U.S. patent application number 15/562776 was filed with the patent office on 2018-09-27 for compositions and methods for increasing, or preventing or reversing loss of, skin pigmentation in a mammalian subject.
The applicant listed for this patent is THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Rosa Ellen COOKSON, Chris NATALE, Todd RIDKY, Jeffrey WINKLER.
Application Number | 20180273482 15/562776 |
Document ID | / |
Family ID | 57072935 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180273482 |
Kind Code |
A1 |
RIDKY; Todd ; et
al. |
September 27, 2018 |
COMPOSITIONS AND METHODS FOR INCREASING, OR PREVENTING OR REVERSING
LOSS OF, SKIN PIGMENTATION IN A MAMMALIAN SUBJECT
Abstract
The present invention includes compounds, compositions
comprising the same, and methods using the compounds and/or
compositions therein, for modulating skin pigmentation in a
mammalian subject. In certain embodiments, the compounds and/or
compositions treat or prevent skin disorders or diseases associated
with hypopigmentation in the subject. In other embodiments, the
compounds and/or compositions comprise an estrogen derivative or
analog that binds to the non-canonical sex steroid hormone receptor
GPER and does not bind to the canonical nuclear estrogen receptor
(ER).
Inventors: |
RIDKY; Todd; (Bryn Mawr,
PA) ; NATALE; Chris; (Philadelphia, PA) ;
WINKLER; Jeffrey; (Wynnewood, PA) ; COOKSON; Rosa
Ellen; (Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA |
Philadelphia |
PA |
US |
|
|
Family ID: |
57072935 |
Appl. No.: |
15/562776 |
Filed: |
April 6, 2016 |
PCT Filed: |
April 6, 2016 |
PCT NO: |
PCT/US2016/026199 |
371 Date: |
September 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62143597 |
Apr 6, 2015 |
|
|
|
62216792 |
Sep 10, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 17/06 20180101;
A61P 17/14 20180101; A61K 8/49 20130101; C07D 405/04 20130101; A61P
17/00 20180101; A61P 17/18 20180101; C07D 221/16 20130101; C07D
491/048 20130101; A61Q 17/04 20130101; A61P 17/16 20180101; A61Q
19/02 20130101 |
International
Class: |
C07D 221/16 20060101
C07D221/16; C07D 405/04 20060101 C07D405/04; C07D 491/048 20060101
C07D491/048; A61K 8/49 20060101 A61K008/49; A61Q 19/02 20060101
A61Q019/02 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
CA163566 awarded by National Institutes of Health/National Cancer
Institute, and under CA174523 awarded by National Institutes of
Health SPORE. The government has certain rights in the invention.
Claims
1. A compound, or a salt, solvate, tautomer, enantiomer or
diastereoisomer thereof, selected from the group consisting of: a
molecule of formula (I): ##STR00045## wherein in (I): R.sub.1 is
selected from the group consisting of .dbd.O, .dbd.N--OH,
.dbd.N--NHC(.dbd.O)(p-methoxy phenyl),
.dbd.N--NHC(.dbd.O)CH(OMe)phenyl, and
.dbd.N--NH(5-iodo-pyrid-2-yl); R.sub.2 is selected from the group
consisting of C.sub.1-C.sub.4 alkyl and C.sub.1-C.sub.4 haloalkyl;
bond a is a single or double bond, such that: if bond a is a double
bond, R.sub.3 and R.sub.4 are H, and if bond a is a single bond,
R.sub.3 is selected from the group consisting of H, --OH, --OAc,
and halo; R.sub.4 is selected from the group consisting of H, --OH,
--OAc, and --S(o-nitrophenyl); or R.sub.3 and R.sub.4 combine to
form a diradical selected from the group consisting of
--CH.sub.2--, --OCH.sub.2O--, --OCH(CH.sub.3)O--, and
--OC(CH.sub.3).sub.2O--; R.sub.5 is selected from the group
consisting of H, benzyl, C.sub.1-C.sub.4 alkyl, and acetyl; R.sub.6
is selected from the group consisting of H, halo, --NO.sub.2,
C.sub.1-C.sub.4 alkyl, --C.ident.CH,
--C.ident.C--Si(CH.sub.3).sub.3 (or --C.ident.C-TMS), --O-benzyl,
--OH, --OAc, C.sub.1-C.sub.4 alkoxy, --COOH, and
--COO(C.sub.1-C.sub.4 alkyl); R.sub.7 is selected from the group
consisting of H, halo, --NO.sub.2, C.sub.1-C.sub.4 alkyl, --OH,
--OAc, and C.sub.1-C.sub.4 alkoxy; R.sub.8 is selected from the
group consisting of H, halo, --NO.sub.2, C.sub.1-C.sub.4 alkyl,
--O-benzyl, --N(R)(R), --SR, --COOH, --COO(C.sub.1-C.sub.4 alkyl),
--OH, --OAc, C.sub.1-C.sub.4 alkoxy, 3-thietyl-methoxy,
--SO.sub.2(morpholino), and --OCH.sub.2CH.dbd.CH.sub.2, wherein
each occurrence of R is independently selected from the group
consisting of H and C.sub.1-C.sub.4 alkyl; R.sub.9 is selected from
the group consisting of H, halo, --NO.sub.2, C.sub.1-C.sub.4 alkyl,
--OH, --OAc, and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and R.sub.9
combine to form a diradical selected from the group consisting of
--OCH.sub.2O--, --OCH(CH.sub.3)O--, --OC(CH.sub.3).sub.2O--,
--O(CH.sub.2).sub.2O--, --O--CH.dbd.CH--, and --CH.dbd.CH--O--;
R.sub.10 is selected from the group consisting of H,
C.sub.1-C.sub.4 alkyl, and halo; wherein each occurrence of benzyl
is independently optionally substituted with at least one group
selected from the group consisting of C.sub.1-C.sub.4 alkyl, --OH,
C.sub.1-C.sub.4 alkoxy, halo, and --NO.sub.2; and a molecule of
formula (II): ##STR00046## wherein in (II): R.sub.1 is selected
from the group consisting of .dbd.O and .dbd.N--OH; R.sub.2 is
C.sub.1-C.sub.4 alkyl; R.sub.5 is selected from the group
consisting of H, benzyl and C.sub.1-C.sub.4 alkyl; R.sub.8 and
R.sub.9 are independently selected from the group consisting of H
and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and R.sub.9 combine to form
a diradical selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--.
2. The compound of claim 1, which is not selected from the group
consisting of G-1 (or G1;
rel-1-[4-(6-bromo-1,3-benzodioxol-5-yl)-3aR,4S,5,9bS-tetrahydro-3H-cyclop-
enta[c]quinolin-8-yl]-ethanone): ##STR00047## and A1-A107.
3. (canceled)
4. The compound of claim 1, which is at least one selected from the
group consisting of A1-A107.
5. The compound of claim 1, or a salt, solvate, tautomer,
enantiomer or diastereoisomer thereof, which is selected from the
group consisting of: a molecule of Formula (I-1): ##STR00048##
wherein in (I-1): R.sub.1 is selected from the group consisting of
.dbd.O and .dbd.N--OH; R.sub.2 is C.sub.1-C.sub.4 alkyl; bond a is
a single or double bond, such that: if bond a is a double bond,
R.sub.3 and R.sub.4 are H, and if bond a is a single bond, R.sub.3
and R.sub.4 are independently selected from the group consisting of
H and --OH, or R.sub.3 and R.sub.4 combine to form a diradical
selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--; R.sub.5 is selected
from the group consisting of H, benzyl and C.sub.1-C.sub.4 alkyl;
R.sub.6 is selected from the group consisting of H and halo;
R.sub.8 and R.sub.9 are independently selected from the group
consisting of H and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and R.sub.9
combine to form a diradical selected from the group consisting of
--OCH.sub.2O--, --OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--; and
a molecule of formula (II): ##STR00049## wherein in (II): R.sub.1
is selected from the group consisting of .dbd.O and .dbd.N--OH;
R.sub.2 is C.sub.1-C.sub.4 alkyl; R.sub.5 is selected from the
group consisting of H, benzyl and C.sub.1-C.sub.4 alkyl; R.sub.8
and R.sub.9 are independently selected from the group consisting of
H and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and R.sub.9 combine to
form a diradical selected from the group consisting of
--OCH.sub.2O--, --OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--.
6-7. (canceled)
8. The compound of claim 5, wherein in (I), (I-1) or (II) at least
one applies: R.sub.2 is selected from the group consisting of
methyl, trifluoromethyl, ethyl, 1-propyl, and 2-propyl; R.sub.5 is
selected from the group consisting of H, benzyl, methyl, ethyl,
1-propyl and 2-propyl.
9. The compound of claim 5, wherein in (I) or (I-1) (a) bond a is a
double bond, and R.sub.3 and R.sub.4 are H, or (b) bond a is a
single bond, and R.sub.3 and R.sub.4 are independently selected
from the group consisting of H and --OH, or (c) bond a is a single
bond, and R.sub.3 and R.sub.4 combine to form
--OC(CH.sub.3).sub.2O--.
10-12. (canceled)
13. The compound of claim 1, wherein in (I) or (I-1) R.sub.3 and
R.sub.4 are --OH and cis to each other.
14. (canceled)
15. The compound of claim 5, wherein in (I), (I-1) or (II) R.sub.5
is selected from the group consisting of H, benzyl, methyl, ethyl,
1-propyl and 2-propyl.
16. The compound of claim 1, wherein R.sub.6 is selected from the
group consisting of H, F, Cl, Br and I.
17. The compound of claim 5, wherein in (I), (I-1) or (II) R.sub.8
and R.sub.9 are independently selected from the group consisting of
H, methoxy, ethoxy, 1-propoxy and 2-propoxy, or R.sub.8 and R.sub.9
combine to form a diradical selected from the group consisting of
--OCH.sub.2O--, --OCH(CH.sub.1)O-- and --OC(CH.sub.3).sub.2O--.
18. (canceled)
19. The compound of claim 1, wherein the molecule of formula (I) is
at least one selected from the group consisting of: ##STR00050##
##STR00051##
20. The compound of claim 1, which is at least one selected from
the group consisting of: CMPD1
(rel-1-((3aS,4R,9bR)-4-(benzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-
-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD2
(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-5-methyl-3a,4,5,-
9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD3
(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-2,3,3a,4,5,9b-he-
xahydro-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD4
(rel-1-((3aS,4R,9bR)-5-benzyl-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,-
9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD5
(rel-1-((3aS,4R,9bR)-4-(2-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta-
[c]quinolin-8-yl)ethan-1-one); CMPD6
(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrah-
ydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one oxime); CMPD7
(rel-1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-3a,4,5,9b-tetrahydro-
-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD8
(rel-1-((3aS,4R,9bR)-4-(6-chlorobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetra-
hydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD9
(rel-1-((6R,6aS,7aS,10aR,10bR)-6-(6-bromobenzo[d][1,3]dioxol-5-yl)-9,9-di-
methyl-6,6a,7,7a,10a,10b-hexahydro-5H-[1,3]dioxolo[4',5':3,4]cyclopenta[1,-
2-c]quinolin-2-yl)ethan-1-one); CMPD10
(rel-1-((1R,2S,3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-1,2-dihydr-
oxy-2,3,3a,4,5,9b-hexahydro-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one);
CMPD11
(rel-1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-2,3,3a,4,5,9b-
-hexahydro-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one); and CMPD12
(rel-1-((4S,5aS,6R,11aR)-4,5,5a,6,11,11a-hexahydro-4,6-methano[1,3]dioxol-
o[4',5':5,6]benzo[1,2-c]acridin-8-yl)ethan-1-one).
21. A pharmaceutical composition comprising at least one
pharmaceutically acceptable carrier and at least one compound, or a
salt, solvate, tautomer, enantiomer or diastereoisomer thereof,
selected from the group consisting of: a molecule of formula (I):
##STR00052## wherein in (I): R.sub.1 is selected from the group
consisting of .dbd.O, .dbd.N--OH, .dbd.N--NHC(.dbd.O)(p-methoxy
phenyl), .dbd.N--NHC(.dbd.O)CH(OMe)phenyl, and
.dbd.N--NH(5-iodo-pyrid-2-yl); R.sub.2 is selected from the group
consisting of C.sub.1-C.sub.4 alkyl and C.sub.1-C.sub.4 haloalkyl;
bond a is a single or double bond, such that: if bond a is a double
bond, R.sub.3 and R.sub.4 are H, and if bond a is a single bond,
R.sub.3 is selected from the group consisting of H, --OH, --OAc,
and halo; R.sub.4 is selected from the group consisting of H, --OH,
--OAc, and --S(o-nitrophenyl); or R.sub.3 and R.sub.4 combine to
form a diradical selected from the group consisting of
--CH.sub.2--, --OCH.sub.2O--, --OCH(CH.sub.3)O--, and
--OC(CH.sub.3).sub.2O--; R.sub.5 is selected from the group
consisting of H, benzyl, C.sub.1-C.sub.4 alkyl, and acetyl; R.sub.6
is selected from the group consisting of H, halo, --NO.sub.2,
C.sub.1-C.sub.4 alkyl, --C.ident.CH,
--C.ident.C--Si(CH.sub.3).sub.3 (or --C.ident.C-TMS), --O-benzyl,
--OH, --OAc, C.sub.1-C.sub.4 alkoxy, --COOH, and
--COO(C.sub.1-C.sub.4 alkyl); R.sub.7 is selected from the group
consisting of H, halo, --NO.sub.2, C.sub.1-C.sub.4 alkyl, --OH,
--OAc, and C.sub.1-C.sub.4 alkoxy; R.sub.8 is selected from the
group consisting of H, halo, --NO.sub.2, C.sub.1-C.sub.4 alkyl,
--O-benzyl, --N(R)(R), --SR, --COOH, --COO(C.sub.1-C.sub.4 alkyl),
--OH, --OAc, C.sub.1-C.sub.4 alkoxy, 3-thietyl-methoxy,
--SO.sub.2(morpholino), and --OCH.sub.2CH.dbd.CH.sub.2, wherein
each occurrence of R is independently selected from the group
consisting of H and C.sub.1-C.sub.4 alkyl; R.sub.9 is selected from
the group consisting of H, halo, --NO.sub.2, C.sub.1-C.sub.4 alkyl,
--OH, --OAc, and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and R.sub.9
combine to form a diradical selected from the group consisting of
--OCH.sub.2O--, --OCH(CH.sub.3)O--, --OC(CH.sub.3).sub.2O--,
--O(CH.sub.2).sub.2O--, --O--CH.dbd.CH--, and --CH.dbd.CH--O--;
R.sub.10 is selected from the group consisting of H,
C.sub.1-C.sub.4 alkyl, and halo; wherein each occurrence of benzyl
is independently optionally substituted with at least one group
selected from the group consisting of C.sub.1-C.sub.4 alkyl, --OH,
C.sub.1-C.sub.4 alkoxy, halo, and --NO.sub.2; and a molecule of
formula (II): ##STR00053## wherein in (II): R.sub.1 is selected
from the group consisting of .dbd.O and .dbd.N--OH; R.sub.2 is
C.sub.1-C.sub.4 alkyl; R.sub.5 is selected from the group
consisting of H, benzyl and C.sub.1-C.sub.4 alkyl; R.sub.8 and
R.sub.9 are independently selected from the group consisting of H
and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and R.sub.9 combine to form
a diradical selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--.
22. The pharmaceutical composition of claim 21, wherein at least
one compound, or a salt, solvate, tautomer, enantiomer or
diastereoisomer thereof, is selected from the group consisting of:
a molecule of Formula (I-1): ##STR00054## wherein in (I-1): R.sub.1
is selected from the group consisting of .dbd.O and .dbd.N--OH;
R.sub.2 is C.sub.1-C.sub.4 alkyl; bond a is a single or double
bond, such that: if bond a is a double bond, R.sub.3 and R.sub.4
are H, and if bond a is a single bond, R.sub.3 and R.sub.4 are
independently selected from the group consisting of H and --OH, or
R.sub.3 and R.sub.4 combine to form a diradical selected from the
group consisting of --OCH.sub.2O--, --OCH(CH.sub.3)O-- and
--OC(CH.sub.3).sub.2O--; R.sub.5 is selected from the group
consisting of H, benzyl and C.sub.1-C.sub.4 alkyl; R.sub.6 is
selected from the group consisting of H and halo; R.sub.8 and
R.sub.9 are independently selected from the group consisting of H
and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and R.sub.9 combine to form
a diradical selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--; and a molecule of
formula (II): ##STR00055## wherein in (II): R.sub.1 is selected
from the group consisting of .dbd.O and .dbd.N--OH; R.sub.2 is
C.sub.1-C.sub.4 alkyl; R.sub.5 is selected from the group
consisting of H, benzyl and C.sub.1-C.sub.4 alkyl; R.sub.7 and
R.sub.8 are independently selected from the group consisting of H
and C.sub.1-C.sub.4 alkoxy, or R.sub.7 and R.sub.8 combine to form
a diradical selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--.
23. The pharmaceutical composition of claim 21, which is formulated
for topical or transdermal administration.
24. The pharmaceutical composition of claim 21, wherein the
compound does not comprise G-1.
25. The pharmaceutical composition of claim 21, wherein the
compound is at least one selected from the group consisting of G-1,
CMPD1-CMPD12, and A1-A107.
26-27. (canceled)
28. The pharmaceutical composition of claim 21, further comprising
at least one selected from the group consisting of a sun-blocking
agent and a sunscreen lotion.
29. (canceled)
30. A method of increasing, or preventing or reversing loss of,
skin pigmentation in a mammalian subject, the method comprising
administering to the subject a therapeutically effective amount of
a compound which is a GPER agonist and does not bind to a canonical
nuclear estrogen receptor.
31. (canceled)
32. The method of claim 30, wherein the canonical nuclear estrogen
receptor comprises at least one selected from the group consisting
of ER.alpha. and ER.beta..
33. The method of claim 30, wherein the compound is a GPER1 or
GPR30 agonist.
34. The method of claim 30, wherein the subject is suffering from
at least one condition selected from the group consisting of
pigmentary changes associated with oral contraceptive use,
pregnancy, and endogenous estrogens in females; solar lentigo;
acne; eczema; chemical, sun, and thermal burn scars; and lupus.
35. The method of claim 30, wherein the compound is administered
topically or transdermally to the subject.
36. The method of claim 35, wherein the compound is formulated as a
pharmaceutical composition for topical or transdermal
administration.
37. The method of claim 36, wherein the pharmaceutical composition
further comprises at least one selected from the group consisting
of a sun-blocking agent and a sunscreen lotion.
38. (canceled)
39. The method of claim 36, wherein at least one applies: the
pharmaceutical composition is essentially free of a skin bleaching
agent; the subject is not further submitted to UV radiation as part
of the treatment to increase, or prevent or reverse loss of, skin
pigmentation.
40. (canceled)
41. The method of claim 30, wherein the compound, or a salt,
solvate, tautomer, enantiomer or diastereoisomer thereof, is at
least one selected from the group consisting of: a molecule of
formula (I): ##STR00056## wherein in (I): R.sub.1 is selected from
the group consisting of .dbd.O, .dbd.N--OH,
.dbd.N--NHC(.dbd.O)(p-methoxy phenyl),
.dbd.N--NHC(.dbd.O)CH(OMe)phenyl, and
.dbd.N--NH(5-iodo-pyrid-2-yl); R.sub.2 is selected from the group
consisting of C.sub.1-C.sub.4 alkyl and C.sub.1-C.sub.4 haloalkyl;
bond a is a single or double bond, such that: if bond a is a double
bond, R.sub.3 and R.sub.4 are H, and if bond a is a single bond,
R.sub.3 is selected from the group consisting of H, --OH, --OAc,
and halo; R.sub.4 is selected from the group consisting of H, --OH,
--OAc, and --S(o-nitrophenyl); or R.sub.3 and R.sub.4 combine to
form a diradical selected from the group consisting of
--CH.sub.2--, --OCH.sub.2O--, --OCH(CH.sub.3)O--, and
--OC(CH.sub.3).sub.2O--; R.sub.5 is selected from the group
consisting of H, benzyl, C.sub.1-C.sub.4 alkyl, and acetyl; R.sub.6
is selected from the group consisting of H, halo, --NO.sub.2,
C.sub.1-C.sub.4 alkyl, --C.ident.CH,
--C.ident.C--Si(CH.sub.3).sub.3 (or --C.ident.C-TMS), --O-benzyl,
--OH, --OAc, C.sub.1-C.sub.4 alkoxy, --COOH, and
--COO(C.sub.1-C.sub.4 alkyl); R.sub.7 is selected from the group
consisting of H, halo, --NO.sub.2, C.sub.1-C.sub.4 alkyl, --OH,
--OAc, and C.sub.1-C.sub.4 alkoxy; R.sub.8 is selected from the
group consisting of H, halo, --NO.sub.2, C.sub.1-C.sub.4 alkyl,
--O-benzyl, --N(R)(R), --SR, --COOH, --COO(C.sub.1-C.sub.4 alkyl),
--OH, --OAc, C.sub.1-C.sub.4 alkoxy, 3-thietyl-methoxy,
--SO.sub.2(morpholino), and --OCH.sub.2CH.dbd.CH.sub.2, wherein
each occurrence of R is independently selected from the group
consisting of H and C.sub.1-C.sub.4 alkyl; R.sub.9 is selected from
the group consisting of H, halo, --NO.sub.2, C.sub.1-C.sub.4 alkyl,
--OH, --OAc, and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and R.sub.9
combine to form a diradical selected from the group consisting of
--OCH.sub.2O--, --OCH(CH.sub.3)O--, --OC(CH.sub.3).sub.2O--,
--O(CH.sub.2).sub.2O--, --O--CH.dbd.CH--, and --CH.dbd.CH--O--;
R.sub.10 is selected from the group consisting of H,
C.sub.1-C.sub.4 alkyl, and halo; wherein each occurrence of benzyl
is independently optionally substituted with at least one group
selected from the group consisting of C.sub.1-C.sub.4 alkyl, --OH,
C.sub.1-C.sub.4 alkoxy, halo, and --NO.sub.2; and a molecule of
formula (II): ##STR00057## wherein in (II): R.sub.1 is selected
from the group consisting of .dbd.O and .dbd.N--OH; R.sub.2 is
C.sub.1-C.sub.4 alkyl; R.sub.5 is selected from the group
consisting of H, benzyl and C.sub.1-C.sub.4 alkyl; R.sub.8 and
R.sub.9 are independently selected from the group consisting of H
and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and R.sub.9 combine to form
a diradical selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--.
