GB2279952A - Naphthopyridine derivative 5alpha-reductase inhibitors - Google Patents

Abstract

Compounds of the Formula I <IMAGE> wherein R1 is hydrogen or C1-6alkyl; n is 1 or 2; and X is hydrogen or a specified substituent, and the salts or esters thereof are useful in the treatment of pathologic conditions that benefit from blockade of isozymes of 5 alpha-reductase.

Description

TITLE OF THE INVENTION 6-SUB S TITUTED-4-AZA-HYDRO-BENZO [F] QUIN OLIN- 3 - ONE DERIVATIVES AS Sa-REDUCTASE INHIBITORS FIELD OF THE INVENTION The present invention provides novel compounds, novel compositions, methods of their use and methods of their manufacture, where such compounds are generally pharmacologically useful as agents in therapies whose mechanism of action rely on the selective inhibition of the isozyme Sa-reductase 1.

BACKGROUND OF THE INVENTION Certain undesirable physiological manifestations, such as acne vulgaris, seborrhea, female hirsutism, androgenetic alopecia (also known as androgenic alopecia and which includes male pattern baldness) and benign pro static hyperplasia, are the result of hyper-androgenic stimulation caused by an excessive accumulation of testosterone or similar androgenic hormones in the metabolic system. Early attempts to provide a chemotherapeutic agent to counter the undesirable results of hyperandrogenicity resulted in the discovery of several steroidal antiandrogens having undesirable hormonal activities of their own. The estrogens, for example, not only counteract the effect of the androgens but have a feminizing effect as well. Non-steroidal antiandrogens have also been developed, for example, 4'-nitro-3'-trifluoromethylisobutyranilide.See Neri, et aL, Endocrinol. 1972, 91 (2). However, these products, though devoid of hormonal effects, compete with all natural androgens for receptor sites, and hence have a tendency to feminize a male host or the male fetus of a female host and/or initiate feed-back effects which would cause hyperstimulation of the testes.

The principal mediator of androgenic activity in some target organs, e.g., the prostate, is 5oc-dihydrotestosterone, formed locally in the target organ by the action of testosterone-5 a-reductase.

Inhibitors of testosterone-5a-reductase will serve to prevent or lessen symptoms of hyperandrogenic stimulation in these organs. It is now known that a second Sa-reductase isozyme exists, which interacts with epidermal tissues, especially in scalp tissues. This form is conventionally designated as Sa-reductase 1, while the isozyme that principally interacts with the prostatic tissues is designated as Sa-reductase 2.

In the treatment of hyperandrogenic disease conditions, e.g.

benign prostatic hyperplasia (BPH), it would be desirable to have one drug entity which is active against both isozymes in the prostate to significantly inhibit dihydrotesterone production, while also having another drug entity which is highly selective for inhibiting the isozyme Sa-reductase 1 associated with the scalp, for use in treating conditions of the skin and scalp, e.g., acne and androgenetic alopecia and female hirsutism. Additionally, a Sa-reductase 1 inhibitor could also be used in combination with a Sa-redustase 2 inhibitor such as finasteride (PROSCARB), which is highly selective for Sa-reductase 2, for combination therapy in the treatment of hyperandrogenic diseases.

Therefore, it is an object of this invention to provide compounds that have sufficient activity in the inhibition of one or both Sa-reductase isozymes. It is an additional object of this invention to provide compounds that are useful in the treatment and/or prevention of benign prostatic hyperplasia. It is an additional object of this invention to provide compounds that are useful in the treatment of female hirsutism, male pattern baldness, acne, seborrhea, androgenetic alopecia, and prostatic cancer. The compounds of the invention have utility in one or more of the afore-mentioned areas.

SUMMARY OF THE INVENTION The compounds of the present invention are those of the general structural Formula I:

or a pharmaceutically acceptable salt or ester thereof, wherein R1 is selected from the group consisting of hydrogen and C1-6 alkyl; n is 1 or 2; X is selected from the group consisting of hydrogen, halogen, cyano, CF3, C1-6 alkyl, C1-6 alkoxy, carboxy, C1-6 alkoxycarbonyl, amino, C1-4 alkylamino, C1-4 dialkylamino, amido, C1-4 alkylamido, C1-4 dialkylamido, C1-6 alkylthio, C1-6 alkylsulfinyl, C1-6 alkylsulfonyl, and -A-R2; A is selected from the group consisting of C1-6 alkylene, C2-6 alkenylene or C2-6 alkynylene; R2 is selected from the group consisting of halogen, hydroxy, CF3, C1-6 alkoxy, carboxy, C1-6 alkoxycarbonyl, amino, C1-4 alkylamino, C1-4 dialkylamino, amido, C1-4 alkylamido, C1-4 dialkylamido, C1-4 alkylsulfonylamino, aminosulfonyl or C1-4 alkylaminosulfonyl;; Alk is Cl l2 straight or branched alkyl; Ar is phenyl; and Het is selected from the group consisting of piperidinyl, piperizinyl, pyrrolidinyl, pyrrolyl, furanyl and thienyl.

