U.S. patent application number 16/919466 was filed with the patent office on 2020-10-22 for novel compounds which activate estrogen receptors and compositions and methods of using the same.
The applicant listed for this patent is Board of Regents of the University of Texas System, The Board of Trustees of the University of Illinois. Invention is credited to Benita Katzenellenbogen, John A. Katzenellenbogen, Sung Hoon Kim, Zeynep Madak-Erdogan, Philip Shaul.
Application Number | 20200331828 16/919466 |
Document ID | / |
Family ID | 1000004932654 |
Filed Date | 2020-10-22 |
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United States Patent
Application |
20200331828 |
Kind Code |
A1 |
Katzenellenbogen; John A. ;
et al. |
October 22, 2020 |
NOVEL COMPOUNDS WHICH ACTIVATE ESTROGEN RECEPTORS AND COMPOSITIONS
AND METHODS OF USING THE SAME
Abstract
Provided are compounds of formulae provided herein. The
compounds may include pathway-preferential estrogens (PaPEs)
derivatives with tissue-selective activities. Also provided are
pharmaceutical compositions comprising the compounds, as well as
methods of treating a disease or condition including administering
the compounds. The disease or condition may include postmenopausal
symptoms, cardiovascular disease, stroke, vascular disease, bone
disease, metabolic disease, arthritis, osteoporosis, obesity,
vasomotor/hot flush, cognitive decline, cancer including breast
cancer.
Inventors: |
Katzenellenbogen; John A.;
(Urbana, IL) ; Katzenellenbogen; Benita; (Urbana,
IL) ; Kim; Sung Hoon; (Champaign, IL) ;
Madak-Erdogan; Zeynep; (Champaign, IL) ; Shaul;
Philip; (Richardson, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the University of Illinois
Board of Regents of the University of Texas System |
Urbana
Austin |
IL
TX |
US
US |
|
|
Family ID: |
1000004932654 |
Appl. No.: |
16/919466 |
Filed: |
July 2, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16067676 |
Jul 2, 2018 |
10703698 |
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PCT/US2017/012586 |
Jan 6, 2017 |
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16919466 |
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62275416 |
Jan 6, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C 49/755 20130101;
C07C 39/367 20130101; C07C 39/12 20130101; A61P 25/00 20180101;
C07C 57/38 20130101; C07C 235/34 20130101; C07C 39/06 20130101;
C07C 39/42 20130101; C07C 39/17 20130101; C07C 2602/10 20170501;
C07C 39/23 20130101; A61P 35/00 20180101; C07C 2602/08 20170501;
C07C 2601/14 20170501; C07C 39/14 20130101; C07C 2603/74 20170501;
C07C 43/196 20130101; C07C 255/47 20130101; C07C 49/733
20130101 |
International
Class: |
C07C 39/17 20060101
C07C039/17; C07C 255/47 20060101 C07C255/47; C07C 39/42 20060101
C07C039/42; C07C 235/34 20060101 C07C235/34; C07C 49/755 20060101
C07C049/755; C07C 43/196 20060101 C07C043/196; C07C 49/733 20060101
C07C049/733; C07C 39/12 20060101 C07C039/12; C07C 39/14 20060101
C07C039/14; C07C 57/38 20060101 C07C057/38; C07C 39/367 20060101
C07C039/367; C07C 39/23 20060101 C07C039/23; A61P 25/00 20060101
A61P025/00; A61P 35/00 20060101 A61P035/00; C07C 39/06 20060101
C07C039/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
AT006288, DK015556, and HL087564 awarded by National Institutes of
Health, and ILLU-698-909 awarded by the U.S. Department of
Agriculture. The government has certain rights in the invention.
Claims
1. A compound of formula (i): ##STR00069## and stereoisomers and
pharmaceutically acceptable salts thereof; wherein n is an integer
from 0 to 4; m is an integer from 0 to 4; X is H, hydroxy, or
C.sub.1-4 alkoxy; R.sub.1 and R.sub.2 are independently H, hydroxy,
C.sub.1-4 alkyl, C.sub.1-4 alkoxy, amino, --S--C.sub.1-4 alkyl, or
halo; R.sub.3 is H, hydroxy, oxo, cyano, halo, C.sub.1-4 alkyl, or
C.sub.1-4 alkoxy; each R.sub.4 is independently hydrogen, hydroxy,
oxo, halo, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy; R.sub.5 is H,
C.sub.1-4 alkynyl, or is absent if a double bond is present; and -
- - is an optional double bond.
2. A compound according claim 1, wherein C.sub.1-4 alkyl or
C.sub.1-4 alkoxy is substituted with one or more of halo, cyano,
amino, hydroxy, and C.sub.1-4 alkoxy,
3. A compound according to any one of claim 1 or 2, wherein R.sub.3
is hydroxyl.
4. A compound according to any one of claims 1-3, wherein at least
one of R.sub.1 and R.sub.2 is C.sub.1-4 alkyl.
5. A compound according to claim 4, wherein both R.sub.1 and
R.sub.2 are C.sub.1-4 alkyl.
6. A compound according to claim 4, wherein at least one of R.sub.1
and R.sub.2 is methyl.
7. A compound according to claim 6, wherein both R.sub.1 and
R.sub.2 are methyl.
8. A compound according to any one of claims 1-7, wherein C.sub.1-4
alkyl is substituted.
9. A compound according to claim 8, wherein the substituent is
hydroxy.
10. A compound according to any one of claims 1-9, wherein R.sub.1
and R.sub.2 are independently selected from H, methyl, ethyl,
chloro, --CH.sub.2OH, and --CH.sub.2OMe.
11. A compound according to any one of claims 1-10, wherein R.sub.3
is selected from H, OH, oxo, chloro, cyano, or methoxy.
12. A compound according to any one of claims 1-11, wherein R.sub.4
is selected from fluoro, chloro, bromo, methoxy, hydroxy, or
oxo.
13. A compound according to any one of claims 1-12, wherein R.sub.5
is H.
14. A compound according to any one of claims 1-12, wherein R.sub.5
is --CCH.
15. A compound according any one of claims 1-11, 13 or 14, wherein
m is 0.
16. A compound according to any one of claims 1-15, wherein n is
1.
17. A compound according to any one of claims 1-15, wherein n is
2.
18. A compound according to any one of claims 1, 3-7, 10, 11, 13,
15-17 wherein the compound is selected from the group consisting
of: ##STR00070##
19. A compound according to claim 1 of formula (ii): ##STR00071##
and stereoisomers and salts thereof wherein R is H or methyl.
20. A compound according to formula (iii): ##STR00072## and
stereoisomers and salts thereof; wherein m is an integer from 0 to
3; R.sub.1 and R.sub.2 are independently H, hydroxy, C.sub.1-4
alkyl, C.sub.1-4 alkoxy, amino, --S--C.sub.1-4 alkyl, or halo;
R.sub.5 is H, hydroxy, or C.sub.1-4 alkyl; and each R.sub.6 is
independently H, hydroxy, halo, or C.sub.1-4 alkyl.
21. A compound according to claim 20, wherein C.sub.1-4 alkyl is
substituted with one or more of halo, cyano, amino, hydroxy, and
C.sub.1-4 alkoxy.
22. A compound according to any one of claim 20 or 21, wherein at
least one of R.sub.1 and R.sub.2 is C.sub.1-4 alkyl.
23. A compound according to claim 22, wherein both R.sub.1 and
R.sub.2 are C.sub.1-4 alkyl.
24. A compound according to claim 22, wherein at least one of
R.sub.1 and R.sub.2 is methyl.
25. A compound according to claim 22, wherein both R.sub.1 and
R.sub.2 are methyl.
26. A compound according to any one of claims 20-25, wherein
C.sub.1-4 alkyl is substituted.
27. A compound according to claim 26, wherein the substituent is
hydroxy.
28. A compound according to any one of claims 20-27, wherein
R.sub.5 is hydroxy.
29. A compound according to any one of claims 20-27, wherein
R.sub.5 is C.sub.1-4 alkyl.
30. A compound according to claim 29, wherein R.sub.5 is
substituted C.sub.1-4 alkyl.
31. A compound according to claim 30, wherein R.sub.5 is
--CH(OH)(CH.sub.3).
32. A compound according to any one of claims 20-31, wherein
R.sub.6 is C.sub.1-4 alkyl.
33. A compound according to claim 32, wherein R.sub.6 is
substituted C.sub.1-4 alkyl.
34. A compound according to claim 33, wherein R.sub.6 is
--CH.sub.2OH.
35. A compound according to any one of claims 18-32, wherein m is
1.
36. A compound according to any one of claims 20-35 selected from
the group consisting of: ##STR00073##
37. A compound having the formula (iv): ##STR00074## wherein
R.sub.1 and R.sub.2 is selected from H, C.sub.1-4 alkyl group,
haloalkyl, hydroxyalkyl, alkyloxyalkyl, cycloalkyloxyalkyl,
alkylthio, alkylthioalkyl, cycloalkylthioalkyl, R'R''N-alkyl where
R' and R'' are independently alkyl, alkylcarbonyl, or cyclic alkyl,
and the parenthesis represents the presence or absence of hydroxyl,
and wherein R.sub.3 and R.sub.4 are independently selected from H,
hydroxy, C.sub.1-4 alkyl, hydroxy-C.sub.1-4alkyl, cyano,
cyanoalkyl, nitro, nitroalkyl, --C(O)-aryl, --C(O)H, alkyl
aldehyde, carboxyl, and carboxyalkyl, or wherein R.sub.3 and
R.sub.4 form a ring of from 4 to 8 member atoms, wherein the ring
is substituted with cyano or hydroxy.
38. A compound according to claim 37, of formula (v): ##STR00075##
wherein R.sub.5 and R.sub.6 are independently selected from
hydroxyl, cyano, hydroxylalkyl, cyanoalkyl, halogenated
hydroxylalkyl, and halogenated cyanoalkyl.
39. A compound according to formula (vi): ##STR00076## and
stereoisomers and pharmaceutically acceptable salts thereof;
wherein n is an integer from 0 to 4; m is an integer from 0 to 4; X
is H, hydroxy, or C.sub.1-4 alkoxy; R.sub.1 and R.sub.2 are
independently H, hydroxy, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, amino,
--S--C.sub.1-4 alkyl, or halo; R.sub.3 is H, hydroxy, oxo, cyano,
halo, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy; each R.sub.4 is
independently hydrogen, hydroxy, oxo, halo, C.sub.1-4 alkyl, or
C.sub.1-4 alkoxy; R.sub.5 is H, alkynyl, or is absent if a double
bond is present; R.sub.6 and R.sub.7 are independently selected
from C.sub.1-4 alkyl and H; and - - - is an optional double
bond.
40. A compound according claim 39, wherein C.sub.1-4 alkyl or
C.sub.1-4 alkoxy is substituted with one or more of halo, cyano,
amino, hydroxy, and C.sub.1-4 alkoxy,
41. A compound according to any one of claim 39 or 40, wherein
R.sub.3 is hydroxyl.
42. A compound according to any one of claims 39-41, wherein at
least one of R.sub.1 and R.sub.2 is C.sub.1-4 alkyl.
43. A compound according to claim 42, wherein both R.sub.1 and
R.sub.2 are C.sub.1-4 alkyl.
44. A compound according to claim 42, wherein at least one of
R.sub.1 and R.sub.2 is methyl.
45. A compound according to claim 44, wherein both R.sub.1 and
R.sub.2 are methyl.
46. A compound according to any one of claims 39-45, wherein
C.sub.1-4 alkyl is substituted.
47. A compound according to claim 46, wherein the substituent is
hydroxy.
48. A compound according to any one of claims 39-47, wherein
R.sub.1 and R.sub.2 are independently selected from H, methyl,
ethyl, chloro, --CH.sub.2OH, and --CH.sub.2OMe.
49. A compound according to any one of claims 39-48, wherein
R.sub.3 is selected from H, OH, oxo, chloro, cyano, or methoxy.
50. A compound according to any one of claims 39-49, wherein
R.sub.4 is selected from fluoro, chloro, bromo, methoxy, hydroxy,
or oxo.
51. A compound according to any one of claims 39-50, wherein
R.sub.5 is H.
52. A compound according to any one of claims 39-50, wherein
R.sub.5 is --CCH.
53. A compound according any one of claims 39-49, 51, or 52,
wherein m is 0.
54. A compound according to any one of claims 39-53, wherein n is
1.
55. A compound according to any one of claims 39-54, wherein n is
2.
56. A compound according to any one of claims 39-55, wherein
R.sub.6 is methyl.
57. A compound according to any one of claims 39-56, wherein
R.sub.7 is H.
58. A compound according to formula (vii): ##STR00077## and
stereoisomers and salts thereof; wherein R.sub.7 and R.sub.8 are
independently H, C.sub.1-5 alkyl, amino, hydroxyl, cyano, amido,
cyclic C.sub.3-8 alkyl, or heterocyclyl.
59. A compound according to claim 58, wherein R.sub.7 and R.sub.8
are selected from C.sub.1-4 alkylaminocarboxy-C.sub.1-4 alkyl,
C.sub.1-4 alkylamino-C.sub.1-4 alkyl, C.sub.1-4
alkylamino-C.sub.1-4 alkyl-amino-carboxy-C.sub.1-4 alkyl, C.sub.1-4
alkyloxy-C.sub.1-4 alkylamino-carboxy-C.sub.1-4 alkyl, C.sub.1-4
alkylthio-C.sub.1-4 alkylamino-carboxy-C.sub.1-4 alkyl, or
C.sub.1-4 alkylthio-C.sub.1-4 alkyl.
60. A compound according to claim 58 or 59, wherein R.sub.7 is
methyl.
61. A compound according to any one of claims 58-60, wherein
R.sub.8 is substituted C.sub.1-5 alkyl.
62. A compound according to claim 61, wherein R.sub.8 is
substituted with --C(O)--R, --C(O)NR.sub.N1R.sub.N2, --C(O)OR,
--NR.sub.N1C(O)R, or --OC(O)R, wherein R.sub.N1, R.sub.N2 and R are
independently selected from H or C.sub.1-4 alkyl.
63. A compound according to claim 62, wherein at least one of
R.sub.N1, R.sub.N2, or R is --(CH.sub.2).sub.n--R.sub.10, wherein n
is an integer of from 2 to 5, and wherein R.sub.10 is --C(O)--R,
--C(O)NR.sub.N1R.sub.N2, --C(O)OR, --NR.sub.N1C(O)R, or --OC(O)R,
wherein R.sub.N1, R.sub.N2 and R are independently selected from H,
aryl, cycloalkyl, or C.sub.1-4 alkyl optionally substituted with OH
or amino.
64. A compound according to any one of claims 58-63, wherein the
compound is ##STR00078##
65. A compound selected from the group consisting of ##STR00079##
##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084##
##STR00085## ##STR00086## ##STR00087## and stereoisomers and
pharmaceutically acceptable salts thereof.
66. A compound according to claim 65, selected from the group
consisting of ##STR00088## and pharmaceutically acceptable salts
thereof.
67. A pharmaceutical composition comprising a compound of any one
of claims 1-66 and a pharmaceutically acceptable excipient.
68. A method of treating a disease or condition in a subject, the
method comprising administering a therapeutically effective amount
of a compound or composition according to any one of claims
1-67.
69. The method of claim 68 wherein the disease or condition is
affected by the extranuclear-initiated pathway of the estrogen
receptor.
70. The method of any one of claim 68 or 69, wherein the disease or
condition is selected from postmenopausal symptoms, cardiovascular
disease, stroke, vascular disease, bone disease, metabolic disease,
arthritis, osteoporosis, obesity, vasomotor/hot flush, cognitive
decline, and cancer.
71. The method of any one of claims 68-70, wherein the disease is
stroke.
72. The method of any one of claims 68-70, wherein the disease is
metabolic disease.
73. The method of any one of claims 68-70, wherein the cancer is
breast cancer.
74. The method of claim 73, wherein the disease is obesity-related
breast cancer.
75. The method of claim 73, wherein the disease is
estrogen-responsive breast cancer.
76. The method of any one of claims 68-70, wherein the disease is
vascular disease.
77. The method of any one of claims 68-70, wherein the disease is
osteoporosis.
78. The method of any one of claims 68-77, wherein the subject is
human.
79. A kit comprising a compound or composition of any one of claims
1-67 and instructions for use.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/067,676, filed Jul. 2, 2018, which is the
U.S. national stage entry, under 35 U.S.C. .sctn. 371, of
International Application Number PCT/US2017/012586, filed Jan. 6,
2017, which claims priority to U.S. Provisional Application No.
62/275,416, filed Jan. 6, 2016, the entire contents of each of
which are hereby incorporated by reference.
BACKGROUND
[0003] Estrogens regulate many essential physiological processes
and are needed for the functional maintenance of many adult target
tissues within and outside of the reproductive system. They can,
however, have deleterious actions in promoting breast and uterine
cancers. This balance of desirable versus undesirable activities in
diverse target tissues offers an intriguing opportunity for the
development of tissue-selective estrogens that provide a net
benefit with minimal risk for menopausal hormone replacement, such
as ones affording bone health, relief from vasomotor symptoms, and
metabolic and vascular protection without stimulation of the breast
or uterus.
[0004] It is now recognized that estrogens act through estrogen
receptors (ERs) by utilizing two distinct signaling pathways, the
direct nuclear-initiated ("genomic") pathway, wherein ER functions
as a chromatin-binding ligand-regulated transcription factor, and
the extranuclear-initiated ("non-genomic") pathway, which involves
kinase cascades initiated by ER action from outside the nucleus.
The activation of specific kinases by the action of estrogens
through extranuclear ER action is generally rapid and often
transient, and its initiation likely requires only the input of a
triggering signal by the ER-hormone complex to initiate a kinase
cascade and cellular activities through the extranuclear-initiated
ER signaling pathway. By contrast, the activation of genes through
the direct nuclear ER signaling pathway appears to require a more
sustained action of ER-hormone complexes, sufficient to effect
dissociation of heat shock proteins, recruit coregulator proteins,
stimulate ER binding to chromatin, alter chromatin architecture and
modify histones, and activate RNA pol II to initiate gene
transcription. ER ligands with potent nuclear ER activity form more
kinetically stable receptor-cofactor complexes, and coactivator
binding can slow ligand dissociation rates by orders of magnitude
(Gee, Mol Endocrinol 13, 1912-1923, 1999). Thus, it seemed possible
that ER ligands preferential for extranuclear over nuclear ER
signaling might be obtained by redesigning the structures of
certain estrogens in ways that would preserve their essential
chemical features, a phenol and often a secondary alcohol, as well
as their overall composition and geometry, but would reduce
considerably their high affinity ER binding.
[0005] There is great medical need for estrogens having favorable
pharmacological profiles, supporting desirable activities for
menopausal women such as bone health, relief from vasomotor
symptoms, and metabolic and vascular protection but lacking
stimulatory activities on the breast and uterus.
SUMMARY
[0006] In an aspect, the present disclosure provides a compound of
formula (i):
##STR00001##
and stereoisomers and pharmaceutically acceptable salts thereof;
wherein [0007] n is an integer from 0 to 4; [0008] m is an integer
from 0 to 4; [0009] X is H, hydroxy, or C.sub.1-4 alkoxy; [0010]
R.sub.1 and R.sub.2 are independently H, hydroxy, C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, amino, --S--C.sub.1-4 alkyl, or halo; [0011]
R.sub.3 is H, hydroxy, oxo, cyano, halo, C.sub.1-4 alkyl, or
C.sub.1-4 alkoxy; [0012] each R.sub.4 is independently hydrogen,
hydroxy, oxo, halo, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy; [0013]
R.sub.5 is H, C.sub.1-4 alkynyl, or is absent if a double bond is
present; and [0014] - - - is an optional double bond.
[0015] In another aspect, the present disclosure provides a
compound according to formula (iii):
##STR00002##
and stereoisomers and salts thereof; wherein [0016] m is an integer
from 0 to 3; [0017] R.sub.1 and R.sub.2 are independently H,
hydroxy, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, amino, --S--C.sub.1-4
alkyl, or halo; [0018] R.sub.5 is H, hydroxy, or C.sub.1-4 alkyl;
and [0019] each R.sub.6 is independently H, hydroxy, halo, or
C.sub.1-4 alkyl.
[0020] In another aspect the present disclosure provides a compound
having the formula (iv):
##STR00003##
wherein [0021] R.sub.1 and R.sub.2 is selected from H, C.sub.1-4
alkyl group, haloalkyl, hydroxyalkyl, alkyloxyalkyl,
cycloalkyloxyalkyl, alkylthio, alkylthioalkyl, cycloalkylthioalkyl,
R'R''N-alkyl where R' and R'' are independently alkyl,
alkylcarbonyl, or cyclic alkyl, and the parenthesis represents the
presence or absence of hydroxyl; and [0022] R.sub.3 and R.sub.4 are
independently selected from H, hydroxy, C.sub.1-4 alkyl,
hydoxy-C.sub.1-4alkyl, cyano, cyanoalkyl, nitro, nitroalkyl,
--C(O)-aryl, --C(O)H, alkyl aldehyde, carboxyl, and carboxyalkyl;
or [0023] R.sub.3 and R.sub.4 form a ring of from 4 to 8 member
atoms, wherein the ring is substituted with cyano or hydroxy.
[0024] In another aspect, the present disclosure provides a
compound according to formula (vi):
##STR00004##
and stereoisomers and pharmaceutically acceptable salts thereof;
wherein [0025] n is an integer from 0 to 4; [0026] m is an integer
from 0 to 4; [0027] X is H, hydroxy, or C.sub.1-4 alkoxy; [0028]
R.sub.1 and R.sub.2 are independently H, hydroxy, C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, amino, --S--C.sub.1-4 alkyl, or halo; [0029]
R.sub.3 is H, hydroxy, oxo, cyano, halo, C.sub.1-4 alkyl, or
C.sub.1-4 alkoxy; [0030] each R.sub.4 is independently hydrogen,
hydroxy, oxo, halo, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy; [0031]
R.sub.5 is H, alkynyl, or is absent if a double bond is present;
[0032] R.sub.6 and R.sub.7 are independently selected from
C.sub.1-4 alkyl and H; and [0033] - - - is an optional double
bond.
[0034] In another aspect, the present disclosure provides a
compound according to formula (vii):
##STR00005##
and stereoisomers and salts thereof; wherein [0035] R.sub.7 and
R.sub.8 are independently H, C.sub.1-5 alkyl, amino, hydroxyl,
cyano, amido, cyclic C.sub.3-8 alkyl, or heterocyclyl.
[0036] In another aspect, the present disclosure provides a
pharmaceutical composition comprising a compound described herein
and a pharmaceutically acceptable excipient.
[0037] In another aspect, the present disclosure provides methods
of using the compounds and compositions described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] The features, objects and advantages other than those set
forth above will become more readily apparent when consideration is
given to the detailed description below. Such detailed description
makes reference to the following drawings, wherein:
[0039] FIG. 1. Structures and molecular and binding properties of
E2 and four PaPEs. MW is molecular weight, cLogP is Log.sub.10 of
the calculated octanol-water partition coefficient, Volume is
molecular volume, Polar Surface Area is a measure of compound
polarity, all obtained using ChemBioDraw Ultra (ver. 13.0.0.3015).
Relative binding affinity (RBA) values were determined by
competitive radiometric binding assays (Carlson et. al.
Biochemistry 36, 14897-14905, 1997). E2 is set at 100 on both ERs.
K.sub.i values calculated as K.sub.i=K.sub.d (for
E2).times.(100/RBA), where K.sub.d of E2 is 0.2 nM (ER.alpha.) and
0.5 nM (ER.beta.). Values are average of 3-4 determinations with
coefficients of variation <0.3.
[0040] FIG. 2. Computational model comparing PaPE-1 and E2 in the
ligand-binding pocket of ER.alpha.. The model of ER.alpha.+E2,
based on a crystal structure (1GWR), has E2 and helical elements
shown in silver/grey and the pocket volume contour in slate blue.
The model for PaPE-1 was generated from the ER.alpha.+E2 structure
by progressive transformation of the ligand structure from E2 to
PaPE-1, partnered with progressive minimization of the ligand and
the ligand-binding domain. The resulting positions of the PaPE-1
ligand and hydrogen bonding residues are shown in orange. For
details, see Methods below.
[0041] FIG. 3. Comparison of gene expression, cell proliferation,
and pathway signaling regulation by PaPE-1 and E2. (A) PaPE-1
preferentially activates extranuclear-initiated (non-genomic) genes
(LRRC54) over direct nuclear (genomic) genes (PgR) compared to E2
in MCF-7 cells. Cells were treated with control vehicle (0.1%
ethanol), 10 nM E2 or 1 .mu.M PaPE-1 for 4 h and gene expression
was monitored by qPCR. (B) MCF-7 cells were pretreated with 1 .mu.M
ICI182,780 (ICI) for 1 h and then treated with Veh (0.1% EtOH), 10
nM E2 or 1 .mu.M PaPE-1 in the presence or absence of ICI for 4 h.
RNA was isolated and subjected to qPCR analysis for the indicated
genes. (C) MCF-7 cells were seeded and after 24 h the cells were
transfected with siCtrl, siER.alpha. or siGPR30 (30 nM) for 72 h.
Cells were then treated with Veh (0.1% EtOH), 10 nM E2 or 1 .mu.M
PaPE-1 for 4 h. RNA was isolated and subjected to qPCR analysis.
(D) MCF-7 cells were treated with Veh, 10 nM E2 or the indicated
concentrations of PaPE-1 for 6 days and proliferation was monitored
by WST assay. (E) PaPE-1 selectively activated mTOR and MAPK
signaling in MCF-7 cells. Cells were treated with control vehicle
(V), or the indicated concentrations of E2 or PaPE-1 for 15 min
(upper panel) and 45 min (lower panel) and Western blots were done
to assess activated signaling proteins and S118 phosphorylation of
ER.alpha.. Total ER.alpha. was monitored, and total ERK2 was used
as a loading control. (F) PaPE-1 induces interaction between
ER.alpha. and Raptor. MCF-7 cells were treated with 10 nM E2 or 1
.mu.M PaPE-1 for 15 min. Cells were crosslinked and incubated with
ER.alpha. and Raptor antibodies overnight and PLA was performed.
Quantitation of signal intensities is shown in panel at the right.
Two-way ANOVA, Bonferroni posttest, * p<0.05, ** p<0.01, ***
p<0.001.
[0042] FIG. 4. PaPE-1 and E2 regulate common as well as distinct
groups of genes in MCF-7 cells. (A) Cells were treated with 10 nM
E2 or 1 .mu.M PaPE-1 for 4 h and 24 h. RNA was isolated and RNA-Seq
was performed. Regulated genes are considered to be those with
P<0.05 and expression fold change >2. (B) PaPE-1 mediated
gene expression changes are sensitive to mTOR and MAPK pathway
inhibitors. Effect of mTOR and MAPK inhibitors on gene regulation
by E2 and PaPE-1 in MCF-7 cells. Cells were pretreated with 1 .mu.M
PP242 or 1 .mu.M AZD6244 for 1 h and then treated with 10 nM E2 or
1 .mu.M PaPE-1 for 4 h in the presence or absence of inhibitors.
RNA was isolated and RNA-Seq performed. (P<0.05, fold change
>2). (C) PaPE-1 does not induce recruitment of ER.alpha. or ERK2
to chromatin but stimulates recruitment of RNA Pol II. MCF-7 cells
were treated with 10 nM E2 or 1 .mu.M PaPE-1 for 45 min. ChIP-Seq
was performed for the indicated proteins. UCSC genome tracks of
cistromes in the presence of E2 or PaPE-1 are shown (right
panel).
