The present invention relates to 17β-substituted-6-azacholesten-3- ones and their use as 5α-testosterone reductase inhibitors.

BACKGROUND OF THE INVENTION

Androgens are responsible for a variety of physiological functions in both males and females. Androgen action is mediated by specific intracellular hormone receptors expressed in androgen responsive cells. Testosterone, the major circulating androgen, is secreted by Leydig cells of the testes under the stimulation of pituitary-derived luteinizing hormone. However, reduction of the 4, 5 double bond of testosterone to dihydrotestosterone (DHT) is required in some target tissues, such as prostate and skin, for androgen action. Steroid 5α-reductases in target tissues catalyze conversion of testosterone to DHT in an NADPH dependent fashion as shown below in Scheme A.

Scheme A

Testosterone Dihydrotestosterone

The requirement for DHT to act as an agonist in these target tissues has been highlighted by studies of steroid 5α-reductase deficient individuals who have vestigial prostate glands and do not suffer from acne vulgaris or male pattern baldness (see McGinley, J. et al., The New England J. of Medicine, 300, 1233 (1979)). Thus, inhibition of the conversion of testosterone to DHT in these target tissues is anticipated to be useful in the treatment of a variety of androgen responsive diseases, e.g., benign prostatic hyperplasia, prostate cancer, acne, male pattern baldness, and hirsutism.

It has recently been discovered that two isozymes of 5α-reductase exist in humans which differ in their tissue distribution, affinity for testosterone, pH profile and sensitivity to inhibitors (see Russell, D.W. et

al, J. Clin. Invest., 89, 293 (1992); Russell, D.W. et al, Nature, 354, 159 (1991)). The steroid 5α-reductase deficient individuals studied by Imperato- McGinley are deficient in the type 2, 5α-reductase enzyme (Russell, D.W. et al, J. Clin. Invest., 90, 799 (1992); Russell, D.W. et al, New England J. Med., 327, 1216 (1992)), which is the predominant isozyme present in the prostate, while the type 1 isozyme is predominant in the skin. The relative value of isozyme specific and dual inhibitors of the two isozymes of 5α- reductase will depend upon the type of disease treated (benign prostatic hyperplasia, prostate cancer, acne, male pattern baldness, or hirsutism) as well as the stage of the disease (prevention versus treatment) and the anticipated side-effects in the intended patients (for example treatment of acne vulgaris in pubescent males).

Because of their valuable therapeutic potential, testosterone 5α- reductase inhibitors (5α-reductase inhibitors) have been the subject of active research worldwide. For example, see: Hsia, S. and Voight, W., /. Invest. Derm., 62, 224 (1973); Robaire, B. et al, J. Steroid Biochem., 8, 307 (1977); Petrow, V. et al, Steroids, 38, 121 (1981); Liang, T. et al, J. Steroid Biochem., 19, 385 (1983); Holt, D. et al, J. Med. Chem., 33, 937 (1990); U.S. Patent No. 4,377,584, U.S. Patent No. 4,760,071, U.S. Patent No. 5,017,568 and published PCT application W094/ 14833. Two particularly promising 5α-reductase inhibitors are MK-906 (Merck), known by the generic name, finasteride, and marketed under the name, PROSCAR™; and SKF-105657 (SmithKline Beecham), shown below in Scheme B.

Scheme B

finasteride (PROSCAR™)

The potent inhibition of bovine adrenal and porcine granulosa cell 3β-hydroxy-Δ5-steroid dehydrogenase/3-keto-Δ5-steroid isomerase (3βHSD) by the 4-azasteroid derivative, 4-MA and not by the drug

finasteride (Tan, C.H.; Fong, C.Y.; Chan, W.K. Biochem. Biophys. Res. Comm., 144, 166 (1987) and Brandt, M.; Levy, M.A. Biochemistry, 28, 140 (1989)) along with the critical role of 3βHSD in steroid biosynthesis (Potts, G.O. et al, Steroids, 32, 257 (1978)), suggests that optimal inhibitors of type 1 and 2 5α-reductase should also be selective versus human adrenal 3βHSD.

The importance of selectivity in 5α-reductase inhibitors has been accented by reports of hepatotoxicity in certain 4-azasteroids such as 4-MA (McConnell, J.D. The Prostate Suppl, 3, 49 (1990) and Rasmusson, G.H. et al. J. Med. Chem. , 27, 1690 (1984)).

SUMMARY OF THE INVENTION One aspect of the present invention includes compounds of

Formula (I);

wherein

R1 and R^ independently represent hydrogen or alkyl and the bond between carbons 1 and 2 is a single or a double bond, or R and R^ may be joined together to form a fused cyclopropane ring;

R3 represents hydrogen, alkyl or halogen;

R ⁴ and R ⁵ represent independently hydrogen or alkyl;

R6 represents alkyl; and the pharmaceutically acceptable salts and solvates thereof.

