WO2017121187A1 - 一种丙型肝炎病毒抑制剂、药物组合物及其应用 - Google Patents

一种丙型肝炎病毒抑制剂、药物组合物及其应用 Download PDF

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WO2017121187A1
WO2017121187A1 PCT/CN2016/106874 CN2016106874W WO2017121187A1 WO 2017121187 A1 WO2017121187 A1 WO 2017121187A1 CN 2016106874 W CN2016106874 W CN 2016106874W WO 2017121187 A1 WO2017121187 A1 WO 2017121187A1
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compound
hepatitis
virus
pharmaceutically acceptable
virus inhibitor
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PCT/CN2016/106874
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English (en)
French (fr)
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王义汉
金剑
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深圳市塔吉瑞生物医药有限公司
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

  • the invention belongs to the technical field of medicine, and in particular relates to a hepatitis C virus inhibitor, a pharmaceutical composition and application thereof.
  • HCV Hepatitis C Virus
  • the encapsulated HCV virion contains a positive-stranded RNA genome that encodes all known virus-specific proteins in a single uninterrupted open reading frame.
  • the open reading frame comprises approximately 9500 nucleotides and encodes a single large polyprotein of approximately 3000 amino acids.
  • Polyproteins include core proteins, envelope proteins E1 and E2, membrane-bound protein P7, and non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B.
  • Harvoni a combination of a fixed-dose new antiviral NS5A inhibitor, Ledipasvir (GS5885) and the NS5B blocker Sofosbuvir, is a heavy hepatitis C treatment approved by Gilead in December 2013. Harvoni is the first fully oral anti-HC program approved for the treatment of genotype 1 hepatitis C infection and does not require the combination of interferon or ribavirin. Harvoni can be used as a single drug or in combination with other oral preparations such as ribavirin.
  • Harvoni produced by the original Gilead in the United States is expensive, and only the price of the drug can not afford the average family. Therefore, it is still necessary to develop a compound having inhibitory activity or better pharmacodynamics against the hepatitis C virus protein NS5A.
  • the present invention discloses a hepatitis C virus inhibitor, a pharmaceutical composition and use thereof, which have better hepatitis C virus protein NS5A inhibitory activity and/or have better pharmacodynamics/pharmacokinetics. performance.
  • a hepatitis C virus inhibitor such as a compound of formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate or a solvent compound,
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 And R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49 , R 50 are each independently hydrogen, deuterium, halogen;
  • the compound of the formula (I) contains at least one ruthenium atom, and the number of ruthenium atoms may be any one of 1 to 50.
  • the strontium isotope content of the cerium in the deuterated position is at least greater than the natural strontium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, and even more preferably greater than 95. %, more preferably greater than 99%.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 And R 32 , R 33 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 , R 44 , R 45 , R 46 , R 47 , R 48 , R 49 and R 50 have a strontium isotope content of at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, and even more preferably greater than 25%.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently hydrazine or hydrogen.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are fluorene.
  • R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 and R 22 are each independently hydrazine or hydrogen.
  • R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 is ⁇ .
  • R 7 and R 8 are ⁇ .
  • R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 and R 31 are each independently hydrazine or hydrogen.
  • R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 and R 31 are ⁇ .
  • R 32 , R 36 , R 37 , R 38 , R 39 and R 40 are each independently hydrazine or hydrogen.
  • R 32 , R 36 , R 37 , R 38 , R 39 and R 40 are fluorene.
  • R 41 is hydrazine
  • R 42 , R 43 and R 44 are oxime.
  • R 45 , R 46 , R 47 , R 48 , R 49 and R 50 are ⁇ .
  • the compound is selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof:
  • the present invention also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a hepatitis C virus inhibitor as described above, or a crystalline form, a pharmaceutically acceptable salt, hydrate or solvate thereof.
  • the pharmaceutically acceptable carrier includes a glidant, a sweetener, a diluent, a preservative, a dye, a colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersant At least one of a disintegrant, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.
  • the pharmaceutical composition is a tablet, a pill, a capsule, a powder, a granule, an ointment, an emulsion, a suspension, a solution, a suppository, an injection, an inhalant, a gel, a microsphere or Aerosol.
  • the pharmaceutical compositions of this invention may be formulated as solid, semi-solid, liquid or gaseous formulations.
  • Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.
  • the pharmaceutical composition of the present invention can be produced by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar-coating method, a pulverization method, an emulsification method, a freeze-drying method, and the like.
  • it further comprises an active compound which is an immunomodulator or an antiviral drug compound.
  • the immunomodulator is an interferon drug compound.
  • the antiviral drug compound is ribavirin, amantadine, other inhibitors of NS5A, helicase in the HCV life cycle, protease, polymerase, metalloproteinase or internal ribosome entry.
  • the invention discloses a use of the hepatitis C virus inhibitor as described above for preparing a hepatitis C treatment Use in inflammatory virus-infected drugs.
  • the invention also discloses a method of treating and/or preventing a hepatitis C virus-associated disease in a subject, the method comprising administering to the subject a compound of formula (I) as hereinbefore described Or a polymorphic form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotope variant, a hydrate or a solvent compound, or a pharmaceutical composition as described above.
  • the present invention also discloses a compound of the formula (I), or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvent thereof, as hereinbefore described, or a drug as hereinbefore described
  • a composition for the treatment and/or prevention of a disease associated with a protein kinase is also disclosed.
  • the hepatitis C virus HCV comprises a plurality of genotypes thereof and a plurality of gene subtypes, preferably 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a, 6a. "
  • halogen means F, Cl, Br, and I unless otherwise specified. More preferably, the halogen atom is selected from the group consisting of F, Cl and Br.
  • deuterated means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted.
  • deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted.
  • deuterated is used interchangeably with “one or more deuterated”.
  • non-deuterated compound means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).
  • the invention also includes isotopically labeled compounds, equivalent to the original compounds disclosed herein.
  • isotopes which may be listed as compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, respectively. , 31 P, 32 P, 35 S, 18 F and 36 Cl. a compound, or an enantiomer, a diastereomer, an isomer, or a pharmaceutically acceptable salt or solvate of the present invention, wherein an isotope or other isotopic atom containing the above compound is within the scope of the present invention .
  • isotopically-labeled compounds of the present invention such as the radioisotopes of 3 H and 14 C, are also among them, useful in tissue distribution experiments of drugs and substrates. ⁇ , ie 3 H and carbon-14, ie 14 C, are easier to prepare and detect and are preferred in isotopes.
  • Isotopically labeled compounds can be prepared in a conventional manner by substituting a readily available isotopically labeled reagent with a non-isotopic reagent using the protocol of the examples.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • a preferred class of salts are the salts of the compounds of the invention with acids.
  • Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid; Amino acids such as amino acid, phenylalanine, aspartic acid, and glutamic acid.
  • a preferred class of salts are the salts of the compounds of the invention with bases, such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium salts).
  • bases such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium salts).
  • amine salts such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butyl
  • solvate refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio.
