U.S. patent application number 11/765548 was filed with the patent office on 2007-10-25 for (hydroxyphenyl)-1h-indole-3-carbaldehyde oxime derivatives as estrogenic agents.
This patent application is currently assigned to Wyeth. Invention is credited to Stephen Marc Bowen, Eric Steven Manas, Richard Eric Mewshaw.
Application Number | 20070249703 11/765548 |
Document ID | / |
Family ID | 34221360 |
Filed Date | 2007-10-25 |
United States Patent
Application |
20070249703 |
Kind Code |
A1 |
Mewshaw; Richard Eric ; et
al. |
October 25, 2007 |
(Hydroxyphenyl)-1H-Indole-3-Carbaldehyde Oxime Derivatives As
Estrogenic Agents
Abstract
This invention provides estrogen receptor modulators of formula
I, having the structure ##STR1## where R.sub.1, R.sub.2, R.sub.3,
and R.sub.4 are as defined in the specification, or a
pharmaceutically acceptable salt thereof.
Inventors: |
Mewshaw; Richard Eric; (King
of Prussia, PA) ; Bowen; Stephen Marc; (Mohnton,
PA) ; Manas; Eric Steven; (Lafayette Hill,
PA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP/WYETH
CIRA CENTRE, 12TH FLOOR
2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Assignee: |
Wyeth
Madison
NJ
07940
|
Family ID: |
34221360 |
Appl. No.: |
11/765548 |
Filed: |
June 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10914747 |
Aug 9, 2004 |
7250440 |
|
|
11765548 |
Jun 20, 2007 |
|
|
|
60494365 |
Aug 12, 2003 |
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|
Current U.S.
Class: |
514/415 ;
514/411 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
19/02 20180101; A61P 15/02 20180101; A61P 15/08 20180101; A61P
35/00 20180101; A61P 35/02 20180101; A61P 17/10 20180101; A61P
13/02 20180101; A61P 1/00 20180101; A61P 25/28 20180101; A61P 3/10
20180101; A61P 19/08 20180101; A61P 3/14 20180101; A61P 9/00
20180101; A61P 15/00 20180101; A61P 17/14 20180101; C07D 209/14
20130101; A61P 13/12 20180101; A61P 1/04 20180101; A61P 19/10
20180101; A61P 5/30 20180101; A61P 3/06 20180101; A61P 39/06
20180101; A61P 7/04 20180101; A61P 13/10 20180101; C07D 209/88
20130101; A61P 25/00 20180101; A61P 13/00 20180101; A61P 1/16
20180101; A61P 13/08 20180101 |
Class at
Publication: |
514/415 ;
514/411 |
International
Class: |
A61K 31/404 20060101
A61K031/404; A61K 31/403 20060101 A61K031/403; A61P 1/00 20060101
A61P001/00; A61P 13/00 20060101 A61P013/00; A61P 15/02 20060101
A61P015/02; A61P 17/10 20060101 A61P017/10; A61P 17/14 20060101
A61P017/14; A61P 19/02 20060101 A61P019/02; A61P 19/08 20060101
A61P019/08; A61P 25/00 20060101 A61P025/00; A61P 3/10 20060101
A61P003/10; A61P 3/14 20060101 A61P003/14; A61P 35/00 20060101
A61P035/00; A61P 9/00 20060101 A61P009/00 |
Claims
1. A method of inhibiting osteoarthritis, hypocalcemia,
hypercalcemia, Paget's disease, osteomalacia, osteohalisteresis,
multiple myeloma or other forms of cancer having deleterious
effects on bone tissues in a mammal in need thereof, comprising
providing to said mammal an effective amount of a compound of the
formula: ##STR8## wherein: R.sub.1 is hydrogen, lower alkyl,
halogen, CN, or lower alkoxy; R.sub.2 is hydrogen, lower alkyl, or
phenyl; or R.sub.1 and R.sub.2 together may form a 5-7 membered
ring; and R.sub.3, and R.sub.4 are each, independently, H, OH,
halogen, CN, phenyl, substituted phenyl, lower alkyl or lower
alkoxy; or a pharmaceutically acceptable salt or prodrug
thereof.
2. A method of inhibiting benign or malignant abnormal tissue
growth in a mammal in need thereof, comprising providing to said
mammal an effective amount of a compound of the formula: ##STR9##
wherein: R.sub.1 is hydrogen, lower alkyl, halogen, CN, or lower
alkoxy; R.sub.2 is hydrogen, lower alkyl, or phenyl; or R.sub.1 and
R.sub.2 together may form a 5-7 membered ring; and R.sub.3, and
R.sub.4 are each, independently, H, OH, halogen, CN, phenyl,
substituted phenyl, lower alkyl or lower alkoxy; or a
pharmaceutically acceptable salt or prodrug thereof.
3. The method of claim 2 wherein the abnormal tissue growth is
prostatic hypertrophy, uterine leiomyomas, breast cancer,
endometriosis, endometrial cancer, polycystic ovary syndrome,
endometrial polyps, benign breast disease, adenomyosis, ovarian
cancer, melanoma, prostrate cancer, cancers of the colon, or CNS
cancers.
4. A method of lowering cholesterol, triglycerides, Lp(a), or LDL
levels; or inhibiting hypercholesteremia; hyperlipidemia;
cardiovascular disease; atherosclerosis; peripheral vascular
disease; restenosis, or vasospasm; or inhibiting vascular wall
damage from cellular events leading toward immune mediated vascular
damage in a mammal in need thereof, comprising providing to said
mammal an effective amount of a compound of the formula: ##STR10##
wherein: R.sub.1 is hydrogen, lower alkyl, halogen, CN, or lower
alkoxy; R.sub.2 is hydrogen, lower alkyl, or phenyl; or R.sub.1 and
R.sub.2 together may form a 5-7 membered ring; and R.sub.3, and
R.sub.4 are each, independently, H, OH, halogen, CN, phenyl,
substituted phenyl, lower alkyl or lower alkoxy; or a
pharmaceutically acceptable salt or prodrug thereof.
5. A method of inhibiting free radical induced disease states in a
mammal in need thereof, comprising providing to said mammal an
effective amount of a compound of the formula: ##STR11## wherein:
R.sub.1 is hydrogen, lower alkyl, halogen, CN, or lower alkoxy;
R.sub.2 is hydrogen, lower alkyl, or phenyl; or R.sub.1 and R.sub.2
together may form a 5-7 membered ring; and R.sub.3, and R.sub.4 are
each, independently, H, OH, halogen, CN, phenyl, substituted
phenyl, lower alkyl or lower alkoxy; or a pharmaceutically
acceptable salt or prodrug thereof.
6. A method of providing cognition enhancement or neuroprotection;
or treating or inhibiting senile dementias, Alzheimer's disease,
cognitive decline, or neurodegenerative disorders in a mammal in
need thereof, comprising providing to said mammal an effective
amount of a compound of the formula: ##STR12## wherein: R.sub.1 is
hydrogen, lower alkyl, halogen, CN, or lower alkoxy; R.sub.2 is
hydrogen, lower alkyl, or phenyl; or R.sub.1 and R.sub.2 together
may form a 5-7 membered ring; and R.sub.3, and R.sub.4 are each,
independently, H, OH, halogen, CN, phenyl, substituted phenyl,
lower alkyl or lower alkoxy; or a pharmaceutically acceptable salt
or prodrug thereof.
7. A method of inhibiting inflammatory bowel disease, ulcerative
proctitis, Crohn's disease, colitis, hot flashes, vaginal or vulvar
atrophy, atrophic vaginitis, vaginal dryness, pruritus,
dyspareunia, dysuria, frequent urination, urinary incontinence,
urinary tract infections, vasomotor symptoms; male pattern
baldness; skin atrophy; acne; type II diabetes; dysfunctional
uterine bleeding; or infertility in a mammal in need thereof,
comprising providing to said mammal an effective amount of a
compound of the formula: ##STR13## wherein: R.sub.1 is hydrogen,
lower alkyl, halogen, CN, or lower alkoxy; R.sub.2 is hydrogen,
lower alkyl, or phenyl; or R.sub.1 and R.sub.2 together may form a
5-7 membered ring; and R.sub.3, and R.sub.4 are each,
independently, H, OH, halogen, CN, phenyl, substituted phenyl,
lower alkyl or lower alkoxy; or a pharmaceutically acceptable salt
or prodrug thereof.
8. A method of inhibiting leukemia, endometrial ablations, chronic
renal or hepatic disease or coagulation diseases or disorders in a
mammal in need thereof, comprising providing to said mammal an
effective amount of a compound of the formula: ##STR14## wherein:
R.sub.1 is hydrogen, lower alkyl, halogen, CN, or lower alkoxy;
R.sub.2 is hydrogen, lower alkyl, or phenyl; or R.sub.1 and R.sub.2
together may form a 5-7 membered ring; and R.sub.3, and R.sub.4 are
each, independently, H, OH, halogen, CN, phenyl, substituted
phenyl, lower alkyl or lower alkoxy; or a pharmaceutically
acceptable salt or prodrug thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 10/914,747, filed Aug. 9, 2004 and claims
benefit to U.S. Provisional Application Ser. No. 60/494,365, filed
Aug. 12, 2003, each of which is incorporated herein by reference in
its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to
(hydroxyphenyl)-1H-indole-3-carbaldehyde oxime derivatives, their
uses as estrogenic agents, and methods of their preparation.
BACKGROUND OF THE INVENTION
[0003] The pleiotropic effects of estrogens in mammalian tissues
have been well documented, and it is now appreciated that estrogens
affect many organ systems [Mendelsohn and Karas, New England
Journal of Medicine 340: 1801-1811 (1999), Epperson, et al.,
Psychosomatic Medicine 61: 676-697 (1999), Crandall, Journal of
Womens Health & Gender Based Medicine 8: 1155-1166 (1999), Monk
and Brodaty, Dementia & Geriatric Cognitive Disorders 11:1 -10
(2000), Hum and Macrae, Journal of Cerebral Blood Flow &
Metabolism 20: 631-652 (2000), Calvin, Maturitas 34: 195-210
(2000), Finking, et al., Zeitschrift fur Kardiologie 89: 442-453
(2000), Brincat, Maturitas 35: 107-117 (2000), Al-Azzawi,
Postgraduate Medical Journal 77: 292-304 (2001)]. Estrogens can
exert effects on tissues in several ways. Probably, the most well
characterized mechanism of action is their interaction with
estrogen receptors leading to alterations in gene transcription.
Estrogen receptors are ligand-activated transcription factors and
belong to the nuclear hormone receptor superfamily. Other members
of this family include the progesterone, androgen, glucocorticoid
and mineralocorticoid receptors. Upon binding ligand, these
receptors dimerize and can activate gene transcription either by
directly binding to specific sequences on DNA (known as response
elements) or by interacting with other transcription factors (such
as AP1), which in turn bind directly to specific DNA sequences
[Moggs and Orphanides, EMBO Reports 2: 775-781 (2001), Hall, et
al., Journal of Biological Chemistry 276: 36869-36872 (2001),
McDonnell, Principles Of Molecular Regulation. p351-361(2000)]. A
class of "coregulatory" proteins can also interact with the
ligand-bound receptor and further modulate its transcriptional
activity [McKenna, et al., Endocrine Reviews 20: 321-344 (1999)].
It has also been shown that estrogen receptors can suppress
NF.kappa.B-mediated transcription in both a ligand-dependent and
independent manner [Quaedackers, et al., Endocrinology 142:
1156-1166 (2001), Bhat, et al., Journal of Steroid Biochemistry
& Molecular Biology 67: 233-240 (1998), Pelzer, et al.,
Biochemical & Biophysical Research Communications 286: 1153-7
(2001)].
[0004] Estrogen receptors can also be activated by phosphorylation.
This phosphorylation is mediated by growth factors such as EGF and
causes changes in gene transcription in the absence of ligand
[Moggs and Orphanides, EMBO Reports 2: 775-781 (2001), Hall, et
al., Journal of Biological Chemistry 276: 36869-36872 (2001)].
[0005] A less well-characterized means by which estrogens can
affect cells is through a so-called membrane receptor. The
existence of such a receptor is controversial, but it has been well
documented that estrogens can elicit very rapid non-genomic
responses from cells. The molecular entity responsible for
transducing these effects has not been definitively isolated, but
there is evidence to suggest it is at least related to the nuclear
forms of the estrogen receptors [Levin, Journal of Applied
Physiology 91: 1860-1867 (2001), Levin, Trends in Endocrinology
& Metabolism 10: 374-377 (1999)].
