CΛRBOXY SUBSTITUTED 3-QXQ-4-ΛZΛSTBRQIPS AWP THB VSB OF

SUCH PRODUCTS AS INHIBITORS OF THE ENZYME 5ALPHA-

RBPUCTΛSB

The present invention relates to a microbiological process for the transformation of 17β-substituted 4- azaandrostan-3-ones and the use of the biotransformation products as inhibitors of the enzyme 5α-reductase.

The class of molecules known as 4-azasteroids are known to be capable of inhibiting specifically the process of 5α-reduction of testosterone. Up to now, a number of examples of inhibitors of the enzyme 5α- reductase having such a structure are known in literature (Rasmusson, 6, H.; Reynolds, 6.F.; Utne T.; Jobson R.B.; Primka R.L.; Broocks J. R.; Berman C. J. Med. Chem. 1984, 27, 1690-1701. Rasmusson, G, H.; Reynolds, G. F.; Steimberg, N. G.; Walton, E.; Patel G. F.; Liang T.; Cacsieri M. A.; Cheung A. H.; Broocks J. R.; Berman C. J. Med. Chem. 1986, 29, 2298-2315; EP 155096, EP 538192, EP468012, EP 484094). Many of the molecules studied from such a point of view have the common feature of having one unsaturation in position 1, as illustrated in the following Formula 1:

wherein R^ is hydrogen or a lower alkyl group, R ₂ is a OH, OR ₃ , NHR3 group, wherein R3 is a straight or branched ^c ι~ ^c 9 alkyl group, cycloalkyl optionally containing one o more heteroatoms selected from S, 0, N, ^C 10" ^C 20 polycycloalkyl, aryl.

The procedures described for the introduction of this unsaturation are numerous: in some cases, phenylselenic anhydride is used as the reactive (Back T.G. J. Org. Chem. 1981, 46, 1442-1446), or said compounds can be prepared either through a sulfenylation reaction (Zoretic P.A. et al. J. Org, Chem. 1978, 43, 1379-1382) or, as reported more recently, through a silylation and oxidation process, vja the formation of an adduct with DDQ (2,3-dichloro-5,6-dicyano-l,4- benzoquinone) . All these procedures require drastic experimental conditions (for example, the reaction temperatures vary, depending on the procedure followed, from -78' to +120 ^* C, specific cautions must be taken for the used solvents, which should be dry, the used reactives are remarkably toxic). Interestingly, though the conversion percentages into the desired product are in some cases satisfactory, the used reactives are markedly coloured and not atoxic, which makes considerably complex the purification procedures to obtain a pharmaceutically acceptable product, to the detriment of the actual yield of the process. Moreover, the environmental problem of the disposal of the reactives used on an industrial scale asks for novel, less polluting methods to carry out this chemical step. Some types of microorganisms are known from literature to be capable of transforming the traditional

steroid moiety (Phytochemistry, 1984, 23(10), 2131-2154; Microbial transformation of steroids and alkaloids, Iizuka H. , Naito A. University of Tokyo Press, 1967; Yamane T. et al. Biotechnology and Bioengineering, 22, 1979, 2133-2141; Sih CJ. et al. Biochem. Biophys. Acta 38, 1969, 378-379; Dodson R.M. et al. J. Am. Chem. Soc. 82, 1960, 4026) but we found no evidences from literature of any type of biotransformation on structures such as 17B-carboxy substituted 4- azaandrostan-3-ones as described in formula 1.

Surprisingly, it has been found that some microorganisms are capable of biotransforming the azasteroidal moiety so as to introduce an unsaturation into position 1 or an hydroxy function into different positions. Thus, using, for example, the known 17-N-t- butylcarbamoyl-4-azaandrostan-3-one as the substrate (Reynolds, G. F.; Stei berg, N. G.; Walton, E.; Patel G. F.; Liang T. Cacsieri M. A.; Cheung A. H.; Broocks J. R.; Berman C J. Med. Chem. 1986, 29, 2298-2315) and Arthrobacter simplex. Nocardia ___> . as the microorganisms, we successfully obtained the corresponding derivative dehydrogenated at position 1, in yields ranging from 20 to 80% (Scheme 1).

Scheme 1

By use of Penicillium g£. , RhϊZOPV-lB , Cunninαhamella we could obtain mixtures of compounds monohydroxylated at different positions.

