|||||||||||I|| US005424463A United States Patent (19) 11 Patent Number: 5,424,463 Lardy et al. 45 Date of Patent: Jun. 13, 1995

(54) DELTA5-ANDROSTENES USEFUL FOR Dehydroepiandrosterone, 1990, Walter de Gruyter & PROMOTING WEIGHT MANTENANCE OR Co. WEIGHT LOSS AND TREATMENT PROCESS Yuan Su and Lardy, Dehydroepiandrosterone-respon sive Enzyme Induction in Rat Liver, J. Biochemistry 75 Inventors: Henry A. Lardy; Ieva L. Reich, both 110:207-213 (1991). of Madison, Wis.; Yong Wei, The Faseb Journal, Abstracts, vol. 2, No. 4, Mar. 15, Washington Boro, N.J. 1988. 73 Assignee: Humanetics Corporation, Chaska, Biochem, IUPAC-IUB Joint Commission on Biochem Minn. ical Nomenclature (JCBN), 1989, The nomenclature of steroids. 21 Appl. No.: 327,843 Schauer, Rohrer, Hanson, Lardy & Stratman, "Beta agonist; in Hormones Thermogenesis and Obesity,' 22 Filed: Oct. 24, 1994 1989, Elsevier, N.Y. pp. 485-501. Lardy, et al., “Dehydroepiandrosterone Induces En Related U.S. Application Data zymes That Permit Thermogenesis and Decrease Meta 63 Continuation of Ser. No. 123,151, Sep. 2, 1993, aban bolic Efficiency,” Hormones, Thermogenesis and Obe doned, which is a continuation-in-part of Ser. No. sity (1989), pp. 415-426. 867,288, Apr. 10, 1992, Pat. No. 5,296,481, which is a Partridge & Lardy, 1989, Cell Bio. 107:203A, Carbohy continuation of Ser. No. 575,156, Aug. 29, 1990, aban drate Chemistry and Metabolism. doned. (List continued on next page) 51) Int. Cl...... A61K 31/58; A61K 31/56 (52) U.S. C...... 552/637; 552/636 Primary Examiner-Marianne M. Cintins 58 Field of Search ...... 552/636, 637; 514/177, Assistant Examiner-T. J. Criares 514/178, 182, 910, 911 Attorney, Agent, or Firm-Faegre & Benson 56) References Cited 57) ABSTRACT U.S. PATENT DOCUMENTS A method for promoting weight control by treating a 4,511,511 4/1985 Lewbart ...... 260/239.55 subject with a therapeutic amount of one of the A5 4,518,595 5/1985 Coleman et al...... 514/178 androstenes listed below to stimulate weight control 4,897,390 1/1990 Ruhe ...... 514/177 without affecting appetite or inducing the synthesis of 4,898,694 2/1990 Schwartz et al. ... 260/.397.5 5,001,119 3/1991 Schwartz et al...... 514/177 sex hormones. A5-Androstenes providing the desired 5,028,631 7/1991 Schwartz et al. . biological activities include: 5,206,008 4/1993 Loria ...... 424/45 A5-Androstene-3,6,7a-diol-17-one 5,227,907 l/1994 Loria ...... 424/93 A5-Androstene-3,3-ol-7,17-dione A5-Androstene-3,3,7a, 1713-triol FOREIGN PATENT DOCUMENTS A5-Androstene-3,3,1713-diol-7-one 0.133995 3/1985 European Pat. Off...... CO7J 1/OO A5-Androstene-3,6-acetoxy-7,16,17-trione 0210665 4/1987 European Pat. Off...... CO7J 1/00 A5-Androstene-3,6, 16a-dihydroxy-7,17-dione 0246650 1 1/1987 European Pat. Off...... CO7J 1/00 A5-Androstene-3o-propionoxy-1613-acetoxy-7, 17 dione - OTHER PUBLICATIONS A5-Androstene-3,6,7a, 1713-triol-16-one Phosphates in Food, Ricardo A. Molins Ph.D., CRC A5-Androstene-3,6, 17(3-diol-7, 16-dione Press, p. 190 (1987). A5-Androstene-3,6, 16a, 1713-triol-7-one. Kalimi, Opoku, Lu, Yanal, Khalid, Regelson & Qureshi, Studies on the Biochemical Action and Mechanism of 6 Claims, No Drawings 5,424,463 Page 2

OTHER PUBLICATIONS Inhibition of Spontaneous Breast Cancer Formation in Nestler, Barlascini, Clore & Blackard, 1989, Hormones, Female C3H (Ary/a) Mice by Long Term Treatment Thermogenesis & Obesity, Dedrdroepiandrosterones with Dehyroepaindrosterone. Effects on Insulin Sensitivity Serum Lipid Levels, and Yen, Allen, Pearson, Acton and Greenberg, Lipids, vol. Body Composition in Normal Men, pp. 405-414. 12, 1977, pp. 409–413, Prevention of obseity in Avy/a Vec, Lopez, The Lancet, Dec. 2, 1989, pp. 1335-1336, Mice By Dehydroepiandrosterone. Antiglucocorticoid Actions of Dehydroepiandroster Gardner, 1974, Academic Press, Inc., A Sensitive Col one and Low Concertrations in Alzeimher's Disease. orimetric Assay for Mitochondrial alpha Glycerophos Lardy, Stratman, 1989, Hormones, Thermogeneis & phate Dehydrogenase. Obesity, pp. 427-439, Up-Regulation of the Immune Lee & Lardy, Mar. 1965, vol. 240, No. 3, The Journal of Response and Resistance to Virus Infection Dehydro Biological Chemistry, Influence of Thyroid Hormones epiandrosterone (DHEA). on L-Alpa-Glycerophosphate Dehydrogenases and Evans, Articles, May 13, 1988, The Steroid and Thy Other Dehydrogenases in Various Organs of the Rat. roid Hormone Receptor Superfamily, pp. 889-895. Dodson, Nicholson & Muir, Dec. 5, 1959, pp. Flood & Roberts, 1988, Elsevier Science Publishers, pp. 6295-6299, Oxidation of Dehydroisoandrosterone at 178-181, Dehydroepiandrosterone Sulfate Improves C-7. Memory in aging mice. Lee, Takemori Lardy, vol. 234, No. 11, Nov. 1959, The Chasalow, Blethen, Tobash Myles & Butler, 1987, Journal of Biological Chemistry, Enhanced Oxidation American Journal of Medical Genetics, Steroid Meta of alpha-Glycerophosphate by Mitochondria of bolic Disturbances in Prader-Willi Syndrome, pp. Thyroid-fed Rats, pp. 3051-3053. 857-864, Gordon, Shantz & Talalay, The Johns Hopkins Univer Bailey, Day, Bray, Lipson & Flatt, Dec. 19, 1984, sity School of Medicine Modulation of Growth, Differ Georg Thieme Venlag Stuttgart Role of Adrenal entiation and Carcinogenesis by Dehydroepiandroster Glands in the Development of Abnormal Glueose and one, pp. 355-382 (1982). Insulin Homeostasis in Genetically Obese (ob/ob) Mice, Effect of Dehydroepiandrosterone Treatment on Liver pp. 357-360. Metabolism in Rats, Margot P. Cleary, The Interna Chasalow, Blethen & Taysi, Oct. & Nov. 1985, pp. tional Journal of Biochemistry, vol. 22, No. 3, pp. 827-843, Steroids, Possible Abnormalities of Steroid 205-120, 1990. Secretion in the Childres with Smith-Lemli-Opitz Syn Anti-Obesity Effect of Two Different Levels of Dehy drome and Their Parents. droepiandrosterone in Lean and Obese Middle-Aged Coleman, Leiter & Schwizer, Jun. 17, 1982, pp. Female Zucker Rats, Margot P. Cleary and Joseph F. 830-833, Rapid Publications, Therapeutic Effects of Zisk, International Journal of Obesity (1986) vol. 10, pp. Dehydroepiandrosterone (DHEA). 193-204. Schwartz & Tannen, vol. 2, No. 12, 1981, pp. Effects of Dietary Dehydroepiandrosterone on Body 1335-1337, Carinogenesis Inhibitation of 7,12-dime Weight and Food Consumption in Lethal Yellow thylbenza)anthracene-and urethan-induced lung (A/A) and White-Bellied Agouti (A/A) Mice tumor formation in A/J mice by long-term treatment (Strain 129/Sv), Lisa D. Staber, Nels H. Granholm, with dehydroepiandrosterone. Peter J. Wilkin, Proceedings of the South Dakota Acad Schwatz, Mar. 1979, Cancer Research, pp. 1129-1131, emy of Science, vol. 62 (1983), pp. 154-162. 5,424,463 1. 2 A5-Androstene-3,6,7a, 1743-triol (3) DELTA5-ANDROSTENES USEFUL FOR A5-Androstene-3,6, 17(3-diol-7-one (4) PROMOTING WEIGHT MAINTENANCE OR A5-Androstene-3,3-acetoxy-7,16,17-trione (5) WEIGHT LOSS AND TREATMENT PROCESS A5-Androstene-3,3,16a-dihydroxy-7,17-dione (6) 5 A5-Androstene-3,3-propionoxy-i6,3-acetoxy-7, 17 This is a continuation of U.S. patent application Ser. dione (7) No. 08/123,151, filed Sep. 2, 1993 which is a continua A5-Androstene-3,3,7a, 17(3-triol-16-one (8) tion-in-part of U.S. patent application Ser. No. 867,288 A5-Androstene-3,6, 1713-diol-7, 16-dione (9) filed Apr. 10, 1992, now U.S. Pat. No. 5,296,481, which A5-Androstene-3,6, 16a, 1713-triol,7-one (10) is a continuation of U.S. patent application Ser. No. 10 and derivatives thereof wherein one or more of the 575,156 filed Aug. 29, 1990, abandoned. hydroxyl or keto substituents is a group convertible thereto by hydrolysis. FIELD OF THE INVENTION Examples of such hydrolyzable groups include hy The invention relates to the use of steroids for effect droxyl groups esterified with an acid selected from the ing a desired biological response. Specifically, the in 15 group consisting of (i) normal or branched, saturated or vention relates to a treatment program using A5-andros unsaturated C2-22 aliphatic acids, (ii) C7-12 aromatic tenes for promoting weight maintenance and/or weight acids, (iii) C3 or larger dicarboxylic acids in which only loss (hereinafter referenced collectively as "weight one of the carboxyl groups is esterified to the hydroxyl control'). group(s) on the steroid, or (iv) inorganic acids such as BACKGROUND sulfuric and phosphoric. The steroid dehydroepiandrosterone (DHEA) is be DETAILED DESCRIPTION OF THE lieved to stimulate various biological responses includ INVENTION INCLUDING A BEST MODE ing (i) inducing the synthesis of various thermogenic The A5-androstenes identified below possess a unique enzymes which are effective for regulating metabolism 25 combination of properties including the ability to pro and thereby promoting weight control without affect mote weight control without affecting appetite and ing caloric intake, and (ii) inducing an increase in the without stimulating the production of sex hormones. production of the sex hormones androgen and estrogen. A5-Androstene-3,6,7a-diol-17-one (1) The ability of DHEA to promote weight control is A5-Androstene-3,6-ol-7,17-dione (2) believed to be mediated through enhanced thermogene 30 A5-Androstene-3,6,7a, 17(3-triol (3) sis (conversion of foodstuffs to heat energy rather than A5-Androstene-3,3,1743-diol-7-one (4) chemical energy such as ATP and/or triacylglyce A5-Androstene-3,3-acetoxy-7,16,17-trione (5) rides). The thermogenic effect mediated by DHEA is A5-Androstene-3,6, 16a-dihydroxy-7,17-dione (6) believed to result from the ability of DHEA to stimulate A5-Androstene-3,6-propionoxy-16,3-acetoxy-7, 17 the synthesis of thermogenic enzymes including mito 35 dione (7) chondrial glycerol 3-phosphate dehydrogenase (G3P A5-Androstene-3,6,7a, 17(3-triol-16-one (8) DH) and cytosolic malic enzyme (ME). Such enzymes A5-Androstene-3,6,17a-diol-7, 16-dione (9) tend to reduce the efficiency of energy metabolism A5-Androstene-3,6,16a, 1748-triol,7-one (10) within the body, and derivatives thereof wherein one or more of the Unfortunately, DHEA is generally considered to be 40 hydroxyl or keto substituents is a group convertible ineffective as a weight controlling therapeutic agent thereto by hydrolysis. because the dosage necessary to achieve weight control Examples of such hydrolyzable groups include hy frequently produces significant adverse side-effects in droxyl groups esterified with an acid selected from the cluding substantial increases in the concentration of sex group consisting of (i) normal or branched, saturated or hormones. 45 unsaturated C2-22 aliphatic acids, (ii) C7-12 aromatic Accordingly, a therapeutic agent possessing the acids, (iii) C3 or larger dicarboxylic acids in which only weight control characteristic of DHEA without the one of the carboxyl groups is esterified to the hydroxyl adverse side-effect of stimulating the synthesis of sex group(s) on the steroid, and (iv) inorganic acids such as hormones would be extremely useful. In addition, the sulfuric and phosphoric. effectiveness of such a therapeutic agent could be signif These steroids may also be administered as a carba icantly enhanced if the agent possessed an increased mate or other such derivative capable of releasing the weight control activity relative to DHEA. specified steroid within the intestinal tract, blood and /or body tissue. The desired biological activity is a SUMMARY OF THE INVENTION function of the steroid moiety. Derivations of a moiety A method for promoting weight maintenance and/or 55 may serve a variety of beneficial functions including weight loss (hereinafter referenced collectively as stabilization of the steroid, flavoring or obscuring the “weight control") which includes the step of treating a natural flavor of the steroid, or affecting the rate of subject with an effective weight controlling amount of absorption of the steroid. a A5-androstene which is effective for stimulating the desired biological response of promoting weight con 60 SYNTHESIS trol while being substantially ineffective for inducing (1) A5-Androstene-3,6,7a-diol-17-one (7a-hydroxy the undesired biological response of synthesising sex DHEA) hormones. A5-Androstene-3,6,7a-diol-17-one (7a-hydroxy A5-androstenes providing the desired beneficial bio DHEA) can be synthesized from commercially avail logical response without the undesired biological re 65 able DHEA acetate by sequentially synthesizing: sponse include: A5-androstene-3,6 hydroxy-17-one acetate (DHEA ace A5-Androstene-3,3,7a-diol-17-one (1) tate) A5-androstene-3,3-hydroxy-7-bromo-17-one ac A5-Androstene-3,6-ol-7,17-dione (2) etate (7-Br DHEA acetate) 5,424,463 3 4. A5-androstene-3,6,7a-dihydroxy-17-one diacetate and purification steps set forth above in connection with (7-OH DHEA diacetate) the synthesis and purification of 7-hydroxy DHEA. A5-androstene-3,6,7a-dihydroxy-17-one (7-hydroxy (3) A5-Androstene-3,6,7a, 17(3-triol (7a-hydroxy DHEA) androstenediol) A5-Androstene-3p3-hydroxy-7-bromo-17-one acetate A5-Androstene-3,6,7a, 1713-triol (3) can be synthe (7-bromo DHEA acetate) can be synthesized from A5 sized from commercially available androstenediol androstene-3,6-hydroxy-17-one acetate (DHEA ace diacetate by sequentially synthesizing: tate) by reacting DHEA acetate with a brominating A5-androstene-3,6, 17(3-diol-diacetate (20) agent, such as dibromantin (1,3-dibromo-5,5-dimethyl A5-androstene-3,6,17a-diol-7-bromo-diacetate (21) hydantoin) or N-bromosuccinimide. The 7-bromo 10 A5-androstene-3,6,7a, 17(3-triol-3,7,17-triacetate (22) DHEA acetate is unstable and must be used immedi A5-androstene-3,6,7a, 1713-triol (3) ately in the next step of the process. A5-Androstene 36,17a-diol-7-bromo-diacetate (21) The 7-bromo DHEA acetate contains an isomeric can be synthesized from the commercially available mixture of 7a-bromo and 7(3-bromo. The isomeric mix A5-androstene-3,6, 17(3-diol-diacetate (20) by reacting ture, may be equilibrated to 7a-bromo DHEA acetate 15 (20) with a brominating agent, such as Dibromantin in accordance with the method described for equilib (1,3-dibromo-5,5-dimethylhydantoin) or N-bromosuc riating a cholesterol derivative in Confalone, P. N., cinimide. The synthesized 7-bromo-androstenediol di Kulesha, I. D., and Uskokovic, M. R. Jour. Org. Chem., acetate (21) is unstable and must be used immediately. vol. 46, pp 1030-1032 (1981). Briefly, the isomeric mix The 7-bromo-androstenediol diacetate (21) contains ture of 7-bromo DHEA acetate is contacted with cold 20 an isomeric mixture of 7a-bromo-androstenediol diace anhydrous LiBr and shielded from light to convert the tate (21a) and 7a-bromo-androstenediol diacetate (21b) product from an isomeric mixture of 7a and 7(3 to which can be equilibrated to a 7a-bromo-andros predominently 7a. tenediol (21a) in accordance with the method described A5-Androstene-3,6,7a-dihydroxy-17-one diacetate in Confalone, P. N. Kulesha, I. D., and Uskokovic, M. (7-hydroxy DHEA diacetate) may be synthesized from 25 R. Jour. Org. Chem., vol. 46, pp 1030-1032 (1981). 7-bromo DHEA acetate by reacting the 7-bromo Briefly, the isomeric mixture of 7-bromo-androstenediol DHEA acetate with a mixture of glacial acetic acid and diacetate (2ia & 21b) is contacted with anhydrous LiBr powdered silver acetate at room temperature in a suit and shielded from light to convert the product from an able solvent, such as a mixture of methylene chloride isomeric mixture of 7a and 7.6 to predominently 7a. and acetone. 