CA2403836A1 - Dipeptide caspase inhibitors and the use thereof - Google Patents

Abstract

The present Invention is directed to novel dipeptides thereof, represented by general Formula (1): where R1-R2 and AA are defined herein. The present invention relates to the discovery that compounds having Formula (1) 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 c11n1ca1 condition in which the loss of cells, tissues or endue organs occurs.

Description

_ ' CA 02403836 2002-10-15 WO 99/47154 PCTNS99/057Z9..
Dipeptide Caspase Inhibitors and the Use Thereof Background of the Invention Field oJ'the Invention This invention is in the field of medicinal chemistry. In particular, the invention relates to dipeptides which are potent inhibitors of caspases. The invention also relates to the use of these dipeptides for reducing or treating apoptotic cell death and/or reducing interleukin 1-~i production.
Description oJBackgrottnd Art 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., Bio1 Rev Cambridge Philos Soc.
26:59-86 (1951); Glucksrnann, A., Archives de Biologie 76:419-437 (1965);
Ellis et al., Dev. 111:591-603 (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 W096/20721).
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)).
Apoptosis is achieved through an endogenous mechanism of cellular suicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and

-2-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); Eilis et al., Ann. Rev Cell Blo. 7: 663 (199I)). 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-S36 (1995)).
Mammalian interleulcin-113 (IL-1D) plays an important role in various pathologic processes, including chronic and acute inflammation and autoimmuue diseases (Oppenheim, J. H. et. ai. Immunology Today. 7, 4S-S6 (1986)). IL-1 is synthesized as a cell associated precursor polypeptide (pro-IL-1 ) that is unable to bind IL-1 receptors and is biologically inactive (Mosley 1S et al., J. Biol. Chem. 262:2941-2944 (I987)). By inhibiting conversion of precursor IL-IB to manwe IL-If3, the activity of interleukin-1 can be inhibited.
Interleulcin-113 converting enzyme (ICE) is a protease responsible for the activation of interleukin-1Li (IL-1B) (Thombetry, N.A., et al., Nature 356:

(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, ICHI, TX/ICH-2, MCH2, MCH3, MCH4, FLICElMACH/MCHS, ICE-LAP6 and ICE,~,III.
2S The proteolytic activity of this family of cysteine professes, whose active site (a cysteine residue) is essential for ICE-mediated apoptosis, appears critical in mediating cell death (Miura et al., Cell 75: 6S3-660 (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 I
summarized these known caspases.

. vwvv ~ ~ / U, ( L '~
CA 02403836 2002-10-15 ' 1~3 ~ ~ SAY 2000

-3-TahlP T
Enz me*

Grou I:
mediators of inflammation cas ase-1ICE

cas ase-4I -II TX ICH-2 cas ase-5IC -III

Grou II:
effectors of tosis cas ase-2ICH-1 mNEDD2 cas ase-3a ain CPP-32 YAMA

cas ase-7Mch-3 ICE-LAP3 CMH-1 Grou III:
activators of a tosis cas ase-6Mch2 --- cas ase-8MACH FLICE MchS

cas ase-9ICE-LAP6 Mch6 Cas ase-10 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 77:1627-164 ( 1991 ); diGiovine et al., Immunology Today 11:13 -(-1990)).
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 (IC2 < 1 N.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.
WO 93/0271 discloses peptide ICE inhibitors with the formula:
Z-Q2-Asp-Q~
wherein Z is an N-terminal protecting group; Q2 is 0 to 4 amino acids such that the sequence Q2-Asp corresponds to at least a portion of the sequence Ala-Tyr-Val-His-Asp (SEQ >D NO:1); Q, comprises an electronegative leaving group. Exemplary

4 ~ 02403836 2002-10-15 dipeptides are Boc-His-Asp-CHiF, Boc-Tyr-Asp-CH2F, Boc-Phe-Asp-CHEF, Ac-His-Asp-CHZF, Ac-Tyr-Asp-CHZF, Ao-Phe-Asp-CHzF, Cbz-His-Asp-CHzF, Cbz-Tyr-Asp-CHiF and Cbz-Phe-Asp-CHZF.
WO 96/03982 discloses aspartic acid analogs as ICE inhibitors with $ the formula:

Rf-N-N CO-CH2-R3 wherein Ri is H or alkyl; R, is a leaving group such as halogen; R, is hcteroaryl-CO or an amino acid residue.
U.S. patent 5,585,357 discloses peptidic ketones as ICE inhibitors with the formula:
C02H Rio I ~N
R~--(AA)n-H O N
O Ra wherein n is 0-2; each AA is independently L-valine or L-alanine; R, is selected from the group consisting of N-benzyloxycarbonyl and other groups;
R" R~, R,o are each independently hydrogen, lower alkyl and other gmups.
Revesz et al. (Tetrahedron Lett. 35, 9693-9696, 1994) report the preparation of ethyl ester tripeptide:
COzEt Z-Vat-Ala-H CH2F
O
as a prodrug of the corresponding acid which is a potent ICE inhibitor.

-5-Summary of the Invention The invention relates to dipeptides of formula I:.

CAyF
R1._AA-H
O
or a pharmaceutically acceptable salt or prodrug thereof; wherein R~ is an N-terminal protecting group; AA is a residue of a non-natural amino acid; RZ is an optionally substituted alkyl or H.
An object of the invention is to provide dipeptide caspase inhibitors and the use thereof.
In accordance with an aspect of the present invention, there is provided a compound of Formula II:
O
H
~. N F
Ri _ R3 Ra O 11 or a pharmaceutically acceptable salt or prodrug thereof, wherein R~ is an N-terminal protecting group; R2 is an optionally substituted alkyl or H; and R3 and R4 independently are haloalkyl, aryl, heterocyclic, heteroaryl, C1_~o alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl or hydroxyalkyl.
In accordance with another aspect of the present invention, there is provided a compound of Fon~nula III:

F
" llI

Sa or a pharmaceutically acceptable salt or prodrug thereof, wherein R~ is an N-terminal protecting group; RZ is an optionally substituted alkyl or H; R5, R.6, R~
and R8 independently are hydrogen, haloalkyl, aryl, heterocyclic, heteroaryl, Ci_,o alkyl, aIkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl or hydroxyalkyl;
provided that at least one of the RS-R8 is other than hydrogen.
In accordance with another aspect of the present invention, there is provided a compound of Formula IV:
R
-- TV
or a pharmaceutically accegtabIe salt or prodrug thereof, wherein R, is an N
terrninal protecting group; Rz is an optionally substituted alkyl or H; R12 is hydrogen or C~_lo alkyl; B is aryl, heteroaryI, saturated or partially unsaturated carbocycle or saturated or partially unsaturated heterocycle which is optionally substituted by hydrogen, halo, Cl-C6 haloalkyl, C6-C,o aryl, C4-C~ cycloalkyl, t ' Ct-C6 alkyl, C2-C6 alkenyl, C2~C6 alkynyl, C6-C,o aryI(Ci-C6)aIkyl, C6-Cla l aryl(C2-C6)alkenyl, C6-CIO aryl(C2-C6)alkynyl, C1-C6 hydroxyalkyl, nitro, amino, cyano, Cl-C6 acylamino, hydroxy, C,-C6 acyloxy, C,-C6 alkoxy, alkylthio, or carboxy.

Sb In accordance with another aspect of the present invention, there is provided a compound of Formula V:
nn n O
H
F
Ria O
Rll Rs Rio V
or a pharmaceutically acceptable salt or prodrug thereof, wherein RI is an N-terrninal protecting group; Rz is an optionally substituted alkyl or H; R,2 is hydrogen or C,_,o alkyl; R9, R,o and R~ 1 are independently hydrogen, C,_,o alkyl, halogen, haIoaIkyl, aryl, heteroaryl, saturated or partially unsaturaxed carbocycle or saturated or partially unsaturated heterocycle;
provided that at least one of R9-R, ~ is halogen, haloalkyl, heteroaryl, saturated or partially unsaturated carbocycle or saturated or partially unsaturated heterocycle.
In accordance with another aspect of the present invention, there is provided a compound selected from the group consisting of:
Z-(3-CN-Ala)-Asp-fmk, Z-( 1-Nal)-Asp-fmk, Z-(3-Me2N-AIa)-Asp-frr~k, ~(2-Abu)-Asp-fmk and Z-Thz-Asp-fmk.

