MXPA01001405A

MXPA01001405A - INHIBITION OF TYPE 3 3&agr;-HYDROXYSTEROID DEHYDROGENASE

Info

Publication number: MXPA01001405A
Application number: MXPA/A/2001/001405A
Authority: MX
Inventors: Fernand Labrie; Sylvain Gauthier; Van Luuthe; Yves Merand; Louis Provencher
Original assignee: Endorechercheinc
Priority date: 1998-08-07
Filing date: 2001-02-07
Publication date: 2002-03-26

Abstract

Novel methods of treating and/or inhibiting development of prostatic cancer, benign prostatic hyperplasia, prostatitis, acne, seborrhea, hirsutism or androgenic alopecia utilize inhibitors of type 3 3&agr;-hydroxysteroid dehydrogenase alone or in combination with other active pharmaceuticals such as inhibitors of type 5 17&bgr;-hydroxysteroid dehydrogenase. Novel inhibitors and pharmaceutical products are also disclosed.

Description

INHIBITION OF 3a-HYDROXYSTEROID-DEHYDROGENASE TYPE 3 FIELD OF THE INVENTION The present invention relates to methods of treatment of sex steroid dependent diseases, based on the use of enzyme inhibitors comprised in the sex steroid biosynthesis from natural precursors. In particular, inhibitors are described which reduce the natural production of androgens such as testosterone and dihydrotetosterone.

BACKGROUND OF THE RELATED ART Many diseases known to be androgen sensitive are known, that is, diseases whose onset or progress is assisted by androgenic activity. They include, but are not limited to, prostate cancer, benign prosthetic hyperplasia, acne, seborrhea, hirsutism, androgenic alopecia, precocious puberty, adrenal hyperplasia, and polycystic ovarian syndrome. Estrogen sensitive diseases are also known, that is, those whose onset or progress is assisted by estrogenic activity. They include, without limitation, breast cancer, endometrial cancer, endometriosis, liomyoma and precocious puberty. Androgenic and estrogenic activity can be suppressed by administering androgen receptor antagonists ("antiandrogen") or estrogen receptor antagonists ("antiestrogen"), respectively. See for example WO 97/26767 and WO 96/26201. It can also reduce androgenic and estrogenic activity by suppressing the biosynthesis or secretions of androgen or estrogen by known methods. See for example WO 90/10462, WO 91/00731, WO 91/00733 and WO 86/01105. In WO 97/11162 the 17β-hydroxysteroid dehydrogenase type 5 is described. Molecular cloning and characterization of human type 3a-hydroxysteroid dehydrogenase type 3 from the human prostate cDNA library has been described by Dufort et al. , Biochemical and Biophysical Research Communications 228, 474-479 (1996). In the United States Provisional Patent Application filed March 11, 1998, serial number 60 / 077,510 describes inhibitors of the 17β-hydroxysteroid dehydrogenase type 5 enzyme. Effective inhibitors of the enzyme are provided by the present invention. human 3-hydroxysteroid dehydrogenase 3-enzyme or effective inhibitors of both human 3a-hydroxysteroid-dehydrogenase type 3 and human 17β-hydroxysteroid dehydrogenase 5, human enzymes as is the discovery that the formation of androgen can be suppressed in this way. It is not considered that the prior art has described or suggested that the inhibition of 3a-hydroxysteroid dehydrogenase type 3 may play a beneficial role in reducing the amount of testosterone and dihydrotestosterone available in target tissues.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to more selectively and effectively inhibit the conversion of 4-androstene-3,17-dione to testosterone and 5α-androstane-3,17-dione to dihydrotestosterone using an inhibitor of 3a-hydroxysteroid dehydrogenase type 3 while inhibiting the inhibition of 17β-hydroxysteroid dehydrogenases type 2 and 4, 3a-hydroxysteroid dehydrogenase type 1 or any other androgen degradation enzyme. It is another object of the present invention to more selectively and effectively inhibit the conversion of 4-androstene-3,17-dione to testosterone and 5-androstane-3,17-dione to dihydrotestosterone using an inhibitor of both the 3a-hydroxysteroid- dehydrogenase type 3 as well as 17a-hydroxysteroid dehydrogenase type 5, while the inhibition of 17β-hydroxysteroid dehydrogenases type 2 or 4, 3a-hydroxysteroid dehydrogenase type 1 or any other androgen degradation enzyme is avoided. . It is another object to provide treatment and prevention regimens for prostate cancer, benign prostatic hyperplasia and prostatitis. It is another object to provide treatment and prevention regimens for androgen-sensitive skin diseases, particularly acne, seborrhea, hirsutism, and androgenic alopecia. In one embodiment, the invention provides a method for inhibiting the conversion of 4-androstene-3,17-dione to testosterone or of 5a-androstane-3,17-dione to dihydrotestosterone in a patient in need of this inhibition, which comprises administering to the patient a therapeutically effective amount of a human 3a-hydroxysteroid dehydrogenase type 3 inhibitor different from the 17-lactone derivative compounds. In another embodiment, the invention provides a method for inhibiting the activity of human type 3a-hydroxysteroid dehydrogenase type 3 which comprises administering to a patient in need of such treatment a therapeutically effective amount of an inhibitor of the 3a-hydroxysteroid dehydrogenase type. 3 human that has the following structure: where the dotted line is an optional pi link; wherein R3 is a portion selected from the group consisting of alkoxy of 1 to 20 carbon atoms, axiloxy of 10 carbon atoms, alkoxycarbonyloxy of 1 to 20 carbon atoms, alkyloxy alkyloxy of 1 to 20 carbon atoms, hydroxyl , (N-alkyl or -H), carbamate and a portion transformed in vi to hydroxyl. wherein R2 and R4 are independently selected from the group consisting of hydrogen, cyano, fluoro, chloro, bromo and nitro (wherein R2 and R4 are not simultaneously hydrogen). wherein R17a is selected from the group consisting of hydrogen, a carbon moiety of 2 to 14 carbon atoms substituted by a radical selected from the group consisting of hydrogen, halogen, carboxyl, amido, alkoxy of 1 to 3 carbon atoms and alkyl from 1 to 5 carbon atoms or R17a and R17 ^ together form a lactone ring of 5 to 7 carbon atoms or is a ketonic oxygen; wherein R17'3 is hydroxyl, acyloxy, alkoxy, alkenyloxy, (N-alkyl or H) amido; or R17a and R17ß together form a lactone ring of 5 to 7 carbon atoms or is a ketonic oxygen; wherein R16a and R16P are independently selected from the group consisting of hydrogen, lower alkyl and benzyl or R16a and R16ß together form a cycloalkene of 5 to 6 carbon atoms. In another embodiment, the invention provides a method for inhibiting the activity of human type 3a-hydroxysteroid dehydrogenase type 3 which comprises administering to a patient in need of this inhibition a therapeutically effective amount of an inhibitor of 3a-hydroxysteroid dehydrogenase type 3 human selected from the group consisting of: EM-1125 In another embodiment, the invention provides a method for determining the effectiveness of a putative inhibitor of the conversion of 4-androstene-3,17-donate to testosterone and 5α-androstane-3,17-dione to dihydrotestosterone, which comprises measuring activity of the 3a-hydroxysteroid dehydrogenase type 3 in the presence of the putative inhibitor and to correlate the effectiveness to a reduction in the activity related to the activity of the dehydrogenase in the absence of the putative inhibitor. In another embodiment, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier diluent and a therapeutically effective amount of an inhibitor of human type 3a-hydroxysteroid dehydrogenase type 3 having the molecular structure: wherein R is a portion selected from the group consisting of alkyloxy of 1 to 20 carbon atoms, acyloxy of 1 to 10 carbon atoms, alkoxycarbonyloxy of 1 to 20 carbon atoms, alkyloxy-alkyloxy of 1 to 20 carbon atoms, hydroxyl, (N-alkyl or -H) carbamate and a portion transformed in vi or hydroxyl; wherein R2 and R4 are independently selected from the group consisting of hydrogen, cyano, fluoro, chloro, bromo and nitro (wherein R2 and R4 are not simultaneously hydrogen); where the dotted line is an optional pi link; wherein R17a is selected from the group consisting of hydrogen, a carbon moiety of 2 to 14 carbon atoms substituted by a radical selected from the group consisting of hydrogen, halogen, carboxyl, amido, alkoxy of 1 to 3 carbon atoms and alkyl from 1 to 5 carbon atoms or R17a and R17p together form a lactone ring of 5 to 7 carbon atoms or is a ketonic oxygen; wherein R17"is selected from the group consisting of hydroxyl, acyloxy, alkoxy, alkenyloxy, (N-alkyl or H) amido, or R17a and R17ß together form a 5- to 7-membered lactone ring or is a ketonic oxygen; In another embodiment, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier diluent of a therapeutically acceptable amount of a human type 3a-hydroxysteroid dehydrogenase type 3 inhibitor having the molecular structure: wherein R, 100 is selected from the group consisting of hydrogen, carboxyl, amido, alkyl of 1 to 5 carbon atoms, halo, nitro, hydroxy and alkoxy of 1 to 3 carbon atoms. In another embodiment, the invention provides an inhibitor of human type 3a-hydroxysteroid dehydrogenase type 3 having the molecular structure: wherein R3 is a portion selected from the group consisting of alkyloxy of 1 to 20 carbon atoms, acyloxy of 1 to 10 carbon atoms, alkoxycarbonyloxy of 1 to 20 carbon atoms, alkyloxy-alkyloxy of 1 to 20 carbon atoms, hydroxyl; (N-alkyl or -H) carbamate and a portion transformed in vivo or hydroxyl; wherein R2 and R4 are independently selected from the group consisting of hydrogen, cyano, fluoro, chloro, bromo and nitro (wherein R2 and R4 are not simultaneously hydrogen); - where the dotted line is an optional pi link; wherein R17 is selected from the group consisting of hydrogen, a carbon moiety of 2 to 14 carbon atoms substituted by a radical selected from the group consisting of hydrogen, halogen, carboxyl, amido, alkoxy of 1 to 3 carbon atoms and alkyl from 1 to 5 carbon atoms or R17a and R17b together form a lactone ring of 5 to 7 carbon atoms or is a ketonic oxygen; wherein R17β is selected from the group consisting of hydroxyl, acyloxy, alkyloxy, alkenyloxy, (N-alkyl or H) amido; or R17a and R17p together form a lactone ring of 5 to 7 carbon atoms or is a ketonic oxygen; wherein R16a and R16β are independently selected from the group consisting of hydrogen, lower alkyl and benzyl or R16a and R16ß together form a cycloalkene of 5 to 6 carbon atoms. In another embodiment, the invention provides a human type 3a-hydroxysteroid dehydrogenase 3 inhibitor having the molecular structure: Wherein R 100 is selected from the group consisting of hydrogen, carboxyl, amido, alkyl of 1 to 5 carbon atoms, halo, nitro, hydroxy and alkoxy of 1 to 3 carbon atoms. In another embodiment, the invention provides a method of treating, or reducing the risk of developing prostate cancer, which comprises administering to a patient in need of this treatment or reduction a therapeutically effective amount of a 3a-hydroxysteroid dehydrogenase inhibitor. human type 3 different from the compounds derived from 17-lactone. In another embodiment, the invention provides a method for treating, or reducing the risk of developing, prosthetic or benign hyperplasia comprising administering to a patient in need of this treatment or risk reduction, a therapeutically effective amount of an inhibitor of the Human type 3 -hydroxysteroid dehydrogenase different from the 17-lactone derivative compounds. In another embodiment, the invention provides a method of treating, or reducing the risk of developing, prostatitis which comprises administering to a patient in need of such treatment or reduction, a therapeutically effective amount of a 3-hydroxysteroid dehydrogenase inhibitor 3 human. In another embodiment, the invention provides a method for treating or reducing the risk of reducing acne, seborrhea, hirsutism or androgenic alopecia comprising administering to the patient, in need of this treatment or reduction as a therapeutically effective amount of an inhibitor of the activity of human type 17β-hydroxysteroid dehydrogenase type 5 of human type 3a-hydroxysteroid dehydrogenase type 3 different from the administration of a compound derived from 17-lactone. The inhibitors of the invention are used to prevent and / or treat certain diseases, analyzed herein, whose onset or progress is stimulated by androgenic activity. One of the most surprising results of our laboratory work is the discovery that the 3a-HSD type 3 that is known as catalytic inability for reactions that affect the position 3 of steroids has now been shown by the applicants that catalyze the reactions that affect the positions 17. This discovery that 3a-HSD type 3 participates in the formation of testosterone and DHT from androstenedione and androstanedione allows the improved suppression of the biosynthesis of these two important androgens by suppressing this new biosynthetic pathway that applicants have discovered. It has been found that 3a-hydroxysteroid dehydrogenase type 3 exhibits activity similar to that of 17β-hydroxysteroid dehydrogenase (which catalyzes the conversion of 4-androstene-dione-3)., 17-dione to testosterone and 5a-androstane-3, 17-dione to dihydrotestosterone), inhibitors that suppress the activity of 17β-hydroxysteroid dehydrogenase of 3a-hydroxysteroid dehydrogenase type 3, with or without combination with inhibitors of 17β -hydroxysteroid dehydrogenase type 5 and / or 5-reductase inhibitors decreases the production of androgens catalyzed by these enzymes. Because androgens formed by reactions catalyzed by these enzymes are precursors to estrogens, the invention has applicability to diseases whose onset or progression is increased by estrogenic activity. With respect to all the doses recommended herein, the attending physician must monitor the patient's individual response, and adjust the dose accordingly. A patient in need of treatment or reduction of risk of onset of a given disease is one who has been diagnosed with the disease or who is susceptible to acquiring the disease. Except where otherwise indicated, the preferred dose of the active compounds of the invention is identical for both therapeutic and prophylactic purposes. The dose for each active component analyzed herein is the same regardless of the particular disease being treated (or prevented). As the methods of medical treatment of the methods of risk reduction of the onset of the disease herein are used, a "3a-hydroxysteroid dehydrogenase type 3 inhibitor" means a compound whose IC50 of inhibition for the enzyme in question ( computed in the same manner as described in conjunction with Table 1 herein) is not greater than 200 n. It is preferred that the IC50 of this inhibitor is not greater than 50 nM. More preferably less than 10 nM. It is also preferred that the undesirable inhibition of 3a-HSD type 1 and 17β-HSD type 2 be less than 90% at 3.10 ~ 7M, preferably less than 80% and more preferably less than 70%. In some embodiments, it is preferred that androgenicity be less than 100% stimulation of Shionogi cells at a concentration of 10 ~ 7M, preferably less than 50%, more preferably less than 20%. Where two or more of the different active agents are analyzed as part of a combination therapy herein (eg, an enzyme inhibitor and an antiandrogen), a plurality of different compounds are administered in place of an inhibitor compound having multiple activities. Except where otherwise noted or as is evident from the context, the doses herein refer to the weight of the active compounds not affected by excipients, diluents, carriers or other pharmaceutical ingredients, although these additional diluents are included in a desirable, as shown in the examples of the present invention. Any dosage form (capsule, tablet, injection or the like) commonly used in the pharmaceutical industry is suitable for use herein and the terms "excipient", "diluent" or "carrier" include these non-active ingredients as typically included , along with active ingredients in dosage forms in the industry. For example, typical capsules, pills, enteric coatings, solid or liquid diluents or excipients, flavorings, preservatives or the like may be included. As noted herein, the hydrocarbon portion term includes, without limitation, straight or branched alkyl, straight or branched alkenyl, straight or branched alkynyl, phenyl, phenylalkyl, phenylalkenyl, phenylalkynyl.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic biosynthesis pathway of active androgens in the human prostate. Figure 2 shows the activity of 17β-HSD and 3a-HSD in intact 293 cells (ATCC CRL 1573) transfected stably with 3a-HSD, in culture. Stably transfected cells with 3a-HSD type 3 were plated in 24-well plates and a density of 10 5 cells / well. 0.1 μM of 4-dione labeled with [C14] and DHT marked with [C14] were added to the recently changed culture medium to assess the activity of 17β-HSD of the enzyme 3a-HSD type 3 [transformation of 4-dione to testosterone] (D)] and the 3a-HSD activity of the 3a-HSD enzyme [DHT transformation to 3a-diol (O)] of the transfected enzyme, respectively. No transfected cells were used as control. After incubation for the indicated periods of time, the media was collected and extracted, and evaluated as described in "Enzymatic assay for types 1, 2, 3 and 5 of 17β-HSD and types 1 and 3 of 3a- HSD ". Figures 3a to 3c show paraffin sections of normal human skin immunostained with 3a-HSD type 3 antibody. The presence of 3a-hydroxysteroid dehydrogenase type 3 can be seen in: a) epithelium and fibroblast b) hair follicle c) glands sweaty DETAILED DESCRIPTION OF THE PREFERRED MODALITIES Prostate cancer is a disease of the prostatic epithelium while benign prostatic hyperplasia (BPH) comprises mainly the stromal compartment of the prostate. Prostatitis, although it is more common in the prostatic epithelium, can be found in both areas of the prostate. The recent results of the applicants show that 17β-hydroxysteroid dehydrogenase -type 5 (17β-HSD type 5) and 3a-hydroxysteroid-dehydrogenase type 3 (3a-HSD type 3) are present in the prostatic epithelium, mainly the cells basal, thus transforming androstenedione into testosterone by the routes shown in Figure 1. This testosterone then diffuses to the luminal epithelial cells, which are androgen dependent, and where prostate cancer grows. With regard to the stromal compartment, 3a-HSD type 3 and 17ß-HSD type 5 are found mainly in fibroblasts that are distributed among muscle cells. It is believed that the growth factors secreted by the fibroblasts stimulate the surrounding cells, thereby leading to BPH. The presence of estrogen receptors in these fibroblasts in the stroma probably provides the basis for the role of estrogens as well as androgens in BPH. While prostate cancer, however, is essentially not just an androgen-sensitive disease. In the prior art, 3a-HSD type 3 was known to convert DHT to androstane-3a, 17β-diol, an inactive metabolite. Applicants have recently discovered the surprising role of 3a-HSD in catalyzing the formation of testosterone and DHT from androstenedione and androstanedione, respectively (see Figure 1). The advantage of inhibiting 3a-HSD type 3 to suppress the formation of testosterone and DHT is believed to be worth more substantially than any reduction in androgen catabolism that could result when 3a-HSD type 3 is inhibited. The presence of numerous enzymes Different that are essential for the formation of androgens in the prosthetic tissues suggests the probability that the combination therapies analyzed in the present (for example, the use of other inhibitors of enzymatic activity, antiandrogens and / or castration) will lead to superior results in relation to the use of an active agent alone. It is believed that this is especially true of those combinations that affect the disease by two or more separate mechanisms (eg inhibit two or more different routes of synthesis, inhibit androgen formation in combination with blocking access to androgen receptors, etc.). ).

Figure 1 applies to each of the cases of prostate cancer, benign prostatic hyperplasia and prostatitis, although the cell types are different between prostate cancer and BPH. In prostate cancer, 17ß-HSD type 5 and 3a-HSD type 3 are present mainly in a cell type (basal cells), while the 5 -reductases are present in the luminal cells, which are located just above the basal cells, thus allowing the diffusion of testosterone from the basal cells to the luminal cells and then the conversion into the more potent androgen, DHT. For fibroblasts located in the stromal compartment, the transformation of androstenedione to testosterone and then to DHT takes place in the same cells. 3a-HSD type 1 is not present in a significant amount in the prostate, but is mainly a liver enzyme. The inhibitors used in the invention (for example, inhibitors of 3-HSD type 3, inhibitors of 17β-HSD type 5, inhibitors of 5α-reductase, etc.), preferably have little or no inhibitory effect on the 3a-HSD. type 1 that acts in a beneficial way to inactivate androgens. In this way, applicants prefer to avoid the inhibition of 3a-HSD type 1, when the invention is practiced, so as not to delay the inactivation of androgens by the liver tissue. It has been found that human type 3a-hydroxysteroid dehydrogenase type 3 that transforms 5a-androstane-3, 17-dione and dihydrotestosterone to androsterone and androstane-3a-17β-diol, respectively, also transforms 4-androstene-3,17-dione to testosterone and 5a-androstane-3, 17-dione to dihydrotestosterone. In prostate tissue, the expression of human type 3a-hydroxysteroid dehydrogenase type 3 is much greater than the expression of human type 17β-hydroxysteroid dehydrogenase type 5, thus implying that a significant proportion of androgens is formed in the prostate, for this reason route (Figure 1). In one of the preferred embodiments, the inhibition of androgen formation, as illustrated in Figure 1, is accomplished by a recent blockade of hydrotestosterone (DHT) production with an effective inhibition of: the activities of both 5a-reductase as of 17ß-hydroxysteroid dehydrogenase type 5. There are two types of 5a-reductase. Both types are expressed in the prostate, however 5a-reductase type 2 is expressed at a higher level. A product of Merck Prosear (finasteride, MK-906), mainly inhibits type 5-reductase 2. Gl 198745 compound. (17ß- {N-2, 5-bis (trifluoromethyl) phenyl}. Carbamoyl-4-aza-5a-androst-l-en-3-one) produced by Glaxo Wellcome, and EM-503 (17- (N, benzoyl, N-phenyl) amino-4-methyl-4-aza-androstane-3-one), an Endorecherche compound, efficiently inhibits both human type 1 and type 2 5 -reductase, thus offering the more efficient possibility of blocking the formation of DHT. However, inhibition of 5a-reductase activity increases testosterone (T) levels. Although weaker than DHT, T also possesses an androgenic effect that will keep the growth of the prostate to a variable degree. In order to achieve a more efficient blockade of androgen formation, it is useful to also inhibit the activity of 17β-hydroxysteroid dehydrogenase which converts 4-dione to T or A-dione to DHT. This activity is catalyzed in the prostate by 17β-HSD type 5, and surprisingly by 3a-HSD type 3. A highly efficient inhibitor for 17β-HSD type 5 has been developed whose molecular structure and synthesis are described below: Synthesis of EM-1401, EM-1404 E SQUEMA A 3-t.rifluoromethanesulfonyloxy-1, 3,5 (10) -estratrien-17 (R) -spiro-2 '- (5', 5'-dimethyl-6'-oxo) etrahydropyran (b). Under an argon atmosphere, a solution of compound a (500 mg, 1.35 mmol) 2,6-lutidine (0.355 ml, 3.05 mmol) and 4-dimethylaminopyridine (33 mg, 0.27 mmol) in dry dichloromethane (25 ml) was cooled to 0 ° C, treated with trifluoromethanesulfonic anhydride (0.308 ml, 1.83 mmol) and stirred for 45 minutes. The reaction mixture was quenched with water and extracted with dichloromethane. The organic phase was washed with 2% hydrochloric acid, saturated sodium bicarbonate and water, dried over magnesium sulfate, filtered and evaporated. The crude oil was purified by flash chromatography (hexanes-ethyl acetate 49-1 to hexanes-ethyl acetate 4-1) to provide trifluoromethane sulfonate b (MS-1399) (540 mg, 80%): IR (CHC13) 2957 , 2872, 1711, 1490, 1426, 1248, 1214, 1141, 926, 846, 621 cm "1; NMR * H (300 MHz, CDCl 3) d 1.03 (s, 3H), 1.28 (s, 3H), 1.29 (s, 3H), 1.35-2.40 (m, 17H), 2.88 (m, 2H), 6.98 (s) , 1H), 7.02 (d, J = 8Hz, 1H), 7.33 (d, J = 8.7Hz, 1H), 13C NMR (75 MHz, CDCI3) d 14.32, 23.22, 25.48, 25.80, 26.89, 27.57, 27.68, 29.37, 31.49, 31.80, 34.69, 37.72, 38.46, 43.66, 47.10, 48.59, 93.43, 116.54, 118.08, 120.80, 121.07, 127.05, 139.31, 140.43, 147.46, 177.70. 3-carboxy-l, 3,5 (10) -estratrien-17 (R) -ßspiro-2 '- (5', 5 '-dimethyl-6' -oxo) tetrahydropyran (EM 1401), Method A: A mixture of compound b (560 mg, 1.12 mmol), potassium acetate (440 mg, 4.48 mmol), palladium acetate (12.6 mg, 0.056 mmol), and 1,1'-) is (diphenylphosphino) ferrocene (125 mg, s; pull ff? ÓxViI Hdno / dA ae Hdil mmaeti? L 1 po l (2 CO IKO p Ó X ld? Carbon for 20 minutes was stirred under a balloon with carbon monoxide at 80 ° C for a period of The reaction mixture was diluted with hydrochloric acid to 0.5 N and extracted with dichloromethane, the organic phase was washed with water, dried over magnesium sulfate, filtered and evaporated. it was purified by flash chromatography dichloromethane-methanol 19-1 to dichloromethane-methanol 4-1) to provide the carboxylic acid EM-1401 (300 mg, 68%): IR (KBr) 2937, 2872, 1718, 1676, 1388, 1314 , 1230, 1180, 1160 cm "1; NMR? H (300 MHz, CDC13 + CD3OD) d 0.75 (s, 3H), 1.01 (s, 6H), 1.10-2.17 (m, 17H), 2.65 (m, 2H ), 7.09 (d, J = 8.1Hz, 1H), 7.48 (s, 1H), 7.51 (d, J = 8.5Hz, 1H) 7.51 ( d, J = 8.5Hz, 1H); 13C NMR (75 MHz, CDC13 + CD3OD) d 13.71, 22.75, 24.98, 25.27, 26.65, 26.87, 28.76, 30.84, 31.46, 34.21, 37.33, 38.22, 43.84, 46.74, 93.92 , 124.84, 126.52, 127.32, 129.91, 136.31, 144.94, 168.70, 178.97. 3-alkoxycarbonyl-l, 3,5 (10) -estrathien-17 (R) -spiro-2 '- (5', 5'-dimethyl-6'-oxo) tetrahydropyran (c). A mixture of compound b, triethylamine (3.25 equivalent), palladium acetate (0.07 equivalent), 1,3-bis (diphenylphosphino) propane (0.06 equivalent), and alcohol (1.5 equivalents in large excess) in DMF (10% P / V) was purged with carbon monoxide for 20 minutes and stirred under a carbon monoxide balloon at 90 ° C for a period of 16 hours. The reaction mixture was cooled to room temperature, diluted with water and extracted with dichloromethane. The organic phase was washed with brine, dried over magnesium sulfate, filtered and evaporated. The reaction mixture was purified by 3 flash chromatographies (2 times with benzene-acetone 4-1 and hexanes-ethyl acetate 7-3) to provide compound c (e.g., EM-1398, R = benzyl, 70%) : IR (CHC13) 2938, 1716, 1293, 1262, 1177, 1152, 1130, 1109, 732 cm "1; NMR tH (300 MHz, CDC13) d 1.02 (s, 3H), 1.28 (s, 3H), 1.29 (s, 3H), 1.34-1.41 (m, 17H), 2.91 (m, 2H), 1.34-1.41 (m, 17H), 2.91 (m, 2H), 5.35 (s, 2H), 7.33-7.45 (m , 6H), 7.79 (s, 1H), 7.83 (d, J = 8.1 Hz, 1H); 13C NMR (75 MHz, CDC13) d 14.39, 23.28, 25.55, 25.74, 27.14, 27.64, 27.75, 29.25, 31.56, 31.93, 34.75, 37.77, 38.56, 44.34, 47.16, 48.82, 66.42, 93.50, 125.34, 126.90, 127.45, 128.05, 128.10, 128.52, 130.23, 136.22, 136.81, 145.49, 166.55, 177.75. 3-carboxy-l, 3,5 (10) -estratrien-17 (R) -spiro-2 '- (5', 5'-dimethyl-6 '-oxo) tetrahydropyran (EM-1401).

Method B: A mixture of compound c (350 mg, 0.72 mmol) and 10% palladium on activated carbon (50 mg) in ethyl acetate (40 ml) was stirred under a hydrogen balloon for a period of 3 hours. The reaction mixture was filtered through celite and evaporated. The crude mixture was purified by flash chromatography (dichloromethane-THF 19-1 to dichloromethane-THF 3-1) to provide the carboxylic acid EM-1401 (240 mg, 84%). A sample was recrystallized from methanol-THF (the characterization was previously described). 3-carboxamido-1,3,5 (10) -estratrien-17 (R) -spiro-2 '- (5', 5'-dimethyl-6'-oxo) tetrahydropyran (d). Under an argon atmosphere, a solution of EM-1401 and pyridine (15 equivalents) in dry dichloromethane (1.6% W / V) was cooled to 0 ° C, treated with oxalyl chloride (6 equivalents) and stirred for 0.5 hours. . The reaction mixture was allowed to reach room temperature and was stirred for a period of 4 hours. The reaction mixture was evaporated, dissolved in dry THF (1.6% W / V), cooled to 0 ° C, treated with 10 equivalents of amine and stirred for 15 minutes. The reaction mixture was quenched with water, extracted with dichloromethane, dried over magnesium sulfate, filtered and evaporated. The crude mixture was purified by flash chromatography (hexanes-acetone 19-1 to hexanes-acetone 3-2) to provide compound d (e.g., EM-1404, RUR2 = H, 65%): IR (CHC13) 3433, 3350, 2941, 2873, 1702, 1664, 1611, 1388, 1310, 1159 cm "1; NMR aH (300 MHz, CDC13 + CD3OD) d 0.73 (s, 3H), 0.99 (s, 6H), 1.10-2.16 (m, 17H), 2.64 (m, 2H), 7.08 (d, J = 8.0 Hz, 1H), 7.30 (s, 1H), 7.32 (d, J "9 Hz, 1H); 13 C NMR (75 MHz, CDC13 + CD3OD) d 13.69, 22.72, 24.96, 25.27, 26.64, 26.84, 28.78, 29.09, 30.81, 31.44, 34.19, 37.31, 38.27, 43.72, 46.74, 93.92, 124.20, 124.93, 127.70, 130.00 , 136.45, 143.88, 170.63, 178.96. The 17ß-HSD type 5 and the 3a-HSD type 3 share 85.5% amino acid identity. The high homology of the primary structure between the 17ß-HSD type 5 and the 3a-HSD type 3 could explain a lower activity of 17ß-HSD found in the activity of the 3a-HSD type 3. However, since the 3a- HSD type 3 is expressed at a much higher level than 17ß-HSD type 5, the unexpected activity of 17β-HSD contributed by 3a-HSD type 3 plays a significant role in the prostate. The inhibition of 3a-HSD type 3 activity in this way is necessary to have an efficient blockade of androgen formation (Figure 1). The inhibitors of 3a-hydroxysteroid dehydrogenase type 3 can be used, according to the invention, alone or as part of a combination therapy with other strategies (listed below) that have beneficial effects on androgen-sensitive diseases through different mechanisms, thus providing synergistic combinations. These combination therapies further include the type 3 inhibitors of the 3a-hydroxysteroid dehydrogenase (and in some embodiments in combination with an inhibitor of the 17β-hydroxy dehydrogenase type 5), one or more of the following strategies.

Strategy 1: Suppression of ovarian or testicular hormone secretion by chemical or surgical castration. This approach is useful for the treatment of diseases that respond adversely to estrogen or androgen, respectively. When surgical or chemical castration is used, chemical castration is preferred using, either an LHRH agonist, an LHRH antagonist and / or an inhibitor of the 17β-hydroxysteroid dehydrogenase type 3 (which as discussed in the present catalytic some testicular androgen formation). Suitable LHRH agonists are reported in U.S. Patent Number 4,659,695, but any LHRH agonist that shows the ability to induce chemical castration can be used since they act through the same mechanisms as originally described (Labrie et al. ., J. Androl, 1: 209-228, 1980). The doses are known in the art. Some suitable LHRH antagonists are reported in U.S. Patent Number 4,666,885, but any LHRH antagonist is acceptable, if used according to the manufacturer's recommendation.

Strategy 2: Use of androgen receptor antagonists or estrogen ("antiandrogen" or "antiestrogen") to prevent the activation of androgen receptors or estrogen by androgens or estrogens, respectively. Strategy 2 is useful against diseases that respond adversely to androgenic or estrogenic activity, respectively. Antiandrogens, and doses for them, are known in the art (for example, Flutamide (N- [4-nitro-3- (trifluoromethyl) phenyl)] -2-methylpropanamide) at a dose of 250 mg, 2 or 3 times a day, Nilutamide at a dose of 150 mg / day, Casodex at a dose of 50 to 750 mg / day. When antiestrogens are used according to the invention, either alone or as part of one of the combination therapies described herein, the attending physician must use, at least initially, the doses recommended by the manufacturer. However, the attending physician must monitor the patient's individual response and metabolism, and adjust the patient's dose accordingly. In fact, it will be true that all the strategies discussed herein and all the active ingredients used in any of the combination therapies of the invention, a preferred antiestrogen is EM-800 reported in PCT / CA 96/00097 (WO 96/26201 ). The molecular structure of EM-800 is: Another preferred antiestrogen of the invention is EM-01538: A selective estrogen receptor modulator (SERM) is a compound that either directly or through its functions as an active metabolite as an estrogen receptor antagonist ("antiestrogen") in breast tissue, provides an estrogen-like effect in body fat, bone tissue and serum cholesterol levels. Other preferred SERMs of the invention include Tamoxifen ((Z) -2- [4- (1, 2-diphenyl-1-butenyl)] - N, -dimethylethanamine) (available from Zeneca, UK), Toremifene (available from Orion- Farmos Pharmaceuticla, Finland, or Schering-Plow), Droloxifene and CP 336,156 (salt of cis-lR- [4'-pi rrolidino-ethoxyphenyl] -2S-phenyl-6-hydroxy-1,2,3,4-tetrahydronaphthalene D - (-) -tartrate) (Pfizer Inc., USA), Raloxifene (Eli Lilly and Co., USA), LY 335563 and LY 353381 (Eli Lilly and Co., USA), Yodoxifene (SmithKine Beecham, USA), Levormeloxifen (3,4-trans-2, 2-dimethyl-3-phenyl-4- [4- (2- (2- (pyrrolidin-1-yl) ethoxy) phenyl] -7-methoxy-chroman) (Novo Nordisk, A / S, Denmark) which is described in Shalmi et al., WO 97/25034, WO 97/25035, WO 97/25037, WO 97/25038, and Korsgaard et al., WO 97/25036), GW5638 (described by Willson at ., Endocrinology, 138 (9), 3901 3911, 1997) and indole derivatives (described by Miller et al., EP 0802183A1) and TSE 424 developed by Wyeth Ayers (EUA) and described in JP10036347 (A merican home products corporation) and non-steroidal estrogen derivatives described in WO 97/32837. There are two types of 5a-reductase. Both types are expressed in the prostate, the 5 -reductase type 2, however, is expressed at a higher level. A product of Merck, Prosear (finasteride, MK-906), mainly inhibits type 5-reductase 2. The compound Gl 198745 (17β- [N-2,5-bis (trifluoromethyl) phenyl] carbamoyl-4-aza-5a -androst-l-en-3-one) produced by Glaxo Wellcome, and EM-503 (17β- (N, benzoyl, N-phenyl) amino-4-methyl-4-aza-androstane-3-one), a composed of Endorecherche, it efficiently inhibits human type 5 and 5-reductase types, thus offering an efficient possibility of blocking the formation of DHT.

Strategy 3: Suppression of androgen conversion, testosterone, to the most potent androgen, dihydrotestosterone (DHT) by inhibiting the activity of testosterone-5 -reductase (for example when administering Prosear, available from Merck Sharp and Dohme Canada, at the recommended dose) . Any other potent 5a-reductase inhibitor can be used. The dose used can be 2 to 20 mg orally daily. The dose should be as recommended by the manufacturer. Strategy 3 is useful against diseases that respond adversely to androgenic activity.

Strategy 4: Use of an aromatase inhibitor to reduce the production of estrogen. Strategy 4 is useful against diseases that respond adversely to estrogenic activity or estrogen receptor-mediated exacerbation of the type of androgen-sensitive diseases that are also estrogen-responsive diseases (eg, benign prostatic hyperplasia). Aromatase inhibitors (and antiestrogens) can also be used to reduce the undesirable estrogenic effects that result from the increased estrogenic levels that may occur during some treatments of androgen-dependent diseases. When aromatase inhibitors are used according to the invention, either alone or as part of one of the combination therapies described herein, the attending physician must initially use the dose recommended by the manufacturer. When administered orally, the dose that is usually effective to provide the desired levels in serum is between 1.0 mg and 20 mg of active ingredient per day per 50 kg of body weight. For example, Arimidex (Zeneca) is taken at the oral dose of 1 mg daily. However, the attending physician must monitor the patient's individual response and metabolism, and therefore adjust the specific dose of the patient. Some aromatase inhibitors include, for example, molecular structures set forth in U.S. Patent Number 5,227,375. Aromatase inhibition can also be achieved, for example, by administering Arimidex (2, 2 '- [5- (1H-1, 2,4-triazol-1-ylmethyl) -1,3-phenylene bis (2-methylpropiononitrile). )) available from Zeneca, UK, at a dose of 1 mg / day. Another aromatase inhibitor may be used according to the manufacturer's recommendations. In general, for androgen-sensitive diseases and estrogen-sensitive diseases, simultaneous treatment with inhibitors of sex steroid biosynthesis inhibitors (enzyme inhibitors that catalyze one or more steps of the estrogen or androgen biosynthesis or the biosynthesis of precursors of estrogen or androgen), and with estrogen receptor antagonists and / or androgen receptor antagonists, is believed to have an additive rather than a redundant effect because they act in a beneficial manner by a different mechanism. Likewise, the activity of two different enzyme inhibitors (enzymes that catalyze one or more different steps of the sexual steroid biosynthesis) is believed to provide an additive effect, especially where the inhibitors affect more than one synthetic route. This approach is believed to achieve a more complete effect. The inhibitors of 3a-hydroxysteroid dehydrogenase type 3 and the inhibitor of the 17β-hydroxysteroid dehydrogenase type 5 of the invention can be used in any combination with any of the strategies 1-4 above, whose effect (increase or decrease the androgenic activity or estrogenic) is consistent with a desirable effect of the disease in question. With this in mind, a list of representative diseases that can be treated, or the risk of which can be reduced, is subsequently set forth in accordance with the present invention. Behind each disease, there are several preferred, indicated therapies or combination therapies for the treatment or reduction of risk, of the particular disease. However, these combinations can be complemented by using one or more of the four strategies listed above, limited only in case a particular disease responds favorably in an adverse manner to estrogenic activity and / or androgenic activity.

A) Prostate cancer (responds adversely to androgenic activity) 1. Inhibitor of 3a-hydroxysteroid-dehydrogenase type 3. 2. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5. 3. 3a-hydroxysteroid dehydrogenase type 3 inhibitor + 17β-hydroxysteroid dehydrogenase inhibitor type 5 + LHRH agonist (or antagonist). 4. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5 + inhibitor of 17β-hydroxysteroid dehydrogenase type 3. 5. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid- dehydrogenase type 5 + inhibitor of 17β-hydroxysteroid dehydrogenase type 3 + LHRH agonist (or antagonist). 6. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5 + LHRH agonist (or antagonist) + antiandrogen. 7. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5 + inhibitor of 17β-hydroxysteroid dehydrogenase type 3 + antiandrogen. 8. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5 + inhibitor of 17β-hydroxysteroid dehydrogenase type 3 + agonist (or antagonist) of LHRH + antiandrogen. 9. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17ß-hydroxysteroid dehydrogenase type 5 + antiandrogen + inhibitor of 5a-reductase + agonist (or antagonist) of LHRH. 10. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17ß-hydroxysteroid dehydrogenase type 5 + LHRH agonist + 5a-reductase inhibitor. 11. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5, + inhibitor of 17β-hydroxysteroid dehydrogenase type 3 + 5a-reductase inhibitor. 12. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5 + inhibitor of 17β-hydroxysteroid dehydrogenase type 3 + antiandrogen + 5a-reductase inhibitor. 13. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5, + inhibitor of 17β-hydroxysteroid dehydrogenase type 3 + agonist (or antagonist) of LHRH + antiandrogen + 5a-reductase inhibitor.

B) Benign prosthetic hyperplasia (responds adversely to both androgenic activity and estrogenic activity) 1. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3. 2. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + antiestrogen or aromatase inhibitor . 3. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + antiandrogen. 4. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + antiandrogen + inhibitor of 5a-reductase + antiestrogen or aromatase inhibitor. 5. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + 5a-reductase inhibitor. 6. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + antiandrogen + 5a-reductase inhibitor. 7. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 5a-reductase + antiestrogen or aromatase inhibitor. 8. Inhibitor of 3-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5. 9. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5 + antiestrogen or aromatase inhibitor. 10. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17ß-hydroxysteroid dehydrogenase type 5 antiandrogen. 11. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17ß-hydroxysteroid dehydrogenase type 5 + antiandrogen + inhibitor of 5a-reductase + antiestrogen or aromatase inhibitor. 12. 3-Hydroxysteroid dehydrogenase type 3 inhibitor + 17β-hydroxysteroid dehydrogenase inhibitor type 5 + 5a-reductase inhibitor. 13. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17ß-hydroxysteroid dehydrogenase type 5 + antiandrogen + 5-reductase inhibitor. 14. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17ß-hydroxysteroid dehydrogenase type 5 + 5a-reductase inhibitor + antiestrogen or aromatase inhibitor.

C) Protastitis (responds adversely to androgenic activity) 1. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + antiandrogen. 2. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + 5a-reductase inhibitor. 3. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + antiandrogen + 5a-reductase inhibitor. 4. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5. 5. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type antiandrogen. 6. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5 + 5a-reductase inhibitor. 7. 3a-hydroxysteroid dehydrogenase inhibitor type 3 + inhibitor of 17ß-hydroxysteroid dehydrogenase type 5 + antiandrogen + 5a-reductase inhibitor.

