.48 g, 40 mmol) in CH2Cl2 (80 ml)-tetrahydrofuran (20 mL) at 0� C. (ice bath) under a nitrogen atmosphere was added 1-hydroxybenzotriazole hydrate (7.12 g, 46.5 mmol) followed by 1-ethyl-3-(3′,3′-dimethyl-l′-aminopropyl)carbodilmide hydrochloride (9.20 g, 48 mmol). After stirring at 0� C. for 15 min., N,O-dimethylhydroxylamine hydrochloride (4.68 g, 48 mmol) and N-methylmorpholine (5.2 mL, 47 mmol) were added. The mixture was allowed to warm to room temperature over 2 hours then stirred at room temperature for 16 hours. The solution was concentrated under vacuum and the residue partitioned between ethyl acetate-5% KHSO4 (200 mL each). The organic phase was washed succesively with 5% KHSO4, saturated sodium bicarbonate and saturated sodium chloride solutions; dried over anhydrous sodium sulfate and evaporated to an oil. Purification of the crude product by flash chromatography on silica gel eluting with EtOAc-hexane (30:70 then 35:65) gave the title product (17.75 g, 98% yield) as a colorless foam. TLC (EtOAc-hexane; 1:1) Rf=0.35.
Part G: (3S)-(9-Fluorenylmethoxycabonyl)Amino-4-Oxobutanoic Acid (tert)-Butyl Ester Semicarbazonyl-4-[2′-(4-Ethyl-Phenoxyacetic Acid)]
To a solution of N-(9-fluorenylmethoxycarbonyl)-L-(N′-methyl-N′-methoxy)aspartamide-β-(tert-butyl) ester (13.20 g, 29 mmol) in anhydrous ether (250 mL) at 0� C. (ice bath) under a nitrogen atmosphere was added dropwise to a 1.0 M solution of LiAlH4 in ether (14.5 mL, 14.5 mmol) at such a rate as to keep the reaction solution temperature between 0-5� C. (addition time 15-20 min). After the addition of the lithium aluminum hydride reagent was complete, the mixture was stirred at 0-5� C. for 1 hr, then quenched by the dropwise addition of 0.3 N KHSO4 solution (100 mL). After adding sufficient 0.3 N KHSO4 solution to dissolve most of the inorganic salts, the mixture was transferred to a seperatory funnel. The organic phase was separated and the aqueous phase back-extracted with ether (100 mL). The combined ether extracts were washed with saturated NaCl solution, dried over anhydrous sodium sulfate and concentrated in vacuo with minimal heating. TLC (EtOAc-hexane): Rf=0.40.
The crude aldehyde was immediately taken up in ethanol(105 mL)-water(45 mL)-tetrahydrofuran(75 mL), placed in an ice bath and treated with sodium acetate (3.20 g, 39 mmol) and semicarbazidyl-4-[2′-(4-ethyl-phenoxyacetic acid)] hydrochloride (8.65 g, 30 mmol). The mixture was stirred at 0� C. (ice bath) under a nitrogen atmosphere for 3 hrs, allowed to warm to room temperature, and stirred overnight (16 hrs). The mixture was concentrated on a rotovap, diluted with water and resulting precipitate collected by suction. The material was dried in vacuo to give 18.36 g of crude product as a white solid. The cnrde product of this reaction was combined with that of a smaller scale reaction (6.34 g) starting with 4.55 g (10 =mol) of N-(9-fluorenylmethoxycarbonyl)-L-(N′-methyl-N′-methoxy)aspartamide-β-(tert-butyl ester) and partitioned between ethyl acetate-tetrahydrofuran(1:1) and 5% KHSO4. The organic phase was washed with 5% KHSO4 and saturated sodium chloride solutions, dried over anhydrous sodium sulfate and evaporated to dryness. The residue was purified by filtration through a pad of silica gel eluting with terahydrofuran/methylene chloride (1:1). The combined product-containing fractions were evaporated to dryness and recrystallized from tetrahydrofuran-Et2O to give pure title product (17.01 g, 69%) as a white solid. TLC (AcOH�MeOH�CH2Cl2, 1:1:40): Rf=0.19.
PREPARATION 3 Assay for Inhibition of ICE/ced-3 Protease Family Activity A. Determination of IC50 Values
Fluorescence enzyme assays detecting the activity of the compounds of Formula I utilizing the recombinant ICE and CPP32 enzymes are performed essentially according to Thomberry et al. (Nature, 356:768:774 (1992)) and Nicholson et al. (Nature, 376:37-43 (1995)) respectively, (herein incorporated by reference) in 96 well microtiter plates. The substrate is Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin (AMC) (SEQ ID NO:1) for the ICE assay and Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (SEQ ID NO:2) for the CPP32, Mch2, Mch3 and Mch5 assays. Enzyme reactions are run in ICE buffer (25 mM HEPES, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, pH 7.5) containing 2 mM DTT at room temperature in duplicate. The assays are performed by mixing the following components:
50 μL compound of Formula I or ICE buffer (control); and
The enzyme and the compound of Formula I to be assayed are allowed to preincubate in the microtitre plate wells for 30 minutes at room temperature prior to the addition of substrate to initiate the reaction. Fluorescent AMC product formation is monitored for one hour at room temperature by measuring the fluorescence emission at 460 nm using an excitation wavelength of 360 nm. The fluorescence change in duplicate (control) wells are averaged and the mean values are plotted as a function of inhibitor concentration to determine the inhibitor concentration producing 50% inhibition (IC50). The results of this assay are set forth below in Table 1.
The reference compound for this assay was Cbz-ValAlaAsp-H and the values are denoted in Table 1 as �Reference�.
For the irreversible inhibition of a ICE/ced-3 Family Protease enzyme with a competitive irreversible inhibitor; using the model represented by the following formulas: The product formation at time t may be expressed as: [ P ] t = [ E ] T  ( [ S ]  K i [ I ]  K s )   ( k s k 3 )  [ 1 -  - k 3  t / ( 1 + K i [ I ]  ( 1 + [ S ] K s ) ) ] Equation 1 where E, I, EI and E-I denote the active enzyme, inhibitor, non-covalent enzyme-inhibitor complex and covalent enzyme-inhibitor adduct, respectively. The Ki value is the overall dissociation constant of the reversible binding steps, and k3 is the irreversible rate constant. The [S] and Ks values are the substate concentration and dissociation constant of the substrate bound to the enzyme, respectively. [E]T is the total enzyme concentration.
The above equations were used to determine the K1 and k3 values of a given inhibitor bound to a ICE/ced-3 family protease. Thus, a continuous assay was run for sixty minutes at various concentrations of the inhibitor and the substrate. The assay was formulated essentially the same as described above for generating the data in Table 1, except that the reaction was initiated by adding the enzyme to the substrate-inhibitor mixture. The Ki and k3 values were obtained by simulating the product AMC formation as a function of time according to Equation 1. The results of this second assay are set forth below in Table 2.
The reference compound for this assay was Cbz-ValAlaAsp-CH2F, which had a Ki (μM) of 0.015 (mICE), 0.820 (CPP32), 0.594 (MCH-2), and 0.018 (MCH-5). Preferred compounds in this assay have a Ki of less than 0.1 μM (100 nM), and more preferably less than 0.01 μM(10 nM).
To this end, representative compounds having a Ki of less than 0.1 μM include the following compounds listed in Table 2A:
(Ki < 0.1 μM)
3-8, 10-13, 15-16, 18-19, 28-35, 40, 42-43, 45-49, 55-61,
63-64, 66, 72, 75-79, 81-82, 84, 89, 91-95, 102-110B
3-13, 15-19, 29-35, 37-38, 40-43, 46-49, 52, 55-61, 64-66,
72, 76, 103-104, 106
3-7, 10-13, 15-16, 18-19, 28-35, 37-43, 45-53, 55-66, 72-75,
78, 83-84, 91-95, 102-108, 110A-110B
3-8, 10-13, 15-16, 18-19, 28-35, 37-43, 45-66, 72, 74-79,
81-84, 89-95, 102-110B
Furthermore, representative compounds having a Ki of less than 0.01 μM include the following compounds listed in Table 2B:
(Ki < 0.01 μM)
3-8, 10-12, 15-16, 33-34, 42, 46, 49, 55-57, 78-79, 82, 93, 95,
4, 29, 38, 42-43, 46, 55, 64, 108
3-7, 10-11, 15-16, 28, 33-34, 38, 42-43, 45-49, 55-59, 64, 72,
75, 78-79, 82, 92-93, 95, 103-104, 106-108. 110A-110B
EXAMPLE 1 (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-4-Oxobutanoic Acid Part A: N-(1-Naphthyl)Oxamic Acid
To a solution of 1-aminonaphthylene (1.43 g, 10 mmol) and triethylamine (1.5 mL, 10.8 mmol) in CH2Cl2 (10 mL) at 0� C. (ice bath) under nitrogen was added dropwise a solution of methyl oxalyl chloride (1.0 mL, 10.9 mmol) in CH2Cl2 (5 mL). When the addition was complete, the mixture was allowed to come to room temperature and stirred for 1 hr. The mixture was concentrated and the residue partitioned between EtOAc-5% KHSO4. The organic phase was washed with 5% KHSO4 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a pink solid. Recrystallization of the crude product from toluene-hexzme gave the N-(1-naphthyl)oxamic acid methyl ester (2.066 g, 90%) as a pink crystalline solid. TLC(EtOAc-hexane) Rf=0.6.
The methyl ester (1.97 g, 8.6 mmol) was taken up in dioxane (10 mL) and treated with 1.0 N LiOH solution (10 mL, 10 mmol) and stirred at room temperature for 1 hr. The mixture was acidified with conc. HCl and extracted with EtOAc. The extract was washed with satuated NaCl solution, dried over anhydrous Na2SO4 and evaporated to a pink solid,. Recrystallization of the crude product from EtOAc-hexane gave the title compound (1.712 g, 85%) as a pink crystalline solid. TLC(AcOH�MeOH�CH2Cl2; 1:1:20) Rf=0.06.
Part B: (3S)-3-[(N-Benzyloxycarbonyl)Leucinyl]Amino-4-Oxobutanoic Acid (tert)-Butyl Ester Semicarbazone
To a solution of (N-benzyloxycarbonyl)leucine N-hydroxysuccinimde ester (1.81 g, 5.0 mmol) in CH2Cl2 (30 mL) at room temperature under nitrogen was added (3S)-amino-4-oxobutanoic acid (tert)-butyl ester semicarbazone, p-toluenesulfonate salt (2.58 g, 6.4 mmol) followed by diisopropyl ethylamine (1.2 mL, 6.9 mmol). After stirring at room temperature for 16 hrs, the mixture was concentrated and the residue partitioned between EtOAc-5% KHSO4. The organic phase was washed with 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to give the title compound (2.798 g) as a pale yellow foam. TLC(MeOH�CH2Cl2; 1:9) Rf=0.52.
Part C: (3S)-3-(Leucinyl)Amino-4-Oxobutanoic Acid (tert)-Butyl Ester Semicarbazone
To a solution of crude (3S)-[(N-benzyloxycarbonyl)leucinyl]amino-4-oxobutanoic acid (tert)-butyl ester semicarbazone (2.798 g, ca.5.0 mmol) in absolute EtOH (40 mL) was added 10% Pd�C (0.40 g) and resulting mixture stirred undera hydrogen atmosphere (ballon) for 1.5 hrs. The mixture was filtered through Celite washing the filter cake with CH2Cl2 and the combined filtrates evaporated to dryness. The residue was chased with CH2Cl2 (2�20 mL) to give the title product (2.113 g) as a colorless foam. TLC(MeOH�CH2Cl2; 1:9) Rf=0.23.
Part D: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-4-Oxobutanoic Acid (tert)-Butyl Ester Semicarbazone
To a solution of N-(1-naphthyl)oxamic acid (0.095 g, 0.44 mmol) and (3S)-3-(leucinyl)amino-4-oxobutanoic acid (tert)-butyl ester semicarbazone (0.180 g, ca 0.41 mmcol) in N-methylpyrrolidone(1.0 mL)-CH2Cl2(1.0 mL) at 0� C. (ice bath) under nitrogen was added hydroxybenzotriazole hydrate (0.100 g) followed by 1-ethyl-3-(3′,3′-dimethyl-1′-aminopropyl)carbodiimide hydrochloride (0.100 g, 0.52 mmol). After stirring at 0� C. for 2 hrs and at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a solid. The solid residue was triturated with Et2O to give the title compound (0.231 g, 97%) as an off-white solid. TLC(MeOH�CH2Cl2; 5:95) Rf=0.32.