SC
The invention relates to the discovery that the compounds represented by Formula I are potent inhibitors of caspases. The invention also relates to the use of the dipeptides 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, Lelangiectasia, and spinobulbar atrophy; treating heart disease including myocardial infarction, congestive heart failure and cardiomyo~thy; treating retinal disorders; treating autoimmune disorders including lupus erythematosus, rheumatoid arthritis, type I diabetes, SjSgren's syndrome and glomerulonephritis; treating polycystic kidney disease and anemia/erythropoiesis; treating immune system disorders, including AIDS and SCIDS; reducing or. preventing cell, tissue and organ damage during transplantation; reducing or preventing cell line death in industrial biotechnology; reducing or preventing alopecia (hair loss); and reducing the premature death of skin cells.
The present invention provides pharmaceutical compositions comprising a compound of Formula I in an effective amount to reduce l apoptotic cell death in an animal.

CA 02403836 2002-10-15 ' ~ V ~l VV
~E~'., 15 MAY 2000 The present invention also provides preservation or storage solutions for mammalian organs or tissue, or growth media for mammalian or yeast cells, wherein an effective amount of a compound of Formula I is included in said solutions or media in order to reduce apoptotic cell death in said organs, tissue or cells.
Detailed Description of the Invention The inhibitors of caspases and apoptotic cell death of the present invention are compounds having the general Formula I:

_ _ CH2F
Rl .AA H
O
or pharmaceutically acceptable salts or prodrugs thereof, wherein:
1 S R, is an N-terminal protecting group including t-butyloxycarbonyl, acetyl, and benzyloxycarbonyl; AA is a residue of a non-natural amino acid;
and RZ is an optionally substituted alkyl or H.
With respect to R2, prefer .-:d alkyl groups are Ct.~ alkyl groups, e.g.
methyl, ethyl, propyl, isopropyl, isobutyl, pentyl and hexyl groups; and substituted Ct~ alkyl groups, e.g. CH20CH3 and CH20COCH3 (AM).
The invention relates to the discovery that the compounds represented by Formula I are potent 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 l which apoptosis plays a role. These conditions are more fully described bel w.
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.
Preferred embodiments of the compounds of Formula I that may be employed as inhibitors of caspases are represented by Formula II:

O
R~N~N CH2F II
H I

or pharmaceutically acceptable salts or ptndtugs thereof wherein R, and R~ are as defined previously with respect to Formula I; and R, and R, independently are haloalkyl, aryl, heterocyclic, heteroaryl, C,-,o alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl and hydroxyalkyl.
Preferred R, is t-butyloxycarbonyl, acetyl or benzyloxycarbonyl.
Preferred Ri is H, Me, Et, t-Bu or AM. Preferred R~ is C,_,a alkyl, haloallcyl, aryl or heteroaryl, and prefen~ed R, is Me.
Another prefen~ed embodiment of the compounds of Formula I that may be employed as inhibitors of caspases are represented by Formula III:
COZRy R1~N N CHZF III
H R~ R8 H O
or pharmaceutically acceptable salts or prodtugs thereof wherein R, and RZ are as defined previously with respect to Formula I; and Rs, R6, R, and R, independently are hydrogen, haloalkyl, aryl, heterocyclic, carbocyelic, heteroaryl, C,.,o alkyl, alkenyl, alkynyl, aratkyl, aralkenyl, aralkynyl or hydroxyalkyl;
provided that at least one of the Rs-Ra is other than hydrogen.
Preferred R, is t-butyloxycarbonyl, acetyl or benzyloxycarbonyl.
Preferred RZ is H, Me, Et, t-Bu or AM. Preferred Rs is hydrogen, C,_,o alkyl, _g-haloalkyl, aryl or heteroaryl; preferred Rs is hydrogen or Me. Preferred R, is C,_,oalkyl, haloalkyl, aryl or heteroaryl; preferred Ra is hydrogen or Me.
Another preferred embodiment of the compounds of Formula I that may be employed as inhibitors of caspases are represented by Formula IV:

O .

Rly B 0 or pharmaceutically acceptable salts or ptodrugs thereof wherein R, and RZ are as defined previously with respect to Formula I; and R,z is hydrogen or C,_,oalkyl;
B is aryl, heteroaryl, saturated or partially unsaturated carbocycle or saturated or partially unsaturated heterocycle, any of which is optionally substitutal by one or more groups selected from hydrogen, halo, C,-C6 haloalkyl, C6-C,o aryl, C~-C, cycloalkyl, C,-C6 alkyl, C~-C6 alkenyl, Ci-C6 aLkynYl, C6-C10 ~YI(C!-C6)alkyl, C6..CIO aTYI~~'C6)~nYl~ Ce-Clo ~YI~Cz-C~~Yh CnCe hydroxyalkyl, nitro, amino, cyano, C,-C6 acylamino, hydroxy, C,-C6 acyloxy, C,-C6 allcoxy, alkylthio, or carboxy.
Preferred R, is t butyloxycarbonyl, acetyl or benzyloxycarbonyl.
Prefen~ed Ri is H, Me, Et, t-Bu or AM. Preferred R,Z is hydrogen, or Me.
Preferred B is optionally substituted phenyl, thienyl, furyl, pyridyl, cyclohexyl, cyclopentyl, cyclobutyl or cyclopropyl.
Other preferred embodiments of the compounds of Formula I that may be employed as inhibitors of caspases are represented by Formula V:

O

R1 'H

R9 Rio _ PCTItJS99/05729 WO 99/47154 ' _g_ or pharmaceutically acceptable salts or prodrugs thereof wherein R,, Rz and R,i are as defined previously with respect to Formula I and IV; and Rq, R,o and R" independently are hydrogen, C,.,o ~Yh halogen, haloalkyl, optionally substituted aryl, heteroaryl, saturated or partially unsaturated carbocycle or saturated or partially unsaturated heterocycle;
provided that at least one of R9-R" is halogen, haloalkyl, heteroaryl, saturated or partially unsaturated carbocycle or saturated or partially unsaturated heterocycle.
Preferred R, is t-butyloxycarbonyl, acetyl or benzyloxycarbonyl.
Preferred RZ is H, Me, Et, t-Bu or AM. Preferred Rn is hydrogen, or Me.
Preferred R9, R,o and R" independently are hydrogen, methyl, chloro, fluoro, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, chlorofluoromethyl, thienyl, furyl, pyridyl, cyclohexyl, cyclopentyl, cyclobutyl and cyclopropyl.
Exemplary preferred inhibitors of caspases and apoptosis having Formula I include, without limitation:
Boc-Phg-Asp-fmk, Boc-(2-F-Phg~Asp-fmk, Boc-(F,-ValrAsp-fink, Boc-(3-F-Val~Asp-fink, Ac-Phg-Asp-fink, Ac-(2-F-Phg~Asp-fink, Ac-(F,-Val)-Asp-fmk, Ac-(3-F-Val)-Asp-fink, Z_Phg-Asp-fink, Z-(2-F-Phg)-Asp-&nk, Z-(F3-Val~Asp-fmk, Z-Chg-Asp-fmk, Z-(2-Fug~Asp-fmk, Z-(4-F-Phg)-Asp-fmk, Z-(4-Cl-Phg)-Asp-fink.
Z-(3-ThgrAsp-fmk, WO 99/47154 PCT/US99/05729' Z-(2-FuarAsp-fink, Z~2-Via)-Asp_~, Z-(3-FuarAsp-fmk, Z-(3-'Tha)-Asp-fcn?c, Z-(3-Cl-AlarAsp-fmk, 2-(3-F-Ala)-Asp-fmk, Z-(F,-Ala)-Asp-fmk, Z-(3-F-3-Me-Ala)-Asp-fink, Z-(3-Cl-3-F-AIa)-Asp-fink, Z-(2-Me-Val)-Asp-fmk, Z-(2-Me-Ala~Asp-fmk, Z-(2-i-Pr-ø-Alal~Asp-fmk.
Z-(3-Ph-(i-Ala)-Asp-fimk.
Z-(3-CN-Ala)-Asp-fmk, Z-( I NaIrAsp-fink, Z-Cha-Asp-fink, Z-(3-CF; Ala)-Asp-fmk.
Z-(4-CF; Phg)-Asp-fmk, Z-(3-Me.IN-Ala)-Asp-fink, Z-(2-Abu)-Asp-fmk, Z-TIe-Asp-fmk, Z-Cpg-Asp->inlc, Z-Cbg-Asp-fink, Z_Thz_Asp-fink, Z-(3-F-Val)-Asp-fink, and Z-(2-Thg)-Asp-fmk.
Where Z is benzyloxy_carbonyl.,..,BOC is tert.-butoxycarbonyl, Ac is acetyl, Phg is phenylgiycine, 2-F-Phg is (2-fluorophenyl)glycine, F3-Val is 4,4,4-trifluoro-valine, 3-F-Val is 3-fluoro-valine, 2-Thg is (2-thienyl)glycine, Chg is cyclohexylglycine, 2-Fug is (2-furyl)glycine, 4-F-Phg is (4-fluorophenyl)glycine. 4-CI-Phg is (4-chlorophenyl)glycine, 3-Thg is (3-thienyl)glycine. 2-Fua is (2-furyl)alaninc, 2-Tha is (2-thienyl)alanine, 3-Fua is (3-furyl~lanine, 3-Tha is (3-thienyl)alanine, 3-CI-Ala is 3-chloroaianine, 3-F-Ala is 3-fluoroalanine, F; Ala is 3,3,3-trifluoroalanine, 3-F-3-Me-Ala is 3-fluoro-3-methylalanine, 3-CI-3-F-Ala is 3-chloro-3-fluoroaIanine, 2-Me-Val is 2-methylvaline, 2-Me-Ala is 2-methylalanine, 2-i-Pr-~i-Ala is 3-amino-2-isopropylpropionic acid, 3-Ph-~i-Ala is 3-amino-3-phenylpropionic acid, 3-CN-Ala is 3-cyanoalanine, 1-Nal is 3-(1-naphthyi~alanine, Cha is cyclohexylalanine, 3-CFj-Ala is 2-amino-4,4,4-trifluorobutyric acid, 4-CF3-Phg is 4-trifluoromethylphenylglycine, 3-Me.IN-Ala is 3-dimethylaminoalanine, 2-Abu is 2-aminobutyric acid, Tle is tert-leucine, Cpg is cyclopentylglycine, Cbg is cyclobutylglycine, and Thz is thioproline. These amino acid derivatives may be enantiomerically pure or racemic mixtures.
Useful aryl groups are C~." aryl, especially C,.,o aryl. Typical Cue" aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, azulenyl, biphenyl, biphenylenyl and fluorenyl groups.
Useful cycloatkyl groups are C,.s cycloalkyl. Typical cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and cycloheptyl.
Useful satiuated or partially saturated carbocyclic groups are cycloalkyl groups as defined above, as well as cycloalkenyl groups, such as cyclopentenyl, cycloheptenyl and cyclooctenyl.
Useful halo or halogen groups include fluoro, chloro, bromo and iodo.
Useful alkyl groups include straight-chained and branched C,.,o alkyl groups, more preferably C,~ alkyl groups. Typical C,.,o 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.
Useful alkenyl groups are CZ., alkenyl groups, preferably Cz~ alkenyl.
Typical CZ~ alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, and sec.-butenyl.