[0006] Two estrogen receptors have been discovered to date. The
first estrogen receptor was cloned about 15 years ago and is now
referred to as ER.alpha. [Green, et al., Nature 320: 134-9 (1986)].
The second was found comparatively recently and is called ER.beta.
[Kuiper, et al., Proceedings of the National Academy of Sciences of
the United States of America 93: 5925-5930 (1996)]. Early work on
ER.beta. focused on defining its affinity for a variety of ligands
and, indeed, some differences with ER.alpha. were seen. The tissue
distribution of ER.beta. has been well mapped in the rodent and it
is not coincident with ER.alpha.. Tissues such as the mouse and rat
uterus express predominantly ER.alpha., whereas the mouse and rat
lung express predominantly ER.beta. [Couse, et al., Endocrinology
138: 4613-4621 (1997), Kuiper, et al., Endocrinology 138: 863-870
(1997)]. Even within the same organ, the distribution of ER.alpha.
and ER.beta. can be compartmentalized. For example, in the mouse
ovary, ER.beta. is highly expressed in the granulosa cells and
ER.alpha. is restricted to the thecal and stromal cells [Sar and
Welsch, Endocrinology 140: 963-971 (1999), Fitzpatrick, et al.,
Endocrinology 140: 2581-2591 (1999)]. However, there are examples
where the receptors are coexpressed and there is evidence from in
vitro studies that ER.alpha. and ER.beta. can form heterodimers
[Cowley, et al., Journal of Biological Chemistry 272: 19858-19862
(1997)].
[0007] The most potent endogenous estrogen is 17.beta.-estradiol. A
large number of compounds have been described that either mimic or
block the activity of 17.beta.-estradiol. Compounds having roughly
the same biological effects as 17.beta.-estradiol are referred to
as "estrogen receptor agonists". Those which block the effects of
17.beta.-estradiol, when given in combination with it, are called
"estrogen receptor antagonists". In reality, there is a continuum
between estrogen receptor agonist and estrogen receptor antagonist
activity and some compounds behave as estrogen receptor agonists in
some tissues but estrogen receptor antagonists in others. These
compounds with mixed activity are called selective estrogen
receptor modulators (SERMS) and are therapeutically useful agents
(e.g. EVISTA) [McDonnell, Journal of the Society for Gynecologic
Investigation 7: S10-S15 (2000), Goldstein, et al., Human
Reproduction Update 6: 212-224 (2000)]. The precise reason why the
same compound can have cell-specific effects has not been
elucidated, but the differences in receptor conformation and/or in
the milieu of coregulatory proteins have been suggested.
[0008] It has been known for some time that estrogen receptors
adopt different conformations when binding ligands. However, the
consequence and subtlety of these changes only recently has been
revealed. The three dimensional structures of ER.alpha. and
ER.beta. have been solved by co-crystallization with various
ligands and clearly show the repositioning of helix 12 in the
presence of an estrogen receptor antagonist, which sterically
hinders the protein sequences required for receptor-coregulatory
protein interaction [Pike, et al., Embo 18: 4608-4618 (1999),
Shiau, et al., Cell 95: 927-937 (1998)]. In addition, the technique
of phage display has been used to identify peptides that interact
with estrogen receptors in the presence of different ligands
[Paige, et al., Proceedings of the National Academy of Sciences of
the United States of America 96: 3999-4004 (1999)]. For example, a
peptide was identified that distinguished between ER.alpha. bound
to the full estrogen receptor agonists 17.beta.-estradiol and
diethylstilbesterol. A different peptide was shown to distinguish
between clomiphene bound to ER.alpha. and ER.beta.. These data
indicate that each ligand potentially places the receptor in a
unique and unpredictable conformation that is likely to have
distinct biological activities.
[0009] As mentioned above, estrogens affect a panoply of biological
processes. In addition, where gender differences have been
described (e.g. disease frequencies, responses to challenge, etc),
it is possible that the explanation involves the difference in
estrogen levels between males and females.
SUMMARY OF THE INVENTION
[0010] This invention relates to
(hydroxyphenyl)-1H-indole-3-carbaldehyde oxime derivatives. In
certain aspects, the invention relates to compounds of formula I:
##STR2## wherein: [0011] R.sub.1 is hydrogen, lower alkyl, halogen,
CN, or lower alkoxy; [0012] R.sub.2 is hydrogen, lower alkyl, or
phenyl; [0013] or R.sub.1 and R.sub.2 together may form a 5-7
membered ring; and [0014] R.sub.3, and R.sub.4 are each,
independently, H, OH, halogen, CN, phenyl, substituted phenyl,
lower alkyl or lower alkoxy; or a pharmaceutically acceptable salt
or prodrug thereof.
[0015] In certain embodiments, the invention concerns a
pharmaceutical composition comprising compound 1 and a
pharmaceutically acceptable carrier.
[0016] In other embodiments, the invention is directed to the use
of such compounds in the treatment or prevention of diseases such
as inflammatory bowel diseases.
DETAILED DESCRIPTIION OF THE INVENTION
[0017] This invention provides estrogenic compounds of formula I,
which is useful in the treatment and/or prevention of diseases such
as inflammatory bowel diseases (including Crohn's disease and
colitis). ##STR3## where R.sub.1 is hydrogen, lower alkyl, halogen,
CN, or lower alkoxy; R.sub.2 is hydrogen, lower alkyl, or phenyl;
or R.sub.1 and R.sub.2 together may form a 5-7 membered ring; and
R.sub.3, and R.sub.4 are each, independently, H, OH, halogen, CN,
phenyl, substituted phenyl, lower alkyl or lower alkoxy; or a
pharmaceutically acceptable salt or prodrug thereof.
[0018] In certain embodiments of compound 1, R.sub.1 is lower
alkyl. In other embodiments, R.sub.2 is hydrogen, or alkyl. Further
embodiments encompass compositions where R.sub.1 and R.sub.2
together form a 6 membered ring. In some embodiments, R.sub.3 is H,
halogen or CN. R.sub.4 is hydrogen in some embodiments. In some
other embodiments, R.sub.4 is fluoro. In some embodiments, R4 is
ortho fluoro.
[0019] Pharmaceutically acceptable salts can be formed from organic
and inorganic acids, for example, acetic, propionic, lactic,
citric, tartaric, succinic, fumaric, maleic, malonic, mandelic,
malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric,
sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic,
toluenesulfonic, camphorsulfonic, and similarly known acceptable
aids when a compound of this invention contains a basic moiety.
Salts may also be formed from organic and inorganic bases, such as
alkali metal salts (for example, sodium, lithium, or potassium)
alkaline earth metal salts, ammonium salts, alkylammonium salts
containing 1-6 carbon atoms or dialkylammonium salts containing 1-6
carbon atoms in each alkyl group, and trialkylammonium salts
containing 1-6 carbon atoms in each alkyl group, when a compound of
this invention contains an acidic moiety.
[0020] The term "alkyl", as used herein, whether used alone or as
part of another group, refers to a substituted or unsubstituted
aliphatic hydrocarbon chain and includes, but is not limited to,
straight and branched chains containing from 1 to 12 carbon atoms,
preferably 1 to 6 carbon atoms, unless explicitly specified
otherwise. For example, methyl, ethyl, propyl, isopropyl, butyl,
i-butyl and t-butyl are encompassed by the term "alkyl."
Specifically included within the definition of "alkyl" are those
aliphatic hydrocarbon chains that are optionally substituted.
[0021] The term "lower alkyl" refers to an alkyl group having 1 to
6 carbon atoms, in some embodiments 1 to 3 carbon atoms.
[0022] The carbon number as used in the definitions herein refers
to carbon backbone and carbon branching, but does not include
carbon atoms of the substituents, such as alkoxy substitutions and
the like.
[0023] The term "alkoxy," as used herein, refers to the group
R--O-- where R is an alkyl group preferably of 1 to 6 carbon
atoms.
[0024] The term "alkenyl", as used herein, whether used alone or as
part of another group, refers to a substituted or unsubstituted
aliphatic hydrocarbon chain and includes, but is not limited to,
straight and branched chains having 2 to 8 carbon atoms and
containing at least one double bond. Preferably, the alkenyl moiety
has 1 or 2 double bonds. Such alkenyl moieties may exist in the E
or Z conformations and the compounds of this invention include both
conformations. Specifically included within the definition of
"alkenyl" are those aliphatic hydrocarbon chains that are
optionally substituted. Heteroatoms, such as 0, S or N--R.sub.1,
attached to an alkenyl should not be attached to a carbon atom that
is bonded to a double bond.
[0025] When used herein the term alkynyl refers to an aliphatic,
straight or branched, hydrocarbon chain having 2 to 7 carbon atoms
that may contain 1 to 3 triple bonds. When used herein, the term
cycloalkyl refers to a monocarbocyclic ring having 3 to 8 carbon
atoms e.g. cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
When used herein, alone or as part of a group, the term aryl refers
to an aromatic 5 to 13 membered mono- or bi-carbocyclic ring such
as phenyl or naphthyl. Preferably, groups containing aryl moieties
are monocyclic having 5 to 7 carbon atoms in the ring. When used
herein alone or as part of a group the term heteroaryl refers to an
aromatic 5 to 13-membered carbon containing mono or bicyclic ring
having one to five heteroatoms which independently may be nitrogen,
oxygen or sulfur. Preferably, groups containing heteroaryl moieties
are monocyclic having 5 to 7 members in the ring where one or two
of the ring members are independently selected from nitrogen,
oxygen or sulfur. Suitable aryl groups include furanyl, thienyl,
pyridinyl, indolyl and quinolyl.
[0026] Examples for a halogen substituented alkyls and alkenyls
include 1-bromo vinyl, 1-fluoro vinyl, 1,2-difluoro vinyl,
2,2-difluorovinyl, 1,2,2-trifluorovinyl, 1,2-dibromo ethane, 1,2
difluoro ethane, 1-fluoro-2-bromo ethane, CF.sub.2CF.sub.3,
CF.sub.2CF.sub.2CF.sub.3, and the like.
[0027] The term "phenyl", as used herein, whether used alone or as
part of another group, refers to a substituted or unsubstituted
phenyl group.
[0028] An optionally substituted alkyl, alkenyl, and phenyl may be
substituted with one or more substituents. Suitable optionally
substituents may be selected independently from nitro, cyano,
--N(R.sub.11)(R.sub.12), halo, hydroxy, carboxy, alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, heteroaryl, alkoxy, aryloxy,
heteroaryloxy, alkylalkoxy, al koxycarbonyl, alkoxyalkoxy,
perfluoroalkyl, perfluoroalkoxy, arylalkyl, alkylaryl,
hydroxyalkyl, alkoxyalkyl, alkylthio,
--S(O).sub.2--N(R.sub.11)(R.sub.12),
--C(.dbd.O)--N(R.sub.11)(R.sub.12), (R.sub.11)(R.sub.12)N-alkyl,
(R.sub.11)(R.sub.12)N-alkoxyalkyl,
(R.sub.11)(R.sub.12)N-alkylaryloxyalkyl, --S(O).sub.s-aryl (where
s=0-2), or --S(O).sub.s-heteroaryl (where s=0-2). In certain
embodiments of the invention, preferred substituents for alkyl,
alkenyl, alkynyl, phenyl and cycloalkyl include nitro, cyano,
--N(R.sub.11)(R.sub.12), halo, hydroxyl, aryl, heteroaryl, alkoxy,
alkoxyalkyl, and alkoxycarbonyl. In certain embodiments of the
invention, preferred substituents for aryl and heteroaryl include
--N(R.sub.11)(R.sub.12), alkyl, halo, perfluoroalkyl,
perfluoroalkoxy, arylalkyl and alkylaryl. R.sub.11 and R.sub.12 are
each independently selected from hydrogen and alkyl.
[0029] The term "lower alkoxy," as used herein, refers to the group
R--O-- where R is an alkyl group of 1 to 6 carbon atoms.
[0030] The term halogen includes bromine, chlorine, fluorine, and
iodine.
[0031] As used in accordance with this invention, the term
"providing," with respect to providing a compound or substance
covered by this invention, means either directly administering such
a compound or substance, or administering a prodrug, derivative, or
analog which will form the effective amount of the compound or
substance within the body.
[0032] The reagents used in the preparation of the compounds of
this invention can be either commercially obtained or can be
prepared by standard procedures described in the literature.