[0043] FIG. 5. PaPE-1, unlike E2, does not change uterus or thymus
weight and does not induce mammary gland ductal branching but, like
E2, PaPE-1 reduces mammary gland adipocyte area. (A) PaPE-1 does
not affect uterus or thymus weight. C57BL/6 mice were
ovariectomized and then were given daily subcutaneous injections of
E2 (5 .mu.g/day) or vehicle (V, Veh), or were implanted with PaPE-1
pellets (5 .mu.g/day and 300 .mu.g/day) for 4 days. Weights of
uterus and thymus were monitored. (B) PaPE-1 stimulates only very
minimal mammary ductal elongation but it greatly reduces adiposity
(adipocyte size) in mammary gland. C57BL/6 mice were ovariectomized
and then pellets of E2 (5 .mu.g/day) or PaPE-1 (5 .mu.g/day and 300
.mu.g/day) were implanted for 3 weeks. Whole mount stain and H and
E stain of mammary gland are shown. (C) Mammary gland adipocyte
area was calculated from the H and E images. Two-way ANOVA,
Bonferroni posttest, * p<0.05, ** p<0.01, *** p<0.001,
**** p<0.0001.
[0044] FIG. 6. PaPE-1, like E2, reduces the increase in body weight
after ovariectomy and reduces adipose stores and the blood
triglyceride level. (A) PaPE-1 is effective in normalizing body
weight after ovariectomy. C57BL/6 mice were ovariectomized and,
after 3 weeks, pellets containing E2 (5 .mu.g/day) or PaPE-1 (300
.mu.g/day) or Veh control were implanted for 3 weeks (n=8/group).
Animals were on normal chow diet. Two-way ANOVA, Bonferroni
posttest, * p<0.05, ** p<0.01, *** p<0.001, ****
p<0.0001, comparing treatments to vehicle (Veh.) (B) Food
consumption for each mouse from A was monitored weekly. (C) Body
composition for each mouse from A was monitored using EchoMRI at
the end of 3 weeks. One-way ANOVA, Newman-Keuls post-test, *
p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. (D) H
and E staining of perigonadal Adipose Tissue (AT). (E) Weights of
various fat depots monitored after 3 weeks of control vehicle or
ligand exposure. (F) Triglycerides were measured in the blood of
animals (n=6/group) at the end of 3 weeks of Veh, E2 or PaPE-1
treatment. (G) H and E staining (upper 2 panels) and Oil Red O
staining (lower panel) of the liver at 3 weeks of treatment. (H)
Gene expression analysis of SREBPc and FASN in liver at 3 weeks
(upper panel) (n=12/group), and time course of FASN and ACACA
expression in livers of E2 and PaPE-1 treated mice over 2 weeks.
Two-way ANOVA, Bonferroni posttest, * p<0.05, ** p<0.01, ***
p<0.001, **** p<0.0001.
[0045] FIG. 7. Gene regulation and signaling pathway activations by
PaPE-1 and E2 in tissues in vivo. (A) C57BL/6 mice were
ovariectomized and after 3 weeks, cholesterol pellets containing E2
(5 .mu.g/day), PaPE-1 (300 .mu.g/day), or vehicle (Veh, cholesterol
alone) were implanted for 3 weeks. Liver, skeletal muscle,
perigonadal fat, pancreas, and uterus were harvested. RNA was
isolated and qPCR performed for the indicated genes. (B) PaPE-1
activates mTOR signaling (monitored by increase in p-S6) in liver
and skeletal muscle. Ovariectomized C57BL/6 mice were injected with
5 .mu.g of E2 or 300 .mu.g of PaPE-1 for 2 h. Indicated tissues
were collected and subjected to Western blot analysis for p-S6 and
p-p42/44 MAPK. .beta.-actin and total ERK2 were used as loading
controls.
[0046] FIG. 8. PaPE-1, like E2, elicits repair of the vascular
endothelium after injury and this is prevented by the antiestrogen
ICI. (A) Carotid artery reendothelialization after an injury that
denudes the endothelial layer in OVX mice treated with PaPE-1 or E2
in the absence or presence of the antiestrogen ICI 182,780 (ICI).
*, p<0.05 vs. basal control. (B) PaPE-1 does not affect uterine
weight in these experiments, whereas E2 increases uterine weight
and this is blocked by ICI. (C) eNOS stimulation by E2 and PaPE-1
in the presence and absence of the antiestrogen ICI 182,780. BAEC
were treated with ligands for 15 min and eNOS activity was
monitored. *, p<0.05 vs. control.
[0047] FIG. 9. Assessment of the activities of PaPE-2, PaPE-3, and
PaPE-4 in MCF-7 cells, in bovine aortic endothelial cells (BAEC)
cells, and in mice. (A) Preferential stimulation of
extranuclear-initiated LRRC54 gene expression vs. direct nuclear
PgR gene expression. MCF-7 cell treatment was with control vehicle
(Veh), 10 nM E2, or 1 .mu.M of the indicated PaPE for 4 h prior to
RNA harvest and qPCR analysis. (B) Proliferation of MCF-7 cells
after treatment with different concentrations of E2 or the PaPE for
6 days. (C) Stimulation of various cell signaling pathway
activations by different concentrations of E2, PaPE-1, or PaPE-4
after 15 min treatment of MCF-7 cells. Level of ER.alpha. is also
shown. (D) Time course of cell signaling pathway activations by
PaPE-2 or PaPE-3, monitored at 15, 45, and 90 min. Level of
ER.alpha. is shown, and total ERK2 is used as a loading control.
(E) Stimulation of NOS activity during 15 min treatment of BAEC
with 10 nM ligand alone or with cotreatment with 1 .mu.M ICI
182,780. (F) The PaPEs and E2 reduce weight gain after ovariectomy
in C57BL/6 mice. Animals were ovariectomized, and after 3 weeks,
animals received pellets of E2 (5 .mu.g/day), the PAPE (300
.mu.g/day), or vehicle, and body weight was monitored over the next
3 weeks. A group of intact non-ovariectomized mice were included
for comparison. Two-way ANOVA, Bonferroni posttest, * p<0.05, **
p<0.01, *** p<0.001, **** p<0.0001, comparing all
treatments to vehicle (Veh.) (G) Food consumption was monitored
over time. (H) Assessment of uterine weight gain in ovariectomized
C57BL/6 mice after 3 weeks of pellets of E2 (5 .mu.g/day) or
PaPE-1, -2, -3 and -4 (300 .mu.g/day). One-way ANOVA, Newman-Keuls
post-test, * p<0.05, ** p<0.01, *** p<0.001, ****
p<0.0001. (I) Fat mass, lean mass, and water mass were measured
by EchoMRI at the end of the 3-week treatment period in mice shown
in Panel F. One-way ANOVA, Newman-Keuls post-test, comparing intact
and all treatments to vehicle (Veh.) * p<0.05, ** p<0.01, ***
p<0.001, **** p<0.0001.
[0048] FIG. 10. Estrogen receptor and coactivator binding and
interaction assays with ligands and ligand dissociation rates. (A)
Comparison of binding affinities of PaPE-1, trans-hydroxytamoxifen
(OH-Tam), and E2 for ER.alpha. (top) and ER.beta. (bottom)
determined by .sup.3H-E2 competition binding assays. (B) Binding of
the coregulator SRC3 to the E2-ER.alpha. or PaPE-1-ER.alpha.
complex. (C) Displacement of SRC3 from the 10 nM E2-ER.alpha.
complex by OH-Tam or PaPE-1. (D) Co-immunoprecipitation assays to
examine the interaction of ER.alpha. and SRC3 in MCF-7 cells after
treatment of cells with control vehicle, 10 nM E2 or 1 .mu.M PaPE-1
for 1 h. After ligand treatment, cell lysates were
immunoprecipitated with antibody to SRC3 or control IgG, and
immunoprecipitates were separated on SDS PAGE gels and blotted for
ER.alpha. using anti-ER.alpha. antibody. (E) Time course of
dissociation of PaPE-1 and E2 (F) from the ER.alpha. ligand-binding
domain. ER.alpha. LBD (2 nM) was equilibrated with 100 nM E2 or 5
.mu.M PaPE-1 for 1 h on ice. Samples held at 5.degree. C. were
assayed by fluorescence polarization for the zero-time point, and
then excess E2 was added to the PaPE-1 or excess OH-Tam to the E2
sample, and the dissociation was followed with time as a change in
anisotropy. For details, see methods below.
[0049] FIG. 11. Proximity ligation assays (PLAs) with E2 and
PaPE-1. PLA was used compare the effects of control vehicle, 10 nM
E2 or 1 .mu.M PaPE-1 on the interaction of: ER.alpha. with mTOR; or
ER.alpha. with PRAS40; or ER.alpha. with pMAPK. MCF-7 cells were
treated with ligands for 15 min and PLA was then conducted.
Quantitation of signal intensity of the PLAs is shown in panels at
the right. One-way ANOVA, Bonferroni posttest, * p<0.05, **
p<0.01, *** p<0.001, **** p<0.0001.
[0050] FIG. 12. Pharmacokinetic (PK) studies for analysis of blood
levels of PaPE-1 after injection or pellet implantation. (A)
Measurement of PaPE-1 level in the blood of ovex mice after SC
injection of 100 .mu.g PaPE-1 was done at the times indicated, up
to 24 h. (B) Measurement of PaPE-1 levels in the blood of ovex mice
over 3 weeks after implantation of a pellet containing 300
.mu.g/day PaPE-1.
[0051] FIG. 13. Effects of PaPE-1 require ER.alpha.. (A) Wild type
(WT) or ER.alpha. knock-out (ERKO) C57BL/6 mice were
ovariectomized, and after 3 weeks, pellets containing E2 (5
.mu.g/day) or PaPE-1 (300 .mu.g/day) were implanted for 3 weeks.
Animals were on normal chow diet. Change in body weight was
monitored. (B) Uterus weights of animals from (A) at the end of 3
weeks of vehicle (Veh) or ligand treatment. (C) Triglyceride levels
in the blood of animals from (A) after 3 weeks of ligand exposure.
(D) Gene expression analysis of FASN and SREBP1c in livers of WT or
ERKO mice treated with Veh, E2 and PaPE-1 for 3 weeks. t-test, *
p<0.05, ** p<0.01, *** p<0.001.
[0052] FIG. 14. Compound 2, which is the same as PaPE-1, activated
the non-genomic gene, LRRC54, to a 1.9 fold higher level than E2.
In contrast to LRRC54 mRNA expression, the level of induction of
the PgR mRNA by compound 2 is almost unchanged from the vehicle
(veh.) level. Hence, it has good preferential activity on the
non-genomic signaling pathway. When the phenolic OH is blocked with
a methyl group, as in compound 5, there is a marked loss of this
non-genomic activity. When the aliphatic alcohol is oxidized to a
ketone (compound 6), the non-genomic activity is lost as well. With
a chlorine in place of the aliphatic alcohol (compound 8), there is
stimulation of the nongenomic gene (LRRC54), but to a lesser degree
than compound 2 (and only 50% compared to E2), and the cyano analog
(compound 9) shows no non-genomic activity. The positional isomer
(compound 10) has quite good selective non-genomic activity, but
the ketone analog (compound 11) is inactive. Compounds 12, 13, and
14 have hydroxyl groups deleted, and while the first two have some
non-genomic activity, all three of them strongly stimulate the
genomic gene PgR.
[0053] FIG. 15. The activities shown in FIG. 14 are shown here as a
radar or star plot, with the red curve representing genomic
activity (stimulation of the PgR gene) and the blue curve
representing the non-genomic activity (stimulation of the LRRC54
gene). Selective non-genomic activity is evident when the points on
the blue curve extend beyond the points on the red curve. From this
it is clear that of the compounds in this figure, compound 2
(PaPE-1) has the greatest non-genomic activity together with very
low genomic activity.
[0054] FIG. 16. Fold change in LRRC54 mRNA (the non-genomic gene)
and PgR mRNA (the genomic gene) with PaPE-1 (compound 2) and
various compounds.
[0055] FIG. 17. The effect of PaPE-1 (compound 2) and various close
analogs on the expression of LRRC54 mRNA (non-genomic) and PgR mRNA
(genomic) genes demonstrated in a radar graph. As shown here,
tetralone (compound 16, also termed PaPE-3) and its open ring
system (compound 15, also termed PaPE-2) have some selectivity for
the non-genomic gene but less than that of compound 2. Indene-1-ol
system (compound 17) loses both LRRC54 and PgR activity.
Indene-1-one system (compound 18) increases expression of both the
non-genomic and genomic genes. 17-Ethynyl estradiol mimic compounds
(compounds 19-21) do not have activity on either gene. When the
alcoholic OH is blocked by a methyl group (compound 22),
stimulation of both genomic and nongenomic gene expression is
lost.
[0056] FIG. 18. Fold change of LRRC54 mRNA (the non-genomic gene)
and PgR mRNA (the genomic gene) with PaPE-1, a racemate, (compound
2) and with the S-enantiomer (compound 3) and R-enantiomer
(compound 4) of compound 2 (PaPE-1). The racemate and S-enantiomer
have equivalent activities; the R-enantiomer has less non-genomic
gene activity.
[0057] FIG. 19. Fold change of LRRC54 mRNA (the non-genomic gene)
and PgR mRNA (the genomic gene) with various additional benzylic
compounds and structurally modified PaPE derivatives.
[0058] FIG. 20. Star plot of the data from FIG. 19. Among benzyl
alcohol-type compounds (compounds 23-28), compounds 24, 26, and 27
showed similar levels of nongenomic gene expression as did
estradiol (E2), but the genomic gene (PgR) expression was slight
higher than the vehicle level. Compound 2 (PaPE-1) showed the
greatest preferential nongenomic gene stimulation. Interestingly,
compound 29 expressed LRRC54 similar level to E2, but it did not
activate PgR above that of vehicle (veh.). Compound 30, derived by
reduction of the ketone (compound 29), elevated both genomic and
nongenomic gene expression to a higher level than does its parent
compound 29.
[0059] FIG. 21. Fold change of LRRC54 and PgR mRNA levels induced
by various biphenyl type analogs lacking portions of the structure
of PaPE-1 (compound 2).
[0060] FIG. 22. Star plot of the data from FIG. 21. From this
series, it can be seen that both the benzylic OH and two methyl
groups flanking the phenolic OH in compound 2 (see FIGS. 14 and 15)
are important to suppress genomic gene expression and stimulate
LRRC54 gene activity. If the methyl groups and OH are eliminated
(compound 31), both activities are lost. If only the methyl groups
are removed from compounds 2 and 6, these derivatives (compounds
32-33) stimulate PgR gene expression at a level close to that of E2
while retaining LRRC54 gene induction similar to (compound 32) or
somewhat less than (compound 33) that of compound 2 (PaPE-1). In
addition to the important roles of the two methyl groups and the
phenolic OH, elimination of the alkyl group bearing the aliphatic
alcohol or its replacement with a phenol (compounds 34-35) causes
loss of activity in stimulating the expression of both LRRC54 and
PgR genes (compound 34) or principally loss of LRRC54 gene
expression (compound 35).
[0061] FIG. 23. Fold change of LRRC54 and PgR mRNA gene expression
induced by various modified versions of estradiol (E2), compounds
36-43.
[0062] FIG. 24. Star plot of the data from FIG. 23. All of the
modifications except that of compound 38 have strong activity on
the genomic gene PgR. Non-genomic gene (LRRC54) activity similar to
that of compound 2 (PaPR-1) is retained with compounds 39 and 41,
and to a less extent with 36 and 37.
[0063] FIG. 25. Compound 40 selectively stimulates LRRC54 gene
expression at femtomolar concentrations, with reduced expression at
nanomolar to micromolar concentrations; genomic gene expression
(PgR) increases only at higher concentrations, demonstrating a
potency separation between non-genomic (highly sensitive) and
genomic (less sensitive) signaling controlling target gene
expression.
[0064] FIG. 26. Fold change of LRRC54 and PgR mRNA gene expression
induced by various additional biphenyl compounds (44-49) to examine
the effect of alkyl chain modifications while retaining the
dimethyl phenol function.
[0065] FIG. 27. Star plot of the data from FIG. 26. From this
series, p- and m-phenethyl alcohol (compounds 44-45) as well as
p-phenethyl bromide (compound 46) compounds preferentially
stimulate LRRC54 better than E2. m- and p-Phenylpropyl alcohol
group (compounds 47-48) stimulate expression of genomic PgR genes
well with compound 47 also having good stimulation of the
non-genomic LRRC54. The m-phenylacetic acid analog (compound 49)
behaves like vehicle.
[0066] FIG. 28. Fold change of LRRC54 and PgR mRNA gene expression
induced by additional indane derivatives (compounds 50-58).
[0067] FIG. 29. Star plot of the data from FIG. 28. .alpha.-Fluoro
(compound 50) and .alpha.-bromo (compound 51) substituted at 2
position of compound 2 (PaPE-1) stimulate both non-genomic LRRC54
and genomic PgR gene expression close to the level that of E2.
Dibromo substituted compound (compound 52) and to some extent also
the methoxy-substituted compound 54 show little expression of both
genes. 2-Naphthol compound (compound 53) has a gene expression
pattern equivalent to that of estradiol (E2). When one methyl group
is deleted (compound 55), the PgR gene is stimulated two fold
greater than that with E2. Addition of polar OH groups onto the
aryl methyl(s) (compounds 56-58) increases the expression of the
non-genomic LRRC54 gene, with compounds 56 and 57 showing
considerable non-genomic gene selectivity.
[0068] FIG. 30. Fold change of LRRC54 and PgR mRNA gene expression
induced by additional biphenyl and cyclofenil mimic compounds
(59-70).
[0069] FIG. 31. Star plot of the data from FIG. 30. Phenolic benzyl
alcohols which have an o,o'-dimethyl phenol (compounds 59 and 60)
or an o,o'-dichloro phenol (compound 64) have selective expression
of the non-genomic LRRC54 gene compared to E2, but the expression
level is less than for compound 2. The dichloro analog of PaPE-1
(compound 63) stimulates the non-genomic gene LRRC54 selectively,
but the activity is two-fold less than that of compound 2.
4-Hydroxy-3-hydroxymethyl-biphenyl (compound 61) stimulates LRRC54
a little better than vehicle (Veh.) and PgR not at all
4,4'-Dihydroxy-3,3',5,5'-tetramethylbiphenyl (compound 65) greatly
suppresses LRRC54 gene expression, with PgR gene expression
equivalent to that of vehicle (Veh.) The two cyclofenil type
compounds (compounds 66-67) stimulate PgR gene selectively. An
additional methyl group at the position ortho to the phenol in
compound 59 (as in Compound 68) suppresses LRRC54 gene expression
compared to that of the parent compound 59. An additional phenolic
group above those in compounds 44 and 47 (as in compounds 69 and
70) does not improve the non-genomic LRRC54 gene response compared
to those of compounds 44 and 47.
[0070] FIG. 32. Comparison of compound 2, hydroxytamoxifen
(HO-TAM), and Raloxifene (RAL) compounds. Raloxifene blocks
expression of both the non-genomic LRRC54 and genomic PgR genes.
HO-TAM shows slight stimulation of the PgR gene but not the
non-genomic LRRC54 gene.
[0071] FIG. 33. Star plot of the data from FIG. 32. Neither
Raloxifene (RAL) nor hydroxytamoxifen (TOT) show selective
activation of the non-genomic gene LRRC54.
[0072] FIG. 34. Characterization of risk factors associated with
tumors and aggressiveness in obese postmenopausal women. (A)
Heatmap of the whole metabolite profiling of 100 serum samples from
63 obese or overweight vs. 37 non-obese postmenopausal women from
Midlife Health Study that fits the criteria (BMI>25 obese or
overweight, 2-3 years into menopause), as measured using GC-MS. For
BMI red indicates higher values, whereas green indicates lower
values. (B) OLINK Biomarker profiling of the same samples as in A.
For BMI red indicates higher values, whereas green indicates lower
values. (C) Cell proliferation assays were performed in both
ER.alpha.-(+) and ER.alpha.-(-) breast cancer cell lines. The serum
from 35 obese and 35 non-obese individuals was used to treat the
cells for 7 days before analysis by the WST1 assay. Three technical
replicates were used. Unpaired t-test was used to assess if serum
from obese vs. non-obese individuals resulted in statistically
significant difference in breast cancer cell line proliferation. *,
p<0.05. Mean.+-.SEM is plotted. (D) Cell migration was tested in
BT474 cells treated with the serum samples of 35 obese and 35
non-obese individuals for 24 hours before measurement of cell
number per field. The mTOR Pathway was found to be activated as
indicated by increased pS6K activity by serum from 35 obese
individuals but not by serum from 35 non-obese individuals in MCF-7
cells. All the assays were performed in triplicate in 96 well
plates. Unpaired t-test was used to assess if serum from obese vs.
non-obese individuals resulted in statistically significant
difference in breast cancer cell line motility and mTOR pathway
activation. ** p<0.01, *** p<0.001. Mean.+-.SEM is plotted.
(E) Correlation analysis (Pearson correlation coefficient) of
biomarkers with the cell phenotype indexes. Those biomarkers that
correlate with MCF-7 proliferation at p-val<0.05 are selected.
Paired t-test with Bonferroni correction was used to identify the
statistically significant difference. * p<0.05, ** p<0.01,
*** p<0.001. F) Correlation analysis (p-val) of biomarkers from
E.
[0073] FIG. 35. Identification of factors associated with cell
proliferation in obese postmenopausal women. (A) Association
analysis of cell proliferation and BMI, estradiol, testosterone and
progesterone. Pearson correlation coefficient (Upper panel) and
p-value (Lower panel) were shown for each comparison. (B) Detailed
association analysis was shown between proliferation and BMI,
estradiol and proliferation, and BMI and estradiol. (C) The
relative concentration of metabolites was measured and shown from
serum samples grouped by proliferation index. Paired t-test with
Bonferroni correction was used to identify the statistically
significant difference. * p<0.05, ** p<0.01, ***
p<0.001.
[0074] FIG. 36. Screening of FFAs for stimulating effects on cell
proliferation. (A) Heatmap of the free fatty acid profiling of the
same samples from FIG. 34A, measured using GC-MS. For BMI and
proliferation red indicates higher values, whereas green indicates
lower values. (B) Important features associated with high and low
proliferation index using Partial Least Squares-Discriminant
Analysis (PLS-DA) in Metaboanalyst. (C) Metabolomics analysis of
Midlife health study-weight loss samples. Initial and final visit
serum samples from 21 postmenopausal women from Midlife Health
Study who met the following criteria was analyzed: BMI>25 at the
initial visit and BMI<25 at the last visit. First visit samples
(when individuals were obese or overweight) are indicated with
red-thick lines. (D) Metabolomics analysis of Komen Tissue Bank
Susceptible Samples. Serum samples are from 23 susceptible
postmenopausal women who were cancer-free at the time of blood
donation who later had breast cancer diagnosis and from 23 women
who are cancer-free as has not reported breast cancer diagnosis
(age, BMI, race matched controls). (E) Cell proliferation of MCF-7
cells in the presence of individual FFAs identified from FIGS. 36A
and B. Cell proliferation was examined after treatment of cells
with free fatty acids at two concentrations (1 and 100 nM). FFA mix
contains all the single free fatty acids tested in this experiment.
Lipid mix (mixture of cholesterol and fatty acids) was purchased
from Sigma. Six replicates were used in each assay, and the
experiment was repeated twice. An unpaired t-test was used to
assess if various free fatty acid (FFA) treatments resulted in
statistically significant stimulation of MCF-7 cell proliferation.
** p<0.01, *** p<0.001, **** p<0.0001. Mean.+-.SEM is
plotted.
[0075] FIG. 37. PaPE-1 suppresses proliferation and pathway
activation in MCF-7 cells and normalizes the oleic acid (OA) level
increased by ovariectomy in mice. (A) Correlation of OA levels in
serum from Midlife health study with MCF-7 cell proliferation. (B)
mTOR pathway activation induced by OA in MCF-7 cells. Cells were
treated with 100 nM oleic acid for 0, 15, 45, and 90 mins.
Phosphorylation and total protein levels of AKT, ERK1/ERK2, S6, and
4EBP1 were examined by western blot analysis. The experiment was
repeated two times and representative blots are shown. (C)
Inhibition of OA-induced mTOR pathway activation by PaPE-1. Oleic
acid stimulates the mTORC1 pathway, and PaPE-1 suppressed the
activation in MCF-7 cells. MCF-7 cells were treated with control
vehicle, and 100 nM Oleic acid, with or without PaPE-1 for 45 mins.
Phosphorylated and total proteins levels of 4EBP1, P70S6K, and S6
were examined by western blot analysis. The experiment was repeated
two times and representative blots are shown. (D) OA effect on
MCF-7 cell proliferation with and without PaPE-1. Dose-response of
OA on cell proliferation was tested alone or in combination with
and without PaPE-1 (1 .mu.M) for 6 days before WST1 assay. The
experiment has six technical replicates and was repeated twice. A
two-way analysis of variance (ANOVA) model was fitted to assess the
contribution of OA dose and inhibitor (Ctrl, and PaPE-1) treatment
on MCF-7 cell proliferation. When the main effects were
statistically significant at .alpha.=0.05, pairwise t-tests with a
Bonferroni correction were employed to identify if treatment were
statistically different from each other. **** p<0.0001.
Mean.+-.SEM is plotted. (E) Inhibition of OA-induced MCF-7 cell
proliferation by ER.alpha. and mTOR targeting agents. Cell
proliferation was stimulated by 100 nM oleic acid and was
suppressed by adding 1 .mu.M 4-OH-Tamoxifen (4-OH-Tam), 1 .mu.M
Fulvestrant (Fulv), mTOR inhibitor 1 .mu.M RAD001, and 1 .mu.M
PaPE-1. In the above cell proliferation experiments, MCF-7 cells
were treated in whole growth medium adding the designated
compounds. The treatment went for 6 days and OD at 450 was measured
by WST1 assay. The data is represented from average of 6 replicates
with standard error of mean. The experiment is repeated twice. A
two-way analysis of variance (ANOVA) model was fitted to assess the
contribution of ligand (Veh or OA) and inhibitor (Ctrl, 4-OH-Tam,
Fulv, RAD001 and PaPE-1) treatment on MCF-7 cell proliferation.
When the main effects were statistically significant at
.alpha.=0.05, pairwise t-tests with a Bonferroni correction were
employed to identify if treatment were statistically different from
each other. **** p<0.0001. Mean.+-.SEM is plotted. (F)
Inhibition of serum-induced MCF-7 cell proliferation by 4-OH-tam,
Fulv and PaPE-1. The MCF-7 cells were treated with Veh, 1 .mu.M
4-OH-tam, 1 .mu.M Fulv, and 1 .mu.M PaPE-1 for six days before
WST-1 assay in serum from 63 obese individuals (Left) and in
standard cell culture (Right). There are 3 technical replicates for
each serum sample and 14 replicates for each treatment in standard
cell culture media. A one-way analysis of variance (ANOVA) model
was fitted to assess the contribution of ligands on serum- or
standard cell culture medium-induced MCF-7 cell proliferation. When
the main effects were statistically significant at .alpha.=0.05,
pairwise t-tests with a Bonferroni correction were employed to
identify if treatment were statistically different from each other.
**** p<0.0001. Mean.+-.SEM is plotted. (G) Restoration of OA
levels with PaPE-1 after ovariectomy mice. Three different mice
models were tested: wildtype C57BL mice under normal diet (N=2 mice
per treatment group, experiment was repeated twice), wild-type
C57BL mice under high-fat diet (N=5 animals per treatment group)
and ob/ob mice (N=4 animals per treatment group) under normal diet.
Mice were ovariectomized at six week old and Alzet slow release
minipumps with Veh or PaPE-1 were implanted subcutaneously for six
weeks. The OA concentration was measured in the serum using GC-MS.
An unpaired t-test was used to assess if PaPE-1 was able to
decrease the relative OA concentration in the serum compared to Veh
treated animals. * p<0.05, ** p<0.01. Mean.+-.SEM is
plotted.
[0076] FIG. 38. Identification of genes induced by OA and PaPE-1's
effect on the gene regulation. (A) RNA-Seq analysis of gene
expression changes induced by OA and OA+PaPE1. Heat map of the
genes with significant changed expression. MCF-7 cells were treated
with Veh or 100 nM OA with and without 1 .mu.M PaPE-1 for 24 h. RNA
was isolated and RNA-Seq was performed using 2 samples from each
treatment group. Differentially regulated genes were determined
with p<0.05 and expression fold change >2. (B) Clusters from
A. regulated by OA and reversed by PaPE-1. The average gene
expression level of the cluster1 (C1) and 2 (C2) identified as
PaPE-1 regulated high-fat genes. In C1, there are 425 genes whose
expression were found stimulated in OA but not in the OA+PaPE-1
treatment. In C2, there are 646 genes whose expression were found
suppressed in the OA but restored by OA+PaPE-1 treatment. (C) Venn
diagram analysis. Venn diagram of the up- and downregulated genes
by OA alone or in combination with PaPE-1. (D) Examples of
OA-regulated genes. Some of the top functions of the involved genes
are presented.
