WO2018171660A1 - 用于抗丙型肝炎病毒感染的含硅化合物 - Google Patents

用于抗丙型肝炎病毒感染的含硅化合物 Download PDF

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Publication number
WO2018171660A1
WO2018171660A1 PCT/CN2018/079965 CN2018079965W WO2018171660A1 WO 2018171660 A1 WO2018171660 A1 WO 2018171660A1 CN 2018079965 W CN2018079965 W CN 2018079965W WO 2018171660 A1 WO2018171660 A1 WO 2018171660A1
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compound
och
hydrogen
formula
group
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PCT/CN2018/079965
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English (en)
French (fr)
Inventor
张寅生
刘保民
黄雨
陈正帮
盖阔
刘戌时
王晓金
徐宏江
Original Assignee
正大天晴药业集团股份有限公司
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Application filed by 正大天晴药业集团股份有限公司 filed Critical 正大天晴药业集团股份有限公司
Priority to CN201880015765.9A priority Critical patent/CN110382487B/zh
Priority to EP18771081.9A priority patent/EP3604295A4/en
Priority to EA201992241A priority patent/EA201992241A1/ru
Priority to US16/494,604 priority patent/US10766912B2/en
Priority to BR112019019724A priority patent/BR112019019724A2/pt
Priority to MX2019011173A priority patent/MX2019011173A/es
Priority to JP2019552128A priority patent/JP2020512331A/ja
Publication of WO2018171660A1 publication Critical patent/WO2018171660A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic System
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/0803—Compounds with Si-C or Si-Si linkages
    • C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
    • C07F7/0812—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring
    • C07F7/0816—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring said ring comprising Si as a ring atom
    • 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
    • A61K31/695—Silicon compounds
    • 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

Definitions

  • the present application belongs to the field of medicinal chemistry, and in particular to a silicon-containing compound for use against hepatitis C virus infection, a process for the preparation thereof, and a pharmaceutical composition containing the same.
  • the application also relates to the use of these compounds and pharmaceutical compositions for the treatment of hepatitis C virus (HCV) infection.
  • HCV hepatitis C virus
  • HCV is a positive-strand RNA virus belonging to the genus Hepatitis C in the Flaviviridae family. At least six major genotypes have been identified, including more than 50 subtypes.
  • the single-stranded HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of approximately 3000 amino acids. In infected cells, the polyprotein is cleaved by cellular and viral proteases at multiple sites, resulting in both structural and non-structural (NS) proteins. In the case of HCV, the formation of mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) is achieved by two viral proteases.
  • Treatment options for chronic HCV infection include: peginterferon- ⁇ combined with ribavirin in the treatment of HCV-infected patients and patients with cirrhosis, if the treatment fails, the interferon-containing treatment regimen is used again, and the sustained virological response rate will be As low as 14%, in addition, interferon-containing treatment regimens have increased toxic side effects in patients with cirrhosis.
  • HCV NS3/4A protease inhibitors NS5A inhibitors and NS5B polymerase inhibitors
  • interferon and ribavirin can be eliminated, which reduces toxicity and shortens the treatment cycle.
  • ABT-267 WO2010144646
  • ABT-530 WO2012051361
  • the application provides a compound of Formula I, or a pharmaceutically acceptable salt, or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof:
  • R 1 and R 2 are each independently selected from hydroxy, alkyl, alkoxy and aryl, or R 1 and R 2 are bonded to form a silicon-containing saturated aliphatic ring containing 1, 2 or 3 silicon atoms;
  • R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of hydrogen and halogen;
  • X is selected from the following groups:
  • Y is selected from the following groups:
  • R 7 and R 8 are each independently selected from -CH(alkyl)(alkoxy), -CH(alkyl) 2 , -CH(alkoxy) 2 , -C(alkyl) 2 (alkoxy) ), -C(alkyl)(alkoxy) 2 , -C(alkyl) 3 and -C(alkoxy) 3 ;
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, and alkoxyalkyl;
  • R a and R b are independently selected from the group consisting of hydrogen, halogen, and alkyl;
  • Each m is independently selected from 0, 1, 2, 3, and 4;
  • n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, or a tautomer, or a stereoisomer, or a deuterated substance And mixtures thereof, as well as one or more pharmaceutically acceptable carriers.
  • the application provides a compound of Formula I, or a pharmaceutically acceptable salt, or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof, or a pharmaceutical composition as described above, in the manufacture of a therapeutic C form Use in drugs for hepatitis virus infection.
  • the application provides a method of treating a hepatitis C virus infection, the method comprising administering to a patient in need of treatment a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt, or tautomer thereof, or Stereoisomers, or deuterated compounds and mixtures thereof, or the above pharmaceutical compositions.
  • the application provides a compound of Formula I, or a pharmaceutically acceptable salt, or tautomer thereof, or a stereoisomer, or a deuterated substance, and mixtures thereof, for use in the treatment of a hepatitis C virus infection, and The above pharmaceutical composition for treating hepatitis C virus infection.
  • the application provides a compound of formula I, or a pharmaceutically acceptable salt, or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof, or a pharmaceutical composition as described above, in the treatment of hepatitis C Use in viral infections.
  • the application provides a compound of Formula I, or a pharmaceutically acceptable salt, or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof:
  • R 1 and R 2 are each independently selected from a hydroxyl group, an alkyl group, an alkoxy group, and an aryl group, or R 1 and R 2 are bonded to form a silicon-containing saturated aliphatic ring (ie, R 1 and R 2 are bonded to the silicon atom to which they are attached). Forming a silicon-containing saturated aliphatic ring together, the silicon-containing saturated aliphatic ring containing 1, 2 or 3 silicon atoms;
  • R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of hydrogen and halogen;
  • X is selected from the following groups:
  • Y is selected from the following groups:
  • R 7 and R 8 are each independently selected from -CH(alkyl)(alkoxy), -CH(alkyl) 2 , -CH(alkoxy) 2 , -C(alkyl) 2 (alkoxy) ), -C(alkyl)(alkoxy) 2 , -C(alkyl) 3 and -C(alkoxy) 3 ;
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, and alkoxyalkyl;
  • Each of R a and R b is independently selected from a halogen and an alkyl group; or each of R a and R b is independently selected from the group consisting of hydrogen, halogen, and alkyl.
  • Each m is independently selected from 0, 1, 2, 3, and 4;
  • n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8.
  • the relative stereochemistry of the 2 and 5 positions of the pyrrole ring to which the benzene ring is attached to N may be cis or trans.
  • the 2- and 5-position configurations on the pyrrole ring include (2S, 5S), (2S, 5R), (2R, 5S), (2R, 5R).
  • the compound of formula I may be a stereoisomer or a mixture of two or more stereoisomers in any ratio.
  • each of R a optionally is substituted on position ortho or silicon atom between the carbon atom.
  • each R b may be any; preferably, X is selected from Y is selected from
  • the alkyl group referred to in the definition of R 1 , R 2 , R 7 , R 8 , R 9 , R 10 , R a and R b is C 1-6 alkyl or C 1-4 alkyl, alkoxy as defined in the definition of R 1 , R 2 , R 7 , R 8 , R 9 and R 10 is C 1-6 alkoxy or C 1-4 alkoxy, R
  • the alkoxyalkyl group referred to in the definition of 9 and R 10 is C 1-6 alkoxy C 1-6 alkyl, C 1-6 alkoxy C 1-4 alkyl or C 1-4 alkoxy C 1-2 alkyl, the aryl group referred to in the definition of R 1 and R 2 is a C 6-12 aryl group;
  • the silicon-containing saturated aliphatic ring involved in the definition of R 1 and R 2 is 3-8 yuan or 4 -6 yuan saturated saturated ring of silicon.
  • R 1 and R 2 are each independently selected from hydroxy, C 1-6 alkyl, C 1-6 alkoxy, and C 6-12 aryl, or R 1 and R 2 are joined to form 3- An 8-membered silicon-containing saturated aliphatic ring containing 1, 2 or 3 silicon atoms.
  • R 1 and R 2 are each independently selected from C 1-6 alkyl, or R 1 and R 2 are joined to form a 3-8 membered silicon-containing saturated aliphatic ring, said 3-8 membered silicon-containing The saturated fat ring contains 1 silicon atom.
  • R 1 and R 2 are each independently selected from methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl, isobutyl, and phenyl, or R 1 and R 2 is connected to form a 3-, 4-, 5-, 6-, 7-, or 8-membered silicon-containing saturated aliphatic ring, wherein the silicon-containing saturated aliphatic ring contains one silicon atom.
  • R 1 and R 2 are each independently selected from methyl, or R 1 and R 2 are joined to form a 4-, 5- or 6-membered silicon-containing saturated aliphatic ring, wherein the silicon-containing saturated fat The ring contains 1 silicon atom.
  • R 3 , R 4 , R 5 , and R 6 are each independently selected from the group consisting of hydrogen, fluorine, and chlorine.
  • R 3 and R 4 are each independently selected from hydrogen and fluorine, and R 5 and R 6 are selected from hydrogen.
  • one of R 3 and R 4 is fluoro, the other is hydrogen, and R 5 and R 6 are selected from hydrogen.
  • R 3 and R 4 are selected from fluorine, and R 5 and R 6 are selected from hydrogen.
  • X is selected from Y is selected from In some embodiments, X is selected from Y is selected from In some embodiments, X is selected from Y is selected from.
  • X is selected from Y is selected from Or X is selected from Y is selected from Preferably, X is selected from Y is selected from Or X is selected from Y is selected from Preferably, X is selected from Y is selected from Or X is selected from Y is selected from
  • X is selected from Y is selected from In some embodiments, X is selected from Y is selected from
  • R 7 and R 8 are each independently selected from -CH(C 1-6 alkyl)(C 1-6 alkoxy), -CH(C 1-6 alkyl) 2 , -CH (C 1-6 alkoxy) 2 , -C(C 1-6 alkyl) 2 (C 1-6 alkoxy), -C(C 1-6 alkyl) (C 1-6 alkoxy) 2 , -C(C 1-6 alkyl) 3 and -C(C 1-6 alkoxy) 3 .
  • R 7 and R 8 are the same.
  • R 7 and R 8 are each independently selected from -CH(CH 3 )(OCH 3 ), -CH(CH 3 ) 2 , -CH(OCH 3 ) 2 , -C(CH 3 ) 2 (OCH 3 ), -C(CH 3 )(OCH 3 ) 2 , -C(CH 3 ) 3 , -C(OCH 3 ) 3 , -CH(CH 2 CH 3 )(OCH 2 CH 3 ), -CH (CH 3 )(OCH 2 CH 3 ), -CH(CH 2 CH 3 )(OCH 3 ), -CH(CH 2 CH 3 ) 2 , -CH(CH 3 )(CH 2 CH 3 ), -CH( OCH 2 CH 3 ) 2 , -CH(OCH 2 CH 3 )(OCH 3 ), -C(CH 2 CH 3 ) 2 (OCH 2 CH 3 ), -C(CH 3 ) 2 (OCH 2 CH 3 ), -C(CH 2 CH
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, halogen, alkyl, and alkoxy. In some embodiments, R 9 and R 10 are each independently selected from hydrogen and alkoxyalkyl.
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, and C 1-6 alkoxy. In some embodiments, R 9 and R 10 are each independently selected from hydrogen and C 1-6 alkoxy C 1-6 alkyl. In some embodiments, R 9 and R 10 are the same.
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, fluoro, chloro, methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl, isobutyl, methoxy. Base, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-butoxy and isobutoxy.
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen, methoxymethyl, methoxyethyl, ethoxymethyl, and ethoxyethyl.
  • R 9 and R 10 are each independently selected from hydrogen and methoxymethyl.
  • both R 9 and R 10 are hydrogen.
  • one of R 9 and R 10 is hydrogen and the other is methoxymethyl.
  • both R 9 and R 10 are methoxymethyl.
  • R 7 and R 8 are each independently selected from -CH(alkyl)(alkoxy), -CH(alkyl) 2, and -CH(alkoxy) 2 . In some embodiments, R 7 and R 8 are each independently selected from -CH(C 1-6 alkyl)(C 1-6 alkoxy), -CH(C 1-6 alkyl) 2, and -CH (C 1-6 alkoxy) 2 . In some embodiments, R 7 and R 8 are both -CH(CH 3 )(OCH 3 ).
  • R 7 and R 8 are each independently selected from -CH(CH 3 )(OCH 3 ) and -CH(CH 3 ) 2 ;
  • R 9 and R 10 are each independently selected from hydrogen and methoxy. methyl.
  • R 7 and R 8 are each independently selected from -CH(CH 3 )(OCH 3 ) and -CH(CH 3 ) 2 ; R 9 and R 10 are selected from hydrogen.
  • R 7 and R 8 are each independently selected from -CH(CH 3 )(OCH 3 ) and -CH(CH 3 ) 2 , wherein -CH(CH 3 )(OCH 3 ) is a chiral carbon
  • the atom is in the R configuration;
  • R 9 and R 10 are each independently selected from the group consisting of hydrogen and methoxymethyl.
  • R 7 and R 8 are each independently selected from -CH(CH 3 )(OCH 3 ) and -CH(CH 3 ) 2 , wherein -CH(CH 3 )(OCH 3 ) is a chiral carbon
  • the atom is in the R configuration; R 9 and R 10 are selected from hydrogen.
  • the chiral carbon atom on the tetrahydropyrrole ring attached to R 9 or R 10 is in the S configuration.
  • each of R a and R b are independently selected from the group consisting of hydrogen, halogen, and C 1-6 alkyl.
  • each of R a and R b are independently selected from halo and C 1-6 alkyl.
  • each of R a and R b are independently selected from the group consisting of fluoro, chloro, methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl, and isobutyl.
  • each of R a and R b are independently selected from the group consisting of hydrogen, fluoro, chloro, methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl, and isobutyl.
  • n is selected from the group consisting of 0 and R b is selected from the group consisting of fluorine.
  • R 1 and R 2 are methyl, or R 1 and R 2 are joined to form a 4-, 5- or 6-membered silicon-containing saturated aliphatic ring containing 1 silicon atom;
  • R 3 and R 4 One is fluorine and the other is hydrogen, or both R 3 and R 4 are fluorine;
  • R 5 and R 6 are hydrogen;
  • X is Y is
  • R 7 and R 8 are both -CH(CH 3 )(OCH 3 );
  • R 9 and R 10 are both hydrogen or both methoxymethyl; n is 0.
  • the application provides a compound of Formula Ia, Formula Ib, Formula Ic or Formula Id, or a pharmaceutically acceptable salt, or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof,
  • the application provides the following compounds, or pharmaceutically acceptable salts, or tautomers thereof, or stereoisomers, or deuterated compounds, and mixtures thereof:
  • the present application provides the following compounds, or pharmaceutically acceptable salts, or tautomers thereof, or stereoisomers, or deuterated compounds and mixtures thereof: Compound 1a, Compound 1b, Compound 2a, Compound 2b, Compound 3a, Compound 4a, Compound 4b, Compound 5a, Compound 5b, Compound 6a, Compound 6b, Compound 7b, Compound 8b, Compound 17b and Compound 18b.
  • the present application provides the following compounds, or pharmaceutically acceptable salts, or tautomers thereof, or stereoisomers, or deuterated compounds and mixtures thereof: Compound 1a, Compound 1b, Compound 2a, Compound 2b, Compound 3a, Compound 4a, Compound 4b, Compound 5a, Compound 5b, Compound 6a, Compound 6b, Compound 7b, Compound 8a, Compound 8b, Compound 9b, Compound 11a, Compound 14b, Compound 15b, Compound 17b and Compound 18b.
  • compositions comprising a therapeutically effective amount of a compound of Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id, or a pharmaceutically acceptable salt thereof, or tautomerous Isomers, or stereoisomers, or deuterated compounds, and mixtures thereof, and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition of the present application may be obtained by combining a compound of the present application, or a pharmaceutically acceptable salt thereof, or a tautomer, or a stereoisomer, or a deuterated substance, and a mixture thereof, with a suitable pharmaceutically acceptable carrier.
  • Prepared by combination for example, can be formulated into solid, semi-solid, liquid or gaseous preparations such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, coagulation Glues, microspheres and aerosols.
  • Typical routes for administration of a compound of the present application, or a pharmaceutically acceptable salt or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof, or a pharmaceutical composition thereof include, but are not limited to, oral, rectal, Transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous.
  • the pharmaceutical composition of the present application can be produced by a method well known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a drag coating method, a grinding method, an emulsification method, a freeze drying method, and the like.
  • the pharmaceutical compositions may be formulated by admixing the active compound withpharmaceutically acceptable carriers such carriers.
  • pharmaceutically acceptable carriers such carriers.
  • These carriers enable the compounds of the present application to be formulated into tablets, pills, troches, dragees, capsules, liquids, gels, slurries, suspensions and the like for oral administration to a patient.
  • Solid oral compositions can be prepared by conventional methods of mixing, filling or tabletting. For example, it can be obtained by mixing the active compound with a solid excipient, optionally milling the resulting mixture, adding other suitable adjuvants if necessary, and then processing the mixture into granules.
  • a solid excipient optionally milling the resulting mixture, adding other suitable adjuvants if necessary, and then processing the mixture into granules.
  • Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.
  • the core of the dragee may optionally be coated according to methods well known in the ordinary pharmaceutical practice, especially using enteric coatings.
  • compositions may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in a suitable unit dosage form.
  • suitable excipients such as fillers, buffers or surfactants can be used.
  • the compound of the present application (including a compound of a specific structure) has hepatitis C virus (HCV) NS5A inhibitory activity and can be used as an HCV NS5A inhibitor for the treatment of hepatitis C virus infection, specifically for hepatitis C virus Treatment of liver diseases such as hepatitis and cirrhosis caused by infection.
  • HCV hepatitis C virus
  • the compound of the present application (including a compound having a specific structure) has hepatitis C virus (HCV) NS5A inhibitory activity, and particularly has excellent inhibitory activity against various genetic subtypes of HCV, and these gene subtypes include 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 6a; also include some chimeric types 1b/3a, 1b/4a, 1b/5a, 1b/6a and the like.
  • HCV hepatitis C virus
  • the compounds of the present application (including compounds of a specific structure) have a good affinity for liver tissue and can be targeted to be distributed in liver tissues.
  • the compounds of the present application (including compounds of specific structure) also have some resistance.
  • the present application provides a compound of Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id, or a pharmaceutically acceptable salt, or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof, in the preparation Use in the treatment of drugs for hepatitis C virus (HCV) infection.
  • HCV hepatitis C virus
  • the present application provides a compound of Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id, or a pharmaceutically acceptable salt or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof, and at least Use of another active compound for the manufacture of a medicament for the treatment of hepatitis C virus (HCV) infection.
  • HCV hepatitis C virus
  • the present application provides a compound comprising Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id, or a pharmaceutically acceptable salt, or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof Use of a pharmaceutical composition for the manufacture of a medicament for the treatment of hepatitis C virus (HCV) infection.
  • HCV hepatitis C virus
  • the present application provides a compound comprising Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id, or a pharmaceutically acceptable salt, or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof, and Use of a pharmaceutical composition of at least one other active compound for the manufacture of a medicament for the treatment of hepatitis C virus (HCV) infection.
  • HCV hepatitis C virus
  • other active compounds include, but are not limited to, other compounds that are resistant to HCV activity.
  • other active compounds include, but are not limited to, immunomodulators and other antiviral agents.
  • the HCV includes a plurality of genotypes thereof and a plurality of gene subtypes, such as 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 6a.
  • the application provides a method of treating a hepatitis C virus infection, the method comprising administering to a patient in need of treatment a therapeutically effective amount of a compound of Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id, or a pharmaceutically acceptable salt thereof Or tautomers, or stereoisomers, or deuterated compounds and mixtures thereof, or the above pharmaceutical compositions.
  • a therapeutically effective amount is from about 0.0001 to 20 mg/kg body weight per day, such as from 0.001 to 10 mg/kg body weight per day.
  • the dosage frequency of a compound of Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id is determined by the needs of the individual patient, for example, one or two times per day, or more times per day. Administration can be intermittent, for example, wherein within a period of several days, the patient receives a daily dose of a compound of Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id, followed by several days or more During this period, the patient does not receive a daily dose of a compound of Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id.
  • the present application provides a compound of Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id for use in the treatment of hepatitis C virus infection, or a pharmaceutically acceptable salt thereof, or a tautomer, or a stereoisomer thereof Or progeny and mixtures thereof, as well as the above pharmaceutical compositions for the treatment of hepatitis C virus infection.
  • the present application provides a compound of Formula I or Formula Ia or Formula Ib or Formula Ic or Formula Id, or a pharmaceutically acceptable salt, or tautomer thereof, or a stereoisomer, or a deuterated compound, and mixtures thereof, or Use of the above pharmaceutical composition for the treatment of hepatitis C virus infection.
  • C mn means having mn carbon atoms in this moiety.
  • C1-6 alkyl means that the alkyl group has from 1 to 6 carbon atoms.
  • C1-6 means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms.
  • any variable eg, R
  • its definition in each case is independent.
  • each R has an independent option.
  • the substituent When a bond of a substituent is cross-linked to two atoms on a ring, the substituent may be bonded to any atom on the ring.
  • a structural unit It is indicated that it can be substituted at any position on the cyclohexyl or cyclohexadiene.
  • halogen means fluoro, chloro, bromo or iodo.
  • alkyl refers to a straight or branched saturated aliphatic hydrocarbon group consisting of a carbon atom and a hydrogen atom, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, ⁇ , ⁇ , etc.
  • the specific alkyl group includes all isomeric forms thereof, for example, the propyl group includes -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , for example, butyl includes -CH 2 CH 2 CH 2 CH 3 ,- CH(CH 3 )(CH 2 CH 3 ), -C(CH 3 ) 3 , -CH 2 CH(CH 3 ) 2 .
  • C1-6 alkyl refers to an alkyl group having from 1 to 6 carbon atoms.
  • C 1-4 alkyl refers to an alkyl group having from 1 to 4 carbon atoms.
  • C1-6 alkoxy refers to an -O-alkyl group having from 1 to 6 carbon atoms.
  • C 1-4 alkoxy refers to an -O-alkyl group having from 1 to 4 carbon atoms.
  • alkoxyalkyl refers to an alkyl group substituted with at least one alkoxy group, that is, at least one hydrogen atom of the alkyl group is substituted with an alkoxy group.
  • alkoxymethyl methoxymethyl, ethoxymethyl, methoxyethyl, ethoxyethyl, methoxypropyl, ethoxypropyl and isopropoxymethyl.
  • 2-methoxy-ethyl 1-, 2- or 3-methoxypropyl, 2-ethoxyethyl.
  • aryl refers to an all-carbon monocyclic or polycyclic fused aromatic ring group having a conjugated ⁇ -electron system, preferably having from 6 to 14 carbon atoms, more preferably from 6 to 12 carbon atoms, most It preferably has 6 carbon atoms.