30 A5-Androstene-3,6,7a, 17.6-triol-3,7,17-triacetate (22) A5-Androstene-3,6,7a-dihydroxy-17-one (7-hydroxy may be synthesized from the 7a-bromo-androstenediol DHEA) may be synthesized from 7-hydroxy DHEA diacetate (21a) by reacting (21a) with a mixture of gla diacetate by dissolving the 7-hydroxy DHEA diacetate cial acetic acid and silver acetate in a suitable solvent, in methanol and reacting the dissolved 7-hydroxy such as a mixture of methylene chloride and acetone. DHEA diacetate with an aqueous solution containing a 35 A5-Androstene-3,3,7a, 17(3-triol (3) may be synthe suitable base such as Na2CO3. sized from the 7a-hydroxy-androstenediol triacetate The synthesized 7-hydroxy DHEA may then be puri (22) by reacting the 7a-hydroxy-androstenediol triace fied by (i) evaporating the methanol in vacuo, (ii) ex tate (22) in methanol with an aqueous solution contain tracting the 7-hydroxy DHEA into an appropriate or ing a suitable base such as Na2CO3. ganic solvent such as dichloromethane, (iii) evaporating The synthesized 7a-hydroxy-androstenediol (3) may the organic solvent in vacuo, (iv) azeotropically drying then be purified by (i) evaporating the methanol in the extracted solids containing the 7-hydroxy DHEA vacuo, (ii) extracting the 7a-hydroxy-androstenediol (3) with a suitable organic solvent such as ethanol, (v) dis into an appropriate organic solvent such as dichloro solving the extracted solids in acetone, and then (vi) methane, (iii) evaporating the organic solvent in vacuo, adding hexane to the acetone solution to produce puri 45 (iv) azeotropically drying the extracted solids contain fied crystals of A5-androstene-3,6,7a-diol-17-one (7- ing the 7a-hydroxy-androstenediol (3) with a suitable hydroxy DHEA). organic solvent such as ethanol, (v) dissolving the ex A second crop of A5-androstene-3,6,7a-diol-17-one tracted solids in acetone, and then (vi) adding hexane to (7a-hydroxy DHEA) crystals may be obtained by cool the acetone solution to produce purified crystals of ing the resultant solution below room temperature. 50 A5-androstene-3,6,7a, 17.6-triol (7a-hydroxy-andros (2) A5-Androstene-3,3-ol-7,17-dione (7-keto DHEA) tenediol) (3). A5-Androstene-3,6-ol-7,17-dione (7-keto DHEA) can A second crop of A5-androstene-3,3,7a, 1713-triol (3) be synthesized from commercially available DHEA crystals may be obtained by cooling the resultant solu acetate by sequentially synthesizing: tion below room temperature. 3,3-acetoxy-A5-androstene-17-one (DHEA acetate) 55 (4) A5-Androstene-3,3,1713-diol-7-one (7-keto-andros 3,3-acetoxy-A5-androstene-7, 17 dione (7-one DHEA tenediol) acetate) A5-Androstene-3,3,17a-diol-7-one (4) can be synthe A5-androstene-3,6-hydroxy-7,17-dione (7-one sized from commercially available androstenediol DHEA) diacetate by sequentially synthesizing: 3,3-Acetoxy-A5-androstene-7,17-dione (7-one DHEA 60 A5-androstene-3,6,17a-diol-7-one-diacetate (41) acetate) can be synthesized from 3p3-acetoxy-A5-andros A5-androstene-3,6,17a-diol-7-one (4) tene-17-one (DHEA acetate) by reacting the DHEA A5-Androstene-3,6,17a-diol-7-one-diacetate (41) can acetate with the oxidizing agent CrO3 in accordance be synthesized from A5-androstene-3,3,1743-diol-diace with the procedure outlined in Fieser, L. F., Jour. Am. tate (androstenediol-diacetate) (40) by reacting the an Chem. Soc., vol 75, pp. 4386-4394 (1953). 65 drostenediol-diacetate (40) with the oxidizing agent A5-Androstene-3,6-hydroxy-7,17-dione (7-one CrO3 in accordance with the procedure outlined in DHEA) can be synthesized from the 7-one DHEA Fieser, L. F. Jour, Am. Chem. Soc., vol. 75, pp acetate and purified by employing the deesterification 4386-4394 (1953). 5,424,463 5 6 A5-Androstene-3,6, 17(3-diol-7-one (7-keto-andros (53) mixture by treating the (53) mixture with tetra-n- tenediol) (4) can be synthesized from androstene butylammonium fluoride in a suitable solvent system 36,17a-diol-7-one-diacetate (41) and purified by em such as ether-dichloromethane-tetrahydrofuran-water. ploying the deesterification and purification steps set 3,3,16-Diacetoxy-A5-androstene-16- phenylseleno forth above with respect to the synthesis and purifica 7,17-dione (54) may be synthesized from the 36 tion of 7a-hydroxy DHEA (1) from 7a-hydroxy acetoxy-6-phenylseleno-A5-androstene-7,17-dione DHEA diacetate. (53a) by submitting (53a) to the seleno-Pummerer reac (5) A5-Androstene-3,3-acetoxy-7,16,17-trione tion outlined in Ikota, N.; Ganem, B. Jour. Org. Chenn., A5-Androstene-3,6-acetoxy-7,16,17-trione (5) may be vol. 43, pp. 1607-1608 (1978). Briefly, the 36-acetoxy synthesized from commercially available DHEA ace O 16-phenylseleno-A5-androstene-7,17-dione (53a) is se tate by sequentially converting: quentially reacted with m-chloroperbenzoic acid and acetic anhydride in a suitable solvent. 3,3-acetoxy-A5 3,3-acetoxy-A5-androstene-17-one (DHEA-acetate) androstene-16-phenylseleno-16-m-chlorobenzoate-7, 17 to dione (54) is also synthesized during this reaction. 36-acetoxy-A5-androstene-7,17-dione (51) 15 3,3,16-diacetoxy-A5,A15-androstadiene-7,17-dione to 3.8-acetoxy-17-hydroxy-A5, 3,3-trimethylsilylacetoxy (57) may be synthesized from 3p3,16-diacetoxy-A5 20 Al6-androstadiene-7-one 17-hydroxy-A5,A16 androstene-16-phenylseleno-7,17-dione (55) by oxida trimethylsilyl ether (52a) androstadiene-7-one tive dehydrogenation. The 3,6-acetoxy-A5-androstene 30% trimethylsilyl ether (52b) 70% 16-phenylseleno-16-m-chlorobenzoate-7,17-dione (54) to present with (55) produces 36-acetoxy-16-m-chloroben 3p3-acetoxy- 36 trimethylsilylacetoxy 16-phenylseleno zoate-A5,A15-androstadiene-7,17-dione (56) which may 16-phenylseleno A5-androstene be separated by chromatography if desired. A5-androstene 7,17-dione 3,3-acetoxy-A5-androstene-7,16,17-trione (5) may be 7,17-dione (53a) synthesized from the 3,3-acetoxy-A5,A15 androstadiene (53b) 25 to 16-m-chlorobenzoate-7,17-dione (56) and/or 36,16 phenylseleno 3,3-acetoxy-16 diacetoxy-A5,A15-androstadiene-7,17-dione (57) by (53a) A5-androstene treating them with triethylamine in methanol. 7,17-dione (6) A5-Androstene-3,6,16a-dihydroxy-7,17-dione (7- to 3.8-acetoxy-A5-androstene 3,3,16-diacetoxy 30 keto-16a-hydroxy DHEA) 16-phenylseleno A5-androstene-16 3f3,16a-dihydroxy-A5-androstene-7,17-dione can be 6-m-chlorobenzoate phenylseleno-7, 17 synthesized by the following sequence from DHEA dione (55) propionate. DHEA propionate can be made by simple 7,17-dione (54) to esterification of DHEA. 3,3-acetoxy-6-m 36,16-diacetoxy-A5,A15 35 chlorobenzoate-A5,A15 androstadiene-7, 17 dione (57) 3p3-propionoxy-A5-androstene-17 one (DHEA-propionate) androstadiene-7,17-dione to (56). 3,3-propionoxy-A5-androstene-7,17-dione (61) to to 3,3-acetoxy-A5-androstene-7,16,17-trione (5) 3,3-propionoxy-17-hydroxy-A5,A16-androstadiene 40 7-one trimethylsilyl ether (62) to 36-Acetoxy-A5-androstene-7,17-dione (51) can be 3p3-propionoxy-16a-hydroxy-A5 synthesized from 3p3-acetoxy-A5-androstene-17-one androstene-7,17-dione (63) to (DHEA-acetate) by reacting DHEA-acetate with the 3,3,16a-dihydroxy-A5 oxidizing agent CrO3 in accordance with the procedure 45 outlined in Fieser, L. F., Jour. Am. Chem. Soc., vol. 75, androstene-7,17-dione (6) pp. 4386-4394 (1953). A mixture of 70% 36-(trimethylsilyl)acetoxy-17 343-Propionoxy-A5-androstene-7,17-dione (61) (NMR hydroxy-A5,A16-androstadiene-7-one trimethylsilyl set forth at table eight) can be synthesized from 3p3-pro ether (52b) and 30%. 3,3-acetoxy-17-hydroxy-A5,A16 SO pionoxy-A5-androstene-17-one (DHEA-propionate) by androstadiene-7-one trimethylsilyl ether (52a) may be reacting the DHEA-propionate with the oxidizing synthesized from the 3,3-acetoxy-A5-androstene 7, 17 agent CrO3 in accordance with the procedure outlined dione (51) by reacting (51) with lithium diisopropyl in Fieser, L. F., Jour. Am. Chem. Soc., vol. 75, pp amide in the presence of trimethylchlorosilane in a suit 4386-4394 (1953). able solvent such as tetrahydrofuran at -78 C. 55 3,3-Propionoxy-17-hydroxy-A5,A16-androstadiene 3£3-Acetoxy-16-phenylseleno-A5-androstene-7, 17 7-one trimethylsilyl ether (62) may be synthesized from dione (53a) and 343-(trimethylsilyl)acetoxy-16-phenyl the 3(3-propionoxy-A5-androstene-7,17-dione (61) by seleno-A5-androstene-7,17-dione (53b) may be synthe reacting (61) with lithium diisopropyl amide in the pres sized from 3p3-acetoxy-17-hydroxy-A5,A16-androstadi ence of trimethylchlorosilane in a suitable solvent such ene-7-one trimethylsilyl ether (52a) and 3.3-(trimethyl as tetrahydrofuran at -78 C. silyl)acetoxy-17-hydroxy-A5,A16-androstadiene-7-one 3p3-Propionoxy-16a-hydroxy-A5-androstene-7, 17 trimethylsilyl ether (52b) respectively by reacting the dione (63) may be synthesized from the 36-propionoxy mixture of (52a) and (52b) with benzeneselenenyl chlo 17-hydroxy-A5,A16-androstadiene-7-one trimethylsilyl ride in the presence of pyridine and a suitable