Heterocyclic substituted benzothiophenes, compositions, and methods

The present invention provides class of compounds of formula I ##STR1## and their pharmaceutically acceptable salts in which R.sup.1 is hydrogen, hydroxy, alkoxy, alkoxycarbonyloxy, alkylcarbonyloxy, arylcarbonyloxy; R.sup.2 is hydrogen, hydroxy, alkoxy, alkoxycarbonyloxy, alkylcarbonyloxy, alkylsulfonyloxy, arylcarbonyloxy, Cl or Br; R.sup.3 and R.sup.4 are independently C.sub.1 -C.sub.4 alkyl, or combine with the nitrogen atom to which they are attached to form a pyrrolidino, piperidino, or hexamethyleneimino ring; X is selected from ##STR2## and Y is --CO--, --CHOH--, or --CH.sub.2 --. The compounds and pharmaceutical compositions containing the compounds, either alone or in combination with either progestin or estrogen, are useful for alleviating the symptoms of osteoporosis, cardiovascular related pathological conditions such as hyperlipidemia, and estrogen-dependent cancer. The compounds of the present invention also are useful for inhibiting uterine fibroid disease and endometriosis in women and aortal smooth muscle cell proliferation, particularly restenosis, in humans.

CROSS-REFERENCE TO RELATED APPLICATIONS 
This application claims the benefit of Provisional Application Serial No. 
60/013,291 filed Mar. 12, 1996. 
FIELD OF THE INVENTION 
The present invention relates to organic compounds having biological 
activity, to compositions comprising the compounds, and to medical methods 
of treatment. More particularly, the present invention concerns a class of 
heterocyclic substituted benzothiophene compounds, to pharmaceutical 
compositions comprising the compounds, and to methods of treating 
conditions associated with post-menopausal syndrome, including 
osteoporosis, cardiovascular disease (particularly hyperlipidemia), 
hormonally dependent cancers (particularly of the breast and uterine) and 
conditions not necessarily associated with post-menopause including 
uterine fibroid disease, endometriosis, and aortal smooth muscle cell 
proliferation. 
BACKGROUND OF THE INVENTION 
"Post-menopausal syndrome" is a term used to describe various pathological 
conditions which frequently affect women who have entered into or 
completed the physiological metamorphosis known as menopause. Although 
numerous pathologies are contemplated by the use of this term, three major 
effects of post-menopausal syndrome are the source of the greatest 
long-term medical concern: osteoporosis, cardiovascular effects such as 
hyperlipidemia, and estrogen-dependent cancer, particularly breast and 
uterine cancer. 
Osteoporosis describes a group of diseases which arise from diverse 
etiologies, but which are characterized by the net loss of bone mass per 
unit volume. The consequence of this loss of bone mass and resulting bone 
fracture is the failure of the skeleton to provide adequate structural 
support for the body. One of the most common types of osteoporosis is that 
associated with menopause. Most women lose from about 20% to about 60% of 
the bone mass in the trabecular compartment of the bone within 3 to 6 
years after the cessation of menses. This rapid loss is generally 
associated with an increase of bone resorption and formation. However, the 
resorptive cycle is more dominant and the result is a net loss of bone 
mass. Osteoporosis is a common and serious disease among post-menopausal 
women. 
There are an estimated 25 million women in the United States, alone, who 
are afflicted with this disease. The results of osteoporosis are 
personally harmful and also account for a large economic loss due its 
chronicity and the need for extensive and long term support 
(hospitalization and nursing home care) from the disease sequelae. This is 
especially true in more elderly patients. Additionally, although 
osteoporosis is not generally thought of as a life threatening condition, 
a 20% to 30% mortality rate is related with hip fractures in elderly 
women. A large percentage of this mortality rate can be directly 
associated with post-menopausal osteoporosis. 
The most vulnerable tissue in the bone to the effects of post-menopausal 
osteoporosis is the trabecular bone. This tissue is often referred to as 
spongy or cancellous bone and is particularly concentrated near the ends 
of the bone (near the joints) and in the vertebrae of the spine. The 
trabecular tissue is characterized by small osteoid structures which 
inter-connect with each other, as well as the more solid and dense 
cortical tissue which makes up the outer surface and central shaft of the 
bone. This inter-connected network of trabeculae gives lateral support to 
the outer cortical structure and is critical to the bio-mechanical 
strength of the overall structure. In post-menopausal osteoporosis, it is, 
primarily, the net resorption and loss of the trabeculae which leads to 
the failure and fracture of bone. In light of the loss of the trabeculae 
in post-menopausal women, it is not surprising that the most common 
fractures are those associated with bones which are highly dependent on 
trabecular support, e.g., the vertebrae, the neck of the weight bearing 
bones such as the femur and the fore-arm. Indeed, hip fracture, collies 
fractures, and vertebral crush fractures are hall-marks of post-menopausal 
osteoporosis. 
At this time, the generally accepted method for treatment of 
post-menopausal osteoporosis is estrogen replacement therapy. Although 
therapy is generally successful, patient compliance with the therapy is 
low primarily because estrogen treatment frequently produces undesirable 
side effects. 
Throughout premenopausal time, most women have less incidence of 
cardiovascular disease than age-matched men. Following menopause, however, 
the rate of cardiovascular disease in women slowly increases to match the 
rate seen in men. This loss of protection has been linked to the loss of 
estrogen and, in particular, to the loss of estrogen's ability to regulate 
the levels of serum lipids. The nature of estrogen's ability to regulate 
serum lipids is not well understood, but evidence to date indicates that 
estrogen can upregulate the low density lipid (LDL) receptors in the liver 
to remove excess cholesterol. Additionally, estrogen appears to have some 
effect on the biosynthesis of cholesterol, and other beneficial effects on 
cardiovascular health. 
It has been reported in the literature that post-menopausal women having 
estrogen replacement therapy have a return of serum lipid levels to 
concentrations to those of the pre-menopausal state. Thus, estrogen would 
appear to be a reasonable treatment for this condition. However, the 
side-effects of estrogen replacement therapy are not acceptable to many 
women, thus limiting the use of this therapy. An ideal therapy for this 
condition would be an agent which would regulate the serum lipid level as 
does estrogen, but would be devoid of the side-effects and risks 
associated with estrogen therapy. 
The third major pathology associated with post-menopausal syndrome is 
estrogen-dependent breast cancer and, to a lesser extent, 
estrogen-dependent cancers of other organs, particularly the uterus. 
Although such neoplasms are not solely limited to a post-menopausal women, 
they are more prevalent in the older, post-menopausal population. Current 
chemotherapy of these cancers has relied heavily on the use of 
anti-estrogen compounds such as, for example, tamoxifen. Although such 
mixed agonist-antagonists have beneficial effects in the treatment of 
these cancers, and the estrogenic side-effects are tolerable in acute 
life-threatening situations, they are not ideal. For example, these agents 
may have stimulatory effects on certain cancer cell populations in the 
uterus due to their estrogenic (agonist) properties and they may, 
therefore, be contraproductive in some cases. A better therapy for the 
treatment of these cancers would be an agent which is an anti-estrogen 
compound having negligible or no estrogen agonist properties on 
reproductive tissues. 
In response to the clear need for new pharmaceutical agents which are 
capable of alleviating the symptoms of, inter alia, post-menopausal 
syndrome, the present invention provides benzothiophene compounds, 
pharmaceutical compositions thereof, and methods of using such compounds 
for the treatment of post-menopausal syndrome and other estrogen-related 
pathological conditions such as those mentioned below. 
