Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-05-20
2001-07-10
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S440000, C549S366000, C549S405000, C549S436000, C564S310000
Reexamination Certificate
active
06258603
ABSTRACT:
This invention relates to non-steroidal ligands which are useful for inducing or suppressing the expression of an exogenous gene in animal and plant cells.
In the field of genetic engineering, precise temporal control of gene expression, that is, the ability to activate or suppress a gene, is a valuable tool in studying, manipulating, and controlling development and other physiological processes (see, for example, Evans and No, PCT International Application No. PCT/US97/05330 and references cited therein). In mammalian systems, applications include inducible gene targeting, overexpression of toxic and teratogenic genes, anti-sense RNA expression, and gene therapy. In plant systems, applications include the control of plant traits, male or female fertility; overexpression of plant protective agents; and production of or modification of desired plant products including both native and non-native materials. For both animals and plants, inducibility can be valuable for foreign protein production, for example, therapeutic proteins, industrial enzymes, polymers, and the like.
It is important that the agent used to control gene expression, which is often referred to as a “gene switch”, be one which is normally absent from the organism in which the gene to be controlled resides. This is to avoid unexpected expression or suppression of the gene. For example, an inducible tetracycline regulated system has been devised and utilized in transgenic mice whereby gene activity is induced in the absence of the antibiotic and suppressed in its presence. Unfortunately, in this case, the pharmacokinetics of tetracycline may interfere with its use as an efficient “on-off” gene switch.
International Patent Application No. PCT/GB96/01195 describes an insect steroid receptor isolated from
Heliothis virescens
(“HEcR”) which is capable of acting as a gene switch responsive to both steroid (e.g., 20-hydroxyecdysone and Muristerone A) and certain non-steroidal inducers. Non-steroidal inducers have a distinct advantage over steroids, in this and many other systems which are responsive to both steroids and non-steroid inducers, for a number of reasons including, for example: lower manufacturing cost, metabolic stability, absence from insects, plants, or mammals, and environmental acceptability. The PCT application describes the utility of two dibenzoylhydrazines, 1,2-dibenzoyl-1-tert-butyl-hydrazine and tebufenozide (N-(4-ethylbenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butyl-hydrazine) as gene switches for the HEcR system and suggests that other dibenzoylhydrazines, such as those disclosed in U.S. Pat. No. 5,117,057 may also function as gene switches in the system. While this may be true, activity of these dibenzoylhydrazines is uncertain. Specifically, U.S. Pat. No. 5,117,057 shows a very broad class of dibenzoylhydrazines, many of which appear to be ineffective as gene switches. GB96/01195 indicates that when 20 such dibenzoylhydrazines were tested, only 7 showed any activity.
International Patent Application No. PCT/EP96/00686 discloses the use of tebufenozide as a chemical ligand for the ecdysone receptor from
Drosophila melanogaster
. This receptor is used to control gene expression in transgenic plants resulting in the control of various traits of agronomic importance
Unfortunately, even though the ligands described in the above-identified references show reporter gene induction activity in isolated cells, no consideration was made for their use in whole organisms such as intact plants, and animals.
Therefore, there remains a continuing need to develop non-steroidal ligands with increased or consistent activity compared to known ligands and which demonstrate that activity in intact plants and animals. We have discovered a limited group of dibenzoylhydrazine derivatives which not only show reporter gene induction activity in isolated cells but also have advantages over the known diacylhydrazines, 1,2-dibenzoyl-1-tert-butyl-hydrazine and tebufenozide, when used in intact plants and animals, due to their improved transport and distribution properties, metabolic stability, residual activity, affinity for the receptor, and lack of adverse effects.
This invention relates to an improvement 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 formula I:
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, 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, SO
Carlson Glenn Richard
Cress Dean Ervin
Dhadialla Tarlochan Singh
Hormann Robert Eugene
Le Dat Phat
Rogerson Thomas D.
Rohm and Haas Company
Schwartzman Robert A.
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