Ligands for modulating the expression of exogenous genes via an ecdysone receptor complex

Abstract

This invention relates to an improved 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. The improvement resides in a select group of non-steroid ligands which show improved activity over known ligands.

We claim:  
     
         1 . In a method to modulate exogenous gene expression comprising contacting an ecdysone receptor complex comprising: 
 a) a DNA binding domain;    b) a ligand binding domain;    c) a transactivation domain; and    d) a ligand;    with a DNA construct comprising:    a) the exogenous gene; and    b) a response element;    wherein:    a) the exogenous gene is under the control of the response element; and    b) binding of the DNA binding domain to the response element in the presence of the ligand results in activation or suppression of the gene;    the improvement comprising:    selecting the ligand from a compound of the formula:                          wherein:    E is a (C 4 -C 6 )alkyl containing a tertiary carbon or a cyano(C 3 -C 5 )alkyl containing a tertiary carbon;    R 1  is H, Me, Et, i-Pr, F, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ,    R 2  is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, NH—CN, or joined with R 3  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;    R 3  is H, Et, or joined with R 2  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;    R 4 , R 5 , and R 6  are independently H, Me, Et, F, Cl, Br, formyl, CF 1 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;    provided that:    a) when R 1  is Me and R 2  is OMe;     then R 3  is H; and the combination R 4 , R 5 , and R 6  is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, or 3,5-di-F;    b) when R 1  is Me and R 2  is OEt;     then R 3  is H and the combination R 4 , R 5 , and R 6  is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl;    c) when R 1  is Et and R 2  is OMe or OEt;     then R 3  is H and the combination R 4 , R 5 , and R 6  is: 
 i) 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl, 3-OMe, 2-Cl-5-Me, 2-Br-5-Me, 2-Cl, 2-Br, or 3-Me; or  
 ii) R 6  is H, R 1  is Me, and R 5  is Et, F, Cl, Br, formyl, CH 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;  
   d) when R 2  is i-Pr;     then R 2  is OMe, or OEt; R 3  is H; and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    e) when R 3  is Et;     then R 2  is H, R 1  is F or Cl, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    f) when R 2  and R 3 , together with the phenyl carbons to which they are attached, form an ethylenedioxy ring;     then R 1  is Me or Et and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    g) when R 2  and R 3 , together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring;     then R 1  is Et and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    h) when R 1  is formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ;     then R 2  is OMe or OEt, R 3  is H, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me; and    i) when R 2  is Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, or NH—CN;     then R 1  is Et, R 3  is H, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me.    
     
     
         2 . A method to modulate exogenous gene expression comprising contacting an ecdysone receptor complex comprising; 
 a) a DNA binding domain;    b) a ligand binding domain;    c) a transactivation domain; and    d) a ligand of the formula:                          wherein:    E is a (C 1 -C 6 )alkyl containing a tertiary carbon or a cyano(C 3 -C 5 )alkyl containing a tertiary carbon;    R 1  is H, Me, Et, i-Pr, F, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN or SCHF 2 ;    R 2  is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, NH—CN, or joined with R 3  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;    R 3  is H, Et, or joined with R 2  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;    R 4 , R 5 , and R 6  are independently H, Me, Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CN, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;    provided that:    a) when R 1  is Me and R 2  is OMe;     then R 3  is H; and the combination R 4 , R 5 , and R 6  is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, or 3,5-di-F;    b) when R 1  is Me an R 2  is OEt;     then R 3  is H and the combination R 4 , R 5 , and R 6  is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl;    c) when R 1  is Et and R 2  is OMe or OEt;     then R 3  is H and the combination R 4 , R 5 , and R 6  is: 
 i) 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl, 3-OMe, 2-Cl-5-Me, 2-Br-5-Me, 2-Cl-2-Br, or 3-Me; or  
 ii) R 6  is H, R 4  is Me, and R 5  is Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;  
   d) when R 1  is i-Pr;     then R 2  is OMe, or OEt; R 3  is H; and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    e) when R 3  is Et;     then R 2  is H, R 1  is F or Cl, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    f) when R 2  and R 3 , together with the phenyl carbons to which they are attached, form an ethylenedioxy ring;     then R 1  is Me or Et and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    g) when R 2  and R 3 , together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring,     then R 1  is Et and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    h) when R 1  is formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ;     then R 2  is OMe or OEt, R 3  is H, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me; and    i) when R 2  is Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H. COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, or NH—CN;     then R 1  is Et, R 3  is H, the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    with a DNA construct comprising:    a) the exogenous gene; and    b) a response element;    wherein:    a) the exogenous gene is under the control of the response element; and    b) binding of the DNA binding domain to the response element in the presence of the ligand results in activation or suppression of the gene.    
     
