Acyl 3-carboxyl aromatic A ring steroid as 5-alpha reductase inhibitors

FIELD OF THE INVENTION The present invention relates to certain novel 17α and 17B substituted acyl 3-carboxy aromatic A ring steroidal compounds, pharmaceutical compositions containing these compounds, and methods for using these compounds to inhibit steroid 5-α-reductase.

DESCRIPTION OF RELATED ART The class of steroidal hormones known as androgens is responsible for the physical characteristics that differentiate males from females. Of the several organs that produce androgens, the testes produce these hormones in the greatest amounts. Centers in the brain exert primary control over the level of androgen production. Numerous physical manifestations and disease states result when ineffective control results in excessive androgen hormone production. For example, acne vulgaris, seborrhea, female hirsutism, male pattern baldness and prostate diseases such as benign prostatic hypertropy are correlated with elevated androgen levels. Additionally, the reduction of androgen levels has been shown to have a therapeutic effect on prostate cancer. Testosterone is the principal androgen secreted by the testes and is the primary androgenic steroid in the plasma of males. It now is known that 5-α-reduced androgens are the active hormones in some tissues such as the prostate and sebaceous gland. Circulating testosterone thus serves as a prohormone for dihydrotestosterone (DHT), its 5-α-reduced analogue, in these tissues but not in others such as muscle and testes. Steroid 5-α-reductase is a nicotinamide adenine dinucleotide phosphate (NADPH) dependent enzyme that converts testosterone to DHT. The importance of this enzyme in male development was dramatically underscored by the discovery of a genetic steroid 5-α-reductase deficiency in male pseudohermaphrodites. Imperato-McGinley, J., ≤t al., (1979), J. Steroid Biochem. 11:637-648.

Recognition of the importance of elevated DHT levels in various disease states has stimulated many efforts to synthesize inhibitors of this enzyme. Among the most potent inhibitors identified to date are 3- carboxy-estra-l,3,5(10) triene steroidal derivatives.

A number of 5-α-reductase inhibiting compounds are known in the art. For example,

1. J. Steroid Biochem.. Vol. 34, Nos. 1-6 pp. 571-575(1989), by M.A. Levy, et al., describes the interaction mechanism between rat prostatic steroid 5-alpha reductase and 3-carboxy-17B-substituted steroids;

2. J. Med. Chem. (1990) Vol. 33, pp. 937-942, by D.A. Holt, et al., describes the new steroid class of A ring aryl carboxylic acids; 3. TIPS (December 1989) Vol. 10, pp. 491-495, by B.W.

Metcalf, et al., describes the effect of inhibitors of steroid 5α-reductase in benign prostatic hyperplasia, male pattern baldness and acne; and

4. EPO Publn. No. 0 343 954 A3, to D. A. Holt, et al., (SmithKline Beckmann) describes steroidal 3-carboxylic acid derivatives as useful 5-α-reductase inhibitors.

However, none of the above references specifically suggests that any of the novel steroidal 17α or 17β-substituted 3-carboxy-estra 1,3,5(10) triene compounds of the present invention would have utility as potent testosterone 5-α-reductase inhibitors.

SUMMARY OF THE INVENTION

This

wherein

Y is oxygen or sulphur; l is hydrogen, a Ci-Cg alkyl group, a C5-C6 cycloalkyl group, a Cg-Cg cycoalkylalkyl group or an aryl group; each of R2 and R3 is, independently, selected from the group consisting of hydrogen, Ci-Cg alkyl, C5-C6 cycloalkyl, Cg-Cg

cycloalkylalkyl and aryl or R2 and R3, taken together with the nitrogen atom, to which they are linked, form a pentatomic or hexatomic saturated heteromonocyclic ring, optionally containing at least one additional heteroatom selected from oxygen and nitrogen; and pharmaceutically acceptable salts, hydrates, solvates and esters thereof. Also included within the scope of this invention are compounds in which the 17 position substituent is in the α position.

The invention also is a method for inhibiting 5-α-reductase activity in mammals, including humans, that comprises administering to a subject an effective amount of a presently invented 5-α-reductase inhibiting compound. Included in the present invention are pharmaceutical compositions comprising a pharmaceutical carrier and compounds useful in the methods of the invention. Also included in the present invention are methods of co-administering the presently invented 5-α-reductase inhibiting compounds with further active ingredients.

DETAILED DESCRIPTION OF THE INVENTION The compounds of this invention that inhibit 5-α-reductase have the following Formula (I):

wherein

Y is oxygen or sulphur; Ri is hydrogen, a Ci-Cg alkyl group, a C5-C6 cycloalkyl group, a

Cg-Cg cycoalkylalkyl group or an aryl group; each of R2 and R3 is, independently, selected from the group consisting of hydrogen, Ci-Cg alkyl, C5-C6 cycloalkyl, CQ-CQ cycloalkylalkyl and aryl or R2 and R3, taken together with the nitrogen atom, to which they are linked, form a pentatomic or hexatomic

saturated heteromonocyclic ring, optionally containing at least one additional heteroatom selected from oxygen and nitrogen; and pharmaceutically acceptable salts, hydrates, solvates and esters thereof.

Also included within the scope of this invention are compounds in which the 17 position substituent is in the α position.

