技术领域technical field
本发明涉及1-乙基-4-{4-[5-羟基-2-(4-羟基苯基)-3-甲基-1H-吲哚-1-基]丁基}哌嗪-2,3-二酮及其制备方法，还涉及其在制备雌激素受体调节剂药物中的应用，。The present invention relates to 1-ethyl-4-{4-[5-hydroxy-2-(4-hydroxyphenyl)-3-methyl-1H-indol-1-yl]butyl}piperazine-2, The 3-diketone and its preparation method also relate to its application in the preparation of estrogen receptor modulator drugs.
背景技术Background technique
模拟雌激素样作用的化合物具有广泛的治疗及预防作用，其中包括：治疗骨质疏松，缓解绝经期症状，治疗痤疮，治疗痛经和功能障碍性子宫出血，前列腺癌和预防心血管疾病。Compounds that mimic estrogen-like effects have a wide range of therapeutic and prophylactic applications, including: treatment of osteoporosis, relief of menopausal symptoms, treatment of acne, treatment of dysmenorrhea and dysfunctional uterine bleeding, prostate cancer and prevention of cardiovascular disease.
研究发现雌激素受体(ER)有两种类型：ERα和ERβ。配体与这两个亚型结合后，以不同的组织特异性发挥生理作用。Studies have found that there are two types of estrogen receptors (ER): ERα and ERβ. After the ligand binds to these two isoforms, it exerts physiological effects with different tissue specificities.
本领域需要的是与雌激素替代疗法一样可产生阳性反应但无不良副作用或副作用减小的化合物，且对机体发挥组织特异性的雌激素样作用。苯基吲哚类衍生物因其具有与雌激素类似的空间结构，可模拟雌激素，在生物体内与雌激素受体结合，发挥生理作用。What is needed in the art is a compound that can produce a positive response like estrogen replacement therapy but without adverse side effects or with reduced side effects, and exert tissue-specific estrogen-like effects on the body. Phenyl indole derivatives have a similar spatial structure to estrogen, can mimic estrogen, and bind to estrogen receptors in vivo to exert physiological effects.
雌激素是人体中一类重要的激素化合物,当妇女进入绝经期后,体内雌激素水平下降,由此引发骨质疏松症、更年期综合征、老年痴呆症和心血管系统等疾病。针对绝经后雌激素水平下降，采取雌激素替代疗法(est rogen replacement therapy,ERT)，能够显著降低绝经后骨质疏松性骨折和冠心病的发生率(Turner RT,Riggs BL,Spelsberg TC.Endocr Rev,1994,15(3):275-300；Mora S,Kershner DW,Vigilance CP,et al.Curr Treat Options Cardiovasc Med,2001,3(1):67-79)。但是,ERT可能诱发乳腺癌和子宫内膜癌(Persson I,Weiderpass E,Bergkvist L,et al.Cancer Causes Control,1999,10(4):253-260)。为克服雌激素致癌变的不良反应,人们又发展了雌、孕激素联合使用的性激素替代疗法(hormone replacement therapy,HRT),但长期的HRT治疗仍可能增加乳腺癌的发生率,即使使用孕激素,并非在所有的情况下均能克服由雌激素所导致的子宫内膜癌的发生,这些不良反应限制了HRT的长期应用。选择性雌激素受体调节剂(selective estrogen receptor modulators,SERMs)对骨骼和心血管系统具有雌激素样作用,而对子宫和乳腺表现出抗雌激素作用的药物。但是临床上使用的他莫西芬和雷洛昔芬能导致子宫内膜癌和热潮红等不良反应(Fisher B,Costantino JP,Wickerham DL,et al.J Nati Cancer Inst,1998,90:1371-1388；Walsh BW,Kuller LH,Wild RA,et al.J Am Med Assoc,1998,279:1445-1451)。Estrogen is an important class of hormone compounds in the human body. When women enter menopause, the level of estrogen in the body decreases, which leads to diseases such as osteoporosis, menopausal syndrome, Alzheimer's disease and cardiovascular system. For postmenopausal decline in estrogen levels, estrogen replacement therapy (est rogen replacement therapy, ERT) can significantly reduce the incidence of postmenopausal osteoporotic fractures and coronary heart disease (Turner RT, Riggs BL, Spelsberg TC. Endocr Rev , 1994, 15(3):275-300; Mora S, Kershner DW, Vigilance CP, et al. Curr Treat Options Cardiovasc Med, 2001, 3(1):67-79). However, ERT may induce breast cancer and endometrial cancer (Persson I, Weiderpass E, Bergkvist L, et al. Cancer Causes Control, 1999, 10(4):253-260). In order to overcome the adverse reactions of estrogen-induced carcinogenesis, people have developed sex hormone replacement therapy (hormone replacement therapy, HRT), which is combined with estrogen and progesterone, but long-term HRT treatment may still increase the incidence of breast cancer, even if progesterone is used , not in all cases can overcome the occurrence of endometrial cancer caused by estrogen, these adverse reactions limit the long-term application of HRT. Selective estrogen receptor modulators (selective estrogen receptor modulators, SERMs) have estrogen-like effects on the bone and cardiovascular system, but exhibit anti-estrogenic effects on the uterus and breast. But clinically used tamoxifen and raloxifene can cause adverse reactions such as endometrial cancer and hot flashes (Fisher B, Costantino JP, Wickerham DL, et al.J Nati Cancer Inst, 1998,90:1371- 1388; Walsh BW, Kuller LH, Wild RA, et al. J Am Med Assoc, 1998, 279:1445-1451).
发明内容Contents of the invention
本发明所解决的技术问题是提供一种如式I所示的化合物、其前体药物和药物活性代谢物，以及上述化合物的立体异构体及其药学上可接受的盐，并提供了其在制备预防和治疗雌激素相关的疾病的药物中的应用。The technical problem solved by the present invention is to provide a compound as shown in formula I, its prodrug and drug active metabolite, as well as the stereoisomer and pharmaceutically acceptable salt thereof, and provide its Application in the preparation of medicines for preventing and treating estrogen-related diseases.
本发明的化合物可以单独给予或优选与药学上可接受的载体或稀释剂，任选根据常规的制药习惯与已知辅剂联合，在药物组合物中给予。化合物经口服给予或经非胃肠道包括静脉内，肌肉内，腹膜内，皮下，直肠和局部途径给予。The compounds of the present invention can be administered alone or preferably in pharmaceutical compositions in combination with pharmaceutically acceptable carriers or diluents, optionally in combination with known adjuvants according to conventional pharmaceutical practices. The compounds are administered orally or parenterally including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes.
