U.S. patent application number 10/721338 was filed with the patent office on 2004-06-17 for caspase inhibitors and the use thereof.
This patent application is currently assigned to Cytovia, Inc.. Invention is credited to Cai, Sui Xiong, Green, Douglas R., Mills, Gordon B., Wang, Yan, Weber, Eckard.
Application Number | 20040116355 10/721338 |
Document ID | / |
Family ID | 26826691 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040116355 |
Kind Code |
A1 |
Cai, Sui Xiong ; et
al. |
June 17, 2004 |
Caspase inhibitors and the use thereof
Abstract
The present invention is directed to novel dipeptide thereof,
represented by the general Formula I: 1 where R.sub.1-R.sub.3, X
and Y are defined herein. The present invention also relates to the
discovery that compounds having Formula I are potent inhibitors of
caspases and apoptotic cell death. Therefore, the inhibitors of
this invention can retard or block cell death in a variety of
clinical conditions in which the loss of cells, tissues or entire
organs occurs.
Inventors: |
Cai, Sui Xiong; (San Diego,
CA) ; Weber, Eckard; (San Diego, CA) ; Wang,
Yan; (San Diego, CA) ; Mills, Gordon B.;
(Houston, TX) ; Green, Douglas R.; (San Diego,
CA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Cytovia, Inc.
|
Family ID: |
26826691 |
Appl. No.: |
10/721338 |
Filed: |
November 26, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10721338 |
Nov 26, 2003 |
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09987417 |
Nov 14, 2001 |
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6716818 |
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09987417 |
Nov 14, 2001 |
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09545565 |
Apr 7, 2000 |
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6355618 |
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60158370 |
Oct 12, 1999 |
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60128545 |
Apr 9, 1999 |
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Current U.S.
Class: |
514/7.3 ;
514/16.4; 514/16.6; 514/18.6; 514/18.9; 514/19.3; 514/20.2;
514/20.7; 514/20.8; 514/21.91; 514/3.8; 546/201; 546/210; 546/229;
546/272.7; 546/277.4; 546/336; 548/338.1; 548/495 |
Current CPC
Class: |
A61P 9/04 20180101; A61P
31/18 20180101; A61P 1/04 20180101; A61P 7/06 20180101; A61P 25/28
20180101; A61P 25/02 20180101; A61P 3/10 20180101; C07D 311/66
20130101; A61P 1/08 20180101; A61P 7/08 20180101; A61P 13/10
20180101; A61P 25/16 20180101; C07C 237/22 20130101; C07D 295/205
20130101; A61P 9/10 20180101; A61P 25/32 20180101; C07C 271/22
20130101; A61P 31/12 20180101; A61P 35/00 20180101; A61P 37/00
20180101; A61P 15/08 20180101; C07C 2601/08 20170501; A61P 1/02
20180101; A61P 17/02 20180101; A61P 27/06 20180101; A61P 17/06
20180101; A61P 1/18 20180101; A61P 43/00 20180101; C07C 311/19
20130101; A61P 1/16 20180101; A61P 31/04 20180101; A61P 37/02
20180101; A61P 39/02 20180101; A61P 27/02 20180101; A61P 17/14
20180101; A61P 29/00 20180101; A61P 25/14 20180101; A61P 7/00
20180101; A61P 25/00 20180101; C07D 311/16 20130101; A61P 13/12
20180101; C07C 2601/14 20170501 |
Class at
Publication: |
514/019 ;
546/201; 546/210; 546/229; 546/272.7; 546/277.4; 546/336;
548/338.1; 548/495 |
International
Class: |
A61K 038/04; C07K
005/04 |
Claims
What is claimed is:
1. A compound having the Formula I: 32or pharmaceutically
acceptable salts or prodrugs thereof, wherein: R.sub.1 is an
optionally substituted alkyl or hydrogen; R.sub.2 is hydrogen or
optionally substituted alkyl; R.sub.3 is an alkyl, saturated
carbocyclic, partially saturated carbocyclic, aryl, saturated
heterocyclic, partially saturated heterocyclic or heteroaryl group,
wherein said group is optionally substituted; X is O, S, NR.sub.4
or (CR.sub.4R.sub.5).sub.n, where R.sub.4 and R.sub.5 are, at each
occurrence, independently selected from the group consisting of
hydrogen, alkyl and cycloalkyl, and n is 0, 1, 2 or 3; or X is
NR.sub.4, and R.sub.3 and R.sub.4 are taken together with the
nitrogen atom to which they are attached to form a saturated
heterocyclic, partially saturated heterocyclic or heteroaryl group,
wherein said group is optionally substituted; or X is
CR.sub.4R.sub.5, and R.sub.3 and R.sub.4 are taken together with
the carbon atom to which they are attached to form a saturated
carbocyclic, partially saturated carbocyclic, aryl, saturated
heterocyclic, partially saturated heterocyclic or oxygen-containing
heteroaryl group, wherein said group is optionally substituted; and
Y is a residue of a natural or non-natural amino acid; provided
that when X is O, then R.sub.3 is not unsubstituted benzyl or
t-butyl; and when X is CH.sub.2, then R.sub.3 is not hydrogen.
2. The compound of claim 1, wherein R.sub.1 is hydrogen, methyl,
ethyl or acetoxymethyl.
3. The compound of claim 1, wherein R.sub.2 is hydrogen,
fluoromethyl, acyloxymethyl, arylacyloxymethyl, aryloxymethyl,
phosphinyloxymethyl, or aminomethyl.
4. The compound of claim 1, wherein Y is valine, isoleucine,
leucine, alanine, phenylalanine, cyclohexylalanine, 2-aminobutyric
acid, phenylglycine or cyclohexylglycine.
5. The compound of claim 1, wherein: R.sub.3 is optionally
substituted alkyl, C.sub.4-C.sub.7 cycloalkyl, saturated
heterocyclic, partially saturated heterocyclic, aryl or heteroaryl;
and X is O, S, NR.sub.4 or (CR.sub.4R.sub.5)n, wherein R.sub.4 and
R.sub.5 are independently hydrogen, alkyl or cycloalkyl, and n is
0, 1, 2 or 3.
6. The compound of claim 1 , wherein X is O, NH or CH.sub.2.
7. The compound of claim 1, wherein R.sub.3 is straight-chained or
branched C.sub.1-6 alkyl.
8. The compound of claim 1, wherein R.sub.3 is straight-chained or
branched C.sub.1-6 alkyl optionally substituted by hydroxy,
carboxy, halogen, C.sub.4-C.sub.7 cycloalkyl, saturated or
unsaturated heterocyclic group, aryl or heteroaryl.
9. The compound of claim 1, wherein R.sub.3 is optionally
substituted benzyl.
10. The compound of claim 1, wherein R.sub.3 is optionally
substituted pyridylmethyl.
11. The compound of claim 1, wherein R.sub.3--X--C(O)-- is an
antioxidant group.
12. The compound of claim 11, wherein said antioxidant group is
33
13. The compound of claim 12, wherein said compound is 34
14. The compound of claim 1, wherein R.sub.3--X--C(O)-- is a
fluorescent group.
15. The compound of claim 14, wherein said fluorescent group is
35
16. The compound of claim 14, wherein said compound is selected
from the group consisting of 36
17. A compound having the Formula II: 37or pharmaceutically
acceptable salts or prodrugs thereof wherein: R.sub.1 is an
optionally substituted alkyl or hydrogen; R.sub.2 is hydrogen or
optionally substituted alkyl; X is O, S, NR.sub.4 or
(CR.sub.4R.sub.5).sub.n, wherein R.sub.4 and R.sub.5 are, at each
occurrence, independently selected from the group consisting of
hydrogen, alkyl, and cycloalkyl, and n is 0, 1, 2 or 3; Y is a
residue of a natural or non-natural amino acid; A is CR.sub.6 or
nitrogen; B is CR.sub.7 or nitrogen; C is CR.sub.8 or nitrogen; D
is CR.sub.9 or nitrogen; E is CR.sub.10 or nitrogen; provided that
not more than three of A, B, C, D and E are nitrogen; and
R.sub.6-R.sub.10 independently are hydrogen, halo, C.sub.1-C.sub.6
haloalkyl, C.sub.6-C.sub.10 aryl, C.sub.4-C.sub.7 cycloalkyl,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.6-C.sub.10 aryl(C.sub.1-C.sub.6)alkyl,
C.sub.6-C.sub.10 aryl(C.sub.2-C.sub.6)alkenyl- , C.sub.6-C.sub.10
aryl(C.sub.2-C.sub.6)alkynyl, C.sub.1-C.sub.6 hydroxyalkyl, nitro,
amino, cyano, C.sub.1-C.sub.6 acylamino, hydroxy, C.sub.1-C.sub.6
acyloxy, C.sub.1-C.sub.6 alkoxy, alkylthio, or carboxy; or one of
R.sub.6 and R.sub.7, or R.sub.7 and R.sub.8, or R.sub.8 and
R.sub.9, or R.sub.9 and R.sub.10 are taken together with the carbon
atoms to which they are attached to form a carbocycle or
heterocycle, selected from the group consisting of --OCH.sub.2O--,
--OCF.sub.2--, --(CH.sub.2).sub.3--, --(CH.sub.2).sub.4--,
--OCH.sub.2CH.sub.2O--, --CH.sub.2N(R.sub.13)CH.sub.2--,
--CH.sub.2CH.sub.2N(R.sub.13)CH.sub.2--,
--CH.sub.2N(R.sub.13)CH.sub.2CH.sub.2--,
--N(R.sub.13)--CH.dbd.CH--, --CH.dbd.CH--N(R.sub.13)--,
--O--CH.dbd.CH--, --CH.dbd.CH--O--, --S--CH.dbd.CH--,
--CH.dbd.CH--S--, --N.dbd.CH--CH.dbd.CH--, --CH.dbd.N--CH.dbd.CH--,
--CH.dbd.CH--N.dbd.CH--, --CH.dbd.CH--CH.dbd.N--- ,
--N.dbd.CH--CH.dbd.N--, and --H.dbd.CH--CH.dbd.CH--; wherein
R.sub.13 is hydrogen, alkyl or cycloalkyl; provided that when X is
O, A is CR.sub.6, B is CR.sub.7, C is CR.sub.8, D is CR.sub.9 and E
is CR.sub.10, then at least one of the R.sub.6-R.sub.10 is not
hydrogen.
18. The compound of claim 17, wherein R.sub.2 is hydrogen,
fluoromethyl, acyloxymethyl, arylacyloxymethyl, aryloxymethyl,
phosphinyloxymethyl, or aminomethyl.
19. The compound of claim 17, wherein R.sub.1 is hydrogen, methyl,
ethyl or acetoxymethyl.
20. The compound of claim 17, wherein Y is valine, isoleucine,
leucine, alanine, phenylalanine, cyclohexylalanine, 2-aminobutyric
acid, phenylglycine or cyclohexylglycine.
21. The compound of claim 17, wherein X is O, A is CR.sub.6, B is
CR.sub.7, C is CR.sub.8, D is CR.sub.9, and E is CR.sub.10.
22. The compound of claim 17, wherein X is O, and one of A, B. C, D
or E is nitrogen.
23. The compound of claim 17, wherein X is CH.sub.2, A is CR.sub.6,
B is CR.sub.7, C is CR.sub.8, D is CR.sub.9 and E is CR.sub.10.
24. A compound having the Formula III: 38or pharmaceutically
acceptable salts or prodrugs thereof, wherein: R.sub.1 is an
optionally substituted alkyl or hydrogen; R.sub.2 is hydrogen or
optionally substituted alkyl; R.sub.3 is an alkyl, saturated
carbocyclic, partially saturated carbocyclic, aryl, saturated
heterocyclic, partially saturated heterocyclic or heteroaryl group,
wherein said group is optionally substituted; and Y is a residue of
a natural or non-natural amino acid.
25. The compound of claim 24, wherein R.sub.1 is hydrogen, methyl,
ethyl or acetoxymethyl.
26. The compound of claim 24, wherein R.sub.2 is hydrogen,
fluoromethyl, acyloxymethyl, arylacyloxymethyl, aryloxymethyl,
phosphinyloxymethyl, or aminomethyl.
27. The compound of claim 24, wherein Y is valine, isoleucine,
leucine, alanine, phenylalanine, cyclohexylalanine, 2-aminobutyric
acid, phenylglycine or cyclohexylglycine.
28. The compound of claim 24, wherein R.sub.3 is straight-chained
or branched C.sub.1-6 alkyl.
29. The compound of claim 24, wherein R.sub.3 is straight-chained
or branched C.sub.1-6 alkyl optionally substituted by hydroxy,
carboxy, halogen C.sub.4-C.sub.7 cycloalkyl, saturated or
unsaturated heterocyclic group, aryl or heteroaryl.
30. The compound of claim 24, wherein R.sub.3 is methylphenyl or
dimethylaminonaphthyl.
31. The compound of claim 1, wherein said compound is selected from
the group consisting of: 2-Chlorobenzyloxycarbonyl-Val-Asp-fmk,
3-Chlorobenzyloxycarbonyl-Val-Asp-fmk,
4-Chlorobenzyloxycarbonyl-Val-Asp-- fmk,
Phenethoxycarbonyl-Val-Asp-fmk,
Cyclohexylmethoxycarbonyl-Val-Asp-fmk- ,
Methoxycarbonyl-Val-Asp-fmk, Ethoxycarbonyl-Val-Asp-fmk,
Isopropyloxycarbonyl-Val-Asp-fmk,
2-Chlorobenzyloxycarbonyl-Ile-Asp-fmk,
3-Chlorobenzyloxycarbonyl-Ile-Asp-fmk,
4-Chlorobenzyloxycarbonyl-Ile-Asp-- fmk, Phenylacetyl-Val-Asp-fmk,
4-Nitrobenzyloxycarbonyl-Val-Asp-fmk,
2,5-Dimethylbenzyloxycarbonyl-Val-Asp-fmk,
3,4-Dichlorobenzyloxycarbonyl-- Val-Asp-fmk,
3,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,
2,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,
2,6-Dichlorobenzyloxycarbonyl-- Val-Asp-fmk,
2,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,
2,4-Dimethylbenzyloxycarbonyl-Val-Asp-fmk,
4-Ethylbenzyloxycarbonyl-Val-A- sp-fmk,
4-Bromobenzyloxycarbonyl-Val-Asp-fmk, 4-Fluorobenzyloxycarbonyl-Va-
l-Asp-fmk, Cyclopentylmethoxycarbonyl-Val-Asp-fmk,
4-Trifluoromethylbenzyl- oxycarbonyl-Val-Asp-fmk,
3-Phenylpropionyl-Val-Asp-fmk, Benzylaminocarbonyl-Val-Asp-fmk,
3-Phenylpropyloxycarbonyl-Val-Asp-fmk,
2,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk,
3,4-Difluorobenzyloxycarbonyl-- Val-Asp-fmk,
4-Morpholinecarbonyl-Val-Asp-fmk, 4-Pyridylmethoxycarbonyl-Va-
l-Asp-fmk, 2-Pyridylmethoxycarbonyl-Val-Asp-fmk,
2,6-Dichlorobenzyloxycarb- onyl-Val-Asp-DCB-methylketone,
Isobutoxycarbonyl-Val-Asp-fmk, Propionyl-Val-Asp-fmk,
Benzyl-glutaryl-Val-Asp-fmk, Glutaryl-Val-Asp-fmk,
3-(2-Phenyloxyphenyl)propionyl-Val-Asp-fmk,
3-(5-Bromo-2-hydroxyphenyl)pr- opionyl-Val-Asp-fmk,
3-Fluorobenzyloxycarbonyl-Val-Asp-fmk,
2-Fluorobenzyloxycarbonyl-Val-Asp-fmk,
3-Methylbenzyloxycarbonyl-Val-Asp-- fmk,
2-Chloro-4-fluorobenzyloxycarbonyl-Val-Asp-fmk, and
2-Naphthylmethoxycarbonyl-Val-Asp-fmk.
32. The compound of claim 24, wherein said compound is selected
from the group consisting of: p-Toluenesulfonyl-Val-Asp-fmk, and
p-Toluenesulfonyl-Phe-Asp-fmk.
33. A pharmaceutical composition, comprising a compound of claim 1,
17 or 24, and a pharmaceutically acceptable carrier.
34. A method of inhibiting cell death of a cell or tissue,
comprising contacting said cell or tissue with an effective amount
of a compound of claim 1, 17 or 24.
