Inhibition of apoptosis using interleukin-1B-converting enzyme (ICE)/CED-3 family inhibitors

Abstract

The present invention provides methods for expanding and increasing survival of hematopoietic cell populations, for prolonging viability of an organ for transplantation, and enhancing bioproduction, using interleukinf-1β-converting enzyme ICE)/CED-3 family inhibitors. Exemplary compounds useful in the methods of the invention are provided herein.

We claim:  
     
         1 . A method for expanding or increasing survival of a cell population comprising contacting the cells with an inhibiting effective amount of a reagent which suppresses the activity of at least one member of the interleukin-1,3-converting enzyme (ICE)/CED-3 family, inhibiting apoptosis in the cell population, thereby expanding or increasing survival of the cell population.  
     
     
         2 . The method of    claim 1   , wherein the cells are differentiated cells.  
     
     
         3 . The method of    claim 1   , wherein the cells are precursor cells.  
     
     
         4 . The method of    claim 1   , wherein the cells are selected from the group consisting of granulocytes, monocytes, erythrocytes, lymphocytes and platelets.  
     
     
         5 . The method of    claim 1   , wherein the contacting is ex vivo.  
     
     
         6 . The method of    claim 1   , wherein the contacting is in vivo.  
     
     
         7 . The method of    claim 1   , wherein the reagent is a nucleic acid.  
     
     
         8 . The method of    claim 7   , wherein the reagent is an ICE antisense sequence.  
     
     
         9 . The method of    claim 1   , wherein the reagent is a protease inhibitor.  
     
     
         10 . The method of    claim 9   , wherein the protease inhibitor is irreversible.  
     
     
         11 . The method of    claim 9   , wherein the protease inhibitor is reversible.  
     
     
         12 . The method of    claim 1   , further comprising contacting the cells with a growth factor.  
     
     
         13 . The method of    claim 9   , wherein the protease inhibitor is a compound of formula 1:  
       
         
           
           
               
               
           
         
       
       wherein: 
 n is 1 or2;  
 R 1  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkl, heteroaryl, (heteroaryl)alkyl or (CH 2 ) m CO 2 R 4 , wherein m=1-4, and R 4  is as defined below;  
 R 2  is a hydrogen atom, chloro, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenyl,(substituted)phenyl, phenylalkyl,  
 (substituted)phenylalkl, heteroaryl, (heteroaryl)alkl or (CH 2 ) p CO 2 R 5 , wherein p=0-4, and R 5  is as defined below;  
 R 3  is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl;  
 R 4  is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl;  
 R 5  is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl;  
 A is a natural and unnatural amino acid;  
 B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl,(substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH 2 ZR 6 , CH 2 OCO(aryl), CH 2 OCO(heteroaryl); or CH 2 OPO(R 7 )R 8    
 where Z is an oxygen or a sulfir atom;  
 R 6  is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; and  
 R 7  and R 8  are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl, and (cycloalkyl) alkyl; and  
 X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl;  
 or a pharmaceutically acceptable salt thereof.  
 
     
     
         14 . The method of    claim 9   , wherein the protease inhibitor is a compound of formula 3:  
       
         
           
           
               
               
           
         
       
       wherein: 
 n is 1 or 2;  
 m is 1 or2;  
 A is R 2 CO—, R 3 —O—CO—, or R 4 SO 2 —;  
 a group of the formula:  
                     
 further wherein:  
 R 1  is a hydrogen atom, alkyl or phenylalkyl;  
 R 2  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;  
 R 3  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl;  
 R 4  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;  
 R 5  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;  
 R 6  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl;  
 R 7  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;  
 R 8  is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids;  
 B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)allyl, or halomethyl;  
 a group of the formula:  
 —CH 2 XR 9 ;  
 wherein R 9  is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom;  
 a group of the formula:  
 —CH 2 —O—CO—(aryl);  
 a group of the formula:  
 —CH 2 —O—CO—(heteroaryl);  
 a group of the formula:  
 —CH 2 —O—PO(R 10 )R 11    
 wherein R 10  and R 11  are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted phenyl) alkyl; and the pharmaceutically-acceptable salts thereof.  
 
     
     
         15 . A method for prolonging organ viability comprising contacting the cells of an organ with an inhibiting effective amount of a reagent which suppresses the activity of at least one member of the interleukin-1β-converting enzyme (ICE)/CED-3 family, thereby prolonging the viability of the organ as compared to an untreated organ.  
     
     
         16 . The method of    claim 15   , wherein the organ is contacted with the reagent ex vivo.  
     
     
         17 . The method of    claim 15   , wherein the organ is contacted with the reagent in vivo.  
     
     
         18 . The method of    claim 15   , wherein the reagent is a nucleic acid.  
     
     
         19 . The method of    claim 15   , wherein the reagent is an ICE antisense sequence.  
     
     
         20 . The method of    claim 15   , wherein the reagent is a protease inhibitor.  
     
     
         21 . The method of    claim 20   , wherein the reagent is an irreversible inhibitor.  
     
     
         22 . The method of    claim 20   , wherein the reagent is a reversible inhibitor.  
     