  • Hydrophilate means a complex formed by the coordination of a compound of the invention with water.
  • the compound of the present invention has excellent inhibitory properties against the hepatitis C virus protein NS5A.
  • the technical solution of the present invention changes the metabolism of the compound in the organism by deuteration, so that the compound has better pharmacokinetic parameter characteristics.
  • the dosage can be changed and a long-acting preparation can be formed to improve the applicability.
  • each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (25 ° C to 100 ° C, preferably 25 ° C to 80 ° C).
  • the reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.
  • a hepatitis C virus inhibitor with the following formula:
  • Step S1 obtaining Compound 2 first according to the following synthetic route
  • Step S2 obtaining compound 4 first according to the following synthetic route
  • Step S3 obtaining compound 7 first according to the following synthetic route
  • Step S4 obtaining compound 12 first according to the following synthetic route
  • Step S5 obtaining compound 12 first according to the following synthetic route
  • Step S6 obtaining compound 14 first according to the following synthetic route
  • Step S7 first obtaining the compound A-1 according to the following synthetic route
  • a hepatitis C virus inhibitor with the following formula:
  • Step S1 obtaining compound 15 first according to the following synthetic route
  • Step S2 obtaining compound 18 first according to the following synthetic route
  • Step S3 obtaining compound 19 first according to the following synthetic route
  • Step S4 obtaining compound 20 first according to the following synthetic route
  • Step S5 first obtaining the compound A-2 according to the following synthetic route
  • a hepatitis C virus inhibitor with the following formula:
  • A-3 was synthesized according to the following route.
  • the compound 20 143 mg was dissolved in 2 mL of anhydrous dichloromethane, and 1 mL of a 4 N hydrogen chloride-dioxane solution was added thereto at room temperature for stirring for 30 minutes.
  • Proline 72 mg, N,N-diisopropylethylamine 119 mg, HATU 154 mg was reacted at room temperature for 20 minutes.
  • the reaction mixture was poured into 20 mL of water, and extracted with EA (20 mL ⁇ 2).
  • a hepatitis C virus inhibitor with the following formula:
  • A-4 was synthesized according to the following route:
  • HCV HCV replication system
  • HCV Replicon System As an evaluation model. Since its first report in Science in 1999, the HCV replication system has become one of the most important tools for studying HCV RNA replication, pathogenicity and viral persistence, for example, the use of replicons has successfully demonstrated the 5' required for HCV RNA replication. - NCR minimum region, and the HCV replication subsystem has been successfully used as an evaluation model for antiviral drugs. The inventors of the present invention verified according to the methods described in Science, 1999, 285 (5424), 110-3, and J. Virol, 2003, 77(5), 3007-19.
  • the inhibitory activities of the recombinant hepatitis C virus genotype 1a and 1b replicons were detected by stable transfection of replicon cells with HCV-1a and HCV-1b. This experiment will use the NS5A inhibitor BMS-790052 as a positive control compound.
  • Step 1 The compound was diluted 1:3 in 8 series points, double-replicated, and added to a 96-well plate.
  • the DMSO was set to no compound control.
  • the final concentration of DMSO in the cell culture was 0.5%.
  • Step 2 HCV-1a and 1b cells were separately suspended in a culture medium containing 10% FBS, and seeded into a 96-well plate containing the compound at a density of 8,000 cells per well. The cells were cultured for 3 days at 5% CO 2 at 37 °C.
  • Step 3 The cytotoxicity of the compound against GT1b replicon was determined using CellTiter-Fluor (Promega).
  • Step 4 Detection of luciferase assay by Bright-Glo (Promega) for anti-hepatitis C virus activity.
  • Step Five using GraphPad Prism data analysis software, the curve fitting and EC 50 values were calculated and the 50 value CC.
  • This infection model is referred to as A novel luciferase and GFP dual reporter virus for rapid and convenient evaluation of HCV replication.
  • Example 1 The compounds of Example 1 - Example 4 were analyzed according to the above procedure to calculate the EC 50 . The results are shown in Table 1.
  • the compound of the present invention can inhibit multiple genotypes of HCV and exerts superior anti-HCV action by inhibiting the mechanism of HCV NS5A protein.
  • Microsomal experiments human liver microsomes: 0.5 mg/mL, Xenotech; rat liver microsomes: 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer Agent (pH 7.4).
  • Preparation of the stock solution A certain amount of the powder of the compound examples 1-4 was accurately weighed and dissolved to 5 mM with DMSO, respectively.
  • phosphate buffer 100 mM, pH 7.4: Mix 150 mL of pre-formed 0.5 M potassium dihydrogen phosphate and 700 mL of 0.5 M potassium dihydrogen phosphate solution, and adjust the mixture with 0.5 M potassium dihydrogen phosphate solution. pH value To 7.4, diluted 5 times with ultrapure water before use, magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.
  • NADPH regeneration system containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride was prepared and placed on wet ice before use.
  • Formulation stop solution acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 ⁇ L of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 ⁇ L of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 25057.5 ⁇ L of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 ⁇ L of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.
  • the corresponding compound had a reaction concentration of 1 ⁇ M and a protein concentration of 0.5 mg/mL.
  • 100 ⁇ L of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min.
  • the plate was centrifuged at 5000 x g for 10 min at 4 °C.
  • 100 ⁇ L of the supernatant was taken into a 96-well plate to which 100 ⁇ L of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.
  • the compounds of the present invention exhibited excellent metabolic stability in both human liver microsomes and rat liver microsome experiments.
  • OBJECTIVE To investigate the pharmacokinetic behavior of the compounds of the present invention after administration of GS5885 and Compound A-1 in rats.
  • SD rat grade SPF grade
  • Weight range 180 ⁇ 220g (actual weight range is 187 ⁇ 197g)
  • Compound A-1 has superior activity to GS5885 and has excellent pharmacokinetic properties as compared with GS5885, and thus is more suitable as a compound for inhibiting hepatitis C virus protein NS5A. It is further suitable for the preparation of a medicament for treating hepatitis C virus infection.

