Use of estrogen antagonists and estrogen agonists in inhibiting pathological conditions

The present invention provides novel methods of inhibiting pathological conditions related to organ systems which respond to estrogen agonists comprising administering to a mammal in need of such treatment an effective amount of a compound of formula I ##STR1##

DETAILED DESCRIPTION OF THE INVENTION 
The present invention concerns methods for inhibiting pathological 
conditions which are susceptible or partially susceptible to inhibition by 
an estrogen, antiestrogen or estrogen agonist. Such conditions include 
uterine cancer, adjuvant breast cancer, breast disorder, male breast 
cancer, migraine, incontinence, vaginal atrophy, bladder infection, senile 
gynecomastia, diabetes, hypoglycemia, failure of would healing, melanoma, 
impotence, inflammatory bowel disease, CNS and GI disorders caused by an 
excess of tackykinins, decreased libido, immune system disorders, 
decreased fertility, pulmonary hypertensive disease, acne, seborrhea, 
autoimmune disease, Turner's syndrome, hirsutism, disorders related to an 
excess of neurokinin and obsessive-compulsive disorders including smoking 
and alcohol abuse. 
Changes in the appearance and texture of the skin with increasing age has 
been proverbial and well documented both quantitatively and qualitatively. 
It is a subject which is highly subjective in its evaluation and its 
ultimate effect on the individual. By in large the effect of the general 
atrophy of skin with age is cosmetic, but can have pathological 
consequences, many of which are psychological in nature, i.e., the feeling 
of getting "old," depression, loss of sexual attractiveness, etc. In some 
cases, the atrophy of the skin in older people can have direct pathologies 
associated with it, e.g., the ability of the skin to repair in wound 
healing. In general, the atrophy of the skin is considered a normal and 
progressive consequence of the aging process and taken with "good grace." 
Despite the normal acceptance of aging, there is a particular time in a 
woman's life, i.e., the menopause, when the progressive aging pattern is 
greatly accelerated, especially with regard to atrophy of the vagina and 
skin. It is often this rapid acceleration and suddenness of change which 
can be contributory to pathological and psychological distress. 
Additionally, vaginal atrophy can lead to discomfort, e.g., itching, 
dryness, and painful intercourse, which can lead to a loss of sexual 
enjoyment and conjugal harmony and in some cases be causal in social 
sequelae such as divorce. 
As mentioned before, the atrophy or aging of the skin can have both 
qualitative and quantitative aspects. The qualitative aspects are: the 
change of smoothness and texture, thus causing a "roughness" in look and 
feel on the outer surface of the skin, the change of elasticity of the 
skin, thus effecting the mechanical properties of the skin, and the 
changes in skin pigmentation. These qualitative changes result in the 
commonly described condition of atrophied skin as: wrinkled, rough, 
withered, and spotty. Quantitatively, skin aging in post-menopausal women 
can be measured as: a decrease in the mitotic rate of keratinocytes, 
changes in dermal thickness, decrease in glycosaminoglycans and soluble 
collagen which are linked to the moisture content of the skin, and the 
decrease in the urinary excretion of hydroxyproline, a measure of decrease 
collagen turnover. The qualitative changes in the skin, i.e., 
sightlessness and mechanical properties, are the result of the 
quantitative changes, i.e., loss or change of the extra-cellular matrix 
components. Therefore, it is possible to evaluate a beneficial effect of a 
therapy for post-menopausal skin atrophy without totally relying on 
subjective analysis, even though a subjective improvement may be the 
ultimate desired effect. In the case of vaginal atrophy, the quantitative 
aspect is the amount of vaginal moisture which is controlled by the amount 
of secretion from glands in the dermis, the qualitative result is 
subjective comfort. 
Currently, there are two major therapies available for the treatment of 
skin and vaginal atrophy in post-menopausal women. The first therapy is 
strictly a cosmetic approach, e.g., the use of make-up, skin moisturizers, 
night cremes, vaginal lubricants, etc. Although this cosmetic therapy does 
not affect the underlying physiological cause of the atrophy, often it 
does achieve some subjective benefit for the individual. The second type 
of therapy involves the treatment of the underlying physiological causes 
with active, medicinal agents, most notably Vitamin A and estrogens. 
Vitamin A is used, its effectiveness is controversial, and it is known to 
have substantial, undesirable side-effects which limit its use. 
At the time of menopause, the levels of estrogen produced by the ovaries 
rapidly decrease. This decrease in estrogen has pronounced effects on the 
skin and vagina causing a rapid acceleration in the natural process of 
atrophy. Estrogen replacement therapy is often beneficial in treating skin 
and vaginal atrophy. However, estrogen replacement therapy has undesired 
side-effects, most serious of which is the potential for the development 
of the threat of cancer. The inclusion of progestinal agents leads to 
undesirable psychological effects. The use of estrogen replacement therapy 
for the sole purpose of treating skin and vaginal atrophy is not common 
because of the negative side-effects. Clearly, an effective and safe agent 
which positively effects the underlying physiology and thus improves the 
qualitative aspects of skin and vaginal properties in post-menopausal 
women would be useful. 
Due to the advancements in medical science, along with education, the 
quality and length of life have both been increased. As a result, the 
population, as a whole, is living longer. As such, situations which have 
been present but not in great numbers are becoming more numerous and 
recognized. 
One of those situations which has been present but possibly not given 
enough weight is sexuality in old age. While there has always been some 
thought that old people are incapable of engaging in sexual activities, 
which is somewhat supported by a definite decline in sexuality for both 
sexes as they get older, sexual activity among the elderly cannot be 
ignored. Therefore, problems associated with such activity also cannot be 
ignored. 
While the male, from the early and middle years, has a relatively steady 
decline in sexual capacity and activity, the same cannot be as 
convincingly said about women. There are reports that the sexual capacity 
of the female does not change much until late in life. (Kinsey, A. C., et 
al., Sexual Behavior in the Human Female, Saunders, P. A., pg. 353 
(1953)). In particular, libido decreases substantially in a considerable 
percentage of cases after the menopause (Lauritzen et al., Estrogen 
Therapy the Benefits and Risks, 3rd International Workshop on Estrogen 
Therapy in Geneva, Oct. 20-21, 1977, Frontiers of Hormone Research, Vol. 
5, pg. 10 (1978)). This belief is supported by Pfeiffer, E., et al., 
Terminus of Sexual Behavior in Middle and Old Age, Journal of the American 
Geriatric Society, pg. 2151-2158 (1972), and Pfeiffer et al., Sexual 
Behavior in Middle Life, American Journal of Psychiatry, 128, 1262-1267 
(1972). In the Pfeiffer studies, a much greater decline in both sexual 
activity and interest among women between the ages of 45 and 71 was found 
as compared to men the same age, and the most dramatic change took place 
between 50-60 years of age, which is, of course, generally the period 
during which women experience menopause or are post-menopausal. In the age 
group of 66-71, 50% of women said they had no or little sexual interest 
compared with 10% of men in the same age group. 
While there has been no direct link between declining estrogen levels and 
declining sexuality, it has been stated that hormonal changes following a 
natural menopause, and certainly following surgical menopause, may 
contribute to the sexual decline in a portion of women. Exactly how 
menopause contributes to the loss of libido is not understood, yet seems 
fairly evident. (Bancroft, Human Sexuality and Its Problems, 2nd ed., pg. 
292-293, (1989)). Therefore, it would be of use to find compounds which 
increase libido in post-menopausal women. 
Contraceptive methods involving the administration of chemical substances 
are widely practiced among women who desire to limit pregnancies. Such 
methods control fertility through various biological mechanisms. Among the 
presently used chemical methods of fertility control, the most important 
are those which act by means of the following: (a) suppression of 
ovulation through inhibition of gonadotropin release; (b) alteration of 
the female reproductive tract to prevent migration of sperm to the site of 
fertilization or, if fertilization occurs, to block implantation of the 
zygote (nidation); (c) spermicidal action or (d) an abortifacient. 
The oral contraceptives are the most prominent chemical contraceptive 
agents. Two types of agents are (a) estrogen combined with a progestin, 
and (b) a progestin alone. The contraceptives of the combined type act 
primarily by suppressing ovulation by negative feedback to prevent 
gonadotropin (LH and FSH) release by the hypothalamus, but alterations in 
the reproductive tract may also contribute to the antifertility effect. 
Such alterations include changes in the cervical mucus (which increase the 
difficulty of sperm migration) and in the endometrium (which decrease the 
likelihood of nidation). The action of a progestin alone in a very low 
oral dose ("mini-pill") appears to involve primarily alterations in the 
female reproductive tract, but ovulation suppression may also occur. 
Although the oral contraceptives are highly effective, their, use is 
associated with unpleasant side effects (such as nausea, depression, 
weight gain, and headache) and an increased long-time risk of severe 
disease (such as thromboembolism, stroke, myocardial infarction, hepatic 
adenoma, gall bladder disease, and hypertension). Bleeding irregularities 
(such as break-through bleeding, spotting, and amenorrhea) are also 
frequent. A progestin, when administered alone, causes an increased 
incidence of changes in menstrual patterns, especially a marked increase 
in the amount and duration of menstrual bleeding. 
Other chemical methods of contraception include the post coital 
administration of estrogens (e.g. diethylstilbestrol, antiprogestins, or 
ethynylestradiol) to prevent nidation or of prostaglandins which act as 
abortifacients. Both of these methods, at present, are limited to 
emergency situations. Still in the very early stages of development are 
immunological methods (vaccination) and methods involving the direct 
control of LHRH secretion from the pituitary by LHRH agonists or 
antagonists. 
Another group of chemical contraceptive agents are the local spermatocides, 
such as nonoxynol or octoxynol, which are placed into the vagina 
immediately prior to coitus in the form of creams, foams, jellies, or 
suppositories. The spermicidal action takes place either in the vagina or 
elsewhere in the reproductive tract. For the latter to occur, the 
spermicidal agent is either absorbed on sperm membranes or is transported 
into the uterus under the influence of uterine contractions. The 
spermicidal methods are not completely reliable in preventing pregnancy 
and are inconvenient to use. 
From the foregoing, it is evident that the presently available methods of 
contraception are inadequate for various reasons. Although many women 
practice contraception in spite of these inadequacies, a need exists in 
medicine for new methods. 
Pulmonary hypertension represents a serious, life threatening spectrum of 
diseases of multiple etiology. These include congenital abnormalities of 
the lung, thorax and diaphragm, congenital or acquired valvular or 
myocardial disease, obstructive lung disease, and can be a complication of 
autoimmune diseases, vasculitis and collagen based diseases (Rubin, Chest. 
104: 236,1993). Patients with pulmonary hypertension frequently present 
with symptoms including dyspnea, fatigue, syncope, and chest pain, and 
have increased pulmonary artery pressure and demonstrate prominence of the 
main pulmonary artery, hilar vessel enlargement and decreased peripheral 
vessels on chest radiographs (Rich. Ann. Internal. Med., 107: 216, 1987). 