35. A method of treating or ameliorating cell death in the central
or peripheral nervous system, retinal neurons, cardiac muscle or
immune system cells of an animal, comprising administering to the
animal in need of such treatment or ameliorating an effective
amount of a compound of claim 1, 17 or 24.
36. The method of claim 35, wherein said cell death is in the
central or peripheral nervous system, and is due to one of: (a) a
condition of ischemia and excitotoxicity selected from the group
consisting of focal ischemia due to stroke and global ischemia due
to cardiac arrest; (b) traumatic injury; (c) viral infection; (d)
radiation-induced nerve cell death; (e) a neurodegenerative
disorder selected from the group consisting of Alzheimer's disease,
Parkinson's Disease, a prion disease, multiple sclerosis,
amyotrophic lateral sclerosis, and spinobulbar atrophy; (f) spinal
cord injury; or (g) acute bacterial meningitis.
37. The method of claim 35, wherein said cell death is in the
central or peripheral nervous system, and is due to expansion of
trinucleotide repeats of specific genes.
38. The method of claim 35, wherein said cell death is due to
Huntington's Disease.
39. The method of claim 35, wherein said cell death is in cardiac
muscle tissue, and is due to myocardial infarction, congestive
heart failure, cardiomyopathy or viral infection of the heart.
40. The method of claim 35, wherein said cell death is in retinal
neurons and is due to increased intraocular pressure, age-related
macular degeneration or retinitis pigmentosa.
41. The method of claim 35, wherein said cell death is in the
immune system, and is due to an immune deficiency disorder selected
from the group consisting of acquired immune deficiency syndrome,
severe combined immune deficiency syndrome and radiation-induced
immune suppression.
42. The method of claim 35, wherein said cell death is due to an
autoimmune disorder selected from the group consisting of lupus
erythematosus, rheumatoid arthritis and type I diabetes.
43. The method of claim 42, wherein said cell death is due to type
I diabetes.
44. A method of treating or preventing polycystic kidney disease,
renal amyloidosis, acute renal failure, cyclosporine A induced
tubular epithelial cell death, hypoxia-induced necrosis of renal
proximal tubules, HIV-induced nephropathy or anemia/erythropoiesis
in an animal, comprising administering to the animal in need of
such treatment an effective amount of a compound of claim 1, 17 or
24.
45. A method of protecting a mammalian organ or tissue from cell
death due to deprivation of normal blood supply, comprising
contacting said organ or tissue with an effective amount of a
compound of claim 1, 17 or 24.
46. The method of claim 45, wherein said organ or tissue is present
in a storage medium prior to transplant into a mammal.
47. The method of claim 45, wherein said tissue is embryonic nigral
tissue.
48. The method of claim 45, wherein said contacting comprises
infusion of said compound into the organ or tissue, or bathing of
said organ or tissue in a storage medium which comprises said
compound.
49. A method of reducing or preventing cell death in a donor organ
or tissue after it has been transplanted into a host due to the
effects of reperfusion injury or due to the effects of host immune
cells, comprising administering to said host in need thereof an
effective amount of a compound of claim 1, 17 or 24.
50. A method of reducing or preventing the death of mammalian sperm
or eggs used in in vitro fertilization procedures, comprising
contacting said sperm or egg with an effective amount of a compound
of claim 1, 17 or 24.
51. A method of extending the lifespan of a mammalian or yeast cell
line, comprising contacting said cell line with a compound of claim
1, 17 or 24.
52. The method of claim 51, wherein said contacting comprises
including said compound in a cell growth medium.
53. A method of treating or ameliorating hair loss or premature
graying of the hair in a manmmal, comprising contacting the hair or
hair follicles of the mammal in need thereof with a compound of
claim 1, 17 or 24.
54. The method of claim 53, wherein hair loss is treated, and said
hair loss is due to male-pattern baldness, radiation, chemotherapy
or emotional stress.
55. A method of treating or ameliorating skin damage of a mammal
due to exposure to high levels of radiation, heat or chemicals,
comprising applying to the skin of the mammal in need thereof with
a compound of claim 1, 17 or 24.
56. The method of claim 55, wherein said compound is applied as
part of an ointment.
57. The method of claim 55, wherein said skin damage is due to
acute over-exposure to the sun, and wherein said treating reduces
blistering and peeling of the skin.
58. A method of treating or ameliorating sepsis or multi-organ
failure in an animal, comprising administering to the animal in
need thereof an effective amount of a compound of claim 1, 17 or
24.
59. A method of treating or ameliorating hepatitis in an animal,
comprising administering to the animal in need thereof an effective
amount of a compound of claim 1, 17 or 24.
60. A method of treating or ameliorating hereditary tyrosinemia
type 1 in an animal, comprising administering to the animal in need
thereof an effective amount of a compound of claim 1, 17 or 24.
61. A method of treating or ameliorating chronic alcohol ingestion
induced buccal mucosa cell death in an animal, comprising
administering to the animal in need thereof an effective amount of
a compound of claim 1, 17 or 24.
62. A method of treating or ameliorating cell death in plants or
flowers, comprising administering to the plants or flowers in need
thereof an effective amount of a compound of claim 1, 17 or 24.
63. A method of treating or ameliorating radiation or
ultraviolet-irradiation induced cell death in an animal, comprising
administering to the animal in need thereof an effective amount of
a compound of claim 1, 17 or 24.
64. A method of treating or ameliorating apoptotic death of bone
marrow cells in myelodysplastic syndromes (MDS), comprising
administering to the animal in need thereof an effective amount of
a compound of claim 1, 17 or 24.
65. A method of treating or ameliorating apoptotic cell death in
acute pancreatitis, comprising administering to the animal in need
thereof an effective amount of a compound of claim 1, 17 or 24.
66. A method of treating or preventing the inflammatory response in
psoriasis or inflammatory bowel disease, comprising administering
to the animal in need thereof an effective amount of a compound of
claim 1, 17 or 24.
67. A method of treating or ameliorating organ apoptosis after burn
injury, comprising administering to the animal in need thereof an
effective amount of a compound of claim 1, 17 or 24.
68. A method of treating or ameliorating small bowel tissue injury
after intestinal ischemia-reperfusion, comprising administering to
the animal in need thereof an effective amount of a compound of
claim 1, 17 or 24.
69. A method of treating, ameliorating. or preventing oral
mucositis, gastrointestinal mucositis, bladder mucositis,
proctitis, bone marrow cell death, skin cell death, or hair loss
resulting from chemotherapy or radiation therapy of cancer in an
animal, comprising administering to the animal in need thereof an
effective amount of a compound of claim 1, 17 or 24.
70. The method of claim 69, wherein said compound is administered
topically or orally.
71. The method of claim 70, wherein said compound is formulated as
part of a mouthwash for the treatment, amelioration or prevention
of oral mucositis.
72. The method of claim 70, wherein said compound is formulated as
part of a slow release buccal lozenge.
73. The method of claim 70, wherein said compound is formulated as
part of a suppository.
74. The method of claim 70, wherein said compound is formulated as
part of a gel.
75. The method of claim 70, wherein said compound is administered
through a bladder catheter for the treatment, amelioration or
prevention of bladder mucositis.
76. The method of claim 70, wherein said compound is administered
as part of an enema for the treatment, amelioration or prevention
of proctitis.
77. The method of claim 70, wherein said compound is formulated as
an oral formulation which is capable of coating the
gastrointestinal surfaces for the treatment, amelioration or
prevention of gastrointestinal mucositis.
78. The method of claim 69, wherein said gastrointestinal mucositis
is esophageal mucositis, gastric mucositis, or intestinal
mucositis.
79. The method of claim 69, wherein said compound is administered
by i.v. injection for the treatment, amelioration or prevention of
bone marrow cell death.
80. The method of claim 69, wherein said compound is administered
as part of a pharmaceutical composition comprising a
pharmaceutically acceptable carrier.
81. The method of claim 69, wherein said compound is administered
after chemotherapy or radiation therapy of cancer in said
animal.
82. The method of claim 69, wherein said compound is administered
during chemotherapy or radiation therapy of cancer in said
animal.
83. The method of claim 69, wherein said compound is administered
prior to chemotherapy or radiation therapy of cancer in said
animal.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention is in the field of medicinal chemistry. In
particular, the invention relates to dipeptide caspase inhibitors
with novel N-terminal blocking groups. The invention also relates
to the use of these caspase inhibitors for reducing or treating
apoptotic cell death and/or reducing interleukin
1-.beta.production.
[0003] 2. Description of Background Art
[0004] Organisms eliminate unwanted cells by a process variously
known as regulated cell death, programmed cell death or apoptosis.
Such cell death occurs as a normal aspect of animal development as
well as in tissue homeostasis and aging (Glucksmann, A., Biol. Rev.
Cambridge Philos. Soc. 26:59-86 (1951); Glucksmann, A., Archives de
Biologie 76:419-437 (1965); Ellis et al., Dev. 112:591-603 (1991);
Vaux et al., Cell 76:777-779 (1994)). Apoptosis regulates cell
number, facilitates morphogenesis, removes harmful or otherwise
abnormal cells and eliminates cells that have already performed
their function. Additionally, apoptosis occurs in response to
various physiological stresses, such as hypoxia or ischemia (PCT
published application WO96/20721).
[0005] There are a number of morphological changes shared by cells
experiencing regulated cell death, including plasma and nuclear
membrane blebbing, cell shrinkage (condensation of nucleoplasm and
cytoplasm), organelle relocalization and compaction, chromatin
condensation and production of apoptotic bodies (membrane enclosed
particles containing intracellular material) (Orrenius, S., J.
Internal Medicine 237:529-536 (1995)).
[0006] Apoptosis is achieved through an endogenous mechanism of
cellular suicide (Wyllie, A. H., in Cell Death in Biology and
Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp.
9-34). A cell activates its internally encoded suicide program as a
result of either internal or external signals. The suicide program
is executed through the activation of a carefully regulated genetic
program (Wylie et al., Int. Rev. Cyt. 68:251 (1980); Ellis et al.,
Ann. Rev. Cell Bio. 7:663 (1991)). Apoptotic cells and bodies are
usually recognized and cleared by neighboring cells or macrophages
before lysis. Because of this clearance mechanism, inflammation is
not induced despite the clearance of great numbers of cells
(Orrenius, S., J. Internal Medicine 237:529-536 (1995)).
[0007] Mammalian interleukin-1.beta.(IL-1.beta.) plays an important
role in various pathologic processes, including chronic and acute
inflammation and autoimmune diseases (Oppenheim et. al. Immunology
Today, 7:45-56 (1986)). IL-1.beta. is synthesized as a cell
associated precursor polypeptide (pro-IL-1.beta.) that is unable to
bind IL-1 receptors and is biologically inactive (Mosley et al., J.
Biol. Chem. 262:2941-2944 (1987)). By inhibiting conversion of
precursor IL-1.beta. to mature IL-1.beta., the activity of
interleukin-1 can be inhibited. Interleukin-1.beta. converting
enzyme (ICE) is a protease responsible for the activation of
interleukin-1.beta. (IL-1.beta.) (Thornberry et al., Nature 356:768
(1992); Yuan et al., Cell 75:641 (1993)). ICE is a
substrate-specific cysteine protease that cleaves the inactive
prointerleukin-1 to produce the mature IL-1. The genes that encode
for ICE and CPP32 are members of the mammalian ICE/Ced-3 family of
genes which presently includes at least twelve members: ICE,
CPP32/Yama/Apopain, mICE2, ICE4, ICH1, TX/ICH-2, MCH2, MCH3, MCH4,
FLICE/MACH/MCH5, ICE-LAP6 and ICE.sub.re1III. The proteolytic
activity of this family of cysteine proteases, whose active site (a
cysteine residue) is essential for ICE-mediated apoptosis, appears
critical in mediating cell death (Miura et al., Cell 75:653-660
(1993)). This gene family has recently been named caspases
(Alnernri et. al. Cell, 87:171 (1996), and Thomberry et. al., J.
Biol. Chem. 272:17907-17911 (1997)) and divided into three groups
according to its known functions. Table 1 summarizes these known
caspases.
1TABLE I Enzyme* Group I: mediators of inflammation Caspase-1 (ICE)
Caspase-4 (ICE.sub.rel-II, TX, ICH-2) Caspase-5 (ICE.sub.rel-III,
TY) Group II: effectors of apoptosis Caspase-2 (ICH-1, mNEDD2)
Caspase-3 (apopain, CPP-32, YAMA) Caspase-7 (Mch-3, ICE-LAP3,
CMH-1) Group III: activators of apoptosis Caspase-6 (Mch2)
Caspase-8 (MACH, FLICE, Mch5) Caspase-9 (ICE-LAP6, Mch6)
Caspase-10
[0008] IL-1 is also a cytokine involved in mediating a wide range
of biological responses including inflammation, septic shock, wound
healing, hematopoiesis and growth of certain leukemias (Dinarello,
C. A., Blood 77:1627-1652 (1991); diGiovine et al., Immunology
Today 11:13 (1990)).
[0009] Many potent caspase inhibitors have been prepared based on
the peptide substrate structures of caspases. However, in contrast
to their potency in vitro, not too many inhibitors with good
efficacy (IC.sub.50<1 .mu.M) in whole-cell models of apoptosis
have been reported (Thormberry, N. A. Chem. Biol. 5:R97-103
(1998)). Therefore the need exists for cell death inhibitors that
are efficacy in whole-cell models of apoptosis and active in animal
model of apoptosis. These inhibitors thus can be employed as
therapeutic agents to treat disease states in which regulated cell
death and the cytokine activity of IL-1 play a role.
[0010] WO 93/05071 discloses peptide ICE inhibitors with the
formula:
Z--Q.sub.2-Asp-Q.sub.1
[0011] wherein Z is an N-terminal protecting group; Q.sub.2 is 0 to
4 amino acids such that the sequence Q.sub.2-Asp corresponds to at
least a portion of the sequence Ala-Tyr-Val-His-Asp (SEQ ID NO:1);
Q.sub.1 comprises an electronegative leaving group.
[0012] WO 96/03982 discloses aspartic acid analogs as ICE
inhibitors with the formula: 2
[0013] wherein R.sub.2 is H or alkyl; R.sub.3 is a leaving group
such as halogen; R.sub.1 is heteroaryl-CO or an amino acid
residue.
[0014] U.S. Pat. No. 5,585,357 discloses peptidic ketones as ICE
inhibitors with the formula: 3
[0015] wherein n is 0-2; each AA is independently L-valine or
L-alanine; R.sub.1 is selected from the group consisting of
N-benzyloxycarbonyl and other groups; R.sub.8, R.sub.9, R.sub.10
are each independently hydrogen, lower alkyl and other groups.
[0016] Mjalli et al. (Bioorg. Med. Chem. Lett. 3:2689-2692 (1993))
report the preparation of peptide phenylalkyl ketones as reversible
inhibitors of ICE, such as: 4
[0017] Thomberry et al. (Biochemistry 33:3934-3940 (1994)) report
the irreversible inactivation of ICE by peptide acyloxymethyl
ketones: 5
[0018] wherein Ar is COPh-2,6-(CF.sub.3).sub.2,
COPh-2,6-(CH.sub.3).sub.2, Ph--F.sub.5 and other groups.
[0019] Dolle et al. (J. Med. Chem. 37:563-564 (1994)) report the
preparation of P.sub.1 aspartate-based peptide
.alpha.-((2,6-dichlorobenz- oyl)oxy)methyl ketones as potent
time-dependent inhibitors of ICE, such as: 6
[0020] Mjalli et al. (Bioorg. Med. Chem. Lett. 4:1965-1968, (1994))
report the preparation of activated ketones as potent reversible
inhibitors of ICE: 7
[0021] wherein X is NH(CH.sub.2).sub.2, OCO(CH.sub.2).sub.2,
S(CH.sub.2).sub.3 and other groups.
[0022] Dolle et al. (J. Med. Chem. 37:3863-3866 (1994)) report the
preparation of
.alpha.-((1-phenyl-3-(trifluoromethyl)-pyrazol-5-yl)oxy)me- thyl
ketones as irreversible inhibitor of ICE, such as: 8
[0023] Mjalli et al. (Bioorg. Med. Chem. Lett. 5:1405-1408 (1995))
report inhibition of ICE by N-acyl-Aspartic acid ketones: 9
[0024] wherein XR.sub.2 is NH(CH.sub.2).sub.2Ph,
OCO(CH.sub.2).sub.2cycloh- exyl and other groups.