DETAILED DESCRIPTION OF THE INVENTION Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts include the following salts:: Acetate Lactobionate Benzenesulfonate Laurate Benzoate Malate Bicarbonate Maleate Bisulfate Mandelate Bitartrate Mesylate Borate Methylbromide Bromide Methylnitrate Calcium Edetate Methyl sulfate Camsylate Mucate Carbonate Napsylate Chloride Nitrate Clavulanate N-methylglucamine Citrate ammonium salt Dihydrochloride Oleate Edetate Oxalate Edisylate Pamoate (Embonate) Estolate Palmitate Esylate Pantothenate Fumarate Phosphateldiphosphate Gluceptate Polygalacturonate Gluconate Salicylate Glutamate Stearate Glycollylarsanilate Sulfate Hexylresorcinate Subacetate Hydrabamine Succinate Hydrobromide Tannate Hydrochloride Tartrate Hydroxynaphthoate Teoclate Iodide Tosylate Isothionate Triethiodide Lactate Valerate The heterocyclic ring represented by the term "Het" may be attached to structural formula I at any heteroatom (N, O or S) or carbon atom in the ring which results in the creation of a stable structure.

The term "pharmacologically effective amount" shall mean that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician; this includes alleviation of the symptoms of the disorder being treated. The novel methods of treatment of this invention are for disorders known to those skilled in the art. The term "mammal" includes humans.

The term "alkyl" shall mean straight or branched chain alkanes of one to ten total carbon atoms, or any specified numbers within this range.

Whenever the term "alkyl" or "aryl" or their prefix root appears in a name of a substituent (e.g. aralkoxyaryloxy) it shall be interpreted as including those limitations given above for "alkyl" or "aryl".

The present invention has the objective of providing methods of treating hyperandrogenic conditions including androgenic alopecia, male pattern baldness, acne vulgaris, seborrhea, and female hirsutism by oral, systemic, parenteral or topical administration of the novel compounds of formula I either alone or in combination with a Sa-reductase 2 inhibitor, or a potassium channel opener, or a retinoic acid or derivative thereof.

Alternatively, treatment may encompass administration of a combination of a compound of formula I with a Sa-reductase 2 inhibitor and another active agent such as a potassium channel opener, or a retinoic acid or derivative therof. The term "treating androgenic alopecia" is intended to include the arresting and/or reversing of androgenic alopecia, and the promotion of hair growth.

The present invention has the further objective of providing methods of treating benign prostatic hyperplasia, prostatitis, and treating and/or preventing prostatic carcinoma by oral, systemic or parenteral administration of the novel compounds of formula I either alone or in combination with a Sa-reductase 2 inhibitor. Alternatively, treatment may encompass administration of a combination of a compound of formula I with a Sa-reductase 2 inhibitor and another active agent such as an al or an ocic adrenergic receptor antagonist.

The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing the present compounds as the active ingredient for use in the treatment of the above-noted conditions can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for systemic administration.

For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.

Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.

The daily dosage of the products may be varied over a range from 0.05 to 1,000 mg per adult human/per day. For oral administration, the compositions are preferably provided in the form of tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.

An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.002 mg./kg. to about 50 mg.lkg. of body weight per day. The range is more particularly from about 0.01 mg./kg. to 7 mg./kg. of body weight per day.

Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

For the treatment of androgenic alopecia, male pattern baldness, acne vulgaris, seborrhea, and female hirsutism, the compounds of the present invention may be administered in a pharmaceutical composition comprising the active compound in combination with a pharmaceutically acceptable carrier adapted for topical administration. Topical pharmaceutical compositions may be, e.g., in the form of a solution, cream, ointment, gel, lotion, shampoo or aerosol formulation adapted for application to the skin. These topical pharmaceutical compositions containing the compounds of the present invention ordinarily include about 0.1% to 15% by weight, and particularly about 5%, of the active compound in admixture with a pharmaceutically acceptable vehicle.