Part E: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-4-Oxobutanoic Acid Semicarbazone
To a suspension of (3S)-3-[N-(N′-(1-naphthyl)oxamyl)leucinyl]amino-4-oxobutanoic acid (tert)-butyl ester semicarbazone (0.212 g, 0.39 mmol) in CH2Cl2(2.0 mL)-anisole(0.5 mL) at room temperature under nitrogen was added trifluoroacetic acid (2.0 mL). The resulting clear solution was stirred at room temperature for 3 hrs, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue was triturated with Et2O to give the title compound (0.181 g, 95%) as an off-white solid. TLC(AcOH�MeOH�CH2Cl2; 1:1:20) Rf=0.16.
Part F: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-4-Oxobutanoic Acid
A suspension of (3S)-3-[N-(N′-(1-naphthyl)oxamyl)leucinyl]amino-4-oxobutanoic acid semicarbazone (0.173 g, 0.36 mmol) in 37% aqueous formaldehyde(1.0 mL)-acetic acid(1.0 mL)-methanol(3.0 mL) was stirred at room temperature under nitrogen for 18 hrs. The resulting clear solution was diluted with water and the resulting white precipitate collected by suction and washed with water. The combined aqueous filtrate was extracted with EtOAc. The extract was washed with water and saturated NaCl solution, dried over anhydrous Na2SO4 and evaporated to a glass. This was combined with the solid which was filtered from the aqueous mixture, taken up in CH2Cl2, filtered through Celite and evaporated to dryness. The crude product was purified by dissolving the residue in CH2Cl2 and precipitating with Et2O-hexane. The precipitate was collected by suction to give the title compound (0.129 g, 84%) as a white solid. TLC(AcOH�MeOH�CH2Cl2; 1:1:20) Rf=0.22. MS(ES) for C22H25N3O6 (MW 427.46): positive 450(M+Na); negative 426(M−H).
EXAMPLE 2 (3RS)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-Fluoro-4-Oxopentanoic Acid Part A: (3RS,4RS)-3-[(N-Benzyloxycarbonyl)Leucinyl]Amino-5-Fluoro-4-Hydroxypentanoic Acid, tert-Butyl Ester
To a solution of (3RS,4RS)-3-amino-5-fluoro-4-hydroxypentanoic acid, tert-butyl ester (0.230 g, 1.1 mmol, prepared as described in Tetrahedron Letters 1994,35, 9693-9696) in CH2Cl2 (2.0 mL) at room temperature under nitrogen was added (N-benzyloxycarbonyl)leucine, N-hydroxysuccinimde ester (0.402 g, 1.1 mmol). After stirring at room temperature for 16 hrs, the mixture was evaporated to dryness and the residue purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:2) to give the title compound (0.332 g, 66%) as a colorless, viscous oil. TLC(EtOAc-hexane; 2:1) Rf=0.51.
Part B: (3RS,4RS)-3-(Leucinyl)Amino-5-Fluoro-4-Hydroxypentanoic Acid, tert-Butyl Ester, p-Toluenesulfonate Salt
To a solution of (3RS,4RS)-3-[(N-benzyloxycarbonyl)leucinyl]amino-5-fluoro-4-hydroxypentanoic acid, tert-butyl ester (0.332 g, 0.734 mmol) in MeOH (100 mL) was added p-toluenesulsufonic acid hydrate (0.140 g, 0.737 mmol) and 10% Pd�C (0.033 g) and resulting mixture stirred under a hydrogen atmosphere (balloon) for 2 hrs. The mixture was filtered through Celite washing the filter cake with CH2Cl2 and the combined filtrates evaporated to dryness. The residue was chased with CH2Cl2 to give the title product (0.371 g) as a colorless foam.
Part C: (3RS,4RS)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-Fluoro-4-Hydroxypentanoic Acid, tert-Butyl Ester
To a solution of N-(1-naphthyl)oxamic acid (0.161 g, 0.749 mmol, see Example 1, Part A) in in N-methylpyrrolidone(1.5 mL)-CH2Cl2(1.5 mL) at room temperature under nitrogen was added O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumn hexafluorophate (0.313 g, 0.823 mmol). After stirring for 0.5 hrs, the mixture was treated with a solution of (3RS,4RS)-3-(leucinyl)amino-5-fluoro-4-hydroxypentanoic acid, tert-butyl ester, p-toluenesulfonate salt (0.371 g, 0.749 mmol) and dilsopropylethylamine (0.39 mL, 2.25 mmol) in N-methylpyrrolidone(2.0 mL)-CH2Cl2(2.0 mL). After stirring at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:1) to give the title compound (0.213 g, 55%) as a colorless foam. TLC(Et2O�CH2Cl2-hexane; 2:1:2, 2 developments) Rf=0.12.
Part D: (3RS)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-Fluoro-4-Oxopentanoic Acid, tert-Butyl Ester
To a solution of (3RS,4RS)-3-[N-(N′-(1-naphthyl)oxamyl)leucinyl]amino-5-fluoro-4-hydroxypentanoic acid, tert-butyl ester (0.163 g, 0.315 mmol) in CH2Cl2 (3.0 mL) at room temperature was added Dess-Martin periodinane (0.160 g, 0.378 mmol). After stirring at room temperature for 0.5 hrs, the mixture was diluted with EtOAc and washed with dilute Na2S2O3, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:3) to give the title compound (0.155 g, 95%) as a white solid. TLC(Et2O�CH2Cl2-hexane; 2:1:2, 2 developments) Rf=0.35. MS(ES) for C27H34FN3O6 (MW 515.57): positive 538(M+Na); negative 514(M−H).
Part E: (3RS)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-Fluoro-4-Oxopentanoic Acid
To a solution of (3RS)-3-[N-(N′-(1-naphthyl)oxamyl)leucinyl]amino-5-fluoro-4-oxopentanoic Acid, tert-butyl ester (0.147 g, 0.285 mmol) in CH2Cl2(1.0 mL)-anisole(0.5 mL) at room temperature under nitrogen was added trifluoroacetic acid (1.0 mL). The resulting clear solution was stirred at room temperature for 1 hr, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue was triturated with Et2O-hexane to give the title compound (0.100 g, 76%) as a white solid. MS(ES) for C23H26FN3O6 (MW 459.47): positive 482(M+Na); negative 458(M−H).
EXAMPLE 3 (3RS)-3-[N-(N-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-Fluoro-4-Oxopentanoic Acid Part A: (3RS,4RS)-3-[(N-Benzyloxycarbonyl)Valinyl]Amino-5-Fluoro-4-Hydroxypentanoic Acid, tert-Butyl Ester
To a solution of (N-benzyloxycarbonyl)valine (0.332 g, 1.32 mmol) in CH2Cl2(7.0 mL) at 0� C. (ice bath) under nitrogen was added hydroxybenzotriazole hydrate (0.219 g) followed by 1-ethyl-3-(3′,3′-dimethyl-1′-aminopropyl)carbodiimide hydrochloride (0.317 g, 1.65 mmol). After stirring at 0� C. for 10 min, the mixture was treated with (3RS,4RS)-3-amino-5-fluoro-4-hydroxypentanoic acid, tert-butyl ester (0.228 g, 1.1 mmol) and the reacton allowed to warm to room temperature. After stirring at room temperature for 24 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography eluting with EtOAc-hexane (1:1) to give the title compound (0.423 g, 87%) as colorless glass. TLC(MeOH�CH2Cl2; 5:95) Rf=0.17.
Part B: (3RS)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-Fluoro-4-Oxopentanoic Acid
Starting with (3RS,4RS)-3-[(N-benzyloxycarbonyl)valinyl]amino-5-fluoro-4-hydroxypentanoic acid, tert-butyl ester and following the methods described in Example 2, Parts B through E gave the title compound as a white solid. MS(ES) for C22H24FN3O6 (MW 445.45): positive 468(M+Na), 484(M+K); negative 444(M−H).
EXAMPLE 4 (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-(2′,6′-Dichlorobenzoyloxy)-4-Oxopentanoic Acid Part A: [(N-Benzyloxycarbonyl)Valinyl]Aspartic Acid, βtert-Butyl, α-Methyl Ester
To a solution of N-benzyloxycarbony)vaine (2.10 g, 8.36 mmol) in CH2Cl2(20 mL) at 0� C. (ice bath) under nitrogen was added hydroxybenzotriazole hydrate (1.74 g) followed by 1-ethyl-3-(3′,3′-imethyl-l′-aminopropyl)carbodiimide hydrochloride (2.40 g, 12.5 mmol). After stirring at 0� C. for 10 min the mixture was treated with aspartic acid, α-tert-butyl, α-methyl ester hydrochloride (2.00 g, 8.34 mmol) and N-methylmorpholine (1.1 mL, 10 mmol), and the reacton allowed to warm to room temperature. After stirring at room temperature for 2.5 hrs, the mixture was concentrated and the residue partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to give the title compound (3.55 g, 97%) as a white solid after tituration with Et2O-hexane. TLC(EtOAc-hexane; 1:1) Rf=0.48.
Part B: (Valinyl)Aspartic Acid, β-tert-Butyl, α-Methyl Ester p-Toluenesulfonate Salt
To a solution of [(N-benzyloxycarbonyl)valinyl]aspartic acid, β-tert-butyl, α-methyl ester (3.55 g, 8.12 mmol) in MeOH (300 mL) was added p-toluenesulsufonic acid hydrate (1.55 g, 8.12 mmol) and 10% Pd�C (0.30 g) and resulting mixture stirred under a hydrogen atmosphere (balloon) for 2 hrs. The mixture was filtered through Celite washing the filter cake with CH2Cl2 and the combined filtrates evaporated to dryness. The residue was chased with CH2Cl2 to give the title product (3.85 g, quantitative) as a colorless foam.
Part C: [N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Aspartic Acid, β-tert-Butyl, α-Methyl Ester
To a solution of N-(1-naphthyl)oxamic acid (0.683 g, 3.18 mmol, see Example 1, Part A) in in N-methylpyrrolidone(7.0 mL)-CH2Cl2(7.0 mL) at room temperature under nitrogen was added O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophate (1.329 g, 3.49 mmol). After stirring for 15 min, the mixture was treated with N-(valinyl)aspartic acid, β-tert-butyl, a-methyl ester p-toluenesulfonate salt (1.506 g, 3.18 mmol) and diisopropylethylamine (1.66 mL, 9.53 mmol). After stirring at room temperature for 2 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:1) to give the title compound (1.153 g, 73%) as a white solid. TLC(EtOAc-hexane; 2:1) Rf=0.48.
Part D: [N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Aspartic Acid, Ptert-Butyl Ester
To a solution of [N-(N′-(1-naphthyl)oxamyl)valinyl] aspartic acid, β-tert-butyl, α-methyl ester (0.490 g, 0.98 mmol) in dioxane (2.4 mL) was added 1.0 N LiOH solution (1.0 mL, 1.0 mmol). After stirring at room temperature for 1 hr, the mixture was acidified with 1.0 N HCl and extracted with EtOAc. The extract was washed with saturated NaCl solution, dried over anhydrous Na2SO4 and evaporated to give the title compound (0.481 g, quantitative) as a white solid. TLC(MeOH�CH2Cl2; 1:9) Rf=0.15.
Part E: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-Diazo-4-Oxopentanoic Acid tert-Butyl Ester
To a solution of [N-(N′-(1-naphthyl)oxamyl)valinyl] aspartic acid, β-tert-butyl ester (0.095 g, 0.20 mmol) and N-methylmorpholine (22 μL, 0.20 mmol) in tetrahydrofuran (2.0 mL) at −10� C. (NaCl/ice bath) under nitrogen was added isobutyl chloroformate (28 μL, 0.22 mmol). After stirring at −10� C. for 0.5 hrs, the resulting mixed anhydride was treated with excess diazomethane/Et2O solution (prepared from 0.072 g, 0.49 mmol of 1-methyl-3-nitro-1-nitrosoguanidine, 1.0 mL 40% KOH/1.0 ml Et2O). After stirring at −10� C. for an additional 1 hr, the mixture was concentrated and the residue purified by flash chromatography on silica gel eluting with CH2Cl2-Et2O-hexane (1:2:2) to give the title compound (0.062 g, 62%) as a white solid. TLC(EtOAc-hexane; 2:1) Rf=0.63.