[0077] FIG. 39. ER.alpha. chromatin binding sites induced by OA and
the effect of PaPE-1 in reversing these changes in the ER.alpha.
cistrome. (A) Recruitment of ER.alpha. to chromatin in the presence
of PaPE-1, OA and OA+PaPE-1. MCF-7 cells were treated with Veh and
100 nM OA with or without 1 .mu.M PaPE-1 for 45 minutes.
ER.alpha.-DNA complexes were pulled down using ER.alpha.
antibodies. Three biological replicates were pooled and sequenced.
Clustering of ER.alpha. binding sites in treatments of Veh (0.1%
EtOH), PaPE-1 (1 .mu.M), OA (100 nM), and OA (100 nM)+PaPE-1 (1
.mu.M) was done using seqMINER software. (B) Validation of effect
of OA and PaPE-1 on the ER.alpha. binding at regulatory region of
PgR, CISH and SREBPlC using ChIP-qPCR. MCF-7 cells were treated
with Veh and 100 nM OA with or without 1 .mu.M PaPE-1 for 45
minutes. ER.alpha.-DNA complexes were pulled down using ER.alpha.
antibodies. Recruitment of ER.alpha. to PgR
(chr11:100,904,522-100,905,458), CISH (chr3:50,642,336-50,643,191)
and SREBP1 (chr17:17,743,329-17,743,912) sites were quantified by
Q-PCR. The experiment was repeated 3 times with at least duplicates
each time. Mean.+-.SEM is plotted. A one-way analysis of variance
(ANOVA) model was fitted to assess the contribution of ligand (Veh
or OA) and inhibitor (Ctrl, PaPE-1) treatment on MCF-7 cell
proliferation. When the main effects were statistically significant
at .alpha.=0.05, pairwise t-tests with a Bonferroni correction were
employed to identify if treatment were statistically different from
each other. * p<0.05, ** p<0.01. Mean.+-.SEM is plotted. (C)
Binding sites whose ER.alpha. occupation increased upon OA
treatment (cluster marked by * in B). The ER.alpha. binding sites
were separated into four clusters of characteristic patterns:
C.sub.1, C2, C3 and C4. (D) Transcription Factor (TF) binding site
enrichment was identified using Seqpos tool from Cistrome/Galaxy
for clusters of C1, C2, C3 and C4. (E) Transcriptional activities
of various TFs. 45 pathway CignalFinder Assay was used to transfect
MCF-7 cells with indicated luciferase construct for 24 h. Cells
were treated with Veh and 100 nM OA with or without 1 .mu.M PaPE-1
for 24 hours before measurement by luciferase assay. The experiment
was replicated two times with technical duplicates. Heatmap of
transcriptional activity from a representative experiment is
plotted using Treeview Java. (F) The example of transcriptional
activity of selected factors from FIGS. 39D and E. The TF activity,
TF motif and statistic are shown in detail for PPAR, LXRa, RXR and
EGRI. Cignal PPAR, LXRa, RXR and EGRI Reporter Assays were used to
transfect MCF-7 cells in duplicate with indicated luciferase
construct for 24 h. The experiment was repeated three times. Cells
were treated with Veh, 1 .mu.M PaPE-1, and 100 nM OA with or
without PaPE-1 for 24 hours before measurement by luciferase assay.
An unpaired t-test was used to assess the impact of treatment (OA)
and inhibitor (PaPE-1) on transcription factor activity in MCF-7
cells. * p<0.05, ** p<0.01, *** p<0.001. Mean.+-.SEM is
plotted.
[0078] FIG. 40. PaPE-1 normalizes the metabolic pathways affected
by OA. (A) Cell metabolic phenotype assay using the Seahorse energy
phenotype kit. Cells treated with Veh, OA, and OA+PaPE-1 for 24
hours were tested for the energy phenotype at basal level (Left)
and under metabolic stress upon inhibition of glycolysis or
mitochondrial activity (Right). Each experiment was replicated
twice with three technical replicates. Results from a
representative experiment are shown. (B) Glycolysis was stimulated
in OA-treated cells but normalized by adding PaPE-1. Cells of the
same treatment as in FIG. 40C were measured using the Glycolysis
stress assay, and ECAR levels were shown at different time points.
Each experiment was replicated twice with three technical
replicates. Results from a representative experiment are shown. (C)
Mitochondrial energy production was measured separately using the
Mitostress kit. Cells were treated in the same way as described
above. Each experiment was replicated twice with three technical
replicates. Results from a representative experiment are shown. (D)
Metabolomics analysis of MCF-7 cells. MCF-7 cells were treated in
triplicate using Veh, 100 nM OA with/without 1 .mu.M PaPE-1 for 24
hours before harvest in cold methanol. Each replicate were pooled
and submitted for whole metabolite analysis. The experiment was
repeated twice. Representative results of metabolites from one of
the experiments were clustered using Cluster3 and visualized using
Treeview Java. (E) Specific metabolic pathways identified by
Metscape plugin of Cytoscape. For glycolysis, pentose phosphate
pathway, fatty acid biosynthesis, TCA cycle, urea cycle and amino
acid metabolism, the levels of affected substrates and their
position in the pathway are shown.
[0079] FIG. 41. Stroke Model in Mice
[0080] FIG. 42. Severity of CNS injury on MRI at 3 and 7 days was
decreased as compared to control.
[0081] FIG. 43. Severity of CNS injury on MRI at 3 and 7 days was
decreased by estradiol and particularly by PaPE-1, as compared to
control.
[0082] FIG. 44. Attenuation in CNS injury by estradiol and
particularly by PaPE-1 is associated with decrease in leukocyte
recruitment to the CNS ipsilateral to the injury.
[0083] FIG. 45. Attenuation in CNS injury by estradiol and
particularly by PaPE-1 is associated with decrease in leukocyte
recruitment to the CNS ipsilateral to the injury.
[0084] FIG. 46. Attenuation in CNS injury by estradiol and
particularly by PaPE-1 is associated with decrease in leukocyte
recruitment to the CNS ipsilateral to the injury.
[0085] FIG. 47. Estradiol and PaPE-1 resulted in improved function
post-stroke as assessed with rotorod testing.
[0086] While the present invention is susceptible to various
modifications and alternative forms, exemplary embodiments thereof
are shown by way of example in the drawings and are herein
described in detail. It should be understood, however, that the
description of exemplary embodiments is not intended to limit the
invention to the particular forms disclosed, but on the contrary,
the intention is to cover all modifications, equivalents and
alternatives falling within the spirit and scope of the invention
as defined by the embodiments above and the claims below. Reference
should therefore be made to the embodiments above and claims below
for interpreting the scope of the invention.
[0087] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
DETAILED DESCRIPTION
[0088] The compositions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
the invention may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements.
[0089] Likewise, many modifications and other embodiments of the
compositions described herein will come to mind to one of skill in
the art to which the invention pertains having the benefit of the
teachings presented in the foregoing descriptions and the
associated drawings. Therefore, it is to be understood that the
invention is not to be limited to the specific embodiments
disclosed and that modifications and other embodiments are intended
to be included within the scope of the appended claims. Although
specific terms are employed herein, they are used in a generic and
descriptive sense only and not for purposes of limitation.
[0090] It is specifically understood that any numerical value
recited herein (e.g., ranges) includes all values from the lower
value to the upper value, i.e., all possible combinations of
numerical values between the lowest value and the highest value
enumerated are to be considered to be expressly stated in this
application. For example, if a concentration range is stated as 1%
to 50%, it is intended that values such as 2% to 40%, 10% to 30%,
or 1% to 3%, etc., are expressly enumerated in this specification.
These are only examples of what is specifically intended.
[0091] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
skill in the art to which the invention pertains. Although any
methods and materials similar to or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described
herein.
Overview
[0092] The present disclosure includes compounds of the claimed
formulae. The compounds may be capable of having interaction with
ER that may be sufficient to activate the extranuclear-initiated
signaling pathway preferentially over the direct nuclear-initiated
pathway. In embodiments, the compound has an affinity for ER that
is about 50,000-fold less than that of the standard estrogen
estradiol, or about 25,000-fold less, or about 10,000-fold less, or
about 5,000-fold less, or about 1,000-fold less. The compounds may
comprise Pathway Preferential Estrogens (PaPEs) that have higher
activity or potency on the extranuclear-initiated pathway of
estrogen receptor action over the direct nuclear-initiated pathway.
The preference of the compounds for activating the
extranuclear-initiated pathway may result in a favorable pattern of
cellular and in vivo biological effects that can be beneficial to
human health. The compounds may elicit a pattern of gene regulation
and cellular and biological processes that may result in minimal or
no stimulation of reproductive tissues, mammary tissues, or breast
cancer cells. Without wishing to be bound by theory, the
stimulation of these tissues is usually considered to be due
largely to the nuclear-initiated actions of estrogens; hence the
compounds have only limited activity compared to the stimulation
effected by the standard estrogen estradiol. In embodiments, the
compounds may result in less than about 20% stimulation of
reproductive tissues, mammary tissues, or breast cancer cells,
compared to the stimulation effected by the standard estrogen
estradiol, or less than about 15%, or less than about 10%, or less
than about 5%, or less than about 2%, or less than about 1%, or
less than about 0.5%, or less than about 0.1%, compared to the
stimulation effected by the standard estrogen estradiol. By
contrast, the compounds may have a favorable pattern of activity on
metabolic tissues (adipose, liver) and the vasculature, reducing
body weight gain and fat accumulation after ovariectomy and
accelerating repair of endothelial damage. The stimulation of these
tissues is considered to be due to a large extent to the
extranuclear-initiated actions of estrogens. Hence, in the
responses in these tissues, the compound may have beneficial health
effects that are the same as that of the standard estrogen
estradiol, or even greater, or they may be about 50% that of
estradiol. This designed ligand structure alteration process
represents a novel approach to govern the balance in utilization of
extranuclear-initiated versus nuclear-initiated pathways of ER
action to obtain tissue-selective/non-nuclear pathway-preferential
estrogens that may prove to be beneficial for postmenopausal
hormone replacement.
[0093] The compounds described herein may represent novel
tissue-selective estrogens. In embodiments, the compounds may
provide favorable actions in metabolic and vascular tissues by
selective activation of signaling pathways critical for ER.alpha.
action in these tissues, yet they fail to activate these pathways
in reproductive tissues that would increase growth of the uterus or
stimulate proliferation of mammary tissue. Without wishing to be
bound by theory, it is thought that the tissue-selective actions of
the compounds results from the greater retention of their activity
through the non-genomic pathway than through the genomic
pathway.
[0094] While the affinity of PaPE-1 and the other PaPEs for ER is
approximately 50,000-fold less than that of E2, non-genomic effects
were stimulated using only a ca. 100-fold excess of PaPE over E2,
an observation suggesting that the non-genomic signaling pathway
might have a lesser dependence on the affinity of ligand for the
receptor than the genomic pathway. Also, whereas PaPE-1, -2, and
-3, which were patterned after E2, all have physical
characteristics (lipophilicity, polar surface area, volume, etc.)
similar to that of E2, PaPE-4 is considerably larger and more polar
than E2 and the other PaPEs, yet PaPE-4 has biological activities
very similar to those of the other three PaPEs, suggesting that the
class of PaPE-like compounds can cover a rather broad range of
physical and structural characteristics.
Definitions
[0095] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. In case of conflict, the present
document, including definitions, will control. Preferred methods
and materials are described below, although methods and materials
similar or equivalent to those described herein can be used in
practice or testing of the present invention. All publications,
patent applications, patents and other references mentioned herein
are incorporated by reference in their entirety. The materials,
methods, and examples disclosed herein are illustrative only and
not intended to be limiting.
[0096] The terms "comprise(s)," "include(s)," "having," "has,"
"can," "contain(s)," and variants thereof, as used herein, are
intended to be open-ended transitional phrases, terms, or words
that do not preclude the possibility of additional acts or
structures. The singular forms "a," "an" and "the" include plural
references unless the context clearly dictates otherwise. The
present disclosure also contemplates other embodiments
"comprising," "consisting of" and "consisting essentially of," the
embodiments or elements presented herein, whether explicitly set
forth or not.
[0097] The modifier "about" used in connection with a quantity is
inclusive of the stated value and has the meaning dictated by the
context (for example, it includes at least the degree of error
associated with the measurement of the particular quantity). The
modifier "about" should also be considered as disclosing the range
defined by the absolute values of the two endpoints. For example,
the expression "from about 2 to about 4" also discloses the range
"from 2 to 4." The term "about" may refer to plus or minus 10% of
the indicated number. For example, "about 10%" may indicate a range
of 9% to 11%, and "about 1" may mean from 0.9-1.1. Other meanings
of "about" may be apparent from the context, such as rounding off,
so, for example "about 1" may also mean from 0.5 to 1.4.
[0098] Definitions of specific functional groups and chemical terms
are described in more detail below. For purposes of this
disclosure, the chemical elements are identified in accordance with
the Periodic Table of the Elements, CAS version, Handbook of
Chemistry and Physics, 75.sup.th Ed., inside cover, and specific
functional groups are generally defined as described therein.
Additionally, general principles of organic chemistry, as well as
specific functional moieties and reactivity, are described in
Organic Chemistry, Thomas Sorrell, University Science Books,
Sausalito, 1999; Smith and March March's Advanced Organic
Chemistry, 5.sup.th Edition, John Wiley & Sons, Inc., New York,
2001; Larock, Comprehensive Organic Transformations, VCH
Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods
of Organic Synthesis, 3.sup.rd Edition, Cambridge University Press,
Cambridge, 1987; the entire contents of each of which are
incorporated herein by reference.
[0099] The term "alkoxy," as used herein, refers to an alkyl group,
as defined herein, appended to the parent molecular moiety through
an oxygen atom. Representative examples of alkoxy include, but are
not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy and
tert-butoxy.
[0100] The term "alkyl," as used herein, means a straight or
branched, saturated hydrocarbon chain containing from 1 to 10
carbon atoms. The term "lower alkyl" or "C.sub.1-C.sub.6-alkyl"
means a straight or branched chain hydrocarbon containing from 1 to
6 carbon atoms. The term "C.sub.1-C.sub.3-alkyl" means a straight
or branched chain hydrocarbon containing from 1 to 3 carbon atoms.
Representative examples of alkyl include, but are not limited to,
methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,
tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,
2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,
and n-decyl.
[0101] The term "alkenyl," as used herein, means a straight or
branched, hydrocarbon chain containing at least one carbon-carbon
double bond and from 1 to 10 carbon atoms.
[0102] The term "alkoxyalkyl," as used herein, refers to an alkoxy
group, as defined herein, appended to the parent molecular moiety
through an alkyl group, as defined herein.
[0103] The term "alkoxyfluoroalkyl," as used herein, refers to an
alkoxy group, as defined herein, appended to the parent molecular
moiety through a fluoroalkyl group, as defined herein.
[0104] The term "alkylene," as used herein, refers to a divalent
group derived from a straight or branched chain hydrocarbon of 1 to
10 carbon atoms, for example, of 2 to 5 carbon atoms.
Representative examples of alkylene include, but are not limited
to, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--.
[0105] The term "alkenylenyl," as used herein, refers to a divalent
group derived from a straight or branched chain hydrocarbon of 1 to
10 carbon atoms, wherein at least one carbon-carbon bond is a
double bond.
[0106] The term "alkylamino," as used herein, means at least one
alkyl group, as defined herein, is appended to the parent molecular
moiety through an amino group, as defined herein.
[0107] The term "amide," as used herein, means --C(O)NR-- or
--NRC(O)--, wherein R may be hydrogen, alkyl, cycloalkyl, aryl,
heteroaryl, heterocycle, alkenyl, or heteroalkyl.
[0108] The term "aminoalkyl," as used herein, means at least one
amino group, as defined herein, is appended to the parent molecular
moiety through an alkylene group, as defined herein.
[0109] The term "amino," as used herein, means --NR.sub.xR.sub.y,
wherein R.sub.x and R.sub.y may be hydrogen, alkyl, cycloalkyl,
aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl. In the case
of an aminoalkyl group or any other moiety where amino appends
together two other moieties, amino may be --NR.sub.x--, wherein
R.sub.x may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl,
heterocycle, alkenyl, or heteroalkyl.
[0110] The term "aryl," as used herein, refers to a phenyl group,
or a bicyclic fused ring system. Bicyclic fused ring systems are
exemplified by a phenyl group appended to the parent molecular
moiety and fused to a cycloalkyl group, as defined herein, a phenyl
group, a heteroaryl group, as defined herein, or a heterocycle, as
defined herein. Representative examples of aryl include, but are
not limited to, indolyl, naphthyl, phenyl, quinolinyl and
tetrahydroquinolinyl.
[0111] The term "cyanoalkyl," as used herein, means at least one
--CN group, is appended to the parent molecular moiety through an
alkylene group, as defined herein.
[0112] The term "cyanofluoroalkyl," as used herein, means at least
one --CN group, is appended to the parent molecular moiety through
a fluoroalkyl group, as defined herein.
[0113] The term "cycloalkoxy," as used herein, refers to a
cycloalkyl group, as defined herein, appended to the parent
molecular moiety through an oxygen atom.
[0114] The term "cycloalkyl," as used herein, refers to a
carbocyclic ring system containing three to ten carbon atoms, zero
heteroatoms and zero double bonds. Representative examples of
cycloalkyl include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
cyclononyl and cyclodecyl. "Cycloalkyl" also includes carbocyclic
ring systems in which a cycloalkyl group is appended to the parent
molecular moiety and is fused to an aryl group as defined herein
(e.g., a phenyl group), a heteroaryl group as defined herein, or a
heterocycle as defined herein. Representative examples of such
cycloalkyl groups include, but are not limited to,
2,3-dihydro-1H-indenyl (e.g., 2,3-dihydro-1H-inden-1-yl and
2,3-dihydro-1H-inden-2-yl), 6,7-dihydro-5H-cyclopenta[b]pyridinyl
(e.g., 6,7-dihydro-5H-cyclopenta[b]pyridin-6-yl), and
5,6,7,8-tetrahydroquinolinyl (e.g.,
5,6,7,8-tetrahydroquinolin-5-yl).
[0115] The term "cycloalkenyl," as used herein, means a
non-aromatic monocyclic or multicyclic ring system containing at
least one carbon-carbon double bond and preferably having from 5-10
carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings
include cyclopentenyl, cyclohexenyl or cycloheptenyl.
[0116] The term "fluoroalkyl," as used herein, means an alkyl
group, as defined herein, in which one, two, three, four, five,
six, seven or eight hydrogen atoms are replaced by fluorine.
Representative examples of fluoroalkyl include, but are not limited
to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl,
difluoromethyl, pentafluoroethyl, and trifluoropropyl such as
3,3,3-trifluoropropyl.
[0117] The term "fluoroalkoxy," as used herein, means at least one
fluoroalkyl group, as defined herein, is appended to the parent
molecular moiety through an oxygen atom. Representative examples of
fluoroalkoxy include, but are not limited to, difluoromethoxy,
trifluoromethoxy and 2,2,2-trifluoroethoxy.
[0118] The term "halogen" or "halo," as used herein, means Cl, Br,
I, or F.
[0119] The term "haloalkyl," as used herein, means an alkyl group,
as defined herein, in which one, two, three, four, five, six, seven
or eight hydrogen atoms are replaced by a halogen.
[0120] The term "haloalkoxy," as used herein, means at least one
haloalkyl group, as defined herein, is appended to the parent
molecular moiety through an oxygen atom.
[0121] The term "halocycloalkyl," as used herein, means a
cycloalkyl group, as defined herein, in which one or more hydrogen
atoms are replaced by a halogen.
[0122] The term "heteroalkyl," as used herein, means an alkyl
group, as defined herein, in which one or more of the carbon atoms
has been replaced by a heteroatom selected from S, O, P and N.
Representative examples of heteroalkyls include, but are not
limited to, alkyl ethers, secondary and tertiary alkyl amines,
amides, and alkyl sulfides.
[0123] The term "heteroaryl," as used herein, refers to an aromatic
monocyclic ring or an aromatic bicyclic ring system. The aromatic
monocyclic rings are five or six membered rings containing at least
one heteroatom independently selected from the group consisting of
N, O and S (e.g. 1, 2, 3, or 4 heteroatoms independently selected
from O, S, and N). The five membered aromatic monocyclic rings have
two double bonds and the six membered aromatic monocyclic rings
have three double bonds. The bicyclic heteroaryl groups are
exemplified by a monocyclic heteroaryl ring appended to the parent
molecular moiety and fused to a monocyclic cycloalkyl group, as
defined herein, a monocyclic aryl group, as defined herein, a
monocyclic heteroaryl group, as defined herein, or a monocyclic
heterocycle, as defined herein. Representative examples of
heteroaryl include, but are not limited to, indolyl, pyridinyl
(including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl,
pyrazinyl, pyridazinyl, pyrazolyl, pyrrolyl, benzopyrazolyl,
1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl,
1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl,
isothiazolyl, thienyl, benzimidazolyl, benzothiazolyl,
benzoxazolyl, benzoxadiazolyl, benzothienyl, benzofuranyl,
isobenzofuranyl, furanyl, oxazolyl, isoxazolyl, purinyl,
isoindolyl, quinoxalinyl, indazolyl, quinazolinyl, 1,2,4-triazinyl,
1,3,5-triazinyl, isoquinolinyl, quinolinyl,
6,7-dihydro-1,3-benzothiazolyl, imidazo[1,2-a]pyridinyl,
naphthyridinyl, pyridoimidazolyl, thiazolo[5,4-b]pyridin-2-yl,
thiazolo[5,4-d]pyrimidin-2-yl.
[0124] The term "heterocycle" or "heterocyclic," as used herein,
means a monocyclic heterocycle, a bicyclic heterocycle, or a
tricyclic heterocycle. The monocyclic heterocycle is a three-,
four-, five-, six-, seven-, or eight-membered ring containing at
least one heteroatom independently selected from the group
consisting of O, N, and S. The three- or four-membered ring
contains zero or one double bond, and one heteroatom selected from
the group consisting of O, N, and S. The five-membered ring
contains zero or one double bond and one, two or three heteroatoms
selected from the group consisting of O, N and S. The six-membered
ring contains zero, one or two double bonds and one, two, or three
heteroatoms selected from the group consisting of O, N, and S. The
seven- and eight-membered rings contains zero, one, two, or three
double bonds and one, two, or three heteroatoms selected from the
group consisting of O, N, and S. Representative examples of
monocyclic heterocycles include, but are not limited to,
azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl,
1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl,
imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl,
isoxazolidinyl, morpholinyl, 2-oxo-3-piperidinyl, 2-oxoazepan-3-yl,
oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl,
oxepanyl, oxocanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl,
pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl,
thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl,
thiazolinyl, thiazolidinyl, thiomorpholinyl,
1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl,
and trithianyl. The bicyclic heterocycle is a monocyclic
heterocycle fused to a phenyl group, or a monocyclic heterocycle
fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused
to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to
a monocyclic heterocycle, or a spiro heterocycle group, or a
bridged monocyclic heterocycle ring system in which two
non-adjacent atoms of the ring are linked by an alkylene bridge of
1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three,
or four carbon atoms. Representative examples of bicyclic
heterocycles include, but are not limited to, benzopyranyl,
benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl,
2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline,
2-azaspiro[3.3]heptan-2-yl, 2-oxa-6-azaspiro[3.3]heptan-6-yl,
azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl),
azabicyclo[3.1.0]hexanyl (including 3-azabicyclo[3.1.0]hexan-3-yl),
2,3-dihydro-1H-indolyl, isoindolinyl,
octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and
tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by
a bicyclic heterocycle fused to a phenyl group, or a bicyclic
heterocycle fused to a monocyclic cycloalkyl, or a bicyclic
heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic
heterocycle fused to a monocyclic heterocycle, or a bicyclic
heterocycle in which two non-adjacent atoms of the bicyclic ring
are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or
an alkenylene bridge of two, three, or four carbon atoms. Examples
of tricyclic heterocycles include, but are not limited to,
octahydro-2,5-epoxypentalene,
hexahydro-2H-2,5-methanocyclopenta[b]furan,
hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane
(1-azatricyclo[3.3.1.13,7]decane), and oxa-adamantane
(2-oxatricyclo[3.3.1,13,7]decane). The monocyclic, bicyclic, and
tricyclic heterocycles are connected to the parent molecular moiety
through any carbon atom or any nitrogen atom contained within the
rings, and can be unsubstituted or substituted.
[0125] The term "hydroxyl" or "hydroxy," as used herein, means an
--OH group.
[0126] The term "hydroxyalkyl," as used herein, means at least one
--OH group, is appended to the parent molecular moiety through an
alkylene group, as defined herein.
[0127] The term "hydroxyfluoroalkyl," as used herein, means at
least one --OH group, is appended to the parent molecular moiety
through a fluoroalkyl group, as defined herein.
[0128] In some instances, the number of carbon atoms in a
hydrocarbyl substituent (e.g., alkyl or cycloalkyl) is indicated by
the prefix "C.sub.x-C.sub.y-", wherein x is the minimum and y is
the maximum number of carbon atoms in the substituent. Thus, for
example, "C.sub.1-C.sub.3-alkyl" refers to an alkyl substituent
containing from 1 to 3 carbon atoms.
[0129] The term "sulfonamide," as used herein, means
--S(O).sub.2NR.sup.d-- or --NR.sup.dS(O)--, wherein R.sup.d may be
hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle,
alkenyl, or heteroalkyl.
[0130] The term "substituents" refers to a group "substituted" on
an aryl, heteroaryl, phenyl or pyridinyl group at any atom of that
group. Any atom can be substituted.
[0131] The term "substituted" refers to a group that may be further
substituted with one or more non-hydrogen substituent groups.
Substituent groups include, but are not limited to, halogen, .dbd.O
(oxo), .dbd.S (thioxo), cyano, nitro, fluoroalkyl,
alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl,
haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl,
heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl,
arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene,
aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino,
aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl,
alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, --COOH,
ketone, amide, carbamate, and acyl. For example, if a group is
described as being "optionally substituted" (such as an alkyl,
alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heteroalkyl,
heterocycle or other group such as an R group), it may have 0, 1,
2, 3, 4 or 5 substituents independently selected from halogen,
.dbd.0 (oxo), .dbd.S (thioxo), cyano, nitro, fluoroalkyl,
alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl,
haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl,
heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl,
arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene,
aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino,
aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl,
alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, --COOH,
ketone, amide, carbamate, and acyl.
[0132] The term "" designates a single bond (-) or a double bond
(=).
[0133] For compounds described herein, groups and substituents
thereof may be selected in accordance with permitted valence of the
atoms and the substituents, such that the selections and
substitutions result in a stable compound, e.g., which does not
spontaneously undergo transformation such as by rearrangement,
cyclization, elimination, etc.
[0134] For the recitation of numeric ranges herein, each
intervening number there between with the same degree of precision
is explicitly contemplated. For example, for the range of 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0135] In accordance with a convention used in the art, the
group:
##STR00006##
is used in structural formulas herein to depict the bond that is
the point of attachment of the moiety or substituent to the core or
backbone structure.
[0136] The term "acceptable" or "pharmaceutically acceptable" with
respect to a compound, formulation, composition, ingredient, salt,
or the like, as used herein, means having no persistent detrimental
effect on the general health of the subject being treated.
[0137] The terms "effective amount" or "therapeutically effective
amount," as used herein, refer to a sufficient amount of an agent
or a compound being administered which will relieve to some extent
one or more of the symptoms of the disease or condition being
treated. The result can be reduction and/or alleviation of the
signs, symptoms, or causes of a disease, or any other desired
alteration of a biological system. For example, an "effective
amount" for therapeutic uses is the amount of the composition
comprising a compound as disclosed herein required to provide a
clinically significant decrease in disease symptoms. An appropriate
"effective" amount in any individual case may be determined using
techniques, such as a dose escalation study.
[0138] The term "subject" or "patient" encompasses mammals and
non-mammals. Examples of mammals include, but are not limited to,
any member of the Mammalian class: humans, non-human primates such
as chimpanzees, and other apes and monkey species; farm animals
such as cattle, horses, sheep, goats, swine; domestic animals such
as rabbits, dogs, and cats; laboratory animals including rodents,
such as rats, mice and guinea pigs, and the like. Examples of
non-mammals include, but are not limited to, birds, fish and the
like. In one embodiment of the methods and compositions provided
herein, the mammal is a human.