  • a monocyclic aromatic ring group is selected from phenyl
  • a bicyclic fused aromatic ring group consists of a phenyl group fused to a 4-6 membered aromatic or non-aromatic carbocyclic ring, including a naphthyl group.
  • saturated fatty ring refers to a carbocyclic ring that is fully saturated and can exist as a single ring, bridged ring or spiro ring. Unless otherwise indicated, the carbocyclic ring is typically a 3-10 membered ring.
  • saturated aliphatic rings include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, bicyclo [2.2.1] heptane, bicyclo [2.2.2] octane Alkane, adamantane, etc.
  • saturated aliphatic ring containing silicon means that 1, 2 or 3 carbon atoms in the above “saturated aliphatic ring” are replaced by silicon atoms.
  • member refers to the number of atoms formed into a ring, for example, "3-8 yuan” or “4-6 yuan” means that the number of atoms in the ring is 3-8 or 4-6, and 4 containing 1 silicon.
  • the number of ring-forming atoms is four, of which three are carbon atoms and one is a silicon atom.
  • pharmaceutically acceptable is for those compounds, materials, compositions and/or dosage forms that are within the scope of sound medical judgment and are suitable for use in contact with human and animal tissues without Many toxic, irritating, allergic reactions or other problems or complications are commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt that retains the biological effectiveness of the free acids and bases of a particular compound without biologically adverse effects.
  • a pharmaceutically acceptable salt for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, or the like can be mentioned. .
  • the pharmaceutically acceptable salts of the present application can be synthesized from the parent compound containing an acid group or a base by conventional chemical methods.
  • such salts are prepared by reacting these compounds in water or an organic solvent or a mixture of the two via a free acid or base form with a stoichiometric amount of a suitable base or acid.
  • a nonaqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.
  • tautomer or "tautomeric form” refers to structural isomers of different energies that are interconvertible via a low energy barrier.
  • proton tautomers also known as proton transfer tautomers
  • proton transfer tautomers include interconversions via proton transfer, such as keto-enol and imine-enamine isomerization.
  • a specific example of a proton tautomer is an imidazole moiety in which a proton can migrate between two ring nitrogens.
  • Valence tautomers include recombination through some recombination of bonding electrons.
  • Non-limiting examples of tautomers include, but are not limited to, versus versus
  • stereoisomer refers to isomers of the same molecular structure of the compound, but which differ in stereostructure, such as enantiomers and diastereomers.
  • an "effective amount” or “therapeutically effective amount” with respect to a pharmaceutical or pharmacologically active agent refers to a sufficient amount of a drug or agent that is non-toxic but that achieves the desired effect.
  • an "effective amount” of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition.
  • the determination of the effective amount will vary from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, and a suitable effective amount in a case can be determined by one skilled in the art based on routine experimentation.
  • the compounds of the present application may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound.
  • radiolabeled compounds can be used, such as deuterium (2 H), tritium (3 H), iodine -125 (125 I) or C-14 (14 C). All isotopic compositional changes of the compounds of the present application, whether radioactive or not, are included within the scope of the present application.
  • substitution with heavier isotopes such as deuterium (ie, 2 H) (eg, formation of deuterated species) may provide certain therapeutic advantages resulting from greater metabolic stability (eg, increased in vivo half-life or reduced dosage requirements), And thus may be preferred in certain circumstances, wherein the hydrazine substitution may be partial or complete, and partial hydrazine substitution means that at least one hydrogen is replaced by at least one hydrazine.
  • deuterium ie, 2 H
  • partial hydrazine substitution means that at least one hydrogen is replaced by at least one hydrazine.
  • the specific position of the deuteration may be that the hydrogen in the methoxy group of the compound of formula I or formula Ia or formula Ib or formula Ic or formula Id is substituted by one or two or three hydrazines; it may also be in the phenyl ring Hydrogen is replaced by one or two or three or four or five hydrazines.
  • deuterated compounds are shown below, but are not limited thereto.
  • the compounds of the present application may contain asymmetrically substituted carbon atoms known as chiral centers. These compounds may, without limitation, be a single stereoisomer (eg, a single enantiomer or a single diastereomer), a mixture of stereoisomers (eg, a mixture of enantiomers or diastereomers). Or in the form of a racemic mixture.
  • Compounds identified herein as single stereoisomers are intended to describe compounds that are substantially free of other stereoisomers. "Substantially free” means that at least 95%, 96%, 97%, 98% or 99% of the compound in the composition is the stereoisomer described.
  • R 7 and R 8 may contain a chiral carbon atom, specifically, when R 7 and R 8 are -CH (C 1-4 Alkyl)(C 1-4 alkoxy), wherein at least one of the chiral carbon atoms is -C*H(C 1-4 alkyl)(C 1-4 alkoxy), wherein C* is The chiral carbon atoms may be in the S configuration or the R configuration, respectively. More specifically, when R 7 and R 8 are -CH(CH 3 )(OCH 3 ), the specific configurations are:
  • stereoisomers of the compounds of the present application can be prepared using a variety of methods known in the art. These methods include, but are not limited to, stereospecific synthesis, chromatographic separation of diastereomers, chromatographic resolution of enantiomers, enantiomeric enantiomeric conversion of enantiomeric mixtures to diastereomers, and subsequent diastereomeric chromatography. Separation and regeneration of individual enantiomers, as well as enzymatic resolution.
  • the compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, combinations thereof with other chemical synthesis methods, and equivalents well known to those skilled in the art. Alternatively, preferred embodiments include, but are not limited to, embodiments of the present application.
  • reaction materials and reaction reagents of the present application can be obtained by purchase or preparation.
  • the compounds of formula I of the present application can be prepared by the following general procedures in the art: wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 R a , R b , X, Y, m, n are as defined above.
  • the method of constructing a fragment can construct a structural fragment first and then construct another different structural fragment.
  • Compound 8 can be prepared as follows:
  • Compound 1 is subjected to a substitution reaction to obtain Compound 2, which is condensed with benzylamine in the presence of a base to form a ring, and a benzyl protecting group is removed to obtain Compound 3, and Compound 3 and Compound 4 are subjected to N-alkylation in the presence of a base.
  • Compound 5 was obtained, and further nitro reduction gave Compound 8.
  • the present application also provides a process for the preparation of the compounds of Formula Ia, Formula Ib, Formula Ic, and Formula Id.
  • the compounds of the formula Ia, the formula Ib, the formula Ic and the formula Id of different configurations can be obtained directly by chromatography column chromatography.
  • Nuclear magnetic resonance chromatography was measured using a Varian VNMR S-400 nuclear magnetic resonance spectrometer; LC/MS was performed using FINNIGAN Thermo LCQ Advantage MAX, Agilent LC 1200 series (column: Waters Symmetry C18, Mm, 5 microns, 35 ° C), using ESI (+) ion mode.
  • TEA triethylamine
  • DMF N,N-dimethylformamide
  • THF tetrahydrofuran
  • h stands for hour, for example "24h” stands for 24 hours
  • room temperature stands for 20-25 °C
  • Boc- represents a tert-butoxycarbonyl group
  • PMB- represents a p-methoxybenzyl group
  • Ms- represents a methylsulfonyl group.
  • Step 1 1-(2,6-Difluoro-4-nitrophenyl)-4,4-dimethyl-1,4-silhapiperidine
  • Step 1 1-(2-Fluoro-4-nitrophenyl)-4,4-dimethyl-1,4-silhapiperidine
  • 1,1-Divinylsilylcyclopentane (19.64 g, 142.4 mmol) was dissolved in 150 mL of n-heptane, benzoyl peroxide (0.06 g, 0.23 mmol) was added and stirred at 40 ° C for 1 h.
  • the dry hydrogen bromide gas was continuously supplied to the reaction liquid until the reaction was completed.
  • the reaction was cooled to room temperature, and the reaction mixture was washed with saturated aqueous sodium carbonate (100 mL ⁇ 3).
  • reaction solution was poured into water (300 mL), stirred for 10 min, suction filtered, washed with water (10 mL ⁇ 3), and dried to give 8-(2,6-difluoro-4-nitrophenyl)-8-aza 5-silane [4.5] decane (2.9 g).
  • Example 5 ((2S,2'S,3R,3'R)-((2S,2'S)-((((2S,5S)-1-(4-(4S,5S)-1-(4-(4,4-dimethylsilyl) Pyridin-1-yl)-3,5-difluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl)) Methyl bis(pyrrolidine-2,1-diyl))bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamate (1a)
  • Step 1 1-(4-((2S,5S)-2,5-bis(4-nitrophenyl)pyrrolidin-1-yl)-2,6-difluorophenyl)-4,4- Dimethylsilylpiperidine
  • Step 2 4,4'-((2S,5S)-1-(4-(4,4-Dimethylsilylpiperidin-1-yl)-3,5-difluorophenyl)pyrrolidine- 2,5-diyl)diphenylamine
  • Step 3 ((2S,2'S,3R,3'R)-((2S,2'S)-((((2S,5S)-1-(4-(4S,5S)-1-(4-(4,4-dimethylsilylpiperidine) -1-yl)-3,5-difluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl)bis(carbonyl)) bis ( Methyl pyrrolidine-2,1-diyl))bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamate (1a)
  • Example 6 ((2S,2'S,3R,3'R)-((2S,2'S)-((((2R,5R)-1-(4-(4,5,5R)-1-(4-(4,4-dimethylsilyl) Pyridin-1-yl)-3,5-difluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl)bis(carbonyl)) double (pyrrolidine-2,1-diyl)) bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (1b)
  • Example 7 ((2S,2'S,3R,3'R)-((2S,2'S)-((((2S,5S)-1-(4-(4S,5S)-1-(4-(4,4-dimethylsilyl) Pyridin-1-yl)-3-fluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl)bis(carbonyl))bis(pyrrolidine) -2,1-diyl)) bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (2a)
  • Step 1 1-(4-((2S,5S)-2,5-bis(4-nitrophenyl)pyrrolidin-1-yl)-2-fluorophenyl)-4,4-dimethyl Silazepidine
  • Step 2 4,4'-((2S,5S)-1-(4-(4,4-Dimethylsilylpiperidin-1-yl)-3-fluorophenyl)pyrrolidine-2,5 -diyl)diphenylamine
  • Methylthiazepine (1.67 g, 3.13 mmol) was dissolved in 20 mL of tetrahydrofuran, and platinum dioxide (0.7 g, 3.13 mmol) was added thereto, and hydrogenation was carried out for 3 hours under normal temperature and normal pressure.
  • Step 3 ((2S,2'S,3R,3'R)-((2S,2'S)-((((2S,5S)-1-(4-(4S,5S)-1-(4-(4,4-dimethylsilylpiperidine) -1-yl)-3-fluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl)bis(carbonyl))bis(pyrrolidine- 2,1-diyl))bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (2a)
  • Example 8 ((2S,2'S,3R,3'R)-((2(2R,5R)-1-(4-(4R,5R)-1-(4-(4,4-dimethylsilyl) Pyridin-1-yl)-3-fluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl)bis(carbonyl))bis(pyrrolidine) -2,1-diyl)) bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (2b)
  • Example 9 ((2S,2'S,3R,3'R)-((2S,2'S)-((((2S,5S)-1-(3,5-difluoro-4-(8-nitrogen) Hetero-5-silhaspiro[4.5]decane-8-yl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl)) double (carbonyl)) bis(pyrrolidine-2,1-diyl)(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (3a)
  • Step 1 8-(4-((2S,5S)-2,5-bis(4-nitrophenyl)pyrrolidin-1-yl)-2,6-difluorophenyl)-8-aza -5-silicon snail [4.5] decane
  • Step 2 4,4'-((2S,5S)-1-(3,5-Difluoro-4-(8-aza-5-siliro[4.5]decane-8-yl)phenyl) Pyrrolidine-2,5-diyl)diphenylamine
  • Step 3 ((2S,2'S,3R,3'R)-((2S,2'S)-((((2S,5S)-1-(3,5-difluoro-4-(8-aza) -5-Silipospiro[4.5]decane-8-yl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl))bis ( Carbonyl)) bis(pyrrolidine-2,1-diyl)(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (3a)
  • Step 1 1-(4-((2S,5S)-2,5-bis(4-chloro-3-nitrophenyl)pyrrolidin-1-yl)-2,6-difluorophenyl)- 4,4-dimethyl-1,4-siperpiperidine
  • Step 2 5,5'-((2S,5S)-1-(4-(4,4-dimethyl-1,4-silhapiperidin-1-yl)-3,5-difluorobenzene Pyrrolidine-2,5-diyl)bis(N-(4-methoxybenzyl)-2-nitroaniline)
  • Step 3 5,5'-((2S,5S)-1-(4-(4,4-Dimethyl-1,4-silhapiperidin-1-yl)-3,5-difluorobenzene Pyrrolidine-2,5-diyl)bis(2-nitroaniline)
  • Step 4 ((2S,2'S,3R,3'R)-((2S,2'S)-((((2S,5S)-1-(4-(4,5,5S)-1-(4-(4,4-dimethyl-1,4) -silhapiperidin-1-yl)-3,5-difluorophenyl)pyrrolidine-2,5-diyl)bis(2-nitro-4,1-phenylene)bis(azane II Methyl) bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester
  • Step 5 ((2S,2'S,3R,3'R)-((2S,2'S)-((((2S,5S)-1-(4-(4S,5S)-1-(4-(4,4-dimethyl-1,4) -silhapiperidin-1-yl)-3,5-difluorophenyl)pyrrolidine-2,5-diyl)bis(2-amino-4,1-phenylene)bis(azanediyl) )) bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester
  • Step 6 (2S, 2'S, 3R, 3'R)-((2S,2'S)-((2S,5S)-1-(4-(4,4-dimethyl-1,4-silica) Piperidin-1-yl)-3,5-difluorophenyl)pyrrolidine-2,5-diyl)bis(1H-benzo[d]imidazol-5,2-diyl))bis(pyrrolidine) -2,1-diyl)) bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (4a)
  • Example 11 ((2S,2'S,3R,3'R)-((2S,2'S)-((2R,5R)-1-(4-(4,4-Dimethylsilylpiperidine)- 1-yl)-3,5-difluorophenyl)pyrrolidine-2,5-diyl)bis(1H-benzo[d]imidazol-5,2-diyl))bis(pyrrolidine-2, 1-Diyl)) bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (4b)
  • Example 12 (2S, 2'S, 3R, 3'R)-((2S,2'S)-((2S,5S)-1-(4-(4,4-dimethyl-1,4-silicon) Heteropiperidin-1-yl)-3-fluorophenyl)pyrrolidine-2,5-diyl)bis(1H-benzo[d]imidazol-5,2-diyl))bis(pyrrolidine-2 ,1-diyl))bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (5a)
  • Example 13 ((2S,2'S,3R,3'R)-((2S,2'S)-((2R,5R)-1-(4-(4,4-Dimethylsilylpiperidine)- 1-yl)-3-fluorophenyl)pyrrolidine-2,5-diyl)bis(1H-benzo[d]imidazol-5,2-diyl))bis(pyrrolidine-2,1-di Methyl) bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (5b)
  • Example 14 ((2S,3R)-1-((S)-2-(6-((2S,5S)-1-(4-(4,4-dimethylsilylpiperidin-1-) -3,5-difluorophenyl)-5-(4-((S)-1-(N-(methoxycarbonyl)-O-methyl-L-threonyl)pyrrolidine-2 -formylamino)phenyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-2-yl)pyrrolidin-1-yl)-3-methoxy-1-oxobutane- 2-based) methyl carbamate (6a)
  • Step 1 1-(4-((2S,5S)-2-(4-Chloro-3-nitrophenyl)-5-(4-nitrophenyl)pyrrolidin-1-yl)-2, 6-difluorophenyl)-4,4-dimethyl-1,4-silhapiperidine
  • Step 2 (S)-2-((4-((2S,5S)-1-(4-(4,4-dimethylsilylpiperidin-1-yl)-3,5-difluorobenzene) 5-(4-nitrophenyl)pyrrolidin-2-yl)-2-nitrophenyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester
  • Step 3 (S)-2-((2-Amino-4-((2S,5S)-5-(4-aminophenyl)-1-(4-(4,4-dimethylsilyl) Tert-butyl ester of pyridin-1-yl)-3,5-difluorophenyl)pyrrolidin-2-yl)phenyl)carbamoyl)pyrrolidine-1-carboxylate
  • Step 4 (S)-2-((2-Amino-4-((2S,5S)-5-(4-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-yl) Amino)phenyl)1-(4-(4,4-dimethylsilylpiperidin-1-yl)-3,5-difluorophenyl)pyrrolidin-2-yl)phenyl)carbamoyl Pyrrolidine-1-carboxylic acid tert-butyl ester
  • Step 5 (S)-2-((4-((2S,5S)-5-(2-((S)-1-(tert-Butoxycarbonyl)pyrrolidin-2-yl)-1H-benzene) And [d]imidazol-6-yl)-1-(4-(4,4-dimethylsilylpiperidin-1-yl)-3,5-difluorophenyl)pyrrolidin-2-yl) Phenyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester
  • Step 6 (S)-N-(4-((2S,5S)-1-(4-(4,4-dimethylsilylpiperidin-1-yl)-3,5-difluorophenyl) -5-(2-((S)-pyrrolidin-2-yl)-1H-benzo[d]imidazol-6-yl)pyrrolidin-2-yl)phenyl)pyrrolidine-2-carboxamide (S)-2-((4-((2S,5S)-5-(2-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-1H-benzo[d] Imidazol-6-yl)-1-(4-(4,4-dimethylsilylpiperidin-1-yl)-3,5-difluorophenyl)pyrrolidin-2-yl)phenyl) tert-Butyl carbamoyl)pyrrolidine-l-carboxylate (0.17 g, 0.
  • Step 7 ((2S,3R)-1-((S)-2-(6-((2S,5S)-1-(4-(4,4-dimethylsilylpiperidin-1-yl) -3,5-difluorophenyl)-5-(4-((S)-1-(N-(methoxycarbonyl)-O-methyl-L-threonyl)pyrrolidine-2- Formylamino)phenyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-2-yl)pyrrolidin-1-yl)-3-methoxy-1-oxobutane-2 -yl)methyl carbamate (6a)
  • Example 15 ((2S,3R)-1-((S)-2-(6-((2R,5R)-1-(4-(4,4-dimethyl-siperpiperidin-1) -yl)-5-(4-((S)-1-(N-(methoxycarbonyl)-O-methyl-L-threonyl)pyrrolidine-2-carboxamido)phenyl)pyrrolidine Methyl-2-phenyl)-benzo[d]imidazol-2-yl)pyrrolidin-1-yl)-3-methoxy-1-oxobutan-2-yl)carbamate (6b)
  • Example 16 ((2S,2'S,3R,3'R)-((2S,2'S)-((2R,5R)-1-(3,5-difluoro-4-(8-aza-)- 5-Siro[4.5]decane-8-yl)phenyl)pyrrolidine-2,5-diyl)bis(1H-benzo[d]imidazol-5,2-diyl))bis(pyrrolidine) -2,1-diyl)) bis-1-oxobutane-1,2-diyl))dicarbamic acid methyl ester (7b)
  • Example 17 ((2S,3R)-1-((S)-2-((4-((2R,5R)-1-(4-(4,4,5R)-1-(4-(4,4-dimethylsilylpiperidin-1) -yl)-3,5-difluorophenyl)-5-(4-(2-((S)-1-(N-(methoxycarbonyl)-O-methyl-L-threonyl) Pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl)pyrrolidin-2-yl)phenyl)carbamoyl)pyrrolidin-1-yl)-3-methoxy-1-oxo Methyl butane-2-yl)carbamate (8b)
  • Step 1 1-(4-((2R,5R)-2-(4-bromophenyl)-5-(4-nitrophenyl)pyrrolidin-1-yl)-2,6-difluorobenzene -4,4-dimethylsilylpiperidine
  • Step 2 1-(2,6-Difluoro-4-((2R,5R)-2-(4-nitrophenyl)-5-(4-(4,4,5,5-tetramethyl) -1,3,2-dioxaborolan-2-yl)pyridin-2-yl)phenyl)pyrrolidin-1-yl)phenyl)-4,4-dimethylsilylpiperidine
  • Step 3 (S)-2-(5-(4-((2R,5R)-1-(4-(4,4-dimethyl-1,4-thiapiperidin-1-yl)-) 3,5-Difluorophenyl))-5-(4-nitrophenyl)pyrrolidin-2-yl)phenyl)-1H-imidazol-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester
  • Step 4 (S)-2-(5-(4-((2R,5R)-5-(4-Aminophenyl)-1-(4-(4,4-dimethyl-)-1,4- Siloxapiperidin-1-yl)pyridin-2-yl)-3,5-difluorophenyl)pyrrolidin-2-yl)phenyl)-1H-imidazol-2-yl)pyrrolidine-1-carboxylate Tert-butyl acid
  • Step 5 (S)-2-((4-((2R,5R)-5-(4-(2-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl))- 5-yl)phenyl)-1-(4-(4,4-dimethyl-1,4-thiapiperidin-1-yl)-3,5-difluorophenyl)pyrrolidine-2- Tert-butyl)phenyl)carbamoyl)-1-carboxylate
  • Step 6 (S)-N-(4-((2R,5R)-1-(4-(4,4-dimethyl-1,4-thiapiperidin-1-yl)-3,5 -difluorophenyl)-5--(2-((S)-pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl)pyrrolidin-2-yl)phenyl)pyrrolidine- 2-formamide
  • Step 7 Methyl ((2S,3R)-1-((S)-2-((4-((2R,5R))-(4-(4,4-dimethyl-)-1,4- Silazepidin-1-yl)-3,5-difluorophenyl)-5-(4-(2-((S)-1-(N-(methoxycarbonyl))-O-methyl- L-threonyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl)pyrrolidin-2-yl)phenyl)carbamoyl)pyrrolidin-1-yl)-3-A Methyl oxy-1-oxobutan-2-yl)carbamate (8b)
  • Example 18 ((2S,3R)-1-((2S,4S)-2-((4-((2R,5R))-(4-(4,4-, -silhapiperidin-1-yl)-3,5-difluorophenyl)-5-(6-fluoro-2-((S)-1-(N-(methoxycarbonyl)-O-- --L-threonyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-5-yl)pyrrolidin-2-yl)phenyl)carboxamide)-4-(methoxy) Methyl pyrrolidin-1-yl)-3-methoxy-1-oxobutan-2-yl)carbamate (17b)
  • Step 1 1-(4-((2R,5R)-2-(4-chloro-2-fluoro-5-nitrophenyl)-5-(4-nitrophenyl)pyrrolidin-1-yl )-2,6-difluorophenyl)-4,4-dimethyl-1,4-silhapiperidine
  • reaction mixture was cooled to room temperature and poured into 6N aqueous hydrochloric acid (200 mL), and ethyl acetate (200 mL ⁇ 2). The organic layer was washed with water (200 mL) and brine (200 mL) Filtration and concentrating, the title compound (m.
  • Step 2 (S)-2-((4-((2R,5R)-1-(4-(4,4-dimethyl-1,4-thiapiperidin-1-yl)-3, 5-Difluorophenyl)-5-(4-nitrophenyl)pyrrolidin-2-yl)-5-fluoro-2-nitrophenyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester
  • Step 3 (S)-2-((2-Amino-4-((2R,5R)-5-(4-aminophenyl)-1-(4-(4,4-dimethyl-1), 4-Sipiperidin-1-yl)-3,5-difluorophenyl)pyrrolidin-2-yl)-5-fluorophenyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester
  • Step 4 (2S,4S)-2-((4-((2R,5R)-5-(5-Amino-4-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-yl) Amido)-2-(4-(4,4-dimethyl-1,4-sialopiperidin-1-yl)-3,5-difluorophenyl)pyrrolidin-2-yl)phenyl ) carbamoyl)-4-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester
  • Step 5 (2S,4S)-2-((4-((2R,5R)-5-(2-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-6-) Fluoro-benzo[d]imidazol-5-yl)-1-(4-(4,4-dimethyl-1,4-sialopiperidin-1-yl)-3,5-difluorophenyl Pyrrolidin-2-yl)phenyl)carbamoyl)-4-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester
  • Step 6 (2S,4S)-N-(4-((2R,5R)-1-(4-(4,4-dimethyl-1,4-thiapiperidin-1-yl)-3) ,5-difluorophenyl)-5-(6-fluoro-2-((S)-pyrrolidin-2-yl)-1H-benzo[d]imidazol-5-yl)pyrrolidin-2-yl Phenyl)-4-(methoxymethyl)pyrrolidine-2-carboxamide
  • Step 1-6 (2S,4S)-N-(4-((2R,5R)-1-(4-(4,4-dimethyl-1,4-thiapiperidin-1-yl)) -3,5-difluorophenyl)-5-(2-((S)-pyrrolidin-2-yl)-1H-benzo[d]imidazol-5-yl)pyrrolidin-2-yl)benzene 4-(methoxymethyl)pyrrolidine-2-carboxamide
  • Test Example 1 Determination of specific compound anti-hepatitis C virus GT1b/3a and GT1b/6a NS5A chimeric replicons and GT1b and GT1a wild-type replicon activities
  • DMEM cell culture solution Invitrogen Fetal bovine serum (FBS) Corning Glutamine (L-Glu) Invitrogen Penicillin-streptomycin Hyclone Non-essential amino acids (NEAA) Invitrogen Dimethyl sulfoxide (DMSO) Sigma CellTiter-Fluor detection reagent Promega Britelite plus detection reagent Perkin Elmer
  • the compound DMSO stock was diluted according to the final concentration of the compound of Table 2 and added to a 96-well assay plate. The final concentration of DMSO was 0.5%. The final concentration of the compound was measured in Table 2.
  • Replicon plasmid DNA is linearized with the corresponding restriction enzymes, and the DNA is transcribed into RNA using T7 RNA polymerase.
  • Replicon RNA prepared in vitro transcription was transfected into Huh-7 cells by electroporation. The transfected cells were seeded at a density of 10,000 cells per well into 96-well assay plates containing diluted compounds. It was then cultured for 3 days in a 5% CO 2 , 37 ° C incubator.
  • Cell viability assay CellTiter-Fluor was added to the 96-well plate of the GT1b wild-type replicon, and the cells were cultured for 1 hour at 37 ° C in a 5% CO 2 incubator. The Fluorescence signal value (RFU) was detected by Envision. Raw data (RFU) was used for compound cytotoxicity calculations.
  • Luminescence signal value was detected with Envi sion in 15 minutes using the luciferase assay reagent Britelite plus, and raw data (RLU) was used for compound activity calculation.
  • Inhibition rate Inhibition% (ZPE-CPD)/(ZPE-HPE)x100%*
  • HPE Hundred percent effect
  • ZPE Zinc percent e effect
  • Percent cell viability (Viability%) and percent inhibition (Inhibition%) were separately introduced into GraphPad Prism software, and the curve was fitted by nonlinear regression method to obtain the corresponding curve of the compound and its cytotoxicity (CC 50 ) and its response to HCV replicon. Inhibitory activity (EC 50 ) value.
  • Test Example 2 Determination of specific compound anti-hepatitis C virus GT1a and GT1b wild-type replicons and GT1b/3a, GT1b/4a, GT1b/5a and GT1b/6a NS5A chimeric replicon activities
  • Huh-7 cells are liver cancer cell lines (derived from AppTec, USA), HCV GT1a and GT1b wild type (wt) replicons, GT1b/3a, GT1b/4a, GT1b/5a and GT1b/6a NS5A chimeric replicons .
  • DMEM medium Invitrogen Fetal bovine serum (FBS) Corning Penicillin-streptomycin Hyclone Non-essential amino acids (NEAA)
  • NEAA Invitrogen Glutamine
  • DMSO Dimethyl sulfoxide
  • Compound Treatment Compound DMSO stock was diluted according to the final concentration of the compound of Table 5 and added to a 96-well assay plate. The final concentration of DMSO was 0.5%. The final concentration of the compound detected is shown in Table 5.
  • Replicon plasmid DNA is linearized with the corresponding restriction enzymes, and the DNA is transcribed into RNA using T7 RNA polymerase.
  • Replicon RNA prepared in vitro transcription was transfected into Huh-7 cells by electroporation. The transfected cells were seeded at a density of 10,000 cells per well into 96-well assay plates containing diluted compounds. It was then cultured for 3 days in a 5% CO 2 , 37 ° C incubator.
  • Cell viability assay CellTiter-Fluor was added to the 96-well plate of the GT1b wild-type replicon, and the cells were cultured for 1 hour at 37 ° C in a 5% CO 2 incubator. The Fluorescence signal value (RFU) was detected by Envision. Raw data (RFU) was used for compound cytotoxicity calculations.
  • Luminescence signal value was detected with Envision in 15 minutes using the luciferase assay reagent Britelite plus, and raw data (RLU) was used for compound activity calculations.
  • the cytotoxicity calculation of the compound is as follows:
  • Inhibition rate Inhibition% (ZPE-CPD)/(ZPE-HPE)x100%*
  • HPE Hundred percent effect
  • ZPE Zero percent effect: Ineffective control cell signal value, with 0.5% DMSO instead of compound.
  • Percent cell viability (Viability%) and percent inhibition (Inhibition%) were separately introduced into GraphPad Prism software, and the curve was fitted by nonlinear regression method to obtain the corresponding curve of the compound and its cytotoxicity (CC 50 ) and its response to HCV replicon. Inhibitory activity (EC 50 ) value.
  • Test Example 3 Determination of specific compound anti-infective hepatitis C virus HCVc (GT2a, JFH-1) activity
  • the main reagents are shown in Table 4 above.
  • the compound DMSO stock was diluted according to the final concentration of the compound of Table 6 and added to a 96-well assay plate. The final concentration of DMSO was 0.5%. The final concentrations of the compounds were tested in Table 6.
  • Drug activity assay The activity of hRLuc was detected by the addition of Renilla luciferase assay reagent. Raw data (RLU) was used for compound activity calculations.
  • Cell viability assay Cell viability was measured by adding alamarBlue assay reagent. Raw data (RFU) was used for compound cytotoxicity calculations.
  • Inhibition rate.% (CPD-VC)/(CC-VC) ⁇ 100%*
  • VC virus Control
  • CC Cell Control: Cell control. Cell control wells, containing only cells, no virus.
  • the percentage of cell viability (% cell viability) and the percentage of compound activity (% inhibition rate) were separately introduced into GraphPad Prism software, and the curve was fitted by nonlinear regression method to obtain the corresponding curve of the compound and its cytotoxicity (CC 50 ) and its pair. HCV replicon inhibitory activity (EC 50) values.
  • Test Example 2 The test results of Test Example 2 and Test Example 3 are shown in Table 7:
  • Test Example 4 Evaluation of specific compound distribution in rat liver tissue
  • Rats can be fed 4 h after dosing.
  • mice 18 male mice were then divided into 2 groups, fasted (without water) overnight, and compound 1a and compound 6b were administered 10 mg/kg by intragastric administration.
  • the test animals were sacrificed at 0.5h, 3h, 8h.
  • mice can be fed 4 h after administration.
  • Test Example 6 Evaluation of drug resistance of specific compounds - Determination of in vitro anti-HCV GT1a wild-type replicon and activity of GT1a-NS5A-Y93H and GT1a-NS5A-L31V mutant replicons of specific compounds
  • Huh-7 cells are liver cancer cell lines (derived from AppTec, USA), HCVGT1a wild-type replicon and GT1a-NS5A-Y93H and GT1a-NS5A-L31V mutant replicons
  • the main reagents are shown in Table 10.
  • DMEM medium Invitrogen Fetal bovine serum (FBS) Corning Penicillin-streptomycin Hyclone Non-essential amino acids (NEAA) Invitrogen Glutamine (L-Glu) Invitrogen Dimethyl sulfoxide (DMSO) Sigma Bright-Glo detection reagent Promega
  • test method transient transfection method detection
  • the compound DMSO stock was diluted according to the final concentration of the compound of Table 11 and added to a 96-well assay plate. The final concentration of DMSO was 0.5%. The final concentrations of the compounds were tested in Table 11.
  • Replicon plasmid DNA is linearized with the corresponding restriction enzymes, and the DNA is transcribed into RNA using T7 RNA polymerase.
  • Replicon RNA prepared in vitro transcription was transfected into Huh-7 cells by electroporation. The transfected cells were seeded at a density of 10,000 cells per well into 96-well assay plates containing diluted compounds. It was then cultured for 3 days in a 5% CO 2 and 37 ° C incubator.
  • Luminescence signal value was detected by Envision within 5 minutes using the luciferase assay reagent Bright-Glo, and raw data (RLU) was used for compound activity calculation.
  • Inhibition rate% (CPD-ZPE)/(HPE-ZPE) ⁇ 100%*
  • the percentage of compound activity was introduced into GraphPad Prism software, and the curve was fitted by a nonlinear regression method to obtain a curve corresponding to the compound and its inhibitory activity (EC50) value for HCV replicon.

Abstract

涉及用于抗丙型肝炎病毒感染的含硅化合物，具体涉及式I所示的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物、其制备方法及包含其的药物组合物。还涉及所述化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物和包含其的药物组合物治疗丙型肝炎病毒感染的用途。

Description

用于抗丙型肝炎病毒感染的含硅化合物
相关申请的交叉引用
本申请要求于2017年03月22日向中国国家知识产权局提交的第201710172925.8号中国专利申请以及于2017年09月19日向中国国家知识产权局提交的第201710845367.7号中国专利申请的优先权和权益，所述专利申请公开的内容通过引用整体并入本文中。
技术领域
本申请属于药物化学领域，具体涉及用于抗丙型肝炎病毒感染的含硅化合物、其制备方法、含有这些化合物的药物组合物。本申请还涉及这些化合物和药物组合物用于治疗丙型肝炎病毒(HCV)感染的用途。
背景技术
HCV是一种正链RNA病毒，属于黄病毒科中的丙型肝炎病毒属，已经鉴定出至少6个主要的基因型，包含50多个亚型。单链HCV RNA基因组长度约为9500个核苷酸，具有单个可读框(ORF)，编码单个约3000个氨基酸的大型多聚蛋白。在感染细胞中，该多聚蛋白在多个位点上被细胞蛋白酶和病毒蛋白酶切割，产生结构和非结构(NS)蛋白。就HCV而言，成熟非结构蛋白(NS2、NS3、NS4A、NS4B、NS5A、NS5B)的形成是通过两种病毒蛋白酶实现的。
慢性HCV感染的治疗方案包括：聚乙二醇干扰素-α与利巴韦林组合治疗HCV感染患者和肝硬化患者，若治疗失败后再次使用含干扰素的治疗方案，持续病毒学应答率将低至14％，此外，含干扰素的治疗方案对肝硬化患者的毒副作用增加。
随着HCV NS3/4A蛋白酶抑制剂、NS5A抑制剂和NS5B聚合酶抑制剂的开发，作为新的疗法可以摆脱干扰素和利巴韦林的使用，既降低了毒性又缩短了治疗周期。这些新一类的抑制剂有ABT-267(WO2010144646)、ABT-530(WO2012051361)等。
发明概述
一方面，本申请提供了式Ⅰ化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物：
Figure PCTCN2018079965-appb-000001
其中：
R 1和R 2分别独立地选自羟基、烷基、烷氧基和芳基，或者R 1和R 2连接形成含硅的饱和脂肪环，所述含硅的饱和脂肪环含有1、2或3个硅原子；
R 3、R 4、R 5和R 6分别独立地选自氢和卤素；
X选自以下基团：
Figure PCTCN2018079965-appb-000002
Y选自以下基团：
Figure PCTCN2018079965-appb-000003
R 7和R 8分别独立地选自-CH(烷基)(烷氧基)、-CH(烷基) 2、-CH(烷氧基) 2、-C(烷基) 2(烷氧基)、-C(烷基)(烷氧基) 2、-C(烷基) 3和-C(烷氧基) 3；
R 9和R 10分别独立地选自氢、卤素、烷基、烷氧基和烷氧基烷基；
各个R a和R b分别独立地选自氢、卤素和烷基；
各个m独立地选自0、1、2、3和4；
n选自0、1、2、3、4、5、6、7和8。
另一方面，本申请提供了药物组合物，所述药物组合物包含治疗有效量的式Ⅰ化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，以及一种或多种药学上可接受的载体。
另一方面，本申请提供了式Ⅰ化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，或上述药物组合物在制备治疗丙型肝炎病毒感染的药物中的用 途。
另一方面，本申请提供了治疗丙型肝炎病毒感染的方法，所述方法包括给予需要治疗的患者治疗有效量的式Ⅰ化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，或上述药物组合物。
另一方面，本申请提供了用于治疗丙型肝炎病毒感染的式Ⅰ化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，以及用于治疗丙型肝炎病毒感染的上述药物组合物。
再一方面，本申请提供了式Ⅰ化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，或上述药物组合物在治疗丙型肝炎病毒感染中的用途。
发明详述
一方面，本申请提供了式Ⅰ化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物：
Figure PCTCN2018079965-appb-000004
其中：
R 1和R 2分别独立地选自羟基、烷基、烷氧基和芳基，或者R 1和R 2连接形成含硅的饱和脂肪环(即，R 1和R 2与它们相连的硅原子一起形成含硅的饱和脂肪环)，所述含硅的饱和脂肪环含有1、2或3个硅原子；
R 3、R 4、R 5和R 6分别独立地选自氢和卤素；
X选自以下基团：
Figure PCTCN2018079965-appb-000005
Y选自以下基团：
Figure PCTCN2018079965-appb-000006
R 7和R 8分别独立地选自-CH(烷基)(烷氧基)、-CH(烷基) 2、-CH(烷氧基) 2、-C(烷基) 2(烷氧基)、-C(烷基)(烷氧基) 2、-C(烷基) 3和-C(烷氧基) 3；
R 9和R 10分别独立地选自氢、卤素、烷基、烷氧基和烷氧基烷基；
各个R a和R b分别独立地选自卤素和烷基；或者各个R a和R b分别独立地选自氢、卤素和烷基。
各个m独立地选自0、1、2、3和4；
n选自0、1、2、3、4、5、6、7和8。
其中N上连有苯环的吡咯环(其中N为1位)的2位和5位的相对立体化学可以是顺式或反式。在一些具体的实施方案中，该吡咯环上2位和5位构型包括(2S,5S)、(2S,5R)、(2R,5S)、(2R,5R)。式Ⅰ化合物可以是一种立体异构体，也可以是两种或两种以上立体异构体任意比例的混合物。
在一些实施方案中，上述式Ⅰ化合物中，各个R a的取代位置任选是在硅原子的邻位或间位的碳原子上。
在一些实施方案中，上述式Ⅰ化合物中，各个R b的取代位置可以是任意的；优选地，X选自
Figure PCTCN2018079965-appb-000007
Y选自
Figure PCTCN2018079965-appb-000008
Figure PCTCN2018079965-appb-000009
在一些实施方案中，上述式Ⅰ化合物中，R 1、R 2、R 7、R 8、R 9、R 10、R a和R b的定义中涉及的烷基为C 1-6烷基或C 1-4烷基，R 1、R 2、R 7、R 8、R 9和R 10的定义中涉及的烷氧基为C 1-6烷氧基或C 1-4烷氧基，R 9和R 10的定义中涉及的烷氧基烷基为C 1-6烷氧基C 1-6烷基、C 1-6烷氧基C 1-4烷基或C 1-4烷氧基C 1-2烷基，R 1和R 2的定义中涉及的芳基为C 6-12芳基；R 1和R 2的定义中涉及的含硅的饱和脂肪环为3-8元或4-6元的含硅的饱和脂肪环。
在一些实施方案中，R 1和R 2分别独立地选自羟基、C 1-6烷基、C 1-6烷氧基和C 6-12芳基，或者R 1和R 2连接形成3-8元含硅的饱和脂肪环，所述3-8元含硅的饱和脂肪环含有1、2或 3个硅原子。
在一些实施方案中，R 1和R 2分别独立地选自C 1-6烷基，或者R 1和R 2连接形成3-8元含硅的饱和脂肪环，所述3-8元含硅的饱和脂肪环含有1个硅原子。
在一些实施方案中，R 1和R 2分别独立地选自甲基、乙基、正丙基、异丙基、叔丁基、正丁基、异丁基和苯基，或者R 1和R 2连接形成3元、4元、5元、6元、7元或8元的含硅的饱和脂肪环，其中所述含硅的饱和脂肪环含有1个硅原子。
在一些实施方案中，R 1和R 2分别独立地选自甲基，或者R 1和R 2连接形成4元、5元或6元含硅的饱和脂肪环，其中所述含硅的饱和脂肪环含有1个硅原子。
在一些实施方案中，R 3、R 4、R 5和R 6分别独立地选自氢、氟和氯。
在一些实施方案中，R 3和R 4各自独立地选自氢和氟，R 5和R 6选自氢。在一些实施方案中，R 3和R 4中的一个为氟，另一个为氢，R 5和R 6选自氢。优选地，R 3和R 4选自氟，R 5和R 6选自氢。
在一些实施方案中，X选自
Figure PCTCN2018079965-appb-000010
Y选自
Figure PCTCN2018079965-appb-000011
Figure PCTCN2018079965-appb-000012
在一些实施方案中，X选自
Figure PCTCN2018079965-appb-000013
Y选自
Figure PCTCN2018079965-appb-000014
在一些实施方案中，X选自
Figure PCTCN2018079965-appb-000015
Y选自
Figure PCTCN2018079965-appb-000016
Figure PCTCN2018079965-appb-000017
在一些实施方案中，X选自
Figure PCTCN2018079965-appb-000018
