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USOORE39049E (19) United States (12) Reissued Patent (10) Patent Number: US RE39,049 E Black et al. (45) Date of Reissued Patent: *Mar. 28, 2006

(54) METHODS FOR INHIBITING BONE LOSS 5,591,753 A 1/1997 Black et al. 5,624,940 A 4/1997 Bryant et al. (75) Inventors: Larry J. Black, Franklin, IN (US); 5,629,425 A 5/1997 LaBell et al. George J. Cullinan, Trafalgar, IN (US) 5,641,790 A 6/1997 Draper 5,646,137 A 7/1997 Black et al. (73) Assignee: Eli Lilly and Company, Indianapolis, 5,731,327 A 3, 1998 Luke IN (US) 5,747,510 A 5/1998 Draper 5,811,120 A 9, 1998 Gibson et al. (*) Notice: This patent is Subject to a terminal dis 5,972,383 A 10, 1999 Gibson et al. claimer. 6,087,378 A 7/2000 Cullinan et al. 6,096,781 A 8, 2000 Cullinan 6,156,786 A 12/2000 Cullinan (21) Appl. No.: 10/375,341 6,395,769 B1 5, 2002 Cullinan (22) Filed: Feb. 27, 2003 6,458,811 B1 10/2002 Arbuthnot et al. 6,713,494 B1 3/2004 Cuffet al. Related U.S. Patent Documents Reissue of: FOREIGN PATENT DOCUMENTS (64) Patent No.: 5,393,763 EP OO62.505 10, 1982 Issued: Feb. 28, 1995 EP O68563 * 1 1983 Appl. No.: 08/180,522 EP 0605193 T 1994 Filed: Jan. 12, 1994 GB 2097788 11, 1982 NO 3O4924 12/1992 U.S. Applications: WO WO93/101.13 5, 1993 (63) Continuation of application No. 07/920,933, filed on Jul. 28, WO WO98, 101.13 5, 1993 1992, now abandoned. WO WO 93/10741 6, 1993 (51) Int. Cl. OTHER PUBLICATIONS A6 IK 3/44 (2006.01) Williams, et al., Journal of Bone and Mineral Research, A6 IK 3/40 (2006.01) 6(1991).* A6 IK 37/06 (2006.01) FDC Reports T&G 11, Mar. 30, 1992.* Jones, et al., J. Med Chem., 27, 1057–1066 (1984).* (52) U.S. Cl...... 514/333; 514/422:514/578; Feldman, et al., Bone and Mineral 7, 245-254 (1989).* 514/443; 514/448 “ Trial Restricted”, Scrip No. 1702 Mar. 20, 1992, (58) Field of Classification Search ...... 514/333, p. 22.* 514/337, 443, 448 Jordan, et al., Research Treatment, 10:31–35 See application file for complete search history. (1987).* Beall, et al., CalcifTissue Int. 36: 123–125 (1984).* (56) References Cited Turner, et al., Journal of Bone and Mineral Research 2, No. U.S. PATENT DOCUMENTS 5, 449 456 (1987).* Williams, et al., Bone and Mineral 14, 205–220 (1991).* 4,133,814 A 1/1979 Jones et al...... 548/525 Turner, et al., Endocrinology, 122, No. 3, 1146–1150 4,185,108 A 1, 1980 Samour et al...... 424,274 (1988).* 4,380,635 A 4, 1983 Peters Love, et al., The New England Journal of Medicine 326, No. 4.418,068 A * 1 1/1983 Jones ...... 514,337 13, 852-856 (1992).*

4.729.999 A * 3/1988 Young ...... 514,227 4,894.373 A * 1/1990 Young ...... 514,239.2 Banks PK, Meyer K. Brodie AM H. Regulation of Ovarian 4,970,237 A * 11/1990 Jensen et al...... 514,651 Biosynthesis by During Proestrus in the 5,011,853 A 4/1991 Olney Rat. Endocrinology 1991;129(3):1295–1304. 5,075,321 A 12/1991 Schreiber Black LJ, Jones CD, Falcone JF. Antagonism of Estrogen 5,118,667 A 6, 1992 Adams Action with a New Benzothiophene Derived . 5,208,031 A 5/1993 Kelly Life Sciences 1983:32:1031–1036. 5,393,763 A 2f1995 Black et al. 5,395,842 A 3, 1995 Labrie et al. (Continued) 5,441,947 A 8/1995 Dodge et al. 5,445,941 A 8/1995 Yang Primary Examiner Edward J. Webman 5,457,116 A 10, 1995 Black et al. (74) Attorney, Agent, or Firm Woodard, Emhardt, 5,457,117 A 10, 1995 Black et al. Moriarty, McNett & Henry LLP 5,461,065. A 10, 1995 Black et al. 5,462,949 A 10, 1995 Jones et al. (57) ABSTRACT 5,464,845 A 11/1995 Black et al. 5,468,773. A 11/1995 Dodge et al. The current invention provides methods and pharmaceutical 5,472,962 A 12/1995 Koizumi et al. formulations that are useful for inhibiting the loss of bone. 5,478,847 A 12/1995 Draper These methods and formulations can be used without the 5,482,949 A 1/1996 Black et al. associated adverse effects of estrogen therapy, and thus serve 5,510,370 A 4, 1996 Hock as an effective and acceptable treatment for osteoporosis. 5,534,527 A 7, 1996 Black et al. 5,567,713 A 10, 1996 Cullinan et al. 32 Claims, No Drawings US RE39,049 E Page 2

OTHER PUBLICATIONS Kleinberg DL, Todd J. Babitsky G. Inhibition by of the lactogenic effect of in primate mammary tissue: Buzdar AU, Marcus C, Holmes F. Hug V, Hortobagyi G. reversal by LY 156758 and tamoxifen. Proc. Phase II Evaluation of LY.156758 in Metastatic Breast Natl Acad. Sci. U.S.A. 1983:80:4144–4148. Cancer. Oncology 1988:45(5):344–345. Knecht M, Brodie AM H., Catt K.J. Aromatase inhibitors Chou Y. Iguchi T. Bern H. Effects of Antiestrogens on Adult prevent granulosa cell differentiation: an obligatory role for and Neonatal Mouse Reproductive Organs. Reprod Toxicol in luteinizing hormone receptor expression. Endo 1992:6(5):439–46. crinology 1985;117(3): 1156–1161. Clemens JA, Bennett DR, Black LJ, Jones CD. Effects of a New Antiestrogen, Keoxifene (LY156758), on Growth of Knecht M. Tsai Morris CH, Catt K.J. Estrogen Dependence Carcinogen-Induced Mammary Tumors and on LH and of Luteinizing Hormone Receptor Expression in Cultured Prolactin Levels. 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Effects of triiodot Gottardis MM, Ricchio ME, Satyaswaroop PG, Jordan VC. hyronine, dexamethasone and estradiol-17? on GH mRNA Effect of steroidal and antiestrogens on the in rat pituitary cells in culture as revealed by in situ growth of a tamoxifen-Stimulated human endometrial car hybridization. Acta Endocrinol 1991;124(1):83–90. cinoma (EnCa101) in athymic mice. Cancer Res McArdle CA, Schomerus E, Groner I, Poch A. Estradiol 1990:50(11):3189 3192. regulates -releasing hormone receptor number, Gray JM, Ziemian L. Antiestrogen binding sites in brain and growth and inositol phosphate production in CT3–1 cells. pituitary of ovariectomized rats. Brain Res Mol. Cell. Endocrinol 1992:87(1-3):95-103. 1992:578(1-2):55–60. Meisel RL, Dohanich GP, McEwen BS, Pfaff DW. Antago Hubert J. F. Vincent A, Labrie F. Estrogenic activity of nism of sexual behavior in female rats by ventromedial phenol red in ray anterior pituitary cell in culture. Biochem. hypothalamic implants of antiestrogen. 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Ortmann O. Emons G. Knuppen R. Catt K.J. Inhibitory Tsai P-S, Uchima F. D.A., Hamamoto ST, Bern HA. Pro actions of keoxifene on luteinizing hormone secretion in liferation and differentiation of prepubertal mouse vaginal pituitary gonadotrophs. Endocrinology epithelial cells in vitro and the specificity of estrogen-in 1988:123(2):962–968. duced growth retardation. In Vitro Cell. Dev. Biol 1991:27 Ortmann O. Sturm R. Knuppen R. Emons G. Weak estro A(6):461–468. genic activity of phenol red in the pituitary gonadotroph: Veldhuis JD, Azimi P. Juchter D, Garmey J. Mechanisms re-evaluation of estrogen and antiestrogen effects. J. Steroid Subserving the bipotential actions of estrogen on ovarian Biochem 1990:35(1):17-22. cells; studies with a selective anti-estrogen, LY 156758, and Osborne CK, Hobbs K. Clark GM. Effect of estrogens and the sparingly metabolizable estrogen , . J. 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O'Connor KM, Newboult E. Comparison of Black et al., “Differential Interaction of Antiestrogens with the biological effects of tamoxifen and a new antioestrogen Cytosol Estrogen Receptors.” Molecular and Cellular Endo (LY 1 17018) on the immature rat uterus, J. Endocri, Vol. 99, crinology, 22:1981, 95-103. 