在用于口服的片剂中，通常加入一般使用的载体包括乳糖和玉米淀粉，以及润滑剂如硬脂酸镁。对胶囊形式的口服药来说，可用的稀释剂包括乳糖和干燥的玉米淀粉。对根据本发明的治疗化合物的口服途径使用来说，被选化合物可以以例如片剂或胶囊的形式，或作为水溶液或混悬液而被给予。对片剂或胶囊形式的口服给药来说，活性药物成分能与可口服，无毒，药学上可接受的惰性载体组合，载体例如有乳糖，淀粉，蔗糖，葡萄糖，甲基纤维素，硬脂酸镁，磷酸二钙，硫酸钙，甘露醇，山梨醇等；对液体形式的口服给药来说，口服药物成分可与任意的可口服，无毒，药学上可接受的惰性载体例如乙醇，甘油，水等组合。此外，还可在混合物中加入适宜的粘合剂，润滑剂，崩解剂和着色剂。适宜的粘合剂包括淀粉，明胶，天然糖如葡萄糖或乳糖，玉米甜味剂，天然和合成的树胶如阿拉伯胶，西黄蓍胶或海藻酸钠，羧甲基纤维素，聚乙二醇，蜡等。适宜的润滑剂包括油酸钠，硬脂酸钠，硬脂酸镁，苯甲酸钠，醋酸钠，氯化钠等。当水性混悬液口服使用时，可将活性成分与乳化剂和混悬剂组合。也可加入某些甜味剂或矫味剂。对肌内，腹膜内，皮下和静脉内使用来说，通常制备成活性成分的无菌溶液，溶液的pH应该适当的调节和缓冲。对静脉内使用来说，应当控制溶质的总浓度以使制剂维持等渗。In tablets for oral administration, commonly used carriers including lactose and corn starch, as well as lubricating agents such as magnesium stearate are usually added. For oral medication in a capsule form, acceptable diluents include lactose and dried cornstarch. For oral route use of therapeutic compounds according to the invention, the selected compound may be administered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension. For oral administration in tablet or capsule form, the active pharmaceutical ingredient can be combined with an orally acceptable, nontoxic, pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, glucose, methylcellulose, hard Magnesium fatty acid, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, etc.; for oral administration in liquid form, oral pharmaceutical ingredients can be mixed with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol , glycerin, water and other combinations. In addition, suitable binders, lubricants, disintegrants and colorants may be added to the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol , wax, etc. Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. When the aqueous suspension is used orally, the active ingredient may be combined with emulsifying and suspending agents. Certain sweetening or flavoring agents may also be added. For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredient are generally prepared, the pH of the solutions should be suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to maintain the formulation isotonic.
本发明的化合物还能以脂质体给药系统的形式例如小型单层囊泡，单层大囊泡和多层囊泡的形式给予。脂质体可由各种磷脂例如胆固醇，硬脂胺或磷脂酰胆碱形成。The compounds of the invention can also be administered in the form of liposomal delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
本发明的化合物还可以通过使用单克隆抗体作为单独载体而给予，其中化合物分子是被偶联的。本发明的化合物还可以与作为目标药物载体的可溶性聚合物偶联。这类聚合物可包括聚乙烯吡咯烷酮，吡喃共聚物，聚羟丙基甲基丙烯酰胺-苯酚，聚羟基-乙基天冬酰胺-苯酚或被棕榈酰基取代的聚氧化乙烯-聚赖氨酸。此外，本发明的化合物可以与一类可生物降解的用于实现药物控释的聚合物偶联，所述聚合物有例如聚乳酸，聚乙醇酸，聚乳酸和聚乙醇酸的共聚物，聚己内酯，聚羟基丁酸，聚原 酸酯类，聚缩醛类，聚二氢吡喃，聚氰基丙烯酸酯和水凝胶的交联或两性嵌段共聚物。The compounds of the invention can also be administered through the use of monoclonal antibodies as separate carriers to which the compound molecules are conjugated. The compounds of the present invention can also be coupled with soluble polymers as desired drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethylene oxide-polylysine substituted with palmitoyl . In addition, the compounds of the present invention can be coupled with a class of biodegradable polymers for achieving controlled drug release, such as polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, poly Cross-linked or amphiphilic block copolymers of caprolactone, polyhydroxybutyrate, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and hydrogels.
本发明的化合物还可与已知的可用于治疗或预防以下疾病的药剂组合使用：骨丢失、骨质疏松、转移性骨病、牙周病、软骨退化，子宫内膜异位，子宫纤维瘤疾病，潮热，LDL胆固醇水平升高，心血管疾病，认知功能缺损，大脑退化疾病，焦虑，由于雌激素缺乏所致的抑郁，炎症，炎性肠疾病，性功能障碍，高血压，视网膜变性和癌症，尤其是骨质疏松。The compounds of the present invention may also be used in combination with known agents useful in the treatment or prevention of bone loss, osteoporosis, metastatic bone disease, periodontal disease, cartilage degeneration, endometriosis, uterine fibroids Illness, hot flashes, elevated LDL cholesterol levels, cardiovascular disease, cognitive impairment, degenerative brain disease, anxiety, depression due to estrogen deficiency, inflammation, inflammatory bowel disease, sexual dysfunction, high blood pressure, retina Degeneration and cancer, especially osteoporosis.
目前公开的化合物与可用于治疗或预防在此公开疾病的药剂的联合也在本发明的范围内。这样的药剂包括以下物质：有机双膦酸盐；组织蛋白酶K抑制剂；雌激素或雌激素受体调节剂；雄激素受体调节剂；破骨细胞质子ATP酶抑制剂；HMG-CoA还原酶抑制剂；整联蛋白受体拮抗剂；成骨细胞合成代谢剂例如PTH；降钙素；维生素D或合成的维生素D类似物；选择性5-羟色胺再摄取抑制剂(SSRIS)；芳香酶抑制剂；及其药学上可接受的盐和混合物。优选的组合是本发明化合物和有机膦酸盐。另一个优选的组合是本发明化合物和组织蛋白K抑制剂。另一个优选的组合是本发明化合物和雌激素。另一个优选的组合是本发明化合物和雄激素受体调节剂。另一个优选的组合是本发明化合物和成骨细胞合成代谢剂。Combinations of the presently disclosed compounds with agents useful in the treatment or prophylaxis of the diseases disclosed herein are also within the scope of the invention. Such agents include the following: organic bisphosphonates; cathepsin K inhibitors; estrogen or estrogen receptor modulators; androgen receptor modulators; osteoclast proton ATPase inhibitors; HMG-CoA reductase Inhibitors; integrin receptor antagonists; osteoblast anabolic agents such as PTH; calcitonin; vitamin D or synthetic vitamin D analogs; selective serotonin reuptake inhibitors (SSRIS); aromatase inhibition agents; and pharmaceutically acceptable salts and mixtures thereof. A preferred combination is a compound of the invention and an organic phosphonate. Another preferred combination is a compound of the invention and a histone K inhibitor. Another preferred combination is a compound of the invention and an estrogen. Another preferred combination is a compound of the invention and an androgen receptor modulator. Another preferred combination is a compound of the invention and an osteoblast anabolic agent.