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Abstract

本发明提供了一种丙型肝炎病毒抑制剂、药物组合物及其应用，所述丙型肝炎病毒抑制剂如为式(I)所示的化合物，或其晶型、药学上可接受的盐、水合物或溶剂化合物。本发明的化合物具有更好的丙肝病毒蛋白NS5A抑制活性，具有更好药效学/药代动力学性能，化合物的适用性好、安全性高，可用于制备治疗丙型肝炎病毒感染的药物，具有良好的市场开发前景。

Description

一种丙型肝炎病毒抑制剂、药物组合物及其应用 技术领域
本发明属于医药技术领域，尤其涉及一种丙型肝炎病毒抑制剂、药物组合物及其应用。
背景技术
HCV(Hepatitis C Virus，丙型肝炎病毒)是一种RNA病毒，其属于黄病毒科(Flaviviridae family)中的丙型肝炎病毒属(Hepacivirus genus)。包裹HCV病毒粒子包含正股RNA基因组，其在单个不间断的开放读码框中编码全部已知的病毒—特异的蛋白质。开放读码框包括大约9500个核苷酸并且编码单个约3000个氨基酸的巨大多蛋白。多蛋白包括芯蛋白，包裹蛋白E1和E2，膜结合蛋白P7，和非结构性蛋白NS2、NS3、NS4A、NS4B、NS5A和NS5B。
HCV感染与进行性肝病状(包括肝硬化和肝细胞癌)有关。固定剂量的新抗病毒NS5A抑制剂Ledipasvir(GS5885)与NS5B阻断剂Sofosbuvir二联复方组合的Harvoni是吉利德2013年12月获准的一个重磅丙肝治疗药物。Harvoni是第一个批准用于治疗基因1型丙肝感染，且不需要联合干扰素或利巴韦林的全口服抗丙肝方案。Harvoni既可以单药使用，也可以和其它口服制剂比如利巴韦林联合使用。
美国吉利德原厂生产的Harvoni价格昂贵，仅仅是药品价格就让普通家庭承受不起，因此，仍需要开发对丙型肝炎病毒蛋白NS5A有抑制活性或更好药效学性能的化合物。
发明内容
针对以上技术问题，本发明公开了一种丙型肝炎病毒抑制剂、药物组合物及其应用，其具有更好的丙肝病毒蛋白NS5A抑制活性和/或具有更好药效学/药代动力学性能。
对此，本发明采用的技术方案为：
一种丙型肝炎病毒抑制剂，如式(I)所示的化合物，或其晶型、药学上可接受的盐、水合物或溶剂化合物，
Figure PCTCN2016106874-appb-000001
其中，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50各自独立地为氢、氘、卤素；
附加条件是R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49和R50中至少一个是氘代的或氘。
优选的，式(I)中化合物的R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49和R50，至少其中一个R含氘，更佳地两个R含氘，更佳地三个R含氘，更佳地四个R含氘，更佳地五个R含氘，更佳地六个R含氘，更佳地七个R含氘，更佳地八个R含氘，更佳地九个R含氘，更佳地十个R含氘，更佳地十一个R含氘，更佳地十二个R含氘，更佳地十三个R含氘，更佳地十四个R含氘，更佳地十五个R含氘，更佳地十六个R含氘，更佳地十七个R含氘，更佳地十八个R含氘，更佳地十九个R含氘，更佳地二十个R含氘，更佳地二十一个R含氘，更佳地二十二个R含氘，更佳地二十三个R含氘，更佳地二十四个R含氘，更佳地二十五个R含氘，更佳地二十六个R含氘，更佳地二十七个R含氘，更佳地二十八个R含氘，更佳地二十九个R含氘，更佳地三十个R含氘，更佳地三十一个R含氘，更佳地三十二个R含氘，更佳地三十三个R含氘，更佳地三十四个R含氘，更佳地三十五个R含氘，更佳地三十六个R含氘，更佳地三十七个R含氘，更佳地三十八个R含氘，更佳地三十九个R含氘，更佳地四十个R含氘，更佳地四十一个R含氘，更佳地四十二个R含氘，更佳地四十三个R含氘，更佳地四十四个R含氘，更佳地四十五个R含氘，更佳地四十六个R含氘，更佳地四十七个R含氘，更佳地四十八个R含氘，更佳地四十九个R含氘，更佳地五十个R含氘。
优选的，式(I)中化合物至少含有一个氘原子，其含氘原子的个数可以是1到50中的任意一个。
优选的，氘在氘代位置的氘同位素含量至少是大于天然氘同位素含量(0.015％)，较佳地大于30％，更佳地大于50％，更佳地大于75％，更佳地大于95％，更佳地大于99％。
进一步优选的，在本发明中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49和R50各氘代位置中氘同位素含量至少是5％，较佳地大于10％，更佳地大于15％，更佳地大于20％，更佳地大于25％，更佳地大于30％，更佳地大于35％，更佳地大于40％，更佳地大于45％，更佳地大于50％，更佳地大于55％，更佳地大于60％，更佳地大于65％，更佳地大于70％，更佳地大于75％，更佳地大于80％，更佳地大于85％，更佳地大于90％，更佳地大于95％，更佳地大于99％。
作为本发明的进一步改进，R1、R2、R3、R4、R5和R6各自独立地为氘或氢。
作为本发明的进一步改进，R1、R2、R3、R4、R5、R6是氘。
作为本发明的进一步改进，R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21和R22各自独立地为氘或氢。
作为本发明的进一步改进，R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22是氘。
作为本发明的进一步改进，R7、R8是氘。
作为本发明的进一步改进，R23、R24、R25、R26、R27、R28、R29、R30和R31各自独立地为氘或氢。
作为本发明的进一步改进，R23、R24、R25、R26、R27、R28、R29、R30、R31是氘。
作为本发明的进一步改进，R32、R36、R37、R38、R39和R40各自独立地为氘或氢。
作为本发明的进一步改进，R32、R36、R37、R38、R39、R40是氘。
作为本发明的进一步改进，R41是氘。
作为本发明的进一步改进，R42、R43、R44是氘。
作为本发明的进一步改进，R45、R46、R47、R48、R49、R50是氘。
作为本发明的进一步改进，所述化合物选自下述化合物或其药学上可接受的盐：
Figure PCTCN2016106874-appb-000002
Figure PCTCN2016106874-appb-000003
Figure PCTCN2016106874-appb-000004
本发明还公开了一种药物组合物，其含有药学上可接受的载体和如上所述的丙型肝炎病毒抑制剂，或其晶型、药学上可接受的盐、水合物或溶剂合物。
作为本发明的进一步改进，所述药学上可接受的载体包括助流剂、增甜剂、稀释剂、防腐剂、染料、着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂中的至少一种。
作为本发明的进一步改进，所述药物组合物为片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球或气溶胶。本发明药物组合物可配制成固态、半固态、液态或气态制剂。
