WO2010107475A1 - Anti-estrogènes purs pour thérapie du cancer du sein - Google Patents

Anti-estrogènes purs pour thérapie du cancer du sein Download PDF

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WO2010107475A1
WO2010107475A1 PCT/US2010/000755 US2010000755W WO2010107475A1 WO 2010107475 A1 WO2010107475 A1 WO 2010107475A1 US 2010000755 W US2010000755 W US 2010000755W WO 2010107475 A1 WO2010107475 A1 WO 2010107475A1
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alkyl
alkenyl
alkynyl
independently
compound
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Shinya Shibutani
Santosh L. Yennurajalingam
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The Research Foundation Of State University Of New York
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/08—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C25/00—Compounds containing at least one halogen atom bound to a six-membered aromatic ring
    • C07C25/24—Halogenated aromatic hydrocarbons with unsaturated side chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/52—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms containing halogen
    • C07C57/58—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms containing halogen containing six-membered aromatic rings
    • C07C57/60—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms containing halogen containing six-membered aromatic rings having unsaturation outside the rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/333—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton
    • C07C67/343—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/62—Halogen-containing esters
    • C07C69/65—Halogen-containing esters of unsaturated acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic System
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C07F7/1804—Compounds having Si-O-C linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/12—Esters of phosphoric acids with hydroxyaryl compounds

Definitions

  • Tamoxifen (TAM, Fig. Ia) is widely used as a first-line endocrine therapy for early-stage breast cancer patients with positive estrogen receptors
  • TAM is a partial ER agonist in uterine tissue [17] and such estrogenic effects may promote endometrial cancer [18, 19].
  • TAM and its metabolites are ⁇ - hydroxylated and undergo O-sulfonation catalyzed by hydroxysteroid sulfotransferase, after which they react with guanine residues in cellular DNA, forming primarily dG-N 2 -TAM and dG-N 2 -N-desmethylTAM adducts [7] .
  • TAM-DNA adducts were detected in the liver of rodents treated with TAM [20-22] and in several tissues of monkeys, including the ovary and uterus [23] .
  • TAM-DNA adducts have been detected in the endometrium of certain women treated with TAM [12, 13, 24] and in human endometrial explants exposed to ⁇ -OHTAM [25] , although there is controversy about such findings in human tissues [26-28] .
  • K-ras mutations were detected frequently in the endometria of women treated with TAM [29] and their mutational specificity was consistent with that of TAM- DNA adducts in mammalian cells [14] .
  • TAM carcinogenicity may be due primarily to DNA damage induced by TAM through this mechanism. Therefore, TAM alternatives free of genotoxic and estrogenic potential are required to diminish such serious side-effects and to increase clinical efficacy.
  • Toremifene (TOR, Fig. 2), the chloroethyl analog of TAM, however, does not promote hepatic DNA adducts [10, 30, 31] and hepatocarcinoma in rats [10, 31] , although the metabolic fate of TOR is similar to that of TAM. Previous studies indicate that this may be due to the steric hindrance caused by a bulky chlorine atom positioned at the ethyl moiety of TOR, which may reduce the ability of hydroxysteroid sulfotransferase to O-sulfonate ⁇ -OHTOR and consequently the amount of TOR ⁇ -sulfate available to react with DNA [32] .
  • the field effect caused by the electron-withdrawing chlorine atom may also diminish the effective elimination of the ⁇ -sulfate group from the ⁇ - carbon and therefore inhibit the formation of the carbocation intermediate that reacts with DNA [32].
  • No K- ras mutation was observed in the endometria of patients receiving TOR [29] .
  • raloxifene . (RAL) and ICI 182,780 (structures shown in Fig. 2) are also currently used in the clinic for full or partial breast cancer therapy.
  • TOR a chlorinated TAM derivative
  • FDA Food and Drug Administration
  • the estrogenic activity of TOR is similar to that of TAM and the clinical efficacy of TOR for breast cancer patients is also similar [33] . Therefore, this drug is not frequently used in the United States.
  • Raloxifene (RAL) was approved by the FDA in 1998 for treating osteoporosis, but it was also found to reduce significantly the incidence of breast cancer in women at high risk of developing the disease [34] .
  • ICI 182,780 blocks the tropic action of 17 ⁇ -estradiol (E2) in the rat uterus and is free of estrogen agonist activity [42].
  • E2 17 ⁇ -estradiol
  • This drug has shown high response rates for advanced breast cancer [43] and was approved for treatment of post-menopausal breast cancer patients who fail to respond to TAM therapy [44].
  • both RAL and ICI 182,780 have two hydroxyl moieties, the hydroxylated compounds can be conjugated rapidly through phase II metabolism and excreted [45, 46] .
  • ICI 182,780 is highly lipophilic and does not ionize under physiological conditions [47].
  • ospemifene (Fig. 2) , lasofoxifene, and apeledoxifene, in addition to arzoxifene, are under clinical trials for treatment of osteoporosis.
  • Clinical trials of GW5638 and SP500263 (Fig. 2) are also being considered for this purpose.
  • GW5638 with its novel carboxylic side chain (see X position in Fig. 2) , is a triphenylethylene compound that has been recognized as having powerful antiestrogenic activity.
  • This compound is a selective estrogen receptor modulator, or SERM, that has estrogen-like actions to preserve bone density in OVX-rats but, unlike TAM, has pure antiestrogenic activity in rodent uterus.
  • SERM selective estrogen receptor modulator
  • GW5638 does not promote tumor growth in rodent breast or endometrial cells [57, 58] .
  • osteoporosis A major health concern for postmenopausal women is osteoporosis, alluded to earlier, which results from a significant reduction in the production of estrogens by the ovaries [61] .
  • Estrogen deficiency results in an imbalance between osteoblastic bone formation and osteoclastic bone resorption, leading to a net bone loss and increased fracture risk.
  • SERM compounds have shown anti-osteoporotic potential in the OVX-rat model. Ospemifene, lasofoxifene, apeledoxifene, and arzoxifene are under clinical trials for the treatment of osteoporosis.
  • idoxifene and levormeloxifene [52- 54] have been discontinued from clinical testing for osteoporosis because of their undesirable effects on the uterus as mentioned earlier.
  • Estrogen has long been regarded as a beneficial factor in preventing cardiovascular diseases by keeping plasma cholesterol levels low in premenopausal women. Postmenopausal women lose this protection due to a reduction in estrogen levels as a result of natural atrophy of the ovaries, resulting in a change in blood- lipid profile. All ER agonists, including TAM, RAL, and GW5638, examined to date that protect against bone loss are effective in suppressing OVX-induced increases in serum cholesterol [62-64], suggesting that the mechanisms by which ER acts in bone and the cardiovascular system are biochemically linked.
  • This invention provides a compound having the structure
  • Ri/ R2/ R4/ R5/ Re/ R?/ Rs/ R9/ Rio/ Rii/ R12/ R13/ Ri4/ and Ri 5 are each, independently, H, halogen, -CN, -NO 2 , Ci-10 alkyl, C 2 - I0 alkenyl, C 2 -I 0 alkynyl, -CO 2 Ri 6 , -CONRi 6 Ri 7 / - SRi 6 , -NRi 6 Ri 7 , -SO 2 Ri 6 , or -ORi 8 , wherein Ri 6 and R i7 are each, independently, H, Ci_i 0 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl;
  • Ri 8 is H, Ci-io alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl,
  • each occurrence of Ri9 is, independently, H, C 1 - 4 alkyl, or aryl; and wherein R 20 and R 2 i are each, independently, H, Ci- 5 alkyl, C 2 _ 5 alkenyl, or C 2 - 5 alkynyl; and wherein R 22 is H, Ci_ 5 alkyl, C2-5 alkenyl, C 2 - 5
  • R 23 and R 24 are each, independently, H, Ci- 5 alkyl, C 2 - 5 alkenyl, C 2 . 5 alkynyl, - (CH 2 ) 1-4-R27, wherein R 2 7 is -CO 2 H, -CONH 2 , -NH 2 , - SH, -SCH 3 , -OH, aryl, or heteroaryl; and wherein R 25 and R2 6 are each, independently,
  • R 28 i s Ci- 10 alkyl or C 2 -io al kenyl ;
  • q is an integer from 1 to 5;
  • X is halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci-10 alkyl, C 2 - I0 alkenyl, or C 2 - I0 alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • This invention provides a process for preparing compound having the structure
  • Ri, R2, R4, Rs, Re, R?, Rs, Rg, Rio, Rn, R12, R13, Ri4, and Ri 5 are each, independently, H, halogen, -CN, -NO 2 , Ci- 1 0 alkyl, C 2 -io alkenyl, C 2 - 1 0 alkynyl, -CO 2 Ri 6 , -CONRi 6 Ri 7 , - SRi 6 , -NR 16 Ri 7 , -SO 2 R 16 , or -ORi 8 , wherein Ri 6 and Ri 7 are each, independently, H, Ci_i 0 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl; Ri 8 is H, Ci-iQ alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl,
  • each occurrence of R 19 is, independently, H, C 1 - 4 alkyl, or aryl; and wherein R 20 and R 2 i are each, independently, H, C 1 - 5 alkyl, C 2 _ 5 alkenyl, or C 2 - 5 alkynyl; and' wherein R 22 is H, C 1 - 5 alkyl, C 2 _ 5 alkenyl, C 2 - 5
  • R 2 3 and R 24 are each, independently,
  • R 27 is -CO 2 H, -CONH 2 , -NH 2 , - SH, -SCH 3 , -OH, aryl, or heteroaryl; and wherein R2 5 and R 26 are each, independently,
  • R 2 ⁇ is Ci- 10 alkyl or C 2 -io alkenyl
  • q is an integer from 1 to 5;
  • X is H, halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci-10 alkyl, C 2 -I 0 alkenyl, or C 2 -I 0 alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • Ri, R 2 , R4, R5, R 6 , R 7 , R 8 , R9, Rio, Rii/ R12/ R13/ Rio and Ri 5 are each, independently, H, halogen, -CN, - NO 2 , Ci-io alkyl, C 2 -io alkenyl, C 2 -io alkynyl, -CO 2 Ri 6 , - CONRi 6 Ri 7 , -SRi 6 , -NR 16 Ri 7 , -SO 2 Ri 6 , or -ORi 8 , wherein R i6 and Ri 7 are each, independently, H, Ci- 10 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl;
  • Ri 8 is H, Ci- 10 alkyl, C 2 -io alkenyl, C 2 - I0 alkynyl, or Si (R 19 ) 3, wherein each occurrence of R 19 is, independently, H, C 1 - 4 alkyl, or aryl;
  • R 3 is Br, I, -OSO 2 H, or -OSO 2 R 3 I, wherein R 3i is Ci- 1 0 alkyl, C 2 -I 0 alkenyl, C 2 - I0 alkynyl, aryl, heteroaryl, or heterocyclyl; and
  • q is an integer from 1 to 5;
  • X is H, halogen , -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , - wherein R 33 and R 34 are each, independently, H, Ci-io alkyl, C 2 -io alkenyl, or C 2 -io alkynyl; and
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • This invention provides a process of preparing a compound having the structure wherein
  • Ri/ R2/ R4/ Rs/ Re/ R?, Ra, Rg/ Rio, Rn, R12, Ri3/ Ri4, and Ri 5 are each, independently, H, halogen, -CN, -NO2, C 1 -I 0 alkyl, C 2 -io alkenyl, C 2 -I 0 alkynyl, -CO 2 Ri6/ -CONRi 6 Ri 7 , - SRi 6 , -NRi 6 Ri 7 , -SO 2 Ri 6 , or -ORi 8 , wherein Ri 6 and Ri 7 are each, independently, H, Ci-10 alkyl, C2-10 alkenyl, C 2 -I 0 alkynyl;
  • Ri 8 is H, Ci- I0 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl,
  • each occurrence of Ri 9 is, independently, H, C 1 - 4 alkyl, or aryl; and wherein R 20 and R 2i are each, independently, H, Ci- 5 alkyl, C 2 . 5 alkenyl, or C 2 . 5 alkynyl; and wherein R 22 is H, C 1 - 5 alkyl, C 2 . 5 alkenyl, C 2 _ 5
  • R 23 and R 24 are each, independently, H, Ci- 5 alkyl, C 2 _ 5 alkenyl, C 2 - 5 alkynyl, -
  • R 27 is -CO 2 H, -CONH 2 , -NH 2 , - SH, -SCH 3 , -OH, aryl, or heteroaryl; and wherein R 25 and R 26 are each, independently,
  • R 2 a is Ci- I0 alkyl or C 2 -I 0 alkenyl
  • R 32 is H, Ci- I0 alkyl, C 2 - I0 alkenyl, or C 2 - I0 alkynyl;
  • q is an integer from 1 to 5;
  • X is H, halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci_i 0 alkyl, C 2 - I0 alkenyl, or C 2 - I0 alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • Ri f R2, R 4 , Rs, Re, Rv/ Rs, R9, Rio, Rn, R12, R13, Ri4, and Ri 5 are each, independently, H, halogen, -CN, - NO 2 , Ci-1 0 alkyl, C2-10 alkenyl, C2-10 alkynyl, -CO2R16, - CONRi 6 Ri 7 , -SR16, -NRi 6 Ri?, -SO 2 Ri 6 , or -ORi 8 , wherein Ri 6 and R17 are each, independently, H,
  • Ri 8 is H, Ci-io alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl, or Si (Ri 9 ) 3, wherein each occurrence of R 19 is, independently, H, C 1 - 4 alkyl, or aryl;
  • R 3 is Br, I, or -OSO 2 R 3I , wherein R 3 i is Ci- 10 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl, aryl, heteroaryl, or heterocyclyl;v and
  • q is an integer from 1 to 5;
  • X is H, halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -
  • R 33 and R 34 are each, independently, H,
  • Ci- 1 0 alkyl C 2 - I0 alkenyl, or C 2 - 1 0 alkynyl
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • This invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising any one of the above compounds, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • This invention provides a method of inhibiting tumor proliferation in a mammal comprising administering to the mammal a therapeutically effective amount of any one of the above compounds so as to inhibit tumor proliferation.