1983, pp. 447-453. Black et al., “Evidence for Biological Action of the Anties Beall, PT, Misra LK, Young RL, Sput HJ, Evans HJ, trogens LY117018 and Tamoxifen by Different Mecha Leblanc. Clomiphene Protects Against Osteoporosis in the nisms,” Endocrinology 109; 1981,987–989. Mature Ovariectomized Rat. Calcif. Tissue Int., 36: 123-125 Black, L. J. "Biological Actions and Binding Properties of a (1984). New Estrogen Antagosist LY 1 17018. In: Hormone Antago Feldman S, Minne HW. Parvizi S, Pfeifer M, Lempert UG, mists, 129–45, 1982 (M. K. Agarwal ed.) Walter de Gruyter Bauss F et al. Antiestrogen and administration and Co., Berlin N.Y. reduce bone mass in the rat. Bone and Mineral Black et al., The Antiestrogenic Action of LY 139481: Spe 1989;7(3):245-254. cies Uniformity Duration of Action and Kinetics of Jones, CD, Jevnikar MG, Pike AJ et al., Antiestrogens. 2. 3H-LY139481 Distribution. In Vivo. Sixty-fifth Annual Structure-Activity Studies in a Series of Meeting of the Endocrine Society, San Antonio, Tex., Jun. 3-Aroyl-2-arylbenzobthiophene Derivatives leading to 8–10, 1983, abs. 93. 6-Hydroxy-2-(4-hydroxyphenyl)benzo 6thien-3-yl 4 Black et al., The Relationship of the Antiestrogenic Efficacy 2-(1-piperidinyl)ethoxyphenylmethanone Hydrochlo of LY.156758 to its and Metabolism Fol ride (LY 156758), a Remarkably Effective Estrogen Antago lowing Oral Administration to Adult Ovariectomized Rats, nist with Only Minimal Intrinsic Estrogenicity; J. Med. Seventh International Congress of Endocrinology, Quebec Chem., vol. 27, 1984, pp. 1057–1066. City, Canada, Jul. 1-7, 1984, abs. 323. Jordan, VC, Phelps E, Lindgren, JU. Effects of anti-estro Black et al., Synthesis and Antiestrogenic Activity of gens on bone in castrated and intact female rats. Breast 3,4-Dihydro-2(4-methoxypehnyl)-1-napthalenylA Cancer Res. and Treat 1987:10:31–35. 2-pyrrolidinyl)ethoxyl phenyl methanone, methane Love et al. Effects of Tamoxifen on Bone Mineral Density sulfonic acid salt, Journal of Medicinal Chemistry 22, 1979, in Postmenopausal Women with Breast Cancer. New 962-966. England Journal of Medicine Mar. 26, Williams, et al., Journal of Bone and Mineral Research, 6 1992:326(13):852-856. (1991). Tamoxifen Increases Spinal Bone Mineral Density in Post Copending U.S. Appl. No. 10/375,274, filed Feb. 27, 2003, menopausal Women. FDC Reports, T&G 11, Mar. 30, 1992. Draper et al. Tamoxifen Trial Restricted, Scrip No. 1702, Mar. 20, 1992, Copending U.S. Appl. No. 10/375,339, filed Feb. 27, 2003, p. 22. Black. Turner et al. Tamoxifen Prevents the Skeletal Effects of Barr's Paragraph IV Certification (pp. 1–29) and 2 page Ovarian Hormone Deficiency in Rats. Journal of Bone and letter (Oct. 9, 2002) enclosing the same. Mineral Research, 1987:2(5):449–456. Eli Lilly and Company’s Supplemental Response To Barr's Turner et al. Tamoxifen Inhibits Osteoclast Mediated Interrogatory No. 12 (served on Jun. 28, 2004). Resorption of Trabecular Bone in Ovarian Hormone—Defi Barr Laboratories Second Supplemental Responses to Eli cient Rats. Endocrinology 1988: 122(3):1146–1150. Lilly's Interrogatories No. 5, 6 and 9 (served on Sep. 20. Williams DC, Paul DC, and Black LJ. Effects of estrogen and tamoxifen on serum osteocalcin levels in ovariecto 2004). mized rats. Bone and Mineral, 14 (1991):205–220. * cited by examiner US RE39,049 E 1. 2 METHODS FOR INHIBITING BONE LOSS those tissues having estrogen receptors. Therefore, some antiestrogens are subject to the same adverse effects asso Matter enclosed in heavy brackets appears in the ciated with estrogen therapy. original patent but forms no part of this reissue specifi The current invention provides methods for inhibiting the cation; matter printed in italics indicates the additions 5 loss of bone without the associated adverse effects of estro made by reissue. gen therapy, and thus serves as an effective and acceptable This application is a continuation of application Ser. No. treatment for osteoporosis. 07/920,933, filed on Jul. 28, 1992, now abandoned. The 2-phenyl-3-aroylbenzothiophene compounds that are the active component in the formulations and methods of BACKGROUND OF THE INVENTION 10 this invention were first developed by C. David Jones and This invention relates to the discovery that a group of Tulio Suarez as anti-fertility agents (see U.S. Pat. No. 2-phenyl-3-aroylbenzothiophenes is useful in the prevention 4,133,814, issued Jan. 9, 1979). Certain compounds in the of bone loss. group were found to be useful in Suppressing the growth of The mechanism of bone loss is not well understood, but mammary tumors. in practical effect, the disorder arises from an imbalance in 15 Jones later found a group of related compounds to be the formation of new healthy bone and the resorption of old useful for antiestrogen and antiandrogen therapy, especially bone, skewed toward a net loss of bone tissue. This bone loss in the treatment of mammary and prostatic tumors (see U.S. includes a decrease in both mineral content and protein Pat. No. 4,418,068, issued Nov. 29, 1983). One of these matrix components of the bone, and leads to an increased compounds, the compound of formula I wherein X is a bond, fracture rate of predominantly, femoral bones and bones in R and R' are hydroxyl, and R is a piperidino ring, was the forearm and vertebrae. These fractures, in turn, lead to an clinically tested for a brief time for the treatment of breast increase in general morbidity, a marked loss of stature and cancer. That compound is called raloxifene, formerly keOX mobility, and, in many cases, an increase in mortality ifene. resulting from complications. 25 Bone loss occurs in a wide range of Subjects, including SUMMARY OF THE INVENTION post-menopausal women, patients who have undergone This invention provides new methods for the treatment of hysterectomy, patients who are undergoing or have under bone loss comprising administering to a human in need of gone long-term administration of corticosteroids, patients treatment an effective amount of a compound of formula I Suffering from Cushing's syndrome, and patients having 30 gonadal dysgensis dysgenesis. (I) Unchecked, bone loss can lead to osteoporosis, a major OCHCH-X-R2 debilitating disease whose prominent feature is the loss of bone mass (decreased density and enlargement of bone spaces) without a reduction in bone volume, producing 35 porosity and fragility. One of the most common types of osteoporosis is found in post-menopausal women affecting an estimated 20 to 25 million women in the United States alone. A significant R feature of post-menopausal osteoporosis is the large and 40 rapid loss of bone mass due to the cessation of estrogen production by the ovaries. Indeed, data clearly Support the wherein ability of estrogens to limit the progression of osteoporotic X is a bond, CH, or CHCH: bone loss, and estrogen replacement is a recognized treat RandR', independently, are hydrogen, hydroxyl, C-C- ment for post-menopausal osteoporosis in the United States 45 alkoxy, C-C-acyloxy, C-C-akoxy-C-C-acyloxy, and many other countries. However, although estrogens R-substituted aryloxy, R-substituted aroyloxy, have beneficial effects on bone, given even at very low R-substituted carbonyloxy, chloro, or bromo: levels, long-term estrogen therapy has been implicated in a R is a heterocyclic ring selected from the group consist variety of disorders, including an increase in the risk of ing of a pyrrolidino, piperidino, or hexamethyleneimino; uterine and breast cancer, causing many women to avoid this 50 R is a C-C-alkyl, C-C-alkoxy, hydrogen, or halo; treatment. Recently suggested therapeutic regimens, which and seek to lessen the cancer risk, Such as administering com R" is C-C-alkoxy or aryloxy; or binations of and estrogen, cause the patient to a pharmaceutically acceptable salt thereof. experience regular withdrawal bleeding, which is unaccept The invention also provides pharmaceutical formulations able to most older women. Concerns over the significant 55 for inhibiting bone loss comprising a compound of formula undesirable effects associated with estrogen therapy, and the I, wherein R, R1, R2, and X are as defined above in an limited ability of estrogens to reverse existing bone loss, amount that increases or retains bone density, together with support the need to develop alternative therapy for bone loss a pharmaceutically acceptable carrier. that generates the desirable effects on bone but does not DETAILED DESCRIPTION OF THE cause undesirable effects. 60 Attempts to fill this need by the use of compounds INVENTION commonly known as antiestrogens, which interact with the The current invention concerns the discovery that a group , have had limited Success, perhaps due to of 2-phenyl-3-aroylbenzothiophenes (benzothiophenes) of the fact that these compounds generally display a mixed formula I are useful in the treatment of osteoporosis. The agonist/antagonist effect. That is, although these compounds 65 benzothiophenes of formula I inhibit the loss of bone that can antagonize estrogen interaction with the receptor, the results from a lack of endogenous estrogen such as occurs in compounds themselves may cause estrogenic responses in women following cessation of menstruation due to natural, US RE39,049 E 3 4 Surgical, or other processes. The reduction of bone density Thus, the method of treatment provided by this invention and mass that more rarely occurs in men is also tied to the is practiced by administering to a human in need of inhibi loss of hormonal regulation and is therefore also a target for tion of bone loss, a dose of a compound of formula I or a therapy according to the methods of the current invention. pharmaceutically acceptable salt thereof, that is effective to The benzothiophenes of formula I are a series of nonste inhibit bone loss. A particular benefit of this method is that roidal compounds that exhibit high affinity for conventional it avoids potentially harmful and unacceptable estrogenic estrogen receptors in primary sex target tissues. However, side effects. The inhibition of bone loss contemplated by the they elicit minimal estrogenic responses in those tissues, and present method includes both medical therapeutic and/or actually serve as potent antagonists of natural estrogens Such prophylactic treatment, as appropriate. as estradiol. In contrast to the report of Feldmann, S. et al., 10 The method also includes the administration of a com "Antiestrogen and antiandrogen administration reduce bone pound of formula I given in combination with estrogen. The mass in the rat'. Bone and Mineral. 7:245 (1989), the term estrogen as used herein refers to any compound which benzothiophenes of formula I are able to antagonize classi approximates the spectrum of activities of the naturally cal estrogenic responses in primary sex target tissues with acting molecule which is commonly believed to be 17 B out significantly reducing bone density when given to intact 15 estradiol. Examples of Such compounds include , or estrogen treated animals, and they prevent bone loss in , ethynyl estradiol, Premarin (a commercial prepara estrogen deficient animals. This dichotomy indicates selec tion of isolated from natural Sources— tive agonist/antagonist actions on specific target cells which Ayerst), and the like. Again, due to the selective agonist/ would appear to be highly desirable in treatment of the antagonist properties of the compounds of the formula I, this menopausal syndrome. Accordingly, the real benefit of the combination provides for the full benefits of estrogen current discovery is that the benzothiophenes of formula I therapy without the concomitant adverse effects associated inhibit the loss of bone but do not elicit significant estrogenic with estrogen therapy alone. responses in the primary sex target tissues. Thus, the current The general chemical terms used in the description of a invention provides a method of inhibiting bone loss com compound of formula I have their usual meanings. For prising administering to a human in need of treatment an 25 amount of a compound of formula I that inhibits bone loss example, the term "C-C-alkyl includes such groups as but does not significantly affect the primary sex target methyl, ethyl, propyl, and isopropyl. tissues. This combination of features allows for long-term The term "C-C-alkoxy” includes such groups as treatment of the chronic ailment with a diminished risk of methoxy, ethoxy, propoxy, butoxy, pentyloxy, and hexyloxy and also includes branched chain structures such as, for developing the undesirable effects of customary estrogen 30 replacement therapy. example, isopropoxy and isobutoxy. The biological action of the benzothiophenes of formula The term "C-C-acyloxy” includes methanoyloxy, I is complex and may be unrelated to the detectable presence ethanoyloxy, propanoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy, and the like and also includes branched chain of the parent compound in the blood. Following oral admin structures Such as, for example, 2.2-dimethylpropanoyloxy, istration of a preferred benzothiophene of this invention, 35 raloxifene (raloxifene hydrochloride), to human subjects in and 3.3-dimethylbutanoyloxy. the clinic, the parent compound was not detected in the The term "C-C-alkoxy-C-C-acyloxy' contemplates, serum of those subjects. It was determined that following for example, methoxyethanoyloxy, methoxypropanoyloxy, oral administration, the compound was extensively conju methoxybutanoyloxy, methoxypentanoyloxy, gated to the glucuronidated form and cleared quickly from 40 methoxy he Xanoyloxy, ethoxyethanoyloxy, the bloodstream. Although no biological endpoints were ethoxypropanoyloxy, ethoxybutanoyloxy, measured in the human recipients, there was concern that the ethoxy pentanoyl oxy, ethoxy he Xanoyloxy, compound was not bioavailable. propoxy ethanoyloxy, prop oxypropanoyloxy, Experiments were undertaken to address the bioavailabil propoxybutanoyloxy, and the like. ity issue in laboratory animals where biological activity 45 It should also be understood that as used herein, refer could be assessed. The animal studies indicated that ralox ences to alkyl and alkoxy structures also include cycloalkyl ifene was maximally active in inhibiting both uterine uptake and cycloalkoxy groups where the number of carbons within of tritiated-estradiol and the normal uterotrophic response to the structure is at least 3. estradiol even under conditions where raloxifene was exten The terms “R-substituted aryloxy” and “R-substituted sively conjugated in the plasma of the animals. Moreover, 50 aroyloxy' include Such groups as phenyloxy, thienyloxy, the conjugate, isolated from the urine of human Subjects furyloxy, naphthyloxy, benzoyloxy, thienoyloxy, furoyloxy, treated with raloxifene, displayed significant antiestrogenic/ naphthoyloxy, and the like, where the R substitution group antiuterotrophic activity when administered intravenously to may be hydrogen, hydroxyl, C-C-alkyl, C-C-alkoxy, or rats, and inhibited the interaction of tritiated-estradiol with halo. rat uterine estrogen receptors in a manner similar to the 55 The term “R-substituted carbonyloxy, where the R' parent compound. These studies suggested the conjugated Substitution group may be C-C-alkoxy or aryloxy, compound may have been converted to the parental form at includes carbonate structures such as methoxycarbonyloxy the site of