Uterine fibrosis (uterine fibroid disease) is an old and ever present 
clinical problem which goes under a variety of names, including uterine 
fibroid disease, uterine hypertrophy, uterine lieomyomata, myometrial 
hypertrophy, fibrosis uteri, and fibrotic metritis. Essentially, uterine 
fibrosis is a condition where there is an inappropriate deposition of 
fibroid tissue on the wall of the uterus. 
This condition is a cause of dysmenorrhea and infertility in women. The 
exact cause of this condition is poorly understood but evidence suggests 
that it is an inappropriate response of fibroid tissue to estrogen. Such a 
condition has been produced in rabbits by daily administrations of 
estrogen for 3 months. In guinea pigs, the condition has been produced by 
daily administration of estrogen for four months. Further, in rats, 
estrogen causes similar hypertrophy. 
The most common treatment of uterine fibrosis involves surgical procedures 
both costly and sometimes a source of complications such as the formation 
of abdominal adhesions and infections. In some patients, initial surgery 
is only a temporary treatment and the fibroids regrow. In those cases a 
hysterectomy is performed which effectively ends the fibroids but also the 
reproductive life of the patient. Also, gonadotropin releasing hormone 
antagonists may be administered, yet their use is tempered by the fact 
they can lead to osteoporosis. Thus, there exists a need for new methods 
for treating uterine fibrosis, and the methods of the present invention 
satisfy that need. 
Endometriosis is a condition of severe dysmenorrhea, which is accompanied 
by severe pain, bleeding into the endometrial masses or peritoneal cavity 
and often leads to infertility. The cause of the symptoms of this 
condition appear to be ectopic endometrial growths which respond 
inappropriately to normal hormonal control and are located in 
inappropriate tissues. Because of the inappropriate locations for 
endometrial growth, the tissue seems to initiate local inflammatory-like 
responses causing macrophage infiltration and a cascade of events leading 
to initiation of the painful response. The exact etiology of this disease 
is not well understood and its treatment by hormonal therapy is diverse, 
poorly defined, and marked by numerous unwanted and perhaps dangerous side 
effects. 
One of the treatments for this disease is the use of low dose estrogen to 
suppress endometrial growth through a negative feedback effect on central 
gonadotropin release and subsequent ovarian production of estrogen; 
however, it is sometimes necessary to use continuous estrogen to control 
the symptoms. This use of estrogen can often lead to undesirable side 
effects and even the risk of endometrial cancer. 
Another treatment consists of continuous administration of progestins which 
induces amenorrhea and by suppressing ovarian estrogen production can 
cause regressions of the endometrial growths. The use of chronic progestin 
therapy is often accompanied by the unpleasant CNS side effects of 
progestins and often leads to infertility due to suppression of ovarian 
function. 
A third treatment consists of the administration of weak androgens, which 
are effective in controlling the endometriosis; however, they induce 
severe masculinizing effects. Several of these treatments for 
endometriosis have also been implicated in causing a mild degree of bone 
loss with continued therapy. Therefore, new methods of treating 
endometriosis are desirable. 
Smooth aortal muscle cell proliferation plays an important role in diseases 
such as atherosclerosis and restenosis. Vascular restenosis after 
percutaneous transluminal coronary angioplasty (PTCA) has been shown to be 
a tissue response characterized by an early and late phase. The early 
phase occurring hours to days after PTCA is due to thrombosis with some 
vasospasms while the late phase appears to be dominated by excessive 
proliferation and migration of aortal smooth muscle cells. In this 
disease, the increased cell motility and colonization by such muscle cells 
and macrophages contribute significantly to the pathogenesis of the 
disease. The excessive proliferation and migration of vascular aortal 
smooth muscle cells may be the primary mechanism to the reocclusion of 
coronary arteries following PTCA, atherectomy, laser angioplasty and 
arterial bypass graft surgery. See "Intimal Proliferation of Smooth Muscle 
Cells as an Explanation for Recurrent Coronary Artery Stenosis after 
Percutaneous Transluminal Coronary Angioplasty," Austin et al., Journal of 
the American College of Cardiology, 8:369-375 (August 1985). 
Vascular restenosis remains a major long term complication following 
surgical intervention of blocked arteries by percutaneous transluminal 
coronary angioplasty (PTCA), atherectomy, laser angioplasty and arterial 
bypass graft surgery. In about 35% of the patients who undergo PTCA, 
reocclusion occurs within three to six months after the procedure. The 
current strategies for treating vascular restenosis include mechanical 
intervention by devices such as stents or pharmacologic therapies 
including heparin, low molecular weight heparin, coumarin, aspirin, fish 
oil, calcium antagonist, steroids, and prostacyclin. These strategies have 
failed to curb the reocclusion rate and have been ineffective for the 
treatment and prevention of vascular restenosis. See "Prevention of 
Restenosis after Percutaneous Transluminal Coronary Angioplasty: The 
Search for a `Magic Bullet`,"Hermans et al., American Heart Journal, 
122:171-187 (July 1991). 
In the pathogenesis of restenosis excessive cell proliferation and 
migration occurs as a result of growth factors produced by cellular 
constituents in the blood and the damaged arterial vessel wall which 
mediate the proliferation of smooth muscle cells in vascular restenosis. 
Agents that inhibit the proliferation and/or migration of smooth aortal 
muscle cells are useful in the treatment and prevention of restenosis. The 
present invention provides for the use of compounds as smooth aortal 
muscle cell proliferation inhibitors and, thus inhibitors of restenosis. 
SUMMARY OF THE INVENTION 
In its principal embodiment, the present invention provides a class of 
substituted benzob!thiophene compounds of formula I 
##STR3## 
or a pharmaceutically acceptable salt thereof in which R.sup.1 is selected 
from the group consisting of hydrogen, hydroxy, C.sub.1 -C.sub.4 alkoxy, 
##STR4## 
(in which Ar is optionally substituted phenyl), and --OSO.sub.2 (C.sub.4 
-C.sub.6 straight chain alkyl). 
R.sup.2 is selected from the group consisting of hydrogen, hydroxy, C.sub.1 
-C.sub.4 alkoxy, 
##STR5## 
##STR6## 
Cl and Br. 
The substituent groups R.sup.3 and R.sup.4 are independently C.sub.1 
-C.sub.4 alkyl, or combine to form, together with the nitrogen atom to 
which they are attached, a piperidino, pyrrolidino, or hexamethyleneimino 
ring; 
X is selected from the group consisting of 
##STR7## 
Y is selected from the group consisting of --CO--, --CHOH--, or --CH.sub.2 
--. 
The present invention further relates to pharmaceutical compositions 
containing compounds of formula I, optionally containing estrogen or 
progestin, and the use of such compounds, alone, or in combination with 
estrogen or progestin, for alleviating the symptoms of post-menopausal 
syndrome, particularly osteoporosis, cardiovascular related pathological 
conditions, and estrogen-dependent cancer. 
The compounds of the present invention also are useful for inhibiting 
uterine fibroid disease and endometriosis in women and aortal smooth 
muscle cell proliferation in humans.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, it has been found that a select 
group of 2-aryl-3-heterocyclic benzob!thiophenes, i.e., the compounds of 
formula I, are useful for the treatment or prevention of the symptoms and 
pathologies of: post-menopausal syndrome: osteoporosis, hyperlipidemia, 
estrogen dependent cancers, uterine fibroids, endometriosis, or restenosis 
in mammals, including humans. 
