U.S. patent application number 09/350648 was filed with the patent office on 2003-06-05 for means for the modulation of processes mediated by retinoid receptors and compounds useful therefor.
Invention is credited to BOEHM, MARCUS F., EICHELE, GREGOR, EVANS, RONALD M., HEYMAN, RICHARD A., MANGELSDORF, DAVID J., THALLER, CHRISTINA.
Application Number | 20030105166 09/350648 |
Document ID | / |
Family ID | 27043936 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030105166 |
Kind Code |
A1 |
EVANS, RONALD M. ; et
al. |
June 5, 2003 |
MEANS FOR THE MODULATION OF PROCESSES MEDIATED BY RETINOID
RECEPTORS AND COMPOUNDS USEFUL THEREFOR
Abstract
In accordance with the present invention, there are provided
methods to modulate processes mediated by retinoid receptors,
employing high affinity, high specificity ligands for such
receptors. In one aspect of the present invention, there are
provided ligands which are more selective for the retinoid X
receptor than is retinoic acid (i.e., rexoids). In another aspect
of the present invention, alternative ligands (other than retinoic
acid) have been discovered which are capable of inducing retinoic
acid receptor mediated processes. In yet another aspect, methods
have been developed for the preparation of such retinoid receptor
ligands from readily available compounds.
Inventors: |
EVANS, RONALD M.; (LA JOLLA,
CA) ; MANGELSDORF, DAVID J.; (DALLAS, TX) ;
HEYMAN, RICHARD A.; (ENCINITAS, CA) ; BOEHM, MARCUS
F.; (SAN DIEGO, CA) ; EICHELE, GREGOR;
(HOUSTON, TX) ; THALLER, CHRISTINA; (HOUSTON,
TX) |
Correspondence
Address: |
STEPHEN E REITER
FOLEY & LARDNER
P O BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Family ID: |
27043936 |
Appl. No.: |
09/350648 |
Filed: |
July 9, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09350648 |
Jul 9, 1999 |
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08472817 |
Jun 7, 1995 |
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5968989 |
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Current U.S.
Class: |
514/725 |
Current CPC
Class: |
C07C 403/20 20130101;
A61K 31/07 20130101; A61K 31/20 20130101; A61K 31/203 20130101;
C07K 14/70567 20130101; C07C 59/80 20130101; A61K 31/232 20130101;
C12P 7/04 20130101; C07C 2601/16 20170501; C07C 57/26 20130101;
C07B 2200/09 20130101; A61K 31/19 20130101; C07C 59/46 20130101;
A61K 31/23 20130101 |
Class at
Publication: |
514/725 |
International
Class: |
A61K 031/07 |
Claims
That which is claimed is:
1. A method for modulating process(es) mediated by retinoid
receptors, said method comprising conducting said process(es) in
the presence of at least one compound of the structure: 17wherein:
unsaturation between carbon atoms C.sup.9 and C.sup.10 has a cis
configuration, and one or both sites of unsaturation between carbon
atoms C.sup.11 through C.sup.14 optionally have a cis
configuration; "Ring" is a cyclic moiety; Z is selected from
carboxyl, carboxaldehyde, hydroxyalkyl, thioalkyl, hydroxyalkyl
phosphate, alkyl ether of a hydroxyalkyl group, alkyl thioether of
a thioalkyl group, esters of hydroxyalkyl groups, thioesters of
hydroxyalkyl group, esters of thioalkyl groups, thioesters of
thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, or carbamate; and
each R is independently selected from H, halogen, alkyl, aryl,
hydroxy, thiol, alkoxy, thioalkoxy, amino, or any of the Z
substituents; or any two or more of the R groups can be linked to
one another to form one or more ring structures.
2. A method according to claim 1 wherein said retinoid receptor is
selected from retinoic acid receptor-alpha, retinoic acid
receptor-beta, or retinoic acid receptor-gamma.
3. A method according to claim 1 wherein said retinoid receptor is
selected from retinoid X receptor-alpha, retinoid X receptor-beta,
or retinoid X receptor-gamma.
4. A method according to claim 1 wherein said process is selected
from in vitro cellular differentiation, in vitro cellular
proliferation, in vitro proliferation of melanoma cell lines, in
vitro differentiation of mouse teratocarcinoma cells (F9 cells), in
vitro differentiation of human epidermal keratinocytes, regulation
of cellular retinol binding protein (CRBP), or in vitro limb
morphogenesis.
5. A method according to claim 1 wherein said process is selected
from the in vivo modulation of lipid metabolism, in vivo modulation
of skin-related processes, or in vivo modulation of malignant cell
development.
6. A method according to claim 1 wherein said compound has the
structure (I): 18wherein: X is
--[CR.sub.2).sub.x--X'--(CR.sub.2).sub.y]--, X' is selected from
--O--, carbonyl, --S--, --S(O)--, --S(O).sub.2--, thiocarbonyl,
--NR"--, or --CR.sub.2--, "Ring" is a cyclic moiety; Z is selected
from carboxyl, carboxaldehyde, hydroxyalkyl, thioalkyl,
hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl group, alkyl
thioether of a thioalkyl group, esters of hydroxyalkyl groups,
thioesters of hydroxyalkyl group, esters of thioalkyl groups,
thioesters of thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, or
carbamate; and each R is independently selected from H, halogen,
alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, or amino; R" is
hydrogen, alkyl, hydroxy, thiol, or alkoxy acyl; x is 0, 1 or 2, y
is 0, 1, or 2, and x+y.ltoreq.2.
7. A method according to claim 1 wherein said compound has the
structure (II): 19wherein: X is
--[(CR.sub.2).sub.x--X'--(CR.sub.2).sub.y]--, X' is selected from
--O--, carbonyl, --S--, --S(O)--, --S(O).sub.2--, thiocarbonyl,
--NR"--, or --CR.sub.2--, "Ring" is a cyclic moiety; Z is selected
from carboxyl, carboxaldehyde, hydroxyalkyl, thioalkyl,
hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl group, alkyl
thioether of a thioalkyl group, esters of hydroxyalkyl groups,
thioesters of hydroxyalkyl group, esters of thioalkyl groups,
thioesters of thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, or
carbamate; and each R is independently selected from H, halogen,
alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or any of
the Z substituents; R" is hydrogen, alkyl, hydroxy, thiol, or
alkoxy acyl; x is 0, 1 or 2, y is 0, 1, or 2, and x+y.ltoreq.2.
8. A method according to claim 1 wherein said compound has the
structure (III): 20wherein: one A is X and the other A is X', X is
--[(CR.sub.2).sub.x--X'--(CR.sub.2).sub.y]--, X' is selected from
--O--, carbonyl, --S--, --S(O)--, --S(O).sub.2--, thiocarbonyl,
--NR"--, or --CR.sub.2--, "Ring" is a cyclic moiety; Z is selected
from carboxyl, carboxaldehyde, hydroxyalkyl, thioalkyl,
hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl group, alkyl
thioether of a thioalkyl group, esters of hydroxyalkyl groups,
thioesters of hydroxyalkyl group, esters of thioalkyl groups,
thioesters of thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, or
carbamate; and each R is independently selected from H, halogen,
alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or any of
the Z substituents; R" is hydrogen, alkyl, hydroxy, thiol, or
alkoxy acyl; x is 0, 1 or 2, y is 0, 1, or 2, and x+y<2.
9. A method according to claim 1 wherein said compound has the
structure (IV): 21wherein: one A is X and the other A is X', B is
X', X is --[(CR.sub.2).sub.x--X'--(CR.sub.2).sub.y]--, X' is
selected from --O--, carbonyl, --S--, --S--(O)--, --S(O).sub.2--,
thiocarbonyl, --NR"--, or --CR.sub.2--, "Ring" is a cyclic moiety;
Z is selected from carboxyl, carboxaldehyde, hydroxyalkyl,
thioalkyl, hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl
group, alkyl thioether of a thioalkyl group, esters of hydroxyalkyl
groups, thioesters of hydroxyalkyl group, esters of thioalkyl
groups, thioesters of thioalkyl groups, aminoalkyl, N-acyl
aminoalkyl, or carbamate; and each R is independently selected from
H, halogen, alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino,
or any of the Z substituents; R" is hydrogen, alkyl, hydroxy,
thiol, or alkoxy acyl; x is 0, 1 or 2, y is 0, 1, or 2, and
x+y.ltoreq.2.
10. A method according to claim 1 wherein, said compound has the
structure (V): 22wherein: X" is
--[(CR.sub.2).sub.a--X'--(CR.sub.2).sub.b]--, X' is selected from
--O--, carbonyl, --S--, --S(O)--, --S(O).sub.2--, thiocarbonyl,
--NR"--, or --CR.sub.2--, "Ring" is a cyclic moiety; Z is selected
from carboxyl, carboxaldehyde, hydroxyalkyl, thioalkyl,
hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl group, alkyl
thioether of a thioalkyl group, esters of hydroxyalkyl groups,
thioesters of hydroxyalkyl group, esters of thioalkyl groups,
thioesters of thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, or
carbamate; and each R is independently selected from H, halogen,
alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or any of
the Z substituents; R" is hydrogen, halogen, alkyl, hydroxy, or
thiol; a is 0, 1, 2, 3 or 4, b is 0, 1, 2, 3, or 4, and a+b is
.gtoreq.2, but .ltoreq.4.
11. A method according to claim 1 wherein said compound has the
structure (VI): 23wherein: Y is
--[(CR.sub.2).sub.c--X'--(CR.sub.2).sub.d]--, X' is selected from
--O--, carbonyl, --S--, --S(O)--, --S(O).sub.2--, thiocarbonyl,
--NR"--, or --CR.sub.2--, "Ring" is a cyclic moiety; Z is selected
from carboxyl, carboxaldehyde, hydroxyalkyl, thioalkyl,
hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl group, alkyl
thioether of a thioalkyl group, esters of hydroxyalkyl groups,
thioesters of hydroxyalkyl group, esters of thioalkyl groups,
thioesters of thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, or
carbamate; and each R is independently selected from H, halogen,
alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or any of
the Z substituents; R" is hydrogen, alkyl, hydroxy, thiol, or
alkoxy acyl; c is 0, 1, 2 or 3, d is 0, 1, 2 or 3, and
c+d.gtoreq.1, but .ltoreq.3.
12. A method according to claim 1 wherein said compound has the
structure (VII): 24wherein: X' ' ' is X" or an unsaturated linking
group having the structure: --[Q.dbd.CR--J]--, wherein Q is
--N.dbd. or --CR.dbd., and J is --CR.dbd.CR--, --N.dbd.CR--,
--CR.dbd.N--, --O--, --S--, or --NR"--, thereby incorporating
C.sup.9 and C.sup.10 of the rexoid compound into an aromatic (or
pseudo-aromatic) ring, X" is --[(CR.sub.2).sub.8--X'--(CR.su-
b.2).sub.b]--, X' is selected from --O--, carbonyl, --S--,
--S(O)--, --S(O).sub.2--, thiocarbonyl, --NR"--, or --CR.sub.2--,
"Ring" is a cyclic moiety; Z is selected from carboxyl,
carboxaldehyde, hydroxyalkyl, thioalkyl, hydroxyalkyl phosphate,
alkyl ether of a hydroxyalkyl group, alkyl thioether of a thioalkyl
group, esters of hydroxyalkyl groups, thioesters of hydroxyalkyl
group, esters of thioalkyl groups, thioesters of thioalkyl groups,
aminoalkyl, N-acyl aminoalkyl, or carbamate; and each R is
independently selected from H, halogen, alkyl, aryl, hydroxy,
thiol, alkoxy, thioalkoxy, amino, or any of the Z substituents; R"
is hydrogen, alkyl, hydroxy, thiol, or alkoxy acyl; a is 0, 1, 2, 3
or 4, b is 0, 1, 2, 3, or 4, and a+b is .gtoreq.2, but
.ltoreq.4.
13. A method according to claim 1 wherein Ring has the following
structure: 25wherein: each R is independently selected from H,
halogen, alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or
any of the Z substituents; any one of C.sup.2, C.sup.3, or C.sup.4
can be replaced with --O--, carbonyl (>CO), --S--, --S(O)--,
--S(O).sub.2--, thiocarbonyl (>CS), or --NR"--; R" is hydrogen,
alkyl, hydroxy, thiol, or alkoxy acyl; and said cyclic moiety
exists as the saturated, 2-ene, 3-ene, 4-ene, or 5-ene
mono-unsaturated isomer, or the 2,4-, 2,5-, or 3,5-diene derivative
thereof; or an aromatic derivative thereof.