  • This invention provides a method of inhibiting and/or reducing the loss of bone mineral density in a mammal comprising administering to the mammal a therapeutically effective amount of any one of the above compounds so as to inhibit and/or reduce loss of bone mineral density.
  • This invention provides a method of delaying tumor formation in a mammal comprising administering to the mammal a therapeutically effective amount of any of the above compounds, or a salt thereof, so as to delay tumor formation .
  • This invention provides a method of prophylaxis against tumor formation in a mammal comprising administering to the mammal a therapeutically effective amount of any of the above compounds, or a salt thereof, so as to provide prophylaxis against tumor formation.
  • FIG. 1a Formation of TAM-DNA adducts via ⁇ - hydroxylation followed by O-sulfonation of TAM metabolites.
  • FIG. 1b Formation of TAM-DNA adducts yia 0- sulfonated TAM metabolites.
  • Figure 5 Effect of antiestrogens on uterine histology in OVX-rats.
  • A Sham; B, E2; C, TAM; D, GW5638; E, SS1020. Magnification: XlO.
  • FIG. 6a Antitumor potential of antiestrogens on DMBA-induced mammary carcinoma.
  • A. 1 mg/kg/day; **, p ⁇ 0.01 and ***, p ⁇ 0.001 versus (vs) Control (t-test) .
  • RAL or GW5638 b) p ⁇ 0.05 vs. RAL, p ⁇ 0.01 vs. TAM or GW5638; c) p ⁇ 0.01 vs. RAL or GW5638, p ⁇ 0.001 vs. TAM; d) p ⁇ 0.01 vs. GW5638, p ⁇ 0.001 vs. TAM or RAL.
  • FIG. 6b Antitumor potential of antiestrogens on DMBA-induced mammary carcinoma.
  • B 0.33 mg/kg/day; **, p ⁇ 0.01 and ***, p ⁇ 0.001 versus (vs) Control (t-test) .
  • Figure 8 HPLC analysis of the dephosphorylation of SS1021 by cytosol of rat intestine. A. No dephosphorylation observed when SS1021 incubated in inactivated cytosol; B. Conversion to SS1020 observed when incubated in cytosol.
  • Figure 9a Antitumor potential of SS1020 in nude mice bearing ER-positive MCF-7 human breast cancer xenograft.
  • A MCF-7; Student t-test; *, p ⁇ 0.05 and **, p ⁇ 0.01 versus Control; a, b, and c) p ⁇ 0.01 vs. TAM; d) p ⁇ 0.001 vs. TAM.
  • Figure 9b Antitumor potential of SS1020 in nude mice bearing ER-negative MDA-MB-231 human breast cancer xenograft.
  • B MDA-MB-231. Student t- test; *, p ⁇ 0.05 and **, p ⁇ 0.01 versus
  • Figure 11 Delaying effect of SS1020 against rat DMBA- induced mammary tumor.
  • FIG. 12 Formation of mammary DMBA-DNA in rats pre- treated with SS1020. Control (corn oil only); ⁇ -NF (73 mg/kg) ; SS1020 (3.3 mg/kg) .
  • Figure 13 Antitumor potential of SS1020 against human
  • MCF-7 breast cancer xenograft in athymic nude mice were treated orally for 4 weeks with TAM or SS1020 at a dose molar equivalent to TAM
  • This invention provides a compound having the structure
  • Ri, R2, R4, R5, Re/ Rv, Rs, Rg, Rio, Rn, R12, R13, Ri4, and Ri 5 are each, independently, H, halogen, -CN, -NO 2 , Ci-10 alkyl, C2-10 alkenyl, C 2 -io alkynyl, -CO 2 Ri 6 , -CONRi 6 Ri 7 , - SRi 6 , -NR 16 Ri 7 , -SO 2 Ri 6 , or -ORi 8 , wherein R i6 and Ri 7 are each, independently, H, Ci- 1 0 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl;
  • Ri 8 is H, Ci- 10 alkyl, C 2 - I0 alkenyl, C 2 -io alkynyl,
  • Ri9 is, independently, H, C 1 - 4 alkyl, or aryl; and wherein R 20 and R 2i are each, independently, H, Ci- 5 alkyl, C 2 - 5 alkenyl, or C 2 - 5 alkynyl; and wherein R 22 is H, C 1 - 5 alkyl, C 2 - 5 alkenyl, C 2 -5
  • R 23 and R 24 are each, independently, H, Ci- 5 alkyl, C 2 _ 5 alkenyl, C 2 -s alkynyl, - (CH 2 ) 1 - 4 -R 27 , wherein R 27 is -CO 2 H, -CONH 2 , -NH 2 , - SH, -SCH 3 , -OH, aryl, or heteroaryl; and wherein R25 and R26 are each, independently,
  • R 28 is Ci-10 alkyl or C 2 -io alkenyl
  • q is an integer from 1 to 5;
  • X is halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci_i 0 alkyl, C 2 -I 0 alkenyl, or C 2 -I 0 alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • the compound has the structure wherein
  • Ri/ R2, R4, Rs, Re, R7, Rs, R9, Rio, Rn, R12, R13, Ri4, and R i5 are each, independently, H, halogen, -CN, -NO2, Ci-1 0 alkyl, C 2 -io alkenyl, C 2 -io alkynyl, -CO 2 Ri 6 , -CONRi 6 Ri7, - SRi 6 , -NRi 6 Ri 7 , -SO 2 Ri 5 , or -ORi 8 , wherein Ri 6 and R i7 are each, independently, H, Ci_i 0 alkyl, C 2 _io alkenyl, C 2 - I0 alkynyl; R 18 is H, Ci- 10 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl,
  • each occurrence of Ri 9 is, independently, H, C 1 - 4 alkyl, or aryl; and wherein R 20 and R 2 i are each, independently, H, Ci- 5 alkyl, C 2 - 5 alkenyl, or C 2 _ 5 alkynyl; and wherein R 22 is H, C 1 - 5 alkyl, C 2 - 5 alkenyl, C 2 _ 5
  • R 23 and R 24 are each, independently, H, Ci- 5 alkyl, C 2 - 5 alkenyl, C 2 - 5 alkynyl, - (CH 2 ) i- 4 -R 27 , wherein R 27 is -CO 2 H, -CONH 2 , -NH 2 , - SH, -SCH 3 , -OH, aryl, or heteroaryl; and wherein R 25 and R 26 are each, independently,
  • R 28 is Ci-io alkyl or C 2 -io alkenyl ;
  • R 3 is Br, I , -OSO 2 H, or -OSO2R31, wherein R 31 is Ci_i 0 alkyl, C 2 - 1 0 alkenyl, C 2 _io alkynyl, aryl, heteroaryl, heterocyclyl, -CO 2 R3 2 , or - (CH 2 ) i-6 ⁇ CO 2 R 32 , wherein R 32 is H, Ci- 10 alkyl, C 2 _io alkenyl, or C 2 - 1 0 alkynyl; and
  • q is an integer from 1 to 5;
  • X is halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci-10 alkyl, C2-10 alkenyl, or C 2 -io alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • the compound has the structure wherein
  • Ri, R2, R4, R5, Re, R?, Ra, Rg, Rio, Rn, R12, R13, Rn, and Ri 5 are each, independently, H, halogen, -CN, -NO 2 , Ci- 10 alkyl, C 2 -io alkenyl, C2-10 alkynyl, -CO 2 RiS, -CONRi 6 Ri 7 , - SRi 6 , -NRi 6 Ri 7 , -SO 2 Ri 6 , or -ORi 8 , wherein Ri 6 and Ri 7 are each, independently, H, Ci- 10 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl; Ri 8 is H, Ci- I0 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl,
  • each occurrence of R 19 is, independently, H, C 1 - 4 alkyl, or aryl; and wherein R 20 and R 2 i are each, independently, H, Ci- 5 alkyl, C 2 - 5 alkenyl, or C 2 - 5 alkynyl; and wherein R 22 is H, C 1 - 5 alkyl, C 2 _ 5 alkenyl, C 2 _ 5
  • R 23 and R 24 are each, independently, H, Ci- 5 alkyl, C 2 _ 5 alkenyl, C 2 - 5 alkynyl, - (CH 2 ) 1-4-R27, wherein R 27 is -CO 2 H, -CONH 2 , -NH 2 , - SH, -SCH 3 , -OH, aryl, or heteroaryl; and wherein R 25 and R2 6 are each, independently,
  • R 28 is Ci- 1 0 alkyl or C 2 -io alkenyl
  • q is an integer from 1 to 5;
  • X is halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci-1 0 alkyl, C 2 - I0 alkenyl, or C 2 -io alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • the compound has the structure wherein
  • Ri, R2, R4, R5, Re, R?, Ra, R9, Rio, Rn, R12, R13, Ru, and R i5 are each, independently, H, halogen, -CN, -NO 2 , Ci-10 alkyl, C 2 -io alkenyl, C2-10 alkynyl, -CO 2 Ri6, -CONRi 6 Ri 7 , - SRi 6 , -NRi 6 Ri 7 , -SO 2 Ri 6 , or -ORi 8 , wherein R i6 and Ri 7 are each, independently, H, Ci_i 0 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl;
  • R 20 and R 2 i are each, independently, H, Ci- 5 alkyl, C 2 _ 5 alkenyl, or C 2 _ 5 alkynyl; and wherein R 22 is H, C 1 -5 alkyl, C 2 - 5 alkenyl, C2-5
  • R 23 and R 24 are each, independently,
  • R 27 is -CO 2 H, -CONH 2 , -NH 2 , - SH, -SCH 3 , -OH, aryl, or heteroaryl; and wherein R25 and R26 are each, independently,
  • R 28 is Ci-10 alkyl or C 2 -io alkenyl ;
  • q is an integer from 1 to 5;
  • X is halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci_i 0 alkyl, C 2 - I0 alkenyl, or C 2 -I 0 alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • the compound has the structure
  • Ri, R2/ R4, Rs, Re, R?, R 8 , R9, Rio, Rn, R12, R13, Ri4, and Ri 5 are each, independently, H, halogen, -CN, -NO2, Ci-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, -CO 2 Ri6, -CONR 16 Ri7, - SRi 6 , -NR 16 Ri 7 , -SO 2 Ri 6 , or -ORi 8 , wherein Ri 6 and R 17 are each, independently, H, Ci_i 0 alkyl, C 2 - 1 0 alkenyl, C 2 - I0 alkynyl;
  • R 20 and R 21 are each, independently, H, Ci- 5 alkyl, C 2 - 5 alkenyl, or C 2 - 5 alkynyl; and wherein R 23 and R 24 are each, independently, H, Ci- 5 alkyl, C 2 _ 5 alkenyl, C 2 _ 5 alkynyl, - (CH 2 ) 1-4- R 27 , wherein R 27 is -CO 2 H, -CONH 2 , -NH 2 , -SH, - SCH 3 , -OH, aryl, or heteroaryl; and wherein R 25 and R 26 are each, independently, H or wherein R 28 is Ci_i 0 alkyl or C 2 - I0 alkenyl;
  • q is an integer from 1 to 5;