As used throughout this specification and the appended claims, the term 
"C.sub.1 -C.sub.6 alkyl" represents a straight or branched alkyl chain 
having from 1 to 6 carbon atoms. Typical C.sub.1 -C.sub.6 alkyl groups 
include methyl, ethyl, n-propyl, n-hexyl, and n-butyl. The term "C.sub.1 
-C.sub.4 alkoxy" represents groups such as methoxy, ethoxy, n-propoxy, and 
n-butoxy. 
"Optionally substituted phenyl" is meant to denote unsubstituted phenyl and 
phenyl substituted with one or two substituent groupos independently 
selected from C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.4 alkoxy, hydroxy, 
nitro, chloro, fluoro, and trichloro- or trifluoromethyl. 
The term "solvate" represents an aggregate that comprises one or more 
molecules of the solute, such as a formula I compound, with a molecule of 
solvent. 
The term "inhibit" is defined to include its generally accepted meaning 
which includes prohibiting preventing, restraining, and slowing, stopping 
or reversing progression, or severity, or such action on a resultant 
symptom. As such, the present invention includes both medical therapeutic 
and/or prophylactic administration, as appropriate. 
As used herein, the term "estrogen" includes steroidal compounds having 
estrogenic activity such as, for example, 17.beta.-estradiol, estrone, 
conjugated estrogen (Premarin.RTM.), equine estrogen, 17-.alpha.ethynyl 
estradiol, and the like. As used herein, the term "progestin" includes 
compounds having progestational activity such as, for example, 
progesterone, norethylnodrel, nongestrel, megestrol acetate, 
norethindrone, and the like. 
While the scope of the chemical compound aspect of the present invention is 
defined by formula I above, specific examples of compounds falling within 
the invention include, but are not necessarily limited to: 
2-(4-hydroxyphenyl)-6-hydroxybenzob!thien-3-yl!2-2-(1-piperidinyl)ethox 
y!pyrimidin-5-yl!methanone, 
2-(4-hydroxyphenyl)-6-hydroxybenzob!thien-3-yl!5-2-(1-piperidinyl)pyrim 
idin-2-yl!methanone, 
2-(4-hydroxyphenyl)-6-hydroxybenzob!thien-3-yl!2-2-(1-piperidinyl)ethox 
y!pyrazin-5-yl!methanone, 
2-(4-hydroxyphenyl)-6-hydroxybenzob!thien-3-yl!2-2-(1-piperidinyl)ethox 
y!pyridazin-5-yl!methanone, and 
2-(4-hydroxyphenyl)-6-hydroxybenzob!thien-3-yl!3-2-(1-piperidinyl)ethox 
y!pyridin-6-yl!methanone. 
Particularly preferred embodiments of this invention are the compounds 
where: R.sub.1 and R.sub.2 are --OH, R.sub.3 and R.sub.4 are taken 
together to form a piperidine ring, X is 2,5 disubstituted pyridine, and Y 
is --CO--, as its hydrochloride salt, for example, 
2-(4-hydroxyphenyl)-6-hydroxy-benzob!thien-3-yl!2-2-(1-piperidinyl)eth 
oxy!pyridin-5-yl!methanone. 
The compounds of the current invention can be made according to procedures 
well known in the art such as those detailed in U.S. Pat. Nos. 4,133,814; 
4,418,068; and 4,380,635, the teachings of which are incorporated herein 
by reference. 
In general, the process of preparing compounds of the present invention 
starts with a benzob!thiophene having a 6-hydroxyl group and a 
2-(4-hydroxyphenyl) group. The starting compound is protected by methods 
detailed in T. Green, et al., "Protective Groups in Organic Synthesis," 
Second Edition, John Wiley & Sons, New York, 1991. The protected compound 
is then acylated, and the resulting product subsequently deprotected to 
form the desired product of formula I, above. Examples of the preparation 
of such compounds are provided in the United States Patents listed above. 
The formula I compounds which are carboxylic esters or sulfonates may be 
prepared by methods described in U.S. Pat. Nos. 5,393,763; 5,482,949 and 
5,482,949. Modifications to the above methods may be necessary to 
accommodate reactive functionalities of particular substituents. Such 
modifications would be both apparent to, and readily ascertained by, those 
skilled in the art of organic chemistry. 
As shown above, the nitrogen containing heterocycle groups defined by the 
group "X" are oriented so that the bond at the top is intended to be read 
as attached to the ethoxy moiety and the bond at the bottom is intended to 
be read as bonded to the "Y" radical; that is: 
##STR8## 
Briefly, compounds of formula II 
##STR9## 
where R.sub.1 and R.sub.2 have the meanings ascribed above, are acylated 
at the 3-position of the benzothiophene nucleus with an activated carboxyl 
compounds of formula IIIa and IIIb under standard Friedel-Crafts 
conditions. 
##STR10## 
where R.sup.3 and R.sup.4 are as defined above. 
The compounds of formula II may be prepared in accordance with the methods 
described in U.S. Pat. No. 4,133,814. It is understood by those skilled in 
the art of organic chemistry that the ligands R.sub.1 and R.sub.2 must be 
compatible with the acylating conditions to form the compounds of formula 
I; thus a preferred intermediate of structure II would a compound where 
R.sub.1 and R.sub.2 are -OMe or other suitably protected hydroxyl group. 
The compounds of formula IIIa and IIIb are prepared from the corresponding 
hydroxy acids by alkylating the hydroxy group with a compound of formula 
IV, 
EQU Z--CH.sub.2 --CH.sub.2 --NR.sub.3 R.sub.4 IV 
in which Z is --Cl or --Br and R.sub.3 and R.sub.4 have their previous 
meanings, in the presence of a strong base, such as K.sub.2 CO.sub.3 or 
Cs.sub.2 CO.sub.3, followed by conversion by techniques well known in the 
art to the corresponding acid anhydride, acid chloride, or the like. 
In general, the conditions for the acylation reaction between compounds of 
type IIIa or IIIb and compounds of formula II involve the use of a Lewis 
acid such as, AlCl.sub.3, BF.sub.3, and the like, in an appropriate 
solvent such as a halogenated hydrocarbon, at temperatures ranging between 
about from 0.degree. C. and 100.degree. C. 
Although shown above, for purposes of illustration, the activated carboxyl 
compounds IIIa and IIIb are acyl chlorides, the activated carboxyl 
compounds employed in the acylation reaction may also be mixed anhydrides, 
and the like, although acid chlorides are preferred. 
In an alternate method for preparing the compounds of formula I, advantage 
is taken of the fact that halogen substituted, nitrogen containing, 
aromatic heterocycles undergo facile substitution reactions. Chloro or 
bromo substituents next to a ring nitrogen can be easily displaced by 
alkyl hydroxy moieties in the presence of a strong base. Thus, a compound 
of formula V can be converted to a compound of formula I by displacement 
of the chlorine with a compound of formula IV, where Z is hydroxyl. 
##STR11## 
The compounds of formula V are prepared by acylation of the 
benzob!thiophene (formula II) with an activated carboxylic acid moiety, 
e.g., an acid chloride, of a chloro-substituted nitrogen heterocyclic 
carboxylic acid under Friedel-Crafts conditions described above. An 
example of this transformation is given below in Preparation 3. 
The above chemical sequences for synthesis of the pyridine compounds are 
utilized for the preparation of other compounds of the present invention 
which contain heterocyclic rings such as derived from pyridazine, 
pyrimidine, and pyrazine. The starting materials for the synthesis are 
known in the art, e.g., the synthesis of 2-chloro-5-carboxy-pyridazine can 
be found in "The Chemistry of Heterocyclic Compound, Pyridazines", Ed. 