action, presumably by the action of ethoxy carbonyl oxy, prop oxy carbonyl oxy, B-glucuronidase. Such conversion may contribute to the but oxy carbonyl oxy, p enty 1 oxy carbonyl oxy, activity of the compound. B-Glucuronidase is fairly ubiqui 60 hexyloxycarbonyloxy, phenyloxy carbonyloxy, tous and is thought to be active in the resorption process of thienyloxycarbonyloxy, furyloxycarbonyloxy, and naphthy bone remodeling, and would presumably be available for loxycarbonyloxy. converting the conjugated compound to the parental form if Preferred methods of this invention comprise the use of required for activity. Therefore, conjugation of the ben compounds of formula I wherein R and R' are other than Zothiophenes of formula I is not considered to be necessarily 65 hydrogen, alkoxy, aryloxy, chloro, or bromo and therefore detrimental to their as an inhibitor of bone represent ester and carbonate configurations. Other preferred loss. methods include the use of formula I compounds wherein R US RE39,049 E 5 6 and R' are the same as one another. Certain R groups also or benzene. The salt normally precipitates out of solution demonstrate preferable characteristics when used in the within about one hour to 10 days and can be isolated by methods of this invention. For example, preferred methods filtration or the solvent can be stripped off by conventional of this invention include the use of formula I compounds CaS. wherein R is piperidino or pyrrolidino, especially piperi Bases commonly used for formation of salts include dino. A further preferred subgroup of the preferred piperi ammonium hydroxide and alkali and alkaline earth metal dino and pyrrolidino compounds include compounds hydroxides, carbonates and bicarbonates, as well as aliphatic wherein R and R' are other than hydrogen and, in particular, and aromatic amines, aliphatic diamines and hydroxy alky those wherein R and R' are hydroxyl. lamines. Bases especially useful in the preparation of addi All of the compounds used in the methods of the current 10 tion salts include ammonium hydroxide, potassium invention can be made according to established procedures, carbonate, Sodium bicarbonate, calcium hydroxide, such as those detailed in U.S. Pat. No. 4,133,814 and U.S. methylamine, diethylamine, ethylene diamine, cyclohexy Pat. No. 4,418,068. In general, the process starts with a lamine and ethanolamine. benzobthiophene having a 6-hydroxyl group and a 2-(4- The pharmaceutically acceptable salts generally have hydroxyphenyl) group. The starting compound is protected, enhanced solubility characteristics compared to the com alkylated, and deprotected to form the formula I compounds 15 pound from which they are derived, and thus are often more wherein R and R' are both hydroxy. The formula I com amenable to formulation as liquids or emulsions. pounds that are ethers, esters, and carbonates may then be The current invention also provides useful pharmaceutical formed if desired. Examples of the preparations of such formulations for inhibiting bone loss comprising a formula compounds are provided in the U.S. patents discussed I compound plus one or more pharmaceutically acceptable above. Specific preparations of yet other derivatized com excipients. Pharmaceutical formulations can be prepared by pounds useful in the current invention are outlined in the procedures known in the art. For example, the compounds Preparations sections below. Modifications to the above can be formulated with common excipients, diluents, or methods may be necessary to accommodate reactive func carriers, and formed into tablets, capsules, Suspensions, tionalities of particular substituents. Such modifications 25 powders, and the like. Examples of excipients, diluents, and would be both apparent to, and readily ascertained by, those carriers that are suitable for such formulations include the skilled in the art. following: fillers and extenders such as starch, Sugars, The compounds used in the methods of this invention mannitol, and silicic derivatives; binding agents such as form pharmaceutically acceptable acid and base addition carboxymethyl cellulose and other cellulose derivatives, salts with a wide variety of organic and inorganic acids and 30 alginates, gelatin, and polyvinyl pyrrolidone; moisturizing bases and include the physiologically acceptable salts which agents such as glycerol, disintegrating agents such as are often used in the pharmaceutical chemistry. Such salts agaragar, calcium carbonate, and Sodium bicarbonate; are also part of this invention. Typical inorganic acids used agents for retarding dissolution Such as paraffin, resorption to form such salts include hydrochloric, hydrobromic, accelerators such as quaternary ammonium compounds; hydroiodic, nitric, Sulfuric, phosphoric, hypophosphoric and 35 Surface active agents such as cetyl alcohol, glycerol the like. Salts derived from organic acids, such as aliphatic monostearate; adsorptive carriers such as kaolin and ben mono and dicarboxylic acids, phenyl Substituted alkanoic tonire; and lubricants such as talc, calcium and magnesium acids, hydroxyalkanoic and hydroxyalkandioic acids, aro Stearate, and solid polyethyl glycols. matic acids, aliphatic and aromatic Sulfonic acids, may also The compounds can also be formulated as elixirs or be used. Such pharmaceutically acceptable salts thus include 40 Solutions for convenient oral administration or as solutions acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, appropriate for parenteral administration, for instance by benzoate, chlorobenzoate, dinitrobenzoate, intramuscular, Subcutaneous or intravenous routes. hydroxybenzoate, methoxybenzoate, methylbenzoate, Additionally, the compounds are well suited to formulation o-acetoxybenzoate, naphthalene-2-benzoate, bromide, as Sustained release dosage forms and the like. The formu isobutyrate, phenylbutyrate, B-hydroxybutyrate, butyne-1,4- 45 lations can be so constituted that they release the active dioate, hexyne-1,4-dioate, caprate, caprylate, chloride, ingredient only or preferably in a particular part of the cinnamate, citrate, formate, fumarate, glycollate, intestinal treat, possibly over a period of time. The coatings, heptanoate, hippurate, lactate, malate, maleate, envelopes, and protective matrices may be made, for hydroxymaleate, malonate, mandelate, mesylate, nicotinate, example, from polymeric Substances or waxes. isonicotinate, nitrate, oxalate, , teraphthalate, 50 The particular dosage of a compound of formula I phosphate, monohydrogenphosphate, dihydrogenphosphate, required to treat or inhibit bone loss according to this metaphosphate, pyrophosphate, propiolate, propionate, invention will depend upon the severity of the disease, its phenylpropionate, Salicylate, sebacate. Succinate, Suberate, route of administration, and related factors that will be sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, decided by the attending physician. Generally, accepted and ben Zene -sulfonate, p-bromo phenyl sulfonate, 55 effective doses will be from about 0.1 to about 1000 mg, and chloroben Zene Sulfonate, ethane Sulfonate, more typically from about 200 to about 600 mg. Such 2-hydroxyethanesulfonate, methanesulfonate, naphthalene dosages will be administered to a Subject in need of treat 1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, ment from once to about three times each day, or more often Xylenesulfonate, tartarate, and the like. as needed to effectively inhibit the bone loss process. In addition, some of the formula I compounds may form 60 It is usually preferred to administer a