WO2018006811A1 - 一种取代的咪唑基化合物及其药物组合物 - Google Patents

一种取代的咪唑基化合物及其药物组合物 Download PDF

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WO2018006811A1
WO2018006811A1 PCT/CN2017/091788 CN2017091788W WO2018006811A1 WO 2018006811 A1 WO2018006811 A1 WO 2018006811A1 CN 2017091788 W CN2017091788 W CN 2017091788W WO 2018006811 A1 WO2018006811 A1 WO 2018006811A1
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compound
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PCT/CN2017/091788
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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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Priority to CN201780004820.XA priority Critical patent/CN108368123B/zh
Publication of WO2018006811A1 publication Critical patent/WO2018006811A1/zh

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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
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered

Definitions

  • the invention belongs to the technical field of medicine, and in particular relates to a substituted imidazolyl compound and a pharmaceutical composition thereof, and the use thereof for preparing a medicament for treating hepatitis C.
  • 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, in China, it is still necessary to develop inhibitory activity or better pharmacodynamics for the hepatitis C virus protein NS5A. Compound.
  • 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 are each independently Is hydrogen, helium, halogen;
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 are each independently a methyl or ethyl group which is deuterated one or more times;
  • Additional conditions are 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 And 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 , X 1 , at least one of X 2 , X 3 , X 4 , X 5 and X 6 is deuterated or contains hydrazine.
  • 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 and R 5 are each independently hydrazine or hydrogen.
  • R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 are each independently hydrazine or hydrogen.
  • R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 and R 24 are each independently hydrazine or hydrogen.
  • R 25 , R 26 , R 27 , R 28 , R 29 and R 30 are each independently hydrazine or hydrogen.
  • R 31 and R 32 are each independently hydrazine or hydrogen.
  • X 1 is a methyl group which is deuterated one or more times.
  • X 4 and X 5 are each independently a methyl group which is deuterated one or more times.
  • R 31 and R 32 are deuterium, and X 4 and X 5 are methyl groups which are deuterated three times.
  • X 1 is a tertiary deuterated methyl group
  • 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 comprises a glidant, a sweetener, a diluent, and an anti-oxidant At least one of a humic agent, a dye, a colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersing agent, a disintegrating agent, 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 present invention discloses the use of a hepatitis C virus inhibitor as described above for the preparation of a medicament for the treatment of hepatitis C virus infection.
  • 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.
  • salts are the compounds and bases of the invention a salt formed, such as an alkali metal salt (such as a sodium or potassium salt), an alkaline earth metal salt (such as a magnesium salt or a calcium salt), an ammonium salt (such as a lower alkanolammonium salt and other pharmaceutically acceptable amine salts),
  • an alkali metal salt such as a sodium or potassium salt
  • an alkaline earth metal salt such as a magnesium salt or a calcium salt
  • an ammonium salt such as a lower alkanolammonium salt and other pharmaceutically acceptable amine salts
  • 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.
  • reaction system was cooled to room temperature, and then DMC was evaporated to dryness under reduced pressure. 5 g of celite was added, and ethyl acetate (100 mL) was added thereto, and 50 mL of water was added thereto, and the mixture was stirred at room temperature for 2 hours, and the mixture was separated by filtration.
  • the intermediate 5 obtained above was dissolved in a mixed solvent of THF-DMSO-water (10 mL + 10 mL + 5 mL), and N-bromosuccinimide (NBS, 2.4 g, 13.76 mmol) was added at room temperature and stirred for 1 hour, TLC The reaction is shown to be complete.
  • the THF in the reaction system was evaporated under reduced pressure, and then diluted with water (100 mL), and ethyl acetate was evaporated, and the organic phase was combined and washed with brine, then dried over anhydrous magnesium sulfate.
  • the intermediate 8 (1.3 g, 2.36 mmol) was dissolved in a mixture of DCM-MeOH (17 mL, 5 mL). The reaction mixture was washed with EtOAc (EtOAc m.
  • N-Moc-L-valine-d 8 (0.35 g, 1.91 mmol) was dissolved in anhydrous DMF (5 mL), cooled in ice, then diisopropylethylamine (DIPEA, 2.77g, 21.45mmol), 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 0.91g, 2.39mmol)
  • DIPEA diisopropylethylamine
  • HATU 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • the above oil was dissolved in 10 mL of MeOH, cooled in an ice bath, and 5 mL of a 1 M aqueous lithium hydroxide solution was added dropwise thereto, and the reaction was allowed to warm for 3 hours after the dropwise addition, and the intermediate disappeared by TLC.
  • the MeOH in the reaction mixture was evaporated under reduced pressure.
  • the aqueous phase was extracted with DCM, and then 4N hydrochloric acid was added dropwise to the aqueous phase to adjust the pH to about 2, and the mixture was extracted with DCM.
  • Body 13 was a colorless foamy solid (0.46 g).
  • the diester intermediate (358 mg, 0.42 mmol) was added with 10 mL of toluene and 1 mL of ethylene glycol methyl ether. The mixture was stirred at room temperature, and 1.64 g (21.26 mmol) of ammonium acetate was added in one portion, followed by heating under reflux in a 110 ° C constant temperature oil bath. After 4 hours, TLC showed the disappearance of the intermediate. The mixture was heated and cooled to room temperature. To the reaction was added ethyl acetate (10 mL), 2M aqueous sodium hydroxide, and the organic layer was dried over anhydrous sodium sulfate.
  • the gray solid intermediate (109 mg, 0.17 mmol) was dissolved in 3 mL of anhydrous DCM, and the mixture was stirred for 2 hours at room temperature by adding 4N hydrogen chloride-dioxane solution until the starting material disappeared.
  • the DCM, hydrogen chloride and dioxane in the reaction system were evaporated to dryness under reduced pressure to give the title compound as a brown solid, EtOAc (3 EtOAc, EtOAc)
  • EtOAc (3 EtOAc, EtOAc)
  • N-Moc-L-phenylglycine 34 mg, 0.16 mmol
  • HATU 62 mg, 0.16 mmol
  • the diester intermediate (145 mg, 0.17 mmol) was added with 5 mL of toluene and 0.5 mL of ethylene glycol methyl ether. The mixture was stirred at room temperature, and ammonium acetate (267 mg, 3.46 mmol) was added in one portion, followed by heating under reflux in a 110 ° C constant temperature oil bath. After 7 hours of reaction, TLC showed the starting material was completely reacted. The mixture was heated to dryness, and the mixture was cooled to room temperature. EtOAc (EtOAc)EtOAc. The crude intermediate was dissolved in 5 mL of dichloromethane, and then stirred, and then stirred and stirred at room temperature overnight ( 839 mg, 8.5 mmol). The above reaction system was filtered, and the residue was washed with methanol until the filtrate was colorless. The combined filtrate was evaporated to dryness.
  • the gray solid intermediate (42 mg, 0.053 mmol) was dissolved in 3 mL of anhydrous dichloromethane, and the mixture was stirred at room temperature for 4 hours with hydrogen chloride-dioxane solution until the starting material disappeared.
  • Dichloromethane, hydrogen chloride and dioxane in the reaction system were evaporated under reduced pressure to give a brown solid intermediate, which was then evaporated to dryness with EtOAc (3 EtOAc)
  • EtOAc 3 EtOAc
  • the above solution was cooled in an ice bath, and N-Moc(d 3 )-L-phenylglycine (13 mg, 0.063 mmol) and HATU (24 mg, 0.063 mmol) were successively added, and the reaction was stirred for 30 minutes.
  • N-Moc-L-valine (0.47 g, 2.69 mmol) was dissolved in anhydrous DMF (5 mL), and cooled in ice-cooling, then diisopropylethylamine (2.90 g, 22.4 (mmol), HATU (1.02 g, 2.69 mmol), stirred at ambient temperature for 2 h.
  • the reaction mixture was poured into 50 mL of water, ethyl acetate was evaporated and evaporated.
  • the diester compound 21 (2.08 g, 7.22 mmol) was added to 15 mL of tetrahydrofuran to dissolve, and the mixture was cooled in an ice bath, and 7.2 mL of a 1 M aqueous sodium hydroxide solution was slowly added dropwise, and the reaction was allowed to warm overnight.
