Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J21/00—Normal steroids containing carbon, hydrogen, halogen or oxygen having an oxygen-containing hetero ring spiro-condensed with the cyclopenta(a)hydrophenanthrene skeleton
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
  • A61K31/566—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol having an oxo group in position 17, e.g. estrone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
  • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
  • A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
  • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/08—Drugs for disorders of the urinary system of the prostate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/08—Antiseborrheics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/10—Anti-acne agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
  • A61P5/26—Androgens

Definitions

• the present invention relates to methods of treatment of sex steroid-dependent diseases based upon the use of inhibitors of enzymes involved in the biosynthesis of sex steroids from natural precursors.
• inhibitors that reduce the natural production of androgens such as testosterone and dihydrotestosterone, are disclosed.
• androgen-sensitive diseases i.e. diseases whose onset or progress is ⁇ aided by androgenic activity
• diseases include but are not limited to prostate cancer, benign prostatic hyperplasia, acne, seborrhea, hirsutism, androgenic alopecia, precocious puberty, adrenal hyperplasia, and polycystic ovarian syndrome.
• Estrogen sensitive diseases i.e. diseases whose onset or progress is aided by estrogenic activity are also known. They include but are not limited to breast cancer, endometrial cancer, endometriosis, leiomyoma, and precocious puberty.
• Androgenic and estrogenic activity may be suppressed by administering androgen receptor antagonists ("antiandrogens”) or estrogen receptor antagonists ("antiestrogens”), respectively. See e.g. WO 94/26767 and WO 96/26201. Androgenic and estrogenic activity may also be reduced by suppressing androgen or estrogen biosynthesis or secretions by known methods. See e.g. WO 90/10462, WO 91/00731, WO 91/00733, and WO 86/01105. Type 5 17 ⁇ -hydroxysteroid dehydrogenase is described in WO 97/11162.
• Inhibitors of human type 5 17 ⁇ -hydroxysteroid dehydrogenase enzyme are disclosed in United States Provisional Patent Application filed in March 11 1998, as serial No 60/077,510.
• Effective inhibitors of human type 3 3 ⁇ -hydroxysteroid dehydrogenase enzyme or effective inhibitors of both human type 3 3 ⁇ -hydroxysteroid dehydrogenase and human type 5 17 ⁇ -hydroxysteroid dehydrogenase enzymes are provided by the present invention, as is the discovery that androgen formation can be suppress thereby.
• the prior art is not believe to have described or suggested that the inhibition of type 3 3 ⁇ -hydroxysteroid dehydrogenase may play a beneficial role in reducing the amount of testosterone and dihydrotestosterone available in target tissues.
• dehydrogenases type 1 3 -hydroxysteroid dehydrogenase, or any other androgen degradation enzyme.
• the invention provides a method of inhibiting conversion of 4- androstene-3,17-dione to testosterone or of 5 ⁇ -androstane-3,17-dione to dihydrotestosterone in a patient in need of such inhibition comprising administering to said patient a therapeutically effective amount of an inhibitor of human type 3 3 ⁇ - hydroxysteroid dehydrogenase other than 17-lactone derivative compounds.
• the invention provides a method of inhibiting activity of human type 3, 3 ⁇ -hydroxysteroid dehydrogenase comprising administering to a patient in need of such treatment a therapeutically effective amount of an inhibitor of human type 3 3 ⁇ -hydroxysteroid dehydrogenase having the following structure:
• R 3 is a moiety selected from the group consisting of O-C20 alkyloxy, C 1 -C acyloxy, C ⁇ -C 2 o alkoxycarbonyloxy, C ⁇ -C 2 o alkyloxy alkyloxy, hydroxyl, (N-alkyl or - carbamate and a moiety transformed in vivo to hydroxyl; wherein R 2 and R 4 are independently selected from the group consisting of hydroge cyano, fluoro, chloro, bromo, and nitro (wherein R 2 and R 4 are not simultaneous hydrogen).
• R 17 is selected from the group consisting of hydrogen, a C2-C ⁇ carbo moiety substituted by a radical selected from the group consisting of hydrogen, haloge carboxyl, amido, C1-C3 alkoxy and C1-C5 alkyl or R ⁇ y ⁇ and R 17 ⁇ together form a C5- lactone ring or is a ketonic oxygen; wherein R 1 ⁇ is hydroxyl, acyloxy, alkoxy, alkenyloxy, (N-alkyl or H) amido; or R 1 and R 17 ⁇ together form a C5-C7 lactone ring or is a ketonic oxygen; wherein R 16 ⁇ and R 16 ⁇ are independently selected from the group consisting hydrogen, lower alkyl, and benzyl, or R 16 ⁇ and R 16P together form a C5-C6 cycloalkene.
• the invention provides a method of inhibiting the activity human type 3 3 ⁇ -hydroxysteroid dehydrogenase comprising administering to a patient need of such inhibition a therapeutically effective amount of an inhibitor of human type 3 ⁇ -hydroxysteroid dehydrogenase selected from the group consisting of:
• the invention provides a method for determining effectiveness of a putative inhibitor of the conversion of 4-androstene-3,17-dione to testosterone and 5 ⁇ -androstane-3,17-dione to dihydrotestosterone, comprising measuring activity of type 3 3 ⁇ -hydroxysteroid deydrogenase in the presence of said putative inhibitor and correlating effectiveness to a reduction in said activity relate to activity of said dehydrogenase in the absence of said putative inhibitor.
• the invention provides a pharmaceutical composition
• a pharmaceutical composition comprisin a pharmaceutically acceptable diluent or carrier and a therapeutically effective amount o an inhibitor of human type 3 3 ⁇ -hydroxysteroid dehydrogenase having the molecular structure:
• R 3 is a moiety selected from the group consisting of C ⁇ -C 2 o alkyloxy, O-Oo acyloxy, C1-C20 alkoxycarbonyloxy, -C20 alkyloxy alkyloxy, hydroxyl, (N-alkyl or -H) carbamate and a moiety transformed in vivo to hydroxyl; wherein R 2 and R 4 are independently selected from the group consisting of hydrogen, cyano, fluoro, chloro, bromo, and nitro (wherein R 2 and R 4 are not simultaneously hydrogen). wherein the dotted line is an optional pi bond; wherein R 17 ° is selected from the group consisting of hydrogen, a C2- 4 carbon moiety substituted by a radical selected from the group consisting of hydrogen, halogen,
• R 1 7 ⁇ is selected from the group cosisting of hydroxyl, acyloxy, alkoxy, alkenyloxy, (N-alkyl or H) amido; or W and R ⁇ together form a C 5 -C 7 lactone ring or is a ketonic oxygen;
• the invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and a therapeutically acceptable amount of an inhibitor of human type 3 3 ⁇ -hvdroxysteroid dehydrogenase having the molecular structure:
• R 1 00 is selected from the group consisting of hydrogen, carboxyl, amido, Q- C 5 alkyl, halo, nitro, hydroxy, and C1-C3 alkoxy.
• the invention provides an inhibitor of human type 3 3 ⁇ -hydroxysteroid dehydrogenase having the molecular structure:
• R 3 is a moiety selected from the group consisting of C1-C20 alkyloxy, C 1 -C 10 acyloxy, Q-C20 alkoxycarbonyloxy, O-C20 alkyloxy alkyloxy, hydroxyl; (N-alkyl or -H) carbamate and a moiety transformed in vivo to hydroxyl; wherein R 2 and R 4 are independently selected from the group consisting of hydrogen, cyano, fluoro, chloro, bromo, and nitro (wherein R 2 and R 4 are not simultaneously hydrogen).
• R 1 ⁇ is selected from the group consisting of hydrogen, a C 2 -Ci 4 carbon moiety substituted by a radical selected from the group consisting of hydrogen, halogen, carboxyl, amido, C1-C3 alkoxy and C1-C5 alkyl or R 1 ⁇ and R 17P together form a C 5 -C 7 lactone ring or is a ketonic oxygen; wherein R 17P is selected from the group consisting of hydroxyl, acyloxy, alkyoxy, alkenyloxy, (N-alkyl or H) amido; or R 17 ⁇ x and R 17 ⁇ together form a C5-C7 lactone ring or is a ketonic oxygen; wherein R 16 ⁇ and R 16 ⁇ are independently selected from the group consisting of hydrogen, lower alkyl, and benzyl, or R 16 ⁇ and R 16p together form a C5-C.6 cycloalkene.
• the invention provides an inhibitor of human type 3 3 ⁇ -hydroxysteroid dehydrogenase having the molecular structure:
• R 100 is selected from the group consisting of hydrogen, carboxyl, amido, Ci- C5 alkyl, halo, nitro, hydroxy, and C1-C3 alkoxy.
• the invention provides a method of treating, or reducing the risk of developing prostate cancer, comprising administering to a patient in need of such treatment or reduction a therapeutically effective amount of an inhibitor of human type 3 3 ⁇ -hydroxysteroid dehydrogenase other than 17-lactone derivative compounds.
• the invention provides a method of treating, or reducing the risk of developing, benign prostatic hyperplasia comprising administering to a patient in need of such treatment or reduction, a therapeutically effective amount of an inhibitor of human type 3 3 ⁇ -hydroxysteroid dehydrogenase other than 17-lactone derivative compounds.
• the invention provides a method of treating, or reducing the risk of developing, prostatitis comprising administering to a patient in need of such treatment or reduction, a therapeutically effective amount of an inhibitor of human type 3 3 ⁇ -hydroxysteroid dehydrogenase.
• the invention provides a method of treating or reducing the risk of developing acne, seborrhea, hirsutism or androgenic alopecia comprising administering to a said patient, in need of such treatment or reduction, a therapeutically effective amount of an inhibitor of human type 5 17 ⁇ -hydroxysteroid dehydrogenase activity of human type 3 3 ⁇ -hydroxysteroid dehydrogenase other than by administering a 17-lactone derivative compound.
• the inhibitors of the invention are used for preventing and/ or treating certain diseases, discussed herein, whose onset or progress is stimulated by androgenic activity.
• type 3 3 ⁇ -HSD which is known for its catalytic activity of reactions affecting the 3 position of steroids has now been shown by Applicants to catalyze reactions affecting the 17 position.
• This discovery that type 3 3 ⁇ -HSD participates in the formation of testosterone and DHT from androstenedione and androstanedione permits enhanced suppression of biosynthesis of these two important androgens by suppressing this new biosynthetic pathway that Applicants have discovered.
• type 3 3 ⁇ -hydroxysteroid dehydrogenase displays activity similar to that of 17 ⁇ -hydroxy steroid dehydrogenase (catalyzing the conversion of 4-androstenedione-3,17-dione to testosterone and 5 ⁇ - androstane-3,17-dione to dihydrotestosterone), inhibitors which suppress the 17 ⁇ - hydroxysteroid dehydrogenase activity of type 3 3 ⁇ -hydroxysteroid dehydrogenase, with or without combination with inhibitors of type 5 17 ⁇ -hydroxy steroid dehydrogenase and/ or inhibitors of 5 ⁇ -reductase diminish the production of androgens catalyzed by these enzymes. Because androgens formed by reactions catalyzed by these enzymes are precursors to estrogens, the invention also has applicability to diseases whose onset or progress is aided by estrogenic activity.
• the attending clinician should monitor individual patient response, and adjust dosage accordingly.
• a patient in need of treatment or reducing the risk of onset of a given disease is one who has either been diagnosed with such disease or one who is susceptible to acquiring such disease.
• the preferred dosage of the active compounds of the invention is identical for both therapeutic and prophylactic purposes.
• the dosage for each active component discussed herein is the same regardless of which particular disease is being treated (or prevented).
• an "inhibitor of type 3 3 ⁇ -hydroxysteroid dehydrogenase” means a compound whose IC50 of inhibition for the enzyme in question (computed in the same manner as described in connection with Table 1 herein) is no higher that 200 nM. It is preferred that IC50 of such inhibitor be no higher than 50 nM, most preferably lower than 10 nM. It is also preferred that undesirable inhibition of 3 -HSD type 1 and 17 ⁇ -HSD type 2 be less than 90% at 3.10 ⁇ , preferably less than 80%, and most preferably less than 70%. In some embodiments, it is preferred that androgenicity be less than 100% of stimulation of Shionogi cells at a concentration of lO ⁇ , preferably less than 50%, most preferably less than 20%.
• a plurality of different compounds are administered rather than a single compound having multiple activities.
• dosages herein refer to weight of active compounds unaffected by pharmaceutical excipients, diluents, carriers or other ingredients, although such additional ingredients are desirably included, as shown in the examples herein.
• Any dosage form (capsule, tablet, injection or the like) commonly used in the pharmaceutical industry is appropriate for use herein, and the terms "excipient”, “diluent” or “carrier” include such non-active ingredients as are typically included, together with active ingredients in such dosage forms in the industry.
• typical capsules, pills, enteric coatings, solid or liquid diluents or excipients, flavorants, preservatives, or the like may be included.
• hydrocarbon moiety includes but are not limited to straight or branched alkyl, straight or branched alkenyl, straight or branched alkynyl, phenyl, phenylalkyl, phenylalkenyl, phenylalkynyl.
• Figure 1 shows a schematic biosynthesis pathway of active androgens in the human prostate.
• Figure 2 shows 17 ⁇ -HSD and 3 ⁇ -HSD activity in intact 293 cells (ATCC CRL 1573) stably transfected with type 3 3 ⁇ -HSD, in culture.
• Cells stably transfected with type 3 3 ⁇ -HSD were seeded into 24-well plates at a density of 10 5 cells/well.
• 0.1 ⁇ M of [ 14 C]-labeled 4- dione and [ 14 C]-labeled DHT were added to freshly changed culture medium to assess the 17 ⁇ -HSD activity of type 3 3 ⁇ -HSD enzyme [transformation of 4-dione to testosterone (D)] and 3 ⁇ -HSD activity of type 3 3 ⁇ -HSD enzyme [transformation of DHT to 3 ⁇ -diol (O)] activity of the transfected enzyme, respectively.
• Non transfected cells were used as control. After incubation for the indicated time periods, the media were collected and extracted, and assayed as described herein "Enzymatic assay for types 1, 2, 3, and 5 17 ⁇ - HSD and types 1 and 3 3 ⁇ -HSD".
• Figures 3a to 3c show paraffin sections of normal human skin immunostained with antibody to type 3 3 ⁇ -HSD.
• the presence of type 3 3 ⁇ -hydroxysteroid dehydrogenase can be seen in: a) epithelium and fibroblast b) hair follicle c) sudoriferous glands.
• Prostate cancer is a disease of the prostatic epithelium while benign prostatic hyperplasia (BPH) mainly involves the stromal compartment of the prostate. Prostatitis, although more common in the prostatic epithelium, can be found in both areas of the prostate.
• BPH benign prostatic hyperplasia
• type 5 17 ⁇ -hydroxysteroid dehydrogenase (type 5 17 ⁇ - HSD) and type 3 3 ⁇ -hydroxysteroid dehydrogenase (type 3 3 ⁇ -HSD) are present in the prostatic epithelium, mainly the basal cells, thus transforming androstenedione into testosterone by the pathways shown in Figure 1.
• Such testosterone then diffuses into the luminal epithelial cells which are androgen-dependent, and where prostate cancer grows.
• Concerning the stromal compartment, type 3 3 ⁇ -HSD and type 5 17 ⁇ -HSD are mainly found in the fibroblasts which are distributed among the muscle cells. It is believed that growth factors secreted by the fibroblasts stimulate the surrounding cells, thus 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 only an androgen-sensitive disease.
• type 3 3 ⁇ -HSD was known to convert DHT into androstane-3 ⁇ , 17 ⁇ -diol, an inactive metabolite.
• Applicants have recently discovered the surprising role of 3 ⁇ -HSD in catalyzing the formation of testosterone and DHT from androstenedione and androstanedione, respectively (See figure 1).
• the advantage of inhibiting type 3 3 ⁇ -HSD to suppress formation of testosterone and DHT is believed to substantially outweigh any reduction in androgen catabolism that might result when type 33 ⁇ -HSD is inhibited.
• Figure 1 applies to each of prostate cancer, benign prostatic hyperplasia, and prostatitis, although the cell types are different between prostate cancer and BPH.
• type 5 17 ⁇ -HSD and type 3 3 ⁇ -HSD are mainly present in one cell type (basal cells) while 5 ⁇ -reductases are present in the luminal cells, which are located just above the basal cells, thus permitting diffusion of testosterone from the basal to the luminal cells and then conversion into the more potent androgen DHT.
• the fibroblasts located in the stromal compartment the transformation of androstenedione to testosterone and then to DHT takes place in the same cells.
• Type 1 3 ⁇ -HSD is not present in a significant amount in the prostate but is mainly a liver enzyme.
• Inhibitors used in the invention e.g., inhibitors of type 3 3 ⁇ -HSD, inhibitors of type 5 17 ⁇ -HSD, inhibitors of 5 ⁇ -reductase, etc.
• inhibition of androgen formation is performed by an efficient blockade of dihydrotestosterone (DHT) production with an effective inhibition of both, 5 ⁇ -reductase and type 5 17 ⁇ -hydroxy steroid dehydrogenase activities.
• DHT dihydrotestosterone
• 5 ⁇ -reductase There are two types of 5 ⁇ -reductase. Both types are expressed in the prostate, type 2 5 ⁇ - reductase, however, is expressed at a higher level.
• a Merck product, Proscar (finasteride, MK-906) inhibits mostly type 25 ⁇ -reductase.
• reaction mixture was cooled at room temperature, diluted with water and extracted with dichloromethane. The organic phase was washed with brine, dried over magnesium sulfate, filtered, and evaporated.
• reaction mixture was evaporated, dissolved in dry THF (1.6% W/V), cooled at 0 °C, treated with 10 equiv of amine and stirred for 15 min.
• the reaction mixture was quenched with water, extracted with dichloromethane, dried over magnesium sulfate, filtered, and evaporated.
• Type 5 17 ⁇ -HSD and type 3 3 ⁇ -HSD share 85.5% amino acid identity.
• the high primary structure homology between type 5 17 ⁇ -HSD and type 3 3 ⁇ -HSD could explain a minor 17 ⁇ -HSD activity found in type 3 3 ⁇ HSD activity.
• type 3 3 ⁇ -HSD is expressed at a much higher level than type 5 17 ⁇ -HSD, the unexpected 17 ⁇ -HSD activity contributed by type 3 3 ⁇ -HSD plays a significant role in the prostate. Inhibition of type 3 3 ⁇ -HSD activity is thus necessary to have an efficient blockade of androgen formation (Figure 1).