D) Acne, seborrhea, hirsutism and androgenic alopecia (it responds adversely to androgenic activity) 1. Inhibitor of 3a-hydroxysteroid-dehydrogenase type 3. 2. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17ß-hydroxysteroid dehydrogenase type 5. 3. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + antiandrogen. 4. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17ß-hydroxysteroid dehydrogenase type 5 + antiandrogen. 5. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + 5a-reductase inhibitor. 6. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase type 5 + 5a-reductase inhibitor. 7. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + antiandrogen + inhibitor of 5a-reductase. 8. Inhibitor of 3a-hydroxysteroid dehydrogenase type 3 + inhibitor of 17β-hydroxysteroid dehydrogenase + antiandrogen + 5-reductase inhibitor.

When the 3a-hydroxysteroid type 3 inhibitors according to the invention are used, either alone or as part of one of the combination therapies described herein, the attending physician will desirably direct the patient's serum concentration of the type inhibitor. 3 between 0.5 ng / ml and 100 ng / ml, preferably between 1 ng / ml and 20 ng / ml, more preferably between 1 ng / ml and 10 ng / ml. The serum concentration can be measured by LC / MS. When administered orally, the dose that is usually effective to provide the desired levels in serum is between 1.0 mg and 1,000 mg of the active ingredient per day per 50 kg of body weight, preferably between 10 mg and 500 mg and of most preferably between 10 mg and 100 mg. However, the dose should vary with the bioavailability of the inhibitor chosen and with the individual response of the patient. For example, when EM-01645, or EM-01667-C, is chosen, an oral dose is preferably between 5 mg and 500 mg per day per 50 kg of body weight, more preferably between 10 mg / day and 300 mg. mg / day, for example between 20 mg / day and 100 mg / day. The attending physician must monitor the patient's individual response and serum levels, it is deemed appropriate, and adjust accordingly to the patient's dose. When administered by injection, usually a lower dose will be appropriate, for example 10 mg to 100 mg per day per 50 kg of body weight. When inhibitors of 17β-hydroxysteroid type 5 are used according to the invention, as a part of one of the combination therapies described herein, the attending physician will desirablely direct the patient's serum concentration of the type 5 inhibitor between 0.5 ng / ml and 100 ng / ml, preferably between 1 ng / ml and 20 ng / ml and more preferably between 1 ng / ml and 10 ng / ml. The serum concentration can be measured by LC / MS. When administered orally, the dose that is usually effective to provide the desired levels in serum is between 1.0 mg and 1000 mg of the active ingredient per day per 50 kg of body weight., preferably between 10 mg and 500 mg and more preferably between 10 mg and 100 mg. However, the dose should vary with the bioavailability of the inhibitor chosen and with the individual response of the patient. For example, when EM-1404 is chosen, the oral dose is preferably between 5 mg and 500 mg per day per 50 kg of body weight, more preferably between 10 mg / day and 300 mg / day, for example between 20 mg / day and 100 mg / day. The attending physician must monitor the patient's individual response and metabolism (serum levels, if deemed appropriate), and adjust the patient's dose accordingly. When administered by injection, usually a lower dose will be appropriate, for example from 10 mg to 100 mg per day per 50 kg of body weight. When inhibitors of 17β-hydroxysteroid type 3 are used according to the invention, as part of one of the combination therapies described herein, the attending physician will desirablely direct the patient's serum concentration of the type 3 inhibitor by 0.5. ng / ml and 100 ng / ml, preferably between 1 ng / ml and 20 ng / ml and more preferably between 1 ng / ml and 10 ng / ml. When administered orally, the dose is preferably between 1.0 mg and 1000 mg of the active ingredient per day per 50 kg of body weight, preferably between 5 mg and 500 mg and more preferably between 10 mg and 100 mg. mg. However, the attending physician must monitor the patient's individual response and metabolism and adjust the patient's dose accordingly. The synthesis of this inhibitor is described later.

Synthesis of inhibitors of 17ß-HSD type 3 MßBr (Ci). THF or Et20.0'C R »CH2 CS-213 = CH2 -or BTN-161-94 E -1324-CS CH2CH2 --or BTN-194-82 Protection of 17ß-alsohol with TBDMS. To a solution of dihydrotestosterone (DHT, e) (5g, 17.2 mmol) in DMF was added imidazole (6 equivalents) and TBDMSC1 (5 equivalents). The reaction was stirred overnight at room temperature. The mixture was poured into ice and filtered. The resulting white precipitate was washed with water, dried over phosphorus pentoxide under reduced pressure for 24 h. A yield of 85 to 90% was obtained. 17β- [(fcer-butyldimethylsilyl) oxy] -5a-androstane-3-one (f). White solid; IR (KBr) v 1719 (C = 0, ketone); NMR XH (CDC13) d -0.001 and 0.005 (s, 6H, Si (CH3) 2), 0.71 (s, 3H, CH3-I8), 0.87 (s, 9H, SiC (CH3) 3), 1.01 (s, 3H, CH3-19), 3.54 (t, J = 8.2 Hz, 1H, CH-17); 13 C NMR (CDCl 3) d -4.80 and -4.47, 11.41, 11.52, 18.11, 21.13, 23.56, 25.87, 28.98, 30.94, 31.36, 35.54, 35.78, 37.13, 38.21, 38.65, 43.36, 44.74, 46.84, 50.55, 54.15, 81.79, 212.03.

Alkylation of carbonyl in position 3. To a solution of compound f (500 mg, 1.23 mmol) in dry THF (100 ml) at 0 ° C was added dropwise 3 equivalents of the commercially available Grignard reagent, in dry THF. The mixture was allowed to react for 3 hours at 0 ° C, then left overnight at room temperature. A solution of saturated NH C1 was added and the crude product was extracted with EtOAc. The organic phase was washed with a saturated solution of NaCl, dried over MgSO 4 and evaporated under reduced pressure. The 3β-alkylated stereoisomer was easily separated from the 3a-alkylated stereoisomer by flash chromatography on silica gel, using a mixture of hexanes and ethyl acetate as eluent. When the Grignard reagent was generated in itself as in the case of ethyl phenylmagnesium bromide, 5 equivalents were prepared. By a well-known procedure, using the corresponding bromide, activated magnesium and iodine. The steroid was then dissolved in dry diethyl ether and added dropwise to the reagent solution. The yields obtained were approximately 60% for the two isomers. 3β-benzyl-17β [(tert-butyldimethylsilyl) oxy] -3α-hydroxy-5-androstane (ga). White solid (24%); GO (KBr) v 3585 and 3460 (OH, alcohol); 1 H NMR (CDC13) d 0. 002 and 0.009 (s, 6H, Si (CH3) 2), 0.69 (s, 3H, CH3-I8), 0. 75 (s, 3H, CH3-19), 0.88 (s, 9H, SiC (CH3) 3) / 2.71 (s, 2H, CH2Ph), 3.54 (t, J = 8.2 Hz, 1H, CH-17), 7.20 to 7.34 (5H, Ph); 13C NMR (CDC13) d -4.82 and -4.50 (SiC (CH3) 3), 11.25, 11.40, 18.08, 20.62, 23.50, . 85, 28.41, 30.91, 31.62, 33.27, 33.81, 35.60, . 84, 37.19, 40.10, 40.84, 43.30, 50.43, 50.69, 54. 43, 71.22, 81.82 (C-17), 126.37, 128.09 (2X), 130.56 (2X), 137.06. 3a-hydroxy-3β- (phenylethyl) -17β [(tert-butyldimethylsilyl) oxy] -5a-androstane (gb). White solid (38%); IR (film) v 3447 (OH, alcohol); RRM XH (CDC13) d 0.018 and 0.025 (s, 6H, Si (CH3) 2), 0.71 (s, 3H, CH3-18), 0.78 (s, 3H, CH3-19), 0.89 (s, 9H, SiC (CH3) 3), 2.73 (m, 2H, Ph-CH2), 3.56 (t, J = 8.1 Hz, 1H, CH-17), 7.18 to 7.31 (5H, Ph); 13 C NMR (CDCl 3) d -4.77 and -4.46 (Si (CH 3) 3), 11.28, 11.44, 18.12 (SiC (CH 3) 3), 20.67, 23.54, 25.89 (SiC (CH 3) 3), 28.52, 29.60, 30.97 , 31.66, 33.31, 33.92, 35.66, 36.04, 37.25, 40.03, 41.05, 43.35, 46.47, 50.76, 54.55, 71.50 (C3), 81.86 (C-17), 125.68, 128.38 (4X), 142.82.

Procedure for hydrolysis of the TBDMS group and oxidation of the resulting alcohol. The silylated ether was dissolved in a methanolic HCl solution (2% v / v) and the resulting mixture was stirred at room temperature for 3 h. Then water was added and the MeOH evaporated under vacuum. The resulting white precipitate was subjected to Jones oxidation without purification. To a stirred solution of crude alcohol in acetone at 0 ° C, Jones reagent (2.7 M chromic acid solution) was added dropwise. After 30 to 45 minutes, the reaction was complete. Isopropanol and water were added and acetone in va cuo was removed. The remaining aqueous layer was extracted with EtOAc. The combined organic phases were washed with brine, dried over MgSO, filtered and evaporated under reduced pressure. The purification was done in silica gel, using HPLC grade solvents, EtOAc and hexanes as eluents. 3β-benzyl-3a-Hydroxy-5α-androstane-17-one (CS-213). White solid (88% for the two steps); IR (KBr) v 3408 (OH, alcohol), 1732 (C = 0, ketone); XH NMR (CDC13) d 0.75 (s, 3H, CH3-19), 0.84 (s, 3H, CH3-I8), 2.69 (s, 2H, CH2Ph), 7.18 to 7.32 (5H, Ph); 13 C NMR (CDCl 3) d 11.18, 13.78, 20.20, 21.71, 28.16, 30.79, 31.52, 33.18, 33.70, 35.64, 35.79, 35.88, 39.97, 40.69, 47.75, 50.39, 51.41, 54.22, 71.12, 126.40, 128.09 (2X) , 130.51 (2X), 136.93, 221.27. 3a-hydroxy-3β-phenylethyl-5a-androstane-17-one (EM-1324-CS). White solid (82% for the two steps); IR (film) v 3486 (OH, alcohol), 1737 (C = 0, ketone); 1H-NMR (CDC13) d 0.79 (s, 3H, CH3-19), 0.86 (s, 3H, CH3-I8), 2.71 (m, 2H, Ph-CH2), 7.18 to 7.30 (5H, Ph); 13C NMR (CDC13) d 11.21, 13.82, 20.26, 21.76, 28.26, 29.54, 30.87, 31.58, 33.27, 33.80, 35.10, 35.84, 36.07, 39.89, 40.90, 46.43, 47.80, 51.49, 54.35, 71.42, 125.69, 128.31 ( 2X), 128.39 (2X), 142.70, 221.31. All active ingredients used in any of the therapies discussed herein can be formulated into pharmaceutical compositions that include one or more of the other active ingredients.

Alternatively, each may be administered separately but in a sufficiently simultaneous manner over time so that a patient eventually has elevated blood levels or otherwise enjoys the benefits of each of the active ingredients (or strategies). ), simultaneously. In some preferred embodiments of the invention, for example one or more active ingredients will be formulated in an individual pharmaceutical composition. In other embodiments of the invention, equipment is provided that includes at least two separate containers wherein, the contents of at least one container differ in part from the contents of at least the other container with respect to the active ingredients contained therein. the same. Two or more different containers are used in these combination therapies of the invention. The combination therapies discussed herein also include the use of an active ingredient of the combination in the manufacture of a medicament for the treatment (or prevention) of the disease in question where the treatment or prevention also includes the other (s) s) active ingredient (s) or combination strategy. Some embodiments of the methods for treating or preventing the disease discussed herein, use the specific inhibitor of 17β-hydroxysteroid dehydrogenase type 5 and / or 3-hydroxysteroid dehydrogenase type 3 inhibitors analyzed herein (ie, the structures Molecules analyzed in the present). LHRH agonists and LHRH antagonists can be used interchangeably to suppress either testicular or ovarian hormone secretions by known techniques, except where preferences are otherwise raised herein. It is desired that the activation of the glucocorticoid receptors be minimized when the active ingredients of the invention are administered. The inhibitors of the 17β-hydroxysteroid dehydrogenase type 3 can be used to provide advantages similar to those provided by the LHRH agonists or antagonists.

PREFERRED INHIBITORS OF 3-HYDROXYSTEROID-DEHYDROGENASE TYPE 3 Exposed in Tables below are lists of compounds that are found to be useful as inhibitors of 3a-hydroxysteroid dehydrogenase type 3. The Tables also include in many cases additional tests of a compound particular in other important parameters such as androgenic and antiandrogenic activity and the effect of a compound on androgen receptors, proliferation of androgen-sensitive cells, and other effects explained more fully later. In the Tables below that which does not include an "apostrophe" (') in its table number, details of the molecular structure of the preferred inhibitors (or comparison compounds) are set forth. The corresponding tables with an "apostrophe" (') in their table number show information a the functional effectiveness of each approved compound. The numbers in the header of the columns correspond to a description at the end of all the tables with respect to what that information is reported in each column and how it is determined. The entries left blank are still undetermined.

TABLE 1 Table 2 LEGEND OF THE TABLES In column 1, the oral bioavailability of the preferred inhibitors of 3a-hydroxysteroid dehydrogenase type 3, expressed in ng / ml. h, was determined as described below in "In Vive Essays on Bioavailability of 3a-hydroxysteroid dehydrogenase type 3 inhibitors, Humana", larger numbers are desirable. ND means that a determination was not made. In column 2, the inhibition of human type 3a-hydroxysteroid dehydrogenase 3 activity expressed by the concentration that produces 50% inhibition of enzyme activity (IC50 in nM) is reported. The manner in which the IC50 is determined is described infra in "II-Enzymatic assay for 17β-HSD types 1, 2, 3 and 5 and 3a-HSD types 1 and 3". White means that no determination was made. The percentage of inhibition of enzymatic activity by the inhibitor at 3.10"7 and 3.10" 6 M is reported in parentheses. In column 3, the inhibition of the activity of human 17β-hydroxysteroid dehydrogenase type 1 expressed by the concentration produced 50% inhibition of enzymatic activity (IC50 sn nM) is reported. The manner in which the IC50 is determined is described in "II-Enzymatic assay for 17β-HSD types 1, 2, 3 and 5 and 3oc-HSD types 1 and 3". Higher IC50 numbers are desirable. White means that no determination was made. The percentage of inhibition of enzymatic activity by the inhibitor at 3.10"7 and 3.10" 6 M is reported in parentheses. In column 4, the inhibition of activity of 17β-hydroxysteroid dehydrogenase type 2 is reported by the concentration produced 50% inhibition of enzymatic activity (IC50 in nM). The manner in which the IC50 is determined is described in "II-Enzymatic assay for 17β-HSD types 1, 2, 3 and 5 and 3a-HSD types 1 and 3". Higher IC50 numbers are desirable. White means that no determination was made. The percentage of inhibition of enzymatic activity by the inhibitor is reported between parentheses at 3.10"7 and 3.10" 6 M. In column 5, the inhibition of activity of 17β-hydroxysteroid dehydrogenase type 3 is reported by the concentration produced 50% inhibition of enzymatic activity (IC50 in nM). The manner in which the IC50 is determined is described in "II-Enzymatic assay for 17β-HSD types 1, 2, 3 and 5 and 3a-HSD types 1 and 3". Minor numbers of IC50 are desirable. White means that no determination was made. The percentage of inhibition of enzymatic activity by the inhibitor at 3.10-7 and 3.10"6 M is reported in parentheses. In column 6, the inhibition of the activity of 3-hydroxysteroid dehydrogenase type 1 is reported by the concentration produced 50% inhibition of enzyme activity (IC50 in nM) The way in which IC50 is determined is described in "II-Enzymatic assay for 17ß-HSD types 1, 2, 3 and 5 and 3a-HSD types 1 and 3"Higher IC 50 numbers are desirable White means that no determination was made Parenthesis reports the percent inhibition of enzyme activity by the inhibitor at 3.10" 7 and 3.10"6 M. In column 7, it is reported (centered numbers) the inhibition of the activity of 17β-hydroxysteroid dehydrogenase type 5 expressed by the concentration that produces 50% inhibition (IC50 in nM) .The way in which the IC50 is determined is described in "II Enzymatic assay for 17ß-HSD types 1, 2, 3 and 5 and 3a-HSD types 1 and 3". Minor numbers for IC50 are desirable. When the ICsor was not determined, the percentage of inhibitions reported in parentheses at 3.10"7 M (left number) and 3.10" 6 M (right number). The percentage of inhibition of enzymatic activity by the inhibitor at 3.10-7 and 3.10"6 M is reported in parentheses. In column 8, the androgenic activity of the preferred inhibitors of 3-hydroxysteroid dehydrogenase type 3 is reported as the percentage of stimulation of proliferation of Shionogi cells at concentrations of 10-7 M (left number) and 10 ~ 6 M (right number) of inhibitor.The manner in which stimulation is determined is described in "III-Activity Androgenic / Antiandrogenic. "Minor numbers are desirable ND means no determination was made In column 9, the antiandrogenic activity of the preferred inhibitors of 3a-hydroxysteroid dehydrogenase type 3 expressed by the concentration producing 50% inhibition (IC50 in nM) of the DHT-induced proliferation of Shionogi cells (numbers centered in brackets) The percent inhibition of DHT-induced proliferation of Shionogi cells at concentrations of 10 ~ 7M (left number) and 10 ~ 6 M (right number) of inhibitor The way in which inhibition is determined is described in "III-Androgenic Activity / Antiandrogenic." Minor numbers are desirable ND means no determination was made In column 10, The estrogenic activity of the preferred inhibitors of 3a-hydroxysteroid dehydrogenase type 3 expressed as the percentage of stimulation of the proliferation of ZR-75-1 cells at concentrations of 10_7M (left number) and 10"6M (right number) of inhibitor. The manner in which the stimulation is determined is described in "IV-Activity Estrogenic / Antiestrogenic. "Minor numbers are desirable.ND means no determination was made.In column 11, the antiestrogenic activity of the preferred inhibitors of 3a-hydroxysteroid dehydrogenase type 3 expressed as the percentage of stimulation of proliferation is reported. induced by E2 of ZR-75-1 cells at a concentration of 10_7M (left number) and 10 ~ 6 (right number) of inhibitor.The manner in which inhibition is determined is described in "IV-Estrogenic / Antiestrogenic Activity" Minor numbers are desirable ND means no determination was made In column 12, binding at the androgen receptor expressed as the percent inhibition of the binding of [3H] R1881 to the 10"8M concentration is reported. (number set to 10 ~ 7M) (left number) and 10 ~ 6M (number set to 10 ~ 5M) (right number) of inhibitor. The manner in which the percent inhibition is determined is described in "V-Androgen Receptor Assays (AR)". Minor numbers are desirable. In column 13, progesterone receptor binding expressed as the percentage inhibition of [3H] R5020 binding at the concentration of 10 ~ 8M (fixed number at 10 ~ 7M) (left number) and 10 ~ is reported. 6M (number set to 10_5M) (right number) of inhibitor. The manner in which the inhibition percentage is determined is described in "VI-Progesterone Receptor Assay". Minor numbers are desirable. In column 14, binding at the glucocorticoid receptor expressed as the percent inhibition of the binding of [6, 7-3H * (N)] -dexametasone at the concentration of 10 ~ 8M (number set at 10"is reported). 7M) (left number) and 10 ~ 6M (fixed number at 10_5M) (right number) of inhibitor.The manner in which the percentage of inhibition is determined is described in "Vil-Glucocorticoid Receptor Assay". 15, the binding in the estrogen receptor is reported as the percentage of inhibition of the binding of [3H] E2 to the concentration of 10 ~ 8M (fixed number to 10 ~ 7M) (left number) and 10"6M (number set to 10 ~ 5M) (right number) of inhibition. The manner in which the percent inhibition is determined is described in "VIII-Estrogen Receptor Assay (ER)".

EFFECTIVENESS OF THE PREFERRED INHIBITORS I. - In Vivo Bioavailability Tests of Human Type 3a-Hydroxysteroid Dehydrogenase 3 Inhibitors 1) Principle Bioavailability tests of 3a-hydroxysteroid dehydrogenase type 3 inhibitors were performed on male Sprague Dawley rats when measuring plasma concentrations of the compounds after the individual oral administration of the compounds. Measurements at various time intervals were for values greater than or equal to 1.0 ng / ml and less than or equal to 50 ng / ml. a) Animals and treatment We obtained from Charles-River, Canada Inc. male Sprague Dawley rats [Crl: CD (SD) Br] weighing 275-350 g and were housed two per cage during the acclimation period and individually during the period of study. The animals were kept under a regime of 12 hours of light: 12 hours of darkness (light at 08:00). The animals received certified food for rodents (Laboratory Diet # 5002, compressed) and tap water at libi t um. The rats were fasted (only with access to water) starting the morning before dosing. Each compound to be tested was administered to three animals as a suspension in 0.4% methylcellulose by oral feeding by tube at a dose of 0.5 mg / rat (1.0 ml / rat). Four to eight new compounds were tested every day and one group of animals received megestrol acetate (MGA) under the same conditions on each dosing day as one. reference. A blood sample of approximately 0.7 ml was collected from the jugular vein of rats under Isoflurane-induced anesthesia at 1, 2, 3, 4, and 7 hours after the priming. The blood samples were immediately transferred into a refrigerated 0.75 ml microcontainer containing EDTA and kept in an ice-water bath until centrifugation at 3000 rpm for 10 minutes. Plasma separation was performed rapidly (less than 50 minutes) after blood collection. An aliquot of 0.25 ml of plasma was then transferred into a borosilicate tube (13 x 100) and quickly frozen on dry ice. Plasma samples were maintained at -80 ° C until the plasma concentration of the inhibitor (s) was measured by LCMS / MS. 2) Measurements of LCMS a.) Apparatus 1. Vacuum distributor 2. LV Turbo-Vap evaporator 3. API III or API-300 mass spectrometer (PE / Sciex) with associated peripherals 4. Automatic injector 5. HPLC pump 6 Infusion pump 7. Calibrated pipettes b) Reagents and solutions 1. Methanol, HPLC grade 2. Water, ultra pure (Super Q) 3. Ethanol, reactive grade 4. N-butyl chloride, HPLC grade 5. Acetone, HPLC grade 6. Male rat plasma (EDTA) 7. Inhibitors of 3a-hydroxysteroid dehydrogenase type 3 in ethanol solution, normal, reference approximately 100 μg / ml. 8. Standard reference, EM 248 Infernal Standard (50 ng / ml solution). 9 Mass gauge solution, propylene glycol (PE / Sciex) c) Mass spectrometer conditions Detector: API-300 mass spectrometer (PE / Sciex) Inferred: Turbo Ion spray inlet (1/5 split) Auxiliary flow: 4.5 l / min (nitrogen) Nebulizer flow: 11 curtain gas: 11 Probe temperature: 460 ° C Pressure: Approximately 3xl0 ~ 5 Torr CAD gas thickness: 3 Count control: 1 Mobile phase: gradient of methanol with 1 mm ammonium formate and water with ammonium formate 1 mm Flow rate: 1 ml / minute d) Analysis parameters of the mass spectrometer for EM-1118 Residency time: 150 msec. Pause time: 30 msec. Duration: 4 minutes Mode of MRM for Analysis of EM-1118: 444.2 and 398. 3 Injection: 10 μl Data management: Update version "Normal computing program API" e) Preparation of normal solutions Reserve solutions were prepared for each inhibitor of 3a-hydroxysteroide dehydrogenase type 3 in methanol and when not in use, solutions in methanol are stored at -20 ° C. Normal calibration curve solutions were prepared for each compound in the male rat plasma as illustrated in Figure 1. A solution of internal normal in methanol containing EM-248 at 50 ng / ml, was prepared from the normal solutions of Reserve of EM-248 stored at -20 ° C.

Volume Volume Concentration of the inhibitor 3a-HSD plasma solution Std 50 ng / ml 90 μl of 1 μg / ml 1.71 ml Std 20 ng / ml 0.8 ml of 50 1.2 ml Std 10 ng / ml ng / ml 0.9 ml Std 5 ng / ml 0.9 ml of 20 0.8 ml Std 2 ng / ml ng / ml 0.9 ml Std 1 ng / ml 0.8 ml 10 0.5 ml Normal 0 ng / ml 0.5 ml White 0.6 ml 5 ng / ml 0.5 ml 0.5 ml 2 ng / ml N / AN / A f) Method of extraction of inhibitors type 3 of rat plasma. Aliquots of rat plasma (0.250 ml) were transferred to 13x100 mm borosilicate tubes. Water (1.0 ml) and normal internal solution (0.1 ml) were added to each sample and vortexed for 2 minutes. A mixture of N-butyl chloride and acetone (v: v, 7: 3) (3 ml) was added to each sample and subjected to stirring for 2 minutes. This step was repeated and the combined organic phases were evaporated to dry under nitrogen in a Turbo Vap evaporator at 35 ° C. The residue was reconstituted with 1 ml of methanol and evaporated in a Turbo Vap evaporator at 35 ° C. The final extract was reconstituted in 0.1 ml of methanol / water (v: v, 75:25) and then transferred in a conical flask for injection into the mass spectrometer. g) Assay The assay procedure was performed by analyzing, in duplicate, rat plasma samples seeded at six different concentrations of inhibitor type 5 (1, 2, 5, 10, 20 and 50 ng / ml). The lower limit of quantification (LOQ) was set at 1.0 ng / ml. Values less than 1.0 ng / ml were expressed as the lower limit of quantification (BLQ). h) Linearity The assay procedures for EM-1118 were found to be linear over the range of 1.0 to 50 ng / ml. The analysis by linear weighted regression (1 / X) and a correlation (r2) of 0.991. i) Calculation of AUC values For all the compounds studied, the area under the plasma contretemps concentration curve (AUC) was determined from time 0 to 7 hours after dosing. The values of AUC0-7 were calculated by the linear trapezoidal method (independent of the model) for each rat and the data were expressed as the mean AUCo- ± SEM (n = 3).

II. Enzymatic assays for 17β-HSD, types 1, 2, 3 and 5 and 3a-HSD types 1 and 3 Enzyme sources: 293 cells transiently transfected with expression vectors encoding 17β-HSD types 1, 2 and 3 (Luu The et al., J. Steroid Biochem, Molec. Biol., 55: 581-587, 1995), 17β-HSD type 5 (as described in WO 97/11162), and 3a-HSD types 1 and 3 (Dufort et al., Biochem. Biophys. Res. Commun. 228: 474-47.9, 1996), using the calcium phosphate process (Kingston et al., In: Current Protocols in Molecular Biology, Edited by EM Ausbel, R. Brent , RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl, John Wiley &Sons, New York, pp. 9.1.1-9.1.9, 1991; Luu-The et al., J. Invest. Dermatol ., 102: 221-226, 1994). For assays using cellular subfractions, the cells were sonically treated in 50 mM sodium phosphate buffer (pH 7.4), containing 20% glycerol and 1 mM EDTA and centrifuged at 10,000 xg for 30 minutes before centrifugation to 100,000 xg for 1 hour to separate the mitochondrial and microsomal fractions, respectively. The cytosolic fractions (100,000 xg of supernatant) were used to determine the type 1 activity while the microsomal fraction (mass of compressed material at 100,000 x g) was used for the measurement of the activities of 17β-HSD types 2 and 3.

Incubation The enzymatic reaction was carried out at 37 ° C in 1 ml of 50 mM sodium phosphate buffer, pH 7.4, containing 20% glycerol, 1 mM EDTA, and 2 mM cofactors (NADPH or NAD +) for 1 hour in the presence of 0.1 μM of substrate labeled with, 14 estrone for 17ß-HSD type 1, DHEA and 4-androstene-3, 17-dione (? 4), for 17ß-HSD types 3 and 5, testosterone for 17ß-HSD type 2 as well as androstran-dione and DHT for activities of 3a-HSD types 1 and 3, an absence or presence of increasing concentration of the preferred inhibitor of the invention, was added to the recently changed culture medium in a 6-well culture plate . After incubation for 1 hour, the steroids were extracted twice with 2 ml of ether. The organic phase was mixed and evaporated to dryness. The steroids were solubilized in 50 μl of dichloromethane, and applied to a thin layer chromatography plate (Merck, Darmstadt, Germany).

(TLC) of silica gel 60, then separated by migration in a toluene-acetone solvent system (4: 1) Substrates were identified in metabolites by comparison with reference steroids and revealed by autoradiography and quantified using the Phosphoimager System (Molecular Dynamics, Sunnyval, CA). Transfection can also be performed with HeLa, SW-13, 293, COS-1 cells as the preferred cell line is 293 cells.

III. Shionogi activity The androgenic / antiandrogenic activity of some preferred compounds has been measured using Shionogi mouse mammary carcinoma cells.

Materials . Minimum essential culture medium (MEM), non-essential amino acids, and fetal calf serum were compared from Flow Laboratories. In order to remove the endogenous steroids, the serum was incubated overnight at 4 ° C with activated charcoal. 1% (Norit A, Fisher), and dextran T-70 0.1% (Pharmacia). A complementary adsorption was carried out for 2 hours at 25 ° C in order to further remove the steroids bound to protein. The serum is also inactivated by a 20 minute incubation at 56 ° C. 5a-dihydrotestosterone (DHT) was obtained from the esteraloids. The antiandrogen, hydroxyflutamide (OH-FLU) was kindly provided by the doctors T.L. Nagabuschan and R. Neri (Schering Corporation, Kenilworth, E.U.A.

Dispersion, cultivation and cell cloning. Male Shionogi mice having androgen sensitive mammary tumors were obtained by Doctors Keishi Matsumoto, Osaka, Japan, and Yvonne Lefebvre, Ottawa, Canada. For the primary culture, the tumors were excised and washed in sterile 25 mM Hepes buffer cooled in ice (137 mM NaCl, 5 mM KCl, 0.7 Na2HP04, 10 mM glucose, pH 7.2). After punching with scissors, the tumor bites were digested for 2 hours at 37 ° C in Hepes buffer containing 3.8 mg / ml collagenase (Clostridium, Boehringer), 1.5 mg / ml hyaluronidase II (Sigma) and fraction V of 3% bovine serum albumin (Schwartz-Mann). The dispersed cells were collected by centrifugation (500 xg for 10 minutes), washed twice by suspension of minimal essential medium (MEM) containing charcoal-treated fetal calf serum, coated with 5% dextran (DCC-FCS), 1% non-essential amino acids, 10 IU / ml penicillin, 50 μg / ml streptomycin, and 100 nM dihydrotestosterone (DHT) (Steraloids). The cells were plated in the same medium at a density of 75,000 cells / ml in 75 cm 2 flasks under a 5% carbon dioxide atmosphere in air at 37 ° C. The medium was changed weekly. The steroids and antiesteroids were dissolved in ethanol and kept in concentrated solutions chosen to produce final concentrations in ethanol less than 0.01% in the culture medium. This concentration of ethanol does not affect cell growth. The cells were subcultured with some confidence, by gentle digestion in a 0.1% pancreatin solution (Flow Laboratories) in Hepes buffer containing 3 M ethylenediaminetetraacetic acid (EDTA) (pH 7.2). The cells were pelleted by centrifugation, redispersed in culture medium, counted in a Coulter counter, and plated back as described above. Cloning was performed with soft agar as described (Stanley et al., Cell 10: 35-44, 1977) in the presence of 100 nM DHT. Measurement of cell growth? _ Sensitivity to steroids and antiesteroids. The cells were placed in 24 cavity plates at a density of 20,000 cells / well. The indicated increasing concentrations of the agents were added to boxes in triplicate, and the cells were cultured for 10-12 days with medium changes every 3-4 days. The cell number was measured by direct count in a Coulter counter. Calculations and statistical analysis. DH50 action values of DHT and glucocorticoids were calculated according to a least squares regression as described (Rodbard, Endocrinology 94: 1427-1431, 1974). The statistical significance was calculated according to a multiple range test (Kramer, Biometrics 12: 307-310, 1956).

IV. Estrogenic / Antiestrogenic Activity The estrogenic / antiestrogenic activity of some preferred compounds have been measured using the human breast cancer cell line ZR-71-1 as described in more detail below. Maintenance of concentrated crops: ZR-75-1 cells (passage 83rd) were obtained from the American Collection of Species (American Type Culture Collection (Rockville, MD)) and routinely cultured in RPMI 1640 phenol red-free supplemented with 1 nM estradiol (E2 ), 2 mM L-glutamine, 1 mM sodium pyruvate, 15 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, 100 IU penicillin / ml, 100 μg streptomycin / ml, and fetal bovine serum at 10% (v / v) (Hyclone, Logan, UT) under a humidified atmosphere of 95% air, 5% C02 at 37 ° C. All media and media supplements were purchased from Sigma. The cells were subcultured weekly by treatment with a pancreatic solution containing EDTA (0.2 g / L). The cell cultures used for the experiments described herein are between passages 89 and 94.

Measurements of cell proliferation. The cells in their logarithmic growth phase were harvested, briefly centrifuged and redispersed in RPMI 1640. The cells were then plated in triplicate in 24-well plastic culture plates, LIMBRO (2 cprV cavity). Since plaque density influences the effect of the hormones on the growth of ZR-75-1 cells, the cells were plated at a density of lx104 cells / well. After 72 hours, the medium was replaced with fresh medium containing the inhibitor at the concentration of 3.10"7 and 10 ~ 6 M in the absence or presence of 0.1 M estradiol (E2). The cells were allowed to grow at 73 ° C for 10 days with medium change (identical composition) every 2 days.In the absence of inhibitors, in the medium containing estradiol 0.1M (E2), the ZR-75 cells -1 had the effect of duplication of approximately 48 hours.After treatment with E2 and / or antiestrogen, the cells were harvested by adding 0.5 ml of a pancreatin solution (Sigma) for 5-10 minutes at 37 ° C before adding 0.5 ml of RPMI 1640 containing carbon-free bovine serum, coated with 5% dextran in order to block the enzymatic action The cell number (0.10 ml aliquot) was determined by measuring the DNA content as described previously (Simard et al., Endocrine logy 126: 3223-3231, 1990).

V. Androgen receptor (AR) assays Tissue preparation. Obtained from Charles-River Inc. (St-Constant, Quebec, Canada) male Sprague-Dawley rats (Crl: CD (SD) Br) weighing 200-300g. The rats underwent gonadectomy under general anesthesia (Isofluoran) and were sacrificed by cervical dislocation 24 hours later. The ventral prostates were removed quickly, cut free of adherent tissue and frozen in dry ice. The prostates were maintained at -80 ° C until the test. All subsequent steps were performed at 0-4 ° C. The prostates were homogenized in 5 vol (p / vol) of buffer A (25 mM Tris-HCl, 1.5 mM EDTA disodium salt, 10 mM monothioglycerol, 10% glycerol and 10 mM sodium molybdate, pH 7.4), using a Polytron PT-10 homogenizer (Brinkman Instrumens, Canada) at a setting of 5 for three periods of 10 seconds, with intervals of 10 seconds for cooling. The homogenate was then centrifuged at 105,000 x g for 60 minutes in a Beckman ultracentrifuge L5-65 (Fullerton, CA). The protein concentration of the cytosol fraction was measured according to the Bradford method (Anal.

Biochem. 72: 248-254, 1976), using bovine serum albumin as a rule. Androgen receptor assay: The androgen binding was measured using the hydroxylapatite (HAP) assay. Briefly, the radioactive steroid [HJR1881 solubilized in ethanol was diluted in buffer A. Then aliquots of the prostate cytosol preparation (0.1 ml) were incubated with [3H] R1881 (0.1 ml, approximately 200,000 cpm) in the presence or absence of the indicated concentrations of unlabeled compounds (0.1 ml, prepared in buffer A containing 10% ethanol) for 16-18 hours at 0-4 ° C. Triamcinolone-acetonide (150 nM) was added in order to mask the progesterone receptors. The bound steroids were separated by incubation for 40 minutes at 0-4 ° C with 0.3 ml of PAH prepared in P buffer (50 mM Tris-HCl, 10 Mm KH2P04, pH 7.4) as follows: 10 g of PAH were washed with buffer P under the supernatant until it reached pH 7.4 and then after centrifugation and decantation of the supernatant, 37.5 ml of P buffer was added. After incubation with HAP and 10 minutes of centrifugation at 1,000 xg, the pellet was washed 3 times with 1 ml of P. buffer. Subsequently, the radioactivity was extracted from the pellet by incubation at room temperature for 60 minutes with 1 ml of EtOH. After centrifugation, the supernatant was decanted into a scintillation flask and the pellet was extracted again with ethanol. Subsequently, 10 ml of the scintillation fluid was added by formulating 989 to the mixed supernatant and the radioactivity was measured in a Beckman counter. Calculations The results were reported as the percentage of inhibitor of the binding of [3H] R1881 at the concentrations of 10 ~ 8 and 10 ~ 6M of the inhibitor.

SAW . Progesterone receptor assay Chemical products. [17a-Methyl-3H] -prommethione (R5020) (84 Ci / mmol) and the corresponding non-labeled compound were compared from New England Nuclear (Lachine, Quebec, Canada). All other chemical compounds were of analytical grade. The concentrated solutions of the unlabeled steroids were kept at 4 ° C in ethanol. The desired solutions of the steroids were then prepared by appropriate dilution in buffer B (10 mM Tris-HCl, 1.5 mM EDTA, 10 mM α-monothioglycerol, pH 7.4) containing 30% ethanol. Preparation of the tissue. Female Sprague-Dawley rats weighing 200-300g were obtained from Charles-River Inc. (St-Constant, Québec, Canada). Those who underwent gonadactomy under general anesthesia (Isoflurane) and sacrificed by cervical dislocation 24 hours later. The uteri were removed quickly, cut free of adherent tissue and frozen in dry ice. The tissues were maintained at -80 ° C until use. Preparation of cytosol. All procedures were performed at 4 ° C. The tissues were pulverized, frozen in dry ice with a Thermovac sprayer. The samples were then homogenized in 10 vol (p / v) buffer A (25 mM Tris-HCl, 1.5 mM EDTA, 10 mM a-monothioglycerol, 10% glycerol, 10 mM sodium molybdate, pH 7.4) using a Polytron PT-10 homogenizer (Brinkmann Instruments, Canada) at a setting of 5 for two periods of 10 seconds, with intervals of 10 seconds for cooling. The homogenate was then centrifuged at 105,000 x g for 90 minutes. The supernatant was used immediately for the assay. Bonding tests. Progesterone binding was measured using the carbon adsorption technique coated with dextran. Incubations were performed at 0-4 ° C for 16-18 hours using 100 μl of cytosol, 100 μl of [3 H] -R5020 (final 5 mM, containing 1,000 nM dexamethasone in order to mask the glucocorticoid receptors) and 100 μl of unlabeled compounds at the indicated concentrations. Each concentration was made in triplicate. The assay was terminated with 300 μl of DCC (0.1% Norit A and Dextran T-70 in buffer B). After 10 minutes of incubation, the tubes were centrifuged at 2,000 x g for 10 minutes and decanted in flasks with 6 ml of BCS scintillation fluid (New England Nuclear, Dupont). The radioactivity was measured in a Bec man counter at a counting efficiency of 35%. Calculations The results were reported as the percentage of the inhibition of the agglutination of [H3] R5020 at the concentrations of 10"8 or 10" 6M of the inhibitors.

VII. Glucocorticoid Receptor Assay Chemicals [6,7-3H (N)] -dexametasone (39 Ci / mmol) was purchased from New England Nuclear (Lachine, Québec, Canada) while unlabeled dexamethasone was obtained from Steraloids (Wilton, NH). All the other chemicals were of analytical grade. The concentrated solutions of the unlabeled steroids were kept at 4 ° C in ethanol. The desired steroid solutions were then prepared by appropriate dilution in buffer B (10 mM Tris-HCl, 1.5 mM EDTA, 10 mM a-monothioglycerol, pH 7.4) containing 30% ethanol. Preparation of tissue. They were obtained from Charles-River Inc. (St-Constant, Québec, Canada) Male Sprague-Dawley rats weighing 200-300g. The rats were sacrificed by cervical dislocation and the liver was rapidly removed, the adherent tissue was cut free and frozen in dry ice. The tissues were maintained at -80 ° C until use. Preparation of cytosol. All procedures were performed at 4 ° C. The tissues were crushed and homogenized in 10 vol (p / v) of buffer A (25 mM Tris-HCl, 1.5 mM EDTA, 10 mM a-monothioglycerol, 10% glycerol, 10 mM sodium molybdate, pH 7. 4) using a Polytron PT-10 homogenizer (Brinkmann Instruments, Canada) at a setting of 5 for two periods of 10 seconds, with intervals of 10 seconds for cooling. The homogenate was then centrifuged at 105, 000 x g for 90 minutes. The supernatant was used immediately for the assay. Bonding tests. The glucocorticoid binding was measured using the carbon adsorption technique coated with dextranol. Incubations were performed at 0-4 ° C for 16-18 hours using 100 μl of cytosol, 100 μl of [3 H] -dexametasone (5 nM final) and 100 μl of the unlabeled compounds at the indicated concentrations. Each concentration was made in triplicate. The tests were terminated with 300 μl of DCC (2.5% Norit A and 0.25% Dextran T-70 in buffer B). After 10 minutes of incubation, the tubes were centrifuged at 2,000 x g for 10 minutes and decanted in flasks with 6 ml BCS liquid scintillation (New England Nuclear, Dupont). The radioactivity was measured in a Beckman counter at a count efficiency of 35%. Calculations The results were reported as the percentage of the inhibition of the agglutination of [H3] -dexametasone at the concentrations of 10-8 and 10 ~ 6M of the inhibitor.

VIII. Estrogen receptor (ER) assay Tissue preparation. Obtained from Charles-River Inc. (St-Constant, Quebec, Canada) Sprague-Dawley rats (Crl: CD (SD) Br) weighing 200-300 g. The rats were subjected to gonadactomy under general anesthesia (Isoflurane) and sacrificed by cervical dislocation 24 hours later. The uteri were removed quickly, cut free of adherent tissue and frozen in dry ice. The uteri were maintained at -80 ° C until the test. All subsequent steps were performed at 0-4 ° C. The uteri were homogenized in 10 vol (p / vol) of buffer A (25 mM Tris-HCl, 1.5 mM EDTA disodium salt, α-monothioglycerol lOmM, 10% glycerol and 10 mM sodium molybdate, pH 7.4), using a Polytron PT-10 homogenizer (Brinkman Instruments, Canada) at a setting of 5 for three periods of seconds, with intervals of 10 seconds for cooling. The homogenate was then centrifuged at 105.00 x g for 60 minutes in a Beckman ultracentrifuge L5-65 (Fullerton, CA). The protein concentration of the cytosol fraction was measured according to the Bradford method (Anal. Biochem. 72: 248-254, 1976), using bovine serum albumin as a rule. Estrogen binding was measured using the carbon adsorption technique coated with dextran as previously described (Asselin et al., Endocrinology, 101: 666-671, 1977, Asselin and Labrie, J. Steroid Biochem., 9: 1079- 1082, 1978). Briefly, [H3] E2 was solubilized in ethanol was diluted in buffer A. Aliquots of the uterine cytosol preparation (0.1 ml) were incubated with [H3] E2 5nM (approximately 200,000 cpm, 0.1 ml) in the presence or absence of the indicated concentrations of the unlabeled compounds (0.1 ml, prepared in buffer A containing 10% ethanol) for 3 hours at room temperature. The unbound steroids were then separated by incubation for 15 minutes at 0 ~ 4 ° C with 0.3 ml of 0.05% Norit-A and 0.05% dextran 7-70 in buffer B (1.5 mM EDTA disodium salt, 10 mM monothioglycerol. , and 10 mM Tris-HCl, pH 7.4) and centrifuged at 3,000 xg for 15 minutes. The aliquots of the supernatants (0.3 ml) were removed for the measurement of radioactivity. After the addition of 10 ml of the disintegration liquid formula 989 (New England Nuclear-DuPont), the radioactivity was measured in a Beckman counter at a count efficiency of 62%.

Calculations The results were reported as the percentage of inhibition of E2 binding at the 10 ~ 8 and 10-6M concentrations of the inhibitor. Primary criteria in the selection of preferred inhibitors include bioavailability, desirable inhibition of 3a-hydroxysteroid dehydrogenase type 3 and 17β-hydroxysteroid dehydrogenase type 5, the degree of undesirable inhibition in 17a-hydroxysteroid dehydrogenase type 2, and androgenicity. It is believed that the methyl groups at the 5 'position in EM 01645 and EM 01667 and the analogous compounds promote the selectivity of the inhibition of 17β-HSD type 5 (against the undesirable type 2 inhibition). It is also believed that the free hydroxy group at position 3 has a beneficial effect also on the substitution of position 2. Applicants have tested a wide range of compounds for effectiveness as inhibitors of 3a-HSD type 3. It is believed, Based on this laboratory work, certain characteristics of the molecular structure analyzed in the present provide favorable characteristics to the steroidal compounds invention. For example, it is believed that an aromatic ring A and a portion at position 3 that is either hydroxyl or a common group of pro-drug that is converted to hydroxyl in vivo are characteristics that favor good inhibition of the 3a-HSD type. 3 when combined with appropriate substitution at position 2 or 4. Substituents at positions 2 and / or 4 are independently selected from the group consisting of hydrogen, cyano, fluoro, bromo and nitro (with the proviso that the substituents in 2 and 4 are not simultaneously hydrogen). Substituents that have worked well in position 2 have tended to work well in position 4 and vice versa. Placing the same substitution in both 2 and 4 may be easier from the manufacturing point of view, and the compounds of that type that have been tried have tended to be good inhibitors of the 3a-HSD type 3. Applicants have found that inhibitors of 3a-HSD type 3 have better selectivity when provided with D-ring substituents such as those described herein in preparation 16 or 17 of a spheroidal core. By "selectivity" it is meant that these preferred D-ring substituents especially those exposed at position 17, tend to suppress undesirable interactions between the inhibitors of the invention and for example enzymes whose inhibition is not desired by receptors whose activation is not desired . Some of the tested parameters (both desirable and undesirable) are exposed in the tables of the present with an "apostrophe" (') after their table number. (See also the detailed explanations after the tables). As can be seen from these tables, the preterred compounds of the invention effectively inhibit the activity of 3a-HSD type 3 while substantially avoiding the numerous undesirable activities for which the applicants tested the same compounds. For example, suitable D-ring substituents tend to reduce undesirable androgenic and estrogenic activities. It has been found that 17-spiro-lactone-17a-benzyl substituents give a good selectivity for 3a-HSD type 3. Not all 3a-HSD type 3 compounds discussed herein are claimed, since some of the compounds they also have good activity against 17β-HSD type 5 and are claimed in a separate patent application by applicants addressed to this separate activity.

"In vitro" inhibition of the transformation of 4-androstenedione (4-dione) to testosterone (T) po, r 3a-HSD type 3. Inhibition of 3a-HSD type was preliminarily determined using the inhibition of DHT transformation to androstane-3a, 17β-diol as described in "II-Enzymatic assay for 17β-HSD types 1, 2 3 and 5 and 3a-HSD types 1 and 3" and reported in column 2 of Tables 1 and 2. To complete these data, Tables 3 and 4 report the inhibition of the transformation of 4-androstenedione (4-dione) to testosterone (T) by 3a-HSD type 3 by some preferred inhibitors.

Enzymatic assay of the transformation of 4-androstenedione (4-dione) to testosterone (T) by 3a-HSD type 3.

Source of enzymes: 3a-HSD type 3, purified human expressed in E. col i. The coding region of the human 3a-HSD type 3 was amplified by PCR and inserted into a pGEX-1ΔT vector (Amersham Pharmacia Biotech, Inc., Quebec, Canada) in order to produce a fusion protein with glutathione. S-transferase. The expression of 3a-HSD type 3 in E. col i, purification of the protein in the affinity column of glutathione-Sepharose (Amersham Pharmacia Biotech), and cleavage of the fusion protein by thrombin were performed as described by the manufacturer.

Incubation: The purified enzyme was incubated to a final volume of 1 ml of 50 mM sodium phosphate buffer (pH 7.5), 20% glycerol, lmM EDTA and 0.1 μM of steroid labeled with [C14] and 1 mM of NADPH. After 2 hours of incubation, the steroids were extracted twice with 1 ml of ether. The organic phases were mixed and evaporated to dryness. The steroids were solubilized in 50 μl of dichloromethane, applied to 60 silica gel TLC plates (Merck, Darmstadt, Germany), before separation by migration in a solvent system of toluene-acetone (4: 1). The substrates and metabolites were identified by comparison with reference steroids and revealed by autoradiography and quantified using the Phosphoimager system (Molecular Dynamics, Sunnyval, CA).

Table 3 See the structures in Table 1 Table 4 Of the compounds in the following tables, the most preferred and their molecular structures are discussed below: EXAMPLES PE SYNTHESIS OF PREFERRED INHIBITORS The IR spectra in the present were taken in a Perkin-Elmer 1600 Series FT-IR spectrophotometer. The proton NMR spectra were recorded on a Brucker AC-F 300 instrument. The following abbreviations have been used: s, singlet, d, doublet, dd, doublet of doublet, t, triplet, q, quadruplet, and m, multiplet. The chemical changes (d) were referenced to chloroform (7.26 ppm for H1 and 77.00 ppm for C13) and were expressed in ppm. The optical rotations were measured at room temperature in a Jasco DIP 360 polarimeter. The mass spectra (MS) were obtained in a V.G. Micromass 16F. Thin layer chromatography (TLC) was performed on 0.25 mm Kieselgel 60F254 plates (E. Merck, Darmstadt, FRG). For the rapid chromatography, Merck-Kieselgel 60 (230-400 A.S.T.M. mesh) was used. Unless otherwise noted, the starting material and the reagent were obtained commercially and used as such or purified by a normal medium. All purified and dried reactive solvents were dried under argon. The anhydrous reactions were carried out under an inert atmosphere, the arrangement was assembled and cooled under argon. The organic solutions were dried over magnesium sulfate, evaporated on a rotary evaporator and under reduced pressure. Start materials and reagents were available from Aldrich Chemical Company, Inc. (Milwaukee, Wisconsin).

LIST OF ABBREVIATIONS DHP 3, 4-dihydro-2H-pyran EDTA Ethylenediaminetetraacetic acid HPLC High pressure liquid chromatography PTSA p-Toluenesulfonic acid THF Tetrahydrofuran THP Tetrahydropyranyl TMS Tetramethylsilyl Example 1 Synthesis of 2-nitro-1,3,5,5 (10) -estratrien-17-spiro-d-lactone derivative This synthesis is described in Scheme 1.

Scheme 1 c BM-1131 R, ».H, R2-Me (cpyerod.EM-U26) EM-1125 R] - R2- Me a. N? N? 2, HN03, AcOH b. TBDMSCI, miduol c d. H2, Pd CßCOj e. 5% HCl, MeOH f.Reictlvo oJoM-i. U3A, Mel Example IA 3-hydroxy-2-nitro-1,3,5 (10-estrathien-17-one (2a).) The title compound was prepared as described by Stubenrauch and Knuppen The procedure is described below. estrone (1.18.004 g, 66.6 mmol) in boiling acetic acid (540 ml) and allowed to cool to 50 ° C. The nitration mixture was prepared from 70% nitric acid (4.5 ml, 70 mmol), water (10 ml) and a little glass of sodium nitrite was heated to 50 ° C and added dropwise to the estrone solution with stirring.After stirring overnight at room temperature, the yellow precipitate was filtered This was obtained by suction and recrystallized from 92% aqueous acetic acid, thereby obtaining the 4-nitro derivative (6,800 g, 32%) as a pale yellow solid, IR (v) 3227 (OH), 2931, 2864, 1723 (C = 0), 1626, 1584, 1523, 1458, 1404, 1374, 1295, 1264, 1245, 1211, 1169, 1085, 1062, 1027, 954, 930, 908, 881, 823, 796, 719 , 654, 588, 556, 530 , 494 cm "1; XH NMR (pyridine-d5) d 0.85 (3H, S, 18'-CH3), 2.85 (2H, D, 6'-CH2), 5.00 (1H, s, OH), 7.11 (1H , d, J = 8.7 Hz, 2'-CH), 7.26 (1H, d, J = 8.7 Hz, l'-CH); 13 C NMR (pyridine-d 5) d 13.8 (C-18), 21.6 (C-15), 24.4 (C-11), 25.7 (C-7), 26.2 (C-12), 32.0 (C-6), 35.9 (C-16), 37.7 (C-8), 44.0 (C-14), 47.9 (C-13), 50.1 (C-9), 115.4 (C-2), 128.4 (Cl), 129.0 (C -10), 131.8 (C-5), 148.4 (C-3), 219.2 (C-17). The filtrate from the above reaction was evaporated under reduced pressure and the residue was recrystallized from EtOH / H20 8.5: 1.5. A brown solid (7.854 g) was obtained which was further purified by flash chromatography on Si02 column (EtOAc / hexanes, gradient 8-20%) to give the pure compound 2a (6.284 g, 30%) as a yellow solid. IR (n): 3300 (OH), 2933, 2864, 1737 (C = 0), 1630, 1562, 1522, 1480, 1431, 1372, 1311, 1252, 1216, 1146, 1084, 1054, 1035, 1008, 905 , 832, 762, 722, 662, 600, 520 cm "1. XH NMR (pyridine-d5) d 0.85 (3H, s, 18'-CH3), 2.76 (2h, d, 6'-CH2), 4.99 ( 1H, s, OH), 6.98 (1H, s, 4'CH), 7.96 (1H, s, 1-CH), 13C NMR (pyridine-d5) d 13.8 (C-18), 21.7 (C-15 ), 25.8 (C-11), 26.1 (C-1), 29.6 (C-12), 31.9 (C-6), 35.9 (C-16), 37.8 (C-8), 43.5 (C-14) , 47.9 (C-13), 50.3 (C-9), 119.8 (C-4), 122.2 (C-1), 132.8 (C-10), 147.8 (C-2), 152.6 (C-3), 219.1 (C-17).

Example IB 3- (tert-butyldimethylsilyloxy) -2-nitro-l, 3,5 (10) -estratrien-17-one (2b). A solution of 2-nitro-estrone (2a, 1118 g, 3.55 mmol), imidazole (0.670 g, 9.84 mmol) and TBDMSCI (0.781 g, 5.18 mmol) in dry DMF (50 mL) was stirred under Ar (g) for the night. The mixture was then poured into ice / water (80 ml). The white precipitate was filtered, washed with water and then dried in vacuo to give (2b) as a yellowish powder (1447 g, 95%). [a] 25D + 123.9 ° (c 1.03, CHC13); IR (NaCl) 2933, 2860, 1736, (S, C = 0), 1617, 1561, 1518, 1492, 1408, 1351, 1291, 1256, 1054, 909, 832, 790, 697 cm "1; 2H NMR 0.24 (6H, s, Si (CH3) 2), 0.92 (3H, s, I8-CH3), 1.01 (9H, s, SiC (CH3) 3), 1.40-1.78 (6H, m), 1.90-2.35 ( 5H, m), 2.37-2.60 (2H, m), 2.90 (2H, m, 6-CH2), 6.67 (1H, s, 4-CH), 7.76 (1H, s, 1-CH); 220.2, 147.2, 143.8, 139.6, 133.3, 122.6, 122.1, 50.3, 47.9, 43.6, 37.8, 35.8, 31.3, 29.4, 26.1, 25.7, 25.6, 21.5, 18.2, 13.8, -4.4.

Example IC 3- (tert-butyldimethylsilyloxy) -17β-hydroxy-2-nitro-17a- (4 '(2"-tetrahydro-2" H-pyranyloxy) -butynyl) -1, 3, 5 (10) - estratrieno (3). To a stirred solution of tetrahydro-2- (butynyloxy) -2H-pyran (1.71 mL, 10.91 mmol) in dry THF (75 mL) under Ar (g) at -35 ° C was added dropwise (MeLi 1.4 M in ether, 7.80 ml, 10.92 mmol). The solution was stirred for 45 minutes, after which a solution of ketone 2b (1294 g, 3.01 mmol) in dry THF (20 ml) was added at -35 ° C. After 75 minutes, ice (20 g) and saturated aqueous NaHCO3 (70 ml) were added to the reaction mixture and the aqueous phase was extracted with EtOAc. The combined organic layers were washed with brine, dried with magnesium sulfate, filtered and then concentrated in vacuo. The crude yellow oil was purified on Si02 (40 g, 2: 8 EtOAc / hexanes) to give compound 3 as a yellow foam (1.617 g, 92%). [a] D25-57.5 ° (c 0.72, CHC13); IR (NaCl) 3423 (broad, OH), 2936, 2870, 2366, 1654, 1630, 1578, 1560, 1527, 1481, 1458, 1438, 1313, 1268, 1121, 1080, 1032, 899, 869, 761, 669 cm "1; 1H NMR 0.23 (6H, s, Si (CH3) 2), 0.87 (3H, s, I8-CH3), 1.00 (9H, s, SiC (CH3) 3) r 1.20-2.35 (20H, m), 2.55 (2H, t, J = 6.9 Hz, CCCH2), 2.84 (2H, m, 6-CH2), 3.55 (2H, m, CH20 of chain), 3.85 (2H, m, CH20 of THP), 4.65 (1H, m, THP CH), 6.65 (1H, s, 4-CH), 7.76 (1H, s, 1-CH); 13C NMR 147.0, 144.2, 139.5, 133.9, 122.6, 122.0, 98.8, 84.5, 83.4, 79.8, 65.8, 62.2, 49.4, 47.1, 43.2, 38.9, 32.6, 30.6, 29.6, 26.7, 26.2, 25.6, 25.4, 22.8, 20.4, 19.4, 18.2, 12.7, -4.4.

Example ID 3- (tert-butyldimethylsilyloxy) -17β-hydroxy-2-nitro-17a- (4 '- (2"-tetrahydro-2'? - pyrynyloxy) -butyl) - 1, 3, 5 (10) - estratrieno (4). A solution of compound 3 (2.00 g, 3.42 mmol) and 5% Pd / CaCO3 (400 mg) in dry MeOH (400 ml) was stirred under H2 (g) atmosphere (balloon) for 1 hour. The mixture was then filtered through celite and the filtrate was evaporated on the rotary evaporator. The residue was purified on silica gel (EtOAc / hexanes 2: 8) to give compound 4 as a foamy solid (1483g, 74%). [a] D25 + 31.3 ° (c 0.90, CHC13); IR (NaCl) 3458 (broad, OH), 2935, 2860, 1616, 1563, 1518 (N02), 1491, 1408, 1348 (N02), 1291, 1256, 1119, 1070, 1023, 925, 893, 832, 784 , 672 cm "1; 1H NMR 0.23 (6H, s, Si (CH3) 2), 0.91 (3H, s, I8-CH3), 1.00 (9H, s, SiC (CH3) 3), 1.20-2.38 ( 26H, m), 2.85 (2H, m, 6-CH2), 3.48 (2H, m, CH20 of chain), 3.83 (2H, m, CH20 of THP), 4.59 (1H, m, CH of THP), 6.65 (1H, s, 4-CH), 7.75 (1H, S, 1-CH); 13C NMR 146.9, 144.1, 139.4, 134.0, 122.4, 121.9, 98.9, 83.2, 67.6, 67.6, 62.4, 62.4, 49.3, 46.6, 36.3, 34.1, 31.3, 30.7, 30.3, 29.5, 26.9, 26.0 ', 25.5, 25.4, 23.3, 20.4, 19.6, 18.1, 14.3, -4.4.

Example SE 17a- (4 'hydroxybutyl) -3, 17β-dihydroxy-2-nitro-1,3,5 (10) -estratriene (5). A solution of 4 (300 mg, 0.510 mmol) in 5% HCl in MeOH (10 mL) was stirred at room temperature and under an argon atmosphere for 12 hours. The reaction mixture was then poured into NaHC03 / ice and MeOH was evaporated under reduced pressure. The aqueous phase was extracted with EtOAc and the organic layers, then washed with brine, dried over MgSO4, filtered and evaporated to dryness. This gave a crude yellow foam (198 mg, 100%). Purification by flash chromatography (column loaded with CH2C12 and then boiled with EtOAc / CH2Cl2 2: 8, 4: 6, 1: -1, 6: 4, 9: 1) gave a compound 82 as a yellow solid (127.0 mg, 64%) Rf 0.21 ((EtOAc / Hexanes 8: 2); mp 184-6 ° C; [a] 26D + 58.8 ° (c.1.00, CHC1); IR (v) 3335, 2934, 2865, 1735, 1719 , 1654, 1630, 1576, 1522, 1479, 1434, 1373, 1305, 1266, 1169, 1112, 1067, 1033, 1000, 896, 874, 762, 659 cm "1; XH NMR 0.90 (3H, s), 3.69 (2H, d, J = 5.7 Hz), 6.84 (1H, s), 7.98 (1H, s), 10.42 (1H, s), 13C NMR 14.3, 19.8, 23.3, 26.1, 26.8, 29.8, 31.2, 33.3, 34.3, 36.1, 39.0, 43.2, 46.5, 49.4, 61.7, 62.8, 83.4, 118.8, 121.4, 131.6, 133.7, 149.2, 152.8.