Therefore, an object of the present invention is a process for the preparation of compounds of formula (1):

wherein R^ is hydrogen or a lower alkyl group, R ₂ is a OH, R3, NHR3 group, wherein R3 is a straight or branched ^c ι" ^c 9 alkyl group, cycloalkyl containing optionally one or more heteroatoms selected from S, 0, N, C ₁₀ -C ₂ o polycycloalkyl, aryl; which process comprises the steps of: a) culturing a microorganism of the genus Arthrobacter or Nocardia in a suitable culture medium; b) inducing the production of dehydrogenase in said microorganism; c) separating the cells of said microorganism; d) incubating said microorganism with a compound of formula (2) :

wherein R^ and R are as defined above; e) recovering the biotransformed product; f) optionally alkylating the steroid nitrogen atom.

The present invention also comprises a process for the monohydroxylation of the steroid moiety of compounds of formula (2):

wherein R.-, is hydrogen or a lower alkyl group, ₂ is a OH, OR ₃ , NHR3 group, wherein R ₃ is a straight or branched ^c _~ ^c _$ alkyl group, cycloalkyl containing optionally one o more heteroatoms selected from S, 0, N, ^c iθ ^-c 20 PθlY ^c Y ^cloa lkyl, aryl; which process comprises the steps of: a) culturing a microorganism of the genus Penicillium ££. , Rhizopus or cunninghamQl- ; b) adding a compound of formula (2):

wherein R^ and R ₂ are as defined above; and subsequently incubating; c) recovering the monohydroxylated derivative obtained in step (b) .

Following a general procedure, bacteria belonging to the genera Arthrobacter or Nocardia are cultured in suitable liquid media containing a carbon source, generally consisting of an hexose, usually glucose, in concentrations ranging from 0.5 to 15 g/1, preferably from 1 to 10 g/1, and of a mixture of complex substances such as peptones, hydrolysates and extracts, for example meat, blood, gelatin peptones; casein or albumin hydrolysates; yeast extracts or autolysates, in a total concentration ranging from 10 to 100 g/1, preferably from 20 to 50 g/1. The culture is carried out at temperatures from 25 to 35'C, preferably at 27-29'C, for a time ranging from 20 to 72 hours (usually 24 hours). After 5-12 hours from the inocule, the culture broth is added with a solution or suspension containing a molecule capable of inducing the production of the enzyme Al dehydrogenase in bacteria. For this purpose, steroid inducers such as progesterone or hydrocortisone can be used in concentrations- ranging from 10 mg to 1 g/1 (preferably from 100 to 500 mg/1). At the end of the

culture, the cells are separated from the liquid by filtration or centrifugation and used. Either the cells in toto or those fractions having an interesting enzymatic activity can be used. It is also possible to keep the cell activity intact for some months, freezing the cell paste at -25*C The transformation reaction is carried out in a buffer at pH 6.00-8.00 (preferably at pH 7.00) and at a temperature from 25 to 35*C, for times between 10 and 120 hours. To obtain a good conversion percentage, it is essential to use a buffer previously saturated with an organic solvent such as CHCI3, tetradecanol, ethyl acetate, cyclohexane, benzene, n- butanol, and preferably to select from said solvents those with log P higher than 1.5, such as CHCI3, to a maximum of 10% v/v. The resulting incubation medium has to be added with an external co-factor such as menadione or phenazine methasulfate, preferably menadione in concentrations ranging from 20 to 250 mg/1. The substrate to transform is added to the reaction mixture in concentrations ranging from 0.1 to 1 g/1, the substrate being added in a single addition, fractionally or continuously.

The reaction is then worked up filtering off the mycelium on Celite and extracting the filtrate with CHCI3. The resulting organic extracts are dried over sodium sulfate, filtered and evaporated under vacuum to give a crude which is subsequently purified by chromatography on silica: by elution with dichloromethane/methanol 95/5, the dehydrogenated product is recovered in yields ranging from 20 to 80%, and by further isocratic elution, the not biotransformed

product is recovered in yields ranging from 60 to 10%.