[0033] The preparation of several representative examples of this
invention are described in the following Schemes 1-3. ##STR4##
##STR5## ##STR6##
[0034] The compounds of this invention are estrogen receptor
modulators useful in the treatment or inhibition of conditions,
disorders, or disease states that are at least partially mediated
by an estrogen deficiency or excess, or which may be treated or
inhibited through the use of an estrogenic agent. The compounds of
this invention are particularly useful in treating a
peri-menopausal, menopausal, or postmenopausal patient in which the
levels of endogenous estrogens produced are greatly diminished.
Menopause is generally defined as the last natural menstrual period
and is characterized by the cessation of ovarian function, leading
to the substantial diminution of circulating estrogen in the
bloodstream. As used herein, menopause also includes conditions of
decreased estrogen production that may be surgically, chemically,
or be caused by a disease state which leads to premature diminution
or cessation of ovarian function.
[0035] Accordingly, the compounds of this invention are useful in
treating or inhibiting osteoporosis and in the inhibition of bone
demineralization, which may result from an imbalance in a
individual's formation of new bone tissues and the resorption of
older tissues, leading to a net loss of bone. Such bone depletion
results in a range of individuals, particularly in post-menopausal
women, women who have undergone bilateral oophorectomy, those
receiving or who have received extended corticosteroid therapies,
those experiencing gonadal dysgenesis, and those suffering from
Cushing's syndrome. Special needs for bone, including teeth and
oral bone, replacement can also be addressed using these compounds
in individuals with bone fractures, defective bone structures, and
those receiving bone-related surgeries and/or the implantation of
prosthesis. In addition to those problems described above, these
compounds can be used in treatment or inhibition for
osteoarthritis, hypocalcemia, hypercalcemia, Paget's disease,
osteomalacia, osteohalisteresis, multiple myeloma and other forms
of cancer having deleterious effects on bone tissues.
[0036] The compounds of this invention are also useful in treating
or inhibiting benign or malignant abnormal tissue growth, including
prostatic hypertrophy, uterine leiomyomas, breast cancer,
endometriosis, endometrial cancer, polycystic ovary syndrome,
endometrial polyps, benign breast disease, adenomyosis, ovarian
cancer, melanoma, prostrate cancer, cancers of the colon, CNS
cancers, such as glioma or astioblastomia.
[0037] The compounds of this invention are cardioprotective and
they are useful in lowering cholesterol, triglycerides, Lp(a), and
LDL levels; inhibiting or treating hypercholesteremia;
hyperlipidemia; cardiovascular disease; atherosclerosis; peripheral
vascular disease; restenosis, and vasospasm; and inhibiting
vascular wall damage from cellular events leading toward immune
mediated vascular damage. These cardiovascular protective
properties are of great importance when treating postmenopausal
patients with estrogens to inhibit osteoporosis and in the male
when estrogen therapy is indicated.
[0038] The compounds of this invention are also antioxidants, and
are therefore useful in treating or inhibiting free radical induced
disease states. Specific situations in which antioxidant therapy is
indicated to be warranted are with cancers, central nervous system
disorders, Alzheimer's disease, bone disease, aging, inflammatory
disorders, peripheral vascular disease, rheumatoid arthritis,
autoimmune diseases, respiratory distress, emphysema, prevention of
reperfusion injury, viral hepatitis, chronic active hepatitis,
tuberculosis, psoriasis, systemic lupus erythematosus, adult
respiratory distress syndrome, central nervous system trauma and
stroke.
[0039] The compounds of this invention are also useful in providing
cognition enhancement, and in treating or inhibiting senile
dementias, Alzheimer's disease, cognitive decline,
neurodegenerative disorders, providing neuroprotection or cognition
enhancement.
[0040] The compounds of this invention are also useful in treating
or inhibiting inflammatory bowel disease, ulcerative proctitis,
Crohn's disease, and colitis; menopausal related conditions, such
as vasomotor symptoms including hot flushes, vaginal or vulvar
atrophy, atrophic vaginitis, vaginal dryness, pruritus,
dyspareunia, dysuria, frequent urination, urinary incontinence,
urinary tract infections, vasomotor symptoms, including hot
flushes, myalgia, arthralgia, insomnia, irritability, and the like;
male pattern baldness; skin atrophy; acne; type II diabetes;
dysfunctional uterine bleeding; and infertility.
[0041] The compounds of this invention are useful in disease states
where amenorrhea is advantageous, such as leukemia, endometrial
ablations, chronic renal or hepatic disease or coagulation diseases
or disorders.
[0042] The compounds of this invention can be used as a
contraceptive agent, particularly when combined with a
progestin.
[0043] When administered for the treatment or inhibition of a
particular disease state or disorder, it is understood that the
effective dosage may vary depending upon the particular compound
utilized, the mode of administration, the condition, and severity
thereof, of the condition being treated, as well as the various
physical factors related to the individual being treated. Effective
administration of the compounds of this invention may be given at
an oral dose of from about 0.1 mg/day to about 1,000 mg/day.
Preferably, administration will be from about 10 mg/day to about
600 mg/day, more preferably from about 50 mg/day to about 600
mg/day, in a single dose or in two or more divided doses. The
projected daily dosages are expected to vary with route of
administration.
[0044] Such doses may be administered in any manner useful in
directing the active compounds herein to the recipient's
bloodstream, including orally, via implants, parenterally
(including intravenous, intraperitoneal and subcutaneous
injections), rectally, intranasally, vaginally, and
transdermally.
[0045] Oral formulations containing the active compounds of this
invention may comprise any conventionally used oral forms,
including tablets, capsules, buccal forms, troches, lozenges and
oral liquids, suspensions or solutions. Capsules may contain
mixtures of the active compound(s) with inert fillers and/or
diluents such as the pharmaceutically acceptable starches (e.g.
corn, potato or tapioca starch), sugars, artificial sweetening
agents, powdered celluloses, such as crystalline and
microcrystalline celluloses, flours, gelatins, gums, etc. Useful
tablet formulations may be made by conventional compression, wet
granulation or dry granulation methods and utilize pharmaceutically
acceptable diluents, binding agents, lubricants, disintegrants,
surface modifying agents (including surfactants), suspending or
stabilizing agents, including, but not limited to, magnesium
stearate, stearic acid, talc, sodium lauryl sulfate,
microcrystalline cellulose, carboxymethylcellulose calcium,
polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan
gum, sodium citrate, complex silicates, calcium carbonate, glycine,
dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate,
lactose, kaolin, mannitol, sodium chloride, talc, dry starches and
powdered sugar. Preferred surface modifying agents include nonionic
and anionic surface modifying agents. Representative examples of
surface modifying agents include, but are not limited to, poloxamer
188, benzalkonium chloride, calcium stearate, cetostearl alcohol,
cetomacrogol emulsifying wax, sorbitan esters, colloidol silicon
dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum
silicate, and triethanolamine. Oral formulations herein may utilize
standard delay or time release formulations to alter the absorption
of the active compound(s). The oral formulation may also consist of
administering the active ingredient in water or a fruit juice,
containing appropriate solubilizers or emulsifiers as needed.
[0046] In some cases it may be desirable to administer the
compounds directly to the airways in the form of an aerosol.
[0047] The compounds of this invention may also be administered
parenterally or intraperitoneally. Solutions or suspensions of
these active compounds as a free base or pharmacologically
acceptable salt can be prepared in water suitably mixed with a
surfactant such as hydroxy-propylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols and mixtures
thereof in oils. Under ordinary conditions of storage and use,
these preparation contain a preservative to prevent the growth of
microorganisms.
[0048] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol and liquid polyethylene glycol),
suitable mixtures thereof, and vegetable oils.
[0049] For the purposes of this disclosure, transdermal
administrations are understood to include all administrations
across the surface of the body and the inner linings of bodily
passages including epithelial and mucosal tissues. Such
administrations may be carried out using the present compounds, or
pharmaceutically acceptable salts thereof, in lotions, creams,
foams, patches, suspensions, solutions, and suppositories (rectal
and vaginal).
[0050] Transdermal administration may be accomplished through the
use of a transdermal patch containing the active compound and a
carrier that is inert to the active compound, is non toxic to the
skin, and allows delivery of the agent for systemic absorption into
the blood stream via the skin. The carrier may take any number of
forms such as creams and ointments, pastes, gels, and occlusive
devices. The creams and ointments may be viscous liquid or
semisolid emulsions of either the oil-in-water or water-in-oil
type. Pastes comprised of absorptive powders dispersed in petroleum
or hydrophilic petroleum containing the active ingredient may also
be suitable. A variety of occlusive devices may be used to release
the active ingredient into the blood stream such as a
semi-permeable membrane covering a reservoir containing the active
ingredient with or without a carrier, or a matrix containing the
active ingredient. Other occlusive devices are known in the
literature.
[0051] Suppository formulations may be made from traditional
materials, including cocoa butter, with or without the addition of
waxes to alter the suppository's melting point, and glycerin. Water
soluble suppository bases, such as polyethylene glycols of various
molecular weights, may also be used.
EXAMPLE 1a
1-(4-Benzyloxyphenyl)-2-methyl-1H-indole
[0052] To a mixture of 2-methylindole (3.28 g, 25 mmol),
4-benzyloxy iodobenzene (7.76 g, 25 mmol), potassium carbonate
(2.65 g, 25 mmol), and N-methylpyrrolidinone (250 mL) was added
copper (I) bromide (0.5 g, 3.5 mmol). The stirred solution was
heated to 180.degree. C. and maintained for 18 hr. The reaction
mixture was cooled, poured onto ice and extracted with ethyl
acetate. After washing the organic layer with water and brine, and
drying over anhydrous MgSO.sub.4 the solvent was removed to yield a
dark liquid. Purification by silica gel chromatography (2% ethyl
acetate-hexanes) provided the title compound as a white solid
(30%): mp 95-96.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta.
2.24 (3H, s), 5.19 (2H, s), 6.38 (1H, s), 6.93-7.04 (3H, m), 7.20
(2H, d, J=1.9Hz), 7.23 (2H, d, J=3.1 Hz), 7.32-7.53 (8H, m); MS m/z
(M+H).sup.+314:
[0053] Anal. for C.sub.22H.sub.19NO 0.1H.sub.2O:
[0054] Calc'd: C, 83.83; H, 6.14; N, 4.44.
[0055] Found: C, 83.84; H, 6.18; N, 4.29.
EXAMPLE 1b
5-Fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole
[0056] 5-Fluoro-2-methylindole (5.0 g, 33.5 mmol) and 4-benzyloxy
iodobenzene (10.40 g, 33.5 mmol) were reacted according to the
procedure used in Example 1a above to afford a white solid (23%):
mp 94-95.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 2.23 (3H,
s), 5.19 (2H, s), 6.39 (1H, s), 6.84-6.94 (2H, m), 7.19-7.29 (3H,
m), 7.34-7.44 (5H, m), 7.51 (2H, d,J=7.1 Hz); MS m/z
(M+H).sup.+332.
[0057] Anal. for C.sub.22H.sub.18NOF:
[0058] Calc'd: C, 79.74; H, 5.47; N, 4.23.
[0059] Found: C, 79.54; H, 5.54; N, 4.21.
EXAMPLE 1c
5-Chloro-1-(4-methoxymethyloxyphenyl)-2-methyl-1H-indole
[0060] 5-Chloro-2-methylindole (4.14 g, 25 mmol) and
4-methoxymethyloxy iodobenzene (6.20 g, 25 mmol) were reacted
according to the procedure used in Example 1a above to afford a
colorless liquid (19%): .sup.1H NMR (DMSO-d.sub.6): .delta. 2.25
(3H, s), 3.44 (2H, s), 5.28 (2H, s), 6.40 (1H, s), 6.96 (1H, d,
J=8.8 Hz), 7.02 (1H, dd, J=2.0 Hz, J=8.8 Hz), 7.22 (2H, d,
J=8.8Hz), 7.36 (2H, d, J=8.8 Hz), 7.55 (1H, d, J=2.0 Hz); MS m/z
(M+H).sup.+302/304 (1 Cl).
[0061] Anal. for C.sub.17H.sub.16CINO.sub.2:
[0062] Calc'd: C, 67.66; H, 5.34; N, 4.64.
[0063] Found: C, 67.49; H, 5.17; N, 4.51.