14. A method according to claim 6 wherein Ring has the following
structure: 26wherein: each R is independently selected from H,
halogen, alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or
any of the Z substituents; any one of C.sup.2, C.sup.3, or C.sup.4
can be replaced with --O--, carbonyl (>CO), --S--, --S(O)--,
--S(O).sub.2--, thiocarbonyl (>CS), or --NR"--; R" is hydrogen,
alkyl, hydroxy, thiol, or alkoxy acyl; and said cyclic moiety
exists as the saturated, 2-ene, 3-ene, 4-ene, or 5-ene
mono-unsaturated isomer, or the 2,4-, 2,5-, or 3,5-diene derivative
thereof, or an aromatic derivative thereof.
15. A method according to claim 7 wherein Ring has the following
structure: 27wherein: each R is independently selected from H,
halogen, alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or
any of the Z substituents; any one of C.sup.2, C.sup.3, or C.sup.4
can be replaced with --O--, carbonyl (>CO), --S--, --S(O)--,
--S(O).sub.2--, thiocarbonyl (>CS), or --NR"--; R" is hydrogen,
alkyl, hydroxy, thiol, or alkoxy acyl; and said cyclic moiety
exists as the saturated, 2-ene, 3-ene, 4-ene, or 5-ene
mono-unsaturated isomer, or the 2,4-, 2,5-, or 3,5-diene derivative
thereof.
16. A method according to claim 8 wherein Ring has the following
structure: 28wherein: each R is independently selected from H,
halogen, alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or
any of the Z substituents; any one of C.sup.2, C.sup.3, or C.sup.4
can be replaced with --O--, carbonyl (>CO), --S--, --S(O)--,
--S(O).sub.2--, thiocarbonyl (>CS), or --NR"--; R" is hydrogen,
alkyl, hydroxy, thiol, or alkoxy acyl; and said cyclic moiety
exists as the saturated, 2-ene, 3-ene, 4-ene, or 5-ene
mono-unsaturated isomer, or the 2,4-, 2,5-, or 3,5-diene derivative
thereof; or an aromatic derivative thereof.
17. A method according to claim 9 wherein Ring has the following
structure: 29wherein: each R is independently selected from H,
halogen, alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or
any of the Z substituents; any one of C.sup.2, C.sup.3, or C.sup.4
can be replaced with --O--, carbonyl (>CO), --S--, --S(O)--,
--S(O).sub.2--, thiocarbonyl (>CS), or --NR"--; R" is hydrogen,
alkyl, hydroxy, thiol, or alkoxy acyl; and said cyclic moiety
exists as the saturated, 2-ene, 3-ene, 4-ene, or 5-ene
mono-unsaturated isomer, or the 2,4-, 2,5-, or 3,5-diene derivative
thereof; or an aromatic derivative thereof.
18. A method according to claim 10 wherein Ring has the following
structure: 30wherein: each R is independently selected from H,
halogen, alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or
any of the Z substituents; any one of C.sup.2, C.sup.3, or C.sup.4
can be replaced with --O--, carbonyl (>CO), --S--, --S(O)--,
--S(O).sub.2--, thiocarbonyl (>CS), or --NR"--; R" is hydrogen,
alkyl, hydroxy, thiol, or alkoxy acyl; and said cyclic moiety
exists as the saturated, 2-ene, 3-ene, 4-ene, or 5-ene
mono-unsaturated isomer, or the 2,4-, 2,5-, or 3,5-diene derivative
thereof; or an aromatic derivative thereof.
19. A method according to claim 11 wherein Ring has the following
structure: 31wherein: each R is independently selected from H,
halogen, alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or
any of the Z substituents; any one of C.sup.2, C.sup.3, or C.sup.4
can be replaced with --O--, carbonyl (>CO), --S--, --S(O)--,
--S(O).sub.2--, thiocarbonyl (>CS), or --NR"--; R" is hydrogen,
alkyl, hydroxy, thiol, or alkoxy acyl; and said cyclic moiety
exists as the saturated, 2-ene, 3-ene, 4-ene, or 5-ene
mono-unsaturated isomer, or the 2,4-, 2,5-, or 3,5-diene derivative
thereof; or an aromatic derivative thereof.
20. A method according to claim 1 wherein said compound is selected
from 9-cis-retinoic acid, 9-phenyl-9-cis-retinoic acid,
4-hydroxy-9-cis-retinoic acid, 4-keto-9-cis-retinoic acid,
9,11-dicis retinoic acid, and 9-cis-locked derivatives of retinoic
acid selected from Structures I-VII as set forth in the
specification, wherein Z is carboxyl and Ring is a .beta.-ionone or
.beta.-ionone-like species having the structure: 32wherein A.sup.4
is selected from >CH.sub.2, >C.dbd.O or >C--OH.
21. A method according to claim 1 wherein Ring has four or five
carbon atoms and is selected from cyclopentane, cyclopentene,
dihydropyran, tetrahydropyran, piperidine, dihydrothiopyran,
tetrahydrothiopyran, dihydrofuran, tetrahydrofuran,
tetrahydrothiophene, pyrrolidine, or derivatives thereof.
22. A method to modulate processes mediated by retinoid receptors,
said method comprising conducting said process in the presence of:
(a) at least one compound of the structure: 33 wherein: each site
of unsaturation in the side chain comprising carbon atoms C.sup.7
through C.sup.14 has a trans configuration; "Ring" is a cyclic
moiety; Z is selected from carboxyl, carboxaldehyde, hydroxyalkyl,
thioalkyl, hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl
group, alkyl thioether of a thioalkyl group, esters of hydroxyalkyl
groups, thioesters of hydroxyalkyl group, esters of thioalkyl
groups, thioesters of thioalkyl groups, aminoalkyl, N-acyl
aminoalkyl, carbamate, and the like; and each R is independently
selected from H, halogen, alkyl, aryl, hydroxy, thiol, alkoxy,
thioalkoxy, amino, or any of the Z substituents; and (b) a
cis/trans isomerase capable of converting at least one of the 9-,
11-, or 13-double bonds from the trans configuration to the
cis-configuration.
23. A method to produce compound(s) of the structure: 34wherein:
unsaturation between carbon atoms C.sup.9 and C.sup.10 has a cis
configuration, and one or both sites of unsaturation between carbon
atoms C.sup.11 through C.sup.14 optionally have a cis
configuration; "Ring" is a cyclic moiety; Z is selected from
carboxyl, carboxaldehyde, hydroxyalkyl, thioalkyl, hydroxyalkyl
phosphate, alkyl ether of a hydroxyalkyl group, alkyl thioether of
a thioalkyl group, esters of hydroxyalkyl groups, thioesters of
hydroxyalkyl group, esters of thioalkyl groups, thioesters of
thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, carbamate, and the
like; and each R is independently selected from H, halogen, alkyl,
aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or any of the Z
substituents; from the corresponding all-trans configuration
material, said method comprising contacting said all-trans
configuration material with a cis/trans isomerase under
isomerization conditions.
24. A method according to claim 23 wherein Ring is a cyclohexyl
ring having the following structure: 35wherein: each R is
independently selected from H, halogen, alkyl, aryl, hydroxy,
thiol, alkoxy, thioalkoxy, amino, or any of the Z substituents; any
one of C.sup.2, C.sup.3, or C.sup.4 can be replaced with --O--,
carbonyl (>CO), --S--, --S(O)--, --S(O).sub.2--, thiocarbonyl
(>CS), or --NR"--; R" is hydrogen, alkyl, hydroxy, thiol, or
alkoxy acyl; and said cyclic moiety exists as the saturated, 2-ene,
3-ene, 4-ene, or 5-ene mono-unsaturated isomer, or the 2,4-, 2,5-,
or 3,5-diene derivative thereof.
25. A method according to claim 23 wherein said contacting is
carried out in vivo.
26. A method according to claim 25 wherein said contacting is
carried out in Schneider cells.
27. A method according to claim 23 wherein said contacting is
carried out in vitro.
28. Composition comprising at least one compound having a structure
selected from: 36wherein: unsaturation between carbon atoms C.sup.9
and C.sup.10 has a cis configuration, and one or both sites of
unsaturation between carbon atoms C.sup.11 through C.sup.14
optionally have a cis configuration; "Ring" is a cyclic moiety,
optionally having one or more substituents thereon; Z is selected
from carboxyl (--COOH), carboxaldehyde (--COH), hydroxyalkyl
[--(CR'.sub.2).sub.n--OH, wherein each R' is independently selected
from hydrogen or a lower alkyl and n falls in the range of 1 up to
about 4], thioalkyl [--(CR'.sub.2).sub.n--S- H, wherein R' and n
are as defined above], hydroxyalkyl phosphate
[--(CR'.sub.2).sub.n--OP(OM).sub.3, wherein R' and n are as defined
above and M is hydrogen, lower alkyl, or a cationic species such as
Na.sup.+, Li.sup.+, K.sup.+, and the like], alkyl ether of a
hydroxyalkyl group [--(CR'.sub.2).sub.n--OR', wherein R' and n are
as defined above], alkyl thioether of a thioalkyl group
[--(CR'.sub.2).sub.n--SR', wherein R' and n are as defined above],
esters of hydroxyalkyl groups [--(CR'.sub.2).sub.n--O--CO--R',
wherein R' and n are as defined above], thioesters of hydroxyalkyl
group [--(CR'.sub.2).sub.n--O--CS--R', wherein R' and n are as
defined above], esters of thioalkyl groups
[--(CR'.sub.2).sub.n--S--CO--R', wherein R' and n are as defined
above], thioesters of thioalkyl groups
[--(CR'.sub.2).sub.n--S--CS--R', wherein R' and n are as defined
above], aminoalkyl [--(CR'.sub.2).sub.n--NR'.sub.- 2, wherein R'
and n are as defined above], N-acyl aminoalkyl
[--(CR'.sub.2).sub.n--NR'--CO--R", wherein R' and n are as defined
above and R" is a lower alkyl or benzyl], carbamate
[--(CR'.sub.2).sub.n--NR'--- CO--OR' or
--(CR'.sub.2).sub.n--O--CO--NR'.sub.2, wherein R' and n are as
defined above]; and each R is independently selected from H,
halogen, alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or
any of the Z substituents, with the proviso thatStructure A is not
9-cis-retinoic acid; or any two or more of the R groups can be
linked to one another to form one or more ring structures;
37wherein: "Ring", Z and R are as defined above; X is
--[(CR.sub.2).sub.x--X'--(CR.sub.2).sub.y]--, X' is selected from
--O--, carbonyl, --S--, --S(O)--, --S(O).sub.2--, thiocarbonyl,
--NR"--, or --CR.sub.2--, R" is hydrogen, alkyl, hydroxy, thiol, or
alkoxy acyl; x is 0, 1 or 2, y is 0, 1, or 2, and x+y.ltoreq.2;
38wherein: X, X', R, R", Z, Ring, x and y are as defined above;
39wherein: one A is X and the other A is X', and X, X', R, R", Z,
Ring, x and y are as defined above; 40wherein: one A is X and the
other A is X', B is X', and X, X', R, R", Z, Ring, x and y are as
defined above; 41wherein: X" is
[--(CR.sub.2).sub.aX'--(CR.sub.2).sub.b]--, X', R, R", Ring and Z
are as defined above, a is 0, 1, 2, 3 or 4, b is 0, 1, 2, 3, or 4,
and a+b is .gtoreq.2, but .ltoreq.4; 42wherein: Y is
--[(CR.sub.2).sub.c--X'--(CR.sub.2).sub.d]--, X', R, R", Ring and Z
are as defined above, c is 0, 1, 2 or 3, d is 0, 1, 2 or 3, and c+d
.gtoreq.1, but .ltoreq.3; and 43wherein: X' ' ' is X" or an
unsaturated linking group having the structure: --[Q.dbd.CR--J]--,
wherein Q is --N.dbd. or --CR.dbd., and J is --CR.dbd.CR--,
--N.dbd.CR--, --CR.dbd.N--, --O--, --S--, or --NR"--, thereby
incorporating C.sup.9 and C.sup.10 of the rexoid compound into an
aromatic (or pseudo-aromatic) ring, and X', X", R, R", Ring, Z, a
and b are as defined above.
29. A composition according to claim 28 wherein Ring is a
cyclohexyl ring having the following structure: 44wherein: each R
is independently selected from H, halogen, alkyl, aryl, hydroxy,
thiol, alkoxy, thioalkoxy, amino, or any of the Z substituents; any
one of C.sup.2, C.sup.3, or C.sup.4 can be replaced with --O--,
carbonyl (>CO), --S--, --S(O)--, --S(O).sub.2--, thiocarbonyl
(>CS), or --NR"--; R" is hydrogen, alkyl, hydroxy, thiol, or
alkoxy acyl; and said cyclic moiety exists as the saturated, 2-ene,
3-ene, 4-ene, or 5-ene mono-unsaturated isomer, or the 2,4-, 2,5-,
or 3,5-diene derivative thereof; or an aromatic derivative thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to intracellular receptors,
and ligands therefor. In a particular aspect, the present
invention, relates to methods for modulating processes mediated by
retinoid receptors.