[0025] Mjalli et al. (Bioorg. Med. Chem. Let. 5:1409-1414 (1995))
report inhibition of ICE by N-acyl-aspartyl aryloxymethyl ketones,
such as: 10
[0026] Dolle et al. (J. Med. Chem. 38:220-222 (1995)) report the
preparation of aspartyl .alpha.-((diphenylphosphinyl)oxy)methyl
ketones as irreversible inhibitors of ICE, such as: 11
[0027] Graybill et al. (Bioorg. Med Chem. Lett. 7:41-46 (1997))
report the preparation of .alpha.-((tetronoyl)oxy)- and
.alpha.-((tetramoyl)oxy)meth- yl ketones as inhibitors of ICE, such
as: 12
[0028] Semple et al. (Bioorg. Med. Chem. Lett. 8:959-964 (1998))
report the preparation of peptidornimetic arrinomethylene ketones
as inhibitors of ICE, such as: 13
[0029] Okamoto et al. (Chem. Pharm. Bull. 47: 11-21 (1999)) report
the preparation of peptide based ICE inhibitors with the P1
carboxyl group converted to an amnide, such as: 14
[0030] EP618223 patent application discloses inhibitors of ICE as
anti-inflasuatory agents:
R--A.sub.1--A.sub.2--X--A.sub.3
[0031] wherein R is a protecting group or optionally substituted
benzyloxy; A.sub.1 is an .alpha.-hydroxy or .alpha.-amtino acid
residue or a radical of formula: 15
[0032] wherein ring A is optionally substituted by hydroxy or
C.sub.1-4 alkoxy and R.sub.a is CO or CS; A.sub.2 is an
.alpha.-hydroxy or .alpha.-amino acid residue or A.sub.1 and
A.sub.2 form together, a pseudo-dipeptide or a dipeptide mimetic
residue; X is a residue derived from Asp; A.sub.3 is
--CH.sub.2--X.sub.1--CO--Y.sub.1, --CH.sub.2--O--Y.sub.2,
--CH.sub.2--S--Y.sub.3, wherein X.sub.1 is O or S; Y.sub.1, Y.sub.2
or Y.sub.3 is cycloaliphatic residue, and optionally substituted
aryl.
[0033] WO99/18781 discloses dipeptides of formula I: 16
[0034] wherein R.sub.1 is an N-terminal protecting group; AA is a
residue of any natural or non-natural .alpha.-amino acid,
.beta.-amino acid, derivatives of an .alpha.-amino acid or
.beta.-amino acid; R.sub.2 is H or CH.sub.2R.sub.4 where R.sub.4 is
an electronegative leaving group, and R.sub.3 is alkyl or H,
provided that AA is not His, Tyr, Pro or Phe. These dipeptides are
surprisingly potent caspase inhibitors of apoptosis in cell based
systems. These compounds are systemically active in vivo and are
potent inhibitors of antiFas-induced lethality in a mouse liver
apoptosis model and have robust neuroprotective effects in a rat
model of ischemic stroke.
[0035] WO 99/47154 disclose dipeptides of formula I: 17
[0036] wherein R.sub.1 is an N-terminal protecting group; AA is a
residue of a non-natural .alpha.-amino acid or .beta.-amino acid;
R.sub.2 is an optionally substituted alkyl or H.
[0037] WO 00/01666 disclosed c-terminal modified oxamyl dipeptides
as inhibitors of the ICE/ced-3 family of cysteine proteases: 18
[0038] wherein A is a natural or unnatural amino acid; B is a
hydrogen atom, a deuterium atom, alkyl, cycloalkyl and other
groups; R.sub.1 is alkyl, cycloalkyl and other groups, R.sub.2 is
hydrogen, lower alkyl and other groups.
SUMMARY OF THE INVENTION
[0039] The invention relates to compound of Formula I: 19
[0040] or pharmaceutically acceptable salts or prodrugs thereof,
wherein:
[0041] R.sub.1 is an optionally substituted alkyl or hydrogen;
[0042] R.sub.2 is hydrogen or optionally substituted alkyl;
[0043] R.sub.3is an alkyl, saturated carbocyclic, partially
saturated carbocyclic, aryl, saturated heteroclyclic, partially
saturated heterocyclic or heteroaryl group, wherein said group is
optionally substituted;
[0044] X is O, S, NR.sub.4, or (CR.sub.4R.sub.5).sub.n, where
R.sub.4 and R.sub.5 are, at each occurrence, independently selected
from the group consisting of hydrogen, alkyl and cycloalkyl, and n
is 0, 1, 2, or 3; or
[0045] X is NR.sub.4, and R.sub.3 and R.sub.4 are taken together
with the nitrogen atom to which they are attached to form a
saturated heterocyclic, partially saturated heterocyclic or
heteroaryl group, wherein said group is optionally substituted;
or
[0046] X is CR.sub.4R.sub.5, and R.sub.3 and R.sub.4 are taken
together with the carbon atom to which they are attached to form a
saturated carbocyclic, partially saturated carbocyclic, aryl,
saturated heterocyclic, partially saturated heterocyclic or
oxygen-containing heteroaryl group, wherein said group is
optionally substituted; and
[0047] Y is a residue of a natural or non-natural amino acid;
[0048] provided that when X is O, then R.sub.3 is not unsubstituted
benzyl or t-butyl; and
[0049] when X is CH.sub.2, then R.sub.3 is not hydrogen.
[0050] The invention relates to the discovery that the compounds
represented by Formula I are inhibitors of caspases. The invention
also relates to the use of the compounds of the invention for
reducing, preventing or treating maladies in which apoptotic cell
death is either a causative factor or a result. Examples of uses
for the present invention include protecting the nervous system
following focal ischemia and global ischemia; treating
neurodegenerative disorders such as Alzheimer's disease,
Huntington's Disease, prion diseases, Parkinson's Disease, multiple
sclerosis, amyotrophic lateral sclerosis, ataxia, telangiectasia,
and spinobulbar atrophy; treating heart disease including
myocardial infarction, congestive heart failure and cardiomyopathy;
treating retinal disorders; treating autoimmune disorders including
lupus erythematosus, rheumatoid arthritis, type I diabetes,
Sjogren's syndrome and glomerulonephritis; treating polycystic
kidney disease and anemia/erythropoiesis; treating immune system
disorders, including AIDS and SCIDS; treating or ameliorating
sepsis, reducing or preventing cell, tissue and organ damage during
transplantation; reducing or preventing cell line death in
industrial biotechnology; reducing or preventing alopecia (hair
loss); and reducing the premature death of skin cells.
[0051] The present invention provides pharmaceutical compositions
comprising a compound of Formula I in an effective amount to reduce
apoptotic cell death in an animal.
[0052] The present invention also provides preservation or storage
solutions for mammalian organs or tissue, or growth media for
mammalian or yeast cells, wherein an effective amount of a compound
of Formula I is included in said solutions or media in order to
reduce apoptotic cell death in said organs, tissue or cells.
[0053] The invention also relates to the use of caspase inhibitors
for treating, ameliorating, and preventing non-cancer cell death
during chemotherapy and radiation therapy and for treating and
ameliorating the side effects of chemotherapy and radiation therapy
of cancer.
[0054] In particular, the invention relates to a method of
treating, ameliorating or preventing oral mucositis,
gastrointestinal mucositis, bladder mucositis, proctitis, bone
marrow cell death, skin cell death and hair loss resulting from
chemotherapy or radiation therapy of cancer in an animal,
comprising administering to the animal in need thereof an effective
amount of a caspase inhibitor.
DETAILED DESCRIPTION OF THE INVENTION
[0055] The inhibitors of caspases and apoptotic cell death of the
present invention are compounds having the general Formula I:
20
[0056] or pharmaceutically acceptable salts or prodrugs thereof,
wherein:
[0057] R.sub.1 is an optionally substituted alkyl or hydrogen;
[0058] R.sub.2 is hydrogen or optionally substituted alkyl;
[0059] R.sub.3 is an alkyl, saturated carbocyclic, partially
saturated carbocyclic, aryl, saturated heterocyclic, partially
saturated heterocyclic or heteroaryl group, wherein said group is
optionally substituted;
[0060] X is O, S, NR.sub.4, or (CR.sub.4R.sub.5).sub.n, where
R.sub.4 and R.sub.5 are, at each occurrence, independently selected
from the group consisting of hydrogen, alkyl and cycloalkyl, and n
is 0, 1, 2, or 3; or
[0061] X is NR.sub.4, and R.sub.3 and R.sub.4 are taken together
with the nitrogen atom to which they are attached to form a
saturated heterocyclic, partially saturated heterocyclic or
heteroaryl group, wherein said group is optionally substituted;
or
[0062] X is CR.sub.4R.sub.5, and R.sub.3 and R.sub.4 are taken
together with the carbon atom to which they are attached to form a
saturated carbocyclic, partially saturated carbocyclic, aryl,
saturated heterocyclic, partially saturated heterocyclic or
oxygen-containing heteroaryl group, wherein said group is
optionally substituted; and
[0063] Y is a residue of a natural or non-natural amino acid;
[0064] provided that when X is 0, then R.sub.3 is not unsubstituted
benzyl or t-butyl; and
[0065] when X is CH.sub.2, then R.sub.3 is not hydrogen.
[0066] With respect to R.sub.1, preferred alkyl groups are
C.sub.1-6 alkyl groups, e.g., methyl, ethyl, propyl, isopropyl,
isobutyl, pentyl and hexyl groups; and substituted C.sub.1-6 alkyl
groups, e.g., CH.sub.2OCH.sub.3 and CH.sub.2OCOCH.sub.3 (AM or
acetoxymethyl).
[0067] Preferred R.sub.2 are alkyl group substituted by
electronegative group or leaving group, including fluoromethyl,
chloromethyl, alkoxymethyl, aryloxymethyl, alkylthiomethyl,
arylthiomethyl, aminomethyl, acyloxymethyl, and arylacyloxymethyl.
Other examples of optional substituents that may be present at the
R.sub.2 alkyl group include, without limitation, 3-pyrazolyloxy
optionally substituted at the 2, 4 and 5-positions with lower
alkyl; 3-(1-phenyl-3-trifluoromethyl)pyra- zolyloxy;
2,6-di(trifluoromethyl)benzoyloxy; 2,6-dimethylbenzoyloxy;
pentafluoro-phenoxy; tetrafluorophenoxy; 2,6-dichlorobenzoyloxy;
2-(3-(2-imidazolyl)naphthyl)oxy; diphenylphosphinyloxy;
tetronyloxy; and tetramoyloxy.
[0068] The R.sub.3 group in compounds of Formula I is designed to
function as the P.sub.3 side chain in a tripeptide. Structure A is
an example of a dipeptide inhibitor of Formula I. In comparison,
structure B is an example of a tripeptide inhibitor. 21
[0069] Preferred X is O, NH and CH.sub.2. With respect to R.sub.3,
preferred alkyl are methyl, ethyl, isopropyl, isobutyl; preferred
substituents on alkyl are hydroxy, carboxy, halogen,
C.sub.4-C.sub.7 cycloalkyl, saturated and partially saturated
heterocyclic, aryl or heteroaryl; preferred cycloalkyl are
cyclopentyl and cyclohexyl; preferred saturated and partially
saturated heterocyclic groups are piperidinyl and morpholinyl;
preferred aryls are phenyl and naphthyl; preferred heteroaryls are
pyridyl, indolyl, furyl and thienyl; preferred substituents in the
aryl and heteroaryl are methyl, ethyl, chloro, fluoro, bromo,
trifluoromethyl, methoxy, hydroxy, carboxy, cyano and nitro.
[0070] With respect to Y, preferred natural and non-natural amino
acid are valine, isoleucine, leucine, proline, alanine,
phenylalanine, methionine, serine, threonine, tryptophan, tyrosine,
2-aminobutyric acid, cyclohexylglycine, phenylglycine,
cyclopentylglycine and t-butylglycine. Especially preferred amino
acids are valine, isoleucine, leucine, alanine, phenylalanine,
cyclohexylalanine, 2-aminobutyric acid, cyclohexylglycine, and
phenylglycine.
[0071] The invention relates to the discovery that the compounds
represented by Formula I are inhibitors of caspases. These
inhibitors slow or block cell death in a variety of clinical
conditions and industrial applications in which the loss of cells,
tissues or entire organs occurs. Therefore, the invention is also
related to methods of treating, preventing or reducing conditions
in which apoptosis plays a role. These conditions: are more fully
described below.
[0072] The methods comprise administering to an animal in need of
such treatment an inhibitor of the present invention, or a
pharmaceutically acceptable salt or prodrug thereof, in an amount
effective to inhibit apoptotic cell death.
[0073] Another group of preferred embodiments of the present
invention that may be employed as inhibitors of caspases are
represented by Formula II: 22
[0074] or pharmaceutically acceptable salts or prodrugs thereof
wherein R.sub.1, R.sub.2, X and Y are as defined previously with
respect to Formula I; and
[0075] A is CR.sub.6 or nitrogen;
[0076] B is CR.sub.7 or nitrogen;
[0077] C is CR.sub.8 or nitrogen;
[0078] D is CR.sub.9 or nitrogen;
[0079] E is CR.sub.10 or nitrogen; provided that not more than
three of A, B, C, D and E are nitrogen; and R.sub.6-R.sub.10
independently are hydrogen, halo, C.sub.1-C.sub.6 haloalkyl,
C.sub.6-C.sub.10 aryl, C.sub.4-C.sub.7 cycloalkyl, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl,
C.sub.6-C.sub.10 aryl(C.sub.1-C.sub.6)alkyl, C.sub.6-C.sub.10
aryl(C.sub.2-C.sub.6)alkenyl- , C.sub.6-C.sub.10
aryl(C.sub.2-C.sub.6)alkynyl, C.sub.1-C.sub.6 hydroxyalkyl, nitro,
amino, cyano, C.sub.1-C.sub.6 acylamino, hydroxy, C.sub.1-C.sub.6
acyloxy, C.sub.1-C.sub.6 alkoxy, alkylthio, or carboxy; or
[0080] one of R.sub.6 and R.sub.7, or R.sub.7 and R.sub.8, or
R.sub.8 and R.sub.9, or R.sub.9 and R.sub.10 are taken together
with the carbon atoms to which they are attached to form a
carbocycle or heterocycle.
[0081] Examples of bridges formed by R.sub.6 and R.sub.7, or
R.sub.7 and R.sub.8, or R.sub.8 and R.sub.9, or R.sub.9 and
R.sub.10 taken together are --OCH.sub.2O--, --OCF.sub.2Q--,
--(CH.sub.2).sub.3--, --CH.sub.2).sub.4--, --OCH.sub.2CH.sub.2O--,
--CH.sub.2N(RI.sub.3)CH.sub.- 2--,
--H.sub.2CH.sub.2N(R.sub.13)CH.sub.2--,
--CH.sub.2N(R.sub.13)CH.sub.2- CH.sub.2--,
--N(R.sub.13)--CH.dbd.CH--, --CH.dbd.CH--N(R.sub.13),
--O--CH.dbd.CH--, --CH.dbd.CH--O--, --S--CH.dbd.CH--,
--CH.dbd.CHS--, --N.dbd.CH--CH.dbd.CH--, --CH.dbd.N--CH.dbd.CH--,
--CH.dbd.CH--N.dbd.CH--- , --CH.dbd.CH--CH.dbd.N--,
--N.dbd.CH--CH.dbd.N--, and --CH.dbd.CH--H.dbd.CH--;
[0082] where R.sub.13 is hydrogen, alkyl or cycloalkyl;
[0083] provided that when X is O, A is CR.sub.6, B is CR.sub.7, C
is CR.sub.8, D is CR.sub.9 and E is CR.sub.10, then at least one of
the R.sub.6-R.sub.10 is not a hydrogen.
[0084] Preferred R.sub.1 is H, Me, Et, t-Bu or AM. Preferred
R.sub.2 is fluoromethyl, acyloxymethyl, arylacyloxymethyl,
aryloxymethyl, phophinyloxymethyl or aminomethyl.
[0085] Another preferred group of the inhibitors of caspases and
apoptotic cell death of the present invention are compoun ds having
the general Formula III: 23
[0086] or pharmaceutically acceptable salts or prodrugs thereof
wherein R.sub.1, R.sub.2, R.sub.3 and Y are as defined previously
with respect to Formula I.
