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

Abstract

The present invention relates to non-steroidal ligands for use in nuclear receptor-based inducible gene expression system, and a method to modulate exogenous gene expression in which an ecdysone receptor complex comprising: a DNA binding domain; a ligand binding domain; a transactivation domain; and a ligand is contacted with a DNA construct comprising: the exogenous gene and a response element; wherein the exogenous gene is under the control of the response element and binding of the DNA binding domain to the response element in the presence of the ligand results in activation or suppression of the gene.

1 - 6 . (canceled) 
     
     
         7 . A method to modulate the expression of one or more exogenous genes in a subject, comprising administering to the subject an effective amount of
 a compound of the formula:   
       
         
           
           
               
               
           
         
       
       wherein: 
       X and X′ are independently O or S; 
       A is unsubstituted or substituted phenyl wherein the substituents are independently 1 to 5H; halo; nitro; cyano; hydroxy; amino (—NR a R b ); alkylaminoalkyl (—(CH 2 ) n NR a R b ); (C 1 -C 6 )alkyl; (C 1 -C 6 )haloalkyl; (C 1 -C 6 )cyanoalkyl; (C 1 -C 6 )hydroxyalkyl; (C 1 -C 6 )alkoxy; phenoxy; (C 1 -C 6 )haloalkoxy; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkenyloxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkoxy; (C 1 -C 6 )alkanoyloxy(C 1 -C 6 )alkyl; (C 2 -C 6 )alkenyl optionally substituted with halo, cyano, (C 1 -C 4 )alkyl, or (C 1 -C 4 )alkoxy; (C 2 -C 6 )alkynyl optionally substituted with halo or (C 1 -C 4 )alkyl; formyl; carboxy; (C 1 -C 6 )alkylcarbonyl; (C 1 -C 6 )haloalkylcarbonyl; benzoyl; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )haloalkoxycarbonyl; (C 1 -C 6 )alkanoyloxy (—OCOR a ); carboxamido (—CONR a R b ); amido (—NR a COR b ); alkoxycarbonylamino (—NR a CO 2 R b ); alkylaminocarbonylamino (—NR a CONR b R a ); mercapto; (C 1 -C 6 )alkylthio; (C 1 -C 6 )alkylsulfonyl; (C 1 -C 6 )alkylsulfonyl(C 1 -C 6 )alkyl; (C 1 -C 6 )alkylsulfoxido (—S(O)R a ); (C 1 -C 6 )alkylsulfoxido(C 1 -C 6 )alkyl-(CH 2 )S(O)R a ); sulfamido (—SO 2 NR a R b ); —SO 3 H; or unsubstituted or substituted phenyl wherein the substituents are independently 1 to 3 halo, nitro, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, or amino; or when one or both of two adjacent positions on the phenyl ring are substituted, the attached atoms may form the phenyl-connecting termini of a linkage selected from the group consisting of (—OCH 2 O—), (—OCH(CH 3 )O—), (—OCH 2 CH 2 O—), (—OCH(CH 3 )CH 2 O—), (—S—CH═N—), (—CH 2 OCH 2 O—), (—O(CH 2 ) 3 —), (═N—O—N═), (—CH═CH—NH—), (—OCF 2 O—), (—NH—CH═N—), (—CH 2 CH 2 O—), and (—(CH 2 ) 4 —); 
       B is:
 (a) unsubstituted or substituted phenyl wherein the substituents are independently 1 to 5H; halo; nitro; cyano; hydroxy; amino (—NR a R b ); alkylaminoalkyl (—(CH 2 ) n NR a R b ); (C 1 -C 6 )alkyl; (C 1 -C 6 )haloalkyl; (C 1 -C 6 )cyanoalkyl; (C 1 -C 6 )hydroxyalkyl; (C 1 -C 6 )alkoxy; phenoxy; (C 1 -C 6 )haloalkoxy; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkenyloxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkoxy; (C 1 -C 6 )alkanoyloxy(C 1 -C 6 )alkyl; (C 2 -C 6 )alkenyl optionally substituted with halo, cyano, (C 1 -C 4 )alkyl, or (C 1 -C 4 )alkoxy; (C 2 -C 6 )alkynyl optionally substituted with halo or (C 1 -C 4 )alkyl; formyl; carboxy; (C 1 -C 6 )alkylcarbonyl; (C 1 -C 6 )haloalkylcarbonyl; -benzoyl; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )haloalkoxycarbonyl; (C 1 -C 6 )alkanoyloxy (—OCOR a ); carboxamido (—CONR a R b ); amido (—NR a COR b ); alkoxycarbonylamino (—NR a CO 2 R b ); alkylaminocarbonylamino (—NR a CONR b R c ); mercapto; (C 1 -C 6 )alkylthio; (C 1 -C 6 )alkylsulfonyl; (C 1 -C 6 )alkylsulfonyl(C 1 -C 6 )alkyl; (C 1 -C 6 )alkylsulfoxido (—S(O)R a ); (C 1 -C 6 )alkylsulfoxido(C 1 -C 6 )alkyl (—CH 2 ) n S(O)R a ); sulfamido (—SO 2 NR a R b ); —SO 3 H; —CH═N—NHC(O)NR a R b ; —CH═N—NHC(O)C(O)NR a R b ; or unsubstituted or substituted phenyl wherein the substituents are independently 1 to 3 halo, nitro, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, or amino; or when one or both of two adjacent positions on the phenyl ring are substituted with C, N, O, or S, these atoms may form the phenyl-connecting termini of a linkage selected from the group consisting of (—OCH 2 O—), (—OCH(CH 3 )O—), (—OCH 2 CH 2 O—), (—OCH(CH 3 )CH 2 O—), (—S—CH═N—), (—CH 2 OCH 2 O—), (—O(CH 2 ) 3 —), (═N—O—N═), (—CH═CH—NH—), (—OCF 2 O—), (—NH—CH═N—), (—CH 2 CH 2 O—), and (—(CH 2 ) 4 —); 
 (b) unsubstituted 6-membered heterocycle or substituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms where the substituents are from one to three of the same or different halo; nitro; hydroxy; (C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy; (C 1 -C 6 )thioalkoxy; carboxy; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )carbocyalkyl; (C 1 -C 6 )alkoxycarbonylalkyl having independently the stated number of carbon atoms in each alkyl group; —CONR a R b ; amino; (C 1 -C 6 )alkylamino; (C 1 -C 6 )dialkylamino having independently the stated number of carbon atoms in each alkyl group; haloalkyl; CH═N—NHC(O)NR a R b ; or —CH═N—NHC(O)C(O)NR a R b ; or 
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl; 
 