BACKGROUND OF THE INVENTION
[0002] A central problem in eukaryotic molecular biology continues
to be the elucidation of molecules and mechanisms that mediate
specific gene regulation in response to exogenous inducers such as
hormones or growth factors. As part of the scientific attack on
this problem, a great deal of work has been done in efforts to
identify exogenous inducers which are capable of mediating specific
gene regulation.
[0003] Although much remains to be learned about the specifics of
gene regulation, it is known that exogenous inducers modulate gene
transcription by acting in concert with intracellular components,
including intracellular receptors and discrete DNA sequences known
as hormone response elements (HREs).
[0004] As additional members of the steroid/thyroid superfamily of
receptors are identified, the search for exogenous inducers for
such newly discovered receptors (i.e., naturally occurring (or
synthetic) inducers) has become an important part of the effort to
learn about the specifics of gene regulation.
[0005] The retinoid members of the steroid/thyroid superfamily of
receptors, for example, are responsive to compounds referred to as
retinoids, which include retinoic acid, retinol (vitamin A), and a
series of natural and synthetic derivatives which have been found
to exert profound effects on development and differentiation in a
wide variety of systems.
[0006] The identification of compounds which interact with retinoid
receptors, and thereby affect transcription of genes which are
responsive to retinoic acid (or other metabolites of vitamin A),
would be of significant value, e.g., for therapeutic
applications.
[0007] Recently, a retinoic acid dependent transcription factor,
referred to as RAR-alpha (retinoic acid receptor-alpha), has been
identified. Subsequently, two additional RAR-related genes have
been isolated; thus there are now at least three different RAR
subtypes (alpha, beta and gamma) known to exist in mice and humans.
These retinoic acid receptors (RARs) share homology with the
superfamily of steroid hormone and thyroid hormone receptors and
have been shown to regulate specific gene expression by a similar
ligand-dependent mechanism [Umesono et al., Nature 336: 262
(1988)]. These RAR subtypes are expressed in distinct patterns
throughout development and in the mature organism.
[0008] More recently, additional novel members of the
steroid/thyroid superfamily of receptors have been identified, such
as, for example, retinoid X receptor-alpha [RXR-.alpha.; see
Mangelsdorf et al., in Nature 345: 224-229 (1990)], retinoid X
receptor-beta [RXR-.beta.; see Hamada et al., Proc. Natl. Acad.
Sci. USA 86: 8289-8293 (1989)], and retinoid X receptor-gamma
[RXR-.gamma.; see Mangelsdorf et al., Genes and Development
6:329-344 (1992)]. While these novel receptors are responsive to
retinoic acid, the primary exogenous inducer(s) for these receptors
have not been identified.
[0009] Although both RAR and RXR respond to retinoic acid in vivo,
the receptors differ in several important aspects. First, RAR and
RXR are significantly divergent in primary structure (e.g., the
ligand binding domains of RARE and RXR.alpha. have only 27% amino
acid identity). These structural differences are reflected in
different relative degrees of responsiveness of RAR and RXR to
various vitamin A metabolites and synthetic retinoids. In addition,
distinctly different patterns of tissue distribution are seen for
RAR and RXR. In contrast to the RARs, which are not expressed at
high levels in the visceral tissues, RXR.alpha. mRNA has been shown
to be most abundant in the liver, kidney, lung, muscle and
intestine. Finally, response elements have recently been identified
in the cellular retinol binding protein type II (CRBPII) and
apolipoprotein AI genes which confer responsiveness to RXR, but not
RAR. Indeed, RAR has also been recently shown to repress
RXR-mediated activation through the CRBPII RXR response element.
These data, in conjunction with the observation that both RAR and
RXR can activate through the RAR response element of the RAP.beta.
promoter, indicate that the two retinoic acid responsive pathways
are not simply redundant, but instead manifest a complex
interplay.
[0010] In view of the related, but clearly distinct nature of these
receptors, the identification of ligands which are more selective
for the retinoid X receptor than is retinoic acid would be of great
value in selectively controlling processes mediated by one or both
of these retinoid receptor types.
[0011] Other information helpful in the understanding and practice
of the present invention can be found in commonly assigned,
co-pending U.S. patent application Ser. Nos. 108,471, filed Oct.
20, 1987 (now issued as U.S. Pat. No. 5,071,773); 276,536, filed
Nov. 30, 1988 (now issued as U.S. Pat. No. 4,981,784); 325,240,
filed Mar. 17, 1989; 370,407, filed June. 22, 1989; and 438,757,
filed Nov. 16, 1989, all of which are hereby incorporated herein by
reference in their entirety.
BRIEF DESCRIPTION OF THE INVENTION
[0012] In accordance with the present invention, we have developed
methods to modulate retinoid receptor mediated processes, employing
high affinity, high specificity ligands for such receptors.
[0013] In a particular aspect of the present invention, there are
provided ligands which are high affinity, high specificity ligands
for retinoid receptors. Thus, in one aspect of the present
invention, there are provided ligands which are more selective for
the retinoid X receptor than is all-trans-retinoic acid. In another
aspect of the present invention, we have discovered alternative
ligands (other than all-trans-retinoic acid) which are capable of
inducing retinoic acid receptor mediated processes.
[0014] In yet another aspect of the present invention, we have
developed methods for the preparation of such retinoid receptor
ligands from readily available retinoid compounds.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 is a transactivation profile of various HPLC
fractions obtained from retinoic acid (RA)-treated S2 cells.
[0016] FIG. 2a is a comparison of the transactivation profile of
all-trans-retinoic acid (RA) on RAR-alpha and RXR-alpha.
[0017] FIG. 2b is a similar comparison to that shown in FIG. 2a,
employing HPLC fraction 18 (instead of RA).
[0018] FIG. 3 presents several activation profiles for analysis of
RXR-alpha or RAR-alpha activation by various retinoic acid isomers.
Panel a. represents experiments done in insect S2 cells, while
panels b. and c. represent experiments done in mammalian CV-1
cells. In the figure, closed circles are used to designate
9-cis-retinoic acid, open circles are used for all-trans-retinoic
acid, open triangles are used for 13-cis-retinoic acid and open
squares are used for 11-cis-retinoic acid.
[0019] FIG. 4 presents the results of saturation binding analysis
of 9-cis-retinoic acid. Cell extracts were incubated with
increasing concentrations of tritiated retinoid in the absence
(total binding) or presence (non-specific binding) of 200-fold
excess non-tritiated retinoid. Non-specific binding was subtracted
from total binding and plotted as specific binding. The data shown
in FIG. 4a represent specific [.sup.2H]-9-cis-retinoic acid binding
to RXR.alpha. (closed circles) or mock (open circles) extracts; or
specific [.sup.3-H]-all-trans-retinoic acid binding to RXRA (open
squares).
[0020] FIG. 4b presents a Scatchard analysis, wherein specific
9-cis-retinoic acid binding to RXR.alpha. in (a) was transformed by
Scatchard analysis and plotted. Linear regression yielded a Kd=11.7
nM (r=0.86).
[0021] FIG. 5 presents a DNA-cellulose column profile of
radiolabelled 9-cis-retinoic acid bound to baculovirus expressed
RXR. In FIG. 5a, sample cell extracts containing RXR.alpha. protein
were labelled with 10 nM [.sup.3H]-9-cis-retinoic acid in the
absence (open squares) or presence (open circles) of 200-fold
excess non-radioactive 9-cis-retinoic acid, and then applied to the
DNA-cellulose column. Fall-through radioactivity was monitored
until a consistent baseline was established. DNA-binding components
were then eluted with a linear salt gradient. The peak radioactive
fractions (labelled 1-15) were then subjected to immunoblot
analysis using an hRXR.alpha.-specific antisera. The peak
radioactive fraction (indicated by an arrow) co-migrated exactly
with the peak amount of RXR.alpha.-specific protein.
[0022] In FIG. 5b, the peak radioactive fraction of the
DNA-cellulose column is shown to contain 9-cis-retinoic acid. The
peak fraction (arrow in (a)) was extracted and analyzed on a
C.sub.18 column developed with mobile phase G. As shown, 0.95% of
the extracted radioactivity co-elutes with authentic 9-cis-retinoic
acid (absorbance peak).
[0023] FIG. 6 is a comparison of the transactivation profile for
RXR-alpha in the presence of 9-cis-retinoic acid employing a
luciferase reporter containing the retinoid response element
derived from either the apolipoprotein A1 gene (APOA13) or cellular
retinol binding protein, type II (CRBPII).
DETAILED DESCRIPTION OF THE INVENTION
[0024] In accordance with the present invention, there is provided
a method for modulating process(es) mediated by retinoid receptors,
said method comprising conducting said process(es) in the presence
of at least one compound of the structure: 1
[0025] wherein:
[0026] unsaturation between carbon atoms C.sup.9 and C.sup.10 has a
cis configuration, and one or both sites of unsaturation between
carbon atoms C.sup.11 through C.sup.14 optionally have a cis
configuration;
[0027] "Ring" is a cyclic moiety, optionally having one or more
substituents thereon;
[0028] Z is selected from carboxyl (--COOH), carboxaldehyde
(--COH), hydroxyalkyl [--(CR'.sub.2).sub.n--OH, wherein each R' is
independently selected from hydrogen or a lower alkyl and n falls
in the range of 1 up to about 4], thioalkyl
[--(CR'.sub.2).sub.n--SH, wherein R' and n are as defined above],
hydroxyalkyl phosphate [--(CR'.sub.2).sub.n--OP(OM).sub.3- ,
wherein R' and n are as defined above and M is hydrogen, lower
alkyl, or a cationic species such as Na.sup.+, Li.sup.+, K.sup.+,
and the like], alkyl ether of a hydroxyalkyl group
[--(CR'.sub.2).sub.n--OR', wherein R' and n are as defined above],
alkyl thioether of a thioalkyl group [--(CR'.sub.2).sub.n--SR',
wherein R' and n are as defined above], esters of hydroxyalkyl
groups [--(CR'.sub.2).sub.n--O--CO--R', wherein R' and n are as
defined above], thioesters of hydroxyalkyl group
[--(CR'.sub.2).sub.n--O--CS--R', wherein R' and n are as defined
above), esters of thioalkyl groups [--(CR'.sub.2).sub.n--S--CO--R',
wherein R' and n are as defined above], thioesters of thioalkyl
groups [--(CR'.sub.2).sub.n--S--CS--R', wherein R' and n are as
defined above], aminoalkyl [--(CR'.sub.2).sub.n--NR'.sub.2, wherein
R' and n are as defined above], N-acyl aminoalkyl
[--(CR'.sub.2).sub.n--NR'--CO--R", wherein R' and n are as defined
above and R" is a lower alkyl or benzyl], carbamate
[--(CR'.sub.2).sub.n--NR'--CO--OR' or --(CR'.sub.2).sub.n--O--C-
O--NR'.sub.2, wherein R' and n are as defined above], and the like;
and
[0029] each R is independently selected from H, halogen, alkyl,
aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or any of the Z
substituents, and the like; or
[0030] any two or more of the R groups can be linked to one another
to form one or more ring structures.
[0031] Exemplary R groups in the latter situation are selected from
alkylene, oxyalkylene, thioalkylene, and the like.
[0032] As employed herein, the term "modulate" refers to the
ability of a ligand for a member of the steroid/thyroid superfamily
to induce expression of gene(s) maintained under hormone expression
control, or to repress expression of gene(s) maintained under such
control.
[0033] As employed herein, the phrase "processes mediated by
retinoid receptors" refers to biological, physiological,
endocrinological, and other bodily processes which are mediated by
receptor or receptor combinations which are responsive to natural
or synthetic retinoids, or natural or synthetic compounds as
defined herein (referred to herein as "rexoids" because of the
ability of many of the compounds described herein to selectively
activate retinoid X receptors). Modulation of such processes can be
accomplished in vitro or in vivo. In vivo modulation can be carried
out in a wide range of subjects, such as, for example, humans,
rodents, sheep, pigs, cows, and the like.
