U.S. patent application number 10/434335 was filed with the patent office on 2003-09-25 for substituted 2-aminobenzamide 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 | 20030181388 10/434335 |
Document ID | / |
Family ID | 26822841 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030181388 |
Kind Code |
A1 |
Cai, Sui Xiong ; et
al. |
September 25, 2003 |
Substituted 2-aminobenzamide caspase inhibitors and the use
thereof
Abstract
The present invention is directed to novel 2-aminobenzamide
thereof, represented by the general Formula I: 1 where
R.sub.1-R.sub.3, X, and A-D 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) ; Wang, Yan; (Austin, TX) ; Weber,
Eckard; (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: |
26822841 |
Appl. No.: |
10/434335 |
Filed: |
May 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10434335 |
May 9, 2003 |
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09527225 |
Mar 16, 2000 |
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60158386 |
Oct 12, 1999 |
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60124675 |
Mar 16, 1999 |
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Current U.S.
Class: |
514/6.9 ;
514/17.7; 514/18.6; 514/18.9; 514/19.3; 514/20.7; 530/330;
530/331 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 1/02 20180101; A61P 19/08 20180101; A61P 39/00 20180101; A61P
1/18 20180101; A61P 43/00 20180101; C07C 2601/14 20170501; A61P
1/00 20180101; A61P 19/02 20180101; A61P 9/00 20180101; A61P 17/02
20180101; A61P 1/04 20180101; A61P 13/12 20180101; A61P 37/00
20180101; A61P 27/02 20180101; C07C 271/24 20130101; A61P 15/08
20180101; C07D 333/38 20130101; A61P 3/10 20180101; A61P 17/16
20180101; C07C 271/28 20130101; A61P 1/16 20180101; A61P 25/32
20180101; A61P 7/00 20180101; C07C 271/22 20130101; A61P 25/00
20180101; A61P 7/06 20180101; C07C 2601/08 20170501; A61P 13/10
20180101; A61P 17/14 20180101; A61P 17/06 20180101; A61P 17/00
20180101 |
Class at
Publication: |
514/17 ; 514/19;
514/18; 530/330; 530/331 |
International
Class: |
A61K 038/08; A61K
038/06; A61K 038/05; C07K 007/06; C07K 005/06; C07K 005/04 |
Claims
What is claimed is:
1. A compound having the Formulae I or II or III: 18or a
pharmaceutically acceptable salt or prodrug thereof, wherein:
R.sub.1 is an optionally substituted alkyl or hydrogen; R.sub.3 is
an N-protecting group; R.sub.2 is hydrogen or optionally
substituted alkyl; Q is an optionally substituted saturated or
partially saturated carbocycle or heterocycle; X is a peptide of
1-4 amino acids or a bond; Y is a peptide of 1-4 amino acids or a
bond; 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; provided that not
more than two of A, B, C or D is nitrogen; and R.sub.6-R.sub.9
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
are taken together with the carbon atoms to which they are attached
to form a carbocycle or heterocycle; E is C.sub.14, nitrogen,
oxygen or sulfur; F is C.sub.15, nitrogen, oxygen or sulfur; G is
C.sub.16, nitrogen, oxygen or sulfur; provided that only one of E,
F, G is nitrogen, oxygen or sulfur and R.sub.14-R.sub.16 are
independently 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.14 and R.sub.15, or R.sub.15 and R.sub.16, are taken together
with the carbon atoms to which they are attached to form a
carbocycle or heterocycle.
2. A compound according to claim 1, wherein R.sub.3 is
t-butyloxycarbonyl, acetyl or benzyloxycarbonyl.
3. A compound according to claim 1, wherein R.sub.1 is H, Me, Et or
acetoxymethyl.
4. A compound according to claim 1, wherein R.sub.2 is hydrogen,
fluoromethyl, acyloxymethyl, arylacyloxymethyl or aminomethyl.
5. A compound according to claim 1, wherein X is a bond.
6. A compound according to claim 1, wherein A, B, C and D are
CH.
7. A compound according to claim 1, wherein A is nitrogen, and B, C
and D are CH.
8. A compound according to claim 1, wherein G is sulfur, and E and
F are CH.
9. A compound according to claim 1, wherein Q is cyclohexyl or
cyclopentyl.
10. A compound according to claim 1, wherein said compound has the
Formula IV: 19or a pharmaceutically acceptable salt or prodrug
thereof, wherein R.sub.2 is hydrogen or optionally substituted
alkyl, wherein the substituent is halo, hydroxy, alkoxy, aryloxy,
alkylthio, arylthio, amino, acyloxy, or arylacyloxy;
R.sub.6-R.sub.9 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
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.2O--, --(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-- and
--CH.dbd.CH--CH.dbd.CH--; wherein R.sub.13 is hydrogen, alkyl or
cycloalkyl; R.sub.10 is hydrogen, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, C.sub.6-C.sub.10
aryl, C.sub.4-C.sub.7 cycloalkyl, C.sub.6-C.sub.10
aryl(C.sub.1-C.sub.6)alkyl, benzyloxy, substituted benzyloxy, or
NR.sub.11R.sub.12; wherein R.sub.11 and R.sub.12 independently are
hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl,
C.sub.6-C.sub.10 aryl, C.sub.4-C.sub.7 cycloalkyl, C.sub.6-C.sub.10
aryl(C.sub.1-C.sub.6)alkyl, or R.sub.11 and R.sub.12 are combined
to form a heterocyclic ring system selected from the group
consisting of pyrrolidine, piperidine, piperazine, and
morpholine.
11. A compound according to claim 10, wherein R.sub.2 is hydrogen,
fluoromethyl, acyloxymethyl, arylacyloxymethyl or aminomethyl.
12. A compound according to claim 10, wherein R.sub.10 is
benzyloxy.
13. A compound according to claim 10, wherein R.sub.1 is H, Me or
acetoxymethyl.
14. A compound according to claim 10, wherein X is a peptide of 1-2
amino acids or a bond.
