U.S. patent application number 09/821673 was filed with the patent office on 2002-10-24 for synthesis and use of retinoid compounds having negative hormone and/or antagonist activities.
Invention is credited to Beard, Richard L., Chandraratna, Roshantha A., Duong, Tien T., Gillett, Samuel J., Johnson, Alan T., Klein, Elliott S., Nagpal, Sunil, Standeven, Andrew M., Teng, Min, Vuligonda, Vidyasagar.
Application Number | 20020156054 09/821673 |
Document ID | / |
Family ID | 27486804 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020156054 |
Kind Code |
A1 |
Klein, Elliott S. ; et
al. |
October 24, 2002 |
Synthesis and use of retinoid compounds having negative hormone
and/or antagonist activities
Abstract
Aryl-substituted and aryl and (3-oxo-1-propenly)-substituted
benzopyran, benzothiopyran, 1,2-dihydroquinoline, and
5,6-dihydronaphthalene derivatives have retinoid negative hormone
and/or antagonist-like biological activities. The invented RAR
antagonists can be administered to mammals, including humans, for
the purpose of preventing or diminishing action of RAR agonists on
the bound receptor sites. Specifically, the RAR agonists are
administered or coadministered with retinoid drugs to prevent or
ameliorate toxicity or side effects caused by retinoids or vitamin
A or vitamin A precursors. The retinoid negative hormones can be
used to potentiate the activities of other retinoids and nuclear
receptor agonists. For example, the retinoid negative hormone
called AGN 193109 effectively increased the effectiveness of other
retinoids and steroid hormones in in vitro transactivation assays.
Additionally, transactivation assays can be used to identify
compounds having negative hormone activity. These assays are based
on the ability of negative hormones to down-regulate the activity
of chimeric retinoid receptors engineered to possess a constitutive
transcription activator domain.
Inventors: |
Klein, Elliott S.; (Marina
del Rey, CA) ; Johnson, Alan T.; (Rancho Santa
Margarita, CA) ; Standeven, Andrew M.; (Corona del
Mar, CA) ; Beard, Richard L.; (Newport Beach, CA)
; Gillett, Samuel J.; (Albany, CA) ; Duong, Tien
T.; (Irvine, CA) ; Nagpal, Sunil; (Irvine,
CA) ; Vuligonda, Vidyasagar; (Irvine, CA) ;
Teng, Min; (Aliso Viejo, CA) ; Chandraratna,
Roshantha A.; (Mission Viejo, CA) |
Correspondence
Address: |
Gabor L. Szekeres
KLEIN & SZEKERES, LLP
Suite 700
4199 Campus Drive
Irvine
CA
92612
US
|
Family ID: |
27486804 |
Appl. No.: |
09/821673 |
Filed: |
March 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09821673 |
Mar 28, 2001 |
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09447082 |
Nov 22, 1999 |
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09447082 |
Nov 22, 1999 |
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09222983 |
Dec 30, 1998 |
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09222983 |
Dec 30, 1998 |
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08871093 |
Jun 9, 1997 |
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08871093 |
Jun 9, 1997 |
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08613863 |
Mar 11, 1996 |
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60019015 |
Sep 1, 1995 |
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60020501 |
Oct 13, 1995 |
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60064853 |
Sep 1, 1995 |
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Current U.S.
Class: |
514/150 ;
514/311; 514/434; 514/456; 514/529; 514/599; 514/617; 534/751;
534/787; 546/171; 546/177; 549/23; 549/398 |
Current CPC
Class: |
C07C 65/19 20130101;
C07C 69/618 20130101; C07D 213/55 20130101; C07C 245/10 20130101;
C07C 65/38 20130101; C07D 333/24 20130101; C07C 69/94 20130101;
C07C 69/76 20130101; C07D 335/06 20130101; C07F 7/081 20130101;
C07C 65/28 20130101; C07C 57/50 20130101; C07C 63/74 20130101; C07C
233/81 20130101; C07D 417/04 20130101; C07C 327/48 20130101; C07C
63/72 20130101; C07D 409/04 20130101; C07C 69/90 20130101; C07D
277/30 20130101; C07C 63/66 20130101; C07D 307/54 20130101; C07C
63/49 20130101; C07C 2602/10 20170501; C07C 63/33 20130101 |
Class at
Publication: |
514/150 ;
514/311; 514/434; 514/456; 514/529; 514/599; 514/617; 534/751;
534/787; 546/171; 546/177; 549/23; 549/398 |
International
Class: |
A61K 031/655; A61K
031/47; A61K 031/382; A61K 031/353; A61K 031/216; A61K 031/165 |
Claims
What is claimed:
1. A compound of the formula 17wherein X is S, O, NR' where R' is H
or alkyl of 1 to 6 carbons, or X is [C(R.sub.1).sub.2].sub.n where
R.sub.1 is H or alkyl of 1 to 6 carbons, and n is an integer
between 0 and 2; R.sub.2 is hydrogen, lower alkyl of 1 to 6
carbons, F, Cl, Br, I, CF.sub.3, fluoro substituted alkyl of 1 to 6
carbons, OH, SH, alkoxy of 1 to 6 carbons, or alkylthio of 1 to 6
carbons; R.sub.3 is hydrogen, lower alkyl of 1 to 6 carbons or F; m
is an integer having the value of 0-3; o is an integer having the
value of 0-3; Z is --C.ident.C----N.dbd.N--, --N.dbd.CR.sub.1--,
--CR.sub.1.dbd.N--, --(CR.sub.1.dbd.CR.sub.1).sub.n'-- - where n'
is an integer having the value 0-5, --CO--NR.sub.1--,
--CS--NR.sub.1--, --NR.sub.1--CO, --NR.sub.1--CS, --COO--, --OCO--;
--CSO--; --OCS--; Y is a phenyl or naphthyl group, or heteroaryl
selected from a group consisting of pyridyl, thienyl, furyl,
pyridazinyl, pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl,
imidazolyl and pyrrazolyl, said phenyl and heteroaryl groups being
optionally substituted with one or two R.sub.2 groups, or when Z is
--(CR.sub.1.dbd.CR.sub.1).sub.n'-- and n' is 3, 4 or 5 then Y
represents a direct valence bond between said
(CR.sub.2.dbd.CR.sub.2).sub.n' group and B; A is (CH.sub.2).sub.q
where q is 0-5, lower branched chain alkyl having 3-6 carbons,
cycloalkyl having 3-6 carbons, alkenyl having 2-6 carbons and 1 or
2 double bonds, alkynyl having 2-6 carbons and 1 or 2 triple bonds;
B is hydrogen, COOH or a pharmaceutically acceptable salt thereof,
COOR.sub.8, CONR.sub.9R.sub.10, --CH.sub.2OH, CH.sub.2OR.sub.11,
CH.sub.2OCOR.sub.11, CHO, CH(OR.sub.12).sub.2, CHOR.sub.130,
--COR.sub.7, CR.sub.7(OR.sub.12).sub.2- , CR.sub.7OR.sub.13O, or
tri-lower alkylsilyl, where R.sub.7 is an alkyl, cycloalkyl or
alkenyl group containing 1 to 5 carbons, R.sub.8 is an alkyl group
of 1 to 10 carbons or trimethylsilylalkyl where the alkyl group has
1 to 10 carbons, or a cycloalkyl group of 5 to 10 carbons, or
R.sub.8 is phenyl or lower alkylphenyl, R.sub.9 and R.sub.10
independently are hydrogen, an alkyl group of 1 to 10 carbons, or a
cycloalkyl group of 5-10 carbons, or phenyl or lower alkylphenyl,
R.sub.11 is lower alkyl, phenyl or lower alkylphenyl, R.sub.12 is
lower alkyl, and R.sub.13 is divalent alkyl radical of 2-5 carbons,
and R.sub.14 is (R.sub.15).sub.r-phenyl, (R.sub.15).sub.r-naphthyl,
or (R.sub.15).sub.r-heteroaryl where the heteroaryl group has 1 to
3 heteroatoms selected from the group consisting of O, S and N, r
is an integer having the values of 0-5, and R.sub.15 is
independently H, F, Cl, Br, I, NO.sub.2, N(R.sub.8).sub.2,
NH(R.sub.8), COR.sub.8, NR.sub.8CON(R.sub.8).sub.2, OH, OCOR.sub.8,
OR.sub.8, CN, an alkyl group having 1 to 10 carbons, fluoro
substituted alkyl group having 1 to 10 carbons, an alkenyl group
having 1 to 10 carbons and 1 to 3 double bonds, alkynyl group
having 1 to 10 carbons and 1 to 3 triple bonds, or a trialkylsilyl
or trialkylsilyloxy group where the alkyl groups independently have
1 to 6 carbons.
2. A compound of claim 1 where Y is phenyl, pyridyl, thienyl or
furyl.
3. A compound of claim 1 where Y is phenyl.
4. A compound of claim 3 where the phenyl ring is 1, 4 (para)
substituted.
5. A compound of claim 1 where Y is naphthyl.
6. A compound of claim 1 where Y is pyridyl.
7. A compound of claim 1 where Y is thienyl or furyl.
8. A compound of claim 1 where Z is
--(CR.sub.1--CR.sub.1).sub.n'--, and n' is 3, 4, or 5 and Y
represents a direct valence bond between said
(CR.sub.1.dbd.CR.sub.1).sub.n' group and B.
9. A compound of claim 1 where R.sub.2 is H, F, or CF.sub.3.
10. A compound of claim 1 where R.sub.3 is H or methyl.
11. A compound of claim 1 where R.sub.14 is (R.sub.15).sub.r-
phenyl.
12. A compound of claim 1 where R.sub.14 is
(R.sub.15).sub.r-heteroaryl.
13. A compound of claim 12 where R.sub.14 is
(R.sub.15).sub.r-heteroaryl where the heteroaryl group is a 5 or
six membered ring having 1 or 2 heteroatoms.
14. A compound of claim 13 where the heteroaryl group is selected
from 2-pyridyl, 3-pyridyl, 2-thienyl and 2-thiazolyl.
15. A compound of claim 1 where the R.sub.15 group is H, CF.sub.3,
F, lower alkyl, lower alkoxy, hydroxy or chlorine.
16. A compound of claim 1 where Z is --C.ident.C--.
17. A compound of claim 1 where Z is --N.dbd.N--.
18. A compound of claim 1 where Z is --CO--NR.sub.1--.
19. A compound of claim 1 where Z is --CS--NR.sub.1--.
20. A compound of claim 1 where Z is --CS--NR.sub.1--.
21. A compound of claim 1 where Z is --COO--.
22. A compound of claim 1 where Z is
--(CR.sub.1.dbd.CR.sub.1).sub.n'-- and n' is 1.
23. A compound of claim 1 where X is [C(R.sub.1).sub.2].sub.n and n
is 1 or 0.
24. A compound of claim 1 where X is S, O, or NR'.
25. A method of treating a pathological condition in a mammal, said
condition associated with a retinoic acid receptor activity, said
method comprising administering to said mammal a retinoid
antagonist or negative hormone capable of binding to a retinoic
acid receptor subtype selected from the group consisting of
RAE.sub..alpha., RAR.sub..beta. and RAR.sub..gamma., said
antagonist or negative hormone being administered in an amount
pharmaceutically effective to provide a therapeutic benefit against
said pathological condition in said mammal.
26. The method of claim 25 wherein the pathological condition is
the toxicity or undesired side effects resulting from
administration of a retinoid compound to said mammal, and wherein
said therapeutic benefit is the prevention or amelioration of said
toxicity or undesired side effects.
27. The method of claim 25 wherein the retinoid antagonist or
negative hormone is administered in order to cure or ameliorate a
pre-existing pathological condition caused by intake of a retinoid
drug or vitamin A or vitamin A precursors by the mammal.
28. The method of claim 25 wherein the retinoid antagonist or
negative hormone is administered topically to block or ameliorate
undesired topical side effects of a retinoid drug administered for
a therapeutic purpose.
29. The method of claim 25 wherein the retinoid antagonist or
negative hormone is administered topically to block or ameliorate
undesired topical side effects of a retinoid drug administered
systemically for a therapeutic purpose.
30. The method of claim 25 wherein the retinoid antagonist or
negative hormone is administered topically to treat a pre-existing
condition or side effect caused by a retinoid drug or vitamin
A.
31. The method of claim 25 wherein the retinoid antagonist or
negative hormone is administered systemically to treat a
pre-existing condition or side effect caused by a retinoid drug or
vitamin A.
32. The method of claim 25 wherein the retinoid antagonist or
negative hormone is administered systemically to block or
ameliorate bone toxicity caused by coadministration of a retinoid
drug or vitamin A.
33. The method of claim 25, wherein the retinoid antagonist or
negative hormone binds to said subtype of retinoid receptor with a
K.sub.d of less than approximately 1 micromolar.
34. The method of claim 25, wherein a retinoid antagonist is
administered.
35. The method of claim 25, wherein the negative hormone or
antagonist has the formula: 18wherein X is S, O, NR' where R' is H
or alkyl of 1 to 6 carbons, or X is [C(R.sub.1).sub.2].sub.n where
R.sub.1 is H or alkyl of 1 to 6 carbons, and n is an integer
between 0 and 2; R.sub.2 is hydrogen, lower alkyl of 1 to 6
carbons, F, Cl, Br, I, CF.sub.3, fluoro substituted alkyl of 1 to 6
carbons, OH, SH, alkoxy of 1 to 6 carbons, or alkylthio of 1 to 6
carbons; R.sub.3 is hydrogen, lower alkyl of 1 to 6 carbons or F; m
is an integer having the value of 0-3; o is an integer having the
value of 0-3; Z is --C.ident.C--, --N.dbd.N--, --N.dbd.CR.sub.1--,
--CR.sub.1.dbd.N--, --(CR.sub.1.dbd.CR.sub.1).sub.n'-- where n' is
an integer having the value 0-5, --CO--NR.sub.1--,
--CS--NR.sub.1--, --NR.sub.1--CO, --NR.sub.1--CS, --COO--, --OCO--;
--CSO--; --OCS--; Y is a phenyl or naphthyl group, or heteroaryl
selected from a group consisting of pyridyl, thienyl, furyl,
pyridazinyl, pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl,
imidazolyl and pyrrazolyl, said phenyl and heteroaryl groups being
optionally substituted with one or two R.sub.2 groups, or when Z is
--(CR.sub.1.dbd.CR.sub.1).sub.n'-- and n' is 3, 4 or 5 then Y
represents a direct valence bond between said
(CR.sub.1.dbd.CR.sub.1).sub.n' group and B; A is (CH.sub.2).sub.q
where q is 0-5, lower branched chain alkyl having 3-6 carbons,
cycloalkyl having 3-6 carbons, alkenyl having 2-6 carbons and 1 or
2 double bonds, alkynyl having 2-6 carbons and 1 or 2 triple bonds;
B is hydrogen, COOH or a pharmaceutically acceptable salt thereof,
COOR.sub.8, CONR.sub.9R.sub.10, --CH.sub.2OH, CH.sub.2OR.sub.11,
CH.sub.2OCOR.sub.11, CHO, CH(OR.sub.12).sub.2, CHOR.sub.13O,
--COR.sub.7, CR.sub.7(OR.sub.12).sub.2- , CR.sub.7OR.sub.13O, or
tri-lower alkylsilyl, where R.sub.7 is an alkyl, cycloalkyl or
alkenyl group containing 1 to 5 carbons, R.sub.8 is an alkyl group
of 1 to 10 carbons or trimethylsilylalkyl where the alkyl group has
1 to 10 carbons, or a cycloalkyl group of 5 to 10 carbons, or
R.sub.8 is phenyl or lower alkylphenyl, R.sub.9 and R.sub.10
independently are hydrogen, an alkyl group of 1 to 10 carbons, or a
cycloalkyl group of 5-10 carbons, or phenyl or lower alkylphenyl,
R.sub.11 is lower alkyl, phenyl or lower alkylphenyl, R.sub.12 is
lower alkyl, and R.sub.13 is divalent alkyl radical of 2-5 carbons,
and R.sub.14 is (R.sub.15).sub.r-phenyl, (R.sub.15).sub.r-naphthyl,
or (R.sub.15).sub.r-heteroaryl where the heteroaryl group has 1 to
3 heteroatoms selected from the group consisting of O, S and N, r
is an integer having the values of 0-5, and R.sub.15 is
independently H, F, Cl, Br, I, NO.sub.2, N(R.sub.8).sub.2,
NH(R.sub.8)COR.sub.8, NR.sub.8CON(R.sub.8).sub.2, OH, OCOR.sub.8,
OR.sub.8, CN, an alkyl group having 1 to 10 carbons, fluoro
substituted alkyl group having 1 to 10 carbons, an alkenyl group
having 1 to 10 carbons and 1 to 3 double bonds, alkynyl group
having 1 to 10 carbons and 1 to 3 triple bonds, or a trialkylsilyl
or trialkylsilyloxy group where the alkyl groups independently have
1 to 6 carbons.
36. A method of claim 35 where in the formula of the antagonist or
negative hormone Y is phenyl, pyridyl, thienyl or furyl.
37. A method of claim 35 where in the formula of the antagonist or
negative hormone Y is phenyl.
38. A method of claim 37 where in the formula of the antagonist or
negative hormone the phenyl ring is 1,4 (para) substituted.
39. A method of claim 35 where in the formula of the antagonist or
negative hormone Y is naphthyl.
40. A method of claim 35 where in the formula of the antagonist or
negative hormone Y is pyridyl.
41. A method of claim 35 where in the formula of the antagonist or
negative hormone Y is thienyl or furyl.
42. A method of claim 35 where in the formula of the antagonist or
negative hormone Z is --(CR.sub.1.dbd.CR.sub.1).sub.n'--, and n' is
3, 4 or 5 and Y represents a direct valence bond between said
(CR.sub.1.dbd.CR.sub.1).sub.n' group and B.
43. A method of claim 35 where in the formula of the antagonist or
negative hormone R.sub.2 is H, F, or CF.sub.3.
44. A method of claim 35 where in the formula of the antagonist or
negative hormone R.sub.3 is H or methyl.
45. A method of claim 35 where in the formula of the antagonist or
negative hormone R.sub.14 is (R.sub.15).sub.r-phenyl.
46. A method of claim 35 where in the formula of the antagonist or
negative hormone R.sub.14 is (R.sub.15).sub.r-heteroaryl.
47. A method of claim 46 where in the formula of the antagonist or
negative hormone R.sub.14 is (R.sub.15).sub.r-heteroaryl where the
heteroaryl group is a 5 or six membered ring having 1 or 2
heteroatoms.
48. A method of claim 47 where in the formula of the antagonist the
heteroaryl group is selected from 2-pyridyl, 3-pyridyl, 2-thienyl
and 2-thiazolyl.
49. A method of claim 35 where in the formula of the antagonist or
negative hormone the R.sub.15, group is H, CF.sub.3, F, lower
alkyl, lower alkoxy, hydroxy or chlorine.
50. A method of claim 35 where in the formula of the antagonist or
negative hormone Z is --C.ident.C--.
51. A method of claim 35 where in the formula of the antagonist or
negative hormone Z is --N.dbd.N--.
52. A method of claim 35 where in the formula of the antagonist or
negative hormone of claim 1 where Z is --CO--NR.sub.1--.
53. A method of claim 35 where in the formula of the antagonist or
negative hormone Z is --CS--NR.sub.1--.
54. A method of claim 53 where in the formula of the antagonist or
negative hormone R.sub.1 is H.
55. A method of claim 35 where in the formula of the antagonist or
negative hormone Z is --COO--.
56. A method of claim 35 where in the formula of the antagonist or
negative hormone Z is --(CR.sub.1.dbd.CR.sub.1).sub.n'-- and n' is
1.
57. A method of claim 35 where in the formula of the antagonist or
negative hormone X is [C(R.sub.1).sub.2].sub.n and n is 1 or 0.
58. A method of claim 35 where in the formula of the antagonist or
negative hormone X is S, O or NR'.
59. A compound of the formula 19wherein X is S, O, NR' where R' is
H or alkyl of 1 to 6 carbons, or X is [C(R.sub.1).sub.2].sub.n
where R.sub.1 is independently H or alkyl of 1 to 6 carbons, and n
is an integer between 0 and 2; R.sub.2 is hydrogen, lower alkyl of
1 to 6 carbons, F, Cl, Br, I, CF.sub.3, fluoro substituted alkyl of
1 to 6 carbons, OH, SH, alkoxy of 1 to 6 carbons, or alkylthio of 1
to 6 carbons; R.sub.3 is hydrogen, lower alkyl of 1 to 6 carbons or
F; m is an integer having the value of 0-3; o is an integer having
the value of 0-3; Y is a phenyl or naphthyl group, or heteroaryl
selected from a group consisting of pyridyl, thienyl, furyl,
pyridazinyl, pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl,
imidazolyl and pyrrazolyl, said phenyl and heteroaryl groups being
optionally substituted with one or two R.sub.2 groups; A is
(CH.sub.2).sub.q where q is 0-5, lower branched chain alkyl having
3-6 carbons, cycloalkyl having 3-6 carbons, alkenyl having 2-6
carbons and 1 or 2 double bonds, alkynyl having 2-6 carbons and 1
or 2 triple bonds; B is hydrogen, COOH or a pharmaceutically
acceptable salt thereof, COOR.sub.8, CONR.sub.9R.sub.10,
--CH.sub.2OH, CH.sub.2OR.sub.11, CH.sub.2OCOR.sub.11, CHO,
CH(OR.sub.12).sub.2, CHOR.sub.13O, --COR.sub.7,
CR.sub.7(OR.sub.12).sub.2, CR.sub.7OR.sub.13O, or tri-lower
alkylsilyl, where R.sub.7 is an alkyl, cycloalkyl or alkenyl group
containing 1 to 5 carbons, R.sub.8 is an alkyl group of 1 to 10
carbons or trimethylsilylalkyl where the alkyl group has 1 to 10
carbons, or a cycloalkyl group of 5 to 10 carbons, or R.sub.8 is
phenyl or lower alkylphenyl, R.sub.9 and R.sub.10 independently are
hydrogen, an alkyl group of 1 to 10 carbons, or a cycloalkyl group
of 5-10 carbons, or phenyl or lower alkylphenyl, R.sub.11 is lower
alkyl, phenyl or lower alkylphenyl, R.sub.12 is lower alkyl, and
R.sub.13 is divalent alkyl radical of 2-5 carbons, and R.sub.14 is
(R.sub.15).sub.r-phenyl, (R.sub.15).sub.r-naphthyl, or
(R.sub.15).sub.r-heteroaryl where the heteroaryl group has 1 to 3
heteroatoms selected from the group consisting of O, S and N, r is
an integer having the values of 0-5, and R.sub.15 is independently
H, F, Cl, Br, I, NO.sub.2, N(R.sub.8).sub.2, N(R.sub.8)COR.sub.8,
NR.sub.8CON(R.sub.8).sub.2, OH, OCOR.sub.8, OR.sub.8, CN, an alkyl
group having 1 to 10 carbons, fluoro substituted alkyl group having
1 to 10 carbons, an alkenyl group having 1 to 10 carbons and 1 to 3
double bonds, alkynyl group having 1 to 10 carbons and 1 to 3
triple bonds, or a trialkylsilyl or trialkylsilyloxy group where
the alkyl groups independently have 1 to 6 carbons. R.sub.16 is H,
lower alkyl of 1 to 6 carbons; R.sub.17 is H, lower alkyl of 1 to 6
carbons, OH or OCOR.sub.11, and p is zero or 1, with the proviso
that when p is 1 then there is no R.sub.17 substituent group, and m
is an integer between 0 and 2.
60. A compound of claim 59 where Y is phenyl, pyridyl, thienyl or
furyl.
61. A compound of claim 59 where Y is phenyl.
62. A compound of claim 61 where the phenyl ring is 1,4 (para)
substituted.
63. A compound of claim 59 where Y is pyridyl.
64. A compound of claim 59 where Y is thienyl or furyl.
65. A compound of claim 59 where R.sub.14 is
(R.sub.15).sub.r-phenyl.
66. A compound of claim 59 where R.sub.14 is
(R.sub.15).sub.r-heteroaryl.
67. A compound of claim 66 where R.sub.14 is
(R.sub.15).sub.r-heteroaryl where the heteroaryl group is a 5 or
six membered ring having 1 or 2 heteroatoms.
68. A compound of claim 67 where the heteroaryl group is selected
from 2-pyridyl, 3-pyridyl, 2-thienyl and 2-thiazolyl.
69. A compound of claim 59 where the R.sub.15 group is H, CF.sub.3,
F, lower alkyl, lower alkoxy, hydroxy or chlorine.
70. A compound of claim 59 where X is [C(R.sub.1).sub.2].sub.n.
71. A compound of claim 70 where R.sub.1 is CH.sub.3 and n is
1.
72. A compound of claim 59 where X is S, O or NR'.
73. A compound of claim 59 where A is (CH.sub.2).sub.q where q is
0-5 and where B is COOH or a pharmaceutically acceptable salt
thereof, COOR.sub.8, or CONR.sub.9R.sub.10.
74. A compound of claim 59 where p is zero.
75. A compound of claim 59 where p is 1.
76. A compound of the formula 20where R.sub.1 is independently H or
alkyl of 1 to 6 carbons; R.sub.2 is hydrogen, lower alkyl of 1 to 6
carbons, F, Cl, Br, I, CF.sub.3, fluoro substituted alkyl of 1 to 6
carbons, OH, SH, alkoxy of 1 to 6 carbons, or alkylthio of 1 to 6
carbons; R.sub.3 is hydrogen, lower alkyl of 1 to 6 carbons or F; m
is an integer having the value of 0-2; o is an integer having the
value of 0-3; A is (CH.sub.2).sub.q where q is 0-5, lower branched
chain alkyl having 3-6 carbons, cycloalkyl having 3-6 carbons,
alkenyl having 2-6 carbons and 1 or 2 double bonds, alkynyl having
2-6 carbons and 1 or 2 triple bonds; B is hydrogen, COOH or a
pharmaceutically acceptable salt thereof, COOR.sub.8,
CONR.sub.9R.sub.10, --CH.sub.2OH, CH.sub.2OR.sub.11,
CH.sub.2OCOR.sub.11, CHO, CH(OR.sub.12).sub.2, CHOR.sub.13O,
--COR.sub.7, CR.sub.7(OR.sub.12).sub.2, CR.sub.7OR.sub.13O, or
tri-lower alkylsilyl, where R.sub.7 is an alkyl, cycloalkyl or
alkenyl group containing 1 to 5 carbons, R.sub.8 is an alkyl group
of 1 to 10 carbons or trimethylsilylalkyl where the alkyl group has
1 to 10 carbons, or a cycloalkyl group of 5 to 10 carbons, or
R.sub.8 is phenyl or lower alkylphenyl, R.sub.9 and R.sub.10
independently are hydrogen, an alkyl group of 1 to 10 carbons, or a
cycloalkyl group of 5-10 carbons, or phenyl or lower alkylphenyl,
R.sub.11 is lower alkyl, phenyl or lower alkylphenyl, R.sub.12 is
lower alkyl, and R.sub.13 is divalent alkyl radical of 2-5 carbons;
R.sub.15 is independently H, F, Cl, Br, I, NO.sub.2,
N(R.sub.8).sub.2, N(R.sub.8)COR.sub.8, NR.sub.8CON(R.sub.8).sub-
.2, OH, OCOR.sub.8, OR.sub.8, CN, an alkyl group having 1 to 10
carbons, fluoro substituted alkyl group having 1 to 10 carbons, an
alkenyl group having 1 to 10 carbons and 1 to 3 double bonds,
alkynyl group having 1 to 10 carbons and 1 to 3 triple bonds, or a
trialkylsilyl or trialkylsilyloxy group where the alkyl groups
independently have 1 to 6 carbons; r is an integer having the
values of 0-5; R.sub.16 is H, lower alkyl of 1 to 6 carbons;
R.sub.17 is H, lower alkyl of 1 to 6 carbons, OH or OCOR.sub.11,
and p is zero or 1, with the proviso that when p is 1 then there is
no R.sub.17 substituent group, and m is an integer having the value
of 0-2.
77. A compound of claim 76 wherein p is 1.
78. A compound of claim 76 where p is zero.
79. A compound of claim 78 where A is (CH.sub.2).sub.q where q is
0-5, and B is COOH or a pharmaceutically acceptable salt thereof,
COOR.sub.8, or CONR.sub.9R.sub.10.
80. A compound of claim 79 where R. is CH.sub.3.
81. A compound of claim 80 where R.sub.2, R.sub.3, R.sub.16 and
R.sub.17 are hydrogen.
82. A compound of claim 81 where R.sub.15 is H or CH.sub.3, and
when R.sub.15 is CH.sub.3 it occupies the 4 position of the phenyl
ring.
83. A compound of claim 82 which is
4-[3-oxo-3-(7,8-dihydro-5-(4-methylphe-
nyl)-8,8dimethyl-2-naphthalenyl)-1-propenyl]-benzoic acid or
4-[3-oxo-3-(7,8-dihydro-5-phenyl-8,8-dimethyl-2-naphthalenyl)-1-propenyl]-
-benzoic acid.
84. A method of identifying retinoid negative hormones, comprising
the following steps: obtaining transfected cells containing a
reporter gene transcriptionally responsive to binding of a
recombinant retinoid receptor, said recombinant retinoid receptor
having at least protein domains located C-terminal to a DNA binding
domain of an intact retinoid receptor; measuring a basal level of
reporter gene expression in untreated transfected cells, said
untreated transfected cells being propagated in the absence of an
added retinoid; treating the transfected cells with a retinoid
compound to be tested for negative hormone activity; measuring a
level of reporter gene expression in treated cells; comparing the
levels of reporter gene expression measured in treated cells and
untreated cells; and identifying as retinoid negative hormones
those retinoid compounds producing a lower level of reporter gene
expression in treated cells compared with the basal level of
reporter gene expression measured in untreated cells.
85. The method of claim 84, wherein the intact retinoid receptor is
a retinoic acid receptor selected from the group consisting of
RAR-.alpha., RAR-.beta. and RAR-.gamma..
86. The method of claim 84, wherein the intact retinoid receptor is
a retinoid X receptor selected from the group consisting of
RAR-.alpha., RXR-.beta. and RXR-.gamma..
87. The method of claim 84, wherein the recombinant retinoid
receptor is selected from the group consisting of RARs and
RXRs.
88. The method of claim 84, wherein the recombinant retinoid
receptor is a chimeric retinoid receptor having a constitutive
transcription activator domain.
89. The method of claim 88, wherein the constitutive transcription
activator domain comprises a plurality of amino acids having a net
negative charge.
90. The method of claim 88, wherein the constitutive transcription
activator domain has an amino acid sequence of a viral
transcription activator domain.
91. The method of claim 90, wherein the viral transcription
activator domain is the herpes simplex virus VP-16 transcription
activator domain.
92. The method of claim 88, wherein the constitutive transcription
activator domain has a net negative charge, and wherein the
recombinant retinoid receptor has deleted therefrom a DNA binding
domain.
93. The method of claim 84, wherein the recombinant retinoid
receptor has a DNA binding domain specific for a cis-regulatory
element other than a retinoic acid responsive element.
94. The method of claim 93, wherein the cis-regulatory element
other than a retinoic acid responsive element is an estrogen
responsive element.
95. The method of claim 84, wherein the transfected cell is
propagated in a growth medium substantially depleted of endogenous
retinoids.
96. The method of claim 95, wherein the growth medium comprises
activated charcoal-extracted serum.
97. The method of claim 84, wherein the reporter gene is the
luciferase gene and wherein the measuring steps comprise
luminometry.
98. The method of claim 84, wherein the reporter gene is the
.beta.-galactosidase gene and wherein the measuring steps comprise
a .beta.-galactosidase assay.
99. The method of claim 84, wherein the transfected cell is a
transfected mammalian cell.
100. The method of claim 99, wherein the transfected mammalian cell
is a transiently transfected mammalian cell.
101. The method of claim 99, wherein the transfected mammalian cell
is a transfected Green monkey cell.
102. The method of claim 99, wherein the transfected mammalian cell
is a transfected human cell.
103. A method of potentiating a pharmacologic activity of a steroid
superfamily receptor agonist administered to a mammal, comprising
coadministering to the mammal with said steroid superfamily
receptor agonist a composition comprising a pharmaceutically
effective dose of a retinoid negative hormone to potentiate the
pharmacologic activity of the steroid superfamily receptor
agonist.
104. The method of claim 103, wherein the pharmacologic activity is
measurable in a reporter gene trans-activation assay in vitro.
105. The method of claim 104, wherein the pharmacologic activity
measurable in the reporter gene transactivation assay is anti-AP-1
activity.
106. The method of claim 103, wherein the pharmacologic activity is
an antiproliferative activity.
107. The method of claim 106, wherein the antiproliferative
activity is measurable in retinal pigment epithelium.
108. The method of claim 103, wherein the steroid superfamily
receptor agonist is selected from the group consisting of a
retinoid receptor agonist, a vitamin D receptor agonist, a
glucocorticoid receptor agonist, a thyroid hormone receptor
agonist, a peroxisome proliferator-activated receptor agonist and
an estrogen receptor agonist.
109. The method of claim 108, wherein the retinoid receptor agonist
is an RAR agonist.
110. The method of claim 109, wherein the RAR agonist is selected
from the group consisting of all-trans-retinoic acid, 13-cis
retinoic acid,
4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino-
]-benzoic acid(Am580), and
(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethy-
l-2-naphthalenyl)-2-propenyl]-benzoic acid (TTNPB).
111. The method of claim 108, wherein the retinoid receptor agonist
is an RXR agonist.
112. The method of claim 111, wherein the RXR agonist is selected
from the group consisting of 9-cis-retinoic acid,
4-[(3,5,5,8,8-pentamethyl-5,6,7,-
8-tetrahydro-2-naphthalenyl)-1-ethenyl]-benzoic acid, and
4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-cyclopropy-
l]-pyridine-5-carboxylic acid.
113. The method of claim 108, wherein the vitamin D receptor
agonist is 1,25-dihydroxyvitamin D.sub.3.
114. The method of claim 108, wherein the glucocorticoid receptor
agonist is dexamethasone.
115. The method of claim 108, wherein the thyroid hormone receptor
agonist is 3,3',5-triiodothyronine.
116. The method of claim 103, wherein the retinoid negative hormone
is an RAR-specific retinoid negative hormone.
117. The method of claim 116, wherein the RAR-specific retinoid
negative hormone has a dissociation constant less than or
approximately equal to 30 nM.
118. The method of claim 117, wherein the RAR-specific retinoid
negative hormone is selected from the group consisting of AGN
193109, AGN 193385, AGN 193389 and AGN 193871.
119. The method of claim 103, wherein the composition comprising a
pharmaceutically effective dose of a retinoid negative hormone is
coadministered at the same time as the steroid superfamily
agonist.
120. The method of claim 119, wherein the composition comprising a
pharmaceutically effective dose of the retinoid negative hormone
and the steroid superfamily agonist are combined prior to
coadministration.
121. The method of claim 103, wherein the composition comprising a
pharmaceutically effective dose of the retinoid negative hormone
and the steroid superfamily agonist are coadministered as separate
compositions.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) of the three following U.S. applications, each
of which was filed as a nonprovisional application and converted to
a provisional application by separate petitions filed on Jan. 31,
1996: application Ser. No. 08/522,778, filed Sep. 1, 1995;
application Ser. No. 08/522,779, filed Sep. 1, 1995; and
application Ser. No. 08/542,648, filed Oct. 13, 1995. The complete
disclosures of these related applications is hereby incorporated
herein by this reference thereto.
FIELD OF THE INVENTION
[0002] The present invention relates to novel compounds having
retinoid negative hormone and/or retinoid antagonist-like
biological activities. More specifically, the invention relates to
4-aryl substituted benzopyran, 4-aryl substituted benzothiopyran,
4-aryl substituted 1,2-dihydroquinoline and 8-aryl substituted
5,6-dihydronaphthalene derivatives which may also be substituted by
a substituted 3-oxo-1-propenyl group. These novel compounds have
retinoid antagonist like-activity and are useful for treating or
preventing retinoid and vitamin A and vitamin A precursor induced
toxicity in mammals and as an adjunct to treatment of mammals with
retinoids to prevent or ameliorate unwanted or undesired side
effects. The invention further relates to the use of retinoid
negative hormones for increasing the biological activities of other
retinoids and steroid hormones and inhibiting the basal activity of
unliganded retinoic acid receptors.
BACKGROUND OF THE INVENTION
[0003] Compounds which have retinoid-like activity are well known
in the art, and are described in numerous United States and other
patents and in scientific publications. It is generally known and
accepted in the art that retinoid-like activity is useful for
treating mammals, including humans, in order to cure or alleviate
the symptoms associated with numerous diseases and conditions.
[0004] Retinoids (vitamin A and its derivatives) are known to have
broad activities, including effects on cell proliferation and
differentiation, in a variety of biological systems. This activity
has made retinoids useful in the treatment of a variety of
diseases, including dermatological disorders and cancers. The prior
art has developed a large number of chemical compounds which have
retinoid-like biological activity, and voluminous patent and
chemical literature exists describing such compounds. The relevant
patent literature includes U.S. Pat. Nos. 4,980,369, 5,006,550,
5,015,658, 5,045,551, 5,089,509, 5,134,159, 5,162,546, 5,234,926,
5,248,777, 5,264,578, 5,272,156, 5,278,318, 5,324,744, 5,346,895,
5,346,915, 5,348,972, 5,348,975, 5,380,877, 5,399,561, 5,407,937,
(assigned to the same assignee as the present application) and
patents and publications cited therein, which particularly describe
or relate to chroman, thiochroman and 1,2,3,4-tetrahydroquinoline
derivatives which have retinoid-like biological activity. In
addition, several applications are pending which are assigned to
the assignee of the present application, and which are directed to
further compounds having retinoid-like activity.
[0005] U.S. Pat. Nos. 4,740,519 (Shroot et al.), 4,826,969 (Maignan
et al.), 4,326,055 (Loeliger et al.), 5,130,335 (Chandraratna et
al.), 5,037,825 (Klaus et al.), 5,231,113 (Chandraratna et al.),
5,324,840 (Chandraratna), 5,344,959 (Chandraratna), 5,130,335
(Chandraratna et al.), Published European Patent Application Nos. 0
176 034 A (Wuest et al.), 0 350 846 A (Klaus et al.), 0 176 032 A
(Frickel et al.), 0 176 033 A (Frickel et al.), 0 253 302 A (Klaus
et al.), 0 303 915 A (Bryce et al.), UK Patent Application GB
2190378 A (Klaus et al.), German Patent Application Nos. DE 3715955
A1 (Klaus et al.), DE 3602473 A1 (Wuest et al., and the articles J
Amer. Acad. Derm. 15:756-764 (1986) (Sporn et al.), Chem. Pharm.
Bull. 33:404-407 (1985) (Shudo et al.), J. Med Chem. 31:2182-2192
(1988) (Kagechika et al.), Chemistry and Biology of Synthetic
Retinoids CRC Press Inc. 1990 pp. 334-335, 354 (Dawson et al.),
describe or relate to compounds which include a tetrahydronaphthyl
moiety and have retinoid-like or related biological activity. U.S.
Pat. No. 4,391,731 (Boller et al.) describes tetrahydronaphthalene
derivatives which are useful in liquid crystal compositions.
[0006] An article by Kagechika et al. in J. Med. Chem 32:834 (1989)
describe certain
6-(3-oxo-1-propenyl)-1,2,3,4-tetramethyl-1,2,3,4-tetrahy-
dronaphthalene derivatives and related flavone compounds having
retinoid-like activity. The articles by Shudo et al. in Chem.
Pharm. Bull. 33:404 (1985) and by Jetten et al. in Cancer Research
47:3523 (1987) describe or relate to further 3-oxo-1-propenyl
derivatives (chalcone compounds) and their retinoid-like or related
biological activity.
[0007] Unfortunately, compounds having retinoid-like activity
(retinoids) also cause a number of undesired side effects at
therapeutic dose levels, including headache, teratogenesis,
mucocutaneous toxicity, musculoskeletal toxicity, dyslipidemias,
skin irritation, headache and hepatotoxicity. These side effects
limit the acceptability and utility of retinoids for treating
disease.
[0008] It is now general knowledge in the art that two main types
of retinoid receptors exist in mammals (and other organisms). The
two main types or families of receptors are respectively designated
as the RARs and RXRs. Within each type there are subtypes: in the
RAR family the subtypes are designated RAR-.alpha., RAR-.beta. and
RAR-.gamma., in RXR the subtypes are: RXR-.alpha., RXB-.beta. and
RXR-.gamma.. Both families of receptors are transcription factors
that can be distinguished from each other based on their ligand
binding specificities. All-trans-RA (ATRA) binds and activates a
class of retinoic acid receptors (RARs) that includes RAR-.alpha.,
RAR-.beta. and RAR-.gamma.. A different ligand, 9-cis-RA (9C-RA),
binds and activates both the RARs and members of the retinoid X
receptor (RXR) family.
[0009] It has also been established in the art that the
distribution of the two main retinoid receptor types, and of the
several subtypes is not uniform in the various tissues and organs
of mammalian organisms. Moreover, it is generally accepted in the
art that many unwanted side effects of retinoids are mediated by
one or more of the RAR receptor subtypes. Accordingly, among
compounds having agonist-like activity at retinoid receptors,
specificity or selectivity for one of the main types or families,
and even specificity or selectivity for one or more subtypes within
a family of receptors, is considered a desirable pharmacological
property.
[0010] Relatively recently compounds have been developed in the art
which bind to RAR receptors without triggering the response or
responses that are triggered by agonists of the same receptors. The
compounds or agents which bind to RAR receptors without triggering
a "retinoid" response are thus capable of blocking (to lesser or
greater extent) the activity of RAR agonists in biological assays
and systems. More particularly, regarding the scientific and patent
literature in this field, published PCT Application WO 94/14777
describes certain heterocyclic carboxylic acid derivatives which
bind to RAR retinoid receptors and are said in the application to
be useful for treatment of certain diseases or conditions, such as
acne, psoriasis, rheumatoid arthritis and viral infections. A
similar disclosure is made in the article by Yoshimura et al. J.
Med. Chem. 38:3163-3173 (1995). Kaneko et al. Med. Chem Res.
1:220-225 (1991); Apfel et al. Proc. Natl. Acad. Sci. USA
89:129-7133 Augusty 1992 Cell Biology; Eckhardt et al. Toxicology
Letters 70:299-308 (1994); Keidel et al. Molecular and Cellular
Biology 14:287-298 (1994); and Eyrolles et al. J. Med Chem.
37:1508-1517 (1994) describe compounds which have antagonist like
activity at one or more of the RAR retinoid subtypes.
[0011] In addition to undesirable side-effects of therapy with
retinoid compounds, there occurs occasionally a serious medical
condition caused by vitamin A or vitamin A precursor overdose,
resulting either from the excessive intake of vitamin supplements
or the ingestion of liver of certain fish and animals that contain
high levels of the vitamin. The chronic or acute toxicities
observed with hypervitaminosis A syndrome include headache, skin
peeling, bone toxicity, dyslipidemias, etc. In recent years, it has
become apparent that the toxicities observed with vitamin A
analogs, i.e., retinoids, essentially recapitulate those of
hypervitaminosis A syndrome, suggesting a common biological cause,
i.e., RAR activation. These toxicities are presently treated mainly
by supportive measures and by abstaining from further exposure to
the causative agent, whether it be liver, vitamin supplements, or
retinoids. While some of the toxicities resolve with time, others
(e.g., premature epiphyseal plate closure) are permanent.
[0012] Generally speaking, specific antidotes are the best
treatment for poisoning by pharmacological agents, but only about
two dozen chemicals or classes of chemicals out of thousands in
existence have specific known antidotes. A specific antidote would
clearly be of value in the treatment of hypervitaminosis A and
retinoid toxicity. Indeed, as increasingly potent retinoids are
used clinically, a specific antidote for retinoid poisoning could
be life saving.
SUMMARY OF THE INVENTION
[0013] The present invention covers compounds of Formula 1 1
[0014] wherein X is S, O, NR' where R' is H or alkyl of 1 to 6
carbons, or
[0015] X is [C(R.sub.1).sub.2].sub.n where R.sub.1 is independently
H or alkyl of 1 to 6 carbons, and n is an integer between 0 and
2;
[0016] R.sub.2 is hydrogen, lower alkyl of 1 to 6 carbons, F, Cl,
Br, I, CF.sub.3, fluoro substituted alkyl of 1 to 6 carbons, OH,
SH, alkoxy of 1 to 6 carbons, or alkylthio of 1 to 6 carbons;
[0017] R.sub.3 is hydrogen, lower alkyl of 1 to 6 carbons or F;
[0018] m is an integer having the value of 0-3;
[0019] o is an integer having the value of 0-3;
[0020] Z is --C.ident.C--,
[0021] --N.dbd.UN--,
[0022] --N.dbd.CR.sub.1--,
[0023] --CR.sub.1.dbd.N,
[0024] --(CR.sub.1.dbd.CR.sub.1).sub.n'-- where n' is an integer
having the value 0-5,
[0025] --CO--NR.sub.1--,
[0026] --CS--NR.sub.1--,
[0027] --NR.sub.1--CO,
[0028] --NR.sub.1--CS,
[0029] --COO--,
[0030] --OCO--;
[0031] --CSO--;
[0032] --OCS--;
[0033] Y is a phenyl or naphthyl group, or heteroaryl selected from
a group consisting of pyridyl, thienyl, furyl, pyridazinyl,
pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl, imidazolyl and
pyrrazolyl, said phenyl and heteroaryl groups being optionally
substituted with one or two R.sub.2 groups, or
[0034] when Z is --(CR.sub.1.dbd.CR.sub.1).sub.n'-- and n' is 3, 4
or 5 then Y represents a direct valence bond between said
(CR.sub.2.dbd.CR.sub.2).sub.n. group and B;
[0035] A is (CH.sub.2).sub.q where q is 0-5, lower branched chain
alkyl having 3-6 carbons, cycloalkyl having 3-6 carbons, alkenyl
having 2-6 carbons and 1 or 2 double bonds, alkynyl having 2-6
carbons and 1 or 2 triple bonds;
[0036] B is hydrogen, COOH or a pharmaceutically acceptable salt
thereof, COOR.sub.8, CONR.sub.9R.sub.10, --CH.sub.2OH,
CH.sub.2OR.sub.11, CH.sub.2OCOR.sub.11, CHO, CH(OR.sub.12).sub.2,
CHOR.sub.13O, --COR.sub.7, CR.sub.7(OR.sub.12).sub.2,
CR.sub.7OR.sub.13O, or tri-lower alkylsilyl, where R.sub.7 is an
alkyl, cycloalkyl or alkenyl group containing 1 to 5 carbons,
R.sub.8 is an alkyl group of 1 to 10 carbons or trimethylsilylalkyl
where the alkyl group has 1 to 10 carbons, or a cycloalkyl group of
5 to 10 carbons, or R.sub.8 is phenyl or lower alkylphenyl, R.sub.9
and R.sub.10 independently are hydrogen, an alkyl group of 1 to 10
carbons, or a cycloalkyl group of 5-10 carbons, or phenyl or lower
alkylphenyl, R.sub.11 is lower alkyl, phenyl or lower alkylphenyl,
R.sub.12 is lower alkyl, and R.sub.13 is divalent alkyl radical of
2-5 carbons, and
[0037] R.sub.14 is (R.sub.15).sub.r-phenyl,
(R.sub.15).sub.r-naphthyl, or (R.sub.15).sub.r-heteroaryl where the
heteroaryl group has 1 to 3 heteroatoms selected from the group
consisting of O, S and N, r is an integer having the values of 0-5,
and
[0038] R.sub.15 is independently H, F, Cl, Br, I, NO.sub.2,
N(R.sub.8).sub.2, N(R.sub.8)COR.sub.8, NR.sub.8CON(R.sub.9).sub.2,
OH, OCOR.sub.8, OR.sub.8, CN, an alkyl group having 1 to 10
carbons, fluoro substituted alkyl group having 1 to 10 carbons, an
alkenyl group having 1 to 10 carbons and 1 to 3 double bonds,
alkynyl group having 1 to 10 carbons and 1 to 3 triple bonds, or a
trialkylsilyl or trialkylsilyloxy group where the alkyl groups
independently have 1 to 6 carbons.
[0039] The present invention further covers compounds of Formula
101 2
[0040] wherein X is S, O, NR' where R' is H or alkyl of 1 to 6
carbons, or
[0041] X is [C(R.sub.1).sub.2].sub.n where R.sub.1 is independently
H or alkyl of 1 to 6 carbons, and n is an integer between 0 and
2;
[0042] R.sub.2 is hydrogen, lower alkyl of 1 to 6 carbons, F, Cl,
Br, I, CF.sub.3, fluoro substituted alkyl of 1 to 6 carbons, OH,
SH, alkoxy of 1 to 6 carbons, or alkylthio of 1 to 6 carbons;
[0043] R.sub.3 is hydrogen, lower alkyl of 1 to 6 carbons or F;
[0044] m is an integer having the value of 0-3;
[0045] o is an integer having the value of 0-3;
[0046] Y is a phenyl or naphthyl group, or heteroaryl selected from
a group consisting of pyridyl, thienyl, furil, pyridazinyl,
pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl, imidazolyl and
pyrrazolyl, said phenyl and heteroaryl groups being optionally
substituted with one or two R.sub.2 groups;
[0047] A is (CH.sub.2).sub.q where q is 0-5, lower branched chain
alkyl having 3-6 carbons, cycloalkyl having 3-6 carbons, alkenyl
having 2-6 carbons and 1 or 2 double bonds, alkynyl having 2-6
carbons and 1 or 2 triple bonds;
[0048] B is hydrogen, COOH or a pharmaceutically acceptable salt
thereof, COOR.sub.8, CONR.sub.9R.sub.10, --CH.sub.2OH,
CH.sub.2OR.sub.11, CH.sub.2OCOR.sub.11, CHO, CH(OR.sub.12).sub.2,
CHOR.sub.13O, --COR.sub.7, CR.sub.7(OR.sub.12).sub.2,
CR.sub.7OR.sub.13O, or tri-lower alkylsilyl, where R.sub.7 is an
alkyl, cycloalkyl or alkenyl group containing 1 to 5 carbons,
R.sub.8 is an alkyl group of 1 to 10 carbons or trimethylsilylalkyl
where the alkyl group has 1 to 10 carbons, or a cycloalkyl group of
5 to 10 carbons, or R.sub.8 is phenyl or lower alkylphenyl, R.sub.9
and R.sub.10 independently are hydrogen, an alkyl group of 1 to 10
carbons, or a cycloalkyl group of 5-10 carbons, or phenyl or lower
alkylphenyl, R.sub.11 is lower alkyl, phenyl or lower alkylphenyl,
R.sub.12 is lower alkyl, and R.sub.13 is divalent alkyl radical of
2-5 carbons, and
[0049] R.sub.14 is (R.sub.15).sub.r-phenyl,
(R.sub.15).sub.r-naphthyl, or (R.sub.15).sub.r-heteroaryl where the
heteroaryl group has 1 to 3 heteroatoms selected from the group
consisting of O, S and N, r is an integer having the values of 0-5,
and
[0050] R.sub.15 is independently H, F, Cl, Br, I, NO.sub.2,
N(R.sub.8).sub.2, N(R.sub.8)COR.sub.8, NR.sub.8CON(R.sub.8).sub.2,
OH, OCOR.sub.8, OR.sub.8, CN, an alkyl group having 1 to 10
carbons, fluoro substituted alkyl group having 1 to 10 carbons, an
alkenyl group having 1 to 10 carbons and 1 to 3 double bonds,
alkynyl group having 1 to 10 carbons and 1 to 3 triple bonds, or a
trialkylsilyl or trialkylsilyloxy group where the alkyl groups
independently have 1 to 6 carbons;
[0051] R.sub.16 is H, lower alkyl of 1 to 6 carbons;
[0052] R.sub.17 is H, lower alkyl of 1 to 6 carbons, OH or
OCOR.sub.11, and
[0053] p is zero or 1, with the proviso that when p is 1 then there
is no R.sub.17 substituent group, and m is an integer between 0 and
2.
[0054] The compounds of the present invention are useful for
preventing certain undesired side effects of retinoids which are
administered for the treatment or prevention of certain diseases or
conditions. For this purpose the compounds of the invention may be
coadministered with retinoids. The compounds of the present
invention are also useful in the treatment of acute or chronic
toxicity resulting from overdose or poisoning by retinoid drugs or
Vitamin A.
[0055] The present invention additionally relates to the use of RAR
antagonists for blocking all or some RAR receptor sites in
biological systems, including mammals, to prevent or diminish
action of RAR agonists on said receptor sites. More particularly,
the present invention relates to the use of RAR antagonists for (a)
the prevention and (b) the treatment of retinoid (including vitamin
A or vitamin A precursor) chronic or acute toxicity and side
effects of retinoid therapy.
[0056] In one particular aspect of the present invention, there is
provided a method of treating a pathological condition in a mammal.
The conditions treated are associated with a retinoic acid receptor
activity. This method involves administering to the mammal a
retinoid antagonist or negative hormone capable of binding to one
of the following retinoic acid receptor subtypes: RAR.sub..alpha.,
RAR.sub.62 and RAR.sub..gamma.. The antagonist or negative hormone
is administered in an amount pharmaceutically effective to provide
a therapeutic benefit against the pathological condition in the
mammal.
[0057] As an antidote to acute or chronic retinoid or vitamin A
poisoning the RAR antagonist can be administered to a mammal
enterally, i.e., intragastric intubation or food/water admixture,
or parenterally, e.g., intraperitoneally, intramuscularly,
subcutaneously, topically, etc. The only requirement for the route
of administration is that it must allow delivery of the antagonist
to the target tissue. The RAR antagonist can be formulated by
itself or in combination with excipients. The RAR antagonist need
not be in solution in the formulation, e.g., in the case of enteral
use.
[0058] As an adjunct to therapy with retinoids and in order to
prevent one or more side effects of the retinoid drug which is
administered, the RAR antagonist can similarly be administered
enterally or parenterally. The RAR antagonist and RAR agonist need
not be administered by the same route of administration. The key is
that sufficient quantities of the RAR antagonist be present
continuously in the tissue of interest during exposure to the RAR
agonist. For the prevention of retinoid toxicity, it is best that
the RAR antagonist be administered concurrently or prior to
treatment with the RAR agonist. In many situations the RAR
antagonist will be administered by a different route than the
agonist. For example undesirable skin effects of an enterally
administered retinoid may be prevented or ameliorated by an RAR
antagonist which is administered topically.