For the treatment of acne vulgaris, androgenic alopecia, male pattern baldness, seborrhea, female hirsutism, benign prostatic hyperplasia, prostatitis and the prevention and/or treatment of prostatic cancer, the compounds of the instant invention can be combined with a therapeutically effective amount of another 5a- reductase inhibitor, such as finasteride, or other Sa-reductase inhibitor compounds having type 2 activity or dual activity for both isozymes, in a single oral, systemic, or parenteral pharmaceutical dosage formulation. Alternatively, a combined therapy can be employed wherein the compound of formula I and the other 5a- reductase inhibitor are administered in separate oral, systemic, or parenteral dosage formulations.Also, for the skin and scalp related disorders of acne vulgaris, androgenic alopecia, male pattern baldness, seborrhea, and female hirsutism, the compounds of the instant invention and another Sa-reductase inhibitor such as finasteride can be formulated for topical administration. For example, a compound of formula I and finasteride can be administered in a single oral or topical dosage formulation, or each active agent can be administered in a separate dosage formulation, e.g., in separate oral dosage formulations, or an oral dosage formulation of finasteride in combination with a topical dosage formulation of a compound of formula I. See, e.g., U.S. Patent No.'s 4,377,584 and 4,760,071 which describe dosages and formulations for Sa-reductase inhibitors.

Furthermore, administration of a compound of the present invention in combination with a therapeutically effective amount of a potassium channel opener, such as minoxidil, cromakalin, pinacidil, a compound selected from the classes of Striazine, thiane-l-oxide, benzopyran, and pyridinopyran derivatives or a pharmaceutically acceptable salt thereof, may be used for the treatment of androgenic alopecia including male pattern baldness.

Therapy may further comprise the administration of a Sa-reductase type 2 inhibitor such as finasteride, or a type 1 and type 2 dual inhibitor, in combination with a compound of the present invention and a potassium channel opener such as minoxidil. The active agents can be administered in a single topical dosage formulation, or each active agent can be administered in a separate dosage formulation, e.g., in separate topical dosage formulations, or an oral dosage formulation of a compound of formula I in combination with a topical dosage formulation of, e.g., minoxidil, or a single oral dosage formulation of a compound of formula I and another 5a- reductase inhibitor, in combination with a topical dosage formulation of, e.g., minoxidil. See, e.g., U.S.Patent No.'s 4,596,812, 4,139,619 and WO 92/02225, published 20 February 1992, for dosages and formulations of calcium channel openers.

Furthermore, for the treatment of acne vulgaris, a combined therapy can be used by administering a therapeutically effective amount of a compound of formula I in combination with a therapeutically effective amount of retinoic acid or a derivative thereof, e.g., an ester or amide derivative thereof, such as e.g., tretinoin or isotretinoin. Optionally, this combined therapy for acne vulgaris may further include a Sa-reductase type 2 inhibitor such as finasteride, or a dual type 1 and type 2 inhibitory compound.

Also, for the treatment of benign prostatic hyperplasia, a combined therapy comprising a administration of a compound of formula I with a Sa-reductase type 2 inhibitor, such as e.g., finasteride, and an alpha adrenergic receptor antagonist, such as e.g., terazosin, doxazosin, prazosin, bunazosin, indoramin or alfuzosin, may be employed. More particularly, the combined therapy can comprise administering a compound of formula I with a Sa-reductase type 2 inhibitor, such as e.g., finasteride, and an alpha' adrenergic receptor antagonist. Compounds which are useful as alpha-lc adrenergic receptor antagonists can be identified according to the procedures described in PCT/US93/09187 (W094/08040, published April 14, 1994).

For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.

The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentration of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.

Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.

Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

The liquid forms in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents which may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired.

Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.

Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations. See, e.g., EP 0 285 382.

The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

The compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

The compounds of the present invention can be prepared readily according to the following reaction Scheme and Examples or modifications thereof using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are themselves known to those of ordinary skill in this art, but are not mentioned in greater detail. There is set forth on the following page a Synthesis Scheme.

The most preferred compounds of the invention are any or all of those specifically set forth in these examples. These compounds are not, however, to be construed as forming the only genus that is considered as the invention, and any combination of the compounds or their moieties may itself form a genus. The following examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. All temperatures are degrees Celsius unless noted otherwise.