42. The method of claim 41, wherein the compound, or a salt,
solvate, tautomer, enantiomer or diastereoisomer thereof, is at
least one selected from the group consisting of: a molecule of
Formula (I-1): ##STR00058## wherein in (I-1): R.sub.1 is selected
from the group consisting of .dbd.O and .dbd.N--OH; R.sub.2 is
C.sub.1-C.sub.4 alkyl; bond a is a single or double bond, such
that: if bond a is a double bond, R.sub.3 and R.sub.4 are H, and if
bond a is a single bond, R.sub.3 and R.sub.4 are independently
selected from the group consisting of H and --OH, or R.sub.3 and
R.sub.4 combine to form a diradical selected from the group
consisting of --OCH.sub.2O--, --OCH(CH.sub.3)O-- and
--OC(CH.sub.3).sub.2O--; R.sub.5 is selected from the group
consisting of H, benzyl and C.sub.1-C.sub.4 alkyl; R.sub.6 is
selected from the group consisting of H and halo; R.sub.8 and
R.sub.9 are independently selected from the group consisting of H
and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and R.sub.9 combine to form
a diradical selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--; and a molecule of
formula (II): ##STR00059## wherein in (II): R.sub.1 is selected
from the group consisting of .dbd.O and .dbd.N--OH; R.sub.2 is
C.sub.1-C.sub.4 alkyl; R.sub.5 is selected from the group
consisting of H, benzyl and C.sub.1-C.sub.4 alkyl; R.sub.7 and
R.sub.8 are independently selected from the group consisting of H
and C.sub.1-C.sub.4 alkoxy, or R.sub.7 and R.sub.8 combine to form
a diradical selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--.
43. The method of claim 41, wherein (I) is at least one selected
from the group consisting of: ##STR00060## ##STR00061##
44. The method of claim 41, wherein the compound is at least one
selected from the group consisting of G1, A1-A107 and
CMPD1-CMPD12.
45. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn. 119(e) to U.S. Provisional Applications No. 62/143,597,
filed Apr. 6, 2015, and No. 62/216,792, filed Sep. 10, 2015, all of
which applications are incorporated herein by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0003] Human skin color ranges in variety from the darkest brown to
the lightest pinkish-white hues. Skin pigmentation in humans
evolved primarily to regulate the amount of ultraviolet (UV)
radiation penetrating the skin, thus controlling the radiation's
biochemical effects. The most important substance that affects the
actual skin color of humans is the pigment melanin.
[0004] Melanin controls the amount of UV radiation from the sun
that penetrates the skin by absorption. While UV radiation can
assist in the production of vitamin D, excessive exposure to UV can
damage health. Melanin is produced within the skin in cells called
melanocytes, and is the main determinant of the skin color of
darker-skinned humans. Both the amount and type of melanin produced
is controlled by a number of genes that operate under incomplete
dominance. Melanocytes residing in the basal epidermis control
human skin color through synthesis of melanin (melanogenesis), a
complex process thought to be primarily regulated by
alpha-melanocyte stimulating hormone (.alpha.MSH). The .alpha.MSH
peptide is secreted centrally by the pituitary, and locally by
surrounding keratinocytes in response to UV damage. .alpha.MSH
binding to the melanocortin receptor 1 (MC1R), a G protein-coupled
receptor (GPCR), activates adenylate cyclase, and increases
cytosolic cAMP. This secondary messenger activates a cascade of
downstream transcriptional events leading to expression of genes
required for melanin synthesis.
[0005] Melanogenesis takes place within small membrane-bound
packages called melanosomes. As the melanosomes become full of
melanin, they move into the slender arms of melanocytes, from where
they are transferred to the keratinocytes. Under normal conditions,
melanosomes cover the upper part of the keratinocytes and protect
them from genetic damage. One melanocyte supplies melanin to about
36 keranocytes according to signals from the keranocytes. They also
regulate melanin production and replication of melanocytes. People
have different skin colors mainly because their melanocytes produce
different amount and kinds of melanin. The genetic mechanism behind
human skin color is mainly regulated by the enzyme tyrosinase,
which creates the color of the skin, eyes, and hair shades.
Differences in skin color are also attributed to differences in
size and distribution of melanosomes in the skin.
[0006] Uneven pigmentation of some sort affects most people,
regardless of bioethnic background or skin color. Skin may appear
lighter or darker than normal, or lack pigmentation at all. There
may be blotchy and uneven areas, patches of brown to gray
discoloration, or freckling. Apart from blood-related conditions
such as jaundice, carotenosis, or argyria, skin pigmentation
disorders generally occur because the body produces either too much
or too little melanin.
[0007] Increased melanin production, also known as
hyperpigmentation, can be caused by distinct phenomena, such as
melasma (darkening of the skin), chloasma (skin discolorations
caused by hormones, which are usually associated with pregnancy,
birth control pills or estrogen replacement therapy), and solar
lentigo (also known as "liver spots" or "senile freckles," which
are darkened spots on the skin caused by aging and the sun). Liver
spots are quite common in adults with a long history of unprotected
sun exposure. Aside from sun exposure and hormones,
hyperpigmentation can be caused by skin damage, such as remnants of
blemishes, wounds or rashes. The most typical cause of darkened
areas of skin, brown spots or areas of discoloration is unprotected
sun exposure. Pregnancy has been long-associated with changes in
skin coloration. Modern physicians recognize several common
patterns of pregnancy-associated hyperpigmentation including
melasma, linea nigra, and pigmentary demarcation lines.
[0008] There is a need in the art to develop novel compositions and
methods for increasing, or preventing or reversing loss of, skin
pigmentation in a mammalian subject, such as a human. Such
compositions and methods would be useful, in non-limiting
embodiments, for treating or preventing skin disorders or diseases
associated with hypopigmentation. The present invention fulfills
this need.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention provides a compound, or a salt, solvate,
tautomer, enantiomer or diastereoisomer thereof. The invention
further provides a pharmaceutical composition comprising at least
one pharmaceutically acceptable carrier and at least one compound
of the invention, or a salt, solvate, tautomer, enantiomer or
diastereoisomer thereof. The invention further provides a method of
increasing, or preventing or reversing loss of, skin pigmentation
in a mammalian subject. The invention further provides a kit for
increasing, or preventing or reversing loss of, skin pigmentation
in a mammalian subject.
[0010] In certain embodiments, the compound of the invention is
selected from the group consisting of:
a molecule of formula (I):
##STR00001##
wherein in (I):
[0011] R.sub.1 is selected from the group consisting of .dbd.O,
.dbd.N--OH, .dbd.N--NHC(.dbd.O)(p-methoxy phenyl),
.dbd.N--NHC(.dbd.O)CH(OMe)phenyl, and
.dbd.N--NH(5-iodo-pyrid-2-yl);
[0012] R.sub.2 is selected from the group consisting of
C.sub.1-C.sub.4 alkyl and C.sub.1-C.sub.4 haloalkyl:
[0013] bond a is a single or double bond, such that: if bond a is a
double bond, R.sub.3 and R.sub.4 are H, and if bond a is a single
bond, R.sub.3 is selected from the group consisting of H, --OH,
--OAc, and halo; R.sub.4 is selected from the group consisting of
H, --OH, --OAc, and --S(o-nitrophenyl); or R.sub.3 and R.sub.4
combine to form a diradical selected from the group consisting of
--CH.sub.2--, --OCH.sub.2O--, --OCH(CH.sub.3)O--, and
--OC(CH.sub.3).sub.2O--;
[0014] R.sub.5 is selected from the group consisting of H, benzyl,
C.sub.1-C.sub.4 alkyl, and acetyl;
[0015] R.sub.6 is selected from the group consisting of H, halo,
--NO.sub.2, C.sub.1-C.sub.4 alkyl, --C.ident.CH,
--C.ident.C--Si(CH.sub.3).sub.3 (or --C.ident.C-TMS), --O-benzyl,
--OH, --OAc, C.sub.1-C.sub.4 alkoxy, --COOH, and
--COO(C.sub.1-C.sub.4 alkyl);
[0016] R.sub.7 is selected from the group consisting of H, halo,
--NO.sub.2, C.sub.1-C.sub.4 alkyl, --OH, --OAc, and C.sub.1-C.sub.4
alkoxy;
[0017] R.sub.8 is selected from the group consisting of H, halo,
--NO.sub.2, C.sub.1-C.sub.4 alkyl, --O-benzyl, --N(R)(R), --SR,
--COOH, --COO(C.sub.1-C.sub.4 alkyl), --OH, --OAc, C.sub.1-C.sub.4
alkoxy, 3-thietyl-methoxy, --SO.sub.2(morpholino), and
--OCH.sub.2CH.dbd.CH.sub.2, wherein each occurrence of R is
independently selected from the group consisting of H and
C.sub.1-C.sub.4 alkyl;
[0018] R.sub.9 is selected from the group consisting of H, halo,
--NO.sub.2, C.sub.1-C.sub.4 alkyl, --OH, --OAc, and C.sub.1-C.sub.4
alkoxy, [0019] or R.sub.8 and R.sub.9 combine to form a diradical
selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.3)O--, --OC(CH.sub.3).sub.2O--,
--O(CH.sub.2).sub.2O--, --O--CH.dbd.CH--, and --CH.dbd.CH--O--;
[0020] R.sub.10 is selected from the group consisting of H,
C.sub.1-C.sub.4 alkyl, and halo;
wherein each occurrence of benzyl is independently optionally
substituted with at least one group selected from the group
consisting of C.sub.1-C.sub.4 alkyl, --OH, C.sub.1-C.sub.4 alkoxy,
halo, and --NO.sub.2; and a molecule of formula (II):
##STR00002##
wherein in (II):
[0021] R.sub.1 is selected from the group consisting of .dbd.O and
.dbd.N--OH;
[0022] R.sub.2 is C.sub.1-C.sub.4 alkyl;
[0023] R.sub.5 is selected from the group consisting of H, benzyl
and C.sub.1-C.sub.4 alkyl:
[0024] R.sub.8 and R.sub.9 are independently selected from the
group consisting of H and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and
R.sub.9 combine to form a diradical selected from the group
consisting of --OCH.sub.2O--, --OCH(CH.sub.3)O-- and
--OC(CH.sub.3).sub.2O--.
[0025] In certain embodiments, the compound is not G-1 (or G1;
rel-1-[4-(6-bromo-1,3-benzodioxol-5-yl)-3aR,4S,5,9bS-tetrahydro-3H-cyclop-
enta[c]quinolin-8-yl]-ethanone):
##STR00003##
In other embodiments, the compound is G-1.
[0026] In certain embodiments, the compound is not selected from
the group consisting of A1-A107. In other embodiments, the compound
is at least one selected from the group consisting of A1-A107. In
yet other embodiments, the compound is not selected from the group
consisting of CMPD1-CMPD12. In yet other embodiments, the compound
is selected from the group consisting of CMPD1-CMPD12.
[0027] In certain embodiments, the compound, or a salt, solvate,
tautomer, enantiomer or diastereoisomer thereof, is selected from
the group consisting of:
a molecule of Formula (I-1):
##STR00004##
wherein in (I-1):
[0028] R.sub.1 is selected from the group consisting of .dbd.O and
.dbd.N--OH;
[0029] R.sub.2 is C.sub.1-C.sub.4 alkyl;
[0030] bond a is a single or double bond, such that: if bond a is a
double bond, R.sub.3 and R.sub.4 are H, and if bond a is a single
bond, R.sub.3 and R.sub.4 are independently selected from the group
consisting of H and --OH, or R.sub.3 and R.sub.4 combine to form a
diradical selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--;
[0031] R.sub.5 is selected from the group consisting of H, benzyl
and C.sub.1-C.sub.4 alkyl;
[0032] R.sub.6 is selected from the group consisting of H and
halo;
[0033] R.sub.8 and R.sub.9 are independently selected from the
group consisting of H and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and
R.sub.9 combine to form a diradical selected from the group
consisting of --OCH.sub.2O--, --OCH(CH.sub.3)O-- and
--OC(CH.sub.3).sub.2O--;
and a molecule of formula (II):
##STR00005##
wherein in (II):
[0034] R.sub.1 is selected from the group consisting of .dbd.O and
.dbd.N--OH;
[0035] R.sub.2 is C.sub.1-C.sub.4 alkyl:
[0036] R.sub.5 is selected from the group consisting of H, benzyl
and C.sub.1-C.sub.4 alkyl;
[0037] R.sub.8 and R.sub.9 are independently selected from the
group consisting of H and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and
R.sub.9 combine to form a diradical selected from the group
consisting of --OCH.sub.2O--, --OCH(CH.sub.3)O-- and
--OC(CH.sub.3).sub.2O--.
[0038] In certain embodiments, the compound is not G-1.
[0039] In certain embodiments, in (I), (I-1) or (II) R.sub.1 is
.dbd.O.
[0040] In certain embodiments, in (I), (I-1) or (II) R.sub.1 is
.dbd.N--OH.
[0041] In certain embodiments, in (I), (I-1) or (II) R.sub.2 is
selected from the group consisting of methyl, trifluoromethyl,
ethyl, 1-propyl, and 2-propyl.
[0042] In certain embodiments, in (1) or (I-1) bond a is a double
bond, and R.sub.3 and R.sub.4 are H.
[0043] In certain embodiments, in (I) or (I-1) bond a is a single
bond, and R.sub.3 and R.sub.4 are independently selected from the
group consisting of H and --OH.
[0044] In certain embodiments, in (I) or (I-1) bond a is a single
bond, and R.sub.3 and R.sub.4 are H.
[0045] In certain embodiments, in (I) or (I-1) bond a is a single
bond, and R.sub.3 and R.sub.4 are --OH.
[0046] In certain embodiments, in (I) or (I-1) R.sub.3 and R.sub.4
are --OH and cis to each other.
[0047] In certain embodiments, in (I) or (I-1) bond a is a single
bond, and R.sub.3 and R.sub.4 combine to form
--OC(CH.sub.3).sub.2O--.
[0048] In certain embodiments, in (I), (I-1) or (II) R.sub.5 is
selected from the group consisting of H, benzyl, methyl, ethyl,
1-propyl and 2-propyl.
[0049] In certain embodiments, R.sub.6 is selected from the group
consisting of H, F, Cl, Br and I.
[0050] In certain embodiments, in (I), (I-1) or (II) R.sub.8 and
R.sub.9 are independently selected from the group consisting of H,
methoxy, ethoxy, 1-propoxy and 2-propoxy.
[0051] In certain embodiments, in (I), (I-1) or (II) R.sub.8 and
R.sub.9 combine to form a diradical selected from the group
consisting of --OCH.sub.2O--, --OCH(CH.sub.3)O-- and
--OC(CH.sub.3).sub.2O--.
[0052] In certain embodiments, (1) is at least one selected from
the group consisting of:
##STR00006## ##STR00007##
[0053] In certain embodiments, the compound is at least one
selected from the group consisting of: CMPD1
(rel-1-((3aS,4R,9bR)-4-(benzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-
-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD2
(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-5-methyl-3a,4,5,-
9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD3
(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-2,3,3a,4,5,9b-he-
xahydro-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one). CMPD4
(rel-1-((3aS,4R,9bR)-5-benzyl-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,-
9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD5
(rel-1-((3aS,4R,9bR)-4-(2-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta-
[c]quinolin-8-yl)ethan-1-one); CMPD6
(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrah-
ydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one oxime); CMPD7
(rel-1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-3a,4,5,9b-tetrahydro-
-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD8
(rel-1-((3aS,4R,9bR)-4-(6-chlorobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetra-
hydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one); CMPD9
(rel-1-((6R,6aS,7aS,10aR,10bR)-6-(6-bromobenzo[d][1,3]dioxol-5-yl)-9,9-di-
methyl-6,6a,7,7a,10a,10b-hexahydro-5H-[1,3]dioxolo[4',5':3,4]cyclopenta[1,-
2-c]quinolin-2-yl)ethan-1-one); CMPD10
(rel-1-((1R,2S,3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-1,2-dihydr-
oxy-2,3,3a,4,5,9b-hexahydro-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one);
CMPD11
(rel-1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-2,3,3a,4,5,9b-
-hexahydro-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one); and CMPD12
(rel-1-((4S,5aS,6R,11aR)-4,5,5a,6,11,11a-hexahydro-4,6-methano[1,3]dioxol-
o[4',5':5,6]benzo[1,2-c]acridin-8-yl)ethan-1-one).
[0054] In certain embodiments, the pharmaceutical composition is
formulated for topical or transdermal administration.
[0055] In certain embodiments, the pharmaceutical composition does
not comprise G-1. In other embodiments, the pharmaceutical
composition comprises at least one compound selected from the group
consisting of G-1, CMPD1-CMPD12, and A1-A107. In yet other
embodiments, the pharmaceutical composition comprises at least one
compound selected from the group consisting of CMPD1-CMPD12. In
other embodiments, the pharmaceutical composition comprises at
least one compound selected from the group consisting of G-11 and
CMPD1-CMPD12.
[0056] In certain embodiments, the pharmaceutical composition
further comprises at least one sun-blocking agent. In certain
embodiments, the pharmaceutical composition further comprises at
least sunscreen lotion.
[0057] In certain embodiments, the method comprises administering
to the subject a therapeutically effective amount of a compound,
wherein the compound is a GPER agonist and does not bind to a
canonical nuclear estrogen receptor. In other embodiments, the
subject is human.
[0058] In certain embodiments, the canonical nuclear estrogen
receptor comprises at least one selected from the group consisting
of ER.alpha. and ER.beta.. In yet other embodiments, the compound
is a GPER1 or GPR30 agonist.
[0059] In certain embodiments, the subject is suffering from at
least one condition selected from the group consisting of
pigmentary changes associated with oral contraceptive use,
pregnancy, and endogenous estrogens in females; solar lentigo;
acne; eczema; chemical, sun, and thermal burn scars; and lupus.
[0060] In certain embodiments, the compound is administered
topically or transdermally to the subject. In other embodiments,
the compound is formulated as a pharmaceutical composition for
topical or transdermal administration. In yet other embodiments,
the pharmaceutical composition further comprises at least one
sun-blocking agent. In yet other embodiments, the pharmaceutical
composition comprises a sunscreen lotion. In yet other embodiments,
the pharmaceutical composition is essentially free of a skin
bleaching agent. In yet other embodiments, the subject is not
further submitted to UV radiation as part of the treatment to
increase, or prevent or reverse loss of, skin pigmentation.
[0061] In certain embodiments, the compound is at least one
selected from the group consisting of G1, A1-A107 and
CMPD1-CMPD12.
[0062] In certain embodiments, the kit comprises at least one
compound or pharmaceutical composition of the invention. In other
embodiments, the kit comprises instructions for topically or
transdermally administering the at least one pharmaceutical
composition to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0064] FIG. 1 comprises a set of images illustrating
pregnancy-associated pigmentation disorder. Images A-C depict
pigmentation disorder of patients with melasma. Image D depicts a
patient with pigmentary demarcation lines. Images E and F depict
typical linea nigra on a mother. Image G depicts typical linea
nigra on a child.
[0065] FIGS. 2A-2F comprise a set of scheme and bar graphs
illustrating melanin production in human melanocytes. FIG. 2A
comprises a schematic illustration of classical regulation of human
melanocyte homeostasis. FIG. 2B comprises a bar graph illustrating
melanin production in response to .alpha.MSH in a dose dependent
manner. FIG. 2C comprises a bar graph illustrating melanin
production in response to estrogen in a dose dependent manner. FIG.
2D comprises a bar graph illustrating melanin production by
melanocytes treated with vehicle, .alpha.MSH, or estrogen. FIG. 2E
comprises a bar graph illustrating melanin production in response
to estrogen using iPS-derived female melanocytes. FIG. 2F comprises
a bar graph illustrating melanin production in response to estrogen
using facial, aged-adult melanocytes. N=3 biologic replicates for
each experiment. Error bars denote .+-.s.d., *p<0.05.
[0066] FIGS. 3A-3C comprise a set of images and bar graphs
illustrating the finding that estrogen increases melanin synthesis.
FIG. 3A comprises a bar graph illustrating melanin content of
primary human melanocytes treated with estrogen (E2), compared to
vehicle-treated controls. FIG. 3B comprises a bar graph and images
illustrating Fontana-Masson (melanin) staining of organotypic skin
treated with vehicle or estrogen. Relative melanin content is
illustrated in the bar graph at the bottom of the Figure. FIG. 3C
comprises a bar graph and images illustrating MITF
(microphthalmia-associated transcription factor)
immunohistochemistry of organotypic skin treated with vehicle or
estrogen. Melanocyte population density is illustrated in the bar
graph at the bottom of the Figure. N=3 biologic replicates for each
experiment. Error bars denote .+-.s.d., *p<0.05, scale bar=50
.mu.m.
[0067] FIGS. 4A-4D comprise a set of images and bar graphs
illustrating the finding that estrogen accesses the classical
melanin production pathway through nonclassical receptors. FIG. 4A
comprises a bar graph illustrating cAMP ELISA from estrogen-treated
melanocytes. FIG. 4B comprises a set of western blot images
demonstrating changes in classical melanin pathway regulators after
estrogen treatment. FIG. 4C comprises a set of western blot images
for estrogen receptors in MCF7 cells and melanocytes. FIG. 4D
comprises a bar graph illustrating melanin content of melanocytes
transduced with control shRNA or shRNA targeting GPER. Cells were
treated with either vehicle or estrogen. N=3 biologic replicates
for each experiment. Error bars denote .+-.s.d., *p<0.05.
[0068] FIGS. 5A-5F comprises a set of bar graphs and table
illustrating hormone receptors in melanocytes. FIG. 5A comprises a
bar graph illustrating relative gene expression of classical
hormone receptors in MCF7 cells and melanocytes, as determined by
qRT-PCR. Ct values were normalized to actin, and set relative to
the expression of androgen receptor (AR) in MCF7 cells. FIG. 5B
comprises a table illustrating RPKM values for classical and
nonclassical estrogen receptors in human melanocytes, by
convention, RPKM values >1 indicate the gene is expressed. FIG.
5C comprises a bar graph illustrating expression of GPER displayed
as 1/Ct value. FIG. 5D comprises a bar graph illustrating relative
expression of transcripts in melanocytes, fibroblasts, and
keratinocytes, as determined by qRT-PCR, displayed relative to the
expression level in melanocytes. FIG. 5E comprises a bar graph
illustrating qRT-PCR showing mRNA knockdown efficiency of the two
hairpins targeting GPER. FIG. 5F comprises a set of bar graphs
illustrating melanin content of melanocytes transduced with
LentiCRISPRV2 with guide RNA targeting GFP or GPER. Cells were
treated with either vehicle or estrogen. Error bars denote
.+-.s.d., *p<0.05.
