GB2264494A - 17b-n-monosubstituted carbamoyl-11-oxo-4-aza-5-a-androst-1-en-3-one testosterone-5-alpha reductase inhibitors - Google Patents

Abstract

Novel 17 beta -N-monosubstituted carbamoyl-11-oxo-aza-5 alpha -androst-1-en-3-ones have the formula: <IMAGE>

Description

TITLE OF THE INVENTION 17P-N-MONOSUBSTITUTED CARBAMOYL-ll-OXO-4-AZA-5- a-ANDROST-EN-3-ONE TESTOSTERONE-5-ALPHA REDUCTASE INHIBITORS BACKGROUND OF THE INVENTION: The present invention is directed to novel ll-oxo, ll-hydroxy, ll-acyl and 11 alkylether 17beta-N-monosubstituted carbamoyl-4-aza-5a-androstl-en-3 one compounds and the use of such compounds as testosterone-5-alpha reductase inhibitors.

The'art reveals that certain undesirable physiological manisfestations including acne vulgaris, seborrhea, female hirsutism, male pattern baldness and benign: prostatic hypertrophy are the result of hyperandrogenic stimulation induced by excessive accumulation of testosterone or similar androgenic hormones. Early attempts to provide chemotherapeutic agents to counter the unwanted physiological effects of hyperandrogenicity resulted in the discovery of several steroidal anti-androgens that unfortunately had hormonal side effects. For example, the estrogens counteract the effects of androgens but also have a feminizing effect.

Non-steroidal anti-androgens such as 4'-nitro 3'-triflouromethylisobutyla'nilide have also been developed. See Neri et al., Endo., 91, (2), (1972).

These compounds may be peripherally active and compete for androgenic receptor sites which could feminize a male host or the male fetus of a female host.

It more recently became known in the art that the princial mediator of androgenic activity in some target organs is 5-a-dihydrotestosterone which is formed locally in the target organ with the catalytic assistance of the enzyme testosterone-5a- reductase.

It therefore has been postulated and demonstrated that inhibitors of testosterone-5a-reductase prevent or diminish the symptoms of hyperandrogenism. See Nayfe et al., Steroids, 14, 269 (1969) and Voight et al., Endocrinology, 92, 1216 (1973). It has also.

been demonstrated that topical application of either testosterone or 5a-dihydro-testosterone causes enlargement of the female hamster flank organ, an androgen dependent sebaceous structure. However, concommitant administration of 4-androsten-30ne 17ss-carboxlyic acid or its methyl ester inhibited the response elicited by testosterone but did not inhibit the response elicited by 5-a-dihydrotestesterone.

These results indicated that these particular compounds were antiandrogenic because they inhibited testosterone-5alpha-reductase. Since this discovery, a number of steroidal 5a-reductase inhibitors have been found. See, U.S. Pat. Nos. 5,032,586; 5,026,882; 5,017,568; 4,970,205; 4,970,204; and 4,954,446.

A number of steroidal derivatives with a hydroxyl functionality at the 11 position (either alpha or beta) are known. See, for example, U.S. Pat. Nos.

4,361,558; 4,430,270; 4,432,905; 4,446,072; and 4,448,725. A steroidal derivative(s) with a hydroxyl functionality at the 11 position is a known Sa-reductase inhibitor. See U.S. Pat. No. 5,041,433.

A number of 4-aza steroid compounds are known.

See, for example, U.S. Pat. Nos. 2,227,876; 3,239,417; and 3,264,301; French Pat. No. 1,465,544; Doorenbos et al., J. Pharm. Sci., 62, (4), 638-640 (1973); J. Pharm. Sci., 60(8), 1234-1235 (1971); J.

Pharm Aci., 63(4) 620-622 (1974).

In addition, U.S. Pat. Nos. 4,377,584; 4,220,775; 4,859,681; and the articles J.Med. Chem., 27, 1690-1701 (1984) and J. Med. Chem., 29, 2998-2315 (1986) by Rasmussen et al. and U.S. Pat. Nos.

4,845,104 and 4,732,897 disclose 4-aza-17P- substituted-5a-androstan-3-ones which are said to be useful in the treatment of hyperandrogenic conditions. Several other U.S. Patents refer to aza-steroids including 5,061,802 (17- aminibenzoyl -4-aza-5a-androst-1-en-3-ones) and 5 , 049, 562 (17ss-Acyl-4-aza-5a-androst-1-ene-3-ones).