[0139] The terms "treat," "treating" or "treatment," as used
herein, include alleviating, abating or ameliorating a disease or
condition symptoms, preventing additional symptoms, ameliorating or
preventing the underlying metabolic causes of symptoms, inhibiting
the disease or condition, e.g., arresting the development of the
disease or condition, relieving the disease or condition, causing
regression of the disease or condition, relieving a condition
caused by the disease or condition, or stopping the symptoms of the
disease or condition either prophylactically and/or
therapeutically. As used herein, "prevent," "preventing" and the
like compared to an appropriate control subject.
[0140] As such, "preventing" means an application that involves a
slowing, stopping or reversing of progression of a disease or
disorder, or the application or administration of a pharmaceutical
composition comprising at least one of any of the compounds
described herein, where the subject has a disease or a symptom of a
disease, where the purpose is to cure, heal, alleviate, relieve,
alter, remedy, ameliorate, improve or affect the disease or
symptoms of the disease. Although there can be overlap between
"treating" and "preventing," it is intended that the latter is a
more drastic (i.e., less subtle) reduction in the disease or
disorder than would be observed during the former. Likewise, it is
intended that "treating" can occur in an individual having a
disease or disorder, whereas "preventing" can occur in an
individual merely susceptible to or not yet exhibiting overt signs
and symptoms of a disease or disorder.
Compounds
[0141] One aspect of the present disclosure provides compounds of
the following formula:
##STR00007## [0142] and stereoisomers and pharmaceutically
acceptable salts thereof; [0143] wherein [0144] n is an integer
from 0 to 4; [0145] m is an integer from 0 to 4; [0146] X is H,
hydroxy, or C.sub.1-4 alkoxy; [0147] R.sub.1 and R.sub.2 are
independently H, hydroxy, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, amino,
--S--C.sub.1-4 alkyl, or halo; [0148] R.sub.3 is H, hydroxy, oxo,
cyano, halo, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy; [0149] each
R.sub.4 is independently hydrogen, hydroxy, oxo, halo, C.sub.1-4
alkyl, or C.sub.1-4 alkoxy; [0150] R.sub.5 is H, C.sub.1-4 alkynyl,
or is absent if a double bond is present; and [0151] - - - is an
optional double bond.
[0152] In embodiments, R.sub.3 is H, OH, oxo, chloro, cyano, or
methoxy. Suitably, R.sub.3 is hydroxyl.
[0153] In embodiments, at least one of R.sub.1 and R.sub.2 is
C.sub.1-4 alkyl. In embodiments, both R.sub.1 and R.sub.2 are
C.sub.1-4 alkyl. In embodiments, at least one of R.sub.1 and
R.sub.2 is methyl. In embodiments, both R.sub.1 and R.sub.2 are
methyl. In embodiments, R.sub.1 and R.sub.2 are independently
selected from H, methyl, ethyl, chloro, --CH.sub.2OH, and
--CH.sub.2OMe.
[0154] In embodiments, a C.sub.1-4 alkyl or C.sub.1-4 alkoxy is
optionally substituted with one or more of halo, cyano, amino,
hydroxy, and C.sub.1-4 alkoxy. Suitably, the substituent is
hydroxy.
[0155] In embodiments, R.sub.4 is selected from fluoro, chloro,
bromo, methoxy, hydroxy, or oxo.
[0156] In embodiments, R.sub.5 is H. In embodiments, R.sub.5 is
--CCH.
[0157] In embodiments, m is 0. In embodiments, n is 1. In
embodiments, n is 2.
[0158] Combinations and permutations of the preferred substituents
identified in paragraphs [00117] to [00122] are explicitly
contemplated.
[0159] In an aspect the present disclosure provides a compound
selected from the group consisting of:
##STR00008##
[0160] In another aspect, the present disclosure provides compounds
of the following formula:
##STR00009## [0161] and stereoisomers and pharmaceutically
acceptable salts thereof; [0162] wherein R is H or methyl.
[0163] In another aspect, the present disclosure provides compounds
of the following formula:
##STR00010## [0164] and stereoisomers and pharmaceutically
acceptable salts thereof; [0165] wherein [0166] m is an integer
from 0 to 3; [0167] R.sub.1 and R.sub.2 are independently H,
hydroxy, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, amino, --S--C.sub.1-4
alkyl, or halo; [0168] R.sub.5 is H, hydroxy, or C.sub.1-4 alkyl;
and [0169] each R.sub.6 is independently H, hydroxy, halo, or
C.sub.1-4 alkyl.
[0170] In embodiments, the C.sub.1-4 alkyl or C.sub.1-4 alkoxy is
substituted with one or more of halo, cyano, amino, hydroxy, and
C.sub.1-4 alkoxy. Suitably, the substituent is hydroxy.
[0171] In embodiments, at least one of R.sub.1 and R.sub.2 is
C.sub.1-4 alkyl. In embodiments, both R.sub.1 and R.sub.2 are
C.sub.1-4 alkyl. In embodiments, at least one of R.sub.1 and
R.sub.2 is methyl. In embodiments, both R.sub.1 and R.sub.2 are
methyl. In embodiments, at least one of R.sub.1 and R.sub.2 is
halogen, and preferably, chlorine.
[0172] In embodiments, R.sub.5 is hydroxy. In embodiments, R.sub.5
is C.sub.1-4 alkyl. Suitably, R.sub.5 is substituted C.sub.1-4
alkyl. In embodiments, R.sub.5 is --CH(OH)(CH.sub.3).
[0173] In embodiments, R.sub.6 is C.sub.1-4 alkyl. Suitably,
R.sub.6 is substituted C.sub.1-4 alkyl. In embodiments, R.sub.6 is
--CH.sub.2OH.
[0174] In embodiments, m is 1.
[0175] Combinations and permutations of the preferred substituents
identified in paragraphs [00127] to [00131] are explicitly
contemplated.
[0176] In an aspect the present disclosure provides a compound
selected from the group consisting of:
##STR00011##
[0177] In another aspect, the present disclosure provides a
compound having the formula:
##STR00012## [0178] wherein R.sub.1 and R.sub.2 is selected from H,
C.sub.1-4 alkyl group, haloalkyl, hydroxyalkyl, alkyloxyalkyl,
cycloalkyloxyalkyl, alkylthio, alkylthioalkyl, cycloalkylthioalkyl,
R'R''N-alkyl where R' and R'' are independently alkyl,
alkylcarbonyl, or cyclic alkyl, and the parenthesis represents the
presence or absence of hydroxyl, and [0179] wherein R.sub.3 and
R.sub.4 are independently selected from H, hydroxy, C.sub.1-4
alkyl, hydoxy-C.sub.1-4alkyl, cyano, cyanoalkyl, nitro, nitroalkyl,
--C(O)-aryl, --C(O)H, alkyl aldehyde, carboxyl, and carboxyalkyl,
or wherein [0180] R.sub.3 and R.sub.4 form a ring of from 4 to 8
member atoms, wherein the ring is substituted with cyano or
hydroxy.
[0181] In embodiments, the C.sub.1-4 alkyl or C.sub.1-4 alkoxy is
substituted with one or more of halo, cyano, amino, hydroxy, and
C.sub.1-4 alkoxy. Suitably, the substituent is hydroxy.
[0182] In embodiments, at least one of R.sub.1 and R.sub.2 is
C.sub.1-4 alkyl. In embodiments, both R.sub.1 and R.sub.2 are
C.sub.1-4 alkyl. In embodiments, at least one of R.sub.1 and
R.sub.2 is methyl. In embodiments, both R.sub.1 and R.sub.2 are
methyl. In embodiments, at least one of R.sub.1 and R.sub.2 is
halogen, and preferably, chlorine.
[0183] In embodiments, R.sub.3 is hydroxy. In embodiments, R.sub.3
is C.sub.1-4 alkyl. Suitably, R.sub.3 is substituted C.sub.1-4
alkyl. In embodiments, R.sub.3 is --CH(OH)(CH.sub.3).
[0184] In embodiments, R.sub.4 is C.sub.1-4 alkyl. Suitably,
R.sub.4 is substituted C.sub.1-4 alkyl. In embodiments, R.sub.4 is
--CH.sub.2OH.
[0185] In embodiments, R.sub.3 and R.sub.4 form a 5 or 6 membered
ring.
[0186] Combinations and permutations of the preferred substituents
identified in paragraphs [00135] to [00139] are explicitly
contemplated.
[0187] In an aspect the present disclosure provides a compound
having the formula:
##STR00013##
wherein R.sub.5 and R.sub.6 are independently selected from
hydroxyl, cyano, hydroxylalkyl, cyanoalkyl, halogenated
hydroxylalkyl, and halogenated cyanoalkyl.
[0188] In another aspect the present disclosure provides compounds
of the following formula:
##STR00014##
and stereoisomers and pharmaceutically acceptable salts thereof;
wherein [0189] n is an integer from 0 to 4; [0190] m is an integer
from 0 to 4; [0191] X is H, hydroxy, or C.sub.1-4 alkoxy; [0192]
R.sub.1 and R.sub.2 are independently H, hydroxy, C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, amino, --S--C.sub.1-4 alkyl, or halo; [0193]
R.sub.3 is H, hydroxy, oxo, cyano, halo, C.sub.1-4 alkyl, or
C.sub.1-4 alkoxy; [0194] each R.sub.4 is independently hydrogen,
hydroxy, oxo, halo, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy; [0195]
R.sub.5 is H, alkynyl, or is absent if a double bond is present;
[0196] R.sub.6 and R.sub.7 are independently selected from
C.sub.1-4 alkyl and H; and [0197] - - - is an optional double
bond.
[0198] In embodiments, R.sub.3 is H, OH, oxo, chloro, cyano, or
methoxy. Suitably, R.sub.3 is hydroxyl.
[0199] In embodiments, at least one of R.sub.1 and R.sub.2 is
C.sub.1-4 alkyl. In embodiments, both R.sub.1 and R.sub.2 are
C.sub.1-4 alkyl. In embodiments, at least one of R.sub.1 and
R.sub.2 is methyl. In embodiments, both R.sub.1 and R.sub.2 are
methyl. In embodiments, R.sub.1 and R.sub.2 are independently
selected from H, methyl, ethyl, chloro, --CH.sub.2OH, and
--CH.sub.2OMe.
[0200] In embodiments, a C.sub.1-4 alkyl or C.sub.1-4 alkoxy is
optionally substituted with one or more of halo, cyano, amino,
hydroxy, and C.sub.1-4 alkoxy. Suitably, the substituent is
hydroxy.
[0201] In embodiments, R.sub.4 is selected from fluoro, chloro,
bromo, methoxy, hydroxy, or oxo.
[0202] In embodiments, R.sub.5 is H. In embodiments, R.sub.5 is
--CCH.
[0203] In embodiments, R.sub.6 is methyl. In embodiments, R.sub.7
is H.
[0204] In embodiments, m is 0. In embodiments, n is 1. In
embodiments, n is 2.
[0205] Combinations and permutations of the preferred substituents
identified in paragraphs [00143] to [00149] are explicitly
contemplated.
[0206] In another aspect, the present disclosure provides compound
of the following formula:
##STR00015##
and stereoisomers and pharmaceutically acceptable salts thereof;
wherein [0207] R.sub.7 and R.sub.8 are independently H, C.sub.1-5
alkyl, amino, hydroxyl, cyano, amido, cyclic C.sub.3-8 alkyl, and
heterocyclyl.
[0208] In embodiments, R.sub.7 and R.sub.8 are selected from
C.sub.1-4 alkylaminocarboxy-C.sub.1-4 alkyl, C.sub.1-4
alkylamino-C.sub.1-4 alkyl, C.sub.1-4 alkylamino-C.sub.1-4
alkyl-amino-carboxy-C.sub.1-4 alkyl, C.sub.1-4 alkyloxy-C.sub.1-4
alkylamino-carboxy-C.sub.1-4 alkyl, C.sub.1-4 alkylthio-C.sub.1-4
alkylamino-carboxy-C.sub.1-4 alkyl, or C.sub.1-4
alkylthio-C.sub.1-4 alkyl.
[0209] In embodiments, R.sub.7 is methyl.
[0210] In embodiments, R.sub.8 is substituted C.sub.1-5 alkyl.
Suitably, R.sub.8 is substituted with --C(O)--R,
--C(O)NR.sub.N1R.sub.N2, --C(O)OR, --NR.sub.N1C(O)R, or --OC(O)R,
wherein R.sub.N1, R.sub.N2 and R are independently selected from H
or C.sub.1-4 alkyl. In embodiments, at least one of R.sub.N1,
R.sub.N2, or R is --(CH.sub.2).sub.n--R.sub.10, wherein n is an
integer of from 2 to 5, and wherein R.sub.10 is --C(O)--R,
--C(O)NR.sub.N1R.sub.N2, --C(O)OR, --NR.sub.N1C(O)R, or --OC(O)R,
wherein R.sub.N1, R.sub.N2 and R are independently selected from H,
aryl, cycloalkyl, or C.sub.1-4 alkyl optionally substituted with OH
or amino.
[0211] Combinations and permutations of the preferred substituents
identified in paragraphs [00152] to [00154] are explicitly
contemplated.
[0212] In an aspect the present disclosure provides a compound
having the formula:
##STR00016##
[0213] In another aspect, the present disclosure provides a
compound selected from the group consisting of:
##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022## ##STR00023## ##STR00024## ##STR00025##
##STR00026##
[0214] The compounds presented herein may possess one or more
stereocenters and each center may exist in the R or S
configuration. The compounds presented herein include all
diastereomeric, enantiomeric, and epimeric forms as well as the
appropriate mixtures thereof. Individual stereoisomers may be
obtained, if desired, by methods known in the art as, for example,
the separation of stereoisomers by chiral chromatographic
columns.
[0215] In the formulas for the compounds of the present disclosure,
variable or undefined substitution positions and stereochemistry
are indicated using common chemical structure drawing conventions
such as "line through a ring system", "squiggly bonds", or a
"straight line bond" in place of a "bold wedge bond" or "dashed
wedge bond", which by convention indicate a substituent coming out
from the indicate plane ("bold wedge bond") or going away from the
indicated plane ("dashed wedge bond").
[0216] For compounds according to the present disclosure, groups
and substituents thereof may be selected in accordance with
permitted valence of the atoms and the substituents, such that the
selections and substitutions result in a stable compound, e.g.,
which does not spontaneously undergo transformation such as by
rearrangement, cyclization, elimination, etc.
[0217] Compounds according to the present disclosure include
compounds that differ only in the presence of one or more
isotopically enriched atoms. For example, compounds may have the
present structures except for the replacement of hydrogen by
deuterium or tritium, or the replacement of a carbon by a .sup.13C-
or .sup.14C-enriched carbon.
[0218] Compounds according to the present disclosure can be in the
form of a salt, e.g., a pharmaceutically acceptable salt. The term
"pharmaceutically acceptable salt" includes salts of the active
compounds that are prepared with relatively nontoxic acids or
bases, depending on the particular substituents found on the
compounds. Suitable pharmaceutically acceptable salts of the
compounds of this invention include acid addition salts which may,
for example, be formed by mixing a solution of the compound
according to the invention with a solution of a pharmaceutically
acceptable acid such as hydrochloric acid, sulfuric acid,
methanesulfonic acid, fumaric acid, maleic acid, succinic acid,
acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid,
carbonic acid or phosphoric acid. Furthermore, where the compounds
of the invention carry an acidic moiety, suitable pharmaceutically
acceptable salts thereof may include alkali metal salts, e.g.
sodium or potassium salts, alkaline earth metal salts, e.g. calcium
or magnesium salts; and salts formed with suitable organic ligands,
e.g. quaternary ammonium salts.
[0219] Neutral forms of the compounds may be regenerated by
contacting the salt with a base or acid and isolating the parent
compound in a conventional manner. The parent form of the compound
differs from the various salt forms in certain physical properties,
such as solubility in polar solvents, but otherwise the salts are
equivalent to the parent form of the compound for the purposes of
this disclosure.
[0220] Various pharmaceutically acceptable salts are well known in
the art and may be used with instant compound such as those
disclosed in Berge S, et al., "Pharmaceutical salts," J. Pharm.
Sci. 66:1-19 (1977) and Haynes D, et al., "Occurrence of
pharmaceutically acceptable anions and cations in the Cambridge
Structural Database," J. Pharm. Sci. 94:2111-2120 (2005), each of
which is incorporated herein by reference as if set forth in its
entirety. For example, the list of FDA-approved commercially
marketed salts includes acetate, benzenesulfonate, benzoate,
bicarbonate, bitartrate, bromide, calcium edetate, camsylate,
carbonate, chloride, citrate, dihydrochloride, edetate, edisylate,
estolate, esylate, fumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,
lactobionate, malate, maleate, mandelate, mesylate, methylbromide,
methylnitrate, methylsulfate, mucate, napsylate, mitrate, pamoate,
pantothenate, phosphate, diphosphate, polygalacturonate,
salicylate, stearate, subacetate, succinate, sulfate, tannate,
tartrate, teoclate and triethiodide.
[0221] In addition to salt forms, the present invention may also
provide compounds according to the present disclosure that are in a
prodrug form. Prodrugs of the compounds are those compounds that
readily undergo chemical changes under physiological conditions to
provide the compounds. Prodrugs can be converted to the compounds
of the present invention by chemical or biochemical methods in an
ex vivo environment. For example, prodrugs can be slowly converted
to the compounds of the present invention when placed in a
transdermal patch reservoir with a suitable enzyme or chemical
reagent.
[0222] Compounds according to the present disclosure can be, for
example, an enantiomerically enriched isomer of a stereoisomer
described herein. Enantiomer, as used herein, refers to either of a
pair of chemical compounds whose molecular structures have a
mirror-image relationship to each other. For example, a compound
may have an enantiomeric excess of at least about 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99%.
[0223] A preparation of a compound according to the present
disclosure may be enriched for an isomer of the compound having a
selected stereochemistry, e.g., R or S, corresponding to a selected
stereocenter. For example, the compound may have a purity
corresponding to a compound having a selected stereochemistry of a
selected stereocenter of at least about 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99%. A compound can, for example,
include a preparation of a compound disclosed herein that is
enriched for a structure or structures having a selected
stereochemistry, e.g., R or S, at a selected stereocenter.
[0224] In some embodiments, a preparation of a compound according
to the present disclosure may be enriched for isomers (subject
isomers) which are diastereomers of the compound. Diastereomer, as
used herein, refers to a stereoisomer of a compound having two or
more chiral centers that is not a mirror image of another
stereoisomer of the same compound. For example, the compound may
have a purity corresponding to a compound having a selected
diastereomer of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99%.
[0225] When no specific indication is made of the configuration at
a given stereocenter in a compound, any one of the configurations
or a mixture of configurations is intended.
[0226] Compounds may be prepared in racemic form or as individual
enantiomers or diastereomers by either stereospecific synthesis or
by resolution. The compounds may, for example, be resolved into
their component enantiomers or diastereomers by standard
techniques, such as the formation of stereoisomeric pairs by salt
formation with an optically active base, followed by fractional
crystallization and regeneration of the free acid. The compounds
may also be resolved by formation of stereoisomeric esters or
amides, followed by chromatographic separation and removal of the
chiral auxiliary. Alternatively, the compounds may be resolved
using a chiral HPLC column. The enantiomers also may be obtained
from kinetic resolution of the racemate of corresponding esters
using lipase enzymes.
[0227] A compound according to the present disclosure can also be
modified by appending appropriate functionalities to enhance
selective biological properties. Such modifications are known in
the art and include those that increase biological penetration into
a given biological system (e.g., blood, lymphatic system, central
nervous system), increase oral availability, increase solubility to
allow administration by injection, alter metabolism, and/or alter
rate of excretion. Examples of these modifications include, but are
not limited to, esterification with polyethylene glycols,
derivatization with pivalates or fatty acid substituents,
conversion to carbamates, hydroxylation of aromatic rings, and
heteroatom substitution in aromatic rings.
Synthesis of Compounds
[0228] A general route for the synthesis of many of these compounds
is shown below:
##STR00027##
[0229] Other methods of synthesizing the compounds of the formulae
herein will be evident to those of ordinary skill in the art.
Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing
the compounds are known in the art and include, for example, those
such as described in R. Larock, Comprehensive Organic
Transformations, VCH Publishers (1989); T. W. Greene and P. G. M.
Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley
and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's
Reagents for Organic Synthesis, John Wiley and Sons (1994); and L.
Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John
Wiley and Sons (1995), and subsequent editions thereof.
Evaluation of Compounds
[0230] Compounds may be analyzed using a number of methods and
assays, including receptor ligand binding kinetic and equilibrium
assay, coregulator binding assays, subcellular distribution
immunohistochemical and proximity ligation assays of receptors and
other proteins that relate to the selective action of estrogens
through nuclear-initiated versus extranuclear-initiated pathways;
cell activity studies including regulation of specific genes and
whole transcriptome (RNA-sequencing, RNA-seq) analyses, receptor,
kinase, and RNA polymerase chromatin binding studies using
chromatin immunoprecipitation (ChIP) and ChIP-sequencing methods,
and cell proliferation assays and signal transduction pathway
analyses that further define the pathways by which ligands for the
estrogen receptor work in target cells; in vivo assays, including
uterine weight assessments and mammary tissue development assays
that can be predictive of compounds that will or will not be
stimulatory to reproductive tissues and might or might not initiate
or promote human breast and uterine cancers; animal body weight and
organ weight studies, and blood and tissue lipid and metabolomics
analyses on animals on different diets that might be related to the
prevention or development of metabolic disorders or metabolic
syndrome in humans; assays in animals of endothelial cell function
involving estrogen-responsive genes regulated by nuclear and
non-nuclear estrogen receptor action, assays of vascular health
including endothelial repair assays and suppression of atheroma and
neointima development in wild type and mutant animals on normal and
high fat diets that are predictive of activity on vascular and
metabolic diseases in humans. Effects of the compounds can be
assayed on metabolic health and glucose utilization in wild type
and mutant animals under various diets that are associated with
diabetes in humans. Compounds can be assayed for their suppression
of injury to the heart ex vivo or brain in vivo under conditions of
enforced ischemia that are models of human diseases of stroke, or
cardiac insufficiency or heart attack. Compounds can be assayed for
their protection of bone mineral density, strength, and
architecture due to low estrogen conditions in mouse or rat models
that are predictive of effects on osteoporosis or osteopenia in
humans. Certain of these methods are described in the examples.
Methods of Use
[0231] In another aspect, a method of treating a disease or
condition in a subject is provided, the method comprising
administering a pharmaceutically effective amount of at least one
compound of any of the claims herein.
[0232] In another aspect, a method of ameliorating a disease or
condition in a subject is provided, the method comprising
administering a pharmaceutically effective amount of at least one
compound of any of the claims herein.
[0233] In another aspect, a method of preventing or slowing the
progress of a disease or condition in a subject is provided, the
method comprising administering a pharmaceutically effective amount
of at least one compound of any of the claims herein.
[0234] In embodiments, the disease or condition is affected by the
extranuclear-initiated pathway of the estrogen receptor.
[0235] The disease or condition is selected from, for example,
postmenopausal symptoms, cardiovascular disease, stroke, vascular
disease, bone disease, metabolic disease, diabetes, arthritis,
osteoporosis, obesity, cognitive decline, vasomotor/hot flush, and
cancer.
[0236] In embodiments, the disease is stroke. In embodiments, the
disease is metabolic disease. In embodiments, the disease is
diabetes. In embodiments, the disease is cancer, such as breast
cancer. In embodiments, the breast cancer is estrogen-responsive
breast cancer or obesity-related breast cancer. In embodiments, the
disease is vascular disease. In embodiments, the disease is
osteoporosis.
Compositions and Routes of Administration
[0237] The present disclosure also provides pharmaceutical
compositions comprising compounds according to the present
disclosure and a pharmaceutically acceptable excipient.
[0238] The pharmaceutical compositions of this disclosure can be
administered by a variety of routes including by way of
non-limiting example, oral, transdermal, subcutaneous, intravenous,
intramuscular and intranasal. Depending upon the intended route of
delivery, the pharmaceutical composition preferably is formulated
as either injectable or oral compositions or as salves, as lotions
or as patches all for transdermal administration.
[0239] The compositions for oral administration can take the form
of bulk liquid solutions or suspensions, or bulk powders. More
commonly, however, the compositions are presented in unit dosage
forms to facilitate accurate dosing. As used herein, "unit dosage
forms" means physically discrete units suitable as unitary dosages
for human subjects and other mammals, each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect, in association with a suitable
pharmaceutical excipient. Typical unit dosage forms include
prefilled, premeasured ampoules or syringes of the liquid
compositions or pills, tablets, capsules or the like in the case of
solid compositions. In such compositions, the combination therapy
is usually a minor component (from about 0.1% to about 50% by
weight or preferably from about 1% to about 40% by weight) with the
remainder being various vehicles or carriers and processing aids
helpful for forming the desired dosing form.
[0240] Liquid forms suitable for oral administration may include a
suitable aqueous or non-aqueous vehicle with buffers, suspending
and dispensing agents, colorants, flavors and the like. Solid forms
may include, for example, any of the following ingredients, or
compounds of a similar nature: a binder such as microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch
or lactose, a disintegrating agent such as alginic acid, Primogel,
or corn starch; a lubricant such as magnesium stearate; a glidant
such as colloidal silicon dioxide; a sweetening agent such as
sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0241] Injectable compositions typically are based upon injectable
sterile saline or phosphate-buffered saline or other injectable
carriers known in the art. As before, the active compound in such
compositions is typically a minor component, often being from about
0.05% to 10% by weight with the remainder being the injectable
carrier and the like.
[0242] Transdermal compositions are typically formulated as a
topical ointment or cream containing the active ingredient(s),
generally in an amount ranging from about 0.01% to about 20% by
weight, preferably from about 0.1% to about 20% by weight,
preferably from about 0.1 to about 10% by weight, and more
preferably from about 0.5% to about 15% by weight. When formulated
as an ointment, the active ingredients will typically be combined
with either a paraffinic or a water-miscible ointment base.
Alternatively, the active ingredients may be formulated in a cream
with, for example an oil-in-water cream base. Such transdermal
formulations are well known in the art and generally include
additional ingredients to enhance the dermal penetration or
stability of the active ingredients or the formulation. All such
known transdermal formulations and ingredients are included within
the scope of this invention.
[0243] The pharmaceutical composition also can be administered by a
transdermal device. Accordingly, transdermal administration can be
accomplished using a patch either of the reservoir or porous
membrane type, or of a solid matrix variety.
[0244] The above-described components for orally administrable,
injectable or topically administrable compositions are merely
representative. Other materials as well as processing techniques
and the like are set forth in Part 8 of Remington's Pharmaceutical
Sciences, 17th ed., 1985, Mack Publishing Company, Easton, Pa.
[0245] The pharmaceutical compositions also can be administered in
sustained release forms or from sustained release drug delivery
systems. A description of representative sustained release
materials can be found in Remington's Pharmaceutical Sciences,
supra.
[0246] In one aspect, the disclosed compounds can be used in
combination with one or more other drugs in the treatment,
prevention, control, amelioration, or reduction of risk of diseases
or conditions for which disclosed compounds or the other drugs can
have utility, where the combination of the drugs together are safer
or more effective than either drug alone. Such other drug(s) can be
administered, by a route and in an amount commonly used therefor,
contemporaneously or sequentially with a compound of the present
disclosure. When a compound of the present disclosure is used
contemporaneously with one or more other drugs, a pharmaceutical
composition in unit dosage form containing such other drugs and a
disclosed compound is preferred. However, the combination therapy
can also include therapies in which a disclosed compound and one or
more other drugs are administered on different overlapping
schedules. It is also contemplated that when used in combination
with one or more other active ingredients, the disclosed compounds
and the other active ingredients can be used in lower doses than
when each is used singly.
[0247] Accordingly, the pharmaceutical compositions include those
that contain one or more other active ingredients, in addition to a
compound of the present disclosure.
[0248] The above combinations include combinations of a disclosed
compound not only with one other active compound, but also with two
or more other active compounds. Likewise, disclosed compounds can
be used in combination with other drugs that are used in the
prevention, treatment, control, amelioration, or reduction of risk
of the diseases or conditions for which disclosed compounds are
useful. Such other drugs can be administered, by a route and in an
amount commonly used therefor, contemporaneously or sequentially
with a compound of the present disclosure. When a compound of the
present disclosure is used contemporaneously with one or more other
drugs, a pharmaceutical composition containing such other drugs in
addition to a disclosed compound is preferred. Accordingly, the
pharmaceutical compositions include those that also contain one or
more other active ingredients, in addition to a compound of the
present disclosure.