[0069] FIGS. 6A-6B comprise a set of bar graphs illustrating the
finding that melanin production is altered by sex steroid
analogs-GPER agonists currently in clinical use. FIG. 6A is a bar
graph illustrating melanin production in response to tamoxifen
(TMX). FIG. 6B is a bar graph illustrating melanin production in
response to ethinyl estradiol (EE2). N=3 biologic replicates for
each experiment. Error bars denote .+-.s.d., *p<0.05.
[0070] FIGS. 7A-7B comprise a set of bar graphs illustrating the
effects of specific GPER agonists on melanin production. FIG. 7A
comprises a bar graph illustrating melanin production in response
to G-1, a specific GPER agonist. FIG. 7B comprises a bar graph
illustrating melanin assay performed on melanocytes lentivirally
transduced with control shRNA or shRNA targeting GPER. These cells
were treated with either vehicle or G-1.
[0071] FIGS. 8A-8C comprise a set of images and bar graphs
illustrating the finding that GPER signaling is sufficient to alter
melanin production in organotypic human tissue. FIG. 8A comprises
an image illustrating organotypic skin treated with vehicle (left)
or G-1 (right). FIG. 8B comprises images and a bar graph
illustrating Fontana-Masson (melanin) staining of organotypic skin
treated with vehicle or G-1. Quantification of melanin content is
shown on the right. FIG. 8C comprises images and a bar graph
illustrating MITF immunohistochemistry of organotypic skin treated
with vehicle or G-1. Quantification of melanocyte population
density is shown on the right. Error bars denote .+-.s.d.,
*p<0.05, scale bar=50 .mu.m.
[0072] FIGS. 9A-9D comprise a set of bar graphs and images
illustrating the finding that topical GPER agonists increase
pigmentation in vivo. FIG. 9A comprises an image illustrating mouse
ear skin treated for 3 weeks with vehicle only on the left ear, and
2% G-1 on the right ear. FIG. 9B comprises a bar graph illustrating
melanin assay on whole ear tissue that was treated with either
vehicle or 2% G-1 for 3 weeks. FIG. 9C comprises a set of images
illustrating Fontana-Masson (melanin) staining of tissue sections
from ears treated with either vehicle or 2% G-1, quantification of
staining on right. FIG. 9D comprises a schematic representation of
selected biological pathways discussed herein. N=3 biologic
replicates for each experiment. Error bars denote .+-.s.d.,
*p<0.05, scale bar=20 .mu.m.
[0073] FIG. 10 comprises a set of western blot images illustrating
the finding that compounds of the invention increase levels of
pCREB in human primary melanocytes. The actin control is
illustrated at the bottom of the images.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The present invention relates, in one aspect, to the
unexpected discovery that compounds of the invention modulate skin
pigmentation in a mammalian subject, such as a human. In certain
embodiments, the compounds of the invention treat or prevent skin
hypopigmentation in a subject. In yet other embodiments, the
compounds of the invention treat or prevent wrinkles and/or
discolorations of premature aging. In yet other embodiments, the
compositions of the invention modulate skin color for esthetic
benefit without exposure to DNA-damaging UV radiation.
[0075] Human pregnancy and oral contraceptive use are commonly
associated with altered epidermal pigmentation, and this suggests
that changes in circulating hormones may regulate melanocyte
homeostasis. However, the specific hormones, receptors, and
downstream pathways mediating these effects have been
undefined.
[0076] As demonstrated herein, physiologically relevant levels of
17.beta.-estradiol (estrogen) promotes skin pigment production.
Altered melanin production is associated with parallel changes in
cAMP, a known regulator of mammalian pigmentation. Normal primary
melanocytes do not express the classical estrogen receptor (ER).
The present studies demonstrate that melanocytes express the
non-canonical steroid hormone receptor G protein-coupled estrogen
receptor 1 (GPER1). Lentiviral-mediated CRISPR-Cas9 ablation of
that protein blocks the pigmentary effects of estrogen, suggesting
that estrogen affect pigmentation through the non-canonical
receptors GPER1 to regulate human melanocyte homeostasis. In
certain aspects, modulation of GPER1 and its associated signaling
pathway elements has therapeutic utility in disorders of epidermal
pigmentation.
[0077] The results presented herein, utilizing both genetic
depletion and specific pharmacologic agonists, establish that
estrogen influences human melanocyte pigment production through
activation of the nonclassical hormone receptor GPER (FIG. 9D). In
one aspect, specific activation of the GPER receptors using
selective agonist compounds is sufficient to increase pigment
synthesis, avoiding the potentially undesirable effects of
classical estrogen receptor activation in other cell types. GPER
has reduced expression in keratinocytes or fibroblasts, which
should limit drug effects to the cells of interest, in contrast to
forskolin, which nonspecifically activates adenylate cyclase in all
cells.
[0078] The present studies indicate that GPER agonist G-1 had an
effect as potent as natural estrogen, but G-1 does not bind to the
canonical nuclear ER. This differential binding behavior should
minimize potential medical concerns associated with exogenous
estrogen treatment. In certain embodiments, the canonical nuclear
ER comprises ER.alpha. and/or ER.beta..
[0079] Specific GPER agonists, such as but not limited to G-1,
represent a useful, novel class of therapeutics for normalizing
disorders of epidermal pigmentation. Myriad genetic and acquired
conditions including common afflictions such as acne, eczema,
vitiligo, ultraviolet (UV) radiation exposure, traumatic injury,
and pregnancy are associated with alterations in skin pigmentation
that can be extensive and long-lasting. A population that could
potentially benefit from modulating skin pigment are people with
naturally light skin, especially those with red hair, who have a
markedly decreased ability to synthesize UV-protective brown
eumelanin as a result of inactivating mutations in MC1R. This large
population is especially susceptible to photodamage, sunburns, and
has an increased lifetime risk of keratinocyte and
melanocyte-derived skin cancers. There is currently no available
therapeutic that promotes protective eumelanin pigment production.
The only method currently available to increase skin melanin is UV
exposure, which is effective at darkening skin but promotes DNA
damage, which leads to premature aging, wrinkles, and skin cancer.
However, the specific activation of GPER alternatively activates
cAMP signaling, bypassing MC1R, to stimulate melanin synthesis, and
can thus be especially useful in this sun-vulnerable population.
Selective GPER activation in skin can potentially be a safe
alternative to intentional UV radiation exposure (via natural
sunlight or tanning beds) for individuals seeking what they
perceive as an esthetically desirable tan.
[0080] In certain non-limiting embodiments, targeting the
receptor(s) described herein can protect skin from wrinkles, and
discolorations of premature aging, and also modulate skin color for
esthetic benefit without exposure to DNA-damaging UV radiation or
potentially toxic skin bleaching agents.
[0081] The invention should not be construed to be limited to the
estrogen derivatives, or any analog thereof, recited herein. In
certain embodiments, the present invention encompasses any estrogen
derivatives, or analogs thereof, that act as agonists towards GPER
and do not bind to the canonical nuclear estrogen receptor.
Definitions
[0082] As used herein, each of the following terms have the meaning
associated with it in this section.
[0083] Unless defined otherwise, all technical and scientific terms
used herein generally have the same meaning as commonly understood
by one of ordinary skill in the art to which this invention
belongs. Generally, the nomenclature used herein and the laboratory
procedures in cell culture, molecular genetics and chemistry are
those well-known and commonly employed in the art.
[0084] As used herein, the articles "a" and "an" refer to one or to
more than one (i.e., to at least one) of the grammatical object of
the article. By way of example, "an element" means one element or
more than one element.
[0085] As used herein, the term ".alpha.MSH" refers to
alpha-melanocyte stimulating hormone.
[0086] As used herein, the term "about" will be understood by
persons of ordinary skill in the art and will vary to some extent
on the context in which it is used. As used herein when referring
to a measurable value such as an amount, a temporal duration, and
the like, the term "about" is meant to encompass variations of
.+-.20% or .+-.10%, .+-.5%, .+-.1%, or .+-.0.1% from the specified
value, as such variations are appropriate to perform the disclosed
methods.
[0087] As used herein, the term "alkoxy" employed alone or in
combination with other terms means, unless otherwise stated, an
alkyl group having the designated number of carbon atoms, as
defined elsewhere herein, connected to the rest of the molecule via
an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy,
2-propoxy (or isopropoxy) and the higher homologs and isomers. A
specific example is (C.sub.1-C.sub.3)alkoxy, such as, but not
limited to, ethoxy and methoxy.
[0088] As used herein, the term "alkyl" by itself or as part of
another substituent means, unless otherwise stated, a straight or
branched chain hydrocarbon having the number of carbon atoms
designated (i.e., C.sub.1-C.sub.10 means one to ten carbon atoms)
and includes straight, branched chain, or cyclic substituent
groups. Examples include methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and
cyclopropylmethyl. A specific embodiment is (C.sub.1-C.sub.4)alkyl,
such as, but not limited to, ethyl, methyl, isopropyl, n-butyl,
isobutyl, t-butyl, and cyclopropylmethyl.
[0089] As used herein, the term "AR" refers to androgen
receptor.
[0090] As used herein, the term "cAMP" refers to cyclic adenosine
monophosphate.
[0091] As used herein, the term "CREB" refers to cAMP response
element-binding protein.
[0092] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated, the animal's health continues to deteriorate. In
contrast, a "disorder" in an animal is a state of health in which
the animal is able to maintain homeostasis, but in which the
animal's state of health is less favorable than it would be in the
absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0093] As used herein, the term "E2" refers to estrogen.
[0094] As used herein, the term "EE2" refers to ethinyl
estradiol.
[0095] As used herein, the terms "effective amount" or
"therapeutically effective amount" or "pharmaceutically effective
amount" of a compound are used interchangeably to refer to the
amount of the compound sufficient to provide a beneficial effect to
the subject to which the compound is administered.
[0096] As used herein, the term "ER" refers to estrogen
receptor.
[0097] As used herein, the term "G1" or "G-1" refers to
rel-1-[4-(6-bromo-1,3-benzodioxol-5-yl)-3aR,4S,5,9bS-tetrahydro-3H-cyclop-
enta[c]quinolin-8-yl]-ethanone), or a salt, solvate, tautomer,
enantiomer or diastereoisomer thereof.
[0098] As used herein, the term "GPCR" refers to a G
protein-coupled receptor.
[0099] As used herein, the term "GPR30 receptor" (also known as
GPER1; CEPR; CMKRL2; DRY12; FEG-1; GPCR-Br; GPER; LERGU; LERGU2;
LyGPR; or mER) refers to a 7-transmembrane (7TM) G protein-coupled
receptor that mediate estrogen-dependent signal transduction. GPR30
is an intracellular protein, found in the endoplasmic reticulum,
which binds estrogen with high affinity (K.sub.d of 6 nM) and
mediates rapid cellular responses including calcium mobilization
and phosphatidylinositol 3,4,5-trisphosphate production in the
nucleus. GPR30 receptor refers to all types of GPR30 receptor,
regardless of the tissue in which such receptor is found and refers
to any variant thereof, including receptors of mammals (such as
humans and domesticated mammals where veterinary applications are
relevant) and variants thereof. Other names which have been used
for GPR30 include CMKRL2, DRY12, FEG-1. GPCR-Br, LERGU, LERGU2,
LyGPR. CEPR and MGC99678, among others.
[0100] As used herein, the term "halo" or "halogen" alone or as
part of another substituent refers to, unless otherwise stated, a
fluorine, chlorine, bromine, or iodine atom.
[0101] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression that can be used to communicate the usefulness of a
compound, composition, assay or method of the invention in a kit
for suppressing or reducing systemic immune response in a subject.
The instructional material of the kit of the invention can, for
example, be affixed to a container which contains the identified
compound, composition, assay, or methods of the invention or be
shipped together with a container that contains the identified
compound, composition, assay, or method. Alternatively, the
instructional material can be shipped separately from the container
with the intention that the instructional material and the
compound, composition, assay, or method be used cooperatively by
the recipient.
[0102] As used herein, the term "MC1R" refers to melanocortin
receptor 1.
[0103] As used herein, the term "MITF" refers to
microphthalmia-associated transcription factor.
[0104] As used herein, the term "modulate" means, with respect to
disease states or conditions associated with binding of a compound
of the present invention to a receptor contemplated in the present
invention, to produce, either directly or indirectly, an
improvement or lessening of a condition or disease state which was,
prior to administration of a compound according to the present
invention, sub-optimal and in many cases, debilitating and even
life threatening. Modulation may occur by virtue of agonist
activity, antagonist activity or mixed agonist/antagonist activity
(depending on the receptor site).
[0105] "Parenteral" administration of a composition includes, e.g.,
subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or
intrasternal injection, or infusion techniques.
[0106] As used herein, the term "pharmaceutical composition" or
"composition" refers to a mixture of at least one compound useful
within the invention with other chemical components, such as
carriers, stabilizers, diluents, dispersing agents, suspending
agents, thickening agents, and/or excipients. The pharmaceutical
composition facilitates administration of the compound to an
organism. Multiple techniques of administering a compound exist in
the art including, but not limited to: intravenous, oral, aerosol,
parenteral, ophthalmic, pulmonary, intracranial, transdermal and
topical administration. In certain embodiments, the administration
comprises topical administration.
[0107] As used herein, the term "pharmaceutically acceptable"
refers to a material, such as a carrier or diluent, which does not
abrogate the biological activity or properties of the composition,
and is relatively non-toxic, i.e., the material may be administered
to an individual without causing undesirable biological effects or
interacting in a deleterious manner with any of the components of
the composition in which it is contained.
[0108] As used herein, the term "pharmaceutically acceptable
carrier" means a pharmaceutically acceptable material, composition
or carrier, such as a liquid or solid filler, stabilizer,
dispersing agent, suspending agent, diluent, excipient, thickening
agent, solvent or encapsulating material, involved in carrying or
transporting a compound useful within the invention within or to
the subject such that it may perform its intended function.
Typically, such constructs are carried or transported from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation, including
the compound useful within the invention, and not injurious to the
subject. Some examples of materials that may serve as
pharmaceutically acceptable carriers include: sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; surface active agents; alginic
acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol; phosphate buffer solutions; and other non-toxic compatible
substances employed in pharmaceutical formulations. As used herein.
"pharmaceutically acceptable carrier" also includes any and all
coatings, antibacterial and antifungal agents, and absorption
delaying agents, and the like that are compatible with the activity
of the compound useful within the invention, and are
physiologically acceptable to the subject. Supplementary active
compounds may also be incorporated into the compositions. The
"pharmaceutically acceptable carrier" may further include a
pharmaceutically acceptable salt of the compound useful within the
invention. Other additional ingredients that may be included in the
pharmaceutical compositions used in the practice of the invention
are known in the art and described, for example in Remington's
Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985,
Easton, Pa.), which is incorporated herein by reference.
[0109] As used herein, the language "pharmaceutically acceptable
salt" refers to a salt of the administered compound prepared from
pharmaceutically acceptable non-toxic acids and/or bases, including
inorganic acids, inorganic bases, organic acids, inorganic bases,
solvates (including hydrates) and clathrates thereof.
[0110] As used herein, a "pharmaceutically effective amount,"
"therapeutically effective amount" or "effective amount" of a
compound is that amount of compound that is sufficient to provide a
beneficial effect to the subject to which the compound is
administered.
[0111] The term "prevent," "preventing" or "prevention" as used
herein means avoiding or delaying the onset of symptoms associated
with a disease or condition in a subject that has not developed
such symptoms at the time the administering of an agent or compound
commences. Disease, condition and disorder are used interchangeably
herein.
[0112] By the term "specifically bind" or "specifically binds" as
used herein is meant that a first molecule preferentially binds to
a second molecule (e.g., a particular receptor or enzyme), but does
not necessarily bind only to that second molecule.
[0113] As used herein, the terms "subject" and "individual" and
"patient" can be used interchangeably and may refer to a human or
non-human mammal or a bird. Non-human mammals include, for example,
livestock and pets, such as ovine, bovine, porcine, canine, feline
and murine mammals. In certain embodiments, the subject is
human.
[0114] As used herein, the term "TMX" refers to tamoxifen.
[0115] As used herein, "topical administration" or "topical
application" refers to a medication applied to body surfaces such
as the skin or mucous membranes.
[0116] The term to "treat," as used herein, means reducing the
frequency with which symptoms are experienced by a patient or
subject or administering an agent or compound to reduce the
severity with which symptoms are experienced. An appropriate
therapeutic amount in any individual case may be determined by one
of ordinary skill in the art using routine experimentation.
[0117] As used herein, the term "treatment" or "treating" is
defined as the application or administration of a therapeutic
agent, i.e., a composition useful within the invention (alone or in
combination with another pharmaceutical agent), to a subject, or
application or administration of a therapeutic agent to an isolated
tissue or cell line from a subject (e.g., for diagnosis or ex vivo
applications), who has a disease or disorder, a symptom of a
disease or disorder or the potential to develop a disease or
disorder, with the purpose to cure, heal, alleviate, relieve,
alter, remedy, ameliorate, improve or affect the disease or
disorder, the symptoms of the disease or disorder or the potential
to develop the disease or disorder. Such treatments may be
specifically tailored or modified, based on knowledge obtained from
the field of pharmacogenomics.
[0118] As used herein, the term "UV" refers to ultraviolet.
[0119] Throughout this disclosure, various aspects of the invention
can be presented in a range format. It should be understood that
the description in range format is merely for convenience and
brevity and should not be construed as an inflexible limitation on
the scope of the invention. Accordingly, the description of a range
should be considered to have specifically disclosed all the
possible subranges as well as individual numerical values within
that range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed subranges such
as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,
from 3 to 6 etc., as well as individual numbers within that range,
for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies
regardless of the breadth of the range.
Compounds and Compositions
[0120] In one aspect, the present invention encompasses an estrogen
derivative or analog that binds to GPER, such as but not limited to
GPER1, and does not bind to the canonical nuclear estrogen receptor
(ER). In certain embodiments, the estrogen derivative or analog is
a GPER agonist.
[0121] In certain embodiments, the compound of the invention, or a
salt, solvate, tautomer, enantiomer or diastereoisomer thereof, is
at least one compound of formula (I):
##STR00008##
wherein in (I):
[0122] R.sub.1 is selected from the group consisting of .dbd.O,
.dbd.N--OH, .dbd.N--NHC(.dbd.O)(p-methoxy phenyl),
.dbd.N--NHC(.dbd.O)CH(OMe)phenyl, and
.dbd.N--NH(5-iodo-pyrid-2-yl);
[0123] R.sub.2 is selected from the group consisting of
C.sub.1-C.sub.4 alkyl and C.sub.1-C.sub.4 haloalkyl (such as, but
not limited to, --CF.sub.3);
[0124] bond a is a single or double bond, such that: [0125] if bond
a is a double bond, R.sub.3 and R.sub.4 are H, and [0126] if bond a
is a single bond, R.sub.3 is selected from the group consisting of
H, --OH, --OAc, and halo; R.sub.4 is selected from the group
consisting of H, --OH, --OAc, and --S(o-nitrophenyl); or R.sub.3
and R.sub.4 combine to form a diradical selected from the group
consisting of --CH.sub.2--, --OCH.sub.2O--, --OCH(CH.sub.3)O--, and
--OC(CH.sub.3).sub.2O--;
[0127] R.sub.5 is selected from the group consisting of H, benzyl,
C.sub.1-C.sub.4 alkyl, and acetyl;
[0128] R.sub.6 is selected from the group consisting of H, halo,
--NO.sub.2, C.sub.1-C.sub.4 alkyl, --C.ident.CH,
--C.ident.C--Si(CH.sub.3).sub.3 (or --C.ident.C-TMS), --O-benzyl,
--OH, --OAc, C.sub.1-C.sub.4 alkoxy, --COOH, and
--COO(C.sub.1-C.sub.4 alkyl);
[0129] R.sub.7 is selected from the group consisting of H, halo,
--NO.sub.2, C.sub.1-C.sub.4 alkyl, --OH, --OAc, and C.sub.1-C.sub.4
alkoxy;
[0130] R.sub.8 is selected from the group consisting of H, halo,
--NO.sub.2, C.sub.1-C.sub.4 alkyl, --O-benzyl, --N(R)(R), --SR,
--COOH, --COO(C.sub.1-C.sub.4 alkyl), --OH, --OAc, C.sub.1-C.sub.4
alkoxy, 3-thietyl-methoxy, --SO.sub.2(morpholino), and
--OCH.sub.2CH.dbd.CH.sub.2, wherein each occurrence of R is
independently selected from the group consisting of H and
C.sub.1-C.sub.4 alkyl;
[0131] R.sub.9 is selected from the group consisting of H, halo,
--NO.sub.2, C.sub.1-C.sub.4 alkyl, --OH, --OAc, and C.sub.1-C.sub.4
alkoxy, [0132] or R.sub.8 and R.sub.9 combine to form a diradical
selected from the group consisting of --OCH.sub.2O--,
--OCH(CH.sub.1)O--, --OC(CH.sub.3).sub.2O--,
--O(CH.sub.2).sub.2O--, --O--CH.dbd.CH--, and --CH.dbd.CH--O--;
[0133] R.sub.10 is selected from the group consisting of H,
C.sub.1-C.sub.4 alkyl, and halo;
wherein each occurrence of benzyl is independently optionally
substituted with at least one group selected from the group
consisting of C.sub.1-C.sub.4 alkyl, --OH, C.sub.1-C.sub.4 alkoxy,
halo, and --NO.sub.2.
[0134] In certain embodiments, the compound of the invention, or a
salt, solvate, tautomer, enantiomer or diastereoisomer thereof, is
at least one compound of formula (I-1):
##STR00009##
wherein in (I-1):
[0135] R.sub.1 is selected from the group consisting of .dbd.O and
.dbd.N--OH;
[0136] R.sub.2 is C.sub.1-C.sub.4 alkyl;
[0137] bond a is a single or double bond, such that: [0138] if bond
a is a double bond, R.sub.3 and R.sub.4 are H, and [0139] if bond a
is a single bond, R.sub.3 and R.sub.4 are independently selected
from the group consisting of H and --OH, or R.sub.3 and R.sub.4
combine to form a diradical selected from the group consisting of
--OCH.sub.2O--, --OCH(CH.sub.3)O-- and --OC(CH.sub.3).sub.2O--;
[0140] R.sub.5 is selected from the group consisting of H, benzyl
and C.sub.1-C.sub.4 alkyl;
[0141] R.sub.6 is selected from the group consisting of H and
halo;
[0142] R.sub.8 and R.sub.9 are independently selected from the
group consisting of H and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and
R.sub.9 combine to form a diradical selected from the group
consisting of --OCH.sub.2O--, --OCH(CH.sub.3)O-- and
--OC(CH.sub.3).sub.2O--.
[0143] In certain embodiments, the compound is not G-1. In other
embodiments, the compound is G-1.