Example 1F 2-Nitro-l, 3,5 (10) -estraetriene-3-ol-17 (R) -es? Iro-2 '- (6'-oxo) tetrahydropyran (EM 1124). To a stirred solution of compound 5 (128 mg, 0.33 mmol) in dry acetone (25 ml) was added slowly a first equivalent of 1.1 of Jones's reagent (1.25 M, 0.29 ml, 0.80 mmol). The orange solution was then stirred for 0.5 hours, then a second equivalent was added. The dark solution was stirred for an additional 0.5 hours and then quenched with isopropanol (precipitating green formed). The mixture was stirred for 10 minutes, then filtered through celite and the filtrate was rotary evaporated. The residue was taken up in EtOAc then washed with NaHCO3, aqueous, saturated, H20, brine, dried (MgSO4), filtered and rotoevaporated. The crude solid was purified by flash chromatography on Si02 (3: 7 EtOAc / hexanes) to give EM-1124 (108 mg, 85%) as a yellow solid. p.f. 213 ° C; [a] 25D + 90.0 * (c 0.70, CHC13); IR (NaCl) 3198, 2934, 2876, 2245, 1720 (s, C = 0, lactone), 1630, 1577, 1522, 1480, 1434, 1378, 1314, 1267, 1234, 1199, 1169, 1151, 1120, 1070 , 1036, 1024, 992, 914, 851, 759, 732, 662, 585 cm "1; XY NMR \ d 1.02 (3H, s, 18-CH3), 1.20-2.23 (16H, m), 2.25-2.65 ( 3H,), 2.90 (2H, m, 6-CH2), 6.85 (1H, s, 4-CH), 7.97 (1H, s, 1-CH), 10.41 (1H, s, OH phenol); 171.9, 152.8, 148.9, 133.3, 131.7, 121.5, 118.9, 93.0, 48.8, 47.1, 43.1, 38.4, 33.9, 31.6, 29.7, 29.4, 27.9, 26 23.4, 15 14.2 Example IG 2-Nitro-1, 3,5 (10) -estratrien-3-ol-17 (R) -spiro-2 '- (5'-methyl-6' -oxo) tetrahydropyran (EM-1126, EM- 1131).

LDA was prepared as follows: to a stirred solution of diisopropylamine (92 μl., 71 mg, 0.70 mmol) in dry THF (5 ml) at -78 ° C under Ar (g) was added n-BuLi (1.2M / hexane, 580 μl, 0.68 mmol) and the solution then at 0 ° C. for 25 minutes, then cooled to -78 ° C. A solution of EM-1124 (66 mg, 0.17 mmol) in dry THF (5 ml) was added and the resulting dark orange solution was then stirred for 30 minutes. Dry HMPA (2 ml) was added and after 15 minutes, Mel (107 μl, 243 mg, 1.71 mmol). The solution was then stirred for an additional 4 hours. The reaction was quenched with saturated, aqueous NH 4 Cl and extracted with EtOAc. The organic phase was washed with aqueous CuS04 (4X), 1M, H20, aqueous 1M Na2SO3, brine, dried (MgSO4), filtered then rotary evaporated to give a crude solid (103 mg). Purification by rapid chromatography on Si02 (EtOAc / hexanes 1: 9 → 2: 8) provided first EM-1126 (11 mg, 16%) followed closely by EM-1131 (34 mg, 34%) both as yellow solids. EM-1126: p.f. 204-6 ° C; [a] 25D + 73.4 ° (c 1.67, CDC13); IR v 3422 (broad, OH), 2937, 2874, 1725 (vs, CO), 1630, 1577, 1525, 1479, 1458, 1432, 1378, 1311, 1269, 1249, 1205, 1188, 1150, 1118, 1088, 1071, 1007, 990, 934, 896, 760, 731, 668, 585, 495 cm "1; 1 NMR 1.03 (3H, s), 1.30 (3H, d, J = 7.1 Hz), 1.31-1.77 (10H , m), 1.89-2.03 (5H, m), 2.15 (1H, td, J = 7.1 Hz, J '= 5.0 Hz), 2.30-2.50 (2H, m), 2.90 (2H, dd, J = 8.3 Hz , J '= 4.9 Hz), 6.85 (1H, s), 7.98 (1H, s), 10.43 (1H, s, OH); C13 NMR 174.8, 152.9, 149.0, 133.4, 131.7, 121.5, 118.9, 93.4, 48.7, 47.1, 43.1, 38.5, 36.2, 34.6, 31.6, 29.7, 28.6, 26.9, 25.9, 25.2, 23.4, 17.4, 14.4.

EM-1131 (5'-epimer of EM-1126, actual configuration not determined: mp 206-8 ° C; [a] 25D + 62.6 ° (c 0.68, CDC13); IR v 3422 (broad, OH), 3192 , 2934, 2876, 2858, 2824, 1721 (vs, CO), 1631, 1578, 1522, 1482, 1458, 1436, 1377, 1314, 1271, 1237, 1204, 1173, 1120, 1103, 1082, 1051, 1019, 1002, 933, 901, 877, 860, 759, 663, 638, 600, 495 cm "1; NMR XH d 1.01 (3H, s), 1.24 (3H, d, J = 7.0 Hz), 1.31-1.80 (10H , m), 1.89-2.20 (6H,), 2.35 (1H, broad s), 2.55 (1H, sextuple, J = 7.5 Hz), 2.89 (2H, t, J = 5.2 Hz), 6.84 (1H, s) , 7.96 (1H, s), 10.41 (H, s, OH); 13C NMR 175.8, 152.8, 148.9, 133.3, 131.6, 121.4, 118.8, 92.5, 77.4, 77.0, 76.6, 48.5, 47.0, 43.0, 38.4, 33.8, 33.4, 31.6, 29.7, 27.16, 26.7, 25.8, 24.3, 23.6, 17.2, 14.3.

Example 1H 2-Nitro-l, 3,5 (10) -estratrien-3-ol-17 (R) -spiro-2 '- (5', 5'-dimethyl-6'-oxo) tetrahydropyran (EM-1125 ). LDA was prepared as follows: to a stirred solution of diisopropylamine (206 μl, 159 mg, 1.57 mmol) in dry THF (12 mml) -7! in the presence of Ar (g) n-BuLi (1.2 M / hexane, 1.28 ml, 1.53 mmol) was added and the solution was then stirred at 0 ° C for 20 minutes, then cooled to -78 ° C. solution of a mixture of EM-1126 and EM-1131 (153 mg, 0.38 mmol) in dry THF (10 ml) and the resulting dark orange solution was stirred for 20 minutes, dry HMPA (4.7 ml) was added and after 15 minutes, Mel (238 μl, 544 mg, 3.83 mmol) The solution was then stirred for 5 minutes, then warmed to -30 ° C and stirred for an additional 1 h.The reaction was quenched with saturated NH C1, and The organic phase was washed with brine (6X), aqueous 1M Na2SO3, dried (MgSO4), filtered and then rotary evaporated to give a crude liquid Purification by rapid chromatography on SiO2 ( EtOAc / hexanes 1: 9 → 2: 8) gave EM 1125 (82 mg, 52%) as a yellow solid, mp 195-7 ° C, [a] 25 D + 72.8 ° (c 1.61, CDC13), IR v 3421 (broad, OH), 3194, 2954, 2927, 2873, 1718 (vs, CO), 1631, 1578, 1523, 1476, 1458, 1438, 1386, 1312, 1298, 1271, 1204, 1151, 1118, 1059, 1032, 1016, 931, 989 , 872, 855, 758, 663, 595 cm "1; C13 NMR 1.02 (3H, s), 1.28 (6H, s), 1.32-1.77 (10H, m), 1.85-2.15 (6H, m), 2.36 (1H, broad s), 2.89 (2H, dd, J = 8.2 Hz, J '= 4.9 Hz), 6.85 (1H, s), 7.97 (1H, s), 10.42 (H, s, OH); NMR 13C d 177.7, 152.8, 149.0, 133.3, 131.7, 121.5, 118.9, 93.4, 48.6, 47.1, 43.1, 38.5, 37.8, 34.7, 31.6, 31.5, 29.7, 27.7 (4), 27.6 (8), 26.7, 25.9, 25.5, 23.3, 14.4 Example 2 Synthesis of 2-cyano-l, 3, 5 (10) -estratrien-30'-17 (R) -spiro-2 '- (5' 5 '-dimethyl-6'-oxo) tetrahydropyran (13) Scheme 2 3-t-butyldimethylsilyloxy-1,3, 5- (10) -estratrien-17-one (7) The ether was prepared from estrone (6) following the method described by Pelletier et al., (Steroids 59: 536-547, 1994). 3-t-butyldimethylsilyloxy-17β-hydroxy-17a-. { 4 '- (2' '-te rahydro-2' 'H-pyranyl) butin-1' -il} -l, 3.5 (10) -estratriene (8). To a solution of HC = C (CH2) 20THP (18.3 ml, 117 mmol) in dry THF (600 ml) at 0 ° C, n-butyllithium (43.7 ml, 109 mmol) was added dropwise and the mixture was added dropwise. it was stirred for 90 minutes. The mixture was cooled to -78 ° C and a solution of TBDMS-estrone 7 (15 g, 39 mmol) in THF (500 ml) was added dropwise. Then, the reaction mixture was allowed to come to room temperature and was allowed to stir for a period of 15 hours. The solvents were evaporated at half volume and 200 ml of water were added. The mixture was extracted with EtOAc (3 x 200 ml), the organic layer was washed with brine, dried (MgSO 4) and evaporated to dryness. The residue was purified on silica gel column chromatography with hexanes / EtOAc (9/1) as an eluent to give 15.1 g (72%) of the product; IR (NaCl cm "1) 3432, 2934, 2858, 1607, 1495, 1287, 1256, 1033, 958, 839; XH NMR (300 MHz, CDC13) d 7.12 (d, 1H, J = 8.4 Hz), 6.62 ( dd, 1H, J = 2.4, 8.4 Hz), 6.54 (d, 1H, J = 2.2 Hz), 4.66 (br, s., 1H), 3.89-3.79 (m, 2H), 3.56-3.50 (m, 2H ), 2.79 (br.

S., 2H), 2.56 (t, 2H, J = 7. O Hz), 2.35-2.17 (m, 3H), 2.07-1.23 (m, 17H), 0.98 (s, 9H), 0.87 (s, 3H , 18-Me), 0.19 (s, 6H); C13 NMR (75 MHz, CDC13) d 153.3, 137.8, 133.0, 126.1, 119.9, 117.1, 98.7, 84.7, 83.2, 80. 0, 65.8, 62.1, 49.5, 47.2, 43.7, 39.4, 39.0, 32.9, 30.6, 29.7, 27.3, 26.4, 25.7, 25.4, 25.4 18. 1, 12.8, -4.4. 3-t-butyldimethylsilyloxy 17β-hydroxy-17a-. { 4 '- (2"-tetrahydro-2'? -pyranyl) butan-1 '-il} -1, 3, 5 (10) -estratriene (9). 5% palladium on activated carbon (1.5 g, 10% by weight) was added to a solution of alkyne 8 (15.1 g, 28 mmol) in EtOAc (500 ml) at room temperature. The flask was purged with H2 three times (vacuum, followed by H2) and allowed to stir under an atmosphere of H2 pressure. The reaction was followed by TLC. After a period of 3 hours, the mixture was filtered with a plug of celite and the solvent was removed under reduced pressure. The crude product was used in the next step without further purification; IR (NaCl, cm "1) 3474, 2935, 2858, 1607, 1570, 1496, 1471, 1286, 1257, 1156, 1137, 1119, 1033, 954, 839, 780; XH NMR (300 MHz, CDC13) d 7.12 (d, 1H, J = 8.4 Hz), 6.62 (dd, 1H, J = 2.1, 8.4 Hz), 6.55 (s, 1H), 4.59 (br, s., 1H), 3.92-3.73 (m, 2H) , 3.55-3.38 (m, 2H), 2.82-2.77 (m, 2H), 2.30-1.33 (m, 26H), 0.97 (s, 9H), 0.90 (s, 3H, 18-Me), 0.18 (s, 6H), C13 NMR (75 MHz, CDC13) d 153.27, 137.81, 133.08, 126.02, 119.87, 117.06, (98.90, 98.84), 83.38, 67.61, 62.33, 49.50, 46.67, 43. 81, 39.58, 36.35, 34.28, 31.60, 30.75, 30.36, 29. 62, 27.51, 26.26, 25.67, 25.47, 23.37, 20.45, 19.66 18.12, 14.35, -4.43. 3-t-Butyldimethylsilyloxy-17β-hydroxy-17a- (4'-hydroxybutan-1'-yl) -1,3,5 (10) -estratriene (10). To a solution of THP-ether 9 (15.1 g, 28 mmol) in MeOH (400 mL), p-toluenesulfonic acid monohydrate (150 mg, 0.8 mmol) was added and the reaction was stirred for a period of 5 hours. A saturated solution of NaHCO3 (100 ml) was added and the volume of the solvent was halved in a rotary evaporator. The mixture was extracted with CH2C12, the organic phase was washed with brine, dried (MgSO4) and evaporated to dryness. The crude product was used in the next step without purification; IR (NaCl, cm "1) 3356, 2931, 2858, 1608, 1496, 1471, 1286, 1256, 954, 839, 780; 1N NMR (300 MHz, CDC13) d 7.12 (d, 1H, j = 8.5 Hz) , 6.61 (dd, 1H, J = 2.5, 8.5 Hz), 6.55 (s, 1H), 3.69 (br, d, 2H, J = 5.2 Hz), 2.82-2.78 (m, 2H), 2.35-2.26 (m , 1H), 2.20-1.94 (m, 2H), 1.90-1.81 (m, 1H), 1.62-1.22 (m, 17H), 0.98 (s, 9H), 0.90 (s, 3H, 18-Me), 0.19 (s, 6H); C13 NMR (75 MHz, CDC13) d 153.27, 137.81, 133.05, 126.02, 119.89, 117.09, 83.59, 62.56, 49.50, 46.69, 48.81, 39.58, 35.98, 34.32, 33.20, 31.61, 29.62, 27.51, 26.26, 25.68, 23.37, 19.74, 18.14, 14.36, -4.40. 3-t-butyldimethylsiloxy-l, 3,5 (10) -estratrien-17 (R) -spiro-2 '- (6'-oxo) -tetrahydropyran (11). To a solution of diol 10 (12.5 g, 27 mmol) in acetone (500 ml) at 0 ° C, a 2.7 M solution of Jone's reagent (15.1 ml, 41 mmol) was added dropwise. The reaction was stirred for 30 minutes. 2-Propanol (100 ml) was added followed by a saturated solution of NaHCO 3 (200 ml). The volume of the solvents was reduced by half by evaporation and the mixture was extracted with EtOAc. The organic phase was washed with brine, dried (MgSO 4) and concentrated under reduced pressure. The residue was purified on silica gel column chromatography with hexanes / acetone (6/1) to give 8.6 g of lactone (68% yield by 3 steps); IR (NaCl, cm "1): 2960, 2930, 2857, 1732, 1607, 1496, 1284, 1264, 1244, 1037, 958, 840; H-NMR (300 MHz, CDC13) d 7.11 (d, 1H, j = 8.4 Hz), 6.61 (dd, 1H, J = 2.3, 8.4 Hz), 6.56 (s, 1H), 2.85-2.79 (m, 2H), 2.58-2.39 (m, 2H), 2.38-2.25 (m, 1H ), 2.21-2.10 (m, 1H), 2.03-1.27 (m, 15H), 1.02 (s, 3H, 18-Me), 0.97 (s, 9H), 0.18 (s, 6H); C13 NMR (75 MHz , CDC13) d 172.00 153.36, 137.63, 132.62, 126.02, 119.92, 117.19, 93.25, 48.88, 47.26, 43.68, 39.05, 33.98, 31.96, 29.50, 29.48, 27.94, 27.46, 25.98, 25.67, 23.48, 18.12, 15.87, 14.30 -4.43. 3-t-butyldimethylsilyloxy-l, 3,5 (10) -estratrien-17 (R) -spiro-2 '(5' -5 '-dimethyl-6'-oxo) tetrahydropyran (12).

In a dry flask of 11 in the presence of argon, lactone 11 (8.6 g, 19 mmmol) was dissolved in dry THF (300 ml) and cooled to 0 ° C. A 1M solution of LiHMDS (47.3 ml, 47.3 mmol) was added dropwise. The mixture was stirred 15 minutes at 0 ° C and cooled to -78 ° C and then methyl iodide (5.9 ml, 79 pmol) was added. The reaction was stirred for 1 hour at this temperature and then allowed to warm to room temperature over a period of 2 hours. A saturated solution of NH 4 Cl (200 ml) was added and the mixture was extracted with EtOAc. The organic layer was washed with a saturated solution of Na2S203 brine, dried (MgSO4) and concentrated under reduced pressure. The residue was purified by column chromatography with hexanes / acetone (5/1) as an eluent to give 7.4 g (81%) of the dimethyl compound; IR (NaCl, cm "1) 2954, 2930, 2858, 1725, 1496, 1287, 1258, 1150, 1137, 956, 840; 1R-NMR (300 MHz, CDC13) d 7.11 (d, 1H, J = 8.5 Hz) , 6.62 (dd, 1H, J = 2.4, 8.5 Hz), 6.55"(d, 1H, J = 2.1 Hz), 2.81-2.78 (m, 2H), 2.36-2.28 (m, 1H), 2.20-1.38 ( m, 16H), 1.28 (s, 3H), 1.27 (s, 3H), 1.02 (s, 3H, 18-Me), 0.97 (s, 9H), 0.lL 6H), C NMR I3J (75 MHz, CDC13) d 177.79, 153.33, 137.62, 132.62, 125.99, 119.90, 117.14, 93.66, 48.67, 47.24, 43.65, 39.06, 37.74, 34.79, 31.96, 31.56, 29.50, 27.73, 27.61, 27.42, 26.01, 25.65, 25.55, 23.26 , 18.11, 14.42, -4.43. 1,3,5 (10) -estratrien-3-ol-17 (R) -spiro-2 '- (5', 5 '-di? Aethyl-6' -oxo) -tetrahydropyran (13): To one solution of silyl ether 12 (7.1 g, 14.7 mmol) in THF (300 ml) at 0 ° C, a 1M solution of TBAF (17.6 ml, 17.6 mmol) was added dropwise and the reaction was stirred for 15 minutes. Water with ice (200 ml) was added to precipitate the compound. The flask was placed in a rotary evaporator to reduce the volume of THF, and then placed in a filler bath. The filtrate was collected by filtration, washed with cold water and dried in an oven (30 ° C) for a period of 24 hours to give 5.4 g (100%) of the compound 3-OH: IR (NaCl, cm "1 ): 3357, 2932, 2871, 1695, 1287, 1158; XH NMR (300 MHz, CDC13) d 7.14 (d, 1H, J = 8.4 Hz), 6.63 (dd, 1H, J = 2.6, 8.4 Hz), 6.55 (d, 1H, J = 2.6 Hz), 4.62 (br.s, 1H, OH), 2.81-2.79 (m, 2H), 2.38-2.39 (m, 1H), 2.20-1.81 (m, 5H), 1.76 -1.31 (m, 11H), 1.29 (s, 3H), 1.28 (s, 3H), 1.01 (s, 3H, 18-Me); C13 NMR (75 MHz, CDC13) d 178.06, 153.52, 138.08, 132.19, 126.42, 115.26, 112.74, 93.80, 48.69, 47.29, 43.65, 39.14, 37.81, 34.84, 31.98, 31.61, 29.53, 27.76, 27.64, 27.39, 26.12, 25.59, 23.29, 14.43.