• Inhibitors of type 3 3 ⁇ -hydroxysteroid dehydrogenase may, in accordance with the invention, be utilized alone or as part of a combination therapy with other strategies (listed below) which have beneficial effects on androgen-sensitive diseases through different mechanisms, thus providing synergistic combinations.
• combination therapies include in addition to type 3 inhibitors of 3 ⁇ -hydroxysteroid dehydrogenase (and in some embodiments in combination with an inhibitor of type 5 17 ⁇ -hydroxy steroid dehydrogenase) one or more of the following strategies:
• Strategy 1 Suppression of ovarian or testicular hormonal secretion by chemical or surgical castration. This approach is useful for the treatment of diseases which respond adversely to estrogen or androgen, respectively.
• chemical castration is preferred utilizing either an LHRH-agonist, an LHRH antagonist and/ or an inhibitor of type 3 17 ⁇ -hydroxy steroid dehydrogenase (which as discussed herein catalyzes some testicular androgen formation).
• Suitable LHRH agonists are reported in US Patent 4,659,695, but any LHRH agonist showing the ability to induce chemical castration can be used since they all act through the same mechanisms as originally described (Labrie et al., J. Androl. 1: 209-228, 1980). Dosages are known in the art.
• Some suitable LHRH antagonists are reported in U.S. Patent 4,666,885 but any LHRH antagonist is acceptable, if used according to the recommendation of the manufacturer.
• Strategy 2 Utilizing androgen or estrogen receptor antagonists ("antiandrogens” or “antiestrogens”) to prevent activation of androgen or estrogen receptors by androgens or estrogens, respectively.
• Antiandrogens, and dosages therefor are known in the art (e.g. Flutamide (N-[4-nitro-3-(trifluoromethyl)phenyl)]-2-methyl propanamide) at a dosage of 250 mg, 2 or 3 times a day, Nilutamide at a dosage of 150 mg/day, Casodex at a dosage of 50 to 750 mg/day.
• EM-800 reported in PCT/ CA96/ 00097 (WO 96/26201) The molecular structure of EM-800 is:
• Another preferred antiestrogen of the invention is EM-01538
• SERMs of the invention include Tamoxifen ((Z)-2-[4-(l,2-diphenyl-l- butenyl)]-N,N-dimethylethanamine) (available from Zeneca, UK), Toremifene (available from Orion-Farmos Pharmaceuticla, Finland, or Schering-Plough), Droloxifene and CP- 336,156 (cis-lR-[4'-pyrrolidino-ethoxyphenyl]-2S-phenyl-6-hydroxy-l,2,3,4,- tetrahydronapthalene D-(-)-tartrate salt) (Pfizer Inc., USA), Raloxifene (Eli Lilly and Co., USA), LY 335563 and LY 353381 (Eli Lilly and Co., USA), Iodoxifene (SmithKline Beecham, USA), Levormeloxifene (3,4-trans-2,2-dimethyl-3-phenyl-4-[4-[
• 5 ⁇ -reductase There are two types of 5 ⁇ -reductase. Both types are expressed in the prostate, type 2 5 ⁇ - reductase, however, is expressed at a higher level.
• a Merck product, Proscar (finasteride, MK-906) inhibits mostly type 25 ⁇ -reductase.
• Strategy 3 Suppression of conversion of the androgen testosterone to the more potent androgen dihydrotestosterone (DHT) by inhibiting the activity of testosterone 5 ⁇ -reductase (e.g. by administering Proscar, available from Merck Sharp and Dohme Canada, at the recommended dosage). Any other potent 5 ⁇ -reductase inhibitor can be used.
• the dosage used can be 2 to 20 mg daily orally. The dosage should be the one recommended by the manufacturer.
• Strategy 3 is useful against diseases that respond adversely to androgenic activity.
• Strategy 4 Utilizing an aromatase inhibitor to reduce estrogen production.
• 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-sensitive diseases (e.g. benign prostatic hyperplasia).
• Aromatase inhibitors (and antiestrogens) may also be used to reduce unwanted estrogenic effects that result from increased estrogenic levels that may occur during some treatments of androgen-dependent diseases.
• aromatase inhibitors are used in accordance with the invention, either alone or as part of one of the combination therapies described herein, the attending clinician should initially use the dosage recommended by the manufacturer.
• the dosage which is usually effective to provide the desired serum levels is between 1.0 mg and 20 mg of active ingredient per day per 50 kg of body weight.
• Arimidex Zeneca
• the attending clinician should monitor individual patient response and metabolism and adjust specific patient dosage accordingly.
• Some aromatase inhibitors include, for example, molecular structures set forth in US patent 5,227,375.
• Aromatase inhibition may also be achieved, for example, by administering Arimidex (2,2'-[5-(lH-l,2,4-triazol-l-ylmethyl)- 1,3-phenylene bis (2-methyl ⁇ ropiononitrile)) available from Zeneca, UK, at a dosage of 1 mg/day. Any other aromatase inhibitor can be used according to the recommendations of the manufacturer.
• sex steroid biosynthesis inhibitors inhibitors of enzymes which catalyze one or more steps of estrogen or androgen biosynthesis or biosynthesis of estrogen or androgen precursors
• estrogen receptor antagonists and/ or androgen receptor antagonists are believed to have additive rather than redundant effect because they are acting in a beneficial manner by a different mechanism.
• the activity of two different enzyme inhibitors are believed to provide additive effect, especially where the inhibitors affect more than one synthetic pathway. Such an approach is believed to achieve a more complete effect.
• the type 3 3 ⁇ -hydroxysteroid dehydrogenase inhibitors and inhibitor of type 5 17 ⁇ -hydroxysteroid dehydrogenase of the invention may be used in any combination with any of the strategies 1-4 above whose effect (increasing or decreasing androgenic or estrogenic activity) is consistent with a desirable effect on the disease in question.
• any of the strategies 1-4 above whose effect (increasing or decreasing androgenic or estrogenic activity) is consistent with a desirable effect on the disease in question.
• set forth below are a list of representative diseases which may be treated, or the risk of which may be reduced, in accordance with the present invention. Beneath each disease, are indicated several preferred therapies or combination therapies for treatment, or risk reduction, of that particular disease. However, these combinations may be supplemented using one or more of the four strategies listed above, limited only by whether a particular disease responds favorably or adversely to estrogenic activity and/ or to androgenic activity.
• Inhibitor of type 3 3 ⁇ -hydroxy steroid dehydrogenase + inhibitor of type 5 17 ⁇ -hydroxysteroid dehydrogenase + LHRH-agonist (or antagonist).