“双膦酸盐”包括双膦酸盐及其药学上可接受的盐类和衍生物。“雌激素”包括但不限于天然存在的雌激素，合成的结合雌激素，口服避孕药和硫酸化雌激素。“雌激素受体调节剂”指干扰或抵制雌激素与受体结合的物质，无论机制如何。“组织蛋白酶K抑制剂”指的是能干扰半胱氨酸蛋白酶组织蛋白酶K活性的化合物。“雄激素受体调节剂”指的是能干扰或抑制雄激素与受体结合的化合物，不论机理如何，包括非那雄胺和其它的5α还原酶抑制剂。“破骨细胞质子ATP酶抑制剂”指的是质子ATP酶的抑制剂，其能在破骨细胞的顶膜上找到，并且已经报道其在骨的再吸收过程中发挥了显著作用。这种质子泵表示了用于设计骨再吸收抑制剂的引人注目的靶标，其可潜在地用于治疗和预防骨质疏松和相关的代谢疾病。“HMG-CoA还原酶抑制剂”指的是3-羟基-3-甲基戊二酰基-CoA还原酶的抑制剂。对HMG-CoA还原酶具有抑制活性的化合物能用本领域已知的测定法容易地确定。“整联蛋白受体拮抗剂”指的是能选择性拮抗，抑制或对抗生理学配体与αγβ3整联蛋白结合的化合物，能选择性拮抗，抑制或对抗生理学配体与αγβ3整联蛋白结合的化合物，能选择性拮抗，抑制或对抗生理学配体与αγβ3整联蛋白和αγβ5整联蛋白结合的化合物，能选择性拮抗，抑制或对抗毛细管上皮细胞表达的特定整联蛋白的活性的化合物。拮抗αγβ3的作用选自抑制骨再吸收，抑制再狭窄，抑制黄斑变性，抑制关节炎和抑制癌症及转移生长。“成骨细胞合成代谢剂”指的是能构建骨的药剂，例如PTH，降钙素能通过抑制破骨细胞的活性来抑制骨的重吸收。“维生素D”包括但不局限于维生素D3和维生素D2,它们 是维生素D的羟基化生物学活性代谢物的天然存在的，生物学失活前体。“合成的维生素D类似物”包括作用类似维生素D的非天然存在的化合物。选择性5-羟色胺再摄取抑制剂通过增加脑中5-羟色胺的数量而发挥作用，其非限制性实例包括氟西汀，帕罗汀，舍曲林，西酞普兰和氟伏沙明，也能用于治疗与雌激素功能有关的疾病。“芳香酶抑制剂”包括抑制芳香酶的化合物，其非限定性的选自：氨鲁米特，来曲唑，福美坦，依西美坦，阿他美坦，法倔唑，氟罗唑，伏氯唑。"Bisphosphonate" includes bisphosphonates and pharmaceutically acceptable salts and derivatives thereof. "Estrogen" includes, but is not limited to, naturally occurring estrogens, synthetic conjugated estrogens, oral contraceptives, and sulfated estrogens. "Estrogen receptor modulator" means a substance that interferes with or counteracts the binding of estrogen to the receptor, regardless of the mechanism. "Cathepsin K inhibitor" refers to a compound that interferes with the activity of the cysteine protease cathepsin K. "Androgen receptor modulators"refers to compounds that interfere with or inhibit the binding of androgens to the receptor, regardless of mechanism, including finasteride and other 5α-reductase inhibitors. "Osteoclast proton ATPase inhibitor" refers to an inhibitor of proton ATPase, which can be found on the apical membrane of osteoclasts and which has been reported to play a significant role in the resorption process of bone. This proton pump represents an attractive target for the design of bone resorption inhibitors, which could potentially be used in the treatment and prevention of osteoporosis and related metabolic diseases. "HMG-CoA reductase inhibitor" refers to an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase. Compounds having inhibitory activity against HMG-CoA reductase can be readily identified using assays known in the art. "Integrin receptor antagonist" means a compound that selectively antagonizes, inhibits or antagonizes the binding of a physiological ligand to αγβ3 integrin, Compounds, compounds capable of selectively antagonizing, inhibiting or antagonizing the binding of physiological ligands to αγβ3 integrin and αγβ5 integrin, compounds capable of selectively antagonizing, inhibiting or antagonizing the activity of specific integrins expressed by capillary epithelial cells. The action of antagonizing αγβ3 is selected from the group consisting of inhibition of bone resorption, inhibition of restenosis, inhibition of macular degeneration, inhibition of arthritis and inhibition of cancer and metastatic growth. "Osteoblast anabolic agent" refers to an agent that builds bone, such as PTH, calcitonin that inhibits bone resorption by inhibiting the activity of osteoclasts. "Vitamin D" includes, but is not limited to, vitamin D3 and vitamin D2, which are naturally occurring, biologically inactive precursors of the hydroxylated biologically active metabolites of vitamin D. "Synthetic vitamin D analogs" include non-naturally occurring compounds that act like vitamin D. Selective serotonin reuptake inhibitors work by increasing the amount of serotonin in the brain, non-limiting examples of which include fluoxetine, parotine, sertraline, citalopram, and fluvoxamine, also It can be used to treat diseases related to estrogen function. "Aromatase inhibitors" include compounds that inhibit aromatase, non-limitingly selected from the group consisting of: aminoglutethimide, letrozole, formestane, exemestane, atamestane, fadrozole, flurazole , vorozole.
有关本发明化合物的术语“给予”及其变体(例如“给予”化合物)的意思是将化合物或化合物的前药引入需要治疗的动物系统中。当本发明的化合物或其前药与一种或多种其它活性剂(例如双膦酸盐化合物等)组合提供时，“给予”及其变体都可以被理解为包括同时和相继引入化合物或其前药以及其它药剂。本发明包括在其范围内的本发明化合物的前药。通常，这种前药是将本发明化合物的官能衍生物，其在体内易于转变为所需的化合物。这样，在本发明的治疗方法中，术语“给予”将包含用具体公开的化合物或可能未被具体公开的化合物，但是其能在给予患者后于体内转化为特定的化合物，以治疗所述的各种疾病。用于选择和制备适宜的前药衍生物的常规方法，在此引入作为参考。这些化合物的代谢物包括将本发明的化合物引入生物环境后产生的活性物质。The term "administering" and variants thereof (eg "administering" a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of an animal in need of treatment. When a compound of the present invention, or a prodrug thereof, is provided in combination with one or more other active agents (e.g., bisphosphonate compounds, etc.), "administration" and variations thereof are understood to include simultaneous and sequential introduction of the compounds or Prodrugs thereof and other agents. The present invention includes within its scope prodrugs of the compounds of the invention. Typically, such prodrugs are functional derivatives of the compounds of the invention which are readily converted in vivo to the desired compound. Thus, in the method of treatment of the present invention, the term "administration" shall include the use of a specifically disclosed compound or a compound which may not be specifically disclosed, but which is converted in vivo to a specific compound after administration to a patient, to treat said various diseases. Routine methods for selecting and preparing suitable prodrug derivatives are incorporated herein by reference. Metabolites of these compounds include active substances produced upon introduction of the compounds of the present invention into the biological environment.