In the formulae of this specification a dotted line ( mi ) indicates a substituent in the α configuration, i.e. below the plane of the ring, and a wedged line ( ^→ - ^→→→ ^Λ ) indicates a substituent in the β- configuration, i.e. above the plane of the ring. The invention includes also the pharmaceutically acceptable salts of the compounds of formula (I) as well as all the possible isomers of formula (I) and their mixtures.

Also the metabolites and the metabolic precursors of the compounds of formula (I) are within the scope of the present invention. In this specification the alkyl groups and the alkyl moiety of the cycloalkylalkyl groups may be straight or branched chain.

A C -CQ alkyl group may be, for example, methyl, ethyl, isopropyl, n-butyl, tert-butyl or tert-butylmethyl (i.e. neopentyl).

A C5-C6 cycloalkyl group is cyclopentyl or cyclohexyl, preferably cyclohexyl.

A Cρ-Cg cycloalkylalkyl group may be, for example, cyclohexylmethyl.

An aryl group may be, for example, phenyl or benzyl.

Rj is, preferably, methyl, ethyl, isopropyl, tert-butyl, cyclohexyl or cyclohexylmethyl.

Preferably each of R2 and R3 is independently, hydrogen, methyl, ethyl, isopropyl, tert-butyl, cyclohexyl, cyclohexylmethyl, phenyl or when

R2 and R3 taken together with the nitrogen atom to which they are linked, form a pentatomic or hexatomic saturated heteromonocyclic ring as defined

Pharmaceutically acceptable salts of the compounds of the invention are salts with pharmaceutically acceptable bases, either inorganic bases such as, for instance, alkali metal, e.g. sodium or potassium, or alkaline-earth metal, e.g. calcium or magnesium, or zinc or aluminium, hydroxides, or organic bases, such as, e.g., aliphatic amines as, e.g., methylamine, diethylamine, trimethylamine, ethylamine, and heterocyclic amines as, e.g., piperidine.

A preferred class of compounds according to the invention are the compounds of formula (I) wherein: Y is oxygen or sulphur;

R ¹ is methyl, ethyl, isopropyl, tert-butyl, cyclohexyl; f ^" / the group N

— N — N — N

\ \ \

C(CH ₃ ) ₃ . CH ₃ . C ₂ H ₅ . and the pharmaceutically acceptable salts thereof. Examples of specific compounds preferred under this invention are:

17β-[N-isopropyl-N-(N-isopropylcarbamoyl) carbamoyl]-estra- l,3,5,(10)-triene-3-carboxylic acid;

17β-[N-cyclohexyl-N-(N-cyclohexylcarbamoyl)carbamoyl]-es tra- l,3,5,(10)-triene-3-carboxylic acid;

17β-[N-tert-butyl-N-(N-tert-butylcarbamoyl)carbamoyl]-es tra- l,3,5,(10)-triene-3-carboxylic acid; 17β-[N-isopropyl-N-(N-isopropylthiocarbamoyl)carbamoyl]-est ra- l,3,5,(10)-triene-3-carboxylic acid;

17β-[N-cyclohexyl-N-(N-cyclohexylthiocarbamoyl)-estra-l, 3,5(10)- triene-3-carboxylic acid; and

17β-[N-methyl-N-(N,N-diethylcarbamoyl)carbamoyl-estra- l,3,5(10)-triene-3-carboxylic acid;

and where appropriate, the pharmaceutically acceptable salts thereof.

Compounds of Formula (I) are included in the pharmaceutical compositions of the invention and used in the methods of the invention. Where a -COOH or -OH group is present, pharmaceutically acceptable esters can be employed, for example methyl, ethyl, pivaloyloxymethyl, and the like for -COOH, and acetate maleate and the like for -OH, and those esters known in the art for modifying solubility or hydrolysis characteristics for use as sustained release or prodrug formulations. The term "α-receptor antagonist", as used herein, refers to a known class of alpha-andrenergic receptor antagonist comounds, such as described in Lafferty, et al. U.S. Patent No. 4,963,547, which are utilized in treating vascular disorders such as diabetes, cardiovascular disease, benign prostatic hypertrophy and ocular hypertension.

Preferred alpha-andrenergic receptor antagonists for use in the compositions and methods of the invention include amsulosin, terazosin, doxazosin, alfuzosin, indoramin, prazosin, 7-chloro-2-ethyl-3,4,5,6- tetrahydro-4-methylthieno[4,3,2-ef][3]-benzazepine and 8-{3-[4-(2- methoxyphenyl)-l-piperazinyl]-propylcarbamoyl}-3-methyl-4-ox o-2- phenyl-4H- 1-benzopyran. By the term "amsulosin" as used herein is meant a compound of the structure

and salts, hydrates and solvates thereof.

Chemically, amsulosin is designated as (-)-(R)-5-[2-[[2-(Q- ethoxyphenoxy)ethyl]am--no]propyl]-2-methoxybenzenesulfonami de. Amsulosin is disclosed in U.S. Patent Number 4,703,063 and claimed in U.S. Patent Number 4,987,152 as being useful in treating lower urinary tract dysfunction.

By the term "terazosin" as used herein is meant a compound of the structure

and salts, hydrates and solvates thereof.

Chemically, terazosin is designated as l-(4-amino-6,7-dimethoxy- 2 quinazolinyl)-4-[(tetrahydro-2-furoyl)carbonyl]piperazine. Terazosin is disclosed in U.S. Patent Number 4,251,532.