[0059] Another aspect of the present invention is a method of
identifying retinoid negative hormones. The method includes the
following steps: obtaining transfected cells containing a reporter
gene transcriptionally responsive to binding of a recombinant
retinoid receptor, the recombinant retinoid receptor having at
least protein domains located C-terminal to a DNA binding domain of
an intact retinoid receptor, measuring a basal level of reporter
gene expression in untreated transfected cells, the untreated
transfected cells being propagated in the absence of an added
retinoid, treating the transfected cells with a retinoid compound
to be tested for negative hormone activity, measuring a level of
reporter gene expression in treated cells, comparing the levels of
reporter gene expression measured in treated cells and untreated
cells, and identifying as retinoid negative hormones those retinoid
compounds producing a lower level of reporter gene expression in
treated cells compared with the basal level of reporter gene
expression measured in untreated cells. In certain preferred
embodiments of this method the intact receptor is an RAR-.alpha.,
RAR-.beta. or RAR-.gamma. subtype. In other embodiments, the intact
receptor is an RAR-.alpha., RAR-.beta. or RAR-.gamma. subtype. The
recombinant receptor can also be either a recombinant RAR or RXR
receptor. In some embodiments, the recombinant receptor is a
chimeric retinoid receptor having a constitutive transcription
activator domain. Such a constitutive transcription activator
domain can comprise a plurality of amino acids having a net
negative charge or have an amino acid sequence of a viral
transcription activator domain, such as the herpes simplex virus
VP-16 transcription activator domain. In embodiments in which the
constitutive transcription activator domain has a net negative
charge, the retinoid receptor can be recombinant and have deleted
therefrom a DNA binding domain, such as a DNA binding domain
specific for a cis-regulatory element other than a retinoic acid
responsive element. These elements include an estrogen responsive
element. The transfected cell is preferably propagated in a growth
medium substantially depleted of endogenous retinoids, such as one
that includes activated charcoal-extracted serum. In this method,
the reporter gene can be the luciferase gene, in which case, the
measuring steps can involve luminometry. The reporter gene can also
be the .beta.-galactosidase gene, in which case the measuring steps
would involve a .beta.-galactosidase assay. The transfected cell
can be a transfected mammalian cell, such as a Green monkey cell or
a human cell.
[0060] Another aspect of the present invention is a method of
potentiating a pharmacologic activity of a steroid superfamily
receptor agonist administered to a mammal. This method involves
coadministering to the mammal with the steroid superfamily receptor
agonist a composition comprising a pharmaceutically effective dose
of a retinoid negative hormone to potentiate the pharmacologic
activity of the steroid superfamily receptor agonist. The
pharmacologic activity is measurable in a reporter gene
trans-activation assay in vitro, such as by measuring anti-AP-1
activity. The pharmacologic activity to be potentiated can be an
antiproliferative activity, such as activity of the type measurable
in retinal pigment epithelium. The steroid superfamily receptor
agonist can be any of the following: a retinoid receptor agonist, a
vitamin D receptor agonist, a glucocorticoid receptor agonist, a
thyroid hormone receptor agonist, a peroxisome
proliferator-activated receptor agonist or an estrogen receptor
agonist. The retinoid receptor agonist can be an RAR agonist, such
as all-trans-retinoic acid or 13-cis retinoic acid. The retinoid
receptor agonist can also be an RXR agonist. A preferred vitamin D
receptor agonist is 1,25-dihydroxyvitamin D.sub.3. A preferred
glucocorticoid receptor agonist is dexamethasone. A preferred
thyroid hormone receptor agonist is 3,3',5-triiodothyronine. The
retinoid negative hormone is an RAR-specific retinoid negative
hormone, which preferably has a dissociation constant less than or
approximately equal to 30 nM. Example of the RAR-specific retinoid
negative hormone include AGN 193109, AGN 193385, AGN 193389 and AGN
193871. The composition comprising a pharmaceutically effective
dose of a retinoid negative hormone can be coadministered at the
same time as the steroid superfamily agonist and be combined prior
to coadministration. These can also be coadministered as separate
compositions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] FIG. 1 shows the chemical structure of AGN 193109.
[0062] FIGS. 2A-2F are a series of line graphs showing that AGN
193109 inhibited ATRA-dependent transactivation at the RARs. FIGS.
2A and 2B represent activity at the RAR-.alpha. receptor; FIGS. 2C
and 2D represent activity at the RAR-.beta. receptor; FIGS. 2E and
2F represent activity at the RAR-.gamma. receptor. In FIGS. 2A, 2C
and 2E, open squares represent retinoic acid treatment and filled
circles represent AGN 193109 treatment. In FIGS. 2B, 2D and 2F the
single lines represent luciferase activity measured after treatment
with 10.sup.-8 M ATRA and variable concentrations of AGN
193109.
[0063] FIGS. 3A and 3B are line graphs representing luciferase
activity detected in CV-1 cells transfected with reporter plasmid
ERE-tk-Luc and expression plasmid ER-RAR-.alpha. and stimulated
with ATRA (FIG. 3A) or AGN 193109 (FIG. 3B) at various
concentrations. Data points represent the mean.+-.SEM of three
independent luciferase determinations. The results of transfections
carried out using different amounts of co-transfected
ER-RAR-.alpha. (0.05, 0.1 and 0.2 .mu.g/well) are indicated in each
figure.
[0064] FIGS. 4A and 4B are line graphs representing luciferase
activity in CV-1 cells transfected with reporter plasmid ERE-tk-Luc
and expression plasmid ER-RAR-.beta. and stimulated with ATRA (FIG.
4A) or AGN 193109 (FIG. 4B) at various concentrations. Data points
represent the mean.+-.SEM of three independent luciferase
determinations. The results of transfections carried out using
different amounts of co-transfected ER-RAR-.beta. (0.05, 0.1 and
0.2 .mu.g/well) are indicated in each figure.
[0065] FIGS. 5A and 5B are line graphs representing luciferase
activity detected in CV-1 cells transfected with reporter plasmid
ERE-tk-Luc and expression plasmid ER-RAR-.gamma. and stimulated
with ATRA (FIG. 5A) or AGN 193109 (FIG. 5B) at various
concentrations. Data points represent the mean.+-.SEM of three
independent luciferase determinations. The results of transfections
carried out using different amounts of co-transfected
ER-RAR-.gamma. (0.05, 0.1 and 0.2 .mu.g/well) are indicated in each
figure.
[0066] FIG. 6 shows ATRA and AGN 193109 dose responses of CV-1
cells cotransfected with the ERE-tk-Luc reporter plasmid and either
the ER-RXR-.alpha. chimeric receptor expression plasmid alone, or
in combination with the RAR-.gamma.-VP-16 expression plasmid.
ER-RXR-.alpha. cotransfected cells were treated with ATRA (square)
and AGN 193109 (diamond). Cells cotransfected with the combination
of ER-RXR-.alpha. and RAR-.gamma.-VP-16 were treated with ATRA
(circle) or AGN 193109 (triangle).
[0067] FIG. 7 shows a line graph representing luciferase activity
measurements recorded in lysates of CV-1 cells transfected with the
ERE-tk-Luc reporter and ER-RAR-.gamma. expression construct and
then treated with ATRA at 10.sup.-8 M and the test compounds at the
concentrations indicated on the horizontal axis. The test compounds
were AGN 193109 (square), AGN 193357 (open diamond), AGN 193385
(circle), AGN 193389 (triangle), AGN 193840 (hatched square) and
AGN 192870 (filled diamond).
[0068] FIG. 8 shows a line graph representing luciferase activity
measurements recorded in lysates of CV-1 cells transfected with the
ERE-tk-Luc reporter and RAR-.gamma.-VP-16 and ER-RAR-.alpha.
expression constructs and then treated with the test compounds at
the concentrations indicated on the horizontal axis. The test
compounds were ATRA (open square), AGN 193109 (open circle), AGN
193174 (open triangle), AGN 193199 (hatched square), AGN 193385
(hatched circle), AGN 193389 (inverted triangle), AGN 193840
(diagonally filled square) and AGN 193871 (half-filled
diamond).
[0069] FIGS. 9A, 9B and 9C schematically diagram a mechanism
whereby AGN 193109 can modulate the interaction between the RAR
(shaded box) and negative coactivator proteins (-) illustrated in
the context of a transactivation assay. FIG. 9A shows that negative
coactivator proteins and positive coactivator proteins (+) are in a
binding equilibrium with the RAR. In the absence of a ligand, basal
level transcription of the reporter gene results. As illustrated in
FIG. 9B, addition of an RAR agonist promotes the association of
positive coactivator proteins with the RAR and results in
upregulated reporter gene transcription. As illustrated in FIG. 9C,
addition of AGN 193109 promotes the association of negative
coactivator proteins with the RAR and prevents reporter gene
transcription.
[0070] FIG. 10 is a bar graph showing the inhibition of TPA-induced
Str-AP1-CAT expression as a function of AGN 191183 concentration
(10.sup.-10 to 10.sup.-12 M) with the AGN 193109 concentration held
constant at 10.sup.-8 M. Results from trials conducted with AGN
191183 alone are shown as hatched bars while stripped bars
represent the results from treatment with the combination of AGN
193109 and AGN 191183.
[0071] FIG. 11 schematically diagrams a mechanism whereby AGN
193109 can potentiate the activities of RARs and other nuclear
receptor family members. As illustrated in the diagram, introduced
RARs (open rectangles having AB-C-DEF domains) have increased
sensitivity to RAR ligands in the anti-AP1 assay because the
negative coactivator protein (ncp), present in limiting supply, is
sequestered onto RARs thereby leading to two populations: RAR+ncp
and RAR-ncp. RAR-ncp has increased sensitivity to ligands. Non-RAR
nuclear factors (shaded rectangles having AB-C-DEF domains) have
increased sensitivity to cognate ligands because ncp has been
sequestered to the RAR by the activity of AGN 193109. The modular
domains of the nuclear receptors are designated using standard
nomenclature as "AB" (ligand independent transactivation domain),
"C" (DNA binding domain), and "DEF" (ligand regulated
transactivation domain and dimerization domain.
[0072] FIG. 12 is a line graph showing the effect of AGN 193109 on
the 1,25-dihydroxyvitamin D.sub.3 dose response in CV-1 cells
transfected with the MT-DR3-Luc reporter plasmid. Transfectants
were treated with 1,25-dihydroxyvitamin D.sub.3 (filled square),
1,25-dihydroxyvitamin D.sub.3 and 10.sup.-8 M AGN 193109 (filled
triangle), and 1,25-dihydroxyvitamin D.sub.3 and 10.sup.-8 M AGN
193109 (filled circle).
[0073] FIG. 13 is a bar graph showing the effect of AGN 193109 (10
nM) coadministration on 1,25-dihydroxyvitamin D.sub.3-mediated
inhibition of TPA induced Str-AP1-CAT activity. Filled bars
represent inhibition of CAT activity in transfected cells treated
with 1,25-dihydroxyvitamin D.sub.3 alone. Open bars represent
inhibition of CAT activity in transfected cells treated with the
combination of 1,25-dihydroxyvitamin D.sub.3 and AGN 193109.
[0074] FIG. 14 is a line graph showing the effect of AGN 193109
alone and in combination with AGN 191183 on HeLa cells
cotransfected with RAR-.gamma. and the RAR responsive MTV-TREp-Luc
reporter construct. Drug treatments illustrated in the graph are:
AGN 193109 alone (square), AGN 193109 in combination with AGN
191183 at 10.sup.-10 M (diamond) and AGN 193109 in combination with
AGN 191183 at a 10.sup.-9 M.
[0075] FIG. 15 is a line graph showing that ECE16-1 cells
proliferated in response to EGF (filled square) but not in response
to defined medium alone (open circle). Cells treated with AGN
193109 alone are represented by the filled triangle. The filled
circles represent results obtained for cells treated with 10 nM AGN
191183 and 0-1000 nM AGN 193109.
[0076] FIG. 16 is a bar graph showing the effect of AGN 193109 on
the proliferation of CaSki cells in the presence or absence of the
AGN 191183 retinoid agonist. All sample groups received 20 ng/ml of
epidermal growth factor (EGF) with the exception of the sample
propagated in defined medium (DM) alone (open bar). Stripped bars
represent samples propagated in the absence of AGN 193109. Filled
bars represent samples propagated in the presence of 1000 nM AGN
193109. The concentrations of AGN 191183 used in the procedure are
shown on the horizontal axis.
[0077] FIG. 17 is a dose response curve showing that AGN 193109
potentiated the antiproliferative activity of ATRA on retinal
pigment epithelium (RPE) cells. Samples treated with ATRA alone are
represented by filled squares. Samples treated with the combination
of ATRA and AGN 193109 (10.sup.-7 M) are represented by filled
circles. The ATRA concentration used for treating the various
samples is given on the horizontal axis.
[0078] FIG. 18 is a dose response curve showing that both 13-cis-RA
and ATRA inhibited RPE cell growth, and that AGN 193109 potentiated
the antiproliferative activity of 13-cis-RA. The various sample
treatments shown in the dose response included 13-cis-RA alone
(filled square), 13-cis-RA in combination with AGN 193109
(10.sup.-6 M) (filled circle), 13-cis-RA in combination with AGN
193109 (10.sup.-8 M) (filled triangle), and ATRA (filled diamond).
The concentrations of 13-cis-RA and ATRA used in the sample
treatments are shown on the horizontal axis.
[0079] FIG. 19 is a dose response curve showing that AGN 193109
potentiated the antiproliferative activity of dexamethasone in
primary RPE cell cultures. The various sample treatments shown in
the dose response included ATRA (filled square), dexamethasone
alone (filled circle), dexamethasone in combination with AGN 193109
(10.sup.-8 M) (filled triangle), and dexamethasone in combination
with AGN 193109 (10.sup.-6 M) (filled diamond). The concentrations
of dexamethasone and ATRA used in the sample treatments are shown
on the horizontal axis.
[0080] FIG. 20 is a dose response curve showing that AGN 193109
potentiated the antiproliferative activity of thyroid hormone (T3)
in primary RPE cell cultures. The various sample treatments shown
in the dose response included ATRA (filled square), T3 alone
(filled circle), T3 in combination with AGN 193109 (10.sup.-8 M)
(filled triangle), T3 in combination with AGN 193109 (10.sup.-6 M)
(filled diamond). The concentrations of T3 and ATRA used in the
sample treatments are shown on the horizontal axis.
DETAILED DESCRIPTION OF THE INVENTION
[0081] Definitions
[0082] For the purposes of the present invention, an RAR antagonist
is defined as a chemical that binds to one or more of the RAR
subtypes with a K.sub.d of less than 1 micromolar (K.sub.d<1
.mu.M) but which does not cause significant transcriptional
activation of that RAR subtypes-regulated genes in a receptor
co-transfection assay. Conventionally, antagonists are chemical
agents that inhibit the activities of agonists. Thus, the activity
of a receptor antagonist is conventionally measured by virtue of
its ability to inhibit the activity of an agonist.
[0083] An RAR agonist is defined as a chemical that binds to one or
more RAR receptor subtype with K.sub.d of less than 1 micromol
(K.sub.d<1 .mu.M) and causes transcriptional activation of that
RAR-subtype-regulated genes in a receptor co-transfection assay.
The term "RAR agonist" includes chemicals that may bind and/or
activate other receptors in addition to RARs, e.g., RXR
receptors.
[0084] As used herein, a negative hormone or inverse agonist is a
ligand for a receptor which causes the receptor to adopt an
inactive state relative to a basal state occurring in the absence
of any ligand. Thus, while an antagonist can inhibit the activity
of an agonist, a negative hormone is a ligand that can alter the
conformation of the receptor in the absence of an agonist. The
concept of a negative hormone or inverse agonist has been explored
by Bond et al. in Nature 374:272 (1995). More specifically, Bond et
al. have proposed that unliganded .beta..sub.2-adrenoceptor exists
in an equilibrium between an inactive conformation and a
spontaneously active conformation. Agonists are proposed to
stabilize the receptor in an active conformation. Conversely,
inverse agonists are believed to stabilize an inactive receptor
conformation. Thus, while an antagonist manifests its activity by
virtue of inhibiting an agonist, a negative hormone can
additionally manifest its activity in the absence of an agonist by
inhibiting the spontaneous conversion of an unliganded receptor to
an active conformation. Only a subset of antagonists will act as
negative hormones. As disclosed herein, AGN 193109 is both an
antagonist and a negative hormone. To date, no other retinoids have
been shown to have negative hormone activity.
[0085] As used herein, coadministration of two pharmacologically
active compounds refers to the delivery of two separate chemical
entities, whether in vitro or in vivo. Coadministration refers to
the simultaneous delivery of separate agents; to the simultaneous
delivery of a mixture of agents; as well as to the delivery of one
agent followed by delivery of the second agent. In all cases,
agents that are coadministered are intended to work in conjunction
with each other.
[0086] The term alkyl refers to and covers any and all groups which
are known as normal alkyl, branched-chain alkyl and cycloalkyl. The
term alkenyl refers to and covers normal alkenyl, branch chain
alkenyl and cycloalkenyl groups having one or more sites of
unsaturation. Similarly, the term alkynyl refers to and covers
normal alkynyl, and branch chain alkynyl groups having one or more
triple bonds.
[0087] Lower alkyl means the above-defined broad definition of
alkyl groups having 1 to 6 carbons in case of normal lower alkyl,
and as applicable 3 to 6 carbons for lower branch chained and
cycloalkyl groups. Lower alkenyl is defined similarly having 2 to 6
carbons for normal lower alkenyl groups, and 3 to 6 carbons for
branch chained and cyclo- lower alkenyl groups. Lower alkynyl is
also defined similarly, having 2 to 6 carbons for normal lower
alkynyl groups, and 4 to 6 carbons for branch chained lower alkynyl
groups.
[0088] The term "ester" as used here refers to and covers any
compound falling within the definition of that term as classically
used in organic chemistry. It includes organic and inorganic
esters. Where B (of Formula 1 or Formula 101) is --COOH, this term
covers the products derived from treatment of this function with
alcohols or thiols preferably with aliphatic alcohols having 1-6
carbons. Where the ester is derived from compounds where B is
--CH.sub.2OH, this term covers compounds derived from organic acids
capable of forming esters including phosphorous based and sulfur
based acids, or compounds of the formula --CH.sub.2OCOR,, where
R.sub.11 is any substituted or unsubstituted aliphatic, aromatic,
heteroaromatic or aliphatic aromatic group, preferably with 1-6
carbons in the aliphatic portions.
[0089] Unless stated otherwise in this application, preferred
esters are derived from the saturated aliphatic alcohols or acids
of ten or fewer carbon atoms or the cyclic or saturated aliphatic
cyclic alcohols and acids of 5 to 10 carbon atoms. Particularly
preferred aliphatic esters are those derived from lower alkyl acids
and alcohols. Also preferred are the phenyl or lower alkyl phenyl
esters.
[0090] Amides has the meaning classically accorded that term in
organic chemistry. In this instance it includes the unsubstituted
amides and all aliphatic and aromatic mono- and di- substituted
amides. Unless stated otherwise in this application, preferred
amides are the mono- and di-substituted amides derived from the
saturated aliphatic radicals of ten or fewer carbon atoms or the
cyclic or saturated aliphatic-cyclic radicals of 5 to 10 carbon
atoms. Particularly preferred amides are those derived from
substituted and unsubstituted lower alkyl amines. Also preferred
are mono- and disubstituted amides derived from the substituted and
unsubstituted phenyl or lower alkylphenyl amines. Unsubstituted
amides are also preferred.
[0091] Acetals and ketals include the radicals of the formula-CK
where K is (--OR).sub.2. Here, R is lower alkyl. Also, K may be
--OR.sub.7O-- where R.sub.7 is lower alkyl of 2-5 carbon atoms,
straight chain or branched.
[0092] A pharmaceutically acceptable salt may be prepared for any
compounds in this invention having a functionality capable of
forming a salt, for example an acid functionality. A
pharmaceutically acceptable salt is any salt which retains the
activity of the parent compound and does not impart any deleterious
or untoward effect on the subject to which it is administered and
in the context in which it is administered.
[0093] Pharmaceutically acceptable salts may be derived from
organic or inorganic bases. The salt may be a mono or polyvalent
ion. Of particular interest are the inorganic ions, sodium,
potassium, calcium, and magnesium. Organic salts may be made with
amines, particularly ammonium salts such as mono-, di- and trialkyl
amines or ethanol amines. Salts may also be formed with caffeine,
tromethamine and similar molecules. Where there is a nitrogen
sufficiently basic as to be capable of forming acid addition salts,
such may be formed with any inorganic or organic acids or
alkylating agent such as methyl iodide. Preferred salts are those
formed with inorganic acids such as hydrochloric acid, sulfuric
acid or phosphoric acid. Any of a number of simple organic acids
such as mono-, di- or tri- acid may also be used.
[0094] Some of the compounds of the present invention may have
trans and cis (E and Z) isomers. In addition, the compounds of the
present invention may contain one or more chiral centers and
therefore may exist in enantiomeric and diastereomeric forms. The
scope of the present invention is intended to cover all such
isomers per se, as well as mixtures of cis and trans isomers,
mixtures of diastereomers and racemic mixtures of enantiomers
(optical isomers) as well. In the present application when no
specific mention is made of the configuration (cis, trans or R or
S) of a compound (or of an asymmetric carbon) then a mixture of
such isomers, or either one of the isomers is intended.
Aryl Substituted Benzopyran, Benzothiopyran, 1.2-Dihydroquinoline
and 5,6-Dihydronaphthalene Derivatives Having Retinoid Antagonist
Like Biological Activity
[0095] With reference to the symbol Y in Formula 1, the preferred
compounds of the invention are those where Y is phenyl, pyridyl,
thienyl or furyl. Even more preferred are compounds where Y is
phenyl or pyridyl, and still more preferred where Y is phenyl. As
far as substitutions on the Y (phenyl) and Y (pyridyl) groups are
concerned, compounds are preferred where the phenyl group is 1, 4
(para) substituted by the Z and A-B groups, and where the pyridine
ring is 2, 5 substituted by the Z and A-B groups. (Substitution in
the 2, 5 positions in the "pyridine" nomenclature corresponds to
substitution in the 6-position in the "nicotinic acid"
nomenclature.) In the preferred compounds of the invention either
there is no optional R.sub.2 substituent on the Y group, or the
optional R.sub.2 substituent is fluoro (F).
[0096] The A-B group of the preferred compounds is
(CH.sub.2).sub.n--COOH or (CH.sub.2).sub.n--COOR.sub.8, where n and
R.sub.8 are defined as above. Even more preferably n is zero and
R.sub.8 is lower alkyl, or n is zero and B is COOH or a
pharmaceutically acceptable salt thereof.
[0097] In the majority of the presently preferred examples of
compounds of the invention X is [C(R.sub.1).sub.2].sub.n where n is
1. Nevertheless, compounds where n is zero (indene derivatives) and
where X is S or O (benzothiopyran and benzopyran derivatives) are
also preferred. When X is [C(R.sub.1).sub.2].sub.n and n is 1, then
R.sub.1 preferably is alkyl of 1 to 6 carbons, even more preferably
methyl.
[0098] The R.sub.2 group attached to the aromatic portion of the
tetrahydronaphthalene, benzopyran, benzothiopyran or
dihydroquinoline moiety of the compounds of Formula 1 is preferably
H, F or CF.sub.3. R.sub.3 is preferably hydrogen or methyl, even
more preferably hydrogen.
[0099] Referring now to the group Z in the compounds of the
invention and shown in Formula 1, in a plurality of preferred
examples Z represents an acetylenic linkage (Z.dbd.--C.ident.C--).
However, the "linker group" Z is also preferred as a diazo group
(Z.dbd.--N.dbd.N--), as an olefinic or polyolefinic group
(Z.dbd.--(CR.sub.1.dbd.CR.sub.1).sub.n'--), as an ester
(Z.dbd.--COO--), amide (Z.dbd.--CO--NR.sub.2--) or thioamide
(Z.dbd.--CS--NR.sub.2--) linkage.
[0100] Referring now to the R.sub.14 group, compounds are preferred
where R.sub.14 is phenyl, 2-pyridyl, 3-pyridyl, 2-thienyl, and
2-thiazolyl. The R.sub.15 group (substituent of the R.sub.14 group)
is preferably H, lower alkyl, trifluoromethyl, chlorine, lower
alkoxy or hydroxy.
[0101] The presently most preferred compounds of the invention are
shown in Table 1 with reference to Formula 2, Formula 3, Formula 4,
Formula 5, and Formula 5a. 3
1TABLE 1 Com- pound For- # mula R.sub.14* Z R.sub.2* R.sub.8* 1 2
4-methylphenyl --C.ident.C-- H Et 1a 2 phenyl --C.ident.C-- H Et 2
2 3-methylphenyl --C.ident.C-- H Et 3 2 2-methylphenyl
--C.ident.C-- H Et 4 2 3,5-dimethyl- --C.ident.C-- H Et phenyl 5 2
4-ethylphenyl --C.ident.C-- H Et 6 2 4-t-butylphenyl --C.ident.C--
H Et 7 2 4-chlorophenyl --C.ident.C-- H Et 8 2 4-methoxy-
--C.ident.C-- H Et phenyl 9 2 4-trifluoro- --C.ident.C-- H Et
methylphenyl 10 2 2-pyridyl --C.ident.C-- H Et 11 2 3-pyridyl
--C.ident.C-- H Et 12 2 2-methyl-5- --C.ident.C-- H Et pyridyl 13 2
3-hydroxy- --C.ident.C-- H Et phenyl 14 2 4-hydroxy --C.ident.C-- H
Et phenyl 15 2 5-methyl-2- --C.ident.C-- H Et thiazolyl 15a 2
2-thiazolyl --C.ident.C-- H Et 16 2 4-methyl-2- --C.ident.C-- H Et
thiazolyl 17 2 4,5-dimethyl-2- --C.ident.C-- H Et thiazolyl 18 2
2-methyl-5- --C.ident.C-- H H pyridyl 19 2 2-pyridyl --C.ident.C--
H H 20 2 3-methylphenyl --C.ident.C-- H H 21 2 4-ethylphenyl
--C.ident.C-- H H 22 2 4-methoxy- --C.ident.C-- H H phenyl 23 2
4-trifluoro- --C.ident.C-- H H methylphenyl 24 2 3,5-dimethyl-
--C.ident.C-- H H phenyl 25 2 4-chlorophenyl --C.ident.C-- H H 26 2
3-pyridyl --C.ident.C-- H H 27 2 2-methylphenyl --C.ident.C-- H H
28 2 3-hydroxy- --C.ident.C-- H H phenyl 29 2 4-hydroxy-
--C.ident.C-- H H phenyl 30 2 5-methyl-2- --C.ident.C-- H H
thiazolyl 30a 2 2-thiazolyl --C.ident.C-- H H 31 2 4-methyl-2-
--C.ident.C-- H H thiazolyl 32 2 4,5-dimethyl-2- --C.ident.C-- H H
thiazolyl 33 2 5-methyl-2- --C.ident.C-- H Et thienyl 33a 2
2-thienyl --C.ident.C-- H Et 34 2 5-methyl-2- --C.ident.C-- H H
thienyl 34a 2 2-thienyl --C.ident.C-- H H 35 2 4-methylphenyl
--CONH-- H Et 36 2 4-methylphenyl --CONH-- H H 37 2 4-methylphenyl
--COO-- H Et 38 2 4-methylphenyl --COO-- H
(CH.sub.2).sub.2Si(CH.sub.3) 39 2 4-methylphenyl --COO-- H H 40 2
4-methylphenyl --CONH-- F Et 41 2 4-methylphenyl --CONH F H 42 2
4-methylphenyl --CSNH-- H Et 43 2 4-methylphenyl --CSNH-- H H 44 2
4-methylphenyl --CH.dbd.CH-- H Et 45 2 4-methylphenyl --CH.dbd.CH--
H H 46a 2 4-methylphenyl --N.dbd.N-- H Et 46b 2 4-methylphenyl
--N.dbd.N-- H H 47 3 4-methylphenyl --C.ident.C-- H Et 48 3
4-methylphenyl --C.ident.C-- H H 49 4 4-methylphenyl --C.ident.C--
H Et 50 4 4-methylphenyl --C.ident.C-- H H 51 5 4-methylphenyl --
-- Et 52 5 4-methylphenyl -- -- H 60 2 4-methylphenyl --C.ident.C--
H H 60a 2 phenyl --C.ident.C-- H H 61 2 4-t-butylphenyl
--C.ident.C-- H H 62 2 4-methylphenyl --CSNH F Et 63 2
4-methylphenyl --CSNH F H 64 5a 4-methylphenyl -- -- Et 65 5a
4-methylphenyl -- -- H 66 2 2-furyl --C.ident.C-- H Et 67 2 2-furyl
--C.ident.C-- H H
Aryl and (3-Oxo-1-Pronenyl)-Substituted Benzopyran. Benzothiopyran.
Dihydroquinoline and 5,6-Dihydronaphthalene Derivatives Having
Retinoid Antagonist-Like Biological Activity
[0102] With reference to the symbol Y in Formula 101, the preferred
compounds of the invention are those where Y is phenyl, pyridyl,
thienyl or furyl. Even more preferred are compounds where Y is
phenyl or pyridyl, and still more preferred where Y is phenyl. As
far as substitutions on the Y (phenyl) and Y (pyridyl) groups are
concerned, compounds are preferred where the phenyl group is 1, 4
(para) substituted by the --CR.sub.16.dbd.CR.sub.17-- and A-B
groups, and where the pyridine ring is 2, 5 substituted by the
--CR.sub.16.dbd.CR.sub.17-- and A-B groups. (Substitution in the
2,5 positions in the "pyridine" nomenclature corresponds to
substitution in the 6-position in the "nicotinic acid"
nomenclature.) In the preferred compounds of the invention there is
no optional R.sub.2 substituent on the Y group.
[0103] The A-B group of the preferred compounds is
(CH.sub.2).sub.n--COOH or (CH.sub.2).sub.n--COOR.sub.8, where n and
R.sub.8 are defined as above. Even more preferably n is zero and
R.sub.8 is lower alkyl, or n is zero and B is COOH or a
pharmaceutically acceptable salt thereof.
[0104] In the presently preferred examples of compounds of the
invention X is [C(R.sub.1).sub.2].sub.n where n is 1. Nevertheless,
compounds where X is S or O (benzothiopyran and benzopyran
derivatives) are also preferred. When X is [C(R.sub.1).sub.2].sub.n
and n is 1, then R.sub.1 preferably is alkyl of 1 to 6 carbons,
even more preferably methyl.
[0105] The R.sub.2 group attached to the aromatic portion of the
tetrahydronaphthalene, benzopyran, benzothiopyran or
dihydroquinoline moiety of the compounds of Formula 101 is
preferably H, F or CF.sub.3. R.sub.3 is preferably hydrogen or
methyl, even more preferably hydrogen.
[0106] Referring now to the R.sub.14 group, compounds are preferred
where R.sub.14 is phenyl, 2-pyridyl, 3-pyridyl, 2-thienyl, and
2-thiazolyl. The R.sub.15 group (substituent of the R.sub.14 group)
is preferably H, lower alkyl, trifluoromethyl, chlorine, lower
alkoxy or hydroxy.
[0107] Preferred compounds of the invention are shown in Table 2
with reference to Formula 102. 4
2TABLE 2 Compund R.sub.15* R.sub.8* 101 CH.sub.3 H 102 CH.sub.3 Et
103 H H 104 H Et
[0108] Biological Activity, Modes of Administration
[0109] As noted above, the compounds of the present invention are
antagonists of one or more RAR receptor subtypes. This means that
the compounds of the invention bind to one or more RAR receptor
subtypes, but do not trigger the response which is triggered by
agonists of the same receptors. Some of the compounds of the
present invention are antagonists of all three RAR receptor
subtypes (RAR-.alpha., RAR-.beta. and RAR-.gamma.), and these are
termed "RAR pan antagonists". Some others are antagonists of only
one or two of RAR receptor subtypes. Some compounds within the
scope of the present invention are partial agonists of one or two
RAR receptor subtypes and antagonists of the remaining subtypes.
The compounds of the invention do not bind to RXR receptors,
therefore they are neither agonists nor antagonists of RXR.
[0110] Depending-on the site and nature of undesirable side effects
which are desired to be suppressed or ameliorated, compounds used
in accordance with the invention may be antagonists of only one or
two of RAR receptor subtypes. Some compounds used in accordance
with the invention may be partial agonists of one or two RAR
receptor subtypes and antagonists of the remaining subtypes. Such
compounds are, generally speaking, usable in accordance with the
invention if the antagonist effect is on that RAR receptor subtype
(or subtypes) which is (are) predominantly responsible for the
overdose poisoning or for the undesired side effect or side
effects. In this connection it is noted that, generally speaking, a
compound is considered an antagonist of a given receptor subtype if
in the below described co-tranfection assays the compound does not
cause significant transcriptional activation of the receptor
regulated reporter gene, but nevertheless binds to the receptor
with a K.sub.d value of less than approximately 1 .mu.M.
[0111] Whether a compound is an RAR antagonist and therefore can be
utilized in accordance with the present invention, can be tested in
the following assays.
[0112] A chimeric receptor transactivation assay which tests for
agonist-like activity in the RAR-.alpha., RAR-.beta., RAR-.gamma.,
RAR-.alpha. receptor subtypes, and which is based on work published
by Feigner P. L. and Holm M. Focus Vol 11, No. 2 (1989) is
described in detail in published PCT Application No. WO94/17796,
published on Aug. 18, 1994. The latter publication is the PCT
counterpart of U.S. application Ser. No. 08/016,404, filed on Feb.
11, 1993, which issued as U.S. Pat. No. 5,455,265. PCT publication
WO94/17796 and the specification of U.S. Pat. No. 5,455,265 are
hereby expressly incorporated by reference. A compound should not
cause significant activation of a reporter gene through a given
receptor subtype (RAR-.alpha., RAR-.beta. or RAR-.gamma.) in this
assay, in order to qualify as an RAR antagonist with utility in the
present invention.
[0113] A holoreceptor transactivation assay and a ligand binding
assay which measure the antagonist/agonist like activity of the
compounds of the invention, or their ability to bind to the several
retinoid receptor subtypes, respectively, are described in
published PCT Application No. WO93/11755 (particularly on pages
30-33 and 37-41) published on Jun. 24, 1993, the specification of
which is also incorporated herein by reference. A description of
the holoreceptor transactivation assay is also provided below.
[0114] Holoreceptor Transactivation Assay
[0115] CV1 cells (5,000 cells/well) were transfected with an RAR
reporter plasmid MTV-TREp-LUC (50 ng) along with one of the RAR
expression vectors (10 ng) in an automated 96-well format by the
calcium phosphate procedure of Heyman et al. Cell 68:397-406. For
RAR-.alpha. and RAR-.gamma. transactivation assays, an
RXR-responsive reporter plasmid CRBPII-tk-LUC (50 ng) along with
the appropriate RXR expression vectors (10 ng) was used
substantially as described by Heyman et al. above, and Allegretto
et al. J. Biol. Chem. 268:26625-26633. For RXR-.beta.
transactivation assays, an RXR-responsive reporter plasmid
CPRE-tk-LUC (50 mg) along with RXR-.beta. expression vector (10 mg)
was used as described in above. These reporters contain DRI
elements from human CRBPII and certain DRI elements from promotor,
respectively (see Mangelsdorf et al. The Retinoids: Biology.
Chemistry and Medicine, pp. 319-349, Raven Press Ltd., New York and
Heyman et al., cited above). A .beta.-galactosidase (50 ng)
expression vector was used as an internal control in the
transfections to normalize for variations in transfection
efficiency. The cells were transfected in triplicate for 6 hours,
followed by incubation with retinoids for 36 hours, and the
extracts were assayed for luciferase and .beta.-galactosidase
activities. The detailed experimental procedure for holoreceptor
transactivations has been described in Heyman et al. above, and
Allegretto et al. cited above. The results obtained in this assay
are expressed in EC.sub.50 numbers, as they are also in the
chimeric receptor transactivation assay. The Heyman et al. Cell
68:397-406, Allegretto et al. J. Biol. Chem. 268:26625-26633, and
Mangelsdorf et al. The Retinoids: Biology Chemistry and Medicine,
pp. 319-349, Raven Press Ltd., New York, are expressly incorporated
herein by reference. The results of ligand binding assay are
expressed in K.sub.d numbers. (See Cheng et al. Biochemical
Pharmacology 22:3099-3108, expressly incorporated herein by
reference.)
[0116] A compound should not cause significant activation of a
reporter gene through a given receptor subtype (RAR-.alpha.,
RAR-.beta. or RAR-.gamma.) in the holoreceptor transactivation
assay assay, in order to qualify as an RAR antagonist with utility
in the present invention. Last, but not least, a compound should
bind to at least one of the RAR receptor subtypes in the ligand
binding assay with a K.sub.d of less than approximately 1
micromolar (K.sub.d<1 .mu.M) in order to be capable of
functioning as an antagonist of the bound receptor subtype,
provided the same receptor subtype is not significantly activated
by the compound.
[0117] Table 3 below shows the results of the holoreceptor
transactivation assay and Table 4 discloses the efficacy (in
percentage) in this assay of the test compound relative to all
trans retinoic acid, for certain exemplary compounds of the
invention. Table 5 shows the results of the ligand binding assay
for certain exemplary compounds of the invention.
3TABLE 3 Holoreceptor Transactivation Assay Compound EC.sub.50
(nanomolar) # RAR.alpha. RAR.beta. RAR.gamma. RXR.alpha. RXR.beta.
RXR.gamma. 18 0.00 0.00 0.00 0.00 0.00 0.00 19 0.00 0.00 0.00 0.00
0.00 0.00 20 0.00 0.00 0.00 0.00 0.00 0.00 21 0.00 0.00 0.00 0.00
0.00 0.00 22 0.00 0.00 0.00 0.00 0.00 0.00 23 0.00 0.00 0.00 0.00
0.00 0.00 24 0.00 0.00 0.00 0.00 0.00 0.00 25 0.00 0.00 0.00 0.00
0.00 0.00 26 0.00 0.00 0.00 0.00 0.00 0.00 27 0.00 0.00 0.00 0.00
0.00 0.00 28 0.00 0.00 0.00 0.00 0.00 0.00 29 0.00 0.00 0.00 0.00
0.00 0.00 30 0.00 0.00 0.00 0.00 0.00 0.00 31 0.00 0.00 0.00 0.00
0.00 0.00 32 0.00 0.00 0.00 0.00 0.00 0.00 34 0.00 0.00 0.00 0.00
0.00 0.00 36 0.00 0.00 0.00 0.00 0.00 0.00 39 0.00 0.00 0.00 0.00
0.00 0.00 41 0.00 0.00 0.00 0.00 0.00 0.00 45 0.00 0.00 0.00 0.00
0.00 0.00 46b 0.00 0.00 0.00 0.00 0.00 0.00 52 0.00 0.00 0.00 0.00
0.00 0.00 60 0.00 0.00 0.00 0.00 0.00 0.00 61 0.00 0.00 0.00 0.00
0.00 0.00 63 0.00 0.00 0.00 0.00 0.00 0.00 101 0.00 0.00 0.00 0.00
0.00 0.00 103 0.00 0.00 0.00 0.00 0.00 0.00 O.O in Table 3
indicates that the compound is less than 20% as active
(efficacious) in this assay than all trans retinoic acid.
[0118]
4TABLE 4 Transactivation Assay Efficacy (% of RA activity) Compound
# RAR.alpha. RAR.beta. RAR.gamma. RXR.alpha. RXR.beta. RXR.gamma.
18 4.00 1.00 0.00 2.00 10.00 1.0 19 0.00 5.0 3.0 0.0 9.0 4.0 20 3.0
4.0 0.00 4.00 0.00 3.0 21 2.00 2.00 2.00 3.00 0.00 3.00 22 0.00
0.00 2.00 1.00 0.00 2.00 23 0.00 6.00 3.00 1.00 0.00 4.00 24 3.00
7.00 4.00 1.00 0.00 3.00 25 2.00 3.00 3.00 5.00 0.00 3.00 26 1.00
6.00 0.00 2.00 0.00 3.00 27 9.00 14.00 6.00 2.00 0.00 4.00 28 2.00
10.00 2.00 2.00 0.00 3.00 29 0.00 6.00 11.00 0.00 6.00 2.00 30 3.00
5.00 1.00 0.00 9.00 3.00 31 4.00 14.00 2.00 1.00 8.00 6.00 32 0.00
2.00 2.00 1.00 0.00 2.00 34 3.00 5.00 2.00 1.00 0.00 3.00 36 1.00
5.00 0.00 1.00 7.00 2.00 39 1.00 7.00 9.00 2.00 0.00 1.00 41 3.00
5.00 6.00 1.00 0.00 3.00 45 2.00 0.00 7.00 3.00 8.00 0.00 46b 4.00
5.00 3.00 2.00 0.00 4.00 52 0.00 15.00 3.00 0.00 0.00 10.00 60 0.00
1.00 4.00 3.00 0.00 3.00 61 2.00 2.00 0.00 1.00 0.00 3.00 63 2.00
2.00 7.00 1.00 0.00 1.00 101 0.00 4.00 2.00 1.00 0.00 3.0 103 4.00
12.0 7.0 0.00 0.0 2.0
[0119]
5TABLE 5 Ligand Binding Assay Compound K.sub.d (nanomolar) #
RAR.alpha. RAR.beta. RAR.gamma. RXR.alpha. RXR.beta. RXR.gamma. 18
24.00 11.00 24.00 0.00 0.00 0.00 19 565 210 659 0.00 0.00 0.00 20
130.00 22.0 34.00 0.00 0.00 0.00 21 16 9 13 0.00 0.00 0.00 22 24.0
17.0 27.0 0.00 0.00 0.00 23 32.00 25.00 31.00 0.00 0.00 0.00 24 699
235 286 0.00 0.00 0.00 25 50 17 20 0.00 0.00 0.00 26 40.00 31.00
36.00 0.00 0.00 0.00 27 69.00 14.00 26.00 0.00 0.00 0.00 28 669 77
236 0.00 0.00 0.00 29 234 48 80 0.00 0.00 0.00 30 683 141 219 0.00
0.00 0.00 31 370 52.00 100.00 0.00 0.00 0.00 32 0.00 89.00 169.00
0.00 0.00 0.00 34 52.00 30.00 17.00 0.00 0.00 0.00 36 13.00 550.00
0.00 0.00 0.00 0.00 39 67.00 38.00 113.00 0.00 0.00 0.00 41 5.1 491
725 0.00 0.00 0.00 45 12.0 2.80 17.0 0.00 0.00 0.00 46b 250 3.70
5.80 0.00 0.00 0.00 52 60.00 63.00 56.00 0.00 0.00 0.00 60 1.5 1.9
3.3 0.00 0.00 0.00 61 96 15 16 0.00 0.00 0.00 63 133 3219 0.00 0.00
0.00 0.00 101 750 143 637 0.00 0.0.0 0.00 103 301 273 261 0.00 0.00
0.00 O.O in Table 5 indicates a value greater than 1000 nM.
[0120] As it can be seen from the test results summarized in Tables
3, 4 and 5, the therein indicated exemplary compounds of the
invention are antagonists of the RAR receptor subtypes, but have no
affinity to RXR receptor subtypes. (Other compounds of the
invention may be antagonist of some but not all RAR receptor
subtypes and agonists of the remaining RAR subtypes.) Due to this
property, the compounds of the invention can be used to block the
activity of RAR agonists in biological assays. In mammals,
including humans, the compounds of the invention can be
coadministered with RAR agonists and, by means of pharmacological
selectivity or site-specific delivery, preferentially prevent the
undesired effects of RAR agonists. The compounds of the invention
can also be used to treat Vitamin A overdose, acute or chronic,
resulting either from the excessive intake of vitamin A supplements
or from the ingestion of liver of certain fish and animals that
contain high level of Vitamin A. Still further, the compounds of
the invention can also be used to treat acute or chronic toxicity
caused by retinoid drugs. It has been known in the art that the
toxicities observed with hypervitaminosis A syndrome (headache,
skin peeling, bone toxicity, dyslipidemias) are similar or
identical with toxicities observed with other retinoids, suggesting
a common biological cause, that is RAR activation. Because the
compounds of the present invention block RAR activation, they are
suitable for treating the foregoing toxicities.
[0121] The compounds of the invention are able to substantially
prevent skin irritation induced by RAR agonist retinoids, when the
compound of the invention is topically coadministered to the skin.
Similarly, compounds of the invention can be administered topically
to the skin, to block skin irritation, in patients or animals who
are administered RAR agonist compounds systemically. The compounds
of the invention can accelerate recovery from preexisting retinoid
toxicity, can block hypertriglyceridemia caused by co-administered
retinoids, and can block bone toxicity induced by an RAR agonist
(retinoid).
[0122] Generally speaking, for therapeutic applications in mammals
in accordance with the present invention, the antagonist compounds
can be admistered enterally or topically as an antidote to vitamin
A, vitamin A precursors, or antidote to retinoid toxicity resulting
from overdose or prolonged exposure, after intake of the causative
factor (vitamin A precursor or other retinoid) has been
discontinued. Alternatively, the antagonist compounds are
coadministered with retinoid drugs in accordance with the
invention, in situations where the retinoid provides a therapeutic
benefit, and where the coadministered antagonist alleviates or
eliminates one or more undesired side effects of the retinoid. For
this type of application the antagonist may be administered in a
site-specific manner, for example as a topically applied cream or
lotion while the coadministered retinoid may be given
enterally.
[0123] For therapeutic applications in accordance with the present
invention the antagonist compounds are incorporated into
pharmaceutical compositions, such as tablets, pills, capsules,
solutions, suspensions, creams, ointments, gels, salves, lotions
and the like, using such pharmaceutically acceptable excipients and
vehicles which per se are well known in the art. For example
preparation of topical formulations are well described in
Remington's Pharmaceutical Science, Edition 17, Mack Publishing
Company, Easton, Pennsylvania. For topical application, the
antagonist compounds could also be administered as a powder or
spray, particularly in aerosol form. If the drug is to be
administered systemically, it may be confected as a powder, pill,
tablet or the like or as a syrup or elixir suitable for oral
administration. For intravenous or intraperitoneal administration,
the antagonist compound will be prepared as a solution or
suspension capable of being administered by injection. In certain
cases, it may be useful to formulate the antagonist compounds by
injection. In certain cases, it may be useful to formulate the
antagonist compounds in suppository form or as extended release
formulation for deposit under the skin or intramuscular
injection.
[0124] The antagonist compounds will be administered in a
therapeutically effective dose in accordance with the invention. A
therapeutic concentration will be that concentration which effects
reduction of the particular condition (such as toxicity due to
retinoid or vitamin A exposure, or side effect of retinoid drug) or
retards its expansion. It should be understood that when
coadministering the antagonist compounds to block retinoid-induced
toxicity or side effects in accordance with the invention, the
antagonist compounds are used in a prophylactic manner to prevent
onset of a particular condition, such as skin irritation.
[0125] A useful therapeutic or prophylactic concentration will vary
from condition to condition and in certain instances may vary with
the severity of the condition being treated and the patient's
susceptibility to treatment. Accordingly, no single concentration
will be uniformly useful, but will require modification depending
on the particularities of the chronic or acute retinoid toxicity or
related condition being treated. Such concentrations can be arrived
at through routine experimentation. However, it is anticipated that
a formulation containing between 0.01 and 1.0 milligrams of
antagonist compound per mililiter of formulation will constitute a
therapeutically effective concentration for topical application. If
administered systemically, an amount between 0.01 and 5 mg per kg
per day of body weight would be expected to effect a therapeutic
result.
[0126] The basis of the utility of RAR antagonists for the
prevention or treatment of RAR agonist-induced toxicity is
competitive inhibition of the activation of RAR receptors by RAR
agonists. The main distinction between these two applications of
RAR antagonists is the presence or absence of preexisting retinoid
toxicity. Most of the examples immediately described below relate
to the use of retinoids to prevent retinoid toxicity, but the
general methods described herein are applicable to the treatment of
preexisting retinoid toxicity as well.
[0127] Description of Experiments Demonstrating the Use of RAR
Antagonists to Prevent or Treat Retinoid Toxicity and/or Side
Effects of Retinoid Drugs
EXAMPLE 1
[0128] Skin Irritation Induced by Topically Applied Agonist is
Treated with Topically Applied Antagonist
[0129] The compound
4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphth-
alen-2-yl)propen-1-yl]benzoic acid, designated AGN 191183, is known
in the prior art as a potent RAR agonist (see for example the
descriptive portion and FIG. 2b of U.S. Pat. No. 5,324,840). (The
"AGN" number is an arbitrarily designated reference number utilized
by the corporate assignee of the present invention for
identification of compounds.)
[0130]
4-[(5,6-dihydro-5,5-dimethyl-8-(phenyl)-2-naphthalenyl)ethynyl]benz-
oic acid (AGN 192869, also designated Compound 60a) is a compound
the preparation of which is described below. This compound is an
RAR antagonist.
[0131] Skin irritation induced by an RAR agonist, AGN 191183,
administered topically, can be blocked by an RAR antagonist, AGN
192869, also administered topically in hairless mice.
[0132] More particularly skin irritation was measured on a
semiquantitative scale by the daily subjective evaluation of skin
flaking and abrasions. A single number, the topical irritation
score, summarizes the skin irritation induced in an animal during
the course of an experiment. The topical irritation score is
calculated as follows. The topical irritation score is the
algebraic sum of a composite flaking score and a composite abrasion
score. The composite scores range from 0-9 and 0-8 for flaking and
abrasions, respectively, and take into account the maximum
severity, the time of onset, and the average severity of the
flaking and abrasions observed.
[0133] The severity of flaking is scored on a 5-point scale and the
severity of abrasions is scored on a 4-point scale, with higher
scores reflecting greater severity. The maximum severity component
of the composite scores would be the highest daily severity score
assigned to a given animal during the course of observation.
[0134] For the time of onset component of the composite score, a
score ranging from 0 to 4 is assigned as follows:
6TABLE 6 Time to Appearance of Flaking or Abrasions of Severity 2
or Greater (days) Time of Onset Score 8 0 6-7 1 5 2 3-4 3 1-2 4
[0135] The average severity component of the composite score is the
sum of the daily flaking or abrasion scores divided by the number
of observation days. The first day of treatment is not counted,
since the drug compound has not had an opportunity to take effect
at the time of first treatment.
[0136] To calculate the composite flaking and abrasion scores, the
average severity and time of onset scores are summed and divided by
2. The result is added to the maximal severity score. The composite
flaking and abrasion scores are then summed to give the overall
topical irritation score. Each animal receives a topical irritation
score, and the values are expressed as the mean.+-.SD of the
individual scores of a group of animals. Values are rounded to the
nearest integer.
[0137] Female hairless mice [Crl:SKH1-hrBR] (8-12 weeks old, n=6)
were treated topically for 5 consecutive days with acetone, AGN
191183, AGN 192869, or some combination of AGN 192869 and 191183.
Doses of the respective compounds are given in Table 7. Treatments
are applied to the dorsal skin in a total volume of 4 ml/kg
(.about.0.1 ml). Mice were observed daily and scored for flaking
and abrasions up to and including 3 days after the last treatment,
i.e., day 8.
7TABLE 7 Experimental Design and Results, Example 1 Dose Dose Molar
Ratio Topical AGN 191183 AGN 192869 (192869: Irritation Group
(mg/kg/d) (mg/kg/d) (191183 Score) A 0 0 -- 0 .+-. 0 B 0.025 0 -- 8
.+-. 2 C 0.025 0.06 2:1 5 .+-. 2 D 0.025 0.30 10:1 2 .+-. 1 E 0.025
1.5 50:1 1 .+-. 0 F 0 1.5 -- 0 .+-. 0
[0138] The topical irritation scores for Example 1 are given in
Table 7. Neither acetone (vehicle) nor AGN 192869 (antagonist) at a
dose of 1.5 mg/kg/d (group F) caused observable topical irritation.
AGN 191183, the RAR agonist, caused modest topical irritation at a
dose of 0.025 mg/kg/d. However, AGN 191183-induced topical
irritation was inhibited in a dose-dependent fashion by AGN 192869,
with nearly complete abrogation of irritation in the presence of a
50-fold molar excess of AGN 192869. This demonstrates that a
topical RAR antagonist blocks skin irritation caused by a topical
RAR agonist. Complete blockade of RAR agonist-induced skin
irritation can be achieved with lower molar ratios of antagonist to
agonist when the RAR antagonists is more potent, such as the
compound
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl]be-
nzoic acid (AGN 193109, also designated in this application as
Compound 60.)
EXAMPLE 2
[0139] Skin Iritation Induced by Orally Applied Agonist is Blocked
with Topically Applied Antagonist
[0140] The potent RAR agonist AGN 191183
(4-[(E)-2-(5,6,7,8-tetrahydro-5,5-
,8,8-tetramethylnaphthalen-2-yl)propen-1-yl]benzoic acid) and the
potent RAR antagonist
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthal-
enyl)ethynyl]benzoic acid (AGN 193109, Compound 60) were used in
this example and body weight of the experimental animals (mice) was
used as a marker of systemic RAR agonist exposure.
[0141] Groups of female hairless mice (8-12 weeks old, n=6) were
treated by intragastric intubation with corn oil or AGN 191183
(0.26 mg/kg) suspended in corn oil (5 ml/kg). Mice were
simultaneously treated topically on the dorsal skin with vehicle
(97.6% acetone/2.4% dimethylsulfoxide) or solutions of AGN 193109
in vehicle (6 ml/kg). Specific doses for the different treatment
groups are give in Table 8. Treatments were administered daily for
4 consecutive days. Mice were weighed and graded for topical
irritation daily as described in Example 1 up to and including 1
day after the last treatment. Percent body weight change is
calculated by subtracting final body weight (day 5) from initial
body weight (day 1), dividing by initial body weight, and
multiplying by 100%. Topical irritation scores are calculated as
described in Example 1.
[0142] Topical irritation scores and weight loss for the different
groups are given in Table 8. Combined treatment with the topical
and oral vehicles, i.e., acetone and corn oil, respectively, caused
no topical irritation or weight loss. Similarly, combined treatment
with the oral vehicle and the topical antagonist AGN 193109
resulted in no topical irritation or weight loss. Oral AGN 191183
by itself induced substantial weight loss and skin irritation. AGN
191183-induced skin irritation was substantially reduced when
combined with the lower dose of AGN 193109 and completely blocked
at the higher dose of AGN 193109. AGN 191183-induced weight loss
was also blocked in a dose-related fashion by topical AGN 193109,
but the blockade was not complete. Thus, topical AGN 193109
preferentially blocked the dermal toxicity of AGN 191183.
