WO1995013077A1 - 7β-SUBSTITUTED-4-AZA-5α-CHOLESTAN-3-ONES AS SELECTIVE 5α-REDUCTASE 1 INHIBITORS - Google Patents

Abstract

7β-substituted 4-aza-5α-cholestan-3-ones and related compounds are selective inhibitors of 5α-reductase 1 useful in the treatment hyperandrogenic conditions.

Description

TITLE OF THE INVENTION

7β-SUBSTITUTED-4-AZA-5α-CHOLESTAN-ONES AS SELECTIVE 5α-REDUCTASE 1 INHIBITORS

This application is a continuation-in-part of International Application No. PCT/US93/04615, which was filed according to the Patent Cooperation Treaty (PCT) and has an international file date of 14 May 1993. Application No. PCT/US93/04615 is itself a continuation-in-part of U.S. Serial No. 886,023, filed 20 May 1992, now abandoned.

BACKGROUND OF THE INVENTION

The present invention is directed to new 7β-substituted-4-aza-5α-cholestan-3-ones and related compounds and the use of such compounds as selective 5α-reductase 1 inhibitors.

DESCRIPTION OF THE PRIOR ART

The art reveals that certain undesirable physiological manifestations, such as acne vulgaris, seborrhea, female hirsutism, male pattern baldness and benign prostatic hypertrophy, are the result of hyperandrogenetic stimulation caused by an excessive accumulation of testosterone (T) or similar androgenic hormones in the metabolic system. Early attempts to provide a chemotherapeutic agent to counter the undesirable results of hyperandrogenicity resulted in the discovery of several steroidal antiandrogens having undesirable hormonal activities of their own. The estrogens, for example, not only counteract the effect of the androgens but have a feminizing effect as well. Non-steroidal antiandrogens have also been developed, for example, 4'-nitro-3'-trifluoromethylisobutyranilide. See Neri, et al., Endo., Vol. 91 , No. 2 (1972). However, these products, though devoid of hormonal effects, are peripherally active, competing with the natural androgens for receptor sites, and hence have a tendency to feminize a male host or the male fetus of a female host. It is now known in the art that the principal mediator of androgenic activity in some target organs is 5α-dihydrotestosterone (DHT), and that it is formed locally in the target organ by the action of testosterone-5α-reductase. It is also known that inhibitors of

testosterone-5α-reductase will serve to prevent or lessen symptoms of hyperandrogenic stimulation.

A number of 4-aza steroid compounds are known in the art . For example, See U.S. Patent Nos. 2,227,876, 3,239,417, 3,264,301 and 3,285,918; French Patent No. 1 ,465,544; Doorenbos and Solomons, J. Pharm. Sci. 62, 4, pp. 638-640 ( 1973); Doorenbos and Brown, J. Pharm. Sci., 60, 8, pp. 1234-1235 (1971 ); and Doorenbos and Kim, J. Pharm. Sci. 63, 4, pp. 620-622 (1974).

In addition, U.S. Patent Nos. 4,377,584, 4,220,775,

4,859,681 , 4,760,071 and the articles J. Med. Chem. 27, p. 1690-1701 (1984) and J. Med. Chem. 29, 2998-2315 (1986) of Rasmusson, et al., U.S. Patent 4,845,104 to Carlin, et al., and U.S. Patent 4,732,897 to Cainelli, et al. describe 4-aza-17β-substituted-5α-androstan-3-ones which are said to be useful in the treatment of DHT-related hyperandrogenic conditions.

However, despite the suggestion in the prior art that hyperandrogenetic diseases are the result of a single 5oc-reductase, there are reports regarding the presence of other 5α-reductase isozymes in both rats and humans. For example, in human prostate, Bruchovsky, et al. (See J. Clin. Endocrinol. Metab. 67, 806-816, 1988) and Hudson (see J. Steroid Biochem. 26, p 349-353, 1987) found different 5α-reductase activities in the stromal and epithelial fractions. Additionally, Moore and Wilson described two distinct human reductases with peaks of activities at either pH 5.5 or pH 7-9. (See J. Biol. Chem. 251, 19, p. 5895-5900, 1976.)

Recently, Andersson and Russell isolated a cDNA which encodes a rat liver 5α-reductase (see J. Biol. Chem. 264 pp. 16249-55 (1989). They found a single mRNA which encodes both the liver and prostatic reductases of rats. The sequence of this rat gene was later used to select a human prostatic cDNA encoding a 5α-reductase termed "5α -reductase 1 ". (See Proc. Nat'l. Acad. Sci. 87, p. 3640-3644, 1990.)

More recently, a second, human prostatic reductase (5α-reductase 2) has been cloned with properties identified with the more abundant form found in crude human prostatic extracts. (See Nature, 354, p. 159-161 , 1991.)

Further, "Syndromes of Androgen Resistance"- The Biology of Reproduction, Vol. 46, p. 168-173 (1992) by Jean O. Wilson indicates that the 5q-reductase 1 enzyme may be associated with hair follicles.

Thus, the art supports the existence of at least two genes for

5α-reductase and two distinct isozymes of 5α-reductase in humans. The isozyme that principally interacts in skin tissues is conventionally designated as 5α-reductase 1 (or 5α-reductase type 1), while the isozyme that principally interacts within the prostatic tissues is designated as 5α-reductase 2 (or 5α-reductase type 2). See, e.g., G. Harris, et al., Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 10787-10791 (Nov. 1992).

In the treatment of hyperandrogenic disease conditions, e.g., benign prostatic hyperplasia (BPH) and/or the prevention and treatment of prostatic cancer, it would be desirable to have one drug entity which is active against both isozymes in the prostate to significantly inhibit dihydrotestosterone production. It would also be desirable to have another drug entity which is selective for inhibiting the isozyme 5α -reductase 1 associated with the scalp, for use in treating conditions of the skin and scalp, e.g., acne vulgaris, male pattern baldness and hirsutism in females. Additionally, a selective 5α-reductase 1 inhibitor could be used in combination with a 5α-reductase 2 inhibitor such as, e.g., finasteride (PROSCAR®), for therapy in the treatment of conditions such as BPH, prostatitis, and/or the prevention and treatment of prostatic cancer, and for the treatment of skin and scalp-related disorders such as acne vulgaris, seborrhea, female hirsutism, and androgenic alopecia. Therefore it is an object of this invention to provide compounds that have sufficient activity in the inhibition of 5α-reductase isozyme 1.

SUMMARY OF THE INVENTION This invention is concerned with novel 7β-substituted-4-aza-5α-cholestan-3-one compounds and processes for their preparation. The instant compounds are inhibitors of 5α-reductase, and particularly are selective for inhibiting 5α-reductase type 1.

The invention is also concerned with the use of the novel compounds either alone or in combination with a 5α-reductase 2 inhibitor, such as finasteride, or in combination with a dual inhibitor of 5oc-reductase 1 and 2, in the treatment of conditions such as acne vulgaris, androgenic alopecia, seborrhea, and female hirsutism, by topical or internal administration.

The invention is further concerned with the use of one of the novel compounds of this invention in combination with an inhibitor of 5α-reductase 2, such as finasteride, or in combination with a dual inhibitor of 5α-reductase 1 and 2, in the treatment of benign prostatic hypertrophy, prostatitis, and the prevention and treatment of prostatic cancer by internal administration.

The invention is still further concerned with pharmaceutical formulations comprising one or more of the novel compounds as active ingredient, either alone or in combination with an inhibitor of 5α-reductase 2 such as finasteride or in combination with a dual inhibitor of 5α-reductase 1 and 2.