15. A compound according to claim 1, wherein said compound is
selected from the group consisting of: 2-(Z-amino)benzoyl-Asp-fmk,
2-(Z-amino)-3-methylbenzoyl-Asp-fmk,
2-(Z-amino)-3,5-dimethylbenzoyl-Asp-- fmk,
2-(Z-amino)-4-chlorobenzoyl-Asp-fmk,
2-(Z-amino)-5-chlorobenzoyl-Asp-- fmk,
2-(Z-amino)-5-fluorobenzoyl-Asp-fmk,
2-(Z-amino)-6-fluorobenzoyl-Asp-- fmk,
cis-2-(Z-amino)-cyclohexanecarboxy-Asp-fmk,
2-(Z-amino)-5-methylbenzo- yl-Asp-fmk,
2-(Z-amino)-6-methylbenzoyl-Asp-fmk, 2-(Z-amino)-6-chlorobenzo-
yl-Asp-fmk, 2-(Z-amino)-3-methoxybenzoyl-Asp-fmk,
3-(Z-amino)thiophene-2-c- arboxyl-Asp-fmk,
3-(methoxycarbonylamino)thiophene-2-carboxyl-Asp-fmk,
cis-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,
trans-2-(Z-amino)cyclopentan- ecarboxyl-Asp-fmk,
2-(Z-amino)benzoyl-Asp-DCB-methylketone,
methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk,
Z-Glu-(2-aminobenzoyl)-Asp-- fmk, and
Z-Val-(2-aminobenzoyl)-Asp-fmk.
16. A pharmaceutical composition, comprising a compound of claim 1,
and a pharmaceutically acceptable carrier.
17. 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.
18. 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 claim1.
19. The method of claim 18, 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.
20. The method of claim 18, wherein said cell death is in the
central or peripheral nervous system, and is due to expansion of
trinucleotide repeats of specific genes.
21. The method of claim 18, wherein said cell death is due to
Huntington's Disease.
22. The method of claim 18, 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.
23. The method of claim 18, wherein said cell death is in retinal
neurons and is due to increased intraocular pressure, age-related
macular degeneration or retinitis pigmentosa.
24. The method of claim 18, 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.
25. The method of claim 18, wherein said cell death is due to an
autoimmune disorder selected from the group consisting of lupus
erythematosus, rheumatoid arthritis and type I diabetes.
26. The method of claim 18, wherein said cell death is due to type
I diabetes.
27. 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.
28. 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.
29. The method of claim 28, wherein said organ or tissue is present
in a storage medium prior to transplant into a mammal.
30. The method of claim 28, 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.
31. 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.
32. 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.
33. A method of extending the lifespan of a mammalian or yeast cell
line, comprising contacting said cell line with a compound of claim
1.
34. The method of claim 33, wherein said contacting comprises
including said compound in a cell growth medium.
35. A method of treating or ameliorating hair loss or premature
graying of the hair in a mammal, comprising contacting the hair or
hair follicles of the mammal in need thereof with a compound of
claim 1.
36. The method of claim 35, wherein hair loss is treated, and said
hair loss is due to male-pattern baldness, radiation, chemotherapy
or emotional stress.
37. 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.
38. The method of claim 37, wherein said compound is applied as
part of an ointment.
39. The method of claim 37, wherein said skin damage is due to
acute over-exposure to the sun, and wherein said treating reduces
blistering and peeling of the skin.
40. 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.
41. 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.
42. 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.
43. 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.
44. 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.
45. 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.
46. 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.
47. 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.
48. 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.
49. 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.
50. 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.
51. 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.
52. The method of claim 51, wherein said compound is administered
topically or orally.
53. The method of claim 52, wherein said compound is formulated as
part of a mouthwash for the treatment, amelioration or prevention
of oral mucositis.
54. The method of claim 52, wherein said compound is formulated as
part of a slow release buccal lozenge.
55. The method of claim 52, wherein said compound is formulated as
part of a suppository.
56. The method of claim 52, wherein said compound is formulated as
part of a gel.
57. The method of claim 52, wherein said compound is administered
through a bladder catheter for the treatment, amelioration or
prevention of bladder mucositis.
58. The method of claim 52, wherein said compound is administered
as part of an enema for the treatment, amelioration or prevention
of proctitis.
59. The method of claim 52, 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.
60. The method of claim 59, wherein said gastrointestinal mucositis
is esophageal mucositis, gastric mucositis, or intestinal
mucositis.
61. The method of claim 51, wherein said compound is administered
by i.v. injection for the treatment, amelioration or prevention of
bone marrow cell death.
62. The method of claim 51, wherein said compound is administered
as part of a pharmaceutical composition comprising a
pharmaceutically acceptable carrier.
63. The method of claim 51, wherein said compound is administered
after chemotherapy or radiation therapy of cancer in said
animal.
64. The method of claim 51, wherein said compound is administered
during chemotherapy or radiation therapy of cancer in said
animal.
65. The method of claim 51, 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 substituted 2-aminobenzamides
and analogs that are inhibitors of caspases. The invention also
relates to the use of these 2-aminobenzamides and analogs 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, J. H.
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, N. A., et al., Nature 356: 768 (1992); Yuan, J., 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, E. S. et. al. Cell, 87, 171
(1996), and Thornberry, N. A. 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.