• Inhibitor of type 3 3 ⁇ -hydroxysteroid dehydrogenase + inhibitor of type 5 17 ⁇ -hy droxysteroid dehydrogenase + antiestrogen or aromatase inhibitor.
• Inhibitor of type 3 3 ⁇ -hy droxysteroid dehydrogenase + inhibitor of type 5 17 ⁇ -hy droxysteroid dehydrogenase + antiandrogen + 5 ⁇ -reductase inhibitor + antiestrogen or aromatase inhibitor.
• Inhibitor of type 3 3 ⁇ -hydroxysteroid dehydrogenase + inhibitor of type 5 17 ⁇ -hy droxysteroid dehydrogenase + 5 ⁇ -reductase inhibitor.
• Inhibitor of type 3 3 -hydroxysteroid dehydrogenase + inhibitor of type 5 17 ⁇ -hy droxysteroid dehydrogenase + antiandrogen + 5 ⁇ -reductase inhibitor.
• Inhibitor of type 3 3 ⁇ -hydroxysteroid dehydrogenase + inhibitor of type 5 17 ⁇ -hy droxysteroid dehydrogenase + 5 ⁇ -reductase inhibitor + antiestrogen or aromatase inhibitor.
• Inhibitor of type 3 3 ⁇ -hydroxysteroid dehydrogenase + antiandrogen + 5 ⁇ - reductase inhibitor. 4. Inhibitor of type 3 3 ⁇ -hydroxysteroid dehydrogenase + inhibitor of type 5 17 ⁇ -hy droxysteroid dehydrogenase.
• Inhibitor of type 3 3 ⁇ -hydroxysteroid dehydrogenase + inhibitor of type 5 17 ⁇ - hydroxysteroid dehydrogenase + antiandrogen.
• Inhibitor of type 3 3 ⁇ -hydroxysteroid dehydrogenase + inhibitor of type 5 17 ⁇ -hy droxysteroid dehydrogenase + 5 ⁇ -reductase inhibitor.
• Inhibitor of type 3 3 ⁇ -hydroxysteroid dehydrogenase + inhibitor of type 5 17 ⁇ -hy droxysteroid dehydrogenase + inhibitor of 5 ⁇ -reductase.
• Inhibitor of type 3 3 ⁇ -hydroxysteroid dehydrogenase + inhibitor of 17 ⁇ -hy droxysteroid dehydrogenase + antiandrogen + inhibitor of 5 ⁇ -reductase When type 3 3 ⁇ -hydroxysteroid inhibitors are used in accordance with the invention, either alone or as part of one of the combination therapies described herein, the attending clinician desirably will target patient serum concentration of the type 3 inhibitor between 0.5 ng/ml and 100 ng/ml, preferably between 1 ng/ml and 20 ng/ml, and most preferably between 1 ng/ml and 10 ng/ml. Serum concentration may be measured by LC/MS.
• the dosage which is usually effective to provide the desired serum levels is between 1.0 mg and 1,000 mg of active ingredient per day per 50 kg of body weight, preferably between 10 mg and 500 mg and most preferably between 10 mg and 100 mg.
• dosage should vary with the bioavailability of the chosen inhibitor and with individual patient response.
• oral dosage is preferably between 5 mg and 500 mg per day per 50 kg body weight, more preferably between 10 mg/day and 300 mg/day, for example between 20 mg/day and 100 mg/day.
• the attending clinician should monitor individual patient response and serum levels, if judged appropriate, and adjust patient dosage accordingly.
• a lesser dosage is usually appropriate, e.g. 10 mg to 100 mg per day per 50 kg of body weight.
• the attending clinician desirably will target patient 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 most preferably between 1 ng/ml and 10 ng/ml. Serum concentration may be measured by LC/MS.
• the dosage which is usually effective to provide the desired serum levels is between 1.0 mg and 1,000 mg of active ingredient per day per 50 kg of body weight, preferably between 10 mg and 500 mg and most preferably between 10 mg and 100 mg.
• dosage should vary with the bioavailability of the chosen inhibitor and with individual patient response.
• oral dosage is preferably between 5 mg and 500 mg per day per 50 kg body weight, more preferably between 10 mg/day and 300 mg/day, for example between 20 mg/day and 100 mg/day.
• the attending clinician should monitor individual patient response and metabolism (serum levels, if judged appropriate) and adjust patient dosage accordingly.
• a lesser dosage is usually appropriate, e.g. 10 mg to 100 mg per day per 50 kg of body weight.
• the attending clinician desirably will target patient serum concentration of the type 3 inhibitor between 0.5 ng/ml and 100 ng/ml, preferably between 1 ng/ml and 20 ng/ml and most preferably between 1 ng/ml and 10 ng/ml.
• the dosage is preferably between 1.0 mg and 1,000 mg of active ingredient per day per 50 kg of body weight, preferably between 5 mg and 500 mg and most preferably between 10 mg and 100 mg.
• the attending clinician should monitor individual patient response and metabolism and adjust patient dosage accordingly. Synthesis of such an inhibitor is described below.
• the 3 ⁇ -alkylated stereoisomer was easily separated from the 3 ⁇ -alkylated stereoisomer by flash chromatography on silica gel, using a mixture of hexanes and ethyl acetate as eluent.
• the Grignard's reagent was generated in situ as in the case of ethylphenyl magnesium bromide, 5 eq. was prepared, by a well-known procedure, using the corresponding bromide, activated magnesium and iodide.
• the steroid was then dissolved in dry diethyl ether and added dropwise to the solution of reagent. The yields obtained were around 60% for the two stereoisomers.
• All of the active ingredients used in any of the therapies discussed herein may be formulated in pharmaceutical compositions which include one or more of the other active ingredients. Alternatively, they may each be administered separately but sufficiently simultaneous in time so that a patient eventually has elevated blood levels or otherwise enjoys the benefits of each of the active ingredients (or strategies) simultaneously.
• one or more active ingredients are to be formulated in a single pharmaceutical composition.
• a kit is provided which includes at least two separate containers wherein, the contents of at least one container differs in whole or in part from the contents of at least one other container with respect to active ingredients contained therein. Two or more different containers are used in these combination therapies of the invention.
• Combination therapies discussed herein also include use of one 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 further includes the other active ingredient(s) or strategy of the combination.