给予本发明药物组合物的典型途径包括但不限于口服、直肠、透黏膜、经肠给药，或者局部、经皮、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。优选口服给药或注射给药。
本发明的药物组合物可以采用本领域周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
作为本发明的进一步改进，其还包含活性化合物，所述活性化合物为免疫调节剂或抗病毒药物化合物。
作为本发明的进一步改进，所述免疫调节剂为干扰素类药物化合物。
作为本发明的进一步改进，所述抗病毒药物化合物为利巴韦林、金刚烷胺、NS5A的其他抑制剂、HCV生命周期中的解旋酶、蛋白酶、聚合酶、金属蛋白酶或内部核糖体进入位点靶标的抑制剂，其中，所述NS5A的其他抑制剂为雷迪帕韦或达卡他伟。
本发明公开了一种如上所述的丙型肝炎病毒抑制剂的用途，用于制备治疗丙型肝 炎病毒感染的药物中的用途。
本发明还公开了一种在受试者中治疗和/或预防与丙型肝炎病毒相关的疾病的方法，所述方法包括向所述受试者给药如前文所述的式(I)化合物或其多晶型、药学上可接受的盐、前药、立体异构体、同位素变体、水合物或溶剂化合物，或者上述的药物组合物。
本发明还公开了前文所述的式(I)化合物或其多晶型、药学上可接受的盐、前药、立体异构体、同位素变体、水合物或溶剂化合物，或者前文所述药物组合物，其用于治疗和/或预防与蛋白激酶相关的疾病。
优选的，所述的丙型肝炎病毒HCV包括其多种基因型以及多种基因亚型，优选1a、1b、2a、2b、3a、3b、4a、5a、6a。“
本文中，如无特别说明，“卤素”指F、Cl、Br、和I。更佳地，卤原子选自F、Cl和Br。
本文中，如无特别说明，“氘代”指化合物或基团中的一个或多个氢被氘所取代；氘代可以是一取代、二取代、多取代或全取代。术语“一个或多个氘代的”与“一次或多次氘代”可互换使用。
本文中，如无特别说明，“非氘代的化合物”是指含氘原子比例不高于天然氘同位素含量(0.015％)的化合物。
本发明还包括同位素标记的化合物，等同于原始化合物在此公开。可以列为本发明的化合物同位素的例子包括氢，碳，氮，氧，磷，硫，氟和氯同位素，分别如2H，3H，13C，14C，15N，17O，18O，31P，32P，35S，18F以及36Cl。本发明中的化合物，或对映体，非对映体，异构体，或药学上可接受的盐或溶剂化物，其中含有上述化合物的同位素或其他其他同位素原子都在本发明的范围之内。本发明中某些同位素标记化合物，例如3H和14C的放射性同位素也在其中，在药物和底物的组织分布实验中是有用的。氚，即3H和碳-14，即14C，它们的制备和检测比较容易，是同位素中的首选。同位素标记的化合物可以用一般的方法，通过用易得的同位素标记试剂替换为非同位素的试剂，用示例中的方案可以制备。
药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸；甲酸、乙酸、三氟乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、苯甲酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘磺酸等有机酸；以及脯氨酸、苯丙氨酸、天冬氨酸、谷氨酸等氨基酸。另 一类优选的盐是本发明化合物与碱形成的盐，例如碱金属盐(例如钠盐或钾盐)、碱土金属盐(例如镁盐或钙盐)、铵盐(如低级的烷醇铵盐以及其它药学上可接受的胺盐)，例如甲胺盐、乙胺盐、丙胺盐、二甲基胺盐、三甲基胺盐、二乙基胺盐、三乙基胺盐、叔丁基胺盐、乙二胺盐、羟乙胺盐、二羟乙胺盐、三羟乙胺盐，以及分别由吗啉、哌嗪、赖氨酸形成的胺盐。
术语“溶剂合物”指本发明化合物与溶剂分子配位形成特定比例的配合物。“水合物”是指本发明化合物与水进行配位形成的配合物。
与现有技术相比，本发明的有益效果为：
第一，本发明化合物对丙型肝炎病毒蛋白NS5A具有优异的抑制性。
第二，本发明的技术方案通过氘化改变了化合物在生物体中的代谢，使化合物具有更好的药代动力学参数特性。在这种情况下，可以改变剂量并形成长效制剂，改善适用性。
第三，用氘取代化合物中的氢原子，由于其氘同位素效应，提高了化合物在动物体内的药物浓度，提高了药物疗效。
第四，用氘取代化合物中的氢原子，由于某些代谢产物被抑制，提高了化合物的安全性。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
下面更具体地描述本发明式(I)结构化合物的制备方法，但这些具体方法不对本发明构成任何限制。本发明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便地制得，这样的组合可由本发明所属领域的技术人员容易地进行。
通常，在制备流程中，各反应通常在惰性溶剂中，在室温至回流温度(25℃～100℃，优选25℃～80℃)下进行。反应时间通常为0.1～60小时，较佳地为0.5～24小时。
实施例1
一种丙型肝炎病毒抑制剂，分子式如下：
Figure PCTCN2016106874-appb-000005
Figure PCTCN2016106874-appb-000006
采用以下步骤合成得到：
步骤S1：按照以下合成路线先得到化合物2；
Figure PCTCN2016106874-appb-000007
取4.0g 2-溴芴加入，分散于175mL冰醋酸中，加入20％硫酸16mL，碘酸钾0.7g，单质碘2.24g，于80℃下加热反应24小时。关闭加热，冰浴冷却反应，加冷水165mL稀释反应混合物，抽滤，滤饼用自来水洗至滤液无色。收集所得固体，60℃真空干燥过夜，加3:1(体积比)的二氯甲烷和甲醇混合物40mL，60℃油浴加热下搅拌打浆1小时，自然冷却过夜，抽滤，固体真空60℃真空干燥过夜得化合物2为白色固体(4.37g)。1H NMR(75MHz,DMSO)δ7.98(s,1H),7.87(s,1H),7.80(s,1H),7.75(d,J＝0.8Hz,2H),7.58(dd,J＝8.2,1.8Hz,1H),3.95(s,2H)。
步骤S2：按照以下合成路线先得到化合物4；
Figure PCTCN2016106874-appb-000008
取化合物2 1.96g，并将N-氟代双本磺酰胺6.66g在氮气保护下加入到40mL无水四氢呋喃溶解，冰盐浴冷却30分钟，之后在20分钟内滴加双三甲基硅胺基钾(1M的四氢呋喃溶液)，滴加完毕自然升温反应3小时。冰浴冷却反应液，滴加0.1mL甲醇(试剂级)，加入正己烷40mL稀释反应液。以上混悬液搅拌30分钟后过滤，滤饼用石油醚(50mL x 3)洗涤，收集滤液浓缩，柱层析(石油醚)得化合物3米白色固体(2.57g)。
