WO1998041211A1 - Methods and compositions for treating polycystic ovary syndrome - Google Patents

Methods and compositions for treating polycystic ovary syndrome Download PDF

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Publication number
WO1998041211A1
WO1998041211A1 PCT/US1998/004955 US9804955W WO9841211A1 WO 1998041211 A1 WO1998041211 A1 WO 1998041211A1 US 9804955 W US9804955 W US 9804955W WO 9841211 A1 WO9841211 A1 WO 9841211A1
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Prior art keywords
aza
methyl
dimethyl
androst
pregn
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PCT/US1998/004955
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French (fr)
Inventor
Joanne Waldstreicher
Georgianna S. Harris
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Merck & Co., Inc.
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Priority claimed from GBGB9800215.7A external-priority patent/GB9800215D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to AU64625/98A priority Critical patent/AU6462598A/en
Publication of WO1998041211A1 publication Critical patent/WO1998041211A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin

Definitions

  • the present invention provides for a novel method of treating polycystic ovary syndrome. Further, the present invention is directed to a method of improving fertility and the response to in vitro fertilization (I.V.F.) by employing a 5 ⁇ -reductase type 1 inhibitor.
  • the present invention also provides for a method of manufacture of a medicament useful for treating polycystic ovary syndrome and a method of manufacture of a medicament useful as an adjunct in I.V.F.
  • the present invention also provides for compositions useful in the method of treating polycystic ovary syndrome and useful as an adjunct in I.V.F.
  • PCOS Polycystic ovary syndrome
  • Stein- Leventhal syndrome is characterized by menstrual irregularity and hirsutism and is a common cause of anovulatory infertility.
  • the biochemical abnormalities are a high concentration of plasma luteinising hormone (LH) or a high LH/follicle stimulating hormone (FSH) ratio and high concentrations of androgens (testosterone and/or androstenedione and/or dehydroepiandrosterone (DHEA)).
  • LH plasma luteinising hormone
  • FSH follicle stimulating hormone
  • DHEA dehydroepiandrosterone
  • the increased androgens can be secreted by the ovary and/or the adrenal gland.
  • Clinical manifestations of PCOS include amenorrhea, hirsutism acanthosis nigricans, acne and obesity. PCOS appears to account for about 75% of anovulatory infertility.
  • the problem is self-perpetuating in part because the atretic follicle becomes an androgenic follicle by a "default" mechanism: because of low aromatase activity in atretic follicles, androstenedione is preferentially metabolized to testosterone and thence to dihydrotestosterone within the ovary.
  • the human ovarian stromal, thecal and granulosa cell compartments each contain 5 ⁇ -reductase activity.
  • the 5 ⁇ - reduced androgens 5 ⁇ -androstane-3,17-dione (5 ⁇ -A) and dihyrotestosterone (DHT) are competitive inhibitors of aromatase activity. This is likely to occur in humans as well, and therefore the 5 ⁇ - reduced metabolites may lead to decreased aromatase activity, increased androgen secretion and follicular atresia.
  • Agarwal et al. "A Mechanism for the Suppression of
  • estrogen Production in Polycystic Ovary Syndrome J. Clin Endocrinology & Metabolism 81(10):3686-3691 (1996), propose that polycystic ovary syndrome follicular fluid contains abnormally high 5 ⁇ - A and/or DHT concentration that can inhibit aromatase activity. They conclude that 5 ⁇ -A is the primary inhibitor of aromatase activity in PCOS follicular fluid.
  • GnRH analogues GnRH analogues
  • oral contraceptives such as prednisone
  • antiandrogens antagonize androgens or decrease the whole H-P- G or H-P- Adrenal axes.
  • None of the treatments currently employed corrects the underlying problem of the hyperandrogenic production by the ovarian follicles.
  • each of the currently employed treatments has significant side effects including: hypoestrogenism (GnRH analogues), menstrual irregularity (spironolactone, an antiandrogen), and headaches, bloating and the rare occurrence of blood clots associated with oral contraceptives.
  • Steroids also have significant side effects such as adrenal suppression, obesity, striae, hypertension, etc.
  • the present invention relates to methods of treating polycystic ovary syndrome. Further, the present invention is directed to a method of improving fertility and the response to in vitro fertilization (I.V.F.) by employing a 5 ⁇ -reductase type 1 inhibitor. It has now been found that a 5 ⁇ -reductase type 1 inhibitors of structural formula I:
  • the enzyme 5 ⁇ -reductase catalyzes the reduction of several androgens including: testosterone (T) to the more potent androgen, 5 ⁇ - dihydrotestosterone (dihydrotestosterone" or DHT), as shown below:
  • 5 ⁇ -reductase also catalyzes the reduction of androstenedione (A) to androstanedione (5 ⁇ -A), as shown below:
  • isozymes of 5 ⁇ - reductase in humans There are two isozymes of 5 ⁇ - reductase in humans. Andersson, et al., Proc. Natl. Acad. Sci. USA, 87:3640-44 (1990); Andersson, et al., Nature, 354, 159-61 (1991).
  • the isozymes usually called Type 1 and Type 2, exhibit differences in their biochemical properties, genetics, and pharmacology. Both isozymes are now the subject of considerable research and it has been found one isozyme (type 1) predominates in he sebaceous glands of facial skin and skin tissue and that the other (type 2) predominates in the prostate.
  • Finasteride (17 ⁇ -(N-tert-butylcarbamoyl)-3-oxo-4-aza-5 ⁇ - androst-l-en-3-one) as shown below, is a potent inhibitor of the human type 2 enzyme.
  • finasteride is known to be useful in the treatment of hyperandrogenic conditions, see e.g., U.S. 4,760,071. Finasteride is currently prescribed for the treatment of benign prostatic hyperplasia (BPH), a condition affecting to some degree the majority of men over age 55. Finasteride's usefulness in the treatment of androgenic alopecia and prostatic cancer is described in the following documents: EP 0 285 382, published 5 October 1988, EP 0 285 383, published 5 October 1988 and Canadian patents 1,302,277 and 1,302,276.
  • BPH benign prostatic hyperplasia
  • type 1 5 ⁇ -reductase inhibitors of the present invention are useful in the treatment of polycystic ovary syndrome and in improving fertility and the response to in vitro fertilization.
  • the present invention further provides for a method for improving fertility and the response to in vitro fertilization (I.V.F.), comprising administration of therapeutically effective amount of compound of structural formula I to the subject. Further, the present invention provides for compositions useful in the methods of the present invention, as well as a method of manufacture of a medicament useful for treating polycystic ovary syndrome or for improving fertility and the response to in vitro fertilization (I.V.F.).
  • the present invention is directed to a method for treating polycystic ovary syndrome in a subject in need thereof by administering to the subject an effective amount of a compound of structural formula I:
  • the C1-C2 and C5-C6 bonds designated with a dotted line each independently represent a single or double bond, provided that when the C5-C6 is a double bond, H a is absent and when the C5-C6 bond is a single bond H a is present and represents hydrogen;
  • Rl is selected from hydrogen and Cl-5 alkyl;
  • R2 is Cl-5alkyl, either straight or branched chain;
  • R3 is C3-7alkyl, either straight or branched chain, optionally having one degree of unsaturation; or a pharmaceutically acceptable salt, or stereoisomer thereof.
  • Still a further aspect of the present invention is a method of improving fertility and the response to in vitro fertilization (I.V.F.), in a subject in need thereof by administering an effective amount of a compound of structural formula I to the subject.
  • the compounds of the present invention may provide a useful adjunct for in vitro fertilization because they may lead to multiple ovarian follicles developing completely through ovulation.
  • Compounds illustrating this subclass include: 7 ⁇ ,20-dimethyl-4-aza-5 ⁇ -pregn-l,17-dien-3-one, 7 ⁇ ,20-dimethyl-4-aza-5 ⁇ -pregn-l-en-3-one, 20-ethyl-7 ⁇ -methyl-4-aza-5 ⁇ -pregn-l-en-3-one, 20-propyl-7 ⁇ -methyl-4-aza-5 ⁇ -pregn-l-en-3-one, 17 ⁇ -n-propyl-7 ⁇ -methyl-4-aza-5 ⁇ -androst-l-en-3-one, 17 ⁇ -n-butyl-7 ⁇ -methyl-4-aza-5 ⁇ -androst-l-en-3-one, 17 ⁇ -isobutyl-7 ⁇ -methyl-4-aza-5 ⁇ -androst-l-en-3-one, 17 ⁇ -tert.-butyl-7 ⁇ -methyl-4-aza-5 ⁇ -androst-l-en-3-one,
  • Compounds illustrating this subclass include: 7 ⁇ ,20-dimethyl-4-aza-5 ⁇ -pregn-l-en-3-one, 20-ethyl-7 ⁇ -methyl-4-aza-5 ⁇ -pregn-l-en-3-one, 20-propyl-7 ⁇ -methyl-4-aza-5 -pregn-l-en-3-one, 17 ⁇ -n-propyl-7 ⁇ -methyl-4-aza-5 ⁇ -androst-l-en-3-one, 17 ⁇ -n-butyl-7 ⁇ -methyl-4-aza-5 ⁇ -androst-l-en-3-one, 17 ⁇ -isobutyl-7 ⁇ -methyl-4-aza-5 ⁇ -androst-l-en-3-one, 17 ⁇ -tert.-butyl-7 ⁇ -methyl-4-aza-5 ⁇ -androst-l-en-3-one, 17 ⁇ -n-pentyl-7 ⁇ -methyl-4-aza-5 ⁇ -androst-l-en-3-one, and 17
  • variable e.g., alkyl, R2, etc.
  • its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
  • alkyl is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, e.g., methyl (Me), ethyl (Et), propyl, butyl, pentyl, and the isomers thereof such as isopropyl (i-Pr), isobutyl (i- Bu), secbutyl (s-Bu), tertbutyl (t-Bu), isopentane, etc.
  • the compounds of the present invention may be administered in the form of pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt is intended to include all acceptable salts such as acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N- methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate, fumarate, phosphate/diphosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate,
  • the subject treated in the methods above is a female mammal, preferably a human being, diagnosed with polycystic ovary syndrome or ovarian or adrenal hyperandrogenism.
  • the subject treated is a human mammal, or preferably a human being, who is infertile due to irregular menstrual cycles and/or undergoing treatment with in vitro fertilization due to idiopathic infertility.
  • terapéuticaally effective amount means the amount the compound of structural formula I that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • composition as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • pharmaceutically acceptable it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • administration of and or “administering a” compound should be understood to mean providing a compound of the invention or a prodrug of a compound of the invention to the individual in need of treatment.
  • the administration of the compound of structural formula I in order to practice the present methods of therapy is carried out by administering an effective amount of the compound of structural formula I to the patient in need of such treatment or prophylaxis.
  • the need for a prophylactic administration according to the methods of the present invention is determined via the use of well known risk factors.
  • the effective amount of an ind vidual compound is determined, in the final analysis, by the physician in charge of the case, but depends on factors such as the exact disease to be treated, the severity of the disease and other diseases or conditions from which the patient suffers, the chosen route of administration other drugs and treatments which the patient may concomitantly require, and other factors in the physician's judgment.
  • the daily dosage of the compound of structural formula I may be varied over a wide range from 0.01 to 1000 mg per adult human per day. Most preferably, dosages range from 0.1 to 100 mg/day.
  • the compositions are preferably provided in the form of tablets containing 0.01 to 1000 mg, particularly 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 3.0, 5.0, 6.0, 10.0, 15.0, 25.0, and 50.0 and 100 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • the dose 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.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • Formulations of the 5 ⁇ -reductase inhibitor employed in the present method for medical use comprise the compound of structural formula I together with an acceptable carrier thereof and optionally other therapeutically active ingredients.
  • the carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient subject of the formulation.
  • the present invention therefor further provides a pharmaceutical formulation comprising the compound of structural formula I together with a pharmaceutically acceptable carrier thereof.
  • the formulations include those suitable for oral, rectal, intravaginal, topical or parenteral (including subcutaneous, intramuscular and intravenous administration). Preferred are those suitable for oral administration.
  • the formulations may be presented in a unit dosage form and may be prepared by any of the methods known in the art of pharmacy. All methods include the step of bringing the active compound in association with a carrier which constitutes one or more ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound in association with a liquid carrier, a waxy solid carrier or a finely divided solid carrier, and then, if needed, shaping the product into desired dosage form.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension or solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup, an elixir, or an emulsion.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients, e.g., binders, lubricants, inert diluents, disintegrating agents or coloring agents.
  • Molded tablets may be made by molding in a suitable machine a mixture of the active compound, preferably in powdered form, with a suitable carrier.
  • 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, carboxymethyl-cellulose, 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.
  • Oral liquid forms such as syrups or suspensions in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl cellulose and the like may be made by adding the active compound to the solution or suspension. Additional dispersing agents which may be employed include glycerin and the like.
  • Formulations for vaginal or rectal administration may be presented as a suppository with a conventional carrier, i.e., a base that is nontoxic and nonirritating to mucous membranes, compatible with the compound of structural formula I, and is stable in storage and does not bind or interfere with the release of the compound of structural formula I.