solvent ether (62) by oxidation with m-chloroperbenzoic acid in such as tetrahydrofuran at -78 C. 65 tetrahydrofuran followed by treatment with a 1N HCl The carbon silylated acetate attached to the C3 car solution. bon atom on the (53b) fraction of the (53) mixture may The final desired product 36,16a-dihydroxy-A5 be desilylated so as to convert (53b) to (53a) within the androstene-7,17-dione (6) may then be synthesized from 5,424,463 7 8 the 3.6-propionoxy-16a-hydroxy-A5-androstene-7, 17 16a-hydroxy-A5-androstene-7,17-dione (63) with a 5% dione (63) by treatment with sulfuric acid in methanol. aqueous sodium carbonate solution in methanol. (7) A5-Androstene-3,6-propionoxy, 166-acetoxy-7, 17 (10) A5-Androstene-3,6,16a, 17(3-triol-7-one -dione (7-Keto-166-acetoxy DHEA propionate) A5-Androstene-3,6,16a, 17(3-triol-7-one (10) can be A5-Androstene-3,6-propionoxy-16p3-acetoxy-7, 17 synthesized from 3(3,16o, 1743-triacetoxy-A5-androstene dione (7) can be synthesized from A5-androstene-3,6- by reacting the 3,9,16a, 17(3-triacetoxy-A5-androstene propionoxy-16a-hydroxy-7,17-dione (63) produced in with the oxidizing agent CrO3 in accordance with the accordance with the reaction sequence set forth above. procedure outlined in Fieser, L. F., Jour. Am. Chem. The A5-androstene-3,6-propionoxy-16a-hydroxy-7, 17 Soc., vol 75, pp. 4386-4394 (1953) with subsequent hy dione is treated in accordance with the Mitsunobu con 10 drolysis of the acetyl groups. ditions set forth in Hughes, D. L.; Reamer, R. A.; Ber Without intending to be unduly limited thereby, it is gan, J. J.; Grabowski, E. J. J. Jour Am. Chem. Soc., vol. believed that the steroids identified above may be modi 110, pp 6487-6491 to invert the configuration and acety fied without loss of biological activity by esterifying late the 16 hydroxy group. one or more of the hydroxyl groups with any of a vari (8) A5-Androstene-3,6,7a, 17.6-triol-16-one 15 ety of organic acids and inorganic acids such as sulfuric A5-Androstene-3,6,7a, 1713-triol-16-one (8) can be or phosphoric acid. synthesized from A5-androstene-3,3,16a-dihydroxy 17-one diacetate (81). The A5-androstene-3,6, 16a-dihy TREATMENT droxy-17-one diacetate (81) starting material can be A subject may be treated with the steroids specified synthesized in accordance with the procedure set forth 20 herein by any of the commonly accepted practices in in Numazawa, M. and Osawa, Y. Steroids, vol. 32, p 519 cluding orally or by injection. While many factors af (1978). fect the dose rate required to attain the desired biologi A5-Androstene-3,6,16a-diacetoxy-7-bromo-17-one cal response, treatment at a dosage rate of about 0.1 to (82) can be synthesized from the A5-androstene-3,6,16a 2 grams, preferably about 0.5 to 2 grams, of the steroid diacetoxy-17-one (81) by reacting A5-androstene 25 per 100 kilograms body weight per day should gener 36,16o-diacetoxy-17-one (81) with a brominating agent, ally be effective for promoting weight control. A dose such as Dibromantin (1,3-dibromo-5,5-dimethylhydan rate of less than 0.1 gram per 100 kilograms bodyweight toin). The A5-androstene-3,6, 16a-diacetoxy-7-bromo per day is generally ineffective for preventing weight gain while a dose rate of greater than about 2 grams per 17-one (82) is unstable and must be used immediately in 30 100 kilograms bodyweight per day increases the cost of the next step of the process. treatment without providing a corresponding benefit in The A5-androstene-3,6,16a-diacetoxy-7-bromo performance. The optimum dose rate to be administered 17-one (82) contains an isomeric mixture of 7a-bromo to a subject is case specific as the optimum dose rate and 7(3-bromo isomers. The isomeric mixture, may be depends upon several factors including current body equilibrated to 7a-bromo in accordance with the 35 composition (percent fat), the desired effect (weight method described for equilibriating a cholesterol deriv gain maintenance versus weight loss), eating habits of ative in Confalone, P. N., Kulesha, I. D., and Us the individual (daily caloric intake), and the like. As kokovic, M. R. Jour. Org. Chem., vol. 46, pp 1030-1032 would be expected, the dose rate provided to a subject (1981). Briefly, the isomeric mixture is contacted with for the purpose of promoting weight loss will be greater cold anhydrous LiBrand shielded from light to convert 40 than that necessary to promote weight maintenance the product from an isomeric mixture of 7a and 7.6 to assuming identical caloric intake under each program. predominently 7a. Without intending to be limited thereby, we believe A5-Androstene-3,6,16d-diacetoxy-7a-hydroxy that the steroids specified herein are metabolic interme 17-one (83) may be synthesized from the A5-androstene diates along the pathway to conversion of DHEA to an 36,16a-diacetoxy-7a-bromo-17-one (82) by reacting the 45 ultimate metabolite(s) which is actually responsible for A5-androstene-3,6,16a-diacetoxy-7a-bromo-17-one (82) mediating an enhanced production of thermogenic en with a mixture of glacial acetic acid and powdered zymes such as glycerol 3-phosphate dehydrogenase and silver acetate at room temperature in a suitable solvent, malic enzyme. such as a mixture of methylene chloride and acetone. A subject may be treated with one of the steroids Also produced in this reaction is a 20% yield of A5 50 specified herein on substantially any time schedule. It is androstene-3,6,7a, 16a-triacetoxy-17-one. believed that the steroids specified herein are effective A5-Androstene-3,3,7a, 1743-trihydroxy-16-one (8) can for promoting weight control while the steroid itself is be synthesized from the A5-Androstene-3,3,16d actively present within the body as well as while the diacetoxy-7a-hydroxy-17-one (83) by dissolving the increased concentration of thermogenic enzyme(s) in A5-Androstene-3,6,16a-diacetoxy-7a-hydroxy-17-one 55 duced by the steroid remain elevated. The in vivo life (83) in a room temperature methanol solution of K2CO3 expectancy of the steroids and the thermogenic en and stirring the solution for two hours. The alkaline zyme(s) induced thereby is not yet fully known. How solution enolizes the 17-keto to form the more stable ever, it is believed that the steroids themselves are not 17-hydroxy-16-one combination. The A5-androstene stored within the body and are removed and/or deacti 3,3,7a, 17-trihydroxy-16-one (8) may then be isolated by 60 vated within days after administration. Accordingly, filtering to remove the insoluble salt, evaporating the for optimum effectiveness, the subject under treatment methanol in vacuo and purifying the compound by should be treated every day or two. For reasons of chromatography. The steroid crystallizes from a hot convenience the subject under treatment may be treated methanol/ether solution upon cooling. less frequently, such as once a week, when less than (9) A5-Androstene-3,6,17(3-diol-7, 16-dione 65 maximum performance is acceptable. A5-Androstene-3,6,17(3-diol-7, 16-dione (9) can be As is apparent from the factors which affect dosage synthesized from 36-propionoxy-16a-hydroxy-A5 and dose rate, each particular subject should be care androstene-7,17-dione (63) by treating 3,3-propionoxy fully and frequently reviewed and the dosage and/or 5,424,463 10 dose rate altered in accordance with the particular situ dichloromethane solution of A5-androstene-3,3,7a-diol ation. 