[0249] The weight ratio of a disclosed compound to the second
active ingredient can be varied and will depend upon the effective
dose of each ingredient. Generally, an effective dose of each will
be used. Thus, for example, when a compound of the present
disclosure is combined with another agent, the weight ratio of a
disclosed compound to the other agent will generally range from
about 1000:1 to about 1:1000, preferably about 200:1 to about
1:200. Combinations of a compound of the present disclosure and
other active ingredients will generally also be within the
aforementioned range, but in each case, an effective dose of each
active ingredient should be used.
[0250] In such combinations disclosed compounds and other active
agents can be administered separately or in conjunction. In
addition, the administration of one element can be prior to,
concurrent to, or subsequent to the administration of other
agent(s).
[0251] Accordingly, the disclosed compounds can be used alone or in
combination with other agents which are known to be beneficial in
the subject indications or other drugs that affect receptors or
enzymes that either increase the efficacy, safety, convenience, or
reduce unwanted side effects or toxicity of the disclosed
compounds. The subject compound and the other agent can be
co-administered, either in concomitant therapy or in a fixed
combination.
[0252] The following formulation examples illustrate representative
pharmaceutical compositions that may be prepared in accordance with
this invention. The present invention, however, is not limited to
the following pharmaceutical compositions.
[0253] Formulation 1--Tablets: A pharmaceutical composition may be
admixed as a dry powder with a dry gelatin binder in an approximate
1:2 weight ratio. A minor amount of magnesium stearate is added as
a lubricant. The mixture is formed into 240-270 mg tablets (80-90
mg of active compound per tablet) in a tablet press.
[0254] Formulation 2--Capsules: A pharmaceutical composition may be
admixed as a dry powder with a starch diluent in an approximate 1:1
weight ratio. The mixture is filled into 250 mg capsules (125 mg of
active compound per capsule).
[0255] Formulation 3--Liquid: A pharmaceutical composition (125 mg)
may be admixed with sucrose (1.75 g) and xanthan gum (4 mg) and the
resultant mixture may be blended, passed through a No. 10 mesh U.S.
sieve, and then mixed with a previously made solution of
microcrystalline cellulose and sodium carboxymethyl cellulose
(11:89, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color
are diluted with water and added with stirring. Sufficient water
may then be added to produce a total volume of 5 mL.
[0256] Formulation 4--Tablets: A pharmaceutical composition may be
admixed as a dry powder with a dry gelatin binder in an approximate
1:2 weight ratio. A minor amount of magnesium stearate is added as
a lubricant. The mixture is formed into 450-900 mg tablets (150-300
mg of active compound) in a tablet press.
[0257] Formulation 5--Injection: A pharmaceutical composition may
be dissolved or suspended in a buffered sterile saline injectable
aqueous medium to a concentration of approximately 5 mg/mL.
[0258] Formulation 6--Topical: Stearyl alcohol (250 g) and a white
petrolatum (250 g) may be melted at about 75.degree. C. and then a
mixture of a pharmaceutical composition (50 g) methylparaben (0.25
g), propylparaben (0.15 g), sodium lauryl sulfate (10 g), and
propylene glycol (120 g) dissolved in water (about 370 g) may be
added and the resulting mixture is stirred until it congeals.
[0259] The following non-limiting examples are intended to be
purely illustrative of some aspects and embodiments, and show
specific experiments that were carried out in accordance with the
disclosure.
EXAMPLES
Materials and Methods
[0260] Cell Culture and siRNA Treatments
[0261] MCF-7 cells were obtained from and grown as recommended by
the American Type Culture Collection. Receptor expression was
verified by qPCR and Western blotting, and gene expression and
proliferative response to estradiol (E2) was monitored regularly.
For experiments with E2 and PaPE treatment, cells were maintained
in phenol red-free tissue culture medium for at least 5 days prior
to use. ER.alpha. knockdown experiments utilized the SMART pool of
4 siRNAs from Dharmacon and were performed as described (see
Madak-Erdogan et. al., Mol Endocrinol 22, 2116-2127, 2008) with 30
nM siCtrl or siER.alpha. or siGPR30 for 72 h. This resulted in
knockdown of the corresponding mRNA and protein by greater than
80%. Primary bovine aortic endothelial cells were harvested,
maintained and employed as previously described in Chambliss, J
Clin Invest 120, 2319-2330, 2010).
Animals and Ligand Treatments
[0262] Studies used wild type and ER.alpha. knockout mice in
C57BL/6 background. All experiments involving animals were
conducted in accordance with National Institutes of Health
standards for the use and care of animals, with protocols approved
by the University of Illinois at Urbana-Champaign and the
University of Texas Southwestern Medical Center. Wild-type C57BL/6
mice were purchased from Jackson Laboratories/National Cancer
Institute. ER.alpha. knockout mice with complete excision of
ER.alpha. and wild type littermates were obtained from Taconic and
were used as described previously in Dupont et al., Development,
127, 4277-4291, 200, and Hewitt et al. FASEB J., 24, 4660-4667,
2010.
[0263] In studies of metabolic parameters and gene expression in
vivo, ligands were administered to ovariectomized recipient mice by
subcutaneous implantation of pellets containing compound mixed with
cholesterol to a total weight of 20 mg. Animals were single-housed
during the study. For 3-week studies, estradiol (E2, Sigma-Aldrich)
dosage (0.125 mg/pellet) was chosen based on previous findings. In
carotid artery reendothelialization experiments, compounds were
delivered at a dose of 6 .mu.g/day by a 4-week (Model 2006) Alzet
minipump, as described (Chambliss, J Clin Invest 120, 2319-2330,
2010). Total body weight and food intake were monitored every week
after ovariectomy. Body fat and lean mass composition were
monitored at the end of 3 weeks using EchoMRI-700 Body Composition
Analyzer (Echo Medical Systems, Houston, Tex.), which enables one
to quantify longitudinal body composition in the live animal.
Western Blotting, and ChIP Assays
[0264] Western blot analysis used specific antibodies for ER.alpha.
(HC-20, Santa Cruz); ERK2 (D-2, Santa Cruz); and pS.sup.6, pS6K,
pmTOR, pRAPTOR, pRICTOR, pMAPK (Cell Signaling).
Coimmunoprecipitation assays used antibodies for SRC3 (Santa Cruz,
C-20) and ER.alpha. (Santa Cruz, F10).
[0265] ChIP assays were carried out as described (Madak-Erdogan et
al. Mol Syst Biol 9, 676 2013 and Madak-Erdogan et al., Mol Cell
Biol 31, 226-236, 2011). Antibodies used were for ER.alpha. (HC20),
ERK2 (Santa Cruz, D2 and C.sub.1-4), and pSer5 RNA Pol II (Santa
Cruz, sc-47701). ChIP DNA was isolated using QIAGEN PCR
purification kit and used for ChIP-seq analysis and quantitative
real-time PCR (qPCR). qPCR was used to calculate recruitment to the
regions studied, as described.
ChIP-Seq Analysis and Clustering
[0266] For genome-wide ChIP-seq, the ChIP DNA was prepared into
libraries according to Illumina Solexa ChIP-seq sample processing
methods, and single read sequencing was performed using the
Illumina HiSeq 2000. Sequences generated were mapped uniquely onto
the human genome (hg18) by Bowtie2. MACS (Model-based Analysis of
ChIP-Seq) algorithm was used to identify enriched peak regions
(default settings) with a p-value cutoff of 6.0e-7 and FDR of 0.01,
as was described in Madak-Erdogan, Mol Syst Biol 9, 676, 2013.
[0267] The seqMINER density array method with a 500-bp window in
both directions was used for the generation of clusters, i.e.,
groups of loci having similar compositional features. BED files for
each cluster were used for further analysis with Galaxy Cistrome
integrative analysis tools (Venn diagram, conservation, CEAS).
RNA-Seq Transcriptional Profiling
[0268] For gene expression analysis, total RNA was extracted from 3
biological replicates for each ligand treatment using Trizol
reagent and further cleaned using the RNAeasy kit (QIAGEN). For
time course studies, MCF-7 cells were treated with Veh (0.1% EtOH),
10 nM E2 or 1 .mu.M PaPE for 4 h and 24 h. For inhibitor studies,
MCF-7 cells were pretreated with Ctrl (0.1% DMSO), 1 .mu.M PP242 or
1 .mu.M AZD6244 for 30 min and then treated with Veh, 10 nM E2 or 1
.mu.M PaPE-1 in the presence or absence of inhibitors for 4 h. Once
the sample quality and replicate reproducibility were verified, 2
samples from each group were subjected to sequencing. RNA at a
concentration of 100 ng/.mu.L in nuclease-free water was used for
library construction. cDNA libraries were prepared with the
mRNA-TruSeq Kit (Illumina, Inc.). Briefly, the poly-A containing
mRNA was purified from total RNA, RNA was fragmented,
double-stranded cDNA was generated from fragmented RNA, and
adapters were ligated to the ends.
[0269] The paired-end read data from the HiSeq 2000 were processed
and analyzed through a series of steps. Base calling and
de-multiplexing of samples within each lane were done with Casava
1.8.2. FASTQ files were trimmed using FASTQ Trimmer (version
1.0.0). TopHat2 (version 0.5) was employed to map paired RNA-Seq
reads to version hg19 of the Homo sapiens reference genome in the
UCSC genome browser in conjunction with the RefSeq genome reference
annotation. Gene expression values (raw read counts) from BAM files
were calculated using StrandNGS (version 2.1) Quantification tool.
Partial reads were considered and option of detecting novel genes
and exons was selected. Default parameters for finding novel exons
and genes were specified. DESeq normalization algorithm using
default values was selected. Differentially expressed genes were
then determined by fold-change and p-value with Benjamini and
Hochberg multiple test correction for each gene for each treatment
relative to the vehicle control. Genes with fold-change >2 and
p-value <0.05 were considered as statistically significant,
differentially expressed. All RNA-Seq datasets have been deposited
with the NCBI under GEO accession number GSE73663.
Motif and GO Category Analysis
[0270] Overrepresented GO biological processes were determined by
the web-based DAVID Bioinformatics Resources database, ClueGO and
web-based GREAT software. Motifs enrichment analysis was done using
Seqpos.
Immunohistochemistry (IHC)
[0271] Hematoxylin and eosin (H&E) staining, and whole mount
staining were performed on paraffin-embedded tissue sections.
Images were quantified by monitoring average cell size from 3
randomly chosen fields in Fiji software
(http://fiji.sc/wiki/index.php/Fiji).
Immunofluorescence Microscopy, Proximity Ligation Assays in Cells
and Data Analysis
[0272] Cells treated with vehicle (0.1% EtOH), 10 nM E2 or 1 .mu.M
PaPE-1 for 15 min were washed in PBS, fixed on glass coverslips and
incubated with antibodies against ER.alpha. (F10, Santa Cruz),
pSer5 RNA Pol II (Santa Cruz, 47701), or RAPTOR (Cell Signaling).
Next day, the proximity ligation assay (PLA) was performed using
the Duolink In Situ kit (Olink Bioscience) according to the
manufacturer's instructions, as described in Zhao et. al.,
Endocrinology, 154, 1349-1360, 2013. Briefly, overnight incubation
with primary antibodies was followed by hybridization with two PLA
probes at 37.degree. C. for 1 h, and then by ligation for 15 min
and amplification for 90 min at 37.degree. C. A coverslip was
mounted on each slide and image acquisition and analysis conducted.
Samples were imaged using a 63.times./1.4 Oil DIC M27 objective in
a Zeiss LSM 700 or 710 laser scanning confocal microscope. Images
were obtained in a sequential manner using a 488 Ar (10 mW) laser
line for PLA signal. The individual channels for DAPI and PLA
signal were obtained using a sequential scanning mode to prevent
bleed-through of the excitation signal. Laser power, gain and
offset were kept constant across the samples and scanned in a high
resolution format of 512.times.512 or 1024.times.1024 pixels with
2/4 frames averaging. Further quantification of the images used
Fiji software (http://fiji.sc/wiki/index.php/Fiji). Briefly, images
were converted to 8 bit for segmentation for each channel, and
images were background subtracted using a rolling-ball method, with
a pixel size of 100 and segmented using the DAPI channel.
Cell Proliferation Assays
[0273] Cells were seeded at 1000 cells/well in 96-well plates. On
the second day, the cells were treated with Veh, E2 or PaPE at the
concentrations indicated and proliferation was assessed using WST-1
reagent (Roche) as described in Madak-Erdogan, Mol Syst Biol 9,
676, 2013.
eNOS Activation
[0274] eNOS activation was assessed in intact primary endothelial
cells by measuring .sup.14C-L-arginine conversion to
.sup.14C-L-citrulline over 15 min using previously reported methods
(Chambliss et. al., J Clin Invest 120, 2319-2330, 2010). Cells were
treated with vehicle (yielding basal activity), E2, or PaPE alone
or with the antiestrogen ICI 182,780 at the concentrations
indicated.
Carotid Artery Reendothelialization
[0275] Carotid artery reendothelialization was studied following
perivascular electric injury in mice by assessing Evans blue dye
uptake 72 h after injury. Endothelial denudation and recovery after
injury in this model have been confirmed by immunohistochemistry
for von Willebrand Factor. At the time of ovariectomy at 8-9 weeks
of age, female mice received intraperitoneal osmotic minipumps
prepared to deliver 6 .mu.g/d E2 or PaPE-1. Carotid artery
denudation was performed 21d later. In select studies additional
treatments included subcutaneous injections of vehicle or ICI
182,780 (360 .mu.g/mouse) administered 3d prior to carotid injury
and on the day of injury. At the end of the study, uteri were also
harvested and weighed.
Pharmacokinetic Analyses
[0276] For short-term studies, ovariectomized C57BL/6 mice were
injected SC with 100 .mu.g of PaPE-1 in 100 .mu.l DMSO. Three mice
were sacrificed at each time point, and 400 .mu.l of blood was
obtained from the abdominal aorta. Samples were centrifuged at 2000
g for 10 min, and serum was collected. A 50 .mu.l portion of each
sample was submitted to the Metabolomics Center at the University
of Illinois for analysis. For longer-term studies, ovariectomized
C57BL/6 mice were implanted SC with a pellet fabricated with 8 mg
PaPE-1 and 12 mg cholesterol. Blood samples (30 .mu.l) were
collected by tail snipping every week until the third week of
treatment. Samples were centrifuged at 2000 g for 10 min and serum
was collected. Serum (10 .mu.l) was mixed with 40 .mu.l of PBS and
submitted to the Metabolomics Center for analysis. For analysis, a
mass standard of PaPE-1 (labeled with three deuterium atoms) was
added to each sample before analysis by liquid chromatography-mass
spectrometry using the 5500 QTrap with Agilent 1200 HPLC.
Ligand Dissociation Assays
[0277] The fluorescence polarization or anisotropy characteristics
of fluorescein attached to C530 in ER.alpha. is sensitive to the
nature of the bound ligand. These differences can be exploited to
detect the dissociation of one ligand and the association of a
second ligand, with the rate of ligand exchange being limited by
the rate of dissociation of the initially bound ligand. PaPE-1
gives an anisotropy value about 20% lower than that of E2 when
bound to ER, and OH-Tam gives an 80% lower anisotropy value than E2
when bound to the ER. Therefore, each ligand pair (PaPE-1/E2 or
E2/OH-Tam) gives a distinct change in anisotropy that can be used
to monitor the rate of dissociation of the initially bound
ligand.
[0278] ER.alpha.-ligand binding domain (LBD), mutated to have one
active cysteine at C530, was site-specifically labeled with
5-iodoacetamidofluorescein and then diluted into t/g buffer (50 mM
Tris, 10% glycerol, pH 8) with 0.01 M mercaptoethanol and 0.03
mg/ml ovalbumin added as a carrier protein to give 2 nM ER. To
minimize homoFRET, a 5-fold excess (10 nM) of unlabeled
ER.alpha.-LBD (10 nM) was added, and the fluorescein-labeled and
unlabeled ER dimers were allowed to exchange at room temperature in
the dark, for 1 h, thereby producing dimers in which essentially
only one monomer is fluorescein labeled. The ER was then bound with
100 nM E2, or 100 nM/RBA of PaPE-1; the RBA of PaPE-1 is 0.002%;
therefore, 100 nM/RBA=50 .mu.M of PaPE was used. These samples were
allowed to complete ligand binding at room temperature in the dark
for 2.5 h.
[0279] The anisotropy was measured on a Spex fluorolog II
cuvette-based fluorimeter under constant wavelength conditions. The
excitation was set at 488 nm and emission at 520 nm, under magic
angle conditions, and three to five time points were taken for a
zero time. To initiate the dissociation of PaPE-1, 300 nM of E2 was
added to the PaPE-1 sample, and the time course of dissociation was
subsequently followed by changes in anisotropy. Due to glycerol
viscosity changes, there is a change in the protein anisotropy
between room temperature and 5.degree. C.; therefore, care was
taken to prechill the protein as well as the cuvette chamber to
5.degree. C.
[0280] To measure the dissociation of E2, 300 nM E2 was added to
the pre-exchanged sample of fluorescein-labeled and unlabeled
apo-ER dimer and allowed to bind for 2.5 h, as above. The cuvette
and chamber were chilled to 5.degree. C., and after taking the zero
time points, E2 dissociation was initiated by adding 5 .mu.M
OH-Tam, and change in anisotropy was followed with time. The data
for both dissociation experiments were fitted to an exponential
decay function by linear regression using Prism 4.
Computational Modeling of the Complex of ER.alpha. with PaPE-1 or
E2
[0281] Starting from the ER.alpha.+E2 crystal structure (PDB code:
1GWR), the structure preparation routine in MOE (MOE: Molecular
Operating Environment, Chemical Computing Group) was used to fill
missing loops, side chains, add explicit hydrogen atoms. A custom
volume visualization code was used to create the binding volume for
the ER+E2 structure, shown in slate blue in FIG. 2B; the red dot is
a structural water. The model of the binding of PaPE-1 was built by
progressive generation of the PaPE-1 ligand structure from that of
E2, coupled with progressive minimization of the ligand-binding
domain: Atoms were first deleted from E2 to open the B-ring and
convert the C ring into an aromatic ring, but the two ortho-methyl
groups on the A-ring were not yet added. At this stage, the
positions of the ligand oxygen atoms and all protein atoms were
fixed, and energy minimization was performed using the MMFF94x
force field with a termination gradient cutoff of 0.1 kcal/(molA)
to obtain a low energy conformation of the PaPE-1 ligand core. All
atoms were then unfixed, and energy minimization was further
performed while constraining protein backbones to optimize
interactions with hydrogen bonding side chains. After the two
A-ring ortho-methyl groups were added, another energy minimization
was performed with constrained backbone atoms, and then a final
unconstrained energy minimization was performed, all to the same
gradient cutoff. The resulting positions of the ligand and hydrogen
bonding residues are shown in yellow in FIG. 2B.
Statistical Analyses
[0282] Data from in vivo animal metabolism studies were analyzed
using either one-way ANOVA to compare different ligand effects or
two-way-ANOVA to compare time dependent changes followed by
Bonferroni post hoc test using GraphPad Prism 6. Data from gene
expression studies were analyzed using t-test.
General Synthetic Procedures
[0283] NMR measurement: .sup.1H NMR spectra were recorded on a
Varian vxr500 MHz, u500, or u400 MHz spectrometer and chemical
shifts are given in .delta.-values [ppm] referenced to the residual
solvent peak chloroform (CDC.sub.3) at 7.26 and methanol
(CD.sub.3OD) at 3.31 ppm. Coupling constants, J, are reported in
Hertz. Materials and solvents were of highest grade available from
commercial sources and were used without further purification.
Molecular weight was determined by using the Waters Quattro ultima
ESI or Waters 70-VSE EI/CI/FD/FI mass spectrometers.
Example 1. Synthesis of
5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol (2)
##STR00028##
[0285] The mixture of 3,5-dimethyl-4-methoxyphenylboronic acid (200
mg, 1.11 mmol), 5-bromo-1-indanone (211 mg, 1.00 mmol),
bis(triphenylphosphine)palladium(II) dichloride (35 mg, 0.05 mmol),
potassium carbonate (307 mg, 2.22 mmol), and deionized (DI) water
(39 mg, 2.22 mmol) in THF (10 mL) was stirred under the argon
atmosphere at 85.degree. C. for 4 hr to 8 hr. After starting
materials disappeared on TLC analysis, ethyl acetate (20 mL) was
added into reaction mixture. When DI water (20 mL) was added to
reaction mixture, an insoluble solid precipitated. The suspended
solid was collected by filtration and more solid was collected
after evaporating the ethyl acetate filtrate. Recrystallization of
the combined solids from ethyl acetate afforded the
5-(4-methoxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one (223
mg, 84%). .sup.1H NMR (500 MHz, CDCl.sub.3) 2.82 (s, 6H), 2.75 (t,
2H, J=6.0 Hz), 3.20 (t, 2H, J=6.0 Hz), 3.79 (s, 3H), 7.31 (s, 2H),
7.58 (d, 1H, J=8.0 Hz), 7.65 (s, 1H), 7.80 (d, 1H, J=8.0 Hz);
.sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 16.55, 26.12, 36.78,
60.07, 122.06, 124.21, 125.06, 126.81, 128.21, 131.74, 135.88,
147.72, 156.10, 157.70, 206.92.
[0286] Boron trifluoride methyl sulfide complex (1.04 g, 0.08 mmol)
was added to the
5-(4-methoxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one (266
mg, 1.00 mmol) solution in DCM (10 mL) and stirred for 6 hr at rt,
followed by evaporation of solvent, addition of water, and
filtration to afford
5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one as a
slightly dark white solid (240 mg, 95%). Upon the demethylation,
the product appears as a blue fluorescent spot on TLC when exposed
to 254 nm UV light. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 2.31
(s, 6H), 2.72 (t, 2H, J=5.5 Hz), 3.17 (t, 2H, J=5.5 Hz), 7.26 (s,
2H), 7.55 (d, 1H, J=8.0 Hz), 7.62 (s, 1H), 7.76 (d, 1H, J=8.0 Hz);
.sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 16.49, 26.11, 36.77,
122.09, 124.22, 124.56, 126.52, 127.91, 128.87, 135.27, 148.19,
153.50, 156.39.
[0287] To the solution of
5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one in
THF-water mixture (200 mg, 0.79 mmol) was added portionwise NaBH4
(50 mg, 1.31 mmol) at rt and the mixture was stirred until starting
material disappeared on silica gel TLC analysis. Upon reduction by
NaBH.sub.4, the blue fluorescence disappeared on silica gel TLC at
254 nm. Compound 2 (189 mg) was obtained in 94% yield after passing
through short silica gel pad by using 35% ethyl acetate in
n-hexane. .sup.1H NMR (500 MHz, CDCl.sub.3+methanol-d.sub.4)
.delta. 1.95-2.05 (m, 1H), 2.31 (s, 6H), 2.48-2.58 (m, 1H),
2.84-2.92 (m, 1H), 3.02-3.16 (m, 1H), 4.66 (s, 1H, phenolic OH),
5.29 (t, 1H, J=5.5 Hz), 7.20 (s, 2H), 7.39-7.46 (m, 3H); .sup.13C
NMR (126 MHz, CDCl.sub.3) .delta. 16.30, 30.06, 36.40, 76.48,
123.47, 123.50, 124.57, 125.83, 127.69, 133.61, 141.83, 143.50,
144.17, 152.07; ESI (m/z) 255.3 (M+.sup.+1, 25%), 237.2
(M+.sup.+1-H.sub.2O, 85%)
Example 2. Synthesis of
S-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol (3:
S-2)
##STR00029##
[0288]
S-5-(4-methoxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol
[0289] The mixture of 98% ee (S)-5-Bromo-2,3-dihydro-1H-inden-1-ol
(100 mg, 0.47 mmol), which was obtained from the asymmetric
reduction of 5-bromo-1-indanone using cat.
(R)-(+)-2-methyl-CBS-oxazaborolidine and 2 eq. diethylaniline
borane, according to the literature (S H Lee et al, J. Org. Chem.
2011, 76, 10011-10019) and 3,5-dimethyl-4-methoxyphenylboronic acid
(101 mg, 0.56 mmol), bis(triphenylphosphine)palladium(II)
dichloride (18 mg, 0.028 mmol), potassium carbonate (155 mg, 1.12
mmol), and DI water (40 mg, 2.22 mmol) in THF (10 mL) was stirred
under the argon atmosphere at 80.degree. C. for 4 hr to 8 hr until
the starting alcohol spot disappeared on silica gel TLC analysis.
After cooling down the temperature of reaction mixture, water (20
mL) was added followed by extraction with ethyl acetate (10
mL.times.3), drying over Na.sub.2SO.sub.4, concentrating the
solvent before loading on silica gel for column chromatography.
Elution with 20% ethyl acetate in n-hexane provided the title
compound as an off-white solid (110 mg, 0.41 mmol, 87%).
Spectroscopic data was identical to that of compound 5 described at
example 4.
S-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol
(S-2)
[0290] To a solution of
S-5-(4-methoxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol (134
mg, 0.5 mmol) in THF (10 mL) was added 1M super-hydride solution in
THF (4 ml, 4.0 mmol) at rt. This solution was refluxed for 24 hr
and after cooling down the temperature, water (10 mL) was added
dropwise with cooling in an ice-bath. This solution was extracted
with ethyl acetate (10 mL.times.3), dried over Na.sub.2SO.sub.4,
concentrated by rotary evaporator to load onto silica gel for
column chromatography. Elution with 35% ethyl acetate in n-hexane
afforded
S-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol as an
off-white solid (81 mg, 0.32 mmol, 64%). Enantiomeric excess was
confirmed by supercritical phase chromatography (SFC) as 98% ee.
Spectroscopic data was identical to that of compound 2.
(Supercritical Fluid Chromatography (SFC) column condition: 15%
MeOH/CO.sub.2. 1 mL/min, Daicel Chiralpak AD column (25
cm.times.4.6 mm), retention time: 10.94 min)
Example 3. Synthesis of
R-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol (4:
R-2)
##STR00030##
[0292] A similar procedure was followed except for the use of
(S)-(+)-2-methyl-CBS-oxazaborolidine as a catalyst to obtain 98% ee
(R)-5-Bromo-2,3-dihydro-1H-inden-1-ol precursor. (Supercritical
Fluid Chromatography (SFC) column condition: 15% MeOH/CO.sub.2. 1
mL/min, Daicel Chiralpak AD column (25 cm.times.4.6 mm), retention
time: 15.18 min)
Example 4.
5-(4-methoxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol (5)
##STR00031##
[0294] Compound 7 (55 mg, 0.21 mmol) in example 1 was treated with
NaBH.sub.4 (15 mg, 0.39 mmol) in the mixed solvent (MeOH-THF=1:1,
v/v, 5 mL). Once starting material disappeared on SiO.sub.2 TLC,
solvent was concentrated by evaporation and passed through a silica
gel pad with EtOAc:n-Hexane (1:1, v/v) to collect compound 5 (48
mg) as an off-white solid. .sup.1H NMR (500 MHz,
CDCl.sub.3+methanol-d.sub.4) .delta.1.97-2.04 (m, 1H), 2.35 (s,
6H), 2.51-2.57 (m, 1H), 2.84-2.90 (m, 1H), 3.08-3.14 (m, 1H), 3.77
(s, 3H), 5.85 (t, 1H, J=5.5 Hz), 5.30 (s, 1H, phenolic OH), 7.23
(s, 2H), 7.12 (d, 1H, J=8.5 Hz), 7.43 (s, 1H), 7.45 (d, 1H, J=8.5
Hz); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 16.50, 30.07,
36.42, 60.03, 76.46, 123.73, 124.59, 126.07, 127.91, 131.33,
137.10, 141.69, 143.91, 144.17, 156.76.
Example 5.