[0034] Exemplary receptors which are responsive to retinoids, and
natural or synthetic compounds as defined herein (i.e., "rexoids"),
include retinoic acid receptor-alpha, retinoic acid receptor-beta,
retinoic acid receptor-gamma, and splicing variants encoded by the
genes for such receptors; retinoid X receptor-alpha, retinoid X
receptor-beta, retinoid X receptor-gamma, and splicing variants
encoded by the genes for such receptors; as well as various
combinations thereof (i.e., homodimers, homotrimers, heterodimers,
heterotrimers, and the like), including combinations of such
receptors with other members of the steroid/thyroid superfamily of
receptors with which the retinoid receptors may interact by forming
heterodimers, heterotrimers, and higher heteromultimers. For
example, the retinoic acid receptor-alpha may form a heterodimer
with retinoid X receptor-alpha, the retinoic acid receptor-beta may
form a heterodimer with retinoid X receptor-alpha, retinoic acid
receptor-gamma may form a heterodimer with retinoid X
receptor-alpha, retinoid X receptor-alpha may form a heterodimer
with thyroid receptor, retinoid X receptor-beta may form a
heterodimer with vitamin D receptor, retinoid X receptor-gamma may
form a heterodimer with retinoic acid receptor-alpha, and the
like.
[0035] As employed herein, the phrase "members of the
steroid/thyroid superfamily of receptors" (also known as "nuclear
receptors" or "intracellular receptors") refers to hormone binding
proteins that operate as ligand-dependent transcription factors,
including identified members of the steroid/thyroid superfamily of
receptors for which specific ligands have not yet been identified
(referred to hereinafter as "orphan receptors"). These hormone
binding proteins have the intrinsic ability to bind to specific DNA
sequences. Following binding, the transcriptional activity of
target gene (i.e., a gene associated with the specific DNA
sequence) is modulated as a function of the ligand bound to the
receptor.
[0036] The DNA-binding domains of all of these nuclear receptors
are related, consisting of 66-68 amino acid residues, and
possessing about 20 invariant amino acid residues, including nine
cysteines.
[0037] A member of the superfamily can be identified as a protein
which contains the above-mentioned invariant amino acid residues,
which are part of the DNA-binding domain of such known steroid
receptors as the human glucocorticoid receptor (amino acids
421-486), the estrogen receptor (amino acids 185-250), the
mineralocorticoid receptor (amino acids 603-668), the human
retinoic acid receptor (amino acids 88-153). The highly conserved
amino acids of the DNA-binding domain of members of the superfamily
are as follows:
1 (SEQ ID No 1) Cys - X - X - Cys - X - X - Asp* - X - Ala* - X -
Gly* - X - Tyr* - X - X - X - X - Cys - X - X - Cys - Lys* - X -
Phe - Phe - X - Arg* - X - X - X - X - X - X - X - X - X - (X - X
-) Cys - X - X - X - X - X - (X - X - X -) Cys - X - X - X - Lys -
X - X - Arg - X - X - Cys - X - X - Cys - Arg* - X - X - Lys* - Cys
- X - X - X - Gly* - Met;
[0038] wherein X designates non-conserved amino acids within the
DNA-binding domain; the amino acid residues denoted with an
asterisk are residues that are almost universally conserved, but
for which variations have been found in some identified hormone
receptors; and the residues enclosed in parenthesis are optional
residues (thus, the DNA-binding domain is a minimum of 66 amino
acids in length, but can contain several additional residues).
[0039] Exemplary members of the steroid/thyroid superfamily of
receptors include steroid receptors such as glucocorticoid
receptor, mineralocorticoid receptor, progesterone receptor,
androgen receptor, vitamin D.sub.3 receptor, and the like; plus
retinoid receptors, such as RAR.alpha., RAR.beta., RAR.gamma., and
the like, plus RXR.alpha., RXR.beta., RXR.gamma., and the like;
thyroid receptors, such as TR.alpha., TR.beta., and the like; as
well as other gene products which, by their structure and
properties, are considered to be members of the superfamily, as
defined hereinabove. Examples of orphan receptors include HNF4
[see, for example, Sladek et al., in Genes & Development 4:
2353-2365 (1990)], the COUP family of receptors [see, for example,
Miyajima et al., in Nucleic Acids Research 16: 11057-11074 (1988),
Wang et al., in Nature 340: 163-166 (1989)], COUP-like receptors
and COUP homologs, such as those described by Mlodzik et al., in
Cell 60: 211-224 (1990) and Ladias et al., in Science 251: 561-565
(1991), the ultraspiracle receptor [see, for example, Oro et al.,
in Nature 347: 298-301 (1990)], and the like.
[0040] Processes capable of being modulated by retinoid receptors,
in accordance with the present invention, include in vitro cellular
differentiation and proliferation, in vitro proliferation of
melanoma cell lines, in vitro differentiation of mouse
teratocarcinoma cells (F9 cells), in vitro differentiation of human
epidermal keratinocytes, limb morphogenesis, regulation of cellular
retinol binding protein (CRBP), and the like. As readily recognized
by those of skill in the art, the availability of ligands for the
retinoid X receptor makes it possible, for the first time, to carry
out assays for the identification of antagonists for said
receptor.
[0041] Processes capable of being modulated by retinoid receptors,
in accordance with the present invention, also include the in vivo
modulation of lipid metabolism, in vivo modulation of skin-related
processes (e.g., acne, aging, wrinkling, skin cancer, and the
like), in vivo modulation of malignant cell development, such as
occurs, for example, in acute promyelocytic leukemia, testicular
cancer, lung cancer, and the like. The ability of compounds of the
invention to modulate such processes is evidenced in a number of
ways. See, for example, FIG. 6 where the ability of RXR-alpha, in
the presence of ligand therefor (e.g., 9-cis-retinoic acid) is
shown to exert a strong effect on the expression of genes under the
control of regulatory elements of apolipoprotein AI. Similarly,
studies with model systems for a variety of disease states (e.g.,
differentiation of HL60 cells as a model for acute promyelocytic
leukemia, proliferation of melanoma cell lines as a model for skin
cancer, differentiation of keratinocytes as a model for
non-malignant skin disorders, and the like), as set forth in the
Examples, demonstrate the ability of retinoid receptors, in the
presence of ligand therefor, e.g., 9-cis-retinoic acid, to exert a
strong effect on such disease states. Such in vivo applications of
the invention process may allow the modulation of various
biological processes with reduced occurrence of undesirable side
effects, and the like.
[0042] In vivo applications of the invention process(es) (and
compositions) can be employed with a wide range of subjects, such
as, for example, humans, rodents, sheep, pigs, cows, and the
like.
[0043] As employed herein, the term "alkyl" refers to "lower
alkyl", i.e., alkyl moieties having in the range of 1 up to about 4
carbon atoms, i.e., methyl groups, ethyl groups, propyl groups,
isopropyl groups, normal-butyl groups, isobutyl groups, sec-butyl
groups, tert-butyl groups, and the like.
[0044] Cyclic moieties contemplated as part of the compounds
employed in the practice of the present invention include 5-, 6-,
and 7-membered carbocyclic, heterocyclic aromatic or heteroaromatic
rings. Included in this definition, for example, are optionally
substituted saturated, mono-unsaturated or polyunsaturated
carbocyclic species, such as, for example, cyclopentane,
cyclopentene, cyclohexane, cyclohex-2-ene, cyclohex-3-ene,
cyclohex-4-ene, and cyclohex-5-ene isomers, and 2,4-, 2,5-, and
3,5-cyclohexadiene variants thereof. Examples of heterocyclic
species contemplated as part of the compounds employed in the
practice of the present invention include dihydrofuran,
tetrahydrofuran, dihydrothiophene, tetrahydrothiophene,
dihydropyran, tetrahydropyran, dihydrothiopyran,
tetrahydrothiopyran, piperidine, pyrrolidine, and the like, as well
as derivatives thereof. Examples of aromatic or heteroaromatic
species contemplated as part of the rexoid compounds of the present
invention include phenyl, tolyl, xylyl, mesityl, benzyl, pyridyl,
thiophenyl, furanyl, and the like, as well as derivatives
thereof.
[0045] Preferred cyclic moieties are typically geminally
di-substituted, mono-unsaturated species. Presently preferred
geminally di-substituted, mono-unsaturated cyclic moieties are the
1,1,5-trisubstituted cyclohex-5-ene structure of naturally
occurring retinoic acid (i.e., the ring structure of .beta.-ionone;
the position of the substituents on the ring are designated
employing the traditional retinoic acid numbering convention for
the ring structure of .beta.-ionone), as well as the
1,1,4,5-tri-substituted cyclohex-5-ene structure provided by
hydroxy- or keto-substituted derivatives of the traditional
.beta.-ionone structure.
[0046] Compounds contemplated for use in the practice of the
present invention include compounds having the structure: 2
[0047] wherein:
[0048] unsaturation between carbon atoms C.sup.9 and C.sup.10 has a
cis configuration, and one or both sites of unsaturation between
carbon atoms C.sup.11 through C.sup.14 optionally have a cis
configuration;
[0049] "Ring" is a cyclic moiety;
[0050] Z is selected from carboxyl, carboxaldehyde, hydroxyalkyl,
thioalkyl, hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl
group, alkyl thioether of a thioalkyl group, esters of hydroxyalkyl
groups, thioesters of hydroxyalkyl group, esters of thioalkyl
groups, thioesters of thioalkyl groups, aminoalkyl, N-acyl
aminoalkyl, carbamate, and the like; and
[0051] R on each of C.sup.7, C.sup.8, C.sup.9, C.sup.10, C.sup.11,
C.sup.12, C.sup.12, C.sup.13, or C.sup.14 is independently selected
from H, halogen, alkyl, aryl, hydroxy, thiol, alkoxy, thioalkoxy,
amino, or any of the Z substituents; or
[0052] any two or more of the R groups can be linked to one another
to form one or more ring structures.
[0053] Presently preferred compounds which are contemplated by the
above generic structure include 9-cis-retinoic acid, as well as
novel derivatives thereof such as 9-phenyl-9-cis-retinoic acid,
4-hydroxy-9-cis-retinoic acid, 4-keto-9-cis-retinoic acid, and the
like.
[0054] In another preferred embodiment of the present invention,
the substituents on C.sup.9 and C.sup.13 are methyl; in yet another
preferred embodiment, the substituents on two or more of the side
chain carbons (i.e., C.sup.7, C.sup.8, C.sup.9, C.sup.10, c.sup.11,
C.sup.12 , C.sup.13 , or C.sup.14) can be linked together to form a
ring structure. For example, the substituents on C.sup.8 and
C.sup.11 can be linked together to form a structure having a
constrained 9-cis double bond (i.e., a 9-cis locked rexoid
derivative), as follows: 3
[0055] wherein:
[0056] X is --[(CR.sub.2).sub.X--X'--(CR.sub.2).gamma.]--,
[0057] X' is selected from --O--, carbonyl (>CO), --S--,
--S(O)--, --S(O).sub.2--, thiocarbonyl (>CS), --NR"--, or
--CR.sub.2--,
[0058] R, Ring and Z are as defined above,
[0059] R" is hydrogen, alkyl, hydroxy, thiol, or alkoxy acyl
(--co--o--alkyl);
[0060] x is 0, 1 or 2,
[0061] y is 0, 1, or 2, and
[0062] x+y.ltoreq.2.
[0063] Such compounds include cyclopentene derivatives, cyclohexene
derivatives, cycloheptene derivatives, dihydrofuran derivatives,
dihydropyrrole derivatives, and the like, wherein the cyclic
structure linking C.sup.8 and C.sup.11 serves to prevent
isomerization of the cis double bond between C.sup.9 and
C.sup.10.
[0064] Especially preferred derivatives of structure I are those
where Z is a carboxyl group, and Ring is a .beta.-ionone-like
species having the structure: 4
[0065] wherein:
[0066] each R is independently defined as provided above;
[0067] any one of C.sup.2, C.sup.3, or C.sup.4 can be replaced with
--0--, carbonyl (>CO), --S--, --S(O)--, --S(O).sub.2--,
thiocarbonyl (>CS), or --NR"--; wherein R" is as defined above;
and
[0068] said cyclic moiety exists as the saturated, 2-ene, 3-ene,
4-ene, or 5-ene mono-unsaturated isomer; the 2,4-, 2,5-, or
3,5-diene derivative thereof; or an aromatic derivative
thereof.
[0069] Especially preferred species for use in the practice of the
present invention are derivatives of structure I where Z is a
carboxyl group, and Ring is a 1,1,5-trisubstituted cyclohex-5-ene
structure or a l,1,4,5-tetrasubstituted cyclohex-5-ene
structure.
[0070] Similarly, the substituents on C.sup.10 and C.sup.13 can be
linked together to form a structure having a constrained 9,
11-di-cis configuration (i.e., a 9-cis locked rexoid derivative),
as follows: 5
[0071] wherein:
[0072] X, X', R, R", Z, Ring, x and y are as defined above.
