WO2018090862A1 - 二氢嘧啶类化合物及其制备方法和用途 - Google Patents

二氢嘧啶类化合物及其制备方法和用途 Download PDF

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WO2018090862A1
WO2018090862A1 PCT/CN2017/110123 CN2017110123W WO2018090862A1 WO 2018090862 A1 WO2018090862 A1 WO 2018090862A1 CN 2017110123 W CN2017110123 W CN 2017110123W WO 2018090862 A1 WO2018090862 A1 WO 2018090862A1
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compound
alkyl
group
cooh
pharmaceutically acceptable
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PCT/CN2017/110123
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English (en)
French (fr)
Inventor
宋帅
蔡家强
田强
曾宏
宋宏梅
邓汉文
唐祖建
段小凡
龙蓉蓉
刘瑶
王利春
王晶翼
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四川科伦博泰生物医药股份有限公司
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Priority to EA201990528A priority Critical patent/EA201990528A1/ru
Priority to MX2019005119A priority patent/MX2019005119A/es
Priority to AU2017359773A priority patent/AU2017359773B2/en
Priority to BR112019005205A priority patent/BR112019005205A2/pt
Priority to ES17870982T priority patent/ES2901401T3/es
Priority to CA3037218A priority patent/CA3037218A1/en
Priority to JP2019513814A priority patent/JP7139568B2/ja
Priority to KR1020197007824A priority patent/KR102496508B1/ko
Application filed by 四川科伦博泰生物医药股份有限公司 filed Critical 四川科伦博泰生物医药股份有限公司
Priority to EP17870982.0A priority patent/EP3508483B1/en
Priority to US16/334,237 priority patent/US10696669B2/en
Priority to MYPI2019001442A priority patent/MY194471A/en
Priority to CN201780057446.XA priority patent/CN109790145B/zh
Priority to PL17870982T priority patent/PL3508483T3/pl
Publication of WO2018090862A1 publication Critical patent/WO2018090862A1/zh
Priority to PH12019550039A priority patent/PH12019550039A1/en
Priority to IL266345A priority patent/IL266345A/en
Priority to US16/859,910 priority patent/US11166954B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5355—Non-condensed oxazines and containing further heterocyclic rings
    • 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
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a dihydropyrimidine compound having antiviral activity, a pharmaceutical composition comprising the same, a preparation method thereof and its prevention or treatment including but not limited to hepatitis A virus, hepatitis B virus, and hepatitis C virus Use in hepatitis, influenza, herpes, and viral diseases of acquired immunodeficiency syndrome (AIDS).
  • a dihydropyrimidine compound having antiviral activity a pharmaceutical composition comprising the same, a preparation method thereof and its prevention or treatment including but not limited to hepatitis A virus, hepatitis B virus, and hepatitis C virus Use in hepatitis, influenza, herpes, and viral diseases of acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • a virus consists of a nucleic acid molecule (DNA or RNA) or a protein (such as a prion).
  • the virus can cause a variety of infectious diseases. Common diseases caused by viruses include, but are not limited to, viral hepatitis A, hepatitis B, hepatitis C, influenza, herpes and acquired immunodeficiency syndrome. (AIDS).
  • antiviral drugs play a role by inhibiting virus attachment, uncoating, viral gene duplication, maturation or release, or by affecting the host's immune system, including reverse transcriptase inhibitors and capsid protein assembly inhibitors. Wait.
  • Hepatitis B virus is a common hepadnavirus-like viral pathogen. Such viruses can cause diseases such as acute hepatitis, chronic hepatitis, liver fibrosis, cirrhosis and liver cancer.
  • Drugs for the treatment of hepatitis B include interferons and nucleoside analogs (such as lamivudine and adefovir dipivoxil).
  • interferon inhibits the replication of hepatitis B virus by reacting with cell surface receptors to produce antiviral proteins.
  • nucleoside analogs act primarily by inhibiting the replication of viral polymerase (reverse transcriptase). The disadvantage of this type of drug is that long-term use can easily cause virus mutation and produce drug resistance.
  • hepatitis B can also be treated with non-nucleoside analogs.
  • heteroaryldihydropyrimidines Boy 41-4109
  • dihydropyrimidines induce misassembly of core proteins, resulting in unstable capsid proteins that accelerate the degradation of core proteins (Biochem. Pharmacol., 2003, 66, 2273-2279).
  • the heteroaryl dihydropyrimidine compound HAP1 (Proc. Natl. Acad. Sci., 2005, 102, 8138-8143) discovered by Zlotnick et al.
  • the dihydropyrimidine compounds of the present invention can inhibit the replication of HBV DNA more efficiently than the disclosed dihydropyrimidine HBV capsid protein assembly regulators (eg, cells).
  • the preferred anti-viral activity of the present invention is about 10 times the activity of the preferred compound (compound of Example 9) of WO2015144093.
  • the compounds of the present invention are free of the cardiotoxicity of the disclosed dihydropyrimidine compounds (such as the hERG inhibitory activity of the preferred compounds of GLS4 and WO2015144093 (compound of Example 9)); and with the disclosed dihydropyrimidine compounds (eg Compared to the compound of Example 5 in WO201403748, the compounds of the invention have a significantly reduced induction of the CYP450 enzyme 3A4 subtype. Furthermore, in the pharmacokinetic tests of rats, Beagle dogs, cynomolgus monkeys, the compounds of the invention exhibit better pharmacokinetic properties (eg better exposure, blood concentration, bioavailability) . At the same time, the preferred compounds of the present invention have good liver targeting properties, and the exposure of the drug to the liver can reach about 10 times of the exposure in plasma, indicating that the compound has liver enrichment ability, thereby facilitating improvement of liver diseases. Efficacy.
  • One aspect of the invention provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug thereof, wherein The compound has the structure of Formula I or Formula Ia:
  • Ar 1 and Ar 2 are each independently selected from a C 6-14 aryl group and a 5-14 membered heteroaryl group, which are optionally selected from one or more selected from the group consisting of halogen, -OH, -CN, -NO 2 , -N Substituent substitution of (R) 2 , C 1-6 alkyl, halo C 1-6 alkyl, C 1-6 alkylthio and C 3-6 cycloalkyl;
  • L is absent or selected from -O-, -S-, and -NR-;
  • R 1 and R 2 are each independently selected from H (including 1 H, 2 H, 3 H), C 1-6 alkyl (eg, deuterated C 1-6 alkyl), and C 3-6 cycloalkyl;
  • R 3 is a quaternary, five-, six- or seven-membered nitrogen heterocyclic ring selected from the following structures:
  • R a , R a ' , R 4 , R 4 ' , R 5 , R 5 ' and R 6 are each independently selected from H, halogen, -OH, -COOH, -CN, -NO 2 , -N(R) 2 , C 1-6 alkyl, halo C 1-6 alkyl, -WC 1-6 alkyl, -C 1-6 alkylene-WR, -WC 1-6 alkylene -W'-R, -WC 2-6 alkenyl, -C 2-6 alkenylene-WR, -WC 2-6 alkenylene-W'-R and C 3-6 cycloalkyl, wherein The alkylene and alkenylene are optionally further separated by one or more W; or R a together with R a ' , R 4 together with R 5 and/or R 4 ' and R 5' together, each occurrence
  • R 6 is attached to the ring carbon atom marked by * and / or ** in the above nitrogen heterocyclic structure
  • R is each independently selected from the group consisting of H, C 1-6 alkyl and C 3-6 cycloalkyl;
  • g is 1 or 2;
  • t is 0, 1, 2 or 3, provided that t is not greater than the number of positions on the corresponding group that can be substituted, and when t is greater than 1, each R 6 may be the same or different.
  • compositions comprising a prophylactically or therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph thereof, A solvate, metabolite, isotopically labeled compound or prodrug and one or more pharmaceutically acceptable carriers, preferably a solid formulation, a liquid formulation or a transdermal formulation.
  • Another aspect of the invention provides a method of preparing a pharmaceutical composition
  • a method of preparing a pharmaceutical composition comprising administering a compound of the invention, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph thereof,
  • the solvate, metabolite, isotopically labeled compound or prodrug is combined with one or more pharmaceutically acceptable carriers.
  • Another aspect of the invention provides a compound of the invention, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof, or Use of a prodrug or a pharmaceutical composition of the invention in the manufacture of a medicament for the prevention or treatment of a viral disease.
  • Another aspect of the invention provides a compound of the invention, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof, or A prodrug or a pharmaceutical composition of the invention for use in preventing or treating a viral disease.
  • Another aspect of the invention provides a method of preventing or treating a viral disease, the method comprising administering to an individual in need thereof an effective amount of a compound of the invention, or a pharmaceutically acceptable salt, ester, stereoisomer thereof, Tautomers, polymorphs, solvates, metabolites, isotopically labeled compounds or prodrugs or pharmaceutical compositions of the invention.
  • the above viral diseases include, but are not limited to, viral hepatitis A, hepatitis B virus, hepatitis C virus, influenza, herpes, and acquired immunodeficiency syndrome (AIDS).
  • viral hepatitis A hepatitis A
  • hepatitis B virus hepatitis B virus
  • hepatitis C virus influenza
  • herpes and acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • Another aspect of the invention provides a method of preparing a compound of the invention, the method comprising the steps of:
  • Hal is selected from the group consisting of F, Cl, Br, and I;
  • the halogenating agent is selected from the group consisting of Cl 2 , Br 2 , I 2 , N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide;
  • Step one is carried out in a protic solvent in the presence of an alkali metal salt
  • Step two is carried out in an aprotic solvent
  • Step 3 is carried out in an aprotic solvent in the presence of an organic base or an inorganic base;
  • R 2 in the compound of the present invention is a C 1-6 alkyl group
  • it can also be synthesized by a method comprising the following steps:
  • R 2 ' is H or C 1-5 alkyl
  • Hal is selected from the group consisting of F, Cl, Br, and I;
  • the halogenating agent is selected from the group consisting of Cl 2 , Br 2 , I 2 , N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide;
  • Step I is carried out in the presence of a Lewis acid in a non-polar solvent
  • Step II is carried out in an aprotic solvent in the presence of an organic or inorganic base
  • Step III is carried out in an aprotic solvent
  • Step IV is carried out in an aprotic solvent in the presence of an organic or inorganic base.
  • Figure 1 Induction of CYP450 enzyme 3A4 subtype by compound 10-227, control compound 3 and rifampicin.
  • alkylene denotes a saturated divalent hydrocarbon group, preferably a saturated divalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms, such as methylene, ethylene, Propylene or butylene.
  • alkenylene denotes a divalent hydrocarbon radical containing one or more double bonds, preferably having 2, 3, 4, 5 or 6 carbon atoms, such as ethenylene, propenylene or Allylene.
  • alkenylene groups the compounds may exist in pure E (ent ought) form, pure Z (zusammen) form, or any mixture thereof.
  • alkyl is defined as a straight or branched chain saturated aliphatic hydrocarbon.
  • an alkyl group has from 1 to 12, such as from 1 to 6 carbon atoms.
  • C1-6 alkyl refers to a linear or branched group of 1 to 6 carbon atoms (eg, methyl, ethyl, n-propyl, isopropyl, n-butyl) Base, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl), which is optionally substituted by one or more (such as 1 to 3) suitable substituents such as halogen (in this case the group)
  • the group is referred to as "haloalkyl” (for example, CF 3 , C 2 F 5 , CHF 2 , CH 2 F, CH 2 CF 3 , CH 2 Cl or -CH 2 CH 2 CF 3
  • C 1-4 alkyl refers to a linear or branched aliphatic hydrocarbon chain of 1 to 4 carbon atoms (ie methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, Sec-butyl or tert-butyl).
  • cycloalkyl refers to a saturated or unsaturated, non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (eg, a monocyclic ring such as cyclopropyl, cyclobutyl, cyclopentyl, Cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, or bicyclic, including spiro, fused or bridged systems (such as bicyclo [1.1.1] pentyl, bicyclo [2.2.1] heptyl, bicyclo [ 3.2.1] Octyl or bicyclo [5.2.0] anthracenyl, decalinyl, etc.), which is optionally substituted by one or more (such as 1 to 3) suitable substituents.
  • bicyclic hydrocarbon ring eg, a monocyclic ring such as cyclopropyl, cyclobutyl, cyclopentyl, Cyclohex
  • C 3-6 cycloalkyl refers to a saturated or unsaturated, non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring of 3 to 6 ring-forming carbon atoms ( For example, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), which is optionally substituted by one or more (such as 1 to 3) suitable substituents, such as methyl substituted cyclopropyl.
  • aryl refers to an all-carbon monocyclic or fused-ring polycyclic aromatic group having a conjugated pi-electron system.
  • C6-14 aryl means an aromatic group containing from 6 to 14 carbon atoms, such as phenyl or naphthyl.
  • the aryl group is optionally substituted with one or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO 2 , C 1-6 alkyl, etc.).
  • aralkyl preferably denotes an aryl-substituted alkyl group, wherein the aryl group and the alkyl group are as defined herein.
  • the aryl group can have from 6 to 14 carbon atoms and the alkyl group can have from 1 to 6 carbon atoms.
  • Exemplary aralkyl groups include, but are not limited to, benzyl, phenylethyl, phenylpropyl, phenylbutyl.
  • heteroaryl refers to a monovalent monocyclic, bicyclic or tricyclic aromatic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 rings.
  • the heteroaryl is selected from the group consisting of thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thia A oxazolyl group or the like, and a benzo derivative thereof; or a pyridyl group, a pyridazinyl group, a pyrimidinyl group, a pyrazinyl group, a triazinyl group or the like, and a benzo derivative thereof.
  • halo or halogen group, as used herein, is defined to include F, Cl, Br or I.
  • alkylthio refers to an alkyl group, as defined above, appended to the parent molecular moiety through a sulfur atom.
  • Representative examples of C1-6 alkylthio include, but are not limited to, methylthio, ethylthio, tert-butylthio, and hexylthio.
  • the 3 to 14 membered nitrogen heterocycle is a group having 3 to 14 carbon atoms and a hetero atom (at least one of which is a nitrogen atom) in the ring, including but not limited to a ternary nitrogen heterocyclic ring (eg, Aziridine), a quaternary nitrogen heterocyclic ring (such as azetidinyl), a five-membered nitrogen heterocyclic ring (such as pyrrolyl, tetrahydropyrrolyl, pyrrolinyl, pyrrolidinone, imidazolyl, imidazolidine) a group, an imidazolinyl group, a pyrazolyl group, a pyrazolinyl group, a six-membered nitrogen heterocyclic ring (such as piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl), a seven-membered nitrogen heterocyclic ring, and the like.
  • substituted means that one or more (eg, 1, 2, 3 or 4) hydrogens on the designated atom are replaced by a selected group, provided that the specified atom is not exceeded in the current situation. Normal valence and the substitution forms a stable compound. The number of alternative groups selected is permissible when such combinations form stable compounds.
  • substituent may be unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted by one or more of the list of substituents, one or more hydrogens on the carbon may be replaced individually and/or together by an optional substituent that is independently selected. If the nitrogen of the substituent is described as being optionally substituted by one or more of the list of substituents, then one or more hydrogens on the nitrogen may each be replaced by an independently selected optional substituent.
  • each substituent may be the same or different than another (other) substituent.
  • one or more means 1 or more than 1, such as 2, 3, 4, 5 or 10 under reasonable conditions.
  • a point of attachment of a substituent may come from any suitable position of the substituent.
  • the invention also includes all pharmaceutically acceptable isotopically-labeled compounds which are identical to the compounds of the invention, except that one or more atoms are of the same atomic number but the atomic mass or mass number differs from the atomic mass prevailing in nature. Or atomic substitution of mass.
  • isotopes suitable for inclusion in the compounds of the invention include, but are not limited to, isotopes of hydrogen (e.g., hydrazine (D, 2 H), hydrazine (T, 3 H)); isotopes of carbon (e.g., 11 C, 13 C) And 14 C); isotope of chlorine (eg 36 Cl); isotope of fluorine (eg 18 F); isotope of iodine (eg 123 I and 125 I); isotopes of nitrogen (eg 13 N and 15 N); isotopes of oxygen (eg 15 O, 17 O and 18 O); phosphorus isotopes (eg 32 P); and sulfur isotopes (eg 35 S).
  • isotopes of hydrogen e.g., hydrazine (D, 2 H), hydrazine (T, 3 H)
  • isotopes of carbon e.g., 11 C, 13 C) And 14 C
  • Certain isotopically-labeled compounds of the invention are useful in drug and/or substrate tissue distribution studies (e.g., assays).
  • the pharmaceutically acceptable solvates of the present invention include those in which the crystallization solvent can be substituted with an isotope, for example, D 2 O, acetone-d 6 or DMSO-d 6 .
  • stereoisomer denotes an isomer formed by at least one asymmetric center.
  • asymmetric center which can produce a racemic mixture, a single enantiomer, a mixture of diastereomers, and Separate diastereomers.
  • Specific individual molecules can also exist as geometric isomers (cis/trans).
  • the compounds of the invention may exist as mixtures (often referred to as tautomers) of two or more different forms in a rapidly balanced structure.
  • tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers Wait.
  • a dihydropyrimidinyl group can exist in equilibrium in the following tautomeric forms: It is to be understood that the scope of the present application covers all such ratios in any ratio (eg, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99). %) isomer or a mixture thereof.
  • Solid line (-), solid wedge can be used in this paper.
  • Virtual wedge Or wavy line The carbon-carbon bonds of the compounds of the invention are depicted.
  • a solid line is used to delineate a bond design that is bonded to an asymmetric carbon atom, including all possible stereoisomers at that carbon atom (eg, specific enantiomers, racemic mixtures, etc.).
  • a wavy line is used to delineate a bond representation linked to an alkenylene group, including all possible stereoisomers at the chemical bond (eg, a particular cis-trans isomer, a mixture of cis-trans isomers in any ratio, or Racemic mixture, etc.).
  • the use of a solid or virtual wedge to characterize the bond to an asymmetric carbon atom indicates the presence of the stereoisomers shown.
  • solid and virtual wedges are used to define relative stereochemistry rather than absolute stereochemistry.
  • the compounds of the invention may be stereoisomers (including cis and trans isomers, optical isomers (eg, R and S enantiomers), diastereomers, Geometric isomers, rotamers, conformers, atropisomers, and mixtures thereof exist.
  • the compounds of the invention may exhibit more than one type of isomerism and consist of a mixture thereof (e.g., a racemic mixture and a diastereomeric pair).
  • the invention encompasses all possible crystalline forms or polymorphs of the compounds of the invention, which may be a single polymorph or a mixture of more than one polymorph in any ratio.
  • the compounds of the invention may exist in free form for treatment or, where appropriate, in the form of their pharmaceutically acceptable derivatives.
  • pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites, isotopically labeled compounds or prodrugs, which are administered to a patient in need thereof
  • the compound of the invention, or a metabolite or residue thereof, can be provided directly or indirectly after the drug.
  • a “compound of the invention” it is also intended to encompass the various derivative forms described above for the compound.
  • the pharmaceutically acceptable salts of the compounds of the present invention include the acid addition salts and base addition salts thereof.
  • Suitable acid addition salts are formed from acids which form pharmaceutically acceptable salts, including suitable inorganic and organic acids. Examples include aspartate, benzoate, bicarbonate/carbonate, hydrogen sulfate/sulfate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate Phosphate, hydrobromide/bromide, hydroiodide/iodide, maleate, malonate, methyl sulfate, naphthylate, nicotinate, nitrate , orotate, oxalate, palmitate and other similar salts.
  • Suitable base addition salts are formed from bases which form pharmaceutically acceptable salts, including suitable inorganic bases and organic bases. Examples include aluminum salts, arginine salts, choline salts, diethylamine salts, lysine salts, magnesium salts, meglumine salts, potassium salts, and other similar salts.
  • esters means an ester derived from a compound of the formulae herein, which includes a physiologically hydrolyzable ester, which is hydrolyzable under physiological conditions to release the free acid or alcohol form of the invention.
  • Compound The compounds of the invention may also be esters per se.
  • the compound of the present invention may exist in the form of a solvate (e.g., hydrate) wherein the compound of the present invention contains a polar solvent as a structural element of the crystal lattice of the compound, particularly such as water, methanol or ethanol.
  • a polar solvent as a structural element of the crystal lattice of the compound, particularly such as water, methanol or ethanol.
  • the amount of polar solvent, particularly water may be present in stoichiometric or non-stoichiometric ratios.
  • metabolites of the compounds of the invention i.e., substances formed in vivo upon administration of a compound of the invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, and the like of the administered compound. Accordingly, the invention includes metabolites of the compounds of the invention, including compounds prepared by contacting a compound of the invention with a mammal for a time sufficient to produce a metabolic product thereof.
  • the invention further includes within its scope prodrugs of the compounds of the invention which are certain derivatives of the compounds of the invention which may or may not be pharmacologically active, which are administered to or into the body It can be converted to a compound of the invention having the desired activity by, for example, hydrolytic cleavage.
  • prodrugs will be functional group derivatives of the compounds which are readily converted in vivo to the desired therapeutically active compound. Additional information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", Volume 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 ( Edited by EBRoche, American Pharmaceutical Association).
  • Prodrugs of the invention may, for example, be known by those skilled in the art as “pro-moiety” (e.g., “Design of Prodrugs", H. Bundgaard (Elsevier, 1985))" It is prepared in place of the appropriate functional groups present in the compounds of the invention.
  • the invention also encompasses compounds of the invention containing a protecting group.
  • a protecting group In any process for preparing a compound of the invention, it may be necessary and/or desirable to protect a sensitive group or reactive group on any of the molecules of interest, thereby forming a chemically protected form of the compound of the invention. This can be achieved by conventional protecting groups, for example, as described in Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene & PGM Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991. Those protecting groups, these references are incorporated herein by reference. Use known in the art The method can remove the protecting group at an appropriate subsequent stage.
  • the term "about” refers to within ⁇ 10% of the stated value, preferably within ⁇ 5%, more preferably within ⁇ 2%.
  • the invention provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof or a pharmaceutically acceptable salt thereof Medicament wherein the compound has the structure of Formula I or Formula Ia:
  • Ar 1 and Ar 2 are each independently selected from a C 6-14 aryl group and a 5-14 membered heteroaryl group, which are optionally selected from one or more selected from the group consisting of halogen, -OH, -CN, -NO 2 , -N Substituent substitution of (R) 2 , C 1-6 alkyl, halo C 1-6 alkyl, C 1-6 alkylthio and C 3-6 cycloalkyl;
  • L is absent or selected from -O-, -S-, and -NR-;
  • R 1 and R 2 are each independently selected from H (including 1 H, 2 H, 3 H), C 1-6 alkyl (eg, deuterated C 1-6 alkyl), and C 3-6 cycloalkyl;
  • R 3 is a quaternary, five-, six- or seven-membered nitrogen heterocyclic ring selected from the following structures:
  • R a , R a ' , R 4 , R 4 ' , R 5 , R 5 ' and R 6 are each independently selected from H, halogen, -OH, -COOH, -CN, -NO 2 , -N(R) 2 , C 1-6 alkyl, halo C 1-6 alkyl, -WC 1-6 alkyl, -C 1-6 alkylene-WR, -WC 1-6 alkylene -W'-R, -WC 2-6 alkenyl, -C 2-6 alkenylene-WR, -WC 2-6 alkenylene-W'-R and C 3-6 cycloalkyl, wherein The alkylene and alkenylene are optionally further separated by one or more W; or R a together with R a ' , R 4 together with R 5 and/or R 4 ' and R 5' together, each occurrence
  • R 6 is attached to the ring carbon atom marked by * and / or ** in the above nitrogen heterocyclic structure
  • R is each independently selected from the group consisting of H, C 1-6 alkyl and C 3-6 cycloalkyl;
  • g is 1 or 2;
  • t is 0, 1, 2 or 3, provided that t is not greater than the number of positions on the corresponding group that can be substituted, and when t is greater than 1, each R 6 may be the same or different.
  • the invention provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof, or a prodrug wherein the compound has the structure of Formula II or Formula IIa:
  • the invention provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof, or Prodrug, wherein Ar 1 is selected from:
  • R c is each independently selected from the group consisting of F, Cl, Br, I, halo C 1-6 alkyl, C 1-6 alkyl, and C 3-6 cycloalkyl;
  • R c is each independently selected from the group consisting of F, Cl, Br, I, C 1-6 alkyl and C 3-6 cycloalkyl;
  • Ar 1 is more preferably from:
  • Ar 1 is particularly preferred from:
  • the invention provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof, or Prodrug, wherein Ar 2 is selected from:
  • R b is each independently selected from H, halogen, halo C 1-6 alkyl, C 1-6 alkyl and C 3-6 cycloalkyl at each occurrence; preferably, R b is present each time Each is independently selected from the group consisting of H, halogen, C 1-6 alkyl, and C 3-6 cycloalkyl;
  • i 0, 1 or 2;
  • Ar 2 is preferably selected from:
  • L is -O-.
  • R 1 and R 2 are each independently selected from H (including 1 H, 2 H, 3 H), methyl, ethyl, n-propyl and isopropyl.