In particular R*, R^, R3 R4 are hydrogen, R^ is hydrogen or methyl, and R ⁶ is alkyl, more particularly CH3CH(CH2)3CH(CH3)2-

Other aspects and advantages of the present invention will become apparent from a review of the detailed description below.

DETAILED DESCRIPTION OF THE INVENTION

By "safe and therapeutically effective amount," as used herein, means a sufficient amount of a drug , pharmaceutical agent or pharmaceutical formulation to abate or inhibit testosterone-5α-reductases without harming the tissues of a mammal, including a human to which the drug is administered.

As used herein, the term "alkyl", alone or in combination, as used herein means a linear or branched chain saturated hydrocarbon group from Cl-Cl2. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, n-hexyl, and the like. As used herein, the terms "halo" and "halogen" means a substitutent which may be fluoro, chloro, bromo, or iodo. The term "room temperature" means from about 20 °C to about 30 °C.

In addition to compounds of Formula (I), other aspects of the invention include: a method of inhibiting testosterone-5α-reductases comprising contacting testosterone-5α-reductases with a compound of Formula (I); a method of treating androgen responsive or mediated disease comprising administering a safe and therapeutically effective amount of a compound of Formula (I) to a patient in need of such treatment; pharmaceutical formulations containing a compound of Formula (I) as an active ingredient; a method of treating androgen responsive or mediated disease comprising administering a safe and therapeutically effective amount of a compound of Formula (I) to a patient in need of such treatment in combination with an anti-androgen such as flutamide; a method of treating benign prostatic hyperplasia comprising administering a safe and therapeutically effective amount of a compound of Formula (I) to a patient in need of such treatment in combination with an alpha 1 adrenergic receptor blocker (e.g. terazosin); a method of treating benign prostatic hyperplasia comprising administering a safe and

O 96/30391

-5- therapeutically effective amount of a compound of Formula (I) to a patient in need of such treatment in combination with an anti-estrogen; and synthetic methods and chemical intermediates used in the preparation of compounds of Formula (I) and pharmaceutical compositions containing them.

During the preparation of the compounds of Formula (I) or a salt thereof it may be necessary or desirable to protect one or more sensitive groups in the molecule to prevent undesirable side reactions. The protecting groups used in the preparation of compounds of Formula (I) may be used in a conventional manner. See, for example, Protective Groups in Organic Chemistry, Ed. J.F.W. McOmie, Plenum Press, London (1973) or Protective Groups in Organic Synthesis, Theodora Green, John Wiley and Sons, New York (1981).

Conventional amino protecting groups may include, for example, arylalkyl groups, such as benzyl and acyl groups, such as N- benzyloxycarbonyl or t-butoxycarbonyl. Thus, compounds of Formula (I) when R4 represents hydrogen may be prepared by deprotection of a corresponding protected compound. Hydroxy groups may be protected, for example, by benzyl, diphenylmethyl or triphenylmethyl groups, acyl groups, such as acetyl, silicon protecting groups, such as triisopropysilyl or t-butyldimethylsilyl groups, or as tetrahydropyran derivatives.

Removal of any protecting groups present may be achieved by conventional procedures known in the art. An arylalkyl group such as benzyl, may be cleaved by hydrogenolysis in the presence of a catalyst, e.g., palladium on charcoal; an acyl group such as N-benzyloxycarbonyl may be removed by hydrolysis with, for example, hydrogen bromide in acetic acid or by reduction, for example by catalytic hydrogenation; silicon protecting groups may be removed, for example, by treatment with fluoride ion or by hydrolysis under acidic conditions; tetrahydropyran groups may be cleaved by hydrolysis under acidic conditions.

As will be appreciated, in any of the general processes of Schemes C and D, it may be desirable or even necessary to protect any sensitive groups in the molecule as described. Thus, a reaction step involving deprotection

of a protected derivative of general Formula (I) or a salt thereof may be carried out subsequent to any of the above described processes of Schemes C and D. Thus, the following reactions may, if necessary or desired be carried out in any appropriate sequence subsequent to any of the processes of Schemes C and D: removal of any protecting groups; and conversion of a compound of Formula (I) or a salt thereof into a pharmaceutical acceptable salt or solvate thereof.

COMPOUNDS Particular compounds of Formula (I) of the present invention include:

7-Methyl-6-azacholest-4-en-3-one, and

6-Azacholest-4-en-3-one.