DETAILED DESCRIPTION OF THE INVENTION

The novel 7β-substituted-4-aza-5α-cholestan-3-one

compounds of this invention have generic formula:

or a stereoisomer or a pharmaceutically acceptable salt or ester thereof, wherein:

R is selected from hydrogen, methyl, ethyl, -OH, -NH₂, and -SCH₃; the dashed lines " - - -" a and b independently represent a single bond or a double bond providing that when b is a double bond, the 5α hydrogen, Ha, is absent;

=Z is selected from:

1) oxo,

2) α-hydrogen and a β-substituent selected from:

a) C₁ -C₄ alkyl,

b) C₂-C₄ alkenyl,

c) CH2COOH,

d) -OH,

e) -COOH,

f) -COO(C₁-C₄ alkyl),

g) -OCONR¹ R² wherein R¹ and R² independently are selected from:

i) H,

ii) C₁-C₄ alkyl,

iii) phenyl, and

iv) benzyl, or

R¹ and R² together with the nitrogen atom to which they are attached represent a 5-6 membered saturated heterocycle, optionally containing one other heteratom selected from -O-, -S- and -N(R')- wherein R' is -H or methyl;

h) C₁-C₄ alkoxy,

i) C₃-C₆ cycloalkoxy,

j) -OC(O)-C _{1 -4} alkyl,

k) halo,

l) hydroxy -C₁ -C₂ alkyl,

m) halo-C₁ -C₂ alkyl,

n) -CF₃, and

o) C₃-C₆ cycloalkyl;

3) =CHR³; wherein R³ is selected from -H and C₁-C₄ alkyl; and 4) spirocyclopropane-R³ of structure:

The 17-substituent cholestane side chain is in the beta configuration. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

The term "C₁-C₄ alkyl" as used herein, is meant to include methyl (Me), ethyl (Et), propyl (Pr), iso-propyl (i-Pr), n-butyl (n-Bu), sec-butyl (s-Bu), iso-butyl (i-Bu) and tert-butyl (t-Bu).

The term " C₂-C₄ alkenyl" as used herein is meant to inclu_de vinyl, allyl, 1-propen-1-yl, 1-propen-2-yl, 1-buten-1-yl, 1 -buten-2-yl, and the like. Included in this invention are all E, Z diastereomers.

The term "C₃-C₆ cycloalkyl" as used herein is meant to include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term "halo" as used herein is meant to include fluoro, chloro, bromo, and iodo.

The term "OC₁-C₄ alkyl" or "C₁ -C₄ alkoxy" as used herein is meant to include methoxy, ethoxy, propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy.

The term "OC₃-C₆ cycloalkyl" or "C₃-C₆ cycloalkoxy" as used herein is meant to include: cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

Representative examples of =Z are where the α-substituent (dashed lines) is hydrogen and the β-substituent (wedge) is, e.g., methyl, ethyl, propyl, allyl, carboxy methyl, hydroxy, methoxy, ethoxy,

cyclopropyloxy, cyclopentyloxy, acetoxy, fluoro, chloro, bromo, trifluoromethyl, fluoromefhyl, chloromefhyl, carboxy, N,N-dimethylcarbamate, hydroxymethyl, and the like.

Representative examples where =Z is an alkenyl substituent, =CH-R³, includes =CH₂, =CH-CH₃, =CH-CH₂CH₃, and the like.

Representative examples wherein =Z is the spirocyclopropyl substituent:

stereoisomers thereof and the like.

Representative examples wherein -NR¹R² represent a heterocycle include: N-piperidinyl, N-morpholinyl, N-piperazinyl, N-(4-methyl)piperazinyl, N-thiomorpholinyl, N-pyrrolidinyl, N-imidazolidinyl and the like.

Representative compounds included in the invention wherein all of the 17- ssbstituents are in the beta configuration are:

7β-ethyl-4-methyl-4-aza-cholest-5-en-3-one,

7β-ethyl-4-methyl-4-aza-cholestane-3-one,

7β-ethyl-4-aza-cholest-5-en-3-one,

7β-ethyl-4-aza-5α-cholestan-3-one,

7β-carboxymethyl-4-aza-cholest-5-en-3-one,

7β-carboxymethyl-4-aza-cholestan-3-one,

7β-propyl-4-methyl-4-aza-cholest-5-en-3-one,

7β-propyl-4-methyl-4-aza-5α-cholestan-3-one,

7β-propyl-4-aza-cholest-5-en-3-one,

7β-propyl-4-aza-5α-cholestan-3-one,

7β-methyl-4-aza-cholest-5-en-3-one,

7β-methyl-4-aza-cholestan-3-one,

4,7β-dimethyl-4-aza-cholest-5-en-3-one,

4,7β-dimethyl-4-aza-5α-cholestan-3-one,

4-methyl-4-aza-5α-cholestan-3,7-dione,

7β-acetoxy-4-methyl-4-aza-5α-cholestan-3-one,

4-methyl-4-aza-cholest-5-en-3,7-dione,

7β-hydroxy-4-methyl-4-aza-5α-cholestane-3-one,

7β-methoxy-4-methyl-4-aza-5α-cholestane-3-one,

7β-hydroxymethyl-4-aza-5α-cholestane-3-one,

7β-bromomethyl-4-aza-5α-cholestane-3-one, 7β-chloromethyl-4-aza-5α-cholestane-3-one,

7β-fluoromethyl-4-aza-5α-cholestane-3-one,

7β-carboxy-4-aza-5α-cholestane-3-one,

7β-trifluoromethyl-4-aza-cholest-5-en-3-one,

7,7-dimethoxy-4-methyl-4-aza-5α-cholestane-3-one,

7β-methoxy-4-methyl-4-aza-cholesta-5-en-3-one,

7β-methoxy-4-methyl-4-aza-cholesta-6-en-3-one,

7β-cyclopropyloxy-4-methyl-4-aza-5α-cholestane-3-one,

7β-cyclopropyloxy-4-methyl-4-aza-cholesta-5,7-dien-3-one,

7β-propylidene-4-methyl-4-aza-5α-cholestane-3-one,

7β-(2-ethyl)spiroethylene-4-methyl-4-aza-5α-cholestane-3-one,

7β-methyl-4-aza-5α-cholest-1-en-3-one,

7β-methyl-5-oxo-A-nor-3,5-seco-cholestanoic acid,

7β-ethyl-5-oxo-A-nor-3,5-seco-cholestanoic acid,

7β-propyl-5-oxo-A-nor-3,5-seco-cholestanoic acid,

7β-i-propyl-5-oxo-A-nor-3,5-seco-cholestanoic acid,

7β-n-butyl-5-oxo-A-nor-3,5-seco-cholestanoic acid,

7β-i-butyl-5-oxo-A-nor-3,5-seco-cholestanoic acid,

7β-s-butyl-5-oxo-A-nor-3,5-seco-cholestanoic acid,

7β-t-butyl-5-oxo-A-nor-3,5-seco-cholestanoic acid,

7β-n-pentyl-5-oxo-A-nor-3,5-seco-cholestanoic acid, and

7β-n-hexyl-5-oxo-A-nor-3,5-seco-cholestanoic acid.

Also included within the scope of this invention are pharmaceutically acceptable salts of the compounds of formula I, where a basic or acidic group is present on the structure. When an acidic substituent is present, i.e. -COOH, there can be formed the ammonium, sodium, potassium, calcium salt, and the like, for use as the dosage form. Where a basic group is present, i.e. amino or a basic heteroaryl radical such as, e.g., 4-pyridyl, an acidic salt, i.e. hydrochloride, hydrobromide, acetate, pamoate, and the like, can be used as the dosage form.

Also, in the case of the -COOH group being present, pharmaceutically acceptable esters can be employed, e.g. acetate, maleate, pivaloyloxymethyl, and the like, and those esters known in the art for modifying solubility or hydrolysis characteristics for use as sustained release or prodrug formulations.

Representative salts include the following salts: 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, hydrochloride, tartrate,

hydroxynaphthoate, teoclate, Iodide, tosylate, isothionate, triethiodide, lactate, and valerate.

In addition, some of the compounds of the instant invention may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of this invention.

The compounds of the present invention have asymmetric centers and may occur as racemates, racemic mixtures and as individual enanti omens or diastereomers, with all isomeric forms being included in the present invention as well as mixtures thereof. Furthermore, some of the crystalline forms for compounds of the present invention may exist as polymorphs and as such are intended to be included in the present invention.

The term "therapeutically effective amount" shall mean that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

The compounds of this invention can be made by procedures outlined in the following Flowsheets. All temperatures are in degrees Celsius.

7-Beta Alkyl Series

The compounds of the instant invention comprising Z as a 7β alkyl group, e.g., methyl, ethyl, isopropyl, allyl, can be prepared by the procedure outlined in The General Flowsheet.