Castle R. N., John Wiley & Sons, NYC, p. 432-433; the synthesis of 
2-chloro-5-cyano pyrimidine can be found in J. Org. Chem., 29, p. 1740; 
the synthesis of 2-carboxy-5-chloro pyrimidine can be found in Collect. 
Czech. Chem. Comm., 37, p. 1721, etc. It would be appearent to those 
skilled in the art of organic chemistry how these compounds could be 
ulitized in the chemistry outlined above to obtain the compounds of 
formula I. 
Other compounds of formula I where Y is a carbinol or methylene can be 
prepared by reduction of the carbonyl group in compounds obtained by the 
processes described above to the carbinol and further, if desired, to 
methylene. Such reductions of the carbonyl group can be accomplished 
step-wise or from the carbonyl to methylene in a single step. 
To obtain compounds of the present invention where Y is --CH(OH)--, the 
carbonyl compound is reduced with LiAlH.sub.4, NaBH.sub.4, or the like in 
appropriate solvents such as chlorocarbons, tetrahydrofuran, ether, etc. 
at temperatures of ranging between about 0.degree. C. and 30.degree. C. 
The carbinol group resulting from such reductions may be further reduced to 
the methylene by the action of silanes, e.g., triethylsilane, in 
appropriate solvents such as, methylene chloride or THF with a strong acid 
such as trifluoroacetic acid, at ambient temperatures. 
Alternatively, the carbonyl compound may be reduced directly to the 
methylene by using LiAlH.sub.4 in a high boiling solvent such as 
propylbenzene at reflux temperatures. 
The following illustrative Examples are provided to enable one skilled in 
the art to practice the present invention. However, these examples are 
illustrative only, and should not be read as limiting the scope of the 
invention as it is defined by the appended claims. 
Preparation 1 
Preparation of 
2-(4-Hydroxyphenyl)-6-hydroxybenzob!thien-3-yl!2-2-(1-piperidinyl)etho 
xy!pyridin-5-yl!methanone 
2-(4-Methoxyphenyl)-6-methoxybenzob!thien-3-yl!2-2-(1-piperidinyl)ethox 
y!-pyridin-5-yl!methanone (1.8 g, 3.58 mmol) of was dissolved in 20 mL of 
dichloroethane and 3.1 mL (35.8 mmol) of condensed BCl.sub.3 was added. 
The reaction mixture was stirred at ambient temperature under a nitrogen 
atmosphere for forty-eight hours. The reaction was quenched with 5 mL of 
MeOH and reaction was stirred for one hour. The reaction volume was 
evaporated to half the orginal volume and chromatographed on a silica gel 
column eluted with a linear gradient starting with CHCl.sub.3 and ending 
with CHCl.sub.3 -MeOH (9:1) (v/v). The desired fractions were determined 
by tlc, combined, and evaporated to dryness. The crude product was again 
chromatographed on a silica gel column eluted with CHCl.sub.3 -MeOH 
(19:1). This yielded 540 mg of the title compound as a yellow-orange, 
amorphous solid. 
Proton magnetic resonance spectrum: Consistent with the proposed structure 
MS: m/e=475 (M+) FD 
Preparation 2 
Preparation of 
2-(4-Methoxyphenyl)-6-methoxybenzob!thien-3-yl!2-2-(1-piperidinyl)etho 
xy!pyridin-5-yl!methanone 
2-(4-Methoxyphenyl)-6-methoxybenzob!thien-3-yl!2-chloropyridin-5-yl!meth 
anone (1 g, 2.44 mmol) was dissolved in 10 mL of benzene and 950 mg (7.32 
mmol) of 2-hydroxyethyl-1-piperidine was added. To the reaction mixture 
was added 85 mg (3.7 mmol) of sodium and the reaction was stirred at 
ambient temperature under a nitrogen atmosphere until all the sodium 
dissolved. The reaction was heated to 70.degree. C. for two and half 
hours, then allowed to cool. To the reaction solution was added 150 mL of 
EtOAc and the solution was washed three times with saturated aqueous 
Na.sub.2 CO.sub.3. The organic layer was separated and evaporated to 
dryness. The crude product was chromatographed on a silica gel column 
eluted with a linear gradient beginning with CHCl.sub.3 and ending with 
CHCl.sub.3 -MeOH (19:1). The desired fraction were determined by tlc, 
combined, and evaporated to dryness. This yielded the title compound as a 
yellow, amorphous solid. 
Proton magnetic resonance spectrum: Consistent with the proposed structure 
Preparation 3 
Preparation of 
2-(4-Methoxyphenyl)-6-methoxybenzob!thien-3-yl!2-chloropyridin-5-yl!met 
hanone 
2-(Methoxyphenyl)-6-methoxybenzob!thiophene (7.8 g, 29 mmol) was dissolved 
in 250 mL of dichloromethane. To this solution was added the 
2-chloro-5-carboxychloropyridine (from Preparation 4) in 20 mL of 
dichloromethane. 31 g (232 mmol) of AlCl.sub.3 was added to the reaction 
in six portions over a hour period. The reaction was allowed to proceed at 
ambient temperature, under a nitrogen atmosphere for three and half hours. 
The reaction was quenched by pouring it over ice and the organic layer 
separated. The organic layer was washed three times with 500 mL of 
saturated, aqueous Na.sub.2 CO.sub.3, Dried by filtration through 
anhydrous Na.sub.2 SO.sub.4 and evaporated to dryness. The crude product 
was chromatographed on a silica gel column eluted with EtOAc-hexane (1:9). 
The desired fractions were determined by tlc, combined, and evaporated to 
dryness. This yielde the title compound as a yellow powder. 
Proton magnetic resonance spectrum: Consistent with the 
proposed structure 
MS: m/e=409 (M+) FD 
Elemental Analysis: Calc for C.sub.22 H.sub.16 ClNO.sub.3 S: C, 64.47; H, 
3.9; N, 3.42; Found: C, 64.77; H, 4.01; N, 3.16 
Preparation 4 
Preparation of 2-Chloro-5-carboxychloropyridine 
2-Hydroxypyridine-5-carboxylic acid (4 g, 29 mmol) was dissolved 150 mL of 
dichloromethane, 50 mL of SOCl.sub.2, and five drops of DMF. The raction 
mixture was heated to reflux under a nitrogen atmosphere for sixteen 
hours. The reaction mixture was evaporated to an oily solid and used 
without further purification or characterization (see Preparation 3). 
Test Procedure 
General Preparation Procedure 
In the examples illustrating the methods, a post-menopausal model was used 
in which effects of different treatments upon circulating lipids were 
determined. 
Seventy-five day old female Sprague Dawley rats (weight range of 200 to 225 
g) are obtained from Charles River Laboratories (Portage, Mich.). The 
animals are either bilaterally ovariectomized (OVX) or exposed to a Sham 
surgical procedure at Charles River Laboratories, and then shipped after 
one week. Upon arrival, they are housed in metal hanging cages in groups 
of 3 or 4 per cage and have ad libitum access to food (calcium content 
approximately 0.5%) and water for one week. Room temperature is maintained 
at 22.2.degree..+-.1.7.degree. C. with a minimum relative humidity of 40%. 
The photoperiod in the room is 12 hours light and 12 hours dark. 