Y选自
Figure PCTCN2018079965-appb-000019
或者X选自
Figure PCTCN2018079965-appb-000020
Y选自
Figure PCTCN2018079965-appb-000021
或者X选自
Figure PCTCN2018079965-appb-000022
Y选自
Figure PCTCN2018079965-appb-000023
优选地，X选自
Figure PCTCN2018079965-appb-000024
Y选自
Figure PCTCN2018079965-appb-000025
或者X选自
Figure PCTCN2018079965-appb-000026
Y选自
Figure PCTCN2018079965-appb-000027
或者X选自
Figure PCTCN2018079965-appb-000028
Y选自
Figure PCTCN2018079965-appb-000029
优选地，X选自
Figure PCTCN2018079965-appb-000030
Y选自
Figure PCTCN2018079965-appb-000031
或者X选自
Figure PCTCN2018079965-appb-000032
Y选自
Figure PCTCN2018079965-appb-000033
或者X选自
Figure PCTCN2018079965-appb-000034
Y选自
Figure PCTCN2018079965-appb-000035
在一些实施方案中，X选自
Figure PCTCN2018079965-appb-000036
Y选自
Figure PCTCN2018079965-appb-000037
或者X选自
Figure PCTCN2018079965-appb-000038
Y选自
Figure PCTCN2018079965-appb-000039
优选地，X选自
Figure PCTCN2018079965-appb-000040
Y选自
Figure PCTCN2018079965-appb-000041
或者X选自
Figure PCTCN2018079965-appb-000042
Y选自
Figure PCTCN2018079965-appb-000043
优选地，X选自
Figure PCTCN2018079965-appb-000044
Y选自
Figure PCTCN2018079965-appb-000045
或者X选自
Figure PCTCN2018079965-appb-000046
Y选自
Figure PCTCN2018079965-appb-000047
在一些实施方案中，X选自
Figure PCTCN2018079965-appb-000048
Y选自
Figure PCTCN2018079965-appb-000049
在一些实施方案中，X选自
Figure PCTCN2018079965-appb-000050
Y选自
Figure PCTCN2018079965-appb-000051
在一些实施方案中，R 7和R 8分别独立地选自-CH(C 1-6烷基)(C 1-6烷氧基)、-CH(C 1-6烷基) 2、-CH(C 1-6烷氧基) 2、-C(C 1-6烷基) 2(C 1-6烷氧基)、-C(C 1-6烷基)(C 1-6烷氧基) 2、-C(C 1-6烷基) 3和-C(C 1-6烷氧基) 3。在一些实施方案中，R 7和R 8相同。
在一些实施方案中，R 7和R 8分别独立地选自-CH(CH 3)(OCH 3)、-CH(CH 3) 2、-CH(OCH 3) 2、-C(CH 3) 2(OCH 3)、-C(CH 3)(OCH 3) 2、-C(CH 3) 3、-C(OCH 3) 3、-CH(CH 2CH 3)(OCH 2CH 3)、-CH(CH 3)(OCH 2CH 3)、-CH(CH 2CH 3)(OCH 3)、-CH(CH 2CH 3) 2、-CH(CH 3)(CH 2CH 3)、-CH(OCH 2CH 3) 2、-CH(OCH 2CH 3)(OCH 3)、-C(CH 2CH 3) 2(OCH 2CH 3)、-C(CH 3) 2(OCH 2CH 3)、-C(CH 2CH 3) 2(OCH 3)、-C(CH 2CH 3)(OCH 2CH 3) 2、-C(CH 3)(OCH 2CH 3) 2、-C(CH 2CH 3)(OCH 3) 2、-C(CH 2CH 3) 3、-C(CH 3)(CH 2CH 3) 2、-C(CH 3) 2(CH 2CH 3)和-C(OCH 2CH 3) 3。
在一些实施方案中，R 9和R 10分别独立地选自氢、卤素、烷基和烷氧基。在一些实施方案中，R 9和R 10分别独立地选自氢和烷氧基烷基。
在一些实施方案中，R 9和R 10分别独立地选自氢、卤素、C 1-6烷基和C 1-6烷氧基。在一些实施方案中，R 9和R 10分别独立地选自氢和C 1-6烷氧基C 1-6烷基。在一些实施方案中，R 9和R 10相同。
在一些实施方案中，R 9和R 10分别独立地选自氢、氟、氯、甲基、乙基、正丙基、异丙基、叔丁基、正丁基、异丁基、甲氧基、乙氧基、正丙氧基、异丙氧基、叔丁氧基、正丁氧基和异丁氧基。在一些实施方案中，R 9和R 10分别独立地选自氢、甲氧基甲基、甲氧基乙基、乙氧基甲基和乙氧基乙基。在一些实施方案中，R 9和R 10分别独立地选自氢和甲氧基甲基。在一些实施方案中，R 9和R 10均为氢。在一些实施方案中，R 9和R 10之一为氢，另一个为甲氧基甲基。在一些实施方案中，R 9和R 10均为甲氧基甲基。
在一些实施方案中，R 7和R 8分别独立地选自-CH(烷基)(烷氧基)、-CH(烷基) 2和-CH(烷氧基) 2。在一些实施方案中，R 7和R 8分别独立地选自-CH(C 1-6烷基)(C 1-6烷氧基)、-CH(C 1-6烷基) 2和-CH(C 1-6烷氧基) 2。在一些实施方案中，R 7和R 8均为-CH(CH 3)(OCH 3)。
在一些实施方案中，R 7和R 8分别独立地选自-CH(CH 3)(OCH 3)和-CH(CH 3) 2；R 9和R 10分别独立地选自氢和甲氧基甲基。
在一些实施方案中，R 7和R 8分别独立地选自-CH(CH 3)(OCH 3)和-CH(CH 3) 2；R 9和R 10选自氢。
在一些实施方案中，R 7和R 8分别独立地选自-CH(CH 3)(OCH 3)和-CH(CH 3) 2，其中-CH(CH 3)(OCH 3)的手性碳原子为R构型；R 9和R 10分别独立地选自氢和甲氧基甲基。
在一些实施方案中，R 7和R 8分别独立地选自-CH(CH 3)(OCH 3)和-CH(CH 3) 2，其中-CH(CH 3)(OCH 3)的手性碳原子为R构型；R 9和R 10选自氢。
在一些实施方案中，与R 9或R 10相连的四氢吡咯环上的手性碳原子为S构型。
在一些实施方案中，各个R a和R b分别独立地选自氢、卤素和C 1-6烷基。
在一些实施方案中，各个R a和R b分别独立地选自卤素和C 1-6烷基。
在一些实施方案中，各个R a和R b分别独立地选自氟、氯、甲基、乙基、正丙基、异丙基、叔丁基、正丁基和异丁基。
在一些实施方案中，各个R a和R b分别独立地选自氢、氟、氯、甲基、乙基、正丙基、异丙基、叔丁基、正丁基和异丁基。
在一些实施方案中，n选自0，R b选自氟。
在一些实施方案中，R 1和R 2为甲基，或者R 1和R 2连接形成含有1个硅原子的4元、5元或6元含硅的饱和脂肪环；R 3和R 4中的一个为氟，另一个为氢，或者R 3和R 4均为氟；R 5和R 6为氢；X为
Figure PCTCN2018079965-appb-000052
Figure PCTCN2018079965-appb-000053
Y为
Figure PCTCN2018079965-appb-000054
R 7和R 8均为-CH(CH 3)(OCH 3)；R 9和R 10均为氢或者均为甲氧基甲基；n为0。
进一步地，本申请提供式Ⅰa、式Ⅰb、式Ⅰc或式Ⅰd的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，
Figure PCTCN2018079965-appb-000055
式Ia、式Ib、式Ic和式Id中的R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R a、R b、X、Y、m、n与上述针对式I中的R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R a、R b、X、Y、m、n的详细描述相同。其他相关描述也与式I中相同。
具体地，本申请提供了如下化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物：
Figure PCTCN2018079965-appb-000056
Figure PCTCN2018079965-appb-000057
Figure PCTCN2018079965-appb-000058
Figure PCTCN2018079965-appb-000059
Figure PCTCN2018079965-appb-000060
Figure PCTCN2018079965-appb-000061
Figure PCTCN2018079965-appb-000062
具体地，本申请提供了如下化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物：化合物1a、化合物1b、化合物2a、化合物2b、化合物3a、化合物4a、化合物4b、化合物5a、化合物5b、化合物6a、化合物6b、化合物7b、化合物8b、化合物17b和化合物18b。
具体地，本申请提供了如下化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物：化合物1a、化合物1b、化合物2a、化合物2b、化合物3a、化合物4a、化合物4b、化合物5a、化合物5b、化合物6a、化合物6b、化合物7b、化合物8a、化合物8b、化合物9b、化合物11a、化合物14b、化合物15b、化合物17b和化合物18b。
本申请另一方面提供了一种药物组合物，该药物组合物包含治疗有效量的式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，以及一种或多种药学上可接受的载体。
本申请的药物组合物可通过将本申请的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物与适宜的药学上可接受的载体组合而制备，例如可配制成固态、半固态、液态或气态制剂，如片剂、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、微球及气溶胶等。
给予本申请的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，或其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。
本申请的药物组合物可以采用本领域众所周知的方法制造，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、冷冻干燥法等等。
对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的载体混合来配制该药物组合物。这些载体能使本申请的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、 液体、凝胶剂、浆剂、悬浮剂等，用于对患者的口服给药。
可以通过常规的混合、填充或压片方法来制备固体口服组合物。例如，可通过下述方法获得：将所述的活性化合物与固体赋形剂混合，任选地碾磨所得的混合物，如果需要则加入其它合适的辅剂，然后将该混合物加工成颗粒，得到了片剂或糖衣剂的核心。适合的辅料包括但不限于：粘合剂、稀释剂、崩解剂、润滑剂、助流剂、甜味剂或矫味剂等。可以根据通常药物实践中公知的方法任选地对糖衣剂的核心进行包衣，尤其使用肠溶包衣。
药物组合物还可适用于肠胃外给药，如合适的单位剂型的无菌溶液剂、混悬剂或冻干产品。能够使用适当的赋形剂，例如填充剂、缓冲剂或表面活性剂。
再一方面，本申请的化合物(包括具体结构的化合物)具有丙型肝炎病毒(HCV)NS5A抑制活性，可作为HCV NS5A抑制剂用于丙型肝炎病毒感染的治疗，具体可用于丙型肝炎病毒感染引起的肝炎、肝硬化等肝病的治疗。
具体地，本申请化合物(包括具体结构的化合物)具有丙型肝炎病毒(HCV)NS5A抑制活性，特别地，具有对HCV的多种基因亚型具有优异的抑制活性，这些基因亚型包括1a、1b、2a、2b、3a、3b、4a、4b、5a、6a；还包括一些嵌合型1b/3a、1b/4a、1b/5a、1b/6a等。
具体地，本申请的化合物(包括具体结构的化合物)对肝脏组织具有较好的亲和力，能靶向性分布于肝脏组织中。
具体地，本申请的化合物(包括具体结构的化合物)还具有一定的耐药性。
本申请提供了式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物在制备治疗丙型肝炎病毒(HCV)感染的药物中的用途。
本申请提供了式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物与至少一种其他活性化合物在制备治疗丙型肝炎病毒(HCV)感染的药物中的用途。
本申请提供了含有式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物的药物组合物在制备治疗丙型肝炎病毒(HCV)感染的药物中的用途。
本申请提供了包含式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物与至少一种其他活性化合物的药物组合物在制备治疗丙型肝炎病毒(HCV)感染的药物中的用途。
在一些实施方式中，其他活性化合物包括但不限于抗HCV活性的其它化合物。在一些实施方式中，其他活性化合物包括但不限于免疫调节剂和其他抗病毒药。
所述的HCV包括其多种基因型以及多种基因亚型，例如1a、1b、2a、2b、3a、3b、4a、4b、5a、6a。
本申请提供了治疗丙型肝炎病毒感染的方法，所述方法包括给予需要治疗的患者治疗有效量的式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，或者上述药物组合物。
本申请所述的式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物的治疗有效量为从约0.0001到20mg/Kg体重/天，例如从0.001到10mg/Kg体重/天。
式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物的剂量频率由患者个体的需求决定，例如，每天1次或2次，或每天更多次。给药可以是间歇性的，例如，其中在若干天的期间内，患者接受式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物的每日剂量，接着在若干天或更多天的期间，患者不接受式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物的每日剂量。
本申请提供了用于治疗丙型肝炎病毒感染的式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，以及用于治疗丙型肝炎病毒感染的上述药物组合物。
本申请提供了式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，或上述药物组合物在治疗丙型肝炎病毒感染中的用途。
有关定义：
除非另有说明，本文所用的下列术语和短语具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。
术语“任选”或“任选地”是指随后描述的事件或情况可能发生或可能不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。
本文所用的C m-n指该部分中具有m-n个碳原子。例如，“C 1-6烷基”指该烷基具有1-6个碳原子。
本文中的数字范围，是指给定范围中的各个整数。例如“C 1-6”是指该基团可具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子、6个碳原子。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被2个R所取代，则每个R都有独立的选项。
当一个取代基的键交叉连接到一个环上的两个原子时，这种取代基可以与这个环上的任 意原子相键合。例如，结构单元
Figure PCTCN2018079965-appb-000063
表示其可在环己基或者环己二烯上的任意一个位置发生取代。
术语“卤素”是指氟、氯、溴或碘。
术语“烷基”是指由碳原子和氢原子组成的直链或支链的饱和脂肪烃基团，例如甲基、乙基、丙基、丁基、戊基、己基、庚基、辛基、壬基、癸基等。所述特定烷基包括其所有同分异构体形式，例如丙基包括-CH 2CH 2CH 3、-CH(CH 3) 2，例如丁基包括-CH 2CH 2CH 2CH 3、-CH(CH 3)(CH 2CH 3)、-C(CH 3) 3、-CH 2CH(CH 3) 2。
术语“C 1-6烷基”指具有1-6个碳原子的烷基。术语“C 1-4烷基”指具有1-4个碳原子的烷基。
术语“C 1-6烷氧基”指具有1-6个碳原子的-O-烷基基团。术语“C 1-4烷氧基”指具有1-4个碳原子的-O-烷基基团。
术语“烷氧基烷基”是指被至少一个烷氧基取代的烷基，也即，该烷基的至少一个氢原子被烷氧基取代。例如甲氧基甲基、乙氧基甲基、甲氧基乙基、乙氧基乙基、甲氧基丙基、乙氧基丙基和异丙氧基甲基。再例如，2-甲氧基-乙基，1-、2-或3-甲氧基丙基，2-乙氧基乙基。
术语“芳基”是指具有共轭的π电子体系的全碳单环或多环稠合的芳香环基团，优选具有6-14个碳原子，更优选具有6-12个碳原子，最优选具有6个碳原子。例如单环芳香环基团选自苯基，双环稠合的芳香环基团由与4-6元芳族或非芳族碳环稠合的苯基组成，包括萘基。
术语“饱和脂肪环”指完全饱和的并且可以以呈单环、桥环或螺环存在的碳环。除非另有指示，该碳环通常为3-10元环。饱和脂肪环非限制性实例包括但不限于环丙烷、环丁烷、环戊烷、环己烷、环庚烷、环辛烷、双环[2.2.1]庚烷、双环[2.2.2]辛烷、金刚烷等。
术语“含硅的饱和脂肪环”指上述“饱和脂肪环”中的1、2或3个碳原子被硅原子替换。
术语“元”是指成环的原子数，例如“3-8元”或“4-6元”是指成环原子数为3-8个或4-6个，而含1个硅的4元饱和脂肪环中，成环原子数为4个，其中3个为碳原子、1个为硅原子。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”指保留了特定化合物的游离酸和碱的生物学效力而没有生物学不良作用的盐。作为药学上可接受的盐，例如，可以提及金属盐、铵盐、与有机碱形成的盐、与无机酸形成的盐、与有机酸形成的盐、与碱性或者酸性氨基酸形成的盐等。
本申请的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。 一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。一般地，优选醚、乙酸乙酯、乙醇、异丙醇或乙腈等非水介质。
本申请的中间体和化合物还可以以不同的互变异构体形式存在，并且所有这样的形式包含于本申请的范围内。术语“互变异构体”或“互变异构体形式”是指可经由低能垒互变的不同能量的结构异构体。例如，质子互变异构体(也称为质子转移互变异构体)包括经由质子迁移的互变，如酮-烯醇及亚胺-烯胺异构化。质子互变异构体的具体实例是咪唑部分，其中质子可在两个环氮间迁移。价互变异构体包括通过一些成键电子的重组的互变。互变异构体的非限制性实例包括但不限于
Figure PCTCN2018079965-appb-000064
与
Figure PCTCN2018079965-appb-000065
与
Figure PCTCN2018079965-appb-000066
术语“立体异构体”指化合物分子的构造相同，但是立体结构不同而产生的异构体，例如対映异构体和非对映异构体。
针对药物或药理学活性剂而言，术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。对于本申请中的口服剂型，组合物中一种活性物质的“有效量”是指与该组合物中另一种活性物质联用时为了达到预期效果所需要的用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
本申请的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氘( 2H)、氚( 3H)、碘-125( 125I)或C-14( 14C)。本申请的化合物的所有同位素组成的变换，无论放射性与否，都包括在本申请的范围之内。
此外，用较重同位素(诸如氘(即 2H))取代(例如形成氘代物)可以提供某些由更高的代谢稳定性产生的治疗优点(例如增加的体内半衰期或降低的剂量需求)，并且因此在某些情形下可能是优选的，其中氘取代可以是部分或完全的，部分氘取代是指至少一个氢被至少一个氘取代。此外，氘代的具体位置可以是式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物中甲氧基中的氢被一个或两个或三个氘取代；也可以是苯环中的氢被一个或两个或三个或四个或五个氘取代。
例示性的氘代化合物如下所示，但不限于此。
Figure PCTCN2018079965-appb-000067
本申请的化合物可以包含被称为手性中心的被不对称取代的碳原子。这些化合物可以，在没有限制的情况下，以单个立体异构体(例如单个对映体或单个非对映体)，立体异构体的混合物(例如对映体或非对映体的混合物)，或外消旋混合物的形式存在。本申请作为单个立体异构体被识别的化合物意图描述以基本上没有其它立体异构体的形式存在的化合物。“基本上没有”是指组合物中至少95％、96％、97％、98％或99％的化合物是所描述的立体异构体。在手性碳的立体化学没有在化合物的化学结构中规定的情况下，该化学结构意图包括含手性中心的任一立体异构体的化合物。例如，式Ⅰ或式Ⅰa或式Ⅰb或式Ⅰc或式Ⅰd的化合物中，R 7和R 8可含有手性碳原子，具体来说，当R 7和R 8为-CH(C 1-4烷基)(C 1-4烷氧基)时，其中至少有一个手性碳原子，为-C*H(C 1-4烷基)(C 1-4烷氧基)，其中C*为手性碳原子，分别可以为S构型或R构型，更具体的，R 7和R 8为-CH(CH 3)(OCH 3)时，具体的构型有：
Figure PCTCN2018079965-appb-000068
本申请的化合物的单独的立体异构体可以使用各种本领域已知的方法来制备。这些方法包括但不限于立体有择合成，非对映体的色谱分离，对映体的色谱拆分，对映体混合物中的对映体转化为非对映体、随后非对映体的色谱分离和单独对映体的再生，以及酶拆分。
本申请的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术人员所熟知的等同替换方式，优选的实施方式包括但不限于本申请的实施例。
本申请的反应原料和反应试剂均可通过购买或者制备获得。
本申请具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本申请的化学变化及其所需的试剂和物料。为了获得本申请的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
本申请的式Ⅰ化合物可以由如下路线用本领域的常用方法来制备：其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R a、R b、X、Y、m、n如上定义。
1、当X选自
Figure PCTCN2018079965-appb-000069
Y选自
Figure PCTCN2018079965-appb-000070
时，
Figure PCTCN2018079965-appb-000071
化合物7和化合物8成环得到化合物9，进一步硝基还原得到化合物10，化合物10与化合物11和/或化合物11’进行酰胺化反应得到化合物12，进一步脱去氨基保护基Cbz得到化合物13，再进一步与化合物14和/或化合物14’进行酰胺化反应得到式Ⅰ化合物。
2、当X选自
Figure PCTCN2018079965-appb-000072
Y选自
Figure PCTCN2018079965-appb-000073
时，
Figure PCTCN2018079965-appb-000074
化合物15进行氨基取代反应得到化合物16，化合物16水解得到化合物16’，化合物16’与化合物17和/或化合物17’进行酰胺化反应得到化合物18，将化合物18中的硝基还原为氨基，再成环得到式Ⅰ化合物。
3、当X选自
Figure PCTCN2018079965-appb-000075
Y选自
Figure PCTCN2018079965-appb-000076
时，
Figure PCTCN2018079965-appb-000077
化合物19先与联硼酸频哪醇酯反应得到化合物20，化合物20与化合物21和/或化合物21’进行偶联反应得到化合物22，化合物22脱除保护基后与化合物23和/或化合物23’进行酰胺化反应得到式Ⅰ化合物。
当X、Y选自不同类型的片段时，可参照上述构建
Figure PCTCN2018079965-appb-000078
Figure PCTCN2018079965-appb-000079
结构片段的方法，可任意依次先构建一种结构片段，再构建另一种不同的结构片段。
其中，化合物8可按照以下方法制备：
Figure PCTCN2018079965-appb-000080
化合物1经取代反应制得化合物2，化合物2在碱的存在与苄胺缩合成环，并脱去苄基保护基得到化合物3，化合物3与化合物4在碱的存在下进行N-烃化反应制得化合物5，再进一步硝基还原得到化合物8。
本申请还提供了式Ⅰa、式Ⅰb、式Ⅰc、式Ⅰd化合物的制备方法。
在制得式Ⅰ化合物的基础上，可直接通过色谱柱层析分离得到不同构型的式Ⅰa、式Ⅰb、 式Ⅰc、式Ⅰd化合物。
也可以分别制备不同的手性中间体片段例如不同构型的化合物10、化合物15、化合物19，最终得到不同构型的式Ⅰa、式Ⅰb、式Ⅰc、式Ⅰd化合物。
具体实施方式
下面的具体实施例，其目的是使本领域的技术人员能更清楚地理解和实施本申请。它们不应该被认为是对本申请范围的限制，而只是本申请的示例性说明和典型代表。本领域技术人员应该理解：还有形成本申请化合物的其它合成途径，下面提供的是非限制性的实施例。
除非另有说明，本申请使用的原料都是市场上直接买到，未经进一步纯化直接使用的。
凡涉及易氧化或易水解的原料的所有操作都在氮气保护下进行。柱层析色谱采用青岛化工有限公司生产的硅胶(200-300目)。薄层色谱采用E.Merck公司生产的预制板(硅胶60PF254,0.25毫米)。手性化合物分离采用色谱柱：Waters XBridge C18，
Figure PCTCN2018079965-appb-000081
毫米，5微米和手性色谱柱：CHIRALPAK IA
Figure PCTCN2018079965-appb-000082
毫米，5微米。核磁共振色谱(NMR)使用Varian VNMRS-400核磁共振仪测定；液质连用(LC/MS)使用FINNIGAN Thermo LCQ Advantage MAX，Agilent LC 1200series(柱子：Waters Symmetry C18，
Figure PCTCN2018079965-appb-000083
毫米，5微米，35℃)，采用ESI(+)离子模式。
除非另有说明，TEA代表三乙胺；DMF代表N,N-二甲基甲酰胺；THF代表四氢呋喃；“h”代表小时，例如“24h”代表24小时；“室温”代表20-25℃；Boc-代表叔丁氧羰基；PMB-代表对甲氧基苄基；Ms-代表甲磺酰基。
实施例1：4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯胺
Figure PCTCN2018079965-appb-000084
步骤1：1-(2,6-二氟-4-硝基苯基)-4,4-二甲基-1,4-硅杂哌啶
4,4-二甲基硅杂哌啶盐酸盐(20.6g,124.4mmol)，三乙胺(51.6g,3373.0mmol)分散于N,N-二甲基甲酰胺(200mL)中，室温搅拌10min，移至冰水浴搅拌，滴加3,4,5-三氟硝基苯(20.0g,113.0mmol)，滴加完毕冰水浴搅拌30min，然后室温搅拌过夜。将反应液倒入1L水中，搅拌10min，抽滤，水洗(10mL×3)，干燥得1-(2,6-二氟-4-硝基苯基)-4,4-二甲基-1,4-硅杂哌啶(31.2g)。
1H NMR(500MHz,DMSO-d 6):δ7.93(dd,J＝10.0Hz,1.0Hz,2H),3.49-3.51(m,4H),0.85-0.88(m,4H),0.12(s,6H).
步骤2：4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯胺
1-(2,6-二氟-4-硝基苯基)-4,4-二甲基-1,4-硅杂哌啶(31.0g,108.3mmol)溶于四氢呋喃(200mL)，加入Pd/C(5.0g,10％,含水量56％)，氢气氛下室温反应过夜。抽滤，滤液浓缩，残留物石油醚打浆，抽滤，干燥，得4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯胺(23.4g)。
1H NMR(500MHz,DMSO-d 6):δ6.10-6.12(m,2H),5.34(s,2H),3.10-3.13(m,4H),0.77-0.79(m,4H),0.08(s,6H).
实施例2：4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3-氟苯胺
Figure PCTCN2018079965-appb-000085
步骤1：1-(2-氟-4-硝基苯基)-4,4-二甲基-1,4-硅杂哌啶
4,4-二甲基硅杂哌啶盐酸盐(19.3g,105.8mmol)，三乙胺(48.2g,349.1mmol)分散于N,N-二甲基甲酰胺(200mL)中，室温搅拌10min，移至冰水浴搅拌，滴加3,4-二氟硝基苯(16.8g,105.8mmol)，滴加完毕冰水浴搅拌30min，室温搅拌过夜。反应液倒入1L水中，搅拌10min，抽滤，大量水洗，收集固体，烘干，得1-(2-氟-4-硝基苯基)-4,4-二甲基-1,4-硅杂哌啶(28.1g)。
1H NMR(300MHz,DMSO-d 6):δ7.91-7.95(m,2H),7.10-7.14(m,1H),3.71-3.73(m,4H),0.85-0.88(m,4H),0.11(s,6H).