取化合物3 2.48g在氮气保护下加20mL无水四氢呋喃溶解，冰盐浴冷却搅拌10分钟。量取3.4mL异丙基氯化镁四氢呋喃溶液(2M)于5分钟内滴加到以上溶液中，搅拌反应27分钟，之后滴加N-甲基-N-甲氧基-2-氯乙酰胺的甲苯溶液(0.92g溶于8mL无水甲苯)。滴加完毕继续在冰盐浴冷却下反应15分钟，撤除冰盐浴，自然升温反应4小时，TLC(薄层色谱)显示反应完毕(PE：EA 5/1，RF值0.3)。向反应液中滴加4N盐酸调pH到1-2左右，加甲基叔丁基醚20mL，饱和食盐水20mL，震荡分液。有机相用无水硫酸镁干燥5分钟过滤，浓缩蒸干四氢呋喃与甲基叔丁基醚，所得油状物加正己烷-二氯甲烷(20:1,22mL)于70℃油浴锅中加热搅拌回流30分钟，之后自然冷却至室温，过滤，滤饼用冷正己烷(5mL x 3)洗涤，真空干燥得化合物4为淡黄色固体(1.51g)。1H NMR(75MHz,CDCl3)δ8.19(d,J＝1.1Hz,1H),8.13(d,J＝ 8.0Hz,1H),7.82(d,J＝1.5Hz,1H),7.68(d,J＝8.4Hz,2H),7.53(d,J＝8.1Hz,1H),4.72(s,2H)。
步骤S3：按照以下合成路线先得到化合物7；
Figure PCTCN2016106874-appb-000009
取1.44g化合物4，并加入0.98g脯氨酸衍生物5、碘化钾66mg，加20mL乙腈室温搅拌分散均匀，加入二异丙基乙基胺540mg，室温搅拌反应5小时，点板显示反应完毕。减压蒸干反应液，加20mL乙酸乙酯溶解所得油状物，先后用1N盐酸，5％氢氧化钠水溶液和饱和食盐水洗，有机相用无水硫酸镁-无水硫酸钠干燥，过滤浓缩蒸干得到化合物6的粗品为褐色油状物。加25mL甲苯溶解以上所得6的粗品，加入醋酸铵6.2g，氮气保护下于95℃油浴锅中加热搅拌反应过夜，撤除油浴，冰浴冷却反应，加乙酸乙酯10mL稀释，先后用水(20mL)、5％的碳酸氢钠(20mL)、饱和食盐水洗。分液，有机相用无水硫酸钠-无水硫酸镁干燥，减压蒸干，加15ml乙酸乙酯搅拌回流30分钟，趁回流滴加正己烷15mL，之后搅拌自然降温至室温，过滤，滤饼用乙酸乙酯-正己烷(1:5，5mL x 3)洗，真空干燥过夜得产物7为棕色粉末状固体(1.32g)。1H NMR(75MHz,CDCl3)δ10.61(s,1H),8.01(s,1H),8.01(s,1H),8.01(s,1H),7.89(d,J＝7.7Hz,1H),7.74(d,J＝1.3Hz,1H),7.57(d,J＝8.1Hz,1H),7.50(d,J＝7.6Hz,1H),7.40(d,J＝7.9Hz,1H),7.31(s,1H),5.11(d,J＝6.0Hz,1H),3.51(d,J＝10.3Hz,1H),3.13(d,J＝10.3Hz,1H),2.82(d,J＝12.3Hz,1H),2.35(t,J＝10.5Hz,1H),1.50(s,9H),0.90(s,1H),0.75–0.49(m,3H)。
步骤S4：按照以下合成路线先得到化合物12；
Figure PCTCN2016106874-appb-000010
取化合物8(768mg)，化合物9(890mg)，加15mL N,N-二甲基甲酰胺室温搅拌溶解，先后加入N-甲基吗啡啉(830mg)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU，1.71g)，室温反应。1小时后向反应液中加水150mL，乙酸乙酯萃取(25mL x 3)，食盐水洗有机相，无水硫酸镁-无水硫酸钠干燥减压蒸干溶剂得到10和11的混合物为红褐色油状物。以上油状物加20mL无水乙醇溶解，115℃油浴锅中封管加热反应48小时。蒸干乙醇，柱层析(石油醚-乙酸乙酯，2:1)纯化得化合 物12为黄褐色油状或泡沫状固体(1.59g)。1H NMR(75MHz,CDCl3)δ10.72(s,1H),7.59(dd,J＝10.9,5.1Hz,1H),7.45–7.19(m,2H),4.54(d,J＝2.6Hz,1H),4.17(s,1H),3.45(d,J＝3.4Hz,1H),2.05–1.7(m,6H),1.54(s,9H)。
步骤S5：按照以下合成路线先得到化合物12；
Figure PCTCN2016106874-appb-000011
取化合物12 1.08g，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物114mg，频哪醇联硼酸酯1.53g，醋酸钾700mg，在氮气保护下加入到带冷凝装置的反应瓶中，搅拌下加入煮沸脱氧的二氧六环30mL。以上混悬液氮气保护于95℃油浴锅中搅拌反应3.5小时。蒸干反应液，柱层析(石油醚/乙酸乙酯，10:1，2:1)得产物13为米白色固体(1.16g)。1H NMR(100MHz,CDCl3)δ10.64(m,1H),8.38–7.36(m,3H),4.57(s,1H),4.16(s,1H),3.50(s,1H),1.90(dd,J＝13.8,9.4Hz,1H),1.76–1.57(m,3H),1.53(s,8H),1.37(s,12H),1.25(s,9H)。
步骤S6：按照以下合成路线先得到化合物14；
Figure PCTCN2016106874-appb-000012
取化合物7 247mg，化合物13 220mg，四三苯基膦钯26mg，碳酸氢钠144mg在氮气保护下加入到带冷凝装置的反应瓶中，搅拌下加入煮沸脱氧的乙二醇二甲醚-水(5:1)混合溶剂5mL，93℃加热回流反应4小时。关闭加热，冷却反应液至室温，加乙酸乙酯20mL稀释，先后用饱和碳酸氢钠(20mL)和饱和食盐水(20mL)洗，无水硫酸钠干燥有机相，浓缩柱层析得产物14 301mg,为淡黄色晶体。1H NMR(75MHz,CDCl3)δ10.73(s,1H),7.97(s,1H),7.88(s,2H),7.75–7.41(m,7H),7.30(s,1H),5.21–5.02(m,1H),4.58(s,1H),4.18(s,1H),3.50(d,J＝10.7Hz,2H),3.15(d,J＝10.2Hz,1H),2.79(d,J＝7.0Hz,1H),2.43–2.27(m,1H),1.97–1.58(m,7H),1.54(s,9H),1.50(s,9H),1.27–1.21(m,9H),0.87(d,J＝12.0Hz,2H),0.76–0.43(m,4H)。
步骤S7：按照以下合成路线先得到化合物A-1；
Figure PCTCN2016106874-appb-000013
取化合物14 155mg，加2mL无水二氯甲烷溶解，室温下加入4N氯化氢-二氧六环溶液搅拌反应30分钟。减压蒸干反应溶剂，加入甲基叔丁基醚(2mL x 3)减蒸得到淡黄色固体，加N,N-二甲基甲酰胺2mL溶解，先后加入N-Moc-d3-L-缬氨酸78mg，N,N-二异丙基乙基胺165微升，HATU 183mg室温反应20分钟。将反应液倒入20mL水中，加乙酸乙酯(20mL x 2)萃取，合并有机相先后用饱和食盐水和饱和碳酸氢钠洗涤，无水硫酸钠干燥柱层析得产物A-1 34mg，为淡黄色粉末状固体。1H NMR(300MHz,DMSO)δ12.20(s,1H),11.86(s,1H),8.06(s,1H),8.00–7.74(m,7H),7.69(s,1H),7.63–7.45(m,2H),7.27(dd,J＝28.6,8.3Hz,2H),5.23–5.14(m,1H),4.65(s,1H),4.54(s,1H),4.15(t,J＝8.0Hz,1H),3.98(t,J＝8.2Hz,1H),3.90–3.70(m,2H),2.65(s,1H),2.40(d,J＝9.3Hz,1H),2.26–2.15(m,1H),2.12–1.90(m,5H),1.76(s,2H),1.68–1.40(m,4H),1.19(d,J＝17.0Hz,9H),1.08–1.01(m,1H),0.94(dd,J＝12.2,6.6Hz,7H),0.89–0.75(m,11H),0.64–0.47(m,4H)；LC-MS(APCI):m/z＝895.6[M+1]+。
