WO2018077260A1 - 作为雌激素受体降解剂的吲哚并取代哌啶类化合物 - Google Patents

作为雌激素受体降解剂的吲哚并取代哌啶类化合物 Download PDF

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WO2018077260A1
WO2018077260A1 PCT/CN2017/108296 CN2017108296W WO2018077260A1 WO 2018077260 A1 WO2018077260 A1 WO 2018077260A1 CN 2017108296 W CN2017108296 W CN 2017108296W WO 2018077260 A1 WO2018077260 A1 WO 2018077260A1
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acid
estrogen receptor
compound
breast cancer
pharmaceutically acceptable
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PCT/CN2017/108296
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English (en)
French (fr)
Inventor
陆剑宇
丁照中
曾旭
贺辉君
陈曙辉
胡利红
董加强
王铁林
Original Assignee
山东罗欣药业集团股份有限公司
罗欣生物科技（上海）有限公司
南京明德新药研发股份有限公司
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Priority to CN201780066789.2A priority Critical patent/CN110139863B/zh
Priority to US16/345,271 priority patent/US10774081B2/en
Publication of WO2018077260A1 publication Critical patent/WO2018077260A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a novel anthracene and substituted piperidine compound, and specifically discloses a compound of the formula (I), a pharmaceutically acceptable salt, hydrate or prodrug thereof, a process for the preparation thereof, a pharmaceutical composition and the like Use of an estrogen receptor degrading agent in the treatment of estrogen receptor positive breast cancer.
  • breast cancer has become the second highest incidence of cancer in the world and the highest incidence of cancer among women.
  • the role of the estrogen-estrogen receptor signaling pathway in the development of breast cancer has been identified; and the estrogen receptor (ER) has also developed into the most important biomarker for breast cancer.
  • ER estrogen receptor
  • breast cancer can be divided into estrogen receptor-positive breast cancer and estrogen receptor-negative breast cancer. Among them, estrogen receptor-positive breast cancer accounts for more than 70% of the total number of breast cancer patients. .
  • Endocrine Therapy which targets the estrogen-estrogen receptor signaling pathway in breast cancer cells, has the least harmful effect and has been used as the first choice for the treatment of estrogen receptor-positive breast cancer.
  • Endocrine therapy mainly includes the following three treatment methods: ovarian suppression therapy, aromatase inhibitor (AI), and selective estrogen receptor modulator (SERM). Ovarian suppression therapy is less effective than the other two treatments because of poor efficacy.
  • aromatase inhibitors first generation, second generation
  • the problem of chemical enzyme inhibitors has been widely used.
  • letrozole has been used as a first-line drug for the treatment of estrogen receptor-positive breast cancer.
  • the selective estrogen receptor modulator (SERM) acts directly on the estrogen receptor to block this signaling pathway, with a significant therapeutic effect and a long history of application.
  • tamoxifen is the most representative selective estrogen receptor modulator.
  • As a first-line drug recommended for its priority tamoxifen has shown significant clinical efficacy in the prevention and treatment of estrogen receptor-positive breast cancer.
  • estrogen Receptor-positive breast cancers are also becoming more prominent in their resistance to aromatase inhibitors and selective estrogen receptor modulators.
  • aromatase inhibitors estrogen receptors can produce corresponding mutations.
  • the mutated estrogen receptor itself maintains an agonistic conformation in the absence of estrogen, allowing it to continue to function as a receptor to promote breast cancer cell proliferation.
  • the resistance mechanism of breast cancer cells to the selective estrogen receptor modulator tamoxifen is more complicated.
  • breast cancer cells can compensate for the estrogen receptor estrogen receptor activation function-2 (AF-2) caused by tamoxifen by activating estrogen receptor activation domain-1 (AF-1) function. The function is missing.
  • breast cancer cells can adapt to the conformation of the estrogen receptor after binding to tamoxifen by regulating the structure or concentration of estrogen receptor coactivator, so that estrogen receptor function is restored, thereby causing drug resistance.
  • AF-2 estrogen receptor estrogen receptor activation function-2
  • AF-1 estrogen receptor activation domain-1
  • Selective estrogen receptor down-regulator has unique advantages in the treatment of breast cancer resistant to both hormones.
  • selective estrogen receptor down-regulation antagonizes estrogen receptor function, which can greatly accelerate the ubiquitination and degradation of estrogen receptors in breast cancer cells (normal or variant), completely blocking estrogen/estrogen Receptor letter No. pathway, to achieve the purpose of inhibiting the growth and proliferation of normal or drug-resistant breast cancer cells.
  • Studies have shown that selective estrogen receptor down-regulation can effectively inhibit the proliferation of hormone-resistant breast cancer cells.
  • Fulvestrant has shown good results in the treatment of hormonal-resistant breast cancer, demonstrating the unique advantages of selective estrogen receptor down-regulation.
  • fulvestrant itself has many problems.
  • fulvestrant because of its poor PK properties, fulvestrant has demonstrated zero oral bioavailability; at the same time, fulvestrant has a higher blood clearance rate. For both of these reasons, this drug can only be administered by intramuscular injection. However, due to its strong lipophilic structure, intramuscularly administered fulvestrant also has serious problems in tissue distribution; its clinical manifestation is that only about 50% of breast cancer patients who use fulvestrant show clinical response. . Therefore, the development of selective estrogen receptor downregulation with oral bioavailability is a much needed medical need.
  • WO2016097071 reports a compound selective estrogen receptor down-regulator (II), which still has problems of molecular instability, high risk of hERG inhibition, and inability to effectively cope with cancer cell metastasis.
  • the present invention discloses a compound of the formula (I), a pharmaceutically acceptable salt, hydrate or prodrug thereof,
  • the present invention also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the above compound, a pharmaceutically acceptable salt, hydrate or prodrug thereof and a pharmaceutically acceptable carrier.
  • the invention also discloses the use of the above compound, a pharmaceutically acceptable salt, hydrate or prodrug thereof or the above pharmaceutical composition for preparing a medicament for treating estrogen receptor positive breast cancer.