  • R 3 is a quaternary, penta, hexa or seven-membered nitrogen heterocycle selected from the group consisting of:
  • R 6 is bonded to the ring carbon atom marked by * and / or ** in the above nitrogen heterocyclic structure.
  • R 7 , R 7 ' , R 7a , R 7a′ are each independently selected from H, C 1-4 alkyl and C 3-6 cycloalkyl at each occurrence;
  • R is selected from the group consisting of H, methyl, ethyl, propyl and cyclopropyl;
  • n 0, 1, 2, 3 or 4;
  • n 1, 2, 3 or 4;
  • j 0, 1, or 2.
  • R 3 is selected from the group consisting of
  • R 3 is selected from the group consisting of
  • the invention provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof, or a pharmaceutically acceptable salt thereof Medicament wherein the compound has the structure:
  • the invention provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof, or a pharmaceutically acceptable salt thereof Medicament wherein the compound has the structure:
  • the invention provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof, or a pharmaceutically acceptable salt thereof Medicine wherein the compound has the following knot Structure:
  • R 1 and R 2 are each independently selected from H (including 1 H, 2 H, 3 H), C 1-6 alkyl (eg, deuterated C 1-6 alkyl), and C 3-6 cycloalkyl, And R 1 is preferably methyl, ethyl, n-propyl, isopropyl or cyclopropyl;
  • Q is -(CR a R a' ) g - or -O-;
  • R a , R a ' , R 4 , R 5 and R 6 are each independently selected from H, halogen (eg F), -OH, -COOH, -CN, -NO 2 , -N(R) at each occurrence. 2 , C 1-6 alkyl, halo C 1-6 alkyl, -WC 1-6 alkyl, -C 1-6 alkylene-WR, -WC 1-6 alkylene-W'- R, -WC 2-6 alkenyl, -C 2-6 alkenylene-WR, -WC 2-6 alkenylene-W'-R and C 3-6 cycloalkyl, wherein the alkylene group The alkenylene group is optionally further separated by one or more W;
  • R b is each independently selected from the group consisting of H, halogen, halogenated C 1-6 alkyl, C 1-6 alkyl, and C 3-6 cycloalkyl;
  • R c is each independently selected from the group consisting of F, Cl, Br, I, halo C 1-6 alkyl, C 1-6 alkyl and C 3-6 cycloalkyl, and R c is preferably Cl Or Br;
  • R 6 is attached to a ring carbon atom marked by * and / or ** in the general structure
  • R is each independently selected from the group consisting of H, C 1-6 alkyl and C 3-6 cycloalkyl;
  • g is 1 or 2;
  • i 0, 1 or 2;
  • n 0, 1, 2, 3 or 4;
  • each R 6 may be the same or different.
  • R b is each independently selected from the group consisting of H, halo, C 1-6 alkyl, and C 3-6 cycloalkyl;
  • R c is each independently selected from the group consisting of F, Cl, Br, I, C 1-6 alkyl, and C 3-6 cycloalkyl, and R c is preferably Cl or Br;
  • the invention provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof, or a pharmaceutically acceptable salt thereof Medicament wherein the compound has the structure:
  • the invention encompasses compounds obtained by any suitable combination of the various embodiments.
  • the invention provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound thereof, or a prodrug wherein the compound is selected from the group consisting of
  • Embodiments of the invention provide methods of making the compounds of the invention, the methods comprising the steps of:
  • Hal is selected from the group consisting of F, Cl, Br, and I;
  • the halogenating agent is selected from the group consisting of Cl 2 , Br 2 , I 2 , N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide;
  • Step 1 in the presence of an alkali metal salt (such as potassium acetate, sodium acetate, potassium carbonate, sodium carbonate, cesium carbonate, sodium hydrogencarbonate, etc.) in a protic solvent (eg 2,2,2-trifluoroethanol, 2,2- In difluoroethanol, 2-fluoroethanol, ethanol, fluoromethanol, hexafluoroisopropanol, etc.);
  • an alkali metal salt such as potassium acetate, sodium acetate, potassium carbonate, sodium carbonate, cesium carbonate, sodium hydrogencarbonate, etc.
  • a protic solvent eg 2,2,2-trifluoroethanol, 2,2- In difluoroethanol, 2-fluoroethanol, ethanol, fluoromethanol, hexafluoroisopropanol, etc.
  • Step two is carried out in an aprotic solvent (for example, carbon tetrachloride, dichloromethane, 1,2-dichloroethane, etc.);
  • an aprotic solvent for example, carbon tetrachloride, dichloromethane, 1,2-dichloroethane, etc.
  • Step 3 in an aprotic solvent eg dichloromethane, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, tert-butyl methyl ether, N,N-dimethylformamide, N , N-dimethylacetamide, N-methylpyrrolidone, etc.
  • an organic base eg N,N-diisopropylethylamine, triethylamine, 1,8-diazabicyclo[5.4.0 ]-7-undecene, 4-dimethylaminopyridine, N-methylmorpholine, N-methylpiperidine, N-methyltetrahydropyrrole, etc.
  • inorganic bases eg potassium acetate, sodium acetate, carbonic acid
  • R 2 in the compound of the formula I or formula Ia of the present invention is a C 1-6 alkyl group, it can also be synthesized by a method comprising the following steps:
  • R 2 ' is H or C 1-5 alkyl
  • Hal is selected from the group consisting of F, Cl, Br, and I;
  • the halogenating agent is selected from the group consisting of Cl 2 , Br 2 , I 2 , N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide;
  • Step I is in a Lewis acid (for example, a triflate (such as indium triflate, ytterbium triflate, etc.), a triflate (such as trimethylsilyl trifluoromethanesulfonate) , boron trifluoride, aluminum trichloride, etc.) in the presence of a non-polar solvent (such as o-xylene, toluene, anisole, etc.);
  • a Lewis acid for example, a triflate (such as indium triflate, ytterbium triflate, etc.), a triflate (such as trimethylsilyl trifluoromethanesulfonate) , boron trifluoride, aluminum trichloride, etc.) in the presence of a non-polar solvent (such as o-xylene, toluene, anisole, etc.);
  • a non-polar solvent such as o-xylene, toluene, anisole
  • Step II is in an organic base (for example, N,N-diisopropylethylamine, triethylamine, 1,8-diazabicyclo[5.4.0]-7-undecene, 4-dimethylaminopyridine, N -methylmorpholine, N-methylpiperidine, N-methyltetrahydropyrrole, etc.) or inorganic bases (eg potassium acetate, sodium acetate, potassium carbonate, sodium carbonate, cesium carbonate, sodium hydrogencarbonate, sodium hydride, uncle In the presence of potassium butoxide, etc., in an aprotic solvent (eg dichloromethane, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, tert-butyl methyl ether, N, N- In dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.);
  • Step III is carried out in an aprotic solvent such as carbon tetrachloride, dichloromethane, 1,2-dichloroethane, etc.;
  • Step IV is in an aprotic solvent (eg dichloromethane, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, tert-butyl methyl ether, N,N-dimethylformamide, N , N-dimethylacetamide, N-methylpyrrolidone, etc.) in an organic base (eg N,N-diisopropylethylamine, triethylamine, 1,8-diazabicyclo[5.4.0 ]-7-undecene, 4-dimethylaminopyridine, N-methylmorpholine, N-methylpiperidine, N-methyltetrahydropyrrole, etc.) or inorganic bases (eg potassium acetate, sodium acetate, carbonic acid) It is carried out in the presence of potassium, sodium carbonate, cesium carbonate, sodium hydrogencarbonate, sodium hydride, potassium t-
  • R 2 in the compound of Formula I or Formula Ia of the present invention is a methyl group
  • it can also be synthesized by a method comprising the following steps:
  • compositions and methods of treatment are provided.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate thereof, A metabolite, an isotopically labeled compound or prodrug, and one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition may further comprise one or more Other therapeutic agents, such as other therapeutic agents for the prevention or treatment of viral diseases.
  • the invention also provides a process for the preparation of a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of the invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate thereof
  • a metabolite, isotopically labeled compound or prodrug is combined with one or more pharmaceutically acceptable carriers or excipients.
  • the present invention provides a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug thereof or the present invention
  • a pharmaceutical composition of the invention in the manufacture of a medicament for the prevention or treatment of a viral disease.
  • the present invention provides a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug thereof or the present invention
  • a pharmaceutical composition of the invention for use in the prevention or treatment of a viral disease.
  • the invention provides a method of preventing or treating a viral disease, the method comprising administering to an individual in need thereof an effective amount of a compound of the invention, or a pharmaceutically acceptable salt, ester, or stereoisod thereof
  • a construct, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug or a pharmaceutical composition of the invention comprising administering to an individual in need thereof an effective amount of a compound of the invention, or a pharmaceutically acceptable salt, ester, or stereoisod thereof.
  • the compounds of the present invention exert an antiviral effect by inhibiting the assembly of capsid proteins.
  • the compounds of the invention are useful in the treatment of any virus involved in the assembly of capsid proteins in the process of affecting a host, including but not limited to hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), influenza virus, herpes virus (HSV) and human immunodeficiency virus (HIV).
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • influenza virus influenza virus
  • HSV herpes virus
  • HSV human immunodeficiency virus
  • viral diseases which can be prevented and treated using the compounds of the present invention include, but are not limited to, viral hepatitis A, hepatitis B virus, hepatitis C virus, influenza, herpes, and acquired immunodeficiency syndrome. (AIDS), and related symptoms or diseases caused by the above diseases (for example, inflammation, liver fibrosis, cirrhosis, liver cancer, etc.).
  • “Pharmaceutically acceptable carrier” in the context of the present invention means a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered, and which is suitable for contacting humans and/or within the scope of sound medical judgment. Tissues of other animals without excessive toxicity, irritation, allergic reactions, or other problems or complications corresponding to a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, minerals. Oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. It is also possible to use physiological saline and an aqueous solution of glucose and glycerin as a liquid carrier, particularly for injection.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, Ethanol and the like.
  • the composition may also contain minor amounts of wetting agents, emulsifying agents or pH buffering agents as needed.
  • Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are as described in Remington's Pharmaceutical Sciences (1990).
  • compositions of the invention may act systemically and/or locally.
  • they may be administered in a suitable route, for example by injection (for example intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, including instillation) or transdermal administration; or by oral, buccal, or oral administration.
  • routes of administration it can be administered in a suitable dosage form.
  • the dosage forms include, but are not limited to, tablets, capsules, troches, hard candy, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions. Injectable solutions, elixirs, syrups.
  • an effective amount refers to an amount of a compound that, to a certain extent, relieves one or more symptoms of the condition being treated after administration.
  • the dosing regimen can be adjusted to provide the optimal desired response. For example, a single administration may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the condition being treated. It is noted that the dose value can vary with the type and severity of the condition and can include single or multiple doses. It is to be further understood that for any particular individual, the particular dosage regimen should be adjusted over time based on individual needs.
  • an effective dose will be from about 0.0001 to about 50 mg per kg body weight per day, for example from about 0.01 to about 10 mg/kg/day (single or divided doses). For a 70 kg person, this would add up to about 0.007 mg/day to about 3500 mg/day, such as from about 0.7 mg/day to about 700 mg/day.
  • a dose level that is not higher than the lower limit of the aforementioned range may be sufficient, while in other cases, a larger dose may still be employed without causing any harmful side effects, provided that the larger The dose is divided into several smaller doses to be administered throughout the day.
  • the amount or amount of the compound of the present invention in the pharmaceutical composition may be from about 0.01 mg to about 1000 mg, suitably from 0.1 to 500 mg, preferably from 0.5 to 300 mg, more preferably from 1 to 150 mg, particularly preferably from 1 to 50 mg, for example, 1.5 mg, 2mg, 4mg, 10mg, 25mg Wait.
  • treating means reversing, alleviating, inhibiting the progression of a condition or condition to which such a term applies or one or more symptoms of such a condition or condition, or Prevention of such a condition or condition or one or more symptoms of such condition or condition.
  • “Individual” as used herein includes human or non-human animals.
  • Exemplary human individuals include a human individual (referred to as a patient) or a normal individual having a disease, such as the disease described herein.
  • “Non-human animals” in the present invention include all vertebrates, such as non-mammals (eg, birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals, and/or domesticated animals (eg, sheep, dogs). , cats, cows, pigs, etc.).
  • the pharmaceutical composition of the present invention may further comprise one or more additional therapeutic or prophylactic agents, which are anti-hepatitis B drugs including, but not limited to, Lamiv Ding, telbivudine, entecavir, adefovir dipivoxil, tenofovir, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.
  • additional therapeutic or prophylactic agents which are anti-hepatitis B drugs including, but not limited to, Lamiv Ding, telbivudine, entecavir, adefovir dipivoxil, tenofovir, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.
  • LC-MS was detected on an Aglient 1200 liquid chromatograph using an Aglient 6120 Quadrupole mass spectrometer at 214 nm and 254 nm.
  • Preparative liquid chromatography using SHIMADZU CBM-20A and Aglient 1260 to prepare a liquid phase meter, C18 OBD 19 ⁇ 150mm 5 ⁇ M preparative column, detection wavelength 214nm, mobile phase A is water, mobile phase B is acetonitrile (add 0.5 ⁇ formic acid), Linear gradient elution as follows:
  • Step 2 6-(Bromomethyl)-4-(2-chloro-4-fluorophenyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylic acid ethyl ester (1 -2) synthesis
  • Step 3 4-(2-Chloro-4-fluorophenyl)-6-((3,3-difluoro-4-hydroxypiperidin-1-yl)methyl)-2-(thiazol-2-yl) Synthesis of ethyl 4-1,4-dihydropyrimidine-5-carboxylate (10-88)
  • Step 1 4-(2-Chloro-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylic acid ethyl ester (3-1) Separation
  • Step 1 Synthesis of benzyl 3,3-difluoro-4-hydroxypiperidine-1-carboxylate (5-2)
  • 3,3-Difluoropiperidin-4-ol hydrochloride (100 mg, 0.73 mmol) was dissolved in dichloromethane (2 mL) and triethylamine (147 mg, 1.46 mmol) A solution of N-(benzyloxycarbonyloxy) succinimide (12 mg, 0.48 mmol) in dichloromethane (2 mL) was evaporated. The title compound was obtained after workup of 100 mg. ESI-MS (m/z): 272.2 [M+H] + .
  • Step 2 Synthesis of 4-(2-ethoxy-2-oxoethoxy)-3,3-difluoropiperidine-1-carboxylic acid benzyl ester (5-3)
  • Step 5 2-((1-((6-(2-Chloro-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidine) Synthesis of 4-yl)methyl)-3,3-difluoropiperidin-4-yl)oxy)acetic acid (10-95)
  • the title compound was obtained in a manner similar to that described in the third step of Example 1 except that the compound (5-5) was used instead of 3,3-difluoropiperidin-4-ol hydrochloride.
  • the title compound 4 mg was obtained by a method similar to that described in the first step of Example 1 (3,3-difluoroazetidine hydrochloride instead of 3,3-difluoropiperidin-4-ol hydrochloride). .
  • Step 2 (R)-1-((6-(2-chloro-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidine Synthesis of 4-yl)methyl)-3-fluoroazetidin-3-carboxylic acid (10-11)
  • Step 1 Synthesis of tert-butyl 3,3-difluoro-4-methoxypiperidine-1-carboxylate (8-2)
  • the title compound 50 mg was obtained by a method similar to that described in the first step of Example 1 (substituting compound (8-3) for 3,3-difluoropiperidin-4-ol hydrochloride).
  • Step 2 (R)-4-(2-Chloro-4-fluorophenyl)-6-((3R,4R)-3-fluoro-4-hydroxytetrahydropyrrol-1-yl)methyl)- Synthesis of ethyl 2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate (10-34)
  • Step 1 Synthesis of (3R,4R)-3-(2-ethoxy-2-oxoethoxy)-4-fluorotetrahydropyrrole-1-carboxylic acid tert-butyl ester (10-b)
  • Step 4 2-(((3R,4R)-1-(((R)-6-(2-chloro-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazole-2-) Synthesis of 3-(6-6-dihydropyrimidin-4-yl)methyl)-4-fluorotetrahydropyrrol-3-yl)oxy)acetic acid (10-36)
  • the title compound 33 mg was obtained by a method similar to the one described in the first step of Example 1 (substituting compound (11-5) for 3,3-difluoropiperidin-4-ol hydrochloride).
  • Step 4 2-((1-((R)-6-(2-Chloro-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)-3,6 Synthesis of dihydropyrimidin-4-yl)methyl)-4,4-difluorotetrahydropyrrol-3-yl)oxy)acetic acid
  • the title compound 25 mg was obtained by a method similar to that described in the first step of Example 1 (substituting compound (12-4) for 3,3-difluoropiperidin-4-ol hydrochloride).
  • Step 2 Synthesis of 3-formylmorpholine-4-carboxylic acid tert-butyl ester (13-3)
  • Step 3 Synthesis of (E)-3-(3-ethoxy-3-oxoprop-1-en-1-yl)morpholine-4-carboxylic acid tert-butyl ester (13-4)
  • Step 6 (E)-3-(4-((R)-6-(2-chloro-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)- Synthesis of 3,6-dihydropyrimidin-4-yl)methyl)morpholin-3-yl)acrylic acid (10-182)
  • Step 1 Synthesis of 3-((2-(tert-butoxy)-2-oxoethoxy)methyl)morpholine-4-carboxylic acid tert-butyl ester (15-2)
  • tert-butyl 3-(hydroxymethyl)morpholine-4-carboxylate 200 mg, 0.99 mmol
  • tetrahydrofuran 6 mL
  • sodium hydride 47.3 mg, 1.2 mmol
  • tert-butyl bromoacetate 192 mg, 0.99 mmol
  • the reaction mixture was poured into 10 mL of water, and the pH was adjusted to 2 to 3 with 1N aqueous hydrochloric acid, and extracted with ethyl acetate.
  • Step 3 2-((4-((R)-6-(2-chloro-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)-3,6 Synthesis of dihydropyrimidin-4-yl)methyl)morpholin-3-yl)methoxy)acetic acid (10-162)
  • the title compound 73 mg was obtained by a method similar to that described in the first step of Example 1 (substituting compound (15-3) for 3,3-difluoropiperidin-4-ol hydrochloride).
  • Step 3 Synthesis of tert-butyl 3,3-difluoro-5-(hydroxymethyl)piperidine-1-carboxylate (16-4)
  • Step 4 5-((2-(tert-Butoxy)-2-oxoethoxy)methyl)-3,3-difluoropiperidine-1-carboxylic acid tert-butyl ester (16-5) synthesis
  • Step 6 2-((1-((R)-6-(2-chloro-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)-3,6 Synthesis of dihydropyrimidin-4-yl)methyl)-5,5-difluoropiperidin-3-yl)methoxy)acetic acid (10-136)
  • Step 1 Synthesis of tert-butyl 3,3-difluoro-5-formylpiperidine-1-carboxylate (17-2)
  • tert-Butyl 3,3-difluoro-5-(hydroxymethyl)piperidine-1-carboxylate (17-1) (50 mg, 0.2 mmol) was dissolved in dichloromethane (2.0 mL). After completely dissolved, the reaction was cooled to 0 ° C, and Dess-Martin reagent (102 mg, 0.24 mmol) was added with stirring. After the addition, the reaction was carried out for 3 h at room temperature. A large amount of a white solid was precipitated from the reaction mixture, and the filtrate was evaporated.
  • Step 5 (E)-3-(1-(((R)-6-(2-chloro-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)-) Synthesis of 3,6-dihydropyrimidin-4-yl)methyl)-5,5-difluoropiperidin-3-yl)acrylic acid (10-116)
  • the title compound 10 mg was obtained by a method similar to the one described in the first step of Example 1 (substituting compound (17-5) for 3,3-difluoropiperidin-4-ol hydrochloride).
  • Step 1 Synthesis of 2-((2-ethoxy-2-oxoethoxy)methyl)-4,4-difluorotetrahydropyrrole-1-carboxylic acid tert-butyl ester (18-2)
  • Step 4 2-((1-((R)-6-(2-Chloro-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)-3,6 Synthesis of dihydropyrimidin-4-yl)methyl)-4,4-difluorotetrahydropyrrole-2-yl)methoxy)acetic acid (10-56)
  • the title compound 25 mg was obtained by a method similar to the one described in the first step of Example 1 (substituting compound (18-4) for 3,3-difluoropiperidin-4-ol hydrochloride).
  • Step 1 Synthesis of 2-(hydroxymethyl)morpholine-4-carboxylic acid tert-butyl ester (19-2)
  • the target compound 70 mg was obtained from the compound (19-2) by the operation similar to the procedure of the procedure of the step of the above.
  • Step 1 Synthesis of 2-(((2-methoxy-2-oxoethyl)(methyl)amino)methyl)morpholine-4-carboxylic acid tert-butyl ester (20-2)
  • Step 4 N-((4-((R)-6-(2-chloro-4-fluorophenyl)-5-ethoxycarbonyl-2-(thiazol-2-yl)-3,6-di Synthesis of Hydropyrimidin-4-yl)methyl)morpholin-2-yl)methyl)-N-methylglycine (10-168)
  • Step 1 Synthesis of (E)-2-acetyl-3-(4-fluorophenyl)but-2-enoic acid ethyl ester (21-1)
  • Step 2 Synthesis of ethyl 4-(4-fluorophenyl)-4,6-dimethyl-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate
  • Step 3 to Step 4 2-((1-(((S)-5-(ethoxycarbonyl))-6-(4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)) -3,6-dihydropyrimidin-4-yl)methyl)-3,3-difluoropiperidin-4-yl)oxy)acetic acid (10-224)
  • the target compound 15 mg was obtained from the compound (21-2') of Example 21, using a procedure similar to that described in Steps 21 to 4 of Example.
  • Example 21 The compound (21-2') of Example 21 was reacted with 2-((4,4-difluorotetrahydropyrrole) after the bromination reaction using a procedure similar to that described in the first step and the third step of Example 1.
  • -3-yl)oxy)acetic acid Compound (12-4) in Example 12 (29 mg, 0.16 mmol)
  • Step 1 Synthesis of (S)-2-formylmorpholine-4-carboxylic acid tert-butyl ester (25-2)
  • Step 2 (R,E)-2-(3-(tert-Butoxy)-3-oxoprop-1-en-1-yl)morpholine-4-carboxylic acid tert-butyl ester (25-3) synthesis
  • Step 4 (E)-3-((R)-4-(((R)-6-(2-chloro-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazole-2) Synthesis of 3-yl-3,6-dihydropyrimidin-4-yl)methyl)morpholin-2-yl)acrylic acid (10-226)
  • Step 2 Synthesis of (S)-2-(methoxy(methyl)carbamoyl)morpholine-4-carboxylic acid tert-butyl ester (39-3)
  • Step 3 Synthesis of (S)-2-acetylmorpholine-4-carboxylic acid tert-butyl ester (39-4)
  • Step 4 (R,E)-2-(4-(tert-Butoxy)-4-oxobut-2-en-2-yl)morpholine-4-carboxylic acid tert-butyl ester (39-5) synthesis
  • Step 6 (E)-3-((R)-4-(((R)-6-(2-chloro-4-fluorophenyl)-5-(methoxycarbonyl)-2-(thiazole-2) Synthesis of 3-yl-3,6-dihydropyrimidin-4-yl)methyl)morpholin-2-yl)but-2-enoic acid (10-246)
  • control compound 1 The structure of the control compound 1 is The structure of the control compound 2 is The structure of the control compound 3 is
  • HBV hepatitis B virus
  • HepG2.2.15 cells in logarithmic growth phase were seeded in 96-well plates at a cell concentration of 40/ ⁇ L. Incubate for 3 days at 37 ° C in a 5% CO 2 incubator; replace with fresh medium (200 ⁇ L/well) before adding the compound.
  • the mother liquor concentration of each of the examples was 200 ⁇ M.
  • the highest concentration was 200 ⁇ M, diluted to a number of different concentrations in DMSO, and 1 ⁇ L of the test compound was placed in the corresponding medium well.
  • the final test concentrations of the compounds were 0.06, 0.24, 0.98, 3.9, 15.6, 62.5, 250, 1000 nM (using Calculate the half effective concentration (EC 50 )).
  • the test results are shown in Table 1-1 and Table 1-2.
  • the tested compounds have strong inhibitory activity against hepatitis B virus (HBV).
  • the anti-HBV virus activity of the compound of the present invention having a single configuration was about 10 times that of the control compound 2.
  • the compound of the present invention has strong inhibitory activity against hepatitis B virus (HBV).
  • the remaining compounds of the invention have inhibitory activities similar to those described above.
  • the test compound was diluted to 30 mM with DMSO, diluted to a maximum concentration of 30 mM, and diluted to a plurality of different concentrations.
  • 0.2 ⁇ L of each compound was added to a 384-well plate, and 2000/50 ⁇ L of HepG2.2.15 was added to each well.
  • the highest concentration of the test compound was 150 ⁇ M; 1 ⁇ L of DMSO was added to the corresponding wells as a control.