17-one was rotovapped to near dryness, azeotropically EXPERIMENTAL dried with absolute ethanol, and then azeotropically dried twice with acetone. Warm acetone was added to Example I the dried extracted solids until the solids were com (Steroid 1) pletely dissolved to form a sixth mixture. Hexane was Synthesis added to the sixth mixture until the mixture began to cloud at which time crystals of A5-androstene-3,6-7a A5-Androstene-3,6,7a-diol-17-one diol-17-one began to form at room temperature. (Step 1) Into a two liter, triple neck, round bottom A second crop of A5-androstene-3,6-7a-diol-17one flask equipped with a magnetic stirrer and a reflux con crystals was obtained by cooling the remaining sixth denser was placed 1000 ml hexane (b. p 69-71), 10 mixture. grams (0.03 mmoles) DHEA acetate and 13.6 grams The crystals melt at 187-189 C. When recrystal (0.16 moles) NaHCO3 to form a first mixture. The first lized from acetone/hexane they melt at 192-193 C. mixture was placed under a N2 atmosphere and heated 15 Example II under constant agitation to reflux. Into the refluxing (Steroid 1) first mixture was added 6.11 grams (0.021 moles) Dibro mantin (1,3-dibromo-A5,5-dimethylhydantoin) as a bro Synthesis minating agent to form a second mixture. The second A5-Androstene-3,6-7(og)-diol-17-one mixture gradually turned orange after which it rapidly turned a pale white/yellow. The second mixture was A5-Androstene-3,6-7(ag)-diol-17-one was manufac refluxed for 30 minutes, cooled to room temperature tured in accordance with the procedure set forth in and filtered through a sintered glass funnel. The residue Example I except that Step 2 was eliminated with the was rinsed with 50 ml dichloromethane and the com dried filtrate from Step 1 simply resolubilized in the 80 bined filtrate rotovapped to dryness at a temperature of 25 ml of dichloromethane in preparation for Step 3. less than 35 C. The residue (A5-androstene-3,6-ol-7- Example III bromo-17-one) is unstable to storage and was used im (Steroid 2) mediately in step two. (Step 2) The residue was resolubilized in 80 ml of Synthesis dichloromethane in a one liter stoppered flask equipped A5-Androstene-3,6-ol-7,17-dione with a magnetic stirrer and placed in an ice bath. Into the resolubilized filtrate was added 8 grams anhydrous (Step 1) Into a 50 ml flask equipped with a magnetic LiBr in 320 ml ice-cold acetone to form a third mixture. stirrer and a water bath was placed 6.5 ml acetic anhy The third mixture was shielded from light and stirred dride, 23 ml acetic acid, 1.7 grams sodium acetate, and continuously for three hours. The resulting mixture 35 2 grams DHEA acetate to form a first mixture. Into the containing predominantly androstene-3,6-ol-7a-bromo first mixture was added 2 grams chromium trioxide 17-one was allowed to warm to room temperature and over a thirty minute period to form a second mixture. used immediately in step three. The first mixture was maintained at a constant tempera (Step 3) Into a 500 ml flask equipped with a magnetic ture of 56-58 C. and continuously agitated during stirrer was placed 320 ml dichloromethane, 80 ml gla addition of the chromium trioxide. The second mixture cial acetic acid, and 26 grams of silver acetate to form a was maintained at 56-58 C. and continuously agitated first suspension. The first suspension was stirred contin for an additional hour after which the second mixture uously for 20 minutes at room temperature. The stirred was cooled and slowly poured under continuous agita first suspension was added under constant agitation into tion into 600 ml of ice water to form a precipitate. The the third mixture of predominantly A5-androstene-3,6- 45 flocculent precipitate was collected on a sintered glass ol-7a-bromo-17-one to form a second suspension. The funnel and washed with water until no longer green. second suspension was constantly stirred for 30 minutes After drying in vacuo over P2O5 the product was dis at room temperature after which the suspension was solved in hot methanol and crystallized to yield substan filtered through a sintered glass funnel to separate a tially pure A5-androstene-3,3-acetoxy-7,17-dione having solid residue. The filtered solid residue was rinsed with 50 a melting point of 184-185° C. 100 ml dichloromethane. The filtrate was washed three (Step 2) The precipitate was resolubilized in 500 ml of times with 1000 ml of water, once with 1000 ml of a 5% methanol in a one liter, triple necked, round bottom NaHCO3 solution, and then twice more with water. flask equipped with a magnetic stirrer and reflux con The organic mixture containing A5-androstene-3,6-17a denser to form a third mixture. The third mixture was diol-17-one diacetate was then rotovapped to dryness. 55 placed under a N2 atmosphere and heated under con (Step 4) The dried extracted solids were resolubilized stant agitation to reflux. Into the third mixture was in 500 ml methanol in a one liter, triple necked flask added 250 ml of a 5% solution of Na2CO3 to form a equipped with a magnetic stirrer and a reflux condenser fourth mixture. The fourth mixture was refluxed under to form a fourth mixture. The fourth mixture was constant agitation for 45 minutes. The methanol was placed under a N2 atmosphere and heated under con rotovapped off and the aqueous fourth mixture care stant stirring to reflux. Into the fourth mixture was fully brought to a pH of 7 with an appropriate amount added 250 ml of a 5% aqueous solution of Na2CO3 to of glacial acetic acid. The neutralized fourth mixture form a fifth mixture. The fifth mixture was refluxed was extracted with two 100 ml portions of dichloro under constant agitation for 45 minutes. The methanol methane, the two dichloromethane portions combined, was rotovapped off and the aqueous fifth mixture care 65 and the dichloromethane evaporated in vacuo. The fully brought to a pH of 7 with an appropriate amount extracted solids were then azeotropically dried first of glacial acetic acid. The neutralized fifth mixture was with absolute ethanol and then with two separate por extracted twice with 100 ml of dichloromethane. The tions of acetone. Methanol was added to the dried ex 5,424,463 11 12 tracted solids until the solids were completely dissolved fourth mixture was added 250 ml of a 5% aqueous solu to form a fifth mixture. Hexane was added to the fifth tion of Na2CO3 to form a fifth mixture. The fifth mix mixture until the mixture began to cloud at which time ture was refluxed under constant agitation for 45 min crystals of A5-androstene-3,6-ol-7, 17 dione formed at utes. The methanol was rotovapped off and the aqueous room temperature. 5 fifth mixture carefully brought to a pH of 7 with an A second crop of A5-androstene-3,6-ol-7,17-dione appropriate amount of glacial acetic acid. The neutral crystals was obtained by cooling the remaining sixth ized fifth mixture was extracted twice with 100 ml di mixture. chloromethane and the combined extract evaporated in The resultant product had a melting point of vacuo. The extracted solids (A5-androstene-3,6,7a, 17(3- 235-238 C. 10 triol) were azeotropically dried with absolute ethanol Example IV and then twice with acetone. Warm acetone was added (Steroid 3) to the dried extracted solids until the solids were com Synthesis pletely dissolved to form a sixth mixture. Hexane was added to the sixth mixture until the mixture began to A5-Androstene-3,6,7a, 1713-triol 5 cloud, at which time crystals of A5-androstene (Step 1) Into a two liter round bottom flask equipped 3,3,7a, 1713-triol formed at room temperature. with a magnetic stirrer and a reflux condenser was A second crop of A5-androstene-3,6,7a, 17(3-triol placed 1000 ml hexane (b. p 69-71), 10 grams (0.03 crystals was obtained by cooling the remaining sixth moles) A5-androstene-3,6-17(3-diol diacetate and 13.6 mixture. grams (0.16 moles) NaHCO3 to form a first mixture. 