By the term doxazosin as used herein is meant a compound of the structure

and salts, hydrates and solvates thereof.

Chemically "doxazosin" is designated as l-(4-amino-6,7- dimethoxy-2-qu-i-- zohnyl)-4-[(2,3-dihydro-l,4-benzodioxin-2- yl)carbonyl]-piperazine.

Doxazosin is disclosed in U.S. Patent Number 4,188,390.

By the term "alfuzosin" as used herein is meant a compound of the structure

and salts, hydrates and solvates thereof.

Chemically alfuzosin is designated as N-[3-[(4-amino-6,7- d--methoxy-2-qu--nazolinyl)methylamino]propyl]tetrahydro-2- furancarboxamide.

Alfuzosin is disclosed in U.S. Patent Number 4,315,007.

By the term "indoramin" as used herein is meant a compound of the structure

and salts, hydrates and solvates thereof.

Chemically indoramin as designated N-[[l-[2-(lH-indol-3- yl)ethyl]-4-piperidinyl]benzamine.

Indoramin is disclosed in U.S. Patent Number 3,527,761.

By the term "prazosin" as used herein is meant a compound of the structure

and salts, hydrates and solvates thereof.

Chemically prazosin is designated as l-(4-amino-6,7-dimethoxy-2- qu--nazolinyl)-4-(2-furanylcarbonyl)piperazine.

Prazosin is disclosed in U.S. Patent Number 3,511,836.

"7-chloro-2-ethyl-3,4,5,6-tetrahydro-4-methylthieno[4,3,2 -efj- [3]benzazepine" as used herein is meant a compound of the structure

and salts, hydrates and solvates thereof.

7-chloro-2-ethyl-3,4,5,6-tetrahydro-4-methylthieno[4,3,2- efl- [3]benzazepine is disclosed in U.S. Patent No. 5,006,521. Additionally, all compounds disclosed in U.S. Patent No. 5,006,521 as alpha- adrenergic receptor antagonist are preferred alpha-adrenergic receptor antagonist as used herein.

"8-{3-[4-(2-methoxyphenyl)-l-piperazinyl]-propylcarbamoyl }-3- methyl-4-oxo-2-phenyl-4H-l-benzopyran" as used herein is meant a compound of the structure

and salts, hydrates and solvates thereof.

8-{3-[4-(2-methoxyphenyl)-l-piperazinyl]-propylcarbamoyl} -3- methyl-4-oxo-2-phenyl-4H-l-benzopyran is disclosed in EPO Publn. No. 0558245 Al, to Leonardi, et al., (Recordati S.A.).

Additionally all compopunds disclosed in EPO Publn. No. 0558245 Al, as alpha-adrenergic receptor antagonists are preferred alpha- adrenergic receptor antagonists as used herein.

Persons skilled in the art can readily determine if a compound other than one specifically referred to herein is a alpha-andrenergic receptor antagonist by utilizing the assay described in Lafferty I. Thus, all such compounds are included within the scope of the term "alpha- andrenergic receptor antagonist" as used herein.

By the term "minoxidi ' as used herein is meant the compound of the structure:

chemically minoxidil is designated as 2,4-pyrimidineadiamine, 6-(l- piperidinyl)-,3-oxide. Minoxidil is the active ingredient in Rogaine® which is sold as topical solution for stimulating hair growth by the Upjohn Company, Kalamazoo, Michigan.

The term "aromatase inhibitor", as used herein, refers to a known class of compounds, steroidal and non-steroidal, which prevent the conversion of androgens to estrogens, such as described in Gormley et al.

International Publication Number WO 92/18132. Aromatase inhibitors are disclosed in Gormley et al. as having utility in treating benign prostatic hyperplasia when used in combination with a 5-α-reductase inhibitor. A preferred aromatase inhibitor for use in the compositions and methods of the invention 4-(5,6,7,8-tetrahydroimidazo-[l,5-α]pyridin-5- yDbenzonitrile (fadrazole). Fadrazole is disclosed in U.S. Patent No. 4,728,645. Additionally, all compounds disclosed in Gormley, et al. International Publication No. WO 92/18132 as having aromatase -tύbiting activity are preferred aromatase inhibitors as used herein. As used herein, when a 5-α-reductase inhibitor, as described herein and a further active ingredient or ingredients are utilized together, said 5-α-reductase inhibitor can be co-administered with said further active ingredient or ingredients. By the term "co-administering" and derivatives thereof as used herein is meant either simultaneous administration or any manner of separate sequential administration of a 5-α-reductase inhibiting compound, as described herein, and a further active ingredient or ingredients, such as other compounds known to treat the disease states of acne vulgaris, seborrhea, female hirsutism, male pattern baldness, benign prostate hypertrophy or prostatic adenocarcinoma or compounds known to have utility when used in combination with 5-α-reductase inhibitors. Preferably, if the administration is not simultaneous, the compounds are administered in a close time proximity to each other. Furthermore, it does not matter if the compounds are administered in the same dosage form, e.g. one compound may be administered topically and another compound may be administered orally.