As seen in the Flowsheet, the starting 3-acetoxy-cholest-5-ene I (see Example 1 for synthesis) is oxidized to the corresponding 5-en-7-one II by treatment with hydrogen t-butyl peroxide and chromium hexacarbonyl in, e.g., acetonitrile at reflux. The C₁-C₄ alkyl group, designated Alk, e.g., methyl can be introduced at this point by a Grignard reaction using, e.g., alkyl magnesium chloride in, e.g., anhydrous tetrahydrofuran (THF) at 0-23°C to produce the 7-alkyl-7-hydroxy adduct III. This is then oxidized with, e.g., aluminum isopropoxide and cyclohexanone (Oppenauer oxidation conditions) in refluxing toluene solvent to produce the 7-alkyl-4,6-dien-3-one IV. This in turn is reduced via a, e.g., metal-ammonia reduction, using lithium, liquid ammonia, THF and toluene at -78°C, quenching the reaction with dibromoethane and ammonium chloride, to selectively yield the 7-beta-alkyl-5-en-3-one V. In the next step the delta-5 double bond is isomerized to the 4-ene by use of DBU (1 ,8-diazabicyclo[5.4.0]undec-7-ene) in, e.g., refluxing THF to produce the 7-beta-alkyl 4-en-3-one, VI. The A Ring is next cleaved by treatment with, e.g., potassium permanganate, sodium periodate in t-butyl alcohol at 80°C to produce the corresponding seco-acid VII.

Treatment of the seco-acid with an appropriate amine, e.g., methylamine hydrochloride and sodium acetate in ethylene glycol at 180°C, yields, e.g., the 4-methyl-4-aza-cholest-5-en-3-one VIII. This in turn is selectively reduced with, e.g., PtO₂ catalyst in a hydrogen atmosphere, to remove the 5-position double bond to produce the 5α-hydrogen

compound IX. The seco-acid VII can be similarly treated with

ammonium acetate in acetic acid (HOAc) to produce the corresponding N-H compound, X, which can then be analogously treated with PtO₂ in a catalytic hydrogenation to produce the corresponding 5α-4N-H

compound XI. Similarly, use of hydroxylamine or hydrazine for ring A closure of the seco acid will afford the corresponding delta-5-4N-X compounds where -X can be -OH or -NH₂, respectively. Reaction of the anion of saturated 4N-compound (generated from the NH precursor by NaH treatment) with methylsulfenyl chloride can provide the

corresponding 4N-X compound where -X is -SCH₃. Thus, R can also be -OH, -NH₂ or SCH₃ in the Formula.

7-Beta-Ethyl-Cholestane Analogues

The 7-ethyl substituent is introduced into the cholestane series as illustrated in Flowsheets C and D by the same analogous procedure as described in the General Flowsheets.

The starting cholesteryl acetate CA is available commercially (Aldrich). This is treated using the analogous chromium hexacarbonyl/-hydrogen t-butylperoxide/acetonitrile oxidation procedure (described in JCS Perkin Trans. 1985, p. 267 by A. J. Pearson) to yield the 3-acetoxy-cholest-5-en-7-one 1. This can be reacted with an alkyl Grignard reagent, e.g., ethyl magnesium chloride to form the adduct 2. This is oxidized under Oppenauer conditions to yield the dienone 3, which then can undergo metal-ammonia reduction to yield the 7β-ethyl-5-en-3-one, 4. This is isomerized using DBU to the 4-en-3-one, 5, which is oxidized to open Ring A to yield the seco-acid 6. This can be treated with amines, e.g., methylamine, to yield the A-ring closed 4-methyl-4-aza compound 7. This in turn can be catalytically hydrogenated to yield the 7-ethyl-5-alpha-4-methyl-4-aza-cholestan-3-one, 8.

Similarly, by treatment of the seco-acid 6 with ammonium acetate/acetic acid, the corresponding 4-NH analog 9, is produced which can be catalytically hydrogenated to yield the 7-beta-ethyl-5α-4-aza-cholestan-3-one, 10.

Following the same procedure but using phenylmagnesium chloride as the Grignard reagent, the corresponding compounds 50 and 51 are produced ("Ph" represents phenyl).

7-Carboxymethyl-Cholestane Series

The 7-carboxy substituent is formed through the corresponding 7-allyl group. As seen in Flowsheet C, 7-oxo-cholesteryl acetate 1 is reacted with allyl Grignard reagent to form the adduct 11 which is oxidized to the dienone 12 by Oppenauer conditions. Metal-ammonia reduction affords the 5-ene analog 13, followed by DBU-catalyzed double bond isomerization to 14. This in turn can be oxidized in a key step to form the 7-carboxymethyl seco-acid 15. Treatment with amines, e.g., ammonia, forms the 4-aza derivative, 16 which is then reduced to the cholestane 17. Use of methylamine in place of ammonia can yield the corresponding 4-methyl analogs of 16 and 17.

7-Propyl-Cholestane Series

The 7-propyl analogs are made starting with the 7-allyl-4-en-3-one 14, which is reduced by hydrogenation using Wilkinson's catalyst to the propyl derivative 18, oxidized to the seco-acid 19, then condensed with amines, e.g., methylamine, to form the 4-methyl analog 20 and then reduced to the cholestane 21. Corresponding treatment with ammonia is shown in Flowsheet E shows the corresponding unsubstituted 4-aza 22 and cholestane 23 analogs.

7-Beta Methyl Cholestane Series

The 7-beta methyl cholestane series is prepared by the analogously same route as described in Flowsheets A and B for the ethyl derivatives.

The methyl Grignard reagent is used to form the adduct 24, followed by Oppenauer oxidation to form 25, metal-ammonia reduction to form 26, double bond isomerization to form 27, seco-acid oxidation to form 28, and treatment by an ammonium salt to form 29, and reduction to form 30. Corresponding treatment with methylamine produces the corresponding 4-methyl-4-aza compounds, 31 and by reduction, 32.

7-BETA ACETOXY CHOLESTANE SERIES

The 7-beta acetoxy series is prepared by the oxidation of starting 33 to the 5-en-7-one 34 by the chromium hexacarbonyl procedure described for 1, or by pyridine-dichromate/t-butyl hydroperoxide oxidation as described in the Examples. Subsequent noble metal, e.g., platinum, ruthenium, catalyzed reduction of 34 yields two products, the reduced 7-oxo compound 35, and 7-beta hydroxy compound 36.

Acylation of 36 with acetic anhydride (Ac₂O) yields the 7-beta acetoxy compound 37. DMAP is dimethylaminopyridine; Py is pyridine; Ac is acetyl.

The 7-beta ethers in the cholestane series are prepared from the 7-beta-ol (7-beta hydroxy derivative). As illustrated in Flowsheet J, the 4-N-methyl-7-beta ol 36 can be reacted with, e.g., methyl iodide and sodium hydride in, e.g., dimethylformamide, to produce the

corresponding methyl ether 37. The other C₁ -C₄ ethers can be prepared in the same manner.

The C₃-C₆ cycloalkyl ethers can be prepared according to the analogous procedure of Steroids, 1972, vol. 19, pp. 639-647 by R. Gardi, et al. For example, 36 can be reacted with 1 ,1-dimethoxycyclohexane to produce the enol ether 38, which can be reduced to the corresponding saturated compound by the use of palladium catalyzed hydrogenation.

The 7-haloalkyl series is made by the procedure illustrated in Flowsheet K.

Starting with the 7-beta-carboxy, 45, this can be treated under Hunsdiecker reaction conditions, i.e. bromination of a mercury metal salt, to yield the 7-bromo derivative 40. The chloro and iodo derivatives can be made in substantially the same fashion.

The haloethyl compounds can be made by starting with the 7-carboxymethyl analog 17 which can be reacted with a reducing agent, e.g. borane, to produce the primary alcohol 41. This in turn can be reacted with triphenylphosphine and carbon tetrabromide to produce the bromoethyl derivative 42.

The halomethyl compounds can be produced starting with the carboxymethyl derivative 17. This is treated with lead tetraacetate under oxidative decarboxylation/halogenation conditions, with a chloride, bromide or iodide salt to yield, e.g. the 7-chloromethyl analog 43. The carboxymethyl compound 17 can be treated with a fluorinating agent (XeF₂) to yield the 7-fluoromethyl analog 44.

The 7-trifluoromethyl derivative can be made from the 7-carboxy derivative 45, by conventional Dast halogenation conditions using SF₄ to yield the 7-trifluoromethyl analog 46.

Flowsheet N illustrates the 7-methylene series. As seen, the Wittig reaction, using e.g. Ph₃PCH(CH₂CH₃), carried out on the 7-oxo compound 35, leads to the 7-(ethyl)methylene compound 47.

Subsequent treatment of 47 with the cyclopropyl forming reagents, CH₂I₂ and zinc, produces the ethyl cyclopropyl spiro compound 48, which is a mixture of stereoisomers.