[0144] In certain embodiments, R.sub.1 is .dbd.O. In other
embodiments, R.sub.1 is .dbd.N--OH.
[0145] In certain embodiments, R.sub.2 is selected from the group
consisting of methyl, trifluoromethyl, ethyl, 1-propyl, and
2-propyl. In other embodiments, R.sub.2 is methyl or
trifluoromethyl.
[0146] In certain embodiments, bond a is a double bond, and R.sub.3
and R.sub.4 are H.
[0147] In certain embodiments, bond a is a single bond, and R.sub.3
and R.sub.4 are independently selected from the group consisting of
H and --OH. In other embodiments, bond a is a single bond, and
R.sub.3 and R.sub.4 are H. In yet other embodiments, bond a is a
single bond, and R.sub.3 and R.sub.4 are --OH. In yet other
embodiments, bond a is a single bond, and R.sub.3 and R.sub.4 are
--OH and cis to each other. In yet other embodiments, bond a is a
single bond, and R.sub.3 and R.sub.4 are --OH and trans to each
other.
[0148] In certain embodiments, bond a is a single bond, and R.sub.3
and R.sub.4 combine to form --OC(CH.sub.3).sub.2O--.
[0149] In certain embodiments, R.sub.5 is selected from the group
consisting of H, benzyl, methyl, ethyl, 1-propyl and 2-propyl. In
certain embodiments, R is selected from the group consisting of H,
benzyl and methyl.
[0150] In certain embodiments, R.sub.6 is selected from the group
consisting of H, F, Cl, Br and I. In certain embodiments, R.sub.5
is selected from the group consisting of H, Cl and Br.
[0151] In certain embodiments, R.sub.8 and R.sub.9 are
independently selected from the group consisting of H and
C.sub.1-C.sub.4 alkoxy. In other embodiments, R.sub.8 and R.sub.9
are independently selected from the group consisting of H, methoxy,
ethoxy, 1-propoxy and 2-propoxy. In yet other embodiments, R.sub.8
and R.sub.9 are independently selected from the group consisting of
H and methoxy.
[0152] In certain embodiments, R.sub.8 and R.sub.9 combine to form
a diradical selected from the group consisting of --OCH.sub.2O--,
--O(CH.sub.2).sub.2O--, --OCH(CH.sub.3)O-- and
--OC(CH.sub.3).sub.2O--.
[0153] In certain embodiments, the compound, or a salt, solvate,
tautomer, enantiomer or diastereoisomer thereof, is at least one
selected from the group consisting of:
##STR00010## ##STR00011##
[0154] In certain embodiments, the compound, or a salt, solvate,
tautomer, enantiomer or diastereoisomer thereof, is at least one
selected from the group consisting of:
TABLE-US-00001 (Ig) ##STR00012## Compound R.sub.6 R.sub.7 R.sub.8
R.sub.9 R.sub.10 R.sub.4 R.sub.3 A1 H H OMe H H H H A2 Cl H H H H H
H A3 Cl H Cl H H H H A4 H H H H H H Fl A5 H H
--OCH.sub.2CH.sub.2O-- H H H A6 Br H --OCH.sub.2CH.sub.2O-- H H H
A7 H H H H H --CH.sub.2-- A8 H H H H H S(o-NO.sub.2)Ph Cl A9 H H Br
H H S(o-NO.sub.2)Ph Cl A10 H H Cl H H S(o-NO.sub.2)Ph Cl A11 F H H
H H S(o-NO.sub.2)Ph Cl A12 H H NO.sub.2 H H S(o-NO.sub.2)Ph Cl A13
NO.sub.2 H H H H S(o-NO.sub.2)Ph Cl A14 Cl H Cl H H S(o-NO.sub.2)Ph
Cl A15 Cl Cl H H H S(o-NO.sub.2)Ph Cl
[0155] In certain embodiments, the compound is A1. In other
embodiments, the compound is A2. In yet other embodiments, the
compound is A3. In yet other embodiments, the compound is A4. In
yet other embodiments, the compound is A5. In yet other
embodiments, the compound is A6. In yet other embodiments, the
compound is A7. In yet other embodiments, the compound is A8. In
yet other embodiments, the compound is A9. In yet other
embodiments, the compound is A10. In yet other embodiments, the
compound is A11. In yet other embodiments, the compound is A12. In
yet other embodiments, the compound is A13. In yet other
embodiments, the compound is A14. In yet other embodiments, the
compound is A15.
[0156] In certain embodiments, the compound is not A1. In other
embodiments, the compound is not A2. In yet other embodiments, the
compound is not A3. In yet other embodiments, the compound is not
A4. In yet other embodiments, the compound is not A5. In yet other
embodiments, the compound is not A6. In yet other embodiments, the
compound is not A7. In yet other embodiments, the compound is not
A8. In yet other embodiments, the compound is not A9. In yet other
embodiments, the compound is not A10. In yet other embodiments, the
compound is not A11. In yet other embodiments, the compound is not
A12. In yet other embodiments, the compound is not A13. In yet
other embodiments, the compound is not A14. In yet other
embodiments, the compound is not A15.
[0157] In certain embodiments, the compound, or a salt, solvate,
tautomer, enantiomer or diastereoisomer thereof, is at least one
selected from the group consisting of:
TABLE-US-00002 (Ih) ##STR00013## Compound R.sub.6 R.sub.7 R.sub.8
R.sub.9 R.sub.10 R.sub.2 R.sub.5 A16 H H CH.sub.3 H H CH.sub.3 H
A17 H CH.sub.3 H H H CH.sub.3 H A18 H H OH H H CH.sub.3 H A19
CH.sub.3 H H H H CH.sub.3 H A20 H H iPr H H CH.sub.3 H A21 H OH H H
H CH.sub.3 H A22 H H Br H H CH.sub.3 H A23 H H Cl H H CH.sub.3 H
A24 H H F H H CH.sub.3 H A25 OH H H H H CH.sub.3 H A26 H H
OCH.sub.3 H H CH.sub.3 H A27 H Cl H H H CH.sub.3 H A28 H Br H H H
CH.sub.3 H A29 H F H H H CH.sub.3 H A30 H H N(CH.sub.3).sub.2 H H
CH.sub.3 H A31 H H SCH.sub.3 H H CH.sub.3 H A32 Cl H H H H CH.sub.3
H A33 F H H H H CH.sub.3 H A34 H H OEt H H CH.sub.3 H A35 Br H H H
H CH.sub.3 H A36 H H COOH H H CH.sub.3 H A37 CH.sub.3 H CH.sub.3 H
CH.sub.3 CH.sub.3 H A38 CH.sub.3 H H CH.sub.3 H CH.sub.3 H A39 H
OCH.sub.3 H H H CH.sub.3 H A40 H H OiPr H H CH.sub.3 H A41
OCH.sub.3 H H H H CH.sub.3 H A42 H H NO.sub.2 H H CH.sub.3 H A43
OEt H H H H CH.sub.3 H A44 H --CH.dbd.CH--O-- H H CH.sub.3 H A45 H
H NEt.sub.2 H H CH.sub.3 H A46 H OH OH H H CH.sub.3 H A47 H
NO.sub.2 H H H CH.sub.3 H A48 Cl H H H Cl CH.sub.3 H A49 OH H OH H
H CH.sub.3 H A50 Cl H H H F CH.sub.3 H A51 H H COOCH.sub.3 H H
CH.sub.3 H A52 H Cl Cl H H CH.sub.3 H A53 H H
--OCH.sub.2CH.dbd.CH.sub.2 H H CH.sub.3 H A54 Cl Cl H H H CH.sub.3
H A55 Cl H Cl H H CH.sub.3 H A56 NO.sub.2 H H H H CH.sub.3 H A57 H
H OAc H H CH.sub.3 H A58 H OH OCH.sub.3 H H CH.sub.3 H A59 H
--OCH.sub.2O-- H H CH.sub.3 H A60 OH H H Br H CH.sub.3 H A61 H
OCH.sub.3 OH H H CH.sub.3 H A62 H H OCH.sub.2Ph H H CH.sub.3 H A63
OH OCH.sub.3 H H H CH.sub.3 H A64 H H --OCH.sub.2Ph(p-Me) H H
CH.sub.3 H A65 OCH.sub.3 H OCH.sub.3 H H CH.sub.3 H A66 H OCH.sub.3
OCH.sub.3 H H CH.sub.3 H A67 Br H OCH.sub.3 H H CH.sub.3 H A68 H H
3-thietyl-methoxy H H CH.sub.3 H A69 OCH.sub.3 H H OMe H CH.sub.3 H
A70 OCH.sub.3 OCH.sub.3 H H H CH.sub.3 H A71 OCH.sub.3 H H Br H
CH.sub.3 H A72 OH Cl H Cl H CH.sub.3 H A73 OCH.sub.2Ph H H H H
CH.sub.3 H A74 H NO.sub.2 Cl H H CH.sub.3 H A75 H OCH.sub.3
OCH.sub.3 OCH.sub.3 H CH.sub.3 H A76 H OH OH H Br CH.sub.3 H A77 H
H --OCH.sub.2Ph(p-Cl) H H CH.sub.3 H A78 --C.ident.CH H
--OCH.sub.2CH.sub.2O-- H CH.sub.3 H A79 Cl H H NO.sub.2 H CH.sub.3
H A80 OH H H NO.sub.2 H CH.sub.3 H A81 H H --OCH.sub.2Ph(o-Cl) H H
CH.sub.3 H A82 H NO.sub.2 OCH.sub.3 H H CH.sub.3 H A83 Cl H Cl H H
CF.sub.3 H A84 H H --OCH.sub.2Ph(p-NO.sub.2) H H CH.sub.3 H A85 Br
H --OCH.sub.2CH.sub.2O-- H CH.sub.3 H A86 Br H --OCH.sub.2O-- H
CH.sub.3 H A87 I H --OCH.sub.2O-- H CH.sub.3 H A88 H OCH.sub.3 OAc
H H CH.sub.3 H A89 H OCH.sub.3 --OCH.sub.2Ph H H CH.sub.3 H A90 Br
--OCH.sub.2O-- H H CH.sub.3 H A91 OAc H OAc H H CH.sub.3 H A92
--C.ident.C-TMS H --OCH.sub.2O-- H CH.sub.3 H A93 H H
--SO.sub.2-morpholino H H CH.sub.3 H A94 H H F H H CH.sub.3 H A95
Br H --OCH.sub.2O-- H CH.sub.3 Me A96 H OEt --OCH.sub.2Ph(p-Cl) H H
CH.sub.3 H A97 NO.sub.2 H --OCH.sub.2O -- H CH.sub.3 H A98 Br H
--OCH.sub.2O -- H CH.sub.3 H A99 H OCH.sub.3 --OCH.sub.2Ph Br H
CH.sub.3 H A100 H OCH.sub.3 --OCH.sub.2Ph(p-Me) Br H CH.sub.3 H
A101 H OCH.sub.3 --OCH.sub.2Ph(o-Cl) Br H CH.sub.3 H A102 Br H
--OCH.sub.2O-- H CH.sub.3 Ac A103 H OCH.sub.3
--OCH.sub.2Ph(m-Cl,p-Cl) Br H CH.sub.3 H A104 H OCH.sub.3
--OCH.sub.2Ph(o-Cl,p-Cl) Br H CH.sub.3 H
[0158] In certain embodiments, the compound is A16. In other
embodiments, the compound is A17. In yet other embodiments, the
compound is A18. In yet other embodiments, the compound is A19. In
yet other embodiments, the compound is A20. In yet other
embodiments, the compound is A21. In yet other embodiments, the
compound is A22. In yet other embodiments, the compound is A23. In
yet other embodiments, the compound is A24. In yet other
embodiments, the compound is A25. In yet other embodiments, the
compound is A26. In yet other embodiments, the compound is A27. In
yet other embodiments, the compound is A28. In yet other
embodiments, the compound is A29. In yet other embodiments, the
compound is A30. In yet other embodiments, the compound is A31. In
yet other embodiments, the compound is A32. In yet other
embodiments, the compound is A33. In yet other embodiments, the
compound is A34. In yet other embodiments, the compound is A35. In
yet other embodiments, the compound is A36. In yet other
embodiments, the compound is A37. In yet other embodiments, the
compound is A38. In yet other embodiments, the compound is A39. In
yet other embodiments, the compound is A40. In yet other
embodiments, the compound is A41. In yet other embodiments, the
compound is A42. In yet other embodiments, the compound is A43. In
yet other embodiments, the compound is A44. In yet other
embodiments, the compound is A45. In yet other embodiments, the
compound is A46. In yet other embodiments, the compound is A47. In
yet other embodiments, the compound is A48. In yet other
embodiments, the compound is A49. In yet other embodiments, the
compound is A50. In yet other embodiments, the compound is A51. In
yet other embodiments, the compound is A52. In yet other
embodiments, the compound is A53. In yet other embodiments, the
compound is A54. In yet other embodiments, the compound is A55. In
yet other embodiments, the compound is A56. In yet other
embodiments, the compound is A57. In yet other embodiments, the
compound is A58. In yet other embodiments, the compound is A59. In
yet other embodiments, the compound is A60. In yet other
embodiments, the compound is A61. In yet other embodiments, the
compound is A62. In yet other embodiments, the compound is A63. In
yet other embodiments, the compound is A64. In yet other
embodiments, the compound is A65. In yet other embodiments, the
compound is A66. In yet other embodiments, the compound is A67. In
yet other embodiments, the compound is A68. In yet other
embodiments, the compound is A69. In yet other embodiments, the
compound is A70. In yet other embodiments, the compound is A71. In
yet other embodiments, the compound is A72. In yet other
embodiments, the compound is A73. In yet other embodiments, the
compound is A74. In yet other embodiments, the compound is A75. In
yet other embodiments, the compound is A76. In yet other
embodiments, the compound is A77. In yet other embodiments, the
compound is A78. In yet other embodiments, the compound is A79. In
yet other embodiments, the compound is A80. In yet other
embodiments, the compound is A81. In yet other embodiments, the
compound is A82. In yet other embodiments, the compound is A83. In
yet other embodiments, the compound is A84. In yet other
embodiments, the compound is A85. In yet other embodiments, the
compound is A86. In yet other embodiments, the compound is A87. In
yet other embodiments, the compound is A88. In yet other
embodiments, the compound is A89. In yet other embodiments, the
compound is A90. In yet other embodiments, the compound is A91. In
yet other embodiments, the compound is A92. In yet other
embodiments, the compound is A93. In yet other embodiments, the
compound is A94. In yet other embodiments, the compound is A95. In
yet other embodiments, the compound is A96. In yet other
embodiments, the compound is A97. In yet other embodiments, the
compound is A98. In yet other embodiments, the compound is A99. In
yet other embodiments, the compound is A100. In yet other
embodiments, the compound is A101. In yet other embodiments, the
compound is A102. In yet other embodiments, the compound is A103.
In yet other embodiments, the compound is A104.
[0159] In certain embodiments, the compound is not A16. In other
embodiments, the compound is not A17. In yet other embodiments, the
compound is not A18. In yet other embodiments, the compound is not
A19. In yet other embodiments, the compound is not A20. In yet
other embodiments, the compound is not A21. In yet other
embodiments, the compound is not A22. In yet other embodiments, the
compound is not A23. In yet other embodiments, the compound is not
A24. In yet other embodiments, the compound is not A25. In yet
other embodiments, the compound is not A26. In yet other
embodiments, the compound is not A27. In yet other embodiments, the
compound is not A28. In yet other embodiments, the compound is not
A29. In yet other embodiments, the compound is not A30. In yet
other embodiments, the compound is not A31. In yet other
embodiments, the compound is not A32. In yet other embodiments, the
compound is not A33. In yet other embodiments, the compound is not
A34. In yet other embodiments, the compound is not A35. In yet
other embodiments, the compound is not A36. In yet other
embodiments, the compound is not A37. In yet other embodiments, the
compound is not A38. In yet other embodiments, the compound is not
A39. In yet other embodiments, the compound is not A40. In yet
other embodiments, the compound is not A41. In yet other
embodiments, the compound is not A42. In yet other embodiments, the
compound is not A43. In yet other embodiments, the compound is not
A44. In yet other embodiments, the compound is not A45. In yet
other embodiments, the compound is not A46. In yet other
embodiments, the compound is not A47. In yet other embodiments, the
compound is not A48. In yet other embodiments, the compound is not
A49. In yet other embodiments, the compound is not A50. In yet
other embodiments, the compound is not A51. In yet other
embodiments, the compound is not A52. In yet other embodiments, the
compound is not A53. In yet other embodiments, the compound is not
A54. In yet other embodiments, the compound is not A55. In yet
other embodiments, the compound is not A56. In yet other
embodiments, the compound is not A57. In yet other embodiments, the
compound is not A58. In yet other embodiments, the compound is not
A59. In yet other embodiments, the compound is not A60. In yet
other embodiments, the compound is not A61. In yet other
embodiments, the compound is not A62. In yet other embodiments, the
compound is not A63. In yet other embodiments, the compound is not
A64. In yet other embodiments, the compound is not A65. In yet
other embodiments, the compound is not A66. In yet other
embodiments, the compound is not A67. In yet other embodiments, the
compound is not A68. In yet other embodiments, the compound is not
A69. In yet other embodiments, the compound is not A70. In yet
other embodiments, the compound is not A71. In yet other
embodiments, the compound is not A72. In yet other embodiments, the
compound is not A73. In yet other embodiments, the compound is not
A74. In yet other embodiments, the compound is not A75. In yet
other embodiments, the compound is not A76. In yet other
embodiments, the compound is not A77. In yet other embodiments, the
compound is not A78. In yet other embodiments, the compound is not
A79. In yet other embodiments, the compound is not A80. In yet
other embodiments, the compound is not A81. In yet other
embodiments, the compound is not A82. In yet other embodiments, the
compound is not A83. In yet other embodiments, the compound is not
A84. In yet other embodiments, the compound is not A85. In yet
other embodiments, the compound is not A86. In yet other
embodiments, the compound is not A87. In yet other embodiments, the
compound is not A88. In yet other embodiments, the compound is not
A89. In yet other embodiments, the compound is not A90. In yet
other embodiments, the compound is not A91. In yet other
embodiments, the compound is not A92. In yet other embodiments, the
compound is not A93. In yet other embodiments, the compound is not
A94. In yet other embodiments, the compound is not A95. In yet
other embodiments, the compound is not A96. In yet other
embodiments, the compound is not A97. In yet other embodiments, the
compound is not A98. In yet other embodiments, the compound is not
A99. In yet other embodiments, the compound is not A100. In yet
other embodiments, the compound is not A101. In yet other
embodiments, the compound is not A102. In yet other embodiments,
the compound is not A103. In yet other embodiments, the compound is
not A104.
[0160] In certain embodiments, the compound of the invention, or a
salt, solvate, tautomer, enantiomer or diastereoisomer thereof, is
at least one compound selected from the group consisting of:
##STR00014##
In other embodiments, the compound is not A105. In other
embodiments, the compound is not A106.
[0161] In certain embodiments, the compound of the invention, or a
salt, solvate, tautomer, enantiomer or diastereoisomer thereof,
is
##STR00015##
In other embodiments, the compound is not A107.
[0162] In certain embodiments, the compound is at least one
selected from the group consisting of: [0163] G1 or G-1
(rel-1-[4-(6-bromo-1,3-benzodioxol-5-yl)-3aR,4S,5,9bS-tetrahydro-3H-cyclo-
penta[c]quinolin-8-yl]-ethanone):
[0163] ##STR00016## [0164] CMPD1
(rel-1-((3aS,4R,9bR)-4-(benzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-
-cyclopenta[c]quinolin-8-yl)ethan-1-one):
[0164] ##STR00017## [0165] CMPD2
(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-5-methyl-3a,4,5,-
9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one):
[0165] ##STR00018## [0166] CMPD3
(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-2,3,3a,4,5,9b-he-
xahydro-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one):
[0166] ##STR00019## [0167] CMPD4
(rel-1-((3aS,4R,9bR)-5-benzyl-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,-
9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one):
[0167] ##STR00020## [0168] CMPD5
(rel-1-((3aS,4R,9bR)-4-(2-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta-
[c]quinolin-8-yl)ethan-1-one):
[0168] ##STR00021## [0169] CMPD6
(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrah-
ydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one oxime):
[0169] ##STR00022## [0170] CMPD7
(rel-1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-3a,4,5,9b-tetrahydro-
-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one):
[0170] ##STR00023## [0171] CMPD8
(rel-1-((3aS,4R,9bR)-4-(6-chlorobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetra-
hydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one):
[0171] ##STR00024## [0172] CMPD9
(rel-1-((6R,6aS,7aS,10aR,10bR)-6-(6-bromobenzo[d][1,3]dioxol-5-yl)-9,9-di-
methyl-6,6a,7,7a,10a,10b-hexahydro-5H-[1,3]dioxolo[4',5':3,4]cyclopenta[1,-
2-c]quinolin-2-yl)ethan-1-one):
[0172] ##STR00025## [0173] CMPD10
(rel-1-((1R,2S,3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-1,2-dihydr-
oxy-2,3,3a,4,5,9b-hexahydro-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one):
[0173] ##STR00026## [0174] CMPD11
(rel-1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-2,3,3a,4,5,9b-hexahy-
dro-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one):
##STR00027##
[0175] In certain embodiments, the compound of the invention, or a
salt, solvate, tautomer, enantiomer or diastereoisomer thereof, is
at least one compound of formula (II):
##STR00028##
wherein:
[0176] R.sub.1 is selected from the group consisting of .dbd.O and
.dbd.N--OH;
[0177] R.sub.2 is C.sub.1-C.sub.4 alkyl;
[0178] R.sub.5 is selected from the group consisting of H, benzyl
and C.sub.1-C.sub.4 alkyl;
[0179] R.sub.8 and R.sub.9 are independently selected from the
group consisting of H and C.sub.1-C.sub.4 alkoxy, or R.sub.8 and
R.sub.9 combine to form a diradical selected from the group
consisting of --OCH.sub.2O--, --OCH(CH.sub.3)O-- and
--OC(CH.sub.3).sub.2O--.