Part F: (3S)-3-[N-(N ′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-Bromo-4-Oxopentanoic Acid tert-Butyl Ester
To a solution of (3S)-3-[N-(N′-(1-naphthyl)oxamyl)valinyl]amino-5-diazo-4-oxopentanoic acid tert-butyl ester (0.135 g, 0.265 mmol) in tetrahydrofuiran (3.0 mL) at 0� C. was added 48% aqueous HBr (30 μL, 0.27 mmol). Gas evolution was observed. After 15 min, the mixture was partitioned between EtOAc-saturated NaHCO3, the organic phase washed with saturated NaCl solution, dried over anhydrous Na2SO4 and evaporated to give the title compound (0.147 g, quantitative) as a white solid. TLC(EtOAc-hexane; 2:1) Rf=0.72.
Part G: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-(2′,6′-Dichlorobenzoyloxy)-4-Oxopentanoic Acid, tert-Butyl Ester
To a solution of (3S)-3-[N-(N′-(1-naphthyl)oxamyl) valinyl]amino-5-bromo-4-oxopentanoic acid tert-butyl ester (0.100 g, 0.18 mmol) and 2,6-dichlorobenzoic acid (0.037 g, 0.20 mmol) in dimethylformamide (1.0 mL) at room temperature under nitrogen was added potassium fluoride (0.031 g, 0.53 mmol). After stirring at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:1) to give the title compound (0.084 g, 70%) as viscous oil. TLC(EtOAc-hexane; 2:1) Rf=0.71.
Part H: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Valinyl]Amino-5-(2′,6′-Dichlorobenzoyloxy)-4-Oxopentanoic Acid
To a solution of (3S)-3-[N-(N′-(1-naphthyl)oxamyl) valinyl]amino-5-(2′,6′-dichlorobenzoyloxy)-4-oxopentanoic acid, tert-butyl ester (0.084 g, 0.125 mmol) in CH2Cl2(1.0 mL)-anisole(0.5 mL) at room temperature under nitrogen was added trifluoroacetic acid (1.0 mL). The resulting clear solution was stirred at room temperature for 1 hr, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue was triturated with Et2O to give the title compound (0.060 g, 78%) as an off-white solid. MS(ES) for C29H27Cl2N3O8 (MW 616.45): positive 638/640(M+Na); negative 614/616(M−H).
EXAMPLES 5-14 Starting with (3S)-3-[N-(N′-(1-naphthyl)oxamyl)valinyl]amino-5-bromo-4-oxopentanoic acid tert-butyl ester (see Example 4, Part F) and following the methods described in Example 4, Parts G through H, the compounds shown below in Table 3 were also prepared:
CH2O(2,6-diF-Ph)
C28H27F2N3O7 555.53
578(M + Na)
554(M − H)
CH2O(2,4,6-triF-Ph)
C28H26F3N3O7 573.52
596(M + Na)
572(M − H)
CH2O(2,3,5,6-tetraF-Ph)
C28H25F4N3O7 591.51
614(M + Na)
590(M − H)
CH2O(6-Me-2-pyron-4-yl)
C28H29N3O9 551.55
574(M + Na)
550(M − H)
CH2O(2-Ph-5,6-
C37H33N3O9 663.68
686(M + Na)
662(M − H)
benzopyran-4-on-3-yl)
CH2OPO(Me)Ph
C29H32N3O8P
582(M + H)
580(M − H)
604(M + Na)
694(M + TFA)
CH2OPOPh2 C34H34N3O8P
666(M + Na)
642(M − H)
CH2O(2-CF3-pyrimidin-4-yl)
C27H26F3N5O7 589.53
612(M + Na)
588(M − H)
CH2O(5-CO2Me-
C27H28N4O10 568.54
591(M + Na)
567(M − H)
isoxazol-3-yl)
CH2OPO(Me)(1-naphthyl)
C33H34N3O8P
654(M + Na)
630(M − H)
744(M + TFA)
EXAMPLE 15 (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-(Diphenylphosphinyloxy)-4-Oxopentanoic Acid Part A: [(N-Benzyloxycarbonyl)Leucinyl]Aspartic Acid, β-tert-Butyl, α-Methyl Ester
To a solution of (N-benzyloxycarbonyl)leucine, N-hydroxysuccinimide ester (4.54 g, 12.5 mmol) and aspartic acid, β-tert-butyl, α-methyl ester hydrochloride (3.00 g, 12.5 mmol) in CH2Cl2 (20 mL) at room temperature under nitrogen was added N-methylmorpholine (1.65 mL, 15 mmol). After stirring at room temperature for 18 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to give the title compound (5.56 g, 99%) as viscous oil. TLC(EtOAc-hexane; 1:1) Rf=0.48.
Part B: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-Bromo-4-Oxopentanoic Acid tert-Butyl Ester
Starting with [(N-benzyloxycarbonyl)leucinyl]aspartic acid, β-tert-butyl, α-methyl ester and following the methods described in Example 4, Parts B through F, gave the title compound as a white solid. TLC(CH2Cl2-Et2O-hexane; 1:2:2) Rf=0.32.
Part C: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-(Diphenylphopshinyloxy)-4-Oxopentanoic Acid, tert-Butyl Ester
To a solution of (3S)-3-[N-(N′-(1-naphthyl)oxamyl) leucinyl]amino-5-bromo-4-oxopentanoic acid tert-butyl ester (0.108 g, 0.187 mmol) and diphenylphosphinic acid (0.046 g, 0.21 mmol) in dimethylformamide (1.0 mL) at room temperature under nitrogen was added potassium fluonde (0.033 g, 0.58 mmol). After stirring at room temperature for 48 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4 saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography on silica gel eluting with CH2Cl2-Et2O-hexane (1:2:2) to give the title compound (0.114 g, 85%) as a white solid. TLC(EtOAc-hexane; 2:1) Rf=0.26.
Part D: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Leucinyl]Amino-5-(Diphenylphosphinyloxy)-4-Oxopentanoic Acid
To a solution of (3S)-3-[N-(N′-(1-naphthyl)oxamyl) leucinyl]amino-5-(diphenylphosphinyloxy)-4-oxopentanoic acid, tert-butyl ester (0.114 g, 0.16 mmol) in CH2Cl2(1.0 mL)-anisole(0.5 mL) at room temperature under nitrogen was added trifluoroacetic acid (1.0 mL). The resulting clear solution was stirred at room temperature for 1 hr, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue was triturated with Et2O-hexane to give the title compound (0.062 g, 59%) as an off-white solid. MS(ES) for C34H34N3O8P (MW 657.66): positive 680(M+Na); negative 656(M−H).
EXAMPLES 16-19 Starting with (3S)-3-[N-(N′-(1-naphthyl)oxamyl)leucinyl]amino-5-bromo-4-oxopentanoic acid tert-butyl ester (see Example 15, Part B) and following the methods descnbed in Example 15, Parts C through D, the compounds shown below in Table 4 were also prepared:
CH2OCO(2,6-diCl-Ph)
C30H29Cl2N3O8 630.48
652/654 (M + Na)
628/630 (M − H)
C29H28F3N3O7 587.55
610(M + Na)
586(M − H)
C29H27F4N3O7 605.54
628(M + Na)
604(M − H)
C30H34N3O8P
596(M + H)
594(M − H)
618(M + Na)
708(M + TFA)
EXAMPLES 20-27 Following the general methods described in Example 4, Parts A through H uting (N-benzyloxycarbonyl)alanine for (N-benzyloxycarbonyl)valine in Part A, the ate oxamic acid for N-(1-naphthyl)oxamic acid in Part C, and the appropiate acid or for 2,6-dichlorobenzoic acid in Part G, the compounds shown below in Table 5 were pared:
(2-Ph)Ph
CH2O(2-F-Ph)
C28H26FN3O7 535.53
558(M + Na)
534(M − H)
CH2OCO(2,6-
C29H25Cl2N3O8 614.44
612/614 (M − H)
di-Cl-Ph)
CH2OPOPh2 C34H32N3O8P
664(M + Na)
640(M − H)
680(M + K)
(2-t-Bu)Ph
C26H30FN3O7 515.54
516(M + H)
514(M − H)
538(M + Na)
554(M + K)
CH2OPOPh2 C32H36N3O8P
644(M + Na)
620(M − H)
666(M + K)
1-naphthyl-CH2 CH2O(2,3,5,6
C27H23F4N3O7 577.48
600(M + Na)
576(M − H)
-tetra-F-Ph)
616(M + K)
1-naphthyl-CH2 CH2OCO(2,6-
C28H25Cl2N3O8 602.42
624/626 (M + Na)
600/602 (M − H)
640/642 (M + K)
714/716 (M + TFA)
1-naphthyl-CH2 CH2OPOPh2 C33H32N3O8P
652(M + Na)
628(M − H)
668(M + K)
EXAMPLE 28 (3S)-3-[N-(N′-(1-Naphthylmethyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid Part A: [(N-Benzyloxycarbonyl)Valinyl]Aspartic Acid, β-tert-Butyl Ester
To a suspension of aspartic acid β-tert-butyl ester (3.784 g, 20 mmol) in acetonitrile (200 mL) at room temperture under nitrogen was added bis(tnmethylsilyl)acetamide (9.9 mL, 40 mmol). After stirring at room temperature for 30 min, the resulting clear solution was treated with (N-benzyloxycarbonyl)valine N-hydroxysuccinimide ester (6.97 g, 20 mmol). After stirring at room temperature for an additional 18 hrs, the mixture was treated with water (20 mL), concentrated on a rotovap and then partitioned betweeen EtOAc/water. The organic phase was washed with water, 5% KHSO4 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. Trituration with Et2O-hexane gave the title compound (8.37 g, 99%) as a white solid. TLC(EtOAc-hexane; 1:1) Rf=0.06.
Part B: (3S)-3-[(N-Benzyloxycarbonyl)Valinyl]Amino-5-Bromo-4-Oxopentanoic Acid tert-Butyl Ester
A solution of [(N-benzyloxycarbonyl)valinyl]aspartic acid, β-tert-butyl ester (8.37 g, 19.9 mmol) and N-methylmorpholine (3.50 mL, 32 mmol) in tetrahydrofuran (100 mL) at −10� C. (NaCl/ice bath) under nitrogen was treated dropwise with isobutyl chloroformate (3.87 mL, 29.8 mmol). After stirring at −10� C. for 20 min, the mixture was filtered (sinctered glass) into a pre-cooled receiver (ice bath) washing the filter cake with additional tetrahydrofuran (approx.30 mL). The combined filtrate was treated with excess diazomethane/Et2O solution (prepared from 7.32 g, 50 mmol of 1-methyl-3-nitro-1-nitrosoguanidine, 40 mL 40% KOH/65 ml Et2O) at 0� C. (ice bath) under nitrogen. After stirring at 0� C. for, 15 min and at room temperature for 30 min, the reaction mixture was again cooled to 0� C. and treated with 48% HBr(10 mL, 60 mmol)/acetic acid(10 mL). After stirring at 0� C. for 15 min and at room temperature for 30 min, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, saturated NaHCO3, and saturated NaCl solutions dried over anhydrous Na2SO4 and evaporated to a dryness. Trituration with hexane gave the crude title compound (9.71 g, 98%) as a white solid. TLC(EtOAc-hexane; 1:1) Rf=0.63.
Part C: (3S)-3-[(N-Benzyloxycarbonyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid tert-Butyl Ester
To a solution of (3S)-3-[(N-benzyloxycarbonyl)valinyl]amino-5-bromo-4-oxopentanoic acid tert-butyl ester (9.71 g, 19.4 mmol) and 2,3,5,6-tetrafluorophenol (3.65 g, 22 mmol) in tetrahydrofuran (20 mL) at room temperature under nitrogen was added potassium fluoride (2.91 g, 50 mmol). After stirring at room temperature for 4 hrs, the mixture was diluted with EtOAc (approx.100 mL), washed with saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:3) to give the title compound (9.19 g, 79%) as a white solid after trituration with Et2O-hexane. TLC(EtOAc-hexane; 1:1) Rf=0.70.