  • X is halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci-10 alkyl, C2-10 alkenyl, or C2-10 alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • the compound has the structure
  • R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , Rg, Rio , Rn , R12 , R13, Ri4 , and Ri 5 are each, independently, H, halogen , -CN, -NO 2 , Ci- 1 0 alkyl, C 2 -io alkenyl, C2-10 alkynyl, -CO 2 Ri6, -CONRi 6 Ri 7 , - SRi 6 , -NRi 6 Ri 7 , -SO 2 Ri 6 , or -ORi 8 , wherein Ri 6 and Ri 7 are each, independently, H, Ci- 10 alkyl, C2-10 alkenyl, C 2 -I 0 alkynyl; Ri ⁇ is H, Ci-io alkyl, C 2 -io alkenyl, C2- 10 alkynyl, or
  • Ri 9 is, independently, H, C 1 - 4 alkyl, or aryl;
  • q is an integer from 1 to 5;
  • X is halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci-10 alkyl, C 2 - I0 alkenyl, or C 2 - I0 alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • the compound has the structure
  • Ri, R 2 , R 4 , R 5 , Re, R?, Rs, Rg, Rio, Rn, R12, R13, R14, and R i5 are each, independently, H, halogen, -CN, -NO2, Ci-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, -CO 2 Ri 6 , -CONRi 6 Ri 7 , - SRi 6 , -NRi 6 Ri 7 , -SO 2 Ri 6 , or -OR i8 , wherein Ri 6 and Ri 7 are each, independently, H, Ci-10 alkyl, C 2 -io alkenyl, C 2 -io alkynyl;
  • Ri 8 is H, Ci- 1 0 alkyl, C 2 -I 0 alkenyl, C 2 -I 0 alkynyl,
  • each occurrence of Ri 9 is, independently, H, C 1 - 4 alkyl, or aryl; and wherein R 20 and R 2 i are each, independently, H,
  • R 23 and R 24 are each, independently, H, Ci- 5 alkyl, C 2 - 5 alkenyl, C 2 - 5 alkynyl, -
  • R 27 is -CO 2 H, -CONH 2 , -NH 2 , - SH, -SCH 3 , -OH, aryl, or heteroaryl; and wherein R 25 and R 26 are each, independently,
  • R 28 is Ci- I0 alkyl or C 2 - I0 alkenyl ;
  • R 32 is H, Ci-io alkyl , C 2 - I0 alkenyl , or C 2 -io alkynyl ; and q is an integer from 1 to 5 ;
  • X is halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci-10 alkyl, C 2 -I 0 alkenyl, or C 2 - I0 alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • X is halogen or OR 33 , or a salt thereof.
  • X is halogen, or a salt thereof.
  • the compound has the structure
  • Ri 3 is H or -ORi 8 ,
  • Ri 8 is H, or .
  • the compound has the structure
  • Ris is H, or H. NH 2 .
  • Ris is H or. or a salt thereof
  • Ris is or , or a salt thereof.
  • the compound has the structure
  • the compound has the structure
  • This invention provides a process for preparing a compound having the structure
  • Ri, R2, Ri, Rs, Re, R?, Re, Rg, Rio, Rn, R12, R13, R14, and Ri 5 are each, independently, H, halogen, -CN, -NO2, Ci-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, -CO 2 Ri6, -CONRi 6 Ri7, - SRi 6 , -NRi 5 Ri 7 , -SO2R16, or -ORi 8 , wherein R 16 and Ri 7 are each, independently, H, Ci-10 alkyl, C2-1 0 alkenyl, C2-10 alkynyl;
  • Ri 8 is H, Ci-io alkyl, C2-1 0 alkenyl, C 2 -io alkynyl,
  • each occurrence of R19 is, independently, H, C 1 - 4 alkyl, or aryl; and wherein R 20 and R 2x are each, independently, H, Ci- 5 alkyl, C 2 - 5 alkenyl, or C 2 - 5 alkynyl; and wherein R 22 is H, C 1 - 5 alkyl, C 2 -5 alkenyl, C2-5
  • R 23 and R 24 are each, independently,
  • R 27 is -CO 2 H, -CONH 2 , -NH 2 , - SH, -SCH 3 , -OH, aryl, or heteroaryl; and wherein R 25 and R 2 ⁇ are each, independently, wherein R 2 a is Ci-10 alkyl or C 2 -io alkenyl;
  • q is an integer from 1 to 5;
  • X is H, halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci- 1 0 alkyl, C 2 - I0 alkenyl, or C 2 - 1 0 alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • Ri, R2, R4, R5, Re, R?, Ra, Rg, Rio, Rn, R12, R13, Ri4, and Ri 5 are each, independently, H, halogen, -CN, - NO 2 , Ci-10 alkyl, C 2 -io alkenyl, C2-10 alkynyl, -CO 2 Ri 6 , -
  • Ri 6 and Ri 7 are each, independently, H, Ci- 10 alkyl, C 2 _io alkenyl, C 2 -io alkynyl; Ri 8 is H, Ci- 10 alkyl, C 2 -I 0 alkenyl, C 2 -I 0 alkynyl, or Si (Ri 9 ) 3, wherein each occurrence of R 19 is, independently, H, C 1 - 4 alkyl, or aryl;
  • R 3 is Br, I, -OSO 2 H, or -OSO 2 R 3I , wherein R 3 i is Ci- I0 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl, aryl, heteroaryl, or heterocyclyl; and
  • ⁇ q is an integer from 1 to 5;
  • X is H, halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -
  • R 33 and R 34 are each, independently, H, Ci-10 alkyl, C 2 - 1 0 alkenyl, or C 2 _io alkynyl; and wherein each occurrence of alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • the process comprises: contacting a compound having the structure
  • Ri, R 2 , R 4 , R5, Re, R7, Re, R9, Rio, Rn, R12, R13, R14, and Ri 5 are each, independently, H, halogen, -CN, - NO 2 , Ci-10 alkyl, C2-10 alkenyl, C 2 -I 0 alkynyl, -CO 2 Ri 6 , -
  • Ri 6 and Ri 7 are each, independently, H, Ci-io alkyl, C 2 - I0 alkenyl, C 2 -I 0 alkynyl;
  • R 18 is H, Ci- 10 alkyl, C 2 - 1 0 alkenyl, C 2 - I0 alkynyl, wherein each occurrence of R 1 9 is, independently, H, C 1 - 4 alkyl, or aryl;
  • R 3 is Br, I, -OSO 2 H, or -OSO 2 R 3I , wherein R 31 is Ci_i 0 alkyl, C 2 - I0 alkenyl, C 2 - 10 alkynyl, aryl, heteroaryl, or heterocyclyl; and
  • q is an integer from 1 to 5;
  • X is halogen, -CN, -NO 2 , -OR33, -SR33, -OSO 2 R 33 , -
  • R 33 and R 34 are each, independently, H, Ci- 10 alkyl, C 2 - I0 alkenyl, or C 2 -I 0 alkynyl; and wherein each occurrence of alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • the process comprises: contacting a compound having the structure
  • Ri/ R2/ R4/ Rs, Re, R?/ Ra, R9, Rio, Rn, R12, Ri3/ Ri4, and R 1S are each, independently, H, halogen, -CN, - NO 2 , Ci-10 alkyl, C2-10 alkenyl, C 2 -io alkynyl, -CO 2 Ri6/ -
  • Ri 6 and Ri 7 are each, independently, H, Ci- 1 0 alkyl, C 2 - I0 alkenyl, C 2 -I 0 alkynyl; Ria is H, Ci- 10 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl, or Si (R19) 3, wherein each occurrence of R 19 is, independently, H, C 1 - 4 alkyl, or aryl;
  • R 3 is Br, I, -OSO 2 H, or -OSO 2 R 3I , wherein R 31 is Ci_i 0 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl, aryl, heteroaryl, or heterocyclyl; and
  • q is an integer from 1 to 5;
  • X is H
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • This invention provides a process of preparing a compound having the structure
  • Ri, R2, R4/ Rs/ Re, R?, R ⁇ / R9/ Rio, Rn, R12, R13, Ri4, and R i5 are each, independently, H, halogen, -CN, -NO2, Ci-10 alkyl, C 2 -io alkenyl, C 2 -io alkynyl, -CO 2 Ri6, -CONRi 6 Ri 7 , - SRi 6 , -NRi 6 Ri 7 , -SO 2 Ri 6 , or -ORi 8 , wherein R i6 and Ri 7 are each, independently, H, Ci-10 alkyl, C 2 - I0 alkenyl, C 2 -I 0 alkynyl;
  • R 18 is H, Ci-iQ alkyl, C 2 -I 0 alkenyl, C 2 -I 0 alkynyl,
  • R19 Si (R19) 3, or wherein each occurrence of R19 is, independently, H, Ci- 4 alkyl, or aryl; and wherein R 2 o and R 21 are each, independently, H, Ci- 5 alkyl, C 2 - 5 alkenyl, or C 2 -5 alkynyl; and wherein R 22 is H, C1-5 alkyl, C2-5 alkenyl, C2-5
  • R 23 and R 24 are each, independently, H, Ci- 5 alkyl, C 2 -5 alkenyl, C 2 -5 alkynyl, - (CH 2 ) 1-4-R27, wherein R 27 is -CO 2 H, -CONH 2 , -NH 2 , -
  • R 2 s is Ci- I0 alkyl or C 2 -io alkenyl
  • R 32 is H, Ci- 10 alkyl, C 2 - I0 alkenyl, or C 2 - I0 alkynyl;
  • q is an integer from 1 to 5;
  • X is H, halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -NR 33 R 34 ,
  • R 33 and R 34 are each, independently, H, Ci- 10 alkyl, C 2 - I0 alkenyl, or C 2 - I0 alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched; comprising: contacting a compound having the structure
  • Ri, R 2 , R 4 , R 5 , Rs, R 7 , Ra, Rg, Rio, Rn, R12, R13, Ri4, and Ri 5 are each, independently, H, halogen, -CN, - NO 2 , Ci- 10 alkyl, C 2 - I0 alkenyl, C 2 -I 0 alkynyl, -CO 2 Ri 6 , - CONRi 6 Ri 7 , -SRi 6 , -NRi 6 Ri 7 , -SO 2 Ri 6 , or -ORi 8 , wherein R i6 and Ri 7 are each, independently, H, Ci- 10 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl;
  • Ri 8 is H, Ci- 10 alkyl, C 2 - I0 alkenyl, C 2 - I0 alkynyl, or Si (Ri9>3, wherein each occurrence of R 1 9 is, independently, H, C 1 - 4 alkyl, or aryl;
  • R 3 is Br, I, or -OSO 2 R 3 I, wherein R 3i is Ci-10 alkyl, C 2 -io alkenyl, C 2 -io alkynyl, aryl, heteroaryl, or heterocyclyl; and
  • q is an integer from 1 to 5;
  • X is H, halogen, -CN, -NO 2 , -OR 33 , -SR 33 , -OSO 2 R 33 , -
  • R 33 and R 34 are each, independently, H, Ci-io alkyl, C 2 - 10 alkenyl, or C 2 -io alkynyl;
  • alkyl, alkenyl, and alkynyl is substituted or unsubstituted, branched or unbranched;
  • X is H.