compound of Solvates with water or organic solvents such as ethanol. formula I in the form of an acid addition salt, as is customary These solvates are also contemplated for use in the methods in the administration of pharmaceuticals bearing a basic of this invention. group Such as the piperidino ring. It is also advantageous to The pharmaceutically acceptable acid addition salts are administer Such a compound by the oral rote to an aging typically formed by reacting a compound of formula I with 65 human (e.g. a post-menopausal female or a male showing an equimolar or excess amount of acid. The reactants are evidence of bone loss by X-ray analysis). For such purposes generally combined in a mutual solvent Such as diethyl ether the following oral dosage forms are available. US RE39,049 E 7 8 FORMULATIONS Formulation 6: Tablets In the formulations which follow, “Active ingredient’ means a compound of formula I. Ingredient Quantity (mg tablet) Formulation 1: Gelatin Capsules Active ingredient O.1-1OOO Cellulose, microcrystalline O-6SO Hard gelatin capsules are prepared using the following: Silicone dioxide, fumed O-6SO Stearate acid O-15 10 Ingredient Quantity (mg/capsule) The components are blended and compressed to form tab Active ingredient O. 1-1000 lets. Starch, NF O-6SO Starch flowable powder O-6SO Alternatively, tablets each containing 0.1-1000 mg of Silicone fluid 350 centistokes O-15 15 active ingredient are made up as follows:

The ingredients are blended, passed through a No. 45 mesh Formulation 7: Tablets U.S. sieve, and filled into hard gelatin capsules. Examples of specific capsule formulations containing raloxifene that have been made include those shown below: Ingredient Quantity (mg tablet) Formulation 2: Raloxifene Capsule Active ingredient O. 1-1000 Starch 45 Cellulose, microcrystalline 35 25 Polyvinylpyrrollidone 4 Ingredient Quantity (mg/capsule) (as 10% solution in water) Sodium carboxymethyl cellulose 4.5 Raloxifene 1 Magnesium Stearate O.S Starch, NF 112 Talc 1 Starch flowable powder 225.3 Silicone fluid 350 centistokes 1.7 30 The active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. Formulation 3: Raloxifene Capsule The solution of polyvinylpyrrolidone is mixed with the 35 resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at Ingredient Quantity (mg/capsule) 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Raloxifene 5 The Sodium carboxymethyl starch, magnesium Stearate, and Starch, NF 108 talc, previously passed through a No. 60 U.S. sieve, are then Starch flowable powder 225.3 40 added to the granules which, after mixing, are compressed Silicone fluid 350 centistokes 1.7 on a tablet machine to yield tablets. Suspensions each containing 0.1-1000 mg of medicament Formulation 4: Raloxifene Capsule per 5 mL dose are made as follows: 45 Formulation 8: Suspensions Ingredient Quantity (mg/capsule) Raloxifene 10 Starch, NF 103 50 Starch flowable powder 225.3 Ingredient Quantity (mg 5 ml) Silicone fluid 350 centistokes 1.7 Active ingredient 0.1-1000 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25 mg Formulation 5: Raloxifene Capsule Benzoic acid solution 0.10 mL. 55 Flavor G.V. Color G.V. Purified water to 5 mL. Ingredient Quantity (mg/capsule) Raloxifene 50 Starch, NF 150 60 The medicament is passed through a No. 45 mesh U.S. sieve Starch flowable powder 397 and mixed with the sodium carboxymethyl cellulose and Silicone fluid 350 centistokes 3.0 syrup to form a smooth paste. The benzoic acid solution, flavor, and color are diluted with some of the water and The specific formulations above may be changed in added, with stirring. Sufficient water is then added to pro compliance with the reasonable variations provided. 65 duce the required volume. A tablet formulation is prepared using the ingredients Illustrative compounds that can be used in the formula below: tions and methods of this invention are shown in Table 1.

US RE39,049 E 11 12 In the following Preparations, the compound numbers PREPARATION 4 correspond to those given in Table 1 Preparation of Compound 4 Preparation 1 Preparation of Compound 1 5 6-(Cyclopropylcarbonyloxy)-2-4- (cyclopropylcarbonyloxy)phenylbenzobthien-3-yl 6-(4-Fluorobenzoyloxy)-2-4-(4-fluorobenzoyloxy) 4-2-(piperidin-1-yl)ethoxyphenylmethanone phenylbenzobthien-3-yl-4-2-(piperidin-1-yl) hydrochloride ethoxyphenylmethanone Raloxifene, 6-hydroxy-2-(4-hydroxyphenyl)benzob 10 Compound 4 was prepared from Compound 3 as thien-3-yl-4-2-(piperidin-1-yl)ethoxyphenyl)methanone described in Preparation 2. hydrochloride, (5.1 g, 10 mmol) was suspended in 250 mL PREPARATION 5 of dry tetrahydrofuran (THF) and 7.1 g (70 mmol) of triethylamine, and approximately 10 mg of 4-(N.N- Preparation of Compound 5 dimethylamino)pyridine were added. The suspension was 15 cooled in an ice bath and placed under an atmosphere of 6-(n-Butanoyloxy)-2-4-(n-butanoyloxy)phenyl nitrogen. 4-Fluorobenzoyl chloride (4.75 g, 30 mmol), dis benzobthien-3-yl-4-2-(piperidin-1-yl)ethoxy solved in 20 mL of dry THF, was slowly added over a twenty phenylmethanone minute period. The reaction mixture was stirred and allowed to slowly warm to room temperature over a period of Compound 5 was prepared using the method of Prepara eighteen hours. It was then filtered, and the filtrate was tion 1, but starting with n-butanoyl chloride, to give 4.12 g evaporated to a gum in vacuo. The crude product thus of final product as an oil. obtained was dissolved in a small volume of chloroform and PMR: consistent with the structure FDMS: m/e=614 chromatographed (HPLC) on a silica gel column eluted with (Mt) a linear gradient of Solvent, starting with chloroform and 25 ending with a mixture of chloroform-methanol (19:1 (v/v)). Preparation 6 The fractions containing the desired product as determined by thin layer chromatography (silica, chloroform-methanol Preparation of Compound 6 (9:1)) were combined and evaporated to a gum. The final product was crystallized from ether to give 3.21 g of 30 6-(n-Butanoyloxy)-2-4-(n-butanoyloxy)phenyl compound 1. benzobthien-3-yl-4-2-(piperidin-1-yl)ethoxy PMR: consistent with the structure FDMS: m/e=717 M+ phenylmethanone hydrochloride Elemental Analysis for CHFNOS: Theor: C, 70.29; Compound 5 (4.12 g) was dissolved in ethyl acetate (50 H, 4.60: N, 1.95 Found: C, 70.05; H, 4.60; N, 1.89 Mol. Wit.: 35 mL), and a solution of HCl in ether was added until the 717. precipitation stopped. The liquid was decanted off, and the PREPARATION 2 white, gummy residue was triturated with diethyl ether and filtered. The residue was dried to give 1.33 g of Compound Preparation of Compound 2 6. 6-(4-Fluorobenzoyloxy)-2-4-(4-fluorobenzoyloxy) 40 PMR: consistent with the structure phenylbenzobthien-3-yl-4-2-(piperidin-1-yl) Elemental Analysis of for CHCINOS: Theor: C, ethoxyphenylmethanone hydrochloride 66.50; H, 6.20; N, 2.15 Found: C, 66.30; H, 6.28; N, 1.98 Compound 1 (5.15g, 7.18 mmol) was dissolved in 25 mL Mo1. Wit.: 650.24. THF, and 150 mL ether was added. Dry HCl gas was bubbled into the Solution, and a white gummy precipitate 45 PREPARATION 7 formed. The liquid was removed by decanting, and the residue was crystallized from ethyl acetate with a small Preparation of Compound 7 amount of ethanol added to effect solution. The product was filtered, washed with ether, and dried to give 4.41 g of 6-(2,2-Dimethylpropanoyloxy)-2-4-(2,2- Compound 2 as a white powder. 50 dimethylpropanoyloxy)phenylbenzobthien-3-yl PMR: consistent with the structure 4-2-(piperidin-1-yl)ethoxyphenylmethanone Elemental Analysis for CHCIFNOS: Theor: C, Compound 7 was prepared using the procedure of Prepa 66.88; H, 4.54; N, 1.86 Found: C, 66.59, H, 4.39; N, 1.60 ration 1, but using 2,2-dimethylpropanoyl chloride. MO1. Wt: 753.5. 