Persons skilled in the art will recognize that stereocenters exist in various compounds of the present Invention. Accordingly, the present Invention includes all possible stereoisomers and geometric isomers. For example, where the compounds contain an asymmetric carbon atom, two enantiomeric forms ("α" and "β" configurations) are possible. As used herein, a black, triangular bond denotes the β configuration and extends above the plane of the page. Likewise, a dashed line from an asymmetric carbon indicates an α bond which extends below the plane of the page. (For a detailed explanation of stereochemical configuration see March, /. Advanced Organic Chemistry, 3rd Ed., ch 4, John Wiley & Sons, New York (1985). The present Invention is intended to include both enantiomeric

forms and any combinations of these forms and where no specific configuration is depicted at the site of an asymmetric carbon, it is to be understood that both enantiomeric forms and mixtures thereof are represented. Additionally, in situations where tautomers of the compounds are possible, the present invention is intended to include all tautomeric forms of the compounds.

Where a compound is desired as a single enantiomer, it may be obtained either by resolution of the final product or by stereospecific synthesis from either isomerically pure starting material or any convenient intermediate. Resolution of the final product, an intermediate or a starting material may be effected by any suitable method known in the art. See, for example, Stereochemistry of Carbon Compounds by E. L. Eliel (Mcgraw Hill, 1962) and Tables of Resolving Agents by S. H. Wilen.

Additionally, compounds of the present Invention may exist in a variety of crystalline forms. Compounds of the present Invention are intended to encompass all possible crystalline forms. Also included in the present Invention are other forms of the compounds including: solvates, hydrates, and the like.

The compounds of the present Invention can be used in the form of an acid addition salt derived from inorganic or organic acids. Where the salt of compounds of the present Invention is to be used for a human or veterinary medicinal application the salt must be pharmaceutically acceptable. However, non-pharmaceutically acceptable salts of compounds of the present Invention may be useful as intermediates in the preparation of a corresponding pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, but are not limited to, salts with inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide and nitrate salts or salts with an organic acid such as the acetate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, palmitoate, salicylate and stearate salts. For further examples of acceptable salts see, "Pharmaceutical Salts," /. Pharm. Scl, 66(1), 1 (1977). Where it is desirable to isolate a compound of the invention as a salt, this may be achieved by treating the free base of

Formula (I) with an appropriate acid, or with creatinine sulfate in a polar, protic solvent, e.g., aqueous ethanol.

PREPARATION OF COMPOUNDS Compounds of the present invention may be prepared by the procedure shown below in Schemes C and D; wherein Rl through R^ are as defined for Formula (I). Compounds contained in brackets and labeled with a lower case "a" are compounds which progress during the synthesis procedure to the next primary compound.

Scheme C

In step 1, the keto group in the 3 position of compounds of Formula (II), is protected by treatment with ethylene glycol in a protic or nonprotic solvent such as toluene in the presence of an acidic dehydrating agent such as tolulenesulfonic acid, yielding compounds of Formula (III). The compounds of Formula (II) are commercially available from: the Aldrich Chemical Company, Inc., 1001 West Saint Paul Avenue, Milwaukee, WI 53233, are explicitly taught in the art, or can be prepared by methods taught in the art from commercially available precursors.

Compounds of Formula (III), in step 2, are treated with ozone in a polar solvent or mixture of polar solvents, such as methylene chloride and methanol, at dry ice /acetone temperatures of about -78°C, and purged with an inert or low reactive gas, such as nitrogen and reduced with a moderately strong organic acid, such as acetic acid and zinc. The mixture is filtered and is then treated with an oxidizing agent such as sodium chlorite and an acid buffer such as sodium hydrogen phosphate monobasic in an alcoholic solvent system such as i-butanol followed by 2-methyl-2- butene. Then using column chromatography the mixture yields the corresponding compound of Formula (rV).

The carboxylic acid of the compounds of Formula (IV), in step 3, are converted to the corresponding acid halide intermediate by treatment with a halogenating agent such as oxalyl chloride, in an aprotic solvent such as methylene chloride. The acid halide intermediate is then treated with a metal azide, such as an alkaline metal azide in a polar solvent or mixture of polar solvents such as acetone and water, to yield the corresponding azide compounds of Formula (IVa).

In step 4, compounds of Formula (V) corresponding to the desired compounds of Formula (I) are heated in an aprotic solvent such as toluene, generating the compounds of Formula (Va). The compounds of Formula (Va) are then converted to the corresponding compounds of Formula (I) with isolation, by standard methods used in the art. For example, the solvent may be removed by in vacuo distillation, the residue acidified then heated in a polar solvent such as tetrahydrofuran, at reflux and purified by chromatography.

Incorporation of other than a hydrogen at position R^ in compounds of Formula (I), (e.g. lower alkyl groups) requires modification of Scheme C. This modification, producing compounds of Formula (I) with a 7 position lower alkyl group is depicted below in Scheme D. Compounds contained in brackets and labeled with a lower case "a" are compounds which progress during the synthesis to the next primary compound.