  • X is halogen, -CN, -NO 2 ,
  • R 33 and R 34 are each, independently, H, Ci-10 alkyl, C 2 - I0 alkenyl, or C 2 _io alkynyl.
  • the compound prepared has the structure
  • the compound prepared has the structure
  • This invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising any one of the above compounds, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • This invention provides a method of inhibiting tumor proliferation in a mammal comprising administering to the mammal a therapeutically effective amount of any one of the above compounds, or a salt thereof, so as to inhibit tumor proliferation.
  • the tumor is an estrogen-induced tumor.
  • the tumor is an ER-positive tumor.
  • the tumor is an ER-negative tumor.
  • This invention provides a method of inhibiting and/or reducing the loss of bone mineral density in a mammal comprising administering to the mammal a therapeutically effective amount of any one of the above compounds, or a salt thereof, so as to inhibit and/or reduce loss of bone mineral density.
  • This invention provides a method of delaying tumor formation in a mammal comprising administering to the mammal a therapeutically effective amount of any of the above compounds, or a salt thereof, so as to delay tumor formation.
  • This invention provides a method of prophylaxis against tumor formation in a mammal comprising administering to the mammal a therapeutically effective amount of any of the above compounds, or a salt thereof, so as to provide prophylaxis against tumor formation.
  • the compounds described herein can be used in inhibiting tumor proliferation in a mammal.
  • the compounds described herein can be used in inhibiting and/or reducing the loss of bone mineral density in a mammal .
  • the compounds described herein can be used in delaying tumor formation in a mammal .
  • the compounds described herein can be used in prophylaxis against tumor formation in a mammal.
  • the compounds of the present invention include all hydrates, solvates, and complexes of the compounds used by this invention. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein.
  • Compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone.
  • the compounds described in the present invention are in racemic form or as individual enantiomers.
  • enantiomers can be separated using known techniques, such as those described in Pure and Applied Chemistry 69, 1469-1474, (1997) IUPAC. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
  • hydrogen atoms are not shown for carbon atoms having less than four bonds to non-hydrogen atoms. However, it is understood that enough hydrogen atoms exist on said carbon atoms to satisfy the octet rule.
  • alkyl includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and may be unsubstituted or substituted.
  • C x -C n as in “Ci-C n alkyl” is defined to include groups having 1, 2, ...., n- 1 or n carbons in a linear or branched arrangement.
  • Ci-C ⁇ as in “Ci-C ⁇ alkyl” is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl, hexyl, and octyl .
  • alkenyl refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon-carbon double bonds may be present, and may be unsubstituted or substituted.
  • C 2 -Ce alkenyl means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and up to 1, 2, 3, 4, or 5 carbon-carbon double bonds respectively.
  • Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl .
  • alkynyl refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present, and may be unsubstituted or substituted.
  • C 2 -C 6 alkynyl means an alkynyl radical having 2 or 3 carbon atoms and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms and up to 3 carbon-carbon triple bonds.
  • Alkynyl groups include ethynyl, propynyl and butynyl .
  • Alkylene alkenylene and alkynylene shall mean, respectively, a divalent alkane, alkene and alkyne radical, respectively. It is understood that an alkylene, alkenylene, and alkynylene may be straight or branched. An alkylene, alkenylene, and alkynylene may be unsubstituted or substituted.
  • aryl is intended to mean ' any stable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include phenyl, p-toluenyl
  • arylalkyl refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an aryl group as described above. It is understood that an "arylalkyl” group is connected to a core molecule through a bond from the alkyl group and that the aryl group acts as a substituent on ' the alkyl group.
  • arylalkyl moieties include, but are not limited to, benzyl (phenylmethyl) , p-trifluoromethylbenzyl (4- trifluoromethylphenylmethyl) , 1-phenylethyl, 2- phenylethyl, 3-phenylpropyl, 2-phenylpropyl and the like.
  • heteroaryl represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S.
  • Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6- membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S.
  • Heteroaryl groups within the scope of this definition include but are not limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyr
  • heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.
  • heterocycle refers to a mono- or poly-cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms.
  • Preferred heteroatoms include N, 0, and/or S, including N-oxides, sulfur oxides, and dioxides.
  • the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation.
  • the heterocycle may be unsubstituted or substituted, with multiple degrees of substitution being allowed.
  • Such rings may be optionally fused to ' one or more of another "heterocyclic" ring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl ring(s) .
  • heterocycles include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1, 3-oxathiolane, and the like.
  • alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise.
  • alkyl, alkenyl, alkynyl, aryl, heterocyclyl and heteroaryl groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups.
  • alternative non-hydrogen groups include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.
  • halogen refers to F, Cl, Br, and I .
  • substituted refers to a functional group as described above in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non- hydrogen or non-carbon atoms, provided that normal valencies are maintained and that the substitution results in a stable compound. Substituted groups also include groups in which one or more bonds to a carbon (s) or hydrogen (s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom.
  • substituents include the functional groups described above, and, in particular, halogens (i.e., F, Cl, Br, and I) ; alkyl groups, such as methyl, ethyl, n- propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4- trifluoromethylphenylmethoxy) ; heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto;
  • substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally.
  • independently substituted it is meant that the (two or more) substituents can be the same or different.
  • substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results .
  • titanium reagent refers to elemental titanium, titanium salts and their complexes wherein the titanium atom can exist at various oxidation states.
  • Suitable titanium reagents include, but are not limited to, TiCl 3 , TiCl 4 , TiCl 3 -THF, and TiCl 3 (DME) i. 5 .
  • reducing agent refers to an agent capable of reducing the oxidation state of another compound.
  • reducing agents include, but are not limited to, aluminium hydrides, such as (i-Bu) 2 AlH, (i-Bu) 3 Al, LiAlH 4 , LiAlH(OMe) 3 , LiAlH(Ot-Bu) 3 , and NaAlH 2 (OCH 2 CH 2 OCH 3 ) 2 ; boron hydrides such as 9-BBN, NaBH 4 , NaBH 4 -CeCl 3 , LiBH 4 , LiEt 3 BH, Li(S-Bu) 3 BH, K(S-Bu) 3 BH, Na(s- Bu) 3 BH, KPh 3 BH, (Ph 3 P) 2 CuBH 4 , Zn (BH 4 ) 2 , Ca (BH 4 ) 2 , Li (n- Bu)BH 3 , NaBH(OMe) 3 , NaBH(OAc) 3 , NaBH 3
  • palladium catalyst refers to elemental palladium and complexes thereof, wherein the palladium atom can be at various oxidation states and can possess one or more ligands.
  • Suitable palladium catalysts include, but are not limited to, tetrakis (triphenylphosphine) palladium (0) , bis (tri-t- butylphosphine) palladium (0) , palladium (II) acetate, bis (tricyclohexylphosphine) palladium (0) , palladium (II) chloride, bis (triphenylphosphine) palladium(II) chloride, tris (dibenzylideneacetone) dipalladium (0) , bis (dibenzylideneacetone) palladium, (1,1'- bis (triphenylphosphino) ferrocene) palladium (II) chloride, and bis (triphenylphosphin
  • EDC N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide
  • TBAF tetra-n-butylammonium fluoride
  • TBS tert-butyldimethylsilyl
  • TMS trimethylsilyl
  • Tf trifluoromethanesulfonyl
  • KHMDS potassium bis (trimethylsilyl) amide or potassium hexamethyldisilazide
  • AIBN 1, 1 ' -azobisisobutyronitrile
  • DCC N, N-dicyclohexylcarbodiimide
  • Ph phenyl
  • OVX ovariectomized
  • the compounds of the instant invention may be in a salt form.
  • a “salt” is the salt of the instant compounds which has been modified by making acid or base salts of the compounds .
  • the salt is pharmaceutically acceptable.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols.
  • the salts can be made using an organic or inorganic acid.
  • Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like.
  • Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium.
  • pharmaceutically acceptable salt in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention.
  • salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al . (1977) "Pharmaceutical Salts", J. Pharm. Sci . 66:1-19) .
  • compositions of this invention may be administered in various forms, including those detailed herein.
  • the treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds.
  • This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously.
  • These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.
  • a "pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human.
  • the carrier may be liquid or solid and is selected with the planned manner of administration in mind.
  • Liposomes are also a pharmaceutically acceptable carrier .
  • the dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.
  • a dosage unit of the compounds may comprise a single compound or mixtures thereof with anti-cancer compounds, or tumor growth inhibiting compounds, or with other compounds also used to treat osteoporosis or cardiovascular disease.
  • the compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions.
  • the compounds may also be administered in intravenous (bolus or infusion) , intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection or other methods, into the cancer, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
  • the compounds can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • a pharmaceutically acceptable carrier suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • the unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration.
  • the compounds can be administered alone but are generally mixed with a pharmaceutically acceptable carrier.
  • This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. In one embodiment the carrier can be a monoclonal antibody.
  • the active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form.
  • suitable solid carriers include lactose, sucrose, gelatin and agar.
  • Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents .
  • suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Oral dosage forms optionally contain flavorants and coloring agents.
  • Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
  • Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
  • the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.
  • Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
  • the compounds can also be administered in the form of liposome delivery systems, ' such as small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
  • the compounds may be administered as components of tissue-targeted emulsions.
  • the compounds may also be coupled to soluble polymers as targetable drug carriers or as a prodrug.
  • Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide- polylysine substituted with palmitoyl residues.
  • the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
  • prodrug refers to any compound that when administered to a biological system generates the compound of the invention, as a result of spontaneous chemical reaction (s), enzyme catalyzed chemical reaction(s), photolysis, and/or metabolic chemical reaction (s).
  • a prodrug is thus a covalently modified analog or latent form of a compound of the invention.
  • the compounds of the subject invention can be converted to prodrugs to optimize absorption and bioavailability.
  • Formation of a prodrug include, but is not limited to, reaction of a free hydroxyl group with a carboxylic acid to form an ester, reaction of a free hydroxyl group with phosphorus oxychloride followed by hydrolysis to form a phosphate, or reaction of a free hydroxyl group with an amino acid to form an amino acid ester, the reagents and process of which have been described previously by Chahdran in WO 2005/046575, which is hereby incorporated by reference.
  • the active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. It can also be administered parentally, in sterile liquid dosage forms.
  • Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours . Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours . Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • liquid dosage form For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • water a suitable oil, saline, aqueous dextrose (glucose) , and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • citric acid and its salts and sodium EDTA are also used.
  • parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol .
  • preservatives such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol .
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.
  • the compounds of the instant invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.
  • Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
  • the compounds and compositions of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.
  • tumor refers to an uncontrolled growth of cells.
  • a tumor may be benign or malignant. Benign tumors are not dangerous to health and are not considered cancerous. Malignant tumors are cancerous; they invade surrounding tissue, are usually capable of producing metastases, may recur after attempted removal, and are likely to cause death of the host unless adequately treated. Left unchecked, malignant cells can eventually spread beyond the original tumor to other parts of the body.
  • Various cancers result in the formation of tumors, including, but not limited to, prostate cancer, breast cancer, endometrial cancer, and colon cancer. In particular, breast cancer is thought to be due to exogenous and endogenous factors .
  • Typical exogenous - factors are environmental mutagens and carcinogens that induce DNA damage.