  • the cells were co-cultured for 4 days at 37 ° C in a 5% CO 2 incubator. After 4 days, 50 ⁇ L of CellTiter-Glo was added to each well to perform a plate reading test, and a half-cytotoxic concentration (CC 50 ) value was calculated.
  • Table 2 The test results are shown in Table 2.
  • hERG human Ether-a-go-go Related Gene
  • IKr inhibition is the most important mechanism for drug-induced QT interval prolongation.
  • the criterion was that if the compound IC 50 > 10 ⁇ M, it was judged that the compound had no inhibitory effect on hERG.
  • test compounds on the hERG potassium channel were tested for the role Predictor TM hERG Fluorescence Polarization Assay. The test results are shown in Table 3 below:
  • Control compound 1 2.85 Control compound 2 1 to 10 10-36 of the tenth embodiment >10 10-40 of the eleventh embodiment >10 10-42 of the twelfth embodiment >10 10-88 of the first embodiment >10 Isomer B of Example 4 >10 Tetraisomer A of the Examples >10 10-93 of the second embodiment >10 10-95 of the fifth embodiment >10 Example twenty-fifth 10-226 >10
  • Control Compound 1 and Control Compound 2 have different degrees of cardiotoxicity (significant inhibition of hERG potassium channel in cardiomyocytes) and thus have a potential risk of inducing arrhythmia; and the present invention tested The compound has no hERG potassium channel inhibition, no obvious cardiotoxicity, and thus has higher safety. Other compounds of the invention have similar safety.
  • Test compounds were administered to male SD rats by intravenous (iv) and gavage (po), respectively.
  • the doses administered by iv and po were 1 mg/kg and 2 mg/kg, respectively, and the iv system was 5% DMSO. : 5% solutol: 90% saline, the vehicle system was 0.5% MC when po was administered.
  • Blood was collected at various time points after iv administration and po administration for PK studies. Plasma samples and liver tissue samples were subjected to protein precipitation and subjected to LC-MS/MS analysis.
  • the mass spectrometer is API 5500, the liquid chromatograph is Waters ACQUITY I CLASS system; the column is Agela ASB C 18 column (2.1mm ⁇ 50mm, 1.9 ⁇ m); the mobile phase A phase is water +0.1% formic acid, and the B phase is acetonitrile.
  • the flow rate was 0.4 mL/min and the column temperature was 40 °C.
  • the ion source is used as the ESI source positive ion mode, and the scanning mode is multiple reaction monitoring (MRM).
  • MRM multiple reaction monitoring
  • the compound of the present invention has a better exposure in the blood of the body at an intravenous dose of 1.00 mg/kg as compared with the control compound 2.
  • AUC INF a higher plasma concentrations
  • C max a higher plasma concentrations
  • the compound of the present invention has a better blood in the body at a dose of 2.00 mg/kg compared to the control compound 2. Exposure (AUC INF ) and higher blood concentration (C max ), thus having better absorption.
  • the compound of the present invention has better exposure in the liver at an intragastric administration dose of 2.00 mg/kg as compared with the control compound 2.
  • the amount (AUC INF ) and the higher blood concentration (C max ) further illustrate that the compounds of the invention (e.g., 10-226 of Example 25) have better absorbency.
  • the compound of the present invention has a bioavailability (F) of 47.6% at an intragastric administration dose of 2.00 mg/kg, which is significantly higher than the bioavailability of the control compound 2.
  • the compound of the present invention (for example, 10-230 of Example 26) is exposed to about 10 times in the liver in vivo, and the blood concentration in the liver is in plasma. About 10 times; while the amount of the control compound 2 exposed in the liver is about 1.5 times that in plasma, and the blood concentration in the liver is about 0.5 times that in plasma.
  • This indicates that the compound of the present invention has excellent exposure (AUC INF ) and blood concentration (C max ) in the liver in vivo, and thus has liver targeting properties.
  • Test compounds were administered to male Beagle dogs by intravenous (iv) and gavage (po), respectively.
  • the doses administered by iv and po were 0.5 mg/kg and 2.5 mg/kg, respectively, and the solvent system was 5% when administered iv.
  • Blood was collected at various time points after iv administration and po administration for PK studies. Plasma samples were subjected to protein precipitation and subjected to LC-MS/MS analysis.
  • the mass spectrometer is API 5500, the liquid chromatograph is Waters ACQUITY I CLASS system; the column is Agela ASB C 18 column (2.1mm ⁇ 50mm, 1.9 ⁇ m); the mobile phase A phase is water +0.1% formic acid, and the B phase is acetonitrile.
  • the flow rate was 0.4 mL/min and the column temperature was 40 °C.
  • the ion source is used as the ESI source positive ion mode, and the scanning mode is multiple reaction monitoring (MRM).
  • MRM multiple reaction monitoring
  • the compound of the present invention has a better exposure in the blood of the body at an intravenous dose of 0.50 mg/kg as compared with the control compound 3. (AUC INF ) and higher plasma concentrations (C max ), thus having better pharmacokinetic parameters.
  • the compound of the present invention (for example, 10-227 of Example 27) was exposed to plasma in an amount of about 14 times that of the control compound 3 at an intragastric administration dose of 2.50 mg/kg.
  • the blood drug concentration was about 6 times that of the control compound 3.
  • the compounds of the invention e.g., 10-227 of Example 27
  • the compound of the present invention has a bioavailability (F) of 75.9% at an intragastric administration dose of 2.50 mg/kg, which is significantly superior to the bioavailability of the control compound 3.
  • Degree (F) 41.7% is significantly superior to the bioavailability of the control compound 3.
  • Test compounds were administered to male cynomolgus monkeys by intravenous (iv) and gavage (po), respectively.
  • the doses administered by iv and po were 0.5 mg/kg and 2.5 mg/kg, respectively, and the solvent system was 5 when iv was administered.
  • Blood was collected at various time points after iv administration and po administration for PK studies. Plasma samples were subjected to protein precipitation and subjected to LC-MS/MS analysis.
  • the mass spectrometer is API 5500, the liquid chromatograph is Waters ACQUITY I CLASS system; the column is Agela ASB C 18 column (2.1mm ⁇ 50mm, 1.9 ⁇ m); the mobile phase A phase is water +0.1% formic acid, and the B phase is acetonitrile.
  • the flow rate was 0.4 mL/min and the column temperature was 40 °C.
  • the ion source is used as the ESI source positive ion mode, and the scanning mode is multiple reaction monitoring (MRM).
  • MRM multiple reaction monitoring
  • the compound of the present invention (for example, 10-227 of Example 27) was compared with the control compound 3 in the cynomolgus PK study at an intravenous dose of 0.50 mg/kg in vivo.
  • AUC INF blood concentration
  • C max blood concentration
  • the compound of the present invention (for example, 10-227 of Example 27) in the cynomolgus PK study, the exposure amount in plasma at the dose of 2.50 mg/kg is About 21 times the control compound 3, the blood concentration was about 33 times that of the control compound 3. It is further illustrated that the compounds of the invention (e.g., 10-227 of Example 27) have better absorbency. Moreover, the compound of the present invention (for example, 10-227 of Example 27) has a bioavailability (F) of 37.2% at an intragastric administration dose of 2.50 mg/kg, which is significantly superior to the bioavailability of the control compound 3. Degree (F) 6.77%.
  • HepG2C3A/pCYP3A4-Luc one C8 cell, was removed from the liquid nitrogen tank, and the cells were resuscitated in a 37 ° C sterile water bath and gently shaken until the ice cubes all melted.
  • the resuscitated cells were transferred to a 15 mL sterile centrifuge tube and 5-10 mL of pre-warmed basal cell culture medium at 37 °C was added. The cells were allowed to settle naturally for 2 minutes and then centrifuged (1000 rpm) for 8 minutes. The supernatant was removed and the cells were resuspended in 10 mL of prewarmed cell culture medium.
  • the cell suspension was transferred to a 10 cm cell culture dish and cultured in a 37 ° C 5% CO 2 incubator. After 24 hours, the original cell culture medium was replaced with the selected cell culture medium.
  • the cells were grown to 80% to 90% culture dishes, the cells were digested and transferred to a 15 mL sterile centrifuge tube. The cells were collected by centrifugation (1000 rpm) for 8 minutes. The supernatant was removed and the cells were resuspended in 3 mL of prewarmed complete medium. The cell suspension was subcultured at a ratio of 1:3 or 1:5.
  • the cell suspension was inoculated into 384-well white cell plates at 25 ⁇ L per well.
  • the cell plates were centrifuged (300 rpm) for 1 minute and placed in a 37 ° C 5% CO 2 incubator for 24 hours.
  • 300 nL of 100X compound was transferred from the compound plate into the cell plate.
  • the cell plates were centrifuged (300 rpm) for one minute and placed in a 37 ° C 5% CO 2 incubator for 72 hours.
  • the cell plate and Bright-Glo luciferase reagent were taken out and equilibrated to room temperature.
  • Bright-Glo luciferase reagent was added to the cell plates (30 ⁇ L per well).
  • the cell plates were centrifuged at 1000 rpm for 1 minute and incubated for 2 minutes at room temperature. The fluorescence signal is measured on a plate reader.
  • the concentration curve of the test compound was prepared using the mapping software Prism 5, and the EC 50 value was calculated.
  • the compound of the present invention (for example, 10-227 of Example 27) has a weaker induction effect on the CYP450 enzyme 3A4 subtype than Comparative Compound 3 and rifampicin, and thus has better safety. Sex.

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Abstract

本发明涉及具有抗病毒活性的二氢嘧啶类化合物、包含其的药物组合物、其制备方法及其在预防或治疗包括但不限于甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、流行性感冒、疱疹和获得性免疫缺陷综合征(AIDS)的病毒性疾病中的用途。

Description

二氢嘧啶类化合物及其制备方法和用途 发明领域
本发明涉及具有抗病毒活性的二氢嘧啶类化合物、包含其的药物组合物、其制备方法及其在预防或治疗包括但不限于甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、流行性感冒、疱疹和获得性免疫缺陷综合征(AIDS)的病毒性疾病中的用途。
发明背景
病毒由一种核酸分子(DNA或RNA)与蛋白质构成或仅由蛋白质构成(如朊病毒)。病毒可引起多种传染性疾病，常见的由病毒引起的疾病包括但不限于甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、流行性感冒、疱疹和获得性免疫缺陷综合征(AIDS)。
目前临床使用的抗病毒药物通过抑制病毒的附着、脱壳、病毒基因复制、成熟或释放，或者通过影响宿主的免疫系统来发挥作用，其主要包括逆转录酶抑制剂和衣壳蛋白装配抑制剂等。
乙型肝炎病毒(HBV)是一种常见的嗜肝性DNA病毒性病原体。这类病毒可以引起急性肝炎、慢性肝炎、肝纤维化、肝硬化和肝癌等疾病。
治疗乙肝的药物包括干扰素和核苷类似物(如拉米夫定和阿德福韦酯)。其中，干扰素通过与细胞表面受体作用使细胞产生抗病毒蛋白，从而抑制乙肝病毒的复制。它的缺点是有效应答率较低，并且需长期注射给药。核苷类似物主要通过抑制病毒多聚酶(逆转录酶)的复制来发挥作用。该类药物的缺点是长期服用易使病毒变异而产生耐药性。
此外，乙型病毒性肝炎还可以使用非核苷类似物进行治疗。Deres等人研究发现杂芳基二氢嘧啶类化合物(Bay41-4109)可以通过抑制病毒衣壳蛋白装配进而阻止HBV病毒复制(Science,2003,299,893-896)。其具体作用机制是二氢嘧啶类化合物诱导核心蛋白错误装配，从而形成不稳定的衣壳蛋白，加速核心蛋白的降解(Biochem.Pharmacol.,2003,66,2273-2279)。Zlotnick等人发现的杂芳基二氢嘧啶类化合物HAP1(Proc.Natl.Acad.Sci.,2005,102,8138-8143)以及广东东阳光药业有限公司报道的杂芳基二氢嘧啶类化合物(GLS4)(Antimicrob.Agents Chemother.,2013,57,5344-5354；WO2015078391,US2016206616以及WO2015144093)也具有抗HBV活性。
虽然上述化合物均不同程度地表现出了抗病毒活性，但它们的活性还不能达到令人满意的程度，并且一些化合物还表现出明显的毒性作用(例如GLS4表现出明显的hERG心脏毒性)。
发明概述
通过深入研究，令人惊奇地发现了本发明的二氢嘧啶类化合物，这类化合物可以比已公开的二氢嘧啶类HBV衣壳蛋白装配调节剂更有效地抑制HBV的DNA复制(例如，细胞水平抗病毒活性，本发明的优选化合物约为WO2015144093中优选化合物(实施例9化合物)活性的10倍)。本发明的化合物没有已公开的二氢嘧啶类化合物所具有的心脏毒性(如GLS4和WO2015144093中优选化合物(实施例9化合物)的hERG抑制活性)；并且与已公开的二氢嘧啶类化合物(如WO201403748中实施例5化合物)相比，本发明的化合物具有显著降低的对CYP450酶3A4亚型的诱导作用。此外，在大鼠、Beagle犬、食蟹猴的药物代谢动力学测试中，本发明的化合物表现出更好的药物代谢动力学性质(如更好的暴露量、血药浓度、生物利用度)。同时，本发明的优选化合物具有很好的肝靶向性质，药物的肝脏中暴露量可以达到血浆中暴露量的约10倍，表明该类化合物具有肝脏富集能力，进而有利于改善对于肝脏疾病的疗效。
本发明的一个方面提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有式I或式Ia的结构：
Figure PCTCN2017110123-appb-000001
其中：
Ar1和Ar2各自独立地选自C6-14芳基和5-14元杂芳基，其任选地被一个或多个选自卤素、-OH、 -CN、-NO2、-N(R)2、C1-6烷基、卤代C1-6烷基、C1-6烷基硫基和C3-6环烷基的取代基取代；
L不存在或者选自-O-、-S-和-NR-；
R1和R2各自独立地选自H(包括1H、2H、3H)、C1-6烷基(例如：氘代C1-6烷基)和C3-6环烷基；
R3为选自下列结构的四元、五元、六元或七元氮杂环系：
Figure PCTCN2017110123-appb-000002
Q选自-(CRaRa’)g-、-NRa-、-O-、-S-、-S(＝O)-和-S(＝O)2-；
Ra、Ra’、R4、R4’、R5、R5’和R6在每次出现时各自独立地选自H、卤素、-OH、-COOH、-CN、-NO2、-N(R)2、C1-6烷基、卤代C1-6烷基、-W-C1-6烷基、-C1-6亚烷基-W-R、-W-C1-6亚烷基-W’-R、-W-C2-6烯基、-C2-6亚烯基-W-R、-W-C2-6亚烯基-W’-R和C3-6环烷基，其中所述亚烷基和亚烯基任选地进一步被一个或多个W间隔；或者Ra与Ra’一起、R4与R5一起和/或R4’与R5’一起，在每次出现时各自独立地形成基团＝CH-W-R；条件是当R3不是四元氮杂环系时，Ra、Ra’、R4、R4’、R5、R5’和R6不同时为氢，并且不为选自-COOH、-C1-6亚烷基-OH和-C1-6亚烷基-C(＝O)OH的基团；且当R3为四元氮杂环系时，R4、R5和R6不同时为氢；
R6连接在上述氮杂环系结构中*和/或**标记的环碳原子上；
W和W’在每次出现时各自独立地选自O、C(＝O)、C(＝O)O、NR、NC(＝O)、N(S＝O)、NS(＝O)2、S、S＝O和S(＝O)2；
R在每次出现时各自独立地选自H、C1-6烷基和C3-6环烷基；
g为1或2；并且
t为0、1、2或3，条件是t不大于对应基团上可被取代的位置的数目，并且当t大于1时，每个R6可以相同或不同。
本发明的另一方面提供药物组合物，其包含预防或治疗有效量的本发明的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药以及一种或多种药学上可接受的载体，所述药物组合物优选是固体制剂、液体制剂或透皮制剂。
本发明的另一方面提供制备药物组合物的方法，所述方法包括将本发明的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药与一种或多种药学上可接受的载体组合。
本发明的另一方面提供本发明的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药或者本发明的药物组合物在制备用于预防或治疗病毒性疾病的药物中的用途。
本发明的另一方面提供本发明的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药或者本发明的药物组合物，其用于预防或治疗病毒性疾病。