U.S. Pat. No. 4,760,071 specifically refers to the compound 17ss(N-t-butylcarbamoyl)-4-aza- 5a-androst-l-en-3-one. There is a need, however, for novel ll-oxo, ll-alkylether, ll-acyl or ll-hydroxyl azasteroids that are inhibitors of testosterone 5 a-reductase. The compounds of this invention are produced via biotransformation in the ubiquitous green algae, Selenastrum capricornutum.

Use of this organism as an environmental barometer of pollutants is known. See Eirkson et al., "Environmental Assessment Technical Assistance Handbook", Government Publication No. PB87-175345, National Technical Information Service (1987).

SUMMARY OF THE INVENTION: The present invention is concerned with novel 17p-N-(monosubstituted)-carbamoyl-ll-oxo-4- aza-5a-androst-1-en-3-one compounds and pharmaceutical compositions and formulations thereof that are useful as testosterone 5a-reductase inhibitors to treat various hyperandrongenic conditions including acne vulgaris, seborrhea, female hirsutism, male pattern baldness, and benign prostatic hypertrophy. This invention is also concerned with a method of treatment for the above conditions using the claimed compounds.

The present invention is concerned with compounds of the formula:

wherein R is hydrogen, methyl or ethyl.

R2 is a hydrocarbon selected from straight and branched chain alkyl of from 1-12 carbons or monocyclic aryl optionally containing 1 or more lower alkyl substitutents of from 1-2 carbon atoms and/or more halogen (C1, F or Br) substituents.

R' is hydrogen or methyl.

R" is hydrogen or methyl.

R"' is hydrogen, a-methyl or methyl.

X = O (oxo) or H and (OR3) wherein R3 is H, C1-13alkyl, Cl-l2alkylcarbonyl, CarboxylC1-12alkylcarbonyl, arylC0~12alkylcarbonyl, or aryl.

A preferred embodiment of the novel compounds of our invention is represented by the formula:

wherein R is hydrogen, methyl or ethyl, X is H and (oR3), R3 is H, and R4 is a branched chain alkyl of from 4-8 carbons Representative compounds of the present invention include the following: : 17ss-(N-tertbutylcarbamoyl)ll-(hydroxyl)-4-aza-4- methyl- 5a-androst-1-en-3-one, 17ss-(N-isobutylcarbamoyl)11-(hydroxyl)-4-aza-4- methyl 5a-androst-l-en-3-one, 17ss-(N-tert-octylcarbamoyl)ll-(hydroxyl)-4-aza-4- methyl -5a-androst-1-en-3-one, 17ss-(N-octylcarbamoyl)ll-(hydroxyl)-4-aza-4-methyl -5a-androst-1-en-3-one, 17ss-(N-l,l-diethylbutylcarbamoyl)ll-(hydroxyl)-4- aza-4-methyl-5a-androst-l-en-3-one, 17ss-(N-neopentylcarbamoyl)ll-(hydroxyl)-4-aza-4- methyl- 5a-androst-1-en-3-one, 17-(N-tert-amylcarbamoyl)ll-(hydroxyl)-4-aza-4- methyl- 5a-androst-1-en-3-one, 17ss-(N-tert-hexylcarbamoyl)ll-(hydroxyl)-4-aza-4- methyl-5-androst-l-en-3-one;; 17ss-(N-tertbutylcarbamoyl)ll-(oxo)-4-aza-4-methyl - 5a-androst-l-en-3-one, 17ss-(N-isobutylcarbamoyl)ll-(-oxo)-4-aza-4-methyl- 5a-androst-l-en-3-one, 17ss-(N-tert-octylcarbamoyl)11-(oxo)-4-aza-4- methyl- 5a-androst-l-en-3-one, 17ss-(N-octylcarbamoyl)ll-(oxo)-4-aza-4-methyl- 5a-androst-1-en-3-one, 17ss-(n-1,1-diethylbutylcarbamoyl)ll-(oxo)-4-aza- 4-methyl-5a-androst-l-en-3-one, 17-(N-neopentylcarbamoyl)ll-(oxo)-4-aza-4-methyl - 5a-androst-l-en-3-one, 17ss-(N-tert-amylcarbamoyl)ll-(oxo)-4-aza-4-methyl - 5a-androst-l-en-3-one, 17ss-(N-tert-hexylcarbamoyl)11-(oxo)-4-aza-4- methyl 5a-androst-l-en-3-one.

and the corresponding compounds wherein the methyl substituent is replaced in each of the above named compounds by a hydrogen atoms or an ethyl group.