Flowsheet O illustrates the synthesis of the 1-ene 7-substituted analogs. For example compound 30 is stirred with DDQ, BSTFA (bis-trimethylsilyltrifluoroacetamide) and trifluoromethyl sulfonic acid in toluene at room temperature for 24 hours, methyl acetoacetate is added and the mixture reluxed for 24 hours and purified by preparative thin layer chromatography on silica gel using 3:1 chloroform/acetone to yield 49.

Another embodiment of this invention is the use of one of the novel compounds of this invention in a method of prevention and/or treatment of hyperandrogenicity through the activity of the 5oc-reductase 1 isozyme.

The activity of the novel compounds as selective 5α-reductase 1 inhibitors is determined by the following Biological assays: Biological Assays

Preparation of Human prostatic and scalp 5oc-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

phenylmethylsulfonyl fluoride, 1 mM dithiothreitol (DTT) containing 0.25 M sucrose using a Potter-Elvehjem homogenizer. A crude nuclear pellet was prepared by centrifugation of the homogenate at 1 ,500xg 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.

5α-reductase assay

The reaction mixture for the type 1 5oc-reductase contained 40 mM potassium phosphate, pH 6.5, 5 μM [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 μM [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 655A autosampler, Applied Biosystems Model 757 variable UV detector, and a Radiomatic Model A 120 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. IC₅₀ values represent the concentration of inhibitor required to decrease enzyme activity to 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.

A compound referrred to herein as a 5α-reductase 2 inhibitor is a compound that shows inhibition of the 5oc-reductase 2 isozyme in the above-described assay, having an IC₅₀ value of about or under 100nM.

A compound referrred to herein as a dual 5α-reductase 1 and 2 inhibitor is a compound that shows inhibition of both the 5α-reductase 1 and 2 isozymes in the above-described assay, having an IC₅₀ value for each of type 1 and type 2 of about or under 100nM.

The present invention has the objective of providing methods of treating the hyperandrogenic conditions of androgenic alopecia including female and male pattern baldness, acne vulgaris, seborrhea, and female hirsutism by oral, systemic, parenteral or topical administration of a therapeutically effective amount of one or more novel compounds of formula I optionally in combination with a 5α-reductase 2 inhibitor or with a dual inhibitor of both 5α-reductase 1 and 2. The term "treating androgenic alopecia" is intended to include the arresting and/or reversing of androgenic alopecia, and the promotion of hair growth.

The present invention has the further objective of providing methods of treating benign prostatic hyperplasia, prostatitis, and treating and/or preventing prostatic carcinoma by oral, systemic or parenteral administration of a therapeutically effective amount of one or more novel compounds of formula I optionally in combination with a 5α-reductase 2 inhibitor or in combination with a dual inhibitor of both 5α-reductase 1 and 2.

In the methods described above, the daily dosage of the compounds of formula I may be varied over a wide range from 0.1 mg to 1 ,000 mg per adult human/per day. An effective amount of one of the novel compounds of this invention is ordinarily from about 0.002 mgs/kg to 50 mgs./kg. of body weight per day, and more particularly the range is from about 0.01 mgs/kg to 7 mgs/kg of body weight per day. The daily dosage of the 5α-reductase 2 inhibitor, e.g., finasteride, is from about 0.01 mg to 50 mg per adult human/per day, and more particularly from about 0.2 mg to 5 mg per adult human/per day. An effective amount of the 5α-reductase 2 inhibitor, e.g., finasteride, is ordinarily from about 0.00015 mgs/kg to about 0.7 mgs/kg of body weight per day, and more particularly the range is from about 0.002 mgs/kg to 0.07 mgs/kg of body weight per day. Effective amounts of a dual 5α-reductase 1 and 2 inhibitor would be in the same ranges as the described above for the compounds of formula I.

The present invention also has the objective of providing suitable systemic, oral, parenteral and topical pharmaceutical

formulations for use in the novel methods of treatment of the present invention. The compositions containing the active ingredient, for use in the treatment of the above-noted hyperandrogenic conditions can be administered in a wide variety of therapeutic dosage forms in

conventional vehicles for systemic administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release

formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the

pharmaceutical arts. For oral administration, for example, the

compositions can be provided in the form of scored or unscored tablets containing 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredients for the symptomatic adjustment of the dosage to the patient to be treated.

For the treatment of androgenic alopecia including female and male pattern baldness, acne vulgaris, seborrhea, and female

hirsutism, the compounds also may be administered in a pharmaceutical composition comprising the active compound in combination with a pharmaceutically acceptable carrier adapted for topical administration. Topical pharmaceutical compositions may be, e.g., in the form of a solution, cream, ointment, gel, lotion, shampoo or aerosol formulation adapted for application to the skin. Topical pharmaceutical compositions useful in the method of treatment of the present invention may include about 0.001% to 15% by weight of the active compound in admixture with a pharmaceutically acceptable carrier.

For the treatment of acne vulgaris, androgenic alopecia, seborrhea, female hirsutism, benign prostatic hyperplasia, prostatitis and the prevention and/or treatment of prostatic cancer, the compounds of the instant invention can be combined with a therapeutically effective amount of a 5α-reductase 2 inhibitor, such as finasteride, or a dual 5α-reductase 1 and 2 inhibitor, in a single oral, systemic, or parenteral pharmaceutical dosage formulation. Alternatively, a combined therapy can be employed wherein the compound of formula I and the 5α-reductase 2 inhibitor or the dual inhibitor are administered in separate oral, systemic, or parenteral dosage formulations. Also, for the skin and scalp related disorders of acne vulgaris, androgenic alopecia including male pattern baldness, seborrhea, and female hirsutism, the compounds of the instant invention and a 5α-reductase 2 inhibitor or a dual inhibitor can be formulated for topical administration. For example, a compound of formula I and finasteride can be administered in a single oral or topical dosage formulation, or each active agent can be administered in a separate dosage formulation, e.g., in separate oral dosage formulations, or an oral dosage formulation of finasteride in combination with a topical dosage formulation of a compound of formula I. See, e.g., U.S. Patent No.'s 4,377,584 and 4,760,071 which describe dosages and formulations for 5α-reductase inhibitors.

Furthermore, for the treatment of acne vulgaris and/or androgenic alopecia, a combined therapy can be used by administering a therapeutically effective amount of a compound of formula I in

combination with a therapeutically effective amount of retinoic acid or a derivative thereof, e.g., an ester or amide derivative thereof, such as, e.g., tretinoin or isotretinoin.

Also, for the treatment of acne vulgaris, androgenic alopecia, seborrhea, female hirsutism, benign prostatic hypeφlasia, prostatitis and the prevention and/or treatment of prostatic cancer, a combined therapy can be used by administering a therapeutically effective amount of a compound of formula I with a therapeutically effective amount of an anti-androgen, such as, e.g., flutamide, spironolactone or casodex.

For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concomitantly, or they each can be

administered at separately staggered times.

Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. The compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a

transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter, arrest or reverse the progress of the condition.

Optimal precision in achieving concentration of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a

consideration of the distribution, equilibrium, and elimination of a drug.

In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of

administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Capsules containing the product of this invention can be prepared by mixing an active compound of the present invention with lactose and magnesium stearate, calcium stearate, starch, talc, or other carriers, and placing the mixture in gelatin capsule. Tablets may be prepared by mixing the active ingredient with conventional tableting ingredients such as calcium phosphate, lactose, corn starch or magnesium stearate. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be

incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.

Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

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

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

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

The compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone,

polyhydroxy butyric acid, polyorthoesters, polyacetals,

polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

The following examples are illustrative of representative embodiments of this invention and should not be construed to be limits on the scope or spirit of the instant invention.

The Rf values cited were carried out on standard thin layer chromatographic Si gel plates. The elution solvent system used is given in the parentheses following the Rf value.

The fast atom bombardment (FAB) mass spectral values are reported as (M+1 ) molecular ion peaks, being the molecular weight plus one atomic mass unit. The electron impact (El) mass spectrum values are reported as molecular ion peaks and are indicated in parentheses, either being (M) or (M+2), the molecular weight, MW, or the MW plus two atomic units. The nuclear magnetic resonance data was taken at 400 MHz in CDCl₃ and is tabulated for unique proton values of each compound at the end of the Examples. The coupling constant J is given in Hertz, Hz. EXAMPLE 1

Synthesis of 7-Oxo-Cholesterol-3-acetate. (1 )

Cholesteryl acetate (CA) is known in the art and can be oxidized to the known 7-oxo-derivative 1 by the analogous procedure described in the JCS Perkins article by Pearson, supra.