  • the structurally-linked trifluoroethyl group of piperidine has stronger electron-withdrawing property, so that the chemical stability of the whole molecule is strong; at the same time, the cyanomethyl structure has more
  • the strong electron-withdrawing ability further reduces the basicity of the azetidine, thereby lowering the risk of hERG inhibition of the compounds of the present invention.
  • In vivo experimental data of animals showed that the plasma AUC of the compound of the present invention was significantly increased at the same dose, and the Brain/Plasma ratio was also significantly improved. It can be seen that the PK properties of the compounds of the invention are superior. It can be reasonably assumed that the compounds of the invention will be used clinically and the dosage will be lower.
  • the compounds of the present invention readily pass through the characteristics of the blood-brain barrier, giving them great potential for brain metastasis for the treatment of ER-positive breast cancer.
  • the compound of the present invention has oral bioavailability and is an estrogen receptor degrading agent which can be developed into an oral preparation.
  • pharmaceutically acceptable salt refers to a salt of a compound of the invention prepared from a compound having a particular substituent found in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting a neutral amount of such a compound with a sufficient amount of a base in a neat solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts.
  • an acid addition salt can be obtained by contacting a neutral form of such a compound with a sufficient amount of an acid in a neat solution or a suitable inert solvent.
  • pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogencarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and an organic acid salt, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and me
  • the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the neutral form of the compound.
  • the parent form of the compound differs from the form of its various salts by certain physical properties, such as differences in solubility in polar solvents.
  • a "pharmaceutically acceptable salt” is a derivative of a compound of the invention wherein the parent compound is modified by salt formation with an acid or with a base.
  • pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acid groups such as carboxylic acids, and the like.
  • Pharmaceutically acceptable salts include the conventional non-toxic salts or quaternary ammonium salts of the parent compound, for example salts formed from non-toxic inorganic or organic acids.
  • non-toxic salts include, but are not limited to, those derived from inorganic acids and organic acids selected from the group consisting of 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, Benzenesulfonic acid, benzoic acid, hydrogencarbonate, carbonic acid, citric acid, edetic acid, ethane disulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, Hydrobromic acid, hydrochloric acid, hydroiodide, hydroxyl, hydroxynaphthalene, isethionethane, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, Pamoic acid, pantothenic acid, phenylacetic acid, phen
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing an acid group or a base by conventional chemical methods.
  • such salts are prepared by reacting these compounds in water or an organic solvent or a mixture of the two via a free acid or base form with a stoichiometric amount of a suitable base or acid.
  • a nonaqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.
  • the compounds provided herein also exist in the form of prodrugs.
  • Prodrugs of the compounds described herein are readily chemically altered under physiological conditions to convert to the compounds of the invention.
  • prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an in vivo setting.
  • Certain compounds of the invention may exist in unsolvated or solvated forms, including hydrated forms.
  • the solvated forms are equivalent to the unsolvated forms and are included within the scope of the invention.
  • Certain compounds of the invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers and individual isomers are included within the scope of the invention.
  • the compounds of the invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including the cis and trans isomers, the (-)- and (+)-p-enantiomers, the (R)- and (S)-enantiomers, and the diastereomeric a conformation, a (D)-isomer, a (L)-isomer, and a racemic mixture thereof, and other mixtures, such as enantiomerically or diastereomeric enriched mixtures, all of which belong to It is within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in the substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the invention.
  • optically active (R)- and (S)-isomers as well as the D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary wherein the resulting mixture of diastereomers is separated and the auxiliary group cleaved to provide pure The desired enantiomer.
  • a diastereomeric salt is formed with a suitable optically active acid or base, followed by conventional methods well known in the art.
  • the diastereomers are resolved and the pure enantiomer is recovered.
  • the separation of enantiomers and diastereomers is generally accomplished by the use of chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (eg, formation of an amino group from an amine). Formate).
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound.
  • radiolabeled compounds can be used, such as tritium (3 H), iodine -125 (125 I) or C-14 (14 C). Alterations of all isotopic compositions of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • pharmaceutically acceptable carrier refers to any formulation or carrier medium that is capable of delivering an effective amount of an active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects to the host or patient, including water, oil, Vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, tackifiers, transdermal enhancers and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical arts. For additional information on vectors, reference is made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are hereby incorporated by reference.
  • an "effective amount” or “therapeutically effective amount” with respect to a pharmaceutical or pharmacologically active agent means a drug that is non-toxic but achieves the desired effect or A sufficient amount of the agent.
  • an "effective amount” of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount will vary from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, and a suitable effective amount in a case can be determined by one skilled in the art based on routine experimentation.
  • active ingredient refers to a chemical entity that is effective in treating a target disorder, disease or condition.
  • the present invention employs the following abbreviations: aq for water; m-CPBA for 3-chloroperoxybenzoic acid; eq for equivalent, equivalent; DCM for methylene chloride; PE for petroleum ether; DMF for N, N-di Methylformamide; DMSO for dimethyl sulfoxide; EtOAc for ethyl acetate; EtOH for ethanol; MeOH for methanol; Cbz for benzyloxycarbonyl, an amine protecting group; Boc for t-butylcarbonyl is an amine Protecting group; AcOH stands for acetic acid; NaCNBH3 stands for sodium cyanoborohydride; THF stands for tetrahydrofuran; Boc2O stands for di-tert-butyl dicarbonate; TFA stands for trifluoroacetic acid; DIPEA stands for diisopropylethylamine; MTBE stands for A Tert-butyl ether; Tf2O stands for triflu
  • High performance liquid chromatography was performed using a Shimadzu LC20AB system equipped with a Shimadzu SIL-20A autosampler and a Shimadzu DAD: SPD-M20A detector using a Xtimate C18 (3 m packing, size 2.1 x 300 mm) column.