  • Typical endogenous factors may be hormones, such as estrogen, that have estrogenic action and induce DNA damage.
  • Such tumors are said to be estrogen-induced tumors.
  • breast cancer tumors may express estrogen receptors ERa and/or ER ⁇ .
  • Tumors expressing any isoform of ER, i.e. ERa or ER ⁇ are said to be ER-positive and tumors not expressing any isoform of ER are said to be ER-negative.
  • tumors expressing ERa are said to be ER ⁇ -positive and tumors not expressing ERa are said to be ER ⁇ -negative.
  • Tumors expressing ER ⁇ are said to be ER ⁇ -positive and tumors not expressing ER ⁇ are said to be ER ⁇ -negative .
  • bone mineral density refers to the density of minerals, such as calcium, found in bones.
  • Bone mineral density can be measured using several different methods. These methods include, but are not limited to, dual-energy X-ray absorptiometry (DEXA) , peripheral dual-energy X-ray absorptiometry (P-DEXA) , dual photon absorptiometry (DPA) , ultrasound, or quantitative computed tomography (QCT) .
  • DEXA dual-energy X-ray absorptiometry
  • P-DEXA peripheral dual-energy X-ray absorptiometry
  • DPA dual photon absorptiometry
  • ultrasound or quantitative computed tomography
  • TAM endometrial cancer
  • endometrial cancer a carcinogenic effect is thought to involve initiation and/or promotion.
  • TAM is a partial ER agonist in uterine tissue [17] and such estrogenic effects may promote endometrial cancer [18, 19] . Therefore, the rational design of safer antiestrogens would encompass lowering both genotoxicity and estrogenic activity in order to circumvent these serious side effects .
  • Toremifene (TOR, Fig. 2), the chloroethyl analog of TAM, does not promote hepatic DNA adducts [10, 30, 31] and hepatocarcinoma in rats [10, 31], although the metabolic fate of TOR is similar to that of TAM. Previous studies indicate that this may be due to the steric hindrance caused by a bulky chlorine atom positioned at the ethyl moiety of TOR, which may reduce the ability of hydroxysteroid sulfotransferase to O-sulfonate ⁇ -OHTOR and consequently the amount of TOR ⁇ -sulfate available to react with DNA [32] .
  • the field effect caused by the electron-withdrawing chlorine atom may also diminish the effective elimination of the ⁇ -sulfate group from the ⁇ - carbon and therefore inhibit the formation of the carbocation intermediate that reacts with DNA [32] .
  • No K- ras mutation was observed in the endometria of patients receiving TOR [29] . Therefore, the non-genotoxicity of TOR may stem from its inability to form DNA adducts as a result of replacing the ethyl group of TAM with a chloroethyl moiety.
  • GW5638 posesses a novel carboxylic acid side chain (see X position in Fig. 2) and has been recognized as having powerful antiestrogenic activity. This compound has estrogen-like actions to preserve bone density in OVX- rats but, unlike TAM, has pure antiestrogenic activity in rodent uterus. Moreover, GW5638 does not promote tumor growth in rodent breast or endometrial cells [57, 58] . However, it was recently found that GW5638 does show estrogenic (uterotrophic) activity equivalent to that of RAL (see Fig. 4) . GW5638 has an ethyl moiety that could potentially undergo ⁇ -hydroxylation followed by O- sulfonation to produce an intermediate capable of reacting with DNA.
  • the moiety at the Y position (Fig. 2) affects the antiestrogenic activity. Since 4-OHTAM is a principal metabolite showing ⁇ 100-fold more antiestrogenic activity than TAM in vitro [65], the 4-OH moiety may confer high antiestrogenic activity.
  • step a the desired benzophenone is reacted with an alkyl phenone of desired length, as indicated by q, and substitution, as indicated by X, in the presence of low- valent titanium (typically synthesized in situ from a • titanium reagent and a reducing agent) to produce the desired triphenylethylene intermediate.
  • low-valent titanium typically synthesized in situ from a • titanium reagent and a reducing agent
  • McMurry reaction to form similar triphenylethylene cores has been described by S ⁇ dervall et al in WO 01/36360, which is hereby incorporated by reference.
  • the triphenylethylene intermediate is then triflated (scheme 1, step b) using triflic anhydride (trifluoromethanesulfonyl anhydride) following the McMurry coupling reaction.
  • triflic anhydride trifluoromethanesulfonyl anhydride
  • the subsequent palladium- catalyzed coupling with an alkyl acrylate is achieved in the presence of Pd(O) or Pd(II) .
  • Hydrolysis of the coupled product yields the desired acrylic acid- functionalized triphenylethylene compound (Scheme 2) .
  • step a the requisite benzophenone and alkyl phenone may be obtained commercially with the desired substituents already in place.
  • substituents are installed onto the phenyl rings of the molecules using standard aryl substitution reactions well known in organic synthesis.
  • the alkyl portion of the alkyl phenone may be unsubstituted or substituted using standard methods well known to the ordinarily skilled artisan.
  • the acrylic acid ester may be replaced by other unsaturated compounds capable of engaging in palladium-catalyzed coupling reactions.
  • unsaturated compounds capable of engaging in palladium-catalyzed coupling reactions.
  • terminal alkenes and alkynes of varying length and substitution may be used.
  • the resulting coupled product may be reduced using well- known reduction chemistry to yield the corresponding saturated product.
  • Phenolic compounds such as the triphenylethylenes where the Y position is OH, may be rapidly conjugated through phase II metabolism and detoxified.
  • the triphenylethylenes can be prepared as prodrugs.
  • the prodrugs should be converted to the active parent during transportation by specific enzymes such as alkaline phosphatase or carboxypeptidase A in the gastrointestinal tract [67] . If the prodrug is efficiently transported into the body and converted to the active drug, the concentrations of the prodrug and its active product should be higher than those obtained with the parent compound alone. Therefore, the prodrug is expected to demonstrate higher efficacy both in inhibiting cancer growth and in preventing cancer development.
  • RAL diphosphate and TAT-59 were designed as a phosphate ester prodrug and has shown improved oral bioavailability [66, 68] .
  • O-Valinyl derivatives have been used as amino acid ester prodrugs [67, 69] .
  • the formation of the phosphate and amino acid ester prodrugs is achieved according to the general procedure shown in Scheme 3. f. DCC, base, protected amino acid g. hydrolysis
  • the phosphate prodrug is formed by reacting a triphenylethylene having a free hydroxyl group with phosphorus oxychloride (POCI3) in the presence of a base, and the resulting product is hydrolyzed to form the desired phosphate prodrug (scheme 3, steps d and e) .
  • POCI3 phosphorus oxychloride
  • the phosphate may also be further alkylated using standard alkylation methods, such as nucleophilic substitution of the deprotonated phosphate.
  • the prodrug can be incubated at 37 0 C for 1 hr in a buffer containing cytosol (0.1 mg protein) of rat intestine.
  • cytosol 0.1 mg protein
  • the amino acid ester prodrug is formed by reacting a triphenylethylene having a free hydroxyl group with an N- protected amino acid in the presence of DCC and a base followed by hydrolysis to form the desired amino acid ester prodrug (scheme 3, steps f and g) .
  • triphenylethylenes of the subject invention are evaluated for activity against breast cancer, osteoporosis, and cardiovascular disease using various in vitro and in vivo assays and experiments, some of which are briefly described hereinafter.
  • Uterotrophic potential Safer antiestrogen alternatives to TAM and RAL should have little or no estrogenic activity in order to minimize the risks of developing uterine and endometrial cancers.
  • the compounds of the invention are subjected to an animal uterotrophic assay for evaluating their estrogenic potential.
  • OVX-rats Sprague Dawley, 6-week-old females
  • the uterine weight is measured 1 day after the final treatment, following an established protocol [62, 70].
  • a dose molar equivalent to TAM (0.33-0.66 mg/kg/day) is human equivalent (20-40 mg/60 kg woman/day)
  • a high dose at a molar equivalent to TAM [10 mg (27 ⁇ mol) /kg/day] suspended in 1.0 ml of corn oil is applied to ensure detection of estrogenic activity of each compound (8 rats/group) .
  • the results are compared with those observed with their counterpart antiestrogens
  • Control rats (8 rats) receive only corn oil orally. The animals are euthanized 1 day after the final treatment by
  • each uterine horn is processed, embedded in paraffin, sectioned into 5 ⁇ m slices and stained with hematoxylin and eosin for histological and morphometric evaluation.
  • Anti-uterotrophic activity is determined by measuring the inhibitory effect of the compounds on the uterine weight of OVX-rats treated with E 2 [62, 70] .
  • OXV-rats (Sprague- Dawley OVX 6-week-old females) are treated subcutaneously with E 2 (0.3 ⁇ g/100 ⁇ l corn oil/rat) for 3 days and simultaneously treated orally for 3 days with the selected compounds (8 rats/group) at doses molar equivalent to TAM (0.1, 1.0, and 10 mg/kg/day) .
  • Antiestrogens TAM, RAL, GW5638, and SP500263 are used as comparisons.
  • the control OVX-rats (8 rats) are treated orally with corn oil.
  • the animals are euthanized by CO2 asphyxiation one day after the final treatment. Uterine wet weights and body weights are then measured to determine the uterine wet-weight/body-weight ratios, as described above for the uterotrophic assay. The data are compared with those obtained from the OVX-rats (8 rats) treated subcutaneously with E 2 (0.3 ⁇ g/100 ⁇ l corn oil/rat, 8 rats) as a positive control.
  • the genotoxic potential of the compounds selected by uterotrophic evaluation is determined by measuring DNA damage in rodents.
  • Rat liver contains a high level of hydroxysteroid sulfotransferase (HST) that actively converts ⁇ -hydroxylated TAM metabolites to their O- sulfate forms, which in turn react with cellular DNA [15] .
  • HST hydroxysteroid sulfotransferase
  • the enzymatic activity of rat HST is at least one order of magnitude higher than that of human HST (SULT 2Al); therefore, the rat is a good model animal to evaluate the genotoxicity of antiestrogens .
  • the selected compounds are administered orally to rats for 7 days, as reported previously [17, 22] .
  • TAM is used as a positive control [22] .
  • Control animals receive corn oil only. Since 20 mg/kg of TAM induce large amounts of hepatic DNA adducts in rats [22], the molar equivalent dose of TAM is applied for the selected compounds (5 rats/group) . Five hours after the final treatment, the animals are then euthanized by CO 2 asphyxiation. The liver is removed quickly, frozen in liquid nitrogen, and stored at -8O 0 C until DNA extraction. DNA adduct analysis using 32 P- postlabeling methods have been established [1, 13] .
  • Anti-breast cancer potential The anti-breast cancer potential of the compounds of the subject invention that lack both estrogenic and genotoxic activities are determined using rats bearing DMBA-induced mammary carcinoma. Selected compounds displaying antitumor activity superior to TAM and RAL are subjected to further evaluation against human MCF-7 breast cancer xenograft in nude mice.
  • DMBA-induced mammary carcinoma in the rat is a widely used animal model to study the factors which control hormone-sensitive breast cancer in humans [62, 70]. Using this model, the antitumor activity of the selected compounds is determined. The results are compared with those observed for antiestrogens (TAM, RAL and ICI 182,780) currently in clinical use or GW5638 as a counterpart of the compounds of the invention. Following an established protocol [70, 71], mammary carcinoma is induced by treating rats (Sprague-Dawley, 8-week-old females) with a single oral dose (50 mg/kg) of DMBA in corn oil and an approximately 8-mm diameter tumor is expected to develop in 8 weeks. The rats are then treated orally for 4 weeks with each compound.
  • TAM and RAL are used as the positive controls. Controls receive corn oil only. The volume of the tumors is recorded twice a week, using the two perpendicular dimensions.
  • Athymic nude mice implanted with human mammary tumor xenograft are widely used to evaluate the antitumor efficacy of antiestrogens against human breast cancer [70, 72].