Dosing Regimen Tissue Collection. After a one week acclimation period 
(therefore, two weeks post-OVX) daily dosing with test compound is 
initiated. 17.alpha.-ethynyl estradiol or the test compound is given 
orally, unless otherwise stated, as a suspension in 1% 
carboxymethylcellulose or dissolved in 20% cyclodextrin. Animals were 
dosed daily for 4 days. Following the dosing regimen, animals are weighed 
and anesthetized with a ketamine: Xylazine (2:1, V:V) mixture and a blood 
sample is collected by cardiac puncture. The animals are then sacrificed 
by asphyxiation with CO.sub.2, the uterus is removed through a midline 
incision, and a wet uterine weight is determined. 
Cholesterol Analysis. Blood samples are allowed to clot at room temperature 
for 2 hours, and serum is obtained following centrifugation for 10 minutes 
at 3000 rpm. Serum cholesterol is determined using a Boehringer Mannheim 
Diagnostics high performance cholesterol assay. Briefly the cholesterol is 
oxidized to cholest-4-en-3-one and hydrogen peroxide. The hydrogen 
peroxide is then reacted with phenol and 4-aminophenazone in the presence 
of peroxidase to produce a p-quinone imine dye, which is read 
spectrophotemetrically at 500 nm. Cholesterol concentration is then 
calculated against a standard curve. The entire assay is automated using a 
Biomek Automated Workstation. 
Uterine Eosinophil Peroxidase (EPO) Assay. Uteri are kept at 4.degree. C. 
until time of enzymatic analysis. The uteri are then homogenized in 50 
volumes of 50 mM Tris buffer (pH-8.0) containing 0.005% Triton X-100. Upon 
addition of 0.01% hydrogen peroxide and 10 mMO-phenylenediamine (final 
concentrations) in Tris buffer, increase in absorbance is monitored for 
one minute at 450 nm. The presence of eosonophils in the uterus is an 
indication of estrogenic activity of a compound. The maximal velocity of a 
15 second interval is determined over the initial, linear portion of the 
reaction curve. 
Source of Compound: 17.alpha.-ethynyl estradiol was obtained from Sigma 
Chemical Co., St. Louis, Mo. 
Hyperlipidemia: 
Data presented in Table 1 show comparative results among ovariectomized 
rats, rats treated with 17-a-ethynyl estradiol(EE.sub.2), and rats treated 
with certain compounds of this invention. Although EE.sub.2 caused a 
decrease in serum cholesterol when orally administered at 0.1 mg/kg/day, 
it also exerted a simulatory effect on the uterus so that EE.sub.2 uterine 
weight was substantially greater than the uterine weight of the 
ovariectomized animals. This uterine response to an estrogen is well 
recognized in the art. 
Not only did the compounds of the present invention reduce serum 
cholesterol compared to the ovariectomized animals, but the uterine weight 
was only minimally increased. Compared to estrogenic compounds known in 
the art, the benefit of serum cholesterol reduction without adversely 
affecting uterine weight is unusual and desirable. 
As expressed in the data below, estrogenicity also was assessed by 
evaluating the response of eosinophil infiltration into the uterus. The 
compounds of this invention did not cause a large increase in the number 
of eosinophils observed in the stromal layer of the ovariectomized, rat 
uteri. EE.sub.2 caused a substantial and expected increase in eosinophil 
infiltration. 
The data presented in Table 1 reflect the response of five or six rats per 
treatment group. 
TABLE 1 
______________________________________ 
Increase in 
Serum Decrease in 
Dose Uterine Weight 
Eosinophil 
Cholesterol 
Compound 
(mg/kg.sup.a) 
(%) (V.sub.max).sup.c 
(%) 
______________________________________ 
EE2.sup.e 
0.1 182.1* 211.1* 87.4* 
Prep. 1 0.1 80.3* 48.0* 57.1* 
1.0 91.4* 70.5* 66.1* 
10.0 80.8* 55.8* 61.7* 
______________________________________ 
.sup.a 17a-Ethynyl estradiol 
.sup.b Uterine Weight % increase versus the ovarierectomized controls 
.sup.c Eosinphil peroxidase Vmaxium 
.sup.d Serum cholesterol decrease versus ovariectomized controls 
*p &lt; .05 
Osteoporosis Test Procedure 
Following the General Preparation Procedure, infra, the rats are treated 
daily for 35 days (6 rats per treatment group) and sacrificed by carbon 
dioxide asphyxiation on the 36th day. The 35 day time period is sufficient 
to allow maximal reduction in bone density, measured as described herein. 
At the time of sacrifice, the uteri are removed, dissected free of 
extraneous tissue, and the fluid contents are expelled before 
determination of wet weight in order to confirm estrogen deficiency 
associated with complete ovariectomy. Uterine weight is routinely reduced 
about 75% in response to ovariectomy. The uteri are then placed in 10% 
neutral buffered formalin to allow for subsequent histological analysis. 
The right femurs are excised and digitilized x-rays generated and analyzed 
by an image analysis program (NIH image) at the distal metaphysis. The 
proximal aspect of the tibiae from these animals are also scanned by 
quantitative computed tomography. 
In accordance with the above procedures, compounds of the present invention 
and ethynyl estradiol (EE.sub.2) in 20% hydroxypropyl .beta.-cyclodextrin 
are orally administered to test animals. 
In summary, ovariectomy of the test animals causes a significant reduction 
in femur density compared to intact, vehicle treated controls. Orally 
administered ethynyl estradiol (EE.sub.2) prevented this loss, but the 
risk of uterine stimulation with this treatment is ever-present. 
The compounds of the present invention prevent bone loss in a general, 
dose-dependent manner. Accordingly, the compounds of the present invention 
are useful for the treatment of post-menopausal syndrome, particularly 
osteoporosis. 
MCF-7 Proliferation Assay 
MCF-7 breast adenocarcinoma cells (ATCC HTB 22) are maintained in MEM 
(minimal essential medium, phenol red-free, Sigma, St. Louis, Mo.) 
supplemented with 10% fetal bovine serum (FBS) (V/V), L-glutamine (2 mM), 
sodium pyruvate (1 mM), HEPES 
{(N-2-hydroxyethyl!piperazine-N'-2-ethanesulfonic acid!10 mM}, 
non-essential amino acids and bovine insulin (1 ug/mL) (maintenance 
medium). Ten days prior to assay, MCF-7 cells are switched to maintenance 
medium supplemented with 10% dextran coated charcoal stripped fetal bovine 
serum (DCC-FBS) assay medium) in place of 10% FBS to deplete internal 
stores of steroids. MCF-7 cells are removed from maintenance flasks using 
cell dissociation medium (Ca++/Mg++ free HBSS (phenol red-free) 
supplemented with 10 mM HEPES and 2 mM EDTA). Cells are washed twice with 
assay medium and adjusted to 80,000 cells/mL. Approximately 100 .mu.L 
(8,000 cells) are added to flat-bottommicroculture wells (Costar 3596) and 
incubated at 37.degree. C. in a 5% CO.sub.2 humidified incubator for 48 
hours to allow for cell adherence and equilibration after transfer. Serial 
dilutions of drugs or DMSO as a diluent control are prepared in assay 
medium and 50 .mu.L transferred to triplicate microcultures followed by 50 
.mu.L assay medium for a final volume of 200 .mu.L. After an additional 48 
hours at 37.degree. C. in a 5% CO.sub.2 humidified incubator, 
microcultures are pulsed with tritiated thymidine (1 uCi/well) for 4 
hours. Cultures are terminated by freezing at -70.degree. C. for 24 hours 
followed by thawing and harvesting of microcultures using a Skatron 
Semiautomatic Cell Harvester. Samples are counted by liquid scintillation 
using a Wallac BetaPlace .beta. counter. The compound in Preparation 1 is 
a potent inhibitor of the growth of MCF-7 cells with an IC.sub.50 of 10 
nM. 
