U.S. patent application number 14/817901 was filed with the patent office on 2016-02-11 for bioavailable diacylhydrazine ligands for modulating the expression of exogenous genes via an ecdysone receptor complex.
This patent application is currently assigned to Intrexon Corporation. The applicant listed for this patent is Intrexon Corporation. Invention is credited to Glenn Richard Carlson, Orestes Chortyk, Robert Eugene HORMANN, Andrew Meyer, Thomas R. Opie, David W. Potter, Colin M. Tice.
Application Number | 20160039750 14/817901 |
Document ID | / |
Family ID | 32965808 |
Filed Date | 2016-02-11 |
United States Patent
Application |
20160039750 |
Kind Code |
A1 |
HORMANN; Robert Eugene ; et
al. |
February 11, 2016 |
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.
Inventors: |
HORMANN; Robert Eugene;
(Melrose Park, PA) ; Potter; David W.; (North
Wales, PA) ; Chortyk; Orestes; (Thompson Station,
TN) ; Tice; Colin M.; (Elkins Park, PA) ;
Carlson; Glenn Richard; (North Wales, PA) ; Meyer;
Andrew; (Maple Glen, PA) ; Opie; Thomas R.;
(North Wales, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Intrexon Corporation |
Blacksburg |
VA |
US |
|
|
Assignee: |
Intrexon Corporation
Blacksburg
VA
|
Family ID: |
32965808 |
Appl. No.: |
14/817901 |
Filed: |
August 4, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11841617 |
Aug 20, 2007 |
9102648 |
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14817901 |
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10787906 |
Feb 26, 2004 |
7456315 |
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11841617 |
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60455741 |
Feb 28, 2003 |
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Current U.S.
Class: |
514/255.06 ;
435/455; 435/468; 435/471; 435/69.1; 514/356; 514/394; 514/406;
514/419; 514/452; 514/546; 514/615; 544/407; 546/322; 548/309.4;
548/374.1; 548/492; 549/365; 560/232; 564/149; 564/150 |
Current CPC
Class: |
A61K 31/222 20130101;
Y02A 50/30 20180101; C07C 243/18 20130101; C07C 317/44 20130101;
A61K 31/404 20130101; A61P 29/00 20180101; C07C 243/38 20130101;
A61K 31/4184 20130101; C07D 317/68 20130101; C12N 9/0069 20130101;
C07D 237/24 20130101; C07D 319/18 20130101; A61K 31/415 20130101;
A61P 31/00 20180101; C07D 239/52 20130101; A61K 31/455 20130101;
C07C 315/02 20130101; C07C 309/59 20130101; C07D 213/87 20130101;
C12P 21/00 20130101; C07D 271/12 20130101; A61P 7/02 20180101; C07C
251/38 20130101; A61K 31/4965 20130101; A61K 31/357 20130101; A61P
3/10 20180101; C07D 319/08 20130101; C07D 319/20 20130101; A61P
33/02 20180101; C07D 405/12 20130101; C07D 209/42 20130101; C07C
241/04 20130101; C07C 243/24 20130101; C07C 317/46 20130101; C07D
213/81 20130101; C07D 231/14 20130101; C07D 235/06 20130101; C12N
15/635 20130101; A61P 9/00 20180101; C07D 235/24 20130101; A61P
5/00 20180101; C07C 281/10 20130101; C07D 231/56 20130101; A61P
19/02 20180101; A61P 43/00 20180101; C07C 251/08 20130101; A61P
35/00 20180101; C07C 255/66 20130101; A61K 31/166 20130101; C07D
213/82 20130101; C07D 241/24 20130101; C07D 213/86 20130101; A61P
23/00 20180101 |
International
Class: |
C07C 241/04 20060101
C07C241/04; A61K 31/357 20060101 A61K031/357; C07D 319/08 20060101
C07D319/08; A61K 31/222 20060101 A61K031/222; C07C 315/02 20060101
C07C315/02; A61K 31/4965 20060101 A61K031/4965; C07D 241/24
20060101 C07D241/24; A61K 31/455 20060101 A61K031/455; C07D 213/86
20060101 C07D213/86; A61K 31/4184 20060101 A61K031/4184; C07D
235/24 20060101 C07D235/24; A61K 31/404 20060101 A61K031/404; C07D
209/42 20060101 C07D209/42; A61K 31/415 20060101 A61K031/415; C07D
231/14 20060101 C07D231/14; A61K 31/166 20060101 A61K031/166 |
Claims
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: ##STR00109##
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.sup.aR.sup.b);
alkylaminoalkyl (--(CH.sub.2).sub.nNR.sup.aR.sup.b);
(C.sub.1-C.sub.6)alkyl; (C.sub.1-C.sub.6)haloalkyl;
(C.sub.1-C.sub.6)cyanoalkyl; (C.sub.1-C.sub.6)hydroxyalkyl;
(C.sub.1-C.sub.6)alkoxy; phenoxy; (C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)alkanoyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; carboxy;
(C.sub.1-C.sub.6)alkylcarbonyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--NR.sup.aCO.sub.2R.sup.b); alkylaminocarbonylamino
(--NR.sup.aCONR.sup.bR.sup.a); mercapto;
(C.sub.1-C.sub.6)alkylthio; (C.sub.1-C.sub.6)alkylsulfonyl;
(C.sub.1-C.sub.6)alkylsulfonyl(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkylsulfoxido (--S(O)R.sup.a);
(C.sub.1-C.sub.6)alkylsulfoxido(C.sub.1-C.sub.6)alkyl-(CH.sub.2)S(O)R.sup-
.a); sulfamido (--SO.sub.2NR.sup.aR.sup.b); --SO.sub.3H; or
unsubstituted or substituted phenyl wherein the substituents are
independently 1 to 3 halo, nitro, (C.sub.1-C.sub.6)alkoxy,
(C.sub.1-C.sub.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.sub.2O--),
(--OCH(CH.sub.3)O--), (--OCH.sub.2CH.sub.2O--),
(--OCH(CH.sub.3)CH.sub.2O--), (--S--CH.dbd.N--),
(--CH.sub.2OCH.sub.2O--), (--O(CH.sub.2).sub.3--),
(.dbd.N--O--N.dbd.), (--CH.dbd.CH--NH--), (--OCF.sub.2O--),
(--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.4--); B is: (a) unsubstituted or substituted
phenyl wherein the substituents are independently 1 to 5H; halo;
nitro; cyano; hydroxy; amino (--NR.sup.aR.sup.b); alkylaminoalkyl
(--(CH.sub.2).sub.nNR.sup.aR.sup.b); (C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)haloalkyl; (C.sub.1-C.sub.6)cyanoalkyl;
(C.sub.1-C.sub.6)hydroxyalkyl; (C.sub.1-C.sub.6)alkoxy; phenoxy;
(C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)alkanoyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; carboxy;
(C.sub.1-C.sub.6)alkylcarbonyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
-benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--NR.sup.aCO.sub.2R.sup.b); alkylaminocarbonylamino
(--NR.sup.aCONR.sup.bR.sup.c); mercapto;
(C.sub.1-C.sub.6)alkylthio; (C.sub.1-C.sub.6)alkylsulfonyl;
(C.sub.1-C.sub.6)alkylsulfonyl(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkylsulfoxido (--S(O)R.sup.a);
(C.sub.1-C.sub.6)alkylsulfoxido(C.sub.1-C.sub.6)alkyl
(--CH.sub.2).sub.nS(O)R.sup.a); sulfamido
(--SO.sub.2NR.sup.aR.sup.b); --SO.sub.3H;
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b;
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b; or unsubstituted or
substituted phenyl wherein the substituents are independently 1 to
3 halo, nitro, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.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.sub.2O--), (--OCH(CH.sub.3)O--),
(--OCH.sub.2CH.sub.2O--), (--OCH(CH.sub.3)CH.sub.2O--),
(--S--CH.dbd.N--), (--CH.sub.2OCH.sub.2O--),
(--O(CH.sub.2).sub.3--), (.dbd.N--O--N.dbd.), (--CH.dbd.CH--NH--),
(--OCF.sub.2O--), (--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.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.sub.1-C.sub.6)alkyl; (C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)thioalkoxy; carboxy;
(C.sub.1-C.sub.6)alkoxycarbonyl; (C.sub.1-C.sub.6)carbocyalkyl;
(C.sub.1-C.sub.6)alkoxycarbonylalkyl having independently the
stated number of carbon atoms in each alkyl group;
--CONR.sup.aR.sup.b; amino; (C.sub.1-C.sub.6)alkylamino;
(C.sub.1-C.sub.6)dialkylamino having independently the stated
number of carbon atoms in each alkyl group; haloalkyl;
CH.dbd.N--NHC(O)NR.sup.aR.sup.b; or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.6)alkyl-1H-indole-2-yl; E is unsubstituted or
substituted (C.sub.4-C.sub.10) branched alkyl wherein the
substituents are independently 1-4 cyano; halo;
(C.sub.5-C.sub.6)cycloalkyl; phenyl; (C.sub.2-C.sub.3)alkenyl;
hydroxy, (C.sub.1-C.sub.6)alkoxy; carboxy;
(C.sub.1-C.sub.6)alkoxycarbonyl; formyl;
(C.sub.1-C.sub.6)trialkylsilyloxy having independently the stated
number of carbon atoms in each alkyl group; --CH.dbd.N--OR.sup.a;
--CH.dbd.N--R.sup.d; --CH.dbd.N--NHC(O)NR.sup.aR.sup.b; or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b; wherein R.sup.a, R.sup.b,
and R.sup.c are independently H, (C.sub.1-C.sub.6)alkyl, or phenyl;
R.sup.d is hydroxy(C.sub.1-C.sub.6)alkyl; and n=1-4; and G is H or
CN; provided that: 1) when E is unsubstituted or substituted
(C.sub.4-C.sub.10) branched alkyl wherein the substituents are
independently 1-4 cyano; halo; (C.sub.2-C.sub.3)alkenyl; carboxy;
or (C.sub.1-C.sub.6)alkoxycarbonyl; then B is: (a) substituted
phenyl which bears at least one --CH.dbd.N--NHC(O)NR.sup.aR.sup.b
or --CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.6)alkyl-1H-indole-2-yl; 2) when E is a substituted
(C.sub.4-C.sub.10) branched alkyl which bears at least one of
phenyl; hydroxy; (C.sub.1-C.sub.6)alkoxy; or formyl; then B is: (a)
substituted phenyl which bears at least one
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.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: ##STR00110## 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.sup.aR.sup.b);
alkylaminoalkyl((CH.sub.2).sub.nNR.sup.aR.sup.b);
(C.sub.1-C.sub.6)alkyl; (C.sub.1-C.sub.6)haloalkyl;
(C.sub.1-C.sub.6)cyanoalkyl; (C.sub.1-C.sub.6)hydroxyalkyl;
(C.sub.1-C.sub.6)alkoxy; phenoxy; (C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)alkanoyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; carboxy;
(C.sub.1-C.sub.6)alkylcarbonyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--NR.sup.aCO.sub.2R.sup.b); alkylaminocarbonylamino
(--NR.sup.aCONR.sup.bR.sup.c); mercapto;
(C.sub.1-C.sub.6)alkylthio; (C.sub.1-C.sub.6)alkylsulfonyl;
(C.sub.1-C.sub.6)alkylsulfonyl(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkylsulfoxido (--S(O)R.sup.a);
(C.sub.1-C.sub.6)alkylsulfoxido(C.sub.1-C.sub.6)alkyl
--(CH.sub.2)S(O)R.sup.a); sulfamido (--SO.sub.2NR.sup.aR.sup.b);
--SO.sub.3H; or unsubstituted or substituted phenyl wherein the
substituents are independently 1 to 3 halo, nitro,
(C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.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.sub.2O--), (--OCH(CH.sub.3)O--), (--OCH.sub.2CH.sub.2O--),
(--OCH(CH.sub.3)CH.sub.2O--), (--S--CH.dbd.N--),
(--CH.sub.2OCH.sub.2O--), (--O(CH.sub.2).sub.3--),
(.dbd.N--O--N.dbd.), (--CH.dbd.CH--NH--), (--OCF.sub.2O--),
(--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.4--); B is: (a) unsubstituted or substituted
phenyl wherein the substituents are independently 1 to 5H; halo;
nitro; cyano; hydroxy; amino (--NR.sup.aR.sup.b); alkylaminoalkyl
(--(CH.sub.2).sub.nNR.sup.aR.sup.b); (C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)haloalkyl; (C.sub.1-C.sub.6)cyanoalkyl;
(C.sub.1-C.sub.6)hydroxyalkyl; (C.sub.1-C.sub.6)alkoxy; phenoxy;
(C.sub.1-C.sub.6)halo alkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)alkanoyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; carboxy;
(C.sub.1-C.sub.6)alkylcarbonyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--NR.sup.aCO.sub.2R.sup.b); alkylaminocarbonylamino
(--NR.sup.aCONR.sup.bR.sup.c); mercapto;
(C.sub.1-C.sub.6)alkylthio; (C.sub.1-C.sub.6)alkylsulfonyl;
(C.sub.1-C.sub.6)alkylsulfonyl(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkylsulfoxido (--S(O)R.sup.a);
(C.sub.1-C.sub.6)alkylsulfoxido(C.sub.1-C.sub.6)alkyl
(--CH.sub.2).sub.nS(O)R.sup.a); sulfamido
(--SO.sub.2NR.sup.aR.sup.b); --SO.sub.3H;
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b;
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b; or unsubstituted or
substituted phenyl wherein the substituents are independently 1 to
3 halo, nitro, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.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.sub.2O--), (--OCH(CH.sub.3)O--),
(--OCH.sub.2CH.sub.2O--), (--OCH(CH.sub.3)CH.sub.2O--),
(--S--CH.dbd.N--), (--CH.sub.2OCH.sub.2O--),
(--O(CH.sub.2).sub.3--), (.dbd.N--O--N.dbd.), (--CH.dbd.CH--NH--),
(--OCF.sub.2O--), (--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.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.sub.1-C.sub.6)alkyl; (C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)thioalkoxy; carboxy;
(C.sub.1-C.sub.6)alkoxycarbonyl; (C.sub.1-C.sub.6)carbocyalkyl;
(C.sub.1-C.sub.6)alkoxycarbonylalkyl having independently the
stated number of carbon atoms in each alkyl group;
--CONR.sup.aR.sup.b; amino; (C.sub.1-C.sub.6)alkylamino;
(C.sub.1-C.sub.6)dialkylamino having independently the stated
number of carbon atoms in each alkyl group; haloalkyl;
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b; or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.6)alkyl-1H-indole-2-yl; E is unsubstituted or
substituted (C.sub.4-C.sub.10) branched alkyl wherein the
substituents are independently 1-4 cyano; halo;
(C.sub.5-C.sub.6)cycloalkyl; phenyl; (C.sub.2-C.sub.3)alkenyl;
hydroxy, (C.sub.1-C.sub.6)alkoxy; carboxy;
(C.sub.1-C.sub.6)alkoxycarbonyl; formyl;
(C.sub.1-C.sub.6)trialkylsilyloxy having independently the stated
number of carbon atoms in each alkyl group; --CH.dbd.N--OR.sup.a;
--CH.dbd.N--R.sup.d; --CH.dbd.N--NHC(O)NR.sup.aR.sup.b; or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b; wherein R.sup.a, R.sup.b,
and R.sup.c are independently H, (C.sub.1-C.sub.6)alkyl, or phenyl;
R.sup.d is hydroxy(C.sub.1-C.sub.6)alkyl; and n=1-4; and G is H or
CN; provided that: 1) when E is unsubstituted or substituted
(C.sub.4-C.sub.10) branched alkyl wherein the substituents are
independently 1-4 cyano; halo; (C.sub.2-C.sub.3)alkenyl; carboxy;
or (C.sub.1-C.sub.6)alkoxycarbonyl; then B is: (a) substituted
phenyl which bears at least one --CH.dbd.N--NHC(O)NR.sup.aR.sup.b
or --CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.6)alkyl-1H-indole-2-yl; 2) when E is a substituted
(C.sub.4-C.sub.10) branched alkyl which bears at least one of
phenyl; hydroxy; (C.sub.1-C.sub.6)alkoxy; or formyl; then B is: (a)
substituted phenyl which bears at least one
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.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: ##STR00111## 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.sup.aR.sup.b); alkylaminoalkyl
(--(CH.sub.2).sub.nNR.sup.aR.sup.b); (C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)haloalkyl; (C.sub.1-C.sub.6)cyanoalkyl;
(C.sub.1-C.sub.6)hydroxyalkyl; (C.sub.1-C.sub.6)alkoxy; phenoxy;
(C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)alkanoyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; carboxy;
(C.sub.1-C.sub.6)alkylcarbonyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--NR.sup.aCO.sub.2R.sup.b); alkylaminocarbonylamino
(--NR.sup.aCONR.sup.bR.sup.c); mercapto;
(C.sub.1-C.sub.6)alkylthio; (C.sub.1-C.sub.6)alkylsulfonyl;
(C.sub.1-C.sub.6)alkylsulfonyl(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkylsulfoxido (--S(O)R.sup.a);
(C.sub.1-C.sub.6)alkylsulfoxido(C.sub.1-C.sub.6)alkyl
--(CH.sub.2).sub.nS(O)R.sup.a); sulfamido
(--SO.sub.2NR.sup.aR.sup.b); --SO.sub.3H; or unsubstituted or
substituted phenyl wherein the substituents are independently 1 to
3 halo, nitro, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.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.sub.2O--), (--OCH(CH.sub.3)O--),
(--OCH.sub.2CH.sub.2O--), (--OCH(CH.sub.3)CH.sub.2O--),
(--S--CH.dbd.N--), (--CH.sub.2OCH.sub.2O--),
(--O(CH.sub.2).sub.3--), (.dbd.N--O--N.dbd.), (--CH.dbd.CH--NH--),
(--OCF.sub.2O--), (--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.4--); B is: (a) unsubstituted or substituted
phenyl wherein the substituents are independently 1 to 5H; halo;
nitro; cyano; hydroxy; amino (--NR.sup.aR.sup.b); alkylaminoalkyl
(--(CH.sub.2).sub.nNR.sup.aR.sup.b); (C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)haloalkyl; (C.sub.1-C.sub.6)cyanoalkyl;
(C.sub.1-C.sub.6)hydroxyalkyl; (C.sub.1-C.sub.6)alkoxy; phenoxy;
(C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C1-C.sub.6)alkanoyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; carboxy;
(C.sub.1-C.sub.6)alkylcarbonyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--NR.sup.aCO.sub.2R.sup.b); alkylaminocarbonylamino
(--NR.sup.aCONR.sup.bR.sup.c); mercapto;
(C.sub.1-C.sub.6)alkylthio; (C.sub.1-C.sub.6)alkylsulfonyl;
(C.sub.1-C.sub.6)alkylsulfonyl(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkylsulfoxido (--S(O)R.sup.a);
(C.sub.1-C.sub.6)alkylsulfoxido(C.sub.1-C.sub.6)alkyl
(--CH.sub.2).sub.nS(O)R.sup.a); sulfamido
(--SO.sub.2NR.sup.aR.sup.b); --SO.sub.3H;
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b;
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b; or unsubstituted or
substituted phenyl wherein the substituents are independently 1 to
3 halo, nitro, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.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.sub.2O--), (--OCH(CH.sub.3)O--),
(--OCH.sub.2CH.sub.2O--), (--OCH(CH.sub.3)CH.sub.2O--),
(--S--CH.dbd.N--), (--CH.sub.2OCH.sub.2O--),
(--O(CH.sub.2).sub.3--), (.dbd.N--O--N.dbd.), (--CH.dbd.CH--NH--),
(--OCF.sub.2O--), (--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.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.sub.1-C.sub.6)alkyl; (C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)thioalkoxy; carboxy;
(C.sub.1-C.sub.6)alkoxycarbonyl; (C.sub.1-C.sub.6)carbocyalkyl;
(C.sub.1-C.sub.6)alkoxycarbonylalkyl having independently the
stated number of carbon atoms in each alkyl group;
--CONR.sup.aR.sup.b; amino; (C.sub.1-C.sub.6)alkylamino;
(C.sub.1-C.sub.6)dialkylamino having independently the stated
number of carbon atoms in each alkyl group; haloalkyl;
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b; or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.6)alkyl-1H-indole-2-yl; E is unsubstituted or
substituted (C.sub.4-C.sub.10) branched alkyl wherein the
substituents are independently 1-4 cyano; halo;
(C.sub.5-C.sub.6)cycloalkyl; phenyl; (C.sub.2-C.sub.3)alkenyl;
hydroxy, (C.sub.1-C.sub.6)alkoxy; carboxy;
(C.sub.1-C.sub.6)alkoxycarbonyl; formyl;
(C.sub.1-C.sub.6)trialkylsilyloxy having independently the stated
number of carbon atoms in each alkyl group; --CH.dbd.N--OR.sup.a;
--CH.dbd.N--R.sup.d; --CH.quadrature.N--NHC(O)NR.sup.aR.sup.b; or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b; wherein R.sup.a, R.sup.b,
and R.sup.c are independently H, (C.sub.1-C.sub.6)alkyl, or phenyl;
R.sup.d is hydroxy(C.sub.1-C.sub.6)alkyl; and n=1-4; and G is H or
CN; provided that: 1) when E is unsubstituted or substituted
(C.sub.4-C.sub.10) branched alkyl wherein the substituents are
independently 1-4 cyano; halo; (C.sub.2-C.sub.3)alkenyl; carboxy;
or (C.sub.1-C.sub.6)alkoxycarbonyl; then B is: (a) substituted
phenyl which bears at least one --CH.dbd.N--NHC(O)NR.sup.aR.sup.b
or --CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.6)alkyl-1H-indole-2-yl; 2) when E is a substituted
(C.sub.4-C.sub.10) branched alkyl which bears at least one of
phenyl; hydroxy; (C.sub.1-C.sub.6)alkoxy; or formyl; then B is: (a)
substituted phenyl which bears at least one
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.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: ##STR00112## 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.sup.aR.sup.b); alkylaminoalkyl
(--(CH.sub.2).sub.nNR.sup.aR.sup.b); (C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)haloalkyl; (C.sub.1-C.sub.6)cyanoalkyl;
(C.sub.1-C.sub.6)hydroxyalkyl; (C.sub.1-C.sub.6)alkoxy; phenoxy;
(C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)alkanoyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; carboxy;
(C.sub.1-C.sub.6)alkylcarbonyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--NR.sup.aCO.sub.2R.sup.b); alkylaminocarbonylamino
(--NR.sup.aCONR.sup.bR.sup.c); mercapto;
(C.sub.1-C.sub.6)alkylthio; (C.sub.1-C.sub.6)alkylsulfonyl;
(C.sub.1-C.sub.6)alkylsulfonyl(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkylsulfoxido (--S(O)R.sup.a);
(C.sub.1-C.sub.6)alkylsulfoxido(C.sub.1-C.sub.6)alkyl
--(CH.sub.2)S(O)R.sup.a); sulfamido (--SO.sub.2NR.sup.aR.sup.b);
--SO.sub.3H; or unsubstituted or substituted phenyl wherein the
substituents are independently 1 to 3 halo, nitro,
(C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.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.sub.2O--), (--OCH(CH.sub.3)O--), (--OCH.sub.2CH.sub.2O--),
(--OCH(CH.sub.3)CH.sub.2O--), (--S--CH.dbd.N--),
(--CH.sub.2OCH.sub.2O--), (--O(CH.sub.2).sub.3--),
(.dbd.N--O--N.dbd.), (--CH.dbd.CH--NH--), (--OCF.sub.2O--),
(--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.4--); B is: (a) unsubstituted or substituted
phenyl wherein the substituents are independently 1 to 5H; halo;
nitro; cyano; hydroxy; amino (--NR.sup.aR.sup.b); alkylaminoalkyl
(--(CH.sub.2).sub.nNR.sup.aR.sup.b); (C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)haloalkyl; (C.sub.1-C.sub.6)cyanoalkyl;
(C.sub.1-C.sub.6)hydroxyalkyl; (C.sub.1-C.sub.6)alkoxy; phenoxy;
(C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)alkanoyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; carboxy;
(C.sub.1-C.sub.6)alkylcarbonyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--NR.sup.aCO.sub.2R.sup.b); alkylaminocarbonylamino
(--NR.sup.aCONR.sup.bR.sup.c); mercapto;
(C.sub.1-C.sub.6)alkylthio; (C.sub.1-C.sub.6)alkylsulfonyl;
(C.sub.1-C.sub.6)alkylsulfonyl(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkylsulfoxido (--S(O)R.sup.a);
(C.sub.1-C.sub.6)alkylsulfoxido(C.sub.1-C.sub.6)alkyl
(--CH.sub.2).sub.nS(O)R.sup.a); sulfamido
(--SO.sub.2NR.sup.aR.sup.b); --SO.sub.3H;
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b;
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b; or unsubstituted or
substituted phenyl wherein the substituents are independently 1 to
3 halo, nitro, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.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.sub.2O--), (--OCH(CH.sub.3)O--),
(--OCH.sub.2CH.sub.2O--), (--OCH(CH.sub.3)CH.sub.2O--),
(--S--CH.dbd.N--), (--CH.sub.2OCH.sub.2O--),
(--O(CH.sub.2).sub.3--), (.dbd.N--O--N.dbd.), (--CH.dbd.CH--NH--),
(--OCF.sub.2O--), (--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.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.sub.1-C.sub.6)alkyl; (C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)thioalkoxy; carboxy;
(C.sub.1-C.sub.6)alkoxycarbonyl; (C.sub.1-C.sub.6)carbocyalkyl;
(C.sub.1-C.sub.6)alkoxycarbonylalkyl having independently the
stated number of carbon atoms in each alkyl group;
--CONR.sup.aR.sup.b; amino; (C.sub.1-C.sub.6)alkylamino;
(C.sub.1-C.sub.6)dialkylamino having independently the stated
number of carbon atoms in each alkyl group; haloalkyl
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b; or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.6)alkyl-1H-indole-2-yl; E is unsubstituted or
substituted (C.sub.4-C.sub.10) branched alkyl wherein the
substituents are independently 1-4 cyano; halo;
(C.sub.5-C.sub.6)cycloalkyl; phenyl; (C.sub.2-C.sub.3)alkenyl;
hydroxy, (C.sub.1-C.sub.6)alkoxy; carboxy;
(C.sub.1-C.sub.6)alkoxycarbonyl; formyl;
(C.sub.1-C.sub.6)trialkylsilyloxy having independently the stated
number of carbon atoms in each alkyl group; --CH.dbd.N--OR.sup.a;
--CH.dbd.N--R.sup.d; --CH.dbd.N--NHC(O)NR.sup.aR.sup.b; or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b; wherein R.sup.a, R.sup.b,
and R.sup.c are independently H, (C.sub.1-C.sub.6)alkyl, or phenyl;
R.sup.d is hydroxy(C.sub.1-C.sub.6)alkyl; and n=1-4; and G is H or
CN; provided that: 1) when E is unsubstituted or substituted
(C.sub.4-C.sub.10) branched alkyl wherein the substituents are
independently 1-4 cyano; halo; (C.sub.7-C.sub.3)alkenyl; carboxy;
or (C.sub.1-C.sub.6)alkoxycarbonyl; then B is: (a) substituted
phenyl which bears at least one --CH.dbd.N--NHC(O)NR.sup.aR.sup.b
or --CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.6)alkyl-1H-indole-2-yl; 2) when E is a substituted
(C.sub.4-C.sub.10) branched alkyl which bears at least one of
phenyl; hydroxy; (C.sub.1-C.sub.6)alkoxy; or formyl; then B is: (a)
substituted phenyl which bears at least one
--CH.dbd.N--NHC(O)NR.sup.aR.sup.b or
--CH.dbd.N--NHC(O)C(O)NR.sup.aR.sup.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.sub.1-C.sub.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.sup.aR.sup.b to
produce a product II, wherein M is Li, Na, or K, and R.sup.a and
R.sup.b are independently (C.sub.1-C.sub.6)alkyl or phenyl; and
##STR00113## 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; ##STR00114## wherein A and B are independently (a)
unsubstituted or substituted phenyl wherein the substituents are
independently 1 to 5H; halo; nitro; cyano; amino
(--NR.sup.aR.sup.b); alkylaminoalkyl
(--(CH.sub.2).sub.nNR.sup.aR.sup.b); (C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)haloalkyl; (C.sub.1-C.sub.6)cyanoalkyl;
(C.sub.1-C.sub.6)alkoxy; phenoxy; (C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy; (C2-C.sub.6)alkenyl
optionally substituted with halo, cyano, (C.sub.1-C.sub.4)alkyl, or
(C.sub.1-C.sub.4)alkoxy; (C.sub.2-C.sub.6)alkynyl optionally
substituted with halo or (C.sub.1-C.sub.4)alkyl; formyl;
(C.sub.1-C.sub.6)haloalkylcarbonyl; benzoyl;
(C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--N(CH.sub.2).sub.nCO.sub.2R.sup.b); alkylaminocarbonylamino
(--N(CH.sub.2).sub.nCONR.sup.bR.sup.c); (C.sub.1-C.sub.6)alkylthio;
sulfamido (--SO.sub.2NR.sup.aR.sup.b); or unsubstituted or
substituted phenyl wherein the substituents are independently 1 to
3 halo, nitro, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.6)alkyl, or
(--NR.sup.aR.sup.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.sub.2O--), (--OCH(CH.sub.3)O--),
(--OCH.sub.2CH.sub.2O--), (--OCH(CH.sub.3)CH.sub.2O--),
(--S--CH.dbd.N--), (--CH.sub.2OCH.sub.2O--),
(--O(CH.sub.2).sub.3--), (.dbd.N--O--N.dbd.), (--CH.dbd.CH--NH--),
(--OCF.sub.2O--), (--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.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.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkeyl; (C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)thioalkoxy; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)carbocyalkyl; --CONR.sup.aR.sup.b;
amino(--NR.sup.aR.sup.b); haloalkyl including CF.sub.3;
-trialkylsilyl (--SiR.sup.aR.sup.bR.sup.c); trityl(C(Ph).sub.3); or
unsubstituted or substituted phenyl wherein the substituents are
independently 1 to 3 halo, nitro, (C.sub.1-C.sub.6)alkoxy,
(C.sub.1-C.sub.6)alkyl, or (--NR.sup.aR.sup.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.sub.1-C.sub.10) straight or branched alkyl wherein the
substituents are independently 1-4 cyano; halo;
(C.sub.5-C.sub.6)cycloalkyl; phenyl; (C.sub.2-C.sub.3)alkenyl;
(C.sub.1-C.sub.6)alkoxy; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)alkanoyloxy (--OCOR.sup.a); formyl;
(C.sub.1-C.sub.6)trialkylsilyloxy having independently the stated
number of carbon atoms in each alkyl group; or
--CH.dbd.N--OR.sup.a; wherein R.sup.a, R.sup.b, and R.sup.e are
independently (C.sub.1-C.sub.6)alkyl or phenyl, and n=1-4.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of biotechnology or
genetic engineering. Specifically, this invention relates to the
field of gene expression. More specifically, this invention relates
to non-steroidal ligands for natural and mutated nuclear receptors
and their use in a nuclear receptor-based inducible gene expression
system and methods of modulating the expression of a gene within a
host cell using these ligands and inducible gene expression
system.
BACKGROUND OF THE INVENTION
[0002] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties. However,
the citation of any reference herein should not be construed as an
admission that such reference is available as "Prior Art" to the
instant application.
[0003] In the field of genetic engineering, precise control of gene
expression is a valuable tool for studying, manipulating, and
controlling development and other physiological processes. Gene
expression is a complex biological process involving a number of
specific protein-protein interactions. In order for gene expression
to be triggered, such that it produces the RNA necessary as the
first step in protein synthesis, a transcriptional activator must
be brought into proximity of a promoter that controls gene
transcription. Typically, the transcriptional activator itself is
associated with a protein that has at least one DNA binding domain
that binds to DNA binding sites present in the promoter regions of
genes. Thus, for gene expression to occur, a protein comprising a
DNA binding domain and a transactivation domain located at an
appropriate distance from the DNA binding domain must be brought
into the correct position in the promoter region of the gene.
[0004] The traditional transgenic approach utilizes a cell-type
specific promoter to drive the expression of a designed transgene.
A DNA construct containing the transgene is first incorporated into
a host genome. When triggered by a transcriptional activator,
expression of the transgene occurs in a given cell type.
[0005] Another means to regulate expression of foreign genes in
cells is through inducible promoters. Examples of the use of such
inducible promoters include the PR1-a promoter, prokaryotic
repressor-operator systems, immunosuppressive-immunophilin systems,
and higher eukaryotic transcription activation systems such as
steroid hormone receptor systems and are described below.
[0006] The PR1-a promoter from tobacco is induced during the
systemic acquired resistance response following pathogen attack.
The use of PR1-a may be limited because it often responds to
endogenous materials and external factors such as pathogens, UV-B
radiation, and pollutants. Gene regulation systems based on
promoters induced by heat shock, interferon and heavy metals have
been described (Wurn et al., 1986, Proc. Natl. Acad. Sci. USA
83:5414-5418; Arnheiter et al., 1990 Cell 62:51-61; Filmus et al.,
1992 Nucleic Acids Research 20:27550-27560). However, these systems
have limitations due to their effect on expression of non-target
genes. These systems are also leaky.
[0007] Prokaryotic repressor-operator systems utilize bacterial
repressor proteins and the unique operator DNA sequences to which
they bind. Both the tetracycline ("Tet") and lactose ("Lac")
repressor-operator systems from the bacterium Escherichia coli have
been used in plants and animals to control gene expression. In the
Tet system, tetracycline binds to the TetR repressor protein,
resulting in a conformational change that releases the repressor
protein from the operator which as a result allows transcription to
occur. In the Lac system, a lac operon is activated in response to
the presence of lactose, or synthetic analogs such as
isopropyl-b-D-thiogalactoside. Unfortunately, the use of such
systems is restricted by unstable chemistry of the ligands, i.e.
tetracycline and lactose, their toxicity, their natural presence,
or the relatively high levels required for induction or repression.
For similar reasons, utility of such systems in animals is
limited.
[0008] Immunosuppressive molecules such as FK506, rapamycin and
cyclosporine A can bind to immunophilins FKBP12, cyclophilin, etc.
Using this information, a general strategy has been devised to
bring together any two proteins simply by placing FK506 on each of
the two proteins or by placing FK506 on one and cyclosporine A on
another one. A synthetic homodimer of FK506 (FK1012) or a compound
resulted from fusion of FK506-cyclosporine (FKCsA) can then be used
to induce dimerization of these molecules (Spencer et al., 1993,
Science 262:1019-24; Belshaw et al., 1996 Proc Natl Acad Sci USA
93:4604-7). Gal4 DNA binding domain fused to FKBP12 and VP16
activator domain fused to cyclophilin, and FKCsA compound were used
to show heterodimerization and activation of a reporter gene under
the control of a promoter containing Gal4 binding sites.
Unfortunately, this system includes immunosuppressants that can
have unwanted side effects and therefore, limits its use for
various mammalian gene switch applications.
[0009] Higher eukaryotic transcription activation systems such as
steroid hormone receptor systems have also been employed. Steroid
hormone receptors are members of the nuclear receptor superfamily
and are found in vertebrate and invertebrate cells. Unfortunately,
use of steroidal compounds that activate the receptors for the
regulation of gene expression, particularly in plants and mammals,
is limited due to their involvement in many other natural
biological pathways in such organisms. In order to overcome such
difficulties, an alternative system has been developed using insect
ecdysone receptors (EcR).
[0010] Growth, molting, and development in insects are regulated by
the ecdysone steroid hormone (molting hormone) and the juvenile
hormones (Dhadialla, et al., 1998. Annu. Rev. Entomol. 43:
545-569). The molecular target for ecdysone in insects consists of
at least ecdysone receptor (EcR) and ultraspiracle protein (USP).
EcR is a member of the nuclear steroid receptor super family that
is characterized by signature DNA and ligand binding domains, and
an activation domain (Koelle et al. 1991, Cell, 67:59-77). EcR
receptors are responsive to a number of steroidal compounds such as
ponasterone A and muristerone A. Recently, non-steroidal compounds
with ecdysteroid agonist activity have been described, including
the commercially available insecticides tebufenozide and
methoxyfenozide that are marketed world wide by Rohm and Haas
Company (see International Patent Application No. PCT/EP96/00686
and U.S. Pat. No. 5,530,028). Both analogs have exceptional safety
profiles to other organisms.
[0011] The insect ecdysone receptor (EcR) heterodimerizes with
Ultraspiracle (USP), the insect homologue of the mammalian RXR, and
binds ecdysteroids and ecdysone receptor response elements and
activate transcription of ecdysone responsive genes. The
EcR/USP/ligand complexes play important roles during insect
development and reproduction. The EcR is a member of the steroid
hormone receptor superfamily and has five modular domains, A/B
(transactivation), C (DNA binding, heterodimerization)), D (Hinge,
heterodimerization), E (ligand binding, heterodimerization and
transactivation and F (transactivation) domains. Some of these
domains such as A/B, C and E retain their function when they are
fused to other proteins.
[0012] Tightly regulated inducible gene expression systems or "gene
switches" are useful for various applications such as gene therapy,
large scale production of proteins in cells, cell based high
throughput screening assays, functional genomics and regulation of
traits in transgenic plants and animals.
[0013] The first version of EcR-based gene switch used Drosophila
melanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) and showed
that these receptors in the presence of steroid, ponasteroneA,
transactivate reporter genes in mammalian cell lines and transgenic
mice (Christopherson K. S., Mark M. R., Baja J. V., Godowski P. J.
1992, Proc. Natl. Acad. Sci. U.S.A. 89: 6314-6318; No D., Yao T.
P., Evans R. M., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:
3346-3351). Later, Suhr et al. 1998, Proc. Natl. Acad. Sci.
95:7999-8004 showed that non-steroidal ecdysone agonist,
tebufenozide, induced high level of transactivation of reporter
genes in mammalian cells through Bombyx mori EcR (BmEcR) in the
absence of exogenous heterodimer partner.
[0014] International Patent Applications No. PCT/US97/05330 (WO
97/38117) and PCT/US99/08381 (WO99/58155) disclose methods for
modulating the expression of an exogenous gene in which a DNA
construct comprising the exogenous gene and an ecdysone response
element is activated by a second DNA construct comprising an
ecdysone receptor that, in the presence of a ligand therefor, and
optionally in the presence of a receptor capable of acting as a
silent partner, binds to the ecdysone response element to induce
gene expression. The ecdysone receptor of choice was isolated from
Drosophila melanogaster. Typically, such systems require the
presence of the silent partner, preferably retinoid X receptor
(RXR), in order to provide optimum activation. In mammalian cells,
insect ecdysone receptor (EcR) heterodimerizes with retinoid X
receptor (RXR) and regulates expression of target genes in a ligand
dependent manner International Patent Application No.
PCT/US98/14215 (WO 99/02683) discloses that the ecdysone receptor
isolated from the silk moth Bombyx mori is functional in mammalian
systems without the need for an exogenous dimer partner.
[0015] U.S. Pat. No. 6,265,173 B1 discloses that various members of
the steroid/thyroid superfamily of receptors can combine with
Drosophila melanogaster ultraspiracle receptor (USP) or fragments
thereof comprising at least the dimerization domain of USP for use
in a gene expression system. U.S. Pat. No. 5,880,333 discloses a
Drosophila melanogaster EcR and ultraspiracle (USP) heterodimer
system used in plants in which the transactivation domain and the
DNA binding domain are positioned on two different hybrid proteins.
Unfortunately, these USP-based systems are constitutive in animal
cells and therefore, are not effective for regulating reporter gene
expression.
[0016] In each of these cases, the transactivation domain and the
DNA binding domain (either as native EcR as in International Patent
Application No. PCT/US98/14215 or as modified EcR as in
International Patent Application No. PCT/US97/05330) were
incorporated into a single molecule and the other heterodimeric
partners, either USP or RXR, were used in their native state.
[0017] Drawbacks of the above described EcR-based gene regulation
systems include a considerable background activity in the absence
of ligands and non-applicability of these systems for use in both
plants and animals (see U.S. Pat. No. 5,880,333). Therefore, a need
exists in the art for improved EcR-based systems to precisely
modulate the expression of exogenous genes in both plants and
animals. Such improved systems would be useful for applications
such as gene therapy, large-scale production of proteins and
antibodies, cell-based high throughput screening assays, functional
genomics and regulation of traits in transgenic animals. For
certain applications such as gene therapy, it may be desirable to
have an inducible gene expression system that responds well to
synthetic non-steroid ligands and at the same is insensitive to the
natural steroids. Thus, improved systems that are simple, compact,
and dependent on ligands that are relatively inexpensive, readily
available, and of low toxicity to the host would prove useful for
regulating biological systems.
[0018] Recently, it has been shown that an ecdysone receptor-based
inducible gene expression system in which the transactivation and
DNA binding domains are separated from each other by placing them
on two different proteins results in greatly reduced background
activity in the absence of a ligand and significantly increased
activity over background in the presence of a ligand (pending
application PCT/US01/09050, incorporated herein in its entirety by
reference). This two-hybrid system is a significantly improved
inducible gene expression modulation system compared to the two
systems disclosed in applications PCT/US97/05330 and
PCT/US98/14215. The two-hybrid system exploits the ability of a
pair of interacting proteins to bring the transcription activation
domain into a more favorable position relative to the DNA binding
domain such that when the DNA binding domain binds to the DNA
binding site on the gene, the transactivation domain more
effectively activates the promoter (see, for example, U.S. Pat. No.
5,283,173). Briefly, the two-hybrid gene expression system
comprises two gene expression cassettes; the first encoding a DNA
binding domain fused to a nuclear receptor polypeptide, and the
second encoding a transactivation domain fused to a different
nuclear receptor polypeptide. In the presence of ligand, the
interaction of the first polypeptide with the second polypeptide
effectively tethers the DNA binding domain to the transactivation
domain. Since the DNA binding and transactivation domains reside on
two different molecules, the background activity in the absence of
ligand is greatly reduced.
[0019] A two-hybrid system also provides improved sensitivity to
non-steroidal ligands for example, diacylhydrazines, when compared
to steroidal ligands for example, ponasterone A ("PonA") or
muristerone A ("MurA"). That is, when compared to steroids, the
non-steroidal ligands provide higher activity at a lower
concentration. In addition, since transactivation based on EcR gene
switches is often cell-line dependent, it is easier to tailor
switching systems to obtain maximum transactivation capability for
each application. Furthermore, the two-hybrid system avoids some
side effects due to overexpression of RXR that often occur when
unmodified RXR is used as a switching partner. In a preferred
two-hybrid system, native DNA binding and transactivation domains
of EcR or RXR are eliminated and as a result, these hybrid
molecules have less chance of interacting with other steroid
hormone receptors present in the cell resulting in reduced side
effects.
[0020] With the improvement in ecdysone receptor-based gene
regulation systems there is an increase in their use in various
applications resulting in increased demand for ligands with higher
activity than those currently exist. U.S. Pat. No. 6,258,603 B1
(and patents cited therein) disclosed dibenzoylhydrazine ligands,
however, a need exists for additional ligands with different
structures and physicochemical properties. We have discovered novel
diacylhydrazine ligands which have not previously been described or
shown to have the ability to modulate the expression of
transgenes.
SUMMARY OF THE INVENTION
[0021] The present invention relates to non-steroidal ligands for
use in nuclear receptor-based inducible gene expression system, and
methods of modulating the expression of a gene within a host cell
using these ligands with nuclear receptor-based inducible gene
expression systems.
[0022] Applicants' invention also relates to methods of modulating
gene expression in a host cell using a gene expression modulation
system with a ligand of the present invention. Specifically,
Applicants' invention provides 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
according to the invention; b) introducing into the host cell a
gene expression cassette comprising i) a response element
comprising a domain to which the DNA binding domain from the first
hybrid polypeptide of the gene expression modulation system binds;
ii) a promoter that is activated by the transactivation domain of
the second hybrid polypeptide of the gene expression modulation
system; and iii) a gene whose expression is to be modulated; and c)
introducing into the host cell a ligand; whereby upon introduction
of the ligand into the host cell, expression of the gene is
modulated. Applicants' invention also provides a method of
modulating the expression of a gene in a host cell comprising a
gene expression cassette comprising a response element comprising a
domain to which the DNA binding domain from the first hybrid
polypeptide of the gene expression modulation system binds; a
promoter that is activated by the transactivation domain of the
second hybrid polypeptide of the gene expression modulation system;
and a gene whose expression is to be modulated; wherein the method
comprises the steps of: a) introducing into the host cell a gene
expression modulation system according to the invention; and b)
introducing into the host cell a ligand; whereby upon introduction
of the ligand into the host, expression of the gene is
modulated.
[0023] FIG. 1. Schematic of switch construct CVBE, and the reporter
construct 6.times.EcRE Lac Z. Flanking both constructs are long
terminal repeats, G418 and puromycin are selectable markers, CMV is
the cytomegalovirus promoter, VBE is coding sequence for amino
acids 26-546 from Bombyx mori EcR inserted downstream of the VP16
transactivation domain, 6.times.EcRE is six copies of the ecdysone
response element, lacZ encodes for the reporter enzyme
.beta.-galactosidase.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Applicants' invention provides ligands for use with ecdysone
receptor-based inducible gene expression system useful for
modulating expression of a gene of interest in a host cell. In a
particularly desirable embodiment, Applicants' invention provides
an inducible gene expression system that has a reduced level of
background gene expression and responds to submicromolar
concentrations of non-steroidal ligand. Thus, Applicants' ligands
and inducible gene expression system and its use in methods of
modulating gene expression in a host cell overcome the limitations
of currently available inducible expression systems and provide the
skilled artisan with an effective means to control gene
expression.
[0025] The present invention is useful for applications such as
gene therapy, large scale production of proteins and antibodies,
cell-based high throughput screening assays, functional genomics,
proteomics, metabolomics, and regulation of traits in transgenic
organisms, where control of gene expression levels is desirable. An
advantage of Applicants' invention is that it provides a means to
regulate gene expression and to tailor expression levels to suit
the user's requirements.
