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

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:
• 5
• wherein:
• E is a (C1-C6)alkyl containing a tertiary carbon or a cyano(C3-C5)alkyl containing a tertiary carbon;
• R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN or SCHF2;
• R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, NH—CN, or joined with R3 and the phenyl carbons to which R2 and R3 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;
• R3 is H, Et, or joined with R2 and the phenyl carbons to which R2 and R3 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;
• R4, R5, and R6 are independently H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, C≡CN, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;
• provided that:
• a) when R1 is Me and R2 is OMe;
• then R3 is H;
• and the combination R4, R5, and R6 is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, or 3,5-di-F;
• b) when R1 is Me an R2 is OEt;
• then R3 is H and the combination R4, R5, and R6 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 R1 is Et and R2 is OMe or OEt;
• then R3 is H and the combination R4, R5, and R6 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) R6 is H, R4 is Me, and R5 is Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;
• d) when R1 is i-Pr;
• then R2 is OMe, or OEt;
• R3 is H;
• and the combination R4, R5, and R6 is 3,5-di-Me;
• e) when R3 is Et;
• then R2 is H, R1 is F or Cl, and the combination R4, R5, and R6 is 3,5-di-Me;
• f) when R2 and R3, together with the phenyl carbons to which they are attached, form an ethylenedioxy ring;
• then R1 is Me or Et and the combination R4, R5, and R6 is 3,5-di-Me;
• g) when R2 and R3, together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring,
• then R1 is Et and the combination R4, R5, and R6 is 3,5-di-Me;
• h) when R1 is formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2;
• then R2 is OMe or OEt, R3 is H, and the combination R4, R5, and R6 is 3,5-di-Me;
• and
• i) when R2 is Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H. COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, or NH—CN;
• then R1 is Et, R3 is H, the combination R4, R5, and R6 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.

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:

• 6
• wherein:
• E is a (C4-C6)alkyl containing a tertiary carbon or a cyano(C3-C5)alkyl containing a tertiary carbon;
• R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2;
• R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, NH—CN, or joined with R3 and the phenyl carbons to which R2 and R3 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;
• R3 is H, Et, or joined with R2 and the phenyl carbons to which R2 and R3 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;
• R4, R5, and R6 are independently H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;
• provided that:
• a) when R1 is Me and R2 is OMe;
• then R3 is H;
• and the combination R4, R5, and R6 is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, or 3,5-di-F;
• b) when R1 is Me and R2 is OEt;
• then R3 is H and the combination R4, R5, and R6 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 R1 is Et and R2 is OMe or OEt;
• then R3 is H and the combination R4, R5, and R6 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) R6 is H, R4 is Me, and R5 is Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;
• d) when R1 is i-Pr;
• then R2 is OMe, or OEt;
• R3 is H;
• and the combination R4, R5, and R6 is 3,5-di-Me;
• e) when R3 is Et;
• then R2 is H, R1 is F or Cl, and the combination R4, R5, and R6 is 3,5-di-Me;
• f) when R2 is R3, together with the phenyl carbons to which they are attached, form an ethylenedioxy ring;
• then R1 is Me or Et and the combination R4, R5, and R6 is 3,5-di-Me;
• g) when R2 is R3, together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring;
• then R1 is Et and the combination R4, R5, and R6 is 3,5-di-Me;
• h) when R1 is formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2;
• then R2 is OMe or OEt, R3 is H, and the combination R4, R5, and R6 is 3,5-di-Me;
• and
• i) when R2 is Me, Et, n-Pr, 1-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H. COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF4, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, or NH—CN;
• then R1 is Et, R4 is H, the combination R4, R5, and R6 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.

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:
• 7
• wherein:
• E is a (C4-C6)alkyl containing a tertiary carbon or a cyano(C3-C5)alkyl containing a tertiary carbon;
• R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2;
• R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2, O-1-Pr, SCN, SCHF2, SOMe, NH—CN, or joined with R3 and the phenyl carbons to which R2 and R3 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;
• R3 is H, Et, or joined with R2 and the phenyl carbons to which R2 and R3 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;
• R4, R5, and R6 are independently H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;
• provided that:
• a) when R1 is Me and R2 is OMe;
• R3 is H;
• and the combination R4, R5, and R6 is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, or 3,5-di-F;
• b) when R1 is Me and R2 is OEt;
• then R3 is H and the combination R4, R5, and R6 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 R1 is Et and R2 is OMe or OEt;
• then R3 is H and the combination R4, R5, and R6 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) R6 is H, R4 is Me, and R5 is Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;
• d) when R1 is i-Pr;
• then R2 is OMe, or OEt;
• R3 is H;
• and the combination R4, R5, and R6 is 3,5-di-Me;
• e) when R3 is Et;
• then R2 is H, R1 is F or Cl, and the combination R4, R5, and R6 is 3,5-di-Me;
• f) when R2 and R3, together with the phenyl carbons to which they are attached, form an ethylenedioxy ring;
• then R1 is Me or Et and the combination R4, R5, and R6 is 3,5-di-Me;
• g) when R2 and R3, together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring;
• then R1 is Et and the combination R4, R5, and R6 is 3,5-di-Me;
• h) when R1 is formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2,
• then R2 is OMe or OEt, R3 is H, and the combination R4, R5, and R6 is 3,5-di-Me;
• and
• i) when R2 is Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl azido, OCF3, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, or NH—CN;
• then R1 is Et, R3 is H, the combination R4, R5, and R6 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.

A method for regulating endogenous or heterologous gene expression in a transgenic organism comprising contacting a ligand of the formula:

• 8
• wherein
• E is (C4-C6)alkyl containing a tertiary carbon or a cyano(C3-C5)alkyl containing a tertiary carbon;
• R1 is Me, Et, i-Pr, F, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2;
• R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2O-i-Pr, SCN, SCHF2, SOMe, NH—CN, or joined with R3 and the phenyl carbons to which R2 and R3 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;
• R3 is H, Et, or joined with R2 and the phenyl carbons to which R2 and R3 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;
• R4, R5, and R6 are independently H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;
• provided that:
• a) when R1 is Me and R2 is OMe;
• than R3 is H;
• and the combination R4, R5, and R6 is 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, or 3,5-di-F;
• b) when R1 is Me and R2 is OEt;
• then R3 is H and the combination R4, R5, and R6 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 R1 is Et and R2 is OMe or OEt;
• then R3 is H and the combination R4, R5, and R6 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) R6 is H, R4 is Me, and R5 is Et, F, Cl, Br, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt;
• d) when R1 is i-Pr;
• then R2 is OMe, or OEt;
• R3 is H;
• and the combination R4, R5, and R6 is 3,5-di-Me;
• c) when R3 is Et;
• then R2 is H, R1 is F or Cl, and the combination R4, R5, and R6 is 3,5-di-Me;
• f) when R2 and R3, together with the phenyl carbons to which they are attached, form an ethylenedioxy ring;
• then R1 is Me or Et and the combination R4, R5, and R6 is 3,5-di-Me;
• g) when R2 and R3, together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring;
• then R1 is Et and the combination R4, R5, and R6 is 3,5-di-Me;
• h) when R1 is formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, SCN, or SCHF2.
• then R2 is OMe or OEt, R3 is H, and the combination R4, R5, and R6 is 3,5-di-Me;
• and
• i) when R2 is Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHCl2, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, O-n-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH═CHCN, allyl, azido, OCF3, OCHF2, O-i-Pr, SCN, SCHF2, SOMe, or NH—CN;
• then R1 is Et, R3 is H, the combination R4, R5, and R6 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.