Following a general procedure, the hydroxylation reaction is carried out using fungal strains of the genera Penicyllium, Rhizopus or Cunninghamella kept on Sabouraud or PDA (potato-dextrose-agar) slants. These microorganisms are cultured in liquid medium containing one simple sugar as the carbon source. Usually glucose is used, in concentrations from 1 to 50 g/1, but equally suitable to the purpose are molasses in amounts from 5 to 80 g/1. The medium is also added with complex nitrogen sources such as corn steep, peptones (meat or soy peptones), hydrolysated (from casein, albumin or gelatin) and yeast extracts or autolysates. It is useful to combine two or more organic nitrogen sources in such amounts as to reach total concentrations of 2-50 g/1, preferably 5-15 g/1. The medium is complemented with mineral salts, essential for a good growth of the microorganisms, such as sodium and potassium phosphates or diphosphates, magnesium sulfate and calcium, sodium, potassium or magnesium chlorides. The concentrations of these mineral salts usually range from 0.1 to 5 g/1, preferably from 0.2 to 2 g/1. Trace elements, such as iron, zinc, manganese, copper, generally exert a stimulating effect on growth and enzymatic activities. Such trace elements are usually provided in the form of sulfates or chlorides, in concentrations of 1-50 mg/1. The pH of medium is adjusted to a range of 6.00-7.00 (preferably 6.4-6.7) by means of an inorganic base. The culture of the microorganisms is effected at a temperature from 23 to 30 ^* C, preferably from 24 to 28 ^* C for times ranging from 24 to 96 hours (usually from 30

to 48). Once cultures with an efficient vegetative growth have been obtained, the fermentation broth is added directly with the substrate to transform. The latter can be added in the form of either a concentrated solution in a suitable organic solvent, or as a powder, in such an amount as to obtain a final concentration of 100 mg/1 to 1 g/1 in the reaction mixture. It can be useful to add the substrate to transform in small amounts, or slowly continuously. The hydroxylation reaction is carried out at the same growth temperature for a time ranging from 12 to 120 hours (preferably from 48 to 72 hours), also depending on the final concentration of the substrate to transform.

When the complete transformation of substrate is reached (tic CHCI3/CH3OH 95/5) the reaction mixture is filtered on celite and the filtrate is extracted with CHCI3. The resulting organic extracts are dried over sodium sulfate, filtered and evaporated under vacuum to give a crude which is subsequently eluted over silica (1/15 ratio); washing subsequently the cake with dichloromethane/methanol 95/5, the mixture of the hydroxylated products is recovered from the filtrate in yields between 60 and 80%. These products can be subsequently recovered by fractional crystallization or by a chromatographic purification over silica (dichloromethane/methanol 95/5 as eluent).

In a preferred embodiment of the invention, the incubation of 17-N-t-butylcarbamoyl-4-azaandrostan-3-one with Penicillium SP. leads to the monohydroxylation of the steroid moiety in 6 and in 15, as shown in the following scheme 2:

The two dehydrogenation and hydroxylation processes described can also be carried out using the above mentioned microorganisms immobilized or using crude homogenates obtained from said microorganisms. The related experimental techniques are easy to carry out and known to those skilled in the art (Glass T.L. et al. -I. Lipjd. Res.. 1982, 23, 352; Fukui S. et al. Acta Biotechnol. 1981, 1, 339; Omata T. et al. ____.. _ . Microbiol. Biotechnol. 1979, 8, 143; Kim M.N. et al. Biotechnol. Letters 1982, 4, 233; Ohloson S. et al. Eur. _ . APPI. Microbiol. Biotechnol. 1979, 7, 103). Moreover, the above mentioned reactions can also be effected using purified enzymes from the same microorganisms. In this case also, the possibility of recovering these dehydrogenase and hydroxylase and using them as

biocatalysts in the native state or supported over of a polymer matrix is widely described in literature (Grunwald J. et al. _[ . ___ . Chem. Soc. 1986,108, 6732; Snjider-Kambers A.M. et al. Reel. Trav. Chim. Pavs-Bas 1991, 110, 226; Santaniello E. et al. Chem. Rev. 92, 1992, 1071-1140).

The following examples further illustrate the invention.