EXAMPLE 1d
5-Bromo-1-(4-methoxymethyloxyphenyl)-2-methyl-1H-indole
[0064] 5-Bromo-2-methylindole (5.59 g, 23.8 mmol) and
4-methoxymethyloxy iodobenzene (7.47 g, 23.8 mmol), were reacted
according to the procedure used in Example 1a above to afford a
colorless liquid (8.4%): .sup.1H NMR (DMSO-d.sub.6): .delta. 2.23
(3H, s), 3.43 (2H, s), 5.26 (2H, s), 6.39 (1H, s), 6.94 (1H, d,
J=8.7 Hz), 7.01 (1H, dd, J=2.2 Hz, J=8.6 Hz), 7.20 (2H, d, J=8.6H
7.34 (2H, d, J=8.6 Hz), 7.53 (1H, d, J=2.2 Hz); MS m/z
(M+H)+346/348 (1 Br).
[0065] Anal. for C.sub.17H.sub.16BrNO.sub.2:
[0066] Calc'd: C, 58.98; H, 4.66; N, 4.05.
[0067] Found: C, 58.72; H, 4.53; N, 4.17.
EXAMPLE 1e
(4E)-9-(4-Benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazole
[0068] Carbazole (8.70 g, 39.7 mmol) and 4-benzyloxy iodobenzene
(12.33 g, 39.7 mmol) were reacted according to the procedure used
in Example 1a above to afford a tan solid (20%): mp 128-129.degree.
C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 1.86 (4H, br s), 2.55 (2H,
br s), 2.72 (2H, br s), 5.16 (2H, s), 7.00-7.03 (2H, m), 7.05-7.08
(1H, m), 7.13-7.16 (2H, m), 7.27-7.30 (2H, m), 7.33-7.36 (1H, m),
7.40-7.43 (2H, m), 7.49 (2H, d, J=7.2 Hz); MS m/z
(M+H).sup.+354.
[0069] Anal. calcd for C.sub.25H.sub.23NO:
[0070] Calc'd: C, 84.95; H, 6.56; N, 3.96.
[0071] Found: C, 84.74; H, 6.76; N, 3.91.
EXAMPLE 1f
1-(4-Benzyloxy-3-fluorophenyl)-2-methyl-1H-indole
[0072] To a mixture of 2-methylindole (6.23 g, 47.5 mmol),
4-benzyloxy-3-fluoro bromobenzene (14.066 g, 50 mmol), potassium
carbonate (5.83 g, 55 mmol), and N-methylpyrrolidinone (500 mL) was
added copper (I) bromide (1.0 g, 7 mmol). The stirred solution was
heated to 180.degree. C. and maintained for 18 hr. The reaction
mixture was cooled, poured onto ice and extracted with ethyl
acetate. After washing the organic layer with water and brine, and
drying over anhydrous MgSO.sub.4 the solvent was removed to yield a
dark liquid, which was purified by silica gel chromatography (2%
ethyl acetate-hexanes) to provide the title compound alley as a
white solid (3.45 gr. 22% yd): mp 70-71.degree. C.; .sup.1H NMR
(DMSO-d.sub.6): .delta. 2.26 (3H, s), 5.28 (2H, s), 6.39 (1H, s),
6.99-7.05 (3H, m), 7.21-7.24 (2H, m), 7.36-7.54 (7H, m)); MS m/z
(M+H).sup.+332:
[0073] Anal. for C.sub.22H.sub.18NOF 0.1 H.sub.2O:
[0074] Calc'd: C, 79.31; H, 5.51; N, 4.20.
[0075] Found: C, 79.29; H, 5.39; N, 4.16
EXAMPLE 2a
1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
[0076] Phosphorous oxychloride (3 mL) was added to anhydrous
dimethylformamide (6 mL) and the mixture was stirred at room
temperature for 15 min. A mixture of
1-(4-benzyloxyphenyl)-2-methyl-1H-indole (0.94 g, 3 mmol) in
dimethylformamide (10 mL) was added and the mixture heated to
80.degree. C. for 18 hr. The reaction mixture was poured onto ice
and adjusted to a pH of 7 by the addition of 2 N NaOH. The mixture
was extracted with ethyl acetate (2.times.100 mL). The organic
layer washed with water and brine and dried over MgSO.sub.4.
Evaporation to provide the product as a dark solid. Purification by
silica gel chromatography (10% EtOAC-hexanes) provided a white
solid (63%): mp 150-152.degree. C.; .sup.1H NMR (DMSO-d.sub.6):
.delta. 2.54 (3H, s), 5.22 (2H, s), 6.99 (1H, d, J=7.9 Hz),
7.17-7.42 (4H, m), 7.45-7.54 (7H, m), 8.17 (1H, d, J=7.5 Hz), 10.19
(1H, s); MS m/z (M+H).sup.+342.
[0077] Anal. for C.sub.23H.sub.19NO.sub.2:
[0078] Calc'd: C, 80.92; H, 5.61; N, 4.10.
[0079] Found: C, 80.76; H, 5.70; N, 3.93.
EXAMPLE 2b
5-Fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
[0080] Phosphorous oxychloride (5 mL) was added to anhydrous
dimethylformamide (10 mL) and the mixture allowed to stir at room
temperature for 15 min whereupon a solution of
5-fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole (1.63 g, 4.9
mmol) in dimethylformamide (10 mL) was added and reacted according
to the procedure used in Example 2a to afford a tan solid (85%): mp
219-220.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 2.53 (3H,
s), 5.22 (2H, s), 6.99-7.06 (2H, m), 7.27 (2H, d, J=8.9 Hz),
7.37-7.53 (7H, m), 7.86 (1H, dd, J=2.5 Hz, J=9.5 Hz); MS m/z
(M+H).sup.+360.
[0081] Anal. for C.sub.23H.sub.18FNO.sub.2.0.5 H.sub.2O:
[0082] Calc'd: C, 74.99; H, 5.20; N, 3.80.
[0083] Found: C, 74.91; H, 4.96; N, 3.59.
EXAMPLE 2c
5-Chloro-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
[0084] Phosphorous oxychloride (5 mL) was added to anhydrous
dimethylformamide (10 mL) and stirred at room temperature for 15
min whereupon a solution of
5-chloro-1-(4-methoxymethyloxyphenyl)-2-methyl-1H -indole (1.22 g,
3.5 mmol) in dimethylformamide (10 mL) was added. The solution was
reacted according to the procedure used in Example 2a to afford a
tan solid (85%): mp >240.degree. C.; .sup.1H NMR (DMSO-d.sub.6):
.delta. 2.52 (3H, s), 6.97-7.02 (3H, m), 7.22 (1H, dd, J=2.3 Hz,
J=8.7 Hz), 7.30 (2H, d, J=8.6 Hz), 8.40 (1H, d, J=2.1 Hz), 10.02
(1H, s), 10.16 (1H, s); MS m/Z (M+H).sup.+286/288 (1 Cl).
[0085] Anal. for C.sub.16H.sub.12CINO.sub.2.0.3 H.sub.2O:
[0086] Calc'd: C, 66.01; H, 4.36; N, 4.81.
[0087] Found: C, 65.85; H, 4.10; N, 4.74.
EXAMPLE 2d
5-Bromo-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
[0088] Phosphorous oxychloride (2.5 mL) was added to anhydrous
dimethylformamide (5 mL) and stirred at room temperature for 15 min
whereupon a solution of
5-bromo-1-(4-methoxymethyloxyphenyl)-2-methyl-1H -indole (0.65 g,
1.9 mmol) in dimethylformamide (10 mL) was added. The solution was
reacted according to the procedure used in Example 2a to afford a
tan solid (51%): mp >250.degree. C.; .sup.1H NMR (DMSO-d.sub.6):
.delta. 2.50 (3H, s), 6.95-7.02 (3 H, m), 7.21 (1H, dd, J=2.4 Hz,
J=8.8 Hz), 7.28 (2H, d, J=8.7 Hz), 8.38 (1H, d J=2.2 Hz), 9.98 (1H,
s), 10.14 (1H, s); MS m/Z (M+H).sup.+318/320 (1 Br).
[0089] Anal. for C.sub.16H.sub.12BrNO.sub.2.0.2 H.sub.20:
[0090] Calc'd: C, 55.99; H, 3.88; N, 4.35.
[0091] Found: C, 55.69; H, 3.95; N, 4.15.
EXAMPLE 2e
(4E)-9-(4-Benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazol-4-one
[0092] To a stirred solution of
(4E)-9-(4-benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazole (1.55 g,
4.4 mmol), tetrahydrofuran (30 mL), and water (20 mL) was added DDQ
(1 g, 4.4 mmol). Stirring was continued for 30 min whereupon the
crude product was filtered and washed with water. The product was
purified by silica gel chromatography (25% EtOAc-hexanes) to give a
white solid (57%): mp 219-220.degree. C.; .sup.1H NMR (CDCI.sub.3):
.delta. 2.18-2.25 (2H, m), 2.62 (2H, t, J=5.9 Hz), 2.79 (2H, t,
J=6.1 Hz), 5.16 (2H, s), 7.11-7.51 (12H, m), 8.30 d, J=7.9 Hz); MS
m/z (M+H).sup.+368.
[0093] Anal. for C.sub.25H.sub.21NO.sub.2.0.5 H.sub.2O:
[0094] Calc'd: C, 79.76; H, 5.86; N, 3.72.
[0095] Found: C, 79.86; H, 5.75; N, 3.53.
EXAMPLE 2f
1-(4-Benzyloxyphenyl)-H-indole-3-carbaldehyde
[0096] 1H-Indole-3-carbaldehyde (3.63 g, 25 mmol) and 4-benzyloxy
iodobenzene (7.76 g, 25 mmol) were reacted according to the
procedure used in Example 1a above to afford a yellow solid (12%):
mp 62-64.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 5.22 (2H,
s), 7.26 (2H, d, J=8.8 Hz), 7.32-7.52 (7H, m), 7.61 (2H, d, J=8.8
Hz), 8.19-8.22 (1H, m), 8.54 (1H, s), 10.02 (1H, s); MS m/z
(M+H).sup.+328.
[0097] Anal. for C.sub.22H.sub.17NO.sub.2.0.1 H.sub.2O:
[0098] Calc'd: C, 80.27; H, 5.27; N, 4.25.
[0099] Found: C, 80.15; H, 5.05; N, 3.97.
EXAMPLE 2g
1-(4-benzyloxy-3-fluorophenyl)-2-methyl-1H-indole-3-carbaldehyde
[0100] Phosphorous oxychloride (3 mL) was added to anhydrous
dimethylformamide (6 mL) and the mixture allowed to stir at room
temperature for 15 min. A mixture of
1-(4-benzyloxy-3-fluorophenyl)-2-methyl-1H-indole (1.03 g, 3.1
mmol) in dimethylformamide (10 mL) was added and the mixture heated
to 80.degree. C. and held for 18 hr. The reaction mixture was
poured onto ice and adjusted to a pH of 7 by the addition of 2 N
NaOH. The mixture was extracted with ethyl acetate (2.times.100
mL), the organic layer washed with water and brine and dried over
MgSO.sub.4, and evaporated to provide the product as a dark solid.
Purification by silica gel chromatography (25% EtOAC-hexanes)
provided a white solid (0.84 gr, 76%): mp 166-168-152.degree. C.;
.sup.1H NMR (DMSO-d.sub.6): .delta. 2.55 (3H, s), 5.30 (2H, s),
7.04 (1H, d, J=6.9 Hz), 7.19-7.28 (2H, m), 7.23-7.52 (7H, m), 7.44
(2H, dd, J=6.0 Hz, J=1.4 Hz ), 7.59 (1H, dd, , J=11.7 Hz, J=2.5),
8.17 (1H, dd, J=7.0 Hz, J=1.0), 10.20 (1H, s) Hz; MS m/z
(M+H).sup.+360:
[0101] Anal. for C.sub.23H.sub.18FNO.sub.2 (0.1 H.sub.2O):
[0102] Calc'd: C, 76.48; H, 5.08; N, 3.88.
[0103] Found: C, 76.39; H, 4.81; N, 3.69
EXAMPLE 3a
1-(4-Benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde oxime
[0104] A solution of
1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde (0.64 g,
1.9 mmol), hydroxylamine hydrochloride (0.25 g, 3,6 mmol), methanol
(25 mL) and pyridine (1 mL) was heated to reflux for 30 min. The
mixture was cooled, the methanol removed by evaporation. The
product was extracted into ethyl acetate (2.times.50 mL) and the
organic layer was washed with water and brine and dried over
MgSO.sub.4. The solvent was evaporated, and the product was
purified by silica gel chromatography (25% ethyl acetate-hexanes)
to provide a white solid (53%): mp 171-173.degree. C.; .sup.1H NMR
(DMSO-d.sub.6): .delta. 2.31 (3H, s), 5.20 (2H, s), 6.94-7. J01
(1H, m), 7.10-7.14 (2H, m), 7.23 (2H, d, J=8.9 Hz), 7.37-7.47 (5H,
m), 7.52 (2H, d, J=7.4 Hz), 8.01-8.03 (1H, m), 8.41 (1H, s), 10.66
(1H, s); MS m/z (M+H).sup.+357.