  • the monocarboxylic acid 22 obtained above was dissolved in 10 mL of anhydrous tetrahydrofuran, and then cooled in ice-cooled, and then triethylamine (0.95 g, 9.38 mmol) and ethyl chloroformate (1.02 g, 9.38 mmol) were successively stirred and reacted for 2 hours.
  • reaction system is filtered out insoluble solids, the filtrate was evaporated to dryness under reduced pressure to give a pale oil, was added 15mL of tetrahydrofuran was cooled to -5 ° C, take NaBH 4 (0.47g, 12.50mmol) was dissolved in deionized water, 5mL, was slowly dropwise added to the above The reaction was stirred at -5 ° C for 2 hours in a light oily tetrahydrofuran solution. To the reaction, 4N hydrochloric acid was added dropwise to the mixture to adjust to pH 2, and the organic layer was dried over anhydrous sodium hydrogen sulfate.
  • the crude product of 24 was dissolved in 10 mL of tetrahydrofuran and 5 mL of methanol, and cooled in an ice bath. Then, 5 mL of a 2.5 M aqueous lithium hydroxide solution was added dropwise thereto, and the mixture was stirred for 4 hours. Methanol and tetrahydrofuran in the system were evaporated under reduced pressure, and the residue was diluted with water (10 mL). The obtained aqueous solution was washed with dichloromethane, and then the mixture was evaporated.
  • the intermediate 36 obtained above was dissolved in a mixed solvent of THF-DMSO-water (6 mL + 6 mL + 3 mL), and N-bromosuccinimide (NBS, 1.03 g, 5.79 mmol) was added at room temperature, and the reaction was stirred for 1 hour. After completion, the THF in the reaction system was evaporated under reduced pressure, and then diluted with water (60 mL), and ethyl acetate was evaporated, and the organic phase was combined and washed with saturated brine, and then dried over anhydrous magnesium sulfate.
  • N-bromosuccinimide N-bromosuccinimide
  • the diester intermediate 39 (374 mg, 0.45 mmol) was added with 10 mL of toluene and 1 mL of ethylene glycol methyl ether, and the mixture was stirred at room temperature, and ammonium acetate (1.72 g, 22.37 mmol) was added in one portion, followed by heating in a constant temperature oil bath at 110 ° C. The reaction was refluxed for 4 hours, and the mixture was evaporated to dryness. EtOAc was evaporated. The crude product was dissolved in 10 mL of dichloromethane, and the active manganese dioxide (1.94 mg, 22.37 mmol) was added in one portion and stirred at room temperature overnight.
  • the diester intermediate 43 (285 mg, 0.34 mmol) was added with 10 mL of toluene and 1 mL of ethylene glycol methyl ether. The mixture was stirred at room temperature, and ammonium acetate (1.30 g, 16.90 mmol) was added in one portion, and the reaction was heated in a constant temperature oil bath at 100 ° C. After overnight, after refluxing at 110 ° C for 1 hour, the heating was turned off, and the temperature was naturally cooled to room temperature. To the reaction was added ethyl acetate (10 mL), 2M aqueous sodium hydroxide solution, and the organic layer was dried over anhydrous sodium sulfate The crude product.
  • the inventors used the HCV Replicon System as an evaluation model. Since its first report in Science in 1999, the HCV replication subsystem has become one of the most important tools for studying HCV RNA replication, pathogenicity and viral persistence, such as the use of replicons. The 5'-NCR minimal region necessary for HCV RNA replication has been successfully demonstrated, 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 GS-5816 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.
  • 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 stock solution A certain amount of the sample of the test example compound and the control compound GS-5816 was accurately weighed and dissolved to 5 mM with DMSO, respectively.
  • phosphate buffer 100 mM, pH 7.4.