  • Suitable bases include: cocoa butter (theobroma oil), polyethylene glycols (such as carbowax and polyglycols), glycol- surfactant combinations, polyoxyl 40 stearate, polyoxyethylene sorbitan fatty acid esters (such as Tween, Myrj, and Arlacel), glycerinated gelatin, and hydrogenated vegetable oils.
  • cocoa butter theobroma oil
  • polyethylene glycols such as carbowax and polyglycols
  • glycol- surfactant combinations such as polyoxyl 40 stearate
  • polyoxyethylene sorbitan fatty acid esters such as Tween, Myrj, and Arlacel
  • glycerinated gelatin When glycerinated gelatin suppositories are used, a preservative such as methylparaben or propylparaben may be employed.
  • 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 0285 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.
  • Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy- ethylaspartamidephenol, or polyethylene-oxide polylysine substituted with palmitoyl residues.
  • 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, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • a drug for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • Formulations suitable for parenteral administration include formulations which comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient. Such formulations suitably comprise a solution or suspension of a compound that is isotonic with the blood of the recipient subject. Such formulations may contain distilled water, 5% dextrose in distilled water or saline and the active compound. Often it is useful to employ a pharmaceutically and pharmacologically acceptable acid addition salt of the active compound that has appropriate solubility for the solvents employed. Useful salts include the hydrochloride isothionate and methanesulfonate salts. Useful formulations also comprise concentrated solutions or solids comprising the active compound which on dilution with an appropriate solvent give a solution suitable for parenteral administration.
  • 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.
  • 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.
  • composition and method of the present invention may further comprise other therapeutically active compounds usually applied in the treatment of the above mentioned pathological conditions, for instance: GnRH analogues, oral contraceptives, antiandrogens, clomiphene, gonadotropins, or steroids, such as prednisone, estrogens and progestins.
  • GnRH analogues for instance: oral contraceptives, antiandrogens, clomiphene, gonadotropins, or steroids, such as prednisone, estrogens and progestins.
  • composition and method of the present invention may further comprise a type 2 5 -reductase inhibitor or a dual 5 ⁇ -reductase inhibitor.
  • Preferred type 2 5 ⁇ -reductase inhibitors for use in the present composition and method include: finasteride and epristeride.
  • a preferred dual inhibitor is: 17 ⁇ -N-(2,5-bis(trifluoromethyl))phenyl carbamoyl-4-aza-5 ⁇ -androst-l-en-3-one.
  • One aspect of the present invention provides a method for treating polycystic ovary syndrome comprising administering to a female mammal in need of treatment an effective amount of a compound of structural formula I.
  • Another aspect of the present invention provides a method of improving fertility and the response to in vitro fertilization (I.V.F.) comprising administering to a female mammal in need of treatment and effective amount of a compound of structural formula I.
  • I.V.F. in vitro fertilization
  • dosages of 0.01 to 10 mg per adult human per day are appropriate for treatment, more preferably 1 to 5 mg/day especially preferred is about 5 mg/day.
  • the compounds of the methods of the present invention can be administered by a variety of routes including oral, rectal, vaginal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal, and such compounds are preferably formulated prior to administration. Therefore, another embodiment of the present invention is a pharmaceutical formulation comprising an effective amount of a compound of structural formula I or a pharmaceutically acceptable salt thereof, a dual 5 ⁇ -reductase inhibitor, and a pharmaceutically acceptable carrier, diluent or excipient therefor.
  • the individual components of the combination can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.
  • the appropriately 7-substituted derivative is prepared according to the procedures of PCT publication WO 93/23420 and to produce (1), the 7 ⁇ - alkyl-substituted pregnenolone acetate.
  • the tertiary carbinol (2) may be alkylated at the 4- position by treatment with sodium hydride and the appropriate Ci-5alkyl iodide in a polar aprotic solvent such as THF or dimethylformamide (DMF).
  • the 4-NH or 4-N-alkyl compound is then dehydrated in the presence of acid, for example, HC1 or acetic acid, in a solvent such as THF or alcohol to produce the 17-ene (3).
  • the 17-ene (3) may be dehydrogenated to form the 1,17-diene (4) by treatment with DDQ in toluene or benzeneselenic anhydride in chlorobenzene, or other known methods, for example as described in U.S. 5,084,574 and 5,021,571.
  • DDQ is preferred for 4-NH compounds and benzeneselenic anhydride is preferred for 4-N-alkyl compounds.
  • the 17-ene (3) may be hydrogenated in the presence of a hydrogenation catalyst, for example Pt ⁇ 2, Pd/C, rhodium on alumina, preferably Pt ⁇ 2, in an appropriate solvent such as an alcohol or acetic acid, preferably methanol, to form the 17-alkyl derivative (5).
  • a hydrogenation catalyst for example Pt ⁇ 2, Pd/C, rhodium on alumina, preferably Pt ⁇ 2, in an appropriate solvent such as an alcohol or acetic acid, preferably methanol
  • the 17-alkyl derivative (5) may be dehydrogenated to form the 1-ene (6) by treatment with DDQ in toluene or benzeneselenic anhydride in chlorobenzene, as described above.
  • the desired 4-N-alkyl substitution may be effected as described previously by treating (2) with the appropriate alkyl iodide, or alternative, the procedure may be carried through with the 4-NH compound, and following after the desired 17-substitution and optional insertion of the 1,2-double bond, the 4-NH compound may be alkylated to the desired 4-N-alkyl compound.
  • the compounds of the present invention may be prepared according to the procedures of Scheme 2.
  • Compound (7) obtained according to procedures in WO 93/23420, is treated with N- phenyl trifluoro methane sulfonamide in a base such as lithium hexamethyldisilazide in THF to form the enol triflate (8).
  • the enol triflate (8) is converted to the desired enyne (9) by treatment with di(triphenylphosphine)palladium diacetate or other appropriate Pdo catalyst with a catalytic amount of cuprous iodide and a mild base such as diisopropylamine or triethylamine in DMF with the appropriate alkyne.
  • the enyne (9) is hyrdrogenated to produce the 17-alkyl derivative (10) by treating with H2 in the presence of 10% Pd/C in an alcoholic or ethyl acetate solvent, preferably ethyl acetate. Insertion of the 1,2-double bond, if desired is accomplished as described in Scheme 1 to produce the 17-alkyl- 1-ene (11).
  • the 5 ⁇ -reductase type 2 inhibitor finasteride that may employed this invention can be prepared as described in U.S. 4,760,071.
  • Rat ovary was obtained from pregnant animals (gestational days 15, 17, 19, 20, 21 and 22 and was pulverized using a freezer mill and homogenized in 20 mM potassium phosphate pH 6.5, 5 mM magnesium sulfate, 25 mM potassium chloride, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 5 mM NADPH, and 0.25 M sucrose using a dounce homogenizer. Glycerol was added to the homogenate to a final concentration of 20%.
  • the reaction mixture contained 33 mM succinic acid 44 mM imidazole, 33 mM diethanolamine (SID), pH 5.5 or pH 7.0, 0.3-15 ⁇ M [7- 3u]-testosterone (T) (specific activity approximately 20-30 Ci/mmol) or [l,2-3H]-progesterone, 1 mM DTT and 0.5 mM NADPH in a final volume of 0.1 mL.
  • the assay was initiated by the addition of enzyme and incubated at 37°C for 20-30 minutes.
  • the reaction was quenched with cyclohexane:ethyl acetate (70:30 v/v) and product peaks were separated by normal phase HPLC. Alternatively, samples were also quenched using 0.05 M phosphoric acid in 10% ethanol.
  • the product profile was analyzed by reverse phase HPLC.
  • the sample was chromatographed on a 25 cm Whatman Partisil 5 ODS-3 column that was equilibrated in methanol:water:acetonitrile (3:3:1) at 1.5 mL/min. Under these conditions, authentic steroids had retention times of 37 min and 24 min for 3 ⁇ - and 3 ⁇ -androstanediol, respectively.
  • Metabolism of testosterone Homogenates of rat ovary from pregnant animals were incubated with 0.3 ⁇ M ⁇ H-testosterone (T) or ⁇ H- progesterone (P) and the products analyzed by HPLC. Product assignments were based on comparison of retention times of unknowns to those of authentic standards on normal phase HPLC. Table 1, below shows representative examples of product distribution using T as substrate.
  • Metabolism of progesterone A representative product profile when P is used as substrate is presented in Table 2 and for the most part mirrors the pattern of products seen with T.
  • Progesterone undergoes 5 ⁇ -reduction to dihydroprogesterone (DHP) and subsequent reduction of DHP by 3 ⁇ -hydroxy steroid dehydrogenase to 3 ⁇ - hydroxy-5 ⁇ -pregnan-20-one.
  • DHP dihydroprogesterone
  • the unknown product at 12.1 minutes most likely represents the same proposed ring hydroxylation step discussed for testosterone metabolism.
  • One obvious difference in P metabolism is conversion to 20 ⁇ -hydroxyP (-17.6 min) by 20 ⁇ -hydroxysteroid dehydrogenase.
  • P is not a substrate for 17 ⁇ -hydroxy steroid dehydrogenase and therefore no product is expected for this enzyme.
  • Specific activity/Isozyme determination The specific activity of the enzyme was determined using T as substrate. The combined contribution of DHT and androstanediol was used for this analysis. As shown in Table 3, there is very little change in the 5 ⁇ - reductase content of the tissues from gestation day 15 to 22.
  • a ratio of 1 or lower suggests that the predominant enzyme is type 1, while a higher ratio indicates that the predominant enzyme is type 2. This study suggests that type 1 5 ⁇ -reductase is present in ovary.
  • Step 2 3-Acetoxy-20- ⁇ erf;-butyldimethylsilyloxy-pregn-5-ene
  • the solvent was removed by rotoevaporation and methylene chloride (0.7 L) was added and warmed to 40°C. Upon cooling to room temperature, the suspension was filtered and the filtrate washed with methylene chloride (0.2 L). The filtrate was rotoevaporated and treated with pyridine (1.35 L) and acetic anhydride (135 mL). After stirring overnight, the solvent was removed by rotoevaporation and the dark orange oil dissolved in methanol (0.6 L). The mixture was heated to 50°C and then cooled to room temperature. The solution was allowed to stand for 3 days and then cooled in an ice bath. The precipitate was filtered, washed with methanol, and dried to yield the title compound. The filtrate was rotoevaporated to a dry gum to yield the crude product.
  • Step 4 20-t;ert;-Butyldimethylsilyloxy-7-methyl-Dregn-5-ene-3.7-diol
  • Step 5 20-£er£-Butyldimethylsilyloxy-7-methyl-pregn-4.6-dien-3-one
  • a solution of 20- ert;-butyldimethylsilyloxy-7-methyl-pregn-5- ene-3,7-diol (298 gm, 0.59 mol, product of Step 4) in toluene (3 L) and cyclohexanone (1.03 L) was azeotroped to remove 750 mL of solvent.
  • a solution of aluminum isopropoxide (121 gm) in toluene (620 mL) was added and the solution azeotroped to remove another 650 mL of solvent.
  • a reflux condenser was added and the solution refluxed overnight.
  • Step 6 20- ert Butyldimethylsilyloxy-7 ⁇ -methyl-pregn-4-en-3-one
  • a slurry of 5% palladium on carbon (7.12 gm) and benzyl alcohol (213 mL) in heptane (356 mL) was refluxed for 20 min.
  • the mixture was cooled to 80°C and a solution of 20-£er -butyldimethyl- silyloxy-7-methyl-pregn-4,6-dien-3-one (71.2 gm, 0.16 mol, product of Step 5) in heptane (427 mL) was added.
  • the slurry was refluxed for 9.5 h.
  • the reaction was cooled to room temperature and filtered through
  • Step 7 20- ert-Butyldimethylsilyloxy-7 ⁇ -methyl-5-oxo-A-nor-3,5- secopregnan-3-oic acid
  • the combined filtrates were rotoevaporated to remove the tert-butanol and washed with methylene chloride.
  • the aqueous solution was acidified to pH ⁇ 3 with 2N hydrochloric acid and extracted with methylene chloride (3x).
  • the organic extracts were combined, washed with 5% sodium bisulfite solution and saturated salt solution, and dried over anhydrous magnesium sulfate.
  • the solvent was removed by rotoevaporation to yield the title compound as a white foam.
  • Step 8 20-£ ⁇ rt Butyldimethylsilyloxy-7 ⁇ -methyl-4-azapregn-5-ene
  • Step 9 20-fer ⁇ Butyldimethylsilyloxy-7 ⁇ -methyl-5 ⁇ -4-azapregnane
  • acetic acid 250 mL
  • platinum oxide 1.8 gm
  • the reaction mixture was stirred overnight under hydrogen (1 atmosphere).
  • the reaction mixture was filtered through a pad of CeliteTM filter aid (trademark for diatomaceous earth) and the filtrate was coevaporated with toluene (3 x 500 mL) to remove all of the acetic acid.