20 Example V The first mixture was placed under a N2 atmosphere and (Steroid 3) heated under constant agitation to reflux. Into the re fluxing first mixture was added 6.11 g (0.021 moles) Synthesis Dibromantin (1,3-dibromo-5,5-dimethylhydantoin) as a brominating agent to form a second mixture. The sec- 25 A5-Androstene-3,3,7(og), 1743-triol ond mixture gradually turned orange after which it A5-Androstene-3,6,7(ag), 17(3-triol was manufactured rapidly turned a pale white/light yellow. The second in accordance with the procedure set forth in Example mixture was refluxed for 30 minutes, cooled to room IV except that Step 2 was eliminated with the dried temperature, and filtered through a sintered glass fun filtrate from Step 1 simply resolublized in the 80 ml of nel. The residue was rinsed with 50 ml dichloromethane 30 methylene chloride in preparation for Step 3. and rotovapped to dryness at a temperature of less than Example VI 35° C. The dry filtrate (A5-androstene-3,6-176-diol-7- (Steroid 4) bromide) is unstable to storage and was used immedi ately in step two. Synthesis (Step 2) The dried filtrate was resolubilized in 80 ml 35 A5-Androstene-3,6,17g-diol-7-one of dichloromethane in a flask equipped with a magnetic stirrer and placed in an ice bath. Into the resolubilized (Step 1) Into a 50 ml flask equipped with a magnetic filtrate was added 8 grams anhydrous LiBr in 320 ml stirrer and a water bath was placed 6.5 ml acetic anhy ice-cold acetone to form a third mixture. The third dride, 23 ml acetic acid, 1.7 grams sodium acetates and mixture was shielded from light and stirred continu- 40 2 grams androstenediol diacetate to form a first mixture. ously for three hours. The resulting mixture of predomi Into the first mixture was added 2 grams chromium nantly A5-androstene-3,6-179-diol-7a-bromide was al trioxide over a thirty minute period to form a second lowed to warm to room temperature and used immedi mixture. The first mixture was maintained at a constant ately. temperature of 56-58 C. and continuously agitated (Step 3) Into a 500 ml flask equipped with a magnetic 45 during addition of the chromium trioxide. The second stirrer was placed 320 ml methylene chloride, 80 ml mixture was maintained at 56-58 C. and continuously glacial acetic acid, and 26 grams silver acetate to form a agitated for an additional hour after which the second first suspension. The first suspension was stirred contin mixture was cooled and slowly poured with continuous uously for 20 minutes at room temperature. The stirred agitation into 600 ml of ice water to form a precipitate. first suspension was added under constant agitation to 50 The flocculent precipitate was filtered through a sin the third mixture of predominantly A5-androstene-3,6- tered glass funnel, washed with water until no longer 17(3-diol-7a-bromide to form a second suspension. The green, and dried in vacuo. second suspension was constantly stirred for 30 minutes (Step 2) The dried precipitate was resolubilized in 500 at room temperature during which the suspension grad ml of methanol in a one liter, round bottom flask ually darkened and was then filtered through a sintered 55 equipped with a magnetic stirrer and reflux condenser glass funnel. The residual solids retained on the glass to form a third mixture. The third mixture was placed filter were rinsed with 10 ml dichloromethane. The under a N2 atmosphere and heated under constant agita filtrate was washed three times with 1000 ml of water, tion to reflux. Into the third mixture was added 250 ml neutralized with 1000 ml of a 5% NaHCO3 solution, and of a 5% aqueous solution of Na2CO3 to form a fourth then washed twice more with water. The resulting 60 mixture. The fourth mixture was refluxed under con organic mixture containing A5-androstene-3,6,7a, 1743 stant agitation for 45 minutes. The methanol was roto triol-3,17-diacetate was then rotovapped to dryness. vapped off and the aqueous fourth mixture carefully (Step 4) The dried extracted solids were resolubilized brought to a pH of 7 with an appropriate amount of in 500 ml methanol within in a one liter, triple necked, glacial acetic acid. The neutralized fourth mixture was round bottom flask equipped with a magnetic stirrer 65 extracted twice with 100 ml portions of dichlorometh and a reflux condenser to form a fourth mixture. The ane and the combined dichloromethane extract evapo fourth mixture was placed under a N2 atmosphere and rated in vacuo. The extracted solids were then azeo heated under constant agitation to reflux. Into the tropically dried first with absolute ethanol and then 5,424,463 13 14 twice with acetone. Methanol was added to the dried 7-one trimethylsilyl ether (52a). The results of the NMR extracted solids until the solids were completely dis analysis are set forth in Tables One & Two. solved to form a fifth mixture. Hexane was added to the (Step 6) Into a fourth 100 ml round bottom flask fifth mixture until the mixture began to cloud at which equipped with a magnetic stirrer and retained within a time crystals of A5-androstene-3,6, 1713-diol-7-one dry-ice bath was placed 2.85 moles of the organic solid formed at room temperature. obtained in Step 5, 20 ml neat tetrahydrofuran and 0.320 The resultant product had a melting point of ml (4.00 mmole) neat pyridine to form a seventh mix 200-202 C. ture. The seventh mixture was cooled to -78 C. and Example VII placed under a N2 atmosphere. Into the seventh mixture (Steroid 5) 10 was added 0.710 grams (3.71 moles) of benzeneselene Synthesis nyl chloride in 4 ml of tetrahydrofuran to form an eighth mixture. A5-Androstene-3,6-acetoxy-7, 16, 17-trione (Step 7) Into a separatory funnel was placed a cosol (Step 1) Into a 50 ml flask equipped with a magnetic vent system of a 0.5N HCl aqueous solution and dichlo stirrer and a water bath was placed 6.5 ml acetic anhy 5 romethane. Into the cosolvent system was added the dride, 23 ml acetic acid, 1.7 grams sodium acetate, and eighth mixture to extract the organic phase. The second 2 grams DHEA acetate to form a first mixture. Into the organic phase was extracted by dichloromethane, se first mixture was added 2 grams chromium trioxide quentially washed with water and a saturated NaHCO3 over a thirty minute period to form a second mixture. aqueous solution, dried over Na2SO4 and the solvent The first mixture was maintained at a constant tempera removed to yield 1.72 grams of an organic oil. The ture of 56-58 C. and continuously agitated during organic oil was separated by chromatography (100 addition of the chromium trioxide. grams silica eluted with hexane-ethyl acetate at 90:10 to (Step 2) The second mixture was maintained at 56-58 C. and continuously agitated for an additional 50:50 with 50 ml fractions) into 1.44 grams of first and hour after which the second mixture was cooled and 25 second organic fractions (Fractions 14-17), diphenyl slowly poured under continuous agitation into 600 ml of diselenide (Fractions 9-11) and unreacted acetoxy-A5 ice water to form a precipitate. The flocculent precipi androstene-7,17-dione (51) (Fractions 18-19). The first tate was collected on a sintered glass funnel and washed and second organic fractions were identified by NMR with water until no longer green. After drying in vacuo as 343-acetoxy-16*-phenylseleno-A5-androstene-7, 17 over P2O5, the product was dissolved in methanol and 30 dione (53a) and 343-(trimethylsilyl)acetoxy-16-phenyl recrystallized to yield substantially pure 3,3-acetoxy seleno-A5-androstene-7,17-dione (53b) respectively. A5-androstene-7,17-dione (51) having a melting point of The results of the NMR analysis are set forth in Tables 184-185 C. Three and Four. (Step 3) Into a second 50 ml round bottom flask (Step 8) Into a fifth 100 ml round bottom flask equipped with a magnetic stirrer and retained within a 35 equipped with a magnetic stirrer was placed 25 ml dry-ice bath was placed 1.00 gram (2.90 mmoles) of the ether, 5 ml dichloromethane and 1.19 mmoles of the 3,3-acetoxy-A5-androstene-7,17-dione (51) and 20 ml second organic fraction obtained in Step 7 (53b) to form neat tetrahydrofuran to form a third mixture, The third a ninth mixture. Into the ninth mixture was added 8 ml mixture was placed under a N2 atmosphere. Into the of a 10% aqueous solution of potassium fluoride and 3 third mixture was added, 1.07 ml (8.43 mmoles) trime ml of a 1M tetra-n-butylammonium fluoride solution in thylchlorosilane to form a fourth mixture. The fourth tertrahydrofuran to form a tenth mixture. The tenth mixture was maintained under the N2 atmosphere and mixture was maintained at 25 C. and continuously cooled to -78 C. agitated for two hours after which the tenth mixture (Step 4) Into a third 25 ml round bottom flask was poured into an ether-hexane-water solvent system equipped with a magnetic stirrer and retained within a 45 to extract the organic phase. The extracted organic dry-ice bath was placed 1.07 ml (7.66 minoles) diisopro phase was washed