Chiral Diacylhydrazine Ligands for Modulating the Expression of Exogenous Genes via an Ecdysone Receptor Complex

Abstract

The present invention provides diacylhydrazine ligands and chiral diacylhydrazine ligands for use with ecdysone receptor-based inducible gene expression systems. Thus, the present invention is useful for applications such as gene therapy, large scale production of proteins and antibodies, cell-based screening assays, functional genomics, proteomics, metabolomics, and regulation of traits in transgenic organisms, where control of gene expression levels is desirable. An advantage of the present invention is that it provides a means to regulate gene expression and to tailor expression levels to suit the user's requirements.

1 - 25 . (canceled) 
     
     
         26 . A process for preparing
 (1) an enantiomerically enriched compound having Formula II:   
       
         
           
           
               
               
           
         
         wherein: 
         A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substituted aryl or optionally substituted heteroaryl; 
         B is optionally substituted aryl or optionally substituted heteroaryl; and 
         R 1  and R 2  are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocycle, optionally substituted aryl or optionally substituted heteroaryl; 
         with the proviso that R 1  does not equal R 2 ; 
         wherein the absolute configuration at the asymmetric carbon atom bearing R 1  and R 2  is predominantly S; or 
         (2) an enantiomerically enriched compound of Formula III: 
       
       
         
           
           
               
               
           
         
         wherein: 
         A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substituted aryl or optionally substituted heteroaryl; 
         B is optionally substituted aryl or optionally substituted heteroaryl; and 
         R 1  and R 2  are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocycle, optionally substituted aryl or optionally substituted heteroaryl; 
         with the proviso that R 1  does not equal R 2 ; 
         wherein the absolute configuration at the asymmetric carbon atom bearing R 1  and R 2  is predominantly R, comprising:
 a) reacting an acyl hydrazine of Formula XI 
 
       
       
         
           
           
               
               
           
         
         with a ketone of Formula XII 
       
       
         
           
           
               
               
           
         
         to form a compound of Formula XIII; 
       
       
         
           
           
               
               
           
         
         wherein R 1  does not equal R 2 ;
 b) reducing the compound of Formula XIII in the presence of a chiral catalyst to form a compound of Formula S-XIV or R-XIV; and 
 
       
       
         
           
           
               
               
           
         
         
           c) reacting the compound of Formula S-XIV or R-XIV with a compound of formula B-CO-LG where LG is a leaving group to form the compound of Formula II or Formula III. 
         
       
     
     
         27 . The process of  claim 26 , wherein B is 3,5-di-methylphenyl. 
     
     
         28 . The process of  claim 26 , wherein A is 2-ethyl-3-methoxyphenyl. 
     
     
         29 . The process of  claim 26 , wherein R 2  is tert-butyl. 
     
     
         30 - 47 . (canceled) 
     
     
         48 . The process of  claim 26 , wherein the chiral catalyst is a chiral phospholane catalyst complex. 
     
     
         49 . The process of  claim 26 , wherein the compound of Formula II or Formula III has an enantiomeric excess of at least 95%. 
     
     
         50 . The process of  claim 26 , wherein the compound of Formula III is:
 (R)—N′-(1-tert-Butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic acid benzyl ester;   (R)—N′-(1-tert-Butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic acid tert-butyl ester;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methoxy-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-chloro-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N′-(2-bromo-benzoyl)-N-(2-tert-butyl-butyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-3-methyl-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-chloro-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-ethyl-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methoxy-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-chloro-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2,6-difluoro-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3,4-dimethoxy-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3,5-difluoro-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzo[1,3]dioxole-5-carbonyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(naphthalene-1-carbonyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(naphthalene-2-carbonyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(thiophene-2-carbonyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2,5-dimethyl-furan-3-carbonyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-chloro-pyridine-3-carbonyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(6-chloro-pyridine-3-carbonyl)-hydrazide;   (R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;   (R)-3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide; or   (R)-3,5-Dimethyl-benzoic acid N′-(4-ethyl-benzoyl)-N-(1-phenethyl-but-3-enyl)-hydrazide.   
     
     
         51 . The process of  claim 50 , wherein the compound of Formula III is:
 (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;   (R)-3,5-dimethyl-benzoic acid N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide;   (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide;   (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide;   (R)-3,5-dimethyl-benzoic acid N′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide;   (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide;   (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide; or   (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide.   
     
     
         52 . The process of  claim 51 , wherein the compound of Formula III is (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide. 
     
     
         53 . The process of  claim 51 , wherein the compound of Formula III is (R)-3,5-dimethyl-benzoic acid N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide. 
     
     
         54 . The process of  claim 51 , wherein the compound of Formula III is (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide. 
     
     
         55 . The process of  claim 51 , wherein the compound of Formula III is (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide. 
     
     
         56 . The process of  claim 51 , wherein the compound of Formula III is (R)-3,5-dimethyl-benzoic acid N′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide. 
     
     
         57 . The process of  claim 51 , wherein the compound of Formula III is (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide. 
     
     
         58 . The process of  claim 51 , wherein the compound of Formula III is (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide. 
     
     
         59 . The process of  claim 51 , wherein the compound of Formula III is (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide. 
     
     
         60 . The process of  claim 26 , wherein the compound of Formula II is:
 (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;   (S)-(3,5-dimethyl-benzoic acid N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-chloro-benzoyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-ethyl-benzoyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2,6-difluoro-benzoyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(thiophene-2-carbonyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2,5-dimethyl-furan-3-carbonyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-chloro-pyridine-3-carbonyl)-hydrazide   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(6-chloro-pyridine-3-carbonyl)-hydrazide; or   (S)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide.