PREPARATION THEREOF, USE THEREOF AS ANTIANDROGENS AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

The present invention relates to the new 3-oxo- and 3-thio-4-aza-5θ-androstenones and -androstanones substituted at the 17-position, having testosterone 5 (- reductase inhibiting activity, to the processes for the preparation thereof, to the therapeutic uses thereof and to the pharmaceutical compositions containing them. Particularly, the present invention relates to compounds of the general formula (A) :

wherein: the carbon-carbon bond at the 1-2-positions can be simple or double;

X is oxygen or sulfur;

R ¹ is hydrogen or linear or branched C-_c ₄ alkyl ; R ¹¹ and R ¹¹¹ , when X is oxygen, are respectively:

-CH-- group and an oxygen atom, which together the carbon atom at the 17-position, form an 0L- or β- epoxyde; or R ¹¹ is a ^β -CH ₂ _NH-R _a or β-CH(CH ₃ )-NH-R , where R is a linear or branched C, -c , alkyl , and R ¹¹¹ is Ci!-hydrogen; or R ¹¹ is β-CO-NH-NH-C(16 ) , where C(16) is the carbon atom at the 16-position, and R ¹¹¹ is & ^_

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hydrogen, or R ¹¹ and R ¹¹¹ form together a =CH ₂ group; and

R ¹¹ and R ¹¹¹ , when X is sulfur, have the same meanings as seen when X is oxygen, and further represent: R ¹¹ a 17&- or 17β-hydroxy group, optionally esterified with a linear or branched C^-Cg carboxylic acid, and R ¹¹¹ is a hydrogen atom; or R ¹¹ is an optionally salified carboxy group, an acetyl group, a 17 - or 17β- CONHR ^IV , where R ^IV is a linear or branched C,-c ₄ alkyl, and R ¹¹¹ is hydrogen; or

R ¹¹ and R ¹¹¹ , together with the carbon atom at the 17- position, form a keto group.

The present invention also relates to the diastereoisomeric and enantiomeric forms, together with the mixtures thereof, of the compounds (A).

Preferred compounds of the above general formula are those in which R ¹ is hydrogen, methyl or ethyl.

Particularly preferred compounds are those in which R ¹ is hydrogen, X is oxygen and: R ¹¹ and R ¹¹¹ form together a =CH group (compounds 5 and 8) ; or

R ¹¹ and R ¹¹¹ , together C(17) are ⁽ X- or β-epoxyde, the carbon-carbon bond at the 1-2 positions being unsaturated (compound 15) or saturated (compound 15 bis) ; or

R ¹¹ is B-CH ₂ _NH-C(CH ₃ ) ₃ and R ¹¹¹ is hydrogen, the carbon-carbon bond at the 1-2 positions being unsaturated (compound 16) or saturated (compound 16 bis) ; or R ¹¹ is β-CH ₂ _NH-CH ₃ and R ¹¹¹ is hydrogen, the carbon-carbon bond at the 1-2 positions being saturated

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(compound 19); or

R ¹¹ is β-CH(CH ₃ )_NH-C(CH ₃ ) ₃ and R ¹¹¹ is hydrogen, the carbon-carbon bond at the 1-2 positions being unsaturated (compound 17) or saturated (compound 17 bis) ; or

R ¹¹ is β-CH(CH ₃ )-NH-CH ₃ and R ¹¹¹ is hydrogen, the carbon-carbon bond at the 1-2 positions being saturated (compound 20); or

R ¹¹ is β-CO-NH-NH-C(16) , and R ¹¹¹ is H, the carbon-carbon bond at the 1-2 positions being unsaturated (compound 18) or saturated (compound 18 bis).

Further particularly preferred compounds are those in which R ¹ is hydrogen, X is sulfur and: R ¹¹ and R ¹¹¹ form together a =CH ₂ group (compounds 6 and 9) ; or

R ¹¹ and R ¹¹¹ are = 0 (compounds 1 and 3); or R ¹¹ and R ¹¹¹ , together C(17 ) are Ot- or β-epoxyde, (compounds 7 and 10); or R ¹¹ is Ql- or β-OH and R ¹¹¹ is hydrogen, (compounds 2 and 4) ; or

R ¹¹ is tt- or β-CO-NH-R ^IV , and R ¹¹¹ is H (compounds 13 and 14).

R ¹¹ is 0- or β-acetyl, and R ¹¹¹ is hydrogen (compound 11); or

R ¹¹ is Ot- or β-COOH and R ¹¹¹ is hydrogen (compound 12).

The availability of drugs capable of specifically inhibiting the process of testosterone 5Wr-reduction can be important for the therapy of pathologies associated with 50v*"eductase hyperactivity, such as for example

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prostatic hypertrophy, prostatic carcinoma, acne and seborrhea, hirsutism, hair loss, [R.E. Gloyna, J.D. Wilson J. Clin. Endocr. 2^, 970 (1969); G.H. Jacobi, J.D. Wilson J. Clin. Endocr. Metab. 4_4, 107 (1977); F.K. Habib, A.L. Tesdale, G.D. Chisholm, A. Busuttil J. Endocrinol. 9_1' ²³ (1981)]. An example of competitive inhibitor of testosterone 50(,-reductase is δ ^" -4- androsten-3-one-17β-carboxylic acid [W. Voigt, S.L. Hsia J. Biol. Chem. 248, 4280 (1973)], whose activity, passing from the in vitro to the in vivo model, appears rather controversial, whereas it has been demonstrated that a competitive in vitro inhibitor such as 170-N,N- diethylcarbamoyl-4-methyl-4aza-50l-androsten-3-one exerts a very good in vivo inhibiting activity [J.R. Brooks et al. Endocrinol. 109, 830 (1981); J.R. Brooks, C. Berman, M. Hichens, R.L. Prinka, G.F. Reynolds, G.H. Rasmusson Proc. Soc. Exp. Biol. Med. 1969, 67 (1982)].

Nowadays a number of compounds, both azasteroidal

[J. Med. Chem. 27_, 1690 (1984); 23, 2298 (1986); Pharmacologist 2!£( ³ )' ^Abs - ²³⁵ (1987); Endocrinology,

130, 685-694 (1992); E.P. 0155096, US-4,377,584 and

4,220,775], and non steroidal (E.P. 0291245A2; J. Med.

Chem., 3j>' 421 (1993)), having testosterone 5 i- reductase inhibiting activity, which are used or potentially applied in the treatment of pathological conditions originating from androgen hyperproduction

(therapy and prevention of benign prostatic hypertrophy, therapy of prostatic carcinoma, therapy of acne and seborrhea, treatment of female hirsutism, treatment of hair loss), are well known.

Recently, the research activity in this field has

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received new impulse from the discovery of the existence of two different genes encoding for two different kinds of 50Creductase, the former of type I and the latter of type II, whose physiological role has not completely been ascertained, but which could act on differently located receptors in the human body (Andersson, S; Russel, D.W. , Proc. Natl. Acad. Sci. U.S.A. (1990, 8 , 3640-3644; Jenkins, E.P.; Andersson, S.; Imperato-Mc Ginley, J. ; Wilson, J.D.; Russel, D.W., J. Clin. Invest. (1992), 9, 293-300).

It has now been found, and it is an object of the present invention, that the compounds of formula (A) have testosterone 5&-reductase inhibiting activity.

Compounds 1 to 10 of the present invention are prepared starting from the well-known azasteroids I and II (J. Med. Chem. 29, 2298 (1986)), according to Scheme 1

SUBSTITUTE SHEET

In a general way, a compound of formula (I)

a) is submitted to the Wittig reaction to give compounds of formula (A), wherein R ¹¹ and R ¹¹¹ form together a =CH ₂ group, and b) the compounds obtained in step a) are treated with a peroxyacid to give compounds of formula (A) , wherein R ¹¹ and R ¹¹¹ are respectively:

-CH ₂ _ group and an oxygen atom, which together the carbon atom at the 17-position, form an - or β- epoxyde ; or alternatively c) is treated with NaH in the presence of trimethylsulfoxonium iodide to obtain compound of formula (A) wherein R ¹¹ and R ¹¹¹ are respectively:

-CH ₂ _ group and an oxygen atom, which together the carbon atom at the 17-position, form an Ot- or β- epoxyde; or d) is submitted to the Wittig reaction and after acid hydrolysis, the intermediate compound (VII)

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is obtained, which is then reacted with an amine of formula R NH _? , wherein R is a linear or branched C,_c ₄ alkyl, and finally treated with NaBH ₄ , or alternatively, compound (VII) is treated with Ti (IV) isopropoxide, reacted with an amine of formula R _a NH ₂ , and finally treated with NaBH ₃ cN, to give compounds of formula (A) , wherein R ¹¹ is -CH ₂ _NH-R _a and R is C-hydrogen; or e) compounds of formula (A) obtained in steps a)-d) are / 1-unsaturated; or f) compounds of formula (I) are _\ 1-unsaturated and submitted to steps a)-d); or g) compounds of formula (A) obtained in any one of the preceding steps are thionated at the 3-position; and, if desired h) compounds of formula (A), obtained in any one of the preceding steps, are converted into compounds of formula (A) wherein R ¹ is C,-c ₄ alkyl. In a first embodiment of the invention, 3-thioxo-4- azaandrostanes (1, 2, 6, 7) are obtained starting from 3-oxo-4-azaandrostan-17-one (I), which is firstly thionated at the 3-position to give the compound (1), of which, subsequently, the keto group at the 17- position is reduced, to give the hydroxy-derivative (2). In another way, the starting compound (I) is transformed into the compound of the invention (5) by means of the Wittig reaction on the carbonyl group at the 17-position. On its turn, (5) is thionated to give (6), which, through the transformation of the double bond at the 17-position, leads to the epoxy-derivative (7).