  • the pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and 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 metabolic stability of human and rat liver microsomes was evaluated by simultaneously testing the compounds of the present invention and their compounds without deuteration.
  • the half-life and liver intrinsic clearance as indicators of metabolic stability are shown in Table 2.
  • the undeuterated compound GS-5816 was used as a control sample in Table 2.
  • the compound of the present invention can significantly improve metabolic stability by comparison with the compound GS-5816 which has not been deuterated, and is thus more suitable as a hepatitis C virus inhibitor.
  • EXPERIMENTAL OBJECTIVE To investigate the pharmacokinetic behavior of the compounds of the present invention after administration of GS-5816 to the compounds of the examples.
  • SD rat grade SPF grade
  • Weight range 180 ⁇ 220g (actual weight range is 187 ⁇ 197g)
  • the experimental results show that the present inventors have found that the compound of the present invention has superior activity and excellent pharmacokinetic properties as compared with GS-5816, and thus is more suitable as a compound for inhibiting the hepatitis C virus protein NS5A, and is suitable for Preparation of a medicament for treating hepatitis C virus infection.

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Abstract

一种取代的咪唑基化合物及其药物组合物，所述化合物如为式（I）所示，或其晶型、药学上可接受的盐、水合物或溶剂化合物。上述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。

Description

一种取代的咪唑基化合物及其药物组合物 技术领域
本发明属于医药技术领域，尤其涉及一种取代的咪唑基化合物及其药物组合物，及其在制备治疗丙型肝炎药物上的用途。
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，目前在我国，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 PCTCN2017091788-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各自独立地为氢、氘、卤素；
X1、X2、X3、X4、X5、X6各自独立地为一次或多次氘代的甲基、乙基；
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、X1、X2、X3、X4、X5和X6中至少一个是氘代的或含氘。
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、X1、X2、X3、X4、X5和X6，至少其中一个R/X含氘，更佳地两个R/X含氘，更佳地三个R/X含氘，更佳地四个R/X含氘，更佳地五个R/X含氘，更佳地六个R/X含氘，更佳地七个R/X含氘，更佳地八个R/X含氘，更佳地九个R/X含氘，更佳地十个R/X含氘，更佳地十一个R/X含氘，更佳地十二个R/X含氘，更佳地十三个R/X含氘，更佳地十四个R/X含氘，更佳地十五个R/X含氘，更佳地十六个R/X含氘，更佳地十七个R/X含氘，更佳地十八个R/X含氘，更佳地十九个R/X含氘，更佳地二十个R/X含氘，更佳地二十一个R/X含氘，更佳地二十二个R/X含氘，更佳地二十三个R/X含氘，更佳地二十四个R/X含氘，更佳地二十五个R/X含氘，更佳地二十六个R/X含氘，更佳地二十七个R/X含氘，更佳地二十八个R/X含氘，更佳地二十九个R/X含氘，更佳地三十个R/X含氘，更佳地三十一个R/X含氘，更佳地三十二个R/X含氘，更佳地三十三个R/X含氘，更佳地三十四个R/X含氘，更佳地三十五个R/X含氘，更佳地三十六个R/X含氘，更佳地三十七个R/X含氘，更佳地三十八个R/X含氘。
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％。
更具体地，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、X1、X2、X3、X4、X5和X6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和R5各自独立地为氘或氢。
作为本发明的进一步改进，R6、R7、R8、R9、R10、R11、R12、R13和R14各自独立地为氘或氢。
作为本发明的进一步改进，R15、R16、R17、R18、R19、R20、R21、R22、R23和R24各自独立地为氘或氢。
作为本发明的进一步改进，R25、R26、R27、R28、R29、R30各自独立地为氘或氢。
作为本发明的进一步改进，R31、R32各自独立地为氘或氢。
作为本发明的进一步改进，X1是一次或多次氘代的甲基。
作为本发明的进一步改进，X4和X5各自独立地为一次或多次氘代的甲基。
更优选地，R31、R32是氘，并且X4和X5是三次氘代的甲基。
更优选地，X1是三次氘代的甲基
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 PCTCN2017091788-appb-000002
Figure PCTCN2017091788-appb-000003
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。
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
制备一种丙型肝炎病毒抑制剂V-1，分子式如下：
Figure PCTCN2017091788-appb-000004
采用以下步骤合成得到：
Figure PCTCN2017091788-appb-000005
步骤1.合成化合物3。
取化合物1(7.11g,25mmol)，化合物2(4.26g,26.3mmol)，加入20mL二甲基甲酰胺(DMF)室温搅拌10分钟分散均匀，之后加入碳酸钾(3.8g,27.5mmol)，于50℃油浴中加热搅拌反应2小时，TLC显示反应完毕。反应混合物倒入200mL水中，乙酸乙酯萃取，合并有机相，2N NaOH水溶液洗，之后无水硫酸钠干燥，过滤蒸干得中间体3为乳白色固体(8.83g，收率97％)。1H NMR(300MHz,CDCl3)δ7.70-7.40(m,3H),7.35-7.05(m,3H),5.10(s,2H),2.92(s,2H),2.65(s,2H),2.13(s,2H)。
步骤2.合成化合物4。
取化合物3(8.83g,24mmol)，特戊酸钯(370mg,1.2mmol)，三(4-氟-苯基)膦(379mg,1.2mmol)，特戊酸(735mg,7.2mmol)和碳酸钾(6.6g,48mmol)在氮气保护下加入到500mL单口反应瓶中，加入通氮气脱氧的二甲基乙酰胺(DMAC，120mL)。以上反应体系搅拌下氮气置换三次，之后于65℃油浴加热搅拌反应3.5小时，TLC显示反应完毕。关闭加热，反应体系冷却至室温，之后减压蒸干DMAC，加入硅藻土5g，加入乙酸乙酯100mL，加水50mL，室温搅拌2小时，过滤分液。有机相蒸干，柱层析得到化合物4为黄色颗粒状固体(3.72g，收率54％)。1H NMR(300MHz,DMSO-d6)δ7.96(d,J＝8.4Hz,1H),7.90(s,1H),7.51(dd,J＝8.3,2.2Hz,1H),7.45(d,J＝2.1Hz,1H),7.35(s,1H),5.14(s,2H),2.95(t,J＝5.9Hz,2H),2.69–2.53(m,2H),2.05(dd,J＝12.2,6.3Hz,2H)。
