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WO2019096241A1 - 抗HBV的四氢异噁唑并[4,3-c]吡啶类化合物 - Google Patents

抗HBV的四氢异噁唑并[4,3-c]吡啶类化合物 Download PDF

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WO2019096241A1
WO2019096241A1 PCT/CN2018/115857 CN2018115857W WO2019096241A1 WO 2019096241 A1 WO2019096241 A1 WO 2019096241A1 CN 2018115857 W CN2018115857 W CN 2018115857W WO 2019096241 A1 WO2019096241 A1 WO 2019096241A1
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Prior art keywords
isomer
pharmaceutically acceptable
iii
acceptable salt
compound
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English (en)
French (fr)
Inventor
吴立方
孙飞
杜金华
丁照中
陈曙辉
张喜全
徐宏江
杨玲
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Chia Tai Tianqing Pharmaceutical Group Co Ltd
Medshine Discovery Inc
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Chia Tai Tianqing Pharmaceutical Group Co Ltd
Medshine Discovery Inc
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Priority to CN201880070173.7A priority Critical patent/CN111247131B/zh
Priority to JP2020526967A priority patent/JP7250015B2/ja
Priority to US16/764,129 priority patent/US11572372B2/en
Priority to EP18879872.2A priority patent/EP3712139A4/en
Priority to SG11202004594SA priority patent/SG11202004594SA/en
Publication of WO2019096241A1 publication Critical patent/WO2019096241A1/zh
Anticipated expiration legal-status Critical
Priority to PH12020550649A priority patent/PH12020550649A1/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/424—Oxazoles condensed with heterocyclic ring systems, e.g. clavulanic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4355—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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

Definitions

  • the invention relates to the field of medicine, in particular to a new class of anti-HBV tetrahydroisoxazo[4,3-c]pyridine compounds, a preparation method thereof and application thereof in preparing medicine for treating hepatitis B.
  • HBV hepatitis B virus
  • WHO statistics about 240 million people worldwide are infected with HBV (hepatitis B virus), causing approximately 680,000 deaths each year, directly or indirectly.
  • China is a big country with hepatitis B and has an infection of more than 70 million people.
  • Long-term infection with HBV can lead to malignant diseases such as liver failure, cirrhosis and liver cancer.
  • nucleoside (acid) drugs such as lamivudine, entecavir, and tenofovir, can inhibit HBV DNA replication, but these drugs can not clear cccDNA, and often rebound after stopping the drug. Patients need long-term medication, and some patients are prone to drug resistance. Interferon drugs can partially activate the patient's immune system and inhibit hepatitis B virus through human autoimmunity. However, these drugs have large side effects, and patients are not well tolerated. What is more serious is the response rate of different populations to interferon therapy. Significant differences, but overall the response rate is low (usually below 30%) (Nat. Rev. Gastro. Hepat. 8 (2011), 275-284).
  • cccDNA a stable covalently closed circular DNA, cccDNA, is formed in the nucleus of the host's liver cells as a template for continuous replication of HBV.
  • All subgenomic RNA (sgRNA) and pregenomic RNA (pgRNA) are formed by cccDNA transcription.
  • the sgRNA is translated into the X protein and the other three envelope proteins, and the pgRNA is translated into the core protein and the viral polymerase.
  • the pgRNA and the core protein self-assemble under the action of the polymerase to form an RNA that encapsulates the nucleocapsid.
  • the pgRNA is reverse transcribed into a negative strand DNA, and thereby the DNA positive strand is further synthesized to form rcDNA.
  • the nucleocapsid-encapsulated rcDNA is re-shelled into the nucleus to further expand the cccDNA; on the other hand, it re-engages with the envelope protein and releases the cells through the endoplasmic reticulum to form a new HBV.
  • the synthesis of nucleocapsids is a critical step in the replication process of HBV genome, and the synthesis of viral DNA can only occur specifically inside the nucleocapsid.