WO 99/4?154 PCTNS99/05729 Useful alkynyl groups are C~~ alkynyl groups, preferably Ci., alkynyl.
Typical C2-4 aIkynyl groups include ethynyl, propynyl, butynyl, and 2-butynyl groups.
Useful arylalkyl groups include any of the above-mentioned C,.,o alkyl groups substituted by any of the above-mentioned C~." aryl groups. Useful values include benzyl, phenethyl and naphthylmethyl. , Useful arylalkenyl groups include any of the above-mentioned C=., alkenyl groups substituted by any of the above-mentioned Cue" aryl groups.
Useful arylaikynyl groups include any of the above-mentioned CZ~
alkynyl groups substituted by any of the above-mentioned C~." aryl groups.
Useful values include phenylethynyl and phenylpmpynyl.
Useful haloalkyl groups include C,_,o 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.
Useful hydroxyalkyl groups include C,.,o all~Yl groups substituted by hydroxy, e.g. hydroxymethyl, hydroxyethyl, hydroxypropyl and hydroxybutyi groups.
Useful alkoxy groups include oxygen substituted by one of the C,.,o alkyl groups mentioned above.
Useful alkylthio groups include sulphur substituted by one of the C,_,o alkyl gmups mentioned above. Also included are the sulfoxides and sulfoxides of such alkylthio groups.
Useful acylamino groups are any C,.~ acyl (alkanoyl) attached to an amino nitrogen, e.g. acetamido, propionamido, butanoylamido, pentanoylamido, hexanoylamido as weh as aryl-substituted C2~ substituted acyl groups.
Useful acyloxy groups are any C,.o acyl (alkanoyl) attached to an oxy (-O-) group, e.g. acetoxy, propionoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy and the like.

Useful amino groups include NH2, -NHR". and -NR"R,s, wherein R"
and R,s are C1_10 alkyl or cycloalkyl groups as defined above.
Useful saturated or partially saturated heterocyclic groups include tetrahydrofuranyl, PY~Yh PiP~~YI. PiP~nYh PY~lidinyl, imidazolidinyl, imidazolinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, isochromanyl, chromanyl, pyrazolidinyl and pyrazolinyl groups.
Useful hetcroaryl groups include any one of the following: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthiinyl, 2X pyrrolyl, pyrrolyl, imidawlyl, pyra~olyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H indolyl, indolyl, indazolyl, purinyl, 4N-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl, naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, carbazolyl, [i-carbolinyl, phenanthridinyl, acrindiuyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl, phenoxazinyl, 1,4-dihydroquinoxaline 2,3-dionyl, 7-aminoisocoumarin-yl, pyrido[1,2-a]pyrimidin-4-onyl, I,2-benzoisoxawl-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.
Certain of the compounds of the present invention may exist as stereoisomers including optical isomers. The W venuon mcwaes au stereoisomers and both the racemic mixtures of such stereoisomers as well as the individual entantiomers that may be separated according to methods that are well known to those of ordinary skill in the art.
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 hydroxide, Tris(hydroxymethyl)aminomethane (TRIS, tromethane) and glucosamine.

WO 99/47154 'PCT/US99I05729 Examples of prodrugs include compounds of Formula I-V wherein Rz is an alkyl group or substituted alkyl gmup such as CHZOCH, and CH~OCOCH, (AM ester). Further, in the cases where AA contains a carboxylic acid group, examples of prodrugs of Formula I-V wherein RZ is H
includes compounds in which either or both carboxyl groups are esterified (e.g. with a C,~ alcohol) or are in the form of the corresponding amides (e.g.
with a C,~ amine). Other prodiugs of the compounds of the invention include C,.~ esters, thioesters and amides of the compounds of the inveation where one of the R,, R" R" Ra, R,~ or B groups are substituted with a hydroxy, thio or amino group. It is anticipated that ester, thioester and amido derivatives will be more Iipophilic .than the parent compound, will be readily taken up by cells in vivo. It is also anticipated that the esters and thioesters will be cleavod in vivo by endogenous esterases to give the parent hydroxy- or mercapto-substituted compound.
The invention is also directed to a method for treating disorders responsive to the inhibition of apoptosis in animals suffering thereof.
Particular preferred embodiments of compounds for use in the method of this invention are represented by previously defined Formula I-V.
The compounds of this invention may be prepared using methods known to those skilled in the art. Specifically, compounds with Formulae I-V
can be prepared as illustrated by exemplary reactions in Scheme 1. The intermediate i was prepared according to Revesz et al. (Tetrahedron Lett. 35, 9693-9696, 1994). Coupling of 1 with a N-protected amino acid, which is either commercial available or which can be prepared from a commercial available compound, such as Z-phenylglycine-0H (Z-Phg-OH) gave amide 2.
Oxidation of 2 by Dens-Martin reagent according to Revesz et al.
(T'etrahedron Lett. 35, 9693-9696,1994) gave 3 as a mixture of diasteriomers.
Acid catalyzed cleavage of the ester gave the free acid 4.