1 TABLE 1 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, no inhibitors with good efficacy
(IC.sub.50<1 .mu.M) in whole-cell models of apoptosis have been
reported (Thornberry, 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; 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] Thornberry 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. Lett., 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)- methyl ketones as inhibitors of ICE, such
as: 12
[0028] Semple et al. (Bioorg. Med. Chem. Lett., 8, 959-964, 1998)
report the preparation of peptidomimetic aminomethylene ketones as
inhibitors of ICE, such as: 13
SUMMARY OF THE INVENTION
[0029] The invention relates to compound of Formulae I, II and III:
14
[0030] wherein
[0031] R.sub.1 is an optionally substituted alkyl or hydrogen;
[0032] R.sub.3 is an N-protecting group;
[0033] R.sub.2 is hydrogen or optionally substituted alkyl;
[0034] A is CR.sub.6 or nitrogen;
[0035] B is CR.sub.7 or nitrogen;
[0036] C is CR.sub.8 or nitrogen;
[0037] D is CR.sub.9 or nitrogen;
[0038] provided that not more than two of A, B, C or D is nitrogen;
and R.sub.6-R.sub.9 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)alkyn- yl, 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
[0039] 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 are taken together with the carbon atoms to
which they are attached to form a carbocycle or heterocycle;
[0040] E is CR.sub.14, nitrogen, oxygen or sulfur;
[0041] F is CR.sub.15, nitrogen, oxygen or sulfur;
[0042] G is C.sub.16, nitrogen, oxygen or sulfur;
[0043] provided that only one of E, F, G is nitrogen, oxygen or
sulfur; where R.sub.14-R.sub.16 are independently 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)alkyn- yl, 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
[0044] one of R.sub.14 and R.sub.15, or R.sub.15 and R.sub.16, are
taken together with the carbon atoms to which they are attached to
form a carbocycle or heterocycle;
[0045] Q represents an optionally substituted saturated or
partially saturated carbocycle or heterocycle;
[0046] X is a peptide of 1-4 amino acids or a bond; and
[0047] Y is a peptide of 1-4 amino acids or a bond.
[0048] The invention relates to the discovery that the compounds
represented by Formulae I, II and III 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 or multi-organ failure in an animal; 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); reducing the premature death of
skin cells; treating or ameliorating apoptotic cell death in acute
pancreatitus; treating or preventing the inflammatory response in
psoriasis or inflammatory bowel disease; and treating or
ameliorating organ apoptosis after burn injury.
[0049] The present invention provides pharmaceutical compositions
comprising a compound of Formulae I, II and III in an effective
amount to reduce apoptotic cell death in an animal.
[0050] 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, II and III is included in said solutions or media in
order to reduce apoptotic cell death in said organs, tissue or
cells.
[0051] 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.
[0052] 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
[0053] The inhibitors of caspases and apoptotic cell death of the
present invention are compounds having the general Formulae I, II
and III: 15
[0054] or pharmaceutically acceptable salts or prodrugs thereof,
wherein:
[0055] R.sub.1 is an optionally substituted alkyl or hydrogen;
[0056] R.sub.3 is a N-protecting group including
t-butyloxycarbonyl, acetyl, and benzyloxycarbonyl;
[0057] R.sub.2 is hydrogen or optionally substituted alkyl;
[0058] A is CR.sub.6 or nitrogen;
[0059] B is CR.sub.7 or nitrogen;
[0060] C is CR.sub.8 or nitrogen;
[0061] D is CR.sub.9 or nitrogen; provided that not more than two
of A, B, C or D is nitrogen; and R.sub.6-R.sub.9 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
[0062] 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 are taken together with the carbon atoms to
which they are attached to form a carbocycle or heterocycle;
[0063] E is C.sub.14, nitrogen, oxygen or sulfur;
[0064] F is C.sub.15, nitrogen, oxygen or sulfur;
[0065] G is C.sub.16, nitrogen, oxygen or sulfur; provided that
only one of E, F, G is nitrogen, oxygen or sulfur; where
R.sub.14-R.sub.16 are independently 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
[0066] one of R.sub.14 and R.sub.15, or R.sub.15 and R.sub.16 are
taken together with the carbon atoms to which they are attached to
form a carbocycle or heterocycle;
[0067] Q represents an optionally substituted saturated or
partially saturated carbocycle or heterocycle;
[0068] X is a peptide of 1-4 amino acids or a bond; And
[0069] Y is a peptide of 1-4 amino acids or a bond. Where X or Y is
one amino acid, it may be any one of the common 20 amino acids
e.g., Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys,
Tyr, Asp, Asn, Glu, Asn, Lys, Arg and His. Where X is a peptide, it
may be Asp-Glu, Asp-Ala, Asp-Phe, Val-Glu, Leu-Glu, Thr-Glu,
Ile-Glu, Tyr-Glu, Trp-Glu. Where Y is a peptide, it may be Glu-His,
Glu-Ile, Glu-Thr, Glu-Val, Glu-Phe, Thr-His, Val-His, Ala-His and
Glu-Pro.
[0070] 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-.sub.6
alkyl groups, e.g. CH.sub.2OCH.sub.3 and CH.sub.2OCOCH.sub.3
(AM).
[0071] The invention relates to the discovery that the compounds
represented by Formulae I, II and III 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] Preferred embodiments of the compounds of Formulae I, II and
III that may be employed as inhibitors of caspases are represented
by Formula IV: 16
[0074] or pharmaceutically acceptable salts or prodrugs thereof
wherein R.sub.1 R.sub.2-, R.sub.6-R.sub.9 and X are as defined
previously with respect to Formula I.
[0075] 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 taken together are
--OCH.sub.2O--, --OCF.sub.2O--, --(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-- and
--CH.dbd.CH--CH.dbd.CH--; where R.sub.13 is hydrogen, alkyl or
cycloalkyl.
[0076] R.sub.10 is hydrogen, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, C.sub.6-C.sub.10
aryl, C.sub.4-C.sub.7 cycloalkyl, C.sub.6-C.sub.10
aryl(C.sub.1-C.sub.6)alkyl, benzyloxy, substituted benzyloxy, or
NR.sub.11R.sub.12, wherein R.sub.11 and R.sub.12 independently are
hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl,
C.sub.6-C.sub.10 aryl, C.sub.4-C.sub.7 cycloalkyl, C.sub.6-C.sub.10
aryl(C.sub.1-C.sub.6)alkyl, or R.sub.11 and R.sub.12 are combined
to form a heterocyclic ring system including pyrrolidine,
piperidine, piperazine, or morpholine.
[0077] Preferred R.sub.1 is H, Me, Et, t-Bu or AM. Preferred
R.sub.2 is fluoromethyl, acyloxymethyl, arylacyloxymethyl and
aminomethyl. Preferred R.sub.10 is benzyloxy and substituted
benzyloxy. Preferred X is a peptide of 1-2 amino acids or a
bond.