     
         23 . the method of    claim 20   , wherein the protease inhibitor is a compound of formula 1:  
       
         
           
           
               
               
           
         
       
       wherein 
 n is 1 or 2;  
 R 1  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH 2 ) m CO 2 R 4 , wherein m=1-4, and R 4  is as defined below;  
 R 2  is a hydrogen atom, chloro, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenyl,(substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH 2 ) p CO 2 R 5 , wherein p=0-4, and R 5  is as defined below;  
 R 3  is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl;  
 R 4  is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkl, phenylalkyl, or (substituted)phenylalkyl;  
 R 5  is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl;  
 A is a natural and unnatural amino acid;  
 B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl,(substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH 2 ZR 6 , CH 2 0CO(aryl), CH 2 0CO(heteroaryl), or CH 2 PO(R 7 )R 8 ; where Z is an oxygen or a sulfur atom;  
 R 6  is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; and  
 R 7  and R 8  are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl and (cycloalkyl) alkyl; and  
 X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl;  
 or a pharmaceutically acceptable salt thereof.  
 
     
     
         24 . The method of    claim 20   , wherein the protease inhibitor is a compound of formula 3:  
       
         
           
           
               
               
           
         
       
       wherein: 
 n is 1 or2;  
 m is 1 or 2;  
 A is R 2 CO—, R 3 —O—CO—, or R 4 SO 2 —;  
 a group of the formula:  
                     
 further wherein:  
 R 1  is a hydrogen atom, alkyl or phenylalkyl;  
 R 2  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;  
 R 3  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl;  
 R 4  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;  
 R 5  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;  
 R 6  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl;  
 R 7  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyI, (substituted phenyl)allyl, heteroaryl, or (heteroaryl)alkyl;  
 R 8  is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids;  
 B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl;  
 a group of the formula:  
 —CH 2 XR 9 ;  
 wherein R 9  is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom;  
 a group of the formula:  
 —CH 2 —O—CO—(aryl);  
 a group of the formula:  
 CH 2 —O—CO—(heteroaryl);  
 a group of the formula:  
 —CH 2 —O—PO(R 10 )R 11    
 wherein R 10  and R 11  are independently selected from a group consisting of alkyl,  
 cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted pheyl) alkyl; and the pharmaceutically-acceptable salts thereof.  
 
     
     
         25 . A method for increasing bioproduction in vitro comprising contacting host cells that produce a product of interest with a reagent which suppresses the activity of at least one member of the ICE/CED-3 family, thereby increasing survival of the cells in vitro.  
     
     
         26 . The method of    claim 25   , wherein the reagent is a nucleic acid.  
     
     
         27 . The method of    claim 25   , wherein the reagent is an ICE antisense sequence.  
     
     
         28 . The method of    claim 25   , wherein the reagent is a protease inhibitor.  
     
     
         29 . The method of    claim 28   , wherein the reagent is an irreversible inhibitor.  
     
     
         30 . The method of    claim 28   , wherein the reagent is a reversible inhibitor.  
     
     
         31 . The method of    claim 25   , wherein the reagent is a compound of formula 1:  
       
         
           
           
               
               
           
         
       
       wherein: 
 n is 1 or 2;  
 R 1  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH 2 ) m CO 2 R 4 , wherein m=1-4, and R 4  is as defined below;  
 R 2  is a hydrogen atom, chloro, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenyl,(substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH 2 ) p CO 2 R 5 , wherein p=0-4, and R 5  is as defined below;  
 R 3  is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl;  
 R 4  is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl;  
 R 5  is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl;  
 A is a natural and unnatural amino acid;  
 B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl,(substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH 2 ZR 6 , CH 2 OCO(aryl), CH 2 OCO heteroaryl), or CH 2 OPO(R 7 )R8; where Z is an oxygen or a sulfur atom;  
 R 6  is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; and  
 R 7  and R 8  are independently selected from the group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenyalkyl, (substituted phenyl) alkyl and (cycloaLkyl) alkyl;  
 X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl;  
 or a pharmaceutically acceptable salt thereof.  
 
     
     
         32 . The method of    claim 25   , wherein the reagent is a compound of formula 3:  
       
         
           
           
               
               
           
         
       
       wherein: 
 n is 1 or2;  
 m is 1 or2;  
 A is R 2 CO—, R 3 —O—CO—, or R 4 SO 2 —;  
 a group of the formula:  
                     
 further wherein:  
 R 1  is a hydrogen atom, alkyl or phenylalkyl;  
 R 2  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;  
 R 3 is alkyl, cycloalkyl, (cycloalkl)alkl, phenylalkyl, or, (substituted phenyl)alkyl;  
 R 4 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;  
 R 5  is alkyl, cycloakyl, (cycloalkyl)alkyl, phenyl, phenylakyl, substituted phenyl, (substituted phenyl)alkl, heteroaryl, or (heteroaryl)alkyl;  
 R 6 is akyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl;  
 R 7  is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;  
 R 8  is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids;  
 B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl;  
 a group of the formula:  
 —CH 2 XR 9 ;  
 wherein R 9  is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom;  
 a group of the formula:  
 —CH 2 —O—CO—(aryl);  
 a group of the formula:  
 —CH 2 —O—CO—(eteroaryl);  
 a group of the formula:  
 —CH 2 —O—PO(R 10 )R 11    
 wherein R 10  and R 11  are independently selected from a group consisting of allyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted phenyl) alkyl; and the pharmaceutically-acceptable salts thereof.