55 PREPARATION 8 PREPARATION 3 Preparation of Compound 3 Preparation of Compound 8 6-(Cyclopropylcarbonyloxy)-2-4- 60 6-(2,2-Dimethylpropanoyloxy)-2-4-(2,2- (cyclopropylcarbonyloxy)phenylbenzobthien-3-yl dimethylpropanoyloxy)phenylbenzobthien-3-yl 4-2-(piperidin-1-yl)ethoxyphenylmethanone 4-2-(piperidin-1-yl)ethoxyphenylmethanone The title compound was prepared using procedures analo hydrochloride gous to those in Preparation 1, but using cyclopropylcarbo nyl chloride, except that the product was not crystallized. 65 Compound 8 was Prepared from Compound 7, as Yield=2.27 g. described in Preparation 2. PMR: consistent with the structure FDMS: m/e=610 M FDMS: m/e=641 (M-HC1-1) US RE39,049 E 13 14 Elemental Analysis of CHCINOS: Theor.: C, 67.29; PREPARATION 14 H, 6.54: N, 2.07 Found: C, 67.02; H, 6.54; N, 1.90 Mol. Wit.: 678.29. Preparation of Compound 14 6-(n-Butoxycarbonyloxy)-2-4-(n- PREPARATION 9 butoxycarbonyloxy)phenylbenzobthien-3-yl 4-2- Preparation of Compound 9 (piperidin-1-yl)ethoxyphenyl)methanone hydrochloride 6-(3.3-Dimethylbutanoyloxy)-2-4-(3.3- dimethylbutanoyloxy)phenylbenzobthien-3-yl 4 Compound 13 was converted to the hydrochloride salt in 2-(piperidin-1-yl)ethoxyphenyl)methanone 10 a manner analogous to that described in Preparation 6. Compound 9 was prepared using the procedures of Prepa PMR: consistent with structure ration 1, but with 3.3-dimethylbutanoyl chloride. Elemental Analysis of CHCINOS: Calc: C, 64.26; H, 6.24; N, 1.97 Found: C, 63.97; H, 6.34; N, 1.98 Mol. Wit.: Preparation 10 710.29. 15 Preparation of Compound 10 PREPARATION 15 6-(3.3-Dimethylbutanoyloxy)-2-4-(3.3- Preparation of Compound 15 dimethylbutanoyloxy)phenylbenzobthien-3-yl 4 6-(Phenyloxycarbonyloxy)-2-4- 2-(piperidin-1-yl)ethoxyphenyl)methanone (phenyloxycarbonyloxy)phenylbenzobthien-3-yl hydrochloride 4-2-(piperidin-1-yl)ethoxyphenylmethanone Compound 10 was prepared from Compound 9 as This compound was prepared in a manner analogous to described in Preparation 2. that described in Preparation 13, but using the appropriate FDMS: m/e=669 (M-HC1-1) acyl ester. Yield=3.59 g of final product as a tan amorphous Elemental Analysis of Cao HsCINOS: Theor.: C, 68.02; 25 powder. H, 6.85: N, 1.98 Found: C, 67.75; H, 6.83; N, 2.04 Mol. Wit.: 706.35. PMR: consistent with structure FDMS: m/e=713 (M+) PREPARATION 16 PREPARATION 11 Preparation of Compound 11 30 Preparation of Compound 16 6-(4-Methylbenzoyloxy)-2-4-(4-methylbenzoyloxy) 6-(Phenyloxycarbonyloxy)-2-4- phenylbenzobthien-3-yl 4-2-(piperidin-1-yl) (phenyloxycarbonyloxy)phenylbenzobthien-3-yl ethoxyphenylmethanone hydrochloride 4-2-(piperidin-1-yl)ethoxyphenylmethanone 35 hydrochloride Compound 11 was prepared from the free base using a Compound 15 was converted to the hydrochloride salt in procedure similar to that of Preparation 2. a manner analogous to that described in Preparation 6. FDMS: m/e=710 (M-HC1-1) PMR: consistent with structure Elemental Analysis of CHCINOS: Theor. C, 70.81; Elemental Analysis of CHCINOS: Calc: C, 67.24; H, H, 5.39: N, 1.88 Found: C, 71.10: H, 5.39; N, 1.94 Mol. Wit.: 40 4.84; N, 1.87 Found: C, 66.94; H, 4.96: N, 1.84 Mol. Wit.: 746.33. 750.27. PREPARATION 12 PREPARATION 17 Preparation of Compound 12 Preparation of Compound 17 45 6-Benzoyloxy-2-4-benzoyloxy)phenylbenzob 6-(Naphthoyloxy)-2-4(1-naphthoyloxy)phenyl thien-3-yl 4-2-(piperidin-1-yl)ethoxy)-phenyl benzobthien-3-yl 4-2-(piperidin-1-yl)ethoxy methanone phenylmethanone Compound 12 was prepared from the appropriate acid Compound 17 was prepared as described in Preparation 1 chloride as described in Preparation 1. 50 using the appropriate acid halide. Yield=3.5 g of a white FDMS: m/e=682 (M+1) amorphous powder. Element Analysis of CHNOS: Calc: C, 73.80; H, PMR: consistent with structure FDMS: m/e=781 (M+) 5.14: N, 2.05 Found: C, 73.27; H, 5.27: N, 1.94 Mol. Wit.: Elemental Analysis of CsoHNOS: Calc: C, 76.80; H, 6818. 55 5.03; N, 1.79 Found: C, 76.53; H, 5.20: N, 1.53 Mol. Wit.: 781.94. PREPARATION 13 Preparation of Compound 13 PREPARATION 18 6-(n-Butoxyoyloxy)-2-4-(n-butoxyoyloxy)phenyl Preparation of Compound 18 benzobthien-3-yl 4-2-(piperidin-1-yl)ethoxy 60 6-(Methoxyethanoyloxy)-2-4-(methoxyethanoyloxy) phenylmethanone phenylbenzobthien-3-yl 4-2-(piperidin-1-yl) Compound 13 was prepared in a manner analogous to that ethoxyphenylmethanone described in Preparation 1, except that n-butylchloroformate Compound 18 was prepared as described in Preparation 1 was used in place of the acid chloride. Yield=6.13 g in form 65 using the appropriate acid halide. Yield=3.61 g of a gummy of oil. solid. PMR: consistent with structure FDMS: m/e=674 (M+1) PRM: consistent with structure FDMS: m/e=618 (M+1) US RE39,049 E 15 16 PREPARATION 19 estradiol (EE), prevented this loss of bone in a dose dependent manner, but it also exerted a stimulatory action on Preparation of Compound 19 the uterus resulting in uterine weights approaching that of an 6-(Methoxyethanoyloxy)-2-4-(methoxyethanoyloxy) intact rat when administered at 100 g/kg. Results are phenylbenzobthien-3-yl 4-2-(piperidin-1-yl) 5 reported as the mean of measurements from thirty rats the ethoxyphenylmethanone hydrochloride standard error of the mean. Compound 19 was prepared from 3.5g of Compound 18 In these studies, raloxifene also prevented bone loss in a as described in Preparation 2. Yield=1.65 g of amorphous dose dependent manner; however, only minimal increase of white powder. 10 uterine weight over the ovariectomized controls was present in these animals. The results of five assays using raloxifene PMR: consistent with structure FDMS: m/e=618 (M+1) are combined in Table 3. Accordingly, each point reflects the Elemental Analysis of CHNOS: Calc: C, 62.43; H, responses of thirty rats and depicts a typical dose response 5.55; N, 2.14 Found: C, 62.23; H, 5.63: N, 2.15. curve for raloxifene in this model. Results are reported as the The following nonlimiting examples illustrate the meth 15 mean + the standard error of the mean. ods and formulations of this invention. EXAMPLE 1. TABLE 2 In the examples illustrating the methods, a model of Bone Density Uterine Weight post-menopausal osteoporosis was used in which effects of (mg/cm/cm) (mg) different treatments upon femur density were determined. ovariectomy control 1703 127 S Seventy-five day old female Sprague Dawley rats (weight (0.5 mL CMC oral) Intact control 22O 4 S45 - 19 range of 225 to 275 g) were obtained from Charles River (0.5 mL CMC oral) Laboratories (Portage, Mich.). They were housed in groups EE 100 g/kg, oral 210 - 4 490 - 11 of 3 and had ad libitum access to food (calcium content 25 approximately 1%) and water. Room temperature was main tained at 22.2+1.7°C. with a minimum relative humidity of 40%. The photoperiod in the room was 12 hours light and 12 TABLE 3 hours dark. Bone Density Uterine Weight One week after arrival, the rats underwent bilateral ova 30 (mg/cm/cm) (mg) riectomy under anesthesia (44 mg/kg Ketamine and 5 mg/kg ovariectomy control 171 3 1275 Xylazine (Butler, Indianapolis, Ind.) administered (0.5 mL CMC oral) intramuscularly). Treatment with vehicle, estrogen, or a Intact control 222 - 3 540 - 22 compound of formula I was initiated on the day of Surgery (0.5 mL CMC oral) railoxifene 0.01 mg/kg, oral 1763 1SOS following recovery from anesthesia. Oral dosage was by railoxifene 0.10 mg/kg, oral 1973 1965 gavage in 0.5 mL of 1% carboxymethylcellulose (CMC). railoxifene 1.00 mg/kg, oral 2013 