DMBA-Induced Mammary Tumor Inhibition 
Estrogen-dependent mammary tumors are produced in female Sprague-Dawley 
rats which are purchased from Harlan Industries, Indianapolis, Ind. At 
about 55 days of age, the rats receive a single oral feeding of 20 mg of 
7,12-dimethylbenza!anthracene (DMBA). About 6 weeks after DMBA 
administration, the mammary glands are palpated at weekly intervals for 
the appearance of tumors. Whenever one or more tumors appear, the longest 
and shortest diameters of each tumor are measured with a metric caliper, 
the measurements are recorded, and that animal is selected for 
experimentation. An attempt is made to uniformly distribute the various 
sizes of tumors in the treated and control groups such that average-sized 
tumors are equivalently distributed between test groups. Control groups 
and test groups for each experiment contain 5 to 9 animals. 
Compounds of Formula I are administered either through intraperitoneal 
injections in 2% acacia, or orally. Orally administered compounds are 
either dissolved or suspended in 0.2 mL corn oil. Each treatment, 
including acacia and corn oil control treatments, is administered once 
daily to each test animal. Following the initial tumor measurement and 
selection of test animals, tumors are measured each week by the 
above-mentioned method. The treatment and measurements of animals continue 
for 3 to 5 weeks at which time the final areas of the tumors are 
determined. For each compound and control treatment, the change in the 
mean tumor area is determined. 
Uterine Fibrosis Test Procedures 
Test 1 
Between 3 and 20 women having uterine fibrosis are administered a compound 
of the present invention. The amount of compound administered is from 0.1 
to 1000 mg/day, and the period of administration is 3 months. 
The women are observed during the period of administration, and up to 3 
months after discontinuance of administration, for effects on uterine 
fibrosis. 
Test 2 
The same procedure is used as in Test 1, except the period of 
administration is 6 months. 
Test 3 
The same procedure is used as in Test 1, except the period of 
administration is 1 year. 
Test 4 
A. Induction of fibroid tumors in guinea pig. 
Prolonged estrogen stimulation is used to induce leiomyomata in sexually 
mature female guinea pigs. Animals are dosed with estradiol 3-5 times per 
week by injection for 2-4 months or until tumors arise. Treatments 
consisting of a compound of the invention or vehicle is administered daily 
for 3-16 weeks and then animals are sacrificed and the uteri harvested and 
analyzed for tumor regression. 
B. Implantation of human uterine fibroid tissue in nude mice. 
Tissue from human leiomyomas are implanted into the peritoneal cavity and 
or uterine myometrium of sexually mature, castrated, female, nude mice. 
Exogenous estrogen are supplied to induce growth of the explanted tissue. 
In some cases, the harvested tumor cells are cultured in vitro prior to 
implantation. Treatment consisting of a compound of the present invention 
or vehicle is supplied by gastric lavage on a daily basis for 3-16 weeks 
and implants are removed and measured for growth or regression. At the 
time of sacrifice, the uteri is harvested to assess the status of the 
organ. 
Test 5 
A. Tissue from human uterine fibroid tumors is harvested and maintained, in 
vitro, as primary nontransformed cultures. Surgical specimens are pushed 
through a sterile mesh or sieve, or alternately teased apart from 
surrounding tissue to produce a single cell suspension. Cells are 
maintained in media containing 10% serum and antibiotic. Rates of growth 
in the presence and absence of estrogen are determined. Cells are assayed 
for their ability to produce complement component C3 and their response to 
growth factors and growth hormone. In vitro cultures are assessed for 
their proliferative response following treatment with progestins, GnRH, a 
compound of the present invention and vehicle. Levels of steroid hormone 
receptors are assessed weekly to determine whether important cell 
characteristics are maintained in vitro. Tissue from 5-25 patients are 
utilized. 
Activity in at least one of the above tests indicates the compounds of the 
present invention are of potential in the treatment of uterine fibrosis. 
Endometriosis Test Procedure 
In Tests 1 and 2, effects of 14-day and 21-day administration of compounds 
of the present invention on the growth of explanted endometrial tissue can 
be examined. 
Test 1 
Twelve to thirty adult CD strain female rats are used as test animals. They 
are divided into three groups of equal numbers. The estrous cycle of all 
animals is monitored. On the day of proestrus, surgery is performed on 
each female. Females in each group have the left uterine horn removed, 
sectioned into small squares, and the squares are loosely sutured at 
various sites adjacent to the mesenteric blood flow. In addition, females 
in Group 2 have the ovaries removed. 
On the day following surgery, animals in Groups 1 and 2 receive 
intraperitoneal injections of water for 14 days whereas animals in Group 3 
receive intraperitoneal injections of 1.0 mg of a compound of the present 
invention per kilogram of body weight for the same duration. Following 14 
days of treatment, each female is sacrificed and the endometrial explants, 
adrenals, remaining uterus, and ovaries, where applicable, are removed and 
prepared for histological examination. The ovaries and adrenals are 
weighed. 
Test 2 
Twelve to thirty adult CD strain female rats are used as test animals. They 
are divided into two equal groups. The estrous cycle of all animals is 
monitored. On the day of proestrus, surgery is performed on each female. 
Females in each group have the left uterine horn removed, sectioned into 
small squares, and the squares are loosely sutured at various sites 
adjacent to the mesenteric blood flow. 
Approximately 50 days following surgery, animals assigned to Group 1 
receive intraperitoneal injections of water for 21 days whereas animals in 
Group 2 receive intraperitoneal injections of 1.0 mg of a compound of the 
present invention per kilogram of body weight for the same duration. 
Following 21 days of treatment, each female is sacrificed and the 
endometrial explants and adrenals are removed and weighed. The explants 
are measured as an indication of growth. Estrous cycles are monitored. 
Test 3 
A. Surgical induction of endometriosis 
Autographs of endometrial tissue are used to induce endometriosis in rats 
and/or rabbits. Female animals at reproductive maturity undergo bilateral 
oophorectomy, and estrogen is supplied exogenously thus providing a 
specific and constant level of hormone. Autologous endometrial tissue is 
implanted in the peritoneum of 5-150 animals and estrogen supplied to 
induce growth of the explanted tissue. Treatment consisting of a compound 
of the present invention is supplied by gastric lavage on a daily basis 
for 3-16 weeks, and implants are removed and measured for growth or 
regression. At the time of sacrifice, the intact horn of the uterus is 
harvested to assess status of endometrium. 
B. Implantation of human endometrial tissue in nude mice. 
Tissue from human endometrial lesions is implanted into the peritoneum of 
sexually mature, castrated, female, nude mice. Exogenous estrogen is 
supplied to induce growth of the explanted tissue. In some cases, the 
harvested endometrial cells are cultured in vitro prior to implantation. 
Treatment consisting of a compound of the present invention supplied by 
gastric lavage on a daily basis for 3-16 weeks, and implants are removed 
and measured for growth or regression. At the time of sacrifice, the uteri 
is harvested to assess the status of the intact endometrium. 