EXAMPLE 2

Synthesis of 7-Ethyl-7-Hydroxy-cholesterol. (2)

To a solution of 1 from Example 1 , being 5.0 g (1 1.32 mmol) in dry tetrahydrofuran at 0°C was added dropwise 56.6 ml ethyl magnesium bromide (1M) over 5-10 minutes. The reaction mixture was then allowed to stir at room temperature for 24 hours, then poured into saturated aqueous ammonium chloride. The THF solvent was removed under vacuum and the aqueous phase extracted with ethyl acetate. The organic layer was washed with brine, dried, concentrated to yield a yellowish-white foam. The Rf value was 0.2 (30% EtOAc/hexane).

Proton NMR confirmed the assigned structure of the title compound 2 which was used in the next step without further purification.

EXAMPLE 3

Synthesis of 7-Ethyl-Cholest-4,6-Dien-3-one, (3)

The above Grignard product 2, 5.13 g (1 1.9 mmol) was dissolved in 50 ml toluene and cyclohexanone and about 40 ml of solvent distilled off under vacuum. To this was added 7.2 g aluminum

isopropoxide and the reaction mixture refluxed overnight for 15 hours. The mixture was cooled, diluted with ethyl acetate, washed with sodium potassium tartarate, brine, and the organic layer was concentrated under vacuum and the residue steam distilled. The residue was extracted with ethyl acetate, the ethyl acetate layer, washed with brine, dried and purified by column chromatography on silica gel, eluting with 5%

EtOAc/hexane to yield the title compound 3. Rf=0.58 (20%

EtOAc/hexane). Mass spec: 412(M=1) by FAB, Calc'd. 41 1.9.

EXAMPLE 4

Synthesis of 7β-ethyl-cholest-5-en-3-one, (4)

To a solution of 3.1 g of 3, from Example 3, in 46 ml ammonia, 10 ml THF, 10 milliliters toluene, was added 449 mg of metallic lithium in small pieces. After stirring the blue solution for 2 hours at -78°C, a solution of 1,2-dibromethane in 2 ml THF was added. After stirring the solution at -78°C for 10 minutes, 2.1 g of ammonium chloride was added and the mixture stirred for 10 minutes. The excess ammonia was removed by evaporation under a nitrogen stream. The reaction mixture was diluted with brine, extracted with ethyl acetate. The organic layer was washed with brine, dried and concentrated to yield crude brown viscous liquid 4 which was used as such in Example 5.

Rf=0.70 (20% EtOAc/hexane). Mass Spec. 412 (El); calculated MW 412.70.

EXAMPLE 5 Synthesis of 7β-ethyl-cholest-4-en-3-one, (5)

To a solution of 4, from Example 4, being 3.1 g in 30 ml THF was added 1.1 ml DBU (1,8-diazabicyclo[5.4,0]undec-7-ene under nitrogen with stirring. The mixture was refluxed for 1.5 hours, then cooled and diluted with NH4CL Then THF solvent was removed under vacuum and the residue extracted with ethyl acetate. The organic layer was then washed with water, brine, dried and concentrated under reduced pressure to yield a crude viscous oil. The titled product 5 was purified by chromatography on silica gel using 10% EtOAc/hexane as eluant. Mass Spec 412 (El), calc'd MW 412.70. Rf=0.6 (20% EtOAc/hexane). EXAMPLE 6

Synthesis of 7-ethyl-17β-(6-methyl-2-heptyl)-5-oxo-A-nor-3,5-secoandrostan-3-oic acid, (6)

To a solution of 1.0 g of 5 in 18 ml t-butyl alcohol at 80°C was added 300 mg sodium carbonate in 1.8 ml water followed by a dropwise addition over 15-20 minutes of a mixture of 2.74 g sodium periodate with 20.3 mg potassium permanganate in 15 ml water. The reaction mixture was heated at 80°C for 2 hours, cooled, filtered, the residue washed with water, and then the filtrate concentrated under vacuum, acidified with aqueous HCl, extracted with ethyl acetate and the organic layer washed with aqueous NaHSO₃, brine, dried and

concentrated to yield crude 6. The proton NMR confirmed the assigned structure. Fast atom bombardment yielded an m/z molecular ion of 434(m+2); calculated 432.69.

EXAMPLE 7 Synthesis of 7-Ethyl-4-methyl-4-aza-cholest-5-en-3-one, (7)

To a solution of 6, 500 mg in 10 ml ethylene glycol was added 1.3 g sodium acetate and 1.0 g methylamine hydrochloride. After stirring the reaction mixture 4 hours at 180°C, the mixture was cooled, diluted with water, extracted with ethyl acetate, dried and concentrated to afford crude title compound 7. Proton NMR confirmed the assigned structure. Rf=0.70 (20% EtOAc/hexane).

Mass Spectral m/z ion (FAB) showed 429

(M+2). calculated, 427.72.

Analysis: Calc. for C₂₉H₄₉ NO

Calc: C; 81.44; H, 1 1.55; N, 3.27

Found: C, 82.19; H, 10.92; N, 3.1 1. EXAMPLE 8

Synthesis of 7-Ethyl-4-methyl-4-Aza-Cholestan-3-one, (8)

To a solution of 7 from Example 7, being 180 mg in 5 ml acetic acid was added 54 mg platinum dioxide and the resulting mixture was evacuated and flushed with hydrogen. The reaction was shaken overnight at room temperature under hydrogen. Filtered, washed solid with EtOAc, combined EtOAc layers were washed with aqueous

NaHCO₃, brine, dried, concentrated to yield the title compound 8.

Mass spectral analysis by FAB yielded m/z ion of 431 (m+2), calculated 429.74.

Analysis for C₂₉ H_{5 1} NO

Calc : C, 81.06; H, 1 1.96, N, 3.26

Found: C, 81.42; H, 12.24; N, 3.16

EXAMPLE 9

Synthesis of 7-Ethyl-4-Aza-Cholest-5-en-3-one. (9)

The seco acid 6, 0.5 g. and ammonium acetate, 0.5 g., in 3.5 ml acetic acid were refluxed for 3 hours. The reaction mixture was cooled, water added and then extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated to yield a residue which was eluted on a silica gel column with 10% EtOAc/hexane to give pure title compound 9, mp. 147-149°C.

Mass Spec. 414 (Mt1). Calc'd; 413.69. Rf=0.45 (30% EtOAc/hexane).

Analysis for C₂₈H₄₉ NO, MW 413.69

Calc: C, 81.30; H, 1 1.45; N, 3.39

Found: C, 81.30; H, 1 1.87; N, 3.45 EXAMPLE 10

Synthesis of 7β-Ethyl-4-aza-5α-cholestan-3-one. (10)

Following the general analogous procedure described in Example 8, 9 was catalytically hydrogenated to yield the titled compound, 10. Chromatography on silica gel with 50% EtOAc:hexane eluant yielded pure product, mp. 169-170°C.

Analysis for C₂₈H₄₉NO, MW=415.17.

Calcd: C; 80.90; H, 11.88; N, 3.37

Found: C; 81.02; H, 12.57; N, 3.47.

Mass Spec: 416 (M+l)

Rf=0.30 (30% EtOAc/hexane).

EXAMPLE 11

Synthesis of 7-Allyl-3,7-dihydroxy-cholest-5-ene, (1 1)

Following the analogous general Grignard procedure of Example 2, allyl magnesium bromide was reacted with Compound 1 in dry THF to yield the titled product 11. Proton NMR confirmed the as signed structure.

Mass Spec 441 (M+1). Calc'd. 440.71. Rf=0.25 (30% EtOAc/hexane).

EXAMPLE 12 Synthesis of 7-allyl-cholest-4.6-dien-3-one. (12)

Following the analogous general Oppenauer oxidation procedure of Example 3, compound 11 was oxidized to yield the titled compound 12. Proton NMR confirmed the assigned structure as well the (FAB) mass spec. 423 (M+1) Calc'd. 422.35. Rf=0.78 (30%

EtOAc/hexane).

EXAMPLE 13

Synthesis of 7-Allyl-cholest-5-en-3-one, (13)

Compound 12, was subjected to the analogous metal-ammonia reduction conditions of Example 4 to yield the title compound 13.

Rf=0.5 (5% EtOAc/hexane). EXAMPLE 14

Synthesis of 7-Allyl-Cholest-4-en-3-one, (14)

Following the general DBU catalyzed isomerization conditions of Example 5, compound 13 was analogously treated to yield the title compound 14.