1995 Body weight was determined at the time of Surgery and railoxifene 10.00 mg/kg, oral 1993 186 - 4 weekly thereafter and the dosage was adjusted with changes in body weight. Vehicle or estrogen treated ovariectomized (ovex) rats and non-ovariectomized (intact) rats were evalu 40 ated in parallel with each experimental group to serve as EXAMPLE 2 negative and positive controls. The rats were treated daily for 35 days (6 rats per Raloxifene was administered alone or in combination treatment group) and sacrificed by decapitation on the 36th with ethynyl estradiol. Rats treated with raloxifene alone day. The 35 day time period was sufficient to allow maximal 45 had uterine weights which were marginally higher than the reduction in bone density, measured as described herein. At overiectomized controls and much less than those of ethynyl the time of sacrifice, the uteri were removed, dissected free estradiol treated rats, which approached those of the intact of extraneous tissue, and the fluid contents were expelled controls. Conversely, raloxifene treatment significantly before determination of wet weight in order to confirm reduced bone loss in ovariectomized rats, and when given in estrogen deficiency associated with complete ovariectomy. combination with ethynyl estradiol it did not appreciably Uterine weight was routinely reduced about 75% in response reduce the protective effect of the estrogen on bone density. to ovariectomy. The uteri were then placed in 10% neutral The results are shown in Table 4. buffered formalin to allow for subsequent histological analy TABLE 4 S1S. 55 The right femurs were excised and scanned at the distal Bone Density Uterine Weight metaphysis 1 mm from the patellar groove with single (mg/cm/cm) (mg) photon absorptiometry. Results of the densitometer mea Experiment A Surements represent a calculation of bone density as a function of the bone mineral content and bone width. Ovariectomy control 1624 142 18 60 (0.5 mL CMC oral) Intact control 219 5 532 - 49 Influence of Raloxifene on Bone Density (0.5 mL CMC oral) EE 100 g/kg, oral 2O2 6 450 - 17 The results of control treatments from five separate EE 100 g/kg + 204 2 315 - 10 experiments are accumulated in Table 2. In Summary, ova railoxifene 0.10 mg/kg, oral riectomy of the rats caused a reduction in femur density of 65 EE 100 g/kg + 2OO 5 2SO - 21 about 25% as compared to intact vehicle treated controls. railoxifene 1 mg/kg, oral Estrogen, administered in the orally active form of ethynyl US RE39,049 E 17

TABLE 4-continued TABLE 5 Bone Density Uterine Weight Bone Density Uterine Weight (mg/cm/cm) (mg) (mg/cm/cm) (mg) Experiment B Ovariectomy control 171 S 126 - 17 (0.5 mL CMC oral) Ovariectomy control 165 8 116 6 Intact control 2O8 4 490 - 6 (0.5 mL CMC oral) (0.5 mL CMC oral) Intact control 22O 4 605 - 69 EE 100 g/kg, oral 212 - 10 SO1 37 (0.5 mL CMC oral) 10 railoxifene 1 mg/kg, oral 2O7 13 1989 EE 100 g/kg, oral 215 - 11 481 24 tamoxifen 1 mg/kg, oral 204 7 216 18 railoxifene 1 mg/kg + 1977 263 17 EE 100 g/kg, oral railoxifene 1 mg/kg 198 11 2O2 5 TABLE 6 15 Epithelial Stromal Myometrial Stromal EXAMPLE 3 Height Eosinophills Thickness Expansion Ovariectomy control 1.24 1.OO 4.42 10.83 The ability of raloxifene to inhibit bone loss was com (0.5 mL CMC oral) pared to that of tamoxifen (SIGMA, St. Louis, Mo.). Intact control 2.71 4.17 8.67 20.67 (0.5 mL CMC oral) Tamoxifen, a well known antiestrogen currently used in the EE 100 g/kg, oral 3.42 5.17 8.92 21.17 treatment of certain cancers, has been shown to inhibit bone railoxifene 1 mg/kg 1.67 3.17 S.42 14.00 loss (see for example, Love, R., et al. 1992 “Effects of tamoxifen 1 mg/kg 2.58 2.83 5.50 14.17 tamoxifen on bone mineral density in post-menopausal women with breast cancer, N Eng J Med 326:852; Turner, 25 R., et al. 1988 “Tamoxifen inhibits osteoclast-mediated EXAMPLE 4 resorption of trabecular bone in ovarian hormone-deficient Other compounds of formula I were administered orally rats'. Endo 122:1146). A relatively narrow range of doses of in the rat assay described in Example 1. Table 7 reports the raloxifene and tamoxifen was administered orally to ova 30 effect of a 1 mg/kg dose of each compound in terms of a riectomized rats as in the previous example. Although both percent inhibition of bone loss and percent uterine weight of these agents displayed the ability to prevent reduction of ga1n. femur density while evoking only modest uterotrophic activity, as identified by gains in uterine weight (Table 5), a TABLE 7 comparison of several histological parameters demonstrated Compound %. Inhibition % Uterine a marked difference between the rats treated with these Number of Bone Loss Weight Gain agents (Table 6). 2 26 26 6 24 19 Increases in epithelial height are a sign of estrogenicity of 8 66 24 therapeutic agents and may be associated with increased 40 10 52 24 11 26 28 incidence of uterine cancer. When raloxifene was adminis 12 60 15 tered as described in Example 1, only at one dose was there 14 121 32 any statistically measurable increase in epithelial height over 16 108 25 18 21 17 the ovariectomized controls. This was in contrast to the 45 27 25 1 results seen with tamoxifen and estrogen. At all doses given, 34 26 -6 tamoxifen increased epithelial height equal to that of an "Percent inhibition of bone loss = (bone density of treated ovex animals - intact rat, about a six-fold increase over the response seen bone density of untreated ovex animals) - (bone density of estrogen treated ovex animals - bone density of untreated ovex animals) x 100. with raloxifene. Estradiol treatment increased epithelial Percent uterine weight gain = (uterine weight of treated ovex animals - height to a thickness greater than intact rats. 50 uterine weight of ovex animals) - (uterine weight of estrogen treated ovex Estrogenicity was also assessed by evaluating the adverse animals - uterine weight of ovex animals) x 100. response of eosinophil infiltration into the stromal layer of the uterus (Table 6). Raloxifene did not cause any increase EXAMPLE 5 in the number of eosinophils observed in the stromal layer 55 Fracture rate as a consequence of osteoporosis is inversely of ovariectomized rats while tamoxifen caused a significant correlated with bone mineral density. However, changes in increase in the response. Estradiol, as expected, caused a bone density occur slowly, and are measured meaningfully large increase in eosinophil infiltration. only over many months or years. It is possible, however, to demonstrate that the formula I compounds, such as Little or no difference was detectable between raloxifene 60 raloxifene, have positive effects on bone mineral density and and tamoxifen effects on thickness of the stroma and myo bone loss by measuring various quickly responding bio metrium. Both agents caused an increase in these measure chemical parameters that reflect changes in skeletal metabo ments that was much less than the effect of estrogen. lism. To this end, in a current test study of raloxifene at least one hundred-sixty patients are enrolled and randomized to A total score of estrogenicity, which was a compilation of 65 four treatment groups: estrogen, two different doses of all four parameters, showed that raloxifene was significantly raloxifene, and placebo. Patients are treated daily for eight less estrogenic than tamoxifen. weeks. US RE39,049 E 19 20 Blood and urine are collected before, during, and at the