Presumably, low levels of AGN 193109 were absorbed systemically and
thus partially blocked the weight loss induced by AGN 191183.
However, such absorption would likely be even less in a species
with less permeable skin, such as humans. Alternatively, the weight
loss inhibition by AGN 193109 could be due to amelioration of the
AGN 191183 induced skin irritation.
8TABLE 8 Experimental Design and Results, Example 2 Dose of Topical
Dose of Oral % Weight Topical AGN 193109 AGN 191183 Gain or
Irritation Group (mg/kg/d) (mg/kg/d) (Loss) Score) A 0 0 1 .+-. 2 0
.+-. 0 B 0 0.26 (21 .+-. 6) 8 .+-. 1 C 0.12 0.26 (9 .+-. 5) 1 .+-.
1 D 0.47 0.26 (3 .+-. 5) 0 .+-. 1 E 0.47 0 3 .+-. 3 0 .+-. 0
[0143] Thus, Example 2 demonstrates that RAR antagonists
administered topically can be used to block preferentially the skin
irritation induced by an RAR agonist administered orally.
EXAMPLE 3
[0144] Topically Applied Antagonist Accelarates Recovery From
Prexisting Retinoid Toxicity
[0145] In this example, weight loss is induced by topical treatment
with the RAR agonist AGN 191183 and then the test animals are
topically treated with either vehicle or the RAR antagonist AGN
193109.
[0146] Female hairless mice (8-12 weeks old, n=5) were treated
topically with AGN 191183 (0.13 mg/kg/d) in vehicle (97.6%
acetone/2.4% DMSO, 4 ml/kg) daily for 2 days. Groups of these same
mice (n=5) were then treated topically either with vehicle or AGN
193109 in vehicle (4 ml/kg) daily for 3 consecutive days beginning
on day 3. Mice were weighed on days 1-5 and on day 8. Body weights
are expressed as the mean .+-.SD. Means were compared statistically
using an unpaired, two-tailed t-test. Differences were considered
significant at P<0.05.
9TABLE 9 Results, Example 3 Treatment Body Weight (g) (days 3-5)
DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 8 vehicle 24.6 .+-. 23.9 .+-.
21.4 .+-. 20.3 .+-. 21.0 .+-. 24.7 .+-. 1.5 1.2 1.2 1.7 1.4 1.0 AGN
193109 23.9 .+-. 23.5 .+-. 21.4 .+-. 22.2 .+-. 22.8 .+-. 25.0 .+-.
1.0 1.2 0.6 0.7 0.8 1.1
[0147] The time course of body weights in Example 3 are given in
Table 9. Body weights of both groups of mice were lowered in
parallel on days 2 and 3 as a result of AGN 191183 treatment on
days 1 and 2. Body weights in the two groups were not significantly
different on days 1, 2, or 3. However, AGN 193109 treatment
significantly increased body weight relative to vehicle treatment
on days 4 and 5. These data indicated that recovery from AGN
191183-induced body weight loss was accelerated by subsequent
treatment with AGN 193109. Body weights were not significantly
different between the two groups of mice on day 8, indicating that
full recovery was achievable in both groups given sufficient time.
Thus, RAR antagonists are effective in alleviating RAR
agonist-induced toxicity even if RAR agonist-induced toxicity
precedes RAR antagonist treatment, i.e., in the RAR agonist
poisoning scenario.
EXAMPLE 4
[0148] Orally Applied Antagonist Blocks Hypertriglyceridemia
Incuded by Orally Coadministered Retinoid Agonist
[0149]
5-[(E)-2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethylnaphthalen-2-yl)p-
ropen-1-yl]-2-thiophencarboxylic acid, is a known RAR/RXR pan-
agonist (see U.S. Pat. No. 5,324,840 column 32) and is designated
AGN 191659. This compound was used orally to induce acute
hypertriglyceridemia in rats, and AGN 193109 Compound 60 was
coadministered orally to block the AGN 191659-induced
hypertriglyceridemia.
[0150] Male Fischer rats (6-7 weeks old, n=5) were treated by
intragastric intubation with corn oil (vehicle), AGN 191659, AGN
193109 or a combination of AGN 191659 and AGN 193109. AGN 191659
and AGN 193109 were given as fine suspensions in corn oil. The
experimental design, including doses, is given in Table 10.
[0151] Blood was withdrawn from the inferior vena cava under carbon
dioxide narcosis. Serum was separated from blood by low speed
centrifugation. Total serum triglycerides (triglycerides plus
glycerol) were measured with a standard spectrophotometric endpoint
assay available commercially as a kit and adapted to a 96-well
plate format. Serum triglyceride levels are expressed as the
mean.+-.SD. Means were compared statistically by one-way analysis
of variance followed by Dunnett's test if significant differences
were found. Differences were considered significant at
P<0.05.
[0152] As shown in Table 10, AGN 191659 by itself caused
significant elevation of serum triglycerides relative to vehicle
treatment. AGN 193109 by itself did not significantly increase
serum triglycerides. Importantly, the combination of AGN 193109 and
AGN 191659 at molar ratios of 1:1 and 5:1 reduced serum
triglycerides to levels that were not signficantly different from
control.
10TABLE 10 Experimental Design and Results, Example 4 Group
Treatment (dose) Serum Triglycerides (mg/dl) A vehicle 55.0 .+-.
3.1 B AGN 193109 (19.6 mg/kg) 52.4 .+-. 6.3 C AGN 191659 (3.7
mg/kg) 122.5 .+-. 27.6 D AGN 193109 (3.9 mg/kg) + 55.7 .+-. 14.7
AGN 191659 (3.7 mg/kg) E AGN 193109 (19.6 mg/kg) + 72.7 .+-. 8.9
AGN 191659 (3.7 mg/kg)
[0153] EXAMPLE 4 demonstrates that an RAR antagonist can be used to
block hypertriglyceridemia induced by a coadministered
retinoid.
EXAMPLE 5
[0154] Parenterally Applied Antagonist Blocks Bone Toxicity Incuded
by Parenterally Coadministered Retinoid Agonist
[0155] Example 5 demonstrates that RAR antagonists can block bone
toxicity induced by an RAR agonist. In this example, AGN 193109 is
used to block premature epiphyseal plate closure caused by a
coadministered RAR agonist, AGN 191183, in guinea pigs.
[0156] Groups of male Hartley guinea pigs (3 weeks old, n=4) were
implanted intraperitoneally with osmotic pumps containing vehicle
(20% dimethylsulfoxide/80% polyethylene glycol-300), AGN 191183
(0.06 mg/ml), or AGN 191183 (0.06 mg/ml) in combination with AGN
193109 (0.34 mg/ml). The osmotic pumps are designed by the
manufacturer to deliver .about.5 .mu.l of solution per hour
continuously for 14 days.
[0157] The animals were euthanized by carbon dioxide asphyxiation
14 days after implantation. The left tibia was was removed and
placed in 10% buffered formalin. The tibias were decalcified by
exposure to a formic acid/formalin solution for 3-4 days, and
paraffin sections were prepared. Sections were stained with
hematoxylin and eosin by standard methods. The proximal tibial
epiphyseal plate was examined and scored as closed or not closed.
Epiphyseal plate closure is defined for this purpose as any
interruption of the continuity of the epiphyseal growth plate
cartilage, i.e., replacement by bone and/or fibroblastic
tissue.
[0158] None of the four vehicle-treated guinea pigs showed
epiphyseal plate closure by the end of the experiment. This was
expected, since the proximal epiphyseal plate of guinea pig tibia
does not normally close until the animals are at least 10 months
old. All four of the AGN 191183-treated guinea pigs showed partial
or complete epiphyseal plate closure. However, none of the guinea
pigs treated with the combination of AGN 191183 and AGN 193109
demonstrated epiphyseal plate closure. Thus, AGN 193109 at a 5-fold
molar excess completely blocked AGN 191183-induced bone toxicity
when these compounds were coadministered parenterally.
[0159] RAR Antagonist Compounds
[0160] The compounds
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-nap-
hthalenyl)ethynyl]benzoic acid (AGN 193109, Compound 60) and
4-[(5,6-dihydro-5,5-dimethyl-8-(phenyl)-2-naphthalenyl)ethynyl]benzoic
acid (AGN 192869, Compound 60a) are examples of RAR antagonists
which were used in the above-described animal tests for blocking
RAR receptors in accordance with the present invention. The
compounds of the following formula (Formula 1) serve as further and
general examples for additional RAR antagonist compounds for use in
accordance with the present invention. 5
[0161] In Formula 1, X is S, O, NR' where R' is H or alkyl of 1 to
6 carbons, or
[0162] X is [C(R.sub.1).sub.2].sub.n where R.sub.1 is H or alkyl of
1 to 6 carbons, and n is an integer between 0 or 1;
[0163] R.sub.2 is hydrogen, lower alkyl of 1 to 6 carbons, F,
CF.sub.3, fluor substituted alkyl of 1 to 6 carbons, OH, SH, alkoxy
of 1 to 6 carbons, or alkylthio of 1 to 6 carbons;
[0164] R.sub.3 is hydrogen, lower alkyl of 1 to 6 carbons or F;
[0165] m is an integer having the value of 0-3;
[0166] m is an integer having the value of 0-3;
[0167] Z is --C.ident.C--,
[0168] --N.dbd.N--,
[0169] --N.dbd.CR.sub.1--,
[0170] --CR.sub.1.dbd.N,
[0171] --(CR.sub.1.dbd.CR.sub.1).sub.n'-- where n' is an integer
having the value 0-5,
[0172] --CO--NR.sub.1--,
[0173] --CS--NR.sub.1--,
[0174] --NR.sub.1--CO,
[0175] --NR.sub.1--CS,
[0176] --COO--,
[0177] --OCO--;
[0178] --CSO--;
[0179] --OCS--;
[0180] Y is a phenyl or naphthyl group, or heteroaryl selected from
a group consisting of pyridyl, thienyl, furyl, pyridazinyl,
pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl, imidazolyl and
pyrrazolyl, said phenyl and heteroaryl groups being optionally
substituted with one or two R.sub.2 groups, or
[0181] when Z is --(CR.sub.1.dbd.CR.sub.1).sub.n'-- and n' is 3, 4
or 5 then Y represents a direct valence bond between said
(CR.sub.2.dbd.CR.sub.2).sub.n' group and B;
[0182] A is (CH.sub.2).sub.q where q is 0-5, lower branched chain
alkyl having 3-6 carbons, cycloalkyl having 3-6 carbons, alkenyl
having 2-6 carbons and 1 or 2 double bonds, alkynyl having 2-6
carbons and 1 or 2 triple bonds;
[0183] B is hydrogen, COOH or a pharmaceutically acceptable salt
thereof, COOR.sub.8, CONR.sub.9R.sub.10, --CH.sub.2OH,
CH.sub.2OR.sub.11, CH.sub.2OCOR.sub.11, CHO, CH(OR.sub.12).sub.2,
CHOR.sub.13O, --COR.sub.7, CR.sub.7(OR.sub.12).sub.2,
CR.sub.7OR.sub.13O, or tri-lower alkylsilyl, where R.sub.7 is an
alkyl, cycloalkyl or alkenyl group containing 1 to 5 carbons,
R.sub.8 is an alkyl group of 1 to 10 carbons or trimethylsilylalkyl
where the alkyl group has 1 to 10 carbons, or a cycloalkyl group of
5 to 10 carbons, or R.sub.8 is phenyl or lower alkylphenyl, R.sub.9
and R.sub.10 independently are hydrogen, an alkyl group of 1 to 10
carbons, or a cycloalkyl group of 5-10 carbons, or phenyl or lower
alkylphenyl, R.sub.11 is lower alkyl, phenyl or lower alkylphenyl,
R.sub.12 is lower alkyl, and R.sub.13 is divalent alkyl radical of
2-5 carbons, and
[0184] R.sub.14 is (R.sub.15).sub.r-phenyl,
(R.sub.15).sub.r-naphthyl, or (R.sub.15).sub.r-heteroaryl where the
heteroaryl group has 1 to 3 heteroatoms selected from the group
consisting of O, S and N, r is an integer having the values of 0-5,
and
[0185] R.sub.15 is independently H, F, Cl, Br, I, NO.sub.2,
N(R.sub.8).sub.2, N(R.sub.8)COR.sub.8, NR.sub.8CON(R.sub.8).sub.2,
OH, OCOR.sub.8, OR.sub.8, CN, an alkyl group having 1 to 10
carbons, fluoro substituted alkyl group having 1 to 10 carbons, an
alkenyl group having 1 to 10 carbons and 1 to 3 double bonds,
alkynyl group having 1 to 10 carbons and 1 to 3 triple bonds, or a
trialkylsilyl or trialkylsilyloxy group where the alkyl groups
independently have 1 to 6 carbons.
[0186] Synthetic Methods--Aryl Substituted Compounds
[0187] The exemplary RAR antagonist compounds of Formula 1 can be
made by the synthetic chemical pathways illustrated here. The
synthetic chemist will readily appreciate that the conditions set
out here are specific embodiments which can be generalized to any
and all of the compounds represented by Formula 1. 6
[0188] Reaction Scheme 1 illustrates the synthesis of compounds of
Formula 1 where the Z group is an ethynyl function (--C.ident.C--)
and X is [C(R.sub.1).sub.2].sub.n where n is 1. In other words,
Reaction Scheme 1 illustrates the synthesis of ethynyl substituted
dihydronaphthalene derivatives of the present invention. In
accordance with this scheme, a tetrahydronaphtalene-1-one compound
of Formula 6 is brominated to provide the bromo derivative of
Formula 7. The compounds of Formula 6 already carry the desired
R.sub.1, R.sub.2 and R.sub.3 substituents, as these are defined
above in connection with Formula 1. A preferred example of a
compound of Formula 6 is
3,4-dihydro-4,4-dimethyl-1(2H)-naphthalenone, which is described in
the chemical literature (Arnold et al. J. Am. Chem. Soc.
69:2322-2325 (1947)). A presently preferred route for the synthesis
of this compound from 1-bromo-3-phenylpropane is also described in
the experimental section of the present application.
[0189] The compounds of Formula 7 are then reacted with
(trimethylsilyl)acetylene to provide the (trimethylsilyl)ethynyl-
substituted 3,4-dihydro-naphthalen-1(2H)-one compounds of Formula
8. The reaction with (trimethylsilyl)acetylene is typically
conducted under heat (approximately 100.degree. C.) in the presence
of cuprous iodide, a suitable catalyst, typically having the
formula Pd(PPh.sub.3).sub.2Cl.sub- .2, an acid acceptor (such as
triethylamine) under an inert gas (argon) atmosphere. Typical
reaction time is approximately 24 hours. The
(trimethylsilyl)ethynyl-substituted
3,4-dihydro-naphthalen-1(2H)-one compounds of Formula 8 are then
reacted with base (potassium hydroxide or potassium carbonate) in
an alcoholic solvent, such as methanol, to provide the ethynyl
substituted 3,4-dihydro-1-naphthalen-1(2H)ones of Formula 9.
Compounds of Formula 9 are then coupled with the aromatic or
heteroaromatic reagent X.sub.1--Y(R.sub.2)-A-B' (Formula 10) in the
presence of cuprous iodide, a suitable catalyst, typically
Pd(PPh.sub.3).sub.2Cl.sub.2, an acid acceptor, such as
triethylamine, under inert gas (argon) atmosphere. Alternatively, a
zinc salt (or other suitable metal salt) of the compounds of
Formula 9 can be coupled with the reagents of Formula 10 in the
presence of Pd(PPh.sub.3).sub.4 or similar complex. Typically, the
coupling reaction with the reagent X.sub.1-Y(R.sub.2)-A-B' (Formula
10) is conducted at room or moderately elevated temperature.
Generally speaking, coupling between an ethynylaryl derivative or
its zinc salt and a halogen substituted aryl or heteroaryl
compound, such as the reagent of Formula 10, is described in U.S.
Pat. No. 5,264,456, the specification of which is expressly
incorporated herein by reference. The compounds of Formula 11 are
precursors to exemplary compounds of the present invention, or
derivatives thereof protected in the B' group, from which the
protecting group can be readily removed by reactions well known in
the art. The compounds of Formula 11 can also be converted into
further precursors to the exemplary compounds by such reactions and
transformations which are well known in the art. Such reactions are
indicated in Reaction Scheme 1 by conversion into "homologs and
derivatives". One such conversion employed for the synthesis of
several exemplary compounds is saponification of an ester group
(when B or B' is an ester) to provide the free carboxylic acid or
its salt.
[0190] The halogen substituted aryl or heteroaryl compounds of
Formula 10 can, generally speaking, be obtained by reactions well
known in the art. An example of such compound is ethyl
4-iodobenzoate which is obtainable, for example, by esterification
of 4-iodobenzoic acid. Another example is ethyl 6-iodonicotinoate
which can be obtained by conducting a halogen exchange reaction on
6-chloronicotinic acid, followed by esterification. Even more
generally speaking, regarding derivatization of compounds of
Formula 11 and/or the synthesis of aryl and heteroaryl compounds of
Formula 10 which can thereafter be reacted with compounds of
Formula 9, the following well known and published general
principles and synthetic methodology can be employed.
[0191] Carboxylic acids are typically esterified by refluxing the
acid in a solution of the appropriate alcohol in the presence of an
acid catalyst such as hydrogen chloride or thionyl chloride.
Alternatively, the carboxylic acid can be condensed with the
appropriate alcohol in the presence of dicyclohexylcarbodiimide and
dimethylaminopyridine. The ester is recovered and purified by
conventional means. Acetals and ketals are readily made by the
method described in March, Advanced Organic Chemistry 2nd Edition,
McGraw-Hill Book Company, p. 810). Alcohols, aldehydes and ketones
all may be protected by forming respectively, ethers and esters,
acetals or ketals by known methods such as those described in
McOmie, Plenum Publishing Press, 1973 and Protecting Groups, Ed.
Greene, John Wiley & Sons, 1981.
[0192] To increase the value of n in the compounds of Formula 10
before affecting the coupling reaction of Reaction Scheme 1 (where
such compounds corresponding to Formula 10 are not available from a
commercial source) aromatic or heteroaromatic carboxylic acids are
subjected to homologation by successive treatment under
Arndt-Eistert conditions or other homologation procedures.
Alternatively, derivatives which are not carboxylic acids may also
be homologated by appropriate procedures. The homologated acids can
then be esterified by the general procedure outlined in the
preceding paragraph.
[0193] Compounds of Formula 10, (or other intermediates or
exemplary compounds) where A is an alkenyl group having one or more
double bonds can be made for example, by synthetic schemes well
known to the practicing organic chemist; for example by Wittig and
like reactions, or by introduction of a double bond by elimination
of halogen from an alpha-halo-arylalkyl-carboxylic acid, ester or
like carboxaldehyde. Compounds of Formula 10 (or other
intermediates or exemplary compounds) where the A group has a
triple (acetylenic) bond can be made by reaction of a corresponding
aromatic methyl ketone with strong base, such as lithium
diisopropylamide, reaction with diethyl chlorophosphate and
subsequent addition of lithium diisopropylamide.
[0194] The acids and salts derived from compounds of Formula 11 (or
other intermediates or exemplary compounds) are readily obtainable
from the corresponding esters. Basic saponification with an alkali
metal base will provide the acid. For example, an ester of Formula
11 (or other intermediates or exemplary compounds) may be dissolved
in a polar solvent such as an alkanol, preferably under an inert
atmosphere at room temperature, with about a three molar excess of
base, for example, lithium hydroxide or potassium hydroxide. The
solution is stirred for an extended period of time, between 15 and
20 hours, cooled, acidified and the hydrolysate recovered by
conventional means.
[0195] The amide may be formed by any appropriate amidation means
known in the art from the corresponding esters or carboxylic acids.
One way to prepare such compounds is to convert an acid to an acid
chloride and then treat that compound with ammonium hydroxide or an
appropriate amine.
[0196] Alcohols are made by converting the corresponding acids to
the acid chloride with thionyl chloride or other means (J. March,
Advanced Organic Chemistry 2nd Edition, McGraw-Hill Book Company),
then reducing the acid chloride with sodium borohydride (March,
Ibid, p. 1124), which gives the corresponding alcohols.
Alternatively, esters may be reduced with lithium aluminum hydride
at reduced temperatures. Alkylating these alcohols with appropriate
alky halides under Williamson reaction conditions (March, Ibid, p.
357) gives the corresponding ethers. These alcohols can be
converted to esters by reacting them with appropriate acids in the
presence of acid catalysts or dicyclohexylcarbodiimide and
dimethylaminopyridine.
[0197] Aldehydes can be prepared from the corresponding primary
alcohols using mild oxidizing agents such as pyridinium dichromate
in methylene chloride (Corey, E. J., Schmidt, G., Tet. Lett. 399,
1979), or dimethyl sulfoxide/oxalyl chloride in methylene chloride
(Omura, K., Swern, D., Tetrahedron 34:1651 (1978)).
[0198] Ketones can be prepared from an appropriate aldehyde by
treating the aldehyde with an alkyl Grignard reagent or similar
reagent followed by oxidation.
[0199] Acetals or ketals can be prepared from the corresponding
aldehyde or ketone by the method described in March, Ibid, p.
810.
[0200] Compounds of Formula 10 (or other intermediates, or
exemplary compounds) where B is H can be prepared from the
corresponding halogenated aromatic or hetero aromatic compounds,
preferably where the halogen is I.
[0201] Referring back again to Reaction Scheme 1, the compounds of
Formula 11 are reacted with sodium bis(trimethylsilyl)amide and
2-[N,N-bis(trifluoromethylsulfonyl)amino]-5-chloropyridine in an
inert ether type solvent, such as tetrahydrofuran, at low
temperatures (-78.degree. C. and 0.degree. C.). This is shown in
Reaction Scheme 1 where the usually unisolated sodium salt
intermediate is shown in brackets as Formula 12. The reaction
results in the trifluoromethylsulfonyloxy derivatives represented
in Formula 13. (Tf=SO.sub.2CF.sub.3). The compounds of Formula 13
are then converted to the exemplary compounds of the invention,
shown in Formula 14, by reaction with an organometal derivative
derived from the aryl or heteroaryl compound R.sub.14H, such that
the formula of the organometal derivative is R.sub.14Met (Met
stands for monovalent metal), preferably R.sub.14Li. (R.sub.14 is
defined as in connection with Formula 1.) The reaction with the
organometal derivative, preferably lithium derivative of the
formula R.sub.14Li is usually conducted in an inert ether type
solvent (such as tetrahydrofuran) in the presence of zinc chloride
(ZnCl.sub.2) and tetrakis(triphenylphosphine)-palladium(0)
(Pd(PPh.sub.3).sub.4). The organolithium reagent R.sub.14Li, if not
commercially available, can be prepared from the compound R.sub.14H
(or its halogen derivative R.sub.14--X.sub.1 where X.sub.1 is
halogen) in an ether type solvent in accordance with known practice
in the art. The temperature range for the reaction between the
reagent R.sub.14Li and the compounds of Formula 13 is, generally
speaking in the range of approximately -78.degree. C. to 50.degree.
C. The compounds of Formula 14 can be converted into further
homologs and derivatives in accordance with the reactions discussed
above.
[0202] The intermediate 7-bromo-tetrahydronaphthalene-1-one
compounds of Formula 7 shown in Reaction Scheme 1 can also be
converted with a Grignard reagent of the formula R.sub.14MgBr
(R.sub.14 is defined as in connection with Formula 1) to yield the
tertiary alcohol of Formula 15. The tertiary alcohol is dehydrated
by treatment with acid to provide the
3,4-dihydro-7-bromonaphthalene derivatives of Formula 16, which
serve as intermediates for the synthesis of additional compounds of
the present invention (see Reaction Schemes 6, 7, and 8). 7
[0203] Referring now to Reaction Scheme 2 a synthetic route to
those compounds is disclosed where with reference to Formula 1 X is
S, O or NR' and the Z group is an ethynyl function (--C.ident.C--).
Starting material for this sequence of the reaction is a
bromophenol, bromothiophenol or bromoaniline of the structure shown
in Formula 17. For the sake of simplifying the present
specification, in the ensuing description X can be considered
primarily sulfur as for the preparation of benzothiopyran
derivatives. It should be kept in mind, however, that the herein
described scheme is also suitable, with such modifications which
will be readily apparent to those skilled in the art, for the
preparation of benzopyran (X=O) and dihydroquinoline (X=NR')
compounds of the present invention. Thus, the compound of Formula
17, preferably para bromothiophenol, para bromophenol or para
bromoaniline is reacted under basic condition with a 3-bromo
carboxylic acid of the Formula 18. In this reaction scheme the
symbols have the meaning described in connection with Formula 1. An
example for the reagent of Formula 18 where R.sub.3 is hydrogen, is
3-bromopropionic acid. The reaction with the 3-bromocarboxylic acid
of Formula 18 results in the compound of Formula 19. The latter is
cyclized by treatment with acid to yield the
6-bromothiochroman-4-one derivative (when X is S) or 6-bromochroman
derivative (when X is O) of Formula 20. The bromo compounds of
Formula 20 are then subjected to substantially the same sequence of
reactions under analogous conditions, which are described in
Reaction Scheme 1 for the conversion of the bromo compounds of
Formula 7 to the compounds of the invention. Thus, briefly
summarized here, the bromo compounds of Formula 20 are reacted with
(trimethylsilyl)acetylene to provide the
6-(trimethylsilyl)ethynyl-substituted thiochroman-4-one or
chroman-4-one compounds of Formula 21. The
6-(trimethylsilyl)ethynyl-substituted thiochroman-4-one compounds
of Formula 21 are then reacted with base (potassium hydroxide or
potassium carbonate) to provide the ethynyl substituted 6-ethynyl
substituted thiochroman-4-ones of Formula 22. Compounds of Formula
22 are then coupled with the aromatic or heteroaromatic reagent
X.sub.1--Y(R.sub.2)-A-B' (Formula 10) under conditions analogous to
those described for the analogous reactions of Reaction Scheme 1,
to yield the compounds of Formula 23.
[0204] The compounds of Formula 23 are then reacted still under
conditions analogous to the similar reactions described in Reaction
Scheme 1 with sodium bis(trimethylsilyl)amide and
2-[N,N-bis(trifluoromethylsulfonyl)am- ino]-5-chloropyridine to
yield the 4-trifluoromethylsulfonyloxy benzothiopyran or benzopyran
derivatives represented in Formula 24. The compounds of Formula 24
are then converted to compounds shown in Formula 25, by reaction
with an organometal derivative derived from the aryl or heteroaryl
compound R.sub.14H, as described in connection with Reaction Scheme
1.
[0205] Similarly to the use of the intermediate
7-bromo-tetrahydronaphthal- ene-1-one compounds of Formula 7 of
Reaction Scheme 1, the intermediate 6-bromothiochroman-4-one
compounds of Formula 20 can also be used for the preparation of
further compounds within the scope of the present invention, as
described below, in Reaction Schemes 6, 7 and 8. The compounds of
Formula 25, can also be converted into further homologs and
derivatives, in reactions analogous to those described in
connection with Reaction Scheme 1. 8
[0206] Reaction Scheme 3 discloses a synthetic route to compounds
where, with reference to Formula 1, X is [C(R.sub.1).sub.2].sub.n,
n is 0 and the Z group is an ethynyl function (--C.ident.C--). In
accordance with this scheme, a 6-bromo-2,3-dihydro-1H-inden-1-one
derivative of Formula 26 is reacted in a sequence of reactions
(starting with reaction with trimethylsilylacetylene) which are
analogous to the reactions described above in connection with
Reaction Schemes 1 and 2, to provide, through intermediates of the
formulas 27-30, the indene derivatives of Formula 31. In a
preferred embodiment within the scope of Reaction Scheme 3, the
starting material is
6-bromo-2,3-dihydro-3,3-dimethyl-1H-inden-1-one that is available
in accordance with the chemical literature (See Smith et al. Org.
Prep. Proced. Int. 1978 10, 123-131). Compounds of Formula 26, such
as 6-bromo-2,3-dihydro-3,3-dimethyl-1H-inden-1-one, can also be
used for the synthesis of still further exemplary compounds for use
in the present invention, as described below. 9
[0207] Referring now to Reaction Scheme 4 a synthetic route to
exemplary compounds is disclosed where, with reference to Formula
1, Z is --(CR.sub.1.dbd.CR.sub.1).sub.n'--, n' is 3, 4 or 5 and Y
represents a direct valence bond between the
(CR.sub.1.dbd.CR.sub.1).sub.n, group and B. This synthetic route is
described for examples where the X group is
[C(R.sub.1).sub.2].sub.n and n is 1 (dihydronaphthalene
derivatives). Nevertheless, it should be understood that the
reactions and synthetic methodology described in Reaction Scheme 4
and further ensuing schemes, is also applicable, with such
modifications which will be readily apparent to those skilled in
the art, to derivatives where X is is S, O, NR' (benzothiopyran,
benzopyran or dihydroquinoline derivatives) or
[C(R.sub.1).sub.2].sub.n and n is 0 (indene derivatives).
[0208] In accordance with Reaction Scheme 4, a
1,2,3,4-tetrahydronaphthale- ne derivative of Formula 32 is reacted
with an acid chloride (R.sub.1COCl) under Friedel Crafts
conditions, and the resulting acetylated product is oxidized, for
example in a Jones oxidation reaction, to yield a mixture of
isomeric 6- and 7-acetyl-1(2H)-naphthalenone derivatives of Formula
33. In a specific preferred example of this reaction, the starting
compound of Formula 32 is
1,2,3,4-tetrahydro-1,1-dimethylnaphthalene (a known compound) which
can be prepared in accordance with a process described in the
experimental section of the present application. The
7-acetyl-1(2H)-naphthalenone derivative of Formula 33 is reacted
with ethylene glycol in the presence of acid to protect the oxo
function of the exocyclic ketone moiety, as a ketal derivative of
Formula 34. The ketal of Formula 34 is thereafter reacted with a
Grignard reagent of the formula R.sub.14MgBr (the symbols are
defined as in connection with Formula 1), to yield the tertiary
alcohol of Formula 35. Thereafter the dioxolane protective group is
removed and the tertiary alcohol is dehydrated by treatment with
acid to provide the 3,4-dihydro-7-acetylnaph- thalene derivatives
of Formula 36. The ketone function of the compounds of Formula 36
is subjeceted to a Homer Emmons (or analogous) reaction under
strongly alkaline conditions with a phosphonate reagent of Formula
37, to yield, after reduction, the aldehyde compounds of Formula
38. Still another Homer Emmons (or analogous) reaction under
strongly alkaline conditions with a reagent of Formula 39 provides
compounds of Formula 40. The latter can be converted into further
homologs and derivatives in accordance with the reactions described
above. A specific example of the Horner Emmons reagent of Formula
37 which is used for the preparation of a preferred compound is
diethylcyanomethylphosphonate; an example of the Homer Emmons
reagent of Formula 39 is diethyl-(E)-3-ethoxycarbonyl-2-meth-
ylallylphosphonate. 10
[0209] Reaction Scheme 5 discloses a synthetic process for
preparing compounds where the Z group is an azo group
(--N.dbd.N--). As in Reaction Scheme 4 this process is described
for examples where the X group is [C(R.sub.1).sub.2].sub.n and n is
1 (dihydronaphthalene derivatives). Nevertheless, it should be
understood that the synthetic methodology described is also
applicable, with such modifications which will be readily apparent
to those skilled in the art, to all azo compounds for use in the
invention, namely to derivatives where X is is S, O, NR'
(benzothiopyran, benzopyran or dihydroquinoline derivatives) or
[C(R.sub.1).sub.2].sub.n and n is 0 (indene derivatives). Thus, a
nitro group is introduced into the starting compound of Formula 6
under substantially standard conditions of nitration, to yield the
3,4-dihydro-7-nitro-1(2H)-naphthalenone derivative of Formula 41.
The latter compound is reduced to the
3,4-dihydro-7-amino-1(2H)-naphthalenone derivative of Formula 42
and is thereafter reacted with a nitroso compound of the formula
ON--Y(R.sub.2)-A-B (Formula 43) under conditions normally employed
(glacial acetic acid) for preparing azo compounds. The nitroso
compound of Formula 43 can be obtained in accordance with reactions
known in the art. A specific example for such compound, which is
used for the synthesis of a preferred compound is ethyl
4-nitrosobenzoate. The azo compound of Formula 44 is thereafter
reacted with sodium bis(trimethylsilyl)amide and
2-[N,N-bis(trifluoromethylsulfon- yl)amino]-5-chloropyridine to
yield the 4-trifluoromethylsulfonyloxy derivatives represented in
Formula 45. The compounds of Formula 45 are then converted to the
azo compounds shown in Formula 46, by reaction with an
organometalic derivative derived from the aryl or heteroaryl
compound R.sub.14H. These latter two reactions, namely the
conversion to the 4-trifluoromethylsulfonyloxy derivatives and
subsequent reaction with the organometal derivative, have been
described above in connection with Reaction Schemes 1, 2 and 3, and
are employed in several presently preferred synthetic processes
leading to exemplary RAR antagonist compounds. 11
[0210] Reaction Scheme 6 discloses a presently preferred synthetic
process for the preparation of compounds where, with reference to
Formula 1, the Z group is COO-- or CONR.sub.1 (R.sub.1 is
preferably H). These ester and amide derivatives are prepared from
the 3,4-dihydro-7-bromonaphthalene derivatives of Formula 16, which
can be obtained as described in Reaction Scheme 1. Thus, the
compounds of Formula 16 are reacted with strong base, such as
t-butyllithium, in an inert ether type solvent, such as
tetrahydrofuiran, at cold temperature, and carbon dioxide
(CO.sub.2) is added to provide the
5,6-dihydro-2-naphthalenecarboxylic acid derivatives of Formula 47.
Compounds of Formula 47 are then reacted with compounds of the
formula X.sub.2-Y(R.sub.2)-A-B (Formula 48) where X.sub.2
reperesent an OH or an NR.sub.1 group, the R.sub.1 preferably being
hydrogen. Those skilled in the art will recognize that the
compounds of Formula 48 are aryl or heteroaryl hydroxy or amino
derivatives which can be obtained in accordance with the
state-of-the-art. The reaction between the compounds of Formula 47
and Formula 48 can be conducted under various known ester or amide
forming conditions, such as coupling of the two in the presence of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and
4-dimethylaminopyridine. Alternatively, the compounds of Formula 47
can be converted into the corresponding acid chloride for coupling
with the compounds of Formula 48 in the presence of base. The amide
or ester compounds of Formula 49 can be converted into further
homologs and derivatives, as described above. Although Reaction
Scheme 6 is described and shown for the example where the X group
of Formula 1 is [C(R.sub.1).sub.2].sub.n and n is 1
(dihydronaphthalene derivatives), the herein described process can
be adapted for the preparation of benzopyran, benzothiopyran,
dihydroquinoline and indene derivatives as well.
[0211] Compounds of the present invention where with reference to
Formula 1, Z is --OCO--, NR.sub.1CO, as well as the corresponding
thioester and thioamide analogs, can be prepared from the
intermediates derived from the compounds of Formula 16 where the
bromo function is replaced with an amino or hydroxyl group and in
accordance with the teachings of U.S. Pat. Nos. 5,324,744, the
specification of which is expressly incorporated herein by
reference. 12
[0212] Reaction Scheme 7 discloses a presently preferred synthetic
process for the preparation of compounds where with reference to
Formula 1, Z is --(CR.sub.1.dbd.CR.sub.1).sub.n'-- and n' is 0.
These compounds of Formula 50 can be obtained in a coupling
reaction between compounds of Formula 16 and a Grignard reagent
derived from the halo compounds of Formula 10. The coupling
reaction is typically conducted in the presence of a zinc salt and
a nickel (Ni(0)) catalyst in inert ether type solvent, such as
tetrahydrofuran. The compounds of Formula 50 can be converted into
further homologs and derivatives, as described above. 13
[0213] Referring now to Reaction Scheme 8 a presently preferred
synthetic process is disclosed for the preparation of compounds
where Z is --(CR.sub.1.dbd.CR.sub.1).sub.n'-- and n' is 1. More
particularly, Reaction Scheme 8 discloses the presently preferred
process for preparing those compounds which are dihydronaphtalene
derivatives and where the Z group represents a vinyl
(--CH.dbd.CH--) function. However, the generic methodology
disclosed herein can be extended, with such modifications which
will be apparent to those skilled in the art, to the analogous
benzopyran, benzothiopyran, dihydroquinoline compounds, and to
compounds where the vinyl group is substituted. Thus, in accordance
with Reaction Scheme 8 the 7-bromo-1(2H)-naphthalenone derivative
of Formula 7 is reacted with a vinyl derivative of the structure
--CH.sub.2.dbd.CH--Y(R.s- ub.2)-A-B (Formula 51) in the presence of
a suitable catalyst, typically having the formula Pd(PPh.sub.3), an
acid acceptor (such as triethylamine) under an inert gas (argon)
atmosphere. The conditions of this reaction are analogous to the
coupling of the acetylene derivatives of Formula 9 with the reagent
of Formula 10 (see for example Reaction Scheme 1), and this type of
reaction is generally known in the art as a Heck reaction. The
vinyl derivative of Formula 51 can be obtained in accordance with
the state of the art, an example for such a reagent used for the
synthesis of a preferred compound to be used in the invention is
ethyl 4-vinylbenzoate.
[0214] The product of the Heck coupling reaction is an ethenyl
derivative of Formula 52, which is thereafter converted into
compounds used in the present invention by treatment with sodium
bis(trimethylsilyl)amide and
2-[N,N-bis(trifluoromethylsulfonyl)amino]-5-chloropyridine to yield
the 4-trifluoromethylsulfonyloxy derivatives of Formula 53, and
subsequent reaction with an organometal derivative derived from the
aryl or heteroaryl compound R.sub.14H, as described above. The
resulting compounds of Formula 54 can be converted into further
homologs and derivatives.
[0215] The compounds of Formula 54 can also be obtained through
synthetic schemes which employ a Wittig or Horner Emmons reaction.
For example, the intermediate of Formula 33 (see Reaction Scheme 4)
can be reacted with a triphenylphosphonium bromide (Wittig) reagent
or more preferably with a diethylphosphonate (Homer Emmons) reagent
of the structure (EtO).sub.2PO--CH.sub.2--Y(R.sub.2)-A-B, as
described for analogous Homer Emmons reactions in U.S. Pat. No.
5,324,840, the specification of which is incorporated herein by
reference. The just mentioned Homer Emmons reaction provides
intermediate compounds analogous in structure to Formula 52, and
can be converted into compounds of Formula 54 by the sequence of
reactions described in Reaction Scheme 8 for the compounds of
Formula 52.
[0216] Synthetic Methods--Aryl and (3-Oxy-1-Propenyl)-Substituted
Compounds
[0217] The exemplary RAR antagonist compounds of Formula 101 can be
made by the synthetic chemical pathways illustrated here. The
synthetic chemist will readily appreciate that the conditions set
out here are specific embodiments which can be generalized to any
and all of the compounds represented by Formula 101. 14
[0218] Reaction Scheme 101 illustrates the synthesis of compounds
of Formula 101 where X is [C(R.sub.1).sub.2].sub.n, n is 1, p is
zero and R.sub.17 is H or lower alkyl. In other words, Reaction
Scheme 101 illustrates the synthesis of compounds of the invention
which are 3,4-dihydronaphthalene derivatives. In accordance with
this scheme, a tetrahydronaphthalene compound of Formula 103 which
is appropriately substituted with the R.sub.3 and R.sub.2 groups
(as these are defined in connection with Formula 101) serves as the
starting material. A preferred example of a compound of Formula 103
is 1,3,3,4-tetrahydro-1,1-dimethyl-n- aphthalene, which is
described in the chemical literature (Mathur et al. Tetrahedron,
1985, 41:1509. A presently preferred route for the synthesis of
this compound from 1-bromo-3-phenylpropane is also described in the
experimental section of the present application.
[0219] The compound of Formula 103 is reacted in a Friedel Crafts
type reaction with an acid chloride having the structure
R.sub.16CH.sub.2COCl (R.sub.16 is defined as in connection with
Formula 101) and is thereafter oxidized with chromium trioxide in
acetic acid to provide the isomeric 6 and 7
acyl-3,4-dihydro-1(2H)-naphthalenone derivatives. Only the 6-acyl
derivative which is of interest from the standpoint of the present
invention, is shown by structural formula (Formula 104) in Reaction
Scheme 101. In the preparation of the presently preferred compounds
of this invention the R, groups represent methyl, R.sub.2, R.sub.3
and R.sub.16 are H, and therefore the preferred intermediate
corresponding to Formula 104 is
3,4-dihydro-4,4-dimethyl-6-acetyl-1(2H)-naphthalenone.
[0220] The exocyclic ketone function of the compound of Formula 104
is thereafter protected as a ketal, for example by treatment with
ethylene glycol in acid, to provide the 1,3-dioxolanyl derivative
of Formula 105. The compound of Formula 105 is then reacted with a
Grignard reagent of the formula R.sub.14MgBr (R.sub.14 is defined
as in connection with Formula 101) to give the
1,2,3,4-tetrahydro-1-hydroxy-naphthalene derivative of Formula 106.
The exocyclic ketone function of the compound of Formula 106 is
then deprotected by treatment with acid and dehydrated to give the
compound of Formula 107.
[0221] An alternate method for obtaining the compounds of Formula
107 from the compounds of Formula 105 is by reacting the compounds
of Formula 105 with sodium bis(trimethylsilyl)amide and
2-[N,N-bis(trifluoromethylsulfon- yl)amino]-5-chloropyridine
(Tf=SO.sub.2CF.sub.3) in an inert ether type solvent, such as
tetrahydrofuran, at low temperatures (-78.degree. C. and 0.degree.
C.). This reaction proceeds through a sodium salt intermediate
which is usually not isolated and is not shown in Reaction Scheme
101. The overall reaction results in a trifluoromethylsulfonyloxy
derivative, which is thereafter reacted with an organometal
derivative derived from the aryl or heteroaryl compound R.sub.14H,
such that the formula of the organometal derivative is R.sub.14Met
(Met stands for monovalent metal), preferably R.sub.14Li, (R.sub.14
is defined as in connection with Formula 101.) The reaction with
the organometal derivative, preferably lithium derivative of the
formula R.sub.14Li is usually conducted in an inert ether type
solvent (such as tetrahydrofuran) in the presence of zinc chloride
(ZnCl.sub.2) and tetrakis(triphenylphosphine)-palladium(0)
(Pd(PPh.sub.3).sub.4). The organolithium reagent R.sub.14Li, if not
commercially available, can be prepared from the compound R.sub.14H
(or its halogen derivative R.sub.14--X.sub.1 where X.sub.1 is
halogen) in an ether type solvent in accordance with known practice
in the art. The temperature range for the reaction between the
reagent R.sub.14Li and the trifluoromethylsulfonyloxy derivative
is, generally speaking, in the range of approximately -78.degree.
C. to 50.degree. C.
[0222] The compounds of the invention are formed as a result of a
condensation between the ketone compound of Formula 107 and an
aldehyde or ketone of Formula 108. In the preparation of the
preferred exemplary compounds of the invention the reagent of
Formula 108 is 4-carboxybenzaldehyde (R.sub.17--H). Examples of
other reagents within the scope of Formula 108 and suitable for the
condensation reaction and for the synthesis of compounds within the
scope of the present invention (Formula 101) are:
5-carboxy-pyridine-2-aldehyde, 4-carboxy-pyridine-2-al- dehyde,
4-carboxy-thiophene-2-aldehyde, 5-carboxy-thiophene-2-aldehyde,
4-carboxy-furan-2-aldehyde, 5-carboxy-furan-2-aldehyde,
4-carboxyacetophenone, 2-acetyl-pyridine-5-carboxylic acid,
2-acetyl-pyridine-4-carboxylic acid,
2-acetyl-thiophene-4-carboxylic acid,
2-acetyl-thiophene-5-carboxylic acid, 2-acetyl-furan-4-carboxylic
acid, and 2-acetyl-furan-5-carboxylic acid. The latter compounds
are available in accordance with the chemical literature; see for
example Decroix et al., J. Chem. Res. (S), 4:134 (1978); Dawson et
al., J. Med. Chem. 29:1282 (1983); and Queguiner et al., Bull Soc.
Chimique de France No. 10, pp. 3678-3683 (1969). The condensation
reaction between the compounds of Formula 107 and Formula 108 is
conducted in the presence of base in an alcoholic solvent.
Preferably, the reaction is conducted in ethanol in the presence of
sodium hydroxide. Those skilled in the art will recognize this
condensation reaction as an aldol condensation, and in case of the
herein described preferred examples (condensing a ketone of Formula
107 with an aldehyde of Formula 108) as a Claisen-Schmidt reaction.
(See March: Advanced Organic Chemistry: Reactions, Mechanisms, and
Structure, pp. 694-695 McGraw Hill (1968). The compounds of Formula
109 are within the scope of the present invention, and can also be
subjected to further transformations resulting in additional
compounds of the invention. Alternatively, the A-B group of Formula
108 may be a group which is within the scope of the invention, as
defined in Formula 101, only after one or more synthetic
transformations of such a nature which is well known and within the
skill of the practicing organic chemist. For example, the reaction
performed on the A-B group may be a deprotection step,
homologation, esterification, saponification, amide formation or
the like.
[0223] Generally speaking, regarding derivatization of compounds of
Formula 109 and/or the synthesis of aryl and heteroaryl compounds
of Formula 108 which can thereafter be reacted with compounds of
Formula 107, the following well known and published general
principles and synthetic methodology can be employed.
[0224] As indicated above, carboxylic acids are typically
esterified by refluxing the acid in a solution of the appropriate
alcohol in the presence of an acid catalyst such as hydrogen
chloride or thionyl chloride. Alternatively, the carboxylic acid
can be condensed with the appropriate alcohol in the presence of
dicyclohexylcarbodiimide and dimethylaminopyridine. The ester is
recovered and purified by conventional means. Acetals and ketals
are readily made by the method described in March, Advanced Organic
Chemistry, 2nd Edition, McGraw-Hill Book Company, p. 810).
Alcohols, aldehydes and ketones all may be protected by forming
respectively, ethers and esters, acetals or ketals by known methods
such as those described in McOmie, Plenum Publishing Press, 1973
and Protecting Groups, Ed. Greene, John Wiley & Sons, 1981.
[0225] To increase the value of n in the compounds of Formula 108
before affecting the condensation reaction of Reaction Scheme 101
(where such compounds corresponding to Formula 108 are not
available from a commercial source) aromatic or heteroaromatic
carboxylic acids may be subjected to homologation (while the
aldehyde group is protected) by successive treatment under
Arndt-Eistert conditions or other homologation procedures.
Alternatively, derivatives which are not carboxylic acids may also
be homologated by appropriate procedures. The homologated acids can
then be esterified by the general procedure outlined in the
preceding paragraph.
[0226] Compounds of Formula 108, (or other intermediates or of the
invention, as applicable) where A is an alkenyl group having one or
more double bonds can be made for example, by synthetic schemes
well known to the practicing organic chemist; for example by Wittig
and like reactions, or by introduction of a double bond by
elimination of halogen from an alpha-halo-arylalkyl- carboxylic
acid, ester or like carboxaldehyde. Compounds of Formula 108 (or
other intermediates or of the invention, as applicable) where the A
group has a triple (acetylenic) bond can be made by reaction of a
corresponding aromatic methyl ketone with strong base, such as
lithium diisopropylamide, reaction with diethyl chlorophosphate and
subsequent addition of lithium diisopropylamide.
[0227] The acids and salts derived from compounds of Formula 109
(or other intermediates or compounds of the invention, as
applicable) are readily obtainable directly as a result of the
condensation reaction, or from the corresponding esters. Basic
saponification with an alkali metal base will provide the acid. For
example, an ester of Formula 109 (or other intermediates or
compounds of the invention, as applicable) may be dissolved in a
polar solvent such as an alkanol, preferably under an inert
atmosphere at room temperature, with about a three molar excess of
base, for example, lithium hydroxide or potassium hydroxide. The
solution is stirred for an extended period of time, between 15 and
20 hours, cooled, acidified and the hydrolysate recovered by
conventional means.
[0228] The amide may be formed by any appropriate amidation means
known in the art from the corresponding esters or carboxylic acids.
One way to prepare such compounds is to convert an acid to an acid
chloride and then treat that compound with ammonium hydroxide or an
appropriate amine.
[0229] Alcohols are made by converting the corresponding acids to
the acid chloride with thionyl chloride or other means (J. March,
Advanced Organic Chemistry, 2nd Edition, McGraw-Hill Book Company),
then reducing the acid chloride with sodium borohydride (March,
Ibid, p. 1124), which gives the corresponding alcohols.
Alternatively, esters may be reduced with lithium aluminum hydride
at reduced temperatures. Alkylating these alcohols with appropriate
alky halides under Williamson reaction conditions (March, Ibid, p.
357) gives the corresponding ethers. These alcohols can be
converted to esters by reacting them with appropriate acids in the
presence of acid catalysts or dicyclohexylcarbodiimide and
dimethylaminopyridine.
[0230] Aldehydes can be prepared from the corresponding primary
alcohols using mild oxidizing agents such as pyridinium dichromate
in methylene chloride (Corey, E. J., Schmidt, G., Tet. Lett., 399,
1979), or dimethyl sulfoxide/oxalyl chloride in methylene chloride
(Omura, K., Swern, D., Tetrahedron, 34:1651 (1978)).
[0231] Ketones can be prepared from an appropriate aldehyde by
treating the aldehyde with an alkyl Grignard reagent or similar
reagent followed by oxidation.
[0232] Acetals or ketals can be prepared from the corresponding
aldehyde or ketone by the method described in March, Ibid, p. 810.
15
[0233] Referring now to Reaction Scheme 102, a synthetic route to
those compounds of the invention is described in which, with
reference to Formula 101 p is zero, R.sub.2 in the aromatic portion
of the condensed ring structure is OH and R.sub.17 is OH. Those
skilled in the art will readily recognize that these compounds are
.beta.-diketones in the enol form. Reaction Scheme 102 also
describes a synthetic route to those compounds of the invention
where p is 1. Those skilled in the art will readily recognize that
the latter compounds are flavones. Thus, in accordance with this
scheme a 1,2,3,4-tetrahydro-6-methoxynaphthalene-1-o- ne derivative
of Formula 110 is reacted with dialkyl zinc (R.sub.1Zn) in the
presence of titanium tetrachloride in a suitable solvent such as
CH.sub.2Cl.sub.2 to replace the oxo function with the geminal
dialkyl group R.sub.1R.sub.1, to yield a compound of Formula 111,
where R.sub.1 is lower alkyl. In preferred embodiments of the
compounds of the invention which are made in accordance with
Reaction Scheme 102 the R.sub.3 group is hydrogen and R.sub.1 are
methyl. Accordingly, the dialkyl zinc reagent is dimethyl zinc, and
the preferred starting material of Formula 110 is
1,2,3,4-tetrahydro-6-methoxynaphthalene-1-one. The latter compound
is commercially available, for example from Aldrich Chemical
Company. The 1,2,3,4-tetrahydro-1,2-dialkyl-6-methoxy naphthalene
derivative of Formula 111 is thereafter oxidized with chromium
trioxide in acetic acid and acetic anhydride to give a
1,2,3,4-tetrahydro-3,4-dialkyl-7-methoxy naphthalen-1-one
derivative of Formula 112. The ketone compound of Formula 112 is
reacted with a Grignard reagent (R.sub.14MgBr, R.sub.14 is defined
as in connection with Formula 101) to yield a
1-hydroxy-1-aryl-3,4-dihydro-3,4-dialkyl-7-methox- y naphthalene
derivative of Formula 113. The hydroxy compound of Formula 113 is
subjected to elimination by heating, preferably in acid, to yield
the dihydronaphthalene compound of Formula 114. The methyl group is
removed from the phenolic methyl ether function of the compound of
Formula 114 by treatment with boron tribromide in a suitable
solvent, such as CH.sub.2Cl.sub.2, and therafter the phenolic OH is
acylated with an acylating agent that introduces the
R.sub.16CH.sub.2CO group, to give a compound of Formula 115. In the
preferred embodiment R.sub.16 is H, and therefore the acylating
agent is acetyl chloride or acetic anhydride. The acetylation
reaction is conducted in a basic solvent, such as pyridine. The
acylated phenol compound of Formula 115 is reacted with aluminum
chloride at elevated temperature, causing it to undergo a Fries
rearrangement and yield the
1aryl-3,4-dialkyl-3,4-dihydro-6-acyl-7-hydrox- y-naphthalene
compound of Formula 116. The phenolic hydroxyl group of the
compound of Formula 116 is acylated with an acylating agent (such
as an acid chloride) that introduces the CO--Y(R.sub.2)-A-B group
to yield a compound of Formula 117. In the acid chloride reagent
Cl--CO--Y(R.sub.2)-A-B (or like acylating reagent) the symbols Y,
R.sub.2 and A-B have the meaning defined in connection with Formula
101. In the preparation of a preferred compound of the invention in
accordance with this scheme this reagent is ClCOC.sub.6H.sub.4COOEt
(the half ethyl ester half acid chloride of terephthalic acid).
[0234] The compound of Formula 117 is reacted with strong base,
such as potassium hydroxyde in pyridine, to yield, as a result of
an intramolecular Claisen condensation reaction, a compound of
Formula 118. The compounds of Formula 118 are within the scope of
the invention and of Formula 101, where there is an OH for the
R.sub.2 substituent in the aromatic portion of the condensed ring
moiety and R.sub.17 is OH. In connection with the foregoing
reaction (intramolecular Claisen condensation) and the previously
mentioned Fries rearrangement it is noted that these probable
reaction mechanisms are mentioned in this description for the
purpose of fully explaining the herein described reactions, and for
facilitating the work of a person of ordinary skill in the art to
perform the herein described reactions and prepare the compounds of
the invention. Nevertheless, the present inventors do not wish to
be bound by reaction mechanisms and theories, and the herein
claimed invention should not be limited thereby.