  • 0-60AB_6 min method Apply a linear gradient, start elution with 100% A (A is 0.0675% TFA in water), and end the elution with 60% B (B is 0.0625% TFA in MeCN solution). The whole process is 4.2 minutes, then eluted with 60% B for 1 minute. The column was equilibrated for 0.8 minutes to reach 100:0 with a total run time of 6 minutes.
  • 10-80AB_6 min method Apply a linear gradient, start elution with 90% A (A is 0.0675% TFA in water), and end the elution with 80% B (B in 0.0625% TFA in acetonitrile). 4.2 minutes, then eluted with 80% B for 1 minute.
  • the column was equilibrated for 0.8 minutes to 90:10 with a total run time of 6 minutes.
  • the column temperature was 50 ° C and the flow rate was 0.8 mL/min.
  • the diode array detector has a scanning wavelength of 200-400 nm.
  • TLC Thin layer chromatography
  • a common solvent for flash column chromatography or thin layer chromatography is a mixture of dichloromethane/methanol, ethyl acetate/methanol and hexane/ethyl acetate.
  • AS-H_3_40_2.35ML Chromatographic conditions Chiralpak AS-H column (specification 250x 4.6mm ID, 5m packing); mobile phase 40% methanol (0.05% DEA)-CO 2 ; flow rate 2.35mL/min, detection wavelength It is 220 nm.
  • OD-H_3_40_2.35M Chromatographic conditions Chiralcel OD-H column (specification 250x 4.6mm ID, 5m packing), mobile phase 40% methanol (0.05% DEA)-CO 2 , flow rate 2.35mL / min, detection wavelength It is 220 nm.
  • AD-H_2_50_2.35ML Chromatographic conditions Chiralpak AD-H column (specification 250x 4.6mm ID, 5mm packing), mobile phase 50% methanol (0.1% MEA)-CO 2 , flow rate 2.35mL / min, detection wavelength It is 220 nm.
  • Preparative SFC analysis was performed on a Waters Thar 80 Pre-SFC system using a Gilson UV detector using Chiralcel OD-H (250x 4.6mm ID, 5m packing) or Chiralpak AD-H (250x size) 4.6mm ID, 5m filler).
  • Chiralcel OD-H 250x 4.6mm ID, 5m packing
  • Chiralpak AD-H 250x size 4.6mm ID, 5m filler
  • the compound is eluted with a low gradient of ethanol-carbon dioxide or methanol-carbon dioxide, with methanol or ethanol containing 0.05% NH 3 ⁇ H 2 O, 0.05% DEA or 0.1% MEA, total run
  • the time is 20-30 minutes.
  • the starting material 1 can be produced by a method reported in the literature (J. Med. Chem., 2010, 53, 5155-5164).
  • the starting material 2 can be produced by a method reported in the literature (J. Org. Chem., 2005, 70, 2372-2375).
  • the starting material 3 can be obtained from a market sales channel.
  • Step B To a solution of starting material 3 (1.2 g, 7.0 mmol, 1.0 eq) in dichloromethane (15.0 mL) was added to boron tribromide (5.2 g, 20.9 mmol, 2.0 mL, 3.0) Eq); The reaction solution was stirred at 25 ° C for 12 hours, the reaction was completed by LCMS, and methanol (20.0 mL) was slowly added dropwise to the mixture, and then the mixture was concentrated. The obtained crude was dissolved in EtOAc (EtOAc) (EtOAc)EtOAc.
  • the starting material 8 can be obtained through a marketing channel.
  • Step A To a three-neck round bottom flask was added 8 (5.00 g, 26.70 mmol, 1.00 eq), triethylamine (6.75 g, 66.75 mmol, 9.25 mL, 2.50 eq) and dichloromethane (100.00 mL) at 0 ° C Next, methanesulfonyl chloride (3.67 g, 32.04 mmol, 2.48 mL, 1.20 eq) was added dropwise under nitrogen, and the mixture was reacted at 0 ° C for two hours. 20 ml of water was added to the mixture, and the layers were separated, and the aqueous phase was extracted three times with dichloromethane (50 ml). The organic phase was combined, washed with EtOAc EtOAc EtOAc m.
  • Step B To a one-neck round bottom flask was added 9 (7.09 g, 26.72 mmol, 1.00 eq) and dimethyl sulfoxide (70.00 mL), potassium cyanide (5.22 g, 80.16 mmol, 3.43 mL, 3.00 eq) in nitrogen The yellow suspension was reacted at 80 ° C for 12 hours. 20 ml of water was added to the reaction mixture, and then extracted with methyl t-butyl ether three times, 50 ml each time, the combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to give product 10 (5.15 g, 26.24 The rate is 98.21%) and is used directly in the next reaction.
  • 1 H NMR 400 MHz, DMSO-d6) ⁇ 4.02-3.89 (m, 2H), 3.64 - 3.48 (m, 2H), 2.86 - 2.79 (m, 3H), 1.38 (s, 9H).
  • Step D To a single-neck round bottom flask was added 7 (200.00 mg, 397.38 umol, 1.00 eq), 11 (63.23 mg, 476.86 umol, 1.20 eq, hydrochloride) and acetonitrile (10.00 mL). Cesium carbonate (284.84 mg, 874.24 umol, 2.20 eq) was added. The yellow suspension was reacted at 60 ° C for 12 hours.
  • reaction solution was filtered, and the filtered cake was washed with ethyl acetate (10 ml), and the filtrate was concentrated and purified by preparative high-performance liquid chromatography (formic acid) to afford product I (30.00mg, 57.45umol, yield 14.46%, purity 99.3%).
  • the MCF-7 cell line was purchased from the European Cell Culture Collection (ECACC). Envision Multi-Label Analyzer (PerkinElmer).
  • MCF-7 cells were seeded in black 384-well plates at 600 cells per well in 30 ml of cell suspension. The cell plates were placed in a carbon dioxide incubator for overnight culture.
  • test compound was diluted 5-fold to a 10th concentration with Epmotion, i.e., diluted from 2.5 millimoles to 1.28 nanomoles, and a double-replica experiment was set. 198 microliters of medium was added to the middle plate, and 2 ⁇ l of each well of the gradient was transferred to the intermediate plate according to the corresponding position. After mixing, 20 ⁇ l of each well was transferred to the cell plate. The cell plates were incubated in a carbon dioxide incubator for 6 days.