  • nucleocapsids The assembly of nucleocapsids is an evolutionary constraint that limits the diversity of HBV, even for subtle molecular interference. Targets for the synthesis and degradation of nucleocapsids are highly promising for the development of new therapies for different hepatitis B virus genotypes and resistant strains. Some anti-HBV compounds associated with nucleocapsids have been reported. NVR 3-778 (WO 2015109130A1), JNJ-56136379, GLS-4JHS and other related compounds are in the clinical research stage.
  • the present invention provides a compound of formula (I), a pharmaceutically acceptable salt thereof or an isomer thereof,
  • ring A is a 6-12 membered aryl group or a 5-6 membered heteroaryl group
  • n 0, 1, 2 or 3;
  • T is N or CR 3 ;
  • R 1 and R 5 are each independently H, F, Cl, Br, I, CN, -OH, -NR a R b , C 1-3 alkoxy or optionally 1, 2 or 3 independently selected Substituting a C 1-3 alkyl group for a substituent of F, Cl, Br, I, -OH, -NH 2 , -CN or -NO 2 ;
  • R 2 and R 4 are each independently H, F, Cl, Br, I, CN, -OH, -NR a R b , C 1-6 alkoxy or optionally 1, 2 or 3 independently selected Substituting a C 1-6 alkyl group for a substituent of F, Cl, Br, I, -OH, -NH 2 , -CN or -NO 2 ;
  • R 3 is F, Br or CN
  • R a , R b , R c and R d are each independently H or C 1-6 alkyl
  • the 5-6 membered heteroaryl contains 1, 2 or 3 heteroatoms or heteroatoms independently selected from -O-, -S-, N or -NH-.
  • the substituent is substituted for C 1-6 alkyl, and other variables are as defined in the present invention.
  • Ring A is phenyl or 5-6 membered heteroaryl, and other variables are as defined herein.
  • Ring A is phenyl, thiazolyl, isothiazolyl, pyrazolyl, pyrrolyl, pyrazinyl, pyrimidinyl or pyridyl, and other variables are as defined herein.
  • said R a , R b , R c and R d are each independently H, -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 or -CH 2 (CH 3 2 )
  • Other variables are as defined by the present invention.
  • each R described above is independently F, Cl, Br, I, CN, C 1-3 alkoxy or C 1-3 alkyl, and other variables are as defined herein.
  • each R described above is independently F, Cl, CN or methoxy, and other variables are as defined herein.
  • n 0, 1 or 2
  • other variables are as defined herein.
  • R 1 and R 5 are each independently H, F, Cl, Br, I, CN, -OH, -OCH 3 , -NH 2 , -CH 3 , -CHF, -CHF 2 or -CF 3 , other variables are as defined by the present invention.
  • R 1 and R 5 above are each independently H, and other variables are as defined herein.
  • each of R 2 and R 4 is independently H, F, Cl, Br, I, CN, -OH, -NH 2 , C 1-3 alkoxy or optionally Two or three substituents independently selected from the group consisting of F, Cl, Br, I are substituted for the C 1-3 alkyl group, and other variables are as defined in the present invention.
  • R 2 and R 4 above are each independently H, F, Cl, CN or optionally 2 substituted by methyl, and other variables are as defined herein.
  • R 2 and R 4 are each independently H, F, Cl, Br, I, CN, -OH, -OCH 3 , -OCH 2 CH 3 , -NH 2 , -CH 3 , -CH 2 CH 3 , -CHF, -CHF 2 or -CF 3 , other variables are as defined in the present invention.
  • the structural unit for R and n and other variables are as defined by the present invention; in some embodiments of the invention, the above structural unit for R and n and other variables are as defined by the present invention.
  • the structural unit for R and other variables are as defined by the present invention.
  • R 1 , R 2 , R 3 , R 4 and R 5 and other variables are as defined in the present invention.
  • R 2 , R 3 and R 4 and other variables are as defined by the present invention.
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof has the structure represented by the formula (I-1):
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof has the structure represented by formula (II-a) or formula (III-a):
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof has the structure represented by formula (II-a-1) or formula (III-a-1):
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof having the formula (II-b), (II-c), (III-b) or (III-c) The structure shown:
  • T 1 and T 2 are each independently N or CH; and R 2 , R 4 , R and n are as defined in the present invention.