步骤2：4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3-氟苯胺
1-(2-氟-4-硝基苯基)-4,4-二甲基-1,4-硅杂哌啶(28.0g,104.3mmol)溶于四氢呋喃(200mL)，加入Pd/C(5.0g,10％,含水量56％)，氢气氛下室温反应过夜。反应液抽滤，滤液浓缩，残留物以石油醚(200mL)打浆，抽滤，干燥，得4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3-氟苯胺(1.2g)。
1H NMR(500MHz,DMSO-d 6):δ6.75-6.79(m,1H),6.25-6.31(m,2H),4.90(s,2H),3.04-3.06(m,4H),0.79-0.81(m,4H),0.08(s,6H)。
实施例3：8-氮杂-5-硅螺[4.5]癸烷盐酸盐
Figure PCTCN2018079965-appb-000086
步骤1：1,1-二乙烯基硅杂环戊烷
1L干燥的三颈瓶中加入1,1-二氯硅杂环戊烷(纯度90％，15.53g，90mmol)和干燥的四氢呋喃(100mL)，氮气保护下降温至0℃，缓慢滴入1M/L的乙烯基溴化镁(220mmol，220mL)，滴加过程控制内温在0℃-5℃。滴加完毕，自然升温至室温搅拌5h。然后降温至0℃，搅拌下缓慢注入水(200mL)，分液，水层以乙醚(60mL×2)萃取，合并有机层，饱和食盐水(100mL)洗涤，无水硫酸钠干燥，过滤，常压蒸除四氢呋喃和乙醚，然后减压蒸馏得到1,1-二乙烯基硅杂环戊烷(4.97g)。
1H NMR(500M Hz,CDCl 3):δ6.27-6.20(m,2H),6.07(d,J＝3.5Hz,1H),6.04(d,J＝4.0Hz,1H),5.83(d,J＝3.5Hz,1H),5.79(d,J＝4.0Hz,1H),1.66-1.63(m,4H),0.75-0.73(m,4H).
步骤2：1,1-二(2-溴乙基)硅杂环戊烷的制备
1,1-二乙烯基硅杂环戊烷(19.64g，142.4mmol)溶于150mL正庚烷中，加入过氧化苯甲酰(0.06g，0.23mmol)，40℃搅拌1h。向反应液中持续通入干燥的溴化氢气体至反应完毕。将反应冷却至室温，饱和碳酸钠水溶液洗涤反应液(100mL×3)。有机层用饱和食盐水洗涤(60mL)，无水硫酸钠干燥。过滤，减压蒸除溶剂，硅胶柱层析(石油醚/乙酸乙酯＝20/1为流动相)，得1,1-二(2-溴乙基)硅杂环戊烷(21.4g)。
1H-NMR(DMSO-d 6,500MHz):δ3.69(t,J＝9.0Hz,4H),1.5(m,8H),0.63(t,J＝3.0Hz,4H).
步骤3：8-苄基-8-氮杂-5-硅螺[4.5]癸烷
1,1-二(2-溴乙基)硅杂环戊烷(21.4g，71.83mmol)，苄胺(9.24g，86.2mmol)，三乙胺(15.99g，158.02mmol)的混合物于氯仿(100mL)中加热回流反应13h，冷却至室温，加入5％NaOH水溶液(150mL)搅拌30min，二氯甲烷萃取(100mL×3)；有机相用饱和食盐水洗涤，无水硫酸钠干燥。减压蒸除溶剂，硅胶柱层析(石油醚/乙酸乙酯＝15/1为流动相)，得8-苄基-8-氮杂-5-硅螺[4.5]癸烷(8.0g)。
1H-NMR(DMSO-d 6,500MHz)：δ7.30(m,4H),7.22(m,1H),3.50(s,2H),2.65(t,J＝6Hz,4H),1.53(m,4H),0.78(t,J＝12.5Hz,4H),0.57(t,J＝1.0Hz,4H).
MS(ESI)m/z：246.2[M+H] +.
步骤4：8-氮杂-5-硅螺[4.5]癸烷盐酸盐
8-苄基-8-氮杂-5-硅螺[4.5]癸烷(8.0g，32.63mmol)溶于80％无水甲醇(100mL)中， 依次加入0.8g的10％Pd-C、36％的浓盐酸(9.92g，97.9mmol),氢气置换三次，加热至50℃加氢还原反应15h。冷却至室温，硅藻土作铺垫过滤。减压蒸除溶剂得白色固体,乙酸乙酯/二氯甲烷(1/1)打浆，抽滤，得8-氮杂-5-硅螺[4.5]癸烷盐酸盐(5.2g)。
1H-NMR(DMSO-d 6,500MHz):δ3.21(t,J＝6.0Hz,4H),1.56(m,4H),1.06(t,J＝6.5Hz,4H),0.64(t,J＝7.0Hz,4H).
MS(ESI)m/z:156.2[M+H] +.
实施例4：3,5-二氟-4-(8-氮杂-5-硅螺[4.5]癸烷-8-基)苯胺
Figure PCTCN2018079965-appb-000087
步骤1：8-(2,6-二氟-4-硝基苯基)-8-氮杂5-硅螺[4.5]癸烷
8-氮杂-5-硅螺[4.5]癸烷盐酸盐(5.0g,26.1mmol)，三乙胺(12.0g,86.1mmol)分散于N,N-二甲基甲酰胺(50mL)中，室温搅拌10min，移至冰水浴搅拌，滴加3，4,5-三氟硝基苯(5.1g,28.7mmol)，滴加完毕冰水浴搅拌30min，室温搅拌过夜。反应液倒入水中(300mL)，搅拌10min，抽滤，水洗(10mL×3)，干燥得8-(2,6-二氟-4-硝基苯基)-8-氮杂5-硅螺[4.5]癸烷(2.9g)。
1H NMR(500MHz,CDCl 3):δ7.70-7.77(m,2H),3.55(t,J＝6.5Hz,4H),1.60-1.63(m,4H),1.00(t,J＝6.5Hz,4H),0.66(t,J＝7.0Hz,4H).
步骤2：3,5-二氟-4-(8-氮杂-5-硅螺[4.5]癸烷-8-基)苯胺
8-(2,6-二氟-4-硝基苯基)-8-氮杂5-硅螺[4.5]癸烷(2.9g,9.3mmol)溶于四氢呋喃(30mL)，加入Pd/C(0.5g,10％,含水量56％)，室温下加氢还原反应2.5h。反应液抽滤，滤液浓缩，残留物以石油醚(30mL)打浆，抽滤，干燥，得3,5-二氟-4-(8-氮杂-5-硅螺[4.5]癸烷-8-基)苯胺(2.45g)。
1H NMR(500MHz,CDCl 3):δ6.14-6.18(m,2H),3.63(s,2H),3.27-3.29(t,J＝6.5Hz,4H),1.59-1.62(m,4H),0.96(t,J＝6.0Hz,4H),0.64-0.66(m,4H).
MS(ESI)m/z:283.1[M+H] +.
实施例5：((2S,2'S,3R,3'R)-((2S,2'S)-(((((2S,5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(4,1-亚苯基))双(氮烷二基))双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(1a)
Figure PCTCN2018079965-appb-000088
步骤1：1-(4-((2S，5S)-2,5-双(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基硅杂哌啶
100mL三口瓶中，(1R，4R)-1,4-双(4-硝基苯基)丁烷-1,4-二基二甲磺酸酯(1.5g，3.0mmol)加入到10mL的N,N-二甲基甲酰胺中，加入4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯胺(7.88g，30.71mmol)，氮气保护下加热至60℃反应24h。反应结束，将反应液倒入200mL的2M盐酸中，过滤收集固体，用二氯甲烷(50mL)复溶，饱和氯化钠水溶液洗涤(30mL×3)，无水硫酸钠干燥，过滤，浓缩，硅胶柱层析(流动相为石油醚：乙酸乙酯＝10:1)，得1-(4-((2S,5S)-2,5-双(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基硅杂哌啶(1.2g)。
1H NMR(500MHz,CDCl 3):δ8.24(d,J＝8.5Hz,4H),7.37(d,J＝8.5Hz,4H),5.78(d,J＝11.5Hz,2H),5.25(d,J＝6.5Hz,2H),3.29(s,4H),2.60(s,2H),2.06(s,2H),0.97(s,4H),0.10(s,6H).
MS(ESI)m/z:553.2[M+H] +.
步骤2：4,4'-((2S，5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)二苯胺
100mL单颈瓶中，1-(4-((2S，5S)-2,5-双(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基硅杂哌啶(2.1g，3.8mmol)溶于30mL的四氢呋喃中，加入二氧化铂(0.43g，1.9mmol)，氢气置换三次，常温常压条件下加氢反应3h。硅藻土做铺垫过滤，残留物经硅胶柱层析提纯(石油醚:乙酸乙酯＝5:1为洗脱液)，得到1.25g粗品，石油醚(15mL)打浆得4,4'-((2S，5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)二苯胺(0.72g)。
1H NMR(500MHz,CDCl 3):δ6.94(d,J＝8.0Hz,4H),6.65(d,J＝8.5Hz,4H),5.82(d,J＝12.0Hz,2H),4.95(d,J＝5.5Hz,2H),3.17(s,4H),2.49(s,2H),1.73(d,J＝5.5Hz,2H),0.82(s,4H),0.07(s,6H).
MS(ESI)m/z:493.1[M+H] +.
步骤3：((2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(4,1-亚苯基))双(氮烷二基)双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(1a)
取一100mL单颈瓶，将4,4'-((2S，5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)二苯胺(0.5g，1.02mmol)溶于10mLN,N-二甲基甲酰胺中，依次加入N-(甲氧羰基)-O-甲基-L-苏氨酰-L-脯氨酸(0.9g,3.05mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.58g,3.05mmol)、1-羟基苯并三唑(0.42g，3.05mmol)、N-甲基吗啉(0.62g，6.1mmol)。加入至55℃反应12h。将反应液倒入水中，过滤，滤饼用二氯甲烷(50mL)复溶，饱和氯化钠水溶液(30mL)洗涤，无水硫酸钠干燥。硅胶柱层析(石油醚：乙酸乙酯＝1：2(v/v))，得化合物1a(0.6g)。
1H NMR(500MHz,DMSO-d 6):δ9.95(s,2H),7.52(d,J＝8.5Hz,4H),7.29(d,J＝8.0Hz,2H),7.14(d,J＝8.5Hz,4H),5.81(d,J＝12.5Hz,2H),5.15(d,J＝6.0Hz,2H),4.44(dd,J＝4.5Hz,8.0Hz,2H),4.28(d,J＝8.0Hz,2H),3.83(m,2H),3.68(d,J＝7.0Hz,2H),3.54(s,6H),3.49(t,J＝6.5Hz,2H),3.23(s,6H),3.04(t,J＝5.5Hz,4H),2.45(m,2H),2.16(m,2H),2.00(d,J＝6.0Hz,2H),1.90(m,4H),1.62(d,J＝5.5Hz,2H),1.15(d,J＝6.0Hz,6H),0.71(t,J＝6.0Hz,4H),0.04(s,6H).
MS(ESI)m/z:1055.3[M+Na] +.
实施例6：((2S,2'S,3R,3'R)-((2S,2'S)-(((((2R,5R)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(4,1-亚苯基))双(氮烷二基)双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(1b)
Figure PCTCN2018079965-appb-000089
参照实施例5中化合物1a的合成步骤1、2、3，得到化合物1b(320mg)。
1H NMR(500MHz,CDCl 3):δ8.92(s,2H),7.45(d,J＝8.5Hz,4H),7.06(d,J＝8.5Hz,4H),5.76(d,J＝12Hz,2H),5.70(d,J＝8Hz,2H),5.01-4.99(m,2H),4.83-4.82(m,2H), 4.70-4.68(m,2H),3.80-3.79(m,2H),3.78-3.77(m,4H),3.69(s,6H),3.38(s,6H),3.33-3.20(m,3H),2.48-2.44(m,4H),2.06-2.01(m,7H),1.74-1.73(m,4H),1.23-1.22(m,6H),0.08(s,6H).
MS(ESI)m/z:1055.5[M+Na] +.
实施例7：((2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3-氟苯基)吡咯烷-2,5-二基)双(4,1-亚苯基))双(氮烷二基)双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(2a)
Figure PCTCN2018079965-appb-000090
步骤1：1-(4-((2S，5S)-2,5-双(4-硝基苯基)吡咯烷-1-基)-2-氟苯基)-4,4-二甲基硅杂哌啶
100mL三口瓶中分别加入(1R，4R)-1,4-双(4-硝基苯基)丁烷-1,4-二基二甲磺酸酯(1.5g，3.1mmol)、N,N-二甲基甲酰胺(15mL)和4-(4,4-二甲基硅杂哌啶-1-基)-3-氟苯胺(7.32g，31mmol)，氮气保护下油浴加热至60℃反应24h。反应液倒入2M盐酸(100mL)中，有固体析出，过滤收集固体，二氯甲烷(50mL)复溶，饱和氯化钠水溶液洗涤(30mL)，无水硫酸钠干燥，过滤，浓缩，硅胶柱层析提纯(石油醚：乙酸乙酯＝10:1为洗脱液)，得1-(4-((2S,5S)-2,5-双(4-硝基苯基)吡咯烷-1-基)-2-氟苯基)-二甲基硅杂哌啶(1.2g)。
1H-NMR(500MHz,CDCl 3):δ8.24(s,4H),7.36(d,J＝8.0Hz,4H),6.71(s,1H),6.00(m,2H),5.29(s,2H),3.11(s,2H),2.63(s,2H),1.96(s,2H),1.58(m,2H),0.85(m,4H),0.07(s,6H).
MS(ESI)m/z:535.1[M+H] +.
步骤2：4,4'-((2S，5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3-氟苯基)吡咯烷-2,5-二基)二苯胺
100mL单颈瓶中1-(4-((2S，5S)-2,5-双(4-硝基苯基)吡咯烷-1-基)-2-氟苯基)-4,4-二甲基硅杂哌啶(1.67g，3.13mmol)溶于20mL的四氢呋喃中，加入二氧化铂(0.7g，3.13mmol)，常温常压条件下加氢反应3h。硅藻土作铺垫过滤，减压蒸溶剂，硅胶柱层析提纯(石油醚:乙酸乙酯＝5:1为洗脱液)。得4,4'-((2S，5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3-氟 苯基)吡咯烷-2,5-二基)二苯胺(1.2g)。
1H NMR(500MHz,CDCl 3):δ6.95(d,J＝8.0Hz,4H),6.64(d,J＝8.5Hz,4H),6.06(m,3H),4.98(d,J＝7.0Hz,2H),3.12(s,2H),2.50(s,2H),1.75(d,J＝5.0Hz,2H),1.26(m,2H),0.88(m,4H),0.12(s,6H).
MS(ESI)m/z:475.1[M+H] +.
步骤3：((2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3-氟苯基)吡咯烷-2,5-二基)双(4,1-亚苯基))双(氮烷二基)双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(2a)
100mL单颈瓶中，4,4'-((2S，5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3-氟苯基)吡咯烷-2,5-二基)二苯胺(0.53g，1.12mmol)溶于10mLN,N-二甲基甲酰胺，依次加入N-(甲氧羰基)-O-甲基-L-苏氨酰-L-脯氨酸(0.97g,3.35mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.64g,3.35mmol)、1-羟基苯并三唑(0.45g，3.35mmol)和N-甲基吗啉(0.68g，6.71mmol)。混合物加热至55℃反应12h。将反应液倒入水中，过滤，滤饼用二氯甲烷复溶，有机相用饱和氯化钠水溶液洗涤，干燥。柱层析提纯(石油醚：乙酸乙酯＝1：2(v/v)为流动相)，得化合物2a(0.5g)。
1H NMR(500MHz,CDCl 3):δ9.93(s,2H),7.50(d,J＝8.5Hz,4H),7.29(d,J＝8.0Hz,2H),7.13(d,J＝8.5Hz,4H),6.71(m,1H),5.96(m,2H),5.13(d,J＝6.0Hz,4H),4.44(m,2H),4.28(m,2H),3.82(m,2H),3.65(m,2H),3.54(s,6H),3.48(m,2H),3.26(s,6H),2.98(m,4H),2.45(m,2H),2.16(m,2H),2.09(m,2H),1.89(m,4H),1.62(d,J＝5.5Hz,2H),1.14(d,J＝6.0Hz,6H),0.73(t,J＝5.5Hz,2H),0.04(s,6H).
MS(ESI)m/z:1015.5[M+H] +.
实施例8：((2S，2'S，3R，3'R)-((2S，2'S)-(((((2R，5R)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3-氟苯基)吡咯烷-2,5-二基)双(4,1-亚苯基))双(氮烷二基)双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(2b)
Figure PCTCN2018079965-appb-000091
Figure PCTCN2018079965-appb-000092
参照实施例7中化合物2a的合成步骤1、2、3，得化合物2b(0.55g)。
1HNMR(500MHz,DMSO-d 6):δ9.94(s,2H),7.51(d,J＝8.5Hz,4H),7.29(d,J＝7.5Hz,2H),7.13(d,J＝8.5Hz,4H),6.70(m,1H),5.96(m,2H),5.13(d,J＝6.0Hz,2H),4.44(dd,J＝5.0Hz,8.0Hz,2H),4.28(t,J＝7.5Hz,2H),.3.69(m,2H),3.54(m,2H),3.49(s,6H),3.47(d,J＝6.5Hz,2H),3.25(s,6H),2.98(m,4H),2.44(m,2H),2.15(m,2H),2.00(d,J＝6.0Hz,2H),1.89(m,4H),1.62(d,J＝5.5Hz,2H),1.13(d,J＝6.5Hz,6H),0.73(t,J＝6.0Hz,4H),0.04(s,6H).
MS(ESI)m/z:1037.50[M+Na] +.
实施例9：((2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(3,5-二氟-4-(8-氮杂-5-硅杂螺[4.5]癸烷-8-基)苯基)吡咯烷-2,5-二基)双(4,1-亚苯基))双(氮烷二基))双(羰基))双(吡咯烷-2,1-二基)(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(3a)
Figure PCTCN2018079965-appb-000093
步骤1：8-(4-((2S，5S)-2,5-双(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-8-氮杂-5-硅螺[4.5]癸烷
100mL单口瓶中，室温下(1R,4R)-1,4-双(4-硝基苯基)丁烷-1,4-二基二甲磺酸酯(0.39g,0.79mmol)及3,5-二氟-4-(8-氮杂-5-硅螺[4.5]癸烷-8-基)苯胺(0.45g,1.59mmol)溶于乙腈(4mL),然后向混合液中加入N,N-二异丙基乙基胺(0.51g,3.95mmol),加热至75℃搅拌过夜。反应液冷至室温后倒入6N盐酸水溶液(50mL)中,乙酸乙酯(20mLx2)萃取。有机相分 别用水(100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥。过滤，浓缩，所得粗品用硅胶柱层析分离异构体(石油醚:乙酸乙酯＝10:1)，得8-(4-((2S，5S)-2,5-双(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-8-氮杂-5-硅螺[4.5]癸烷(0.25g)。
1H NMR(500MHz,DMSO-d 6):δ8.22(d,J＝8.5Hz,4H),7.52(d,J＝8Hz,4H),5.88(d,J＝11.5Hz,2H),5.50-5.49(m,2H),3.07-3.04(m,4H),1.75-1.74(m,2H),1.52-1.49(m,4H),1.25-1.23(m,2H),0.81-0.78(m,4H),0.55-0.52(m,4H).
MS(ESI)m/z 579.5[M+H] +.
步骤2：4,4'-((2S，5S)-1-(3,5-二氟-4-(8-氮杂-5-硅螺[4.5]癸烷-8-基)苯基)吡咯烷-2,5-二基)二苯胺
100mL单口瓶中，将8-(4-((2S，5S)-2,5-双(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-8-氮杂-5-硅螺[4.5](0.25g,0.43mmol)溶于四氢呋喃(4mL)中，再向该溶液中加入二氧化铂(0.1g,0.43mmol)，氢气置换三次，然后在氢气氛围下室温反应过夜。反应液经硅藻土过滤，滤液浓缩得4,4'-((2S，5S)-1-(3,5-二氟-4-(8-氮杂-5-硅螺[4.5]癸烷-8-基)苯基)吡咯烷-2,5-二基)二苯胺(0.19g)。
MS(ESI)m/z：519.5[M+H] +.
步骤3：((2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(3,5-二氟-4-(8-氮杂-5-硅杂螺[4.5]癸烷-8-基)苯基)吡咯烷-2,5-二基)双(4,1-亚苯基))双(氮烷二基))双(羰基))双(吡咯烷-2,1-二基)(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(3a)
100mL单口瓶中，室温下，4,4'-((2S，5S)-1-(3,5-二氟-4-(8-氮杂-5-硅螺[4.5]癸烷-8-基)苯基)吡咯烷-2,5-二基)二苯胺(0.14g,0.27mmol)及N-(甲氧羰基)-O-甲基-L-苏氨酰-L-脯氨酸(0.23g,0.81mmol)溶于N,N-二甲基甲酰胺(5mL)，然后依次加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(0.16g,0.81mmol),1-羟基苯并三唑(0.11g,0.81mmol)及N-甲基吗啉(0.14g,1.35mmol)。然后加热至60℃,反应1小时。反应结束后，将反应液倒入水(50mL)中，并用乙酸乙酯(100mL)萃取。有机相分别用水(100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥。过滤，浓缩，纯化，得化合物3a(0.14g)。
1H NMR(500MHz,DMSO-d 6):δ9.95(s,2H),7.52(d,J＝10Hz,4H),7.31(d,J＝10Hz,2H),7.14(d,J＝5Hz,4H),5.80(d,J＝10Hz,2H),5.15(s,2H),4.45-4.42(m,2H),4.29-4.26(m,2H),3.85-3.81(m,2H),3.69-3.65(m,2H),3.53(s,6H),3.49-3.45(m,2H),3.25(s,6H),3.08-3.05(m,4H),2.44(s,2H),2.17-2.12(m,2H),2.01-1.96(m,2H),1.91-1.84(m,4H),1.62-1.61(m,2H),1.51-1.50(m,4H),1.15-1.13(m,6H),0.81-0.79(m,4H),0.56-0.53(m,4H).
MS(ESI)m/z 1081.3[M+Na] +.