  • RPMI 1640 medium fetal bovine serum, PBS, 16% paraformaldehyde, Triton, blocking solution, estrogen receptor antibody, near-infrared goat anti-rabbit secondary antibody, DRAQ5 dye.
  • the MCF-7 cell line was purchased from the European Cell Culture Collection (ECACC). Odyssey infrared fluorescence scanning imaging system.
  • MCF-7 cells were seeded in black 384-well plates at 3200 cells per well in 30 ml of cell suspension. The cell plates were incubated in a carbon dioxide incubator for 4 days.
  • test compound was diluted 5-fold with Epmotion to the 10th concentration, that is, fulvestrant was diluted from 0.25 mmol to 0.128 nm, and the other compounds were diluted from 2.5 mmol to 1.28 nm, and a double-replica experiment was set. 198 microliters of medium was added to the middle plate, and 2 ⁇ l of each well of the gradient was transferred to the intermediate plate according to the corresponding position. After mixing, 20 ⁇ l of each well was transferred to the cell plate. The cell plates were incubated in a carbon dioxide incubator for 20 hours.
  • mice On the 21st day of administration, three mice were randomly selected from the drug-administered group, and plasma samples of the mice were collected at different time points, and the mice were euthanized near Tmax, and samples were collected for tumor, brain and breast. .
  • the plasma and tissue sample collection process is as follows:
  • Plasma Blood (60 ⁇ l) was collected using a 1.5 mL EDTA-K2 anticoagulation tube, and immediately centrifuged at 7000 rpm, 4 ° C for 10 minutes to obtain plasma (30 ⁇ l), and the plasma sample was stored at -80 ° C for drug concentration determination.
  • Tissue Brains were collected using a 5 mL cryotube and immediately thawed into liquid nitrogen for rapid freezing. After sampling, they were stored at -80 °C for drug concentration determination to compare distribution.
  • the compound of formula (I) has better PK properties, better brain tissue distribution and better penetrating blood-brain barrier.

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Abstract

本发明公开了一类作为雌激素受体降解剂的吲哚并取代哌啶类化合物，具体公开了式(I)所示化合物、其药学上可接受的盐、水合物或前药、其制备方法、药物组合物及其作为作为雌激素受体降解剂在治疗雌激素受体阳性乳腺癌中的用途。

Description

作为雌激素受体降解剂的吲哚并取代哌啶类化合物
相关申请的交叉引用
本申请主张2016年10月28日提交的中国专利申请CN201610968449.6的优先权，其内容在此并入本申请。
技术领域
本发明涉及新的吲哚并取代哌啶类化合物，具体公开了式(I)所示化合物、其药学上可接受的盐、水合物或前药、其制备方法、药物组合物及其作为作为雌激素受体降解剂在治疗雌激素受体阳性乳腺癌中的用途。
背景技术
根据WHO统计，乳腺癌已成为全球发病率第二高的癌症，也是女性中发病率最高的癌症。经过多年的研究，已经确定了雌激素-雌激素受体信号通路在乳腺癌发展中的作用；而雌激素受体(ER)也已经发展成了乳腺癌最重要的生物标记物。以雌激素受体表达为判别指标，乳腺癌可以划分为雌激素受体阳性乳腺癌及雌激素受体阴性乳腺癌；其中，雌激素受体阳性的乳腺癌占乳腺癌患者总数的70％以上。
