Diacylhydrazine ligands for modulating the expression of exogenous genes in mammalian systems via an ecdysone receptor complex

Patent metadata

Field	Value
Publication number	US-9272986-B2
Application number	US-201414254291-A
Country	US
Kind code	B2
Filing date	Apr 16, 2014
Priority date	Feb 10, 2003
Publication date	Mar 1, 2016
Grant date	Mar 1, 2016

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.

First claim

What is claimed is:

1. A method of modulating the expression of a target gene in a host cell, wherein the host cell includes a first gene expression cassette comprising a first polynucleotide encoding a first polypeptide comprising:
(i) a transactivation domain;
(ii) a DNA-binding domain; and
(iii) a Group H nuclear receptor ligand binding domain;
a second gene expression cassette comprising:
(i) a response element capable of binding to said DNA binding domain;
(ii) a promoter that is activated by the transactivation domain; and
(iii) said target gene;

the method comprising contacting said host cell with a compound of the formula:

wherein X and X′ are independently O or S;
Y is:
(a) substituted or unsubstituted phenyl wherein the substituents are independently 1-5H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 2 -C 4 )alkenyl, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, hydroxy, amino, cyano, or nitro; or
(b) substituted or unsubstituted 2-pyridyl, 3-pyridyl, or 4-pyridyl, wherein the substituents are independently 1-4H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 2 -C 4 )alkenyl, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, hydroxy, amino, cyano, or nitro;
R 1 and R 2 are independently: H; cyano; cyano-substituted or unsubstituted (C 1 -C 7 ) branched or straight-chain alkyl; cyano-substituted or unsubstituted (C 2 -C 7 ) branched or straight-chain alkenyl; cyano-substituted or unsubstituted (C 3 -C 7 ) branched or straight-chain alkenylalkyl; or together the valences of R 1 and R 2 form a (C 1 -C 7 ) cyano-substituted or unsubstituted alkylidene group (R a R b C═) wherein the sum of non-substituent carbons in R a and R b is 0-6;
R 3 is H, methyl, ethyl, n-propyl, isopropyl, or cyano;
R 4 , R 7 , and R 8 are independently: H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 2 -C 4 )alkenyl, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, hydroxy, amino, cyano, or nitro; and
R 5 and R 6 are independently: H, (C 1 -C 4 )alkyl, (C 2 -C 4 )alkenyl, (C 3 -C 4 ) alkenylalkyl, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, (C 1 -C 4 )alkoxy, hydroxy, amino, cyano, or nitro;
wherein the number of carbon atoms, excluding those of cyano substitution, for either or both of groups R 1 or R 2 is greater than 4, and the number of carbon atoms, excluding those of cyano substitution, for the sum of groups R 1 , R 2 , and R 3 is 10, 11, or 12.

2. The method of claim 1 , wherein the compound is of the specified formula and:
X and X′ are 0;
Y is:
(a) substituted or unsubstituted phenyl wherein the substituents are independently 1-5H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, cyano, or nitro; or
(b) substituted or unsubstituted 2-pyridyl, 3-pyridyl, or 4-pyridyl, wherein the substituents are independently 1-4H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, cyano, or nitro;
R 3 is H, methyl, ethyl, or cyano;
R 4 , R 7 , and R 8 are independently: H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, cyano, or nitro; and
R 5 and R 6 are independently: H, (C 1 -C 4 )alkyl, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, (C 1 -C 4 )alkoxy, hydroxy, amino, cyano, or nitro.

3. The method of claim 2 , wherein the compound is of the specified formula and:
Y is:
(a) substituted or unsubstituted phenyl wherein the substituents are independently 1-5H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl; or
(b) substituted or unsubstituted 3-pyridyl, wherein the substituents are independently 1-4H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl;
R 1 and R 2 are independently: H; cyano; cyano-substituted or unsubstituted (C 1 -C 7 ) branched or straight-chain alkyl; cyano-substituted or unsubstituted (C 2 -C 7 ) branched or straight-chain alkenyl; cyano-substituted or unsubstituted (C 3 -C 7 ) branched or straight-chain alkenylalkyl; or together the valences of R 1 and R 2 form a (C 1 -C 7 ) cyano-substituted or unsubstituted alkylidene group (R a R b C═) wherein the sum of non-substituent carbons in R a and R b is 0-3;
R 3 is methyl;
R 4 , R 7 , and R 8 are independently selected from: H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl; and
R 5 and R 6 are independently: H, (C 1 -C 4 )alkyl, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, or (C 1 -C 4 )alkoxy.