步骤3.合成化合物5。
以上所得化合物4(3.72g，13.07mmol)，醋酸钯(147mg,0.65mmol)，2-双环己基膦-2',6'-二甲氧基联苯(Sphos,538mg,1.31mmol)，乙烯基三氟硼酸钾(5.25g,39.21mmol) 与碳酸钾(5.42g,39.21mmol)在氮气保护下加入到250mL单口反应瓶中，加入130mL通氮气脱氧的正丙醇。以上反应体系氮气置换三次，在120℃油浴中加热回流反应12小时，LC-MS监控显示原料消失，关闭加热，并于冷却反应至室温，减压蒸干正丙醇。向以上所得糊状物中加入50mL乙酸乙酯，室温搅拌30分钟，过滤，滤液减压蒸干柱层析得到化合物5为黄色颗粒状固体。1H NMR(400MHz,CDCl3)δ7.70(d,J＝8.1Hz,1H),7.62(s,1H),7.58(s,1H),7.43(d,J＝8.2Hz,1H),7.22(s,1H),6.73(dd,J＝17.6,10.9Hz,1H),5.81(d,J＝17.6Hz,1H),5.32(d,J＝11.3Hz,1H),5.12(s,2H),2.97(t,J＝6.0Hz,2H),2.74–2.57(m,2H),2.14(dd,J＝12.6,6.3Hz,2H)。
步骤4.合成化合物6。
以上所得中间体5加THF-DMSO-水的混合溶剂(10mL+10mL+5mL)溶解，室温下加入N-溴代丁二酰亚胺(NBS,2.4g，13.76mmol)搅拌反应1小时，TLC显示反应完毕。减压蒸去反应体系中的THF，之后加水100mL稀释，乙酸乙酯萃取，合并有机相，饱和食盐水洗，之后用无水硫酸镁干燥，过滤蒸干溶剂得中间体。以上中间体的粗品加20mL DCM溶解，加入活性二氧化锰(24g,275.2mmol)室温搅拌反应过夜，TLC显示反应完毕。过滤去除不溶性固体，滤液加5mL甲醇，减压蒸去DCM至大量橙黄色固体析出，停止减压蒸馏，过滤收集固体。母液加DCM 20mL，之后减压蒸馏至橙黄色固体析出，停止减压蒸馏，过滤收集固体。重复以上操作一次，合并三次所得固体即为中间体6(1.72g,收率35％)。1H NMR(300MHz,CDCl3)δ8.01(d,J＝8.2Hz,1H),7.91–7.77(m,2H),7.66(s,2H),5.18(s,2H),4.45(s,2H),2.99(t,J＝6.0Hz,2H),2.73–2.57(m,2H),2.28–2.07(m,2H)。
步骤5.合成化合物8。
取中间体6(1.41g,3.8mmol)，氨基酸7(1.09g,4.2mmol)加乙腈(8mL)搅拌分散均匀，加入二异丙基乙胺(517mg,4.0mmol)室温搅拌反应过夜，TLC显示反应完毕。减压蒸干反应液，柱层析得中间体8为灰色粉末状固体(1.3g,收率62％)。1H NMR(500MHz,CDCl3)δ7.92(t,J＝7.6Hz,1H),7.83(d,J＝8.2Hz,1H),7.73(s,1H),7.65(d,J＝3.3Hz,2H),5.68–5.03(m,4H),4.50–4.37(m,1H),3.84–3.60(m,1H),3.53–3.30(m,5H),3.24(dd,J＝10.8,7.8Hz,1H),2.99(t,J＝6.0Hz,2H),2.73–2.63(m,2H),2.56(dd,J＝19.8,7.2Hz,2H),2.19–2.12(m,2H),1.45(s,9H)。
步骤6.合成化合物9。
取中间体8(1.3g,2.36mmol)加入DCM-MeOH的混合溶剂(17mL+5mL)溶解，一次加入吡啶三溴化氢(0.832g,2.60mmol)室温搅拌反应2.5小时，TLC显示反应完毕。先后用4N盐酸和饱和食盐水洗反应液，之后无水硫酸钠干燥，过滤蒸干得中间体9为黄绿色泡沫状固体(1.46g)。
步骤7.合成化合物11。
取焦谷氨酸乙酯10(3.97g,15.4mmol)，氮气保护下加30mL无水四氢呋喃溶解，用恒温低温反应浴冷却至-40℃，缓慢滴加甲基氯化镁的四氢呋喃溶液(3M,5.4mL)，滴加完毕搅拌反应并自然升温至室温。TLC显示原料完全消失，冰浴冷却下向反应中滴加4N盐酸至体系pH到2左右，分液，四氢呋喃相用饱和食盐水洗之后减压蒸干柱层析得中间体11为无色油状物(3.80g,收率91％)。1H NMR(400MHz,CDCl3)δ5.10(d,J＝6.9Hz,1H),4.36–4.05(m,3H),2.54(dd,J＝15.4,7.8Hz,2H),2.21–1.72(m,5H),1.44(s,9H),1.28(t,J＝7.1Hz,3H)。
步骤8.合成化合物12。
上所得中间体11加DCM(20mL)溶解，之后加入三氟乙酸10mL，室温搅拌反应至体系无气泡产生，减压蒸干得无色油状物。所得油状物加无水乙醇60mL溶解，加入5％的Pd/C(380mg)，通入4个大气压的氢气室温反应过夜。LC-MS显示原料消失，撤掉氢气，过滤去除Pd/C，滤液蒸干得中间体12(4.92g)。1H NMR(400MHz,CDCl3)δ11.29(s,1H),4.49(dd,J＝9.3,4.3Hz,1H),4.30(q,J＝7.1Hz,2H),3.96–3.85(m,1H),2.53–2.40(m,1H),2.34–2.17(m,2H),1.74–1.60(m,1H),1.51(d,J＝6.5Hz,2H),1.32(t,J＝7.2Hz,3H)。
步骤9.合成化合物13。
取中间体12(0.84g)，N-Moc-L-缬氨酸-d8(0.35g,1.91mmol)加无水DMF(5mL)溶解，冰浴冷却，之后先后加入二异丙基乙胺(DIPEA,2.77g,21.45mmol)，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU,0.91g,2.39mmol)，自然升温搅拌反应2小时。将反应液倒入50mL水中，乙酸乙酯萃取，有机相减压蒸干得到中间体的粗品为一油状物。以上油状物加入MeOH 10mL溶解，冰浴冷却，滴加1M的氢氧化锂水溶液5mL，滴加完毕自然升温反应3小时，TLC显示中间体消失。减压蒸去反应液中的MeOH，水相用DCM萃取，之后向水相滴加4N盐酸调pH至2左右，DCM萃取，合并有机相饱和食盐水洗无水硫酸钠干燥，过滤蒸干得中间体13为无色泡沫状固体(0.46g)。1H NMR(300MHz,CDCl3)δ5.24(s,1H),4.65(s,1H),4.56(t,J＝8.6Hz,1H),3.67(s,3H),2.48–2.38(m,2H),2.09–2.01(m,2H),1.85–1.75(m,1H),1.30(d,J＝6.7Hz,3H)。
步骤10.合成化合物V-1。
取中间体9(408mg,0.65mmol)，二肽衍生物13(325mg,1.10mmol)，加入2-甲基-四氢呋喃3mL室温搅拌至溶清，一次性加入碳酸铯(169mg,0.52mmol)，室温搅拌10分钟，之后将反应体系转移至50℃恒温油浴中搅拌反应24小时，TLC显示中间体9消失。关闭加热，将反应体系降至室温，加乙酸乙酯10mL稀释，之后饱和碳酸氢钠洗。有机相减压蒸干柱层析得灰色泡沫状固体二酯中间体(358mg)。
取二酯中间体(358mg,0.42mmol)加10mL甲苯与1mL乙二醇甲醚，室温搅拌溶清，一次性加入醋酸铵1.64g(21.26mmol)，之后在110℃恒温油浴中加热回流反应4小时，TLC显示中间体消失。关闭加热，自然降温至室温，向反应中加入乙酸乙酯10mL，2M氢氧化钠水溶液洗，分液，有机相经无水硫酸钠干燥之后减压浓缩得到的粗品中间体。以上粗品加DCM 10mL溶解，搅拌下一次性加入活性二氧化锰(2.22g,25.50mmol)室温反应过夜。过滤以上反应体系，滤渣用甲醇洗至滤液无色。合并滤液蒸干，柱层析得化合物为灰色固体(109mg)。
灰色固体中间体(109mg,0.17mmol)加3mL无水DCM溶解，室温下加入4N氯化氢-二氧六环溶液搅拌反应2小时至原料消失。减压蒸干反应体系中的DCM，氯化氢以及二氧六环，得到中间体为褐色固体，加无水DMF 3mL，之后加入二异丙基乙基胺(176mg,1.32mmol)搅拌溶清，冰浴冷却以上溶液，先后加入N-Moc-L-苯甘氨酸(34mg,0.16mmol)和HATU(62mg,0.16mmol)，搅拌反应30分钟。以上反应液加入到30mL水中，乙酸乙酯萃取，合并蒸干柱层析得V-1为黄色粉末状固体(67mg)。LC-MS(APCI):m/z＝889.9(M+1)+，1H NMR(500MHz,MeOD)δ8.46–7.21(m,12H),5.59(s,1H),5.34–5.09(m,3H),4.35–4.02(m,1H),3.96–3.34(m,8H),3.29–2.89(m,2H),2.75–2.54(m,1H),2.45(s,1H),2.40–2.11(m,2H),1.99(s,1H),1.60(d,J＝6.6Hz,2H),1.31(s,1H),1.15(s,1H)。
实施例2
制备一种丙型肝炎病毒抑制剂V-2，分子式如下：