Schtme 1 Naox Phg-OH + PhCH~OCOCl Z-Phg-OH
HZO
COZBu-t CHEF 1 COZBU-t H;N
~ F Dess-Martin OH Z-Phg-~ 2 EDCr, HOBT, DMAP OH

COzBu-t ~2H
TFA
CHI F Z-Phg-N
Z-Phg-~ H O
O

Other examples of non-natural amino acids that may be used in this process include, without limitation, the enantiomeric and racemic forms of 2-methylvaline, 2-methylalanine, (2-i-propyl)=~3-alanine, phenylglycine, 4-methylphenylglycine, 4-isopropylphenylglycine, 3-bromophenylglycine, 4-bromophenylglycine, 4-chlorophenylglycine, 4-methoxyphenylglycine, 4-ethoxyphenylglycine, 4-hydroxyphenylglycine, 3-hydroxyphenylglycine, 3,4-dihydroxyphenylglycine. 3,5-dihydroxyphenylgiycine, 2,5-dihydrophenylglycine, 2-fluorophenylglycine, 3-fluorophenylglycine, 4-fluorophenylglycine, 2,3-difluorophenylglycine, 2,4-difluorophenylglycine, 2,5-difluorophenylglycine, 2,6-difluorophenylglycine, 3,4-difluorophenylglycine, 3,5-difluorophenylglycine, 2-{trifluoromethyl)phenylglycine, 3-(trifluoromethyl)phenylglycine, 4-(trifluoromethyl)phenylglycine, 2-{2-thienyl~lycine, 2-(3-thienyl)glycine, 2-{2-furyl)glycine, 3-pyridylglycine, 4-fluorophenylalanine, 4-chlorophenylalanine, 2-bromophenylalanine, 3-bromophenylalanine, 4-bromophenylalanine, 2-naphthylalanine, 3-(2-quinoyl)alanine, 3-(9-anthracenyl~lanine, 2-amino-3-phenylbutanoic acid, 3-chlorophenylalanine, 3-(2-thienyl)alanine, 3-(3-thienyl~lanine, 3-phenylserine, 3-(2-pyridyl)serine, 3-(3-pyridyl)serine, 3-(4-pyridyl)serine, 3-(2-thienyl)serine, 3-(2-furyl)serine, WO 99/47154 PGTNS99/05729.

3-(2-thiazolyl)alanine, 3-{4-thiazolyl~lanine, 3-(1,2,4-triazol-1-yl)-alanine, (1,2,4-triazol-3-yl)-alanine, hexafluomvaline. 4,4,4-trifluorovaline, 3-fluorovaline, 5,5,5-trifluoroleucine, 2-amino-4,4,4-trifluorobutyric acid, 3-chloroalanine, 3-fluoroalanine, 2-amino-3-flurobutyric acid, 3-fluoronorleucine, 4,4,4-trifluorothreonine, L-allylglycine, tcrt-Leucine, propargylglycine, vinylglycine, S-methylcysteine, cyclopentylglycine, cyclohexylglycine, 3-hydroxynorvaline, 4-azaleucine, 3-hydroxyleucine, 2-amino-3-hydroxy-3-methylbutanoic acid, 4-thiaisoleucine, acivicin, ibotenic acid, quisqalic acid, 2-indanylglycine. 2-aminoisobutyric acid, 2-cyclobutyl-2-IO phenylglycine, 2-isopropyl-2-phenylglycine, 2-methylvaline, 2,2-diphenylglycine, I-amino-I-cyclopropanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cyclohexanecarboxylic acid, 3-amino-4,4,4-trifluorobutyric acid, 3-phenylisoserine, 3-amino-2-hydroxy-5-methylhexanoic acid, 3-amino-2-hydroxy-4-phenylbutyric acid, 3-amino-3-{4-I S bromophenyl)propionic acid, 3-amino-3-(4-chlorophenyl~ropionic acid, 3-amino-3-(4-methoxyphenyl)propionic acid, 3-amino-3-(4-fluorophenyl~ropionic acid, 3-amino-3-(2-fluorophenyl)propionic acid, 3-amino-3-(4-nitrophenyl)propionic acid, and 3-amino-3-(1-naphthyl)propionic acid. These non-natural amino acids are commercial available from the 20 following commercial suppliers including Aldrich, Sigma, Fluka, Lancaster, ICN, TCI, Advanced ChemTech, Oakwood Products, Indofine Chemical Company, NSC Technology, PCR Research Chemicals, Bachem, Acros Organics, Celgene, Bionet Research, Tyger Scientific, Tocris, Research Plus, Ash Stevens, Kanto, Chiroscience, and Peninsula Lab. The following amino 25 acids can be synthesized according to literature procedures: 3,3,3-trifluoroalanine {Sakai, T.; et al.. Tetrahedron 1996, 51, 233) and 3,3-difluotnalanine {D'Orchymont, H. Synthesis 1993, 10, 961). Other N-protecting groups can be used in the place of Z include Acetyl (Ac), tert-butoxycarbonyl (Boc), methoxycarbonyl or ethoxycarbonyl.
30 An important aspect of the present invention is the discovery that compounds having Formulae I-V are potent 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.
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 S 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, 1 S including but not limited to Alzheimer's disease (Mattson er al. Brain Res.
807: I67-176 (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:

(I990)).
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.
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. Frxp. Med, !84:2067-2072 (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 WO 99/47154 PCTIUS99/OS?22 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 vlvo 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 (HT 1 ) (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. Farp.
Biol.
Med. 117: 450-454 (1998)).
The cell death inhibitors of the inv~tion can be used to reduce or prevent cell death of retinal neurons (Kertner et al. J. Neurosci. 18: 4656-(1998) and Miller et al. Am. J. Yet. Res. 59: I49-I52 (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.
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 Gaklcai Shi, 40: 276-83 (1998)), acute renal failure (Liebetthal et at. Sernin Nephrol. 18: 505-518 ( 1998)), marine tubular epithelial cell death induced by cyclosporine A (Ortiz et al. Kidney Internatio»al Supp. 68: S25-S29 (1998)) and HIV-induced nephropathy (Conaldi et al. J. Clin. Invest 102: 2041-2049 (1998)).
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)).
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-( 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)).

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)).
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)).
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 deficicncy syndrome (SCIDS) and related diseases. The cell death inhibitors can also be used to treat radiation-induced immune suppression.
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 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.
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 WO 99/d7154 PGTNS99/057Z9 these cell death inhibitors in the growth medium in a concentration range of 1-100 M.
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.
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.
Goldberg et al. (Nature Generics 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 I-ID. 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.

WO 99/47154 PCTNS99/05729, 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 with 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 mgllcg, 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, and immune system disorders. Preferably, about 0.01 to about 10 mg/kg is orally administered Lo 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 I S preferably, from about 0.01 to about 5 mg/kg.
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.
In a topical formulation, the compound may be present at a concentration of about 0.01 to 100 mg per gram of carrier.
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 catriers 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 and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally. such as suppositories. as well as suitable solutions for administration WO 99!47154 PCTNS99/05729 by injection 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.
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, furnaric acid, malefic 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.
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.
The pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose. For example, adminisu~ation may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, 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.
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.

p~~S~/057Z9 Suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations andlor 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, tragaeanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcelluiose, 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.
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 glycero! or sorbitol. '/he 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, andlor 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.
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.
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, andlor dextran. Optionally, the suspension may also contain stabilizers.
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.
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 ~i-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.

rc~rius99rosrig 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 corticostctoid 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.
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 1$ soft pacaWn), branched chain fats or oils, animal fats and high molecular weight alcohol (greater than C,~. The preferred carriers are those in which the active ingredient is soluble. Emulsifiers, stabili~rs, humectants and antioxidants may also be included as well as agents imparting color or fragrance, if desired. Additionally, traasdermal penetration enhancers can be employed in these topical formulations. Examples of such enhancers can be found in U.S. Patent Nos. 3,989,816 and 4.444,762.
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.
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.

