Background of the Inventiαn

This invention relates to novel inhibitors of sex steroid activity such as antiandrogen compounds having effective antagonistic capability while substantially lacking agonistic effects. More particularly, cer¬ tain preferred embodiments of the invention relate to certain dihydrotes- tosterone analogs which have high affinity for androgen receptors but do not activate such receptors and/or which inhibit the production of sex steroids or their precursors.

Brief Descri ^p tion of the Prior Art

During the treatment of certain sex steroid-dependent diseases, it is important to greatly reduce or, if possible, eliminate certain sex steroid-induced effects. For this purpose, it is desirable both to block receptor sites stimulated by sex steroids and also to reduce the amount of sex steroid available to act at these sites. For example, alternative or concurrent therapy to administration of antiandrogens could involve attempts to block the production of androgens such that none is available to activate receptor sites. However, prior art methods for blocking an-

TE SHEET

drogen production and/or reducing androgen concentration insufficiently inhibit androgen-induced functions. Moreover, it is possible that even in the total absence of androgen, unoccupied androgen receptors .-__ay be bio¬ logically active. Hence, antiandrogens may produce greater therapeutic results than therapy which only inhibits androgen production.

Antiandrogens may have a significant therapeutic effect in slowing or stopping the progress of androgen-dependent diseases such as prostate cancer. Known antiandrogens such as cyproterone acetate, ≤pironolactone, flutamide and anandron have been used in clinical studies of androgen- responsive diseases. The nonsteroida.1 compounds flutamide and anandron are pure effective antiandrogens (i.e., do not activate androgen recep¬ tors) , but cause an increase in serum concentration of luteinizing hormo¬ ne (LH) and testosterone (Neri and Peets, J. Steroid Biochem. 6, 815-819, 1975; Seguin et al., Mol. Cell. Endocrinol. 21, 37-41, 1981; Neumann et Jacobi, In: Clinics in Oncology, Vol. 1, pp. 41-64, 1982, Ed. B.J.A. Furr, Eastbourne; tf.B. Saunders) . Moreover, nonsteroidal antiandrogens of the prior art possess a relatively low affinity for the androgen receptor (Simard et al., Mol. Cell. Endocrinol. 44, 261-270, 1986). On the other hand, steroidal antiandrogens (i.e., cyproterone acetate and spironolactone) , while having better receptor affinity, may possess intrinsic androgenic activity, thus undesirably functioning as agonists (Poyet and Labrie, Mol. Cell. Endocrinol. 42, 283-288, 1985; Labrie, C. et al., J. Steroid Biochem. 28, 379-384, 1987; uthy et al., J. Steroid Biochem. 31, 845-852, 1988; Plante et al., J. Steroid Biochem. 31, 61-64, 1988).

There is, therefore, a need in the art for antiandrogens which more effectively block peripheric androgen receptors with neither activity on central nervous system nor progestationai, nor intrinsic androgenic or glucocorticoid activity.

Certain nonsteroidal compounds which are stated to have antiandro- genic effect are described by Furr et al., J. Endocr. 113, R7-R9.. 1987.

In U.S. ?at. Ho. 4,329.364, it _is disclosed that the antiandrogen. 4'-nitro-3'-trifluoromethyl isobuty anilide may be used fcr treatment of prostatic cancer.

F. Labrie et al.. The Prostate 4, 579-594, 1983, disclose that use of a combination therapy of an LHRH agonist (Buserelin) and an antiandro¬ gen (Anandron) to treat advanced prostate cancer in previously untreated patients affects simultaneous elimination of androgens of both testicular and adrenal origin.

F. Labrie et al., J. steroid Biochem. 19, 99-1007, 1983, disclose the treatment of prostate cancer by the combined administration of an HRH agonist and an antiandrogen. Labrie et al. disclose animal and cli¬ nical data in support of the proposition that the combined LHRH/anti- aadrogen treatment neutralizes the stijmilatory influence of all androgens on the development and growth of androgen-dependent prostatic cancer.

In U.S. Pat. No. 4.659,695, a method of treatment of prostate cancer is disclosed for susceptible male animals including humans whose testi¬ cular hormonal secretions are blocked by surgical or chemical means, e.g., by use of an LEHH agonist, e.g., [D-Trp«, des-Gly-NH^'jLHSH ethyl- amide. The treatment includes administering an antiandrogen, e.g., fluta¬ mide in association with at least one inhibitor of sex steroid biosynthe¬ sis, e.g., a inoglutethimide and or ketoconazole.

. Labrie et al., in: Genitourinary Cancer, eds. T.L. Ratliff and W.J. Catalona, Martinus Nijhoff Publishers, Boston, pp. 157-200, 1987 and in: Important Advances in Oncology, eds. V.T. De Vita, S. Hellman and S.A. Rosenberg, J.B. Lippincott Company, Philadelphia, pp. 193-217, 1985 descr ⁱ be the role of peripheral formation of androgens from inactive adrenal steroid precursors and the need to use a pure antiandrogen for the treatment of androgen-sensitive diseases.

SUBSTITUTE SHEET

C. Labrie et al. , J. Steroid Biochem. 13 , 379-384, 1987, describe the potent stimulatory effect of the adrenal precursors dehydroepiandros- terone and androstenedione on the growth of the prostate in the rat.

U.S. Pat. No. 4,472,382 discloses a cethod of treating prostate cancer using the combination of an antiandrogen and an LHSH agonist.

In U.S. Pat. No. 4,386,080, it is disclosed that new amide deriva¬ tives, and more particularly novel acyianilides, possess antiandrogenic properties.

In U.S. Pat. No. 3,995,060, TI.S Pat. No. 4,161,540 and U.S. Pat. No. 4,139,638, it is disclosed that certain 4'-substituted and 3'-, 4 ^• -disubstituted anilides have antiandrogenic properties.

EP Pat. No. 138504, EP Pat. No. 166509, EP Pat No. 124369, EP Pat. No. 160508, EP Pat. No. 163 416, U.S. Pat. No. 4,732,312, U.S. Pat. No. 4,760,061, U.S Pat. No. 4,751,240, U.S. Patent 4,659,516 and akeling A.E. and Bowler J., J. Endocr. 112, R7-R10, 1987, and _l___ Steroid Biochem. 30, 141-147, 1988 disclose that certain long chain substitutions onto an estrogenic nucleus may result in compositions exhibiting antiestrogenic activity.

Chang et al., Biochemistry 21, 4102-4109, 1982, disclose the use of teStOSterone' 17p-Vn»ττri «yιr»r-ιτ*ιy" ^" | -" . ~ ^~ ~ -Hi »τm'πnr*Kτ-τnpy ^* l atn-i nA-S-aphaross 4B in the purification of androgen receptor.

De Larminat et al., The Prostate 5: 123-140, 1984, disclose the use of dihydrotestosterone- and testosterone-7c-undecanoyl agarose in the purification of androgen receptor.

Gyorki et al., J. Steroid Biochem. 25, 355-358, 1986, and Macaulay and arne, J. Steroid Biochem. 26, 535-538. 1987, disclose the use of 7α-carboxyethinyl testosterone linked to Seøharose 4B in the purification of androgen receptor.

Salman et al., J. Steroid Biochem. 25, 383-391, 1987, disclose the use of I7α-hexyr_yi r.ortestosterone Sepharose in the purification of androgen receptor.

Grunweil et al., Steroids 27, 759-771, 1976, and Solo et ai., Steroids 40, 503-614, 1982, disclose the synthesis of a series of 7α-alkyltestosterone derivatives and describe their biological activities.

For a number of years, there has been search for compounds which can efficiently inhibit androgen and/or estrogen formation without causing adverse effects to healthy tissues. More particularly, the inhibition of 17β-hydroxysteroid dehydrogenase, which is involved in the biosynthesis of testosterone, androst-5-ene-3β,17β-diol and estradiol, has been studied by some workers. Some affinity-label inhibitors for human placental estradiol 17β-dehydrogenase have been described (C.C. Chin and J.C. Warren, J. Biol. Chem. 250, 7682-7686, 1975; Y.M. Bhatnagar et al., J. Biol. Chem. 253, 811-815, 1978; C.C. Chin et al., J. Biol. Chem. 255, 3560-3664, 1980; J.L. Thomas and R.C. Strickler, J. Biol. Chem. 258, 1587-1590, 1983).

B. Tobias et al., J. Biol. Chem. 257, 2783-2786, 1982 and R.J. Auchus and D.F. Covey, Biochemistry 25, 7295-7300, 1986 disclose, respec¬ tively, the use of 17β-propynyl-substittιted progestins and propynyl- substituted 3-hydroxy-14,15-secoestra-l,3,5(10)-trien-17-one as inhibitors of the 17β-estradiol dehydrogenase.

Thomas J.L. et al., J. Biol. Chem. 258, 11500, 1983 have described that 16-oethylene estradiol and 16-methylene estrone are inhibitors of 17β-HSD activity.

French patent publication number 2,528,434 relates to the pharmaceu ^¬ tical use of llβ- and 2-substituted 19-nor-steroids.

T

Objects of the Invention

It is an object of the present invention to provide methods of inhi¬ biting sex steroid activity. Such methods may be useful in the treatment of sex steroid-related diseases.

It is another object of the invention to provide a steroidal pure antiandrogen for therapeutic use.

It is another object of the invention to provide compositions capa¬ ble of inhibiting sex steroid synthesis, especially androgen synthesis.

It is anotheS object to provide an antiandrogen having good affinity for androgen receptors, but substantially lacking undesirable agonistic activity regarding these receptors and substantially lacking hormonal activity.

It is another object of the invention to provide a therapeutic anti¬ androgenic composition -useful in the treatment of androgen-related disea¬ ses. These diseases include, but are not limited to, prostate cancer, acne vulgaris, hirsutism, precocious puberty, benign prostatic hyperpla- sia, seborrhea, androgenic alopecia and sexual deviants. Control of androgen activity may also be useful in male contraception.

It is another effect of the invention to provide inhibitors of sex steroid production useful in the treatment of both estrogen- and andro¬ gen-related diseases. Estrogen-related diseases include but are not limi¬ ted to breast cancer, uterine cancer, ovarian cancer, endometriosis, uterine fibroma, precocious puberty and benign prostatic hyperplasia.

Summar ^y of the Invention

The present invention provides a method of inhibiting sex steroid activity in a warm-blooded animal, including humans, comprising administering to said animal a therapeutically effective amount of a

compound having an androgenic nucleus substituted at a ring carbon with at least one side chain represented by the formula -R ¹ [-5- ³ —3 L-G wherein: x is an integer from 0 to 6, wherein at least one of L and G is a polar moiety distanced from said ring carbon by at least three interven¬ ing atoms, and wherein:

R ¹ and R ¹ are independently either absent or selected from the group consisting of straight- or branched-chain alkylene, straight- or branch- ed-chain alkynylene, straight- or branched-chain alkenyiene, phenylene, ^■ and fluoro-substituted analogs of the foregoing;

B is either absent or selected from the group consisting of -0-, -S-. -Se-, -SO-, -SO,-, -NR»-, -SiR*,-. -CR ^s OR»-, -NR ^* »CC~, -NR'CS-, -CONR ³ -, -CSHR ^J -, -COO-, -COS-, -SCO-, -CSS-, -SCS-, -OCO- and phenylene (R* being hydrogen or lower alkyl) ;

L is either a moiety which together with G, forms a heterocyclic ring having at least one nitrogen atom or is selected from the group consisting of lower alkyl, -C0NR*-, -CSHR*-, -NR'CO-, -NR'CS-, -NR«C0NR*-

NR« It -NR«C-NR*-, -S0,NR*-, -CSS-, -SCS-, -(H0)R*-. -(P0)R»-, -NR*CCO-,

-NR«SO,-, -0-, -NR*-, -S-, -SO- and -SO,- (R* and R« being independently selected from the group consisting of hydrogen and lower alkyl; and R' being selected from the group consisting of hydrogen, nitrile and nitro) ; and

G is selected from the group consisting of hydrogen, lover alkyl, lower alkenyl, lower alkynyl, (C,-C ₇ )cycloalkyl, bromo(lower) lkyl, chlo-

STITUTE SHEET

1

8

ro(lower)alkyl, uoroiiower) lkyl, iodo(lower)alkyl, cyano(lower) lkyl, carboxy(lower)alkyl, (lower) lkoxycarbonyl(lower) lkyl. (C _β -C, _a )aryl, ( ^c τ" ^c ιι)arylal yl, di(lower)alkylamino(lower)alkyl, fluoro-substituted analogs of the foregoing, and a moiety which together with L forms a heterocyclic ring having at least one nitrogen atom.

The invention further provides a method of inhibiting sex steroid activity in a wapn-blooded animal, including humans, comprising adminis¬ tering a therapeutically effective- amount of at least one compound having, as part„ξj>f its molecular structure, a substituted or unsubstitut¬ ed nucleus of formula I:

wherein the dotted lines represent optional double bonds; a is a carbon or oxygen atom: b is carbon or nitrogen atom; ?■.- - and R ¹³ are independently hydrogen or lover altyl; said compound further having, as a substituent to said nucleus in at least one position selected from the group consisting of 6α, 7 , 14α,

15o, 16o, 17α and 17β, a side chain of the formula -R ¹ [-B-R ³ -] _χ L-G, wherein: x is an integer from 0 to 6, wherein Lis separated from said andro¬ genic nucleus by at least 3 atoms, and wherein:

R* and R» are independently either absent or selected from the group consisting of straight- or branched-chain alkylene, straight- cr branch¬ ed-chain aikynyiene, straight- or branched-chain alkenyiene, phenylene and fiuoro-substituted analogs of the foregoing;

B is either absent cr selected from the group consisting of -0-, -S-, -Se-, -SO-, -SO,-, -NR ³ -, -SiR ³ ,-, -CR ³ OR ³ -, -NR'CO-, -NR CS-, -CONR-, -CSNR-, -C00-, -COS-, -SCO-, -CSS-, -SCS-, -OC0- and phenylene CR' being hydrogen or lower alkyl) ;

L is either a moiety which together with G. forms a heterocyclic -ring having at least one nitrogen atom or is selected from the group consisting of lower alkylene -COHR*-, -CSHR*-, -NR»CO-, -NR»CS-,

NR«

-NR ^S CONR*-, -NR«C-NR*-, -SO,NR*-. -CSS-. -SCS-, -OTO)R*-, -(PO)R*-, -NR'COO-, -NR.SO,-, -0-, -NR*-, -S-, -SO- and -SO,- (R* and R« being independently selected from the group consisting of hydrogen and lower alkyl; and R« being selected from the group consisting of hydrogen, nitrile and nitro) ; and

G ⁱ s selected from the group consisting of hydrogen, lower alkyl, lower alkenyl, lower alkynyl, <C,-C _T )cycloalk7l, halo(lower)alkyl, carboxy(lower)alkyl, (lover)alkoxycarbonyl(lower)alkyl, ( _C| -C _lβ )aryl, (C,-C _xl )arylalkyl, di(lover)alkylamino(lover)al yl, fiuoro-substituted analogs of the foregoing and a moiety which together with L forms a heterocyclic ring having at least one nitrogen atom.

As used herein, the term "sex steroid activity inhibitor" includes any compound which suppresses the activity of sex steroids by any mecha¬ nism including, for example, inhibition of sex steroid synthesis or anta¬ gonistic blocking of sex steroid receptors. "Androgen activity inhibi¬ tors'- and "estrogen activity inhibitors" are sex steroid inhibitors capa¬ ble of inhibiting the activity of androgens and estrogens, respectively.

SUBSTITUT

Inhibitors include, but are not limited to antiandrogens which block androgen receptors, thereσy making them unavailable to androgen compounds which could otherwise activate those receptors. Androgen activity inhi¬ bitors also include compounds which inhibit the formation of compounds capable of activating androgen receptors such as inhibitors of production of natural androgens (e.q. dihydrotestosterone) or inhibitors of produc¬ tion of precursors of natural androgens. One mechanism by which these androgen production inhibitors may operate is by blocking enzymes vhich catalyze production of natural androgens or their precursors (e.g. enzy¬ mes such as aromatase, 17 -hydroxysteroid dehydrogenase, 3β-hydroxyste- roid dehydro enase, 5α-reductase and the like).

As used herein, the term "androgenic nucleus" includes any compound which, in the absence of the side chain substituent specified herein, is capable of acting as an androgen as determined by a weight increase of at least 35 percent over a seven-day period of the prostates of castrated rats treated with the compound in question (15 milligrams twice daily per 100 grams of body weight) versus a control group of castrated rats. Treatment should start on the day of castration. The precise test, other than any parameters set forth in this paragraph, is that reported in Labrie et al., J. Steroid Biochem. 28, 379-384, 1987.

The present invention further provides for the treatment of sex ste¬ roid-related diseases by the methods of administering therapeutically effective amounts of sex-steroid activity inhibitors as disclosed herein (with or without pharmaceutical carriers or diluents). Sex steroid-rela¬ ted diseases include any disease whose onset, maintenance or progress is, at least in part, dependent upon biological activities induced by sex steroids such as androgens and estrogens. For example, androgen-dependent diseases include but are not limited to prostate cancer, acne vulgaπs.

hirsutism, precocious puberty, benign prostatic hyperplasia, seborrhea, androgen alopecia and sexual deviance. Control of androgenic activity nay also be useful in male contraception.

The invention further provides a pharmaceutical composition compris¬ ing a pharmaceutically acceptable diluent or carrier and a therapeutical¬ ly effective amount of a sex steroid activity inhibitor having an andro¬ genic nucleus substituted at a ring carbon with at least one side chain represented by the formula -R ¹ [-B-R ³ -] L-G wherein: x is an integer from 0 to 6, wherein at least one of L and G is a polar moiety distanced from said ring carbon by at least three interven¬ ing atoms, and wherein:

R ¹ and R ³ are independently either absent or selected from the group consisting of straight- or branched-chain alkylene, straight- or branch¬ ed-chain alkynylene, straight- or branched-chain alkenylene, phenylene and fiuoro-substituted analogs of the foregoing; -

B is either absent or selected from the group consisting of -O-, -S-, -Se-, -SO-, -SO,-, -NR ³ -, -SiR ³ ,-, -CR OR ³ -, -NR CC-, -NR ³ CS- _f -C0NR ³ -, -CSNR ³ -, -C00-, -COS-, -SCO-, -CSS-, -SCS-, -OC0- and phenylene (R ³ being hydrogen or lover alkyl) ;

L is either a moiety which together with G, forms a heterocyclic ring having at least one nitrogen atom or is selected from the group

NR« consisting of -CONR*-, -CSNR*-, -NR«C0-, -NR*CS-, -NR«C0NR*-, -NR*C-NR*-, -S0,NR*-, -CSS-, -SCS-, -(N0)R*-, -(PO)R ⁴ -, -NR»C00-, -NR*S0,-, -0-, -NR ⁴ -, -S-, -SO- and -SO,- (R* and R ^s being independently selected from the group consisting of hydrogen and lower alkyl; and R, being selected from the group consisting of hydrogen, nitrile and nitro) ; and

SUBSTITUTE SHEET

91/00732

12

G is selected from the group consisting of hydrogen, lower alkyl, lower alkenyi, lower alkynyl, (C ₃ -C _τ )cycloalkyl, halo(lower)alkyl, carbo¬ xy(lower)alkyl, (lower) lkoxycarbonyl(lower)alkyl, (C,-C ₁₀ )aryl, (C _τ - C )arylalkyl, di(lower)alkylamino(lower)alkyl, fiuoro-substituted ana¬ logs of the foregoing and a moiety which together with L forms a hetero- i. cyclic ring having at least one nitrogen atom.

The invention further provides a sex steroid activity inhibiting compound having an androgenic nucleus substituted at a ring carbon with at least one side chain represented by the formula -R ^l [-B-R ³ -] L-G ^: wherein: x is an integer from 0 to 6, wherein at least one of L and G is a polar moiety distanced from said ring carbon by at least 8 atoms, and -wherein:

R ¹ and R- are independently either absent or selected from the group consisting of straight- or branched-chain alkylene, straight- or branch¬ ed-chain alkynylene, straight- or branched-chain alkenylene, phenylene and fiuoro-substituted analogs of the foregoing;

B is either absent or selected from the group consisting of -0-, -S-, -Se-, -SO-, -SO,-, - -- , -SiR ³ ,-, -CR'OR ³ -, -NR C0-, -NR'CS-, -CONR ³ -, -CSNR ³ -, -COO-, -COS-, -SCO-, -CSS-, -SCS-, -OCO- and phenylene (R ³ being hydrogen or lover alkyl) ;

L is either a moiety which together with G, forms a heterocyclic ring having at least one nitrogen atom or is selected from the group

NR« il consisting of -CONR*- , -CSNR*- , -NR«CO- , -NR»CS- , -NR»CONR*- , -NR*C-NR*- .

-SO _j NR*-, -CSS-, -SCS-, -(N0)R ⁴ -, -(PO)R*-, -NR ^s COO-, -NR*SO,- _f -0-, -NR*-, -S-, -SO- and -SO,- (R* and R ⁵ being independently selected from the group consisting of hydrogen and lower alkyl; and R* being selected from the group consisting of hydrogen, nitrile and nitro) ; and

G is selected from the group consisting of hydrogen, lower alkyl, lower alkenyl, lower alkynyl, (C,-C _τ )cycloalkyl, halo(lower)alkyl, carbo¬ xy(lower)alkyl, (lower)alkoxycarbonyl(lower)alkyl, (C ₈ -C ₁₀ )aryl, (C _γ -C _jj iarylal l, di(lower)alkylamino(lover)alkyl, fiuoro-substituted analogs of the foregoing and a moiety which together with L forms a heterocyclic ring having at least one nitrogen atom.

The following conventions apply to structural formulae set forth herein. Unless specifically designated to the contrary, substituents may have either α or β stereochemistry or, where valence permits may repre¬ sent one substituent in α position and another in β position. Presence of optional double bonds are independent of each other. All structures include salts thereof. Atoms of any androgenic nucleus for which no sub¬ stituent is shown or described may optionally be substituted or unsubsti¬ tuted so long as such substitution does not prevent the nucleus from functioning as an "androgenic nucleus" as defined herein. Those atoms having a defined substitutent may optionally be further substituted by other substituents where their valence permits such further substitution. As used herein, the term "lover", vhen describing a chemical moiety means a moiety having 8 or fever atoms. For instance, a "lower alkyl" means a Cj to C, alkyl. Any moiety of more than two atoms may be straight- or branched-chain unless otherwise specified.

TE SHEET

WO 91/00732 PCT/CA9

14

BRIEF DESCRIPTION OF TΗE DRAWINGS

Figure 1 is a graph illustrating the antiandrogenic activity of one preferred antiandrogen of the invention.

Figure 2 is a graph illustrating that the antiandrogen which is the subject of Figure 1 is also a good inhibitor of sex steroid synthesis as compared to other known synthesis inhibitors.

Figure 3 is a graph showing the inhibitory effect of different concentra¬ tions of 17β-hydroxy-17o-(12-iododecynyl)-4-androsten-3-one ("ΞM 150") on the activity of 17β-hydroxysteroid dehydrogenase (an enzyme which catalyses various reactions involved in sex steroid synthesis).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Preferred methods of treating of sex steroid-related diseases, especially androgen-related diseases, and preferred methods of blocking androgen receptors comprise administering to a patient in need of such treatment, a therapeutically effective amount of a sex steroid-activity inhibitors comprising an androgenic nucleus substituted with a side chain of the formula -RϊJ-B-R ³ -] L-G as defined above.

Preferred androgenic nuclei suitable for use in accordance with the invention are dihydrotestosterone and derivatives thereof, as well as testosterone and its derivatives. Other suitable androgenic nuclei include but are not limited to those which (as reported in the references set forth below) ^• effect more than the threshold increase in prostate

weight of castrated rats (Labrie C et al. , J. Steroid Biochem. 28: 379-384 , 1987 ; Labrie C et al. . Endocrinology 123 : 1412-1417 , 1988 ; Plante et al. , J. Steroid Biochem. 31: 64-74, 1988) .

Some preferred androgenic nuclei are those of the general structure:

wherein R ^{1 Q} and R ¹³ are independently hydrogen or lower alkyl.

In some preferred embodiments, a and b of structure I are carbon atoms or a is carbon and b is nitrogen. Preferably, the nucleus is substituted in the 17β position with hydroxyl or ( C _j -C , _e ) alkanoyloxy .

Other preferred androgenic nuclei have the structure:

wherein R ^{l β} and R ¹³ are independently hydrogen or lower alkyl;

.»,<«> is selected from the group consisting of hydrogen, hydroxyl. lower alkanoyloxy, lower alkyl, lower alkenyl, lower alkynyl.

HEET

halo (lower) lkyl , halo(lower) alkenyl , halo (lover) alkynyl and fiuoro- substituted aromatic ring.

R ^{1 T} is selected from the group consisting of hydroxyl , (Cj-C _{ϊ β} ) alkanoyloxy, (C ₃ -C _r )alkenoyloxy, (C ₃ -C ₇ )alkynoyloxy, aroyloxy, alkenoyloxy, cycloalkenyloxy, 1-alkyloxy-alkyloxy, 1-alkyloxycycloalkyloxy, alkylsilyloxy, carboxyl, alkanoyl, or R ⁱ * ^(σ) and

»ι.Cβ> together are represented by the formula:

Other preferred androgenic nuclei have the structure:

wherein: R* ^τ is selected from the group consisting of hydrogen, hydroxyl, lower alkanoyloxy, lower alkyl, lower alkenyl, lower alkynyl.

halo(lower)alkyl, halo(lower)alkenyi, halo(lower) lkynyi and fiuoro- substituted aromatic ring.

(S3) R ^1"1 is selected from the group consisting of hydroxyl,

(CJ-C _{J J} )alkanoyloxy, (C,-C ₇ )alkenoyloxy, (C,-C _τ )alkynoyloxy, aroyloxy, alkenoyloxy, cycloalken loxy, 1-alkyloxy-alkyloxy, 1-alkyloxycycloalkylo¬ xy, alkylsilyloxy, carboxyl, alkanoyl, or R ^1T and R ^{1T β} together are represented by the formula:

The AB-ring junction, in preferred embodiments, has trans configura¬ tion.

When sex steroid activity inhibitors are administered in accordance with the invention, they are preferably administered as a dosage from about 1 mg to about 2000 mg of active expedient (i.e. sex steroid activi¬ ty inhibitor) , per day per 50 kg of body weight, most preferably from about 10 mg to about 100 mg per day per kg of body weight.

The sex steroid activity inhibitors are preferably prepared as phar¬ maceutical compositions together with pharmaceutically acceptable car¬ riers and diluents. When prepared for parenteral injection, an inhibitor of sex steroid activity is preferably added at a concentration between about 1 mg/ml and about 100 mg/ml (preferably about 2 mg/ml to about 10 mg/ml) into a carrier preferably selected from the group consisting of saline, water, aqueous ethanol, aqueous dimethylsulfoxide and oil.

When a pharmaceutical composition of the invention is prepared for oral ingestion, the composition preferably included at least one inhibi¬ tor of sex steroid activity wherein the total concentration of all such inhibitors in said pharmaceutical composition is from about 1% to about 95% of the composition (by weight) , and preferably from about 5% to about 20%. The composition preferably further includes a pharmaceutically acceptable diluent, for example, starch or lactose with or without tar- trazine. Slow release pharmaceutical products comprising the novel inhi¬ bitors of sex steroid activity may be incorporated into slow release ^•■ pharmaceutical products which, other than addition of the novel inhibi¬ tors, may be prepared by known methods and administered orally as well as parenterally.

It is preferred that at least one side chain -R ¹ [-B-R ³ -] L-G be substituted onto an androgenic nucleus at either the 6α, 7o, 14 , 15o, 16c, 17o or 17β position (for locating these positions. See structural formula I above) . Especially preferred are the 15α, 17α and particularly 7α positions. In the above side-chain structure, L is preferably separat¬ ed from the androgenic nucleus by at least 3 intervening and preferably 6 atoms. A polar moiety (G, L or both) is preferably separated from the androgenic nucleus by at least 8 intervening atoms.

In certain embodiments of the invention, R ^1T ^ substituents are hydroxyl or one of its ester derivatives, such as acetate, oenanthate, cypionate and trans-4-n-butyl-cyclohexanecarboxylate. It is also preferr¬ ed that the side chain R ¹ [-B-R ¹ -] L-G have between about 7 and 30 carbon

19

a ^t oms ^, in _certain embodiments , therapeutic composi _t ions may comprise one or more sex steroid activity inhibitors represented by the formula V below:

G

vherein *» _is preferably absent, hydrogen or me _t hyl in _β configura _t ion, vherein R„ _{is hydrogen or lower alkyl>} ^^ _{?referred seχ} ^^ _& ^_

vity inhibitors include those of formula VI below (or i _t s 17β-es _t er deri _¬ vatives ⁾ which illustrates one preferred side chain at the 7α position.-

wherein y _is preferably an integer from 4 to 20; vherein L is preferably -CONR*-, -CSNR*-, -».«-. -NR«CS- or -CH,- «.

«* »' being hydrogen or methyl) and G is preferably n-propyl. n-butyl, n-pentyl or halo(lower)alkyl; wherein the AB-ring juction is preferably _t rans _; and the dotted line represents an optional double bond;

SUBSTIT

1/00732

20

wherein R ^1T is preferably hydroxyl or alkanoyloxy; wherein R ^iT is hydrogen or lower alkyl or R ¹⁷ or R ^ιr ^ together are represented by the formula:

Structures V and VI represent preferred antiandrogens for use in the treatment of prostate cancer and other androgen-related diseases where the combination of androgen receptor blocking and androgen synthesis inhibition is desirable.

The inhibitors of sex steroid activity may be used in combination with surgical or chemical castration and/or other inhibitors of sex steroid activity, especialy those which act upon other enzymes involved in synthesis of sex steroids-or their precursors.

Another preferred androgen-activity inhibitor is one of the general formula V below (or its 17β-ester derivatives) which illustrates that positions 10 and 13 (for locating these positions, see structural formula V above) are preferably methylated in the β configuration, and that a 17β hydroxyl is also

vherein the AB-ring junction is trans, the dotted lines in the A-ring represents optional pi bonds; y is an integer from 4 to 20, L is selected from the group consisting of -CONR*-, -CSNR*-, -NR--C0-, -NR--CS- or -CH,- (R- ^* .and R* being hydrogen or methyl) and G either is n-propyl, n-butyl, n-pentyl or haloalkyl.

EXAMPLES OF SYNTHESIS OF PREFERRED INHIBITORS OF SEX STEROID ACTIVITY

INSTRUMENTATION

The IR spectra were taken on a Perkin-Elmer 1310 spectrophotometer. Proton NMR spectra were recorded on a Varian EM-360A (60 MHz, when speci¬ fied) or a Varian XL-200 (MHz) instrument. The following abreviations have been used: s, singlet; d, doublet; dd, doublet of doublet: t, ^" triplet; q, quadruplet; and m, ultiplet. Chemical shifts are reported in δ vaiues in ppm relative to tetramethysilane (TMS) as internal standard. Mass spectra (MS) vere obtained on a V.G. Micromass 16F machine. Thin- layer chromatography (TLC) vas performed on 0.25 mm Kieselgel 60F254 plates (E. Merck, Darmstadt, FRG) . For flash chromatography, Merck- ieselgel 60(230-400 mesh A.S.T.M.) vas used. All solvents used in chromatography has been distilled. Unless otherwise note, starting material and reactant vere obtained commercially and vere used as such or purified by standard means. All solvents and reactants purified and dried vere stored under argon. Anhydrous reactions vere performed under an inert atmosphere, the set-up assembled and cooled under argon. Organic solutions vere dried over magnesium sulfate, evaporated on a rotatory evaporator and under reduced pressure. Anhydrous solvents were prepared in the following way.

SOLVENT DISTILLED OVER

AMINΞ, DIMETHYLFORMAMIDE CaH,

TE SHEET

22

HEXANE, ZICHLOROMETHANE P ₅ Os

ACETONE K,C0 ₃

BENZENE LiAlH _λ

TOLUENE Na

ETHER, TΞTRAHYDROFURAN LiAlH _t , Na Benzophenone

Synthesis of N-butyl, N-metfayl-11-(17'β-hydruxy- '-androsten-3'-on-7'α- yl) undecanamide (5, x-10) (Scheme 1)

17β-acetoαcy-7α-Cll'-hydroay undecanyl)-4-androste_tt-3-one (2)

Under argon atmosphere, in a flame dried apparatus with magnetic stirrer, a solution of 11-bromo undecanol tetrahydropyranyl ether (25 g, 74 mmol) in anhydrous THF (150 ml) was added dropwise to iodine-activated magnesium (1.9 g).The mixture vas kept at room temperature overnight and then vas cooled to -30*C and anhydrous cuprous chloride (0.3 g) vas added quickly. After 45 min of strirring at this temperature, commercial 4,6-androstadien-17β-ol-3-one acetate (1) (10 g, 30.5 mmol) in anhydrous THF (100 ml) vas added dropwise during 4 h. After 35 min, acetic acid (6 ml) and water (100 ml) vas added. The mixture vas alloved to reach room temperature and vas stirred overnight. Aftervards, the organic compound vas extracted vith ether (3X) . The organic layers vere vashed vith vater, dried on magnesium sulfate and evaporated. The residue was dissolved in acetic acid (35 ml) and water (100 ml) and kept 48 h at

room temperature. And then, the organic compounds were extracted with ether (3X). The organic layers were washed with saturated sodium bicar¬ bonate solution and water, dried on magnesium sulfate and evaporated. The product was purified by Silica gel dry column chromatography (Kieselgel, 60F254, Merk, 0.063- 0.200 mm, 150 g) . Elution with a mixture of methyl- ene chloride and ethyl acetate (20:1 v/v) gave 17β-acetoxy-7α-(ll'- h-ydroxy-undecanyl)-4-androsten-3-one (2a, 1.46 g, 2.8 mmol, 9.22) as a colorless oil; IR υ neat 3450, 1740, 1685, 1620 and 1245 cm" ¹ ; NMR max

0.84 (s, 3H, 18'-CH,), 1.21 (s, 3H,~ 19'-CH ₃ ) , 2.05 (s,3H, OC0CH ₃ ), 3.61 t, 2H, J=6.59 Hz, H-C.l'), 4.61 (t, IH, J=7.69 Hz, H-C.17) and 5.73 (s, IH, H-C.4) and 17β-acetoxy-7β-(11'-hydroxy undecanyl)-4-androsten-3-one (2b, 0.9 g, 1.7 mmol, 5.52) as a colorless oil.

ll-(17'β-aceteB_y-4 ^, -androstea-3 ^, -on-7 ^, o-yl) undecanoic acid (3)

To 17β-acetoxy-7c-(11'-hydroxy undecanyl)-4-androsten-3-one (2a, 800 mg, 1.6 mmol) dissolved in acetone (50 ml) and cooled to 0°C was added under stirring during 5 -it ^" -- , a solution of Jones' reagent (8N chromic acid solution) (0.283 ml). After 15 min. isopropanol (0.5 ml) was added followed by water and the mixture vas extracted with ethyl acetate (3X) . The organic layers were washed with brine, dried on magnesium sulfate and evaporated to dryness under reduced pressure. The crude ll-(17^-acetoxy-4'-androsten-3'-on-7O-yl) undecanoic acid (3) (740 mg) was used in the next step without purification.