本发明的化合物是雌激素受体的配体，可用于治疗或预防与雌激素功能相关的各种疾病，其中包括：骨丢失，骨折，骨质疏松症，转移性骨病，软骨退化，乳腺癌，子宫内膜异位，潮热，前列腺肥大，前列腺癌，卵巢癌，绝经综合症或与接触环境化学品或天然激素失衡有关的不良生殖作用。本发明还包含可用于治疗骨质疏松或其它骨病的药物组合物，治疗包括给予治疗有效量的本发明化合物，该组合物含有或不含有药学上可接受的载体或稀释剂。本发明的适宜组合物包括含有本发明化合物和药学上可接受载体例如盐水的水溶液，pH在一定水平上，例如为7.4。溶液可以通过局部推注而被引入到患者的血流中。The compounds of the present invention are ligands for estrogen receptors and are useful in the treatment or prevention of various diseases associated with estrogen function, including: bone loss, fractures, osteoporosis, metastatic bone disease, cartilage degeneration, breast cancer, endometriosis, hot flashes, enlarged prostate, prostate cancer, ovarian cancer, menopausal syndrome, or adverse reproductive effects associated with exposure to environmental chemicals or natural hormone imbalances. The present invention also includes a pharmaceutical composition useful for treating osteoporosis or other bone diseases. The treatment includes administering a therapeutically effective amount of the compound of the present invention, and the composition contains or does not contain a pharmaceutically acceptable carrier or diluent. Suitable compositions of the invention include aqueous solutions comprising a compound of the invention and a pharmaceutically acceptable carrier, such as saline, at a pH of, for example, 7.4. The solution can be introduced into the patient's bloodstream by local bolus injection.
当本发明的化合物被给予人类受试中，日剂量将通常由处方医师确定，剂量一般根据患者个体的年龄，体重和反应以及患者症状的严重程度而变化。在一个示例性应用中，将适宜量的化合物给予接受治疗的哺乳动物。当用于所指示的作用时，本发明的口服剂量将为约0.01mg每kg体重每天(mg/kg/天)到约100mg/kg/天，优选0.01到10mg/kg/天，最优选0.1到5.0mg/kg/天。对口服给药来说，组合物优选以片剂的形式被提供，其中片剂包含0.01，0.05，0.1，0.5，1.0，2.5，5.0，10.0，15.0，25.0，50.0，100和500mg的活性成分，用于调节受治患者症状的剂量。药物一般包含约0.01mg到约500mg的活性成分，优选约1mg到约100mg活性成分。对于静脉注射，在恒速输注期间，最优选的剂量将为约0.1到约10mg/kg/分钟。本发明的化合物可以以每日一次的剂量给予，或者是可以将每日总剂量分为每日两次，三次或四次的剂量给予。此外，本发明的优选化合物可以以鼻内药物的形式通过局部使用适宜的鼻内载 体，或通过经皮途径，使用本领域普通技术人员已知的经皮贴剂形式而给予。对于以经皮给药系统的形式进行的给药来说，剂量给药在整个给药方案中当然将是连续的而不是间断的。When the compounds of this invention are administered to human subjects, the daily dosage will generally be determined by the prescribing physician and will generally vary according to the age, weight and response of the individual patient and the severity of the patient's symptoms. In one exemplary application, an appropriate amount of the compound is administered to a mammal receiving treatment. When used for the indicated effects, the oral dosage of the present invention will be about 0.01 mg per kg body weight per day (mg/kg/day) to about 100 mg/kg/day, preferably 0.01 to 10 mg/kg/day, most preferably 0.1 to 5.0mg/kg/day. For oral administration, the composition is preferably presented in the form of a tablet comprising 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100 and 500 mg of the active ingredient , used to adjust the dosage for the symptoms of the treated patients. The medicament generally contains from about 0.01 mg to about 500 mg of active ingredient, preferably from about 1 mg to about 100 mg of active ingredient. For intravenous injection, the most preferred dosage will be from about 0.1 to about 10 mg/kg/minute during a constant rate infusion. The compounds of the present invention may be administered in a once daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. In addition, preferred compounds of the present invention may be administered in the form of intranasal pharmaceuticals by topical use of suitable intranasal vehicles, or by transdermal routes using transdermal patches known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
本发明的化合物能与其它可用于治疗雌激素介导的状况的药剂组合使用。这种组合的各个成分能在治疗期间以不同的次数分别或同时以分开的形式或单一组合的形式给予。因此本发明能被理解为包含所有这样的同时或交替治疗的方案，而且术语“给予”也相应的按此解释。本发明化合物与其它可用于治疗组织蛋白酶介导状况的药剂的组合的范围，原则上包括与能用于治疗与雌激素功能有关疾病的任意药物组合物的任意组合。The compounds of the invention can be used in combination with other agents useful in the treatment of estrogen-mediated conditions. The individual components of the combination can be administered separately or simultaneously at different times during the treatment period, either in separate form or in a single combination. The present invention is therefore to be understood as encompassing all such regimens of simultaneous or alternate treatment, and the term "administering" is to be construed accordingly. The scope of combinations of the compounds of the invention with other agents useful in the treatment of cathepsin-mediated conditions includes in principle any combination with any pharmaceutical composition useful in the treatment of diseases related to estrogen function.
因此本发明还可以包括与第二种药剂组合的使用，其中第二种药剂选自：有机双磷酸盐化合物，组织蛋白酶K抑制剂，雌激素，雌激素受体调节剂，雄激素受体调节剂，破骨细胞质子ATP酶抑制剂，HMG-CoA还原酶抑制剂，整联蛋白受体拮抗剂，成骨细胞合成代谢剂，降钙素，维生素D，合成的维生素D类似物，选择性5-羟色胺再摄取抑制剂，芳香酶抑制剂，及其药学上可接受的盐和混合物。The present invention therefore also includes use in combination with a second agent selected from the group consisting of: organobisphosphonate compounds, cathepsin K inhibitors, estrogens, estrogen receptor modulators, androgen receptor modulators agent, osteoclast proton ATPase inhibitor, HMG-CoA reductase inhibitor, integrin receptor antagonist, osteoblast anabolic agent, calcitonin, vitamin D, synthetic vitamin D analog, selective Serotonin reuptake inhibitors, aromatase inhibitors, and pharmaceutically acceptable salts and mixtures thereof.
本发明的化合物能与其它可用于治疗由雌激素介导的状况的药剂组合使用。这种组合的每个成分都能在治疗期间以不同的次数分别给予或同时以分开的形式或单一组合的形式给予。因此本发明应被理解为包含所有这样的同时或交替治疗的方案，而且术语“给予”也应按此解释。应该理解，本发明化合物与其它可用于治疗雌激素介导状况的药剂的组合的范围原则上包括与能用于治疗与雌激素功能有关疾病的任意药物组合物的任意组合。The compounds of the invention can be used in combination with other agents useful in the treatment of estrogen-mediated conditions. Each component of the combination can be administered separately at different times during the treatment period or simultaneously in separate form or in a single combination. The invention is therefore to be understood to encompass all such regimens of simultaneous or alternating treatment and the term "administration" is to be construed accordingly. It should be understood that the scope of combinations of the compounds of the present invention with other agents useful in the treatment of estrogen-mediated conditions includes in principle any combination with any pharmaceutical composition useful in the treatment of diseases related to estrogen function.