[0073] Such compounds include cyclopentene derivatives, cyclohexene
derivatives, cycloheptene derivatives, dihydrofuran derivatives,
dihydropyrrole derivatives, and the like, wherein the cyclic
structure linking C.sup.10 and C.sup.13 serves to hinder
isomerization of the cis double bond between C.sup.9 and C .sup.10,
and prevent isomerization of the cis double bond between C.sup.11
and C.sup.12.
[0074] Especially preferred derivatives of Structure II are those
where Z is a carboxyl group, and the Ring is a 1,1,5-trisubstituted
cyclohex-5-ene structure or a 1,1,4,5-tetrasubstituted
cyclohex-5-ene structure.
[0075] Similarly, at least two of the substituents on C.sup.8,
C.sup.11, and/or C.sup.14 can be linked together to form a
structure having a constrained 9, 13-di-cis configuration (i.e., a
9-cis locked rexoid derivative), shown below as Structure 6
[0076] wherein:
[0077] one A is X and the other A is X', and
[0078] X, X', R, R", Z, Ring, x and y are as defined above. Those
of skill in the art recognize that the junction between the two
bridging groups (A) can only occur through an atom with a valence
of three or four (i.e., through carbon or nitrogen), so as to
accomodate the bonds required to link the fused rings together.
[0079] Similarly, at least two of the substituents on C.sup.8,
C.sup.11, and/or C.sup.14 can be linked together, and further
linked to C.sup.5 of Ring, or to a substituent on C.sup.5 to form a
structure having a constrained 9, 13-di-cis configuration (i.e., a
9-cis locked rexoid derivative), shown below as Structure IV: 7
[0080] wherein:
[0081] one A is X and the other A is X',
[0082] B is X', and
[0083] X, X', R, R", Z, Ring, x and y are as defined above. As
noted above with respect to Structure III, those of skill in the
art recognize that the junction(s) between the bridging groups (A)
and (B) can only occur through an atom with a valence of three or
four (i.e., through carbon or nitrogen), so as to accomodate the
bonds required to link the fused rings together.
[0084] Such compounds include cyclopentene derivatives, cyclohexene
derivatives, cycloheptene derivatives, dihydrofuran derivatives,
dihydropyrrole derivatives, and the like, wherein the cyclic
structures linking C.sup.8, C.sup.11 and/or C.sup.13 serves to
prevent isomerization of the cis double bonds at carbon 9 and
carbon 13.
[0085] Especially preferred derivatives of Structures III and IV
are those where Z is a carboxyl group, and Ring is a
1,1,5-trisubstituted cyclohex-5-ene structure or a
1,1,4,5-tetrasubstituted cyclohex-5-ene structure.
[0086] Similarly, the substituents on C.sup.10 and C.sup.11 can be
linked together to form a structure having a constrained 9-cis
double bond (i.e., a 9-cis locked rexoid derivative) , as follows:
8
[0087] wherein:
[0088] X" is --[(CR.sub.2).sub.a--X'--(CR.sub.2).sub.b]--,
[0089] X', R, R", Ring and Z are as defined above,
[0090] a is 0, 1, 2, 3 or 4,
[0091] b is 0, 1, 2, 3, or 4, and
[0092] a+b is .gtoreq.2, but .ltoreq.4.
[0093] Such compounds include cyclopentene derivatives, cyclohexene
derivatives, cycloheptene derivatives, dihydrofuran derivatives,
dihydropyrrole derivatives, and the like, wherein the cyclic
structure linking C.sup.10 and C .sup.11 serves to prevent
isomerization of the cis double bond between C.sup.9 and
C.sup.10.
[0094] Especially preferred derivatives of Structure V are those
where Z is a carboxyl group, and Ring is a 1,1,5-trisubstituted
cyclohex-5-ene structure or a 1,1,4,5-tetrasubstituted
cyclohex-5-ene structure.
[0095] Similarly, the substituents on C.sup.7 and C.sup.9 can be
linked together, and the substituents on C.sup.10 and C12 can be
linked together to form a structure having a constrained 9-cis
double bond (i.e., a 9-cis locked rexoid derivative), as follows:
9
[0096] wherein:
[0097] Y is --[(CR.sub.2).sub.c--X'--(CR.sub.2).sub.d]--,
[0098] X', R, R", Ring and Z are as defined above,
[0099] c is 0, 1, 2 or 3,
[0100] d is 0, 1, 2 or 3, and
[0101] c+d .gtoreq.1, but .ltoreq.3.
[0102] Such compounds include cyclopentene derivatives, cyclohexene
derivatives, cycloheptene derivatives, dihydrofuran derivatives,
dihydropyrrole derivatives, and the like, wherein the cyclic
structures linking C.sup.7 and C.sup.9, and C .sup.10 and C.sup.12
serve to prevent isomerization of the cis double bond between
C.sup.9 and C.sup.10.
[0103] Especially preferred derivatives of Structure VI are those
where Z is a carboxyl group, and Ring is a 1,1,5-trisubstituted
cyclohex-5-ene structure or a 1,1,4,5-tetrasubstituted
cyclohex-5-ene structure.
[0104] Similarly, the substituents on C.sup.9 and C.sup.10 can be
linked together to form a structure having a constrained C-9 double
bond (i.e., a 9-cis locked rexoid derivative), as follows: 10
[0105] wherein:
[0106] X'" is X" or an unsaturated linking group having the
structure:
[0107] --[Q.dbd.CR--J]--,
[0108] wherein Q is --N.dbd.or --CR.dbd., and J is --CR.dbd.CR--,
--N.dbd.CR--, --CR.dbd.N--, --O--, --S--, or --NR"--,
[0109] thereby incorporating C.sup.9 and C.sup.10 of the rexoid
compound into an aromatic (or pseudo-aromatic) ring, and
[0110] X', X", R, R", Ring, Z, a and b are as defined above.
[0111] Such compounds include cyclohexene derivatives, cycloheptene
derivatives, benzene derivatives, pyridine derivatives, furan
derivatives, thiophene derivatives, pyrrole derivatives, oxazole
derivatives, thiazole derivatives, imidazole derivatives, pyrazole
derivatives, and the like, wherein the cyclic structure linking
C.sup.9 and C.sup.10 serves to prevent isomerization of the
C.sup.9--C.sup.10 double bond; however, rotation about the 8--9
and/or 10--11 single bonds can still occur.
[0112] Especially preferred derivatives of Structure VII are those
where Z is a carboxyl group, and Ring is a 1,1,5-trisubstituted
cyclohex-5-ene structure or a 1,1,4,5-tetrasubstituted
cyclohex-5-ene structure.
[0113] In addition to the structures set forth above, those of
skill in the art can readily identify additional means to constrain
the basic cis-configuration containing rexoid compounds employed in
the practice of the present invention.
[0114] In accordance with a preferred embodiment of the present
invention, the cyclic moiety has the .beta.-ionone structure set
forth above. Especially preferred are the 1,1,5-trisubstituted
cyclohex-5-ene structure (characteristic of .beta.-ionone) as well
as the closely related 1,1,4,5-tetrasubstituted cyclohex-5-ene
structure from which many rexoid compounds according to the present
invention can be prepared.
[0115] In accordance with a particularly preferred embodiment of
the present invention, the compounds employed in the invention
process are selected from 9-cis-retinoic acid and derivatives
thereof as contemplated by Structure A set forth above, as well as
9-cis-locked derivatives of retinoic acid as set forth in
Structures I-VII above. Examples of specific compounds contemplated
for use in the practice of the present invention are compounds
wherein Z is carboxy, Ring is the 1,1,5-trisubstituted
cyclohex-5-ene structure characteristic of .beta.-ionone (or the
closely related 1,1,4,5-tetrasubstituted cyclohex-5-ene), and
having a side chain structure(s) as described above for Structures
I-VII.
[0116] "Rexoid" derivatives as described above can be prepared
employing a variety of synthetic methods, which are readily
available (and well known) to those of skill in the art. See, for
example, the methods described in Chemistry and Biology of
Synthetic Retinoids, Dawson and Okamura, eds., CRC Press, Inc.
(1990), especially Chapter 4, by Ito (found at pages 78-97), and
Chapter 9, by de Lera et al. (found at pages 202-227) can readily
be adapted for the preparation of the compounds described herein.
The contents of this publication are hereby incorporated by
reference herein. See also Asato et al., J. Am. Chem. Soc. 108:
5032 (1986); Sheves et al., J. Am. Chem. Soc. 108: 6440 (1986);
Akita et al., J. Am. Chem. Soc. 102: 6370 (1980); Derguini and
Nakanishi, Photobiochem, and Photobiophys. 13: 259 (1986), the
entire contents of each of which is hereby incorporated by
reference herein.
[0117] In accordance with another embodiment of the present
invention, there is provided a method for modulating processes
mediated by retinoid receptors, said method comprising conducting
said process in the presence of:
[0118] (a) at least one compound of the structure: 11
[0119] wherein:
[0120] each site of unsaturation in the side chain comprising
carbon atoms C.sup.7 through C.sup.14 has a trans
configuration;
[0121] "Ring", Z, and R are as previously described, and
[0122] (b) a cis/trans isomerase capable of converting at least the
9-double bond from the trans configuration to the
cis-configuration.
[0123] As employed herein, the term "cis/trans isomerase" refers to
enzymes which promote a change of geometrical configuration at a
double bond. Examples of such enzymes include maleate isomerase,
maleylacetoacetate isomerase, retinal isomerase, maleylpyruvate
isomerase, linoleate isomerase, furylfuramide isomerase, and the
like.
[0124] In accordance with yet another embodiment of the present
invention, there is provided a method to produce compound(s) of the
structure: 12
[0125] wherein:
[0126] unsaturation between carbon atoms C.sup.9 and C.sup.10 has a
cis configuration, and one or both sites of unsaturation between
carbon atoms C.sup.11 through C.sup.14 optionally have a cis
configuration;
[0127] "Ring" is a cyclic moiety;
[0128] Z is selected from carboxyl, carboxaldehyde, hydroxyalkyl,
thioalkyl, hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl
group, alkyl thioether of a thioalkyl group, esters of hydroxyalkyl
groups, thioesters of hydroxyalkyl group, esters of thioalkyl
groups, thioesters of thioalkyl groups, aminoalkyl, N-acyl
aminoalkyl, carbamate, and the like; and
[0129] each R is independently selected from H, halogen, alkyl,
aryl, hydroxy, thiol, alkoxy, thioalkoxy, amino, or any of the Z
substituents;
[0130] from the corresponding all-trans configuration material,
said method comprising contacting said all-trans configuration
material with a cis/trans isomerase under isomerization
conditions.
[0131] In accordance with still another embodiment of the present
invention, there are provided novel compositions comprising
compound(s) of Structure A (excluding previously identified
compounds such as retinoic acid as well as constrained compounds
selected from Structures I-VII, as set forth above. Examples of
such compounds include 9-phenyl-9-cis-retinoic acid,
4-hydroxy-9-cis-retinoic acid, 4-keto-9-cis-retinoic acid, and the
like. Presently preferred compounds are those wherein Z is carboxyl
and Ring is a 1,1,5-trisubstituted cyclohex-5-ene structure or a
1,1,4,5-tetrasubstituted cyclohex-5-ene structure.
[0132] The invention compounds can be employed for both in vitro
and in vivo applications. For in vivo applications, the invention
compounds can be incorporated into a pharmaceutically acceptable
formulation for administration. Those of skill in the art can
readily determine suitable dosage levels when the invention
compounds are so used.
[0133] As employed herein, the phrase "suitable dosage levels"
refers to levels of compound sufficient to provide circulating
concentrations high enough to effect activation of retinoid
receptor(s). Such a concentration typically falls in the range of
about 10 nM up to 2 .mu.M; with concentrations in the range of
about 100 nM up to 200 nM being preferred.
[0134] In accordance with a particular embodiment of the present
invention, compositions comprising at least one 9-cis-retinoic
acid-like compound (as described above), and a pharmaceutically
acceptable carrier are contemplated. Exemplary pharmaceutically
acceptable carriers include carriers suitable for oral,
intravenous, subcutaneous, intramuscular, intracutaneous, and the
like administration. Administration in the form of creams, lotions,
tablets, dispersible powders, granules, syrups, elixirs, sterile
aqueous or non-aqueous solutions, suspensions or emulsions, and the
like, is contemplated.
[0135] For the preparation of oral liquids, suitable carriers
include emulsions, solutions, suspensions, syrups, and the like,
optionally containing additives such as wetting agents, emulsifying
and suspending agents, sweetening, flavoring and perfuming agents,
and the like.
[0136] For the preparation of fluids for parenteral administration,
suitable carriers include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil and corn oil, gelatin, and injectable organic
esters such as ethyl oleate. Such dosage forms may also contain
adjuvants such as preserving, wetting, emulsifying, and dispersing
agents. They may be sterilized, for example, by filtration through
a bacteria-retaining filter, by incorporating sterilizing agents
into the compositions, by irradiating the compositions, or by
heating the compositions. They can also be manufactured in the form
of sterile water, or some other sterile injectable medium
immediately before use.