  • OVX- nude mice (nu/nu-BALB/c, 8-week-old females) supplemented with an estrogen pellet (E 2 , 0.72 mg/pellet) given subcutaneously are injected with MCF-7 cells (2 x 10 7 cells in 0.2 ml saline) subcutaneously into the shoulder region; the tumors are then allowed to grow for 6 weeks (up to ⁇ 6 mm in diameter) .
  • the nude mice (8 mice/group) are then treated orally for 4 weeks with the selected compounds.
  • Body weight is also measured twice a week to monitor the growth of the animals. Since it is often observed that drug metabolism in mice is faster than in rats, a dose (10 mg/kg) higher than that applied to the rat tumor model is used. To explore the dose response effects, the efficacy of lower molar doses equivalent to TAM (1.0 and 3.0 mg/kg/day, p.o.) are also examined for the selected compounds, and the results are compared with those for TAM and RAL. Mice from which the estrogen pellets are removed before starting antiestrogen treatment are used as negative controls. As a further control, 8 mice receive corn oil only. Following an established protocol [73], as described for the DMBA- rats, the volume of the tumors are measured twice a week. TV and RTV are estimated from equations 1 and 2 above, respectively. Because studies using nude mice have previously been performed [68], the anti-breast cancer potential of the compounds of the invention is readily determined.
  • ER ⁇ may play an important role in the antitumor action of the selected compounds. Other mechanisms such as apoptosis and cell arrest may also be involved in enhancing their antitumor potential.
  • ER-binding specificity of the compounds of the invention is determined using an ER binding assay. The level of cell- cycle arrest and apoptosis induced by the compounds is also determined.
  • the differential binding affinity of antiestrogen to ERa and ER ⁇ may affect transcriptional regulation [57]; thereby, the antitumor activity of antiestrogens may vary.
  • RBA relative binding affinity
  • results are compared with those observed for other antiestrogens (TAM, 4-OHTAM, TOR, RAL, ICI 182,780, GW5638, GW7604, and SP500263) .
  • the results obtained from this assay may suggest the contribution of ERa and ER ⁇ to the antitumor potency of antiestrogens.
  • certain antiestrogens could act as estrogen agonists or antagonists to mimic estrogen's positive effects, depending on the specific tissue.
  • human and mouse genetic studies suggest a predominant role for ERa in bone metabolism [74, 75] . Different affinities of antiestrogen to ERa and ER ⁇ may also affect the results of anti-osteoporotic efficacy- tests .
  • Cell-cycle and apoptosis A mechanism other than an antiestrogenic one may also contribute to improving the antitumor potential of antiestrogens.
  • weak cell-cycle arrest and apoptosis have been observed in cultured breast cancer cells exposed to TAM [76, 77] .
  • flow cytometric analysis and other techniques the stages of the cell- cycle and apoptosis in cultured human breast cancer cells exposed to the compounds of the invention as well as in mammary tumors of rodents treated with the compounds are determined.
  • MDA-MB-231 cells Human MCF-7 breast cancer cells express both ERa and ER ⁇ but not the apoptotic factor caspase-3; in contrast, MDA- MB-231 cells do not express ERa but they do express caspase-3. Therefore, if the compounds of the invention do have an apoptotic effect, it should be observable in MDA-MB-231 cells.
  • TAM as a positive control, alterations to the cell-cycle and apoptosis generated by the compounds with varying exposure times (1, 3, and 5 days) and several doses (0.01, 0.1, 1.0, 10, 25, 50 and 100 ⁇ M) are evaluated. Cell viability is determined by using a trypan blue exclusion assay to measure the percentage of viable cells under a field microscope.
  • both types of human breast cancer cells exposed to antiestrogens are harvested from culture media, fixed in paraformaldehyde, incubated with DNA-labeling solution, and stained using propidium iodide (PI) working solution with RNase type2A. If the cell-cycle and apoptosis stages in mammary tumors taken from rodents treated with antiestrogens can be determined, a more precise effect of the compounds could be investigated.
  • mammary tumors are collected from rats bearing DMBA-induced mammary carcinoma or from nude mice bearing MCF-7 human breast tumor xenograft.
  • Caspase-3 functions as a major mediator of apoptotic execution [78]. Involvement of caspase-3 in antiestrogen- induced anti-breast cancer potential is determined using Western-blot analysis [79] and immunohistochemical analysis. Immunohistochemical analysis is performed on BrdU-labeled (brown-stained nuclei) cells using caspase-3 antibody and an ABC kit. The slides are counter-stained with hematoxylin for identification of tumor morphology. The percentage of caspase-3 positive nuclei in the mammary tumor is determined using a semiquantitative histomorphometric evaluation.
  • MDA-MB-231 cells expressing caspase-3 are also implanted in nude mice for exploring the contribution of apoptosis to the antitumor potential of antiestrogens. Since MDA- MB-231 cells are ER ⁇ -negative, OVX and estrogen pellet implantation are not required for this experiment. Intact nude mice are injected with MDA-MB-231 cells (2 x 10 7 cells in 0.2 ml saline) subcutaneously into the shoulder region for the implantation, following an established protocol [81] .
  • the nude mice When the tumors grow to ⁇ 6 mm in diameter, the nude mice are treated orally for 4 weeks with compounds of the invention at a dose molar equivalent to TAM (1.0, 3.0 or 10 mg/kg/day, 8 mice/dose) .
  • TAM 1.0, 3.0, and 10 mg/kg/day
  • Eight control mice receive corn oil only. If apoptosis is primarily involved in the antitumor potential, then growth of MDA-MB-231 xenograft should be reduced more effectively than that of MCF-7 xenograft.
  • MDA-MB-231 xenograft collected from nude mice are determined using flow cytometry and Western-blot analysis, as described earlier. If apoptosis is a major factor in suppressing the growth of mammary tumors, then it should be induced more in MDA-MB-231 xenograft expressing caspase-3, as compared with MCF-7. Results obtained from studies determining apoptotic potential provide insight into the anti-breast cancer mechanism.
  • Typical exogenous factors are environmental mutagens and carcinogens that induce DNA damage.
  • Typical endogenous factors may be hormones that have estrogenic action and induce DNA damage. Such factors may be involved in initiating and/or promoting mammary tumor.
  • Compounds having the capability of preventing development of mammary tumors could be used as preventive agents. Therefore, the preventive activities of the selected compounds are determined in the following two animal models.
  • Chemical carcinogen (DMBA) -induced mammary tumor in rats is used as a model to evaluate the preventive effect against a typical exogenous factor.
  • E2 Hormone (E2)- induced mammary tumor in rats is used as a model to evaluate the preventive effect against a typical endogenous factor.
  • the incidence of mammary tumors may parallel the frequency of carcinogen-induced DNA adducts in the mammary gland, as observed with ⁇ -NF [82] .
  • treatment with TAM prior to a single DMBA injection has been shown to reduce the formation of mammary DMBA- DNA adducts in rats [82] .
  • Such treatment lowered the incidence of mammary carcinoma in mice [83, 84].
  • TAM or its metabolite is thought to inhibit the bioactivation of DMBA and/or to increase its rate of detoxification, thus lowering the level of adduct formation, as demonstrated by ⁇ -NF and ⁇ -NF [82,85].
  • RAL with its higher antiestrogenic activity, did not show such a preventive effect on mammary tumor in mice [84] , although this drug significantly reduced the incidence of breast cancer in women at high risk of developing the disease [34] .
  • the preventive mechanism of each antiestrogen may involve other factors.
  • the carcinogen-induced rodent mammary tumor model has been widely used to explore the preventive effects of many compounds, including antiestrogens .
  • antiestrogens include antiestrogens, antiestrogens, antiestrogens, antiestrogens, antiestrogens, antiestrogens, antiestrogens, antiestrogens, antiestrogens, antiestrogens .
  • ⁇ -NF post-initiation (promotion) stage in DMBA-induced carcinogenesis
  • Rats (10 rats/dose) are treated orally with the compounds of the invention at molar equivalent doses to TAM (1.0, 3.0, or 10 mg/kg/day) 7 days prior to oral treatment with a single dose (50 mg/kg) of DMBA.
  • the second protocol is used to determine the effect of antiestrogen on the post-initiation stage of carcinogenesis.
  • Oral treatment with compounds at molar equivalent doses to TAM (1.0, 3.0. or 10 mg/kg/day) of rats (10 rats/dose) start 3 weeks after a single dose (50 mg/kg) of DMBA and continue for 3 weeks.
  • the control rats receive a vehicle (corn oil) only.
  • TAM 1.0, 3.0, 10 mg/kg/day
  • RAL will be used as a negative control [84] .
  • Pretreatment with ⁇ -NF [85] or ⁇ -NF [82] reduced the formation of DMBA-DNA adducts in rodents and/or the incidence of DMBA-induced mammary tumor; therefore, ⁇ -NF or ⁇ -NF (29 or 73 mg/kg/day) at doses molar equivalent to TAM (40 or 100 mg/kg/day) are also used.
  • ⁇ -NF or ⁇ -NF 29 or 73 mg/kg/day
  • TAM 40 or 100 mg/kg/day
  • DMBA-DNA adducts in the mammary gland (as a target organ) and the liver (as a major organ metabolizing DMBA) are determined as an end-point marker using 32 P-postlabeling analysis.
  • Procedures for DNA extraction and 32 P-postlabeling analysis have been established [87] .
  • Rats (5 rats/dose) are treated orally with the compounds at doses molar equivalent to TAM (1.0, 3.0, or 10 mg/kg/day) 7 days prior to a single oral dose (50 mg/kg) of DMBA.
  • Positive controls (5 rats) receive corn oil only prior to a single oral dose of DMBA.
  • Negative controls (5 rats) receive corn oil only without DMBA treatment. The results are compared with those observed with TAM or RAL (1.0, 3.0, or 10 mg/kg/day) . Since pretreatment with ⁇ -NF [85] or ⁇ -NF [82] has been shown to reduce the formation of DMBA-DNA adducts in rodents, ⁇ -NF or ⁇ -NF (29 or 73 mg/kg/day) at doses molar equivalent to TAM (40 or 100 mg/kg/day) are used as a positive control. The rats are euthanized one day after the DMBA treatment by CO 2 asphyxiation. Mammary gland and liver are collected for analysis of DMBA-DNA adducts.
  • DNA is isolated from mammary gland or liver using phenol/chloroform extraction or a commercially available kit and digested using micrococcal nuclease and spleen phosphodiesterase to produce normal (dN3'P) and adducted deoxynucleoside 3' -monophosphate (dX3'P) .
  • dN3'P normal
  • dX3'P deoxynucleoside 3' -monophosphate
  • nuclease Pl enrichment adducted nucleotides are enriched during this process (nuclease Pl enrichment), labeled with 32 P using [ ⁇ - 32 P]ATP and T4 polynucleotide kinase, and resolved by 30% non-denaturing polyacrylamide gel electrophoresis (PAGE) or by using an HPLC/radioisotope detector.
  • Active diol epoxide forms of DMBA [ ( ⁇ )anti-DMBADE and ( ⁇ ) syn-DMBADE] are prepared, following an established method [88] , and reacted with 2' -deoxyguanosine monophosphate (dGMP) or 2'- deoxyadenosine monophosphate (dAMP) in a buffer [89] .
  • dGMP 2' -deoxyguanosine monophosphate
  • dAMP 2'- deoxyadenosine monophosphate
  • DMBA-DNA adducts are used as markers for 32 P- postlabeling analysis.
  • the limit of detection for a 5 ⁇ g DNA sample is approximately 3 adducts in 10 9 nucleotides for 32 P-postlabeling/PAGE and 3 adducts in 10 10 nucleotides for 32 P-postlabeling/HPLC.
  • LC/MS/MS liquid chromatography electrospray tandem mass spectrometry
  • LC/MS/MS LC/MS/MS
  • DNA is digested using micrococcal nuclease and spleen phosphodiesterase and incubated with alkaline phosphatase.
  • the resulting nucleosides are subjected to LC/MS/MS.