Compounds of Formula (VI) in Scheme D are treated, in Step 1, with an oxidizing agent such as chromium trioxide in the presence of an amine such as 3,5-dimethylpyrazole in an apolar aprotic solvent such as methylene chloride to yield compounds of Formula (VII). The enone of the compounds of Formula (VII), in Step 2, are converted to the exocyclic olefinic compounds of Formula (VDI) by treatment with an olefination reagent such as trimethylsilylmethylmagnesium chloride in a polar aprotic solvent such as tetrahydrofuran. In Step 3, compounds of Formula (Vm) are reduced to the compounds of Formula (IX) by treatment with a metal catalyst such as tristriphenylphosphine rhodium(I) chloride under an atmosphere of hydrogen in a polar aprotic solvent like ethyl acetate yielding the corresponding compounds of Formula (IX). The compounds of Formula (IX) are then converted to compounds of Formula (I) via Steps 4, 5 and 6 in reactions similar to Steps 2, 3 and 4 as seen in Scheme C.

It will be appreciated by those skilled in the art that in certain cases where R^ is halogen some Steps in the procedures shown in Schemes C and D are incompatible with survival of the halogen functional group. In these cases, the R^ halogen is either introduced subsequent to the incompatible Step or is present in a protected form. An example is where the R3 halogen is introduced by reacting a compound of Formula (I) with a halogenated succinimide, such as N-bromosuccinimide.

A double bond may be inserted between the carbon in the 1 position and the carbon in the 2 position by conventional means such as dehydrogenation with 2,3-dichloro-5, 6-dicyano-l,4-benzoquinone by refluxing in an aprotic solvent to produce a compound of Formula (I) that is unsarurated in the 1, 2 position. A compound of Formula (I) with a double bond in the 1, 2 position may then be treated with the anion of trimetylsulfoxonium iodide, prepared by deprotonation with a base such as sodium hydride, in an aprotic solvent to give a compound of Formula (I) herein Rl and R^ taken together form a cyclopropane ring.

Persons skilled in the art will quickly realize that during the herein described syntheses of 6-azaandrostenones that the various substituents on compounds herein described may be exchanged for other substituents

herein described. This exchange and the ease of the exchange is dependent on the site in the synthesis where the exchange is attempted. Furthermore, persons skilled in the art will also realize that the addition of the double bond between carbons 1 and 2 may be added at various steps in the synthesis and that this addition and the ease of the addition is dependent on the site in the systhesis where the addition is attempted. Persons skilled in the art will also realize the exchange of substitutents and the addition of the double bond between carbons 1 and 2 herein described is merely an example of one place in the syntheses this exchange or addition can be achieved and should not be construed as a limitation of the present Invention.

The compound of Formula (I) and the intermediate compounds, shown in Schemes I and II may be purified by any convenient means known in the art, e.g., chromatography or crystallization.

STEROID 5α-REDUCTASES

In Vitro Assay Enzyme activities may be determined using microsomes derived from: 1) prostate tissue from benign prostatic hyperplasia (BPH) patients; 2) recombinant baculovirus infected SF9 cells that express human type 1 5α- reductase; 3) prostate tissue from the rat; or 4) recombinant baculovirus infected SF9 cells that express human type 2 5α-reductase. Microsomes were prepared by homogenization of the tissue or cells, followed by differential centrifugation of the homogenate. Microsome extracts were incubated with varying concentrations of [l,2,6,7-3H]-testosterone, lmM NADPH, and varying amounts of the compounds of Formula I, i.e. a test compound, in buffer containing a NADPH regenerating system capable of maintaining NADPH concentrations for a period of time within the range 0.5-240 minutes. Corresponding incubations were carried out with no test compound as a control study. For type 1 IC5 ₀ measurements, assay components except testosterone were preincubated for 10 minutes at pH 7.0, and following the addition of lOOnM testosterone the assays were allowed to proceed for 10-120 minutes.

For type 2 IC50 measurements, assay components except testosterone were preincubated for 20 minutes at pH 6.0, and following the addition of 8nM testosterone the assays were allowed to proceed for 20-40

minutes. The percentage of conversion of testosterone to DHT in the presence of test compounds compared to the corresponding conversion in the control study was estimated using high pressure liquid chromatography (HPLC) with radiochemical detection. The results of these assays appear as K-50's reported in Table 1.

TABLE 1 5-REDUCTASE in vitro INHIBITORY ACTIVITY

Compound IC50 Human IC50 Human Type 1 Type 2

7α-methyl-6-azacholest-4-en-3-one +++ +++

7 β-methy l-6-azacholest-4-en-3-one + + + + + +

6-azacholest-4-en-3-one +++ +++

+++ = < 10 nM

+ + = 10 - 100 nM

+ = >100 nM

IN VIVO EVALUATION OF STEROID 5α-REDUCTASE INHIBITORS

The in vivo activity of steroid 5α-reductase inhibitors may be determined in both acute and chronic rat models. The acute model utilizes castrated male rats that receive testosterone (1 mg) subcutaneously and test compound (10 mg/kg) p.o., at 0.5 hr. and 4.5 hr. prior to sacrifice, respectively. Levels of DHT in the serum and prostate indicate the ability of the test compound to inhibit steroid 5α-reductase in an acute rat model. Known steroid 5α-reductase inhibitors were tested in parallel to ensure consistency of the assay method.