[0137] The invention will now be described in greater detail by
reference to the following non-limiting examples.
EXAMPLES
Example 1
Identification of Compound(s) that Activate RXR
[0138] In order to acertain if retinoic acid can be converted to a
product that binds directly to RXR, thereby resulting in modulation
of transcription, a strategy was developed to identify retinoic
acid metabolites that might modulate the transcriptional properties
of RXR. The identification of any such active metabolite would
allow one to further determine whether this metabolite was capable
of directly binding to the receptor protein.
[0139] Accordingly, the Drosophila melanogaster Schneider cell line
(S2) was incubated with or without all-trans-retinoic acid (RA) for
a period of 24 hours. Prior to the addition of retinoic acid,
Drosophila melanogaster Schneider cell line (S2) cells were grown
in Schneider Drosophila medium (GIBCO) supplemented with
penicillin, streptomycin and 12% heat inactivated FCS (Irvine
Scientific). One hundred tissue culture flasks (75 cm.sup.2) were
set up with 10.sup.7 cells and 12 ml of medium/flask. Twenty four
hours later, either all-trans-retinoic acid (or ethanol solvent
control) was added to each flask to a final concentration of
5.times.10.sup.-6 M in reduced light conditions. Cells were
harvested 24 hours later by centrifugation for 5 minutes at 800 g.
Cells were washed twice with PBS and the resultant pellets were
frozen at -80.degree. C. until extraction.
[0140] In parallel, CV-1 cells were set up on 64 tissue culture
dishes (150 mm) at 2.times.10 .sup.6 cells and 25 ml of
medium/dish. Cells were treated with retinoic acid and harvested as
with the S2 cells except that the CV-1 cells (which are adherent)
were washed in their dishes with PBS and scraped with a rubber
policeman prior to centrifugation and freezing.
[0141] Following incubation, the cell pellets were collected,
organically extracted and chromatographically fractionated by HPLC.
The various HPLC fractions were assayed for their ability to
produce a ligand dependent increase in transcriptional activity
mediated by RXR. This assay system involves transfecting cells with
the cDNA for the RXR receptor and a luciferase reporter molecule
which is under control of a promoter containing a RXR response
element (RXRE) [see Mangelsdorf et al., Cell 66:555 (1991)]. The
addition of a ligand capable of activating RXR results in an
increase in luciferase activity.
[0142] Schneider cells, CV-1 cells and mouse tissues were extracted
as described by C. Thaller and G. Eichele in Nature Vol. 327:625
(1987). Mouse tissue was used to determine if any RXR ligand is
present in vivo. In the case of tissue extractions, 2.10.sup.5 dpm
internal standard [11,12--.sup.3H]-all-trans-retinoic acid (New
England Nuclear) or 9-cis-retinoic acid (generated by isomerization
with light) were added to the homogenate. Extracts were
fractionated on a Waters Novapak 300 mm C.sub.18 analytical column
at a flow rate of 1 ml min.sup.-1. The mobile phase (G) was a 1:1
mixture of:
[0143] A [CH.sub.3CN/CH.sub.3OH/2% aqueous CH.sub.3COOH (3:1:1)]
and
[0144] E [CH.sub.3CN/CH.sub.3OH/2% aqueous CH.sub.3COOH
(11:3:10)].
[0145] Other mobile phases used have the following
compositions:
[0146] C: CH.sub.3CN/CH.sub.3OH/H.sub.2/CH.sub.3COOH
(80:10:10:1),
[0147] H: mix CH.sub.3OH/10 mM ammonium acetate (9:1) with equal
volume of CH.sub.3OH/10 mM ammonium acetate (3:1).
[0148] Methyl esters of retinoic acid isomers and/or metabolites
contained in the HPLC fractions were generated as described in
Wedden et al. [Meth. Enzymol. 190:201 (1990)]. Reference standards
used were from Aldrich, Sigma or kindly provided by
Hoffmann-LaRoche. Authentic 9-cis-retinol, 9-cis-retinoic acid and
9-cis-methylretinoate were either synthesized from 9-cis-retinal
(see E. J. Corey et al., J. Am. Chem. Soc. 90:5616 (1968); C. D. B.
Bridges & R. A. Alvares (Meth. Enzymol. 81:463 (1982)] or
generated by photoisomerization of the all-trans isomer followed by
fractionation of the resulting isomers by HPLC.
[0149] Photoisomerization of all-trans-retinoic acid is carried out
employing standard isomerization techniques which are well known to
those of skill in the art. For example, retinoic acid can be
dissolved in a polar organic solvent such as ethanol, placed in a
quartz cuvette, and irradiated with a variety of wavelengths of
light (such as fluorescent light). Temperature at which irradiation
is carried out is not critical; accordingly, irradiation can be
carried out at room temperature. Irradiation time is also not
critical; typical irradiation times are in the range of about 0.5-2
hours.
[0150] The various HPLC fractions were diluted 1:100 and assayed
for their ability to modulate the transcriptional properties of
RXR.
[0151] Cotransfection Assay in CV-1 Cells
[0152] A monkey kidney cell line, CV-1, was used in the cis-trans
assay. Cells were transfected with two DNA transfection vectors.
The trans-vector allowed efficient production of retinoid receptor
(e.g., RAR or RXR) in these cells, which do not normally express
these receptors. The cis-vector contains an easily assayable gene,
in this case the firefly luciferase, coupled to a
retinoid-responsive promoter. Addition of retinoic acid or an
appropriate synthetic retinoid results in the formation of a
retinoid-receptor complex that activates the luciferase gene,
causing light to be emitted from cell extracts. The level of
luciferase activity is directly proportional to the effectiveness
of the retinoid-receptor complex in activating gene expression.
This sensitive and reproducible cotransfection approach permits the
identification of retinoids that interact with the different
receptor isoforms.
[0153] Cells were cultured in DMEM supplemented with 10% charcoal
resin-stripped fetal bovine serum, and experiments were conducted
in 96-well plates. The plasmids were transiently transfected by the
calcium phosphate method [Umesono and Evans, Cell 57:1139-1146
(1989); Berger et al., J. Steroid Biochem. Molec. Biol. 41:733-738
(1992)] by using 10 ng of a pRS (Rous sarcoma virus promoter)
receptor-expression plasmid vector, 50 ng of the reporter
luciferase (LUC) plasmid, 50 ng of pRS.beta.-GAL
(.beta.-galactosidase) as an internal control, and 90 ng of carrier
plasmid pGEM. Cells were transfected for 6 hours and then washed to
remove the precipitate. The cells were then incubated for 36 hours
with or without retinoid. After the transfection, all subsequent
steps were performed on a Beckman Biomek Automated Workstation.
Cell extracts were prepared as described by Berger et al. supra,
then assayed for luciferase and .beta.-galactosidase activities.
All determinations were performed in triplicate in two independent
experiments and were normalized for transfection efficiency by
using .beta.-galactosidase as the internal control. Retinoid
activity was normalized relative to that of retinoic acid and is
expressed as potency (EC50), which is the concentration of retinoid
required to produce 50% of the maximal observed response, and
efficacy (%), which is the maximal response observed relative to
that of retinoic acid at 10.sup.-5 M.
[0154] The receptor expression vectors used in the cotransfection
assay have been described previously [pRShRAR-.alpha.: Giguere et
al., Nature 330:624-629 (1987); pRShRAR-.beta. and pRShRAR-.gamma.:
Ishikawa et al., Mol. Endocrinol. 4:837-844 (1990); retinoid X
receptor-alpha (RXR-.alpha.) [see Mangelsdorf et al., in Nature
345: 224-229 (1990)], retinoid X receptor-beta (RXR-.beta.) and
retinoid x receptor-gamma (RXR-.gamma.) [see Mangelsdorf et al.,
Genes and Development 6:329-344 (1992)]. A basal reporter plasmid
.DELTA.MTV-LUC [Hollenberg and Evans, Cell 55:899-906
(1988)]containing two copies of the TRE-palindromic response
element 5'-TCAGGTCATGACCTGA-3' [SEQ ID No 2; see Umesono et al.,
Nature 336:262-265 (1988)] was used in all transfections for the
retinoid receptors.
[0155] The bacterial expression vector for PET-8c-RAR-.alpha. used
in the competitive binding assay has been reported [Yang et al.,
Proc. Natl. Acad. Sci. USA 88:3559-3563 (1991)]. Similar expression
vectors employing the PET-8c vector system [Studier et al., Methods
in Enzymology 185:60-69 (1990)] were constructed for RAR-.beta. and
RAR-.gamma..
[0156] The transactivation profile of RXR-alpha with the various
HPLC fractions containing various retinoic acid isomers and/or
metabolites is shown in FIG. 1. These data reveal two distinct
regions of activity, one relatively early (fraction 7) and a second
broader region of activity (fractions 16-21) that elutes
considerably later. The all-trans-retinoic acid coelutes in
fractions 20 and 21 (FIG. 1) and is the major U.V. absorbing
material present in the cell extracts. However, the activity
profile demonstrates that, in addition to all-trans-retinoic acid,
there are active components that must be derived from, or induced
by, all-trans-retinoic acid that activate RXR.
[0157] To identify potential compounds that would be as effective
or more active than all-trans-retinoic acid, one must take into
account not only the activity of the individual fractions, but also
their concentrations. All active fractions were therefore reassayed
over a broad range of concentrations, taking into account the
relative concentrations of the individual fractions. To determine
the relative concentrations of the fractions, the following initial
assumptions were made: 1) the active fractions are retinoic acid
metabolites and 2) the molar extinction coefficient of the various
active fractions is relatively similar (i.e., within a factor of
two). This assumption is supported by values reported in the
literature for a large number of retinoids. A comparison of the
transactivation profile of all-trans-retinoic acid (i.e., fraction
20) on RAR-alpha and RXR-alpha is shown in FIG. 2a. Although the
maximal activation (i.e., efficacy) of RAR and RXR with retinoic
acid is similar, RAR is more sensitive by a factor of approximately
10 fold (i.e., 10 fold more potent). In contrast, analysis of the
various fractions produced as describes above demonstrates that
fraction 18 is considerably more active on RXR than RAR (see FIG.
2b). These data suggest that a metabolic product present in S2
cells pretreated with retinoic acid is a more potent activator of
the RXR subfamily than the RAR subfamily.
Example 2
Identification of 9-cis Retinoic Acid as a Transactivator of
RXR
[0158] Two observations suggest that fraction 18 (peak X, see FIG.
1) is a cellular metabolite of all-trans-retinoic acid. First,
extracts of Schneider cells grown in the absence of
all-trans-retinoic acid do not exhibit peak X. Second, when cells
are exposed to all-trans-retinoic acid, X appears in a
time-dependent fashion.
[0159] Therefore, to chemically identify X, fraction 18 was
subjected to chemical derivatization, high performance liquid
chromatography (HPLC) and gas chromatography/mass spectrometry
(GC/MS). It was found that upon methylation with diazomethane, the
retention time of peak X shifts dramatically (i.e., from 10.2
minutes to 19.5 minutes under the HPLC conditions used). This
indicates that the compound(s) corresponding to peak X has a free
carboxyl group. When methylated X was analyzed by GC/MS, the
electron impact mode revealed that X gives rise to a molecular ion
at m/z 314, corresponding to that of a retinoic acid methyl ester.
This suggests that X is a stereoisomer of retinoic acid. To
determine which isomer X represents, the retention time of X was
compared with that of 9-cis-, 11-cis- and 13-cis-retinoic acid. It
was found that X coelutes with authentic 9-cis-retinoic acid.
Furthermore, the methyl ester of X coelutes with
9-cis-methylretinoate, and when the methyl ester of X is reduced to
the alcohol with lithium aluminum hydride, the resulting product
coelutes with authentic 9-cis-retinol.
[0160] For GC/MS analysis, methylated retinoic acid isomers were
dissolved in hexane. The sample was injected via a falling needle
injector (280.degree. C.) into a 30 m.times.0.32 mm fused silica
DB5 capillary column (J+J scientific) inserted directly into the
ion source of a VG Trio-1000 mass spectrometer operating in
electron impact mode (70 eV). The sample was eluted with a
temperature gradient (200-300.degree. C., 10.degree. C.
min.sup.-1).
[0161] Finally, the mass spectrum of authentic 9-cis-retinoic acid
methyl ester and that of methylated peak X are found to be
identical. Taken together these analyses establish that peak X
represents 9-cis-retinoic acid. Although earlier work indicated the
presence of 9-cis-retinol in fish liver, it was not clear whether
9-cis-retinoic acid existed in vivo (i.e., whether 9-cis-retinoic
acid is a physiological compound). To find out if 9-cis-retinoic
acid exists in vivo, mouse liver and kidney tissues were extracted.