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof having the formula (II-b-1), (II-c-1), (III-b-1) Or the structure shown in (III-c-1):
  • T 1 and T 2 are each independently N or CH; and R 2 , R 4 , R and n are as defined in the present invention.
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof having the formula (II-d), (II-e), (II-f), (II-k) , (III-d), (III-e), (III-f) or (III-k) structure:
  • R 2 , R 4 , R and n are as defined in the present invention.
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof has the formula (II-d-1), (II-e-1), (II-f-1) , (II-k-1), (III-d-1), (III-e-1), (III-f-1) or (III-k-1) structure:
  • R 2 , R 4 , R and n are as defined in the present invention.
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof having the structure represented by formula (II-m) or (III-m):
  • R 2 and R 4 are as defined in the present invention.
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof has the structure represented by the formula (II-m-1) or (III-m-1):
  • R 2 and R 4 are as defined in the present invention.
  • R 2 , R 4 , R and n are as defined in the present invention.
  • R 2 , R 4 , R and n are as defined in the present invention.
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof which has the formula (II-g-1) to (II-i-1) or the formula (III-g-1) ) ⁇ (III-i-1) structure:
  • R 2 , R 4 , R and n are as defined in the present invention.
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof has the formula (II-j-1) to (II-n-1) or the formula (III-j-1) ) ⁇ (III-n-1) structure:
  • R 2 , R 4 , R and n are as defined in the present invention.
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof is selected from the group consisting of:
  • the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof is selected from the group consisting of:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof, and a pharmaceutically acceptable carrier.
  • the present invention provides the use of the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof, and the above pharmaceutical composition for the preparation of an anti-hepatitis B drug.
  • the present invention provides the use of the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof and the above pharmaceutical composition for preventing or treating hepatitis B.
  • the present invention provides a method of treating or preventing hepatitis B comprising administering to a mammal, preferably a human, in need of such treatment or prevention a therapeutically effective amount of the above compound, a pharmaceutically acceptable salt thereof or Isomers thereof and pharmaceutical compositions thereof as described above.
  • the present invention provides the above compound, a pharmaceutically acceptable salt thereof or an isomer thereof, and a pharmaceutical composition thereof, for use in the treatment or prevention of hepatitis B.
  • the present invention relates to a new class of HBV inhibitors which differ in the structure of the parent core from the prior art.
  • the compounds of the present invention exhibit inhibition of HBV DNA replication activity.
  • pharmaceutically acceptable as used herein is intended to mean that those compounds, materials, compositions and/or dosage forms are within the scope of sound medical judgment and are suitable for use in contact with human and animal tissues. Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of a compound of the invention prepared from a compound having a particular substituent found in the present invention and a relatively non-toxic acid or base.
  • a relatively acidic functional group is contained in the compound of the present invention
  • a base addition salt can be obtained by contacting a sufficient amount of a base with such a compound in a neat solution or a suitable inert solvent.
  • an acid addition salt can be obtained by contacting a sufficient amount of an acid with such a compound in a neat solution or a suitable inert solvent.
  • Certain specific compounds of the invention contain both basic and acidic functional groups which can be converted to any base or acid addition salt.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing an acid group or a base by conventional chemical methods.
  • such salts are prepared by reacting these compounds in water or an organic solvent or a mixture of the two via a free acid or base form with a stoichiometric amount of a suitable base or acid.
  • the compounds of the invention may exist in specific geometric or stereoisomeric forms. All such compounds are contemplated by the present invention, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers , (D)-isomer, (L)-isomer, and racemic mixtures thereof and other mixtures, such as enantiomerically or diastereomeric enriched mixtures, all of which belong to the present Within the scope of the invention. Additional asymmetric carbon atoms may be present in the substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the invention.
  • enantiomer or “optical isomer” refer to stereoisomers that are mirror images of one another.
  • diastereomer refers to a stereoisomer in which the molecule has two or more chiral centers and the molecules are in a non-mirrored relationship.