A compound of formula (I) may be obtained by a process comprising reacting a compound of formula (II)

wherein Y, R*, R ² , R3 are as defined above, with carbon monoxide (CO) in the presence of a Ci-Cβ alkyl alcohol, so obtaining a 3 position Ci-Cgalkyl ester compound of formula (I), wherein Y, Rl, R ² , R3 are as defined above followed by selective hydrolysis, into a corresponding compound of formula (I), wherein Y, R*, R ² , R3 _are as defined above, and/or, if desired, converting a compound of formula (I) into a salt thereof, or, converting a salt of a compound of formula (I) into the corresponding free compound, and/or, if desired, separating a mixture of isomers of a compound of formula (I) into the single isomers.

The reaction of a compound of formula (II) with carbon monoxide in the presence of a Ci-Cg alkyl alcohol, according to the process may be carried out, e.g., by treating a solution of the compound of formula (H) in a suitable organic solvent, preferably dimethylformamide (DMF), with an organic base such as, for example, triethylamine (TEA), a palladium complex such as, for example, bis (triphenylphosphine) palladium (II) chloride, and a Ci-Cg alkyl alcohol.

Optionally the palladium complexes, can be formed in situ, by adding, separately, a phosphine, such as triphenylphosphine, and a palladium salt, such as, for example, palladium (II) acetate or palladium (II) chloride.

Then the reaction mixture is purged with carbon monoxide (CO) for some minutes and then stirred under a CO balloon for a time varying from one hour to 48 hours, at a temperature ranging from 0°C. to 90°C. Analogous procedure is described, e.g., in Tetr. Lett. 26 (8), 1109-12, (1985).

The conversion of a 3 position Ci-Cgalkyl ester compound of formula (I), wherein the Y, Rl, R ² , R3 are as defined above into a corresponding compound of formula (I), wherein Y, R*, R ² , R^ are as

defined above e.g., in a suitable solvent, such as, for example, methanol, ethanol, tetrahydrofurane, dioxane, in the presence of an aqueous concentrate solution of an alkali metal hydroxide such as, for example, potassium hydroxide, sodium hydroxide or, preferably, lithium hydroxide, for a time varying from some hours to some days, at a temperature ranging from about 0°C. to the reflux temperature of the solvent, optionally under an inert atmosphere of nitrogen.

Standard procedures may be used for converting a compound of formula (I) into a pharmaceutically acceptable salt thereof as well as for obtaining a free commpound from the corresponding salt and for separating a mixture of isomers of formula (I) into the single isomers.

A compound of formula (II) wherein Y, R*, R ² , R^ are as defined above, may be

wherein Y, R , R ² , R^ are as defined above according to the following procedure.

A compound of formula (VI) is dissolved in a suitable organic solvent such as, for example, tetrahydrofurane (THF), in the presence of a suitable organic proton donor, such as, for example, tert-butanol or aniline; then a reducing metal amine solution, e.g. a lithium/liquid ammonia solution, is added to the obtained mixture and then the reaction mixture is stirred at a temperature ranging from about - 100°C. to about - 30°C, preferably at about - 78°C, for a time of about 2 or 3 hours. Then the reaction is quenched with an organic lithium scavenger, such as, for example, bromobenzene, dibromoetane or, preferably, isoprene and the solvent is removed in vacuo.

The solid residue is redissolved in an organic solvent such as, for example, tetrahydrofurane or diethyl ether, and treated with an N- aryltrifluoroalkylsulfonimide, preferably with N- phenyltrifluoromethylsulfonimide, at a temperature ranging from about

-20°C. to about -30°C, for a time varying from about 2 hours to about 24 hours.

The method is performed as in Tetr.Lett, 1983, 24, 979-982. A compound of formula (II), wherein Y, Rl, R ² , R^ are as defined above, may be obtained from a corresponding compound of formula (VI), wherein Y, Rl, R ² , R^ are as defined above, e.g. by adding to a solution containing a compound of formula (VI), as defined above, and an organic hindered base such as, 2,6-di-tert-butyl-4-methylpyridine, in a suitable organic solvent such as, e.g., methylene chloride, a trifluorosulphonic anhydride, preferably trifluoromethanesulfphonic anhydride, according to the procedure reported in Synthesis 438-440, 1979.

The compounds of formula (VI) can be in their turn synthesized using known procedures, for instance according to the three pathways a), b) and c)

(XI)

According to reaction (a), a compound of formula (VI) wherein Y, Rl, R ² , R3 are as defined above, provided that R and R ² are not hydrogen and R^ is hydrogen, may be obtained reacting a compound of formula (VTI), wherein Y is as defined above, with a carbodiimmide of formula (VIII) wherein R and R ² are as defined above.

According to reaction (b), a compound of formula (VI), wherein R* is as defined above, provided that it is not hydrogen, and Y, R ² and R^ are as defined above, maybe obtained from a compound of formula (IX), wherein R ² and Bp are as defined above, by alkylation with a compound of formula (X) wherein R* is as defined above provided that it is not hydrogen, and X is a halogen atom, e.g., iodine.

According to reaction (c) a compound of formula (VI), wherein B? is hydrogen and Y, R , R ² are as defined above, provided that R ² is not hydrogen, may be obtained from a compound of formula (XI), wherein R is as defined above, by reaction with a compound of formula (XII) wherein R ² and Y are as defined above, provided that R ² is not hydrogen.

Compounds of formula (VII), wherein Y=S, formula (IX) and formula (XI) may be obtained reacting a compound of formula (XIII)

wherein A may be any suitable activating group of the carboxy function which is useful in the formation of amidic and peptidic linkages with the

appropriate reagent as indicated in the following. The suitable activating group ma be for instance one of the following groups.