PCTNS99IQ57Z9~
Lotions may be conveniently prepared by dissolving the active ingredient, in a suitable high molecular weight alcohol such as propylene glycol or polyethylene glycol.
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 procxed 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.
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 l~alth 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.
When the compounds are to be administered to Plants, they may be applied to the leaves andlor stems andlor tlo~rs 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.
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 z ~(s)-Png~ Asp-I~x Step A. Z (S)-Phenylglycine. To a solution of (S)-{+)-phenylglycine $ (2.0 g, 13.2 mmol) in 2 N NaOH aqueous solution (30 mL) was added benzyl chloroformate (2.3 mL, 16.2 mmol) at room temperature. The resulting solution was stirred at room temperature for 5 h, and acidified to pH = 1. The resulting white solid was collected by filtration, washed with water and dried in vacuo to yield the title compound (2.5 g, 8.8 mmol, G7%). 'H NMR
(CDCl3): 8 8.05 (d, J = 8.4, 1H), 7.40-7.28 (m, 10H), 5.12 (d, J = 8.4, 1H), 5.03 (s, 2H).
Step B. tent-Butyl 5 Jluoro-3-(Z (S) phenyl-Gly.,amidoJ-4-hydroxypentanoate. To a solution of Z-(S)-phenylglycine (205 mg, 0.72 mmol) in THF (10 mL) was added 1-(2-dimethylaminopropyl)-3_ ethylcarbodiimide (EDCI) (148 mg, 0.77 mmol), 1-hydroxybenzotriazole hydrate (HOBT) (112 mg, 0.73 mmol) and dimethylaminopyridine (DMAP) (61 mg, 0.50 mrtwl). The resulting mixture was stirred at rt for 5 min, to which was then added a solution of tert-butyl 3-amino-5-fluoro-4-hydroxypentanoate (98 mg, 0.47 mmol) in THF (5 mL). The resulting mixture was stin-ed at rt for three days. The THF was evaporated in vacuo to give a residue, which was dissolved in 50 mL of ethyl acetate. The solution was washed with 2N HCI, saturated NaHC03, brine, dried over NaZSO"
concentrated In vacuo and the residue was purified by flash chromatography (EtOAclHexane 615) to give the title compound as a colorless oil (200 mg, 0.42 mmol, 90%). 'H NMR (CDCI,): S 7.36-7.26 (m, 10H), 6.74-6.49 (m, 1H), 5.98 (ra, 1H~ 5.14-5.02 (m, 3H), 4.50-3.91 (m, 4H), 2.68-2.51 (m, 2H), 1.41-1.33 (m, 9H).
Step C. Z ((S)-PhgJ Asp(Ot-Bu) fmk To a suspension of Dens-Martin periodinane (0.67 g, 1.59 mmol) in dichloromethane ( 1 S mL) was added a solution of tent-butyl 5-fluoro-3-(Z-phenyl-Gly-amidor4-hydroxypentanoate (150 mg, 0.32 mmol) in dichloromethane (5 mL). The mixture was refluxed for 20 h, cooled to rt, and 25 mL of saturated sodium bicarbonate aqueous - ' pCTNS99/057Z9~

solution containing 1.0 g of Na~SiO, was added. The resulting mixture was stirred for 1 h, extracted with 1:1 hexane/EtOAc (3 x 25 mL). The combincd organic phase was washed with water and brine, dried over NaiSO, and concentrated in vacuo to yield the title compound as a hydroscopic white solid g . (140 mg, 0.30 mmol> 94%). 'H NMR (CDCL,): 8 7.37-7.31 (m, IOH), 6.84 (d, J = 6.9, 1 H), .6.03-5.96 (m, 1 H). 5.23-4.65 (m, 6H), 3.03-2.58 (m, 2H), 1.40, 1.29 (2s, 9H).
Step D. Z-[(S)-PhgJ Asp-fm~ To a solution of Z-[(S~-Phg]-Asp(Ot Bu~fmk (140 mg, 0.30 mmol) in 5 mL of CH=Ch was added 1 mL of TFA.
The resulting solution was allowed to stir at rt for 1 h. The solvent was evaporated in vacuo .to give a residue, which was purified by flash chromatography (acetone) to give the title compound as a white solid (110 mg, 0.26 mmol, 87%). 'H NMR (DMSO-d6): 8 8.80 (m, 1H), 8.00 (s, 1H), 7.97-7.67 (m, 1 H), 7.47-7.20 (m, 1 OH), 5.26-4.94 (m, 2H), 5.03 (s, 2H), 4.54 (s,1H), 2.68-2.56 (m, 2H).
Fxample 2 Z-('Z Me-Val)-Asp-Jmk The title compound was prepared from DL-2-methylvaline in four-steps as described in Example 1. 'H NMR (DMSO-d6): 8 8.00 (brs, 1H), 7.35 (s, SH), 4.99 (s, 2H), 5.10-4.53 (m, 3H), 2.88-2.48 (m, 2H), 1.95 (s, 1H), 1.30, 1.27 (2s, 3H), 0.86-0.80 (m, 6H).
Example 3 Z-(Z Me Ala) Asp-Jmk The title compound was prepared from Z-DL-2-methylalanine in three-steps as described in Example 1. 'H NMR (DMSO-d6): 8 8.00 (br s, 1 H), 7.35 (s, 5 H), 4.99 (s, 2 H), 5.10-4.53 (m, 3 H), 2.88-2.48 (m, 2 H), 1.95 (s, H), 1.30, 1.27 (2s, 3 H), 0.86-0.80 (m, 6H).

. PCT/US99/05729 Example ~
Z (2-i-Pr-~-Ala)~Asp-fmk Step A. Ethyl 2-isopropylcyanoacetate. A mixture of ethyl cyanoacetate (2.0 mL, 18.7 mmol), KiCO~ . (4.0 g, 28.9 mmol) and 2-iodopropane (3.2 mL, 32.0 mmol) in acetone (30 mL) was refluxed for 48 h.
It was diluted with 1:1 hexane/EtOAc (120 mL), washed with water and brine, dried over NazSO, and concentrated in vacuo to yield the title compound as a light yellow oil (2.7 g, 17.4 mmol, 93%). 'H NMR (CDCh): S 4.27 (q, J = 7.2, 2H), 3.39 (d, J = 5.7, 1H), 2.42 (m, 1H), 1.32 (t, J = 7.2, 3H), 1.13 (d, J =
7.2, 3H), 1.10 (d, J = 6.6, 3H).
Step B. 3-amino-2-isopropylpropionic acid hydrochloride. A mixture of ethyl 2-isopropylcyanoacetate (2.7 g, I7.4 mmol), concentrated HCl (3.0 mL) and PtOZ (107 mg) in ethanol (50 mL) was shaken under hydrogen atmosphere (48.5 psi) for 24 h. The catalyst was filtered out. The filtrate was concentrated in vacuo to about 5 mL and 2N HCl (15 mL) was added. The resulting solution was washed with 1:1 hexanelEtOAc (2 x 25 mL). The aqueous solution was then refluxed for 24 h, cooled to room temperature, washed with 1:1 hexanelEtOAc (25 mL), concentrated in vacuo to yield the product as a sticky liquid (1.8 g, 10.7 mmol, 61%). 'H NMR (DMSO-d6):
S 12.74 (s, 1 H), 8.03 (s, 2H), 3.01-2.79 (m, 2H), 2.55-2.48 (m, 1 H), 2.00 (m, 1 H), 0.90-0.87 m, 6H).
The title compound was then prepared in four-steps as described in Example 1 from 3-amino-2-isopropylpropionic acid hydrochloride (2-i=Pr-(i-alanine). 'H NMR (DMSO-d6): 8 8.33 (br s,1H), 7.31 (s, SH), 7.21 (br s,1H), 4.98 (s, 2H), 5.22-4.97 (m, 1 H), 4.64-4.50 (m, 1 H), 3.16 (m, 2H), 2.62 (m, 2H), 2.25 (m, 1 H), 1.70 (m, 1 H), 0.89-0.82 (m, 6H).

pGTIUS99/05729 Exarrrple S
Z (F3-Yal) Asp-Jmk The title compound was prepared in four-steps as described in Example S 1 from 4,4,4-trifluoro-DL-valine (Fz-Val). 'H NMR (DMSO-d6): b 12.53 (br s, 1H), 8.74-7.76 (m, 2H), 7.34 (s, SH), 5.24-4.32 (m, 4H), 5.05 (s, 2H), 2.92-.
2.58 (m, 3H), 1.24-0.85 (m, 3H).
Fxamplt 6 ~(2-Tlrg) Asp-Imk The title compound was prepared in four-steps as described in Example t from 2-thienyl-DL-glycine (2-'Ihg). 'H NMR (DMSO-d6): S 8.80 (m, 1H), 8.08 (br s, 1H), 7.45-6.94 (m, 8H), 5.51 (d, J = 7.8, 1H), 5.04 (s, 2H), 5.20 4.80 (m, 2H), 4.55 (m, 1H), 2.62 (m, 2H).
Example 7 Z-(2-F-Phg) Asp-jmk The title compound was prepared in four-steps as described in Example 1 from 2-fluorophenyl-DL-glycine (2-F-Phg). 'H NMR (DMSO-d6): 512.45 (br s,1H), 8.73 (s, 1H), 8.10 (s,1H), 7.33-7.16 (m, 9H), 5.46 (d, J = 6.9,1H), 5.04 (s, 2H), 5.20-4.32 (m, 3H), 2.70-2.40 (m, 2H).
Exa'nPl~ 8 ~(L-2-Tha)-AsP-I~
The title compound was prepared in four-steps as described in Example 1 from 3-(2-thienyl)-L-alanine (2-Tha). 'H NMR (DMSO-d6): 8 8.62 (br s, 1 H), 7.65 (s, 1 H), 7.33-6.90 (m, 7H), 4.99 (s, 2H), 5.20-4.85 (m, 1 H), 4.85 (br s, 1H), 4.23 (br s, 1H), 3.19-2.9b (m, 3H), 2.66 (br s, 2H)..