[0105] Anal. for C.sub.23H.sub.20N.sub.2O.sub.2.0.5 H.sub.2O:
[0106] Calc'd: C, 75.60; H, 5.79; N, 7.67.
[0107] Found: C, 75.12; H, 5.53; N, 7.34.
EXAMPLE 3b
5-Fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
oxime
[0108] A mixture of
5-fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
(0.97 g, 2.7 mmol), hydroxylamine hydrochloride (0.25 g, 3,6 mmol),
methanol (25 mL) and pyridine (1 mL) was reacted according to the
procedure in Example 3a to afford white solid (88%): mp
183-185.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 2.30 (3H,
s), 5.20 (2H, s) d, 6.94-6.97 (2H, m), 7.23 (2H, d, J=8.9 Hz),
7.37-7.46 (5H, m), 7.52 (2H, d, J=6.8 Hz), 7.71 (1H, d, J=10.1 Hz),
8.41 (1H, s), 10.72 (1H, s); MS m/z (M+H).sup.+375.
[0109] Anal. for C.sub.23H.sub.19FN.sub.2O.sub.2:
[0110] Calc'd: C, 73.78; H, 5.11 N, 7.48.
[0111] Found: C, 73.55; H, 5.03; N, 7.35.
EXAMPLE 3e
(4E)-9-(4-benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazol-4-one
oxime
[0112] A mixture of
(4E)-9-(4-benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazol-4-one
(1.00 g, 2.7 mmol), hydroxylamine hydrochloride (0.56 g, 8.1 mmol),
methanol (25 mL) and pyridine (1 mL) was reacted according to the
procedure in Example 3a to afford a white solid (51%): mp
241-243.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 1.83-1.92
(2H, m), 2.65 (2H, t, J=5.9 Hz), 2.72 (2H, t, J=6.0 Hz), 5.20 (2H,
s), 7.09-7.14 (3H, m), 7.22 (2H, d, J=8.8 Hz), 7.36-7.46 (5H, m),
7.51 (2H, d, J=6.9 Hz), 8.00-8.02 (1H, m), 10.46 (1H, s); MS m/z
(M-H).sup.-379.
[0113] Anal. for C.sub.25H.sub.24N.sub.2O.sub.2:
[0114] Calc'd: C, 78.51; H, 5.80 N, 7.32.
[0115] Found: C, 78.24; H, 5.52; N, 7.26.
EXAMPLE 3f
1-(4-Benzyloxyphenyl)-H-indole-3-carbaldehyde oxime
[0116] A mixture of 1-(4-benzyloxyphenyl)-H-indole-3-carbaldehyde
(0.50 g, 1.5 mmol), hydroxylamine hydrochloride (0.25 g, 3,6 mmol),
methanol (25 mL) and pyridine (1 mL) was reacted according to the
procedure in Example 3a to afford a yellow solid (61%): mp
152-154.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 5.20 (2H,
s), 7.19-7.53 (12H, m), 7.86 (1H, s), 8.10 (1H, d, J=7.5 Hz), 10.75
(1H, s
[0117] MS m/z (M+H).sup.+383. Anal. for
C.sub.22H.sub.18N.sub.2O.sub.2:
[0118] Calc'd: C, 77.17; H, 5.30; N, 8.18.
[0119] Found: C, 77.49; H, 5.44; N, 7.85.
EXAMPLE 3g
1-(4-benzyloxy-3-fluorophenyl)-2-methyl-1H-indole-3-carbaldehyde
oxime
[0120] To a stirred solution of
1-(4-benzyloxy-3-fluorophenyl)-2-methyl-1H-indole-3-carbaldehyde
(0.81 g, 2.3 mmol), hydroxylamine hydrochloride (0.25 g, 3,6 mmol)
and methanol (25 mL) was added pyridine (1 mL). The reaction
mixture was heated to reflux and held for 30 min. The mixture was
cooled, the methanol removed by evaporation and the residue taken
up in ethyl acetate/water. The product was extracted into ethyl
acetate (2.times.50 mL), the ethyl acetate washed with water and
brine and dried over MgSO.sub.4. The solvent was evaporated, and
the crude product was purified by silica gel chromatography (25%
ethyl acetate-hexanes) to provide a white solid (0.7 gr, 83%): mp
167-168.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 2.32 (3H,
s), 5.29 (2H, s), 6.99-7.01 (1H, m), 7.10-7.17 (2H, m), 7.25-7.28
(1H, m), 7.37-7.42 (1H, m), 7.43-7.49 (3H, m), 7.49-7.54 (2H, m),
8.01-8.04 (1H, m), 8.41 (1H, s), 10.67 (1H, s); MS m/z
(M+H).sup.+375:
[0121] Anal. for C.sub.23H.sub.19FN.sub.2O.sub.2:
[0122] Calc'd: C, 73.78; H, 5.11; N, 7.48.
[0123] Found: C, 73.53; H, 5.06; N, 7.32
EXAMPLE 4
1-(4-Hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde oxime
[0124] To a stirred solution of dichloromethane (20 mL),
Pd(OAc).sub.2 (50 mg, 0.2 mmol), and triethylsilane (2 mL) was
added triethylamine (1 mL), and the mixture was stirred at room
temperature for 15 min. To the mixture was added
1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde oxime (0.36
g, 1 mmol) in dichloromethane (10 mL), and stirred for 90 min.
Ethyl acetate (25 mL) and ammonium chloride (25 mL of 10% solution)
were added and the product extracted into ethyl acetate (2.times.25
mL). The combined organic layers were washed with water (3.times.25
mL) and brine (25 mL) and dried over MgSO.sub.4. After removing the
solvent, a liquid was obtained, which was dissolved in anhydrous
tetrahydrofuran (10 mL). To this mixture was added tetrabutyl
ammonium fluoride (3 mL). After stirring for 30 min, the mixture
was extracted with ethyl acetate and water. The organic layer was
washed with water and brine and dried over MgSO.sub.4. After
evaporation of the solvent afforded the crude solid which was
purified by silica gel chromatography (25% ethyl acetate-hexanes)
to give a pale yellow solid (83%): mp 171-173.degree. C.; .sup.1H
NMR (DMSO-d.sub.6): .delta. 2.29 (3H, s), 6.95 (4H, d, J=7.7 Hz),
7.09-7.13 (2H, m), 7.22 (2H, d, J=8.7 Hz), 7.98-8.01 (1H, m), 8.40
(1H, s), 9.89 (1H, s), 10.63 (1H, s); MS m/z (M+H).sup.+267.
[0125] Anal. for C.sub.16H.sub.14N.sub.2O.sub.2:
[0126] Calc'd: C, 72.17; H, 5.30; N, 10.52.
[0127] Found: C, 71.78; H, 5.23; N, 10.37.
EXAMPLE 5
5-Fluoro-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
oxime
[0128]
5-Fluoro-1-(4-benzyloxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
oxime (0.49 g, 1.3 mmol) was reacted according to the procedure
used in example 4 to afford a white solid (67%): mp 162-163.degree.
C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 2.29 (3H, s), 6.94-6.97
(4H, m) d, 7.23 (2H, d, J=8.6 Hz), 7.70 (2H, d, J=10.1 Hz), 8.39
(1H, s), 9.92 (1H, s), 10.70 (1H, s); MS m/z (M+H).sup.+285.
[0129] Anal. for C.sub.16H.sub.13FN.sub.2O.sub.2:
[0130] Calc'd: C, 67.60; H, 4.61; N, 9.85.
[0131] Found: C, 67.44; H, 4.67; N, 9.79.
EXAMPLE 6
5-Chloro-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
oxime
[0132] A mixture of
5-chloro-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
(0.63 g, 2.2 mmol), hydroxylamine hydrochloride (0.30 g, 4.3 mmol),
methanol (25 mL) and pyridine (1 mL) was reacted according to the
procedure used in Example 4 to afford a white solid (68%): mp
212-213.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 2.29 (3H,
s), 6.96 (3H, d, J=8.7 Hz), 7.12 (1H, dd, J=2.2 Hz, J=8.7 Hz), 7.24
(2H, d, J=8.7 Hz), 8.03 (1H, d, J=2.1 Hz), 8.40 (1H, s), 9.93 (1H,
s), 10.75 (1H, s); MS m/Z (M+H).sup.+301/303 (1 Cl).
[0133] Anal. for C.sub.16H.sub.13CIN.sub.2O.sub.2:
[0134] Calc'd: C, 63.90; H, 4.36 N, 9.31.
[0135] Found: C, 63.63; H, 4.12; N, 9.17.
EXAMPLE 7
5-Bromo-1-(4-hydroxyphenyl)-2-methyl-1H-indole -3-carbaldehyde
oxime
[0136] A mixture of
5-bromo-1-(4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde (0.16
g, 0.5 mmol), hydroxylamine hydrochloride (0.10 g, 1.4 mmol),
methanol (10 mL) and pyridine (0.2 mL) was reacted according to the
procedure used in Example 4 to afford a white solid (66%): mp
209-211.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 2.29 (3H,
s), 6.92-6.97 (3H, m), 7.20-7.25 (3H, m), 8.19 (1H, d, J=1.9 Hz),
8.39 (1H, s), 9.94 (1H, br s), 10.76 (1H, s); MS m/z
(M+H).sup.+345/347 (1 Br).
[0137] Anal. for C.sub.16H.sub.13BrN.sub.2O.sub.2.0.5 H.sub.2O;
[0138] Calc'd: C, 54.26; H, 3.98 N, 7.91.
[0139] Found: C, 54.26; H, 3.68; N, 7.65.
EXAMPLE 8
(4E)-9-(4-Hydroxyphenyl)-1,2,3,9-tetrahydro-4H-carbazol-4-one
oxime
[0140]
(4E)-9-(4-Benzyloxyphenyl)-1,2,3,9-tetrahydro-4H-carbazol-4-one
oxime (0.23 g, 0.6 mmol) was reacted according to the procedure
used in Example 4 to afford a tan solid (40%): mp 227-229.degree.
C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 1.87-1.91 (2H, m), 2.63
(2H, t, J=5.9 Hz), 2.71 (2H, t, J=6.1 Hz), 6.95 (2H, d, J=8.6 Hz),
7.09-7.13 (3H, m), 7.26 (2H, d, J=8.5 Hz), 7.98-8.00 (1H, m), 9.85
(1H, s), 10.43 (1H, s); MS m/z (M+H).sup.+293.
[0141] Anal. for C.sub.18H.sub.16N.sub.2O.sub.2:
[0142] Calc'd: C, 73.95; H, 5.52; N, 9.58.
[0143] Found: C, 73.61; H, 5.39; N, 9.42.
EXAMPLE 9
1-(4-Hydroxyphenyl)-1H-indole-3-carbaldehyde oxime
[0144] 1-(4-Benzyloxyphenyl)-H-indole-3-carbaldehyde oxime (0.31 g,
0.9 mmol) was reacted according to the procedure used in Example 4
to afford a yellow solid (18%): mp 95-97.degree. C.; .sup.1H NMR
(DMSO-d.sub.6): .delta. 6.94-6.98 (2H, m), 7.19-7.29 (2H, m),
7.36-7.45 (4H, m), 8.31 (1H, s), 9.80-9.83 (1H, m), 10.72 (1H, s),
11.44 (1H, s): MS m/z (M+H).sup.+253.
[0145] Anal. for C.sub.15H.sub.12N.sub.2O.sub.2:
[0146] Calc'd: C, 71.42; H, 4.79; N, 11.10.
[0147] Found: C, 71.85; H, 5.36; N, 9.70.
EXAMPLE 10
1-(3-Fluoro-4-hydroxyphenyl)-2-methyl-1H-indole-3-carbaldehyde
oxime
[0148] To a stirred solution of dichloromethane (10 mL),
Pd(OAc).sub.2 (50 mg, 0.2 mmol), and triethylsilane (3 mL) was
added triethylamine (1.5 mL), and the mixture was stirred at
ambient temperature for 15 min. To the mixture was added
1-(4-benzyloxy-3-fluorophenyl)-2-methyl-1H-indole-3-carbaldehyde
oxime (0.52 g, 1.4 mmol) in dichloromethane (15 mL), and stirring
was continued for 4 hr. Ethyl acetate (25 mL) and ammonium chloride
(25 mL of 10% solution) were added and the product extracted into
ethyl acetate (2.times.25 mL). The combined organics were washed
with water (3.times.25 mL) and brine (25 mL) and dried over
MgSO.sub.4. After removing the solvent, a liquid was obtained,
which was dissolved in anhydrous tetrahydrofuran (10 mL). To this
stirring mixture was added tetrabutyl ammonium fluoride (3 mL).