Also included as representative compounds are any of the above indicated compounds having the N-branched chain substituent replaced by a methyl ethyl, propyl, i-propyl, butyl, phenyl; 2, 3 or 4 toxyl, xylyl, 2-bromo or 2-chlorophenyl, 2-6-dichloro, or a 2,6-dibromophenyl substitutent.

In a more preferred embodiment, the compound 17ss(N-t-butylcarbamoyl)-11&alpha;-(hydroxyl)-4-aza- 5a-androst-1-en-3-one is produced by biotransformat ion using the algal organism Selenastrum capri cornutum.

DETAILED DESCRIPTION OF THE INVENTION: The present invention is concerned with novel 17ss-N-(monosubstituted)-carbamoyl-ll-oxo- 4-aza-5a-androst-1-en-3-one compounds and pharmaceutical compositions and formulations thereof that are useful as testosterone Sa-reductase inhibitors to treat various hyperandrongenic conditions including acne vulgaris, seborrhea, female hirsutism, male pattern baldness, and benign prostatic hypertrophy. The present invention is concerned with compounds of the formula:

wherein R is hydrogen, methyl or ethyl.

R2 is a hydrocarbon selected from straight and branched chain alkyl of from 1-12 carbons or monocyclic aryl optionally containing 1 or more lower alkyl substitutents of from 1-2 carbon atoms and/or more halogen (C1. F or Br) substituents.

R' is hydrogen or methyl.

R" is hydrogen or methyl.

R"' is hydrogen, a-methyl or methyl.

X = O (oxo) or H and (OR3) wherein R3 is H, C1-13alkyl Cl-l2alkylcarbonyl, CarboxylC112alkylcarbonyl, arylC0-12alkylcarbonyl, or aryl.

A preferred embodiment of the novel compounds of the present invention is represented by the formula:

wherein R is hydrogen, methyl or ethyl, X is H and (OR3), R3 is H, and R4 is a branched chain alkyl of from 4-8 carbons. Representative compounds of the invention include the following: 17ss-(N-tertbutylcarbamoyl)11&alpha;-(hydroxyl)-4-aza-4- methyl-5a-androst-l-en3-one.

17ss-(N-isobutylcarbamoyl)11&alpha;-(hydroxyl)-4-aza-4- methyl-5a-androst-l-en-3-one 17ss-(N-tert-octylcarbamoyl)lla-(hydroxyl)-4-aza-b methyl-5a-androst-i-en-3-one 17p-(N-octylcarbamoyl)lla-(hydroxyl)-4-aza-4- methyl-5a-androst-l-en-3-one.

17p-(N-l,l-diethylbutylcarbamoyl)lla-(hydroxyl)-4- aza-4-methyl-5a-androst-l-en-3-one 17ss-(N-neopentylcarbamoyl)lla-(hydroxyl)-4-aza-4- methyl-5a-androst-l-en-3-one, 17ss-(N-tert-amylcarbamoyl)lla-(hydroxyl)-4-aza-4- methyl-5a-androst-l-en-3-one, 17ss-(N-tert-hexylcarbamoyl)lla-(hydroxyl)-4-aza-4- methyl-5a-androst-1-en-3-one;; 17ss-(N-tertbutylcarbamoyl)ll-(oxo)-4-aza-4-methyl- 5a-androst-1-en-3-one, 17ss-(N-isobutylcarbamoyl)ll-(oxo)-4-aza-4-methyl- 5a-androst-l-en-3-one, 17ss-(N-tert-octylcarbamoyl)ll-(oxo)-4-aza-4-methyl- 5a-androst-1-en-3-one, 17ss-(N-octylcarbamoyl)ll-(oxo)-4-aza-4-methyl- 5a-androst-l-en-3-one, 17ss-(N-l,l-diethylbutylcarbamoyl)ll-(oxo)-4-aza-4- methyll5a-androst-l-en-3-one, 17ss-(N-neopentylcarbamoyl)ll-(oxo)-4-aza-4-methyl- 5a-androst-1-en-3-one, 17ss-(N-tert-amylcarbamoyl)ll-(oxo)-4-aza-4-methyl- 5a-androst-l-en-3-one, 17ss-(N-tert-hexylcarbamoyl)ll-(oxo)-4-aza-4-methyl- 5a-androst-1-en-3-one.- and the corresponding compounds wherein the methyl substituent is replaced in each of the above named compounds by a hydrogen atom or an ethyl group.

Also included as representative compounds are any of the above indicated compounds having the N-branched chain substituent replaced by a methyl ethyl, propyl, i-propyl, hlltyl, phenyl; 2, 3 or 4 toxyl, xylyl, 2-bromo or 2-chlorophenyl, 2-6- dichloro, or a 2,6-dibromophenyl substitutent.