[0180] In certain embodiments, the compound of formula (II) is
[0181] CMPD12
(rel-1-((4S,5aS,6R,11aR)-4,5,5a,6,11,11a-hexahydro-4,6-methano[1,3-
]dioxolo[4',5':5,6]benzo[1,2-c]acridin-8-yl)ethan-1-one):
##STR00029##
[0182] In certain embodiments, the compound useful within the
methods of the invention, or a salt, solvate, enantiomer or
diastereoisomer thereof, is at least one GPER agonist recited in
U.S. Patent Application Publications No. US 2008/0167334 and US
2011/0092533, all of which are incorporated herein in their
entireties by reference:
##STR00030##
wherein:
[0183] X is .dbd.N--, O, S, or N--R, with the proviso that when X
is N--R and R is a bond, N together with R.sup.1 forms a 5- to
7-membered optionally substituted heterocyclic group;
[0184] R is a bond, H, --OH, --NO.sub.2, optionally substituted
C.sub.1-C.sub.6 hydrocarbyl (such as optionally substituted alkyl),
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted heterocycle, optionally substituted
--C(O)--(C.sub.1-C.sub.6) alkyl (amide), optionally substituted
--C(O)--O--(C.sub.1-C.sub.6) alkyl (urethane), optionally
substituted --C(O)--NH(C.sub.1-C.sub.6) alkyl (urea), optionally
substituted --C(O)--N(C.sub.1-C.sub.6)dialkyl, optionally
substituted --C(O)--NH(aryl), optionally substituted
--C(O)--N(diaryl), optionally substituted --C(O)--NH(heteroaryl),
optionally substituted --C(O)--N(diheteroaryl), optionally
substituted --C(O)--NH(heterocycle) or optionally substituted
--C(O)--N(diheterocycle);
[0185] R.sup.1, R.sup.2 and R.sup.5 are each independently selected
from H, --OH, --NO.sub.2, halogen, C.sub.1-C.sub.6 optionally
substituted carboxylic acid group, optionally substituted
O--(C.sub.1-C.sub.6)alkyl, optionally substituted C.sub.1-C.sub.6
hydrocarbyl optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocycle, optionally
substituted --C(O)--(C.sub.1-C.sub.6) alkyl (ketone), optionally
substituted --C(O)--O--(C.sub.1-C.sub.6) alkyl (ester), optionally
substituted O--C(O)--(C.sub.1-C.sub.6) alkyl (ester), optionally
substituted --C(O)--NH(C.sub.1-C.sub.6) alkyl (urea), optionally
substituted --C(O)--N(C.sub.1-C.sub.6)dialkyl, optionally
substituted --C(O)--NH(aryl), optionally substituted
--C(O)--N(diaryl), optionally substituted --C(O)--NH(heteroaryl),
optionally substituted --C(O)--N(diheteroaryl), optionally
substituted --C(O)--NH(heterocycle) or optionally substituted
--C(O)--N(diheterocycle);
[0186] R.sup.3 and R.sup.4 are each independently selected from H,
--OH, --NO.sub.2, halogen, C.sub.1-C.sub.6 optionally substituted
carboxylic acid group, optionally substituted
O--(C.sub.1-C.sub.6)alkyl, optionally substituted C.sub.1-C.sub.6
hydrocarbyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocycle, optionally
substituted --C(O)--(C.sub.1-C.sub.6) alkyl (ketone), optionally
substituted --C(O)--O--(C.sub.1-C.sub.6) alkyl (ester), optionally
substituted O--C(O)--(C.sub.1-C.sub.6) alkyl (ester), optionally
substituted --C(O)--NH(C.sub.1-C.sub.6) alkyl (urea), optionally
substituted --C(O)--N(C.sub.1-C.sub.6)dialkyl, optionally
substituted --C(O)--NH(aryl), optionally substituted
--C(O)--N(diaryl), optionally substituted --C(O)--NH(heteroaryl),
optionally substituted --C(O)--N(diheteroaryl), optionally
substituted --C(O)--NH(heterocycle) or optionally substituted
--C(O)--N(diheteroaryl) or R.sup.3 and R.sup.4 together form a 5-
or 6-membered optionally substituted carbocyclic (which may be
saturated or unsaturated), optionally substituted aryl, optionally
substituted heteroaryl or optionally substituted heterocyclic
group;
[0187] R.sup.6 and R.sup.7 are each independently absent or are
selected from H, --OH, --NO.sub.2, halogen, C.sub.1-C.sub.6
optionally substituted carboxylic acid group, optionally
substituted O--(C.sub.1-C.sub.6)alkyl, optionally substituted
C.sub.1-C.sub.6 hydrocarbyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted
heterocycle, optionally substituted --C(O)--(C.sub.1-C.sub.6) alkyl
(ketone), optionally substituted --C(O)--O--(C.sub.1-C.sub.6) alkyl
(ester), optionally substituted O--C(O)--(C.sub.1-C.sub.6) alkyl
(ester), optionally substituted --C(O)--NH(C.sub.1-C.sub.6) alkyl
(urea), optionally substituted --C(O)--N(C.sub.1-C.sub.6)dialkyl,
optionally substituted --C(O)--NH(aryl), optionally substituted
--C(O)--N(diaryl), optionally substituted --C(O)--NH(heteroaryl),
optionally substituted --C(O)--N(diheteroaryl), optionally
substituted --C(O)--NH(heterocycle) or optionally substituted
--C(O)--N(diheteroaryl), or together R.sup.6 and R.sup.7 form a 4-,
5-, 6- or 7-membered optionally substituted carbocyclic (which may
be saturated or unsaturated), optionally substituted aryl,
optionally substituted heteroaryl or optionally substituted
heterocyclic group, or a 5- to 9-membered optionally substituted
carbocyclic or heterocyclic bicyclic group, with the proviso that
R.sup.7 is not absent when both R.sup.7' and R.sup.7'' are also
absent;
[0188] R.sup.6' is absent, H, C.sub.1-C.sub.6 optionally
substituted hydrocarbyl group (such as H, CH.sub.3 or
CH.sub.2CH.sub.3) or together with R.sup.6 forms .dbd.O;
[0189] R.sup.7' is absent, H, optionally substituted hydrocarbyl
group (such as H, CH.sub.3 or CH.sub.2CH.sub.3), or together with R
forms .dbd.O;
[0190] R.sup.7'' is absent, H, --OH, halogen, optionally
substituted O--(C.sub.1-C.sub.6)alkyl, optionally substituted
C.sub.1-C.sub.6 hydrocarbyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted
heterocycle, optionally substituted --C(O)--(C.sub.1-C.sub.6) alkyl
(ketone), optionally substituted --C(O)--O--(C.sub.1-C.sub.6) alkyl
(ester), optionally substituted O--C(O)--(C.sub.1-C.sub.6) alkyl
(ester), optionally substituted --C(O)--NH(C.sub.1-C.sub.6) alkyl
(urea), optionally substituted --C(O)--N(C.sub.1-C.sub.6)dialkyl,
optionally substituted --C(O)--NH(aryl), optionally substituted
--C(O)--N(diaryl), optionally substituted --C(O)--NH(heteroaryl),
optionally substituted --C(O)--N(diheteroaryl), optionally
substituted --C(O)--NH(heterocycle) or optionally substituted
--C(O)--N(diheteroaryl);
[0191] R.sup.8' is absent (when the carbon to which R.sup.8' is
attached and the carbon to which R.sup.6 is attached form an
optional double bond), H, CH.sub.3 or CH.sub.2CH.sub.3;
[0192] R.sup.10, R.sup.11, R.sup.12 and R.sup.13 are each
independently H, --OH, --NO.sub.2, halogen, C.sub.1-C.sub.6
optionally substituted carboxylic acid group, optionally
substituted O--(C.sub.1-C.sub.6)alkyl, optionally substituted
C.sub.1-C.sub.6 hydrocarbyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted
heterocycle, optionally substituted --C(O)--(C.sub.1-C.sub.6) alkyl
(ketone), optionally substituted --C(O)--O--(C.sub.1-C.sub.6) alkyl
(ester), optionally substituted O--C(O)--(C.sub.1-C.sub.6) alkyl
(ester), optionally substituted --C(O)--NH(C.sub.1-C.sub.6) alkyl
(urea), optionally substituted --C(O)--N(C.sub.1-C.sub.6)dialkyl,
optionally substituted --C(O)--NH(aryl), optionally substituted
--C(O)--N(diaryl), optionally substituted --C(O)--NH(heteroaryl),
optionally substituted --C(O)--N(diheteroaryl), optionally
substituted --C(O)--NH(heterocycle) or optionally substituted
--C(O)--N(diheteroaryl);
[0193] R.sup.14 is H, --OH, --NO.sub.2, halogen, C.sub.1-C.sub.6
optionally substituted carboxylic acid group, optionally
substituted O--(C.sub.1-C.sub.6)alkyl, optionally substituted
C.sub.1-C.sub.6 hydrocarbyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted
heterocycle, optionally substituted --C(O)--(C.sub.1-C.sub.6) alkyl
(ketone), optionally substituted --C(O)--O--(C.sub.1-C.sub.6) alkyl
(ester), optionally substituted O--C(O)--(C.sub.1-C.sub.6) alkyl
(ester), optionally substituted --C(O)--NH(C.sub.1-C.sub.6) alkyl
(urea), optionally substituted --C(O)--N(C.sub.1-C.sub.6)dialkyl,
optionally substituted --C(O)--NH(aryl), optionally substituted
--C(O)--N(diaryl), optionally substituted --C(O)--NH(heteroaryl),
optionally substituted --C(O)--N(diheteroaryl), optionally
substituted --C(O)--NH(heterocycle) or optionally substituted
--C(O)--N(diheterocycle) or together with the carbon to which
R.sup.7 is attached forms a 5-, 6- or 7-membered optionally
substituted carbocyclic (which may be saturated or unsaturated),
optionally substituted aryl, optionally substituted heteroaryl or
optionally substituted heterocyclic ring;
[0194] Y is optionally substituted (CH.sub.2).sub.n group where n
is 0, 1 or 2, optionally substituted .dbd.CH-- group, a C.dbd.O
group, O, S, optionally substituted N--(C.sub.1-C.sub.6)alkyl,
optionally substituted N-aryl, optionally substituted N-heteroaryl,
optionally substituted N-heterocycle, optionally substituted
N--C(O)--(C.sub.1-C.sub.6)alkyl, optionally substituted
N--C(O)-aryl, optionally substituted N--C(O)-heteroaryl, optionally
substituted N--C(O)-heterocycle;
[0195] Z is optionally substituted (CH.sub.2).sub.n group where n
is 1 or 2, optionally substituted .dbd.CH-- group, a C.dbd.O group,
O, S, optionally substituted N--(C.sub.1-C.sub.6)alkyl, optionally
substituted N-aryl, optionally substituted N-heteroaryl, optionally
substituted N-heterocycle, optionally substituted
N--C(O)--(C.sub.1-C.sub.6)alkyl, optionally substituted
N--C(O)-aryl, optionally substituted N--C(O)-heteroaryl, optionally
substituted N--C(O)-heterocycle.
[0196] In certain embodiments, the compound of the invention, or a
salt, solvate, enantiomer or diastereoisomer thereof, is at least
one GPER agonist recited in PCT Patent Application No. WO
2016/014847, which is incorporated herein in its entirety by
reference:
##STR00031##
wherein in (i)-(iv):
[0197] Ring A is an aromatic or heteroaromatic five or six membered
ring containing one or more heteroatom such as N, O, or S;
[0198] R.sub.1 is independently selected from SO.sub.2NH.sub.2,
SO.sub.2NR.sub.aR.sub.b, COOH, CONH.sub.2 and CONR.sub.aR.sub.b,
and H. In R.sub.1, each occurrence of R.sub.a and R.sub.b is
independently selected from H, alkyl (C.sub.1-C.sub.6), alkenyl
(C.sub.2-C.sub.6), alkynyl (C.sub.2-C.sub.6), alkoxy
(C.sub.2-C.sub.6), cycloalkyl (C.sub.3-C.sub.7), alkylthio,
alkylaryl, and aromatic and heteroaromatic rings. The aromatic and
heteroaromatic rings can be further substituted with electron
withdrawing and donating groups. R.sub.a and R.sub.b can form a
cyclic ring (C.sub.3-C.sub.7) or an aromatic ring optionally
containing one or more heteroatoms. Such aromatic rings can be
further substituted with electron withdrawing groups such as
halogens, --COOH, --CN, --NO.sub.2 and the like, or electron
donating groups such as alkyl groups;
[0199] R.sub.2 is H, halogen, or a heteroatom such as N, O, or
S;
[0200] Ring A is an aromatic or heteroaromatic ring (5 or 6
membered);
[0201] Ring B is a six membered saturated or aromatic ring
containing N at the indicated position. The nitrogen of ring B
optionally can be substituted with an alkyl, aryl, or alkaryl
substituent;
[0202] Ring C is an independently a substituted or unsubstituted
carbocyclic ring, bicyclic ring, aromatic ring, fused aromatic
rings, or a heteroaromatic ring. Additionally, when it is a
carbocyclic ring, it may contain one or more double bonds and one
or more heteroatoms such as N, O, or S. It may also have an
.alpha.-.beta. unsaturated ketone function;
[0203] R.sub.3 is selected independently from H, halogen, --OH, CN,
(C.sub.1-C.sub.6) alkyl, (C.sub.2-C.sub.6) alkenyl,
(C.sub.2-C.sub.6) alkynyl, (C.sub.1-C.sub.6) alkoxy,
(C.sub.3-C.sub.7) cycloalkyl, (C.sub.1-C.sub.6) alkylthio,
NR.sub.aR.sub.b, R.sub.aR.sub.b or haloalkyl (e.g., CF.sub.3). In
R.sub.3, each occurrence of R.sub.a and R.sub.b is independently
hydrogen or (C.sub.1-C.sub.6) alkyl, or R.sub.a and R.sub.b form a
saturated or unsaturated heterocyclic ring containing from 3-7 ring
atoms, which ring may optionally contain another heteroatom
selected from N, O, and S, and may be optionally substituted by
from 1-3 groups which may be the same or different and are selected
from (C.sub.1-C.sub.4) alkyl, phenyl, and benzyl; and m is 1-4:
[0204] Rings B and C are in certain embodiments cis fused:
[0205] Rings D and B is directly connected or connected through a
spacer (C.sub.1-C.sub.2). When connected directly, they can be cis
or trans with respect to the fusion of Ring B and C. Ring D is an
aromatic or heteroaromatic ring containing one or more heteroatoms
such as N, O, or S. It can be optionally substituted with R.sub.5
and R.sub.6 groups selected independently, or with --R.sub.6R.sub.5
or R.sub.5R.sub.6;
[0206] R.sub.5 is independently selected from H, halogen, electron
donating groups, and electron withdrawing groups such as alkyl,
haloalkyl, alkoxy, --NO.sub.2, --SF.sub.5, --CN, and the like;
[0207] R.sub.6 can be NHC(O)OR.sub.c, OC(O)NHR.sub.c,
C(O)O(CH.sub.2).sub.nR.sub.c, OC(O)(CH.sub.2).sub.nR.sub.c,
C(O)NHR.sub.c, or NHC(O)R.sub.c, where n=0-4; or R.sub.6 can be
alkyl, branched alkyl (C.sub.1-C.sub.10), alkynyl
(C.sub.1-C.sub.10), a carbocyclic ring, alkenyl (C.sub.1-C.sub.10),
halogen, CN, COOH, CONH.sub.2, --OH, or NH.sub.2. R.sub.c is alkyl,
branched alkyl (C.sub.1-C.sub.10), alkoxy, alkylamino, acyl,
alkynyl (C.sub.1-C.sub.8) or alkenyl (C.sub.1-C.sub.6). R.sub.6 can
also be X(CH.sub.2).sub.n-E, wherein X is NH, O, S, C.ident.C, or
HC.dbd.CH, and n=0-2, and E is independently a substituted or
unsubstituted carbocyclic ring, bicyclic ring, aromatic ring, fused
aromatic rings or heteroaromatic ring;
[0208] in certain embodiments, R.sub.1 is selected from the group
consisting of carboxyl, carboxamide, carboxyalkyl, carboxyaryl,
cyano, nitro, hydroxyl, sulfonyl, sulfonamide, alkylsulfonamide,
arylsulfonamide, alkylsulfonyl, aralkylsulfonamide,
trifluoromethylsulfonamide, trifluoromethylsulfonyl carboxamide,
and sulfonylcarbamide. In certain embodiments, R.sub.1 is
sulfonamide, alkylsulfonamide, or arylsulfonamide. R.sub.2 can be a
3-, 4-, 5-, or 6-membered saturated or aromatic carbon ring or ring
system optionally containing one or two heteroatoms selected from
N, O, and S, the ring or ring system optionally substituted with
one or more substituents selected from the group consisting of
cyano, halo, acyl, acyloxy, alkyl, alkoxy, heteroalkyl, alkylester,
alkylamido, alkylamino, aryl, aryloxy, arylalkyl, arylester, azido,
alkylhalo, alkenyl, alkynyl, alkyl ether, nitro, thiohalo, and
thiocyano. R.sub.3 can be H, or a C.sub.1-C.sub.5 alkyl or
cycloalkyl group optionally substituted with one or more of cyano,
nitro, and one or more aromatic or heteroaromatic groups containing
N, O, or S. A and D can be independently selected from CH,
CH.sub.2, N, and O, and the bond joining them is a single or double
bond as appropriate for the selected atoms. X, Y, and Z are
independently selected from no atom (i.e., they are absent), CH,
C-halogen, N, O, and S;
[0209] in certain embodiments, R.sub.2 is a substituent represented
by --R.sub.dR.sub.eRf or by --R.sub.dCOR.sub.eRf; wherein R.sub.d,
R.sub.e, and R.sub.f are independently selected from a 3-, 4-, 5-,
or 6-membered saturated or aromatic carbon ring or ring system
optionally containing one or two heteroatoms selected from N, O,
and S, the ring or ring system optionally substituted with one or
more substituents selected from the group consisting of cyano,
halo, acyl, acyloxy, alkyl, alkoxy, heteroalkyl, alkylester,
alkylamido, alkylamino, aryl, aryloxy, arylalkyl, arylester, azido,
alkylhalo, alkenyl, alkynyl, alkyl ether, nitro, thiohalo, and
thiocyano;
[0210] Compounds of the invention may be prepared according to
methods described herein, methods known in the art, and/or methods
described in certain references, such as but not limited to: PCT
Application Publications No. WO 2004/072046 and No. WO 2016/014847;
U.S. application Ser. No. 10/511,083; U.S. Patent Application
Publications No. US 2008/0167334 and US 2011/0092533; and Burai, et
al., 2010, Org. & Biomol. Chem. 8:2252-2259; all of which are
included herein in their entireties by reference.
[0211] The compounds of the invention may possess one or more
stereocenters, and each stereocenter may exist independently in
either the (R) or (S) configuration. In certain embodiments,
compounds described herein are present in optically active or
racemic forms. The compounds described herein encompass racemic,
optically active, regioisomeric and stereoisomeric forms, or
combinations thereof that possess the therapeutically useful
properties described herein. Preparation of optically active forms
is achieved in any suitable manner, including by way of
non-limiting example, by resolution of the racemic form with
recrystallization techniques, synthesis from optically active
starting materials, chiral synthesis, or chromatographic separation
using a chiral stationary phase. A compound illustrated herein by
the racemic formula further represents either of the two
enantiomers or mixtures thereof, or in the case where two or more
chiral center are present, all diastereomers or mixtures
thereof.
[0212] In certain embodiments, the compounds of the invention exist
as tautomers. All tautomers are included within the scope of the
compounds recited herein.
[0213] Compounds described herein also include isotopically labeled
compounds wherein one or more atoms is replaced by an atom having
the same atomic number, but an atomic mass or mass number different
from the atomic mass or mass number usually found in nature.
Examples of isotopes suitable for inclusion in the compounds
described herein include and are not limited to .sup.2H, .sup.3H,
.sup.11C, .sup.13C, .sup.14C, .sup.36Cl, .sup.18F, .sup.123I,
.sup.125I, .sup.13N, .sup.15N, .sup.15O, .sup.17O, .sup.18O,
.sup.32P, and .sup.35S. In certain embodiments, substitution with
heavier isotopes such as deuterium affords greater chemical
stability. Isotopically labeled compounds are prepared by any
suitable method or by processes using an appropriate isotopically
labeled reagent in place of the non-labeled reagent otherwise
employed.
[0214] In certain embodiments, the compounds described herein are
labeled by other means, including, but not limited to, the use of
chromophores or fluorescent moieties, bioluminescent labels, or
chemiluminescent labels.
[0215] In all of the embodiments provided herein, examples of
suitable optional substituents are not intended to limit the scope
of the claimed invention. The compounds of the invention may
contain any of the substituents, or combinations of substituents,
provided herein.
[0216] In certain embodiments, the invention further provides
pharmaceutical compositions comprising at least one compound of the
invention and at least one pharmaceutically acceptable carrier. In
other embodiments, the pharmaceutical composition is formulated for
topical and/or transdermal application. Topical formulations of the
compounds contemplated within the invention may be used for skin
darkening, as well as for treating skin conditions or diseases
associated with hypopigmentation. These conditions or diseases
include, but are not limited to, acne, eczema, chemical, sun and
thermal burn scars, and lupus.
[0217] In certain embodiments, the invention provides topical
formulations comprising at least one sun-blocking agent and at
least one compound of the invention. In other embodiments, the
topical formulation comprises a formulated sunblock or sunscreen
lotion and at least one compound of the invention. In yet other
embodiments, the at least one sun-blocking agent allows for
protection against UV light damage caused by the natural sun light,
and the one or more compounds of the invention allow for darkening
of the skin. In yet other embodiments, the melanin production
triggered by the at least one compound of the invention further
protects the skin against UV light damage caused by the natural sun
light. In yet other embodiments, the topical formulation protects
against UV-induced skin damage and/or aging.
[0218] The present invention also pertains to kits useful within
any of the methods of the invention described herein. Such kits
comprise components useful in any of the methods described herein,
including for example, compositions and methods for modulating skin
pigmentation in a mammalian subject, such as a human, one or more
containers (e.g., test tube, cell culture dish, cell culture plate,
cell culture flask, cell culture bag) for containing a component of
any of the embodiments of the invention described elsewhere herein,
and instructional materials.
Salts
[0219] The compounds described herein may form salts with acids or
bases, and such salts are included in the present invention. The
term "salts" embraces addition salts of free acids or bases that
are useful within the methods of the invention. The term
"pharmaceutically acceptable salt" refers to salts that possess
toxicity profiles within a range that affords utility in
pharmaceutical applications. In certain embodiments, the salts are
pharmaceutically acceptable salts. Pharmaceutically unacceptable
salts may nonetheless possess properties such as high
crystallinity, which have utility in the practice of the present
invention, such as for example utility in process of synthesis,
purification or formulation of compounds useful within the methods
of the invention.
[0220] Suitable pharmaceutically acceptable acid addition salts may
be prepared from an inorganic acid or from an organic acid.