Example 1 Dehvdroαenation of 17-t-butvlcarbamovl-4-azaandrostan-3- one with A. simplex

The microorganism is kept on slants containing 9 ml of a medium having the following composition: Ingredients Concentration (g/1)

Meat extract (Difco) 1.500

Yeast extract (Difco) 3.000

Peptone (Difco) 6.000

Casein hydrolysate (Costantino) 4.000 Dextrose (Cerestar) 1.000

Agar (Difco) 18.000

The growth takes place in 18-20 hours at a temperature of 28*C. Transplants are carried out every 20-30 days. For the vegetative culture, 300 ml flasks are prepared containing 50 ml each of a medium with the following composition:

Ingredients Concentration (g/1)

Dextrose (Cerestar) 1.000

Bacteriologic peptone (Costantino) 6.000 Casein hydrolysate (Costantino) 4.000

Yeast autolysate (Costantino) 3.000

pH (6.10-6.30) is adjusted to 7.00 by means of IN NaOH and the medium is sterilised at 121 ^* C for 15 minutes. The inocule is effected with 0.50 ml/flask of a suspension obtained washing a slant with 5 ml of saline solution. The flasks are then incubated at 28"C on a rotary shaker at 200 rpm for 17 hours. The subsequent step is carried out in a fer enter of 3 1 total volume, containing 1.7 1 of the above indicated medium and inoculated at the rate of 6% (120 ml). The operative conditions are as follows: 28*C, aeration 0.5 vvm at 200-500 rpm. After 6 hours, 1.7 ml of a suspension of hydrocortisone in 2 ml of acetone are added (at a temperature of 60 ^* C) and culture is carried out for a further 18 hours. After 24 total hours, the cells are recovered by centrifugation at 4.800 xg (20 minutes) and suspended again in 0.1 M phosphate buffer, pH 7.00, saturated with CHC1 ₃ at the rate of 50 g/1 humid weight. The cell suspension is distributed into 300 ml flasks (20 ml/flask), each of them being added with 0.125 ml of a solution prepared from 84 mg of 17β-t-butylcarbamoyl- 4-azaandrostan-3-one and 8.7 mg of menadione in 2 ml of methanol (substrate final concentration 260 mg/1). The cultures are incubated at a temperature of 30 ^* C for 72 hours over rotary shaker at 250 rpm. The reaction is

then worked up filtering the reaction broth on celite and extracting the filtrate with CHCI3 (1:1 v/v to the aqueous phase) . The organic extracts are dried over sodium sulfate, filtered and evaporated to dryness to give a crude which is subsequently purified by chromatography over silica: gradient elution with CH ₂ Cl ₂ /MeOH 95/5 allows to recover the desired product in 60% yields and the starting product in 20% yields. iH-NMR (60 MHz): 0.70 (s, 3H), 0.97 (s, 3H), 1.40 (s, 9H), 3.1-3.6 (m, 1H), 5.2 (m, 1H), 6.3 (m, 1H), 5.8 (d, J = 9Hz, 1H), 6.7 (d, J = 9Hz, 1H). M.p. 253-254 ^# C.

Example 2 Hvdroxvlation of 17-t-butvlcarbamovl-4-azaandrostan-3- one with Penicillium SP.

The fungus is kept on "EMMON'S modification of SABOURAUD'S AGAR" slants, in thermo-stat at a temperature of 26-27 ^* C for 6-7 days until obtaining an abundantly sporulated mycelium. Subsequently the slants can be kept at 5*C for 2-3 months. The vegetative culture is carried out in the same medium as that used for the conservation, free from agar, distributed into 300 ml flasks at the rate of 50 ml/flask. In order to carry out the inoculation, 1.0-1.5 cm^ of mycelium for each flask to inoculate are withdrawn sterilely from a slant by means of a spatula. The felt is finely ground in Potter with about 1 ml of saline solution and the resulting suspension is distributed into the flasks, which are incubated at 27 ^* C on a rotary shaker at 140 rpm for 48 hours. The primary vegetative culture is used to inoculate a medium having the following composition:

Ingredients Concentration (g/1)