EXAMPLE 1 To a suspension of aluminum trichloride (13.4 g, 100.8 mmol) in dichloromethane (50 mL) at -500C was added 4-Chloro- phenylacetyl chloride (10.lg, 53.4 mmol) over 20 minutes. Propene was then bubbled through the reaction mixture over 10 minutes to yield a yellow suspension. The resulting mixture was then warmed to ambient temperature and stirred for 18 hours. The reaction was quenched with ice water (500 mL) and extracted with dichloromethane (x2). The combined dichloromethane extracts were washed with water, 2N HCl, water, sat. aq. sodium bicarbonate, water and dried over anhydrous sodium sulfate. Removal of solvent yielded a brown syrup (11.3 g). The syrup was purified using a Waters Prep 500 (Porasil 15-20 ,u, hexane:ethyl acetate 10:1) in three batches to yield 1(2.5 g).

EXAMPLE 2 A solution of the product of Example 1 (500 mg, 2.6 mmol), acrylamide (370 mg, 5.2 mmol), p-toluenesulfonic acid (50 mg) and dimethylacetamide (1.0 mL) was heated at 80"C for 16 hours then at 1700C for 3 hours. The mixture was diluted with dichloromethane and filtered. The filtrate was washed with water and concentrated to remove solvent. The residue was triturated with ether and filtered to collect 2 as a slightly tan solid: 210 mg.

EXAMPLE 3 A solution of the product of Example 2 (600 mg), trifluoroacetic acid (4 mL), triethylsilane (0.60 mL) and dichloromethane (6 mL) was stirred at ambient temperature for 7 days.

Water was added and the resulting mixture extracted with dichloromethane (x2) and dried over anhydrous magnesium sulfate.

Removal of solvent gave a nearly colorless solid. After trituration with ether, 3 (256 mg) was obtained as a colorless solid.

EXAMPLE 4 A solution of the product of Example 3 (100 mg) in THF (5 mL) under nitrogen was treated with sodium hydride (30 mg). After 30 minutes methyl iodide (1 mL, excess) was added and the resulting grey mixture was stirred for 2 hours. The reaction was quenched with 1M ammonium chloride and extracted with dichloromethane (x4). The combined organic extracts were washed with water (x2), brine and dried over anhydrous sodium sulfate and concentrated to a yellow oil. The product was purified by column chromatography (silica gel, Hexanes:Isopropanol 85:15) to yield 4 (75 mg) as a mixture of isomers.

The diastereomeric mixture was separated into two pairs of enantiomers 5 and 6 by HPLC (Dynamax 5 Rm silica, 21 mm x 250 mm, Hexane:EtOH 98:2, 10 ml/min, 220 nm). Stereochemistry of the 7-methyl group was determined by nOe experiment.

Further resolution of the enatiomeric pairs was achieved using chiral HPLC (ChiralCel OD, 10 mm x 250 mm, Hexanes:EtOH 95:5, 30"C) to yield the individual enatiomers 7, 8, 2, and 10.

BIOLOGICAL ASSAYS Sa-reductase assay The reaction mixture contained in a final volume of 100 Cllis: 40 mM buffer (human scalp, potassium phosphate, pH 6.5; human prostatic Sa-reductase, sodium citrate, pH 5.5), 0.3-10 RM14C-T (or 3H-T), 1 mM DTT, and 500 RM NADPH. Typically, the assay is initiated by the addition of 50-100 ,ug prostatic homogenate or 75-200 ,ug scalp homogenate and incubated at 370C.

After 10-50 min the reaction is quenched by extraction with 250 Rl of a mixture of 70% cyclohexane: 30% ethyl acetate containing 10 ,tLg each DHT and T. The aqueous and organic layers are separated by centrifugation at 14,000 rpm in an Eppendorf microfuge. The organic layer is subjected to normal phase HPLC (10 cm Whatman partisil 5 silica column equilibrated in 1 ml/min 70% cyclohexane: 30% ethyl acetate; retention times DHT, 6.8-7.2 min; androstanediol, 7.6-8.0; T, 9.1-9.7 min). The HPLC system consists of a Waters Model 680 Gradient System equipped with a Hitachi Model 655A autosampler, Applied Biosystems Model 757 variable UV detector, and a Radiomatic Model A120 radio-activity analyzer.The conversion of T to DHT is monitored using the radioactivity flow detector by mixing the HPLC effluent with one volume of Flo Scint 1 (Radiomatic). Under the conditions described, the production of DIlT is linear for at least 25 min. The only steroids observed with the human prostate and scalp preparations were T, DHT and androstanediol.