The novel compounds of formula I and II of the present invention are prepared by adding varying concentrations of a non-oxidized or non-hydroxylated 4-azasteroid (also termed "non-biotransformed precursor") to cellular preparations or cultures of the commonly known and ubiquitous green algae Selenastrum capricornutum. This green algae is a unicellular or colonial organism from the Phylum Chlorophyta, Order Chlorococcales. It occurs worldwide. Selenastrum capricornutum is available at the American Type Culture Collection at 12301 Parklawn Drive, Rockville, MD 20852 (ATCC 22662).

The University of Texas at Austin also maintains a culture collection of this species (1648).

Scheme 1 illustrates a synthesis of compounds of the instant invention.

0 II o c-Ennz II Selsnas trum capr C-NHR1 'Continious Flow systenl Continuous Flow Systerri R I C1 base /0 11 - hydroxyl (2) acylhalide / (R5C-XD Cl) base (NaH) C2) C,~,, alkyl hal 0 11 C1.12 alkyl ha3 C-NHR2 0 RJ-C- II R5-C-o < C-NEEP R R''' Ct-t2 % < R''' I R 0 11 - acyl R 11 - alkylethsr

Scheme 1 The non-biotransformed precursor is biotransformed in a continuous-flow Selenastrum capricornutum system. The ll-hydroxyl product may be purified directly or alkylated with a C1~12 alkyl halide -or acylated with an acylhalide to yield compounds claimed in the instant invention.

Conditions for culturing and maintaining S.

capricornutum are described in the literature. The organism is readily available and may easily be cultured and maintained under a variety of conditions. See Eirkson et al., "Environmental Assessment Technical Assistance Handbook, Government Document No. PB87-175345, pp. 3-30 (1987); U.S. Environmental Protection Agency, "Algal Assay Procedure:Bottle Test", U.S. E.P.A., Corvalis, Oregon (1971); U.S. Environmental Protection Agency, "Toxic Substances Control Act Guidelines, Final Rule, Federal Register 50, 39323, 39330 (1985); Miller et al.," The Selenastrum capricornutum Printz Algal Assay Bottle Test", EPA-600/9-78-018, U.S. Environmental Protection Agency, Corvalis, Oregon (1978); and Stein, J.R., Ed. "Handbook of Phycological Methods. Culture Methods and Growth Measurements." Cambridge University Press, Cambridge, UK (1973).

The precursor 4-azasteroids used as starting materials in the process for producing the claimed oxidized derivatives are partially soluble in the aqueous cellular system. For example, the azasteroid Finasteride (17ss(N-t-butylcarbamoyl)-4-aza-5a- androst-l-en-3-one) reaches its saturation point in water at sixty(60) milligrams/liter(mg/L). The term "oxidation" in this application refers to the process of adding an oxygen atom or a hydroxyl molecule to the starting (precursor) 4-azasteroids and includes oxidation at the ll-position. It further includes oxidation to the lla (hydroxyl) compounds.

Saturated solutions of the precursor azasteroids may be added to cultures of the organism Selenastrum capricornutum which oxidizes the respective 4azasteroid over time to achieve substantially complete conversion of the starting material to the ll-oxo or ll-hydroxyl compounds claimed in the instant invention. The ll-hydroxyl compound may be further derivatized to an alkyl or aryl ether or an acyl derivative by conventional means (See Scheme 1).

If live cultures of the organism Selenastrum capricornutum are used to produce the compounds claimed in the instant invention, the cell batches can be monitered over daily or weekly periods to determine cell density and overall health and to ensure successful biotransformation to the claimed products. The term "biotransformation" retains its ordinary meaning and refers to a chemical modification of a particular molecule or biomolecule to a distinct product via the use of a microorganism such as green algae. In this specification, the term also includes cellular extracts of Selenastrum capricornutum, and enzymatic preparations derived therefrom, which hydroxylate or oxygenate the starting 4-azasteroids. The term "non-biotransformed precursor" refers to the starting 4-azasteroids used in the process to produce the claimed compounds.The term "hydroxylate" refers to hydroxylation or the addition of -OH to a carbon atom on the starting azasteroid. The term "oxygenate" refers to oxidation of a carbon atom on the azasteroid to the respective ketone or oxygen radical or oxygen anion. It is understood that an intermediate ketone (isolated or unisolated) or stable ketone or oxygen radical or anion may be further reduced (or reacted with H2O.or other hydrogen source) either in the'intact cellular system, the cellular extract, or in an.enzymatic preparation derived from said cells or cellular extract, to yield the hydroxyl derivative. In addition, the hydroxyl compound may be dehydrogenated to form the respective ketone.