针对乳腺癌细胞内雌激素-雌激素受体信号通路的内分泌疗法(Endocrine Therapy，ET)因其危害性最小，疗效显著，已经成为治疗雌激素受体阳性乳腺癌的首选疗法。内分泌疗法主要包含以下三种治疗方法：卵巢抑制疗法，芳香化酶抑制剂(Aromatase inhibitor,AI)，选择性雌激素受体调节剂(Selective estrogen receptor modulator,SERM)。卵巢抑制疗法因为疗效不理想，患者满意度低而应用较其他两种疗法少。早期的芳香化酶抑制剂(第一代，第二代)靶点选择性低，毒副作用大；经过多年的研究，第三代芳香化酶抑制剂大大提高了其选择性，解决了早期芳香化酶抑制剂的问题而得到了广泛的应用。其中，来曲唑等已作为一线药物用于治疗雌激素受体阳性乳腺癌。选择性雌激素受体调节剂(SERM)直接作用于雌激素受体以阻断此信号通路，疗效显著，应用历史较长。其中，他莫昔芬是最具有代表性的选择性雌激素受体调节剂。作为优先推荐使用的一线药物，他莫昔芬用于预防和治疗雌激素受体阳性乳腺癌表现出显著的临床疗效。
尽管芳香化酶抑制剂来曲唑以及选择性雌激素受体调节剂他莫昔芬在治疗雌激素受体阳性乳腺癌方面表现了良好的疗效，但是，随着两类药物的应用，雌激素受体阳性乳腺癌针对芳香化酶抑制剂以及选择性雌激素受体调节剂的耐药性问题也表现的越来越突出。大量的研究表明，乳腺癌对上述两种荷尔蒙疗法的耐药机理是不完全相同的。对于芳香化酶抑制剂，雌激素受体可产生相应的变异。变异后的雌激素受体可在无雌激素存在的条件下本身保持激动的构象，使得其继续发挥受体功能以促进乳腺癌细胞增殖。乳腺癌细胞对于选择性雌激素受体调节剂他莫昔芬的耐药机理较为复杂多样。首先，乳腺癌细胞可通过激活雌激素受体激活功能区-1(AF-1)功能来补偿因他莫昔芬导致的雌激素受体雌激素受体激活功能区-2(AF-2)的功能缺失。同时，乳腺癌细胞可通过调节雌激素受体共激活因子结构或者浓度来适应与他莫昔芬结合后的雌激素受体的构象，使得雌激素受体功能恢复，从而引起耐药。
在治疗耐上述两种荷尔蒙疗法的乳腺癌上，选择性雌激素受体下调剂(Selective estrogen receptor down-regulator,SERD)体现出了特有的优越性。机理上，选择性雌激素受体下调剂拮抗雌激素受体功能，可大大加速乳腺癌细胞内(正常的或者变异的)雌激素受体的泛素化降解，彻底阻断雌激素/雌激素受体信 号通路，达到抑制正常的或者耐药性乳腺癌细胞生长增殖的目的。研究表明，选择性雌激素受体下调剂可有效抑制耐荷尔蒙的乳腺癌细胞的增殖。作为唯一上市的选择性雌激素受体下调剂氟维司群(Fulvestrant)在治疗耐荷尔蒙疗法的乳腺癌上表现出了良好的效果，验证了选择性雌激素受体下调剂的独特优势。但是，氟维司群本身存在很多问题。首先，因为其PK性质很差，氟维司群变现出了零口服生物利用度；同时，氟维司群又有较高的血液清除率。由于以上两种原因，这个药物只能通过肌肉注射给药。但是，因为其强亲酯性结构，肌肉注射给药的氟维司群在组织分布上也存在严重问题；其临床变现为只有约50％应用氟维司群的乳腺癌患者表现出了临床响应。因此，研发有口服生物利用度的选择性雌激素受体下调剂是亟需的医疗需求。
WO2016097071报道了化合物选择性雌激素受体下调剂(II)，其仍然存在分子不稳定、hERG抑制风险高、无法有效应对癌细胞转移的问题。
Figure PCTCN2017108296-appb-000001
发明内容
本发明公开了式(I)所示化合物、其药学上可接受的盐、水合物或前药，
Figure PCTCN2017108296-appb-000002
本发明还公开了一种药物组合物，其含有治疗有效量的上述的化合物、其药学上可接受的盐、水合物或前药和药学上可接受的载体。
本发明还公开了上述的化合物、其药学上可接受的盐、水合物或前药或上述的药物组合物在制备治疗雌激素受体阳性乳腺癌药物中的应用。
技术效果
本发明中的化合物与现有技术相比，哌啶结构上相连的三氟乙基基团具有更强的吸电子性质，从而使得整个分子的化学稳定性根强；同时，氰甲基结构有着更强的吸电子能力，使得氮杂环丁烷碱性进一步降低，进而使本发明化合物的hERG抑制风险降得更低。动物体内实验数据显示，在同等剂量下，本发明化合物血浆AUC显著升高，其Brain/Plasma比也明显提高。由此可见，本发明化合物的PK性质更优。可以合理推测，本发明化合物应用于临床，剂量将会更低。再者，本发明化合物容易透过血脑屏障的特性，使其具备用于治疗ER阳性乳腺癌的脑转移巨大潜力。本发明化合物有口服生物利用度，是可以发展成为口服制剂的雌激素受体降解剂类药物。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机氨或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐(参见Berge et al.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66:1-19(1977))。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
优选地，以常规方式使盐与碱或酸接触，再分离母体化合物，由此再生化合物的中性形式。化合物的母体形式与其各种盐的形式的不同之处在于某些物理性质，例如在极性溶剂中的溶解度不同。
本文所用的“药学上可接受的盐”属于本发明化合物的衍生物，其中，通过与酸成盐或与碱成盐的方式修饰所述母体化合物。药学上可接受的盐的实例包括但不限于：碱基比如胺的无机酸或有机酸盐、酸根比如羧酸的碱金属或有机盐等等。药学上可接受的盐包括常规的无毒性的盐或母体化合物的季铵盐，例如无毒的无机酸或有机酸所形成的盐。常规的无毒性的盐包括但不限于那些衍生自无机酸和有机酸的盐，所述的无机酸或有机酸选自2-乙酰氧基苯甲酸、2-羟基乙磺酸、乙酸、抗坏血酸、苯磺酸、苯甲酸、碳酸氢根、碳酸、柠檬酸、依地酸、乙烷二磺酸、乙烷磺酸、富马酸、葡庚糖、葡糖酸、谷氨酸、乙醇酸、氢溴酸、盐酸、氢碘酸盐、羟基、羟萘、羟乙磺酸、乳酸、乳糖、十二烷基磺酸、马来酸、苹果酸、扁桃酸、甲烷磺酸、硝酸、草酸、双羟萘酸、泛酸、苯乙酸、磷酸、多聚半乳糖醛、丙酸、水杨酸、硬脂酸、亚乙酸、 琥珀酸、氨基磺酸、对氨基苯磺酸、硫酸、单宁、酒石酸和对甲苯磺酸。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。一般地，优选醚、乙酸乙酯、乙醇、异丙醇或乙腈等非水介质。
除了盐的形式，本发明所提供的化合物还存在前药形式。本文所描述的化合物的前药容易地在生理条件下发生化学变化从而转化成本发明的化合物。此外，前体药物可以在体内环境中通过化学或生化方法被转换到本发明的化合物。