After stirring for 30 min., the mixture was taken up in ethyl
acetate/water and shaken and the layers separated. The organic
portion was washed with water and brine and dried over MgSO.sub.4.
After evaporation of the solvent, the crude solid was purified by
silica gel chromatography (25% ethyl acetate-hexanes) to give a
pale yellow solid (0.22 g, 56%): mp 182-184.degree. C.; .sup.1H NMR
(DMSO-d.sub.6): .delta. 2.31 (3H, s), 6.98-7.01 (1H, m), 7.07-7.17
(4H, m), 7.35 (1H, dd, J=11.7 Hz, J=2.4 Hz), 7.99-8.03 (1H, m),
8.40 (1H, s), 10.35 (1H, br s), 10.64 (1H, s); MS m/z
(M+H).sup.+285:
[0149] Anal. for C.sub.16H.sub.13FN.sub.2O.sub.2 (0.3
H.sub.2O):
[0150] Calc'd: C, 66.34; H, 4.57 N, 9.30.
[0151] Found: C, 66.38; H, 4.61; N, 9.85.
EXAMPLE 11
Selectivity for ER.alpha. or ER.beta. Receptors
[0152] Representative examples of the invention were evaluated for
their ability to compete with 17.beta.-estradiol for both ER.alpha.
and ER.beta.. This test procedure provides the methodology for one
to determine whether a particular commpound binds to the estrogen
receptor (and is therefore "estrogenic") and whether there is
selectivity for ER.alpha. or ER.beta.. The values are shown in the
Table infra and are reported as IC.sub.50s. 17.beta.-estradiol is
included as a standard reference for comparison. The procedure used
is briefly described below. A crude lysate of E. coli expressing
the estrogen receptor ligand binding domains (D, E, & F) of
human ER.alpha. or ER.beta. was prepared. Both receptors and
compounds were diluted in 1.times. Dulbecco's PBS (DPBS)
supplemented with 1 mM EDTA. Using a high binding masked microtiter
plate, 100 uL of receptor (1 uG/well) was combined with 2 nM
[.sup.3H]-17.beta.-estradiol and various concentrations of
compound. After between 5 and 15 hours at room temperature, the
plates were washed with DPBS/1 mM EDTA and bound radioactivity
determined by liquid scintillation counting. The IC.sub.50 is
defined as the concentration of compound that decreases total
17.beta.-estradiol binding by 50%. The results obtained are
described in the table below. TABLE-US-00001 TABLE 1
1-(4'-Hydroxy-phenyl)-1H-indole-3-carbaldehyde Oxime Derivatives
##STR7## Ex. R1 R2 R3 R4 ER.sub..beta.(nM) ER.sub..alpha.(nM) 4 Me
H H H 12 270 5 Me H F H 6.2 91 6 Me H Cl H 29 145 7 Me H Br H 93.5
235 8 --CH.sub.2CH.sub.2CH.sub.2-- H H 10 153 9 H H H H 202 1905 10
Me H H F 8 298
[0153] The results obtained in the standard pharmacologic test
procedure demonstrate that the compounds of this invention are
estrogenic compounds, some with strong preferential affinity for
the ER.beta. receptor. The compounds of this invention range from
having high preferential affinity for ER.beta. over ER.alpha. to
almost equal affinity for both receptors. Thus, compounds of this
invention will span a range of activity based, at least partially,
on their receptor affinity selectivity profiles. Additionally,
since each novel receptor ligand complex is unique and thus its
interaction with various coregulatory proteins is unique, compounds
of this invention will display different modulatory behavior
depending on the cellular context they are in. For example, in some
cell-types, it is possible for a compound to behave as an estrogen
agonist while in other tissues, an antagonist. Compounds with such
activity have sometimes been referred to as SERMs (Selective
Estrogen Receptor Modulators). Unlike many estrogens, however, many
of the SERMs do not cause increases in uterine wet weight. These
compounds are antiestrogenic in the uterus and can completely
antagonize the trophic effects of estrogen agonists in uterine
tissue. These compounds, however, act as estrogen agonists in the
bone, cardiovascular, and central nervous systems. Due to this
tissue selective nature of these compounds, they are useful in
treating or preventing in a mammal disease states or syndromes
which are caused or associated with an estrogen deficiency (in
certain tissues such as bone or cardiovascular) or an excess of
estrogen (in the uterus or mammary glands).
[0154] Even beyond such cell-specific modulation, compounds of this
invention also have the potential to behave as agonists on one
receptor type while behaving as antagonists on the other. For
example, it has been demonstrated that compounds can be an
antagonist on ER.beta. while being an agonist on ER.alpha. (Meyers,
Marvin J.; Sun, Jun; Carlson, Kathryn E.; Katzenellenbogen, Benita
S.; Katzenellenbogen, John A. J. Med. Chem. (1999), 42(13),
2456-2468). Such ERSAA (Estrogen Receptor Selective Agonist
Antagonist) activity provides for pharmacologically distinct
estrogenic activity within this series of compounds.
[0155] Standard pharmacological test procedures are readily
available to determine the activity profile of a given test
compound. The following briefly summarizes several representative
test procedures. Standard pharmacological test procedures for SERMs
are also provided in U.S. Pat. Nos. 4,418,068 and 5,998,402.
EXAMPLE 12
Rat Uterotrophic/Antiuterotrophic Test Procedure
[0156] The estrogenic and antiestrogenic properties of the
compounds can be determined in an immature rat uterotrophic assay
(4 day) that (as described previously by L. J. Black and R. L.
Goode, Life Sciences, 26, 1453 (1980). Immature Sprague-Dawley rats
(female, 18 days old) were tested in groups of six. The animals are
treated by daily ip injection with 10 uG compound, 100 uG compound,
(100 uG compound+1 uG 17.beta.-estradiol) to check
antiestrogenicity, and 1 uG 17.beta.-estradiol, with 50% DMSO/50%
saline as the injection vehicle. On day 4 the animals are
sacrificed by CO.sub.2 asphyxiation and their uteri removed and
stripped of excess lipid, any fluid removed and the wet weight
determined. A small section of one horn is submitted for histology
and the remainder used to isolate total RNA in order to evaluate
complement component 3 gene expression.
EXAMPLE 13
6-Week Ovariectomized Rat Test Procedure--Bone and
Cardioprotection
[0157] Female Sprague Dawley CD rats, ovx or sham ovx, are obtained
1 day after surgery from Taconic Farm (weight range 240-275 g).
They are housed 3 or 4 rats/cage in a room on a 12/12 (light/dark)
schedule and provided with food (Purina 5K96C rat chow) and water
ad libitum. Treatment for all studies begin 1 day after the animals
arrival and dosed 7 days per week as indicated for 6 weeks. A group
of age matched sham operated rats not receiving any treatment serve
as an intact, estrogen replete control group for each study.
[0158] All treatments are prepared in 1% tween 80 in normal saline
at defined concentrations so that the treatment volume is 0.1
mL/100 g body weight. 17.beta.-estradiol is dissolved in corn oil
(20 .mu.g/mL) and delivered subcutaneously, 0.1 mL/rat. All dosages
are adjusted at three week intervals according to group mean body
weight measurements.
[0159] Five weeks after the initiation of treatment and one week
prior to the termination of the study, each rat is evaluated for
bone mineral density (BMD). The total and trabecular density of the
proximal tibia are evaluated in anesthetized rats using an XCT-960M
(pQCT; Stratec Medizintechnik, Pforzheim, Germany). The
measurements are performed as follows: Fifteen minutes prior to
scanning, each rat is anesthetized with an intraperitoneal
injection of 45 mg/kg ketamine, 8.5 mg/kg xylazine, and 1.5 mg/kg
acepromazine.
[0160] The right hind limb is passed through a polycarbonate tube
with a diameter of 25 mm and taped to an acrylic frame with the
ankle joint at a 90.degree. angle and the knee joint at
180.degree.. The polycarbonate tube is affixed to a sliding
platform that maintains it perpendicular to the aperture of the
pQCT. The platform is adjusted so that the distal end of the femur
and the proximal end of the tibia would be in the scanning field. A
two dimensional scout view is run for a length of 10 mm and a line
resolution of 0.2 mm. After the scout view is displayed on the
monitor, the proximal end of the tibia is located. The pQCT scan is
initiated 3.4 mm distal from this point. The pQCT scan is 1 mm
thick, has a voxel (three dimensional pixel) size of 0.140 mm, and
consists of 145 projections through the slice.
[0161] After the pQCT scan is completed, the image is displayed on
the monitor. A region of interest including the tibia but excluding
the fibula is outlined. The soft tissue is automatically removed
using an iterative algorithm. The density of the remaining bone
(total density) is reported in mg/cm.sup.3. The outer 55% of the
bone is peeled away in a concentric spiral. The density of the
remaining bone (Trabecular density) is reported in mg/cm.sup.3. One
week after BMD evaluation the rats are euthanized by carbon dioxide
suffocation and blood collected for cholesterol determination. The
uteri are removed and the weights taken. Total cholesterol is
determined using a Boehringer-Mannheim Hitachi 911 clinical
analyzer using the Cholesterol/HP kit. Statistics were compared
using one-way analysis of variance with Dunnet's test.
EXAMPLE 14
MCF-7/ERE Antiproliferative Test Procedure
[0162] Stock solutions of test compounds (usually 0.1 M) are
prepared in DMSO and then diluted 10 to 100-fold with DMSO to make
working solutions of 1 or 10 mM. The DMSO stocks are stored at
either 4.degree. C. (0.1 M) or -20.degree. C. (<0.1M). MCF-7
cells are passaged twice a week with growth medium [D-MEM/F-12
medium containing 10% (v/v) heat-inactivated fetal bovine serum, 1%
(v/v) Penicillin-Streptomycin, and 2 mM glutaMax-1]. The cells are
maintained in vented flasks at 37.degree. C. inside a 5%
CO.sub.2/95% humidified air incubator. One day prior to treatment,
the cells are plated with growth medium at 25,000/well into 96 well
plates and incubated at 37.degree. C. overnight.
[0163] The cells are infected for 2 hr at 37.degree. C. with 50
.mu.l/well of a 1:10 dilution of adenovirus 5-ERE-tk-luciferase in
experimental medium [phenol red-free D-MEM/F-12 medium containing
10% (v/v) heat-inactived charcoal-stripped fetal bovine serum, 1%
(v/v) Penicillin-Streptomycin, 2 mM glutaMax-1, 1 mM sodium
pyruvate]. The wells are then washed once with 150 .mu..lamda. of
experimental medium. Finally, the cells are treated for 24 hr at
37.degree. C. in replicates of 8 wells/treatment with 150
.mu..lamda./well of vehicle (.ltoreq.0.1% v/v DMSO) or compound
that is diluted .gtoreq.1000-fold into experimental medium.
[0164] Initial screening of test compounds is done at a single dose
of 1 .mu.M that is tested alone (agonist mode) or in combination
with 0.1 nM 17.beta.-estradiol (EC.sub.80; antagonist mode). Each
96 well plate also includes a vehicle control group (0.1% v/v DMSO)
and an agonist control group (either 0.1 or 1 nM
17.beta.-estradiol). Dose-response experiments are performed in
either the agonist and/or antagonist modes on active compounds in
log increases from 10.sup.-14 to 10.sup.-5 M. From these
dose-response curves, EC.sub.50 and IC.sub.50 values, respectively,
are generated. The final well in each treatment group contains 5
.mu.l of 3.times.10.sup.-5 M ICI-182,780 (10.sup.-6 M final
concentration) as an ER antagonist control.
[0165] After treatment, the cells are lysed on a shaker for 15 min
with 25 .mu.l/well of 1X cell culture lysis reagent (Promega
Corporation). The cell lysates (20 .mu.l ) are transferred to a 96
well luminometer plate, and luciferase activity is measured in a
MicroLumat LB 96 P luminometer (EG & G Berthold) using 100
.mu.l/well of luciferase substrate (Promega Corporation). Prior to
the injection of substrate, a 1 second background measurement is
made for each well. Following the injection of substrate,
luciferase activity is measured for 10 seconds after a 1 second
delay. The data are transferred from the luminometer to a Macintosh
personal computer and analyzed using the JMP software (SAS
Institute); this program subtracts the background reading from the
luciferase measurement for each well and then determines the mean
and standard deviation of each treatment.