[0026] The present invention pertains to compounds of the general
formula:
##STR00001##
[0027] wherein X and X' are independently O or S;
[0028] A is unsubstituted or substituted phenyl wherein the
substituents are independently 1 to 5H; halo; nitro; cyano;
hydroxy; amino (--NR.sup.aR.sup.b); alkylaminolkyl
(--(CH.sub.2).sub.nNR.sup.aR.sup.b); (C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)haloalkyl; (C.sub.1-C.sub.6)cyanoalkyl;
(C.sub.1-C.sub.6)hydroxyalkyl; (C.sub.1-C.sub.6)alkoxy; phenoxy;
(C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)alkanoyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; carboxy;
(C.sub.1-C.sub.6)alkylcarbonyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--NR.sup.aCO.sub.2R.sup.b); alkylaminocarbonylamino
(--NR.sup.aCONR.sup.bR.sup.c); mercapto;
(C.sub.1-C.sub.6)alkylthio; (C.sub.1-C.sub.6)alkylsulfonyl;
(C.sub.1-C.sub.6)alkylsulfonyl(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkylsulfoxido (--S(O)R.sup.a);
(C.sub.1-C.sub.6)alkylsulfoxido(C.sub.1-C.sub.6)alkyl
--(CH.sub.2)S(O)R.sup.a); sulfamido (--SO.sub.2NR.sup.aR.sup.b);
--SO.sub.3H; or unsubstituted or substituted phenyl wherein the
substituents are independently 1 to 3 halo, nitro,
(C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.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.sub.2O--), (--OCH(CH.sub.3)O--), (--OCH.sub.2CH.sub.2O--),
(--OCH(CH.sub.3)CH.sub.2O--), (--S--CH.dbd.N--),
(--CH.sub.2OCH.sub.2O--), (--O(CH.sub.2).sub.3--),
(.dbd.N--O--N.dbd.), (--C.dbd.CH--NH--), (--OCF.sub.2O--),
(--N--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.4);
[0029] B is [0030] (a) unsubstituted or substituted phenyl wherein
the substituents are independently 1 to 5H; halo; nitro; cyano;
hydroxy; amino (--NR.sup.aR.sup.b); alkylaminolkyl
(--(CH.sub.2).sub.nNR.sup.aR.sup.b); (C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)haloalkyl; (C.sub.1-C.sub.6)cyanoalkyl;
(C.sub.1-C.sub.6)hydroxyalkyl; (C.sub.1-C.sub.6)alkoxy; phenoxy;
(C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)alkanoyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; carboxy;
(C.sub.1-C.sub.6)alkylcarbonyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--NR.sup.aCO.sub.2R.sup.b); alkylaminocarbonylamino
(--NR.sup.aCONR.sup.bR.sup.e); mercapto;
(C.sub.1-C.sub.6)alkylthio; (C.sub.1-C.sub.6)alkylsulfonyl;
(C.sub.1-C.sub.6)alkylsulfonyl(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkylsulfoxido (--S(O)R.sup.a);
(C.sub.1-C.sub.6)alkylsulfoxido(C.sub.1-C.sub.6)alkyl
(--CH.sub.2).sub.nS(O)R.sup.a); sulfamido
(--SO.sub.2NR.sup.aR.sup.b); --SO.sub.3H; or unsubstituted or
substituted phenyl wherein the substituents are independently 1 to
3 halo, nitro, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.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.sub.2O--), (--OCH(CH.sub.3)O--),
(--OCH.sub.2CH.sub.2O--), (--OCH(CH.sub.3)CH.sub.2O--),
(--S--CH.dbd.N--), (--CH.sub.2OCH.sub.2O--),
(--O(CH.sub.2).sub.3--), (.dbd.N--O--N.dbd.), (--C.dbd.CH--NH--),
(--OCF.sub.2O--), (--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.4--); [0031] (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.sub.1-C.sub.6)alkyl; (C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)thioalkoxy; carboxy;
(C.sub.1-C.sub.6)alkoxycarbonyl; (C.sub.1-C.sub.6)carboxyalkyl;
(C.sub.1-C.sub.6)alkoxycarbonylalkyl having independently the
stated number of carbon atoms in each alkyl group;
--CONR.sup.aR.sup.b; amino; (C.sub.1-C.sub.6)alkylamino;
(C.sub.1-C.sub.6)dialkylamino having independently the stated
number of carbon atoms in each alkyl group; haloalkyl including
CF.sub.3; --C.dbd.N--NHC(O)NR.sup.aR.sup.b; or
--C.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b; or [0032] (c)
5-benzimidazolyl; 1-trityl-5-benzimidazolyl;
3-trityl-5-benzimidazolyl; 1H-indazole-3-yl;
1-trityl-1H-indazole-3-yl; or
1-(C.sub.1-C.sub.6)alkyl-1H-indole-2-yl;
[0033] E is unsubstituted or substituted (C.sub.4-C.sub.10)
branched alkyl wherein the substituents are independently 1-4
cyano; halo; (C.sub.5-C.sub.6)cycloalkyl; phenyl;
(C.sub.2-C.sub.3)alkenyl; hydroxy, (C.sub.1-C.sub.6)alkoxy;
carboxy; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)alkanoyloxy (--OCOR.sup.a); formyl;
(C.sub.1-C.sub.6)trialkylsilyloxy having independently the stated
number of carbon atoms in each alkyl group; --C.dbd.N--OR.sup.a;
--C.dbd.N--R.sup.d; --C.dbd.N--NHC(O)NR.sup.aR.sup.b; or
--C.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b;
[0034] wherein R.sup.a, R.sup.b, and R.sup.e are independently H,
(C.sub.1-C.sub.6)alkyl, or phenyl; R.sup.d is
hydroxy(C.sub.1-C.sub.6)alkyl; and n=1-4; and
[0035] G is H or CN;
[0036] provided that: [0037] 1) when E is unsubstituted or
substituted (C.sub.4-C.sub.10) branched alkyl wherein the
substituents are independently 1-4 cyano; halo;
(C.sub.2-C.sub.3)alkenyl; carboxy; or
(C.sub.1-C.sub.6)alkoxycarbonyl; [0038] then B is [0039] (a)
substituted phenyl which bears at least one
--C.dbd.N--NHC(O)NR.sup.aR.sup.b or
--C.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b group; [0040] (b) substituted
6-membered heterocycle having 1-3 nitrogen atoms and 3-5 nuclear
carbon atoms which bears at least one haloalkyl group; or [0041]
(c) 5-benzimidazolyl; 1-trityl-5-benzimidazolyl;
3-trityl-5-benzimidazolyl; 1H-indazole-3-yl;
1-trityl-1H-indazole-3-yl; or
1-(C.sub.1-C.sub.6)alkyl-1H-indole-2-yl; [0042] wherein R.sup.a,
R.sup.b are independently H, (C.sub.1-C.sub.6)alkyl, or phenyl; or
[0043] 2) when E is a substituted (C.sub.4-C.sub.10) branched alkyl
which bears at least one of phenyl; hydroxy,
(C.sub.1-C.sub.6)alkoxy; or formyl; [0044] then B is [0045] (a)
substituted phenyl which bears at least one
--C.dbd.N--NHC(O)NR.sup.aR.sup.b or
--C.dbd.N--NHC(O)C(O)NR.sup.aR.sup.b group; [0046] (b) substituted
or unsubstituted 6-membered heterocycle having 1-3 nitrogen atoms
and 3-5 nuclear carbon atoms; or [0047] (c) 5-benzimidazolyl;
1-trityl-5-benzimidazolyl; 3-trityl-5-benzimidazolyl;
1H-indazole-3-yl; 1-trityl-1H-indazole-3-yl; or
1-(C.sub.1-C.sub.6)alkyl-1H-indole-2-yl;
[0048] wherein R.sup.a and R.sup.b are independently H,
(C.sub.1-C.sub.6)alkyl, or phenyl.
[0049] Compounds of the general formula are preferred when X and X'
are O and G is H.
[0050] Compounds of the present invention most preferred are the
following:
TABLE-US-00001 ##STR00002## Compound A B E RG-100864 4-Cl--Ph Ph
t-Bu RG-101013 4-Et--Ph 2-NO.sub.2--Ph t-Bu RG-101542
4-CH.sub.3--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-102125 4-Et--Ph
3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-100801 2,6-di-F--Ph 3-Cl,
5-Cl--Ph t-Bu RG-101202 2-CH.sub.3, 3-Cl--Ph 3-Cl--Ph t-Bu
RG-101248 2-Cl, 3-OMe--Ph 2-Cl-5-CH.sub.3--Ph t-Bu RG-101664
2-CH.sub.3, 3-Cl--Ph 3-CH.sub.3-4-Br--Ph t-Bu RG-101862 4-Et--Ph
3,5-di-CH.sub.3-4-Cl--Ph t-Bu RG-101863 4-Et--Ph
3,4-di-CH.sub.3-5-Cl--Ph t-Bu RG-101057 4-OCH.sub.3--Ph
2-Cl-4-F--Ph t-Bu RG-101774 4-Et--Ph 3-CH.sub.3, 5-Cl--Ph t-Bu
RG-102592 4-Et--Ph 2-Et--Ph t-Bu RG-101376 4-OCH.sub.3--Ph 3-Cl,
5-Cl--Ph t-Bu RG-101398 4-Et--Ph 2-NO.sub.2-5-CH.sub.3--Ph t-Bu
RG-100875 4-CH.sub.2CH--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
RG-100694 2-CH.sub.3, 3-OMe--Ph 3-CH.sub.3--Ph t-Bu RG-101759
4-Br--Ph 3-Cl, 5-Cl--Ph t-Bu RG-100915 2-CH.sub.3, 3-NO.sub.2--Ph
3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-100763 2-CH.sub.3,
3-CH.sub.3--Ph 2,5-di-OCH.sub.3--Ph t-Bu RG-101178 2-CH.sub.3,
3-CH.sub.3--Ph 2-OCH.sub.3-5-Cl--Ph t-Bu RG-100568 2-NO.sub.2,
3-OMe--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-100764 2-CH.sub.3,
3-CH.sub.3--Ph 3-OMe, 5-OMe--Ph t-Bu RG-101864 3-Cl, 4-Et--Ph
3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-100342 4-CH(OH)CH.sub.3--Ph 3-F,
5-F--Ph t-Bu RG-101316 2-CH.sub.3, 3-NMe.sub.2--Ph 3-Cl, 5-Cl--Ph
t-Bu RG-100814 2-CH.sub.3, 3-Ac--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
RG-100749 2-CH.sub.3, 3-OAc--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
RG-101734 2-CH.sub.3, 3-I--Ph 3-CH3, 5-CH.sub.3--Ph t-Bu RG-101408
2-CH.sub.3, 3-OMe--Ph 3-Cl, 5-Br--Ph t-Bu RG-101670 2-CH.sub.3,
3-Oi-Pr--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-100127 2-CH.sub.3,
3-OCH3--Ph 2-Cl-3-pyridyl t-Bu RG-100766 2-CH.sub.3, 3-OMe--Ph
2-OCH.sub.3-5-CH.sub.3--Ph t-Bu RG-100603 2-CH.sub.3, 3-OMe--Ph
2,5-F--Ph t-Bu RG-101062 2-CH.sub.3, 3-OMe--Ph 2-Et--Ph t-Bu
RG-101353 2-CH.sub.3, 3-OMe--Ph 3-CH.sub.3, 5-Br--Ph t-Bu RG-100767
2-CH.sub.3, 3-OMe--Ph 3-OMe, 5-CH.sub.3--Ph t-Bu RG-100848
2-CH.sub.3, 3-OMe--Ph 2-OCH.sub.3-4-Cl--Ph t-Bu RG-101692
2-CH.sub.3, 3-OCF.sub.3--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
RG-100768 2-CH.sub.3, 3-OMe--Ph 3-OCH.sub.3-4-CH.sub.3--Ph t-Bu
RG-101585 3-OCH.sub.3, 4-CH3--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
RG-100769 2-CH.sub.3, 3-OMe--Ph 2-OCH.sub.3-4-CH.sub.3--Ph t-Bu
RG-100394 2-CH.sub.3, 3-OCH.sub.3--Ph 2,6-di-Cl-4-pyridyl t-Bu
RG-100569 2-CH.sub.3, 3-OMe--Ph 2-NO.sub.2-5-CH.sub.3--Ph t-Bu
RG-100929 2-CH.sub.3, 3-OMe--Ph 2-F-4-Cl--Ph t-Bu RG-101048
3,4-OCH.sub.2O--Ph 2-Cl-4-F--Ph t-Bu RG-102240 2-Et, 3-OMe--Ph
3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-101691 2-CH.sub.3, 3-Et--Ph
3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-101531 3-CH.sub.2CH.sub.2O-4-Ph
3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-101382 2-CH.sub.3, 3-OMe--Ph
3,5-di-Cl-4-F--Ph t-Bu RG-100448 2-CH.sub.3, 3,4-OCH.sub.2O--Ph
4-F--Ph t-Bu RG-100698 2-Et, 3,4-OCH.sub.2O--Ph 2-OCH.sub.3--Ph
t-Bu RG-101889 3,4-di-Et--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
RG-100812 2-Et, 3-OMe--Ph 4-F--Ph t-Bu RG-100725 2-Et, 3-OMe--Ph
2-OCH.sub.3--Ph t-Bu RG-100524 2-CH.sub.3, 3-OMe--Ph
2-OCH.sub.3-4-F--Ph t-Bu RG-100667 2-Et, 3-OCH.sub.3--Ph
2-Cl-6-CH.sub.3-4-pyridyl t-Bu RG-100778 2-Et, 3-OMe--Ph 3-OMe,
5-OMe--Ph t-Bu RG-101528 2-I, 3-OMe--Ph 3-CH.sub.3, 5-CH.sub.3--Ph
t-Bu RG-100492 3,4-ethylenedioxy-Ph 2-OCH.sub.3--Ph t-Bu RG-101887
3,4-(CH.sub.2).sub.4--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-115496
2-Et, 3-OMe--Ph 2,3-OCH.sub.2O--Ph t-Bu RG-100901 2-F, 4-Et--Ph
4-F--Ph t-Bu RG-100699 2-Et, 3-OMe--Ph 3,4-methylenedioxy-Ph t-Bu
RG-100425 2-CH.sub.3, 3,4-ethylenedioxy- 4-F--Ph t-Bu Ph RG-101511
3,4-OCH(CH.sub.3)O--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-101659
2-Et, 3,4-OCH(CH.sub.3)O--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
RG-100360 2-CH.sub.3, 3,4-ethylenedioxy- 3-OCH.sub.3--Ph t-Bu Ph
RG-101509 3-OCH(CH.sub.3)CH.sub.2O-4-Ph 3-CH.sub.3, 5-CH.sub.3--Ph
t-Bu RG-101340 2-Br, 3,4-ethylenedioxy-Ph 3-CH.sub.3,
5-CH.sub.3--Ph t-Bu RG-101494 2-Et, 3,4-ethylenedioxy-Ph
3-CH.sub.3, 5-Cl--Ph t-Bu RG-101036 2-Et, 3,4-ethylenedioxy-Ph
3-CH.sub.3--Ph t-Bu RG-100690 2-Et, 3,4-ethylenedioxy-Ph
2-OCH.sub.3--Ph t-Bu RG-100691 2-Et, 3,4-ethylenedioxy-Ph
3-OCH.sub.3--Ph t-Bu RG-101312 3-S--C.dbd.N-4-Ph 3-CH.sub.3,
5-CH.sub.3--Ph t-Bu RG-101218 2-Et, 3-OMe--Ph 2-OCH.sub.3-4-Cl--Ph
t-Bu RG-100779 2-Et, 3-OMe--Ph 2,5-di-OCH.sub.3--Ph t-Bu RG-101088
2-CH.sub.3, 4,5- 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu methylenedioxy-Ph
RG-101016 3-CH.sub.2OCH.sub.2O-4-Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
RG-100216 2-CH.sub.3, 3-OCH.sub.2OCH.sub.2-4-Ph 2-OCH.sub.3--Ph
t-Bu RG-100574 2-Et, 3-OCH.sub.2OCH.sub.2-4-Ph 4-F--Ph t-Bu
RG-101171 2-Cl 4,5-methylenedioxy- 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
Ph RG-100620 2,3,6-tri-F--Ph 2-Cl-4-F--Ph t-Bu RG-115033 2-Et,
3-OMe--Ph 2,6-F--Ph t-Bu RG-115515 2-Et, 3-OMe--Ph 3-F--Ph t-Bu
RG-115038 2-Et, 3-OMe--Ph 3-Br--Ph t-Bu RG-115330 2-Et, 3-OMe--Ph
2-NO.sub.2--Ph t-Bu RG-115627 2-Et, 3-OMe--Ph 2,3-F--Ph t-Bu
RG-115329 2-Et, 3-OMe--Ph 3,4,5-tri-OCH.sub.3--Ph t-Bu RG-115088
2-Et, 3-OMe--Ph 3-CF.sub.3, 5-F--Ph t-Bu RG-115327 2-Et, 3-OMe--Ph
3-CN--Ph t-Bu RG-115534 2-Vinyl, 3-OMe--Ph 2,4-di-Cl-5-F--Ph t-Bu
RG-115046 2-Et, 3-OCH.sub.2OCH.sub.2-4-Ph Ph t-Bu RG-115025 2-Et,
3-OMe--Ph 3-CH.sub.3, 5-CH.sub.3--Ph --C(CH.sub.3).sub.2C(O)OEt
RG-115143 2-Et, 3-OMe--Ph 3-CH.sub.3, 5-CH.sub.3--Ph
--C(CH.sub.3).sub.2CH.sub.2OH RG-115407 2-Et, 3-OMe--Ph 3-CH.sub.3,
5-CH.sub.3--Ph --C(CH.sub.3).sub.2CHO RG-115006 2-Et, 3-OMe--Ph
3-CH.sub.3, 5-CH.sub.3--Ph --C(CH.sub.3).sub.2CH.sub.2OCH.sub.3
RG-115258 2-Et, 3-OMe--Ph 3-CH.sub.3, 5-CH.sub.3--Ph
--C(CH.sub.3).sub.2CH.dbd.NOH RG-115378 2-NH.sub.2, 3-OMe--Ph
3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-115223 2-Et, 3-OMe--Ph
3-CH.sub.2OAc, 5-CH.sub.3--Ph t-Bu RG-115310 2-Et, 3-OMe--Ph
3-CH.sub.3, 5-CH.sub.3--Ph --C(CH.sub.3).sub.2CH.sub.2OC(O)CH.sub.3
RG-115567 2-CH.sub.3, 3-OH--Ph 2,3,4-F--Ph t-Bu RG-115443
2-CH.sub.3, 3-OH--Ph 3-Cl-5-OCH.sub.3-4-pyridyl t-Bu RG-115261
2-CH.sub.3, 3-OH--Ph 2,6-di-Cl-4-pyridyl t-Bu RG-115595 2-CH.sub.3,
3-OH--Ph 3-OCH.sub.3-4-pyridyl t-Bu RG-115220 2-CH.sub.3, 3-OH--Ph
3,5-di-OCH.sub.3-4-CH.sub.3--Ph t-B RG-115102 2-CH.sub.3,
3-CH.sub.2CH.sub.2CH.sub.2O-4- 2-OCH.sub.3--Ph t-Bu Ph RG-115302
2-Et, 3-OMe--Ph 2,4-di-Cl-5-F--Ph t-Bu RG-115539 2-CH.sub.3,
3-CH.sub.2CH.sub.2CH.sub.2O-4- 2,4-di-Cl-5-F--Ph t-Bu Ph RG-115499
2-CH.sub.3, 3-CH.sub.2CH.sub.2CH.sub.2O-4- 2-F, 5-CH.sub.3--Ph t-Bu
Ph RG-115055 2-CH.sub.3, 3-CH.sub.2CH.sub.2CH.sub.2O-4-
3,5-di-OCH.sub.3-4-CH.sub.3--Ph t-Bu Ph RG-115508 2-Et,
3,4-ethylenedioxy-Ph 2,5-F--Ph t-Bu RG-115580 2-Et,
3,4-ethylenedioxy-Ph 2,3,4-F--Ph t-Bu RG-115337 2-Et,
3,4-ethylenedioxy-Ph 2,3,4,5--Ph t-Bu RG-115280 2-Et,
3,4-ethylenedioxy-Ph 3-CF.sub.3-4-F--Ph t-Bu RG-115297 2-Et,
3,4-ethylenedioxy-Ph 2,6-di-Cl-4-pyridyl t-Bu RG-115244 2-Et,
3,4-ethylenedioxy-Ph 2-OCH.sub.3--Ph t-Bu RG-115684 2-Et,
3,4-ethylenedioxy-Ph 2,4-di-Cl-5-F--Ph t-Bu RG-115514 2-Et,
3,4-ethylenedioxy-Ph 2-F, 4-Cl--Ph t-Bu RG-115557 2-CH.sub.3,
3-OAc--Ph 3,5-di-OCH.sub.3-4-CH.sub.3--Ph t-Bu RG-115253 2-Et,
3-OMe--Ph 2-OCH.sub.3-5-Cl--Ph t-Bu RG-115085 2-Et,
3,4-OCH.sub.2O--Ph 2-OCH.sub.3-4-Cl--Ph t-Bu RG-115551 2-CH.sub.3,
3-CH.sub.2CH.sub.2CH.sub.2O-4- 2-OCH.sub.3-5-Cl--Ph t-Bu Ph
RG-115162 2-Et, 3-OMe--Ph 2-NO.sub.2-5-CH.sub.3--Ph t-Bu RG-115647
2-Et, 3-OMe--Ph 2-NO.sub.2-4-Cl--Ph t-Bu RG-115257 2-Et, 3-OMe--Ph
2-NO.sub.2-5-Cl--Ph t-Bu RG-115664 2-CH.sub.3,
3-CH.sub.2CH.sub.2CH.sub.2O-4- 2-NO.sub.2-5-CH.sub.3--Ph t-Bu Ph
RG-115171 Benzo[1,2,5]oxadiazole-5- 2-OCH.sub.3-4-Cl--Ph t-Bu yl
RG-115480 2-Vinyl, 3-OMe--Ph 2-Cl, 5-NO.sub.2--Ph t-Bu RG-115095
2-Vinyl, 3-OMe--Ph 2-OCH.sub.3-4-Cl--Ph t-Bu RG-115106 2-Et,
3-OCH.sub.3--Ph 1-methyl-1H-indole-2-yl t-Bu RG-115130 2-Et,
3,4-ethylenedioxy-Ph 3,5-di-OCH.sub.3-4-CH.sub.3--Ph t-Bu RG-115532
2-Cl, 3-CH.sub.2OCH.sub.2O-4-Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
RG-115167 2-F, 4-Et--Ph 3-NO.sub.2--Ph t-Bu RG-115269 2-F, 4-Et--Ph
3-OCH.sub.3--Ph t-Bu RG-115441 2-Cl, 3-CH.sub.2OCH.sub.2O-4-Ph
3,5-di-OCH.sub.3-4-CH.sub.3--Ph t-Bu RG-115128 2-F, 4-Et--Ph
2,6-di-Cl-4-pyridyl t-Bu RG-115077 2-F, 4-Et--Ph
3,5-di-OCH.sub.3-4-CH.sub.3--Ph t-Bu RG-115259 2-F, 4-Et--Ph
3,4,5-F--Ph t-Bu RG-115674 2-F, 4-Et--Ph 3-CH.sub.3--Ph t-Bu
RG-115422 2-F, 4-Et--Ph 2-OCH.sub.3--Ph t-Bu RG-115086 2-F,
4-Et--Ph 2-NO.sub.2-5-F--Ph t-Bu RG-115592 2-F, 4-Et--Ph
2-OCH.sub.2CF.sub.3, 5-OCH.sub.3--Ph t-Bu RG-115112 2-F, 4-Et--Ph
2-Cl-6-CH.sub.3-4-pyridyl t-Bu RG-115050 2-F, 4-Et--Ph
2,6-di-OCH.sub.3-3-pyridyl t-Bu RG-115689 3-NH--C.dbd.C-4-Ph
3-CH.sub.3, 5-CH.sub.3--Ph t-Bu RG-115199 2-Et, 3-OMe--Ph
2-S(O)CH.sub.3--Ph t-Bu RG-115352 3,4-OCF.sub.2O--Ph 2-NO.sub.2--Ph
t-Bu RG-115256 3,4-OCF.sub.2O--Ph 3-CH.sub.3, 5-CH.sub.3--Ph t-Bu
RG-115683 3,4-OCF.sub.2O--Ph 3-OCH.sub.3--Ph t-Bu RG-115648 2-Et,
3-OMe--Ph 3-Br--Ph --C(CH.sub.3).sub.2CN RG-115306 2-CH.sub.2OMe,
3-OMe--Ph 3,5-di-Cl--Ph t-Bu RG-115625 2-Et, 3-OMe--Ph
3-CH.dbd.NOH, 5-CH.sub.3--Ph t-Bu RG-115429 2-Et, 3-OMe--Ph
3-CH.dbd.NNHCONH.sub.2, 5-CH.sub.3--Ph t-Bu RG-115613 2-Et,
3-OMe--Ph 3-CH.dbd.NNHCOCONH.sub.2, t-Bu 5-CH.sub.3--Ph RG-115043
2-Et, 3-OMe--Ph 3-CH.sub.3, 5-CH.sub.3--Ph --C(CH.sub.3).sub.2CN
RG-115690 2-Et, 3-OMe--Ph 3,5-di-OCH.sub.3-4-CH.sub.3--Ph
--C(CH.sub.3).sub.2CN RG-115065 2-Et, 3-OCH.sub.2OCH.sub.2-4-Ph
2-OCH.sub.3--Ph t-Bu RG-115229 2-CH.sub.3,
3-CH.sub.2CH.sub.2CH.sub.2O-4- 2,4,5F--Ph t-Bu Ph RG-115575
2-CH.sub.3, 3-CH.sub.2CH.sub.2CH.sub.2O-4- 3,4,5-F--Ph t-Bu Ph
RG-115278 2-CH.sub.3, 3-CH.sub.2CH.sub.2CH.sub.2O-4- 3-F--Ph t-Bu
Ph RG-115260 2-Et, 3,4-OCH.sub.2O--Ph 3-CF.sub.3--Ph t-Bu RG-115118
2-CH.sub.3, 3-CH.sub.2CH.sub.2CH.sub.2O-4- 4-F--Ph t-Bu Ph
RG-115416 2-CH.sub.3, 3-CH.sub.2CH.sub.2CH.sub.2O-4- 3,4-F--Ph t-Bu
Ph RG-115207 2-CH.sub.3, 3-CH.sub.2CH.sub.2CH.sub.2O-4-
3,5-di-F--Ph t-Bu Ph RG-115518 2-CH.sub.3,
3-CH.sub.2CH.sub.2CH.sub.2O-4- 2,3,4,5-tetra-F--Ph t-Bu Ph
RG-115611 2-Et, 3-OCH.sub.2OCH.sub.2-4-Ph 4-CH.sub.3--Ph t-Bu
RG-115191 2-Et, 3-OMe--Ph 3,5-di-OCH.sub.3-4-OAc--Ph t-Bu RG-115116
2-Et, 3-OMe--Ph 3,5-di-OCH.sub.3--OH--Ph t-Bu RG-115637 2-CH.sub.3,
3,4-ethylenedioxy- 3,5-di-OCH.sub.3-4-CH.sub.3--Ph t-Bu Ph
RG-115517 2-CH.sub.3, 3,4-ethylenedioxy- 2,6-di-OCH.sub.3-3-pyridyl
t-Bu Ph RG-115536 2-CH.sub.3, 3,4-ethylenedioxy-
2,6-di-Cl-4-pyridyl t-Bu Ph RG-115350 2-CH.sub.3,
3,4-ethylenedioxy- 3-F--Ph t-Bu Ph RG-115169 2-CH.sub.3,
3,4-ethylenedioxy- 3-CF.sub.3, 5-F--Ph t-Bu Ph RG-115384
2-CH.sub.3, 3,4-ethylenedioxy- 2-NO.sub.2-5-CH.sub.3--Ph t-Bu Ph
RG-115783 2-ethyl, 3-methoxy 4,6-dimethyl-pyridyl t-Bu RG-115856
2-CH.sub.3, 3,4-ethylenedioxy- 3,5-di-CH.sub.3--Ph
--CH(Et)C(CH.sub.3).sub.3 Ph RG-115857 2-CH.sub.3,
3,4-ethylenedioxy- 3,5-di-OCH.sub.3-4-CH.sub.3--Ph
--CH(Et)C(CH.sub.3).sub.3 Ph RG-115864 2-CH.sub.3,
3,4-ethylenedioxy- 3,5-di-CH.sub.3--Ph --CH(n-Pr)C(CH.sub.3).sub.3
Ph RG-115865 2-CH.sub.3, 3,4-ethylenedioxy-
3,5-di-OCH.sub.3-4-CH.sub.3--Ph --CH(n-Pr)C(CH.sub.3).sub.3 Ph
RG-115858 2-CH.sub.2CH.sub.3, 3,4- 3,5-di-CH.sub.3--Ph
--CH(Et)C(CH.sub.3).sub.3 ethylenedioxy-Ph RG-115859
2-CH.sub.2CH.sub.3, 3,4- 3,5-di-OCH3-4-CH3--Ph
--CH(Et)C(CH.sub.3).sub.3 ethylenedioxy-Ph RG-115866
2-CH.sub.2CH.sub.3, 3,4- 3,5-di-CH.sub.3--Ph
--CH(n-Pr)C(CH.sub.3).sub.3 ethylenedioxy-Ph RG-115867
2-CH.sub.2CH.sub.3, 3,4- 3,5-di-OCH.sub.3-4-CH.sub.3--Ph
--CH(n-Pr)C(CH.sub.3).sub.3 ethylenedioxy-Ph RG-115834 2-CH.sub.3,
3-OCH.sub.3--Ph 2-methoxy-6-trifluoromethyl-3- --C(CH.sub.3).sub.3
pyridyl RG-115835 2-CH.sub.3, 3-OCH.sub.3--Ph
1-methyl-2-oxo-6-trifluoromethyl-
--C(CH.sub.3).sub.3 3-pyridyl RG-115849 2-CH.sub.3, 3-OCH.sub.3--Ph
2,6-dimethoxy-4-pyrimidinyl --C(CH.sub.3).sub.3 RG-115850
2-CH.sub.3, 3-OCH.sub.3--Ph 3,6-dimethoxy-4-pyridazinyl
--C(CH.sub.3).sub.3 RG-115861 2-CH.sub.3, 3-OCH.sub.3--Ph
3,6-dichloro-4-pyridazinyl --C(CH.sub.3).sub.3 RG-115862
2-CH.sub.3, 3-OCH.sub.3--Ph 4-pyridazinyl --C(CH.sub.3).sub.3
RG-115863 2-CH.sub.3, 3-OCH.sub.3--Ph 3-oxo-6-methoxy-4-pyridazinyl
(or --C(CH.sub.3).sub.3 regioisomer) RG-115819 2-CH.sub.3,
3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph --CH(Et)C(CH.sub.3).sub.3
RG-115820 2-CH.sub.3, 3-OCH.sub.3--Ph
3,5-di-OCH.sub.3-4-CH.sub.3--Ph --CH(Et)C(CH.sub.3).sub.3 RG-115823
2-CH.sub.3, 3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--CH(n-Pr)C(CH.sub.3).sub.3 RG-115824 2-CH.sub.3, 3-OCH.sub.3--Ph
3,5-di-OCH.sub.3-4-CH.sub.3--Ph --CH(n-Pr)C(CH.sub.3).sub.3
RG-115832 2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--CH(Et)C(CH.sub.3).sub.3 RG-115831 2-CH.sub.2CH.sub.3,
3-OCH.sub.3--Ph 3,5-di-OCH.sub.3-4-CH.sub.3--Ph
--CH(Et)C(CH.sub.3).sub.3 RG-115830 2-CH.sub.2CH.sub.3,
3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph --CH(n-Pr)C(CH.sub.3).sub.3
RG-115829 2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph
3,5-di-OCH.sub.3-4-CH.sub.3--Ph --CH(n-Pr)C(CH.sub.3).sub.3
RG-103309 2-CH.sub.3, 3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--CH(Et)C(CH.sub.3).sub.3 RG-115595 2-CH.sub.3, 3-OH--Ph
3-OCH.sub.3-4-pyridyl --C(CH.sub.3).sub.3 RG-100021
4-CH(OH)CH.sub.3--Ph 3,5-di(CH.sub.2OH)--Ph --C(CH.sub.3).sub.3
RG-115199 2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 2-S(O)CH.sub.3--Ph
--C(CH.sub.3).sub.3 RG-100150 4-C(O)CH.sub.3--Ph
3,5-di-CO.sub.2H--Ph --C(CH.sub.3).sub.3 RG-115517 2-CH.sub.3,
3,4-ethylenedioxy- 2,6-di-OCH.sub.3-3-pyridyl --C(CH.sub.3).sub.3
Ph RG-115280 2-CH.sub.2CH.sub.3, 3,4- 3-CF.sub.3-4-F-phenyl
--C(CH.sub.3).sub.3 ethylenedioxy-Ph RG-101523 2-F,
4-CH.sub.2CH.sub.3--Ph 3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3
RG-115555 2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 2-SO.sub.3H--Ph
--C(CH.sub.3).sub.3 RG-102408 2-CH.sub.3,
3-CH.sub.2CH.sub.2CH.sub.2O-4- 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.3 Ph RG-103451 4-CH.sub.2CH.sub.3--Ph
3,5-di-CH.sub.3--Ph --CH(CH.sub.3)C(CH.sub.3).sub.3 RG-101036
2-CH.sub.2CH.sub.3, 3,4- 3-CH.sub.3--Ph --C(CH.sub.3).sub.3
ethylenedioxy-Ph RG-103361 2,3-di-CH.sub.3--Ph Ph --CH(Et)(n-Bu)
RG-104074 2,3-di-CH.sub.3--Ph 3-CH.sub.3--Ph --CH(Et)(t-Bu)
RG-115009 2-CH.sub.3, 3,4-ethylenedioxy- 3,5-di-OCH.sub.3, 4-OH--Ph
--C(CH.sub.3).sub.3 Ph RG-115068 2-F, 3-CH.sub.2OCH.sub.2O-4-Ph
3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3 RG-115064 2-CH.sub.3,
3,4-ethylenedioxy- 2-S(O)CH.sub.3--Ph --C(CH.sub.3).sub.3 Ph
RG-115092 2-CH.sub.3, 3,4-ethylenedioxy- 3,5-di-OCH.sub.3,
4-CH.sub.3--Ph --C(CH.sub.3).sub.2CN Ph RG-115311
2-CH.sub.2CH.sub.3-3-OCH.sub.3--Ph 6-CH.sub.3-2-pyridyl-
--C(CH.sub.3).sub.3 RG-115609 2-CH.sub.3, 3,4-ethylenedioxy-
2-NO.sub.2-3,5-di-OCH.sub.3, 4-CH.sub.3--Ph --C(CH.sub.3).sub.3 Ph
RG-102317 2-CH.sub.3, 3,4-ethylenedioxy- 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.3 Ph RG-102125 4-CH.sub.2CH.sub.3--Ph
3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3 RG-102398
2-CH.sub.3-3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3
RG-115836 4-CH.sub.2CH.sub.3--Ph 3,5-di-CH.sub.3--Ph --CH(Et)(t-Bu)
RG-115837 4-CH.sub.2CH.sub.3--Ph 2-OCH.sub.3-3-pyridyl
--CH(Et)(t-Bu) RG-115840 4-CH.sub.2CH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--CH(n-Bu)(t-Bu) RG-115841 4-CH.sub.2CH.sub.3--Ph 3,5-di-OCH.sub.3,
4-CH.sub.3--Ph --CH(n-Bu)(t-Bu) RG-115842 4-CH.sub.2CH.sub.3--Ph
2-OCH.sub.3-3-pyridyl --CH(n-Bu)(t-Bu) RG-115846
4-CH.sub.2CH.sub.3--Ph 3,5-di-CH.sub.3--Ph --CH(Ph)(t-Bu) RG-115847
4-CH.sub.2CH.sub.3--Ph 3,5-di-OCH.sub.3, 4-CH.sub.3--Ph
--CH(Ph)(t-Bu) RG-115848 4-CH.sub.2CH.sub.3--Ph
2-OCH.sub.3-3-pyridyl --CH(Ph)(t-Bu) RG-115719 2-CH.sub.2CH.sub.3,
3-OCH.sub.3--Ph 5-benzimidazolyl --C(CH.sub.3).sub.3 RG-115718
2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 1- (or
3-)trifyl-5-benzimidazolyl --C(CH.sub.3).sub.3 RG-115721
2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph
5-methyl-1-phenyl-1H-pyrazole-3- --C(CH.sub.3).sub.3 yl RG-115716
2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph
3-chloro-6-methylsulfanyl-pyrazine- --C(CH.sub.3).sub.3 2-yl
RG-115723 2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 1H-indazole-3-yl
--C(CH.sub.3).sub.3 RG-115722 2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph
1-trityl-1H-indazole-3-yl --C(CH.sub.3).sub.3 RG-115717
2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 5-methoxycarbonyl-2-pyridyl
--C(CH.sub.3).sub.3 RG-115550 2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph
pyrazine-2-yl --C(CH.sub.3).sub.3 RG-115665 2-CH.sub.2CH.sub.3,
3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.2CH.sub.2OSi(CH.sub.3)2tBu RG-115511
2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.2CH.dbd.NCH.sub.2CH.sub.2OH RG-115653
2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.2CH.dbd.NNHC(O)NH.sub.2 RG-115597
2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.2CH.dbd.NNHC(O)C(O)NH.sub.2 RG-115044
2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.2COOH RG-115172 2-CH.sub.2S(O)CH.sub.3,
3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3 RG-115408
2-CH.sub.2C(O).sub.2CH.sub.3, 3-OCH3--Ph 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.3 RG-115497 2-CH.sub.2NMe.sub.2, 3-OCH.sub.3--Ph
3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3 RG-115079
2-CH.sub.2NHCH.sub.3, 3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.3 RG-102021 2-CH.dbd.CH.sub.2, 3-OCH.sub.3--Ph--
3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3 RG-115117 2-CH.sub.2OMe,
3-OCH.sub.3--Ph-- 3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3 RG-115358
2-CH.sub.2SCH.sub.3, 3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.3 RG-115003 2-CH.sub.2OCH.sub.2CH.dbd.CH.sub.2,
3- 3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3 OCH.sub.3--Ph RG-115490
2-CH.sub.2Cl, 3-OCH.sub.3--Ph-- 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.3 RG-115371 2-CH.sub.2OH, 3-OCH.sub.3--Ph--
3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3 RG-115225 2-CH.sub.2OAc,
3-OCH.sub.3--Ph 3,5-di-CH.sub.3--Ph --C(CH.sub.3).sub.3 RG-115160
2-CH.sub.2F, 3-OCH.sub.3--Ph-- 3,5-di-CH.sub.3--Ph
--C(CH.sub.3).sub.3 RG-115851 2-CH.sub.3, 3-OCH.sub.3
3,5-di-CH.sub.3 --CH(n-Bu)(t-Bu) RG-115852 2-CH.sub.3, 3-OCH.sub.3
3,5-di-OCH.sub.3, 4-CH.sub.3 --CH(n-Bu)(t-Bu) RG-115091
2-CH.sub.2CH.sub.3, 3-OCH.sub.3--Ph 5-Methyl-pyrazine-2-yl-
--C(CH.sub.3).sub.3
[0051] Because the compounds of the general formula of the present
invention may contain a number of stereogenic carbon atoms, the
compounds may exist as enantiomers, diastereomers, stereoisomers,
or their mixtures, even if a stereogenic center is explicitly
specified.
[0052] The present invention also pertains to a process for the
preparation of a compound of formula (IV) comprising the steps of:
[0053] i reacting a compound of formula (I) with a base selected
from NaH, KH, or an amide MNR.sup.aR.sup.b to produce a product II,
wherein M is Li, Na, or K, and R.sup.a and R.sup.b are
independently (C.sub.1-C.sub.6)alkyl or phenyl; and
[0053] ##STR00003## [0054] 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;
##STR00004##
[0054] wherein A and B are independently [0055] (a) unsubstituted
or substituted phenyl wherein the substituents are independently 1
to 5H; halo; nitro; cyano; amino (--NR.sup.aR.sup.b);
alkylaminoalkyl (--(CH.sub.2).sub.nNR.sup.aR.sup.b);
(C.sub.1-C.sub.6)alkyl; (C.sub.1-C.sub.6)haloalkyl;
(C.sub.1-C.sub.6)cyanoalkyl; (C.sub.1-C.sub.6)alkoxy; phenoxy;
(C.sub.1-C.sub.6)haloalkoxy;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkenyloxy(C.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkoxy(C.sub.1-C.sub.6)alkoxy;
(C.sub.2-C.sub.6)alkenyl optionally substituted with halo, cyano,
(C.sub.1-C.sub.4)alkyl, or (C.sub.1-C.sub.4)alkoxy;
(C.sub.2-C.sub.6)alkynyl optionally substituted with halo or
(C.sub.1-C.sub.4)alkyl; formyl; (C.sub.1-C.sub.6)haloalkylcarbonyl;
benzoyl; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)haloalkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); carboxamido (--CONR.sup.aR.sup.b); amido
(--NR.sup.aCOR.sup.b); alkoxycarbonylamino
(--N(CH.sub.2).sub.nCO.sub.2R.sup.b); alkylaminocarbonylamino
(--N(CH.sub.2)CONR.sup.bR.sup.c); (C.sub.1-C.sub.6)alkylthio;
sulfamido (--SO.sub.2NR.sup.aR.sup.b); or unsubstituted or
substituted phenyl wherein the substituents are independently 1 to
3 halo, nitro, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.6)alkyl, or
(--NR.sup.aR.sup.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.sub.2O--), (--OCH(CH.sub.3)O--),
(--OCH.sub.2CH.sub.2O--), (--OCH(CH.sub.3)CH.sub.2O--),
(--S--CH.dbd.N--), (--CH.sub.2OCH.sub.2O--),
(--O(CH.sub.2).sub.3--), (.dbd.N--O--N.dbd.), (--C.dbd.CH--NH--),
(--OCF.sub.2O--), (--NH--CH.dbd.N--), (--CH.sub.2CH.sub.2O--), and
(--(CH.sub.2).sub.4--); or [0056] (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.sub.1-C.sub.6)alkyl;
(C.sub.1-C.sub.6)alkeyl; (C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)thioalkoxy; (C.sub.1-C.sub.6)alkoxycarbonyl;
(C.sub.1-C.sub.6)carbocyalkyl; --CONR.sup.aR.sup.b; amino
(--NR.sup.aR.sup.b); haloalkyl including CF.sub.3; -trialkylsilyl
(--SiR.sup.aR.sup.bR.sup.c); trityl(C(Ph).sub.3); or unsubstituted
or substituted phenyl wherein the substituents are independently 1
to 3 halo, nitro, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.6)alkyl,
or (--NR.sup.aR.sup.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.sub.1-C.sub.10) straight
or branched alkyl wherein the substituents are independently 1-4
cyano; halo; (C.sub.5-C.sub.6)cycloalkyl; phenyl;
(C.sub.2-C.sub.3)alkenyl; (C.sub.1-C.sub.6)alkoxy;
(C.sub.1-C.sub.6)alkoxycarbonyl; (C.sub.1-C.sub.6)alkanoyloxy
(--OCOR.sup.a); formyl; (C.sub.1-C.sub.6)trialkylsilyloxy having
independently the stated number of carbon atoms in each alkyl
group; or --C.dbd.N--OR.sup.a; wherein R.sup.a, R.sup.b, and
R.sup.c are independently (C.sub.1-C.sub.6)alkyl or phenyl, and
n=1-4.
DEFINITIONS
[0057] When an R.sup.x group is specified, wherein x represents a
letter a-g, and the same R.sup.x group is also specified with an
alkyl group chain length such as "(C.sub.1-C.sub.3)", it is
understood that the specified chain length refers only to the cases
where R.sup.x may be alkyl, and does not pertain to cases where
R.sup.x may be a non-alkyl group, such as H or aryl.
[0058] The term "alkyl" includes both branched and straight chain
alkyl groups. Typical alkyl groups include, for example, methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, isooctyl,
nonyl, and decyl.
[0059] The term "halo" refers to fluoro, chloro, bromo or iodo.
[0060] The term "haloalkyl" refers to an alkyl group substituted
with one or more halo groups such as, for example, chloromethyl,
2-bromoethyl, 3-iodopropyl, trifluoromethyl, and
perfluoropropyl.
[0061] The term "cycloalkyl" refers to a cyclic aliphatic ring
structure, optionally substituted with alkyl, hydroxy, or halo,
such as cyclopropyl, methylcyclopropyl, cyclobutyl,
2-hydroxycyclopentyl, cyclohexyl, and 4-chlorocyclohexyl.
[0062] The term "hydroxyalkyl" refers to an alkyl group substituted
with one or more hydroxy groups such as, for example, hydroxymethyl
and 2,3-dihydroxybutyl.
[0063] The term "alkylsulfonyl" refers to a sulfonyl moiety
substituted with an alkyl group such as, for example, mesyl, and
n-propylsulfonyl.
[0064] The term "alkenyl" refers to an ethylenically unsaturated
hydrocarbon group, straight or branched chain, having 1 or 2
ethylenic bonds such as, for example, vinyl, allyl, 1-butenyl,
2-butenyl, isopropenyl, and 2-pentenyl.
[0065] The term "haloalkenyl" refers to an alkenyl group
substituted with one or more halo groups.
[0066] The term "alkynyl" refers to an unsaturated hydrocarbon
group, straight or branched, having 1 or 2 acetylenic bonds such
as, for example, ethynyl and propargyl.
[0067] The term "alkylcarbonyl" refers to an alkylketo
functionality, for example acetyl, n-butyryl and the like.
[0068] The term "heterocyclyl" or "heterocycle" refers to an
unsubstituted or substituted; saturated, partially unsaturated, or
unsaturated 5 or 6-membered ring containing one, two or three
heteroatoms, preferably one or two heteroatoms independently
selected from the group consisting of oxygen, nitrogen and sulfur.
Examples of heterocyclyls include, for example, pyridyl, thienyl,
furyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, pyrrolyl,
indolyl, tetrahydrofuryl, pyrrolidinyl, piperidinyl,
tetrahydropyranyl, morpholinyl, piperazinyl, dioxolanyl, and
dioxanyl.
[0069] The term "alkoxy" includes both branched and straight chain
alkyl groups attached to a terminal oxygen atom. Typical alkoxy
groups include, for example, methoxy, ethoxy, n-propoxy,
isopropoxy, and tert-butoxy.
[0070] The term "haloalkoxy" refers to an alkoxy group substituted
with one or more halo groups such as, for example chloromethoxy,
trifluoromethoxy, difluoromethoxy, and perfluoroisobutoxy.
[0071] The term "alkylthio" includes both branched and straight
chain alkyl groups attached to a terminal sulfur atom such as, for
example methylthio.
[0072] The term "haloalkylthio" refers to an alkylthio group
substituted with one or more halo groups such as, for example
trifluoromethylthio.
[0073] The term "alkoxyalkyl" refers to an alkyl group substituted
with an alkoxy group such as, for example, isopropoxymethyl.
[0074] "Silica gel chromatography" refers to a purification method
wherein a chemical substance of interest is applied as a
concentrated sample to the top of a vertical column of silica gel
or chemically-modified silica gel contained in a glass, plastic, or
metal cylinder, and elution from such column with a solvent or
mixture of solvents.
[0075] "Flash chromatography" refers to silica gel chromatography
performed under air, argon, or nitrogen pressure typically in the
range of 10 to 50 psi.
[0076] "Gradient chromatography" refers to silica gel
chromatography in which the chemical substance is eluted from a
column with a progressively changing composition of a solvent
mixture.
[0077] "Rf" is a thin layer chromatography term which refers to the
fractional distance of movement of a chemical substance of interest
on a thin layer chromatography plate, relative to the distance of
movement of the eluting solvent system.
[0078] "Parr hydrogenator" and "Parr shaker" refer to apparatus
available from Parr Instrument Company, Moline Ill., which are
designed to facilitate vigorous mixing of a solution containing a
chemical substance of interest with an optional solid suspended
catalyst and a pressurized, contained atmosphere of a reactant gas.
Typically, the gas is hydrogen and the catalyst is palladium,
platinum, or oxides thereof deposited on small charcoal particles.
The hydrogen pressure is typically in the range of 30 to 70
psi.
[0079] "Dess-Martin reagent" refers to
(1,1,1-triacetoxy)-1,1-dihydro-1,2-benziodoxol-3(1H)-one as a
solution in dichloromethane available from Acros Organics/Fisher
Scientific Company, L.L.C.
[0080] "PS-NMM" refers to a --SO.sub.2NH(CH.sub.2).sub.3-morpholine
functionalized polystyrene resin available from Argonaut
Technologies, San Carlos, Calif.
[0081] "AP-NCO" refers to an isocyante-functionalized resin
available from ArgonautTechnologies, San Carlos, Calif.
[0082] "AP-trisamine" refers to a
polystyrene-CH.sub.2NHCH.sub.2CH.sub.2NH(CH.sub.2CH.sub.2NH.sub.2).sub.2
resin available from Argonaut Technologies, San Carlos, Calif.
[0083] The term "isolated" for the purposes of the present
invention designates a biological material (nucleic acid or
protein) that has been removed from its original environment (the
environment in which it is naturally present). For example, a
polynucleotide present in the natural state in a plant or an animal
is not isolated, however the same polynucleotide separated from the
adjacent nucleic acids in which it is naturally present, is
considered "isolated". The term "purified" does not require the
material to be present in a form exhibiting absolute purity,
exclusive of the presence of other compounds. It is rather a
relative definition.
[0084] A polynucleotide is in the "purified" state after
purification of the starting material or of the natural material by
at least one order of magnitude, preferably 2 or 3 and preferably 4
or 5 orders of magnitude.
[0085] A "nucleic acid" is a polymeric compound comprised of
covalently linked subunits called nucleotides. Nucleic acid
includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid
(DNA), both of which may be single-stranded or double-stranded. DNA
includes but is not limited to cDNA, genomic DNA, plasmids DNA,
synthetic DNA, and semi-synthetic DNA. DNA may be linear, circular,
or supercoiled.
[0086] A "nucleic acid molecule" refers to the phosphate ester
polymeric form of ribonucleosides (adenosine, guanosine, uridine or
cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine,
deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"),
or any phosphoester anologs thereof, such as phosphorothioates and
thioesters, in either single stranded form, or a double-stranded
helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are
possible. The term nucleic acid molecule, and in particular DNA or
RNA molecule, refers only to the primary and secondary structure of
the molecule, and does not limit it to any particular tertiary
forms. Thus, this term includes double-stranded DNA found, inter
alia, in linear or circular DNA molecules (e.g., restriction
fragments), plasmids, and chromosomes. In discussing the structure
of particular double-stranded DNA molecules, sequences may be
described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the non-transcribed
strand of DNA (i.e., the strand having a sequence homologous to the
mRNA). A "recombinant DNA molecule" is a DNA molecule that has
undergone a molecular biological manipulation.
[0087] The term "fragment" will be understood to mean a nucleotide
sequence of reduced length relative to the reference nucleic acid
and comprising, over the common portion, a nucleotide sequence
identical to the reference nucleic acid. Such a nucleic acid
fragment according to the invention may be, where appropriate,
included in a larger polynucleotide of which it is a constituent.