In particular, the compounds of formula (VII) wherein Y is sulfur, may be, e.g., obtained from compounds of formula (XIH) according to known procedures.

One procedure may involve, for example, reacting a compound of formula (XIII) wherein A is chlorine, with gaseous hydrogen sulfide in the presence of dimethylthioformamide, in a solvent such as, for example, CH2CI2, at room temperature for a time varying from, e.g., ten minutes to some hours under vigorous stirring, analogue to the method described in Synthesis, 671-2 (1985). Another procedure may involve, e.g., reacting a compound of formula (XIII) wherein A is

that is the S-2 pyridylthioate derivative, with an excess of sodium hydrogen sulfide monohydrate. The reaction may be performed in a solvent such as, for example, methylene chloride, tetrahydrofurane, acetonitrile, at a temperature ranging from, e.g., about 0°C. to about 50° C, for a time varying, e.g., from about one hour to about 48 hours.

A compound of formula (VII) wherein Y is sulphur may also be synthetized according to the general methods described in the literature for the synthesis of thiocarboxylic acids, for example in analogous way as described in Houben Weyl, Bd E 5 pages 832-842, or by Duns F. in Barton and Ollis, Comprehensive Organic chemistry, Vol. 3 Pergamon Press, Oxford, 1979, pages 20-32.

A compound of formula (IX), as defined above, may be obtained reacting a compound of formula (XIII), as defined above, with an urea of formula (XTV),

wherein R2 and R3, as defined above.

A compound of formula (XI), may be obtained reacting a compound of formula (XIII), with an amine of formula (XV)

Rl-NH ₂ (XV)

wherein R^ is as defined above.

A compound of formula (XIII) may be obtained from a compound of formula (VII) wherein Y is oxygen by well known procedures. The compounds of formulae (III), (IV), (V), (VII), wherein Y is oxygen, (VIII), OI), (XII), (XIV) and (XV) are commercially available compounds or can be prepared by known procedures from known compounds.

Formula I compounds in which the 17 position substitutent is in the α position are prepared from compounds which contain the corresponding β substituent by the General Method below.

General Method A

To a stirrred solution of a substituent 17β steroidal 5α-reductase inhibiting compound of Formula (I) in an appropriate solvent, preferably ethylene glycol or dimethyl sulfoxide, is added a base such as a hydroxide or alkoxide base, preferably sodium hydroxide, potassium hydroxide or sodium methoxide, at a temperature over 100°C preferably at reflux temperatures to yield the corresponding α epimer, after isolation and work up. In determining the appropriate solvent for conducting the epimerization, dimethyl sulfoxide or other non-reactive high boiling solvents are preferred when the starting 17B 5α-reductase inhibiting steroidal compound contains reactive substituents or reactive unsaturated bonds that are, for example, subject to nucleophilic attack and ethylene glycol, or other reactive high boiling solvents can be used

when the reactivity of the substituents or any unsaturated bonds of the starting 17B 5α-reductase inhibiting steroidal compound is not a consideration.

By the term "solvent" or "appropriate solvent" as used herein and the in the claims is meant a solvent such as methylene chloride, ethylene chloride, chloroform, ethylene glycol, carbon tetrachloride, tetrahydrofuran (THF), ethyl ether, toluene, ethyl acetate, hexane, dimethylsulfoxide (DMSO), N,N'-dimethyl-N,N'-propylene urea, N- methyl-2-pyrrolidinone, methanol, isopropylalcohol, dimethylformamide (DMF), water, pyridine, quinoline or ethanol.

Pharmaceutically acceptable salts, hydrates and solvates of Formula (I) compounds are formed, where appropriate, by methods well known to those of skill in the art.

Because the presently invented pharmaceutically active compounds inhibit steroid 5-α-reductase activity, they have therapeutic utility in treating diseases and conditions wherein decreases in DHT activity produces the desired therapeutic effect. Such diseases and conditions include acne vulgaris, seborrhea, female hirsutism, male pattern baldness, prostate diseases such as benign prostatic hypertrophy, and prostatic adenocarcinoma.

In determining potency in inhibiting the human 5α-reductase enzyme, the following procedure is employed:

Preparation of membrane particulates used as source for recombinant steroid 5α-reductase isozvme 1.

Chinese hamster ovary (CHO) cells containing expressed, recombinant human steroid 5α-reductase isoenzyme 1 (Andersson, S., Berman, D.M., Jenkins, E.P., and Russell, D.W. (1991) Nature 354 159-161) are homogenized in 20 mM potassium phosphate, pH 6.5, buffer containing 0.33 M sucrose, 1 mM dithiothreitol, and 50 uM NADPH (buffer A) using a Dounce glass-to-glass hand homogenizer (Kontes Glass Co., Vineland, N.J.). Membrane particulates are isolated by centrifugation (100,000 x g at 4°C for 60 minutes) and resuspended in 20 mM potassium phosphate, pH 6.5, containing 20% glycerol, 1 mM dithiothreitol, and 50 μM NADPH (buffer B). The suspended particulate solution is stored at -80°C.

Preparation of prostatic membrane particulates used as source for steroid 5α-reductase isozvme 2.