  • the detection-- limit is approximately 1 adduct in 10 8 bases using 25 ⁇ g of DNA. If the amount of DMBA-DNA adduct formed in the mammary gland parallels the incidence of developing mammary tumors generated by antiestrogens, this assay can be used to predict the preventive efficacy of compounds and also used as a screening system to find highly effective preventive agents.
  • CYP 1 family enzymes CYPlAl, 1A2 and IBl
  • AhR aryl hydrocarbon receptor
  • pretreatment with ⁇ -NF a potent inhibitor of CYP 1 family enzymes, reduced the formation of DMBA- DNA adducts in rodents [91] . If pretreatment with the selected compounds shows a preventive effect against DMBA-induced mammary tumor, then expression of CYPl enzymes is determined.
  • a fraction of mammary gland (as a target organ) and liver (primary site of DMBA metabolism) collected for studies for DMBA-DNA adduct formation are used for determination of CYP 1 and AhR expression using Western-blot analysis and relative reverse transcriptase-polymerase chain reaction (RT-PCR) .
  • RT-PCR relative reverse transcriptase-polymerase chain reaction
  • ⁇ -NF a potent inducer of phase II enzymes
  • the expression of GSTs and UDP-GT, typical phase II enzymes [92] is also determined.
  • RNA/DNA/protein isolation reagent such as TRI - REAGENT® (MRC)
  • MRC a monophase solution comprising phenol and guanidine thiocyanate
  • proteins are resolved on polyacrylamide gels and transferred to a hydrophobic polyvinylidene difluoride membrane, such as HYBONDTM-P membrane (Amersham Pharmacia Biotech) .
  • Membranes are blocked 8 h at room temperature with shaking in Tris-buffered saline plus Tween (TBST) and then incubated with antibody to CYPlAl, CYP1A2, CYPlBl, AhR, GSTs, or UDP-GT in TBST plus milk powder overnight at 4°C with shaking. ⁇ -Actin is used as a loading control. After washing, the membranes is incubated in TBST plus milk powder containing horseradish peroxidase-conjugated secondary IgG A. Membranes are washed three times in TBST, and the proteins are visualized using an enhanced chemiluminescence system, such as ECL PLUSTM (Amersham Pharmacia Biotech) , and detected by autoradiography. Immunoquantitation is obtained by densitometric scanning of the resulting autoradiographs using a molecular imager.
  • RNA/DNA/protein isolation reagent such as TRI REAGENT® (MRC)
  • MRC RNA/DNA/protein isolation reagent
  • First- strand complementary DNAs (cDNAs) are synthesized from total RNA using the Reverse Transcription System (Promega) .
  • PCR is performed using the primer sequences for CYPlAl, CYP1A2, CYPlBl, ⁇ -actin [93], for AhR [94], and for GSTs and UDP-GT [92, 95] .
  • the PCR products are fractionated through an agarose gel and visualized by dual staining with ethidium bromide and SYBR green.
  • the bands are analyzed by video densitometry; the areas of the peaks are calculated in arbitrary units.
  • the relative value is generated by calculating the ratio of the
  • hormone (E 2 ) -induced mammary tumor ACI rats have been used as model animals for studying human sporadic breast cancer [96] .
  • mammary tumors Following this protocol [96], mammary tumors have been successfully induced in approx. 3 months in ACI rats implanted under the skin with a pellet containing 2.5 mg of E 2 .
  • Oral treatment with selected compounds at molar equivalent doses to TAM (1.0, 3.0, or 10 mg/kg/day) of rats (10 rats per dose) are started 7 days prior to implanting the estrogen pellet under light isoflurane anesthesia and continue until - the experiment is completed. Controls (10 rats) receive a placebo pellet. Tumor size and number are recorded once a week during the course of the study. The results are then compared with those observed with TAM or RAL (1.0, 3.0, or 10 mg/kg/day) . If a preventive effect is observed against E 2 -induced rat mammary tumors, the expression of mammary CYP 1, AhR, and PR is monitored during the period of antiestrogen treatment.
  • Rats (10 rats/dose) are treated orally with selected compounds at doses molar equivalent to TAM (1.0, 3.0, or 10 mg/kg/day) prior to being implanted with an E 2 pellet.
  • Controls (10 rats) receive a placebo pellet.
  • Five rats are then euthanized 1.5 and 3.0 months after the pellet implantaion by CO 2 asphyxiation.
  • the fourth mammary gland is collected for analysis of CYP 1, AhR, and PR expression using Western blot and RT-PCR, as described earlier. The results are- compared with those observed with TAM or RAL (1.0, 3.0, or 10 mg/kg/day) .
  • the preventive activity may be associated with the inhibitory effect of antiestrogen on CYP 1, AhR, and/or PR expression.
  • Expression of ERa and ER ⁇ in ACI rats treated with selected compounds are also monitored using Western-blot analysis [79] .
  • the skin containing the third mammary gland is collected from ACI rats 1.5 and 3 months after the E 2 - or placebo-pellet implantation.
  • the mammary gland is fixed in 10% neutral buffered formalin for 3 days and then dissected free from the skin and processed as a whole mount.
  • the gland is defatted in ethanol, acetone, chloroform and ethanol again, 3 days for each solvent.
  • the gland is stained with hematoxylin and washed with distilled water.
  • the stained gland is cleaned up under a stereomicroscope, dehydrated in ethanol, cleared in xylene and mounted. Photographs are taken using a digital color camera mounted on a microscope.
  • antiestrogens have the capability of inhibiting the development of E 2 -stimulated mammary gland, then the increase in size and number of end buds by E 2 treatment observed in normal mammary gland [100] should be reduced.
  • the preventive potential of compounds of the invention Taken together with results obtained from the DMBA-induced mammary tumor model, the preventive potential of compounds of the invention, and the preventive mechanism are evaluated. If the compounds protect against development of mammary tumors induced by the endogenous or exogenous carcinogen, such antiestrogens could provide potential benefits for women in preventing the disease.
  • Antiestrogens act as selective estrogen receptor modulators (SERMs) , exerting a broad spectrum of effects on tissues, including maintenance of bone density and cardiovascular protection [101] .
  • TAM a first-generation SERM, revealed the efficacy of estrogen in preventing bone loss [102-104].
  • RAL Several other antiestrogens, including RAL, were more effective than TAM in preventing bone loss (35, 39, 62, 105] . If the compounds of the invention have a similar or higher preventive effect than RAL, such drugs could provide more benefits for patients to protect against osteoporosis.
  • ⁇ CT micro-computed tomography
  • serum cholesterol levels are also determined as a biomarker in - - the cardiovascular - system. -- ⁇ - - -
  • OVX-rat is widely used as an osteopenic animal model that mimics the development of estrogendeficiency-induced osteopenia in humans [111, 112].
  • OVX-rats have been known to lose bone mineral content (BMC) [70] .
  • BMC bone mineral content
  • OVX-rats, aged 13 weeks (Sprague- Dawley, females) are treated orally once daily for 3 months with each compound.
  • Doses molar equivalent to TAM 1.0, " 3.0, or 10 mg/kg/day in corn oil
  • 10 rats per a dose are used.
  • TAM and RAL are used as the positive control.
  • E 2 (0.0073 ⁇ mol/kg/day) is administered subcutaneously to one group of OVX-rats as a positive control.
  • Sham rats are treated with corn oil as a control.
  • the vehicle volume is adjusted to 1.0 ml/kg rat body weight.
  • Body weight is measured weekly.
  • Three-dimensional bone quantity, quality, and architecture are monitored once every month for 3 months under a light anesthesia using a ⁇ CT.
  • Blood is also taken from the tail before treatment of the compound and then once every month for 3 months to determine the serum alkaline phosphatase activity and total cholesterols .
  • the animals are euthanized by CO2 asphyxiation one day after the final treatment. Blood and tissues are taken and stored in a -80 0 C deep freezer.
  • uteri are dissected free of fat, and the wet weights measured for determination of uterotrophic potential. A portion of each uterus is used for preparation of formalin-fixed tissue for pathological determination .
  • normal Sprague-Dawley rats (10 rats per dose) are also treated orally once daily for 3 months with the selected compounds at doses molar equivalent to TAM (3.0 or 10 mg/kg/day) .
  • the body weight is measured twice a week to monitor the growth of the animals.
  • the animals are then euthanized by CO 2 asphyxiation one day after the final treatment.
  • Liver and uteri are dissected and subjected to pathological evaluation. The final pathological evaluation is performed. If abnormal effects are found in comparison with the control, such compounds are excluded from the candidate compounds .
  • a ⁇ CT scanner provides a high resolution imaging technique to quantify changes in bone morphology over time, including bone mineral density (BMD) and BMC [113] . Since bone metabolism is more active in trabecular bone than cortical bone, osteopenic abnormality generally appears on trabecular bone earlier than cortical bone [106] .
  • BMD bone mineral density
  • BMC BMC
  • cortical and trabecular bone quantity and quality, and micro-architecture of the distal femur and the femoral diaphysis are measured. Three-dimensional images are also acquired and a Gaussian filter is used to partly suppress noise in the volumes [107] .
  • tissue volume (TV), bone volume, (BV), bone volume fraction (BV/TV) , bone surface (BS) , the structure model index (SMI, an estimation of the plate-rod characteristic of the structure) , connectivity density (Conn. D), mean trabecular thickness (Tb. Th), mean trabecular separation (Tb. Sp), mean trabecular number (Tb. N) and the geometrical degree of anisotropy (DA, ratio between the -maximal and minimal - radii of the mean intercept length ellipsoid) , and tissue mineral density (TMD, a measure of tissue mineralization) .
  • TMD tissue mineral density
  • alkaline phosphatase levels have been used as a biomarker of bone turnover [HO].
  • alkaline phosphatase levels increase in both OVX- rats [116] and postmenopausal women [117], indicating an increase in bone turnover.
  • Blood is taken from OVX-rats once every month during the 3 months of antiestrogen treatment.
  • Serum total alkaline phosphatase activity is determined by commercially available kits [116] . Since this assay is based on colorimetric estimation of p- nitrophenol formed after the breakdown of p- nitrophenylphosphate by the enzyme, both hepatic and bone alkaline phosphatases can be measured together.
  • Serum cholesterol levels increase in postmenopausal women and can be reversed by hormone replacement therapy [118] . Serum cholesterol levels also increase in OVX-rats [62]. Therefore, serum cholesterol level is accepted as a biomarker of estrogen action in the cardiovascular system.
  • E 2 , TAM and RAL suppress the rise in serum cholesterol associated with ovariectomy [119, 120].
  • E 2 lowers cholesterol by upregulating the hepatic low- density lipoprotein (LDL) receptor, resulting in an increased removal of serum cholesterol from the circulation [121] .
  • LDL low- density lipoprotein
  • HDL rat high-density lipoprotein
  • serum HDL and LDL cholesterols are also determined. Serum total cholesterol, LDL and HDL levels in OVX-rats treated with selected compounds are determined once every month by commercially . available kits [120] . If antiestrogen treatment does not increase the serum cholesterol level, such compounds may help prevent cardiovascular disease.
  • the compounds and compositions of the invention are useful for preventing and/or inhibiting the proliferation of tumors, particularly estrogen-induced tumors, as well as tumors expressing ERa. Furthermore, the compounds and compositions of the invention are also useful for the treatment of osteoporosis.
  • GW5638 Most triphenylethylene antiestrogens are currently prepared using many synthetic steps. Therefore, the yield is low, which contributes to the high cost of the drugs. For example, GW5638 has been prepared through 9 steps [73] . To expedite the synthesis, GW5638 was prepared in only 3 steps with good overall yield ( ⁇ 45%) according to the general procedure shown in schemes 1 and 2. The triphenylethylene core was synthesized according to a strategy similar to that described by Gaulthier and co- workers [123] . The McMurry reaction [124] was carried out with commercially available 4-bromo benzophenone and propiophenone in the presence of TiCl 4 and Zn.
  • SSlOlO was synthesized according to scheme 5.