  • the residue was dissolved in chloroform and filtered again through a pad of CeliteTM filter aid to remove residual catalyst.
  • the solvent was removed by roto-evaporation to yield the title compound which was taken directly on to the next step without any further purification.
  • Step 12 20-Hvdroxy-7 ⁇ .20-dimethyl-4-aza-5 ⁇ -pregnan-3-one To a solution of 7 ⁇ -methyl-4-aza-5 ⁇ -pregnane-3,20-dione
  • Step 13 7 ⁇ .20-Dimethyl-4-aza-5 -pregn-17-en-3-one
  • the titled compound was synthesized in the same fashion as 7 ⁇ ,20-dimethyl-4-aza-5 ⁇ -pregna-l-ene-3-one, starting with 7 ⁇ ,20- dimethyl-4-aza-5 ⁇ -pregna-17-en-3-one with the exception it was purified by recrystallization in ethyl acetate. 400 MHz !H NMR (CDCI3) : ⁇ 0.85
  • Step 3 20-ethyl-4.7 ⁇ -dimethyl-4-aza-5 ⁇ -pregn-17-en-3-one
  • the titled compound was synthesized in a similar fashion to 7 ⁇ ,20-dimethyl-4-aza-5 ⁇ -pregna-17-ene-3-one and taken forward without any purification.
  • the titled compound was synthesized in a similar fashion to 20-Hydroxy-7 ⁇ ,20-dimethyl-4-aza-5 ⁇ -pregnan-3-one using 2M allylmagnesium chloride in tetrahydrofuran in place of the methylmagnesium bromide. No further purification was done prior to the following step.
  • Step 2 20-Allyl-20-hydroxy-4,7 ⁇ -dimethyl-4-aza-5 ⁇ -pregnan-3- one
  • the titled compound was synthesized in a fashion similar to 20-ethyl-20-Hydroxy-4,7 ⁇ -dimethyl-4-aza-5 ⁇ -pregnan-3-one. 400 MHz iH
  • Step 3 20-propyl-4.7 ⁇ -dimethyl-4-aza-5 ⁇ -pregnan-3-one
  • 20-allyl-20-Hydroxy-4,7 ⁇ -dimethyl-4-aza-5 ⁇ - pregnan-3-one 29.0 mg., 0.075 mmol
  • 10% palladium on carbon 5.0 mg.
  • a mixture of ethyl acetate- ethanol 5.0 mL., 1:1
  • the reaction was then filtered through CeliteTM and the solvent evaporated in vacuo.
  • an oral composition of a compound of this invention 3 mg of 7 ⁇ ,20-dimethyl-4-aza-5 ⁇ -pregna-l- en-3-one is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.
  • EXAMPLE 10 Oral Composition
  • 0.5 mg of a compound of structural formula I is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.
  • an oral composition of a compound of this invention 2.5 mg of a compound of structural formula I is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.
  • an oral composition of a compound of this invention 6 mg of a compound of structural formula I is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.
  • the silicone fluid and 7 ⁇ ,20-dimethyl-4-aza-5 ⁇ -pregna-l-en-3-one are mixed together and the colloidal silicone dioxide is added to increase viscosity.
  • the material is then dosed into a subsequently heat sealed polymeric laminate comprised of the following: polyester release liner, skin contact adhesive composed of silicone or acrylic polymers, a control membrane which is a polyolefin (e.g. polyethylene, polyvinyl acetate or polyurethane), and an impermeable backing membrane made of a polyester multilaminate.
  • the resulting laminated sheet is then cut into 10 cm2 patches. For 100 Patches.
  • the polyethylene glycol 1000 and polyethylene glycol 4000 are mixed and melted.
  • the 5 ⁇ -reductase type 1 inhibitor is mixed into the molten mixture, poured into molds and allowed to cool. For 1000 suppositories.
  • the 7 ⁇ ,20-dimethyl-4-aza-5 ⁇ -pregna-l-en-3-one, magnesium sulfate heptahydrate and buffering agents are dissolved in the water for injection with stirring, and the resulting solution is filtered, filled into ampules, sealed and sterilized by autoclaving. For 1000 Ampules.
  • Samples of human tissue were pulverized using a freezer mill and homogenized in 40 mM potassium phosphate, pH 6.5, 5 mM magnesium sulfate, 25 mM potassium chloride, 1 mM phenylmethyl- sulfonyl fluoride, 1 mM dithiothreitol (DTT) containing 0.25 M sucrose using a Potter-El vehj em homogenizer.
  • a crude nuclear pellet was prepared by centrifugation of the homogenate at 1,500 x g for 15 min. The crude nuclear pellet was washed two times and resuspended in two volumes of buffer. Glycerol was added to the resuspended pellet to a final concentration of 20%.
  • the enzyme suspension was frozen in aliquots at -80°C. The prostatic and scalp reductases were stable for at least 4 months when stored under these conditions.
  • EXAMPLE 18 5 ⁇ - Reductase Assay
  • the reaction mixture for the type 1 5 ⁇ -reductase contained 40 mM potassium phosphate, pH 6.5, 5 mM [7-3H]-testosterone, 1 mM dithiothreitol and 500 ⁇ M NADPH in a final volume of 100 ⁇ L.
  • the reaction mixture for the type 2 5 ⁇ -reductase contained 40 mM sodium citrate, pH 5.5, 0.3 mM [7- 3 H]-testosterone, 1 mM dithiothreitol and 500 ⁇ M NADPH in a final volume of 100 ⁇ L.
  • the assay was initiated by the addition of 50-100 ⁇ g prostatic homogenate or 75-200 ⁇ g scalp homogenate and incubated at 37°C. After 10-50 min the reaction was quenched by extraction with 250 ⁇ L of a mixture of 70% cyclohexane: 30% ethyl acetate containing 10 ⁇ g each DHT and T. The aqueous and organic layers were separated by centrifugation at 14,000 rpm in an Eppendorf microfuge.
  • the organic layer was 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 min; T, 9.1-9.7 min).
  • HPLC system consisted of a Waters Model 680 Gradient System equipped with a Hitachi Model 655 ⁇ Autosampler, Applied Biosystems Model 757 variable UV detector, and a Radiomatic Model A120 radioactivity analyzer.
  • the conversion of T to DHT was 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 DHT was linear for at least 25 min.
  • the only steroids observed with the human prostate and scalp preparations were T, DHT and androstanediol.
  • 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 5 ⁇ -reductase type 1 and type 2 inhibition.
  • a compound referred to herein as a 5 ⁇ -reductase 2 inhibitor is a compound that shows inhibition of the 5 ⁇ -reductase 2 isozyme in the above-described assay, having an IC50 value of about or under 100 nM.
  • the compounds are tested in the above-described assay for 5 ⁇ -reductase type 1 and type 2 inhibition, and were found to have IC50 values under about 100 nM for inhibition of the type 1 isozyme.
  • Compounds found to have IC50 values of under about 50 nM for inhibition of the type 1 isozyme are called type 1 inhibitors.
  • Compounds called "dual inhibitors" additionally had IC50 S under about 200 nM for inhibition of the type 2 isozyme.

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Abstract

The present invention provides for a method of treating polycystic ovary syndrome in a subject in need of such treatment comprising the administration of a therapeutically effective amount of a compound of structural formula (I) to the subject. The present invention further provides for a method for improving fertility and the response to in vitro fertilization (I.V.F), comprising administration of therapeutically effective amount of compound of structural formula (I) to the subject. Further, the present invention provides for compositions useful in the methods of the present invention, as well as a method of manufacture of a medicament useful for treating polycystic ovary syndrome or for improving fertility or the response to in vitro fertilization (I.V.F.).

Description

TITLE OF THE INVENTION
METHODS AND COMPOSITIONS FOR TREATING POLYCYSTIC
OVARY SYNDROME
CROSS-REFERENCE TO RELATED APPLICATIONS Not applicable
BACKGROUND OF THE INVENTION
The present invention provides for a novel method of treating polycystic ovary syndrome. Further, the present invention is directed to a method of improving fertility and the response to in vitro fertilization (I.V.F.) by employing a 5α-reductase type 1 inhibitor. The present invention also provides for a method of manufacture of a medicament useful for treating polycystic ovary syndrome and a method of manufacture of a medicament useful as an adjunct in I.V.F. The present invention also provides for compositions useful in the method of treating polycystic ovary syndrome and useful as an adjunct in I.V.F.
Polycystic ovary syndrome (PCOS), also known as Stein- Leventhal syndrome, is characterized by menstrual irregularity and hirsutism and is a common cause of anovulatory infertility. The biochemical abnormalities are a high concentration of plasma luteinising hormone (LH) or a high LH/follicle stimulating hormone (FSH) ratio and high concentrations of androgens (testosterone and/or androstenedione and/or dehydroepiandrosterone (DHEA)). The increased androgens can be secreted by the ovary and/or the adrenal gland. Clinical manifestations of PCOS include amenorrhea, hirsutism acanthosis nigricans, acne and obesity. PCOS appears to account for about 75% of anovulatory infertility.
The chronic anovulation typical of PCOS results in an increased number of atretic follicles (which become cysts) and increased interstitial tissue in the stroma of the ovaries.
Under normal conditions, women produce a single dominant follicle that participates in a single ovulation each menstrual cycle. The process begins when a cohort of primordial follicles is recruited to initiate growth. Successive recruitment gives rise to the primary, secondary, tertiary and graafian follicles present in the ovaries. The ability to become a dominant follicle is not a characteristic shared by all follicles, and those that lack the property die by atresia due to increased androgens. In the human female, only about 400 of the original 7 million follicles survive atresia and give rise to dominant follicles.
In patients with PCOS, the process of folliculogenesis does not proceed normally. The initial steps, recruitment and growth to the small graafian stages, are functioning in PCOS, but the terminal step, the selection of dominant follicles that can ovulate, does not occur regularly. Viable follicles seldom develop beyond about the 6 mm stage. In some unexplained way, this condition leads to the accumulation of large numbers of small graafian follicles (commonly referred to as cysts) in which the theca interstitial cells (TIC) produce abnormally large amounts of androgen, but the granulosa cells (GC) fail to express the aromatase enzyme and aromatize the androgen substrate to estradiol. Consequently, a state of continued hyperandrogenism results. The problem is self-perpetuating in part because the atretic follicle becomes an androgenic follicle by a "default" mechanism: because of low aromatase activity in atretic follicles, androstenedione is preferentially metabolized to testosterone and thence to dihydrotestosterone within the ovary.
The human ovarian stromal, thecal and granulosa cell compartments each contain 5α-reductase activity. In the rat the 5α- reduced androgens 5α-androstane-3,17-dione (5α-A) and dihyrotestosterone (DHT) are competitive inhibitors of aromatase activity. This is likely to occur in humans as well, and therefore the 5α- reduced metabolites may lead to decreased aromatase activity, increased androgen secretion and follicular atresia. Agarwal et al. "A Mechanism for the Suppression of
Estrogen Production in Polycystic Ovary Syndrome" J. Clin Endocrinology & Metabolism 81(10):3686-3691 (1996), propose that polycystic ovary syndrome follicular fluid contains abnormally high 5α- A and/or DHT concentration that can inhibit aromatase activity. They conclude that 5α-A is the primary inhibitor of aromatase activity in PCOS follicular fluid.
Stewart et al. "5α-reductase activity in polycystic ovary syndrome" The Lancet 335:431-433 (1990) investigated the hypothesis that in PCOS increased cortisol metabolism stimulates corticotropin- mediated androgen excess. They proposed that enhanced activity of 5α- reductase is the fundamental defect in many patients with PCOS. The enzyme abnormality is proposed to mediate both hirsutism and enhanced hepatic cortisol metabolism. A concomitant increase in corticotropin secretion in women with PCOS is hypothesized to keep plasma cortisol concentrations normal, but at the expense of androgen excess. This hypothesis likely provides another mechanism, via the adrenal, in which the 5α-reductase activity contributes to PCOS.
Presently polycystic ovary syndrome is treated with GnRH analogues, oral contraceptives, steroids (such as prednisone) and/or antiandrogens. These antagonize androgens or decrease the whole H-P- G or H-P- Adrenal axes. None of the treatments currently employed corrects the underlying problem of the hyperandrogenic production by the ovarian follicles. Further, each of the currently employed treatments has significant side effects including: hypoestrogenism (GnRH analogues), menstrual irregularity (spironolactone, an antiandrogen), and headaches, bloating and the rare occurrence of blood clots associated with oral contraceptives. Steroids also have significant side effects such as adrenal suppression, obesity, striae, hypertension, etc.
The present invention relates to methods of treating polycystic ovary syndrome. Further, the present invention is directed to a method of improving fertility and the response to in vitro fertilization (I.V.F.) by employing a 5α-reductase type 1 inhibitor. It has now been found that a 5α-reductase type 1 inhibitors of structural formula I:
Figure imgf000006_0001
are useful for the treatment of polycystic ovary syndrome and for improving fertility and the response to in vitro fertilization.