本发明的另一方面提供预防或治疗病毒性疾病的方法，所述方法包括向需要其的个体给药有效量的本发明的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药或者本发明的药物组合物。
上述病毒性疾病包括但不限于甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、流行性感冒、疱疹和获得性免疫缺陷综合征(AIDS)。
本发明的另一方面提供制备本发明的化合物的方法，所述方法包括以下步骤：
Figure PCTCN2017110123-appb-000003
其中：
Hal选自F、Cl、Br和I；
卤化试剂选自Cl2、Br2、I2、N-氯代琥珀酰亚胺、N-溴代丁二酰亚胺和N-碘代丁二酰亚胺；
其余各基团如上述所定义；
步骤一在碱金属盐存在下于质子性溶剂中进行；
步骤二在非质子性溶剂中进行；并且
步骤三在非质子性溶剂中，在有机碱或无机碱的存在下进行；
当本发明的化合物中R2为C1-6烷基时，其也可通过包括以下步骤的方法合成：
Figure PCTCN2017110123-appb-000004
其中：
R2’为H或C1-5烷基；
Hal选自F、Cl、Br和I；
卤化试剂选自Cl2、Br2、I2、N-氯代琥珀酰亚胺、N-溴代丁二酰亚胺和N-碘代丁二酰亚胺；
其余各基团如上述所定义；
步骤I在路易斯酸存在下于非极性溶剂中进行；
步骤II在有机碱或无机碱的存在下，在非质子性溶剂中进行；
步骤III在非质子性溶剂中进行；并且
步骤IV在非质子性溶剂中，在有机碱或无机碱的存在下进行。
附图简要说明
图1：化合物10-227、对照化合物3和利福平对CYP450酶3A4亚型的诱导作用。
定义
除非在下文中另有定义，本文中所用的所有技术术语和科学术语的含义与本领域技术人员通常所理解的相同。本文中使用的技术指在本领域中通常所理解的技术，包括那些对本领域技术人员理解的技术的变化或等同技术的替换。虽然相信以下术语对于本领域技术人员可以理解，但仍然阐述以下定义以更好地解释本发明。
术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它变体形式为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤。
如本文中所使用，术语“亚烷基”表示饱和二价烃基，优选表示具有1、2、3、4、5或6个碳原子的饱和二价烃基，例如亚甲基、亚乙基、亚丙基或亚丁基。
如本文中所使用，术语“亚烯基”表示包含一个或多个双键的二价烃基，其优选具有2、3、4、5或6个碳原子，例如亚乙烯基、亚丙烯基或亚烯丙基。当本发明的化合物含有亚烯基时，所述化合物可以纯E(异侧(entgegen))形式、纯Z(同侧(zusammen))形式或其任意混合物形式存在。
如本文中所使用，术语“烷基”定义为直链或支链饱和脂肪族烃。在一些实施方案中，烷基具有1至12个，例如1至6个碳原子。例如，如本文中所使用，术语“C1-6烷基”指1至6个碳原子的线性或支化的基团(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基或正己基)，其任选地被1或多个(诸如1至3个)适合的取代基如卤素取代(此时该基团被称作“卤代烷基”)(例如CF3、C2F5、CHF2、CH2F、CH2CF3、CH2Cl或-CH2CH2CF3等)。术语“C1-4烷基”指1至4个碳原子的线性或支化的脂肪族烃链(即甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基)。
如本文中所使用，术语“环烷基”指饱和或不饱和的非芳族单环或多环(诸如双环)烃环(例如单环，诸如环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基，或双环，包括螺环、稠合或桥连系统(诸如双环[1.1.1]戊基、双环[2.2.1]庚基、双环[3.2.1]辛基或双环[5.2.0]壬基、十氢化萘基等)，其任选地被1或多个(诸如1至3个)适合的取代基取代。所述环烷基具有3至15个碳原子。例如，术语“C3-6环烷基”指3至6个成环碳原子的饱和或不饱和的非芳族单环或多环(诸如双环)烃环(例如环丙基、环丁基、环戊基或环己基)，其任选地被1或多个(诸如1至3个)适合的取代基取代，例如甲基取代的环丙基。
如本文中所使用，术语“芳基”指具有共轭π电子系统的全碳单环或稠合环多环芳族基团。例如，如本文中所使用，术语“C6-14芳基”意指含有6至14个碳原子的芳族基团，诸如苯基或萘基。芳基任选地被1或多个(诸如1至3个)适合的取代基(例如卤素、-OH、-CN、-NO2、C1-6烷基等)取代。
术语“芳烷基”优选表示芳基取代的烷基，其中所述芳基和所述烷基如本文中所定义。通常，所述芳基可具有6-14个碳原子，并且所述烷基可具有1-6个碳原子。示例性芳烷基包括但不限于苄基、苯基乙基、苯基丙基、苯基丁基。
如本文中所使用，术语“杂芳基”指一价单环、双环或三环芳族环系，其具有5、6、7、8、9、10、11、12、13或14个环原子，特别是1或2或3或4或5或6或9或10个碳原子，且其包含至少一个可以相同或不同的杂原子(所述杂原子是例如氧、氮或硫)，并且，另外在某些情况下可为苯并稠合的。特别地，杂芳基选自噻吩基、呋喃基、吡咯基、噁唑基、噻唑基、咪唑基、吡唑基、异噁唑基、异噻唑基、噁二唑基、三唑基、噻二唑基等，以及它们的苯并衍生物；或吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基等，以及它们的苯并衍生物。
如本文中所使用，术语“卤代”或“卤素”基团定义为包括F、Cl、Br或I。
如本文中所使用，术语“烷基硫基”意指通过硫原子连接至母核分子部分的如上文所定义的烷基。C1-6烷基硫基的代表性实例包括但不限于甲硫基、乙硫基、叔丁硫基及己硫基。
如本文中所使用，术语“氮杂环系”指饱和或不饱和的单环或双环基团，其在环中具有2、3、4、5、6、7、8、9、10、11、12或13个碳原子和至少一个氮原子，其还可任选地包含一个或多个(例如1个、2个、3个或4个)选自N、O、C＝O、S、S＝O和S(＝O)2的环成员；所述氮杂环系通过氮原子与分子的其余部分连接。特别地，3至14元氮杂环系为在环中具有3-14个碳原子及杂原子(其中至少一个为氮原子)的基团，其包括但不限于三元氮杂环系(如氮丙啶基)、四元氮杂环系(如氮杂环丁烷基)、五元氮杂环系(如吡咯基、四氢吡咯基、吡咯啉基、吡咯烷酮基、咪唑基、咪唑烷基、咪唑啉基、吡唑基、吡唑啉基)、六元氮杂环系(如哌啶基、吗啉基、硫吗啉基、哌嗪基)、七元氮杂环系等。
术语“取代”指所指定的原子上的一个或多个(例如1个、2个、3个或4个)氢被所选择的基团代替，条件是未超过所指定的原子在当前情况下的正常原子价并且所述取代形成稳定的化合物。所选的替代基团数量是当这种组合形成稳定的化合物时才允许的。
如果取代基被描述为“任选地被取代”，则取代基可(1)未被取代或(2)被取代。如果取代基的碳被描述为任选地被取代基列表中的一个或多个取代，则碳上的一个或多个氢可单独和/或一起被独立地选择的任选的取代基替代。如果取代基的氮被描述为任选地被取代基列表中的一个或多个取代，则氮上的一个或多个氢可各自被独立地选择的任选的取代基替代。
如果取代基被描述为“独立地选自”，则各取代基可与另一(其他)取代基相同或不同。
如本文中所使用，术语“一个或多个”意指在合理条件下的1个或超过1个，例如2个、3个、4个、5个或10个。
除非指明，否则如本文中所使用，取代基的连接点可来自取代基的任意适宜位置。
当取代基的键显示为穿过环中键而未指定位置时，则这样的取代基可键连至该可取代的环中的任一适当的成环原子。
本发明还包括所有药学上可接受的同位素标记的化合物，其与本发明的化合物相同，除了一个或多个原子被具有相同原子序数但原子质量或质量数不同于在自然界中占优势的原子质量或质量数的原子替代。适合包含入本发明的化合物中的同位素的实例包括(但不限于)氢的同位素(例如氘(D，2H)、氚(T，3H))；碳的同位素(例如11C、13C及14C)；氯的同位素(例如36Cl)；氟的同位素(例如18F)；碘的同位素(例如123I及125I)；氮的同位素(例如13N及15N)；氧的同位素(例如15O、17O及18O)；磷的同位素(例如32P)；及硫的同位素(例如35S)。某些同位素标记的本发明的化合物可用于药物和/或底物组织分布研究(例如分析)中。本发明的药学上可接受的溶剂合物包括其中结晶溶剂可被同位素取代的那些，例如，D2O、丙酮-d6或DMSO-d6。
术语“立体异构体”表示由于至少一个不对称中心形成的异构体。在具有一个或多个(例如1个、2个、3个或4个)不对称中心的化合物中，其可产生外消旋混合物、单一对映异构体、非对映异构体混合物和单独的非对映异构体。特定个别分子也可以几何异构体(顺式/反式)存在。类似地，本发明的化合物可以两种或更多种处于快速平衡的结构不同的形式的混合物(通常称作互变异构体)存在。互变异构体的代表性实例包括酮-烯醇互变异构体、苯酚-酮互变异构体、亚硝基-肟互变异构体、亚胺-烯胺互变异构体等。例如，二氢嘧啶基团在溶液中可以下列互变异构形式平衡存在：
Figure PCTCN2017110123-appb-000005
要理解，本申请的范围涵盖所有这样的以任意比例(例如60％、65％、70％、 75％、80％、85％、90％、95％、96％、97％、98％、99％)的异构体或其混合物。
本文中可使用实线(——)、实楔形
Figure PCTCN2017110123-appb-000006
虚楔形
Figure PCTCN2017110123-appb-000007
或波浪线
Figure PCTCN2017110123-appb-000008
描绘本发明的化合物的碳-碳键。使用实线以描绘键连至不对称碳原子的键表示，包括该碳原子处的所有可能的立体异构体(例如，特定的对映异构体、外消旋混合物等)。使用波浪线以描绘键连至亚烯基的键表示，包括该化学键处的所有可能的立体异构体(例如，特定的顺反异构体、任意比例的顺反异构体的混合物、或外消旋混合物等)。使用实或虚楔形以描绘键连至不对称碳原子的键表示，存在所示的立体异构体。当存在于外消旋混合物中时，使用实及虚楔形以定义相对立体化学，而非绝对立体化学。除非另外指明，否则本发明的化合物可以为立体异构体(其包括顺式及反式异构体、光学异构体(例如R及S对映异构体)、非对映异构体、几何异构体、旋转异构体、构象异构体、阻转异构体及其混合物)的形式存在。本发明的化合物可表现一种以上类型的异构现象，且由其混合物(例如外消旋混合物及非对映异构体对)组成。
本发明涵盖本发明化合物的所有可能的结晶形式或多晶型物，其可为单一多晶型物或多于一种多晶型物的任意比例的混合物。
还应当理解，本发明的化合物可以游离形式存在用于治疗，或适当时，以其药学上可接受的衍生物形式存在。在本发明中，药学上可接受的衍生物包括但不限于，药学上可接受的盐、酯、溶剂合物、代谢物、同位素标记的化合物或前药，在将它们向需要其的患者给药后，能够直接或间接提供本发明的化合物或其代谢物或残余物。因此，当在本文中提及“本发明的化合物”时，也意在涵盖化合物的上述各种衍生物形式。
本发明的化合物的药学上可接受的盐包括其酸加成盐及碱加成盐。
适合的酸加成盐由形成药学可接受盐的酸来形成，包括适当的无机酸和有机酸。实例包括天冬氨酸盐、苯甲酸盐、碳酸氢盐/碳酸盐、硫酸氢盐/硫酸盐、延胡索酸盐、葡庚糖酸盐、葡糖酸盐、葡糖醛酸盐、六氟磷酸盐、氢溴酸盐/溴化物、氢碘酸盐/碘化物、顺丁烯二酸盐、丙二酸盐、甲基硫酸盐、萘甲酸盐(naphthylate)、烟酸盐、硝酸盐、乳清酸盐、草酸盐、棕榈酸盐及其它类似的盐。
适合的碱加成盐由形成药学可接受盐的碱来形成，包括适当的无机碱和有机碱。实例包括铝盐、精氨酸盐、胆碱盐、二乙胺盐、赖氨酸盐、镁盐、葡甲胺盐、钾盐及其它类似的盐。
适合的盐的综述参见Stahl及Wermuth的“Handbook of Pharmaceutical Salts:Properties,Selection,and Use”(Wiley-VCH,2002)。用于制备本发明的化合物的药学上可接受的盐的方法为本领域技术人员已知的。
如本文中所使用，术语“酯”意指衍生自本申请中各个通式化合物的酯，其包括生理上可水解的酯，可在生理条件下水解以释放游离酸或醇形式的本发明的化合物。本发明的化合物本身也可以是酯。
本发明的化合物可以溶剂合物(例如：水合物)的形式存在，其中本发明的化合物包含作为所述化合物晶格的结构要素的极性溶剂，特别是例如水、甲醇或乙醇。极性溶剂特别是水的量可以化学计量比或非化学计量比存在。
在本发明的范围内还包括本发明的化合物的代谢物，即在给药本发明的化合物时体内形成的物质。这样的产物可由例如被给药的化合物的氧化、还原、水解、酰胺化、脱酰胺化、酯化、酶解等产生。因此，本发明包括本发明的化合物的代谢物，包括通过使本发明的化合物与哺乳动物接触足以产生其代谢产物的时间的方法制得的化合物。
本发明在其范围内进一步包括本发明的化合物的前药，其为自身可具有药理学活性或无药理学活性的本发明的化合物的某些衍生物，其当被给药至身体中或其上时可通过例如水解裂解转化成具有期望活性的本发明的化合物。通常这样的前药会是所述化合物的官能团衍生物，其易于在体内转化成期望的治疗活性化合物。关于前药的使用的其他信息可参见“Pro-drugs as Novel Delivery Systems”，第14卷，ACS Symposium Series(T.Higuchi及V.Stella)及“Bioreversible Carriers in Drug Design,”Pergamon Press,1987(E.B.Roche编辑，American Pharmaceutical Association)。本发明的前药可例如通过用本领域技术人员已知作为“前-部分(pro-moiety)(例如“Design of Prodrugs”，H.Bundgaard(Elsevier,1985)中所述)”的某些部分替代本发明的化合物中存在的适当官能团来制备。
本发明还涵盖含有保护基的本发明的化合物。在制备本发明的化合物的任何过程中，保护在任何有关分子上的敏感基团或反应基团可能是必需的和/或期望的，由此形成本发明的化合物的化学保护形式。这可以通过常规的保护基实现，例如，在Protective Groups in Organic Chemistry,ed.J.F.W.McOmie,Plenum Press,1973；和T.W.Greene & P.G.M.Wuts,Protective Groups in Organic Synthesis,John Wiley & Sons,1991中所述的那些保护基，这些参考文献通过援引加入本文。使用本领域已知的 方法，在适当的后续阶段可以移除保护基。
如本文中所使用，术语“约”是指在所述数值的±10％范围内，优选±5％范围内，更优选±2％范围内。
发明详细描述
化合物及其制备方法
在一个实施方案中，本发明提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有式I或式Ia的结构：
Figure PCTCN2017110123-appb-000009
其中：
Ar1和Ar2各自独立地选自C6-14芳基和5-14元杂芳基，其任选地被一个或多个选自卤素、-OH、-CN、-NO2、-N(R)2、C1-6烷基、卤代C1-6烷基、C1-6烷基硫基和C3-6环烷基的取代基取代；
L不存在或者选自-O-、-S-和-NR-；
R1和R2各自独立地选自H(包括1H、2H、3H)、C1-6烷基(例如：氘代C1-6烷基)和C3-6环烷基；
R3为选自下列结构的四元、五元、六元或七元氮杂环系：
Figure PCTCN2017110123-appb-000010
Q选自-(CRaRa’)g-、-NRa-、-O-、-S-、-S(＝O)-和-S(＝O)2-；
Ra、Ra’、R4、R4’、R5、R5’和R6在每次出现时各自独立地选自H、卤素、-OH、-COOH、-CN、-NO2、-N(R)2、C1-6烷基、卤代C1-6烷基、-W-C1-6烷基、-C1-6亚烷基-W-R、-W-C1-6亚烷基-W’-R、-W-C2-6烯基、-C2-6亚烯基-W-R、-W-C2-6亚烯基-W’-R和C3-6环烷基，其中所述亚烷基和亚烯基任选地进一步被一个或多个W间隔；或者Ra与Ra’一起、R4与R5一起和/或R4’与R5’一起，在每次出现时各自独立地形成基团＝CH-W-R；条件是当R3不是四元氮杂环系时，Ra、Ra’、R4、R4’、R5、R5’和R6不同时为氢，并且不为选自-COOH、-C1-6亚烷基-OH和-C1-6亚烷基-C(＝O)OH的基团；且当R3为四元氮杂环系时，R4、R5和R6不同时为氢；
R6连接在上述氮杂环系结构中*和/或**标记的环碳原子上；
W和W’在每次出现时各自独立地选自O、C(＝O)、C(＝O)O、NR、NC(＝O)、N(S＝O)、NS(＝O)2、S、S＝O和S(＝O)2；
R在每次出现时各自独立地选自H、C1-6烷基和C3-6环烷基；
g为1或2；并且
t为0、1、2或3，条件是t不大于对应基团上可被取代的位置的数目，并且当t大于1时，每个R6可以相同或不同。
在优选的实施方案中，本发明提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有式II或式IIa的结构：
Figure PCTCN2017110123-appb-000011
在优选的实施方案中，本发明提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中Ar1选自：
Figure PCTCN2017110123-appb-000012
其中Rc在每次出现时各自独立地选自F、Cl、Br、I、卤代C1-6烷基、C1-6烷基和C3-6环烷基；
优选地，Rc在每次出现时各自独立地选自F、Cl、Br、I、C1-6烷基和C3-6环烷基；
Ar1更优选自：
Figure PCTCN2017110123-appb-000013
Ar1特别优选自：
Figure PCTCN2017110123-appb-000014
在优选的实施方案中，本发明提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中Ar2选自：
Figure PCTCN2017110123-appb-000015
Rb在每次出现时各自独立地选自H、卤素、卤代C1-6烷基、C1-6烷基和C3-6环烷基；优选地，Rb在每次出现时各自独立地选自H、卤素、C1-6烷基和C3-6环烷基；并且
i为0、1或2；
Ar2优选自：
Figure PCTCN2017110123-appb-000016
在优选的实施方案中，L为-O-。
在优选的实施方案中，R1和R2各自独立地选自H(包括1H、2H、3H)、甲基、乙基、正丙基和异丙基。
在优选的实施方案中，R3为选自下列结构的四元、五元、六元或七元氮杂环系：
Figure PCTCN2017110123-appb-000017
R6连接在上述氮杂环系结构中*和/或**标记的环碳原子上。
在优选的实施方案中，Ra、Ra’、R4、R4’、R5、R5’和R6在每次出现时各自独立地选自H、F、Cl、Br、-(CR7R7a)mOH、-O-C1-6烷基、-(CR7R7a)mCOOH、-C(R7’)＝C(R7a’)(CR7R7a)mCOOH和-(CR7R7a)m-W-(CR7’R7a’)nCOOH，所述-(CR7R7a)m-W-(CR7’R7a’)nCOOH优选为-(CR7R7a)mO(CR7’R7a’)nCOOH、-(CR7R7a)mNR(CR7’R7a’)nCOOH或-(CR7R7a)mS(＝O)j(CR7’R7a’)nCOOH；或者Ra与Ra’一起、R4与R5一起和/或R4’与R5’一起，在每次出现时各自独立地形成基团＝CH-W-R；
R7、R7’、R7a、R7a’在每次出现时各自独立地选自H、C1-4烷基和C3-6环烷基；
R选自H、甲基、乙基、丙基和环丙基；
m为0、1、2、3或4；
n为1、2、3或4；并且
j为0、1或2。
在更优选的实施方案中，Ra、Ra’、R4、R4’、R5、R5’和R6在每次出现时各自独立地选自H、F、-OH、-CH2OH、-OCH3、-COOH、-CH2COOH、-(CH2)2COOH、-(CH2)3COOH、-CH＝CHCOOH、-OCH2COOH、-SCH2COOH、-N(CH3)CH2COOH、-CH2OCH2COOH、-CH2SCH2COOH、-CH2N(CH3)CH2COOH、-C(CH3)＝CHCOOH和-CH＝C(CH3)COOH。
在特别优选的实施方案中，Ra、Ra’、R4、R4’、R5、R5’和R6在每次出现时各自独立地选自H、F、-OH、-CH2OH、-OCH3、-COOH、-CH2COOH、-(CH2)2COOH、-(CH2)3COOH、-CH＝CHCOOH、-OCH2COOH、-SCH2COOH、-N(CH3)CH2COOH、-CH2OCH2COOH、-CH2SCH2COOH和-CH2N(CH3)CH2COOH。
在优选的实施方案中，R3选自：
Figure PCTCN2017110123-appb-000018
在更优选的实施方案中，R3选自：
Figure PCTCN2017110123-appb-000019
在部分实施方案中，本发明提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有以下结构：
Figure PCTCN2017110123-appb-000020
在部分实施方案中，本发明提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有以下结构：
Figure PCTCN2017110123-appb-000021
在部分实施方案中，本发明提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有以下结 构：
Figure PCTCN2017110123-appb-000022
其中：
R1和R2各自独立地选自H(包括1H、2H、3H)、C1-6烷基(例如：氘代C1-6烷基)和C3-6环烷基，并且R1优选为甲基、乙基、正丙基、异丙基或环丙基；
Q为-(CRaRa’)g-或-O-；
Ra、Ra’、R4、R5和R6在每次出现时各自独立地选自H、卤素(例如F)、-OH、-COOH、-CN、-NO2、-N(R)2、C1-6烷基、卤代C1-6烷基、-W-C1-6烷基、-C1-6亚烷基-W-R、-W-C1-6亚烷基-W’-R、-W-C2-6烯基、-C2-6亚烯基-W-R、-W-C2-6亚烯基-W’-R和C3-6环烷基，其中所述亚烷基和亚烯基任选地进一步被一个或多个W间隔；
Rb在每次出现时各自独立地选自H、卤素、卤代C1-6烷基、C1-6烷基和C3-6环烷基；
Rc在每次出现时各自独立地选自F、Cl、Br、I、卤代C1-6烷基、C1-6烷基和C3-6环烷基，并且Rc优选为Cl或Br；
R6连接在通式结构中*和/或**标记的环碳原子上；
W和W’在每次出现时各自独立地选自O、C(＝O)、C(＝O)O、NR、NC(＝O)、N(S＝O)、NS(＝O)2、S、S＝O和S(＝O)2；
R在每次出现时各自独立地选自H、C1-6烷基和C3-6环烷基；
g为1或2；
i为0、1或2；
m为0、1、2、3或4；并且
t为0、1或2，条件是当t大于1时，每个R6可以相同或不同。
在部分实施方案中，Rb在每次出现时各自独立地选自H、卤素、C1-6烷基和C3-6环烷基；
在部分实施方案中，Rc在每次出现时各自独立地选自F、Cl、Br、I、C1-6烷基和C3-6环烷基，并且Rc优选为Cl或Br；
在部分实施方案中，本发明提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有以下结构：
Figure PCTCN2017110123-appb-000023
本发明涵盖对各个实施方案进行任意适当组合所得的化合物。
在优选的实施方案中，本发明提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物选自：
Figure PCTCN2017110123-appb-000024
Figure PCTCN2017110123-appb-000025
Figure PCTCN2017110123-appb-000026
Figure PCTCN2017110123-appb-000027
Figure PCTCN2017110123-appb-000028
Figure PCTCN2017110123-appb-000029
本发明的实施方案提供制备本发明的化合物的方法，所述方法包括以下步骤：
Figure PCTCN2017110123-appb-000030
其中：
Hal选自F、Cl、Br和I；
卤化试剂选自Cl2、Br2、I2、N-氯代琥珀酰亚胺、N-溴代丁二酰亚胺和N-碘代丁二酰亚胺；
其余各基团如上述所定义；
步骤一在碱金属盐(例如乙酸钾、乙酸钠、碳酸钾、碳酸钠、碳酸铯、碳酸氢钠等)存在下于质子性溶剂(例如2,2,2-三氟乙醇、2,2-二氟乙醇、2-氟乙醇、乙醇、氟甲醇、六氟异丙醇等)中进行；
步骤二在非质子性溶剂(例如四氯化碳、二氯甲烷、1,2-二氯乙烷等)中进行；并且
步骤三在非质子性溶剂(例如二氯甲烷、1,2-二氯乙烷、乙腈、四氢呋喃、2-甲基四氢呋喃、乙醚、叔丁基甲基醚、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮等)中，在有机碱(例如N,N-二异丙基乙胺、三乙胺、1,8-二氮二环[5.4.0]-7-十一烯、4-二甲氨基吡啶、N-甲基吗啉、N-甲基哌啶、N-甲基四氢吡咯等)或无机碱(例如乙酸钾、乙酸钠、碳酸钾、碳酸钠、碳酸铯、碳酸氢钠、氢化钠、叔丁醇钾等)的存在下进行。
当本发明的式I或式Ia的化合物中R2为C1-6烷基时，其也可通过包括以下步骤的方法合成：
Figure PCTCN2017110123-appb-000031
其中：
R2’为H或C1-5烷基；
Hal选自F、Cl、Br和I；
卤化试剂选自Cl2、Br2、I2、N-氯代琥珀酰亚胺、N-溴代丁二酰亚胺和N-碘代丁二酰亚胺；
其余各基团如上述所定义；
步骤I在路易斯酸(例如三氟甲磺酸盐(如三氟甲磺酸铟、三氟甲磺酸铋等)、三氟甲磺酸酯(如三氟甲磺酸三甲基硅酯)、三氟化硼、三氯化铝等)存在下于非极性溶剂(例如邻二甲苯、甲苯、苯甲醚等)中进行；
步骤II在有机碱(例如N,N-二异丙基乙胺、三乙胺、1,8-二氮二环[5.4.0]-7-十一烯、4-二甲氨基吡啶、N-甲基吗啉、N-甲基哌啶、N-甲基四氢吡咯等)或无机碱(例如乙酸钾、乙酸钠、碳酸钾、碳酸钠、碳酸铯、碳酸氢钠、氢化钠、叔丁醇钾等)的存在下，在非质子性溶剂(例如二氯甲烷、1,2-二氯乙烷、乙腈、四氢呋喃、2-甲基四氢呋喃、乙醚、叔丁基甲基醚、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮等)中进行；
步骤III在非质子性溶剂(例如四氯化碳、二氯甲烷、1,2-二氯乙烷等)中进行；并且
步骤IV在非质子性溶剂(例如二氯甲烷、1,2-二氯乙烷、乙腈、四氢呋喃、2-甲基四氢呋喃、乙醚、叔丁基甲基醚、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮等)中，在有机碱(例如N,N-二异丙基乙胺、三乙胺、1,8-二氮二环[5.4.0]-7-十一烯、4-二甲氨基吡啶、N-甲基吗啉、N-甲基哌啶、N-甲基四氢吡咯等)或无机碱(例如乙酸钾、乙酸钠、碳酸钾、碳酸钠、碳酸铯、碳酸氢钠、氢化钠、叔丁醇钾等)的存在下进行。
在优选的实施方案中，当本发明的式I或式Ia的化合物中R2为甲基时，其也可通过包括以下步骤的方法合成：
Figure PCTCN2017110123-appb-000032
其中各基团如上述所定义，并且步骤I-IV如上述进行。
药物组合物和治疗方法
本发明提供药物组合物，其包含预防或治疗有效量的本发明的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药以及一种或多种药学上可接受的载体或赋形剂。在另种实施方案中，所述药物组合物还可包含一种或多种 其它治疗剂，例如用于预防或治疗病毒性疾病的其它治疗剂。
本发明还提供制备药物组合物的方法，所述方法包括将本发明的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药与一种或多种药学上可接受的载体或赋形剂组合。
本发明提供本发明的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药或者本发明的药物组合物在制备用于预防或治疗病毒性疾病的药物中的用途。
本发明提供本发明的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药或者本发明的药物组合物，其用于预防或治疗病毒性疾病。
在另种实施方案中，本发明提供预防或治疗病毒性疾病的方法，所述方法包括向需要其的个体给药有效量的本发明的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药或者本发明的药物组合物。
本发明的化合物通过对衣壳蛋白装配的抑制作用来发挥抗病毒作用。因此，本发明的化合物可用于治疗在影响宿主的过程中涉及到衣壳蛋白装配的任意病毒，其包括但不限于甲型肝炎病毒(HAV)、乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、流行性感冒病毒、疱疹病毒(HSV)和人类免疫缺陷病毒(HIV)。
因此，可使用本发明的化合物进行预防和治疗的病毒性疾病包括但不限于甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、流行性感冒、疱疹和获得性免疫缺陷综合征(AIDS)，以及由上述疾病引发的相关症状或疾病(例如炎症、肝纤维化、肝硬化和肝癌等)。
本发明中“药学上可接受的载体”是指与治疗剂一同给药的稀释剂、辅剂、赋形剂或媒介物，并且其在合理的医学判断的范围内适于接触人类和/或其它动物的组织而没有过度的毒性、刺激、过敏反应或与合理的益处/风险比相应的其它问题或并发症。