Figure PCTCN2017091788-appb-000006
采用以下步骤合成得到：
Figure PCTCN2017091788-appb-000007
步骤1.合成化合物14。
取中间体12(1.50g)，N-Moc-(d3)-L-缬氨酸(0.76g,4.29mmol)加无水DMF(5mL)溶解，冰浴冷却，之后先后加入二异丙基乙胺(2.77g,21.45mmol)，HATU(1.96g,5.15mmol)，自然升温搅拌反应2小时。将反应液倒入50mL水中，乙酸乙酯萃取，有机相减压蒸干得到油状中间体。向该油状物中加入THF 23mL溶解，冰浴冷却，滴加1M的氢氧化锂水溶液12mL，滴加完毕自然升温反应3小时，TLC显示原料消失。减压蒸去反应液中的THF，水相用二氯甲烷萃取，之后向水相滴加4N盐酸调pH至2左右，二氯甲烷萃取，合并有机相饱和食盐水洗无水硫酸钠干燥，过滤蒸干得中间体14为无色泡沫状固体(0.9g)。1H NMR(500MHz,CDCl3)δ5.49(d,J＝9.2Hz,1H),4.67–4.61(m,1H),4.52(t,J＝8.5Hz,1H),4.22–4.15(m,1H),2.27(dd,J＝8.7,4.2Hz,2H),2.05(d,J＝11.5Hz,2H),1.77(d,J＝12.4Hz,1H),1.32(d,J＝6.6Hz,3H),1.00(d,J＝6.5Hz,3H),0.97(d,J＝6.5Hz,3H),0.93(dd,J＝7.7,5.5Hz,1H)。
步骤2.合成化合物V-2。
取中间体9(373mg,0.65mmol)，二肽衍生物14(292mg,1.01mmol)，加入5mL 2-甲基-四氢呋喃室温搅拌至溶清，一次性加入碳酸铯(154mg,0.47mmol))，室温搅拌10分钟，之后将反应体系转移至40℃恒温油浴中搅拌反应24小时，TLC显示中间体9消失。关闭加热，过滤，蒸干柱层析得二酯中间体为灰色泡沫状固体(145mg)。
取二酯中间体(145mg,0.17mmol)加5mL甲苯与0.5mL乙二醇甲醚，室温搅拌溶清，一次性加入醋酸铵(267mg,3.46mmol)，之后在110℃恒温油浴中加热回流反应7小时，TLC显示原料反应完全。关闭加热，自然降温至室温，向反应中加入乙酸乙酯10mL，饱和碳酸氢钠水溶液洗，分液，有机相经无水硫酸钠干燥之后减压浓缩得到中间体的粗品。该中间体粗品加二氯甲烷5mL溶解，搅拌下一次性加入活性二氧化锰(839mg,8.5mmol)室温反应过夜。过滤以上反应体系，滤渣用甲醇洗至滤液无色。合并滤液蒸干，柱层析得灰色固体中间体(42mg)。
灰色固体中间体(42mg,0.053mmol)加3mL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2小时至原料消失。减压蒸干反应体系中的二氯甲烷，氯化氢以及二氧六环，得到褐色固体中间体，加无水DMF 3mL，之后加入二异丙基乙基胺(82mg,0.63mmol)搅拌溶清，冰浴冷却以上溶液，先后加入N-Moc(d3)-L-苯甘氨酸(13mg,0.063mmol)和HATU(24mg,0.063mmol)，搅拌反应30分钟。以上反应液加入到30mL水中，乙酸乙酯萃取，合并蒸干柱层析得V-2为黄色粉末状固体(18mg)。LC-MS(APCI):m/z＝889.9(M+1)+，1H NMR(400MHz,MeOD)δ8.44–6.89(m,12H),5.57(s,1H),5.34–4.98(m,3H),4.35–3.93(m,2H),3.72–3.35(m,2H),3.29–2.80(m,4H),2.77–2.03(m,5H),1.96(s,1H),1.58(d,J＝6.6Hz,2H),1.28(s,1H),1.14–1.01(m,4H),0.98–0.80(m,3H)。
实施例3
制备一种丙型肝炎病毒抑制剂V-3，分子式如下：
Figure PCTCN2017091788-appb-000008
采用以下步骤合成得到：
Figure PCTCN2017091788-appb-000009
步骤1.合成化合物15。
取镁屑(1.36g,57mmol)，单质碘(50mg)氮气保护下加入到40mL无水乙醚中。搅拌下向体系中滴加0.9mL氘代碘甲烷，加热使乙醚微沸，体系红褐色逐渐消失，变为乳白色混悬液，之后缓慢滴加2mL氘代碘甲烷，滴加完毕加热保持乙醚微沸反应2小时，之后 冷却得氘代甲基碘化镁格氏试剂。取焦谷氨酸乙酯10(4.15g,16.1mmol)，氮气保护下加30mL无水四氢呋喃溶解，用恒温低温反应浴冷却至-40℃，缓慢滴加以上氘代甲基碘化镁的乙醚溶液(15mL)，滴加完毕搅拌反应并自然升温至室温。待原料完全消失后，冰浴冷却下向反应中滴加4N盐酸至体系pH到2左右，分液，四氢呋喃相用饱和食盐水洗之后减压蒸干柱层析得中间体15为无色油状物(2.63g,收率59％)。1H NMR(300MHz,CDCl3)δ5.10(d,J＝7.4Hz,1H),4.35–4.07(m,3H),2.53(dd,J＝8.5,6.9Hz,2H),2.12(dd,J＝13.1,6.3Hz,1H),1.87(tt,J＝14.3,7.2Hz,1H),1.44(s,9H),1.28(t,J＝7.1Hz,3H)。
步骤2.合成化合物16。
取中间体15(0.61g,2.2mmol)加6mL DCM溶解，之后加入氘代三氟乙酸3mL，室温搅拌反应至体系无气泡产生，减压蒸干得无色油状物。所得油状物加氘代无水乙醇6mL溶解，加入5％的Pd/C(60mg,重水，乙醇-d各洗三次)，通入4个大气压的氘气室温反应过夜。LC-MS显示原料消失，撤掉氘气，过滤去除Pd/C，滤液蒸干得中间体16。1H NMR(300MHz,CDCl3)δ4.51(dd,J＝9.3,4.2Hz,1H),4.38–4.24(m,2H),2.64–2.19(m,2H),1.59–1.19(m,5H)。
步骤3.合成化合物17。
取以上所得中间体16，N-Moc-L-缬氨酸(0.47g,2.69mmol)加无水DMF(5mL)溶解，冰浴冷却，之后先后加入二异丙基乙胺(2.90g,22.4mmol)，HATU(1.02g,2.69mmol)，自然升温搅拌反应2小时。将反应液倒入50mL水中，乙酸乙酯萃取，有机相减压蒸干得到一油状物。以上油状物加入无水甲醇10mL溶解，冰浴冷却，滴加1M的氢氧化锂水溶液5mL，滴加完毕自然升温反应3小时，TLC显示原料消失。减压蒸去反应液中的甲醇，水相用二氯甲烷萃取，之后向水相滴加4N盐酸调pH至2左右，二氯甲烷萃取，合并饱和食盐水洗无水硫酸钠干燥，过滤蒸干得中间体17为无色泡沫状固体(0.45g)。1H NMR(500MHz,CDCl3)δ4.56(t,J＝8.7Hz,1H),4.20(t,J＝8.9Hz,1H),3.67(s,3H),2.45–2.36(m,2H),2.05(d,J＝7.7Hz,2H),1.01(d,J＝6.7Hz,3H),0.97(d,J＝6.7Hz,3H),0.94(d,J＝7.0Hz,1H)。
步骤4.合成化合物V-3。
与实施例1步骤10所述相似方法，不同点在于使用化合物17代替化合物13，从而制得目标化合物V-3。LC-MS(APCI):m/z＝889.9(M+1)+，1H NMR(500MHz,MeOD)δ8.36–7.09(m,12H),5.61–5.53(m,1H),5.30–5.01(m,3H),4.42–4.02(m,2H),3.83–3.34(m,7H),3.31–3.07(m,2H),2.99–2.33(m,3H),2.08(dd,J＝19.7,12.3Hz,1H),1.98(s,1H),1.29(s,1H),1.14–0.79(m,6H)。
实施例4
制备一种丙型肝炎病毒抑制剂V-4，分子式如下：
Figure PCTCN2017091788-appb-000010
采用以下步骤合成得到：
Figure PCTCN2017091788-appb-000011
步骤1.合成化合物18。
取中间体12(1.64g)，N-Moc-L-缬氨酸(0.82g,4.67mmol)加5mL无水DMF溶解，冰浴冷却，之后先后加入二异丙基乙胺(3.02g,23.35mmol)，HATU(2.13g,5.60mmol)，自然升温搅拌反应2小时。将反应液倒入50mL水中，用乙酸乙酯萃取，有机相减压蒸干得到红色油状物中间体。以上油状物加入无水甲醇23mL溶解，冰浴冷却，滴加1M的氢氧化锂水溶液12mL，滴加完毕自然升温反应3小时，待中间体消失后，减压蒸去反应液中的甲醇，水相用二氯甲烷萃取，之后向水相滴加4N盐酸调pH至2左右，二氯甲烷萃取，合并饱和食盐水洗无水硫酸钠干燥，过滤蒸干得中间体18为无色泡沫状固体(1.01g)。1H NMR(400MHz,CDCl3)δ5.54(d,J＝9.4Hz,1H),4.70–4.59(m,1H),4.51(t,J＝8.8Hz,1H),4.18(t,J＝9.2Hz,1H),3.66(s,3H),2.33–2.22(m,2H),2.10–1.99(m,2H),1.77(d,J＝12.0Hz,1H),1.32(d,J＝6.7Hz,3H),1.00(d,J＝6.7Hz,3H),0.97(d,J＝6.7Hz,3H),0.92(dd,J＝6.0,4.4Hz,1H)。