[0087] Preferred R.sub.1 is H, Me, Et, t-Bu or AM. Preferred
R.sub.2 is fluoromethyl, acyloxymethyl, arylacyloxymethyl,
aryloxymethyl, phophinyloxymethyl or aminomethyl. Preferred R.sub.3
is optionally substituted alkyl or aryl. Preferred Y is valine,
isoleucine, leucine, alanine, phenylalanine, cyclohexylalanine,
2-aminobutyric acid, cyclohexylglycine or phenylglycine.
[0088] Exemplary preferred inhibitors of caspases having Formular I
include, without limitation:
[0089] 2-Chlorobenzyloxycarbonyl-Val-Asp-fmk,
[0090] 3-Chlorobenzyloxycarbonyl-Val-Asp-fmk,
[0091] 4-Chlorobenzyloxycarbonyl-Val-Asp-fmk,
[0092] Phenethoxycarbonyl-Val-Asp-fmk,
[0093] Cyclohexylmethoxycarbonyl-Val-Asp-fmk,
[0094] Methoxycarbonyl-Val-Asp-fmk,
[0095] Ethoxycarbonyl-Val-Asp-fmk,
[0096] Isopropyloxycarbonyl-Val-Asp-fmk,
[0097] 2-Chlorobenzyloxycarbonyl-Ile-Asp-fmk,
[0098] 3-Chlorobenzyloxycarbonyl-Ile-Asp-fmk,
[0099] 4-Chlorobenzyloxycarbonyl-Ile-Asp-fmk,
[0100] Phenylacetyl-Val-Asp-fmk,
[0101] 4-Nitrobenzyloxycarbonyl-Val-Asp-fmk,
[0102] 2,5-Dimethylbenzyloxycarbonyl-Val-Asp-fmk,
[0103] 3,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,
[0104] 3,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,
[0105] 2,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,
[0106] 2,6-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,
[0107] 2,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk,
[0108] 2,4-Dimethylbenzyloxycarbonyl-Val-Asp-fmk,
[0109] 4-Ethylbenzyloxycarbonyl-Val-Asp-fmk,
[0110] 4-Bromobenzyloxycarbonyl-Val-Asp-fmkk,
[0111] 4-Fluorobenzyloxycarbonyl-Val-Asp-fmk,
[0112] Cyclopentylmethoxycarbonyl-Val-Asp-fmk,
[0113] 4-Trifluoromethylbenzyloxycarbonyl-Val-Asp-fmk,
[0114] 3-Phenylpropionyl-Val-Asp-fmk,
[0115] Benzylarninocarbonyl-Val-Asp-fmk,
[0116] 3-Phenylpropyloxycarbonyl-Val-Asp-fmk,
[0117] 2,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk,
[0118] 3,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk,
[0119] 4-Morpholinecarbonyl-Val-Asp-fmk,
[0120] 4-Pyridylmethoxycarbonyl-Val-Asp-fmk,
[0121] 2-Pyridylmethoxycarbonyl-Val-Asp-fmk,
[0122] 2,6-Dichlorobenzyloxycarbonyl-Val-Asp-DCB-methylketone,
[0123] Isobutoxycarbonyl-Val-Asp-fmk,
[0124] Propionyl-Val-Asp-fmk,
[0125] Benzyl-glutaryl-Val-Asp-fmk,Glutaryl-Val-Asp-fmk,
[0126] 3-(2-Phenyloxyphenyl)propionyl-Val-Asp-fmk,
[0127] 3-(5-Bromo-2-hydroxyphenyl)propionyl-Val-Asp-fmk,
[0128] 3-Fluorobenzyloxycarbonyl-Val-Asp-fmk,
[0129] 2-Fluorobenzyloxycarbonyl-Val-Asp-fmk,
[0130] 3-Methylbenzyloxycarbonyl-Val-Asp-fmk,
[0131] 2-Chloro-4-fluorobenzyloxycarbonyl-Val-Asp-fmk,
[0132] 2-Naphthylmethoxycarbonyl-Val-Asp-fmk,
[0133] p-Toluenesulfonyl-Val-Asp-fmk, and
[0134] p-Toluenesulfonyl-Phe-Asp-fmk.
[0135] where fmk is fluoromethylketone and DCB is
2,6-dichlorobenzoyloxy.
[0136] Useful aryl groups are C.sub.6-14 aryl, especially
C.sub.6-10 aryl. Typical C.sub.6-14 aryl groups include phenyl,
naphthyl, phenanthrenyl, anthracenyl, indenyl, azulenyl, biphenyl,
biphenylenyl and fluorenyl groups.
[0137] Useful cycloalkyl groups are C.sub.3-8 cycloalkyl. Typical
cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl and cycloheptyl.
[0138] Useful saturated or partially saturated carbocyclic groups
are cycloalkyl groups as defined above, as well as cycloalkenyl
groups, such as cyclopentenyl, cycloheptenyl and cyclooctenyl.
[0139] Useful halo or halogen groups include fluorine, chlorine,
bromine and iodine.
[0140] Useful alkyl groups include straight-chained and branched
C.sub.1-10 alkyl groups, more preferably C.sub.1-6 alkyl groups.
Typical C.sub.1-10 alkyl groups include methyl, ethyl, propyl,
isopropyl, butyl, sec-butyl, tert-butyl, 3-pentyl, hexyl and octyl
groups. Also contemplated is a trimethylene group substituted on
two adjoining positions on the benzene ring of the compounds of the
invention.
[0141] Useful arylalkyl groups include any of the above-mentioned
C.sub.1-10 alkyl groups substituted by any of the above-mentioned
C.sub.6-14 aryl groups. Useful values include benzyl, phenethyl and
naphthylmethyl.
[0142] Useful haloalkyl groups include CI-1.sub.0 alkyl groups
substituted by one or more fluorine, chlorine, bromine or iodine
atoms, e.g., fluoromethyl, difluoromethyl, trifluoromethyl,
pentafluoroethyl, 1,1-difluoroethyl, chloromethyl,
chlorofluoromethyl and trichloromethyl groups.
[0143] Useful alkoxy groups include oxygen substituted by one of
the C.sub.1-10 alkyl groups mentioned above.
[0144] Useful alkylthio groups include sulphur substituted by one
of the C.sub.1-10 alkyl groups mentioned above. Also included are
the sulfoxides and sulfones of such alkylthio groups.
[0145] Useful acylamino groups are any C.sub.1-6 acyl (alkanoyl)
attached to an amino nitrogen, e.g., acetamido, propionamido,
butanoylamido, pentanoylamido, hexanoylarnido as well as
aryl-substituted C.sub.2-6 substituted acyl groups.
[0146] Useful acyloxy groups are any C.sub.1-6 acyl (alkanoyl)
attached to an oxy (--O--) group, e.g., formyloxy, acetoxy,
propionoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy and the
like.
[0147] Useful arylacyloxy groups include any of the aryl groups
mentioned above substituted on any of the acyloxy groups mentioned
above, e.g., 2,6-dichlorobenzoyloxy, 2,6-difluorobenzoyloxy and
2,6-di-(trifluoromethyl)-benzoyloxy groups.
[0148] Useful amino groups include --NH.sub.2, --NHR.sub.11, and
--NR.sub.11R.sub.12, wherein R.sub.11 and R.sub.12 are C.sub.1-10
alkyl or cycloalkyl groups as defined above.
[0149] Useful saturated or partially saturated heterocyclic groups
include tetrahydrofuranyl, pyranyl, piperidinyl, piperizinyl,
pyrrolidinyl, imidazolidinyl, imidazolinyl, indolinyl,
isoindolinyl, quinuclidinyl, morpholinyl, isochromanyl, chromanyl,
pyrazolidinyl pyrazolinyl, tetronoyl and tetramoyl groups.
[0150] Useful heteroaryl groups include any one of the following:
thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl,
furyl, pyranyl, isobenzofuranyl, chromenyl, coumarinyl, xanthenyl,
phenoxanthiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl,
pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,
4H-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl,
naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl, acrindinyl, perimidinyl,
phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,
isoxazolyl, furazanyl, phenoxazinyl,
1,4-dihydroquinoxaline-2,3-dione, 7-aminoisocoumarin,
pyrido[1,2-a]pyrimidin4-one, 1,2-benzoisoxazol-3-yl,
benzimidazolyl, 2-oxindolyl and 2-oxobenzimidazolyl. Where the
heteroaryl group contains a nitrogen atom in a ring, such nitrogen
atom may be in the form of an N-oxide, e.g., a pyridyl N-oxide,
pyrazinyl N-oxide, pyrimidinyl N-oxide and the like.
[0151] Optional substituents include one or more alkyl; halo;
haloalkyl; cycloalkyl; hydroxy; carboxy; phosphinyloxy; aryl
optionally substituted with one or more lower alkyl, halo, amino,
alkylamino, dialkylamino, alkoxy, hydroxy, carboxy, haloalkyl or
heteroaryl groups; aryloxy optionally substituted with one or more
lower alkyl, halo, amino, alkylamino, dialkylamino, aikoxy,
hydroxy, carboxy, haloalkyl or heteroaryl groups; aralkyl;
heteroaryl optionally substituted with one or more lower alkyl,
halo, amino, alkylamino, dialkylamino, alkoxy, hydroxy, carboxy,
haloalkyl and aryl groups; heteroaryloxy optionally substituted
with one or more lower alkyl, halo, amino, alkylamino,
dialkylamino, alkoxy, hydroxy, carboxy, haloalkyl and aryl groups;
alkoxy; alkylthio; arylthio; amino; alkylamino; dialkylamino;
acyloxy; arylacyloxy optionally substituted with one or more lower
alkyl, halo, amino, alkylamino, dialkylamino, alkoxy, hydroxy,
carboxy, haloalkyl and aryl groups; diphenylphosphinyloxy
optionally substituted with one or more lower alkyl, halo, amino,
alkylamino, dialkylamino, alkoxy, hydroxy, carboxy, or haloalkyl
groups; heterocyclo optionally substituted with one or more lower
alkyl, halo, amino, alkylamino, dialkylamino, alkoxy, hydroxy,
carboxy, haloalkyl and aryl groups; heterocycloalkyloxy optionally
substituted with one or more lower alkyl, halo, amino, alkylamino,
dialkylamino, alkoxy, hydroxy, carboxy, haloalkyl and aryl groups;
partially saturated heterocycloalkyl optionally substituted with
one or more lower alkyl, halo, amino, alkylamino, dialkylamino,
alkoxy, hydroxy, carboxy, haloalkyl and aryl groups; or partially
saturated heterocycloalkyloxy optionally substituted with one or
more lower alkyl, halo, amino, alkylamino, dialkylamino, alkoxy,
hydroxy, carboxy, haloalkyl and aryl groups.
[0152] Certain of the compounds of the present invention may exist
as stereoisomers including optical isomers. The invention includes
all stereoisomers and both the racemic mixtures of such
stereoisomers as well as the individual enantiomers that may be
separated according to methods that are well known to those of
ordinary skill in the art.
[0153] Examples of pharmaceutically acceptable addition salts
include inorganic and organic acid addition salts such as
hydrochloride, hydrobromide, phosphate, sulphate, citrate, lactate,
tartrate, maleate, fumarate, mandelate and oxalate; and inorganic
and organic base addition salts with bases such as sodium hydroxy
and Tris(hydroxymethyl)aminometha- ne (TRIS, tromethane).
[0154] Examples of prodrugs include compounds of Formula I wherein
R.sub.1 is an alkyl group or substituted alkyl group such as
CH.sub.2OCH.sub.3 and CH.sub.2OCOCH.sub.3 (AM ester).
[0155] The invention is also directed to a method for treating
disorders responsive to the inhibition of caspases in animals
suffering thereof. Particular preferred embodiments of compounds
for use in the method of this invention are represented by
previously defined Formula I.
[0156] The compounds of this invention may be prepared using
methods known to those skilled in the art. Specifically, compounds
with Formula I can be prepared as illustrated by exemplary
reactions in Scheme 1. The intermediate 1 was prepared according to
Revesz et al. (Tetrahedron Lett. 35:9693-9696 (1994)). Coupling of
1 with a N-protected amino acid, such as
(2-chlorobenzyloxycarbonyl)-Val, which was prepared from
2-chlorobenzyl chloroformate and Valine, gave amide 2. Oxidation of
2 by Dess-Martin reagent according to Revesz et al. (Tetrahedron
Lett. 35:9693-9696 (1994)) gave 3 as a mixture of diastereomers.
The oxidation also can be done using other agents such as
pyridinium chlorochromate (PCC) or pyridinium dichromate (PDC).
Acid catalyzed cleavage of the ester gave the free acid 4. 24
[0157] Other N-protected amino acid can be prepared as illustrated
by exemplary reactions in Scheme 2-4. 25 26
[0158] Compounds of Formula III with substituted-sulfonyl as
N-protecting group can be prepared similar to what described in
Scheme. 1. Examples of sulfonyl protected amino acids which can be
used for the preparation of novel caspase inhibitors of Formula Im
are shown in Scheme 5. 27
[0159] Other N-protecting group with special function also can be
used. For instance, an antioxidant such as Trolox can be introduced
as the protecting group. The compound can be prepared as shown in
Scheme 6. Alternatively, the compound can be prepared as shown in
Scheme 7. The compound will combine the property of a caspase
inhibitor with an antioxidant, which might be more efficacious as a
neuroprotectant for the treatment of stroke (Pierre-Etienne
Chabrier et al., PNAS 96:10824-10829 (1999)). 28
[0160] A fluorescent dye also can be introduced as the protecting
group, such as the compounds shown in Scheme 8. 29
[0161] These compounds can inhibit caspase and resulted in the
attachment of the fluorescent dye to the caspase. Therefore these
molecules should be useful for the labeling of caspase and
detection of caspase activity in the cells. These compounds can be
prepared as illustrated by exemplary reactions in Scheme 9. 30
[0162] Preferred fluorescent protecting groups of formula
R.sub.3--X--C(O)-- include, for example: 31
[0163] An important aspect of the present invention is the
discovery that compounds having Formula I are inhibitors of
caspases. Therefore, these inhibitors are expected to slow or block
cell death in a variety of clinical conditions in which the loss of
cells, tissues or entire organs occurs.
[0164] The cell death inhibitors of the present invention can be
used to reduce or prevent cell death in the nervous system (brain,
spinal cord, and peripheral nervous system) under various
conditions of ischemia and excitotoxicity, including, but not
limited to, focal ischemtia due to stroke and global ischemia due
to cardiac arrest, as well as spinal cord injury (Emery et al., J.
Neurosurgery 89:911-920 (1998)). One particular usage is to treat
the effects of oxygen deprivation which can occur during the birth
of infants in high-risk labors or drowning. The cell death
inhibitors can also be used to reduce or prevent cell death in the
nervous system due to traumatic injury (such as head trauma), viral
infection or radiation-induced nerve cell death (for example, as a
side-effect of cancer radiotherapy), as well as acute bacterial
meningitis (Braun et al., Nat Med 5:298-302 (1999)). The cell death
inhibitors can also be used to reduce or prevent cell death in a
range of neurodegenerative disorders, including but not limited to
Alzh&imer's disease (Mattson et al., Brain Res. 807:167-176
(1998)), Huntington's Disease, Parkinson's Disease, multiple
sclerosis, amyotrophic lateral sclerosis, and spinobulbar atrophy.
The in vivo neuroprotective properties of cell death inhibitors of
the invention can be tested in a rat transient focal brain ischemia
model (Xue et al., Stroke 21:166 (1990)). The cell death inhibitors
may also be used to treat or ameliorate cell death in acute
bacterial meningitis (Braun et al., Nat Med 5:298-302 (1999))
[0165] The cell death inhibitors of the invention can be used to
reduce or prevent cell death in any condition which potentially
results in the death of cardiac muscle (Black et al., J. Mol. Cel.
Card. 30:733-742 (1998) and Maulik et al., Free Radic. Biol. Med.
24:869-875 (1998)). This includes myocardial infarction due to
myocardial ischemia and reperfusion, congestive heart failure and
cardiomyopathy. One particular application is to reduce or prevent
myocardial cell death as occurs in certain viral infections of the
heart.
[0166] The in vivo activity of the cell death inhibitors of the
invention can be tested using the "mouse liver apoptosis" model
described by Rodriguez et al. (J. Exp. Med. 184:2067-2072 (1996)).