The enzyme 5α-reductase catalyzes the reduction of several androgens including: testosterone (T) to the more potent androgen, 5α- dihydrotestosterone (dihydrotestosterone" or DHT), as shown below:
5α-reductase
Figure imgf000006_0002
NADPH
Figure imgf000006_0003
testosterone dihydrotestosterone
5α-reductase also catalyzes the reduction of androstenedione (A) to androstanedione (5α-A), as shown below:
Figure imgf000006_0004
androstenedione androstanedione
and the reduction of progesterone to dihydroprogesterone, as shown below:
Figure imgf000007_0001
progesterone dihydroprogesterone
There are two isozymes of 5 α- reductase in humans. Andersson, et al., Proc. Natl. Acad. Sci. USA, 87:3640-44 (1990); Andersson, et al., Nature, 354, 159-61 (1991). The isozymes, usually called Type 1 and Type 2, exhibit differences in their biochemical properties, genetics, and pharmacology. Both isozymes are now the subject of considerable research and it has been found one isozyme (type 1) predominates in he sebaceous glands of facial skin and skin tissue and that the other (type 2) predominates in the prostate.
Finasteride (17β-(N-tert-butylcarbamoyl)-3-oxo-4-aza-5α- androst-l-en-3-one) as shown below, is a potent inhibitor of the human type 2 enzyme.
Figure imgf000007_0002
finasteride
Under the tradename PROSCAR®, finasteride is known to be useful in the treatment of hyperandrogenic conditions, see e.g., U.S. 4,760,071. Finasteride is currently prescribed for the treatment of benign prostatic hyperplasia (BPH), a condition affecting to some degree the majority of men over age 55. Finasteride's usefulness in the treatment of androgenic alopecia and prostatic cancer is described in the following documents: EP 0 285 382, published 5 October 1988, EP 0 285 383, published 5 October 1988 and Canadian patents 1,302,277 and 1,302,276. There have been reports (e.g., Ciotta et al., Fertility & Sterility 64(2): 299-306,1996 and Fruzetti et al., J. Clin. Endocrin. Metab. 79: 703-706, 1994) in the literature of administration of the type 2 inhibitor finasteride to women with hirsutism, with no observed menstrual changes. Further, it has been reported that women with type 2 5α-reductase deficiency have normal menstrual cycles. This suggests that the type 2 enzyme does not predominate in the ovary.
Haning, Jr., et al., J. Steroid Molec. Biol. 59(2): 199-204 (1996) have recently reported that the human ovary apparently expresses mRNA for 5α-reductase type 1.
It has been presently found that the type 1 5α-reductase inhibitors of the present invention are useful in the treatment of polycystic ovary syndrome and in improving fertility and the response to in vitro fertilization.
SUMMARY OF THE INVENTION
The present invention provides for a method of treating polycystic ovary syndrome in a subject in need of such treatment comprising the administration of a therapeutically effective amount of a compound of structural formula I:
Figure imgf000008_0001
to the subject. The present invention further provides for a method for improving fertility and the response to in vitro fertilization (I.V.F.), comprising administration of therapeutically effective amount of compound of structural formula I to the subject. Further, the present invention provides for compositions useful in the methods of the present invention, as well as a method of manufacture of a medicament useful for treating polycystic ovary syndrome or for improving fertility and the response to in vitro fertilization (I.V.F.).
BRIEF DESCRIPTION OF THE DRAWINGS Not applicable.
DETAILED DESCRIPTION OF THE INVENTION
In one embodiment, the present invention is directed to a method for treating polycystic ovary syndrome in a subject in need thereof by administering to the subject an effective amount of a compound of structural formula I:
Figure imgf000009_0001
wherein: the C1-C2 and C5-C6 bonds designated with a dotted line each independently represent a single or double bond, provided that when the C5-C6 is a double bond, Ha is absent and when the C5-C6 bond is a single bond Ha is present and represents hydrogen; Rl is selected from hydrogen and Cl-5 alkyl; R2 is Cl-5alkyl, either straight or branched chain; and R3 is C3-7alkyl, either straight or branched chain, optionally having one degree of unsaturation; or a pharmaceutically acceptable salt, or stereoisomer thereof. Still a further aspect of the present invention is a method of improving fertility and the response to in vitro fertilization (I.V.F.), in a subject in need thereof by administering an effective amount of a compound of structural formula I to the subject. The compounds of the present invention may provide a useful adjunct for in vitro fertilization because they may lead to multiple ovarian follicles developing completely through ovulation.
Another aspect of the present invention is the use of a compound of structural formula I for the manufacture of a medicament useful to treat polycystic ovary syndrome in a subject in need thereof. Still a further aspect of the present invention is the use of a compound of structural formula I for the manufacture of a medicament useful to improve fertility and the response to in vitro fertilization in a subject in need thereof.
In one class of the instant invention are employed compounds of formula I wherein the C5-C6 bond is a single bond and Ha is present.
In a sub-class of the compounds of this class are compounds wherein R^ is methyl.
Compounds illustrating this sub-class are:
7β,20-dimethyl-4-aza-5α-pregn-17-en-3-one,
7β,20-dimethyl-4-aza-5α-pregn-l,17-dien-3-one,
20-ethyl-4,7β-dimethyl-4-aza-5α-pregn-17-en-3-one, 20-ethyl-4,7β-dimethyl-4-aza-5α-pregnan-3-one ,
7β,20-dimethyl-4-aza-5α-pregnan-3-one,
7β,20-dimethyl-4-aza-5α-pregn-l-en-3-one,
20-ethyl-4,7β-dimethyl-4-aza-5α-pregnan-3-one,
20-propyl-4,7β-dimethyl-4-aza-5α-pregnan-3-one, 20-ethyl-4,7β-dimethyl-4-aza-5α-pregn-l-en-3-one,
4,7β,20-trimethyl-4-aza-5α-pregn-l-en-3-one,
20-propyl-4,7β-dimethyl-4-aza-5α-pregn-l-en-3-one,
20-ethyl-7β-methyl-4-aza-5α-pregn-l-en-3-one,
20-propyl-7β-methyl-4-aza-5α-pregnan-3-one, 20-propyl-7β-methyl-4-aza-5α-pregn-l-en-3-one, β-n-propyl-7β-methyl-4-aza-5α-androst-l-en-3-one, β-n-propyl-4,7β-dimethyl-4-aza-5α-androst-l-en-3-one, β-n-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one, β-isobutyl-7β-methyl-4-aza-5α-androst-l-en-3-one, β-tert.-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one, β-n-butyl-4,7β-dimethyl-4-aza-5α-androst-l-en-3-one, β-isobutyl-4,7β-dimethyl-4-aza-5α-androst-l-en-3-one, β-tert.-butyl-4,7β-dimethyl-4-aza-5α-androst-l-en-3-one, β-n-pentyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 7β-isopentyl-7β-methyl-4-aza-5α-androst-l-en-3-one, β-(5-methylhexyl)-7β-methyl-4-aza-5α-androst-l-en-3-one, β-(5-methylhexyl)-4,7β-dimethyl-4-aza-5α-androst-l-en-3-one, 7β-n-propyl-7β-methyl-4-aza-5α-androstan-3-one, 7β-n-propyl-4,7β-dimethyl-4-aza-5α-androstan-3-one, 7β-n-butyl-7β-methyl-4-aza-5α-androstan-3-one, 7β-n-butyl-4,7β-dimethyl-4-aza-5α-androstan-3-one, 7β-(5-methylhexyl)-7β-methyl-4-aza-5α-androstan-3-one, and 7β-(5-methylhexyl)-4,7β-dimethyl-4-aza-5α-androstan-3-one.
Compounds further illustrating this sub-class are: β,20-dimethyl-4-aza-5α-pregn-17-en-3-one, β,20-dimethyl-4-aza-5α-pregn-l,17-dien-3-one, 0-ethyl-4,7β-dimethyl-4-aza-5 -pregn-17-en-3-one, 0-ethyl-4,7β-dimethyl-4-aza-5α-pregnan-3-one , β,20-dimethyl-4-aza-5α-pregnan-3-one, β,20-dimethyl-4-aza-5α-pregn-l-en-3-one, 0-ethyl-4,7β-dimethyl-4-aza-5α-pregnan-3-one, 0-propyl-4,7β-dimethyl-4-aza-5α-pregnan-3-one, 7β-n-propyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 7β-n-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 7β-(5-methylhexyl)-7β-methyl-4-aza-5α-androst-l-en-3-one, 7β-n-propyl-7β-methyl-4-aza-5α-androstan-3-one, 7β-n-butyl-7β-methyl-4-aza-5α-androstan-3-one, and 7β-(5-methylhexyl)-7β-methyl-4-aza-5α-androstan-3-one. In a further subclass of the present invention are employed compounds wherein the C1-C2 bond is a double bond and Rl is hydrogen. Compounds illustrating this subclass include:
7β,20-dimethyl-4-aza-5α-pregn-l,17-dien-3-one, 7β,20-dimethyl-4-aza-5α-pregn-l-en-3-one, 20-ethyl-7β-methyl-4-aza-5 -pregn-l-en-3-one, 20-propyl-7β-methyl-4-aza-5α-pregn-l-en-3-one, 17β-n-propyl-7β-methyl-4-aza-5α-androst- l-en-3-one, 17β-n-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-isobutyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-tert.-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-n-pentyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-isopentyl-7β-methyl-4-aza-5α-androst-l-en-3-one,
17β-(5-methylhexyl)-7β-methyl-4-aza-5α-androst-l-en-3-one.
In yet another subclass of this class of the present invention are employed compounds wherein R^ is C3-6 alkyl. Compounds illustrating this sub-class are:
7β,20-dimethyl-4-aza-5α-pregn-17-en-3-one,
7β,20-dimethyl-4-aza-5α-pregn-l,17-dien-3-one,
20-ethyl-4,7β-dimethyl-4-aza-5α-pregn-17-en-3-one,
20-ethyl-4,7β-dimethyl-4-aza-5α-pregnan-3-one , 7β,20-dimethyl-4-aza-5α-pregnan-3-one,
7β,20-dimethyl-4-aza-5α-pregn-l-en-3-one,
20-ethyl-4,7β-dimethyl-4-aza-5α-pregnan-3-one,
20-propyl-4,7β-dimethyl-4-aza-5α-pregnan-3-one,
20-ethyl-4,7β-dimethyl-4-aza-5α-pregn-l-en-3-one, 4,7β,20-trimethyl-4-aza-5α-pregn-l-en-3-one,
20-propyl-4,7β-dimethyl-4-aza-5α-pregn-l-en-3-one,
20-ethyl-7β-methyl-4-aza-5α-pregn-l-en-3-one,
20-propyl-7β-methyl-4-aza-5α-pregnan-3-one,
20-propyl-7β-methyl-4-aza-5α-pregn-l-en-3-one, 17β-n-propyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-n-propyl-4,7β-dimethyl-4-aza-5α-androst-l-en-3-one, 17β-n-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-isobutyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-tert.-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-n-butyl-4,7β-dimethyl-4-aza-5α-androst-l-en-3-one, 17β-isobutyl-4,7β-dimethyl-4-aza-5α-androst-l-en-3-one, 17β-tert.-butyl-4,7β-dimethyl-4-aza-5 -androst-l-en-3-one, 17β-n-pentyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-isopentyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-(5-methylhexyl)-7β-methyl-4-aza-5α-androst-l-en-3-one,
17β-(5-methylhexyl)-4,7β-dimethyl-4-aza-5α-androst-l-en-3-one, 17β-n-propyl-7β-methyl-4-aza-5α-androstan-3-one, 17β-n-propyl-4,7β-dimethyl-4-aza-5α-androstan-3-one, 17β-n-butyl-7β-methyl-4-aza-5α-androstan-3-one, and 17β-n-butyl-4,7β-dimethyl-4-aza-5α-androstan-3-one.
Further illustrating this subclass are compounds wherein R2 is methyl.
In a further subclass of the present invention are employed compounds wherein the C1-C2 bond is a double bond, Rl is hydrogen, R^ is methyl, and R^ is C3.6 alkyl.
Compounds illustrating this subclass include: 7β,20-dimethyl-4-aza-5α-pregn-l,17-dien-3-one, 7β,20-dimethyl-4-aza-5α-pregn-l-en-3-one, 20-ethyl-7β-methyl-4-aza-5α-pregn-l-en-3-one, 20-propyl-7β-methyl-4-aza-5α-pregn-l-en-3-one, 17β-n-propyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-n-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-isobutyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-tert.-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one,
17β-n-pentyl-7β-methyl-4-aza-5α-androst-l-en-3-one, and 17β-isopentyl-7β-methyl-4-aza-5α-androst-l-en-3-one.
Still further illustrating this subclass are compounds wherein R3 is fully saturated. Compounds illustrating this subclass include: 7β,20-dimethyl-4-aza-5α-pregn-l-en-3-one, 20-ethyl-7β-methyl-4-aza-5α-pregn-l-en-3-one, 20-propyl-7β-methyl-4-aza-5 -pregn-l-en-3-one, 17β-n-propyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-n-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-isobutyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-tert.-butyl-7β-methyl-4-aza-5α-androst-l-en-3-one, 17β-n-pentyl-7β-methyl-4-aza-5α-androst-l-en-3-one, and 17β-isopentyl-7β-methyl-4-aza-5α-androst-l-en-3-one.