步骤2.合成化合物19。
取中间体9(654mg,1.04mmol)，二肽衍生物18(200mg,0.67mmol)，加入2-甲基-四氢呋喃4mL室温搅拌至溶清，一次性加入碳酸铯(114mg,0.35mmol)，室温搅拌10分钟，之后将反应体系转移至50摄氏度恒温油浴中搅拌反应24小时，待中间体9反应完全后，关闭加热，将反应体系降至室温，加二氯甲烷10mL稀释，之后水洗。有机相减压蒸干柱层析得二酯中间体19为灰色泡沫状固体(433mg)。LC-MS(APCI):m/z＝835.4(M+1)+。
步骤3.合成化合物20。
取二酯中间体19(433mg,0.52mmol)加10mL甲苯与1mL乙二醇甲醚，室温搅拌溶清，一次性加入醋酸铵(2.00g，25.99mmol)，之后在110摄氏度恒温油浴中加热回流反应4小时，关闭加热，自然降温至室温，向反应中加入乙酸乙酯10mL，2M氢氧化钠水溶液 洗，分液，有机相经无水硫酸钠干燥之后减压浓缩得到一中间体粗品。以上粗品加二氯甲烷10mL溶解，搅拌下一次性加入活性二氧化锰(2.26g,25.99mmol)室温反应过夜。过滤以上反应体系，滤渣用甲醇洗至滤液无色。合并滤液蒸干，柱层析得化合物20为灰色固体(204mg)。LC-MS(APCI):m/z＝792.4(M+1)+。
步骤4.合成化合物V-4。
中间体20(134mg,0.17mmol)加3mL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1.5小时至原料消失。减压蒸干反应体系中的二氯甲烷，氯化氢以及二氧六环，得到褐色固体中间体，加无水二甲基甲酰胺3mL，之后加入二异丙基乙基胺(269mg,1.69mmol)搅拌溶清，冰浴冷却以上溶液，先后加入N-Moc(d3)-L-苯甘氨酸(43mg,0.20mmol)和HATU(77mg,0.20mmol),搅拌反应30分钟。以上反应液加入到30mL水中，乙酸乙酯萃取，合并蒸干柱层析得V-4为黄色粉末状固体。LC-MS(APCI):m/z＝886.8(M+1)+，1H NMR(400MHz,MeOD)δ8.47–7.22(m,12H),5.60(s,1H),5.33–5.03(m,3H),4.43–4.03(m,2H),3.95–3.58(m,3H),3.56–2.86(m,11H),2.78–2.07(m,5H),2.00(d,J＝9.5Hz,1H),1.76–1.29(m,5H),1.22–0.81(m,8H)。
实施例5
制备一种丙型肝炎病毒抑制剂V-5，分子式如下：
Figure PCTCN2017091788-appb-000012
采用以下步骤合成得到：
Figure PCTCN2017091788-appb-000013
步骤1.合成化合物22。
取二酯化合物21(2.08g,7.22mmol)加入15mL四氢呋喃溶解，冰浴冷却，缓慢滴加1M氢氧化钠水溶液7.22mL，滴加完毕自然升温反应过夜。减压蒸干四氢呋喃，水相用二氯甲烷洗，之后用4N盐酸调pH至2左右，用二氯甲烷萃取，合并有机相饱和食盐水洗， 之后无水硫酸钠干燥。过滤蒸干，得到中间体22为无色油状物1.71g。
步骤2.合成化合物23。
以上所得单羧酸22加无水四氢呋喃10mL溶解，冰浴冷却，先后加入三乙胺(0.95g,9.38mmol)和氯甲酸乙酯(1.02g,9.38mmol)搅拌反应2小时。过滤出去反应体系中不溶性固体，滤液减压蒸干得浅色油状物，加四氢呋喃15mL冷却至-5摄氏度，取NaBH4(0.47g,12.50mmol)加去离子水5mL溶解，缓慢滴加到以上浅色油状物的四氢呋喃溶液中保持-5摄氏度搅拌反应2小时。-5摄氏度向反应中滴加4N盐酸调pH至2，分液，有机相经饱和碳酸氢钠水溶液洗无水硫酸钠干燥得中间体23为无色油状物1.25g。
步骤3.合成化合物24。
将化合物23溶于20mL无水二氯甲烷，室温下先后加入2，6-二叔丁基吡啶(1.84g,9.62mmol)、三氟甲磺酸银(1.85g,7.22mmol)，搅拌分散均匀后滴加氘代碘甲烷(1.39g,9.62mmol)，滴加完毕室温反应过夜，反应完成后，过滤去除不溶性固体。滤液加盐蒸干得到24的粗品为淡红色油状物。
步骤4.合成化合物25。
将24的粗品加10mL四氢呋喃和5mL甲醇溶解，冰浴冷却，之后滴加2.5M的氢氧化锂水溶液5mL,搅拌自然升温反应4小时。减压蒸干体系中的甲醇和四氢呋喃，剩余物加水10mL稀释。所得水溶液用二氯甲烷洗涤，之后用4N盐酸调pH至2，二氯甲烷萃取，合并有机相经无水硫酸钠干燥减压蒸干得25为无色油状物664mg。1H NMR(400MHz,CDCl3)δ4.44–4.21(m,1H),3.70–3.62(m,1H),3.48–3.10(m,3H),2.45–2.20(3,2H),2.18–1.75(m,1H),1.49(s,5H),1.43(s,4H)。
步骤5.合成化合物26。
取中间体6(442mg,1.19mmol)，氨基酸25(312mg,1.19mmol)加5mL 2-甲基四氢呋喃搅拌分散均匀，加入碳酸铯(213mg,0.65mmol)室温搅拌反应过夜，待反应完成后，减压蒸干反应液，柱层析得中间体26为黄色粉末状固体(519mg)。1H NMR(500MHz,CDCl3)δ7.91(d,J＝7.3Hz,1H),7.83(dd,J＝8.0,3.5Hz,1H),7.73(s,1H),7.65(s,2H),5.62–5.19(m,2H),5.16(s,2H),4.44(dd,J＝33.4,8.1Hz,1H),3.86–3.68(m,1H),3.52–3.34(m,2H),3.23(dd,J＝10.7,7.7Hz,1H),2.99(t,J＝5.9Hz,2H),2.75–2.63(m,2H),2.54(dd,J＝17.5,10.4Hz,2H),2.17(dd,J＝12.7,6.1Hz,2H),2.04(s,1H),1.44(9H)。
步骤6.合成化合物27。
取中间体26(517mg,0.94mmol)加入二氯甲烷-甲醇(5mL-2mL)的混合溶剂溶解，一次加入吡啶三溴化氢(314g,0.98mmol)室温搅拌反应4.5小时，反应完全后，先后用4N盐酸和饱和食盐水洗反应液，之后无水硫酸钠干燥，过滤蒸干得黄绿色泡沫状固体的中间体，该黄绿色中间体和化合物18(150mg,0.52mmol)加入到5mL 2-甲基四氢呋喃中搅拌分散均匀，一次性加入碳酸铯(86mg,0.26mmol)室温搅拌10分钟，之后将反应转移到50摄氏度恒温油浴中加热搅拌反应24小时。反应加10mL乙酸乙酯稀释，水洗，浓缩柱层析得褐色固体二酯中间体232mg。LC-MS(APCI):m/z＝837.7(M+1)+。
向上述二酯中间体(232mg,0.277mmol)加甲苯-乙二醇甲醚(5/1(V/V),6mL)搅拌至溶清，一次加入醋酸铵(1.068g,13.86mmol)，之后在100摄氏度恒温油浴中加热搅拌反应过夜。反应完成后冷却至室温，加10mL乙酸乙酯稀释，冰浴下滴加2M NaOH水溶液调pH至8～9，分液，有机相经无水硫酸钠干燥浓缩蒸干得到中间体粗品。
将该粗品加10mL二氯甲烷溶解，加入二氧化锰(1.205g,13.86mmol)室温搅拌反应过夜。反应完成后过滤，滤渣用甲醇洗至滤液无色。滤液合并蒸干，柱层析得化合物27(78mg)。LC-MS(APCI):m/z＝795.7(M+1)+。
步骤7.合成化合物V-5。
中间体27(78mg,0.0981mmol)加3mL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1小时。减压蒸干反应溶剂，得到褐色固体中间体，加无水DMF 3mL，之后加入二异丙基乙基胺(142mg,1.10mmol)搅拌溶清，冰浴冷却以上溶液，先后加N-Moc(d3)-L-苯甘氨酸(127mg,0.981mmol)和HATU(45mg,0.118mmol)，搅拌反应30分钟。以上反应液加入到30mL水中，乙酸乙酯萃取，合并蒸干柱层析得V-5为黄色粉末状固体(41mg)。LC-MS(APCI):m/z＝886.9(M+1)+，1H NMR(400MHz,MeOD)δ8.39–6.91(m,12H),5.66–5.50(m,1H),5.29–4.97(m,3H),4.42–3.96(m,2H),3.86–3.54(m,5H),3.27–2.81(m,2H),2.67–2.46(m,1H),2.46–2.05(m,4H),1.95–1.90(m,1H),1.60(t,J＝13.8Hz,2H),1.35(td,J＝7.6,4.0Hz,2H),1.28(s,1H),1.07–0.76(m,7H)。
实施例6
制备一种丙型肝炎病毒抑制剂V-6，分子式如下：
Figure PCTCN2017091788-appb-000014
采用以下步骤合成得到：
Figure PCTCN2017091788-appb-000015
Figure PCTCN2017091788-appb-000016
步骤1.合成化合物30。