In this model, mice are treated intravenously (IV) with an antiFas
antibody which induces massive apoptosis in the liver and other
organs, leading to generalized organ failure and death. This model
is useful for indirectly testing the systemic bioavailability of
the cell death inhibitors of the invention, as well as their in
vivo anti-apoptotic properties. The cell death inhibitors of the
invention therefore can be used to reduce or prevent apoptosis of
liver cells (Jones et al., Hepatology 27:163242 (1998)) such as in
sepsis (Jaeschke et al., J. Immunol. 160:3480-3486 (1998)) and
hereditary tyrosinemia type 1 (HT1) (Kubo et al., Prov. Natl. Acad.
Sci. USA 95:9552-9557 (1998)). The cell death inhibitors of the
invention also can be used to treat hepatitis (Suzuki, Proc. Soc.
Exp. Biol. Med. 217:450-454 (1998)).
[0167] The cell death inhibitors of the invention can be used to
reduce or prevent cell death of retinal neurons (Kermer et al., J.
Neurosci. 18:4656-4662 (1998) and Miller et al., Am. J. Vet. Res.
59:149-152 (1998)) as can occur in disorders which increase
intraocular pressure (such as glaucoma) or retinal disorders
associated with the aging process (such as age-related macular
degeneration). The inhibitors can also be used to treat hereditary
degenerative disorders of the retina, such as retinitis
pigmentosa.
[0168] The cell death inhibitors of the invention can also be used
to reduce or prevent cell death in the kidney. This includes renal
amyloidosis (Hiraoka et al., Nippon Jinzo Gakkai Shi. 40:276-83
(1998)), acute renal failure (Lieberthal et al., Semin Nephrol.
18:505-518 (1998)), murine tubular epithelial cell death induced by
cyclosporine A (Ortiz et al., Kidney International Supp. 68:S25-S29
(1998)) and HIV-induced nephropathy (Conaldi et al., J. Clin.
Invest. 102:2041-2049 (1998)).
[0169] The cell death inhibitors of the invention can also be used
to reduce or prevent cell death of buccal mucosa due to chronic
alcohol ingestion (Slomiany et al., Biochem. Mol. Biol. Int.
45:1199-1209 (1998)).
[0170] The cell death inhibitors of the invention can also be used
to reduce or prevent cell death in plants (Richberg et al., Curr.
Opin. Plant Biol. 1:480-485 (1998)), such as plant cell death due
to pathogens (Pozo et al., Curr. Biol. 8:1129-1132 (1998) and
Greenberg et al., Cell 77:551-563 (1994)).
[0171] The cell death inhibitors of the invention can also be used
to reduce or prevent cell death due to radiation and
ultraviolet-irradiation (Sheikh et al., Oncogene 17:2555-2563
(1998)).
[0172] The cell death inhibitors of the invention can also be used
to reduce or prevent apoptotic death of bone marrow cells in
myelodysplastic syndromes (MDS) (Mundle et al., Am. J. Hematol.
60:36-47 (1999)).
[0173] The cell death inhibitors of the invention can also be used
to reduce or prevent premature death of cells of the immune system,
and are particularly useful in treating immune deficiency
disorders, such as acquired immune deficiency syndrome (AIDS),
severe combined immune deficiency syndrome (SCIDS) and related
diseases. The cell death inhibitors can also be used to treat
radiation-induced immune suppression.
[0174] Transplantation of human organs and tissues is a common
treatment for organ failure. However, during the transplantation
process, the donor organ or tissue is at risk for cell death since
it is deprived of its normal blood supply prior to being implanted
in the host. This ischemic state can be treated with cell death
inhibitors by infusion into the donor organ or tissue, or by direct
addition of the cell death inhibitors to the organ/tissue storage
medium. For example, it was reported that treatment of the
embryonic nigral cell suspension with Ac-YVAD-cmk (SEQ ID NO:2), a
caspase-I inhibitor, mitigated DNA fragmentation and reduced
apoptosis in transplants. It also increased survival of
dopaminergic neurons grafted to hemiparkinsonian rats, and thereby
substantially improved functional recovery (Schierle et al., Nat.
Med. 5:97-100 (1999)). Cell death inhibitors may also be used to
reduce or prevent cell death in the donor organ/tissue after it has
been transplanted to protect it from the effects of reperfusion
injury and /or effects of host immune cells which kill their
targets by triggering apoptosis. The cytoprotective effects of cell
death inhibitors can also be used to prevent the death of human or
animal sperm and eggs used in in vitro fertilization procedures.
These inhibitors can be used during the harvesting process and can
also be included in the storage medium.
[0175] Mammalian cell lines, insect cells and yeast cells are
commonly used to produce large amounts of recombinant proteins
(such as antibodies, enzymes or hormones) for industrial or
medicinal use. The lifespan of some of these cell lines is limited
due to growth conditions, the nature of the recombinant molecule
being expressed (some are toxic) and other unknown factors. The
lifespans of industrial cell lines can be extended by including
these cell death inhibitors in the growth medium in a concentration
range of 1-100 .mu.M.
[0176] The factors governing hair growth and loss are largely
unknown. There is some evidence, however, that hair follicle
regression (referred to as catagen) may be due at least partially
to apoptosis. Therefore, it is contemplated that the cell death
inhibitors of the present invention can be used to treat hair loss
that occurs due to various conditions, including but not limited to
male-pattern baldness, radiation-induced or chemotherapy-induced
hair loss, and hair loss due to emotional stress. There is also
evidence that apoptosis may play a role in the loss of hair color.
Therefore, it is contemplated that the cell death inhibitors of the
present invention can also be used in treating or preventing cases
of premature graying of the hair.
[0177] The death of skin epithelial cells can occur after exposure
to high levels of radiation, heat or chemicals. It is contemplated
that the cell death inhibitors of the present invention can be used
to treat, reduce or prevent this type of skin damage. In one
particular application, the cell death inhibitors can be applied as
part of a topical formulation, e.g., an ointment, to treat acute
over-exposure to the sun and to prevent blistering and peeling of
the skin.
[0178] Goldberg et al. (Nature Genetics 13: 442-449 (1996))
reported recently that huntingtin, a protein product of
Huntington's disease (HD) gene, can be cleaved by CPP32 but not
ICE. The mutation underlying HD is an expansion of a CAG
trinucleotide at the 5' end of the HD gene. The trinucleotide
expansion exceeding 36 repeats is associated with the clinical
presentation of HD. The CAG expansion promotes cleavage of
huntingtin by CPP32, thus links the role of CPP32 in the apoptotic
cell death in HD. Compounds of the present invention with CPP32
inhibiting activity will be useful in blocking CPP32 induced
apoptotic cell death, thus in preventing and treating HD and other
disorders characterized by expansion of trinucleotide repeats such
as myotonic dystrophy, fragile X mental retardation, spinobulbar
muscular atrophy, spinocerebellar atoxia type I and Dentato-Rubro
pallidoluysian atrophy.
[0179] The invention relates to a method of treating, ameliorating
or preventing oral mucositis, gastrointestinal mucositis, bladder
mucositis, proctitis, bone marrow cell death, skin cell death and
hair loss resulting from chemotherapy or radiation therapy of
cancer in an animal, comprising administering to the animal in need
thereof an effective amount of a cell death inhibitor of the
present invention.
[0180] When animals are treated with chemotherapeutic agents and/or
radiation to kill cancer cells, an unwanted side effect is the
apoptotic death of rapidly dividing non-cancer cells. Such
non-cancer cells include cells of the gastrointestinal tract, skin,
hair, and bone marrow cells. According to the present invention,
caspase inhibitors are administered to such non-cancer cells to
prevent apoptosis of such cells. In a preferred embodiment, the
caspase inhibitors are administered locally, e.g., to the
gastrointestinal tract, mouth, skin or scalp to prevent apoptosis
of the gastrointestinal, mouth, skin or hair cells but allowing for
the death of the cancer cells. Thus, in one example, it is possible
to treat brain cancer with chemotherapy or radiation therapy and
protect the outer skin, hair cells, gastrointestinal tract and bone
marrow by local administration of a caspase inhibitor. In the case
of oral mucositis, the caspase inhibitor can be applied, for
example, in the form of a mouth wash or mouth rinse, in a gel, or.
in the form of an oral slow release lozenge to prevent activation
of caspases and apoptotic cell death induced by the
chemotherapeutic agent or by radiation. In the case of
gastrointestinal mucositis, the caspase inhibitor can be applied in
a form such that it is not absorbed systemically or in a form that
coats the surface of the gastrointestinal tract, or a suppository
formulation for the treatment of gastrointestinal mucositis. In the
case of proctitis, the capsase inhibitor may be applied as part of
an enema or suppository. In the case of bladder mucositis, the
caspase inhibitor may be applied though a bladder catheter. For
prevention of radiation or chemotherapy-induced hair loss, the
caspase inhibitor can be applied, for example, to the scalp in the
form of a hair rinse, hair gel, shampoo or hair conditioner.
Importantly, the caspase inhibitor can be applied prior to the
administration of the chemotherapeutic agent or radiation, thus
preventing the onset of the damaging effects of the
chemotherapeutic agent or radiation to the normal cells.
[0181] Compositions within the scope of this invention include all
compositions wherein the compounds of the present invention are
contained in an amount which is effective to achieve its intended
purpose. While individual needs vary, determination of optimal
ranges of effective amounts of each component is within the skill
of the art. Typically, the compounds may be administered to
mammals, e.g., humans, orally at a dose of 0.0025 to 50 mg/kg, or
an equivalent amount of the pharmaceutically acceptable salt
thereof, per day of the body weight of the mammal being treated for
apoptosis-mediated disorders, e.g., neuronal cell death, heart
disease, retinal disorders, polycystic kidney disease, immune
system disorders and sepsis. Preferably, about 0.01 to about 10
mg/kg is orally administered to treat or prevent such disorders.
For intramuscular injection, the dose is generally about one-half
of the oral dose. For example, for treatment or prevention of
neuronal cell death, a suitable intramuscular dose would be about
0.0025 to about 25 mg/kg, and most preferably, from about 0.01 to
about 5 mg/kg.
[0182] The unit oral dose may comprise from about 0.01 to about 50
mg, preferably about 0.1 to about 10 mg of the compound. The unit
dose may be administered one or more times daily as one or more
tablets each containing from about 0.1 to about 10, conveniently
about 0.25 to 50 mg of the compound or its solvates.
[0183] In a topical formulation, the compound may be present at a
concentration of about 0.01 to 100 mg per gram of carrier. In a
preferred embodiment, the compound is present at a concentration of
about 0.07-1.0 mg/ml, more preferably, about 0.1-0.5 mg/ml, most
preferably, about 0.4 mg/ml.
[0184] In addition to administering the compound as a raw chemical,
the compounds of the invention may be administered as part of a
pharmaceutical preparation containing suitable pharmaceutically
acceptable carriers comprising excipients and auxiliaries which
facilitate processing of the compounds into preparations which can
be used pharmaceutically. Preferably, the preparations,
particularly those preparations which can be administered orally or
topically and which can be used for the preferred type of
administration, such as tablets, dragees, slow release lozenges and
capsules, mouth rinses and mouth washes, gels, liquid suspensions,
hair rinses, hair gels, shampoos and also preparations which can be
administered rectally, such as suppositories, as well as suitable
solutions for administration by injection, topically or orally,
contain from about 0.01 to 99 percent, preferably from about 0.25
to 75 percent of active compound(s), together with the
excipient.
[0185] Also included within the scope of the present invention are
the non-toxic pharmaceutically acceptable salts of the compounds of
the present invention. Acid addition salts are formed by mixing a
solution of the particular cell death inhibitors of the present
invention with a solution of a pharmaceutically acceptable
non-toxic acid such as hydrochloric acid, fumaric acid, maleic
acid, succinic acid, acetic acid, citric acid, tartaric acid,
carbonic acid, phosphoric acid, oxalic acid, and the like. Basic
salts are formed by mixing a solution of the particular cell death
inhibitors of the present invention with a solution of a
pharmaceutically acceptable non-toxic base such as sodium
hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate
Tris and the like.
[0186] The pharmaceutical compositions of the invention may be
administered to any animal which may experience the beneficial
effects of the compounds of the invention. Foremost among such
animals are mammals, e.g., humans, although the invention is not
intended to be so limited.
[0187] The caspase inhibitors and pharmaceutical compositions
thereof may be administered by any means that achieve their
intended purpose. For example, administration may be by parenteral,
subcutaneous, intravenous, intramuscular, intraperitoneal,
transdermal, buccal, intrathecal, intracranial, intranasal or
topical routes. Alternatively, or concurrently, administration may
be by the oral route. The dosage administered will be dependent
upon the age, health, and weight of the recipient, kind of
concurrent treatment, if any, frequency of treatment, and the
nature of the effect desired. In general, the caspase inhibitors
are administered locally to the tissues that are to be protected
from apoptosis and separately from the chemotherapeutic agent. For
example, cisplatin may be administered by i.v. injection to treat a
cancer such as brain, lung, breast, liver, kidney, pancreatic,
ovarian, prostatic cancer, and the caspase inhibitor administered
locally to treat, ameliorate, or prevent apototic cell death in the
mouth or gastrointestinal tract, such as a mouth wash for the
treatment of oral mucositis; and IV injectable aqueous solution for
the treatment of bone marrow cell death; and an oral formulation
suitable for coating the gastrointestinal surfaces or an emema or
suppository formulation for the treatment of gastrointestinal
mucositis including proctitis. The caspase inhibitors may also be
applied through a bladder catheter for the treatment, amelioration
or prevention of bladder mucositis. Alternatively or concurrently,
the caspase inhibitors may be applied topically to the skin and/or
scalp to treat, ameliorate or prevent apoptotic cell death of hair
and skin cells. In a further embodiment, the chemotherapeutic agent
or radiation may be applied locally to treat a localized cancer
such as brain, lung, breast, liver, kidney, pancreatic, ovarian,
prostatic cancer, and the caspase inhibitor administered
systemically, e.g., by i.v. injection, to treat, ameliorate or
prevent apoptotic cell death of the gastrointestinal tract cells,
mouth epithelial cells, bone marrow cells, skin cells and hair
cells. In the case of oral mucositis in brain cancer treatment, for
example, a caspase inhibitor that does not penetrate the
blood-brain barrier can be applied, for example, systemically by
i.v. injection followed by irradiation of the brain tumor. This
would protect the oral mucosa from the harmful effects of radiation
but the caspase inhibitor would not protect the brain tumor from
the therapeutic effects of radiation. Importantly, the caspase
inhibitor can be applied prior to administration of the radiation,
thus preventing the onset of the damaging effects of the radiation
to the normal mucosa cells.
[0188] The pharmaceutical preparations of the present invention are
manufactured in a manner which is itself known, for example, by
means of conventional mixing, granulating, dragee-making,
dissolving, or lyophilizing processes. Thus, pharmaceutical
preparations for oral use can be obtained by combining the active
compounds with solid excipients, optionally grinding the resulting
mixture and processing the mixture of granules, after adding
suitable auxiliaries, if desired or necessary, to obtain tablets or
dragee cores.
[0189] Suitable excipients are, in particular, fillers such as
saccharides, for example lactose or sucrose, mannitol or sorbitol,
cellulose preparations and/or calcium phosphates, for example
tricalcium phosphate or calcium hydrogen phosphate, as well as
binders such as starch paste, using, for example, maize starch,
wheat starch, rice starch, potato starch, gelatin, tragacanth,
methyl cellulose, hydroxypropylmethylcellulose, sodium
carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,
disintegrating agents may be added such as the above-mentioned
starches and also carboxymethyl-starch, cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof, such as
sodium alginate. Auxiliaries are, above all, flow-regulating
agents. and lubricants, for example, silica, talc, stearic acid or
salts thereof, such as magnesium stearate or calcium stearate,
and/or polyethylene glycol. Dragee cores are provided with suitable
coatings which, if desired, are resistant to gastric juices. For
this purpose, concentrated saccharide solutions may be used, which
may optionally contain gum arabic, talc, polyvinyl pyrrolidone,
polyethylene glycol and/or titanium dioxide, lacquer solutions and
suitable organic solvents or solvent mixtures. In order to produce
coatings resistant to gastric juices, solutions of suitable
cellulose preparations such as acetylcellulose phthalate or
hydroxypropymethyl-cellulose phthalate, are used. Dye stuffs or
pigments may be added to the tablets or dragee coatings, for
example, for identification or in order to characterize
combinations of active compound doses.