Example 3 Synthesis of EM-01667 Scheme 3 EM-01667 a. PhSeCI, CHCI3 b. NCS, CHCI3 3-Hydroxy-2-phenylselenenyl-estra-l, 3,5 (10) -triene-17 (R) -spiro-2 '(5', 5 'dimethyl-6' -oxo) tetrahydropyran (14) A solution of 3-hydroxy-estra-l, 3, 5 (10) -triene-17 (R) -spiro-2'- (5 ', 5'-dimethyl-6'-oxo) tetrahydropyran (406 mg, 1.10 mmol) (13) and phenylselenenyl chloride (253 mg, 1.32 mmol) in dry CHC13 (24 ml) under Ar (g) was stirred at 0 ° C for one hour, then at room temperature overnight. The resulting yellow solution was poured into ice / H20 and then extracted with CH2C12 (3x). The combined organic phase was dried (cotton plug) then rotary evaporated to give a crude foamy solid. Purification by flash chromatography (Si02) using EtOAc / hexane 1: 9 as eluent gave 7 (353 mg, 61%) with the 4-isomer (86 mg, 15%). Compound 7: [a] 25D + 77.7 ° (c 1.14, CHC13) IR v 3366, 3050, 2965, 2928, 2869, 1709, 1603, 1576, 1550, 1458, 1438, 1384, 1349, 1310, 1294, 1262 , 1202, 1157, 1141, 1114, 1065, 1017, 984, 892, 845, 736, 689, 665, 593, 555, 498, 460 cm "1; XH NMR (CDC13) (d) 1.02 (3H, s) , 1.27 (9) (s, 3H), 1.28 (4) (s, 3H), 1.27-1.80 (11H, s), 1.88-2.28 (6H, m), 2.87 (2H, t, J = 4.8 Hz) , 6.24 (1H, s, OH), 6.80 (1H, s), 7.21 (5H, broad s), 7.52 (1H, s), ppm; C13 NMR (CDC13) (d) 14.4, 23.3, 25.5, 26.1, 27.2, 27.6, 27.7, 29.5, 31.5, 31.8, 34.7, 37.7, 38.9, 43.4, 47.2, 48.6, 93.6, 116.6, 114.7, 126.5, 129.2 (6), 129.3 '(4), 131.2, 133.3, 134.7, 141.4 154.4, 177.8 ppm. 2-Chloro-3-hydroxy-estra-l, 3,5 (10) -triene-17 (R) -spiro-2 '- (5', 5 '-dimethyl-6' -oxo) tetrahydropyran (EM-01667 ). A solution of 14 (116 mg, 0.22 mmol) and N-chlorosuccinimide (44 mg, 0.33 mmol) in dry CHC13 (15 ml) under Ar (g) at 0 ° C was stirred for 30 minutes. The solution was poured into ice / H20 then extracted with CH2C12 (3x). The combined organic phase was dried (cotton plug) then rotary evaporated to give a crude solid. Purification by rapid chromatography (Si02) using EtOAc / toluene 1:29 = 1:19 as eluent gave EM-01667 (51 mg, 57%) as a white solid: Rf 0.28 EtOAc / hexane 3: 7); p.f. 241 ° C; [aJ25D + 62.6 ° (c 1.09 CDC13); IR (v) 3253 (broad, OH), 2936, 2873, 1685 (CO), 1608, 1501, 1458, 1418, 1388, 1314, 1300, 1263, 1223, 1160, 1016, 987, 884, 844, 737, 673.598 cm "1 XH NMR (CDC13) (d) 1.01 (3H, s), 1.28 (6H, s), 1.25-1.75 (11H, m), 1.85-2.28 (6H, m), 2.80 (2H, dd, J '= 8.7 Hz, J' '= 3.9 Hz), 5.33 (1H, broad s, PH), 6.73 (1H, s), 7.19 (1H, s) ppm; C13 NMR (CDC13) (d) 14.4 , 23.3, 25.6, 26.1, 27.2, 27.7, 27.8, 29.0, 31.6, 31.8, 34.8, 37.8, 38.8, 43.4, 47.2, 48.6, 93.6, 116.0, 117.1, 125.6, 133.5, 137.1, 149.0, 177.8 ppm.

Example 4 Synthesis of EM-01728 Scheme 4 to. PhCH.MgCI, THF b PhSßCI c. NCS 17-benzyl-l, 3,5- (10) estratriene-3, 17β-diol (15) To a solution of estrone (5.0 g, 18.5 mmol) in dry THF (200 ml) at 0 ° C under Ar (g) ), benzyl magnesium chloride (2M in THF, 65 ml) was added, and the solution was stirred overnight. The saturated, aqueous NH 4 Cl solution was added at 0 ° C and the solution was extracted with CH 2 Cl 12 (3 times), washed with brine, dried with MgSO 4, filtered and then evaporated. The product was purified by rapid chromatography (RP-C18, H20 / MeOH / CH3CN 30: 30: 40-10: 30: 60) to give 1 as a white solid (4.0 g, 60%) and estrone: 1.5 g, 30 % recovery). Compound 1: Rf: 0.25 (2.98, MeOH / CH2Cl2); IR (v) 3284, 2926, 2856, 1725, 1686, 1655, 1606, 1561, 1499, 1439, 1378, 1343, 1321, 1286, 1252, 1221, 1156, 1082, 1029, 1016, 930, 915, 888, 872, 820, 786, 732, 700, 646, 586 cm "1; 1H NMR (CD30D) (d) 0.94 (3H, s, H18), 1.32-2.35 (13H, m), 2.67 (1H, d, J = 13.5 Hz, CH-Ph), 2.78 (2H, m, H6), 2.87 (1H, d, J = 13.5 Hz, CH-Ph), 6.49 (1H, d, J = 2.4 Hz, H4), 6.56 ( 1H, dd, J = 8.3 Hz, J '= 2.5 Hz, H2), 7.10 (1H, d, J = 8.3 Hz, Hl), 7.18-7.30 (5H, m, Ph), ppm, C13 NMR (CD3OD) (d) 15.3, 24.1, 27.7, 28.8, 30.8, 32.4, 32.8, 41.4, 43.6, 45.2, 50.9, 84.5, 113.7, 116.1, 126.9, 127.2, 128.7, 132.3, 136.6, 138.8, 140.3, 155.9 ppm. 17-benzyl-2-phenylenyl-1, 3,5, (10) estratriene-3,17-diol (16) To a stirred solution of 1 (508 mg, 1.40 mmol) in MeOH / CHCl 3 (1:10, 33 ml) at 0 ° C PhSeCl (322 mg, 1.68 mmol) was added. After two hours, the orange solution had turned yellow and poured into ice / H20, extracted with CH2C12, dried (MgSO4), filtered and then evaporated. The crude solid was purified by rapid chromatography (Si02, EtOAc / toluene 1:29) to give 2 as a beige solid (456 mg, 63%) and the 4-phenylenyl isomer (71 mg, 10%). Compound 2: NMR 1.00 (3H, S, H18), 1.30-2.38 (14H, m), 2.82 (2H, dd, J = 78.1 Hz, J '= 13.2 Hz, CH2-Ph), 2.90-2.93 ( 2H, m, H6), 6.26 (1H, broad s, OH), 6.85 (1H, s, Hl), 7.21-7.38 (10H, m), 7.59 (1H, s, H4), C13 NMR (d) 14.5 , 23.4, 26.4, 27.4, 29.7, 31.3, 33.7, 39.4, 42.4, 43.4, 43.7, 46.7, 49.5, 82.9, 111.2, 114.7, 126.3, 126.5, 128.1, 129.2, 129.3, 131.0, 131.3, 133.7, 134.8, 138.2, 141.6 154.4 ppm. 17-benzyl-2-chloro-l, 3,5, (10) estratrien-3, 17β-diol (EM-01728) To a stirred solution of 2 (155 mg, 0.30 mmol) in CHC13 (15ml) at 0 ° C in the presence of Ar (g) was added N-chlorosuccinimide (48 mg, 0.36 mmol). After one hour, the reaction was treated as in 2 and purification by rapid chromatography (Si02, EtOAc / toluene 1:29? 1:19) to give EM-01728 as a white solid (74 mg, 62%). Rf 0.18 (EtOAc / toluene 1:19); p.f. 220 ° C; [a] 25D + 74.8 ° (c 0.96, acetone-d6); IR (v) 3544 (OH), 3284 (broad, OH), 3023, 2931, 2849, 1601, 1497, 1454, 1338, 1285, 1257, 1201, 1084, 1015, 979, 918, 885, 796, 755, 702, 675, 642, 560, 532, 504 cm "1; H NMR (CD3OD) (d) 0.95 (3H, s, H18), 1.33-1.75 (15H, m), 2.68 (1H, d, J = 15.5 Hz, CH-Ph), 2.76 (1H, dd, J = 8.3 Hz, J '= 3.6 Hz, H6), 2.88 (1H, d, J = 15.5 Hz, CH-Ph), 6.60 (1H, s, H4), 7.16 (1H, s, Hl), 7.17-7.31 ( 5H, m, Ph) ppm; C13 NMR (acetone-d6) (d) 15.2, 24.0, 27.3, 28.3, 29.9, 32.1, 33.5, 40.7, 43.5, 44.6, 48.0, 50.3, 83.6, 117.5, 118.3, 126.6, 127.5, 128.5, 132.2, 134.2, 137.8 , 140.6, 151.3 ppm.

Example 5 Synthesis of EM-01831 and EM-01832 Scheme 5 to. NaH, Mel b. TMSI c. PhSeCI d. NCS e. LÍAIH4 16, 16-dimethyl-3-methoxy-1, 3,5 (10) -estratrien-17-one (17) To a solution of 3-methoxy-estra-1, 3, 5 (10) triene-17-one (10.00 g, 35 mmol) in anhydrous THF (500 ml) in the presence of Ar (g) at room temperature was added NaH (60% in oil, 2.55 g, 105 mmol) and iodomethane (22 ml, 350 mmol). The solution was refluxed overnight. Then more NaH (2.55 g, 105 mmol) and iodomethane (22 ml, 350 mol) were added and the solution was refluxed for another 24 hours. The resulting solution was rapidly cooled with ethanol (100 ml) and then water on ice (300 ml). This solution was extracted using ethyl acetate (2 x 300 ml), washed with brine (2 x 300 ml) dried with MgSO 4 and evaporated under reduced pressure to give a yellow solid. Purification by rapid chromatography on silica gel using ethyl acetate / hexane (1:19) as eluent gave 3 (10.19 g, 93%) as a white solid. IR (v) 2933, 2869, 1731 (CO), 1609, 1502, 1467, 1382, 1315, 1258, 1240, 1153, 1037, 1020, 902, 850, 816, 781 cm "1; XH NMR (d) 0.94 (3H, s, H18), 1.09 (3H, s, 16-Me), 1.22 (3H, s, 16-Me), 1.40-2.42 (11H, m), 2.90 (2H, dd, J = 8.0 Hz, J '= 3.3 Hz, H6), 3.79 (3H, s, OMe), 6.65 (1H, d, J = 2.7 Hz, H4), 6.73 (1H, dd, J = 8.4 Hz, J' = 2.7 Hz, H2 ), 7.21 (1H, d, J = 8.5 Hz, Hl) ppm; C13 NMR (d) 14.4, 25.8, 26.0, 26.7, 27.3, 29.7, 32.3, 37.6, 37.9, 44.2, 45.3, 47.2, 49.0, 55.2, 111.5, 113.9, 126.3, 132.2, 137.7, 157.6, 225.1 ppm. 16, 16-dimethyl-l, 3,5 (10) -estratrien-3-ol-17-one (18) A solution of 3-methoxy-estra-1, 3, 5 (10) triene-17-one- 16-Dimethyl, 17 (6.00 g, 19 mmol) and iodotrimethylsilane (27 mL, 190 mmol) in anhydrous CH2C12 (1000 mL) in the presence of Ar (g) was refluxed overnight. The resulting solution was poured into ice / H20 (600 mL), extracted with CH2C12 (3 x 600 mL), dried MgSO4, filtered and then evaporated under reduced pressure. The crude brown solid was purified by rapid chromatography on silica gel using ethyl acetate / toluene (1: 9) as eluent to give 4 as a white solid (3.50g, 62%). IR (v) 3361 (br, s, OH), 3027, 2923, 2876, 1860 (), 1717 (CO, 1620, 1584, 1499, 1460, 1355, 1286, 1248, 1152, 1099, 1023, 909, 875 , 816, 787, 735, 647, 571, 516 cm "1 XR NMR (d) 0.91 (3H, s, H18), 1.06 (3H, s, 16-Me), 1.18 (3H, s, 16-Me) , 1.23-2.35 (11H, m), 2.81 (2H, t, J = 4.5 Hz, H6), 6.56 (1H, d, J = 2.3 Hz, H4), 6.62 (1H, dd, J = 8.5 Hz, J '= 2.6 Hz, H2), 7.09 (1H, d, J = 8.4 Hz, Hl) ppm; C13 NMR (d) 14.4, 25.7, 25.8, 26.6, 27.2, 27.1, 29.4, 32.2, 37.5, 37.0, 44.0, 45.3, 47.1, 49.1, 112.7, 115.2, 126.2, 131.3, 137.7, 154.2, 226.3 ppm. 16, 16-dimethyl-2-phenyl-selenyl-1, 3,5 (10) -estratrien-3-ol-17-one (19) To a solution of 18 (2.00 g, 6.70 mmol) in MeOH / CHCl3 (1:10, 550 ml) at 0 ° C PhSeCl was added (1.56 g, 8.15 mmol). The reaction mixture was stirred overnight at room temperature. The resulting yellow solution was then poured into ice / H20 (500 ml), extracted with CH2C12 (2 x 500 ml), dried with MgSO4, filtered and then evaporated under reduced pressure. The product was purified by flash chromatography on silica gel using ethyl acetate / toluene (0.3: 9.7) to give 5 as a white foam (2.44 g, 80%) and the 2-chloro isomer (100 mg, 5%). Compound 5: XH NMR (d) 0.94 (3H, s, H18), 1.08 (3H, s, 16-Me), 1.21 (3H, s, 16-Me), 1.24-2.36 '(11H, m), 2.92 (2H, dd, J = 8.4 Hz, J' = 3.6 Hz, H6) 6.21 (1H, s, OH), 6.82 (1H, s, H4), 7.17-7.23 (5H, m, Ph), 7.54 (1H, s, Hl) ppm. 2-chloro-16,16-dimethyl-l, 3,5 (10) -estrathien-3-ol-17-one (EM-01831) To a stirred solution of 5 (680 mg, 1.50 mmol) in anhydrous CHC13 ( 200 ml) at room temperature and in the presence of Ar (g) was added N-chlorosuccinimide (246 mg, 1.84 mmol). The mixture was then stirred at -30 ° C for one hour. The reaction was quenched with saturated aqueous NH C1 (300 mL), extracted with CH2C12 (2 x 300 mL), dried with Na2SO4, filtered and then evaporated under reduced pressure. The crude product was purified by flash chromatography using methanol / ethyl acetate / hexane (0.5: 1: 9) as the eluent to give EM-01831 as a yellow solid (178 mg, 33%). IR (v) 3321 (br, s, OH), 2922, 2853, 1724 (CO), 1606, 1502, 1468, 1414, 1380, 1340, 1260, 1215, 1019, 885, 830, 738, 673, 626 cm "1; XH NMR (d) 0.93 (3H, s, H18), 1.08 (3H, s, 16-Me), 1.21 (3H, s, 16-Me), 1.26-1.35 (11H, m), 2.84 (2H, dd, J = 9.0, J '= 4.3 Hz, H6), 5.33 (lH, s, OH), 6.75 (1H, s, H4), 7.20 (1H, s, Hl) ppm; C13 NMR (d) 14.4, 25.7, 26.0, 26.5, 27.3, 29.0, 32.2 37. 5, 37.6, 43.9, 45.3, 47.1, 49.0, 116.0, 117.2, 125.7, 133.4, 137.0, 149.1, 225.0 ppm. 2-Chloro-16,16-dimethyl-1,3,5 (10) -estratrien-3,17-diol (EM01832) To a stirred solution of EM-01831 (200 mg, 0.60 mmol) in anhydrous THF (20 ml ) at -78 ° C in the presence of Ar (g) was added LiAlH4 (33 mg, 0.86 mmol). The reaction temperature was slowly allowed to return to room temperature for 24 hours. The reaction was cooled to 0 ° C, more LiAlH4 (23 mg, 0.60 mmol) was added and the mixture was stirred for another 2 hours. The reaction mixture was quenched with 1M aqueous Rochelle salt (50 ml) then extracted with ethyl acetate (3 x 50 ml). The organic layer was washed with brine (50 ml), dried with MgSO 4, filtered and then evaporated under reduced pressure. The product was purified by flash chromatography on silica gel using ethyl acetate / toluene (1:19) as eluent to give EM-01832 as a white solid (145 mg, 72%). IR (v) 3557 (s, OH), 3388, (s, OH), 3186 (br, s, OH), 2921, 2861, 1602, 1576, 1486, 1454, 1430, 1409, 1382, 1344, 1256, 1207, 1128, 1069, 1029, 981, 880, 798, 733, 677, 584, 534 cm "1; 1 H NMR (d) 0.81 (3 H, s, H18), 1.01 (3H, s, 16-Me), 1.06 (3H, s, 16-Me), 1.24-2.20 (11H, m), 2.74 (2H, dd, J = 8.4 Hz, J '3.7 Hz), 3.23 (1H , s, H17), 6.59 (1H, s, H4), 7.13 (1H, s, Hl) ppm; NMR C13 (d) 11.5, 25.3, 26.2, 27.2, 29.1, 32.3, 37.7, 37.9, 39.0, 41.2, 43.8, 45.4, 46.8, 89.8, 115.9, 117.0, 125.7, 134.0, 137.3, 148.9 ppm.

Example 5 Derivatives 3-hydroxy of 2-cyano-l, 3, 5 (10) -estratrien-17-spiro- (dimethyl-d-lactone) Scheme 5 Example 5A 2-formyl-l, 3,5 (10) -estratrien-3-ol-17 (R) -spiro-2 '- (5', 5'-dimethyl-6'-oxo) tetrahydropyran (20). Lactone 13 (1.0 g, 2.72 mmol) was dissolved in dry 1,2-dichloroethane (9 ml) under an argon atmosphere. SnCl4 (0.16 ml, 1.37 mmol) and Bu3N (0.52 ml, 2.18 mmol) were successively added. The mixture was stirred at room temperature for 20 minutes. Formaldehyde (0.23 g, 7.84 mmol) was added and the mixture was stirred at reflux for 6 hours. The reaction mixture was poured into aqueous acid (pH = 2) and extracted with CH2C12. The organic layers were washed with brine solution, dried (Na 2 SO 4) filtered and concentrated in vacuo. The crude product was purified by rapid chromatography on silica gel, eluting with (95: 5 to 80:20) hexanes / acetone to yield 0.74 g (69%) of the product; IR (NaCl, cm "1): 3164, 2937, 2872, 1716, 1652, 1571, 1487, 1466, 1386, 1298, 1152, 1017, 914, 731; XH-NMR (CDC13) 1.00 (s, 3H), 1.26 (s, 6H), 1.23-2.40 (m, 17H), 2.80-2.90 (m, 2H), 6.66 (s, 1H), 7.39 (s, 1H), 9.79 (s, 1H), 10.77 (s, 1H) ), 13C NMR (CDC13) d 14.3, 23.2, 25.4, 25.9, 26.8, 27.6, 27.7, 30.0, 31.4, 31.6, 34.6, 37.7, 38.6, 42.9, 47.0, 48.5, 93.4, 116.9, 118.9, 130.3, 132.2, 147.8, 159.2, 177.7, 196.0.

Example 5B 2-oximino-l, 3,5 (10) -estratrien-3-ol-17 (R) spiro-2 '- (5', 5'-dimethyl-6'-oxo) tetrahydropyran (21). Under an argon atmosphere, a solution of compound 20 (215 mg, 0.54 mmol) in anhydrous-pyridine ethanol 1-1 (4 ml) was treated with hydroxylamine hydrochloride. (56.6 mg, 0.814 mmol) and stirred at room temperature for 25 minutes. The reaction mixture was evaporated, diluted with water and extracted 3 times with dichloromethane. The combined organic phase was washed with brine, dried over sodium sulfate, filtered and evaporated to provide enzyme 113 (127 mg, 98%); NMR aH (300 MHz, CDC13) d 1.02 (s, 3H), 1.29 (s, 6H), 1.43-1.70 (m, 10H), 1.89-2.12 (m, 6H), 2.22-2.37 (m, 1H), 2.80-2.87 (m, 2H), 6.69 (s, 1H), 7.05 (s, 1H), 8.15 (s broad, 1H), 8.20 (s, 1H), 9.61 (s, 1H).

Example 5C 3-Acetoxy-2-cyano-1, 3,5- (10) -estratrien-17 (R) -spiro-2 '- (5', 5'-dimethyl-6'-oxo) tetrahydropyran (22a ). A solution of compound 21 (180 mg, 044 mmol) and acetic anhydride (125 μL, 1.32 mmol) in pyridine (3.5 mL) was refluxed for 1 hour. The reaction mixture was evaporated, diluted with dichloromethane, and washed 3 times with water, 1 time with saturated sodium bicarbonate and 1 time with brine. The organic phase was dried over magnesium sulfate, filtered and evaporated. The crude mixture was purified by flash chromatography (dichloromethane to dichloromethane-ethyl acetate 19-1) to give acetate 22a (145 mg, 76%): IR (CHC13) 2933, 2872, 2229, 1773, 1718, 1613, 1494, 1183 cm "1; NMR XH (300 MHz, CDC13) d 1.02 (s, 3H), 1.28 (s, 6H), 1.34-1.89 (m, 11H), 1.94-2.33 (m, 6H), 2.37 (s, 3H), 2.89- 2.94 (m, 2H), 6.95 (s, 1H), 7.55 (s, 1H).

Example 5D 2-cyano-1, 3,5 (10) -estratrien-3-ol-17 (R) -spiro-2 '- (5', 5 '-dimethyl-6'-oxo) tetrahydropyran (22b). A solution of compound 22a (60 mg, 0.14 mmol) in methanol (5 ml) was treated with 10% potassium carbonate. (0.5 ml) and stirred 30 minutes. The reaction mixture was acidified to pH 2 with IN hydrochloric acid and extracted 3 times with dichloromethane. The combined organic phase was washed with water, saturated sodium bicarbonate and brine, and dried over magnesium sulfate, filtered and evaporated. The crude mixture was recrystallized from aqueous ethanol to give phenol 22b (28mg, 52%). IR (CDC13) 3334, 2932, 2868, 1692, 1312, 1206, 1159 cm "1; XH NMR (300 MHz, CDC13) d 0.97 (s, 3H), 1.29 (s, 6H), 1.26-2.14 (m, 16H), 2.21-2.28 (m, 1H), 2.82-2.86 (m, 2H), 6.69 (s, 1H), 6.91 (s, 1H), 7.35 (s, 1H), C13 NMR (75 MHz, CDC13) d 14.41, 23.30, 25.62, 26.92, 27.69, 27.78, 29.84, 31.62, 31.79, 34.82, 37.84, 38.70, 43.18, 47.23, 48.67, 93.52, 97.01, 116.29, 116.69, 129.44 133.69, 144.86, 155.73, 177.88.