6-(4-hydroxy-3,5-dimethylphenyl)-1,2,3,4-tetrahydronaphthalen-1-
-ol (16)
##STR00032##
[0296] Compound 16 was synthesized according to the method
described in example 1 with 3,5-dimethyl-4-methoxyphenylboronic
acid (200 mg, 1.11 mmol) and 6-bromo-1-tetralone (225 mg, 1.00
mmol). The yield was comparable to that of compound 2. .sup.1H NMR
(500 MHz, CDCl.sub.3) .delta. 1.78-1.85 (m, 1H), 1.91-1.97 (m, 1H),
1.98-2.05 (m, 1H), 2.31 (s, 6H), 2.75-2.82 (m, 1H), 2.85-2.92 (m,
1H), 3.02-3.16 (m, 1H), 4.83 (t, 1H, J=5.5 Hz), 7.20 (s, 2H), 7.27
(d, 1H, J=1.5 Hz), 7.38 (dd, 1H, J, J=1.5, 8.5 Hz), 7.47 (d, 1H,
J=8.5 Hz); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 16.30, 19.09,
29.68, 32.63, 68.24, 123.50, 124.95, 126.58, 127.53, 129.25,
133.24, 137.31, 137.60, 140.66, 152.09.
Example 6. 4'-(1-hydroxyethyl)-3,5-dimethyl-[1,1'-biphenyl]-4-ol
(15)
##STR00033##
[0298] Compound 15 was synthesized according to the method
described at example 1 with 3,5-dimethyl-4-methoxyphenylboronic
acid (200 mg, 1.11 mmol) and 4-bromo-acetophenone (199 mg, 1.00
mmol). The yield was comparable to that of compound 2. .sup.1H NMR
(500 MHz, CDCl.sub.3) .delta.1.53 (d, 3H, J=5.0 Hz), 2.31 (s, 3H),
4.94 (q, 1H, J=5.0 Hz), 4.83 (t, 1H, J=5.5 Hz), 7.22 (s, 2H), 7.24
(d, 2H, J=8.5 Hz), 7.41 (d, 2H, J=8.5 Hz); .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta. 16.31, 25.34, 70.49, 123.54, 125.98, 126.49,
127.06, 127.37, 127.54, 133.19, 140.61, 152.09, 144.19, 152.09.
Example 7. Diphenolic acid-NHEtNHAc (71)
##STR00034##
[0300] Diphenolic Acid NHS Ester.
[0301] To the solution of diphenolic acid (285 mg, 1.00 mmol) in
DMF (1 mL) was added N-hydroxysuccinic acid (121 mg, 1.05 mmol),
cat. amount of DMAP, and DCC (217 mg, 1.05 mmol). After stirring
for 1 hr, EtOAc (10 mL) was added to precipitate the urea which was
removed by filtration. The filtrate was washed with water (2
mL.times.4), sat. NaHCO.sub.3, and dried over Na.sub.2SO.sub.4, and
evaporation provided the NHS ester which is used without further
purification. .sup.1H NMR (400 MHz, CDCl.sub.3+methanol-d.sub.4)
.delta. 1.49 (s, 3H), 2.26-34 (m, 2H), 2.34-2.43 (m, 2H), 2.75 (s,
4H), 6.66 (d, 4H, J=8.0 Hz), 6.94 (d, 4H, J=8.0 Hz); .sup.13C NMR
(126 MHz, CDCl.sub.3+methanol-d.sub.4) .delta. 25.72, 27.39, 30.19,
36.49. 44.53, 115.14, 128.32, 139.81, 154.86, 169.27, 169.57. ESI
(m/z) 384.3 (M+.sup.+1)
Diphenolic Acid-NHEtNHAc (71)
[0302] The reaction of NHS ester (38 mg, 0.1 mmol) with mono
acetylated ethylene diamine (35 mg, 0.35 mmol) in DMF (500 ul) at
rt. After sonicating (Branson sonicator Model 2210) the reaction
mixture for 30 min at rt, added DI water (10 mL), and extracted
with EtOAc (5 mL.times.3), washed with water (5 mL.times.3) again,
dried over Na.sub.2SO.sub.4, concentrated to load 1 mm SiO.sub.2
preparative thin layer chromatography. Rf 0.25 band developed with
5% MeOH in DCM was scraped out from Prep. TLC, followed by placing
the SiO.sub.2 chunk into sintered glass filter to extract out title
compound with 20% MeOH in DCM to provide 28 mg of title compound.
.sup.1H NMR (500 MHz, CDCl.sub.3+methanol-d.sub.4) .delta.1.48 (s,
3H), 1.85-1.94 (m, 5H), 2.28 (q, 2H, J=5 Hz), 3.12-3.22 (m, 4H),
6.66 (d, 4H, J=8.0 Hz), 6.96 (d, 4H, J=8.0 Hz); .sup.13C NMR (126
MHz, CDCl.sub.3+methanol-d.sub.4) .delta. 18.11, 26.61, 31.86,
36.35, 41.57, 43.42, 48.58, 118.81, 132.36, 144.36, 158.61. 176.42,
179.65; ESI (m/z) 371.2 (M+.sup.+1)
Example 8.
1-ethynyl-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-
-1-ol (19)
##STR00035##
[0304] Compound 19 was synthesized by the reaction of compound 6
(80 mg, 0.32 mmol) with ethynylmagnesium bromide (1.2 mL.times.2,
0.5 M THF) in THF (10 mL) at -78.degree. C. for 2 hr. After the
reaction temperature rose to rt, water was carefully added dropwise
to the reaction mixture, and 0.1 N HCl solution was added to
acidify the reaction solution. The reaction mixture was extracted
with EtOAc (5 mL.times.3), dried over Na.sub.2SO.sub.4, and
concentrated to be loaded on a 1 mm SiO.sub.2 prep TLC. Prep. TLC
was developed with 20% EtOAc in n-hexane and the band at Rf 0.3 was
scraped out to extract compound 19 (26 mg). .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 2.32 (s, 6H), 2.48-2.52 (m, 1H), 2.60-2.64 (m,
1H), 2.66 (s, 1H, OH), 2.92-3.01 (m, 1H), 3.17-3.22 (m, 1H), 4.74
(s, 1H, phenolic OH), 7.20 (s, 2H), 7.25 (s, 1H), 7.42 (d, 1H,
J=8.5 Hz), 7.57 (d, 1H, J=8.5 Hz); .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta. 16.30, 29.80, 43.53, 73.20, 76.19, 86.13,
123.46, 123.56, 126.32, 127.75, 128.00, 133.43, 142.71, 143.75,
143.88, 152.22.
Example 9.
4-(1-chloro-2,3-dihydro-1H-inden-5-yl)-2,6-dimethylphenol (8)
##STR00036##
[0306] Compound 8 was synthesized by treating compound 2 (25 mg,
0.1 mmol) with SOCl.sub.2 (40 mg, 0.34 mmol) in DCM (1 mL) at
0.degree. C. for 2 hr. Washing with 5% NaHCO.sub.3 and evaporation
of the solvent afforded compound 8 (24 mg). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 2.09-2.13 (m, 1H), 2.29 (s, 6H), 2.42-2.56 (m,
1H), 2.86-2.94 (m, 1H), 3.09-3.18 (m, 1H), 4.62 (s, 1H, phenolic
OH), 6.20 (dd, 1H, J, J=3.0 Hz, 3.0 Hz), 7.18 (s, 2H), 7.37 (d, 1H,
J=8.4 Hz), 7.41 (s, 1H), 7.42 (d, 1H, J=8.4 Hz); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 16.29, 30.05, 32.72, 78.39, 123.26,
123.48, 125.83, 127.66, 127.77, 133.47, 139.54, 142.43, 145.32,
152.14.
Example 10.
5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-indene-1-carbonitrile
(9)
##STR00037##
[0308] Compound 8 (14 mg, 0.51 mmol) was treated with potassium
cyanide (50 mg, 1.56 mmol) and TBA-CN (100 mg) at DMF (1 mL) at
55.degree. C. for 4 hr. The reaction mixture was poured into water
(10 mL) and extracted with EtOAc (5 mL.times.3), dried over
Na.sub.2SO.sub.4, concentrated under vacuum to load on a 0.2 mm
SiO.sub.2 TLC plate (20 cm.times.20 cm). TLC plate was developed by
15% EtOAc in n-hexane solvent, the Rf 0.5 band was scraped off and
extracted to afford compound 9 (5 mg). .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 2.32 (s, 6H), 2.42-2.47 (m, 1H), 2.60-2.65 (m,
1H), 3.00-3.04 (m, 1H), 3.12-3.17 (m, 1H), 4.14 (t, 1H, J=8.0 Hz),
4.66 (s, 1H, phenolic OH), 7.20 (s, 2H), 7.43-7.47 (m, 3H).
Example 11.
4-(hydroxymethyl)-3',5'-dimethyl-[1,1'-biphenyl]-3,4'-diol (60)
##STR00038##
[0310] Compound 60 was synthesized according to the methods used in
example 1 to synthesize compound 2, using
3,5-dimethyl-4-methoxyphenylboronic acid (200 mg, 1.11 mmol) and
4-bromo-2-methoxybenzaldehyde (215 mg, 1.00 mmol). The yield was
comparable to that of compound 2. .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 2.31 (s, 6H), 4.70 (s, 1H, phenolic OH), 4.91 (s, 2H), 7.04
(dd, 1H, J, J=1.5 Hz, 8.5 Hz), 7.07 (d, 1H, J=8.5 Hz), 7.09 (d, 1H,
J=1.5 Hz), 7.22 (s, 2H), 7.27 (s, 1H); .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta. 16.28, 64.88, 115.03, 118.65, 123.00, 123.52,
127.48, 132.81, 142.93, 152.26, 156.56.
Example 12.
3'-(hydroxymethyl)-3,5-dimethyl-[1,1'-biphenyl]-4,4'-diol (59)
##STR00039##
[0312] Compound 59 was synthesized according to the methods used in
example 1 to synthesize compound 2, using
3,5-dimethyl-4-methoxyphenylboronic acid (200 mg, 1.11 mmol) and
5-bromo-2-methoxybenzaldehyde (215 mg, 1.00 mmol). The yield was
comparable to that of compound 2. .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 2.31 (s, 6H), 4.70 (s, 1H, phenolic OH), 4.93 (s, 2H), 6.94
(d, 1H, J=8.5 Hz), 7.15 (s, 2H), 7.21 (d, 1H, J=1.5 Hz), 7.39 (dd,
1H, J, J=1.5 Hz, 8.5 Hz); .sup.13C NMR (126 MHz, CDCl.sub.3)
.delta. 16.29, 65.16, 117.08, 123.51, 124.91, 126.33, 127.17,
127.96, 133.09, 133.55, 151.66, 155.31.
Example 13.
5-(3,5-dichloro-4-hydroxyphenyl)-2,3-dihydro-1H-inden-1-ol (63)
##STR00040##
[0314] Compound 63 was synthesized according to the methods used in
example 1 to synthesize compound 2, using
3,5-dichloro-4-methoxyphenylboronic acid (200 mg, 1.11 mmol) and
5-bromo-1-indanone (211 mg, 1.00 mmol). The yield was comparable to
that of compound 2. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.
2.01-2.05 (m, 1H), 2.52-2.59 (m, 1H), 2.85-2.91 (m, 1H), 3.09-3.15
(m, 1H), 5.30 (t, 1H, J=5.5 Hz), 5.94 (s, 1H, phenolic OH), 7.37
(d, 1H, J=8.5 Hz), 7.38 (s, 1H), 7.47 (d, 1H, J=8.5 Hz), 7.48 (s,
2H); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 30.04, 36.38,
76.35, 121.65, 123.46, 124.96, 125.76, 127.11, 135.29, 139.04,
144.59, 144.89, 147.31.
Example 14.
3,5-Dichloro-3'-(hydroxymethyl)-[1,1'-biphenyl]-4,4'-diol (64)
##STR00041##
[0316] Compound 64 was synthesized according to the methods used in
example 1 to synthesize compound 2, with
3,5-dichloro-4-methoxyphenylboronic acid (200 mg, 1.11 mmol),
5-bromo-2-methoxybenzaldehyde (215 mg, 1.00 mmol). The yield was
comparable to that of compound 2. .sup.1H NMR (500 MHz,
CDCl.sub.3+methanol-d.sub.4) .delta. 4.75 (s, 2H), 7.04 (dd, 1H, J,
J=1.5 Hz, 8.5 Hz), 6.83 (d, 1H, J=8.5 Hz), 7.19 (d, 1H, J=2.0 Hz),
7.24 (dd, 1H, J, J=2.0, 8.5 Hz), 7.35 (s, 2H); .sup.13C NMR (126
MHz, CDCl.sub.3+methanol-d.sub.4) .delta. 63.16, 116.48, 122.02,
126.48, 126.56, 127.18, 130.36, 134.47, 139.95, 147.33, 155.80.
Example 15.
3'-fluoro-4'-(hydroxymethyl)-3,5-dimethyl-[1,1'-biphenyl]-4-ol
(25)
##STR00042##
[0318] Compound 25 was synthesized according to the methods used in
example 1 to synthesize compound 2, using
3,5-dimethyl-4-methoxyphenylboronic acid (200 mg, 1.11 mmol) and
4-bromo-2-fluorobenzaldehyde (203 mg, 1.00 mmol). The yield was
comparable to that of compound 2. .sup.1H NMR (500 MHz,
CDCl.sub.3+methanol-d.sub.4) .delta. 2.32 (s, 6H), 4.79 (s, 2H),
4.81 (s, 1H, phenolic OH), 7.21 (s, 2H), 7.24 (d, 1H, J=11.5 Hz),
7.33 (d, 1H, J=8.0 Hz), 7.43 (t, 1H, J=8.0 Hz); .sup.13C NMR (126
MHz, CDCl.sub.3+methanol-d.sub.4) .delta. 16.29, 59.60 (d,
J.sub.CF=3.9 Hz), 113.61 (d, J.sub.CF=22.4 Hz), 122.56 (d,
J.sub.CF=2.8 Hz), 123.76, 125.80 (d, J.sub.CF=14.5 Hz), 127.46,
129.85 (d, J.sub.CF=4.9 Hz), 131.90, 143.16 (d, J.sub.CF=7.8 Hz),
152.60, 161.82 (d, J.sub.CF=245.9 Hz).
Example 16. 3'-(3-hydroxypropyl)-3,5-dimethyl-[1,1'-biphenyl]-4-ol
(47)
##STR00043##
[0320] The mixture of
4'-hydroxy-3',5'-dimethyl-[1,1'-biphenyl]-3-carbaldehyde (40 mg,
0.18 mmol), and malonic acid (19 mg, 0.18 mmol), 11 .mu.L of the
mixture of pyridine and piperidine (10:1, v/v) was heated up at
130.degree. C. for 3 hr. The reaction mixture was diluted with 1 mL
EtOAc and loaded onto a 1 mm SiO.sub.2 Preparative TLC place (20
cm.times.20 cm) and developed with 40% EtOAc in n-hexane solvent.
Rf 0.45 band was scraped off to afford
(E)-3-(4'-hydroxy-3',5'-dimethyl-[1,1'-biphenyl]-3-yl)acrylic acid
(35 mg) after extraction using a sintered glass filter and 20% MeOH
in DCM. Subsequently, reduction of the acid (20 mg, 0.07 mmol) with
LAH (8.5 mg, 0.22 mmol) in THF (1 mL) at the temperature of
-78.degree. C. for addition and then 0.degree. C. for a reaction,
and a typical workup with Rochelle salt, and separation with 45%
EtOAc in n-hexane afforded compound 47 (18 mg).
(E)-3-(4'-hydroxy-3',5'-dimethyl-[1,1'-biphenyl]-3-yl)acrylic
acid
##STR00044##
[0322] .sup.1H NMR (400 MHz, CDCl.sub.3+methanol-d.sub.4) .delta.
2.28 (s, 6H), 6.45 (d, 1H, J=16.0 Hz), 7.17 (s, 2H), 7.37 (t, 1H,
J=7.5 Hz), 7.41 (d, 1H, J=7.5), 7.51 (d, 1H, J=7.5 Hz); .sup.13C
NMR (100 MHz, CDCl.sub.3+methanol-d.sub.4) .delta. 16.41, 118.14,
124.12, 126.39, 126.71, 127.47, 129.01, 129.37, 132.39, 134.79,
142.06, 146.26, 152.62, 169.96.
##STR00045##
[0323] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.94 (quintet, 2H,
J=6.5 Hz), 2.31 (s, 6H), 2.76 (t, 2H, J=6.5 Hz), 3.70 (t, 2H, J=6.5
Hz), 7.12 (d, 1H, J=8.0 Hz), 7.21 (s, 2H), 7.31 (t, 1H, J=8.0 Hz),
7.36 (d, 1H, J=8.0); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta.
16.33, 32.41, 34.51, 62.55, 123.62, 124.59, 126.85, 127.15, 127.59,
128.92, 133.55, 141.47, 142.35, 152.13.
Example 17. 4'-(3-hydroxypropyl)-3,5-dimethyl-[1,1'-biphenyl]-4-ol
(48)
##STR00046##
[0325] Using the same method to make compound 47, compound 48 (13
mg) was prepared from
4'-hydroxy-3',5'-dimethyl-[1,1'-biphenyl]-4-carbaldehyde (40 mg,
0.18 mmol) and malonic acid (21 mg, 0.2 mmol) in the mixture of
pyridine and piperidine (10:1, v/v) and subsequent reduction using
LAH in THF (1 ml).
##STR00047##
[0326] .sup.1H NMR (400 MHz, CDCl.sub.3+methanol-d.sub.4) .delta.
2.23 (s, 6H), 6.36 (d, 1H, J=16.0 Hz), 7.23 (s, 2H), 7.50 (s, 4H),
7.64 (d, 1H, J=16.0 Hz).
##STR00048##
[0327] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.94 (quintet, 2H,
J=6.5 Hz), 2.30 (s, 6H), 2.75 (t, 2H, J=6.5 Hz), 3.72 (t, 2H, J=6.5
Hz), 7.22 (s, 2H), 7.25 (d, 2H, J=8.0 Hz), 7.47 (d, 2H, J=8.0);
.sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 16.28, 31.90, 34.46,
62.57, 123.49, 126.73, 126.80, 126.97, 127.46, 128.95, 139.01,
140.29, 151.92.
Example 18. 4-(2,3-dihydro-1H-inden-5-yl)-2,6-dimethylphenol
(13)
##STR00049##
[0329] Compound 14 (24 mg, 0.1 mmol) was dissolved into M (mL
containing cat. amount of 10% Pd/C and shaken under 30 PSI H.sub.2
in a Parr shaker at rt for 1 hr. Filtration through a sintered
glass filter and evaporation provided compound 13 (21 mg). .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. 2.109 (q, 2H, J=7.5 Hz), 2.31 (s,
6H), 2.92 (d, 2H, J=7.5 Hz), 2.96 (d, 2H, J=7.5 Hz), 4.62 (s, 1H,
Phenolic OH), 7.20 (s, 2H), 7.25 (d, 1H, J=8.5 Hz), 7.31 (d, 1H,
J=8.5 Hz), 7.40 (s, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta.
16.29, 25.79, 32.76, 33.13, 123.05, 123.25, 123.36, 127.61, 142.83,
144.98, 151.74, 154.50.
Example 19. 5-(3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol
(12)
##STR00050##
[0330]
5-(4-Trifluoromethansulfonyloxy-3,5-dimethylphenyl)-2,3-dihydro-1H--
inden-1-one
##STR00051##
[0332] Compound 6 (50 mg, 1.98 mmol) was reacted with p-nitrophenyl
trifluoromethansulfonate (58 mg, 2.14 mmol) over K.sub.2CO.sub.3
(27.6 mg, 0.2 mmol) in DMF (1 mL) at 55.degree. C. for 2 hr. The
reaction solution was poured into water (10 mL), extracted with
EtOAc (10 mL.times.3), washed with sat. NaHCO.sub.3 aqueous
solution, concentrated to load onto silica gel for column
chromatography. Elution with 30% EtOAc in n-hexane solvent afforded
5-(4-Trifluoromethansulfonyloxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden--
1-one (40 mg) as a colorless solid. (Rf=0.4).sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 2.46 (s, 6H), 2.78 (t, 2H, J=5.5 Hz), 3.22 (t,
2H, J=5.5 Hz), 7.36 (s, 2H), 7.56 (d, 1H, J=8.5 Hz), 7.65 (s, 1H),
7.83 (d, 1H, J=8.5 Hz); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta.
17.56, 26.19, 36.83, 118.85 (q, J.sub.CF=320.6), 124.56, 125.56,
127.13, 132.42, 136.33, 141.27, 146.65, 147.20, 156.59, 208.24;
.sup.19F NMR (470 MHz, CDCl.sub.3) .delta.-73.78.
5-(3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one
##STR00052##
[0334] The triflated compound (25 mg, 0.07 mmol) was dissolved into
MeOH. Et.sub.3N (20 .mu.L) and catalytic amount of 10% Pd/C were
added, and hydrogen (30 PSI) was applied. After shaking for 1 hr
using a Parr Reactor, the reaction mixture was passed through a
sintered glass filter to remove Pd/C and evaporated under vacuum to
provide 5-(3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one (15 mg).
This residue used without further purification. .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 2.40 (s, 6H), 2.74 (t, 2H, J=5.5 Hz), 3.19
(t, 2H, J=5.5 Hz), 7.06 (s, 1H), 7.25 (s, 2H), 7.59 (d, 1H, J=8.0
Hz), 7.66 (s, 1H), 7.81 (d, 1H, J=8.0 Hz); .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta. 21.64, 26.11, 36.77, 124.19, 125.34, 125.63,
127.04, 130.21, 136.08, 138.76, 140.43, 148.23, 156.05, 206.91.
5-(3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol (12)
##STR00053##
[0336] Subsequently, treatment of
5-(3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one (15 mg, 0.06
mmol) with NaBH.sub.4 (5 mg, 0.13 mmol) in THF-water (1:1, v/v, 1
mL), followed by extraction with EtOAc (5 mL.times.3), drying over
Na.sub.2SO.sub.4, and passing through silica gel pad afforded 12 mg
12 as colorless solid, after solvent evaporation. .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 1.97-2.05 (m, 1H), 2.39 (s, 6H), 2.51-2.58
(m, 1H), 2.85-2.91 (m, 1H), 3.09-3.15 (m, 1H), 5.30 (t, 1H, J=5.5
Hz), 7.01 (s, 1H), 7.21 (s, 2H), 7.47 (s, 3H); .sup.13C NMR (126
MHz, CDCl.sub.3) .delta. 21.66, 30.07, 36.43, 76.49, 123.95,
124.60, 125.44, 126.29, 129.13, 138.48, 141.57, 142.18, 144.17,
144.16.
Example 20.
2-fluoro-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol
(50)
##STR00054##
[0338] Compound 6 (40 mg, 0.16 mmol) was treated with Accufluor (55
mg, 1.74 mmol) in MeOH at reflux temperature for 4 hr. Once
starting material disappeared on SiO.sub.2 TLC analysis (10% EtOAc
in n-Hexane), the temperature of the reaction solution was cooled
down to rt and NaBH.sub.4 (10 mg, 0.26 mmol) was added to the
reaction mixture. The mixture was evaporated to load onto silica
gel for column chromatography. Elution with 20% EtOAc in n-Hexane
afforded 50 as a colorless solid (28 mg). .sup.1H NMR (500 MHz,
CDCl.sub.3+methanol-d.sub.4) .delta. 2.23 (s, 6H), 3.02-3.22 (m,
2H), 5.07 (dd, 1H, J.sub.H-H, J.sub.H-F=4.2, 17.9 Hz), 5.24 (ABXF,
1H, J, J, J=2.0, 4.4, 53.9 Hz), 7.12 (s, 2H), 7.35 (s, 1H), 7.40
(s, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 16.44, 36.77
(d, J=22.2 Hz), 75.86, 95.06 (d, J=182.5 Hz), 123.56, 124.39,
124.79, 126.27, 127.43, 132.91, 139.11, 139.70, 152.56.
Example 21. 4'-(2-hydroxyethyl)-3,5-dimethyl-[1,1'-biphenyl]-4-ol
(45)
##STR00055##
[0340] Compound 45 was synthesized according to the method
described in example 1, using 3,5-dimethyl-4-methoxyphenylboronic
acid (200 mg, 1.11 mmol) and 3-bromo-phenethylalcohol (201 mg, 1.00
mmol). The yield was comparable to that of compound 2.
[0341] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 2.31 (s, 6H), 2.90
(t, 2H, J=6.5 Hz), 3.90 (t, 2H, J=6.5 Hz), 7.21 (s, 2H), 7.27 (d,
2H, J=8.0 Hz), 7.49 (d, 2H, J=8.0); .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta. 16.33, 39.01, 63.94, 123.57, 127.16, 127.48,
129.57, 133.26, 136.87, 139.63, 152.04.
Example 22. 3'-(2-hydroxyethyl)-3,5-dimethyl-[1,1'-biphenyl]-4-ol
(44)
##STR00056##
[0343] Compound 44 was synthesize according to the methods
described at example 1, using 3,5-dimethyl-4-methoxyphenylboronic
acid (200 mg, 1.11 mmol) and 4-bromo-phenethylalcohol (201 mg, 1.00
mmol). The yield was comparable to that of compound 2. .sup.1H NMR
(500 MHz, CDCl.sub.3) .delta. 2.32 (s, 6H), 2.93 (t, 2H, J=6.5 Hz),
3.91 (t, 2H, J=6.5 Hz), 4.82 (s, 1H, Phenolic OH), 7.16 (d, 1H,
J=8.0 Hz), 7.22 (s, 2H), 7.35 (d, 2H, J=8.0 Hz), 7.41 (d, 2H,
J=8.0); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 16.33, 39.53,
63.99, 123.61, 125.22, 127.43, 127.62, 127.74, 129.15, 133.38,
138.99, 141.70, 152.18.
Example 23.
4-(1-methoxy-2,3-dihydro-1H-inden-5-yl)-2,6-dimethylphenol (22),
5-(4-hydroxy-3-methyl-5-methoxymethylphenyl)-2,3-dihydro-1H-inden-1-
-ol (64), and 4-(1H-inden-6-yl)-26-dimethylphenol (14)
##STR00057##
[0345] Compound 2 (400 mg) was treated with Activated charcoal
(Norit, 4 g) in MeOH for 50 min by frequently heating at boiling
temperature, followed by filtration, evaporation of filtrate to
afforded the mixture of 2, 22, 64, and 14.
4-(1-methoxy-2,3-dihydro-1H-inden-5-yl)-2,6-dimethylphenol (22)
[0346] Compound 22 (80 mg) was obtained from the mixture by
SiO.sub.2 column chromatography with the eluent of 25% EtOAc in
n-Hexane (rf=0.8). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.
2.17-2.23 (m, 1H), 2.34 (s, 6H), 2.38-2.47 (m, 1H), 2.88-2.94 (m,
1H), 3.15-3.21 (m, 1H), 3.49 (s, 3H), 4.91 (ABq, 1H, J=4.0, 6.5
Hz), 5.03 (s, 1H, phenolic OH), 7.25 (s, 2H), 7.43 (d, 1H, J=7.5
Hz), 7.47 (s, 1H), 7.48 (d, 1H, J=7.5 Hz); .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta. 16.37, 30.48, 32.32, 56.23, 84.64, 123.49,
123.76, 125.39, 125.49, 127.67, 133.68, 140.95, 141.93, 144.94,
152.14.
5-(5-Methoxymethyl-4-hydroxy-3-methylphenyl)-2,3-dihydro-1H-inden-1-ol
(64)
[0347] Compound 64 (10 mg) was collected from the reaction by using
HPLC column chromatography (Supelco semi prep Silica column, 10
.mu.m particle size, L.times.I.D. 25 cm.times.10 mm, 5% IPA in
n-Hexane, 4 ml/min, retention time: 11.79 min). .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 1.97-2.03 (m, 1H), 2.36 (s, 3H), 2.52-2.60
(m, 1H), 2.84-2.91 (m, 1H), 3.05-3.14 (mm, 1H), 3.46 (s, 3H), 4.73
(s, 2H, benzylic CH.sub.2), 5.29 (q, 1H, J=5.5 Hz), 7.09 (s, 1H),
7.33 (s, 1H), 7.41 (d, 1H, J=8.5 Hz), 7.42 (s, 1H), 7.45 (d, 1H,
J=8.5 Hz), 7.66 (s, 1H, Phenolic OH).
4-(1H-inden-6-yl)-2,6-dimethylphenol (14)
##STR00058##
[0348] Compound 14 (15 mg) was isolated from the mixture obtained
from the treatment of Activated charcoal, by chromatography using
the mixed solvent (25% EtOAc in Hexane, Rf=0.9). The mixture of
Compound 2 (35 mg, 0.14 mmol), cat. amount of p-toluene sulfonic
acid monohydrate in benzene was refluxed under a system equipped
with a Dean-Stark trap for 4 hr. After washing with sat.