本发明的某些化合物可以以非溶剂化形式或者溶剂化形式存在，包括水合物形式。一般而言，溶剂化形式与非溶剂化的形式相当，都包含在本发明的范围之内。
本发明的某些化合物可以具有不对称碳原子(光学中心)或双键。外消旋体、非对映异构体、几何异构体和单个的异构体都包括在本发明的范围之内。
除非另有说明，用楔形键和虚线键
Figure PCTCN2017108296-appb-000003
表示一个立体中心的绝对构型，用
Figure PCTCN2017108296-appb-000004
表示一个立体中心的相对构型。当本文所述化合物含有烯属双键或其它几何不对称中心，除非另有规定，它们包括E、Z几何异构体。同样地，所有的互变异构形式均包括在本发明的范围之内。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(3H)，碘-125(125I)或C-14(14C)。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
术语“药学上可接受的载体”是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂或载体介质代表性的载体包括水、油、蔬菜和矿物质、膏基、洗剂基质、软膏基质等。这些基质包括悬浮剂、增粘剂、透皮促进剂等。它们的制剂为化妆品领域或局部药物领域的技术人员所周知。关于载体的其他信息，可以参考Remington:The Science and Practice of Pharmacy,21st Ed.,Lippincott,Williams&Wilkins(2005)，该文献的内容通过引用的方式并入本文。
针对药物或药理学活性剂而言，术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或 药剂的足够用量。对于本发明中的口服剂型，组合物中一种活性物质的“有效量”是指与该组合物中另一种活性物质联用时为了达到预期效果所需要的用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
术语“活性成分”、“治疗剂”，“活性物质”或“活性剂”是指一种化学实体，它可以有效地治疗目标紊乱、疾病或病症。
本发明所使用的所有溶剂是市售的，无需进一步纯化即可使用。反应一般是在惰性氮气下、无水溶剂中进行的。质子核磁共振数据记录在Bruker Avance III 400(400MHz)分光仪上，化学位移以四甲基硅烷低场处的(ppm)表示。质谱是在安捷伦1200系列加6110(&1956A)上测定。LC/MS或Shimadzu MS包含一个DAD：SPD-M20A(LC)和Shimadzu Micromass 2020检测器。质谱仪配备有一个正或负模式下操作的电喷雾离子源(ESI)。
本发明采用下述缩略词：aq代表水；m-CPBA代表3-氯过氧苯甲酸；eq代表当量、等量；DCM代表二氯甲烷；PE代表石油醚；DMF代表N，N-二甲基甲酰胺；DMSO代表二甲亚砜；EtOAc代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；Cbz代表苄氧羰基，是一种胺保护基团；Boc代表叔丁基羰基是一种胺保护基团；AcOH代表乙酸；NaCNBH3代表氰基硼氢化钠；THF代表四氢呋喃；Boc2O代表二-叔丁基二碳酸酯；TFA代表三氟乙酸；DIPEA代表二异丙基乙基胺；MTBE代表甲基叔丁基醚；Tf2O代表三氟甲磺酸酐，TEA代表三乙胺，LAH代表四氢锂铝，IBX代表2-碘酰基苯甲酸，TFAA代表三氟乙酸酐，MsCl代表三氟甲磺酰氯，TsCl代表对甲基苯磺酰氯。
用配有Shimadzu SIL-20A自动进样器和日本岛津DAD：SPD-M20A探测器的岛津LC20AB系统进行高效液相色谱分析，采用Xtimate C18(3m填料，规格为2.1x 300mm)色谱柱。0-60AB_6分钟的方法：应用线性梯度，以100％A(A为0.0675％TFA的水溶液)开始洗脱，并以60％B(B为0.0625％TFA的MeCN溶液)结束洗脱，整个过程为4.2分钟，然后以60％B洗脱1分钟。将色谱柱再平衡0.8分钟达到100:0，总运行时间为6分钟。10-80AB_6分钟的方法：应用线性梯度，以90％A(A为0.0675％TFA的水溶液)开始洗脱，并以80％B(B为0.0625％TFA的乙腈溶液)结束洗脱，整个过程为4.2分钟，然后以80％B洗脱1分钟。将色谱柱再平衡0.8分钟达到90:10，总运行时间为6分钟。柱温为50℃，流速为0.8mL/min。二极管阵列检测器扫描波长为200-400nm。
在Sanpont-group的硅胶GF254上进行薄层色谱分析(TLC)，常用紫外光灯照射检出斑点，在某些情况下也采用其他方法检视斑点，在这些情况下，用碘(10g硅胶中加入约1g碘并彻底混合而成)、香草醛(溶解大约1g香草醛于100mL 10％H2SO4中制得)、茚三酮(从Aldrich购得)或特殊显色剂(彻底混合(NH4)6Mo7O24·4H2O、5g(NH4)2Ce(IV)(NO3)6、450mL H2O和50mL浓H2SO4而制得)展开薄层板，检视化合物。采用Still,W.C.；Kahn,M.；and Mitra,M.Journal of Organic Chemistry,1978,43,2923-2925.中所公开技术的类似方法，在Silicycle的40-63μm(230-400目)硅胶上进行快速柱色谱。快速柱色谱或薄层色谱的常用溶剂是二氯甲烷/甲醇、乙酸乙酯/甲醇和己烷/乙酸乙酯的混合物。
在Gilson-281Prep LC 322系统上采用吉尔森UV/VIS-156探测器进行制备色谱分析,所采用的色谱柱是Agella Venusil ASB Prep C18，5m、150x 21.2mm；Phenomenex Gemini C18、5m、150x 30mm；Boston Symmetrix C18，5m、150x 30mm；或者Phenomenex Synergi C18、4m、150x 30mm。在流速约为25 mL/min时，用低梯度的乙腈/水洗脱化合物，其中水中含有0.05％HCl、0.25％HCOOH或0.5％NH3·H2O，总运行时间为8-15分钟。