Such fragments comprise, or alternatively consist of,
oligonucleotides ranging in length from at least 6, 8, 9, 10, 12,
15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54,
57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200,
300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a
nucleic acid according to the invention.
[0088] As used herein, an "isolated nucleic acid fragment" is a
polymer of RNA or DNA that is single- or double-stranded,
optionally containing synthetic, non-natural or altered nucleotide
bases. An isolated nucleic acid fragment in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA or synthetic DNA.
[0089] A "gene" refers to an assembly of nucleotides that encode a
polypeptide, and includes cDNA and genomic DNA nucleic acids.
"Gene" also refers to a nucleic acid fragment that expresses a
specific protein or polypeptide, including regulatory sequences
preceding (5' non-coding sequences) and following (3' non-coding
sequences) the coding sequence. "Native gene" refers to a gene as
found in nature with its own regulatory sequences. "Chimeric gene"
refers to any gene that is not a native gene, comprising regulatory
and/or coding sequences that are not found together in nature.
Accordingly, a chimeric gene may comprise regulatory sequences and
coding sequences that are derived from different sources, or
regulatory sequences and coding sequences derived from the same
source, but arranged in a manner different than that found in
nature. A chimeric gene may comprise coding sequences derived from
different sources and/or regulatory sequences derived from
different sources. "Endogenous gene" refers to a native gene in its
natural location in the genome of an organism. A "foreign" gene or
"heterologous" gene refers to a gene not normally found in the host
organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, or chimeric genes. A "transgene" is a gene
that has been introduced into the genome by a transformation
procedure.
[0090] "Heterologous" DNA refers to DNA not naturally located in
the cell, or in a chromosomal site of the cell. Preferably, the
heterologous DNA includes a gene foreign to the cell.
[0091] The term "genome" includes chromosomal as well as
mitochondrial, chloroplast and viral DNA or RNA.
[0092] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength (see Sambrook et al., 1989
infra). Hybridization and washing conditions are well known and
exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T.
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor (1989), particularly
Chapter 11 and Table 11.1 therein (entirely incorporated herein by
reference). The conditions of temperature and ionic strength
determine the "stringency" of the hybridization.
[0093] Stringency conditions can be adjusted to screen for
moderately similar fragments, such as homologous sequences from
distantly related organisms, to highly similar fragments, such as
genes that duplicate functional enzymes from closely related
organisms. For preliminary screening for homologous nucleic acids,
low stringency hybridization conditions, corresponding to a T.sub.m
of 55.degree., can be used, e.g., 5.times.SSC, 0.1% SDS, 0.25%
milk, and no formamide; or 30% formamide, 5.times.SSC, 0.5% SDS).
Moderate stringency hybridization conditions correspond to a higher
T.sub.m, e.g., 40% formamide, with 5.times. or 6.times.SCC. High
stringency hybridization conditions correspond to the highest
T.sub.m, e.g., 50% formamide, 5.times. or 6.times.SCC.
[0094] Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of
the hybridization, mismatches between bases are possible. The term
"complementary" is used to describe the relationship between
nucleotide bases that are capable of hybridizing to one another.
For example, with respect to DNA, adenosine is complementary to
thymine and cytosine is complementary to guanine. Accordingly, the
instant invention also includes isolated nucleic acid fragments
that are complementary to the complete sequences as disclosed or
used herein as well as those substantially similar nucleic acid
sequences.
[0095] In a specific embodiment of the invention, polynucleotides
are detected by employing hybridization conditions comprising a
hybridization step at T.sub.m of 55.degree. C., and utilizing
conditions as set forth above. In a preferred embodiment, the
T.sub.m is 60.degree. C.; in a more preferred embodiment, the
T.sub.m is 63.degree. C.; in an even more preferred embodiment, the
T.sub.m is 65.degree. C.
[0096] Post-hybridization washes also determine stringency
conditions. One set of preferred conditions uses a series of washes
starting with 6.times.SSC, 0.5% SDS at room temperature for 15
minutes (min), then repeated with 2.times.SSC, 0.5% SDS at
45.degree. C. for 30 minutes, and then repeated twice with
0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30 minutes. A more
preferred set of stringent conditions uses higher temperatures in
which the washes are identical to those above except for the
temperature of the final two 30 min washes in 0.2.times.SSC, 0.5%
SDS was increased to 60.degree. C. Another preferred set of highly
stringent conditions uses two final washes in 0.1.times.SSC, 0.1%
SDS at 65.degree. C. Hybridization requires that the two nucleic
acids comprise complementary sequences, although depending on the
stringency of the hybridization, mismatches between bases are
possible.
[0097] The appropriate stringency for hybridizing nucleic acids
depends on the length of the nucleic acids and the degree of
complementation, variables well known in the art. The greater the
degree of similarity or homology between two nucleotide sequences,
the greater the value of T.sub.m for hybrids of nucleic acids
having those sequences. The relative stability (corresponding to
higher T.sub.m) of nucleic acid hybridizations decreases in the
following order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids of
greater than 100 nucleotides in length, equations for calculating
T.sub.m have been derived (see Sambrook et al., supra, 9.50-0.51).
For hybridization with shorter nucleic acids, i.e.,
oligonucleotides, the position of mismatches becomes more
important, and the length of the oligonucleotide determines its
specificity (see Sambrook et al., supra, 11.7-11.8).
[0098] In a specific embodiment of the invention, polynucleotides
are detected by employing hybridization conditions comprising a
hybridization step in less than 500 mM salt and at least 37 degrees
Celsius, and a washing step in 2.times.SSPE at at least 63 degrees
Celsius. In a preferred embodiment, the hybridization conditions
comprise less than 200 mM salt and at least 37 degrees Celsius for
the hybridization step. In a more preferred embodiment, the
hybridization conditions comprise 2.times.SSPE and 63 degrees
Celsius for both the hybridization and washing steps.
[0099] In one embodiment, the length for a hybridizable nucleic
acid is at least about 10 nucleotides. Preferable a minimum length
for a hybridizable nucleic acid is at least about 15 nucleotides;
more preferably at least about 20 nucleotides; and most preferably
the length is at least 30 nucleotides. Furthermore, the skilled
artisan will recognize that the temperature and wash solution salt
concentration may be adjusted as necessary according to factors
such as length of the probe.
[0100] The term "probe" refers to a single-stranded nucleic acid
molecule that can base pair with a complementary single stranded
target nucleic acid to form a double-stranded molecule.
[0101] As used herein, the term "oligonucleotide" refers to a
nucleic acid, generally of at least 18 nucleotides, that is
hybridizable to a genomic DNA molecule, a cDNA molecule, a plasmid
DNA or an mRNA molecule. Oligonucleotides can be labeled, e.g.,
with .sup.32P-nucleotides or nucleotides to which a label, such as
biotin, has been covalently conjugated. A labeled oligonucleotide
can be used as a probe to detect the presence of a nucleic acid.
Oligonucleotides (one or both of which may be labeled) can be used
as PCR primers, either for cloning full length or a fragment of a
nucleic acid, or to detect the presence of a nucleic acid. An
oligonucleotide can also be used to form a triple helix with a DNA
molecule. Generally, oligonucleotides are prepared synthetically,
preferably on a nucleic acid synthesizer. Accordingly,
oligonucleotides can be prepared with non-naturally occurring
phosphoester analog bonds, such as thioester bonds, etc.
[0102] A "primer" is an oligonucleotide that hybridizes to a target
nucleic acid sequence to create a double stranded nucleic acid
region that can serve as an initiation point for DNA synthesis
under suitable conditions. Such primers may be used in a polymerase
chain reaction.
[0103] "Polymerase chain reaction" is abbreviated PCR and means an
in vitro method for enzymatically amplifying specific nucleic acid
sequences. PCR involves a repetitive series of temperature cycles
with each cycle comprising three stages: denaturation of the
template nucleic acid to separate the strands of the target
molecule, annealing a single stranded PCR oligonucleotide primer to
the template nucleic acid, and extension of the annealed primer(s)
by DNA polymerase. PCR provides a means to detect the presence of
the target molecule and, under quantitative or semi-quantitative
conditions, to determine the relative amount of that target
molecule within the starting pool of nucleic acids.
[0104] "Reverse transcription-polymerase chain reaction" is
abbreviated RT-PCR and means an in vitro method for enzymatically
producing a target cDNA molecule or molecules from an RNA molecule
or molecules, followed by enzymatic amplification of a specific
nucleic acid sequence or sequences within the target cDNA molecule
or molecules as described above. RT-PCR also provides a means to
detect the presence of the target molecule and, under quantitative
or semi-quantitative conditions, to determine the relative amount
of that target molecule within the starting pool of nucleic
acids.
[0105] A DNA "coding sequence" is a double-stranded DNA sequence
that is transcribed and translated into a polypeptide in a cell in
vitro or in vivo when placed under the control of appropriate
regulatory sequences. "Suitable regulatory sequences" refer to
nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, polyadenylation recognition sequences, RNA
processing site, effector binding site and stem-loop structure. The
boundaries of the coding sequence are determined by a start codon
at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxyl) terminus. A coding sequence can include, but is not
limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA
sequences, and even synthetic DNA sequences. If the coding sequence
is intended for expression in a eukaryotic cell, a polyadenylation
signal and transcription termination sequence will usually be
located 3' to the coding sequence.
[0106] "Open reading frame" is abbreviated ORF and means a length
of nucleic acid sequence, either DNA, cDNA or RNA, that comprises a
translation start signal or initiation codon, such as an ATG or
AUG, and a termination codon and can be potentially translated into
a polypeptide sequence.
[0107] The term "head-to-head" is used herein to describe the
orientation of two polynucleotide sequences in relation to each
other. Two polynucleotides are positioned in a head-to-head
orientation when the 5' end of the coding strand of one
polynucleotide is adjacent to the 5' end of the coding strand of
the other polynucleotide, whereby the direction of transcription of
each polynucleotide proceeds away from the 5' end of the other
polynucleotide. The term "head-to-head" may be abbreviated
(5')-to-(5') and may also be indicated by the symbols
(.rarw..fwdarw.) or (3'.rarw.5'5'.fwdarw.3').
[0108] The term "tail-to-tail" is used herein to describe the
orientation of two polynucleotide sequences in relation to each
other. Two polynucleotides are positioned in a tail-to-tail
orientation when the 3' end of the coding strand of one
polynucleotide is adjacent to the 3' end of the coding strand of
the other polynucleotide, whereby the direction of transcription of
each polynucleotide proceeds toward the other polynucleotide. The
term "tail-to-tail" may be abbreviated (3')-to-(3') and may also be
indicated by the symbols (.rarw..fwdarw.) or
(5'.fwdarw.3'3'.rarw.5').
[0109] The term "head-to-tail" is used herein to describe the
orientation of two polynucleotide sequences in relation to each
other. Two polynucleotides are positioned in a head-to-tail
orientation when the 5' end of the coding strand of one
polynucleotide is adjacent to the 3' end of the coding strand of
the other polynucleotide, whereby the direction of transcription of
each polynucleotide proceeds in the same direction as that of the
other polynucleotide. The term "head-to-tail" may be abbreviated
(5')-to-(3') and may also be indicated by the symbols
(.fwdarw..fwdarw.) or (5'.fwdarw.3'5'.fwdarw.3').
[0110] The term "downstream" refers to a nucleotide sequence that
is located 3' to reference nucleotide sequence. In particular,
downstream nucleotide sequences generally relate to sequences that
follow the starting point of transcription. For example, the
translation initiation codon of a gene is located downstream of the
start site of transcription.
[0111] The term "upstream" refers to a nucleotide sequence that is
located 5' to reference nucleotide sequence. In particular,
upstream nucleotide sequences generally relate to sequences that
are located on the 5' side of a coding sequence or starting point
of transcription. For example, most promoters are located upstream
of the start site of transcription.
[0112] The terms "restriction endonuclease" and "restriction
enzyme" refer to an enzyme that binds and cuts within a specific
nucleotide sequence within double stranded DNA.
[0113] "Homologous recombination" refers to the insertion of a
foreign DNA sequence into another DNA molecule, e.g., insertion of
a vector in a chromosome. Preferably, the vector targets a specific
chromosomal site for homologous recombination. For specific
homologous recombination, the vector will contain sufficiently long
regions of homology to sequences of the chromosome to allow
complementary binding and incorporation of the vector into the
chromosome. Longer regions of homology, and greater degrees of
sequence similarity, may increase the efficiency of homologous
recombination.
[0114] Several methods known in the art may be used to propagate a
polynucleotide according to the invention. Once a suitable host
system and growth conditions are established, recombinant
expression vectors can be propagated and prepared in quantity. As
described herein, the expression vectors which can be used include,
but are not limited to, the following vectors or their derivatives:
human or animal viruses such as vaccinia virus or adenovirus;
insect viruses such as baculovirus; yeast vectors; bacteriophage
vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name
but a few.
[0115] A "vector" is any means for the cloning of and/or transfer
of a nucleic acid into a host cell. A vector may be a replicon to
which another DNA segment may be attached so as to bring about the
replication of the attached segment. A "replicon" is any genetic
element (e.g., plasmid, phage, cosmid, chromosome, virus) that
functions as an autonomous unit of DNA replication in vivo, i.e.,
capable of replication under its own control. The term "vector"
includes both viral and nonviral means for introducing the nucleic
acid into a cell in vitro, ex vivo or in vivo. A large number of
vectors known in the art may be used to manipulate nucleic acids,
incorporate response elements and promoters into genes, etc.
Possible vectors include, for example, plasmids or modified viruses
including, for example bacteriophages such as lambda derivatives,
or plasmids such as pBR322 or pUC plasmid derivatives, or the
Bluescript vector. For example, the insertion of the DNA fragments
corresponding to response elements and promoters into a suitable
vector can be accomplished by ligating the appropriate DNA
fragments into a chosen vector that has complementary cohesive
termini. Alternatively, the ends of the DNA molecules may be
enzymatically modified or any site may be produced by ligating
nucleotide sequences (linkers) into the DNA termini. Such vectors
may be engineered to contain selectable marker genes that provide
for the selection of cells that have incorporated the marker into
the cellular genome. Such markers allow identification and/or
selection of host cells that incorporate and express the proteins
encoded by the marker.
[0116] Viral vectors, and particularly retroviral vectors, have
been used in a wide variety of gene delivery applications in cells,
as well as living animal subjects. Viral vectors that can be used
include but are not limited to retrovirus, adeno-associated virus,
pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr,
adenovirus, geminivirus, and caulimovirus vectors. Non-viral
vectors include plasmids, liposomes, electrically charged lipids
(cytofectins), DNA-protein complexes, and biopolymers. In addition
to a nucleic acid, a vector may also comprise one or more
regulatory regions, and/or selectable markers useful in selecting,
measuring, and monitoring nucleic acid transfer results (transfer
to which tissues, duration of expression, etc.).
[0117] The term "plasmid" refers to an extra chromosomal element
often carrying a gene that is not part of the central metabolism of
the cell, and usually in the form of circular double-stranded DNA
molecules. Such elements may be autonomously replicating sequences,
genome integrating sequences, phage or nucleotide sequences,
linear, circular, or supercoiled, of a single- or double-stranded
DNA or RNA, derived from any source, in which a number of
nucleotide sequences have been joined or recombined into a unique
construction which is capable of introducing a promoter fragment
and DNA sequence for a selected gene product along with appropriate
3' untranslated sequence into a cell.
[0118] A "cloning vector" is a "replicon", which is a unit length
of a nucleic acid, preferably DNA, that replicates sequentially and
which comprises an origin of replication, such as a plasmid, phage
or cosmid, to which another nucleic acid segment may be attached so
as to bring about the replication of the attached segment. Cloning
vectors may be capable of replication in one cell type and
expression in another ("shuttle vector").
[0119] Vectors may be introduced into the desired host cells by
methods known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, lipofection (lysosome fusion), use of a
gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992,
J. Biol. Chem. 267: 963-967; Wu and Wu, 1988, J. Biol. Chem. 263:
14621-14624; and Hartmut et al., Canadian Patent Application No.
2,012,311, filed Mar. 15, 1990).
[0120] A polynucleotide according to the invention can also be
introduced in vivo by lipofection. For the past decade, there has
been increasing use of liposomes for encapsulation and transfection
of nucleic acids in vitro. Synthetic cationic lipids designed to
limit the difficulties and dangers encountered with
liposome-mediated transfection can be used to prepare liposomes for
in vivo transfection of a gene encoding a marker (Feigner et al.,
1987, PNAS 84:7413; Mackey, et al., 1988. Proc. Natl. Acad. Sci.
U.S.A. 85:8027-8031; and Ulmer et al., 1993, Science
259:1745-1748). The use of cationic lipids may promote
encapsulation of negatively charged nucleic acids, and also promote
fusion with negatively charged cell membranes (Feigner and Ringold,
1989, Science 337: 387-388). Particularly useful lipid compounds
and compositions for transfer of nucleic acids are described in
International Patent Publications WO95/18863 and WO96/17823, and in
U.S. Pat. No. 5,459,127. The use of lipofection to introduce
exogenous genes into the specific organs in vivo has certain
practical advantages. Molecular targeting of liposomes to specific
cells represents one area of benefit. It is clear that directing
transfection to particular cell types would be particularly
preferred in a tissue with cellular heterogeneity, such as
pancreas, liver, kidney, and the brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting (Mackey, et
al., 1988, supra). Targeted peptides, e.g., hormones or
neurotransmitters, and proteins such as antibodies, or non-peptide
molecules could be coupled to liposomes chemically.
[0121] Other molecules are also useful for facilitating
transfection of a nucleic acid in vivo, such as a cationic
oligopeptide (e.g., WO95/21931), peptides derived from DNA binding
proteins (e.g., WO96/25508), or a cationic polymer (e.g.,
WO95/21931).
[0122] It is also possible to introduce a vector in vivo as a naked
DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and
5,580,859). Receptor-mediated DNA delivery approaches can also be
used (Curiel et al., 1992, Hum. Gene Ther. 3: 147-154; and Wu and
Wu, 1987, J. Biol. Chem. 262: 4429-4432).
[0123] The term "transfection" means the uptake of exogenous or
heterologous RNA or DNA by a cell. A cell has been "transfected" by
exogenous or heterologous RNA or DNA when such RNA or DNA has been
introduced inside the cell. A cell has been "transformed" by
exogenous or heterologous RNA or DNA when the transfected RNA or
DNA effects a phenotypic change. The transforming RNA or DNA can be
integrated (covalently linked) into chromosomal DNA making up the
genome of the cell.
[0124] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
or "recombinant" or "transformed" organisms.
[0125] The term "genetic region" will refer to a region of a
nucleic acid molecule or a nucleotide sequence that comprises a
gene encoding a polypeptide.
[0126] In addition, the recombinant vector comprising a
polynucleotide according to the invention may include one or more
origins for replication in the cellular hosts in which their
amplification or their expression is sought, markers or selectable
markers.
[0127] The term "selectable marker" means an identifying factor,
usually an antibiotic or chemical resistance gene, that is able to
be selected for based upon the marker gene's effect, i.e.,
resistance to an antibiotic, resistance to a herbicide,
colorimetric markers, enzymes, fluorescent markers, and the like,
wherein the effect is used to track the inheritance of a nucleic
acid of interest and/or to identify a cell or organism that has
inherited the nucleic acid of interest. Examples of selectable
marker genes known and used in the art include: genes providing
resistance to ampicillin, streptomycin, gentamycin, kanamycin,
hygromycin, bialaphos herbicide, sulfonamide, and the like; and
genes that are used as phenotypic markers, i.e., anthocyanin
regulatory genes, isopentanyl transferase gene, and the like.
[0128] The term "reporter gene" means a nucleic acid encoding an
identifying factor that is able to be identified based upon the
reporter gene's effect, wherein the effect is used to track the
inheritance of a nucleic acid of interest, to identify a cell or
organism that has inherited the nucleic acid of interest, and/or to
measure gene expression induction or transcription. Examples of
reporter genes known and used in the art include: luciferase (Luc),
green fluorescent protein (GFP), chloramphenicol acetyltransferase
(CAT), .beta.-galactosidase (LacZ), .beta.-glucuronidase (Gus), and
the like. Selectable marker genes may also be considered reporter
genes.
[0129] "Promoter" refers to a DNA sequence capable of controlling
the expression of a coding sequence or functional RNA. In general,
a coding sequence is located 3' to a promoter sequence. Promoters
may be derived in their entirety from a native gene, or be composed
of different elements derived from different promoters found in
nature, or even comprise synthetic DNA segments. It is understood
by those skilled in the art that different promoters may direct the
expression of a gene in different tissues or cell types, or at
different stages of development, or in response to different
environmental or physiological conditions. Promoters that cause a
gene to be expressed in most cell types at most times are commonly
referred to as "constitutive promoters". Promoters that cause a
gene to be expressed in a specific cell type are commonly referred
to as "cell-specific promoters" or "tissue-specific promoters".
Promoters that cause a gene to be expressed at a specific stage of
development or cell differentiation are commonly referred to as
"developmentally-specific promoters" or "cell
differentiation-specific promoters". Promoters that are induced and
cause a gene to be expressed following exposure or treatment of the
cell with an agent, biological molecule, chemical, ligand, light,
or the like that induces the promoter are commonly referred to as
"inducible promoters" or "regulatable promoters". It is further
recognized that since in most cases the exact boundaries of
regulatory sequences have not been completely defined, DNA
fragments of different lengths may have identical promoter
activity.
[0130] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site (conveniently defined for example, by
mapping with nuclease S1), as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0131] A coding sequence is "under the control" of transcriptional
and translational control sequences in a cell when RNA polymerase
transcribes the coding sequence into mRNA, which is then trans-RNA
spliced (if the coding sequence contains introns) and translated
into the protein encoded by the coding sequence.
[0132] "Transcriptional and translational control sequences" are
DNA regulatory sequences, such as promoters, enhancers,
terminators, and the like, that provide for the expression of a
coding sequence in a host cell. In eukaryotic cells,
polyadenylation signals are control sequences.
[0133] The term "response element" means one or more cis-acting DNA
elements which confer responsiveness on a promoter mediated through
interaction with the DNA-binding domains of the first chimeric
gene. This DNA element may be either palindromic (perfect or
imperfect) in its sequence or composed of sequence motifs or half
sites separated by a variable number of nucleotides. The half sites
can be similar or identical and arranged as either direct or
inverted repeats or as a single half site or multimers of adjacent
half sites in tandem. The response element may comprise a minimal
promoter isolated from different organisms depending upon the
nature of the cell or organism into which the response element will
be incorporated. The DNA binding domain of the first hybrid protein
binds, in the presence or absence of a ligand, to the DNA sequence
of a response element to initiate or suppress transcription of
downstream gene(s) under the regulation of this response element.
Examples of DNA sequences for response elements of the natural
ecdysone receptor include: RRGG/TTCANTGAC/ACYY (see Cherbas L., et.
al., (1991), Genes Dev. 5, 120-131); AGGTCAN.sub.(n)AGGTCA, where
N.sub.(n) can be one or more spacer nucleotides (see D'Avino P P.,
et. al., (1995), Mol. Cell. Endocrinol, 113, 1-9); and
GGGTTGAATGAATTT (see Antoniewski C., et. al., (1994). Mol. Cell
Biol. 14, 4465-4474).
[0134] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0135] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from a nucleic acid or polynucleotide. Expression may
also refer to translation of mRNA into a protein or
polypeptide.
[0136] The terms "cassette", "expression cassette" and "gene
expression cassette" refer to a segment of DNA that can be inserted
into a nucleic acid or polynucleotide at specific restriction sites
or by homologous recombination. The segment of DNA comprises a
polynucleotide that encodes a polypeptide of interest, and the
cassette and restriction sites are designed to ensure insertion of
the cassette in the proper reading frame for transcription and
translation. "Transformation cassette" refers to a specific vector
comprising a polynucleotide that encodes a polypeptide of interest
and having elements in addition to the polynucleotide that
facilitate transformation of a particular host cell. Cassettes,
expression cassettes, gene expression cassettes and transformation
cassettes of the invention may also comprise elements that allow
for enhanced expression of a polynucleotide encoding a polypeptide
of interest in a host cell. These elements may include, but are not
limited to: a promoter, a minimal promoter, an enhancer, a response
element, a terminator sequence, a polyadenylation sequence, and the
like.
[0137] For purposes of this invention, the term "gene switch"
refers to the combination of a response element associated with a
promoter, and an EcR based system which in the presence of one or
more ligands, modulates the expression of a gene into which the
response element and promoter are incorporated.
[0138] The terms "modulate" and "modulates" mean to induce, reduce
or inhibit nucleic acid or gene expression, resulting in the
respective induction, reduction or inhibition of protein or
polypeptide production.
[0139] The plasmids or vectors according to the invention may
further comprise at least one promoter suitable for driving
expression of a gene in a host cell. The term "expression vector"
means a vector, plasmid or vehicle designed to enable the
expression of an inserted nucleic acid sequence following
transformation into the host. The cloned gene, i.e., the inserted
nucleic acid sequence, is usually placed under the control of
control elements such as a promoter, a minimal promoter, an
enhancer, or the like. Initiation control regions or promoters,
which are useful to drive expression of a nucleic acid in the
desired host cell are numerous and familiar to those skilled in the
art. Virtually any promoter capable of driving these genes is
suitable for the present invention including but not limited to:
viral promoters, bacterial promoters, animal promoters, mammalian
promoters, synthetic promoters, constitutive promoters, tissue
specific promoter, developmental specific promoters, inducible
promoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4,
GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI,
alkaline phosphatase promoters (useful for expression in
Saccharomyces); AOX1 promoter (useful for expression in Pichia);
.beta.-lactamase, lac, ara, tet, trp, lP.sub.L, lP.sub.R, T7, tac,
and trc promoters (useful for expression in Escherichia coli);
light regulated-, seed specific-, pollen specific-, ovary
specific-, pathogenesis or disease related-, cauliflower mosaic
virus 35S, CMV 35S minimal, cassava vein mosaic virus (CsVMV),
chlorophyll a/b binding protein, ribulose 1,5-bisphosphate
carboxylase, shoot-specific, root specific, chitinase, stress
inducible, rice tungro bacilliform virus, plant super-promoter,
potato leucine aminopeptidase, nitrate reductase, mannopine
synthase, nopaline synthase, ubiquitin, zein protein, and
anthocyanin promoters (useful for expression in plant cells);
animal and mammalian promoters known in the art include, but are
not limited to, the SV40 early (SV40e) promoter region, the
promoter contained in the 3' long terminal repeat (LTR) of Rous
sarcoma virus (RSV), the promoters of the E1A or major late
promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus
(CMV) early promoter, the herpes simplex virus (HSV) thymidine
kinase (TK) promoter, a baculovirus IE1 promoter, an elongation
factor 1 alpha (EF1) promoter, a phosphoglycerate kinase (PGK)
promoter, a ubiquitin (Ubc) promoter, an albumin promoter, the
regulatory sequences of the mouse metallothionein-L promoter and
transcriptional control regions, the ubiquitous promoters (HPRT,
vimentin, .alpha.-actin, tubulin and the like), the promoters of
the intermediate filaments (desmin, neurofilaments, keratin, GFAP,
and the like), the promoters of therapeutic genes (of the MDR, CFTR
or factor VIII type, and the like), pathogenesis or disease
related-promoters, and promoters that exhibit tissue specificity
and have been utilized in transgenic animals, such as the elastase
I gene control region which is active in pancreatic acinar cells;
insulin gene control region active in pancreatic beta cells,
immunoglobulin gene control region active in lymphoid cells, mouse
mammary tumor virus control region active in testicular, breast,
lymphoid and mast cells; albumin gene, Apo AI and Apo AII control
regions active in liver, alpha-fetoprotein gene control region
active in liver, alpha 1-antitrypsin gene control region active in
the liver, beta-globin gene control region active in myeloid cells,
myelin basic protein gene control region active in oligodendrocyte
cells in the brain, myosin light chain-2 gene control region active
in skeletal muscle, and gonadotropic releasing hormone gene control
region active in the hypothalamus, pyruvate kinase promoter, villin
promoter, promoter of the fatty acid binding intestinal protein,
promoter of the smooth muscle cell .alpha.-actin, and the like. In
addition, these expression sequences may be modified by addition of
enhancer or regulatory sequences and the like.
[0140] Enhancers that may be used in embodiments of the invention
include but are not limited to: an SV40 enhancer, a cytomegalovirus
(CMV) enhancer, an elongation factor 1 (EF1) enhancer, yeast
enhancers, viral gene enhancers, and the like.
[0141] Termination control regions, i.e., terminator or
polyadenylation sequences, may also be derived from various genes
native to the preferred hosts. Optionally, a termination site may
be unnecessary, however, it is most preferred if included. In a
preferred embodiment of the invention, the termination control
region may be comprise or be derived from a synthetic sequence,
synthetic polyadenylation signal, an SV40 late polyadenylation
signal, an SV40 polyadenylation signal, a bovine growth hormone
(BGH) polyadenylation signal, viral terminator sequences, or the
like.
[0142] The terms "3' non-coding sequences" or "3' untranslated
region (UTR)" refer to DNA sequences located downstream (3') of a
coding sequence and may comprise polyadenylation [poly(A)]
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor.
[0143] "Regulatory region" means a nucleic acid sequence that
regulates the expression of a second nucleic acid sequence. A
regulatory region may include sequences which are naturally
responsible for expressing a particular nucleic acid (a homologous
region) or may include sequences of a different origin that are
responsible for expressing different proteins or even synthetic
proteins (a heterologous region). In particular, the sequences can
be sequences of prokaryotic, eukaryotic, or viral genes or derived
sequences that stimulate or repress transcription of a gene in a
specific or non-specific manner and in an inducible or
non-inducible manner. Regulatory regions include origins of
replication, RNA splice sites, promoters, enhancers,
transcriptional termination sequences, and signal sequences which
direct the polypeptide into the secretory pathways of the target
cell.
[0144] A regulatory region from a "heterologous source" is a
regulatory region that is not naturally associated with the
expressed nucleic acid. Included among the heterologous regulatory
regions are regulatory regions from a different species, regulatory
regions from a different gene, hybrid regulatory sequences, and
regulatory sequences which do not occur in nature, but which are
designed by one having ordinary skill in the art.
[0145] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from post-transcriptional processing of the
primary transcript and is referred to as the mature RNA. "Messenger
RNA (mRNA)" refers to the RNA that is without introns and that can
be translated into protein by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to RNA transcript that includes the mRNA and so
can be translated into protein by the cell. "Antisense RNA" refers
to a RNA transcript that is complementary to all or part of a
target primary transcript or mRNA and that blocks the expression of
a target gene. The complementarity of an antisense RNA may be with
any part of the specific gene transcript, i.e., at the 5'
non-coding sequence, 3' non-coding sequence, or the coding
sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA,
or other RNA that is not translated yet has an effect on cellular
processes.
[0146] A "polypeptide" is a polymeric compound comprised of
covalently linked amino acid residues. Amino acids have the
following general structure:
##STR00005##
[0147] Amino acids are classified into seven groups on the basis of
the side chain R: (1) aliphatic side chains, (2) side chains
containing a hydroxylic (OH) group, (3) side chains containing
sulfur atoms, (4) side chains containing an acidic or amide group,
(5) side chains containing a basic group, (6) side chains
containing an aromatic ring, and (7) proline, an imino acid in
which the side chain is fused to the amino group. A polypeptide of
the invention preferably comprises at least about 14 amino
acids.
[0148] A "protein" is a polypeptide that performs a structural or
functional role in a living cell.
[0149] An "isolated polypeptide" or "isolated protein" is a
polypeptide or protein that is substantially free of those
compounds that are normally associated therewith in its natural
state (e.g., other proteins or polypeptides, nucleic acids,
carbohydrates, lipids). "Isolated" is not meant to exclude
artificial or synthetic mixtures with other compounds, or the
presence of impurities which do not interfere with biological
activity, and which may be present, for example, due to incomplete
purification, addition of stabilizers, or compounding into a
pharmaceutically acceptable preparation.
[0150] A "substitution mutant polypeptide" or a "substitution
mutant" will be understood to mean a mutant polypeptide comprising
a substitution of at least one (1) wild-type or naturally occurring
amino acid with a different amino acid relative to the wild-type or
naturally occurring polypeptide. A substitution mutant polypeptide
may comprise only one (1) wild-type or naturally occurring amino
acid substitution and may be referred to as a "point mutant" or a
"single point mutant" polypeptide. Alternatively, a substitution
mutant polypeptide may comprise a substitution of two (2) or more
wild-type or naturally occurring amino acids with 2 or more amino
acids relative to the wild-type or naturally occurring polypeptide.
According to the invention, a Group H nuclear receptor ligand
binding domain polypeptide comprising a substitution mutation
comprises a substitution of at least one (1) wild-type or naturally
occurring amino acid with a different amino acid relative to the
wild-type or naturally occurring Group H nuclear receptor ligand
binding domain polypeptide.
[0151] Wherein the substitution mutant polypeptide comprises a
substitution of two (2) or more wild-type or naturally occurring
amino acids, this substitution may comprise either an equivalent
number of wild-type or naturally occurring amino acids deleted for
the substitution, i.e., 2 wild-type or naturally occurring amino
acids replaced with 2 non-wild-type or non-naturally occurring
amino acids, or a non-equivalent number of wild-type amino acids
deleted for the substitution, i.e., 2 wild-type amino acids
replaced with 1 non-wild-type amino acid (a substitution+deletion
mutation), or 2 wild-type amino acids replaced with 3 non-wild-type
amino acids (a substitution+insertion mutation).
[0152] Substitution mutants may be described using an abbreviated
nomenclature system to indicate the amino acid residue and number
replaced within the reference polypeptide sequence and the new
substituted amino acid residue. For example, a substitution mutant
in which the twentieth (20.sup.th) amino acid residue of a
polypeptide is substituted may be abbreviated as "x20z", wherein
"x" is the amino acid to be replaced, "20" is the amino acid
residue position or number within the polypeptide, and "z" is the
new substituted amino acid. Therefore, a substitution mutant
abbreviated interchangeably as "E20A" or "Glu20Ala" indicates that
the mutant comprises an alanine residue (commonly abbreviated in
the art as "A" or "Ala") in place of the glutamic acid (commonly
abbreviated in the art as "E" or "Glu") at position 20 of the
polypeptide.
[0153] A substitution mutation may be made by any technique for
mutagenesis known in the art, including but not limited to, in
vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J.
Biol. Chem. 253: 6551; Zoller and Smith, 1984, DNA 3: 479-488;
Oliphant et al., 1986, Gene 44: 177; Hutchinson et al., 1986, Proc.
Natl. Acad. Sci. U.S.A. 83: 710), use of TAB.RTM. linkers
(Pharmacia), restriction endonuclease digestion/fragment deletion
and substitution, PCR-mediated/oligonucleotide-directed
mutagenesis, and the like. PCR-based techniques are preferred for
site-directed mutagenesis (see Higuchi, 1989, "Using PCR to
Engineer DNA", in PCR Technology: Principles and Applications for
DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp.
61-70).
[0154] "Fragment" of a polypeptide according to the invention will
be understood to mean a polypeptide whose amino acid sequence is
shorter than that of the reference polypeptide and which comprises,
over the entire portion with these reference polypeptides, an
identical amino acid sequence. Such fragments may, where
appropriate, be included in a larger polypeptide of which they are
a part. Such fragments of a polypeptide according to the invention
may have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200, 240,
or 300 amino acids.
[0155] A "variant" of a polypeptide or protein is any analogue,
fragment, derivative, or mutant which is derived from a polypeptide
or protein and which retains at least one biological property of
the polypeptide or protein. Different variants of the polypeptide
or protein may exist in nature. These variants may be allelic
variations characterized by differences in the nucleotide sequences
of the structural gene coding for the protein, or may involve
differential splicing or post-translational modification. The
skilled artisan can produce variants having single or multiple
amino acid substitutions, deletions, additions, or replacements.
These variants may include, inter alia: (a) variants in which one
or more amino acid residues are substituted with conservative or
non-conservative amino acids, (b) variants in which one or more
amino acids are added to the polypeptide or protein, (c) variants
in which one or more of the amino acids includes a substituent
group, and (d) variants in which the polypeptide or protein is
fused with another polypeptide such as serum albumin. The
techniques for obtaining these variants, including genetic
(suppressions, deletions, mutations, etc.), chemical, and enzymatic
techniques, are known to persons having ordinary skill in the art.
A variant polypeptide preferably comprises at least about 14 amino
acids.
[0156] A "heterologous protein" refers to a protein not naturally
produced in the cell.
[0157] A "mature protein" refers to a post-translationally
processed polypeptide; i.e., one from which any pre- or propeptides
present in the primary translation product have been removed.
"Precursor" protein refers to the primary product of translation of
mRNA; i.e., with pre- and propeptides still present. Pre- and
propeptides may be but are not limited to intracellular
localization signals.
[0158] The term "signal peptide" refers to an amino terminal
polypeptide preceding the secreted mature protein. The signal
peptide is cleaved from and is therefore not present in the mature
protein. Signal peptides have the function of directing and
translocating secreted proteins across cell membranes. Signal
peptide is also referred to as signal protein.
[0159] A "signal sequence" is included at the beginning of the
coding sequence of a protein to be expressed on the surface of a
cell. This sequence encodes a signal peptide, N-terminal to the
mature polypeptide, that directs the host cell to translocate the
polypeptide. The term "translocation signal sequence" is used
herein to refer to this sort of signal sequence. Translocation
signal sequences can be found associated with a variety of proteins
native to eukaryotes and prokaryotes, and are often functional in
both types of organisms.
[0160] The term "homology" refers to the percent of identity
between two polynucleotide or two polypeptide moieties. The
correspondence between the sequence from one moiety to another can
be determined by techniques known to the art. For example, homology
can be determined by a direct comparison of the sequence
information between two polypeptide molecules by aligning the
sequence information and using readily available computer programs.
Alternatively, homology can be determined by hybridization of
polynucleotides under conditions that form stable duplexes between
homologous regions, followed by digestion with
single-stranded-specific nuclease(s) and size determination of the
digested fragments.
[0161] As used herein, the term "homologous" in all its grammatical
forms and spelling variations refers to the relationship between
proteins that possess a "common evolutionary origin," including
proteins from superfamilies (e.g., the immunoglobulin superfamily)
and homologous proteins from different species (e.g., myosin light
chain, etc.) (Reeck et al., 1987, Cell 50:667). Such proteins (and
their encoding genes) have sequence homology, as reflected by their
high degree of sequence similarity. However, in common usage and in
the instant application, the term "homologous," when modified with
an adverb such as "highly," may refer to sequence similarity and
not a common evolutionary origin.
[0162] Accordingly, the term "sequence similarity" in all its
grammatical forms refers to the degree of identity or
correspondence between nucleic acid or amino acid sequences of
proteins that may or may not share a common evolutionary origin
(see Reeck et al., 1987, Cell 50: 667).
[0163] In a specific embodiment, two DNA sequences are
"substantially homologous" or "substantially similar" when at least
about 50% (preferably at least about 75%, and most preferably at
least about 90 or 95%) of the nucleotides match over the defined
length of the DNA sequences. Sequences that are substantially
homologous can be identified by comparing the sequences using
standard software available in sequence data banks, or in a
Southern hybridization experiment under, for example, stringent
conditions as defined for that particular system. Defining
appropriate hybridization conditions is within the skill of the
art. See, e.g., Sambrook et al., 1989, supra.
[0164] As used herein, "substantially similar" refers to nucleic
acid fragments wherein changes in one or more nucleotide bases
results in substitution of one or more amino acids, but do not
affect the functional properties of the protein encoded by the DNA
sequence. "Substantially similar" also refers to nucleic acid
fragments wherein changes in one or more nucleotide bases does not
affect the ability of the nucleic acid fragment to mediate
alteration of gene expression by antisense or co-suppression
technology. "Substantially similar" also refers to modifications of
the nucleic acid fragments of the instant invention such as
deletion or insertion of one or more nucleotide bases that do not
substantially affect the functional properties of the resulting
transcript. It is therefore understood that the invention
encompasses more than the specific exemplary sequences. Each of the
proposed modifications is well within the routine skill in the art,
as is determination of retention of biological activity of the
encoded products.
[0165] Moreover, the skilled artisan recognizes that substantially
similar sequences encompassed by this invention are also defined by
their ability to hybridize, under stringent conditions
(0.1.times.SSC, 0.1% SDS, 65.degree. C. and washed with
2.times.SSC, 0.1% SDS followed by 0.1.times.SSC, 0.1% SDS), with
the sequences exemplified herein. Substantially similar nucleic
acid fragments of the instant invention are those nucleic acid
fragments whose DNA sequences are at least 70% identical to the DNA
sequence of the nucleic acid fragments reported herein. Preferred
substantially nucleic acid fragments of the instant invention are
those nucleic acid fragments whose DNA sequences are at least 80%
identical to the DNA sequence of the nucleic acid fragments
reported herein. More preferred nucleic acid fragments are at least
90% identical to the DNA sequence of the nucleic acid fragments
reported herein. Even more preferred are nucleic acid fragments
that are at least 95% identical to the DNA sequence of the nucleic
acid fragments reported herein.
[0166] Two amino acid sequences are "substantially homologous" or
"substantially similar" when greater than about 40% of the amino
acids are identical, or greater than 60% are similar (functionally
identical). Preferably, the similar or homologous sequences are
identified by alignment using, for example, the GCG (Genetics
Computer Group, Program Manual for the GCG Package, Version 7,
Madison, Wis.) pileup program.
[0167] The term "corresponding to" is used herein to refer to
similar or homologous sequences, whether the exact position is
identical or different from the molecule to which the similarity or
homology is measured. A nucleic acid or amino acid sequence
alignment may include spaces. Thus, the term "corresponding to"
refers to the sequence similarity, and not the numbering of the
amino acid residues or nucleotide bases.
[0168] A "substantial portion" of an amino acid or nucleotide
sequence comprises enough of the amino acid sequence of a
polypeptide or the nucleotide sequence of a gene to putatively
identify that polypeptide or gene, either by manual evaluation of
the sequence by one skilled in the art, or by computer-automated
sequence comparison and identification using algorithms such as
BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al.,
(1993) J. Mol. Biol. 215: 403-410; see also
www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more
contiguous amino acids or thirty or more nucleotides is necessary
in order to putatively identify a polypeptide or nucleic acid
sequence as homologous to a known protein or gene. Moreover, with
respect to nucleotide sequences, gene specific oligonucleotide
probes comprising 20-30 contiguous nucleotides may be used in
sequence-dependent methods of gene identification (e.g., Southern
hybridization) and isolation (e.g., in situ hybridization of
bacterial colonies or bacteriophage plaques). In addition, short
oligonucleotides of 12-15 bases may be used as amplification
primers in PCR in order to obtain a particular nucleic acid
fragment comprising the primers. Accordingly, a "substantial
portion" of a nucleotide sequence comprises enough of the sequence
to specifically identify and/or isolate a nucleic acid fragment
comprising the sequence.
[0169] The term "percent identity", as known in the art, is a
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" and "similarity" can be readily calculated by
known methods, including but not limited to those described in:
Computational Molecular Biology (Lesk, A. M., ed.) Oxford
University Press, New York (1988); Biocomputing: Informatics and
Genome Projects (Smith, D. W., ed.) Academic Press, New York
(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M.,
and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence
Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press
(1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,
J., eds.) Stockton Press, New York (1991). Preferred methods to
determine identity are designed to give the best match between the
sequences tested. Methods to determine identity and similarity are
codified in publicly available computer programs. Sequence
alignments and percent identity calculations may be performed using
the Megalign program of the LASERGENE bioinformatics computing
suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the
sequences may be performed using the Clustal method of alignment
(Higgins and Sharp (1989) CABIOS. 5:151-153) with the default
parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default
parameters for pairwise alignments using the Clustal method may be
selected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5.
[0170] The term "sequence analysis software" refers to any computer
algorithm or software program that is useful for the analysis of
nucleotide or amino acid sequences. "Sequence analysis software"
may be commercially available or independently developed. Typical
sequence analysis software will include but is not limited to the
GCG suite of programs (Wisconsin Package Version 9.0, Genetics
Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX
(Altschul et al., J. Mol. Biol. 215:403-410 (1990), and DNASTAR
(DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA). Within
the context of this application it will be understood that where
sequence analysis software is used for analysis, that the results
of the analysis will be based on the "default values" of the
program referenced, unless otherwise specified. As used herein
"default values" will mean any set of values or parameters which
originally load with the software when first initialized.
[0171] "Synthetic genes" can be assembled from oligonucleotide
building blocks that are chemically synthesized using procedures
known to those skilled in the art. These building blocks are
ligated and annealed to form gene segments that are then
enzymatically assembled to construct the entire gene. "Chemically
synthesized", as related to a sequence of DNA, means that the
component nucleotides were assembled in vitro. Manual chemical
synthesis of DNA may be accomplished using well-established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the genes can be tailored for optimal gene expression based on
optimization of nucleotide sequence to reflect the codon bias of
the host cell. The skilled artisan appreciates the likelihood of
successful gene expression if codon usage is biased towards those
codons favored by the host. Determination of preferred codons can
be based on a survey of genes derived from the host cell where
sequence information is available.
[0172] As used herein, two or more individually operable gene
regulation systems are said to be "orthogonal" when; a) modulation
of each of the given systems by its respective ligand, at a chosen
concentration, results in a measurable change in the magnitude of
expression of the gene of that system, and b) the change is
statistically significantly different than the change in expression
of all other systems simultaneously operable in the cell, tissue,
or organism, regardless of the simultaneity or sequentially of the
actual modulation. Preferably, modulation of each individually
operable gene regulation system effects a change in gene expression
at least 2-fold greater than all other operable systems in the
cell, tissue, or organism. More preferably, the change is at least
5-fold greater. Even more preferably, the change is at least
10-fold greater. Still more preferably, the change is at least 100
fold greater. Even still more preferably, the change is at least
500-fold greater. Ideally, modulation of each of the given systems
by its respective ligand at a chosen concentration results in a
measurable change in the magnitude of expression of the gene of
that system and no measurable change in expression of all other
systems operable in the cell, tissue, or organism. In such cases
the multiple inducible gene regulation system is said to be "fully
orthogonal". The present invention is useful to search for
orthogonal ligands and orthogonal receptor-based gene expression
systems such as those described in co-pending U.S. application Ser.
No. 09/965,697, which is incorporated herein by reference in its
entirety.
[0173] The term "modulate" means the ability of a given
ligand/receptor complex to induce or suppress the transactivation
of an exogenous gene.
[0174] The term "exogenous gene" means a gene foreign to the
subject, that is, a gene which is introduced into the subject
through a transformation process, an unmutated version of an
endogenous mutated gene or a mutated version of an endogenous
unmutated gene. The method of transformation is not critical to
this invention and may be any method suitable for the subject known
to those in the art. For example, transgenic plants are obtained by
regeneration from the transformed cells. Numerous transformation
procedures are known from the literature such as agroinfection
using Agrobacterium tumefaciens or its T.sub.1 plasmid,
electroporation, microinjection of plant cells and protoplasts, and
microprojectile transformation. Complementary techniques are known
for transformation of animal cells and regeneration of such
transformed cells in transgenic animals. Exogenous genes can be
either natural or synthetic genes and therapeutic genes which are
introduced into the subject in the form of DNA or RNA which may
function through a DNA intermediate such as by reverse
transcriptase. Such genes can be introduced into target cells,
directly introduced into the subject, or indirectly introduced by
the transfer of transformed cells into the subject. The term
"therapeutic gene" means a gene which imparts a beneficial function
to the host cell in which such gene is expressed. Therapeutic genes
are not naturally found in host cells.
[0175] The term "ecdysone receptor complex" generally refers to a
heterodimeric protein complex consisting of two members of the
steroid receptor family, ecdysone receptor ("EcR") and
ultraspiracle ("USP") proteins (see Yao, T. P., et. al. (1993)
Nature 366, 476-479; Yao, T.-P., et. al., (1992) Cell 71, 63-72).
The functional ecdysteroid receptor complex may also include
additional protein(s) such as immunophilins. Additional members of
the steroid receptor family of proteins, known as transcriptional
factors (such as DHR38, betaFTZ-1 or other insect homologs), may
also be ligand dependent or independent partners for EcR and/or
USP. The ecdysone receptor complex can also be a heterodimer of
ecdysone receptor protein and the vertebrate homolog of
ultraspiracle protein, retinoic acid-X-receptor ("RXR") protein.
Homodimer complexes of the ecdysone receptor protein or USP may
also be functional under some circumstances.
[0176] An ecdysteroid receptor complex can be activated by an
active ecdysteroid or non-steroidal ligand bound to one of the
proteins of the complex, inclusive of EcR, but not excluding other
proteins of the complex.