       E is unsubstituted or substituted (C 4 -C 10 ) branched alkyl wherein the substituents are independently 1-4 cyano; halo; (C 5 -C 6 )cycloalkyl; phenyl; (C 2 -C 3 )alkenyl; hydroxy, (C 1 -C 6 )alkoxy; carboxy; (C 1 -C 6 )alkoxycarbonyl; formyl; (C 1 -C 6 )trialkylsilyloxy having independently the stated number of carbon atoms in each alkyl group; —CH═N—OR a ; —CH═N—R d ; —CH═N—NHC(O)NR a R b ; or —CH═N—NHC(O)C(O)NR a R b ; 
       wherein R a , R b , and R c  are independently H, (C 1 -C 6 )alkyl, or phenyl; R d  is hydroxy(C 1 -C 6 )alkyl; 
       and n=1-4; and 
       G is H or CN; 
       provided that:
 1) when E is unsubstituted or substituted (C 4 -C 10 ) branched alkyl wherein the substituents are independently 1-4 cyano; halo; (C 2 -C 3 )alkenyl; carboxy; or (C 1 -C 6 )alkoxycarbonyl; 
 then B is: 
 (a) substituted phenyl which bears at least one —CH═N—NHC(O)NR a R b  or —CH═N—NHC(O)C(O)NR a R b  group; 
 (b) substituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms which bears at least one haloalkyl group; or 
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl; 
 2) when E is a substituted (C 4 -C 10 ) branched alkyl which bears at least one of phenyl; hydroxy; (C 1 -C 6 )alkoxy; or formyl; 
 then B is: 
 (a) substituted phenyl which bears at least one —CH═N—NHC(O)NR a R b  or —CH═N—NHC(O)C(O)NR a R b  group; 
 (b) substituted or unsubstituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms; or 
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl. 
 
     
     
         8 . A method for regulating endogenous or heterologous gene expression in a transgenic subject comprising contacting a ligand with an ecdysone receptor complex within the cells of the subject, wherein the cells further contain a DNA binding sequence for the ecdysone receptor complex when in combination with the ligand and wherein formation of an ecdysone receptor complex-ligand-DNA binding sequence complex induces expression of the gene, and where the ligand is
 a compound of the formula:   
       
         
           
           
               
               
           