4. The method of claim 1 , wherein the compound is selected from the group consisting of:
a) 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-heptyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide,
b) 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-heptyl)-N′-(4-ethyl-benzoyl)-hydrazide,
c) 3,5-Dimethoxy-4-methyl-benzoic acid-N-(1-tert-butyl-heptyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide,
d) 3,5-Dimethoxy-4-methyl-benzoic acid-N-(1-tert-butyl-heptyl)-N′-(4-ethyl-benzoyl)-hydrazide,
e) 2-Methoxy-nicotinic acid N-(1-tert-butyl-heptyl)-N′-(4-ethyl-benzoyl)-hydrazide,
f) 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-3,4,4-trimethyl-pent-2-enyl)-N-(3-methoxy-2-methyl-benzoyl)-hydrazide,
g) 3,5-Dimethyl-benzoic acid N-(1-butyl-2,2-dimethyl-pentyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide, and
h) 3,5-Dimethyl-benzoic acid N-(1-butyl-2,2-dimethyl-pent-4-enyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide.

5. A method to modulate the expression of one or more exogenous genes in a subject, wherein a host cell within said subject comprises a gene switch comprising a ligand binding domain that binds said ligand, the method comprising administering to the subject an effective amount of a ligand of the formula:

wherein X and X′ are independently O or S;
Y is:
(a) substituted or unsubstituted phenyl wherein the substituents are independently 1-5H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 2 -C 4 )alkenyl, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, hydroxy, amino, cyano, or nitro; or
(b) substituted or unsubstituted 2-pyridyl, 3-pyridyl, or 4-pyridyl, wherein the substituents are independently 1-4H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 2 -C 4 )alkenyl, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, hydroxy, amino, cyano, or nitro;
R 1 and R 2 are independently: H; cyano; cyano-substituted or unsubstituted (C 1 -C 7 ) branched or straight-chain alkyl; cyano-substituted or unsubstituted (C 2 -C 7 ) branched or straight-chain alkenyl; cyano-substituted or unsubstituted (C 3 -C 7 ) branched or straight-chain alkenylalkyl; or together the valences of R 1 and R 2 form a (C 1 -C 7 ) cyano-substituted or unsubstituted alkylidene group (R a R b C═) wherein the sum of non-substituent carbons in R a and R b is 0-6;
R 3 is H, methyl, ethyl, n-propyl, isopropyl, or cyano;
R 4 , R 7 , and R 8 are independently: H, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 2 -C 4 )alkenyl, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, hydroxy, amino, cyano, or nitro; and
R 5 and R 6 are independently: H, (C 1 -C 4 )alkyl, (C 2 -C 4 )alkenyl, (C 3 -C 4 ) alkenylalkyl, halo (F, Cl, Br, I), (C 1 -C 4 )haloalkyl, (C 1 -C 4 )alkoxy, hydroxy, amino, cyano, or nitro;
wherein the number of carbon atoms, excluding those of cyano substitution, for either or both of groups R 1 or R 2 is greater than 4, and the number of carbon atoms, excluding those of cyano substitution, for the sum of groups R 1 , R 2 , and R 3 is 10, 11, or 12.

6. A method for regulating endogenous or heterologous gene expression in a transgenic subject comprising contacting a ligand with

Assignees

• Intrexon Corp

Inventors

• Hormann Robert Eugene
• Tice Colin M
• Chortyk Orestes
• Smith Howard
• Meteyer Thomas

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• C07F7/1896

  Compounds having one or more Si-O-acyl linkages · CPC title

• C07D319/20

  with substituents attached to the hetero ring · CPC title

• C07D311/66

  with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2 · CPC title

• C12N2830/002

  inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor · CPC title