Inhibition studies Compounds were dissolved in 100% ethanol. The compound to be tested was pre-incubated with the enzyme (either 5areductase type 1 or 2) prior to initiation by addition of substrate testosterone. IC50 values represent the concentration of inhibitor required to decrease enzyme conversion of testosterone to dihydrotestosterone by 50% of the control. IC50 values were determined using a 6 point titration where the concentration of the inhibitor was varied from 0.1 to 1000 nM. Representative compounds of this invention were tested in the above described assay for 5areductase type 1 and type 2 inhibition.

A compound referred to herein as a Sa-reductase 2 inhibitor is a compound that shows inhibition of the Sa-reductase 2 isozyme in the above-described assay, having an ICs0 value of about or under 100 nM.

A compound referred to herein as a dual Sa-reductase type 1 and 2 inhibitor is a compound that shows inhibition of both the type 1 and type 2 isozymes, having an IC50 value of about or under 100 nM for each isozyme.

Human Dermal Papilla Cell Assav The dermal papilla is a small group of cells at the base of each hair follicle, and it is presently thought that these cells are stem cells that form the basis for hair growth. These cells have been shown to have 5 alpha reductase activity, and it is therefore possible to test inhibitors of 5 alpha reductase in these cell culture systems.

Isolated and cultured dermal papilla cells are prepared according to the methods of Messenger, A.G., The Culture of Dermal Papilla Cells From Human Hair Follicles, Br. J. Dermatol.

110:685-689, 1984 and Itami, S. et. aL, Sos-Reductase Activity In Cultured Human Dermal Papilla Cells From Beard Compared With Reticular Dermal Fibroblasts, J. Invest. Dermatol. 94: 150-152, 1990.

Beard dermal papilla cells and occipital scalp hair of two different individuals are used throughout the study. All experiments are performed at confluency after the fourth to sixth subculture.

Confluent monolayers are. rinsed twice with phosphate-buffered saline, scraped from dishes by rubber policemen, and collected into a centrifuge tube. The cell suspensions are centrifuged at 1,500 rpm for 10 min at 4"C. The pellets are resuspended in 20 mM Tris-HCl buffer, pH 7.5, at 40C, containing 250 mM sucrose, 1 mM MgC12, and 2 mM CaC12, by vortexing and 10 passes through a 25-gauge needle. The crude homogenate is further homogenized by a teflonglass homogenizer, and is used as the cell homogenate. For the study of subcellular localization of Sa-reductase, the cell homogenate is centrifuged at 800 x g for 10 min to yield a crude nuclear pellet.

The resultant supernatant is centrifuged at 10,000 x g for 15 min to produce a crude mitochondrial pellet. The supernatant is centrifuged at 100,000 x g for 60 min to yield a microsomal pellet and cytosol.

Each particulate fraction is washed twice and resuspended in the buffer.

A standard incubation mixture will consist of 50 nM [3H]-testosterone, 1 mM NADPH, 100 mM sodium citrate, pH 5.5 or 100 mM Tris-HCl, pH 7.5, and 50 ml of the cell homogenate, in a final volume of 100 ml. Each tube contains 50-100 mg of cellular protein. Incubation is carried out at 370C for 30 min. During this incubation, the reaction is proportional to the time. For the study of optimum pH, citrate buffer is used at pH 4.5-6.5, and the Tris HCl buffer at pH 7.0-9.0. The protein content is determined by the method of Lowry, et. al., Protein Measurement With The Folin Phenol Reagent J. Biol. Chem. 193:265-275, 1951.

After incubation, the reaction is stopped by adding 4 times volume of chloroform-methanol (2/1:V/V) containing 110 mg each of carrier steroids. The extracted steroids are analyzed by thinlayer chromatographyl as previously described by Gomez, et. al., In Vitro Metabloism Of Testosterone-4-14C and D-androstene-3, 17dione-4-14C In Human Skin. Biochem. 7:24-32, 1968, and the purity of each steroid is determined by the recrystallization method. The activity of Sa-reductase is expressed by the sum of dihydrotestosterone, andro-stanediol and androstanedione formed.

[1,2-3H]-testosterone (55.2 Ci/mrnol) is obtainable from New England Nuclear Corporation (Boston, MA) and unlabeled steroids can be purchased from Sigma Chemical Company (St. Louis, MO).