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The corresponding /_\ unsaturated compounds (3, 4,

8, 9, 10) are obtained dehydrogenating (I) and (5) according to conventional methods in the chemistry of steroids, obtaining the known compound (II) and the compound of the invention (8), respectively, which are then transformed into thio-derivatives (3) and (9) and their final respective reduction and epoxydation to give (4) and (10).

Compounds of formula (A), wherein X is sulfur, R ¹ is hydrogen or linear or branched C._c ₄ alkyl;

R is an optionally salified carboxy group, an acetyl group, a 17X- or 17β-CONHR ^IV , where R ^IV is a linear or branched ^C _τ -C ₄ alkyl, and R III is hydrogen; are prepared by a process comprising that a compound of formula (III)

a) is thionated at the 3-position to give compounds of formula (A) wherein R is an acetyl group and R ¹¹¹ is hydrogen, R TT; if desired b) is converted to carboxy group, which can optionally be salified, said carboxy group; if desired c) is reacted, after activation of said carboxy group, with an amine R ^IV NH ₂ , wherein R is linear or branched ^c ι ^-c 4 alkyl; and, if desired d) compounds of formula (A) obtained in any one of the

SUBSTITUTE SHEET

preceding steps are converted into compounds of formula (A) wherein R ¹ is C ₁ -c ₄ alkyl.

In a second preferred embodiment the compounds (11), (12), (13), and (14) are prepared starting from the well known azasteroid (III) (J. Med. Chem., 29, 2298 (1986)); and in an alternative way, according to Scheme 2, the compounds (13) and (14) may also be prepared from the well known azasteroids (IV) and (V), which are disclosed in .EP 0 155 096.

SUBSTITUTE SHEET

SCHEME 2

»>4 »BΛSsateiR '

12

In the above Scheme, thionation and /_\ 1- dehydrogenation reactions are as described for the above Scheme 1.

The remaining reactions, namely obtaining the 17- 5 carboxy group and its transformation into an amide group, are well known to the man skilled in the art.

Among the chemical steps used to obtain the compounds of the present invention, the thionation process herein used should be briefly commented. It is

10 well known that Lawesson's reagent is an effective thionating reagent for carboxamides, ketones, lactones, lactams and 3-keto-carboxyl derivatives [Reviews: R.A. Cherkasov, G.A. Kutyrev, A.N. Pudvik, Tetrahedron (1985), 41 (13), 2567-2624; 4JL (22), 5061-5087].

15 With respect to other thionating methods [Steliu, Mrami, J. Am. Chem. Soc. , 104 , 3104 (1982): D.H.R. Barton, L.S.L. Choi, R.H. Hesse, M.H. Pahet, C. Wilshire, J.C.S. Perkin I, (1979), 1166], this reagent proved to be capable of leading to the desired products

20 with high conversion percentages and high chemoselectivity degree.

Some preferred embodiments of the present invention are herein illustrated.

Compounds of formula (A), wherein X is S, are

25 obtained by treating the appropriate starting compounds with Lawesson's reagent, namely compounds (I), (II), (5), (8), (III), (IV), (V). Lawesson's reagent, which is [2 ,4-bis (4-methoxyphenyl)-l,3 ,2 ,4-dithiaphosphetan- 2 ,4-disulfide] , in a ratio ranging from 1 to 2

30 equivalents per mol of substrate, is added to a solution of the lactam, in a concentration ranging from

SUBSTITUTE SHEET

0.2 to 1 M, in an anhydrous apolar solvent, under stirring at a temperature ranging from 0° to 60°C. The reaction mixture is left under stirring for a time interval ranging from 1 to 12 hours. The reaction mixture is then under vacuum concentrated and the residue is purified through silica chromatography (ratio 1:60, elution in dichloromethane/methanol gradient); the so obtained product (with yields ranging from 30 to 80%) may further be purified by crystallization from methanol/water or acetonitrile.

Compounds of formula (A), wherein X is sulfur, R ¹¹ and R ¹¹¹ are a hydroxyl group and a hydrogen atom respectively, are obtained by reducing compounds (1) and (3) with NaBH ₄ . NaBH ₄ , solid or in 1M NaOH solution, in a ratio ranging from 1 to 3 equivalents per mol of substrate, is added to a solution of the substrate in a concentration ranging from 0.1 to 1 M, in an alcoholic solvent, preferably methanol, under stirring at a temperature ranging from 5 to 25°C. The reaction mixture is maintained under stirring for a time interval ranging from 0.5 to 2 hours, then pH is adjusted to 4 with IN HCl and the organic solvent is evaporated off. The aqueous phase is extracted three times with an equal amount of dichloromethane, dried over Na ₂ S0 ₄ , filtered and evaporated to dryness to give the desired product, with yields higher than 90%. These products may subsequently be crystallized from acetonitrile.

Compounds of formula (A), wherein X is oxygen, R ¹¹ and R ¹¹¹ form an epoxy group, may directly be prepared from compound (I) according to the following procedure:

SUBSTITUTE SHEET

NaH (from 2 to 10 mol per mol of compound (I)) is added to anhydrous dimethyl sulfoxide (DMSO) (final concentration ranging from 0.5 to 5 M) . The obtained suspension is warmed up to 75°C until complete dissolution (about 45 minutes). A 0.54 M trimethylsulfoxonium iodide solution (0.72 mol per mol of NaH) in DMSO is added to the so obtained solution and subsequently a 0.5 M solution of compound (I) is added. The reaction mixture is maintained under stirring at a temperature ranging from -5 to 20°C for a time interval ranging from 1 to 5 hours. The reaction mixture is worked up by diluting with water (1:6) and extracting with dichloromethane. The organic phase is evaporated under vacuum to dryneεs to give a crude which is subsequently purified by silica gel chromatography (1:80 ratio, elution with dichloromethane/ ethanol 95/5) to give the desired product with yields ranging from 60 to 95%.

Alternatively, the above compounds (15 and 15 bis)

may be prepared starting from compounds (5) and (8) by epoxidation with meta-chloroperbenzoic acid (from 1 to 5 mol per mol of substrate) in a dichloromethane solution under stirring at a temperature ranging from

SUBSTITUTE SHEET

15

-5 to 25°C for a time interval ranging from 1 to 7 hours. The reaction mixture is worked up by subsequent 5% NH ₃ and aqueous sodium thiosulfate washings until negative reaction for oxidants. Then the organic phase 5 is dried over a ₂ sθ , evaporated to dryness to give a crude which is subsequently crystallized from diethyl ether thus obtaining the desired product with yields ranging from 50 to 77 - Compounds of formula (A), wherein X is sulfur, R ¹ and

10 R ¹¹ form an epoxy group, are obtained by treating compounds (6) and (9), dissolved into dichloromethane in a concentration ranging from 0.05 to 0.1 M, under stirring at a temperature ranging from -5 to 25°C with from 1 to 2 equivalents of an organic peroxyacid,

15 preferably 3-chloroperbenzoic acid. After a time interval ranging from 0.5 to 2 hours, the reaction mixture is worked up washing the organic phase with the same amount of 5% aqueous ammonia, then with a seturated sodium thiosulfate solution. The organic

20 phase is then dried on Na ₂ sθ ₄ , filtered and evaporated to dryness to give a crude which is subsequently purified by silica gel chromatography (1:60 ratio, elution with dichloromethane/methanol gradient) to give the desired product with yields ranging from 50 to 77%.

25 Compounds of formula (A), wherein X is oxygen, R ¹¹ and R form a methylene group, are obtained by means of the Wittig reaction on compound (I). Accordingly, methyltriphenylphosphonium bromide, from 0.5 to 1 mol per mol of base, is added under vigorous stirring at a

30 temperature ranging from -20 to 0°C to a suspension of a strong base, such as n-BuLi, NaOH or, preferably,

SUBSTITUTE SHEE-

potassium t-butoxide in a concentration ranging from 0.5 to 0.05 M in an anhydrous aprotic solvent, preferably diglyme. After 30 minutes, a suspension of the ketone (I) (from 1 to 0.4 mol per mol of phosphonium salt) in diglyme in a concentration ranging from 0.5 to 0.2 M is added. The reaction mixture is maintained under stirring in nitrogen atmosphere for a time interval ranging from 4 to 18 hours. After neutralization with 6N HCl, the reaction mixture is extracted with dichloromethane and the organic phases are then dried on Na ₂ sθ ₄ and evaporated to dryness. The so obtained crude is crystallized from methanol/water to give the desired product with yields ranging from 60 to 75%. According to the present invention, the passage from the androstanic nucleus to the androst-1-enic nucleus is generally carried out by well known methods of _\ 1-dehydrogenation of steroids.

Particularly, J_\ 1-dehydrogenation of compounds (5) and (13) may be carried out, for example, by treating the substrate solution, in concentration ranging from 0.02 to 0.1 M in an anhydrous aprotic solvent, such as toluene, diglyme or, preferably, chlorobenzene, with phenylselenic anhydride (from 1 to 1.5 equivalents per mol of substrate). The reaction mixture is then warmed to 110°C for a time interval ranging from 6 to 18 hours, then the solvent is removed under vacuum. The so obtained crude is purified by silica gel chromatography (1:60 ratio, elution with dichloromethane/acetone gradient) to give the desired product with yields from 50 to 85%.