Mass Spec. 425 (M+1) by FAB. Calc'd.: 424.37

Rf=0.45 (5% EtOAc/hexane). EXAMPLE 15

Synthesis of 7-Propyl-cholest-4-en-3-one, (18)

1.0 g. of the 7-allyl-enone 14, 5 ml. EtOAc and 50 mg.

triphenylphosphine rhodium chloride (Wilkinson's catalyst) were allowed to stir two hours (under H₂ atmosphere). The reaction products were filtered through 25 ml. silica gel, and evaporated to dryness to yield fairly pure title product, 18, as confirmed by proton NMR.

Mass Spec. 427 (M+1). Calc'd.: 426.39 Rf=0.15 (5% EtOAc/hexane). EXAMPLE 16

Synthesis of 7-Propyl-5-oxo-A-nor-3,5-seco-cholestanoic acid. (19)

Following the general procedure of Example 6 for the oxidative Ring A cleavage, compound 18 (7-propyl analogue) was analogously treated to yield the above-titled seco-acid 19. The assigned structure was confirmed by proton NMR. Mass Spec: 447 (M+1) (FAB). Calc'd.: 446.38 Rf=0.1 (20% EtOAc/hexane).

EXAMPLE 17

Synthesis of 7-Propyl-4-methyl-4-aza-cholest-5-en-3-one, (20)

Following the general procedure of Example 7, compound 19, was analogously treated with methylamine hydrochloride and sodium acetate in ethylene glycol to yield the above-titled liquid product 20. The assigned structure was confirmed by proton NMR.

Mass Spec. 442 (M+1) (FAB), Calc'd.: 441.74 C,H,N analysis for C H N

O as 0.2 H20, MW=441.74;

Calcd: C, 80.91; H, 1 1.63; N, 3.15.

Found: C, 81.00; H, 12.06; N, 2.93.

Rf=0.3 (20% EtOAc/hexane).

EXAMPLE 18

Synthesis of 7-Propyl-4-methyl-4-aza-5α-cholestan-3-one, (21 )

Following the analogous general procedure of Example 8, compound 20 was catalytically hydrogenated in HOAc to yield the title liquid compound 21. Proton NMR confirmed the assigned structure. Mass spec. 444 (M+1) (FAB), C,H,N analysis for C H N;

Calcd: C, 81.19; H, 12.05; N, 3.16. MW=443.41.

Found: C, 80.78; H, 12.06; N, 3.22.

Rf=0.17 (20% EtOAc/hexane). EXAMPLE 19

Synthesis of 7-Propyl-4-aza-cholest-5-en-3-one, (22)

Following the analogous procedure of Example 9,

compound 19 was treated with ammonium acetate in acetic acid to yield the titled compound, 22. Recrystalhzed from EtOAc/Et₂O to yield a white crystalline solid, mp. 91-94°C, C,H,N analysis as the 0.25 H₂O hydrate: Calc'd MW 427.39

Calcd: C, 80.59; H, 1 1.54; N, 3.24.

Found: C, 80.59; H, 1 1.69; N, 3.36.

Mass Spec. 428 (M+1 ). EXAMPLE 20

Synthesis of 7-Propyl-4-aza-5α-cholestan-3-one, (23)

Following the analogous procedure described in Example 8, compound 22 was catalytically hydrogenated to yield the title compound

23, mp. 65-68°C.

Analysis for C,H,N, calc'd as 0.25 H2θ hydrate:

Calcd: C, 80.21; H, 1 1.95; N, 3.23.

Found: C, 80.20; H, 12.14; N, 3.07.

Mass Spec. = 430 (M+1 ) calc'd MW 429.40.

Rf=0.12 (20% EtOAc/hexane).

EXAMPLE 21 Synthesis of 7-Methyl-7-Hydroxy-cholesterol, (24)

Following the analogous Grignard procedure of Example 1 , cholesteryl acetate-7-one 1 was reacted with methyl magnesium bromide under standard Grignard conditions to yield title compound 24, a solid. NMR confirmed the assigned structure and mass spectral analysis confirmed the molecular weight.

EXAMPLE 22

Synthesis of 7-Methyl-Cholest-4,6-Dien-3-one, (25)

Following the analogous procedure of Example 2, the above

Grignard product 24, was subjected to Oppenauer oxidation conditions to yield the title compound, 7β-methyl-cholest-4,6-dien-3-one, 25.

EXAMPLE 23

Synthesis of 7β-methyl-cholest-5-en-3-one, (26)

Following the analogous procedure of Example 4 for the metal-ammonia reduction, 25 was similarly treated with lithium in ammonia/THF/toluene to yield title compound 26. EXAMPLE 24

Synthesis of 7β-methyl-cholest-4-en-3-one, (27)

Following the general isomerization procedure of Example 5 using DBU in THF, 26 was analogously treated to yield the title compound 27.

EXAMPLE 25

Synthesis of 7-methyl-17β-(2,6-Dimefhylhexyl)-5-oxo-A-nor-3,5-secoandrostan-3-oic acid. (28)

Following the general procedure of Example 6 for the oxidative Ring A cleavage, compound 27 was analogous treated to yield the above titled seco-acid 28. The proton NMR confirmed the assigned structure.

EXAMPLE 26 Synthesis of 7-Methyl-4-aza-cholest-5-en-3-one, (29)

Following the general procedure of Example 9, compound

28 was analogously treated with ammonium chloride in acetic acid to yield the above-titled product 29.

Mass Spectral m/z ion (FB) showed 400.2 (M+1)

(M+2).calculated, 399.

EXAMPLE 27

Synthesis of 7-Methyl-4-Aza-Cholestan-3-one. (30)

Following the analogous general procedure of Example 8, compound 29 was catalytically hydrogenated in HOAc to yield the title compound 30.

Mass spectral analysis by El yielded m/z ion of 401 calculated 401. EXAMPLE 28

Synthesis of 7-Methyl-4-methyl-4-Aza-Cholest-5-en-3-one, (31)

The seco acid 28, was treated analogously as in Example 7 to give pure title compound 31.

Mass Spec. 414 (M+1 ) by FAB, calc'd., 413.

EXAMPLE 29 Synthesis of 7β-Methyl-4-methyl-4-aza-5α-cholestan-3-one, (32)

Following the general analogous procedure described in Example 8, 31 was catalytically hydrogenated to yield the titled compound, 32. Chromatography on silica gel with 30% EtOAc/hexane, eluant yielded pure product.

Mass Spec. (El) 415, calc'd., 415.

EXAMPLE 30

Synthesis of 4-methyl-4-aza-cholest-5-en-3,7-dione, (34)

An oxidation procedure is carried out on 4-methyl-4-aza-cholest-5-en-3-one 33 to yield the title compound, 34. (See USP

3,264,301 by Doorenboos and J. Org. Chem. 1961. Vol. 26, p. 4548.) The compound 33 was heated at 70°C with a mixture of pyridinium dichromate/t-butyl hydroperoxide in benzene over a 3-4 hour period to produce 34.

EXAMPLE 31

Synthesis of 7β-Acetoxy-4-methyl-4-aza-5α-cholestan-3-one, (37)

Compound 34 is hydrogenated by the analogous procedure of Example 8 to produce the 7-H analog 35, and the 7β-ol, 36. Acylation of 36 with acetic anhydride, in the presence of pyridine, 4-dimethylaminopyridine in methylene chloride at 23°C for 24 hrs.

produces the title compound 37. EXAMPLE 32

Synthesis of 7-Beta Methyl-4-aza-5α-cholest-1 -en-3-one, (49)

To a solution of 280 mg. (0.698 mmol) of 30 in 4 milliliters toluene, was added 178.8 mg. DDQ, 0.7186 mg. BSTFA and 8.163 mg. triflic acid and the reaction contents allowed to stir at room temperature for 24 hours. Methyl acetoacetate, 8.1 mg., was added and the reaction refluxed for 24 hours. The contents were cooled, diluted with ethyl acetate, washed with aqueous sodium carbonate, aqueous sodium bisulfite, brine, dried over magnesium sulfate and concentrated to yield an oil. The crude compound was purified by preparative TLC on silica gel, eluting with 3: 1 CHCl₃/acetone to yield pure 49, whose proton NMR confirmed the assigned structure.

The following Table lists the unique proton NMR values (400 MHz in CDCI₃) for each compound. The data are reported as: s = singlet, d = doublet, m = multiplet, J = coupling constant. The absoφtion values are given in delta (δ) scale with a reference point signal from tetramethylsilane, and are illustrated for the C-18, C-19 and C-21 angular ring methyl protons and protons associated with unique portions of the molecule.