[0177] The ecdysone receptor complex includes proteins which are
members of the steroid receptor superfamily wherein all members are
characterized by the presence of an amino-terminal transactivation
domain, a DNA binding domain ("DBD"), and a ligand binding domain
("LBD") separated by a hinge region. Some members of the family may
also have another transactivation domain on the carboxy-terminal
side of the LBD. The DBD is characterized by the presence of two
cysteine zinc fingers between which are two amino acid motifs, the
P-box and the D-box, which confer specificity for ecdysone response
elements. These domains may be either native, modified, or chimeras
of different domains of heterologous receptor proteins.
[0178] The DNA sequences making up the exogenous gene, the response
element, and the ecdysone receptor complex may be incorporated into
archaebacteria, procaryotic cells such as Escherichia coli,
Bacillus subtilis, or other enterobacteria, or eucaryotic cells
such as plant or animal cells. However, because many of the
proteins expressed by the gene are processed incorrectly in
bacteria, eucaryotic cells are preferred. The cells may be in the
form of single cells or multicellular organisms. The nucleotide
sequences for the exogenous gene, the response element, and the
receptor complex can also be incorporated as RNA molecules,
preferably in the form of functional viral RNAs such as tobacco
mosaic virus. Of the eucaryotic cells, vertebrate cells are
preferred because they naturally lack the molecules which confer
responses to the ligands of this invention for the ecdysone
receptor. As a result, they are insensitive to the ligands of this
invention. Thus, the ligands of this invention will have negligible
physiological or other effects on untransformed cells, or the whole
organism.
[0179] The term "subject" means an intact plant or animal or a cell
from a plant or animal. It is also anticipated that the ligands
will work equally well when the subject is a fungus or yeast. When
the subject is an intact animal, preferably the animal is a
vertebrate, most preferably a mammal.
[0180] The ligands of the present invention, when used with the
ecdysone receptor complex which in turn is bound to the response
element linked to an exogenous gene, provide the means for external
temporal regulation of expression of the exogenous gene. The order
in which the various components bind to each other, that is, ligand
to receptor complex and receptor complex to response element, is
not critical. Typically, modulation of expression of the exogenous
gene is in response to the binding of the ecdysone receptor complex
to a specific control, or regulatory, DNA element. The ecdysone
receptor protein, like other members of the steroid receptor
family, possesses at least three domains, a transactivation domain,
a DNA binding domain, and a ligand binding domain. This receptor,
like a subset of the steroid receptor family, also possesses less
well-defined regions responsible for heterodimerization properties.
Binding of the ligand to the ligand binding domain of ecdysone
receptor protein, after heterodimerization with USP or RXR protein,
enables the DNA binding domains of the heterodimeric proteins to
bind to the response element in an activated form, thus resulting
in expression or suppression of the exogenous gene. This mechanism
does not exclude the potential for ligand binding to either EcR or
USP, and the resulting formation of active homodimer complexes
(e.g. EcR+EcR or USP+USP). Preferably, one or more of the receptor
domains can be varied producing a chimeric gene switch. Typically,
one or more of the three domains may be chosen from a source
different than the source of the other domains so that the chimeric
receptor is optimized in the chosen host cell or organism for
transactivating activity, complementary binding of the ligand, and
recognition of a specific response element. In addition, the
response element itself can be modified or substituted with
response elements for other DNA binding protein domains such as the
GAL-4 protein from yeast (see Sadowski, et. al. (1988) Nature, 335,
563-564) or LexA protein from E. coli (see Brent and Ptashne
(1985), Cell, 43, 729-736) to accommodate chimeric ecdysone
receptor complexes. Another advantage of chimeric systems is that
they allow choice of a promoter used to drive the exogenous gene
according to a desired end result. Such double control can be
particularly important in areas of gene therapy, especially when
cytotoxic proteins are produced, because both the timing of
expression as well as the cells wherein expression occurs can be
controlled. The term "promoter" means a specific nucleotide
sequence recognized by RNA polymerase. The sequence is the site at
which transcription can be specifically initiated under proper
conditions. When exogenous genes, operatively linked to a suitable
promoter, are introduced into the cells of the subject, expression
of the exogenous genes is controlled by the presence of the ligand
of this invention. Promoters may be constitutively or inducibly
regulated or may be tissue-specific (that is, expressed only in a
particular type of cell) or specific to certain developmental
stages of the organism.
[0181] Another aspect of this invention is a method to modulate the
expression of one or more exogenous genes in a subject, comprising
administering to the subject an effective amount, that is, the
amount required to elicit the desired gene expression or
suppression, of a ligand comprising a compound of the present
invention and wherein the cells of the subject contain: [0182] a)
an ecdysone receptor complex comprising: [0183] 1) a DNA binding
domain; [0184] 2) a binding domain for the ligand; and [0185] 3) a
transactivation domain; and [0186] b) a DNA construct comprising:
[0187] 1) the exogenous gene; and [0188] 2) 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.
[0189] A related aspect of this invention is a method for
regulating endogenous or heterologous gene expression in a
transgenic subject comprising contacting a ligand comprising a
compound of the present invention with an ecdysone receptor within
the cells of the subject wherein the cells contain a DNA binding
sequence for the ecdysone receptor and wherein formation of an
ecdysone receptor-ligand-DNA binding sequence complex induces
expression of the gene.
[0190] Another aspect of the present invention is a method for
producing a polypeptide comprising the steps of:
[0191] a) selecting a cell which is substantially insensitive to
exposure to a ligand comprising a compound of the present
invention;
[0192] b) introducing into the cell: [0193] 1) a DNA construct
comprising: [0194] i) an exogenous gene encoding the polypeptide;
and [0195] ii) a response element; wherein the gene is under the
control of the response element; and [0196] 2) an ecdysone receptor
complex comprising: [0197] i) a DNA binding domain; [0198] ii) a
binding domain for the ligand; and [0199] iii) a transactivation
domain; and
[0200] c) exposing the cell to the ligand.
[0201] As well as the advantage of temporally controlling
polypeptide production by the cell, this aspect of the invention
provides a further advantage, in those cases when accumulation of
such a polypeptide can damage the cell, in that expression of the
polypeptide may be limited to short periods. Such control is
particularly important when the exogenous gene is a therapeutic
gene. Therapeutic genes may be called upon to produce polypeptides
which control needed functions, such as the production of insulin
in diabetic patients. They may also be used to produce damaging or
even lethal proteins, such as those lethal to cancer cells. Such
control may also be important when the protein levels produced may
constitute a metabolic drain on growth or reproduction, such as in
transgenic plants.
[0202] Numerous genomic and cDNA nucleic acid sequences coding for
a variety of polypeptides are well known in the art. Exogenous
genetic material useful with the ligands of this invention include
genes that encode biologically active proteins of interest, such
as, for example, secretory proteins that can be released from a
cell; enzymes that can metabolize a substrate from a toxic
substance to a non-toxic substance, or from an inactive substance
to an active substance; regulatory proteins; cell surface
receptors; and the like. Useful genes also include genes that
encode blood clotting factors, hormones such as insulin,
parathyroid hormone, luteinizing hormone releasing factor, alpha
and beta seminal inhibins, and human growth hormone; genes that
encode proteins such as enzymes, the absence of which leads to the
occurrence of an abnormal state; genes encoding cytokines or
lymphokines such as interferons, granulocytic macrophage colony
stimulating factor, colony stimulating factor-1, tumor necrosis
factor, and erythropoietin; genes encoding inhibitor substances
such as alpha.sub.1-antitrypsin, genes encoding substances that
function as drugs such as diphtheria and cholera toxins; and the
like. Useful genes also include those useful for cancer therapies
and to treat genetic disorders. Those skilled in the art have
access to nucleic acid sequence information for virtually all known
genes and can either obtain the nucleic acid molecule directly from
a public depository, the institution that published the sequence,
or employ routine methods to prepare the molecule.
[0203] For gene therapy use, the ligands described herein may be
taken up in pharmaceutically acceptable carriers, such as, for
example, solutions, suspensions, tablets, capsules, ointments,
elixirs, and injectable compositions. Pharmaceutical preparations
may contain from 0.01% to 99% by weight of the ligand. Preparations
may be either in single or multiple dose forms. The amount of
ligand in any particular pharmaceutical preparation will depend
upon the effective dose, that is, the dose required to elicit the
desired gene expression or suppression.
[0204] Suitable routes of administering the pharmaceutical
preparations include oral, rectal, topical (including dermal,
buccal and sublingual), vaginal, parenteral (including
subcutaneous, intramuscular, intravenous, intradermal, intrathecal
and epidural) and by naso-gastric tube. It will be understood by
those skilled in the art that the preferred route of administration
will depend upon the condition being treated and may vary with
factors such as the condition of the recipient.
[0205] The ligands described herein may also be administered in
conjunction with other pharmaceutically active compounds. It will
be understood by those skilled in the art that pharmaceutically
active compounds to be used in combination with the ligands
described herein will be selected in order to avoid adverse effects
on the recipient or undesirable interactions between the compounds.
Examples of other pharmaceutically active compounds which may be
used in combination with the ligands include, for example, AIDS
chemotherapeutic agents, amino acid derivatives, analgesics,
anesthetics, anorectal products, antacids and antiflatulents,
antibiotics, anticoagulants, antidotes, antifibrinolytic agents,
antihistamines, anti-inflamatory agents, antineoplastics,
antiparasitics, antiprotozoals, antipyretics, antiseptics,
antispasmodics and anticholinergics, antivirals, appetite
suppressants, arthritis medications, biological response modifiers,
bone metabolism regulators, bowel evacuants, cardiovascular agents,
central nervous system stimulants, cerebral metabolic enhancers,
cerumenolytics, cholinesterase inhibitors, cold and cough
preparations, colony stimulating factors, contraceptives,
cytoprotective agents, dental preparations, deodorants,
dermatologicals, detoxifying agents, diabetes agents, diagnostics,
diarrhea medications, dopamine receptor agonists, electrolytes,
enzymes and digestants, ergot preparations, fertility agents, fiber
supplements, antifungal agents, galactorrhea inhibitors, gastric
acid secretion inhibitors, gastrointestinal prokinetic agents,
gonadotropin inhibitors, hair growth stimulants, hematinics,
hemorrheologic agents, hemostatics, histamine H.sub.2 receptor
antagonists, hormones, hyperglycemic agents, hypolipidemics,
immunosuppressants, laxatives, leprostatics, leukapheresis
adjuncts, lung surfactants, migraine preparations, mucolytics,
muscle relaxant antagonists, muscle relaxants, narcotic
antagonists, nasal sprays, nausea medications nucleoside analogues,
nutritional supplements, osteoporosis preparations, oxytocics,
parasympatholytics, parasympathomimetics, Parkinsonism drugs,
Penicillin adjuvants, phospholipids, platelet inhibitors, porphyria
agents, prostaglandin analogues, prostaglandins, proton pump
inhibitors, pruritus medications psychotropics, quinolones,
respiratory stimulants, saliva stimulants, salt substitutes,
sclerosing agents, skin wound preparations, smoking cessation aids,
sulfonamides, sympatholytics, thrombolytics, Tourette's syndrome
agents, tremor preparations, tuberculosis preparations, uricosuric
agents, urinary tract agents, uterine contractants, uterine
relaxants, vaginal preparations, vertigo agents, vitamin D analogs,
vitamins, and medical imaging contrast media. In some cases the
ligands may be useful as an adjunct to drug therapy, for example,
to "turn off" a gene that produces an enzyme that metabolizes a
particular drug.
[0206] For agricultural applications, in addition to the
applications described above, the ligands of this invention may
also be used to control the expression of pesticidal proteins such
as Bacillus thuringiensis (Bt) toxin. Such expression may be tissue
or plant specific. In addition, particularly when control of plant
pests is also needed, one or more pesticides may be combined with
the ligands described herein, thereby providing additional
advantages and effectiveness, including fewer total applications,
than if the pesticides are applied separately. When mixtures with
pesticides are employed, the relative proportions of each component
in the composition will depend upon the relative efficacy and the
desired application rate of each pesticide with respect to the
crops, pests, and/or weeds to be treated. Those skilled in the art
will recognize that mixtures of pesticides may provide advantages
such as a broader spectrum of activity than one pesticide used
alone. Examples of pesticides which can be combined in compositions
with the ligands described herein include fungicides, herbicides,
insecticides, miticides, and microbicides.
[0207] The ligands described herein can be applied to plant foliage
as aqueous sprays by methods commonly employed, such as
conventional high-liter hydraulic sprays, low-liter sprays,
air-blast, and aerial sprays. The dilution and rate of application
will depend upon the type of equipment employed, the method and
frequency of application desired, and the ligand application rate.
It may be desirable to include additional adjuvants in the spray
tank. Such adjuvants include surfactants, dispersants, spreaders,
stickers, antifoam agents, emulsifiers, and other similar materials
described in McCutcheon's Emulsifiers and Detergents, McCutcheon's
Emulsifiers and Detergents/Functional Materials, and McCutcheon's
Functional Materials, all published annually by McCutcheon Division
of MC Publishing Company (New Jersey). The ligands can also be
mixed with fertilizers or fertilizing materials before their
application. The ligands and solid fertilizing material can also be
admixed in mixing or blending equipment, or they can be
incorporated with fertilizers in granular formulations. Any
relative proportion of fertilizer can be used which is suitable for
the crops and weeds to be treated. The ligands described herein
will commonly comprise from 5% to 50% of the fertilizing
composition. These compositions provide fertilizing materials which
promote the rapid growth of desired plants, and at the same time
control gene expression.
Host Cells and Non-Human Organisms of the Invention
[0208] As described above, ligands for modulating gene expression
system of the present invention may be used to modulate gene
expression in a host cell. Expression in transgenic host cells may
be useful for the expression of various genes of interest. The
present invention provides ligands for modulation of gene
expression in prokaryotic and eukaryotic host cells. Expression in
transgenic host cells is useful for the expression of various
polypeptides of interest including but not limited to antigens
produced in plants as vaccines, enzymes like alpha-amylase,
phytase, glucanes, and xylanse, genes for resistance against
insects, nematodes, fungi, bacteria, viruses, and abiotic stresses,
antigens, nutraceuticals, pharmaceuticals, vitamins, genes for
modifying amino acid content, herbicide resistance, cold, drought,
and heat tolerance, industrial products, oils, protein,
carbohydrates, antioxidants, male sterile plants, flowers, fuels,
other output traits, therapeutic polypeptides, pathway
intermediates; for the modulation of pathways already existing in
the host for the synthesis of new products heretofore not possible
using the host; cell based assays; functional genomics assays,
biotherapeutic protein production, proteomics assays, and the like.
Additionally the gene products may be useful for conferring higher
growth yields of the host or for enabling an alternative growth
mode to be utilized.
[0209] Thus, the present invention provides ligands for modulating
gene expression in an isolated host cell according to the
invention. The host cell may be a bacterial cell, a fungal cell, a
nematode cell, an insect cell, a fish cell, a plant cell, an avian
cell, an animal cell, or a mammalian cell. In still another
embodiment, the invention relates to ligands for modulating gene
expression in an host cell, wherein the method comprises culturing
the host cell as described above in culture medium under conditions
permitting expression of a polynucleotide encoding the nuclear
receptor ligand binding domain comprising a substitution mutation,
and isolating the nuclear receptor ligand binding domain comprising
a substitution mutation from the culture.
[0210] In a specific embodiment, the isolated host cell is a
prokaryotic host cell or a eukaryotic host cell. In another
specific embodiment, the isolated host cell is an invertebrate host
cell or a vertebrate host cell. Preferably, the host cell is
selected from the group consisting of a bacterial cell, a fungal
cell, a yeast cell, a nematode cell, an insect cell, a fish cell, a
plant cell, an avian cell, an animal cell, and a mammalian cell.
More preferably, the host cell is a yeast cell, a nematode cell, an
insect cell, a plant cell, a zebrafish cell, a chicken cell, a
hamster cell, a mouse cell, a rat cell, a rabbit cell, a cat cell,
a dog cell, a bovine cell, a goat cell, a cow cell, a pig cell, a
horse cell, a sheep cell, a simian cell, a monkey cell, a
chimpanzee cell, or a human cell. Examples of preferred host cells
include, but are not limited to, fungal or yeast species such as
Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida,
Hansenula, or bacterial species such as those in the genera
Synechocystis, Synechococcus, Salmonella, Bacillus, Acinetobacter,
Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas,
Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus,
Methanobacterium and Klebsiella; plant species selected from the
group consisting of an apple, Arabidopsis, bajra, banana, barley,
beans, beet, blackgram, chickpea, chili, cucumber, eggplant,
favabean, maize, melon, millet, mungbean, oat, okra, Panicum,
papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus,
potato, pumpkin, rice, sorghum, soybean, squash, sugarcane,
sugarbeet, sunflower, sweet potato, tea, tomato, tobacco,
watermelon, and wheat; animal; and mammalian host cells.
[0211] In a specific embodiment, the host cell is a yeast cell
selected from the group consisting of a Saccharomyces, a Pichia,
and a Candida host cell.
[0212] In another specific embodiment, the host cell is a
Caenorhabdus elegans nematode cell.
[0213] In another specific embodiment, the host cell is an insect
cell.
[0214] In another specific embodiment, the host cell is a plant
cell selected from the group consisting of an apple, Arabidopsis,
bajra, banana, barley, beans, beet, blackgram, chickpea, chili,
cucumber, eggplant, favabean, maize, melon, millet, mungbean, oat,
okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple,
Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash,
sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato,
tobacco, watermelon, and wheat cell.
[0215] In another specific embodiment, the host cell is a zebrafish
cell.
[0216] In another specific embodiment, the host cell is a chicken
cell.
[0217] In another specific embodiment, the host cell is a mammalian
cell selected from the group consisting of a hamster cell, a mouse
cell, a rat cell, a rabbit cell, a cat cell, a dog cell, a bovine
cell, a goat cell, a cow cell, a pig cell, a horse cell, a sheep
cell, a monkey cell, a chimpanzee cell, and a human cell.
[0218] Host cell transformation is well known in the art and may be
achieved by a variety of methods including but not limited to
electroporation, viral infection, plasmid/vector transfection,
non-viral vector mediated transfection, Agrobacterium-mediated
transformation, particle bombardment, and the like. Expression of
desired gene products involves culturing the transformed host cells
under suitable conditions and inducing expression of the
transformed gene. Culture conditions and gene expression protocols
in prokaryotic and eukaryotic cells are well known in the art (see
General Methods section of Examples). Cells may be harvested and
the gene products isolated according to protocols specific for the
gene product.
[0219] In addition, a host cell may be chosen which modulates the
expression of the inserted polynucleotide, or modifies and
processes the polypeptide product in the specific fashion desired.
Different host cells have characteristic and specific mechanisms
for the translational and post-translational processing and
modification [e.g., glycosylation, cleavage (e.g., of signal
sequence)] of proteins. Appropriate cell lines or host systems can
be chosen to ensure the desired modification and processing of the
foreign protein expressed. For example, expression in a bacterial
system can be used to produce a non-glycosylated core protein
product. However, a polypeptide expressed in bacteria may not be
properly folded. Expression in yeast can produce a glycosylated
product. Expression in eukaryotic cells can increase the likelihood
of "native" glycosylation and folding of a heterologous protein.
Moreover, expression in mammalian cells can provide a tool for
reconstituting, or constituting, the polypeptide's activity.
Furthermore, different vector/host expression systems may affect
processing reactions, such as proteolytic cleavages, to a different
extent. The present invention also relates to a non-human organism
comprising an isolated host cell according to the invention. In a
specific embodiment, the non-human organism is a prokaryotic
organism or a eukaryotic organism. In another specific embodiment,
the non-human organism is an invertebrate organism or a vertebrate
organism.
[0220] Preferably, the non-human organism is selected from the
group consisting of a bacterium, a fungus, a yeast, a nematode, an
insect, a fish, a plant, a bird, an animal, and a mammal More
preferably, the non-human organism is a yeast, a nematode, an
insect, a plant, a zebrafish, a chicken, a hamster, a mouse, a rat,
a rabbit, a cat, a dog, a bovine, a goat, a cow, a pig, a horse, a
sheep, a simian, a monkey, or a chimpanzee.
[0221] In a specific embodiment, the non-human organism is a yeast
selected from the group consisting of Saccharomyces, Pichia, and
Candida.
[0222] In another specific embodiment, the non-human organism is a
Caenorhabdus elegans nematode.
[0223] In another specific embodiment, the non-human organism is a
plant selected from the group consisting of an apple, Arabidopsis,
bajra, banana, barley, beans, beet, blackgram, chickpea, chili,
cucumber, eggplant, favabean, maize, melon, millet, mungbean, oat,
okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple,
Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash,
sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato,
tobacco, watermelon, and wheat.
[0224] In another specific embodiment, the non-human organism is a
Mus musculus mouse.
Gene Expression Modulation System of the Invention
[0225] The present invention relates to a group of ligands that are
useful in an ecdysone receptor-based inducible gene expression
system. As presented herein, a novel group of ligands provides an
improved inducible gene expression system in both prokaryotic and
eukaryotic host cells. Thus, the present invention relates to
ligands that are useful to modulate expression of genes. In
particular, the present invention relates to ligands having the
ability to transactivate a gene expression modulation system
comprising at least one gene expression cassette that is capable of
being expressed in a host cell comprising a polynucleotide that
encodes a polypeptide comprising a Group H nuclear receptor ligand
binding domain. Preferably, the Group H nuclear receptor ligand
binding is from an ecdysone receptor, a ubiquitous receptor, an
orphan receptor 1, a NER-1, a steroid hormone nuclear receptor 1, a
retinoid X receptor interacting protein-15, a liver X receptor 13,
a steroid hormone receptor like protein, a liver X receptor, a
liver X receptor .alpha., a farnesoid X receptor, a receptor
interacting protein 14, and a farnesol receptor. More preferably,
the Group H nuclear receptor ligand binding domain is from an
ecdysone receptor.
[0226] In a specific embodiment, the gene expression modulation
system comprises a gene expression cassette comprising a
polynucleotide that encodes a polypeptide comprising a
transactivation domain, a DNA-binding domain that recognizes a
response element associated with a gene whose expression is to be
modulated; and a Group H nuclear receptor ligand binding domain
comprising a substitution mutation. The gene expression modulation
system may further comprise a second gene expression cassette
comprising: i) a response element recognized by the DNA-binding
domain of the encoded polypeptide of the first gene expression
cassette; ii) a promoter that is activated by the transactivation
domain of the encoded polypeptide of the first gene expression
cassette; and iii) a gene whose expression is to be modulated.
[0227] In another specific embodiment, the gene expression
modulation system comprises a gene expression cassette comprising
a) a polynucleotide that encodes a polypeptide comprising a
transactivation domain, a DNA-binding domain that recognizes a
response element associated with a gene whose expression is to be
modulated; and a Group H nuclear receptor ligand binding domain
comprising a substitution mutation, and b) a second nuclear
receptor ligand binding domain selected from the group consisting
of a vertebrate retinoid X receptor ligand binding domain, an
invertebrate retinoid X receptor ligand binding domain, an
ultraspiracle protein ligand binding domain, and a chimeric ligand
binding domain comprising two polypeptide fragments, wherein the
first polypeptide fragment is from a vertebrate retinoid X receptor
ligand binding domain, an invertebrate retinoid X receptor ligand
binding domain, or an ultraspiracle protein ligand binding domain,
and the second polypeptide fragment is from a different vertebrate
retinoid X receptor ligand binding domain, invertebrate retinoid X
receptor ligand binding domain, or ultraspiracle protein ligand
binding domain. The gene expression modulation system may further
comprise a second gene expression cassette comprising: i) a
response element recognized by the DNA-binding domain of the
encoded polypeptide of the first gene expression cassette; ii) a
promoter that is activated by the transactivation domain of the
encoded polypeptide of the first gene expression cassette; and iii)
a gene whose expression is to be modulated.
[0228] In another specific embodiment, the gene expression
modulation system comprises a first gene expression cassette
comprising a polynucleotide that encodes a first polypeptide
comprising a DNA-binding domain that recognizes a response element
associated with a gene whose expression is to be modulated and a
nuclear receptor ligand binding domain, and a second gene
expression cassette comprising a polynucleotide that encodes a
second polypeptide comprising a transactivation domain and a
nuclear receptor ligand binding domain, wherein one of the nuclear
receptor ligand binding domains is a Group H nuclear receptor
ligand binding domain comprising a substitution mutation. In a
preferred embodiment, the first polypeptide is substantially free
of a transactivation domain and the second polypeptide is
substantially free of a DNA binding domain. For purposes of the
invention, "substantially free" means that the protein in question
does not contain a sufficient sequence of the domain in question to
provide activation or binding activity. The gene expression
modulation system may further comprise a third gene expression
cassette comprising: i) a response element recognized by the
DNA-binding domain of the first polypeptide of the first gene
expression cassette; ii) a promoter that is activated by the
transactivation domain of the second polypeptide of the second gene
expression cassette; and iii) a gene whose expression is to be
modulated.
[0229] Wherein when only one nuclear receptor ligand binding domain
is a Group H ligand binding domain comprising a substitution
mutation, the other nuclear receptor ligand binding domain may be
from any other nuclear receptor that forms a dimer with the Group H
ligand binding domain comprising the substitution mutation. For
example, when the Group H nuclear receptor ligand binding domain
comprising a substitution mutation is an ecdysone receptor ligand
binding domain comprising a substitution mutation, the other
nuclear receptor ligand binding domain ("partner") may be from an
ecdysone receptor, a vertebrate retinoid X receptor (RXR), an
invertebrate RXR, an ultraspiracle protein (USP), or a chimeric
nuclear receptor comprising at least two different nuclear receptor
ligand binding domain polypeptide fragments selected from the group
consisting of a vertebrate RXR, an invertebrate RXR, and a USP (see
co-pending applications PCT/US01/09050, PCT/US02/05235, and
PCT/US02/05706, incorporated herein by reference in their
entirety). The "partner" nuclear receptor ligand binding domain may
further comprise a truncation mutation, a deletion mutation, a
substitution mutation, or another modification.
[0230] Preferably, the vertebrate RXR ligand binding domain is from
a human Homo sapiens, mouse Mus musculus, rat Rattus norvegicus,
chicken Gallus gallus, pig Sus scrofa domestica, frog Xenopus
laevis, zebrafish Danio rerio, tunicate Polyandrocarpa misakiensis,
or jellyfish Tripedalia cysophora RXR.
[0231] Preferably, the invertebrate RXR ligand binding domain is
from a locust Locusta migratoria ultraspiracle polypeptide
("LmUSP"), an ixodid tick Amblyomma americanum RXR homolog 1
("AmaRXR1"), a ixodid tick Amblyomma americanum RXR homolog 2
("AmaRXR2"), a fiddler crab Celuca pugilator RXR homolog ("CpRXR"),
a beetle Tenebrio molitor RXR homolog ("TmRXR"), a honeybee Apis
mellifera RXR homolog ("AmRXR"), an aphid Myzus persicae RXR
homolog ("MpRXR"), or a non-Dipteran/non-Lepidopteran RXR
homolog.
[0232] Preferably, the chimeric RXR ligand binding domain comprises
at least two polypeptide fragments selected from the group
consisting of a vertebrate species RXR polypeptide fragment, an
invertebrate species RXR polypeptide fragment, and a
non-Dipteran/non-Lepidopteran invertebrate species RXR homolog
polypeptide fragment. A chimeric RXR ligand binding domain for use
in the present invention may comprise at least two different
species RXR polypeptide fragments, or when the species is the same,
the two or more polypeptide fragments may be from two or more
different isoforms of the species RXR polypeptide fragment.
[0233] In a preferred embodiment, the chimeric RXR ligand binding
domain comprises at least one vertebrate species RXR polypeptide
fragment and one invertebrate species RXR polypeptide fragment.
[0234] In a more preferred embodiment, the chimeric RXR ligand
binding domain comprises at least one vertebrate species RXR
polypeptide fragment and one non-Dipteran/non-Lepidopteran
invertebrate species RXR homolog polypeptide fragment.
[0235] In a specific embodiment, the gene whose expression is to be
modulated is a homologous gene with respect to the host cell. In
another specific embodiment, the gene whose expression is to be
modulated is a heterologous gene with respect to the host cell.
[0236] The ligands for use in the present invention as described
below, when combined with the ligand binding domain of the nuclear
receptor(s), which in turn are bound to the response element linked
to a gene, provide the means for external temporal regulation of
expression of the gene. The binding mechanism or the order in which
the various components of this invention bind to each other, that
is, for example, ligand to ligand binding domain, DNA-binding
domain to response element, transactivation domain to promoter,
etc., is not critical.
[0237] In a specific example, binding of the ligand to the ligand
binding domain of a Group H nuclear receptor and its nuclear
receptor ligand binding domain partner enables expression or
suppression of the gene. This mechanism does not exclude the
potential for ligand binding to the Group H nuclear receptor (GHNR)
or its partner, and the resulting formation of active homodimer
complexes (e.g. GHNR+GHNR or partner+partner). Preferably, one or
more of the receptor domains is varied producing a hybrid gene
switch. Typically, one or more of the three domains, DBD, LBD, and
transactivation domain, may be chosen from a source different than
the source of the other domains so that the hybrid genes and the
resulting hybrid proteins are optimized in the chosen host cell or
organism for transactivating activity, complementary binding of the
ligand, and recognition of a specific response element. In
addition, the response element itself can be modified or
substituted with response elements for other DNA binding protein
domains such as the GAL-4 protein from yeast (see Sadowski, et al.
(1988) Nature, 335: 563-564) or LexA protein from Escherichia coli
(see Brent and Ptashne (1985), Cell, 43: 729-736), or synthetic
response elements specific for targeted interactions with proteins
designed, modified, and selected for such specific interactions
(see, for example, Kim, et al. (1997), Proc. Nati. Acad. Sci., USA,
94: 3616-3620) to accommodate hybrid receptors. Another advantage
of two-hybrid systems is that they allow choice of a promoter used
to drive the gene expression according to a desired end result.
Such double control can be particularly important in areas of gene
therapy, especially when cytotoxic proteins are produced, because
both the timing of expression as well as the cells wherein
expression occurs can be controlled. When genes, operably linked to
a suitable promoter, are introduced into the cells of the subject,
expression of the exogenous genes is controlled by the presence of
the system of this invention. Promoters may be constitutively or
inducibly regulated or may be tissue-specific (that is, expressed
only in a particular type of cells) or specific to certain
developmental stages of the organism.
[0238] The ecdysone receptor is a member of the nuclear receptor
superfamily and classified into subfamily 1, group H (referred to
herein as "Group H nuclear receptors"). The members of each group
share 40-60% amino acid identity in the E (ligand binding) domain
(Laudet et al., A Unified Nomenclature System for the Nuclear
Receptor Subfamily, 1999; Cell 97: 161-163). In addition to the
ecdysone receptor, other members of this nuclear receptor subfamily
1, group H include: ubiquitous receptor (UR), orphan receptor 1
(OR-1), steroid hormone nuclear receptor 1 (NER-1), retinoid X
receptor interacting protein 15 (RIP-15), liver X receptor .beta.
(LXR.beta.), steroid hormone receptor like protein (RLD-1), liver X
receptor (LXR), liver X receptor .alpha. (LXR.alpha.), farnesoid X
receptor (FXR), receptor interacting protein 14 (RIP-14), and
farnesol receptor (HRR-1
[0239] In particular, described herein are novel ligands useful in
a gene expression modulation system comprising a Group H nuclear
receptor ligand binding domain comprising a substitution mutation.
This gene expression system may be a "single switch"-based gene
expression system in which the transactivation domain, DNA-binding
domain and ligand binding domain are on one encoded polypeptide.
Alternatively, the gene expression modulation system may be a "dual
switch"- or "two-hybrid"-based gene expression modulation system in
which the transactivation domain and DNA-binding domain are located
on two different encoded polypeptides.
[0240] An ecdysone receptor-based gene expression modulation system
of the present invention may be either heterodimeric or
homodimeric. A functional EcR complex generally refers to a
heterodimeric protein complex consisting of two members of the
steroid receptor family, an ecdysone receptor protein obtained from
various insects, and an ultraspiracle (USP) protein or the
vertebrate homolog of USP, retinoid X receptor protein (see Yao, et
al. (1993) Nature 366, 476-479; Yao, et al., (1992) Cell 71,
63-72). However, the complex may also be a homodimer as detailed
below. The functional ecdysteroid receptor complex may also include
additional protein(s) such as immunophilins. Additional members of
the steroid receptor family of proteins, known as transcriptional
factors (such as DHR38 or betaFTZ-1), may also be ligand dependent
or independent partners for EcR, USP, and/or RXR. Additionally,
other cofactors may be required such as proteins generally known as
coactivators (also termed adapters or mediators). These proteins do
not bind sequence-specifically to DNA and are not involved in basal
transcription. They may exert their effect on transcription
activation through various mechanisms, including stimulation of
DNA-binding of activators, by affecting chromatin structure, or by
mediating activator-initiation complex interactions. Examples of
such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70,
SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the
promiscuous coactivator C response element B binding protein,
CBP/p300 (for review see Glass et al., Curr. Opin. Cell Biol.
9:222-232, 1997). Also, protein cofactors generally known as
corepressors (also known as repressors, silencers, or silencing
mediators) may be required to effectively inhibit transcriptional
activation in the absence of ligand. These corepressors may
interact with the unliganded ecdysone receptor to silence the
activity at the response element. Current evidence suggests that
the binding of ligand changes the conformation of the receptor,
which results in release of the corepressor and recruitment of the
above described coactivators, thereby abolishing their silencing
activity. Examples of corepressors include N--CoR and SMRT (for
review, see Horwitz et al. Mol Endocrinol. 10: 1167-1177, 1996).
These cofactors may either be endogenous within the cell or
organism, or may be added exogenously as transgenes to be expressed
in either a regulated or unregulated fashion. Homodimer complexes
of the ecdysone receptor protein, USP, or RXR may also be
functional under some circumstances.
[0241] The ecdysone receptor complex typically includes proteins
that are members of the nuclear receptor superfamily wherein all
members are generally characterized by the presence of an
amino-terminal transactivation domain, a DNA binding domain
("DBD"), and a ligand binding domain ("LBD") separated from the DBD
by a hinge region. As used herein, the term "DNA binding domain"
comprises a minimal polypeptide sequence of a DNA binding protein,
up to the entire length of a DNA binding protein, so long as the
DNA binding domain functions to associate with a particular
response element. Members of the nuclear receptor superfamily are
also characterized by the presence of four or five domains: A/B, C,
D, E, and in some members F (see U.S. Pat. No. 4,981,784 and Evans,
Science 240:889-895 (1988)). The "A/B" domain corresponds to the
transactivation domain, "C" corresponds to the DNA binding domain,
"D" corresponds to the hinge region, and "E" corresponds to the
ligand binding domain. Some members of the family may also have
another transactivation domain on the carboxy-terminal side of the
LBD corresponding to "F".
[0242] The DBD is characterized by the presence of two cysteine
zinc fingers between which are two amino acid motifs, the P-box and
the D-box, which confer specificity for ecdysone response elements.
These domains may be either native, modified, or chimeras of
different domains of heterologous receptor proteins. The EcR
receptor, like a subset of the steroid receptor family, also
possesses less well-defined regions responsible for
heterodimerization properties. Because the domains of nuclear
receptors are modular in nature, the LBD, DBD, and transactivation
domains may be interchanged.
[0243] Gene switch systems are known that incorporate components
from the ecdysone receptor complex. However, in these known
systems, whenever EcR is used it is associated with native or
modified DNA binding domains and transactivation domains on the
same molecule. USP or RXR are typically used as silent partners. It
has previously been shown that when DNA binding domains and
transactivation domains are on the same molecule the background
activity in the absence of ligand is high and that such activity is
dramatically reduced when DNA binding domains and transactivation
domains are on different molecules, that is, on each of two
partners of a heterodimeric or homodimeric complex (see
PCT/US01/09050).
Method of Modulating Gene Expression of the Invention
[0244] The present invention also relates to methods of modulating
gene expression in a host cell using a gene expression modulation
system according to the invention. Specifically, the present
invention provides 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 according to the
invention; and b) introducing into the host cell a ligand; wherein
the gene to be modulated is a component of a gene expression
cassette comprising: i) a response element comprising a domain
recognized by the DNA binding domain of the gene expression system;
ii) a promoter that is activated by the transactivation domain of
the gene expression system; and iii) a gene whose expression is to
be modulated, whereby upon introduction of the ligand into the host
cell, expression of the gene is modulated.
[0245] The invention also provides 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
according to the invention; b) introducing into the host cell a
gene expression cassette according to the invention, wherein the
gene expression cassette comprises i) a response element comprising
a domain recognized by the DNA binding domain from the gene
expression system; ii) a promoter that is activated by the
transactivation domain of the gene expression system; and iii) a
gene whose expression is to be modulated; and c) introducing into
the host cell a ligand; whereby upon introduction of the ligand
into the host cell, expression of the gene is modulated.
[0246] The present invention also provides a method of modulating
the expression of a gene in a host cell comprising a gene
expression cassette comprising a response element comprising a
domain to which the DNA binding domain from the first hybrid
polypeptide of the gene expression modulation system binds; a
promoter that is activated by the transactivation domain of the
second hybrid polypeptide of the gene expression modulation system;
and a gene whose expression is to be modulated; wherein the method
comprises the steps of: a) introducing into the host cell a gene
expression modulation system according to the invention; and b)
introducing into the host cell a ligand; whereby upon introduction
of the ligand into the host, expression of the gene is
modulated.
[0247] Genes of interest for expression in a host cell using
methods disclosed herein may be endogenous genes or heterologous
genes. Nucleic acid or amino acid sequence information for a
desired gene or protein can be located in one of many public access
databases, for example, GENBANK, EMBL, Swiss-Prot, and PIR, or in
many biology related journal publications. Thus, those skilled in
the art have access to nucleic acid sequence information for
virtually all known genes. Such information can then be used to
construct the desired constructs for the insertion of the gene of
interest within the gene expression cassettes used in the methods
described herein.
[0248] Examples of genes of interest for expression in a host cell
using methods set forth herein include, but are not limited to:
antigens produced in plants as vaccines, enzymes like
alpha-amylase, phytase, glucanes, and xylanse, genes for resistance
against insects, nematodes, fungi, bacteria, viruses, and abiotic
stresses, nutraceuticals, pharmaceuticals, vitamins, genes for
modifying amino acid content, herbicide resistance, cold, drought,
and heat tolerance, industrial products, oils, protein,
carbohydrates, antioxidants, male sterile plants, flowers, fuels,
other output traits, genes encoding therapeutically desirable
polypeptides or products that may be used to treat a condition, a
disease, a disorder, a dysfunction, a genetic defect, such as
monoclonal antibodies, enzymes, proteases, cytokines, interferons,
insulin, erthropoietin, clotting factors, other blood factors or
components, viral vectors for gene therapy, virus for vaccines,
targets for drug discovery, functional genomics, and proteomics
analyses and applications, and the like.
Measuring Gene Expression/Transcription
[0249] One useful measurement of the methods of the invention is
that of the transcriptional state of the cell including the
identities and abundances of RNA, preferably mRNA species. Such
measurements are conveniently conducted by measuring cDNA
abundances by any of several existing gene expression
technologies.
[0250] Nucleic acid array technology is a useful technique for
determining differential mRNA expression. Such technology includes,
for example, oligonucleotide chips and DNA microarrays. These
techniques rely on DNA fragments or oligonucleotides which
correspond to different genes or cDNAs which are immobilized on a
solid support and hybridized to probes prepared from total mRNA
pools extracted from cells, tissues, or whole organisms and
converted to cDNA.
[0251] Oligonucleotide chips are arrays of oligonucleotides
synthesized on a substrate using photolithographic techniques.
Chips have been produced which can analyze for up to 1700 genes.
DNA microarrays are arrays of DNA samples, typically PCR products,
that are robotically printed onto a microscope slide. Each gene is
analyzed by a full or partial-length target DNA sequence.
Microarrays with up to 10,000 genes are now routinely prepared
commercially. The primary difference between these two techniques
is that oligonucleotide chips typically utilize 25-mer
oligonucleotides which allow fractionation of short DNA molecules
whereas the larger DNA targets of microarrays, approximately 1000
base pairs, may provide more sensitivity in fractionating complex
DNA mixtures.
[0252] Another useful measurement of the methods of the invention
is that of determining the translation state of the cell by
measuring the abundances of the constituent protein species present
in the cell using processes well known in the art.
[0253] Where identification of genes associated with various
physiological functions is desired, an assay may be employed in
which changes in such functions as cell growth, apoptosis,
senescence, differentiation, adhesion, binding to a specific
molecules, binding to another cell, cellular organization,
organogenesis, intracellular transport, transport facilitation,
energy conversion, metabolism, myogenesis, neurogenesis, and/or
hematopoiesis is measured.
[0254] In addition, selectable marker or reporter gene expression
may be used to measure gene expression modulation using the present
invention.
[0255] Other methods to detect the products of gene expression are
well known in the art and include Southern blots (DNA detection),
dot or slot blots (DNA, RNA), northern blots (RNA), RT-PCR (RNA),
western blots (polypeptide detection), and ELISA (polypeptide)
analyses. Although less preferred, labeled proteins can be used to
detect a particular nucleic acid sequence to which it
hybidizes.
[0256] In some cases it is necessary to amplify the amount of a
nucleic acid sequence. This may be carried out using one or more of
a number of suitable methods including, for example, polymerase
chain reaction ("PCR"), ligase chain reaction ("LCR"), strand
displacement amplification ("SDA"), transcription-based
amplification, and the like. PCR is carried out in accordance with
known techniques in which, for example, a nucleic acid sample is
treated in the presence of a heat stable DNA polymerase, under
hybridizing conditions, with one pair of oligonucleotide primers,
with one primer hybridizing to one strand (template) of the
specific sequence to be detected. The primers are sufficiently
complementary to each template strand of the specific sequence to
hybridize therewith. An extension product of each primer is
synthesized and is complementary to the nucleic acid template
strand to which it hybridized. The extension product synthesized
from each primer can also serve as a template for further synthesis
of extension products using the same primers. Following a
sufficient number of rounds of synthesis of extension products, the
sample may be analyzed as described above to assess whether the
sequence or sequences to be detected are present.
[0257] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention.
EXAMPLES
General Methods
[0258] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A
Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, N.Y. (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan,
and L. W. Enquist, Experiments with Gene Fusions, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel,
F. M. et al., Current Protocols in Molecular Biology, Greene
Publishing Assoc. and Wiley-Interscience (1987).
[0259] Materials and methods suitable for the maintenance and
growth of bacterial cultures are well known in the art. Techniques
suitable for use in the following examples may be found as set out
in Manual of Methods for General Bacteriology (Phillipp Gerhardt,
R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A.
Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society
for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, Second
Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All
reagents, restriction enzymes and materials used for the growth and
maintenance of host cells were obtained from Aldrich Chemicals
(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL
(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.)
unless otherwise specified.
[0260] Manipulations of genetic sequences may be accomplished using
the suite of programs available from the Genetics Computer Group
Inc. (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),
Madison, Wis.). Where the GCG program "Pileup" is used the gap
creation default value of 12, and the gap extension default value
of 4 may be used. Where the CGC "Gap" or "Bestfit" program is used
the default gap creation penalty of 50 and the default gap
extension penalty of 3 may be used. In any case where GCG program
parameters are not prompted for, in these or any other GCG program,
default values may be used.
[0261] The meaning of abbreviations is as follows: "h" means
hour(s), "min" means minute(s), "sec" means second(s), "d" means
day(s), "mL" means microliter(s), "mL" means milliliter(s), "L"
means liter(s), ".mu.M" means micromolar, "mM" means millimolar,
"M" means molar, "mol" means moles, "mmol" means millimoles,
".mu.g" means microgram(s), "mg" means milligram(s), "A" means
adenine or adenosine, "T" means thymine or thymidine, "G" means
guanine or guanosine, "C" means cytidine or cytosine, "x g" means
times gravity, "nt" means nucleotide(s), "aa" means amino acid(s),
"bp" means base pair(s), "kb" means kilobase(s), "k" means kilo,
".mu." means micro, ".degree. C." means degrees Celsius, "C" in the
context of a chemical equation means Celsius, "THF" means
tetrahydrofuran, "DME" means dimethoxyethane, "DMF" means
dimethylformamide, "NMR" means nuclear magnetic resonance, "psi"
refers to pounds per square inch, and "TLC" means thin layer
chromatography.
Example 1
Preparation of Compounds
[0262] The compounds of the present invention may be made according
to the following synthesis routes.
1.1 Preparation of 3,5-Dimethyl-benzoic acid
N-tert-butyl-N'-(4-ethyl-2-fluoro-benzoyl)-hydrazide
(RG-101523)
##STR00006##
[0264] To a 3-neck, 2 L round bottom flask was added 173.71 g (1.0
mol, 97%) of 2-amino-2-methyl-1-propanol in 300 mL of dry methylene
chloride. The flask was equipped with a magnetic stir bar and
thermometer and was placed into a dry ice/acetone bath and cooled
to 0.degree. C. From a separatory funnel, a solution of
4-ethylbenzoyl chloride (168.5 g, 1.0 mol), dissolved in about 300
mL of methylene chloride was slowly added, while maintaining the
reaction temperature below 5.degree. C. The mixture was allowed to
stir at room temperature overnight. Solid propanol amine-HCl was
filtered off and the filter cake was washed with methylene
chloride. The combined methylene chloride extracts were
concentrated partially on a rotary evaporator and used directly in
the next step. The intermediate amide solution generated in the
first step was cooled in an ice bath and DMF (0.5 mL) was added.
125 g (1.04 mol) of SOCl.sub.2 in 50 mL of methylene chloride from
a separatory funnel was added drop-wise at a controlled rate,
keeping the reaction temperature at 0-5.degree. C. The reaction was
stirred at room temperature for an additional 2-3 hours. The
reaction mixture was cooled in an ice bath and 25% NaOH was added
to make the aqueous layer basic (pH=11-12). The mixture was
transferred to a large separatory funnel, the methylene chloride
layer was separated, and the aqueous layer was extracted twice with
chloroform. The combined organic phases were dried and evaporated
to yield 201 g of
2-(4-ethyl-phenyl)-4,4-dimethyl-4,5-dihydro-oxazole as a yellow
viscous oil. .sup.1H NMR (CDCl.sub.3, 300 MHz), .delta. (ppm): 7.8
(d, 2H), 7.2 (d, 2H), 4.088 (s, 2H), 2.68 (q, 2H), 1.375 (s, 6H),
1.24 (t, 3H).
##STR00007##
[0265] The 4-ethylphenyloxazoline (2.03 g, 10 mmol) was dried in a
vacuum oven at 60.degree. C. for 2-3 hours, dissolved in 50 mL of
dry THF, and charged to a 300 mL round bottom flask equipped with a
thermometer, nitrogen inlet, and magnetic stir bar. The mixture was
cooled under nitrogen to -70.degree. C. in a dry ice/acetone bath.
Butyl lithium in hexane (7.5 mL, 0.012 mmol) was added in two
portions and warmed to -25.degree. C. over 2 hours. The mixture was
cooled again to -65.degree. C. and N-fluorobenzenesulfonimide (3.79
g, 0.012 mmol) was added in three portions. The mixture was allowed
to warm to room temperature and was stirred overnight. The reaction
mixture was quenched with 100 mL of saturated NH.sub.4Cl, and
transferred to a separatory funnel with ethyl ether washes. 25%
NaOH was added slowly and mixed until an aqueous phase with pH=10
was achieved. The aqueous phase was extracted with ether and the
ether was washed with a small volume of water. The ether extracts
were dried over MgSO.sub.4 and evaporated to give 2.51 g of
2-(2-fluoro-4-ethyl-phenyl)-4,4-dimethyl-4,5-dihydro-oxazole as a
brown oil. In a second experiment, a rate of 70% fluorination was
achieved (highest) with a reactant ratio of 1:1.5:1.5 oxazoline:
BuLi: N-fluorobenzenesulfonimide.
[0266] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. (ppm): 7.0 (d,
2H), 7.78 (t, 1H), 4.1 (s, 2H), 2.7 (q, 2H), 1.39 (s, 6H), 1.2 (t,
3H).
##STR00008##
[0267] DMSO (4 mL) and CH.sub.3I (2 mL) were added to 2.31 g of
oxazoline in a round bottom flask, and the mixture was stirred
overnight at room temperature. Methyl iodide was removed in vacuo
on a rotary evaporator. Aqueous KOH (4.4 g in 35 mL of water) was
added and the mixture was refluxed for 8 hours. The reaction
mixture and water washes were transferred to a separatory funnel;
the neutral components were removed with a chloroform extraction.