The term enzyme or enzymatic preparation refers to oxygenases which may be responsible for adding oxygen to the 4-azasteroid substrate. The term oxygenase encompasses the well known class of enzymes, the monooxygenases which are also called hydroxylases. For example, it is known that the hydroxylase cytochrome P-450 hydroxylates corticosterone to the llss(hydroxyl) compound.

The term "aryl" shall mean a mono- or polycyclic system composed of 5- and 6- membered aromatic rings containing 0, 1, 2, 3 or 4 heteroatoms chosen from N, O or S and either unsubstituted or substituted with halogens, C1-12 alkyloxy groups, or hydroxyl groups.

The term "alkyl" shall mean straight or branched chain alkane, alkene or alkyne.

The term "halogen" shall include flourine, chlorine, bromine and iodine.

The term "oxy" shall mean an oxygen atom.

The term "hydroxyl" shall mean an (OH) group.

The term "oxo" shall mean an oxygen atom attached to a carbon atom which is attached to two carbon atoms or C(CO)C.

The term "batch system" refers to a vessel to which algae, algal nutrient solution and precursor azasteroid are added wherein the algal biotransformed solution is filtered and extracted or purified by HPLC. The term "continuous flow system" refers to an enclosed packed algal bed where the in-flow contains nutrients, algae, and starting material and the "out-flow" contains essentially filtered biotransformed product in solution which may then be further purified.

The algae growth and density or effects thereon may be compared to a control batch that does not contain the starting 4-azasteroid. Additional healthy cells may be added to the batch over time to ensure efficient and further biotransformation to the desired target compound. Algal cell concentrations may be monitered over a two week period, or longer if desired, at several day intervals. The concentration of starting azasteroid can be varied and may include either saturated or unsaturated concentrations in the particular aqueous culture system. The cell system can be monitered for both changes in cell density and growth and for changes in the morphological characteristics of the batch. The temperature of the system may range from 1" to 30"C.

Advantageously, compounds claimed in the instant invention may be produced in a continuous flow system. This system is initially charged with algae packed on a solid bed. The "in-flow" initially contains nutrients to promote algal growth. When the desired biomass density is achieved, the precursor 4-azasteroid is added to the in-flow stream. The "out-flow" is then monitered for biotransformed product (such as 17ss(N-t-butylcarbamoyl)-ll- a(hydroxyl)-4-aza-5a-androst-l-en-3-one) The biotransformed product may readily be purified and collected at this stage. The rate or volume-of inflow may also by controlled to maximize bioconversion or biotransformation. Nutrients may be added to maintain a healthy algae culture. Additional algae may be added to the inflow if necessary.

Advantageously, the pH of the aqueous system is approximately 7.0 and the temperature is room temperature or 24 + 2"C.

Production of the hydroxylated .or oxygenated azasteroid products may be monitered by High Performance Liquid Chromatography (HPLC) over time.

Production of the hydroxylated or oxygenated compounds claimed in the instant invention may be monitered both by peak loss of the particular starting 4-azasteroid used and by the concommitant peak rise in the target compounds claimed in the '.*st; invention. The HPLC elution profile may show -ndllction of the oxygenated compound claimed in the instant invention as the major component. Once complete loss or disappearance of starting 4-azasteroid has occured, or after a majority of starting compound loss has occured, the target products may readily be purified and isolated via extraction and purification technology to yield the compounds claimed in the instant invention.

In a batch system, the aqueous algal solution is filtered to remove cellular solids and then extracted with an organic solvent to obtain converted oxidized product. Polypropylene cartridges or other suitable filters may be used as the filtering system. To remove any bound target compounds claimed in the instant invention from the cellular solids or membranes, the filtered solids may be washed with a solvent such as methanol.

Alternatively, the aqueous solution from a batch system or the effluent from the continuous stream system may be directly added-to a HPLC system.

Advantageously, the extract or combined extracts or initial HPLC discharge can be further purified by HPLC to obtain pure oxidized 4-azasteroids. The purified compounds may then be analyzed by mass spectrometry and by proton or carbon Nuclear Magnetic Resonance (NMR) to determine the structures of the target compounds. Mass spectrometry will indicate the addition of the oxygen or hydroxyl moiety. NMR will also reflect the addition of the oxygen or hydroxyl atom or molecule. The reactions and purifications can be run at any scale to achieve desired quantities of the claimed compounds.