These tissues were selected because they contain a broad spectrum
of retinoid metabolites and also express RXR. Prior to extraction,
radiolabeled 9-cis-retinoic acid was added to the kidney homogenate
to serve as an internal standard. Extracts were first fractionated
on a reverse phase column (Waters Novo pak 300 mm C.sub.18
analytical column at a flow rate of 1 ml/min) using mobile phase
G.
[0162] Fractions from the kidney extracts containing radioactive
internal standard were rechromatographed on a second C.sub.18
column using mobile phase H. This procedure gave a small, but
distinct absorbance peak which co-migrated with authentic
9-cis-retinoic acid.
[0163] Similarly, liver extract was fractionated on a reverse phase
column and eluted with mobile phase G. However under the conditions
employed, 9-cis-retinoic acid eluted with all-trans-retinol (which
is abundantly present in the liver). To separate these two
retinoids, this fraction was methylated with diazomethane and then
reanalyzed by HPLC employing mobile phase C. This approach resulted
in a distinct peak coeluting with the authentic methyl ester of
9-cis-retinoic acid.
[0164] To rule out the possibility that 9-cis-retinoic acid had
formed during the extraction procedure from all-trans-retinoic
acid, liver tissue homogenate was spiked with tritiated
all-trans-retinoic acid. Subsequent HPLC fractionation revealed
that 94% of the radioactivity still resided in all-trans-retinoic
acid, approximately 5% in 13-cis-retinoic acid and 1% or less in
9-cis-retinoic acid. Based on peak area integration the
concentrations of 9-cis-retinoic acid in liver and kidney are
estimated to be .about.4 ng, and .about.4 ng, respectively, per g
of wet weight. This indicates that endogenous 9-cis-retinoic acid
is not formed from all-trans-retinoic acid during extraction. In
conclusion, these experiments establish that 9-cis-retinoic acid is
a naturally occurring retinoic acid isomer.
Example 3
Transactivation Profile of Retinoid Isomers on RXR and RAR
[0165] The establishment that peak X represents a stereoisomer of
all-trans-retinoic acid suggested that the various retinoid isomers
may have different retinoid receptor activation profiles. To
further analyze the ability of retinoic acid isomers to modulate
the transcriptional properties of RXR-alpha and RAR-alpha, the four
major photoisomers of all-trans-retinoic acid were identified and
assayed for the ability to transactivate RXR and RAR. FIG. 3 shows
the dose response curves for 13-cis-, 11l-cis-, 9-cis- and
all-trans-retinoic acid for both RAR-alpha and RXR-alpha.
[0166] Of the four major isomers of retinoic acid, 9-cis-retinoic
acid is seen to be the most potent and efficacious activator of
RXR-alpha in both insect S2 cells (see FIG. 3A) and mammalian CV-1
cells (see FIG. 3B). The maximal response (EC50 value) is 10.sup.-8
M and 5.times.10.sup.-8 M, respectively. The observed rank order of
potency for the different isomers is the same in both cell lines.
9-cis-retinoic acid is approximately 40 fold more potent as an
activator of RXR than 11-cis-, 13-cis- or all-trans-retinoic acid.
These transactivation data strongly suggest that 9-cis-retinoic
acid is an endogenous RXR-alpha activator.
[0167] In contrast, 9-cis-retinoic acid is equipotent to
all-trans-retinoic acid as an activator of RAR-alpha (FIG. 3C). The
EC50 value for 9-cis-retinoic acid on RAR-alpha is
2.times.10.sup.-7 M. 9-cis-retinoic acid is the most potent
RXR-alpha ligand to be tested to date.
[0168] Similarly, transactivation of other isoforms of RXR (i.e.,
RXR-beta, RXR-gamma) and RAR (i.e., RAR-beta, RAR-gamma) by
9-cis-retinoic acid was also examined. 9-cis-retinoic acid was also
found to be a potent activator of these isoforms as well, as shown
in Table 1:
2 TABLE 1 EC.sub.50* (nM) Receptor All-trans-retinoic Acid
9-cis-retinoic Acid RAR-.alpha. 3861 .+-. 13 327 .+-. 30 RAR-.beta.
152 .+-. 12 95 .+-. 13 RAR-.gamma. 48 .+-. 8 61 .+-. 5 RXR-.alpha.
1174 .+-. 26 255 .+-. 17 RXR-.beta. 1841 .+-. 26 218 .+-. 17
RXR-.gamma. 1369 .+-. 26 254 .+-. 19 *Mean .+-. SEM
Example 4
9-cis Retinoic Acid Binds Directly to RXRs
[0169] The ability of 9-cis-retinoic acid to transactivate
RXR-alpha suggested testing to see whether 9-cis-retinoic acid was
also capable of binding directly to RXRs. RXR-alpha was expressed
in baculovirus and was shown to have biochemical properties that
were identical to the mammalian expressed protein. The baculovirus
expressed protein had a molecular weight of 51,000, reacted
specifically with RXR-alpha antibody and was capable of binding in
vitro to DNA sequences that have been previously shown to be
specific RXR response elements [i.e. CRBPII, see Mangelsdorf et
al., Cell 66:555 (1991); apolipoprotein AI gene, see Rottman et
al., Mol. Cell Biol. 11:3814 (1991)].
[0170] To characterize the ligand binding characteristics of
9-cis-retinoic acid to baculovirus-derived RXR, saturation binding
analysis was carried out (see FIG. 4). Radiolabelled 9-cis-retinoic
acid binds specifically to RXR-alpha in a saturable manner.
Scatchard analysis suggests a single high affinity binding site
with a Kd value of 11.7 nM (see FIG. 4b). Under identical binding
conditions [.sup.3H]-all-trans-ret- inoic acid did not bind to
RXR-alpha (see FIG. 4a). In addition, 9-cis-retinoic acid was also
capable of binding specifically to RAR-alpha as a high affinity
ligand. 9-cis-retinoic acid did not bind to mock baculovirus
extracts (i.e., control extracts from cells that do not express
RXRs).
[0171] Similarly, binding studies were also carried out with other
isoforms of RXR (i.e., RXR-beta, RXR-gamma), other isoforms of RAR
(i.e., RAR-beta, RAR-gamma), and cellular reinoic acid binding
protein (CRABP) with all-trans-retinoic acid and 9-cis-retinoic
acid. While all-trans-retinoic acid is known to bind to each of
these "receptors", 9-cis-retinoic acid was also found to bind to
the other isoforms of retinoid receptors (but not to the cellular
retinoic acid binding protein, CRABP), as shown in Table 2:
3 TABLE 2 Kd (nM) Receptor All-trans-retinoic Acid 9-cis-retinoic
Acid RAR-.alpha. 0.4 0.3 RAR-.beta. 0.4 0.2 RAR-.gamma. 0.2 0.8
RXR-.alpha. No binding 1.5 RXR-.beta. No binding 2.1 RXR-.gamma. No
binding 1.9 CRABP 20 >100
[0172] The properties of many members of the steroid hormone
receptor superfamily have been characterized and defined using DNA
cellulose chromatography [see, for example, Pike and Haussler,
Proc. Natl. Acad. Sci. USA 76:5485 (1979) and Pike et al. , J.
Biol. Chem. 258:1289 (1983)]. Receptors, such as the VDR, have been
shown in the presence of their cognate ligand to bind to
DNA-cellulose [see, for example, Allegretto et al., J. Biol. Chem.
262:1312 (1987)] with high affinity and the ligand-receptor complex
elutes with a salt gradient. A DNA-cellulose column profile of the
baculovirus expressed RXR that had been prelabeled with
[.sup.3H]-9-cis-retinoic acid is shown in FIG. 5. The two different
profiles represent 1) the total amount of [.sup.3H]-9-cis-retinoic
acid bound and 2) the level of binding that remains in the presence
of 200-fold excess of cold (i.e. non-labeled 9-cis-retinoic
acid).
[0173] There is a peak of radioactivity (marked in the Figure by an
arrow) that elutes off the DNA-cellulose column at 0.15 M KC1. This
elution profile is similar to that seen with RAR.alpha. in the
presence of [.sup.3H]-all-trans-retinoic acid. A 200 fold excess of
cold ligand (i.e. non-specific) is capable of competing greater
than 90% of the total radioactivity bound, demonstrating that the
radioactivity in the peak fractions is 9-cis-retinoic acid
specifically bound to RXR.
[0174] The radioactivity eluted off the column was extracted with
organic solvent and subjected to HPLC analysis.
[0175] Inspection of FIG. 5b makes it clear that the radioactivity
bound to RXR co-chromatographs with authentic 9-cis-retinoic acid.
This observation further confirms that 8 .sup.3H]-9-cis-retinoic
acid is the species bound to RXR.
[0176] To demonstrate that the protein contained in the peak
fractions is indeed RXR, these fractions (labelled 1-15 in FIG. 5a)
were subjected to immunoblot analysis using an RXR.alpha. specific
polyclonal antiserum (see FIG. 5a, top). All fractions containing
radioactivity display a distinct RXR.alpha. band at a M.sub.r of
51,000. When a similar experiment was conducted with a baculovirus
mock extract, no specific radioactivity was retained on the column.
Taken together, these data strongly suggest that 9-cis-retinoic
acid is capable of binding specifically to RXR.
[0177] Protein samples were resuspended in 2.times.sample buffer
[Laemelli, Nature Vol. 227:680 (1970)] and boiled for 5 minutes
prior to loading onto a 9% SDS polyacrylamide gel. After
electrophoretic separation the gels were electroblotted onto
nitrocellulose membranes (Scheicher and Schuell) for 8 hours at 30
volts using a Hoeffer electro-transfer apparatus. Membranes were
then incubated in 10% isopropanol, 10% acetic acid for 15 minutes,
washed 5 minutes in deionized H.sub.2O and 5 minutes in T-TBS
buffer (10 mM Tris pH 7.5, 150 mM NaCl and 0.5% Triton X-100). The
membranes were blocked in 5% nonfat milk in T-TBS for 1 hour. The
remainder of the protocol was adapted from the Amersham ECL
(Enhanced Chemiluminescence) Western blotting detection system kit.
The primary antibody was a rabbit polyclonal serum raised against a
synthetic peptide corresponding to amino acids 214-229 of
hRXR.alpha. [Kliewer et al., Proc. Natl. Acad. Sci. USA
89:1448-1452 (1992)]. The primary antiserum was diluted 1:5000 in
T-TBS. The secondary antibody (Donkey anti rabbit IgG conjugated to
horseradish peroxidase, Amersham) was used at a dilution of
1:2500.
Example 5
Effects of Topical Application of 9-cis-retinoic Acid (Compared
with All-trans-retinoic Acid) on Horn-filled Utriculus Size in the
Rhino Mouse
[0178] All-trans-retinoic acid is known to influence cell
differentiation and exert profound therapeutic benefits in the
treatment of keratinization disorders [Elias et al., Arch.
Dermatol. Vol. 117:160-180 (1981)]. Mezick et al. [see J. Invest.
Derm. Vol. 83:110-113 (1984)] demonstrated that topical treatment
of rhino mice (hr hr) with all-trans-retinoic acid could reduce
keratinized pilosebaceous structures (horn-filled utriculus). This
animal test model was used to evaluate the "antikeratinizing"
effects of 9-cis-retinoic acid. Results are summarized in Table
3:
4 TABLE 3 Pilosebaceous structure size (% red'n) Vehicle Control
178 .mu.m 9-cis-retinoic acid, 0.1% 52 .mu.m (-74%) 0.01% 72 .mu.m
(-64%) All-trans-retinoic acid, 0.1% 44 .mu.m (-78%) 0.01% 50 .mu.m
(-75%)
[0179] 9-cis-retinoic acid reduced the mean utriculi diameter after
14 days of topical application. These results demonstrate that
topical application of 9-cis-retinoic acid over a 14 day period can
reduce keratinized pilosebaceous structures (horn-filled utriculus)
in Rhino mouse skin. Reduction in the mean utriculi diameter by
9-cis-retinoic acid was comparable to that observed with
all-trans-retinoic acid.
Example 6
Effects of 9-cis-retinoic Acid (Compared with All-trans-retinoic
Acid) on Differentiation of HL60 Cells
[0180] Retinoids are known to differentiate human promyelocytic
leukemia cells. Differentiation of HL60 cells (a model system for
promyelocytic leukemia) can be assessed by Nitro Blue Tetrazolium
(NBT) dye reduction (superoxide anion generation) and by
measurement of up-regulation of the gene encoding the .beta.
subunit of the leukocyte adherence receptor, CD18 (J.B.C. vol. 263
No. 27, pp. 13863-13867).