The aqueous mixture was acidified with 6N HCl to pH 1-2, and
extracted with ether. Ether extracts were dried over MgSO.sub.4 and
evaporated to yield 1.2 g of a white solid, comprised of both
2-fluoro-4-ethylbenzoic acid and 4-ethylbenzoic acid. The product
mixture was dissolved in KOH and the pH was adjusted with 2N HCl to
pH=7. With vigorous stirring and careful monitoring with a pH
meter, the mixture was acidified to pH=5 with 0.1N HCl.
4-Ethylbenzoic acid precipitated first, which was filtered through
Whatman #541 paper, acidification continued with 0.1N HCl to
pH=4.9, and at 0.1 unit increments until pH=4.3, each time
filtering the solids through Whatman #541 paper. Finally, the
mixture was acidified to pH=2.5 and filtered. Precipitates of
decreasing pH contain increasing rations of 2-fluoro-4-ethylbenzoic
acid, the last two fractions contain 98-100% desired product.
Extraction of the remaining aqueous phase with ether recovers more
2-fluoro-4-ethylbenzoic acid. The product was air-dried as drying
in a vacuum oven results in substantial product losses due to
volatility. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.=7.95 (t, 1H),
7.1 (d, 1H), 7.0 (d, 1H), 2.71 (q, 2H), 1.27 (t, 3H).
4-Ethylbenzoic acid: .sup.1H NMR (CDCl.sub.3) .delta. (ppm): 8.1
(d, 2H), 7.3 (t, 2H), 2.71 (q, 2H), 1.27 (t, 3H).
##STR00009##
[0268] 1.31 g (1.3 mmol) of thionyl chloride, 1 drop of DMF and 1.0
g (5.95 mmol) of acid were added to 30 mL of toluene with stirring.
The mixture was refluxed for 4 hours. After this period, the
toluene and unreacted thionyl chloride were removed by
distillation. The resulting 2-fluoro-4-ethylbenzoyl chloride was
used without further purification.
##STR00010##
[0269] 0.150 g of 3,5-dimethyl-benzoic acid N-tert-butyl-hydrazide
(1 eq, 0.68 mmol) and 0.110 mL (1.2 eq, 0.77 mmol) of
2-fluoro-4-ethyl benzoyl chloride were weighed into a 1 oz. vial. A
small stirbar was added followed by 2 mL of methylene chloride. The
mixture was stirred until the hydrazone dissolved. The stirring was
stopped and 2 mL of a 1 M potassium carbonate (K.sub.2CO.sub.3)
solution was added. The mixture was allowed to stir overnight. At
the end of this period, 1 mL of water and 1 mL of methylene
chloride were added. The aqueous phase was removed and the organic
phase was washed twice with 1 M potassium carbonate solution. The
organic phase was removed and dried over magnesium sulfate. The
organic phase was filtered thru a pad of basic alumina and the
solvent removed. The product, 3,5-dimethylbenzoic acid
N-tert-butyl-N'-(2-fluoro-4-ethylbenzoyl)-hydrazide, was purified
by trituration with 1:1 ether: hexane. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. (ppm): 7.7 (m, 2H), 7.6 (t, 1H), 7.0 (m, 3H),
2.6 (q, 2H), 2.3 (s, 3H), 2.1 (s, 6H), 1.5 (s, 9H), 1.1 (t,
3H).
1.2 Preparation of 5-chloro-4H-benzo[1,3]dioxine-6-carboxylic
acid
##STR00011##
[0271] 5-chloro and 7-chloro isomers were separated by silica gel
cartridge chromatography. The mixture was dissolved in
CHCl.sub.3/CH.sub.3OH and added to the top of a large cartridge.
The 5-chloro isomer eluted with 2:3 ether: hexane and the
7-chloroisomer began to elute with 3:2 ether: hexane and completed
elution with neat ether. .sup.1H NMR (DMSO-d.sub.6, 300 MHz)
.delta. (ppm): 7.75 (d, 1H), 6.95 (d, 1H), 5.3 (s, 2H), 4.9 (s,
2H).
1.3 Preparation of 2-fluoro-4-hydroxy-benzoic acid
##STR00012##
[0273] To a stirred solution of 2-fluoro-4-hydroxybenzonitrile
(20.00 g, 145.9 mmol) in 160 mL of water, was added 50% aqueous
sodium hydroxide (40.00 g, 500.0 mmol). The mixture was heated to
reflux for 4 hours, cooled to room temperature, poured into iced
concentrated hydrochloric acid, and extracted with ether. The
product was extracted into saturated aqueous sodium bicarbonate and
the ether layer discarded. This aqueous extract was acidified with
concentrated hydrochloric acid and extracted with ether. The
organic extract was dried over magnesium sulfate, filtered, and
evaporated to give a white solid (22.90 g) of
2-fluoro-4-hydroxybenzoic acid in 100% yield. .sup.1H NMR (300 MHz,
CD.sub.3COCD.sub.3) .delta. (ppm): 9.80 (b, 1H), 7.87 (t, 1H), 6.77
(dd, 1H), 6.66 (dd, 1H). .sup.19F-NMR (300 MHz, CD.sub.3COCD.sub.3)
.delta. (ppm): -108.13 (s, decoupled).
2-Fluoro-4-hydroxybenzonitrile: .sup.1H-NMR (300 MHz,
CD.sub.3COCD.sub.3) .delta. (ppm): 7.61 (t, 1H), 6.81 (m, 2H), 5.80
(b, 1H). .sup.19F-NMR (300 MHz, CD.sub.3COCD.sub.3) .delta. (ppm):
-108.82 (s, decoupled)
1.4 Preparation of 5-fluoro-4H-benzo[1,3]dioxine-6-carboxylic acid,
methyl ester and 6-fluoro-4H-benzo{1,3}dioxine-7-carboxylic acid,
methyl ester
##STR00013##
[0275] 1.6 g methyl 2-fluoro-4-hydroxy-benzoate (7.54 mmol), 0.157
g of p-toluenesulfonic acid (0.9 mmol), 50 mL of toluene and 1.2 g
of paraformaldehyde (40 mmol) were combined and refluxed for 3
hours after which time TLC (1:1 ethyl acetate: CH.sub.2Cl.sub.2)
showed the absence of the starting material. Occasionally it became
necessary to cool the reaction and scrape off unreacted
paraformaldehyde from the walls of the reaction flask. The reaction
flask was vented to the hood exhaust. After the mixture was
filtered to remove the solid paraformaldehyde, the solid was washed
twice with 100 mL of toluene. The toluene washes were combined with
the filtered liquid. The organic liquid was washed three times with
75 mL of 5% aqueous NaOH. 50 mL of methanol was added to the
organic phase and the solvent was removed on the rotary evaporator
to yield a thick syrup which gradually formed a somewhat tacky
white solid. Proton and .sup.19F NMR showed the presence of two
isomers in a ratio of approximately 7:3. These isomers could be
separated by careful chromatography on silica gel using a hexane to
85% hexane-15% ether gradient. The desired
3,4-methylenedioxy-2-fluoro benzoic acid, methyl ester eluted first
as a white solid. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm):
3.85 (s, 3H), 4.85 (s, 2H), 5.20 (s, 2H), 6.60 (d, 1H), 7.80 (t,
1H). .sup.19F NMR (ppm, CDCl.sub.3) -115 (s); Rf=0.4 (1:1
CH.sub.2Cl.sub.2: EtOAc).
[0276] The 4,5-methylenedioxy isomer eluted shortly thereafter,
also as a white solid. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
(ppm): 3.90 (s, 3H), 4.85 (s, 2H), 5.25 (s, 2H), 6.65 (d, 1H), 7.60
(d, 1H). .sup.19F NMR (ppm, CDCl.sub.3) -108 (s); Rf=0.32 (1:1
CH.sub.2Cl.sub.2: EtOAc).
1.5 Preparation of 5-fluoro-4H-benzo{1,3}dioxin-6-carboxylic acid
and 6-fluoro-4H-benzo{1,3}dioxin-7-carboxylic acid
##STR00014##
[0278] 5-Fluoro-4H-benzo[1,3]dioxine-6-carboxylic acid methyl ester
(2.02 g), water (1 mL), methanol (20 mL) and sodium hydroxide (1 mL
of a 50% NaOH solution) were added to a flask equipped with a
condenser and magnetic stirbar. The stirring was started and the
mixture was refluxed for 2 hours. At this point, the TLC
(1:1:CH.sub.2Cl.sub.2/ethyl acetate) showed no starting ester was
present. The solvent was removed, leaving a white solid. The solid
was taken up in water and the aqueous layer was washed three times
with 50 mL of ether. The aqueous layer was then acidified with
dilute hydrochloric acid causing the formation of a white
precipitate. This white solid was collected on a sintered glass
filter funnel and washed well with de-ionized water. The solid was
dried under vacuum at 60.degree. C. overnight and used in the
following reaction without further purification.
1.6 Preparation of 5-fluoro-4H-benzo{1,3}dioxine-6-carboxylic acid
N'-tert-butyl-hydrazide and 5-fluoro-4H-[1,3]dioxine-6-carboxylic
acid
N-tert-butyl-N'-(5-fluoro-4H-benzo[1,3]dioxine-6-carbonyl)-hydrazide
##STR00015##
[0280] 1.6 g of 5-fluoro-4H-benzo{1.3}dioxin-6-carboxylic acid (8.1
mmol), 30 mL of toluene and 1 drop of DMF were combined in a 100 mL
flask equipped with magnetic stirbar, scrubber and condenser. 0.59
mL of thionyl chloride (0.96 g, 9.7 mmol) was added and the mixture
was heated to reflux and held at reflux for 4 hours. After this
period, the mixture was cooled slightly and the condenser was
replaced with a distillation head. The excess thionyl chloride was
distilled off. The mixture was cooled to 20.degree. C. and the
toluene was removed using a rotary evaporator. NOTE: It is
advisable to use care during the toluene removal. The
5-fluoro-4H-benzo[1,3]dioxine-6-carbonyl chloride starts to distill
under vacuum if the temperature exceeds 27.degree. C.
[0281] 50% NaOH (0.648 g, 8.1 mmol) of was dissolved in 3 mL of
water and added to a reaction flask containing a magnetic stirbar
and rubber septum for reagent addition. 1.00 (8.1 mmol) g of
tert-butyl hydrazine hydrochloride was added. The mixture was
stirred for 5 min at room temperature and then cooled to -5.degree.
C. 5-Fluoro-4H-benzo[1,3]dioxine-6-carbonyl chloride (8.1 mmol) was
dissolved in 25 mL of dichloromethane and was added simultaneously
with a second portion of 0.648 g (8.1 mmol) of 50% NaOH in 3 mL of
water. The reaction temperature was kept below -2.degree. C. during
the addition. The mixture was stirred at -5.degree. C. to
-2.degree. C. for 30 min. After this time, the mixture was allowed
to warm to room temperature and stirred for 30 min. 50 mL of
dichloromethane and 50 mL of water were added to the reaction
mixture. The layers were separated and the organic layer was washed
three times with 50 mL of water. The organic layer was then dried
over MgSO.sub.4 and filtered. Removal of the solvent yielded 2.4 g
of a yellow syrup, which appeared to be approximately 85% of the
desired product by NMR analysis. The pure product,
5-fluoro-4H-benzo[1,3]dioxine-6-carboxylic acid
N'-tert-butyl-hydrazide, was isolated as a pale, yellow solid by
careful column chromatography on silica gel using a dichloromethane
to 4:1 dichloromethane/ethyl acetate gradient. .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 0.85 (s, 9H), 4.58, (s, 2H),
4.90 (s, 2H), 6.40 (d, 1H), 7.50 (t, 1H) and 7.70 (br s, 1H).
.sup.19F NMR (CDCl3), .delta. ppm: (s, -190, s). Rf=0.35 (1:1
CH.sub.2Cl.sub.2: ethyl acetate).
1.7 Preparation of 2-Bromomethyl-3-methoxy-benzoic acid methyl
ester
##STR00016##
[0283] Into a 2 L 3-neck round bottom flask was added 75 g (0.42
mol) of 2-methyl-3-methoxy methyl benzoate, 500 mL of CCl.sub.4,
80.1 g (0.45 mol) of NBS, and 1 g of AIBN. The mixture was stirred
and refluxed gently for 2 hours. The reaction mixture was cooled
and ca. 600 mL of CH.sub.2Cl.sub.2 and 500 mL of water were added.
The mixture was stirred to dissolve floating solids, transferred to
a 2 L separatory funnel, and then shaken. The organic layer was
separated and the water extracted with CH.sub.2Cl.sub.2. The
aqueous fractions were discarded and the organic phase extracted
with 400-500 mL of water to remove the NBS (note: solubility of
succinimide is 1 g/3 g water, solubility of NBS is 1.47 g/100 mL
water). The water extractions were repeated, the organic phase
dried with MgSO.sub.4 and charcoal, and the solvent evaporated in 2
portions, to yield methyl-3-methoxy-2-bromomethylbenzoate. TLC:
Rf=0.58, single spot (1:1 ethyl acetate: hexane). .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 7.5 (d, 1H), 7.3 (t, 1H), 7.05
(d, 1H), 5.06 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H).
1.8 Preparation of 4-methoxy-3H-isobenzofuran-1-one
##STR00017##
[0285] In a 500 mL round bottom flask was added 15.15 g (0.0585
mol) of 2-bromomethyl, 3-methoxy methyl benzoate, 29.3 g (0.293
mol) of CaCO.sub.3, 150 mL of dioxane and 150 mL of water. The
flask was placed into an oil bath and the mixture heated with
stirring at 85.degree. C. for 3.5 to 4 hours. The CaCO.sub.3 was
filtered off and washed with ethyl acetate and water. To the
filtrate was added ethyl acetate (200 mL) and water (50 mL) and the
mixture then shaken in separatory funnel. The water phase was
extracted twice with ethyl acetate (50 mL). The ethyl acetate
extracts were combined, extracted once with water, dried over
MgSO.sub.4, and evaporated. This yielded 9.2 g of white crystals of
7-methoxybenzolactone (95% yield). .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. (ppm): 7.5 (m, 2H), 7.1 (m, 1H), 5.26 (s, 2H), 3.93
(s, 3H). TLC: Rf=0.46 (1:1 EtOAC: hexane).
1.9 Preparation of 2-cyanomethyl-3-methoxybenzoic acid
##STR00018##
[0287] Into a 500 mL 3-neck round bottom flask was added 10 g
(61.75 mmol) of 2-bromomethyl, 3-methoxy methyl benzoate, 4.0 g
(81.6 mmol) of NaCN, 0.30 g (2 mmol) of NaI, 100 mL of CH.sub.3CN,
and 50 mL of DMF. The reaction mixture was heated and refluxed for
10 hours. The precipitate (NaBr) was filtered off, and the solution
was concentrated on an evaporator. 300 mL of water and 200 mL of
ether were added and then shaken in a separatory funnel. The water
was extracted twice with 100 mL of ether. The ether fractions were
dried over MgSO.sub.4, and concentrated to yield methyl
3-methoxy-2-cyanomethylbenzoate (95-100% yield). This ester (0.053
mmol, 10.51 g) was stirred vigorously in 100 mL of CH.sub.3OH.
Ba(OH).sub.2H2O (0.079 mmol, 14.97 g) was added and the mixture
stirred at room temperature overnight. The CH.sub.3OH was removed
on a rotary evaporator. 150 mL of water, 200 mL of
CH.sub.2Cl.sub.2, and 50 mL of 6N HCl were added, and then stirred
in a flask to dissolve all residues. The mixture was transferred to
a separatory funnel, acidified with 6N HCl to pH 1-2. The
CH.sub.2Cl.sub.2 phase was separated and the aqueous phase
extracted twice with 50 mL of CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 extracts were combined, dried over MgSO.sub.4 and
charcoal, filtered, and evaporated to yield 8.8 g of a white solid,
2-cyanomethyl-3-methoxybenzoic acid, (87%).
[0288] Methyl 3-methoxy-2-cyanomethylbenzoate: .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 7.6 (d, 1H), 7.4 (t, 1H), 7.1
(d, 1H), 4.18 (s, 2H), 3.94 (s, 3H), 3.926 (s, 3H). TLC (1:1 ethyl
acetate: hexane) 0.55.
[0289] 2-Cyanomethyl-3-methoxybenzoic acid: .sup.1H NMR (300 Mhz,
CDCl.sub.3) .delta. (ppm): 7.55 (d, 1H), 7.45 (t, 1H), 7.3 (d, 1H),
4.121 (d, 2H), 3.91, (s, 3H). TLC (1:1 ethyl acetate: hexane), Rf
0.36 streak.
1.10 Preparation of 3-methoxy-2-methylsulfanylmethyl-benzoic acid
and pentafluorophenyl 2-(methylthiomethyl 3-methoxybenzoate
##STR00019##
[0291] Methyl 2-bromomethyl-3-methoxy benzoate was stirred in
methanol at room temperature with 1.02 eq. of sodium
methylmercaptide. After 30 min the reaction was complete based on
GC analysis. The mixture was poured into water and extracted twice
with ethyl acetate. The combined organic layers were stripped under
vacuum leaving methyl 2-(methylthiomethyl)-3-methoxybenzoate as a
pale yellow oil in about 86% yield. GC: DB-5, 30 m, film: 0.25 um,
t.sub.init=1.00 T=120-280 C@20 C/min; Rt=6:30 area %=98. .sup.1H
NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.46 (d, 1H), 7.26 (t,
1H), 7.03 (d, 1H), 4.18 (s, 1H), 3.90 (s, 2H), 3.89 (s, 3H), 2.04
(s, 3H).
[0292] Methyl 2-(methylthiomethyl)-3-methoxy benzoate was heated to
reflux with 1.5 eq of NaOH in 10% aqueous methanol for 1.5 hr. The
solution was added drop-wise to excess 10% sulfuric acid. The
precipitate was filtered and dried in air giving ca. 92% yield of
2-(methylthiomethyl)-3-methoxybenzoic acid. TLC (2:1 ethyl acetate:
hexane) indicated one spot, Rf 0.50.
[0293] 2-(Methylthiomethyl)-3-methoxybenzoic acid was dissolved in
ethyl acetate and added to a solution of 1.1 eq. of
pentafluorophenol and 1.1 eq. of dicyclohexycarbodiimide in ethyl
acetate. After 1 hr the mixture was filtered and the mother liquors
were stripped under vacuum. The yellow oily residue was
crystallized from hexane to give the product, pentafluorophenyl
2-(methylthiomethyl 3-methoxybenzoate, in 100% yield. TLC (1:2
ethyl acetate: hexane) indicated one spot, Rf 0.58.
1.11 Preparation of 3-Methoxy-2-methoxymethyl-benzoic acid
##STR00020##
[0295] To a 100 mL flask containing 10.1 g (0.039 mol) of methyl
2-bromomethyl-3-methoxybenzoate in 50 mL of CH.sub.3OH, were added
19 g of a 25% wt. solution of NaOMe (4.74 g, 0.087 mol). The
reaction was stirred at room temperature for 2 hours and then
evaporated on a rotary evaporator to remove the CH.sub.3OH. About
200 mL of water were added to the residue and the resulting
solution was extracted with CHCl.sub.3. The CHCl.sub.3 extract was
dried and evaporated to yield 6.27 g of crude methyl
3-methoxy-2-methoxymethylbenzoate (77% yield). .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 7-7.4 (multiple, 3H), 4.783
(s, 2H), 3.897 (s, 3H), 3.864 (s, 3H), 3.37 (s, 3H). 6.27 g of
methyl 3-methoxy-2-methoxymethylbenzoate was stirred with a 20%
aqueous KOH solution (6.7 g, 0.12 mol in 34 g of solution) at
50.degree. C. in an oil bath for 4-5 hours, and then at room
temperature for 16 hours. The reaction mixture was acidified with
3N HCl to pH 2 and extracted with CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 extract was dried and evaporated to yield 5.35 g
of 3-methoxy-2-methoxymethylbenzoic acid (92% yield). .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 7.65 (1H, d), 7.40 (t, 1H),
7.10 (d, 1H), 4.83 (s, 2H), 3.88 (s, 3H), 3.46 (s, 3H).
1.12 Preparation of
N'-t-butyl-3-methoxy-2-methoxymethylphenylhydrazide
##STR00021##
[0297] To 4.30 g (0.0219 m) of 3-methoxy-2-methoxymethylbenzoic
acid in 50 mL of ethyl acetate in a round bottom flask, was added
8.88 g of a 50% wt solution of pentafluorophenyl phenol, followed
by 21.91 mL of DCC solution (0.0219 m). After stirring for 2 hr at
room temperature, TLC showed a spot for the intended
pentafluorophenyl ester product at Rf=0.64 (1:1 ethyl acetate:
hexanes), while the starting acid was Rf=0.39.
[0298] A small volume of ethyl acetate (30 mL) and a teaspoon of
anhydrous MgSO.sub.4 was added and then filtered to remove the DCC
and DCU. The filtrate was evaporated to yield 9.4 g of product.
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 3.39 (s, 3H), 3.90
(s, 3H), 4.81 (s, 2H). NMR analysis of the starting material
indicated the following spectrum: .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. (ppm): 4.83 (s, 2H), 3.88 (s, 3H), 3.4 (s, 3H).
[0299] The remaining DCC and DCU were removed by column
chromatography on silica gel. The products eluted in the 6 and 8%
ethyl acetate/hexane fractions. The yield of 7.08 g, contained some
DCC and DCU. 7.08 g (0.02 mol) of the pentafluorophenyl ester in 60
mL of CH.sub.2Cl.sub.2 was stirred with 3.67 g (0.029 mol) of
t-butylhydrazine HCl and 12 g of K.sub.2CO.sub.3 in 60 mL of water
at room temperature overnight. 60 mL of CH.sub.2Cl.sub.2 and 50 mL
of water were added and then shaken in separatory funnel. The
organic phase was dried over MgSO.sub.4 and then evaporated to
yield 5.6 g of t-butyl hydrazide. .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. (ppm): 7.4 (d, 2H). 7.0 (t, 1H), 4.627 (s, 2H), 3.874 (s,
3H), 3.465 (s, 3H), 1.184 (s, 9H). TLC: (1:1 ethyl acetate:
hexanes), Rf=0.16. The product can be further purified by
trituration with hexanes.
1.13 Preparation of 3-methoxy-2-methoxymethyl-benzoic acid
##STR00022##
[0301] 1.0 g (0.0061 mol) of lactone was refluxed with 20 mL of
7.5% NaOH and 20 mL of CH.sub.3OH for 7 hours. The methanol was
removed on a rotary evaporator, set up a Dean Stark, refluxed in
toluene to azeotropically remove water, toluene was removed in
vacuo, and the residue was dried in a vacuum oven. .sup.1H NMR
(DMSO, 300 MHz) .delta. (ppm): 3.74 (s, 3H), 4.47 (s, 2H), 6.9 (d,
1H), 7.1 (t, 1H), 7.2 (d, 1H).
[0302] The sodium carboxylate was dissolved in 15 mL of DMF and
then CH.sub.3I (0.87 g, 0.0061 mol) was added and the mixture was
stirred at room temperature overnight. 50 mL of saturated
NH.sub.4Cl was added to quench the reaction. 250 mL of water was
added (solution is basic at this point), and extracted with ether
to remove the neutral and basic substances. The remaining aqueous
solution was acidified with 3N HCl to pH=2 and the desired
carboxylic acid extracted with ether. The ether extracts were dried
and evaporated to yield 0.55 g of
3-methoxy-2-hydroxymethylbenzoate, sodium salt and
3-methoxy-2-methoxymethylbenzoic acid. .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. (ppm): 3.456 (s, 3H), 3.884 (s, 3H), 4.832 (s, 2H),
7.1 (d, 1H), 7.4 (t, 1H), 7.6 (d, 1H).
1.14 Preparation of 2-allyloxymethyl-3-methoxy-benzoic acid
##STR00023##
[0304] 1.0 g (0.005 mol) of sodium
3-methoxy-2-hydroxymethylbenzoate was combined in a 200 mL flask
with 1.68 g (0.01 mol) of allyl iodide and 50 mL of dioxane and
refluxed for 2 hr. The mixture was stirred at room temperature
overnight. The reaction mixture was concentrated on an evaporator.
Water were added, and aqueous 5% NaOH to pH=10-11, and the mixture
was extracted with ether. The ether was evaporated to give a
diallyl product (0.34 g). .sup.1H NMR indicated complex allyl
signals in addition to the aromatic protons. The water solution was
acidified with 3N HCl and extracted twice with 100 mL of ether to
yield 3-methoxy-2-allyloxybenzoic acid and allyl
3-methoxy-2-allyloxybenzoate (0.34 g). .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. (ppm): 3.879 (s, 3H), 4.11 (d, 2H), 5.3 (q, 2H),
5.9-6.0 (m, 1H). TLC: (1:1 ethyl acetate: hexane): diallyl, Rf
0.60, monoallyl, Rf 0.31, streak.
1.15 Preparation of methyl 3-methoxy-2-allyloxymethyl benzoate
##STR00024##
[0306] Into a 25 mL round bottom flask, was added 0.96 g (0.0165 m)
of allyl alcohol and 3 mL of DMF. While cooling the flask in an ice
bath, 0.80 g of a 60% dispersion of NaH (0.020 m, 0.48 g) was
added, with magnetic stirring. The reaction mixture was stirred for
45 min at room temperature. The flask was placed in the ice bath
and 2 g of DMF and 3.89 g (0.015 m) of methyl
2-bromomethyl-3-methoxy benzoate were added in small portions. The
reaction was allowed to stir at room temperature for 4-5 hours. The
reaction was transferred to a separatory funnel with 150 mL of
ethyl ether and 50 mL of water. The reaction mixture was shaken,
the ether phase separated and the water phase again extracted with
50 mL of ether. The ether phase was extracted with water (20 mL),
dried with MgSO.sub.4 and concentrated to yield 2.7 g of a pale,
yellow oil (76% yield). .sup.1H NMR (CDCL.sub.3, 300 MHz) .delta.
(ppm): 7.3-7.0 (m, 3H), 5.9-6.0 (m, 1H), 5.1-5.3 (2d, 2H), 4.8 (d,
2H), 4.02 (d, 2H), 3.90 (s, 3H), 3.88 (s, 3H). TLC (1:1 ethyl
acetate/hexane), Rf 0.58.
1.16 Preparation of 2-allyloxymethyl-3-methoxybenzoic acid
##STR00025##
[0308] Into a 200 mL round bottom flask containing 5.40 g (0.0229
m) of 3-methoxy-2-allyloxymethyl benzoate, was added 40 mL of
methyl-alcohol. With magnetic stirring, 6.50 g (0.034 m) of barium
hydroxide monohydrate was added. The reaction was stirred for 4
hours in a 45.degree. C. water bath. The reaction flask was
transferred to a rotary evaporator and the methanol was removed
under vacuum. H.sub.2O (150 mL) was added to the residue in the
flask and the mixture was stirred until most of the residue
dissolved. The reaction mixture was transferred with water (50-100
mL) to a large beaker. The mixture was acidified with 6 HCl (to
pH=1) and transferred to a separatory funnel. The reaction mixture
was extracted three times with 100 mL of ethyl acetate with salting
out. Ethyl acetate extract was dried and evaporated to yield 4.38
(g) of viscous product, 2-allyloxymethyl-3-methoxybenzoic acid (98%
yield). .sup.1H NMR (CDCL.sub.3, 300 MHz) .delta. (ppm): 7.55 (d,
1H), 7.40 (t, 1H), 7.1 (d, 1H), 6.0-5.9 (m, 1H), 5.4-5.2 (2d, 2H),
4.87 (d, 2H), 4.10 (d, 2H), 3.878 (s, 3H). TLC (1:1 ethyl
acetate:hexane) Rf 0.38.
1.17 Preparation of pentafluorophenyl
2-allyloxymethyl-3-methoxybenzoate
##STR00026##
[0310] Into a 200 mL round bottom flask was added 6.6 g (0.0297
mol) of 2-allyloxymethyl-3-methoxy benzoic acid and 40 mL of ethyl
acetate. 24.05 g of a 25% pentafluorophenol (6.01 g, 0.0327 mol)
solution in ethyl acetate was added while stirring. The reaction
flask was placed into a water bath and while stirring small
portions of DCC (6.2 g, 0.030 mol) were added. The stirring
continued overnight at room temperature. The reaction was filtered
through two Whatman #541 filters to remove the DCU precipitate. The
ethyl acetate solution was concentrated to yield 12.8 g (110%
yield), indicating presence of DCC and DCU. This was confirmed by
TLC (1:1 ethyl acetate: hexane), which indicated a Rf of 0.72 plus
other less polar compounds (I.sub.2 stain indicates about 85%
purity). .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.7 (d,
1H), 7.45 (t, 1H), 7.15 (d, 1H), 6.0-5.9 (m, 1H), 5.3-5.1 (2d, 2H),
4.88-4.83 (d, 2H), 4.06 (d, 2H), 3.90 (s, 3H).
1.18 Preparation of 2-allyloxymethyl-3-methoxy-benzoic acid
N'-tert-butyl-hydrazide
##STR00027##
[0312] Into a round bottom flask containing 18.9 g (0.048 m) of
pentafluorophenyl ester in 50 mL of CH.sub.2Cl.sub.2, was added 9.1
g (0.73 m) of t-butylhydrazine hydrochloride, and then 20.16 g
(0.146 m) of K.sub.2CO.sub.3 in 50 mL of H.sub.2O. The mixture was
stirred at room temperature for 24 hours. 50 mL of H.sub.2O were
added, the CH.sub.2Cl.sub.2 layer was separated, and H.sub.2O phase
extracted twice with 100 mL of CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 fraction was dried with MgSO.sub.4, and
concentrated to yield 9.75 g of
N-2-allyloxymethyl-3-methoxyphenyl-N'-t-butylhydrazide. .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 1.151 (s, 9H), 3.87 (s, 3H),
4.12 (d, 2H), 4.68 (s, 2H), 5.15-5.35 (q, 2H), 5.19 (m, 1H), 7.0
(t, 1H), 7.4 (d, 2H). TLC: (1:1, ethyl acetate: hexane)
Rf=0.25.
1.19 Preparation of 3,5-dimethyl-benzoic acid
N'-(2-allyloxymethyl-3-methoxy-benzoyl)-N-tert-butyl-hydrazide
(RG-115003)
##STR00028##
[0314] To a flask containing 2.0 g (0.0068 mol) of
2-allyloxymethyl-3-methoxy-benzoic acid N'-tert-butyl-hydrazide
dissolved in 15 mL of CH.sub.2Cl.sub.2, was added 1.27 g (0.0075
mol) of 3,5-dimethylbenzoyl chloride in 10 mL of CH.sub.2Cl.sub.2
and 2.84 g of K.sub.2CO.sub.3 (0.02 mol) in 30 mL of H.sub.2O. The
mixture was stirred at room temperature for 24 hours. The reaction
mixture was diluted and partitioned, and the organic phase was
dried and solvent was removed in vacuio. The product was purified
by silica gel chromatography; eluting in 25% ethyl acetate: hexane
fractions, to yield 2.40 g of pure 3,5-dimethyl-benzoic acid
N'-(2-allyloxymethyl-3-methoxy-benzoyl)-N-tert-butyl-hydrazide.
[0315] .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.2 (t, 1H)
7.1 (s, 1H), 7.05 (d, 2H), 6.95 (m, 2H), 5.9 (m, 1H), 5.2-5.3 (q,
2H), 4.5 (d, 2H), 3.9 (m, 2H), 3.80 (s, 3H), 2.245 (s, 6H), 1.547
(s, 9H).
1.20 Preparation of 3,5-dimethyl-benzoic acid
N-tert-butyl-N'-(2-hydroxymethyl-3-methoxy-benzoyl)-hydrazide
(RG-115371)
##STR00029##
[0317] 1.57 g of allyl ether were dissolved in 50 mL of CH.sub.3OH.
600 mg of Pd/C and 20 drops of 1% HClO.sub.4/H.sub.2O were added
and refluxed for 4 hours. CH.sub.2Cl.sub.2 (70 mL) and a teaspoon
of anhydrous MgSO.sub.4 were added and then filtered. The filtrate
was evaporated to dryness to yield 1.62 g of crude benzylic
alcohol. The product was purified by column chromatography on
silica gel and eluted with 50-60% ethyl acetate/hexanes to yield
1.25 g of 3,5-dimethyl-benzoic acid
N-tert-butyl-N'-(2-hydroxymethyl-3-methoxy-benzoyl)-hydrazide as a
white solid. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.1
(t, 1H), 7.05 (s, 2H), 6.98 (s, 1H), 6.9 (d, 1H), 6.5 (d, 1H), 4.2
(q, 2H), 3.79 (s, 3H), 2.23 (s, 6H), 1.57 (s, 9H). TLC (1:1 ethyl
acetate: hexane) Rf=0.20.
1.21 Preparation of 3,5-dimethyl-benzoic acid
N-tert-butyl-N'-(2-chloromethyl-3-methoxy-benzoyl)-hydrazide
(RG-115490)
##STR00030##
[0319] To a 50 mL round bottom flask, was added 400 mg (0.00315
mol) of oxalyl chloride and 5 mL of CH.sub.2Cl.sub.2. The mixture
was stirred and then cooled in acetone/dry ice bath to -70.degree.
C. 616-620 mg (0.0079 mol) of DMSO in 5 mL of CH.sub.2Cl.sub.2 was
slowly added and stirred for 30 min at -70.degree. C. 405 mg
(0.00105 mol) of RG-115371 in 4 mL of CH.sub.2Cl.sub.2 was added
and stirred for 30 min at -70.degree. C. The dry ice bath was
removed and the mixture was allowed to warm to room temperature
over 30 min. The mixture was cool again to -70.degree. C. and then
1.60 g (0.158 mol) of triethylamine was added and the mixture was
allowed to warm to room temperature. 6 mL of water was added to
quench the reaction. CH.sub.2Cl.sub.2 was added to the flask and
transferred to a separatory funnel with a total of 100 mL of
CH.sub.2Cl.sub.2. 50 mL of water were added and the aqueous layer
was again extracted with CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2
extract was extracted with dilute (0.05-0.1 N) HCl/H.sub.2O to
remove the Et.sub.3N and DMSO. The CH.sub.2Cl.sub.2 extract was
dried and concentrated to yield about 0.44 g of product. TLC:
Rf=0.47. The product can be purified by silica gel column
chromatography, eluting with 35-40% ethyl acetate in hexanes.
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 8.0 (s, 1H),
6.9-7.2 (t, s, s, d, 5H), 6.35 (d, 1H), 4.5 (d, 1H), 4.1 (d, 1H),
3.85 (s, 3H), 2.28 (s, 6H), 1.59 (s, 9H).
1.22 Preparation of 3,5-dimethyl-benzoic acid
N-tert-butyl-N'-(2-iodomethyl-3-methoxy-benzoyl)-hydrazide
##STR00031##
[0321] To a 50 mL flask containing 400 mg of the RG-115490, was
added 10 mL of CH.sub.3CN, 1 mL of DMF, and 100 mg of NaI. The
mixture was refluxed for 4 hours. The reaction was poured into 250
mL of ether and 75 mL of water in a separatory funnel. The mixture
was shaken vigorously and the ether layer extracted with ca. 50 mL
of water. The ether extracts were dried over MgSO.sub.4 and
charcoal, filtered, and the solvent removed to yield 250 mg of a
yellow solid. TLC Rf=0.50 (1:1 ethyl acetate: hexane). .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 6.9-7.3 (m, 6H), 6.3 (d, 1H),
4.3 (d, 1H), 4.2 (d, 1H), 3.88 (s, 3H), 2.28 (s, 6H), 1.62 (s,
9H).
1.23 Preparation of 3,5-dimethyl-benzoic acid
N-tert-butyl-N'-(2-methylaminomethyl-3-methoxy-benzoyl)-hydrazide
(RG-115079)
##STR00032##
[0323] Into a small flask, containing 300 mg of the RG-115490
dissolved in 15 mL of dioxane (99.8% anhydrous, Aldrich), was added
CH.sub.3NH.sub.2 in dioxane (4 eq). The reaction was refluxed for 2
hours. The solvent was removed on a rotovap, redissolved in
CH.sub.2Cl.sub.2, filtered, and the CH.sub.2Cl.sub.2 solubles were
concentrated. The methylamine was obtained after column
chromatography by elution with ethyl acetate, then 9:1 ethyl
acetate: methanol with about 0.2% triethylamine, after having run
the column with 4:1 ethyl acetate: hexane, 0.2% triethylamine. The
total yield of the product was 183 mg. TLC: Rf 0.23 (1:1 ethyl
acetate: hexane+triethylamine). .sup.1H NMR (CDCl.sub.3, 300 MHz),
.delta. (ppm): 7.3-6.9 (m, 6H), 3.80 (s, 3H), 3.6 (d, 1H), 2.8 (d,
1H), 2.4 (s, 3H), 2.28 (s, 6H), 1.59 (s, 9H).
1.24 Preparation of 3,5-dimethyl-benzoic acid
N-tert-butyl-N'-(2-dimethylaminomethyl-3-methoxy-benzoyl)-hydrazide
(RG-115079)
##STR00033##
[0325] 250 mg (0.0006 mol) of the RG-115490 was added to a 20 mL
vial. 3 mL of THF and 0.31 mL of a 2 M dimethylamine/THF solution
(Aldrich) was then added. The mixture was stirred for 4 hr at room
temperature. The solvent was removed on a rotovap and the solid was
triturated with hexane while stirring at room temperature.
3,5-Dimethyl-benzoic acid
N-tert-butyl-N'-(2-dimethylaminomethyl-3-methoxy-benzoyl)-hydrazide:
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.1-6.9 (m, 6H),
3.93 (s, 3H), 2.68 (s, 3H), 2.54 (s, 3H), 2.24 (s, 6H), 1.61 (s,
9H).
1.25 Preparation of 3,5-dimethyl-benzoic acid
N-tert-butyl-N'-(2-acetoxymethyl-3-methoxy-benzoyl)-hydrazide
(RG-115225)
##STR00034##
[0327] In a 20 mL vial containing 200 mg of RG-115371 in 4 mL of
anhydrous CH.sub.2Cl.sub.2, was added 200 mg of Et.sub.3N and 10 mg
of CH.sub.3COCl. The mixture was stirred at room temperature
overnight. TLC indicated an incomplete reaction. 100 mg of acetyl
chloride and some pyridine were added and refluxed for 1 hour. The
reaction mixture was poured into CH.sub.2Cl.sub.2 and extracted
with aqueous, dilute K.sub.2CO.sub.3, then dilute aqueous HCl. The
CH.sub.2Cl.sub.2 extract was dried and concentrated, to yield a
crude acetate. The material was purified by silica gel column
chromatography, eluting with 1:1 ethyl acetate: hexane. .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 7.3-6.9 (m, 6H), 6.4 (d, 1H),
4.8 (d, 1H), 4.6 (d, 1H), 3.795 (s, 3H), 2.3 (s, 6H), 2.03 (s, 3H),
1.58 (s, 9H).
1.26 Preparation of 2-methanesulfinylmethyl-3-methoxy-benzoic acid
N'-tert-butyl-N'-(3,5-dimethyl-benzoyl)-hydrazide (RG-115172)
##STR00035##
[0329] 3-Methoxy-2-methylsulfanylmethyl-benzoic acid
N'-tert-butyl-N'-(3,5-dimethyl-benzoyl)-hydrazide in
CH.sub.2Cl.sub.2 was stirred at room temperature with 1.0 eq of
m-chloroperbenzoic acid. The reaction was complete within 5 min as
indicated by TLC. The reaction mixture was washed with saturated
NaHCO.sub.3 and the organic layer was stripped under vacuum. The
residue was mixed with 1-2 mL of 1:1 ether: hexane and the solution
was removed with a pipette, leaving the product which was then
dried under vacuum.
1.27 Preparation of 2-methanesulfonylmethyl-3-methoxy-benzoic acid
N'-tert-butyl-N'-(3,5-dimethyl-benzoyl)-hydrazide (RG-115408)
##STR00036##
[0331] RG-115172 was dissolved in ethylene dichloride. 1.2 eq. of
m-chloroperbenzoic acid was added and the mixture was heated to
reflux. The reaction was complete by the time the mixture reached
reflux. After cooling to ambient temperature, the solution was
washed with saturated NaHCO.sub.3. The organic layer was stripped
under vacuum. The residue was mixed with 1-2 mL of ether and the
solution was removed with a pipette, leaving the product which was
dried under vacuum.
1.28 Preparation of 2,4,6-trimethyl-pyridine 1-oxide
##STR00037##
[0333] In a 500 mL round bottom flask equipped with a magnetic
stirrer and thermometer were added 36.7 g (164 mmol) of 77%
3-chloroperbenzoic acid (Aldrich) and 200 mL of methylene chloride.
This slurry was cooled to 5.degree. C. and a solution of 16.6 g
(137 mmol) of collidine (Aldrich) in 50 mL of methylene chloride
was added over 30 min while maintaining the temperature at
5-10.degree. C. The mixture was then allowed to warm to room
temperature over 1 hr and then stirred overnight. The crude
reaction mixture was transferred slowly to a beaker containing 200
g of basic alumina, which resulted in a slight warming of the
mixture. The mixture was stirred and filtered and the alumina was
mixed with 300 mL of 2:1 CHCl.sub.3:CH.sub.3OH. The solvent was
removed on a rotary evaporator at room temperature. The alumina was
washed with ether and the solvent was removed to yield 24.7 g of a
clear liquid, which solidified to give a white, waxy solid. This
yield was slightly high due to the presence of some salt. TLC
(silica gel developed with methanol) showed a single major spot
(Rf=0.45) along with a minor spot (Rf=0.55). The major spot was the
desired N-oxide. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm):
6.96 (s), 2.51 9(s) and 2.28 (s). The minor spot corresponded to
the starting collidine. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
(ppm): 6.78 (s), 2.48 (s) and 2.26 (s).
1.29 Preparation of (4,6-dimethyl-pyridin-2-yl)-methanol
##STR00038##
[0335] Under an atmosphere of nitrogen, 18.1 g (137 mmol) of the
collidine N-oxide was dissolved in 200 mL of methylene chloride
dried over molecular sieves. The mixture was cooled to 5.degree. C.
Trifluoroacetic anhydride (71.9 g, 49.8 ml, 343 mmol) was added
drop-wise in portions to maintain the reaction mixture at
5-10.degree. C. After addition of the trifluoroacetic anhydride,
the mixture was allowed to warm to room temperature and then
stirred at room temperature overnight. Subsequent TLC (reverse
phase, methanol/water, 7:3) showed the absence of the starting
N-oxide. The solvent was removed to yield the acetate product as a
yellow, waxy solid. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
(ppm): 2.5 (s, 3H), 2.8 (s, 3H), 5.65 (s, 2H), and 7.45 (m, 2H).
The mixture was cooled in an ice bath and 100 mL of a 10% solution
of KOH in methanol was added. The pH of the solution was checked,
and if the solution was not basic, additional KOH was added to make
the solution basic. The solution was then stirred at 10-15.degree.
C. for 30 min and then stirred at room temperature for 6 hours. The
solvent was removed to yield 7.4 g of a yellow-brown syrup. If
desired, the alcohol could be purified by careful chromatography,
using silica gel and eluting with ethyl acetate/chloroform (4:1).
The alcohol was isolated as a pale, yellow oil. TLC: Rf is 0.55 in
silica gel, developed with methanol/ethyl acetate, 1:1. .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 6.67 (s, 2H), 4.67 (s, 2H),
2.50 (s, 3H) and 2.31 (s, 3H). In most cases, the crude alcohol was
sufficiently pure to be used for the subsequent oxidation
reaction.
1.30 Preparation of 4,6-dimethyl-2-pyridinecarboxylic acid
##STR00039##
[0337] 4,6-Dimethyl-2-pyridinemethanol (7.5 g, 29.2 mmol) was added
to 100 mL of water and stirred at 0-5.degree. C. A solution of 5.3
g (32.1 mmol) of potassium permanganate in 100 mL of water was
added portion-wise over 30 min while maintaining the temperature at
5-10.degree. C. This resulted in the formation of a black solid.
The mixture was stirred at 5-10.degree. C. for an additional 30 min
and then allowed to stir at room temperature for 30 min. The
mixture was filtered and the manganese dioxide washed with
methanol. The methanol washings were combined with the water
extracts and the solvent was removed. The resulting tan solid was
redissolved in water and washed with chloroform. The water layer
was separated and the water removed to yield 5.4 g of
4,6-dimethyl-2-pyridinecarboxylic acid. The product was
characterized by HPLC/MS.
1.31 Preparation of pyrazine-2-carboxylic acid
N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide
(RG-115550)
##STR00040##
[0339] Into a 20 mL vial, containing a stirred mixture of 0.120 g
(0.003 mol) of NaH in 6 mL of DMF, was slowly added 0.238 g (0.001
mol) of 2-ethyl-3-methoxy-benzoic acid N'-tert-butyl-hydrazide. The
reaction was stirred for 1 hr at room temperature. 0.278 g (0.001
mol) of pentafluorophenyl ester pyrazine-2-carboxylic acid
pentafluorophenyl ester in 2 mL of DMF was slowly added. The
reaction was stirred for 24 hours. The reaction was washed out with
ethyl acetate into a separatory funnel containing 100 mL of water
and 100 mL of ethyl ether. The reaction mixture was shaken and the
organic phase was dried over MgSO.sub.4 and concentrated to dryness
to yield pyrazine-2-carboxylic acid
N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide: .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): (note--a 1:3 reaction ratio of
hydrazide to NaH gave highest yield of product (100%), lesser
ratios, such as 1:2, only yielded 70%; DMF was a better solvent
than DMSO). The progress of the reaction was monitored by following
the intensity of OCH.sub.3 signals in .sup.1H NMR. .sup.1H NMR (300
MHz, CDCl3) .delta. ppm: 9.05 (1H), 8.6 (s, 1H), 8.45 (s, 1H), 8.4
(s, 1H), 7.1 (t, 1H), 6.9 (d, 1H), 6.45 (d, 1H), 3.8 (s, 3H), 2.4
(m, 1H), 1.95 (m, 1H), 1.62 (s, 9H), 0.95 (t, 3H).
Pyrazine-2-carboxylic acid pentafluorophenyl ester. .sup.1H NMR
(300 MHz, CDCl3) .delta. ppm: 9.49 (s, 1H), 8.95 (d, 1H), 8.87 (d,
1H).
TABLE-US-00002 TABLE 1 Optimization of the preparation of RG-115550
##STR00041## ##STR00042## base/ solvent Temp Time yield comment NaH
(1 eq) 20 16 40 DMF NaH (2 eq) 20 16 65-70 DMF NaH (3 eq) 20 16 100
Pentafluorophenyl ester free of DMF DCC-derived urea NaH (2 eq) 20
16 45 DMSO ##STR00043##
TABLE-US-00003 TABLE 2 Preparation of heterocyclic diacylhydrazines
by the pentafluorophenyl ester method. ##STR00044## R NMR yield
.sup.1H NMR (300 MHz, CDCl.sub.3) ##STR00045## ca. 30%
Diacylhydrazine: 9.1 (s, 1H), 8.4 (d, 1H), 7.85 (d, 1H), 7.1 (t,
1H), 6.9 (d, 1H), 6.3 (d, 1H), 4.0 (s, 3H), 3.82 (s, 3H), 2.4 (m,
1H), 2.1 (m, 1H), 1.7 (s, 9H), 0.95 (t, 3H) R-Pentafluorophenyl
ester: 9.45 (s, 1H), 8.6 (d, 1H), 8.4 (d, 1H), 4.04 (s, 3H)
##STR00046## ca. 20% Diacylhydrazine: Isomer 1: 9.4 (br, 1H), 7.95
(s, 1H), 7.7 (s, 1H), 7.3 (m, 10H), 7.1 (m, 5H), 6.85 (t, 1H), 6.75
(d, 1H), 6.5 (d, 1H), 6.05 (d, 1H), 3.75 (s, 3H), 2.1 (m, 1H), 1.9
(m, 1H), 1.57 (s, 9H), 0.8 (t, 3H), Isomer 2: 8.6 (br, 1H), 7.9 (s,
1H), 7.8 (s, 1H), 7.3 (m, 10H), 7.1 (m, 5H), 6.9 (t, 1H), 6.75 (d,
1H), 6.5 (d, 1H), 6.05 (d, 1H), 3.75 (s, 3H), 2.1 (m, 1H), 1.9 (m,
1H), 1.57 (s, 9H), 0.85 (t, 3H) R-pentafluorophenyl ester: 8.15 (s,
1H), 8.05 (d, 1H), 7.9 (d, 1H), 7.1-7.5 (m, 15H) ##STR00047## 100%
Diacylhydrazine: 7.75 (s, 1H), 7.55 (d, 1H), 7.35 (d, 1H), 7.3 (m,
1H), 7.15 (t, 1H), 7.0 (t, 1H), 6.8 (d, 1H), 6.6 (s, 1H), 6.4 (d,
1H), 3.9 (s, 3H), 3.75 (s, 3H), 2.2 (m, 1H), 1.9 (m, 1H), 1.62 (s,
9H), 0.85 (t, 3H) R-pentafluorophenyl ester: 7.75 (d, 1H), 7.65 (s,
1H), 7.5 (br s, 2H), 7.2 (m, 1H), 4.09 (s, 3H) ##STR00048## 100%
Diacylhydrazine: 7.85 (s, 1H), 7.65 (s, 1H), 7.5 (m, 3H), 7.4 (m,
2H), 7.15 (t, 1H), 6.9 (d, 1H), 6.65 (d, 1H), 3.81 (s, 3H), 2.6 (m,
1H), 2.5 (s, 3H), 2.2 (m 1H), 1.6 (s, 9H), 1.1 (t, 3H)
R-pentafluorophenyl ester: 8.25 (s, 1H), 7.6 (m, 3H), 7.5 (m, 2H),
2.62 (s, 3H) ##STR00049## 100% Diacylhydrazine: 8.5 (s, 1H), 7.95
(s, 1H [NH]), 7.1 (t, 1H), 6.87 (d, 1H), 6.3 (d, 1H), 3.85 (s, 3H),
2.55 (s, 3H), 2.5 (m, 1H), 2.3 (m, 1H), 1.64 (s, 9H), 1.05 (t, 3H).