[0235] The compounds of Formula 118 are reacted with strong acid,
such as sulfuric acid, in a suitable protonic solvent, such as
acetic acid, to yield the flavone compounds of Formula 119. The
compounds of Formula 119 are also compounds of the invention,
within the scope of Formula 101 where p is 1. Both the compounds of
Formula 118 and Formula 119 can be subjected to further reactions
and transformations to provide further homologs and derivatives, as
described above in connection with Reaction Scheme 101. This is
indicated in Reaction Scheme 102 as conversion to homologs and
derivatives. 16
[0236] Referring now to Reaction Scheme 103 a synthetic route is
shown leading to those compounds of the invention where, with
reference to Formula 101 X is S, O or NR', p is zero and R.sub.17
is H or lower alkyl. However, by applying the generic principles of
synthesis shown in Reaction Scheme 102 the presently shown
synthetic process can be modified or adapted by those of ordinary
skill in the art to also obtain compounds of the invention where X
is S, O or NR' and p is 1, or where X is S, O or NR' and p is zero,
the R.sub.2 group in the aromatic portion of the condensed ring
moiety is OH and R.sub.17 is OH.
[0237] The starting compound of Reaction Scheme 103 is a phenol,
thiophenol or aniline derivative of Formula 120. In the presently
preferred compounds of the invention the R.sub.2 and R.sub.16
groups are both hydrogen, and the preferred starting compounds of
Formula 120 are 3-ethenyl-thiophenol or 3-ethenyl-phenol which are
known in the chemical literature (Nuyken, et al. Polym. Bull
(Berlin) 11:165 (1984). For the sake of simplifying the present
specification, in the ensuing description X can be considered
primarily sulfur as for the preparation of benzothiopyran
derivatives of the present invention. It should be kept in mind,
however, that the herein described scheme is also suitable, with
such modifications which will be readily apparent to those skilled
in the art, for the preparation of benzopyran (X=O) and
dihydroquinoline (X=NR') compounds within the scope of the present
invention. Thus, the compound of Formula 120 is reacted under basic
condition with a 3-bromo carboxylic acid of the Formula 121. In
this reaction scheme the symbols have the meaning described in
connection with Formula 101. An example for the reagent of Formula
121 where R.sub.3 is hydrogen, is 3-bromopropionic acid. The
reaction with the 3-bromocarboxylic acid of Formula 121 results in
the compound of Formula 122. The latter is cyclized by treatment
with acid to yield the 7-ethenyl-thiochroman-4-one derivative (when
X is S) or 7-ethenyl-chroman derivative (when X is 0) of Formula
123. The 7-ethenyl-thiochroman-4-one or 7-ethenyl-chroman-4-one
derivative of Formula 123 is oxidized in the presence of
Pd(II)Cl.sub.2 and CuCl.sub.2 catalysts to provide the
corresponding 7-acyl (ketone) compound of Formula 124. Those
skilled in the art will recognize the latter reaction as a Wacker
oxidation. The exocyclic ketone group of the compound of Formula
124 is protected in the form of a ketal, for example by treatment
with ethylene glycol in acid, to provide the 1,3-dioxolanyl
derivative of Formula 125. Thereafter the compound of Formula 125
is subjected to a sequence of reactions analogous to those
described for the compounds of Formula 105 in Reaction Scheme 101.
Thus, the 1,3-dioxolanyl derivative of Formula 125 is reacted with
a Grignard reagent of the formula R.sub.14MgBr to give the tertiary
alcohol of Formula 126, which is thereafter dehydrated in acid to
provide the benzothiopyran (X is S), benzopyran (X is O) or
dihydroquinoline (X is NR') derivative of Formula 127. The ketone
compound of Formula 127 is then reacted in the presence of base
with the reagent of Formula 108 in an aldol condensation
(Claisen-Schmidt) reaction to provide compounds of the invention of
Formula 128. The compounds of Formula 128 can be converted into
further homologs and derivatives, as described above in connection
with Reaction Schemes 101 and 102.
SPECIFIC EXAMPLES
[0238] 2-hydroxy-2-methyl-5-phenylpentane
[0239] To a mixture of magnesium turnings 13.16 g (0.541 mol) in
200 ml of anhydrous Et.sub.2O was added 100.0 g (0.492 mol) of
1-bromo-3-phenyl propane as a solution in 100 ml of Et.sub.2O.
After of 5-10 ml of the solution had been added, the addition was
stopped until the formation of the Grignard reagent was in
progress. The remaining bromide was then added over 1 hour. The
Grignard reagent was stirred for 20 minutes at 35.degree. C. and
then 31.64 g (0.541 mol) of acetone was added over a 45 minute
period. The reaction was stirred overnight at room temperature
before being cooled to 0.degree. C. and acidified by the careful
addition of 20% HCl. The aqueous layer was extracted with Et.sub.2O
(3.times.200 ml) and the combined organic layers washed with water,
and saturated aqueous NaCl before being dried over MgSO.sub.4.
Removal of the solvent under reduced pressure and distillation of
the residue afforded 63.0 g (72%) of the product as a pale-yellow
oil, bp 99-102.degree. C./0.5 mm Hg. 1H NMR (CDCl.sub.3): .delta.
7.28-7.18 (5H, m), 2.63 (2H, t, J=7.5 Hz), 1.68 (2H, m), 1.52 (2H,
m), 1.20 (6H,s).
[0240] 1,2,3,4-tetrahydro-1,1-dimethylnaphthalene
[0241] A mixture of P.sub.2O.sub.5 (55.3 g, 0.390 mol) in 400 ml of
methanesulfonic acid was heated to 105.degree. C. under argon until
all of the solid had dissolved. The resulting solution was cooled
to room temperature and 2-hydroxy-2-methyl-5-phenylpentane (63.0 g,
0.354 mol) added slowly with stirring. After 4 hours the reaction
was quenched by carefully pouring the solution onto 1 L of ice. The
resulting mixture was extracted with Et.sub.2O (4.times.125 ml)and
the combined organic layers washed with water, saturated aqueous
NaHCO.sub.3, water, and saturated aqueous NaCl before being dried
over MgSO.sub.4. Concentration of the solution under reduced
pressure, followed by distillation afforded 51.0 g (90%) of the
product as a clear colorless oil, bp. 65-67.degree. C./1.1 mmHg. 1H
NMR (CDCl.sub.3): .delta. 7.32 (1H, d, J=7.4 Hz), 7.16-7.05 (3H,
m), 2.77 (2H, t, J=6.3 Hz), 1.80 (2H, m), 1.66 (2H, m), 1.28 (6H,
s).
[0242] 3,4-dihydro-4,4-dimethyl-1(2H)-naphthalenone (Compound
A)
[0243] A solution of 350 ml of glacial acetic acid and 170 ml of
acetic anhydride was cooled to 0.degree. C. and CrO.sub.3, 25.0 g
(0.25 mol) carefully added in small portions. The resulting mixture
was stirred for 30 minutes before 120 ml of benzene was added.
1,2,3,4-tetrahydro-1,1-dim- ethylnaphthalene was added slowly as a
solution in 30 ml of benzene. Upon completing the addition the
reaction was stirred for 4 hours at 0.degree. C. The solution was
diluted with H.sub.2O (200 ml) and extracted with Et.sub.2O
(5.times.50 ml). The combined organic layers were washed with
water, saturated aqueous NaCO.sub.3, and saturated aqueous NaCl,
before being dried over MgSO.sub.4. Removal of the solvents under
reduced pressure, and distillation afforded 16.0 g (74%) of the
product as a pale-yellow oil, bp 93-96.degree. C./0.3 mm Hg 1H NMR
(CDCl.sub.3): .delta. 8.02 (1H, dd, J=1.3, 7.8 Hz), 7.53 (1H, m ),
7.42 (1H, d, J=7.9 Hz), 7.29 (1H, m), 2.74 (2H, t, J=6.8 Hz), 2.02
(2H, t, J=6.8 Hz), 1.40 (6H, s).
[0244] 3,4-dihydro-4,4-dimethyl-7-bromo-1(2H)-naphthalenone
(Compound B)
[0245] A 100 ml three-necked flask, fitted with an efficient reflux
condenser and drying tube, and addition funnel, was charged with a
mixture of AICl.sub.3 9.5g (71.4 mmol) and 3 ml of
CH.sub.2Cl.sub.2. The 3,4-dihydro-4,4-dimethyl-1(2H)-naphthalenone
(5.0 g, 28.7 mmol), was added dropwise with stirring (Caution:
Exothermic Reaction!) to the mixture at room temperature. Bromine,
5.5 g (34.5 mmol), was then added very slowly, and the resulting
mixture stirred for 2 hours at room temperature. (Note: if stirring
stops, the mixture can be warmed to 70.degree. C. until stirring
resumes.) The reaction was then quenched by the slow addition of
ice-cold 6M HCl. The mixture was extracted with Et.sub.2O and the
combined organic layers washed with water, saturated aqueous
NaHCO.sub.3, and saturated NaCl, before being dried over
MgSO.sub.4. Removal of the solvent under reduced pressure, and
distillation of the residue afforded 5.8 g (80%) of the product as
a pale-yellow oil which solidified on standing, bp: 140.degree.
C./0.4 mm Hg. 1H NMR (CDCl.sub.3): .delta. 8.11 (1H, d, J=3.0 Hz),
7.61 (1H, dd, J=3.0, 9.0 Hz), 7.31 (1H, d, J=9.0 Hz), 2.72 (2H, t,
J=6.0 Hz), 2.01 (2H, t, J=6.0 Hz), 6H, s).
[0246]
1,2,3,4-tetrahydro-1-hydroxy-1-(4-methylphenyl)-4,4-dimethyl-7-brom-
onaphthalene (Compound C)
[0247] To a mixture of magnesium turnings (648.0 mg, 27.0 mmol) in
25 ml of THF was added a solution of 4-bromotoluene (5.40 g, 31.8
mmol) in 10 ml of THF in two portions. The reaction was initiated
by the addition of 2 ml of the solution, followed by the slow
addition of the remaining solution via an addition funnel. The
mixture was stirred at room temperature for 1 hour, and then the
solution was transferred to a second flask using a canula. To the
resulting Grignard reagent was added 4.0 g (15.9 mmol) of
3,4-dihydro-4,4-dimethyl-7-bromo-1(2H)-naphthalenone (Compound B)
as a solution in 15 ml of THF. The resulting solution was heated to
reflux overnight, cooled to room temperature, and the reaction
quenched by the careful addition of ice-cold 10% HCl. Extraction
with Et.sub.2O was followed by washing of the combined organic
layers with H.sub.2O and saturated aqueous NaCl, then drying over
MgSO.sub.4. Removal of the solvent under reduced pressure provided
an oil which afforded the product as a colorless solid after column
chromatography (hexanes/EtOAc, 96:4). 1H NMR (CDCl.sub.3): .delta.
7.36 (1H, dd, J=2.1, 7.6 Hz), 7.26 (3H, m), 7.12 (3H, s), 2.34 (3H,
s), 2.24-2.04 (2H, m), 1.81 (1H, m), 1.55 (1H, m), 13.5 (3H, s),
1.30 (3H, s).
[0248]
3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-7-bromonaphthalene
(Compound D)
[0249] A flask equipped with a Dean-Stark trap was charged with 3.4
g of (9.85 mmol) of
1,2,3,4-tetrahydro-1-hydroxy-1-(4-methylphenyl)-4,4-dimeth-
yl-7-bromonaphthalene (Compound C) and 40 ml of benzene. A
catalytic amount of p-toluenesulfonic acid monohydrate was added
and the resulting solution heated to reflux for 2 hours. Upon
cooling to room temperature, Et.sub.2O was added and the solution
washed with H.sub.2O, saturated aqueous NaHCO.sub.3, and saturated
aqueous NaCl then dried over MgSO.sub.4. Removal of the solvents
under reduced pressure, and column chromatography (100%
hexane/silica gel) afforded the title compound as a colorless
solid. 1H NMR (CDCl.sub.3): .delta. 7.32 (1H, dd, J=2.1, 8.2 Hz),
7.21 (5H, m), 7.15 (1H, d, J=2.1 Hz), 5.98 (1H, t, J=4.7 Hz), 2.40
(3H, s), 2.32 (2H, d, J=4.7 Hz), 1.30 (6H, s).
[0250] 7-Ethenyl-3,4-dihydro-4,4-dimethylnaphthalen-1(2H)-one
(Compound E)
[0251] To a solution (flushed for 15 minutes with a stream of
argon) of 7 g (27.6 mmol) of
3,4-dihydro-4,4-dimethyl-7-bromo-1(2H)-naphthalenone (Compound B)
in 150 ml of triethylamine was added 0.97 g (1.3 mmol) of
bis(triphenylphosphine)palladium(II) chloride and 0.26 g (1.3 mmol)
of cuprous iodide. The solution mixture was flushed with argon for
5 minutes and then 39 ml (36.6 mmol) of (trimethylsilyl)acetylene
was added. The reaction mixture was sealed in a pressure tube and
placed in a preheated oil bath (100.degree. C.) for 24 hours. The
reaction mixture was then filtered through Celite, washed with
Et.sub.2O and the filtrate concentrated in vacuo to give crude
7-(trimethylsilyl)ethynyl-3,4-dihydro-
-4,4-dimethylnaphthalen-1(2H)-one. To a solution of this crude
TMS-acetylenic compound in 50 ml of methanol was added 0.6 g (4.3
mmol) of K.sub.2CO.sub.3. The mixture was stirred for 8 hours at
ambient temperature and then filtered. The filtrate was
concentrated in vacuo, diluted with Et.sub.2O, washed with water,
10% HCl and brine, dried over MgSO.sub.4 and concentrated in vacuo.
Purification by column chromatography (silica, 10% EtOAc-hexane)
yielded the title compound as a white solid. PMR (CDCl.sub.3):
.delta. 1.39 (6H, s), 2.02 (2H, t, J=7.0 Hz), 2.73 (2H, t, J=7.0
Hz), 3.08 (1H, s), 7.39 (1H, d, J=8.2 Hz), 7.61 (1H, dd, J=1.8, 8.2
Hz), 8.14 (1H, d, J=9 1.8 Hz).
[0252] Ethyl-4-iodobenzoate
[0253] To a suspension of 10 g (40.32 mmol) of 4-iodobenzoic acid
in 100 ml absolute ethanol was added 2 ml thionyl chloride and the
mixture was then heated at reflux for 3 hours. Solvent was removed
in vacuo and the residue was dissolved in 100 ml ether. The ether
solution was washed with saturated NaHCO.sub.3 and saturated NaCl
solutions and dried (MgSO.sub.4). Solvent was then removed in vacuo
and the residue Kugelrohr distilled (100.degree. C.; 0.55 mm) to
give the title compound as a colorless oil, PMR (CDCl.sub.3):
.delta. 1.42 (3H, t, J.about.7 Hz), 4,4 (2H, q, J.about.7 Hz), 7.8
(4H).
[0254] 6-iodonicotinic acid
[0255] Sodium iodide (20.59 g, 137.40 mmol) was cooled to
-78.degree. C. under argon and then hydriodic acid (97.13 g, 759.34
mmol) was added. The cooling bath was removed and the suspension
was stirred for 5 minutes. To this mixture was added
6-chloronicotinic acid (22.09 g, 140.20 mmol) and the resulting
mixture was slowly warmed to ambient temperature with stirring. The
mixture was heated to reflux at 125.degree. C. for 24 hours, cooled
to ambient temperature and poured into acetone (500 ml) at
0.degree. C. The yellow solid was collected by filtration and
washed with 200 ml of 1N aqueous NaHSO.sub.3 solution.
Recrystallization from methanol (crystals were washed with ethyl
ether) afforded the title compound as white crystals: mp
177-179.degree. C. [lit. mp 187-192, Newkome et al. "Reductive
Dehalogenation of Electron-Poor Heterocycles: Nicotinic Acid
Derivatives" J. Org. Chem. 51:953-954 (1986). 1H NMR (DMSO-d6):
.delta. 8.81 (1H, dd, J=0.8, 2.4 Hz), 8.01 (1H, dd, J=0.8, 8.2 Hz),
7.91 (1H, dd, J=2.4, 8.2 Hz).
[0256] Ethyl 6-iodonicotinoate
[0257] To a suspension of 6-iodonicotinic acid (23.38 g, 94.20
mmol) in dichloromethane (100 ml) was added a solution of
1-(3-dimethylaminopropyl- )-3-ethylcarbodiimide hydrochloride
(19.86 g, 103.6 mmol) in dichloromethane (250 ml). To this mixture
was added ethanol (12.40 g, 269.27 mmol) followed by
dimethylaminopyridine (1.15 g, 9.41 mmol). The mixture was heated
at 50.degree. C. for 24.5 hours, concentrated in vacuo, and diluted
with water (200 ml) then extracted with ethyl ether (550 ml). The
combined organic phases were washed with saturated aqueous NaCl,
dried (MgSO.sub.4) and concentrated to a yellow solid. Purification
by flash chromatography (silica, 10% EtOAc-hexane) afforded the
title compound as white needles: mp 48-49.degree. C.; 1H NMR
(CDCl.sub.3): .delta. 8.94 (1H, d, J=2.1 Hz), 7.91 (1H, dd, J=2.1,
8.2 Hz), 7.85 (1H, d, J=8.2 Hz), 4.41 (2H, q, J=7.1 Hz), 1.41 (3H,
t, J=7.1 Hz).
[0258] Ethyl
4-[(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-naphthalenyl)ethy-
nyl]benzoate (Compound F)
[0259] To a solution of 4 g (21.7 mmol) of
7-ethynyl-3,4-dihydro-4,4-dimet- hylnaphthalen-1(2H)-one (Compound
E ) flushed for 15 minutes with a stream of argon, and 6 g (21.7
mmol) of ethyl 4-iodobenzoate in 100 ml of triethylamine was added
5 g (7.2 mmol) of bis(triphenylphosphine)palladiu- m(II) chloride
and 1.4 g (7.2 mmol) of cuprous iodide. The mixture was flushed
with argon for 5 minutes and then stirred at ambient temperature
for 18 hours. The reaction mixture was filtered through Celite and
the filtrate was concentrated in vacuo. Purification by flash
chromatography (silica, 10% EtOAc-hexane) yielded the title
compound as a white solid. PMR (CDCl.sub.3): .delta. 1.41 (3H, t,
J=7.2 Hz), 1.41 (6H, s), 2.04 (2H, t, J=6.5 Hz), 2H, t, J=6.5 Hz),
4.40 (2H, q, J=7.2 Hz), 7.44 (1H, d, J=8.2 Hz), 7.59 (2H, d, J=8.4
Hz), 7.68 (1H, dd, J=1.8, 8.2 Hz), 8.04 (2H, d, J=8.4 Hz), 8.15
(1H, d, J=1.8 Hz).
[0260] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-
-naphthalenyl)ethynyl]benzoate (Compound G)
[0261] To a cold solution (-78.degree. C.) of 291.6 mg (1.59 mmol)
of sodium bis(trimethylsily)amide in 5.6 ml of THF was added a
solution of 500.0 mg (1.44 mmol) of ethyl
4-[(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-
-naphthalenyl)ethynyl]benzoate (Compound F) in 4.0 ml of THF. The
reaction mixture w as stirred at '178.degree. C. for 35 minutes and
then a solution of 601.2 mg (1.59 mmol) of
5-chloro(2-bis-triflouromethylsulfony- l)imide in 4.0 ml of THF was
added. After stirring at -78.degree. C. for 1 hour, the solution
was warmed to 0.degree. C. and stirred for 2 hours. The reaction
was quenched by the addition of saturated aqueous NH.sub.4Cl. The
mixture was extracted with EtOAc (50 ml) and the combined organic
layers were washed with 5% aqueous NaOH, water, and brine. The
organic phase was dried over Na.sub.2SO.sub.4 and then concentrated
in vacuo to a yellow oil. Purification by column, chromatography
(silica, 7% EtOAc-hexanes) yielded the title compound as a
colorless solid. 1H NMR (CDCl.sub.3): .delta. 8.04 (2H, dd, J=1.8,
8.4 Hz), 7.60 (2H, dd, J=1.8, 8.4 Hz), 7.51 (2H, m), 7.32 (1H, d,
J=8.0 Hz), 4.40 (2H, q, J=7.1 Hz), 6.02 (1H, t, J=5.0 Hz), 2.44
(2H, d, J=5.0 Hz), 1.43 (3H, t, J=7.1 Hz), 1.33 (6H, s).
[0262] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthaleny-
l)ethenyl]benzoate (Compound 1)
[0263] A solution of 4-lithiotoluene was prepared by the addition
of 189.9 mg (1.74 ml, 2.96 mmol) of t-butyl lithium (1.7M solution
in hexanes) to a cold solution (-78.degree. C.) of 253.6 mg (1.482
mmol) of 4-bromotoluene in 2.0 ml of THF. After stirring for 30
minutes a solution of 269.4 mg (1.977 mmol) of zinc chloride in 3.0
ml of THF was added. The resulting solution was warmed to room
temperature, stirred for 30 minutes, and added via cannula to a
solution of 472.9 mg (0.988 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-napht-
halenyl)ethynyl]benzoate (Compound G) and 50 mg (0.04 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 4.0 ml of THF. The
resulting solution was heated at 50.degree. C. for 45 minutes,
cooled to room temperature and diluted with sat. aqueous
NH.sub.4Cl. The mixture was extracted with EtOAc (40 ml) and the
combined organic layers were washed with water and brine. The
organic phase was dried over Na.sub.2SO.sub.4 and concentrated in
vacuo to a yellow oil. Purification by column chromatography
(silica, 5% EtOAc-hexanes) yielded the title compound as a
colorless solid. 1H NMR (d6-acetone): .delta. 1.35 (6H, s), 1.40
(3H, t, J=7.1 Hz), 2.36 (2H, d, J=4.7 Hz), 2.42 (3H,s), 4.38 (2H,
q, J=7.1 Hz), 5.99 (1H, t, J=4.7 Hz), 7.25 (5H, m), 7.35 (2H, m),
7.52 (2H, d, J=8.5 Hz), 7.98 (2H, d, J=8.5 Hz).
[0264] Ethyl
4-[(5.6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenylethynyl]b-
enzoate (Compound 1a)
[0265] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 203.8 mg (0.43 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 58.2 mg (0.36 ml, 0.69 mmol) of
phenyllithium (1.8M solution in cyclohexane/Et.sub.2O), 116.1 mg
(0.85 mmol) of zinc chloride and 13.8 mg (0.01 mmol) of
tetrakis(triphenylphosphine)palladium- (0). PMR (CDCl.sub.3):
.delta. 1.36 (6H, s), 1.40 (3H, t, J=7.1Hz), 2.37 (2H, d, J=4.7
Hz), 4.38 (2H, q, J=7.1 Hz), 6.02 (1H, t, J=4.7 Hz), 7.20 (1H, d,
J=1.5 Hz), 7.27 (1H, m), 7.39 (6H, m), 7.52 (2H, d, J=8.2 Hz), 7.98
(2H, d, J=8.2 Hz).
[0266] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(3-methylphenyl)-2-naphthaleny-
l)ethynyl]benzoate (Compound 2)
[0267] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 250.0 mg (0.522 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 284.8 mg (2.090 mmol) of zinc
chloride, 24 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(O) in 2.0 ml of THF, and
3-methylphenyl lithium (prepared by adding 201.2 mg (1.86 ml, 3.14
mmol) of tert-butyllithium (1.7M solution in pentane) to a cold
solution (-78.degree. C.) of 274.0 mg (1.568 mmol) of
3-methylbromobenzene in 2.0 ml of THF). 1H NMR (CDCl.sub.3):
.delta. 7.99 (2H, d, J=8.4 Hz), 7.51 (2H, d, J=8.4 Hz), 7.39-7.14
(7H, m), 5.99 (1H, t, J=4.7 Hz), 4.37 (2H, q, J=7.1 Hz), 2.60 (3H,
s), 2.35 (2H, d, J=4.7 Hz), 1.39 (3H, t, J=7.1 Hz), 1.34 (6H,
s).
[0268] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(2-methylphenyl)-2-naphthaleny-
l)ethynyl]benzoate (Compound 3)
[0269] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 200.0 mg (0.418 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 199.4 mg (1.463 mmol) of zinc
chloride, 24 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 4.0 ml of THF, and
2-methylphenyl lithium (prepared by adding 133.9 mg (1.23 ml, 2.09
mmol) of tert-butyllithium (1.7M solution in pentane) to a cold
solution (-78.degree. C.) of 178.7 mg (1.045 mmol) of
2-methylbromobenzene in 2.0 ml of THF). 1H NMR (CDCl.sub.3):
.delta. 7.97 (2H, d, J=8.4 Hz), 7.50 (2H, d, J=8.4 Hz), 7.49-7.19
(6H, m), 6.81 (1H, d, J=1.6 Hz), 5.89 (1H, t, J=4.5 Hz), 4.36 (2H,
q, J=7.1 Hz), 2.43-2.14 (2H, dq, J=3.7, 5.4 Hz), 2.15 (3H, s),
1.39-1.34 (9H, m).
[0270] Ethyl
4-r(5,6-dihydro-5,5-dimethyl-8-(3,5-dimethylphenyl)-2-naphtha-
lenyl)ethynyl]benzoate (Compound 4)
[0271] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 250.0 mg (0.522 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 249.0 mg (1.827 mmol) of zinc
chloride, 24 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and
3,5-dimethylphenyl lithium (prepared by adding 167.7 mg (1.54 ml,
2.62 mmol) of tert-butyllithium (1.7M solution in pentane) to a
cold solution (-78.degree. C.) of 249.0 mg (1.305 mmol) of
3,5-dimethylbromobenzene in 2.0 ml of THF). 1H NMR (CDCl.sub.3):
.delta. 7.98 (2H, d, J=8.4 Hz), 7.52 (2H, d, J=8.4 Hz), 7.40-7.33
(2H, m), 7.20 (1H, d, J=1.6 Hz), 7.00 (1H, s), 6.97 (2H, s), 5.97
(1H, t, J=4.8 Hz), 4.37 (2H, q, J=7.1 Hz), 2.36 (6H, s), 2.34 (2H,
d, J=4.8 Hz), 1.39 (3H, t, J=7.1 Hz), 1.37 (6H, s).
[0272] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-ethylphenyl)-2-naphthalenyl-
)ethynyl]benzoate (Compound 5)
[0273] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 250.0 mg (0.522 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 249.0 mg (1.827 mmol) of zinc
chloride, 24 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and
4-ethylphenyl lithium (prepared by adding 167.7 mg (1.54 ml, 2.62
mmol) of tert-butyllithium (1.7M solution in pentane) to a cold
solution (-78.degree. C.) of 244.0 mg (1.305 mmol) of
4-ethylbromobenzene in 2.0 ml of THF). 1H NMR (CDCl.sub.3): .delta.
7.99 (2H, d, J=8.4 Hz), 7.51 (2H, d, J=8.4 Hz), 7.42-7.24 (7H, m),
5.99 (1H, t, J=4.7 Hz), 4.37 (2H, q, J=7.1 Hz), 2.71 (2H, q, J=7.6
Hz), 2.35 (2H, d, J=4.7 Hz), 1.39 (3H, t, J=7.1 Hz), 1.34 (6H,
s).
[0274] Ethyl
4-[(5,6-dihydro-5,5-dimethyI-8-(4-(1,1-dimethylethyl)phenyl)--
2-naphthalenyl)ethynyl]benzoate (Compound 6)
[0275] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl-
]benzoate (Compound 1), 250.0 mg (0.52 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 142.4 mg (1.045 mmol) of zinc
chloride and 4-tert-butylphenyl lithium (prepared by adding 100.6
mg (0.97 ml, 1.57 mmol) of tert-butyllithium (1.5M solution in
pentane) to a cold solution (-78.degree. C.) of 167.0 mg (0.78
mmol) of 4-tert-butylbromobenzene in 1.0 ml of THF). 1H NMR
(CDCl.sub.3): .delta. 7.99 (2H, d, J=8.4 Hz), 7.55 (2H, d, J=8.4
Hz), 7.28-7.45 (7H, m), 6.02 (1H, t, J=4.9 Hz), 4.38 (2H, q, J=7.2
Hz), 2.36 (2H, d, J=4.9 Hz), 1.59 (3H, s), 1.40 (3H, t, J=7.2 Hz),
1.39 (9H, s), 1.35 (6H, s).
[0276] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-chlorophenyl)-2-naphthaleny-
l)ethynyl]benzoate (Compound 7)
[0277] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 250.0 mg (0.522 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 249.0 mg (1.827 mmol) of zinc
chloride, 24 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and
4-chlorophenyl lithium (prepared by adding 167.7 mg (1.54 ml, 2.62
mmol) of tert-butyllithium (1.7M solution in pentane) to a cold
solution (781.LAMBDA.[.degree. C.) of 252.4 mg (1.305 mmol) of
4-chloro-1-bromobenzene in 2.0 ml of THF). 1H NMR (CDCl.sub.3):
.delta. 7.98 (2H, d, J=8.4 Hz), 7.53 (2H, d, J=8.4 Hz), 7.40-7.27
(6H, m), 7.12 (1H, d, J=1.6 Hz), 6.00 (1H, t, J=4.8 Hz), 4.37 (2H,
q, J=7.1 Hz), 2.35 (2H, d, J=4.8 Hz), 1.40 (2H, t, J=7.1 Hz), 1.34
(6H, s).
[0278] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2-naphthalen-
yl)ethynyl]benzoate (Compound 8)
[0279] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 250.0 mg (0.522 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 249.0 mg (1.827 mmol) of zinc
chloride, 24 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and
4-methoxyphenyl lithium (prepared by adding 167.7 mg (1.54 ml, 2.62
mmol) of tert-butyllithium (1.7M solution in pentane) to a cold
solution (-78.degree. C.) of 244.1 mg (1.305 mmol) of
4-methoxy-1-bromobenzene in 2.0 ml of THF). 1H NMR (CDCl.sub.3):
.delta. 7.98 (2H, d, J=8.5 Hz), 7.52 (2H, d, J=8.6 Hz), 7.40-7.21
(5H, m), 6.95 (2H, d, J=8.7 Hz), 5.97 (1H, t, J=4.7 Hz), 4.37 (2H,
q, J=7.1 Hz), 4.34 (3H, s), 2.34 (2H, d, J=4.7 Hz), 1.39 (3H, t,
J=7.1 Hz), 1.34 (6H, s).
[0280] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-trifluoromethylphenyl)-2-na-
phthalenyl)ethynyl]benzoate (Compound 9)
[0281] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 250.0 mg (0.522 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 249.0 mg (1.827 mmol) of zinc
chloride, 24 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and
4-trifluoromethylphenyl lithium (prepared by adding 167.7 mg (1.54
ml, 2.62 mmol) of tert-butyllithium (1.7M solution in pentane) to a
cold solution (-78.degree. C.) of 296.6 mg (1.305 mmol) of
4-trifluoromethylbromobenzene in 2.0 ml of THF). 1H NMR
(CDCl.sub.3): .delta. 7.98 (2H, d, J=8.5 Hz), 7.67 (2H, d, J=8.3
Hz), 7.54-7.36 (6H, m), 7.10 (1H, d, J=1.6 Hz), 6.06 (1H, t, J=4.8
Hz), 4.37 (2H, q, J=Hz), 2.38 (2H, d, J=4.8 Hz), 1.39 (3H, t, J=7.1
Hz), 1.35 (6H, s).
[0282] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(2-pyridyl)-2-naphthalenyl)eth-
ynyl]benzoate (Compound 10)
[0283] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 250.0 mg (0.52 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 142.4 mg (1.045 mmol) of zinc
chloride and 2-lithiopyridine (prepared by the addition of 100.6 mg
(0.97 ml, 1.57 mmol) of tert-butyllithium (1.5M solution in
pentane) to a cold solution (-78.degree. C.) of 123.8 mg (0.784
mmol) of 2-bromopyridine in 1.0 ml of THF). 1H NMR (d6-acetone):
.LAMBDA.[.delta. 8.64 (1H, m), 7.99 (2H, d, J=8.5 Hz), 7.85 (1H,
ddd, J=1.8, 7.7, 9.5 Hz), 7.58 (2H, d, J=8.4 Hz), 7.50 (1H, d,
J=7.7 Hz), 7.47 (2 H, d, J=1.1 Hz), 7.35 (2H, m), 6.32 (1H, t,
J=4.8 Hz), 4.34 (2H, q, J=7.2 Hz), 2.42 (2H, d, J=7.4 Hz), 1.35
(3H, t, J=7.0 hz), 1.35 (6H, s).
[0284] Ethyl
4-[(5.6-dihydro-5.5-dimethyl-8-(3-pyridyl)-2-naphthalenyl)eth-
ynyl]benzoate (Compound 11)
[0285] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 170.0 mg (0.35 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 142.4 mg (1.045 mmol) of zinc
chloride and 3-lithiopyridine (prepared by the addition of 100.2 mg
(0.92 ml, 1.56 mmol) of tert-butyllithium (1.5M solution in
pentane) to a cold solution (-78.degree. C.) of 123.8 mg (0.784
mmol) of 3-bromopyridine in 1.0 ml of THF). 1H NMR (CDCl.sub.3):
.delta. 8.63-8.61 (2H, dd, J=1.7 Hz), 7.99 2H, d, J=8.4 Hz), 7.67
(1H, dt, J=7.9 Hz), 7.52 (2H, d, J=8.4 Hz), 7.43-7.34 (3H, m), 7.10
(1H, d, J=1.6 Hz), 6.07 (1H, t, J=4.7 Hz), 4.37 (2H, q, J=7.1 Hz),
2.40 (2H, d, J=4.7 Hz), 1.390 (3H, t, J=7.1 Hz), 1.36 (6H, s).
[0286] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(2-methyl-5-pyridyl)-2-naphtha-
lenyl)ethynyl]benzoate (Compound 12)
[0287] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenlyl)-2-naphthalenyl)eth-
ynyl]benzoate (Compound 1), 250.0 mg (0.522 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 142.4 mg (1.045 mmol) of zinc
chloride and 2-methyl-5-lithiopyridine (prepared by the addition of
100.5 mg (0.92 ml, 1.57 mmol) of tert-butyllithium (1.7 M solution
in pentane) to a cold solution (-78.degree. C.) of 134.8 mg (0.784
mmol) of 2-methyl-5-bromopyridine in 1.0 ml of THF). 1H NMR
(CDCl.sub.3): .delta. 8.50 (1H, d, J=2.2 Hz), 7.99 (2H, d, J=8.3
Hz), 7.56 (1H, dd, J=2.3, 8.0 Hz), 7.53 (2H, d, J=8.4 Hz), 7.43
(1H, dd, J=2.3, 8.0 Hz), 7.37 (2H, d, J=8.0 Hz), 7.21 (1H, d, J=8.1
Hz), 7.11 (1H, d, J=1.5 Hz), 6.04 (1H, t, J=4.7 Hz), 4.38 (2H, q,
J=7.2 Hz), 2.63 (3H, s), 2.38 (2H, d, J=4.6 HKz), 1.40 (3H, t,
J=7.1 Hz), 1.35 (6H, s).
[0288] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(3-((2,2-dimethylethyl)-dimeth-
ylsiloxyvphenyl)-2-naphthalenyl)ethynyl]benzoate (Compound H)
[0289] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethyn-
yl]benzoate (Compound G), 150.0 mg (0.314 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 150.0 mg (1.10 mmol) of zinc
chloride, 24 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and
3-((2,2-dimethylethyl)dimethylsiloxy)phenyl lithium (prepared by
adding 100.2 mg (0.92 ml, 1.564 mmol) of tert-butyllithium (1.7M
solution in pentane) to a cold solution (-78.degree. C.) of 226.0
mg (0.787 mmol) of
3-((2,2-dimethylethyl)dimethylsiloxy)bromobenzene in 2.0 ml of
THF). 1H NMR (CDCl.sub.3): .delta. 7.98 (2H, d, J=8.4 Hz), 7.51
(2H, d, J=8.4 Hz), 7.40-7.22 (4H, m), 6.95 (1H, d, J=7.6 Hz),
6.84-6.82 (2H, m), 6.00 (1H, t, J=4.7 Hz), 4.37 (2H, q, J=7.1 Hz),
2.35 (2H, d, J=4.7 Hz), 1.39 (3H, t, J=7.1 Hz), 1.34 (3H, s), 0.99
(9H, s), 0.23 (6H, s,).
[0290] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-((2,2-dimethylethyl)-dimeth-
ylsiloxyphenyl)-2-naphthalenyl)ethynyl]benzoate (Compound 1)
[0291] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 210.0 mg (0.439 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 209.0 mg (1.53 mmol) of zinc
chloride, 24 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and
4-((2,2-dimethylethyl)dimethylsiloxy)phenyl lithium (prepared by
adding 140.3 mg (1.30 ml, 2.19 mmol) of tert-butyllithium (1.7M
solution in pentane) to a cold solution (-78.degree. C.) of 315.0
mg (1.09 mmol) of 4-((2,2-dimethylethyl)dimethylsiloxy)bromobenzene
in 2.0 ml of THF). 1H NMR (CDCJ.sub.3): .delta. 7.98 (2H, d, J=8.4
Hz), 7.51 (2H, d, J=8.4 Hz), 7.39-7.25 (3H, m), 7.21 (2H, d, J=8.5
Hz), 5.87 (2H, d, J=8.5 Hz), 5.96 (1H, t, J=4.7 Hz), 4.37 (2H, q,
J=7.1 Hz), 2.33 (2H, d, J=4.7 Hz), 1.39 (3H, t, J=7.1 Hz), 1.33
(6H, s), 1.01 (9H, s), 0.25 (6H, s).
[0292] Ethyl
4-[(5.6-dihydro-5,5-dimethyl-8-(3-hydroxyphenyl)-2-naphthalen-
yl)ethynyl]benzoate (Compound 13)
[0293] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(3-((2,2dieth-
ylethyl)-dimethylsiloxy)-phenyl)-2-naphthalenyl)ethynyl]benzoate
(Compound H) 60.0 mg (0.114 mmol) in 1.0 ml of THF at room
temperature was added 91.5 mg (0.35 ml, 0.35 mmol) of
tetrabutylamonium flouride (1 M solution in THF). After stirring
overnight, the solution was diluted with EtOAc and washed with
H.sub.2O and saturated aqueous NaCl, before being dried over
MgSO.sub.4. Removal of the solvents under reduced pressure,
followed by column chromatography (4:1, Hexanes:EtOAc) afforded the
title compound as a colorless solid. 1H NMR (CDCl.sub.3): .delta.
7.98 (2H, d, J=7.8 Hz), 7.52 (2H, d, J=8.3 Hz), 7.39-7.21 (4H, m),
6.93 (1H, d, J=7.5 Hz), 6.84 (1H, d, 7.1 Hz), 6.83 (1H, s), 6.01
1H, t, J=4.7 Hz), 4.91 (1H, s), 4.39 (2H, q, J=7.1 Hz), 2.35 (2H,
d, J=4.7 Hz), 1.39 (3H, t, J=7.1 Hz), 1.34 (6H, s).
[0294] Ethyl
4-[(5,6-dihydro-5,5-dimethyI-8-(4-hydroxynhenyl)-2-naphthalen-
yl)ethenyl]benzoate (Compound 14)
[0295] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-((2,2-dime-
thylethyl)-dimethylsiloxy)phenyl)-2-naphthalenyl)ethynyl]benzoate
(Compound I) 50.0 mg (0.095 mmol) in 1.0 ml of THF at room
temperature was added 73.2 mg (0.29 ml, 0.29 mmol) of
tetrabutylamonium fluoride (1 M solution in THF). After stirring
overnight, the solution was diluted with EtOAc and washed with
H.sub.2O and saturated aqueous NaCl, before being dried over
MgSO.sub.4. Removal of the solvents under reduced pressure,
followed by column chromatography (4:1, Hexanes:EtOAc) afforded the
title compound as a colorless solid. 1H NMR (CDCl.sub.3): .delta.
7.98 (2H, d, J=8.2 Hz), 7.52 (2H, d, J=8.3 Hz), 7.41-7.20 (5H, m),
6.88 (2H, d, J=8.4 Hz), 5.96 (1H, t, J=4.5 Hz), 4.37 (2H, q, J=7.1
Hz), 2.34 (2H, d, J=4.5 Hz), 1.39 (3H, t, J=7.1 Hz), 1.34 (6H,
s).
[0296] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(5-methylthiazol-2-yl)-2-napht-
halenyl)ethynyI]benzoate (Compound 15)
[0297] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 264.0 mg (0.552 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 150.0 mg (1.10 mmol) of zinc
chloride, 14 mg (0.012 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 4.0 ml of THF, and
5-methylthiazol-2-yl lithium (prepared by adding 53.2 mg (0.53 ml,
0.83 mmol) of n- butyllithium (1.55 M solution in hexanes) to a
cold solution (-78.degree. C.) of 82.0 mg (0.83 mmol) of
5-methylthiazole in 5.0 ml of THF). 1H NMR (CDCl.sub.3): .delta.
7.99 (2H, d, J=7.8 Hz), 7.88 (1H, d, J=1.5 Hz), 7.55 (2H, d, J=7.8
Hz), 7.54 (1H, s), 7.45 (1H, dd, J=1.5, 8.0 Hz), 7.35 (1H, d, J=7.9
Hz), 6.48 (1H, t, J=4.8 Hz), 4.38 (2H, q, J=7.1 Hz), 2.51 (3H, s),
2.38 (2H, d, J=4.8 Hz), 1.40 (3H, s), 1.32 (6H, s).
[0298] Ethyl
4-r(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)e-
thenyl]benzoate (Compound 15a)
[0299] A solution of 2-lithiothiazole was prepared by the addition
of 41.2 mg (0.42 ml, 0.63 mmol) of n-butyl-lithium (1.5M solution
in hexanes) to a cold solution (-78.degree. C.) of 53.4 mg (0.63
mmol) of thiazole in 1.0 ml of THF. The solution was stirred at for
30 minutes and then a solution of 113.9 mg (0.84 mmol) of zinc
chloride in 1.5 ml of THF was added. The resulting solution was
warmed to room temperature, stirred for 30 minutes and then the
organozinc was added via cannula to a solution of 200.0 mg (0.42
mmol) of ethyl 4-[(5,6-dihydro-5,5-dimethyl-8-(trifluorome-
thylsulfonyl)oxy-2-naphthalenyl)ethynyl]benzoate (Compound G) and
12.4 mg (0.01 mmol) of tetrakis(triphenylphosphine)palladium(0) in
1.5 ml of THF. The resulting solution was heated at 50.degree. C.
for 45 minutes, cooled to room temperature and diluted with sat.
aqueous NH.sub.4Cl. The mixture was extracted with EtOAc (40 ml)
and the combined organic layers were washed with water and brine.
The organic phase was dried over Na.sub.2SO.sub.4 and concentrated
in vacuo to a yellow oil. Purification by column chromatography
(silica, 20% EtOAc-hexanes) yielded the title compound as a
colorless oil. PMR (CDCl.sub.3): .delta. 1.35 (6H, s), 1.40 (3H, t,
J=7.1 Hz), 2.42 (2H, d, J=4.8 Hz), 4.38 (2H, q, J=7.1 Hz), 6.57
(1H, t, J=4.8 Hz), 7.33 (1H, d, J=3.3 Hz), 7.36 (1H, d, J=8.0 Hz),
7.46 (1H, dd, J=1.7 , 8.1 Hz), 7.55 (2H, d, J=8.4 Hz), 7.87 (1H, d,
J=1.7 Hz), 7.92 (1H, d, J=3.3 Hz), 8.00 (2H, d, J=8.4 Hz).
[0300] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylthiazol-2-yl)-2-napht-
halenyl)ethynyl]benzoate (Compound 16)
[0301] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5dimethyl-8-4-methylphenyl)-2-naphthalenyl)ethyny-
l]benzoate (Compound 1), 295.0 mg (0.617 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 168.0 mg (1.23 mmol) of zinc
chloride, 16 mg (0.014 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 6.0 ml of THF, and
4-methylthiazol-2-yl lithium (prepared by adding 59.6 mg (0.60 ml,
0.93 mmol) of n- butyllithium (1.55 M solution in hexanes) to a
cold solution (-78.degree. C.) of 92.0 mg (0.93 mmol) of
4-methylthiazole in 6.0 ml of THF). 1H NMR (CDCl.sub.3): .delta.
8.00 (2H, d, J=8.4 Hz), 7.80 (1H, d, J=1.7 Hz), 7.55 (2H, d, J=8.4
Hz), 7.45 (1H, dd, J=1.7, 8.0 Hz), 7.35 (1H, d, J=8.0 Hz), 6.87
(1H, s), 6.52 (1H, t, J=4.7 Hz), 4.37 (2H, q, J=7.2 Hz), 2.54 (3H,
s), 2.39 (2H, d, J=4.7 Hz), 1.40 (3H, t, J=7.2 Hz), 1.33 (3H,
s).
[0302] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4,5-dimethylthiazol-2-yl)-2-n-
aphthalenyl) ethynyl] benzoate (Compound 17)
[0303] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 200.0 mg (0.418 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 110.0 mg (0.84 mmol) of zinc
chloride, 12 mg (0.011 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and
4,5-dimethylthiazol-2-yl lithium (prepared by adding 40.2 mg (0.39
ml, 0.63 mmol) of n-butyllithium (1.55 M solution in hexanes) to a
cold solution (-78.degree. C.) of 71.0 mg (0.63 mmol) of
4,5-dimethylthiazole in 2.0 ml of THF). 1H NMR (CDCl.sub.3):
.delta. 8.00 (2H, d, J=8.4 Hz), 7.82 (1H, d, J=1.7 Hz), 7.54 (2H,
d, J=8.4 Hz), 7.43 (1H, dd, J=1.7, 8.0 Hz), 7.33 91H, d, J=8.0 Hz),
6.45 (1H, t, J=4.9 Hz), 4.38 (2H, q, J=7.1 Hz), 2.41 (3H, s), 2.40
(3H, s), 2.37 (2H, d, J=4.9 Hz), 1.40 (3H, t, J=7.1 Hz), 1.32 (6H,
s).
[0304]
4-[(5,6-Dihydro-5,5-dimethyl-8-(2-methyl-5-pyridyl)-2-naphthalenyl)-
ethynyl]benzoic acid (Compound 18)
[0305] A solution of 81.7 mg (0.194 mmol) of ethyl
4-[(5,6-dihydro-5,5-dim-
ethyl-8-(2-methyl-5-pyridyl)-2-naphthalenyl)ethynyl]benzoate
(Compound 12) and 40.7 mg (0.969 mmol) of LiOH--H.sub.2O in 3 ml of
THF/water (3:1, v/v), was stirred overnight at room temperature.
The reaction was quenched by the addition of saturated aqueous
NH.sub.4Cl and extracted with EtOAc. The combined organic layers
were washed with water and brine, dried over Na.sub.2SO.sub.4 and
concentrated in vacuo to give the title compound as a colorless
solid. 1H NMR (d6-DMSO): .delta. 8.41 (1H, d, J=1.9 Hz), 7.90 (2H,
d, J=8.3 Hz), 7.63 (1H, dd, J=2.3, 7.9 Hz), 7.59 (2H, d, J=8.3 Hz),
7.49 (2H, m), 7.33 (1H, d, J=7.8 Hz), 6.95 (1H, s), 6.11 (1H, t,
J=4.5 Hz), 2.52 (3H, s), 2.37 (2H, d, J=4.6 Hz), 1.31 (6H, s).
[0306]
4-[(5,6-Dihydro-5,5-dimethyl-8-(2-pyridyl)-2-naphthalenyl)ethynyl]b-
enzoic acid (Compound 19)
[0307] A solution of 80.0 mg (0.196 mmol) of ethyl
4-[(5,6-dihydro-5,5-dim-
ethyl-8-(2-pyridyl)-2-naphthalenyl)ethynyl]benzoate (Compound 10)
and 20.6 mg (0.491 mmol) of LiOH--H.sub.2O in 3 ml of THF/water
(3:1, v/v), was stirred overnight at room temperature. The reaction
was quenched by the addition of saturated aqueous NH.sub.4Cl and
extracted with EtOAc. The combined organic layers were washed with
water and brine, dried over Na.sub.2SO.sub.4 and concentrated in
vacuo to give the title compound as a colorless solid. 1H NMR
(d6-DMSO): .delta. 8.64 (1H, m), 7.94 (2H, d, J=8.3 Hz), 7.87 (1H,
dt, J=1.7, 7.8 Hz), 7.58 (2H, d, J=8.3 Hz), 7.50 (1H, d, J=8.2 Hz),
7.47 (2H, s), 7.37 (1H, m), 7.25 (1H, s), 6.30 (1H, t, J=4.6 Hz),
2.39 (2H, d, J=4.6 Hz), 1.31 (6H, s).
[0308]
4-[(5,6-Dihydro-5,5-dimethyl-8-(3-methylphenyl)-2-naphthalenyl)ethe-
nyl]benzoic acid (Compound 20)
[0309] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(3-methylphen-
yl)-2-naphthalenyl)ethynyl]benzoate (Compound 2) 30.0 mg (0.071
mmol) in 3 ml of EtOH and 2 ml of THF was added 28.0 mg (0.70 mmol,
0.7 ml) of NaOH (1.0 M aqueous solution). The solution was heated
to 50.degree. C. for 2 hours, cooled to room temperature, and
acidified with 10% HCl. Extraction with EtOAc, followed by drying
over Na.sub.2SO.sub.4, and removal of the solvents under reduced
pressure afforded the title compound as a colorless solid. 1H NMR
(DMSO): .delta. 7.90 (2H, d, J=8.5 Hz), 7.59 (2H, d, J=8.5 Hz),
7.46 (2H, s), 7.32-7.13 (4H, m), 7.10 (1H, s), 6.98 (1H, t, J=4.5
Hz), 2.34 (3H, s), 2.31 (2H, d, J=4.5 Hz), 1.30 (6H, s).
[0310]
4-[(5,6-Dihydro-5,5-dimethyl-8-(4-ethylphenyl)-2-naphthalenyl)ethyn-
yl]benzoic acid (Compound 21)
[0311] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-ethylpheny-
l)-2-naphthalenyl)ethynyl]benzoate (Compound 5) 47.0 mg (0.108
mmol) in 3 ml of EtOH and 2 ml of THF was added 28.0 mg (0.70 mmol,
0.7 ml) of NaOH (1.0 M aqueous solution). The solution was heated
to 50.degree. C. for 2 hours, cooled to room temperature, and
acidified with 10% HCl. Extraction with EtOAc, followed by drying
over Na.sub.2SO.sub.4, and removal of the solvents under reduced
pressure afforded the title compound as a colorless solid. 1H NMR
(DMSO): .delta. 7.90 (2H, d, J=8.3 Hz), 7.59 (2H, d, J=8.3 Hz),
7.46 (2H, s), 7.29-7.21 (4H, m), 7.02 (1H, s), 6.01 (1H, t, J=4.5
Hz), 2.64 (2H, q, J=7.5 Hz), 2.33 (2H, d, J=4.5 Hz), 1.29 (6H, s),
1.22 (3H, t, J=7.5 Hz)
[0312]
4-[(5,6-Dihydro-5,5dimethyl-8(4-methoxyphenyl)-2-naphthalenyl)ethyn-
yl]benzoic acid (Compound 22)
[0313] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methoxyphe-
nyl)-2-naphthalenyl)ethynyl]benzoate (Compound 8) 80.0 mg (0.183
mmol) in 3 ml of EtOH and 2 ml of THF was added 40.0 mg (1.00 mmol,
1.0 ml) of NaOH (1.0 M aqueous solution). The solution was heated
to 50.degree. C. for 2 hours, cooled to room temperature, and
acidified with 10% HCl. Extraction with EtOAc, followed by drying
over Na.sub.2SO.sub.4, and removal of the solvents under reduced
pressure afforded the title compound as a colorless solid. 1H NMR
(DMSO): .delta. 7.90 (2H, d, J=8.3 Hz), 7.60 (2H, d, J=8.3 Hz),
7.45 (2H, s), 7.24 (2H, d, J=8.6 Hz), 7.02-6.89 (3H, m), 5.98 (1H,
t, J=4.4 Hz), 3.79 (3H, s), 2.31 (2H, d, J=4.7 Hz), 1.29 (6H,
s).
[0314]
4-[(5,6-Dihydro-5,5dimethyl-8-(4-trifluoromethylphenyl)-2-naphthale-
nyl)ethynyl]benzoic acid (Compound 23)
[0315] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-trifiuorom-
ethylphenyl)-2-naphthalenyl)ethynyl]benzoate (Compound 9) 70.0 mg
(0.148 mmol) in 3 ml of EtOH and 2 ml of THF was added 60.0 mg
(1.50 mmol, 1.50 ml) of NaOH (1.0 M aqueous solution). The solution
was heated to 50.degree. C. for 2 hours, cooled to room
temperature, and acidified with 10% HCl. Extraction with EtOAc,
followed by drying over NaSO.sub.4, and removal of the solvents
under reduced pressure afforded the title compound as a colorless
solid. 1H NMR (DMSO): .delta. 7.90 (2H, d, J=8.3 Hz), 7.80 (2H, d,
J=8.1 Hz), 7.61-7.47 (6H, m), 6.97 (2H, s), 6.16 (1H, t, J=4.5 Hz),
2.37 (2H, d, J=4.6 Hz), 1.30 (6H, s).
[0316]
4-[(5,6-Dihydro-5,5-dimethyl-8-(3,5-dimethylphenyl)-2-naphthalenyl)-
ethenyl]benzoic acid (Compound 24)
[0317] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(3,5-dimethyl-
phenyl)-2-naphthalenyl)ethynyl]-benzoate (Compound 4) 90.0 mg
(0.207 mmol) in 3 ml of EtOH and 2 ml of THF was added 48.0 mg
(1.20 mmol, 1.20 ml) of NaOH (1.0 M aqueous solution). The solution
was heated to 50.degree. C. for 2 hours, cooled to room
temperature, and acidified with 10% HCl. Extraction with EtOAc,
followed by drying over Na.sub.2SO.sub.4, and removal of the
solvents under reduced pressure afforded the title compound as a
colorless solid. 1H NMR (DMSO): .delta. 7.90 (2H, d, J=8.2 Hz),
7.59 (2H, d, J=8.2 Hz), 7.45 (2H, s), 7.00 (1H, s), 6.97 (1H, s),
5.97 (1H, t, J=4.5 Hz), 2.31 (2H, d, J=4.5 Hz), 2.30 (6H, s), 1.29
(6H, s).