The chronic model also utilizes castrated male rats that are dosed daily with testosterone (20 μg/rat) subcutaneously and with test compound (0.01-10 mg/kg) p.o. for 7 days. The animals are then sacrificed and their prostates weighed. Reduction in the size of testosterone- stimulated prostate weight demonstrated activity of the test compound. Known steroid 5α-reductase inhibitors were tested in parallel to ensure consistency of the assay method.

UTILITY The steroid 5α-reductase inhibitors of the present invention are useful in the treatment of androgen responsive diseases, e.g., benign and malignant diseases of the prostate, especially benign prostatic hyperplasia. For correlation of in vitro, rat in vivo and human clinical data relating to an inhibitor of 5α-reductase, see Stoner, J. Steroid Biochem. Molec. Biol., 37, 375 (1990); Brooks, et al., Steroids, 47, 1 (1986) and Rasmusson, J. Med. Chem., 29, 2298 (1986)). They are also useful in the treatment of prostatitis, prostate cancer, androgen mediated diseases of the skin, such as acne, hirsutism and male pattern baldness. Other hormone related diseases, e.g., polycystic ovary disease, would be expected to respond to treatment with these inhibitors.

The amount of compound of Formula (I) required to be a safe and therapeutically effective amount as an 5α-reductase inhibitor will, of course, vary with the individual mammal being treated and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being treated, the route of administration, the nature of the Formulation, the mammal's body weight, surface area, age and general condition, and the particular compound to be administered. However, a suitable safe and therapeutically effective 5α-reductase inhibitory dose is in the range of about 0.01 to about 5 mg/kg body weight per day, preferably in the range of about 0.05 to about 2 mg/kg per day.

The total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day, or by intravenous infusion for a selected duration. Dosages above or below the range cited above are within the scope of the present invention and may be administered to the individual patient if desired and necessary. For example, for a 75 kg mammal, a dose range would be about 5 to about 150 mg per day, and a typical dose would be about 20 mg per day. If discrete multiple doses are indicated, treatment might typically be 5 mg of a compound of Formula (I) given 4 times per day.

The compounds of Formula (I) may also be administered in a topical Formulation, e.g., ointment, cream, gel, or lotion, in cases of

dermatological disorders such as acne vulgaris. A safe and therapeutically effective topical Formulation contains from about 0.25% to about 10% by weight, of a compound of Formula (I) which is applied at the rate of about 0.1 g to about 1.0 g per square centimeter of infected skin area. Typically a dose is about 1 gram of a 1% ointment, cream, gel, or lotion of a compound of Formula (I) gently rubbed onto the square centimeter of skin in need of treatment.

FORMULATIONS Formulations of the present invention for medical use comprise an active compound, i.e., a compound of 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 thereof.

The present invention, therefore, further provides a pharmaceutical formulation comprising a compound of Formula (I) together with a pharmaceutically acceptable carrier thereof. The formulations include those suitable for oral, rectal or parenteral (including subcutaneous, intramuscular and intravenous) administration. Preferred are those suitable for oral or parenteral administration.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier and then, if necessary, shaping the product into desired unit 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, an emulsion or a draught.

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, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.

A syrup or suspension may be made by adding the active compound to a concentrated, aqueous solution of a sugar, e.g., sucrose, to which may also be added any accessory ingredients. Such accessory ingredient(s) may include flavoring, a preservative, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, e.g., as a polyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppository with a conventional carrier, e.g., cocoa butter or Witepsol S55 (trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Formulations suitable for parenteral administration conveniently 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 pharmaceutically and pharmacologically acceptable acid addition salt of a compound of Formula (I) that is isotonic with the blood of the recipient. Thus, such formulations may conveniently contain distilled water, 5% dextrose in distilled water or saline and a pharmaceutically and pharmacologically acceptable acid addition salt of a compound of the Formula (I) that has an appropriate solubility in these solvents, for example the hydrochloride, isothionate and methanesulfonate salts, preferably the latter. Useful Formulations also comprise concentrated solutions or solids containing the compound

of Formula (I) which upon dilution with an appropriate solvent give a solution suitable for parental administration.

Parenteral formulations of the present Invention must be in a sterile form. Any of the various methods known to persons skilled in the art employed to prepare sterile parenteral preparations that will not degrade components of the present Invention are suitable for use in the sterile preparation of the formulations

Topical Formulations include ointments, creams, gels and lotions which may be prepared by conventional methods known in the art of pharmacy. In addition to the ointment, cream, gel, or lotion base and the active ingredient, such topical formulation my also contain preservatives, perfumes, and additional active pharmaceutical agents.