     
         3 . 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 ligand of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 E is a (C 4 -C 6 )alkyl containing a tertiary carbon or a cyano(C 3 -C 5 )alkyl containing a tertiary carbon;  
 R 1  is H, Me, Et, i-Pr, F, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ;  
 R 2  is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, NH—CN, or joined with R 3  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;  
 R 3  is H, Et, or joined with R 2  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;  
 R 4 , R 5 , and R 6  are independently H, Me, Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;  
 provided that:  
 a) when R 1  is Me and R 2  is OMe;  
  then R 3  is H; and the combination R 4 , R 5 , and R 6  is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, or 3,5-di-F;  
 b) when R 1  is Me and R 2  is OEt;  
  then R 3  is H and the combination R 4 , R 5 , and R 6  is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl;  
 c) when R 1  is Et and R 2  is OMe or OEt;  
  then R 3  is H and the combination R 4 , R 5 , and R 6  is: 
 i) 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl, 3-OMe, 2Cl-5-Me, 2-Br-5-Me, 2-Cl, 2-Br, or 3-Me; or  
 ii) R 6  is H, R 4  is Me, and R 5  is Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;  
 
 d) when R 1  is i-Pr;  
  then R 2  is OMe, or OEt; R 3  is H; and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;  
 e) when R 3  is Et;  
  then R 2  is H, R 1  is F or Cl, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;  
 f) when R 2  is R 3 , together with the phenyl carbons to which they are attached, form an ethylenedioxy ring;  
  then R 1  is Me or Et and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;  
 g) when R 2  is R 3 , together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring;  
  then R 1  is Et and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;  
 h) when R 1  is formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ;  
  then R 2  is OMe or OEt, R 3  is H, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me; and  
 i) when R 2  is Me, Et, n-Pr, 1-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H. COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 4 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, or NH—CN;  
  then R 1  is Et, R 4  is H, the combination R 4 , R 5 , and R 6  is 3,5-di-Me;  
 wherein the cells of the subject contain:  
 a) an ecdysone receptor complex comprising: 
 1) a DNA binding domain;  
 2) a binding domain for the ligand; and  
 3) a transactivation domain; and  
 
 b) a DNA construct comprising: 
 1) the exogenous gene; and  
 2) a response element; and  
 wherein:  
 
 a) the exogenous gene is under the control of the response element; and  
 b) binding of the DNA binding domain to the response element in the presence of the ligand results in activation or suppression of the gene.  
 
     
     
         4 . A method for producing a polypeptide comprising the steps of: 
 a) selecting a cell which is substantially insensitive to exposure to a ligand of the formula:                          wherein:    E is a (C 4 -C 6 )alkyl containing a tertiary carbon or a cyano(C 3 -C 5 )alkyl containing a tertiary carbon;    R 1  is H, Me, Et, i-Pr, F, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ;    R 2  is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-1-Pr, SCN, SCHF 2 , SOMe, NH—CN, or joined with R 3  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;    R 3  is H, Et, or joined with R 2  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;    R 4 , R 5 , and R 6  are independently H, Me, Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;    provided that:    a) when R 1  is Me and R 2  is OMe;     R 3  is H; and the combination R 4 , R 5 , and R 6  is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, or 3,5-di-F;    b) when R 1  is Me and R 2  is OEt;     then R 3  is H and the combination R 4 , R 5 , and R 6  is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl;    c) when R 1  is Et and R 2  is OMe or OEt;     then R 3  is H and the combination R 4 , R 5 , and R 6  is: 
 i) 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4-or 2,5-di-Cl, 3-OMe, 2-Cl-5-Me, 2-Br-5-Me, 2-Cl, 2-Br, or 3-Me; or  
 ii) R 6  is H, R 4  is Me, and R 5  is Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;  
   d) when R 1  is i-Pr;     then R 2  is OMe, or OEt; R 3  is H; and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    e) when R 3  is Et;     then R 2  is H, R 1  is F or Cl, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    f) when R 2  and R 3 , together with the phenyl carbons to which they are attached, form an ethylenedioxy ring;     then R 1  is Me or Et and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    g) when R 2  and R 3 , together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring;     then R 1  is Et and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    h) when R 1  is formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ,     then R 2  is OMe or OEt, R 3  is H, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me; and    i) when R 2  is Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, or NH—CN;     then R 1  is Et, R 3  is H, the combination R 4 , R 5 , and R 6  is 3,5-di-Me;    b) introducing into the cell: 
 1) a DNA construct comprising: 
 a) an exogenous gene encoding the polypeptide; and  
 b) a response element;  
 wherein the gene is under the control of the response element; and  
 
 2) an ecdysone receptor complex comprising: 
 a) a DNA binding domain;  
 b) a binding domain for the ligand; and  
 c) a transactivation domain; and  
 
   c) exposing the cell to the lignad.    
     