While pulmonary hypertension has multiple etiologies, primary pulmonary 
hypertension appears to involve an autoimmune component and has been 
reported as a complication in patients with mixed connective tissue 
disease, rheumatoid arthritis, Sjogren's syndrome, systemic sclerosis and 
lupus (Sato, Hum. Path, 24: 199, 1993). Primary pulmonary hypertension 
occurs in females 1.7 times more frequently than males with the greatest 
predominance between the third and fourth decades of life (Rich, Ann. 
Internal. Med., 107: 216, 1987). The increased incidence of primary 
pulmonary hypertension in women of child bearing age as well as the 
clinical observations that the disease can be exacerbated by pregnancy and 
oral contraceptives (Miller, Ann. Rheum. Dis. 46: 159, 1987; and cited in 
Farhat et al., J. PET., 261: 686, 1992) suggests a role for estrogen in 
the disease process. To this extent, Farhat et al. have demonstrated that 
estradiol potentiates the vasopressor response to a thromboxane mimetic in 
perfused rat lungs (J. PET, 261: 686, 1992). However, the role of estrogen 
in pulmonary hypertension is complex and may be dependent on the etiology 
of the disease process. In a rat model of pulmonary hypertension induced 
by injection of monocrotaline pyrrole (Reindel, Tax, Appl. Pharm., 106: 
179,1990) progressive pulmonary hypertension, right ventricular 
hypertrophy and interstitial edema around the large airways and blood 
vessels becomes apparent, similar to the pathology observed in man. 
Estradiol treatment decreased right ventricular hypertrophy and prevented 
interstitial edema in this model (Farhat et al., Br. J. Pharm., 110: 719, 
1993) as well as attenuating the hypoxic vasoconstrictive response in 
isolated sheep lungs (Gordon et al., J. Appl. Physiol., 61: 2116, 1986). 
Current therapy for pulmonary hypertension is inadequate and is largely 
dependent on the use of vasodilators, diuretics, and anticoagulants 
(Rubin, Drugs, 43: 37, 1992; Palevsky, JAMA, 265:1014, 1991). Vasodilators 
are effective in only a small subpopulation of patients with primary 
pulmonary hypertension and is complicated by systemic hypotensive 
responses. Prostacyclin infusion and high dose calcium channel blockers 
are also being used with limited efficacy. Heart-lung and single lung 
transplantation have been used on patients which do not respond to 
vasodilator therapy, however, due to surgical morbidity and mortality, 
this approach is usually limited to those patients who continue to 
deteriorate despite aggressive therapy at centers experienced in 
management of this disease. Patients frequently die of right heart failure 
and those individuals which have signs of right heart failure have a mean 
survival of 6-12 months (Rubin, Drugs, 43: 37,1992). 
Therefore, pulmonary hypertensive diseases are characterized by inadequate 
therapies, necessity of organ transplantation and poor prognosis, and a 
need exits for new therapies. 
Acne and seborrhea are two general classes of skin diseases which are 
marked by an abnormal function (usually hyperactivity) of the sebaceous 
glands in the skin. The subject of this invention is the use of compounds 
to inhibit acne and seborrhea. 
Acne vulgaris is a disease of the pilosebaceous unit in the skin and is 
chronic and inflammatory in nature. It is characterized by comedos 
(blackheads), papules, pustules, cysts, and nodules. The areas of the body 
most commonly affected by the disease are those which have the most 
sebaceous glands, i.e., the face, neck, back, and chest. Acne is a very 
common disease in both men and women and usually appears at the beginning 
of puberty. Although the disease is usually mild and resolves itself by 
the time most people reach their midtwenties, it can in many instances be 
disfiguring and a source of great physiological distress. In some extreme 
cases, acne can be the source of severe infection and even 
life-threatening. 
The etiology and pathogenesis of the disease begins with cohesive 
hyperkeratosis in which cornified cells adhere and block the follicular 
canal between the sebaceous gland and the surface of the skin. The 
sebaceous gland under hormonal control (testosterone and 
dihydrotestosterone) are stimulated to enlarge and produce increasing 
amounts of sebaceous secretions (principally in the form of 
triacylglycerols). These sebaceous secretions are trapped in the blocked, 
follicular canal and build up to form a closed comedo. At this stage, 
common, indigenous skin bacteria (principally, Propionibacterum Acnes) 
begin to metabolize the triacylglycerols to free fatty acids. These 
liberated fatty acids are inflammatory and results in the formation of a 
papule. This papule is often raised and is typical of an inflammatory 
lesion, i.e., red, edematous, and painful. The papule may continue to 
expand and rupture the follicle wall, thus forming a pustule or cyst. The 
pustule stage is very painful and unsightly and is often a site for 
secondary infection by opportunistic bacteria such as Staphofius. The 
pustules and cysts often lead to the scarring and disfigurement seen in 
severe cases of acne. 
There are several drugs available for the treatment of acne. For mild 
cases, benzoyl peroxide is used and is often moderately effective. Benzoyl 
peroxide is thought to work by inhibiting cohesive hyperkeratosis and by 
suppressing P. Aches although benzoyl peroxide is effective in mild cases 
of acne, it suffers from several drawbacks: first, it must be applied 
topically and does not always penetrate to the pilosebaceous unit where 
the acne lesion initiates, second, it can cause skin irritation which can 
exacerbate the disease. Another moderately effective drug is vitamin A 
(retenoic acid, Retin A) which is used topically. Vitamin A inhibits 
cohesive hyperkeratosis; however, being a topical preparation it suffers 
from some of the same drawbacks as benzoyl peroxide and in addition it can 
cause a deterioration of the protective stratum corneum if used 
extensively. Yet another group of commonly used drugs for the treatment of 
acne are antibiotics. These can be used either topically or systemically. 
The most commonly used antibiotics are tetracyclines and erythromycin and 
to a lesser extent minocycline, ampicillin, clindamycin, trimethoprim, and 
sulfamethoxazole. These antibiotics inhibit P. Aches and other secondary 
bacterial infections. There are two major drawbacks to the prolonged use 
of antibiotics for acne; first, the continued long exposure to antibiotics 
often lead to formation of resistant bacterial strains both in the skin 
and systemically, and second continued use of antibiotics may lead to 
sensitization of the patient to the antibiotic. A newer drug used for acne 
is Isotretinoin (Accutane, 13-cis-retenoic acid). This drug works like 
vitamin A; however, it can be used systemically. The side-effects of 
isotretinoin are often: cheilitis, a rise in serum triglycerides, elevated 
sedimentation rates, and most importantly, isotretinoin is a teratogen in 
humans and therefore cannot be used if there is a question of pregnancy 
during treatment. All of the above drugs have some positive effect in the 
treatment of acne, but each has its limiting side-effects. 
Hormonal therapy is also effective for the treatment of acne in women. In 
many cases, the administration of estrogens has a positive effect in 
treating acne. Estrogens counteract the effect of endogenous androgens and 
therefore, decrease sebaceous excretion. However, since the use of 
unopposed estrogen administration in women with a uterus poses the 
potential for the development of endometrial cancer, a cyclic therapy of 
estrogen and a progestin are used for the treatment of acne. Typically, 
women are prescribed the normal birth control protocols for acne 
treatment. Although, these protocols are often effective for acne, in many 
cases these regiments contain progestins which have significant androgenic 
activity. This androgenic activity exacerbates the disease. Additionally, 
it is well known that progestinal agents are the cause of many negative, 
psychological side-effects. Clearly, a better hormonal agent would be 
beneficial. 
Seborrhea or seborrheic dermatitis is another group of skin diseases 
thought to be associated with abnormal function of the sebaceous glands. 
It occurs in areas where there are large numbers of sebaceous glands and 
is characterized by flasking of the skin and red, mildly inflammatory 
patches. Seborrhea is most common in the hair (a form of dandruff), scalp 
margins, eyebrows, naso-labial folds, external ear canals, postier 
auricular fold, and presternal area. Generally, mild seborrhea is 
controlled by topical medication such as glucocorticoids and LDH in Nivea 
oil. However, more severe cases are more difficult to control. 
There are several conditions in which the ovaries do not develop and in 
consequence puberty does not occur. Gonadal dysgenesis results in a severe 
disease state known as Turner's Syndrome resulting from the absence of a 
second sex chromosome (X chromosome monosomy). The syndrome is associated 
with the female phenotype, shortness of stature, sexual infantilism, and 
various somatic abnormalities. Several typical features are observed in 
these patients including distinct facial features, square chest, and short 
broad neck with webbing. Additional anomalies include cubitus valgus, 
congenital lymphedema of the feet and hands, renal abnormalities, high 
arched palate, skeletal anomalies, pigmented nevi, keloid formation, 
abnormal nails, and recurrent otitis media. Cardiovascular abnormalities 
include bicuspid aortic valves, partial anomalous venous drainage, and 
hypoplastic left-sided heart syndrome (Miller, M. J., et al. J. Pedriatr., 
102: 47-50, (1983), Mazzanti, I. et al. Helv. Paediatr. Acta, 43: 25-31, 
(1988), Van Egmond, H. et al. Br. Heart J., 60: 69-71, (1988)). Renal 
abnormalities include rotation of the kidney, horseshoe kidney, 
duplication of renal pelvis and ureter, and hydronephrosis secondary to 
ureteropelvic obstruction. 
Skeletal maturation is normal or slightly delayed in childhood but lags in 
adolescence as a result of gonadal steroid deficiency. Typically, fishnet 
appearance caused by localized rarefications occurs. Bone mineral content 
reduction occurs as early as 8 years of age as well as later in puberty. 
Changes of the spine, vertebral hypoplasia, and scoliosis are also common. 
Abnormalities of the carpal, wrist, knee and pelvis are also noted. The 
shortness of stature, including uterine growth retardation, is not evident 
until after the first 3 years of life after which growth velocity 
decelerates appreciably (Park, E., et al. Pediatr. Res. 17: 1-7, (1983), 
Lyon, A. J. et al. Arch. Dis. Child., 60: 932-935, (1985)). In general, 
the patients suffer from sexual infantilism with genital ducts and 
external genitalia being immature. As a result, ovarian development is 
retarded. 
Current therapy is directed towards correcting stature, somatic anomalies 
and inducing secondary sexual characteristics. Recent data indicated 
growth hormone is a viable therapy for stature improvement (Rosenfeld, R. 