twice with water, once with a satu pylamine, 3.60 ml of a 1.94M solution of n-butyllithium rated NaCl aqueous solution, dried over Na2SO4 and in hexane (6.96 mmoles), and 4 ml of tetrahydrofuran to the solvent removed to yield 0.594 grams of 3,3- form lithium diisopropyl amide. The solution (LDA acetoxy-16-phenylseleno-A5-androstene-7,17-dione solution) was prepared under a N2 atmosphere at -78 (53a). C. The LDA solution was warmed slightly to dissolve (Step 9) Into a sixth 100 ml round bottom flask any solids therein and then added, under a N2 atmo equipped with a magnetic stirrer was placed 25ml di spheres to the fourth mixture via cannula to form a fifth chloromethane and 1.10 mmoles of 3,3-acetoxy-16 mixture. phenylseleno-A5-androstene-7,17-dione (53a) to form (Step 5) The fifth mixture was removed from the 55 an eleventh mixture. The eleventh mixture was cooled dry-ice bath and allowed to warm to room temperature to O C. and maintained under continuous agitation. Into for 15 minutes at which time 1.25 ml triethylamine was the eleventh mixture was added 0.296 grams (1.2 added to the fifth mixture to form a sixth mixture. Into mmoles) m-chloroperbenzoic acid (70%) followed five a separatory funnel was placed 40 ml hexane, 40 ml of a minutes later by 0.40 ml dimethylsulfide to form a thir saturated NaHCO3 aqueous solution, and the sixth mix teenth mixture. The thirteenth mixture was washed 3 ture. The organic phase was extracted with hexane, times with 50 ml of a cold NaHCO3 aqueous solution washed with a saturated NaCl aqueous solution, dried and filtered through Na2SO4. over Na2SO4 and the solvent removed to yield 1.42 (Step 10) Into a seventh 250 ml round bottom flask grams of a dry organic solid. The organic solid was equipped with a magnetic stirrer was placed 1.2 ml identified by NMR (CDCl3) as an approximately 70:30 65 acetic anhydride, 1.2 ml pyridine and the liquid phase of mixture of 36-(trimethylsilyl)acetoxy-17-hydroxy the thirteenth mixture to form a fourteenth mixture. A5,A16-androstadiene-7-one trimethylsilyl ether (52b) The fourteenth mixture was continuously agitated at and 3,3-acetoxy-17-hydroxy-A5,A16-androstadiene room temperature for an hour after which 60 ml of a 5,424,463 15 16 saturated NaHCO3 aqueous solution was added to form bath. Into the first mixture was placed 2.95 mmoles of a fifteenth mixture having separate organic and inor m-chloroperbenzoic acid (80-90%) to form a second ganic layers. The fifteenth mixture was maintained mixture. The second mixture was warmed to 25 C. and under continuous agitation until bubbling ceased. The maintained under constant agitation for 10 min. organic layer was separated from the inorganic layer by (Step 2) The second mixture was continuously agi drawing off the organic layer in a separatory funnel. tated and 40 ml of a 1N HCl aqueous solution added to The separated organic layer was washed with a satu form a third mixture which was maintained under con rated NaHCO3 aqueous solution, dried over Na2SO4 stant agitation for 20 min. The third mixture was parti and the solvent removed to yield 0.713 grams of a dry tioned between ether and water and separated by de organic solid. The organic solid was identified by NMR 10 canting the ether phase from the water phase. The ether (CDCl3) as a 90:10 mixture of 3,3,16-diacetoxy-16 phase was washed with a saturated NaHCO3 aqueous phenylseleno-A5-androstene-7,17-dione (55) and 3p3 solution, then a saturated NaCl aqueous solution, and acetoxy-A5-androstene-16-phenylseleno-16-m- dried in vacuo to yield an organic solid. The organic chlorobenzoate-7,17-dione (54). The results of an NMR solid was purified by chromatography (140gm of silica analysis of the (55) fraction in the mixture are set forth 15 in Table Five. eluted with 60:40 to 70:30 ethyl acetate-hexane with 25 (Step 11) Into a round bottom flask equipped with a ml fractions) to give 0.586 gm of a first organic com magnetic stirrer was placed 25 ml carbon tetrachloride pound. The first organic compound was identified by and 1.10 mmoles of the 90:10 mixture of 3(3,16 NMR as 3,3-propionoxy-16a-hydroxy-A5-androstene diacetoxy-6-phenylseleno-A5-androstene-7,17-dione 7,17-dione (63). The results of the NMR analysis are set (55) and 36-acetoxy-A5-androstene-16-phenylseleno 20 forth in Table Ten. 16-m-chlorobenzoate-7,17-dione (54) to form a six (Step 3) In a second round bottom flask equipped teenth mixture. Into the sixteenth mixture was added with a magnetic stirrer was placed 0.155 mmoles of 0.12 ml pyridine, 10 mg benzeneseleninic acid, and 19 36-propionoxy-16a-hydroxy-A5-androstene-7,17-dione mg diphenyldiselenide to form a seventeenth mixture. 25 (63) dissolved in 6 ml of methanol to form a third mix The seventeenth mixture was maintained under vigor ture. To this third mixture was added 1.5 ml of an aque ous agitation and 3.4 ml of a 15% H2O2 aqueous solution ous solution of 6N sulfuric acid to form a fourth mixture added to form an eighteenth mixture. The eighteenth which was maintained at 25 C. for 18 hours. The fourth mixture was agitated continuously for 45 minutes after mixture was then partitioned between ethyl acetate and which the eighteenth mixture was washed twice with a water and separated by decanting the ethyl acetate saturated NaHCO3 aqueous solution and dried in vacuo 30 phase from the water phase. The ethyl acetate was to yield 0.390 grams of an organic solid. The organic evaporated to yield an organic product which was puri solid was separated by chromatography (50 grams silica fied by chromatography (silica preparative plate eluted gel eluted with 50% hexane-ethyl acetate with 25 ml three times with 60% ethyl acetate-hexane) to yield 18 fractions) to yield 0.236 grams of an organic fraction mg of an organic compound which was then dissolved (Fractions 9-10). The organic fraction was identified by 35 in and crystallized from methanol to give 6 mg of sub NMR as pure 3,3,16-diacetoxy-A5,A15-androstadiene stantially pure material having a melting point of 7,17-dione (57). The results of the NMR analysis are set 235-239 C. The organic compound was identified by forth in Table Six. NMR as 3,3,16a-dihydroxy-A5-androstene-7,17-dione (Step 12) Into a round bottom flask equipped with a (6). The results of the NMR analysis are set forth in magnetic stirrer was placed 1.03 mmoles of 36,16 40 Table Eleven. diacetoxy-A5,A15-androstadiene-7,17-dione (57) and 15 Example IX ml methanol to form a nineteenth mixture. Into the (Steroid 7) nineteenth mixture was placed 0.500 ml triethylamine to form a twentieth mixture. The twentieth mixture was Synthesis placed under a N2 atmosphere and maintained under 45 A5-Androstene-3,6-propionoxy-1613-acetoxy-7,17-dione constant agitation for 16 hours. The twentieth mixture was then extracted between H2O and CH2Cl2. The (Step 1) Into a round bottom flask equipped with a organic layer was washed twice with a saturated NaH magnetic stirrer was placed 1.52 mmoles of 3,3-pro CO3 aqueous solution, and then dried and evaporated pionoxy-16a-hydroxy-A5-androstene-7,17-dione, 3.0 under reduced pressure to yield 0.281 grams of a yellow 50 mmoles of triphenylphosphine, 6.0 mmoles of acetic organic solid. The organic solid was identified by NMR acid in 11 ml of tetrahydrofuran. This first mixture was as a mixture of organic compounds including about placed under N2 and cooled to 0°C. in an ice bath. To 65% 36-acetoxy-A5-androstene-7,16,17-trione (5). The the first mixture was added 3.0 mmoles of diethylazodi results of the NMR analysis are set forth in Table carboxylate dropwise to form a second mixture. The Seven. 