[0318]
4-[(5,6-Dihydro-5,5-dimethyl-8-(4-chlorophenyl)-2-naphthalenyl)ethe-
nyl]benzoic acid (Compound 25)
[0319] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-chlorophen-
yl)-2-naphthalenyl)ethynyl]benzoate (Compound 7) 80.0 mg (0.181
mmol) in 3 ml of EtOH and 2 ml of THF was added 48.0 mg (1.20 mmol,
1.20 ml) of NaOH (1.0 M aqueous solution). The solution was heated
to 50.degree. C. for 2 hours, cooled to room temperature, and
acidified with 10% HCl. Extraction with EtOAc, followed by drying
over Na.sub.2SO.sub.4, and removal of the solvents under reduced
pressure afforded the title compound as a colorless solid. 1H NMR
(DMSO): .delta. 7.90 (2H, d, J=8.3 Hz), 7.60 (2H, d, J=8.3 Hz),
7.51-7.48 (4H, m), 7.34 (2H, d, J=8.4 Hz), 6.97 (1H, s), 6.07 (1H,
t, J=4.5 Hz), 2.34 (2H, d, J=4.6 Hz), 1.29 (6H, s).
[0320]
4-[(5,6-Dihydro-5,5-dimethyl-8-(3-pyridyl)-2-naphthalenyl)ethynyl]b-
enzoic acid (Compound 26)
[0321] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(3-pyridyl)-2-
-naphthalenyl)ethynyl]benzoate (Compound 11) 45.0 mg (0.110 mmol)
in 3 ml of EtOH and 2 ml of THF was added 48.0 mg (1.20 mmol, 1.20
ml) of NaOH (1.0 M aqueous solution). The solution was heated to
50.degree. C. for 2 hours, cooled to room temperature, and
acidified with 10% HCl. Extraction with EtOAc, followed by drying
over Na.sub.2SO.sub.4, and removal of the solvents under reduced
pressure afforded the title compound as a colorless solid. 1H NMR
(DMSO): .delta. 8.60 (1H, d, J=4.6 Hz), 8.55 (1H, s), 7.90 (2H, d,
J=8.3 Hz), 7.76 (1H, d, J=7.5 Hz), 7.60 (2H, d, J=8.3 Hz),
7.51-7.46 (3H, m), 6.94 (1H, s), 6.14 (1H, t, J=4.5 Hz), 2.37 (2H,
d, J=4.5 Hz), 1.31 (6H, s).
[0322]
4-[(5,6-Dihydro-5,5-dimethyl-8-(2-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoic acid (Compound 27)
[0323] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(2-methylphen-
yl)-2-naphthalenyl)ethynyl]-benzoate (Compound 3) 80.0 mg (0.190
mmol) in 3 ml of EtOH and 2 ml of THF was added 60.0 mg (1.50 mmol,
1.50 ml) of NaOH (1.0 M aqueous solution). The solution was heated
to 50.degree. C. for 2 hours, cooled to room temperature, and
acidified with 10% HCl. Extraction with EtOAc, followed by drying
over Na.sub.2SO.sub.4, and removal of the solvents under reduced
pressure afforded the title compound as a colorless solid. 1H NMR
(DMSO): .delta. 7.89(2H, d, J=8.4 Hz), 7.57 (2H, d, J=8.4 Hz), 7.46
(2H, s), 7.29-7.14 (4H, m), 6.59 (1H, s), 5.90 (1H, t, J=4.7 Hz),
2.39 (2H, m), 2.60 (3H, s), 1.39 (3H, s), 1.29 (3H, s).
[0324]
4-[(5,6-Dihydro-5,5-dimethyl-8-(3-hydroxyphenyl)-2-naphthalenyl)eth-
ynyl]benzoic acid (Compound 28)
[0325] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(3-((2,2-dime-
thylethyl)-dimethylsiloxy)phenyl)-2-naphthalenyl)ethynyl]benzoate
(Compound H) 40.0 mg (0.076 mmol) in 3 ml of EtOH and 2 ml of THF
was added 40.0 mg (1.00 mmol, 1.00 ml) of NaOH (1.0 M aqueous
solution). The solution was heated to 50.degree. C. for 2 hours,
cooled to room temperature, and acidified with 10% HCl. Extraction
with EtOAc, followed by drying over Na.sub.2SO.sub.4, and removal
of the solvents under reduced pressure afforded the title compound
as a colorless solid. 1H NMR (d6-acetone): .delta. 7.90 (2H, d,
J=8.3 Hz), 7.49 (2H, d, J=8.4 Hz), 7.35 (2H, s), 7.15-7.07 (2H, m),
6.77-6.69 (3H, m), 5.92 (1H, t, J=4.7 Hz), 2.25 (2H, d, J=4.7 Hz),
1.23 (6H, s).
[0326]
4-[(5,6-Dihydro-5,5-dimethyl-8-(4-hydroxyphenyl)-2-naphthalenyl)eth-
ynyl]benzoic acid (Compound 29)
[0327] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-((2,2-dime-
thylethyl)-dimethylsiloxy)phenyl)-2-naphthalenyl)ethynyl]benzoate
(Compound I)75.0 mg (0.143 mmol) in 3 ml of EtOH and 2 ml of THF
was added 60.0 mg (1.50 mmol, 1.50 ml) of NaOH (1.0 M aqueous
solution). The solution was heated to 50.degree. C. for 2 hours,
cooled to room temperature, and acidified with 10% HCl. Extraction
with EtOAc, followed by drying over Na.sub.2SO.sub.4, and removal
of the solvents under reduced pressure afforded the title compound
as a colorless solid. 1H NMR (d6-acetone): .delta. 8.01 (2H, d,
J=8.3 Hz), 7.59 (2H, d, J=8.4 Hz), 7.45 (2H, s), 7.20-7.17 (3H, m),
6.92-6.89 (2H, m), 5.97 (1H, t, J=4.7 Hz), 2.35 (2H, d, J=4.7 Hz),
1.34 (6H, s).
[0328]
4-[(5,6-Dihydro-5,5-dimethyl-8-(5-methylthiazol-2-yl)-2-naphthaleny-
l)ethynyl]benzoic acid (Compound 30)
[0329] To a solution of ethyl
4-[5,6-dihydro-5,5-dimethyl-8-(5-methylthiaz-
ol-2-yl)-2-naphthalenyl]ethynylbenzoate (Compound 15) (100 mg, 0.23
mmol) and 4 ml of EtOH at room temperature was added aqueous NaOH
(1 ml, 1 M, 1 mmol). The resulting solution was warmed to
50.degree. C. for 1 hour and concentrated in vacuo. The residue was
suspended in a solution of CH.sub.2Cl.sub.2 and ether (5:1) and
acidified to pH 5 with 1M aqueous HCl. The layers were separated
and the organic layer was washed with brine, dried
(Na.sub.2SO.sub.4), filtered and the solvents removed under reduced
pressure to give the title compound as a white solid. 1H NMR
(d6-DMSO): .delta. 7.96 (1H, d, J=1.7 Hz), 7.95 (2H, d, J=8.0 Hz),
7.65 (2H, d, J=8.0 Hz), 7.64 (1H, s), 7.53 (1H, dd, J=1.7, 8.0 Hz),
7.46 (1H, d, J=8.0 Hz), 6.59 (1H, t, J=4.5 Hz), 2.50 (3H, s), 2.39
(2H, d, J=4.5 Hz), 1.27 (6H, s).
[0330]
4-[(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl-
]benzoic acid (Compound 30a)
[0331] A solution of 33.9 mg (0.08 mmol) of ethyl
4-[(5,6-dihydro-5,5-dime-
thyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl]benzoate (Compound 15a)
and 8.5 mg (0.20 mmol) of LiOH--H.sub.2O in 3 ml of THF/water (3:1,
v/v), was stirred overnight at room temperature. The reaction was
quenched by the addition of sat. aqueous NH.sub.4Cl and extracted
with EtOAc. The combined organic layers were washed with water and
brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo to
give the title compound as a colorless solid. PMR (d.sub.6-DMSO):
.delta. 1.29 (6H, s), 2.42 (2H, d, J=4.6 Hz), 6.68 (1H, t, J=4.6
Hz), 7.51 (2H, m), 7.62 (2H, d, J=8.2 Hz), 7.77 (1H, d, J=3.3 Hz),
7.93 (2H, d, J=8.2 Hz), 7.98 (1H, d, J=3.3 Hz).
[0332]
4-[(5,6-Dihydro-5,5-dimethyl-8-(4-methylthiazol-2-yl)-2-naphthaleny-
l)ethynyl]benzoic acid (Compound 31)
[0333] To a solution of ethyl
4-[5,6-dihydro-5,5-dimethyl-8-(4-methylthiaz-
ol-2-yl)-2-naphthalenyl]ethynylbenzoate (Compound 16) (145.0 mg,
0.34 mmol) and 4 ml of EtOH at room temperature was added aqueous
NaOH (1 ml, 1 M, 1 mmol). The resulting solution was warmed to
50.degree. C. for 1 hour and concentrated in vacuo. The residue was
suspended in a solution of CH.sub.2Cl.sub.2 and ether (5:1) and
acidified to pH 5 with 1M aqueous HCl. The layers were separated
and the organic layer was washed with brine, dried
(Na.sub.2SO.sub.4), filtered and the solvents removed under reduced
pressure to give the title compound as a white solid. 1H NMR
(d6-DMSO): .delta. 7.94 (2H, d, J=8.1 Hz), 7.87 (1H, d, J=1.6 Hz),
7.63 (2H, d, J=8.3 Hz), 7.50 (1H, dd, J=1.6, 8.1 Hz), 7.45 (1H, d,
J=8.1 Hz), 7.27 (1H, s), 6.58 (1H, t, J=4.8 Hz), 2.43 (3H, s), 2.37
(2H, d, J=4.8 Hz), 1.26 (6H, s).
[0334]
4-[(5,6-Dihydro-5,5-dimethyl-8-(4,5-dimethylthiazol-2-yl)-2-naphtha-
lenyl)ethynyl]benzoic acid (Compound 32)
[0335] To a solution of ethyl
4-[5,6-dihydro-5,5-dimethyl-8-(4,5-dimethylt-
hiazol-2-yl)-2-naphthalenyl]ethynylbenzoate (Compound 17) (58.0 mg,
0.13 mmol) and 4 ml of EtOH at room temperature was added aqueous
NaOH (1 ml, 1 M, 1 mmol). The resulting solution was warmed to
50.degree. C. for 1 hour and concentrated in vacuo. The residue was
suspended in a solution of CH.sub.2Cl.sub.2 and ether (5:1) and
acidified to pH 5 with 1M aqueous HCl. The layers were separated
and the organic layer was washed with brine, dried
(Na.sub.2SO.sub.4), filtered and the solvents removed under reduced
pressure to give the title compound as a white solid. 1H NMR
(d6-DMSO): .delta. 7.94 (2H, d, J=8.4 Hz), 7.86 (1H, d, J=1.6 Hz),
7.61 (2H, d, J=8.3 Hz), 7.50 (1H, dd, J=1.6, 8.0 Hz), 7.45 (1H, d,
J=8.0 Hz), 6.51 (1H, t, J=4.9 Hz), 2.37 (3H, s), 2.36 (2H, d, J=4.6
Hz), 2.32 (3H, s), 1.26 (6H, s).
[0336] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(5-methyl-2-thienyl)-2-naphtha-
lenyl) ethynyl]benzoate (Compound 33)
[0337] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 170.0 mg (0.366 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 202.0 mg (1.48 mmol) of zinc
chloride, 24 mg (0.022 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and
5-methyl-2-lithiothiophene (prepared by adding 58.6 mg (0.36 ml,
0.915 mmol) of n-butyllithium (2.5 M solution in hexanes) to a cold
solution (-78.degree. C.) of 89.8 mg (0.915 mmol) of
2-methylthiophene in 2.0 ml of THF). 1H NMR (CDCl.sub.3): .delta.
8.00 (2H, d, J=8.3 Hz), 7.59 (1H, d, J=1.7 Hz), 7.55 (2H, d, J=8.2
Hz), 7.43 (1H, dd, J=1.7, 8.0 Hz), 7.35 (1H, d, J=8.0 Hz), 6.87
(1H, d, J=3.5 Hz), 6.74 (1H, d, J=2.8 Hz), 6.15 (1H, t, J=4.8 Hz),
4.38 (2H, q, J=7.1 Hz), 2.52 (3H, s), 2.32 (2H, d, J=4.8 Hz). 1.40
(3H, t, 7.1 Hz), 1.32 (6H, s).
[0338] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(2-thienyl)-2-naphthalenyl)eth-
ynyl]benzoate (Compound 33a)
[0339] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 250.0 mg (0.52 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 186.8 mg (1.37 mmol) of zinc
chloride 37.1 mg (0.03 mmol) of
tetrakis(triphenylphosphine)palladium(0) and 2-lithiothiophene
(prepared by the addition of 65.9 mg (0.69 ml, 1.03 mmol) of
n-butyllithium (1.5M solution in hexane) to a cold solution
(-78.degree. C.) of 86.5 mg (1.03 mmol) of thiophene in 1.0 ml of
THF). PMR (CDCl.sub.3): .delta. 1.33 (6H, s), 1.36 (3H, t, J=7.1
Hz), 2.38 (2H, d, J=4.7 Hz), 4.34 (2H, q, J=7.2 Hz), 6.25 (1H, t,
J=4.7 Hz), 7.13 (2H, m), 7.47 (4H, m), 7.62 (2H, d, J=8.5 Hz), 8.00
(2H, d, J=8.5 Hz).
[0340]
4-[(5,6-Dihydro-5.5-dimethyl-8-(5-methyl-2-thienyl)-2-naphthalenyl)-
ethynyl]benzoic acid (Compound 34)
[0341] To a solution of ethyl
4-[5,6-dihydro-5,5-dimethyl-8-(5-methyl-2-th-
ienyl)-2-naphthalenyl]ethynylbenzoate (Compound 33) (35.0 mg, 0.082
mmol) in 2 ml of EtOH and 1 ml THF at room temperature was added
aqueous NaOH (1 ml, 1 M, 1 mmol). The resulting solution was
stirred at room temperature overnight and then acidified with 10%
HCl. Extraction with EtOAc, followed by drying over
Na.sub.2SO.sub.4, and removal of the solvents under reduced
pressure afforded the title compound as a colorless solid. 1H NMR
(d6-acetone): .delta. 8.03 (2H, d, J=8.6 Hz), 7.63 (2H, d, J=8.6
Hz), 7.54-7.48 (3H, m), 6.89 (1H, m), 6.18 (1H, t, J=4.7 Hz), 2.49
(3H, s), 2.35 (2H, d, J=4.7 Hz), 1.32 (6H, s).
[0342]
4-[(5,6-dihydro-5,5-dimethyl-8-(2-thienyl)-2-naphthalenyl)ethynyl]b-
enzoic acid (Compound 34a)
[0343] Employing the same general procedure as for the preparation
of
4-[(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl]benzo-
ic acid (Compound 30a), 70.0 mg (0.17 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(2-thienyl)-2-naphthalenyl)ethynyl]benzoat-
e (Compound 33a) was converted into the title compound (colorless
solid) using 17.8 mg (0.42 mmol) of LiOH in H.sub.2O. PMR
(d.sub.6-DMSO): .delta. 1.27 (6H, s), 2.33 (2H, d, J=4.9 Hz), 6.23
(1H, t, J=4.9 Hz), 7.14 (2H, m), 7.38-7.56 (4H, m), 7.61 (2H, d,
J=8.3 Hz), 7.92 (2H, d, J=8.3 Hz).
[0344]
5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenecarboxylic
acid (Compound K)
[0345] A solution of
3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-7-bromona- phthalene
(Compound D) (250.0 mg, 0.764 mmol) in 2.0 ml of THF was cooled to
-78.degree. C. and 1.0 ml of t-butyllithium (1.68 mmol, 1.7 M
solution in pentane) was added slowly. After stirring for 1 hour at
-78.degree. C. gaseous CO.sub.2 (generated by evaporation of
Dry-Ice, and passed though a drying tube) was bubbled through the
reaction for 1 hour. The solution was then allowed to warm to room
temperature and the reaction was quenched by the addition of 10%
HCl. Extraction with EtOAc was followed by washing the combined
organic layers with H.sub.2O and saturated aqueous NaCl, and drying
over MgSO.sub.4. Removal of the solvents under reduced pressure and
washing of the solid with hexanes afforded the title compound as a
colorless solid. 1H NMR (CDCl.sub.3): .delta. 7.94 (1H, dd, J=1.8,
8.1 Hz), 7.76 (1H, d, J=1.8 Hz), 7.45 (1H, d, J=8.1 Hz), 7.24 (4H,
m), 6.01 (1H, t, J=4.7 Hz), 2.40 (3H, s), 2.36 (2H, d, J=4.7 Hz),
1.35 (6H, s).
[0346] Ethyl
4-[[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalen-
yl)carbonyl]amino]-benzoate (Compound 35)
[0347] A solution of 170.0 mg (0.58 mmol)
5,6-dihydro-5,5-dimethyl-8-(4-me-
thylphenyl)-2-naphthalenecarboxylic acid (Compound K) 115.0 mg
(0.70 mmol) of ethyl 4-aminobenzoate, 145.0 mg (0.76 mmol) of
1-(3-dimethylaminopropy- l)-3-ethylcarbodiimide hydrochloride, and
92.4 mg (0.76 mmol) of 4-dimethylaminopyridine in 6.0 ml of DMF was
stirred overnight at room temperature. Ethyl acetate was added and
the resulting solution washed with H.sub.2O, saturated aqueous
NaHCO.sub.3, and saturated aqueous NaCl, then dried over
MgSO.sub.4. After removal of the solvent under reduced pressure,
the product was isolated as a colorless solid by column
chromatography (10 to 15% EtOAc/hexanes). 1H NMR (CDCl.sub.3):
.delta. 8.02 (2H, d, J=8.7 Hz), 7.72 (2H, m), 7.65 (2H, d, J=8.7
Hz), 7.52 (1H, d, J=1.8 Hz), 7.48 (1H, d, J=8.0 Hz), 7.25 (4H, m),
6.15 (1H, t, J=4.9 Hz), 4.36 (2H, q, J=7.1 Hz), 2.40 (3H, s), 2.38
(2H, d, J=4.9 Hz), 1.39 (3H, t, J=7.1 Hz), 1.37 (6H, s).
[0348]
4-[[(5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)car-
bonyl]amino]-benzoic acid (Compound 36)
[0349] To a solution of 26.5 mg (0.06 mmol) ethyl
4[[(5,6-dihydro-5,5-dime-
thyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl]amino]-benzoate
(Compound 35) in 3.0 ml EtOH and 4.0 ml of THF was added 240.1 mg
NaOH (6.00 mmol, 3.0 ml of a 2M aqueous solution). After stirring
at room temperature for 72 hours, the reaction was quenched by the
addition of 10% HCl. Extraction with EtOAc, and drying of the
organic layers over MgSO.sub.4, provided a solid after removal of
the solvent under reduced pressure. Crystallization from CH.sub.3CN
afforded the title compound as a colorless solid. 1H NMR (d6-DMSO):
.delta. 10.4 (1H, s), 7.91-7.81 (5H, m), 7.54 (1H, d, J=8.1 Hz),
7.45 (1H, d, J=1.7 Hz), 7.23 (4H, s), 6.04 (1H, t, J=4.7 Hz), 2.35
(5H, s), 1.33 (6H, s).
[0350] Ethyl
4-[[(5,6dihydro-5,5-dimethyl-8-(4-methyl-phenyl)-2-naphthalen-
yl)carbonyl]oxy]-benzoate (Compound 37)
[0351] A solution of 25.0 mg (0.086 mmol)
5,6-dihydro-5,5-dimethyl-8-(4-me-
thylphenyl)-2-naphthalenecarboxylic acid (Compound K) 17.5 mg
(0.103 mmol) of ethyl 4-hydroxybenzoate, 21.4 mg (0.112 mmol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and
12.6 mg (0.103 mmol) of 4-dimethylaminopyridine in 2.0 ml of DMF
was stirred overnight at room temperature. Ethyl acetate was added
and the resulting solution washed with H.sub.2O, saturated aqueous
NaHCO.sub.3, and saturated aqueous NaCl, before being dried over
MgSO.sub.4. After removal of the solvent under reduced pressure,
the product was isolated by column chromatography as a pale-yellow
solid (10% EtOAc/hexanes). 1H NMR (CDCl.sub.3): .delta. 8.08 (2H,
d, J=8.1 Hz), 8.05 (1H, dd, J=1.8, 8.1 Hz), 7.89 (1H, d, J=1.8 Hz),
7.50 (2H, d, J=8.1 Hz), 7.22 (5H, m), 6.05 (1H, t, J=4.7 Hz), 4.37
(2H, q, J=7.1 Hz), 2.39 (2H, d, J=4.7 Hz), 2.38 (3H, s), 1.39 (3H,
t, J=7.1 Hz), 1.37 (6H, s).
[0352] 2-Trimethylsilylethyl
4-[[(5,6-dihydro-5,5-dimethyl-8-(4-methylphen-
yl)-2-naphthalenyl)carbonyl]oxy]-benzoate (Compound 38)
[0353] A solution of 93.5 mg (0.320 mmol)
5,6-dihydro-5,5-dimethyl-8-(4-me-
thylphenyl)-2-naphthalenecarboxylic acid (Compound K) 76.0 mg
(0.319 mmol) of 2-trimethylsilylethyl-4-hydroxybenzoate, 80.0 mg
(0.417 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride, and 51.0 mg (0.417 mmol) of 4-dimethylaminopyridine
in 4.0 ml of DMF was stirred overnight at room temperature. Ethyl
acetate was added and the resulting solution washed with H.sub.2O,
saturated aqueous NaHCO.sub.3, and saturated aqueous NaCl, before
being dried over MgSO.sub.4. After removal of the solvent under
reduced pressure, the product was isolated as a colorless solid by
column chromatography (5% EtOAc/hexanes). 1H NMR (CDCl.sub.3):
.delta. 8.08 (2H, d, J=8.8 Hz), 8.05 (1H, dd, J=1.8, 8.1 Hz), 7.50
(1H, d, J=8.1 Hz), 7.26-7.18 (6H, m), 6.05 (1H, t, J=4.7 Hz), 4.42
(2H, t, J=8.4 Hz), 2.40 (2H, d, J=4.7 Hz), 2.39 (3H, s), 1.38 (6H,
s), 0.09 (9H, s).
[0354]
4-[[(5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)car-
bonyl]oxy]-benzoic acid (Compound 39)
[0355] A solution of 110.0 mg (0.213 mmol) 2-trimethylsilylethyl
4[[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl]o-
xy]-benzoate (Compound 38) and 167.3 mg of tetrabutylammonium
flouride (0.640 mmol, 0.64 ml of a 1M solution in THF) in 2.0 ml
THF was stirred at room temperature for 22 hours. Ethyl acetate was
added and the resulting solution washed with H.sub.2O and saturated
aqueous NaCl then dried over MgSO.sub.4. Removal of the solvents
under reduced pressure and washing of the residual solid with EtOAc
and CH.sub.3CN afforded the title compound as a colorless solid. 1H
NMR (d6-acetone): .delta. 8.10 (2H, d, J=8.8 Hz), 8.06 (1H, dd,
J=2.0, 8.1 Hz), 7.82 (1H, d, J=1.9 Hz), 7.64 (1H, d, J=8.1 Hz),
7.35 (2H, d, J=8.6 Hz), 7.25 (4H, m), 6.08 (1H, t, J=4.7 Hz), 2.42
(2H, d, J=4.7 Hz), 2.35 (3H, s), 1.39 (6H, s).
[0356] Ethyl
2-fluoro-4-[[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-n-
aphthalenyl)carbonyl]amino]-benzoate (Compound 40)
[0357] A solution of 115.0 mg (0.41 mmol)
5,6-dihydro-5,5-dimethyl-8-(4-me-
thylphenyl)-2-naphthalenecarboxylic acid (Compound K) 89.0 mg (0.49
mmol) of ethyl 2-fluoro-4-aminobenzoate, 102.0 mg (0.53 mmol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and
65.0 mg (0.53 mmol) of 4-dimethylaminopyridine in 5.0 ml of DMF was
stirred at 50.degree. C. for 1 hour and then overnight at room
temperature. Ethyl acetate was added and the resulting solution
washed with H.sub.2O, saturated aqueous NaHCO.sub.3, and saturated
aqueous NaCl, before being dried over MgSO.sub.4. After removal of
the solvent under reduced pressure, the product was isolated as a
colorless solid by column chromatography (20% EtOAc/hexanes). 1H
NMR (CDCl.sub.3): .delta. 7.96 (1H, s), 7.89 (1H, t, J=8.4 Hz),
7.70 (2H, m), 7.52 (1H, d, J=1.9 Hz), 7.45 (1H, d, J=8.1 Hz), 7.23
(5H, m), 6.04 (1H, t, J=4.8 Hz), 4.36 (2H, q, J=7.1 Hz), 2.38 (3H,
s), 2.35 (2H, d, J=4.8 Hz), 1.39 (3H, t, J=7.1 Hz), 1.36 (6H,
s).
[0358]
2-Fluoro-4-[[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphtha-
lenyl)carbonyl]amino]-benzoic acid (Compound 41)
[0359] To a solution of 41.6 mg (0.091 mmol) ethyl
2-fluoro-4-[[(5,6-dihyd-
ro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl]amino]-benzoate
(Compound 40) in 2.0 ml EtOH and 2.0 ml of THF was added 40.0 mg
NaOH (1.00 mmol, 1.0 ml of a 1 M aqueous solution). After stirring
at room temperature for overnight, the reaction was quenched by the
addition of 10% HCl. Extraction with EtOAc, and drying of the
organic layers over MgSO.sub.4, provided a solid after removal of
the solvent under reduced pressure. Crystallization from CH.sub.3CN
afforded the title compound as a pale-yellow solid. 1H NMR
(d6-acetone): .delta. 9.84 (1H, s), 7.94-7.83 (3H, m), 7.64 (1H,
dd, J=2.0 Hz), 7.53 (2H, d, J=8.1 Hz), 7.23 (4H, s), 6.04 (1H, t,
J=4.7 Hz) 2.38 (2H, d, J=4.7 Hz), 2.36 (3H, s), 1.35 (6H, s).
[0360] Ethyl
4[[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthaleny-
l)thiocarbonyl]amino]-benzoate (Compound 42)
[0361] A solution of 110.0 mg (0.25 mmol) ethyl
4-[[(5,6-dihydro-5,5-dimet-
hyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl]amino]-benzoate
(Compound 35) and 121.0 mg (0.30 mmol) of
[2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4--
diphosphetane-2,4-disulfide] (Lawesson's Reagent) in 12.0 ml of
benzene was refluxed overnight. Upon cooling to room temperature,
the mixture was filtered and the filtrate concentrated under
reduced pressure. The title compound was isolated by column
chromatography (10 to 25% EtOAc/hexanes) as a yellow solid. 1H NMR
(CDCl.sub.3): .delta. 8.92 (1H, s), 8.06 (2H, t, J=8.5 Hz),
7.88-7.70 (3H, m), 7.42 (2H, d, J=8.1 Hz), 7.18 (4H, m), 6.03 (1H,
t, J=4.7 Hz), 4.37 (2H, q, J=7.1 Hz), 2.38 (3H, s), 2.36 (2H, d,
J=4.7 Hz), 1.56 (3H, t, J=7.1 Hz), 1.35 (6H, s).
[0362]
4-[[(5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)thi-
ocarbonyl]amino]-benzoic acid (Compound 43)
[0363] To a solution of 84.0 mg (0.184 mmol) ethyl
4[[(5,6-dihydro-5,5-dim-
ethyl-8-(4-methylphenyl)-2-naphthalenyl)thiocarbonyl]amino]-benzoate
(Compound 42) in 2.0 ml EtOH and 2.0 ml of THF was added 60.0 mg
NaOH (1.50 mmol, 1.5 ml of a 1 M aqueous solution). After stirring
at room temperature overnight, the reaction was quenched by the
addition of 10% HCl. Extraction with EtOAc, and drying of the
organic layers over MgSO.sub.4, provided a solid after removal of
the solvent under reduced pressure. Crystallization from CH.sub.3CN
afforded the title compound as a yellow solid. 1H NMR (d6-acetone):
.delta. 10.96 (1H, s), 8.05 (4H, m), 7.72 (1H, dd, J=2.0, 8.0 Hz),
7.54 (1H, s), 7.46 (1H, d, J=8.1 Hz), 7.20 (4H, m), 6.04 (1H, t,
J=4.7 Hz), 2.38 (2H, d, J=4.7 Hz), 2.33 (3H, s), 1.35 (6H, s).
[0364] 2-acetyl-6-bromonaphthalene (Compound L)
[0365] To a cold (10.degree. C.) mixture of 44.0 g (0.212 mol) of
2-bromonaphthalene and 34.0 g (0.255 mol) of aluminum chloride in
400 ml of nitrobenzene was added 21.0 g (267 mmol) of acetyl
chloride. The mechanically stirred reaction mixture was warmed to
room temperature, and heated to 40.degree. C. for 18 hours. After
cooling to 0.degree. C. in an ice bath, the reaction was quenched
by the addition of 12M HCl (70 ml). The layers were separated and
the organic phase was washed with water and dilute aqueous
Na.sub.2CO.sub.3. Kugelrohr distillation, followed by
recrystallization from 10% EtOAc-hexane yielded 23 g of the title
compound as a tan solid. 1H NMR (CDCl.sub.3): .delta. 8.44 (1H, br
s), 8.04-8.10 (2H, m), 7.85 (1H, d, J=8.5 Hz), 7.82 (1H, d, J=8.8
Hz), 7.64 (1H, d, J=8.8 Hz), 2.73 (3H, s).
[0366] 6-bromo-2-naphthalenecarboxylic acid (Compound M)
[0367] To a solution of sodium hypochlorite (62 ml, 5.25% in water
(w/w), 3.6 g, 48.18 mmol) and sodium hydroxide (6.4 g, 160.6 mmol)
in 50 ml of water was added a solution of
2-acetyl-6-bromonaphthalene (Compound L) 4 g, (16.06 mmol) in 50 ml
of 1,4-dioxane. The yellow solution was heated to 70.degree. C. in
an oil bath for 2 hours, cooled to ambient temperature, and
extracted with ethyl ether (2.times.50 ml). The aqueous layers were
diluted with NaHSO.sub.3 solution (until KI indicator solution
remained colorless) and then acidified (pH <2) with 1N sulfuric
acid to give a white precipitate. The mixture was extracted with
ethyl ether, and the combined organic phase washed with saturated
aqueous NaCl, dried (MgSO.sub.4) and concentrated to give 3.54 g
(88%) of the title compound as a solid. 1H NMR (DMSO-d6): .delta.
8.63 (1H, br s), 8.32 (1H, d, J=2.0 Hz), 8.10 (1H, d, J=8.8 Hz),
8.00-8.05 (2H, m), 7.74 dd, J=2.0, 8.8 Hz).
[0368] Ethyl 6-bromo-2-naphthalenecarboxylate (Compound N)
[0369] To a solution of 6-bromo-2-naphthalenecarboxylic acid
(Compound M) 3.1 g, (12.43 mmol) in ethanol (30 ml, 23.55 g, 511.0
mmol) was added 18M sulfuric acid (2 ml). The solution was refluxed
for 30 minutes, cooled to room temperature, and the reaction
mixture partitioned between pentane (100 ml) and water (100 ml).
The aqueous phase was extracted with pentane (100 ml) and the
combined organic layers washed with saturated aqueous NaCl (100
ml), dried (MgSO.sub.4), and concentrated to yield an off-white
solid. Purification by flash. chromatography (silica, 10%
EtOAc-hexane) afforded the title compound as a white solid. 1H NMR
(CDCl.sub.3): .delta. 8.58 (1H, br s), 8.10 (1H, dd, J=1.7, 9 Hz),
8.06 (1H, d, J=2 Hz), 7.83 (1H, d, J=9 Hz), 7.80 (1H, d, J=9 Hz),
7.62 (1H, dd, J=2, 9 Hz).
[0370] Ethyl
(E)-4-[2-(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-naphthaleny-
l)ethenyl]-benzoate (Compound O)
[0371] To a solution of 520.0 mg (2.00 mmol) of
3,4-dihydro-4,4-dimethyl-7- -bromo-1(2H)-naphthalenone (Compound B)
and 510.0 mg (2.90 mmol) of ethyl 4-vinylbenzoate in 4.0 ml of
triethylamine (degassed by sparging with argon for 25 minutes), was
added 124.0 mg (0.40 mmol) of tris(2-methylphenyl) phosphine,
followed by 44.0 mg (0.20 mmol) of palladium(II)acetate. The
resulting solution was heated to 95.degree. C. for 2.5 hours,
cooled to room temperature, and concentrated under reduced
pressure. Purification by column chromatography (10% EtOAc/hexanes)
afforded the title compound as a colorless solid. 1H NMR
(CDCl.sub.3): .delta. 8.19 (1H, d, J=2.0 Hz), 8.03 (2H, d, J=8.4
Hz), 7.69 (1H, dd, J=2.0, 8.2 Hz), 7.57 (2H, d, J=8.4 Hz), 7.45
(1H, d, J=8.2 Hz), 7.20 (2H, s), 4.39 (2H, q, J=7.1 Hz), 2.76 (2H,
t, J=6.5 Hz), 2.04 (2H, t, J=6.5 Hz), 1.41(3H, t, J=7.1 Hz, and 6H,
s).
[0372] Ethyl
(E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl-
)oxy-2-naphthalenyl)ethenyl]-benzoate (Compound P)
[0373] To a cold (-78.degree. C.) solution of 440.0 mg (2.40 mmol)
of sodium bis(trimethylsilyl)amide in 10.0 ml of THF was added
700.0 mg (2.00 mmol) of ethyl
(E)-4-[2-(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-na-
phthalenyl)ethenyl]-benzoate (Compound O) as a solution in 25.0 ml
of THF. After stirring at -78.degree. C. for 1.5 hours, 960.0 mg
(2.40 mmol) of 2[N,N-
bis(trifluoromethylsulfonyl)amino]-5-chloropyridine was added in
one portion. After 30 minutes the solution was warmed to 0.degree.
C. and stirred for 3 hours. The reaction was quenched by the
addition of saturated aqueous NH.sub.4Cl, and extracted with EtOAc.
The combined extracts were washed with 5% aqueous NaOH, dried
(Na.sub.2SO.sub.4), and the solvents removed under reduced
pressure. The title compound was isolated as a colorless solid by
column chromatography (7% EtOAc/hexanes). 1H NMR (CDCl.sub.3):
.delta. 8.04 (1H, d, J=8.4 Hz), 7.57 (2H, d, J=8.4 Hz), 7.52 (1H,
s), 7.49 (1H, d, J=8.0 Hz), 7.33 (1H, d, J=8.0 Hz), 7.20 (1H, d,
J=16.4 Hz), 7.10 (1H, d, J=16.4 Hz), 6.00 (1H, t, J=4.9 Hz), 4.39
(2H, d, J=7.1 Hz), 2.43 (2H, d, J=4.9 Hz), 1.41 (3H, t, J=7.1 Hz),
1.32 (6H, s).
[0374]
Ethyl(E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphth-
alenyl)ethenyl]-benzoate (Compound 44)
[0375] A solution of 4-lithiotoluene was prepared at -78.degree. C.
by the addition of 130.7 mg of t-butyllithium (2.04 mmol; 1.20 ml
of a 1.7M solution in pentane) to a solution of 374.5 mg (2.20
mmol) of 4-bromotoluene in 2.5 ml of THF. After 30 minutes a
solution of 313.4 mg (2.30 mmol) of ZnCl.sub.2 in 2.0 ml of THF was
added. The resulting solution was warmed to room temperature,
stirred for 1.25 hour and then added via canula to a solution of
285.0 mg (0.590 mmol) of ethyl
(E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-napht-
halenyl)ethenyl]-benzoate (Compound P) and 29.0 mg (0.025 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF. The
resulting solution was stirred at room temperature for 1 hour and
then at 55.degree. C. for 2 hours. Upon cooling to room temperature
the reaction was quenched by the addition of saturated aqueous
NH.sub.4Cl. The mixture was extracted with EtOAc, and the combined
extracts were washed with 5% aqueous NaOH, saturated aqueous NaCl,
and dried over Na.sub.2SO.sub.4 before being concentrated under
reduced pressure. The title compound was isolated by column
chromatography (10% EtOAC/hexanes) as a colorless solid. 1H NMR
(CDCl.sub.3): .delta. 7.96 (2H, d, J=8.1 Hz), 7.47 (2H, d, J=8.1
Hz), 7.43-7.16 (7H, m), 7.07 (1H, d, J=16.3 Hz), 6.93 (1H, d,
J=16.3 Hz), 5.97 (1H, t, J=4.7 Hz), 4.39 (2H, q, J=7.0 Hz), 2.41
(3H, s), 2.33 (1H, d, J=4.7 Hz), 1.38 (3H, t, J=7.0 Hz), 1.33 (6H,
s).
[0376]
(E)-4-[2-(5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthaleny-
l)ethenyl]-benzoic acid Compound 45
[0377] To a solution of 65.0 mg (0.190 mmol) of ethyl
(E)4-[2-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethen-
yl]-benzoate (Compound 44) in 4.0 ml of THF was added 30.0 mg of
LiOH (0.909 mmol, 1.0 ml of a 1.1M solution) and 1.0 ml of MeOH.
The solution was heated to 55.degree. C. for 3 hours, cooled to
room temperature, and concentrated under reduced pressure. The
residue was dissolved in H.sub.2O and extracted with hexanes. The
aqueous layer was acidified to pH 1 with 10% HCl, and extracted
with Et.sub.2O. The combined organic layers were washed with
saturated aqueous NaCl, diluted with EtOAc to give a clear
solution, and dried over Na.sub.2SO.sub.4. The solvents were
removed under reduced pressure to give the title compound as a
colorless solid. 1H NMR (d6-DMSO): .delta. 7.86 (2H, d, J=8.4 Hz),
7.66 (2H, d, J 8.4 Hz), 7.58 (1H, dd, J=1.7, 8.1 Hz), 7.41 (1H, d,
J=8.1 Hz), 7.28 (1H, d, J=16.5 Hz), 7.23 (4H, s), 7.08 (1H, d,
J=1.7 Hz), 7.07 (1H, d, J=16.5 Hz), 5.97 (1H, t, J=4.6 Hz), 2.35
(3H, s), 2.31 (1H, d, J=4.6 Hz), 1.29 (6H, s).
[0378] Ethyl
4-[2-(1,1-dimethyl-3-(4-methylphenyl)-5-indenyl)ethynyl]benzo- ate
(Compound 47)
[0379] A solution of 32.0 mg (0.187 mmol) of 4-bromotoluene in 1.0
ml THF was cooled to -78.degree. C. and 24.0 mg of t-butyllithium
(0.375 mmol, 0.22 ml of a 1.7 M solution in pentane) was slowly
added. The yellow solution was stirred for 30 minutes at which time
29.8 mg (0.219 mmol) of ZnCl.sub.2 was added as a solution in 1.0
ml THF. The resulting solution was warmed to room temperature and
after 30 minutes added to a second flask containing 29.0 mg (0.062
mmol) of ethyl 4-[2-(1,1-dimethyl-3-(trif-
luoromethylsulfonyl)oxy-5-indenyl) ethynyl]benzoate (Compound FF)
and 2.9 mg (0.003 mmol) of tetrakis(triphenylphosphine)palladium
(0) in 1.0 ml THF. The resulting solution was warmed to 50.degree.
C. for 1 hour and then stirred at room temperature for 4 hours. The
reaction was quenched by the addition of saturated aqueous
NH.sub.4Cl, and then extracted with Et.sub.2O. The combined organic
layers were washed with water, saturated aqueous NaCl, and dried
over MgSO.sub.4 before being concentrated under reduced pressure.
The title compound was isolated as a colorless oil by column
chromatography (10% Et.sub.2O/hexanes). 1H NMR (300 MHz,
CDCl.sub.3): .delta. 8.03 (2H, d, J=8.5 Hz), 7.66 (1H, s), 7.58
(2H, d, J=8.5 Hz), 7.50 (2H, d, J=8.0 Hz), 7.46 (1H, d, J=7.9 Hz),
7.38 (1H, d, J=7.7 Hz), 7.28 (2H, d, J=9 Hz), 6.43 (1H, s), 4.40
(2H, q, J=7.2 Hz), 2.43 (3H, s), 1.41 (3H, t; +6H, s).
[0380]
4-[2-(1,1-dimethyl-3-(4-methylphenyl)-5-indenyl)ethynyl]benzoic
acid (Compoound 48)
[0381] To a solution of 10.0 mg (0.025 mmol) of ethyl
4-[2-(1,1-dimethyl-3-(4-methylphenyl)-5-indenyl)ethynyl]benzoate
(Compound 47) in 0.5 ml THF/H.sub.2O (3:1 v/v) was added 5.2 mg
(0.12 mmol) LiOH H.sub.2O. After stirring at room temperature for
48 hours the solution was extracted with hexanes and the aqueous
layer was acidified with saturated aqueous NH.sub.4Cl. Solid NaCl
was added and the resulting mixture extracted with EtOAc. The
combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure to give the title compound as a
colorless solid. 1H NMR (300 MHz, d.sub.6-DMSO): .delta. 7.95 (2H,
d, J=8.3 Hz), 7.65 (2H, d, J=8.3 Hz), 7.57 (2H, m), 7.49 (3H, m),
7.30 (2H, d, J=7.9 Hz), 6.61 (1H, s), 2.36 (3H, s), 1.36 (6H,
s).
[0382] 3-(4-bromothiophenoxy)propionic acid
[0383] To a solution of 1.44 g (35.7 mmol) of NaOH in 20.0 ml
degassed H.sub.2O (sparged with argon) was added 6.79 g (35.7 mmol)
of 4-bromothiophenol. The resulting mixture was stirred at room
temperature for 30 minutes. A second flask was charged with 2.26 g
(16.3 mmol) of K.sub.2CO.sub.3 and 15 ml of degassed H.sub.2O. To
this solution was added (in portions) 5.00 g (32.7 mmol) of
3-bromopropionic acid. The resulting potassium carboxylate solution
was added to the sodium thiolate solution, and the resulting
mixture stirred at room temperature for 48 hours. The mixture was
filtered and the filtrate extracted with benzene, and the combined
organic layers were dicarded. The aqueous layer was acidified with
10% HCl and extracted with EtOAc. The combined organic layers were
washed with saturated aqueous NaCl, dried over MgSO.sub.4, and
concentrated under reduced pressure. The resulting solid was
recrystallized from Et.sub.2O--hexanes to give the title compound
as off-white crystals. 1H NMR (CDCl.sub.3): .delta. 7.43 (2H, d,
J=8.4 Hz), 7.25 (2H, d, J=8.4 Hz), 3.15 (2H, t, J=7.3 Hz), 2.68
(2H, t, J=7.3 Hz).
[0384] 2,3-dihydro-6-bromo-(4H)-1-benzothiopyran-4-one
[0385] A solution of 3.63 g (13.9 mmol) of
3-(4-bromothiophenoxy)propionic acid in 60 ml methanesulfonic acid
was heated to 75.degree. C. for 1.5 hours. After cooling to room
temperature the solution was diluted with H.sub.2O and extracted
with EtOAc. The combined organic layers were washed with 2N aqueous
NaOH, H.sub.2O, and saturated aqueous NaCl and then dried over
MgSO.sub.4. Removal of the solvent under reduced pressure afforded
a yellow solid from which the product was isolated by column
chromatography (3% EtOAc-hexanes) as a pale-yellow solid. 1H NMR
(CDCl.sub.3): .delta. 8.22 (1H, d, J=2.1 Hz), 7.48 1H, dd,
J=2.1,8.3 Hz), 7.17 (1H, d, J=8.5 Hz), 3.24 (2H, t, J=6.4 Hz), 2.98
(2H, t, J=6.7 Hz).
[0386]
2,3-dihydro-6-(2-trimethylsilylethynyl)-(4H)-1-benzothiopyran-4-one
[0387] A solution of 1.00 g (4.11 mmol)
2,3-dihydro-6-bromo-(4H)-1-benzoth- iopyran-4-one and 78.3 mg (0.41
mmol) CuI in 15.0 ml THF and 6.0 ml Et.sub.2NH was sparged with
argon for 5 minutes. To this solution was added 2.0 ml (1.39 g,
14.2 mmol) of (trimethylsilyl)acetylene followed by 288.5 mg (0.41
mmol) of bis(triphenylphosphine)palladium(II) chloride. The
resulting dark solution was stirred at room temperature for 3 days
and then filtered through a pad of Celite, which was washed with
EtOAc. The filtrate was washed with H.sub.2O and saturated aqueous
NaCl before being dried over MgSO.sub.4. The title compound was
isolated as an orange oil by column chromatography (4%
EtOAc--hexanes). 1H NMR (CDCl.sub.3): .delta. 8.13 (1H, d, J=1.9
Hz), 7.36 (1H, dd, J=2.1, 8.2 Hz), 7.14 (1H, d, J=8.2 Hz), 3.19
(2H, d, J=6.3 Hz), 2.91 (2H, d, J=6.3 Hz), 0.21 (9H, s).
[0388] 2,3-dihydro-6-ethynyl-(4H)-1-benzothiopyran-4-one
[0389] A solution containing 600.0 mg (2.25 mmol) of
2,3-dihydro-6-(2-trimethylsilylethynyl)-(4H)-1-benzothiopyran-4-one
and 100.0 mg (0.72 mmol) K.sub.2CO.sub.3 in 15 ml MeOH was stirred
at room temperature for 20 hours. The solution was diluted with
H.sub.2O and extracted with Et.sub.2O. The combined organic layers
were washed with H.sub.2O and saturated aqueous NaCl before being
dried over MgSO.sub.4. Removal of the solvents under reduced
preesure afforded the title compound as an orange solid. 1H NMR
(CDCl.sub.3): .delta. 8.17 (1H, d, J=1.8 Hz), 7.40 (1H, dd, J=1.8,
8.2 Hz), 7.19 (1H, d, J=8.2 Hz), 3.22 (2H, t, J=6.3 Hz), 3.08 (1H,
s) 2.94 (2H, t, J=6.3 Hz).
[0390] Ethyl
4-[2-(6-(2,3-dihydro-(4H)-1-benzothiopyran-4-onyl))ethynyl]be-
nzoate
[0391] A solution of 405.0 mg (2.15 mmol)
2,3-dihydro-6-ethynyl-(4H)-1-ben- zothiopyran-4-one and 594.0 mg
(2.15 mmol) of ethyl 4-iodobenzoate in 15 ml Et.sub.3N and 3 ml THF
was sparged with argon for 15 minutes. To this solution was added
503.0 mg (0.72 mmol) of bis(triphenylphosphine)palladi- um(II)
chloride and 137.0 mg (0.72 mmol) CuI. This solution was stirred
for 20 hours at room temperature and then filtered through a pad of
Celite, which was washed with EtOAc. Removal of the solvents under
reduced pressure afforded a brown solid. Column chromatography (3%
EtOAc-hexanes) afforded the title compound as an orange solid. 1H
NMR (d.sub.6-acetone): .delta. 8.15 (1H, d, J=2.0 Hz), 8.02 (2H, d,
J=8.5 Hz), 7.69 (2H, d, J=8.5 Hz), 7.61 (1H, dd, J=2.1, 8.3 Hz),
7.40 (1H, d, J=8.2 Hz), 4.35 (2H, q, J=7.1 Hz), 3.40 (2H, t, J=6.3
Hz), 2.96 (2H, t, J=6.3 Hz), 1.37 (3H, t, J=7.1 Hz).
[0392] Ethyl
4-[2-(6-(4-(trifluoromethylsulfonyl)oxy-(2H)-1-benzothiopyran
nyl))ethynyl]benzoate
[0393] To a solution of 221.9 mg (1.21 mmol) of sodium
bis(trimethylsilyl)amide in 3.0 ml THF cooled to -78.degree. C. was
added 370.0 mg (1.10 mmol) of ethyl
4-[2-(6-(2,3-dihydro-(4H)-1-benzothiopyran--
4-onyl))ethynyl]benzoate in 4.0 ml THF. After 30 minutes, a
solution of
2-[N,N-bis(trifluoromethylsulfonyl)amino]-5-chloropyridine in 4.0
ml THF was slowly added. The reaction was slowly warmed to room
temperature and after 5 hours quenched by the addition of saturated
aqueous NH.sub.4Cl. The mixture was extracted with EtOAc, and the
combined organic layers were washed with 5% aqueous NaOH, H.sub.2O,
and saturated aqueous NaCl before being dried over MgSO.sub.4.
Removal of the solvents under reduced pressure, followed by column
chromatography (4% EtOAc-hexanes) afforded the title compound as a
pale-yellow solid. 1H NMR (d.sub.6-acetone): .delta. 8.12 (2H, d,
J=8.5 Hz), 7.66 (2H, d, J=8.5 Hz), 7.56 (1H, d, J=1.7 Hz), 7.49
(1H, dd, J=1.7, 8,1 Hz), 7.40 (1H, d, J=8.1 Hz), 6.33 (1H, t, J=5.7
Hz), 4.35 (2H, q, J=7.1 Hz), 3.82 (2H, d, J=5.7 Hz), 1.37 (3H, t,
J=7.1 Hz).
[0394] Ethyl
4-[2-(6-(4-(4-methylphenyl)-(2H)-1-benzothiopyranyl))ethynyl]-
benzoate (Compound 49)
[0395] To a solution of 120.8 mg (0.70 mmol) of 4-bromotoluene in
2.0 ml THF at -78.degree. C. was added 88.4 mg (1.38 mmol, 0.81 ml
of a 1.7 M solution in pentane) of t-butyllithium. After 30 minutes
a solution of 131.6 mg (0.97 mmol) ZnCl.sub.2 in 2.0 ml THF was
added and the reulting pale-yellow solution warmed to room
temperature. Stirring for 40 minutes was followed by addition of
this solution to a second flask containing 129.2 mg (0.28 mmol) of
ethyl 4-[2-(6-(4-(trifluoromethylsulfonyl)oxy-(2H-
)-1-benzothiopyranyl))ethynyl]benzoate, 14.0 mg (0.012 mmol)
tetrakis(triphenylphosphine)palladium (0), and 2.0 ml THF. The
resulting solution was heated to 50.degree. C. for 5 hours, cooled
to room temperature, and quenched by the addition of saturated
aqueous NH.sub.4Cl. The mixture was extracted with EtOAc, and the
combined organic layers were washed with H.sub.2O and saturated
aqueous NaCl, then dried (MgSO.sub.4) and concentrated to an orange
oil. The title compound was isolated as a colorless solid by column
chromatography (3 to 5% EtOAc-hexanes). 1H NMR (d.sub.6-acetone):
.delta. 7.98 (2H, d, J=8.3 Hz), 7.58 (2H, d, J=8.2 Hz), 7.44-7.38
(2H, m), 7.26-7.15 (5H, m), 6.14 (1H, t, J=5.8 Hz), 4.34 (2H, q,
J=7.1 Hz), 3.53 (2H, d, J=5.8 Hz), 2.37 (2H, s), 1.35 (3H, t, J=7.1
Hz).
[0396]
4-[2-(6-(4-(4-methylphenyl)-(2H)-1-benzothiopyranyl))ethynyl]-benzo-
ic acid (Compound 50)
[0397] To a solution of 29.0 mg (0.07 mmol) ethyl
4-[2-(6-(4-(4-methylphen-
yl)-(2H)-1-benzothiopyranyl))ethynyl]benzoate (Compound 49) in 2.0
ml THF and 2.0 ml EtOH was added 160.0 mg (4.00 mmol, 2.0 ml of a 2
M aqueous solution). The resulting solution was stirred at
35.degree. C. for 2 hours, and then cooled to room temperature and
stirred an additional 2 hours. The reaction was quenched by the
addition of 10% aqueous HCl and extracted with EtOAc. The combined
organic layers were washed with H.sub.2O and saturated aqueous
NaCl, and dried over Na.sub.2SO.sub.4 Removal of the solvents under
reduced pressure afforded a solid which was washed with CH.sub.3CN
and dried under high vacuum to give the title compound as a
pale-yellow solid. 1H NMR (d.sub.6-DMSO): .delta. 7.90 (2H, d,
J=8.4 Hz), 7.59 (2H, d, J=8.4 Hz), 7.40 (4H, m), 7.25-7.13 (4H, m),
7.02 (1H, d, J=1.7 Hz), 6.11 (1H, t, J=5.7 Hz), 3.54 (2H, d, J=5.7
Hz), 2.34 (3H, s).
[0398] 3,4-Dihydro-4,4-dimethyl-7-acetyl-1(2H)-naphthalenone
(Compound R): and
3,4-dihydro-4,4-dimethyl-6-acetyl-1(2H)-naphthalenone (Compound
S)
[0399] To a cold (0.degree. C.) mixture of aluminum chloride (26.3
g, 199.0 mmol) in dichloromethane (55 ml) was added acetylchloride
(15 g, 192 mmols) and 1,2,3,4-tetrahydro-1,1-dimethylnaphthalene
(24.4 g, 152 mmols) in dichloromethane (20 ml) over 20 minutes. The
reaction mixture was warmed to ambient temperature and stirred for
4 hours. Ice (200 g) was added to the reaction flask and the
mixture diluted with ether (400 ml). The layers were separated and
the organic phase washed with 10% HCl (50 ml), water (50 ml), 10%
aqueous sodium bicarbonate, and saturated aqueous NaCl (50 ml)
before being dried over MgSO.sub.4. Ths solvent was removed by
distillation to afford a yellow oil which was dissolved in benzene
(50 ml).
[0400] To a cold (0.degree. C.) solution of acetic acid (240 ml)
and acetic anhydride (120 ml) was added chromiumtrioxide (50 g, 503
mmols) in small portions over 20 minutes under argon. The mixture
was stirred for 30 mins at 0.degree. C. and diluted with benzene
(120 ml). The benzene solution prepared above was added with
stirring via an addition funnel over 20 minutes. After 8 hours, the
reaction was quenched by careful addition of isopropanol (50 ml) at
0.degree. C., followed by water (100 ml). After 15 minutes, the
reaction mixture was diluted with ether (1100 ml) and water (200
ml), and then neutralized with solid sodium bicarbonate (200 g).