SUBSTITUTE SHEET

Alternatively, a suspension of the substrate in anhydrous dioxane, in a concentration ranging from 0.1 to 0.4 M, is added with DDQ (2 ,3-dichloro-5,6-dicyano- 1,4-benzoquinone) (from 1 to 1.5 mol per mol of substrate), then is treated with N,0- bis (trimethylsilyl)trifluoroacetamide (from 4 to 6 mol per mol of substrate) . The reaction mixture is maintained to stand for 4 hours under stirring at room temperature, then at 110°C for a time interval ranging from 8 to 24 hours. The reaction is worked up by diluting it in a 1:2 ratio with dichlorometane and washing it with 1% sodium hydrosulfite. The precipitate is filtered off and the organic phase is washed with 2N HCl, dried over a ₂ sθ ₄ and evaporated to dryness. The so obtained crude may be purified by crystallization from acetonitrile or ethanol/water to give the desired product with yields ranging from 60 to 85%.

Compounds of formula (A), wherein X is sulfur, R ¹¹ and are a carboxyl group and a hydrogen atom, are obtained by treating a solution of the compound (11), in a concentration ranging from 1 to 2 M, which has been obtained by thionating the known azasteroid (III), in pyridine under stirring at the temperature of 90°C with a slow addition (30 minutes) of iodine (from 1 to 1.2 mol per mol of substrate). The reaction mixture is refluxed for a time interval ranging from 1 to 2 hours, then cooled to 10°C. The so formed precipitate is recovered by filtration and washed with pyridine and ether. The so obtained complex is dissolved into a 10% KOH 1/1 methanol/water solution at a concentration ranging from 5 to 20% and maintained under stirring for

SUBSTITUTE SHEET

a time interval ranging from 10 minutes to 1 hour. The organic solvent is then evaporated under vacuum and the residue is taken up with water to a concentration ranging from 10 to 20% and acidified with 6N HCl. The so formed precipitate is recovered by filtration and dried at 80°C (12 mmHg) for 12 hours to give the desired product (12) with yields ranging from 65 to 78%.

On its turn compound (12) may be used to obtain the compounds of the present invention wherein X is sulfur and R TT and RTT ^J T- are a carbamoyl group and a hydrogen atom, respectively.

Accordingly, pyridine (from 1.2 to 1.6 mol per mol of substrate) is added to a solution of substrate (12) in a concentration ranging from 0.1 to 0.3 M in a anhydrous aprotic solvent such as THF, ether, preferably toluene. Then, oxalyl chloride (from 1.1 to 1.3 mol per mol of substrate) is added at a temperature ranging from -5 and 10°C. The reaction temperature is maintained for a time interval ranging from 0.5 to 2 hours, then an amine of formula R ¹ NH2, wherein R ^IV is as above defined, (in a ratio from 2 to 7 mol per mol of substrate) . The reaction mixture is then warmed to a temperature ranging from 40 to 70°C for 30 minutes. The reaction mixture is worked up by diluting it in a 1:2 ratio with water and adjusting pH to neutrality. The organic phase is separated and the aqueous phase is extracted twice with an equal volume of dichloromethane. The gathered organic phases are then dried on Na ₂ sθ ₄ , filtered and evaporated to dryness to give a crude which is purified by silica gel

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chromatography (1:60 ratio, elution with dichloromethane/methanol gradient) to give the desired product with yields ranging from 30 to 70%.

Alternatively, compounds (13) and (14) are obtained by thionating compounds (IV) and (V), disclosed in EP 0 155 096, with Lawesson's reagent.

Compounds of formula (A) , wherein R is B-CH__NH-

Ra and R ¹¹¹ is hydrogen may be obtained from compound

(I) by the Wittig reaction, (see scheme 3) accordingly, methoxymethyltriphenylphosphonium chloride, from 1 to 4 mol per mol of compound (I), in an aprotic solvent, such as ether THF, diglyme, under stirring and at a temperature ranging from -20 to 0°C is added to a 1.6 M n-BuLi hexane solution or a potassium t-butoxide solution (from 1 to 2 equivalents per mol of phosphonium salt). The reaction mixture is maintained under stirring for a time interval ranging from 20 minutes to 1 hour. Then a IM ether solution of compound

(I) is added and the temperature is left to raise to room temperature under stirring for a time interval ranging from 3 to 18 hours. After neutralization with IM HCl, the reaction mixture is extracted with ethyl acetate and the organic phases are then dried on Na_S0 ₄ and evaporated to dryness. The crude so obtained is purified by silica gel chromatography (1:80 ratio, elution with 1/1 petroleum ether/ethyl acetate) recovering the intermediate enol-ether (VI) with yields ranging from 60 to 87%. The intermediate is then hydrolyzed with a catalytic amount of sulfuric acid in THF under stirring at reflux temperature at a concentration ranging from 0.2 to 0.01 M, for a time

SUBSTITUTE SHEET

interval ranging from 6 to 18 hours. The reaction mixture is then neutralized with aqueous NaHCO-. and extracted with ether. The organic layer is dried on

Na ₂ sθ ₄ and evaporated to dryness to give the desired product (VII) with yields ranging from 75 to 92%. This last is transformed into the final compound by reaction in alcoholic solution, under stirring at room temperature for a time interval ranging from 1 to 3 hours, with the desired amine R -NH ₀ (from 1 to 3 mol per mol of aldehyde). Then the intermediate Schiff base (VIII) is reduced with a 2.4 M NaBH ₄ alcoholic solution (from 2 to 6 equivalents) and maintained under stirring at room temperature for a time interval ranging from 2 to 8 hours. The reaction mixture is neutralized with IN HCl, the organic phase is evaporated under vacuum and extracted with dichloromethane. The organic phase is dried over Na ₂ so ₄ , evaporated to dryness and the crude is purified through silica chromatography to give the desired compound with yields ranging from 40 to 70%. The subsequent transformation of this product into the corresponding /_\ 1-unsaturated compound is carried out by conventional processes of j 1-dehydrogenation already described for compounds (5) and (13) to give the desired compounds with yields ranging from 60 to 78%.

In a third preferred embodiment, compounds (15), (16) and (18) are also prepared starting from (I) and (II). Particularly, (15), (16) and (16 bis) are prepared according to the following Scheme 3.

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SUBSTITUTE SHEET

22

Alternatively, compounds (19 and 16 bis)

10 may be prepared by reacting the intermediate aldehyde (VII) with titanium (IV) isopropylate (from 1 to 2 mol per mol of aldehyde) and the proper amine (from 2 to 4 mol per mol of aldehyde) under stirring at room temperature for a time interval ranging from 1 to 2

15 hours. Then the reaction mixture is diluted 1:10 with ethanol and reduced with NaBH-CN (from 1 to 3 equivalents) at room temperature for a time interval ranging from 10 and 18 hours. The reaction mixture is acidified with IN HCl and extracted with

20 dichloromethane. After the usual purification, compounds (19 and 16 bis) are obtained with yields ranging from 60 and 80%.

As shown in scheme 4, compounds of formula (A), wherein R ¹¹ is β-CH(CH ₃ )NH-Ra and R ¹¹¹ is hydrogen, and

25 particularly compound (17), may be obtained from compound (XI) in the same way as compound (VII) (the same weight ratios and reaction conditions) to give the intermediates (XIII) with yields ranging from 50 to 82%. The intermediates are then oxidized according to

30 the classical Oppenauer reaction to give, after crystallization from hexane, the intermediates (XIV)

SUBSTITUTE SHEET

with yields ranging from 50 to 65%. Secosteroids (XV) are obtained through a process which comprises their heath oxidation (80°C) with a 26% NaI0 ₄ and 2% KMn0 ₄ water solution (5.8 ml per mmol of substrate (XIV)) which is slowly added to a 0.17 M solution of compound (XIV) in 7.5:1 t-butanol/water containing 2.5% Na-,C0 ₃ . The reaction mixture is refluxed for a time interval ranging from 1 to 2.5 hours, then is cooled to 30°C and after 1 hour is filtered under vacuum. The filtrate is then concentrated under vacuum to about 1/3 of the starting volume and acidified with 6M HCl (pH 3), the precipitate so obtained is recovered by filtration to give the intermediates (XV) with yields ranging from 40 to 75%. Compounds (XV) are transformed into the intermediates (XVI) by heating a 0.36 M solution of the secosteroid in ethylene glycol saturated with an excess of liquid ammonia or the proper amine R NH_ (about 26 mol per mol of secosteroid). The reaction is heated to 180°C within 45 minutes then is maintained at this temperature for 15 minutes. Then, the reaction mixture is cooled to room temperature, diluted with water and acidified to pH 3 with concentrated HCl. Compounds (XVI) wherein R ¹ is hydrogen are recovered by filtration; those wherein R ¹ is alkyl are extracted with chloroform with yields ranging from 50 to 75%. Compound (17 bis) is obtained from compound (XVI) by catalytic hydrogenation (10% Pd/C or 5% Pt/C w/w) from 0.07 M methanol or acetic acid solutions bubbling hydrogen at atmospheric pressure or at 3 atm at the temperature of 60°C. After a time interval ranging from 30 minutes to 2 hours the reaction mixtures is worked

SUBSTITUTE SHEET

up, degassing hydrogen under vacuum and filtrating off the catalyst. Compound (17 bis) is isolated by evaporating under vacuum the filtrate and subsequent crystallization from acetonitrile with yields ranging from 80 to 93%.