         
       
       wherein: 
       X and X′ are independently O or S; 
       A is unsubstituted or substituted phenyl wherein the substituents are independently 1 to 5H; halo; nitro; cyano; hydroxy; amino (—NR a R b ); alkylaminoalkyl((CH 2 ) n NR a R b ); (C 1 -C 6 )alkyl; (C 1 -C 6 )haloalkyl; (C 1 -C 6 )cyanoalkyl; (C 1 -C 6 )hydroxyalkyl; (C 1 -C 6 )alkoxy; phenoxy; (C 1 -C 6 )haloalkoxy; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkenyloxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkoxy; (C 1 -C 6 )alkanoyloxy(C 1 -C 6 )alkyl; (C 2 -C 6 )alkenyl optionally substituted with halo, cyano, (C 1 -C 4 )alkyl, or (C 1 -C 4 )alkoxy; (C 2 -C 6 )alkynyl optionally substituted with halo or (C 1 -C 4 )alkyl; formyl; carboxy; (C 1 -C 6 )alkylcarbonyl; (C 1 -C 6 )haloalkylcarbonyl; benzoyl; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )haloalkoxycarbonyl; (C 1 -C 6 )alkanoyloxy (—OCOR a ); carboxamido (—CONR a R b ); amido (—NR a COR b ); alkoxycarbonylamino (—NR a CO 2 R b ); alkylaminocarbonylamino (—NR a CONR b R c ); mercapto; (C 1 -C 6 )alkylthio; (C 1 -C 6 )alkylsulfonyl; (C 1 -C 6 )alkylsulfonyl(C 1 -C 6 )alkyl; (C 1 -C 6 )alkylsulfoxido (—S(O)R a ); (C 1 -C 6 )alkylsulfoxido(C 1 -C 6 )alkyl —(CH 2 )S(O)R a ); sulfamido (—SO 2 NR a R b ); —SO 3 H; or unsubstituted or substituted phenyl wherein the substituents are independently 1 to 3 halo, nitro, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, or amino; or when one or both of two adjacent positions on the phenyl ring are substituted, the attached atoms may form the phenyl-connecting termini of a linkage selected from the group consisting of (—OCH 2 O—), (—OCH(CH 3 )O—), (—OCH 2 CH 2 O—), (—OCH(CH 3 )CH 2 O—), (—S—CH═N—), (—CH 2 OCH 2 O—), (—O(CH 2 ) 3 —), (═N—O—N═), (—CH═CH—NH—), (—OCF 2 O—), (—NH—CH═N—), (—CH 2 CH 2 O—), and (—(CH 2 ) 4 —); 
       B is:
 (a) unsubstituted or substituted phenyl wherein the substituents are independently 1 to 5H; halo; nitro; cyano; hydroxy; amino (—NR a R b ); alkylaminoalkyl (—(CH 2 ) n NR a R b ); (C 1 -C 6 )alkyl; (C 1 -C 6 )haloalkyl; (C 1 -C 6 )cyanoalkyl; (C 1 -C 6 )hydroxyalkyl; (C 1 -C 6 )alkoxy; phenoxy; (C 1 -C 6 )halo alkoxy; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkenyloxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkoxy; (C 1 -C 6 )alkanoyloxy(C 1 -C 6 )alkyl; (C 2 -C 6 )alkenyl optionally substituted with halo, cyano, (C 1 -C 4 )alkyl, or (C 1 -C 4 )alkoxy; (C 2 -C 6 )alkynyl optionally substituted with halo or (C 1 -C 4 )alkyl; formyl; carboxy; (C 1 -C 6 )alkylcarbonyl; (C 1 -C 6 )haloalkylcarbonyl; benzoyl; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )haloalkoxycarbonyl; (C 1 -C 6 )alkanoyloxy (—OCOR a ); carboxamido (—CONR a R b ); amido (—NR a COR b ); alkoxycarbonylamino (—NR a CO 2 R b ); alkylaminocarbonylamino (—NR a CONR b R c ); mercapto; (C 1 -C 6 )alkylthio; (C 1 -C 6 )alkylsulfonyl; (C 1 -C 6 )alkylsulfonyl(C 1 -C 6 )alkyl; (C 1 -C 6 )alkylsulfoxido (—S(O)R a ); (C 1 -C 6 )alkylsulfoxido(C 1 -C 6 )alkyl (—CH 2 ) n S(O)R a ); sulfamido (—SO 2 NR a R b ); —SO 3 H; —CH═N—NHC(O)NR a R b ; —CH═N—NHC(O)C(O)NR a R b ; or unsubstituted or substituted phenyl wherein the substituents are independently 1 to 3 halo, nitro, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, or amino; or when one or both of two adjacent positions on the phenyl ring are substituted with C, N, O, or S, these atoms may form the phenyl-connecting termini of a linkage selected from the group consisting of (—OCH 2 O—), (—OCH(CH 3 )O—), (—OCH 2 CH 2 O—), (—OCH(CH 3 )CH 2 O—), (—S—CH═N—), (—CH 2 OCH 2 O—), (—O(CH 2 ) 3 —), (═N—O—N═), (—CH═CH—NH—), (—OCF 2 O—), (—NH—CH═N—), (—CH 2 CH 2 O—), and (—(CH 2 ) 4 —); 
 (b) unsubstituted 6-membered heterocycle or substituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms where the substituents are from one to three of the same or different halo; nitro; hydroxy; (C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy; (C 1 -C 6 )thioalkoxy; carboxy; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )carbocyalkyl; (C 1 -C 6 )alkoxycarbonylalkyl having independently the stated number of carbon atoms in each alkyl group; —CONR a R b ; amino; (C 1 -C 6 )alkylamino; (C 1 -C 6 )dialkylamino having independently the stated number of carbon atoms in each alkyl group; haloalkyl; —CH═N—NHC(O)NR a R b ; or —CH═N—NHC(O)C(O)NR a R b ; or 
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl; 
 