The ether layer was washed with water (100 ml), and saturated
aqueous NaCl (2.times.100 ml), and dried over MgSO.sub.4. Removal
of the solvent under reduced pressure afforded a mixture of the
isomeric diketones which were separated by chromatography (5%
EtOAc/hexanes). (Compound R): 1H NMR (CDCl.sub.3): .delta. 8.55
(1H, d, J=2.0 Hz), 8.13 (1H, dd, J=2.0, 8.3 Hz), 7.53 (1H, d, J=8.3
Hz), 2.77 (2H, t, J=6.6 Hz), 2.62 (3H, s), 2.05 (2H, t, J=6.6 Hz),
1.41 (6H, s). (Compound S): 1H NMR (CDCl.sub.3): .delta. 8.10 (1H,
d, J=8.1 Hz), 8.02 (1H, d, J=1.6 Hz), 7.82 (1H, dd, J=1.6, 8.1 Hz),
2.77 (2H, t, J=7.1 Hz), 2.64 (3H, s), 2.05 (2H, t, J=7.1 Hz), 1.44
(6H, s).
[0401]
3,4-Dihydro-4.4-dimethyl-7-(2-(2-methyl-1,3-dioxolanyl))-1(2H)-naph-
thalenone (Compound T)
[0402] A mixture of 3,4-dihydro-4,4-dimethyl-7-acetyl-1
(2H)-naphthalenone (Compound R) (140.0 mg, 0.60 mmol), ethylene
glycol (55.0 mg, 0.90 mmol), p-toluenesulfonic acid monohydrate (4
mg) and benzene (25 ml) was refluxed using a Dean-Stark apparatus
for 12 hours. The reaction was quenched by the addition of 10%
aqueous sodium bicarbonate, and extracted with ether (2.times.75
ml). The combined organic layers were washed with water (5 ml), and
saturated aqueous NaCl (5 ml), and dried over MgSO.sub.4. Removal
of the solvent under reduced pressure afforded the title compound
as an oil. 1H NMR (CDCl.sub.3): .delta. 8.13 (1H, d, J=2.0 Hz),
7.64 (1H, dd, J=2.0, 8.2 Hz), 7.40 (1H, d, J=8.2 Hz), 3.97-4.10
(2H, m), 3.70-3.83 (2H, m), 2.73 (2H, t, J=6.5 Hz), 2.01 (2H, t,
J=6.5 Hz), 1.64 (3H, s), 1.39 (6H, s).
[0403]
1,2,3,4-Tetrahydro-1-hydroxy-1-(4-methylphenyl)-4,4-dimethyl-7-(2-(-
2-methyl-1,3-dioxolanyl))naphthalene (Compound U)
[0404] To a solution of 195.4 mg (1.00 mmol)
p-tolulylmagnesiumbromide (1.0 ml; 1M solution in ether) in 2 ml
THF was added a solution of
3,4-dihydro-4,4-dimethyl-7-(2-(2-methyl-1,3-dioxolanyl))-1(2H)-naphthalen-
one (Compound T) 135.0 mg, 0.52 mmol) in 5 ml THF. The solution was
refluxed for 16 hours, cooled to room temperature, and diluted with
ether (50 ml). The solution was washed with water (5 ml), saturated
aqueous NH.sub.4Cl (5 ml), and dried over MgSO.sub.4. Removal of
the solvents under reduced pressure and column chromatography (5%
EtOAc 1 hexanes) afforded the title compound as a solid. 1H NMR
(CDCl.sub.3): .delta. 7.37 (2H, d), 7.21 (1H, s), 7.13 (2H, d,
J=8.5 Hz), 7.08 (2H, d, J=8.5 Hz), 3.88-3.99 (2H, m), 3.58-3.75
(2H, m), 2.34 (3H, s), 2.12-2.30 (2H, m), 1.79-1.90 (1H, m), 1.57
(3H, s), 1.48-1.58 (1H, m), 1.38 (3H, s), 1.31 (3H, s).
[0405]
3,4-Dihydro-1-(4-methylphenyl)-4,4-dimethyl-7-acetylnaphthalene
(Compound V)
[0406] A mixture of
1,2,3,4-tetrahydro-1-hydroxy-1-(4-methylphenyl)-4,4-di-
methyl-7-(2-(2-methyl-1,3-dioxolanyl))naphthalene (Compound U)
130.0 mg (0.38 mmol), p-toluenesulfonic acid monohydrate (4 mg) and
benzene (5 ml) was refluxed for 16 hours. Upon cooling to room
temperature, the reaction mixture was diluted with ether (100 ml)
and washed with 10% aqueous sodium bicarbonate, water, and
saturated aqueous NaCl. The organic layer was dried over MgSO.sub.4
and the solvents were removed under reduced pressure to give the
title compound as a solid. 1H NMR (CDCl.sub.3): .delta. 7.83 (1H,
dd, J=1.8,8.0 Hz), 7.66 (1H, d, J=1.8 Hz), 7.45 (1H, d, J=8.0 Hz),
7.25 (2H, d, J=8.5 Hz), 7.22 (2H, d, J=8.5 Hz), 6.03 (1H, t, J=6.3
Hz), 2.47 (3H, s), 2.41 (3H, s), 2.37 (2H, d, J=6.3Hz), 1.36 (6H,
s).
[0407]
(E)-3-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)--
2-butenenitrile (Compound W)
[0408] To a slurry of NaH (48.0 mg, 2.00 mmol) in THF (6 ml), was
added diethylcyanomethylphosphonate (450.0 mg, 2.50 mmol). After 40
mins, a solution of
3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-7-acetylnaphthale- ne
(Compound V) 95.0 mg, (0.33 mmol) in THF (4 ml) was added. The
mixture was stirred for 16 hours, diluted with ether (100 ml), and
washed with water, and saturated aqueous NaCl before being dried
over MgSO.sub.4. Removal of the solvents under reduced pressure,
and column chromatography (3% EtOAc/hexanes) afforded the title
compound as a solid. 1H NMR (CDCl.sub.3): .delta. 7.39 (1H, d,
J=1H), 7.32 (1H, dd, J=2.0, 8.1Hz), 7.20-7.25 (4H, brs), 7.15 (1H,
d, J=2.0 Hz), 6.03 (1H, t, J=6.0 Hz), 5.44 (1H, s), 2.42 (3H, s),
2.36 (2H, d, J=6.0 Hz), 2.35 (3H, s), 1.35 (6H, s).
[0409]
(E)-3-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)--
2-butenal (Compound X)
[0410] To a cold solution (-78.degree. C.) of
(E)-3-(5,6-dihydro-5,5-dimet-
hyl-8-(4-methylphenyl)-2-naphthalenyl)-2-butenenitrile (Compound W)
84.0 mg, 0.29 mmol) in dichloromethane (4 ml) was added 0.50 ml
(0.50 mmol) of diisobutylaluminumhydride (1M solution in
dichloromethane). After stirring for 1 hour, the reaction was
quenched at -78.degree. C. by adding 2-propanol (1 ml) diluted with
ether (100 ml). Upon warming to room temperature, the solution was
washed with water, 10% HCl, and saturated aqueous NaCl. The organic
layer was dried over MgSO.sub.4 and the solvent removed under
reduced pressure to give the title compound as an oil. 1H NMR
(CDCl.sub.3): .delta. 10.12 (1H, d, J=7.9 Hz), 7.43 (2H, s),
7.19-7.28 (5H, m), 6.27 (1H, d, J=7.9 Hz), 6.03 (1H, t, J=4.8 Hz),
2.47 (3H, s), 2.42 (3H, s), 2.37 (2H, d, J=4.8 Hz), 1.37 (6H,
s).
[0411] Ethyl
(E,E,E)-3-methyl-7-(5.6-dihydro-5.5-dimethyl-8-(4-methylpheny-
l)-2-naphthalenyl)-2,4,6-octatrienoate (Compound 51)
[0412] To a cold (-78.degree. C.) solution of
diethyl-(E)-3-ethoxycarbonyl- -2-methylallylphosphonate [prepared
in accordance with J. Org. Chem. 39:821 (1974)] 264.0 mg, (1.00
mmol) in THF (2 ml) was added 26.0 mg (0.41 mmol, 0.65 ml)of
n-butyllithium in hexanes (1.6 M solution) followed immediately by
the addition of (E)-3-(5,6-dihydro-5,5-dimethyl-8-
-(4-methylphenyl)-2-naphthalen-yl)-2-butenal (Compound X) 82.0 mg,
0.26 mmol) in THF (3 ml). After 1 hour, the reaction mixture was
diluted with ether (60 ml), washed with water (5 ml), saturated
aqueous NaCl (5 ml) and dried over MgSO.sub.4. After removal of the
solvents under reduced pressure, the title compound was isolated as
an oil by column chromatography (5% EtOAc/hexanes, followed by HPLC
using 1% EtOAc/hexanes). 1H NMR (acetone-d6): .delta. 7.36-7.43
(2H, m), 7.18-7.27 (4H, m), 7.17 (1H, d, J=1.7 Hz), 7.08 (1H, dd,
J=11.2, 15.2 Hz), 6.46 (1H, d, J=11.2 Hz), 6.38 (1H, d, J=15.2 Hz),
5.98 (1H, t, J=4.7 Hz), 5.78 (1H, s), 4.10 (2H, q, J=7.1 Hz), 2.35
(3H, s), 2.33 (3H, s), 2.32 (2H, d, J=4.7 Hz), 2.12 (3H, s), 1.31
(6H, s), 1.22 (3H, t, J=7.1 Hz).
[0413]
(E,E,E)-3-methyl-7-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-n-
aphthalenyl)-2.4,6-octatrienoic acid (Compound 52)
[0414] To a solution of ethyl
(E,E,E)-3-methyl-7-(5,6-dihydro-5,5-dimethyl-
-8-(4-methylphenyl)-2-naphthalenyl)-2,4,6-octatrienoate (Compound
51) 85.0 mg, 0.20 mmol) in THF (1 ml) and methanol (1 ml) was added
12.0 mg (0.50 mmol) of LiOH (0.5 ml, 1M solution). The mixture was
stirred for 6 hours, diluted with ether (60 ml), acidified with 10%
HCl (1 ml). The solution was washed with water, and saturated
aqueous NaCl, before being dried over MgSO.sub.4. Removal of the
solvents under reduced pressure afforded the title compound as a
solid, which was purified by recrystallization from acetone. 1H NMR
(acetone-d6): .delta. 7.35-7.45 (2H, m), 7.19-7.28 (4H, m), 7.17
(1H, d, J=1.8Hz), 7.09 (1H, dd, J=11.5, 15.1 Hz), 6.48 (1H, d,
J=11.5 Hz), 6.42 (1H, d, J=15.1 Hz), 5.99 (1H, t, J=4.7 Hz), 5.82
(1H, s), 2.36 (3H, s), 2.33 (2H, d, J=4.7Hz), 2.32 (3H, s), 2.13
(3H, s), 1.32 (6H, s).
[0415] 3,4-dihydro-4,4-dimethyl-7-nitro-1(2H)-naphthalenone
(Compound Y)
[0416] To 1.7 ml (3.0 g, 30.6 mmol, 18M) H.sub.2SO.sub.4 at
-5.degree. C. (ice-NaCl bath) was slowly added 783.0 mg (4.49 mmol)
of 3,4-dihydro-4,4-dimethyl-1(2H)-naphthalenone. A solution of
426.7 mg (6.88 mmol, 0.43 ml, 16M) HNO.sub.3, and 1.31 g (0.013
mol, 0.74 ml, 18 M) H.sub.2SO.sub.4 was slowly added. After 20
minutes, ice was added and the resulting mixture extracted with
EtOAc. The combined extracts were concentrated under reduced
pressure to give a residue from which the title compound, a pale
yellow solid, was isolated by column chromatography (10%
EtOAC/hexanes). 1H NMR (CDCl.sub.3): .delta. 8.83 (1H, d, J=2.6
Hz), 8.31 (1H, dd, J=2.8, 8.9 Hz), 7.62 (1H, d, J=8.7 Hz), 2.81
(2H, t, J=6.5 Hz), 2.08 (2H, t, J=6.5 Hz), 1.45 (6H, s).
[0417] 3,4-dihydro-4,4-dimethyl-7-amino-1(2H)-naphthalenone
(Compound Z)
[0418] A solution of 230.0 mg (1.05 mmol)
3,4-dihydro-4,4-dimethyl-7-nitro- -1(2H)-naphthalenone (Compound Y)
in 5.0 ml of EtOAc was stirred at room temperature with a catalytic
amount of 10% Pd-C under 1 atm of H.sub.2 for 24 hours. The
catalyst was removed by filtration through a pad of Celite, and the
filtrate concentrated under reduced pressure to give the title
compound as a dark green oil. 1H NMR (CDCl.sub.3): .delta. 7.30
(1H, d, J=2.7 Hz), 7.22 (1H, d, J=8.4 Hz), 6.88 (1H, dd, J=2.7, 8.5
Hz), 2.70 (2H, t, J=6.6 Hz), 1.97 (2H, t, J=6.6 HZ), 1.34 (6H,
s).
[0419] Ethyl
4-[(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-naphthalenyl)azo]-
-benzoate (Compound AA)
[0420] To a solution of 198.7 mg (1.05 mmol)
3,4-dihydro-4,4-dimethyl-7-am- ino-1(2H)-naphthalenone (Compound Z)
in 5.0 ml glacial acetic acid was added 180.0 mg (1.00 mmol) of
ethyl 4-nitrosobenzoate. The resulting solution was stirred
overnight at room temperature, and then concentrated under reduced
pressure. The product was isolated from the residual oil as a red
solid, by column chromatography (15% EtOAc-hexanes). 1H NMR
(CDCl.sub.3): .delta. 8.57 (1H, d, J=2.0 Hz), 8.19 (2H, d, J=8.4
Hz), 8.07 (1H, d, J=8.0 Hz), 7.94 (2H, d, J=8.4 Hz), 7.58 (1H, d,
J=8.6 Hz), 4.41 (2H, q, J=7.1 Hz), 2.79 (2H, t, J=6.6 Hz), 2.07
(2H, t, J=7.02 Hz), 1.44 (6H, s), 1.42 (3H, t, J=7.1 Hz).
[0421] Ethyl
4-[(5,6-dihydro-5,5dimethyl-8-(trifluoromethylsulfonyl)oxy-2--
naphthalenyl)azo]-benzoate (Compound BB)
[0422] To a solution of 90.4 mg sodium bis(trimethylsilyl)amide
(0.48 mmol, 0.48 ml of a 1.0 M THF solution) in 2.0 ml THF at
-78.degree. C., was added 153.0 mg (0.437 mmol) of ethyl
4-[(5,6,7,8-tetrahydro-5,5-dimet-
hyl-8-oxo-2-naphthalenyl)azo]-benzoate (Compound AA) in 2.0 ml THF.
The dark red solution was stirred at -78.degree. C. for 30 minutes
and then 204.0 mg (0.520 mmol) of
2-[N,N-bis(trifluoromethylsulfonyl)amino]-5-chlo- ropyridine was
added as a solution in 2.0 ml THF. The reaction mixture was allowed
to warm to room temperature and after 3 hours it was quenched by
the addition of H.sub.2O. The organic layer was concentrated to a
red oil under reduced pressure. The product was isolated by column
chromatography (25% EtOAc/hexanes) as a red oil. 1H NMR
(CDCl.sub.3): .delta. 8.21 (2H, d, J=8.6 Hz), 7.96 (2H, d, J=8.6
Hz), 7.94 (2H, m), 7.49 (1H, d, J=8.2 Hz), 6.08 (1H, t, J=2.5 Hz),
4.42 (2H, q, J=7.1 Hz), 2.49 (2H, d, J=4.8 Hz), 1.44 (3H, t, J=7.1
Hz), 1.38 (6H, s).
[0423] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthaleny-
l)azo]-benzoate (Compound 46a)
[0424] A solution of 4-lithiotoluene was prepared by the addition
of 62.9 mg (0.58 ml, 0.98 mmol) of t-butyl lithium (1.7 M solution
in pentane) to a cold solution (-78.degree. C.) of 84.0 mg (0.491
mmol) of 4-bromotoluene in 1.0 ml of THF. After stirring for 30
minutes a solution of 107.0 mg (0.785 mmol) of zinc chloride in 2.0
ml of THF was added. The resulting solution was warmed to room
temperature, stirred for 30 minutes, and added via cannula to a
solution of 94.7 mg (0.196 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-napht-
halenyl)azo]-benzoate (Compound BB) and 25 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF. The
resulting solution was heated at 50.degree. C. for 1.5 hours,
cooled to room temperature and diluted with sat. aqueous
NH.sub.4Cl. The mixture was extracted with EtOAc (40 ml) and the
combined organic layers were washed with water and brine. The
organic phase was dried over Na.sub.2SO.sub.4, concentrated in
vacuo, and the title compound isolated as a red solid by column
chromatography (25% EtOAc-hexanes) 1H NMR (CDCl.sub.3): .delta.
8.21 (2H, d, J=8.6 Hz), 7.96 (2H, d, J=8.6 Hz), 7.94 (2H, m), 7.49
(1H, d, J=8.2 Hz), 6.08 (1H, t, J=2.5 Hz), 4.42 (2H, q, J=7.1 Hz),
2.49 (2H, d, J=4.8 Hz), 1.44 (3H, t, J=7.1 Hz), 1.38 (6H, s).
[0425]
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)azo]-
-benzoic acid (Compound 46b)
[0426] To a solution of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphen-
yl)-2-naphthalenyl)azo]-benzoate (Compound 46a) 16.5 mg, 0.042
mmol) in THF (2 ml) and ethanol (1 ml) was added 80.0 mg (2.00
mmol) of NaOH (2.0 ml, 1M aqueous solution). The mixture was
stirred for 12 hours at room temperature, acidified with 10% HCl,
and extracted with EtOAc. The combined organic layers were washed
with water, and saturated aqueous NaCl, then dried over MgSO.sub.4.
Removal of the solvents under reduced pressure, and
recrystallization of the residue from EtOAC/hexane, afforded the
title compound as a red solid. 1H NMR (acetone-d6): .delta. 8.19
(2H, d, J=8.4 Hz), 7.92 (2H, d, J=8.5 hz), 7.88 (2H, dd, J=2.1, 6.1
Hz), 7.66 (1H, s), 7.64 (2H, d, J=2.3 Hz), 7.28 (4H, d, J=3.0 Hz),
6.09 (1H, t, J=2.5 Hz), 2.42 (2H, d, J=4.8 Hz), 2.39 (3H, s), 1.40
(6H, s).
[0427]
6-(2-Trimethylsilyl)ethynyl-2,3-dihydro-3,3-dimethyl-1H-inden-1-one
(CompoundCC)
[0428] To a solution of 815.0 mg (3.41 mmol)
6-bromo-2,3-dihydro-3,3-dimet- hyl-1H-inden-1-one (See Smith et al.
Org. Prep. Proced. Int. 1978 10 123-131) in 100 ml of degassed
Et.sub.3N (sparged with argon for 20 min) was added 259.6 mg (1.363
mmol) of copper(I) iodide, 956.9 mg (1.363 mmol) of
bis(triphenylphosphine)palladium(II)chloride, and 3.14 g (34.08
mmol) of (trimethylsilyl)acetylene. This mixture was heated at
70.degree. C. for 42 hours, cooled to room temperature, and
filtered through a pad of silica gel and washed with ether. The
filtrate was washed with water, 1 M HCl, water, and finaly with
saturated aqueous NaCl before being dried over MgSO.sub.4.
Concentration of the solution under reduced pressure, followed by
column chromatography (silica gel; 10% Et.sub.2O--hexanes) afforded
the title compound as a brown oil. 1H NMR (300 MHz, CDCl.sub.3):
.delta. 7.79(1H, d, J=1.4 Hz), 7.69 (1H, dd, J=1.6, 8.3 Hz), 7.42
(1H, d, J=8.5 Hz), 2.60 (2H, s), 1.41 (6H, s), 0.26 (9H, s).
[0429] 6-Ethynyl-2,3-dihydro-3.3-dimethyl-1H-inden-1-one (Compound
DD)
[0430] To a solution of 875.0 mg (3.41 mmol)
6-(2-trimethylsilyl)ethynyl-2-
,3-dihydro-3,3-dimethyl-1H-inden-1-one (Compound CC) in 28 ml of
MeOH, was added 197.3 mg (1.43 mmol) of K.sub.2CO.sub.3 in one
portion. After stirring for 6 hours at room temperature the mixture
was filtered though a pad of Celite and the filtrate concentrated
under reduced pressure. The residual oil was placed on a silica gel
column and eluted with 5% EtOAc-hexanes to give the title product
as a colorless oil. 1H NMR (300 MHz, CDCl3): .delta. 7.82 (1H, s),
7.72 (1H, dd, J=1.6, 7.8 Hz), 7.47 (1H, d, J=8.4 Hz), 3.11 (1H, s),
2.61 (2H, s), 1.43 (6H, s).
[0431] Ethyl
4-[2-(5,6-dihydro-5,5-dimethyl-7-oxo-2-indenyl)ethynyl]benzoa- te
(Compound EE)
[0432] A solution of 280.0 mg (1.520 mmol)
6-ethynyl-2,3-dihydro-3,3-dimet- hyl-1H-inden-1-one (Compound DD)
and 419.6 mg (1.520 mmol) ethyl 4-iodobenzoate in 5 ml Et.sub.3N
was sparged with argon for 40 minutes. To this solution was added
271.0 mg (1.033 mmol) of triphenylphosphine, 53.5 mg (0.281 mmol)
of copper(I) iodide, and 53.5 mg (0.076 mmol) of
bis(triphenylphosphine)palladium(II) chloride. The resulting
mixture was heated to reflux for 2.5 hours, cooled to room
temperature, and diluted with Et.sub.2O. After filtration through a
pad of Celite, the filtrate was washed with H.sub.2O, 1 M HCl,
H.sub.2O, and saturated aqueous NaCl, then dried over MgSO.sub.4,
and concentrated under reduced pressure. The title compound was
isolated as a pale-yellow solid by column chromatography (15%
EtOAc-hexanes). 1H NMR (300 MHz, d6-acetone): .delta. 8.05 (2H, d,
J=8.6 Hz), 7.87 (1H, dd, J=1.4, 8.1 Hz), 7.75 (2H, m), 7.70 (2H, d,
J=8.5 Hz), 4.36 (2H, q, J=7.1 Hz), 2.60 (2H, s), 1.45 (6H, s), 1.37
(3H, t, J=7.1 Hz).
[0433] Ethyl
4-[2-(1,1-dimethyl-3-(trifluoromethyl-sulfonyl)oxy-5-indenyl)-
ethynyl]benzoate (Compound FF)
[0434] A solution of 88.0 mg (0.48 mmol) of sodium
bis(trimethylsilyl)amid- e in 0.5 ml THF was cooled to -78.degree.
C. and 145.0 mg (0.436 mmol) of ethyl
4-[2-(5,6-dihydro-5,5-dimethyl-7-oxo-2-indenyl)ethynyl]benzoate
(Compound EE) was added as a solution in 1.0 ml THF. After 30
minutes 181.7 mg (0.480 mmol) of
2-(N,N-bis(trifluoromethansulfonyl)amino)-5-chlo- ro-pyridine was
added as a solution in 1.0 ml THF. The reaction was allowed to
slowly warm to room temperature and quenched after 5 hours by the
addition of saturated aqueous NH.sub.4Cl. The mixture was extracted
with EtOAc, and the combined organic layers washed with 5% aqueous
NaOH, H.sub.2O, and saturated aqueous NaCl, then dried (MgSO.sub.4)
and concentrated under reduced pressure. The product was isolated
as a colorless solid by column chromatography (10%
Et.sub.2O-hexanes). 1H NMR (300 MHz, d6-acetone): .delta. 8.05 (2H,
d, J=8.3 Hz), 7.69 (2H, d, J=8.4 Hz), 7.63 (2H, s), 7.55 (1H, s),
4.36 (2H, q, J=7.1 Hz), 1.44 (6H, s), 1.37 (3H, t, J=7.1 Hz).
[0435]
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoic acid (Compound 60)
[0436] A solution of 142.6 mg (0.339 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl]be-
nzoate (Compound 1) and 35.6 mg (0.848 mmol) of LiOH-H.sub.2O in 12
ml of THF/water (4:1, v/v), was stirred overnight at room
temperature. The reaction mixture was extracted with hexanes, and
the hexane fraction extracted with 5% aqueous NaOH. The aqueous
layers were combined and acidified with 1M HCl, and then extracted
with EtOAc and Et.sub.2O. The combined organic layers were dried
over Na.sub.2SO.sub.4 and concentrated in vacuo to give the title
compound as a colorless solid. 1H NMR (d.sub.6-DMSO): .delta. 7.91
(2H, d, J=8.4 Hz), 7.60 (2H, d, J=8.4 Hz), 7.47 (2H, s), 7.23 (4H,
q, J=8.1 Hz), 7.01 (1H, s), 6.01 (1H, t, J=4.6 Hz), 2.35 (3H, s),
2.33 (2H, d, J=4.8 Hz), 1.30 (6H, s).
[0437]
4-[(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethynyl]benzoi-
c acid (Compound 60a)
[0438] Employing the same general procedure as for the preparation
of
4-[(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl]benzo-
ic acid (Compound 30a), 27.0 mg (0.07 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethynyl]benzoate
(Compound 1 a) was converted into the title compound (colorless
solid) using 5.9 mg (0.14 mmol) of LiOH in H.sub.2O. PMR
(d.sub.6-DMSO): 67 1.31 (6H, s), 2.35 (2H, d, J=4.5 Hz), 6.05 (1H,
t, J=J=J=4.5 Hz), 7.00 (1H, s), 7.33 (2H, d, J=6.2 Hz), 7.44 (4H,
m), 7.59 (2H, d, J=8.1 Hz), 7.90 (2H, d, J=8.1 Hz).
[0439]
4-[(5,6-Dihydro-5,5-dimethyl-8-(4-(1,1-dimethylethyl)phenyl)-2-naph-
thalenyl)ethynyl]benzoic acid (Compound 61)
[0440] A solution of 80.0 mg (0.173 mmol) of ethyl
4-[(5,6-dihydro-5,5-dim-
ethyl-8-(4-(1,1-dimethylethyl)phenyl)-2-naphthalenyl)ethynyl]benzoate
(Compound 6) and 18.1 mg (0.432 mmol) of LiOH--H.sub.2O in 6 ml of
THF/water (3:1, v/v), was stirred overnight at room temperature.
The reaction mixture was extracted with hexanes, and the remaining
aqueous layer acidified with 1M HCl, and then extracted with EtOAc.
The combined organic layers were dried over Na.sub.2SO.sub.4 and
concentrated in vacuo to give the title compound as a colorless
solid. 1H NMR (d.sub.6-DMSO): .delta. 7.82 (2H, d, J=8.2 Hz), 7.44
(6H, m), 7.25 (2H, d, J=8.3 Hz), 7.02 (1H, s), 6.01 (1H, t, J=4.6
Hz), 2.32 (2H, d, J=4.7 Hz), 1.32 (9H, s), 1.29 (6H, s).
[0441] Ethyl
2-fluoro-4-[[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-n-
aphthalenyl)thiocarbonyl]amino]-benzoate (Compound 62)
[0442] A solution of 54.4 mg (0.119 mmol) ethyl
2-fluoro-4-[[(5,6-dihydro--
5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl]amino]-benzoate
(Compound 40) and 57.7 mg (0.143 mmol) of
[2,4-bis(4-methoxyphenyl)-1,3-d-
ithia-2,4-diphosphetane-2,4-disulfide] (Lawesson's Reagent) in 12.0
ml of benzene was refluxed overnight. Upon cooling to room
temperature, the mixture was filtered and the filtrate concentrated
under reduced pressure. The title compound was isolated by column
chromatography (10 to 25% EtOAc/hexanes) as a yellow solid. 1H NMR
(CDCl.sub.3): .delta. 9.08 (1H, s), 7.92 (1H, br s), 7.90 (1H, t,
J=8.2 Hz), 7.66 (1H, dd, J=2.0, 6.0 Hz), 7.38 (3H, m), 7.18 (4H,
m), 6.01 (1H, t, J=4.7 Hz), 4.35 (2H, q, J=7.1 Hz), 2.36 (3H, s),
2.33 (2H, d, J=4.7 Hz), 1.38 (3H, t, J=7.1 Hz), 1.33 (6H, s).
[0443]
2-fluoro-4-[[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphtha-
lenyl)thiocarbonyl]amino]-benzoic acid (Compound 63)
[0444] To a solution of 46.5 mg (0.098 mmol) ethyl
2-fluoro-4-[[(5,6-dihyd-
ro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)thiocarbonyl]amino]-benz-
oate (Compound 62) in 1.0 ml EtOH and 1.0 ml of THF was added 55 mg
NaOH (1.4 mmol) and 1.0 ml of H.sub.2O. After stirring at room
temperature for overnight EtOAc was added, and the reaction
quenched by the addition of 10% HCl. Extraction with EtOAc was
followed by washing of the combined organic layers with H.sub.2O,
saturated aqueous NaCl, and drying over MgSO.sub.4. Removal of the
solvent under reduced pressure provided a solid which after
crystallization from CH.sub.3CN afforded the title compound as a
pale-yellow solid. 1H NMR (d.sub.6-acetone): .delta. 11.05 (1H, s),
8.02 (1H, m), 7.99 (1H, t, J=8.3 Hz), 7.75 (1H, m), 7.69 (1H, dd,
J=2.0, 6.1 Hz), 7.52 (1H, s), 7.46 (1H, d, J=8.1 Hz), 7.21 (4H, m),
6.04 (1H, t, J=4.8 Hz), 2.37 (2H, d, J=4.8 Hz), 2.33 (3H, s), 1.36
(6H, s).
[0445] Ethyl
5',6'-dihydro-5',5'-dimethyl-8'-(4-methylphenyl)-[2,2'-binaph-
thalene]-6-carboxylate (Compound 64)
[0446] A solution of
3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-7-bromona- phthalene
Compound D) 0.45 g, 1.40 mmol) and THF (2.1 ml) was added to
magnesium turnings (0.044 g, 1.82 mmol) at room temperature under
argon. Two drops of ethylene dibromide were added, and the
solution, which slowly became cloudy and yellow, was heated to
reflux for 1.5 hours. In a second flask was added zinc chloride
(0.210 g, 1.54 mmol), which was melted under high vacuum, cooled to
room temperature and dissolved in THF (3 ml). The Grignard reagent
was added to the second flask and, after 30 minutes at room
temperature, a solution of ethyl 6-bromo-2-naphthalinecar- boxylate
(Compound N) 0.293 g, (1.05 mmol) and THF (2 ml) were added. In a
third flask was prepared a solution of Ni(PPh.sub.3).sub.4 and THF
as follows: To a solution of NiCl.sub.2(PPh.sub.3).sub.2 (0.82 g,
1.25 mmol) and PPh.sub.3 (0.66 g, 2.5 mmol) in THF (3.5 ml) was
added a 1M solution of diisobutylaluminum hydride and hexanes (2.5
ml, 2.5 mmol), and the resulting solution diluted with THF to a
total volume of 15 ml and stirred at room temperature for 15
minutes. Three 0.60 ml aliquots of the Ni(PPh.sub.3).sub.4 solution
were added at 15 minutes intervals to the second flask. The
resulting suspension was stirred at room temperature for 2 hours.
The reaction was quenched by the addition of 5 ml 1N aqueous HCl
and stirred for 1 hour before extracting the products with ethyl
acetate. The organic layers were combined, washed with brine, dried
(MgSO.sub.4), filtered and the solvent removed in-vacuo. The
residue was crystalized from hexanes to give 130 mg of pure
material. The mother liquor was concentrated under reduced pressure
and the residue purified by silica gel chromatography
(95:5-hexanes:ethyl acetate) to give an additional 170 mg of the
title compound (overall yield=300 mg, 64%) as a colorless solid. 1H
NMR (CDCl.sub.3) .delta. 8.57 (s, 1H), 8.05 (dd, 1H, J=1.7, 8.0
Hz), 7.84-7.95 (overlapping d's, 3H), 7.66 (dd, 1H, J=1.7, 8.5 Hz),
7.58 (dd, 1H, J=2.0, 8.0 Hz), 7.48 (d, 1H, J=8.0 Hz), 7.43 (d, 1H,
J=2.0 Hz), 7.32 (d, 2H, J=8.0 Hz), 7.21 (d, 2H, J=8.0 Hz), 6.04 (t,
1H, J=4.8 Hz), 4.44 (q, 2H, J=7.1 Hz), 2.40 (s, 3H), 2.39 (d, 2H,
J=4.8 Hz), 1.45 (t, 3H, J=7.1 Hz), 1.39 (s, 6H).
[0447]
5',6'-Dihydro-5',5'-dimethyl-8'-(4-methylphenyl)-[2.2'-binaphthalen-
e]-6-carboxylic acid (Compound 65)
[0448] A solution of ethyl
5',6'-dihydro-5',5'-dimethyl-8'-(4-methylphenyl-
)-[2,2'-binaphthalene]-6-carboxylate (Compound 64) 0.19 g, 0.43
mmol), EtOH (8 ml) and 1N aqueous NaOH (2 ml) was heated to
60.degree. C. for 3 hours. The solution was cooled to 0.degree. C.
and acidified with 1N aqueous HCl. The product was extracted into
ethyl acetate, and the organic layers combined, washed with water,
brine, dried (MgSO.sub.4), filtered and the solvent removed
in-vacuo. The residue was recrystalized from THF/ethyl acetate at
0.degree. C. to give 35 mg of pure material. The mother liquor was
concentrated under reduced pressure and the residue purified by
silica gel chromatography (100% ethyl acetate) to give an
additional 125 mg of the title compound (overall yield=160 mg, 90%)
as a colorless solid. 1H NMR (DMSO-d.sub.6) .delta. 8.57 (s, 1H),
8.11 (d, 1H, J=8.7 Hz), 7.96-7.82 (overlapping d's, 3H), 7.65 (d,
2H, J=7.6 Hz), 7.50 (d, 1H, J=7.9 Hz), 7.28 (s, 1H), 7.26 (d, 2H,
J=8.3 Hz), 7.21 (d, 2H, J=8.3 Hz), 6.01 (t, 1H, J=4.5 Hz), 3.34 (br
s, 1H), 2.31 (s, 3H), 2.31 (d, 2H, J=4.5 Hz), 1.31 (s, 6H).
[0449] Ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(2-furyl)-2-naphthalenyl)ethyn-
yl]benzoate (Compound 66)
[0450] Employing the same general procedure as for the preparation
of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethy-
nyl]benzoate (Compound 1), 250.0 mg (0.52 mmol) of ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthaleny-
l)ethynyl]benzoate (Compound G) was converted into the title
compound (colorless solid) using 142.4 mg (1.045 mmol) of zinc
chloride, 24.1 mg (0.02 mmol) of
tetrakis(triphenylphosphine)palladium(0) and 2-lithiofuran
(prepared by the addition of 53.4 mg (0.52 ml, 0.78 mmol) of
n-butyllithium (1.5M solution in hexane) to a cold solution
(-78.degree. C.) of 53.4 mg (0.784 mmol) of furan in 1.0 ml of
THF). PMR (CDCl.sub.3): .delta. 1.32 (6H, s), 1.41 (3H, t, J=7.1
Hz), 2.35 (2H, d, J=5.0 Hz), 4.39 (2H, q, J=7.1 Hz), 6.41 (1H, t,
J=5.0 Hz), 6.50 (2H, s), 7.36 (1H, d, J=8.0 Hz), 7.45 (1H, dd,
J=1.7, 8.0 Hz), 7.49 (1H, s), 7.57 (2H, d, J=8.2 Hz), 7.63 (1H, d,
J=1.7 Hz), 8.02 (2H, d, J=8.2 Hz).
[0451]
4-[(5,6-dihydro-5,5-dimethyl-8-(2-furyl)-2-naphthalenyl)ethynyl]ben-
zoic acid (Compound 67)
[0452] Employing the same general procedure as for the preparation
of
4-[(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl]benzo-
ic acid (Compound 30a), ethyl
4-[(5,6-dihydro-5,5-dimethyl-8-(2-furyl)-2-n-
aphthalenyl)ethynyl]benzoate (Compound 66) was converted into the
title compound (colorless solid) using 16.0 mg (0.38 mmol) of LiOH
in H.sub.2O. PMR (d.sub.6-DMSO): .delta. 1.26 (6H, s), 2.33 (2H, d,
J=4.9 Hz), 6.41 (1H, t, J=4.9 Hz), 6.60 (2H, m), 7.45-7.53 (3H, m),
7.64 (2H, d, J=8.3 Hz), 7.75 (1H, d, J=1.6 Hz), 7.93 (2H, d, J=8.3
Hz).
[0453] 3,4-dihydro-4,4-dimethyl-7-acetyl-1(2H)-naphthalenone
(Compound 100C) and
3,4-dihydro-4,4-dimethyl-6-acetyl-1(2H)-naphthalenone (Compound
100D)
[0454] To a cold (0.degree. C.) mixture of aluminum chloride (26.3
g, 199.0 mmols) in dichloromethane (55 ml) was added acetylchloride
(15 g, 192 mmols) and 1,2,3,4-tetrahydro-1,1-dimethylnaphthalene
(24.4 g, 152 mmols) in dichloromethane (20 ml) over 20 minutes. The
reaction mixture was warmed to ambient temperature and stirred for
4 hours. Ice (200 g) was added to the reaction flask and the
mixture diluted with ether (400 ml). The aqueous and organic layers
were separated and the organic phase was washed with 10% HCl (50
ml), water (50 ml), 10% aqueous sodium bicarbonate, and saturated
aqueous NaCl (50 ml) and then dried over MgSO.sub.4. The solvent
was removed by distillation to afford a yellow oil which was
dissolved in benzene (50 ml).
[0455] To a cold (0.degree. C.) solution of acetic acid (240 ml)
and acetic anhydride (120 ml) was added chromium trioxide (50 g,
503 mmols) in small portions over 20 minutes under argon. The
mixture was stirred for 30 minutes at 0.degree. C. and diluted with
benzene (120 ml). The benzene solution prepared above was added
with stirring via an addition funnel over 20 minutes. After 8
hours, the reaction was quenched by the careful addition of
isopropanol (50 ml) at 0.degree. C., followed by water (100 ml).
After 15 minutes, the reaction mixture was diluted with ether (1100
ml) and water (200 ml), and then neutralized with solid sodium
bicarbonate (200 g). The ether layer was washed with water (100
ml),and saturated aqueous NaCl (2.times.100 ml), and dried over
MgSO.sub.4. Removal of the solvent under reduced pressure afforded
a mixture of the isomeric diketones which were separated by
chromatography (5% EtOAc/hexanes). (Compound 100C): 1H NMR
(CDCl.sub.3): .delta. 8.55 (1H, d, J=2.0 Hz), 8.13 (1H, dd, J=2.0,
8.3 Hz), 7.53 (1H, d, J=8.3 Hz), 2.77 (2H, t, J=6.6 Hz), 2.62 (3H,
s), 2.05 (2H, t, J=6.6 Hz), 1.41 (6H, s). (Compound 100D): 1H NMR
(CDCl.sub.3): .delta. 8.10 (1H, d, J=8.1 Hz), 8.02 (1H, d, J=1.6
Hz), 7.82 (1H, dd, J=1.6, 8.1 Hz), 2.77 (2H, t, J=7.1 Hz), 2.64
(3H, s), 2.05 (2H, t, J=7.1 Hz), 1.44 (6H, s).
[0456]
3.4-dihydro-4,4-dimethyl-6-(2-(2-methyl-1,3-dioxolanyl))-1(2H)-naph-
thalenone (Compound 100E)
[0457] A solution of 1.80 g (8.34 mmol) of a 1:5 mixture of
3,4-dihydro-4,4-dimethyl-7-acetyl-1(2H)-naphthalenone (Compound
100C); and 3,4-dihydro-4,4-dimethyl-6-acetyl-1(2H)-naphthalenone
(Compound 100D) in 50 ml benzene was combined with 517.7 mg (8.34
mmol) of ethylene glycol and 20.0 mg (0.11 mmol) of
p-toluenesulfonic acid monohydrate. The resulting solution was
heated to reflux for 18 hours, cooled to room temperature, and
concentrated under reduced pressure. The title compound was
isolated by column chromatography (10% EtOAc-hexanes) ras a
colorless oil. 1H NMR (CDCl.sub.3): .delta. 8.01 (1H, d, J=8.2 Hz),
7.51 (1H, s), 7.43 (1H, dd, J=1.7, 6.4 Hz), 4.07 (2H, m), 3.79 (2H,
m), 2.74 (2H, t, J=6.5 Hz), 2.904 (2H, t, J=7.1 Hz), 1.67 (3H, s),
1.46 (6H, s).
[0458]
1,2,3,4-tetrahydro-1-hydroxy-1-(4-methylphenyl)-4,4-dimethyl-6-(2-(-
2-methyl-1,3-dioxolanyl))naphthalene (Compound 100F)
[0459] To a solution of 496.2 mg (2.54 mmol)
p-tolylmagnesiumbromide in 20 ml THF (2.54 ml; 1M solution in
ether) was added a solution of
3,4-dihydro-4,4-dimethyl-6-(2-(2-methyl-1,3-dioxolan-yl))-1(2H)-naphthale-
none (Compound 100E, 200.0 mg, 0.769 mmol) in THF (5 ml). The
solution was refluxed for 16 hours, cooled to room temperature, and
washed with water, saturated aqueous NH.sub.4Cl, and dried over
MgSO.sub.4. Removal of the solvents under reduced pressure and
column chromatography (10% EtOAc/hexanes) afforded the title
compound as a colorless solid. 1H NMR (CDCl.sub.3): .delta. 7.49
(1H, d, J=1.7 Hz), 7.19 (2H, m), 7.10 (2H, d, J=7.9 Hz), 7.04 (1H,
d, J=8.2 Hz), 4.05 (2H, m), 3.80 (2H, m), 2.34 (3H, s), 2.21 (1H,
m), 2.10 (1H, m), 1.88 (1H, m), 1.65 (3H, s), 1.54 (1H, m), 1.39
(3H, s), 1.33 (3H, s).
[0460]
3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-6-acetylnaphthalene
(Compound 100G)
[0461] A solution of
1,2,3,4-tetrahydro-1-hydroxy-1-(4-methylphenyl)-4,4-d-
imethyl-6-(2-(2-methyl-1,3-dioxolanyl))naphthalene (Compound 100F
160.0 mg, 0.52 mmol), p-toluenesulfonic acid monohydrate (4 mg) and
30 ml benzene was refluxed for 12 hours. After cooling to room
temperature, the reaction mixture was diluted with ether (100 ml)
and washed with 10% aqueous sodium bicarbonate, water, and
saturated aqueous NaCl. The organic layer was dried over MgSO.sub.4
and the solvents were removed under reduced pressure to give the
title compound, which was isolated by column chromatography (10%
EtOAc-hexanes) as a yellow oil. 1H NMR (CDCl.sub.3): .delta. 7.97
(1H, d, J=1.8 Hz), 7.67 (1H, dd, J=1.7, 6.4 Hz), 7.22 (4H, s), 7.13
(1H, d, J=8.1 Hz), 6.10 (1H, t, J=4.5 Hz), 2.59 (3H, s), 2.40 (3H,
s), 2.38 (2H, d, J=4.7 Hz), 1.38 (6H, s).
[0462]
4-[3-oxo-3-(7,8-dihydro-5-(4-methylphenyl)-8,8-dimethyl-2-naphthale-
nyl)-1-propenyl]-benzoic acid (Compound 101)
[0463] To a solution of 78.7 mg (0.272 mmol)
3,4-dihydro-1-(4-methylphenyl- )-4,4-dimethyl-6-acetylnaphthalene
(Compound 100G) in 4.0 ml of MeOH was added 53.1 mg (0.354 mmol) of
4-carboxy benzaldehyde, and 80. mg (2.00 mmol; 2.0 ml of 1M aqueous
NaOH). The resulting solution was stirred at room temperature for
12 hours, concentrated under reduced pressure, and the residual oil
dissolved in EtOAc. The solution was treated with 10% HCl, and the
organic layer was washed with H.sub.2O, and saturated aqueous NaCl,
then dried over Na.sub.2SO.sub.4. Removal of the solvents under
reduced pressure gave the title compound as a colorless solid which
was purified by recrystallization from CH.sub.3CN. 1H NMR
(acetone-d6): .delta. 8.00 (7H, m), 7.83 (1H, d, J=15.6 Hz), 7.24
(4H, s), 7.13 (1H, d, J=8.1 Hz), 6.12 (1H, t, J=4.5 Hz), 2.42 (2H,
d, J=4.8 Hz), 2.38 (3H, s), 1.41 (6H, s).
[0464] 3,4-dihydro-1-phenyl-4,4-dimethyl-6-acetylnaphthalene
(Compound 100H)
[0465] To a solution of 508.0 mg (1.95 mmol) of
3,4-dihydro-4,4-dimethyl-6-
-(2-(2-methyl-1,3-dioxolanyl))-1(2H)-naphthalenone (Compound 100E)
in 10 ml of THF was added 496.2 mg (2.54 mmol; 2.54 ml of a 1 M
solution in Et.sub.2O) of phenylmagnesium bromide. The resulting
solution was heated to reflux for 8 hours, H.sub.2O was added and
heating continued for 30 minutes. The THF was removed under reduced
pressure and the aqueous residue was extracted with EtOAc. The
combined organic layers were dried (MgSO.sub.4), concentrated under
reduced pressure, and the title compound isolated from the residue
by column chromatography (10% EtOAc-hexanes) as a colorless oil. 1H
NMR (CDCl.sub.3): .delta. 7.97 (1H, d, J=1.8 Hz), 7.67 (1H, dd,
J=2.1, 8.0 Hz), 7.34 (5H, m), 7.10 (1H, d, J=8.1 Hz), 6.12 (1H, d,
J=4.6 Hz), 2.59 (3H, s), 2.39 (2H, d, J=4.8 Hz), 1.38 (6H, s).
[0466]
4-[3-oxo-3-(78dihydro-5-phenyl-8,8-dimethyl-2-naphthalenyl)-1-prope-
nyl]-benzoic acid (Compound 103)
[0467] To a solution of 115.0 mg (0.42 mmol) of
3,4-dihydro-1-phenyl-4,4-d- imethyl-6-acetylnaphthalene (Compound
100H) and 65.0 mg (0.43 mmol) of 4-formyl-benzoic acid in 5.0 ml
EtOH and 1.0 ml THF, was added 120.0 mg (3.00 mmol; 3.0 ml of a 1 M
aqueous solution) of NaOH. The resulting yellow solution was
stirred at room temperature for 12 hours. The solution was
acidified with 6% aqueous HCl and extracted with EtOAc. The
combined organic layers were dried (MgSO.sub.4), concentrated under
reduced pressure, and the title compounds was isolated by column
chromatography (50% EtOAc-hexanes) as a pale yellow solid. 1H NMR
(CDCl.sub.3): .delta.8.13 (2H, d, J=7.7 Hz), 8.04 (1H, s), 7.81
(1H, d, J=15.5 Hz), 7.75 (3H, m), 7.60 (1H, d, J=15.5 Hz), 7.35
(5H, m), 7.14 (1H, d, J=8.1 Hz), 6.15 (1H, t, J=4.2 Hz), 2.41 (2H,
d, J=4.2 Hz), 1.41 (6H, s).
Method of Potentiating Nuclear Receptor Agonists
[0468] Overview and Introduction
[0469] We have discovered that a subset of retinoid antagonists
which exhibit negative hormone activity can be used for
potentiating the biological activities of other retinoids and
steroid receptor superfamily hormones. These other retinoids and
steroid receptor superfamily hormones can be either endogenous
hormones or pharmaceutical agents. Thus, for example, when used in
combination with a retinoid negative hormone, certain activities of
pharmaceutical retinoid agonists can be rendered more active in
eliciting specific biological effects. Advantageously, this
combination approach to drug administration can minimize
undesirable side effects of pharmaceutical retinoids because lower
dosages of the pharmaceutical retinoids can be used with improved
effectiveness.
[0470] More particularly, we have discovered that AGN 193109, a
synthetic retinoid having the structure shown in FIG. 1, exhibits
unique and unexpected pharmacologic activities. AGN 193109 exhibits
high affinity for the RAR subclass of nuclear receptors without
activating these receptors or stimulating transcription of retinoid
responsive genes. Instead, AGN 193109 inhibits the activation of
RARs by retinoid agonists and therefore behaves as a retinoid
antagonist.
[0471] Additionally, we have discovered that retinoid negative
hormones can be used without coadministration of a retinoid agonist
or steroid hormone to control certain disease symptoms. More
specifically, the retinoid negative hormone disclosed herein can
down-regulate the high level basal transcription of genes that are
responsive to unliganded RARs. If, for example, uncontrolled
cellular proliferation results from the activity of genes
responsive to unliganded RARs, then that gene activity can be
reduced by the administration of a retinoid negative hormone that
inactivates RARs. Consequently, cellular proliferation dependent on
the activity of unliganded RARs can be inhibited by the negative
hormone. Inhibition of unliganded RARs cannot be achieved using
conventional antagonists.
[0472] Significantly, we have discovered that AGN 193109 can both
repress RAR basal activity and can sometimes potentiate the
activities of other retinoid and steroid receptor superfamily
hormone agonists. In the context of the invention, a hormone
agonist is said to be potentiated by a negative hormone such as AGN
193109 if, in the presence of the negative hormone, a reduced
concentration of the agonist elicits substantially the same
quantitative response as that obtainable with the agonist alone.
The quantitative response can, for example, be measured in a
reporter gene assay in vitro. Thus, a therapeutic retinoid that
elicits a desired response when used at a particular dosage or
concentration is potentiated by AGN 193109 if, in combination with
AGN 193109, a lower dosage or concentration of the therapeutic
retinoid can be used to produce substantially the same effect as a
higher dosage or concentration of the therapeutic retinoid when
that therapeutic retinoid is used alone. The list of agonists that
can be potentiated by coadministration with AGN 193109 includes RAR
agonists, vitamin D receptor agonists, glucocorticoid receptor
agonists and thyroid hormone receptor agonists. More particularly,
specific agonists that can be potentiated by coadministration
include: ATRA, 13-cis retinoic acid, the synthetic RAR agonist AGN.
191183, 1,25-dihydroxyvitamin D.sub.3, dexamethasone and thyroid
hormone (3,3',5-triiodothyronine). Also disclosed herein is a
method that can be used to identify other hormones that can be
potentiated by coadministration with AGN 193109.
[0473] Thus, AGN 193109 behaves in a manner not anticipated for a
simple retinoid antagonist, but as a negative hormone that can
potentiate the activities of various members of the family of
nuclear receptors. We also disclose a possible mechanism that can
account for both negative hormone activity and the ability of AGN
193109 to potentiate the activities of other nuclear receptor
ligands. This mechanism incorporates elements known to participate
in retinoid-dependent signalling pathways and additionally
incorporates a novel negative regulatory component.
[0474] Those having ordinary skill in the art will appreciate that
RARs, which are high affinity targets of AGN 193109 binding, are
transcription factors that regulate the expression of a variety of
retinoid responsive genes. Cis-regulatory DNA binding sites for the
RARs have been identified nearby genes that are transcriptionally
regulated in a retinoid-dependent fashion. RAR binding to such DNA
sites, known as retinoic acid response elements (RAREs), has been
well defined. Importantly, the RAREs bind heterodimers consisting
of one RAR and one RXR. The RXR component of the heterodimer
functions to promote a high affinity interaction between the
RAR/RXR heterodimer and the RARE (Mangelsdorf et al. The Retinoid
Receptors in The Retinoids: Biology Chemistry and Medicine, 2nd
edition, eds. Sporn et al., Raven Press, Ltd., New York 1994, the
disclosure of which is hereby incorporated by reference).
[0475] As detailed below, our findings related to the negative
hormone activity of AGN 193109 are consistent with a mechanism
involving the interaction of a putative Negative Coactivator
Protein (NCP) with the RAR. According to the proposed mechanism,
this interaction is stabilized by AGN 193109.
[0476] Our results further indicated that AGN 193109 can modulate
intracellular availability of NCP for interaction with nuclear
receptors other than RARs that are occupied by AGN 193109. It
follows that AGN 193109 can potentiate transcriptional regulatory
pathways involving nuclear receptors that share with the RARs the
ability to bind the NCP. In this regard, AGN 193109 exhibits the
ability to modulate a variety of nuclear receptor pathways, an
activity that would not be predicted for a conventional retinoid
antagonist. Accordingly, AGN 193109 is useful as an agent for
potentiating the activity of nuclear receptor ligands, including
both endogenous hormones and prescribed therapeutics. This specific
embodiment illustrates the more general principle that any nuclear
receptor negative hormone will potentiate the activity of other
nuclear receptors that competitively bind the NCP.
[0477] Although other materials and methods similar or equivalent
to those described herein can be used in the practice or testing of
the present invention, the preferred methods and materials are now
described. General references for methods that can be used to
perform the various nucleic acid manipulations and procedures
described herein can be found in Molecular Cloning: A Laboratory
Manual (Sambrook et al. eds. Cold Spring Harbor Lab Publ. 1989) and
Current Protocols in Molecular Biology (Ausubel et al. eds., Greene
Publishing Associates and Wiley-Interscience 1987). The disclosures
contained in these references are hereby incorporated by reference.
A description of the experiments and results that led to the
creation of the present invention follows.
[0478] Example 6 describes the methods used to demonstrate that AGN
193109 bound each of three RARs with high affinity but failed to
activate retinoid dependent gene expression.
EXAMPLE 6
AGN 193109 Binds RARs With High Affinity But Does Not Transactivate
Retinoid-Dependent Gene Expression
[0479] Human RAR-.alpha., RAR-.beta.and RAR-.gamma. receptors were
separately expressed as recombinant proteins using a baculovirus
expression system essentially according to the method described by
Allegretto et al. in J. Biol. Chem. 268:26625 (1993). The
recombinant receptor proteins were separately employed for
determining AGN 193109 binding affinities using the [.sup.3H]-ATRA
displacement assay described by Heyman et al. in Cell 68:397
(1992). Dissociation constants (Kds) were determined according to
the procedure described by Cheng et al. in Biochemical Pharmacology
22:3099 (1973).