Anhydrous dextrose 5. 000

Atomized Corn Steep 2 . 000

Soy peptone 1. 000

Urea 0 . 300

Monobasic potassium phosphate 1 . 000

Magnesium sulfate.7H ₂ 0 0. 300

Iron sulfate.7H ₂ 0 0.005

Zinc sulfate.7H ₂ 0 0. 005

Manganese sulfate.H ₂ 0 0. 001

Copper sulfate.5H 0 0.001

The final pH is adjusted to 6.5 with IN KOH, distributed into 300 ml flasks at the rate of 50 ml/flask and sterilised at 121 ^* C for 20 minutes. The flasks inoculated with 10% of vegetative culture are incubated at a temperature of 27"C (140 rpm) for 24 hours. After this time, in case large pellets or mycelium growth confluent into a single block are present, homogenization in Blendor is effected for some seconds. The finely dispersed mycelium is added with 0.35 ml/flask of a solution of 42 mg/1 o^ 17β-t- butylcarbamoyl-4-azaandrostan-3-one in ethanol (final concentration in the flask 270 mg/1 of substrate) and the whole is incubated again in the above defined conditions. After 24 hours each flask is added with a further 0.7 ml of the same solution, incubating again at 27 ^# C for a further ^{^} 72 hours. At the end of the transformation the reaction mixture is filtered on celite and the filtrate is extracted with CHCI3 (3 times

for an equal volume of the aqueous phase) . The organic extracts are dried over sodium sulfate, filtered and evaporated to dryness to give a crude which is subsequently eluted on silica (1/15 ratio to the crude): subsequent washings with CH ₂ Cl ₂ /MeOH 95/5 allow to recover in 90% yields the mixture of monohydroxylated products which are subsequently separated by crystallization from ethyl acetate. Three products have been obtained having the following spectrum characteristics: 1st product:

¹ H-NMR (500 MHz; CDC1 ₃ ; diagnostic signals) 6: 0.7 (s, 3H), 0.95 (s, 3H), 1.45 (s, 9H), 2.4 (m, 2H), 2.88 (d, 1H), 3.55 (m, 1H), 5.1 (s, 1H), 6.8 (s, 1H, exchangeable);

MS (m/e): 390.30 (m+), 375.30 (m-15), 372.25 (β-18), 357.40 (m-33), 318.30 (m-72), 290.25 (m-100); assignable to 17β-t-butylcarbamoγl-6α-hγdroxy-4-azaan- drostan-3-one; 2nd and 3rd product:

¹ H-NMR (500 MHz; CDC1 ₃ ; diagnostic signals) 6: 0.92-1.04 (dd, 6H), 1.37 (d, 9H), 2.42 (m, 2H), 3.05 (m, 1H), 4.3 and 5.05 (2m, 1H total), 5.22 (s, 1H, exchangeable), 5.5 (s, 1H, exchangeable); MS (m/e): 390.30 (m+), 372.25 (m-18), 357.25 (m-33), 331.25 (m-59), 314.20 (m-73), 290.30 (m-100); assignable to 17β-t-butylcarbamoyl-15α-hydroxy-4-azaan- drostan-3-one and to 17β-t-butylcarbamoyl-15β-hydroxy-4- azaandrostan-3-one. The three monohydroxylated products obtained by biotransformation of the azasteroid moiety using

Penicillium SP. have biological activity on the enzyme 5α-reductase. For this purpose, the enzymatic in vitro method described in WO/9413691, published on June 23, 1994 has been used. Unexpectedly, a strong inhibiting activity on the enzyme 5α-reductase was evidenced for the products 2 and 3, their concentration inhibiting by 50% the enzymatic activity (IC-^ _Q ) being superimposable to that of Pinasteride, used as the control product, whereas product 1 turned out to be poorly active.

Therefore, the present invention also provides the use of the monohydroxylated compounds as therapeutical agents, particularly for the preparation of a medicament having inhibiting activity on testosterone 5α-reductase.

The present invention also provides pharmaceutical compositions for the oral, parenteral and topical administrations, containing the above mentioned compounds together with conventional, pharmaceutically acceptable carriers and excipients.

Examples of oral pharmaceutical compositions are tablets, capsules, sachets and suspensions; examples of parenteral compositions are freeze-dried ampoules or sterile suspensions; examples of topical compositions are creams, ointments, gels, aerosols or foams.

The compositions of the invention are prepared with conventional methods, as for example those described in Remington's Pharmaceutical Sciences Handbook, Mack Pub. XVII Ed. N.Y. , U.S.A.

The daily dosage and mode of administration will be decided by the physician, depending on the disease to treat, the severity of the symptoms and the conditions of the patient (age, sex and weight).