Test 4 
A. Tissue from human endometrial lesions is harvested and maintained in 
vitro as primary nontransformed cultures. Surgical specimens are pushed 
through a sterile mesh or sieve, or alternately teased apart from 
surrounding tissue to produce a single cell suspension. Cells are 
maintained in media containing 10% serum and antibiotic. Rates of growth 
in the presence and absence of estrogen are determined. Cells are assayed 
for their ability to produce complement component C3 and their response to 
growth factors and growth hormone. In vitro cultures are assessed for 
their proliferative response following treatment with progestins, GnPa, a 
compound of the invention, and vehicle. Levels of steroid hormone 
receptors are assessed weekly to determine whether important cell 
characteristics are maintained in vitro. Tissue from 5-25 patients is 
utilized. 
Activity in any of the above assays indicates that the compounds of the 
present invention are useful in the treatment of endometriosis. 
Inhibition of Aortal Smooth Cell Proliferation/Restenosis 
Test Procedure 
Compounds of the present invention have capacity to inhibit aortal smooth 
cell proliferation. This can be demonstrated by using cultured smooth 
cells derived from rabbit aorta, proliferation being determined by the 
measurement of DNA synthesis. Cells are obtained by explant method as 
described in Ross, J. of Cell Bio. 50: 172 (1971). Cells are plated in 96 
well microtiter plates for five days. The cultures become confluent and 
growth arrested. The cells are then transferred to Dulbecco's Modified 
Eagle's Medium (DMEM) containing 0.5-2% platelet poor plasma, 2 
mML-glutamine, 100 U/ml penicillin, 100 mg ml streptomycin, 1 mC/ml .sup.3 
H-thymidine, 20 ng/ml platelet-derived growth factor, and varying 
concentrations of the present compounds. Stock solution of the compounds 
is prepared in dimethyl sulphoxide and then diluted to appropriate 
concentration (0.01-30 mM) in the above assay medium. Cells are then 
incubated at 37.degree. C. for 24 hours under 5% CO.sub.2 /95% air. At the 
end of 24 hours, the cells are fixed in methanol. .sup.3 H thymidine 
incorporation in DNA is then determined by scintillation counting as 
described in Bonin, et al., Exp. Cell Res. 181:475-482 (1989). 
Inhibition of aortal smooth muscle cell proliferation by the compounds of 
the present invention are further demonstrated by determining their 
effects on exponentially growing cells. Smooth muscle cells from rabbit 
aortae are seeded in 12 well tissue culture plates in DMEM containing 10% 
fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml 
streptomycin. After 24 hours, the cells are attached and the medium is 
replaced with DMEM containing 10% serum, 2 mM L-glutamine, 100 U/ml 
penicillin, 100 mg/ml streptomycin, and desired concentrations of the 
compounds. Cells are allowed to grow for four days. Cells are treated with 
trypsin and the number of cells in each culture is determined by counting 
using a ZM-Coulter counter. 
Activity in the above assays indicates that the compounds of the present 
invention are of potential in the treatment of restenosis. 
The present invention also provides a method of alleviating post-menopausal 
syndrome in women which comprises the aforementioned method using 
compounds of Formula I and further comprises administering to a woman an 
effective amount of estrogen or progestin. These treatments are 
particularly useful for treating osteoporosis and lowering serum 
cholesterol because the patient will receive the benefits of each 
pharmaceutical agent while the compounds of the present invention would 
inhibit undesirable side-effects of estrogen and progestin. Activity of 
these combination treatments in any of the post-menopausal tests, infra, 
indicates that the combination treatments are useful for alleviating the 
symptoms of post-menopausal symptoms in women. 
Various forms of estrogen and progestin are commercially available. 
Estrogen-based agents include, for example, ethynyl estrogen (0.01-0.03 
mg/day), mestranol (0.05-0.15 mg/day), and conjugated estrogenic hormones 
such as Premarin.RTM. (Wyeth-Ayerst; 0.3-2.5 mg/day). Progestin-based 
agents include, for example, medroxyprogesterone such as Provera.RTM. 
(Upjohn; 2.5-10 mg/day), norethylnodrel (1.0-10.0 mg/day), and 
nonethindrone (0.5-2.0 mg/day). A preferred estrogen-based compound is 
Premarin, and norethylnodrel and norethindrone are preferred 
progestin-based agents. 
The method of administration of each estrogen- and progestin-based agent is 
consistent with that which is known in the art. For the majority of the 
methods of the present invention, compounds of Formula I are administered 
continuously, from 1 to 3 times daily. However, cyclical therapy may 
especially be useful in the treatment of endometriosis or may be used 
acutely during painful attacks of the disease. In the case of restenosis, 
therapy may be limited to short (1-6 months) intervals following medical 
procedures such as angioplasty. 
As used herein, the term "effective amount" means an amount of compound of 
formula I which is capable of alleviating the symptoms of the various 
pathological conditions herein described. The specific dose of a compound 
administered according to this invention will, of course, be determined by 
the particular circumstances surrounding the case including, for example, 
the compound administered, the route of administration, the state of being 
of the patient, and the pathological condition being treated. Atypical 
daily dose will contain a nontoxic dosage level of from about 0.1 mg to 
about 1000 mg/day of a compound of the present invention, and more 
particularly will be from about 20 mg to about 200 mg/day. 
The compounds of this invention form pharmaceutically acceptable acid and 
base addition salts with a wide variety of organic and inorganic acids and 
bases and include the physiologically acceptable salts which are often 
used in pharmaceutical chemistry. Such salts are also part of this 
invention. Typical inorganic acids used to form such salts include 
hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, 
hypophosphoric and the like. Salts derived from organic acids, such as 
aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, 
hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and 
aromatic sulfonic acids, may also be used. Such pharmaceutically 
acceptable salts thus include acetate, phenylacetate, trifluoroacetate, 
acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, 
hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, 
naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, 
.beta.-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate, 
caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate, 
heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, 
mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, 
phthalate, teraphthalate, phosphate, monohydrogenphosphate, 
dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, 
phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, 
bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzene-sulfonate, 
p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 
2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, 
naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartarate, 
and the like. A preferred salt is the hydrochloride salt. 
The pharmaceutically acceptable acid addition salts are typically formed by 
reacting a compound of formula I with an equimolar or excess amount of 
acid. The reactants are generally combined in a mutual solvent such as 
diethyl ether or benzene. The salt normally precipitates out of solution 
within about one hour to 10 days and can be isolated by filtration or the 
solvent can be stripped off by conventional means. 
Bases commonly used for formation of salts include ammonium hydroxide and 
alkali and alkaline earth metal hydroxides, carbonates, as well as 
aliphatic and primary, secondary and tertiary amines, aliphatic diamines. 
Bases especially useful in the preparation of addition salts include 
ammonium hydroxide, potassium carbonate, methylamine, diethylamine, 
ethylene diamine and cyclohexylamine. 
The pharmaceutically acceptable salts generally have enhanced solubility 
characteristics compared to the compound from which they are derived, and 
thus are often more amenable to formulation as liquids or emulsions. 
It is usually preferred to administer a compound of formula I in the form 
of an acid addition salt, as is customary in the administration of 
pharmaceuticals bearing a basic group, such as the piperidino ring. It is 
also advantageous to administer such a compound by the oral route. For 
such purposes the following oral dosage forms are available. 
The compounds of this invention can be administered by a variety of routes 
including oral, rectal, transdermal, subucutaneus, intravenous, 
intramuscular, and intranasal. These compounds preferably are formulated 
prior to administration, the selection of which will be decided by the 
attending physician. Thus, another aspect of the present invention is a 
pharmaceutical composition comprising an effective amount of a compound of 
Formula I, or a pharmaceutically acceptable salt thereof, optionally 
containing an effective amount of estrogen or progestin, and a 
pharmaceutically acceptable carrier, diluent, or excipient. 