Frozen human prostates are thawed and minced into small pieces ( Brinkmann Polytron (Sybron Corp., Westbury, New York). The solution is sonicated for 3 to 5 minutes with a Sonifier (Branson Sonic Power Co.) followed by hand homogenization in a Dounce hand homogenizer. Prostatic particles are obtained by differential centrifugation at 600 or 1000 x g for 20 minutes and 140,000 x g for 60 minutes at 4°C. The pellet obtained from the 140,000 x g centrifugation was washed with 5 to 10 tissue volumes of the buffer described above and centrifuged at 140,000 x g. The resulting pellet is suspended in buffer B and the particulate suspension was stored at -80°C.

Preparation of membrane particulates used as source for recombinant steroid 5-α-reductase isozvme 2.

Chinese hamster ovary (CHO) cells containing expressed, recombinant human steroid 5-α-reductase isozyme 2 are homogenized in 20 mM potassium phosphate, pH 6.5, buffer containing 0.33 M sucrose, ImM dithiothreitol, and 50 uM NADPH (buffer A) using a Douce hand homogenizer. Membrane particulates containing the recombinant human enzyme are isolated by centrifugation (100,000 x g at 4°C for 60 minutes) and resuspended in 20 mM potassium phosphate, pH 6.5 containing 20% glycerol, ImM dithiothreitol, and 50 uM NADPH (buffer B). The suspended particulate solution is stored at -80°C until used.

Assay for enzvmes activities and inhibitors potency.

A constant amount of [ CJtestosterone (50 to 55 mCi/mmol) in ethanol and varying amounts of potential inhibitor in ethanol are deposited in test tubes and concentrated to dryness in vacuo. To each tube is added buffer, 10 μL (recombinant isoenzyme 1 or isoenzyme 2) or 20 μL (isoenzyme 2 from human prostate tissue) of 10 mM NADPH and an aliquot of a steroid 5α-reductase preparation to a final volume of 0.5 mL. Assays for human steroid 5α-reductase isoenzyme 1 are conducted with a sample of the recombinant protein expressed in CHO cells in 50 mM phosphate buffer, pH 7.5 while assays of isoenzyme 2 are conducted with a suspension of human prostatic particulates and/or recombinant protein expressed in CHO cells in 50 mM citrate buffer at pH 5.0.

After incubating the solution at 37°C for 20 or 30 minutes the reaction is quenched by the addition of 4 mL ethyl acetate and 0.25 μmol each of testosterone, 5α-dihydrotestosterone, androstanediol, and androstanedione as carriers. The organic layer is removed to a second test tube and evaporated to dryness in a Speed Vac. The residue is dissolved in 40 μL chloroform, spotted on an individual lane of a 20 x 20 cm prechannelled silica gel TLC plate (Si 250F-PA, Baker Chemical) and developed twice with acetone:chloroform (1:9). The radiochemical content in the bands of the substrate and the products are determined with a BIOSCAN Imaging Scanner (Bioscan Inc., Washington, D.C). The percent of recovered radiolabel converted to product is calculated, from which enzyme activity is determined. All incubations are conducted such that no more than 20% of the substrate (testosterone) is consumed. The experimentally obtained data is computer fit to a linear function by plotting the reciprocal of the enzyme activity (1/velocity) against the variable inhibitor concentration; apparent inhibition constants (Kj _>a pp) are determined by the Dixon analysis (Dixon, M. (1953). The value for the inhibition constant (Ki) is calculated from known procedures (Levy, M. (1989), Biochemistry.22:2815-2824).

All of the compounds within the scope of this invention are useful in inhibiting steroid 5-α-reductase in a mammal, including humans, in need thereof. The pharmaceutically active compounds of the present invention are preferably incorporated into convenient dosage forms such as capsules, tablets, or injectable preparations. Solid or liquid pharmaceutical carriers are employed. Solid carriers include, starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline, and water. Similarly, the carrier or diluent may include any prolonged release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies widely but, preferably, will be from about 25 mg to about 1 g per dosage unit. When a liquid carrier is used, the preparation will preferably be in the form of a syrup, elixir, emulsion,

soft gelatin capsule, sterile injectable liquid such as an ampoule, or an aqueous or nonaqueous liquid suspension.

The pharmaceutical preparations are made following conventional techniques of a pharmaceutical chemist involving mixing, granulating, and compressing, when necessary, for tablet forms, or mixing, filling and dissolving the ingreidents, as appropriate, to give the desired oral or parenteral products.

Doses of the presently invented pharmaceutically active compounds in a pharmaceutical dosage unit as described above will be an efficacious, nontoxic quantity preferably selected from the range of 0.01 - 1000 mg/kg of active compound, preferably 0.1 - 100 mg/kg. When treating a human patient in need of steroid 5-α-reductase inhibition, the selected dose is administered preferably from 1-6 times daily, orally or parenterally. Preferred forms of parenteral administration include topically, rectally, transdermally, by injection and continuously by infusion. Oral dosage units for human administration preferably contain from 0.1 to 500 mg of active compound. Oral administration, which uses lower dosages is preferred. Parenteral administration, at high dosages, however, also can be used when safe and convenient for the patient.

The method of this invention of inhibiting steroid 5-α-reductase activity in mammals, including humans, comprises administering to a subject in need of such inhibition an effective steroid 5-α-reductase inhibiting amount of a pharmaceutically active compound of the present invention.