NaHCO.sub.3 aqueous solution, evaporation of the solvent afford the
compound 14 (28 mg). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 2.33
(s, 6H), 3.47 (s, 2H), 7.06 (s, 1H), 7.25 (s, 2H), 6.57 (dt, 1H, J,
J=1.9, 5.5 Hz), 6.92 (d, 1H, J=5.5 Hz), 7.26 (s, 2H), 7.43 (d, 1H,
J=8.0 Hz), 7.47 (d, 1H, J=8.0 Hz), 7.67 (s, 1H); .sup.13C NMR (126
MHz, CDCl.sub.3) .delta. 16.34, 39.38, 121.19, 122.52, 123.51,
125.30, 127.66, 132.04, 134.17, 134.40, 137.99, 143.64, 144.57,
151.80.
Example 24. Synthesis of
(1S,3aR,5S,7aS)-5-(4-hydroxy-3,5-dimethylphenyl)-7a-methyloctahydro-1H-in-
den-1-ol (43)
##STR00059##
[0349]
(1S,7aS)-1-hydroxy-7a-methyl-1,2,3,6,7,7a-hexahydro-5H-inden-5-one
[0350] This compound was prepared by the reaction of
(S)-7a-methyl-2,3,7,7a-tetrahydro-1H-indene-1,5(6H)-dione (0.4 g,
2.43 mmol) with NaBH.sub.4 (23.0 mg, 0.62 mmol) by titrating MeOH
at -78.degree. C., according to the literature (E. J. Schweiger et
al, Tetrahedron letters, Vol. 38, No. 35, pp. 6127-6130, 1997).
(1S,7aS)-5-(4-methoxy-3,5-dimethylphenyl)-7a-methyl-2,3,7,7a-tetrahydro-1H-
-inden-1-ol
[0351] To the solution of 2,6-dimethyl-4-bromo-anisole (777 mg, 3.6
mmol) was added dropwise n-BuLi (1.6 M in n-hexane, 2.25 mL) at
-78.degree. C. This mixture was stirred for 1 hr before adding
dropwise a
(1S,7aS)-1-hydroxy-7a-methyl-1,2,3,6,7,7a-hexahydro-5H-inden-5-one
(150 mg, 0.90 mmol) solution in THF (5 mL) through a cannula needle
at -78.degree. C. The reaction mixture was stirred for 4 more hr at
rt, followed by addition of water (20 mL), extraction with ethyl
acetate (10 mL.times.3), drying over Na.sub.2SO.sub.4, and
concentration under vacuum to load onto SiO.sub.2 for column
chromatography. Elution with 20% ethyl acetate in n-hexane afforded
(1S,7aS)-5-(4-methoxy-3,5-dimethylphenyl)-7a-methyl-2,3,7,7a-tetrahydro-1-
H-inden-1-ol (130 mg). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.
1.02 (s, 3H), 1.48-1.55 (m, 1H), 2.02-2.08 (m, 1H), 2.31 (s, 6H),
2.41-2.48 (m, 1H), 2.58-2.68 (m, 2H), 3.74 (s, 3H), 4.08 (t, 1H,
J=7.5 Hz), 5.42 (s, 1H), 6.54 (d, 1H, J=1.5 Hz), 7.16 (s, 2H);
.sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 15.05, 16.50, 25.85,
34.27, 38.50, 44.92, 59.97, 82.21, 119.41, 119.59, 126.00, 130.81,
136.90, 137.81, 146.80, 156.72.
(1S,3aR,5S,7aS)-5-(4-hydroxy-3,5-dimethylphenyl)-7a-methyloctahydro-1H-ind-
en-1-ol
[0352] Hydrogen (30 PSI) in a Parr shaker was applied to a methanol
solution of
(1S,7aS)-5-(4-methoxy-3,5-dimethylphenyl)-7a-methyl-2,3,7,7a-tetrahydro-1-
H-inden-1-ol (110 mg, 0.39 mmol) containing a cat. amount of 10%
Pd/C for 2 hr at rt. The system was purged with nitrogen before
passage through a celite pad to remove Pd/C to afford title
compound (102 mg). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.92
(s, 3H), 1.07-1.03 (m, 1H), 1.05-1.75 (m, 7H), 1.83-1.85 (m, 1H),
2.09-2.15 (m, 1H), 2.28 (s, 6H), 2.36-2.44 (m, 2H), 3.72 (s, 3H),
3.78 (t, 1H, J=7.5 Hz), 6.87 (s, 2H); .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta. 10.69, 16.41, 25.70, 30.12, 30.71, 33.73,
37.18, 42.76, 44.52, 45.62, 59.92, 82.05, 127.42, 130.67, 142.79,
155.21.
[0353] After evaporating and drying the MeOH solvent, the residue
was re-dissolved into DCM (10 mL) to which was added a boron
trifluoride methyl sulfide (350 mg, 2.7 mmol) at rt, and the
reaction was continuously stirred for 6 hr before adding MeOH
dropwise at 0.degree. C., followed by extraction with ethyl acetate
(10 mL.times.3), drying over Na.sub.2SO.sub.4, and evaporation of
solvent under vacuum to afford the title compound (81 mg). .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. 0.87 (s, 3H), 0.91-0.94 (m, 1H),
1.00-1.05 (m, 1H), 1.38-1.78 (m, 8H), 1.95-1.97 (m, 1H), 2.03-2.09
(m, 1H), 2.23 (s, 6H), 2.35-2.42 (m, 1H), 3.74 (t, 1H, J=8.0 Hz),
6.85 (s, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 10.69,
16.25, 25.72, 30.32, 30.74, 33.90, 37.23, 42.77, 44.38, 45.69,
82.09, 123.04, 127.23, 139.27, 150.53.
Example 25.
2-Bromo-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one
(51) and
2,2-dibromo-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-o-
ne (52)
##STR00060##
[0355] The mixture of copper (II) bromide (110 mg, 0.49 mmol) and
5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one (40 mg,
0.16 mmol) in MeOH was refluxed until starting material disappeared
on SiO.sub.2 TLC analysis (20% EtOAc in n-Hexane). The reaction
mixture was concentrated under vacuum and re-dissolved into EtOAc
to load onto a Silica gel column. First and second fraction with
25% EtOAc in n-Hexane provided
2-bromo-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1--
one (45 mg) and
2,2-dibromo-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one
(62 mg), respectively, and subsequently treatment with NaBH.sub.4
in THF-water (1:1) afforded 51 (39 mg) (Rf=0.45) and 52 (56 mg)
(Rf=0.38), respectively, as well.
2,2-dibromo-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one
[0356] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 2.33 (s, 6H), 4.11
(s, 2H), 4.93 (s, 1H, phenolic OH), 7.28 (s, 2H), 7.52 (s, 1H),
7.66 (d, 1H, J=8.0 Hz), 7.94 (d, 1H, J=8.0 Hz); .sup.13C NMR (126
MHz, CDCl.sub.3) .delta. 16.32, 52.70, 57.62, 123.62, 124.06,
127.10, 127.15, 128.01, 128.07, 131.52, 148.05, 150.32, 153.65,
192.57.
2-bromo-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-one
[0357] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 2.34 (s, 6H), 3.45
(ABq. 1H, J, J=18.0 Hz, 3.0 Hz), 3.87 (ABq, 1H, J, J=18.0 Hz, 7.5
Hz), 4.70 (ABq, 1H, J, J=7.5, 3.5 HZ), 7.29 (s, 2H), 7.59 (s, 1H),
7.62 (d, 1H, J=8.5 Hz), 7.87 (d, 1H, J=8.5 Hz); .sup.13C NMR (126
MHz, CDCl.sub.3) .delta. 16.32, 38.30, 44.79, 124.01, 124.20,
125.62, 127.41, 128.03, 131.82, 131.88, 149.34, 152.10, 153.45.
2,2-dibromo-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol
(52)
[0358] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 2.32 (s, 6H), 3.95
(d, 1H, J=16.5 Hz), 4.10 (d, 1H, J=16.5 Hz), 4.70 (s, 1H, phenolic
OH), 5.20 (d, 1H, J=9.0 Hz), 7.21 (s, 2H), 7.40 (s, 1H), 7.46 (d,
1H, J=7.6 Hz), 7.50 (d, 1H, J=7.50 Hz).
2-bromo-5-(4-hydroxy-3,5-dimethylphenyl)-2,3-dihydro-1H-inden-1-ol
(51)
[0359] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 2.35 (s, 6H),
3.39-3.52 (m, 2H), 4.69 (s, 1H, phenolic OH), 4.94-4.96 (m, 1H),
5.01-5.04 (m, 1H), 7.23 (s, 2H), 7.22 (s, 1H), 7.48 (s, 2H).
Example 26.
5-(4-hydroxy-3-(hydroxymethyl)phenyl)-2,3-dihydro-1H-inden-1-ol (57
and
5-(4-hydroxy-3,5-(dihydroxymethyl)phenyl)-2,3-dihydro-1H-inden-1-ol
(58)
##STR00061##
[0361] Compound 32 (30 mg, 0.13 mmol) was suspended in DI water (1
mL). Formaldehyde solution 35 wt. % (105 .mu.l) and NaOH (20 mg,
0.5 mmol) were added to the suspension. This solution was
continuously stirred for 8 hr at 85.degree. C. 1 N HCl was titrated
to adjust pH at .about.5.5. Solution was extracted with EtOAc (5
mL.times.3). The organic layer was dried over Na.sub.2SO.sub.4,
concentrated under vacuum, and loaded onto a SiO.sub.2 prep. TLC
(0.1 mm) plate. Scraping off the Rf 0.55 and 0.45 bands and
extraction of each of these bands provided compound 57 (12 mg) and
58 (18 mg), respectively.
5-(4-hydroxy-3-(hydroxymethyl)phenyl)-2,3-dihydro-1H-inden-1-ol
(57)
[0362] .sup.1H NMR (500 MHz, CDCl.sub.3-CD.sub.3OD) .delta.
1.93-2.01 (m, 1H), 2.42-2.50 (m, 1H), 2.78-2.84 (m, 1H), 3.02-3.08
(m, 1H), 4.79 (s, 2H, benzylic CH.sub.2), 5.22 (t, 1H, J=5.5 Hz),
6.87 (d, 1H, J=8.0 Hz), 7.26 (s, 1H), 7.27-7.40 (m, 4H).
5-(4-hydroxy-3,5-(dihydroxymethyl)phenyl)-2,3-dihydro-1H-inden-1-ol
(58)
[0363] .sup.1H NMR (500 MHz, CDCl.sub.3-CD.sub.3OD) .delta.
1.84-1.95 (m, 1H), 2.36-2.2.42 (m, 1H), 2.72-2.79 (m, 1H),
2.98-3.02 (m, 1H), 4.72 (s, 4H, benzylic CH.sub.2), 5.17 (t, 1H,
J=5.5 Hz), 7.22 (s, 2H), 7.30 (d, 1H, J=8.0 Hz), 7.31 (s, 1H), 7.33
(d, 1H, J=8.0 Hz).
Example 27. 5-(4-hydroxy-3-methylphenyl)-2,3-dihydro-1H-inden-1-ol
(55) and
5-(4-hydroxy-3-(hydroxymethyl)-5-methylphenyl)-2,3-dihydro-1H-inden-1-
-ol (56)
##STR00062##
[0365] As described for the synthesis of compounds 57 and 58,
compound 56 (18 mg) was prepared by the reaction of compound 55 (24
mg, 0.1 mmol), formaldehyde wt. % solution (80 .mu.l), and NaOH (30
mg) in DI water at 85.degree. C.
[0366] Compound 55 was synthesized according to the method
described in example 1, using 3-methyl-4-methoxyphenylboronic acid
(199 mg, 1.20 mmol) and 5-bromo-2,3-dihydro-1H-inden-1-one (211 mg,
1.00 mmol). The yield was comparable to that of compound 2.
5-(4-hydroxy-3-methylphenyl)-2,3-dihydro-1H-inden-1-ol (55)
[0367] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.96-2.04 (m, 1H),
2.12 (s, 3H), 2.52-2.59 (m, 1H), 2.83-2.92 (m, 1H), 3.04-3.08 (m,
1H), 4.92 (s, 1H, phenolic OH), 5.24 (t, 1H, J=5.5 Hz), 6.82 (d,
1H, J=8.5 Hz), 7.32 (d, 1H, J=8.5 Hz), 7.35 (s, 1H), 7.41 (s, 2H),
7.43 (d, 1H, J=8.5 Hz).
5-(4-hydroxy-3-(hydroxymethyl)-5-methylphenyl)-2,3-dihydro-1H-inden-1-ol
(56)
[0368] .sup.1H NMR (500 MHz, CDCl.sub.3-CD.sub.3OD) .delta.
1.91-1.97 (m, 1H), 2.68 (s, 3H), 2.43-2.49 (m, 1H), 2.75-2.84 (m,
1H), 3.02-3.08 (m, 1H), 4.82 (s, 2H, benzylic CH.sub.2), 5.21 (t,
1H, J=5.5 Hz), 7.05 (s, 1H), 7.25 (s, 1H), 7.34 (d, 1H, J=8.5 Hz),
7.36 (s, 1H), 7.38 (d, 1H, J=8.5 Hz); .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta. 15.99, 30.00, 64.44, 76.00, 123.21, 124.14,
124.59, 124.87, 125.57, 125.84, 129.29, 132.81, 141.48, 143.51,
144.15, 154.21.
Example 28.
3-(hydroxymethyl)-3',5,5'-trimethyl-[1,1'-biphenyl]-4,4'-diol
##STR00063##
[0369]
3-(Hydroxymethyl)-3',5,5'-trimethyl-[1,1'-biphenyl]-4,4'-diol
(68)
[0370] Compound 68 was obtained from the reaction of methyl
4,4'-dihydroxy-3',5,5'-trimethyl-[1,1'-biphenyl]-3-carboxylate (60
mg, 0.21 mmol) with LAH (16 mg, 0.42 mmol) in dried diethyl ether
(5 mL) at 0.degree. C. and then rt for 2 hr, followed by quenching
with 10% NaOH aqueous solution, adjusting pH to 5.5, extracting
with EtOAc (5 mL.times.3) drying over Na.sub.2SO.sub.4, and
evaporating to afford compound 68 (12 mg). 68: .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 2.32 (s, 6H), 2.34 (s, 3H), 4.93 (s, 2H),
7.06 (s, 1H), 7.16 (s, 2H), 7.29 (s, 1H).
Methyl
4,4'-dimethoxy-3',5,5'-trimethyl-[1,1'-biphenyl]-3-carboxylate
##STR00064##
[0372] Methyl
4,4'-dimethoxy-3',5,5'-trimethyl-[1,1'-biphenyl]-3-carboxylate (295
mg) was obtained from the reaction as described method at example 1
with 3,5-dimethyl-4-methoxyphenylboronic acid (200 mg, 1.11 mmol)
and 4-bromo-phenethylalcohol (259 mg, 1.00 mmol). .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 2.38 (s, 6H), 2.40 (s, 3H), 3.79 (s, 3H),
3.89 (s, 3H), 3.97 (s, 3H), 7.24 (s, 2H), 7.54 (d, 1H, J=1.5 Hz),
7.84 (d, 1H, J=1.5 Hz); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta.
16.49, 52.49, 60.05, 61.84, 124.84, 127.64, 127.74, 131.45, 133.19,
133.76, 135.62, 136.62, 156.89, 157.70, 167.23.
Methyl
4,4'-dihydroxy-3',5,5'-trimethyl-[1,1'-biphenyl]-3-carboxylate
##STR00065##
[0374] Methyl
4,4'-dihydroxy-3',5,5'-trimethyl-[1,1'-biphenyl]-3-carboxylate (125
mg) was obtained from the reaction of methyl
4,4'-dimethoxy-3',5,5'-trimethyl-[1,1'-biphenyl]-3-carboxylate (157
mg, 0.05 mmol) with BF.sub.3--SMe.sub.2 (600 mg, 4.6 mmol) at rt in
DCM, as described in Example 1. .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 2.36 (s, 6H), 2.38 (s, 3H), 3.99 (s, 3H), 4.65 (brs, 1H),
7.18 (s, 2H), 7.53 (d, 1H, J=1.5 Hz), 7.83 (d, 1H, J=1.5 Hz);
[.sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 16.13, 16.30, 52.56,
111.93, 123.56, 125.29, 127.12, 129.99, 132.59, 135.32, 151.79,
159.21, 171.34.]
Example 29.
3'-(2-hydroxyethyl)-3,5-dimethyl-[1,1'-biphenyl]-4,4'-diol (69)
##STR00066##
[0376] As described for the synthesis of compound 68, compound 69
prepared from the Suzuki coupling reaction with
3,5-dimethyl-4-methoxyphenylboronic acid (200 mg, 1.11 mmol) and
methyl 2-(5-bromo-2-methoxyphenyl)acetate (259 mg, 1.00 mmol),
deprotection of the methyl groups on the phenols with
BF.sub.3--SMe.sub.2, and subsequent reduction of the methyl
carboxylate (65 mg, 0.23 mmol) to the alcohol with LAH to afford
compound 69 (43 mg). .sup.1H NMR (500 MHz, CDCl.sub.3-CD.sub.3OD)
.delta. 2.30 (s, 6H), 2.96 (t, 2H, J=6.5 Hz), 4.03 (t, 2H, J=6.5
Hz), 6.95 (d, 1H, J=10.5 Hz), 7.16 (s, 2H), 7.24 (d, 1H, J=2.5 Hz),
7.32 (dd, 1H, J, J=10.5 Hz, 2.5 Hz).
Example 30.
4-(((1r,3r)-adamantan-2-ylidene)(4-bromophenyl)methyl)-2,6-dimethylphenol
##STR00067##
[0378] The mixture of titanium(IV) chloride THF complex (1.0 g, 3.0
mmol) and zinc powder (290 mg, 6.15 mmol) in THF (50 ml) was
refluxed for 1.5 hr and cooled down to rt. To the mixture was added
the mixed solution of
(4-bromophenyl)(4-hydroxy-3,5-dimethylphenyl)methanone (308 mg,
1.01 mmol) and 2-adamantanone (150 mg, 1.00 mmol) at rt. This
mixture was refluxed under a hot oil bath for 4 hr before pouring
into the mixture of EtOAc (100 ml) and 10% K.sub.2CO.sub.3 aqueous
solution (200 ml) and stirred until the color of aqueous solution
turned to white. The organic layer was separated and washed aqueous
solution with EtOAc (50 mL.times.2) and combined into previously
collected organic solution, followed by dried over
Na.sub.2SO.sub.4, concentration under vacuum, and chromatography
with the Combiflash (Teledyne Co., LTD.). Gradient elution with
n-Hexane and EtOAc (n-Hexane:EtOAc, 100:0 to 90:10 over 30 min)
afforded the title compound (280 mg) as an off-white solid. .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. 61.87 (brs, 10H), 2.00 (s, 2H),
2.20, (s, 6H), 2.71 (S, 1H), 2.81 (s, 1H), 4.65 (brs, 1H, phenolic
OH), 6.72 (s, 2H), 7.01 (d, 2H, J=8.0 Hz), 7.38 (d, 2H, J=8.0 Hz);
.sup.13C NMR (126 MHz, CDCl.sub.3) 16.27, 28.39, 34.68, 37.36,
39.80, 119.94, 122.74, 129.49, 129.92, 131.25, 131.46, 134.68,
142.56, 146.91. 150.93.
Example 31.
4-((4-Bromophenyl)(cyclohexylidene)methyl)-2,6-dimethylphenol
##STR00068##
[0380] Title compound (235 mg) was obtained from the reaction of
(4-bromophenyl)(4-hydroxy-3,5-dimethylphenyl)methanone (308 mg,
1.01 mmol), cyclohexanone (98 mg, 1.00 mmol), Titanium(IV) chloride
THF complex (1.0 g, 3.0 mmol), and zinc powder (290 mg, 6.15 mmol)
in THF (50 mL) as described above. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 61.60 (brs, 6H), 2.20, (s, 6H), 2.24 (t, 4H,
J=6.5 Hz), 4.59 (brs, 1H, phenolic OH), 6.70 (s, 2H), 6.99 (d, 2H,
J=8.0 Hz), 7.39 (d, 2H, J=8.0 Hz); .sup.13C NMR (126 MHz,
CDCl.sub.3) 16.20, 27.01, 28.86, 32.64, 32.75, 120.02, 122.63,
130.20, 131.18, 131.71, 133.32, 134.78, 139.42, 142.68, 150.91.
Example 32: Pathway Preferential Estrogen, PaPE-1, has Impeded
Estrogen Receptor Binding and Coactivator Interactions
[0381] The relative binding affinity (RBA) values of E2 and PaPE-1
(FIG. 1) were obtained by a competitive radiometric binding assay
using purified full-length human ER.alpha. and ER (Carlson et. al.,
Biochemistry 36, 14897-14905, 1997). The RBAs for E2 are defined as
100, and relative to E2, PaPE-1 binds 50,000-fold less well to
ER.alpha. and ERO. The equivalent K.sub.D values are 10 and 25
.mu.M for PaPE-1 on ER.alpha. and ERO, respectively, compared to
the sub-nanomolar K.sub.D values for E2 (FIG. 10A). Thus, the ER
binding affinities of PaPE-1 for both ERs was lowered while
preserving as much as possible the physical and functional group
attributes of E2.
[0382] Using a time-resolved Forster resonance energy transfer
(tr-FRET) assay, the binding of the steroid receptor coactivator 3
(SRC3) to ER.alpha. was monitored. (Jeyakumar et. al. J Biol Chem,
286, 12971-12982, 2011, Jeyakumar et. al., Anal Biochem 386, 73-78,
2009). In coactivator titration assays (FIG. 10B), SRC3 bound with
high affinity to ER.alpha. complexes with E2, but showed no binding
to ER.alpha. complexes with PaPE-1. Notably, however, the
antagonist trans-hydroxytamoxifen (OH-Tam) reversed the ER-SRC3
interaction promoted by E2; PaPE-1 also reversed the ER-SRC3
interaction, but only at much higher concentrations, commensurate
with its lower ER.alpha. binding affinity (FIG. 10C). The
E2-elicited interaction of ER.alpha. with SRC3 was also observed by
coimmunoprecipitation of the complexes from MCF-7 cells, whereas no
coimmunoprecipitated complexes were observed for ER.alpha. and SRC3
after exposure to PaPE-1 (FIG. 10D).
Example 33: PaPE-1 Regulates a Subset of Estrogen-Modulated Genes
and Activates Kinases, but does not Stimulate Breast Cancer Cell
Proliferation
[0383] PaPE-1 was selective in activating non-genomic genes, as
shown by LRRC54 stimulation, but was essentially without activity
on the direct ER gene target PgR (FIG. 3A). Activation of LRRC54
gene expression by PaPE-1 was blocked by treatment with the
antiestrogen (Fulvestrant) ICI 182,780 (FIG. 3B) and by knockdown
of ER.alpha. (FIG. 3C). By contrast, knockdown of GPR30 did not
have any effect on the gene stimulation (FIG. 3C). PaPE-1 also did
not stimulate proliferation of MCF-7 cells, whereas E2 potently
stimulated proliferation (FIG. 3D).
[0384] When activation of major signaling pathways in MCF-7 cells
was monitored, it was observed that PaPE-1 was very efficient in
activating mTOR and MAPK signaling (FIG. 3E), as seen by increased
pP70S6K and p4EBP1 associated with mTORC1 activation, increased
pSGK1 associated with mTORC2 activation, and increased pMAPK.
Elevations of pAKT and pSREBP1 were also more notable with PaPE-1
compared with E2. However, PaPE-1 did not induce any detectable
Ser18 phosphorylation of ER.alpha., which was observed with E2
(FIG. 3E) and is associated with mitogenic activity of
ER.alpha..
[0385] The mTOR pathway is the major signaling system that senses
the nutrient state of the environment and modulates metabolic
functions in the cell. mTOR kinase is present in two distinct
complexes, mTORC1 and mTORC2, which are distinguished by the
presence of RAPTOR and RICTOR scaffolding proteins, respectively.
It is thought that mTORC1 primarily modulates cell metabolism,
whereas mTORC2 is principally involved in regulating the
cytoskeleton and cell proliferation. To further characterize the
mTOR activation by PaPE-1, proximity ligation assays (PLA) in MCF-7
cells were performed and it was found that ER.alpha. interacted
with RAPTOR to a greater extent in the presence of PaPE-1 than E2
(FIG. 3F). No interaction was detected between ER.alpha. and mTOR,
or ER.alpha. and proline-rich Akt substrate of 40 kDa (PRAS40),
which is important in Akt and mTOR signaling, suggesting that
ER.alpha. modulated the mTOR pathway through direct interactions
with Raptor (FIG. 11).
Example 34: PaPE-1 Regulates Metabolism-Related Genes in an mTOR
and MAPK Activity-Dependent Manner, but does not Cause Recruitment
of ER.alpha. to Chromatin
[0386] RNA-Seq analysis was performed to compare genes that were
changed in their expression level by PaPE-1 and/or E2. MCF-7 cells
were treated with 10 nM E2 or 1 .mu.M PaPE-1 for 4 h and 24 h, and
compared genes regulated by each compound at these two time points
(FIG. 4A). At both times, E2 regulated more genes than did PaPE,
and the magnitude of gene regulation by E2 was generally higher
than that by PaPE. At 4 h, E2 regulated nearly 1500 genes, whereas
PaPE-1 modulated expression of only 500 genes (FIG. 4A upper Venn
diagram). At 24 h, about 3000 genes were regulated by E2, whereas
PaPE-1 regulated 2200 genes (FIG. 4A lower Venn diagram). At both 4
h and 24 h, three quarters of the genes regulated by PaPE-1 were
also targets of E2. Notably, only E2 upregulated mitosis genes and
downregulated apoptosis genes at 24 h, consistent with the
observation that PaPE-1 did not stimulate MCF-7 cell
proliferation.
[0387] Pathway-selective inhibitors were used to assess the effect
of mTOR pathway or MAPK pathway activation on PaPE- and E2-mediated
gene regulation. The mTOR pathway inhibitor PP242 blocked the
regulation of 60% of PaPE-regulated genes, whereas only 40% of E2
regulated genes were affected by this inhibitor. Similarly, the
MAPK inhibitor AZD6244 blocked almost 50% of PaPE-1 regulated
genes, whereas only 23% of E2 regulated genes were blocked by the
same inhibitor (FIG. 4B). Examination of enriched gene ontology
(GO) functions revealed that these inhibitors blocked E2 regulation
of cell migration, immune, cell cycle and angiogenesis related
genes, whereas they blocked PaPE-1 regulation of genes involved in
nucleotide metabolism, inflammatory response, ncRNA processing,
amino acid transport, and glycoprotein metabolism.
[0388] To investigate the roles of ER.alpha., ERK2 and active RNA
Pol II recruitment in PaPE-1-mediated transcriptional events,
ChIP-Seq analysis was performed. ChIP-Seq (FIG. 4C, revealed that
ER.alpha. was recruited to chromatin upon E2 treatment of cells,
whereas ER.alpha. was not recruited to chromatin in the presence of
PaPE-1. PaPE-1 induced recruitment of ERK2 to proximal promoter
regions of genes, whereas E2 caused recruitment of ERK2 to enhancer
regions together with ER.alpha.. Distinct RNA Pol II recruitment
sites were observed with PaPE-1, further suggesting that PaPE-1
induces transcriptional events through recruitment of RNA Pol II
without affecting ER.alpha. or ERK2 recruitment to enhancer regions
of target genes. These distinct patterns of ER.alpha., ERK2, and
pSer5 RNA polymerase II binding after cell treatment with Veh, E2,
and PaPE-1 are shown for the TFF1/pS2 gene and LRRC54/TSKU gene in
FIG. 4C, right.
[0389] The in vivo biological activities of PaPE-1 were
characterized in ovariectomized female mice. As is known with E2,
PaPE-1 was cleared rapidly after subcutaneous injection (t/2 ca. 1
h, FIG. 12A); so, to provide more prolonged exposure for in vivo
studies, administration by ALZET minipump or by a pellet containing
PaPE-1 was used, both implanted subcutaneously (FIG. 12B).