       E is unsubstituted or substituted (C 4 -C 10 ) branched alkyl wherein the substituents are independently 1-4 cyano; halo; (C 5 -C 6 )cycloalkyl; phenyl; (C 2 -C 3 )alkenyl; hydroxy, (C 1 -C 6 )alkoxy; carboxy; (C 1 -C 6 )alkoxycarbonyl; formyl; (C 1 -C 6 )trialkylsilyloxy having independently the stated number of carbon atoms in each alkyl group; —CH═N—OR a ; —CH═N—R d ; —CH═N—NHC(O)NR a R b ; or —CH═N—NHC(O)C(O)NR a R b ; 
       wherein R a , R b , and R c  are independently H, (C 1 -C 6 )alkyl, or phenyl; R d  is hydroxy(C 1 -C 6 )alkyl; 
       and n=1-4; and 
       G is H or CN; 
       provided that:
 1) when E is unsubstituted or substituted (C 4 -C 10 ) branched alkyl wherein the substituents are independently 1-4 cyano; halo; (C 2 -C 3 )alkenyl; carboxy; or (C 1 -C 6 )alkoxycarbonyl; 
 then B is: 
 (a) substituted phenyl which bears at least one —CH═N—NHC(O)NR a R b  or —CH═N—NHC(O)C(O)NR a R b  group; 
 (b) substituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms which bears at least one haloalkyl group; or 
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl; 
 2) when E is a substituted (C 4 -C 10 ) branched alkyl which bears at least one of phenyl; hydroxy; (C 1 -C 6 )alkoxy; or formyl; 
 then B is: 
 (a) substituted phenyl which bears at least one —CH═N—NHC(O)NR a R b  or —CH═N—NHC(O)C(O)NR a R b  group; 
 (b) substituted or unsubstituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms; or 
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl. 
 
     
     
         9 . The method of  claim 8 , wherein the ecdysone receptor complex is a chimeric ecdysone receptor complex and the DNA construct further comprises a promoter. 
     
     
         10 . The method of  claim 8 , wherein the subject is a plant. 
     
     
         11 . The method of  claim 8 , wherein the subject is a mammal. 
     
     
         12 . A method of modulating the expression of a gene in a host cell comprising the steps of:
 a) introducing into the host cell a gene expression modulation system comprising:
 i) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide sequence that encodes a first hybrid polypeptide comprising:
 (a) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and 
 (b) an ecdysone receptor ligand binding domain; 
 
 ii) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising:
 (a) a transactivation domain; and 
 (b) a chimeric retinoid X receptor ligand binding domain; and 
 
 iii) a third gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide sequence comprising:
 (a) a response element recognized by the DNA-binding domain of the first hybrid polypeptide; 
 (b) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and 
 (c) a gene whose expression is to be modulated; and 
 
   b) introducing into the host cell
 a compound of the formula: 
   
       
         
           
           
               
               
           
         