[0078] Exemplary preferred inhibitors of caspases having Formulae
I-IV include, without limitation:
[0079] 2-(Z-amino)benzoyl-Asp-fmk,
[0080] 2-(Z-amino)-3-methylbenzoyl-Asp-fmk,
[0081] 2-(Z-amino)-3,5-dimethylbenzoyl-Asp-fmk,
[0082] 2-(Z-amino)-4-chlorobenzoyl-Asp-fmk,
[0083] 2-(Z-amino)-5-chlorobenzoyl-Asp-fmk,
[0084] 2-(Z-amino)-5-fluorobenzoyl-Asp-fmk,
[0085] 2-(Z-amino)-6-fluorobenzoyl-Asp-fmk,
[0086] cis-2-(Z-amino)-cyclohexanecarboxyl-Asp-fmk,
[0087] 2-(Z-amino)-5-methylbenzoyl-Asp-fmk,
[0088] 2-(Z-amino)-6-methylbenzoyl-Asp-fmk,
[0089] 2-(Z-amino)-6-chlorobenzoyl-Asp-fmk,
[0090] 2-(Z-amino)-3-methoxybenzoyl-Asp-fmk,
[0091] 3-(Z-amino)thiophene-2-carboxyl-Asp-fmk,
[0092] 3-(methoxycarbonylamino)thiophene-2-carboxyl-Asp-fmk,
[0093] cis-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,
[0094] trans-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,
[0095] 2-(Z-amino)benzoyl-Asp-DCB-methylketone,
[0096] methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk,
[0097] Z-Glu-(2-aminobenzoyl)-Asp-fmk, and
[0098] Z-Val-(2-aminobenzoyl)-Asp-fmk.
[0099] where Z is benzyloxycarbonyl, fmk is fluoromethylketone and
DCB is 2,6-dichlorobenzoyloxy.
[0100] 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.
[0101] Useful cycloalkyl groups are C.sub.3-8 cycloalkyl. Typical
cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl and cycloheptyl.
[0102] Useful saturated or partially saturated carbocyclic groups
are cycloalkyl groups as defined above, as well as cycloalkenyl
groups, such as cyclopentenyl, cycloheptenyl and cyclooctenyl.
[0103] Useful halo or halogen groups include fluorine, chlorine,
bromine and iodine.
[0104] 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.
[0105] 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.
[0106] Useful haloalkyl groups include C.sub.1-10 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.
[0107] Useful alkoxy groups include oxygen substituted by one of
the C.sub.1-10 alkyl groups mentioned above.
[0108] 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.
[0109] Useful acylamino groups are any C.sub.1-6 acyl (alkanoyl)
attached to an amino nitrogen, e.g. acetamido, propionamido,
butanoylamido, pentanoylamido, hexanoylamido as well as
aryl-substituted C.sub.2-6 substituted acyl groups.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] Useful heteroaryl groups include any one of the following:
thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl,
furyl, pyranyl, isobenzofuranyl, chromenyl, 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]pyrimidin-4-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.
[0115] Optional substituents include one or more alkyl; halo;
haloalkyl; cycloalkyl; aryl optionally substituted with one or more
lower alkyl, halo, haloalkyl or heteroaryl groups; aryloxy
optionally substituted with one or more lower alkyl, halo,
haloalkyl or heteroaryl groups; aralkyl; heteroaryl optionally
substitued with one or more lower alkyl, haloalkyl and aryl groups;
heteroaryloxy optionally substitued with one or more lower alkyl,
haloalkyl and aryl groups; alkoxy; alkylthio; arylthio; amino;
acyloxy; arylacyloxy optionally substitued with one or more lower
alkyl, halo alkyl and aryl groups; Diphenylphosphinyloxy optionally
substituted with one or more lower alkyl, halo or haloalkyl groups;
heterocyclo optionally substitued with one or more lower alkyl,
halo alkyl and aryl groups; heterocycloalkyloxy optionally
substitued with one or more lower alkyl, halo alkyl and aryl
groups; partially unsaturated heterocycloalkyl optionally
substitued with one or more lower alkyl, halo alkyl and aryl
groups; or partially unsaturated heterocycloalkyloxy optionally
substitued with one or more lower alkyl, halo alkyl and aryl
groups. Particular examples of such optional substituents that may
be present at R.sub.2 include, without limitation, 3-pyrazolyloxy
optionally substituted at the 2, 4 and 5-positions with lower
alkyl; 3-(1-phenyl-3-trifluoromethyl)pyrazolyloxy;
2,6-di(trifluoromethyl)benzoy- loxy; 2,6-dimethylbenzoyloxy,
pentafluorophenoxy; 2,6-dichlorobenzoyloxy;
2-(3-(2-imidazolyl)naphthyl)oxy; diphenylphosphinyloxy;
tetronyloxy; and tetramoyloxy.
[0116] 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.
[0117] 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).
[0118] Examples of prodrugs include compounds of Formulae I-IV
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).
[0119] 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 Formulae I-IV.
[0120] The compounds of this invention may be prepared using
methods known to those skilled in the art. Specifically, compounds
with Formulae I-IV 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 2-aminobenzoic acid, which is either
commercially available or which can be prepared from a commercially
available 2-aminobenzoic acid, such as 2-Z-aminobenzoic acid, gave
amide 2. Oxidation of 2 by Dess-Martin reagent according to Revesz
et al. (Tetrahedron Lett. 35, 9693-9696, 1994) gave 3. Acid
catalyzed cleavage of the ester gave the free acid 4. 17
[0121] An important aspect of the present invention is the
discovery that compounds having Formulae I-IV 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.
[0122] 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 ischemia 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). 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
Alzheimer'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.
[0123] 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.
[0124] 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. (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: 1632-42 (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)); treat or ameliorate apoptotic cell death in acute
pancreatitus; and treat or ameliorate organ apoptosis after burn
injury.