R-pentafluorophenyl ester: 8.8 (s, 1H), 2.62 (s, 3H) Procedure for
pentafluorophenyl ester formation: Heterocyclic carboxylic acid and
pentafluorphenol are dissolved in anhydrous dioxane, ethyl acetate,
dimethoxyethane, or THF under and N.sub.2 atmosphere. One
equivalent of dicyclohexylcarbodiimide (DCC) is added. The reaction
is stirred at room temperature overnight. A trace of water is then
added to quench any remaining DCC. The DCC-derived urea (DCU) is
removed by filtration on Celite, the filtrate is washed with dilute
NaHCO.sub.3 to remove remaining pentafluorophenol, and the filtrate
is evaporated to dryness. The product is purified by trituration or
chromatography on silica gel. It is thought that the level of trace
DCC or urea in the pentafluorophenyl ester may be critically
detrimental to the success of the NaH amide coupling reation.
1.32 Preparation of 1H-Indazole-3-carboxylic acid
N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide
(RG-115723)
##STR00050##
[0341] Into a 10 mL round bottom flask, was added 0.328 g (0.001
mol) of pentafluorophenyl ester of indazole-3-carboxylic acid,
0.244 g (0.001 mol) of triphenylmethane, 0.227 g (0.001 mol) of
2,3-dichloro, 5,6-dicyano, 1,4-benzoquinone and 4 mL of dry
toluene. The reaction mixture was refluxed for 7-8 hours. The
reaction mixture was washed out with ethyl acetate (60 mL) into a
separatory funnel and extracted with water (20 mL). The organic
phase was dried and concentrated to yield 0.45 g of
1-trityl-1H-indazole-3-carboxylic acid pentafluorophenyl ester. NMR
indicated the presence of the product, but TLC also showed the
presence of the starting pentafluorophenyl ester (Rf 57) and
product (Rf 70). The product was purified by column chromatography
on silica gel, eluted with 5% ethyl acetate/hexane to yield 0.30 g
of pure product. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm):
8.2 (d, 1H), 7.4-7.0 (m, 17H), 6.52 (d, 1H).
##STR00051##
[0342] 0.17 g (0.16 mmol) of 1-trityl-1H-indazole-3-carboxylic acid
pentafluorophenyl ester in 3 mL of CH.sub.2Cl.sub.2 was added to a
100 mL flask with 20 mL of CH.sub.2Cl.sub.2, containing 2% TFA and
1% H.sub.2O. The reaction was stirred at room temperature for 90
min. TLC indicated 50% reaction. An additional 15 mL of the
TFA--H.sub.2O--CH.sub.2Cl.sub.2 was added and stirring continued
for 1 hr. The reaction was transferred to a separatory funnel and
washed with ca. 0.5 M K.sub.2CO.sub.3/H.sub.2O. The
CH.sub.2Cl.sub.2 phase was dried and concentrated to give crude
1H-indazole-3-carboxylic acid
N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide (0.15 g).
TLC: product, Rf 0.33; starting material, Rf=0.68. The product was
purified by column chromatography, eluting with 45% ethyl
acetate/hexane. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm):
8.6 (s, 1H), 8.2 (s, 1H), 7.4-7.2 (m, 3H), 7.1 (t, 1H), 6.9 (d,
1H), 6.7 (d, 1H), 3.80 (s, 3H), 2.4 (m, 1H), 2.0 (m, 1H), 1.652 (s,
9H), 0.85 (t, 3H).
1.33 Preparation of 3H-benzoimidazole-5-carboxylic acid
N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide
(RG-115718)
##STR00052##
[0344] About 120 mg of 3-trityl-3H-benzoimidazole-5-carboxylic acid
N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide were
dissolved in 35 mL of CH.sub.3OH and added into a hydrogenation
bottle, together with 2 drops of glacial acid and 0.20 g Pd/C.
Hydrogenation was conducted by shaking the bottle for 6 hours and
then remaining under H.sub.2 pressure for 16 hours. The Pd/C was
removed by filtration and the methanol removed by an evaporator.
The residue was stirred with CH.sub.2Cl.sub.2 and the
CH.sub.2Cl.sub.2 was decanted. Evaporation of the CH.sub.2Cl.sub.2
yielded a solid product identified by NMR, as triphenylmethane. The
residue was stirred with dilute KOH/H.sub.2O and extracted with
ethyl acetate. The ethyl acetate was dried and concentrated on a
rotary evaporator to yield the product
3H-benzoimidazole-5-carboxylic acid
N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide. NMR analysis
of the product indicated the absorbances for the methoxy and
t-Butyl groups occurred at 3.69 and 1.61 vs. 3.77 and 1.59 for the
starting material.
1.34 Preparation of
N'-2-Ethyl-3-methoxybenzoyl-N-t-butyl-N--(N-methylindole-2-carbonyl)
hydrazide
[0345] N'-2-Ethyl-3-methoxybenzoyl-N-t-butyl hydrazide (150 mg, 0.6
mmol) was dissolved in 2 mL of DMF. Potassium t-butoxide (80 mg,
0.7 mmol) was added and magnetically stirred for about 5 min.
N-Methylindole-2-carboxylic acid pentafluorophenyl ester was added
and the mixture was heated to 100.degree. C. After 3 hours the
reaction was complete as indicated by TLC. The mixture was cooled
to ambient temperature and poured into 10 mL of water. Two
extractions with methylene chloride were combined and evaporated.
The residue was mixed with about 2 mL of 1:1 ethyl ether: hexane.
The mother liquors were removed by pipette and the residue dried
under vacuum. The product was a tan solid weighing 140 mg. LC MS
analysis confirmed the structure and estimated the purity (UV
detection) at 91%. (Yield=52%).
1.35 Preparation of 2,6-dimethoxy-nicotinic acid
N-tert-butyl-N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydra-
zide (RG-115517)
##STR00053##
[0347] 2.0 g (10.92 mmol) of 2,6-dimethoxyisonicotinic acid was
dissolved in 100 mL of toluene and then 1 drop of dimethyl
formamide was added. 1.55 g (13.1 mmol, 0.98 mL) of thionyl
chloride was added and the solution was refluxed for 4 hours. The
toluene and excess thionyl chloride were removed under vacuum and
2,6-dimethoxyisonicotinoyl chloride was used without further
purification.
##STR00054##
[0348] In a 1 oz vial, with a stirbar, 1 mL of 1 M K.sub.2CO.sub.3
was added. 0.250 g (1.2 mmol) of
5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid
N'-tert-butyl-hydrazide was dissolved in 2 mL of methylene chloride
and added to the aqueous solution. 2,6-dimethyl-isonicotinoyl
chloride was then added and the mixture was allowed to stir at room
temperature overnight. The aqueous layer was removed and the
organic layer was washed twice with 2 mL of a 1 M K.sub.2CO.sub.3
solution followed by 2 mL of water. The water layer was removed and
the organic layer was dried over MgSO.sub.4. The organic layer was
filtered and then removed. The product, 2,6-dimethoxy-nicotinic
acid
N-tert-butyl-N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydra-
zide, was purified by trituration with 1:1 ether: hexane or
chromatography. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm):
1.6 (s, 9H), 1.9 (s, 3H), 3.9 (s s, 6H), 4.2 (m, 4H), 6.2 (m, 1H),
6.7 (d, 1H), 7.7 (d, 1H), 8.3 (m, 1H).
1.36 Preparation of 4-Hydroxy-3,5-dimethoxy-benzoic acid
N-tert-butyl-N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydra-
zide (RG-115009)
##STR00055##
[0350] 4-Acetoxy-3,5-dimethoxy-benzoic acid (1.45 g) was heated
with thionyl chloride (0.86 g) in 3 mL of dimethoxyethane. After
1.5 hours the mixture was stripped under vacuum leaving 1.60 g of
4-acetoxy-3,5-dimethoxy-benzoyl chloride as an oil. .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 2.26 (s, 3H), 3.89 (s, 9H),
7.34 (s, 2H).
##STR00056##
[0351] 4-Acetoxy-3,5-dimethoxy-benzoyl chloride (250 mg) and
5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid
N'-tert-butyl-hydrazide (204 mg) were dissolved in 3 mL of
dichloromethane and stirred at ambient temperature with 1.5 mL of a
1 M aqueous sodium carbonate solution. After two hours the phases
were separated and the organic phase was evaporated. The solid
residue was washed with 1:1 ether: hexane leaving 360 mg of acetic
acid
44N-tert-butyl-N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hyd-
razinocarbonyl]-2,6-dimethoxy-phenyl ester.
[0352] .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 1.60 (m,
15H), 2.06 (s, 3H), 2.32 (s, 3H), 3.77 (s, 6H), 4.2 (m, 4H), 6.08
(d, 1H), 6.63 (d, 1H), 6.74 (s, 2H).
##STR00057##
[0353] Acetic acid
4-[N-tert-butyl-N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hy-
drazinocarbonyl]-2,6-dimethoxy-phenyl ester (300 mg) was dissolved
in methanol with 28% aqueous ammonia (750 mg). The mixture was
stirred at ambient temperature over the weekend. The precipitate
was filtered to provide 110 mg of white solid
4-hydroxy-3,5-dimethoxy-benzoic acid
N-tert-butyl-N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydra-
zide. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 1.59 (s,
15H), 2.02 (s, 3H), 3.85 (s, 6H), 4.21 (m, 4H), 5.6-5.7 (broad s,
1H), 6.22 (d, 1H), 6.58 (d, 1H), 6.81 (s, 2H).
1.37 Preparation of Compounds RG-115613 and RG-115429
##STR00058##
[0355] Into a 100 mL round bottom flask containing 2.50 g (10 mmol)
of 2-ethyl-3-methoxy-benzoyl-N'-tert-butyl-hydrazide was added 15
mL of methylene chloride, 2.60 g (10.5 mmol) of
3-bromomethyl-5-methylbenzoyl chloride in 5 mL of methylene
chloride and a solution of 2.76 g (20 mmol) of potassium carbonate
in 15 mL of water. The reaction mixture was stirred overnight at
room temperature, then diluted with 20 mL of methylene chloride and
transferred to a separatory funnel. The methylene chloride layer
was separated and dried, and the solvent was removed in vacuo. The
crude product was purified by column chromatography to yield 4.01 g
of N-(3-bromomethyl-5-methyl-benzoyl)-N-tert-butyl-N'-(2-ethyl-3-m-
ethoxy-benzoyl) hydrazide (87% yield). .sup.1H NMR (CDCL.sub.3, 300
MHz) .delta. (ppm): 7.41 (s, 1H), 7.1 (m, 3H), 7.02 (t, 1H), 6082
(d, 1H), 6.08 (d, 1H), 4.41 (s, 2H), 3.78 (s, 3H), 2.4 (m, 1H),
2.31 (s, 3H), 2.25 (m, 1H), 1.60 (s, 9H), 1.01 (t, 3H).
##STR00059##
[0356] To 4.00 g (8.68 mmol) of
N-(3-bromomethyl-5-methyl-benzoyl)-N-tert-butyl-N'-(2-ethyl-3-methoxy-ben-
zoyl) hydrazide, contained in a 250 mL round bottom flask, were
added 40 mL of dioxane, 40 mL of water, and 4.34 g of calcium
carbonate. The reaction flask was placed into an 85.degree. C. oil
bath and the reaction was stirred and heated for 18 hours. The
reaction mixture was cooled, transferred to a larger flask with
ethyl acetate and most of the dioxane was evaporated. The reaction
mixture was shaken with about 100 mL of ethyl acetate and filtered.
The ethyl acetate layer was separated and the aqueous layer
extracted twice with ethyl acetate. Ethyl acetate extract was dried
and evaporated to yield 2.07 g of
N-(3-hydroxymethyl-5-methyl-benzoyl)-N-tert-butyl-N'-(2-ethyl-3-methoxy-b-
enzyl) hydrazide (60% yield). .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. (ppm): 7.78 (s, 1H), 7.1-7.4 (3s, 3H), 6.96 (t, 1H), 6.8
(d, 1H), 6.08 (d, 1H), 4.53 (s, 2H), 3.77 (s, 3H), 2.35 (m, 1H),
2.32 (s, 3H), 2.2 (m, 1H), 1.60 (s, 9H), 0.96 (t, 3H).
##STR00060##
[0357] To 2.00 g (5.02 mmole) of
N-(3-hydroxymethyl-5-methyl-benzoyl)-N-tert-butyl-N'-(2-ethyl-3-methoxy-b-
enzoyl)hydrazide placed in a 250 mL round bottom flask, were added
100 mL of methylene chloride and 1.16 g of pyridinium
chlorochromate. The reaction mixture was refluxed for about 1 hour,
at which time TLC (1:1 ethyl acetate: hexane) indicated the
formation of the product (R.sub.f=0.5). The reaction mixture was
concentrated to about 20 mL and then chromatographed on silica gel.
Elution with 30-35% ethyl acetate in hexane yielded 1.75 g (88%)
3-formyl-5-methyl-benzoic acid
N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide as a white
solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. (ppm): 9.93 (s,
1H), 7.6-7.8 (3s, 3H), 7.0 (t, 1H), 6.82 (d, 1H), 6.19 (d, 1H),
3.77 (s, 3H), 2.42 (s, 3H), 2.3 (m, 1H), 2.0 (m, 1H), 1.62 (s, 9H),
0.90 (t, 3H).
##STR00061##
[0358] To 100 mg (0.25 mmoles) of
N-(3-formyl-5-methyl-benzoyl)-N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-
hydrazide placed in a 20 mL vial, were added 2 mL of methanol, 112
mg of semicarbazide hydrochloride, 0.2 g triethylamine and a drop
of glacial acetic acid. The reaction mixture was magnetically
stirred on a hot plate adjusted to 50.degree. C. for about 3 hours,
then at room temperature for 48 hours. The solvent was evaporated
with a stream of nitrogen and the resulting residue was dissolved
in 20 mL of chloroform and extracted with dilute HCl. The
chloroform extract was dried, the solvent was removed in vacuo, and
the residue was dried in a vacuum oven at 60.degree. C. The residue
was cooled and triturated with hexane to yield 81 mg of
semicarbazide of
N-(3-formyl-5-methyl-benzoyl)-N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)
hydrazide. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. (ppm): 8.9
(broad s, 1H), 8.6 (broad s, 1H), 7.3-7.5 (3s, 3H), 7.03 (t, 1H),
6.8 (d, 1H), 6.38 (d, 1H), 3.76 (s, 3H), 3.26 (d, 1H), 2.4 (s, 3H),
1.95 (m, 1H), 1.57 (s, 9H), 0.95 (t, 3H).
##STR00062##
[0359] To 100 mg (0.25 mmol) of
N-(3-formyl-5-methyl-benzoyl)-N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-
hydrazide placed in a 20 mL vial, was added 2 mL of methanol, 103
mg of oxamic hydrazide hydrochloride, and a drop of glacial acetic
acid. The reaction mixture was magnetically stirred on a hot plate
adjusted to 50.degree. C. for about 3 hours, then at room
temperature for 48 hours. The solvent was evaporated with a stream
of nitrogen and the resulting residue was dried in a vacuum oven at
60.degree. C. The residue was cooled and triturated with hexane to
yield 70 mg of oxamic hydrazone of
N-(3-formyl-5-methyl-benzoyl)-N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)
hydrazide. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. (ppm): 8.5
(s, 1H), 8.6 (d, 1H), 7.9 (d, 1H), 7.55 (s, 1H), 7.5 (s, 1H), 7.3
(s, 1H), 7.0 (t, 1H), 6.95 (d, 1H), 3.73 (s, 3H), 2.2 (m, 1H), 2.35
(s, 3H), 1.95 (m, 1H), 1.51 (s, 9H), 0.80 (t, 3H).
1.38 Preparation of 3,5-dichloro-4-fluorobenzoic acid
[0360]
(N-(3-methoxy-2-methylbenzoyl)-N'-(3,5-dichloro-4-fluorobenzoyl)-N'-
-tert-butylhydrazine can be prepared according to U.S. Pat. No.
5,530,028. Briefly, the product of Example 8 and
3,5-dichloro-4-fluorobenzoyl chloride can be prepared in accordance
with Example 9 to yield
(N-(3-methoxy-2-methylbenzoyl)-N'-(3,5-dichloro-4-fluorobenzoyl)-N'-tert--
butylhydrazine.
##STR00063##
[0361] 3,5-dichloro-4-fluorobenzoyl chloride can be prepared as
follows: To a round bottom flask with nitrogen purge through a 10%
aqueous NaOH trap, was added 3,5-dichloro-4-flurobenzotriflouride
(5.00 g, 21.46 mmol, Aldrich), concentrated sulfuric acid (4.30 g,
42.92 mmol), and finally chlorosulfonic acid (5.15 g, 43.78 mmol).
The reaction began to bubble immediately. After the bubbling
subsided, the mixture was heated to 80.degree. C. for 1 hr, cooled
to room temperature, and added cautiously to stirred ice water.
This was extracted twice with methylene chloride. The combined
extracts were washed with water and brine, dried over magnesium
sulfate, filtered, and evaporated to give a white solid (2.19 g) of
3,5-dichloro-4-fluorobenzoic acid in 49% yield. .sup.1H-NMR
(CD.sub.3COCD.sub.3, 300 MHz) .delta. (ppm): 7.95 (d, 2H).
##STR00064##
[0362] 3,5-dichloro-4-fluorobenzoic acid was refluxed with >1
equivalent of thionyl chloride and one drop of DMF neat or as a
solution in CHCl.sub.3. The solvent and volatile by-products were
removed in vacuo to provide 3,5-dichloro-4-fluorbenzoyl
chloride.
1.39 Preparation of 3,5-dimethoxy-4-methyl-2-nitrobenzoic acid
N-t-butyl-N'-(5-methy-lbenzo-1,4-dioxan-6-carbonyl)-hydrazide
(RG-115609)
##STR00065##
[0364] 3,5-Dimethoxy-4-methylbenzoic acid was slurried with 2.7 eq.
Of acetic anhydride and 0.05 molar equivalents of concentrated
sulfuric acid in dichloromethane. After cooling to 10.degree. C.,
1.05 eq. Of 70% nitric acid was added drop-wise while the
temperature was maintained below 15.degree. C. After 30 min the
mixture was poured into water and extracted twice with ethyl
acetate. The combined organic extracts were concentrated until a
thick slurry was present. The slurry was filtered and the solid
washed with ice-cold dichloromethane. Further concentration of the
mother liquors gave a second crop. The total yield of
3,5-dimethoxy-4-methyl-2nitrobenzoic acid was about 80%. .sup.1H
NMR: (acetone-d6, 300 MHz) .delta. (ppm): 7.34 (s, 1H), 4.00 (s,
3H), 3.85 (s, 3H), 2.23 (s, 3H).
##STR00066##
[0365] 3,5-Dimethoxy-4-methyl-2nitrobenzoic acid was stirred with
1.1 eq. Of thionyl chloride at ambient temperature in
dimethoxyethane until the reaction was complete. The solvent and
excess thionyl chloride were distilled at atmospheric pressure and
the residue dissolved in dichloromethane. This solution was added
to a mixture of 1 M aqueous potassium carbonate and
5-methylbenzo-1,4-dioxan-6-benzoic acid-N'-t-butyl-hydrazide in
dichloromethane. After 3 hr, water and dichloromethane were added.
The organic phase was removed and stripped under vacuum. The
residue was triturated with 1:1 (wt: wt) ether: hexane to provide
3,5-dimethoxy-4-methyl-2-nitrobenzoic acid
N-t-butyl-N'-(5-methy-lbenzo-1,4-dioxan-6-carbonyl)-hydrazide (ca
94% yield). TLC (1:1 ethyl acetate: hexane) indicated one spot, Rf
0.53. .sup.1H NMR: (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.84 (s,
1H), 7.02 (t, 1H), 6.86 (d, 1H), 6.82 (s, 1H), 6.11 (s, 1H), 3.90
(m, 4H), 3.79 (s, 6H), 2.16 (s, 3H), 1.60 (s, 9H).
1.40 Preparation of 3,5-dimethyl-benzoic acid
N'-(2,3-dimethyl-benzoyl)-N-(1-ethyl-2,2-dimethyl-propyl)-hydrazide
(RG-103309)
##STR00067##
[0367] T-butylcarbazate (35.15 g, 266 mmol) and 200 mL of
CH.sub.2Cl.sub.2 were added to a round bottom flask. Potassium
carbonate (55.2 g, 0.4 moles) dissolved in 350 mL of water was
added to the flask, and the mixture was stirred for 15 minutes with
ice chilling. 2,3 dimethylbenzoyl chloride (44.9 g, 266 mmol) in
ca. 200 mL of CH.sub.2Cl.sub.2 was added drop-wise from a 500 mL
separatory funnel over 30 minutes. The reaction was allowed to stir
overnight and then the reaction mixture was poured into a 1 L
separatory funnel and the CH.sub.2Cl.sub.2 phase was separated.
Then ca. 150 mL of water was added, and the mixture was extracted
twice with 150 mL of CHCl.sub.3. The combined organic phase was
back-extracted with 100 mL of water, then with 1N HCl (250 mL), to
remove the hydrazide. The organic phase was dried, stirred with
charcoal, and the solvent removed in vacuo to yield a light tan
solid (71.5 g) of N'-(2,3-dimethyl-benzoyl)-hydrazinecarboxylic
acid, tert-butyl ester. .sup.1H NMR (300 MHz, CHCl.sub.3) .delta.
(ppm): 7.7 (br, 1H), 7.22 (m, 2H), 7.1 (t, 1H), 7.85 (br, 1H), 2.35
(s, 3H), 2.3 (s, 3H), 1.5 (s, 9H).
##STR00068##
[0368] N'-(2,3-Dimethyl-benzoyl)-hydrazinecarboxylic acid,
tert-butyl ester (70.3 g, 266 moles) was placed in to a 500 mL
round bottom flask. With gentle stirring, 200 mL of trifluoroacetic
acid (296 g, 2.6 moles) was slowly added, resulting in a vigorous
evolution of gas. The reaction mixture was then stirred at room
temperature for 2 hours. Water (ca. 100 mL) was then added slowly
to the mixture. The mixture was slowly added to 1 L of a cold 2 M
K.sub.2CO.sub.3 solution, contained in a 2 L beaker, while stirring
slowly (evolution of gas). About 200 mL of a 10% NaOH solution and
250 mL of CH.sub.2Cl.sub.2 were added. The reaction mixture was
transferred to a large separatory funnel and gently shaken (gas
evolution). The aqueous phase was extracted with CHCl.sub.3 and the
extracts dried and evaporated to yield a white solid, which was
dried in a 50.degree. C. vacuum oven to yield 31.72 g (73% yield)
of 2,3-dimethyl-benzoic acid hydrazide. .sup.1H NMR (CDCl.sub.3,
300 MHz) .delta. (ppm): 7-7.3 (m, 4H), 4.00 (br s, 2H), 2.271 (s,
6H).
##STR00069##
[0369] In a 200 mL round bottom flask, 7.90 g (48 mmol) of
2,3-dimethyl-benzoic acid hydrazide was dissolved in 60 mL of
methanol and 3 drops of glacial acetic acid were then added. To the
reaction mixture was added 6.00 g (52.6 mmol) of
2,2-diemthylpentan-3-one, and the reaction was stirred at room
temperature for 24 hours. The product hydrazone was not isolated,
but subjected directly to reduction. Glacial acetic acid (10 mL)
and sodium cyanoborohydride (3.2 g, 50.95 mmol) were added to the
reaction mixture, which was then stirred at room temperature for 24
hours. About 50 mL of 10% aqueous NaOH solution was added and most
of the CH.sub.3OH was removed on a rotary evaporator. The reaction
was diluted with water (100 mL) and the product was extracted with
CH.sub.2Cl.sub.2. The organic extract was dried and evaporated to
yield 11.87 g (94%) of 2,3-dimethyl-benzoic acid
N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. (.pi..pi..mu.): 7.0-7.3 (m, 4H), 2.32 (s, 3H),
2.296 (s, 3H), 1.7 (m, 1H), 1.3 (m, 1H), 1.16 (t, 3H, 0.979 (s,
9H). TLC: Rf=0.57 (1:1 ethyl acetate: hexane), indicated >90%
purity. Further purification can be achieved by silica gel
chromatography and elution of product with 20% ethyl acetate in
hexanes.
##STR00070##
[0370] 2,3-dimethyl-benzoic acid
N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide (0.59 g, 2.25 mmol) was
dissolved in 15 mL of CH.sub.2Cl.sub.2 in a small round-bottom
flask. Aqueous K.sub.2CO.sub.3 solution (0.70 g in 150 mL of
H.sub.2O) was added. 3,5-dimethylbenzoyl chloride (0.45 g, 2.7
mmol) dissolved in 10 mL of CH.sub.2Cl.sub.2 was added and the
reaction mixture was stirred at room temperature for 24 hours. The
reaction mixture was transferred to a separatory funnel and
extracted with CH.sub.2Cl.sub.2. (In another experiment, this was
washed with weak NaOH to get rid of excess acid and acid chloride).
The extract was dried and evaporated to give about 1 g of a white
solid, which was purified by silica gel chromatography. Elution
with 15% ethyl acetate in hexane yielded pure product of
3,5-dimethyl-benzoic acid
N'-(2,3-dimethyl-benzoyl)-N-(1-ethyl-2,2-dimethyl-propyl)-hydrazide
(0.62 g, 70%).
[0371] .sup.1H NMR (500 MHz, CDCl.sub.3) 6=(ppm): 6.95-7.4 (m, 7H),
4.61 (m, 1H), 2.2-2.4 (multiple s, 9H), 1.81 (s, 3H), 1.6-1.8 (m,
2H), 1.3 (br t, 3H), 1.08 (multiple br s, 9H).
1.41 Preparation of 3,5-dimethyl-benzoic acid
N-(1-ethyl-2,2-dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide
(RG-115819)
##STR00071##
[0373] 10.34 g (57.5 mmol) of 3-methoxy-2-methyl-benzoic acid
hydrazide (lot CPO 10925) was dissolved in 100 mL of methanol and
stirred. 9.80 g (86.3 mmol) of 2,2-dimethyl-3-pentanone was added
followed by 3 drops of glacial acetic acid. The mixture was allowed
to stir for 48 hours. The crude mixture was then brought to pH=3.
3.84 g (61.2 mmol) of NaCNBH.sub.3 was then added. The slurry was
allowed to stir at room temperature overnight, and then the
methanol was removed. 100 mL of 10% NaOH and 100 mL methylene
chloride were added. The mixture was shaken and the methylene
chloride layer removed. The aqueous layer was washed twice with 75
mL of methylene chloride. The methylene chloride layers were
combined and dried. Removal of the solvent yielded the desired
product as a pale, yellow liquid. The product could also be
obtained by reduction of the hydrazide with 10% Pd on carbon.
Purification was accomplished by column chromatography on silica
gel, eluting with 3:2 hexane: ether. .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. (ppm): 1.0 (s, 9H), 1.1 (t, 3H) 1.2 (m, 1H), 1.5 (m
2H), 2.1 (s, 3H), 3.8, (s, 3H), 4.9 (br s, 1H), 6.6 (d, 1H),
7.0-7.2 (m, 2H).
[0374] TLC: Rf=0.45 (1:1 hexane: ether).
##STR00072##
[0375] 6.2 g (22.1 mmol) of 3-methoxy-2-methyl-benzoic acid
(1-ethyl-2,2-dimethyl-propyl)-hydrazide was dissolved in 35 mL of
ethyl acetate and cooled to 0.degree. C. 74 mL of a 1N aqueous
K.sub.2CO.sub.3 was added and the mixtures stirred. 5.6 g (33.6
mmol) of 3,5-dimethylbenzoyl chloride was dissolved in 40 mL ethyl
acetate and this solution was added to the hydrazide mixture over
15 min. The mixture was allowed to warm to room temperature and
stirred overnight. The aqueous layer was then removed and the
organic layer was washed with 75 mL of a 1N aqueous K.sub.2CO.sub.3
solution and then with 100 mL of water. The water layer was removed
and the organic layer was dried and removed to yield an off-white
solid. This material was triturated three times with 25 mL of 1:1
hexane: ether to yield the final product in 98.7% purity. .sup.1H
NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 0.7-1.5 (m, 15H), 2.1 (s,
9H), 3.7 (s, 3H), 6.8-7.1, (m, 6H).; TLC: Rf=0.62 (1:1 hexane:
ether).
1.42 Preparation of 3,5-dimethoxy-4-methyl-benzoic acid
N-(1-ethyl-2,2-dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide
(RG-115820)
[0376] ##STR00073## [0377] RG-115820
[0378] Into a 20 mL vial was added 161 mg (0.75 mmol) of 3,5
dimethoxy, 4-methyl benzoyl chloride, a 5 mL solution of
3-methoxy-2-methyl-benzoic acid
N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide, and 1.5 mL of aqueous
25% K.sub.2CO.sub.3. The reaction mixture was stirred at room
temperature for 24 hours. The reaction mixture was transferred to a
separatory funnel with CH.sub.2Cl.sub.2 and shaken with dilute
aqueous NaHCO.sub.3. The organic phase was dried, concentrated and
chromatographed on silica. 100 mg of pure product,
3,5-dimethoxy-4-methyl-benzoic acid
N-(1-ethyl-2,2-dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide-
, was eluted with 25% ethyl acetate/hexane. TLC: Rf=0.54 (1:1 ethyl
acetate: hexane); .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm):
6.6-7.2 (m, 4H), 6.25 (d, 1H), 4.6 (d, 1H), 3.8-3.95 (br s, 9H),
2.1 (br s, 3H), 1.9 (s, 3H), 1.6 (br, 2H), 1.3 (m, 3H), 0.9-1.2 (br
d, 9H).
1.43 Preparation of 2,2-dimethyl-heptan-3-ol
##STR00074##
[0380] 20 g (0.232 mol) of pivaldehyde dissolved in 600 mL of THF
was added to a 2 L 3-neck round bottom flask equipped with a
magnetic stir bar, thermometer and rubber stopper. The vessel was
maintained under N.sub.2. The reaction mixture was cooled to
-65.degree. C. in a dry ice/acetone bath. 112 mL (0.279 mol) of a
2.5 M BuLi solution in hexane was slowly added in 5 mL portions
with a 20 mL glass syringe, maintaining the temperature below
-55.degree. C. The reaction was stirred at -60.degree. C. for one
hour, then allowed to warm to -5.degree. C. over one hour. The
reaction was cooled again to -60.degree. C. and slowly quenched
with NH.sub.4Cl/H.sub.2O solution, maintaining the temperature
below -50.degree. C. 100 mL of water were added and the reaction
was allowed to warm to room temperature. The THF was removed on a
rotary evaporator with a bath temperature of 25.degree. C. until an
oil was observed. The product was extracted with ethyl ether, and
the ether was dried and evaporated carefully to yield 31.0 g of
2,2-dimethyl-heptan-3-ol that was used directly in a subsequent
oxidation reaction. .sup.1H NMR (CDCl.sub.3, 500 MHz) .delta.
(ppm): 3.2 (m, 1H), 1.2-1.7 (m, 3H), 0.93 (m, 3H), 0.89 (s,
9H).
1.44 Preparation of 2,2-dimethyl-heptan-3-one
##STR00075##
[0382] 2,2-Dimethyl-heptan-3-ol (0.23 mol) was dissolved in 350 mL
of CH.sub.2Cl.sub.2 in a 500 mL round bottom flask with a magnetic
stirbar. The flask was partially cooled with ice. 76.6 g (0.355
mol) of pyridinium chlorochromate was added, while vigorously
stirring. The reaction turned black and warmed up slightly. The
reaction mixture was stirred at room temperature for 24 hours. The
solution was decanted away from the black sludge, which was rinsed
with hexane. The organic extracts were combined and chromatographed
directly on silica gel. (Note: only silica has been found to trap
and remove the reduced non-reacted chromium compounds). The
product, 2,2-dimethyl-heptan-3-one, eluted with
CH.sub.2Cl.sub.2/hexane and in a subsequent 10% ethyl
acetate/hexane fraction to yield 29.19 g of product at 88% yield.
.sup.1H NMR (CDCl.sub.3, 500 MHz) .delta. (ppm): 2.48 (t, 2H), 1.54
(m, 2H), 1.28 (m, 2H), 1.13 (s, 9H), 0.90 (m, 3H).
1.45 Preparation of 3-methoxy-2-methyl-benzoic acid
N'-(1-tert-butyl-pentyl)-hydrazide
##STR00076##
[0384] 2.84 g (20 mmol) of 2,2-dimethyl-heptan-3-one, 3.60 g (20
mmol) of 2-methyl, 3-methoxy benzoic acid hydrazide, 20 drops of
glacial acetic acid and 40 mL of 100% ethyl alcohol were refluxed
for 4 hours and then stirred at room temperature for 24 hours. TLC
indicated only a 35% reaction. Accordingly, the reaction mixture
was refluxed for an additional 6 hours. TLC indicated ca. 80%
reaction (TLC Rf=0.57, starting hydrazide, Rf=0.08, 1:1 ethyl
acetate:hexane). To the reaction mixture was added, 3.5 mL of
glacial acetic acid and 1.89 g (30 mmol) of NaCNBH.sub.3. The
mixture was stirred at room temperature for 2 hours and refluxed
for 1 hour. 50 mL of water was added and 15% NaOH was added until
the reaction mixture was basic. Most of the alcohol was removed on
a rotary evaporator and the product was extracted with CHCl.sub.3,
to yield 4.28 g of crude material. TLC indicated the product
hydrazide at Rf 0.54 (1:1 ethyl acetate:hexane). Purification by
gradient chromatography on silica yielded 3.03 g of product, which
eluted in a 25-40% ethyl acetate/hexane fraction. Drying in a
vacuum oven at 55.degree. C. eliminated volatile materials,
yielding 2.69 g of 3-methoxy-2-methyl-benzoic acid
N'-(1-tert-butyl-pentyl)-hydrazide. .sup.1H NMR (CDCl.sub.3, 500
MHz) .delta. (ppm): 7.2 (t, 1H), 7.05 (br, 1H[NH]), 6.9 (m, 2H),
4.9 (br, 1H), 3.84 (s, 3H), 2.5 (m, 1H), 2.3 (s, 3H), 1.2-1.8 (m,
6H), 0.97 (s, 9H), 0.92 (t, 3H).
##STR00077##
[0385] 17.85 g (90.98 mmol) of 4-methoxybenzyl carbazate was
dissolved in 50 mL of CH.sub.2Cl.sub.2 in a 250 mL flask and then
cooled in ice water. 21.42 g (155 mmol) of potassium carbonate
dissolved in 80 mL of water was added. While the reaction mixture
was being stirred in the ice bath, 17.0 g (79.95 mmol) of
5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl chloride in 60 mL
of CH.sub.2Cl.sub.2 were slowly added. The reaction mixture was
stirred at room temperature overnight and then transferred to a
separatory funnel with 200 mL of CH.sub.2Cl.sub.2 and 200 mL of
H.sub.2O. After shaking, a floating white precipitate was filtered
off, washed with water, dried in a vacuum oven to give 29.1 g of
N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)hydrazinecarboxylic
acid 4-methoxy-benzyl ester. The CH.sub.2Cl.sub.2 solution was
dried and concentrated to give 5.19 g of a residue consisting of
the original acid chloride, 4-methoxybenzyl carbazate, and some
product. TLC Rf=0.38 (streak 1:1 ethyl acetate: hexane). .sup.1H
NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.4-6.7 (m, 6H), 5.139 (s
2H), 4.279 (s 4H), 3.81 (s, 3H), 2.303 (s, 3H).
##STR00078##
[0386] In a 500 mL volume flask, was combined 18.6 g (0.0499 moles)
of
N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinecarboxyli-
c acid 4-methoxy-benzyl ester, 72 mL of concentrated HCl, and 108
mL of dioxane. The flask was placed into an 80.degree. C. oil bath
and mechanically stirred for 2 hours. The reaction mixture was
cooled with ice water, then poured onto ice water and transferred
to a separatory funnel. The reaction mixture--H.sub.2O solution was
then extracted twice with 150 mL of CH.sub.2Cl.sub.2 to remove the
acids and neutrals (the starting material). The aqueous phase was
made basic (pH 12) with a 20% NaOH solution and extracted 4 times
with 150 mL of ethyl acetate. The ethyl acetate extract was dried
over MgSO.sub.4 and concentrated to yield 4.5 g of
5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid hydrazide.
.sup.1H NMR (CDCL.sub.3, 300 MHz) .delta. (ppm): 7.0 (s, 1H), 6.85
(d, 1H), 6.74 (d, 1H), 4.28 (m, 4H), 2.781 (s, 3H).
1.46 Preparation of
5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid
N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide
##STR00079##
[0388] 0.86 g (4.1 mmol) of
-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid hydrazide
and 1.14 g (10 mmol) of 2,2 dimethyl pentan-3-one, 30 mL of ethyl
alcohol and 20 drops of glacial acetic acid were refluxed for 6
hours. TLC indicated ca. a 60% conversion to
5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid
(1-ethyl-2,2-dimethyl-propylidene)-hydrazide (Rf=0.40, 1:1 ethyl
acetate: hexane). The reaction was cooled, 3 mL of glacial acetic
acid followed by 0.63 g (10 mmol) of sodium cyanoborohydride were
added, and the reaction was stirred at room temperature for 3
hours. Most of the alcohol was removed on a rotary evaporator. 30
mL of water was added, followed by the addition of 10%
NaOH/H.sub.2O until the reaction mixture was basic. The mixture was
extracted extensively with ethyl acetate. The ethyl acetate extract
was dried and evaporated to give 1.2 g of crude material. The
product was purified by column chromatography on silica gel,
eluting with 20-30% ethyl acetate/hexane. About 0.46 g of pure
5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid
N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide was obtained. TLC:
Rf=0.46 (1:1 ethyl acetate: hexane). .sup.1H NMR (CDCl.sub.3, 500
MHz) .delta. (ppm): 7.1 (br, 1H[NH]), 6.85 (d, 1H), 6.71 (d, 1H),
4.8 (br, 1H), 4.29 (m, 2H), 4.25 (m, 2H), 2.4 (m, 1H), 2.29 (s,
3H), 1.7 (m, 1H), 1.3 (m, 1H), 1.15 (t, 3H), 0.98 (s, 9H).
1.47 Preparation of RG-115858, 3,5-dimethyl-benzoic acid
N'-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-N-(1-ethyl-2,2-dime-
thyl-propyl)-hydrazide
##STR00080##
[0390] 2.38 g (18 mmol) of t-butyl carbazate were dissolved in 50
mL of CH.sub.2Cl.sub.2 in a 250 mL round bottom flask and cooled to
0.degree. C. An aqueous K.sub.2CO.sub.3 solution was prepared (4.15
g K.sub.2CO.sub.3/35 mL H.sub.2O) and added to the reaction mixture
which was again cooled to 0.degree. C. 3.63 g (16 mmol) of
5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl chloride were
dissolved in 40 mL of CH.sub.2Cl.sub.2 and added from a separatory
funnel, drop-wise over 15 min. The reaction mixture was stirred at
room temperature for 3 days. The reaction mixture was transferred
to a separatory funnel with CH.sub.2Cl.sub.2 and H.sub.2O. The
water phase was thoroughly extracted with CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 extract was then extracted with 0.5N HCl, dried,
and evaporated. The residue was further dried in a vacuum oven to
yield 5.15 g of a tan solid of
N'-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinecarboxylic
acid tert-butyl ester. TLC (1:1 ethyl acetate: hexane) gave a
single spot at Rf=0.43 and NMR indicated a very pure product:
.sup.1H NMR (CDCl.sub.3, 500 MHz) .delta. (ppm): 7.5 (br, 1H), 7.0
(br, 1H), 6.75 (d, 2H), 4.28 (br, 4H), 2.76 (m, 2H), 1.5 (s, 9H),
1.18 (t, 3H).
##STR00081##
[0391] 5.15 g (16 mmol) of
N'-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinecarboxylic
acid tert-butyl ester were added to a 200 mL round bottom flask.
About 20 mL of trifluoroacetic acid were added and the reaction
mixture was stirred at room temperature for 24 hours. Then about 40
mL of water were added, followed by the slow addition of cold 10%
NaOH/H.sub.2O, with stirring, until the acid was neutralized (pH
14). The reaction mixture was transferred to a separatory funnel
and extracted with ethyl acetate by shaking gently (caution: gas
evolution). The ethyl acetate extract was dried and evaporated to
yield 5.51 g of a pale, viscous yellow semi-solid. The material was
then placed in a 50.degree. C. vacuum oven for about 1 hour to
yield 4.62 g of 5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic
acid hydrazide. The t-Boc cleavage is best accomplished with neat
trifluoroacetic acid; use of adjunctive solvents always resulted in
much lower yields. .sup.1H NMR (CDCl.sub.3, 500 MHz) .delta. (ppm):
7.0 (br, 1H), 6.83 (m, 1H), 6.71 (m, 1H), 4.28 (br s, 4H), 2.76 (m,
2H), 1.6 (br, 2H), 1.17 (t, 3H).
##STR00082##
[0392] 1.12 g (5.1 mmol) of
5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid hydrazide,
1.37 g (12 mmol) of 2,2 dimethyl pentanone-3, 30 mL of ethanol, and
20 drops of glacial acetic acid were refluxed for 6 hours to
generate 5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid
(1-ethyl-2,2-dimethyl-propylidene)-hydrazide, which was used in
situ. To the cooled reaction mixture, was added 3 mL of glacial
acetic acid and 0.63 g (10 mmol) of NaCNBH.sub.3. The reaction was
stirred at room temperature for 24 hours. 25 mL of water were added
and most of the alcohol was removed on a rotary evaporator. Then
10% NaOH/H.sub.2O was added until the reaction mixture was basic.
The product was extracted with ethyl acetate, which was then dried
and evaporated to give 1.61 g of residue. Pure
5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid
N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide was obtained (ca. 0.77
g) by column chromatography on silica gel, eluting with 25% ethyl
acetate/hexane. TLC: Rf=0.53, 1:1 ethyl acetate: hexane). .sup.1H
NMR (CDCl.sub.3, 500 MHz) .delta. (ppm): 7.1 (br s, 1H), 6.8 (d,
1H), 6.7 (d, 1H), 4.27 (m, 4H), 2.8 (m, 2H), 2.4 (m, 1H), 1.7 (m,
1H), 1.3 (m, 1H), 1.2 (t, 3H), 1.15 (t, 3H), 0.97 (s, 9H).
##STR00083##
[0393] 0.214 g (0.70 mmol) of
5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid
N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide, 151 mg (0.9 mmol) of
3,5 dimethylbenzoyl chloride, 7 mL of 25% K.sub.2CO.sub.3/H.sub.2O
and 7 mL of CH.sub.2Cl.sub.2 were added to a 20 mL vial and stirred
at room temperature for 24 hours. The reaction mixture was
transferred to a separatory funnel, and dilute NaHCO.sub.3 and
CH.sub.2Cl.sub.2 were added. The CH.sub.2Cl.sub.2 layer was
separated and the water layer extracted twice with
CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2 extracts were dried over
MgSO.sub.4 and evaporated to yield 0.59 g of a white residue.
Purification by column chromatography and elution with 15 mL of 20%
ethyl acetate/hexane yielded about 350 mg of 3,5-dimethyl-benzoic
acid
N'-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-N-(1-ethyl-2,2-dime-
thyl-propyl)-hydrazide (95% pure by TLC: Rf=0.56, 1:1 ethyl
acetate:hexane). .sup.1H NMR (CDCl.sub.3, 500 MHz) .delta. (ppm):
7.05 (s, 1H), 7.0 (s, 2H), 6.6 (d, 1H), 6.27 (d, 1H), 4.65 (d, 1H),
4.25 (s, 4H), 2.9 (m, 1H), 2.3 (s, 6H), 2.0 (m, 1H), 1.55-1.7 (m,
2H), 1.25 (m, 3H), 0.9-1.2 (3s, 9H), 0.9 (t, 3H).
[0394] The following compounds were prepared in a similar
manner:
[0395] 3,5-Dimethoxy-4-methyl-benzoic acid
N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide.
TLC: Rf=0.45, 3:2 (hexane: acetone).
[0396] RG-115851, 3,5-dimethyl-benzoic acid
N-(1-tert-butyl-pentyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide.
.sup.1H NMR (CDCl.sub.3, 500 MHz), .delta. (ppm): 7.7 (s, 1H),
7.22, 7.1 (2 br s, 1H), 7.08 (s, 2H), 7.0 (s, 1H), 6.87 (m, 1H),
6.28 (m, 1H), 4.7 (m, 1H), 3.78 (s, 3H), 2.28 (s, 6H), 1.8 (s, 3H),
1.3-1.6 (br m, 6H), 1.2, 1.1, 0.95 (3s, 9H), 0.95 (m, 3H); TLC
Rf=0.56 (1:1 ethyl acetate:hexane).
[0397] RG-115852,3,5-dimethoxy-4-methyl-benzoic acid
N-(1-tert-butyl-pentyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide.
.sup.1H NMR (CDCl.sub.3, 500 MHz), .delta. (ppm): 7.05 (t, 1H), 7.0
(s, 1H), 6.85 (d, 1H), 6.65 (s, 2H), 6.25 (d, 1H), 4.7 (d, 1H),
3.89 (s, 3H), 3.78 (s, 6H), 2.10 (s, 3H), 1.86 (s, 3H), 1.3-1.6 (br
m, 6H), 1.06, 0.99 (2s, 9H), 0.94 (t, 3H); TLC Rf=0.55 (1:1 ethyl
acetate:hexane).
[0398] 3,5-Dimethyl-benzoic acid
N-(1-ethyl-2,2-dimethyl-propyl)-N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxin-
e-6-carbonyl)-hydrazide. .sup.1H NMR (CDCl.sub.3, 500 MHz), .delta.
(ppm): 7.05 (s, 2H), 7.0 (s, 1H), 6.6 (d, 1H), 6.3 (d, 1H), 4.6 (d,
1H), 4.25 (m, 4H), 2.25 (s, 6H), 1.85 (s, 3H), 1.5-1.8 (br, 2H),
1.3 (t, 3H), 1.0-1.2 (2s, 9H); TLC Rf=0.52 (1:1 ethyl
acetate:hexane).
[0399] 3,5-Dimethoxy-4-methyl-benzoic acid
N-(1-ethyl-2,2-dimethyl-propyl)-N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxin-
e-6-carbonyl)-hydrazide. .sup.1H NMR (CDCl.sub.3, 500 MHz), .delta.
(ppm): 6.8 (br s, 1H), 6.62 (s, 1H), 6.6 (d, 1H), 6.27 (d, 1H), 4.6
(d, 1H), 4.25 (m, 4H), 3.84, 3.78 (2s, 6H), 2.1 (s, 3H), 1.87 (s,
3H), 1.6 (br, 2H), 1.3 (t, 3H), 0.9-1.2 (m, 9H); TLC Rf=0.45 (1:1
ethyl acetate: hexane).