[0480] AGN 193109 was also tested for its ability to transactivate
RARs in CV-1 cells transiently cotransfected with RAR expression
vectors and a retinoid responsive reporter gene construct. Receptor
expression vectors pRShRAR-.alpha. (Giguere et al. Nature 330:624
(1987)), pRShRAR-.beta. (Benbrook et al. Nature 333:669 (1988)) and
pRShRAR-.gamma. (Ishikawa et al. Mol. Endocrinol. 4:837 (1990))
were separately cotransfected with the .DELTA.MTV-TREp-Luc reporter
plasmid. Use of this luciferase reporter plasmid has been disclosed
by Heyman et al. in Cell 68:397 (1992). The .DELTA.MTV-TREp-Luc
plasmid is essentially identical to the .DELTA.MTV-TREp-CAT
reporter construct described by Umesono et al. in Nature 336:262
(1988), except that the chloramphenicol acetyltransferase (CAT)
reporter gene was substituted by a polynucleotide sequence encoding
firefly luciferase. Transfection of green monkey CV-1 cells was
carried out using the calcium phosphate coprecipitation method
described in Molecular Cloning: A Laboratory Manual (Sambrook et
al. eds. Cold Spring Harbor Lab Publ. 1989). CV-1 cells were plated
at a density of 4.times.10.sup.4/well in 12 well multiwell plates
and transiently transfected with a calcium phosphate precipitate
containing 0.7 .mu.g of reporter plasmid and 0.1 .mu.g of receptor
plasmid according to standard laboratory procedures. Cells were
washed after 18 hours to remove the precipitate and refed with
Dulbecco's modified Eagle's medium (DMEM) (Gibco), containing 10%
activated charcoal extracted fetal bovine serum (Gemini
Bio-Products). Cells were treated with vehicle alone (ethanol) or
AGN 193109 (10.sup.-9 to 10.sup.-6 M) for 18 hours. Cell lysates
were prepared in 0.1 M KPO.sub.4 (pH 7.8), 1.0% TRITON X-100, 1.0
mM DTT, 2 mM EDTA. Luciferase activity was measured as described by
de Wet et al. in Mol. Cell. Biol. 7:725 (1987), using firefly
luciferin (Analytical Luminescence Laboratory) and an EG&G
Berthold 96-well plate luminometer. Reported luciferase values
represented the mean.+-.SEM of triplicate determinations.
[0481] The results presented in Table 11 indicated that AGN 193109
bound each of RAR-.alpha., RAR-.beta. and RAR-.gamma. with high
affinity, but did not activate retinoid-dependent gene expression.
More specifically, AGN 193109 bound each of the three receptors
with Kd values in the 2-3 nM range. Despite this tight binding, AGN
193109 failed to activate gene expression when compared with
inductions stimulated by ATRA. Accordingly, the half-maximal
effective concentration of AGN 193109 (EC.sub.50) was ummeasurable.
Although not presented in the Table, we also found that AGN 193109
had no measurable affinity for the RXRs.
11TABLE 11 AGN 193109 Binding and Transactivation of the RARs
RAR-.alpha. RAR-.beta. RAR-.gamma. EC.sub.50 (nM) No Activity No
Activity No Activity K.sub.d (nM) 2 2 3
[0482] Example 7 describes the methods used to demonstrate that AGN
193109 is an antagonist of ATRA dependent gene expression.
EXAMPLE 7
AGN 193109-Dependent Inhibition of RAR Transactivation by ATRA
[0483] The ability of AGN 193109 to antagonize ATRA mediated RAR
activation was investigated in CV-1 cells cotransfected by the
calcium phosphate coprecipitation method of Sambrook et al.
(Molecular Cloning: A Laboratory Manual Cold Spring Harbor Lab
Publ. 1989). Eukaryotic expression vectors pRShRAR-.alpha. (Giguere
et al. Nature 330:624 (1987)), pRShRAR-.beta. (Benbrook et al.
Nature 333:669 (1988)) and pRShRAR-.gamma. (Ishikawa et al. Mol.
Endocrinol. 4:837 (1990)) were cotransfected with the
.DELTA.-MTV-Luc reporter plasmid described by Hollenberg et al.
(Cell 55:899 (1988)). Notably, the reporter plasmid contained two
copies of the TRE-palindromic response element. Calcium phosphate
transfections were carried out exactly as described in Example 6.
Cells were dosed with vehicle alone (ethanol), ATRA (10.sup.-9 to
10.sup.-6 M), AGN 193109 (10.sup.-9 to 10.sup.-6 M), or 10.sup.-8 M
ATRA in combination with AGN 193109 (10.sup.-9 to 10.sup.-6 M) for
18 hours. Cell lysates and luciferase activity measurements were
also performed as in Example 6.
[0484] The results of these procedures are presented in FIGS. 2A
through 2F where luciferase values represent the mean.+-.SEM of
triplicate determinations. More specifically, the results presented
in FIGS. 2A, 2C and 2E indicated that stimulation of transfected
cells with ATRA led to dose responsive increases in luciferase
activity. This confirmed that ATRA activated each of the three RARs
in the experimental system and provided a comparative basis for
detecting the activity of an antagonist. The graphic results
presented in FIGS. 2B, 2D and 2F indicated that cotreatment of
transfected cells with 10 nM ATRA and increasing concentrations of
AGN 193109 led to an inhibition of luciferase activity. In
particular, equal doses of AGN 193109 and ATRA gave greater than
50% inhibition relative to ATRA alone for all three RAR subtypes.
Comparison of the ATRA dose response in the presence of different
concentrations of AGN 193109 indicated that ATRA was competitively
inhibited by AGN 193109. Notably, the horizontal axes on all of the
graphs shown in FIG. 2 represents the log of the retinoid
concentration. These results proved that AGN 193109 was a potent
RAR antagonist.
[0485] We next performed experiments to elucidate the mechanism
underlying the antagonist activity of AGN 193109. Those having
ordinary skill in the art will appreciate that nuclear receptor
activation is believed to involve a conformational change of the
receptor that is induced by ligand binding. Indeed, the results of
protease protection assays have confirmed that nuclear hormone
agonists and antagonists cause receptor proteins to adopt different
conformations (Keidel et al. Mol. Cell. Biol. 14:287 (1994); Allan
et al. J. Biol. Chem. 267:19513 (1992)). We used such an assay to
determine if AGN 193109 and ATRA caused RAR-.alpha. to adopt
different conformations. AGN 193583, an RAR-.alpha.-selective
antagonist, was included as a positive control that is known to
confer an antagonist-specific pattern of protease sensitivity.
[0486] Example 8 describes one method that was used to detect
conformational changes in RAR-.alpha. resulting from AGN 193109
binding. As presented below, the results of this procedure
unexpectedly indicated that AGN 193109 led to a pattern of trypsin
sensitivity that was substantially identical to that induced by
ATRA, an RAR agonist, and unlike that induced by a model RAR
antagonist. This finding suggested that AGN 193109 possessed
properties distinct from other retinoid antagonists.
EXAMPLE 8
Protease Protection Analysis
[0487] A plasmid constructed in the vector pGEM3Z (Pharmacia) and
containing the RAR-.alpha. cDNA (Giguere et al. Nature 330:624
(1987)), was used in connection with the TNT-coupled reticulocyte
lysate in vitro transcription-translation system (Promega) to
prepare [.sup.35S]-methionine labeled RAR-.alpha.. Limited
proteolytic digestion of the labeled protein RAR-.alpha.was carried
out according to the method described by Keidel et al. in Mol.
Cell. Biol. 14:287 (1994). Aliquots of reticulocyte lysate
containing [.sup.35S]-methionine labeled RAR-cc were incubated with
either ATRA, AGN 193583 or AGN 193109 on ice for 45 minutes in a
total volume of 9 .mu.l. The retinoid final concentration for all
trials was 100 nM for ATRA and AGN 193109, and 1000 nM for AGN
193583. The difference between the final concentrations of the
retinoids was based on the approximate 10-fold difference in
relative affinities of ATRA and AGN 193109 (having Kd at
RAR-.alpha. of 2 and 10 nM, respectively) and AGN 193583 (having Kd
at RAR-.alpha. of .gtoreq.100 nM). After ligand binding, 1 .mu.l of
appropriately concentrated trypsin was added to the mixture to give
final concentrations of 25, 50 or 100 .mu.g/ml. Samples were
incubated at room temperature for 10 minutes and trypsin digestion
stopped by addition of SDS-sample buffer. Samples were subjected to
polyacrylamide gel electrophoresis and autoradiographed according
to standard procedures.
[0488] Both the agonist and antagonist led to distinct patterns of
trypsin sensitivity that were different from the result obtained by
digestion of the unliganded receptor. Autoradiographic results
indicated that trypsin concentrations of 25, 50 and 100 .mu.g/ml
completely digested the radiolabeled RAR-.alpha. in 10 minutes at
room temperature in the absence of added retinoid. Prebinding of
ATRA led to the appearance of two major protease resistant species.
Prebinding of the RAR-.alpha.x-selective antagonist AGN 193583 gave
rise to a protease resistant species that was of lower molecular
weight than that resulting from ATRA prebinding. This result
demonstrated that a retinoid agonist and antagonist led to
conformational changes detectable by virtue of altered trypsin
sensitivities. Surprisingly, prebinding of AGN 193109 gave rise to
a protease protection pattern that was indistinguishable from that
produced by prebinding of ATRA.
[0489] The results presented above confirmed that AGN 193109 bound
the RAR-.alpha. and altered its conformation. Interestingly, the
nature of this conformational change more closely resembled that
which resulted from binding of an agonist (ATRA) than the
alteration produced by antagonist (AGN 193583) binding. Clearly,
the mechanism of AGN 193109 dependent antagonism was unique.
[0490] We considered possible mechanisms that could account for the
antagonist activity of AGN 193109. In particular, we used a
standard gel shift assay to test whether AGN 193109 perturbed
RAR/RXR heterodimer formation or inhibited the interaction between
RAR and its cognate DNA binding site.
[0491] Example 9 describes a gel electrophoretic mobility-shift
assay used to demonstrate that AGN 193109 neither inhibited RAR/RXR
dimerization nor inhibited binding of dimers to a target DNA.
EXAMPLE 9
Gel Shift Analysis
[0492] In vitro translated RAR-.alpha. was produced essentially as
described under Example 8, except that .sup.35S-labeled methoinine
was omitted. In vitro translated RXR-.alpha. was similarly produced
using a pBluescript(II)(SK)-based vector containing the RXR-.alpha.
cDNA described by Mangelsdorf, et al. in Nature 345:224-229 (1990)
as the template for generating in vitro transcripts. The labeled
RAR-.alpha. and RXR-.alpha., alone or in combination, or prebound
with AGN 193109 (10.sup.-6 M) either alone or in combination, were
allowed to interact with an end-labeled DR-5 RARE double-stranded
probe having the sequence 5'-TCAGGTCACCAGGAGGTCAGA-3' (SEQ ID NO:
1). The binding mixture was electrophoresed on a non-denaturing
polyacrylamide gel and autoradiographed according to standard
laboratory procedures. A single retarded species appearing on the
autoradiograph that was common to all the lanes on the gel
represented an undefined probe-binding factor present in the
reticulocyte lysate. Only the RAR/RxR combination gave rise to a
retinoid receptor-specific retarded species. Neither RAR alone nor
RXR alone bound the probe to produce this shifted species. The
presence of AGN 193109 did not diminish this interaction.
[0493] These results indicated that AGN 193109 did not
substantially alter either the homo- or hetero-dimerization
properties of RAR-.alpha.. Further, AGN 193109 did not inhibit the
interaction of receptor dimers with a DNA segment containing the
cognate binding site.
[0494] In view of the unique properties which characterized AGN
193109, we proceeded to investigate whether this antagonist could
additionally inhibit the activity of unliganded RARs. The
receptor/reporter system used to make this determination
advantageously exhibited high level constitutive activity in the
absence of added retinoid agonist. More specifically, these
procedures employed the ER-RAR chimeric receptor and ERE-tk-Luc
reporter system. The ERE-tk-Luc plasmid includes the region -397 to
-87 of the estrogen responsive 5'-flanking region of the Xenopus
vitellogenin A2 gene, described by Klein-Hitpass, et al. in Cell
46:1053-1061 (1986), ligated upstream of the HSV thymidine kinase
promoter and luciferase reporter gene of plasmid tk-Luc. The ER-RAR
chimeric receptors consisted of the estrogen receptor DNA binding
domain fused to the "D-E-F" domain of the RARs. Those having
ordinary skill in the art appreciate this "D-E-F" domain functions
to bind retinoid, to provide a retinoid inducible transactivation
function and to provide a contact site for heterodimerization with
RXR. Thus, luciferase expression in this reporter system was
dependent on activation of the transfected chimeric receptor
construct.
[0495] Example 10 describes the method used to demonstrate that AGN
193109 inhibited basal gene activity attributable to unliganded
RARs. These procedures were performed in the absence of added
retinoid agonist. The results presented below provided the first
indication that AGN 193109 exhibited negative hormone activity.
EXAMPLE 10
Repression of Basal Gene Activity of a Retinoid-Regulated Reporter
in Transiently Cotransfected Cell Lines
[0496] CV-1 cells were co-transfected with the ERE-tk-Luc reporter
plasmid and either ER-RAR-.alpha., ER-RAR-.beta. or ER-RAR-.gamma.
expression plasmids. The ERE-tk-Luc plasmid contained the
estrogen-responsive promoter element of the Xenopus laevis
vitellogenin A2 gene and was substantially identical to the
reporter plasmid described by Klein-Hitpass et al. in Cell 46:1053
(1986), except that the CAT reporter gene was substituted by a
polynucleotide sequence encoding luciferase. The ER-RAR-.alpha.,
ER-RAR-.beta. and ER-RAR-.gamma. chimeric receptor-encoding
polynucleotides employed in the co-transfection have been described
by Graupner et al. in Biochem. Biophys. Res. Comm. 179:1554 (1991).
These polynucleotides were ligated into the pECE expression vector
described by Ellis et al. in Cell 45:721 (1986) and expressed under
transcriptional control of the SV-40 promoter. Calcium phosphate
transfections were carried out exactly as described in Example 6
using 0.5 .mu.g/well of reporter plasmid and either 0.05 .mu.g,
0.10 .mu.g or 0.2 .mu.g/well of receptor plasmid. Cells were dosed
with vehicle alone (ethanol), ATRA (10.sup.-9 to 10.sup.-6 M), or
AGN 193109 (10.sup.-9 to 10.sup.-6 M) for 18 hours. Cell lysates
and luciferase activity measurements were performed as described in
Example 6.
[0497] The results presented in FIGS. 3A, 4A and 5A confirmed that
ATRA strongly induced luciferase expression in all transfectants.
Basal level expression of luciferase for the three transfected
chimeric RAR isoforms ranged from approximately 7,000 to 40,000
relative light units (rlu) and was somewhat dependent on the amount
of receptor plasmid used in the transfection. Thus, the three
chimeric receptors were activatable by ATRA, as expected. More
specifically, all three receptors bound ATRA and activated
transcription of the luciferase reporter gene harbored on the
ERE-tk-Luc plasmid.
[0498] FIGS. 3B, 4B and 5B present AGN 193109 dose response curves
obtained in the absence of any exogenous retinoid agonist.
Interestingly, ER-RAR-.alpha. (FIG. 3B) was substantially
unaffected by AGN 193109, while the ER-RAR-.beta. and
ER-RAR-.gamma. chimeric receptors (FIGS. 4B and 5B, respectively)
exhibited an AGN 193109 dose responsive decrease in luciferase
reporter activity.
[0499] We further investigated the negative hormone activity of AGN
193109 by testing its ability to repress gene expression mediated
by a chimeric RAR-.gamma. receptor engineered to possess a
constitutive transcription activator domain. More specifically, we
used a constitutively active RAR-.gamma. chimeric receptor fused to
the acidic activator domain of HSV VP-16, called RAR-.gamma.-VP-16,
in two types of luciferase reporter systems. The first consisted of
the ERE-tk-Luc reporter cotransfected with ER-RARs and
ER-RXR-.alpha.. The second utilized the .DELTA.MTV-TREp-Luc
reporter instead of the ERE-tk-Luc reporter.
[0500] Example 11 describes the method used to demonstrate that AGN
193109 could suppress the activity of a transcription activator
domain of an RAR. The results presented below proved that AGN
193109 could suppress RAR-dependent gene expression in the absence
of an agonist and confirmed that AGN 193109 exhibited negative
hormone activity.
EXAMPLE 11
Repression of RAR-VP-16 Activity in Transiently Transfected
Cells
[0501] CV-1 cells were transiently cotransfected according to the
calcium phosphate coprecipitation technique described under Example
6 using 0.5 .mu.g/well of the ERE-tk-Luc luciferase reporter
plasmid, 0.1 .mu.g/well of the ER-RXR-.alpha. chimeric reporter
expression plasmid, and either 0 .mu.g or 0.1 .mu.g/well of the
RAR-.gamma.-VP-16 expression plasmid. The chimeric receptor
ER-RXR-.alpha. consisted of the hormone binding domain (amino acids
181 to 458) of RXR-.alpha. (Mangelsdorf, et al. Nature 345:224-229
(1990)) fused to the estrogen receptor DNA binding domain
(Graupner, et al. Biochem. Biophys. Res. Comm. 179:1554 (1991)) and
was expressed from the SV-40 based expression vector pECE described
by Ellis, et al. in Cell 45:721 (1986). RAR-.gamma.-VP-16 is
identical to the VP16RAR-.gamma.1 expression plasmid described by
Nagpal et al. in EMBO. J. 12:2349 (1993), the disclosure of which
is hereby incorporated by reference, and encodes a chimeric protein
having the activation domain of the VP-16 protein of HSV fused to
the amino-terminus of full length RAR-.gamma.. At eighteen hours
post-transfection, cells were rinsed with phosphate buffered saline
(PBS) and fed with DMEM (Gibco-BRL) containing 10% FBS (Gemini
Bio-Products) that had been extracted with charcoal to remove
retinoids. Cells were dosed with an appropriate dilution of AGN
193109 or ATRA in ethanol vehicle or ethanol alone for 18 hours,
then rinsed with PBS and lysed using 0.1 M KPO.sub.4 (pH 7.8), 1.0%
TRITON X-100, 1.0 mM DTT, 2 mM EDTA. Luciferase activity was
measured according to the method described by de Wet, et al. in
Mol. Cell. Biol. 7:725 (1987), using firefly luciferin (Analytical
Luminescence Laboratory) and an EG&G Berthold 96-well plate
luminometer. Luciferase values represented the mean.+-.SEM of
triplicate determinations.
[0502] As shown in FIG. 6, CV-1 cells transfected with the
ERE-tk-Luc reporter 5 construct and the ER-RAR-.alpha.chimeric
expression plasmid exhibited a weak activation of luciferase
activity by ATRA, likely due to isomerization of ATRA to 9C-RA, the
natural ligand for the RXRs (Heyman et al. Cell 68:397 (1992).
Cells transfected with the same mixture of reporter and chimeric
receptor plasmids but treated with AGN 193109 did not exhibit any
effect on luciferase activity. As AGN 193109 does not bind to the
RXRs, this latter result was expected. CV-1 cells similarly
transfected with the ERE-tk-Luc reporter but with substitution of
an ER-RAR chimeric receptor expression plasmid for ER-RXR-.alpha.
exhibited a robust induction of luciferase activity following ATRA
treatment.
[0503] In contrast, inclusion of the RAR-.gamma.-VP-16 expression
plasmid with the ER-RXR-.alpha. and ERE-tk-Luc plasmids in the
transfection mixture resulted in a significant increase in the
basal luciferase activity as measured in the absence of any added
retinoid. This increase in basal luciferase activity observed for
the ER-RXR-.alpha./RAR-.gamma.-- VP-16 cotransfectants, when
compared to the result obtained using cells transfected with
ER-RXR-.alpha. alone, indicated that recombinant ER-RXR-.alpha. and
RAR-.gamma.-VP-16 proteins could heterodimerize. Interaction of the
heterodimer with the cis-regulatory estrogen responsive element led
to a targeting of the VP-16 activation domain to the promoter
region of the ERE-tk-Luc reporter. Treatment of such triply
transfected cells with ATRA led to a modest increase of luciferase
activity over the high basal level. However, treatment of the
triple transfectants with AGN 193109 resulted in a dose dependent
decrease in luciferase activity. Importantly, FIG. 6 shows that AGN
193109 treatment of cells cotransfected with ER-RXR-.alpha. and
RAR-.gamma.-VP-16 led to repression of luciferase activity with
maximal inhibition occurring at approximately 10.sup.-8 M AGN
193109.
[0504] Our observation that AGN 193109 repressed the constitutive
transcriptional activation function of RAR-.gamma.-VP-16 in the
presence of an RXR was explained by a model wherein binding of AGN
193109 to the RAR induced a conformational change in the RAR which
stabilizes a negative conformation that facilitates the binding of
a trans-acting negative coactivator protein. When the AGN
193109/RAR complex is bound by the NCP, the RAR is incapable of
upregulating transcription of genes that are ordinarily responsive
to activated RARs. Our model further proposes that the
intracellular reservoir of NCP is in limiting concentration in
certain contexts and can be depleted by virtue of AGN 193109
stimulated complexation with RARs.
[0505] The results presented in FIG. 6 additionally indicated that
even at 10.sup.-6 M AGN 193109, the ER-RAR-.alpha. and
RAR-.gamma.-VP-16 proteins could interact to form heterodimers
competent for activating transcription of the reporter gene. More
specifically, cells transfected with ER-RAR-.alpha. and
RAR-.gamma.-VP-16 and treated with AGN 193109 at a concentration
(10.sup.-8-10.sup.-6 M) sufficient to provide maximal inhibition,
gave luciferase activity readings of approximately 16,000 rlu.
Conversely, cells transfected only with ER-RXR-.alpha. and then
treated with AGN 193109 at a concentration as high as 10.sup.-6 M
exhibited luciferase expression levels of only approximately 8,000
rlu. The fact that a higher level of luciferase activity was
obtained in cells that expressed both ER-RXR-.alpha. and
RAR-.gamma.-VP-16, even in the presence of 10.sup.-6 M AGN 193109
demonstrated the persistence of an interaction between the two
recombinant receptors. The repression of RAR-.gamma.-VP-16 activity
by AGN 193109 suggested that modulation of NCP interaction can be
codominate with VP-16 activation. Accordingly, we realized that it
may be possible to modulate the expression of genes which are not
ordinarily regulated by retinoids in an AGN 193109 dependent
manner.
[0506] Candidates for AGN 193109 regulatable genes include those
that are activated by transcription factor complexes which consist
of non-RAR factors that associate or heterodimerize with RARs,
wherein the non-RAR factor does not require an RAR agonist for
activation. While stimulation with an RAR agonist may have
substantially no effect on the expression of such genes,
administration with AGN 193109 can promote formation of inactive
transcription complexes comprising AGN 193109/RAR/NCP.
Consequently, addition of the AGN 193109 retinoid negative hormone
can down-regulate transcription of an otherwise
retinoid-insensitive gene.
[0507] This same mechanism can account for the observation that AGN
193109 can repress the activity of the tissue transglutaminase
(TGase) gene in HL-60 cells. A retinoid response element consisting
of three canonical retinoid half sites spaced by 5 and 7 base pairs
has been identified in the transcription control region of this
gene. While TGase can be induced by RXR-selective agonists, it is
not responsive to RAR-selective agonists. The TGase retinoid
response element is bound by an RAR/RXR heterodimer (Davies et al.
in Press). Interestingly, AGN 193109 is able to repress TGase
activity induced by RXR agonists. This AGN 193109 mediated
repression can be accounted for by the ability of this negative
hormone to sequester NCPs to the RAR component of the heterodimer,
thereby repressing the activity of the associated RXR.
[0508] We have also obtained results which support conclusions
identical to those presented under Example 11 by employing
RAR-.gamma.-VP-16 and expression constructs and the
.DELTA.MTV-TREp-Luc reporter plasmid instead of the
RAR-.gamma.-VP-16 and ER-RAR-.alpha. expression constructs in
combination with the ERE-tk-Luc reporter plasmid. Consistent with
the results presented above, we found that RAR-.gamma.-VP-16
activity at the .DELTA.MTV-TREp-Luc reporter was inhibited by AGN
193109. Therefore, AGN 193109 repressed RAR-.gamma.-VP-16 activity
when this chimeric receptor was directly bound to a retinoic acid
receptor response element instead of indirectly bound to an
estrogen response element in the promoter region of the reporter
plasmid. These findings demonstrated that an assay for identifying
agents having negative hormone activity need not be limited by the
use of a particular reporter plasmid. Instead, the critical feature
embodied by an experimental system useful for identifying retinoid
negative hormones involves detecting the ability of a compound to
repress the activity of an RAR engineered to contain a constitutive
transcription activation domain.
[0509] Generally, retinoid negative hormones can be identified as
the subset of retinoid compounds that repress within a transfected
cell the basal level expression of a reporter gene that is
transcriptionally responsive to direct or indirect binding by a
retinoid receptor or a chimeric receptor that includes at least the
domains of the retinoid receptor located C-terminal to the DNA
binding domain of that receptor. This approach has been adapted to
a screening method useful for identifying retinoid negative
hormones. In the various embodiments of the invented screening
method, the structure of the receptor for which a negative hormone
is sought is variable. More specifically, the retinoid receptor can
be either of the RAR or the RXR subtype. The receptor can
optionally be engineered to include a constitutive transcription
activator domain. The retinoid receptor used to screen for negative
hormones optionally contains a heterologous DNA binding domain as a
substitute for the DNA binding domain endogenous to the native
receptor. However, when a second receptor is used in the screening
method, and where the second receptor can dimerize with the
retinoid receptor for which a negative hormone is sought, then that
retinoid receptor may not require a DNA binding domain because it
can be linked to the transcription control region of the reporter
gene indirectly through dimerization with the second receptor which
is itself bound to the transcription control region.
[0510] In the practice of the screening method, the ability of a
compound to repress the basal expression of a reporter is typically
measured in an in vitro assay. Basal expression represents the
baseline level of reporter expression in transfected cells under
conditions where no exogenously added retinoid agonist is present.
Optionally, steps may be taken to remove endogenous retinoid
ligands from the environment of the transfected cells via
procedures such as charcoal extraction of the serum that is used to
culture cells in vitro.
[0511] Examples of reporter genes useful in connection with the
screening method include those encoding luciferase, beta
galactosidase, chloramphenicol acetyl transferase or cell surface
antigens that can be detected by immunochemical means. In practice,
the nature of the reporter gene is not expected to be critical for
the operability of the method. However, the transcriptional
regulatory region of the reporter construct must include one or
more cis-regulatory elements that are targets of transcription
factors for which negative hormones are being sought. For example,
if one desires to identify RAR negative hormones, then the
transcriptional regulatory region of the reporter construct could
contain a cis-regulatory element that can be bound by an
RAR-containing protein. In this example, there should be
correspondence between the DNA binding domain of the RAR and the
cis-regulatory element of the transcriptional regulatory region of
the reporter construct. Thus, if a chimeric RAR having a
constitutive transcription activator domain and a DNA binding
domain that can bind cis-regulatory estrogen responsive elements is
employed in the screening method, then the transcriptional
regulatory region of the reporter construct should contain an
estrogen responsive element.
[0512] Examples of cis-regulatory elements that directly bind
retinoid receptors (RARES) useful in connection with the reporter
assay are disclosed by Mangelsdorf et al. in The Retinoid Receptors
in The Retinoids: Biology, Chemistry and Medicine, 2nd edition,
eds. Spom et al., Raven Press, Ltd., New York (1994), the
disclosure of which has been incorporated by reference hereinabove.
Examples of cis-regulatory elements that indirectly bind chimeric
receptors include DNA binding sites for any DNA binding protein for
which the DNA binding domain of the protein can be incorporated
into a chimeric receptor consisting of this DNA binding domain
attached to a retinoid receptor. Specific examples of heterologous
DNA binding domains that can be engineered into chimeric receptors
and that will recognize heterologous cis-regulatory elements
include those recognizing estrogen responsive elements. Thus, the
retinoid receptor portion of a chimeric receptor useful in
connection with the screening method need not contain the DNA
binding of the retinoid receptor but must contain at least the
ligand binding domain of the retinoid receptor.
[0513] A further example of indirect retinoid receptor binding to
the cis-regulatory element includes the use of a protein that can
bind the cis-regulatory element and dimerize with a retinoid
receptor. In this case, the retinoid receptor associates with the
cis-regulatory element only by association with the protein
responsible for DNA binding. An example of such a system would
include the use of a fusion protein consisting of a heterologous
DNA binding domain fused to an RXR, containing at least the domain
of the RXR responsible for dimerization with RARs. Cointroduced
RARs can dimerize with such a fusion protein bound to the
cis-regulatory element. We anticipate that any cis-regulatory
element-binding protein that dimerizes with RARs to result in an
indirect association of the RAR with the cis-regulatory element
will also be suitable for use with the negative hormone screening
method.
[0514] In a preferred embodiment of the screening method, retinoid
negative hormones are identified as those retinoids that repress
basal expression of an engineered RAR transcription factor having
increased basal activity. Although not essential for operability of
the screening method, the engineered RAR employed in the following
Example included a constitutive transcription activating domain.
Use of this chimeric receptor advantageously provided a means by
which the basal expression of a reporter gene could be elevated in
the absence of any retinoid. Although we have employed transient
transfection in the procedures detailed above, stably transfected
cell lines constitutively expressing the chimeric receptor would
also be useful in connection with the screening method.
[0515] As disclosed in the following Example, a chimeric retinoid
receptor having a constitutive transcription activator domain was
heterodimerizable with a second receptor engineered to contain a
DNA binding domain specific for an estrogen responsive
cis-regulatory element. In this case the chimeric retinoid receptor
having a constitutive transcription activator domain associates
with the cis-regulatory region controlling reporter gene expression
indirectly via binding to a second receptor that binds a DNA target
sequence. More particularly, the second receptor was engineered to
contain a DNA binding domain that recognized an estrogen responsive
element. Advantageously, the reporter gene having an estrogen
responsive element in the upstream promoter region was unresponsive
to retinoid agonists in the absence of the transfected chimeric
receptor having the constitutive transcription activator domain.
Accordingly, all reporter gene activity was attributed to the
transfected receptors. The combination use of the estrogen
responsive element DNA binding domain and the estrogen responsive
element cis-regulatory element are intended to be illustrative
only. Those having ordinary skill in the art will realize that
other combinations of engineered receptors having specificity for
non-RARE cis-regulatory elements will also be useful in the
practice of the invented screening method.
[0516] Cells useful in connection with the screening method will be
eukaryotic cells that can be transfected. The cells may be animal
cells such as human, primate or rodent cells. We have achieved very
good results using CV-1 cells, but reasonably expect that other
cultured cell lines could also be used successfully. Any of a
number of conventional transfection methods known in the art can be
used to introduce an expression construct encoding the chimeric
retinoid receptor having a constitutive transcription activator
domain.
[0517] The constitutive transcription activator domain will consist
of a plurality of amino acids which will likely have an overall
acidic character as represented by a negative charge under neutral
pH conditions. For example, the constitutive transcription
activator domain may have an amino acid sequence which is also
found in viral transcription factors. One example of a viral
transcription factor having a constitutive transcription activator
domain is the herpes simplex virus 16. However, other viral or
synthetic transcription activator domains would also be useful in
the construction of expression constructs encoding the chimeric
retinoid receptor having a constitutive transcription activator
domain.
[0518] As described below, we have developed a generalized
screening method useful for identifying retinoid negative hormones.
This screening method provides a means for distinguishing simple
antagonists from negative hormones. Table 12 lists several retinoid
compounds which exhibit potent affinity for RAR-.gamma. yet, with
the exception of ATRA, did not transactivate this receptor in a
transient cotransfection transactivation assay. We therefore tested
these compounds to determine which were RAR-.gamma. antagonists and
which, if any, of these antagonists exhibited negative hormone
activity.
[0519] Example 12 describes the method used to identify retinoid
compounds that were antagonists, and the subset of antagonists that
exhibited negative hormone activity.
EXAMPLE 12
Assay for Retinoid Negative Hormones
[0520] 4.times.10.sup.4 CV-1 cells were transfected by the calcium
phosphate coprecipitation procedure described in Molecular Cloning.
A Laboratory Manual (Sambrook et al. eds. Cold Spring Harbor Lab
Publ. 1989) using 0.5 .mu.g ERE-tk-Luc reporter plasmid and 0.1
.mu.g ER-RAR-.gamma. (Graupner et al. Biochem. Biophys. Res. Comm.
179:1554 (1991)) chimeric expression plasmid. After 18 hours, cells
were rinsed with PBS and fed with DMEM (Gibco-BRL) containing 10%
activated charcoal extracted FBS (Gemini Bio-Products). Cells were
treated with 10.sup.-8 M ATRA in ethanol or ethanol alone. In
addition, ATRA treated cells were treated with 10.sup.-9,
10.sup.-8, 10.sup.-7 or 10.sup.-6 M of the compounds listed in
Table 12. After 18 hours, cells were rinsed in PBS and lysed in 0.1
M KPO.sub.4 (pH 7.8), 1.0% TRITON X-100, 1.0 mM DTT, 2 mM EDTA.
Luciferase activities were measured as described by deWet et al. in
Mol. Cell. Biol. 7:725 (1987).
12 TABLE 12 Compound Kd (nM) @ RAR-.gamma..sup.a EC.sub.50 (nM) @
RAR-.gamma..sup.b ATRA 12 17 AGN 193109 6 na (Compound 60) AGN
193174 52 na (Compound 34a) AGN 193199 30 na AGN 193385 25 na
(Compound 23) AGN 193389 13 na (Compound 25) AGN 193840 40 na AGN
193871 30 na (Compound 50) .sup.aRelative affinity (Kd) determined
by competition of .sup.3H-ATRA binding to baculovirus expressed
RAR-.gamma. and application of the Cheng-Prussof equation.
.sup.bEC.sub.50 measured in CV-1 cells transiently cotransfected
with .DELTA.MTV-TREp-Luc and RS-RAR-.gamma.. "na" denotes no
activity.
[0521] As indicated by the results presented in part in FIG. 7 and
in Table 12, with the exception of ATRA, all of the compounds
listed in Table 12 were retinoid antagonists at RAR-.gamma..
[0522] The RAR-.gamma. antagonists identified in Table 12 were next
screened to determine which, if any, were also retinoid negative
hormones. 4.times.10.sup.4 CV-1 cells were transfected according to
the calcium phosphate procedure described in Molecular Cloning: A
Laboratory Manual (Sambrook et al. eds. Cold Spring Harbor Lab
Publ. 1989) using 0.5 .mu.g ERE-tk-Luc reporter plasmid and 0.1
.mu.g ER-RAR-.alpha. (Graupner et al. Biochem. Biophys. Res. Comm.
179:1554 (1991)) and 0.2 .mu.g RAR-.gamma.-VP-16 (Nagpal et al.
EMBO J. 12:2349 (1993)) chimeric expression plasmids. After 18
hours, cells were rinsed with PBS and fed with DMEM (Gibco-BRL)
containing 10% activated charcoal extracted FBS (Gemini
Bio-Products). Cells were treated with 10.sup.-9, 10.sup.-8,
10.sup.-7 or 10.sup.-6 M of each of the compounds listed in Table
12. Treatment with ethanol vehicle alone served as the negative
control. After 18 hours, cells were rinsed in PBS and lysed in 0.1
M KPO.sub.4 (pH 7.8), 1.0% TRITON X-100, 1.0 mM DTT, 2 mM EDTA.
Luciferase activities were measured as previously by deWet et al.
in Mol. Cell. Biol. 7:725 (1987).
[0523] As shown in FIG. 8, the retinoid antagonists of Table 12
could be separated into two classes by virtue of their effect on
the constitutive transcription activation function of the
RAR-.gamma.-VP-16 chimeric retinoid receptor. One group, which
included AGN 193174, AGN 193199 and AGN 193840, did not repress
RAR-.gamma.-VP-16 activity even though they were ATRA antagonists.
In contrast AGN 193109, AGN 193385, AGN 193389 and AGN 193871
exhibited a dose dependent repression of RAR-.gamma.-VP-16
constitutive activity. Therefore, while the compounds of both
groups were RAR-.gamma. antagonists, only those of the second group
exhibited negative hormone activity. This assay advantageously
distinguished retinoid negative hormones from simple retinoid
antagonists.
[0524] The foregoing experimental results proved that AGN 193109
met the criteria that define a negative hormone. More specifically,
the results presented under Example 11 demonstrated that AGN 193109
had the capacity to exert inhibitory activity at the RARs even in
the absence of exogenously added retinoid ligands. As such, this
compound possessed biological activities that did not depend upon
blockade of the interaction between the RARs and agonists such as
ATRA and AGN 191183. These findings led us to conclude that AGN
193109 stabilized interactions between RARs and NCPs. As diagrammed
in FIG. 9, NCP/RAR/PCP interactions exist in an equilibrium state.
An agonist serves to increase PCP interactions and decrease NCP
interactions, while an inverse agonist or negative hormone
stabilizes NCP and decreases PCP interactions. As indicated
previously, our experimental results suggested that the
intracellular availability of NCP for other receptors can be
modulated by AGN 193109 administration. More specifically, we
discovered that AGN 193109 can promote complexation of NCP with
RARs, thereby reducing the intracellular reservoir of NCP available
for interaction with transcription factors other than the RARs.
[0525] We next examined the effect of AGN 193109 on
agonist-mediated inhibition of AP-1 dependent gene expression. In
Endocr. Rev. 14:651 (1993), Pfhal disclosed that retinoid agonists
can down-regulate gene expression by a mechanism that involved
inhibition of AP-1 activity. We postulated that AGN 193109 could
have had either of two effects when used in combination with a
retinoid agonist in a model system designed to measure AP-1
activity. First, AGN 193109 could conceivably have antagonized the
effect of the agonist, thereby relieving the agonist-dependent
inhibition of AP-1 activity. Alternatively, AGN 193109 could have
potentiated the agonist's activity, thereby exaggerating the
agonist-dependent inhibition of AP-1 activity.
[0526] Example 13 describes the methods used to demonstrate that
AGN 193109 potentiated the anti-AP-1 activity of a retinoid
agonist. As disclosed below, the AGN 191183 retinoid agonist weakly
inhibited AP-1 dependent gene expression. The combination of AGN
193109 and the retinoid agonist strongly inhibited AP-1 dependent
gene expression. By itself, AGN 193109 had substantially no
anti-AP-1 activity.
EXAMPLE 13
AGN 193109 Potentiates the Anti-AP-1 Activity of a Retinoid
Agonist
[0527] HeLa cells were transfected with 1 .mu.g of the Str-AP1-CAT
reporter gene construct and 0.2 .mu.g of plasmid pRS-hRAR.alpha.,
described by Giguere et al. in Nature 33:624 (1987), using
LIPOFECTAMINE (Life Technologies, Inc.). Str-AP1-CAT was prepared
by cloning a DNA fragment corresponding to positions -84 to +1 of
the rat stromelysin-1 promoter (Matrisian et al., Mol. Cell. Biol.
6:1679 (1986)) between the HindIII-BamHI sites of pBLCAT3 (Luckow
et al., Nucl. Acids Res. 15:5490 (1987)). This sequence of the
stromelysin-1 promoter contains an API motif as its sole enhancer
element (Nicholson et al., EMBO J. 9:4443 (1990). The promoter
sequence was prepared by annealing two synthetic oligonucleotides
having sequences:
13 5'-AGAAGCTTATGGAAGCAATTATGAGTCAGTTTGCGGGTGACTCTGCA
AATACTGCCACTCTATAAAAGTTGGGCTCAGAAAGGTGGACCTCGAGGAT CCAG-3' (SEQ ID
NO:2), and 5'-CTGGATCCTCG AGGTCCACCTTTCTGAGCCCAACTTTTATAGAGTG
GCAGTATTTGCAGAGTCACCCGCAAACTGACTCATAATTGCTTCCATAAG CTTCT-3' (SEQ ID
NO:3).
[0528] Procedures involving transfection, treatment with
appropriate compounds and measurement of CAT activity were carried
out as described by Nagpal et al. in J. Biol. Chem. 270:923 (1995),
the disclosure of which is hereby incorporated by reference.
[0529] The results of these procedures indicated that AGN 193109
potentiated the anti-AP-1 activity of the retinoid agonist, AGN
191183. More specifically, in the concentration range of from
10.sup.-12 to 10.sup.-10 M, AGN 191183 did not inhibit the
TPA-induced Str-AP1-CAT expression. Treatment with AGN 193109 in
the concentration range of from 10.sup.-10 to 10.sup.-8 M did not
substantially inhibit AP-1 mediated reporter activity. However, the
results presented in FIG. 10 indicated that stimulation of the
transfectants with the combination of AGN 193109 (10.sup.-8 M) and
AGN 191183 in the concentration range of from 10.sup.-12 to
10.sup.-8 M substantially inhibited TPA-induced Str-AP1-CAT
expression by an amount of from 12% to 21%. Therefore, AGN 193109
potentiated the anti-AP-1 activity of AGN 191183 under conditions
where this retinoid agonist ordinarily did not inhibit AP-1
activity.
[0530] We reasoned that AGN 193109 potentiated the agonist-mediated
repression of AP-1 activity by a mechanism that likely involved AGN
193109-dependent sequestration of NCPs onto RARs. RARs belong to a
superfamily of nuclear receptors that also includes receptors for
1,25-dihydroxyvitamin D.sub.3, glucocorticoid, thyroid hormone,
estrogen and progesterone. It was a reasonable assumption that the
ability to bind NCPs may be shared among different members of the
nuclear receptor superfamily. This led us to speculate that AGN
193109 could potentiate the anti-AP-1 activity of one or more of
the ligands that interact with this superfamily of nuclear
receptors.
[0531] The results presented in the preceding Example clearly
indicated that AGN 193109 potentiated the anti-AP-1 activity of a
retinoid agonist. More specifically, AGN 193109 lowered the
threshold dose at which the anti-AP-1 activity of AGN 191183 could
be detected. Since AGN 193109 has substantially no anti-AP-1
activity by itself, its effect on nuclear receptor agonists was
synergistic. We also found that the AGN 193109 negative hormone
potentiated the anti-AP-1 activity of 1,25-dihydroxyvitamin
D.sub.3, the natural ligand for the vitamin D.sub.3 receptor.
[0532] The observed synergy between AGN 193109 and AGN 191183 in
the preceding Example necessarily implied that the anti-AP-1
activity of the retinoid agonist and the AGN 193109-mediated
potentiation of that activity must result from different
mechanisms. If the mechanisms of action of the two agents were
identical, then it follows that the effectiveness of the
combination of AGN 193109 and the agonist would have been additive.
Instead, the combination was shown to be more effective than either
agent alone, an effect that could not have been predicted in
advance of this finding.
[0533] Significantly, the AGN 193109-mediated potentiation of the
RAR agonist was performed using an approximately 100-fold molar
excess of AGN 193109 over that of the retinoid agonist.
Accordingly, the majority of RAs should have been bound by AGN
193109 leaving very few RARs available for agonist binding. In
spite of this fact, the population of RARs that were not bound by
AGN 193109 were able to bind retinoid agonist and vigorously
stimulate an agonist-dependent response measurable as an inhibition
of reporter gene expression. Thus, our data suggested possible
heterogeneity of RARs that are induced by AGN 193109.
[0534] The negative hormone activity of AGN 193109, attributed to
its ability to promote the interaction of RARs and NCPs, provided a
basis for understanding the synergy between AGN 193109 and retinoid
agonists. Our results were fully consistent with a model in which
AGN 193109 treatment of cells promoted binding of RARs and NCPs,
thereby reducing the number of free NCP and free RAR within the
cell. This results in the generation of two populations of RARs
that are functionally distinct. The first population is represented
by RARs associated with NCPs. Such AGN 193109/RAR/NCP complexes
cannot be activated by retinoid agonists. The second population
consists of RARs that are not bound by NCP, and that remain
available for interaction with agonists. This latter population is
designated "RAR*" to indicate free RARs in an environment
substantially depleted of NCP.
[0535] The RAR*s have decreased probabilities of association with
NCP through equilibrium binding and have an increased sensitivity
to retinoid agonists measurable, for example, as anti-AP-1
activity. This is so because, while the intracellular reservoir of
NCP is depleted by virtue of AGN 193109 administration, the
reservoir of PCP has not been depleted. Accordingly, free RAR*s can
bind a retinoid agonist and interact with PCP factors in an
environment substantially depleted of NCP. The ability of AGN
193109 to increase the sensitivity of other nuclear receptors to
their respective agonists can be attributed to the ability of these
different nuclear receptors to interact with the same NCPs that
interact with AGN 193109/RAR complexes. This model of AGN
193109-mediated modulation of NCP availability for nuclear receptor
family members is schematically represented in FIG. 11.
[0536] This mechanistic model led us to predict that AGN 193109
could modulate the activities of nuclear receptor ligands other
than retinoid agonists. As illustrated in the following Example, we
confirmed that AGN 193109 potentiated the activity of
1,25-dihydroxyvitamin D.sub.3 in an in vitro transactivation
assay.
[0537] Example 14 describes the methods used to demonstrate that
AGN 193109 enhanced the activity of 1,25-dihydroxyvitamin D.sub.3
in a transactivation assay.
EXAMPLE 14
AGN 193109 Potentiates 1,25-Dihydroxyvitamin D.sub.3 Activity
[0538] Hela cells were transfected using the cationic
liposome-mediated transfection procedure described by Felgner et
al. in Proc. Natl. Acad. Sci. USA 84:7413 (1987). 5.times.10.sup.4
cells were plated in 12-well multiwell plates and grown in DMEM
supplemented with 10% FBS. Cells were cotransfected in serum-free
medium using 2 .mu.g/well of LIPOFECTAMINE reagent (Life
Technologies, Inc.) with 0.7 .mu.g of the reporter plasmid
MTV-VDRE-Luc, containing two copies of the 1,25-dihydroxyvitamin
D.sub.3 response element 5'-GTACAAGGTTCACGAGGTTCACGTCTTA-3' (SEQ ID
NO: 4) from the mouse osteopontin gene (Ferrara et al. J. Biol.
Chem. 269:2971 (1994)) ligated into the reporter plasmid
.DELTA.MTV-Luc (Heyman et al. in Cell 68:397 (1992)), and 0.3 .mu.g
of the plasmid pGEM3Z (Pharmacia, Inc.) as carrier DNA to bring the
final concentration of DNA to 1.0 .mu.g per well. After six hours
of transfection, cells were fed with growth medium containing
charcoal extracted FBS at a final concentration of 10%. Eighteen
hours after transfection cells were treated with vehicle alone
(ethanol) or AGN 193109 in ethanol at a final concentration of
either 10.sup.-8 or 10.sup.-7 M. Six hours later
1,25-dihydroxyvitamin D.sub.3 was added in ethanol to a final
concentration of from 10.sup.-10 to 10.sup.-7 M. Cells were lysed
and harvested eighteen hours following 1,25-dihydroxyvitamin
D.sub.3 treatment. Luciferase activity was measured as described
above. This experimental system allowed a convenient method of
monitoring and quantitating 1,25-dihydroxyvitamin D.sub.3-dependent
gene expression.
[0539] The results presented in FIG. 12 indicated that, when
compared with the result obtained using 1,25-dihydroxyvitamin
D.sub.3 alone, AGN 193109 coadministered with 1,25-dihydroxyvitamin
D.sub.3 shifted the dose response curve to the left. This confirmed
that AGN 193109 potentiated the effectiveness of
1,25-dihydroxyvitamin D.sub.3 in the in vitro transactivation
assay. More specifically, FIG. 12 graphically illustrates that an
AGN 193109 concentration as low as 10-100 nM rendered the
1,25-dihydroxyvitamin D.sub.3 approximately 10 fold more active.
While a 1,25-dihydroxyvitamin D.sub.3 concentration of 10.sup.-8 M
was required to produce a luciferase expression of approximately
2,000 rlu, only one-tenth as much 1,25-dihydroxyvitamin D.sub.3 was
required to produce the same luciferase output when the vitamin was
coadministered with AGN 193109 at a concentration of
10.sup.-8-10.sup.-7 M. Although not shown on the graph in FIG. 12,
substantially identical results were obtained using AGN 193109
concentrations of 10.sup.-9 M and 10.sup.-8 M. Thus,
coadministration with AGN 193109 substantially reduced the amount
of 1,25-dihydroxyvitamin D.sub.3 that was required to produce a
similar effect in the absence of the negative hormone.
[0540] Interestingly, when the above procedure was repeated with
cotransfection of a vitamin D receptor (VDR) expression plasmid,
there was a coincident decrease in the ability of AGN 193109 to
potentiate the activity of 1,25-dihydroxyvitamin D.sub.3. We
interpreted this result as indicating that over-expression of VDRs
could affect the ability of AGN 193109 to potentiate the activity
of 1,25-dihydroxyvitamin D.sub.3. Thus, the intracellular
concentration of a ligand receptor, which may differ in a
tissue-specific fashion, can influence the ability of AGN 193109 to
potentiate the activity of a ligand that binds the receptor. This
was again consistent with a model in which titratable NCPs
contributed to the regulation of the Vitamin D.sub.3 response, and
supported the model set forth above.
[0541] As illustrated in the following Example, we also confirmed
that AGN 193109 potentiated the anti-AP-1 activity of
1,25-dihydroxyvitamin D.sub.3. Our model for the activity of AGN
193109 action explains this observation by invoking that NCPs
avidly associate with RARs in the presence of this drug. Endogenous
vitamin D receptors present in HeLa cells likely were rendered more
sensitive to the 1,25-dihydroxyvitamin D.sub.3 ligand, with the
consequence of exaggerating the ability of this ligand to inhibit
expression from the Str-AP1--CAT reporter.
[0542] Example 15 describes the methods used to demonstrate that
AGN 193109 potentiated the anti-AP-1 activity of
1,25-dihydroxyvitamin D.sub.3.
Example 15
AGN 193109 Potentiates the Anti-AP-1 Activity of
1.25-Dihydroxyvitamin D.sub.3
[0543] HeLa cells were transfected with 1 .mu.g of Str-AP1-CAT
using LIPOFECTAMINE according to the method described by Nagpal et
al. in J. Biol. Chem. 270:923 (1995). Transfected cells were
treated with AGN 193109 alone (10.sup.-9 to 10.sup.-7 M),
1,25-dihydroxyvitamin D.sub.3 alone (10.sup.-12 to 10.sup.-7 M) or
1,25-dihydroxyvitamin D.sub.3 (10.sup.-12 to 10.sup.-7 M) in the
presence of 10.sup.-8 M AGN 193109.
[0544] The results of these procedures indicated that AGN 193109
potentiated the ability of 1,25-dihydroxyvitamin D.sub.3 to inhibit
TPA-induced AP-1 activity. When used alone in the concentration
range of from 10.sup.-9 to 10.sup.-7 M, AGN 193109 had no
detectable anti-AP-1 activity. The results presented in FIG. 13
indicated that 1,25-dihydroxyvitamin D.sub.3 repressed
TPA-stimulated activity only in the 10.sup.-8 and 10.sup.-7 M
concentration range. Analysis of 1,25-dihydroxyvitamin D.sub.3
mediated repression of TPA stimulated CAT activity in the presence
of 10.sup.-8 M AGN 193109 indicated that anti-AP-1 activity was
detectable at 10.sup.-10 and 10.sup.-9 M 1,25-dihydroxyvitamin
D.sub.3 and an increase in activity at 10.sup.-8 and 10.sup.-7 M
doses compared to 1,25-dihydroxyvitamin D.sub.3 treatment alone.
This AGN 193109 dependent modulation of 1,25-dihydroxyvitamin
D.sub.3 mediated anti-AP-1 activity was consistent with our model
in which NCP sequestration to RARs made the NCP unavailable for
interaction with other nuclear receptor family members.
Accordingly, the receptors were rendered more sensitive to the
1,25-dihydroxyvitamin D.sub.3 treatment.
[0545] The mechanisms underlying RAR mediated transactivation and
anti-AP-1 activity are likely different. This conclusion was based
on our observation that high doses of AGN 193109 completely
inhibited transactivation without substantially inhibiting anti-AP
1 activity. We therefore wished to gain additional evidence to
support our model for RAR* formation mediated by AGN 193109
treatment. To accomplish this, we investigated whether AGN 193109
could potentiate the activity of the RAR specific agonist AGN
191183 in an in vitro transactivation assay.
[0546] Example 16 describes the methods used to demonstrate that
AGN 193109 potentiated the activity of the RAR specific agonist,
AGN 191183. The results of this procedure indicated that, under
particular circumstances, AGN 193109 enhanced the potency of the
RAR specific retinoid, and provided strong evidence that AGN 193109
promoted RAR* formation.
Example 16
Potentiation of Retinoid Effectiveness by AGN 193109
Coadministration
[0547] Hela cells were transfected using the cationic
liposome-mediated transfection procedure described by Felgner et
al. in Proc. Natl. Acad. Sci. USA 84:7413 (1987). 5.times.10.sup.4
cells were plated in 12 well multiwell plates and grown in DMEM
supplemented with 10% FBS. Cells were cotransfected in serum free
medium using LIPOFECTAMINE reagent (2 ug/well, Life Technologies,
Inc.) with 0.7 .mu.g of the reporter plasmid MTV-TREp-Luc,
containing two copies of the TREpal response element
5'-TCAGGTCATGACCTGA-3' (SEQ ID NO: 5) inserted into the reporter
plasmid .DELTA.MTV-Luc (Heyman et al. in Cell 68:397 (1992)), and
0.1 .mu.g of the RAR-.gamma. expression plasmid pRShRAR-.gamma.
(Ishikawa et al. Mol. Endocrinol. 4:837 (1990)). After six hours of
transfection, cells were fed with growth medium containing charcoal
extracted FBS at a final concentration of 10%. Eighteen hours after
transfection, cells were treated with vehicle alone (ethanol) or
AGN 193109 in ethanol at a final concentration of from 10.sup.-11
to 10.sup.-8 M. Six hours later, AGN 191183 was added in ethanol to
a final concentration of either 0, 10.sup.-10 or 10.sup.-9 M. Cells
were harvested after eighteen hours of AGN 191183 treatment and
luciferase activity was measured as described above.
[0548] Preliminary experiments indicated that 10.sup.-9 M AGN
193109 was relatively ineffective at inhibiting the response to of
10.sup.-9 M AGN 191183 in HeLa cells. This contrasted with the
ability of 10.sup.-9 M AGN 193109 to inhibit 10.sup.-8 M ATRA in
CV-1 cells (FIG. 2).