使用本发明化合物的剂量方案将根据多种因素进行选择，这包括患者的类型，种属，年龄，体重，性别和医学状况；受治状况的严重程度；给药途径；患者的肾和肝功能；以及所用的特定化合物或其盐。普通技术医师，兽医或临床医师可容易的确定和开具需要预防，抗击或阻止状况发展的有效药量。Dosage regimens for the compounds of this invention will be selected based on a variety of factors, including the type, species, age, weight, sex and medical condition of the patient; severity of the condition being treated; route of administration; renal and hepatic function of the patient ; and the specific compound or salt thereof used. A physician, veterinarian or clinician of ordinary skill can readily determine and prescribe an effective amount of the drug needed to prevent, combat or arrest the development of the condition.
在本发明的方法中，在此详细描述的化合物能形成活性成分，并根据给药形式即口服片剂，胶囊，酏剂，糖浆剂等而与适当选择的适宜的药学稀释剂，赋形剂或载体(在此统称为‘载体’物质)混合，并符合常规的药学习惯。In the method of the present invention, the compound described in detail herein can form the active ingredient, and according to the form of administration, i.e. oral tablet, capsule, elixir, syrup, etc., is mixed with an appropriate pharmaceutical diluent, excipient or carrier (collectively referred to herein as 'carrier' substances), and conform to conventional pharmaceutical practices.
本发明化合物的药学上可接受的盐类包括由无机或有机酸形成的常规无毒盐。常规的无毒盐包括源自无机酸例如盐酸，氢溴酸，硫酸，氨基磺酸，磷酸，硝酸等的盐，以及由有机酸例如醋酸，丙酸，琥珀酸，乙醇酸，硬脂酸，乳酸，苹果酸，酒石酸，柠檬酸，抗坏血酸，双羟萘酸，马来酸，羟基马来酸，苯乙酸，谷氨酸，苯甲酸，水扬酸，对氨基苯磺酸，2-乙酰氧基-苯甲酸，反丁烯二酸，甲苯磺酸，甲磺酸，乙烷二磺酸，草酸，羟乙磺酸，三氟醋酸等制备的盐类。本发明化合物的药学上接受的盐类能由包含酸性或碱性部分的本发明化合物经常规的化学方 法合成。通常，碱性化合物的盐类能通过离子交换色谱法制备，或将游离碱与化学计量或过量的所期望的成盐无机或有机酸在适宜的溶剂或溶剂的各种组合中进行反应来制备。类似的，酸性化合物的盐类可通过与适宜的无机或有机碱进行反应来形成。Pharmaceutically acceptable salts of the compounds of this invention include conventional non-toxic salts formed with inorganic or organic acids. Conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc., and those derived from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, Lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxy -Salts prepared from benzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, etc. Pharmaceutically acceptable salts of the compounds of this invention can be synthesized from compounds of this invention which contain acidic or basic moieties by conventional chemical methods. In general, salts of basic compounds can be prepared by ion exchange chromatography or by reacting the free base with stoichiometric or excess amounts of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents . Similarly, salts of acidic compounds may be formed by reaction with a suitable inorganic or organic base.
本发明的化合物能根据以下的一般方案使用适宜的物质来制备，并且通过随后的具体实施例进一步的举例说明。以下制备步骤的条件和方法的各种已知变化也能用于制备这些化合物。所有的温度均为摄氏度，除非另有指明。Compounds of the present invention can be prepared according to the following general schemes using appropriate materials and are further illustrated by the specific examples that follow. Various known variations of the conditions and methods of the following preparative steps can also be used to prepare these compounds. All temperatures are in degrees Celsius unless otherwise indicated.
下面的流程描述了1-乙基-4-{4-[5-羟基-2-(4-羟基苯基)-3-甲基-1H-吲哚-1-基]丁基}哌嗪-2,3-二酮的制备。The following scheme describes 1-ethyl-4-{4-[5-hydroxy-2-(4-hydroxyphenyl)-3-methyl-1H-indol-1-yl]butyl}piperazine- Preparation of 2,3-diketones.
流程1Process 1
本发明所述化合物制备方法简单，收率稳定，制备的化合物能较好的预防和治疗雌激素相关的疾病。The preparation method of the compound of the invention is simple, the yield is stable, and the prepared compound can better prevent and treat estrogen-related diseases.
具体实施方式detailed description
以下述实例详细叙述本发明。但是,应当明白,本发明不限于具体叙述的下述实例。The present invention is described in detail with the following examples. However, it should be understood that the present invention is not limited to the examples specifically described below.
实施例1：1-乙基-4-{4-[5-羟基-2-(4-羟基苯基)-3-甲基-1H-吲哚-1-基]丁基}哌嗪-2,3-二酮的制备Example 1: 1-ethyl-4-{4-[5-hydroxyl-2-(4-hydroxyphenyl)-3-methyl-1H-indol-1-yl]butyl}piperazine-2 , Preparation of 3-diketone
步骤A)：4-硝基苄氧基苯的制备Step A): Preparation of 4-nitrobenzyloxybenzene
将4-硝基苯酚(20.0g,0.152mol)、无水碳酸钾(131.1g,0.950mol)置于1000mL烧瓶中，丙酮作溶剂，滴加氯化苄(12.0g,0.095mol)，回流反应24h，减压蒸除溶剂，剩余物加水搅拌，抽滤，干燥，得到淡黄色固体19.8g，收率90.8％。m.p.105-106℃。ESI-MS:m/z 139([M+H]+)。Put 4-nitrophenol (20.0g, 0.152mol) and anhydrous potassium carbonate (131.1g, 0.950mol) in a 1000mL flask, use acetone as solvent, add dropwise benzyl chloride (12.0g, 0.095mol), and reflux reaction After 24 hours, the solvent was evaporated under reduced pressure, the residue was stirred with water, filtered with suction, and dried to obtain 19.8 g of a light yellow solid with a yield of 90.8%. mp105-106°C. ESI-MS: m/z 139 ([M+H]+ ).
步骤B)：4-苄氧基苯胺的制备Step B): Preparation of 4-benzyloxyaniline
将4-硝基苄氧基苯(10.0g,0.044mol)、氯化亚锡(50g,0.220mol)置于1000mL圆底烧瓶中，乙醇作溶剂，N2保护下，45℃反应24h。减压蒸除溶剂，用600mL碳酸钠溶液将残留物转移到1000mL烧杯中，充分搅 拌至不再有气泡产生，抽滤，滤饼加乙醇搅拌，抽滤，滤液旋蒸，得白色固体7.86g，收率90.3％。m.p.51-52℃。ESI-MS:m/z 200([M+H]+)。4-Nitrobenzyloxybenzene (10.0g, 0.044mol) and stannous chloride (50g, 0.220mol) were placed in a 1000mL round-bottomed flask with ethanol as the solvent, and reacted at 45°C for 24h under the protection of N2 . Evaporate the solvent under reduced pressure, transfer the residue to a 1000mL beaker with 600mL of sodium carbonate solution, stir well until there are no more bubbles, filter with suction, stir the filter cake with ethanol, filter with suction, and rotary evaporate the filtrate to obtain 7.86g of white solid , yield 90.3%. mp51-52°C. ESI-MS: m/z 200 ([M+H]+ ).