[0400] 3,5-Dimethyl-benzoic acid
N-(1-tert-butyl-butyl)-N'-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbo-
nyl)-hydrazide. .sup.1H NMR (CDCl.sub.3, 500 MHz), .delta. (ppm):
7.05 (s, 2H), 7.0 (s, 1H), 6.6 (d, 1H), 6.3 (d, 1H), 4.7 (d, 1H),
4.2 (m, 4H), 2.3 (s, 6H), 1.8 (s, 3H), 1.3-1.7 (br m, 4H), 1.1,
1.15 (2s, 9H), 0.95 (t, 3H).
[0401] 3,5-Dimethoxy-4-methyl-benzoic acid
N-(1-tert-butyl-butyl)-N'-(5-methyl-2,3-dihydrobenzo[1,4]dioxine-6-carbon-
yl)-hydrazide. .sup.1H NMR (CDCl.sub.3, 500 MHz), .delta. (ppm):
6.75 (br s, 1H), 6.62 (s, 1H), 6.6 (d, 1H), 6.25 (d, 1H), 4.7 (t,
1H), 4.25 (m, 4H), 3.78, 3.84 (2s, 6H), 2.85 (br, 1H), 2.37 (m,
1H), 2.07 (s, 3H), 1.86 (s, 3H), 1.3-1.7 (br m, 4H),
[0402] 3,5-Dimethoxy-4-methyl-benzoic acid
N'-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-N-(1-ethyl-2,2-dime-
thyl-propyl)-hydrazide. .sup.1H NMR (CDCl.sub.3, 500 MHz), .delta.
(ppm): 6.8 (br s, 1 h), 6.65 (s, 1H), 6.6 (d, 1H), 6.25 (d, 1H),
4.6 (d, 1H), 4.25 (2s, 4H), 3.79-3.84 (2s, 6H), 2.9 (br, 1H), 2.35
(br, 1H), 2.1 (s, 3H), 1.3-1.9 (br m, 2H), 1.3 (t, 3H), 1.1-1.3 (m,
9H), 0.94 (t, 3H); TLC Rf=0.48 (1:1 ethyl acetate: hexane).
[0403] 3,5-Dimethyl-benzoic acid
N-(1-tert-butyl-butyl)-N'-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbon-
yl)-hydrazide. .sup.1H NMR (CDCl.sub.3, 500 MHz), .delta. (ppm):
7.05 (s, 2H), 7.0 (s, 1H), 6.59 (d, 1H), 6.18 (d, 1H), 4.7 (d, 1H),
4.27, 4.25 (s, 4H), 2.85 (m, 1H), 2.3 (s, 6H), 2.1 (m, 1H), 1.3-1.8
(br m, 4H), 1.1, 1.15 (2s, 9H), 0.95 (t, 6H); TLC Rf=0.53 (1:1
ethyl acetate: hexane).
[0404] 3,5-Dimethoxy-4-methyl-benzoic acid
N-(1-tert-butyl-butyl)-N'-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbon-
yl)-hydrazide. .sup.1H NMR (CDCl.sub.3, 500 MHz), .delta. (ppm):
6.75 (br s, 1H), 6.62 (s, 1H), 6.6 (d, 1H), 6.2 (d, 1H), 4.7 (m,
1H), 4.25 (br m, 4H), 3.84, 3.78 (2s, 6H), 2.4 (m, 1H), 1.95 (m,
1H), 2.1 (s, 3H), 1.2-1.8 (br m, 4H), 1.1-0.95 (m, 9H), 0.95 (m,
6H). TLC: 0.54 (1:1 ethyl acetate: hexane); TLC Rf=0.54 (1:1 ethyl
acetate: hexane).
1.48 Preparation of RG-115665
##STR00084##
[0406] Benzyl carbazate (25 g, 0.15 mol) was dissolved in 50 mL of
DMF in a round bottom flask. The solution was heated to
95-100.degree. C. From two separate addition funnels, ethyl
2-bromoisobutyrate (58.5 g, 0.3 mol) and pyridine (29.7 g, 0.375
mol, 30 mL) were added drop-wise separately and simultaneously over
30-120 minutes. Heating was continued if necessary to propel the
reaction to completion. The reaction was monitored by TLC (30%
ethyl acetate in hexanes, I.sub.2 visualization). The mixture was
allowed to cool, and then poured onto ice water. The aqueous
mixture was extracted with ethyl ether, and the solvent was removed
in vacuo. 2-(N'-benzyloxycarbonyl-hydrazino)-2-methyl-propionic
acid ethyl ester was isolated, optionally after silica gel
chromatography. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. (ppm):
7.36 (br s, 5H), 6.6 (br s, 1H), 5.15 (br s, 2H), 4.2 (q, 2H), 1.3
(s, 6H), 1.25 (t, 3H). .sup.1H NMR analysis alone is insufficient
to ascertain the extent of the reaction.
##STR00085##
[0407] 2-(N'-benzyloxycarbonyl-hydrazino)-2-methyl-propionic acid
ethyl ester (28 g, 0.1 mol) was dissolved in 50 mL CH.sub.2Cl.sub.2
and cooled on ice. A solution of 20.7 g K.sub.2CO.sub.3 in 30 mL of
water was added. A solution of 3,5-dimethylbenzoyl chloride (17 g,
0.1 mol) in 50 mL of CH.sub.2Cl.sub.2 was added drop-wise over a
period of 1 hour, maintaining the temperature at 0-5.degree. C. The
mixture was stirred for 1 hour on an ice bath, and then at room
temperature overnight. TLC indicated the reaction was complete. The
aqueous layer was removed in a separatory funnel and the organic
phase was washed with water and then brine, and then dried over
Na.sub.2SO.sub.4. The solvent was removed on a rotary evaporator.
The residue was slurried in hexane, filtered, and then air-dried.
2-[N'-Benzyloxycarbonyl-N-(3,5-dimethyl-benzoyl)-hydrazino]-2-methyl-prop-
ionic acid ethyl ester was obtained as a white solid (35 g), giving
a single spot by TLC. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
(ppm): 7.4 (s, 1H), 7.3 (m, 3H), 7.15 (m, 2H), 7.1 (s, 2H), 7.0 (s,
1H), 5.2 (d, 1H), 5.0 (d, 1H), 4.2 (m, 2H), 2.25 (s, 6H), 1.78 (s,
3H), 1.64 (s, 3H), 1.28 (t, 3H).
##STR00086##
[0408] To a 500 mL round bottom flask was added 20.62 g (0.05 mol)
of
2-[N'-benzyloxycarbonyl-N-(3,5-dimethyl-benzoyl)-hydrazino]-2-methyl-prop-
ionic acid methyl ester and 200 mL of dry THF. The mixture was
stirred and the flask was cooled in dry ice, and then 0.87 g (0.04
mol) of LiBH.sub.4 was added with stirring at room temperature. The
reaction mixture was refrigerated and more LiBH.sub.4 was added
(1.3 g) and the reaction was refrigerated for 2 days. The reaction
mixture was warmed to room temperature, and 100 mL of ether were
added and the total mixture was poured slowly into 150 mL of water
in a separatory funnel After the bubbling subsided, the mixture was
agitated and then shaken gently. The ether layer was separated and
the water extracted twice with 100 mL of et2o. The total ether
extract was extracted with water, washed with brine, and evaporated
to yield 20.04 g of product. The product was purified by
chromatography. The product eluted with 30-35% ethyl acetate:
hexane to yield 11.6 g of
N'-(3,5-dimethyl-benzoyl)-N'-(2-hydroxy-1,1-dimethyl-ethyl)-hydrazinecarb-
oxylic acid benzyl ester, .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
(ppm): 7.3-6.9 (3s, 8H), 5.1 (d, 1H), 4.9 (d, 1H), 4.1 (d, 1H), 4.1
(d, 1H), 3.6 (d, 1H), 2.25 (s, 6H), 1.48 (s, 3H), 1.41 (s, 3H), as
well as 4 g of unreacted starting material.
##STR00087##
[0409] To a round bottom flask was added 4.00 g (0.0108 mol) of
N'-(3,5-dimethyl-benzoyl)-N'-(2-hydroxy-1,1-dimethyl-ethyl)-hydrazinecarb-
oxylic acid benzyl ester, 3.27 g (0.048 mol) of imidazole and 20 mL
of DMF. The flask was cooled in an ice bath and then 4.08 g (0.027
mol) of t-butyl, dimethylsilyl chloride was slowly added as the
temperature was maintained below 25.degree. C. The ice bath was
removed and the reaction stirred overnight at room temperature. The
reaction mixture was then poured into 200 mL of water and extracted
three times with 100 mL of ether. The ether extract was washed with
water, dried over MgSO.sub.4, and concentrated to yield 7.12 g of
product. The product was cleaned up by column chromatography.
Unreacted t-butyl, dimethylsilyl chloride was eluted with hexane
and 10% CH.sub.2Cl.sub.2/hexane. The product eluted with 20-100%
CH.sub.2Cl.sub.2/hexane to yield 5.13 g of
N'-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-N'-(3,5-dimeth-
yl-benzoyl)-hydrazinecarboxylic acid benzyl ester. .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 7.3-6.7 (m, 8H), 5.0 (s, 2H),
4.1 (d, 1H), 3.4 (d, 1H), 2.15 (d, 6H), 1.54 (d, 3H), 1.25 (d, 3H),
0.82 (s, 9H), 0.01 (s, 6H).
##STR00088##
[0410] Into a 200 mL round bottom flask, was added 4.62 g (0.0095
mol) of
N'-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-N'-(3,5-dimeth-
yl-benzoyl)-hydrazinecarboxylic acid benzyl ester, 100 mL of dry
CH.sub.2Cl.sub.2, 2.5 g of Et.sub.3N, and 1.66 g (0.0143 mol) of
Et.sub.3SiH. The reaction mixture was cooled in an ice bath, and
then 100 mg of palladium acetate was added in 3 portions over 30
min. The reaction was then allowed to warm to room temperature. As
TLC indicated no product, only the starting material (20% ethyl
acetate/hexane, Rf 4.3), the reaction was warmed gently with a heat
gun and then stirred at room temperature for 30 min. TLC indicated
the product (20% ethyl acetate/hexane, Rf 0.43).
[0411] The reaction mixture was stirred with some MgSO.sub.4 and
then filtered. The filter cake was then washed with
CH.sub.2Cl.sub.2. The total CH.sub.2Cl.sub.2 fraction was shaken
with saturated NH.sub.4Cl, then with H.sub.2O. The CH.sub.2Cl.sub.2
was dried and evaporated to yield 3.78 g of 3,5-Dimethyl-benzoic
acid
N-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-hydrazide.
The product was purified by column chromatography, eluted with
5-10% ethyl acetate/hexane. The product fractions were combined,
concentrated, and placed in warm (50.degree. C.) vacuum oven to
yield 2.96 g of solid 3,5-Dimethyl-benzoic acid
N-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-hydrazide.
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.0 (s, 2H), 6.9
(s, 1H), 3.80 (s, 2H), 2.23 (s, 6H), 1.40 (s, 6H), 0.82 (s, 9H),
0.08 (s, 6H).
##STR00089##
[0412] Into a 250 mL round bottom flask, was added 3.71 g (0.010
mol) of 3,5-dimethyl-benzoic acid
N-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-hydrazide
and 50 mL of CH.sub.2Cl.sub.2. 2.11 g (0.106 mol) of
2-ethyl-3-methoxybenzoyl chloride and a K.sub.2CO.sub.3 solution
(4.15 g in 20 mL of H.sub.2O) were added. The reaction mixture was
stirred at room temperature overnight. The reaction mixture was
transferred to a separatory funnel and the aqueous layer extracted
twice with 50 mL of CH.sub.2Cl.sub.2. The organic phase was dried
and concentrated to give 5.52 g of a syrupy product. The product
was purified by column chromatography, eluting in 10% ethyl acetate
in hexane. Further purification was achieved by triturating the
product with heptane, placing the mixture into the freezer, and
then either decanting the yellow solution or the more preferred
method of rapidly filtering through a cold Buchner filter for 1-2
hours to obtain a white solid product, 2-ethyl-3-methoxy-benzoic
acid
N'42-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-N'-(3,5-dimethy-
l-benzoyl)-hydrazide (2.9 g). .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. (ppm): 7.0-6.8 (m, 5H), 6.2 (d, 1H), 4.1 (d, 1H), 3.69 (s,
3H), 3.5 (d, 1H), 2.19 (s, 6H), 1.62 (s, 3H), 1.40 (s, 3H), 0.9 (t,
3H), 0.76 (s, 9H).
##STR00090##
[0413] 2.78 g (0.00526 mol) of 2-ethyl-3-methoxy-benzoic acid
N'42-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethylFN'-(3,5-dimethyl-
-benzoyl)-hydrazide were dissolved in 24 mL of THF. The mixture was
cooled in an ice bath and 6.0 mL (0.006 mol) of a 1 M tetrabutyl
ammonium fluoride solution in THF were added. The reaction was
stirred at room temperature for 5-6 hours, and then 100 mL of
Et.sub.2O were added and the reaction mixture was poured into ice
water in a separatory funnel. The aqueous layer was further
extracted twice with 25 mL of Et.sub.2O and the total ether layer
was dried and concentrated. TLC of the product showed a new product
(Rf 0.20) below some of the starting material. The product was
purified by chromatography, eluting with 40-50% ethyl
acetate/hexane to yield 1.42 g of pure 3,5-dimethyl-benzoic acid
N'-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-N-(2-hydroxy-1,1-dimethyl-ethyl)--
hydrazide. (67% yield). TLC: Rf=0.20. .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. (ppm): 7.1-6.8 (m, 5H), 6.0 (d, 1H), 4.3 (d, 1H), 3.78
(s, 3H), 3.5 (d 1H), 2.4 (d, 1H) 2.29 (s, 6H), 2.2 (d, H), 1.56 (s,
3H), 1.45 (s, 3H), 1.00 (t, 3H).
##STR00091##
[0414] 1.09 g (0.0027 mol) of 3,5-dimethyl-benzoic acid
N'-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-N-(2-hydroxy-1,1-dimethyl-ethyl)--
hydrazide were dissolved in 100 mL of CH.sub.2Cl2 and 1.80 g
(0.0082 mol) of pyridinium chlorochromate were added. The reaction
mixture was refluxed for 2 hours. After cooling, the total reaction
mixture was poured onto a silica chromatography column to purify
the product, 3,5-dimethyl-benzoic acid
N-(1,1-dimethyl-2-oxo-ethyl)-N'-[1-(2-ethyl-3-methoxy-phenyl)-vinyThhydra-
zide. A white crystalline solid (1.04 g) was eluded with 30-35%
ethyl acetate/hexane. TLC indicated high purity (>95%), Rf=0.46
(1:1 ethyl acetate: hexane). .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. (ppm): 9.6 (s, 1H), 7.15 (s, 2H), 7.1 (m, 2H), 6.9 (d, 1H),
6.3 (d, 1H), 3.806 (s, 3H), 2.304 (s, 6H), 2.2-2.4 (m, 2H), 1.576
(s, 3H), 1.429 (s, 3H), 1.01 (t, 3H).
##STR00092##
[0415] Into a 20 mL vial, was added 55 mg of 3,5-dimethyl-benzoic
acid
N'-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-N-(2-hydroxy-1,1-dimethyl-ethyl)--
hydrazide, 2 mL of CH.sub.2Cl.sub.2, 200 mg of Et.sub.3N. The
reaction mixture was stirred and then 17 mg of acetyl chloride were
added. After stirring at room temperature for 2 hours, the reaction
was warmed at 40.degree. C. for 30 min. After cooling, more
CH.sub.2Cl.sub.2 was added, and then transferred to separatory
funnel and shaken with dilute K.sub.2CO.sub.3. The CH.sub.2Cl.sub.2
layer was dried with MgSO.sub.4. TLC showed a major spot at Rf 38.
The product was cleaned up by chromatography by eluting with 30%
ethyl acetate in hexane. This yielded about 40 mg of acetic acid
2-{N-(3,5-dimethyl-benzoyl)-N'-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-hydra-
zino}-2-methyl-propyl ester. .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. (ppm): 7.1-6.8 (m, 6H), 6.1 (d, 1H), 4.7-4.4 (q, 2H), 3.79
(s, 3H), 2.29 (s, 6H), 2.12 (s, 3H), 1.75 (s, 3H), 1.49 (s, 3H),
0.98 (t, 3H).
##STR00093##
[0416] Into a flask containing 1.5 g (0.0036 mol, 80% pure) of
2-[N'-benzyloxycarbonyl-N-(3,5-dimethyl-benzoyl)-hydrazino]-2-methyl-prop-
ionic acid ethyl ester, was added 20 mL of dry CH.sub.2Cl.sub.2,
1.5 mL of Et.sub.3N, and 1.27 g (0.010 mol) of Et.sub.3SiH. While
stirring, small portions of a total of 0.010 g of Pd(OAc).sub.2 was
added and the reaction was stirred for 2 hours at room temperature.
To the reaction mixture was added 100 mL of CH.sub.2Cl.sub.2 and
some MgSO.sub.4 to aid in the filtration/removal of the Pd product.
The CH.sub.2Cl.sub.2 solution was shaken with saturated NH.sub.4Cl,
CH.sub.2Cl.sub.2 dried, and evaporated to yield 1.61 g of
2-[N-(3,5-dimethyl-benzoyl)-hydrazino]-2-methyl-propionic acid
ethyl ester. The product was purified by chromatography, elution
with 21-24% ethyl acetate in hexane. .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. (ppm): 7.045 (s 2H), 7.0 (s 1H), 4.4 (s, 2H), 2.324 (s
6H), 2.236 (s 3H), 1.487 (s, 6H).
##STR00094##
[0417] To a flask containing 1.6 g (0.0057 mol) of
2-[N-(3,5-Dimethyl-benzoyl)-hydrazino]-2-methyl-propionic acid
ethyl ester in 30 mL of CH.sub.2Cl.sub.2 was added 3.5 g of
Et.sub.3N and 1.20 g (0.0060 mol) of 2-ethyl-3methoxybenzoyl
chloride. The reaction mixture was refluxed for 3 hours and then
evaporated to dryness. The residue was redissolved with 100 mL of
CH.sub.2Cl.sub.2 and extracted twice with 50 mL of dilute aqueous
K.sub.2CO3. The CH.sub.2Cl.sub.2 extract was dried and evaporated
to a residue, which was then triturated with 6% Et.sub.2O in
hexane. A white solid product,
24N-(3,5-dimethyl-benzoyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazino]-2-me-
thyl-propionic acid ethyl ester, was filtered off and dried in a
warm (50%) vacuum oven. TLC: product, Rf=0.40, starting material,
Rf=0.35, 1:1 ethyl acetate: hexane. .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. (ppm): 7.70 (s 1H), 7.2-6.8 (m-5H), 6.2 (d 1H), 4.2 (q
2H), 3.801 (s 3H), 2.4 (q 2H), 2.291 (s 6H), 1.882 (s 3H), 1.557 (s
3H), 1.291 (t, 3H), 1.036 (t, 3H).
##STR00095##
[0418]
2-[N-(3,5-Dimethyl-benzoyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazin-
o]-2-methyl-propionic acid was prepared by oxidation of
2-ethyl-3-methoxy-benzoic acid
N'-(3,5-dimethyl-benzoyl)-N'-(1,1-dimethyl-2-oxo-ethyl)-hydrazide
with KMnO.sub.4. The corresponding ester could not be saponified to
the acid shown, even with 40% NaOH/CH.sub.3OH, or 50% aqueous
NaOH+EtOH with reflux.
##STR00096##
[0419] 50 mg (0.000126 mol) of 2-ethyl-3-methoxy-benzoic acid
N'-(3,5-dimethyl-benzoyl)-N'-(1,1-dimethyl-2-oxo-ethyl)-hydrazide
was weighed into a 20 mL vial. 20 mg of hydroxylamine-HCl dissolved
in 0.5 mL of CH.sub.3OH was then added. 40 mg of triethylamine was
added and the reaction mixture stirred at room temperature
overnight. The reaction mixture was concentrated to dryness with
N.sub.2, re-dissolved with 5 mL of CH.sub.2Cl.sub.2 and 5 mL of
0.1N HCl/H.sub.2O. The CH.sub.2Cl.sub.2 layer was separated. TLC
showed the product, 2-ethyl-3-methoxy-benzoic acid
N'-(3,5-dimethyl-benzoyl)-N'-(2-hydroxyimino-1,1-dimethyl-ethyl)-hyd-
razide, had a Rf of 0.27 (1:1 ethyl acetate: hexane) and was about
85% pure. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.1-6.8
(m, 5H), 6.2 (d, 1H), 3.79 (s, 3H), 2.29 (s, 6H), 1.76 (s, 3H),
1.65 (s, 3H), 0.98 (t, 3H).
##STR00097##
[0420] 50 mg of 2-ethyl-3-methoxy-benzoic acid
N'-(3,5-dimethyl-benzoyl)-N'-(1,1-dimethyl-2-oxo-ethyl)-hydrazide,
40 mg of semicarbazide, 40 mg of Et.sub.3N and 2 mL of CH.sub.3OH
were refluxed for 2 hours, concentrated to dryness, redissolved
with CH.sub.2Cl.sub.2 and diluted with (0.5N) HCl. The
CH.sub.2Cl.sub.2 extract was dried and evaporated to give the
semicarbazide of 2-ethyl-3-methoxy-benzoic acid
N'-(3,5-dimethyl-benzoyl)-N'-(1,1-dimethyl-2-oxo-ethyl)-hydrazide.
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.4-6.8 (m, 5H),
6.2 (d, 1H), 3.767 (s, 3H), 2.203 (s, 6H), 1.717 (s, 3H), 1.474 (s,
3H), 0.913 (t, 3H).
##STR00098##
[0421] 50 mg of 2-ethyl-3-methoxy-benzoic acid
N'-(3,5-dimethyl-benzoyl)-N'-(1,1-dimethyl-2-oxo-ethyl)-hydrazide
aldehyde, 2 mL of CH.sub.3OH, 0.5 mL of a 0.5% glacial acetic acid
solution with CH.sub.3OH and 26 mg of oxamic hydrazide, and 40 mg
of Et.sub.3N were refluxed for 2 hours. The solvents were removed
on an evaporator and the residue redissolved with CH.sub.2Cl.sub.2
and water. The CH.sub.2Cl.sub.2 extract was dried and evaporated.
TLC showed the presence of the product, the oxamic carbazide of
2-ethyl-3-methoxy-benzoic acid
N'-(3,5-dimethyl-benzoyl)-N'-(1,1-dimethyl-2-oxo-ethyl)-hydrazide,
which had a Rf of 0.50 while the starting aldehyde had a Rf of 0.74
(in 100% ethyl acetate). .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
(ppm): 8.1 (s 1H), 7.1-6.8 (m, 6H), 6.1 (d, 1H), 3.667 (s, 3H), 2.3
(m, 1H), 2.19 (s, 6H), 2.00 (m, 1H), 1.581 (s, 3H), 1.511 (s, 3H),
0.802 (t, 3H).
##STR00099##
[0422] 50 mL of 2-ethyl-3-methoxy-benzoic acid
N'-(3,5-dimethyl-benzoyl)-N'-(1,1-dimethyl-2-oxo-ethyl)-hydrazide
were added to a 20 mL flask, with 45 g of aminoethanol in 2 mL of
CH.sub.3OH, and then refluxed for 2 hours. After cooling, the
CH.sub.3OH was removed on the evaporator and the residue was
chromatographed. The product 3,5-dimethyl-benzoic acid
N'-[1-(2-ethyl-3-methoxy-phenyl)-vinyl]-N-(2-hydroxymethoxyimino-1,1-dime-
thyl-ethyl)-hydrazide was eluted with 40% ethyl acetate/hexane.
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.2-6.9 (m, 5H),
4.1 (m, 2H), 3.83 (s, 3H), 3.7 (m, 2H), 2.75 (m, 2H), 2, 338 (s,
6H), 1.36 (s, 3H), 1.21-1.87 (m, 6H).
##STR00100##
[0423] To a flask containing 0.94 g (0.002 mol) of methyl ether
N'-(3,5-dimethyl-benzoyl)-N'-(2-methoxy-1,1-dimethyl-ethyl)-hydrazinecarb-
oxylic acid benzyl ester, was added 10 mL of CH.sub.2Cl.sub.2, 0.87
g of Et.sub.3SiH, 0.10 g of palladium acetate, and 1 g of
Et.sub.3N. The reaction mixture was stirred at room temperature for
over 10 hours. More CH.sub.2Cl.sub.2 (10-20 mL) was added, and the
mixture was filtered to remove the palladium. The brown
CH.sub.2Cl.sub.2 solution was treated with MgSO.sub.4 and charcoal,
then filtered and evaporated. The evaporation yielded 0.87 g of a
red, oily solid. TLC indicated the presence of the product; Rf=0.44
in 1:1 ethyl acetate: hexane. The product was purified by
chromatography, eluted with 19-20% ethyl acetate/hexane to yield
401 mg (80%) of 3,5-dimethyl-benzoic acid
N-(2-methoxy-1,1-dimethyl-ethyl)-hydrazide product. .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. (ppm): 7.1 (s, 2H), 7.0 (s, 1H),
3.682 (s, 2H), 3.377 (s, 3H), 2.319 (s, 6H), 1.495 (s, 3H).
##STR00101##
[0424] To a 20 mL vial containing 90 mg of 3,5-dimethyl-benzoic
acid N-(2-methoxy-1,1-dimethyl-ethyl)-hydrazide 1462 (0.00036), was
added 2 mL of CH.sub.2Cl.sub.2, 145 mg (0.00072 mol) of
2-ethyl-3-methoxybenzoyl chloride, 0.5 K.sub.2CO.sub.3 in 3 mL of
H.sub.2O. The reaction was stirred at room temperature overnight.
The reaction mixture was transferred to separatory funnel with 10
mL of K.sub.2CO.sub.3 and 50 mL of CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 extract was dried and evaporated to dryness. The
product, 2-ethyl-3-methoxy-benzoic acid
N'-(3,5-dimethyl-benzoyl)-N'-(2-methoxy-1,1-dimethyl-ethyl)-hydrazide,
was purified by chromatography, eluting with 25% ethyl acetate in
hexane to yield 105 mg. TLC: Rf=0.44 (1:1 ethyl acetate: hexane).
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. (ppm): 7.8 (s, 1H),
7.1-6.8 (m, 5H), 6.2 (d, 1H), 4.0 (d, 1H), 3.84 (d, 1H), 3.77 (s,
3H), 3.387 (s, 3H), 2.27 (s, 6H), 2.4-2.1 (m, 2H), 1.728 (s, 3H),
1.503 (s, 3H) 0.98 (t 3H).
1.49 Preparation of Compound RG-101494
[0425]
N-(5-ethyl-1,4-benzodioxan-6-carbonyl)-N'-(tert-butyl)-N'-(3-chloro-
-5-methylbenzoyl)hydrazine can be prepared in accordance with U.S.
Pat. No. 5,530,028. Briefly, the product of Example 17 is treated
by the method of Example 5 and then the method of Example 8. The
resulting product is treated with 3-methyl-5-chlorobenzoyl chloride
[(K. Knoevenagel, Chemische Berichte 28: 2045 (1895); Slootmaekers,
P. J., Verbeerst, R., Bull. Soc. Chm. Belg. 77: 273-285 (1968)]
according to the method of Example 9.
1.50 Preparation of Compound RG-102240
[0426]
N-(3-methoxy-2-ethylbenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butyl-
hydrazine can be prepared in accordance with Example 12 of U.S.
Pat. No. 5,530,028.
1.51 Preparation of Compound RG-102317
[0427] N-(5-methyl-1,4-benzo
dioxan-6-carbonyl)-N'-(tert-butyl)-N'-(3,5-dimethylbenzoyl)hydrazine
can be prepared in accordance with Example 3 of U.S. Pat. No.
5,530,021.
1.52 Preparation of Compound RG-115092
[0428]
N-(5-methyl-1,4-benzodioxan-6-carbonyl)-N'-(2-cyano-2-propyl)-N'-(3-
,5-dimethoxy-4-methylbenzoyl)hydrazine can be prepared by a method
directly analogous to Examples 802 and 809 of U.S. Pat. No.
5,117,057 but using N-5-methyl-1,4-benzodioxan-6-carbohydrazine
(for preparation see U.S. Pat. No. 5,530,021, Example 2) and
3,5-methoxy-4-methylbenzoyl chloride.
1.53 Preparation of Compound RG-115575
[0429] 3,4,5-Trifluoro-benzoic acid
N-tert-butyl-N'-(5-methyl-chroman-6-carbonyl)-hydrazide can be
prepared by analogy to Example 11 of U.S. Pat. No. 5,530,021, but
using 3,4,5-trifluorobenzoyl chloride.
1.54 Preparation of Compound RG-115637
[0430] 5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid
N'-tert-butyl-N'-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide can be
prepared by analogy to Example 3 of U.S. Pat. No. 5,530,021, but
using 3,5-dimethoxy-4-methylbenzoyl chloride.
Example 2
Determination of Physical and Transport Properties
2.1 Determination of LC log P (experimental)
[0431] 1000 ppm solutions for each of a set of log P standards
(compounds for which log P is known experimentally; see Table 3)
and for each test compound are prepared. Liquid chromatography
retention times (RT) are measured for each substance using the
conditions described below. A linear equation is derived relating
RT to log P is developed from the data for the log P standards. The
log P for the test compound is calculated from the log P/retention
time equation.
Chromatogaphic Conditions:
TABLE-US-00004 [0432] Column: MetaChem Polaris A-18 3u 50 .times.
3.0 mm; part no C2001-050x030 Solvent Gradient: time (min.)
methanol (%) water (%) 0.0 25 75 7.0 99 1 8.0 25 75 Temperature:
(.degree. C.): 30 Detector Type: UV or DAD (diode array detector):
200-220 nm
2.2 Determination of C log P
[0433] Clog P can be calculated according to standard calculations
known to those of skill in the art. Exploring QSAR: Fundamentals
and Applications in Chemistry and Biology. Corwin Hansch, Albery
Leo, American Chemical Society, Washington, D. C., 1995
2.3 Determination of Water Solubility
[0434] Aqueous solutions are prepared as follows in triplicate: 50
.mu.l of a 10,000 ppm solution (2,000 .mu.g of solid dissolved in
200 .mu.l of methanol) of the substrate in methanol or DMSO is
added to 1 mL of de-ionized water in a 2 dram or smaller vial with
magnetic stirring. Stirring is continued overnight at ambient
temperature. The slurry is taken up into a syringe with a luer tip.
The contents are passed through a new 13 mm 0.2 .mu.M Acrodisc
filter (tuffryn or glass fiber) into an autosampler bottle. For
preparation of calibration standard solutions: dilutions of the
10,000 ppm solution were prepared at 10, 5, 1, 0.5, and 0.2 ppm.
The water solubility of most diacylhydrazines falls within this
concentration range. For more soluble materials, dilution of the
samples into this range is preferable to increasing the calibration
range because the non-linearity of the response results in less
useful calibration curves. However a shift in the range of
calibration standards is required for very insoluble compounds.
[0435] Chromatography of the samples was then preformed. For most
diacylhydrazines the following conditions were adequate for the
measurement. Other columns and gradients may be substituted as
appropriate.
TABLE-US-00005 Column MetaChem Polaris A-18 3.mu. 50 .times. 3.0
mm; part no C2001-050x030 (or MetaChem Inertsil 5.mu. ODS3 50 mm
.times. 2.1 mm) Solvent Gradient time (min.) methanol (%) water (%)
0.00 25 75 4.50 99 1 6.00 99 1 Temperature (.degree. C.) 30
[0436] Analysis of the test samples is conducted as follows: Each
solubility replicate is analyzed in duplicate. While any suitable
analysis method is acceptable, these results were obtained by LC/MS
on a Micromass Platform II in the electrospray negative ion mode
using SIM (single ion monitoring). Standard curves are obtained
from duplicate injections of the standards. The concentration of
the substrate is determined by calculation from the equation
relating concentration and response.
2.4 Determination of Cell Permeation Coefficients
[0437] The method to determine cell permeation coefficients is
known to those of skill in the art. MI-QSAR: Predicting Caco-2 Cell
Permeation Coefficients of Organic Molecules using
Membrane-Interaction QSAR Analysis. Kulkarni, Amit; Han, Yi;
Hopfinger, A. J.; Journal of Chemical Information and Computer
Sciences (2002) 42: 331-342. Table 2 represents the physical and
transport properties of the compounds of the present invention.
TABLE-US-00006 TABLE 2 Physical and Transport Properties of
Compounds MI- Exp. QSAR MI- LC LogP Water P(caco2) .times. QSAR
Compound (exp) C logP Sol. (.mu.M) 10{circumflex over ( )}6 cm/sec
Log BB RG-115009 1.9 2.19 30.4 NA NA RG-115613 2.8 0.82 NA NA NA
RG-101523 4.35 4.46 13.4 9.3 -0.63 RG-101382 4.4 4.96 NA NA NA
RG-101494 4.3 4.43 NA NA NA RG-102240 4.2 4.18 9.8 11.4 -1.00
RG-102317 3.6 4.21 10.1 0.13 0.13 RG-103309 5.89 2.9 NA NA
RG-115092 3.5 2.8 NA NA NA RG-115517 3.15 2.68 36.7 0.04 0.04
RG-115575 4.1 4.22 21.6 12 12.0 RG-115637 3.54 3.6 13.7 6.1 6.1 NA
= not assayed
TABLE-US-00007 TABLE 3 Retention Times (RT) and logP for
Diacylhydrazine standards RT Compound (min.) logP ##STR00102## 2.59
2.9 ##STR00103## 3.29 3.2 ##STR00104## 3.82 3.7 ##STR00105## 3.81
3.5 ##STR00106## 4.84 4.2 ##STR00107## 5.52 5.02 ##STR00108## 4.64
4.2
2.5 Aqueous Solubility
[0438] Equilibrium solubility was measured in pH 7.4 aqueous
buffer. The buffer was prepared by adjusting the pH of a 0.07 M
solution of NaH.sub.2PO.sub.4 to pH 7.4 with 10 N NaOH. The buffer
had an ionic strength of 0.15. At least 1 mg of powder was combined
with 1 mL of buffer to make mg/mL mixture. These samples were
shaken for .gtoreq.2 hours and left to stand overnight at room
temperature. The samples were then filtered through a 0.45-.mu.m
Nylon syringe filter that was first saturated with the sample. The
filtrate was sampled twice, consecutively. The filtrate was assayed
by HPLC against standards prepared in methanol.
TABLE-US-00008 TABLE 4 Solubility of Compounds Solubility (mg/mL)
Compound pH 7.4 RG-115280 0.0012 RG-102125 0.0006 RG-102398
.ltoreq.0.0002 RG-100150 .gtoreq.1.0 RG-115595 0.026 RG-103309
.ltoreq.0.0002 RG-115555 .gtoreq.1.0 RG-115199 0.0064 RG-115823
0.0003 RG-101523 0.0010 RG-102240 0.0007 RG-102317 0.0043 RG-115517
0.014 RG-100021 .gtoreq.1.0
2.6 Partition Coefficients
[0439] The partition coefficient, Log (D), between water-saturated
1-octanol and pH 7.4 buffer was determined for the test compounds.
The buffer was prepared as described in section 2. A 12 ml aliquot
of a 10 mM stock solution in DMSO was introduced to a vial
containing 0.60 mL of octanol and 0.60 mL of buffer at room
temperature. Testosterone was also added to a final concentration
of 100 .mu.M as an internal control. The solution was vortexed for
60 minutes and centrifuged at 10,000 rpm for 10 minutes. The
organic and aqueous layers were removed. Serial dilutions of the
organic layer were made with 50% methanol except for the initial
dilution, which was made in 100% methanol. Serial dilutions of the
aqueous layer were made in the pH 7.4 buffer. The diluted samples
were then assayed by LC/MS for the compound as well as for
testosterone. The Log of the ratio of peak area responses was
calculated to obtain the Log (D). Typical Log D values for
testosterone are from 3.0-3.3.
TABLE-US-00009 TABLE 5 Log (D) of Compounds Log(D) Compound
Octanol/pH 7.4 RG-115280 3.9 RG-102125 3.2 RG-102398 3.5 RG-100150
-2.1 RG-115595 2.1 RG-103309 3.0 RG-115555 0.0 RG-115199 2.0
RG-115823 3.3 RG-101523 2.9 RG-102240 3.4 RG-102317 3.6 RG-115517
2.9 RG-100021 0.8
2.7 Bi-Directional Permeability, CACO-2
[0440] Caco-2 monolayers were grown to confluence on
collagen-coated, microporous, polycarbonate membranes in 12-well
Costar Transwell plates. Details of the plates and their
certification are shown below. The permeability assay buffer was
Hank's Balanced Salt Solution containing 10 mM HEPES and 15 mM
glucose at a pH of 7.0.+-.0.2. The dosing solution concentration
was 10 .mu.M in assay buffer. At each time point, 1 and 2 hours, a
200-mL aliquot was taken from the receiver chamber and replaced
with fresh assay buffer. Cells were dosed on the apical side
(A-to-B) or basolateral side (B-to-A) and incubated at 37.degree.
C. with 5% CO.sub.2 and 90% relative humidity. Each determination
was performed in duplicate. The important experimental parameters
are outlined below. Permeability through a cell-free (blank)
membrane was studied to determine non-specific binding and free
diffusion of the compound through the device. Lucifer yellow flux
was also measured for each monolayer after being subjected to the
test compounds to ensure no damage was inflicted to the cell
monolayers during the flux period.
[0441] All samples were assayed by LC/MS using electrospray
ionization. Typical LC/MS conditions are as follows:
Liquid Chromatography
[0442] Column: Keystone Hypersil BDS C18 30.times.2.0 mm i.d., 3
.mu.m, with guard column
M.P. Buffer: Ammonium Formate Buffer, pH 3.5
[0443] Aqueous Reservoir (A): 90% water, 10% buffer Organic
Reservoir (B): 90% acetonitrile, 10% buffer
Flow Rate: 300 .mu.L/min
[0444] Gradient Program (typically):
TABLE-US-00010 Time Grad. Curve % A % B TE3 TE4 -0.1 0 100 0 close
1.2 1 60 40 close 3.0 1 0 100 3.1 0 100 0 4 0 100 0 close
Total run time: 4.5 min Autosampler: 10 .mu.L injection volume
Autosampler wash: water/acetonitrile/2-propanol:1/1/1; with 0.2%
formic acid
Mass Spectrometer
(Typical Operating Conditions)
Interface: Electrospray ("Turbo Ionspray")
Mode: Single Ion Monitoring
[0445] Gases: Neb Gas=8, Curtain Gas=10, Turbo Ionspray Gas=8000
mL/min.
TEM: 350.degree. C.
[0446] Voltages: IS 4500, OR 25, RNG 200, QO 10, IQ1-12, ST-15,
RQ0-12, DF-200, CEM (per age) Method: 4.5 minute duration.
[0447] The apparent permeability, Papp, and percent recovery were
calculated as follows:
Papp=(dC.sub.r/dt).times.V.sub.r/(A.times.C.sub.0) (1)
Percent
Recovery=100.times.((V.sub.r.times.C.sub.r.sup.final)+(V.sub.d.t-
imes.C.sub.d.sup.final))/(V.sub.d.times.C.sub.0) (2)
where, [0448] dC.sub.r/dt is the cumulative concentration in the
receiver compartment versus time in M s.sup.-1. [0449] V.sub.r is
the volume of the receiver compartment in cm.sup.3. [0450] V.sub.d
is the volume of the donor compartment in cm.sup.3. [0451] A is the
area of the cell monolayer (1.13 cm.sup.2 for 12-well Transwell).
[0452] C.sub.0 is the concentration of the dosing solution in M.
[0453] C.sub.r.sup.final is the cumulative receiver concentration
in M at the end of the incubation period. [0454] C.sub.d.sup.final
is the concentration of the donor in M at the end of the incubation
period.
TABLE-US-00011 [0454] Plates: TW12 TW12 Seed Date: Jun. 11, 2002
(KW) Jun. 18, 2002 (PSK) Passage: 62 61 Age (days): 27 22
Certification Acceptance Criteria TEER Value (.OMEGA. cm.sup.2):
506 504 450-650 .OMEGA. cm.sup.2 Lucifer Yellow, 0.14 0.12 <0.4
.times. 10.sup.-6 cm/s Papp .times. 10.sup.-6 cm/s: Atenolol, Papp
.times. 10.sup.-6 cm/s: 0.20 0.18 <0.5 .times. 10.sup.-6 cm/s
Propranolol, 20 19 15-25 .times. 10.sup.-6 cm/s Papp .times.
10.sup.-6 cm/s: Digoxin, Papp .times. 10.sup.-6 cm/s: 1.7 1.8 none
Digoxin, Papp .times. 10.sup.-6 cm/s: 12 16 none
Experimental Parameters
Dosing Concentration: 10 .mu.M
Replicates: 2
[0455] Direction: apical-to-basolateral, basolateral-to-apical Time
Points: 1 and 2 hours
TABLE-US-00012 TABLE 6 Recovery and Permeability (10.sup.-6 cm/s)
of Compounds Papp.sup.B-A Percent Recovery.sup.(C) Papp.sup.(D)
Papp, A-to-B Papp, B-to-A Papp.sup.A-B Absorption Significant
Compound Blank A-to-B B-to-A Blank Rep. 1 Rep. 2 Avg Rep. 1 Rep. 2
Avg Ratio.sup.(B) Potential.sup.(A) Efflux.sup.(B) RG-115280 41 46
99 1.88 1.24 1.30 1.27 1.50 1.48 1.49 1.2 High No RG-102125 62 84
63 21.9 25.7 26.3 26.0 20.7 19.7 20.2 0.8 High No RG-102398 94 85
90 34.7 27.0 26.8 26.9 25.9 28.2 27.1 1.0 High No RG-100150 117 102
107 36.9 0.19 0.18 0.18 0.33 0.34 0.33 1.8 Low No RG-115595 103 95
104 33.2 19.6 18.9 19.2 29.8 31.7 30.7 1.6 High No RG-103309 70 74
75 23.6 25.2 23.8 24.5 32.5 32.5 32.5 1.3 High No RG-115555 110 98
106 35.8 0.18 0.18 0.18 1.55 1.67 1.61 8.9 Low Yes RG-115199 82 85
87 23.0 31.1 30.2 30.6 31.0 30.9 30.9 1.0 High No RG-115823 77 64
63 24.2 17.4 17.8 17.6 18.8 15.4 17.1 1.0 High No RG-101523 95 95
88 29.4 24.2 26.6 25.4 23.6 25.4 24.5 1.0 High No RG-102240 78 94
87 28.4 32.3 31.4 31.8 23.3 23.7 23.5 0.7 High No RG-102317 87 91
86 27.9 28.6 27.3 28.0 21.0 21.2 21.1 0.8 High No RG-115517 96 91
95 31.6 28.4 29.3 28.8 25.2 27.6 26.4 0.9 High No RG-100021 113 90
94 43.0 0.21 0.22 0.22 0.91 0.95 0.93 4.3 Low No .sup.(A)Absorption
Potential Classification: Papp(A-to-B) .gtoreq.1.0 .times.
10.sup.-6 cm/s High Papp(A-to-B) >0.5 .times. 10.sup.-6 cm/s,
Papp <1.0 .times. 10.sup.-6 cm/s Medium Papp(A-to-B) <0.5
.times. 10.sup.-6 cm/s Low .sup.(B)Efflux considered significant
if: Papp (B-to-A) .gtoreq.1.0 .times. 10.sup.-6 cm/s and Ratio
Papp(B-to-A)/Papp(A-to-B) .gtoreq.3.0 .sup.(C)Low recoveries caused
by non-specific binding, etc. can affect the measured permeability
.sup.(D)A low rate of diffusion (<20 .times. 10.sup.-6 cm/s)
through the cell-free membrane indicates a lack of free diffusion,
which may affect the measured permeability.
Example 3
Biological Testing of Compounds
[0456] The ligands of the present invention are useful in various
applications including gene therapy, expression of proteins of
interest in host cells, production of transgenic organisms, and
cell-based assays.
27-63 Assay
Gene Expression Cassette
[0457] GAL4 DBD (1-147)-CfEcR(DEF)/VP16AD-.beta.RXREF-LmUSPEF:
[0458] The wild-type D, E, and F domains from spruce budworm
Choristoneura fumiferana EcR ("CfEcR-DEF"; SEQ ID NO: 1) were fused
to a GAL4 DNA binding domain ("Gal4DBD1-147"; SEQ ID NO: 2) and
placed under the control of a phosphoglycerate kinase promoter
("PGK"; SEQ ID NO: 3). Helices 1 through 8 of the EF domains from
Homo sapiens RXR.beta. ("HsRXR.beta.-EF"; nucleotides 1-465 of SEQ
ID NO: 4) and helices 9 through 12 of the EF domains of Locusta
migratoria Ultraspiracle Protein ("LmUSP-EF"; nucleotides 403-630
of SEQ ID NO: 5) were fused to the transactivation domain from VP16
("VP16AD"; SEQ ID NO: 6) and placed under the control of an
elongation factor-la promoter ("EF-1.alpha."; SEQ ID NO: 7). Five
consensus GAL4 response element binding sites ("5.times.GAL4RE";
comprising 5 copies of a GAL4RE comprising SEQ ID NO: 8) were fused
to a synthetic TATA minimal promoter (SEQ ID NO: 9) and placed
upstream of the luciferase reporter gene (SEQ ID NO: 10).
Stable Cell Line
[0459] CHO cells were transiently transfected with transcription
cassettes for GAL4 DBD (1-147) C:fEcR(DEF) and for VP16AD
13RXREF-LmUSPEF controlled by ubiquitously active cellular
promoters (PGK and EF-la, respectively) on a single plasmid. Stably
transfected cells were selected by Zeocin resistance. Individually
isolated CHO cell clones were transiently transfected with a GAL4
RE-luciferase reporter (pFR Luc). 27-63 clone was selected using
Hygromycin.
Treatment with Ligand
[0460] Cells were trypsinized and diluted to a concentration of
2.5.times.10.sup.4 cells mL. 100 .mu.L of cell suspension was
placed in each well of a 96 well plate and incubated at 37.degree.
C. under 5% CO.sub.2 for 24 h. Ligand stock solutions were prepared
in DMSO and diluted 300 fold for all treatments. Dose response
testing consisted of 8 concentrations ranging from 33 .mu.M to 0.01
.mu.M.
Reporter Gene Assay
[0461] Luciferase reporter gene expression was measured 48 h after
cell treatment using Bright-Glo.TM. Luciferase Assay System from
Promega (E2650). Luminescence was detected at room temperature
using a Dynex MLX microtiter plate luminometer.
Z3 Assay
Stable Cell Line
[0462] Dr. F. Gage provided a population of stably transformed
cells containing CVBE and 6.times.EcRE as described in Suhr, S. T.,
Gil, E. B., Senut M. C., Gage, F. H. (1998) Proc. Natl. Acad. Sci.
USA 95, 7999-804. Human 293 kidney cells, also referred to as
HEK-293 cells, were sequentially infected with retroviral vectors
encoding first the switch construct CVBE, and subsequently the
reporter construct 6.times.EcRE Lac Z. The switch construct
contained the coding sequence for amino acids 26-546 from Bombyx
mori EcR (BE) (Iatrou) inserted in frame and downstream of the VP16
transactivation domain (VBE). A synthetic ATG start codon was
placed under the control of cytomegalovirus (CVBE) immediate early
promoter and flanked by long terminal repeats (LTR). The reporter
construct contained six copies of the ecdysone response element
(EcRE) binding site placed upstream of LacZ and flanked on both
sides with LTR sequences (6.times.EcRE).
[0463] Dilution cloning was used to isolate individual clones.
Clones were selected using 450 ug/mL G418 and 100 ng/mL puromycin.
Individual clones were evaluated based on their response in the
presence and absence of test ligands. Clone Z3 was selected for
screening and SAR purposes.
[0464] Human 293 kidney cells stably transformed with CVBE and
6.times.EcRE LacZ were maintained in Minimum Essential Medium
(Mediates, 10-010-CV) containing 10% FBS (Life Technologies,
26140-087), 450 gum G418 (Mediates, 30-234-CR), and 100 gnome
promising (Sigma, P-7255), at 37.degree. C. in an atmosphere
containing 5% CO.sub.2 and were subculture when they reached 75%
confluence.
Treatment with Ligand
[0465] Z3 cells were seeded into 96-well tissue culture plates at a
concentration of 2.5.times.10.sup.3 cells per well and incubated at
37.degree. C. in 5% CO.sub.2 for twenty-four hours. Stock solutions
of ligands were prepared in DMSO. Ligand stock solutions were
diluted 100 fold in media and 50 .mu.L of this diluted ligand
solution (33 .mu.M) was added to cells. The final concentration of
DMSO was maintained at 0.03% in both controls and treatments.