[0166] The luciferase data are transformed by logarithms, and the
Huber M-estimator is used to down-weight the outlying transformed
observations. The JMP software is used to analyze the transformed
and weighted data for one-way ANOVA (Dunnett's test). The compound
treatments are compared to the vehicle control results in the
agonist mode, or the positive agonist control results (0.1 nM
17.beta.-estradiol) in the antagonist mode. For the initial single
dose experiment, if the compound treatment results are
significantly different from the appropriate control (p<0.05),
then the results are reported as the percent relative to the
17.beta.-estradiol control [i.e., ((compound-vehicle
control)/(17.beta.-estradiol control-vehicle control)).times.100].
The JMP software is also used to determine the EC.sub.50 and/or
IC.sub.50 values from the non-linear dose-response curves.
EXAMPLE 15
Inhibition of LDL Oxidation--Antioxidant Activity
[0167] Porcine aortas are obtained from an abattoir, washed,
transported in chilled PBS, and aortic endothelial cells are
harvested. To harvest the cells, the intercostal vessels of the
aorta are tied off and one end of the aorta clamped. Fresh, sterile
filtered, 0.2% collagenase (Sigma Type I) is placed in the vessel
and the other end of the vessel then clamped to form a closed
system. The aorta is incubated at 37.degree. C. for 15-20 minutes,
after which the collagenase solution is collected and centrifuged
for 5 minutes at 2000.times.g. Each pellet is suspended in 7 mL of
endothelial cell culture medium consisting of phenol red free
DMEM/Ham's F12 media supplemented with charcoal stripped FBS (5%),
NuSerum (5%), L-glutamine (4 mM), penicillin-streptomycin (100
U/ml, 100 .mu.g/ml) and gentimicin (75 .mu.g/ml), seeded in 100 mm
petri dish and incubated at 37.degree. C. in 5% CO.sub.2. After 20
minutes, the cells are rinsed with PBS and fresh medium added, this
was repeated again at 24 hours. The cells are confluent after
approximately 1 week. The endothelial cells are routinely fed twice
a week and, when confluent, trypsinized and seeded at a 1:7 ratio.
Cell mediated oxidation of 12.5 .mu.g/mL LDL is allowed to proceed
in the presence of the compound to be evaluated (5 .mu.M) for 4
hours at 37.degree. C. Results are expressed as the percent
inhibition of the oxidative process as measured by the TBARS
(thiobarbituric acid reactive substances) method for analysis of
free aldehydes (Yagi K., Biochem Med 15:212-216 (1976)).
EXAMPLE 16
D12Hypothalmic Cell Test Procedure
[0168] D12 rat hypothalamic cells are subcloned from the RCF17
parental cell line and stored frozen. They are routinely grown in
DMEM:F12 (1:1), glutaMAX-1 (2 mM), penicillin (100
U/ml)-streptomycin (100 mg/ml), plus 10% fetal bovine serum (FBS).
The cells are plated in phenol red-free medium (DMEM:F12, glutaMAX,
penicillin-streptomycin) containing 2-10% charcoal stripped FBS at
a subconfluent density (1-4.times.10 6 cells/150 mm dish). The
cells are refed 24 h later with medium containing 2% stripped
serum. To test for agonist activity, cells are treated with 10 nM
17b-estradiol or various doses of test compound (1 mM or a range
from 1 pM to 1 mM). To test for antagonist activity the cells are
treated with 0.1 nM 17.beta.-estradiol in the absence or presence
of varying doses (100 pM to 1 mM) of test compound. Control dishes
are also treated with DMSO as a negative control. Forty-eight hours
after hormone addition, the cells are lysed and binding test
procedure performed.
[0169] For each binding test procedure 100-150 mg protein is
incubated with 10 nM .sup.3H-R5020+100-fold excess R5020 in a 150
ml volume. Triplicate reactions (three with R5020, three without
R5020) are prepared in a 96 well plate. The protein extract is
added first followed by .sup.3H-R5020 or .sup.3H-R5020+100.times.
unlabeled R5020. The reaction is performed for 1-2 hr at room
temperature . The reaction is stopped by the addition of 100 ml
cold 5% charcoal (Norit SX-4), 0.5% dextran 69 K (Pharmacia) in TE
pH 7.4 . After 5 min at room temperature, the bound and unbound
ligand are separated by centrifugation (5 min, 1000 RCF, 4.degree.
C.). The supernatant solution (.about.150 ml) is removed and
transferred to a scintillation vial. Following the addition of
scintillation fluid (Beckman Ready Protein+), the samples are
counted for 1 min in a scintillation counter.
EXAMPLE 17
Progesterone Receptor in the CNS Preoptic Area
[0170] Sixty (60) day old female Sprague-Dawley rats are
ovariectomized. The animals are housed in an animal care facility
with a 12-h light, 12-h dark photoperiod and free access to tap
water and rodent chow.
[0171] Ovariectomized animals are randomly divided into groups that
are injected with vehicle (50% DMSO, 40% PBS, 10% ethanol vehicle),
17.beta.-estradiol (200 ng/kg) or the compound to be tested.
Additional animals are injected with the test compound 1 hr prior
to injection of 17.beta.-estradiol to evaluate the antagonistic
properties of this compound. Six hrs after s.c. injection, animals
are euthanized with a lethal dose of CO.sub.2 and their brains
collected and frozen.
[0172] Tissue collected from animals is cut on a cryostat at
-16.degree. C. and collected on Silane-coated microscope slides.
The section-mounted slides are then dried on a slide warmer
maintained at 42.degree. C. and stored in desiccated slide boxes at
-80.degree. C. Prior to processing, the desiccated slide boxes are
slowly warmed to room temperature (-20.degree. C. for 12-16 hrs;
4.degree. C. for 2 hrs; room temperature for 1 hr) to eliminate
condensation formation on slides and thus minimize tissue and RNA
degradation. The dry slides are loaded into metal racks, postfixed
in 4% paraformaldehyde (pH 9.0) for 5 min and processed as
previously described.
[0173] A plasmid containing a 815 bp fragment of the rat PR cDNA 9
(ligand binding domain) is linearized and used to generate a S
35-UTP labeled probe that is complimentary to a portion of the rat
PR mRNA. Processed section-mounted slides are hybridized with 20 ml
of hybridization mix containing the riboprobe (4-6.times.106
DPM/slide) and 50% formamide and incubated overnight in a
55.degree. C. humidified chamber. In the morning, the slides are
placed in metal racks that are immersed in 2.times.SSC (0.15M NaCl,
0.015M sodium citrate; pH 7.0)/10 mM DTT. The racks are all
transferred to a large container and washed in 2.times.SSC/10 mM
DTT for 15 min at RT with gentle agitation. Slides are then washed
in RNase buffer at 37.degree. C. for 30 min, treated with RNase A
(2 mg/ml) for 30 min at 37.degree. C., and washed for 15 min in
room temperature 1.times.SSC. Subsequently, the slides are washed
(2.times.30 min) in 65.degree. C. in 0.1.times.SSC to remove
nonspecific label, rinsed in room temperature 0.1.times.SSC for 15
min and dehydrated with a graded series of alcohol: ammonium
acetate (70%, 95%, and 100%). Air dried slides are opposed to x-ray
film for 3 days and then photographically processed. The slides
from all animals are hybridized, washed, exposed and
photographically processed together to eliminate differences due to
interassay variation in conditions.
EXAMPLE 18
Rat Hot Flush--CNS Effects
[0174] Ovariectomized-female, 60 day-old Sprague-Dawley rats are
obtained following surgery. The surgeries are done a minimum of 8
days prior to the first treatment. The animals are housed
individually under 12 h light/dark cycle and given standard rat
chow and water ad libitum.
[0175] Two control groups are included in every study. Doses are
prepared based on mg/kg mean group body weight in either 10% DMSO
in sesame oil (sc studies) or in 1.0% tween 80 in saline (po
studies). Animals are administered test compounds at doses ranging
from 0.01 to 10 mg/kg mean group body weight. Vehicle and ethinyl
estradiol (EE) controls (0.1 mg/kg, sc or 0.3 mg/kg, po) control
groups are included in each test. When the compounds are tested for
their antagonist activity, EE is coadministered at 0.1 or 0.3 mg/kg
for sc or po studies, respectively. The test compounds are
administered up to the day tail skin temperature is measured.
[0176] After the acclimation period of four days, the animals are
treated once daily with the compound(s) of interest. There are 10
animals/treatment group. Administration of the compound is either
by sc injection of 0.1 ml in the nape of the neck or po in a volume
of 0.5 ml. On the 3rd day of treatment, a morphine pellet (75 mg
morphine sulfate) is implanted subcutaneously. On the 5th day of
treatment, one or two additional morphine pellets are implanted. On
the eighth day, approximately half of the animals are injected with
Ketamine (80 mg/kg, intramuscularly) and a thermocouple, connected
with to a MacLab Data Acquisition System (API Insturments, Milford,
Mass) is taped on the tail approximately one inch from the root of
the tail. This system allowed the continuous measurement of tail
skin temperature. Baseline temperature is measured for 15 min, then
naloxone (1.0 mg/kg) is given sc (0.2 ml) to block the effect of
morphine and tail skin temperature is measured for one hour
thereafter. On the ninth day, the remaining of the animals are set
up and analyzed similarly.
EXAMPLE 19
Vasomotor Function in Isolated Rat Aortic Rings
[0177] Sprage-Dawley rats (240-260 grams) are divided into 4
groups: [0178] 1. Normal non-ovariectomized (intact) [0179] 2.
Ovariectomized (ovex) vehicle treated [0180] 3. Ovariectomized
17.beta.-estradiol treated (1 mg/kg/day) [0181] 4. Ovariectomized
animals treated with test compound (i.e., 1 mg/kg/day)
[0182] Animals are ovariectomized approximately 3 weeks prior to
treatment. Each animal receives 1 mg/kg/day of either
17.beta.-estradiol sulfate or test compound suspended in distilled,
deionized water with 1% tween-80 by gastric gavage. Vehicle treated
animals received an appropriate volume of the vehicle used in the
drug treated groups.
[0183] Animals are euthanized by CO.sub.2 inhalation and
exsanguination. Their thoracic aortas are rapidly removed and
placed in 37.degree. C. physiological solution with the following
composition (mM): NaCl (54.7), KCl (5.0), NaHCO.sub.3 (25.0),
MgCl.sub.2 2H.sub.2O (2.5), D-glucose (11.8) and CaCl.sub.2 (0.2)
gassed with CO.sub.2--O.sub.2, 95%/5% for a final pH of 7.4. The
advantitia is removed from the outer surface and the vessel is cut
into 2-3 mm wide rings. Rings are suspended in at 10 mL tissue bath
with one end attached to the bottom of the bath and the other to a
force transducer. A resting tension of 1 gram is placed on the
rings. Rings are equilibrated for 1 hour, signals are acquired and
analyzed.
[0184] After equilibration, the rings are exposed to increasing
concentrations of phenylephrine (10.sup.-8 to 10.sup.-4 M) and the
tension recorded. Baths are then rinsed 3 times with fresh buffer.
After washout, 200 mM L-NAME is added to the tissue bath and
equilibrated for 30 minutes. The phenylephrine concentration
response curve is then repeated.
EXAMPLE 20
Eight Arm Radial Arm Maze--Cognition Enhancement
[0185] Male Sprague-Dawley, CD rats (Charles River, Kingston, N.Y.)
weighing 200-250 g on arrival are used. For one week, the rats are
housed, six per cage, with standard laboratory chow and water
available ad libitum. Housing is in a colony room maintained at
22.degree. C. and had a 12 hour light/dark cycle with lights on at
6:00 AM. Following habituation to the facility, animals are
individually housed and maintained at 85% of free-feeding weight.
Once stable weights are attained, the rats are acclimated to the
8-arm radial maze.