Compounds of formula (A) , wherein R ¹ and R ¹¹¹ are respectively: β-CH(CH, )-NH-R ₌ , where R is a linear or branched C. -c . alkyl, and R ¹¹¹ is Ot-hydrogen; are prepared by a process which comprises that: a compound of formula (XI)

(XI)

a) is reacted with an amine of formula R NH , wherein _Q i _{s a} linear or branched C,-C ₄ alkyl, and finally treated with NaBH ₄ , or alternatively, the intermediate compound is treated with Ti (IV) isopropoxide, reacted with an amine of formula R _a NH ₂ , and finally treated with NaBH^CN, b) the compound from step a) is reacted with aluminum isopropoxide; c) the compound from step b) is treated with an oxidant to give secosteroid d) secosteroid is reacted with liquid ammonia to obtain azasteroid; e) azasteroid is hydrogenated to obtain compounds of

SUBSTITUTE SHEET

^^a-^-^εi

25

formula (A) wherein R and R ¹¹¹ are as above defined; and, if desired f) compounds of formula (A) obtained in steps d)-e) are /\ 1-unsaturated and/or g) compounds of formula (A) obtained in steps d)-f) are thionated at the 3-position; and, if desired h) compounds of formula (A) obtained in any one of the preceding steps are converted into compounds of formula (A) wherein R ¹ is C ₁ _c ₄ alkyl.

10 In a fourth preferred embodiment, compound (17) has been prepared starting from 5-pregnen-3β-ol-20-one according the following Scheme 4

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SCHEME 4

(XIII)

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Scheme 4 (continued)

NHiBu

(XV) (XVI)

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Alternatively, compounds (17 bis and 20)

)

may be prepared from the known azasteroid (III) according to the same method for the preparation of compound (16 bis). Using the already described technique, which uses titanium (IV) isopropoxide as dehydrating agent and NaBH ₃ CN as reducing agent. The desired product is obtained after purification by silica gel chromatography (1:10 ratio, elution with dichloromethane/methanol gradient) with yields ranging from 30 to 77%. Compounds of formula (A) wherein R and R ¹¹ are respectively: β-CO-NH-NH-C(16 ) , where C(16) is the carbon atom at the 16-position, and hydrogen, are prepared by a process comprising that : a compound of formula (I)

SUBSTITUTE SHEET

a) is reacted with benzaldehyde to obtain a 16- benzylydene intermediate; b) said intermediate is submitted to the Wittig reaction to obtain the 17-formyl-16-benzylydene intermediate c) the intermediate of step b) is treated with an oxidant to convert the 17-formyl group into 17-carboxyl group; d) 17-carboxyl group is then reacted with hydrazine to give a 17-hydrazine derivative; e) said derivative of step d) is ozonized and the intermediate acylhydrazone is then treated with NaBH. , to give a compound of formula (A) wherein R and R ¹¹¹ are as above defined; f) compound of formula (A) obtained may be _\ 1- unsaturated and if desired g) compound of formula (A) obtained in step e) and f) is thionated at the 3-position; and, if desired h) compounds of formula (A) obtained in any one of the preceding steps are converted into compounds of formula (A) wherein R ¹ is C ₁ -c ₄ alkyl.

In a fifth preferred embodiment, compound (18) is prepared according the following Scheme 5:

SUBSTITUTE SHEET

SCHEME 5

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Scheme 5 (continued)

(18 bis)

SUBSTITUTE SHEET

It is understood that the above Schemes, together with the relevant considerations and descriptions, are applicable also to the compounds of formula (A) wherein R ¹ is different from hydrogen, particularly with R ¹ methyl or ethyl. The skilled in the art can easily apply the above mentioned Schemes, considerations and descriptions, to the preparation of said compounds.

The compounds (A) of the present invention are testosterone 5#<-reductase inhibiting agents therefore they are useful as therapeutic agents.

A further object of the present invention is the use of compounds of formula (A) for the manufacturing of a medicament useful for the treatment of hyperandrogenic conditions, particularly, prostatic hypertrophy, prostatic carcinoma, acne vulgaris, seborrhea, female hirsutism, male hair loss.

Compounds of formula (A) inhibit 5 -reductase of [ ¹⁴ C]-testosterone in rat prostate (Liang. et Al. , Endocrinology 117,571,1985). A quote of fresh rat prostate homogenate containing about 100 μg of protein is incubated in 250 μl of Krebs-Ringer buffer in the presence of a NADPH generating system (NADP disodium salt 11.76x10 " M and glucose-6-phosphate dehydrogenase 3.5xl0~ ² I.U.) and of [ ¹⁴ C]-testosterone (3.16xl0 ^~6 M, specific activity about 56.9 mCi/mol, Amersham, England) as labelled substrate. Vials not containing proteins are used for the blank determination. The incubation is carried out in a 37°C thermostated stirring bath for two hours under a 98/2 0-/C0 ₉ flow. At the end of incubation, the reaction is stopped placing the samples in ice. For a correct quantitative

SUBSTITUTE SHEET

evaluation of the two main 5^<-reduced metabolites which are formed by the prostate activity in these conditions (DHT and 3&-diol), tritium labelled DHT and 3<*-diol (about 5000 dpm) are added to each sample before the extraction with the scope to evaluate the recovery. The formed metabolites are twice extracted with 5.5 ml of diethyl ether. The extracts, after dissolution into 200 ul of ethanol, containing non labelled DHT and 3Λ-diol, as reference standards, are separated by thin layer chromatography on silica gel plates (Merck 60 F 254, DC), eluted three times with a 11:1 dichloromethane/diethyl ether mixture at a temperature of 4°C. DHT and 30.-diol spots are detected with iodine vapours, whereas those of testosterone by UV light exposition (UV absorption of the 4-5 double bond conjugated with the 3-keto group). The silica gel areas where DHT and 30(-diol respectively have been evidenced are scratched and put into tubes for count. After the addition of 0.5 ml of water, in order to inactivate the steroid bond with silica, and 6 ml of scintillation liquid, the samples are put on a horizontal shaker and shaken for 15 minutes. After decanting the silica gel, the samples are counted in a liquid scintillation spectrometer (Packard 300 C) . The values in dpm, obtained through a standard calibration curve, are corrected on the recovery percentage calculated on the tritiated steroids, added to each sample before the extraction. The results are expressed in pg of steroid formed within 2 hours of incubation per mg of protein. In each experiment IC ₅₀ of the tested compound was evaluated by using different concentrations of the

SUBSTITUTE SHEET

substance starting from 10 ^J till 10 M.

Compounds of formula (A) showed IC _5Q ranging from 50 to 100 nM. For comparison purpose, finasteride, disclosed in EP 0155096, shows IC ₅ _ _σ f 80nM. Further, compounds of formula (A) inhibit testosterone induced increase of prostate and seminal vesicles in the castrated rat (Di Salle E. et Al., J.Steroid Biochem. Biol. Molec. Biol., 41,765,1992).

22 days male Crl:CD BR rats are castrated under ether anaesthesia and then left without treatment for 7 days. After seven days the treatment is started and is continued for seven days according to the following procedure: - a control group of only castrated animals; - a group receiving 0.3 mg/kg s.c. testosterone for seven days; the other groups receiving 0.3 mg/kg s.c. testosterone and 20 mg/kg os of the tested compounds for seven days. At the end of the treatment, after sacrifice, the prostates and seminal vesicles were removed and weighted. The results are expressed as the percent decrease of the two organs with respect to the group treated with testosterone only. Compounds of formula (A) show percent decrease of the prostate ranging from 40 to 60% compared with the 50% decrease of finasteride; moreover, the former showed seminal vesicle percent decrease ranging from 50 to 70% compared with 60% of finasteride. The present invention relates also to oral, parenteral and topical pharmaceutical compositions

SUBSTITUTE SHEET

containing a compound of the invention in admixture with conventional excipients and vehicles.

Examples of oral pharmaceutical compositions are tablets, capsules, sachets and suspensions; examples of parenteral pharmaceutical compositions are liophilized vials or sterile suspensions; examples of topical pharmaceutical compositions are creams, ointments, gels, aerosol or foam formulation.

The compositions according to the invention are prepared by conventional methods, such as for example those described in Remington's Pharmaceutical Sciences Handbook, Mack Pub. XVII ed. N.Y. U.S.A.

The daily oral dosage ranges from 5 to 50 mg; the unit doses may contain from 2.5 to 25 mg of active ingredient.

For the topical administration the concentration of the active ingredient ranges from 0.1 to 10%, preferably 5%, the daily applications may be one or two. The following examples further illustrate the invention.

EXAMPLE 1 Compound (1)

578 mg (2 mmol) of compound (I) were dissolved at room temperature into 5 ml of anhydrous dichloromethane. Then, 455 mg (1.03 mmol) of Lawesson's reagent were added at room temperature under stirring. The reaction mixture was stirred in the dark for 4 hours, thereafter the reaction mixture was concentrated under vacuum to a volume of about 1 ml, loaded on a silica gel column (20 g) and eluted with a 95/5

SUBSTITUTE SHEET

dichloromethane/methanol mixture. An oil was obtained, which, after crystallization from acetonitrile, gave 300 mg of product (0.98 mmol).