         wherein X and X′ are independently O or S; 
         A is unsubstituted or substituted phenyl wherein the substituents are independently 1 to 5H; halo; nitro; cyano; hydroxy; amino (—NR a R b ); alkylaminoalkyl (—(CH 2 ) n NR a R b ); (C 1 -C 6 )alkyl; (C 1 -C 6 )haloalkyl; (C 1 -C 6 )cyanoalkyl; (C 1 -C 6 )hydroxyalkyl; (C 1 -C 6 )alkoxy; phenoxy; (C 1 -C 6 )haloalkoxy; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkenyloxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkoxy; (C 1 -C 6 )alkanoyloxy(C 1 -C 6 )alkyl; (C 2 -C 6 )alkenyl optionally substituted with halo, cyano, (C 1 -C 4 )alkyl, or (C 1 -C 4 )alkoxy; (C 2 -C 6 )alkynyl optionally substituted with halo or (C 1 -C 4 )alkyl; formyl; carboxy; (C 1 -C 6 )alkylcarbonyl; (C 1 -C 6 )haloalkylcarbonyl; benzoyl; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )haloalkoxycarbonyl; (C 1 -C 6 )alkanoyloxy (—OCOR a ); carboxamido (—CONR a R b ); amido (—NR a COR b ); alkoxycarbonylamino (—NR a CO 2 R b ); alkylaminocarbonylamino (—NR a CONR b R c ); mercapto; (C 1 -C 6 )alkylthio; (C 1 -C 6 )alkylsulfonyl; (C 1 -C 6 )alkylsulfonyl(C 1 -C 6 )alkyl; (C 1 -C 6 )alkylsulfoxido (—S(O)R a ); (C 1 -C 6 )alkylsulfoxido(C 1 -C 6 )alkyl —(CH 2 ) n S(O)R a ); sulfamido (—SO 2 NR a R b ); —SO 3 H; or unsubstituted or substituted phenyl wherein the substituents are independently 1 to 3 halo, nitro, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, or amino; or when one or both of two adjacent positions on the phenyl ring are substituted, the attached atoms may form the phenyl-connecting termini of a linkage selected from the group consisting of (—OCH 2 O—), (—OCH(CH 3 )O—), (—OCH 2 CH 2 O—), (—OCH(CH 3 )CH 2 O—), (—S—CH═N—), (—CH 2 OCH 2 O—), (—O(CH 2 ) 3 —), (═N—O—N═), (—CH═CH—NH—), (—OCF 2 O—), (—NH—CH═N—), (—CH 2 CH 2 O—), and (—(CH 2 ) 4 —); 
         B is:
 (a) unsubstituted or substituted phenyl wherein the substituents are independently 1 to 5H; halo; nitro; cyano; hydroxy; amino (—NR a R b ); alkylaminoalkyl (—(CH 2 ) n NR a R b ); (C 1 -C 6 )alkyl; (C 1 -C 6 )haloalkyl; (C 1 -C 6 )cyanoalkyl; (C 1 -C 6 )hydroxyalkyl; (C 1 -C 6 )alkoxy; phenoxy; (C 1 -C 6 )haloalkoxy; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkenyloxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkoxy; (C1-C 6 )alkanoyloxy(C 1 -C 6 )alkyl; (C 2 -C 6 )alkenyl optionally substituted with halo, cyano, (C 1 -C 4 )alkyl, or (C 1 -C 4 )alkoxy; (C 2 -C 6 )alkynyl optionally substituted with halo or (C 1 -C 4 )alkyl; formyl; carboxy; (C 1 -C 6 )alkylcarbonyl; (C 1 -C 6 )haloalkylcarbonyl; benzoyl; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )haloalkoxycarbonyl; (C 1 -C 6 )alkanoyloxy (—OCOR a ); carboxamido (—CONR a R b ); amido (—NR a COR b ); alkoxycarbonylamino (—NR a CO 2 R b ); alkylaminocarbonylamino (—NR a CONR b R c ); mercapto; (C 1 -C 6 )alkylthio; (C 1 -C 6 )alkylsulfonyl; (C 1 -C 6 )alkylsulfonyl(C 1 -C 6 )alkyl; (C 1 -C 6 )alkylsulfoxido (—S(O)R a ); (C 1 -C 6 )alkylsulfoxido(C 1 -C 6 )alkyl (—CH 2 ) n S(O)R a ); sulfamido (—SO 2 NR a R b ); —SO 3 H; —CH═N—NHC(O)NR a R b ; —CH═N—NHC(O)C(O)NR a R b ; or unsubstituted or substituted phenyl wherein the substituents are independently 1 to 3 halo, nitro, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, or amino; or when one or both of two adjacent positions on the phenyl ring are substituted with C, N, O, or S, these atoms may form the phenyl-connecting termini of a linkage selected from the group consisting of (—OCH 2 O—), (—OCH(CH 3 )O—), (—OCH 2 CH 2 O—), (—OCH(CH 3 )CH 2 O—), (—S—CH═N—), (—CH 2 OCH 2 O—), (—O(CH 2 ) 3 —), (═N—O—N═), (—CH═CH—NH—), (—OCF 2 O—), (—NH—CH═N—), (—CH 2 CH 2 O—), and (—(CH 2 ) 4 —); 
 (b) unsubstituted 6-membered heterocycle or substituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms where the substituents are from one to three of the same or different halo; nitro; hydroxy; (C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy; (C 1 -C 6 )thioalkoxy; carboxy; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )carbocyalkyl; (C 1 -C 6 )alkoxycarbonylalkyl having independently the stated number of carbon atoms in each alkyl group; —CONR a R b ; amino; (C 1 -C 6 )alkylamino; (C 1 -C 6 )dialkylamino having independently the stated number of carbon atoms in each alkyl group; haloalkyl; —CH═N—NHC(O)NR a R b ; or —CH═N—NHC(O)C(O)NR a R b ; or 
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl; 
 