Reporter Gene Assays
[0466] Reporter gene expression was evaluated 48 hours after
treatment of cells, .beta.-galactosidase activity was measured
using Gal Screen.TM. bioluminescent reporter gene assay system from
Tropix (GSY1000). Fold induction activities were calculated by
dividing relative light units ("RLU") in ligand treated cells with
RLU in DMSO treated cells. Luminescence was detected at room
temperature using a Dynex MLX microtiter plate luminometer.
[0467] A schematic of switch construct CVBE, and the reporter
construct 6.times.EcRE Lac Z is shown in FIG. 1. Flanking both
constructs are long terminal repeats, G418 and puromycin are
selectable markers, CMV is the cytomegalovirus promoter, VBE is
coding sequence for amino acids 26-546 from Bombyx mori EcR
inserted downstream of the VP16 transactivation domain,
6.times.EcRE is six copies of the ecdysone response element, lacZ
encodes for the reporter enzyme .beta.-galactosidase.
13B3 Assay
Gene Expression Cassette
[0468] GAL4 DBD-CfEcR(DEF)/VP16AD-MmRXRE: The wild-type D, E, and F
domains from spruce budworm Choristoneura fumiferana EcR
("CfEcR-DEF"; SEQ ID NO: 1) were fused to a GAL4 DNA binding domain
("Gal4DBD1-147"; SEQ ID NO: 2) and placed under the control of the
SV40e promoter of pM vector (PT3119-5, Clontech, Palo Alto,
Calif.). The D and E domains from Mus Musculus RXR ("MmRXR-DE"; SEQ
ID NO: 11) were fused to the transactivation domain from VP16
("VP16AD"; SEQ ID NO: 6) and placed under the control of the SV40e
promoter of the pVP16 vector (PT3127-5, Clontech, Palo Alto,
Calif.).
Stable Cell Line
[0469] CHO cells were transiently transfected with transcription
cassettes for GAL4 DBD-CfEcR(DEF) and for VP16AD-MmRXRE controlled
by SV40e promoters. Stably transfected cells were selected using
Hygromycin. Individually isolated CHO cell clones were transiently
transfected with a GAL4 RE-luciferase reporter (pFR-Luc,
Stratagene, La Jolla, Calif.). The 13B3 clone was selected using
Zeocin.
Treatment with Ligand
[0470] Cells were trypsinized and diluted to a concentration of
2.5.times.10.sup.4 cells mL. 100 .mu.L of cell suspension was
placed in each well of a 96 well plate and incubated at 37.degree.
C. under 5% CO.sub.2 for 24 h. Ligand stock solutions were prepared
in DMSO and diluted 300 fold for all treatments. Dose response
testing consisted of 8 concentrations ranging from 33 .mu.M to 0.01
.mu.M.
Reporter Gene Assay
[0471] Luciferase reporter gene expression was measured 48 h after
cell treatment using Bright-Glo.TM. Luciferase Assay System from
Promega (E2650). Luminescence was detected at room temperature
using a Dynex MLX microtiter plate luminometer.
[0472] The results of the assays are shown in Tables 7 and 8. Each
assay was conducted in two separate wells, and the two values were
averaged. Fold inductions were calculated by dividing relative
light units ("RLU") in ligand treated cells with RLU in DMSO
treated cells. EC.sub.50s were calculated from dose response data
using a three-parameter logistic model. Relative Max FI was
determined as the maximum fold induction of the tested ligand (an
embodiment of the invention) observed at any concentration relative
to the maximum fold induction of GS-.TM.-E ligand
(3,5-dimethyl-benzoic acid
N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide) observed at
any concentration.
TABLE-US-00013 TABLE 7 Biological Assay Results for Compounds: Fold
Induction 13B3 Assay 13B3 Assay 27-63 Assay 27-63 Assay Z3 Assay Z3
Assay Compound 3.3 uM 33 uM 3.3 uM 33 uM 3.3 uM 33 uM RG-100021 0 1
RG-100127 365 49 1239 245 RG-100150 1 1 RG-100216 8037 53 1312 398
RG-100342 611 2 1287 427 RG-100360 1111 745 1627 1353 870 891
RG-100394 178 1339 11 RG-100425 747 1156 1099 1128 592 RG-100448 2
66 847 423 RG-100492 1002 4 1378 143 RG-100524 1211 991 146
RG-100568 2 4122 615 2286 276 1193 RG-100569 1710 329 884 841 754
457 RG-100574 0 151 21 389 RG-100603 570 453 RG-100620 13982 710
428 RG-100667 1191 1480 238 RG-100690 1094 500 1136 1047 779 475
RG-100691 643 1378 1209 843 565 RG-100694 3385 2078 1057 1004 1294
1288 RG-100698 434 1089 591 RG-100699 2296 398 1413 415 RG-100725
581 1096 1050 511 RG-100749 0 288 874 145 RG-100763 2442 107 1609
151 RG-100764 3814 915 369 RG-100766 391 931 1993 921 474 RG-100767
4504 682 2097 2171 825 371 RG-100768 709 1738 1852 1246 425
RG-100769 1014 386 1595 1556 1096 542 RG-100778 2344 2159 2312 365
RG-100779 2979 947 304 RG-100801 16 1341 1244 153 RG-100812 202
1587 399 RG-100814 423 1151 2441 2165 410 1279 RG-100848 3391 578
1181 1151 885 26 RG-100864 318 187 1 1184 63 840 RG-100875 23 1143
34 1592 1006 1081 RG-100901 882 1527 185 RG-100915 133 525 532 1292
RG-100929 359 1080 988 490 RG-101013 1084 406 927 988 1039 846
RG-101016 4693 308 1347 394 RG-101036 4582 908 1721 1459 767 404
RG-101036 4582 908 1721 1459 767 404 RG-101048 701 19 1453 283
RG-101057 33 842 30 1008 632 930 RG-101062 319 1127 391 RG-101088
1400 6 1743 302 RG-101171 8453 395 1357 268 RG-101178 699 1200 431
RG-101202 5 7 195 1 RG-101218 1983 1097 1223 886 398 RG-101248 415
146 1033 375 RG-101312 1874 600 292 RG-101316 1280 1045 350
RG-101340 675 879 1624 1549 866 432 RG-101353 1347 718 3583 3835
530 439 RG-101376 25656 7393 1293 415 RG-101382 13058 1012 2704
2568 725 426 RG-101398 159 1138 403 RG-101408 326 2445 1886 780 398
RG-101494 755 613 717 294 832 384 RG-101509 1144 1303 328 RG-101511
1912 744 1421 1207 947 487 RG-101523 831 718 2427 2745 704 596
RG-101528 259 3027 748 1515 RG-101531 274 1130 428 RG-101542 439
1151 324 RG-101585 1015 154 1495 330 RG-101659 1556 1185 2283 1896
954 876 RG-101664 1 0 6 1 RG-101670 2297 738 1190 929 RG-101691
5245 630 2249 2073 654 1089 RG-101692 4180 170 1798 591 435 860
RG-101734 918 442 987 951 RG-101759 623 137 796 158 RG-101774 987
526 1807 1359 631 429 RG-101862 3279 293 717 250 RG-101863 3187 207
1705 636 832 374 RG-101864 5959 349 1807 1464 796 494 RG-101887
2462 542 1142 1107 1004 334 RG-101889 378 1085 245 RG-102021 4081
2951 417 RG-102125 762 315 1164 1043 359 473 RG-102125 762 315 1164
1043 359 473 RG-102317 2425 814 2551 2504 416 1501 RG-102398 8125
795 2875 3181 535 1972 RG-102408 25 249 RG-102592 194 909 7 1265
746 574 RG-103309 924 95 2537 1201 1155 591 RG-103361 504 2262 171
244 RG-103451 576 661 3326 3865 67 118 RG-104074 544 5378 189 200
RG-115006 4180 3146 415 1071 RG-115009 19 2547 35 472 RG-115025 8
1700 2288 2243 269 489 RG-115033 386 12 2 1256 24 388 RG-115038
4119 4970 600 321 RG-115043 835 547 466 1588 RG-115046 1076 18 1069
754 RG-115050 2027 894 424 1446 RG-115055 1356 1350 4499 2725 573
617 RG-115064 2 415 1 3 RG-115065 880 859 1095 878 RG-115068 2828
1500 54 802 RG-115077 932 199 294 RG-115085 236 1143 1149 627
RG-115086 433 RG-115088 542 1048 561 228 RG-115092 2322 2409 2869
3106 351 302 RG-115095 RG-115102 1425 109 1154 971 88 865 RG-115106
68 RG-115112 618 RG-115116 2 276 RG-115118 979 769 1063 914 90 1160
RG-115128 110 RG-115130 987 511 4436 4096 412 930 RG-115143 1 2032
59 1736 111 1045 RG-115162 755 320 1814 1464 334 772 RG-115167 73
RG-115169 405 443 RG-115171 RG-115191 3 386 RG-115199 349 RG-115199
349 RG-115207 7260 7959 1332 1279 354 508 RG-115220 5 1143 298
RG-115223 8 3437 273 1935 323 1299 RG-115229 599 709 2829 1423 1032
RG-115244 2404 573 2203 1847 283 816 RG-115253 471 848 910 832
RG-115256 647 RG-115257 820 354 1130 1320 297 691 RG-115258 144
3745 1973 2212 382 1120 RG-115259 3513 2981 91 RG-115260 13 526
RG-115261 31 RG-115269 112 RG-115278 1950 1050 1250 906 158 1225
RG-115280 0 9 422 376 RG-115280 0 9 422 376 RG-115297 1364 304 1544
946 443 604 RG-115302 521 648 940 815 404 RG-115306 5 RG-115310 2
3044 199 960 RG-115311 RG-115327 3785 279 325 RG-115329 5644 259
430 RG-115330 1995 3577 633 432 RG-115337 631 1010 1080 1053 682
RG-115350 450 499 RG-115352 7 RG-115378 1778 2424 1493 1407 488
1464 RG-115384 2753 2277 1713 1282 337 RG-115407 2476 2612 1611
1515 391 879 RG-115416 3618 2737 2412 1867 164 1116 RG-115422 204
RG-115429 18 1843 3 1724 RG-115441 118 RG-115443 RG-115480
RG-115496 874 RG-115499 1182 731 2092 1536 173 641 RG-115508 310
191 1195 969 199 680 RG-115514 4009 515 1616 1427 383 658 RG-115515
1996 1306 420 RG-115517 8397 11953 408 RG-115517 8397 11953 408
RG-115518 1644 926 1640 1126 232 803 RG-115532 908 738 530
RG-115534 211 168 249 RG-115536 483 488 RG-115539 20 592 1076 534
339 RG-115550 1 0 21 RG-115551 290 1150 1068 470 RG-115555 1
RG-115557 426 RG-115567 RG-115575 3085 865 282 785 RG-115580 298
1104 1031 615 RG-115592 RG-115595 6 1381 44 1067 RG-115595 6 1381
44 1067 RG-115609 558 4217 90 383 RG-115611 745 RG-115613 180 1726
991 1469 RG-115625 3 715 265 1559 RG-115627 1291 10058 378 362
RG-115637 1109 636 4159 2741 211 RG-115647 2169 98 817 815 96 510
RG-115648 9 RG-115664 1363 333 1928 1768 209 620 RG-115674 442 319
RG-115683 RG-115684 151 498 RG-115689 13 RG-115690 930 571
RG-115716 3 0 66 RG-115717 0 0 9 RG-115718 0 2 0 RG-115719 0 1 271
RG-115721 0 0 2 RG-115722 0 0 1 RG-115723 0 0 17 RG-115819 1970
2371 1433 722 RG-115820 2861 1971 1413 701 RG-115823 2093 1025 1440
1050 RG-115824 2675 948 895 737 RG-115829 2605 45 1441 319
RG-115830 2287 353 1604 983 RG-115831 2063 1435 1481 544 RG-115832
2063 1435 1564 621 RG-115834 1900 1837 RG-115835 3 1895 RG-115836
1822 823 RG-115837 1474 1156 RG-115840 1612 263 RG-115841 1407 437
RG-115842 1269 447 RG-115846 1643 645 RG-115847 2729 848 RG-115848
1346 1156 RG-115849 1 231 RG-115850 1 23 RG-115856 1760 RG-115857
328 RG-115858 182 RG-115859 1056 RG-115861 8 593 RG-115862 1 243
RG-115863 1 804 RG-115864 1255 RG-115865 76 RG-115866 3 RG-115867
654 RG-115003 0 301 1276 RG-115044 0 1 1 2 1 7 RG-115079 3 0 1 0
RG-115091 1 61 RG-115117 1 221 7 RG-115160 3 3 3 157 RG-115172 1 0
1 216
RG-115225 3 8 3 571 RG-115358 1 584 33 769 RG-115371 1 1092 249
1774 RG-115408 1 0 1 3 RG-115490 2 844 123 814 RG-115497 1 0 1 20
RG-115511 1 98 1 1155 10 570 RG-115597 0 4667 2 2065 942 RG-115653
6 0 1 832 666 RG-115665 1 0 2 0 6 74 RG-115783 3 2765 1362
TABLE-US-00014 TABLE 8 Biological Assay Results for Compounds: EC50
Relative Max FI 13B3 EC50 13B3 assay Rel 27-63 assay 27-63 assay Z3
assay Z3 assay Compound (.mu.M) Max FI EC50 (.mu.M) Rel Max FI EC50
(.mu.M) Rel Max FI RG-100127 6.704 0.689 4.677 RG-100216 6.972
0.685 1.778 RG-100342 17.694 0.674 10.233 RG-100360 0.588 0.825
0.615 0.873 0.257 1.073 RG-100394 1.223 RG-100425 0.370 0.671 0.099
1.180 RG-100448 5.288 0.609 2.818 RG-100492 11.757 0.916 8.128
RG-100524 0.263 1.002 RG-100568 44.284 1.035 4.132 1.197 3.311
0.855 RG-100569 0.185 0.773 0.287 0.720 0.098 0.835 RG-100574 4.000
0.371 1.122 RG-100620 2.399 0.869 RG-100667 0.347 0.890 RG-100690
0.268 0.506 0.200 0.727 0.240 1.008 RG-100691 0.330 0.863 0.257
0.945 RG-100694 0.405 0.632 0.400 0.767 0.389 1.157 RG-100698 1.000
1.025 RG-100699 3.963 0.939 2.089 RG-100725 0.330 0.726 RG-100749
2.692 0.951 RG-100763 5.301 0.856 2.138 RG-100764 2.344 0.900
RG-100766 3.419 0.944 0.174 1.194 RG-100767 1.056 0.920 0.334 1.218
0.251 0.996 RG-100768 0.333 1.039 0.214 1.129 RG-100769 0.337 0.685
0.196 0.735 0.219 1.109 RG-100778 0.678 1.342 0.513 1.000 RG-100779
0.178 0.707 RG-100801 2.076 0.936 0.891 RG-100812 0.912 0.954
RG-100814 37.888 0.843 1.500 1.157 1.148 RG-100848 1.645 0.666
0.322 0.765 0.240 0.996 RG-100864 1.155 17.269 0.716 2.239
RG-100875 4.019 0.853 8.920 1.004 2.042 0.818 RG-100901 0.316 0.918
RG-100915 4.449 0.552 1.000 0.912 RG-100929 1.047 0.770 0.631
RG-101013 2.256 0.539 2.140 0.606 1.479 0.720 RG-101016 4.201 0.703
1.995 RG-101036 0.223 0.545 0.177 0.724 0.054 1.049 RG-101036 0.223
0.545 0.177 0.724 0.054 1.049 RG-101048 8.415 0.708 19.055
RG-101057 4.970 0.569 6.617 0.648 4.467 0.697 RG-101062 2.570 0.992
RG-101088 9.947 1.159 4.074 RG-101171 4.019 0.877 3.236 RG-101178
1.318 0.887 RG-101202 0.369 0.007 1.738 RG-101218 0.128 0.645 0.316
0.998 RG-101248 5.036 0.790 1.445 RG-101312 5.623 0.955 RG-101316
1.585 0.896 RG-101340 0.106 0.364 0.240 0.964 0.234 1.126 RG-101353
0.675 1.163 0.266 1.168 0.107 1.078 RG-101376 1.361 0.654 0.537
0.777 RG-101382 0.588 0.915 0.306 1.188 0.066 0.951 RG-101398 0.282
0.712 RG-101408 0.336 1.185 0.148 1.118 RG-101494 0.148 0.822 0.036
0.916 0.079 1.093 RG-101509 1.175 0.882 RG-101511 0.794 0.811 0.714
0.762 0.468 1.122 RG-101523 2.374 0.772 0.165 0.943 0.242 RG-101528
29.303 1.111 1.047 0.923 RG-101531 0.085 RG-101542 0.324 0.793
RG-101585 5.057 0.893 4.898 RG-101659 0.562 0.867 0.347 1.009 0.214
RG-101664 >50 0.002 21.081 RG-101670 0.646 0.874 0.468 0.853
RG-101691 0.406 0.918 0.250 0.969 0.174 1.068 RG-101692 0.728 1.004
0.788 1.008 0.309 0.975 RG-101734 0.145 0.988 RG-101759 0.877 0.336
0.331 0.639 RG-101774 0.327 0.657 0.359 0.795 0.155 0.943 RG-101862
1.050 0.861 0.269 0.866 RG-101863 0.728 0.837 1.003 1.043 0.257
0.953 RG-101864 0.288 0.460 0.343 0.750 0.245 1.075 RG-101887 0.861
0.579 0.351 0.675 0.257 0.930 RG-101889 3.300 RG-102021 4.045 1.136
0.513 0.920 RG-102125 0.479 0.190 0.721 0.174 RG-102125 0.479 0.190
0.721 0.174 RG-102240 0.5 1 0.288 1 0.286 1 RG-102317 0.345 0.102
0.948 RG-102398 0.627 0.838 0.325 1.017 0.095 1.027 RG-102408 0.091
0.662 0.054 1.114 RG-102592 4.922 0.684 7.770 0.798 3.890 0.699
RG-103309 0.146 1.486 3.024 0.975 0.078 0.950 RG-103361 28.000
0.758 0.526 0.938 RG-103451 0.208 0.742 0.314 0.884 0.083 1.054
RG-104074 0.865 0.812 0.307 1.122 RG-115006 3.496 0.890 0.275 1.031
RG-115009 18.908 0.917 3.307 0.916 RG-115025 39.254 0.186 2.449
1.523 0.347 0.385 RG-115033 1.000 0.643 18.101 0.868 16.218 0.575
RG-115038 1.743 1.172 0.550 1.064 RG-115043 2.669 0.852 0.178 0.943
RG-115046 5.657 0.739 3.548 RG-115050 0.762 0.724 0.282 0.808
RG-115055 0.303 0.894 0.318 1.341 0.043 0.936 RG-115064 >50
0.000 >50 0.031 RG-115065 3.000 0.746 1.479 RG-115068 0.926
0.722 21.000 0.804 RG-115077 0.847 1.509 0.389 0.911 RG-115085
0.807 0.683 0.195 RG-115086 0.692 0.433 RG-115088 1.827 0.709 0.501
0.958 RG-115092 0.184 1.176 0.213 1.092 0.037 0.917 RG-115102 0.930
0.546 0.491 0.667 0.139 0.858 RG-115106 2.570 0.457 RG-115112 0.309
0.618 RG-115116 >50 1.172 3.311 0.960 RG-115118 0.689 0.530
0.394 0.612 0.234 0.969 RG-115128 0.245 0.538 RG-115130 0.512 0.093
1.375 0.036 RG-115143 >50 0.651 7.777 1.112 7.413 0.844
RG-115162 0.811 0.630 0.424 0.653 0.069 0.988 RG-115167 0.240 0.533
RG-115169 0.497 0.618 0.347 0.956 RG-115191 >50 1.513 2.399
0.934 RG-115199 3.467 0.734 RG-115207 0.618 0.707 0.593 0.704 0.257
0.923 RG-115220 >50 0.125 0.871 0.591 RG-115223 >50 1.346
4.685 1.167 1.905 0.925 RG-115229 2.250 0.418 1.153 0.735 0.427
RG-115244 0.369 0.663 0.169 0.785 0.085 0.959 RG-115253 0.107 0.742
0.269 RG-115256 0.692 0.527 RG-115257 0.886 0.742 0.698 0.663 0.204
0.885 RG-115258 27.111 1.334 1.879 1.279 0.646 0.882 RG-115259
2.704 0.702 0.427 0.617 RG-115261 29.512 0.498 RG-115269 0.437
0.552 RG-115278 0.630 0.823 0.885 0.720 0.257 0.893 RG-115280
11.000 0.254 RG-115280 11.000 0.254 RG-115297 1.361 0.737 0.330
0.986 0.066 0.872 RG-115302 2.000 0.624 1.044 0.521 0.275 RG-115306
3.467 0.424 RG-115310 >50 0.676 2.630 0.781 RG-115327 2.754
0.662 RG-115329 2.570 0.892 RG-115330 5.690 0.711 0.186 0.860
RG-115337 0.621 0.927 0.363 0.822 0.095 RG-115350 0.795 0.711 0.309
0.935 RG-115352 45.709 0.265 RG-115378 1.762 0.655 0.758 0.863
0.324 0.947 RG-115384 0.320 0.426 0.113 0.645 0.166 0.904 RG-115407
7.000 1.048 1.056 0.932 0.347 0.906 RG-115416 0.938 12.104 0.637
0.721 0.091 1.869 RG-115422 0.398 0.769 RG-115429 8.676 1.065 9.574
0.880 RG-115441 0.126 0.759 RG-115496 1.175 0.878 RG-115499 0.328
0.489 0.336 0.705 0.170 0.954 RG-115508 0.849 0.805 1.033 0.719
0.537 0.787 RG-115514 0.541 0.550 0.170 0.720 0.056 0.970 RG-115515
0.617 0.975 RG-115517 0.355 0.675 0.089 0.923 RG-115517 0.355 0.675
0.089 0.923 RG-115518 1.253 0.648 1.053 0.866 0.257 0.834 RG-115532
0.518 0.835 0.129 0.933 RG-115534 2.754 0.463 RG-115536 0.781 0.734
0.126 0.950 RG-115539 5.000 0.955 1.177 0.595 0.151 RG-115551 0.852
0.684 0.398 RG-115555 >50 0.006 RG-115557 1.698 0.566 RG-115575
0.271 0.641 0.102 0.886 RG-115580 0.375 0.760 0.182 RG-115595 5.623
0.686 RG-115595 5.623 0.686 RG-115609 13.782 0.883 1.386 0.625
RG-115611 1.585 0.703 RG-115613 5.937 0.734 0.589 0.964 RG-115625
25.322 0.931 0.389 0.950 RG-115627 0.921 0.854 0.813 0.840
RG-115637 0.088 1.002 0.117 1.305 0.018 0.947 RG-115647 0.832 0.662
0.372 0.505 0.145 0.923 RG-115648 21.380 0.182 RG-115664 0.323
0.497 0.359 0.635 0.126 0.846 RG-115674 1.723 0.875 0.229 0.995
RG-115689 100.000 0.411 RG-115690 0.632 1.181 0.209 0.998 RG-115819
0.025 1.045 0.042 0.915 RG-115820 0.193 1.270 0.203 0.887 RG-115823
0.011 1.069 0.020 0.933 RG-115824 0.036 1.218 0.046 0.806 RG-115829
0.035 1.264 0.058 0.937 RG-115830 0.036 1.049 0.045 1.020 RG-115831
0.096 1.366 0.102 0.937 RG-115832 0.035 1.075 0.037 1.002 RG-115834
1.170 0.733 RG-115835 20.000 0.731 RG-115836 0.098 0.808 RG-115837
1.114 0.569 RG-115840 0.110 0.776 RG-115841 2.015 0.610 RG-115842
1.196 0.524 RG-115846 0.095 0.846 RG-115847 1.100 1.120 RG-115848
1.291 0.494 RG-115849 >33 0.007 RG-115850 >33 0.001 RG-115851
0.02 1 RG-115852 0.07 1 RG-115856 0.003 0.879 RG-115857 0.013 1.140
RG-115858 0.005 0.915 RG-115859 0.007 1.218 RG-115861 4.308 0.215
RG-115862 >33 0.048 RG-115863 >33 0.154 RG-115864 0.004 0.979
RG-115865 0.010 1.246 RG-115044 >50 0.87 >33 0.00 >50 1.08
RG-115079 0.02 0.20 >50 0.02 RG-115117 >50 0 3.93 0.36
RG-115160 >50 0 >50 0.21 RG-115172 >50 0 >50 0.32
RG-115225 0.58 0.08 >50 0.32 RG-115358 >50 0.20 9.45 0.48
RG-115371 >50 1.44 3.02 0.76 RG-115490 >50 1.01 6.35 0.44
RG-115497 >50 0 >50 0.07 RG-115511 >50 0.07 12.00 0.74
38.99 0.66 RG-115597 9.83 1.26 RG-115408 >50 0 >50 0.01
RG-115653 19.77 0.53 RG-115665 >50 0 5.49 0.01 12.20 0.09
RG-115783 ~15 1.42 3.05 0.93 RG-115866 0.010 0.999
Example 4
Biological (In Vivo) Testing of Compounds
[0473] Applicants' ligands are useful in various applications
including gene therapy, expression of proteins of interest in host
cells, production of transgenic organisms, and cell-based assays.
In vivo induction of a reporter enzyme with various ligands of the
present invention was evaluated in a C57BL/6 mouse model system
containing a gene switch.
Gene Expression Cassettes
[0474] The wild-type D, E, and F domains from spruce budworm
Choristoneura fumiferana EcR ("CfEcR-DEF"; SEQ ID NO: 1) were
mutated [V107 (gtt).fwdarw.I107 (aft) and Y127 (tac).fwdarw.E127
(gag)] and fused to a GAL4 DNA binding domain ("Gal4DBD1-147"; SEQ
ID NO: 2). Helices 1 through 8 of the EF domains from Homo sapiens
RXR.beta. ("HsRXR.beta.-EF"; nucleotides 1-465 of SEQ ID NO: 4) and
helices 9 through 12 of the EF domains of Locusta migratoria
Ultraspiracle Protein ("LmUSP-EF"; nucleotides 403-630 of SEQ ID
NO: 5) were fused to the transactivation domain from VP16
("VP16AD"; SEQ ID NO: 6), which regulates a reporter gene human
secreted alkaline phosphatase ("SEAP", SEQ ID NO: 12) that was
placed under the control of a 6.times.GAL4 response element (SEQ ID
NO: 13) and a transthyretin promoter (SEQ ID NO: 14). Each element
of the gene switch was on a separate plasmid. Receptor expression
was under the control of a CMV promoter (SEQ ID NO: 15). Induction
was evaluated by the amount of reporter protein expressed in the
presence of ligand.
Electroporation of Gene Switch
[0475] SEAP expression in serum of mice was evaluated after
electroporation of the gene switch into mouse quadriceps. Mice were
anesthetized with 2 .mu.L/g of a mixture of ketamine (100 mg/mL)
and xylazine (20 mg/mL) Animals were then shaved, DNA vectors
injected into the muscle in a volume of 2.times.50 .mu.L
polyglutamic acid (12 mg/mL water), electrode conductivity gel
applied, and an electrode caliber (1 cm.times.1 cm; model 384) was
placed on hind leg. The muscle was electroporated with 200 V/cm, 8
times, for 20 msec/pulse, at 1 sec time intervals. The transverse
electrical field direction was reversed after the animals received
half of the pulses. Electroporation was performed with an ECM 830
electroporator from BTX Molecular Delivery Systems.
Treatment with Ligand
[0476] In some experiments mice received an intraperitoneal
injection (IP) of 2.6 .mu.mol of ligand in 50 .mu.L of DMSO 3 days
after electroporation of the gene switch. In other experiments the
concentration of ligand was decrease to 26 nmol/50 .mu.L of
DMSO/mouse. SEAP expression was evaluated 2-11 days after ligand
administration. In other experiments ligand was administered in
rodent chow. The chow was prepared by dissolving 2 g of ligand in
20 mL of acetone and adding it to 1 kg of LabDiet 5010 autoclavable
chow from Purina Mills. This was thoroughly mixed in a Hobart mixer
and then mixed for an additional 15 min in a Cross Blend mixer
Animals received chow ad libitum for 1, 2, or 3 days. All values
are the average from four animals. Background SEAP in sera from
animals treated with vector alone without ligand addition was 0-11
ng/mL serum.
Reporter Assay
[0477] Mouse serum was obtained by centrifugation of blood acquired
by retroorbital bleeding with a small glass capillary tube. SEAP
quantification was determined using a Clontech Great Escape
chemiluminescence kit and by comparison with the Clontech SEAP
standard.
[0478] Table 9: In vivo evaluation of ligand-mediated induction of
a mutated ecdysone receptor-based gene switch. SEAP expression in
serum of mice was evaluated after electroporation of the gene
expression cassettes into mouse quadriceps. Mice received an IP
injection of 2.6 mmol of ligand 3 days after electroporation. SEAP
expression was evaluated 2-11 days after ligand administration.
Each dose group was composed of four animals. Percentage of
GS.TM.-E ligand induction was determined by averaging SEAP
expression from four animals divided by the average SEAP expression
induced with GS.TM.-E ligand and then multiplying by 100.
[0479] Table 10: Induction of gene switch expression with low
concentrations of ligand. SEAP expression in serum of mice was
evaluated after electroporation of the gene expression cassettes
into mouse quadriceps. Mice received an IP injection of 26 nmol of
ligand or 130 nmol GS.TM.-E ligand 3 days after electroporation.
SEAP expression was evaluated 2-7 days after ligand administration.
Values are the average from 4 animals.
[0480] Table 11: Induction of SEAP in C57BL/6 mice with GS.TM.-E
ligand or RG-103309 administered in rodent chow. SEAP expression in
serum of mice was evaluated after electroporation of the gene
expression cassettes into mouse quadriceps. Mice received GS.TM.-E
ligand or RG-103309 in their feed (2 g/kg) 3 days after
electroporation. Feed was administered ad libitum for 1, 2, or 3
days. After each interval ligand-treated feed was removed and
animals received untreated feed. Values are the average from 4
animals.
TABLE-US-00015 TABLE 9 In vivo evaluation of ligand-mediated
induction of a mutated ecdysone receptor-based gene switch.
Secreted Alkaline Phosphatase (percentage of GS .TM.-E ligand
induction) Compound. Day 2 Day 3 Day 11 RG-101382 92 81 890
RG-102317 112 116 61 RG-101523 85 79 1,116 RG-101494 136 138 ND
RG-115613 2 1 ND RG-115575 74 78 69 RG-115637 12 7 0 RG-115517 4 3
ND RG-115092 8 2 ND RG-115009 4 3 ND GS .TM.-E ligand 100 100 100
RG-103309 251 298 ND RG-103451 76 73 371 RG-115819 172 215 3,008
RG-115820 82 63 399 RG-115823 129 183 2,652 RG-115824 102 101 415
RG-115832 147 189 4,183 RG-115831 118 121 105 RG-115830 120 158
3,558 RG-115829 72 83 687 ND--not determined.
TABLE-US-00016 TABLE 10 Induction of gene switch expression with
low concentrations of ligand. Secreted Alkaline Phosphatase (ng/ml
mouse sera) Compound Day 2 Day 3 Day 7 GS .TM.-E ligand(130 652
1,780 139 nmol) RG-103309 (26 nmol) 3,428 2,800 143 RG-115819 (26
nmol) 5,984 4,096 453 RG-115823 (26 nmol) 3,788 2,705 373 RG-115832
(26 nmol) 2,349 1,807 149 RG-115830 (26 nmol) 6,835 5,339 590
RG-115856 (26 nmol) 2,292 2,350 ND RG-115857 (26 nmol) 574 401 ND
RG-115858 (26 nmol) 13,661 11,820 ND RG-115864 (26 nmol) 6,722
5,652 ND ND--not determined.
TABLE-US-00017 TABLE 11 Induction of gene switch expression with
ligands administered in rodent`s feed. Secreted Alkaline
Phosphatase (ng/ml mouse sera) .sup.1 Compound (dose period) Day 2
Day 3 Day 7 GS .TM.-E ligand (1 day) 7,302 7,670 3,784 GS .TM.-E
ligand (2 day) 13,046 15,831 8,816 GS .TM.-E ligand (3 day) 8,064
11,392 8,372 RG-103309 (1 day) 9,003 14,850 5,172 RG-103309 (2 day)
6,971 16,518 7,460 RG-103309 (3 day) 11,126 20,373 11,549
[0481] In addition, one of ordinary skill in the art is also able
to predict that the ligands disclosed herein will also work to
modulate gene expression in various cell types described above
using gene expression systems based on group H and group B nuclear
receptors.
Sequence CWU 1
1
1811054DNAChoristoneura fumiferana 1cctgagtgcg tagtacccga
gactcagtgc gccatgaagc ggaaagagaa gaaagcacag 60aaggagaagg acaaactgcc
tgtcagcacg acgacggtgg acgaccacat gccgcccatt 120atgcagtgtg
aacctccacc tcctgaagca gcaaggattc acgaagtggt cccaaggttt
180ctctccgaca agctgttgga gacaaaccgg cagaaaaaca tcccccagtt
gacagccaac 240cagcagttcc ttatcgccag gctcatctgg taccaggacg
ggtacgagca gccttctgat 300gaagatttga agaggattac gcagacgtgg
cagcaagcgg acgatgaaaa cgaagagtct 360gacactccct tccgccagat
cacagagatg actatcctca cggtccaact tatcgtggag 420ttcgcgaagg
gattgccagg gttcgccaag atctcgcagc ctgatcaaat tacgctgctt
480aaggcttgct caagtgaggt aatgatgctc cgagtcgcgc gacgatacga
tgcggcctca 540gacagtgttc tgttcgcgaa caaccaagcg tacactcgcg
acaactaccg caaggctggc 600atggcctacg tcatcgagga tctactgcac
ttctgccggt gcatgtactc tatggcgttg 660gacaacatcc attacgcgct
gctcacggct gtcgtcatct tttctgaccg gccagggttg 720gagcagccgc
aactggtgga agaaatccag cggtactacc tgaatacgct ccgcatctat
780atcctgaacc agctgagcgg gtcggcgcgt tcgtccgtca tatacggcaa
gatcctctca 840atcctctctg agctacgcac gctcggcatg caaaactcca
acatgtgcat ctccctcaag 900ctcaagaaca gaaagctgcc gcctttcctc
gaggagatct gggatgtggc ggacatgtcg 960cacacccaac cgccgcctat
cctcgagtcc cccacgaatc tctagcccct gcgcgcacgc 1020atcgccgatg
ccgcgtccgg ccgcgctgct ctga 10542441DNASaccharomyces cerevisiae
2atgaagctac tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag
60tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac
120tctcccaaaa ccaaaaggtc tccgctgact agggcacatc tgacagaagt
ggaatcaagg 180ctagaaagac tggaacagct atttctactg atttttcctc
gagaagacct tgacatgatt 240ttgaaaatgg attctttaca ggatataaaa
gcattgttaa caggattatt tgtacaagat 300aatgtgaata aagatgccgt
cacagataga ttggcttcag tggagactga tatgcctcta 360acattgagac
agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt
420caaagacagt tgactgtatc g 4413538DNAMus musculus 3tcgagggccc
ctgcaggtca attctaccgg gtaggggagg cgcttttccc aaggcagtct 60ggagcatgcg
ctttagcagc cccgctggca cttggcgcta cacaagtggc ctctggcctc
120gcacacattc cacatccacc ggtagcgcca accggctccg ttctttggtg
gccccttcgc 180gccaccttct actcctcccc tagtcaggaa gttccccccc
gccccgcagc tcgcgtcgtg 240caggacgtga caaatggaag tagcacgtct
cactagtctc gtgcagatgg acagcaccgc 300tgagcaatgg aagcgggtag
gcctttgggg cagcggccaa tagcagcttt gctccttcgc 360tttctgggct
cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag
420gggcggggcg ggcgcgaagg tcctcccgag gcccggcatt ctcgcacgct
tcaaaagcgc 480acgtctgccg cgctgttctc ctcttcctca tctccgggcc
tttcgacctg cagccaat 5384720DNAHomo sapiens 4gcccccgagg agatgcctgt
ggacaggatc ctggaggcag agcttgctgt ggaacagaag 60agtgaccagg gcgttgaggg
tcctggggga accgggggta gcggcagcag cccaaatgac 120cctgtgacta
acatctgtca ggcagctgac aaacagctat tcacgcttgt tgagtgggcg
180aagaggatcc cacacttttc ctccttgcct ctggatgatc aggtcatatt
gctgcgggca 240ggctggaatg aactcctcat tgcctccttt tcacaccgat
ccattgatgt tcgagatggc 300atcctccttg ccacaggtct tcacgtgcac
cgcaactcag cccattcagc aggagtagga 360gccatctttg atcgggtgct
gacagagcta gtgtccaaaa tgcgtgacat gaggatggac 420aagacagagc
ttggctgcct gagggcaatc attctgttta atccagatgc caagggcctc
480tccaacccta gtgaggtgga ggtcctgcgg gagaaagtgt atgcatcact
ggagacctac 540tgcaaacaga agtaccctga gcagcaggga cggtttgcca
agctgctgct acgtcttcct 600gccctccggt ccattggcct taagtgtcta
gagcatctgt ttttcttcaa gctcattggt 660gacaccccca tcgacacctt
cctcatggag atgcttgagg ctccccatca actggcctga 7205635DNALocusta
migratoria 5tgcatacaga catgcctgtt gaacgcatac ttgaagctga aaaacgagtg
gagtgcaaag 60cagaaaacca agtggaatat gagctggtgg agtgggctaa acacatcccg
cacttcacat 120ccctacctct ggaggaccag gttctcctcc tcagagcagg
ttggaatgaa ctgctaattg 180cagcattttc acatcgatct gtagatgtta
aagatggcat agtacttgcc actggtctca 240cagtgcatcg aaattctgcc
catcaagctg gagtcggcac aatatttgac agagttttga 300cagaactggt
agcaaagatg agagaaatga aaatggataa aactgaactt ggctgcttgc
360gatctgttat tcttttcaat ccagaggtga ggggtttgaa atccgcccag
gaagttgaac 420ttctacgtga aaaagtatat gccgctttgg aagaatatac
tagaacaaca catcccgatg 480aaccaggaag atttgcaaaa cttttgcttc
gtctgccttc tttacgttcc ataggcctta 540agtgtttgga gcatttgttt
ttctttcgcc ttattggaga tgttccaatt gatacgttcc 600tgatggagat
gcttgaatca ccttctgatt cataa 6356271DNAherpes simplex virus 7
6atgggcccta aaaagaagcg taaagtcgcc cccccgaccg atgtcagcct gggggacgag
60ctccacttag acggcgagga cgtggcgatg gcgcatgccg acgcgctaga cgatttcgat
120ctggacatgt tgggggacgg ggattccccg gggccgggat ttacccccca
cgactccgcc 180ccctacggcg ctctggatat ggccgacttc gagtttgagc
agatgtttac cgatgccctt 240ggaattgacg agtacggtgg ggaattcccg g
27171167DNAHomo sapiens 7tgaggctccg gtgcccgtca gtgggcagag
cgcacatcgc ccacagtccc cgagaagttg 60gggggagggg tcggcaattg aaccggtgcc
tagagaaggt ggcgcggggt aaactgggaa 120agtgatgtcg tgtactggct
ccgccttttt cccgagggtg ggggagaacc gtatataagt 180gcagtagtcg
ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt
240gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg
cgtgccttga 300attacttcca cctggctcca gtacgtgatt cttgatcccg
agctggagcc aggggcgggc 360cttgcgcttt aggagcccct tcgcctcgtg
cttgagttga ggcctggcct gggcgctggg 420gccgccgcgt gcgaatctgg
tggcaccttc gcgcctgtct cgctgctttc gataagtctc 480tagccattta
aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg
540taaatgcggg ccaggatctg cacactggta tttcggtttt tgggcccgcg
gccggcgacg 600gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg
cctgcgagcg cggccaccga 660gaatcggacg ggggtagtct caagctggcc
ggcctgctct ggtgcctggc ctcgcgccgc 720cgtgtatcgc cccgccctgg
gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg 780aaagatggcc
gcttcccggc cctgctccag ggggctcaaa atggaggacg cggcgctcgg
840gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc
tcagccgtcg 900cttcatgtga ctccacggag taccgggcgc cgtccaggca
cctcgattag ttctggagct 960tttggagtac gtcgtcttta ggttgggggg
aggggtttta tgcgatggag tttccccaca 1020ctgagtgggt ggagactgaa
gttaggccag cttggcactt gatgtaattc tcgttggaat 1080ttgccctttt
tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt
1140tttttcttcc atttcaggtg tcgtgaa 1167819DNAArtificial sequenceGAL4
response element 8ggagtactgt cctccgagc 1996DNAArtificial
sequencesynthetic promoter 9tatata 6101653DNAArtificial
Sequenceluciferase gene 10atggaagacg ccaaaaacat aaagaaaggc
ccggcgccat tctatcctct agaggatgga 60accgctggag agcaactgca taaggctatg
aagagatacg ccctggttcc tggaacaatt 120gcttttacag atgcacatat
cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 180gttcggttgg
cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta
240tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt
tatcggagtt 300gcagttgcgc ccgcgaacga catttataat gaacgtgaat
tgctcaacag tatgaacatt 360tcgcagccta ccgtagtgtt tgtttccaaa
aaggggttgc aaaaaatttt gaacgtgcaa 420aaaaaattac caataatcca
gaaaattatt atcatggatt ctaaaacgga ttaccaggga 480tttcagtcga
tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat
540tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa
ttcctctgga 600tctactgggt tacctaaggg tgtggccctt ccgcatagaa
ctgcctgcgt cagattctcg 660catgccagag atcctatttt tggcaatcaa
atcattccgg atactgcgat tttaagtgtt 720gttccattcc atcacggttt
tggaatgttt actacactcg gatatttgat atgtggattt 780cgagtcgtct
taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac
840aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa
aagcactctg 900attgacaaat acgatttatc taatttacac gaaattgctt
ctgggggcgc acctctttcg 960aaagaagtcg gggaagcggt tgcaaaacgc
ttccatcttc cagggatacg acaaggatat 1020gggctcactg agactacatc
agctattctg attacacccg agggggatga taaaccgggc 1080gcggtcggta
aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa
1140acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat
tatgtccggt 1200tatgtaaaca atccggaagc gaccaacgcc ttgattgaca
aggatggatg gctacattct 1260ggagacatag cttactggga cgaagacgaa
cacttcttca tagttgaccg cttgaagtct 1320ttaattaaat acaaaggata
tcaggtggcc cccgctgaat tggaatcgat attgttacaa 1380caccccaaca
tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt
1440cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga
gatcgtggat 1500tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg
gaggagttgt gtttgtggac 1560gaagtaccga aaggtcttac cggaaaactc
gacgcaagaa aaatcagaga gatcctcata 1620aaggccaaga agggcggaaa
gtccaaattg taa 165311786DNAMus musculus 11aagcgggaag ctgtgcagga
ggagcggcag cggggcaagg accggaatga gaacgaggtg 60gagtccacca gcagtgccaa
cgaggacatg cctgtagaga agattctgga agccgagctt 120gctgtcgagc
ccaagactga gacatacgtg gaggcaaaca tggggctgaa ccccagctca
180ccaaatgacc ctgttaccaa catctgtcaa gcagcagaca agcagctctt
cactcttgtg 240gagtgggcca agaggatccc acacttttct gagctgcccc
tagacgacca ggtcatcctg 300ctacgggcag gctggaacga gctgctgatc
gcctccttct cccaccgctc catagctgtg 360aaagatggga ttctcctggc
caccggcctg cacgtacacc ggaacagcgc tcacagtgct 420ggggtgggcg
ccatctttga cagggtgcta acagagctgg tgtctaagat gcgtgacatg
480cagatggaca agacggagct gggctgcctg cgagccattg tcctgttcaa
ccctgactct 540aaggggctct caaaccctgc tgaggtggag gcgttgaggg
agaaggtgta tgcgtcacta 600gaagcgtact gcaaacacaa gtaccctgag
cagccgggca ggtttgccaa gctgctgctc 660cgcctgcctg cactgcgttc
catcgggctc aagtgcctgg agcacctgtt cttcttcaag 720ctcatcgggg
acacgcccat cgacaccttc ctcatggaga tgctggaggc accacatcaa 780gccacc
786121560DNAHomo sapiens 12atgctgctgc tgctgctgct gctgggcctg
aggctacagc tctccctggg catcatccca 60gttgaggagg agaacccgga cttctggaac
cgcgaggcag ccgaggccct gggtgccgcc 120aagaagctgc agcctgcaca
gacagccgcc aagaacctca tcatcttcct gggcgatggg 180atgggggtgt
ctacggtgac agctgccagg atcctaaaag ggcagaagaa ggacaaactg
240gggcctgaga tacccctggc catggaccgc ttcccatatg tggctctgtc
caagacatac 300aatgtagaca aacatgtgcc agacagtgga gccacagcca
cggcctacct gtgcggggtc 360aagggcaact tccagaccat tggcttgagt
gcagccgccc gctttaacca gtgcaacacg 420acacgcggca acgaggtcat
ctccgtgatg aatcgggcca agaaagcagg gaagtcagtg 480ggagtggtaa
ccaccacacg agtgcagcac gcctcgccag ccggcaccta cgcccacacg
540gtgaaccgca actggtactc ggacgccgac gtgcctgcct cggcccgcca
ggaggggtgc 600caggacatcg ctacgcagct catctccaac atggacattg
acgtgatcct aggtggaggc 660cgaaagtaca tgtttcgcat gggaacccca
gaccctgagt acccagatga ctacagccaa 720ggtgggacca ggctggacgg
gaagaatctg gtgcaggaat ggctggcgaa gcgccagggt 780gcccggtatg
tgtggaaccg cactgagctc atgcaggctt ccctggaccc gtctgtgacc
840catctcatgg gtctctttga gcctggagac atgaaatacg agatccaccg
agactccaca 900ctggacccct ccctgatgga gatgacagag gctgccctgc
gcctgctgag caggaacccc 960cgcggcttct tcctcttcgt ggagggtggt
cgcatcgacc atggtcatca tgaaagcagg 1020gcttaccggg cactgactga
gacgatcatg ttcgacgacg ccattgagag ggcgggccag 1080ctcaccagcg
aggaggacac gctgagcctc gtcactgccg accactccca cgtcttctcc
1140ttcggaggct accccctgcg agggagctcc atcttcgggc tggcccctgg
caaggcccgg 1200gacaggaagg cctacacggt cctcctatac ggaaacggtc
caggctatgt gctcaaggac 1260ggcgcccggc cggatgttac cgagagcgag
agcgggagcc ccgagtatcg gcagcagtca 1320gcagtgcccc tggacgaaga
gacccacgca ggcgaggacg tggcggtgtt cgcgcgcggc 1380ccgcaggcgc
acctggttca cggcgtgcag gagcagacct tcatagcgca cgtcatggcc
1440ttcgccgcct gcctggagcc ctacaccgcc tgcgacctgg cgccccccgc
cggcaccacc 1500gacgccgcgc acccgggtta ctctagagtc ggggcggccg
gccgcttcga gcagacatga 156013117DNAArtificialresponse element
13gcggagtact gtcctccgag cggagtactg tcctccgagc ggagtactgt cctccgagcg
60gagtactgtc ctccgagcgg agtactgtcc tccgagcgga gtactgtcct ccgagcg
11714136DNAMus musculus 14cttttgttga ctaagtcaat aatcagaatc
agcaggtttg gagtcagctt ggcagggatc 60agcagcctgg gttggaagga gggggtataa
aagccccttc accaggagaa gccgtcacac 120agatccacaa gctcct
13615659DNACytomegalovirus 15tcaatattgg ccattagcca tattattcat
tggttatata gcataaatca atattggcta 60ttggccattg catacgttgt atctatatca
taatatgtac atttatattg gctcatgtcc 120aatatgaccg ccatgttggc
attgattatt gactagttat taatagtaat caattacggg 180gtcattagtt
catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
240gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
atgttcccat 300agtaacgcca atagggactt tccattgacg tcaatgggtg
gagtatttac ggtaaactgc 360ccacttggca gtacatcaag tgtatcatat
gccaagtccg ccccctattg acgtcaatga 420cggtaaatgg cccgcctggc
attatgccca gtacatgacc ttacgggact ttcctacttg 480gcagtacatc
tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
540caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
ccattgacgt 600caatgggagt ttgttttggc accaaaatca acgggacttt
ccaaaatgtc gtaacaact 6591617DNAArtificial sequenceSynthetic
response element of the ecdysone receptor 16rrggttcant gacacyy
171713DNAArtificial sequenceSynthetic response element of the
ecdysone receptor 17aggtcanagg tca 131815DNAArtificial
sequenceSynthetic response element of the ecdysone receptor
18gggttgaatg aattt 15
* * * * *
References