G., et al. J. Pediatr., 113: 393 (1988)). Patients not treated with 
estrogen often develop a severe form of osteoporosis similar to that 
experienced by females after menopause. Fractures and vertebral collapse 
are common. Steroid hormone therapy is normally deferred until after 15 
years of age as it is believed treatment at an earlier age may result in 
premature maturation of the skeleton and thus a decrease in height. In 
fact, pharmacological doses of estrogen can accelerate bone maturation and 
resulting in epiphyseal fusion at an early age without concomitant 
increases in height. Other studies have shown low-dose estrogen allows 
patients to develop breasts without causing any changes in height 
(Alexander, R. L. et al., Clin. Res. 26: 174A (1978)). However, studies 
indicate a number of cases of endometrial cancer in patients with gonadal 
dysgenesis as a result of estrogen therapy (Levine, L. S., Pediatrics, 62: 
1178-1183 (1979)). 
Given the adverse side effects of estrogen in Turner's Syndrome patients, a 
need exists for a bone sparing agent which does not posses significant 
uterotrophic consequences. 
In the United States, one in every four women require medical attention for 
breast symptomatology. While much rarer, males also encounter breast 
disorders. Such disorders include galactorrhea, gynecomastia, hypertrophy, 
polythelia, mastodynia/mastalgia, hyperprolactinermia, and generally 
non-fibrocystic, non-cancerous mascopathias. 
Breast pain is common and estimated to be present in 50% of women. Normally 
the etiology is unclear. The discomfort generally is classified as (1) 
cyclic mastalgia or mastodynia occurring immediately prior to the menses; 
(2) changes in the breast such as duct ectasia and sclerosing adenosis, or 
(3) referred pain such as costochondritis. 
Gynecomastia is enlargement of the glandular breast tissue in male humans 
(the female counterpart is hypertrophy). This enlargement is localized to 
the aureoles and can be unilateral or more commonly bilateral. The 
condition is usually benign in nature; however, it can be the source of 
severe psychological disturbance to the patient. Gynecomastia is most 
commonly found in males at the time of puberty, but can occur at any age. 
Gynecomastia can have many underlying causes, e.g., Klinefelter's syndrome 
(XXY chromosomal abnormality), liver disorders, estrogen therapy for 
prostatic carcinoma, tumors of various endocrine organs, and certain drugs 
(digitalis and Dilantin). The common relationship between all these causes 
and the resulting gynecomastia is the production of abnormal amounts of 
estrogens. Currently, treatment of this disease is limited to three 
therapies: 1) Determination and treatment of the underlying cause; 2) 
Surgical removal of the breast tissue; and 3) Treatment with 
diethylstilbestrol and radiation. Determination and treatment of the 
underlying cause of gynecomastia is not always possible. Surgery and 
treatment with diethylstilbestrol and radiation is not always successful 
and entails great expense and risk. Clearly, a more effective and safer 
therapy would be useful. 
Galactorrhea is the production of breast milk in the male or female when 
not immediately associated with pregnancy. The highly inappropriate and 
rare response in the male breast is accompanied by severe psychological 
discomfort to the male patient. It is thought to be caused by an 
overproduction of estrogen and prolactin excess. Surgical treatment is 
usually the therapy of choice if the underlying cause can not be 
determined or treated. A safer and less costly therapy would be useful. 
Diabetes mellitus is a systemic disease characterized by disorders in the 
actions of insulin and other regulatory hormones in the metabolism of 
carbohydrates, fats and proteins, and in the structure and function of 
blood vessels. The primary symptom of diabetes is hyperglycemia, often 
accompanied by glucosuria, the presence in urine of large amounts of 
glucose, and polyuria, the excretion of large volumes of urine. Additional 
symptoms arise in chronic or long standing diabetes. These symptoms 
include degeneration of the walls of blood vessels. Although many 
different organs are affected by these vascular changes, the nerves, eyes 
and kidneys appear to be the most susceptible. As such, long-standing 
diabetes mellitus, even when treated with insulin, is a leading cause of 
blindness. 
There are two recognized types of diabetes. Type I diabetes is of juvenile 
onset, ketosis-prone, develops early in life with much more severe 
symptoms and has a near-certain prospect of later vascular involvement. 
Control of this type of diabetes is difficult and requires exogenous 
insulin administration. Type II diabetes mellitus is of adult onset, 
ketosis-resistent, develops later in life, is milder and has a more 
gradual onset. 
One of the most significant advancements in the history of medical science 
came in 1922 when Banting and Best demonstrated the therapeutic effects of 
insulin in diabetic dogs. However, even today, a clear picture of the 
basic biochemical defects of the disease is not known, and diabetes 
remains a serious health problem. It is believed that two percent of the 
United States population is afflicted with some form of diabetes. The 
introduction of orally effective hypoglycemic agents was an important 
development in the treatment of hyperglycemia. Oral hypoglycemic agents 
are normally used in the treatment of adult onset diabetes. 
Observations in animal models on glucose metabolism for type II diabetes 
and in humans suggest that sex steroids play a permissive role in the 
phenotypic expression of hyperglycemia. These observations have prompted 
studies on the effects of androgens and estrogens on blood glucose levels. 
Testosterone administration to intact or ovariectomized female rats 
resulted in marked insulin resistance which correlated to morphological 
changes in muscle, Holmang, et al., Am. J. Physiol., 259, E555-560 (1990); 
Holmang, et al., Am. J. Physiol., 262, E851-855 (1992). In streptozotocin 
diabetic rats, implanted testosterone antagonized the ability of residual 
insulin to maintain glycemic control, Le et al., Endocrinology, 116, 
2450-2455 (1985). In contrast, glucosuria disappeared in castrated 
diabetic KK mice and reappeared when androgens were replaced in these 
mice, Nonaka, et al., Jpn. J. Vet. Sci., 50, 1121-1123 (1988): Higuichi, 
et al., Exp. Anim., 38, 25-29 (1989). 
Results from estrogen administrations also support the hypothesis that the 
balance between androgens and estrogens is critical to the development of 
hyperglycemia. Daily estradiol administrations to diabetic KK mice 
normalized the blood glucose levels and eliminated glucosuria, Toshiro, et 
al., Jpn. J. Vet. Sci., 51, 823-826 (1989). Estradiol also lowered the 
blood glucose levels of C57BL/6J-ob/ob mice, Dubuc, Proc. Soc. Exp. Biol. 
Med., 180, 468473 (1985) and C57BL/KsJ-db/db mice, Garris, Anatomical 
Record, 225, 310-317 (1989). 
In climacteric women, anxiety, depression, tension and irritability begin 
during the perimenopause and can be correlated to reduce estrogen levels 
and estrogen replacement therapy has been recommended for the treatment of 
these symptoms (Malleson J., Lancet, 2: 158, (1953); Wilson et. al., J. 
Am. Geriatric Soc., 11: 347 (1963)). The mechanism for protective effects 
of estrogen in this case in unknown, but may be related to potential 
effects of estrogen on biogenic amines such as serotonin (Aylward M., Int. 
Res. Communications System Med. Sci., 1: 30 (1973)). To this regard 
circulating serotonin is reduced in post-menopausal women (Gonzales G., 
et. al., Maturitas 17: 23-29 (1993)), and serotonin (as well as several 
other biogenic amines) have a putative role in behavioral depression. 
Phillips and Sherwin (Psychoneuroendocrinology, 17: 485-495 (1992)) 
reported that in surgically menopausal women given estrogen, scores in 
immediate and delayed recall tests are greater than in similar women not 
given estrogen. Two potential hypotheses might explain this effect. There 
is some evidence that partial estrogen agonists (or anti-estrogens) such 
as tamoxifen interact with the muscarinic receptor (Ben-Baruch G., et. 
al., Molec. Pharmacol. 21: 287-293 (1982)), and muscarinic agonists 
(M.sub.2) are known to produce positive effects in a number of memory 
associated tasks and may have clinical relevance in Alzheimer's Disease. 
Another interesting possibility may be linked to neurokinins such as 
Substance P, which are known to have neurotrophic as well as 
memory-promoting effects (Thoenen H., Trends in Neuroscience, 14: 165-170 
(1991); Huston J. et. al., Neurosci. Biobehav. Rev. 13: 171-180 (1989)), 
thus, through an effect either at a neurotransmitter receptor in the CNS 
or at a neuropeptide receptor, a tissue selective estrogen 
agonist/antagonist could produce memory and cognitive enhancing effects. 
Such an activity would most relevantly be assessed in man, but a variety 
of animal models (i.e. maze learning, extinction etc.) are available for 
preclinical testing. 
Perhaps the most frequent CNS related problem in climacteric women is the 
occurrence of hot flushes. While this undoubtedly is a somatic effect 
mediated by effects on the microvasculature, current evidence points 
strongly in the direction of CNS initiated effect (Lomax P., et. al., 
Pharmac. Ther. 57: 347-358 (1993)). Therefore, a tissue selective estrogen 
agonist/antagonist might offer the ideal therapy providing the desired 
effect in the absence of untoward side effects on reproductive tissue. 
Obsessive-compulsive disorder is one of the rarer psychiatric illnesses, 
although minor obsessional symptoms probably occur in one-sixth of the 
population (Encyclopedia of Medicine, American Medical Association; 
Current Diaqnosis, W. B. Saunders Company, 1985). It is characterized by 
one or both of two symptoms. The first, comprises recurrent, intrusive 
ruminative thoughts that the patient may realize are senseless but of 
which he cannot stop thinking. The most common of these are thoughts of 
violence, contamination, doubt, or personal illness. Normally, the patient 
does not believe these thoughts are true reflections of reality. However, 
some patients become convinced that their obsessive ruminations are true, 
and suffer from psychotic delusions. 
The second comprises repetitive, ritualistic behaviors that the patient 
recognizes are needless but that he cannot keep himself from performing. 
Hand washing, counting, checking rituals, and touching rituals are 
examples of such rituals. The carrying out of the ritual is not constant, 
but fluctuates and mirrors anxiety levels. There normally are intense 
feelings of panic and anxiety if the patient is prevented from completing 
a ritual. 
While appearing depressed, a review of the history of obsessive-compulsive 
patients normally reveals that obsessions and compulsions precede the 
onset of dysphoric mood states and that depressed feelings are related to 
the impact the obsessive-compulsive behavior has on life. In severe cases, 
the patient will be incapacitated, completely overtaken by the distraction 
of constant obsessive ruminations or the demand to complete endless 
compulsive rituals. 
Consumptive disorders include those disorders in which the intake, normally 
oral, of the amount of a substance is outside a normal range, often to an 
extent where health is impaired. Examples of such are eating or appetite 
disorders (obesity, bulimia, pica, anorexia nervosa, and psychogenic 
rumination) and substance abuse or overuse (smoking, nicotine dependence, 
alcoholism, alcohol abuse). 
It is well known that the chronic administration of nicotine results in 
tolerance and, eventually, dependence. The use of tobacco has become 
extremely widespread in all countries, despite the well known adverse 
effects of the use of tobacco in all its forms. Thus, it is clear that 
tobacco use is extremely habit-forming, if not addictive, and that its use 
provides sensations to the user which are pleasant and welcome, even 
though the user is fully aware of the drastic long term ill effects of its 
use. 