[0125] 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.
[0126] 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)).
[0127] 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)).
[0128] 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)).
[0129] 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)).
[0130] 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)).
[0131] 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.
[0132] 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. 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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 caspase 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.
[0139] The cell death inhibitors of the present invention may also
be used to treat or prevent the inflammatory response in psoriasis
or inflammatory bowel disease.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] Possible pharmaceutical preparations which can be used
rectally include, for example, 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.
[0151] 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 glycol-400 (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.
[0152] 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.
[0153] 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 .beta.-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.
[0154] 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.
[0155] 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.
[0156] 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 paraffin by weight.
[0157] Lotions may be conveniently prepared by dissolving the
active ingredient, in a suitable high molecular weight alcohol such
as propylene glycol or polyethylene glycol.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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).
[0162] 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).
[0163] 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. Metoclopramide 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.
[0164] 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.
[0165] The compositions 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.
[0166] 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-(Z-Amino)benzoyl-Asp-fmk
[0167] Step A. 2-Z-Aminobenzoic acid. To a solution of
2-aminobenzoic acid (0.30 g, 2.2 mmol) in pyridine (2 mL) was added
benzyl chloroformate (0.6 mL, 4.2 mmol) at 0.degree. C. The mixture
was then stirred at room temperature for 1 h, diluted with EtOAc
(50 mL), washed with 2N HCl, water and brine, dried over
Na.sub.2SO.sub.4 and concentrated in vacuo. The crude solid was
washed with 4:1 hexane/EtOAc twice and dried in vacuo to yield the
title compound as a white solid (270 mg, 1.0 mmol, 45%). .sup.1H
NMR (DMSO-d.sub.3): .delta. 11.49 (br s, 1H), 8.22 (d, J=7.5, 1 H),
7.95 (d, J=7.5, 1H), 7.51 (t, J=7.5, 1H), 7.39-7.32 (m, 5 H), 7.05
(d, J=7.5, 1 H), 5.15 (s, 2 H).
[0168] Step B. tert-Butyl
5-fluoro-3-[2-Z-aminobenzoylamido]-4-hydroxypent- anoate. A mixture
of 2-Z-aminobenzoic acid (90 mg, 0.33 mmol),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI,
61 mg, 0.40 mmol), 1-hydroxybenzotriazole hydrate (HOBT, 73 mg,
0.38 mmol), dimethylaminopyridine (DMAP, 22 mg, 0.18 mmol) and
tert-butyl 3-amino-5-fluoro-4-hydroxypentanoate (79 mg, 0.38 mmol)
in THF (10 mL) was stirred at room temperature for 20 h, diluted
with 1:1 hexane/EtOAc (75 mL), washed with water, 2N HCl, water, 2N
NaOH and brine, dried over Na.sub.2SO.sub.4 and cocentrated in
vacuo. The residue was purified by chromatography (3:2
Hexane/EtOAc) to yield the title compound as a yellow hydroscopic
solid (52 mg, 0.11 mmol, 33%).
[0169] Step C. 2-(Z-Amino)benzoyl-Asp(OBu-t)-fmk. A mixture of
periodinane (0.41 g, 0.97 mmol) and tert-butyl
5-fluoro-3-[2-Z-aminobenzoylamido]-4-h- ydroxypentanoate (52 mg,
0.11 mmol) in dichloromethane (15 mL) was refluxed for 20 h, cooled
to room temperature, and 25 mL of saturated sodium bicarbonate
aqueous solution containing 1.0 g of Na.sub.2S.sub.2O.sub.3 was
added. The resulting mixture was stirred for 2 h, diluted with 1:1
hexane/EtOAc (75 mL), washed with water and brine, dried over
Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was
purified by chromatography (3:2 hexane/EtOAc) to yield the title
compound as a hydroscopic yellow solid (45 mg, 0.10 mmol, 91%).
.sup.1H NMR (CDCl.sub.3): .delta. 10.48 (s, 1H), 8.42 (d, J=8.7,
1H), 7.51-7.33 (m, 7H), 7.07 (m, 1 H), 5.30-4.98 (m, 1H), 5.21 (s,
2H), 3.11-2.83 (m, 2 H), 1.44 (s, 9 H).
[0170] Step D. 2-(Z-Amino)benzoyl-Asp-fmk. To a solution of
2-(Z-Amino)benzoyl-Asp(OBu-t)-fmk (45 mg, 0.10 mmol) in 5 mL of
CH.sub.2Cl.sub.2 was added 1 mL of TFA. The resulting solution was
allowed to stir at rt for 1 hr, diluted with EtOAc (75 ml), washed
with water, aqueous Na.sub.2HPO.sub.4 to pH 5 and then brine, dried
over Na.sub.2SO.sub.4 and concentrated in vacuo to give the title
compound as a white solid (15 mg, 0.037 mmol, 38%). .sup.1H NMR
(DMSO-d.sub.6): .delta. 10.63 (s, 1H), 9.08 (s, 1 H), 8.14 (d,
J=7.8, 1 H), 7.76 (d, J=7.8, 1 H), 7.54 (t, J=7.8, 1 H), 7.41-7.35
(m, 5 H), 7.15 (t, J=7.8, 1H), 5.16 (s, 2H), 5.26-4.95 (m, 2H),
4.83 (m, 1H), 3.00-2.64 (m, 2 H).
EXAMPLE 2
2-(Z-Amino)-6-methylbenzoyl-Asp-fmk
[0171] The title compound was prepared in four-steps as described
in Example 1 from 2-amino-6-methylbenzoic acid. .sup.1H NMR
(DMSO-d.sub.6): .delta. 8.85 (s, 1H), 8.68 (s, 1H), 7.49-7.01 (m,
8H), 5.12 (s, 2H), 4.82 (m, 1H), 5.26-4.95 (m, 2H), 3.00-2.64 (m.
2H), 2.26 (s, 3H).
EXAMPLE 3
2-(Z-Amino)-5-methylbenzoyl-Asp-fmk
[0172] The title compound was prepared in four-steps as described
in Example 1 from 2-amino-5-methylbenzoic acid. .sup.1H NMR
(DMSO-d.sub.6): .delta. 10.48 (s, 1H), 9.10 (d, J=9, 1H), 8.00 (d,
J=8.7, 1H), 7.89 (s, 1H), 7.58 (s, 1H), 7.41-7.34 (m, 5H), 5.14 (s,
2H), 4.83 (m, 1H), 5.39-4.41 (m, 2H), 2.94-2.80 (m. 2H), 2.30 (s,
3H).
EXAMPLE 4
2-(Z-Amino)-3-methylbenzoyl-Asp-fmk
[0173] The title compound was prepared in four-steps as described
in Example 1 from 2-amino-3-methylbenzoic acid. .sup.1H NMR
(DMSO-d.sub.6): .delta. 9.05 (s, 1H), 8.73 (s, 1H), 7.38-7.20 (m,
8H), 5.08 (s, 2H), 4.72 (m, 1H), 5.32 (bs, 2H), 2.81-2.66 (m. 2H),
2.21 (s, 3H).
EXAMPLE 5
2-(Z-Amino)-3-methoxybenzoyl-Asp-fmk
[0174] The title compound was prepared in four-steps as described
in Example 1 from 2-amino-3-methoxybenzoic acid. .sup.1H NMR
(DMSO-d.sub.6): .delta. 8.73 (bs, 1H), 8.64 (bs, 1H), 7.57-7.06 (m,
9H), 5.06 (s, 2H), 4.72 (bs, 1H), 5.26-4.97 (m, 2H), 3.78 (s, 3H),
2.78-2.66 (m. 2H).
EXAMPLE 6
2-(Z-Amino)-5-fluorobenzoyl-Asp-fmk
[0175] The title compound was prepared in four-steps as described
in Example 1 from 2-amino-5-fluorobenzoic acid. .sup.1H NMR
(DMSO-d.sub.6): .delta. 10.40 (bs, 1H), 9.13 (bs, 1H), 8.07 (q,
J=5.1, 1H), 7.61 (d, J=6.6, 1H), 7.46-7.33 (m, 6H), 5.15 (s, 2H),
4.81 (bs, 1H), 2.84-2.72 (m. 2H).
EXAMPLE 7
cis-2-(Z-Amino)cyclohexanecarboxyl-Asp-fmk
[0176] The title compound was prepared in four-steps as described
in Example 1 from cis-2-aminocyclohexanecarboxylic acid. .sup.1H
NMR (DMSO-d.sub.6): .delta. 8.28 (bs, 1H), 7.39-7.09 (m, 5H), 4.98
(s, 2H), 4.52-4.45 (m, 1H), 3.99 (bs, 1H), 2.62-2.53 (m, 4H),
1.77-1.23 (m, 8H).
EXAMPLE 8
2-(Z-Amino)-3,5-dimethylbenzoyl-Asp-fmk
[0177] The title compound was prepared in four-steps as described
in Example 1 from 2-amino-3,5-dimethylbenzoic acid. .sup.1H NMR
(DMSO-d.sub.6): .delta. 8.05 (s, 1H), 7.42-7.17 (m, 8H), 5.15 (s,
2H), 5.19-5.03 (m, 2H), 4.87 (m, 1H), 2.30 (s, 3H), 2.26 (s,
3H).
EXAMPLE 9
2-(Z-Amino)-5-chlorobenzoyl-Asp-fmk
[0178] The title compound was prepared in four-steps as described
in Example 1 from 2-amino-5-chlorobenzoic acid. .sup.1H NMR
(DMSO-d.sub.6): .delta. 10.56 (s, 1H), 9.19 (s, 1H), 8.15 (d,
J=9.0, 1H), 7.84 (s, 1H), 7.61 (d, J=9.0, 1H), 7.41-7.37 (m, 5H),
5.16 (s, 2H), 4.81 (m, 1H), 5.41-4.80 (m, 2H), 2.84-2.73 (m,
2H).
EXAMPLE 10
2-(Z-Amino)-6-chlorobenzoyl-Asp-fmk
[0179] The title compound was prepared in four-steps as described
in Example 1 from 2-amino-6-chlorobenzoic acid. .sup.1H NMR
(DMSO-d.sub.6): .delta. 9.17 (d, J=4.2, 1H), 8.95 (s, 1H),
7.74-7.24 (m, 8H), 5.50-5.21 (m, 2H), 5.15 (s, 2H), 4.85-4.78 (m,
1H), 2.98-2.65 (m, 2H).
EXAMPLE 11
2-(Z-Amino)-4-chlorobenzoyl-Asp-fmk
[0180] 5 The title compound was prepared in four-steps as described
in Example 1 from 2-amino-3,5-dimethylbenzoic acid. .sup.1H NMR
(DMSO-d.sub.6): .delta. 10.84 (s, 1H), 9.19 (s, 1H), 8.24 (s, 1H),
7.81 (d, J=8.4, 1H), 7.42-7.24 (m, 6H), 5.18 (s, 2H), 5.25-5.20 (m,
2H), 4.82 (m, 1H), 2.94-2.63 (m, 2H).
EXAMPLE 12
3-(Z-Amino)thiophene-2-carboxyl-Asp-fmk
[0181] Step A. 3-(Z-Amino)thiophene-2-carboxylic acid. The mixture
of methyl 3-aminothiophene-2-carboxylate (0.2 g, 1.27 mmol) in 2N
NaOH (10 mL) was heated at 90.degree. C. for 15 min, then cooled to
0.degree. C. To the resulting solution was added benzyl
chloroformate (1.5 mL, 10.5 mmol) and THF (10 mL). The mixture was
then stirred at room temperature for 1 h, washed with 3:1
hexane:ethyl acetate (2.times.15 mL). The aqueous phase was
acidified with 2N HCl to pH.about.1-2, extracted with ethyl acetate
(3.times.15 mL), washed with water and brine, dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to yield the title
compound as a white solid (70 mg).