取化合物28(4.0g,16.99mmol)溶于30mL四氢呋喃(THF)，冰水浴下加入三乙胺(TEA，2.58g,25.48mmol)，缓慢滴加氯甲酸氯乙酯(2.77g,25.48mmol)，滴毕撤冰水浴室温搅30分钟。过滤，滤液旋干得29，加20mL THF溶解，冰水浴下滴加NaBD4(1.42g,33.98mmol)的4mL D2O溶液，室温搅拌30分钟。依次向反应液加入7mL 4N HCl溶液、10mL饱和食盐水、10mL乙酸乙酯，分液，有机相依次用饱和碳酸氢钠、饱和食盐水洗涤，无水硫酸钠干燥、过滤、浓缩得30为淡黄色固体(2.69g)。1H NMR(500MHz,CDCl3)δ7.51(t,J＝4.1Hz,1H),7.46(d,J＝8.5Hz,1H),7.14(dd,J＝8.5,2.6Hz,1H)。
步骤2.合成化合物33。
取化合物30(2.68g，12.02mmol)溶于24mL THF，冰水浴下分批加入60％NaH(577mg，13mmol)，分批加入对甲苯磺酰氯(2.29g，12.02mmol))，室温搅拌30分钟。加入1mL水淬灭，加入5mL饱和食盐水，分液，浓缩有机相得化合物31的粗品为淡黄色固体(4.50g)。取化合物31(4.50g,11.91mmol)和化合物32(1.93g,11.91mmol)溶于15mL DMF，加入碳酸钾(2.47g,17.87mmol)，50摄氏度下搅拌2小时。将反应液倒入100mL水中。水相用乙酸乙酯/石油醚(1:1)萃取，合并有机相，依次用1N NaOH溶液、饱和食盐水洗涤，无水硫酸钠干燥，过滤，浓缩得到白色固体粉末为化合物(33 3.86g)。
步骤3.合成化合物34。
取化合物33(3.86g,10.50mmol)，特戊酸钯(149mg,0.53mmol)，三(4-氟-苯基)膦(166mg,0.53mmol)，特戊酸(322mg,3.15mmol)和碳酸钾(2.90g,21mmol)在氮气保护下加入到500mL单口反应瓶中，加入通氮气脱氧的二甲基乙酰胺(DMAC，60mL)。以上反应体系搅拌下氮气置换三次，之后于70摄氏度油浴加热搅拌反应3.5小时，反应完成后关闭加热，反应体系冷却至室温，之后减压蒸干DMAC，加入硅藻土5g，加入乙酸乙酯50mL，加水50mL，室温搅拌2小时，过滤分液。有机相蒸干，柱层析得到化合物34为黄色颗粒状固体(1.75mg)。1H NMR(400MHz,CDCl3)δ7.66(d,J＝8.4Hz,1H),7.62–7.60(m,1H),7.55–7.53(m,1H),7.37(dd,J＝8.4,2.2Hz,1H),7.18(d,J＝2.1Hz,1H),3.00–2.94(m,2H),2.69–2.60(m,2H),2.20–2.10(m,2H)。
步骤4.合成化合物35。
以上所得化合物34(1.75g，6.10mmol)，醋酸钯(69mg,0.31mmol)，2-双环己基膦-2',6'-二甲氧基联苯(Sphos,251mg,0.61mmol)，乙烯基三氟硼酸钾(2.45g,18.31mmo l)与碳酸钾(2.52g,18.31mmol)在氮气保护下加入到100mL单口反应瓶中，加入通氮气脱氧的61mL正丙醇。以上反应体系氮气置换三次，在120摄氏度油浴中加热回流反应12小时，LC-MS监控显示原料消失，关闭加热，冰浴冷却反应至室温，减压蒸干正丙醇。向以上所得糊状物中加入50mL乙酸乙酯，室温搅拌30分钟，过滤，滤液减压蒸干柱层析得到化合物35为黄色颗粒状固体(1.54g)。1H NMR(500MHz,CDCl3)δ7.70(d,J＝8.1Hz,1H),7.62(s,1H),7.58(s,1H),7.43(d,J＝8.1Hz,1H),7.22(d,J＝1.8Hz,1H),6.73(dd,J＝17.6,10.9Hz,1H),5.81(d,J＝17.5Hz,1H),5.32(d,J＝10.9Hz,1H),3.01–2.93(m,2H),2.69–2.60(m,2H),2.20–2.10(m,2H)。
步骤5.合成化合物36。
以上所得中间体36加THF-DMSO-水的混合溶剂(6mL+6mL+3mL)溶解，室温下加入N-溴代丁二酰亚胺(NBS,1.03g，5.79mmol)搅拌反应1小时，反应完成后减压蒸去反应体系中的THF，之后加水60mL稀释，乙酸乙酯萃取，合并有机相，饱和食盐水洗，之后用无水硫酸镁干燥，过滤蒸干溶剂得中间体的粗品。该中间体的粗品加20mL二氯甲烷溶解，加入活性二氧化锰(9.60g,86.95mmol)室温搅拌反应过夜，过滤去除不溶性固体，滤液加5mL甲醇，减压蒸去二氯甲烷至大量橙黄色固体析出，停止减压蒸馏，过滤收集固体。母液加二氯甲烷20mL，之后减压蒸馏至橙黄色固体析出，停止减压蒸馏，过滤收集固体。重复以上操作一次，合并三次所得固体即为中间体36(1.72g,收率为35％)。1H NMR(400MHz,CDCl3)δ8.03(dd,J＝8.2,1.9Hz,1H),7.89–7.81(m,2H),7.67(s,2H),4.47(s,2H),3.01(dd,J＝7.0,5.2Hz,2H),2.69(dd,J＝7.2,5.9Hz,2H),2.18(p,J＝6.4Hz,2H)。
步骤6.合成化合物37。
取中间体36(500mg,1.34mmol)，氨基酸7(347mg,1.34mmol)加6mL 2-甲基四氢呋喃搅拌分散均匀，加入碳酸铯(240mg,0.74mmol)室温搅拌反应过夜，减压蒸干反应液，柱层析得中间体37为黄色泡沫状固体598mg。1H NMR(400MHz,CDCl3)7.91(t,J＝6.4Hz,1H),7.82(dd,J＝8.1,3.2Hz,1H),7.72(s,1H),7.63(s,2H),5.46–5.16(m,2H),4.48–4.37(m,1H),3.80–3.68(m,1H),3.48–3.29(m,5H),3.22(dd,J＝10.7,7.7Hz,1H),2.97(t,J＝6.0Hz,2H),2.70–2.60(m,2H),2.55–2.45(m,2H),2.15–2.09(m,2H),1.48–1.40(9H)。
步骤7.合成化合物38。
取中间体37(598mg,1.08mmol)加入二氯甲烷-甲醇(8mL+3mL)的混合溶剂溶解，一次加入吡啶三溴化氢(0.382g,1.19mmol)室温搅拌反应2.5小时，先后用4N盐酸和饱和食盐水洗反应液，之后无水硫酸钠干燥，过滤蒸干得中间体38为黄绿色泡沫状固体(684mg)。LC-MS(APCI):m/z＝632.5(M+1)+。
步骤8.合成化合物39。
取中间体38(684mg,1.08mmol)，二肽衍生物18(208mg,0.73mmol)，加入2-甲基-四氢呋喃5mL室温搅拌至溶清，一次性加入碳酸铯(118mg,0.36mmol)，室温搅拌10分钟，之后将反应体系转移至50摄氏度恒温油浴中搅拌反应24小时，关闭加热，过滤，蒸干柱层析得二酯中间体39为灰色泡沫状固体(374mg)。LC-MS(APCI):m/z＝836.7(M+1)+。
步骤9.合成化合物40。
取二酯中间体39(374mg,0.45mmol)加10mL甲苯与1mL乙二醇甲醚，室温搅拌溶清，一次性加入醋酸铵(1.72g，22.37mmol)，之后在110摄氏度恒温油浴中加热回流反应4小时，关闭加热，自然降温至室温，向反应中加入乙酸乙酯10mL，2M氢氧化钠水溶液洗，分液，有机相经无水硫酸钠干燥之后减压浓缩得到中间体的粗品。该粗品加二氯甲烷10mL溶解，搅拌下一次性加入活性二氧化锰(1.94mg,22.37mmol)室温反应过夜。过滤以上反应体系，滤渣用甲醇洗至滤液无色。滤液合并蒸干，柱层析得化合物40为灰色固体(145mg)。LC-MS(APCI):m/z＝794.8(M+1)+。
步骤10.合成化合物V-6。
中间体40(145mg,0.18mmol)加3mL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1小时。减压蒸干反应溶剂，得到褐色固体中间体，加无水DMF 3mL，之后加入二异丙基乙基胺(302mg,1.83mmol)搅拌溶清，冰浴冷却以上溶液，先后加入N-Moc-L-苯甘氨酸(46mg,0.22mmol)和HATU(84mg,0.22mmol)，搅拌反应30分钟。以上反应液加入到30mL水中，乙酸乙酯萃取，合并蒸干柱层析得V-6为黄色粉末状固体(115mg)。LC-MS(APCI):m/z＝885.9(M+1)+，1H NMR(500MHz,MeOD)δ8.45–7.16(m,12H),5.60(s,1H),5.23(dt,J＝16.8,7.9Hz,1H),4.40–4.02(m,2H),3.92–3.60(m,5H),3.60–3.39(m,2H),3.36–3.24(m,7H),3.16–2.89(m,1H),2.68(s,1H),2.54–2.09(m,3H),1.96(d,J＝26.0Hz,1H),1.73–1.55(m,2H),1.31(s,1H),1.18–0.80(m,7H)。
实施例7
制备一种丙型肝炎病毒抑制剂V-7，分子式如下：
Figure PCTCN2017091788-appb-000017
采用以下步骤合成得到：
Figure PCTCN2017091788-appb-000018
步骤1.合成化合物41。