[0190] Other pharmaceutical preparations which can be used orally
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer such as glycerol or
sorbitol. The push-fit capsules can contain the active compounds in
the form of granules which may be mixed with fillers such as
lactose, binders such as starches, and/or lubricants such as talc
or magnesium stearate and, optionally, stabilizers. In soft
capsules, the active compounds are preferably dissolved or
suspended in suitable liquids, such as fatty oils, or liquid
paraffin. In addition, stabilizers may be added.
[0191] Possible pharmaceutical preparations which can be used
rectally include, for example, enemas or suppositories, which
consist of a combination of one or more of the active compounds
with a suppository base. Suitable suppository bases are, for
example, natural or synthetic triglycerides, or paraffin
hydrocarbons. In addition, it is also possible to use gelatin
rectal capsules which consist of a combination of the active
compounds with a base. Possible base materials include, for
example, liquid triglycerides, polyethylene glycols, or paraffin
hydrocarbons.
[0192] Suitable formulations for parenteral administration include
aqueous solutions of the active compounds in water-soluble form,
for example, water-soluble salts and alkaline solutions. In
addition, suspensions of the active compounds as appropriate oily
injection suspensions may be administered. Suitable lipophilic
solvents or vehicles include fatty oils, for example, sesame oil,
or synthetic fatty acid esters, for example, ethyl oleate or
triglycerides or polyethylene glycol400 (the compounds are soluble
in PEG-400). Aqueous injection suspensions may contain substances
which increase the viscosity of the suspension include, for
example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
Optionally, the suspension may also contain stabilizers.
[0193] In accordance with one aspect of the present invention,
compounds of the invention are employed in topical and parenteral
formulations and are used for the treatment of skin damage, such as
that caused by exposure to high levels of radiation, including
ultraviolet radiation, heat or chemicals.
[0194] One or more additional substances which have therapeutic
effects on the skin may also be incorporated in the compositions.
Thus, the composition may also contain one or more compounds
capable of increasing cyclic-AMP levels in the skin. Suitable
compounds include adenosine or a nucleic acid hydrolysate in an
amount of about 0.1-1% and papaverine, in an amount of about
0.5-5%, both by weight based on the weight of the composition. Also
suitable are 0-adrenergic agonists such as isoproterenol, in an
amount of about 0.1-2% or cyclic-AMP, in an amount of about 0.1-1%,
again both by weight based on the weight of the composition. Other
suitable types of additional active ingredients which may be
incorporated in the compositions of this invention include any
compounds known to have a beneficial effect on skin. Such compounds
include-retinoids such as Vitamin A, in an amount of about
0.003-0.3% by weight and chromanols such as Vitamin E or a
derivative thereof in an amount of about 0.1-10% by weight, both
based on the weight. of the composition. Additionally,
anti-inflammatory agents and keratoplastic agents may be
incorporated in the cosmetic composition. A typical
anti-inflammatory agent is a corticosteroid such as hydrocortisone
or its acetate in an amount of about 0.25-5% by weight, or a
corticosteroid such as dexamethasone in an amount of about
0.025-0.5% by weight, both based on the weight of the composition.
A typical keratoplastic agent is coal tar in an amount of about
0.1-20% or anthralin in an amount of about 0.05-2% by weight, both
based on the weight of the composition.
[0195] The topical compositions of this invention are formulated
preferably as oils, creams, lotions, ointments and the like by
choice of appropriate carriers. Suitable carriers include vegetable
or mineral oils, white petrolatum (white soft paraffin), branched
chain fats or oils, animal fats and high molecular weight alcohol
(greater than C.sub.12). The preferred carriers are those in which
the active ingredient is soluble. Emulsifiers, stabilizers,
humectants and antioxidants may also be included as well as agents
imparting color or fragrance, if desired. Additionally, transdermal
penetration enhancers can be employed in these topical
formulations. Examples of such enhancers can be found in U.S. Pat.
Nos. 3,989,816 and 4,444,762.
[0196] Creams are preferably formulated from a mixture of mineral
oil, self-emulsifying beeswax and water in which mixture the active
ingredient, dissolved in a small amount of an oil such as almond
oil, is admixed. A typical example of such a cream is one which
includes about 40 parts water, about 20 parts beeswax, about 40
parts mineral oil and about 1 part almond oil.
[0197] Ointments may be formulated by mixing a solution of the
active ingredient in a vegetable oil such as almond oil with warm
soft paraffin and allowing the mixture to cool. A typical example
of such an ointment is one which includes about 30% almond oil and
about 70% white soft paraffm by weight.
[0198] Lotions may be conveniently prepared by dissolving the
active ingredient, in a suitable high molecular weight alcohol such
as propylene glycol or polyethylene glycol.
[0199] In addition, these compositions may include other medicinal
agents, growth factors, wound sealants, carriers, etc., that are
known or apparent to those skilled in the art. The compositions of
the invention are administered to a warm-blooded animal, such as
human, already suffering from a skin damage, such as a burn, in an
amount sufficient to allow the healing process to proceed more
quickly than if the host were not treated. Amounts effective for
this use will depend on the severity of the skin damage and the
general state of health of the patient being treated. Maintenance
dosages over a prolonged period of time may be adjusted as
necessary. For veterinary uses, higher levels may be administered
as necessary.
[0200] In the case of an animal suffering from decreased hair
growth, the compositions of the invention are administered in an
amount sufficient to increase the rate of hair growth. Amounts
effective for this use will depend on the extent of decreased hair
growth, and the general state of health of the patient being
treated. Maintenance dosages over a prolonged period of time may be
adjusted as necessary. For veterinary uses, higher levels may be
administered as necessary.
[0201] When the compounds are to be administered to plants, they
may be applied to the leaves and/or stems and/or flowers of the
plant, e.g., by spraying. The compounds may be spayed in
particulate form or dissolved or suspended in an appropriate
carrier, e.g., in water or an oil-water emulsion. The compounds may
also be combined with the soil of the plant. In this embodiment,
the compounds are taken up by the roots of the plant.
[0202] In a preferred embodiment, the caspase inhibitor is
formulated as part of a mouthwash for the treatment, amelioration
or prevention of oral mucositis. Such mouthwashes are aqueous
solutions of the caspase inhibitor which may also contain alcohol,
glycerin, synthetic sweeteners and surface-active, flavoring and
coloring agents. They may also contain anti-infective agents such
as hexetidine and cetylpyridinium chloride. The mouthwashes may
also contain topical anesthetics (e.g., benzocaine, cocaine,
dyclonine hydrochloride, lidocaine, proparacaine hydrochloride or
teracaine hydrochloride), for example, for relieving pain of
radiation or chemotherapy-induced sores. The mouth washes may have
either acidic or basic pH. See Remington's Pharmaceutical Sciences,
A. R. Gennaro (ed.), Mack Publishing Company, pp. 1045, 1046, 1526
and 1965 (1990).
[0203] In another preferred embodiment, the caspase inhibitor is
formulated as an oral formulation which is capable of coating the
gastrointestinal surfaces for the treatment, amelioration or
prevention of gastrointestinal mucositis. Examples of
gastrointestinal mucositis include esophageal mucositis, gastric
mucositis, and intestinal mucositis. Such formulations may comprise
gastric antacids such as aluminum carbonate, aluminum hydroxide
gel, bismuth subnitrate, bismuth subsalicylate, calcium carbonate,
dihydroxyaluminum sodium carbonate, magaldrate, magnesium
carbonate, magnesium hydroxide, magnesium oxide, sodium
bicarbonate, milk of bismuth, dihydroxyaluminum aminoacetate,
magnesium phosphate, magnesium trisilicate and mixtures thereof.
Other additives include without limitation H.sub.2-receptor
antagonists, digestants, anti-emetics, adsorbants, and
miscellaneous agents. See Remington's Pharmaceutical Sciences, A.
R. Gennaro (ed.), Mack Publishing Company, pp. 774-778 (1990).
[0204] Chemotherapy agents such as cisplatin and radiation therapy
often induce early and late onset emesis in the patient. Thus, in
one embodiment an antiemetic is coadminstered together with the
caspase inhibitor to avoid emesis and retain contact of the caspase
inhibitor with the gastrointestinal tract. Examples of such
antiemetics include without limitation compounds that block the
dopaminergic emetic receptors such as metoclopramide and
trimethobenzamide, and cannabinoids. Metocloprarnide may be
administered orally prior to and/or during chemotherapy/radiation
therapy/caspase inhibitor therapy to prevent the early emesis
response and then later by intranasal administration according to
U.S. Pat. Nos. 5,760,086 and 4,536,386 to prevent delayed onset
emesis. During the period after chemotherapy/radiation therapy,
both the caspase inhibitor and the antiemetic may be coadministered
to treat, ameliorate or prevent gastrointestinal mucositis.
[0205] In a further embodiment, the caspase inhibitor may be
formulated as an IV injectable solution for the treatment,
amelioration or prevention of bone marrow cell death.
[0206] The compositions of the invention may be administered to a
warm-blooded animal, such as human, already suffering from
chemotherapy or radiation therapy-induced non-cancer cell death,
or, more preferably, before or during therapy with chemotherapy or
radiation.
[0207] The following examples are illustrative, but not limiting,
of the method and compositions of the present invention. Other
suitable modifications and adaptations of the variety of conditions
and parameters normally encountered in clinical therapy and which
are obvious to those skilled in the art are within the spirit and
scope of the invention.
EXAMPLE 1
2-Chlorobenzyloxycarbonyl-Val-Asp-fmk
[0208] Step A. 2-Chlorobenzyl chlorofornate. To a solution of
2-chlorobenzyl alcohol (1.0 g, 7.0 mmol) in diethyl ether (15 ml)
at 0.degree. C. was added N,N-diisopropylethyl amine (2.4 ml, 14.0
mmol), and phosgene solution in toluene (7.5 ml, 14.0 mmol). The
mixture was allowed to warm up to room temperature in 2 h while
stirring, then it was filtered. The diethyl ether was removed by
rotary evaporator, and the solution of 2-chlorobenzyl chloroformate
in toluene was carried on for the next step reaction.
[0209] Step B. 2-Chlorobenzyloxycarbonyl-Val. To a solution of
L-valine (0.5 g, 4.3 mmol) in 2 N NaOH aqueous solution (10 ml) was
added 2-chlorobenzyl chloroformate (14.0 mmol) at room temperature.
The resulting solution was stirred at room temperature for 12 h,
and then was diluted with 20 ml of ethyl acetate, washed with 2N
NaOH, 2N HCl and brine, dried over Na.sub.2SO.sub.4 and
concentrated in vacuo. The title compound was obtained as white
solid (0.94 g, 3.29 mmol, 77%). .sup.1H NMR (DMSO-d.sub.6): 12.24
(bs, 1H), 7.61 (d, J=8.4, 1H), 7.50 (m, 2H), 7.37 (m, 2H), 5.12 (s,
2H), 3.87 (dd, J=8.7, 6.0, 1H), 2.06 (m, 1H), 0.89 (m, 6H).
[0210] Step C. t-Butyl
5-fluoro-3-(2-chlorobenzyloxycarbonyl-valinamido)-4-
-hydroxypentanoate. To a solution of 2-chlorobenzyl-oxycarbonyl-Val
(216 mg, 0.76 mmol) in THF (10 ml) was added
1-(3-dimethylaminopropyl)-3-ethyl- carbodiimide hydrochloride
(EDCI) (159 mg, 0.83 mmol), 1-hydroxybenzotriazole hydrate (HOBT)
(116 mg, 0.77 mmol) and 4-(dimethylamino)pyridine (DMAP) (46 mg,
0.38 mmol). The resulting mixture was stirred at room temperature
for 5 min, to which was then added a solution of t-butyl
3-amino-5-fluoro-4-hydroxypentanoate (157 mg, 0.75 mmol) in THF (5
ml). The resulting mixture was stirred at room temperature for 12
h, and diluted with ethyl acetate (20 ml), washed with 1N HCl,
saturated NaHCO.sub.3, brine and dried over Na.sub.2SO.sub.4.
Evaporation of solvent, followed by flash chromatography
(EtOAc/Hexane 2/3) gave the title compound as a colorless oil (80
mg, 0.17 mmol, 22%). .sup.1H NMR (CDCl.sub.3): 7.38 (m, 2H), 7.27
(m, 2H), 7.06-6.84 (m, 1H), 5.56 (m, 1H), 5.22 (s, 2H), 4.514.21
(m, 3H), 4.01 (m, 3H), 2.63 (m, 2H), 2.12 (m, 1H), 1.43 (m, 9H),
0.96 (m, 6H).
[0211] Step D. 2-Chlorobenzyloxycarbonyl-Val-Asp(OBu-t)-fmk. To a
suspension of Dess-Martin periodinane (0.35 g, 0.835 mmol) in
dichloromethane (15 ml) was added a solution of t-butyl
5-fluoro-3-(2-chlorobenzyloxycarbonyl-valinamido)-4-hydroxypentanoate
(80 mg, 0.17 mmol) in dichloromethane (5 ml). The mixture was
refluxed for 12 h, cooled to room temperature, then it was diluted
with 25 ml of ethyl acetate, washed with saturated Na.sub.2SO.sub.3
aqueous solution, brine, and then dried over Na.sub.2SO.sub.4.
Evaporation of the solvent, followed by flash chromatography
(EtOAc/Hexane 1/2) gave the title compound as a pale white solid
(56 mg, 0.12 mmol, 72%). .sup.1H NMR (CDCl.sub.3): 7.39 (m, 2H),
7.28 (m, 2H), 5.43 (m, 1H), 5.25-4.84 (m, 5H), 4.05 (m, 1H), 2.96
(m, 1H), 2.77 (m, 1H), 2.14 (m, 1H), 1.42 (s, 9H), 0.96 (m,
6H).
[0212] Step E. 2-Chlorobenzyloxycarbonyl-Val-Asp-fmk. To a solution
of 2-chlorobenzyloxycarbonyl-Val-Asp(OBu-t)-fmk (56 mg, 0.12 mmol)
in 3 ml of CH.sub.2Cl.sub.2 at room temperature was added 1 ml of
TFA. The resulting solution was allowed to stir for 3 h, and then
diluted with 20 ml of ethyl acetate, washed with saturated
Na.sub.2HPO.sub.4, brine, and dried over Na.sub.2SO.sub.4. The
solvent was evaporated in vacuo to give the title compound as a
white solid (39 mg, 0.09 mmol, 77%). .sup.1H NMR (CDCl.sub.3): 8.61
(m, 1H), 8.15 (m, 1H), 7.49 (m, 2H), 7.37 (m, 2H), 5.21 (m, 1H),
5.11 (s, 2H), 4.59 (m, 2H), 3.84 (m, 1H), 2.65 (m, 2H), 1.98 (m,
1H), 0.86 (m, 6H).
EXAMPLE 2
3-Chlorobenzyloxycarbonyl-Val-Asp-fmk
[0213] The title compound was prepared in five steps as described
in Example 1 from 3-chlorobenzyl alcohol. .sup.1H NMR (CDCl.sub.3):
7.55 (bs, 1H), 5.48 (m, 1H), 4.90 (m, 4H), 4.02 (m, 2H), 3.85 (m,
1H), 3.08 (m, 1H), 2.76 (m, 1H), 1.93 (m, 1H), 1.71 (m, 7H), 1.23
(m, 4H), 0.95 (m, 6H).
EXAMPLE 3
Phenethoxycarbonyl-Val-Asp-fmk
[0214] The title compound was prepared in five steps as described
in Example 1 from phenethyl alcohol. .sup.1H NMR (CDCl.sub.3): 7.87
(m, 2H), 7.23 (m, 5H), 5.74 (d, J=8.4, 1H), 4.87 (m, 2H), 4.26 (m,
3H), 3.99 (m, 1H), 2.89 (m, 4H), 2.09 (m, 1H), 0.91 (m, 6H).
EXAMPLE 4
4-Chlorobenzyloxycarbonyl-Val-Asp-fmk
[0215] The title compound was prepared in five steps as described
in Example 1 from 4-chlorobenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.51 (m, 1H), 7.42 (m, 5H), 5.21 (m, 1H), 5.03 (s,
2H), 4.60 (m, 1H), 3.83 (m, 1H), 2.65 (m, 2H), 1.93 (m, 1H), 0.85
(m, 6H).
EXAMPLE 5
Cyclohexylmethoxycarbonyl-Val-Asp-fmk
[0216] The title compound was prepared in five steps as described
in Example 1 from cyclohexylmethanol. .sup.1H NMR (CDCl.sub.3):
7.55 (bs, 1H), 5.48 (m, 1H), 4.90 (m, 4H), 4.02 (m, 2H), 3.85 (m,
1H), 3.08 (m, 1H), 2.76 (m, 1H), 1.93 (m, 1H), 1.71 (m, 7H), 1.23
(m, 4 H ), 0.95 (m, 6H).