Fetal calf serum is obtainable from Hazleton (Lenaxa, Kansas). All other chemicals are of reagent grade.

Fuzzv Rat Acne Model Adult fuzzy rats are a variety of rat that has stunted hair growth, brown colored seborrhea covering their entire back skin and abnormally increased sebum production after puberty that has been demonstrataed to be due to circulating androgens. 0.1, 0.05 and 0.025% solutions of a selected Sa-reductase inhibitor of interest are prepared in a vehicle of propylene glycol, isopropanol, isopropyl myristate and water (50/30/2/18%), and is topically applied onto the backs of adult male fuzzy rats, 0.2 ml per animal daily for 4 weeks.

Controls receive the vehicle alone and 5 of them are castrated. After 2 weeks seborrhea will be dose-dependently depleted and after 4 weeks bromodeoxyuridine (BrdU, 200 mg/kg) is intraperitoneally injected 2 hours before sacrifice. The skin tissues are incubated with EDTA (20 mM) in phosphate buffer, 1.5 hours at 370C. The pilosebaceous unit attached to the epidermis is stripped from the dermis and fixed with formalin for immuno-staining of BrdU. DNA synthesis cells showing a BrdU-positive nucleus are located in the outer glandular border. The number of S-phase cells per lobe is determined with a micro-image apparatus. Using formalin fixed skin, frozen serial sections are stained with 1% osmium and the size of the lobes is measured.A positive inhibitor of skin Sa-reductrase will induce suppression of sebum production by inhibiting the rate of glandular cell turnover, and showing reduced lobular size.

While the invention has been described and illustrated with reference to certain preferred embodiments thereof, those skilled in the art will appreciate that various changes, modifications and substitutions can be made therein without departing from the spirit and scope of the invention. For example, effective dosages other than the preferred dosages as set forth herein above may be applicable as a consequence of variations in the responsiveness of the mammal being treated for any of the indications for the compounds of the invention indicated above. Likewise, the specific pharmacological responses observed may vary according to and depending upon the particular active compound selected or whether there are present pharmaceutical carriers, as well as the type of formulation and mode of administration employed, and such expected variations or differences in the results are contemplated in accordance with the objects and practices of the present invention. It is intended, therefore, that the invention be limited only by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable.

Claims (4)

WHAT IS CLAIMED IS:

1. A compound of the Formula I

or a pharmaceutically acceptable salt or ester thereof, wherein R1 is selected from the group consisting of hydrogen and C1-6 alkyl; n is 1 or 2; X is selected from the group consisting of hydrogen, halogen, cyano, CF3, C1-6 alkyl, C1-6 alkoxy, carboxy, C1-6 alkoxycarbonyl, amino, C1-4 alkylamino, C1-4 diallcylamino, amido, C1-4 alkylamido, C1-4 dialkylamido, C1-6 alkylthio, C1-6 alkylsulfinyl, C1-6 alkylsulfonyl, and -A-R2; A is selected from the group consisting of C1-6 alkylene, C2-6 alkenylene or C2-6 alkynylene;; R2 is selected from the group consisting of halogen, hydroxy, CF3, C1-6 alkoxy, carboxy, C1-6 alkoxycarbonyl, amino, C1-4 alkylamino, C1-4 dialkylamino, amido, C1-4 alkylamido, C1-4 dialkylamido, C1-4 alkylsulfonylamino, aminosulfonyl or C1-4 alkylaminosulfonyl; Alk is C1-12 straight or branched alkyl; Ar is phenyl; and Het is selected from the group consisting of piperidinyl, piperizinyl, pyrrolidinyl, pyrrolyl, furanyl and thienyl.

2. A method for inhibiting Sa-reductase or the isozymes thereof, comprising the step of administering to a mammal in need of such inhibition a pharmacologically effective amount of the compound as claimed in Claim 1.

3. A method for treating, in a mammal in need thereof, the hyperandrogenic conditions of acne, androgenic alopecia, male pattern baldness, female hirsutism, benign prostatic hyperplasia, prostatitis, and the treatment and prevention of prostatic cancer, comprising the step of administering to said mammal a pharmacologically effective amount of the compound as claimed in Claim 1.

4. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmacologically effective amount of a compound as claimed in Claim 1, sufficient to treat a hyperandrogenic condition in a mammal in need of such treatment.