[0182] The title compound was then prepared in three steps as
described in Example 1 (B-D). .sup.1H NMR (DMSO-d.sub.6): .delta.
10.43 (s, 1H), 8.74 (s, 1H), 7.81 (d, J=5.4, 1H), 7.73 (d, J=5.4,
1H), 7.44-7.35 (m, 5H), 5.18 (s, 2H), 5.32-5.04 (m, 2H), 4.79 (m,
1H), 2.88-2.67 (m, 2H).
EXAMPLE 13
3-(Methoxycarbonylamino)thiophene-2-carboxyl-Asp-fmk
[0183] The title compound was prepared in four-steps as described
in Example 12 from methyl 3-aminothiophene-2-carboxylate and methyl
chloroformate. .sup.1H NMR (DMSO-d.sub.6): .delta. 10.34 (s, 1H),
8.70 (s, 1H), 7.79 (d, J=5.7, 1H), 7.72 (d, J=5.7, 1H), 5.31-4.80
(m, 3H), 3.70 (s, 3H), 2.96-2.73 (m, 2H).
EXAMPLE 14
Cis-2-(Z-Amino)cyclopentanecarboxyl-Asp-fmk
[0184] The title compound was prepared in four-steps as described
in Example 1 from cis-2-aminocyclopentanecarboxylic acid. .sup.1H
NMR (DMSO-d.sub.6): .delta. 8.35 (s, 1H), 7.34-7.28 (m, 5H), 7.09
(m, 1H), 5.13-4.50 (m, 5H), 4.11 (m, 1H), 2.81 (m, 1H), 2.73-2.51
(m, 2H), 1.91-1.40 (m, 6H).
EXAMPLE 15
Trans-2-(Z-Amino)cyclohexanecarboxyl-Asp-fmk
[0185] The title compound was prepared in four-steps as described
in Example 1 from trans-2-aminocyclohexanecarboxylic acid. .sup.1H
NMR (DMSO-d.sub.6): .delta. 12.48 (s, 1H), 8.26-8.15 (m, 1H),
7.38-7.17 (m, 5H), 5.18-4.47 (m, 5H), 2.67-2.50 (m, 2H), 2.19 (m,
1H), 1.83-1.06 (m, 10H).
EXAMPLE 16
Z-Glu-(2-aminobenzoyl)-Asp-fmk
[0186] Step A. Z-Glu(OBu-t)-2-aminobenzoic acid. To a solution of
Z-Glu(OBu-t)OH (272 mg, 0.81 mmol) in THF (5 mL) was added
N-methylmorpholine (110 .mu.L, 1.1 mmol) at -45.degree. C.,
followed by isobutyl chloroformate (105 .mu.L, 0.81 mmol). The
mixture was stirred at -45.degree. C. for 30 min. and a solution of
anthranllic acid (127 mg, 0.93 mmol) in THF (5 mL) was added,
followed by more N-methylmorpholine (200 .mu.L, 1.82 mmol). The
resulting mixture was stirred overnight and the cooling bath was
allowed to slowly warm to room temperature. After diluted with
EtOAc (100 mL), the mixture was washed with 2N HCl, water,
saturated NaHCO.sub.3, water, 2N HCl, water and brine, dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to yield the product as
a highly hydroscopic white solid (330 mg, 0.72 mmol, 89%). .sup.1H
NMR (DMSO-d.sub.6): .delta. 11.79 (s, 1H), 8.59 (d, J=8.6, 1H),
8.00 (t, J=6.0, 1H), 7.60 (t, J=8.6, 1H), 7.39-7.31 (m, 5H), 7.17
(t, J=7.8, 1H), 5.16-4.99 (m, 2H), 4.08 (m, 1H), 2.33 (m, 2H),
2.08-1.75 (m, 2H), 1.38 (s, 9H).
[0187] Step B. tert-Butyl
5-fluoro-3-[Z-Glu(OBu-t)-(2-aminobenzoyl)amido]--
4-hydroxypentanoate. A mixture of Z-Glu(OBu-t)-2-aminobenzoic acid
(330 mg, 0.72 mmol), EDCI (129 mg, 0.67 mmol), HOBT (104 mg, 0.68
mmol), DMAP (46 mg, 0.38 mmol) and tert-butyl
3-amino-5-fluoro4-hydroxypentanoate (136 mg, 0.66 mmol) in THF (6
mL) was stirred at room temperature for 20 h. After diluted with
1:1 hexane/EtOAc (75 mL), the mixture was washed with water, 2N
HCl, water, 2N NaOH and brine, dried over Na.sub.2SO.sub.4 and
concentrated in vacuo. The residue was purified by chromatography
(3:1 then 3:2 hexane/EtOAc) to yield the title compound as a white
solid (45 mg, 0.068 mmol, 10%).
[0188] Step C. Z-Glu(OBu-t)-[2-aminobenzoyl]-Asp(OBu-t)-fmk. The
title compound was synthesized by a similar procedure as described
in Step C, Example 1 in 58% yield.
[0189] Step D. Z-Glu-[2-aminobenzoyl]-Asp-fmk. The title compound
was synthesized by a similar procedure as described in Step D,
Example 1 in 14% yield. .sup.1H NMR (DMSO-d.sub.6): .delta. 11.46
(s, 1H), 9.18 (s, 1H), 8.57-7.20 (m, 6H), 5.36-4.84 (m, 5H), 4.04
(br s, 1H), 2.95-1.81 (m, 6H).
EXAMPLE 17
Z-Val-(2-Aminobenzoyl)-Asp-fmk
[0190] The title compound was synthesized as described in Example
16 from Z-Val. .sup.1H NMR (DMSO-d.sub.6): .delta. 11.34-11.25 (m,
1H), 9.17-7.17 (m, 11H), 5.42-4.30 (m, 5H), 3.95-3.75 (m, 1H),
2.95-2.57 (m, 2H), 1.92 (m, 1H), 0.91-0.84 (m, 6H).
EXAMPLE 18
2-(Z-Amino)benzoyl-Asp-DCB-methylketone
[0191] 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 potassium 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).