EXAMPLE 6
Ethoxycarbonyl-Val-Asp-fmk
[0217] The title compound was prepared in four steps as described
in Example 1 from L-valine and ethyl chloroformate. .sup.1H NMR
(DMSO-d.sub.6): 8.43 (s, 1H), 7.19 (m, 1H), 5.14(bs, 2H),
4.66-4.53(m, 1H), 4.01(q, J=6.9, 2H), 3.83-3.76 (m, 1H), 2.73-2.67
(m, 2H), 1.96-1.88 (m, 1H), 1.16 (t, J=6.9, 3H), 0.86-0.82 (m,
6H).
EXAMPLE 7
Benzylcarbonyl-Val-Asp-fmk
[0218] The title compound was prepared in four steps as described
in Example 1 from phenylacetyl chloride. .sup.1H NMR
(DMSO-d.sub.6): 8.55 (m, 1H), 8.21 (m, 1H), 7.21 (m, 5H), 5.14 (m,
2H), 4.62 (m, 2H), 4.14 (m, 1H), 2.66 (m, 2H), 1.93 (m, 1H), 0.83
(m, 6H).
EXAMPLE 8
4-Nitrobenzyloxycarbonyl-Val-Asp-fmk
[0219] The title compound was prepared in four steps as described
in Example 1 from 4-nitrobenzyl chloroformate. .sup.1H NMR
(DMSO-d.sub.6): 12.5 (s,I H), 8.66-8.54 (m, 1H), 8.23 (m, 2H), 7.64
(m, 2H), 5.19 (m, 4H), 4.66-4.54 (m, 2H), 3.82 (m, 1H), 2.76 (m,
2H), 1.95 (m, 1H), 0.87 (m, 6H).
EXAMPLE 9
2,5-Dimethylbenzyloxycarbonyl-Val-Asp-fmk
[0220] The title compound was prepared in five steps as described
in Example 1 from 2,5-dimethylbenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.50 (m, 1H), 7.42 (m, 1H), 7.06 (m, 2H), 4.99 (s,
2H), 4.64-4.56 (m, 1H), 3.83 (m, 1H), 2.97 (m, 1H), 2.67 (m, 2H),
1.89 (m, 1H), 0.86 (m, 6H).
EXAMPLE 10
3,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk
[0221] The title compound was prepared in five steps as described
in Example 1 from 3,4-dichlorobenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.50 (m, 1H), 7.65-7.35 (m, 4H), 5.03 (m, 3H), 4.59
(m, 1H), 3.84 (m, 1H), 2.67 (m, 2H), 1.94 (m, 1H), 0.86 (m,
6H).
EXAMPLE 11
3,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk
[0222] The title compound was prepared in five steps as described
in Example 1 from 3,5-dichlorobenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.54 (m, 1H), 7.56-7.34 (m, 3H), 5.05 (m, 3H),
4.63-4.55 (m, 1H), 3.86 (m, 1H), 2.73 (m, 2H), 1.95 (m, 1H), 0.86
(m, 6H).
EXAMPLE 12
2,5-Dichlorobenzyloxycarbonyl-Val-Asp-fmk
[0223] The title compound was prepared in five steps as described
in Example 1 from 2,5-dichlorobenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.54 (m, 1H), 7.51 (m, 3H), 5.26-5.08 (m, 3H),
4.65-4.55 (m, 1H), 3.87 (m, 1H), 2.73-2.60 (m, 2H), 1.98 (m, 1H),
0.86 (m, 6H).
EXAMPLE 13
2,6-Dichlorobenzyloxycarbonyl-Val-Asp-fmk
[0224] The title compound was prepared in five steps as described
in Example 1 from 2,6-dichlorobenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.50 (m, 1H), 7.55-7.41 (m, 3H), 5.25 (m, 3H),
4.644.51 (m, 1H), 3.78 (m, 1H), 2.71 (m, 2H), 1.92 (m, 1H), 0.84
(m, 6H).
EXAMPLE 14
2,4-Dimethylbenzyloxycarbonyl-Val-Asp-fmk
[0225] The title compound was prepared in five steps as described
in Example 1 from 2,4-dimethylbenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.53 (m, 1H), 7.39 (m, 1H), 7.28-7.18 (m, 3H), 5.26
(m, 1H), 4.99 (m, 2H); 4.66-4.53 (m, 1H), 3.80 (m, 1H), 2.78-2.72
(m, 1H), 2.59 (m, 3H), 1.92 (m, 1H), 1.16 (m, 3H), 0.85 (m,
6H).
EXAMPLE 15
4-Ethylbenzyloxycarbonyl-Val-Asp-fmk
[0226] The title compound was prepared in five steps as described
in Example 1 from 4-ethylbenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.50 (m, 1H), 7.23 (m, 4H), 4.98 (m, 3H), 4.63 (m,
1H), 3.84 (m, 1H), 2.68 (m, 2H), 1.91 (m, 1H), 0.85 (m, 6H).
EXAMPLE 16
4-Chlorobenzyloxycarbonyl-Ile-Asp-fmk
[0227] The title compound was prepared in five steps as described
in example 1 from 4-chlorobenzyl alcohol. .sup.1H NMR (CDCl.sub.3):
8.61 (m, 1H), 8.52 (m, 1H), 7.48 (m, 1H), 7.39 (m, 4H), 5.19 (m,
2H), 5.02 (s, 2H), 4.54 (m, 1H), 3.87 (m, 1H), 2.73 (m, 2H), 1.68
(m, 1H), 1.39 (m, 1H), 1.17 (m, 1H), 0.81 (m, 6H).
EXAMPLE 17
4-Bromobenzyloxycarbonyl-Val-Asp-fmk
[0228] The title compound was prepared in five steps as described
in example 1 from 4-bromobenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 7.56 (d, J=8.1 Hz, 1H), 7.42 (m, 1H), 7.32 (d,
J=8.1 Hz, 1H), 5.21 (m, 1H), 5.00 (s, 2H), 4.62 (m, 2H), 3.83 (m,
1H), 3.60 (m, 1H), 2.71 (m, 2H), 1.76 (m, 1H), 0.84 (m, 6H).
EXAMPLE 18
4-Fluorobenzyloxycarbonyl-Val-Asp-fmk
[0229] The title compound was prepared in five steps as described
in Example 1 from 4-fluorobenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.63 (m, 1H), 7.92 (m, 1H), 7.55 (m, 4H), 5.03 (m,
3H), 4.61 (m, 2H), 3.83 (m, 1H), 2.73 (m, 2H), 1.95 (m, 1H), 0.95
(m, 6H).
EXAMPLE 19
2,4-Dichlorobenzyloxycarbonyl-Val-Asp-fmk
[0230] The title compound was prepared in five steps as described
in Example 1 from 2,4-dichlorobenzyl alcohol. .sup.1H NMR
(CDCl.sub.3): 8.51 (m, 1H), 7.52 (m, 3H), 5.08 (m, 3H), 4.59 (m,
2H), 3.85 (m, 1H), 2.69 (m, 2H), 1.94 (m, 1H), 0.95 (m, 6H).
EXAMPLE 20
Cyclopentylmethoxycarbonyl-Val-Asp-fmk.
[0231] The title compound was prepared in five steps as described
in Example 1 from cyclopentylmethanol. .sup.1H NMR (DMSO-d.sub.6):
7.22 (bs, 1H), 5.21 (m, 1H), 5.09 (m, 1H), 4.61 (m, 2H), 3.84 (m,
3H), 2.73 (m, 2H), 2.10 (m, 1H), 1.92 (m, 1H), 1.49 (m, 6H), 1.23
(m, 2H), 0.84 (m, 6H).
EXAMPLE 21
4-Trifluoromethylbenzyloxycarbonyl-Val-Asp-fmk
[0232] The title compound was prepared in four steps as described
in Example 1 from 4-trifluoromethylbenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.63 (m, 1H), 8.18 (m, 1H), 7.74 (d, J=7.2 Hz, 2H),
7.57(d, J=8.1, 2H), 5.14 (m, 4H), 4.64 (m, 1H), 3.81 (m, 1H), 2.72
(m, 2H), 1.94 (m, 1H), 0.86 (m, 6H).
EXAMPLE 22
3-Phenylpropionyl-Val-Asp-fmk
[0233] The title compound was prepared in four steps as described
in Example 2 from 3-phenylpropionyl chloride. .sup.1H NMR
(DMSO-d.sub.6): 12.29 (bs, 1H), 8.39 (m, 1H), 7.82 (m, 1H), 7.21(m,
5H), 5.25 (m, 2H), 4.39 (m, 1H), 4.03 (m, 1H), 2.80 (m, 3H), 2.54
(m, 3H), 1.89 (m, 1H), 0.80 (m, 6H).
EXAMPLE 23
Benzylaminocarbonyl-Val-Asp-fmk
[0234] The title compound was prepared in five steps as described
in Example 1 from benzyl amine. .sup.1H NMR (DMSO-d.sub.6): 8.55
(m, 1H), 7.25 (m, 5H), 6.57 (m, 1H), 6.20 (m, 1H), 5.16 (m, 1H),
4.22 (m, 5H), 2.61 (m, 2H), 1.89 (m, 1H), 0.87 (m, 6H).
EXAMPLE 24
3-Phenylpropyloxycarbonyl-Val-Asp-fmk
[0235] The title compound was prepared in five steps as described
in Example 1 from 3-phenyl-1-propanol. .sup.1H NMR (DMSO-d.sub.6):
8.61 (m, 1H), 8.18 (m, 1H), 7.21 (m, 5H), 5.17 (m, 1H), 4.53 (m,
1H), 3.83 (m, 4H), 2.71 (m, 4H), 1.85 (m, 3H), 0.85 (m, 6H).
EXAMPLE 25
2,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk
[0236] The title compound was prepared in five steps as described
in Example 1 from 2,4-difluorobenzyl alcohol. .sup.1H NMR
(CDCl.sub.3): 7.33 (m, 1H), 6.83 (m, 2H), 5.60 (m, 1H), 5.11 (m,
3H), 4.88 (m, 2H), 4.03 (m, 1H), 3.05 (m, 1H), 2.79 (m, 1H), 2.05
(m, 1H), 0.94 (m, 6H).
EXAMPLE 26
3,4-Difluorobenzyloxycarbonyl-Val-Asp-fmk
[0237] The title compound was prepared in five steps as described
in Example 1 from 3,4-difluorobenzyl alcohol. .sup.1H NMR
(CDCl.sub.3): 8.63 (m, 1H), 8.18 (m, 1H), 7.52 (m, 1H), 7.27 (m,
2H), 5.07 (m, 3H), 4.51 (m, 2H), 3.99 (m, 1H), 2.70 (m, 2H), 1.95
(m, 1H), 0.85 (m, 6H).
EXAMPLE 27
4-Morpholinecarbonyl-Val-Asp-fmk
[0238] The title compound was prepared in four steps as described
in Example 1 from 4-morpholinecarbonyl chloride. .sup.1H NMR
(CDCl.sub.3): 10.61 (bs, 1H), 7.58 (m, 1H), 5.19 (d, J=8.1 Hz, 1H),
4.95 (m, 3H), 4.42 (m, 114), 3.71 (m, 4H), 3.42 (m, 4H), 2.94 (m,
2H), 2.19 (m, 1H), 0.98 (m, 6H).
EXAMPLE 28
4-Pyridylmethoxycarbonyl-Val-Asp-fmk
[0239] The title compound was prepared in five steps as described
in Example 1 from 4-pyridylcarbinol. .sup.1H NMR (CDCl.sub.3): 8.56
(m, 2H), 8.15 (m, 2H), 7.23 (m, 1H), 6.47 (m, 1H), 5.10 (s, 2H),
4.35 (m, 3H), 4.08 (m, 1H), 2.61 (m, 2H) 1.46 (m, 1H), 0.94 (m,
6H).
EXAMPLE 29
2-Pyridylmethoxycarbonyl-Val-Asp-fmk
[0240] The title compound was prepared in five steps as described
in Example 1 from 2-pyridylcarbinol. .sup.1H NMR (CDCl.sub.3): 8.59
(m, 1H), 7.28 (m, 3H), 7.03 (m, 1H), 5.58 (m, 1H), 5.11 (m, 5H),
4.06 (m, 1H), 2.83 (m, 2H), 2.17 (m, 1H), 0.93 (m, 6H).
EXAMPLE 30
2,6-Dichlorobenzyloxycarbonyl-Val-Asp-DCB-methylketone
[0241] Step A. Z-Asp(OBu-t)-DCB-methylketone. To a solution of
Z-Asp(OBu-t)-bromomethylketone (500 mg, 1.24 mmol) in DMF (10 ml)
was added potassian fluoride (320 mg, 5.50 mmol), and
2,6-dichlorobenzoic acid (348 mg, 1.82 mmol). The mixture was
stirred at room temperature for 12 h, and then was diluted with 25
ml of ethyl acetate, washed with aqueous NH.sub.4Cl and brine,
dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The title
compound was obtained as white solid (0.78 g, 2.62 mmol, 69%).
.sup.1H NMR (CDCl.sub.3): 7.34 (m, 8H), 5.96 (d, J=8.7, 1H), 5.21
(d, J=6.6, 2H), 5.16 (s, 2H), 4.70 (m, 1H), 2.88 (m, 2H), 1.27 (s,
9H).
[0242] Step B. Asp(OBu-t)-DCB-methylketone-N-hydrochloride. To a
solution of Z-Asp(OBu-t)-DCB-methylketone (572 mg, 1.14 mmol) in
ethanol (15 ml) was added Pd/C (50 mg) and 6N HCl (0.2 ml). The
mixture was stirred at room temperature under H.sub.2 atmosphere (1
atm) for 12 h, then it was filtered and concentrated. The title
compound was obtained as pale while solid (416 mg, 1.04 mmol, 90%).
.sup.1H NMR (CDCl.sub.3): 7.27 (m, 3H), 5.28 (m, 2H), 4.94 (m, 1H),
3.27 (m, 2H), 1.42 (s, 9H).
[0243] Step C.
2,6-Dichlorobenzyloxycarbonyl-Val-Asp(OBu-t)-DCB-methylketo- ne. To
a solution of 2,6-dichlorobenzyloxycarbonyl-Val (200 mg, 0.60 mmol)
in THF (10 ml) was added
Asp(OBu-t)-DCB-methylketone-N-hydrochloride (250 mg, 0.60 mmol),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI,
126 mg, 0.66 mmol), 1-hydroxybenzotriazole hydrate (HOBT, 92 mg,
0.60 mmol) and 4-(dimethylamino)pyridine (DMAP, 29 mg, 0.28 mmol).
The resulting mixture was stirred at room temperature for 12 h, and
diluted with ethyl acetate (20 ml), washed with 1N HCl, saturated
NaHCO.sub.3, brine and dried over Na.sub.2SO.sub.4. Evaporation of
solvent, followed by flash chromatography (EtOAc/Hexane 2/3) gave
the title compound as a colorless oil (85 mg, 0.18 mmol, 21%).
.sup.1H NMR (CDCl.sub.3): 7.35 (m, 6H), 7.27 (m, 1H), 5.41 (m, 3H),
5.15 (m, 2H), 4.41 (m, 1H), 2.96 (m, 2H), 2.28 (m, 1H), 1.44 (m,
9H), 0.94 (m, 6H).
[0244] Step D. 2,
6-Dichlorobenzyloxycarbonyl-Val-Asp-DCB-methylketone. To a solution
of 2,6-dichlorobenzyloxycarbonyl-Val-Asp(OBu-t)-DCB-methylketo- ne
(85 mg, 0.18 mmol) in CH.sub.2Cl.sub.2 (3 ml) at room temperature
was added TFA (1 ml). The resulting solution was allowed to stir
for 4 h, and then diluted with 20 ml of ethyl acetate, washed with
saturated Na.sub.2HPO.sub.4, brine, and dried over
Na.sub.2SO.sub.4. The solvent was evaporated in vacuo to give the
title compound as a white solid (27 mg, 0.04 mmol, 25%). .sup.1H
NMR (CDCl.sub.3): 9.28 (bs, 1H), 8.11 (m, 1H), 7.49 (m, 1H), 7.30
(m, 6H), 5.37(m, 4H), 4.32 (m, 2H), 3.73 (m, 2H), 2.19 (m, 1H),
1.25 (m, 3H), 0.98 (m, 3H).