[0181] The EC-50 for 9-cis-retinoic acid-mediated differentiation,
as determined by NBT after 6 days treatment, was 0.2 .mu.M compared
to 2 .mu.M for all-trans-retinoic acid. Maximal effects
(efficacies) were comparable, and CD18 was up-regulated by both
ligands. Alpha-interferon potentiated both all-trans-retinoic acid
and 9-cis-retinoic acid-mediated differentiation, as determined by
NBT.
[0182] HL60R cells have been shown to be resistant to
differentiation by all-trans-retinoic acid, probably related to a
mutation in the retinoic acid receptor-alpha gene. This cell line
was found to be resistant to differentiation (NBT) by both
all-trans-retinoic acid and 9-cis-retinoic acid at concentrations
up to 10 .mu.M.
[0183] 9-cis-retinoic acid effects differentiation of HL60 cells as
evidenced by NBT and up-regulation of CD18. Compared with all-trans
retinoic acid, 9-cis retinoic acid is more potent with similar
efficacy.
Example 7
Effects of 9-cis-retinoic Acid (Compared with All-trans-retinoic
Acid) on in Vitro Proliferation of Melanoma Cell Lines
[0184] All-trans-retinoic acid and several synthetic analogs
(retinoids) have been shown to prevent the development of benign
and malignant, chemically induced epithelial tumors in vivo [Sporn
et al., Fed. Proc. Vol. 35:1332-1338 (1976)]. Lotan et al. (J.
Natl. Cancer, Vol. 60:1035-1041, 1978) found that
all-trans-retinoic acid inhibited the growth of several tumor cell
lines in vitro. In view of these earlier findings, it was of
interest to evaluate the growth inhibitory properties of
9-cis-retinoic acid.
[0185] 9-cis-retinoic acid inhibited the growth of the murine
melanoma cell line Clone M3 in a concentration dependent manner, as
follows:
5 % Growth inhibition (Conc added) 1 .mu.M 0.01 .mu.M
9-cis-retinoic acid -85% -49% all-trans-retinoic acid -94% -48%
[0186] Similarly, 9-cis retinoic acid inhibited the growth of the
human primary metastatic melanoma cell line c81-46c in a
concentration dependent manner.
6 % Growth inhibition (Conc added) 1 .mu.M 0.01 .mu.M
9-cis-retinoic acid -45% -28% all-trans-retinoic acid -44% -17%
[0187] In summary, 9-cis-retinoic acid has been shown to inhibit
the in vitro proliferation of murine melanoma cell line Clone M3
and human metastatic melanoma cell line c81-46c in a concentration
dependent manner. 9-cis-retinoic acid has an equal inhibitory
effect on these cells as compared to all-trans-retinoic acid.
Example 8
Effects of 9-cis-retinoic Acid (Compared with All-trans-retinoic
Acid) on Differentiation of F9 Cells
[0188] Retinoids are known to differentiate mouse teratocarcinoma
cells (F9). Differentiation of F9 cells is specifically associated
with irreversible changes in morphology and induction of the
biochemical marker alkaline phosphatase (ALP) and tissue
plasminogen activator (tPA) (Biochem. J. Vol. 274:673-678).
[0189] Both all-trans-retinoic acid and 9-cis-retinoic acid induced
differentiation of F9 cells into partial endoderm-like cells as
indicated by irreversible changes in cellular morphology.
All-trans-retinoic acid was 40 times more potent than
9-cis-retinoic acid in inducing ALP, maximal responses were
similar.
[0190] The response of tissue plasminogen activator factor was less
for 9-cis-retinoic acid than for all-trans-retinoic acid. At a
concentration of 1 .mu.M of 9-cis-retinoic acid (or
all-trans-retinoic acid), increased cellular activities of tPA by
0.48.+-.0.05 and 0.80.+-.0.08, respectively were observed. This
effect was concentration-dependent.
[0191] In summary, 9-cis-retinoic acid promoted differentiation of
F9 cells as evidenced by changes in morphology and marker enzyme
activities. Compared with all-trans-retinoic acid, 9-cis-retinoic
acid was less potent with regard to both enzyme markers. Efficacy
was comparable with ALP but indeterminate for tPA.
Example 9
Effects of 9-cis-retinoic Acid (Compared with All-trans-retinoic
Acid) on Differentiation of Keratinocytes
[0192] Retinoids are known to inhibit squamous cell differentiation
of cultured normal human epidermal keratinocytes (NHEK534 cell
line), as judged by morphological alterations and inhibition of
induction of transglutaminase (Type I) (J. Biol. Chem. Vol.
261:15097, 1986; Lab. Invest. Vol. 56:654, 1987).
[0193] Both all-trans-retinoic acid and 9-cis-retinoic acid
inhibited squamous cell differentiation in a concentration
dependent manner as judged by morphological changes and by
transglutaminase activity. The EC50s for inhibition of
differentiation by all-trans-retinoic acid and 9-cis-retinoic acid
were identical (20.+-.2.8 nM). 9-cis retinoic acid and
all-trans-retinoic acid EC50s and potencies were nearly identical
for effects on transglutaminase activities.
[0194] In summary, like all-trans-retinoic acid, 9-cis-retinoic
acid inhibits morphological differentiation of NHEK534 cells and
induction of transglutaminase activity.
Example 10
Synthesis of 9-phenyl-9-cis-retinoic Acid
[0195] To a solution of 44 mg (0.10 mmole) of the following
phosphonate reagent: 13
[0196] in THF (0.5 ml) at room temperature was added NaH (60% in
oil, 5 mg; 0.13 mmole) and the mixture stirred at that temperature
for 10 minutes. To this, 26 mg (0.08 mmole) of the aldehyde: 14
[0197] in THF (0.5 ml) was added at room temperature and the
mixture allowed to stir for 30 minutes. Aqueous workup in the usual
manner (NH.sub.4Cl (aq), H.sub.2O, brine, MgSO.sub.4) gave a
mixture of 9-phenyl-9-cis ester and 9-phenyl-9,13-dicis ester (30
mg, 92%) (the calculated ratio of 9-cis:9,13-dicis=4:1).
[0198] ethyl ester of 9-phenyl-9-cis-retinoic acid 15
[0199] ethyl ester of 9-phenyl-9.13-dicis-retinoic acid 16
[0200] To a mixture of 9-cis and 9,13-dicis ester (20 mg, 0.05
mmole) in methanol (0.7 ml) and H.sub.2O (0.7 ml) at 25.degree. C.
was added KOH (14.3 mg, 0.25 mmole). Consequently, the mixture was
heated to 70.degree. C. for 2 hours. The reaction was then cooled
down to 0.degree. C., diluted with 10 ml of diethyl ether), and
acidified with HCl (0.12M in HCl, 2.17 ml). Aqueous workup in the
usual manner (H.sub.2O, brine, MgSO.sub.4) gave a mixture of 9-cis
and 9,13-dicis acid. Flash column chromatography (silica, 13% ethyl
acetate in benzene) gave pure 9-phenyl-9-cis retinoic acid (14.5
mg. 100%).
[0201] The .sup.1HNMR spectrum of 9-phenyl-9-cis retinoic acid is
as follows:
[0202] .sup.1HNMR (400 mHz), CDCl.sub.3) .delta.7.4-7.3 (m, 5H,
aromatic), 7.20 (dd, J=16, 12 Hz, 1H, olefinic), 6.60 (d, J=16 Hz,
1 H, olefinic), 6.38 (d, J=16 Hz, 1 H, olefinic), 6.25 (d, J =12
Hz, 1H, olefinic), 6.15 (d, J=16 Hz, 1 H, olefinic), 5.80 (s. 1H,
olefinic), 2.48 (s. 3H, CH.sub.3l, 2.05 (t. J=5Hz, 2H, CH.sub.2),
1.79 (s, 3H, CH.sub.3), 1.70-1.40 (m, 4H, CH.sub.2--CH.sub.2), 1.00
(s, 6H, 2.times.CH.sub.3).
[0203] 9-phenyl-9-cis RA : TLC Rf 0.23 (13% ethyl acetate in
Benzene)
Example 11
Synthesis of 4-hydroxy-9-cis-retinoic Acid
[0204] To a solution of 9-cis-retinoic acid (51 mg, 0.17 mmole) in
1.4-dioxane (2 ml) was added SeO.sub.2 (19 mg, 0.17 mmole) at
60.degree. C. The solution was allowed to stir at that temperature
for 3 hours. The reaction mixture was then filtered through a
silica bed. The filtrate was concentrated and the residue subjected
to flash column chromatography (silica, 75% ether in petroleum
ether) to afford 4-OH-9-cis-retinoic acid (21 mg., 40% yield),
which is characterized as follows: Oil; TLC Rf=0.25 (silica, 75%
ether in petroleum ether); .sup.1HNMR (400 MHz, CDCl.sub.3)
.delta.7.08 (dd, J=16, 12 Hz, 1H, olefinic); 6.64 (d, J=16 Hz, 1H,
olefinic), 6.21 (d, J=16 HZ, 1H, olefinic), 6.20 (d, J=16 Hz, 1H,
olefinic); 6.04 (d, J=12 Hz, olefinic), 5.79 (s, 1H, olefinic),
4.02 (t, J=5 Hz, 1H, CH--O), 2.18 (s, 3H, CH.sub.3), 2.02 (s, 3H,
CH.sub.3), 1.82 (s, 3H, CH.sub.3), 2.0-1.6 (m, 4H,
CH.sub.2--CH.sub.2), 1.05, 1.03 (2.times.s, 2.times.3H,
2.times.CH.sub.3).
Example 12
Synthesis of 4-keto-9-cis-retinoic Acid
[0205] To a solution of 4-hydroxy-9-cis-retinoic acid (16 mg, 0.05
mmole) in CH.sub.2Cl.sub.2 (1.5 ml) was added Dess-Martin reagent
[see Dess and Martin in J. org. Chem. 48:4155 (1983)] (42 mg, 0.1
mmole) in one portion at 25.degree. C. After stirring for 5
minutes, the mixture was diluted with 10 ml of ether and to this
was added saturated aqueous NaHCO.sub.3 (5 ml) containing
Na.sub.2SO.sub.3 (55 mg). The mixture was stirred for 20 minutes to
dissolve the solid and the layers separated. The ether layer was
washed with H.sub.2O (2.times.5 ml), brine (5 ml) and dried
(MgSO.sub.4). The solvent was recovered under reduced pressure and
residue was subjected to flash column chromatography (silica, 60%
ether in Hexane) to give 4-keto-9-cis-retinoic acid (14 mg. 90%),
characterized as follows: TLC rf=0.6 (silica, 80% ether in hexane);
.sup.1HNMR (400 mHz, CDCl.sub.3) .delta.7.05 (dd, J=16, 12 Hz, 1H,
olefinic), 6.82 (d, J=16 Hz, 1 H, olefinic), 6.32 (d, J=16 Hz, 1H,
olefinic), 6.30 (d, J=16 Hz, 1H, olefinic), 6.20 (d, J=12 Hz, 1H,
olefinic), 5.80 (s, 1H, olefinic), 2.5 (t, J=7 Hz, 2H,
CH.sub.2--CO), 2.31 (s, 3H, CH.sub.3), 2.01 (s, 3H, CH.sub.3), 1.9
(s, 3H, CH.sub.3), 1.89 (m, 2H, CH.sub.2), 1.20 (s, 6H, 2
.times.CH.sub.3).
Example 13
In Vitro Evaluation of 9-phenyl-9cis-retinoic Acid,
4-hydroxy-9-cis-retinoic Acid and 4-keto-9-cis-retinoic Acid
[0206] The potency and efficacy of the compounds described in
Examples 10, 11 and 12 were determined (as described in Example
1--under the heading "Cotransfection Assay in CV-1 Cells". The
results are presented in Table 4:
7 TABLE 4 9-phenyl-9-cis- 4-hydroxy-9-cis- 4-keto-9-cis-
9-cis-retinoic acid retinoic acid retinoic acid retinoic acid
Potency Potency Potency Potency Receptor (nM) Efficacy (nM)
Efficacy (nM) Efficacy (nM) Efficacy RXR.alpha. 88 170% 210 76%
1700 161% 520 104% RXR.beta. 61 106% 44 88% 650 143% 1300 105%
RXR.gamma. 360 137% 290 77% 1700 115% 1100 133% RAR.alpha. 99 94%
>10,000 <2% 380 65% 200 50% RAR.beta. 22 97% 880 39% 160 71%
26 67% RAR.gamma. 43 108% 250 59% 180 81% 55 107%
[0207] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
Sequence CWU 1
1
* * * * *