• Some embodiments of the methods of treating or preventing disease discussed herein utilize the specific type 5 17 ⁇ -hy droxysteroid dehydrogenase inhibitor and/ or type 3 3 ⁇ -hy droxysteroid dehydrogenase inhibitors discussed herein (i.e. the molecular structures discussed herein).
• LHRH agonists and LHRH antagonists may be used interchangeably to suppress either testicular or ovarian hormonal secretions by known techniques, except where preferences are otherwise stated herein. It is desired that activation of glucocorticoid receptors be minimized when administering the active ingredients of the invention.
• Inhibitors of type 3 17 ⁇ -hy droxysteroid dehydrogenase may be used to provide advantages similar to those provided by LHRH agonists or antagonists.
• IC50 The manner in which IC50 was determined is described in " II- Enzymatic assay for types 1, 2, 3 and 5 17 ⁇ -HSD and types 1 and 3 3 ⁇ -HSD". Lower numbers for IC50 are desirable. When IC50 was not determined, the percentage of inhibition is reported in parentheses at 3.10" ⁇ (left number) and S.IO ⁇ M (right number). In parentheses is reported the percentage of inhibition of enzymatic activity by the inhibitor at 3.10" 7 and 3.10- 6 M.
• 3 ⁇ -hydroxysteroid dehydrogenase inhibitors expressed as the percentage of stimulation of the proliferation of ZR-75-1 cells at concentrations of 10- 7 M (left number) and lO ⁇ M (right number) of inhibitor.
• the manner in which the stimulation is determined is described in "IV- Estrogenic/ Antiestrogenic Activity" Lower numbers are desirable. ND means that a determination was not done.
• the assays of the bioavailability of type 3 3 ⁇ -hydroxysteroid dehydrogenase inhibitors were performed in male Sprague Dawley rats by measuring the plasma concentrations of the compounds after single oral administration of the compounds. The 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.
• Each compound to be tested was administered to three animals as a suspension in 0.4% methylcellulose by oral gavage at a dose of 0.5 mg/rat (1.0 ml/ rat).
• Four to eight new compounds were tested each day and one group of animals received megestrol acetate (MGA) under the same conditions on each dosing day as a reference.
• MAA megestrol acetate
• Microtainer 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. One aliquot of 0.25 ml of plasma was then transferred into a borosilicate tube
• Plasma samples were kept at -80°C until measurement of plasma concentration of the inhibitor(s) by
• a solution of internal standard in methanol containing EM-248 at 50 ng/mL was prepared from stock standard solutions of EM-248 stored at -20°C.
• the final extract was reconstituted into 0.1 mL of methanol/ water (v:v, 75:25) and then transferred into a conical vial for injection into the mass spectrometer.
• the assay procedure was performed by analyzing, in duplicate, rat plasma samples spiked at six different Type 5 inhibitor concentrations (1, 2, 5, 10, 20 and 50 ng/mL).
• the lower limit of quantitation (LOQ) was established at 1.0 ng/mL. Values lower than 1.0 ng/mL were expressed as below limit of quantification (BLQ).
• Enzyme sources 293 cells transiently transfected with expression vectors encoding types 1, 2 and 3 17 ⁇ -HSD (Luu-The et al, J. Steroid Biochem. Molec. BioL, 55: 581-587, 1995) type 5 17 ⁇ -HSD (described in WO 97/11162), and types 1 and 3 3 ⁇ -HSD (Dufort et al. Biochem. Biophys.
• cells were sonicated in 50 mM sodium phosphate buffer (pH 7.4), containing 20% glycerol and 1 mM EDTA and centrifuged at 10 000 xg for 30 min before centrifugation for 100 000 x g for 1 h to separate the mitochondrial and microsomal fractions, respectively.
• the cytosol fractions (100 000 x g supernatant) was used to determine type 1 activity while the microsomal fraction (pellet at 100 000 x g) was used for measurement of types 2 and 3 17 ⁇ -HSD activities.
• estrone for types 1 17 ⁇ -HSD, DHEA and 4- androstene-3,17-dione ( ⁇ 4), for type 3 and 5 17 ⁇ -HSD, testosterone for type 2 17 ⁇ -HSD as well as,androstanedione and DHT for types 1 and 3 3 ⁇ -HSD activities, in absence or presence of increasing concentration of preferred inhibitor of the invention, was added to freshly changed culture medium in a 6- well culture plate. After incubation for 1 h, the steroids were extracted twice with 2 ml of ether. The organic phase were pooled and evaporated to dryness.
• the steroids were solubilized in 50 ⁇ l of dichloromethane, applied to Silica gel 60 thin layer chromatography (TLC) plate (Merck, Darmstad, Germany) then separated by migration in the toluene-acetone (4:1) solvent system. Substrates and metabolites were identified by comparison with reference steroids and revealed by autoradiography and quantitated using the Phosphoimager System (Molecular Dynamics, Sunny val, CA). Transfection could be also performed with HeLa, SW-13, 293, COS-1 cells, the preferred cell line is 293 cells.
• Androgenic/ antiandrogenic activity of some preferred compounds has been measured using the Shionogi mouse mammary carcinoma cells.
• MEM minimal essential culture medium
• non-essential amino acids fetal calf serum
• fetal calf serum purchased from Flow Laboratories.
• serum was incubated overnight at 4 °C with 1% activated charcoal (Norit A, Fisher) and 0.1% Dextran T-70 (Pharmacia).
• a 2-h supplementary adsorption was performed at 25°C in order to further remove protein-bound steroids.
• Serum was also inactivated by a 20-min incubation at 56°C.
• DHT 5 ⁇ -dihydrotestosterone
• the antiandrogen hydroxy flutamide was kindly supplied by Drs. T.L. Nagabuschan and R. Neri (Schering Corporation, Kenilworth,
• the tumor minces were digested for 2 h at 37°C in Hepes buffer containing 3.8 mg/ml collagenase (Clostridium, Boehringer), 1.5 mg/ml hyaluronidase II (Sigma), and 3% bovine serum albumin fraction V (Schwartz-Mann).
• Dispersed cells were collected by centrifugation (500 x g for 10 min), washed twice by suspension in minimal essential medium (MEM) containing 5% dextran-coated charcoal-treated fetal calf serum (DCC-FCS), 1% non-essential amino acids, 10 IU/ml penicillin, 50 ⁇ g/ml streptomycin, and 100 nM dihydrotestosterone (DHT) (Steraloids).