Cigarette smoking is the most dominant cause of preventable morbidity and 
early demise in developed countries. On average, smokers die several years 
earlier than nonsmokers and have an increased risk of fatal heart disease, 
lung cancer, cancers of the mouth, throat, esophagus, pancreas, kidney, 
bladder, and cervix, peptic ulcers and of fractures of the hip, wrist, and 
vertebrae. Olfaction and taste are impaired in smokers, and facial 
wrinkles are increased. Diabetic patients who smoke may have an increased 
risk of proteinuria. 
Smoking cessation provides benefits, even late in life, such as reducing 
the risk of death or myocardial infarction in persons with coronary artery 
disease, reducing the progression of carotid atherosclerosis, and with 
reversal of chronic bronchitis. 
Children of persons who smoke have lower birth weights, more frequent 
respiratory infections, less efficient pulmonary function, and a higher 
incidence of chronic ear infections than children of non-smokers and are 
more likely to become smokers themselves. Exposure to passive smoke has 
been shown to increase the risk of cervical cancer, lung cancer, and heart 
disease and to promote endothelial damage and platelet aggregation. 
Recently, vigorous campaigns against the use of tobacco have taken place, 
and it is now common knowledge that the cessation of smoking brings with 
it numerous unpleasant withdrawal symptoms, which include irritability, 
anxiety, restlessness, lack of concentration, lightheadedness, insomnia, 
tremor, increased hunger and weight gain, and, of course, a craving for 
tobacco. 
Alcohol abuse and alcohol dependence (i.e., alcoholism) are serious public 
health problems of modern society. In the United States alone, an 
estimated 13 million adults exhibit symptoms of alcohol dependence due to 
excessive alcohol intake, and an additional 7 million abuse alcohol 
without showing symptoms of dependence according to United States 
government projections from studies conducted in the mid-1980s. Alcohol 
dependence and abuse are very expensive as it is estimated that it cost 
the United States well over $200 billion in 1991 with no prospect of 
falling or leveling off. The social and psychological damages inflicted on 
individuals as a consequence of alcohol-abuse, e.g., children born with 
fetal alcohol syndrome (FAS) and victims of alcohol-related accidental 
death, homicide, suicide, etc., are immense. 
While it is generally accepted that alcoholism and alcohol abuse are 
affiliations with staggering international economic, social, medical, and 
psychological repercussions, success in preventing or otherwise 
ameliorating the consequences of these problems has been an elusive goal. 
Only very recently the public view that alcoholism and alcohol abuse are 
remedial solely by moral imperatives has been changed to include an 
awareness of alcoholism and alcohol abuse as physiological aberrations 
whose etiology may be understood and for which therapy may be found 
through scientific pursuits. Both alcohol abuse and dependence arise as a 
result of different, complex, and as yet incompletely understood 
processes. At present, alcohol research is in the mainstream of scientific 
efforts. 
This invention provides methods for inhibiting obsessive-compulsive and 
consumptive disorders. 
Hirsutism (hypertrichosis) is characterized by excessive growth of hair. In 
women, hirsutism refers specifically to excessive growth of hair in a male 
pattern and distribution. Clinically, hirsutism in women is seen as a 
growth of terminal hair on the face (particularly on the upper lip), the 
chin, chest, back, and lower abdomen (escutcheon). This growth of hair is 
often seen as unsightly and can be the cause of embarrassment and 
psychological distress. Hirsutism is a common occurrence at the menopause, 
but can occur any time after puberty. The etiology of the condition has 
been linked to over production of androgens by either the ovaries or 
adrenal glands or both. 
Hirsutism in women can be treated in a variety of ways. Cosmetic treatment 
of the condition, including'shaving, plucking of hairs, and bleaching, 
while effective in improving the appearance of the patient, are only 
palliative and must be constantly re-applied. Glucocorticoid steroids are 
often effective; however, they have the potential of serious side-effects 
such as Cushing's Syndrome. Oral contraceptives can be effective; however, 
care must be taken because certain progestins used in common oral 
contraceptive regiments may actually contribute hirsutism because of their 
androgenic side-effects. Climetidine and Spironolactone have shown some 
effectiveness in the treatment of hirsutism; however, each of these can 
have unwanted side-effects. Clearly, a more effective and better tolerated 
agent would be useful. 
Alopecia (hair loss) can occur in women for a variety of reasons, and 
includes female pattern alopecia. Female pattern alopecia is characterized 
by chronic and progressive hair loss often beginning around thirty years 
of age and accelerating at menopause. The hair loss is usually confined to 
the central scalp in a diffuse pattern. This loss of hair is cosmetically 
damaging and often psychologically disturbing to the patient. The etiology 
of the condition has been linked to an elevated level of androgens and the 
subsequent response of androgen sensitive hair follicles. Treatment of the 
condition is primarily cosmetic in nature, e.g., wigs, hair styles which 
cover the effected area, etc. The drug, Spironolactone, has been used, but 
does have side-effects. Clearly, an effective treatment for this condition 
would be useful. 
Macrophages play a central role in host defense through a variety of 
effector mechanisms involving both membrane related and secretory events 
(Gordon et. al., Curr. Opin. Immunol., 4, 25, 1992; Fuller, Brit. Med. J., 
48, 65, 1992). Phagocytosis, chemotaxis and antigen presentation are 
membrane related processes involved in immunologic defense mechanisms 
necessary for host survival. The importance of macrophages in defense 
against microbes, immune surveillance, destruction of tumor cells, and in 
the clearing of senescent erythrocytes has been documented in man and in 
animal models characterized by the selective elimination of macrophages 
(Claassen et. al., J. Immunol Meth., 134, 153, 1990). Macrophages also 
contribute to host defense through secretion of bacteriostatic and 
bactericidal proteins, cytokines and lipid mediators, as well as oxygen 
and nitrogen reactive intermediates. The secretory capacity of the 
macrophage is central to its function as these cells secrete over 100 
distinct mediators and are located in every organ (Nathan, J. Clin. 
invest., 79, 319, 1987). 
While aberrant activation of macrophage functions is associated with 
autoimmune diseases as well as both chronic and acute inflammatory 
processes, the reciprocal condition, suppression of macrophage effector 
functions, is associated with reoccurring infections of both opportunistic 
and non-opportunistic pathogens and contributes to increased morbidity and 
mortality. Populations associated with an immunocompromised state include 
burn patients, transplants, HIV infected individuals, cancer patients 
undergoing chemotherapy and surgical patients, notably those with a higher 
risk of infection as observed in thoracoabdominal patients. 
Current therapeutic approaches to these patients includes the use of 
intravenous infusion of macrophage derived cytokines notably the colony 
stimulating factors G-CSF, GM-CSF, and M-CSF (Nemunaitis, Transfusion 33: 
70, 1993). Supportive therapy with antibiotics and fluids is also used, 
however, the limitations of these approaches are demonstrated by the 
continued problems of infection in immunocompromised patients and the 
emergence of more deadly strains of antibiotics resistant organisms. 
Furthermore, infections of immunocompromised patients with opportunistic 
pathogens including Pneumocystis and Cryptococcal infections remain 
significant and result in complications despite various antibiotic 
protocols. Clearly, novel therapeutics which can selectively enhance 
macrophage effector functions to augment host defense would play a central 
role in the clinical management of these patients. 
Estrogen has been reported to increase select macrophage effector functions 
including Fc mediated phagocytosis, class II antigen expression, and IL-1 
secretion. These observations coupled with the known propensity of women 
to be more resistent to a variety of infections (Ahmed et al., Am. J. 
Path., 121, 531, 1985) suggests that estrogen-like compounds may enhance 
macrophage effector functions and thus be beneficial in disease states 
associated with depressed host defense such as bladder infections or 
depressed wound healing. 
The term "inhibit" is defined to include its generally accepted meaning 
which includes prophylactically treating a subject to prevent the 
occurrence of one or more of these disease states, holding in check the 
symptoms of such a disease state, and/or treating such symptoms. Thus, the 
present methods include both medical therapeutic and/or prophylactic 
treatment, as appropriate. 
The methods of this invention are practiced by administering to an 
individual in need of treatment an effective amount of a compound formula 
I. 
Compounds of formula I are described as being effective in treatment of 
prostate disease, breast cancer, osteoporosis, endometriosis, 
cardiovascular disease and hypercholesterolemia in commonly owned U.S. 
patent application Ser. No. 08/369,954 which is hereby incorporated by 
reference. 
The terms C.sub.1 -C.sub.3 chloroalkyl and C.sub.1 -C.sub.3 fluoroalkyl 
include methyl, ethyl, propyl and isopropyl substituted to any desired 
degree with chlorine or fluorine atoms, from one atom to full 
substitution. The term C.sub.5 -C.sub.7 cycloalkyl includes cyclopentyl, 
cyclohexyl and cycloheptyl. 
Halo means chloro, bromo, iodo and fluoro. Aryl (Ar) includes phenyl and 
naphthyl optionally substituted with one to three substituents 
independently selected from R.sup.4 as defined above. DTT means 
dithiothreitol. DMSO means dimethyl sulfoxide. EDTA means ethylene diamine 
tetra acetic acid. 
Estrogen agonists are herein defined as chemical compounds capable of 
binding to the estrogen receptor sites in mammalian tissue, and mimicking 
the actions of estrogen in one or more tissues. 
Estrogen antagonists are herein defined as chemical compounds capable of 
binding to the estrogen receptor sites in mammalian tissue, and blocking 
the actions of estrogen in one or more tissues. 
One of ordinary skill will recognize that certain substituents listed in 
this invention will be chemically incompatible with one another or with 
the heteroatoms in the compounds, and will avoid these incompatibilities 
in selecting compounds of this invention. Ukewise certain functional 
groups may require protecting groups during synthetic procedures which the 
chemist of ordinary skill will recognize. 
The chemist of ordinary skill will recognize that certain compounds of this 
invention will contain atoms which may be in a particular optical or 
geometric configuration. All such isomers are included in this invention; 
exemplary levorotatory isomers in the cis configuration are preferred. 
Likewise, the chemist will recognize that various pharmaceutically 
acceptable esters and salts may be prepared from certain compounds of this 
invention. All of such esters and salts are included in this invention. 