Part D: (3S,4RS)-3-[(N-Benzyloxycarbonyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
To a solution of (3S)-3-[(N-benzyloxycarbonyl)valinyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-oxopentanoic acid tert-butyl ester (9.19 g, 15.7 mmol) in MeOH(200 mL)/tetrahydrofuran(200 mL) at 0� C. under nitrogen was added sodium borohydride (0.594 g, 15.7 mmol). After stirring at 0� C. for 1 hr, the mixture was concentrated and the residue partitioned between EtOAc-half saturated NH4Cl solution. The organic phase was washed with saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:3) to give the title compound (7.99 g, 87%) as a white solid. TLC(EtOAc-hexane; 1:1) Rf=0.54.
Part E: (3S,4RS)-3-(Valinyl)Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
To a solution of (3S,4RS)-3-[(N-benzyloxycarbonyl)valinyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (7.99 g, 13.6 mmol) in MeOH (130 mL) was added 10% Pd�C (0.80 g) and resulting mixture stirred under a hydrogen atmosphere (balloon) for 2 hrs. The mixture was filtered through Celite washing the filter cake with CH2Cl2 and the combined filtrates evaporated to dryness. The residue purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:3) then methanol to give the title compound (5.13 g, 83%) as a viscous oil. TLC(EtOAc-hexane; 1:1) Rf=0.07.
Part F: (3S,4RS)-3-[N-(N′-(1-Naphthylmethyl)Oxamyl) Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
To a solution of N-(1-naphthylmethyl)oxamic acid (0.051 g, 0.22 mmol, prepared from 1-naphthylmethylamine by the method described in Example 1, Part A) in in N-methylpyrrolidone(1.0 mL)-CH2Cl2(1 .0 mL) at room temperature under nitrogen was added O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophate (0.092 g, 0.24 mmol). After stirring for 15 min, the mixture was treated with (3S,4RS)-3-(valinyl)amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.100 g, 0.22 mmol) and diusopropylethylamine (115 μL, 0.66 mmol). After stirring at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to give the crude title compound (0.157 g, 100%) as a viscous oil. TLC(EtOAc-hexane; 1:1) Rf=0.44.
Part G: (3S)-3-[N-(N′-(1-Naphthylmethyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid tert-Butyl Ester To a solution of (3S,4RS)-3-[N-(N′-(1-naphthylmethyl)oxamyl)valinyl]-amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.157 g, ca 0.22 mmol) in dimethylsulfoxide (5 mL) at room temperature under nitrogen was added Dess-Martin Periodinane (0.600 g, 1.42 mmol). After stirring at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with saturated NaHCO3, and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue (0.175 g) was purified by flash chromatography on silica gel eluting with EtOAc-hexane (3:7) to give the title compound (0.111 g, 77%) as a white solid. TLC(EtOAc-hexane; 1:1)Rf=0.58.
Part H: (3S)-3-[N-(N′-(1-Naphthylmethyl)Oxamyl)Valinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid
To a solution of (3S)-3-[N-(N′-(1-naphthylmethyl)oxamyl)valinyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-oxopentanoic acid, tert-butyl ester (0.108 g, 0.16 mmol) in CH2Cl2(2.0 mL)-anisole(0.1 mL)-water(0.05 mL) at room temperature under nitrogen was added trifluoroacetic acid (2.0 mL). The resulting clear solution was stirred at room temperature for 2 hr, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue was triturated with Et2O to give the title compound (0.098 g, 100%) as a white solid. MS(ES) for C29H27F4N3O7 (MW 605.54): positive 628(M+Na); negative 604(M−H).
EXAMPLES 29-74 Starting with (3S,4RS)-3-(valinyl)amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (see Example 28, Part E) and following the methods described in Example 28, Parts F through H, the compounds shown below in Table 6 were also prepared:
PhCH2 C25H25F4N3O7 555.48
556(M + H)
Ph(CH2)2 C26H27F4N3O7 569.51
592(M + Na)
568(M − H)
C31H29F4N3O7 631.58
C24H23F4N3O7 541.46
564(M + Na)
540(M − H)
C30H27F4N3O7 617.55
640(M + Na)
616(M − H)
730(M + TFA)
(2-PhCH2)Ph
(3-PhO)Ph
C30H27F4N3O8 633.55
634(M + H)
632(M − H)
656(M + Na)
4-Cl-1-naphthyl
C28H24ClF4N3O7 625.96
648/650 (M + Na)
624/626 (M − H)
C32H27F4N3O7 641.57
642(M + H)
C25H23F4N5O7 581.48
C28H33F4N3O7 599.58
600(M + H)
598(M − H)
C24H22F5N3O7 559.45
582(M + Na)
558(M − H)
672(M + TFA)
(2-CF3)Ph
C25H22F7N3O7 609.45
632(M + Na)
608(M − H)
722(M + TFA)
C28H31F4N3O7 597.56
620(M + Na)
596(M − H)
710(M + TFA)
(4-n-heptyl)Ph
C31H37F4N3O7 639.64
662(M + Na)
638(M − H)
(2-CH3O)Ph
C25H25F4N3O8 571.48
594(M + Na)
570(M − H)
(2-PhO)Ph
746(M + TFA)
C28H29F4N3O7 595.55
C23H22F4N4O7 542.44
543(M + H)
541(M − H)
C23H18F8N4O7 614.40
615(M + H)
613(M − H)
C22H21F4N5O7 543.43
544(M + H)
542(M − H)
634(M + K)
(2-Cl)Ph
C24H22ClF4N3O7 575.90
598/600 (M + Na)
574/576 (M − H)
(2-Br)Ph
C24H22BrF4N3O7 620.35
644/642 (M + Na)
620/618 (M − H)
734/732 (M + TFA)
(2-I)Ph
C24H22F4IN3O7 667.35
690(M + Ma)
666(M − H)
706(M + K)
780(M + TFA)
(2,6-di-F)Ph
C24H22F6N3O7 577.44
690(M + TFA)
(2,5-di-t-Bu)Ph
C32H39F4N3O7 653.67
654(M + H)
652(M − H)
676(M + Na)
688(M + Cl)
692(M + K)
766(M + TFA)
C27H27F4N3O7 581.52
620(M + K)
(3,4,5-tri-
C28H31F4N3O10 645.56
646(M + H)
644(M − H)
MeO)PhCH2 668(M + Na)
684(M + K)
C19H21F4N3O7 479.38
502(M + Na)
478(M − H)
592(M + TFA)
C25H33F4N3O7 563.55
586(M + Na)
562(M − H)
602(M + K)
676(M + TFA)
C26H35F4N3O7 577.57
C24H29F4N3O7 547.50
548(M + H)
546(M − H)
570(M + Na)
660(M + TFA)
586(M + K)
5-Ph-3-pyrazolyl
C27H25F4N5O7 607.52
630(M + Na)
606(M − H)
646(M + K)
(2-F-4-I)Ph
C24H21F5IN3O7 685.34
686(M + H)
684(M − H)
708(M + Na)
720(M + Cl)
724(M + K)
C24H19F8N3O7 613.41
614(M + H)
612 (M − H)
tetra-F)Ph
636(M + Na)
726 (M + TFA)
652(M + K)
(2,3,4,6-
612(M − H)
726(M + TFA)
C24H19Cl4F4N3O7 679.23
700/702/704(M + Na)
676/678/680 (M − H)
tetra-Cl)Ph
716/718/ 720(M + K)
790/792/ 794 (M + TFA)
(2,3,4,5,6-penta-
C24H18F9N3O7 631.40
670(M + K)
666(M + Cl)
C30H28F4N4O7 632.57
633(M + H)
631(M − H)
655(M + Na)
745(M + TFA)
PHCH2(Ph)N
C31H30F4N4O7 646.59
647(M + H)
645(M − H)
669(M + Na)
681 (M + Cl)
685(M + K)
C25H25F4N3O7 571.48
684(M + TFA)
EXAMPLE 75 (3S)-3-[N-(N′-(2-tert-Butylphenyl)Oxamyl)Alaninyl]Amino-5-(2′,3 ′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid Part A: [(N-Benzyloxycarbonyl)Alaninyl]Aspartic Acid, β-tert-Butyl Ester
To a suspension of aspartic acid ,-tert-butyl ester (3.784 g, 20 mmol) in ylformamide (150 mL) at room temperture under nitrogen was added bis(trimethylsilyl)-trifluoroacetamide (10.6 mL, 40 mmol). After stirring at room temperature for 30 min, the resulting clear solution was treated with (N-benzyloxycarbonyl)alanine N-hydroxysuccinimide ester (6.406 g, 20 mmol). After stirring at ronum temperature for an additional 48 hrs, the mixture was treated with water (20 mL), stirred for 15 min and then partitioned betweeen EtOAc/water. The organic phase was washed with water, 5% KHSO4 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue was dissolved in Et2O and extracted with saturated NaHCO3. The aqueous extract was acidified (pH 2.0) with concentrated HCl and extracted with EtOAc. The EtOAc extract was washed with saturated NaCl solution, dried over anhydrous Na2SO4 and evaporated to a give the title compound (6.463 g, 82%) as a white foam. TLC(EtOAc-hexane-AcOH; 70:30:2) Rf=0.50.
Part B: (3S,4RS)-3-(Alaninyl)Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
Starting with [(N-benzyloxycarbonyl)alaninyl]aspartic acid, β-tert-butyl ester and following the methods described in Example 28, Parts B through E gave the title compound as a colorless, viscous oil. TLC(EtOAc-hexane; 1:1) Rf=0.06.
Part C: (3S,4RS)-3-[N-(N′-(2-tert-Butylphenyl)Oxamyl) Alaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
To a solution of N-(2-tert-butylphenyl)oxamic acid (0.041 g, 0.19 mmol, prepared from 2-tert-butylaniline by the method described in Example 1, Part A) in CH2Cl2(6.0 mL) at 0� C. under nitrogen was added hydroxybenzotriazole hydrate (0.030 g) followed by 1-ethyl-3-(3′,3′-dimethyl-l′-aminopropyl)-carboduimide hydrochloride (0.050 g, 0.26 mmol). After stirring at 0� C. for 10 min, the mixture was treated with (3S,4RS)-3-(alaninyl)amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.079 g, 0.19 mmol) and N-methylmorpholine (22 μL, 0.20 mmol). After stirring at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to give the crude title compound (0.090 g, 77%) as a viscous oil. TLC(EtOAc-hexane; 1:1) Rf=0.70.
Part D: (3S)-3-[N-(N′-(2-tert-Butylphenyl)Oxamyl)Alaninyl]Amino-5-(2′, 3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid tert-Butyl Ester
To a solution of (3S,4RS)-3-[N-(N′-(2-tert-butylphenyl)oxamyl)alaninyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.0.092 g, ca 0.15 mmol) in CH2Cl2 (6.5 mL) at room temperature under nitrogen was added iodobenzene diacetate (0.188 g, 0.58 mmol) folowed by a catalytic amount of 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO, 0.0046 g, 0.03 mrimol). After stirring at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue (0.096 g) was purified by preparative layer chromatography on silica gel eluting with EtOAc-hexane (3:7) to give the title compound (0.071 g, 77%) as a colorless glass. TLC(EtOAc-hexane; 2:3) Rf=0.60.
Part E: (3S)-3-[N-(N′-(2-tert-Butylphenyl)Oxamyl)AlaninylAmino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid
To a solution of (3S)-3-[N-(N′-(2-tert-butylphenyl)oxamyl)alaninyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-oxopentanoic acid, tert-butyl ester (0.071 g, 0.11 mmol) in CH2Cl2(2.5 mL)-anisole(0.05 mL) at room temperature under nitrogen was added trifluoroacetic acid (1.5 mL). The resulting clear solution was stirred at room temperature for 1 hr, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue (0.061 g) was purified by preparative layer chromatography on silica gel eluting with MeOH�CH2Cl2 (1:9) to give the title compound (0.044 g, 69%) as a colorless glass. MS(ES) for C26H27F4N3O7 (MW 569.51): positive 570(M+H); negative 568(M−H).