• MEM minimal essential medium
• DCC-FCS dextran-coated charcoal-treated fetal calf serum
• DHT dihydrotestosterone
• Estrogenic/ antiestrogenic activity of some preferred compounds has been measured using the ZR-71-1 human breast cancer cell line as described in more detail below.
• ZR-75-1 cells (83 rd passage) were obtained from the American Type Culture Collection (Rockville, MD) and routinely cultured in phenol red free RPMI 1640 supplemented with 1 nM estradiol (E 2 ), 2 mM L glutamine, 1 mM sodium pyruvate, 15 mM N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid, 100 IU penicillin/ ml, 100 ⁇ g streptomycin/ ml, and 10% (v/v) fetal bovine serum (Hy clone, Logan, UT) under a humidified atmosphere of 95% air, 5% C0 2 , at 37 °C. All media and medium supplments were purchased from Sigma. Cells were subcultured weekly by treatment with a pancreatic solution containing EDTA (0.2 g/L). The cell cultures used for the experiments herein describied were between passages 89 and 94.
• E 2 estradiol
• 2
• Androgen Receptor Assay was measured using the hydroxylapatite assay (HAP).
• HAP hydroxylapatite assay
• the radioactive steroid [3HJR1881 solubilized in ethanol was diluted into buffer A. Aliquots of prostate cytosol preparation (0.1 ml) were then incubated with 8 nM [3HJR1881 (0.1 ml, -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 h at 0-4°C.
• Triamcinolone acetonide Triamcinolone acetonide
• HAP (150 nM) was added in order to mask progesterone receptors. Unbound steroids were separated by incubation for 40 min at 0-4°C with 0.3 ml HAP prepared in buffer P (50 mM, Tris-HCl, 10 mM KH 2 P0 , pH 7.4) as follows: 10 g HAP were washed with buffer P until the supernatant reached a pH of 7.4 and then following centrifugation and decantation of the supernatant, 37.5 ml of buffer P were added. After incubation with HAP and 10 minutes of centrifugation at 1,000 x g, the pellet was washed 3 times with 1 ml buffer P.
• the radioactivity was extracted from the pellet by incubation at room temperature for 60 minutes with 1 ml EtOH. After centrifugation, the supernatant was decanted into a scintillation vial and the pellet was extracted again with ethanol. Thereafter, 10 ml Formula-989 scintillation liquid was added to pooled supernatant and the radioactivity was measured in a Beckman counter.
• Binding Assays Progesterone binding was measured using the dextran-coated charcoal adsorption technique. Incubations were performed at 0-4°C. for 16-18 h using 100 ⁇ l of cytosol, 100 ⁇ l of [ 3 H]-R5020 (5 nM final, which contained 1,000 nM of dexamethasone in order to mask the glucocorticoid receptors) and 100 ⁇ l of unlabeled compounds at the indicated concentrations. Each concentration was done in triplicate. Assay was ended with 300 ⁇ l of DCC (1% Norit A and 0.1% Dextran T-70 in Buffer B). After 10 min of incubation, tubes were centrifuged at 2,000 x g for 10 min. and decanted in vials with 6 ml of DCC (1% Norit A and 0.1% Dextran T-70 in Buffer B). After 10 min of incubation, tubes were centrifuged at 2,000 x g for 10 min. and decanted in vials with 6
• Tissue preparation Male Sprague-Dawley rats weighing 200-300g were obtained from Charles-River Inc. (St-Constant, Quebec, Canada). The rats were killed by cervical dislocation and the liver were rapidly removed, dissected free from adhering tissue and frozen on dry-ice. Tissues were kept at -80°C until use.
• Binding Assays Glucocorticoid binding was measured using the dextran-coated charcoal adsorption technique. Incubations were performed at 0-4°C. for 16-18 h using 100 ⁇ l of cytosol, 100 ⁇ l of [ 3 H]-Dexamethasone (5 nM final) and 100 ⁇ l of unlabeled compounds at the indicated concentrations. Each concentration was done in triplicate. Assay was ended with 300 ⁇ l of DCC (2.5% Norit A and 0.25% Dextran
• Estrogen binding was measured using the dextran-coated charcoal adsorption technique as described previously (Asselin et al., Endocrinology, 101: 666-671, 1977; Asselin and Labrie, J. Steroid Biochem., 9: 1079-1082, 1978). Briefly, [3H]E 2 solubilized in ethanol were diluted into buffer A. Aliquots of uterine cytosol preparation (0.1 ml) were incubated with 5 nM PHJE 2 ( ⁇ 200,000 cpm, 0.1 ml) in the presence or absence of the indicated concentrations of unlabeled compounds (0.1 ml, prepared in buffer A containing 10% ethanol) for 3 h at room temperature. Unbound steroids were then separated by incubation for 15 min at 0-4°C with 0.3 ml 0.5 % Norit-A and 0.05 % Dextran T-70 in buffer B
• Primary criteria in selecting preferred inhibitors include bioavailability , desirable inhibition of type 3 3 ⁇ -hydroxysteroid dehydrogenase and type 5 17 ⁇ -hydroxysteroid dehydrogenase, extent of undesirable inhibition on type 2 17 ⁇ -h droxysteroid dehydrogenase and androgenicity. It is believed that the methyl groups in 5' position in EM 01645 and EM 01667 and analogous compounds promote selectivity of type 5 17 ⁇ -HSD inhibition (versus undesirable type 2 inhibition). It is also believed that free hydroxy group in 3-position has a benific effect as well as the substitution in position 2.
• inhibitors of type 3 3 ⁇ -HSD have better selectivity when provided with D-ring substituents such as those described herein at the 16 or 17 position of a steroidal nucleus.
• selectivity it is meant that these preferred D-ring substituents, especially those set forth at 17 position, tend to suppress undesirable interactions between the inhibitors of the invention and, for example, enzymes whose inhibition is not desired or receptors whose activation is not desired.
• preferred compounds of the invention effectively inhibit activity of type 3 3 ⁇ -HSD while substantially avoiding numerous undesirable activities for which Applicants tested the same compounds.
• appropriate D-ring substituents tend to reduce undesirable androgenic or estrogenic activities.
• 17-spiro-lactone and 17 ⁇ -benzyl substituents give a good selectivity for type 3 3 ⁇ -HSD.