The remedies for the conditions and diseases for use in the methods of this 
invention can be prepared by the methods commonly employed using 
conventional, organic or inorganic additives, such as an excipient (e.g., 
sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, 
calcium phosphate or calcium carbonate), a binder (e.g., cellulose, 
methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, 
polyvinylprrolidone, gelatin, gum arabic, polyethyleneglycol, sucrose or 
starch), a disintegrator (e.g., starch, carboxymethylcellulose, 
hydroxypropylstarch, low substituted hydroxypropylcellulose, sodium 
bicarbonate, calcium phosphate or calcium citrate), a lubricant (e.g., 
magnesium stearate, light anhydrous silicic acid, talc or sodium lauryl 
sulfate), a flavoring agent (e.g., citric acid, menthol, glycine or orange 
powder), a preservative (e.g., sodium benzoate, sodium bisulfite, 
methylparaben or propylparaben), a stabilizer (e.g., citric acid, sodium 
citrate or acetic acid), a suspending agent (e.g., methylcellulose, 
polyvinylpyrrolidone or aluminum stearate), a dispersing agent (e.g., 
hydroxypropylmethylcellulose), a diluent (e.g., water), and base wax 
(e.g., cocoa butter, white petrolatum or polyethylene glycol). The amount 
of the active ingredient in the medical composition may be at a level that 
will exercise the desired therapeutical effect; for example, about 0.1 mg 
to 50 mg in unit dosage for both oral and parenteral administration. 
The active ingredient may be usually administered once to four times a day 
with a unit dosage of 0.1 mg to 50 mg in human patients, but the above 
dosage may be properly varied depending on the age, body weight and 
medical condition of the patient and the type of administration. A 
preferred dose is 0.25 mg to 25 mg in human patients. One dose per day is 
preferred. 
Compounds used in the methods invention are readily prepared by the 
reactions illustrated in the schemes below. 
Certain compounds of formula I are conveniently prepared from an 
unsaturated intermediate 
##STR10## 
by hydrogenation with a noble metal catalyst in a reaction inert solvent. 
Pressure and temperatures are not critical and hydrogenation is normally 
accomplished in a few hours at room temperatures at 20-80 psi hydrogen 
pressure. 
The hydrogenated product is isolated, purified if desired, and the ether 
group is cleaved with an acidic catalyst in a reaction inert solvent at a 
temperature between 0.degree. C. to 100.degree. C. depending on the acidic 
catalyst used. Hydrogen bromide at elevated temperatures, boron tribromide 
and aluminum chloride at 0.degree. C. to ambient temperature have been 
found to be effective for this reaction. 
The product, Formula I is isolated and purified by standard procedures. 
Intermediates of Formula II where A is CH.sub.2, and B, D and E are CH are 
described in U.S. Pat. No. 3,274,213; J. Med. Chem 10, 78 (1967); J. Med. 
Chem 10, 138 (1967); and J. Med. Chem. 12, 881 (1969), the disclosures of 
which are herein incorporated by reference. They can also be prepared by 
procedures described below. 
The preparation of the compounds of Formula I where e=1, A=CH.sub.2, 
Z.sup.1 =OCH.sub.2 CH.sub.2, G=cycloalkylamine, B=CH is shown in Scheme 1. 
Compounds 1-2, where D and E are CH are made by alkylation of 
4-bromophenol with the corresponding N-chloroethylamine using potassium 
carbonate as base in a polar aprotic solvent like dimethylformamide at 
elevated temperatures. A preferred temperature is 100.degree. C. Compounds 
1-2 where D or E or both are N are synthesized using a nucleophilic 
displacement reaction performed on dibromides (1-1) using hydroxy ethyl 
cycloalkylamines under phase transfer conditions to afford bromo amines 
(1-2). Synthesis, 77, 573 (1980). Following halogen metal exchange using 
n-butyllithium or magnesium metal, bromo amines (1-2) yield the 
corresponding lithium or magnesium reagents which are allowed to react at 
low temperature in the presence of cesium chloride preferably (without 
cesium chloride the reaction also proceeds) with 6-methoxy-1-tetralone to 
afford either carbinols (1-3) or styrenes (1-4) after acidic workup. 
Treatment of either carbinols (1-3) or styrenes (1-4) with a brominating 
agent such as pyridinium bromide perbromide. affords bromo styrenes (1-5). 
Aryl or heteroaryl zinc chlorides or aryl or heteroaryl boronic acids 
react with bromides (1-5) in the presence of a palladium metal catalyst 
like tetrakis triphenyl phosphine palladium (0) to yield diaryl styrenes 
(1-6). [Pure & Applied Chem. 63, 419,(1991) and Bull. Chem. Soc. Jpn. 61, 
3008-3010, (1988)] To prepare the preferred compounds the substituted 
phenyl zinc chlorides or substituted phenylboronic acids are used in this 
reaction. The aryl zinc chlorides are prepared by quench of the 
corresponding lithium reagent with anhydrous zinc chloride. The aryl 
boronic acids, that are not commercially available, are prepared by 
quenching the corresponding aryl lithium reagent with trialkyl borate, 
preferably the trimethyl or triisopropyl borate, followed by aqueous acid 
workup. Acta Chemica Scan. 47, 221-230 (1993). The lithium reagents that 
are not commercially available are prepared by halogen metal exchange of 
the corresponding bromide or halide with n-butyl or t-butyllithium. 
Alternately, the lithium reagent is prepared by heteroatom facilitated 
lithiations as described in Organic Reactions, Volume 27, Chapter 1. 
Catalytic hydrogenation of 1-6 in the presence of palladium hydroxide on 
charcoal, for example, affords the corresponding dihydro methoxy 
intermediates which were subsequently demethylated using boron tribromide 
at 0.degree. C. in methylene chloride or 20 48% hydrogen bromide in acetic 
acid at 80-100.degree. C. to afford target structures (1-7). These 
compounds are racemic and can be resolved into the enantiomers via high 
pressure liquid chromatography using a column with a chiral stationary 
phase like the Chiralcel OD columns. Alternately optical resolution can be 
carried out by recrystallization of the diastereomeric salts formed with 
optically pure acids like 1,1'-binapthyl-2,2'-diyl hydrogen phosphate (see 
Example 8). 
The cis compounds (1-7) can be isomerized to the trans compounds on 
treatment with base (see Example 2). 
When D and/or E is nitrogen the intermediates (Formula II) and compounds of 
Formula I may be prepared from the corresponding dihalopyridines or 
pyrimidines as illustrated in Scheme 1 and as fully described for 
6-phenyl-5-[6-(2-pyrrolidin-1-yl-ethoxy) 
pyridin-3-yl]-5,6,7,8-tetrahydronaphthalen-2-ol in Example 6. 
The methyl ether of the compound of Formula I where e=1,-- A=CH.sub.2, 
Z.sup.1 =OCH.sub.2 CH.sub.2, G=pyrrolidine, D,E, B=CH, Y=Ph can also be 
conveniently prepared by a first step of hydrogenation of nafoxidine 
(Upjohn & Co., 700 Portage Road, Kalamazoo, Mich. 49001) in a reaction 
inert solvent in the presence of a nobel metal catalyst. Pressure and 
temperature are not critical; the reaction is conveniently run in ethanol 
at room temperature for approximately 20 hours at 50 psi. 
The second step is cleavage of the methoxy group which is accomplished 
conveniently at room temperature with an acidic catalyst such as boron 
tribromide in a reaction inert solvent or at 80-100.degree. C. with 
hydrogen bromide in acetic acid. The product is then isolated by 
conventional methods and converted to an acid salt if desired. 
##STR11## 
Compounds of formula I wherein B is nitrogen are prepared by the procedures 
illustrated in Scheme 2 and 3 and Examples 3-5 and 10-12. 
The synthesis of compounds of Formula I where B=N is shown in Scheme 2. 
Aryl acid chlorides (2-1) on treatment with primary amines afford aryl 
secondary amides (2-2), which are reduced with lithium aluminum hydride in 
ethereal solvents to yield secondary amines (2-3). Subsequent acylation of 
(2-3) with aroyl acid chlorides leads to tertiary amides (2-4), which are 
cyclized in hot phosphorus oxychloride to yield dihydro isoquinolinium 
salts (2-5). Reduction with sodium borohydride to alkoxytetrahydro 
isoquinolines; followed by boron tribromide demethylation in methylene 
chloride affords the target structures. 
##STR12## 
The synthesis of the compounds of Formula I where B.dbd.N is also described 
below in Scheme 3. Secondary amines (3-1) on acylation with benzyloxyaroyl 
chlorides (3-2) afford tertiary amides (3-3) which on cyclization with hot 
phosphorous oxychloride yield dihydro isoquinoline salts (3-4). Sodium 
borohydride reduction of (3-4) followed by debenzylation with aqueous 
hydrochloric acid affords isoquinolines (3-5), which are alkylated with 
the appropriately functionalized chlorides and demethylated with boron 
ibromide to yield the desired target structures. 
##STR13## 
Although the free-base form of formula I compounds can be used in the 
ethods of the present invention, it is preferred to prepare and use a 
pharmaceutically acceptable salt form. Thus, the compounds used in the 
methods of this invention form pharmaceutically acceptable acid and base 
addition salts with a wide variety of inorganic and, preferably, organic 
acids 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, terephthalate, phosphate, monohydrogenphosphate, 
dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, 
phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, 
bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzenesulfonate, 
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 citrate 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. 
The pharmaceutically acceptable salts of formula I compounds 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. 
Once prepared, the free base or salt form of formula I compounds can be 
administered to an individual in need of treatment for the methods herein 
described. The following nonlimiting test examples illustrate the methods 
of the present invention. 
For the methods of the present invention, compounds of Formula I are 
administered continuously, or from 1 to 4 times daily. 
As used herein, the term "effective amount" means an amount of compound of 
the methods of the present invention which is capable of inhibiting the 
symptoms of the 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 severity of the 
pathological condition being treated. A typical daily dose will contain a 
nontoxic dosage level of from about 0.25 mg to about 100 mg/day of a 
compound of the present invention. Preferred daily doses generally will be 
from about 1 mg to about 40 mg/day. 
The compounds of this invention can be administered by a variety of routes 
including oral, rectal, transdermal, subucutaneous, intravenous, 
intramuscular, and intranasal. These compounds preferably are formulated 
prior to administration, the selection of which will be decided by the 
attending physician. Typically, a formula I compound, or a 
pharmaceutically acceptable salt thereof, is combined with a 
pharmaceutically acceptable carrier, diluent or excipient to form a 
pharmaceutical formulation. 
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/or salt must be compatible 
with the other ingredients of the formulation, and not deleterious to the 
recipient thereof. 
Pharmaceutical formulations containing a compound of formula I can be 
prepared by procedures known in the art using well known and readily 
available ingredients. For example, the compounds of formula I 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 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. 
In the formulations which follow, "active ingredient" means a compound of 
formula I, or a salt thereof. Formulation 1: Gelatin Capsules 
Hard gelatin capsules are prepared using the following: 
______________________________________ 
Ingredient Quantity (mg/capsule) 
______________________________________ 
Active ingredient 0.25-100 
Starch, NF 0-650 
Starch flowable powder 
0-50 
Silicone fluid 350 centistokes 
0-15 
______________________________________ 
A tablet formulation is prepared using the ingredients below: 
Formulation 2: Tablets 
______________________________________ 
Ingredient Quantity (mg/tablet) 
______________________________________ 
Active ingredient 
0.25-100 
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 0.25-100 mg of active ingredient are 
made up as follows: Formulation 3: Tablets 
______________________________________ 
Ingredient Quantity (mg/tablet) 
______________________________________ 
Active ingredient 0.25-100 
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 0.25-100 mg of medicament per 5 ml dose are 
made as follows: Formulation 4: Suspensions 
______________________________________ 
Ingredient Quantity (mg/5 ml) 
______________________________________ 
Active ingredient 0.25-100 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 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 
20 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. 