用带有Agilent1260自动进样器和Agilent DAD：1260检测器的Agilent 1260Infinity SFC系统进行SFC分析。色谱柱采用Chiralcel OD-H 250x 4.6mm I.D.,5um或者Chiralpak AS-H 250x 4.6mm I.D.，5m或者Chiralpak AD-H 250x 4.6mm I.D.，5m。OD-H_5_40_2.35ML的色谱条件：Chiralcel OD-H色谱柱(规格为250x 4.6mm I.D.,m填料)，流动相为40％乙醇(0.05％DEA)-CO2；流速为2.35mL/min；检测波长为220nm。AS-H_3_40_2.35ML色谱条件：Chiralpak AS-H色谱柱(规格为250x 4.6mm I.D.,5m填料)；流动相为40％甲醇(0.05％DEA)-CO2；流速为2.35mL/min，检测波长为220nm。OD-H_3_40_2.35M色谱条件：Chiralcel OD-H色谱柱(规格为250x 4.6mm I.D，5m填料)，流动相为40％甲醇(0.05％DEA)-CO2，流速为2.35mL/min，检测波长为220nm。AD-H_2_50_2.35ML色谱条件：Chiralpak AD-H色谱柱(规格为250x 4.6mm I.D，5mm填料),流动相为50％甲醇(0.1％MEA)-CO2，流速为2.35mL/min，检测波长为220nm。
在使用Gilson UV检测器的Waters Thar 80Pre-SFC系统上进行制备型SFC分析，所采用的色谱柱为Chiralcel OD-H(规格为250x 4.6mm I.D，5m填料)或者Chiralpak AD-H(规格为250x 4.6mm I.D，5m填料)。在流速约为40-80mL/min时，用低梯度的乙醇-二氧化碳或者甲醇-二氧化碳洗脱化合物，其中甲醇或乙醇含有0.05％NH3·H2O、0.05％DEA或者0.1％MEA，总运行时间为20-30分钟。
化合物经手工或者
Figure PCTCN2017108296-appb-000005
软件命名，市售化合物采用供应商目录名称。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1：中间体7
Figure PCTCN2017108296-appb-000006
其中，起始原料1可以通过文献(J.Med.Chem.,2010,53,5155-5164)报道的方法来制备。
其中，起始原料2可以通过文献(J.Org.Chem.,2005,70,2372-2375)报道的方法来制备。
其中，起始原料3可从市场销售渠道获得。
步骤A：在25℃下，向起始原料1(14.0g，40.2mmol，1.0eq.)的二氧六环(140.0mL)溶液中加入DIPEA(15.6g，120.5mmol，3.0eq.)和起始原料2(18.0g，80.3mmol，2.0eq.)；反应混合物在氮气保护下75℃搅拌12小时。LCMS检测到产物生成。反应液用水(100mL)稀释，乙酸乙酯(150mL*2)萃取，萃取有机相用盐水(100mL)洗，无水硫酸钠干燥，过滤，真空浓缩得粗残余物。粗残余物经硅胶柱层析初步分离纯化(石油醚：乙酸乙酯＝5:1到3:1)，得中间体6(6.5g,粗品)为液体，直接用于下一步反应。
步骤B：在-60℃下，向起始原料3(1.2g,7.0mmol,1.0eq)的二氯甲烷(15.0mL)溶液中加入三溴化硼(5.2g,20.9mmol,2.0mL,3.0eq)；反应液在25℃搅拌12小时，LCMS监测反应完成，甲醇(20.0mL)缓慢滴加到反应液中，然后将混合液浓缩。得到的粗品溶解到乙酸乙酯(100mL)中，用水(80mL)洗涤，无水硫酸钠干燥，过滤，浓缩得到中间体4(700.0mg,4.4mmol,产率：63.5％)。
步骤C：在25℃，向中间体4(250.0mg,1.6mmol,1.0eq)的乙腈(7.0mL)溶液中加入碳酸铯(772.8mg,2.4mmol,1.5eq)和1,2-二溴乙烷(445.6mg,2.4mmol,179.0uL,1.5eq)。反应液在60℃搅拌12小时。TLC(石油醚：乙酸乙酯＝4：1)显示反应完成。将反应液倒入水(40mL)中，用乙酸乙酯(40mL)萃取，无水硫酸钠干燥，过滤，浓缩。粗品用pre-TLC(石油醚：乙酸乙酯＝4：1)纯化；得到白色固体产品中间体5(150.0mg,565.9umol,产率：35.8％)。
步骤D：在25℃，向中间体5(150.0mg,565.9umol,1.0eq)的甲苯(5.0mL)溶液中加入中间体6(187.4mg,565.9umol,1.0eq)和醋酸(101.9mg,1.7mmol,97.1uL,3.0eq)；反应液在90℃搅拌5小时；LCMS显示反应完成，将反应液倒入水(50mL)中，用乙酸乙酯萃取(50mL)，无水硫酸钠干燥，过滤，浓 缩。粗品通过pre-TLC(石油醚：乙酸乙酯＝4：1)纯化。得到中间体7(200.0mg,403.7umol,产率：71.3％)；LCMS：m/z 495.2(M+1)。
实施例2：化合物(I)
Figure PCTCN2017108296-appb-000007
其中，起始原料8可以通过市场销售渠道获得。
步骤A：向三口圆底烧瓶中加入8(5.00g,26.70mmol,1.00eq)，三乙胺(6.75g,66.75mmol,9.25mL,2.50eq)和二氯甲烷(100.00mL)，在0℃下，甲烷磺酰氯(3.67g,32.04mmol,2.48mL,1.20eq)于氮气下滴加，该混合液在0℃下反应两小时。向混合物中加入20毫升水，分液，水相用二氯甲烷萃取三次，每次50毫升。合并有机相，并用50毫升饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液浓缩得到产品9(7.08g,粗品)，直接用于下一步。
步骤B:向单口圆底烧瓶中加入9(7.09g,26.72mmol,1.00eq)和二甲基亚砜(70.00mL)，氰化钾(5.22g,80.16mmol,3.43mL,3.00eq)在氮气下加入，该黄色悬浮液于80℃下反应12小时。向反应液中加入20毫升水，然后用甲基叔丁基醚萃取三次，每次50毫升，合并的有机相用无水硫酸钠干燥，过滤，浓缩得到产品10(5.15g,26.24mmol,产率98.21％)，直接用于下一步反应。1H NMR(400MHz,DMSO-d6)δ4.02-3.89(m,2H),3.64–3.48(m,2H),2.86-2.79(m,3H),1.38(s,9H)。