Examples of inorganic acids include sulfate, hydrogen sulfate,
hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric,
and phosphoric acids (including hydrogen phosphate and dihydrogen
phosphate). Appropriate organic acids may be selected from
aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,
carboxylic and sulfonic classes of organic acids, examples of which
include formic, acetic, propionic, succinic, glycolic, gluconic,
lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,
fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,
4-hydroxybenzoic, phenylacetic, mandelic, embonic (or pamoic),
methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,
sulfanilic, 2-hydroxyethanesulfonic, trifluoromethanesulfonic,
p-toluenesulfonic, cyclohexylaminosulfonic, stearic, alginic,
(3-hydroxybutyric, salicylic, galactaric, galacturonic acid,
glycerophosphonic acids and saccharin (e.g., saccharinate,
saccharate). Salts may be comprised of a fraction of one, one or
more than one molar equivalent of acid or base with respect to any
compound of the invention.
[0221] Suitable pharmaceutically acceptable base addition salts of
compounds of the invention include, for example, ammonium salts and
metallic salts including alkali metal, alkaline earth metal and
transition metal salts such as, for example, calcium, magnesium,
potassium, sodium and zinc salts. Pharmaceutically acceptable base
addition salts also include organic salts made from basic amines
such as, for example, N,N'-dibenzylethylene-diamine,
chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine
(or N-methylglucamine) and procaine. All of these salts may be
prepared from the corresponding compound by reacting, for example,
the appropriate acid or base with the compound.
Methods
[0222] In one aspect, the invention includes a method of modulating
skin pigmentation in a mammalian subject, such as a human. In
certain embodiments, the method of the invention treats or prevents
skin hypopigmentation in the subject. In other embodiments, the
compound is administered topically or transdermally to the
subject.
[0223] In certain embodiments, the method comprises administering
to the subject a therapeutically effective amount of a compound
that binds to a non-classical hormone receptor present on a
melanocyte and does not bind to a canonical sex steroid hormone
receptor, such as the estrogen receptor. In other embodiments, the
non-classical hormone receptor comprises GPER. In yet other
embodiments, the non-classical hormone receptor comprises GPER1. In
yet other embodiments, the compound is a GPER agonist and
increases, or prevents further loss of, skin pigmentation.
[0224] In certain embodiments, the subject is suffering from a
condition comprising at least one selected from the group
consisting of pigmentary changes associated with oral contraceptive
use, pregnancy, and endogenous estrogens in females (such as
melasma and chloasma); solar lentigo; acne; eczema; chemical, sun,
and thermal burn scars; and lupus.
Formulations/Administration
[0225] The relative amounts of the active ingredient, the
pharmaceutically acceptable carrier, and any additional ingredients
in a pharmaceutical composition of the invention will vary,
depending upon the identity, size, and condition of the subject
treated. By way of example, the composition may comprise between
about 0.005% and about 100% (w/w) of the active agent, or any
fractions or multiples thereof.
[0226] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions that are suitable for ethical administration to
humans, it will be understood by the skilled artisan that such
compositions are generally suitable for administration to animals
of all sorts. Modification of pharmaceutical compositions suitable
for administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design and
perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the
pharmaceutical compositions of the invention is contemplated
include, but are not limited to, humans and other primates, mammals
including commercially relevant mammals such as cattle, pigs,
horses, sheep, cats, and dogs.
[0227] In certain embodiments, the compositions comprising a
compound contemplated within the invention are formulated using one
or more pharmaceutically acceptable excipients or carriers. In
certain embodiments, the pharmaceutical compositions of the
invention comprise a therapeutically effective amount of at least
one compound contemplated within the invention and a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers, which are useful, include, but are not limited to,
glycerol, water, saline, ethanol and other pharmaceutically
acceptable salt solutions such as phosphates and salts of organic
acids. Examples of these and other pharmaceutically acceptable
carriers are described in Remington's Pharmaceutical Sciences
(1991, Mack Publication Co., New Jersey).
[0228] The carrier may be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity may be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of the action of microorganisms may be achieved by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In
many cases, isotonic agents, for example, sugars, sodium chloride,
or polyalcohols such as mannitol and sorbitol, are included in the
composition. Prolonged absorption of the injectable compositions
may be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate or
gelatin.
[0229] Formulations may be employed in admixtures with conventional
excipients, i.e., pharmaceutically acceptable organic or inorganic
carrier substances suitable for oral, parenteral, nasal,
intravenous, subcutaneous, enteral, or any other suitable mode of
administration, known to the art. In certain embodiments, the
administration comprises topical administration. The pharmaceutical
preparations may be sterilized and if desired mixed with auxiliary
agents, e.g., lubricants, preservatives, stabilizers, wetting
agents, emulsifiers, salts for influencing osmotic pressure
buffers, coloring, flavoring and/or aromatic substances and the
like. They may also be combined where desired with other active
agents, e.g., other analgesic agents.
[0230] As used herein, "additional ingredients" include, but are
not limited to, one or more of the following: excipients; surface
active agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" that may be
included in the pharmaceutical compositions of the invention are
known in the art and described, for example in Genaro, ed. (1985,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa.), which is incorporated herein by reference.
[0231] The composition of the invention may comprise a preservative
from about 0.005% to 2.0% by total weight of the composition. The
preservative is used to prevent spoilage in the case of exposure to
contaminants in the environment. Examples of preservatives useful
in accordance with the invention included but are not limited to
those selected from the group consisting of benzyl alcohol, sorbic
acid, parabens, imidurea and combinations thereof. An illustrative
preservative is a combination of about 0.5% to 2.0% benzyl alcohol
and 0.05% to 0.5% sorbic acid.
[0232] The composition may include an antioxidant and a chelating
agent that inhibit the degradation of the compound. Selected
antioxidants for some compounds are BHT, BHA, .alpha.-tocopherol
and ascorbic acid in the exemplary range of about 0.01% to 0.3%,
such as BHT in the range of 0.03% to 0.1% by weight by total weight
of the composition. For example, the chelating agent is present in
an amount of from 0.01% to 0.5% by weight by total weight of the
composition. Illustrative chelating agents include edetate salts
(e.g. disodium edetate) and citric acid in the weight range of
about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight
by total weight of the composition. The chelating agent is useful
for chelating metal ions in the composition which may be
detrimental to the shelf life of the formulation. While BHT and
disodium edetate are illustrative antioxidant and chelating agent
respectively for some compounds, other suitable and equivalent
antioxidants and chelating agents may be substituted as would be
known to those skilled in the art.
[0233] The composition comprising a compound contemplated within
the invention can be administered to a mammal as frequently as
several times daily, or it may be administered less frequently,
such as once a day, once a week, once every two weeks, once a
month, or even less frequently, such as once every several months
or even once a year or less.
[0234] It is understood that the amount of compound dosed per day
may be administered, in non-limiting examples, every day, every
other day, every 2 days, every 3 days, every 4 days, or every 5
days. For example, with every other day administration, a 0.5-5 mg
per day dose may be initiated on Monday with a first subsequent
0.5-5 mg per day dose administered on Wednesday, a second
subsequent 0.5-5 mg per day dose administered on Friday, and so on.
The frequency of the dose will be readily apparent to the skilled
artisan and will depend upon any number of factors, such as, but
not limited to, the type and severity of the disease being treated,
the type and age of the animal, and so forth.
Topical Administration
[0235] An obstacle for topical administration of pharmaceuticals is
the stratum corneum layer of the epidermis. The stratum corneum is
a highly resistant layer comprised of protein, cholesterol,
sphingolipids, free fatty acids and various other lipids, and
includes cornified and living cells. One of the factors that limit
the penetration rate (flux) of a compound through the stratum
corneum is the amount of the active substance that can be loaded or
applied onto the skin surface. The greater the amount of active
substance applied per unit of area of the skin, the greater the
concentration gradient between the skin surface and the lower
layers of the skin, and in turn the greater the diffusion force of
the active substance through the skin. Therefore, a formulation
containing a greater concentration of the active substance is more
likely to result in penetration of the active substance through the
skin, and more of it, and at a more consistent rate, than a
formulation having a lesser concentration, all other things being
equal.
[0236] Formulations suitable for topical administration include,
but are not limited to, liquid or semi-liquid preparations such as
liniments, lotions, oil-in-water or water-in-oil emulsions such as
creams, ointments or pastes, and solutions or suspensions. Such
formulations may be applied to the skin directly or through the use
of swabs, applicators, spatulas and the like, as well as in the
form of transdermal patches. In certain embodiments, the patch
minimizes loss of pharmaceuticals through washing, friction,
scratching and/or rubbing of the skin. In other embodiments, the
patch increases absorption of the pharmaceutical through the skin,
while minimizing the exposure of the skin to the
pharmaceutical.
[0237] Topically administrable formulations may, for example,
comprise from about 0.005% to about 10% (w/w) active ingredient,
although the concentration of the active ingredient may be as high
as the solubility limit of the active ingredient in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0238] Enhancers of permeation may be used. These materials
increase the rate of penetration of drugs across the skin. Typical
enhancers in the art include ethanol, glycerol monolaurate, PGML
(polyethylene glycol monolaurate), dimethylsulfoxide, and the like.
Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol,
laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar
lipids, or N-methyl-2-pyrrolidone.
[0239] One acceptable vehicle for topical delivery of some of the
compositions of the invention may contain liposomes. The
composition of the liposomes and their use are known in the art
(for example, U.S. Pat. No. 6,323,219).
[0240] In alternative embodiments, the pharmaceutical composition
of the invention may be optionally combined with other ingredients
such as adjuvants, anti-oxidants, chelating agents, surfactants,
foaming agents, wetting agents, emulsifying agents, viscosifiers,
buffering agents, preservatives, and the like. In other
embodiments, a permeation or penetration enhancer is included in
the composition and is effective in improving the percutaneous
penetration of the active ingredient into and through the stratum
corneum with respect to a composition lacking the permeation
enhancer. Various permeation enhancers, including oleic acid, oleyl
alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids,
dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are
known to those of skill in the art. In another aspect, the
composition may further comprise a hydrotropic agent, which
functions to increase disorder in the structure of the stratum
corneum, and thus allows increased transport across the stratum
corneum. Various hydrotropic agents such as isopropyl alcohol,
propylene glycol, or sodium xylene sulfonate, are known to those of
skill in the art.
[0241] The topically active pharmaceutical composition of the
invention should be applied in an amount effective to affect
desired changes. As used herein, "amount effective" shall mean an
amount sufficient to cover the region of skin surface where a
change is desired. An active compound should be present in the
amount of from about 0.0001% to about 15% by weight volume of the
composition; or in an amount from about 0.0005% to about 5% of the
composition; or in an amount of from about 0.005% to about 1% of
the composition. Such compounds may be synthetically-or naturally
derived.
Controlled Release Formulations and Drug Delivery Systems
[0242] Controlled- or sustained-release formulations of a
pharmaceutical composition of the invention may be made using
conventional technology. In some cases, the dosage forms to be used
can be provided as slow or controlled-release of one or more active
ingredients therein using, for example, hydropropylmethyl
cellulose, other polymer matrices, gels, permeable membranes,
osmotic systems, multilayer coatings, microparticles, liposomes, or
microspheres or a combination thereof to provide the desired
release profile in varying proportions. Suitable controlled-release
formulations known to those of ordinary skill in the art, including
those described herein, can be readily selected for use with the
pharmaceutical compositions of the invention. Thus, single unit
dosage forms suitable for topical administration, such as
liniments, lotions, oil-in-water or water-in-oil emulsions such as
creams, ointments or pastes, transdermal patches, and solutions or
suspensions that are adapted for controlled-release are encompassed
by the present invention.
[0243] Most controlled-release pharmaceutical products have a
common goal of improving drug therapy over that achieved by their
non-controlled counterparts. Ideally, the use of an optimally
designed controlled-release preparation in medical treatment is
characterized by a minimum of drug substance being employed to cure
or control the condition in a minimum amount of time. Advantages of
controlled-release formulations include extended activity of the
drug, reduced dosage frequency, and increased patient compliance.
In addition, controlled-release formulations can be used to affect
the time of onset of action or other characteristics, such as blood
level of the drug, and thus can affect the occurrence of side
effects.
[0244] Most controlled-release formulations are designed to
initially release an amount of drug that promptly produces the
desired therapeutic effect, and gradually and continually release
of other amounts of drug to maintain this level of therapeutic
effect over an extended period of time. In order to maintain this
constant level of drug in the body, the drug must be released from
the dosage form at a rate that will replace the amount of drug
being metabolized and excreted from the body.
[0245] Controlled-release of an active ingredient can be stimulated
by various inducers, for example pH, temperature, enzymes, water,
or other physiological conditions or compounds. The term
"controlled-release component" in the context of the present
invention is defined herein as a compound or compounds, including,
but not limited to, polymers, polymer matrices, gels, permeable
membranes, liposomes, or microspheres or a combination thereof that
facilitates the controlled-release of the active ingredient.
[0246] In certain embodiments, the formulations of the present
invention may be, but are not limited to, short-term, rapid-offset,
as well as controlled, for example, sustained release, delayed
release and pulsatile release formulations.
[0247] The term sustained release is used in its conventional sense
to refer to a drug formulation that provides for gradual release of
a drug over an extended period of time, and that may, although not
necessarily, result in substantially constant blood levels of a
drug over an extended time period. The period of time may be as
long as a month or more and should be a release that is longer that
the same amount of agent administered in bolus form.
[0248] For sustained release, the compounds may be formulated with
a suitable polymer or hydrophobic material which provides sustained
release properties to the compounds. As such, the compounds for use
the method of the invention may be administered in the form of
microparticles, for example, by injection or in the form of wafers
or discs by implantation.
[0249] In certain embodiments of the invention, the compounds of
the invention are administered to a patient, alone or in
combination with another pharmaceutical agent, using a sustained
release formulation.
[0250] The term delayed release is used herein in its conventional
sense to refer to a drug formulation that provides for an initial
release of the drug after some delay following drug administration
and that may, although not necessarily, includes a delay of from
about 10 minutes up to about 12 hours.
[0251] The term pulsatile release is used herein in its
conventional sense to refer to a drug formulation that provides
release of the drug in such a way as to produce pulsed plasma
profiles of the drug after drug administration.
[0252] The term immediate release is used in its conventional sense
to refer to a drug formulation that provides for release of the
drug immediately after drug administration.
[0253] As used herein, short-term refers to any period of time up
to and including about 8 hours, about 7 hours, about 6 hours, about
5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour,
about 40 minutes, about 20 minutes, or about 10 minutes and any or
all whole or partial increments thereof after drug administration
after drug administration.
[0254] As used herein, rapid-offset refers to any period of time up
to and including about 8 hours, about 7 hours, about 6 hours, about
5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour,
about 40 minutes, about 20 minutes, or about 10 minutes, and any
and all whole or partial increments thereof after drug
administration.
[0255] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures, embodiments, claims, and
examples described herein. Such equivalents were considered to be
within the scope of this invention and covered by the claims
appended hereto. For example, it should be understood, that
modifications in reaction conditions, including but not limited to
reaction times, reaction size/volume, and experimental reagents,
such as solvents, catalysts, pressures, atmospheric conditions,
e.g., nitrogen atmosphere, and reducing/oxidizing agents, with
art-recognized alternatives and using no more than routine
experimentation, are within the scope of the present
application.
[0256] It is to be understood that wherever values and ranges are
provided herein, all values and ranges encompassed by these values
and ranges, are meant to be encompassed within the scope of the
present invention. Moreover, all values that fall within these
ranges, as well as the upper or lower limits of a range of values,
are also contemplated by the present application.
[0257] The following examples further illustrate aspects of the
present invention. However, they are in no way a limitation of the
teachings or disclosure of the present invention as set forth
herein.
EXAMPLES
[0258] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only, and the invention is not limited to these
Examples, but rather encompasses all variations that are evident as
a result of the teachings provided herein.
Materials and Methods
[0259] Unless otherwise noted, all cell lines, biological
materials, chemical materials, reagents and resins were obtained
from commercial suppliers and used without purification.
Melanocyte Culture
[0260] Primary melanocytes were extracted from fresh discarded
surgical human foreskin specimens. After overnight incubation in
Dispase, the epidermis was separated from the dermis and treated
with trypsin for 10 minutes. Cells were pelleted and plated on
selective MC Medium 254 (Invitrogen) with Human Melanocyte Growth
Supplement, and 1% penicillin and streptomycin. Lightly pigmented
primary melanocytes were utilized for experiments assaying estrogen
and GPER agonist effects. 17.beta.-Estradiol (E8875), and
.alpha.MSH (M4135) were purchased from Sigma-Aldrich. G-1
(10008933) was purchased from Cayman Chemical. These compounds were
diluted to working stock solutions in Medium 254.
Melanin Assay
[0261] 2.times.10.sup.5 melanocytes were seeded uniformly on 6-well
tissue culture plates. Cells were treated with vehicle controls,
sex steroids, or hormone derivatives for 4 days. Cells were then
trypsinized, counted, and spun down at 300.times.g for 5 minutes.
The resulting cell pellet was solubilized in 1M NaOH, and boiled
for 5 minutes. The optical density of the resulting solution was
read at 450 nm using an EMax microplate reader (Molecular Devices).
The absorbance was normalized to the number of cells in each
sample, and relative amounts of melanin were set based on vehicle
treated controls. For tissue melanin assays, tissue was weighed
prior to boiling in 1M NaOH for 20 minutes. Samples were spun down
to eliminate insoluble materials, and then the optical density of
the sample was measured and normalized to the weight of tissue.
cAMP ELISA
[0262] cAMP ELISA was performed on primary human melanocytes using
the Cyclic AMP XP Assay Kit (Cell Signaling Technology, #4339)
following manufacturer instructions.
Western Blot Analyses and Antibodies
[0263] Adherent cells were treated with 1 .mu.M doses of E2
overnight, washed once with DPBS, and lysed with 1% NP-40 buffer
(150 mM NaCl, 50 mM Tris, pH 7.5, 1 mM EDTA, and 1% NP-40)
containing 1.times. protease inhibitors (Roche) and 1.times.
phosphatase inhibitors (Roche). Lysates were quantified (Bradford
assay), normalized, reduced, denatured (95.degree. C.) and resolved
by SDS gel electrophoresis on 4-15% Tris/Glycine gels (Bio-Rad).
Resolved protein was transferred to PVDF membranes (Millipore)
using a Semi-Dry Transfer Cell (Bio-Rad), blocked in 5% dry milk in
TBS-T and probed with primary antibodies recognizing MITF (Cell
Signaling Technology. #12590, 1:1000), pCREB (Cell Signaling
Technology, #9198, 1:1000), CREB (Cell Signaling Technology, #9104,
1:1000), and .beta.-Actin (Cell Signaling Technology, #3700,
1:4000). After incubation with the appropriate secondary antibody,
proteins were detected using either Luminata Crescendo Western HRP
Substrate (Millipore) or ECL Western Blotting Analysis System (GE
Healthcare).
Melanin Staining
[0264] Formalin-fixed paraffin embedded tissue was sectioned at 5
uM and collected on superfrost plus slides (Fisher), and subjected
to Fontana-Masson stain for melanin. Briefly, sections were
deparaffinized, rehydrated, and incubated in the following
solutions: 2.5% aqueous silver nitrate for 10 min, 0.1% aqueous
gold chloride for 15 min, and 5% aqueous sodium thiosulfate for 5
min. Distilled deionized water was used for rinsing and incubations
were done at room temperature except for silver nitrate at
60.degree. C. Slides were counterstained with 0.1% nuclear fast red
Kemechtrot for 5 min, dehydrated, cleared, and coverslipped using
MM24 mounting media (Leica). All staining reagents were from
Polyscientific R & D Corporation.
Immunohistochemistry
[0265] Formalin fixed paraffin embedded (FFPE) human skin tissue
sections from organotypic tissue was stained for MITF protein
expression using a primary antibody to MITF (Leica Biosystems,
NCL-L-MITF, 1:15). Staining was performed following the
manufacturer protocol for high temperature antigen unmasking
technique for immunohistochemical demonstration on paraffin
sections.
Quantification of Melanin Staining
[0266] Tissue sections from organotypic culture were stained using
methods described elsewhere herein. In terms of quantitation,
briefly, 20.times. photomicrograph images of representative tissue
sections were taken using the Zeiss Axiophot microscope. Tiff files
of the images were saved and transferred to Adobe PHOTOSHOP.RTM.
where pixels corresponding to Fontana-Masson staining and epidermal
counter stain were selected using the color selection tool. Images
corresponding to the single specific color were then analyzed using
FIJI (Image J) to determine the number of pixels in each sample.
The numbers of pixels representing Fontana-Masson staining were
normalized to the total amount epidermal counter staining. Final
ratios Fontana-Masson staining in the epidermis were set relative
to amount of staining in vehicle treated controls.
Quantitative RT/PCR
[0267] mRNA was extracted from melanocytes according to the RNeasy
Mini Kit protocol (Qiagen), and reverse transcribed to cDNA using
the High Capacity RNA-to cDNA kit (Applied Biosystems).
Quantitative PCR of the resulting cDNA was carried out using Power
SYBR Green Master Mix (Applied Biosystems) and gene-specific
primers, in triplicate, on a ViiA 7 Real-Time PCR System (Life
Technologies).
[0268] The following primers were used for detection; B-Actin
forward: 5'-CAT GTA CGT TGC TAT CCA GGC-3' (SEQ ID NO: 1). B-Actin
reverse: 5'-CTC CTT AAT GTC ACG CAC GAT-3' (SEQ ID NO:2); ER-A
forward: 5'-AAA GGT GGG ATA CGA AAA GAC C-3' (SEQ ID NO:3); ER-A
reverse: 5'-AGC ATC CAA CAA GGC ACT GA-3' (SEQ ID NO:4); ER-B
forward: 5'-GGC TGC GAG AAA TAA CTG CC-3' (SEQ ID NO:5); ER-B
reverse: 5'-AAT GCG GAC ACG TGC TTT TC-3' (SEQ ID NO:6); PGR
forward: 5'-AGG TCT ACC CGC CCT ATC TC-3' (SEQ ID NO:7); PGR
reverse: 5'-AGT AGT TGT GCT GCC CTT CC-3' (SEQ ID NO:8); AR
forward: 5'-GTG CTG TAC AGG AGC CGA AG-3' (SEQ ID NO:9); AR
reverse: 5'-GTC AGT CCT ACC AGG CAC TT-3' (SEQ ID NO: 10); GPER
forward: 5'-ACA GAG GGA AAA CGA CAC CT-3' (SEQ ID NO: 11); GPER
reverse: 5'-AAT TTT CAC TCG CCG CTT CG-3' (SEQ ID NO: 12). Relative
expression was determined using the 2-[delta][delta] Ct method
followed by normalization to the AR receptor transcript levels in
MCF7 cells.