[0390] PaPE-1 is a tissue-specific modulator of ER and mTOR
signaling, with preferential estrogen-like activity in
non-reproductive (metabolic and vascular) tissues. E2-like effects
of PaPE-1 were seen on body weight, liver, adipose tissues, and
vasculature vs absence of E2-like effects of PaPE-1 on uterus,
thymus and mammary gland growth. Mice at 8 weeks of age were
ovariectomized, and after 2 weeks, they were treated with control
Veh, E2 or PaPE-1 for 3 weeks. While E2 was very effective in
increasing growth of the uterus, PaPE-1 did not exhibit any
stimulatory effects on uterine weight (FIG. 5A). As is well known,
it was observed that E2 induced marked involution of the thymus,
but PaPE-1, even at a high dose, had no effect on thymus size (FIG.
5A). Likewise, PaPE-1 did not change mammary gland ductal
morphology from that of vehicle control mice, whereas E2 elicited
marked ductal growth (elongation and branching) (FIG. 5B). Of
interest, however, both E2 and PaPE-1 reduced mammary gland
adiposity that develops in mice after ovariectomy. This was
associated with reduced adipocyte area after E2 or PaPE-1 treatment
(FIG. 5C).
[0391] Effects of PaPE-1 and E2 were seen on body weight and
metabolic effects in liver and adipose tissues. Body weights of the
mice increased after ovariectomy, as is well-known, and this
increase in weight was suppressed by PaPE-1 and by E2 over the
3-week period monitored (FIG. 6A). Body weights were statistically
lower in E2- and PaPE-1-treated mice vs. Veh control treated
animals (two-way ANOVA with Bonferroni post-test, E2 p<0.05;
PaPE-1 p<0.01), and the PaPE-1 and E2 groups were not
statistically different from one another. The reduction in body
weight gain that was observed with PaPE-1 was not due to a change
in food consumption (FIG. 6B). Because of the known anorexigenic
effect of E2, there was an early drop in food intake in E2-treated
animals; however, this was not observed in PaPE-1-treated animals,
suggesting that this was not a determinant of differing body
weights and changes in the fat depots (FIG. 6B).
[0392] A major impact of PaPE-1 was on fat depots of the animals,
as observed in EchoMRI measurements. Both E2 and PaPE-1 greatly
reduced fat mass, but total lean mass and water mass were increased
only by E2 and were not affected by PaPE-1, further indicating that
PaPE-1 worked by changing overall adiposity rather than lean mass
of the animals (FIG. 6C). H&E and Oil Red O staining of
perigonadal adipose tissue (FIG. 6D) highlighted the similar and
marked estrogenic effects of both PaPE-1 and E2 on this tissue.
PaPE-1 and E2 decreased the weight of all of the fat depots
examined (FIG. 6E). Perigonadal, perirenal, and subcutaneous
adipose tissues were reduced by both ligands, but to a larger
extent by E2 at the dosages tested. Mesenteric adipose tissue was
similarly reduced by both ligands, and both ligands also decreased
triglyceride levels in blood (FIG. 6F).
[0393] A very dramatic liver phenotype was observed when
histological changes in the liver were monitored. Removal of
estrogens by OVX increased lipid droplet accumulation, and E2 or
PaPE-1 decreased hepatocyte lipid content, as observed in Oil Red O
staining of liver sections (FIG. 6G). Changes in fatty acid
synthesis pathways were verified by monitoring two biomarkers of
hepatic steatosis, FASN and SREBP-1c, and found that the level of
expression of these genes was reduced following 3 weeks of E2 or
PaPE-1 treatment (FIG. 6H). Expression of SREBP-1 transcriptional
targets, FASN and the gene encoding the enzyme acetyl-CoA
carboxylase alpha (ACACA), which catalyzes the carboxylation of
acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid
synthesis, was decreased by 24 h of PaPE-1 or E2 treatment, and
remained low over the 2 weeks monitored (FIG. 6H).
[0394] Gene expression and signaling pathway activations by PaPE-1
vs. E2 were studied in multiple tissues in vivo. PaPE-1 was found
to induce tissue-specific molecular changes in gene expression and
signaling pathway activation patterns. To characterize these
changes in the various tissues harvested from animals in long-term
studies, an analysis of the expression of genes reported in the
literature to be altered in liver, skeletal muscle, perigonadal
fat, pancreas, and uterus was performed. PaPE-1 and E2 generated
quite similar profiles in metabolic tissues, namely in liver and
skeletal muscle; in stark contrast, only E2 induced gene expression
changes in uterus (FIG. 7A), consistent with the selective
stimulatory actions of PaPE-1 in non-reproductive tissues.
[0395] To gain more mechanistic insight, the signaling pathways
activated by E2 and PaPE-1 were profiled in different tissues (FIG.
7B). E2 and PaPE-1 had similar pathway effects in non-reproductive
tissues, whereas E2 and PaPE-1 had contrasting effects in
reproductive tissues (uterus and mammary gland), with only E2 being
stimulatory. Furthermore, in metabolic tissues like liver and
skeletal muscle, both E2 and PaPE-1 were highly effective in
inducing mTOR signaling, as observed by S6 phosphorylation.
[0396] PaPE-1 activity is lost in ER.alpha.-knockout mice. A select
number of effects of E2 and PaPE-1 on physiologic processes and
gene expression were compared in wild type and ER.alpha.-knockout
mice (ERKO) (FIG. 13). OVX mice were treated with E2 or PaPE-1 for
3 weeks, and as shown in FIG. 13A, the ability of E2 or PaPE-1 to
diminish the body weight gain after ovariectomy was lost in the
ERKO mice. E2 stimulation of uterine growth was, as expected, not
observed in ERKO mice (FIG. 13B), nor was there a decrease in blood
triglycerides after E2 or PaPE-1 (FIG. 13C). FIG. 13D shows that
ER.alpha. is a mediator of the suppressive effect of PaPE-1 or E2
on FASN and SREBPc genes seen in the liver of wild type mice that
was not observed in ERKO mice.
[0397] The vascular effects of E2 and PaPE-1 were studied.
Estrogens have potential beneficial actions on vascular cells, as
exemplified by in vivo studies of carotid artery
reendothelialization following perivascular electric injury in
female mice. Following ovariectomy, mice were treated with vehicle,
E2 or PaPE-1 for 18 days, at which time E2 and PaPE-1-treated mice
were also administered a single dose of vehicle or the antiestrogen
ICI 182,780. Three days later, carotid artery denudation was
performed, and the mice received a second dose of vehicle or ICI
while E2 or PaPE-1 was continued, and 72 h after denudation Evans
blue dye was administered systemically to assess the remaining area
of denudation. FIG. 8A, upper panel, shows Evans blue dye uptake by
the intimal surface of the carotid artery. E2 and PaPE-1 caused
similar marked endothelial repair, as indicated by the minimal area
of remaining denudation, and the responses elicited by E2 and
PaPE-1 were fully prevented by the antiestrogen ICI 182,780 (ICI,
Fulvestrant). Summary data for the different treatment groups are
shown in FIG. 8A, lower panel. In the same animals, uterine weight,
which was greatly elevated by E2, was unaffected by PaPE-1, and as
expected, the antiestrogen ICI blocked uterine stimulation by E2
(FIG. 8B).
[0398] To evaluate direct effects on endothelium, endothelial NO
synthase (eNOS) activation was assessed by measuring the conversion
of .sup.14C-L-arginine to .sup.14C-L-citrulline by intact primary
bovine endothelial cells in culture. eNOS was activated by E2, as
previously observed (Chambliss, J Clin Invest 120, 2319-2330,
2010), and there was a comparable response to PaPE-1; the effects
of both E2 and PaPE-1 were fully attenuated by ICI (FIG. 8C).
Example 35: Characterization of Additional PaPEs
[0399] To further exemplify compounds that preferentially activate
ER non-genomic signaling, results from PaPE-2 and PaPE-3 are
presented, two ligands that are structurally related to PaPE-1 by
being altered forms of the steroidal ligand, estradiol, but having
variations in the structure of what was originally the estradiol
D-ring, namely ring cleavage in PaPE-2 and ring enlargement in
PaPE-3 (FIG. 1). To further diversify the structures of PaPEs, an
additional ligand, PaPE-4 was also studied; this PaPE is derived
from a non-steroidal estrogen, bisphenol A (BPA), and it retains
the core bisphenol structure of BPA but has been modified to have
reduced ER binding affinity by replacing one of the methyl groups
with a polar bis-amide substituent. The ER binding and physical
properties of PaPE-2, -3, and -4 are given in FIG. 1.
[0400] All of the PaPEs showed similar biological activities in
vitro and in vivo. They all caused preferential non-genomic vs.
genomic gene stimulation (LRRC54>PgR) compared to E2, which
stimulated expression of both genes very well (FIG. 9A). In
contrast to E2, none of the PaPEs stimulated proliferation of MCF-7
cells over a broad concentration range tested (FIG. 9B). All of the
PaPEs increased activation of MAPK, mTOR and AKT signaling
pathways, as monitored by pMAPK, pP70S6 and pSREBP1, and pAKT in
these cells (FIGS. 9C and 9D). In endothelial cells, PaPE-2, -3,
and -4, like E2, also increased NOS activity (FIG. 9E), as was seen
with PaPE-1 (FIG. 8C), and the NOS stimulation was fully blocked
with the antiestrogen, ICI. In vivo, the four PaPEs and E2 reduced
body weight gain after ovariectomy (FIG. 9F), with no change in
food consumption (FIG. 9G). The four PaPEs also did not elicit any
increase in uterine weight, which, in contrast, was markedly
increased by E2 (FIG. 9H). The reduction in body weight with the
four PaPEs was largely due to a change in body fat mass, with
little or no change in lean mass or water mass, as monitored by
EchoMRI. E2 reduced fat mass more markedly, and did increase lean
mass and water mass (FIG. 9I). These differential effects may
account for the fact that the body weight of E2-treated animals
matched that of the PaPE-treated animals.
Example 36: Investigation of ER Ligand Binding Dynamics Couple with
Signal Transduction Pathways
[0401] A computational model showing structural details of the
accommodation of PaPE-1 in the ligand-binding pocket of ER.alpha.
in comparison to E2 is presented in FIG. 2. Helices 3 and 6
constrict the A-ring end of the binding pocket, and E2
(silver/gray) forms hydrogen bonds to both GLU353 and ARG394,
further stabilized by a crystallographic water (red dot) bridging
GLU and ARG. By contrast, the ortho-methyl groups of PaPE-1 (in
yellow) introduce steric clashes with H3 and H6, inducing a
significant shift in ligand positioning. While the A-ring OH of
PaPE1 maintains a hydrogen bond to GLU353 and the crystallographic
water still bridges between GLU and ARG, direct ligand contact to
ARG394 is broken (ER structure in yellow). At the D-ring end of the
pocket, there is also a subtle shift in the positioning of HIS524.
Overall, it appears that the reduced volume of the PaPE-1 ligand
and the increased planarity due to the aromatic C-ring mimetic
result in fewer van der Waals contacts throughout the central
region of the pocket. All of these changes with respect to the
ER-E2 complex are consistent with the greatly lower binding
affinity of PaPE-1. Nevertheless, the overall shape of the
ligand-binding pocket is not altered in a major way by the binding
of PaPE-1, suggesting that PaPE-1 can still form a structurally
competent, though short-lived complex with ER.
[0402] The dissociation rates of PaPE-1 and E2 were measured for
ER.alpha. (FIGS. 10E and F), and they differ by nearly 2000 fold;
the half-life of the ER-E2 complex is nearly 30 hours, whereas that
of the PaPE-1 complex is less than 1 minute.
Example 37. Activation of Non-Genomic Genes by PaPEs
[0403] Compounds that have cause preferential activation of the
LRRC54 gene to a higher level than the PgR gene are considered to
have a preference for activation of the extranuclear-initiated or
non-genomic pathway. In each case, this preference is apparent from
the histograms showing the fold change in stimulation of the two
genes as well as from the star or radar plots, where the activation
of LRRC54 is plotted as points on a blue curve and the activation
of PgR is plotted as points on a red curve. (FIGS. 14-33).
[0404] In the histograms, a compound shows pathway preference for
activation of non-genomic genes when the fold activation of the
non-genomic gene (LRRC54) is higher relative to the activation by
estradiol (E2) compared to the activation of the genomic gene (PgR)
relative to the activation by estradiol (E2). On the star or radar
plots, preferential activation of the non-genomic gene is evident
when the point on the blue curve extends further from the center of
the plot than the point on the red curve.
[0405] Examples of compounds showing clear evidence of preference
for activation of the non-genomic pathway are: Compounds 2, 3, 4;
15, 16, 18; 24, 26, 29-30; 32; 41; 44-47; 56-58; 59-60; 63-64. At
the appropriate concentration (picomolar), compound 40 also shows
preference for the non-genomic pathway (FIG. 25).
[0406] Examples of compounds showing little or no preference for
activation of the non-genomic pathway are: Compounds 5-14; 17,
19-22; 23, 25, 27-28; 31-35; 36-40, 42-43; 48-49; 50-55;
61,62,65-67.
[0407] The list of ER.alpha. and ER.beta. RBA value for selected
compounds. RBA (relative binding affinity) values are a measure of
binding affinity relative to that of estradiol, which has an RBA
values of 100.
TABLE-US-00001 Compound ER.alpha. ER.beta. 2 0.001 .+-. 0.001 0.002
.+-. 0.001 8 0.001 .+-. 0 0.006 .+-. 0.001 9 0.003 .+-. 0.001 0.002
.+-. 0.001 10 0.008 .+-. 0.002 <0.001 12 0.001 .+-. 0 0.002 .+-.
0.001 15 0.002 .+-. 0.001 0.005 .+-. 0.003 16 0.002 .+-. 0.001
0.004 .+-. 0.003 17 0.003 .+-. 0.001 0.002 .+-. 0.001 19 0.004 .+-.
0.001 <0.001 20 <0.001 <0.001 21 0.019 .+-. 0.004 0.012
.+-. 0.002 23 0.002 .+-. 0.001 0.002 .+-. 0.001 24 0.005 .+-. 0.001
0.004 .+-. 0 25 0.003 .+-. 0 0.009 .+-. 0 26 <0.001 0.002 .+-.
0.001 27 0.003 .+-. 0.001 0.003 .+-. 0 28 0.008 .+-. 0.002 0.013
.+-. 0.002 30 0.002 .+-. 0.001 0.002 .+-. 0.001 32 0.005 .+-. 0.001
0.190 .+-. 0.04 35 0.011 .+-. 0.003 0.755 .+-. 0.20 40 0.302 .+-.
0.04 0.058 .+-. 0.01 41 0.004 .+-. 0.001 0.002 .+-. 0 42 0.004 .+-.
0 0.002 .+-. 0.001 43 <0.001 0.001 44 0.001 0.001 45 0.001 0.002
55 0.002 0.004 56 <0.001 <0.001 60 <0.001 ~0.001 63 0.005
0.006
Example 38. Effect of PaPe-1 on Breast Cancer Cells
[0408] Identification of Factors from Obese Postmenopausal Women
that Drive Breast Cancer Cell Proliferation and Aggressiveness
[0409] To identify factors from serum that are associated with
obesity, a total of 100 samples (37 non-obese, 63 obese-subjects)
was used from the original study named "Midlife Health Study".
Serum was collected from women who are residents of the Baltimore
metropolitan region, which includes Baltimore City and several of
its surrounding counties. BMI>25 was used as the cut-off to
categorize an individual as overweight and obese. All the subjects
were 2-3 years postmenopausal women. Whole metabolite profiling and
OLINK biomarker profiling was performed for inflammation, CVD and
oncology panels of these 100 samples (FIGS. 34A and B). These
analysis showed that serum metabolite composition and protein
composition to some extent stratified the samples based on the BMI
of the subjects. Since breast cancer incidence follow-up for this
study was not available, the serum from each individual was used to
derive a proliferation index in ER.alpha.(+) MCF-7 and T47D cells
and ER.alpha.(-) MDA-MB-231 cells (FIG. 34C). Cell proliferation
was increased when ER.alpha.(+) but not ER.alpha.(-) cells were
treated with serum from obese individuals. There was a
statistically significant linear correlation between in vitro cell
proliferation index and serum donor's BMI but not with estradiol,
testosterone or progesterone levels (FIGS. 35A and B). A motility
index was used for BT474 cells and mTOR pathway activation in MCF-7
cells as surrogates for measuring breast cancer aggressiveness
(FIG. 34D). Since the original sample set is from cancer-free
women, the levels of several well-known cancer serum biomarkers
were measured to verify the ability of any biomarker identified of
predicting the breast cancer outcome. For example, lower glycerol,
phenylalanine, glutamate and isoleucine levels were found in the
blood from breast cancer patients compared to healthy counterparts.
Those serum samples associated with a higher proliferation index
had a lower level of these metabolites compared with those samples
associated with a lower proliferation index (FIG. 35C). Thus, the
evaluation of cell proliferation, motility, and mTOR pathway
activation was informative regarding breast cancer outcomes and was
used to identify biomarkers that are associated with increased
breast cancer aggressiveness.
[0410] Next, a correlation analysis was perforrmed between the
serum metabolite and biomarker levels and the cell proliferation
index from ER.alpha.(+) cells. A heatmap was used to visualize
correlation coefficients of metabolites and protein biomarkers that
correlated with MCF-7 proliferation with a p-val<0.05. This
analysis showed that several free fatty acids (FFAs), including
oleic acid/9-octadecenoic acid (OA) and palmitic acid (hexadecanoic
acid), leptin (LEP), cytokines CCL3 and IL-20, were positively
correlated with MCF-7 and T47D cell proliferation (FIGS. 34E and
F).
Serum-Induced Proliferation of ER.alpha.(+) Breast Cancer Cells is
Associated with FFAs
[0411] To identify particular FFAs that might increase ER.alpha.
(+) cell proliferation, the same serum samples were profiled for
free fatty acids (FIGS. 36A and 2B), and verified that those
samples that caused higher proliferation in breast cancer cells had
higher levels of all FFAs analyzed. In addition, metabolite
profiling was performed in a subset of samples from the Midlife
health study, which included samples from 21 postmenopausal women
who were obese at the beginning of the study that later lost weight
and were non-obese by the end of the study. Initial samples
(labeled with thick, red lines in the hierarchical tree) and final
serum samples (labeled with black in the hierarchical tree) were
profiled from these individuals and observed a reduction in FFAs
levels after weight loss (FIG. 36C). Finally, A sample set obtained
from Komen Tissue Bank, which included samples from postmenopausal
women who donated blood when they were cancer-free and later had a
diagnosis of breast cancer was used. Serum samples from age, BMI
and race matched control samples were also used. This analysis
showed that obese postmenopausal women who developed breast cancer
had higher levels of FFAs in their serum as compared to the healthy
controls (FIG. 36D). A follow-up screen was performed by testing
the ability of each FFA to affect the proliferation of the ER (+)
MCF-7 cell line. (FIG. 36E). It was found that unsaturated fatty
acids and particularly OA was very efficient in increasing cell
proliferation in MCF-7 cells. By contrast, the other FFAs were not
as effective as OA in inducing cell proliferation (FIG. 36E).
OA Increases Breast Cancer Cell Proliferation by Activating
ER.alpha. and mTOR Signaling
[0412] In order to verify OA as active component of FFA mixture in
the serum abe to induce cell proliferation, an in vitro
proliferation assay was performed and the assay validated that the
high level of OA is significantly associated with higher
proliferation index in breast cancer cells (FIG. 37A). Since it was
found that sera from obese individuals also increases mTOR pathway
activation (FIG. 34C), a time course experiment was performed using
OA, and it was found that OA treatment caused a robust activation
of mTOR pathway downstream targets Akt, S6 and 4EBP1 (FIG. 37B).
ERK1/ERK2 was also activated but this activation was earlier and
transient (FIG. 37B). Next, PaPE-1 was used and showed that PaPE-1
modulated ER.alpha.-mTOR signaling crosstalk, to see if OA worked
through ER.alpha.-mTOR signaling. Treatment of the cells with
PaPE-1 decreased OA-induced mTOR pathway activation (FIG. 37C) and
OA-induced cell proliferation (FIG. 37D). To further confirm that
OA-induced cell proliferation was through ER.alpha. and mTOR
pathways, OA treatments were performed in the presence of
4-OH-Tamoxifen (R--OH-Tam), Fulvestrant (Fulv), an ER.alpha.
antagonist, and RAD001, an mTOR pathway inhibitor. All of the
tested agents blocked OA-induced cell proliferation (FIG. 37E). To
ensure that serum from obese individuals also induced MCF-7 cells
proliferation through ER.alpha. and mTOR pathways, cell
proliferation assays were performed in the presence of 4-OH-Tam,
Fulv and PaPE-1. Notably, PaPE-1 was the most effective agent in
inhibiting serum-induced proliferation of MCF-7 cells (FIG. 37F
left panel). However, in normal cell culture conditions, 4-OH-Tam
and Fulv were better agents (FIG. 37F right panel), suggesting that
treatment with the serum from obese individuals makes MCF-7 cells
more susceptible to growth inhibitory effects of PaPE-1. Finally,
to ensure that the high level of OA was independent from the diet
but associated only with obesity status, both wild-type mice, that
were either on normal or high-fat diet, and leptin-deficient
(ob/ob) mice on normal diet were ovariectomized, and subcutaneously
implanted with Alzet minipumps that delivered Veh or PaPE-1 over 6
weeks. PaPE-1 treatment reduced ovariectomy-induced weight gain
without changing food intake. Ovariectomy-associated decrease in
estrogen level causes an increase in OA level in serum in the
ovariectomized animals compared to the intact animals (FIG. 37G,
left panel). In all three models, treatment with PaPE-1 decreased
serum levels of OA, suggesting that in addition to direct effects
through ER.alpha.-mTOR signaling in breast cancers cells, PaPE-1
might prevent obesity-associated breast cancer by reducing serum
level of OA systemically (FIG. 37G).
OA Induces Gene Expression Changes in Breast Cancer Cells that are
Blocked by PaPE-1
[0413] To identify gene expression changes associated with
OA-induced ER.alpha. and mTOR pathway modulation, MCF-7 cells were
treated with vehicle (Ctrl), 100 nM OA or 100 nM OA in the presence
of 1 .mu.M PaPE-1 (FIG. 38A). OA upregulated about 350 genes, and
activation of 80% of these upregulated genes was blocked by PaPE-1.
On the other hand, about 500 genes were downregulated by OA, and
PaPE-1 was able to restore expression of about 60% of these genes
(FIGS. 38B and C). GO term analysis showed that OA upregulated
those genes that were involved in cell proliferation, energy
reserve metabolic process, epithelial cell migration and
angiogenesis. On the other hand, OA treatment downregulated those
genes that were involved in glucose and fatty acid metabolism and
inhibitors of epithelial cell proliferation. (FIG. 38 4D)
FFA Treatment Induces Recruitment of ER.alpha. to Chromatin
[0414] To test if any of the gene expression changes induced by OA
treatment occurred through direct ER.alpha. recruitment to
chromatin, a ChIP-Seq experiment was performed which showed that
ER.alpha. was recruited to novel chromatin sites upon OA treatment
and most of this recruitment was blocked by PaPE-1 treatment (FIG.
39A). The pattern of ER.alpha. recruitment to various sites
including a classic ER.alpha. binding site of PgR was verified
(FIG. 39B). To understand the nature of ER.alpha. recruitment to
chromatin in the presence of OA, binding sites that were occupied
by ER.alpha. upon OA treatment were studied (FIGS. 39A and C).
There were four main clusters that were named C1, C2, C3 and C4
(FIG. 39C). The majority of these binding events were associated
with genes whose expression was increased upon OA treatment. Then
transcription factor binding motif enrichment analysis was
performed using Seqpos tool from Cistrome/Galaxy. Interestingly,
none of the clusters, except C2, had any enrichment of EREs, which
suggested a potential tethering mode of recruitment for ER.alpha.
to these sites (FIG. 39D). Next, the transcriptional activity of
some of these factors was analyzed using a luciferase-based system
called Cignal finder assay (FIG. 39E). Exposure to OA increased
transcriptional activities of PPAR, LXR and RXR (FIGS. 39E and F),
suggesting that ER.alpha. might be recruiting and partnering with
these nuclear receptors to regulate gene expression, resulting in
changes that are essential for metabolic processes and survival of
breast cancer cells.
OA Treatment Causes Metabolic Reprogramming in Breast Cancer
Cells
[0415] The gene expression, ER.alpha. ChIP-Seq and transcription
factor activity assays pointed out a potential change in metabolic
pathways in MCF-7 cells upon OA exposure. Therefore cell metabolic
phenotyping assays were performed, which revealed that in the
presence of OA, the cells adopted an energetic phenotype and coped
with the metabolic stress better by increasing their aerobic and
glycolytic metabolic potential (FIG. 40A). The cells were more
glycolytic (FIG. 40B), and their mitochondrial metabolism was
increased (FIG. 40C), and OA-induced glycolytic and aerobic
respiration was reversed by PaPE-1 treatment. Next, parallel
metabolomics experiments were performed in MCF-7 cells that were
consistent with findings from cell phenotype experiments (FIG.
40D), had increased metabolites for the pentose pathway (FIG. 40E)
and certain aminoacids including proline (FIG. 40E). OA
downregulated many of the TCA cycle metabolites except malate and
fumarate, suggesting an increase in malate shunt from the cytosol,
which is consistent with the increase in certain amino acid levels
as well as increased mitochondrial activity (FIG. 40E). Finally,
fatty acid biosynthetic pathways were overall downregulated up
until linoleate synthesis, suggesting a negative feed back loop due
to high levels of extracellular OA (FIG. 40E). Overall, these
analyses suggest that OA treatment causes significant metabolic
reprogramming of breast cancer cells which can be reversed by
PaPE-1 treatment.
Example 39. Effect of PaPE-1 on Severity of Stroke Injury and
Outcome in Mice
[0416] Female 8 week-old C57BL/6 mice were ovariectomized. Then,
following a 2 week washout period subcutaneous pellet implants were
implanted containing vehicle, estradiol, or PaPE-1 (randomized,
n=9-10/group). One week later the mice underwent cerebral
ischemia-reperfusion injury invoked by middle cerebral artery
occlusion for 30 min followed by a 90 min reperfusion period. One
cohort received rotorod training prior to 45-min transient middle
cerebral artery occlusion (tMCAo), with MRI and rotorod assessed
through 2 weeks post-tMCAo. Splenic leukocyte subpopulations and
uterine weights were quantified. Another cohort was subjected to
45-min tMCAo to quantify leukocytes subpopulations in the brain,
spleen, and blood 3d post-stroke. MRI were performed 3 and 7 days
post-stroke, and rotorod testing was performed throughout a 13 day
post-stroke period. (FIG. 41).
[0417] Mice experienced a 53% decline in rotorod performance from
baseline at 2d post-tMCAo. PaPE-1 improved functional recovery to
85% (p=0.005) and 82% (p=0.001) of baseline and at 6 d and 13 d
post-tMCAo, respectively, while estradiol treatment improved
function only at d13 (p=0.005) post-stroke. Compared with vehicle,
both PaPE-1 and estradiol significantly reduced infarct volumes at
3d and 7d post-tMCAo. Further, PaPE-1 and estradiol treatment also
reduced the numbers of leukocytes infiltrated in the brain 3 days
post-stroke by 93% (p=0.002) and 63% (p=0.019), respectively.
Compared with vehicle, however, estradiol caused an increase in
uterine wet weight whereas PaPE-1 had no effect.
[0418] With E2 and PaPE-1 treatment, the severity of CNS injury on
MRI at 3 and 7 days was decreased compared with control treatment
(FIGS. 42 and 43). The degree of swelling associated with the
injury was also attenuated by E2 and PaPE-1 3 days post-stroke
(FIGS. 42 and 43). The attenuation in CNS injury with E2 and PaPE-1
was associated with a decrease in leukocyte recruitment to the CNS
ipsilateral to the injury (FIGS. 44-46). E2 and PaPE-1 additionally
resulted in improved function post-stroke assessed with rotorod
testing (FIG. 47).
[0419] The PaPE-1 cumulative findings indicate that non-nuclear
estrogen receptor activation with PaPE-1 affords protection from
stroke injury and its adverPaPE-1 sequelae in mice.
[0420] All patents, publications and references cited herein are
hereby fully incorporated by reference. In case of conflict between
the present disclosure and incorporated patents, publications, and
references, the present disclosure should control.
* * * * *
References