步骤C)：1-(4-苄氧基苯基)-1-丙酮的制备Step C): Preparation of 1-(4-benzyloxyphenyl)-1-propanone
将1-(4-羟基苯基)-1-丙酮(20.0g,0.146mol)、无水碳酸钾(100.7g,0.730mol)置于1000mL圆底烧瓶中，丙酮作溶剂，滴加氯化苄(22.3g,0.176mol)，回流反应24h，减压蒸除溶剂，剩余物加水搅拌，抽滤，干燥，得白色固体30.4g，收率为91.5％。m.p.101-102℃。ESI-MS:m/z 241([M+H]+)。Put 1-(4-hydroxyphenyl)-1-propanone (20.0g, 0.146mol) and anhydrous potassium carbonate (100.7g, 0.730mol) in a 1000mL round-bottomed flask, use acetone as solvent, add benzyl chloride dropwise (22.3g, 0.176mol), reflux reaction for 24h, evaporate the solvent under reduced pressure, add water to the residue, stir, filter with suction, and dry to obtain 30.4g of white solid with a yield of 91.5%. mp101-102°C. ESI-MS: m/z 241 ([M+H]+ ).
步骤D)：1-(4-苄氧基苯基)-2-溴-1-丙酮的制备Step D): Preparation of 1-(4-benzyloxyphenyl)-2-bromo-1-propanone
将1-(4-苄氧基苯基)-1-丙酮(10.0g,0.058mol)置于500mL圆底烧瓶中，1,4-二氧六环、无水甲醇作溶剂，缓慢滴加液溴(7.0g,0.061mol)，室温反应至溶液透明澄清，加水搅拌1h，抽滤，干燥，得白色固体15.5g，收率89.6％。m.p.74-75℃。ESI-MS:m/z 320([M+H]+)。Put 1-(4-benzyloxyphenyl)-1-propanone (10.0g, 0.058mol) in a 500mL round bottom flask, 1,4-dioxane and anhydrous methanol as solvents, slowly drop Bromine (7.0 g, 0.061 mol) was reacted at room temperature until the solution was transparent and clear, added water and stirred for 1 h, filtered with suction, and dried to obtain 15.5 g of white solid with a yield of 89.6%. mp74-75°C. ESI-MS: m/z 320 ([M+H]+ ).
步骤E)：1-(4-苄氧基苯基)-2-(4-苄氧基苯基氨基)-1-丙酮的制备Step E): Preparation of 1-(4-benzyloxyphenyl)-2-(4-benzyloxyphenylamino)-1-propanone
将4-苄氧基苯胺(7.8g,0.039mol)、1-(4-苄氧基苯基)-2-溴-1-丙酮(10.5g,0.033mol)置于500mL圆底烧瓶中，乙醇作溶剂，加入三乙胺(6.6g,0.065mol)，回流反应5h。自然冷却至室温，抽滤，得乳白色固体12.3g，收率85.3％。m.p.125-126℃。ESI-MS:m/z 438([M+H]+)。Place 4-benzyloxyaniline (7.8g, 0.039mol), 1-(4-benzyloxyphenyl)-2-bromo-1-propanone (10.5g, 0.033mol) in a 500mL round bottom flask, ethanol As a solvent, triethylamine (6.6g, 0.065mol) was added, and the reaction was refluxed for 5h. Naturally cooled to room temperature, and suction filtered to obtain 12.3 g of a milky white solid with a yield of 85.3%. mp125-126°C. ESI-MS: m/z 438 ([M+H]+ ).
步骤F)：5-苄氧基-2-(4-苄氧基苯基)-3-甲基-1H-吲哚的制备Step F): Preparation of 5-benzyloxy-2-(4-benzyloxyphenyl)-3-methyl-1H-indole
将1-(4-苄氧基苯基)-2-(4-苄氧基苯基氨基)-1-丙酮(12.3g,0.028mol)、4-苄氧基苯胺(14.0g,0.070mol)置于500mL烧瓶中，乙二醇单乙醚作溶剂，回流反应6h。减压蒸除溶剂，剩余物加乙醇搅拌，抽滤，得棕色固体9.5g，收率80.5％。m.p.151-152℃。ESI-MS:m/z 420([M+H]+)。1H-NMR(400MHz,CDCl3)δ7.89(s,1H),7.45(m,14H),7.28(d,J＝2.6Hz,1H),7.11(d,J＝8.4Hz,2H),6.97(dd,J1＝8.5Hz,J2＝1.5Hz,1H),5.18(s,2H),5.15(s,2H),2.43(s,3H)。1-(4-benzyloxyphenyl)-2-(4-benzyloxyphenylamino)-1-propanone (12.3g, 0.028mol), 4-benzyloxyaniline (14.0g, 0.070mol) Place in a 500mL flask, use ethylene glycol monoethyl ether as a solvent, and reflux for 6 hours. The solvent was evaporated under reduced pressure, the residue was stirred with ethanol, and filtered with suction to obtain 9.5 g of a brown solid with a yield of 80.5%. mp151-152°C. ESI-MS: m/z 420 ([M+H]+ ).1 H-NMR (400MHz, CDCl3 ) δ7.89(s, 1H), 7.45(m, 14H), 7.28(d, J=2.6Hz, 1H), 7.11(d, J=8.4Hz, 2H), 6.97 (dd, J1 =8.5 Hz, J2 =1.5 Hz, 1H), 5.18 (s, 2H), 5.15 (s, 2H), 2.43 (s, 3H).