Lentiviral Vectors
[0269] The following shRNAs were expressed from the GIPZ vector
(Open Biosystems): shPAR7.3 (V3LHS_364596, 5'-TGT GGT AGA GAA GAG
CTG G-3'; SEQ ID NO: 13), shGPER.1 (V2LHS_132008, 5'-TCC TTC TCC
TCT TTA ACT C-3'; SEQ ID NO: 14), shGPER.3 (V3LHS_390319, 5'-TGA
TGA AGT ACA GGT CGG G-3'; SEQ ID NO: 15). Guide RNAs were designed
using software tools provided on the website www dot
genome-engineering dot org/. Guide RNAs were subsequently cloned
into lentiCRISPRv2 (Addgene #52961) according to the accompanying
protocol. Guide RNA sequences are as follows: lentiCRISPR GFP,
5'-GAA GTT CGA GGG CGA CAC CC-3' (SEQ ID NO: 16); lentiCRISPR
GPER.1, 5'-ACAGGCCGATCACGTACTGC-3' (SEQ ID NO: 17); lentiCRISPR
GPER.2, 5'-GAG CAC CAG CAG TAC GTG AT-3' (SEQ ID NO: 18).
Synthesis of G-1.
[0270] G-1 (.+-.)
1-(4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta-
[c]quinolin-8-yl)ethan-1-one was prepared according to the
following protocol. 6-bromopiperonal (1.110 g, 4.85 mmol) and
4-aminoacetophenone (656 mg, 4.85 mmol) were dissolved in anhydrous
acetonitrile (16.2 mL, 0.3M) and allowed to stir at 25.degree. C.
under argon. After approximately 1.5 h, trifluoroacetic acid (350
.mu.L, 4.61 mmol) was added and the reaction was allowed to stir at
25.degree. C. for 45 min. Freshly prepared cyclopentadiene (1.63
mL, 19.4 mmol) was added dropwise to the reaction mixture. After 2
h at 25.degree. C. the reaction mixture was concentrated in vacuo.
The crude product was purified by silica gel chromatography using
30% EtOAc in hexanes as eluent to provide racemic
1-(4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta-
[c]quinolin-8-yl)ethan-1-one (1.05 g, 53%). The G-1 was >95%
pure as determined by high pressure liquid chromatography analysis.
.sup.1H NMR (500 MHz, CDCl) .delta. 7.70 (s, 1H), 7.61 (dd. J=5.0,
2.5 Hz, 1H), 7.09 (s, 1H), 7.01 (s, 1H), 6.61 (d, J=8.5 Hz, 1H),
5.99 (d, J=6.0 Hz, 2H), 5.93 (m, 1H), 5.67 (d, J=4.5 Hz, 1H), 4.97
(d, J=3.0 Hz, 1H), 4.12 (d, J=9.0 Hz, 1H), 3.19 (q, J=8.5 Hz, 1H),
2.54-2.48 (m, 4H), 1.84-1.79 (m, 1H). .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta. 196.5, 150.0, 147.5, 147.4, 133.8, 133.5,
130.4, 130.0, 128.5, 127.6, 125.0, 115.1, 112.9, 112.8, 107.6,
101.8, 56.1, 45.3, 42.0, 31.3, 26.0.
Topical G-1 Treatment.
[0271] 2% (w/v) G-1 was prepared in DMSO. 20 .mu.L of this solution
was applied daily to the right ear, with vehicle only applied to
the left ear, of 4-week-old C57BL/6 mice. These studies were
performed without inclusion/exclusion criteria, randomization, or
blinding. Based on a two-fold anticipated effect, this experiment
was performed with 3 biological replicates.
Statistical Analysis
[0272] * denotes a P-value of less than 0.05 in an unpaired,
two-tailed Students T-Test.
Example 1: Preparation of 3-D Organotypic Skin Cultures
[0273] Organotypic skin grafts containing MCs were established
using modifications to previously detailed methods (Ridky, et al.,
Nature Med. 2010, 16, 1450-1455; Chudnovsky, et al., Nature Gen.
2005, 37, 745-749). The Keratinocyte Growth Media (KGM) used for
keratinocyte-only skin grafts was replaced with modified Melanocyte
Xenograft Seeding Media (MXSM). MXSM is a 1:1 mixture of KGM,
lacking cholera toxin, and Keratinocyte Media 50/50 (Gibco)
containing 2% FBS, 1.2 mM calcium chloride, 100 nM Et-3 (endothelin
3), 10 ng/mL rhSCF (recombinant human stem cell factor), and 4.5
ng/mL r-basic FGF (recombinant basic fibroblast growth factor).
1.5.times.10.sup.5 melanocytes and 5.0.times.10.sup.5 keratinocytes
were suspended in 80 .mu.L MXSM, seeded onto the dermis, and
incubated at 37.degree. C. for 8 days at the air-liquid
interface.
Example 2: Estrogen Increases Melanin Synthesis
[0274] To examine whether sex steroids directly influence melanin
synthesis, normal primary human melanocytes was treated with
estrogen (17.beta.-estradiol), which resulted in a dose dependent
increase in melanin production (FIG. 2C). 25 nM estrogen were used
for subsequent experiments, a medically-relevant concentration
observed during second and third trimester pregnancy. After 4 days,
melanin production was markedly increased (208.+-.27%) (FIG. 3A).
The change in melanin production was similar in magnitude to
changes observed with .alpha.MSH (FIGS. 2B & 2D). This
melanin-promoting estrogen effect was consistent with involvement
of estrogen in melanin synthesis. To examine the effects of
estrogen on melanocyte homeostasis in the context of intact human
epidermis, architecturally-faithful 3-D organotypic skin was
established utilizing normal primary epidermal keratinocytes and
melanocytes within the framework of native stroma and intact
basement membrane. After 1 week, estrogen-treated skin displayed a
3-fold increase in melanin content (FIG. 3B), without any increase
in total melanocyte number or density (FIG. 3C).
[0275] To determine whether female cells also responded similarly,
female iPS-derived melanocytes were treated with estrogen.
Responses similar to those observed with the male cells were noted
(FIG. 2E).
[0276] To determine whether melanocytes isolated from body sites
other than foreskin also responded similarly to estrogen,
melanocytes from adult facial skin were treated with this sex
hormone; responses observed were similar to those observed with the
foreskin melanocytes (FIG. 2F).
Example 3: Primary Human Melanocytes do not Express Nuclear
Estrogen Receptor (ER), and Respond to Estrogen Via Altered cAMP
Signaling
[0277] To determine the mechanisms through which estrogen mediates
its pigment effects, components of the canonical pigment production
pathway were examined. An increase in cAMP upon estrogen treatment
(FIG. 4A) was observed. This suggests that estrogen accesses the
canonical pigment production pathway downstream of MC1R. Consistent
with this, pCREB and MITF proteins were similarly induced (FIG.
4B).
[0278] Without wishing to be limited by any theory, these data
suggest that estrogen, ] and .alpha.-MSH converge on the canonical
melanin production pathway at the level of adenylate cyclase to
modulate melanin synthesis. In certain embodiments,
pregnancy-associated hyperpigmentation is not observed all over the
body, but is characteristically limited to specific areas where
melanocyte density or UV radiation exposure is highest, including
the face, genital, and areolar regions.
[0279] As steroid hormones are not predicted to signal through
MC1R, which natural ligand is the peptide hormone .alpha.MSH,
efforts were put forth to identify the specific receptors through
which estrogen mediates its effects on melanin synthesis. Classical
estrogen (ER) receptor was not detected in melanocytes using
qRT-PCR, despite robust expression in the MCF7 ER/PR positive
breast cell line (FIG. 5A). RNAseq studies in primary human
melanocytes failed to detect ER transcripts (FIG. 5B). No ER
protein was detected via western blotting of melanocyte protein
lysates, although this receptor was readily apparent in MCF7
extracts (FIG. 4C).
Example 4: Estrogen Signaling in Melanocytes is Dependent on the
Nonclassical Membrane Bound G-Protein Coupled Sex Hormone Receptor
GPER
[0280] Since MC1R is a G protein-coupled receptor (GPCR), it was
hypothesized that an alternative GPCR may be binding estrogen to
mediate pigment effects. To identify possible candidates, whole
transcriptome RNAseq data from primary human MCs to identify other
expressed GPCRs were analyzed. The membrane-bound, G
protein-coupled estrogen receptor (GPER) was expressed (FIG. 5B).
Primary human MCs were analyzed for expression of GPER by qRT-PCR,
and its expression was verified (FIG. 5C). Notably, GPER expression
was markedly lower in other skin cell types including keratinocytes
and fibroblasts (FIG. 5D).
[0281] shRNA-mediated knockdown was utilized to establish the
necessity of GPER to mediate estrogen effects in MCs (FIG. 5E).
GPER depletion using either of two independent hairpins completely
eliminated the melanocyte pigmentation response to estrogen (FIG.
4D).
Example 5: Specific Activation of GPER Alters Pigment Production in
Human Skin Tissue
[0282] To complement these genetic studies establishing that
estrogen pigment effects are mediated through nonclassical sex
steroid receptors, small molecule steroid analogs were utilized
with specific agonist activity on ER and GPER. Tamoxifen, an ER
antagonist, is associated with development of melasma in breast
cancer patients. The mechanistic basis for this clinical
observation was previously unknown and somewhat perplexing, given
that tamoxifen blocks estrogen receptor function and thus might
have been predicted to limit melanin synthesis. However, tamoxifen
acts as a GPER agonist. Tamoxifen treatment resulted in a
dose-dependent increase in melanin production to levels comparable
to those observed with native estrogen (FIG. 7A), consistent with
the model in which GPER activation promotes melanin synthesis. Most
oral contraceptives incorporate an estrogen derivative (ethinyl
estradiol) for the estrogen component. Treatment of primary human
MCs with ethinyl estradiol also resulted in a dose dependent
increase in melanin production, indistinguishable from native
estrogen at an equal concentration (FIG. 7B).
[0283] To determine whether GPER signaling was sufficient to
increase melanin production, the specific GPER agonist G-1, which
does not bind to the classical estrogen receptor ER, was used. G-1
treatment resulted in a dose-dependent increase in melanin
production (FIG. 8A), and GPER was necessary for this effect (FIG.
8B). The same effect was seen upon G-1 treatment of human
organotypic skin (FIGS. 9A-9C).
Example 6: Topical Delivery of a GPER-Specific Synthetic Estrogen
Analog In Vivo Increases Skin Melanin
[0284] To demonstrate that GPER was sufficient to promote melanin
production in vivo, a 2% G-1 was formulated in a vehicle for
topical delivery. The right ears of mice were treated with G-1 over
3 weeks, and saw increased pigment in the G-1 treated ears compared
to vehicle (FIG. 9A). Melanin content is G-1 treated tissue was
1.6-fold higher than controls, a cosmetically significant change,
and in line with the magnitude of melanin change seen in vitro
(FIG. 10B). Melanin content was also analyzed using melanin
staining of tissue sections and noted similar melanin increases
(FIG. 10C).
Example 7: Compounds of the Invention Induce pCREB in Human Primary
Melanocytes
General Methods
[0285] Solvents used for extraction and purification were HPLC
grade from Fisher Scientific. Unless otherwise indicated, all
reactions were run under an inert atmosphere of argon. Anhydrous
tetrahydrofuran, dichloromethane and toluene were obtained via
passage through an activated alumina column. Commercial reagents
were used as received.
[0286] VWR pre-coated silica gel plates (250 .mu.m, 60 F254) were
used for analytical TLC. Spots were visualized using 254 nm
ultraviolet light with either potassium permanganate or vanillin
stains as visualizing agents. Chromatographic purifications were
performed on Sorbent Technologies silica gel (particle size 32-63
microns).
[0287] .sup.1H and .sup.13C NMR spectra were recorded at 500 MHz
and 125 MHz, respectively in CDCl.sub.3 on a Bruker AM-500 or
DRX-500 spectrometer. Chemical shifts are reported relative to
internal chloroform (.delta.=7.26 for .sup.1H, .delta.=77.00 for
.sup.13C).
[0288] Infrared spectra were recorded on a NaCl plate using a
Perkin-Elmer 1600 series Fourier transform spectrometer.
[0289] High-resolution mass spectra were obtained at the University
of Pennsylvania Mass Spectrometry Service Center on an Autospec
high resolution double-focusing electrospray ionization/chemical
ionization spectrometer with either DEC 11/73 or OPUS software data
system.
[0290] Melting points were obtained on a Thomas Hoover capillary
melting point apparatus and are uncorrected.
1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3-
H-cyclopenta[c]quinolin-8-yl)ethan-1-one, G-1
##STR00032##
[0292] A solution of scandium trifluoromethanesulfonate (370 mg,
0.742 mmol) in CH.sub.3CN (2.8 mL) was added dropwise to a solution
of 4-aminoacetophenone (1.0 g, 7.42 mmol), 6-bromopiperonal (1.7 g,
7.42 mmol) and freshly distilled cyclopentadiene (3.1 mL, 37.2
mmol) in CH.sub.3CN (45 mL). After 3 h stirring at rt the solution
was concentrated in vacuo and chromatography (4:1 hexanes/EtOAc)
followed by recrystallization (hexanes/EtOAc) gave
tetrahydroquinoline G-1 (2.8 g, 6.81 mmol, 92%) as a white solid
and the single diastereoisomer as a racemic mixture of enantiomers.
The spectroscopic data is consistent with that previously reported
in Burai. et al., 2010, Org. Biomol. Chem. 8:2252-2259.
[0293] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.74-7.68 (m,
1H), 7.63 (dd, J=8.3, 2.0 Hz, 1H), 7.10 (s, 1H), 7.05 (s, 1H), 6.61
(d, J=8.3 Hz, 1H), 6.01 (dd, J=7.4, 1.2 Hz, 2H), 5.97-5.92 (m, 1H),
5.72-5.65 (m, 1H), 4.99 (d, J=3.4 Hz, 1H), 4.13 (d, J=8.8 Hz, 1H),
3.99 (s, 1H), 3.25-3.15 (m, 1H), 2.56-2.50 (m, 1H), 2.51 (s, 3H),
1.88-1.78 (m, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) .delta.:
196.6, 150.0, 147.6, 147.5, 133.8, 133.6, 130.5, 130.1, 128.7,
127.7, 125.2, 115.2, 113.1, 113.0, 107.7, 101.9, 56.2, 45.4, 42.0,
31.4, 26.1. mp: 177-178.degree. C.
1-((3aS,4R,9bR)-4-(benzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclo-
penta[c]quinolin-8-yl)ethan-1-one, CMPD1
##STR00033##
[0295] A solution of scandium trifluoromethanesulfonate (164 mg,
0.333 mmol) in CH.sub.3CN (1.3 mL) was added dropwise to a solution
of 4-aminoacetophenone (450 mg, 3.33 mmol), piperonal (500 mg, 3.33
mmol) and freshly distilled cyclopentadiene (1.4 mL, 16.7 mmol) in
CH.sub.3CN (20 mL). After 3 h stirring at rt the solution was
concentrated in vacuo and chromatography (4:1 hexanes/EtOAc)
followed by precipitation from a 4:1 mixture of hexanes/EtOAc gave
tetrahydroquinoline CMPD-1 (670 mg, 2.00 mmol, 60%) as a white
solid and the single diastereoisomer as a racemic mixture of
enantiomers. The spectroscopic data is consistent with that
reported in PCT Int. Appl. WO 2007/019180 A2 to Prossnitz, et
al.
[0296] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.70 (s, 1H),
7.61 (dd, J=8.4, 2.0 Hz, 11), 6.91 (s, 1H), 6.87 (dd, J=8.1, 1.7
Hz, 1H), 6.82 (d, J=8.2 Hz, 1H), 6.59 (d, J=8.5 Hz, 1H), 5.98 (s,
2H), 5.94-5.90 (m, 1H), 5.69-5.65 (m, 1H), 4.64 (d, J=3.4 Hz, 1H),
4.16 (s, 1H), 4.10 (d, J=8.5 Hz, 1H), 2.99-2.92 (m, 1H), 2.59-2.51
(m, 1H), 2.51 (s, 3H), 1.91-1.83 (m, 1H). .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta.: 196.6, 150.1, 147.9, 146.9, 135.9, 133.8,
130.6, 130.1, 128.4, 127.8, 125.0, 119.4, 114.9, 108.3, 106.9,
101.1, 57.2, 46.0, 45.6, 31.5, 26.1. mp: 158-160.degree. C.
1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-5-methyl-3a,4,5,9b-tet-
rahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one, CMPD2
##STR00034##
[0298] Sodium hydride (60% w.r.t dispersion in oil; 8.3 mg, 0.206
mmol) was added to a solution of G-1 (50 mg, 0.122 mmol) in DMF
(2.0 mL) at 0.degree. C. After stirring for 30 min methyl iodide
(12 .mu.L, 0.182 mmol) was added and the solution was warmed to rt
and stirred for a further 2 h. The solution was diluted with EtOAc
(30 mL) and washed with water (3.times.10 mL). The organic phase
was dried (Na.sub.2SO.sub.4), concentrated in vacuo and
chromatography (8:1 hexanes/EtOAc) gave tetrahydroquinoline CMPD2
(35 mg, 0.0824 mmol, 68%) as a colorless oil and a racemic mixture
of enantiomers. The spectroscopic data is consistent with that
reported in PCT Int. Appl. WO 2007/019180 A2 to Prossnitz, et
al.
[0299] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.77-7.71 (m,
2H), 6.98 (s, 1H), 6.74-6.68 (m, 1H), 6.66 (s, 1H), 6.07 (dd,
J=5.5, 2.7 Hz, 1H), 5.98-5.92 (m, 2H), 5.57 (dd, J=5.0, 2.0 Hz,
1H), 4.95 (d, J=5.8 Hz, 1H), 4.01 (d, J=7.9 Hz, 1H), 3.11-3.01 (m,
1H), 2.79 (s, 3H), 2.53 (s, 3H), 2.33-2.22 (m, 1H), 2.08 (dd,
J=16.9, 6.9 Hz, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) .delta.:
196.6, 151.0, 147.4, 147.2, 134.6, 132.9, 130.4, 129.6, 128.6,
127.0, 124.2, 114.3, 112.4, 112.2, 108.9, 101.7, 62.7, 43.9, 40.9,
39.0, 33.5, 26.0.
1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-2,3,3a,4,5,9b-hexahydr-
o-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one, CMPD3
##STR00035##
[0301] Hydrogen gas was bubbled through a mixture of G-1 (50 mg,
0.122 mmol) and palladium on carbon (10% by wt, 5.0 mg) in EtOAc
(2.0 mL) for 5 min and the mixture was then stirred under a
hydrogen atmosphere for 2 h. The mixture was filtered through
Celite.RTM. and chromatography (4:1 hexanes/EtOAc) gave
tetrahydroquinoline CMPD3 (45 mg, 0.109 mmol, 89%) as a white solid
and a racemic mixture of enantiomers.
[0302] .sup.1H NMR (500 MHz. CDCl.sub.3) .delta.: 7.81 (s, 1H),
7.63 (dd, J=8.5, 2.1 Hz, 1H), 7.12 (s, 1H), 7.03 (s, 1H), 6.56 (d,
J=8.2 Hz, 1H), 6.00 (d, J=1.5 Hz, 1H), 5.99 (d, J=1.5 Hz, 1H), 4.95
(d, J=3.1 Hz, 1H), 4.09 (s, 1H), 3.51-3.44 (m, 1H), 2.66-2.59 (m,
1H), 2.52 (s, 3H), 2.20 (ddt, J=12.8, 9.2, 7.0 Hz, 1H), 1.97-1.90
(m, 1H), 1.65-1.44 (m, 3H), 1.29-1.22 (m, 1H). .sup.13C NMR (126
MHz, CDCl.sub.3) .delta.: 196.7, 149.4, 147.5, 147.4, 134.1, 130.3,
128.3, 127.7, 125.4, 114.2, 113.0, 112.9, 107.9, 101.8, 55.8, 42.7,
40.1, 34.2, 26.1, 24.1, 23.0. HRMS: (ES.sup.+) Calculated for
C.sub.21H.sub.21NO.sub.3Br [M+H].sup.+: 414.0705; Found: 414.0693.
FTIR (thin film): 3327, 1658, 1596, 1503, 1747, 1234 cm.sup.-1. mp:
166-168 OC.
1-((3aS,4R,9bR)-5-benzyl-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tet-
rahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one, CMPD4
##STR00036##
[0304] Sodium hydride (60% w.r.t dispersion in oil; 8.3 mg, 0.206
mmol) was added to a solution of G-1 (50 mg, 0.122 mmol) in DMF
(2.0 mL) at 0.degree. C. After stirring for 30 min benzyl bromide
(22 .mu.L, 0.183 mmol) was added and the solution was stirred for a
further 2 h. The solution was diluted with EtOAc (30 mL) and washed
with water (3.times.10 mL). The organic phase was dried
(Na.sub.2SO.sub.4), concentrated in vacuo and chromatography (7:1
hexanes/EtOAc) gave tetrahydroquinoline CMPD4 (42 mg, 0.0838 mmol,
69%) as a brown oil and a racemic mixture of enantiomers.
[0305] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.84-7.79 (m,
1H), 7.62 (dd, J=8.7, 2.3 Hz, 1H), 7.29 (dd, J=8.2, 6.4 Hz, 2H),
7.24-7.17 (m, 3H), 6.93 (s, 1H), 6.64-6.60 (m, 2H), 6.02 (dq,
J=4.3, 2.1 Hz, 1H), 5.91-5.86 (m, 2H), 5.51 (dt, J=4.9, 2.4 Hz,
1H), 5.08 (d, J=6.1 Hz, 1H), 4.53 (d, J=16.8 Hz, 1H), 4.14-4.05 (m,
2H), 3.12 (tt, J=7.6, 5.5 Hz, 1H), 2.50 (s, 3H), 2.41-2.26 (m, 2H).
.sup.13C NMR (126 MHz, CDCl.sub.3) .delta.: 196.6, 149.8, 147.6,
147.0, 137.7, 134.8, 132.5, 130.2, 129.8, 128.7, 128.7, 127.1,
127.0, 126.7, 124.1, 115.0, 112.4, 112.2, 109.3, 101.7, 61.9, 54.1,
44.6, 40.6, 34.2, 26.0. HRMS: (ES.sup.+) Calculated for
C.sub.28H.sub.25NO.sub.3Br [M+H].sup.+: 502.1018; Found: 502.1020.
FTIR (thin film): 1665, 1597,1503, 1476, 1233, 1037 cm.sup.-1.