EXAMPLES 76-87 Starting with (3S,4RS)-3-(alaninyl)amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (see Example 75, Part B) and following the methods described in Example 75, Parts C through E, the compounds shown below in Table 7 were also prepared:
C23H18F7N3O7 581.40
C28H23F4N3O7 589.50
C29H25F4N3O7 603.53
604(M + H)
602(M − H)
C28H23F4N3O8 605.50
5,6,7,8-tetrahydro-1-naphthyl
C26H25F4N3O7 567.49
590(M + Na)
566(M − H)
C26H21F4N3O7 563.46
608(M + K)
C22H19F4N3O7 513.40
552(M + K)
512(M − H)
C22H17F6N3O7 549.38
572(M + Na)
548(M − H)
662(M + TFA)
(4-MeO)Ph
C23H21F4N3O8 543.43
582(M + K)
642(M + K)
EXAMPLE 88 (3S)-3-[N-(N′-(2′-Phenoxyphenyl)Oxamyl)Cyclohexylalaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)4-Oxopentanoic Acid Part A: (3S)-3-(N-Benzyloxycarbonyl)Amino-5-Bromo-4-Oxopentanoic Acid tert-Butyl Ester
A solution of (N-benzyloxycarbonyl)aspartic acid, β-tert-butyl ester (2.28 g, 7.06 mmol) and N-methylmorpholine (0.85 mL, 7.7 mmol) in tetrahydrofuran (40 mL) at −10� C. (NaCl/ice bath) under nitrogen was treated dropwise via syringe with isobutyl chloroformate (1.1 mL, 8.5 mmol). After stirring at −10� C. for 20 min, the mixture was filtered (sinctered glass) into a pre-cooled receiver (ice bath) washing the filter cake with additional tetrahydrofuran (approx.10 mL). The combined filtrate was treated with excess diazomethane/Et2O solution (prepared from 3.10 g, 21 mmol of 1-methyl-3-nitro-1-nitrosoguanidine, 20 mL 40% KOH/10 ml Et2O) at 0� C. (ice bath) under nitrogen. After stirring at 0� C. for 15 min and at room temperature for 30 min, the reaction mixture was again cooled to 0� C. and treated with 48% HBr(2.0 mL, 12 mmol)/acetic acid(2.0 mL). After stirring at 0� C. for 15 min and at room temperature for 15 min, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, saturated NaHCO3, and saturated NaCl solutions dried over anhydrous Na2SO4 and evaporated to a dryness. Trituration with hexane gave the crude title compound (3.32 g) as a yellow oil. TLC(EtOAc-hexane; 1:1) Rf=0.60 (intermediate diazoketone Rf=0.52).
Part B: (3S,4RS)-3-(N-Benzyloxycarbonyl)Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
To a solution of (3S)-3-(N-benzyloxycarbonyl)amino-5-bromo-4-oxopentanoic acid tert-butyl ester (0.857 g, 2.14 mmol) and 2,3,5,6-tetrafluorophenol (0.410 g, 2.45 mmol) in dimethylfonnamide (5.0 mL) at room temperature under nitrogen was added potassium fluoride (0.40 g, 6.9 mmol). After stirring at room temperature for 16 hrs, the mixture was diluted with EtOAc, washed with saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a to give the crude tetrafluorophenoxymethyl Icetone (1.08 g, 98%) as a yellow, viscous oil. TLC(EtOAc-hexane; 1:1) Rf=0.57.
To a solution of the above crude ketone (1.08 g, ca 2.14 mmol) in ethanol (10 mL) at 0� C. under nitrogen was added sodium borohydride (0.057 g, 1.5 mmol). After stirring at 0� C. for 1 hr, the excess reducing agent was discharged by treatment with acetone (1.0 mL), the mixture concentrated and the residue partitioned between EtOAc-half saturated NH4Cl solution. The organic phase was washed with saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:3) to give the title compound (1.012 g, 94%) as a colorless oil. TLC(EtOAc-hexane; 1:1) Rf=0.48.
Part C: (3S,4RS)-3-[(N-9-Fluorenylmethoxycarbonyl)Cyclohexylalaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
To a solution of (3S,4RS)-3-(N-benzyloxycarbonyl)amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (1.012 g, 2.08 mmol) in MeOH (25 mL) was added 10% Pd�C (0.30 g) and resulting mixture stirred under a hydrogen atmosphere (balloon) for 4 hrs. The mixture was filtered through Celite washing the filter cake with CH2Cl2 and the combined filtrates evaporated to give the crude amine (0.682 g, 93%) as a viscous oil. TLC(MeOH�CH2Cl2; 5:95) Rf=0.21.
To a solution of (N-9-fluorenylmethoxycarbonyl) cyclohexylalanine (0.763 g, 1.94 mmol) in CH2Cl2(10 mL) at 0� C. (ice bath) under nitrogen was added hydroxybenzotriazole hydrate (0.282 g) followed by 1-ethyl-3-(3′,3′-dimethyl-1′-aminopropyl)carbodiimide hydrochloride (0.447 g, 2.33 mmol). After stirring at 0� C. for 10 min, the mixture was treated with the above crude amine (0.682 g, ca 1.93 mmol) and the reacton allowed to warm to room temperature. After stirring at room temperature for 3 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography eluting with EtOAc-hexane (1:2) to give the title compound (1.028 g, 73%) as yellow foam. TLC(EtOAc-hexane; 1:2) Rf=0.20.
Part D: (3S,4RS)-3-[Cyclohexylalaninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
A mixture of (3S,4RS)-3-[(N-9-fluorenylmethoxycarbonyl)cyclohexylalaninyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (1.028 g, 1.4 mmol) and 10% piperidine/dimethylformamide (3.0 mL) was stirred at room temperature under nitrogen for 2 hrs. The mixture was diluted with CH2Cl2, washed with water and saturated NaHCO3 solution, dried over anhydrous anhydrous Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography eluting with isopropanol-CH2Cl2 (7:93) to give the title compound (0.561 g, 78%) as a white solid. TLC(MeOH�CH2Cl2; 5:95) Rf=0.43.
Part E: (3S,4RS)-3-[N-(N′-(2′-Phenoxyphenyl)Oxamyl)Cyclohexylalaninyl]-Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
To a solution of N-(2-phenoxyphenyl)oxamic acid (0.064 g, 0.25 mmol, prepared from 2-phenoxyaniline by the method described in Example 1, Part A) and (3S,4RS)-3-[cyclohexylalaninyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic Acid tert-butyl ester (0.124 g, 0.245 mmol) in CH2Cl2(5.0 mL) at 0� C. (ice bath) under nitrogen was added hydroxybenzotriazole hydrate (0.051 g) followed by 1-ethyl-3-(3′,3′-dimethyl-1′-aminopropyl)carbodiimide hydrochloride (0.061 g, 0.32 mmol). After stirring at 0� C. for 10 min and at room temperature for 18 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to give the crude title compound (0.194 g) as yellow foam. TLC(EtOAc-hexane; 1:2) Rf=0.40.
Part F: (3S)-3-[N-(N′-(2′-Phenoxyphenyl)Oxamyl)Cyclohexylalaninyl]-Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxoxypentanoic Acid tert-Butyl Ester
To a solution of crude (3S,4RS)-3-[N-(N′-(2′-phenoxyphenyl)oxamyl) cyclohexylalaninyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.194 g, ca 0.245 mmol) in CH2Cl2 (5 mL) at room temperature under nitrogen was added Dess-Martin Periodinane (0.150 g, 0.35 mmol). After stirring at room temperature for 2 hrs, the mixture was diluted with EtOAc, washed with 1.0 M Na2S2O3, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:3) to give the title compound (0.142 g, 80%) as a colorless, viscous oil. TLC(EtOAc-hexane; 1:2) Rf=0.50.
Part G: (3S)-3-[N-(N′-(2′-Phenoxyphenyl)Oxamyl)Cyclohexylalaninyl]-Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxoxypentanoic Acid
To a solution of (3S)-3-[N-(N′-(2′-phenoxyphenyl)oxamyl)cyclohexylalaninyl]amino-5-(2′,3′,5′, 6′-tetrafluorophenoxy)-4-oxopentanoic acid, tert-butyl ester (0.142 g, 0.19 mmol) in CH2Cl2(2.0 mL) at room temperature under nitrogen was added trifluoroacetic acid (1.0 mL). The resulting clear solution was stirred at room temperature for 0.5 hr, evaporated to dryness and chased with toluene-CH2Cl2 (1:1) to give the title compound (0.123 g, 93%) as a white foam. MS(ES) for C34H33F4N308 (MW 687.64): positive 688(M+H), 710(M+Na), 726(M+K); negative 686(M−H), 800(M+TFA).
EXAMPLES 89-91 Starting with (3S,4RS)-3-[cyclohexylalaninyl]amino-5-(2′, 3′,5′,6′-tetrafluoro-phenoxy)-4-hydroxypentanoic acid tert-butyl ester (see Example 88, Part D) and following the methods described in Example 88, Parts E through G, the compounds shown below in Table 8 were also prepared:
C34H33F4N3O7 671.64
672(M + H)
670(M − H)
694(M + Na)
784(M + TFA)
C35H35F4N3O7 685.67
798(M + TFA)
C32H31F4N3O7 645.61
668(M + Na)
758(M + TFA)
EXAMPLE 92 (3S)-3-[N-(N′-(5,6,7,8-Tetrahydro-1-Naphthyl)Oxamyl)-Cyclohexylalaninyl]Amino-5-(2′,6′-Dichlorobenzoyloxy)-4-Oxopentanoic Acid Part A: Aspartic Acid, β-tert-Butyl, α-Methyl Ester p-Toluenesulfonate Salt
To a solution of N-(benzyloxycarbonyl)-L-aspartic acid, β-tert-butyl ester 10.57 g, 32.7 mmol) in methanol(20 mL)-CH2Cl2(30 mL) at 0� C. (ice bath) was added wise a 2.0 M solution of (trimethylsilyl)diazomethane in hexanes (20 mL, 40 mmol). After stirring at 0� C. for 45 min, the excess reagent was quenced with formic acid (1.0 mL). The mixture was washed with saturated NaHCO3 solution, dried over anhydrous Na2SO4 and evaporated to a pale yellow oil (11.34 g).
The crude product (11.34 g, ca 32.7 mmol) was taken up in methanol (100 mL), treated with p-toluenesulfonic acid mono hydrate (6.20 g, 32.6 mmol) and 10% Pd�C (0.5 g) and stirred under a hydrogen atmosphere (balloon) for 3 hrs. The mixture was filtered through Celite and concentrated to give the title compound as a white solid (12.68 g).
Part B: [(N-Benzyloxycarbonyl)Cyclohexylalaninyl]Aspartic Acid, β-tert-Butyl, α-Methyl Ester
To a solution of (N-benzyloxycarbonyl)-cyclohexylalanine dicyclohexylamine salt (0.866 g, 1.77 mmol) in CH2Cl2(10 mL) at 0� C. (ice bath) under nitrogen was added hydroxybenzotriazole hydrate (0.100 g) followed by 1-ethyl-3-(3′,3′-dimethyl-1′-aminopropyl)carbodiimide hydrochloride (0.41 g, 2.14 mmol). After stirring at 0� C. for 10 min, the mixture was treated with aspartic acid, β-tert-butyl, α-methyl ester p-toluenesulfonate salt (0.665 g, 1.77 mmol) and N-methylmorpholine (0.2 mL, 1.8 mmol), and the reacton allowed to warm to room temperature. After stirring at room temperature for 2.5 hrs, the mixture was concentrated and the residue partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to an oil. Purification by flash chromatography on silica gel eluting with EtOAc-hexane (1:3) gave the title compound (0.764 g, 88%) as a viscous oil. TLC(EtOAc-hexane; 1:2) Rf=0.46.
Part C: (3S)-3-[N-(N ′-(5,6,7,8-Tetrahydro-1-Naphthyl)Oxamyl)-Cyclohexylalaninyl]Amino-5-(2′,6′-Dichlorobenzoyloxy)-4-Oxopentanoic Acid
Starting with [(N-benzyloxycarbonyl)cyclohexyl-alaninyl]aspartic acid, β-tert-butyl, α-methyl ester and following the general methods described in Example 4, Parts B through H, gave the title compound as a white solid. MS(ES) for C33H37Cl2N3O8 (MW 674.58): positive 696/698(M+Na). negative 672/674(M−H), 786/788(M+TFA).