Examplt 9 Z-j(R)-PhgJ Asp-fmk The title compound was prepared in four-steps as described in Example 1 from (Rrphenylglycine ((RrPhg) as a white solid. 'H NMR (DMSO-ds): 8 8.80 (s, 1H), 8.00 (m, 1 H), 7.39-7.33 (m, 10 H), 5.27 (m, 1 H), 5.03 (s, 2 H), 4.SS (s, l H).
Example 10 Z (3-Ph-,l3-Alaj Asp-jmk The title compound was prepared in four-steps as described in Example 1 from (DL)-3-amino-3-phenylpropionic acid (3-ph-~i-Ala) as a white solid.
'H NMR (DMSO-d~: b 8.54 (s, 1H), 7.92 (m, 1 H), 7.30 (m, 10 H), 5.04-4.45 (m, 6 H), 2.60-2.58 (m, 2 H).
Exanrplc 11 ~(4-Cl-Phi .4sp-Jrmk The title compound was prepared in four-steps as described in Example 1 from (DL)-2-(4-chlorophenyl)glycine (4-CI-Phg) as a brown solid. 'H NMR
(DMSO-db): 8 8.78 (s, 1H), 8.06 (m,1 H), 7.40-7.33 (m, 9 H), 5.28-5.26 (m, 2 H), 5.03 (s, 2 H), 4.54 (s, 1 H), 2.64 (m, 2 H).
Example 12 Z-(4 F Phg) Asp-fmk The title compound was prepared in four-steps as described in Example 1 from (DL~2-(4-fluorophenyl)giycine (4-F-Phg) as a brown solid ( 109 mg, 0.25 mmol). 'H NMR (DMSO-db): 8 8.04 (s. 1H), 7.93 (s, 1 H), 7.43-7.33 (m, 9 H), 5.27 (m, 2 H), 5.07 (s, 2 H), 4.54 (s, 1 H), 2.88-2.72 (m, 2 H).

_ . PCTlUS99/05729 F.~cample 13 Z j(L)-2-ThgJ Asp-fmk The title compound was prepared in four-steps as described in Example 1 from (L)-(2-thienyl)glycine ((L)-2-Thg) as a brown solid. 'H NMR (DMSO-d6): b 8.80 (s, 1H), 8.10 (s, 1 H), 7.87-7.85 (m, 1 H), 7.34 (m, 5 H), 7.05-

6.97 (m, 2 H), 5.51-5.49 (m, 1 H), 5.05 (s, 2 H), 4.74 (s, 2~H), 4.59-4.56 (m, 1 H), 2.78-2.65 (m, 2 H).
Fxample 14 Z (3-The Asp-fmk The title compound was prepared in four-steps as described in Example 1 from (DL~(3-thienyl)glycine (3-Thg) as a solid. 'H NMR (DMSO-db): 8 8.79 (s, IH), 8.00 (s, 1 H), 7.34 (m, 5 H~ 7.13 (s, 1 H), 5.33 (m, 2 H), 5.04 (s, 2 H), 4.56-4.36 (m, l H), 2.70-2.59 (m, 2 H).
Exa~»ple 1 S
~((L) 3-Thgj Asp-fmk The title compound was prepared in four-steps as described in Example 1 from (Ll-(3-thienyl)glycine ((L~3-Thg) as a solid. 'H NMR (DMSO-db): 8 8.77 (s, 1 H), 7.99 (s, l H), 7.34 (m, 5 H), 7.14 (s, l H), 5.33 (m, 1 H), 5.04 (s, 2 H), 4.56 (m, 1 H).
Fxamplt l6 ~j(L~3-CN-AIa~.Asp-fmk The title compound was prepared in four-steps as described in Example 1 from (L)-3-cyano-alanine ((Lr3-CN-Ala) as a brown solid. 'H NMR
(DMSO-db): S 8.59 (s, 1 H), 7.97-7.82 (m, 1 H), 7.34 (m, 5 H), 5.06 (s, 2 H), 4.54 (s, 1 H), 4.38 (s, 1 H), 2.93-2.81 (m, 2 H), 2.61 (m, 2 H).

rcrnls99ros7z9 Fxample 17 Z j(L)-1-Nal) Asp-Jmk The title compound was prepared in four-steps as described in Example 1 from (L)-3-(1-naphthylralanine as a brown solid. 'H NMR (DMSO-dd): 8 8.19-8.17 (m, IH), 7.93-7.29 (m, 14 H), 4.92 (s, 2 H), 4.61-4.36 (s, 3 H), 3.45 (m, 2 H), 2.72 (m, 2 H).
Example 18 Z j(L)-Cka~Asp~'mk The title compound was prepared in four-steps as described in Example 1 from (L)-3-cyclohexylalanine as a brown solid. 'H NMR (DMSO-d6): 8

7.33 (m, 5 H), 5.02 (s, Z H), 4.02 (s, 1 H), 2.73 (m, 2 H), 1.61-1.12 (m, 13 H).
Fxampk 19 ~-j(L) 3-C1 AIaJ Asp-Jmk The title compound was prepared in four-steps as described in Example 1 from (L)-3-chloroalanine as a brown solid. 'H NMR (DMSO-db): S 7.92 (s, 1 H), 7.63 (s, IH), 7.34 (m, 5 H), 5.04 (s, 2 H), 2.72 (m, 2 I->).
F,xample l0 Z (3-F Ala~Asp-Jmk The title compound was prepared in four-steps as described in Example 1 from (DL~3-fluoroalanine as a brown solid. 'H NMR (DMSO-d6): 8 7.95-7.66 (m, IH), 7.35 (m, 5 H), 5.05 (s, 2 H), 4.63-4.4? (m, I H).

Example 21 Z (3-CF,-Ala~Asp-fmk The title compound was prepared in four-steps as described in Example 1 from 2-amino-4,4,4-trifluorobutyric acid (3-CF3-Ala) as a brown solid. 'H
NMR (DMSO-db): 8 7.86-7.84 (d, 1 H), 7.62-7.59 (d, 1H), 7.34 (m, 5 H), 5.05 (s, 2 H), 4.57 (s, 1 H), 2.72-2.65 (m, 2 H).
Example 22 Z (4 CF,-Phgj-Asp-jmk The title compound was prepared in four-steps as described in Example 1 from 4-trifluoromethylphenylglycine (4-CF,-Phg) as a solid. 'H NMR
(DMSO-db): S 8.86 (s, 1 H), 8.18 (s, 1 I~, 7.70-7.64 (m, 4 I-~, 7-34 (m, 5 H), 5.42 (s, 1 H), 5.04 (s, 2 H), 4.55 (s, 1 H).
Example 23 Z-(3-Me~IV-AIarAsp-jmk The title compound was prepared in four-steps as described in Example 1 from (DL)-dimethylamino-alanine (3-MeiN-Ala) as a solid. 'H NMR
(DMSO-db): 8 7.40-7.34 (m, 5 H), 5.06-4.99 (m, 3 H), 4.57-4.37 (m, 1 H), 2.60 (s,10 H).
Example 24 2~(3-F 3-Me Alaj Asp-~'mk The title compound was prepared in four-steps as described in Example 1 from 2-amino-3-fluorobutyric acid (3-F-3-Me-Ala) as a solid. 'H NMR
(DMSO-dd): 8 8.54 (s, 1 H), 7.74-7.68 (m, 1 H), 7.34 (m, 5 H), 5.03 (s, 2 H), 4.54 (s, 1 H), 2.55 (m, 2 H), 1.39-1.22 (m, 3 H).