conclusion of treatment. In addition, an assessment of the uterine epithelium is made at the beginning and at the conclusion of the study. Estrogen administration and pla cebo serve as the positive and negative controls, respec tively. The patients are healthy post-menopausal (Surgical or natural) women, age 45–60 who would normally be con sidered candidates for estrogen replacement in treatment for osteoporosis. This includes women with an intact uterus, 10 R who have had a last menstrual period more than six months, but less than six years in the past. wherein Patients who have received any of the following medica X is a bond, CH, or CHCH: tions systematically at the beginning of the study are 15 RandR', independently, are hydrogen, hydroxyl, C-C- excluded from the study: vitamin D, corticosteroids, alkoxy, C-C-acyloxy, C-C-akoxy-C-C-acyloxy, hypolipidemics, thiazides, antigout agents, salicylates, R-substituted aryloxy, R-substituted aroyloxy, phenothiazines, Sulfonates, tetracyclines, neomycin, and R-substituted carbonyloxy, chloro, or bromo: antihelmintics. Patients who have received any estrogen, R is a heterocyclic ring selected from the group consist progestin, or androgen treatment more recently than three ing of pyrrolidino, piperidino, and hexamethylene months prior to the beginning of the study; patients who imino; have ever received calcitonin, fluoride, or bisphosphonate R is C-C-alkyl, C-C-alkoxy, hydrogen, or halo; and therapy; patients who have diabetes mellitus; patients who R" is C-C-alkoxy or aryloxy; or a pharmaceutically have a cancer history anytime any time within the previous acceptable salt thereof, that is effective to inhibit post five years; patients with any undiagnosed or abnormal 25 menopausal bone loss in a human said woman. genital bleeding; patients with active, or a history of throm 2. A method of claim 1 wherein the human woman has boembolic disorders; patients who have impaired liver or been diagnosed as suffering from post-menopausal kidney function; patients who have abnormal thyroid func osteoporosis. tion; patients who are poor medical or psychiatric risks; or 3. A method of claim 1 wherein the human is a post patients who consume an excess of alcohol or abuse drugs. 30 menopausal female. Patients in the estrogen treatment group receive 0.625 4. A method of claim 1 wherein the human is a male. mg/day and the two raloxifene groups receive dosages of 5. A method of claim 1 wherein the compound is admin 200 and 600 mg/day, all groups receiving oral capsule istered prophylactically. formulations. Calcium carbonate, 648 mg tablets, is used as 6. A method of claim 1 wherein X is a bond. calcium Supplement with all patients taking 2 tablets each 35 7. A method of claim 1 wherein R is piperidino or morning during the course of the study. pyrrolidino. 8. A method of claim 6 wherein R is piperidino or The study is a double-blind design. The investigators and pyrrolidino. the patients do not know the treatment group to which the patient is assigned. 9. A method of claim 6 wherein R is piperidino. 40 10. A method of claim 6 wherein R is pyrrolidino. A baseline examination of each patient includes quanti 11. A method of claim 1 wherein R and R' are both tative measurement of urinary calcium, creatinine, hydroxyl, C-C-acyloxy, C-C-alkoxy-C-C-acyloxy, hydroxyproline, and pyridinoline crosslinks. Blood samples R-substituted aroyloxy, or R-substituted carbonyloxy. are measured for serum levels of osteocalcin, bone-specific 12. A method of claim 11 wherein R and R' are both alkaline phosphatase, raloxifene, and raloxifene metabolites. 45 hydroxyl. Baseline measurements also include examination of the 13. A method of claim 11 wherein R and R' are both uterus including uterine biopsy. C-C-acyloxy. During Subsequent visits to the investigating physician, 14. A method of claim 13 wherein R and R' are both measurements of the above parameters in response to treat n-butanoyloxy, 2,2-dimethylpropanoyloxy, or 3,3- ment are repeated. The biochemical markers listed above 50 dimethylbutanoyloxy. that are associated with bone resorption have all been shown 15. A method of claim 11 wherein R and R' are both to be inhibited by the administration of estrogen as com R-substituted carbonyloxy. pared to an untreated individual. Raloxifene is also expected 16. A method of claim 15 wherein R and R' are both to inhibit the markers in estrogen deficient individuals as an methoxycarbonyloxy or phenyloxycarbonyloxy. 55 17. A method of claim 11 wherein R and R' are both indication that raloxifene is effective in inhibiting bone loss R-substituted aroyloxy. from the time that treatment is begun. 18. A method of claim 17 wherein R and R' are both Subsequent longer term studies can incorporate the direct benzoyloxy, methylbenzoyloxy, or naphthoyloxy. measurement of bone density by the use of a photon absorp 19. A method of claim 8 wherein R and R' are both tiometry and the measurement of fracture rates associated 60 hydroxyl, C-C-acyloxy, C-C-alkoxy-C-C-acyloxy, with therapy. R-substituted aroyloxy, or R-substituted carbonyloxy. We claim: 20. A method of claim 9 wherein R and R' are both 1. A method of inhibiting post-menopausal bone loss in a hydroxyl. postmenopausal woman in need of treatment to prevent or to 21. A method of claim 10 wherein R and R' are both treat post-menopausal Osteoporosis comprising administer 65 hyroxyl. ing to a human said woman in need of said treatment an 22. A method of claim 8 wherein R and R' are both amount of a compound of Formula I C-C-acyloxy. US RE39,049 E 21 22 23. A method of claim 22 wherein R and R' are both 30. A method of claim 1 wherein the compound of n-butanoyloxy, 2,2-dimethylpropanoyloxy, or 3.3- formula I is administered in an amount of 600 to 1000 mg. dimethylbutanoyloxy. 31. A method of claim 20 wherein the human woman has been diagnosed as suffering from post-menopausal 24. A method of claim 8 wherein R and R' are both osteoporosis. R*-substituted carbonyloxy. 32. A method of claim 20 wherein the human is a 25. A method of claim 24 wherein R and R' are both post-menopausal woman. methoxycarbonyloxy or phenyloxycarbonyloxy. 33. A method of claim 20 wherein the compound is 26. A method of claim 8 wherein R and R' are both administered prophylactically. R-substituted aroyloxy. 34. A method of claim 1 wherein the administration of a 27. A method of claim 26 wherein R and R' are both 10 compound of formula I does not significantly affect the benzoyloxy, methylbenzoyloxy, or naphthoyloxy. primary six target tissues. 28. A method of claim 1 wherein the compound of 35. A method of claim 1 wherein a compound of formula formula I is administered in an amount of 0.1 to 1000 mg. I is administered in combination with estrogen. 29. A method of claim 1 wherein the compound of formula I is administered in an amount of 200 to 600 mg. k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : RE 39,049 E Page 1 of 1 APPLICATIONNO. : 10/375341 DATED : March 28, 2006 INVENTOR(S) : Black et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

In column 22, line 11, please change “six” to -sex

Signed and Sealed this Second Day of January, 2007 WDJ

JON. W. DUDAS Director of the United States Patent and Trademark Office

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : RE 39,049 E Page 1 of 1 APPLICATIONNO. : 10/375341 DATED : March 28, 2006 INVENTOR(S) : Larry J. Black and George J. Cullinan

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Title Page; In section (75) Inventors, please add an additional inventor: --Michael W. Draper, Carmel, IN (US)--

Signed and Sealed this Eighth Day of September, 2009

David J. Kappos Director of the United States Patent and Trademark Office