实施例10：(2S，2'S，3R，3'R)-((2S，2'S)-(((2S，5S)-1-(4-(4,4-二甲基-1,4-二氮杂环辛烷-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(1H-苯并[d]咪唑-5,2-二基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(4a)
Figure PCTCN2018079965-appb-000094
步骤1：1-(4-((2S，5S)-2,5-双(4-氯-3-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基-1,4-硅杂哌啶
100mL单口瓶中，室温下(1R，4R)-1,4-双(4-氯-3-硝基苯基)丁烷-1,4-二基二甲磺酸酯(2.5g,4.49mmol)及4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯胺(9.2g,35.88mmol)溶于N,N-二甲基甲酰胺(30mL)，加热至60℃过夜。反应冷至室温，倒入1N盐酸(50mL)，并用乙酸乙酯(20mLx2)萃取。有机相合并、干燥、过滤及浓缩得到粗品，经硅胶柱层析(石油醚:乙酸乙酯＝10:1)纯化，得到1-(4-((2S，5S)-2,5-双(4-氯-3-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基-1,4-硅杂哌啶(2.5g)。
1H NMR(500MHz,CDCl 3):δ＝7.70(d,J＝1Hz,2H),7.52(d,J＝8.5Hz,2H),7.34-7.28(m,2H),6.02-5.99(m,1H),5.98-5.93(m,1H),5.22(d,J＝6.5Hz,2H),3.14-3.13(m,4H),2.60-2.50(m,2H),1.90-1.892(m,2H),0.86-0.85(m,4H),0.08(s,6H).
MS(ESI)m/z 621.1[M+H] +.
步骤2：5,5'-((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(N-(4-甲氧基苄基)-2-硝基苯胺)
100mL单口瓶中，1-(4-((2S，5S)-2,5-双(4-氯-3-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基-1,4-硅杂哌啶(2.0g,3.22mmol)加入4-甲氧基苄胺(3.53g,25.74mmol)， 油浴加热至145℃反应1h。反应液冷至室温，用乙酸乙酯(100mL)稀释，依次用2N盐酸(50mLx2)和饱和食盐水(20mL)洗涤。有机相干燥、过滤、浓缩得粗品，经硅胶柱层析(石油醚:乙酸乙酯＝10:1)纯化，得到5,5'-((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(N-(4-甲氧基苄基)-2-硝基苯胺)(2.1g)。MS(ESI)m/z 845.3[M+Na] +.
步骤3：5,5'-((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(2-硝基苯胺)
100mL单口瓶中，5,5'-((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(N-(4-甲氧基苄基)-2-硝基苯胺)(2.1g,2.55mmol)溶于三氟乙酸(20mL)，室温搅拌1h。反应液浓缩除去溶剂，用乙酸乙酯(20mL)稀释，依次用饱和碳酸氢钠(20mL)及饱和食盐水(10mL)洗。有机相干燥、过滤、浓缩得粗品，经硅胶柱层析(石油醚:乙酸乙酯＝3:1)纯化，得到5,5'-((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(2-硝基苯胺)(1.5g).
MS(ESI)m/z 583.1[M+H] +.
步骤4：((2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(2-硝基-4,1-亚苯基)双(氮烷二基))双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯
100mL单口瓶中，5,5'-((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(2-硝基苯胺)(1.4g,2.4mmol)及N-(甲氧羰基)-O-甲基-L-苏氨酰-L-脯氨酸(2.08g,7.2mmol)溶于吡啶(15mL)，冰浴下滴加三氯氧磷(1.11g,7.2mmol)，继续反应15min。反应液用乙酸乙酯(30mL)稀释，依次用1N盐酸(20mLx2)、饱和碳酸氢钠(20mL)及饱和食盐水(20mL)洗。有机相干燥、过滤、浓缩得粗品，经硅胶柱层析(石油醚:乙酸乙酯＝2:3)纯化，得到((2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(2-硝基-4,1-亚苯基)双(氮烷二基))双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(1.6g)。
MS(ESI)m/z 1145.5[M+Na] +.
步骤5：((2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(2-氨基-4,1-亚苯基)双(氮烷二基))双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯
100mL单口瓶中，((2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(2-硝基-4,1-亚苯基)双(氮 烷二基))双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(1.6g,1.42mmol)溶于四氢呋喃(20mL)，加入二氧化铂(200mg,0.88mmol)，氢气条件下室温反应1h。反应液经硅藻土过滤，滤液浓缩得到((2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(2-氨基-4,1-亚苯基)双(氮烷二基))双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(1.2g，)。
MS(ESI)m/z 1085.4[M+Na] +.
步骤6：(2S，2'S，3R，3'R)-((2S，2'S)-(((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(1H-苯并[d]咪唑-5,2-二基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(4a)
100mL单口瓶中，(2S，2'S，3R，3'R)-((2S，2'S)-(((((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2,5-二基)双(2-氨基-4,1-亚苯基)双(氮烷二基))双(羰基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(1.2g,1.13mmol)加入乙酸(10mL)，油浴加热至70℃,反应2h。反应液浓缩除去溶剂,残留物用乙酸乙酯(30mL)稀释，依次用饱和碳酸氢钠(30mL)和饱和食盐水(20mL)洗。有机相干燥、过滤、浓缩得粗品，经硅胶柱层析(二氯甲烷:甲醇＝20：1)及prep-HPLC纯化，得到化合物4a(88mg)。
1H NMR(500MHz,CDCl 3):δ＝10.39(s,1H),10.26(s,1H),7.73-7.71(m,1H),7.53-7.50(m,1H),7.33-7.30(m,1H),7.15-7.15(m,3H),5.80-5.75(m,2H),5.72-5.68(m,2H),5.55-5.51(m,2H),5.22-5.17(m,2H),4.64(s,1H),4.57(s,1H),3.80-3.78(m,3H),3.77-3.74(m,8H),3.73-3.72(m,1H),3.31-3.09(m,3H),3.10-3.07(m,4H),2.98-2.90(m,4H),2.62-2.57(m,2H),2.30-2.27(m,4H),2.20-2.10(m,2H),1.75-1.72(m,2H),1.29-1.27(m,2H),1.18-1.14(m,5H),0.77-0.74(m,4H),0.04(s,6H).
MS(ESI)m/z 1049.4[M+Na] +.
实施例11：((2S，2'S，3R，3'R)-((2S，2'S)-(((2R，5R)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3，5-二氟苯基)吡咯烷-2,5-二基)双(1H-苯并[d]咪唑-5,2-二基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(4b)
Figure PCTCN2018079965-appb-000095
参照实施例10中化合物的4a的合成方法合成得到((2S，2'S，3R，3'R)-((2S，2'S)-(((2R，5R)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3，5-二氟苯基)吡咯烷-2,5-二基)双(1H-苯并[d]咪唑-5,2-二基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(4b)(0.7g)。
1H NMR(500MHz,DMSO-d 6):δ12.08(s,1H),12.00(s,1H),7.49(d,J＝8.5Hz,1H),7.41(d,J＝8.0Hz,2H),7.19(s,1H),7.08(m,4H),5.87(t,J＝6.0Hz,2H),5.35(d,J＝7.5Hz,2H),5.16(t,J＝3.5Hz,2H),4.29(d,J＝4.5Hz,2H),3.85(d,J＝6.0Hz,4H),3.55(s,6H),3.48(d,J＝5.5Hz,2H),3.18(m,6H),3.00(m,4H),2.54(m,2H),2.20(s,4H),2.02(m,4H),1.69(s,2H),1.04(m,6H),0.69(s,4H),0.02(s,6H).
MS(ESI)m/z:1027.4[M+H] +.
实施例12：(2S，2'S，3R，3'R)-((2S，2'S)-(((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3-氟苯基)吡咯烷-2,5-二基)双(1H-苯并[d]咪唑-5,2-二基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(5a)
Figure PCTCN2018079965-appb-000096
参照实施例10中化合物的4a的合成方法合成得到(2S，2'S，3R，3'R)-((2S，2'S)-(((2S，5S)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3-氟苯基)吡咯烷-2,5-二基)双(1H-苯并[d]咪唑-5,2-二基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯5a(44mg)。
1H-NMR(500MHz,CDCl 3):δ＝10.40(s,1H),10.26(s,1H),7.72-7.04(m,1H),7.56-7.55(m,1H),7.31-7.30(m,1H),7.17-7.08(m,3H),6.59(s,1H),6.08-5.95(m,2H),5.76-5.68(m,2H),5.57-5.50(m,2H),5.28-5.21(m,2H),4.64-4.61(s,1H),4.60-4.57(s,1H),3.82-3.80(m,4H),3.79-3.67(m,9H),3.63-3.57(m,1H),3.33-3.26(m,3H),3.08-2.95(m,9H),2.67-2.54(m,2H),2.30-2.22(m,4H),2.18-2.13(m,2H),1.85-1.80(m,3H),1.20-1.03(m.3H),0.81-0.79(m,4H),0.04(s,6H)。
MS(ESI)m/z 1031.3[M+Na] +.
实施例13：((2S，2'S，3R，3'R)-((2S，2'S)-(((2R，5R)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3-氟苯基)吡咯烷-2,5-二基)双(1H-苯并[d]咪唑-5,2-二基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(5b)
Figure PCTCN2018079965-appb-000097
参照实施例10中化合物的4a的合成方法合成得到((2S，2'S，3R，3'R)-((2S，2'S)-(((2R，5R)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3-氟苯基)吡咯烷-2,5-二基)双(1H-苯并[d]咪唑-5,2-二基))双(吡咯烷-2,1-二基))双(3-甲氧基-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(5b)(0.4g)。
1H NMR(DMSO-d 6,500MHz):δ12.04(s,1H),11.98(s,1H),7.47(d,J＝8.0Hz,2H),7.39(d,J＝8.5Hz,2H),7.07(m,4H),6.65(m,1H),6.02(d,J＝7.0Hz,2H),5.32(d,J＝5.0Hz,2H),5.15(d,J＝7.0Hz,2H),4.29(s,2H),3.85(s,4H),3.55(s,6H),3.48(m,2H),3.27(s,6H),2.94(m,4H),2.54(m,2H),2.21(d,J＝5.5Hz,4H),2.01(m,4H),1.70(s,2H),1.05(t,J＝6.0Hz,6H),0.71(t,J＝3.0Hz,4H),0.02(s,6H).
MS(ESI)m/z:1009.5[M+H] +.
实施例14：((2S,3R)-1-((S)-2-(6-((2S,5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)-5-(4-((S)-1-(N-(甲氧基羰基)-O-甲基-L-苏氨酰基)吡咯烷-2-甲酰氨基)苯基)吡咯烷-2- 基)-1H-苯并[d]咪唑-2-基)吡咯烷-1-基)-3-甲氧基-1-氧代丁烷-2-基)氨基甲酸甲酯(6a)
Figure PCTCN2018079965-appb-000098
步骤1：1-(4-((2S,5S)-2-(4-氯-3-硝基苯基)-5-(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基-1,4-硅杂哌啶
(1R，4R)-1-(4-氯-3-硝基苯基)-4-(4-硝基苯基)丁烷-1,4-二基二甲磺酸酯(2.00g,4.5mmol)，4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯胺(9.20g,36.0mmol)，N，N-二异丙基乙胺(5.85g,45.0mmol)的混合物于N,N-二甲基甲酰胺(45mL)中加热至60℃油浴搅拌反应过夜。反应结束，反应液倒入2M/盐酸(200mL)中，搅拌10min，乙酸乙酯(60mL×2)萃取，有机相分别以2M盐酸(60mL)和饱和食盐水洗(60mL)，无水硫酸钠干燥，浓缩，残留物经硅胶柱层析(流动相石油醚：乙酸乙酯＝15:1)，得1-(4-((2S,5S)-2-(4-氯-3-硝基苯基)-5-(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基-1,4-硅杂哌啶(1.31g)。
1H NMR(500MHz,DMSO-d 6)：δ8.21(d,J＝8.5Hz,2H),7.96(d,J＝2.0Hz,1H),7.75(d,J＝8.5Hz,1H),7.48-7.55(m,3H),5.92(d,J＝12.0Hz,2H),5.43-5.50(m,2H),3.04(t,J＝6.0Hz,4H),1.94-2.03(m,2H),1.72-1.79(m,2H),0.72(t,J＝6.0Hz,4H),0.03(s,6H).
MS(ESI)m/z:587.1.
步骤2：(S)-2-((4-((2S,5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)-5-(4-硝基苯基)吡咯烷-2-基)-2-硝基苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯
1-(4-((2S,5S)-2-(4-氯-3-硝基苯基)-5-(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基-1,4-硅杂哌啶(1.47g,2.49mmol)，(S)-2-氨基甲酰基吡咯烷-1-羧酸叔丁酯(0.81g,3.75mmol)，碳酸铯(1.23g,3.75mmol)，三(二亚苄基丙酮)二钯(0.12g,0.12mmol)，4,5-双二苯基膦-9,9-二甲基氧杂蒽(0.42g,0.75mmol)分散于1,4-二氧六环(40mL)中，N 2氛围，100℃油浴搅拌反应4h。反应液冷却至室温，抽滤，滤液倒入200mL水中，乙酸乙酯(60mL×2)萃取，水洗(60mL×3)，干燥，浓缩，残留物硅胶柱层析纯化(流动相石油醚：乙酸乙酯＝10:1)，(S)-2-((4-((2S,5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)-5-(4-硝基苯基)吡咯烷-2-基)-2-硝基苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯(1.1g)。
1H NMR(500MHz,DMSO-d 6)：δ10.52(s,1H),8.21-8.28(m,2H),7.85-7.86(m,1H),7.49-7.66(d,J＝8.5Hz,1H),7.50-7.58(m,3H),5.91(d,J＝12.5Hz,2H),5.45(dd,J＝8.0Hz,J＝34.5Hz,2H),4.30(s,1H),3.34-3.43(m,2H),3.02-3.10(m,4H),2.42-2.45(m,1H),2.13-2.28(m,1H),1.72-1.97(m,6H),1.3(s,9H),0.69-0.72(m,4H),0.03(s,6H).
MS(ESI)m/z:765.3.
步骤3：(S)-2-((2-氨基-4-((2S，5S)-5-(4-氨基苯基)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯
(S)-2-((4-((2S,5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)-5-(4-硝基苯基)吡咯烷-2-基)-2-硝基苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯(1.00g,1.30mmol)溶于四氢呋喃(15mL)，加入氧化铂(0.1g)，氢气氛围室温搅拌反应6h。反应液抽滤，滤液浓缩，残留物以甲基叔丁基醚(15mL)打浆，(S)-2-((2-氨基-4-((2S，5S)-5-(4-氨基苯基)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯(0.58g，63％)。
1H NMR(500MHz,DMSO-d 6)：δ9.24(s,1H),7.06(d,J＝9.0Hz,1H),6.84-6.85(m,2H),6.50-6.58(m,3H),6.43(d,J＝7.5Hz,1H),5.81(d,J＝12.5Hz,2H),4.95(s,4H),4.79-4.93(m,2H),4.24(s,1H),3.34-3.43(m,2H),3.03-3.08(m,4H),2.35-2.41(m,2H),2.13-2.22(m,1H),1.89-1.91(m,2H),1.76-1.79(m,1H),1.57-1.61(m,2H),1.32-1.41(m,9H),0.71-0.73(m,4H),0.04(s,6H).
MS(ESI)m/z:705.3.
步骤4：(S)-2-((2-氨基-4-((2S，5S)-5-(4-((S)-1-(叔丁氧基羰基)吡咯烷-2-甲酰氨基)苯基)1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯
(S)-2-((2-氨基-4-((2S，5S)-5-(4-氨基苯基)-1-(4-(4,4-二甲基硅杂哌啶-1-基) -3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯(0.40g,0.57mmol)，L-Boc-脯氨酸(0.13g,0.60mmol)，2-(7-氧化苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(0.33g,0.86mmol)，N,N-二异丙基乙胺(0.20mL,1.14mmol)，溶于N,N-二甲基甲酰胺(10mL)，室温搅拌反应2h。反应液倒入水(50mL)中，搅拌10min，抽滤，水洗(5mL×3)滤饼，收集固体，干燥得(S)-2-((2-氨基-4-((2S，5S)-5-(4-((S)-1-(叔丁氧基羰基)吡咯烷-2-甲酰氨基)苯基)1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯(0.46g)。
1H NMR(500MHz,DMSO-d 6)：δ9.92(s,1H),9.15-9.23(m,1H),7.53(d,J＝8.0Hz,1H),7.14-7.15(m,2H),7.07(d,J＝8.0Hz,1H),6.57-6.61(m,2H),5.81(d,J＝12.5Hz,2H),5.06(dd,J＝7.5Hz,J＝34.0Hz,2H),4.380-4.93(m,2H),4.19-4.25(m,2H),3.33-3.41(m,4H),3.03-3.05(m,4H),2.43-2.47(m,2H),2.19-2.20(m,2H),1.80-1.89(m,6H),1.64-1.67(m,2H),1.36-1.41(m,18H),0.70-0.72(m,4H),0.03(s,6H).
MS(ESI)m/z:902.6.
步骤5：(S)-2-((4-((2S，5S)-5-(2-((S)-1-(叔丁氧基羰基)吡咯烷-2-基)-1H-苯并[d]咪唑-6-基)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯
(S)-2-((4-((2S，5S)-5-(2-((S)-1-(叔丁氧基羰基)吡咯烷-2-基)-1H-苯并[d]咪唑-6-基)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯(0.45g,0.50mmol)，乙酸(2.7mL)溶于甲苯(9mL)，55℃油浴搅拌反应5h。反应液以饱和碳酸钠水溶液调pH至碱性，乙酸乙酯萃取(30mL×2)，无水硫酸钠干燥，过滤，浓缩，残留物以硅胶柱层析纯化(流动相石油醚：乙酸乙酯＝10:1)，得(S)-2-((4-((2S，5S)-5-(2-((S)-1-(叔丁氧基羰基)吡咯烷-2-基)-1H-苯并[d]咪唑-6-基)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯(0.21g)。
1H NMR(500MHz,DMSO-d 6)：δ9.95(s,1H),7.55(d,J＝8.0Hz,2H),7.46(d,J＝7.5Hz,1H),7.17-7.25(m,3H),7.06(d,J＝8.5Hz,1H),5.82-5.86(m,2H),5.25(dd,J＝5.5Hz,J＝48.5Hz,2H),4.85-4.95(m,1H),4.41-4.42(m,2H),3.39-.356(m,4H),3.02-3.04(m,4H),2.15-2.20(m,2H),1.80-1.99(m,8H),1.68-1.70(m,2H),1.29-1.40(m,18H),0.68-0.71(m,4H),0.02(s,6H).
MS(ESI)m/z:884.4.
步骤6：(S)-N-(4-((2S,5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)-5-(2-((S)-吡咯烷-2-基)-1H-苯并[d]咪唑-6-基)吡咯烷-2-基)苯基)吡咯烷-2-甲酰胺(S)-2-((4-((2S，5S)-5-(2-((S)-1-(叔丁氧基羰基)吡咯烷-2-基)-1H-苯并[d]咪唑-6-基)-1-(4-(4,4-二甲基硅杂哌 啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-羧酸叔丁酯(0.17g,0.23mmol)，溶于四氢呋喃(8mL)，浓盐酸(2mL,24mL)，室温反应1h。反应液浓缩，残留物加水(10mL)稀释，乙酸乙酯洗涤，水相饱和碳酸钠水溶液调pH至碱性，二氯甲烷和甲醇10:1混合溶液萃取，有机相水洗，干燥，浓缩得(S)-N-(4-((2S,5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)-5-(2-((S)-吡咯烷-2-基)-1H-苯并[d]咪唑-6-基)吡咯烷-2-基)苯基)吡咯烷-2-甲酰胺(0.15g)。
1H NMR(500MHz,DMSO-d 6)：δ9.92(s,1H),7.61(d,J＝8.5Hz,2H),7.46(d,J＝7.5Hz,1H),7.16-7.23(m,3H),7.03(d,J＝8.5Hz,1H),5.85(d,J＝12.5Hz,2H),5.29(dd,J＝5.5,45.0Hz,2H),4.31-4.32(m,1H),3.66-3.69(m,2H),3.02(t,J＝6.0Hz,4H),2.87-2.94(m,4H),1.95-2.01(m,4H),1.73-1.78(m,3H),1.63-1.67(m,4H),1.23-1.25(m,3H),0.68(t,J＝6.0Hz,4H),0.02(s,6H).
MS(ESI)m/z:684.2.
步骤7：((2S,3R)-1-((S)-2-(6-((2S,5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)-5-(4-((S)-1-(N-(甲氧基羰基)-O-甲基-L-苏氨酰基)吡咯烷-2-甲酰氨基)苯基)吡咯烷-2-基)-1H-苯并[d]咪唑-2-基)吡咯烷-1-基)-3-甲氧基-1-氧代丁烷-2-基)氨基甲酸甲酯(6a)
(S)-N-(4-((2S,5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)-5-(2-((S)-吡咯烷-2-基)-1H-苯并[d]咪唑-6-基)吡咯烷-2-基)苯基)吡咯烷-2-甲酰胺(0.14g,0.21mmol)，(2S,3R)-3-甲氧基-2-((甲氧基羰基)氨基)丁酸(0.09g,0.47mmol)，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(0.16g,0.41mmol)，N,N-二异丙基乙胺(0.21g,1.64mmol)，N,N-二甲基甲酰胺(5mL)，室温反应3h。反应结束将反应液倒入水中(30mL)，搅拌10min，抽滤，水洗(10mL×3)，收集得到粗品，制备纯化后得((2S,3R)-1-((S)-2-(6-((2S,5S)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)-5-(4-((S)-1-(N-(甲氧基羰基)-O-甲基-L-苏氨酰基)吡咯烷-2-甲酰氨基)苯基)吡咯烷-2-基)-1H-苯并[d]咪唑-2-基)吡咯烷-1-基)-3-甲氧基-1-氧代丁烷-2-基)氨基甲酸甲酯(6a)(0.065g)。
1H NMR(500MHz,DMSO-d 6):δ11.97-12.29(m,1H),9.95-10.00(m,1H),7.53(d,J＝8.0Hz,2H),7.43(dd,J＝8.5Hz,J＝34.5Hz,1H),7.30(d,J＝8.0Hz,1H),7.16-7.27(m,4H),7.04(dd,J＝8.0Hz,J＝25.5Hz,1H),5.84(dd,J＝3.5Hz,J＝12.5Hz,2H),5.14-5.34(m,3H),4.43-4.46(m,1H),4.27-4.30(m,2H),3.83-3.84(m,2H),3.66-3.68(m,1H),3.47-3.55(m,7H),3.16-3.28(m,6H),3.02(t,J＝6.0Hz,4H),2.49-2.51(m,1H),2.14-2.24(m,3H),1.97-2.07(m,4H),1.85-1.93(m,2H),1.63-1.70(m,2H),1.23-1.25(m,2H),1.06-1.15(m,6H),0.70(t,J＝5.0Hz,4H),0.03(s,6H).
MS(ESI)m/z:1030.4.