Formulations of the present Invention may be packaged as articles of manufacture comprising a safe and therapeutically effective amount of a compound of Formula (I) and its physiologically acceptable salts and solvates and one or more optional accessory ingredient(s) utilized in the art of pharmaceutical formulations. The packaging material may also have labeling and information relating to the pharmaceutical composition printed thereon. Additionally an article of manufacture may also have a brochure, report, notice, pamphlet, or leaflet containing product information. This form of pharmaceutical product information is sometimes, in the pharmaceutical industry, called the "package insert." A package insert may be attached to or included with a an article of manufacture. The package insert and any article of manufacture labeling provides information relating to the pharmaceutical formulation contained therein. This information and labeling provides various forms of information utilized by health care professionals and patients that describes the formulation, its dosage and various other parameters required by regulatory agencies, such as the United States Food and Drug Administration.

In addition to the aforementioned ingredients, the formulations of this invention may further include one or more optional accessory ingredient(s) utilized in the art of pharmaceutical formulations, e.g., diluents, buffers, flavoring agents, binders, surface active agents,

thickeners, lubricants, suspending agents, preservatives (including antioxidants) and the like.

EXAMPLES The following examples illustrate various aspects of the present

Invention, but should not be construed as limitations. The symbols, conventions and nomenclature are consistent with those used in the contemporary chemical literature, for example the journal of the American Chemical Society.

Unless otherwise noted all starting materials were obtained from commercial suppliers and used without further purification. All reactions involving oxygen or moisture-sensitive compounds were performed under a dry N2 atmosphere. All reactions and chromatography fractions were analyzed by thin-layer chromatography on silica gel plates, visualized with UV light and I ₂ stain.

COMPOUNDS

Example 1.

7-Carboxy-secocholest-3,5-dione-3-ethylene ketal, (Formula IV).

Ozone is bubbled through a CH2Cl2/MeOH solution (140 mL/40 mL) containing cholest-5-en-3-one ethylene ketal (4.65 g, 10.8 mmoL), the starting material prepared as in /. Org. Chem. 1952, 17, 1341, cholest-5-ene- 3-one-3-ethylene-ketal, is cooled to -78 °C until the characteristic blue color persisted (~ 5 minutes). Nitrogen is then bubbled through the solution to dissipate any excess ozone. The reaction mixture is transferred to an ice-water bath and activated zinc dust (3.55 g, 54.3 mmoL) is added. Glacial acetic acid (4 mL) is added dropwise and upon stirring an additional 45 minutes the zinc metal is removed via vacuum filtration. The bulk of the solvent is removed in vacuo , dissolved in CH2CI2 and washed with H2O. Drying over MgSθ4 and concentration afforded the

crude ketoaldehyde which is taken directly on into the next step. The crude ketoaldehyde is dissolved in 100 mL of t-butanol and then 2-methyl- 2-butene (6.82 g, 97.2 mmoL) is added. An aqueous solution (30 mL) of sodium chlorite (3.91 g, 43.2 mmoL) and sodium hydrogen phosphate monobasic (3.24 g, 27 mmoL) is added. After 30 minutes the bulk of the t- butanol is removed in vacuo and the remaining residue dissolved in CH2CI2. The organics are washed with H2O, dried (MgSθ4) and concentrated. Purification via column chromatography (eluent: hexane/ethyl acetate (3:2)) gave 7-carboxy-secocholest-3,5-dione-3-ethylene ketal as a white solid; m.p. 78-82 °C. Anal. Calcd. for C29H48O5; C, 73.07; H, 10.15. Found: C, 73.21; H, 10.19. Example 2.

6-Azacholest-4-en-3-one- (Formula I).

To a CH2CI2 solution (6 mL) containing oxalyl chloride (295 mg, 2.33 mmoL) cooled to 0 °C is added CH2CI2 solution (5 mL) containing 7- carboxy-secocholest-3,5-dione-3-ethylene ketal (616 mg, 1.30 mmoL), prepared as in example 1 and pyridine (205 mg, 2.59 mmoL) added to it. After 5h the solvent is removed in vacuo and the residue pumped on under high vacuum overnight. The resulting solid is dissolved in 8 mL of acetone, cooled to 0 °C and 3 mL of an aqueous solution of sodium azide (505 mg, 7.77 mmoL) added. After 50 minutes most of the acetone is removed in vacuo and the remaining material poured into ethyl acetate (50 mL). The organics are washed with H2O, dried (MgSθ4) and concentrated yielding the crude acyl azide (the IR showed the characteristic azide stretch at 2130 cm ^~ l). This material is taken directly into the next step. The acyl azide is dissolved in 8 mL of toluene and heated to 80 °C for 1.5h. The toluene is removed in vacuo and the residual oil pumped on overnight under high vacuum. The resulting oil is dissolved in 10 mL of THF followed by addition of 3.25 mL of a 4.0M aqueous HC1 solution. The reaction is heated to 65 °C for 2h, cooled, poured into 2N NaOH and the

organics extracted with ethyl acetate (2 X 40 mL). Drying over MgSθ4 and concentration yielded an oil which is purified via column chromatography (eluent: CH2C-2/MeOH (9:1)) affording 6-azacholest-4-en- 3-one as a white solid; m.p. 220-225 °C deco p. Anal. Calcd. for C26H43NO; C, 80.98; H, 11.24; N, 3.63. Found: C, 80.73; H, 11.30; N, 3.60.