  • wedge-shaped dashed keys Represents the absolute configuration of a solid center with straight solid keys
  • straight dashed keys Indicates the relative configuration of the stereocenter, using wavy lines Indicates a wedge solid key Or wedge-shaped dotted key Or with wavy lines Represents a straight solid key And straight dashed keys
  • the terms "enriched in one isomer”, “isomer enriched”, “enriched in one enantiomer” or “enantiomeric enriched” refer to one of the isomers or pairs
  • the content of the oligo is less than 100%, and the content of the isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or greater than or equal to 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the two isomers or the relative percentages of the two enantiomers. For example, if one of the isomers or enantiomers is present in an amount of 90% and the other isomer or enantiomer is present in an amount of 10%, the isomer or enantiomeric excess (ee value) is 80%. .
  • optically active (R)- and (S)-isomers as well as the D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary wherein the resulting mixture of diastereomers is separated and the auxiliary group cleaved to provide pure The desired enantiomer.
  • a diastereomeric salt is formed with a suitable optically active acid or base, followed by conventional methods well known in the art.
  • the diastereomers are resolved and the pure enantiomer is recovered.
  • the separation of enantiomers and diastereomers is generally accomplished by the use of chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (eg, formation of an amino group from an amine). Formate).
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound.
  • radiolabeled compounds can be used, such as tritium (3 H), iodine -125 (125 I) or C-14 (14 C).
  • hydrogen can be replaced by heavy hydrogen to form a deuterated drug.
  • the bond composed of barium and carbon is stronger than the bond composed of common hydrogen and carbon.
  • deuterated drugs have reduced side effects and increased drug stability. Enhance the efficacy and prolong the biological half-life of the drug. Alterations of all isotopic compositions of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • pharmaceutically acceptable carrier refers to any formulation or carrier medium that is capable of delivering an effective amount of an active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects to the host or patient, including water, oil, Vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, tackifiers, transdermal enhancers and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical arts.
  • excipient generally refers to the carrier, diluent and/or vehicle required to formulate an effective pharmaceutical composition.
  • treating means administering a compound or formulation described herein to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:
  • an "effective amount” or “therapeutically effective amount” with respect to a pharmaceutical or pharmacologically active agent refers to a sufficient amount of a drug or agent that is non-toxic but that achieves the desired effect.
  • an "effective amount” of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount will vary from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, and a suitable effective amount in a case can be determined by one skilled in the art based on routine experimentation.
  • active ingredient refers to a chemical entity that is effective in treating a target disorder, disease or condition.
  • substituted means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, and may include variants of heavy hydrogen and hydrogen, as long as the valence of the particular atom is normal and the substituted compound is stable. of.
  • Oxygen substitution does not occur on the aromatic group.
  • optionally substituted means that it may or may not be substituted, and unless otherwise specified, the kind and number of substituents may be arbitrary on the basis of chemically achievable.
  • C mn herein is that the moiety has an integer number of carbon atoms in a given range.
  • C1-6 means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.
  • C 1-3 means that the group may have 1 carbon atom, 2 carbon atoms, and 3 carbon atoms.
  • any variable eg, R
  • its definition in each case is independent.
  • the group may optionally be substituted with at most two R, and each case has an independent option.
  • combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.
  • substituents When the listed substituents are not indicated by which atom is attached to the substituted group, such a substituent may be bonded through any atom thereof, for example, a pyridyl group as a substituent may be passed through any one of the pyridine rings. A carbon atom is attached to the substituted group.
  • the listed linking group does not indicate its direction of attachment, its connection direction is arbitrary, for example, The medium linking group L is -MW-, and at this time, -MW- can be connected in the same direction as the reading order from left to right to form ring A and ring B. It is also possible to connect the ring A and the ring B in a direction opposite to the reading order from left to right. Combinations of the linking groups, substituents and/or variants thereof are permissible only if such combinations result in stable compounds.
  • hetero denotes a hetero atom or a hetero atomic group (ie, a radical containing a hetero atom), including atoms other than carbon (C) and hydrogen (H), and radicals containing such heteroatoms, including, for example, oxygen (O).