Advantageously, the compounds of the instant invention may be prepared by passing a saturated aqueous solution of 4-azasteroid, such as finasteride, through a large algal(Selenastrum capricornutum) bed in a continuous flow stream system to produce a ll-a-hydroxyl 4-azasteroid, such as 17ss(N-t-butylcarbamoyl)lla(hydroxyl)-4-aza-5a-androst -l-en-3-one. The reaction or biotransformation may be run at various scales to produce either small or large quantities of the ll-hydroxyl azasteroids.

The compounds of the present invention, prepared in accordance with the method described above, are antiandrogens by virtue of their ability to specifically inhibit testosterone 5-a-reductase found in prostatic tissue.

Additionally, the present invention is concerned with providing an alternative method of treating the hyperandrogenic conditions of acne vulgaris, seborrhea, and female hirsutism by topical administration, and a method of treating all of the above conditions as well as benign prostatic hypertrophy, by parenteral administration, of the novel compounds of the present invention.

The present invention is thus concerned with providing suitable topical and parenteral pharmaceutical formulation for use in the novel methods of treatment of the present invention.

Compositions containing the compounds of the present invention as the active ingredient for use in the treatment of the various androgenic conditions may be administered in a wide variety of therapeutic dosage forms in conventional vehicles for systemic administration, as for example, by oral administration in the form of tablets, capsules, solutions, or suspensions, or by intravenous injection. 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 a 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.The liquid forms may be 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.

For the treatment of acne vulgaris, seborrhea, female hirsutism, the compounds of the present invention are administered in the formula or pharmaceutical composition comprising the active compound in combination with a pharmacologically acceptable carrier adapted for topical administration. These topical pharmaceutical compositions may be in the form of a cream, ointment, gel or aerosol formulation adapted for application to the skin. These topical pharmaceutical compositions containing the compounds of the present invention ordinarily include about 0.1% to 15% of the active compound in admixture with about 85% to 99.9% of the vehicle.

The method of preparing the novel compounds of the present invention, already described above in general terms, may be further illustrated by the following example.

EXAMPLE 1 Production of 17P-(N-t-butylcarbamoyl)-lla (hvdroxvl)-4-aza-501-androst-l-en-3-one.

Finasteride (17P-(N-t-butylcarbamoyl)- 4-aza-5a-androst-l-en-3-one), a 4-azasteroid, was added to a cellular batch of the commonly found green algal organism, Selenastrum capricornutum at four different concentrations: (1) 8.7 mg/L; (2) 15.5 mg/L; (3) 28.0 mg/L and (4) 49.0 mg/L(approaching saturation concentration). The biotransformation was allowed to proceed over a fourteen (14) day period in each concentration batch. A control batch was also used to moniter the algal system over the same period of time for comparative purposes.

The green algae were cultured in media prepared according to the formula described in Table 1. This forumulation is further described in Miller et al., "The Selenastrum capricornutum Printz Algal Assay Bottle Test." EPA-600/9-78-018, U.S.

Environmental Protection Agency, Corvalis, OR. (1978).

Table 1. Formula for the preparation of media used in the culture of the green alga, Selenastrum capricornutum Salts Amount (g) A. Macronutrients NaNO3 12.750 MgSO4-7H20 7.350 K2HPO4 0.522 NaHCO3 7.50 MgCl2#6H2O 6.08 CaC12-2H2O 2.20 B. Micronutrients H3BO3 0.0928 MnCl2#4H2O 0.208 ZnCl2 0.00164 FeCl3.6H20 0.0799 CoC12R6Hv0 0. 000714b Na2MoO4#2H2O 0.000363C CuCl2#2H2O 0. 000006d Na2EDTA#2H2O 0.150 aZnC12 - Weigh out 164 mg and dilute to 100 ml. Add 1 ml of this solution to the micronutrient stock solution.

bCoCl2.6H20 - Weigh out 71.4 mg and dilute to 100 ml. Add 1 ml of this solution to the micronutrient stock solution.

cNa2MoO4#2H20 - Weigh out 36.6 mg and dilute to 10 ml. Add 1 ml of this solution to the micronutrient stock solution.

dCuC12-2H20 - Weigh out 60.0 mg and dilute to 1,000 ml. Take 1 ml of this solution and dilute to 10 ml. Take 1 ml of the second dilution and add to the micronutrient stock solution.