实施例2
一种丙型肝炎病毒抑制剂，分子式如下：
Figure PCTCN2016106874-appb-000014
采用以下步骤合成得到：
步骤S1：按照以下合成路线先得到化合物15；
Figure PCTCN2016106874-appb-000015
取化合物8 500mg，加入15mL重水分散均匀，加入氘代浓盐酸210微升密封，140℃条件下密封搅拌微波反应2小时关闭微波反应装置，自然降温至室温，加2N氢氧化钠水溶液4mL，二氯甲烷(10mL x 2)萃取，有机相饱和食盐水洗，浓缩，柱层 析(石油醚-乙酸乙酯，2:1)得15为棕褐色固体，236mg。LC-MS(APCI):m/z＝192.2[M+1]+。
步骤S2：按照以下合成路线先得到化合物18；
Figure PCTCN2016106874-appb-000016
取化合物15(188mg)，化合物9(239mg)，加1mL N,N-二甲基甲酰胺室温搅拌溶解，先后加入N-甲基吗啡啉(200mg)和2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU，414mg)，室温反应。6.5小时后向反应液中加水10mL，乙酸乙酯萃取(5mL x 2)，食盐水洗有机相，无水硫酸镁-无水硫酸钠干燥减压蒸干溶剂得到16和17的混合物为红褐色油状物。以上油状物加10mL无水乙醇溶解，130℃油浴锅中封管加热反应过夜。蒸干乙醇，柱层析(石油醚-乙酸乙酯，2:1)纯化得化合物18为黄褐色油状或泡沫状固体(241mg)。1H NMR(75MHz,CDCl3)δ10.69(s,1H),4.53(s,1H),4.15(s,1H),3.45(s,1H),2.10–1.80(m,2H),1.57–1.19(m,13H)。
步骤S3：按照以下合成路线先得到化合物19；
Figure PCTCN2016106874-appb-000017
取化合物18 239mg，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物25mg，频哪醇联硼酸酯338mg，醋酸钾154mg，在氮气保护下加入到带冷凝装置的反应瓶中，搅拌下加入煮沸脱氧的二氧六环5mL。以上混悬液氮气保护于95℃油浴锅中搅拌反应1.5小时。蒸干反应液，柱层析(石油醚/乙酸乙酯，10:1，2:1)得产物19为米白色固体(208mg)。1H NMR(100MHz,CDCl3)δ10.66(s,1H),4.55(s,1H),4.14(s,1H),3.49(s,1H),2.00–1.81(m,3H),1.72(s,2H),1.63(s,9H),1.35(s,9H),1.27(s,3H),1.24(s,6H)。
步骤S4：按照以下合成路线先得到化合物20；
Figure PCTCN2016106874-appb-000018
取化合物7 216mg，化合物13 194mg，四三苯基膦钯23mg，碳酸氢钠117mg在氮气保护下加入到带冷凝装置的反应瓶中，搅拌下加入煮沸脱氧的乙二醇二甲醚-水(5:1)混合溶剂3mL，93摄氏度加热回流反应5小时。关闭加热，冷却反应液至室温，加乙酸乙酯20mL稀释，先后用饱和碳酸氢钠(20mL)和饱和食盐水(20mL)洗，无水硫酸钠干燥有机相，浓缩柱层析得产物20 201mg,为淡黄色晶体。1H NMR(75MHz,CDCl3)δ10.72(s,1H),7.99(s,1H),7.88(s,1H),7.75–7.65(m,1H),7.65–7.51(m,2H),7.46(td,J＝7.2,2.9Hz,1H),7.31(s,1H),5.17–5.07(m,1H),4.59(s,1H),4.18(s,1H),3.50(d,J＝10.7Hz,1H),3.14(d,J＝10.3Hz,1H),2.80(d,J＝11.8Hz,1H),2.35(t,J＝10.6Hz,1H),2.05(d,J＝9.8Hz,2H),1.97–1.83(m,2H),1.75(s,2H),1.69–1.58(m,2H),1.54(s,9H),1.38(s,2H),1.31–1.19(m,9H),0.98–0.78(m,4H),0.75-0.50(m,6H)。
步骤S5：按照以下合成路线先得到化合物A-2；
Figure PCTCN2016106874-appb-000019
取化合物20 155mg，加2mL无水二氯甲烷溶解，室温下加入4N氯化氢-二氧六环溶液搅拌反应30分钟。减压蒸干反应溶剂，加入甲基叔丁基醚(2mL x 3)减蒸得到淡黄色固体，加N,N-二甲基甲酰胺2mL溶解，先后加入N-Moc-L-缬氨酸98mg，N,N-二异丙基乙基胺164mg，HATU 203mg室温反应20分钟。将反应液倒入20mL水中，加乙酸乙酯(20mL x 2)萃取，合并有机相先后用饱和食盐水和饱和碳酸氢钠洗涤，无水硫酸钠干燥柱层析得产物A-2 28mg，为淡黄色粉末状固体。1H NMR(75MHz,DMSO)δ12.21(s,1H),11.88(s,1H),8.08(s,1H),7.97(s,2H),7.89(s,2H),7.82(d,J＝7.8Hz,1H),7.71(s,1H),7.33(d,J＝8.3Hz,1H),7.24(d,J＝8.4Hz,1H),5.25–5.15(m,1H),4.67(s,1H),4.56(s,1H),4.17(t,J＝7.9Hz,1H),4.00(t,J＝8.1Hz,1H),3.83(d,J＝9.7Hz,1H),3.72(d,J＝9.5Hz,1H),3.55(d,J＝1.6Hz,6H),2.67(s,1H),2.51(s,2H),2.42(d,J＝8.2Hz,1H),2.28–2.15(m,1H),2.10–1.86(m,6H),1.52(dd,J＝29.0,10.6Hz,3H),1.04–0.76(m,16H),0.75–0.48(m,4H)；LC-MS(APCI):m/z＝892.3[M+1]+。
实施例3
一种丙型肝炎病毒抑制剂，分子式如下：
Figure PCTCN2016106874-appb-000020
在实施例2的基础上，按照以下路线合成A-3
Figure PCTCN2016106874-appb-000021