EXAMPLES 93-99 Starting with [(N-benzyloxycarbonyl)cyclohexyl-alaninyl]aspartic acid, β-tert-butyl, α-methyl ester (see Example 92, Part B), and following the general methods described in Example 4, Parts B through H, the compounds shown below in Table 9 were also prepared:
CH2O(2,3,5,6-
C32H35F4N3O7 649.64
672(M + Na)
648(M − H)
tetra-F-Ph)
C33H42N3O8P
752(M + TFA)
CH2OPOPh2 C38H44N3O8P
724(M + Na)
700(M + H)
740(M + K)
C36H42N3O8P
698(M + Na)
674(M − H)
714(M + K)
788(M + TFA)
CH2OPOPh2 C41H44N3O8P
760(M + Na)
736(M − H)
776(M + K)
850(M + TFA)
C40H42N3O8P
684(M + Na)
660(M − H)
700(M + K)
774(M + TFA)
CH2OPOPh2 C35H40N3O8P
746(M + Na)
722(M − H)
762(M + K)
836(M + TFA)
EXAMPLE 100 (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Homoprolinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid Part A: [N-(1-Naphthyl)Oxamyl]Homoproline
To a solution of N-(1-naphthyl)oxamic acid (0.108 g, 0.50 mmol, see Example 1, Part A) in in N-methylpyrrolidone(1.0 mL)-CH2Cl2(1.0 mL) at room temperature under nitrogen was added O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophate (0.209 g, 0.55 mmol). After stirring for 20 min, the mixture was treated with homoproline methyl ester (0.072 g, 0.50 mmol) and diisopropylethylamine (0.26 mL, 1.5 mmol). After stirring at room temperature for 4 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to give crude [N-(1-naphthyl)oxamyl]homoproline (0.156 g, 92%) as a colorless glass. TLC (EtOAc-hexane; 1:1) Rf=0.70.
To a solution of the crude methyl ester (0.156 g, ca 0.46 mmol) in dioxane(0.75 mL)-water(0.25 mL) was added 1.0 N LiOH solution (0.5 mL, 0.5 mmol). After stirring at room temperature for 1 hr, the mixture was acidified with 1.0 N HCl and extracted with EtOAc. The extract was washed with saturated NaCl solution, dried over anhydrous Na2SO4 and evaporated to give the title compound (0.105 g, 70%) as a white solid after trituration with Et2O.
Part B: (3S,4RS)-3-[N-(N′-(1-Naphthyl)Oxamyl)Homoprolinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
To a solution of [N-(1-naphthyl)oxamyl]homoproline (0.483 g, 1.48 mmol) in in N-methylpyrrolidone(0.5 mL)-CH2Cl2(14 mL) at 0� C. under nitrogen was added O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophate (0.676 g, 1.78 mmol). After stirring for 20 min, the mixture was treated with a solution of (3S,4RS)-3-amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.540 g, 1.54 mmol, see Example 49, Part C) in CH2Cl2 (4.0 mL) followed by diusopropylethylamine (0.50 mL, 2.9 mmol). After stirring at 0� C. for 3 hrs and at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. Purification by flash chromatography on silica gel eluting with EtOAc-hexane (1:2) gave the title compound (0.268 g, 27%) as a tan foam. TLC (EtOAc-hexane; 1:1) Rf=0.39.
Part C: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Homoprolinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxoypentanoic Acid tert-Butyl Ester
To a solution of (3S,4RS)-3-[N-(N′-(1-naphthyl)oxamyl)homoprolinyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)4-hydroxypentanoic acid tert-butyl ester (0.251 g, 0.38 mmol) in CH2Cl2 (4 mL) at room temperature under nitrogen was added Dess-Martin Periodinane (0.201 g, 0.475 mmol). After stirring at room temperature for 30 min, the mixture was diluted with EtOAc, washed with 1.0 M Na2S2O3, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue was purified by flash chromatography on silica gel eluting with CH2Cl2-Et2O-hexane (1:2:2) then EtOAc-hexane (1:2) to give the title compound (0.160 g, 64%) as a white foam. TLC(EtOAc-hexane; 1:1)Rf=0.57.
Part D: (3S,4RS)-3-[N-(N ′-(1-Naphthyl)Oxamyl)Homoprolinyl]Amino-5-(2 ′,3 ′,5 ′,6′-Tetrafluorophenoxy)-4-Oxoypentanoic Acid
To a solution of (3S)-3-[N-(N′-(1-naphthyl)oxamyl)homoprolinyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-oxopentanoic acid tert-butyl ester (0.152 g, 0.23 mmol) in CH2Cl2(1.0 mL)-anisole(0.4 mL) at room temperature under nitrogen was added trifluoroacetic acid (1.0 mL). The resulting clear solution was stirred at room temperature for 1 hr, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue was triturated with hexane to give the title compound (0.103 g, 74%) as an off-white solid. TLC(MeOH�CH2Cl2; 1:9) Rf=0.33. MS(ES) for C29H25F4N3O7 (MW 603.53): positive 626(M+Na); negative 602(M−H).
EXAMPLE 101 (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Indoline-2-Carbonyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid Part A: [N-(1-Naphthyl)Oxamyl]Indoline-2-Carboxylic Acid Ethyl Ester
To a solution of N-(1-naphthyl)oxamic acid (2.37 g, 11 mmol, see Example 1, Part A) in in N-methylpyrrolidone(7.0 mL)-CH2Cl2(40 mL) at 0� C. (ice bath) under nitrogen was added 1,1′-carbonylduimidazole (1.96 g, 12.1 mmol). After stirring at 0� C. for 1.5 hrs and at room temperature for 0.5 hrs, (S)-indoline-2-carboxylic acid ethyl ester hydrochloride (1.25 g, 5.5 mmol) and diisopropylethylamine (1.1 mL, 6.4 mmol) was added. After stirring at room temperature for 18 hrs, the mixture was diluted with EtOAc, washed successively with 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified by flash chromatography on silica gel eluting with CH2Cl2-Et2O-hexane (1:1:3) to give the title compound (0.472 g, 22%) as a pale yellow oil. TLC(CH2Cl2-Et2O-hexane; 1:1:3) Rf=0.48.
Part B: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)lndoline-2-Carbonyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid
Starting from [N-(1-naphthyl)oxamyl]indoline-2-carboxylic Acid ethyl ester, and following the methods described in Example 100, Parts A through D, the title compound was also prepared. MS(ES) for C32H23F4N3O7 (MW 637.54): positive 660(M+Na),676(M+K); negative 636(M−H),672(M+Cl), 750(M+TFA).
EXAMPLE 102 (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Cyclohexylglycinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid Part A: (3S,4RS)-3-[(N-9-Fluorenylmethoxycarbonyl)Cyclohexylglycinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
To a solution of (N-9-fluorenylmethoxycarbonyl) cyclohexylglycine (0.514 g, 1.35 mmol) and (3S,4RS)-3-amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.479 g, 1.36 mmol, see Example 88, Part C) in CH2Cl2(10 mL) at 0� C. (ice bath) under nitrogen was added O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophate (0.619 g, 1.62 mmol) and diisopropylethylamine (0.47 mL, 2.7 mmol). After stirring at 0� C. for 3 hrs, the reaction was allowed to warm to room temperature. After stirring at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography eluting with EtOAc-hexane (1:2) to give the title compound (0.481 g, 50%) as a white solid. TLC(EtOAc-hexane; 1:2) Rf=0.42.
Part B: (3S,4RS)-3-[Cyclohexylglycinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
A solution of (3S,4RS)-3-[(N-9-fluorenylmethoxycarbonyl)cyclohexylglycinyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.478 g, 0.67 mmol) in piperidine(0.1 mL)/dimethylformamide(2.0 mL) was stirred at room temperature under nitrogen for 1 hr. The mixture was diluted with EtOAc, washed with water and saturated NaCl solution, dried over anhydrous anhydrous Na7SO4 and evaporated to dryness. The residue was purified by flash chromatography eluting with EtOAc-hexane (1:2) to give the title compound (0.121 g, 45%) as a white solid. TLC(MeOH�CH2Cl2; 5:95) Rf=0.38.
Part C: (3S,4RS)-3-[N-(N′-(1-Naphthyl)Oxamyl)Cyclohexylglycinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
To a solution of N-(1-naphthyl)oxamic acid (0.088 g, 0.41 mmol, see Example 1, Part A) and (3S,4RS)-3-(cyclohexylglycinyl)amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.110 g, 0.27 mmol) in N-methylpyrrolidone(0.5 mL)-CH2Cl2(3.0 mL) at 0� C. under nitrogen was added O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophate (0.125 g, 0.32 mmol) and diusopropylethylamine (90 μL, 0.54 mmol). After stirring at 0� C. for 3 hrs and at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to dyness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:2) to give the title compound (0.094 g, 50%) as a white foam. TLC(EtOAc-hexane; 1:1) Rf=0.50.
Part D: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Cyclohexylglycinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxoypentanoic Acid tert-Butyl Ester
To a solution of (3S,4RS)-3-[N-(N′-(1-naphthyl)oxamyl)cyclohexylglycinyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.082 g, 0.12 mmol) in CH2Cl2(1 mL)-CH3CN(2 mL)-DMSO(0.2 mL) at room temperature under nitrogen was added Dess-Martin Periodinane (0.145 g, 0.34 mmol). After stirring at room temperature for 1 hr, the mixture was diluted with EtOAc, washed with 1.0 M Na2S2O3, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:2 then 1:1) to give the title compound (0.068 g, 83%) as a tan foam. TLC(EtOAc-hexane; 1: 1) Rf=0.63.
Part E: (3S,4RS)-3-[N-(N′-(1-Naphthyl)Oxamyl) Cyclohexylglycinyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxoypentanoic Acid
To a solution of (3S)-3-[N-(N′-(1-naphthyl)oxamyl)_cyclohexylglycinyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-oxopentanoic acid tert-butyl ester (0.065 g, 0.23 mmol) in CH2Cl2(1.0 mL)-anisole(0.2 mL) at room temperature under nitrogen was added trifluoroacetic acid (1.0 mL). The resulting clear solution was stirred at room temperature for 30 min, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue was triturated with Et2O to give the title compound (0.034 g, 56%) as an off-white solid. TLC(MeOH�AcOH�CH2Cl2; 1:1:32) Rf=0.45. MS(ES) for C31H29F4N3O7 (MW 631.58): positive 654(M+Na); negative 630(M−H).
EXAMPLES 103-109 Starting from (3S,4RS)-3-amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxy-pentanoic acid tert-butyl ester (see Example 88, Part C) and following the general methods described in Example 102, Parts A through E, the compounds shown below in Table 10 were also prepared:
644(M + K)
640(M + Cl)
718(M + TFA)
(t-butyl)glycine
606(M + H)
(t-butyl)alanine
C20H29F4N3O7 619.57
620(M + H)
618(M − H)
642(M + Na)
732(M + TFA)
658(M + K)
C31H23F4N3O7 625.53
626(M + H)
624(M − H)
648(M + Na)
660(M + Cl)
664(M + K)
738(M + TFA)
C32H25F4N3O7 639.56
640(M + H)
674(M + Cl)
678(M + K)
712(M + TFA)
C33H27F4N3O7 653.59
626(M + Na)
602(M + H)
EXAMPLE 110 (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Methioninyl(Sulfoxide)]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxopentanoic Acid Part A: (3S,4RS)-3-[N-(N′-(1-Naphthyl)Oxamyl)Methioninyl]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Hydroxypentanoic Acid tert-Butyl Ester
Starting from (N-9-fluorenylmethoxycarbonyl) methionine and following the methods described in Example 102, Parts A through C, the title compound was also prepared. TLC(EtOAc-hexane; 1:2) Rf=0.39.