步骤C：向单口圆底烧瓶中加入10(3.15g,16.05mmol,1.00eq)，再加入12M盐酸(8.03mL,6.00eq)并于30℃下反应30分钟。该混合物减压浓缩得到产品11(2.00g,粗品,盐酸盐)，直接用于下一步。1H NMR(400MHz,METHANOL-d4)δ4.27-4.15(m,2H),4.05-3.91(m,2H),3.40-3.19(m,2H),2.90(d,J＝6.8Hz,1H),2.80-2.72(m,1H)。
步骤D：向单口圆底烧瓶中加入7(200.00mg,397.38umol,1.00eq)，11(63.23mg,476.86umol,1.20eq,盐酸盐)和乙腈(10.00mL)，氮气氛围下，向混合物中加入碳酸铯(284.84mg,874.24umol,2.20eq)。该黄色悬浮液于60℃下反应12小时。反应液过滤，滤饼用10毫升乙酸乙酯洗涤，滤液浓缩并用制备级高效液相色谱法分离(甲酸)得到产品I(30.00mg,57.45umol,产率14.46％,纯度99.3％)。1H NMR(400MHz,CHLOROFORM-d)δ7.62-7.51(m,2H),7.26(br d,J＝6.8Hz,1H),7.19-7.09(m,2H),6.43(br d,J＝10.4Hz, 2H),5.28(s,1H),3.94(t,J＝5.2Hz,2H),3.65-3.52(m,3H),3.30-3.19(m,1H),3.16-3.08(m,3H),3.03-2.92(m,1H),2.89-2.77(m,3H),2.69-2.61(m,3H),1.19(d,J＝6.4Hz,3H)；MS(ESI,M+):541.1
实验例1：体外评价
(1)MCF-7细胞增殖抑制实验：
实验材料：
RPMI 1640培养基，胎牛血清，Promega CellTiter-Glo试剂。MCF-7细胞系购自欧洲细胞培养物保藏所(ECACC)。Envision多标记分析仪(PerkinElmer)。
实验方法：
将MCF-7细胞种于黑色384孔板中，每30毫升细胞悬液中600个细胞每孔。细胞板置于二氧化碳培养箱中过夜培养。
将待测化合物用Epmotion进行5倍稀释至第10个浓度，即从2.5毫摩稀释至1.28纳摩，设置双复孔实验。向中间板中加入198微升培养基，再按照对应位置，转移2微升每孔的梯度稀释化合物至中间板，混匀后转移20微升每孔到细胞板中。细胞板置于二氧化碳培养箱中培养6天。
向细胞板中加入每孔25微升的Promega CellTiter-Glo试剂，室温孵育10分钟使发光信号稳定。采用PerkinElmer Envision多标记分析仪读数。
数据分析：
利用方程式(Max-Ratio)/(Max-Min)*100％将原始数据换算成抑制率，IC50的值即可通过四参数进行曲线拟合得出。(XLFIT5中205模式得出，iDBS)
(2)MCF-7细胞内ER降解
实验材料：
RPMI 1640培养基，胎牛血清,PBS,16％多聚甲醛，Triton，封闭液，雌激素受体抗体，近红外羊抗兔二抗，DRAQ5染料。MCF-7细胞系购自欧洲细胞培养物保藏所(ECACC)。Odyssey红外荧光扫描成像系统。
实验方法：
将MCF-7细胞种于黑色384孔板中，每30毫升细胞悬液中3200个细胞每孔。细胞板置于二氧化碳培养箱中培养4天。
将待测化合物用Epmotion进行5倍稀释至第10个浓度，即氟维司群从0.25毫摩稀释至0.128纳摩，其他化合物从2.5毫摩稀释至1.28纳摩，设置双复孔实验。向中间板中加入198微升培养基，再按照对应位置，转移2微升每孔的梯度稀释化合物至中间板，混匀后转移20微升每孔到细胞板中。细胞板置于二氧化碳培养箱中培养20小时。
向细胞板中加入每孔50微升的8％多聚甲醛，室温孵育30分钟后，用PBS洗两次，拍干后加入50ul含0.1％Triton的PBS，室温孵育15分钟，然后用PBS洗五次，拍干后加入50微升封闭液室温孵育1个小时，拍干后加入50ul含0.1％雌激素受体抗体的封闭液，4摄氏度过夜。第二天拍掉一抗后PBS洗五次，然后加入含0.1％近红外羊抗兔二抗和0.05％DRAQ5染料的封闭液，室温孵育1个小时后，用PBS洗五遍，拍干后用Odyssey红外荧光扫描成像系统读数。
数据分析：
利用方程式(Max-Ratio)/(Max-Min)*100％将原始数据换算成抑制率，IC50的值即可通过四参数进行曲线拟合得出。(XLFIT5中205模式得出，iDBS)
表1：本发明化合物体外筛选试验结果
化合物 MCF-7抗细胞增殖(nM) MCF-7细胞内ER降解IC50(nM)
氟维司群 1.77 0.71
(I) 1.17 1.13
结论：本发明化合物体外活性比已经上市的雌激素受体下调剂氟维司群更为优异。
实验例2：药物组织分布评价
在给药第21天，从给药组中随机挑选三只小鼠，挑取不同时间点对小鼠的血浆进行样品收集，并在Tmax附近安乐死小鼠，对肿瘤、脑、乳腺进行样品收集。
血浆和组织样品收集过程如下：
血浆：使用1.5mL的EDTA-K2抗凝管收集血液(60μl)，立即在7000rpm、4℃条件下离心10分钟得到血浆(30μl)，将血浆样品保存在-80℃下用于药物浓度测定。
组织：使用5mL的冻存管收集大脑，立即丢入液氮中进行速冻，在取样结束后保存在-80℃下用于药物浓度测定以比较分布。
测定结果总结在表2中。
表2组织分布评价
Figure PCTCN2017108296-appb-000008
结论：同等剂量下，式(I)化合物的PK性质更优，有更好脑组织分布，穿透血脑屏障性能更优。

Claims (3)

  1. 式(I)所示化合物、其药学上可接受的盐、水合物或前药，
    Figure PCTCN2017108296-appb-100001
  2. 一种药物组合物，其含有治疗有效量的根据权利要求1所述的化合物、其药学上可接受的盐、水合物或前药和药学上可接受的载体。
  3. 根据权利要求1所述的化合物、其药学上可接受的盐、水合物或前药或根据权利要求2所述的药物组合物在制备治疗雌激素受体阳性乳腺癌药物中的应用。
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