¹ H-NMR (60 MHz): ζ 3.4-2.9 (m, 3H) , 0.5 - 2.6 (complex system), 0.95 (s, overlapping complex system) IR (CHC1 ₃ ): 1735.7, 1518.5

MS m/e: 306 (M+l), 305 (M) , 290 (M-15), 275 (M-30) Elemental analysis for C.„H-, ₇ 0 N S calculated: C-70.81, H-8.85, N-4.59, S-10.49 found: C=70.86, H-8.82, N-4.61, S-10.45

EXAMPLE 2 Compound (2)

600 mg (1.96 mmol) of compound (1) were dissolved under stirring at room temperature in 10 ml of methanol. 112 mg (2.94 mmol) of NaBH. were added and the reaction mixture was maintained under stirring at room temperature for 3 hours. The reaction mixture was then neutralized with IN HCl, the organic solvent was evaporated under vacuum and the aqueous phase was extracted with dichloromethane. The organic phase was dried on Na ₂ S0 ₄ , evaporated under vacuum to give a crude, which was crystallized from acetonitrile to give 550 mg of product (1.79 mmol).

¹ H-NMR (60 MHz): ζ~ 3-3.8 (m, 4H) , 0.6 - 2.3 (complex system), 0.9 and 1.1 (2s, overlapping complex system)

- IR (CHC1 ₃ ) _: 3204 (broad), 1518

- MS m/e: 307 (M) , 292 (M-15), 289 (M-18), 277 (M-30)

Elemental analysis for C, ₈ H _?Q ONS calculated: C-70.35, H-9.44, N-4.56, S-10.42 found: C-70.38, H-9.42, N-4.60 , S-10.40

SUBSTITUTE SHEET

EXAMPLE 3 Compound (3)

According to the method described for compound (1), starting from 800 mg (2.78 mmol) of compound (II), 500 mg (1.65 mmol) of product were obtained after crystallization.

¹ H-NMR (60 MHz): S 6.9 (d, J-9Hz, 1H) , 6.5 (d, J=9Hz, 1H) 3.3 - 3 (m, 1H) , 2.8 - 0.6 (complex system) IR (CHC1 ₃ ) _: 1736;1514 - MS m/e: 303 (M) , 288 (M-15), 273 (M-30); Elemental analysis for C. _R H ₂ ,-ONS calculated: C-71.28, H-8.25, N=4.62, S-10.56 found: C-71.31, H-8.23, N-4.59, S-10.54

EXAMPLE 4 Compound (4)

According to the method described for compound (2), starting from 700 mg (2.29 mmol) of compound (3), 560 mg (1.83 mmol) of product were obtained after crystallization. ¹ H-NMR (60 MHz): 6 ^" 6.9 (d, J-9Hz, 1H) , 6.1 (d, J-9Hz, 1H) , 3.3 - 3.8 (m, 2H) , 0.6 - 2.1 (complex system), 0.8 and 1 (2s, overlapping complex system)

- IR (CHC1 ₃ ): 3200 (broad); 1512

- MS m/e: 305 (M) , 290 (M-15), 287 (M-18), 275 (M-30) - Elemental analysis for calculated: C-70.82, H=8.85, N=5.24, S-10.49 found: C-70.80, H-8.87, N-5.23, S-10.46

EXAMPLE 5 Compound (5) 5 ml of a suspension of 500 mg (1.73 mmol) of compound (I) in diglyme were added to a stirred suspension of

SUBSTITUTE SHEET

800 mg, (7.13 mmol) of potassium ter-butoxide, 1.51 g (4.23 mmol) of methyltriphosphonium bromide in 15 ml of diglyme at the temperature of 0°C in nitrogen atmosphere. The reaction mixture was stirred at 0°C under nitrogen flow for 2 hours, then at room temperature for 8 hours. pH was adjusted to neutrality with 6N HCl and the reaction mixture was extracted with dichloromethane (3x10 ml). The organic phases were collected and dried over Na ₂ S0 ₄ , evaporated to dryness under vacuum to give a crude, which was crystallized from methanol/water to give 350 mg of product (1.22 mmol)

¹ H-NMR (60 MHz): 5 ^" 5.05 (s, 2H) , 3 - 3.2 (m, 1H) , 0.6 - 2.6 (complex system), 0.8 and 0.9 (2s, overlapping complex system)

- IR (CHC1 ₃ ): 1654,3

- MS m/e: 287 (M), 273 (M-14), 272 (M-15), 257 (M-30)

Elemental analysis for C, _g H _2q NO calculated: C-79.44, H-10.10, N-4.87 found: C-79.41, H-10.11, N-4.85

EXAMPLE 6 Compound (6)

According to the method described for compound (1), starting from 700 mg (2.44 mmol) of compound (5), 450 mg (1.57 mmol) of product were obtained after crystallization.

¹ H-NMR (60 MHz): S 5.1 (s, 2H) , 2.8 - 3.2 (m, 3H) , 0.6 - 2.6 (complex system), 0.8 and 0.9 (2s, overlapping complex system)

- IR (CHC1 ₃ ): 1519

- MS m/e: 303 (M) , 289 (M-14), 288 (M-15), 273 (M-30)

SUBSTITUTE SHEE-

Elemental analysis for C- _jo H _q NS calculated: C-75.25, H-9.57, N-4.62, S-10.56 found: C-75.23, H-9.55, N-4.60, S-10.59

EXAMPLE 7 Compound (7)

800 mg (2.64 mmol) of compound (6) were dissolved into 30 ml of anhydrous dichloromethane. Then, 959 mg (5.5 mmol) of> 3-chloroperbenzoic acid were added. The reaction mixture was stirred at room temperature for 2 hours. Then the reaction mixture was washed with 5% NH ₃ (3x20 ml) and with a saturated sodium thiosulfate solution (1x20 ml). The organic phase was cautiously evaporated under vacuum to give a crude, which was purified by silica gel chromatography eluting with 95/5 dichloromethane/methanol to give 650 mg of product (2.03 mmol)

¹ H-NMR (60 MHz): δ ^" 3 - 3.2 (m, 3H) , 2.8 (m, 2H) , 0.8 - 2.6 (complex system) - IR (CHC1 ₃ ): 1518 - MS m/e: 319 (M) , 304 (M-15), 301 (M-18), 289 (M-30) Elemental analysis for C._H _2Q ONS calculated: C-71.47, H-9.09, N-4.39, S-10.03 found: C-71.46, H-9.07, N-4.41, S-10.05

EXAMPLE 8 Compound (8)

623 mg (2.17 mmol) of compound (5) were dispersed into

30 ml of dioxane with 494 mg (2.17 mmol) of DDQ under stirring at room temperature. Then 2.29 g (8.89 mmol) of N,0-bis(trimethylsilyl)trifluoroacetamide were added to the reaction mixture under vigorous stirring. After

4 hours under stirring at room temperature, the

SUBSTITUTE SHEET

reaction mixture was heated to 110°C an kept at this temperature under stirring for 16 hours. Then the reaction mixture was diluted with 12 ml of dichloromethane and added with 1 ml of a 1% sodium bisulfite solution; the so formed precipitate was filtered off, the organic phase was washed with 3.8 ml of 2N HCl and evaporated under vacuum to dryness to give a crude, which was purified by crystallizing from methanol/water to give 550 mg of product (1.93 mmol) ¹ H-NMR (60 MHz): 5 ^" 6.9 (d, J-9Hz, 1H) , 6.15 (d, J=9Hz, 1H) 5.82 (m, exchangeable), 5.0 ( , 2H) , 3 - 3.2 (m, 1H) , 0.8 - 2.6 (complex system) IR (CHC1 ₃ ): 1650

MS m/e: 286 (M+l), 285 (M+), 271 (M-14), 270 (M- 15), 255 (M-30)

Elemental analysis for C, _g H ₂₇ NO calculated: C-80.00, H-9.47, N-4.91 found: C-79.90, H-9.45, N-4.89

EXAMPLE 9 Compound (9)

According to the method described for compound (1), starting from 680 mg (2.18 mmol) of compound (8), 420 mg (1.39 mmol) of product were obtained after crystallization. ^-H-NMR (60 MHz): S ^" 6.9 (d, J-9Hz, 1H) , 6 (d, J-9Hz, 1H), 5.05 (m, 2H), 3 - 3.3 (m, 1H) , 0.8 - 2.6 (complex system)

- IR (CHC1 ₃ ): 1515

- MS m/e: 301 (M+), 287 (M-14), 286 (M-15), 271 (M-30) - Elemental analysis for C. _g H _?7 NS calculated: C-75.74, H-8.97, N-4.65, S-10.63

SUBSTITUTE SHEET

found: C-75 . 76 , H=8 . 95 , N-4 . 63 , S=10 . 62

EXAMPLE 10 Compound (10)

According to the method described for compound (6), starting from 1 g (3.3 mmol) of compound (9), 450 mg (1.42 mmol) of product were obtained after chromatographic purification.

¹ H-NMR (60 MHz): 6 ^* 7.05 (d, 1H) , 6.1 (d, 1H) , 3 - 3.2 (m, 1H), 2.8 (m, 2H) , 2.5 - 0.6 (complex system)

- IR (CHC1 ₃ ): 1514

- MS m/e: 317 (M+), 303 (M-14), 302 (M-15), 287 (M-30)

Elemental analysis for C. _H ₂₇ 0NS calculated: C-71.92, H=8.52, N=4.41, S=10.09 found: C-71.90, H-8.54, N-4.40, S=10.07 EXAMPLE 11

Compound 11

According to the method described for compound (1), starting from 800 mg (2.52 mmol) of compound (III), 460 mg (1.38 mmol) of product were obtained after crystallization.