WO 99/47154 PGT/US99/05729~

Example 2S
Z ((L)-CHgJ Asp-Jmk The title compound was prepared in four-steps as described.in Example 1 from (L)-cyclohexylglycine ((L)-Chg) as a yellow solid. 'H NMR (DMSO-db): 8 8.38 (s, 1 H), 7.33 (m, 5 H), 5.00 (s, 2 H), 4.56-4.44 (m, 1 H), 3.86-3.80 (m, 1 H), 2.67 (m, 2 H), I .62-1.56 (m, I I H).
I 0 Example 26 Z (Z-Fug) ,4sp-jrnk The title compound was prepared in four-steps as described in Example 1 from 2-furyl-Gly. 'H NMR (DMSO-d6): 8 8.59 (br s, I H), 8.09-7.47 (m, 3 H), 7.32-7.26 (m, 5 H), 6.94 (m, 1 H), 6.41-6.33 (m, 2 H), 5.36 (m, 1 H), 5.20-4.95 (m, 2 H), 5.04 {s, 2 H), 5.46 (m, 1 H), 2.56-2.40 (m, 2H).
Eacample 27 Z (3-F Val) Asp-fmk The title compound was prepared in four-steps as described in Example 1 from 3-fluoro-Val. 'H NMR (DMSO-d6): b 8.57 (br s, 1 H), 7.85-7.57 (m, 1 H), 7.39 {s, 5 H), 7.08 (s, 2 H), 4.59 (m, I H), 4.33 (m, 1 H), 2.65 (m, 2 H), 1.35 (d, J = 21.9, 6H).
Example 28 ~(2 Abu) Asp-Jmk The title compound was prepared in four-steps as described in Example 1 from 2-aminobutyric acid (Abu) as a white solid. 'H NMR (DMSO-d6), 8 _ . PC'fNS99105729 0.831 (t, 3H, J=7), I .60 (m, 2H), 2.90 (m, I H), 4.47 (m, 1 H), 5.01 (m, 2H), 5.20 (m, 1 H), 7.34 (s, SH), 7.48 (m, 1 H), 8.23 (m, I H).
Example 29 ~Tle Asp jmk The tide compound was prepared in four-steps~as described in Example 1 from ten-Leucine (Tle). 'H NMR (DMSO-d6): b 8.51 (br s, 1 H), 7.36 (br s, SH), 5.04 (br s, 2H), 5.30-4.95 (m, 2H), 4.63 (br s, 1H), 3.89 (s, 1H), 2.80-2.50 (m, 2H), 0.91 (s, 9H).
Fxampk 30 z-cpg Asp-I~
The tide compound was prepared in four-steps as described in Example 1 from cyclopentylglycine (Cpg). 'H NMR (DMSO-dd): 8 7.37-7.28 (m, SH), 6.85 (d, J = 7.5, I H), 5.12-5.03 (m, 2H), 5.09 (s, 2H), 4.84 (br s, 1 H), 4.07 (m, 1H), .2.90-2.75 (m, 2H), 2.28 (m, 1H), 1.80-1.30 (m, 8H).

. CA 02403836 2002-10-15 1 V I
~'~~S 15 MAY 2000 Example 31 Enzyme Activity The activity of Cbz-Phg-Asp-fmk as an inhibitor of caspase-3 (CPP32) was measured in a fluorometric enzyme assay. Recombinant caspase-3 protein was prepared by expressing DNA clones encoding these enzymes in an insect host cell (sue cells) using baculovirus as the vector. See, Webb, N.R. et al., "Expression of proteins using recombinant Baculovirus," Techniques 2:173-_. 188 (1990). 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.
Caspase-3 activity was measured using the following buffer conditions: 100 mM HEPES pH 7.5, with 10% sucrose, 1 °!o CHAPS. The peptide substrate for caspase-3 is Asp-Glu-Vat-Asp-AMC (SEQ m 10:2).
Substrate was used at the K", concentrations of 5 EtM. The assay for enzyme activity was typically carried out at 30°C for 30 minutes.
Table II lists the IC2 of Cbz-Phg-Asp-fmk and other dipeptides as - inhibitors of caspase-3.

WO 99147154 ~ PCTNS99I05729 Table 11: Activity ojDipeptide as Inhibitor ojCaspase-3 Name Caspase-3 IC,o (1tM) Z-[(S~-Phg]-Asp-fink 0.10 Z-(2-Me-Val)-Asp-fink 2.3 Z-(2-Me-Ala)-Asp-fink 1.4 Z-(2-i-Pr- -Ala)-Asp-fink1.4 Z-(F3-Vai)-Asp-$nk 0.60 Z-(2-Thg)-Asp-fmk 0.28 ?r(2-F-Phg~Asp-fmk 0.36 Z-(L-Tha)-Asp-fink 0.15 Z-[(RrPhg]-Asp-fink 0.39 Z-(2-Fug)-Asp-fink 0.44 Z-(3-Ph- -Ala)-Asp-fmk 4-7 Z-(4-Cl-Phg)-Asp-fmk 0.27 Z-(4-F-Phg)-Asp-fmk 1.2 Z-[(LrChgJ-Asp-fmk 0.10 Z-(3-F-V al}-Asp-fink 0.44 Z-[(L)-2-Thg]-Asp-fmk 0.51 Z-(3-Tlig)-Asp-f~ 0.34 Z-[(L)-3-Thg]-Asp-fink 0.26 Z-[(L)-3-CN-Ala]-Asp-8nk0.53 Z-[(L~1-Nal]-Asp-fmk 1.0 Z-[(L)-3-CI-Ala]-Asp-fmk1.0 Z-[(LrChg]-Asp-fink 0.90 Z-(3-F-Ala~Asp-fmk 0.9 Z-(3-CF,-Ala)-Asp-fmk 2.6 Z-(4-CF3-Phg)-Asp-fmk 1.2 Z-(3-MeiN-Ala)-Asp-fink9.6 Z-(3-F-3-Me-AlarAsp-fink0.48 Z-2-Abu-Asp-fmk 0.10 Fxample 32 In Vivo Activity Cbz-Phg-Asp-fmk was examined as a protectant against lethality in mice caused by an anti-Fas antibody (clone Jo2) (Rodriguez, I. et al J. Exp.
Med 184:2067-2072 (1996). Swiss Webster female mice weighing between 16-20 g were used. Food and water were given ad libium. Purified hamster CA 02403836 2002-10-15 ' rcrius99ros7a9 anti-mouse Fas monoclonal antibody (clone Jo2, commercially available) 1 mg/ml in phosphate buffered saline was dosed i.v. at 6 p.g! mouse (80 ~tl).
Cbz-Phg-Asp-fink was dissolved in O.OSM Tris base adjusted to pH 8.5 with HCI. The concentration of Cbz-Phg-Asp-fmlc was 1-10 mg~ml dosed as a single bolus i.v. in a volume of 1-5 p.Ug body weight. Five groups of mice were injected with 6 ug of Fas antibody per mouse. S minutes following the antibody injection the mice received either Tris vehicle control or 1, 2.5, 10 or 50 mg/kg of Cbz-Phg-Asp-fmk. The survival of the mice was followed throughout 24 hours.
Table III lists the percent survival of mice treated with Cbz-Phg-Asp-fmk.
Table Ill: Percent survival over time due to administration oJCbz Phg Asp jmk Vehicle 1 2.5 10 control mg/kgliv. mg/kgl'~.v.mg/lcP,/~~.v.mg/kg/i.v.

1 h 0% 100% 100% 100% 100%

3 h - 14.3% 28.6% 42.9% 100%

~ h - 0% 0% 14.3% 42.9%

The results in Table III show that a compound of the present invention is a potent inhibitor of antiFas induced lethality in mice.
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 and publications cited herein are fully incorporated by reference herein in their entirety.

SEQUENCE LISTING
<110> Cytovia Inc.
<120> Dipeptide Caspase Inhibitors and the Use Thereof <130> 184-324 <140> 2,323,439 <141> 1999-03-16 <150> US 60/078,051 <151> 1998-03-16 <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 1 Ala Tyr Val His Asp <210> 2 <211> 4 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: peptide <400> 2 Asp Glu Val Asp

Claims (50)

EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A compound of Formula II:
or a pharmaceutically acceptable salt or prodrug thereof, wherein R1 is an N-terminal protecting group; R2 is an optionally substituted alkyl or H; and R3 and R4 independently are haloalkyl, aryl, heterocyclic, heteroaryl, C1-10 alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl or hydroxyalkyl.

2. A compound according to claim 1, wherein said compound is selected from the group consisting of:
Z-(2-Me-Val)-Asp-fmk and Z-(2-Me-Ala)-Asp-fmk;
wherein Z is benzyloxycarbonyl and fmk represents a fluoromethyl group bonded to the carboxy terminal of the amino acid.

3. A compound of Formula III:
or a pharmaceutically acceptable salt or prodrug thereof, wherein R1 is an N-terminal protecting group; R2 is an optionally substituted alkyl or H; R5, R6, R7, and R8 independently are hydrogen, haloalkyl, aryl, heterocycliC1 heteroaryl, C1-10 alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl or hydroxyalkyl; provided that at least one of the R5-R8 is other than hydrogen.