Example 3.

Cholest-5-en-3,7-dione-3-ethylene ketal, (Formula VII).

To a 2L 4-neck flask equipped with a mechanical stirrer and addition funnel is added 61.8 g (618 mmoL) of chromium trioxide followed by 800 mL of CH2CI2. The solution is cooled to 0 °C via an ice-water bath and 3,5- dimethylpyrazole (59.4 g, 618 mmoL) is added with stirring in one portion. The dark solution is stirred for 30 minutes and then cholest-5-en-3-one ethylene ketal, (26.47 g, 61.8 mmoL), (prepared as in /. Org. Chem. 1952, 17, 1341) in 300 mL of CH2CI2 is added at a steady rate via the addition funnel. The reaction is slowly allowed to warm to room temperature and after stirring, celite and 200 mL of 2N NaOH is added. After 30 minutes the dark sludge is filtered through a large plug of florisil covered by glass wool. The florisil is washed thoroughly with CH2CI2 and then the bulk of the

CH2CI2 removed in vacuo. The residual material is poured into H2O and extracted with CH2CI2 (2X500 mL). Drying over MgSθ4 and concentration yielded a dark sludge which is passed through silica gel (eluent: CH2θ2/hexane/ethyl acetate (6:3:1) (2X)) affording cholest-5-en-3,7-dione- 3-ethylene ketal as a light yellow solid; m.p. 144-145 °C. Anal. Calcd. for C29H46O3; C, 78.68; H, 10.47. Found: C, 78.82; H, 10.51.

Example 4.

7-Methylene-cholest-5,7-dien-3-one ethylene ketal, (Formula VIII).

To a THF solution (100 mL) containing cholest-5-en-3,7-dione-3- ethylene ketal (10.0 g, 22.7 mmoL), prepared as in example 3, is added 84

mL of a 1.0M ether solution of trimethylsilylmethylmagnesium chloride at room temperature. After stirring overnight an additional 50 mL of the 1.0M ether solution of trimethylsilylmethylmagnesium chloride is added. Upon stirring for a total of 2 days the solution is cooled to 0 °C via an ice- water bath and then carefully quenched with H2O. Once quenched the mixture is poured into saturated NaHSθ4. The organic layer is separated and the aqueous layer extracted with ethyl acetate (200 mL). The combined organic layers are dried (MgSθ4) and concentrated. The residual material is purified via column chromatography (eluent: hexane/ethyl acetate (9:1)) yielding 7-methylene-cholest-5,7-dien-3-one ethylene ketal as a white solid; m.p. 142-145 °C. Anal. Calcd. for C30H48O2; C, 81.76; H, 10.98. Found: C, 81.68; H. 10.94.

Example 5.

7α,β-Methyl-cholest-5-en-3-one ethylene ketal, (Formula IX).

A flask containing 7-methylene-cholest-5,7-dien-3-one ethylene ketal (2.1 g, 4.77 mmoL), prepared as in example 4, and tristriphenylphosphine rhodium(I) chloride (442 mg, 0.48 mmoL, 10 mole%) in 70 mL of ethyl acetate is evacuated and then filled and evacuated 4X with hydrogen gas. The reaction is placed under a balloon of H2 and upon stirring overnight the heterogeneous mixture is filtered through a plug of florisil. The florisil is rinsed with ethyl acetate and concentrated. Purification via column chromatography (eluent: hexane/ethyl acetate (9:1)) gave 7α,β-methyl-cholest-5-en-3-one ethylene ketal as a 2.2:1 diastereomeric mixture; m.p. 102-105 °C. Anal. Calcd. for C30H50O2; C, 81.39; H, 11.38. Found: C, 81.48; H, 11.39.

Example 6.

7-Carboxy-7-α,β-methyl-secocholest-3,5-dione-3-ethylene ketal, (Formula X).