实施例15：((2S，3R)-1-((S)-2-(6-((2R，5R)-1-(4-(4,4-二甲基-硅杂哌啶-1-基)-5- (4-((S)-1-(N-(甲氧羰基)-O-甲基-L-苏氨酰基)吡咯烷-2-甲酰氨基)苯基)吡咯烷-2-基)-苯并[d]咪唑-2-基)吡咯烷-1-基)-3-甲氧基-1-氧代丁烷-2-基)氨基甲酸甲酯(6b)
Figure PCTCN2018079965-appb-000099
化合物6b的制备参考实施例化合物6a的制备方法，合成得到化合物6b(0.06g)。
1H NMR(500MHz,DMSO-d 6):δ11.98-12.28(m,1H),9.96-10.00(m,1H),7.53(d,J＝8.5Hz,1H),7.44(dd,J＝8.0Hz,J＝39.0Hz,1H),7.29(d,J＝4.0Hz,1H),7.12-7.24(m,4H),7.05(m,1H),5.84(dd,J＝6.0Hz,J＝12.0Hz,2H),5.12-5.34(m,3H),4.43-4.46(m,1H),4.26-4.34(m,2H),3.83-3.84(m,2H),3.66-3.68(m,1H),3.463.54(m,7H),3.10-3.25(m,6H),3.02(t,J＝6.0Hz,4H),2.47-2.49(m,1H),2.15-2.20(m,3H),1.87-1.92(m,6H),1.62-1.69(m,2H),1.23-1.25(m,2H),0.99-1.14(m,6H),0.69(t,J＝6.0Hz,4H),0.02(s,6H).
MS(ESI):m/z 1030.4.
实施例16：((2S,2'S,3R,3'R)-((2S,2'S)-(((2R,5R)-1-(3,5-二氟-4-(8-氮杂-5-硅螺[4.5]癸烷-8-基)苯基)吡咯烷-2,5-二基)双(1H-苯并[d]咪唑-5,2-二基))双(吡咯烷-2,1-二基))双-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(7b)
Figure PCTCN2018079965-appb-000100
参照实施例10中化合物4a的制备方法制备得到((2S,2'S,3R,3'R)-((2S,2'S)-(((2R,5R)-1-(3,5-二氟-4-(8-氮杂-5-硅螺[4.5]癸烷-8-基)苯基)吡咯烷-2,5-二基)双(1H-苯并[d]咪唑-5,2-二基))双(吡咯烷-2,1-二基))双-1-氧代丁烷-1,2-二基))二氨基甲酸甲酯(7b)
实施例17：((2S,3R)-1-((S)-2-((4-((2R,5R)-1-(4-(4,4-二甲基硅杂哌啶-1-基)-3,5-二氟苯基)-5-(4-(2-((S)-1-(N-(甲氧基羰基)-O-甲基-L-苏氨酰基)吡咯烷-2-基)-1H-咪唑-5-基)苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-基)-3-甲氧基-1-氧代丁烷-2-基)氨基甲酸甲酯(8b)
Figure PCTCN2018079965-appb-000101
步骤1：1-(4-((2R,5R)-2-(4-溴苯基)-5-(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基硅杂哌啶
250mL单口瓶中(1S,4S)-1-(4-溴苯基)-4-(4-硝基苯基)丁烷-1,4-二基二甲磺酸酯(12g,23.03mmol)、4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯胺(47.2g,184.2mmol)和N,N-二异丙基乙基胺(23.8g,184.2mmol)的混合物于N,N-二甲基甲酰胺(100mL)中加热至60℃搅拌5小时。反应结束后，将反应液倒入2N稀盐酸溶液(300mL)中，并用乙酸乙酯(300mL)萃取。有机相分别用6N稀盐酸溶液(100mL)，水(100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥。过滤，浓缩。所得粗品用硅胶柱层析纯化(石油醚:乙酸乙酯＝25:1)，得1-(4-((2R,5R)-2-(4-溴苯基)-5-(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基硅杂哌啶(4.7g)。
MS(ESI)m/z：586.0.
步骤2：1-(2,6-二氟-4-((2R,5R)-2-(4-硝基苯基)-5-(4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)吡啶-2-基)苯基)吡咯烷-1-基)苯基)-4,4-二甲基硅杂哌啶
反应瓶中依次加入1-(4-((2R,5R)-2-(4-溴苯基)-5-(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基硅杂哌啶(3g,5.13mmol)，联硼酸频那醇酯(1.56g,6.16mmol)，醋酸钾(1.51g,15.39mmol)和[1-1’-(二苯基膦基)二茂铁]二氯化钯(0.19g,0.26mmol)，氮气保护下加入N,N-二甲基甲酰胺(75mL)，混合物加热到100℃搅拌9小时。反应结束后，将反应液倒入冰水(200mL)中，并用乙酸乙酯(300mL)萃取。有机相分别用水(100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥。过滤，浓缩，所得粗品用硅胶柱层析纯化(石油醚:乙酸乙酯＝12:1)，得1-(2,6-二氟-4-((2R,5R)-2-(4-硝基苯基)-5-(4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)吡啶-2-基)苯基)吡咯烷-1-基)苯基)-4,4-二甲基硅杂哌啶(1.54g)。
MS(ESI):m/z 634.2.
步骤3：(S)-2-(5-(4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基))-5-(4-硝基苯基)吡咯烷-2-基)苯基)-1H-咪唑-2-基)吡咯烷-1-羧酸叔丁酯
反应瓶中依次加入1-(2,6-二氟-4-((2R,5R)-2-(4-硝基苯基)-5-(4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)吡啶-2-基)苯基)吡咯烷-1-基)苯基)-4,4-二甲基硅杂哌啶(1.3g,2.05mmol)，(S)-2-(5-溴-1H-咪唑-2-基)吡咯烷-1-羧酸叔丁酯(0.71g,2.26mmol)，碳酸钾(0.85g,6.15mmol)和四(三苯基磷)钯(0.12g,0.103mmol)，氮气保护下向瓶中加入二氧六环(65mL)和水(13mL)，混合物加热到90℃搅拌4小时。反应结束后，将反应液倒入水(100mL)中，并用乙酸乙酯(200mL)萃取。有机相分别用水(100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥。过滤，浓缩，所得粗品用硅胶柱层析纯化(石油醚:乙酸乙酯＝2:1)，得(S)-2-(5-(4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基))-5-(4-硝基苯基)吡咯烷-2-基)苯基)-1H-咪唑-2-基)吡咯烷-1-羧酸叔丁酯(0.8g)。
MS(ESI)：m/z 743.2.
步骤4：(S)-2-(5-(4-((2R，5R)-5-(4-氨基苯基)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)吡啶-2-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)-1H-咪唑-2-基)吡咯烷-1-羧酸叔丁酯
反应瓶中依次加入(S)-2-(5-(4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基))-5-(4-硝基苯基)吡咯烷-2-基)苯基)-1H-咪唑-2-基)吡咯烷-1-羧酸叔丁酯(0.8g,1.08mmol)，四氢呋喃(20mL)和二氧化铂(0.08g)，室温常压加氢还原反应搅拌2小时。反应结束后，反应液用硅藻土过滤，滤液浓缩，所得粗品用柱层析纯化(石油醚: 乙酸乙酯＝2:1)，得(S)-2-(5-(4-((2R，5R)-5-(4-氨基苯基)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)吡啶-2-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)-1H-咪唑-2-基)吡咯烷-1-羧酸叔丁酯(0.46g)。
1H NMR(500M,DMSO-d 6):δ11.78(s,1H),7.65(d,J＝5Hz,2H),7.37(s,1H),7.15(d,J＝5Hz,2H),6.86(d,J＝10Hz,2H),6.52(d,J＝10Hz,2H),5.82(d,J＝10Hz,2H),5.12(d,J＝5Hz,1H),5.02(d,J＝10Hz,1H),4.94(brs,2H),4.83-4.76(m,1H),3.52-3.50(m,1H),3.37-3.34(m,1H),3.03(t,J＝5Hz,4H),2.47-2.39(m,2H),2.21-2.14(m,1H),2.03-1.96(m,2H),1.86-1.83(m,1H),1.66-1.58(m,2H),1.39(s,3H),1.23(s,1H),1.16(s,5H),0.72-0.69(m,4H),0.02(s,6H)；
MS(ESI)m/z:713.3.
步骤5：(S)-2-((4-((2R，5R)-5-(4-(2-((S)-1-(叔丁氧基羰基)吡咯烷-2-基)-5-基)苯基)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)-1-羧酸叔丁酯
反应瓶中依次加入(S)-2-(5-(4-((2R，5R)-5-(4-氨基苯基)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)吡啶-2-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)-1H-咪唑-2-基)吡咯烷-1-羧酸叔丁酯(0.36g,0.51mmol)，(叔丁氧基羰基)-L-脯氨酸(0.13g,0.61mmol)，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(0.29g,0.765mmol)，N,N-二异丙基乙基胺(0.13g,1.02mmol)和N,N-二甲基甲酰胺(20mL)，室温搅拌3小时。反应结束后，将反应液倒入水(100mL)中，并用乙酸乙酯(200mL)萃取。有机相分别用水(100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥。过滤，浓缩，所得粗品用硅胶柱层析纯化(石油醚:乙酸乙酯＝2:1)，得(S)-2-((4-((2R，5R)-5-(4-(2-((S)-1-(叔丁氧基羰基)吡咯烷-2-基)-5-基)苯基)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)-1-羧酸叔丁酯(0.27g)。
1H NMR(500M,DMSO-d 6):δ11.85(s,1H),9.94(d,J＝5Hz,1H),7.66(d,J＝10Hz,2H),7.55(d,J＝5Hz,2H),7.37(s,1H),7.17(d,J＝5Hz,4H),5.83(d,J＝15Hz,2H),5.18(s,2H),4.84-4.76(m,1H),4.26-4.18(m,1H),3.53(s,1H),3.42-3.41(m,1H),3.38-3.34(m,2H),3.03(t,J＝5Hz,4H),2.47-2.43(m,2H),2.21-2.16(m,2H),2.03-2.01(m,2H),1.88-1.76(m,4H),1.68-1.64(m,2H),1.39(s,6H),1.28(s,6H),1.23(s,1H),1.16(s,5H),0.70(t,J＝5Hz,4H),0.02(s,6H)；
LC-MS(ESI)：m/z 732.7.
步骤6：(S)-N-(4-((2R，5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5--(2-((S)-吡咯烷-2-基)-1H-咪唑-5-基)苯基)吡咯烷-2-基)苯基)吡咯烷-2-甲酰胺
将(S)-2-((4-((2R，5R)-5-(4-(2-((S)-1-(叔丁氧基羰基)吡咯烷-2-基)-5- 基)苯基)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)-羧酸(0.2g,0.22mmol)溶于四氢呋喃(5mL)中，然后向其中滴加浓盐酸(2.5mL)，滴加完毕之后室温搅拌2小时。反应结束后，减压蒸馏除去溶剂，所得浓缩液用饱和碳酸钠溶液调节pH至碱性。然后用二氯甲烷/甲醇溶液(200mL,二氯甲烷:甲醇＝10:1)萃取，有机相用无水硫酸钠干燥。过滤，浓缩得标题化合物(0.15g)。
MS(ESI)m/z 710.3.
步骤7：甲基((2S，3R)-1-((S)-2-((4-((2R，5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5-(4-(2-((S)-1-(N-(甲氧基羰基)-O-甲基-L-苏氨酰基)吡咯烷-2-基)-1H-咪唑-5-基)苯基)吡咯烷-2-基)苯基)氨基甲酰基)吡咯烷-1-基)-3-甲氧基-1-氧代丁烷-2-基)氨基甲酸甲酯(8b)
反应瓶中依次加入(S)-N-(4-((2R，5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5-(2-((S)-吡咯烷-2-基)-1H-咪唑-5-基)苯基)吡咯烷-2-基)苯基)吡咯烷-2-甲酰胺(0.13g,0.183mmol)，N-(甲氧基羰基)-O-甲基-L-苏氨酸(0.081g,0.421mmol)，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(0.16g,0.421mmol)，N,N-二异丙基乙基胺(0.189g,1.464mmol)和N,N-二甲基甲酰胺(6mL)，混合液室温搅拌3小时。反应结束后，将反应液倒入水(150mL)中，并用乙酸乙酯(200mL)萃取。有机相分别用水(100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥。过滤，浓缩，所得粗品用制备液相纯化，得到标题化合物(0.06g)。
1H NMR(500M,DMSO-d 6):δ11.69(s,1H),9.97(s,1H),7.65(d,J＝5Hz,2H),7.53(d,J＝10Hz,2H),7.40(s,1H),7.32(d,J＝5Hz,1H),7.25-7.21(m,1H),7.15-7.14(m,4H),5.82(d,J＝10Hz,2H),5.17(s,2H),5.06(s,1H),4.44(s,1H),4.29-4.26(m,2H),3.84-3.80(m,2H),3.70-3.65(m,1H),3.57(s,1H),3.54(s,5H),3.49-3.40(m,3H),3.27(s,1H),3.24(s,3H),3.17(s,2H),3.04-3.01(m,4H),2.46(s,2H),2.18-2.13(m,3H),2.01-1.95(m,3H),1.91-1.86(m,2H),1.66-1.63(m,2H),1.19-1.13(m,3H),1.09-1.05(m,3H),0.71-0.69(m,4H),0.02(s,6H)；
LC-MS(ESI)m/z 1056.4[M+H] +
实施例18：((2S,3R)-1-((2S,4S)-2-((4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5-(6-氟-2-((S)-1-(N-(甲氧基羰基)-O-甲基-L-苏氨酰基)吡咯烷-2-基)-1H-苯并[d]咪唑-5-基)吡咯烷-2-基)苯基)甲酰胺基)-4-(甲氧甲基)吡咯烷-1-基)-3-甲氧基-1-氧代丁烷-2-基)氨基甲酸甲酯(17b)
Figure PCTCN2018079965-appb-000102
步骤1：1-(4-((2R,5R)-2-(4-氯-2-氟-5-硝基苯基)-5-(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基-1,4-硅杂哌啶
单口瓶中加入(1S,4S)-1-(4-氯-2-氟-5-硝基苯基)-4-(4-硝基苯基)丁烷-1,4-二基二甲磺酸酯(15g,27.7mmol)，4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯胺(14.3g,55.46mmol)，乙腈(60mL)和N,N-二异丙基乙基胺(18g,138.5mmol)，加热至75℃过夜反应。反应结束后，将反应液冷却至室温后倒入6N盐酸水溶液(200mL)中,乙酸乙酯(200mLx2)萃取。有机相分别用水(200mL)和饱和食盐水(200mL)洗涤，无水硫酸钠干燥。过滤，浓缩，所得粗品用硅胶柱层析分离(石油醚:乙酸乙酯＝10:1)，得到标题化合物(1.5g)。
LC-MS(ESI)m/z 605.8[M+H] +。
步骤2：(S)-2-((4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5-(4-硝基苯基)吡咯烷-2-基)-5-氟-2-硝基苯基)氨基甲酰基)吡咯烷-1-甲酸叔丁酯
单口瓶中加入1-(4-((2R,5R)-2-(4-氯-2-氟-5-硝基苯基)-5-(4-硝基苯基)吡咯烷-1-基)-2,6-二氟苯基)-4,4-二甲基-1,4-硅杂哌啶(1g,1.66mmol)，(S)-2-氨基甲酰基吡咯烷-1-羧酸叔丁酯(0.53g,2.48mmol)，三(二亚苄基丙酮)二钯(0.23g,0.248mmol)，4,5-双二苯基膦-9,9-二甲基氧杂蒽(0.287g,0.496mmol)，碳酸铯(0.81g,2.48mmol)和二氧六环(12.5ml)，氮气保护下100℃反应12小时。硅藻土过滤，浓缩，所得粗品用硅胶柱层析纯化(石油醚:乙酸乙酯＝5:1)，得到标题化合物(0.54g)。
LC-MS(ESI)m/z 783.8[M+H] +。
步骤3：(S)-2-((2-氨基-4-((2R,5R)-5-(4-氨基苯基)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)-5-氟苯基)氨基甲酰基)吡咯烷-1-甲酸叔丁酯
单口瓶中加入(S)-2-((4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5-(4-硝基苯基)吡咯烷-2-基)-5-氟-2-硝基苯基)氨基甲酰基)吡咯烷-1-甲酸叔丁酯(0.54g,0.69mmol)，四氢呋喃(10mL)和二氧化铂(0.054g)，氢气氛围下室温反应过夜。硅藻土过滤，浓缩得标题化合物(0.5g)。
LC-MS(ESI)m/z 723.5[M+H] +。
步骤4：(2S,4S)-2-((4-((2R，5R)-5-(5-氨基-4-((S)-1-(叔丁氧羰基)吡咯烷-2-甲酰胺基)-2-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)-4-(甲氧基甲基)吡咯烷-1-甲酸叔丁酯
单口瓶中加入(S)-2-((2-氨基-4-((2R,5R)-5-(4-氨基苯基)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)-5-氟苯基)氨基甲酰基)吡咯烷-1-甲酸叔丁酯(0.22g,0.297mmol)，(2S，4S)-1-(叔丁氧基羰基)-4-(甲氧基甲基)吡咯烷-2-甲酸(0.085g,0.327mmol)，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(0.17g,0.446mmol)，N,N-二异丙基乙基胺(0.077g,0.594mmol)和N,N-二甲基甲酰胺(6mL)，室温反应1小时。反应液倒入水(150mL)中，过滤，得到标题化合物(0.32g)。
LC-MS(ESI)m/z 986.8[M+Na] +。
步骤5：(2S,4S)-2-((4-((2R,5R)-5-(2-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-6-氟-苯并[d]咪唑-5-基)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)-4-(甲氧基甲基)吡咯烷-1-甲酸叔丁酯
单口瓶中加入(2S,4S)-2-((4-((2R，5R)-5-(5-氨基-4-((S)-1-(叔丁氧羰基)吡咯烷-2-甲酰胺基)-2-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)-4-(甲氧基甲基)吡咯烷-1-甲酸叔丁酯(0.31g,0.203mmol)和醋酸(2ml)，加热到72℃反应30分钟，反应液用饱和碳酸氢钠溶液调节pH至碱性，乙酸乙酯萃取，有机相分别用水和饱和食盐水洗涤，无水硫酸钠干燥，过滤，浓缩得标题化合物(0.26g)。
LC-MS(ESI)m/z 946.9[M+H] +。
步骤6：(2S,4S)-N-(4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5-(6-氟-2-((S)-吡咯烷-2-基)-1H-苯并[d]咪唑-5-基)吡咯烷-2-基)苯基)-4-(甲氧基甲基)吡咯烷-2-甲酰胺
单口瓶中加入(2S,4S)-2-((4-((2R,5R)-5-(2-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-6-氟-苯并[d]咪唑-5-基)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)吡咯烷-2-基)苯基)氨基甲酰基)-4-(甲氧基甲基)吡咯烷-1-甲酸叔丁酯(0.25g,0.26mmol)，浓盐酸(3mL)和四氢呋喃(3mL)，室温搅拌1小时，反应液用饱和碳酸钠溶液调节pH至碱 性，二氯甲烷和甲醇的混合溶液(二氯甲烷:甲醇＝10:1)萃取，无水硫酸钠干燥，过滤，浓缩得标题化合物(0.18g)。
LC-MS(ESI)m/z 746.6[M+H] +。
步骤7：
单口瓶中加入(2S,4S)-N-(4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5-(6-氟-2-((S)-吡咯烷-2-基)-1H-苯并[d]咪唑-5-基)吡咯烷-2-基)苯基)-4-(甲氧基甲基)吡咯烷-2-甲酰胺(0.18g,0.24mmol)，N-(甲氧基羰基)-O-甲基-L-苏氨酸(0.11g,0.55mmol)，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(0.18g,0.48mmol)，N,N-二异丙基乙基胺(0.25g,1.92mmol)和N,N-二甲基甲酰胺(6ml)，室温搅拌1小时。反应液倒入水(150mL)中，乙酸乙酯(200mL)萃取。有机相分别用水(100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥。过滤，浓缩，所得粗品用制备液相纯化，得标题化合物(0.083g)。
1H NMR(500M,DMSO-d 6)δ12.23(brs,1H),9.98(s,1H),7.54-7.53(m,2H),7.37-7.29(m,2H),7.20-7.18(m,2H),7.12(s,1H),6.99(s,1H),5.83-5.81(m,2H),5.47-5.45(m,1H),5.22(s,1H),5.11(s,1H),4.42-4.38(m,1H),4.26-4.24(m,2H),4.10-4.08(m,1H),3.81(s,2H),3.54(s,6H),3.45-3.39(m,6H),3.26-3.24(m,6H),3.08-3.03(m,6H),2.44-2.37(m,1H),2.30(s,1H),2.19(s,2H),2.02-1.97(m,2H),1.74(s,1H),1.66-1.59(m,2H),1.25-1.23(m,2H),1.13(s,3H),0.96(s,3H),0.70(s,4H),0.02(s,6H)。
LC-MS(ESI)m/z 1092.9[M+H] +。
实施例19：((2S,3R)-1-((2S,4S)-2-((4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5-(2-((S)-1-(N-(甲氧基羰基)-O-甲基-L-苏氨酰基)吡咯烷-2-基)-1H-苯并[d]咪唑-5-基)吡咯烷-2-基)苯基)甲酰胺基)-4-(甲氧甲基)吡咯烷-1-基)-3-甲氧基-1-氧代丁烷-2-基)氨基甲酸甲酯(18b)
Figure PCTCN2018079965-appb-000103
步骤1-6：(2S,4S)-N-(4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5-(2-((S)-吡咯烷-2-基)-1H-苯并[d]咪唑-5-基)吡咯烷-2-基)苯基)-4-(甲氧基甲基)吡咯烷-2-甲酰胺
将起始原料由(1S,4S)-1-(4-氯-2-氟-5-硝基苯基)-4-(4-硝基苯基)丁烷-1,4-二基二甲磺酸酯替换成(1S,4S)-1-(4-氯-3-硝基苯基)-4-(4-硝基苯基)丁烷-1,4-二基二甲磺酸酯，参考实施例18中的步骤1-6，得到目标化合物。
步骤7：