The invention also provides for the use of a compound of Formula (I) in the manufacture of a medicament for use in the inhibition of steroid 5-α-reductase.

The invention also provides for a pharmaceutical composition for use in the treatment of benign prostate hypertrophy which comprises a compound of Formula I and a pharmaceutically acceptable carrier.

The invention also provides for a pharmaceutical composition for use in the treatment of prostatic adenocarcinoma which comprises a compound of Formula I and a pharmaceutically acceptable carrier. The invention also provides for a process for preparing a pharmaceutical composition containing a pharmaceutically acceptable carrier or diluent and a compound of Formula I which comprises

bringing the compound of Formula I into association with the pharmaceutically acceptable carrier or diluent.

No unacceptable toxicological effects are expected when compounds of the invention are administered in accordance with the present invention.

In addition, the pharmaceutically active compounds of the present invention can be co-administered with further active ingredients, such as other compounds known to treat the disease states of acne vulgaris, seborrhea, female hirsutism, male pattern baldness, benign prostate hypertrophy or prostatic adenocarcinoma or compounds known to have utility when used in combination with 5-α-reductase inhibitors.

Particularly preferred is the co-administration of a 5-α-reductase inhibitor, as disclosed herein, and minoxidil for use in the treatment of male pattern baldness. Particularly preferred is the co-administration of a 5α-reductase inhibitor, as disclosed herein, and a α-receptor antagonist for use in the treatment of benign prostatic hypertrophy.

Preferred is the co-administration of a 5-α-reductase inhibitor, as disclosed herein, and an aromatase inhibitor for use in the treatment of benign prostatic hypertrophy. Preferred is the co-adrninistration of a 5- α-reductase inhibitor, as disclosed herein, a α -receptor antagonist and an aromatase inhibitor for use in the treatment of benign prostatic hypertrophy.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following Examples are, therefore, to be construed as merely illustrative and not a limitation of the scope of the present invention in any way.

EXAMPLE 1 l-(3-hydroxy-estra-l,3,5(10)-triene-17β-carbonyl)-l,3-diiso - propylurea (VI): Y=0, Rι=iPr, R2=iPR, R3=H]

To a stirred solution of 3-hyrdroxy-estra-l,3,5(10)-triene-17β- carboxylic acid (50 g) in ethylacetate (1.5 1) and triethylamine (33 ml) N,N-diisopropylcarbodiimide (32.4 ml) is added dropwise during 5 minutes and then the reaction mixture is refluxed for 1 h.

The reaction mixture is cooled and filtered on buckner; the filtrate is washed with IN HC1, 0.5N NaHCθ3, brine and anhydrified over

sodium sulphate and concentrated to about 150 ml; by cooling the title product precipitates and it is filtered by suction filtration, washed with isoproplic ether.

Following an anallogous procedure the below listed compounds can be prepared: l-(3-hydroxy-estra-l,3,5(10)-triene-17β-carbonyl)-l,3- dicyclohexylurea) l-(3-hydroxy-estra-l,3,5(10)-triene-17β-carbonyl)-l,3- ditertbutylurea)

EXAMPLE 2 l-(3-hydroxy-estra-l,3,5(10)-triene-17β-carbonyl)-l,3-diiso propyl- thiourea [(VI):Y=S,Rι=iPr,R2=iPr]

A solution of 2-pyridyl 3-hyrdroxy-estra-l,3,5(10)triene-17β- carbothioate

(3.0 g) in tetrahydrofurane (70 ml) is treated with 70% sodium hydrogen sulphide monohydrate (3.9 g) and the mixture is heated under reflux for 2 hours. After acidification with IN hydrochloric acid, the mixture is extracted thoroughly with methylene chloride.

The combined organic extracts are washed with water till neutrality, dried over sodium sulphate and evaporated to dryness.

Further purification by flash chromatography on silica gel affords (3-hydroxy-estra-l,3,5(10)-triene-17β-carbothioic aάd[(VII) Y=S].

A solution of the thioacid so obtained in ethyl acetate is treated first with triethylamine and then with N,N'"diisopropylcarbodiimide and the mixture is stirred for 5 hours at room temperature.

The reaction mixture is directly chromatographed on a column of silica gel, so obtaining the title compound.

Following an analogous procedure, the compounds listed below can be obtained: l-(3-hydroxy-estra-l,3-5(10)triene-17β-carbonyl)-l,3-dicycl ohexyl- thiourea

l-(3-hydroxy-estra-l,3,5(10)-triene-17β-carbonyl)-l,3- ditertbutylthiourea.

EXAMPLE S 3-([(trifluoromethyl)sulfonyl]oxy)- 17β-[N-isopropyl-N-(N- isopropylcarbamoyl)carbamoyl]-estra-l,3,5(10)-triene [(II) Y=0, Rι=iPr,

R ₂ =iPR, R ₃ =H]

To a stirred solution of 17β-[N-isopropyl-N-(N- isopropylcarbamoyl)carbamoyl]-estra-l,3,5(10)-triene-3-hydro xy and 2,6- ditert-butyl-4-methylpyridine in methylene chloride maintained under inert atmosphere of nitrogen at room temperature, the trifluoroacetic anhydride is added dropwise.

After stirring for 30 minutes, the reaction mixture is diluted with methylene chloride and washed with saturated aqueous sodium bicarbonate, with IN HCl, with water until neutrality and anhydrified over sodium sulphate.