¹ H-NMR (60 MHz): ζ 6.5 - 6.2 (m, 1H) , 3.4 - 2.8 ( , 3H), 2.8-0.7 (complex system), 2.15, 0.9 and 0.7 (3s, overlapping complex system)

- IR (CHC1 ₃ ): 1698,1518 - MS m/e: 333 (M+), 318 (M-15), 303 (M-30), 290 (M-43) Elemental analysis for C _2Q H ₃₁ NOS calculated: C-72.07, H-9.31, N-4.20, S-9.60 found: C-72.09, H=9.30, N-4.18, S=9.61

EXAMPLE 12 Compound (12)

1.74 g (6.85 mmol) of iodine were slowly added (within

SUBSTITUTE SHEET

15 minutes) to a solution of 2.1 (6.3 mmol) of compound (11) in 5.4 ml of pyridine under stirring at 90°C. The reaction mixture was then refluxed for 1 hour, then cooled down to room temperature. The so formed precipitate was isolated by filtration and washed with

4 ml of pyridine and 4 ml of ethyl ether. Subsequently, the solid was dissolved into 20 ml of methanol/water and acidified to pH 2 with 6N HCl. The so formed precipitate (1.47 g, 4.41 mmol) was isolated by filtration and essiccated for 16 hours at 60°C."

IR (CHC1 ₃ ): 1719, 1514.

- MS m/e: 335 (M+), 320 (M-15), 317 (M-18), 305 (M-30),

290 (M-45).

Elemental analysis for C-,QH _2Q 0 ₂ N S calculated: C-68.05, H=8.65, N=4.18, S-9.57 found : C-68.03, H=8.64, N-4.15, S-9.59

EXAMPLE 13

Compound (13), R ^IV - t-butyl

1.135 g (3.38 mmol) of compound (12) were dissolved into 17 ml of anhydrous toluene and 375 ul of pyridine.

The reaction mixture was then cooled down to 10°C and 537 mg (4.23 mmol) of oxalyl chloride into 3 ml of toluene were slowly added. The reaction mixture was maintained under stirring for 1 hours at 10°C then a solution of 1.78 ml of t-butylamine in toluene was added. The reaction temperature was raised to 40°C and kept for 30 minutes. Thereafter the reaction mixture was poured into 16 g of ice and neutralized. The organic phase was separated and the aqueous phase was extracted with dichloromethane (2x15 ml). The organic phases were collected, dried on Na ₂ sθ ₄ and evaporated

SUBSTITUTE SHEE-

> s^W>iSSϊ ^" '

43

to dryness under vacuum to give a crude which was purified by silica gel chromatography (50 g) eluting with a 95/5 dichloromethane/methanol mixture, thus obtaining 1.05 g (2.7 mmol) of product. 5 ¹ H-NMR (60 MHz): fS~ 2.8 - 3.2 (m, 3H) , 2.3 - 0.7 (complex system), 0.65, 0.85 and 1.3 (3s, overlapping complex system)

IR (CHC1 ₃ ): 1670,9, 1513

MS m/e: 390 (M+), 375 (M-15), 360 (M-30), 335 (M- 10 55), 318 (M-72), 290 (M-100)

Elemental analysis for ^C 23 ^H 3« ^N ₂ ^OS calculated: C=70.76, H=9.74, N=7.18, S=8.20 found: C=70.74, H-9.75, N-7.15, S-8.19

EXAMPLE 14 15 Compound (14)

According to the method described for compound (8), starting from 500 mg (1.28 mmol) of compound (13), 273 mg (0.7 mmol) of product were obtained after crystallization. 20 ¹ H-NMR (60 MHz): £ ^" 7.27 (m, 1H) , 6.3-6 (m, 1H) , 2.8 - 3 ( , 3H) , 2.3 - 0.7 (complex system), 0.7, 1.1 and 1.4 (3s, overlapping complex system) IR (CHC1 ₃ ) _: 1670.3, 1514

MS m/e: 388 (M+), 373 (M-15), 358 (M-30), 333 (M- 25 55), 316 (M-72), 288 (M-100)

Elemental analysis for ^C 23H _? 6 ^N 2 ^0S calculated: C-71.13, H-9.28, N-7.21, S-8.25 found: C-71.11, H=9.29, N-7.19, S-8.23

EXAMPLE 15 30 Alternatively to Examples 13 and 14, compounds (13) and (14) may be directly obtained from the well known

SUBSTITUTE SHEET

compounds (IV) and (V), using the method described for compound (1) with yields, after purification by silica chromatography and crystallization from acetonitrile, of 80 and 65%, respectively. EXAMPLE 16

Compound (15)

A 81% solution of 1.37 g of NaH (35.77 mmol) in 21 ml of dimethyl sulfoxide was warmed up to 75°C for 45. Then the reaction mixture was cooled down to -5°C, diluted with 21 ml of tetrahydrofurane and 21 ml of 5.8 g (26 mmol) of trimethylsulfoxonium iodide in 48 ml of DMSO and a solution of 1.98 g (6.9 mmol) of 3,17-dioxo- 4-aza-50(-androst-l-ene in tetrahydrofurane were quickly added. The reaction mixture was left under stirring at a temperature of 5°C for 2 hours. The reaction mixture was poured into 300 ml of water and the organic phase was separated. The aqueous phase was extracted with dichloromethane (3x150 ml) and the collected organic phases were dried over Na ₂ sθ ₄ and evaporated under vacuum to dryness to give 2.3 g of a crude. Silica gel cromatography (1:80 ratio, elution with 95/5 dichloromethane/methanol) gave 1.9 g of the desired product.

^-H-NMR (60 MHz): S 6.8 (d, J=9 Hz, 1H) , 5.82 (m, exchanging overlapping d, J-9 Hz, 2H) , 3.3 (m, 1H) , 2.8 (m, 2H) , 2.3-0.6 (complex system).

IR (CHC1 ₃ )« 3400, 1670

MS m/e: 302 (M+l), 301 (M), 286 (M-15), 271 (M-30)

Elemental analysis for C- _Q H.> ₇ 0 _? N; calculated C-75.74; H=8.97; N-4.65; found C-75.73; H-8.99; N-4.67.

SUBSTITUTE SHEET

EXAMPLE 17 Compound (16) and (16 bis) a) 10 ml of a 1.6 M n-BuLi hexane solution were added to a suspension of 4.61 g (13.45 mmol) of methoxymethyltriphenylphosphonium chloride in ether, under vigorous stirring at -20°C in nitrogen atmosphere. After 20 minutes 10 ml of a diethyl ether solution containing 3.24 g (11.22 mmol) of 3,17-dioxo- 4-aza-5θ(-androstane was added. The reaction mixture was stirred for 2 hours at -20°C, then at room temperature for 3 hours. After this time, the reaction mixture was neutralized with IM HCl and extracted with ether (3x30 ml). The organic phase was dried over Na _? sθ ₄ and evaporated under vacuum to give 4 g of crude which were purified through silica chromatography (1:80 ratio, elution with 1/1 petroleum ether/ethyl acetate). 3.07 g (9.7 mmol, 86% yield) of methoxyenolether (VI) were obtained. Compound (VI) was subsequently used without further purification for the next step. ¹ H-NMR (60 MHz): S 5.9 (s, 1H) , 4.5 (m exchangeable, 1H), 3.55 (s, 3H), 3.3 - 3 (m, 1H) , 2.3 - 0.9 (complex system) . b) 3-oxo-4-aza-5W-17β-formylandrostane (VII) was prepared by acid hydrolysis of the preceding compound: a solution of 10 ml of tetrahydrofurane containing 634 mg (2 mmol) of methoxyenolether (VI) was treated with 0.1 M H ₂ S0 ₄ (5 ml) and refluxed for 12 hours. The reaction mixture was then neutralized with aqueous

NaHC0 ₃ , the organic phase was evaporated under vacuum and the aqueous phase was extracted with diethyl ether

(3x10 ml). The organic extracts were dried over Na ₂ S0 ₄

SUBSTITUTE SHEET

and evaporated under vacuum to give 545 mg (1.79 mmol, 90% yield) of the desired product. The so obtained product was used in the next step without further purification. Only for analytic purpose, a sample was purified through silica gel chromatography (1:80, 1/1 petroleum ether/ethyl acetate).

¹ H-NMR (60 MHz): S 9.9 (S, 1H) , 5.4 (m, 1H, exchangeable), 3.3 - 3 (m, 1H) , 2.3 - 0.8 (complex system) .

I.R. (CHC1 ₃ ): 3400, 1735, 1660.

MS m/e: 304 (M+l), 303 (M) , 288 (M-15), 274 (M-29)

Elemental analysis for C,gH ₂ g0 ₂ N: calculated C-75.24, H-9.57, N-4.62; found C-75.28, H-9.58, N-4.64. 20 ml of a methanol solution containing 3 g ( 10 mmol ) of 3-oxo-4-aza-50 -17β-formylandrostane , under stirring at room temperature was added with 864 mg ( 12 mmol ) of t-butylamine . After 2 hours , 10 ml of a methanol solution containing 0.9 g (24 mmol) of NaBH ₄ were slowly added. The reaction mixture was stirred for 2 hours at room temperature, then was neutralized with IN

HCl, then the organic phase was vacuum evaporated and the residue extracted with dichloromethane (3x10 ml).

The organic phase was dried over Na_so ₄ , evaporated under vacuum and the crude was subsequently purified through silica chromatography (1:80) by elution with

9/1 dichloromethane/methanol to give 2.5 g (6.94 mmol;

70% yield) of pure 17β-t-butylamino-3-oxo-4-aza-5 - androstane (16 bis). ¹ H-NMR (60 MHz): $ 5.5 (m, 2H, exchanging), 3.3 - 3.0

(m, 1H) , 2.8 - 0.6 (complex system singlet 1.6, 0.9,

SUBSTITUTE SHEET

,^et&~ ^~ -~-A

47

0 . 7 ) .