[0186] The structure of the maze is an adaptation from that of
Peele and Baron (Pharmacology, Biochemistry, and Behavior,
29:143-150, 1988). The maze is elevated to a height of 75.5 cm and
composed of a circular area surrounded by 8 arms radiating away
from the center, equidistant from one another. Each arm is 58 cm
long.times.13 cm high. A clear plexiglass cylinder is lowered to
enclose the animal in the center portion of the maze prior to the
start of each session. Each arm of the maze is equipped with 3 sets
of photocells interfaced to a data acquisition unit, which in turn
is interfaced to a computer. The photocells are used to track the
movement of the rat in the maze. Pellet feeders located above food
cups at the end of each arm, dispensed two 45 mg chocolate pellets
when the outer photocell of the arm is activated for the first time
in a given session. The maze is located in a testing room with
black and white geometric posters on each wall to serve as visual
cues. During all training and testing procedures, white noise is
audible (.about.70 db).
[0187] The training procedure consists of five phases, each with
daily sessions lasting 5 or 10 minutes. A 10 second delay is
imposed between the time the rat is placed in the center portion of
the maze and when the cylinder is raised to begin the session.
During Phase 1, food-restricted pairs of rats are placed on the
maze for 10 minutes with 45 mg chocolate food pellets scattered
throughout the 8 arms of the maze. During Phase II, each rat is
placed individually on the maze for a 10 minute period, with
pellets scattered from the middle photocell to the food cup of each
arm. During Phase III, each rat is placed on the maze for a 10
minute period, with food pellets located only in and around the
food cups in each arm. In Phase IV, each rat is allowed 10 minutes
to collect two pellets from each arm. Re-entry into an arm is
considered an error. Rats are trained daily in this manner until
they achieved criterion performance with less than or equal to 2
total errors on three consecutive days of training. Total
habituation and training time is approximately 3 weeks.
[0188] Test compound is prepared in phosphate buffered saline and
administered in a volume of 1 ml/kg. Scopolamine HBr (0.3 mg/kg
s.c.) served as the impairing agent, producing an increase in error
rate (loss of memory). Test compound is given intraperitoneally
simultaneously with scopolamine, 30 minutes prior to the first maze
exposure on any given test day.
[0189] To assess the test compound, an 8.times.8 balanced latin
square for repeated measures is designed, in order to achieve a
high experimental efficiency with the least amount of animals.
Eight experimental sessions, two per week, are conducted with the 8
treatments (vehicle, scopolamine, 3 doses of test compound in
combination with scopolamine) randomized within each session. Each
treatment followed every other treatment the same number of times.
Therefore, the residual effect of every treatment could be
estimated and removed from the direct treatment effect. Following
ANOVA, multiple comparisons are performed using Dunnett's two-sided
test on adjusted means.
[0190] Animals that did not make 4 correct choices within 5 minutes
during the first exposure, or that had not made a total of 8
choices by the end of the 2nd exposure, are considered to have
"timed-out" for that session. Any animal that "timed-out" following
administration of more than one dose of the test compound is
excluded from the analysis.
EXAMPLE 21
Neuroprotection
Inhibition of Time-Dependent Death of Cells in Primary Cortical
Neuron Cultures
[0191] Primary cortical neurons were produced from rat brains that
were 0-1 day old using a variation of methods described by Monyer
et al. 1989, Brain Research 483:347-354. Dispersed brain tissue was
grown in DMEM/10% PDHS (pregnant donor horse serum) for three days
and then treated with cytosine arabinoside (ARC) for two days to
remove contaminating glial cells. On day 5, the ARC media was
removed and replaced with DMEM/10% PDHS. The neuronal cells were
cultured for a further 4-7 days before use.
[0192] Control primary neuronal cultures show progressive cell
death between days 12 and 18 in culture. Twelve cultures were
evaluated on days 12 and 16 for levels of the enzyme lactate
dehydrogenase (LD) after adding test compound to 6 cultures
maintained in DMEM and 10% PDHS on day 9 and maintaining the
remaining cultures as controls. LD was assayed using a variation of
the method by Wroblewski et al. 1955, Proc. Soc. Exp. Biol. Med.
90:210-213. LD is a cytosolic enzyme which is commonly used in both
clinical and basic research to determine tissue viability. An
increase in media LD is directly related to cell death.
Neuroprotection Against Cytotoxicity Induced by Hypoglycemia
[0193] C6 glioma cells obtained from ATCC were plated in RPMI media
with FBS at a concentration of 1.times.10<6 > cells/ml in
FALCON 25 cm.sup.2 tissue culture flasks. Four hours prior to the
onset of hypoglycemia, the maintenance media was discarded,
monolayers were washed twice in the appropriate media and then
incubated for four hours at 37.degree. C. in either serum free or
serum free plus test compound. Kreb's Ringer Phosphate buffer was
used to wash the monolayers twice before the addition of
appropriate glucose treatment. RPMI medium contains 2 mg
glucose/ml; flasks were divided into groups of 6 each receiving
100% glucose (2 mg/ml), 80% glucose (1.6 mg/ml), 60% glucose (1.2
mg/ml) or 0% glucose (buffer) or supplemented with test compound.
All flasks were incubated for 20 hours and then evaluated for
total, live, and dead cell number utilizing trypan blue.
Neuroprotection Against Excitotoxic Amino Acids
[0194] Five culture dishes containing SK-N-SH neuroblastoma cells
were treated with test compound and 5 culture dishes were treated
with RPMI media. Four hours later, all cell were treated with NMDA
(500 mu M) for 5 minutes. Total live cells and dead cells were then
determined.
Neuroprotection Against Oxygen-Glucose Deprivation
[0195] Analysis of pyknotic nuclei to measure apoptosis: Cortical
neurons are prepared from E18 rat fetus and plated in 8-well
chamber slides precoated with poly-D-lysine (10 ng/ml) and serum at
a density of 100,000 cells/well. Cells are plated in high glucose
DMEM containing 10% FCS and kept in the incubator at 37.degree. C.
with 10% CO.sub.2/90% air. On the next day, serum is removed by
replacing culture media with high glucose DMEM containing B27
supplement and cells are kept in the incubator without further
media change until the day of experiment. On day 6, slides are
divided into two groups; control group and OGD group. Cells in
control group receive DMEM with glucose and custom B27 (without
antioxidants). Cells in OGD group receive no-glucose DMEM with
custom B27, which has been degassed under vacuum for 15 min. Cells
are flushed with 90% N.sub.2/10% CO.sub.2 for 10 min in an airtight
chamber and incubated at 37.degree. C. for 6 hrs. After 6 hrs, both
control and OGD cells are subject to replacement of media
containing either vehicle (DMSO) or test compound in
glucose-containing DMEM with custom B27. Cells are returned to
normoxic incubator at 37.degree. C. After 24 hrs, cells are fixed
in 4% PFA for 10 min at 4.degree. C. and stained with Topro
(Fluorescent nuclear binding dye). Apoptosis is assessed using
Laser Scanning Cytometer by measuring pyknotic nuclei.
[0196] Measurement of LDH release as an indication of cell death:
Cortical neurons are prepared from E18 rat fetus and plated in
48-well culture plates precoated with poly-D-lysine (10 ng/ml) and
serum at a density of 150,000 cells/well. Cells are plated in high
glucose DMEM containing 10% FCS and kept in the incubator at
37.degree. C. with 10% CO.sub.2/90% air. On the next day, serum is
removed by replacing culture media with high glucose DMEM
containing B27 supplement. On day 6, cells are divided into two
groups; control group and OGD group. Cells in control group receive
DMEM with glucose and custom B27 (without antioxidants). Cells in
OGD group receive no-glucose DMEM with custom B27, which has been
degassed under vacuum for 15 min. Cells are flushed with 90%
N.sub.2/10% CO.sub.2 for 10 min in an airtight chamber and
incubated at 37.degree. C. for 6 hrs. After 6 hrs, both control and
OGD cells are subject to replacement of media containing either
vehicle (DMSO) or test compound in glucose-containing DMEM with
custom B27. Cells are returned to normoxic incubator at 37.degree.
C. After 24 hrs, cell death is assessed by measuring cellular
release of LDH (lactate dehydrogenase) into the culture medium. For
LDH assay, an aliquot of 50 .mu.l culture medium is transferred
into the 96 well plate. After the addition of 140 .mu.l 0.1M
potassium phosphate buffer (pH 7.5) and 100 .mu.l 0.2 mg/ml NADH,
the plate is let sit in the dark at room temperature for 20 min.
The reaction is initiated by the addition of 10 .mu.l of sodium
pyruvate. The plate is read immediately at 340 nM in a Thermomax
plate reader (Molecular Devices). The absorbance, an index of NADH
concentration, is recorded every 6 seconds for 5 minutes and the
slope indicating the rate of NADH disappearance is used to
calculate LDH activity.
LDHActivity(U/ml)=(.DELTA.A/min)(TCF)(20)(0.0833)/(0.78) [0197]
where: 0.0833=proportionality constant [0198] 0.78=instrument light
path length (cm)
EXAMPLE 22
HLA Rat Test Procedure--Crohn's Disease and Inflammatory Bowel
Disorders
[0199] Male HLA-B27 rats are obtained from Taconic and provided
unrestricted access to a food (PMI Lab diet 5001) and water. At the
start of the study, rats are 22-26 weeks old.
[0200] Rats are dosed subcutaneously once per day for seven days
with one of the formulations listed below. There are five rats in
each group and the last dose is administered two hours before
euthanasia. [0201] vehicle (50% DMSO/50% Dulbecco's PBS) [0202]
17.alpha.-ethinyl-17.beta.-estradiol (10 .mu.g/kg) [0203] test
compound
[0204] Stool quality is observed daily and graded according to the
following scale: Diarrhea=3; soft stool=2; normal stool=1. At the
end of the test procedure, serum is collected and stored at
-70.degree. C. A section of colon is prepared for histological
analysis and an additional segment is analyzed for myeloperoxidase
activity.
[0205] The following method is used to measure myeloperoxidase
activity. Colon tissue is harvested and flash frozen in liquid
nitrogen. A representative sample of the entire colon is used to
ensure consistency between samples. The tissue is stored at
-80.degree. C. until use. Next, the tissue is weighed
(approximately 500 mg) and homogenized in 1:15 w/v of 5 mM
H.sub.2KPO.sub.4 (pH 6) washing buffer. The tissue is spun down at
20,000.times.g in a Sorvall RC 5B centrifuge for 45 minutes at
2-8.degree. C. Supernatant is then discarded. Tissue is resuspended
and homogenized in 2.5 ml (1:5 w/v) of 50 mM H.sub.2KPO.sub.4 with
10 mM EDTA and 0.5% Hex Ammonium Bromide to help solubilize the
intracellular MPO. Tissue is frozen in liquid Nitrogen, thawed in a
37.degree. C.-water bath and sonicated for 15 seconds to ensure
membrane lysis. This procedure is repeated 3 times. Samples are
then kept on ice for 20 minutes and centrifuged at 12,000.times.g
for 15 minutes at 2-8.degree. C. The supernatant is analyzed
following these steps.
[0206] The test mixture is prepared by adding 2.9 ml of 50 mM
H.sub.2KPO.sub.4 with 0.167 O-Dianisidine/ml with 0.0005%
H.sub.2O.sub.2 into a reaction tube. When hydrogen peroxide is
degraded, O-Dianisidine is oxidized and absorbs at 460 nm in a
concentration dependent manner. The mixture is heated to 25.degree.
C. One hundred (100) .mu.L of the tissue supernatant is added to
the reaction tube, incubated for one minute at 25.degree. C., then
1 ml is transferred to a disposable plastic cuvette. OD is measured
every 2 minutes reaction time at 460 nm against a blank containing
2.9 ml of the reaction mixture and 100 .mu.l of the 0.5% ammonium
bromide solution.
[0207] Enzyme activity units are quantified by comparison of
absorbence @ 460 to a standard curve prepared with purified human
MPO 31.1 UnitsNial. The MPO is reconstituted and serially diluted
using 50 mM H.sub.2KPO.sub.4 with 10 mM EDTA and 0.5% Hex Ammonium
Bromide to four known concentrations. Sample absorbencies are
compared against this curve to determine activity.
[0208] Histological analysis is performed as follows. Colonic
tissue is immersed in 10% neutral buffered formalin. Each specimen
of colon is separated into four samples for evaluation. The
formalin-fixed tissues are processed in a vacuum infiltration
processor for paraffin embedding. The samples are sectioned at 5
.mu.m and then stained with hematoxylin and eosin (H&E) for
blinded histologic evaluations using a scale modified after
Boughton-Smith. After the scores are completed the samples are
unblinded, and data are tabulated and analyzed by ANOVA linear
modeling with multiple mean comparisons.
[0209] All patents, publications, and other documents cited herein
are hereby incorporated by reference in their entirety.
* * * * *