         E is unsubstituted or substituted (C 4 -C 10 ) branched alkyl wherein the substituents are independently 1-4 cyano; halo; (C 5 -C 6 )cycloalkyl; phenyl; (C 2 -C 3 )alkenyl; hydroxy, (C 1 -C 6 )alkoxy; carboxy; (C 1 -C 6 )alkoxycarbonyl; formyl; (C 1 -C 6 )trialkylsilyloxy having independently the stated number of carbon atoms in each alkyl group; —CH═N—OR a ; —CH═N—R d ; —CH□N—NHC(O)NR a R b ; or —CH═N—NHC(O)C(O)NR a R b ; 
         wherein R a , R b , and R c  are independently H, (C 1 -C 6 )alkyl, or phenyl; R d  is hydroxy(C 1 -C 6 )alkyl; and n=1-4; and 
         G is H or CN; 
         provided that: 
         1) when E is unsubstituted or substituted (C 4 -C 10 ) branched alkyl wherein the substituents are independently 1-4 cyano; halo; (C 2 -C 3 )alkenyl; carboxy; or (C 1 -C 6 )alkoxycarbonyl; 
         then B is: 
         (a) substituted phenyl which bears at least one —CH═N—NHC(O)NR a R b  or —CH═N—NHC(O)C(O)NR a R b  group; 
         (b) substituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms which bears at least one haloalkyl group; or 
         (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl; 
         2) when E is a substituted (C 4 -C 10 ) branched alkyl which bears at least one of phenyl; hydroxy; (C 1 -C 6 )alkoxy; or formyl; 
         then B is: 
         (a) substituted phenyl which bears at least one —CH═N—NHC(O)NR a R b  or —CH═N—NHC(O)C(O)NR a R b  group; 
         (b) substituted or unsubstituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms; or
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl. 
 
       
     
     
         13 . A method for producing a polypeptide comprising the steps of:
 a) selecting a cell which is substantially insensitive to exposure to
 a compound of the formula: 
   
       
         
           
           
               
               
           
         