  • the McMurry reaction was carried out with commercially available 4- bromo benzophenone and chloropropiophenone .
  • the bromo olefin 3 was obtained as a mixture of E and Z isomers which were separated by column chromatography.
  • the subsequent palladium-catalyzed coupling reaction of (Z)- bromo olefin 3 with t-butyl acrylate was achieved in the presence of Pd (PPh 3 ) 2 and 1, 2, 2, 6, 6-pentamethylpiperidine (PMP) in DMF solvent to form the coupled acrylate 4.
  • Acrylate 4 was hydrolyzed in the presence of trifluoroacetic acid (TFA) to yield SSlOlO.
  • TFA trifluoroacetic acid
  • SS1020 was synthesized according to scheme 6.
  • the McMurry reaction was carried out with a monosilylated dihydroxy benzophenone and chloropropiophenone.
  • the resulting product 5 was obtained as a mixture of E and Z isomers and the mixture was subsequently triflated using triflic anhydride to give triflate 6.
  • Triflate 6 was purified by column chromatography to give the (Z) -triflate .
  • the palladium-catalyzed coupling reaction of (Z) -triflate 6 with t-butyl acrylate was achieved in the presence of Pd(PPh 3 J 2 and 1, 2, 2, 6, 6-pentamethylpiperidine (PMP) in DMF solvent to form the coupled acrylate 7.
  • PMP Pd(PPh 3 J 2 and 1, 2, 2, 6, 6-pentamethylpiperidine
  • Acrylate 7 was hydrolyzed in the presence of TFA and the silyl group was cleaved using tetrabut
  • SS1022 (valinyl derivative of SS1020)
  • the valinyl derivative of SS1020 was obtained according to established procedures [128] and is shown in scheme 8. Reaction of the t-butyl ester of SS1020 with protected valine in the presence of N,N-dicyclohexylcarbodiimide (DCC) and pyridine followed by hydrolysis gave the desired valinyl product SS1022.
  • DCC N,N-dicyclohexylcarbodiimide
  • Sensitive 32 P postlabeling/PAGE analysis [87] has been used for determination of the level of DNA adducts generated by antiestrogens.
  • TAM TAM
  • three rats were treated orally for 7 days with SS1020 or TOR at a dose molar equivalent to TAM (20 mg/kg/day) .
  • Rats receiving corn oil only were used as the control.
  • a high level ( ⁇ 1 adduct/105 nucleotides) of hepatic DNA adducts was observed with TAM while no DNA adducts were detected with SS1020 or TOR (Fig. 3) and the control.
  • SS1020 may be a compound free of genotoxic effects.
  • the uterotrophic potential of SS1020 was determined using ovariectomized (OVX) rats and compared with those of antiestrogens being used clinically or considered for clinical trials.
  • OVX-rats (Sprague-Dawley, 6-week old females) were treated orally for 3 days with each compound and the uterine wet weight [mg/g body weight (B.W.)] was measured one day after the final treatment.
  • a dose molar equivalent of TAM [10 mg (27 ⁇ mol) /kg/day] suspended in 0.5 ml of corn oil was used for each antiestrogen (4 rats/group) .
  • the uterine weight of the untreated OVX-rats was 0.255 mg/gB.W.
  • TAM had high uterotrophic activity, showing 54 % of that observed for E 2 -treated OVX-rats; even using 0.1 and 1.0 mg/kg TAM, the uterotrophic activities were still 33 % and 34 %, respectively.
  • 4-OHTAM recognized as a principal antiestrogenic TAM "metabolite, showed uterotrophic activity (45 %) similar to TAM.
  • TOR 46 %
  • idoxifene 47 %
  • ospemifene 41 %
  • RAL showed partial estrogenic activity (14 %) similar to that observed with GW5638 (16 %) , GW7604 (17 %) , or SP500263 (15 %) , indicating that these antiestrogens still have weak estrogenic activity.
  • the uterotrophic activity of SS1020 was significantly lower than that of RAL, GW5638 or GW7065 (see Fig. 4) .
  • SS1020 did not have significant - uterotrophic activity, indicating that it lacks estrogenic function.
  • Uterine tissue obtained from the uterotrophic assay was fixed in 4% neutral buffered formalin. The tissue was embedded in paraffin, and stained with hematoxylin and eosin. Samples for histopathological examination were taken from the midpoint of each uterine horn.
  • the uterine thickness was more than twice that of the sham (5A) and marked hypertrophy was observed in uterine epithelial cells.
  • the uterine thickness of TAM- or GW5638-treated OVX-rats (5C & 5D) was 1.9 and 1.6 times higher than that of the sham; epithelial hypertrophy was also observed.
  • the uterine histology of OVX-rats treated with SS1020 (5E) was not significantly different from that of the sham-treated rats .
  • mammary carcinoma was successfully induced in rats treated with DMBA (50 mg/kg, p.o.) .
  • Rats bearing mammary tumors (5 rats per a dose) were treated orally for 4 weeks with TAM or SS1020 at the equivalent molar dose of TAM (1.0 mg/kg body weight/day).
  • Controls (8 rats) received vehicle only. No significant effect was observed on the growth of rats treated with SS1020.
  • Tumor volume was recorded, using the two perpendicular dimensions and normalized to 100% on the first day of dosing. With SS1020, the tumor volume did not increase, as indicated by statistical analysis (t-test; ***, p ⁇ 0.001 versus the control) (Fig.
  • SS1021 (100 ⁇ M) was incubated at 37 °C for 1 hr in a buffer including cytosol (0.1 mg protein) of rat intestine.
  • cytosol 0.1 mg protein
  • the cleavage of the phosphate group of SS1021 was confirmed using HPLC analysis (Fig. 8B) .
  • Fig. 8A the conversion was not observed.
  • SS1020 was administered orally for 4 weeks to athymic nude mice (5 mice/group) bearing an ER-positive MCF-7 human breast cancer xenograft (Fig. 9a) .
  • SS1020 at an equivalent molar dose of TAM no tumor growth was observed during the period of treatment (t-test; *, p ⁇ 0.05 and **, p ⁇ 0.01 vs. the control) .
  • SS1020 has a much stronger antitumor potential than TAM.
  • Estrogen receptor binding assay for antiestrogens The ERa binding potential of SS1020 and other antiestrogens was determined using an established method [57] . The binding curve was fitted using a single binding site competition model and PRISM® statistical analysis software (Fig. 10) . The IC 50 of 4-OHTAM was similar to that of E 2 and approximately 50 times higher than that of TAM, as reported earlier [65]. The relative binding affinity (RBA) of RAL was 44% of that of E 2 and 16 times higher than that of TAM. Therefore, 4-OHTAM and RAL have strong binding potentials for ERa. The RBA of GW5638 was 0.06, relative to that of E 2 .
  • the RBAs of the 4- hydroxylated forms were 70 and 60 times, respectively, higher than that of GW5638. This indicates that 4-hydroxylated compounds have at least one or two orders of magnitude higher binding activities to ERa, as observed with 4-OHTAM.
  • SS1020 Preventive activity of SS1020 against hormone-induced mammary tumors
  • SS1020 was given to female ACI rats implanted under the skin with an E2 (2.5 mg) pellet and the development of mammary tumors was monitored.
  • SS1020 was dietary given ad libitum after implanting the pellet using phytoestrogen-free AIN-76A diet containing 50 ppm SS1020 (approximately equivalent to 3.0 mg/kg/day) .
  • 3 rats developed a palpable mammary tumor in 13 weeks (Fig. 15) .
  • no tumors were observed in ACI rats given SS1020. Therefore, SS1020 has preventive effect against hormone-induced mammary tumors, indicating that this compound can be used as a safer prophylactic agent for women at high risk of developing breast cancer.
  • Rats (3 rats) were treated orally with SS1020 [3.3 mg (8.1 ⁇ mol)/kg], ⁇ -NF [73 mg (270 ⁇ mol)/kg], or vehicle (corn oil) 7 days prior to a single oral dose (50 mg/kg) of DMBA.
  • the level of mammary DMBA-DNA adduct was determined using 32 P-postlabeling/PAGE analysis.
  • the level of DMBA-DNA adduct in the control rats was 1.0 ⁇ 0.32 adducts/10 8 nucleotides (Fig. 12) .
  • Pretreatment of rats with SS1020 significantly reduced the formation of DMBA- DNA adducts by 41% (p ⁇ 0.05, t-test) .
  • ⁇ -NF also suppressed adduct formation
  • the molar dose of SS1020 (8.1 ⁇ mol/kg) used was 33 times lower than that of ⁇ -NF (270 ⁇ mol/kg) .
  • a low dose of SS1020 is able to reduce the formation of mammary DMBA-DNA adduct, predicting that this compound has preventive potency against breast cancer.
  • Determination of osteoporotic potential of antiestrogens To determine the osteoporotic potential of SS1020, OVX- rats (5 rats/group) were treated orally for 4 weeks with SS1020 at a dose equivalent to 10 mg/kg/day TAM.
  • Bone mineral density was measured using an X-ray bone densitometer (Model pDEXA, Norland Medical Systems Inc.) .
  • OVX-rats treated with vehicle lost approximately 10% of BMD in the femur, lumbar and whole body (Table 1), as reported previously [105] .
  • the BMD of OVX-rats treated subcutaneously with E 2 (1.2 ⁇ g/kg/day) was similar to that of the sham-treated rats.
  • the BMD of TAM-treated OVX-rats tended to decrease while the BMD of SS1020-treated OVX-rats was not significantly different from that of the sham.
  • the results indicate that SS1020 may have higher anti- osteoporotic potential than TAM.
  • Rats were treated with a single dose of SS1020 or RAL at the equivalent molar dose of RAL (10 mg/kg, p.o.). Controls received corn oil only. Rats were euthanized by CO 2 asphyxiation and blood was collected 1, 2, 4, 6, 8, 10, and 16 h after the treatment. The serum was incubated with ⁇ -glucuronidase/sulfatase in a buffer, extracted by methanol, and evaporated to dryness. Analysis of SS1020 and RAL was performed using HPLC/UV and LC/MS/MS, respectively.
  • the serum concentration of SS1020 was much higher than that observed with RAL (Fig. 14) .
  • the area under curve of SS1020 was 32 times higher than that of RAL. Hence, SS1020 has higher bioavailability than RAL.
  • the compounds and compositions of the invention are useful for preventing and/or inhibiting the proliferation of tumors, particularly estrogen-induced tumors, as well as tumors expressing ER " . Furthermore, the compounds and compositions of the invention are also useful for the treatment of osteoporosis. The compounds and compositions of the invention may also prove to be useful for the prevention and/or treatment of other cancers, such as prostate cancer.
  • NSABP National Surgical Adjuvant Breast and Bowel Project
  • MCF-7 cells in vitro and in vivo. Cancer Res. 58, 3833- 3838 (1998) .
  • Raloxifene (LY139481 HCI) prevents bone loss and reduces serum cholesterol without causing uterine hypertrophy in ovariectomized rats. J. Clin. Invest. 93, 63-69 (1994) .

Abstract

La présente invention porte sur un composé présentant une structure dans laquelle R1-R15, q et X sont définis dans la description, ou sur un sel de celui-ci. L'invention porte également sur un procédé de préparation du composé ci-dessus, sur une composition pharmaceutique comprenant le composé ci-dessus, sur une méthode d'inhibition de prolifération de tumeur chez un mammifère, et sur une méthode d'inhibition et/ou de réduction de perte de densité minérale osseuse chez un mammifère.
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JP2018111665A (ja) * 2017-01-13 2018-07-19 二村　芳弘 ヒト型エストロゲン受容体活性化作用を呈するキノリン誘導体
CN111094227A (zh) * 2017-07-28 2020-05-01 里科瑞尔姆Ip控股有限责任公司 丙烯酸类似物
JP2020529403A (ja) * 2017-07-28 2020-10-08 リキュリウム アイピー ホールディングス リミテッド ライアビリティー カンパニー アクリル酸類似体
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