Nutrient soltuions were prepared from analytical or reagent grade chemicals as concentrated stock solutions, filter-sterilized, and stored in the dark at 4"C. Macronutrient stock solution were prepared by dissolving the weight in grams of chemical listed in Section A of Table 1 into 500 ml of deionied water. The micronutrient stock solution was prepared in accordance with the instructions at the bottom of Table 1. Culture media was prepared by adding one ml of each macronutrient stock solution and one ml of the micronutrient stock solution to one liter of deionized laboratory tapwater. Culture media in control solutions exhibited a pH of approximately 7.0, hardness of 51 mg/L (as CaCO3), alkalinity of 12 mg/L (as CaCO3), and a conductivity of 210 umhos/cm.

Cultures of S. capricornutum were initiated daily by transferring a four-ml aliquot of an established culture (3-10 days old) to 200 ml of autoclaved media in 500-ml Erlenmeyer flasks.

Cultures were incubated at a temperature of 24+/-2OC under continuous fluorescent lighting (400+/foot-candles) on a shaker table. The established culture (30 mls at 7 days old) was then centrifuged to concentrate the algal cells and remove culture media. Cells were then resuspended in fresh media to determine the cell concentration. The algal cell concentration was then adjusted to approximately 105 cells/ml to achieve an initial algal cell concentration of 103 cells/ml in the biotransformation vessel. One ml of this algal solution was added to 99 ml of an almost saturated (50 mg/L) solution of finasteride in a 250 ml flask. The flask was then placed on a gyratory shaker operating at 100 rpm in an environmental chamber.

A comparison of the cell concentrations in each transformation batch over time indicated no major statistical differences between cell numbers in the control or in each batch after a ten to fourteen day period. Between the first biotransformation day and the tenth day several variations in cell numbers were detected in the various concentration batches.

For example, after five days cell numbers diminished in the 28 mg/L batch and in the 49 mg/L batch but cell numbers normalized to control levels after a ten day period. Algal cell densities were determined from microscopic examination of single subsamples of the cultures using a Palmer Maloney counting cell (0.1-ml capacity) and a Whipple square micrometer for enumerating algal cells. The counting procedure entailed viewing a maximum of approximately 100 Whipple square sample (represents a total of 100 x 100 = 100,000 "small squares") across the diameter of the Palmer-Maloney cell. See American Public Health Association, "Standard Methods for the Examination of Water and Wastewater, 15th Edition. American Public Health Association, Washington, D.C. (1980).

At day zero, the measured concentrations for each starting concentration was 12.0 mg/L; 14.0 mg/L; 24.0 mg/L; and 39.0 mg/L. The measured concentration of the starting component in the algal solution was determined by comparing its Ultraviolet (UV) response to the known UV response of the starting azasteroid at various prepared concentrations. The biotransformation was allowed to proceed at each concentration over a fourteen day period and after this period less than 1 mg/L of the starting compound was detected in each batch. The biotransformation reaction was monitered by HPLC and purified on C18 Sep Pack cartridges.The elution prvX > after the full two week period indicated that the target compound, 17P-(N-t-butyl-carbamoyl)-lla-(hydroxyl) 4-aza-5alpha-andros-l-en-3-one, represented (on average) 68 percent of the product blend, 17.3 micrograms of the above lla- (hydroxyl) azasteroid was isolated, purified, and characterized.

Thermospray (TSP) mass spectroscopy was performed on the major biotransformation product.

The results indicated a molecular ion (M+H]+ at m/z 389 which is 16 atomic units greater than the starting reactant's (finasteride) molecular ion of m/z 373 [M+H]+. 16 atomic units Qr Daltons (Da) is the molecular weight of an additional oxygen moiety.

Proton (1H) NMR was used to confirm the location (carbon atom point of attachment on the azasteroid skeleton) and stereoposition (alpha or beta, where the 17-beta position is defined as the axial position of a classical steroidal structure and the 17-alpha position is defined as the equatorial position on the same steroidal three dimensional skeleton) of the hydroxyl group on the biotransformed product. 1H NMR showed a new HC-OH multiplet at 4.09 ppm- relative to the starting reactant's NMR spectrum. In addition, downfield shifts of 1.10 parts per million (ppm) for the C-l hydrogen and 0.11 ppm for the C-19 hydrogens occured relative to the starting material. The downfield displacements were indicative of a new nearby substituent. These results demonstrated that the hydroxylation had occurred at the Il- position.