EXAMPLE 31
Isobutoxycarbonyl-Val-Asp-fmk
[0245] The title compound was prepared in four-steps as described
in Example 1 from L-valine and isobutyl chloroformate. .sup.1H NMR
(DMSO-d.sub.6): .delta. 8.47 (m, 1H), 7.23 (m, 1H), 5.24-4.52 (m,
3H), 3.81-3.72 (m, 3H), 2.73-2.55 (m, 2H), 1.94-1.79 (m, 2H),
0.89-0.83 (m, 12H).
EXAMPLE 32
Methoxycarbonyl-Val-Asp-fmk
[0246] The title compound was prepared in four-steps as described
in Example 1 from L-valine and methyl chloroformate. .sup.1H NMR
(DMSO-d.sub.6): .delta. 8.49 (s, 1H), 7.28 (m, 1H), 5.16-4.54 (m,
3H), 3.81 (m, 1H), 3.53 (s, 3H), 2.72-2.56 (m, 2H), 1.93 (m, 1H),
0.85 (m, 6H).
EXAMPLE 33
Isopropyloxycarbonyl-Val-Asp-fmk
[0247] The title compound was prepared in four-steps as described
in Example 1 from L-valine and isopropyl chloroformate. .sup.1H NMR
(DMSO-d.sub.6): .delta. 8.43 (s, 1H), 7.11 (m, 1H), 5.18-4.54 (m,
4H), 3.76 (m, 1H), 2.69-2.67 (m, 2H), 1.91 (m, 1H), 1.17 (d, J=6,
6H), 0.83 (m, 6H).
EXAMPLE 34
Propionyl-Val-Asp-fmk
[0248] The title compound was prepared in four-steps as described
in Example 1 from L-valine and propionyl chloride. .sup.1H NMR
(DMSO-d.sub.6): .delta. 8.63-7.82 (m, 2H), 5.37-5.02 (m, 1H),
4.70-3.95 (m, 3H), 2.89-2.56 (m, 2H), 2.21-2.10 (m, 2H), 1.91 (m,
1H), 0.98 (t, J=7.2, 3H), 0.86-0.82 (m, 6H).
EXAMPLE 35
Benzyl-glutaryl-Val-Asp-fmk
[0249] Step A. Benzyl monoglutarate. A solution of glutaric
anhydride (460 mg, 4.03 mmol) in benzyl alcohol (1 ml) was heated
at 70.degree. C. overnight. The mixture was purified by
chromatography (3:1 hexane/EtOAc) to yield the product as an oil
(0.4 g, 1.8 mmol, 45%). .sup.1H NMR (CDCl.sub.3): .delta. 7.36 (br
s, 5H), 5.12 (s, 2H), 2.48-2.41 (m, 4H), 2.00-1.96 (m, 2H).
[0250] Step B. Benzyl-glutaryl-Val-OBu-t. A mixture of benzyl
monoglutarate (0.4 g, 1.8 mmol), Val-OBu-t (382 mg, 1.82 mmol), EDC
(335 mg, 1.75 mmol), HOBT (265 mg, 1.73 mmol) and DMAP (372 mg,
3.04 mmol) in THF (15 ml) was stirred at room temperature for 17 h.
It was worked up and purified by chromatography to yield the title
compound as a colorless oil (370 mg, 0.98 mmol, 54%). .sup.1H NMR
(CDCl.sub.3): .delta. 7.39-7.30 (m, 5H), 5.96 (d, J=8.4, 1H), 5.12
(s, 2H), 4.45 (m, 1H), 2.47-1.94 (m, 7H), 1.46 (s, 9H), 0.95-0.87
(m, 6H).
[0251] Step C. Benzyl-glutaryl-Val-OH. To a solution of
benzyl-glutaryl-Val-OBu-t (370 mg, 0.98 mmol) in methylenechloride
(3 ml) was added TFA (1 ml). The mixture was stirred at room
temperature for 8 h. It was worked up to yield the title compound
as a colorless oil (200 mg, 0.62 mmol, 63%). .sup.1H NMR
(CDCl.sub.3): .delta. 7.36 (br s, 5H), 6.43 (d, J=8.7, 1H), 5.13
(s, 2H), 4.55 (m, 1H), 2.49-2.21 (m, 5H), 2.00 (m, 2H), 0.99-0.94
(m, 6H).
[0252] Step D. Benzyl-glutaryl-Val-Asp-fmk. The title compound was
prepared from benzyl-glutaryl-Val-OH in three steps by a similar
procedure as described in Example 1 as a white solid. .sup.1H NMR
(CDCl.sub.3): .delta. 7.35 (br s, 5H), 6.72 (s, 1H), 5.56 (s, 1H),
5.11 (s, 2H), 4.92-4.22 (m, 4H), 3.13-2.68 (m, 2H), 2.43-1.93 (m,
6H), 0.92 (d, J=5.1, 6H).
EXAMPLE 36
Glutaryl-Val-Asp-fmk
[0253] A solution of benzyl-glutaryl-Val-Asp(OBu-t)-fmk (78 mg,
0.15 mmol) in 30% AcOH solution of HBr (2 ml) was stirred at room
temperature for 4 h. The solvent was removed in vacuo. To the
residue was added acetone (1 ml), then EtOAc (10 ml) and hexane (20
ml). The solvents were then removed in vacuo to yield the title
compound as a brown solid (36 mg, 0.10 mmol, 67%). .sup.1H NMR
(DMSO-d.sub.6): .delta. 8.60-7.87 (m, 2H), 5.36-4.42 (m, 4H),
2.94-2.57 (m, 2H), 2.33-1.69 (m, 7H), 0.83 (s, 6H).
EXAMPLE 37
3-(2-Phenyloxyphenyl)propionyl-Val-Asp-fmk
[0254] Step A. 3-(2-Phenyloxyphenyl)propionic acid. A mixture of
3-(2-hydroxyphenyl)propionic acid (0.57 g, 3.4 mmol), phenylbromide
(0.4 ml, 3.37 mmol), and K.sub.2CO.sub.3 (2.5 g) in acetone (10 ml)
was stirred at room temperature for 2 days. The mixture was diluted
with 1:1 hexane/EtOAc (100 ml), washed with water and brine, dried
over sodium sulfate and concentrated in vacuo. The residue was
purified by chromatography (5:1 then 4:1 hexane/EtOAc) to yield
phenyl 3-(2-phenyloxyphenyl)propionate (0.45 g, 1.30 mmol,
77%).
[0255] A mixture of phenyl 3-(2-phenyloxyphenyl)propionate (0.45 g,
1.30 mmol), 2N NaOH (20 ml) and MeOH (10 ml) was stirred at room
temperature overnight, which was then neutralized to pH-2 with
concentrated HCl and extracted with EtOAc (100 ml). The organic
phase was washed with brine and dried over sodium sulfate and
concentrated in vacuo to yield 3-(2-phenyloxyphenyl)propionic acid
as an oil. This was used for next step without further
purification.
[0256] Step B. 3-(2-Phenyloxyphenyl)propionyl-Val-Asp-fmk. The
title compound was prepared in a similar procedure as described in
Examples 36 and 1 from crude 3-(2-phenyloxyphenyl)propionic acid
synthesized above. .sup.1H NMR (DMSO-d.sub.6): .delta. 8.62-8.50
(m, 1H), 7.47-6.85 (m, 9H), 5.36-5.04 (m, 4H), 4.63-4.02 (m, 2H),
5.83 (br s, 6H), 1.85 (br s, 1H), 0.80 (s, 6H).
EXAMPLE 38
3-(5-Bromo-2-hydroxyphenyl)propionyl-Val-Asp-fmk
[0257] A mixture of 3-(2-phenyloxyphenyl)propionyl-Val-Asp-fmk (30
mg, 30 mg, 0.06 mmol) in 30% acetic acid solution of HBr (2 ml) was
stirred at room temperature for 6 h. The solvent was removed in
vacuo and 1:1 EtOAc/hexane was added to the residue. Evaporation of
the solvent yielded the title compound as a brown solid (18 mg,
0.037 mmol, 62%). .sup.1H NMR (acetone-d.sub.6): .delta. 7.45-6.74
(m, 4H), 5.03-4.35 (m, 4H), 3.03-2.73 (m, 6H), 0.97-0.87 (m,
6H).
EXAMPLE 39
3-Fluorobenzyloxycarbonyl-Val-Asp-fmk
[0258] The title compound was prepared in five steps as described
in Example 1 from 3-fluorobenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.64 (m, 1H), 7.95 (m, 1H), 7.45 (m, 2H), 7.19 (m,
3H), 5.25 (m, 1H), 5.06 (s, 2H), 4.58 (m, 2H), 3.85 (m, 1H), 2.73
(m, 2H), 1.99 (m, 1H), 0.86 (m, 6H).
EXAMPLE 40
2-Fluorobenzyloxycarbonyl-Val-Asp-fmk
[0259] The title compound was prepared in five steps as described
in Example 1 from 2-fluorobenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 7.36 (m, 2H), 7.12 (m, 3H), 5.68 (m, 1H), 5.17 (s,
2H), 4.34 (m, 3H), 3.97 (m, 2H), 2.62 (m, 2H), 2.06 (m, 1H), 0.91
(m, 6H).
EXAMPLE 41
3-Methylbenzyloxycarbonyl-Val-Asp-fmk
[0260] The title compound was prepared in five steps as described
in Example 1 from 3-methylbenzyl alcohol. .sup.1H NMR
(DMSO-d.sub.6): 8.52 (m, 1H), 7.92 (m, 1H), 7.43 (m, 1H), 7.17 (m,
4H), 5.25 (m, 1H), 4.99 (s, 2H), 4.56 (m, 2H), 3.83 (m, 1H), 2.71
(m, 2H), 2.29 (s, 3H), 1.93 (m, 1H), 0.85 (m, 6H).
EXAMPLE 42
2-Chloro-4-fluorobenzyloxycarbonyl-Val-Asp-fmk
[0261] The title compound was prepared in five steps as described
in Example 2 from 2-chloro-4-fluorobenzyl alcohol. .sup.1H NMR
(CDCl.sub.3): 8.63 (m, 1H), 7.42 (m, 4H), 5.15 (m, 3H), 4.61 (m,
2H), 4.02 (bs, 1H), 3.81 (m, 1H), 2.63 (m, 2H), 1.90 (m, 1H), 0.83
(m, 6H).
EXAMPLE 43
2-Naphthalenemethoxycarbonyl-Val-Asp-fmk
[0262] The title compound was prepared in five steps as described
in Example 2 from 2-naphthalenemethanol. .sup.1H NMR
(DMSO-d.sub.6): 7.80 (m, 4H), 7.46 (m, 3H), 6.99 (m, 1H), 5.59 (m,
1H), 5.25 (s, 2H), 4.46 (m, 3H), 4.03 (m, 2H), 2.62 (m, 2H), 2.1
1(m, 1H), 0.94 (m, 6H).
EXAMPLE 44
p-Toluenesulfonyl-Val-Asp-fmk
[0263] Step A. p-Toluenesulfonyl-Val. The title compound was
prepared by a similar procedure as described in Example 1 step B in
13% yield from valine and p-toluenesulfonyl chloride. .sup.1H NMR
(DMSO-d.sub.6): 7.90 (d, J=9.3, 1H), 7.63 (d, J=8.1, 2H), 7.32 (d,
J=8.1, 2H), 3.47 (m, 1H), 2.35 (s, 3H), 1.90 (m, 1H), 0.82-0.76 (m,
6H).
[0264] Step B. tert-Butyl
5-fluoro-3-[p-toluenesulfonyl-Valineamido]-4-hyd- roxypentanoate.
The title compound was prepared by a similar procedure as described
in Example 1 step C in 37% yield. .sup.1H NMR (CDCl.sub.3): 7.73
(d, J=8.4, 2H), 7.30 (d, J=7.2, 2H), 6.72-6.50 (m, 1H), 5.28-5.08
(m, 1H), 4.32-3.81 (m, 4H), 3.45 (m, 1H), 2.65-2.45(m, 2H), 2.43,
2.41 (2s, 3H), 2.05 (m, 1H), 1.45, 1.43 (2s, 9H), 0.88-0.79 (m,
6H).
[0265] Step C. p-Toluenesulfonyl-Val-Asp(OBu-t)-fmnk. The title
compound was prepared by a similar procedure as described in
Example 1 step D in 92% yield. .sup.1H NMR (CDCl.sub.3): 7.74-7.70
(m, 2H), 6.93 (m, 1H), 7.31-7.27 (m, 2H), 7.03 (d, J=7.8, 1H),
6.96(d, J=8.1, 1H), 5.26-4.61 (m, 3H), 3.55-3.47 (m, 1H), 2.98-2.48
(m, 2H), 2.11 (m, 1H), 2.43, 2.41 (2s, 3H), 1.45, 1.42 (2s, 9H),
0.87-0.81 (m, 6H).
[0266] Step D. p-Toluenesulfonyl-Val-Asp-fnk. The title compound
was prepared by a similar procedure as described in Example 1 step
E in 31% yield. .sup.1H NMR (DMSO-d.sub.6): 8.53-8.43 (m, 1H), 7.81
(br s, 1H), 7.63 (d, J=7.4, 2H), 7.32 (d, J=7.4, 2H), 5.02-4.28 (m,
4H), 2.18-2.40 (m, 2H), 2.34 (s, 3H), 1.79 (m, 1H), 0.77-0.74 (m,
6H).
EXAMPLE 45
p-Toluenesulfonyl-Phe-Asp-fmk
[0267] The title compound was prepared by a similar procedure as
described in Example 1 in three steps from p-toluenesulfonyl-Phe
and t-butyl 3-amino-5-fluoro-4-hydroxypentanoate. .sup.1H NMR
(CD.sub.3OD): 7.61 (d, J=6.9, 2H), 7.29-7.13 (m, 7H), 4.56-3.94 (m,
4H), 3.01-2.78 (m, 2H), 2.44 (s, 3H), 2.51-2.37 (m, 2H).
EXAMPLE 46
Enzyme Activity
[0268] The activity of 2-chlorobenzyloxycarbonyl-Val-Asp-fmk as an
inhibitor of caspase-3 was measured in a fluorometric enzyme assay.
Enzyme activity was measured using synthetic peptide substrates
attached to a fluorogenic leaving group. Cleavage of the synthetic
substrate by the enzyme results in a fluorescent signal which is
read in a spectrofluorometer or in a fluorometric microtiter plate
reader.
[0269] 12 concentrations of the testing compound ranged from 30 pM
to 10 .mu.M were tested in the enzyme assay. The enzyme reaction
was conducted in the presence of 2 ng rCaspase 3 (purchased from
PharMingen, a Becton division company, San Diego, Calif.), various
concentrations of testing compound, 10 .mu.M caspase 3 substrate
Ac-DEVD-AMC (SEQ ID NO:3) (purchased from Quality Controlled
Biochemicals, Inc. Hopkinton, Mass.) and caspase buffer (20 mM
PIPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS and 10%
sucrose, pH 7.2) in a total volume of 100 .mu.l. The enzyme
reaction was carried out in a 96-well plate and incubated at
37.degree. C. for 30 minutes. The plate was then read with a
fluorescence plate reader (EG&G WALLAG 1420-002) using
excitation filter at 355nm/emission filter at 460 nm. The data was
analyzed using GraphPrism software. Other inhibitors were tested
using the same procedure and the results is summarized in Table
II.
2TABLE II Activity of Dipeptide as Inhibitor of Caspase-3 Caspase-3
IC.sub.50 Name (nM) 2-Chlorobenzyloxycarbonyl-Val-Asp-fmk 36
3-Chlorobenzyloxycarbony- l-Val-Asp-fmk 36
Phenethoxycarbonyl-Val-Asp-fmk 110
4-Chlorobenzyloxycarbonyl-Val-Asp-fmk 34 Cyclohexylmethoxycarbony-
l-Val-Asp-fmk 72 Ethoxycarbonyl-Val-Asp-fmk 58
[0270] As shown in Table II, the dipeptides are potent inhibitor of
caspase-3.
[0271] Having now fully described this invention, it will be
understood by those of ordinary skill in the art that the same can
be performed within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any embodiment thereof. All patents, patent
applications and publications cited herein are fully incorporated
by reference herein in their entirety.
* * * * *