[0549] The results presented in FIG. 14 supported the prediction
that AGN 193109 stimulated the formation of RAR*. Consistent with
our characterization of the antagonist and negative hormone
activities of AGN 193109, treatment with AGN 193109 resulted in a
biphasic dose response curve. The lowest doses of AGN 193109
(10.sup.-11 and 10.sup.10 M) resulted in a stimulation of
luciferase activity over that of AGN 191183 alone. This effect
suggests that RAR*s are generated by AGN 193109. Curiously, this
was also seen for AGN 193109 treatment alone, suggesting that
RAR*'s can respond to an endogenous ligand. AGN 191183 is a
synthetic retinoid agonist and, like ATRA, activates transcription
through the RARs. Substitution of AGN 191183 for ATRA in Example 7
would give qualitatively similar results (i.e., AGN 193109 would
antagonize the effect of 10 nM AGN 191183). Example 16 illustrates
that, while AGN 193109 can function as an antagonist of RAR
agonists, dosing conditions could easily be identified wherein AGN
193109 coadministration potentiated activation mediated by an RAR
agonist. It is important to note that the doses of the compounds
used in Example 16 are substantially lower than the doses employed
in the procedure described under Example 7. We proposed that AGN
193109 treatment could lead to RAR heterogeneity RARs versus RAR*s.
The apparent heterogeneity (i.e., ability to potentiate) appears to
have different windows in transactivation versus AP-1 repression.
The reason that the curves are biphasic is because, with increasing
amounts of AGN 193109, there is proportionately less RAR available
to bind the agonist. This doesn't appear to be the case for AP-1
repression and we are left to speculate that this difference must
reflect two distinct mechanisms for transactivation and AP-1
repression by the same receptor species.
[0550] Clinical results have confirmed that some retinoids are
useful for inhibiting the growth of premalignant and malignant
cervical lesions. Exemplary studies supporting this conclusion have
been published by Graham et al. in West. J. Med 145:192 (1986), by
Lippman et al. in J. Natl. Cancer Inst. 84:241 (1992), and by
Weiner et al. in Invest. New Drugs 4:241 (1986)).
[0551] Similar conclusions are supported by the results of in vitro
studies that used cultured cells to quantitate the
antiproliferative effects of various retinoids. More specifically,
Agarwal et al. in Cancer Res. 51:3982 (1991) employed the ECE16-1
cell line to model the early stages of cervical dysplasia and
demonstrated that retinoic acid could inhibit epidermal growth
factor (EGF) dependent cellular proliferation.
[0552] Example 17 describes the methods used to demonstrate that
AGN 193109 can antagonize the activity of the AGN 191183 retinoid
agonist which inhibited proliferation of the ECE16-1 cell line.
Example 17
AGN 193109 Antagonizes the Antinroliferative Effect of Retinoids in
ECE16-1 Cells
[0553] ECE16-1 cells were seeded at a density of 1.times.10.sup.4
cells per cm.sup.2 in complete medium containing DMEM:F12 (3:1),
nonessential amino acids, 5% FBS, 5 .mu.g/ml transferrin, 2 nM of
3,3',5 triiodothyronine (thyroid hormone or "T.sub.3"), 0.1 nM
cholera toxin, 2 mM L-glutamine, 1.8.times.10.sup.-4 M adenine and
10 ng/ml EGF. Cells were allowed to attach to plates overnight and
then shifted to defined medium containing DMEM:F12 (3:1), 2 mM
L-glutamine, nonessential amino acids, 0.1% bovine serum albumin,
1.8.times.10.sup.-4 M adenine, 5 .mu.g/ml transferrin, 2 nM
T.sub.3, 50 .mu.g/ml ascorbic acid, 100 ug/ml streptomycin, 100
units/ml penicillin and 50 .mu.g/ml gentamicin. Defined medium (DM)
was supplemented with 10 ng/ml EGF. EGF treated cells received 10
nM of the AGN 191183 retinoid agonist in combination with either 0,
0.1, 1.0, 10, 100 or 1000 nM AGN 193109 or 1000 nM AGN 193109
alone. After three days of treatment, cells were harvested as
described by Hembree et al. in Cancer Res. 54:3160 (1994) and cell
numbers determined using a COULTER counter.
[0554] The results presented in FIG. 15 demonstrated that ECE16-1
cells proliferated in response to EGF but not in defined medium
alone. This confirmed the findings published by Andreatta-van Leyen
et al. in J. Cell. Physio. 160:265 (1994), and by Hembree et al. in
Cancer Res. 54:3160 (1994). Addition of 10 nM AGN 191183 and 0 nM
AGN 193109 completely inhibited EGF mediated proliferation. Thus,
AGN 191183 was a potent antiproliferative retinoid. Increasing the
AGN 193109 concentration from 0 nM to 10 nM antagonized the AGN
191183 mediated growth inhibition by approximately 50%. A ten-fold
molar excess of AGN 193109 completely reversed the
antiproliferative effect of AGN 191183. Treatment of cells with
1000 nM AGN 193109 alone had no effect on the EGF mediated
proliferation increase. These results proved that AGN 193109
antagonized the antiproliferative effect of a retinoid but had
substantially no antiproliferative activity of its own when used to
treat cells representing cervical epithelium that is sensitive to
growth inhibition by retinoids such as AGN 191183. Notably, there
was no evidence that AGN 193109 potentiated the antiproliferative
activity of the AGN 191183 agonist using the ECE16-1 model
system.
[0555] In contrast to the model system represented by the ECE16-1
cell line, there are other examples where cellular proliferation
associated with cervical dysplasia cannot be inhibited by retinoid
agonists. For example, Agarwal et al. in Cancer Res. 54:2108 (1994)
described the use of CaSki cells as a model for cervical tumors
that are unresponsive to retinoid therapy. As disclosed below,
rather than inhibiting cell proliferation, retinoid treatment had
substantially no effect on the growth rate of CaSki cells. The
following Example addressed the effect of the AGN 193109 negative
hormone on the proliferation rates of this cell line. The results
unexpectedly proved that AGN 193109 can inhibit the proliferation
of cervical tumor cells that are unresponsive to the
antiproliferative effects of retinoid agonists.
[0556] Example 18 describes the methods used to demonstrate that
AGN 193109 inhibited the growth of a cervical tumor cell line that
did not respond to the antiproliferative effects of other retinoids
such as AGN 191183. Significantly, AGN 193109 displayed
antiproliferative activity in the absence of added retinoid
Example 18
AGN 193109 Inhibits the Proliferation Rate of CaSki Cervical
Carcinoma-Derived Cell Line
[0557] We tested the effect of EGF on CaSki cell proliferation,
either alone or in combination with the AGN 191183 retinoid agonist
and/or the AGN 193109 negative hormone at a concentration of
10.sup.-6 M. Cell proliferation assays were performed as described
above for studies involving ECE16-1 cells. EGF was added to the
retinoid treated cultures to give a final concentration of 20
ng/ml. Cells were treated with AGN 191183 (10.sup.-10 to 10.sup.-6
M) in the presence or absence of 10.sup.-6 M AGN 193109 for a total
of three days. The media was replaced with fresh media and each of
the two retinoid compounds, as appropriate, every day. Cell numbers
were determined using a COULTER counter as described above.
[0558] The results presented in FIG. 16 indicated that CaSki cells
were substantially refractory to the effects of a retinoid agonist
and that AGN 193109 exhibited antiproliferative activity in the
absence of added retinoid. The presence of EGF in the culture media
stimulated CaSki cell growth. This conclusion was based on
comparison of the stripped bar representing no AGN 191183 and the
open bar representing defined growth media ("DM") alone. AGN 191183
treatment had no antiproliferative activity on the CaSki tumor cell
line. We discounted any slight increase in the cellular
proliferation rate associated with the retinoid agonist, because a
ten thousand fold increase in the retinoid agonist concentration
was associated with only roughly a 20% increase in the
proliferation rate. Thus, the AGN 191183 agonist had substantially
no effect on the proliferation rate of CaSki cells.
[0559] The results presented in FIG. 16 also indicated that AGN
193109 inhibited proliferation of the CaSki cervical epithelial
cell line. This conclusion was based on comparison of the
measurements appearing as the "0" AGN 191183 black bar and the "0"
AGN 191183 stripped bar. Thus, AGN 193109 was capable of
stimulating a biological response in the absence of added retinoid
agonist when used to treat cervical tumor cells that were not
growth inhibited by retinoid agonists such as AGN 191183.
[0560] Our discovery that the AGN 193109 negative hormone could
inhibit cellular proliferation was consistent with a model in which
unliganded RAR mediated the expression of genes that were required
for proliferation. While an RAR agonist such as AGN 191183 had
substantially no effect, or perhaps promoted cellular proliferation
slightly, AGN 193109 had an antiproliferative effect. The AGN
193109 negative hormone likely bound RARs thereby promoting NCP
association and causing the RARs to adopt an inactive conformation.
According to our model, this repressed gene activity that was
positively regulated by unliganded RARs. This ability of AGN 193109
to down-regulate the activity of unliganded RARs likely resulted
from its ability to promote the association of RARs and NCPs.
[0561] Those having ordinary skill in the art will appreciate that
some retinoid agonists are useful for controlling the undesirable
consequences of cell growth that follows retinal detachment. After
retinal detachment the retinal pigment epithelium (RPE)
dedifferentiates, proliferates and migrates into the subretinal
space. This process can negatively impact the success of surgical
procedures aimed at retinal reattachment. Campochiaro et al. in
Invest. Opthal & Vis. Sci. 32:65 (1991) have demonstrated that
RAR agonists such as ATRA exhibited an antiproliferative effect on
the growth of primary human RPE cultures. Retinoid agonists have
also been shown to decrease the incidence of retinal detachment
following retinal reattachment surgery (Fekrat et al. Opthamology
102:412 (1994)). As disclosed in the following Example, we analyzed
the ability of the AGN 193109 negative hormone to suppress growth
in primary human RPE cultures.
[0562] Example 19 describes the methods used to demonstrate that
AGN 193109 potentiated the antiproliferative effect of a retinoid
antagonist in a primary culture of human retinal pigment
epithelium.
Example 19
AGN 193109 Potentiates the Antiproliferative Activity of ATRA
[0563] Primary cultures of human retinal pigment epithelium (RPE)
were established according to the method described by Campochiaro
et al. in Invest. Opthal & Vis. Sci. 32:65 (1991).
5.times.10.sup.4 cells were plated in 16-mm wells of 24-well
multiwell plates in DMEM (Gibco) containing 5% FBS. Cells were mock
treated with ethanol vehicle alone, ATRA (10.sup.-10 to 10.sup.-6
M) in ethanol, AGN 193109 (10.sup.-10 to 10.sup.-6 M) in ethanol,
or ATRA (10.sup.-10 to 10.sup.-6 M) and 10.sup.-6 M AGN 193109.
Cells were fed with fresh media containing the appropriate
concentrations of these compounds every two days for a total of
five days of treatment. Cells were removed from the plates by
gentle digestion with trypsin and the number of cells was counted
with an electronic cell counter.
[0564] The results presented in FIG. 17 indicated that AGN 193109
dramatically potentiated the antiproliferative activity of ATRA on
RPE cells. Treatment of primary RPE cells with ATRA led to a dose
dependent decrease in RPE cell proliferation with an approximately
40% decrease at 10.sup.-6 M ATRA relative to control cultures. AGN
193109 treatment did not substantially alter the growth rate of the
RPE cells at any concentration tested in the procedure.
Unexpectedly, the combination of ATRA (10.sup.-11 to 10.sup.-6 M)
and 10.sup.-6 M AGN 193109 had a stronger antiproliferative
activity than ATRA alone. Thus, AGN 193109 cotreatment potentiated
the antiproliferative effect of ATRA. More specifically, the
results shown in the Figure indicated that the antiproliferative
effect of 10.sup.-8 M ATRA was obtainable using only 10.sup.-10 M
ATRA in combination with 10.sup.-7 M AGN 193109. Thus, the AGN
193109 negative hormone advantageously enhanced the
antiproliferative activity of ATRA by approximately 100 fold.
[0565] In an independent experiment, comparison of the
antiproliferative effect of ATRA (10.sup.-11 to 10.sup.-6 M) with
that of ATRA and 10.sup.-6 M AGN 193109 again demonstrated the
apparent increase in sensitivity of primary RPE cells to ATRA in
the presence of AGN 193109. In this system, AGN 193109 neither
functioned as a retinoid antagonist nor exhibited an
antiproliferative effect when used alone. However, AGN 193109
coadministration potentiated the antiproliferative activity of the
retinoid agonist.
[0566] AGN 193109 was tested for its ability to potentiate the
anti-proliferative effect of 13-cis retinoic acid (13-cis RA) in
primary RPE cultures using conditions and techniques to measure RPE
cell proliferation described above. Notably, 13-cis RA is
clinically significant. More particularly, 13-cis RA is useful in
the treatment of several disease states, including acne (Peck et
al. N. Engl. J. Med. 300:329 (1977); Jones et al. Br. J. Dermatol.
108:333 (1980)), and squamous cell carcinoma of the skin and cervix
in combination treatment with interferon 2.alpha. (Lippman et al.
J. Natl. Cancer Inst. 84:241 (1992); Moore et al. Seminars in
Hematology 31:31 (1994)).
[0567] The results presented in FIG. 18 indicated that both 13-cis
RA (10.sup.-12 to 10.sup.-6 M) and ATRA (10.sup.-12 to 10.sup.-6M)
effectively inhibited RPE cell growth. Notably, the 13-cis isomer
was approximately two orders of magnitude less effective in this
assay when compared with ATRA. Similar to the results obtained
using coadministration of AGN 193109 and ATRA (above),
coadministration of AGN 193109 (either 10.sup.-8 or 10.sup.-6M)
with 13-cis RA (10.sup.-12 to 10.sup.-6M) dramatically increased
the potency of 13-cis RA in mediating repression of RPE cell
proliferation. In contrast to treatment with 13-cis RA alone,
coadministration of AGN 193109 enhanced the potency of 13-cis RA.
Thus, AGN 193109 potentiated the antiproliferative activity of
13-cis RA.
[0568] We next tested the ability of AGN 193109 to potentiate the
activities of other nuclear receptor hormones in primary RPE cell
cultures. Dexamethasone, a synthetic glucocorticoid receptor
agonist, is one member of a class of compounds that have been used
clinically for their potent anti-inflammatory and immunosuppressive
properties. Thyroid hormone (T3; 3,3',5'-Triiodothyronine) is a
natural thyroid hormone receptor agonist used primarily for hormone
replacement therapy in the treatment of hypothyroidism. Methods
used in these experiments were identical to those described above
for procedures employing ATRA and 13-cis RA.
[0569] The results of these procedures indicated that
coadministration of AGN 193109 and the nuclear receptor agonists
potentiated the antiproliferative activities of the nuclear
receptor agonists. More specifically, the results presented in FIG.
19 showed that single-agent treatment of RPE cells with either
dexamethasone (10.sup.-11 to 10.sup.-6M) or ATRA (10.sup.-12 to
10.sup.-6M) was substantially unable to inhibit RPE cell
proliferation. However, treatment of RPE cells with dexamethasone
(10.sup.-11 to 10.sup.-6M) and either 10.sup.-8 or 10.sup.-6M AGN
193109 repressed RPE cell proliferation to an extent that
approximated the inhibition caused by treatment with ATRA.
Similarly, the results presented in FIG. 20 indicated that AGN
193109 potentiated the antiproliferative activity of thyroid
hormone. Similar to the results obtained using dexamethasone, the
proliferation of RPE cells was refractory to single-agent treatment
with thyroid hormone (10.sup.-11 to 10.sup.-6M). However,
co-treatment of RPE cells with thyroid hormone (10.sup.-11 to
10.sup.-6M) and AGN 193109 (either 10.sup.-8 or 10.sup.-6M)
inhibited RPE cell proliferation in a thyroid hormone dependent
manner. We concluded that AGN 193109 rendered primary RPE cultures
sensitive to the anti-proliferative effects of these nuclear
receptor agonists. The mechanism by which AGN 193109 mediated these
effects likely involved modulation of NCP/RAR interactions.
[0570] We additionally examined the effect of AGN 193109 on the
expression of marker genes in other experimental systems that were
sensitive to retinoid agonists. Both the MRP8 and stromelysin genes
are known to be inhibited by retinoid agonists in a variety of
biological systems. For example, Wilkinson et al. in J. Cell Sci.
91:221 (1988) and Madsen et al. in J. Invest. Dermatol. 99:299
(1992) have disclosed that MRP8 gene expression was elevated in
psoriasis. Conversely, MRP8 gene expression was repressed by the
retinoid agonist AGN 190168 in human psoriatic skin (Nagpal et al.,
submitted 1995), in human keratinocyte raft cultures (Chandraratna
et al. J. Invest. Dermatol. 102:625 (1994)) and in cultured human
newborn foreskin keratinocytes (Thacher et al. J. Invest. Dermatol.
104:594 (1995)). Nagpal et al. in J. Biol. Chem. 270:923 (1995)
have disclosed that stromelysin mRNA levels were repressed by
retinoid agonists such as AGN 190168 in cultured human newborn
foreskin keratinocytes. We analyzed the regulated expression of
these genes following treatment of cultured human newborn foreskin
keratinocytes with either the AGN 191183 retinoid agonist or AGN
193109.
[0571] Example 20 describes the methods used to demonstrate that
AGN 193109 inhibited MRP-8 expression in cultured
keratinocytes.
Example 20
AGN 193109 Inhibits MRP-8 Expression in Keratinocytes
[0572] Primary foreskin keratinocytes were isolated according to
the procedure described by Nagpal et al. in J. Biol. Chem. 270:923
(1995) and cultured in keratinocyte growth medium (KGM) that was
purchased from Clonetics. After 3 days of treatment with AGN 191183
(10.sup.-7 M) or AGN 193109 (10.sup.-6 M), total cellular RNA was
isolated from treated and control keratinocytes according to
standard methods. The mRNA was reverse transcribed into cDNA which
then served as the template in a PCR amplification protocol using
primers specific for either the glyceraldehyde phosphate
dehydrogenase (GAPDH) housekeeping gene or MRP-8. The GAPDH primers
had the sequences 5'-CCACCCATGGCAAATTCCATGGCA-3' (SEQ ID NO: 6) and
5'-TCTAGACGGCAGGTCAGGTCCACC-3' (SEQ ID NO: 7). The MRP-8 primers
had the sequences 5'-ACGCGTCCGGAAGACCTGGT-3' (SEQ ID NO: 8) and
5'-ATTCTGCAGGTACATGTCCA-3' (SEQ ID NO: 9). An aliquot from the
MRP-8 amplification reaction (10 .mu.l) was removed after every
cycle of PCR amplification starting from 12 cycles and ending at 21
cycles. Similarly, an aliquot of the GAPDH amplification reaction
was removed after every PCR cycle starting at 15 cycles and ending
at 24 cycles. The samples were electrophoresed on 2% agarose gels
and the separated amplification products detected by ethidium
bromide staining. The staining intensity of the amplification
products served as a quantitative measure of the amount of starting
mRNA specific for the given primer set.
[0573] The results of this procedure indicated that both AGN 191183
and AGN 193109 independently inhibited MRP-8 expression in
keratinocytes. The intensity of the stained GAPDH amplification
product was substantially equivalent in the lanes of the gel
representing starting material isolated from control, AGN 191183,
and AGN 193109 treated keratinocytes. Weak bands representing the
GAPDH amplification product were first detectable in lanes
corresponding to samples removed after 18 cycles of PCR
amplification. The equivalent staining intensities among the
various lanes of the gel indicated that equivalent masses of
starting material were used for all samples. Accordingly,
differences in the intensities of stained bands representing MRP-8
amplification products were indicative of differences in MRP-8 mRNA
expression among the various starting samples. As expected, the
MRP-8 amplified signal was inhibited in AGN 191183 (10.sup.-7 M)
treated cultures relative to an untreated control. AGN 193109
(10.sup.-6 M) treatment of cultured keratinocytes also repressed
MRP8 expression as judged by lower intensity of stained
amplification product.
[0574] As illustrated in the following Example, AGN 193109 also
inhibited expression of a second marker gene in keratinocytes.
Nagpal et al. in J. Biol. Chem. 270:923 (1995) disclosed that
stromelysin mRNA expression was down-regulated by RAR specific
agonists in cultured newborn human foreskin keratinocytes.
Nicholson et al. (EMBO J. 9:4443 (1990)) disclosed that an AP-1
promoter element played a role in the retinoid-dependent negative
regulation of the stromelysin-1 gene. Thus, it was of interest to
determine whether AGN 193109 could alter the expression of this
gene.
[0575] Example 21 describes the methods used to demonstrate that
AGN 193109 inhibited stromelysin-1 gene expression in the absence
of an exogenously added retinoid agonist.
Example 21
AGN 193109 Inhibits Stromelysin-1 Expression in Cultured
Keratinocytes
[0576] Primary foreskin keratinocytes were either mock treated or
treated for 24 hours with the RAR agonist AGN 191183 (10.sup.-7 M),
or AGN 193109 (10.sup.-6 M). Total RNA prepared from mock-treated
and retinoid-treated keratinocytes was reverse transcribed and the
resulting cDNA was PCR amplified using .beta.-actin or
stromelysin-1 oligo primers exactly as described by Nagpal et al.
in J. Biol. Chem. 270:923 (1995)), the disclosure of which has been
incorporated by reference. A sample (10 .mu.l) from the PCR
amplification reaction was removed after every three cycles
starting from 18 cycles of PCR amplification. The sample was
electrophoresed on a 2% agarose gel and detected after ethidium
bromide staining.
[0577] Results of these procedures indicated that AGN 193109
inhibited stromelysin-1 gene expression in the absence of an
exogenously added retinoid agonist. More specifically,
ethidium-stained bands representing .beta.-actin amplification
products were easily detectable the agarose gels after 18 cycles of
PCR. While all band intensities increased with additional cycles of
the amplification reaction, stained bands were somewhat less
intense in samples representing AGN 191183 treated cells. This
indicated that a slightly lesser amount of RNA must have been
present in the starting samples corresponding to cells treated with
AGN 191183. The results also indicated that stromelysin-1 mRNA was
detectable in mock-treated keratinocytes starting at 33 cycles of
PCR amplification. As expected, stromelysin-1 mRNA expression was
inhibited after AGN 191183 (10.sup.-7 M) treatment as judged by the
weaker band intensity on when compared with samples derived from
mock-treated samples. When normalized to the intensities of the
M-actin amplification products, and consistent with the results
obtained in measurements of MRP-8 expression, AGN 193109 (10.sup.-6
M) treatment of keratinocytes resulted in down-regulation of
stromelysin-1 mRNA levels. Indeed, the down-regulation stimulated
by AGN 193109 treatment was indistinguishable from the
down-regulation caused by treatment of keratinocytes with the RAR
agonist AGN 191183.
[0578] As disclosed herein, AGN 193109 can have any of three
possible effects with respect to modulating the activity of a
coadministered steroid superfamily agonist. First, AGN 193109 may
have no effect. Second, AGN 193109 may antagonize the effect of the
agonist, thereby leading to a decrease in the activity of the
agonist. Finally, AGN 193109 may potentiate the activity of the
agonist, thereby leading to a stimulation of the measured effect
produced by the agonist.
[0579] Compounds having activities that can be modulated by AGN
193109 include retinoid receptor agonists and agonists which bind
to other members of the steroid receptor superfamily. This latter
category of agonists includes vitamin D receptor agonists,
glucocorticoid receptor agonists and thyroid hormone receptor
agonists. Peroxisome proliferator-activated receptors, estrogen
receptor and orphan receptors having presently unknown ligands may
also be potentiated by AGN 193109. In the case where the steroid
superfamily agonist is an RAR agonist, AGN 193109 may either
antagonize or potentiate the activity of that agonist. In the case
where the agonist used in combination with AGN 193109 is a compound
that can bind to a nuclear receptor other than an RAR,
coadministration of AGN 193109 will either have no effect or will
sensitize of the system to the agonist so that the activity of the
agonist is potentiated.
[0580] A generalized exemplary procedure for determining which of
the three possible activities AGN 193109 will have in a particular
system follows. This description illustrates each of the possible
outcomes for AGN 193109 coadministration with a steroid receptor
superfamily agonist. Biological systems useful for assessing the
ability of AGN 193109 to modulate the activity of a nuclear
receptor agonist include but are not limited to: established tissue
culture cell lines, virally transformed cell lines, ex-vivo primary
culture cells and in vivo studies utilizing living organisms.
Measurement of the biological effect of AGN 193109 in such systems
could include determination of any of a variety of biological
endpoints. These endpoints include: analysis of cellular
proliferation, analysis of programmed cell death (apoptosis),
analysis of the differentiation state of cells via gene expression
assays, analysis of the ability of cells to form tumors in nude
mice and analysis of gene expression after transient or stable
introduction of reporter gene constructs.
[0581] For illustrative purposes, an mRNA species designated as
mRNA "X" is expressed from gene "X" in primary cultured "Y" cells
isolated from the organ "Z." Under standard culture conditions,
where several "Y" cell genetic markers are maintained, including
expression of gene "X", addition of a retinoid agonist leads to a
decrease in the abundance of "X" mRNA. Analysis of gene X
expression can be assessed via isolation of cellular mRNA and
measurement of the abundance of X mRNA levels via polymerase chain
reaction, ribonuclease protection or RNA blotting procedures such
as Northern analyses. After isolation from organ Z, primary Y cells
are cultured in an appropriate growth medium. The primary cultures
are then plated into tissue culture plates for expansion of the
cell population. This step facilitates separation of the cells into
four sample groups so that various doses of the retinoid agonist
and AGN 193109 can be delivered. The first group will be a control,
receiving vehicle only. The second group will receive the RAR
agonist, retinoic acid, delivered in ethanol, in amounts sufficient
to provide final concentrations in the range of from 10.sup.-11 to
10.sup.-6 M. The lowest dose may need to be empirically determined
depending on the sensitivity of the system. Such determinations
fall within the scope of routine experimentation for one having
ordinary skill in the art. The third group will receive both the
nuclear receptor agonist at the same doses used for treating the
cells of group 2, and a constant dose of AGN 193109. The dose of
AGN 193109 used for treating the cells of group 3 will also need to
be determined empirically, but should approximate the affinity
constant (Kd) of AGN 193109 for the RAR subtypes (i.e., at least
10.sup.-8 M). The fourth group will receive AGN 193109 at doses
minimally including that used for agonist coadministration in group
3. An alternative to this dosing regimen would substitute AGN
193109 for the retinoid agonist described in the foregoing example,
as specified in group 2, and a constant dose of retinoid agonist in
place of AGN 193109, as specified in groups 3 and 4. After a
suitable incubation period, cells should be harvested in a manner
suitable for determination of the biological endpoint being
measured as an indicator of agonist activity.
[0582] For example, analysis of the effect of AGN 193109 on
retinoic acid dependent regulation of gene expression would involve
comparison of the abundance of the MRNA species X in the mRNA pool
harvested from cells treated according to each of the four
protocols described above. RNA derived from control cells will
serve to determine the baseline expression of X mRNA and will
represent a condition corresponding to no repression. Comparison of
this level with that measured in the mRNA pool derived from cells
treated with retinoic acid will allow for determination of the
effect of this agonist on gene expression. Quantitated levels of
the repression of specific mRNAs resulting from retinoic acid
treatment can then be compared with mRNA abundances from cells
treated in parallel with either AGN 193109 alone or AGN 193109 in
combination with retinoic acid. While this generalized example
illustrates an analysis of the effect of coadministered AGN 193109
on the expression of a gene repressed by a retinoid agonist, the
example could alternatively have described analysis of the effect
of coadministered AGN 193109 on a gene that was induced by a
retinoid agonist. The critical feature for determining whether AGN
193109 will behave as an agonist, as a negative hormone or have no
effect in a particular system will involve quantitative comparison
of the magnitude of the effect in the presence and absence of AGN
193109.
[0583] An example in which AGN 193109 potentiated the activity of a
coadministered agonist would be a case in which AGN 193109
cotreatment with retinoic acid resulted in a level of X mRNA
expression that is further repressed relative to the level measured
in cells treated with retinoic acid alone. More specifically,
comparison of the dose response curve of the biological effect
(i.e., repression of X mRNA abundance) plotted on the Y-axis versus
the dose of the agonist (logarithmic scale) on the X-axis would
allow comparison of agonist-mediated repression of X mRNA abundance
in the presence and absence of AGN 193109 cotreatment. The ability
of AGN 193109 to sensitize the biological response to the agonist,
thereby potentiating the activity of the agonist, will be indicated
by a leftward shift in the dose response curve. More specifically,
in the presence of AGN 193109 less agonist would be required to
obtain the same biological effect obtainable using the agonist
alone.
[0584] An example of AGN 193109 mediating antagonism of a
coadministered agonist would be a case in which AGN 193109
cotreatment with retinoic acid resulted in a level of X MRNA
expression that is less repressed compared to that measured in
cells treated with retinoic acid alone. Comparison of dose response
curves of X mRNA repression versus log dose of agonist in the
presence and absence of AGN 193109 will demonstrate a shift to the
right in the dose response curve. More specifically, in the
presence of AGN 193109, more agonist will be necessary to obtain
the same biological effect obtainable with single agent treatment
with the agonist alone.
[0585] The above examples wherein AGN 193109 mediates either
antagonism or potentiation describe experimental outcomes for
coadministration of AGN 193109 with a retinoid agonist. If,
however, the agonist coadministered with AGN 193109 is an agonist
capable of binding and activating a member of the steroid receptor
superfamily other than an RAR, then instead of antagonizing the
agonist, it becomes possible that AGN 193109 would have no effect
on the activity of the agonist. If AGN 193109 cotreatment with such
an agonist results in a level of mRNA expression which is equal to
that measured in cells treated with agonist alone, then AGN
193109's ability to affect the availability of NCPs via promotion
of RAR:NCP associations will be silent in this system. This would
be an example wherein AGN 193109 has no effect on a coadministered
agonist.
[0586] Example of Antagonism
[0587] The method disclosed in the above generalized example for
determining the effect of AGN 193109 coadministered with a retinoid
agonist is exemplified by the procedure described under Example 7.
CV-1 cells cotransfected with one of the three retinoic acid
receptors and the retinoid agonist inducible MTv-TREp-Luc reporter
construct were dosed with either ethanol (control, group 1), AGN
193109 at final concentrations of from 10.sup.-9 to 10.sup.-6 M
(group 2), AGN 193109 at final concentrations of from 10.sup.-9 to
10.sup.-6 M coadministered with retinoic acid at 18.sup.-8 M (group
3), or retinoic acid (10.sup.-8 M, group 4). Comparison of the
luciferase activity of group 1 with that of group 4 allowed
determination of the level of retinoid agonist induced expression
of the luciferase reporter gene in the absence of added AGN 193109.
Comparison of luciferase reporter gene expression in cells of group
3 with that measured in cells of group 4 indicated that AGN 193109
behaved as an antagonist of the retinoid agonist in this
system.
[0588] Example of Antagonism
[0589] The method disclosed in the generalized example for
determining the effect of AGN 193109 coadministered with a retinoid
agonist was similarly used to determine in Example 17 that AGN
193109 functioned as an antagonist of a-retinoid agonist-mediated
repression of EGF-stimulated cellular proliferation in ECE-16-1
transformed cervical epithelial cells. In this procedure,
treatments of ECE-16-1 cells included a control sample treated with
EGF alone (group 1), a sample treated with the combination of EGF
and AGN 193109 at a final concentration of 10.sup.-6 M (group 2), a
sample treated with the combination of EGF and AGN 193109 at final
concentrations of from 10.sup.-10 to 10.sup.-6 M coadministered
with a single dose of the retinoid agonist AGN 191183 at 10.sup.-8
M (group 3), and a sample treated with the combination of EGF and
AGN 191183 at 10.sup.-8 M (group 4). After three days of treatment,
cellular proliferation rates were determined. Determination that
the cells had been stimulated to proliferate by EGF was possible
because an additional control treatment was included wherein cells
were exposed to defined medium that did not contain EGF. Comparison
of the number of cells in group 1 with the number of cells in group
4 allowed for determination that RAR agonist AGN 191183 repressed
the EGF-stimulated proliferation of ECE-16-1 cells. Comparison of
group 3 with group 4 indicated that AGN 193109 antagonized the
activity of the RAR agonist in this system.
[0590] Example of Potentiation
[0591] The method disclosed in the generalized example for
determining the effect of AGN 193109 coadministered with a retinoid
agonist was also used in Example 14 to determine that AGN 193109
potentiated the activity of a nuclear receptor agonist in HeLa
cells transfected with the 1,25-dihydroxyvitamin D.sub.3 inducible
MTV-VDRE-Luc reporter gene. Treatments of transfected cells
included vehicle alone (control, group 1), 1,25-dihydroxyvitamin
D.sub.3 at final concentrations of from 10.sup.-10 to 10.sup.-7 M
(group 2), 1,25-dihydroxyvitamin D.sub.3 at final concentrations of
from 10.sup.-10 to 10.sup.-7 M coadministered with AGN 193109 at a
final concentration of either 10.sup.-8 or 10.sup.-7 M (group 3),
and AGN 193109 as a single agent treatment at a final concentration
of either 10.sup.-8 or 10.sup.-7 M (group 4). Comparison of the
luciferase activity measured in group 1 (control) cells with that
of group 2 cells allowed for determination that
1,25-dihydroxyvitamin D.sub.3 stimulated luciferase activity was
dose-dependent. Comparison of luciferase activity measured in cells
of group 4 (AGN 193109 single agent treatment) with that measured
in cells of group 3 (AGN 193109 coadministration) similarly allowed
for determination of dose-dependent 1,25-dihydroxyvitamin D.sub.3
stimulated luciferase activity in the presence of a given
concentration of AGN 193109. In this instance, the zero value
represented the luciferase activity in cells treated with AGN
193109 alone (group 4). Such a dosing regimen allowed for
comparison of three 1,25-dihydroxyvitamin D.sub.3 dose response
curves. Comparison of the dose response curve of
1,25-dihydroxyvitamin D.sub.3 in the absence of AGN 193109 with the
curve representing coadministration of AGN 193109 (either 10.sup.-8
or 10.sup.-7 M) demonstrated potentiation of the agonist activity
as evidenced by a leftward shift in the half-maximal response.
[0592] Example of Potentiation
[0593] The method disclosed in the generalized example for
determining the effect of AGN 193109 coadministered with a retinoid
agonist was further used to determine in Example 19 that AGN 193109
potentiated the antiproliferative activity of an RAR agonist in
primary cultures of human retinal pigment epithelium cells.
Treatments of cells included: ethanol vehicle alone (group 1),
retinoic acid at final concentrations of from 10.sup.-10 to
10.sup.-6 M (group 2), retinoic acid at final concentrations of
from 10.sup.-10 to 10.sup.-6 M coadministered with 10.sup.-6 M AGN
193109 (group 3), and AGN 193109 alone at final concentrations of
from 10.sup.-10 to 10.sup.-6 M (group 4). Comparison of assay
results obtained using cells of groups 1 and 2 allowed for
determination of the dose dependent inhibition of proliferation of
these cells by retinoic acid. Similarly, comparison of results
obtained using cells of group 3 with those of group 1 allowed for
determination of the dose dependent inhibition of proliferation of
these cells by retinoic acid in the presence of coadministered AGN
193109. Group 4 demonstrated the inability of AGN 193109 to
substantially alter the proliferation rate of these cells when used
as a single treatment agent. Comparison of the dose response curves
of retinoic acid mediated repression of cellular proliferation
generated in groups 2 and 3 provided the basis for the conclusion
that AGN 193109 sensitized primary RPE cells to the
antiproliferative effects of the RAR agonist, thereby potentiating
the activity of the RAR agonist.
[0594] As indicated above, Agarwal et al., in Cancer Res. 54:2108
(1994)), showed that CaSki cell growth, unlike the growth of HPV
immortalized ECE-16-1 cells, was not inhibited by treatment with
retinoid agonists. As disclosed herein, we unexpectedly found that
CaSki cell growth was inhibited by AGN 193109 in the absence of a
retinoid agonist. The following Example illustrates how AGN 193109
can be used to inhibit the growth of CaSki cell tumors in vivo.
Example 22
Inhibition of CaSki Cell Tumor Growth in Nude Mice Following
Administration of AGN 193109
[0595] 1.times.10.sup.6 CaSki cells are injected into each of a
panel of nude mice. Tumor formation is assessed using techniques
that will be familiar to one having ordinary skill in the art.
After injection, mice are randomly divided into control and test
groups. The control group receives a placebo. The test group is
administered with AGN 193109. Animals administered with the placebo
receive intragastric intubation of corn oil. The test animals
receive 20 .mu.Mol/kg AGN 193109 in corn oil daily for the period
of the treatment. Tumor volume is measured in cubic milliliters
using graduated calipers. Tumor volume is plotted as function of
time. Mice receiving AGN 193109 exhibit tumors which are
significantly reduced in their growth rate as compared to tumors in
control mice as judged by tumor size and number over the period of
the study. This result provides an in vivo demonstration that AGN
193109 inhibits the growth of an advanced cervical carcinoma that
is resistant to therapy comprising administration of a retinoid
agonist.
[0596] As indicated above, CaSki cells are a model of cervical
tumors that are not responsive to retinoid agonist therapy.
However, herein we have disclosed that CaSki cell growth was
inhibited by AGN 193109 in the absence of treatment with a retinoid
agonist. The ability of AGN 193109 to inhibit the proliferation of
CaSki cells suggested that AGN 193109 could be used to
therapeutically treat cervical carcinomas that are insensitive to
retinoid agonist therapy. The following Example illustrates one
method that can be used to assess the therapeutic potential of AGN
193109 in the treatment of a cervical carcinoma.
Example 23
Assessing the Therapeutic Potential of AGN 193109 in Patients
Having Cervical Carcinoma
[0597] A patient presenting with an advanced cervical carcinoma is
first identified. A cervical biopsy is obtained according to
methods that will be familiar to one having ordinary skill in the
art. Cells from the explanted tumor are propagated in tissue
culture according to standard techniques to provide cell numbers
sufficient to allow division into three sample groups. Culture
conditions described by Agarwal et al. in Cancer Res. 54:2108
(1994) are employed for this purpose. The first group is reserved
as a control and receives vehicle alone (ethanol). The second group
is treated with the RAR agonist retinoic acid at a concentration of
from 10.sup.-10 to 10.sup.-6 M. The third group is treated with AGN
193109 at doses ranging from 10.sup.-10 to 10.sup.-6 M. Cells are
fed with fresh growth medium daily and are provided with the
retinoids described above as appropriate for each sample group.
Cells are counted after three days using an electric cell counter.
Comparison of the number of cells in control cultures with the
number of cells in retinoic acid treated cultures indicates the RAR
agonist does not substantially inhibit the growth rate of the
cultured cervical carcinoma cells. In contrast, cells treated with
AGN 193109 exhibit a dose-dependent decrease in cell number when
compared with cell counts in the control group. This result,
wherein AGN 193109 treatment inhibits cultured cervical carcinoma
cell proliferation, indicates that AGN 193109 will be a useful
therapeutic agent for treating cervical carcinoma patients having
metastatic disease.
[0598] Cervical carcinoma patients having undergone surgery for the
removal of primary tumors and who present with metastatic disease
are enlisted in a randomized clinical study seeking to demonstrate
the therapeutic benefit of AGN 193109 in this indication. Patients
are divided into two groups. The first group is a control group
while members of the second group are treated with AGN 193109. AGN
193109 is combined with a pharmaceutically acceptable excipient to
produce a composition suitable for systemic administration, all
according to techniques that will be familiar to one having
ordinary skill in the art. The control group is administered a
placebo formulation and the experimental group is administered with
the formulation containing the AGN 193109 negative hormone. Dosing
of patients is at the maximum tolerated dose and is performed every
other day for a period of from three months to one year. The
outcome of the study is quantified via measurement of disease-free
survival over time. Individuals receiving AGN 193109 display a
significant increase in disease-free survival, including a
disproportionate number of patients displaying complete remission
of their metastatic disease. This result indicates that AGN 193109
has therapeutic utility for in vivo treatment of cervical
carcinomas that are unresponsive to the antiproliferative effects
of retinoid agonists, such as retinoic acid.
[0599] As disclosed above, AGN 193109 potentiated the
antiproliferative activity of RAR agonists in primary cultures of
human retinal pigment epithelium cells. Accordingly,
coadministration of AGN 193109 with an RAR agonist in vivo is
reasonably expected to increase the therapeutic index of the
agonist because a lesser amount of the RAR agonist will be required
to obtain the same therapeutic endpoint. Additionally, AGN 193109
has been demonstrated to sensitize primary cultures of human
retinal pigment epithelium cells to the antiproliferative effects
of glucocorticoid and thyroid hormone receptor agonists. The
following rabbit model of PVR will be utilized in two separate
studies to demonstrate the increased therapeutic index obtained via
coadministration of AGN 193109 with an RAR agonist (13-cis retinoic
acid) or a thyroid hormone receptor agonist, respectively. Notably,
the rabbit model of retinal redetachment published by Sen et al. in
Arch. Opthalmol. 106:1291 (1988), has been used to demonstrate that
retinoid agonists which inhibit proliferation of primary RPE cells
in vitro also inhibit the frequency of retinal detachment in vivo
(Araiz et al. Invest. Opthalmol 34:522 (1993)). Thus, with respect
to their use as therapeutics in the prevention of retinal
detachment, a correlation between the in vitro and in vivo
activities of retinoid agonists has already been established. The
following Examples illustrate how AGN 193109 can be used in
therapeutic applications directed at preventing retinal
detachment.
Example 24
Use of AGN 193109 to Increase the Therapeutic Potential of Steroid
Superfamily Receptor Agonists in the Treatment of Proliferative
Vitreoretinopathy (PVR)
[0600] In a first study, human RPE cells are injected into the
vitreous cavity of rabbit eyes according to the method described by
Sen et al. in Arch. Opthalmol. 106:1291 (1988). After intravitreal
injection, the rabbits are divided into five groups. The first
group (control) will receive vehicle alone by intravitreal
injection. The second group receives retinoic acid as single agent
treatment (100 .mu.g) by intravitreal injection. The third group
receives AGN 193109 as a single agent treatment (100 .mu.g) by
intravitreal injection. The fourth group receives by intravitreal
injection the RAR agonist (retinoic acid) at a dose one-tenth the
amount administered to group 2 (10 .mu.g). The fifth group receives
the combination of AGN 193109 (100 .mu.g) and retinoic acid (10
.mu.g) by intravitreal injection. Animals receive a single
intravitreal injection of the appropriate treatment one day after
intravitreal injection of human RPE cells. Rabbits are examined by
indirect ophthalmoscopy on days 7, 14 and 28, and are graded for
the frequency and severity of tractional retinal detachment.
Rabbits from the group injected with 100 .mu.g retinoic acid
exhibit a significantly reduced frequency and severity of retinal
detachment compared to control rabbits or rabbits receiving either
AGN 193109 or retinoic acid (10 .mu.g) alone. Rabbits in the group
administered with the combination of AGN 193109 and retinoic acid
(10 .mu.g) exhibit significantly reduced frequency and severity of
retinal detachment as compared to those in groups either control,
AGN 193109 or retinoic acid (10 .mu.g). This result demonstrates
that AGN 193109 improves the therapeutic index of the RAR agonist
retinoic acid in an in vivo model of PVR.
[0601] In a second study, rabbits are first provided with an
injection of human RPE cells into the vitreous cavity of the eye,
and then divided into four groups. The first group (control)
receives vehicle alone by intravitreal injection. The second group
receives thyroid hormone as single agent treatment (100 .mu.g) by
intravitreal injection. The third group is administered with AGN
193109 as a single agent treatment (100 .mu.g) by intravitreal
injection. The fourth group is administered with the combination of
AGN 193109 (100 .mu.g) and thyroid hormone (100 .mu.g). Rabbits are
examined by indirect ophthalmoscopy on days 7, 14 and 28, and
graded for the frequency and severity of tractional retinal
detachment. Comparison of the frequency and severity of retinal
detachment in the four groups demonstrates that single agent
treatment with either AGN 193109 or thyroid hormone does not
inhibit retinal detachment when compared with control rabbits. In
contrast, the group of rabbits administered with the combination of
AGN 193109 and thyroid hormone exhibit significantly reduced
incidence and severity of retinal detachment. This result
demonstrates that AGN 193109 improves the therapeutic index of
thyroid hormone in an in vivo model of PVR.
[0602] The following Example illustrates how AGN 193109 can be used
to enhance the therapeutic index of an RAR agonist used to treat
human patients following retinal reattachment surgery.
Example 25
Increasing the Therapeutic Index of RAR Agonist 13-cis Retinoic
Acid
[0603] A population of adult volunteers having retinal detachment
resulting from PVR is first identified. Individuals undergo
surgical repair of the detachments using techniques that are
standard in the art. The patients are then divided into five
groups. The control group consists of patients who undergo surgical
repair of the retinal detachment and do not receive any retinoid
compound. The second group receives 40 mg oral 13-cis retinoic acid
twice daily for four weeks postoperatively. The third group
receives 40 mg oral AGN 103109 twice daily for four weeks
postoperatively. The fourth group receives 4 mg oral 13-cis
retinoic acid twice daily for four weeks postoperatively. The fifth
group receives 40 mg oral AGN 193109 in combination with 4 mg oral
13-cis retinoic acid twice daily for four weeks postoperatively.
The treatment protocol and assessment of drug efficacy is performed
essentially as described by Fekrat et al. in Ophthalmology 102:412
(1995).
[0604] The frequency and severity of retinal redetachment in
postoperative patients in all five groups is monitored over a
period of nine months using ophthalmologic examination techniques
that will be familiar to those of ordinary skill in the art.
Patients receiving 40 mg oral 13-cis retinoic acid exhibit
significantly reduced incidence of retinal redetachment when
compared with control patients, patients receiving 4 mg oral 13-cis
retinoic acid twice daily or patients receiving 40 mg oral AGN
193109 twice daily. Examination of the patient group receiving the
combination of 40 mg oral AGN 193109 and 4 mg oral 13-cis retinoic
acid twice daily for four weeks postoperatively demonstrates the
therapeutic outcome in this patient group is equal to or better
than those patients receiving 40 mg oral 13-cis retinoic acid twice
daily for four weeks postoperatively. This result demonstrates that
the AGN 193109 negative hormone improves the therapeutic index of
an RAR agonist by virtue of decreasing the frequency and severity
of retinal redetachment in PVR patients.
[0605] Generalized Assay for Identifying Nuclear Receptor Negative
Hormones
[0606] We have demonstrated above that AGN 193109 can function as a
negative hormone capable of repressing the basal transcriptional
activity of RAR nuclear receptors. Further, we have described an
assay using CV-1 cells co-transfected with the ERE-tk-Luc
luciferase reporter plasmid and the ER-RXR-.alpha. and
RAR-.gamma.-VP-16 receptor expression plasmids for distinguishing
RAR ligands that are simple antagonists from those having negative
hormone activity.
[0607] We have concluded that RAR negative hormones mediate
repression of RAR-mediated transcriptional activity by promoting
increased interaction between the RAR and NCPs. Further, we have
demonstrated that AGN 193109 can potentiate the effects of agonists
of other nuclear receptors in a manner consistent with the mutual
sharing of NCPs between members of the steroid superfamily of
nuclear receptors. As such, ligands can be designed and screened to
identify compounds having negative hormone activity at these
non-RAR nuclear receptors.
[0608] Our method of RAR negative hormone screening based on the
use of CV-1 cells co-transfected with the ERE-tk-Luc luciferase
reporter plasmid and the ER-RXR-.alpha. and RAR-.gamma.-VP-16
receptor expression plasmids can be adapted generally such that the
RAR-.gamma. moiety of the RAR-.gamma.-VP-16 plasmid is converted to
that of peroxisome proliferator-activated receptors (PPAR), vitamin
D receptor (VDR), thyroid hormone receptor (T3R) or any other
steroid superfamily nuclear receptor capable of heterodimerizing
with RXR. CV-1 cells co-transfected with such plasmids would
express high basal levels of luciferase activity. Ligands capable
of binding the ligand binding domain of the receptor substituted
for the RAR-.gamma. moiety can be easily screened for negative
hormone activity by measuring their ability to repress luciferase
activity.
[0609] For steroid superfamily nuclear receptors that do not
heterodimerize with RXR (e.g., glucocorticoid and estrogen
receptors) the same end result can be achieved using GR-VP-16 or
ER-VP-16 receptors and a luciferase reporter plasmid consisting of
the appropriate glucocorticoid or estrogen response element fused
to a heterologous promoter element and luciferase or other reporter
gene. An essential feature of a generalized negative hormone
screening assay is the inclusion of at least the ligand binding
domain of the particular nuclear receptor for which inverse
agonists are to be screened and a method for localizing the nuclear
receptor ligand binding domain to the promoter of a reporter gene.
This could be achieved using the receptors's natural DNA binding
site, or alternatively by construction of a chimeric receptor
having a heterologous DNA binding domain and corresponding use of a
reporter gene which is under control of a DNA regulatory element
which is recognized by the heterologous DNA binding domain. In a
preferred embodiment, the plasmid expressing the nuclear receptor
for which inverse agonists are to be screened would express this
nuclear receptor as a fusion protein containing a constitutive
activation domain, such as the HSV VP-16 activation domain, in
order to provide allow high basal activity. This high basal
activity would effectively increase assay sensitivity, thereby
allowing analysis of nuclear receptor ligands which repress basal
transcriptional activity in the absence of added nuclear receptor
agonist.
[0610] The following Example illustrates one method that can be
used to screen for compounds having negative hormone activity at
the thyroid hormone receptor.
Example 26
Method of Identifying Thyroid Hormone Receptor Negative
Hormones
[0611] CV-1 cells are co-transfected with the luciferase reporter
plasmid ERE-tk-Luc and the plasmids ER-RAR-.alpha. and T3R-VP-16.
T3R-VP-16 is identical to the plasmid RAR-.gamma.-VP-16, except the
RAR-.gamma. moiety of RAR-.gamma.-VP-16 has been substituted by the
thyroid hormone receptor cDNA. As such, T3R-VP-16 expresses a
fusion protein containing the activation domain of HSV VP-16 in
frame with the N-terminus of the thyroid hormone receptor. Standard
transfection and cell culture methods are employed for this
purpose. After transfection, cells are rinsed and fed with growth
medium containing 10% fetal calf serum which has been extracted
with activated charcoal. Cells are treated with vehicle alone
(ethanol), thyroid hormone (10.sup.-9 to 10.sup.-10 M), or compound
TR-1 (10.sup.-9 to 10.sup.-6 M). TR-1 is a synthetic thyroid
hormone receptor ligand which exhibits strong affinity for the
thyroid hormone receptor in competition binding studies, but which
does not activate transfected thyroid hormone receptor in transient
cotransfection transactivation assays using a thyroid hormone
responsive reporter gene and a thyroid hormone receptor expression
plasmid. Further, TR-1 is capable of antagonizing thyroid hormone
mediated transactivation and as such is a thyroid receptor
antagonist.
[0612] Analysis of luciferase activity from CV-1 cell transfected
with ERE-tk-Luc, ER-RXR.alpha. and T3R-VP-16 demonstrates a high
basal level of luciferase reporter activity in vehicle-treated
cells. Cells treated with thyroid hormone show a slight increase of
luciferase activity in a dose dependent manner. Cells treated with
TR-1 exhibit a dose dependent decrease in luciferase activity. This
indicates that TR-1 exhibits thyroid receptor inverse agonist
activity, presumably due to the increased interaction of a NCP with
the thyroid hormone receptor.
[0613] The proliferation rate of human primary retinal pigment
epithelium cells is repressed by treatment with RAR agonists. The
therapeutic value of this observation has been demonstrated in
post-operative use retinoid therapy after retinal reattachment
surgery. We have above demonstrated the AGN 193109 RAR negative
hormone can sensitize primary RPE cells to the antiproliferative
effect of ATRA and 13-cis retinoic acid in coadministration
procedures. Further, AGN 193109 was also shown to sensitize RPE
cells to the antiproliferative effects of other nuclear receptor
agonists. More specifically, AGN 193109 sensitized RPE cells to the
antiproliferative effects of the glucocorticoid agonist,
dexamethasone, and the thyroid hormone agonist
3,3',5-triiodothyronine, T3. This data was consistent with our
working model wherein AGN 193109 modulated the availability of NCPs
that were shared between the members of the nuclear receptor
family. Treatment of RPE cells with the thyroid hormone receptor
inverse agonist TR-1 will similarly alter the availability of
shared NCPs such that coadministration with a non-thyroid receptor
agonist, such as the RAR agonist 13-cis retinoic acid will lead to
an increased antiproliferative effect upon the RPE cultures as
compared to 13-cis retinoic acid as a single agent treatment.
[0614] The following Example illustrates one method that can be
used to render primary RPE cells more sensitive to the
antiproliferative activity of an RAR agonist. Notably, this Example
further illustrates how the activity of RAR agonists can be
potentiated by coadministration with a negative hormone.
Example 27
Sensitizing Primary Retinal Pigment Epithelium Cells to the
Antiproliferative Effects of RAR Agonists by Coadministration of
the TR-1 Thyroid Hormone Inverse Agonist
[0615] Human primary RPE cells are obtained and cultured according
to standard methods. The cultured cells are divided into four
groups and treated as follows. Group 1 receives vehicle alone
(ethanol). Group 2 is treated with 13-cis retinoic acid at
concentrations ranging from 10.sup.-11 to 10.sup.-6 M. Group 3 is
treated with the thyroid hormone inverse agonist TR-1 at
concentrations ranging from 10.sup.-11 to 10.sup.-1 M. Group 4 is
co-treated with 13-cis retinoic acid at concentrations ranging from
10.sup.-11 to 10.sup.-6 M TR-1. Cells are refed with fresh growth
medium and re-treated with the appropriate compound every two days
for a total of five days of treatment. The proliferation rate over
the duration of the experiment is quantitated via measurement of
the cell number in the cultures using an electric cell counter.
[0616] TR-1 treated cells (Group 3) exhibits rates of cellular
proliferation which are essentially the same as control (Group 1)
cells and there is no effect of this inverse agonist upon the
measured growth rate of the cultures. Cells treated with 13-cis
retinoic acid (Group 2) exhibit a dose dependent decrease in cell
number. Comparison of the dose dependent decrease in cellular
proliferation of Group 4 cells (13-cis RA and TR-1
coadministration) with that obtained in Group 3 demonstrates the
ability of TR-1 thyroid hormone receptor inverse agonist
coadministration to sensitize RPE cultures to the antiproliferative
effect of 13-cis retinoic acid as measured by the shift in the dose
response curve of this RAR agonist to the left in Group 4 as
compared to Group 2 cells.
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