在本发明的药物组合物中可使用的药学上可接受的载体包括但不限于无菌液体，例如水和油，包括那些石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、芝麻油等。当所述药物组合物通过静脉内给药时，水是示例性载体。还可以使用生理盐水和葡萄糖及甘油水溶液作为液体载体，特别是用于注射液。适合的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽糖、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙二醇、水、乙醇等。所述组合物还可以视需要包含少量的湿润剂、乳化剂或pH缓冲剂。口服制剂可以包含标准载体，如药物级的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。适合的药学上可接受的载体的实例如在Remington’s Pharmaceutical Sciences(1990)中所述。
本发明的药物组合物可以系统地作用和/或局部地作用。为此目的，它们可以适合的途径给药，例如通过注射(如静脉内、动脉内、皮下、腹膜内、肌内注射，包括滴注)或经皮给药；或通过口服、含服、经鼻、透粘膜、局部、以眼用制剂的形式或通过吸入给药。对于这些给药途径，可以采用适合的剂型给药。
所述剂型包括但不限于片剂、胶囊剂、锭剂、硬糖剂、散剂、喷雾剂、乳膏剂、软膏剂、栓剂、凝胶剂、糊剂、洗剂、软膏剂、水性混悬剂、可注射溶液剂、酏剂、糖浆剂。
如本文中所使用的术语“有效量”指被给药后会在一定程度上缓解所治疗病症的一或多种症状的化合物的量。
可调整给药方案以提供最佳所需响应。例如，可以单次给药，可随时间施用数个分剂量，或可根据治疗情况按比例减少或增加剂量。要注意，剂量值可随病况的类型及严重性而变化，且可包括单次或多次剂量。要进一步理解，对于任何特定个体，具体的给药方案应是根据个体需要判断来随时间调整。
所给药的本发明的化合物的量会取决于所治疗的个体、病症或病况的严重程度、给药的频率、化合物的处置及处方医师的判断。一般而言，有效剂量在每日每kg体重约0.0001至约50mg，例如约0.01至约10mg/kg/日(单次或分次给药)。对70kg的人而言，这会合计为约0.007mg/日至约3500mg/日，例如约0.7mg/日至约700mg/日。在一些情况下，不高于前述范围的下限的剂量水平可以是足够的，而在其它情况下，仍可在不引起任何有害副作用的情况下采用较大剂量，条件是首先将所述较大剂量分成数个较小剂量以在一整天中给药。
本发明的化合物在药物组合物中的含量或用量可以是约0.01mg至约1000mg，适合地是0.1-500mg，优选0.5-300mg，更优选1-150mg，特别优选1-50mg，例如1.5mg、2mg、4mg、10mg、25mg 等。
除非另外说明，否则如本文中所使用，术语“治疗(treating)”意指逆转、减轻、抑制这样的术语所应用的病症或病况或者这样的病症或病况的一或多种症状的进展，或预防这样的病症或病况或者这样的病症或病况的一或多种症状。
如本文所使用的“个体”包括人或非人动物。示例性人个体包括患有疾病(例如本文所述的疾病)的人个体(称为患者)或正常个体。本发明中“非人动物”包括所有脊椎动物，例如非哺乳动物(例如鸟类、两栖动物、爬行动物)和哺乳动物，例如非人灵长类、家畜和/或驯化动物(例如绵羊、犬、猫、奶牛、猪等)。
在另种实施方案中，本发明的药物组合物还可以包含一种或多种另外的治疗剂或预防剂，所述的治疗剂或预防剂为抗乙肝药物，其包括但不限于拉米夫定、替比夫定、恩替卡韦、阿德福韦酯、替诺福韦、替诺福韦二吡呋酯富马酸盐以及替诺福韦艾拉酚胺富马酸盐。
实施例
以下结合实施例进一步描述本发明，但提供这些实施例不以任何方式限制本发明的范围，其中的条件可适当任意组合。
除非另外说明，均使用市售的无水溶剂和HPLC级溶剂而不经进一步的纯化。
用Bruker仪器(400MHz)在环境温度下记录1H NMR光谱，使用TMS为内标。化学位移(δ)以ppm为单位给出，耦合常数(J)以赫兹(Hz)为单位给出。1H NMR波谱峰的裂分重数缩写如下：s(单峰)、d(双峰)、t(三重峰)、q(四重峰)、m(多重峰)、br(宽峰)。
LC-MS采用Aglient 1200液相色谱仪联用Aglient 6120 Quadrupole型质谱仪，在214nm及254nm下检测。制备液相色谱法使用SHIMADZU CBM-20A及Aglient 1260型制备液相仪，C18 OBD 19×150mm 5μM制备柱，检测波长214nm，流动相A为水，流动相B为乙腈(添加0.5‰甲酸)，按下表进行线性梯度洗脱：
时间(min) A％ B％
0 90 10
15 40 60
30 10 90
本发明中的缩写具有以下含义：
缩写 含义 缩写 含义
DAST 二乙胺基三氟化硫 HOAc 乙酸
DEA 二乙胺 IPA 异丙醇
EA 乙酸乙酯 MeOH 甲醇
EtOH 乙醇 PE 石油醚
HEX 己烷 TLC 薄层色谱法
实施例一 4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-羟基哌啶-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(10-88)的合成
Figure PCTCN2017110123-appb-000033
步骤一：4-(2-氯-4-氟苯基)-6-甲基-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(1-1)的合成
在室温下，将乙酰乙酸乙酯(4.7g，36.0mmol)、噻唑-2-甲脒盐酸盐(5.4g，36.0mmol)，2-氯-4-氟苯甲醛(5.8g，36.0mmol)和乙酸钾(6.0g，60.0mmol)加入到2,2,2-三氟乙醇(100mL)中，加热至回流，反应16小时。将反应液降至室温，减压蒸除溶剂，后处理得标题化合物(6.0g)。ESI-MS(m/z):380.1[M+H]+。
步骤二：6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(1-2)的合成
将化合物(1-1)(5.7g，15.0mmol)溶于四氯化碳(60mL)中，升温至50℃，一次性加入N-溴代丁二酰亚胺(2.7g，15.0mmol)，反应30min。将反应液降至室温，过滤移除不溶物，浓缩得粗品纯化得标题化合物(6.0g)。ESI-MS(m/z):458.0[M+H]+。
步骤三：4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-羟基哌啶-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(10-88)的合成
在室温下，将化合物(1-2)(68mg，0.14mmol)、3,3-二氟哌啶-4-醇盐酸盐(34mg，0.2mmol)和N,N-二异丙基乙基胺(50mg，0.4mmol)加入到二氯甲烷(3mL)中，在室温下反应过夜，将反应液浓缩得粗品纯化得标题化合物(10-88)(27mg)。
其结构表征如下：
1H NMR(400MHz，DMSO-d6):δ9.54(s,1H),7.99(d,J＝3.14Hz,1H),7.94(d,J＝3.14Hz,1H),7.44-7.40(m,2H),7.19(td,J＝8.44,2.64Hz,1H),6.05(s,1H),5.62(t,J＝5.40Hz,1H),4.00-3.92(m,4H),3.76(s,1H),3.06-2.67(m,4H),1.86(d,J＝3.52Hz,1H),1.74(d,J＝3.08Hz,1H),1.04(t,J＝7.12Hz,3H).ESI-MS(m/z):515.2[M+H]+。
实施例二 4-(2-溴-4-氟苯基)-6-((3,3-二氟-4-羟基哌啶-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(10-93)的合成
Figure PCTCN2017110123-appb-000034
根据以上反应路线，采用与实施例一中类似的操作(除在步骤一中用2-溴-4-氟苯甲醛代替2-氯-4-氟苯甲醛)，制备得到标题化合物(27mg)。
其结构表征如下：
1H NMR(400MHz，DMSO-d6):δ9.54(s,1H),7.99(d,J＝3.14Hz,1H),7.94(d,J＝3.14Hz,1H),7.44-7.40(m,2H),7.19(td,J＝8.44,2.64Hz,1H),6.05(s,1H),5.62(t,J＝5.40Hz,1H),4.00-3.92(m,4H),3.76(s,1H),3.06-2.67(m,4H),1.86(d,J＝3.52Hz,1H),1.74(d,J＝3.08Hz,1H),1.04(t,J＝7.12Hz,3H).ESI-MS(m/z):559.2[M+H]+。
实施例三 (S)-4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-羟基哌啶-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯异构体A和(S)-4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-羟基哌啶-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯-异构体B的合成
Figure PCTCN2017110123-appb-000035
步骤一：4-(2-氯-4-氟苯基)-6-甲基-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(3-1)的分离
将化合物(3-1)(10g)通过手性色谱法分离，分离条件如下：分离柱CHIRALPAK IE 0.46cm I.D.× 15cm L，流动相：MeOH/DEA＝100/0.1(V/V)，流速1.0ml/min，波长UV 254nm，温度35℃。
分离得到(S)-4-(2-氯-4-氟苯基)-6-甲基-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(3-2)4.7g，ee％＝99.9％，Rt＝2.642min。ESI-MS(m/z):380.1[M+H]+；和
(R)-4-(2-氯-4-氟苯基)-6-甲基-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(3-2’)4.5g，ee％＝99.9％，Rt＝4.783min。ESI-MS(m/z):380.1[M+H]+。
步骤二至步骤三
以化合物(3-2)为原料，采用与实施例一步骤二和步骤三中类似的操作，制备得到(S)-4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-羟基哌啶-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(3-4)。ESI-MS(m/z):515.2[M+H]+。
步骤四：化合物(3-4)的分离
将化合物(3-4)(340mg)通过手性色谱法分离，分离条件如下：分离柱CHIRALPAK IE 0.46cm I.D.×15cm L，流动相：HEX:IPA＝100/0.1(V/V)，流速1.0ml/min，波长UV 254nm，温度35℃。其中，Rt＝5.961min的产物为异构体A，ee％＝99.3％，122mg，其结构表征如下：
1H NMR(400MHz，DMSO-d6):δ9.54(s,1H),7.99(d,J＝3.14Hz,1H),7.94(d,J＝3.14Hz,1H),7.44-7.40(m,2H),7.19(td,J＝8.44,2.64Hz,1H),6.05(s,1H),5.62(t,J＝5.40Hz,1H),4.00-3.92(m,4H),3.76(s,1H),3.06-2.67(m,4H),1.86(d,J＝3.52Hz,1H),1.74(d,J＝3.08Hz,1H),1.04(t,J＝7.12Hz,3H).ESI-MS(m/z):515.2[M+H]+；和
其中Rt＝7.130min的产物为异构体B，ee％＝99.5％，131mg，其结构表征如下：
1H NMR(400MHz，DMSO-d6):δ9.54(s,1H),7.99(d,J＝3.14Hz,1H),7.94(d,J＝3.14Hz,1H),7.44-7.40(m,2H),7.19(td,J＝8.44,2.64Hz,1H),6.05(s,1H),5.62(t,J＝5.40Hz,1H),4.00-3.92(m,4H),3.76(s,1H),3.06-2.67(m,4H),1.86(d,J＝3.52Hz,1H),1.74(d,J＝3.08Hz,1H),1.04(t,J＝7.12Hz,3H).ESI-MS(m/z):515.2[M+H]+。
实施例四 (R)-4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-羟基哌啶-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯-异构体A和(R)-4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-羟基哌啶-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯-异构体B的合成
Figure PCTCN2017110123-appb-000036
以化合物(3-2’)为原料，采用与实施例三中类似的操作，制备得到目标产物。其中Rt＝8.171min的产物为异构体A，ee％＝99.1％，137mg，其结构表征如下：
1H NMR(400MHz，DMSO-d6):δ9.54(s,1H),7.99(d,J＝3.14Hz,1H),7.94(d,J＝3.14Hz,1H),7.44-7.40(m,2H),7.19(td,J＝8.44,2.64Hz,1H),6.05(s,1H),5.62(t,J＝5.40Hz,1H),4.00-3.92(m,4H),3.76(s,1H),3.06-2.67(m,4H),1.86(d,J＝3.52Hz,1H),1.74(d,J＝3.08Hz,1H),1.04(t,J＝7.12Hz,3H).ESI-MS(m/z):515.2[M+H]+；和
其中Rt＝7.088min的产物为异构体B，ee％＝99.4％，128mg，其结构表征如下：
1H NMR(400MHz，DMSO-d6):δ9.54(s,1H),7.99(d,J＝3.14Hz,1H),7.94(d,J＝3.14Hz,1H),7.44-7.40(m,2H),7.19(td,J＝8.44,2.64Hz,1H),6.05(s,1H),5.62(t,J＝5.40Hz,1H),4.00-3.92(m,4H),3.76(s,1H),3.06-2.67(m,4H),1.86(d,J＝3.52Hz,1H),1.74(d,J＝3.08Hz,1H),1.04(t,J＝7.12Hz,3H).ESI-MS(m/z):515.2[M+H]+。
实施例五 2-((1-((6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-3,3-二氟哌啶-4-基)氧基)乙酸(10-95)的合成
Figure PCTCN2017110123-appb-000037
步骤一：3,3-二氟-4-羟基哌啶-1-甲酸苄酯(5-2)的合成
将3,3-二氟哌啶-4-醇盐酸盐(5-1)(100mg，0.73mmol)溶于二氯甲烷(2mL)中，加入三乙胺(147mg，1.46mmol)，在冰浴降温下，滴加N-(苄氧羰基氧基)琥珀亚胺(12mg，0.48mmol)的二氯甲烷溶液(2mL)，将反应物升至室温反应1h。后处理得标题化合物100mg。ESI-MS(m/z):272.2[M+H]+。
步骤二：4-(2-乙氧基-2-氧代乙氧基)-3,3-二氟哌啶-1-甲酸苄酯(5-3)的合成
将化合物(5-2)(100mg，0.37mmol)溶于四氢呋喃(2mL)中，在冰浴下加入氢化钠(18mg，0.74mmol)，将反应物升至室温反应1小时。将反应物置于冰浴中，滴加溴乙酸乙酯(94mg，0.56mmol)的四氢呋喃溶液，滴加完成后升至室温继续搅拌5小时。减压蒸除溶剂，得到标题化合物150mg。ESI-MS(m/z):358.2[M+H]+。
步骤三：2-((1-((苄氧基)羧基)-3,3-二氟哌啶-4-基)氧基)乙酸(5-4)的合成
在室温下，将化合物(5-3)(132mg，0.37mmol)溶于四氢呋喃(1mL)中，加入氢氧化钠(89mg，2.22mmol在0.3mL H2O中)，在室温下反应过夜。将反应体系倒入水中后处理得到标题化合物80mg。ESI-MS(m/z):328.2[M-H]-。
步骤四：2-((3,3-二氟哌啶-4-基)氧基)乙酸(5-5)的合成
在室温下，将化合物(5-4)(80mg，0.24mmol)溶于甲醇(2mL)中，加入钯/碳(10％，10mg)，使反应物在氢气环境中于室温下反应3小时。滤除不溶物，减压蒸除溶剂，得到标题化合物40mg。ESI-MS(m/z):196.2[M+H]+。
步骤五：2-((1-((6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-3,3-二氟哌啶-4-基)氧基)乙酸(10-95)的合成
除了用化合物(5-5)代替3,3-二氟哌啶-4-醇盐酸盐外，采用与实施例一步骤三所描述类似的方法制备得到标题化合物10mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ12.66(s,1H),9.54(d,J＝2.4Hz,1H),8.07-7.82(m,2H),7.53-7.33(m,2H),7.18(td,J＝8.5,2.7Hz,1H),6.05(s,1H),4.30-4.09(m,2H),4.06-3.89(m,4H),3.84(s,1H),3.10-2.81(m,3H),2.72(s,1H),2.00(s,1H),1.85(s,1H),1.04(t,J＝7.0Hz,3H).ESI-MS(m/z):573.2[M+H]+。
实施例六 (R)-4-(2-氯-4-氟苯基)-6-((3,3-二氟氮杂环丁烷-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(10-7)的合成
Figure PCTCN2017110123-appb-000038
采用与实施例一步骤三所描述类似的方法(用3,3-二氟氮杂环丁烷盐酸盐代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物4mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ9.46(s,1H),8.06-7.99(m,1H),7.95(d,J＝3.1Hz,1H),7.46-7.36(m,2H),7.19(td,J＝8.5,2.6Hz,1H),6.03(s,1H),4.15(s,2H),3.97(t,J＝7.1Hz,2H),3.85(t,J＝12.6Hz,4H),1.05(t,J＝7.1Hz,3H).ESI-MS(m/z):471.1[M+H]+。
实施例七 (R)-1-((6-(2-氯-4-氟苯基)-5-乙氧羰基-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-3-氟氮杂丁烷-3-羧酸(10-11)的合成
Figure PCTCN2017110123-appb-000039
步骤一：3-氟氮杂环丁烷-3-羧酸的合成
将1-(叔丁氧基羰基)-3-氟氮杂丁烷-3-羧酸(30mg，0.14mmol)溶于二氯甲烷(2mL)中，在室温下加入三氟乙酸(0.5mL)，反应持续1小时。减压蒸除溶剂，得到标题化合物的三氟乙酸盐33mg。ESI-MS(m/z):120.1[M+H]+。
步骤二：(R)-1-((6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-3-氟氮杂环丁烷-3-羧酸(10-11)的合成
采用与实施例一步骤三所描述类似的方法(用3-氟氮杂环丁烷-3-羧酸三氟乙酸盐代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物6mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ9.47(s,1H),8.01(d,J＝3.08Hz,1H),7.95(d,J＝3.09Hz,1H),7.40(dd,J＝15.82,6.61Hz,2H),7.21-7.15(m,1H),6.02(s,1H),4.12(s,2H),3.96(dd,J＝14.10,7.12Hz,2H),3.91-3.81(m,2H),3.71(dd,J＝20.97,9.47Hz,2H),1.05(t,J＝7.06Hz,3H).ESI-MS(m/z):497.0[M+H]+。
实施例八 (4R)-4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-甲氧基哌啶-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(10-98)的合成
Figure PCTCN2017110123-appb-000040
步骤一：3,3-二氟-4-甲氧基哌啶-1-羧酸叔丁酯(8-2)的合成
向25mL烧瓶中加入3,3-二氟-4-羟基哌啶-1-羧酸叔丁酯(8-1)(100mg，0.42mmol)和四氢呋喃(5mL)，在氮气保护下，降温至0℃，向其中加入氢化钠(20mg，0.5mmol)，反应30分钟，再向其中加入碘甲烷(120mg，0.84mmol)，反应16小时。将反应液缓慢倒入水中后处理得到标题化合物的粗品100mg，将其在未经纯化下直接用于下一步反应。ESI-MS(m/z):252.2[M+H]+。
步骤二：3,3-二氟-4-甲氧基哌啶三氟乙酸盐(8-3)的合成
向25mL烧瓶中加入化合物(8-2)(100mg，0.4mmol)和二氯甲烷(6mL)，将反应物降温至0℃，然后向其中加入三氟乙酸(2mL)，反应3小时。减压蒸除溶剂后得粗品120mg。ESI-MS(m/z):152.2[M+H]+。
步骤三：(4R)-4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-甲氧基哌啶-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(10-98)的合成
采用与实施例一步骤三所描述类似的方法(用化合物(8-3)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物50mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ9.54(s,1H),8.00(dd,J＝3.1,1.7Hz,1H),7.94(d,J＝3.1Hz,1H),7.47-7.35(m,2H),7.18(td,J＝8.5,2.6Hz,1H),6.05(s,1H),4.06-3.91(m,4H),3.60(d,J＝5.0Hz,1H),3.41(d,J＝1.6Hz,3H),3.12-2.93(m,1H),2.92-2.76(m,1H),2.67(s,1H),1.94(s,1H),1.77(s,1H),1.04(t,J＝7.1Hz,3H).ESI-MS(m/z):529.2[M+H]+。
实施例九 (R)-4-(2-氯-4-氟苯基)-6-(((3R,4R)-3-氟-4-羟基四氢吡咯-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(10-34)的合成
Figure PCTCN2017110123-appb-000041
步骤一：(3R,4R)-4-氟四氢吡咯-3-醇(9-2)的合成
在冰浴下，将(3R,4R)-3-羟基-4-氟四氢吡咯-1-甲酸叔丁酯(9-1)(60mg，0.3mmol)溶于二氯甲烷(1mL)中，加入三氟乙酸(0.3mL)，在室温下反应1.5h。减压蒸除溶剂，得标题化合物的三氟乙酸盐60mg。ESI-MS(m/z):106.1[M+H]+。
步骤二：(R)-4-(2-氯-4-氟苯基)-6-(((3R,4R)-3-氟-4-羟基四氢吡咯-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(10-34)的合成
采用与实施例一步骤三所描述类似的方法(用化合物(9-2)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物30mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ7.86(t,J＝2.6Hz,1H),7.59(s,2H),7.49-7.33(m,1H),7.16(ddd,J＝8.3,4.3,2.6Hz,1H),7.02(d,J＝9.0Hz,1H),6.18(d,J＝2.3Hz,1H),5.19(d,J＝50.5Hz,1H),4.93-4.26(m,4H),4.06(qd,J＝7.1,1.7Hz,2H),3.53(s,2H),1.10(td,J＝7.1,4.5Hz,3H).ESI-MS(m/z):483.2[M+H]+。
实施例十 2-(((3R,4R)-1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-4-氟四氢吡咯-3-基)氧基)乙酸(10-36)的合成
Figure PCTCN2017110123-appb-000042
步骤一：(3R,4R)-3-(2-乙氧基-2-氧代乙氧基)-4-氟四氢吡咯-1-甲酸叔丁酯(10-b)的合成
将(3R,4R)-3-羟基-4-氟四氢吡咯-1-甲酸叔丁酯(10-a)(100mg，0.49mmol)溶于四氢呋喃(5mL)中，在冰浴降温下，加入氢化钠(40mg,60％在油中，0.98mmol)，升至室温反应2h。在冰浴降温下，加入溴乙酸乙酯(124mg，0.74mmol)，室温反应过夜。加入饱和氯化铵(3mL)，然后加入二氯甲烷(15mL)稀释，用饱和食盐水洗涤，无水硫酸钠干燥。过滤移除干燥剂，减压蒸除溶剂，得到标题化合物120mg。ESI-MS(m/z):292.2[M+H]+。
步骤二：2-(((3R,4R)-1-(叔丁氧羰基)-4-氟四氢吡咯-3-基)氧基)乙酸(10-c)的合成
将化合物(10-b)(120mg，0.41mmol)溶于四氢呋喃与水的混合溶液中(v:v＝1:1，3mL)中，加入一水合氢氧化锂(104mg，2.47mmol)，在室温下反应3.5h。用柠檬酸水溶液调节pH至5，加入二氯甲烷(15mL)，用饱和食盐水洗涤，无水硫酸钠干燥。过滤移除无水硫酸钠，减压蒸除溶剂，得到标题化合物100mg。ESI-MS(m/z):263.2[M+H]+。
步骤三：2-(((3R,4R)-4-氟四氢吡咯-3-基)氧基)乙酸(10-d)的合成
在室温下，将化合物(10-c)(100mg，0.38mmol)溶于二氯甲烷(1mL)中，加入三氟乙酸(0.3mL)，在室温下反应1.5h。减压蒸除溶剂，得到标题化合物的三氟乙酸盐60mg。ESI-MS(m/z):164.1[M+H]+。
步骤四：2-(((3R,4R)-1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-4-氟四氢吡咯-3-基)氧基)乙酸(10-36)的合成
采用与实施例一步骤三所描述类似的方法(用化合物(10-d)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物13mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ12.66(s,1H),9.47(d,J＝12.6Hz,1H),7.98(dd,J＝5.1,3.2Hz,1H),7.93(dd,J＝3.2,1.9Hz,1H),7.41(ddd,J＝9.5,6.4,3.0Hz,2H),7.19(tt,J＝8.5,2.0Hz,1H),6.04(d,J＝1.4Hz,1H),5.15(d,J＝51.7Hz,1H),4.40-3.77(m,9H),3.12-2.87(m,2H),1.04(td,J＝7.1,2.1Hz,3H).ESI-MS(m/z):541.2[M+H]+。
实施例十一 (4R)-4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-羟基四氢吡咯-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(10-40)的合成
步骤一：2,2-二氟乙烯基对甲苯磺酸酯(11-2)的合成
将2,2,2-三氟乙基对甲苯磺酸酯(11-1)(2.57g，10.0mmol)溶于四氢呋喃(15mL)中，将反应液在氮气保护下降温到-78℃，缓慢滴加正丁基锂(2.5M在己烷中，8mL，20mmol)。将反应液在-78℃下搅拌40分钟，缓慢滴加水(4.5g,25mmol)和四氢呋喃(10mL)。将反应液缓慢升至室温。加水(60mL)，用乙酸乙酯萃取(30mL×2)，收集有机相，将其用饱和氯化钠溶液洗涤(50mL×2)，无水硫酸钠干燥，过滤移除无水硫酸钠，减压浓缩后通过柱色谱法纯化，得到目标化合物2.4g。ESI-MS(m/z):235.1[M+H]+。
步骤二：1-苄基-4,4-二氟四氢吡咯-3-基对甲基苯磺酸酯(11-3)的合成
将化合物(11-2)(2340mg，10.0mmol)和N-甲氧甲基-N-三甲基硅甲基-苄胺(9500mg，40.0mmol)于130℃油浴锅中搅拌5分钟，滴加三氟乙酸(110mg，1.1mmol)。将反应物继续搅拌1小时，冷却到室温，加乙酸乙酯稀释，纯化处理得到标题化合物3.0g。ESI-MS(m/z):368.1[M+H]+。
步骤三：1-苄基-4,4-二氟四氢吡咯-3-醇(11-4)的合成