The foam which is obtained is purified by flash chromatography on silica gel, so obtaining the title compound.

Following an analogous procedure the below listed compounds can be prepared:

3-([(trifluoromethyl)sulfonyl]oxy)-17β-[N-isopropyl-N-(N - isopropylthiocarbamoyl)carbamoyl]-estra-l,3,5(10)-triene;

3-([(trifluoromethyl)sulfonyl]oxy)-17β-[N-cyclohexyl-N-( N- cyclohexylcarbamoyl]-estra-l,3,5(10)-triene; 3-([(trifluoromethyl)sulfonyl]oxy)-17β-[N-cyclohexyl-N-(N- cyclohexylthiocarbamoyl)carbamoyl]-estra-l,3,5(10)-triene;

3-([(trifluoromethyl)sulfonyl]oxy)-17β-CN-tert-butyl-N-t ert- butylcarbamoyl)carbamoyl]-estra- 1,3 ,5( 10)- triene and

3-([(trifluoromethyl)sulfonyl]oxy)-17β-[N-methyl-N-(N,N- diethylcarbamoyl)carbamoyl]-estra-l,3,5(10)-triene.

EXAMPLE 4 17β-[N-isopropyl-N-(N-isopropylcarbamoyl)carbamoyl]-estra- l,3,5(10)-triene-3-carboxylate [(I): Y=0, R ₁₌ iPr, R ₂ =iPr, R ₃ =H]

To a solution of 3-{[trifluoromethyl)sulfonyl]oxy}-17β-[N-isopropyl- N-(N-isopropylcarbamoyl)carbamoyl]estra l,3,5(10)triene in

dimethylformamide, methanol and triethylamine, bis(triphenylphosphine) palladium (II) acetate is added; the mixture is purged with carbon monoxide and then is stirred overnight at room temperature under a carbon monoxide atmosphere. Ethyl acetate is then added and the organic solution is washed with water until neutral, anhydrified over sodium sulphate and the solvent is removed under vacuum. The crude is purified by flash chromatography on silica gel so obtaining the title product.

Following an analogous procedure the below listed compounds can be prepared:

Methyl 17β-[N-isopropyl-N-(N-isopropylthiocarbamoyl)carbamoyl]- estra- 1,3,5( 10)-triene-3-carboxylate;

Methyl 17β-[N-cyclohexyl-N-(N-cyclohexylcarbamoyl)carbamoyl]- estra-l,3,5(10)-triene-3-carboxylate; Methyl 17β-[N-cyclohexyl-N-(N- cyclohexylthiocarbamoyl)carbamoyl]-estra-l,3,5(10)-triene-3- carboxylate;

Methyl 17β-[N-tert-butyl-N-(N-tert-butylcarbamoyl)carbamoyl]- estra- 1,3,5( 10)-triene-3-carboxylate and

Methyl 17β-[N-methyl-N-(N,N-diethylcarbamoyl)carbamoyl]-estra- l,3,5(10)-triene-3-carboxylate.

EXAMPLE 5 17β-[N-isopropyl-N-(N-isopropylcarbamoyl)carbamoyl]-estra- l,3,5(10)-triene-3-carboxylate acid [(I):Y=0, Rι=iPr, R2=iPr, R3=H A mixture of methyl 17β-[N-isopropyl-N-(N- isopropylcarbamoyl)carbamoyl]-estra-l,3,5(10)-triene-3-carbo xylate, methanol and aqueous lithium hydroxide is stirred at room temperature for 5 days.

The methanol is evaporated under vacuum and water is added; the mixture is acidified with IN HCl and extracted with methylene chloride; the organic layers are washed with brine, water until neutrality, anhydrified over sodium sulphate and the solvent removed under vacuum.

The yellow solid obtained is purified by flash cromatography on silica gel so obtaining the title compound.

Following an analogous procedure the below listed compounds can be prepared:

17β-[N-isopropyl-N-(N-isopropylthiocarbamoyl) carbamoyl]-estra- l,3,5,(10)-triene-3-carboxylic acid;

17β-[N-cyclohexyl-N-(N-cyclohexylcarbamoyl)carbamoyl]-es tra- l,3,5,(10)-triene-3-carboxylic acid; 17β-[N-cyclohexyl-N-(N-cyclohexylthiocarbamoyl)carbamoyl]- estra-l,3,5,(10)-triene-3-carboxylic acid;

17β-[N-tert-butyl-N-(N-tert-butylcarbamoyl)carbamoyl]-es tra- l,3,5,(10)-triene-3-carboxylic acid and

17β-[N-methyl-N-(N,N-diethylcarbamoyl)carbamoyl]-estra- l,3,5(10)-triene-3-carboxylic acid.

FORMULATION EXAMPLE Scored tablets for oral use, eachcontaining 250 mg of the active substance, are manufactured as follows:

Composition (for 10,000 tablets)

17β-[N-isopropyl-N-(N-isopropylcarbamoyl) 2500 g carbamoyll-estra- 1 ,3 ,5 ,( 10)-triene-3-carboxylic acid corn starch 275 g talc powder 187 g calcium stearate 38 g

The active substance was granulated with a 4%w/v aqueous solution of methyl cellulose.

To the dried granules a mixture of the remainder of the ingredients is added and the final mixture compressed into tablets of proper weight.