Part B: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Methioninyl(Sulfoxide)]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxoypentanoic Acid tert-Butyl Ester
To a solution of (3S,4RS)-3-[N-(N′-(1-naphthyl)oxamyl)methioninyl]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-hydroxypentanoic acid tert-butyl ester (0.251 g, 0.37 mmol) in CH2Cl2(4.0 mL) at room temperature under nitrogen was added Dess-Martin Periodinane (0.203 g, 0.48 mmol). After stirring at room temperature for 1 hr, the mixture was diluted with EtOAc, washed with 1.0 M Na2S2O3, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexane (1:2 then 1:1) followed by MeOH�CH2Cl2 (5:95 then 1:9) to give a mixture of two isomeric sulfoxides (0.225 g); TLC(MeOH�CH2Cl2; 1:9) Rf's 0.48 and 0.43. The mixture was re-chromatograhed on silica gel eluting with isopropanol-CH2Cl2 (2.5% to 5% to 10%) to give sulfoxide isomer A (less polar, 0.051 g), sulfoxide isomer B (more polar, 0.086 g) and a mixture of isomers A and B (0.040 g). Both isomers have virtually identical mass spectra. MS(ES) for C32H33F4N3O8S (MW 695.68): positive 718(M+Na); negative 694(M−H).
Part C: (3S ,4RS)-3-[N-(N′-(1-Naphthyl)Oxamyl)Methioninyl(Sulfoxide)]Amino-5-(2′,3′,5′,6′-Tetrafluorophenoxy)-4-Oxoypentanoic Acid
To a solution of (3S)-3-[N-(N′-(1-naphthyl)oxamyl) methioninyl(sulfoxide)]amino-5-(2′,3′,5′,6′-tetrafluorophenoxy)-4-oxopentanoic acid tert-butyl ester (isomer A, 0.046 g, 0.07 mmol) in CH2Cl2(2.0 mL)-anisole(0.1 mL) at room temperature under nitrogen was added trifluoroacetic acid (1.0 mL). The resulting clear solution was stirred at room temperature for 30 min, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue was triturated with Et2O-hexane to give the title compound, isomer A (0.034 g, 81%) as an off-white solid. TLC(MeOH�AcOH�CH2Cl2; 1:1:32) Rf=0.20. MS(ES) for C28H25F4N3O8S (MW 639.57): positive 640(M+H),662(M+Na),678(M+K); negative 638(M−H), 752(M+TFA). Under the same conditions sulfoxide isomer B (0.081 g, 0.12 mmol) gave the title compound, isomer B (0.055 g, 74%). MS(ES) for C28H25F4N3O8S (MW 639.57): positive 640(M+H),662(M+Na),678(M+K); negative 638(M−H),674(M+Cl), 752(M+TFA).
EXAMPLE 111 (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Homoprolinyl]Amino-4-Oxobutanoic Acid Part A: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Homoprolinyl]Amino-4-Oxobutanoic Acid (tert)-Butyl Ester Semicarbazone
To a solution of [N-(1-naphthyl)oxamyl]homoproline (0.103 g, 0.32 mmol, see Example 100, Part A) in CH2Cl2 (3.0 mL) at 0� C. under nitrogen was added was added hydroxybenzotriazole hydrate (0.058 g) followed by 1-ethyl-3-(3′,3′-dimethyl-1′-aminopropyl)carbodiimide hydrochloride (0.91 g, 0.47 mmol). After stirring at 0� C. for 10 min, the mixture was treated with (3S)-amino-4-oxobutanoic acid (tert)-butyl ester semicarbazone, p-toluenesulfonate salt (0.127 g, 0.32 mmol) and N-methylmorpholine (42 μL, 0.38 mmol). After stirring at 0� C. for 2 hrs, the mixture was concentrated and the residue partitioned between EtOAc-5% KHSO4. The organic phase was washed with 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to give the crude title compound (0.119 g, 70%) as a colorless glass.
Part B: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Homoprolinyl]Amino-4-Oxobutanoic Acid Semicarbazone
To a solution of (3S)-3-[N-(N′-(1-naphthyl)oxamyl)homoprolinyl]amino-4-oxobutanoic acid semicarbazone tert-butyl ester (0.119 g, 0.21 mmol) in CH2Cl2(2.0 mL)-anisole(0.05 mL)-water(0.05 mL) at room temperature under nitrogen was added trifluoroacetic acid (0.32 mL). The resulting clear solution was stirred at room temperature for 18 hrs, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue was triturated with Et2O to give the title compound (0.079 g, 74%) as a white solid.
Part C: (3S)-3-[N-(N′-(1-Naphthyl)Oxamyl)Homoprolinyl]Amino-4-Oxobutanoic Acid
A suspension of (3S)-3-[N-(N′-(1-naphthyl)oxamyl)homoprolinyl]amino-4-oxobutanoic acid semicarbazone (0.079 g, 0.16 mmol) in 37% aqueous formaldehyde(0.6 mL)-acetic acid(0.6 mL)-methanol(1.8 mL) was stirred at room temperature under nitrogen for 18 hrs. The resulting clear solution was diluted with water and mixture concentrated on a rotovap. The aqueous solution was then frozen and lyophilized. The residue was taken up in methanol, filtered through Celite and filtrate evaporated to dryness. Trituration of the residue with Et2O gave the title compound (0.037 g, 53%) as a white solid. MS(ES) for C22H23N3O6 (MW 425.44): positive 448(M+Na); negative 424(M−H).
EXAMPLE 112 (3S)-3-[N-(N′-(2-(1H-Tetrazol-5-yl)Phenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid Part A: 2-(1′-Phenylmethyl-5′-Tetrazolyl)Aniline Hydrochloride
A solution of 2-cyano-acetanilide (0.801 g, 5.0 mmol) and tri-n-butyltin azide (2.05 mL, 7.5 mmol) in anhydrous toluene (10 mL) was heated at reflux for 48 hrs. The mixture was allowed to cool to room temperature and treated with 2.0 N HCl in Et2O (5.0 mL). The resulting precipate was collected by suction, washed with hexane and dried in vacuo to give 2-(1H-tetrazol-5-yl)acetanilide (0.917 g, 90%) as a white solid.
To a suspension of 2-(1H-tetrazol-5-yl)acetanilide (0.203 g, 1.0 mmol) in tetrahydrofuran (2.0 mL) at 0� C. under nitrogen was added triethylamine (0.170 mL, 1.2 mmol) and benzyl bromide (0.125 mL, 1.05 mmol). After stirring at 0� C. for 3 hrs and at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to a dryness. The residue was triturated with hexane to give 2-(1′-phenylmethyl-5′-tetrazolyl)acetanilide (0.218 g, 74%) as a white solid. 1H-NMR indicates that the product is a single regioisomer. Assignment of regiochemistry should be considered tentative. 1H-NMR(CDCl3): δ2.22 ppm (3H,s), 5.84 (2H,s), 7.16 (1H, dt, J=7.8, 1.5 Hz), 7.40 (6H, m), 8.19 (1H, dd, J=7.8, 1.5 Hz), 8.63 (1H, d, J=8.4 Hz). 10.58 (1H, bs).
A mixture of 2-(1 ′-phenylmethyl-5′-tetrazolyl)acetanilide (0.216 g, 0.74 mmol) and 10% aqueous HCl (3.0 mL) was refluxed for 18 hrs. The mixture was evaporated to dryness and the residue triturated with Et2O to give the title compound (0.187 g, 88%) as a white solid.
Part B: N-[2-(l ′-Phenylmethyl-5′-Tetrazolyl)Phenyl]Oxamic Acid
To a solution of 2-(1′-phenylmethyl-5′-tetrazolyl)aniline hydrochloride (0.177 g, 0.615 mmol), 4-dimethylaminopyridine (0.008 g, 0.065 mmol) and triethylamine (0.19 mL, 1.4 mmol) in CH2Cl2 (1.0 mL) at 0� C. (ice bath) under nitrogen was added methyl oxalyl chloride (62 μL, 0.67 mmol). After stirring at 0� C. for 2 hrs, the mixture was allowed to come to room temperature, stirred for 18 hrs and then partitioned between EtOAc-5% KHSO4. The organic phase was washed with saturated NaCl solution, dried over anhydrous Na2SO4 and evaporated to dryness.
The crude methyl ester (0.207 g, ca 0.615 mmol) was taken up in dioxane (2.0 mL) and treated with 1.0 N LiOH solution (0.68 mL, 0.68 mmol) and stirred at room temperature for 1 hr. The mixture was acidified with 1.0 N HCl and extracted with EtOAc. The extract was washed with saturated NaCl solution, dried over anhydrous Na2SO4 and evaporated to dryness. Trituration of the crude product with hexane gave the title compound (0.121 g, 61%) as a white solid.
Part C: (3S)-3-[N-(N′-(2-(1′-Phenylmethyl-5′-Tetrazolyl)Phenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid Semicarbazone tert-Butyl Ester
To a solution of N-[2-(1′-phenylmethyl-5′-tetrazolyl)phenyl]oxamic acid (0.065 g, 0.20 mmol) in CH2Cl2(2.0 mL) at 0� C. under nitrogen was added hydroxybenzotriazole hydrate (0.037 g) followed by 1-ethyl-3-(3′,3′-dimethyl-1′-aminopropyl)carboduimide hydrochloride (0.058 g, 0.30 mmol). After stirring at 0� C. for 10 min, the mixture was treated with (3S)-3-(valinyl)amino-4-oxobutanoic acid (tert)-butyl ester semicarbazone (0.066 g, 0.20 mmol, prepared by the method described for the corresponding leucine analogue in Example 1, Parts B and C) and N-methylmorpholine (26 μL, 0.24 mmol). After stirring at room temperature for 16 hrs, the mixture was partitioned between EtOAc-water. The organic phase was washed with water, 5% KHSO4, saturated NaHCO3 and saturated NaCl solutions, dried over anhydrous Na2SO4 and evaporated to give the crude title compound (0.090 g, 62%) as a colorless glass.
Part D: (3S)-3-[N-(N′-(2-(1′H-5′-Tetrazolyl)Phenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid Semicarbazone tert-Butyl Ester
To a solution of crude (3S)-3-[N-(N′-(2-(I′-phenylmethyl-5′-tetrazolyl)phenyl)oxamyl)valinyl]amino-4-oxobutanoic acid semicarbazone tert-butyl ester (0.089 g, ca.0.14 mmol) in MeOH (1.0 mL) was added 10% Pd�C (0.009 g) and resulting mixture stirred under a hydrogen atmosphere (balloon) for 48 hrs. The mixture was filtered through Celite washing the filter cake with CH2Cl2 and the combined filtrates evaporated to dryness. The residue was triturated with Et2O to give the title product (0.060 g, 79%) as a white solid.
Part E: (3S)-3-[N-(N′-(2-(1′H-5′-Tetrazolyl)Phenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid Semicarbazone
To a solution of (3S)-3-[N-(N′-(2-(1′H-5′-tetrazolyl)phenyl)oxamyl)valinyl]amino-4-oxobutanoic acid tert-butyl ester (0.058, 0.11 mmol) in CH2Cl2(1.0 mL)-anisole(0.05 mL) at room temperature under nitrogen was added 6.0 M HCl/AcOH (1.0 mL). The resulting solution was stirred at room temperature for 18 hrs, evaporated to dryness and chased with toluene-CH2Cl2 (1:1). The residue was triturated with Et2O to give the title compound (0.048 g, 92%) as a white solid.
Part F: (3S)-3-[N-(N′-(2-(1′H-5 ′-Tetrazolyl)Phenyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid
A solution of (3S)-3-[N-(N′-(2-(1′H-5′-tetrazolyl)phenyl)oxamyl)valinyl]amino-4-oxobutanoic acid semicarbazone (0.048 g, 0.10 mmol) in 37% aqueous formaldehyde(0.4 mL)-acetic acid(0.4 mL)-methanol(1.2 mL) was stirred at room temperature under nitrogen for 18 hrs. The resulting clear solution was diluted with water and mixture concentrated on a rototvap. The aqueous solution was then frozen and lyophilized. The residue was taken up in methanol, filtered through Celite and filtrate evaporated to dryness. Trituration of the residue with Et2O gave the title compound (0.025 g, 59%) as a white solid. MS(ES) for C18H21N7O6 (MW 431.41): positive 454(M+Na); negative 430(M−H).