将化合物(11-3)(500mg，1.35mmol)溶于甲醇(5mL)中，加入镁屑(326mg，13.6mmol)，将反应物在室温下搅拌2小时，加入冰水(20mL)，滴加浓盐酸直到固体全部溶解，用乙酸乙酯萃取，收集水相，用饱和氢氧化钠调节pH＝7，乙酸乙酯萃取，收集有机相，用饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤移除无水硫酸钠，减压浓缩，将粗品通过制备硅胶色谱法纯化(石油醚/乙酸乙酯＝3/2)，得到目标化合物240mg。ESI-MS(m/z):214.2[M+H]+。
步骤四：4,4-二氟四氢吡咯-3-醇(11-5)的合成
将化合物(11-4)(100mg，0.47mmol)溶于甲醇(2mL)中，加入钯/碳(8mg，10％Pd，0.047mmol)，将反应物于氢气环境中在室温下搅拌过夜，过滤移除钯/碳，减压浓缩，得到目标化合物50mg。ESI-MS(m/z):124.1[M+H]+。
步骤五：(4R)-4-(2-氯-4-氟苯基)-6-((3,3-二氟-4-羟基四氢吡咯-1-基)甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(10-40)的合成
采用与实施例一步骤三所描述类似的方法(用化合物(11-5)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物33mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ9.36(s,1H),7.84(d,J＝3.1Hz,1H),7.48(s,1H),7.31(s,1H),7.14(dt,J＝8.5,2.5Hz,1H),6.94(s,1H),6.20(d,J＝6.9Hz,1H),4.45-3.87(m,6H),3.20(s,2H),2.85(s,1H),1.13(td,J＝7.1,4.7Hz,3H).ESI-MS(m/z):501.0[M+H]+。
实施例十二 2-((1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-4,4-二氟四氢吡咯-3-基)氧基)乙酸(10-42)的合成
Figure PCTCN2017110123-appb-000044
步骤一：2-((1-苄基-4,4-二氟四氢吡咯-3-基)氧基)乙酸乙酯(12-2)的合成
将1-苄基-4,4-二氟四氢吡咯-3-醇(12-1)(100mg，0.47mmol)溶于四氢呋喃(5mL)中，在冰浴降温下，加入氢化钠(40mg 60％在油中，0.94mmol)，升至室温反应2h。在冰浴降温下，加入溴乙酸乙酯(119mg，0.71mmol)，在室温下反应过夜，反应完成。加入饱和氯化铵(3mL)，加入二氯甲烷(15mL)稀释，用饱和食盐水洗涤，无水硫酸钠干燥。过滤移除无水硫酸钠，减压蒸除溶剂得到标题化合物123mg，将其在未经纯化下直接用于下一步反应。ESI-MS(m/z):300.1[M+H]+。
步骤二：2-((1-苄基-4,4-二氟四氢吡咯-3-基)氧基)乙酸(12-3)的合成
将化合物(12-2)(123mg，0.41mmol)溶于四氢呋喃与水的混合溶液中(v:v＝1:1，3mL)中，加入一水合氢氧化锂(104mg，2.47mmol)，在室温下反应3.5h，用柠檬酸水溶液调节pH至5，加入二氯甲烷(15mL)，用饱和食盐水洗涤，无水硫酸钠干燥。过滤移除干燥剂，减压蒸除溶剂，将粗品通过制备色谱法纯化，得到标题化合物50mg。ESI-MS(m/z):272.1[M+H]+。
步骤三：2-((4,4-二氟四氢吡咯-3-基)氧基)乙酸(12-4)的合成
将化合物(12-3)(50mg，0.18mmol)溶于甲醇(2mL)中，加入钯/碳(5mg，10％Pd，0.018mmol)，将反应物于氢气环境中在室温下搅拌过夜，过滤移除钯/碳，减压浓缩得到目标化合物30mg。ESI-MS(m/z):182.1[M+H]+。
步骤四：2-((1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-4,4-二氟四氢吡咯-3-基)氧基)乙酸的合成
采用与实施例一步骤三所描述类似的方法(用化合物(12-4)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物25mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ7.87(dd,J＝3.1,1.3Hz,1H),7.49(s,1H),7.33(t,J＝7.4Hz,1H),7.14(dd,J＝8.5,2.6Hz,1H),6.96(t,J＝8.3Hz,1H),6.19(d,J＝2.3Hz,1H),4.44(d,J＝16.3Hz,1H),4.26(dd,J＝16.3,2.3Hz,4H),4.08-4.01(m,2H),3.43(s,4H),1.12(t,J＝7.1Hz,3H).ESI-MS(m/z):559.2[M+H]+。
实施例十三 (E)-3-(4-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-3-基)丙烯酸(10-182)的合成
Figure PCTCN2017110123-appb-000045
步骤一：3-(羟甲基)吗啉-4-甲酸叔丁酯(13-2)的合成
向50mL三口瓶中加入吗啉-3基甲醇盐酸盐(1.0g，6.5mmol)、三乙胺(1.64g，16mmol)和二氯甲烷(10mL)，在氮气保护下搅拌，降温至0℃，再加入二碳酸二叔丁酯(2.1g，10mmol)，加入完毕后，将反应物升至室温反应3小时。将反应液缓慢倒入水中，用二氯甲烷萃取，收集有机相，用饱和食盐水洗涤，无水硫酸钠干燥。过滤移除无水硫酸钠，减压蒸除溶剂，纯化得到目标化合物1.32g。ESI-MS(m/z):162.0[M+H]+。
步骤二：3-甲酰基吗啉-4-甲酸叔丁酯(13-3)合成
向50mL三口瓶中加入化合物(13-2)(500mg，2.3mmol)和二氯甲烷(10mL)，在室温下向其中加入戴斯-马丁试剂(1.3g，3.0mmol)，搅拌3小时。将反应液缓慢倒入碳酸氢钠水溶液中，用二氯甲烷萃取，收集有机相，用饱和食盐水洗涤，无水硫酸钠干燥，过滤移除无水硫酸钠，减压蒸除溶剂，得到粗品520mg。ESI-MS(m/z):160.0[M+H]+。
步骤三：(E)-3-(3-乙氧-3-氧代丙-1-烯-1-基)吗啉-4-甲酸叔丁酯(13-4)的合成
向50mL三口瓶中加入三乙基膦酰乙酸酯(600mg，3.3mmol)和四氢呋喃(10mL)，在氮气保护下搅拌，降温至0℃，再加入氢化钠(320mg，6.6mmol)，加入完毕后反应10min，再向其中滴加化合物(13-3)(520mg，3.3mmol)的四氢呋喃(5mL)溶液，滴加完毕后，升至室温反应16h。将反应液缓慢倒入水中，用乙酸乙酯萃取，收集有机相，用饱和食盐水洗涤，无水硫酸钠干燥。过滤移除干燥剂，减压蒸除溶剂，得到粗品620mg。ESI-MS(m/z):186.0[M+H]+。
步骤四：(E)-3-(4-(叔丁氧羰基)吗啉-3-基)丙烯酸(13-5)的合成
在室温下，向50mL烧瓶中加入化合物(13-4)(620mg，2.2mmol)、无水乙醇(10mL)和水(10mL)，搅拌，再向其中加入氢氧化钠(260mg，6.6mmol)，在室温下反应4h。然后向反应物中加入水(20mL)淬灭反应，旋转蒸发移除乙醇，用甲基叔丁基醚萃取，将水相用1N的盐酸水溶液调节pH至2～3，将水相再用乙酸乙酯萃取，收集有机相，用饱和食盐水洗涤，无水硫酸钠干燥。过滤移除无水硫酸钠，减压蒸除溶剂，得到粗品460mg，将其在未经纯化直接用于下一步反应。ESI-MS(m/z):158.0[M+H]+。
步骤五：(E)-3-(吗啉-3-基)丙烯酸(13-6)的合成
向25mL烧瓶中加入4N盐酸在二氧六环中的溶液(5mL)，将其降温至0℃，然后向其中滴加化合物(13-5)(460mg，1.8mmol)的乙酸乙酯(5mL)溶液，在室温下反应2h。减压蒸除溶剂，得到目标化合物的盐酸盐300mg。ESI-MS(m/z):158.0[M+H]+。
步骤六：(E)-3-(4-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-3-基)丙烯酸(10-182)的合成
采用与实施例一步骤三所描述类似的方法(用化合物(13-6)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到目标化合物47mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ12.47(s,1H),9.69(d,J＝28.7Hz,1H),8.11-7.91(m,2H),7.53-7.27(m,2H),7.23-7.10(m,1H),6.66(ddd,J＝35.0,15.8,8.8Hz,1H),6.11(dd,J＝28.6,15.7Hz,1H),6.03(d,J＝14.0Hz,1H),4.02-3.85(m,4H),3.84-3.70(m,2H),3.70-3.57(m,1H),3.46-3.35(m,2H),2.84(dd,J＝38.9,12.6Hz,1H),2.48-2.41(m,1H),1.03(t,J＝7.1Hz,3H).ESI-MS(m/z):535.1[M+H]+。
实施例十四 (E)-3-(4-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-180)的合成
Figure PCTCN2017110123-appb-000046
根据以上反应路线，以吗啉-2基甲醇盐酸盐为起始原料，采用与实施例十三中类似的操作，制备得到标题化合物22mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ12.40(s,1H),9.64(d,J＝2.4Hz,1H),8.02(d,J＝2.92Hz,2H),7.95(d,J＝3.12Hz,2H),7.21-7.16(m,1H),6.83-6.72(m,1H),6.05(d,1H),6.00-5.91(m,1H),4.27-4.22(m,1H),4.00-3.85(m,5H),3.70-3.61(m,1H),3.07-2.94(m,1H),2.84-2.66(m,1H),2.42-2.67(m,1H),2.17-2.04(m,1H),1.04(t,J＝7.0Hz,3H).ESI-MS(m/z):535.1[M+H]+。
实施例十五 2-((4-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-3-基)甲氧基)乙酸(10-162)
Figure PCTCN2017110123-appb-000047
步骤一：3-((2-(叔丁氧基)-2-氧代乙氧基)甲基)吗啉-4-甲酸叔丁酯(15-2)的合成
向50mL三口瓶中加入3-(羟甲基)吗啉-4-甲酸叔丁酯(200mg，0.99mmol)和四氢呋喃(6mL)，在室温下向其中加入氢化钠(47.3mg，1.2mmol)，搅拌30分钟，然后加入溴乙酸叔丁酯(192mg，0.99mmol)，在室温下反应3小时。将反应液缓慢倒入10mL水中，用1N的盐酸水溶液调节pH至2～3，用乙酸乙酯萃取，收集有机相，用饱和食盐水洗，无水硫酸钠干燥，过滤移除无水硫酸钠，减压蒸除溶剂，得到粗品300mg。ESI-MS(m/z):232.0[M+H]+。
步骤二：2-(吗啉-3-基甲氧基)乙酸三氟乙酸盐(15-3)的合成
向25mL烧瓶中加入化合物(15-2)(300mg，0.9mmol)和二氯甲烷(6mL)，降温至0℃，然后向其中加入三氟乙酸(2mL)，在室温下反应3小时。减压蒸除溶剂，得到粗品250mg。ESI-MS(m/z):176.0[M+H]+。
步骤三：2-((4-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-3-基)甲氧基)乙酸(10-162)的合成
采用与实施例一步骤三所描述类似的方法(用化合物(15-3)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到目标化合物73mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ12.70(s,1H),9.79(d,J＝22.9Hz,1H),8.01(t,J＝2.8Hz,1H),7.93(t,J＝3.0Hz,1H),7.50-7.37(m,2H),7.18(qd,J＝8.3,2.6Hz,1H),6.04(d,J＝1.4Hz,1H),4.32(dd,J＝20.0,17.6Hz,1H),4.09-3.91(m,4H),3.87-3.63(m,3H),3.62-3.50(m,3H),3.46(dd,J＝11.2,8.2Hz,1H),2.87–2.66(m,2H),2.48-2.39(m,1H),1.05(td,J＝7.0,1.5Hz,3H).ESI-MS(m/z):553.1[M+H]+。
实施例十六 2-((1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-5,5-二氟哌啶-3-基)甲氧基)乙酸(10-136)的合成
Figure PCTCN2017110123-appb-000048
步骤一：1-叔丁基3-甲基5-氧代哌啶-1,3-二羧酸酯(16-2)的合成
在室温下，将1-叔丁基3-甲基5-羟基哌啶-1,3-二羧酸酯(16-1)(1.0g，3.86mmol)溶于二氯甲烷(20mL)中，待完全溶解后，将反应物降温至0℃，在搅拌下加入戴斯-马丁试剂(3.27g,7.71mmol)，加入完毕后，在室温下搅拌过夜。反应液中有大量白色固体析出，过滤，将滤液依次用水(50mL)和饱和碳酸钠水溶液(50mL)洗涤，将有机相用无水硫酸钠干燥，减压蒸除溶剂得到标题化合物1.0g。ESI-MS(m/z):202.1[M+H-56]+。
步骤二：1-叔丁基3-甲基5,5-二氟哌啶-1,3-二羧酸酯(16-3)的合成
将化合物(16-2)(1.0g，3.89mmol)溶于二氯甲烷(20mL)中，搅拌溶解后将反应物降温至-70℃，缓慢滴加DAST(1.9g，11.67mmol)，滴加完毕后，将反应物升至室温反应4.5h。LC-MS监测至原料反应完全后，将反应液用饱和碳酸氢钠水溶液(20mL)淬灭，用二氯甲烷萃取，干燥、纯化得到标 题化合物550mg。ESI-MS(m/z):224.1[M+H-56]+。
步骤三：3,3-二氟-5-(羟甲基)哌啶-1-羧酸叔丁酯(16-4)的合成
在室温下，将化合物(16-3)(500mg，1.79mmol)加入到甲醇(8mL)中，将反应物降温至0℃，分批缓慢加入硼氢化钠(272mg，7.16mmol)，加入完毕后，室温反应过夜，LC-MS监测至原料未反应完全，补加硼氢化钠(136mg，3.58mmol)，继续反应过夜直至原料反应完全。向反应液加水(20mL)淬灭，用乙酸乙酯萃取，合并有机相，用水洗涤，无水硫酸钠干燥，减压蒸除溶剂得到标题化合物500mg。ESI-MS(m/z):196.1[M+H-56]+。
步骤四：5-((2-(叔丁氧基)-2-氧代乙氧基)甲基)-3,3-二氟哌啶-1-羧酸叔丁酯(16-5)的合成
在室温下，将化合物(16-4)(150mg，0.60mmol)加入到四氢呋喃(4mL)中，将反应物降温至0℃，缓慢加入氢化钠(48mg，1.2mmol)，加入完毕后，在0℃搅拌30min，然后加入溴乙酸叔丁酯(140mg，0.72mmol)的四氢呋喃(1.0mL)溶液，在室温下反应过夜，LC-MS监测原料未反应完全，补加溴乙酸叔丁酯(35mg，0.18mmol)，继续反应过夜直至原料反应完全。向反应液加入饱和氯化铵水溶液(5mL)淬灭，加水(20mL)稀释，用乙酸乙酯萃取，合并有机相，用水洗涤，干燥纯化得到标题化合物120mg。ESI-MS(m/z):254.1[M+H-112]+。
步骤五：2-((5,5-二氟哌啶-3-基)甲氧基)乙酸(16-6)的合成
在室温下，将化合物(16-5)(60mg，0.16mmol)加入到二氯甲烷(3mL)中，将反应物降温至0℃，加入三氟乙酸(1.0mL)，加入完毕后，在室温下反应1h，LC-MS监测原料未反应完全，继续反应过夜直至原料反应完全，减压蒸除溶剂，得到标题化合物的三氟乙酸盐60mg。ESI-MS(m/z):210.1[M+H]+。
步骤六：2-((1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-5,5-二氟哌啶-3-基)甲氧基)乙酸(10-136)的合成
采用与实施例一步骤三所描述类似的方法(用化合物(16-5)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物19mg。
其结构表征如下：
1H NMR(400MHz,氯仿-d)δ7.93(dd,J＝9.67,3.06Hz,1H),7.54(s,1H),7.46-7.31(m,1H),7.13(ddd,J＝8.55,2.63,1.35Hz,1H),6.96(d,J＝8.50Hz,1H),6.24(d,J＝3.93Hz,1H),4.28(d,J＝16.74Hz,1H),4.14(d,J＝16.81Hz,1H),3.94(t,J＝16.64Hz,2H),3.80(t,J＝8.68Hz,1H),3.65(t,J＝8.64Hz,1H),3.56-3.43(m,1H),2.94(d,J＝53.06Hz,4H),2.65(s,1H),2.45(s,1H),2.13(dd,J＝26.84,13.90Hz,1H),1.92(d,J＝9.36Hz,1H),1.13(td,J＝7.10,3.76Hz,3H).ESI-MS(m/z):587.2[M+H]+。
实施例十七 (E)-3-(1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-5,5-二氟哌啶-3-基)丙烯酸(10-116)的合成
Figure PCTCN2017110123-appb-000049
步骤一：3,3-二氟-5-甲酰基哌啶-1-羧酸叔丁酯(17-2)的合成
在室温下，将3,3-二氟-5-(羟甲基)哌啶-1-羧酸叔丁酯(17-1)(50mg，0.2mmol)溶于二氯甲烷(2.0mL)中，待完全溶解后，将反应物降温至0℃，在搅拌下加入戴斯-马丁试剂(102mg,0.24mmol)，加入完毕后在室温下反应3h。反应液中有大量白色固体析出，过滤，将滤液在减压下蒸除溶剂后得到标题化合物50mg。
步骤二：(E)-5-(3-乙氧基-3-氧代丙-1-烯-1-基)-3,3-二氟哌啶-1-羧酸叔丁酯(17-3)的合成
在室温下，将化合物(17-2)(50mg，0.2mmol)溶于二氯甲烷(2.0mL)中，待溶解完全后，在搅拌下加入乙氧甲酰基亚甲基三苯基膦(70mg,0.2mmol)，加入完毕后在室温下反应过夜。将反应液纯化得到标题化合物30mg。ESI-MS(m/z):264.1[M+H-56]+。
步骤三：(E)-3-[1-(叔丁氧羰基)-5,5-二氟哌啶-3-基)丙烯酸(17-4)的合成
在室温下，将化合物(17-3)(30mg，0.1mmol)溶于四氢呋喃(4.0mL)和水(2mL)中，待溶解完全后，在搅拌下加入氢氧化锂(19mg,0.47mmol)，加入完毕后在室温下反应4h。将反应液加水稀释， 用1N盐酸调节pH＝3-4，用乙酸乙酯萃取，合并有机相，用水洗涤，干燥后得到标题化合物30mg。
步骤四：(E)-3-(5,5-二氟哌啶-3-基)丙烯酸三氟乙酸盐(17-5)的合成
在室温下，将化合物(17-4)(30mg，0.1mmol)加入到二氯甲烷(1.5mL)中，降温至0℃，加入三氟乙酸(0.5mL)，加入完毕后，在室温下反应1.5h。将反应液减压蒸除溶剂后得到标题化合物30mg。ESI-MS(m/z):192.1[M+H]+。
步骤五：(E)-3-(1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-5,5-二氟哌啶-3-基)丙烯酸(10-116)的合成
采用与实施例一步骤三所描述类似的方法(用化合物(17-5)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物10mg。
其结构表征如下：
1H NMR(400MHz,氯仿-d)δ7.90(s,1H),7.36(s,1H),7.14(dt,J＝8.53,2.80Hz,1H),7.02-6.86(m,2H),6.25(d,J＝8.50Hz,1H),5.90(dd,J＝23.97,15.72Hz,1H),4.20(d,J＝15.93Hz,1H),4.04(q,J＝5.70,5.05Hz,3H),3.11(d,J＝62.19Hz,3H),2.71(s,2H),2.36(s,2H),1.14(td,J＝7.06,5.31Hz,3H).ESI-MS(m/z):569.2[M+H]+。
实施例十八 2-((1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-4,4-二氟四氢吡咯-2-基)甲氧基)乙酸(10-56)的合成
Figure PCTCN2017110123-appb-000050
步骤一：2-((2-乙氧基-2-氧代乙氧基)甲基)-4,4-二氟四氢吡咯-1-甲酸叔丁酯(18-2)的合成
将4,4-二氟-2-(羟甲基)四氢吡咯-1-甲酸叔丁酯(18-1)(80mg，0.34mmol)溶于四氢呋喃(5mL)中，在冰浴降温下，加入氢化钠(27mg 60％in oil，0.68mmol)，升至室温下反应2h。在冰浴降温下，加入溴乙酸乙酯(85mg，0.51mmol)，在室温下反应4h。加入饱和氯化铵(3mL)，加入二氯甲烷(15mL)稀释，用饱和食盐水洗涤，无水硫酸钠干燥。后处理得到标题化合物100mg，将其在未经纯化下直接用于下一步反应。
步骤二：2-((1-(叔丁氧羰基)-4,4-二氟四氢吡咯-2-基)甲氧基)乙酸(18-3)的合成
将化合物(18-2)(100mg，0.31mmol)溶于四氢呋喃与水的混合溶液(v:v＝1:1，3mL)中，加入一水合氢氧化锂(45mg，1.86mmol)，在室温下反应3.5h。用柠檬酸水溶液调节pH至5，加入二氯甲烷(15mL)，用饱和食盐水洗涤，无水硫酸钠干燥后处理得到标题化合物80mg。
步骤三：2-((4,4-二氟四氢吡咯-2-基)甲氧基)乙酸(18-4)的合成
在室温下，将化合物(18-3)(80mg，0.27mmol)溶于二氯甲烷(1mL)中，加入三氟乙酸(0.3mL)，在室温下反应1.5h。减压蒸除溶剂，得到标题化合物的三氟乙酸盐60mg。
步骤四：2-((1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-4,4-二氟四氢吡咯-2-基)甲氧基)乙酸(10-56)的合成
采用与实施例一步骤三所描述类似的方法(用化合物(18-4)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物25mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ12.52(s,1H),9.54(s,1H),7.96(dd,J＝27.4,3.2Hz,2H),7.51-7.27(m,2H),7.19(td,J＝8.5,2.8Hz,1H),6.01(d,J＝12.5Hz,1H),4.21(d,J＝2.7Hz,1H),4.01-3.84(m,3H),3.66-3.44(m,3H),3.34-3.26(m,4H),3.14-2.96(m,1H),2.33-2.07(m,1H),1.08-0.98(m,3H).ESI-MS(m/z):573.2[M+H]+。
实施例十九 2-((4-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)甲氧基)乙酸(10-160)的合成
Figure PCTCN2017110123-appb-000051
步骤一：2-(羟甲基)吗啉-4-羧酸叔丁酯(19-2)的合成
向50mL三口瓶中加入吗啉-2-基甲醇盐酸盐(19-1)(500mg，3.3mmol)、三乙胺(0.82g，8mmol)和二氯甲烷(10mL)，在氮气保护下搅拌，降温至0℃，再加入二碳酸二叔丁酯(1.1g，5mmol)，加入完毕后，在室温下反应3小时。将反应液缓慢倒入水中，用二氯甲烷萃取(30mL×3)，收集有机相，用饱和食盐水洗涤，无水硫酸钠干燥。过滤移除无水硫酸钠，减压蒸除溶剂，纯化得到目标化合物0.6g。ESI-MS(m/z):162.0[M+H]+。
步骤二至步骤四
采用与实施例十五步骤一至步骤三相似的操作，由化合物(19-2)制备得到目标化合物70mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ12.60(s,1H),9.66(d,J＝2.7Hz,1H),8.07-7.90(m,2H),7.49-7.35(m,2H),7.18(td,J＝8.5,2.5Hz,1H),6.05(s,1H),4.04(s,1H),4.01-3.80(m,6H),3.68(s,1H),3.63-3.43(m,3H),2.78(ddd,J＝56.5,46.6,11.4Hz,2H),2.41-2.25(m,1H),2.15(dt,J＝31.9,10.6Hz,1H),1.05(t,J＝7.0Hz,3H).ESI-MS(m/z):553.1[M+H]+。
实施例二十 N-((4-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)甲基)-N-甲基甘氨酸(10-168)的合成
Figure PCTCN2017110123-appb-000052
步骤一：2-(((2-甲氧基-2-氧代乙基)(甲基)氨基)甲基)吗啉-4-甲酸叔丁酯(20-2)的合成
将2-甲酰基吗啉-4-甲酸叔丁酯(20-1)(117mg，0.5mmol)和肌氨酸甲酯盐酸盐(84mg，0.6mmol)溶于甲醇中(3mL)，在冰浴降温下，加入冰乙酸(0.2mL)，然后分批加入氰基硼氢化钠(76mg，1.2mmol)，升至室温反应2h。加入乙酸乙酯(20mL)，搅拌10min，过滤移除不溶物，将滤液浓缩，得到标题化合物98mg。ESI-MS(m/z):303.2[M+H]+。
步骤二：N-((4-(叔丁氧羰基)吗啉-2-基)甲基)-N-甲基甘氨酸(20-3)的合成
将化合物(20-2)(98mg，0.3mmol)溶于甲醇和水(v:v＝1:1，4mL)的混合溶剂中，加入氢氧化锂一水合物(84mg，2mmol)，搅拌过夜。用1N盐酸溶液调节pH＝2，减压蒸除溶剂，得到标题化合物80mg，将其在未经纯化下用于下一步反应。ESI-MS(m/z):289.2[M+H]+。
步骤三：N-甲基-N-((吗啉-2-基甲基)甘氨酸(20-4)的合成
在室温下，将化合物(20-3)(80mg，0.28mmol)溶于二氯甲烷(3mL)中，加入三氟乙酸(1mL)，在室温下反应3h。减压蒸除溶剂，得到标题化合物的三氟乙酸盐94mg。ESI-MS(m/z):189.2[M+H]+。
步骤四：N-((4-(((R)-6-(2-氯-4-氟苯基)-5-乙氧羰基-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)甲基)-N-甲基甘氨酸(10-168)的合成
采用与实施例一步骤三所描述类似的方法(用化合物(20-4)代替3,3-二氟哌啶-4-醇盐酸盐)制备得到标题化合物9mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ9.66(d,1H),8.03-8.01(m,1H),7.95-7.94(m,1H),7.44-7.39(m, 2H),7.22-7.17(m,1H),6.05(d,1H),3.99-3.82(m,5H),3.70-3.68(m,1H),3.63-3.54(m,1H),3.25(d,1H),3.16(d,1H),2.92-2.56(m,4H),2.34(d,3H),2.35-2.24(m,1H),2.14-1.98(m,1H),1.04(t,J＝7.1Hz,3H).ESI-MS(m/z):566.1[M+H]+。
实施例二十一 2-((1-(((S)-5-(乙氧羰基)-6-(4-氟苯基)-6-甲基-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-3,3-二氟哌啶-4-基)氧基)乙酸(10-224)的合成
Figure PCTCN2017110123-appb-000053
步骤一：(E)-2-乙酰基-3-(4-氟苯基)丁-2-烯酸乙酯(21-1)的合成
在室温下，将乙酰乙酸乙酯(3.12g，24.0mmol)、4-氟苯乙炔(2.88g，24.0mmol)和三氟甲磺酸铟(216mg，0.384mmol)加入到邻二甲苯(15mL)中，加热至120℃，保温反应2小时，LC-MS检测反应完成。将反应物降至室温，减压蒸除溶剂得粗品6.0g。ESI-MS(m/z):251.1[M+H]+。
步骤二：4-(4-氟苯基)-4,6-二甲基-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸乙酯的合成
将噻唑-2-甲脒盐酸盐(3.03g，18.5mmol)、碳酸氢钠(3.15g，37.5mmol)加入到N-甲基吡咯烷酮(40mL)中，升温至120℃，向反应液中逐滴加入化合物(21-1)(3.12g，12.5mmol)，保温反应1小时，LC-MS检测反应完成。将反应物降至室温，加入乙酸乙酯(60mL)，用水和饱和食盐水洗涤，无水硫酸钠干燥。过滤移除无水硫酸钠，减压蒸除溶剂得粗品。将粗品通过快速硅胶柱色谱法(石油醚:乙酸乙酯＝10:1)纯化得标题化合物，其为黄色固体(2.11g)。取上述产物350mg，将其通过手性制备柱色谱法分离，分离条件如下：分离柱CHIRALPAK IC 0.46cm I.D.×15cm L，流动相：己烷/IPA/DEA＝90/10/0.1(V/V)，流速1.0ml/min，波长UV 254nm，温度35℃。
分离得到(S)-4-(4-氟苯基)-4,6-二甲基-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸乙酯(21-2)172mg，ee％＝99.3％，Rt＝3.555min。ESI-MS(m/z):360.1[M+H]+；和