In another class of the present invention are employed compounds wherein the C5-C6 bond is a double bond and Ha is absent.
When any variable (e.g., alkyl, R2, etc.) occurs more than one time in any constituent or in formula I, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
As used herein "alkyl" is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, e.g., methyl (Me), ethyl (Et), propyl, butyl, pentyl, and the isomers thereof such as isopropyl (i-Pr), isobutyl (i- Bu), secbutyl (s-Bu), tertbutyl (t-Bu), isopentane, etc.
The compounds of the present invention may be administered in the form of pharmaceutically acceptable salts.
The term "pharmaceutically acceptable salt" is intended to include all acceptable salts such as acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N- methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate, fumarate, phosphate/diphosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide, tannate, hydro chloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, isothionate, triethiodide, lactate, panoate, valerate, and the like which can be used as a dosage form for modifying the solubility or hydrolysis characteristics or can be used in sustained release or pro-drug formulations.
The subject treated in the methods above is a female mammal, preferably a human being, diagnosed with polycystic ovary syndrome or ovarian or adrenal hyperandrogenism. Alternatively the subject treated is a human mammal, or preferably a human being, who is infertile due to irregular menstrual cycles and/or undergoing treatment with in vitro fertilization due to idiopathic infertility.
The term "therapeutically effective amount" means the amount the compound of structural formula I that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The terms "administration of and or "administering a" compound should be understood to mean providing a compound of the invention or a prodrug of a compound of the invention to the individual in need of treatment. The administration of the compound of structural formula I in order to practice the present methods of therapy is carried out by administering an effective amount of the compound of structural formula I to the patient in need of such treatment or prophylaxis. The need for a prophylactic administration according to the methods of the present invention is determined via the use of well known risk factors. The effective amount of an ind vidual compound is determined, in the final analysis, by the physician in charge of the case, but depends on factors such as the exact disease to be treated, the severity of the disease and other diseases or conditions from which the patient suffers, the chosen route of administration other drugs and treatments which the patient may concomitantly require, and other factors in the physician's judgment.
Generally, the daily dosage of the compound of structural formula I may be varied over a wide range from 0.01 to 1000 mg per adult human per day. Most preferably, dosages range from 0.1 to 100 mg/day. For oral administration, the compositions are preferably provided in the form of tablets containing 0.01 to 1000 mg, particularly 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 3.0, 5.0, 6.0, 10.0, 15.0, 25.0, and 50.0 and 100 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
The dose 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, when administered via intranasal routes, transdermal routes, by rectal suppositories, or through a continual intravenous solution, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
Formulations of the 5α-reductase inhibitor employed in the present method for medical use comprise the compound of structural formula I together with an acceptable carrier thereof and optionally other therapeutically active ingredients. The carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient subject of the formulation. The present invention, therefor further provides a pharmaceutical formulation comprising the compound of structural formula I together with a pharmaceutically acceptable carrier thereof.
The formulations include those suitable for oral, rectal, intravaginal, topical or parenteral (including subcutaneous, intramuscular and intravenous administration). Preferred are those suitable for oral administration.
The formulations may be presented in a unit dosage form and may be prepared by any of the methods known in the art of pharmacy. All methods include the step of bringing the active compound in association with a carrier which constitutes one or more ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound in association with a liquid carrier, a waxy solid carrier or a finely divided solid carrier, and then, if needed, shaping the product into desired dosage form.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension or solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup, an elixir, or an emulsion.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients, e.g., binders, lubricants, inert diluents, disintegrating agents or coloring agents. Molded tablets may be made by molding in a suitable machine a mixture of the active compound, preferably in powdered form, with a suitable carrier. 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, carboxymethyl-cellulose, 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.
Oral liquid forms, such as syrups or suspensions in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl cellulose and the like may be made by adding the active compound to the solution or suspension. Additional dispersing agents which may be employed include glycerin and the like. Formulations for vaginal or rectal administration may be presented as a suppository with a conventional carrier, i.e., a base that is nontoxic and nonirritating to mucous membranes, compatible with the compound of structural formula I, and is stable in storage and does not bind or interfere with the release of the compound of structural formula I. Suitable bases include: cocoa butter (theobroma oil), polyethylene glycols (such as carbowax and polyglycols), glycol- surfactant combinations, polyoxyl 40 stearate, polyoxyethylene sorbitan fatty acid esters (such as Tween, Myrj, and Arlacel), glycerinated gelatin, and hydrogenated vegetable oils. When glycerinated gelatin suppositories are used, a preservative such as methylparaben or propylparaben may be employed.
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 0285 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.
Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy- ethylaspartamidephenol, or polyethylene-oxide polylysine substituted with palmitoyl residues. Furthermore, 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, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
Formulations suitable for parenteral administration include formulations which comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient. Such formulations suitably comprise a solution or suspension of a compound that is isotonic with the blood of the recipient subject. Such formulations may contain distilled water, 5% dextrose in distilled water or saline and the active compound. Often it is useful to employ a pharmaceutically and pharmacologically acceptable acid addition salt of the active compound that has appropriate solubility for the solvents employed. Useful salts include the hydrochloride isothionate and methanesulfonate salts. Useful formulations also comprise concentrated solutions or solids comprising the active compound which on dilution with an appropriate solvent give a solution suitable for parenteral administration.
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 pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds usually applied in the treatment of the above mentioned pathological conditions, for instance: GnRH analogues, oral contraceptives, antiandrogens, clomiphene, gonadotropins, or steroids, such as prednisone, estrogens and progestins.
The composition and method of the present invention may further comprise a type 2 5 -reductase inhibitor or a dual 5α-reductase inhibitor. Preferred type 2 5α-reductase inhibitors for use in the present composition and method include: finasteride and epristeride. A preferred dual inhibitor is: 17β-N-(2,5-bis(trifluoromethyl))phenyl carbamoyl-4-aza-5α-androst-l-en-3-one.
One aspect of the present invention provides a method for treating polycystic ovary syndrome comprising administering to a female mammal in need of treatment an effective amount of a compound of structural formula I.
Another aspect of the present invention provides a method of improving fertility and the response to in vitro fertilization (I.V.F.) comprising administering to a female mammal in need of treatment and effective amount of a compound of structural formula I.
In particular, when a type 2 5α-reductase inhibitor or a dual 5α-reductase inhibitor is employed, dosages of 0.01 to 10 mg per adult human per day are appropriate for treatment, more preferably 1 to 5 mg/day especially preferred is about 5 mg/day.
The compounds of the methods of the present invention can be administered by a variety of routes including oral, rectal, vaginal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal, and such compounds are preferably formulated prior to administration. Therefore, another embodiment of the present invention is a pharmaceutical formulation comprising an effective amount of a compound of structural formula I or a pharmaceutically acceptable salt thereof, a dual 5α-reductase inhibitor, and a pharmaceutically acceptable carrier, diluent or excipient therefor. In accordance with the method of the present invention, the individual components of the combination can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly. It will be understood that the scope of combinations of the compounds of this invention with other agents useful for treating polycystic ovary syndrome and with other agents useful in improving fertility and the response to in vitro fertilization (I.V.F.) by employing a 5α-reductase type 1 inhibitor. The compounds of this invention can be prepared as shown in Scheme 1.
Figure imgf000021_0001
Starting with commercially available pregnenolone acetate, the appropriately 7-substituted derivative is prepared according to the procedures of PCT publication WO 93/23420 and to produce (1), the 7β- alkyl-substituted pregnenolone acetate. Treating (1) with the appropriate C2-6alkyl Grignard in tetrahydrofuran (THF), produces the tertiary carbinol (2). The tertiary carbinol (2) may be alkylated at the 4- position by treatment with sodium hydride and the appropriate Ci-5alkyl iodide in a polar aprotic solvent such as THF or dimethylformamide (DMF). The 4-NH or 4-N-alkyl compound is then dehydrated in the presence of acid, for example, HC1 or acetic acid, in a solvent such as THF or alcohol to produce the 17-ene (3). The 17-ene (3), in turn, may be dehydrogenated to form the 1,17-diene (4) by treatment with DDQ in toluene or benzeneselenic anhydride in chlorobenzene, or other known methods, for example as described in U.S. 5,084,574 and 5,021,571. DDQ is preferred for 4-NH compounds and benzeneselenic anhydride is preferred for 4-N-alkyl compounds.
Alternatively, the 17-ene (3) may be hydrogenated in the presence of a hydrogenation catalyst, for example Ptθ2, Pd/C, rhodium on alumina, preferably Ptθ2, in an appropriate solvent such as an alcohol or acetic acid, preferably methanol, to form the 17-alkyl derivative (5). The 17-alkyl derivative (5), in turn, may be dehydrogenated to form the 1-ene (6) by treatment with DDQ in toluene or benzeneselenic anhydride in chlorobenzene, as described above. The desired 4-N-alkyl substitution may be effected as described previously by treating (2) with the appropriate alkyl iodide, or alternative, the procedure may be carried through with the 4-NH compound, and following after the desired 17-substitution and optional insertion of the 1,2-double bond, the 4-NH compound may be alkylated to the desired 4-N-alkyl compound.
Processes for inserting the 1,2-double bond in a 3-oxo-4- azasteroid are described in U.S. Patents 5,084,574 and 5,021,571. The formation of a 7-β bond is described in U.S. Patents 4,220,775 5,237,064. SCHEME 2
Figure imgf000023_0001
Alternatively, the compounds of the present invention may be prepared according to the procedures of Scheme 2. Compound (7), obtained according to procedures in WO 93/23420, is treated with N- phenyl trifluoro methane sulfonamide in a base such as lithium hexamethyldisilazide in THF to form the enol triflate (8). The enol triflate (8) is converted to the desired enyne (9) by treatment with di(triphenylphosphine)palladium diacetate or other appropriate Pdo catalyst with a catalytic amount of cuprous iodide and a mild base such as diisopropylamine or triethylamine in DMF with the appropriate alkyne. The enyne (9) is hyrdrogenated to produce the 17-alkyl derivative (10) by treating with H2 in the presence of 10% Pd/C in an alcoholic or ethyl acetate solvent, preferably ethyl acetate. Insertion of the 1,2-double bond, if desired is accomplished as described in Scheme 1 to produce the 17-alkyl- 1-ene (11). The 5α-reductase type 2 inhibitor finasteride that may employed this invention can be prepared as described in U.S. 4,760,071.
The following examples are not intended to be limitations on the scope of the instant invention in any way, and they should not be so construed. Furthermore, examples are not to be construed as forming the only methods and compositions that are considered as the invention. Those skilled in the art will readily understand that known variations of the conditions, processes, methods and compositions of the following preparative procedures can be used.
EXAMPLE 1
Distribution of 5α-reductase in Pregnant rat tissues
Enzyme Preparation:
Rat ovary was obtained from pregnant animals (gestational days 15, 17, 19, 20, 21 and 22 and was pulverized using a freezer mill and homogenized in 20 mM potassium phosphate pH 6.5, 5 mM magnesium sulfate, 25 mM potassium chloride, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 5 mM NADPH, and 0.25 M sucrose using a dounce homogenizer. Glycerol was added to the homogenate to a final concentration of 20%.
Enzyme Assay:
The reaction mixture contained 33 mM succinic acid 44 mM imidazole, 33 mM diethanolamine (SID), pH 5.5 or pH 7.0, 0.3-15 μM [7- 3u]-testosterone (T) (specific activity approximately 20-30 Ci/mmol) or [l,2-3H]-progesterone, 1 mM DTT and 0.5 mM NADPH in a final volume of 0.1 mL. The assay was initiated by the addition of enzyme and incubated at 37°C for 20-30 minutes. The reaction was quenched with cyclohexane:ethyl acetate (70:30 v/v) and product peaks were separated by normal phase HPLC. Alternatively, samples were also quenched using 0.05 M phosphoric acid in 10% ethanol. The product profile was analyzed by reverse phase HPLC.
Product Assignments Products produced in the enzymatic assays were assigned based on mobility on normal or reverse phase HPLC compared to authentic standards. The normal phase HPLC system consisted of a 25 cm Whatman Partisil 5 silica column equilibrated in cyclohexane: ethyl acetate (65:35) at 1 mL/min. Under these conditions, retention times were as follows: dihydrotestosterone (DHT), 7 min; androstenedione, 8 min; 3α/β-androstane diol, 9.5 min; testosterone (T), 11 min. For assays using progesterone (P) as substrate, the conditions were modified to cyclohexane: ethyl acetate (78:22) at 1.5 mL/min. Under these conditions, retention times for authentic steroids were as follows: progesterone 9.8 min; 3α-/3β-androstane diol, 8 min; 20α-hydroxyprogesterone, 17 min. Variations ± 3.5 min in retention times for the products were seen, but similar shifts were observed in the retention times of standards. The absolute assignment of 3α- versus 3β-androstanediol was made on the basis of mobility on reverse phase HPLC. The sample was chromatographed on a 25 cm Whatman Partisil 5 ODS-3 column that was equilibrated in methanol:water:acetonitrile (3:3:1) at 1.5 mL/min. Under these conditions, authentic steroids had retention times of 37 min and 24 min for 3α- and 3β-androstanediol, respectively.