反应瓶中加入(2S,4S)-N-(4-((2R,5R)-1-(4-(4,4-二甲基-1,4-硅杂哌啶-1-基)-3,5-二氟苯基)-5-(2-((S)-吡咯烷-2-基)-1H-苯并[d]咪唑-5-基)吡咯烷-2-基)苯基)-4-(甲氧基甲基)吡咯烷-2-甲酰胺(0.30g,0.41mmol)，N-(甲氧基羰基)-O-甲基-L-苏氨酸(0.18g,0.95mmol)，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(0.31g,0.82mmol)，N,N-二异丙基乙胺(0.56mL,3.28mmol)，N,N-二甲基甲酰胺(5mL)，混合物在室温搅拌反应45分钟。反应结束将反应液倒入水中(50mL)，搅拌5分钟，乙酸乙酯(30mL)萃取，有机相水洗，干燥，浓缩，残留物制备纯化得化合物18b(0.26g)。
1H NMR(500MHz,CDCl 3)δ＝10.33-10.41(brs,1H),8.81(s,1H),7.44-7.46(m,3H),7.28-7.32(m,1H),7.05-7.10(m,3H),5.70-5.78(m,4H),5.46-5.50(m,1H),5.14-5.17(m,1H),5.03-5.04(m,1H),4.78-4.81(m,1H),4.70-4.72(m,1H),4.61-4.63(m,1H),4.02-4.06(m,1H),3.76-3.77(m,3H),3.70-3.71(m,6H),3.42-3.51(m,2H),3.6(s,6H),3.26-3.27(m,3H),3.12(t,J＝6.0Hz,4H),2.48-2.61(m,3H),2.09-2.37(m,5H),1.71-1.79(m,5H),1.15-1.23(m,6H),0.76-0.79(m,4H),0.05(s,6H)。
LC-MS(ESI)m/z1074.8[M+H] +。
试验例1：测定具体化合物抗丙型肝炎病毒GT1b/3a和GT1b/6a NS5A嵌合复制子及GT1b和GT1a野生型复制子活性
1、实验材料
细胞：Huh 7细胞系及HCV GT1b/3a和GT1b/6a NS5A嵌合复制子及GT1b和GT1a野生型复制子RNA。
化合物：用100％DMSO配制成10mM母液暂存氮气柜。
主要试剂见表1：
表1 试剂列表
试剂名称 供应商
DMEM细胞培养液 Invitrogen
胎牛血清(FBS) Corning
谷氨酰胺(L-Glu) Invitrogen
青霉素-链霉素 Hyclone
非必需氨基酸(NEAA) Invitrogen
二甲基亚砜(DMSO) Sigma
CellTiter-Fluor检测试剂 Promega
Britelite plus检测试剂 Perkin Elmer
2、实验方法
化合物处理：根据表2化合物检测终浓度，对化合物DMSO母液进行稀释并加入96孔实验板中。DMSO终浓度为0.5％。化合物检测终浓度见表2。
表2
Figure PCTCN2018079965-appb-000104
准备细胞：复制子质粒DNA用相应的限制性内切酶线性化后，用T7RNA聚合酶将DNA转录成RNA。用电击穿孔法将体外转录制备的复制子RNA转染至Huh-7细胞内。转染后的细 胞以每孔10000个细胞的密度接种到含有稀释好化合物的96孔实验板中。随后置于5％CO 2、37℃培养箱中培养3天。
细胞活性检测:在GT1b野生型复制子的96孔板中加入细胞生长荧光滴定检测试剂CellTiter-Fluor，37℃、5％CO 2培养箱培养细胞1小时后，用Envision检测Fluorescence信号值(RFU)，原始数据(RFU)用于化合物细胞毒性计算。
药物活性检测：加入用荧光素酶检测试剂Britelite plus，在15分钟内用Envi sion检测Luminescence信号值(RLU)，原始数据(RLU)用于化合物活性计算。
数据处理：化合物细胞毒性计算，所用公式如下：
细胞活性Viability％＝(CPD-HPE)/(ZPE-HPE)x100％*
化合物活性计算，所用公式如下
抑制率Inhibition％＝(ZPE-CPD)/(ZPE-HPE)x100％*
*CPO:化合物孔的信号值；
HPE(Hundred percent effect)：100％有效作用对照孔信号值，孔中只有DMEM培养液；
ZPE(Zero percent e effect)：无效作用对照孔信号值，用0.5％DMSO代替化合物。
将细胞活力百分数(Viability％)、抑制百分数(Inhibition％)分别导入GraphPad Prism软件，采用非线性回归方法拟合曲线，得出化合物对应的曲线及其细胞毒性(CC 50)和其对HCV复制子的抑制活性(EC 50)数值。
3、实验结果见表3
表3.化合物抗HCV GT1b/3a和GT1b/6a NS5A嵌合复制子及GT1b和GT1a野生型复制子活性EC 50值和细胞毒性CC 50值
Figure PCTCN2018079965-appb-000105
Figure PCTCN2018079965-appb-000106
试验例2：测定具体化合物抗丙型肝炎病毒GT1a和GT1b野生型复制子以及GT1b/3a、GT1b/4a、GT1b/5a和GT1b/6a NS5A嵌合复制子活性
1、实验材料
细胞：Huh-7细胞为肝癌细胞株(来源于美国AppTec公司)，HCV GT1a和GT1b野生型(wt)复制子，GT1b/3a、GT1b/4a、GT1b/5a和GT1b/6a NS5A嵌合复制子。
化合物：用100％DMSO配制成10mM母液存放于-40℃冰箱保存。
主要试剂见表4：
表4.主要试剂
试剂名称 供应商
DMEM培养液 Invitrogen
胎牛血清(FBS) Corning
青霉素-链霉素 Hyclone
非必需氨基酸(NEAA) Invitrogen
谷氨酰胺(L-Glu) Invitrogen
二甲基亚砜(DMSO) Sigma
CellTiter-Fluor检测试剂 Promega
Britelite plus检测试剂 Perkin Elmer
Renilla Luciferase检测试剂 Promega
alamarBlue检测试剂 Invitrogen
2、实验方法-瞬时转染法
化合物处理：根据表5化合物检测终浓度，对化合物DMSO母液进行稀释并加入96孔实验板中。DMSO终浓度为0.5％。化合物检测终浓度见表5。
表5
Figure PCTCN2018079965-appb-000107
Figure PCTCN2018079965-appb-000108
Figure PCTCN2018079965-appb-000109
准备细胞：复制子质粒DNA用相应的限制性内切酶线性化后，用T7RNA聚合酶将DNA转录成RNA。用电击穿孔法将体外转录制备的复制子RNA转染至Huh-7细胞内。转染后的细胞以每孔10000个细胞的密度接种到含有稀释好化合物的96孔实验板中。随后置于5％CO 2、37℃培养箱中培养3天。
细胞活性检测:在GT1b野生型复制子的96孔板中加入细胞生长荧光滴定检测试剂CellTiter-Fluor，37℃、5％CO 2培养箱培养细胞1小时后，用Envision检测Fluorescence信号值(RFU)，原始数据(RFU)用于化合物细胞毒性计算。
药物活性检测：加入用荧光素酶检测试剂Britelite plus，在15分钟内用Envision检测Luminescence信号值(RLU)，原始数据(RLU)用于化合物活性计算。
数据处理：
化合物细胞毒性计算，所用公式如下：
细胞活性Viability％＝(CPD-HPE)/(ZPE-HPE)x100％*
化合物活性计算，所用公式如下
抑制率Inhibition％＝(ZPE-CPD)/(ZPE-HPE)x100％*
*CPO:化合物孔的信号值；
HPE(Hundred percent effect)：100％有效作用对照孔信号平均值，孔中只有DMEM培养液；
ZPE(Zero percent effect)：无效作用对照孔信号值，用0.5％DMSO代替化合物。
将细胞活力百分数(Viability％)、抑制百分数(Inhibition％)分别导入GraphPad Prism软件， 采用非线性回归方法拟合曲线，得出化合物对应的曲线及其细胞毒性(CC 50)和其对HCV复制子的抑制活性(EC 50)数值。
试验例3：测定具体化合物抗感染性丙型肝炎病毒HCVcc(GT2a，JFH-1)活性
1、实验材料
细胞：Huh7.5.1细胞和丙型肝炎病毒HCVcc(GT2a，JFH-1)感染系统。
化合物：用100％DMSO配制成10mM母液暂存氮气柜。
主要试剂见上表4。
2、试验方法
化合物处理：根据表6化合物检测终浓度，对化合物DMSO母液进行稀释并加入96孔实验板中。DMSO终浓度为0.5％。化合物检测终浓度见表6。
表6 化合物检测终浓度
Figure PCTCN2018079965-appb-000110
病毒感染：Huh7.5.1细胞接种于96孔细胞培养板中，细胞在37℃和5％CO2培养箱中培养过夜后备用。将化合物及HCVcc病毒液(MOI＝0.1)同时加入细胞中，病毒感染培养3天。平行的细胞毒性试验和病毒抑制方法相同，但无病毒感染。
药物活性检测：加入Renilla luciferase检测试剂检测hRLuc的活性。原始数据(RLU)用于化合物活性计算。
细胞活性检测：加入alamarBlue检测试剂检测细胞活力。原始数据(RFU)用于化合物细胞毒性计算。
数据处理：化合物细胞毒性计算，所用公式如下：
细胞活力.％＝CPD/CC×100％*
化合物活性计算，所用公式如下：
抑制率.％＝(CPD-VC)/(CC-VC)×100％*
*CPD：化合物孔的信号值。
VC(Virus Control)：病毒对照。DMSO对照孔的平均信号，包含细胞和病毒。
CC(Cell Control)：细胞对照。细胞对照孔，只包含细胞，没有病毒。
将细胞活力百分数(细胞活力％)、化合物活性百分数(抑制率％)分别导入GraphPad Prism软件，采用非线性回归方法拟合曲线，得出化合物对应的曲线及其细胞毒性(CC 50)和其对HCV复制子的抑制活性(EC 50)数值。
3、试验例2和试验例3的试验结果见表7：
表7
Figure PCTCN2018079965-appb-000111
ND：未检测
试验例4：具体化合物在大鼠肝脏组织分布评价
18只雄性大鼠随即分为两组，每组9只，禁食(不禁水)过夜，分别灌胃给予化合物1a、化合物6b 10mg/kg，给药后，于0.5h、3h、8h放血处死受试动物，取血1mL左右，EDTA-K2抗凝，立即将血样放置冰中，30min内转移到4℃，4000rpm，10min条件下离心分离血浆。同时于各个时间点取整个肝脏，用冰冷的生理盐水冲洗，吸干，称重，生理盐水(W/V＝1：5)稀释匀浆。收集全部血浆和肝脏匀浆液后立即于-20℃保存待测。
大鼠给药后4h可以进食。
所有样品/样本完全融化后，涡旋混匀；移取50μL样品至1.5mL离心管中，分别加入300μL的IS-D(20ng/ml地西泮乙腈)溶液(空白样品加入200μL乙腈)；将样品涡旋10min后，在 12000rpm、4℃条件下离心10min；于96孔板中对应加入80μL的纯水后从离心管中分别移取80μL上清液至96孔板中，涡旋3min，放置于4℃直至LC-MS/MS进样分析。血浆及肝脏药物浓度见表8。
表8.化合物在大鼠血浆及肝脏浓度数据。
Figure PCTCN2018079965-appb-000112
试验例5：具体化合物在小鼠肝脏组织分布评价
18只雄性小鼠随即分为2组，禁食(不禁水)过夜，分别灌胃给予化合物1a、化合物6b 10mg/kg，给药后，于0.5h、3h、8h放血处死受试动物，取血1mL左右，EDTA-K2抗凝，立即将血样放置冰中，30min内转移到4℃，4000rpm，10min条件下离心分离血浆。同时于各个时间点取整个肝脏，用冰冷的生理盐水冲洗，吸干，称重，生理盐水(W/V＝1：5)稀释匀浆。收集全部血浆和肝脏匀浆液后立即于-20℃保存待测。
小鼠给药后4h可以进食。
所有样品/样本完全融化后，涡旋混匀；移取50μL样品至1.5mL离心管中，分别加入200μL的IS-D(20ng/ml地西泮乙腈)溶液(空白样品加入200μL乙腈)；将样品涡旋10min后，在12000rpm、4℃条件下离心10min；于96孔板中对应加入80μL的纯水后从离心管中分别移取80μL上清液至96孔板中，涡旋3min，放置于4℃直至LC-MS/MS进样分析。血浆及肝脏药物浓度见表9。
表9.化合物在小鼠血浆及肝脏浓度数据
Figure PCTCN2018079965-appb-000113
试验例6：具体化合物的耐药性评价-------测定具体化合物的体外抗HCV GT1a野生型复制子以及GT1a-NS5A-Y93H和GT1a-NS5A-L31V突变复制子的活性
1、实验材料
细胞：Huh-7细胞为肝癌细胞株(来源于美国AppTec公司)，HCVGT1a野生型复制子及GT1a-NS5A-Y93H和GT1a-NS5A-L31V突变复制子
化合物：用100％DMSO配制成10mM母液存放于氮气柜保存。
主要试剂见表10。
表10.主要试剂
试剂名称 供应商
DMEM培养液 Invitrogen
胎牛血清(FBS) Corning
青霉素-链霉素 Hyclone
非必需氨基酸(NEAA) Invitrogen
谷氨酰胺(L-Glu) Invitrogen
二甲基亚砜(DMSO) Sigma
Bright-Glo检测试剂 Promega
2、试验方法：瞬时转染法检测
化合物处理：根据表11化合物检测终浓度，对化合物DMSO母液进行稀释并加入96孔实验板中。DMSO终浓度为0.5％。化合物检测终浓度见表11。
表11 化合物检测终浓度
Figure PCTCN2018079965-appb-000114
准备细胞：复制子质粒DNA用相应的限制性内切酶线性化后，用T7RNA聚合酶将DNA转录成RNA。用电击穿孔法将体外转录制备的复制子RNA转染到Huh-7细胞内。转染后的细胞以每孔10,000个细胞的密度接种到含有稀释好化合物的96孔实验板中。随后置于5％CO2和37℃培养箱中培养3天。
化合物活性检测：加入用荧光素酶检测试剂Bright-Glo，在5分钟内用Envision检测Luminescence信号值(RLU)，原始数据(RLU)用于化合物活性计算。
数据处理：
化合物活性计算，所用公式如下：
抑制率％＝(CPD-ZPE)/(HPE-ZPE)×100％*
*CPD：化合物孔的信号值；
HPE(Hundred percent effect)：100％有效作用对照孔信号平均值，孔中只有DMEM培养液；ZPE(Zero percent effect)：无效作用对照孔信号平均值，用0.5％DMSO代替化合物。
将化合物活性百分数(抑制率％)导入GraphPad Prism软件，采用非线性回归方法拟合曲线，得出化合物对应的曲线及其对HCV复制子的抑制活性(EC50)数值。
3、试验结果见表12：
表12.
Figure PCTCN2018079965-appb-000115
NT：未检测。

Claims (18)

  1. 式Ⅰ化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物：
    Figure PCTCN2018079965-appb-100001
    其中：
    R 1和R 2分别独立地选自羟基、烷基、烷氧基和芳基，或者R 1和R 2连接形成含硅的饱和脂肪环，所述含硅的饱和脂肪环含有1、2或3个硅原子；
    R 3、R 4、R 5和R 6分别独立地选自氢和卤素；
    X选自以下基团：
    Figure PCTCN2018079965-appb-100002
    Y选自以下基团：
    Figure PCTCN2018079965-appb-100003
    R 7和R 8分别独立地选自-CH(烷基)(烷氧基)、-CH(烷基) 2、-CH(烷氧基) 2、-C(烷基) 2(烷氧基)、-C(烷基)(烷氧基) 2、-C(烷基) 3和-C(烷氧基) 3；
    R 9和R 10分别独立地选自氢、卤素、烷基、烷氧基和烷氧基烷基；
    各个R a和R b分别独立地选自氢、卤素和烷基；
    各个m独立地选自0、1、2、3和4；
    n选自0、1、2、3、4、5、6、7和8。
  2. 如权利要求1所述的化合物，其为式Ia的化合物、式Ib的化合物、式Ic的化合物或式Id的化合物：
    Figure PCTCN2018079965-appb-100004
    其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R a、R b、X、Y、m和n如权利要求1所定义。
  3. 如权利要求1或2所述的化合物，其中R 1和R 2分别独立地选自C 1-6烷基，或者R 1和R 2连接形成3-8元含硅的饱和脂肪环，所述3-8元含硅的饱和脂肪环中含有1个硅原子。
  4. 如权利要求1或2所述的化合物，其中R 1和R 2分别独立地选自甲基、乙基、正丙基、异丙基、叔丁基、正丁基、异丁基和苯基，或者R 1和R 2连接形成3元、4元、5元或6元的含硅的饱和脂肪环，其中所述含硅的饱和脂肪环含有1个硅原子。
  5. 如权利要求1-4中任一项所述的化合物，其中R 3、R 4、R 5和R 6分别独立地选自氢、氟和氯；优选地，R 3和R 4各自独立地选自氢和氟，R 5和R 6选自氢；优选地，R 3和R 4中的一个为氟，另一个为氢，R 5和R 6选自氢；优选地，R 3和R 4选自氟，R 5和R 6选自氢。
  6. 如权利要求1-5中任一项所述的化合物，X选自
    Figure PCTCN2018079965-appb-100005
    Y选自
    Figure PCTCN2018079965-appb-100006
    或者X选自
    Figure PCTCN2018079965-appb-100007
    Y选自
    Figure PCTCN2018079965-appb-100008
    或者X选自
    Figure PCTCN2018079965-appb-100009
    Y选自
    Figure PCTCN2018079965-appb-100010
    或者X选自 Y选自
    Figure PCTCN2018079965-appb-100012
    或者X选自
    Figure PCTCN2018079965-appb-100013
    Y选自
    Figure PCTCN2018079965-appb-100014
    Figure PCTCN2018079965-appb-100015
    优选地，X选自
    Figure PCTCN2018079965-appb-100016
    Y选自
    Figure PCTCN2018079965-appb-100017
    或者X选自
    Figure PCTCN2018079965-appb-100018
    Y选自
    Figure PCTCN2018079965-appb-100019
    或者X选自
    Figure PCTCN2018079965-appb-100020
    Y选自
    Figure PCTCN2018079965-appb-100021
    或者X选自
    Figure PCTCN2018079965-appb-100022
    Y选自
    Figure PCTCN2018079965-appb-100023
    或者X选自
    Figure PCTCN2018079965-appb-100024
    Y选自
    Figure PCTCN2018079965-appb-100025
    优选地，X选自
    Figure PCTCN2018079965-appb-100026
    Y选自
    Figure PCTCN2018079965-appb-100027
    或者X选自
    Figure PCTCN2018079965-appb-100028
    Y选自
    Figure PCTCN2018079965-appb-100029
    或者X选自
    Figure PCTCN2018079965-appb-100030
    Y选自
    Figure PCTCN2018079965-appb-100031
    或者X选自
    Figure PCTCN2018079965-appb-100032
    Y选自
    Figure PCTCN2018079965-appb-100033
    或者X选自
    Figure PCTCN2018079965-appb-100034
    Y选自
    Figure PCTCN2018079965-appb-100035
    Figure PCTCN2018079965-appb-100036
  7. 如权利要求1-5中任一项所述的化合物，其中X选自
    Figure PCTCN2018079965-appb-100037
    Figure PCTCN2018079965-appb-100038
    Y选自
    Figure PCTCN2018079965-appb-100039
    或者，X选自
    Figure PCTCN2018079965-appb-100040
    Y选自
    Figure PCTCN2018079965-appb-100041
  8. 如权利要求1-7中任一项所述的化合物，其中R 7和R 8分别独立地选自-CH(CH 3)(OCH 3)、-CH(CH 3) 2、-CH(OCH 3) 2、-C(CH 3) 2(OCH 3)、-C(CH 3)(OCH 3) 2、-C(CH 3) 3、-C(OCH 3) 3、-CH(CH 2CH 3)(OCH 2CH 3)、-CH(CH 3)(OCH 2CH 3)、-CH(CH 2CH 3)(OCH 3)、-CH(CH 2CH 3) 2、-CH(CH 3)(CH 2CH 3)、-CH(OCH 2CH 3) 2、-CH(OCH 2CH 3)(OCH 3)、-C(CH 2CH 3) 2(OCH 2CH 3)、-C(CH 3) 2(OCH 2CH 3)、-C(CH 2CH 3) 2(OCH 3)、-C(CH 2CH 3)(OCH 2CH 3) 2、-C(CH 3)(OCH 2CH 3) 2、-C(CH 2CH 3)(OCH 3) 2、-C(CH 2CH 3) 3、-C(CH 3)(CH 2CH 3) 2、-C(CH 3) 2(CH 2CH 3)和-C(OCH 2CH 3) 3。
  9. 如权利要求1-8中任一项所述的化合物，其中R 9和R 10分别独立地选自氢、氟、氯、甲基、乙基、正丙基、异丙基、叔丁基、正丁基、异丁基、甲氧基、乙氧基、正丙氧基、异丙氧基、叔丁氧基、正丁氧基、异丁氧基、甲氧基甲基、甲氧基乙基、乙氧基甲基和乙氧基乙基。
  10. 如权利要求1-7中任一项所述的化合物，其中R 7和R 8分别独立地选自-CH(CH 3)(OCH 3)和-CH(CH 3) 2；R 9和R 10分别独立地选自氢和甲氧基甲基。
  11. 如权利要求1-10中任一项所述的化合物，其中R a和R b分别独立地选自氢、氟、氯、甲基、乙基、正丙基、异丙基、叔丁基、正丁基和异丁基。
  12. 如权利要求1-11中任一项所述的化合物，其中R 1和R 2为甲基，或者R 1和R 2连接形成含有1个硅原子的4元、5元或6元含硅的饱和脂肪环；R 3和R 4中的一个为氟，另一个为氢，或者R 3和R 4均为氟；R 5和R 6为氢；X为
    Figure PCTCN2018079965-appb-100042
    Figure PCTCN2018079965-appb-100043
    Y为
    Figure PCTCN2018079965-appb-100044
    R 7和R 8均为-CH(CH 3)(OCH 3)；R 9和R 10均为氢或者均为甲氧基甲基；n为0。
  13. 如权利要求1或2所述的化合物，选自下列化合物：
    Figure PCTCN2018079965-appb-100045
    Figure PCTCN2018079965-appb-100046
    Figure PCTCN2018079965-appb-100047
    Figure PCTCN2018079965-appb-100048
    Figure PCTCN2018079965-appb-100049
    Figure PCTCN2018079965-appb-100050
  14. 药物组合物，其包含治疗有效量的权利要求1-13中任一项所述的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，以及一种或多种药学上可接受的载体。
  15. 权利要求1-13中任一项所述的化合物或其药学上可接受的盐、或互变异构体、或立体异 构体、或氘代物及其混合物，或权利要求14所述的药物组合物在制备治疗丙型肝炎病毒感染的药物中的用途。
  16. 治疗丙型肝炎病毒感染的方法，所述方法包括给予需要治疗的患者治疗有效量的权利要求1-13中任一项所述的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，或权利要求14所述的药物组合物。
  17. 用于治疗丙型肝炎病毒感染的权利要求1-13中任一项所述的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，或权利要求14所述的药物组合物。
  18. 权利要求1-13中任一项所述的化合物或其药学上可接受的盐、或互变异构体、或立体异构体、或氘代物及其混合物，或权利要求14所述的药物组合物在治疗丙型肝炎病毒感染中的用途。
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