       
       wherein X and X′ are independently O or S; 
       A is unsubstituted or substituted phenyl wherein the substituents are independently 1 to 5H; halo; nitro; cyano; hydroxy; amino (—NR a R b ); alkylaminoalkyl (—(CH 2 ) n NR a R b ); (C 1 -C 6 )alkyl; (C 1 -C 6 )haloalkyl; (C 1 -C 6 )cyanoalkyl; (C 1 -C 6 )hydroxyalkyl; (C 1 -C 6 )alkoxy; phenoxy; (C 1 -C 6 )haloalkoxy; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkenyloxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkoxy; (C 1 -C 6 )alkanoyloxy(C 1 -C 6 )alkyl; (C 2 -C 6 )alkenyl optionally substituted with halo, cyano, (C 1 -C 4 )alkyl, or (C 1 -C 4 )alkoxy; (C 2 -C 6 )alkynyl optionally substituted with halo or (C 1 -C 4 )alkyl; formyl; carboxy; (C 1 -C 6 )alkylcarbonyl; (C 1 -C 6 )haloalkylcarbonyl; benzoyl; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )haloalkoxycarbonyl; (C 1 -C 6 )alkanoyloxy (—OCOR a ); carboxamido (—CONR a R b ); amido (—NR a COR b ); alkoxycarbonylamino (—NR a CO 2 R b ); alkylaminocarbonylamino (—NR a CONR b R c ); mercapto; (C 1 -C 6 )alkylthio; (C 1 -C 6 )alkylsulfonyl; (C 1 -C 6 )alkylsulfonyl(C 1 -C 6 )alkyl; (C 1 -C 6 )alkylsulfoxido (—S(O)R a ); (C 1 -C 6 )alkylsulfoxido(C 1 -C 6 )alkyl —(CH 2 )S(O)R a ); sulfamido (—SO 2 NR a R b ); —SO 3 H; or unsubstituted or substituted phenyl wherein the substituents are independently 1 to 3 halo, nitro, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, or amino; or when one or both of two adjacent positions on the phenyl ring are substituted, the attached atoms may form the phenyl-connecting termini of a linkage selected from the group consisting of (—OCH 2 O—), (—OCH(CH 3 )O—), (—OCH 2 CH 2 O—), (—OCH(CH 3 )CH 2 O—), (—S—CH═N—), (—CH 2 OCH 2 O—), (—O(CH 2 ) 3 —), (═N—O—N═), (—CH═CH—NH—), (—OCF 2 O—), (—NH—CH═N—), (—CH 2 CH 2 O—), and (—(CH 2 ) 4 —); 
       B is:
 (a) unsubstituted or substituted phenyl wherein the substituents are independently 1 to 5H; halo; nitro; cyano; hydroxy; amino (—NR a R b ); alkylaminoalkyl (—(CH 2 ) n NR a R b ); (C 1 -C 6 )alkyl; (C 1 -C 6 )haloalkyl; (C 1 -C 6 )cyanoalkyl; (C 1 -C 6 )hydroxyalkyl; (C 1 -C 6 )alkoxy; phenoxy; (C 1 -C 6 )haloalkoxy; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkenyloxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkoxy; (C 1 -C 6 )alkanoyloxy(C 1 -C 6 )alkyl; (C 2 -C 6 )alkenyl optionally substituted with halo, cyano, (C 1 -C 4 )alkyl, or (C 1 -C 4 )alkoxy; (C 2 -C 6 )alkynyl optionally substituted with halo or (C 1 -C 4 )alkyl; formyl; carboxy; (C 1 -C 6 )alkylcarbonyl; (C 1 -C 6 )haloalkylcarbonyl; benzoyl; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )haloalkoxycarbonyl; (C 1 -C 6 )alkanoyloxy (—OCOR a ); carboxamido (—CONR a R b ); amido (—NR a COR b ); alkoxycarbonylamino (—NR a CO 2 R b ); alkylaminocarbonylamino (—NR a CONR b R c ); mercapto; (C 1 -C 6 )alkylthio; (C 1 -C 6 )alkylsulfonyl; (C 1 -C 6 )alkylsulfonyl(C 1 -C 6 )alkyl; (C 1 -C 6 )alkylsulfoxido (—S(O)R a ); (C 1 -C 6 )alkylsulfoxido(C 1 -C 6 )alkyl (—CH 2 ) n S(O)R a ); sulfamido (—SO 2 NR a R b ); —SO 3 H; —CH═N—NHC(O)NR a R b ; —CH═N—NHC(O)C(O)NR a R b ; or unsubstituted or substituted phenyl wherein the substituents are independently 1 to 3 halo, nitro, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, or amino; or when one or both of two adjacent positions on the phenyl ring are substituted with C, N, O, or S, these atoms may form the phenyl-connecting termini of a linkage selected from the group consisting of (—OCH 2 O—), (—OCH(CH 3 )O—), (—OCH 2 CH 2 O—), (—OCH(CH 3 )CH 2 O—), (—S—CH═N—), (—CH 2 OCH 2 O—), (—O(CH 2 ) 3 —), (═N—O—N═), (—CH═CH—NH—), (—OCF 2 O—), (—NH—CH═N—), (—CH 2 CH 2 O—), and (—(CH 2 ) 4 —);
 (b) unsubstituted 6-membered heterocycle or substituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms where the substituents are from one to three of the same or different halo; nitro; hydroxy; (C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy; (C 1 -C 6 )thioalkoxy; carboxy; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )carbocyalkyl; (C 1 -C 6 )alkoxycarbonylalkyl having independently the stated number of carbon atoms in each alkyl group; —CONR a R b ; amino; (C 1 -C 6 )alkylamino; (C 1 -C 6 )dialkylamino having independently the stated number of carbon atoms in each alkyl group; haloalkyl —CH═N—NHC(O)NR a R b ; or —CH═N—NHC(O)C(O)NR a R b ; or 
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl; 
 
 
       E is unsubstituted or substituted (C 4 -C 10 ) branched alkyl wherein the substituents are independently 1-4 cyano; halo; (C 5 -C 6 )cycloalkyl; phenyl; (C 2 -C 3 )alkenyl; hydroxy, (C 1 -C 6 )alkoxy; carboxy; (C 1 -C 6 )alkoxycarbonyl; formyl; (C 1 -C 6 )trialkylsilyloxy having independently the stated number of carbon atoms in each alkyl group; —CH═N—OR a ; —CH═N—R d ; —CH═N—NHC(O)NR a R b ; or —CH═N—NHC(O)C(O)NR a R b ; 
       wherein R a , R b , and R c  are independently H, (C 1 -C 6 )alkyl, or phenyl; R d  is hydroxy(C 1 -C 6 )alkyl; 
       and n=1-4; and 
       G is H or CN; 
       provided that:
 1) when E is unsubstituted or substituted (C 4 -C 10 ) branched alkyl wherein the substituents are independently 1-4 cyano; halo; (C 7 -C 3 )alkenyl; carboxy; or (C 1 -C 6 )alkoxycarbonyl; 
 then B is: 
 (a) substituted phenyl which bears at least one —CH═N—NHC(O)NR a R b  or —CH═N—NHC(O)C(O)NR a R b  group; 
 (b) substituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms which bears at least one haloalkyl group; or 
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl; 
 2) when E is a substituted (C 4 -C 10 ) branched alkyl which bears at least one of phenyl; hydroxy; (C 1 -C 6 )alkoxy; or formyl; 
 then B is: 
 (a) substituted phenyl which bears at least one —CH═N—NHC(O)NR a R b  or —CH═N—NHC(O)C(O)NR a R b  group; 
 (b) substituted or unsubstituted 6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear carbon atoms; or 
 (c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl; 1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or 1-(C 1 -C 6 )alkyl-1H-indole-2-yl; 
 b) introducing into the cell:
 1) a DNA construct comprising:
 i) an exogenous gene encoding the polypeptide; and 
 ii) a response element; 
 
 
 wherein the gene is under the control of the response element; and
 2) a DNA construct encoding an ecdysone receptor complex comprising:
 i) a DNA binding domain; 
 ii) an ecdysone receptor ligand binding domain; and 
 iii) a transactivation domain; and 
 
 
 c) exposing the cell to the ligand. 
 