Results:
Metabolism of testosterone: Homogenates of rat ovary from pregnant animals were incubated with 0.3 μM ^H-testosterone (T) or ^H- progesterone (P) and the products analyzed by HPLC. Product assignments were based on comparison of retention times of unknowns to those of authentic standards on normal phase HPLC. Table 1, below shows representative examples of product distribution using T as substrate.
Table 1. Metabolism of T by pregnant rat ovary at gestation day 19
Figure imgf000026_0001
Three products were observed which migrated at ~6.9 min, ~7.8 min and ~9.3 min. These products were identified as DHT, A (androstenedione) and 3-androstane diol (Adiol). Although 3α- and 3β-androstane diol co- migrate by normal phase HPLC, these two steroids are readily separated by reverse phase HPLC. Using this methodology, the product from the enzymatic reaction was defined as 3α-androstane diol. Two non-5 - reduced products were also detected: androstenediol (~7.8 min), the product of 17β-hydroxysteroid dehydrogenase, and an unidentified metabolite (~13.6 min). The unidentified metabolite most likely represents a ring hydroxylation product.
Metabolism of progesterone: A representative product profile when P is used as substrate is presented in Table 2 and for the most part mirrors the pattern of products seen with T.
Table 2. Metabolism of P by pregnant rat ovary at gestation day 19
Figure imgf000026_0002
3α-hydP - 3α-hydroxy-5α-pregnan-20-one 20α-hydP - 20α-hydroxyprogesterone
Progesterone undergoes 5α-reduction to dihydroprogesterone (DHP) and subsequent reduction of DHP by 3 α-hydroxy steroid dehydrogenase to 3α- hydroxy-5α-pregnan-20-one. The unknown product at 12.1 minutes most likely represents the same proposed ring hydroxylation step discussed for testosterone metabolism. One obvious difference in P metabolism is conversion to 20α-hydroxyP (-17.6 min) by 20α-hydroxysteroid dehydrogenase. P is not a substrate for 17 β-hydroxy steroid dehydrogenase and therefore no product is expected for this enzyme.
Specific activity/Isozyme determination: The specific activity of the enzyme was determined using T as substrate. The combined contribution of DHT and androstanediol was used for this analysis. As shown in Table 3, there is very little change in the 5α- reductase content of the tissues from gestation day 15 to 22.
Table 3. Steroid 5α-reductase specific activity determination in pregnant rat ovary 1
Figure imgf000028_0001
assay conducted with 15 μM T
An initial attempt was made to define the isozyme content of the tissues by comparing the 5α-reductase activity at pH 5.5 and 7.0. Under these assay conditions, the ratio of activity at the two pH's can predict isozyme content. This assessment is based on the difference in pH optimum of 7.0 and 5.5 for the type 1 and type 2 5α-reductases, respectively. The results of these studies are presented in Table 4. Table 4. Isozyme content of rat ovary obtained on gestation day 21 as determined by pH/activity analysis
Figure imgf000029_0001
A ratio of 1 or lower suggests that the predominant enzyme is type 1, while a higher ratio indicates that the predominant enzyme is type 2. This study suggests that type 1 5α-reductase is present in ovary.
EXAMPLE 2 7β.20-Dimethyl-4-aza-5α-pregna-17-en-3-one Step 1: 3-Acetoxy-pregn-5-en-20-ol
Sodium borohydride (21 gm) was added to a solution of pregnenolone acetate (100 g, 0.28 mol) in absolute ethanol (1 L) and methylene chloride (0.4 L) at -10°C. After stirring overnight at 4°C, another amount of sodium borohydride (10.5 gm) was added and the reaction stirred at room temperature overnight. The reaction mixture was quenched by pouring into 5% sodium phosphate monobasic (2 L) and extracted with methylene chloride. The organic extracts were dried over anhydrous magnesium sulfate and filtered through a pad of anhydrous sodium sulfate. The solvent was removed by rotoevaporation to give the title compound.
Step 2: 3-Acetoxy-20-^erf;-butyldimethylsilyloxy-pregn-5-ene
Imidazole (203.7 gm, 2.28 mol) was added to a stirred suspension of 3-acetoxy-pregn-5-en-20-ol (361 gm, 1 mol, product of Step 1) in dimethylformamide (3.7 L). t-Butyldimethylsilyl chloride (228.9 mg, 1.52 mol) was added over a 10-15 min period. The mixture was stirred at room temperature for 3 days. The dimethylformamide was removed by decantation and methanol (50 mL) was added to it. Water (4 L) was added and the solution extracted with ethyl acetate (2 x 4 L). The precipitate remaining behind after decantation was dissolved in ethyl acetate and added to the above ethyl acetate extracts. The combined solvent extracts were washed with water, saturated salt solution, and dried over anhydrous magnesium sulfate. The solvent was removed by rotoevaporation and the product purified by column chromatography on silica gel eluted with 2:1 hexane-methylene chloride followed by 1:1 hexane-methylene chloride. The title compound was isolated as a mixture of 20 - and β-isomers.
Step 3: 3-Acetoxy-20-fer^butyldimethylsilyloxy-pregn-5-en-7-one
To a solution of 3-acetoxy-20- er>i-butyldimethylsilyloxy- pregn-5-ene (337 gm, 0.71 mol, product of Step 2) in methyl ethyl ketone (4 L) was added N-hydroxyphthalimide (115.8 gm, 0.71 mol) and dibenzoyl peroxide (1.1 gm, 4.4 mmol). Air was bubbled through the reaction as the reaction was refluxed for 7.5 hr. Additional N-hydroxyphthalimide (9 gm) and dibenzoyl peroxide (0.1 gm) were added and reflux continued for 5 hr. The solvent was removed by rotoevaporation and methylene chloride (0.7 L) was added and warmed to 40°C. Upon cooling to room temperature, the suspension was filtered and the filtrate washed with methylene chloride (0.2 L). The filtrate was rotoevaporated and treated with pyridine (1.35 L) and acetic anhydride (135 mL). After stirring overnight, the solvent was removed by rotoevaporation and the dark orange oil dissolved in methanol (0.6 L). The mixture was heated to 50°C and then cooled to room temperature. The solution was allowed to stand for 3 days and then cooled in an ice bath. The precipitate was filtered, washed with methanol, and dried to yield the title compound. The filtrate was rotoevaporated to a dry gum to yield the crude product.
Step 4: 20-t;ert;-Butyldimethylsilyloxy-7-methyl-Dregn-5-ene-3.7-diol
A solution of 3-acetoxy-20- ert butyldimethylsilyloxy-pregn- 5-en-7-one (279 gm, 0.57 mol, product of Step 3) in tetrahydrofuran (5.6 L) was cooled to 4°C. A 3M solution of methyl magnesium chloride in tetrahydrofuran (1.037 L, 3.1 mol) was added at such a rate as to keep the temperature < 0°C. The ice bath was removed and the reaction allowed to warm to room temperature overnight. The reaction was cooled in an ice bath and quenched with a 20% solution of ammonium chloride (3 L). The organic layer was removed and the aqueous layer extracted with ethyl acetate. The organic layers were combined, washed with saturated salt solution, and dried over anhydrous magnesium sulfate. The solution was filtered through a pad of anhydrous sodium sulfate and the solvent removed by rotoevaporation to yield the title compound.
Step 5: 20-£er£-Butyldimethylsilyloxy-7-methyl-pregn-4.6-dien-3-one A solution of 20- ert;-butyldimethylsilyloxy-7-methyl-pregn-5- ene-3,7-diol (298 gm, 0.59 mol, product of Step 4) in toluene (3 L) and cyclohexanone (1.03 L) was azeotroped to remove 750 mL of solvent. A solution of aluminum isopropoxide (121 gm) in toluene (620 mL) was added and the solution azeotroped to remove another 650 mL of solvent. A reflux condenser was added and the solution refluxed overnight. The solution was cooled to 40°C and Supercell™ (125 gm) and water (125 mL) were added. After stirring for 10 min, the mixture was filtered and the solids washed with toluene (550 mL). The solvent was removed by rotoevaporation to yield a orange liquid which was purified by column chromatography on silica gel eluted with hexane, followed by 5-10% ethyl acetate in hexanes. The title compound was isolated as a mixture of 20α- and 20β-isomers.
Step 6: 20- ert Butyldimethylsilyloxy-7β-methyl-pregn-4-en-3-one A slurry of 5% palladium on carbon (7.12 gm) and benzyl alcohol (213 mL) in heptane (356 mL) was refluxed for 20 min. The mixture was cooled to 80°C and a solution of 20-£er -butyldimethyl- silyloxy-7-methyl-pregn-4,6-dien-3-one (71.2 gm, 0.16 mol, product of Step 5) in heptane (427 mL) was added. The slurry was refluxed for 9.5 h. The reaction was cooled to room temperature and filtered through
SOLKA FLOK filter aid which was subsequently washed with hexane. The filtrate was extracted with acetonitrile which was subsequently back-extracted with hexane. The heptane and hexane extracts were combined, washed with saturated sodium sulfate and saturated salt solutions, and dried over anhydrous magnesium sulfate. The solution was filtered through a pad of anhydrous sodium sulfate and the solvent removed by rotoevaporation. The title compound was purified by column chromatography on silica gel eluted with 7% ethyl acetate in hexanes.
Step 7: 20- ert-Butyldimethylsilyloxy-7β-methyl-5-oxo-A-nor-3,5- secopregnan-3-oic acid
To a solution of 20- er^butyldimethylsilyloxy-7β-methyl- pregn-4-en-3-one (73.57 gm, 0.165 mol, product of Step 6) in tert-butanol (0.96 L) was added a solution of sodium carbonate (25.8 gm) in water (120 mL). The mixture was heated to 80°C with stirring. A warm solution of sodium periodate (244 gm) and potassium permanganate (1.91 gm) in water (0.96 L) was slowly added and then the reaction refluxed for 2 h. The reaction was cooled to room temperature and filtered through a pad of SuperCell™. The filter cake was washed with water (2 x 190 mL). The combined filtrates were rotoevaporated to remove the tert-butanol and washed with methylene chloride. The aqueous solution was acidified to pH ~ 3 with 2N hydrochloric acid and extracted with methylene chloride (3x). The organic extracts were combined, washed with 5% sodium bisulfite solution and saturated salt solution, and dried over anhydrous magnesium sulfate. The solvent was removed by rotoevaporation to yield the title compound as a white foam.
Step 8: 20-£βrt Butyldimethylsilyloxy-7β-methyl-4-azapregn-5-ene
To a solution of 20- er^butyldimethylsilyloxy-7β-methyl-5- oxo-A-nor-3,5-secopregnan-3-oic acid (26 gm., 56 mmol, product of Step 7) in ethylene glycol (500 mL) under nitrogen was added anhydrous ammonium acetate (50 gm). The mixture was heated at 180°C for 5 h, cooled to room temperature, and diluted with water (3.5 L). After stirring for 1 hr, the solid was filtered and the aqueous layer was extracted with methylene chloride (500 mL). The organic layer was dried over anhydrous magnesium sulfate and the solvent removed by rotoevaporation. The residue was combined with the filtered solid and dried in a vacuum oven overnight to give the title compound.
Step 9: 20-fer^Butyldimethylsilyloxy-7β-methyl-5α-4-azapregnane To a solution of 20- er7 butyldimethylsilyloxy-7β-methyl-4- azapregn-5-ene (23.9 g, 53.6 mmol, product of Step 8) in acetic acid (250 mL) was added platinum oxide (1.8 gm). The mixture was stirred overnight under hydrogen (1 atmosphere). The reaction mixture was filtered through a pad of Celite™ filter aid (trademark for diatomaceous earth) and the filtrate was coevaporated with toluene (3 x 500 mL) to remove all of the acetic acid. The residue was dissolved in chloroform and filtered again through a pad of Celite™ filter aid to remove residual catalyst. The solvent was removed by roto-evaporation to yield the title compound which was taken directly on to the next step without any further purification.
Step 10: 20-Hvdroxy-7β-methyl-5α-4-azapregnan-3-one
To a slurry of crude 20- er -butyldimethylsilyloxy-7β-methyl- 5α-4-azapregnane (25.2 g, product of Step 9) in acetonitrile (300 mL) was added an aqueous solution of hydrofluoric acid (12 mL). After stirring for 8 hr at room temperature, the reaction mixture was cooled to 0°C and saturated sodium bicarbonate solution was slowly added. The mixture was extracted with methylene chloride (3 x 500 mL) and the combined extracts washed with water, saturated salt solution and dried over anhydrous sodium sulfate. The solvent was removed by rotoevaporation to give the title compound which was used without purification in the subsequent reaction.