- I . R . (CHC1 ₃ ) _: 1690 , 3390 .

- MS m/e: 361 (M+l), 360 (M) , 345 (M-15), 288 (M-72).

- Elemental analysis for C ₂₃ H ₄₀ ON ₂ :

5 calculated C-76.66, H=ll.ll, N-7.77 ; found C=76.64, H=11.14, N-7.80 c) 687 mg (1.961 mmol) of 20-t-butylaminomethylene-3- oxo-4-aza-50-androstane were dissolved into 50 ml of diethylene glycol dimethyl ether with 980 mg (2.72 10 mmol) of phenylselenic anhydride. The reaction mixture was refluxed to 120°C for 10 hours, then the solvent was removed by evaporation under vacuum. The so obtained crude was purified through silica chromatography (1:80 ratio, elution with 9/1 15 dichloromethane/methanol) to give 581 mg (1.62 mmol; 85% yield) of 17β-t-butylamino-3-oxo-4-aza-5 -androst- 1-ene (16) .

^■ " ^■ H-NMR (60 MHz): S 6.82 (d, J-9Hz, 1H) , 5.82 (m, exchangeable overlapping d, J-9Hz, 3H) , 3.3 ÷ 3 (m, 20 1H) , 2.8 ÷ 0.6 (complex system). I.R. (CHC1 ₃ ) - 3410, 1669

MS m/e: 359 (M+l), 358 (M) , 343 (M-15), 300 (M-58) Elemental analysis for ^C 23H _3o N ₂ 0: calculated C-77.09, H-10.61, N-7.82; 25 found C-77.10, H=10.63, N-7.85

EXAMPLE 18 Compound (18)

30 ml of a 10% KOH/ethanol solution containing 3 g

(10.38 mmol) of 3 ,17-dioxo-4-aza-50^-androstane was

30 stirred in the dark at room temperature. Then 3.3 g

(31.14 mmol) of benzaldehyde were added and the

SUBSTITUTE SHEET

reaction mixture was stirred for 6 hours. Thereafter, 30 g of ice-water were added and the reaction mixture was concentrated under vacuum to one third of its volume. The reaction mixture was then extracted with dichloromethane (3x20 ml) and the organic phases were dried over Na ₂ S0 ₄ and evaporated under vacuum. The so obtained crude was purified through silica chromatography (1:80) eluting with 95/5 dichloromethane/methanol. 3.52 g (9.34 mmol; 90% yield) of 3 ,17-dioxo-16-benzylydene-4-aza-5D(-androstane were obtained.

¹ H-NMR (60 MHz): S ^" 7.5 (s, 5H) , 7.1 (s, 1H) , 3.3 - 3.1 (m, 1H) , 2.9 - 0.9 (complex system).

- I.R. (CHCl ₃ ) = 1710, 1655 - MS m/e: 377 (M) , 362 (M-15), 286 (M-91), 234 (M-143) Elemental analysis for C_,-H 0 _? N: calculated C-79.57, H=8.22, N-3.71; found C-79.6, H=8.23, N=3.73

According to the method described in Example 17, the Wittig reaction was carried out, and the subsequent acid hydrolysis of the methoxyenolether intermediate gave compound (XX).

Starting from 2.5 g (6.63 mmol) of compound (XVIII), 2.13 g (5.25 mmol; 79% yield) of intermediate (XIX) were obtained.

- I.R. (CHC1 ₃ ) = 1660

- MS m/e: 405 (M) , 390 (M-15), 374 (M-31), 314 (M-91).

Elemental analysis for C ₂₇ H ₃₅ 0 ₂ N: calculated C-80.00, H-8.64, N-3.45; found C-80.03, H-8.65, N-3.48

Starting from 2 g (4.33 mmol) of intermediate (XIX),

SUBSTITUTE SHEET

1.65 g (4.23 mmol; 86% yield) of 17B-formyl-16- benzylydene-3-oxo-4-aza-5&-androstane (XX) were obtained.

¹ H-NMR (60 MHz): ζ 9.8 (S, 1H) , 7.5 (s, 5H) , 7 (s, 1H) 3.3 ÷ 3.1 (m, 1H) , 2.9 ÷ 0.6 (complex system).

I.R. (CHC1 ₃ ) = 1740, 1660

MS m/e: 392 (M+l), 391 (M) , 376 (M-15), 362 (M- 29), 301 (M-91).

Elemental analysis for ^c ₂ gH ₃₃ 0 ₂ N: calculated C=79.79, H-8.43, N-3.58; found C-79.81, H-8.41, N=3.61

3.06 g (7.82 mmol) of compound (XX) were dissolved into 15 ml of dioxane under stirring in the dark at room temperature. 5 ml of an aqueous solution containing 2.66 g of AgN0 ₃ were added and after 1 minute 5 ml of an aqueous solution containing 2.92 g of KOH. After 30 minutes the reaction mixture was filtered on Celite^ ^R ^ and dioxane was evaporated off the filtrate. The aqueous phase was acidified with 2N HCl and extracted with dichloromethane (3x20 ml). The organic phase was dried over Na ₂ S0 ₄ and evaporated under vacuum to give 2.6 g (6.41 mmol; 82% yield) of compound (XXI).

- I.R. (CHC1 ₃ ) = 3100, 1760, 1660

- MS m/e: 407 (M) , 392 (M-15), 389 (M-18), 358 (M-49) - Elemental analysis for C _2g H ₃₃ 0 ₃ N: calculated C-76.65, H-8.10, N-3.43; found C-76.68, H-8.14, N-3.47.

Acid (XXI) was used in the next step without further purification 13.75 g (33.8 mmol) of compound (XXI) were dissolved into 170 ml of anhydrous toluene and 3.75 ml of pyridine. The reaction mixture was then cooled down

SUBSTITUTE SHEET

»«& _! tøtøj-3r$-»'SSϊ3* ^!w ϊ ^L

50

to 10°C and 5.37 g (42.35 mmol) of oxalyl chloride into 10 ml of toluene were slowly added. The reaction temperature was kept 1 hour at 10°C, then 16 ml of a toluene solution containing 5.37 g (168 mmol) of 5 hydrazine were added. The reaction mixture was maintained under stirring at 40°C for 30 minutes, then the reaction mixture was added with 160 g of ice and neutralized with IN HCl and the organic phase was separated. The aqueous phase was extracted with

10 dichloromethane (150 ml) and the organic phases were collected, dried over Na ₂ S0 ₄ and evaporated under vacuum to give a crude which was subsequently purified by silica gel chromatography (1:40) eluting with a 9/1 dichloromethane/methanol mixture, thus obtaining 14.11

15 g (29.4 mmol; 87% yield) of 16-benzylydene-17β-N- hydrazinylcarbamoyl-3-oxo-4-aza-50tc-androstane (XXII) .

- I.R. (CHC1 ₃ ) = 3600, 1680

- MS m/e: 421 (M) , 406 (M-15), 390 (M-31), 391 (M-30).

Elemental analysis for ^C 26 ^H 35°2 ^{N :} 20 calculated C=74.10, H=8.31, N-9.97; found C-74.12, H-8.35, N-9.94.

550 mg (1.03 mmol) of compound (XI) were dissolved into 10 ml of methanol and cooled down to -70°C. Then the product was ozonized until a persistent blue colour was 25 observed. The 0 ₃ excess was removed by N ₂ bubbling and the solution was added with a suspension of triphenylphosphine polymer bound (2.06 meq of reducing agent) in methanol. The reaction mixture was stirred for 2 hours, then the product was isolated by 30 filtration (accurately washing the filter with 30 ml of methanol). This solution was directly used in the next

SUBSTITUTE SHEET

step without further purification: the filtrate obtained from the preceding reaction was stirred at room temperature and added with 5 ml of a methanol solution containing 151 mg of NaBH ₄ After 3 hours at room temperature, the reaction mixture was neutralized with IN HCl, methanol was evaporated under vacuum and aqueous phase extracted with dichloromethane (3x10 ml). The organic phase was dried on Na ₂ S0 ₄ , evaporated under vacuum to give a crude which was subsequently purified by silica gel chromatography (1:50), elution with 9/1 dichloromethane/methanol to give 0.221 g (0.67 mmol; 65% yield) of product (18 bis).

¹ H-NMR (60 MHz): S 5.8 (m, 1H exchangeable), 3.3 - 3 (m, 1H) , 2.9 - 0.6 (complex system). - I.R. (CHC1-) = 1680

MS m/e: 331 (M) , 316 (M-15), 301 (M-30). Elemental analysis for C, _g H- _g O N,: calculated C=68.88, H=8.76, N-12.68; found C-68.91, H=8.78, N-12.70 The unsaturation reaction was carried out according to the procedure described for compound (16 bis). Starting from 750 mg (2.26 mmol) of compound (18 bis), 480 mg of compound (18) were obtained after chromatographic purification. ¹ H-NMR (60 MHz): S 6.82 (d, J-lOHz, 1H) , 5.9 (m, exchangeable overlapped to d, J-lOHz, 2H) , 3.3 ÷ 3.1 (m, 1H) , 2.9 * 0.6 (complex system).

- I.R. (CHC1 ₃ ) = 1660, 1690

- MS m/e: 330 (M+l), 329 (M) , 314 (M-15), 229 (M-30).