1/00732

24

N-butyl, N-iDetbyl-ll-(17 ^, β-acetoxy-4 ^, -androsten-3 ^, -on-7 ^, α-yl) undecana-

To a solution of the above undecanoic acid derivative 3 (390 mg, 0.78 mmol) in anhydrous methylene chloride (8 ml) cooled at -10 ^* *C was added, under stirring, triisobutylamine (240 μl) and isobutylchlorofor- mate (140 μl) . After 30 min, N-methylbutylamine (1.8 ml) was added and the mixture was stirred at room temperature for 1 h. Methylene chloride was added. The organic solution was washed with IN hydrochloric acid, water, saturated sodium bicarbonate solution and finally with water, dried on magnesium sulfate and evaporated to dryness. The residue was chromatographed on silica gel (Kieselgel, 60F254, Merck, 0.063-0.200 mm, 20 g) . Elution with a mixture of diethyl ether and methylene chloride (1:20, v/v) gave N-butyl, N-mβthyl-ll-(17 ^, β-acetoxy- ^l -androsten-3 ^, -on- 7'e-yl) undecanamide 4 (230 mg, 0.39 mmol, 46% for the alcohol

( 2a )) as a colorless oil; IR v neat 1740, 1680, 1640 and 1240 max cm-*; HM 0.84 (s, 3H, 18'-CH,), 0.95 (t, 3H, J-6.93 Hz, N-(CH ,CH,) , 1.21 (s, 3H, 19'-CH,), 2.04- (s, 3H, OCOCH,), 2.91 and 2.97 (2s, 3H, N-CH,), 3.26 and 3.36 (2t, 2H, J-7.86 Hz, N-CH,C,H _T ) , 4.61 (t, IH, J-8.42 Hz, H-C.17') and 5.72 (s, IH, H-C.4').

N-butyl, N-metfayl-ll-(17'β-hydroιy— '-androsten-3'-on-7'c-yl) undec¬ anamide (5) (EM 101)

The above acetoxy amide 4 (170 mg, 0.29 mmol) was dissolved in methanol (20 ml) and 6% potassium carbonate (2 ml) and heated at 65"C for

200 min. After cooling, acetic acid (1 ml) and water (150 ml) were added and the mixture was extracted with ethyl acetate (3X) . The organic layers were washed with water, dried on magnesium sulfate and evaporated to dryness. The residue was purified by Silica gel dry column chromatography (Kieselgel, 60F254, Merk, 0.063-0.200 mm, 20 g) . Elution with a mixture of diethyl ether and methylene chloride (1:9, v/v) gave N-butyl-N-methyl- 11-(17'β-hydroxy-4'-androsten-3 ^! -on-7'α-yl) undecanamide (EM 101, 94 mg,

0.17 mmol, 58%) as a colorless oil: IR \> (neat) 3400, 1670 and 1640

_ max car ⁱ ; NMR 0.80 (s, 3H, 18'-CH ₃ ), 0.95 (t,3H, J-6.75 Hz. N-(CH,) ,CH ₃ ) , 1.21 (s, 3H, 19'-CH ₃ ), 2.91 and 2.97 (2s, 3H, N-CH,), 3.25 and 3.35 (2t, 2H, J=7.3Hz, N-CH,C ₃ H ₇ ), 3.67 (t, IH, J-8.18, H-C.17') and 5.72 (s, IH, H-C.4').

N-butyl, H-netbyl-11-(17*β-benzoyluιy- '-androsten-3 ^* -oa-7'α-yl) undec¬ anamide (6)

The 17β-alcohol 5 obtained previously (55 mg, 0.10 mmol) are dissolved in pyridine (1 ml) and benzoyl chloride (0.1 ml) and kept under stirring overnight at room temperature. Then, the mixture was poured in ice-water and extracted with ether (3x) . The organic layers were washed with IN HCl, water, saturated sodium bicarbonate solution and finally with water, dried on magnesium sulfate and evaporated to dryness. The residue was purified by Silica gel dry column chromatography (Kieselgel, 60F254, Merk, 0.063-0.200 mm, 10 g) . Elution with a mixture of diethyl ether and methylene chloride (1:20 v/v) gave the N-butyl, N-methyl-11-

(17β-benzoyloxy- '-androsten-3'on-7 ¹ α-yl) undecanamide (6, R ^» C,H _s C0)

(45 mg, 0.07 mmol, 70%) as a colorless oil.

TE SHEET

SCHEME 1

α 7G-H b 7α-H

TABLE 1

Ester of N, '-dialkyl-ll-(17'β-bydroxy- •-androsten-3'-on-7'α-yl) alkylamide

By analogous methods to those described above and using the same or other tetrahydropyranyloxy bromoalkane, the same or other dialkylamine and the same or other acid chloride, the following compounds, described in Table 1, are synthesized.

Efficacy of an antiandrogen synthesized in accordance with Example 1

Compound 5 ("EM 101") shown in Scheme 1 above is itself an androgen activity inhibitor as well as intermediate in the synthesis of compounds 6. EM 101 has been tested both for efficacy in acting as an antiandrogen by blocking androgen receptors without substantially activating those receptors, and for efficacy in inhibiting 17β-hydroxysteroid dehydrogena¬ se, an enzyme which catalyzes reactions involved in the synthesis of both androgens and estrogens (hereinafter referred to as "17β-HSD").

The efficacy of EM 101 as an antiandrogen is shown in Figure 1. Human mammary cancer cells ZR-75-1 contain androgen receptors. They secrete the gross cystic disease fluid protein (GCDFP-15) and this secre¬ tion is stimulated by androgens. ZR-75-1 cells were seeded at 9000 cells/plate in multiwell dishes in medium containing 2% dextran-coated charcoal-treated fetal calf serum. Three days after plating, the medium vas changed and the compounds to be tested vere added to triplicate dishes. Increasing concentrations of EM 101 vere added in medium contain¬ ing 0.1 nM dihydrotestosterone (DHT) . This concentration of DHT in the absence of antiandrogen causes about a 3-fold increase in GCDFP-15 secre¬ tion. Medium was changed every 2-3 days and collected after 12 days of incubation (48 hours after the last change). GCDFP-15 was measured by radioimmunoassay. Cells were collected and the total DNA content was measured by fluorometry. GCD5P-15 was expressed as pg GCDF?-15/μg DNA. As shown in Figure 1, increasing concentrations of EM 101 significantly

inhibited the DHT-induced GCDFP-15 secretion, thus indicating a antian¬ drogenic action of ΞM 101 in this in vitro system.

To compare the effect of ΞM 101 to known inhibitors of 170-HSD (16- methylene-Ξ ₁ and 16-methylene-E.), rat ovaries were homogenized in KH,P0 _t (20 mM), ΞDTA (1 mM) and glycerol (20%) at pH 7.5, and 1000 g pellets were discarded. A reaction vessel was prepared containing 100 μl of homo- genate, NAD* (1 mM) , NADP* (1 mM) , [ ³ H] estradiol (10 mM) , an inhibitor (either EM 101, 16-methylene E, or 16-methylene-E,) at different concen¬ trations indicated on the X-axis of Figure 2, and the volume was complet¬ ed to 1 ml vith phosphate buffer [KH,P0 ₄ (12.5 mM) , EDTA (1 mM) pH 7.5]. The reaction was allowed to proceed at 37*C for 20 min. After methylene chloride extraction (2X) , the organic phase was dried on magnesium sulfa¬ te and evaporated under a stream of nitrogen. The residue vas dissolved in methanol and separated by thin layer chromatography on aluminium-coat¬ ed silica-gel plates (benzene acetone 4:1). Spots were cut, dissolved in ethanol and counted using Formula 963 as scintillation fluid. The conver¬ sion of estradiol (E,) into estrone (E,) (a 17β-HSD-catalyzed reactions) was measured. As shown in Figure 2, increasing concentrations of EM 101 inhibited this conversion more rapidly and completely than did the two known 17β-HSD inhibitors 16-methylene-E, and 16-methylene-E,.