Step 11: 7β-Methyl-5α-4-azapregnane-3.20-dione
To a stirred solution of 20-hydroxy-7β-methyl-5α-4- azapregnan-3-one (22.3 gms, 67 mmol, product of Step 10) in dry methylene chloride under nitrogen (110 mL) was added 4-methyl morpholine N-oxide (11.8 gms, 100 mmol) followed by 4A molecular sieves (33 gm). To this mixture was added tetrapropylammonium perruthenate (1.2 gm). After stirring at room temperature for 4 h, the reaction mixture was poured through pad of silica gel in a 300 mL sintered glass funnel which was subsequently eluted with 4:1 ethyl acetate/methylene chloride (5 L). The solvent was removed by rotoevaporation and the title compound recrystallized.
Step 12: 20-Hvdroxy-7β.20-dimethyl-4-aza-5α-pregnan-3-one To a solution of 7β-methyl-4-aza-5α-pregnane-3,20-dione
(1.24 g., 3.73 mmol., product of Step 11) in tetrahydrofuran (20 mL.) was added methylmagnesiumbromide in diethyl ether (3.73 mL., 11.2 mmol) at room temperature. The reaction was stirred for 45 minutes under a nitrogen atmosphere and then quenched with saturated ammonium chloride solution and diluted with ethyl acetate (500 mL.). The organic phase was washed with water (500 mL, x 2) and brine solution (300 mL.). It was dried over sodium sulfate, filtered and the solvent evaporated in vacuo to give a white foam. The foam was flash chromatographed on silica gel using methanol in methylene chloride (1:19) as the mobile phase to yield a white foam. The foam was then recrystallized in methylene chloride and hexane (1:4) to yield the titled compound as white crystals. Rf = 0.35, 5% methanol : methylene chloride. 400 MHz lH NMR (CDCI3) : δ 0.82 (s, 3H); 0.87 (s, 3H); 1.16(s, 3H); 1.27 (s, 3H); 3.04
(dd, 1H).
Step 13: 7β.20-Dimethyl-4-aza-5 -pregn-17-en-3-one
A mixture of 20-Hydroxy-7β-methyl-4-aza-5α-pregnan-3-one (0.810 g., 2.35 mmol, product of Step 12), 2M hydrochloric acid (35 mL.) and tetrahydrofuran (THF, 35 mL) was refluxed at 70°C for 3 hours. THF was then evaporated in vacuo and the aqueous phase was basified using 2.5 M sodium hydroxide. The aqueous phase was then extracted with methylene chloride (200 mL) three times. The organic phases were combined and washed water (500 mL.) and brine (300 mL.). The organic phase was then dried with sodium sulfate, filtered and the solvent evaporated in vacuo to give a yellow oil. The oil was recrystallized in methylene chloride and hexane (1:3) to give a yellow solid.
EXAMPLE 3 7β.20-dimethyl-4-aza-5α-pregnan-3-one To a solution of 7β,20-dimethyl-4-aza-5α-pregna-17-en-3-one (730 mg., 2.22 mmol, the product of Example 1) and methanol (40 mL) was added platinum oxide (250 mg). This mixture was stirred under a hydrogen atmosphere overnight. It was then filtered through Celite™ diatomaceous earth and the solvent was removed under vacuum. The crude residue was chromatographed using 10 % 2-propanol in hexane as the mobile phase to yield the titled compound as a white solid. 400 MHz !H NMR (CDCI3) : δ 0.66 (s, 3H); 0.83 (d, 3H); 0.85 (s, 3H); 0.91 (d,
3H); 0.99 (d, 3H); 3.05 (dd, 1H). Mass spec. = 332 (M+l)
EXAMPLE 4 7β.20-dimethyl-4-aza-5α-pregn-l-en-3-one
To a solution of 7β,20-dimethyl-4-aza-5α-pregna-3-one (500 mg., 1.51 mmol, the product of Example 2) in dry toluene (15 mL.) was added 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (410 mg, 1.81 mmol), bis(trimethyl silyl)trifluoroacetamide (1.6 mL, 6.04 mmol) and triflic acid (0.00625 mL., 0.068 mmol). The mixture was stirred under nitrogen atmosphere overnight, followed by addition of methyl acetoacetate 90.032 mL., 0.30 mmol). The mixture was then refluxed overnight. The reaction mixture was poured into water (100 mL) containing sodium bicarbonate (800 mg.) and sodium sulfite (300 mg) and extracted with methylene chloride (3 x 100 mL). The organic phases were combined and washed with water (200 mL) and brine (100 mL). The organic phase was dried over sodium sulfate, filtered and the solvent evaporated in vacuo. The residue was purified by flash chromatography on silica gel eluded with 15% acetone in methylene chloride and recrystallization methyl ethyl ketone (MEK) to yield titled compound. 400 MHz ΪH NMR (CDCI3) : δ 0.67 (s, 3H); 0.82 (d, 3H); 0.89 (s, 3H); 0.92 (d, 3H); 1.01 (d, 3H);
3.34 (dd, 1H); 5.78 (dd, 1H); 6.78 (d, 1H). Mass spec. = 330 (M+l)
EXAMPLE 5 7β.20-dimethyl-4-aza-5α-pregn-1.17-dien-3-one
The titled compound was synthesized in the same fashion as 7β,20-dimethyl-4-aza-5α-pregna-l-ene-3-one, starting with 7β,20- dimethyl-4-aza-5α-pregna-17-en-3-one with the exception it was purified by recrystallization in ethyl acetate. 400 MHz !H NMR (CDCI3) : δ 0.85
(s, 3H); 0.90 (s, 3H); 1.04 (d, 3H); 1.54 (s, 3H); 1.68 (s, 3H); 3.34 (dd, IH); 5.78 (dd, IH); 6.78 (d, IH). Mass spec. = 328 (M+l)
EXAMPLE 6 20-ethyl-4.7β-dimethyl-4-aza-5α-pregn-17-en-3-one Step 1: Preparation of 20-Ethyl-20-hydroxy-7β-methyl-4-aza-5α- pregnan-3-one The titled compound was synthesized in a similar fashion to
20-Hydroxy-7β,20-dimethyl-4-aza-5α-pregnane-3-one using 3M ethylmagnesium bromide in diethyl ether in place of the methylmagnesium bromide. 400 MHz *H NMR (CDCI3) : δ 0.82 (s, 3H);
0.84 (t, 3H); 0.86 (s, 3H); 0.99 (d, 3H); 1.23 (s, 3H); 3.03 (dd, IH). Mass spec. = 343 (M-18)
Step 2: Preparation of 20-Ethyl-20-hydroxy-4,7β-dimethyl-4-aza-
5α-pregnan-3-one
To a slurry of sodium hydride (8.0 mg., 0.2 mmol) and 20- Ethyl-20-hydroxy-7β-methyl-4-aza-5α-pregnan-3-one (63.0 mg., 0.17 mmol, product of Step 1) in tetrahydrofuran was added methyl iodide (15.0 μL., 2.55 mmol). The solution was allowed to stir under a nitrogen atmosphere at room temperature overnight. The reaction was quenched with water and extracted with ethyl acetate (2 x 100 mL.). The organic phase was washed with water (100 mL) and brine ( 100 mL) and dried over sodium sulfate. The solvent was evaporated in vacuo and the residue purified via flash chromatography on silica gel eluding with 10 % acetone in methylene chloride to yield the titled compound as a white foam. 400 MHz IH NMR (CDCI3) : δ 0.81-0.84 (t, 3H); .083 (s, 3H); 0.85 (s, 3H); 1.03 (d, 3H); 1.22 (s, 3H); 2.9 (s, 3H); 2.99 (dd, IH). Mass spec. = 375 (M+)
Step 3: 20-ethyl-4.7β-dimethyl-4-aza-5α-pregn-17-en-3-one The titled compound was synthesized in a similar fashion to 7β,20-dimethyl-4-aza-5α-pregna-17-ene-3-one and taken forward without any purification.
EXAMPLE 7
20-ethyl-4.7β-dimethyl-4-aza-5α-pregnan-3-one
The titled compound was synthesized in a similar fashion to 7β,20-dimethyl-4-aza-5α-pregnane-3-one, starting with the product of Example 5. 400 MHz 3-H NMR (CDCI3) : δ 0.64 (d, 3H); 0.79 (d, 3H); 0.83 (s, 3H); 0.88 (d, 3H); 1.04 (d, 3H); 2.89 (s, 3H); 3.0 (dd, IH). Mass spec. = 359 (M+)
EXAMPLE 8 20-propyl-4.7β-dimethyl-4-aza-5 -pregnan-3-one Step 1: 20-Allyl-20-hvdroxy-7β-methyl-4-aza-5α-pregnan-3-one
The titled compound was synthesized in a similar fashion to 20-Hydroxy-7β,20-dimethyl-4-aza-5α-pregnan-3-one using 2M allylmagnesium chloride in tetrahydrofuran in place of the methylmagnesium bromide. No further purification was done prior to the following step.
Step 2: 20-Allyl-20-hydroxy-4,7β-dimethyl-4-aza-5α-pregnan-3- one
The titled compound was synthesized in a fashion similar to 20-ethyl-20-Hydroxy-4,7β-dimethyl-4-aza-5α-pregnan-3-one. 400 MHz iH
NMR (CDCI3) : δ 0.82 (s, 3H); 0.85 (s, 3H); 1.03 (d, 3H); 1.26 (s, 3H); 2.89
(s,3H); 3.00 (dd, IH); 5.05 (dd, 2H); 5.78 (m, IH).
Step 3: 20-propyl-4.7β-dimethyl-4-aza-5α-pregnan-3-one A slurry of 20-allyl-20-Hydroxy-4,7β-dimethyl-4-aza-5α- pregnan-3-one (29.0 mg., 0.075 mmol), 10% palladium on carbon (5.0 mg.) and a mixture of ethyl acetate- ethanol (5.0 mL., 1:1) was stirred for 48 hours under a hydrogen atmosphere at room temperature. The reaction was then filtered through Celite™ and the solvent evaporated in vacuo. The residue was purified via HPLC on a Waters 19 x 300 mm 8μ silica Nova Pak column using a 5 to 10 % 2-propanol hexane linear gradient at a 20 mL. per minute flow rate to yield the titled compound. 400 MHz IH NMR (CDCI3) : δ 0.65 (s, 3H); 0.80 (m, 9H); 1.02 (d, 3H); 2.89
(s, 3H); 3.00 (dd, IH). Mass spec. = 373 (M+)
EXAMPLE 9
Oral Composition
As a specific embodiment of an oral composition of a compound of this invention, 3 mg of 7β,20-dimethyl-4-aza-5α-pregna-l- en-3-one is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.
EXAMPLE 10 Oral Composition As a specific embodiment of an oral composition of a compound of this invention, 0.5 mg of a compound of structural formula I is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.
EXAMPLE 11
Oral Composition
As a specific embodiment of an oral composition of a compound of this invention, 2.5 mg of a compound of structural formula I is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.
EXAMPLE 12 Oral Composition
As a specific embodiment of an oral composition of a compound of this invention, 6 mg of a compound of structural formula I is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.
EXAMPLE 13 Transdermal Patch Formulation Ingredient Amount 7β,20-dimethyl-4-aza-5α-pregna-l- en-3-one 40 g
Silicone fluid 45 g
Colloidal silicone dioxide 2.5 g
The silicone fluid and 7β,20-dimethyl-4-aza-5α-pregna-l-en-3-one are mixed together and the colloidal silicone dioxide is added to increase viscosity. The material is then dosed into a subsequently heat sealed polymeric laminate comprised of the following: polyester release liner, skin contact adhesive composed of silicone or acrylic polymers, a control membrane which is a polyolefin (e.g. polyethylene, polyvinyl acetate or polyurethane), and an impermeable backing membrane made of a polyester multilaminate. The resulting laminated sheet is then cut into 10 cm2 patches. For 100 Patches.
EXAMPLE 14 Suppository
Ingredient Amount
7β,20-dimethyl-4-aza-5α-pregna-l- 25 g en-3-one
Polyethylene glycol 1000 1481 g
Polyethylene glycol 4000 494 g
The polyethylene glycol 1000 and polyethylene glycol 4000 are mixed and melted. The 5α-reductase type 1 inhibitor is mixed into the molten mixture, poured into molds and allowed to cool. For 1000 suppositories.
EXAMPLE 15
Iniectable solution
Ingredient Amount
7β,20-dimethyl-4-aza -5α-pregna-l- 5 g en-3-one
Buffering agents q.s.
Propylene glycol 400 mg
Water for injection 600 mL The 7β,20-dimethyl-4-aza-5 -pregna-l-en-3-one and buffering agents are dissolved in the propylene glycol at about 50°C. The water for injection is then added with stirring and the resulting solution is filtered, filled into ampules, sealed and sterilized by autoclaving. For 1000 Ampules.
EXAMPLE 16 Injectable solution
Ingredient Amount
7β,20-dimethyl-4-aza-5α-pregna-l- 5 g en-3-one
Buffering agents q.s.