- Elemental analysis for ^c _ι QH ₂₇ 0 ₂ N, : calculated C=69.30, H-8.20, N=12.76;

SUBSTITUTE SHEET

found C-69 . 31 , H=8 . 22 , N=12 . 74 .

EXAMPLE 19 Compounds (17) and (17 bis)

According to the method described for compound (VII) to obtain compound (16 bis), using the same reactant weight ratio and the same reaction conditions, compound (17) was prepared starting from 20 g (63.19 mmol) of 5- pregnen-3β-ol-20-one. 19.32 mg (51.8 mmol; 82% yield) of compound (XIII) were obtained after chromatographic purification.

^■ • ^• H-NMR (60 MHz): cT 5.8 (m, exchangeable 2 H), 5.3 (s, 1 H) , 2.4 -r 0.6 (complex system).

- I.R. 3600.

- MS m/e= 373 (M) , 358 (M-15), 343 (M-30), 301 (M-72) - Elemental analysis for C-.. H _4T 0 _N calculated C = 80.42, H- 11.52, N - 3.75; found C - 80.44, H- 11.56, N= 3.79 20.51 g (55 mmol) of compound (XIII) were dissolved into 400 ml of toluene and 45,8 ml of cyclohexanone, then the temperature was raised up to 100°C and 100 ml of a toluene solution containing 10 g of aluminum isopropoxide were added. The reaction mixture was refluxed for 4 hours, then neutralized with 5% H ₂ S0 ₄ The reaction mixture was filtrated on Celite' ^R ^ and the filtrate was extracted with toluene (2x50 ml). The organic phase was dried on Na ₂ S0 ₄ and under vacuum evaporated to give a crude which was subsequently purified by crystallization from hexane to give 12 g (32.4 mmol; 59% yield) of product (XIV)'. ¹ H-NMR (60 MHz): δ ^" 5.7 (s, 1H) , 5.4 (m, 1H exchangeable) 2.4 - 0.6 (complex system)

SUBSTITUTE SHEET

-«*->>.8-SET ^*

53

- IR (CHC1 ₃ )- 1660

- MS m/e- 371 (M) , 356 (M-15), 314 (M-57), 299 (M-72) Elemental analysis for C- c H ₄₁ o N: calculated C- 80.86, H= 11.05, N= 3.77; found C= 80.89, H= 11.08, N- 3.79 3.79 mg (10.23 mmol) of compound (XIV) were dissolved into 60 ml of butanol with 8 ml of an aqueous solution containing 1.68 g of Na ₂ co ₃ . The solution was refluxed and under vigorous stirring 60 ml of a warm aqueous

10 solution containing 16 g of NaI0 ₄ and 120 mg of KMn0 ₄ were added within 30 minutes. The reaction mixture was refluxed for 1 hour, then was cooled to 30°C and after 20 minutes was filtered under vacuum (washing the filtrate with 10 ml of water). The filtrate was

15 concentrated under vacuum to about 1/3 of the starting volume and acidified with 6N HCl (pH 3). The so obtained precipitate was isolated by filtration giving 2.7 mg of crude (XV) .

IR (CHC1 ₃ ): 1710, 1660

20 MS- 391 (M), 376 (M-15), 373 (M-18) Compound (XV) was used in the next reaction without further purification: 2.15 g (5.5 mmol) of compound (XV) were dissolved into 15 ml of ethylene glycol. The solution was cooled down to -20°C and 2.5 ml of liquid

25 ammonia were added. Then the reaction temperature was raised up to 180°C and kept for 15 minutes. After this time the reaction mixture was cooled down to room temperature, water was added and pH was adjusted to 3 with concentrated HCl. The so obtained solid was

30 isolated by filtration, washing the filter with water (5 ml). The so obtained solid was purified through

SUBSTITUTE SHEET

silica chromatography (1:50) eluting with 9/1 dichloromethane/methanol to give 1.2 g (3.23 mmol; 59% yield) of product (XVI). IR (CHC1 ₃ ) _: 1650 - Elemental analysis for C ₂₄ H ₄₀ 0 N ₂ ; calculated: C- 77.42, H- 10.75, N- 7.52; found: C- 77.44, H- 10.77, N- 7.54.

1.2 g (3.23 mmol) of compound (XVI) were dissolved into 46 ml of methanol with 1.2 g of 10% Pd/C. Air was removed and hydrogen was bubbled for 3 minutes. Then the reaction mixture was refluxed under vigorous stirring while bubbling hydrogen. After 10 hours the reaction mixture was filtrated on Celite and the filtrate was evaporated to dryness. 1.15 g (3.07 mmol; 95% yield) of compound (17 bis) were obtained.

^■ " ^■ H-NMR (60 MHz): S 5.4 (m, 1 H, exchangeable), 3.3 - 3.2 (m, 1 H), 2.9 - 0.9 (complex system) MS m/e- 374 (M) . 359 (M-15) Elemental analysis for C_ ₄ H ₄₂ 0 N _? ; calculated C- 77.00, H- 11.22, N- 7.48; found C- 77.40, H- 11.24, N= 7.51.

Compound (17) was obtained from compound (17 bis) according to the procedure described for compound (16 bis) (Example 17). Starting from 2 g (5.34 mmol) of compound (17 bis), 1.59 g (4.27 mmol; 80% yield) of compound (17) were obtained after chromatographic purification.

¹ H-NMR (60 MHz): S 6.82 (d, J= 9 H„ l _H ) , 5.82 (m, exchangeable overlapping d, J= 9 Hz- 3H) , 3.4 (m, 1H) , 2.8 : 0.6 (complex system)

IR (CHC1 ₃ )= 3330, 1650.

SUBSTITUTE SHEET

MS m/e- 373 (M+l), 372 (M) , 357 (M-15), 314 (M-58) Elemental analysis for C ₂₄ H ₄₀ 0 N ₂ , calculated: C= 77.42, H= 10.75, N= 7.52 found : C- 77.46, H= 10.76, N= 7.50. EXAMPLE 20

Compound (19)

400 mg (1.32 mmol) of compound (VII) were dissolved into 524 ul of titanium (IV) isopropoxide. The solution was cooled to 0°C and added with 142 ul of methylamine. The reaction was allowed to stand under stirring at room temperature for 1 hour, then was diluted with 3 ml of methanol and added with 88 mg of NaBH,CN. The reaction mixture was stirred at room temperature for 18 hours. Finally, the reaction mixture was acidified with IN HCl and extracted with dichloromethane

(3x25ml). The organic phases were dried on Na ₂ S0 ₄ and evaporated to dryness to give a crude, which was purified by silica gel chromatography (1:10) eluting with 95/5 dichloromethane/methanol to give 272 mg of the desired product (65% yield).

¹ H-NMR (60 MHz): S 0.65 (s, 3H) , 0.9 (s, 3H) , 0.5*3.6 (complex system), 2.7 (ε, 3H) , 3.5 (m, 2H) , 4.2 (m, 2H) .

IR (Nujol): 1660 - MS (m/e) - 318 (M+), 303 (M-15), 275 (M-43), 260 (M-58)

Elemental analysis for ^C _2Q H, ₄ ON ₂ calculated: C - 75.46; H - 10.69, N - 8.81 found: C - 75.44, H = 10.70, N - 8.83 EXAMPLE 21

Compound (20)

SUBSTITUTE SHEET

3.9 g (12.3 mmol) of compound (III) were dissolved into 4.59 ml of titanium (IV) isopropoxide. The solution was cooled down to 0°C and added with 380 mg of methylamine. After 30 minutes the reaction was diluted with 20 ml of ethanol and added with 510 mg of NaBH-CN. The reaction mixture was stirred at room temperature for 24 hours. Finally, the reaction mixture was acidified with concentrated HCl and diluted 1:1 with dichloromethane then directly purified by silica gel chromatography (1:10) eluting with 8/2 dichloromethane/methanol to give 50% yield of the desired product.

¹ H-NMR (60 MH ₂ ) _: δ ^" 0.8 (s, 3H) , 0.9 (s, 3H) 0.8÷3.6 (complex system), 2.7 (s, 3H) , 5.8÷6.5 (m, 2H) - IR (Nujol) : 1654

MS: m/e: 332 (M) , 317 (M-15), 276 (M-56) Elemental analysis for C„-H-, ₆ N ₂ 0 calculated C - 75.90; H - 10.84; N - 8.43 found C - 75.92; H - 10.84; N - 8.45 EXAMPLE 22

Compound (15 bis)

1.13 g (3.93 mmol) of compound (5) were dissolved into 71 ml of dichloromethane under stirring at room temperature. 2.457 g of 55% 3-chloroperbenzoic acid were added. The reaction mixture was stirred at room temperature for 2 hours. Then the reaction mixture was washed with 5% NH ₃ (3x70 ml) and with a saturated sodium thiosulfate solution until negative reaction for oxidants. The organic phase was evaporated to dryness to give 1.3 g of a crude, which was twice crystallized from ether to give 920 mg of the desired product (77%

SUBSTITUTE SHEET

S„ ^v *- ~

57

yield) .

¹ H-NMR (60 MH ₂ ) _: 0.85 (s, 3H) , 1 (s, 3H) , 0.8÷3.4

(complex system), 2.8 (m, 2H)

IR (CHC1 ₃ ) : 1653.7

MS m/e - 304 (M+l), 303 (M ⁺ ), 288 (M-15), 273 (M- 30)

Elemental analysis for ^c _τq H _2g 0 ₂ N calculated: C - 75.25; H - 9.57; N - 4.62 found: C - 75.24; H - 9.55, N - 4.64

SUBSTITUTE SHEET