EXAMPLE 113 (3S)-3-[N-(N′-(1-Adamantanyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid Part A: (3S)-3-[N-(9-Fluorenylmethoxycabonyl)Valinyl]Amino-4-Oxobutanoic Acid (tert-Butyl) Ester Semicarbazonyl-4-[2′-(4-Ethyl-Phenoxyacetyl)]Aminomethylpolystrene
Aminomethylpolystryene resin (10.0 g, 100-200 mesh, 0.71 meq/g) was placed in a 200 mL filter tube equipped with a vacuum stopcock and glass frit and washed successively with CH2Cl2(50 mL)/dimethylformamide(50 mL), diisopropylethylamine(5 mL)/dimethylformamide(30 mL), dimethylformamide (2�50 mL) and tetrahydrofuran (30 mL). The resin was suspended in tetrahydrofuran(20 mL)/N-methylpyrolidinone(20 mL) with nitrogen agitation through the bottom of the frit and treated with diiospropylethylamine (1.9 mL, 10.9 mmol) and (3S)-3-(9-fluorenylmethoxycabonyl)amino-4-oxobutanoic acid (tert-butyl) ester semicarbazonyl-4-[2′-(4-ethyl-phenoxyacetic acid)] (2.24 g, 3.56 mmol). After all of the solid had dissolved (approx. 10 min), the mixture was treated with pyBOP [benzotriazolyloxy-tris(N-pyrolidinyl)phosphonium hexafluorophosphate, 2.78 g, 5.34 mmol) in one portion. After mixing by nitrogen agitation for 3 hrs, the supernatant was removed by suction and the resin washed succesively with tetrahydrofuran (2�50 mL), dimethylformamide (3�50 mL) and CH2Cl2 (2�50 mL). Unreacted amine groups were capped by treatment with a mixture of acetic anhydride(10 mL)/dimethylformamide(30 mL)/diisopropylethylamine(1.0 mL). After mixing by nitrogen agitation for 1 hr, the supernatant was removed by suction and the resin washed with dimethylformamide(4�50 mL).
The resin was treated with piperidine(10 mL)/ dimethylformamide(40 ml,) and mixed by nitrogen agitation for 1 hr. The supernatant was removed by suction and the resin washed with dimethylformamide(4�50 mL) and tetrahydrofuran (50 mL).
The resin was suspended in tetrahydrofuran(20 mL)/N-methylpyrolidinone(20 mL), treated with N-(9-fluorenylmethoxycabonyl)valine (3.63 g, 10.7 mmol), diisopropylethylamine (5.7 mL, 32.7 mmol) and pyBOP (8.34 g, 16.0 mmol) and mixed by nitrogen agitation for 2.5 hrs. The supematant was removed by suction and the resin washed succesively with dimethylformamide (3�40 mL) and CH2Cl2 (3�40 mL), methanol (2�40 mL) and Et2O (2�40 mL). The resin was dried in vacuo to give the title product (12.69 g, quanitative). Based on the starting semicarbazone-acid, the resin loading was calculated as approximately 0.28 meq/g.
Part B: (3S)-3-[N-(N′-(1-Adamantanyl)Oxamyl)Valinyl]Amino-4-Oxobutanoic Acid
An aliquot of the Part A resin (0.125 g, ca 0.035 mmol) was placed in a 6 mL Supelco� fitration tube equipped with a 201 μm polyethylene frit, treated with piperidine-dimethylformamide (1.0 mL, 1:4 v/v) and mixed on an orbital shaker for 1 hr. The supernatant was removed by suction and the resin washed with dimethylformamide (4�1.0 mL) and CH2Cl, (3�1.0 mL). The resin was treated with 0.5M iPr2NEt in N-methylpyrolidinone (0.40 mL, 0.20 mmol), (1-adamantanyl)oxamic acid (0.0246 g, 0.11 mmol) and 0.25M O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophate in N-methylpyrolidinone (0.40 mL, 0.10 mmol). The mixture was mixed on an orbital shaker under an nitrogen atmosphere for 16 hrs. The supernatant was removed by suction and the resin washed succesively with dimethylformamide (3�1.0 mL) and CH2Cl2 (3�1.0 mL), methanol (2�1.0 mL) and Et2O(2�1.0 mL).
The resin was treated with 1.0 mL of CH2Cl2 and allowed to re-swell for 15 min. The solvent was removed by suction and the resin treated with trifluoroacetic acid-CH2Cl2-anisole (1.0 mL, 4:3:1 v/v/v). After mixing on an orbital shaker under nitrogen for 5.5 hrs, the supernatant was removed by suction and the resin washed with CH2Cl2 (4�1.0 mL). The resin was treated with 37% aqueous formaldehyde-acetic acid-tetrahydrofuran-trifluoroacetic acid (1.0 mL, 1:1:5:0.025 v/v/v/v) and mixed on an orbital shaker under nitrogen for 4.5 hrs. The supematant was collected by suction, the resin washed with tetrahydrofuran (3�0.5 mL). The combined filtrates were blown down under nitrogen. The residue was taken up in methanol (0.5 mL), filtered and applied directly to a 3 mL Supelco� LC-18 reverse phase extraction tube which had been pre-conditioned with water, and eluted successively with 3 mL each of 10% MeOH-water, 30% MeOH-water, 60% MeOH-water and 90% MeOH-water. The product-containing fractions (TLC) were combined and evaporated to dryness to give the title compound (0.0114 g, 77%) as a colorless glass. TLC(AcOH�MeOH�CH2Cl2; 1:1:20) Rf=0.23. MS(ES) for C21H31N3O6 (MW 421.49): positive 444(M+Na), 460(M+K); negative 420(M−H), 534(M+TFA).
EXAMPLES 114-127 Starting with (3S)-3-[N-(9-fluorenylmethoxycabonyl)valinyl]amino-4-oxobutanoic acid (tert-butyl) ester semicarbazonyl-4-[2′-(4-ethyl-phenoxyacetyl)]aminomethylpolystrene (see Example 113, Part A) and following the methods described in Example 113, Part B, the compounds shown below in Table 11 were also prepared:
C17H21N3O6 363.37
386(M + Na)
362(M − H)
402(M + K)
PhCH2 C18H23N3O6 377.40
400(M + Na)
376(M − H)
Ph(CH2)2 C19H25N3O6 391.42
414(M + Na)
390(M − H)
430(M + K)
504(M + TFA)
C18H20F3N3O6 431.37
454(M + Na)
430(M − H)
C21H29N3O6 419.48
442(M + Na)
418(M − H)
458(M + K)
532(M + TFA)
C23H25N3O6 439.47
462(M + Na)
438(M − H)
478(M + K)
552(M + TFA)
C24H27N3O6 453.49
476(M + Na)
452(M − H)
492(M + K)
566(M + TFA)
C23H25N3O7 455.47
478(M + Na)
454(M − H)
494(M + K)
568(M + TFA)
C21H23N3O6 413.43
436(M + Na)
412(M − H)
452(M + K)
526(M + TFA)
C21H22ClN3O6 447.87
470/472 (M + Na)
446/448 (M − H)
486/488 (M + K)
5,6,7,8-tetrahydro-1-
C21H27N3O6 417.46
440(M + Na)
416(M − H)
456(M + K)
530(M + TFA)
(1-naphthyl)CH2 C22H25N3O6 427.46
450(M + Na)
426(M − H)
466(M + K)
540(M + TFA)
Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention, Accordingly, the invention is limited only by the claims.
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS5869519Dec 16, 1996Feb 9, 1999Idun Pharmaceuticals, Inc.C-terminal modified (n-substituted)-2-indolyl dipeptides as inhibitors of the ICE/ced-3 family of cysteine proteasesUS5877197Sep 12, 1997Mar 2, 1999Karanewsky; Donald S.C-terminal modified (N-substituted)-2-indolyl dipeptides as inhibitors of the ICE/ced-3 family of cysteine proteasesUS5968927Sep 12, 1997Oct 19, 1999Idun Pharmaceuticals, Inc.Tricyclic compounds for the inhibition of the ICE/ced-3 protease family of enzymesUS6197750Oct 22, 1998Mar 6, 2001Idun Pharmaceuticals, Inc.C-terminal modified oxamyl dipeptides as inhibitors of the ICE/ced-3 family of cysteine proteasesEP0618223A2Mar 3, 1994Oct 5, 1994Sandoz Ltd.Peptides inhibiting interleukin 1-b�ta release useful as antiinflammatory agentsEP0623592A1Apr 27, 1994Nov 9, 1994Sterling Winthrop Inc.Peptide analogs as irreversible interleukin-1beta protease inhibitorsWO1997022619A2Dec 20, 1996Jun 26, 1997Vertex PharmaINHIBITORS OF INTERLEUKIN-1β CONVERTING ENZYMENon-Patent CitationsReference1Gagliardini et al. Prevention of Vertebrate Neuronal Death by the crmA Gene, Science vol. 263, Feb. 11, 1994 pp. 826-828.2Hugunin et al., "Protease activity of m-vitro transcribed and translated caenorhabditis elegans cell death gene (ced-3) product" J. Biol. Chem. 271(7):3517-3512, Feb. 16, 1996.3Ilugunin et al., "Protease activity of m-vitro transcribed and translated caenorhabditis elegans cell death gene (ced-3) product" J. Biol. Chem., Feb. 16, 1996, 271(7) 3517-12 abstract only, 1996.4Juan et al., "Molecular characterization of mouse and rat CPP32 beta gene encoding a cysteine protease resembling interleukin-1 beta convesting enzyme and CED-3" Oncogene 13(4):749-755, Aug. 15, 1996.5Juan TS et al., "Molecular characterization of mouse and rat CPP32 beta gene encoding a cysteine protease resembling interleukin-1 beta convesting enzyme and CED-3" Oncogene, 13(4)749-55, Aug. 15, 1996 (Abstract only).6Marx. Cell Death Studies Yield Cancer Clues; Science vol. 259, Feb. 5, 1993, pp. 760-761.7Sleath et al., Substrate Specificity of the Protease that Processes Human Interleukin-1.beta., The Journal of Biological Chemistry, vol. 265, No. 24, pp. 14526-14528, Aug. 25, 1990.8Sleath et al., Substrate Specificity of the Protease that Processes Human Interleukin�1.beta., The Journal of Biological Chemistry, vol. 265, No. 24, pp. 14526-14528, Aug. 25, 1990.Referenced byCiting PatentFiling datePublication dateApplicantTitleUS6949516May 5, 2003Sep 27, 2005Cytovia, Inc.Dipeptide apoptosis inhibitors and the use thereofUS7183260Aug 25, 2004Feb 27, 2007Idun Pharmaceuticals, Inc.C-terminal modified oxamyl dipeptides as inhibitors of the ICE/ced-3 family of cysteine proteasesUS7692038 *Dec 6, 2007Apr 6, 2010Pfizer, Inc.Crystalline forms* Cited by examinerClassifications U.S. Classification514/21.91, 546/278.4, 548/517, 544/360, 548/527International ClassificationA61K31/16, A61P25/28, C07D239/00, C07C237/22, C07K5/078, C07D235/30, C07D211/60, C07C235/80, A61P7/06, A61K38/05, A61K31/33, C07F9/32, C07C, C07D311/32, A61P9/10, A61K38/06, C07C317/50, A61P43/00, C07D261/18, C07D239/34, C07F9/46, A61K38/00, C07K5/06, A61P37/06, C07D309/30, C07C23/22, C07K5/062, A61P29/00, C07D209/42, C07D237/22Cooperative ClassificationC07D261/18, C07D309/30, C07F9/3264, C07C2103/74, C07C237/22, C07D239/34, C07D209/42, C07D235/30, C07D311/32, C07D211/60, C07C2101/14European ClassificationC07D235/30, C07F9/32C1, C07D309/30, C07D211/60, C07D239/34, C07D311/32, C07D261/18, C07D209/42, C07C237/22Legal EventsDateCodeEventDescriptionMay 1, 2013ASAssignmentFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:IDUN PHARMACEUTICALS, INC.;REEL/FRAME:030330/0658Owner name: CONATUS PHARMACEUTICALS, INC., CALIFORNIAEffective date: 20130411Oct 4, 2010FPAYFee paymentYear of fee payment: 8Oct 4, 2006FPAYFee paymentYear of fee payment: 4Mar 23, 2004CCCertificate of correctionRotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google