Magnesium sulfate heptahydrate 100 mg
Water for injection 880 mL
The 7β,20-dimethyl-4-aza-5α-pregna-l-en-3-one, magnesium sulfate heptahydrate and buffering agents are dissolved in the water for injection with stirring, and the resulting solution is filtered, filled into ampules, sealed and sterilized by autoclaving. For 1000 Ampules.
EXAMPLE 17
Preparation of Human Prostatic and Scalp 5α-Reductases
Samples of human tissue were pulverized using a freezer mill and homogenized in 40 mM potassium phosphate, pH 6.5, 5 mM magnesium sulfate, 25 mM potassium chloride, 1 mM phenylmethyl- sulfonyl fluoride, 1 mM dithiothreitol (DTT) containing 0.25 M sucrose using a Potter-El vehj em homogenizer. A crude nuclear pellet was prepared by centrifugation of the homogenate at 1,500 x g for 15 min. The crude nuclear pellet was washed two times and resuspended in two volumes of buffer. Glycerol was added to the resuspended pellet to a final concentration of 20%. The enzyme suspension was frozen in aliquots at -80°C. The prostatic and scalp reductases were stable for at least 4 months when stored under these conditions.
EXAMPLE 18 5 α- Reductase Assay The reaction mixture for the type 1 5α-reductase contained 40 mM potassium phosphate, pH 6.5, 5 mM [7-3H]-testosterone, 1 mM dithiothreitol and 500 μM NADPH in a final volume of 100 μL. The reaction mixture for the type 2 5α-reductase contained 40 mM sodium citrate, pH 5.5, 0.3 mM [7-3H]-testosterone, 1 mM dithiothreitol and 500 μM NADPH in a final volume of 100 μL. Typically, the assay was initiated by the addition of 50-100 μg prostatic homogenate or 75-200 μg scalp homogenate and incubated at 37°C. After 10-50 min the reaction was quenched by extraction with 250 μL of a mixture of 70% cyclohexane: 30% ethyl acetate containing 10 μg each DHT and T. The aqueous and organic layers were separated by centrifugation at 14,000 rpm in an Eppendorf microfuge. The organic layer was 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 min; T, 9.1-9.7 min). The HPLC system consisted of a Waters Model 680 Gradient System equipped with a Hitachi Model 655α Autosampler, Applied Biosystems Model 757 variable UV detector, and a Radiomatic Model A120 radioactivity analyzer. The conversion of T to DHT was 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 DHT was 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 5α-reductase 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 5α-reductase type 1 and type 2 inhibition. A compound referred to herein as a 5α-reductase 2 inhibitor is a compound that shows inhibition of the 5α-reductase 2 isozyme in the above-described assay, having an IC50 value of about or under 100 nM.
The compounds are tested in the above-described assay for 5α-reductase type 1 and type 2 inhibition, and were found to have IC50 values under about 100 nM for inhibition of the type 1 isozyme. Compounds found to have IC50 values of under about 50 nM for inhibition of the type 1 isozyme are called type 1 inhibitors. Compounds called "dual inhibitors" additionally had IC50 S under about 200 nM for inhibition of the type 2 isozyme.
While the invention has been described and illustrated with reference to certain particular 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 particular 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 defined by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable.

Claims

WHAT IS CLAIMED IS:
1. A method of treating polycystic ovary syndrome in a subject in need of such treatment comprising administration to the subject of an effective amount of a compound of structural formula I:
Figure imgf000043_0001
wherein: the C1-C2 and C5-C6 bonds designated with a dotted line each independently represent a single or double bond, provided that when the C5-C6 is a double bond, Ha is absent and when the C5-C6 bond is a single bond Ha is present and represents hydrogen; Rl is selected from hydrogen and Cl-5 alkyl; R2 is Cl-5alkyl, either straight or branched chain; and R3 is C3-7alkyl, either straight or branched chain, optionally having one degree of unsaturation; or a pharmaceutically acceptable salt thereof.
2. The method of Claim 1 wherein the compound of structural formula I is selected from:
7╬▓,20-dimethyl-4-aza-5╬▒-pregn-17-en-3-one,
7╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one,
20-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-17-en-3-one,
20-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one , 7╬▓,20-dimethyl-4-aza-5╬▒-pregnan-3-one,
7╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one,
20-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one,
20-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, ,7╬▓,20-trimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5 -pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-(5-methylhexyl)-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one. ╬▓,20-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one , ╬▓,20-dimethyl-4-aza-5╬▒-pregnan-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, and 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androstan-3-one. ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one , ╬▓,20-dimethyl-4-aza-5╬▒-pregnan-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5oc-pregn-l-en-3-one, ,7╬▓,20-trimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0 -propyl-7 ╬▓-methyl-4-aza-5╬▒-pregnan-3 -one, 0-propyl-7╬▓-methyl-4-aza-5 -pregn-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-7╬▓-methyl-4-aza-5 -androst-l-en-3-one, 7╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isopentyl-7╬▓-methyl-4-aza-5 -androst-l-en-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one, 17╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one,
17╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one,
17╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 20-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 20-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 17╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-tert.-butyl-7╬▓-methyl-4-aza-5 -androst-l-en-3-one, 17╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, and 17╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one.
3. The method of Claim 1 wherein the compound of structural formula I is 7╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one.
4. The method of Claim 1 wherein the subject is a female human.
5. The method of Claim 1 wherein the compound of structural formula I is administered at a dose of 0.01 to 1000 mg per day.
6. The method of Claim 7 wherein the compound of structural formula I is administered at a dose of 0.1 to 100 mg per day.
7. A method of improving the response to in vitro fertilization which comprises the administration to a subject in need thereof of an effective amount of a compound of structural formula I:
Figure imgf000047_0001
wherein: the C1-C2 and C5-C6 bonds designated with a dotted line each independently represent a single or double bond, provided that when the C5-C6 is a double bond, Ha is absent and when the C5-C6 bond is a single bond Ha is present and represents hydrogen; Rl is selected from hydrogen and Cχ-5 alkyl; R2 is Cl-5alkyl, either straight or branched chain; and R3 is C3-7alkyl, either straight or branched chain, optionally having one degree of unsaturation; or a pharmaceutically acceptable salt thereof.
8. The method of Claim 7 wherein the compound of structural formula I is selected from: 7╬▓,20-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one , ╬▓,20-dimethyl-4-aza-5╬▒-pregnan-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, ,7╬▓,20-trimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst- l-en-3-one, 7╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7 ╬▓-isopentyl-7╬▓-methyl-4- aza-5╬▒-andro st- l-en-3 -one , 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5 -androstan-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5 -androstan-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-(5-methylhexyl)-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one. ╬▓,20-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one , ╬▓,20-dimethyl-4-aza-5╬▒-pregnan-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst- l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, and 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androstan-3-one. ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one , ╬▓,20-dimethyl-4-aza-5 -pregnan-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, ,7╬▓,20-trimethyl-4-aza-5 -pregn-l-en-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, ╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, ╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, ╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, ╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, ╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, ╬▓-isobutyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst- l-en-3-one, 7╬▓-(5-methylhexyl)-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, ╬▓,20-dimethyl-4-aza-5 -pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5 -pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5 -androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, and 17╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one.
9. The method of Claim 7 wherein the compound of structural formula I is 7╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one.
10. The method of Claim 7 wherein the subject is a female human.
11. The method of Claim 7 wherein the compound of structural formula I is administered at a dose of 0.01 to 1000 mg per day.
12. The method of Claim 7 wherein the compound of structural formula I is administered at a dose of 0.1 to 100 mg per day.
13. A method of improving fertility in a female human which comprises the administration to a subject in need thereof of an effective amount of a compound of structural formula I:
Figure imgf000051_0001
wherein: the C1-C2 and C5-C6 bonds designated with a dotted line each independently represent a single or double bond, provided that when the C5-C6 is a double bond, Ha is absent and when the C5-C6 bond is a single bond Ha is present and represents hydrogen; Rl is selected from hydrogen and Ci-5 alkyl; R2 is Cχ-5alkyl, either straight or branched chain; and R3 is C3-7alkyl, either straight or branched chain, optionally having one degree of unsaturation; or a pharmaceutically acceptable salt thereof.
14. The method of Claim 13 wherein the compound of structural formula I is selected from: 7╬▓,20-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, 7╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, 20-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, 20-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one , 7╬▓,20-dimethyl-4-aza-5╬▒-pregnan-3-one, 7╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 20-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 20-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 20-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 4,7╬▓,20-trimethyl-4-aza-5╬▒-pregn-l-en-3-one, 20-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 20-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 20-propyl-7╬▓-methyl-4-aza-5╬▒-pregnan-3-one, 20-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 17╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst- l-en-3-one, 17╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-isobutyl-4,7╬▓-dimethyl-4-aza-5 -androst-l-en-3-one, 17╬▓-tert.-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-(5-methylhexyl)-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-n-propyl-7╬▓-methyl-4-aza-5 -androstan-3-one, 7╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-(5-methylhexyl)-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one. ╬▓,20-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one , ╬▓,20-dimethyl-4-aza-5╬▒-pregnan-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst- l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, and 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androstan-3-one. ╬▓,20-dimethyl-4-aza-5 -pregn-l,17-dien-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-17-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one , ╬▓,20-dimethyl-4-aza-5╬▒-pregnan-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregnan-3-one, 0-ethyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn- l-en-3-one, ,7╬▓,20-trimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregnan-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isobutyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-(5-methylhexyl)-4,7╬▓-dimethyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-propyl-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androstan-3-one, 7╬▓-n-butyl-4,7╬▓-dimethyl-4-aza-5╬▒-androstan-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l,17-dien-3-one, ╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one, 0-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 0-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one, 7╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-n-butyl-7╬▓-methyl-4-aza-5 -androst-l-en-3-one, 7╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 7╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, 17╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one,
17╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one,
7╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one,
20-ethyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one,
20-propyl-7╬▓-methyl-4-aza-5╬▒-pregn-l-en-3-one,
17╬▓-n-propyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one,
17╬▓-n-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one,
17╬▓-isobutyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one,
17╬▓-tert.-butyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one,
17╬▓-n-pentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one, and
17╬▓-isopentyl-7╬▓-methyl-4-aza-5╬▒-androst-l-en-3-one.
15. The method of Claim 13 wherein the compound of structural formula I is 7╬▓,20-dimethyl-4-aza-5╬▒-pregn-l-en-3-one.
16. A composition comprising a pharmaceutically acceptable carrier, and a therapeutically effective amount of a compound of structural formula I:
Figure imgf000055_0001
wherein: the C1-C2 and C5-C6 bonds designated with a dotted line each independently represent a single or double bond, provided that when the C5-C6 is a double bond, Ha is absent and when the C5-C6 bond is a single bond Ha is present and represents hydrogen; Rl is selected from hydrogen and Cχ-5 alkyl; R2 is Cχ-5alkyl, either straight or branched chain; and R3 is C3-7alkyl, either straight or branched chain, optionally having one degree of unsaturation; or a pharmaceutically acceptable salt thereof.
17. The use of a compound of structural formula I:
Figure imgf000056_0001
wherein: the C1-C2 and C5-C6 bonds designated with a dotted line each independently represent a single or double bond, provided that when the C5-C6 is a double bond, Ha is absent and when the C5-C6 bond is a single bond Ha is present and represents hydrogen; Rl is selected from hydrogen and Cχ-5 alkyl; R2 is Cχ-5alkyl, either straight or branched chain; and R3 is C3-7alkyl, either straight or branched chain, optionally having one degree of unsaturation; or a pharmaceutically acceptable salt thereof; for the preparation of a medicament useful to treat polycystic ovary syndrome.
18. The use of a compound of structural formula I:
Figure imgf000057_0001
wherein: the C1-C2 and C5-C6 bonds designated with a dotted line each independently represent a single or double bond, provided that when the C5-C6 is a double bond, Ha is absent and when the C5-C6 bond is a single bond Ha is present and represents hydrogen; Rl is selected from hydrogen and Cχ-5 alkyl; R2 is Cχ-5alkyl, either straight or branched chain; and R3 is C3-7alkyl, either straight or branched chain, optionally having one degree of unsaturation; or a pharmaceutically acceptable salt thereof; for the preparation of a medicament useful to improve the response to in vitro fertilization.
19. The use of a compound of structural formula I:
Figure imgf000057_0002
wherein: the C1-C2 and C5-C6 bonds designated with a dotted line each independently represent a single or double bond, provided that when the C5-C6 is a double bond, Ha is absent and when the C5-C6 bond is a single bond Ha is present and represents hydrogen; Rl is selected from hydrogen and Cχ-5 alkyl; R2 is Cχ-5alkyl, either straight or branched chain; and R3 is C3-7alkyl, either straight or branched chain, optionally having one degree of unsaturation; a pharmaceutically acceptable salt thereof, preparation of a medicament useful to improve fertility.
PCT/US1998/004955 1997-03-17 1998-03-13 Methods and compositions for treating polycystic ovary syndrome WO1998041211A1 (en)

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