The total active ingredients in such formulations comprises from 0.1% to 
99.9% by weight of the formulation. By "pharmaceutically acceptable" it is 
meant the carrier, diluent, excipients and salt must be compatible with 
the other ingredients of the formulation, and not deleterious to the 
recipient thereof. 
Pharmaceutical formulations of the present invention can be prepared by 
procedures known in the art using well known and readily available 
ingredients. For example, the compounds of formula I, with or without an 
estrogen or progestin compound, can be formulated with common excipients, 
diluents, or carriers, and formed into tablets, capsules, suspensions, 
powders, and the like. Examples of excipients, diluents, and carriers that 
are suitable for such formulations include the following: fillers and 
extenders such as starch, sugars, mannitol, and silicic derivatives; 
binding agents such as carboxymethyl cellulose and other cellulose 
derivatives, alginates, gelatin, and polyvinyl-pyrrolidone; moisturizing 
agents such as glycerol; disintegrating agents such as calcium carbonate 
and sodium bicarbonate; agents for retarding dissolution such as paraffin; 
resorption accelerators such as quaternary ammonium compounds; surface 
active agents such as cetyl alcohol, glycerol monostearate; adsorptive 
carriers such as kaolin and bentonite; and lubricants such as talc, 
calcium and magnesium stearate, and solid polyethyl glycols. 
The compounds also can be formulated as elixirs or solutions for convenient 
oral administration or as solutions appropriate for parenteral 
administration, for example, by intramuscular, subcutaneous or intravenous 
routes. Additionally, the compounds are well suited to formulation as 
sustained release dosage forms and the like. The formulations can be so 
constituted that they release the active ingredient only or preferably in 
a particular physiological location, possibly over a period of time. The 
coatings, envelopes, and protective matrices may be made, for example, 
from polymeric substances or waxes. 
Compounds of formula I, alone or in combination with a pharmaceutical agent 
of the present invention, generally will be administered in a convenient 
formulation. The following formulation examples only are illustrative and 
are not intended to limit the scope of the present invention. 
Formulations 
In the formulations which follow, "active ingredient" means a compound of 
formula I, or a salt or solvate thereof. 
Formulation 1: Gelatin Capsules 
Hard gelatin capsules are prepared using the following: 
______________________________________ 
Ingredient Quantity (mg/capsule) 
______________________________________ 
Active ingredient 0.1-1000 
Starch, NF 0-650 
Starch flowable powder 
0-650 
Silicone fluid 350 centistokes 
0-15 
______________________________________ 
The formulation above may be changed in compliance with the reasonable 
variations provided. 
A tablet formulation is prepared using the ingredients below: 
Formulation 2: Tablets 
______________________________________ 
Ingredient Quantity (mg/tablet) 
______________________________________ 
Active ingredient 
2.5-1000 
Cellulose, microcrystalline 
200-650 
Silicon dioxide, fumed 
10-650 
Stearate acid 5-15 
______________________________________ 
The components are blended and compressed to form tablets. 
Alternatively, tablets each containing 2.5-1000 mg of active ingredient are 
made up as follows: 
Formulation 3: Tablets 
______________________________________ 
Ingredient Quantity (mg/tablet) 
______________________________________ 
Active ingredient 25-1000 
Starch 45 
Cellulose, microcrystalline 
35 
Polyvinylpyrrolidone 
4 
(as 10% solution in water) 
Sodium carboxymethyl cellulose 
4.5 
Magnesium stearate 0.5 
Talc 1 
______________________________________ 
The active ingredient, starch, and cellulose are passed through a No. 45 
mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone 
is mixed with the resultant powders which are then passed through a No. 14 
mesh U.S. sieve. The granules so produced are dried at 
50.degree.-60.degree. C. and passed through a No. 18 mesh U.S. sieve. The 
sodium carboxymethyl starch, magnesium stearate, and talc, previously 
passed through a No. 60 U.S. sieve, are then added to the granules which, 
after mixing, are compressed on a tablet machine to yield tablets. 
Suspensions each containing 0.1-1000 mg of medicament per 5 ml dose are 
made as follows: 
Formulation 4: Suspensions 
______________________________________ 
Ingredient Quantity (mg/5 ml) 
______________________________________ 
Active ingredient 0.1-1000 mg 
Sodium carboxymethyl cellulose 
50 mg 
Syrup 1.25 mg 
Benzoic acid solution 
0.10 mL 
Flavor q.v. 
Color q.v. 
Purified water to 5 mL 
______________________________________ 
The medicament is passed through a No. 45 mesh U.S. sieve and mixed with 
the sodium carboxymethyl cellulose and syrup to form a smooth paste. The 
benzoic acid solution, flavor, and color are diluted with some of the 
water and added, with stirring. Sufficient water is then added to produce 
the required volume. 
An aerosol solution is prepared containing the following ingredients: 
Formulation 5: Aerosol 
______________________________________ 
Ingredient Quantity (% by weight) 
______________________________________ 
Active ingredient 0.25 
Ethanol 25.75 
Propellant 22 Chlorodifluoromethane 
70.00 
______________________________________ 
The active ingredient is mixed with ethanol and the mixture added to a 
portion of the propellant 22, cooled to 30.degree. C., and transferred to 
a filling device. The required amount is then fed to a stainless steel 
container and diluted with the remaining propellant. The valve units are 
then fitted to the container. 
Suppositories are prepared as follows: 
Formulation 6: Suppositories 
______________________________________ 
Ingredient Quantity (mg/suppository) 
______________________________________ 
Active ingredient 
250 
Saturated fatty acid glycerides 
2,000 
______________________________________ 
The active ingredient is passed through a No. 60 mesh U.S. sieve and 
suspended in the saturated fatty acid glycerides previously melted using 
the minimal necessary heat. The mixture is then poured into a suppository 
mold of nominal 2 g capacity and allowed to cool. 
An intravenous formulation is prepared as follows: 
Formulation 7: Intravenous Solution 
______________________________________ 
Ingredient Quantity 
______________________________________ 
Active ingredient 50 mg 
Isotonic saline 1,000 mL 
______________________________________ 
The solution of the above ingredients is intravenously administered to a 
patient at a rate of about 1 mL per minute. 
Formulation 8: Combination Capsule I 
______________________________________ 
Ingredient Quantity (mg/capsule) 
______________________________________ 
Active ingredient 
50 
Premarin 1 
Avicel pH 101 50 
Starch 1500 117.50 
Silicon Oil 2 
Tween 80 0.50 
Cab-O-Sil 0.25 
______________________________________ 
Formulation 9: Combination Capsule II 
______________________________________ 
Ingredient Quantity (mg/capsule) 
______________________________________ 
Active ingredient 
50 
Norethylnodrel 
5 
Avicel pH 101 82.50 
Starch 1500 90 
Silicon Oil 2 
Tween 80 0.50 
______________________________________ 
Formulation 10: Combination Tablet 
______________________________________ 
Ingredient Quantity (mg/capsule) 
______________________________________ 
Active ingredient 
50 
Premarin 1 
Corn Starch NF 50 
Povidone, K29-32 
6 
Avicel pH 101 41.50 
Avicel pH 102 136.50 
Crospovidone XL10 
2.50 
Magnesium Stearate 
0.50 
Cab-O-Sil 0.50 
______________________________________