取中间体36(427mg,1.14mmol)，氨基酸24(300mg,1.34mmol)加6mL 2-甲基四氢呋喃搅拌分散均匀，加入碳酸铯(205mg,0.63mmol)室温搅拌反应过夜，减压蒸干反应液，柱层析得中间体41为黄色泡沫状固体(440mg)。1H NMR(400MHz,CDCl3)δ8.00–7.88(m,1H),7.83(dd,J＝8.2,3.0Hz,1H),7.72(s,1H),7.64(s,2H),5.46–5.16(m,2H),4.43(dt,J＝21.4,8.1Hz,1H),3.82–3.61(m,3H),3.45–3.33(m,2H),3.23(dd,J＝10.8,7.7Hz,1H),2.98(t,J＝6.0Hz,2H),2.69–2.61(m,2H),2.57(dd,J＝17.7,10.8Hz,2H),2.21–2.10(m,3H),1.50–1.38(9H)。
步骤2.合成化合物42。
取中间体41(440mg,0.79mmol)加入二氯甲烷-甲醇(6mL+2mL)的混合溶剂溶解，一次加入吡啶三溴化氢(279mg,0.87mmol)室温搅拌反应2.5小时，先后用4N盐酸和饱和食盐水洗反应液，之后无水硫酸钠干燥，过滤蒸干得中间体42为黄绿色泡沫状固体(491mg)。LC-MS(APCI):m/z＝635.5(M+1)+。
步骤3.合成化合物43。
取中间体42(491mg,0.78mmol)，二肽衍生物25(150mg,0.52mmol)，加入2-甲基-四氢呋喃5mL室温搅拌至溶清，一次性加入碳酸铯(86mg,0.26mmol)，室温搅拌10分钟，之后将反应体系转移至50摄氏度恒温油浴中搅拌反应24小时，关闭加热，过滤，蒸干柱层析得二酯中间体43为灰色泡沫状固体(285mg)。LC-MS(APCI):m/z＝846.6(M+1)+。
步骤4.合成化合物44。
取二酯中间体43(285mg,0.34mmol)加10mL甲苯与1mL乙二醇甲醚，室温搅拌溶清，一次性加入醋酸铵(1.30g，16.90mmol)，在100摄氏度恒温油浴中加热反应过夜，之后110摄氏度回流1小时，关闭加热，自然降温至室温，向反应中加入乙酸乙酯10mL，2M氢氧化钠水溶液洗，分液，有机相经无水硫酸钠干燥之后减压浓缩得到中间体的粗品。以上粗品加二氯甲烷10mL溶解，搅拌下一次性加入活性二氧化锰(1.47mg,16.90mmol)室温反应过夜。过滤以上反应体系，滤渣用甲醇洗至滤液无色。滤液合并蒸干，柱层析得化合物44为灰色固体(145mg)。LC-MS(APCI):m/z＝804.8(M+1)+。
步骤5.合成化合物V-7。
中间体44(88mg,0.11mmol)加3mL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1小时。减压蒸干反应溶剂，得到褐色固体中间体，加无水DMF 3mL，之后加入二异丙基乙基胺(142mg,1.10mmol)搅拌溶清，冰浴冷却以上溶液，先后加入N-Moc(d3)-L-苯甘氨酸(28mg,0.13mmol)和HATU(50mg,0.13mmol)，搅拌反应30分钟。以上反应液加入到30mL水中，乙酸乙酯萃取，合并蒸干柱层析得V-7为黄色粉末状固体(39mg)。LC-MS(APCI):m/z＝896.3(M+1)+。1H NMR(400MHz,MeOD)δ8.40–6.88(m,12H),5.55(d,J＝19.3Hz,1H),5.42–5.04(m,2H),4.43–3.97(m,2H),3.88–3.59(m,3H),3.52–3.33(m,2H),3.18–2.83(m,1H),2.74–1.86(m,7H),1.69–1.47(m,1H),1.08(dd,J＝22.4,6.4Hz,3H),0.95(s,1H),0.89(dd,J＝14.3,6.5Hz,3H)。
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GS-5816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7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。
表1实施例0-1与对照品Ombitasvir的抗HCV基因型复制子活性对比表
编号 GT1a EC50(nM) GT1b EC50(nM) CC50(nM)
GS-5816 0.022 0.017 >2
实施例1 0.010 0.012 >2
实施例2 0.016 0.009 >2
实施例3 0.011 0.008 >2
实施例4 0.009 0.008 >2
实施例5 0.017 0.012 >2
实施例6 0.010 0.009 >2
实施例7 0.011 0.013 >2
实验结果表明，本发明的化合物可抑制HCV的多个基因型。与未氘代的化合物GS-5816相比，本发明的化合物对GT1a和GT1b复制子表现出更优良的抑制活性，并通过抑制HCV NS5A蛋白的机制，发挥了更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：精密称取一定量的待测实施例化合物和对照化合物GS-5816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，并根据以下公式计算t1/2和CLint，其中V/M即等于1/蛋白浓度。
Figure PCTCN2017091788-appb-000019
对本发明化合物及其没有氘代的化合物同时测验比较，评价其在人和大鼠肝微粒体的代谢稳定性。作为代谢稳定性的指标的半衰期及肝固有清除率如表2所示。表2中采用未经氘代的化合物GS-5816作为对照样品。如表2所示，通过与未经氘代的化合物GS-5816对照，本发明化合物可以显著改善代谢稳定性，进而更适于作为丙型肝炎病毒抑制剂。
表2实施例D-1与GS-5816对照样的代谢稳定性对比表
Figure PCTCN2017091788-appb-000020
(4)大鼠药代动力学实验。
实验目的：研究大鼠给予GS-5816、实施例化合物后，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℃烘箱内烘干后，置于4℃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GS5816 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GS-5816(3mg/kg)、实施例化合物(3mg/kg)后的药代动力学相关参数。
实验结果表明，与GS-5816相比，本发明人发现本发明化合物具有更优的活性，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 (10)

  1. 一种取代的咪唑基化合物，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 PCTCN2017091788-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各自独立地为氢、氘、卤素；
    X1、X2、X3、X4、X5、X6各自独立地为一次或多次氘代的甲基、乙基；
    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、X1、X2、X3、X4、X5和X6中至少一个是氘代的或含氘。
  2. 根据权利要求1所述的咪唑基化合物，其特征在于：R1、R2、R3、R4和R5各自独立地为氘或氢。
  3. 根据权利要求1所述的咪唑基化合物，其特征在于：R6、R7、R8、R9、R10、R11、R12、R13和R14各自独立地为氘或氢。
  4. 根据权利要求1所述的咪唑基化合物，其特征在于：R15、R16、R17、R18、R19、R20、R21、R22、R23和R24各自独立地为氘或氢。
  5. 根据权利要求1所述的咪唑基化合物，其特征在于：R25、R26、R27、R28、R29、R30各自独立地为氘或氢。
  6. 根据权利要求1所述的咪唑基化合物，其特征在于：R31、R32各自独立地为氘或氢。
  7. 根据权利要求1所述的咪唑基化合物，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 PCTCN2017091788-appb-100002
    Figure PCTCN2017091788-appb-100003
  8. 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。
  9. 根据权利要求8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。
  10. 一种如权利要求1～8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。
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