Ozone is bubbled through a CH2Cl2/MeOH solution (60 mL/20 mL) containing 7α,β-methyl-cholest-5-en-3-one ethylene ketal, prepared as in example 5, (1.89 g, 4.28 mmoL) at -78 °C for 8 minutes. Nitrogen is then bubbled through the solution to dissipate any excess ozone. The flask is transferred to an ice-water bath and activated zinc dust (1.4 g, 21.4 mmoL) added. Glacial acetic acid (2 mL) is added dropwise and 30 minutes later the mixture filtered to remove the zinc metal. The bulk of the solvent is removed in vacuo, the residual material dissolved in CH2CI2 and then washed with H2O. Drying over MgSθ4 and concentration yielded the crude ketoaldehyde. This material is then taken directly into the next reaction. The aldehyde is dissolved in 40 mL of t-butanol and to this solution is added 2-methyl-2-butene (2.7 g, 38.5 mmoL). Then 10 mL of an aqueous solution containing sodium chlorite (1.5 g, 17.1 mmoL) and sodium hydrogen phosphate monobasic (1.3 g, 10.7 mmoL) added. After 30 minutes the bulk of the t-butanol is removed in vacuo. The remaining material is dissolved in CH2CI2 and washed with H2O. The aqueous layer is extracted with CH2CI2 and the combined organic layers dried (MgSθ4). Concentration and purification via column chromatography (eluent: ethyl acetate/hexane/CH2θ2 (2:2:1)) afforded 7-carboxy-7-α,β-methyl- secocholest-3,5-dione-3-ethylene ketal as a white solid; m.p. 98-102 °C. Anal. Calcd. for C30H50O5; C, 73.43; H, 10.27. Found: C, 73.23; H, 10.23.

Example 7.

7α-Methyl-6-azacholest-4-en-3-one and 7β-methyl-6-azacholest-4-en-3-one, (Formula I).

To a CH2CI2 solution containing oxalyl chloride (180 mg, 1.43 mmoL) cooled to 0 °C is added a CH2CI2 solution (4 mL) containing 7- carboxy-7-α,β-methyl-secocholest-3,5-dione-3-ethylene ketal (350 mg, 0.71 mmoL) and pyridine (113 mg, 1.43 mmoL). After 5h the solvent is removed in vacuo and the residual oil dissolved in 6 mL of acetone. The solution is cooled in an ice-water bath followed by addition of an aqueous solution (2 mL) of sodium azide (279 mg, 4.28 mmoL). After 40 minutes the mixture is poured into H2O and the organics extracted with CH2CI2 (2X25 mL). Drying over MgSθ4 and concentration yielded the acyl azide (ER showed the characteristic azide stretch at 2130 cm ^~ l). The crude acyl azide is dissolved in 6 mL of toluene and heated to 80 °C for 1.5h. The toluene is removed in vacuo and the product pumped on under high vacuum overnight. The residual oil is dissolved in 7 mL of THF followed by addition 1.8 mL of a aqueous 4.0M HC1 solution. After being heated to 65 °C for lh 45 minutes the bulk of the THF is removed in vacuo. The residual oil is dissolved in ethyl acetate and washed with 2N NaOH. Following an aqueous wash and drying over MgS04 the solvent is removed in vacuo affording 7β-methyl-6-azacholest-4-en-3-one and 7a- methyl-6-azacholest-4-en-3-one as a 2.3:1 diastereomeric mixture; yield 128 mg (45%). The diastereomers are separated on a Chiral AD semiprep HPLC column (eluent: hexane (+ 0.1% diethyl amine)/ethanol (87:13)). α- Diastereomer: m.p. 204-206 °C; Anal. Calcd for C27H45NO; C, 81.14; H, 11.35; N, 3.50. Found: C, 81.00; H, 11.29; N, 3.46. β-Diastereomer: m.p. 193- 194 °C. High resolution mass spectrum Calcd. for C27H45NO: 400.3580. Found: 400.3579.

PHARMACEUTICAL FORMULATIONS

1. Transdermal System - For 1000 Patches

Ingredients Amount

Active compound (Formula (I)) 40 g Silicone fluid 450 g

Colloidal silicon dioxide 25 g

The silicon fluid and active compound are mixed together and the colloidal silicon 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 sq. cm patches.

.. Oral Tablet - For 1000 Tablets

Ingredients Amount

Active compound (Formula (I)) 20 g

Starch 20 g

Magnesium Stearate i g

The active compound and the starch are granulated with water and dried. Magnesium stearate is added to the dried granules and the mixture is thoroughly blended. The blended mixture is compressed into tablets.

3. Suppository - For 1000 Suppositories

Ingredients Amount

Active compound (Formula (I)) 25 g

Theobromine sodium salicylate 250 g

Witepsol S55 1725 g

The inactive ingredients are mixed and melted. The active compound is then distributed in the molten mixture, poured into molds and allowed to cool.

4. Injection - For 1000 Ampules

Ingredients Amount

Active Compound (Formula (I)) 5

Buffering Agents q.s. Propylene glycol 400 mg

Water for injection 600 mL

The active compound 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.

5. Capsule - For 1000 Capsules

Ingredients Amount

Active Compound (Formula (I)) 20 g

Lactose 450 g

Magnesium stearate 5 g

The finely ground active compound is mixed with the lactose and stearate and packed into gelatin capsules.