The numbering of the 4-aza steroid is given by the following structure:

TABLE

Compound

No. 18-CH₃ 19-CH₃ 21 -CH₃ Others

2 s 0.660 s 1.030 d 0.940 6H s 6.120

0.662 1.060 J=7 (values given for second isomer)

3 s 0.755 s 1.061 d 0.915 4H and 6H s

J=7 5.61, 5.97

4 s 0.720 s 1.110 d 0.930 4 CH₂ m 2.83 -

J=7 3.28

5 s 0.730 s 1.12 d 0.930 4H s 5.74

J=7

6 s θ.66 s 0.963 d 0.894

J=7

7 s 0.692 s 0.977 d 0.908 N-CH₃ s 3.153

J=7

8 s 0.690 s 0.830 d 0.900 N-CH₃ s 2.93

J=7

9 s 0.653 s 0.991 d 0.903 6H d 4.91 J=4

J=7

10 s 0.675 s 0.808 d 0.893 5H, m, 2.97 -

J=7 3.13

11 s 0.66 s 0.90 d 0.915 allylic H m (5.8-

J=7 5.94) Compound

No. 18-CH₃ 19-CH₃ 21 -CH₃ Others

12 s 0.78 s 1.07 d 0.96 allylic H m (5.73-

J=7 5.85)

13 s 0.70 s 1.08 d 0.90 6H, s (5.23)

J=7

14 s 0.73 s 1.13 d 0.93 4H s 5.72

J=7

18 s 0.71 s 1.13 d 0.93 4H s 5.71

J=7

19 s 0.65 s 0.963 d 0.91

J=7

20 s 0.691 s 0.974 d 0.902 (6H) - d , 4.92

J=7 (J=4) (N-CH₃) s

3.16

21 s 0.665 s 0.795 d 0.883

J=7 (N-CH₃) s 2.92

5H m (2.96-3.00)

22 s 0.680 s 1.01 d 0.890 (6H) d 4.86

J=7 J=4 23 s 0.680 s 0.808 d 0.884 5H m (3.0-3.1)

J=7

24 s, 0.68, s, 0.94, d 0.91 6H, s, 5.19, 5.21

0.69 1.04 J=7 Compound

No. 18-CH₃ 19-CH₃ 21 -CH₃ Others

25 s, 0.76 s, 1.07 d 0.92 4H, 6H 5.59,

J=7 5.92

27 s, 0.70 s, 1.15 d 0.92 7-CH₃, d, 1.04,

J=7 J=6.5 4H, s, 5.71

28 s, 0.69 s, 1.12 d 0.92 7-CH₃, d, 0.96,

J=7 J=6.5

29 s, 0.69 s, 1.04 d 0.91 7-CH₃, d, 0.97,

J=7 J=6.5 6H, d, 4.59,

J=3.0

30 s, 0.67 s, 0.835 d 0.91 7-CH₃, d, 1.00,

J=7 J=6.5 5H, dd,

J=3.3, 12.63

31 s, 0.69 s, 1.00 d 0.95 7-CH₃, d, 1.05,

J=7 J=6.5 6H, d,

J=3.0

32 s, 0.68 s, 0.825 d 0.91 , 7-CH₃, d, 1.05,

J=7H J=6.5 4-CH₃, s,

3.92

33 s, 0.69 s, 1.23 d 0.91 C6 - s, 5.42

J=7 N-CH₃, s, 3.14

Mass Spec

(EI)=413 Compound

No. 18-CH₃ 19-CH₃ 21 -CH₃ Others

49 s, 0.69 s, 0.90 d 0.915 C-7CH₃, 1.02,

J=7 d, J=6, C-2, 1H,

5.79, dd J=2.5

J=9.1

50 s, 0.62 s, 1.01 d 0.86 C-5, 1H, 3.08,

J=7 dd J= 3.87

J=12.9

C-7Ph, 5H, m,

7.1-7.3

51 s, 0.63 s, 1.02 d 0.8 C-5, 1-H, 3.2, dd

J=7 J= 5.88

J=10.5

C-7Ph, 5H, m,

7.08-7.3

Claims

WHAT IS CLAIMED IS:

1. The use of a 5α-reductase 2 inhibitor and a compound of formula I

or a stereoisomer or a pharmaceutically acceptable salt or ester thereof, wherein:

R is selected from hydrogen, methyl, ethyl, -OH, -NH₂, and -SCH₃; the dashed lines " - - - " a and b independently represent a single bond or a double bond providing that when b is a double bond, the 5oc hydrogen, Ha, is absent;

=Z is selected from:

1) oxo,

2) α-hydrogen and a β-substituent selected from:

a) C₁ -C₄ alkyl,

b) C₂-C₄ alkenyl,

c) CH₂COOH,

d) -OH,

e) -COOH,

f) -COO(C₁ -C₄ alkyl),

g) -OC(O)NR¹R² wherein R¹ and R² independently are selected from: i) H,

ii) C₁-C₄ alkyl,

iii) phenyl, and

iv) benzyl, or

R¹ and R² together with the nitrogen atom to which they are attached represent a 5-6 membered saturated heterocycle, optionally containing one other heteratom selected from -O-, -S- and -N(R')- wherein R' is -H or methyl;

h) C₁-C₄ alkoxy,

i) C₃-C₆ cycloalkoxy,

j) -OC(O)-C_{1 -4} alkyl,

k) halo,

l) hydroxy -C₁-C₂ alkyl,

m) halo-C₁-C₂ alkyl,

n) -CF₃, and

o) C3-C6 cycloalkyl;

3) =CHR₃; wherein R³ is selected from -H and C₁-C₄ alkyl; and

4) spirocyclopropane-R³ of structure:

for the preparation of a medicament useful for inhibiting the biosynthetic conversion of testosterone to dihydrotestosterone in a mammal in need of such inhibition.

2. The use of a 5oc-reductase 2 inhibitor and a compound of formula I

or a stereoisomer or a pharmaceutically acceptable salt or ester thereof, wherein:

R is selected from hydrogen, methyl, ethyl, -OH, -NH₂, and -SCH₃; the dashed lines " - - - " a and b independently represent a single bond or a double bond providing that when b is a double bond, the 5α hydrogen,

Ha, is absent;

=Z is selected from:

1) oxo,

2) α-hydrogen and a β-substituent selected from:

a) C₁-C₄ alkyl,

b) C₂-C₄ alkenyl,

c) CH₂COOH,

d) -OH,

e) -COOH,

f) -COO(C₁-C₄ alkyl),

g) -OC(O)NR¹R² wherein R¹ and R² independently are selected from:

i) H,

ii) C₁-C₄ alkyl,

iii) phenyl, and

iv) benzyl, or

R¹ and R² together with the nitrogen atom to which they are attached represent a 5-6 membered saturated heterocycle, optionally containing one other heteratom selected from -O-, -S- and -N(R')- wherein R' is -H or methyl; h) C₁-C₄ alkoxy,

i) C₃-C₆ cycloalkoxy,

j) -OC(O)-C _{1 -4} alkyl,

k) halo,

l) hydroxy -C₁ -C₂ alkyl,

m) halo-C₁-C₂ alkyl,

n) -CF₃, and

o) C₃-C₆ cycloalkyl;

3) =CHR₃; wherein R³ is selected from -H and C₁-C₄ alkyl; and

4) spirocyclopropane-R³ of structure:

for the preparation of a medicament useful for treating acne vulgaris, androgenic alopecia, seborrhea, female hirsutism, benign prostatic hypeφlasia, prostatitis, prostatic cancer and the prevention of prostatic cancer in a mammal in need of such treatment.