(R)-4-(4-氟苯基)-4,6-二甲基-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸乙酯(21-2’)171mg，ee％＝98.1％，Rt＝4.873min。ESI-MS(m/z):360.1[M+H]+。
步骤三至步骤四：2-((1-(((S)-5-(乙氧羰基)-6-(4-氟苯基)-6-甲基-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-3,3-二氟哌啶-4-基)氧基)乙酸(10-224)
采用与实施例一步骤二和步骤三中所描述的类似操作，通过化合物(21-2)在溴化反应后与2-((3,3-二氟哌啶-4-基)氧基)乙酸(实施例五中化合物5-5)反应得到目标化合物15mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ9.35(s,1H),8.01(d,J＝3.2Hz,1H),7.92(d,J＝3.2Hz,1H),7.41(dd,J＝8.8,5.5Hz,2H),7.11(t,J＝8.8Hz,2H),4.18-4.06(m,2H),3.89-3.53(m,6H),3.04-2.60(m,4H),2.44(s,1H),1.97(s,1H),1.80(s,3H),0.92(td,J＝7.1,1.4Hz,3H).ESI-MS(m/z):553.2[M+H]+。
实施例二十二 2-((1-(((R)-5-(乙氧羰基)-6-(4-氟苯基)-6-甲基-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-3,3-二氟哌啶-4-基)氧基)乙酸(10-225)的合成
Figure PCTCN2017110123-appb-000054
采用与实施例二十一步骤三至步骤四中所描述的类似操作，由实施例二十一中化合物(21-2’)制备得到目标化合物15mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ9.35(s,1H),8.01(d,J＝3.2Hz,1H),7.92(d,J＝3.2Hz,1H),7.41 (dd,J＝8.8,5.5Hz,2H),7.11(t,J＝8.8Hz,2H),4.21-4.11(m,2H),3.94-3.54(m,6H),3.14-2.70(m,4H),2.45(s,1H),1.97(s,1H),1.80(s,4H),0.92(td,J＝7.1,1.4Hz,3H).ESI-MS(m/z):553.2[M+H]+。
实施例二十三 2-((1-(((S)-5-(乙氧羰基)-6-(4-氟苯基)-6-甲基-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-4,4-二氟四氢吡咯-3-基)氧基)乙酸(10-211)的合成
Figure PCTCN2017110123-appb-000055
采用与实施例一步骤二和步骤三中所描述的类似操作，通过实施例二十一中化合物(21-2)在溴化反应后与2-((4,4-二氟四氢吡咯-3-基)氧基)乙酸(实施例十二中化合物(12-4))(29mg，0.16mmol)反应得到标题化合物2mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ7.85(s,1H),7.50-7.35(m,3H),6.99(t,J＝8.6Hz,2H),4.22-4.12(m,1H),3.94-3.84(m,3H),3.30-3.17(m,3H),3.08-2.93(m,2H),2.27-1.99(m,1H),1.91(s,3H),1.68-1.48(m,1H),0.97(t,J＝7.0Hz,3H).ESI-MS(m/z):539.2[M+H]+。
实施例二十四 2-((1-(((R)-5-(乙氧羰基)-6-(4-氟苯基)-6-甲基-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-4,4-二氟四氢吡咯-3-基)氧基)乙酸(10-212)的合成
Figure PCTCN2017110123-appb-000056
采用与实施例一步骤二和步骤三中所描述的类似操作，通过实施例二十一中化合物(21-2’)在溴化反应后与2-((4,4-二氟四氢吡咯-3-基)氧基)乙酸(实施例十二中化合物(12-4))(29mg，0.16mmol)反应得到标题化合物4mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ7.85(s,1H),7.51-7.35(m,3H),6.99(t,J＝8.6Hz,2H),4.23-4.09(m,1H),3.94-3.82(m,3H),3.33-3.17(m,3H),3.13-2.90(m,2H),2.27-1.98(m,1H),1.91(s,3H),1.68-1.45(m,1H),0.97(t,J＝7.0Hz,3H).ESI-MS(m/z):539.2[M+H]+。
实施例二十五 (E)-3-((R)-4-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-226)的合成
Figure PCTCN2017110123-appb-000057
步骤一：(S)-2-甲酰基吗啉-4-甲酸叔丁酯(25-2)的合成
在冰浴下，将(S)-2-(羟甲基)吗啉-4-甲酸叔丁酯(25-1)(1.0g，4.6mmol)在二氯甲烷(10mL)中溶解，分批加入戴斯-马丁试剂(2.9g，6.9mmol)，在15℃下搅拌4h。反应液中有大量固体析出，过滤，弃去滤饼，向滤液加入饱和硫代硫酸钠溶液，搅拌30min，静置分层，将有机层用饱和碳酸氢钠溶液和食盐水洗涤，无水硫酸钠干燥，减压浓缩980mg。将粗品在未经纯化下直接用于下一步反应。
步骤二：(R,E)-2-(3-(叔丁氧基)-3-氧代丙-1-烯-1-基)吗啉-4-甲酸叔丁酯(25-3)的合成
在室温下，将NaH(60％，182mg，4.55mmol)在干燥四氢呋喃(7mL)中分散，搅拌5min后，缓慢滴加二乙基膦酰乙酸叔丁酯(1.21g，4.78mmol)的干燥四氢呋喃(3mL)溶液，在室温下搅拌2h。然后将上述反应液加入化合物(25-2)(980mg，4.55mmol)的干燥四氢呋喃(5mL)溶液中，滴加完毕后， 在室温下搅拌过夜。减压蒸除溶剂，将残渣通过柱色谱法纯化，得无色油状物(520mg)，在室温下静置会析出固体，HPLC检测表明，Z/E<1/35，ee(对映体过量)值为94％。ESI-MS(m/z):214.1[M+1-100]+。
步骤三：(R,E)-3-(吗啉-2-基)丙烯酸三氟乙酸盐(25-4)的合成
在室温下，将化合物(25-3)(520mg，1.66mmol)溶于二氯甲烷(10mL)中，加入三氟乙酸(5mL)，在室温下反应3h。过滤移除不溶物，将滤液浓缩得标题化合物粗品423mg，将其在未经纯化下直接用于下一步反应。ESI-MS(m/z):158.1[M+H]+。
步骤四：(E)-3-((R)-4-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-226)的合成
在室温下，将(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸乙酯(400mg，0.87mmol)、化合物(25-4)(423mg，1.66mmol)和N,N-二异丙基乙基胺(451mg，3.49mmol)加入到二氯甲烷(10mL)中，在室温下反应过夜，将反应液浓缩得粗品。将所述粗品通过制备液相色谱法纯化得到标题化合物200mg。Z/E<1/35，ee值为95.5％。
其结构表征如下：
1H NMR(400MHz，DMSO-d6)δ12.50(s,1H),9.63(s,1H),8.02(d,J＝3.12Hz,1H),7.95(d,J＝3.16Hz,1H),7.44-7.40(m,2H),7.18(td,J＝8.48,2.64Hz,1H),6.73(dd,J＝15.80,4.08Hz,1H),6.05(s,1H),5.93(dd,J＝15.80,1.76Hz,1H),4.24-4.21(m,1H),3.98-3.92(m,5H),3.68(td,J＝10.92,1.72Hz,1H),2.95(d,J＝11.12Hz,1H),2.83(d,J＝10.92Hz,1H),2.40(td,J＝11.16,2.68Hz,1H),2.07(t,J＝10.64Hz,1H),1.04(t,J＝7.08Hz,3H).ESI-MS(m/z):535.2[M+H]+。
实施例二十六 (E)-3-((R)-1-(((R)-6-(2-氯-4-氟苯基)-5-(乙氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-5,5-二氟哌啶-3-基)丙烯酸(10-230)的合成
Figure PCTCN2017110123-appb-000058
将实施例十七的化合物(608mg)通过手性色谱法分离，分离条件如下：分离柱CHIRALPAK IG0.46cm I.D.×15cm L，流动相：己烷/EtOH/HOAc＝75/25/0.1(V/V/V)，流速1.0ml/min，波长UV 254nm，温度35℃。分离得到标题化合物277mg，ee％＝99.5％，Rt＝15.656min，其结构表征数据如下：
1H NMR(400MHz,DMSO-d6)δ12.32(brs,1H),9.55(s,1H),8.00(d,J＝3.12Hz,1H),7.94(d,J＝3.12Hz,1H),7.45-7.41(m,2H),7.19(td,J＝8.48Hz,2.68Hz,1H),6.76(dd,J＝15.85Hz,6.64Hz,1H),6.06(s,1H),5.82(dd,J＝15.85Hz,1.16Hz,1H),4.08(d,J＝16.53Hz,1H),4.01(d,J＝16.53Hz,1H),3.98-3.92(m,2H),3.26-3.13(m,1H),2.90(brd,J＝11.08Hz,1H),2.79-2.69(m,2H),2.29(t,J＝10.80Hz,2H),1.94-1.77(m,1H),1.04(t,J＝7.12Hz,3H).ESI-MS(m/z):569.2[M+H]+。
实施例二十七 (E)-3-((R)-4-(((R)-6-(2-氯-4-氟苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-227)的合成
Figure PCTCN2017110123-appb-000059
在室温下，将(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯(400mg，0.90mmol)和实施例二十五中的化合物(25-4)(488mg，1.80mmol)在二氯甲烷(10mL)中溶解，然后加入N,N-二异丙基乙胺(696mg，5.40mmol)，在室温下反应过夜。将反应液浓缩得粗品。粗品经制备液相色谱法纯化，得标题化合物205mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ12.44(s,1H),9.68(s,1H),7.98(dd,J＝27.6,3.1Hz,2H),7.48-7.36(m,2H),7.18(td,J＝8.5,2.6Hz,1H),6.73(dd,J＝15.8,4.1Hz,1H),6.04(s,1H),5.93(dd,J＝15.8,1.6Hz,1H),4.23(d,J＝9.3Hz,1H),4.01-3.90(m,3H),3.68(t,J＝10.2Hz,1H),3.52(s,3H),2.94 (d,J＝11.0Hz,1H),2.82(d,J＝11.1Hz,1H),2.41(dd,J＝11.0,8.6Hz,1H),2.08(t,J＝10.7Hz,1H)。ESI-MS(m/z):521.1[M+H]+。
实施例二十八 (E)-3-((R)-4-(((R)-6-(2-溴-4-氟苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-229)的合成
Figure PCTCN2017110123-appb-000060
在室温下，将(R)-6-(溴甲基)-4-(2-溴-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯(400mg，0.82mmol)和实施例二十五中的化合物(25-4)(443mg，1.63mmol)溶于二氯甲烷中(10mL)，然后加入N,N-二异丙基乙胺(635mg，4.92mmol)，在室温下反应过夜。将反应液浓缩得粗品。粗品经制备液相色谱法纯化，得标题化合物200mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ12.49(s,1H),9.68(s,1H),8.01(d,J＝3.1Hz,1H),7.95(d,J＝3.1Hz,1H),7.57(dd,J＝8.6,2.6Hz,1H),7.38(dd,J＝8.7,6.2Hz,1H),7.22(td,J＝8.5,2.6Hz,1H),6.73(dd,J＝15.8,4.1Hz,1H),6.02(s,1H),5.93(dd,J＝15.8,1.7Hz,1H),4.27-4.19(m,1H),4.01-3.89(m,3H),3.68(t,J＝10.2Hz,1H),3.52(s,3H),2.94(d,J＝11.0Hz,1H),2.83(d,J＝11.3Hz,1H),2.41(dd,J＝11.1,8.4Hz,1H),2.08(t,J＝10.6Hz,1H)。ESI-MS(m/z):567.1[M+H]+。
实施例二十九 (E)-3-((R)-4-(((R)-6-(2-溴-3-氟苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-236)的合成
Figure PCTCN2017110123-appb-000061
采用与实施例二十七中所描述的类似操作，用(R)-4-(2-溴-3-氟苯基)-6-(溴甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯代替(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯，得标题化合物25mg。
其结构表征如下：
1H NMR(400MHz,DMSO-d6)δ9.70(s,1H),8.02(d,J＝3.2Hz,1H),7.95(d,J＝3.2Hz,1H),7.39(m,J＝7.9,5.5Hz,1H),7.31-7.17(m,2H),6.74(dd,J＝15.8,4.1Hz,1H),6.09(s,1H),5.93(d,J＝15.9Hz,1H),4.27-4.19(m,1H),4.02(m,J＝16.8,9.4Hz,1H),3.95(d,J＝7.7Hz,2H),3.68(td,J＝11.4,2.5Hz,1H),3.51(s,3H),2.99-2.92(m,1H),2.83(d,J＝11.4Hz,1H),2.42(td,J＝11.4,3.3Hz,1H),2.09(t,J＝10.6Hz,1H),1.41(s,1H).ESI-MS(m/z):567.0[M+H]+。
实施例三十 (E)-3-((R)-4-(((R)-6-(2-氯-3,4-二氟苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-237)的合成
Figure PCTCN2017110123-appb-000062
采用与实施例二十七中所描述的类似操作，用(R)-6-(溴甲基)-4-(2-氯-3,4-二氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯代替(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧 酸甲酯，得标题化合物25mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ7.79(d,J＝3.1Hz,1H),7.45(d,J＝3.1Hz,1H),7.12-6.94(m,2H),6.78(dd,J＝15.7,4.0Hz,1H),6.11(s,1H),6.06(d,J＝15.6Hz,1H),4.53(s,1H),4.31(d,J＝15.8Hz,1H),4.13(d,J＝15.3Hz,1H),4.01(s,2H),3.54(s,3H),3.27(d,J＝70.2Hz,2H),2.76(s,1H).ESI-MS(m/z):539.2[M+H]+。
实施例三十一 (E)-3-((R)-4-(((R)-6-(2-溴-3,4-二氟苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-238)的合成
Figure PCTCN2017110123-appb-000063
采用与实施例二十七中所描述的类似操作，用(R)-4-(2-溴-3,4-二氟苯基)-6-(溴甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯代替(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯，得标题化合物70mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ9.66(s,1H),7.85(d,J＝3.1Hz,1H),7.47(d,J＝3.1Hz,1H),7.12–7.01(m,2H),6.92-6.87(m,1H),6.19(s,1H),6.13-6.09(m,1H),4.50-4.35(m,1H),4.20-4.05(m,3H),4.00-3.80(m,2H),3.62(s,3H),3.05-2.75(m,2H),2.71-2.55(m,1H),2.30-2.15(m,1H).ESI-MS(m/z):584.0[M+H]+。
实施例三十二 (E)-3-((R)-4-(((S)-6-(3,4-二氟-2-甲基苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-239)的合成
Figure PCTCN2017110123-appb-000064
采用与实施例二十七中所描述的类似操作，以(S)-6-(溴甲基)-4-(3,4-二氟-2-甲基苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯代替(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯，得标题化合物22mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ7.93(dd,J＝3.2,1.5Hz,1H),7.72(t,J＝2.7Hz,1H),7.02(m,J＝7.2,3.3Hz,2H),6.82(m,J＝15.8,4.2Hz,1H),6.16-5.96(m,1H),5.91(s,1H),4.37(d,J＝9.8Hz,1H),4.20-3.75(m,4H),3.60(s,3H),3.14-2.64(m,2H),2.55(d,J＝2.4Hz,3H),2.60-2.41(m,J＝11.7,5.8Hz,1H),2.29-2.20(m,J＝10.6Hz,1H).ESI-MS(m/z):519.2[M+H]+。
实施例三十三 (E)-3-((R)-4-(((R)-6-(2-氯-3-氟苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-240)的合成
Figure PCTCN2017110123-appb-000065
采用与实施例二十七中所描述的类似操作，用(R)-6-(溴甲基)-4-(2-氯-3-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯代替(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯，得标题化合物36mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ7.84(d,J＝3.1Hz,1H),7.48(s,1H),7.22-7.16(m,J＝16.5Hz,2H),7.06(s,1H),6.89(d,J＝15.7Hz,1H),6.25(s,1H),6.11(d,J＝15.7Hz,1H),4.50(s,1H),4.22(d,J＝15.6Hz,1H),4.03(dd,J＝31.8,9.3Hz,3H),3.60(s,3H),3.04(s,2H),2.70(s,1H),2.35(s,1H).ESI-MS(m/z):521.2[M+H]+。
实施例三十四 (E)-3-((R)-4-(((R)-6-(2,4-二氯苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-241)的合成
Figure PCTCN2017110123-appb-000066
采用与实施例二十七中所描述的类似操作，用(R)-6-(溴甲基)-4-(2,4-二氯苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯代替(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯，得标题化合物400mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ9.64(s,1H),7.86(d,J＝3.1Hz,1H),7.47(d,J＝3.1Hz,1H),7.42(d,J＝2.1Hz,1H),7.28(d,J＝6.5Hz,1H),7.19(dd,J＝8.4,2.1Hz,1H),6.93(dd,J＝15.7,4.1Hz,1H),6.22(s,1H),6.12(dd,J＝15.7,1.8Hz,1H),4.40(d,J＝9.9Hz,1H),4.12-4.01(m,2H),3.96-3.85(m,2H),3.62(s,3H),2.89-2.75(m,2H),2.60(td,J＝10.9,2.8Hz,1H),2.23(t,J＝10.7Hz,1H).ESI-MS(m/z):537.2[M+H]+。
实施例三十五 (E)-3-((R)-4-(((S)-6-(4-氟-2-甲基苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-242)的合成
Figure PCTCN2017110123-appb-000067
采用与实施例二十七中所描述的类似操作，以(S)-6-(溴甲基)-4-(4-氟-2-甲基苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯代替(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯，得标题化合物80mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ9.59(s,1H),7.82(d,J＝3.1Hz,1H),7.44(s,1H),7.13(t,J＝7.1Hz,1H),6.90(t,J＝4.1Hz,2H),6.80(t,J＝3.6Hz,1H),6.12(dd,J＝15.7,1.2Hz,1H),5.96(s,1H),4.40(s,1H),4.05(d,J＝11.3Hz,2H),3.93(d,J＝16.0Hz,2H),3.61(s,3H),2.81(s,2H),2.63(s,3H),2.57(s,1H),2.20(s,1H).ESI-MS(m/z):501.2[M+H]+。
实施例三十六 (E)-3-((R)-4-(((R)-6-(2-氯-4-氟苯基)-5-(甲氧羰基)-2-(4-甲基噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-243)的合成
Figure PCTCN2017110123-appb-000068
采用与实施例二十七中所描述的类似操作，用(R)-6-(溴甲基)-4-(2-甲基-4-氟苯基)-2-(4-甲基噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯代替(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯，得标题化合物400mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ9.65(s,1H),7.29-7.25(m,1H),7.13(dd,J＝8.6,2.6Hz,1H),7.01(s,1H),6.93-6.88(m,2H),6.19(s,1H),6.10(d,J＝15.7Hz,1H),4.38(s,1H),4.06(d,J＝12.5Hz,2H),3.90-3.86(m,2H),3.60(s,3H),2.81(s,2H),2.60(s,1H),2.45(s,3H),2.20(s,1H).ESI-MS(m/z):535.1[M+H]+。
实施例三十七 (E)-3-((R)-4-(((R)-6-(2-氯-4-氟苯基)-5-(甲氧羰基)-2-(4-(三氟甲基)噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-244)的合成
Figure PCTCN2017110123-appb-000069
采用与实施例二十七中所描述的类似操作，用(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(4-(三氟甲基)噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯代替(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯，得标题化合物80mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ9.70(s,1H),7.86(s,1H),7.27(s,1H),7.17(dd,J＝8.6,2.6Hz,1H),6.96(ddd,J＝11.3,10.6,3.4Hz,2H),6.24(s,1H),6.18(d,J＝15.4Hz,1H),4.38(d,J＝10.4Hz,1H),4.19-4.10(m,1H),4.02(d,J＝11.1Hz,1H),3.83(dd,J＝13.3,9.8Hz,2H),3.63(s,3H),2.95(d,J＝10.8Hz,1H),2.66(d,J＝11.6Hz,1H),2.51-2.33(m,2H).ESI-MS(m/z):589.1[M+H]+。
实施例三十八 (E)-3-((R)-4-(((S)-6-(2-(二氟甲基)-4-氟苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丙烯酸(10-245)的合成
Figure PCTCN2017110123-appb-000070
采用与实施例二十七中所描述的类似操作，以(S)-6-(溴甲基)-4-(2-(二氟甲基)-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯代替(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯，得标题化合物70mg。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ9.68(s,1H),7.83(d,J＝3.1Hz,1H),7.69-7.55(m,1H),7.46(d,J＝2.9Hz,1H),7.40(d,J＝7.5Hz,1H),7.20(s,1H),7.08(t,J＝7.5Hz,1H),6.96(dd,J＝15.7,3.9Hz,1H),6.15(dd,J＝15.7,1.5Hz,1H),6.08(s,1H),4.40(d,J＝5.0Hz,1H),4.16(d,J＝17.6Hz,1H),4.00(d,J＝11.4Hz,1H),3.93-3.80(m,2H),3.62(s,3H),2.93(d,J＝9.2Hz,1H),2.65(d,J＝9.7Hz,1H),2.46(t, J＝11.1Hz,1H),2.36(d,J＝9.6Hz,1H).ESI-MS(m/z):537.2[M+H]+。
实施例三十九 (E)-3-((R)-4-(((R)-6-(2-氯-4-氟苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丁-2-烯酸(10-246)的合成
Figure PCTCN2017110123-appb-000071
步骤一：(S)-4-(叔丁氧羰基)吗啉-2-甲酸(39-2)的合成
室温下，将(S)-2-(羟甲基)吗啉-4-甲酸叔丁酯(39-1)(5g，23.01mmol)溶于丙酮(250mL)中，加入饱和碳酸氢钠溶液(75mL)，冰浴降温至0℃，加入溴化钠(474mg，4.6mmol)和四甲基哌啶氮氧化物(65mg，0.46mmol)，再缓慢加入三氯异氰尿酸(10.7g，46.03mmol)，室温反应过夜。加入异丙醇(15mL)搅拌30min，抽滤，弃去滤饼，将滤液浓缩，加入饱和碳酸钠溶液(75mL)，乙酸乙酯(50mL×2)萃取，弃去有机相，将水相用6N盐酸中和，乙酸乙酯(50mL×3)萃取，合并有机相，无水硫酸钠干燥，滤除干燥剂，滤液浓缩得标题化合物3g，不经纯化直接用于下一步反应。ESI-MS(m/z):176.1[M+1-56]+。
步骤二：(S)-2-(甲氧(甲基)氨基甲酰基)吗啉-4-甲酸叔丁酯(39-3)的合成
室温下，将化合物(39-2)(2g，8.65mmol)溶于二氯甲烷(20mL)中，加入2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(3.95g，10.38mmol)，室温反应30min。加入N,N-二异丙基乙基胺(2.57g，19.89mmol)和N,O-二甲基羟胺盐酸盐(1.01g，10.38mmol)，反应过夜。加入水(20mL)，二氯甲烷(20mL×3)萃取，合并有机相，将有机相依次用0.05N盐酸(20mL)、饱和碳酸氢钠溶液、水和饱和氯化钠溶液洗，无水硫酸钠干燥，滤除干燥剂，将滤液浓缩得标题化合物(2g)，不经纯化直接用于下一步反应。ESI-MS(m/z):219.1[M+1-56]+。