• Not all of the type 3 3 ⁇ -HSD compounds discussed herein are claimed because some of the compounds also have good activity against type 5 17 ⁇ -HSD and are claimed in a separate patent application by Applicants directed to this separate activity.
• Purified human type 33 ⁇ -HSD expressed over in E. coli The coding region of human type 3 3 ⁇ -HSD was amplified by PCR and inserted in a pGEX-l ⁇ T(Amersham Pharmacia Biotech, Inc., Quebec , Canada) vector in order to produce a fusion protein with glutathion-S- transferase. Expression over of the type 3 3 ⁇ -HSD in E. coli, purification of the protein on the glutathione-Sepharose 4B affinity column (Amersham Pharmacia Biotech), and cleavage of the fusion protein by thrombin were performed as described by the manufacturer.
• the purified enzyme was incubated in a final volume of 1 ml of 50 mM sodium phosphate buffer (pH 7.5), 20% glycerol, 1 mM EDTA and O.l ⁇ M of [ 14 C]-labeled steroid and 1 mM of NADPH. After 2 h incubation, the steroids were extracted twice with 1 ml of ether. The organic phases were pooled and evaporated to dryness. The steroids were solubilized in 50 ⁇ l of dichloromethane, applied to Silica gel 60 TLC plates (Merck, Darmstad, Germany), before separation by migration in the toluene- acetone (4:1) solvent system. Substrates and metabolites were identified by comparison with reference steroids and revealed by autoradiography and quantified using the Phosphoimager System (Molecular Dynamics, Sunny val, CA). Table 3
• Kieselgel 60F254 plates E. Merck, Darmstadt, FRG.
• Merck-Kieselgel 60 230-400 mesh A.S.T.M.
• starting material and reactant were obtained commercially and were used as such or purified by standard means. All solvents and reactants purified and dried were stored under argon.
• Estrone (1, 18.004 g, 66.6 mmol) was dissolved in boiling acetic acid (540 mL) and allowed to cool down to 50°C.
• the nitrating mixture was prepared from 70% nitric acid (4.5 mL, 70 mmol), water (10 mL) and a few crystal of sodium nitrite, warmed up to 50 °C and added dropwise to the solution of estrone with stirring. After stirring overnight at room temperature, the yellow precipitate was filtered by suction and recrystallized from 92% aqueous acetic acid. 4-nitro derivative (6.800 g, 32%) was thus obtained as a pale yellow solid.
• Example IB 3-(tert-butyldimethylsilyloxy)-2-nitro-l,3,5(10)-estratrien-17-one (2b).
• Example 1C 3-(tert-butyldimethylsilyloxy)-17 ⁇ -hydroxy-2-nitro-17 ⁇ -(4 , -(2"- tetrahydro-2"H-pyranyloxy)-butynyl)-l,3,5(10)-estratriene (3).
• tetrahydro-2-(butynyloxy)-2H-pyran (1.71 mL, 10.91 mmole) in dry THF (75 mL) under Ar (g) at -35 °C was dropwise added
• EM-1131 (5'-epimer of EM-1126, real configuration not determined): M.p. 206-8 °C; [ ⁇ o +62.6 ° (c 0.68, CDC1 3 ); IR v 3422 (br, OH), 3192,
• LDA was prepared as follows: To a stirred solution of diisopropylamine (206 ⁇ L, 159 mg, 1.57 mmol) in dry THF (12 mL) at -78 °C under Ar(g) was added n-BuLi (1.2 M/Hexane, 1,28 mL, 1.53 mmol) and the solution was then stirred at 0 °C for 20 min. then cooled down to -78 °C.
• estrone 5.0 g, 18.5 mmol
• benzyl magnesium chloride 2M in THF, 65 mL
• Aqueous saturated NH C1 solution was added at 0°C and the solution was extracted with CH 2 C1 2 (3 times), washed with brine, dried with MgS0 , filtered and then evaporated.
• the product was purified by flash chromatography (RP- C18, 30:30:40 - 10:30:60, H 2 0/MeOH/CH3CN) to give 1 as a white solid (4.0 g, 60%) and estrone: (1.5g, 30% recovery).
• 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%).
• 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 MgS0 , 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 white solid (145 mg, 72%).
• Example 5B 2-oximino-l,3,5(10)-estratrien-3-ol-17(R)-spiro-2 , -(5 , ,5 , -dimethyl-6'- oxo)tetrahydropyran (21). Under argon atmosphere, a solution of compound 20 (215 mg, 0.54 mmol) in anhydrous ethanol-pyridine 1-1 (4 mL) was treated with hydroxylamine hydrochloride (56.6 mg, 0.814 mmol) and stirred at room temperature for 25 min. The reaction mixture was evaporated, diluted with water, and extracted 3 times with dichloromethane.
• Example 5C 3-acetoxy-2-cyano-l,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethyl-6'- oxo)tetrahydropyran (22a).
• a solution of compound 21 (180 mg, 0.44 mmol) and acetic anhydride (125 ⁇ L, 1.32 mmol) in pyridine (3.5 mL) was refluxed for 1 h.
• 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.
• compositions utilizing a preferred active compound EM-2330 (an inhibitor of type 3 3 ⁇ -HSD).
• EM-2330 an inhibitor of type 3 3 ⁇ -HSD
• Other compounds of the invention or combination thereof may be used in place of (or in addition to) EM-02318 and EM-02200.
• concentration of active ingredient may be varied over a wide range compatible with the preferred dosages discussed herein, and depending on preferred frequency of administration.
• the amounts and types of other ingredients that may be included are well known in the art.
• composition suitable for injection is Composition suitable for injection
• composition suitable for use as topical lotion is provided.
• composition suitable for use as topical gel is
• composition suitable for use as topical gel is
• inhibitors of type 3 3 ⁇ -hydroxysteroid dehydrogenase may be substituted for EM-2330 in the above formulations.
• two or more type 3 3 ⁇ -hydroxysteroid inhibitors may be included together, (or one inhibitor of type 3 3 ⁇ -HSD plus on inhibitor of type 5 17 ⁇ -HSD) in which case the combined weight percent of the two is preferably double what is shown in the above examples for EM-2330 alone, with a corresponding reduction in the weight percent of the most prevalent excipient (e.g., water, lactose, ethanol or the like).
• the most prevalent excipient e.g., water, lactose, ethanol or the like.
• Other active ingredients of preferred combinations herein may be added in like manner.

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