[0192] Step B. Asp(OBu-t)-DCB-methylketone-HCl 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 white 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).
[0193] Step C. 2-(Z-Amino)benzoyl-Asp(OBu-t)-DCB-methylketone To a
solution of 2-(Z-amino)benzoic acid (140 mg, 0.52 mmol) in THF (5
mL) was added N-methylmorpholine (65 .mu.L, 0.59 mmol), followed by
2-methylpropyl chloroformate (70 .mu.L, 0.54 mmol) at -45.degree.
C. After 30 min., a solution of Asp(OBu-t)-DCB-methylketone-HCl
(121 mg, 0.27 mmol) in THF (5 mL) was added to the solution. The
resulting mixture was further stirred overnight and the cooling
bath was allowed to slowly warm to room temperature. It was then
diluted with 1:1 hexane/EtOAc (100 mL), washed with water, 2N NaOH
and brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo.
The residue was purified by chromatography (3:1 hexane/EtOAc) to
yield the title compound as a white solid (365 mg, 0.05 mmol, 19%).
.sup.1H NMR (CDCl.sub.3): 10.90 (s, 1H), 8.49 (d, J=7.5, 1H), 7.87
(dd, J=8.1, 1.8, 1H), 7.56-7.32 (m, 9H), 7.08 (t, J=6.9, 1H), 5.23
(s, 2H), 5.28 (m, 1H), 4.90 (d, J=1.8, 2H), 3.16-2.91 (m, 1H), 1.43
(m, 9H).
[0194] Step D. 2-(Z-Amino)benzoyl-Asp-DCB-methylketone A solution
of 2-(Z-amino)benzoyl-Asp(OBu-t)-DCB-methylketone (35 mg) and TFA
(1 mL) in methylenechloride (3 mL) was stirred at room temperature
for 2 h. The mixture was diluted with EtOAc (70 mL), washed with
saturated Na.sub.2HPO.sub.4 to pH.about.5, and further washed with
water, and brine, dried over Na.sub.2SO.sub.4 and concentrated in
vacuo to yield the title compound as a white solid (10 mg, 0.016
mmol, 33%). .sup.1H NMR (CDCl.sub.3): 10.94 (s, 1H), 8.49 (d,
J=8.4, 1H), 7.87 (dd, J=8.1, 1.5, 1H), 7.53 (m, 1H), 7.45-7.33 (m,
8H), 7.07 (m, 1H), 5.22 (s, 2H), 5.10-5.07 (m, 1H), 4.90 (m, 2H),
3.11 (dd, J=8.4, 19.0, 1H), 2.95 (dd, J=1.8, 19.0, 1H)).
EXAMPLE 19
Methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk
[0195] Step A. tert-Butyl
5-fluoro-3-(2-aminobenzoylamido)-4-hydroxypentan- oate.HCl. A
mixture of tert-butyl 5-fluoro-3-(2-Z-aminobenzoylamido)-4-hyd-
roxypentanoate (80 mg, 0.174 mmol), Pd-C (23 mg) and 6N HCl (0.087
mL) in ethanol (5 mL) was stirred under hydrogen atmosphere at room
temperature for 2 h. The mixture was filtered and the solvent was
evaporated to yield the title product. It was used in next step
without further purification.
[0196] Step B. tert-Butyl
5-fluoro-3-(methoxycarbonyl-Val-2-aminobenzoylam-
ido)-4-hydroxypentanoate. To a solution of methoxycarbonyl-Val-OH
(31 mg, 0.17 mmol) in THF (5 mL) was added N-methylmorpholine (38
.mu.L, 0.34 .mu.mmol) at -45.degree. C., followed by isobutyl
chloroformate (45 .mu.L, 0.034 mmol). The mixture was stirred at
-45.degree. C. for 30 min and a solution of tert-butyl
5-fluoro-3-(2-aminobenzoylamido)-4-hydroxype- ntanoate.HCl in THF
(5 mL) was added, followed by more N-methylmorpholine (50 .mu.L,
0.45 mmol). The resulting mixture was stirred overnight and the
cooling bath was allowed to slowly warm to room temperature. After
dilution with ethyl acetate (50 mL), the mixture was washed with
water and brine, dried over Na.sub.2SO.sub.4 and concentrated in
vacuo. The residue was purified by chromatography (3:2 hexane:ethyl
acetate) to yield the title compound as a white hydroscopic solid
(20 mg, 0.041 mmol, 24%). .sup.1H NMR (CDCl.sub.3): .delta.
11.36-11.24 (m, 1H), 8.54 (d, J=8.7, 1H), 7.55-7.05 (m, 4H), 5.40
(m, 1H), 4.89-3.85 (m, 6H), 3.71 (d, J=1.8, 3H), 2.84-2.61 (m, 2H),
2.29 (m, 1H), 1.46-1.44 (m, 9H), 1.05-0.98 (m, 6H).
[0197] Step C, D. Methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk. The
title compound was synthesized with a similar procedure as
described in Step C and D of Example 1. .sup.1H NMR (DMSO-d.sub.6):
.delta. 12.52 (s, 1H), 11.27 (d, J=6.0, 1H), 9.18 (m, 1H),
7.88-7.54 (m, 4H), 7.20 (t, J=7.5, 1H), 5.39-4.44 (m, 3H), 3.83 (m,
1H), 3.57 (d, J=2.4, 3H), 2.96-2.65 (m, 2H), 2.18 (m, 1H), 0.92 (t,
J=6.3, 6H).
EXAMPLE 20
Enzyme Activity
[0198] The activity of 2-(Z-amino)benzoyl-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.
[0199] 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 (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 355 nm/emission filter
at 460 nm. The data was analyzed using GraphPrism software to give
an IC.sub.50 value of 0.2 .mu.M.
[0200] 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.
Sequence CWU 1
1
1 1 4 PRT Homo sapiens MOD RES (1)..(1) N-terminal acetyl 1 Asp Glu
Val Asp 1
* * * * *