If hydroxylation had occured at the alternative nearby position, the six position, then the normal C-S hydrogen would appear with reduced multiplicity and would be displaced downfield. In fact, the C-S hydrogen signal was unchanged. Likewise, if hydroxlation had occured at the C-9 position, then the new multiplet at 4.09 ppm would not have appeared. The lla configuration for the hydroxyl was indicated because of the modest 0.1 ppm displacement of the C-19 signal and the unchanged chemical shift for the hydrogens on C-18 relative to spectrum for the starting azasteroid finasteride. An 1113 hydroxyl causes a 0.25 ppm downfield displacement of both angular(axial) methyl peaks. The biotransformed product, l713(N-t-butyl- carbamoyl )-lla- (hydroxyl )-4-aza-5a-androst-l-en-3- one, demonstrated inhibition of the prostate enzyme testosterone 5-a-reductase. As indicated in Scheme 1, this compound may be further derivatized to alkyl or aryl ethers or acyl compounds. The acyl derivatives may readily be used as radiolabeled 4-azasteroidal probes.

Claims (10)

WHAT IS CLAIMED IS:

1. A compound of the formula:

wherein the double line indicates a double bond which can be present; R is hydrogen, methyl or ethyl; R2 is a hydrocarbon selected from straight and branched chain alkyl of from 1-12 carbons or monocyclic aryl optionally containing 1 or more lower alkyl substitutents of from 1-2 carbon atoms and/or more halogen substituents; R' is hydrogen or methyl; R" is hydrogen or methyl; R"' is hydrogen, a-methyl or 13-methyl; X = oxo or H and (OR3); wherein R3 is H, C1-13 alkyl, C1-12 alkylcarbonyl, Carboxy C1-12alkylcarbonyl, arylC0-12 alkylcarbonyl, or aryl.

2. A compound according to Claim 1 wherein X is ll-H,(hydroxyl).

3. A compound according to Claim 1 wherein X is ll-ssH,a(hydroxyl).

4. A compound according to Claim 1 wherein X is oxo.

5. A compound according to Claim 3 comprising: 17p-(N-tertiarybutylcarbamoyl)-lla-(hydroxyl)-4- aza-4-methyl-5a-androst-l-en-3-one; 17ss-(N-isobutylcarbamoyl)-lla-(hydroxyl)-4-aza- 4-methyl-5a-androst-l-en-3-one; 17ss-(N-tertiaryoctylcarbamoyl)-lla-(hydroxyl)-4- aza-4-methyl-5a-androst-l-en-3-one; 17ss-(N-octylcarbamoyl)-11&alpha;-(hydroxyl)-4-aza-4- methyl-5a-androst-l-en-3-one; 17ss-(N-diethylbutylcarbamoyl)-lla-(hydroxyl)-4- aza-4-methyl-5a-androst-l-en-3-one; 17ss-(N-neopentylcarbamoyl)-11&alpha;-(hydroxyl)-4- aza-4-methyl-5a-androst-1-en-3-one; l713-(N-tertiaryamylcarbamoyl)-lloc-(hydroxyl )-4-aza- 4-methyl-5a-androst-l-en-3-one; 17ss-(N-tertiaryhexylcarbamoyl)-lla-(hydroxyl)-4- aza-4-methyl-5a-androst-l-en-3-one.

6. A..-c^mpound according to Claim 4 comprising: 17ss-(N-tertiarybutylcarbamoyl)-ll-(o o)-4-aza-4- methyl-5a-androst-l-en-3-one; 17ss-(N-isobutylcarbamoyl)-11-(oxo)-4-aza-4- methyl-5a-androst-l-en-3-one; 17ss-(N-tertiaryoxtylcarbamoyl5-11-(oxo)-4-aza-4- methyl-5a-androst-l-en-3-one; 17ss-(N-octylcarbamoyl)-11-(oxo)-4-aza-4- methyl-5a-androst-l-en-3-one; 17ss-(N-diethylbutylcarbamoyl)-11-(oxo)-4-aza- 4-methyl5a-androst-l-en-3-one; 17ss-(N-neopentylcarbamoyl)-11-(oxo)-4-aza-4- methyl-5a-androst-l-en-3-one; 17ss-(N-tertiaryamylcarbamoyl)-ll-(oxo)-4-aza-4- methyl-5a-androst-l-en-3-one; 17ss-(N-tertiaryhexylcarbamoyl)-11-(oxo)-4-aza-4- methyl-5a-androst-l-en-3-one.

7. A compound according to Claim 5 comprising 17ss(N-t-butylcarbamoyl)-lla (hydroxyl)-4-aza-5a-androst-l-en-3-one.

8. The use of a compound according to any of claims 1 to 7 in treating the hyperandrogenic condition of acne vulgaris, seborrhea, female hirsutism, and benign prostatic hypertrophy.

9. The use of a compound according to any of claims 1 to 7 for inhibiting testosterone Sa-reductase.

10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the compound of Claim l.