30

Example 2

Synthesis 17β-hydroxy-17α-(tt-iodoalkyιgrl)-4-androsten-3- ne (9) (Scheme 2)

(±)3,3-et±ylenedioay-17β-tetrahydrupyιauylu y-17αr^tfayπyl-5-androstene (8)

A mixture of ethisterone (7) (9.5 g, 30.4 mmol), ethylene glycol (3.34 g, 3 ml, 53.8 mmol) and p-toluenesulfonic acid (50 mg, 0.29 mmol) dissolved in 500 ml of dry benzene was refluxed (Dean-Stark) for 24 h under nitrogen. Then, a mixture of ether and dichloromethane (1:1, I D was added and the resulting solution washed successively with sodium carbonate (2 x 100 ml, 52 aqueous) and with water (4 x 200 ml) . The organic phase was dried, filtered and concentrated to dryness. The crude 3,3-ethylenedio^r-17α-ethynyl-5-androsten-17β-ol (9.73 g, 90% of crude dioxolane) was used without any further purification in the next step.

A mixture of crude dioxolane (9.73 g, 27.3 mmol) in dry dichlorome¬ thane (500 ml), 2,3-dihydropyran (6.9 g, 7.5 ml, 82.2 mmol), and cataly¬ tic pyridinium p-toluenesulfonate (100 g, 0.4 mmol) was stirred at room temperature for 36 h. Then, ether (500 ml) was added and the resulting solution was washed successively vith sodium carbonate (2 x 100 ml, 52 aqueous) and vith vater (4 x 200 ml). The organic phase vas dried, filtered and evaporated to give 12.74 g of crude material. The residue was purified by flash chromatography (hexane: acetone, 95:5) to give compound 7 (7.7 g, 58%); max: 3300 and 3230 (≡C-H) , 2090

(CΞC) and 1150-980 (C- ) cm" ¹ ; »H-NMR (60 MHz) 5.35 - 4.80 (2H,m,-C-CH-, -OCH0CH,-). 3.88 (4H,s,-0CH ₃ CH ₅ 0-) , 2.50 (IH,s,-C≡C-H) , 1.00 (3H.S.19- CH,), 0.88 (3H,s,18-CH,); MS m e (70eV) : 440 (M*). Further elution gave the corresponding enone (2.94 g, 24%).

(±)3,3-Et_hylenedioxy^l7β-tetrahydropyra_αylαxy-17oc- (5*-iodopentyπyl)-5- androstene (8, n-3)

To a solution of butyllithium ^" (2.84 ml of a 1.6 M solution in 'hexane, 4.5 mmol) in dry tetrahydrofuran (THF, 30 ml) was added dropwise a solution of diprotected ethisterone 7 (500 mg, 1.13 mmol) in dry THF (10 ml) at -40*C. The reaction mixture vas then alloved to warm up to -10'C and stirred for 1 h. At this temperature, a solution of 1,3-di- iodopropane (1.61 g, 627 μl, 5.4 mmol) in dry THF (5 ml) was added in one portion. The cooling bath was removed and the reaction mixture was stirred at room temperature for 15 h. Then, the solution vas diluted with 100 ml of ether and was washed with water (6 x 30 ml) , dried, filtered and concentrated to an oil. The residue was purified by flash chromatography (hexane: acetone, 95:5) followed by preparative thin-layer chromatography (TLC) (benzene: acetone, 95:5, Rf 0.68) to give compound 8 (n-3) (302 mg, 432); 1H-NMR 5.35 - 4.80 (2H,m,-C-CH-, -0CH0CH,-) , 3.88 (4H,s,-OCH,CH,0-), 3.23 (2H,t,j- 6.0 Hz,-CH,I) , 1.02 (3H,s,19-CH,) , 0.89 (3H,s,18-CH ₃ ).

32

17β-Hydroxy-17α-(5 ^, -iodopentynyl)-4-androste__r-3-one (9 , n-3)

To a solution of the oily tetrahydropyranyl ethers 8 (n-3) (38 mg, 6.25 x lO ^-1 mmol) in ethanol (5 ml) was added oxalic acid (2 ml, 2% aqueous) . The reaction mixture was heated at reflux for 2.5 h. Then, most of the ethanol was evaporated and the residue, transferred into a separatory funnel with ether (40 ml) and water (20 ml) was washed thoroughly with water. The ethereal phase was dried, filtered and concen¬ trated to an oil. The residue was purified by preparative TLC (benzene: acetone, 95:5, Rf 0.26) to give 17β-hydroxy-17α-(5'-iodopentynyl)-4- androsten-3-one (9, n-3) as colorless oil; IR (neat) . max: 3600-3150 (OH), 2230 (CΞC), 1660 (C-O) and 1610 (C ^« C) cm" ¹ ; *H-HH 5.74 (lH.s, -CH-C-), 5.29 (2H,t,J- 6.6 Hz, -CH ₃ I) , 1.20 (3H.S.19-CH,) , 0.88 (3H,s,18- CH,); MS m e (70eV): 480 (M*).

(:U3,3-Ethylenedi_oxy--17p-tetrahydcopy^ 5-anάrostene (8, n-6)

The preparation of this derivative vas done as described for alkyl iodide 8 (n-3) (vide supra) with the following quantities: acetylene 7 (570 mg, 1.29 mmol), butyllithium (2.07 ml of a 2.5 M solution in hexane, 5.17 mmol), 1,6-diiodohexane (2.1 g, 6.2 mmol) , tetrahydrofuran (50 ml) . The crude material was purified by flash chromatography (hexane: acetone, 95:5) to give compound 8 (n-6) (260 mg, 30.5%) as colorless oil; IR (neat) v .max 2220 (CΞC) and 1150-980 (C-O) cm- ¹ ; ¹ H-NMR 5.35 (lH,m,-C-CH-), 5.15 and 4.94 (IH, 2m,-0CH0CH,-) , 3.95 (4H,

a, -0CH,CH,0-) , 3.50 (lH,m,-0CH0CHH-) . 3.20 (2H,t,J= 6 ,96 Hz ,-CH.I) , 2.58 C lH .d of m, J= 13.5 Hz .-OCHOCHH-) , 1.04 and 1.03 (3H, 2s , 19-CH ₃ ) , 0.88 (3H,s , 18-CH, ) ; MS m/e (70eV) : 650 (M > .

17β-Hydroxy-17α-(8 ^, -iodooctynyl)-4-androsten-3-one (9 , n-6)

The hydrolysis of compound 8 (n-6) was done as described for tetrahydropyranyl ethers 8 (n-3) (vide supra) with the following quantities: tetrahydropyranyl ethers 8 (n-6) (24 mg, 3.69 x 10" - mmol), oxalic acid (1.5 ml, 22 aqueous), ethanol (5 ml). The crude material was purified by preparative TLC (hexane: acetone, 9:1, Rf 0.17) to give 17β-hydroxy-17α-(8 ^, -iodooctynyl)-4-androsten-3-one (9, n-6) (18 mg, 932) as colorless oil; IR (neat) v max 3600-3150 (OH), 2225 (CSC), 1660 (C-O) and 1610 (C-C) ar - ', *H-SBR: 5.74 (1H.S.-CH-C-) , 3.17 (2H,t,J- 6.96 Hz, -CH,I), 1.20 (3H,s,19-CH _a ) , 0.88 (3H,s,18-CH,) ; MS m e (70eV): 522 (M*).

(±)3,3-E ^* t±ylenediocry-17β-tetxakydιupyxauylux>^17tt-(_L2 ^, -iodododecyιιyl)- 5-androstene (8, n-10)

The preparation of this derivative was realized as described for alkyl iodide 8 (n-3) (vide supra) with the following quantities: acetylene 7 (500 mg, 1.13 mmol), butyllithium (2.84 ml of a 1.6 M solution in hexane, 4.54 mmol), 1,10-diiododecane (2.15 g, 5.45 mmol), tetrahydrofuran (45 ml) . ^* The crude material was purified by flash chromatography (hexane: acetone, 96:4) to give compound 8 (n-10)

SCHEME 2

pyr. p-toluene sut onσte

nβuϋ. !(CH ₂ ) _n l

(208 mg, 26%) as colorless oil; IR (neat) υ max 2240 (CΞC) and 1150-980 (C-O) cm- ¹ ; ⁱ H- MR: 5.36 (lH,m,-C=CH-) , 5.18 and 4.97 (lH,2m,-0CH0CH,-) , 3.95 (4H,m,-0CH.CH _: 0-), 3.50 (lH,m,-0CH0CHH-) , 3.19 (2H,t,J= 6,96 Hz,-CH,I), 2.58 (lH,m,-OCHOCHH-),1.0 and 1.03 (3H,2s,19-CH ₃ ) , 0.88 (3H,s,18-CH ₃ ); MS m/e (70eV) : 706 (M ^*» ) .