取化合物20 143mg，加2mL无水二氯甲烷溶解，室温下加入1mL 4N氯化氢-二氧六环溶液搅拌反应30分钟。减压蒸干反应溶剂，加入甲基叔丁基醚(2mL x 3)减蒸得到淡黄色固体，加N,N-二甲基甲酰胺5mL溶解，先后加入N-Moc-d3-L-缬氨酸72mg，N,N-二异丙基乙基胺119mg，HATU 154mg室温反应20分钟。将反应液倒入20mL水中，加EA(20mL x 2)萃取，合并有机相先后用饱和食盐水和饱和碳酸氢钠洗涤，无水硫酸钠干燥柱层析得产物A-3 100mg，为淡黄色粉末状固体。1H NMR(75MHz,DMSO)δ12.19(s,1H),11.86(s,1H),8.07(s,1H),7.97(s,1H),7.89(s,2H),7.82(d,J＝8.3Hz,1H),7.69(s,1H),7.38–7.26(m,1H),7.22(d,J＝8.5Hz,1H),5.25–5.14(m,1H),4.67(s,1H),4.55(s,1H),4.16(t,J＝7.9Hz,1H),4.02–3.94(m,1H),3.86–3.77(m,1H),3.72(d,J＝9.8Hz,1H),2.67(s,1H),2.41(d,J＝8.9Hz,1H),2.30–2.15(m,1H),2.10–1.86(m,5H),1.56(d,J＝11.8Hz,1H),1.47(d,J＝8.4Hz,1H),1.25(d,J＝6.9Hz,2H),1.04–0.75(m,16H),0.70(d,J＝5.4Hz,1H),0.58(dd,J＝12.7,6.6Hz,3H)；LC-MS(APCI):m/z＝898.4[M+1]+。
实施例4
一种丙型肝炎病毒抑制剂，分子式如下：
Figure PCTCN2016106874-appb-000022
在实施例2的基础上，按照以下路线合成A-4：
Figure PCTCN2016106874-appb-000023
取化合物20 172mg，加2mL无水二氯甲烷溶解，室温下加入1mL 4N氯化氢-二氧六环溶液搅拌反应30分钟。减压蒸干反应溶剂，加入甲基叔丁基醚减压蒸馏得到淡黄色固体，加N,N-二甲基甲酰胺5mL溶解，先后加入N-Moc-L-缬氨酸-d8 89mg，N,N-二异丙基乙基胺143mg，HATU 189mg室温反应20分钟。将反应液倒入20mL水中，加入乙酸乙酯萃取，合并有机相先后用饱和食盐水、饱和碳酸氢钠洗涤，无水硫酸钠干燥浓缩柱层析得产物A-4 120mg，为淡黄色粉末状固体。1H NMR(75MHz,DMSO)δ12.75–12.00(br,2H),8.08(s,1H),7.96(d,J＝8.8Hz,2H),7.88(d,J＝11.1Hz,3H),7.72(s,1H),7.32(s,1H),7.22(s,1H),5.19(d,J＝7.3Hz,1H),4.67(s,1H),4.55(s,1H),4.03(dd,J＝14.3,7.1Hz,1H),3.77(dd,J＝25.9,9.3Hz,2H),3.34(s,6H),2.67(s,1H),2.42(s,1H),2.21(t,J＝9.9Hz,1H),2.08(s,1H),1.93(s,1H),1.63–1.42(m,3H),1.19(dd,J＝16.6,9.5Hz,4H),0.83(s,1H),0.71(s,2H),0.60(d,J＝8.7Hz,3H)；LC-MS(APCI):m/z＝908.5[M+1]+。
实施例5
对以上实施例的化合物进行生物活性评价。
为了验证本文所述的化合物对HCV的作用，发明人采用HCV复制子系统(HCV
Replicon System)作为评价模型。自Science1999年首次报道以来，HCV复制子系统已经成为研究HCV RNA复制、致病性和病毒持续性的最重要的工具之一，例如已经利用复制子成功地证明了HCV RNA复制所必须的5'-NCR最小区域，并且HCV复制子系统已经成功地被用作抗病毒药物的评价模型。本发明的发明人按照Science，1999，285(5424)，110-3，以及J.Virol，2003，77(5)，3007-19所描述的方法进行验证。
(1)检测化合物抗HCV 1a和1b基因型复制子活性
应用HCV-1a和HCV-1b稳定转染复制子细胞检测化合物丙型肝炎病毒基因型1a和1b复制子的抑制活性。本实验将以NS5A抑制剂BMS-790052作为阳性对照化合物。
步骤一：对化合物进行1:3系列稀释8个浓度点，双复孔，加入96孔板中。设置DMSO为无加化合物对照。细胞培养液中的DMSO最终浓度为0.5％。
步骤二：将HCV-1a和1b细胞分别悬浮在含10％FBS的培养液中，以每孔8,000 个细胞的密度种到含有化合物的96孔板中。细胞在5％CO2、37℃条件下培养3天。
步骤三：用CellTiter-Fluor(Promega)测定化合物对GT1b复制子细胞毒性。
步骤四：用Bright-Glo(Promega)检测荧光素酶测定化合物抗丙型肝炎病毒活性。
步骤五：采用GraphPad Prism软件分析数据，拟合曲线并计算EC50值和CC50值。
对实施例1～实施例4的化合物按照如上步骤进行分析计算EC50值和CC50值，结果如表1所示。
(2)测定化合物抗感染性病型肝炎病毒(GT2a)活性
该感染模型参考已报道文献(A novel luciferase and GFP dual reporter virus for rapid and convenient evaluation of HCV replication.Virus Res 2011.155:406)。用HCVcc(MOI＝0.2)感染Huh-7.5.1细胞后，化合物处理3天(8个浓度，3倍稀释,双复孔)。抗病毒活性将以荧光素酶法测定。同时测定化合物对Huh7.5.1细胞活性。本实验将以NS5A抑制剂BMS-790052作为阳性对照化合物。采用GraphPad软件计算EC50。
对实施例1-实施例4的化合物按照上述步骤进行分析计算EC50，结果如表1所示。
表1 实施例1-4的生物活性评价分析表
Figure PCTCN2016106874-appb-000024
如表1所示，证明本发明的化合物可抑制HCV的多个基因型，并通过抑制HCV NS5A蛋白的机制，发挥了优越的抗丙肝病毒作用。
(3)代谢稳定性评价
微粒体实验：人肝微粒体：0.5mg/mL，Xenotech；大鼠肝微粒体：0.5mg/mL，Xenotech；辅酶(NADPH/NADH)：1mM，Sigma Life Science；氯化镁：5mM，100mM磷酸盐缓冲剂(pH为7.4)。
储备液的配制：精密称取一定量的化合物实施例1-4的粉末，并用DMSO分别溶解至5mM。
磷酸盐缓冲液(100mM，pH7.4)的配制：取预先配好的0.5M磷酸二氢钾150mL和700mL的0.5M磷酸氢二钾溶液混合，再用0.5M磷酸氢二钾溶液调节混合液pH值 至7.4，使用前用超纯水稀释5倍，加入氯化镁，得到磷酸盐缓冲液(100mM)，其中含100mM磷酸钾，3.3mM氯化镁，pH为7.4。
配制NADPH再生系统溶液(含有6.5mM NADP，16.5mM G-6-P，3U/mL G-6-P D，3.3mM氯化镁)，使用前置于湿冰上。
配制终止液：含有50ng/mL盐酸普萘洛尔和200ng/mL甲苯磺丁脲(内标)的乙腈溶液。取25057.5μL磷酸盐缓冲液(pH7.4)至50mL离心管中，分别加入812.5μL人肝微粒体，混匀，得到蛋白浓度为0.625mg/mL的肝微粒体稀释液。取25057.5μL磷酸盐缓冲液(pH7.4)至50mL离心管中，分别加入812.5μL SD大鼠肝微粒体，混匀，得到蛋白浓度为0.625mg/mL的肝微粒体稀释液。