4. A compound according to claim 3, wherein said compound is selected from the group consisting of:
Z-(2-i-Pr-.beta.-Ala)-Asp-fmk and Z-(3-Ph-(i-Ala)-Asp-fmk wherein Z is benzyloxycarbonyl and fmk represents a fluoromethyl group bonded to the carboxy terminal of the amino acid.

5. A compound of Formula IV:
or a pharmaceutically acceptable salt or prodrug thereof, wherein R, is an N-terminal protecting group; R2 is an optionally substituted alkyl or H; R12 is hydrogen or C1-10 alkyl; B is aryl, heteroaryl, saturated or partially unsaturated carbocycle or saturated or partially unsaturated heterocycle which is optionally substituted by hydrogen, halo, C1-C6 haloalkyl, C6-C10 aryl, C4-C7 cycloalkyl, C1-C6 alkyl, alkenyl, C2-C6 alkynyl, C6-C10 aryl (C6-C10 alkyl, C6-C10 aryl (C2-C6) alkenyl, C6-C10 aryl (C2-C6) alkynyl, C1-C6 hydroxyalkyl, nitro, amino, cyano, C1-C6 acylamino, hydroxy, C1-C6 acyloxy, C1-C6 alkoxy, alkylthio, or carboxy.

(. A compound according to claim 5, wherein R1 is t-butyloxycarbonyl, acetyl or benzyloxycarbonyl.

7, A compound according to claim 5, wherein R2 is H, Me or acetoxymethyl.

8 A compound according to claim 5, wherein B is an optionally substituted phenyl or cyclohexyl.

9. A compound according to claim 5, wherein R12 is H.

10 A compound according to claim 5, wherein said compound is selected from the group consisting of:
Boc-Phg-Asp-fmk Boc-(2-F-Phg)-Asp-fmk Ac-Phg-Asp-fmk Ac-(2-F-Phg)-Asp-fmk Z-Phg-Asp-fmk Z-(2-F-Phg)-Asp-fmk Z-Chg-Asp-fmk Z-(2-Fug)-Asp-fmk Z-(4-F-Phg)-Asp-fmk Z-(4-Cl-Phg)-Asp-fmk Z-(3-Thg)-Asp-fmk Z-(4-CF3-Phg) -Asp-fmk Z-Cpg-Asp-fmk Z-Cbg-Asp-fmk and Z-(2-Thg)-Asp-fmk wherein Z is benzyloxycarbonyl and fmk represents a fluoromethyl group bonded to the C-terminal ketone of the amino acid residue.

11 A compound of Formula V
or a pharmaceutically acceptable salt or prodrug thereof, wherein R, is an N-terminal protecting group; R2 is an optionally substituted alkyl or H; R12 is hydrogen or C1-10 alkyl; R9, R10, and R11 are independently hydrogen, C1-10 alkyl, halogen, haloalkyl, aryl, heteroaryl, saturated or partially unsaturated carbocycle or saturated or partially unsaturated heterocycle; provided that at least one of R9-R10 is halogen, haloalkyl, heteroaryl, saturated or partially unsaturated carbocycle or saturated or partially unsaturated heterocycle.

12. A compound according to claim 11, wherein R1 is t-butyloxycarbonyl, acetyl or benzyloxycarbonyl.

13. A compound according to claim 11, wherein R2 is H, Me or acetoxymethyl.

14. A compound according to claim 11, wherein R12 is H.

15. A compound according to claim 11, wherein R12 and R9 are H, R,o is alkyl or haloalkyl, and R11 is CF3.

16. A compound according to claim 11, wherein R12 is H, R9 and R10 independently are hydrogen, alkyl or haloalkyl, and R11 is F or heteroaryl.

17. A compound according to claim 11, wherein said compound is selected from the group consisting of:
Boc-(F3-Val)-Asp-fmk, Boc- (3-F-Val)-Asp-fmk, Ac-(F3-Val)-Asp-fmk, Ac-(3-F-Val)-Asp-fmk, Z-(3-F-Val)-Asp-fmk, Z-(3-Cl-Ala)-Asp-fmk, Z-(3-F-Ala)-Asp-fmk, Z-(F3-Ala)-Asp-fmk, Z-(3-F-3-Me-Ala)-Asp-fmk, Z-(3-Cl-3-F-Ala)-Asp-fmk, Z-(3-CF3-Ala)-Asp-fmk, Z-Tle-Asp-fmk, Z-(F3-Val)-Asp-fmk, Z-(2-Fua)-Asp-fmk, Z-(2-Tha)-Asp-fmk, Z-(3-Fua)-Asp-fmk, Z-(3-Tha)-Asp-fmk, Z-Cha-Asp-fmk;
wherein Z is benzyloxycarbonyl and fmk represents a fluoromethyl group bonded to the carboxy terminal of the amino acid.

18. A compound selected from the group consisting of:
Z-(3-CN-Ala)-Asp-fmk, Z-(1-Nal)-Asp-fmk, Z-(3-Me2N-Ala)-Asp-fmk, Z-(2-Abu)-Asp-fmk and Z-Th2-Asp-fmk;
wherein Z is benzyloxycarbonyl and fmk represents a fluoromethyl group bonded to the carboxy terminal of the amino acid.

19. A pharmaceutical composition, comprising a compound of claim 1, 3, 5,11 or 18 and a pharmaceutically acceptable carrier.

20. 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, 3, 5,11 or 18.

21. A method of treating or ameliorating cell death in the central or peripheral nervous system, retinal neurons, cardiac muscle or immune system cells of an animal, comprising administering to the animal in need of such treatment or ameliorating an effective amount of a compound of claim 1, 3, 5, 11 or 18.

22. The method of claim 21, 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; or (f) spinal cord injury.

23. The method of claim 21, wherein said cell death is in the central or peripheral nervous system and is due to expansion of trinucleotide repeats of specific genes.

24. The method of claim 21, wherein said cell death is due to Huntington's Disease.

25. The method of claim 21, 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.

26. The method of claim 21, wherein said cell death is in retinal neurons and is due to increased intraocular pressure, age-related macular degeneration or retinitis pigmentosa.

27. The method of claim 21, 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.

28. The method of claim 21, wherein said cell death is due to an autoimmune disorder selected from the group consisting of lupus erythematosus, rheumatoid arthritis and type I diabetes.

29. The method of claim 21, wherein said cell death is due to type I diabetes.

30. A method of treating or preventing polycystic kidney disease, renal amyloidosis, acute renal failure, cyclosporine A induced murin tubular epithelial cell death, HIV-induced nephropathy or anemia/erythropoiesis in an animal, comprising administering to the animal in need of such treatment or preventing an effective amount of a compound of claim 1, 3, 5, 11 or 18.

31. 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, 3, 5, 11 or 18.

32. The method of claim 31, wherein said organ or tissue is present in a storage medium prior to transplant into a mammal.

33. The method of claim 31, 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.

34. 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 host immune cells, comprising administering to said host in need thereof an effective amount of a compound of claim 1, 3, 5, 11 or 18.

35. 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, 3, 5, 11 or 18.

36. A method of extending the lifespan of a mammalian or yeast cell line, comprising contacting said cell line with a compound of claim 1, 3, 5, 11 or 18.

37. The method of claim 36, wherein said contacting comprises including said compound in a cell growth medium.

38. 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, 3, 5, 11 or 18.

39. The method of claim 38, wherein hair loss is treated, and said hair loss is due to male-pattern baldness, radiation, chemotherapy or emotional stress.

40. 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, 3, 5, 11 or 18.

41. The method of claim 40, wherein said compound is applied as part of an ointment.

42. The method of claim 40, wherein said skin damage is due to acute over-exposure to the sun, and wherein said treating reduces blistering and peeling of the skin.

43. A method of treating or ameliorating sepsis in an animal, comprising administering to the animal in need thereof an effective amount of a compound of claim 1, 3, 5, 11 or 18.

44. 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, 3, 5, 11 or 18.

45. 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, 3, 5, 11 or 18.

46. 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, 3, 5, 11 or 18.

47. 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, 3, 5, 11 or 18.

48. A method of treating or ameliorating radiation or ultraviolet-in adiation induced cell death in an animal, comprising administering to the animal in need thereof an effective amount of a compound of claim 1, 3, 5, 11 or 18.

49. 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, 3, 5, 11 or 18.

50. A compound according to claim 1, wherein R3 and R4 independently are haloalkyl, aryl, heteroaryl, C1-10 alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl or hydroxyalkyl.