17β-Hydro_x_y-17α-(12'-iodododecynyl)-4-androstett-3-on e ("EM 150", 9, n-10)

The hydrolysis of compound 8 (n-10) was realized as described for tetrahydropyranyl ethers 8 (n-3) (vide supra) with the following quantities: tetrahydropyranyl ethers 3 (n-10) (100 mg, 0.14 mmol), oxalic acid (2 ml, 22 aqueous), ethanol (7 ml). The crude material was purified by column chromatography (toluene: acetone, 96:4) to give 17β- hydroxy-17α-(12'-iodododecynyl-4-androsten-3-one) ("EM 150", 9, n-10)

(63 mg, 772) as colorless oil; IR (neat) ^~ J max 3600-3150 (OH), 2225

(CΞC), 1660 (C-O) and 1610 (C-C) em" ^' ; ^l H-NMR 5.74 (lH,s,-CH-C-) , 3.19

(2H,t,J- 6.96 Hz, -CH,I), 1.20 (3H,s,19-CH,) , 0.88 (3H,s,18-CH ₃ ) ; MS m e

(70eV): 578 (M*).

17β^ydrαsy-17o-(10*-ioόx_iβ7πyl)-4-aιιdrosten-3-on β (9, n ^« 8)

To a solution of butyllithium (1.45 ml of a 2.5 M solution in hexane, 3.6 mmol) in dry tetrahydrofuran (THF, 20 ml) was added dropwise a solution of diprotected ethisterone 7 (400 mg, 0.91 mmol) in dry THF (7 ml) at -40'C. The reaction mixture was then allowed to stir for

1.5 h. At -35"C, a solution of 1,8-diiodooctane (1.6 g, 870 μl, 4.37 mmol) in dry THF (5 ml) was added in one portion. Ihe cooling bath was removed and the reaction mixture was stirred at room temperature for 17 h. Then, the solution was diluted with 100 ml of ether and was washed with water (6 x 30 ml) , dried, filtered and concentrated to yield a diasteromeric mixture of 3,3-ethylenedioxy-17β-tetrahydropyranyloxy-17α- (10*-iododecynyl)-5- androstene (8, n-8) as an oil which was directly hydrolyzed.

To a solution of the oily tetrahydropyranyl ethers in ethanol (20 -ml) was added aqueous oxalic acid (3 ml, 2% aqueous). The reaction mixtu¬ re was heated at reflux for 2.5 h. Then, most of the ethanol was evapora¬ ted and the residue transferred into a separatory funnel with ether (100 ml) and was washed thoroughly with water. The ethereal phase was dried, filtered and concentrated to an oil. The residue was purified by flash chromatography (toluene:acetone, 97:3) to yield the 17β-hydroxy-17α-(10'- iodoάecynyl)-4-androsten-3-one (9, n-8) (170 mg, 342).

In a similar way, compounds 9 with n equals to 9, 11 and 12 were synthesized at respective yields of 30, 25 and 352 by using respectively dϋodononane, diiodoundecane and diiodododecane as reagents.

Efficacy of compounds Synthesized in A ^~m v ^~m ' ^~ ]' ^~ - ^~ "-' ^t~ with Example 2

Compound "EM 150" synthesized above has been tested and found to be an effective inhibitor of the activity of the 17β-hydroxysteroid dehydro¬ genase, an enzyme vhich catalyzes reactions involved in the synthesis of both androgens and estrogens. In order to test this inhibition, the ef¬ fect of the compound on 17β-HSD conversion of estradiol to estrone was

measured. The reaction was followed by monitoring the formation of NADH at 340 na (the rate of conversion of the cofactor NAD to NADH varies directly with the rate of estradiol conversion to estrone) . The ability of compounds of the invention to inhibit this reaction is indicative of their ability to inhibit the estrogen-forming reverse reaction and various androgen-forming reactions which are also catalyzed by 17β-hydro- xysteroid dehydrogenase (Thomas J.L. et al., J. Biol. Chem. 258: 11500:11504, 1983). The 17β-hydroxysteroid dehydrogenase (17β-HSD) was purified to homogeneity from human placenta. A reaction vessel was prepa¬ red containing 1 μg 17β-HSD, 5 mM NAD, 20 μM 17β-estradiol. The concen¬ trations of the tested compound is indicated along the X-axis in figure 3 in 1.0 ml of a mixture of Tris-HCl (50 mM) , EDTA (2 mM) , NaN ₃ (5 mM) . The pH was 7.5. The reaction was allowed to proceed at 25"C for 15 min. Formation of NADH vas measured at 340 nm. As shown in figure 3, the compound EM 150 significantly decreases the activity of 17β-hydroxyste- roid dehydrogenase.

38

Example 3 Synthesis of N,N, dialkyl-n-(17'β-hydrααcy--4•-androsten-3'-on-17'-yl)- (u-D-alkylaπ de (11) (Schema 3)

Ethyl-7-(17'β-hydroxy- '-androstenr-3'-on-17'α-yl)-6-heptynoate (10, n-3, R-CH,CH ₃ )

A. To a suspention of sodium hydride (55 mg (602 in mineral oil) , 1.37 mmol) in dry THF (3 ml) vas added dropvise diethylmalonate (274 mg, 260 *μl, 1.71 mmol) at 0*C. The reaction mixture vas vanned up to room tempe¬ rature and vas stirred 30 min. Then, this solution vas added dropvise (using a seringe) to a solution of alkyl iodide 8 (n-3) (208 mg, 0.34 mmol) in THF (4 ml) and the resulting mixture vas stirred at room tempe¬ rature for 17 h. The reaction vas diluted vith ether (100 ml) and vashed vith vater (5 x 30 ml) , dried, filtered and concentrated to an oil. The residue vas partly purified by flash chromatography (hexane: acetone, 9:1) to give 170 mg, 782 of the desired malonate contaminated vith some diethylmalonate (as shown by --H-NMR spectroscopy) which was used as such in the next step.

B. A solution containing the malonate (170 mg, 0.265 mmol), lithium chloride (225 mg, 5.3 mmol) and water (96 mg, 96 μi, 5.3 mmol) in dimethylformamide (DMF, 7 ml) was stirred at 155*C for 20 h. Then, ethanol (5 ml) and oxalic acid (7 ml, 22 aqueous) were added and the resulting solution was heated at 90*C for 2 h. The reaction mixture was diluted with ether (100 ml) and washed thoroughly with water (7 x 30 ml).

The ethereal phase was dried, filtered and concentrated to an oil. The residue was purified by flash chromatography (hexane: acetone, 4:1) to afford ethyl-7-(17'β-hydroxy-4•-androsten-3'-on-17'α-yl)-6-hept ynoate (10, n-3, R=CH,CK ₃ ) (57 mg, 382) as colorless oil; IR (neat) υ max 3600-3200 (OH), 1725 (C=0,ester), 1660 (C=0,enone) and 1610 (C=C) cm- - -, ⁱ H-NMR 5.73 (lH,s,-CH«C-) , 4.11 (2H,q,J- 6.96 Hz, -OCH,CH ₃ ), 1.24 (3H,t, J- 6,96 Hz,-0CH,CH,). 1.20 (3H,s,19-CH ₃ ) , 0.88 (3H,s,18-CH,) ; MS m/e (70eV): 440 (M*).

Ethyl-10-(17 ^* β-bydroxy- '-androsten-3*-on-17*α-yl)-9-decynoate (10, n-6, R-CH _j CH _j )

The preparation of this ester was realized as described for ester 10 (n-3) (vide supra) with the following quantities: A. alkyl iodide 8 (n-6) (130 mg, 0.2 mmol), sodium hydride (30 mg (602 in mineral oil), 0.75 mmol), diethylmalonate (132 mg, 125 μl, 0.82 mmol), THF (7 ml), 25*C, 12 h. The crude material was used as such in part B; B. crude malonate (0,2 mmol), lithium chloride (100 mg, 2.36 mmol), water (23 mg, 23 μl, 1.27 mmol), DMF (7 ml), 155 ^*» C, 20 h; and ethanol (5 ml), oxalic acid (7ml, 22 aqueous), 90*C, 2h. The crude material was purified by preparative TLC (hexane: acetone, 4:1, Rf 0.25) to give ethyl-10-(17 ^* β- hydroxy-4 ^, -androsten-3'-on-17 ^t α--yl)-9-decynoate (10, n-6, R-CH,CH,) (23 mg, 242) as colorless oil; IR (neat) \> max 3650-3150 (OH), 2220 (CΞC), 1722 (C-O,ester), 1660 (C-C,enone) and 1610 (C-C) cm' ¹ ; ^l H-NMR 5.75 (1H,S,-CH-C-), 4.13 (2H,q,J- 7.32 Hz, -0CH,CH ₃ ). 1.26 (3H,t,J- 7.32 Hz.-OCH _j CH,), 1.21 (3H,s,19-CH ₃ ) , 0.89 (3H,s,18-CH ₃ ) ; MS m/e (70eV): 482 (M*) along with 11 mg, 10% of the corresponding malonate (Rf 0.2).