样品的孵育：用含70％乙腈的水溶液将相应化合物的储备液分别稀释至0.25mM，作为工作液，备用。分别取398μL的人肝微粒体或者大鼠肝微粒体稀释液加入96孔孵育板中(N＝2)，分别加入2μL 0.25mM的的工作液中，混匀。
代谢稳定性的测定：在96孔深孔板的每孔中加入300μL预冷的终止液，并置于冰上，作为终止板。将96孔孵育板和NADPH再生系统置于37℃水浴箱中，100转/分钟震荡，预孵5min。从孵育板每孔取出80μL孵育液加入终止板，混匀，补充20μL NADPH再生系统溶液，作为0min样品。再向孵育板每孔加入80μL的NADPH再生系统溶液，启动反应，开始计时。相应化合物的反应浓度为1μM，蛋白浓度为0.5mg/mL。分别于反应10、30、90min时，各取100μL反应液，加入终止板中，涡旋3min终止反应。将终止板于5000×g，4℃条件下离心10min。取100μL上清液至预先加入100μL蒸馏水的96孔板中，混匀，采用LC-MS/MS进行样品分析。
数据分析：通过LC-MS/MS系统检测相应化合物及内标的峰面积，计算化合物与内标峰面积比值。通过化合物剩余量的百分率的自然对数与时间作图测得斜率，并根据公式计算t1/2和CLint，其中V/M即等于1/蛋白浓度。
Figure PCTCN2016106874-appb-000025
对实施例1～实施例3的化合物按照上述步骤进行分析，结果如表2所示。
表2 实施例1～实施例4的化合物代谢稳定性的测定结果表
Figure PCTCN2016106874-appb-000026
Figure PCTCN2016106874-appb-000027
如表2所示，本发明化合物在人肝微粒体与大鼠肝微粒体实验中都表现出优异的代谢稳定性。
(4)大鼠药代动力学实验
实验目的：研究大鼠给予GS5885、化合物A-1后，考察本发明化合物的药代动力学行为。
实验动物：
种类及品系：SD大鼠等级：SPF级
性别及数量：雄性，6只
体重范围：180～220g(实际体重范围为187～197g)
来源：上海西普尔必凯实验动物有限公司
实验及动物合格证号：SCXK(沪)2013-0016
实验过程：
在血样采集之前，预先在EDTA-K2抗凝管中加入20L的2M氟化钠溶液(酯酶抑制剂)，于80度烘箱内烘干后，置于4度冰箱存放。
大鼠，雄性，体重187～197g，随机分为2组，于实验前一天下午开始禁食过夜但可自由饮水，给药后4h给食物。A组给予GS5885 3mg/kg，B组给予式A-1化合物3mg/kg，分别于给药后15min、30min、1、2、3、5、8、10h从大鼠眼眶静脉取血100-200L左右，置于经EDTA-K2抗凝的0.5mL的Eppendorf管中，立即混匀，抗凝后，尽快将试管轻轻颠倒混匀5-6次后,血取好后放置在冰盒中，30min内把血样本在4000rpm，10min，4℃条件下离心分离血浆，收集全部血浆后立即于-20℃保存。所有时间点样品采集后测定每个时间点的血浆中的血药浓度。
根据上述所得的给药后平均血药浓度-时间数据，采用Winnonin软件，按非房室统计矩理论求算雄性SD大鼠分别i.g给予GS5885(3mg/kg)、式A-1化合物(3mg/kg)后的药代动力学相关参数，详见表3。
表3 大鼠给予GS5885及A-1化合物后药代参数
PK参数 GS5885 A-1
Tmax(hr) 4.00±0.00 6.00±3.46
Cmax(ng/mL) 252.3±41.7 268.2±83.0
t1/2(hr) 3.17±0.46 6.41±3.38
MRT(hr) 7.74±0.09 9.17±2.25
如表3所示，与GS5885相比，本发明人发现化合物A-1具有与GS5885更优的活性，并且具有优异的药代动力学性质，因此更适合作为抑制丙型肝炎病毒蛋白NS5A的化合物，进而适合制备治疗丙型肝炎病毒感染的药物。
应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围，实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则份数和百分比为重量份和重量百分比；以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (11)

  1. 一种丙型肝炎病毒抑制剂，其特征在于：如式(I)所示的化合物，或其晶型、药学上可接受的盐、水合物或溶剂化合物，
    Figure PCTCN2016106874-appb-100001
    其中，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50各自独立地为氢、氘、卤素；
    附加条件是R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49和R50中至少一个是氘代的或氘。
  2. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：R1、R2、R3、R4、R5和R6各自独立地为氘或氢。
  3. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21和R22各自独立地为氘或氢。
  4. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：R42、R43、R44是氘。
  5. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：R45、R46、R47、R48、R49、R50是氘。
  6. 根据权利要求1所述的丙型肝炎病毒抑制剂，其特征在于：所述化合物选自下述化合物或其药学上可接受的盐：
    Figure PCTCN2016106874-appb-100002
    Figure PCTCN2016106874-appb-100003
    Figure PCTCN2016106874-appb-100004
  7. 一种药物组合物，其特征在于：其含有药学上可接受的载体和如权利要求1～9任意一项所述的丙型肝炎病毒抑制剂，或其晶型、药学上可接受的盐、水合物或溶剂合物。
  8. 根据权利要求10所述的药物组合物，其特征在于：其还包含活性化合物，所述活性化合物为免疫调节剂或抗病毒药物化合物。
  9. 一种如权利要求1～9任意一项所述的丙型肝炎病毒抑制剂的用途，其特征在于：用于制备治疗丙型肝炎病毒感染的药物中的用途。
  10. 一种在受试者中治疗和/或预防与丙型肝炎病毒相关的疾病的方法，所述方法包括向所述受试者给药如权利要求1～6任意一项所述的式(I)化合物或其多晶型、药学上可接受的盐、前药、立体异构体、同位素变体、水合物或溶剂化合物，或者权利要求7或8中任一项的药物组合物。
  11. 根据权利要求1～6任意一项所述的式(I)化合物或其多晶型、药学上可接受的盐、前药、立体异构体、同位素变体、水合物或溶剂化合物，或者权利要求7或8中任一项的药物组合物，其用于治疗和/或预防与蛋白激酶相关的疾病。
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