Antiestrogens are compounds that prevent estrogens from expressing their 
effects on estrogen dependent target tissues consequently antagonizing a 
variety of estrogen-dependent processes. However, most antiestrogents such 
as tamoxifen are not pure antagonists, since they exhibit some 
estrogenicity. The methods below enable the skilled practitioner to 
determine the estrogen and antiestrogen effect of the compounds of this 
invention. U.S. Pat. No. 4,859,585, incorporated herein by reference 
claims two alternative general protocols by which a substance may be 
characterized as an estrogen agonist and/or estrogen antagonist. 
Methods to Determine Estrogenic and Antiestrogenic Potential 
Uterine weight test. Compounds of Formula I are given orally to immature 
female Sprague-Dawley (SD) rats (20 days old; 40 g body wt; Charles River 
Wiga, Sulzfeld, F. R. G.) for 3 consecutive days to test estrogenic 
activity. In addition to each dose of a compound of formula I, a standard 
dose of 1 mg/kg estradiol is administered orally to juvenile SD rats to 
determine the antiestrogenic effect of the compounds. The compounds are 
suspended in 0.25% agar for the administration. The animals are killed on 
day 4, the uteri removed, cleared of any intrauterine fluid and 
subsequently weighed in a dry condition. Estrogenic activity is estimated 
by the increase in uterine weight. (uteroptropic effect) initiated by the 
respective daily doses of compounds of formula 1. The antiestrogenic 
effect of the compounds is tested by the reduction of the uterine weight 
(anti uterotropic effect) in the presence of 1 mg/kg estradiol. 
Estrogen receptor-binding assay. Estrogen receptors (ER) are measured in 
the cytosol of uterine tissue of female immature white New Zealand rabbits 
(3 months of age). The uteri are separated from surrounding fatty tissue, 
rinsed in ice-cold phosphate-buffered saline, and immediately transferred 
into liquid nitrogen. The frozen uterine tissue is put into a capped 
Teflon cylinder pre-cooled in liquid nitrogen that is vibrated (501 {7.) 
for at least 30 sec. in a microdismembrator (Braun, Melsungen, F. R. G.) 
in the presence of a tungsten carbide bal 1. The resulting power is mixed 
with units (1:4/w:v) of Trisbuffer (0.01 M `1` r-is, 0.001 EDTA, pH 7.5), 
homogenized with a Dounce homogenizer and centrifuged at 105,000 g for 1 
hr. The supernatant (cytosol) is decanted and the protein concentration 
adjusted to 5 mg protein/ml. The protein concentration is measured 
according to Lowry et al. [8]. Aliquots of cytosol are pipetted into 
plastic tubes, 2.5.times.10.sup.-9 M [17.beta.-.sup.3 H]estradiol, and a 
range of concentrations of unlabeled estradiol and antiestrogens of 
formula I are added. The relative binding affinity of the antiestrogens to 
the estrogen receptor is carried out with the dextran-charcoal method at 
2.degree. C. as described by Deyleesch ouwer et al. [10]. 
All steps are carried out in triplicate. The relative binding affinity is 
defined as the ratio of the concentrations of radioinert 
17.beta.-estradiol to the compound of formula I that is necessary to 
achieve a 50% inhibition of the specific [17&3 H]estradiol binding. Bound 
radioactivity at the highest concentration of 17/3-estradiol 
(2,5.times.1.sup.-7 M) is taken as unspecific binding and subtracted from 
all values. 
Procedures for evaluating compounds of this invention for treatment of skin 
and vaginal atrophy are described in U.S. Pat. No. 5,461,064, which is 
incorporated herein by reference. 
Skin Atrophy 
Three to twenty women, who are post-menopausal and in good health, are 
selected. Additionally, these women are selected on the basis of their 
presenting several signs of rapid dermal atrophy, such as a rapid increase 
in the number of facial wrinkles or crow's feet, rapid change in the 
pigmentation of the skin, i.e. "age spots", or other complaints of rapid 
dermal aging. It should be remembered by the attending physician that 
these criterion may be highly subjective to the patient and that some 
consideration must be taken into account in patient selection. Also, 
dermal atrophy may be the result of other factors such as UV damage from 
the sun or other environmental insults and that such patients who are 
suffering from these effects would be excluded. 
The first component of the study is qualitative and subjective one, i.e., 
an evaluation of improvement in the patient's appearance. Such an 
evaluation requires an initial benchmark for future comparison. Some 
initial benchmarks might be in the form of a standardized set of questions 
as to how the patient views her own appearance, photographs of the 
patient, or a psychological profile of the patient's self-image. The 
second component is quantitative; these include the measurement of urinary 
excretion of hydroxyproline, moisture content of the skin, 
glycosaminoglyeans in the skin, and changes in resilience and pliability 
of the skin. Methods for determining these factors are found in. "The 
Menopause", Ed. R. J. Beard, University Press, Chapter 7 (1977) and 
"Methods in Skin Research", Ed. Skerrow, D. and Skerrow C. J., John Wiley 
& Sons Ltd., Chp. 22, "Analysis of Sebaceous Upids", p. 587-608 (1985), 
and further references cited therein, all herein incorporated by 
reference. Again, an initial benchmark of these quantitative factors is 
obtained. 
The women, thus selected and initially evaluated, are placed in a clinical 
protocol of receiving 20-100 mg of a compound of this invention by oral 
administration either as a single or split dose. Alternatively, these 
patients are placed in a protocol for topical administration to areas of 
the skin most effected by the atrophy. This topical protocol includes the 
use of a suitable formulation containing 5-50% (by weight) of an active 
compound of this invention applied to the affected area once or twice a 
day. Either of these protocols continues two to twelve months. Subsequent 
evaluations, both quantitative and qualitative, are made at appropriate 
intervals. 
A positive result is an improvement in the overall qualitative index of the 
patient's appearance and/or an improvement in the quantitative parameters, 
e.g., an increase in the urinary excretion of hydroxyproline signifying an 
increase in turnover and synthesis of collagen, an increase in moisture 
content, glycosaminoglycans, pliability, or resilience of the skin. 
Vaginal Atrophy 
Three to twenty women suffering from vaginal atrophy associated with 
menopause are selected. These women are in general good health. Since the 
nature of this disorder is highly idiosyncratic and subjective, evaluation 
of the effectiveness of treatment would necessarily be subjective in 
nature. These patients are asked to keep a daily log noting such details 
as vaginal itching and scaling and the degree of comfort in sexual 
intercourse. These women are placed on a clinical protocol similar to that 
described above for atrophy of the skin. Particular emphasis is placed on 
the use of vaginal suppositories containing 5-25% of an active compound of 
this invention. 
A positive result is an improvement in the comfort of sexual intercourse 
and/or a decrease in vaginal itching or scaling. 
Utility of the compounds described herein is exhibited by the positive 
results observed in one or both of the above assays. 
Procedures for evaluating the utility of a compound of this invention for 
increasing the libido of post-menopausal women are described in U.S. Pat. 
No. 5,439,931, which is incorporated herein by reference. 
Assay 1 
Animals used are ovariectomized or ovariectomized/adrenalectomized 
Sprague-Dawley rats (Specific Pathogen Free-Anticimex, Stockholm) weighing 
250-300 gms. They are housed in a room maintained at a temperature of 
24.degree. C. under reversed lighting (10 hours dark). Food and water (or 
saline) are available ad libitum. A compound of the invention is 
administered to one group of rats, and the other group is maintained as a 
control, and behavioral observations are made by placing each female with 
2 cage-adapted, sexually experienced males for a 5-minute period during 
which about 20 mounts occur. The following measures are recorded: 
1. Proportion of mounts by the male which elicited a lordosis 
response--L/M; 
2. Lordosis intensity measured on a 3 point scale; 
3. Lordosis duration (in seconds); 
4. Female acceptance ratio--No. of mounts divided by No. of refused 
mounting attempts plus mounts, a measure of the female's willingness to 
accept the male when he attempts to mount her. 
Activity of the compound is shown through positive impact on any one of the 
4 observations, as compared to control. 
Assay 2 
Five to fifty women are selected for the clinical study. These women are 
post-menopausal, i.e., have ceased menstruating for between 6 and 12 
months prior to the study's initiation, are in good general health, and 
suffer from self-described loss of libido especially noted after 
menopause. Because of the idiosyncratic and subjective nature of this 
symptom, the study has a placebo control group, i.e., the women are 
divided into two groups, one of which receive the active agent of this 
invention and the other receives a placebo. Women in the test group 
receive between 50-200 mg of the drug per day by the oral route. They 
continue this therapy for 3-12 months. Accurate records are kept as to the 
level of libido of the women in both groups and at the end of study these 
results are compared. 
Activity of the compounds of the invention is illustrated by positive 
effects in at least one of the above assays. 
Test methods for measuring the ability of a compound of this invention to 
inhibit fertility in women are described in U.S. Pat. No. 5,462,949, which 
is incorporated herein by reference. 
Assay 1 
Between five and fifty young adult virgin female rats weighing 200-300 g. 
each are separated into groups having the same number of rats. One of the 
groups serves as the control group and the other groups as experimental 
groups, each such experimental group receiving raloxifene at a particular 
dose level. Raloxifene is prepared in corn oil such that the daily 
administration is in 0.1 ml. of vehicle. The designated quantity of 
raloxifene in the vehicle is administered to each rat within the defined 
group subcutaneously (sc) daily. Alternatively, administration may be made 
via oral gavage or an intramuscular route. The control group receives only 
the vehicle. Administration of the vehicle or the combination of 
raloxifene and vehicle is continued on a daily basis for 15 days. On the 
5th day of treatment, one or two adult male rats weighing at least 250 g 
are added to each group, and cohabitation is continued until the 15th day 
at which time the male rats are withdrawn from the group. Each group of 
female rats then is maintained for an additional seven days after which 
the rats are sacrificed and examined for the presence of viable or 
resorbing fetuses. 
The number of animals that exhibit evidence of pregnancy over the number of 
animals in the group multiplied by one hundred is the pregnancy ratio 
percentage (PRP). A compound is considered active when the PRP is 0 to 
20%. A PRP of 40% constitutes marginal activity, and anything higher is 
inactive. 
Assay 2 
Between five and fifty young adult virgin female rats weighing 200-300 g. 
each are separated into groups having the same number of female rats, and 
paired with male rats. One of the groups serves as the control group and 
the other groups as experimental groups. Vaginal Smears are performed on 
the females daily until sperm and vaginal plugs are found, which coincides 
with the day of vaginal estrus and is designated day one of pregnancy. 