     
     
         14 . A process for the preparation of a compound of formula (IV) comprising the steps of:
 i reacting a compound of formula (I) with a base selected from NaH, KH, or an amide MNR a R b  to produce a product II, wherein M is Li, Na, or K, and R a  and R b  are independently (C 1 -C 6 )alkyl or phenyl; and   
       
         
           
           
               
               
           
         
         ii reacting the product (II) of step (i) with a compound of formula (III) wherein R is phenyl substituted with three to five of the same or different chloro, fluoro, or trifluoromethyl; 
       
       
         
           
           
               
               
           
         
       
       wherein 
       A and B are independently
 (a) unsubstituted or substituted phenyl wherein the substituents are independently 1 to 5H; halo; nitro; cyano; amino (—NR a R b ); alkylaminoalkyl (—(CH 2 ) n NR a R b ); (C 1 -C 6 )alkyl; (C 1 -C 6 )haloalkyl; (C 1 -C 6 )cyanoalkyl; (C 1 -C 6 )alkoxy; phenoxy; (C 1 -C 6 )haloalkoxy; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkenyloxy(C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy(C 1 -C 6 )alkoxy; (C2-C 6 )alkenyl optionally substituted with halo, cyano, (C 1 -C 4 )alkyl, or (C 1 -C 4 )alkoxy; (C 2 -C 6 )alkynyl optionally substituted with halo or (C 1 -C 4 )alkyl; formyl; (C 1 -C 6 )haloalkylcarbonyl; benzoyl; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )haloalkoxycarbonyl; (C 1 -C 6 )alkanoyloxy (—OCOR a ); carboxamido (—CONR a R b ); amido (—NR a COR b ); alkoxycarbonylamino (—N(CH 2 ) n CO 2 R b ); alkylaminocarbonylamino (—N(CH 2 ) n CONR b R c ); (C 1 -C 6 )alkylthio; sulfamido (—SO 2 NR a R b ); or unsubstituted or substituted phenyl wherein the substituents are independently 1 to 3 halo, nitro, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, or (—NR a R b ); or when one or both of two adjacent positions on the phenyl ring are substituted, the attached atoms may form the phenyl-connecting termini of a linkage selected from the group consisting of (—OCH 2 O—), (—OCH(CH 3 )O—), (—OCH 2 CH 2 O—), (—OCH(CH 3 )CH 2 O—), (—S—CH═N—), (—CH 2 OCH 2 O—), (—O(CH 2 ) 3 —), (═N—O—N═), (—CH═CH—NH—), (—OCF 2 O—), (—NH—CH═N—), (—CH 2 CH 2 O—), and (—(CH 2 ) 4 —); or 
 (b) unsubstituted 5- or 6-membered heterocycle or substituted 5 or 6-membered heterocycle having 1-3 nitrogen atoms where the substituents are from one to four of the same or different halo; nitro; (C 1 -C 6 )alkyl; (C 1 -C 6 )alkeyl; (C 1 -C 6 )alkoxy; (C 1 -C 6 )thioalkoxy; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )carbocyalkyl; —CONR a R b ; amino(—NR a R b ); haloalkyl including CF 3 ; -trialkylsilyl (—SiR a R b R c ); trityl(C(Ph) 3 ); or unsubstituted or substituted phenyl wherein the substituents are independently 1 to 3 halo, nitro, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkyl, or (—NR a R b ); or when two adjacent positions are substituted, these positions may form a benzo ring fusion; and 
 
       E is phenyl, or unsubstituted or substituted (C 1 -C 10 ) straight or branched alkyl wherein the substituents are independently 1-4 cyano; halo; (C 5 -C 6 )cycloalkyl; phenyl; (C 2 -C 3 )alkenyl; (C 1 -C 6 )alkoxy; (C 1 -C 6 )alkoxycarbonyl; (C 1 -C 6 )alkanoyloxy (—OCOR a ); formyl; (C 1 -C 6 )trialkylsilyloxy having independently the stated number of carbon atoms in each alkyl group; or —CH═N—OR a ;
 wherein R a , R b , and R e  are independently (C 1 -C 6 )alkyl or phenyl, and n=1-4.