步骤G)：1-(4-溴丁基)-5-苄氧基-2-(4-苄氧基苯基)-3-甲基-1H-吲哚的制备Step G): Preparation of 1-(4-bromobutyl)-5-benzyloxy-2-(4-benzyloxyphenyl)-3-methyl-1H-indole
将氢化钠(1.1g,0.048mol)置于250mL圆底烧瓶中，加入N,N-二甲基甲酰胺(15mL)，冰浴下搅拌10min，滴加溶有5-苄氧基-2-(4-苄氧基苯基)-3-甲基-1H-吲哚(10.0g,0.024mol)的N,N-二甲基甲酰胺溶液，滴毕，冰浴下继续搅拌30min。同温下滴加含有1,4-二溴丁烷(7.7g,0.036mol)的N,N-二甲基甲酰胺溶液，滴毕，升温至室温，反应2.5h。将反应液倒入冰水中，二氯甲烷萃取(3×50mL)，饱和氯化钠洗涤，无水硫酸钠干燥，减压蒸除溶剂，柱层析得浅黄色固体8.0g。收率60.6％。m.p.89-90℃。ESI-MS:m/z 554([M+H]+),576([M+Na]+)。1H-NMR(400MHz,DMSO-d6)δ7.42(m,14H),7.17(d,J＝8.7Hz,1H),7.10(d,J＝2.3Hz,1H),6.88(dd,J1＝8.8Hz,J2＝2.4Hz,1H),5.17(s,2H),5.13(s,2H),4.04(t,J＝6.5Hz,2H),3.31(t,J＝6.0Hz,2H),2.10(s,3H),1.54(m,4H)。Sodium hydride (1.1g, 0.048mol) was placed in a 250mL round bottom flask, N,N-dimethylformamide (15mL) was added, stirred for 10min under ice cooling, and 5-benzyloxy-2- (4-benzyloxyphenyl)-3-methyl-1H-indole (10.0g, 0.024mol) in N,N-dimethylformamide solution, after dropping, continue to stir for 30min under ice bath. At the same temperature, N,N-dimethylformamide solution containing 1,4-dibromobutane (7.7g, 0.036mol) was added dropwise. After the drop was completed, the mixture was warmed to room temperature and reacted for 2.5h. The reaction solution was poured into ice water, extracted with dichloromethane (3×50 mL), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and evaporated to remove the solvent under reduced pressure. Column chromatography gave 8.0 g of a light yellow solid. Yield 60.6%. mp89-90°C. ESI-MS: m/z 554 ([M+H]+ ), 576 ([M+Na]+ ).1 H-NMR (400MHz, DMSO-d6 ) δ7.42 (m, 14H), 7.17 (d, J = 8.7Hz, 1H), 7.10 (d, J = 2.3Hz, 1H), 6.88 (dd, J1 = 8.8Hz, J2 = 2.4Hz, 1H), 5.17(s, 2H), 5.13(s, 2H), 4.04(t, J = 6.5Hz, 2H), 3.31(t, J = 6.0Hz, 2H ), 2.10(s,3H), 1.54(m,4H).
步骤H)：1-乙基-4-{4-[5-羟基-2-(4-羟基苯基)-3-甲基-1H-吲哚-1-基] 丁基}哌嗪-2,3-二酮的制备Step H): 1-Ethyl-4-{4-[5-hydroxy-2-(4-hydroxyphenyl)-3-methyl-1H-indol-1-yl]butyl}piperazine-2 , Preparation of 3-diketone
将1-(4-溴丁基)-5-苄氧基-2-(4-苄氧基苯基)-3-甲基-1H-吲哚(1.0g,1.8mmol)，1-乙基哌嗪-2,3-二酮(0.5g,3.6mmol)，三乙胺(0.4g,3.6mmol)置于100mL茄型瓶中，正丁醇作溶剂，回流反应4h。旋蒸。剩余物用乙酸乙酯与乙醇的混合溶液溶解，加入10％Pd/C(0.2g,0.4mmol)，45℃反应24h。滤除Pd/C，滤液旋蒸，柱层析，得白色油状物0.22g，收率49.1％。ESI-MS:m/z 436([M+H]+)。1H-NMR(400MHz,DMSO-d6)δ9.69(s,1H),8.70(s,1H),7.41(d,J＝7.2Hz,1H),7.39(d,J＝7.7Hz,2H),7.20(d,J＝8.4Hz,2H),7.09(d,J＝2.3Hz,1H),6.86(dd,J1＝8.7Hz,J2＝2.5Hz,1H),4.02(t,J＝6.7Hz,2H),3.30(m,4H),3.16(m,2H),2.32(m,2H),2.09(s,3H),1.54(m,4H),1.24(m,3H)。1-(4-bromobutyl)-5-benzyloxy-2-(4-benzyloxyphenyl)-3-methyl-1H-indole (1.0g, 1.8mmol), 1-ethyl Piperazine-2,3-dione (0.5g, 3.6mmol) and triethylamine (0.4g, 3.6mmol) were placed in a 100mL eggplant-shaped bottle, and n-butanol was used as a solvent, and the reaction was refluxed for 4h. Rotary evaporation. The residue was dissolved in a mixed solution of ethyl acetate and ethanol, added with 10% Pd/C (0.2 g, 0.4 mmol), and reacted at 45° C. for 24 h. Pd/C was filtered off, the filtrate was rotary evaporated, and column chromatography gave 0.22 g of a white oily substance with a yield of 49.1%. ESI-MS: m/z 436 ([M+H]+ ).1 H-NMR (400MHz, DMSO-d6 ) δ9.69(s, 1H), 8.70(s, 1H), 7.41(d, J=7.2Hz, 1H), 7.39(d, J=7.7Hz, 2H ), 7.20 (d, J = 8.4Hz, 2H), 7.09 (d, J = 2.3Hz, 1H), 6.86 (dd, J1 = 8.7Hz, J2 = 2.5Hz, 1H), 4.02 (t, J =6.7Hz, 2H), 3.30(m, 4H), 3.16(m, 2H), 2.32(m, 2H), 2.09(s, 3H), 1.54(m, 4H), 1.24(m, 3H).
药物组合物pharmaceutical composition
在下述制剂中，“活性成分”是指式1化合物，或其盐或溶剂化物。In the following formulations, "active ingredient" refers to the compound of formula 1, or a salt or solvate thereof.
实施例2：明胶胶囊Embodiment 2: gelatin capsule
实施例3：片剂Embodiment 3: tablet
实施例4：片剂Embodiment 4: tablet
将活性组分，淀粉和纤维素通过45目U.S.筛并充分混合，将所得粉末与聚乙烯吡咯烷酮混合，然后通过14目U.S.筛，将这样得到的颗粒在50-60℃干燥并通过18目U.S.筛。将羧甲基纤维素钠，硬脂酸镁和滑石， 先通过60目U.S.筛，然后加入至上述颗粒中，混合后，在压片机上压制成片剂。Pass the active ingredient, starch and cellulose through a 45-mesh U.S. sieve and mix thoroughly. The resulting powder is mixed with polyvinylpyrrolidone and then passed through a 14-mesh U.S. sieve. The granules thus obtained are dried at 50-60° C. and passed through a 18-mesh U.S. screen. The sodium carboxymethylcellulose, magnesium stearate and talc, first passed through a 60-mesh U.S. sieve, are then added to the above granules, mixed and compressed on a tablet machine to form tablets.
实施例5：悬浮剂Embodiment 5: suspending agent
将药物通过45目U.S.筛并与羧甲基纤维素钠及糖浆混合以形成均匀糊状物，将苯甲酸溶液，矫味剂，和着色剂用一些水稀释，并边搅拌边加入，然后加入足够量水以达到所需的体积。Pass the drug through a 45 mesh U.S. sieve and mix with sodium carboxymethylcellulose and syrup to form a homogeneous paste. Dilute the benzoic acid solution, flavor, and color with some water and add with stirring, then add Sufficient water to achieve desired volume.