Example 5E 3-Alkyloxy-2-cyano-1, 3,5 (10) -es ratrien-17 (R) -spiro-2 '- (5', 5'-dimethyl-6'-oxo) etrahydropyran (22c) . Under an argon atmosphere, a suspension of compound 22b, alkyl iodide (5 equivalent) and cesium carbonate (1.5 equivalent) in anhydrous acetonitrile (1% W / V) was stirred for 16 hours with reflux conditioning, if necessary. The reaction mixture was quenched with brine and extracted 3 times with dichloromethane. The combined organic phase was washed with brine, dried over magnesium sulfate, filtered and evaporated. The crude mixture was purified by flash chromatography (dichloromethane to dichloromethane-ethyl acetate 10-1) and recrystallization (methanol) to provide compound 22c (for example EM-1396, R = (CH 2) 2 OCH 3, 75%); IR (CHDC13) d 1.01 (s, 3H), 1.28 (s, 6H), 1.20-1.75 (m, 10H), 1.80-2.20 (m, 6H), 2.20-2.35 (m, 1H), 2.80-2.95 ( m, 2H), 3.47 (s, 3H), 3.79 (t, J = 4.8 Hz, 2H), 4.17 (t, J = 4.8 Hz, 2H), 6.67 (s, 1H), 7.44 (s, 1H); C13 NMR (75 Mhz, CDC13) d 14.41, 23.25, 25.58, 25.90, 26.96, 27.66, 27.74, 30.24, 31. 59, 31.78, 34.79, 37.80, 38.68, 43.16, 47.20, 48. 60, 59.55, 68.78, 70.71, 93.43, 99.55, 112.80, 116.98, 130.72, 133.31, 144.09, 158.29, 177.70.

Example 6 EXAMPLES OF PHARMACEUTICAL COMPOSITIONS The following are exemplified, by way of example and not limitation, various pharmaceutical compositions using a preferred active compound EM-2330 (a 3a-HSD type 3 inhibitor). Other compounds of the invention or combination thereof may be used in place of (or in addition to) EM-02318 and EM-02200. The concentration of the active ingredient can be varied over a broad range compatible with the preferred doses discussed herein, and depending on the preferred frequency of administration. The amounts and types of other ingredients that may be included are well known in the art.

EXAMPLE A Composition suitable for injection EXAMPLE B Composition suitable for use in topical lotion EXAMPLE C Composition suitable for use as a topical gel EXAMPLE D Tablet EXAMPLE E Gelatin capsule EXAMPLE F Composition suitable for use as a topical gel Other inhibitors of 3a-hydroxysteroid dehydrogenase type 3 can be substituted for EM-2330 in the above formulations. In some embodiments, two or more 3a-hydroxysteroid type 3 inhibitors may be co-included together (or a 3a-HSD type 3 inhibitor plus an inhibitor of 17β-HSD type 5) in which case the percent in which The combined weight of the two is preferably double that of the previous examples for EM-2330 only, with a corresponding reduction in the weight percent of the majority of the dominant excipient (eg water, lactose, ethanol) and similar). Other active ingredients of preferred combinations can be added in the same way herein.

The invention has been described in terms of the preferred embodiments and examples, but is not limited in this way. Those skilled in the art will readily recognize the broader applicability and scope of the invention which is limited only by the patent claims herein.

Claims

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A method to treat, or reduce the risk of developing, androgen-sensitive diseases, by inhibiting the conversion of 4-androstene-3,17-dione to testosterone or of 5a-androstane-3, 17-dione to dihydrotestosterone in a patient in need of this inhibition, which comprises administering to the patient a therapeutically effective amount of a inhibitor of human type 3a-hydroxysteroid dehydrogenase type 3. The method according to claim 1, further comprising administering an inhibitor of 17β-hydroxysteroid dehydrogenase type 5. 3. A method for treating or reducing the risk of developing androgen-sensitive diseases, by inhibiting the activity of 17β-hydroxysteroid- 3a-hydroxysteroid dehydrogenase type 3 human dehydrogenase comprising administering to a patient in need of this treatment a therapeutically effective amount of a human type 3a-hydroxysteroid dehydrogenase type 3 inhibitor having the following structure: where the dotted line is an optional pi link; wherein R3 is a portion selected from the group consisting of alkyloxy of 1 to 20 carbon atoms, acyloxy of 1 to 10 carbon atoms, alkoxycarbonyloxy of 1 to 20 carbon atoms, alkyloxy-alkyloxy of 1 to 20 carbon atoms, hydroxyl, (N-alkyl or -H) carbamate and a portion transformed in vivo to hydroxyl; wherein R2 and R4 are independently selected from the group consisting of hydrogen, cyano, fluoro, chloro, bromo, and nitro (wherein R2 and R4 are not simultaneously hydrogen); wherein R2 is not a cyano group when R17 is hydrogen or lower alkyl, R17β is hydroxyl, acyloxy, or alkoxy or R17 and R17 | i together are a ketonic oxygen, and both of R and R, 16β are independently hydrogen or lower alkyl; wherein R17a is selected from the group consisting of hydrogen, a portion of 2 to 14 carbon atoms substituted by a radical selected from the group consisting of hydrogen, carboxyl halogen, amido, alkoxy of 1 to 3 carbon atoms and alkyl of 1 at 5 carbon atoms or R17 and R1713 together form a lactone ring of 5 to 7 carbon atoms or is a ketone oxygen; wherein R17P is hydroxyl acyloxy, alkoxy, alkenyloxy, (N-alkyl or H), amido; or R17a and R1713 together form a lactone ring of 5 to 7 carbon atoms or is a ketonic oxygen; wherein R16a and R16β are independently selected from the group consisting of hydrogen, lower alkyl and benzyl, or R16a and R16 ^ together form a cycloalkene of 5 to 6 carbon atoms; where the inhibitor is not 4. The method according to claim 3, wherein the inhibitor of human type 3a-hydroxysteroid dehydrogenase type 3 has the molecular structure: where n is an integer of 1-2; wherein the dotted lines are independently optional pi links; wherein X and Y are independently selected from the group consisting of -H, alkyl of 1 to 3 carbon atoms and alkenyl of 2 to 3 carbon atoms. 5. The method according to claim 4, wherein R3 is hydroxy. The method according to claim 4, wherein at least one of X, or Y is methyl. The method according to claim 4, wherein both X and Y are methyl. The method according to claim 4, wherein R is chloro or cyano. The method according to claim 3, wherein the inhibitor of human type 3a-hydroxysteroid dehydrogenase type 3 has the molecular structure: wherein either: i) R17ß is a hydroxyl, R17a is a portion of 2 to 4 carbon atoms substituted by a radical selected from the group consisting of hydrogen, halogen, carboxyl, amido, alkoxy of 1 to 3 carbon atoms and alkyl of 1 to 5 carbon atoms and R16a and R16P are hydrogen; or ii) R17 ^ is a hydroxyl, R17a is hydrogen and R16a and R16P are either lower alkyl, benzyl or together they are a cycloalkane of 5 to 6 carbon atoms; or iii) R17a and R17 ^ are together a ketonic oxygen and R16a and R16β are either lower alkyl, benzyl, or together they are a cycloalkane of 5 to 6 carbon atoms. The method according to claim 9, wherein R17a is a benzyl group. 11. The method according to claim 10, wherein R3 is hydroxy. 12. The method according to claim 10, wherein R2 is chloro or cyano. A method for inhibiting the activity of human type 3a-hydroxysteroid dehydrogenase type 3 which comprises administering to a patient in need of this inhibitor a therapeutically effective amount of a human 3a-hydroxysteroid dehydrogenase type 3 inhibitor selected from the group it consists of: EM-01667-C 14. A method to determine the effectiveness of a putative inhibitor of the conversion of 4-androstene-3, 17-dione to testosterone and 5a-androstane-3, 17-dione to dihydrotestosterone, which comprises measuring the activity of 3a-hydroxysteroid- dehydrogenase type 3 in the presence of the putative inhibitor and correlate the effectiveness to a reduction in activity relative to the activity of the dehydrogenase in the absence of the putative inhibitor. The method according to claim 14, wherein the method comprises the following steps: a) providing culture medium with recombinant host cells transformed or transfected with a recombinant vector comprising a promoter sequence and a nucleotide sequence coding for the 3a -hydro-dehydrogenase type 3 human; b) providing the media with both the putative inhibitor and a substrate which, in the absence of the inhibitor, undergoes a conversion of human type 3a-hydroxysteroid dehydrogenase type 3; and c) measure the conversion. 16. A pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and a therapeutically effective amount of a human 3a-hydroxysteroid dehydrogenase type 3 inhibitor having the molecular structure: wherein R3 is a portion selected from the group consisting of alkyloxy of 1 to 20 carbon atoms, acyloxy of 1 to 10 carbon atoms, alkoxycarbonyloxy of 1 to 20 carbon atoms, alkyloxy-alkyloxy of 1 to 20 carbon atoms, hydroxyl, (N-alkyl or -H) carbamate and a portion transformed in vi to hydroxyl; wherein R2 and R4 are independently selected from the group consisting of hydrogen, cyano, fluoro, chloro, bromo, and nitro (wherein R2 and R4 are not simultaneously hydrogen); wherein R2 is not a cyano group when R17a is hydrogen or lower alkyl, R17 ^ is hydroxy, acyloxy, or alkoxy or R17a and R17 ^ together are a ketonic oxygen, and both of R16a and R16 »3 are independently hydrogen or lower alkyl; wherein R2 or R4 is not halo or N02 when hydrogen, R17 ^ is hydroxyl or R17a R17β together is a ketonic oxygen, and R16a and Rl6 »3 are both hydrogen wherein the dotted line is an optional pi bond; wherein R16a and R163 are independently selected from the group consisting of hydrogen, lower alkyl and benzyl, or R16a and R16β together form a cycloalkene of 5 to 6 carbon atoms; wherein R17a is selected from the group consisting of hydrogen, a portion of 2 to 14 carbon atoms substituted by a radical selected from the group consisting of hydrogen, halogen, carboxyl, amido, alkoxy of 1 to 3 carbon atoms and alkyl of 1 to 5 carbon atoms or R17a and R1713 together form a lactone ring of 5 to 7 carbon atoms or is a ketonic oxygen; wherein R 1 P is selected from the group consisting of hydroxyl, acyloxy, alkoxy, alkenyloxy, (N-alkyl or H) amido; or R17a and R17ß together form a lactone ring of 5 to 7 carbon atoms or is a ketone oxygen. 17. The pharmaceutical composition according to claim 16, wherein R17ot is a phenyl or propyl group. 18. The pharmaceutical composition according to claim 16, wherein R3 is hydroxy. 19. The pharmaceutical composition according to claim 16, wherein R2 is chloro or cyano. 20. A pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and a therapeutically acceptable amount of a human 3a-hydroxysteroid dehydrogenase type 3 inhibitor having the molecular structure: wherein R 100 is selected from the group consisting of hydrogen, carboxyl, amido, alkyl of 1 to 5 carbon atoms, halo, nitro, hydroxy and alkoxy of 1 to 3 carbon atoms. 21. A pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and a therapeutically acceptable amount of a human 3a-hydroxysteroid dehydrogenase type 3 inhibitor selected from the group consisting of 22. An inhibitor of human type 3a-hydroxysteroid dehydrogenase type 3 that has the molecular structure: wherein R3 is a portion selected from the group consisting of alkyloxy of 1 to 20 carbon atoms, acyloxy of 1 to 10 carbon atoms, alkoxycarbonyloxy of 1 to 20 carbon atoms, alkyloxy-alkyloxy of 1 to 20 carbon atoms, hydroxyl; (N-alkyl or -H) carbamate and a portion transformed in vi to hydroxyl; wherein R2 and R4 are independently selected from the group consisting of hydrogen, cyano, fluoro, chloro, bromo, and nitro (wherein R2 and R4) are not simultaneously hydrogen); wherein R2 is not a cyano group when R17a is hydrogen or lower alkyl, R17β is hydroxy, acyloxy, or alkoxy or R17a and R17β together are a ketonic oxygen, and both of R and R, 16β are independently hydrogen or lower alkyl; wherein R2 or R4 is not halo or N02 when R17a is hydrogen, R, 17βp is hydroxyl or R, 17a and R .1J7β together are a ketonic oxygen and R16a and R16'3 are both hydrogen; where the dotted line is an optional pi link; wherein R17a is selected from the group consisting of hydrogen, a portion of 2 to 14 carbon atoms substituted by a radical selected from the group consisting of hydrogen, halogen, carboxyl, amido, alkoxy of 1 to 3 carbon atoms and alkyl of 1 to 5 carbon atoms or R17a and R17ß together form a lactone ring of 5 to 7 carbon atoms or is a ketonic oxygen; wherein R17p is hydroxyl, acyloxy, alkoxy, alkenyloxy, (N-alkyl or H) amido; or R17a and R17 (3 together form a lactone ring of 5 to 7 carbon atoms or is a ketonic oxygen, wherein R16a and R16β are independently selected from the group consisting of hydrogen, lower alkyl and benzyl, or R16a and R16 ( 3 together form a cycloalkene of 5 to 6 carbon atoms 23. The inhibitor according to claim 22, wherein R17 is a phenyl or propyl group 24. The inhibitor according to claim 22, wherein R3 is hydroxy. inhibitor according to claim 22, wherein R2 is chloro or cyano 26. An inhibitor of human type 3a-hydroxysteroid dehydrogenase type 3 having the molecular structure: wherein R 100 is selected from the group consisting of hydrogen, carboxyl, amido, alkyl of 1 to 5 carbon atoms, halo, nitro, hydroxy and alkoxy of 1 to 3 carbon atoms. 27. A method for treating or reducing the risk of developing prostate cancer, comprising administering to a patient in need of this treatment or reducing a therapeutically effective amount of an inhibitor of the 17β-hydroxysteroid dehydrogenase activity of the 3a-hydroxysteroid Human dehydrogenase type 3 different from the compounds derived from 17-lactone. The method according to claim 27, further comprising administering a therapeutically effective amount of an inhibitor of human type 17β-hydroxysteroid dehydrogenase type 5. The method according to claim 27, wherein the prostate cancer is treated with the inhibitor which further comprises administering a therapeutically effective amount of an effective LHRH agonist (or antagonist) to reduce testicular secretion of sex steroids. 30. The method according to claim 28, further comprising administering a therapeutically effective amount of an antiandrogen. 31. The method according to claim 29, further comprising administering a therapeutically effective amount of an antiandrogen. 32. The method according to claim 26, further comprising administering a therapeutically effective amount of a 5a-reductase inhibitor. The method according to claim 27, further comprising administering a therapeutically effective amount of a 5a-reductase inhibitor. 34. The method according to claim 28, further comprising administering a therapeutically effective amount of a 5a-reductase inhibitor. 35. The method according to claim 29, which further comprises administering a therapeutically effective amount of a 5a-reductase inhibitor. 36. The method according to claim 30, further comprising a therapeutically effective amount of a 5a-reductase inhibitor. 37. The method according to claim 31, further comprising a therapeutically effective amount of a 5a-reductase inhibitor. 38. The method according to claim 26, further comprising a therapeutically effective amount of a 17β-hydroxysteroid dehydrogenase type 3. 39. The method according to claim 27, further comprising a therapeutically effective amount of a 17β-hydroxysteroid dehydrogenase type 3. The method according to claim 29, further comprising a therapeutically effective amount of a 17β-hydroxysteroid dehydrogenase type 3. 41. The method according to claim 31, further comprising a therapeutically effective amount of a 17β-hydroxysteroid- type 3 dehydrogenase. The method according to claim 35, further comprising a therapeutically effective amount of a 17β-hydroxysteroid dehydrogenase type 3. 43. The method according to claim 36, further comprising a therapeutically effective amount of a 17β- Hydroxysteroid dehydrogenase type 3. 44. The method according to the claim 37, which further comprises a therapeutically effective amount of a 17β-hydroxysteroid dehydrogenase type 3. 45. The method according to claim 27, further comprising a therapeutically effective amount of an antiandrogen. 46. A method for treating, or reducing the risk of developing, benign prosthetic hyperplasia comprising administering to a patient in need of this treatment or reduction, a therapeutically effective amount of an inhibitor of the activity of 17β-hydroxysteroid dehydrogenase of the 3a Human type 3 -hydroxysteroid dehydrogenase different from administering a compound derived from 17-lactone. 47. The method according to claim 46, further comprising administering to the patient the therapeutically effective amount of an inhibitor of human type 17β-hydroxysteroid dehydrogenase type 5. 48. The method according to claim 46, further comprising administering to the patient a therapeutically effective amount of an agent selected from the group consisting of an antiestrogen or an aromatase inhibitor. 49. The method according to claim 47, further comprising administering to the patient a therapeutically effective amount of an agent selected from the group consisting of an antiestrogen or an aromatase inhibitor. 50. The method according to claim 48, further comprising administering to the patient a therapeutically effective amount of an antiandrogen. 51. The method according to claim 49, further comprising administering to the patient a therapeutically effective amount of an antiandrogen. 52. The method according to claim 50, further comprising administering to the patient a therapeutically effective amount of a 5a-reductase inhibitor. 53. The method according to claim 51, further comprising administering to the patient a therapeutically effective amount of a 5-reductase inhibitor. 54. The method according to claim 48, further comprising administering to the patient a therapeutically effective amount of a 5a-reductase inhibitor. 55. The method according to claim 49, further comprising administering to the patient a therapeutically effective amount of a 5a-reductase inhibitor. 56. The method according to claim 48, further comprising administering to the patient a therapeutically effective amount of a 5a-reductase inhibitor and an antiestrogen or aromatase inhibitor. 57. The method according to claim 49, further comprising administering to the patient a therapeutically effective amount of a 5a-reductase inhibitor and an antiestrogen or aromatase inhibitor. 58. The method according to claim 48, further comprising administering to the patient a therapeutically effective amount of an inhibitor of 5a-reductase, an antiandrogen and of an antiestrogen or an aromatase inhibitor. 59. The method according to claim 49, further comprising administering to the patient a therapeutically effective amount of an inhibitor of 5a-reductase, an antiandrogen and of an antiestrogen or an aromatase inhibitor. 60. A method to treat, or reduce the risk of developing prostatitis, that. comprises administering to a patient in need of this treatment or reduction, a therapeutically effective amount of an inhibitor of the 17β-hydroxysteroid dehydrogenase activity of human type 3a-hydroxysteroid dehydrogenase type 3. 61. The method according to claim 60, further comprising administering a therapeutically effective amount of an inhibitor of human type 17β-hydroxysteroid dehydrogenase type 5. 62. The method according to claim 60, further comprising administering to the patient a therapeutically effective amount of an antiandrogen. 63. The method according to claim 61, further comprising administering to the patient a therapeutically effective amount of an antiandrogen. 64 The method according to claim 60, further comprising administering to the patient a therapeutically effective amount of a 5a-reductase inhibitor. 65. The method according to claim 61, further comprising administering to the patient a therapeutically effective amount of a 5a-reductase inhibitor. 66. The method according to claim 62, further comprising administering to the patient a therapeutically effective amount of a 5a-reductase inhibitor. 67. The method according to claim 63, further comprising administering to the patient a therapeutically effective amount of a 5a-reductase inhibitor. 68. A method for treating or reducing the risk of developing acne, seborrhea, hirsutism or androgenic alopecia comprising administering to the patient, in need of this treatment or reduction, a therapeutically effective amount of an inhibitor of 17β-hydroxysteroid dehydrogenase activity human type 3a of the 3a-hydroxysteroid dehydrogenase type 3 different from administering a compound derived from 17-lactone. 69. The method according to claim 68, further comprising administering to the patient a therapeutically effective amount of an inhibitor of human type 17β-hydroxysteroid dehydrogenase type 5. 70. The method according to claim 68, further comprising administering to the patient a therapeutically effective amount of an antiandrogen. 71. The method according to claim 69, which further comprises administering to the patient a therapeutically effective amount of an antiandrogen. 72. The method according to claim 68, further comprising administering to the patient a therapeutically effective amount of a 5a-reductase inhibitor. 73. The method according to claim 69, further comprising administering to a patient a therapeutically effective amount of the 5a-reductase inhibitor. 74. The method according to claim 70, further comprising administering to the patient a therapeutically effective amount of the 5a-reductase inhibitor. 75. The method according to claim 71, further comprising administering to the patient a therapeutically effective amount of the 5a-reductase inhibitor.