91/00732

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Methyl-14-(17'β-hydroxy-4•-androsten-3'-on-17'α yl)-13-tetradecynoate (10, n-10, R=CH,CH ₃ )

The preparation of this ester was done as described for ester 10 (n-3) (vide supra) with the following quantities: A. alkyl iodide 8 (n-10) (150 mg, 0.21 mmol), sodium hydride (34 mg (602 in mineral oil), 0,85 mmol), dimethylmalonate (127 mg, 110 μl, 1 mmol), THF (10 ml), 25*C, 18 h; B. crude malonate (0,21 mmol), lithium chloride (182 mg, 4.3 mmol), water (77 mg, 77μl, 4.3 mmol), DMF (7 ml), 155*C, 20 h; and ethanol (5 lal), oxalic acid (7 ml, 22 aqueous), 90*C, 2 h. The crude material was purified by flash chromatography (hexane: acetone, 85:15) to give methyl- 14-(17'β-hydroxy-4'-androsten-3'-on-17•α-yl)-13-tetradec ynoate (10, n-10, R-CH ₃ CH,) (47 mg, 422) as colorless oil; IR (neat) v max 3650-3150 (OH), 2225 (CΞC), 1730 (C-O,ester) , 1665 (C-0,enone) and 1610 (C-C) cm" ³ ; ⁱ H-NMR 5.74 (lH,s,-CH-C-) , 3.67 (3H,s,-OCH ₃ ) , 1.20 (3H.s, 19-CH,), 0.88 (3H,s,18-CH,); MS m e (70eV) : 524 (M*).

lH ^j utyl-l_H__eti__yl-7-(17*β-hydrox7- '-androstea-3'-ON-17'α-yl)-6-beptyπ- amide (11, n-3)

A. The ethyl ester 10 (n-3, R-CH,CH ₃ ) (30 mg, 6.8 x 10' ³ mmol) was dissolved in dry ethanol (3 al) , anhydrous potassium carbonate (20 mg, 0.144 mmol) was added and the resulting solution allowed to stir under argon at room temperature overnight (16 h) . The ethanol was evaporated, ether (20 ml) andi water (5 ml) were added, and the mixture separated into neutral and alkaji soluble fractions. The alkaline fraction was brought

to pK2 with hydrochloric acid and extracted with ether (3 x 5 mi) . The combined ethereal phases were vashed with water (3 x 10 ml) , dried, filtered and concentrated to give the crude acid which was i___mediately converted to the amide.

B. A solution of crude acid (6.8 x 10' ³ mmol) in dry dichloroεethane (5 ml) was treated with tributylamine (37.3 mg, 48 μl, 0.2 mmol) and iso- butyl chloroformate (27.4 mg, 26μl, 0.2 mmol) at 0*C for 50 min. Then, N-methylbutylamine (35.3 mg, 48 μl, 0.4 mmol) was added and the mixture -was stirred at 0"C for 50 min. The reaction mixture was diluted with ether (10 ml) and was washed successively with a solution of hydrochloric acid (2 x 5 ml, 1% aqueous) and with water (5 x 5 ml). The organic phase was dried, filtered and concentrated to an oil. The residue was purified by preparative TLC (benzene: acetone, 9:1, Rf 0.08) to give N-butyl-N- methyl-7-(17'β-hydroxy- '-androsten-3'-on-17'α-yl)-6-heptynamide (11, n-3) (15 mg, 462) as colorless oil; IR (neat) υ max 3550-3200 (OH) , 2220 (CΞC ⁾ , 1660 (C-O,enone) and 1635 (C-O.amide) cm-*; ^X H-NMR 5.74 (lH,s, -CH-C-), 3.35 and 3.25 (2H,2t,J- 7.32 Hz,-NCH _a -) , 2.96 and 2.50 (3H,2s, -NCH ₃ ). 1.20 (3H,s,19-CH ₃ ), 0.95 (3H,t,J- 6,6 Hz,-CH,CH ₃ ), 0.88 (3H,s, 18-CH,); MS m/e (70eV):

N-butyl-N-eethyl-10-(17*β-hydroxy-4•-androsten-3•-on -17*α-yl)-9-decyn- aa de (11, n-6)

The preparation of this amide was done as described for amide 11 (n-3 ⁾ (vide supra) with the following quantities: A. ester 10 (n-6) (16.5 mg, 3.4 x lO' ² mmol), potassium carbonate (10 mg, 7.2 x 10-' mmol).

91/00732

42

methanol (2 al) , room temperature, 2 h. The crude material was used as such in part B; Ξ. crude acid (3.4 x 10" ³ πmol) , tributylamine (14 eg, 18 μl, 7.5 x 10" ^*3 mmol), isobutyl chloroformate (10.5 mg, 10 μl, 7.7 x 10" ¹ mmol), dichloromethane (3 ml), O'C, 30 min.; N-methylbutylamine (14.7 mg, 20 ul, 0.168 saol) , 0*C, 50 min. The residue was purified by preparation TLC (hexane: acetone, 7:3, Rf 0.12) to give N-butyl-N-methyl- 10-(17 ^, β-hydroxy-4 ^l -androsten-3 ^, -on-17'α-yl)-9-decynamide (11, n-6) (9.3 rag, 522) as colorless oil; IR (neat) v max 3600-3150 (OH). 2220 (C≡C), 1660(C=O,enone) and 1630 (C-O,amide) cm" ¹ ; ^l H-NMR 5.73 (lH.s, -CH-C-), 3.35 and 3.25 (2H,2t,J- 7.32 Hz.-NCH ₃ -) . 2.96 and 2.90 (3H,2s,-NCH ₃ ), 1.20 (3H,s,19-CH ₃ ) . 0.95 (3H.t,J- 6,96 Hz,-CH,CH ₃ ), 0.88 (3H,s,18-CH ₃ ) ; MS m/e (70eV) : 523 (M ^φ ) and the corresponding methyl ester (6.4 mg. 402, Rf 0.22).

N-butyl-N-aethyl-1 -(17*β-hydroxy- «-androsten-3'-on-17*α-yl)-13- tetradecyna de (11, n-10)

A. To a solution of ester 10 (n-10) (24 mg, 4.58 x 10" ³ mmol) in methanol (5 ml) vas added sodium hydroxide (0.5 ml, 52 aqueous) . The reaction mixture was stirred at room temperature for 45 min. Then, ether (30 ml) was added and the resulting solution was vashed successively with hydrochloric acid (2 x 5 ml, 102 aqueous) and with water (4 x 10 ml). The ethereal phase was dried, filtered and concentrated to an oil. The residue was purified by preparative TLC (hexane: acetone, 7:3, Rf 0.24) to give 5.6 mg, 242 of acid. B. The preparation of the title amide vas realized as described for amide 11 (n-3) (vide suora) with the

following quantities: acid (5.6 mg, 1.1 x 10" ³ mmol), tributylaaine (14 mg, 18 μl, 7.5 x 10" ³ mmol), isobutyl chloroformate (10.4 mg, 10 μl, 7.7 x 1C" ⁵ mmol) , άichloromethane (3 ml), 0 ^β C, 30 min.; N-methylbutylamine

(14.7 mg, 20 μl, 0.168 mmol), 0°C, 50 min. Tne residue was purified by preparative TLC (hexane: acetone, 7:3, Rf 0.35) to give N-butyl-N-raethyl-

14-(17'β-hydroxy-4'-androsten-3'-on-17 ^• α-yl)-13-tetradecynamide (11, n-10) (5.2 mg, 822) as colorless oil; IR (neat) v max 3600-3100 (0H),

2220 (C≡C), 1660 (C=0,enone) and 1630 (C-O,amide) cm" ³ ; ³ H-ffifR 5.74

(lH.s.-CH-C-). 3.36 and 3.26 (2H,2t ^" ,J= 7.32 Hz,-NCH.-) , 2.97 and 2.91

(3H,2s,-NCH _j ), 1.20 (3H,s,18-CH ₃ ) , 0.95 (3H,t,J= 6,77 Hz,-CH,CK ₃ ), 0.88

(3H,s,19-CH ₃ ); MS m/e (70eV) : 579 (M*).

By analogous methods to those described and using the same ester 10 as starting material, the following syntheses are performed with different amines, and resulting compounds are summarized in Table 2.

44

TABLE 2

N,H' -dialkyl-tr- ⁽ 17 • β-hydrιxιy- ' -androsten-3 • -on-17 • α-yl) -(w-1) -alkylamide 11

n

*!

3

3

3

6

6

6

10

10

10

SCHEME 3

-CO

1) 2CO ₃ .C2H5OH

2) CICOϊiBu. NBu ₃

3) HN(CH3)C ₄ H

BSTITUTE SHEET