The male rats are removed, and the experimental groups of female rats are 
administered raloxifene via oral garage, an intramuscular route, or by 
subcutaneous injection. The administration continues on a daily basis 
until the twelfth day of pregnancy at which time all the female rats are 
sacrificed and examined for the presence of implantation sites. A compound 
is considered active when the PRP, as defined above, is 60% or lower. 
Utility of the compounds described herein is exhibited by activity in at 
least one of the above assays. 
Procedures for determining the effectiveness of compounds of this invention 
in treatment of pulmonary hypertensive disease are described in U.S. Pat. 
No. 5,447,941, which is incorporated herein by reference. 
Assay 1 
The procedure as set out in Farhat et al., J PET, 261: 686 (1992) (herein 
incorporated by reference) is carried out. Four to thirty rats are 
sacrificed. The lungs are exsanguinated by perfusion via the hepatic 
pulmonary vein. The pulmonary artery is cannulated as is the trachea to 
maintain ventilation and the pulmonary cannula is connected to the 
perfusion line and the whole ventilated lung is removed and suspended in a 
perfusion chamber. The effects of vasoconstrictor substances on perfusion 
pressure of the isolated perfused lung is measured using a Statham 
pressure transducer. The increase in perfusion pressure (vasoconstriction) 
induced by thromboxane mimetics in the presence of estradiol is determined 
and the ability to block the thromboxane effects with a compound of 
formula I or the estradiol potentiation of the thromboxane effects will be 
determined. 
Activity of compounds of formula I is illustrated by a reduction in 
pulmonary perfusion pressure increase following thromboxane mimetic 
stimulation. 
Assay 2 
Between five and fifty rats are administered a single IV dose of 
monocrotaline pyrrole, (3.5 mg/kg) and pulmonary disease is evaluated by 
histopathology, accumulation of fluorescein conjugated dextran in 
bronchial alveolar lavage fluid (as a measurement of pulmonary edema), and 
measurement of artery pressure using a Standtham P23ID pressure transducer 
(Reindel et al., Tax and Applic. Pharm. 106:179-200 (1990), incorporated 
herein by reference. A compound of formula I is administered and the 
effect on the rats are evaluated. 
Activity of compounds of formula I is illustrated by a reduction in uptake 
of fluorescein conjugated dextran from bronchial alveolar lavage fluids of 
animals treated with a compound of formula I, indicating a reduction in 
pulmonary edema. Rat lungs will also be removed from thorax, perfused with 
modified Karnovskys fixative and processed for histopathology. A reduction 
in thickening of the arterial walls in treated rats is evidence for the 
protective role of compounds of formula I as is a decrease in pulmonary 
artery pressure. 
Assay 3 
Five to fifty women are selected for the clinical study. The women suffer 
from a pulmonary hypertensive disease. Because of the idiosyncratic and 
subjective nature of these disorders, the study has a placebo control 
group, i.e., the women are divided into two groups, one of which receives 
a compound of formula I as the active agent and the other receives a 
placebo. Women in the test group receive between 50-200 mg of the drug per 
day by the oral route. They continue this therapy for 3-12 months. 
Accurate records are kept as to the number and severity of the symptoms in 
both groups and at the end of the study these results are compared. The 
results are compared both between members of each group and also the 
results for each patient are compared to the symptoms reported by each 
patient before the study began. 
Utility of the compounds of formula I is illustrated by the positive impact 
they have in at least one of the assays described above. 
The utility of a compound of this invention for inhibiting acne or 
seborrhea is tested by the procedures described in U.S. Pat. No. 
5,439,923, incorporated herein by reference. 
Assay 1 
Each of from between two and twenty patients selected for the clinical 
evaluation is placed in a comfortable environment, i.e., comfortable 
temperature, humidity, lighting, etc. These patients have refrained from 
strenuous exercise and consumption of spicy foods for the twelve hours 
prior to the evaluation. An area of the body which contains a large number 
of sebaceous glands affected by seborrhea or acne, such as the forehead, 
is wiped with a gauze pad to remove accumulated lipids. A patch of the 
skin is taped off, forming a rectangle sized 2.5 by 1.8 cm. A pad of 
cigarette paper or other suitable absorbent material sixed 2.5 by 1.8 cm 
is placed on the test area of the skin. The absorbent material must have 
first been defatted with ether prior to the placement on the test area to 
remove background lipids. The pad is the held in place with a bandage. 
After fifteen minutes the pad is replaced with a fresh pad (test pad). 
This procedure removes the background lipids in the skin so the true rate 
of lipid production by the sebaceous glands may be determined. The test 
pad is left in place for three to six hours and then removed. The test pad 
is then extracted with ether to remove the lipids and the ether 
evaporated. The residual lipids are then weighed. The result is expressed 
as the number of sebaceous lipids (mg) per 10 cm.sup.2 per hour. The 
patient then takes either 30-400 mg/day of the active ingredient by the 
oral route, or applies a topical formulation containing 5-20% by weight of 
the active ingredient daily, both for three to nine weeks. The above 
described test pad methodology is repeated several times throughout 
administration of the active ingredient to monitor progress. This assay 
may also be performed on animals to verify utility. A positive effect is 
reflected by a decrease of the rate of sebaceous gland lipid production. 
Assay 2 
Between two and twenty patients are enrolled in this clinical protocol and 
are initially evaluated by direct observation of the skin and lesions 
thereon. This is done by choosing one cm.sup.2 sections of affected skin 
and the number and type of lesion (comedos, seborrheic lesions, etc.) is 
noted. The areas normally used are the cheeks, scalp or back. The patient 
then takes either 30-400 mg/day of the active ingredient by the oral 
route, or applies a topical formulation containing 5-20% by weight of the 
active ingredient daily, both for three to nine weeks. The areas of the 
skin being evaluated are checked during the period of administration. Care 
must be taken to evaluate the same areas and in order to accomplish this, 
a small mark or marks may be made on the skin by a permanent marker. A 
positive result is reflected by a reduction in the number and/or severity 
of the lesions in the monitored areas of the skin. 
Utility of the compounds described herein is exhibited by the positive 
results observed in one or both of the above assays. 
Utility of the compounds of this invention for treating Turners Syndrome is 
determined by a procedure described in U.S. Pat. No. 5,441,966, 
incorporated herein by reference. 
Test Procedure 
Five to thirty females are selected for the clinical study. The females are 
between the age of twelve and eighteen and exhibit characteristics of 
Turner's Syndrome, but are in good general health otherwise. The study has 
a placebo control group, i.e., the females are divided into two groups, 
one of which receives the active agent of this invention and the other 
receives a placebo. Females in the test group receive between 10-100 mg of 
the active agent per day by the oral route. They continue this therapy for 
3-12 months. Accurate records are kept as to the number and severity of 
the above mentioned symptoms in both groups and at the end of the study 
these results are compared. The results are compared both between members 
of each group and also the results for each patient are compared to the 
symptoms reported by each patient before the study began. 
Utility of the compounds of the invention is illustrated by the positive 
impact they have on one or more of the symptoms when used in a study as 
above. 
EP 0 659 419 A1 incorporated herein by reference provides methods for 
evaluating compounds of the present invention for breast disorders. 
Assay 1 
Five to fifty women are selected for the clinical study. The women have a 
history of a breast disorder as described herein, but are in good general 
health. Because of the subjective nature of these disorders, the study has 
a placebo control group, i.e., the women are divided into two groups, one 
of which receive the active agent of this invention and the other receive 
a placebo. Women in the test group receive between 50-200 mg of the drug 
per day by the oral route. They continue this therapy for 3-12 months. 
Accurate records are kept as to the status of the breast disorders in both 
groups and at the end of the study these results are compared. The results 
are compared both between members of each group and also the results for 
each patient are compared to the symptoms reported by each patient before 
the study began. 
Utility of the compounds of the invention is illustrated by the positive 
impactthey have on the disorder or a symptom or symptoms thereof when used 
in a study as above. 
Assay 2 
Between three and twenty male patients suffering from gynecomastia or 
galactorrhea are selected. Initial measurement of breast size and evidence 
of lactation is noted. The patients receive 30-100 mg of an active 
compound of this invention per day as a single or divided dose via the 
oral route. This treatment is continued for 3-12 months. At appropriate 
intervals, further measurements of breast size or evidence of lactation 
are being made. 
Utility of the compounds of the invention is illustrated by the positive 
impact on the disorder or its symptoms. 
A method for determining hypoglycemic activity of the compounds of this 
invention is set forth in EP 0 635 264 A2, incorporated herein by 
reference. 
Five to 6 month old male, inbred viable yellow obese-diabetic mice are 
used. Male viable yellow mice are obese, hyperglycemic, hyperinsulinemic 
and insulin resistant. 
Mice are housed 6 per plastic cage with bedding and fed water and Purina 
Formulab Chow 5008 (Purina Mills, St. Louis, Mo.) ad libitum. The 
temperature of the animal rooms is maintained at 23.+-.2.degree. C. Lights 
in the animal rooms were on from 0600 to 1800 h. 
Antiestrogens are tested at various doses as admixtures of diets. Each dose 
of an antiestrogen is tested on 6 mice housed in the same cage. Compounds 
are mixed in pulverized chow and repelletized. Mice serving as controls 
are given repelletized diet without any test compound. Blood samples are 
collected from the tail vein immediately before and weekly after the start 
of a test. Blood glucose concentrations are determined by the glucose 
oxidase method with a model 300 Alpkem Rapid Flow nalyzer (Clackamaus, 
Oreg.). 
Reduction of blood glucose concentration below the levels of the control is 
indicative of an effective antiestrogen with utility in treatment of 
diabetes and hyperglycemia. 
The effect of compounds of the present invention on central nervous system 
(CNS) disorders in post-menopausal women may be evaluated by a method 
described in EP 0 659 413 A2, incorporated herein by reference. 
Test Procedure 
Five to fifty women are selected for the clinical study. The women are 
post-menopausal, i.e., have ceased menstruating for between 6 and 12 
months prior to the studys initiation, are in good general health, and 
suffer from one or more of the above-mentioned CNS disorders. Because of 
the idiosyncratic and subjective nature of these disorders, the study has 
a placebo control group, i.e., the women are divided into two groups, one 
of which receive the active agent of this invention and the other receive 
a placebo. Women in the test group receive between 50-100 mg of the drug 
per day by the oral route. They continue this therapy for 3-12 months. 
Accurate records are kept as to the number and severity of the above 
mentioned disorders in both groups and at the end of the study these 
results are compared. The results are compared both between members of 
each group and also the results for each patient are compared to the 
disorders reported by each patient before the study began. 
Utility of the compounds of the invention is illustrated by the positive 
impact they have on one or more of the CNS symptoms/disorders when used in 
a study as above. 