     
         5 . A method for regulating endogenous or heterologous gene expression in a transgenic organism comprising contacting a ligand of the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 E is (C 4 -C 6 )alkyl containing a tertiary carbon or a cyano(C 3 -C 5 )alkyl containing a tertiary carbon;  
 R 1  is Me, Et, i-Pr, F, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ;  
 R 2  is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 O-i-Pr, SCN, SCHF 2 , SOMe, NH—CN, or joined with R 3  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;  
 R 3  is H, Et, or joined with R 2  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;  
 R 4 , R 5 , and R 6  are independently H, Me, Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;  
 provided that:  
 a) when R 1  is Me and R 2  is OMe;  
  than R 3  is H; and the combination R 4 , R 5 , and R 6  is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, or 3,5-di-F;  
 b) when R 1  is Me and R 2  is OEt;  
 then R 3  is H and the combination R 4 , R 5 , and R 6  is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5di-Cl;  
 c) when R 1  is Et and R 2  is OMe or OEt;  
  then R 3  is H and the combination R 4 , R 5 , and R 6  is: 
 i) 3,5-di-OMe-4-Me, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl, 3-OMe, 2-Cl-5-Me, 2-Br-5-Me, 2-Cl, 2-Br, or 3-Me; or  
 ii) R 6  is H, R 4  is Me, and R 5  is Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;  
 
 d) when R 1  is i-Pr;  
  then R 2  is OMe, or OEt; R 3  is H; and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;  
 c) when R 3  is Et;  
  then R 2  is H, R 1  is F or Cl, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;  
 f) when R 2  and R 3 , together with the phenyl carbons to which they are attached, form an ethylenedioxy ring;  
  then R 1  is Me or Et and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;  
 g) when R 2  and R 3 , together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring;  
  then R 1  is Et and the combination R 4 , R 5 , and R 6  is 3,5-di-Me;  
 h) when R 1  is formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 .  
  then R 2  is OMe or OEt, R 3  is H, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me; and  
 i) when R 2  is Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, or NH—CN;  
  then R 1  is Et, R 3  is H, the combination R 4 , R 5 , and R 6  is 3,5-di-Me;  
 with an Ecdysone receptor complex within the cells of the organism 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.  
 
     
     
         6 . The method of    claim 2    wherein the ligand is of the specified formula and E is t-butyl; R 1  is Me, Et, i-Pr, or F; R 2  is OH, OMe, OEt, or joined with R 3  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a phenyl carbon; R 3  is H, Et or joined with R 2  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a phenyl carbon; and R 4 , R 5 , and R 6  are independently Me, F, Cl, CH 2 OH, or OMe.  
     
     
         7 . The method of    claim 3    wherein the ligand is of the specified formula and E is t-butyl; R 1  is Me, Et, i-Pr, or F; R 2  is OH, OMe, OEt, or joined with R 3  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a phenyl carbon; R 3  is H, Et or joined with R 2  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a phenyl carbon; and R 4 , R 5 , and R 6  are independently Me, F, Cl, CH 2 OH, or OMe.  
     
     
         8 . The method of    claim 4    wherein the ligand is of the specified formula and E is t-butyl; R 1  is Me, Et, i-Pr, or F; R 2  is OH, OMe, OEt, or joined with R 3  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a phenyl carbon; R 3  is H, Et or joined with R 2  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a phenyl carbon; and R 4 , R 5 , and R 6  are independently Me, F, Cl, CH 2 OH, or OMe.  
     
     
         9 . The method of    claim 5    wherein the ligand is of the specified formula and E is t-butyl; R 1  is Me, Et, i-Pr, or F; R 2  is OH, OMe, OEt, or joined with R 3  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a phenyl carbon; R 4  is H, Et or joined with R 2  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a phenyl carbon; and R 4 , R 5 , and R 6  are independently Me, F, Cl, CH 2 OH, or OMe.  
     
     
         10 . The method of    claim 2    wherein the ligand is of the specified formula and E is t-butyl, R 1  is Et, R 2  is OEt, R 3  is H, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me.  
     
     
         11 . The method of    claim 3    wherein the ligand is of the specified formula and E is t-butyl, R 1  is Et, R 2  is OEt, R 3  is H, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me.  
     
     
         12 . The method of    claim 4    wherein the ligand is of the specified formula and E is t-butyl, R 1  is Et, R 2  is OEt, R 3  is H, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me.  
     
     
         13 . The method of    claim 5    wherein the ligand is of the specified formula and E is t-butyl, R 1  is Et, R 2  is OEt, R 3  is H, and the combination R 4 , R 5 , and R 6  is 3,5-di-Me.  
     
     
         14 . The method of    claim 2    wherein the ecdysone receptor complex is a chimeric ecdysone receptor complex and the DNA construct further comprises a promoter.  
     
     
         15 . The method of    claim 3    wherein the ecdysone receptor complex is a chimeric ecdysone receptor complex and the DNA construct further comprises a promoter.  
     
     
         16 . The method of    claim 4    wherein the ecdysone receptor complex is a chimeric ecdysone receptor complex and the DNA construct further comprises a promoter.  
     
     
         17 . The method of    claim 5    wherein the ecdysone receptor complex is a chimeric ecdysone receptor complex and the DNA construct further comprises a promoter.  
     
     
         18 . The method of    claim 3    wherein the subject is a plant  
     
     
         19 . The method of    claim 3    wherein the subject is a mammal.