实施例6：气溶胶Example 6: Aerosol
将活性成分与乙醇混合，并将所得混合物加入至部分的抛射剂22中，冷却至30℃，并转移至容器中。然后将所需量加入至不锈钢容器中并用剩余喷射剂稀释，然后安装阀门装置。The active ingredient is mixed with ethanol, and the resulting mixture is added to a portion of the propellant 22, cooled to 30°C, and transferred to a container. The required amount is then added to the stainless steel container and diluted with the remainder of the propellant before installing the valve assembly.
实施例7：栓剂Embodiment 7: suppositories
将活性组分通过60目U.S.筛并将其悬浮于预先熔化的饱和脂肪酸甘油酯类化合物中，然后将混合物倾入至标准的2g腔栓剂模中并冷却。实施例8：可注射制剂The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in pre-melted saturated fatty acid glycerides. The mixture is then poured into standard 2g cavity suppository molds and allowed to cool. Example 8: Injectable Formulations
将以上溶液静脉内注射给药至患者，速度约1mL每分钟。The above solution was administered intravenously to the patient at a rate of about 1 mL per minute.
药理药效实验Pharmacological efficacy experiment
实施例9：雌激素受体结合实验Embodiment 9: Estrogen receptor binding experiment
雌激素受体配体结合试验设计为采用闪烁亲近检测，使用含氚的雌二醇和重组表达的雌激素受体。在杆状病毒表达系统中生产全长重组人类ERα及ERβ蛋白。ERα及ERβ提取物在含有6mMα-单硫代甘油的磷酸缓冲盐水中以1:400稀释。将200μL等分样的稀释受体制备物加入96 孔Flashplate板每一孔中。用Saran Wrap覆盖培养板，于4℃温育过夜。第二天早上将20μL含有10％牛血清白蛋白的磷酸缓冲盐水等分试样加入至该96孔板的每一孔中，且使其在4℃温育2h。然后用200μL含有20mM Tris(pH7.2)，1mM EDTA，10％丙三醇，50mM KCl及6mMα-单硫代甘油的缓冲液洗涤培养板。为在这些受体包被的培养板中进行测试，加入178μL相同缓冲液至96孔板的每一孔中。然后将20μL 10nM 3H-雌二醇溶液加入至该培养板的每一孔中。The estrogen receptor ligand binding assay was designed using a scintillation proximity assay using tritiated estradiol and recombinantly expressed estrogen receptor. Full-length recombinant human ERα and ERβ proteins were produced in a baculovirus expression system. ERα and ERβ extracts were diluted 1:400 in phosphate buffered saline containing 6 mM α-monothioglycerol. A 200 [mu]L aliquot of the diluted receptor preparation was added to each well of a 96-well Flashplate. Plates were covered with Saran Wrap and incubated overnight at 4°C. The next morning a 20 μL aliquot of phosphate buffered saline containing 10% bovine serum albumin was added to each well of the 96-well plate and allowed to incubate at 4°C for 2h. The plate was then washed with 200 μL of a buffer containing 20 mM Tris (pH 7.2), 1 mM EDTA, 10% glycerol, 50 mM KCl and 6 mM α-monothioglycerol. For testing in these receptor-coated plates, 178 [mu]L of the same buffer was added to each well of a 96-well plate. Then 20 μL of 10 nM 3H-estradiol solution was added to each well of the culture plate.
在0.01nM到1000nM的浓度范围内评估测试化合物。测试化合物储液应在100％DMSO中制备为试验中测试所期望终浓度的100X。在96孔板测试孔中的DMSO量不应超过1％。最终加入至测试中的为配制于100％DMSO中的2μL测试化合物等分试样。密封这种板并使其在室温平衡3h。在为计数96孔板装备的闪烁计数器中计数板。Test compounds were evaluated over a concentration range of 0.01 nM to 1000 nM. Test compound stocks should be prepared in 100% DMSO at 100X the final concentration expected to be tested in the assay. The amount of DMSO in the test wells of the 96-well plate should not exceed 1%. Final additions to the assay were 2 [mu]L aliquots of test compounds in 100% DMSO. The plate was sealed and allowed to equilibrate at room temperature for 3h. Plates were counted in a scintillation counter equipped for counting 96-well plates.
实验结果表明，1-乙基-4-{4-[5-羟基-2-(4-羟基苯基)-3-甲基-1H-吲哚-1-基]丁基}哌嗪-2,3-二酮对雌激素受体ERα及ERβ结合实验抑制率IC50分别为6000μM和3.9μM。因此，本发明所述化合物均表现出与雷洛昔芬类似的生物活性。The experimental results showed that 1-ethyl-4-{4-[5-hydroxy-2-(4-hydroxyphenyl)-3-methyl-1H-indol-1-yl]butyl}piperazine-2 , 3-diketone inhibited the estrogen receptor ERα and ERβ binding assay IC50 were 6000μM and 3.9μM. Therefore, the compounds of the present invention all exhibit similar biological activities to raloxifene.
实施例16：对HOB细胞中IL-6和GM-CSF生产的作用Example 16: Effects on IL-6 and GM-CSF Production in HOB Cells
将人体破骨细胞HOB铺板在96孔皿内使其在常规HOB培养基(Ham氏F12，补充有28mM HEPES，Ph7.4，10％FCS，1.1mM CaCl2，2mM谷酰胺和1％抗生素-抗真菌剂)中的密度为7×103个细胞/孔。次日，细胞用化合物或载体处理(0.2％DMSO)处理30分钟，随后加入IL-1β(1ng/mL)和TNF-α(10ng/mL)。培养持续18至24小时。利用市售ELISA试剂盒测定培养基中IL-6和GM-CSF水平。Human osteoclast HOB was plated in a 96-well dish in regular HOB medium (Ham's F12, supplemented with 28mM HEPES, Ph7.4, 10% FCS, 1.1mM CaCl2 , 2mM glutamine and 1% antibiotic- antifungal agent) at a density of 7×103 cells/well. The next day, cells were treated with compound or vehicle treatment (0.2% DMSO) for 30 minutes, followed by the addition of IL-1β (1 ng/mL) and TNF-α (10 ng/mL). The cultivation lasts for 18 to 24 hours. The levels of IL-6 and GM-CSF in the culture medium were measured using commercially available ELISA kits.
实验结果表明，在载体处理组中HOB细胞中的IL-6的浓度为0.189ng/mL，在使用本发明化合物组中IL-6的浓度为0.110ng/mL；同时本发明化合物对HOB细胞中GM-CSF的抑制浓度为IC50＝29.4μM，因此本发明化合物显示出对IL-6和GM-CSF具有抑制作用。The experimental results show that the concentration of IL-6 in the HOB cells in the carrier treatment group is 0.189ng/mL, and the concentration of IL-6 in the compound group using the present invention is 0.110ng/mL; The inhibitory concentration of GM-CSF is IC50 =29.4 μM, so the compound of the present invention shows inhibitory effect on IL-6 and GM-CSF.
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