Methods for evaluating the effect of compounds of this invention in 
treating obsessive-compulsive and consumptive disorders are described in 
EP 0 659 428 A1, incorporated herein by reference. 
Assay 1 
In order to demonstrate the in vivo effect of the compounds on alcohol 
consumption, experiments are designed to test the effect on free choice 
ethanol intake in golden hamsters. Hamsters are chosen based on previous 
reports that they are receptive to and give preference to high ethanol 
intake when compared with several other mammalian species. Kulkosky and 
Cornell (Pharmacol. Biochem. & Behav. 11: 439-44, 1979) concluded that the 
species differences in ethanol intake and preferences were correlated with 
differences in ethanol metabolism. 
The animals used for the experiments described herein are two to six male 
adult golden hamsters. Animals are maintained on a light/dark cycle of 14 
hours of light per day and for a 6-week acclimation period. Animals have 
access to food and water ad libitum. 
For the experiment, the animals are maintained as described above in a 
single large cage with four 250 ml calibrated drinking bottles. The 
drinking bottles are fitted with stainless steel straight sipper tubes 
used to measure fluid consumption to the nearest 5 ml. Spillage from the 
drinking tubes is caught by 2 oz. jars fitted with glass funnels and 
positioned under the sipper tubes. Fluid consumption by the hamsters is 
measured once every 3 days so that the consumption volumes are large 
enough to obtain reasonably accurate measurements. 
After a 6-week acclimation period, the body weights of the animals are 
taken, and water intake is noted. Water in 2 of the 4 drinking bottles is 
then replaced by a 15% ethanol solution and consumption of water and 
aqueous ethanol are measured for a period of 2 weeks. Within 2 to 3 days 
after the beginning of this free choice phase of feeding, the hamsters 
establish an explicit preference for aqueous ethanol over water and the 
initial preference ratio (aqueous ethanol intake divided by water intake) 
is noted. 
As a control, the animals are then fed with 0.2 ml water twice daily, using 
a stainless steel animal feeding needle. Water feeding does not seem to 
have any effect on the animals drinking behavior as measured by total 
fluid intake. After 6 days, the same group of hamsters are fed a compound 
of formula I via a liquid mixture for a period of 3 to 12 weeks. Activity 
of the compounds of formula I is illustrated by the preference ratio being 
lower during administration of said compound of the invention than the 
initial preference ratio. 
Assay 2 
Five to fifty women are selected for the clinical study. The women are 
post-menopausal, i.e., have ceased menstruating for between 6 and 12 
months prior to the study's initiation, are in good general health, and 
suffer from either obsessive-compulsive or a consumptive disorder. Because 
of the idiosyncratic and subjective nature of these disorders, the study 
has a placebo control group, i.e., the women are divided into two groups, 
one of which receives raloxifene as the active agent and the other 
receives a placebo. Women in the test group receive between 50-200 mg of 
the drug per day by the oral route. They continue this therapy for 3-12 
months. 
Accurate records are kept as to the number and severity of the symptoms in 
both groups and at the end of the study these results are compared. The 
results are compared both between members of each group and also the 
results for each patient are compared to the symptoms reported by each 
patient before the study began. 
Utility of the compounds of formula I is illustrated by the positive impact 
they have on one or more of the disorders/symptoms when used in an assay 
described above. 
EP 0 659 414 A2, incorporated herein by reference describes methods for 
evaluating compounds of the invention for inhibition of hirsutism and 
alopecia. 
Hirsutism 
Three to twenty women suffering from hirsutism are selected. These patients 
are initially scored for the extent and severity of hirsutism. The 
clinical evaluation is made by the methods described in the reference 
"Methods of Skin Research," John Wiley and Sons, pp 308-318 (1985), and 
the references cited therein. The patients receive 10-400 mg of an active 
compound of this invention per day as a single or split dose by oral 
administration. Alternatively, they apply a 10%, by weight of active 
ingredient, creme or lotion once or twice a day to the affected areas. The 
patient continues this protocol for six months. At appropriate intervals, 
re-evaluation by one of the methods described above would be made. 
Alopecia 
Three to twenty women suffering from female pattern alopecia ate selected. 
These patients are initially scored for the extent and severity of the 
alopecia. This clinical evaluation is made by the methods described in 
"Methods of Skin Research," John Wiley and Sons, pp 308-318 (1985) and 
Habif, T., "Clinical Dermatology," C. V. Mosby Co., Chapter 23, pp 493-504 
(1985); and references therein, herein incorporated by reference. 
Especially helpful in these evaluations is the "hair pluck" procedure and 
measurement of anagen to telogen ratio. The patients receive 10-400 mg of 
an active compound of this invention per day as a single or split dose by 
oral administration. Alternatively, the patients apply a 5-10% (by weight 
of a compound of this invention) as a creme, lotion, or shampoo to the 
affected area, once to twice a day. This protocol continues for six 
months. At appropriate intervals, re-evaluation by one of the methods 
described in the above references is made. A positive result is exhibited 
by an increase in the anagen to telogen ratio or an increase in the number 
of terminal hairs in the affected scalp region. 
Utility of the compounds of the invention is illustrated by the positive 
impact they have on one or more of the symptoms when used in a study as 
above. 
Assays which show the ability of compounds of the invention to increase 
macrophage function are described in EP 0 659 425 A1, incorporated herein 
by reference. 
Assay 1 
The procedure as set out in Friedman et al., J. Clin. Invest., 75, 162-167 
(1985) (herein incorporated by reference) is carried out, with certain 
modifications. Between five and one hundred mice are administered oral 
doses in the range of 1-10 mg/kg of a compound of formula 1 on a daily 
basis. Following the administration, macrophages are harvested and changes 
in both immune (Fc mediated) and non-immune phagocytosis are quantitated 
by using fluorescein conjugated yeast particles prepared based on 
Ragsdale, J. Immunol. Meth., 123:259 (1989). For immune mediated 
phagocytosis, fluorescein conjugated yeast is preincubated with mouse sera 
to promote opsonization. Increase in fluorescence uptake by macrophages is 
quantitated by an increase in fluorescent emission using excitation and 
emission wavelengths of 482 and 520 nm, respectively. This procedure is 
used with ex vivo or in vitro macrophage cultures and changes in 
fluorescence units quantitated. 
An increase in fluorescent units, as compared to control indicates activity 
of compounds of formula 1. 
Assay 2 
The procedure as set out in Zuckerman et al., Cell immunol, 103:207, 
(1986); J. Immunol., 140:978 (1988) (herein incorporated by reference) is 
carried out. The ability to induce class II antigens and consequently 
promote antigen presentation is determined on ex vivo primary peritoneal 
macrophages and in vitro with the murine macrophage cell line P388D1. 
Between five and one hundred mice are dosed with a compound of formula 1 
macrophages are harvested and probed with antibodies against class II 
antigens of the D haplotype. Increased class II antigen expression is 
determined by flow cytometry using the appropriate secondary antibodies. 
In vitro studies evaluate the effects of the compounds in increasing the 
basal level and gamma interferon inducible expression of class II antigen 
by flow cytometry. An increase in class II expression reflect an increase 
in macrophage activation. 
Assay 3 
The procedure as set out in Seow et al., J. Immunol. Meth., 98, 113 (1987) 
(herein incorporated by reference) is carried out. The assay is used to 
evaluate increases in macrophage effector functions which uses 
measurements of 2-deoxyglucose uptake. Macrophages ex vivo and in vivo are 
plated in 96 well plates at 10.sup.5 cells per well and incubated in 
phosphate buffered saline in the presence of 0.78 uCi/ml of 
3H-deoxyglucose, and a compound of formula 1 is placed in the wells. 
Reduction in the amount of extracellular glucose reflects the uptake of 
this non-metabolizable glucose analog and consequently provides an 
independent assay for the determination of the state of macrophage 
activation mediated by the compound of formula 1. Increase in deoxyglucose 
uptake by the compound demonstrates the ability of the compounds to 
increase the state of macrophage activation. 
Assay 4 
The procedure as set out in Zuckerman, Circ Shock, 29, 279 (1989) (herein 
incorporated by reference) is carried out to illustrate the ability of the 
compounds of formula 1 to protect in murine sepsis and endotoxin lethality 
models. Between five and one hundred mice are dosed orally with 1-10 mg/kg 
with a compound of formula 1 for 1 week prior to sepsis challenge. 
Challenge is performed using a bolus IV endotoxin injection under 
condition in which an LD100 is achieved (200 .mu.g lipopolysaccharide). 
Exogenous glucocorticoids such as dexamethasone at 20 mg/kg serve as a 
positive control in creasing survival. The effects of the compound of 
formula 1 is also determined using a sepsis model involving cecal ligation 
and puncture. Sepsis by both Gram positive and Gram negative organisms 
results in an LD100 by 48 hours despite the use of antibiotics. An 
increase in the number of surviving animals or in survival time, as 
compared to control, demonstrates the activity of the compounds. 
Assay 5 
The ability of the compounds of formula 1 to increase the secretion of 
cytokines such as TNF is quantitated in vivo by sera measurements using 
commercially available TNF ELISAs specific for mouse TNF. Between five and 
one hundred mice are orally dosed with 1-10 mg/kg of a compound of formula 
1 for one week prior to injection of a lethal or sublethal dose of 
lipopolysaccharide (200 and 1 .mu.g, respectively). At one hour post LPS 
injection the mice are bled and the basal and LPS inducible amounts of 
serum TNF determined. Routinely, TNF levels below 10 pg/ml are observed 
prior to LPS injection and achieve levels of 5-20 ng/ml following LPS. The 
ability of the compounds to modulate the basal or inducible levels of TNF 
is determined. An increase in basal TNF without triggering massive 
systemic TNF release in compound treated mice demonstrates the activity of 
the compounds in promoting cytokyne secretion. Finally, ex vivo and in 
vitro measurements of TNF release from peritoneal macrophages exposed to 
1-5 .mu.M of a compound in vitro is also performed by ELISA to determine 
the extent of cytokine increase mediated by a compound of formula 1. 
Assay 6 
Five to fifty women are selected for the clinical study. The women are 
immunosuppressed. Because of the idiosyncratic and subjective nature of 
these disorders, the study has a placebo control group, i.e., the women 
are divided into two groups, one of which receives a compound of formula 1 
as the active agent and the other receives a placebo. Women in the test 
group receive between 50-200 mg of the drug per day. They continue this 
therapy for 3-12 months. Accurate records are kept as to the number and 
severity of the symptoms in both groups and at the end of the study these 
results are compared. The results are compared both between members of 
each group and also the results for each patient are compared to the 
symptoms reported by each patient before the study began. 
Utility of the compounds of formula 1 in increasing macrophage function is 
illustrated by the positive impact they have in at least one of the assays 
described above. Such compounds are useful in combating infections and 
promote wound healing.