55 second mixture was warmed to 25 C. and maintained at Example VIII this temperature for 18 hr. It was then partitioned be tween ether-hexane and water. The ether-hexane layer (Steroid 6) was washed with waters water, saturated NaHCO3 Synthesis aqueous solution, saturated NaCl aqueous solution, 60 dried over Na2SO4 and evaporated to give an organic 3,3,16a-dihydroxy-A5-androstene-7,17-dione solid which was purified by chromatography on 130gm (Step 1) Into a round bottom flask equipped with a silica eluted with ethyl acetate-hexane (20:80 to 50:50) magnetic stirrer was placed 2.80 mmoles of 3,3-pro with 25 ml fractions and crystallized from dichlorome pionoxy-17-hydroxy-A5,A16-androstadiene-7-one tri thane-hexane to yield 0.267 gm of an organic com methylsilyl ether (62) synthesized in accordance with 65 pound. The organic compound was identified by NMR the procedure set forth in Example VII and identified aS 36-propionoxy-16p3-acetoxy-A5-androstene-7, 17 by NMR (See Table Nine) and 40 ml of tetrahydrofuran dione containing ~20% of the 16a epimer. The results to form a first mixture which was cooled to 0 in an ice of the NMR analysis are set forth in Table Twelve. 5,424,463 17 18 (Step 2) Conversion to A5-androstene-3,6,166-dihy two hours. The alkaline solution enolized the 17-keto to droxy-7,17-dione can be done as described in Example form the more stable 17-hydroxy-16-one compound. VIII. A5-Androstene-3,6,7a, 17-trihydroxy-16-one (8) was Example X isolated from the fifth mixture by filtering to remove the (Steroid 8) insoluble salts evaporating the methanol in vacuo, puri Synthesis fying the organic residue over silica gel and crystalliz ing the organic compound from methanol-ethyl ether A5-Androstene-3,3,7a, 179-triol-16-one solution. The crystallized organic fraction (180 mg, (Step 1) Into a 100 ml round bottomed flask was 56%; mp:) 230 C.) was identified by NMR as A5 placed 3 grams (7.7 mmoles) of 3,3,16d-diacetoxy androstene-3,6,7a, 17-trihydroxy-16-one (8). The results DHEA (prepared in accordance with the procedure set of the NMR analysis are set forth in Table Fifteen. forth in Numazawa, M. and Osawa, Y. Steroids, vol. 32, Example XI p 519 (1978)) and 3.5 grams NaHCO3 in 50 ml of hexane (Steroid 9) to form a first mixture, The first mixture was stirred and heated to reflux under a N2 atmosphere. To the first 15 Synthesis mixture was added 1.6 grams dibromantin (1,3- A5-Androstene-3,6, 1713-diol-7, 16-dione dibromo-5,5-dimethylhydantoin) to form a second mix te. (Step 1) Into a round bottom flask equipped with a (Step 1) The second mixture was stirred, refluxed for magnetic stirrer was added 1.05 grams (2.80 mmoles) 30 minutes, and then cooled to room temperature. The A5-androstene-3,6-propionoxy-16a-hydroxy-7,17-dione refluxed second mixture was filtered to remove solids (63), 80 ml methanol and 40 ml of a 5% aqueous Na2 and washed with CH2Cl2. The resultant filtrate was CO3 solution while stirring rapidly to form a first mix concentrated to near dryness in vacuo using a water ture. The first mixture was stirred for 42 hours after bath maintained below 35 C. which the methanol was evaporated and a combination (Step 3) The dried filtrate was resolubilized in 21 ml 25 of 100 ml of water and 2 ml of acetic acid added to form of toluene in a one liter stoppered flask equipped with a a second mixture. A solid material was filtered from the magnetic stirrer and placed in an ice bath. Into the second mixture, resolubilized in methanol and then resolubilized filtrate was added 2.1 grams anhydrous crystalized to yield 0.324 gm of an organic compound. LiBr in 80 ml ice-cold acetone to form a third mixture. A small sample of the organic compound was again The third mixture was shielded from light and stirred recrystalized from methanol to produce a purified sam continuously for three hours at 0° C. The resulting ple having a melting point of 215-218 C. The first mixture containing predominantly 7a-bromo was used organic compound was identified by NMR as andros immediately in step four. tene-3,3,1713-diol-7, 16-dione. The results of the NMR (Step 4) Into a 500 ml flask equipped with a magnetic analysis are set forth in Table Sixteen. stirrer was placed 80 ml dichloromethane, 21 ml glacial 35 acetic acid, and 6.7 grams of silver acetate to form a first TABLE ONE suspension. The first suspension was stirred continu NMR Results ously for 20 minutes at room temperature. The stirred 3,3-acetoxy-17-hydroxy-A5,A16-androstadiene first suspension was added under constant agitation into 7-one-trimethylsilyl ether (52a) the warmed third mixture to form a second suspension. Conditions Peak Significance (8) CDCl3 0.21 s, O-SiMe3 The second suspension was constantly stirred for 30 270 MHz minutes at room temperature after which the suspension 0.87 s, 18-CH3 was filtered through a sintered glass funnel to separate 125 s, 19-CH3 a solid residue. The filtrate was concentrated to yield an 2.07 s, CH3COO oily residue. 45 2.75 ddd, J = 14, 6, 3, Hz 15-H (Step 5) To the oily residue was added 300 ml H2O 4.54 dd, J = 3, 1.5 Hz and sufficient NaHCO3 to achieve a fourth mixture with 16-H a neutral pH. The fourth mixture was extracted five 4.73 tt, J = 11, 5 Hz times with 150 ml of ethyl acetate, the organic layers 3a-H combined, washed with brine, dried over MgSO4 and 5.76 d, Js-2 Hz concentrated to dryness. 6-H (Step 6) The crude organic phase was separated by chromatography (silica gel eluted with ethyl acetate: TABLE TWO pet ether at 1:3, 1:2 and 1:1) to yield 700 mg (20%) of a NMR Results first organic fraction and 1.5 grams (48%) of a second 55 3{3-(trimethylsilyl)acetoxy-17-hydroxy organic fraction. After crystallization from diethyl A5,A16-androstadiene-7-one trimethylsilyl ether (52b) ether the first (mp 170°-172 C.) and second (mp Conditions Peak Significance 155-158 C.) organic fractions were identified by (8) CDCl3 0.13 s, C-SiMe3 NMR as A5-androstene-3,6,7a, 16a-triacetoxy-17-one 270 MHz and the corresponding A5-androstene-3,3,16a 60 0.21 s, O-SiMes diacetoxy-7a-ol-17-one (83) respectively. The results of 0.87 s, 18-CH3 1.25 s, 19-CH3 the NMR analysis of these two compounds are set forth 90 s, CH2-Si in Tables Thirteen and Fourteen. 2.75 ddd, J = 14, 6, 3, Hz (Step 7) Into a flask equipped with a magnetic stirrer So-H was added 400 mg (1 mmole) of the A5- Androstene 65 4.54 dd, J = 3, 1.5 Hz 3g,16a-diacetoxy-7a-hydroxy-17-one (83) and 342 mg 16-H 4.73 tt, J = 11, 5 Hz of K2CO3 in 25 ml of methanol at room temperature to 3a-H form a fifth mixture. The fifth mixture was stirred for 5.76 d, J-2 Hz

5,424,463 23 24 moting the synthesis of sex hormones comprising A5 TABLE SIXTEEN androstene-3,6-acetoxy-7,16,17-trione. NMR Results 2. A biologically active steroid effective for promot 3,3,17g-dihydroxy-A5 ing weight control without suppressing appetite or pro androstene-7, 6-dione (9) 5 moting the synthesis of sex hormones comprising A5 Conditions Peak Significance androstene-3,3,16a-dihydroxy-7,17-dione. (8) CDCl3 0.75 s, 18-CH3 3. A biologically active steroid effective for promot 270 MHz ing weight control without suppressing appetite or pro 1.26 s, 19-CH3 moting the synthesis of sex hormones comprising A5 3.5 ddd, J = 19, 7, 10 androstene-3,6-propionoxy-166-acetoxy-7,17-dione. 1.5 Hz 4. A biologically active steroid effective for promot 15d-H ing weight control without suppressing appetite or pro 3.72 tt, J = 10, 4.5 Hz moting the synthesis of sex hormones comprising A5 3o-H androstene-3,6,7a, 17(3-triol-16-one. 3.78 broads 15 5. A biologically active steroid effective for promot 17d-H ing weight control without suppressing appetite or pro 5.77 d, J = 1.5 Hz moting the synthesis of sex hormones comprising A5 6-H androstene-3,6,17a-diol-7, 16-dione. 6. A biologically active steroid effective for promot 20 ing weight control without suppressing appetite or pro We claim: moting the synthesis of sex hormones comprising A5 1. A biologically active steroid effective for promot androstene-3,6,16a, 17,3-triol,7-one. ing weight control without suppressing appetite or pro ck ck ck k sk

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65 UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT No. 5,424,463 DATED June 13, 1995 INVENTOR(S) :dy et al it is certified that error appears in the above-indentified patent and that said Letters Patent is hereby Corrected as shown below:

Col. 24, Line18, “17o” should be -17B

Signed and Sealed this Twenty-ninth Day of October 1996

BRUCELEHMAN Attesting Officer Commissioner of Patents and Trademarks