步骤三：(S)-2-乙酰基吗啉-4-甲酸叔丁酯(39-4)的合成
室温下，将化合物(39-3)(2g，7.29mmol)溶于无水四氢呋喃(40mL)中，氮气保护下降温至-20℃，滴加甲基溴化镁(3M，7.29mL，21.87mmol)，-20℃反应4h。加入饱和氯化铵(20mL)，乙酸乙酯(20mL×3)萃取，合并有机相，有机相依次用0.05N盐酸(20mL)、饱和碳酸氢钠溶液、水和饱和氯化钠溶液洗涤，无水硫酸钠干燥，滤除干燥剂，将滤液浓缩得标题化合物(1.5g)，不经纯化直接用于下一步反应。ESI-MS(m/z):174.1[M+1-56]+。
步骤四：(R,E)-2-(4-(叔丁氧基)-4-氧代丁-2-烯-2-基)吗啉-4-甲酸叔丁酯(39-5)的合成
采用与实施例二十五中步骤二所描述的类似操作，以化合物(39-4)代替(S)-2-甲酰基吗啉-4-甲酸叔丁酯，得标题化合物(1.1g)。ESI-MS(m/z):172.1[M+1-100-56]+。
步骤五：(R,E)-3-(吗啉-2-基)丁-2-烯酸三氟乙酸盐(39-6)的合成
采用与实施例二十五中步骤三所描述的类似操作，以化合物(39-5)代替(R,E)-2-(3-(叔丁氧基)-3-氧代丙-1-烯-1-基)吗啉-4-甲酸叔丁酯，得标题化合物(491mg)。ESI-MS(m/z):172.1[M+H]+。
步骤六：(E)-3-((R)-4-(((R)-6-(2-氯-4-氟苯基)-5-(甲氧羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)吗啉-2-基)丁-2-烯酸(10-246)的合成
采用与实施例二十七中所描述的类似操作，以化合物(39-6)代替(R,E)-3-(吗啉-2-基)-丙烯酸三氟乙酸盐，得标题化合物(180mg)。
其结构表征如下：
1H NMR(400MHz,CDCl3)δ7.85(d,J＝3.1Hz,1H),7.51(d,J＝3.0Hz,1H),7.33(dd,J＝8.6,2.0Hz,1H),7.14(dd,J＝8.5,2.6Hz,1H),6.95(td,J＝8.3,2.6Hz,1H),6.18(s,1H),6.04(s,1H),4.37-4.22(m,2H),4.10-4.07(m,2H),3.62(s,3H),3.27(s,1H),2.83(s,1H),2.43(s,1H),2.08(s,3H).ESI-MS(m/z):535.1[M+H]+。
其他化合物均可参考上述实施例中类似的方法合成。
在下列药理学实验中，将本申请背景技术中提及的化合物GLS4(对照化合物1)、WO2015144093中实施例9的化合物(对照化合物2)和WO2014037480中实施例5的化合物(对照化合物3)，与本申 请的实施例化合物进行比较，以充分地展示本申请实施例化合物的优势。
●对照化合物1的结构为
Figure PCTCN2017110123-appb-000072
对照化合物2的结构为
Figure PCTCN2017110123-appb-000073
对照化合物3的结构为
Figure PCTCN2017110123-appb-000074
实验例1：生物活性检测
测试本发明的化合物对乙型肝炎病毒(HBV)的抑制作用。在病毒-细胞水平测试本发明的化合物的细胞毒性以及对病毒(HBV)核酸(DNA)复制水平的影响。
试验方法
将对数生长期的HepG2.2.15细胞接种于96孔板中，细胞浓度为40个/μL。于37℃，5％CO2培养箱中培养3天；加入化合物前用新培养基(200μL/孔)更换。各实施例化合物的母液浓度为200μM。以200μM为最高浓度，用DMSO稀释为多个不同浓度，取1μL待测化合物置于相应培养基孔，化合物的最终测试浓度为0.06、0.24、0.98、3.9、15.6、62.5、250、1000nM(用于计算半数有效浓度(EC50))。测试结果如表1-1和表1-2中所示。
表1-1
化合物编号 EC50(nM)
实施例六的10-7 102.3
实施例九的10-34 13.2
实施例十的10-36 98.1
实施例十一的10-40 6.2
实施例十二的10-42 164.5
实施例一的10-88 24.7
实施例四的异构体B 34.7
实施例四的异构体A 35.1
实施例五的10-95 314.4
实施例十六的10-136 110.4
实施例十五的10-162 213.1
实施例十四的10-180 23.5
实施例十三的10-182 61.8
由表1-1可见，所测试的化合物对乙型肝炎病毒(HBV)具有较强的抑制活性。
表1-2
化合物编号 EC50(nM)
对照化合物2 89
实施例二十五的10-226 9.2
实施例二十七的10-227 10.0
实施例二十八的10-229 8.3
实施例二十六的10-230 11.2
实施例三十的10-237 10.9
实施例三十一的10-238 10.2
实施例三十二的10-239 28.9
实施例三十三的10-240 21.1
由表1-2可见，本发明具有单一构型的化合物的抗HBV病毒活性为对照化合物2的约10倍。本发明化合物对乙型肝炎病毒(HBV)具有较强的抑制活性。
本发明的其余化合物具有与上述类似的抑制活性。
实验例2：细胞毒性检测
将待测化合物用DMSO稀释为30mM，以30mM为最高浓度，三倍稀释为多个不同的浓度，取0.2μL各浓度的化合物于384孔板中，每孔加入2000个/50μL的HepG2.2.15细胞，待测化合物最高终浓度为150μM；将1μL DMSO加入相应孔中作为对照。于37℃，5％CO2培养箱中共培养4天。4天后每孔加入50μL CellTiter-Glo，进行读板检测，计算得到致半数细胞毒性浓度(CC50)值。测试结果如表2中所示。
表2
化合物编号 CC50(μM)
实施例六的10-7 129.5
实施例九的10-34 79.6
实施例十的10-36 >150.0
实施例十一的10-40 73.5
实施例十二的10-42 >150.0
实施例一的10-88 61.1
实施例四的异构体B 51.3
实施例四的异构体A 43.9
实施例五的10-95 131.3
实施例十六的10-136 122.3
实施例十五的10-162 >150.0
实施例十四的10-180 >150.0
实施例十三的10-182 >150.0
实施例二十五的10-226 >150.0
实施例二十六的10-230 137.0
实施例二十七的10-227 >150.0
实施例二十八的10-229 >150.0
实施例三十的10-237 >150.0
实施例三十一的10-238 >150.0
实施例三十二的10-239 >150.0
实施例三十三的10-240 >150.0
所测试的化合物细胞毒性较低，安全性较高。本发明的其他化合物具有类似的安全性。
实验例3：对hERG抑制作用的测试
在心肌细胞中，hERG(human Ether-a-go-go Related Gene)编码的钾通道介导延迟整流钾电流(IKr)。IKr抑制是药物导致QT间期延长最重要的机制。在hERG测试中，判定标准为若化合物IC50>10μM，则判定化合物对hERG无抑制作用。
采用PredictorTM hERG Fluorescence Polarization Assay，检测化合物对hERG钾离子通道的作用。试验结果如下表3所示：
表3
化合物编号 IC50值(μM)
对照化合物1 2.85
对照化合物2 1～10
实施例十的10-36 >10
实施例十一的10-40 >10
实施例十二的10-42 >10
实施例一的10-88 >10
实施例四的异构体B >10
实施例的四异构体A >10
实施例二的10-93 >10
实施例五的10-95 >10
实施例二十五的10-226 >10
实施例二十七的10-227 >10
实施例二十六的10-230 >10
由以上数据看出，对照化合物1和对照化合物2都具有不同程度的心脏毒性(对心肌细胞中hERG钾离子通道具有明显抑制作用)，因而具有诱发心律失常的潜在风险；而所测试的本发明中的化合物无hERG钾离子通道抑制作用，无明显心脏毒性，因而具有更高的安全性。本发明的其他化合物具有类似的安全性。
实验例4：大鼠体内的药物代谢动力学(PK)研究
分别通过静脉(iv)和灌胃(po)向雄性SD大鼠给药测试化合物，通过iv和po给药的剂量分别是1mg/kg和2mg/kg，iv给药时溶媒系统为5％DMSO:5％solutol:90％生理盐水，po给药时溶媒系统为0.5％MC。iv给药和po给药后在多个时间点收集血液，用于PK研究。血浆样品和肝组织样品经蛋白沉淀处理后进行LC-MS/MS分析。质谱仪为API 5500，液相色谱仪为Waters ACQUITY I CLASS系统；色谱柱为Agela ASB C18柱(2.1mm×50mm,1.9μm)；流动相A相为水+0.1％甲酸，B相为乙腈；流速为0.4mL/min，柱温为40℃。采用离子源为ESI源正离子模式，扫描方式为多重反应监测(MRM)。试验结果如下表所示：
表4
Figure PCTCN2017110123-appb-000075
由表4数据可知，与对照化合物2相比，本发明的化合物(例如实施例二十五的10-226)在1.00mg/kg的静脉给药剂量下在体内血液中具有更好的暴露量(AUCINF)和更高的血药浓度(Cmax)，因而有更好的药代动力学参数。
表5 血浆中的药代动力学参数
Figure PCTCN2017110123-appb-000076
由表5可以看出，与对照化合物2相比，本发明的化合物(例如实施例二十五的10-226)在2.00mg/kg的灌胃给药剂量下在体内血液中具有更好的暴露量(AUCINF)和更高的血药浓度(Cmax)，因而具有更好的吸收性。
表6 肝中的药代动力学参数
Figure PCTCN2017110123-appb-000077
由表6数据可知，与对照化合物2相比，本发明的化合物(例如实施例二十五的10-226)在2.00mg/kg的灌胃给药剂量下在体内肝脏中具有更好的暴露量(AUCINF)和更高的血药浓度(Cmax)，进一步说明本发明的化合物(例如实施例二十五的10-226)具有更好的吸收性。并且，本发明的化合物(例如实施例二十五的10-226)在2.00mg/kg的灌胃给药剂量下，生物利用度(F)为47.6％，明显高于对照化合物2的生物利用度(F)20.9％。
表7
Figure PCTCN2017110123-appb-000078
由表7可以看出，本发明的化合物(例如实施例二十六的10-230)在体内肝脏中的暴露量是血浆中的约10倍，在体内肝脏中的血药浓度是血浆中的约10倍；而对照化合物2在体内肝脏中的暴露量是血浆中的约1.5倍，在体内肝脏中的血药浓度是血浆中的约0.5倍。这说明本发明的化合物在体内肝脏中具有优异的暴露量(AUCINF)和血药浓度(Cmax)，因而具有肝靶向性。
实验例5：Beagle犬体内的药物代谢动力学(PK)研究
分别通过静脉(iv)和灌胃(po)向雄性Beagle犬给药测试化合物，通过iv和po给药的剂量分别是0.5mg/kg和2.5mg/kg，iv给药时溶媒系统为5％DMSO:5％solutol:90％生理盐水，po给药时溶媒系统为0.5％MC。iv给药和po给药后在多个时间点收集血液，用于PK研究。血浆样品经蛋白沉淀处理后进行LC-MS/MS分析。质谱仪为API 5500，液相色谱仪为Waters ACQUITY I CLASS系统；色谱柱为Agela ASB C18柱(2.1mm×50mm,1.9μm)；流动相A相为水+0.1％甲酸，B相为乙腈；流速为0.4mL/min，柱温为40℃。采用离子源为ESI源正离子模式，扫描方式为多重反应监测(MRM)。试验结果如下表所示：
表8
Figure PCTCN2017110123-appb-000079
由表8数据可知，与对照化合物3相比，本发明的化合物(例如实施例二十七的10-227)在0.50mg/kg的静脉给药剂量下在体内血液中具有更好的暴露量(AUCINF)和更高的血药浓度(Cmax)，因而有更好的药代动力学参数。
表9
Figure PCTCN2017110123-appb-000080
由表9可以看出，本发明的化合物(例如实施例二十七的10-227)在2.50mg/kg的灌胃给药剂量下在血浆中的暴露量是对照化合物3的约14倍，血药浓度是对照化合物3的约6倍。进一步说明本发明的化合物(例如实施例二十七的10-227)具有更好的吸收性。并且，本发明的化合物(例如实施例二十七的10-227)在2.50mg/kg的灌胃给药剂量下，生物利用度(F)为75.9％，明显优于对照化合物3的生物利用度(F)41.7％。
实验例6：食蟹猴体内的药物代谢动力学(PK)研究
分别通过静脉(iv)和灌胃(po)向雄性食蟹猴给药测试化合物，通过iv和po给药的剂量分别是0.5mg/kg和2.5mg/kg，iv给药时溶媒系统为5％DMSO:5％solutol:90％生理盐水，po给药时溶媒系统为0.5％MC。iv给药和po给药后在多个时间点收集血液，用于PK研究。血浆样品经蛋白沉淀处理后进行LC-MS/MS分析。质谱仪为API 5500，液相色谱仪为Waters ACQUITY I CLASS系统；色谱柱为Agela ASB C18柱(2.1mm×50mm,1.9μm)；流动相A相为水+0.1％甲酸，B相为乙腈；流速为0.4mL/min，柱温为40℃。采用离子源为ESI源正离子模式，扫描方式为多重反应监测(MRM)。试验结果如下表所示：
表10
Figure PCTCN2017110123-appb-000081
由表10数据可知，与对照化合物3相比，本发明的化合物(例如实施例二十七的10-227)在食蟹猴PK研究中，在0.50mg/kg的静脉给药剂量下在体内血液中具有更好的暴露量(AUCINF)和更高的血药浓度(Cmax)，因而有更好的药代动力学参数。
表11
Figure PCTCN2017110123-appb-000082
Figure PCTCN2017110123-appb-000083
由表11可以看出，本发明的化合物(例如实施例二十七的10-227)在食蟹猴PK研究中，在2.50mg/kg的灌胃给药剂量下在血浆中的暴露量是对照化合物3的约21倍，血药浓度是对照化合物3的约33倍。进一步说明本发明的化合物(例如实施例二十七的10-227)具有更好的吸收性。并且，本发明的化合物(例如实施例二十七的10-227)在2.50mg/kg的灌胃给药剂量下，生物利用度(F)为37.2％，明显优于对照化合物3的生物利用度(F)6.77％。
实验例7：CYP450酶诱导研究
细胞复苏
从液氮罐中取出HepG2C3A/pCYP3A4-Luc,C8细胞一支，在37℃无菌水浴中复苏细胞，轻轻摇晃直至冰块全部融化。将复苏的细胞转移到15mL无菌离心管中，加入5-10mL 37℃预热的基础细胞培养基。细胞自然沉降2分钟后，离心(1000rpm)8分钟。去除上清液，用10mL预热的细胞培养基重新悬浮细胞。将细胞悬浮液转移至10cm细胞培养皿中，置于37℃5％CO2培养箱中培养。24小时后，用选择细胞培养基替换原细胞培养基。
细胞扩增
待细胞生长至80％～90％培养皿，将细胞消化，并转移到15mL无菌离心管中。离心(1000rpm)8分钟，收集细胞。去除上清液，用3mL预热的完全培养基重新悬浮细胞。将细胞悬浮液以1:3或1:5的比例进行传代培养。
CYP3A4诱导实验
第一天准备细胞板。在384孔白色细胞板中加入5μL 1×Matrigel，600rpm离心1分钟。取出培养皿，去除培养基，用1mL PBS清洗并吸出，再加入2mL 0.25％胰酶，置于培养箱中2-3分钟，待细胞完全消化，加入5mL细胞培养基终止，转移至离心管中。离心(1000rpm)8分钟。去除上清液，重新悬浮细胞并进行细胞计数，将悬浮液稀释至4×105个细胞/毫升。将细胞悬浮液接种到384孔白色细胞板，每孔25μL。将细胞板离心(300rpm)1分钟，并置于37℃5％CO2培养箱中培养24小时。第二天，从化合物板中转移300nL 100X化合物到细胞板中。将细胞板离心(300rpm)一分钟，并置于37℃5％CO2培养箱中培养72小时。第五天，将细胞板及Bright-Glo荧光素酶试剂取出，平衡至室温。将Bright-Glo荧光素酶试剂加入到细胞板中(每孔30μL)。将细胞板在1000rpm下离心1分钟，室温孵育2分钟。读板仪上测量荧光信号。
数据处理
利用作图软件Prism 5制作受试化合物浓度曲线，计算EC50值。
表12
化合物编号 实施例二十七的10-227 对照化合物3 利福平
EC50(μM) 122.8 5.641 1.828
由表12可知，与对照化合物3和利福平相比，本发明的化合物(例如实施例二十七的10-227)对CYP450酶3A4亚型的诱导作用较弱，因而具有更好的安全性。
由附图1可知，与10μM浓度下的对照化合物3和利福平相比，实施例二十七的10-227在10μM的浓度下对CYP450酶3A4亚型的诱导作用较弱，约为利福平的29％；而对照化合物3在同样浓度下对CYP450酶3A4亚型的诱导作用与利福平相近，说明本发明的化合物(例如实施例二十七的10-227)具有更好的安全性。
除本文中描述的那些外，根据前述描述，本发明的多种修改对本领域技术人员而言会是显而易见的。这样的修改也意图落入所附权利要求书的范围内。本申请中所引用的各参考文献(包括所有专利、专利申请、期刊文章、书籍及任何其它公开)均以其整体援引加入本文。

Claims (20)

  1. 化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有式I或式Ia的结构：
    Figure PCTCN2017110123-appb-100001
    其中：
    Ar1和Ar2各自独立地选自C6-14芳基和5-14元杂芳基，其任选地被一个或多个选自卤素、-OH、-CN、-NO2、-N(R)2、C1-6烷基、卤代C1-6烷基、C1-6烷基硫基和C3-6环烷基的取代基取代；
    L不存在或者选自-O-、-S-和-NR-；
    R1和R2各自独立地选自H(包括1H、2H、3H)、C1-6烷基(例如：氘代C1-6烷基)和C3-6环烷基；
    R3为选自下列结构的四元、五元、六元或七元氮杂环系：
    Figure PCTCN2017110123-appb-100002
    Q选自-(CRaRa’)g-、-NRa-、-O-、-S-、-S(＝O)-和-S(＝O)2-；
    Ra、Ra’、R4、R4’、R5、R5’和R6在每次出现时各自独立地选自H、卤素、-OH、-COOH、-CN、-NO2、-N(R)2、C1-6烷基、卤代C1-6烷基、-W-C1-6烷基、-C1-6亚烷基-W-R、-W-C1-6亚烷基-W’-R、-W-C2-6烯基、-C2-6亚烯基-W-R、-W-C2-6亚烯基-W’-R和C3-6环烷基，其中所述亚烷基和亚烯基任选地进一步被一个或多个W间隔；或者Ra与Ra’一起、R4与R5一起和/或R4’与R5’一起，在每次出现时各自独立地形成基团＝CH-W-R；条件是当R3不是四元氮杂环系时，Ra、Ra’、R4、R4’、R5、R5’和R6不同时为氢，并且不为选自-COOH、-C1-6亚烷基-OH和-C1-6亚烷基-C(＝O)OH的基团；且当R3为四元氮杂环时，R4、R5和R6不同时为氢；
    R6连接在上述氮杂环系结构中*和/或**标记的环碳原子上；
    W和W’在每次出现时各自独立地选自O、C(＝O)、C(＝O)O、NR、NC(＝O)、N(S＝O)、NS(＝O)2、S、S＝O和S(＝O)2；
    R在每次出现时各自独立地选自H、C1-6烷基和C3-6环烷基；
    g为1或2；并且
    t为0、1、2或3，条件是t不大于对应基团上可被取代的位置的数目，并且当t大于1时，每个R6可以相同或不同。
  2. 权利要求1的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有式II或式IIa的结构：
    Figure PCTCN2017110123-appb-100003
  3. 权利要求1或2的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中Ar1选自：
    Figure PCTCN2017110123-appb-100004
    其中Rc在每次出现时各自独立地选自F、Cl、Br、I、卤代C1-6烷基、C1-6烷基和C3-6环烷基；
    Ar1特别优选自：
    Figure PCTCN2017110123-appb-100005
  4. 权利要求3的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，
    其中Rc在每次出现时各自独立地选自F、Cl、Br、I、C1-6烷基和C3-6环烷基。
  5. 权利要求1-4中任一项的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中Ar2选自：
    Figure PCTCN2017110123-appb-100006
    Rb在每次出现时各自独立地选自H、卤素、卤代C1-6烷基、C1-6烷基和C3-6环烷基；并且
    i为0、1或2；
    Ar2优选自：
    Figure PCTCN2017110123-appb-100007
  6. 权利要求5的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，
    其中Rb在每次出现时各自独立地选自H、卤素、C1-6烷基和C3-6环烷基。
  7. 权利要求1-6中任一项的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，
    其中：
    Ra、Ra’、R4、R4’、R5、R5’和R6在每次出现时各自独立地选自H、F、Cl、Br、-(CR7R7a)mOH、-O-C1-6烷基、-(CR7R7a)mCOOH、-C(R7’)＝C(R7a’)(CR7R7a)mCOOH和-(CR7R7a)m-W-(CR7’R7a’)nCOOH，所述-(CR7R7a)m-W-(CR7’R7a’)nCOOH优选为-(CR7R7a)mO(CR7’R7a’)nCOOH、-(CR7R7a)mNR(CR7’R7a’)nCOOH或-(CR7R7a)mS(＝O)j(CR7’R7a’)nCOOH；或者Ra与Ra’一起、R4与R5一起和/或R4’与R5’一起，在每次出现时各自独立地形成基团＝CH-W-R；优选地，Ra、Ra’、R4、R4’、R5、R5’和R6在每次出现时各自独立地选自H、F、-OH、-CH2OH、-OCH3、-COOH、-CH2COOH、-(CH2)2COOH、-(CH2)3COOH、-CH＝CHCOOH、-OCH2COOH、-SCH2COOH、-N(CH3)CH2COOH、-CH2OCH2COOH、-CH2SCH2COOH、-CH2N(CH3)CH2COOH、-C(CH3)＝CHCOOH和-CH＝C(CH3)COOH；
    R7、R7’、R7a、R7a’在每次出现时各自独立地选自H、C1-4烷基和C3-6环烷基；
    R选自H、甲基、乙基、丙基和环丙基；
    m为0、1、2、3或4；
    n为1、2、3或4；并且
    j为0、1或2。
  8. 权利要求1-7中任一项的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中R3选自：
    Figure PCTCN2017110123-appb-100008
    Figure PCTCN2017110123-appb-100009
  9. 权利要求1-8中任一项的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中R3选自：
    Figure PCTCN2017110123-appb-100010
  10. 权利要求1-9中任一项的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有以下结构：
    Figure PCTCN2017110123-appb-100011
  11. 化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有以下结构：
    Figure PCTCN2017110123-appb-100012
    其中：
    R1和R2各自独立地选自H(包括1H、2H、3H)、C1-6烷基(例如：氘代C1-6烷基)和C3-6环烷基；
    Q为-(CRaRa’)g-或-O-；
    Ra、Ra’、R4、R5和R6在每次出现时各自独立地选自H、卤素、-OH、-COOH、-CN、-NO2、-N(R)2、C1-6烷基、卤代C1-6烷基、-W-C1-6烷基、-C1-6亚烷基-W-R、-W-C1-6亚烷基-W’-R、-W-C2-6烯基、-C2-6亚烯基-W-R、-W-C2-6亚烯基-W’-R和C3-6环烷基，其中所述亚烷基和亚烯基任选地进一步被一个或多个W间隔；
    Rb在每次出现时各自独立地选自H、卤素、卤代C1-6烷基、C1-6烷基和C3-6环烷基；
    Rc在每次出现时各自独立地选自F、Cl、Br、I、卤代C1-6烷基、C1-6烷基和C3-6环烷基；
    R6连接在通式结构中*和/或**标记的环碳原子上；
    W和W’在每次出现时各自独立地选自O、C(＝O)、C(＝O)O、NR、NC(＝O)、N(S＝O)、NS(＝O)2、S、S＝O和S(＝O)2；
    R在每次出现时各自独立地选自H、C1-6烷基和C3-6环烷基；
    g为1或2；
    i为0、1或2；
    m为0、1、2、3或4；并且
    t为0、1或2，条件是当t大于1时，每个R6可以相同或不同。
  12. 权利要求11的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，
    其中Rb在每次出现时各自独立地选自H、卤素、C1-6烷基和C3-6环烷基；
    Rc在每次出现时各自独立地选自F、Cl、Br、I、C1-6烷基和C3-6环烷基。
  13. 权利要求11或12的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物具有以下结构：
    Figure PCTCN2017110123-appb-100013
  14. 权利要求1-13中任一项的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药，其中所述化合物选自：
    Figure PCTCN2017110123-appb-100014
    Figure PCTCN2017110123-appb-100015
    Figure PCTCN2017110123-appb-100016
    Figure PCTCN2017110123-appb-100017
    Figure PCTCN2017110123-appb-100018
    Figure PCTCN2017110123-appb-100019
  15. 药物组合物，其包含预防或治疗有效量的权利要求1-14中任一项的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药以及一种或多种药学上可接受的载体，所述药物组合物优选是固体制剂、液体制剂或透皮制剂。
  16. 制备药物组合物的方法，所述方法包括将权利要求1-14中任一项的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药与一种或多种药学上可接受的载体组合。
  17. 权利要求1-14中任一项的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、代谢物、同位素标记的化合物或前药或者权利要求15的药物组合物在制备用于预防或治疗病毒性疾病的药物中的用途。
  18. 权利要求17的用途，其中所述药物为通过口服、静脉内、动脉内、皮下、腹膜内、肌内或经皮途径给药的药物。
  19. 权利要求17的用途，其中所述病毒性疾病选自甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、流行性感冒、疱疹和获得性免疫缺陷综合征(AIDS)。
  20. 制备权利要求1-14中任一项的化合物的方法，所述方法包括以下步骤：
    Figure PCTCN2017110123-appb-100020
    或者
    Figure PCTCN2017110123-appb-100021
    其中：
    Hal选自F、Cl、Br和I；
    卤化试剂选自Cl2、Br2、I2、N-氯代琥珀酰亚胺、N-溴代丁二酰亚胺和N-碘代丁二酰亚胺；
    其余各基团如权利要求1-14中任一项所定义；
    步骤一在碱金属盐存在下于质子性溶剂中进行；
    步骤二在非质子性溶剂中进行；并且
    步骤三在非质子性溶剂中，在有机碱或无机碱的存在下进行；
    当权利要求1-14中任一项的化合物中的R2为C1-6烷基时，其也可通过包括以下步骤的方法合成：
    Figure PCTCN2017110123-appb-100022
    或者
    Figure PCTCN2017110123-appb-100023
    其中：
    R2’为H或C1-5烷基；
    Hal选自F、Cl、Br和I；
    卤化试剂选自Cl2、Br2、I2、N-氯代琥珀酰亚胺、N-溴代丁二酰亚胺和N-碘代丁二酰亚胺；
    其余各基团如上述所定义；
    步骤I在路易斯酸存在下于非极性溶剂中进行；
    步骤II在有机碱或无机碱的存在下，在非质子性溶剂中进行；
    步骤III在非质子性溶剂中进行；并且
    步骤IV在非质子性溶剂中，在有机碱或无机碱的存在下进行。
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