1-((5S,6aS,7R,12aR)-5,6,6a,7,12,12a-hexahydro-5,7-methano[1,3]dioxolo[4',5-
':4,5]benzo[1,2-c]acridin-9-yl)ethan-1-one, CMPD12
##STR00037##
[0307] G-1 (20 mg, 0.0486 mmol), sodium tertbutoxide (6.5 mg,
0.0680 mmol), JohnPhos (1.5 mg, 4.86 .mu.mol) and
tris(dibenzylideneacetone)dipalladium (2.2 mg, 2.43 .mu.mol) were
added to a vial which was evacuated and back-filled with argon 3
times. Toluene (0.5 mL) and benzylamine (6.0 .mu.L, 0.0584 mmol)
were then added and the mixture was heated to 110.degree. C. for 24
h. The mixture was cooled to rt, filtered through Celite.RTM. and
chromatography (2:1 hexanes/EtOAc) gave tetrahydroquinoline CMPD12
(10 mg, 0.0300 mmol, 62%) as a yellow oil and a racemic mixture of
enantiomers.
[0308] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.66 (d, J=2.4
Hz, 1H), 7.53 (dd, J=8.4, 2.0 Hz, 1H), 6.67 (s, 1H), 6.48 (d, J=8.5
Hz, 1H), 6.42 (s, 1H), 5.84 (d. J=1.2 Hz, 1H), 5.79 (d, J=1.2 Hz,
1H), 5.11 (s, 1H), 4.60 (d, J=5.5 Hz, 1H), 3.35 (ddd, J=11.9, 6.9,
3.7 Hz, 1H), 2.98 (t, J=5.2 Hz, 1H), 2.80 (q. J=5.6 Hz, 1H), 2.70
(td, J=12.4, 6.1 Hz, 1H), 2.45 (s, 3H), 2.09-2.02 (m, 1H),
2.01-1.95 (m, 1H), 1.75 (ddd, J=12.5, 3.8, 2.0 Hz, 1H). .sup.13C
NMR (126 MHz, CDCl.sub.3) .delta.: 196.6, 146.4, 146.2, 145.9,
139.9, 131.1, 128.5, 127.8, 127.6, 126.6, 114.0, 107.3, 106.1,
100.7, 53.4, 46.1, 40.3, 37.9, 37.6, 36.0, 26.0. HRMS: (ES.sup.+)
Calculated for C.sub.21H.sub.20NO.sub.3 [M+H].sup.+: 334.1443;
Found: 334.1445. FTIR (thin film): 3348, 1651, 1593, 1481, 1359,
1259 cm.sup.-1.
1-((3aS,4R,9bR)-4-(2-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]qui-
nolin-8-yl)ethan-1-one, CMPD5
##STR00038##
[0310] A solution of scandium trifluoromethanesulfonate (80 mg,
0.162 mmol) in CH.sub.3CN (0.7 mL) was added dropwise to a solution
of 4-aminoacetophenone (220 mg, 1.62 mmol), 2-bromobenzaldehyde
(300 mg, 1.62 mmol) and freshly distilled cyclopentadiene (0.68 mL,
8.10 mmol) in CH.sub.3CN (10 mL). After 1 h stirring at rt the
solution was concentrated in vacuo and chromatography (4:1
hexanes/EtOAc) followed by recrystallization from 5:2 mixture
hexanes/Et.sub.2O gave tetrahydroquinoline CMPD5 (530 mg, 1.44
mmol, 89%) as a white solid and the single diastereoisomer as a
racemic mixture of enantiomers.
[0311] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.74-7.72 (m,
1H), 7.64 (dd, J=8.3, 2.2 Hz, 1H), 7.60 (dd, J=3.2, 1.5 Hz, 1H),
7.59 (dd, J=2.9, 1.5 Hz, 1H), 7.37 (td, J=7.5, 1.2 Hz, 1H), 7.19
(td, J=7.5, 1.8 Hz, 1H), 6.63 (d, J=8.3 Hz, 1H), 5.94 (dtd, J=5.9,
2.9, 1.5 Hz, 1H), 5.69-5.65 (m, 1H), 5.08 (d, J=2.9 Hz, 1H), 4.17
(d, J=9.0 Hz, 1H), 4.07 (s, 1H), 3.30 (qd, J=8.7, 3.4 Hz, 1H),
2.59-2.53 (m, 1H), 2.52 (s, 3H), 1.81-1.71 (m, 1H). .sup.13C NMR
(126 MHz, CDCl.sub.3) .delta.: 196.5, 150.1, 140.3, 133.8, 133.1,
130.4, 130.0, 128.9, 128.6, 127.7, 127.7, 127.6, 125.2, 122.9,
115.2, 56.3, 45.4, 41.8, 31.4, 26.1. HRMS: (ES.sup.+) Calculated
for C.sub.20H.sub.19NOBr [M+H].sup.+: 368.0650; Found: 368.0633.
FTIR (thin film): 3323, 1660, 1597, 1355, 1272, 1227 cm.sup.-1. mp:
137-139.degree. C.
1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3-
H-cyclopenta[c]quinolin-8-yl)ethan-1-one oxime, CMPD6
##STR00039##
[0313] Hydroxylamine hydrochloride (13 mg, 0.190 mmol) was added to
a solution of G-1 (60 mg, 0.146 mmol) and pyridine (15 mg, 0.190
mmol) in dioxane (1.5 mL) and the solution was heated to 80.degree.
C. for 16 h. The solution was cooled to rt and purification through
preparative TLC (2% MeOH in CH.sub.2Cl.sub.2) gave oxime CMPD6 (18
mg, 0.0422 mmol, 29%) as an off-white amorphous solid and a racemic
mixture of enantiomers.
[0314] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.39 (s, 1H),
7.29 (d, J=8.5 Hz, 1H), 7.14 (s, 1H), 7.04 (s, 1H), 6.62 (d, J=8.5
Hz, 1H), 6.01 (d, J=1.2 Hz, 1H), 5.99 (d, J=1.2 Hz, 1H), 5.94-5.89
(m, 1H), 5.69-5.65 (m, 1H), 4.93 (d, J=3.7 Hz, 1H), 4.12 (d, J=8.5
Hz, 1H), 3.79 (s, 1H), 3.19 (qd, J=8.7, 3.2 Hz, 1H), 2.56 (dd,
J=13.9, 9.3 Hz, 1H), 2.27 (s, 3H), 1.88-1.78 (m, 1H). .sup.13C NMR
(126 MHz, CDCl.sub.3) .delta.: 156.1, 147.6, 147.4, 146.6, 134.2,
133.9, 130.4, 127.4, 126.9, 125.9, 124.4, 115.9, 113.1, 112.9,
108.0, 101.8, 56.6, 45.9, 42.2, 31.4, 12.0. HRMS: (ES.sup.+)
Calculated for C.sub.21H.sub.20N.sub.2O.sub.3Br [M+H].sup.+:
427.0657; Found: 427.0641. FTIR (thin film): 3348, 3191, 1721,
1607, 1503, 1475, 1235 cm.sup.-1.
1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-3a,4,5,9b-tetrahydro-3H-cy-
clopenta[c]quinolin-8-yl)ethan-1-one, CMPD7
##STR00040##
[0316] A solution of scandium trifluoromethanesulfonate (80 mg,
0.162 mmol) in CH.sub.3CN (0.7 mL) was added dropwise to a solution
of 4-aminoacetophenone (220 mg, 1.62 mmol), 6-bromoveratraldehyde
(400 mg, 1.62 mmol) and freshly distilled cyclopentadiene (0.68 mL,
8.10 mmol) in CH.sub.3CN (10 mL). After 1 h stirring at rt the
solution was concentrated in vacuo and chromatography (2:1
Et.sub.2O/hexanes) followed by precipitation from 1:10
CH.sub.2Cl.sub.2/MeOH gave tetrahydroquinoline CMPD7 (520 mg, 1.23
mmol, 80%) as a white solid and the single diastereoisomer as a
racemic mixture of enantiomers.
[0317] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.74-7.71 (m,
1H), 7.64 (dd, J=8.4, 2.0 Hz, 1H), 7.10 (s, 1H), 7.05 (s, 1H), 6.65
(dd, J=8.2, 1.8 Hz, 1H), 5.95 (ddt, J=5.8, 3.1, 1.4 Hz, 1H),
5.71-5.66 (m, 2H), 4.99 (d, J=3.4 Hz, 1H), 4.15 (d, J=8.5 Hz, 1H),
4.06 (s, 1H), 3.91 (s, 3H), 3.89 (s, 2H), 3.24-3.15 (m, 1H),
2.60-2.54 (m, 1H), 2.52 (s, 3H), 1.86-1.78 (m, 1H). .sup.13C NMR
(126 MHz, CDCl.sub.3) .delta.: 196.6, 150.0, 148.7, 148.6, 134.0,
132.3, 130.4, 130.1, 128.8, 127.7, 125.4, 115.7, 115.3, 112.7,
110.4, 56.2, 56.2, 56.1, 45.5, 42.1, 31.5, 26.1. HRMS: (ES.sup.+)
Calculated for C.sub.22H.sub.23NO.sub.3Br [M+H].sup.+: 428.0861;
Found: 428.0874. FTIR (thin film): 3340, 1662, 1598, 1506, 1286,
1209 cm.sup.-1. mp: 185-187.degree. C.
1-((3aS,4R,9bR)-4-(6-chlorobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro--
3H-cyclopenta[c]quinolin-8-yl)ethan-1-one, CMPD8
##STR00041##
[0319] A solution of scandium trifluoromethanesulfonate (80 mg,
0.162 mmol) in CH.sub.3CN (0.7 mL) was added dropwise to a solution
of 4-aminoacetophenone (220 mg, 1.62 mmol), 6-chloropiperonal (300
mg, 1.62 mmol) and freshly distilled cyclopentadiene (0.68 mL, 8.10
mmol) in CH.sub.3CN (10 mL). After 1 h stirring at rt the solution
was concentrated in vacuo and chromatography (1:2 to 1:1
Et.sub.2O/hexanes) followed by precipitation from Et.sub.2O gave
tetrahydroquinoline CMPD8 (490 mg, 1.32 mmol, 81%) as a white solid
and the single diastereoisomer as a racemic mixture of
enantiomers.
[0320] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.71 (d, J=1.2
Hz, 1H), 7.62 (dd, J=8.5, 2.1 Hz, 1H), 7.10 (s, 1H), 6.87 (s, 1H),
6.61 (d, J=8.5 Hz, 1H), 6.01 (d, J=1.5 Hz, 1H), 6.00 (d, J=1.5 Hz,
1H), 5.94 (ddt, J=5.8, 2.7, 1.4 Hz, 1H), 5.69-5.66 (m, 1H), 5.04
(d, J=3.7 Hz, 1H), 4.13 (d, J=8.5 Hz, 1H), 3.99 (s, 1H), 3.18 (dtd,
J=10.1, 8.5, 3.4 Hz, 1H), 2.56-2.49 (m, 1H), 2.51 (s, 3H), 1.83
(dddd, J=16.2, 8.5, 2.7, 1.5 Hz, 1H). .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta.: 196.6, 150.0, 147.2, 147.0, 133.9, 132.2,
130.5, 130.1, 128.8, 127.7, 125.2, 124.3, 115.2, 110.1, 107.2,
101.9, 53.8, 45.5, 42.0, 31.5, 26.1. HRMS: (ES.sup.+) Calculated
for C.sub.21H.sub.19NO.sub.3Cl [M+H].sup.+: 368.1053; Found:
368.1062. FTIR (thin film): 3324, 1660, 1596, 1504, 1476, 1287,
1235 cm.sup.-1. mp: 174-176.degree. C.
1-((1R,2S,3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-1,2-dihydroxy-2,-
3,3a,4,5,9b-hexahydro-1H-cyclopenta[c]quinolin-8-yl)ethan-1-one,
CMPD10
##STR00042##
[0322] 4-methylmorpholine N-oxide (86 mg, 0.730 mmol) and osmium
tetroxide (1% in water, 0.10 mL) were sequentially added to a
solution of G-1 (250 mg, 0.608 mmol) in acetone (13 mL) at
0.degree. C. After 4 hr stirring sat. aqueous
Na.sub.2S.sub.2O.sub.3 (10 mL) was added and the acetone was
removed in vacuo. The aqueous phase was extracted with EtOAc
(3.times.20 mL) and the combined organic phases were washed with
brine (20 mL), dried (Na.sub.2SO.sub.4) and concentrated in vacuo.
Chromatography (100% EtOAc) gave diol CMPD10 (250 mg, 0.562 mmol,
92%) as a white solid and the single diastereoisomer as a racemic
mixture of enantiomers.
[0323] .sup.1H NMR (500 MHz, CDCl.sub.1) .delta.: 7.90 (s, 1H),
7.68 (dd, J=8.2, 2.1 Hz, 1H), 7.07 (s, 1H), 7.04 (s, 1H), 6.63 (d,
J=8.5 Hz, 1H), 6.02 (d, J=1.5 Hz, 1H), 6.00 (d, J=1.5 Hz, 1H), 4.76
(d, J=3.4 Hz, 1H), 4.13-4.10 (m, 2H), 4.07 (t, J=3.7 Hz, 1H), 3.48
(dd, J=8.9, 3.4 Hz, 1H), 3.36 (qd, J=8.7, 3.4 Hz, 1H), 2.53 (s,
3H), 1.97 (ddd, J=13.7, 9.8, 6.4 Hz, 1H), 1.36 (ddd, J=13.4, 8.7,
4.0 Hz, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) .delta.: 197.1,
150.4, 147.5, 132.9, 130.0, 128.8, 128.1, 123.5, 115.0, 113.2,
113.1, 107.8, 101.9, 81.7, 72.3, 60.4, 56.8, 46.2, 38.7, 30.8,
26.1. HRMS: (ES.sup.+) Calculated for C.sub.21H.sub.20NO.sub.5BrNa
[M+Na].sup.+: 468.0423; Found: 468.0406. FTIR (thin film): 3315,
1655, 1593, 1475, 1286, 1235 cm.sup.-1. mp: 163-165.degree. C.
1-((6R,6aS,7aS,10aR,10bR)-6-(6-bromobenzo[d][1,3]dioxol-5-yl)-9,9-dimethyl-
-6,6a,7,7a,10a,10b-hexahydro-5H-[1,3]dioxolo[4',5':3,4]cyclopenta[1,2-c]qu-
inolin-2-yl)ethan-1-one, CMPD9
##STR00043##
[0325] p-Toluenesulfonic acid monohydrate (3.0 mg, 0.0162 mmol) was
added to a solution of CMPD10 (24 mg, 0.0530 mmol) and
2,2-dimethoxypropane (0.12 mL, 0.971 mmol) in acetone (0.7 mL) at
rt. After stirring for 30 min the solution was concentrated in
vacuo, redissolved in EtOAc (10 mL) and washed with sat. aqueous
NaHCO.sub.3 (10 mL). The aqueous phase was extracted with EtOAc
(2.times.10 mL) and the combined organic phases were dried
(Na.sub.2SO.sub.4). Chromatography (1:2 hexanes/Et.sub.2O) gave
ketal CMPD9 (20 mg, 0.0415 mmol, 77%) as a colorless oil and as a
racemic mixture of enantiomers.
[0326] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.95 (s, 1H),
7.66 (dd. J=8.5, 2.1 Hz, 1H), 7.05 (s, 1H), 7.03 (s, 1H), 6.58 (d,
J=8.2 Hz, 1H), 6.00 (d, J=1.5 Hz, 1H), 5.97 (d, J=1.5 Hz, 1H), 5.01
(d, J=2.7 Hz, 1H), 4.86 (d, J=5.5 Hz, 1H), 4.61 (t, J=5.5 Hz, 1H),
4.24 (s, 1H), 3.49 (d, J=6.7 Hz, 1H), 2.99 (dtd, J=12.5, 6.3, 2.7
Hz, 1H), 2.52 (s, 3H), 1.74 (td, J=13.7, 5.5 Hz, 1H), 1.53-1.49 (m,
1H), 1.47 (s, 3H), 1.31 (s, 3H). .sup.13C NMR (126 MHz, CDCl.sub.3)
.delta.: 196.4, 149.2, 147.7, 147.6, 133.3, 130.9, 128.4, 128.2,
118.9, 114.6, 113.3, 113.1, 109.4, 107.5, 101.9, 87.4, 79.4, 54.7,
46.4, 38.0, 31.1, 26.1 (2C), 23.8. HRMS: (ES.sup.+) Calculated for
C.sub.24H.sub.24NO.sub.5BrNa [M+Na].sup.+: 508.0736; Found:
508.0755. FTIR (thin film): 3330, 1664, 1596, 1503, 1475, 1235
cm.sup.-1.
1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-2,3,3a,4,5,9b-hexahydro-1H-
-cyclopenta[c]quinolin-8-yl)ethan-1-one, CMPD11
##STR00044##
[0328] Hydrogen gas was bubbled through a mixture of CMPD7 (70 mg,
0.164 mmol) and palladium on carbon (10% by wt, 7.0 mg) in EtOAc
(2.7 mL) for 5 min and the mixture was then stirred under a
hydrogen atmosphere for 6 h. The mixture was filtered through
Celite.RTM. and chromatography (2:1 Et.sub.2O/hexanes) gave
tetrahydroquinoline CMPD11 (65 mg, 0.152 mmol, 93%) as a yellow
solid and a racemic mixture of enantiomers.
[0329] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.84-7.80 (m,
1H), 7.65 (dd, J=8.2, 2.1 Hz, 1H), 7.13 (s, 1H), 7.04 (s, 1H), 6.60
(d, J=8.2 Hz, 1H), 4.96 (d, J=3.1 Hz, 1H), 4.16 (s, 1H), 3.91 (s,
3H), 3.89 (s, 3H), 3.53-3.46 (m, 1H), 2.64 (dtd, J=11.0, 7.8, 3.2
Hz, 1H), 2.53 (s, 3H), 2.27-2.17 (m, 1H), 1.94 (dtd, J=12.8, 5.3,
2.9 Hz, 1H), 1.71-1.43 (m, 3H), 1.30-1.21 (m, 1H). .sup.13C NMR
(126 MHz, CDCl.sub.3) .delta.: 196.7, 149.5, 148.6, 148.5, 132.8,
130.4, 128.4, 127.7, 125.7, 115.6, 114.3, 112.6, 110.6, 56.2, 56.2,
55.7, 42.9, 40.1, 34.3, 26.1, 24.2, 23.1. HRMS: (ES.sup.+)
Calculated for C.sub.22H.sub.25NO.sub.3Br [M+H].sup.+: 430.1018;
Found: 430.1021. FTIR (thin film): 3344, 1660, 1598, 1506, 1209
cm.sup.-1. mp: 167-168.degree. C.
[0330] Compounds of the invention were evaluated for their ability
to increase levels of pCREB in human primary melanocytes, as
demonstrated for other compounds elsewhere herein. The
corresponding western blots are illustrated in FIG. 11. The actin
control is illustrated at the bottom of the images. All assays were
performed using a 100 nM concentration and a 30 minute treatment of
primary human melanocytes.
Example 8: In Vivo Studies
[0331] Tissue generated elsewhere herein is orthotopically
xenografted onto immunocompromised mice, allowing analysis of human
skin in vivo. Hormones are administered subcutaneously or topically
to demonstrate the necessity of GPER to mediate hormone-induced
changes in pigmentation. The effects of the specific agonists are
evaluated.
[0332] To demonstrate that this mechanism is not restricted to
foreskin-derived melanocytes, melanocytes are isolated from various
body sites and donors of different age, sex, and race. 3-D
organotypic cultures are established with melanocytes from these
sources, and the necessity and sufficiency of GPER signaling is
evaluated.
Example 9: Evaluation of Mechanism Through which GPER Influences
Pigmentation
[0333] To analyze the mechanism through which estrogen influences
pigmentation downstream of GPER the G.alpha. subunit activated by
the receptor is identified. To accomplish this, agonist-promoted
[.sup.35S]GTP.gamma.S exchange assays are used, followed by
immuno-enrichment of specific G protein subgroups. G
protein-coupled receptor signaling drives guanine nucleotide
exchange on the G.alpha. subunit, switching GDP for GTP.
[.sup.35S]GTP.gamma.S is an isotopic, non-hydrolysable analogue of
GTP. Membrane preparations from primary human melanocytes are
isolated and stimulated with GPER agonist, as well as vehicle
controls, in the presence of [.sup.35S]GTP.gamma.S. Next, Gs and Gi
subunits are immunoprecipitated from the samples, and
[.sup.35S]GTP.gamma.S binding is measured using a scintillation
spectrophotometer. Melanocytes with ablated GPER are used as
negative controls in these assays.
[0334] Both the Gs and Gi subunits influence adenylate cyclase
activity, which functions to produce cAMP. To examine the mechanism
through which GPER is regulating the canonical pigmentation pathway
downstream of G protein signaling, adenylate cyclase activity is
analyzed in the presence of estrogen using cAMP ELISA, relative to
vehicle-treated controls. Western analysis is used to examine
downstream changes in pCREB and MITF relative to vehicle-treated
controls.
[0335] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0336] While the invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
Sequence CWU 1
1
18121DNAArtificial SequenceChemically Synthesized 1catgtacgtt
gctatccagg c 21221DNAArtificial SequenceChemically Synthesized
2ctccttaatg tcacgcacga t 21322DNAArtificial SequenceChemically
Synthesized 3aaaggtggga tacgaaaaga cc 22420DNAArtificial
SequenceChemically Synthesized 4agcatccaac aaggcactga
20520DNAArtificial SequenceChemically Synthesized 5ggctgcgaga
aataactgcc 20620DNAArtificial SequenceChemically Synthesized
6aatgcggaca cgtgcttttc 20720DNAArtificial SequenceChemically
Synthesized 7aggtctaccc gccctatctc 20820DNAArtificial
SequenceChemically Synthesized 8agtagttgtg ctgcccttcc
20920DNAArtificial SequenceChemically Synthesized 9gtgctgtaca
ggagccgaag 201020DNAArtificial SequenceChemically Synthesized
10gtcagtccta ccaggcactt 201120DNAArtificial SequenceChemically
Synthesized 11acagagggaa aacgacacct 201220DNAArtificial
SequenceChemically Synthesized 12aattttcact cgccgcttcg
201319DNAArtificial SequenceChemically Synthesized 13tgtggtagag
aagagctgg 191419DNAArtificial SequenceChemically Synthesized
14tccttctcct ctttaactc 191519DNAArtificial SequenceChemically
Synthesized 15tgatgaagta caggtcggg 191620DNAArtificial
SequenceChemically Synthesized 16gaagttcgag ggcgacaccc
201720DNAArtificial SequenceChemically Synthesized 17acaggccgat
cacgtactgc 201820DNAArtificial SequenceChemically Synthesized
18gagcaccagc agtacgtgat 20
* * * * *