3. The use of Claim 2 wherein the androgenic alopecia is male pattern alopecia.

4. The use of Claim 1 wherein the compound of formula

I is

5. The use of Claim 4 wherein =Z is α-hydrogen and the β-substituent is C₁ -C₄alkyl or C₂-C₄alkenyl.

6. The use of Claim 1 wherein the compound of formula I is selected from the group consisting of:

7β-ethyl-4-methyl-4-aza-cholest-5-en-3-one,

7β-ethyl-4-methyl-4-aza-cholestane-3-one,

7β-ethyl-4-aza-5α-cholestan-3-one,

7β-carboxymethyl-4-aza-cholest-5-en-3-one,

7β-carboxymethyl-4-aza-cholestan-3-one,

7β-propyl-4-methyl-4-aza-cholest-5-en-3-one,

7β-propyl-4-methyl-4-aza-5α-cholestan-3-one,

7 β-propyl-4-aza-5α-cholestan-3-one,

7β-methyl-4-aza-cholest-5-en-3-one,

7β-methyl-4-aza-cholestan-3-one,

4,7β-dimethyl-4-aza-cholest-5-en-3-one,

4,7β-dimethyl-4-aza-5α-cholestan-3-one,

4-methyl-4-aza-5α-cholestan-3,7-dione,

7β-acetoxy-4-methyl-4-aza-5α-cholestan-3-one,

7β-hydroxy-4-methyl-4-aza-5α-cholestane-3-one,

7β-methoxy-4-methyl-4-aza-5α-cholestane-3-one,

7β-hydroxymethyl-4-aza-5α-cholestane-3-one,

7β-bromomethyl-4-aza-5α-cholestane-3-one,

7β-chloromethyl-4-aza-5α-choIestane-3-one,

7β-fluoromethyl-4-aza-5α-cholestane-3-one,

7β-carboxy-4-aza-5α-cholestane-3-one,

7β-trifluoromethyl-4-aza-cholest-5-en-3-one,

7β-methoxy-4-methyl-4-aza-cholesta-5-en-3-one,

7β-cyclopropyloxy-4-methyl-4-aza-5α-cholestane-3-one,

7β-propylidene-4-methyl-4-aza-5α-cholestane-3-one,

7β-(2-ethyl)spiroethylene-4-methyl-4-aza-5α-cholestane-3-one,

7β-methyl-4-aza-5α-cholest-1 -en-3-one;

and the pharmaceutically acceptable salts thereof.

7. The use of Claim 6 wherein the compound of formula I is selected from the group consisting of: 7β-ethyl-4-methyl-4-azacholestan-3-one; 7β-propyl-4-methyl-4-aza-5α-cholestan-3-one; 4,7β-dimethyl-4-aza-5α-cholestan-3-one; and the pharmaceutically acceptable salts thereof.

8. The use of Claim 7 wherein the compound of formula I is 4,7β-dimethyl-4-aza-5α-cholestan-3-one or a pharmaceutically acceptable salt thereof.

9. The use of Claim 1 wherein the 5α-reductase 2 inhibitor is finasteride.

10. The use of Claim 9 wherein the compound of formula I is 4,7β-dimethyl-4-aza-5α-cholestan-3-one or a pharmaceutically acceptable salt thereof.

11. The use of retinoic acid or a derivative thereof and a compound of formula I

or a stereoisomer or a pharmaceutically acceptable salt or ester thereof, wherein:

R is selected from hydrogen, methyl, ethyl, -OH, -NH₂, and -SCH₃;

the dashed lines " - - - " a and b independently represent a single bond or a double bond providing that when b is a double bond, the 5α hydrogen, Ha, is absent;

=Z is selected from: 1) oxo,

2) α-hydrogen and a β-substituent selected from:

a) C₁ -C₄ alkyl,

b) C₂-C₄ alkenyl,

C) CH₂COOH,

d) -OH,

e) -COOH,

f) -COO(C₁-C₄ alkyl),

g) -OC(O)NR¹R² wherein R¹ and R² independently are selected from:

i) H,

ii) C₁ -C₄ alkyl,

iii) phenyl, and

iv) benzyl, or

R¹ and R² together with the nitrogen atom to which they are attached represent a 5-6 membered saturated heterocycle, optionally containing one other heteratom selected from -O-, -S- and -N(R')- wherein R' is -H or methyl;

h) C₁-C₄ alkoxy,

i) C₃-C₆ cycloalkoxy,

j) -OC(O)-C_{1 -4} alkyl,

k) halo,

l) hydroxy -C₁ -C₂ alkyl,

m) halo-C₁-C₂ alkyl,

n) -CF₃, and

0) C₃-C₆ cycloalkyl;

3) =CHR³; wherein R³ is selected from -H and C₁ -C₄ alkyl; and

4) spirocyclopropane-R³ of structure:

for the preparation of a medicament useful for treating acne vulgaris and androgenic alopecia in a mammal in need of such treatment.

12. The use of an anti-androgen and a compound of formula I

or a stereoisomer or a pharmaceutically acceptable salt or ester thereof, wherein:

R is selected from hydrogen, methyl, ethyl, -OH, -NH₂, and -SCH₃; the dashed lines " - - - " a and b independently represent a single bond or a double bond providing that when b is a double bond, the 5α hydrogen,

Ha, is absent;

=Z is selected from:

1) oxo,

2) α-hydrogen and a β-substituent selected from:

a) C₁-C₄ alkyl,

b) C₂-C₄ alkenyl,

c) CH₂COOH,

d) -OH,

e) -COOH,

f) -COO(C₁-C₄ alkyl),

g) -OC(O)NR¹R² wherein R¹ and R² independently are selected from:

i) H,

ii) C₁ -C₄ alkyl,

iii) phenyl, and

iv) benzyl, or R¹ and R² together with the nitrogen atom to which they are attached represent a 5-6 membered saturated heterocycle, optionally containing one other heteratom selected from -O-, -S- and -N(R')- wherein R' is -H or methyl;

h) C₁-C₄ alkoxy,

i) C₃-C₆ cycloalkoxy,

j) -OC(O)-C_{1 -4} alkyl,

k) halo,

l) hydroxy -C₁ -C₂ alkyl,

m) halo-C₁ -C₂ alkyl,

n) -CF₃, and

0) C₃-C₆ cycloalkyl;

3) =CHR³; wherein R³ is selected from -H and C₁-C₄ alkyl; and

4) spirocyclopropane-R³ of structure:

for the preparation of a medicament useful for treating acne vulgaris, androgenic alopecia, seborrhea, female hirsutism, benign prostatic hypeφlasia, prostatitis, prostatic cancer and preventing prostatic cancer in a mammal in need of such treatment.

13. The use of Claim 12 wherein the anti-androgen is selected from flutamide, spironolactone and casodex.

14. A pharmaceutical composition comprising (i) a pharmaceutically acceptable carrier, (ii) a therapeutically effective amount of a first compound selected from a 5α-reductase 2 inhibitor, retinoic acid or a derivative thereof and an anti-androgen, and (iii) a therapeutically effective amount of a second compound of formula I

or a stereoisomer or a pharmaceutically acceptable salt or ester thereof, wherein:

R is selected from hydrogen, methyl, ethyl, -OH, -NH₂, and -SCH₃; the dashed lines " - - - " a and b independently represent a single bond or a double bond providing that when b is a double bond, the 5α hydrogen,

Ha, is absent;

=Z is selected from:

1) oxo,

2) α-hydrogen and a β-substituent selected from:

a) C₁-C₄ alkyl,

b) C₂-C₄ alkenyl,

c) CH₂COOH,

d) -OH,

e) -COOH,

f) -COO(C₁-C₄ alkyl),

g) -OC(O)NR¹R₂ wherein R¹ and R² independently are selected from:

i) H,

ii) C₁-C₄ alkyl,

iii) phenyl, and

iv) benzyl, or

R¹ and R² together with the nitrogen atom to which they are attached represent a 5-6 membered saturated heterocycle, optionally containing one other heteratom selected from -O-, -S- and -N(R')- wherein R' is -H or methyl; h) C₁ -C₄ alkoxy,

i) C₃-C₆ cycloalkoxy,

j) -OC(O)-C_{1 -4} alkyl,

k) halo,

l) hydroxy -C₁ -C₂ alkyl,

m) halo-C₁-C₂ alkyl,

n) -CF₃, and

o) C₃-C₆ cycloalkyl;

3) =CHR³; wherein R³ is selected from -H and C₁-C₄ alkyl; and 4) spirocyclopropane-R³ of structure:

15. The pharmaceutical composition of Claim 14 wherein the 5 α- reductase 2 inhibitor is finasteride and the anti-androgen is selected from flutamide, spironolactone and casodex.

16. The pharmaceutical composition of Claim 14 wherein the second compound is 4,7β-dimethyl-4-aza-5α-cholestan-3-one or a pharmaceutically acceptable salt thereof.

17. The pharmaceutical composition of Claim 14 wherein the first compound is a 5α-reductase 2 inhibitor.

18. The pharmaceutical composition of Claim 17 wherein the 5α-reductase 2 inhibitor is finasteride and the second compound is 4,7β-dimethyl-4-aza-5α-cholestan-3-one or a pharmaceutically

acceptable salt thereof.