  • the number of atoms on the ring is generally defined as the number of elements of the ring.
  • “5-7 membered ring” refers to a “ring” that is arranged around 5-7 atoms.
  • ring means substituted or unsubstituted cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aryl or heteroaryl. So-called rings include single rings, interlocking rings, spiral rings, parallel rings or bridge rings. Unless otherwise specified, the ring optionally contains from 1 to 3 heteroatoms.
  • 5- to 7-membered ring includes, for example, phenyl, pyridine, and piperidinyl; on the other hand, the term “5- to 7-membered heterocycloalkyl ring” includes pyridyl and piperidinyl, but does not include phenyl.
  • ring also includes ring systems containing at least one ring, each of which "ring” independently conforms to the above definition.
  • hydrocarbyl refers to a functional group containing only two atoms of carbon and hydrogen
  • hydrocarbyl or its subordinate concept (such as alkyl, alkenyl, alkynyl, aryl, etc.) itself or as another
  • a portion of a substituent means a linear, branched or cyclic hydrocarbon radical or a combination thereof, and may be fully saturated (eg, alkyl), unitary or polyunsaturated (eg, alkenyl, alkynyl, aryl).
  • C 1 -C 12 represents 1 to 12 carbons, and C 1-12 is selected from C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 and C 12 ; C 3-12 are selected from C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 and C 12 .
  • Hydrocarbyl includes, but is not limited to, aliphatic hydrocarbyl groups including chain and cyclic, including but not limited to alkyl, alkenyl, alkynyl groups including, but not limited to, 6-12 members.
  • An aromatic hydrocarbon group such as benzene, naphthalene or the like.
  • hydrocarbyl refers to a straight or branched chain of atoms or a combination thereof, which may be fully saturated, mono- or polyunsaturated, and may include divalent and multivalent radicals.
  • saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, isobutyl, cyclohexyl, (cyclohexyl).
  • a homolog or isomer of a methyl group, a cyclopropylmethyl group, and an atomic group such as n-pentyl, n-hexyl, n-heptyl, n-octyl.
  • the unsaturated hydrocarbon group has one or more double or triple bonds, and examples thereof include, but are not limited to, a vinyl group, a 2-propenyl group, a butenyl group, a crotyl group, a 2-isopentenyl group, and a 2-(butadienyl group). , 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers body.
  • alkyl is used to denote a straight or branched saturated hydrocarbon group, which may be monosubstituted (eg, -CH 2 F) or polysubstituted (eg, -CF 3 ), and may be monovalent (eg, Methyl), divalent (such as methylene) or polyvalent (such as methine).
  • alkyl group include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl). , t-butyl), pentyl (eg, n-pentyl, isopentyl, neopentyl) and the like.
  • halo or “halogen”, by itself or as part of another substituent, denotes a fluorine, chlorine, bromine or iodine atom.
  • haloalkyl is intended to include both monohaloalkyl and polyhaloalkyl.
  • halo(C 1 -C 4 )alkyl is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Wait.
  • examples of haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.
  • alkoxy represents attached through an oxygen bridge
  • C 1-6 alkoxy groups include C 1, C 2, C 3 , C 4, C 5 , and C 6 alkoxy groups.
  • alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentyloxy and S- Pentyloxy.
  • aryl denotes a polyunsaturated, aromatic hydrocarbon substituent which may be monosubstituted or polysubstituted, which may be monovalent, divalent or polyvalent, which may be monocyclic or polycyclic ( For example, 1 to 3 rings; at least one of which is aromatic), they are fused together or covalently linked.
  • heteroaryl refers to an aryl group (or ring) containing one, two, three, four heteroatoms, or containing one, two, three, four to form any number of intervening heteroatoms.
  • the heteroatoms are selected from the group consisting of B, N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized.
  • a heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
  • Non-limiting examples of aryl or heteroaryl groups include phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, phenyl-oxazolyl, isomerism Azyl, thiazolyl, isothiazolyl, furyl, thienyl, pyridyl, pyrimidinyl, benzothiazolyl, indolyl, benzimidazolyl, indolyl, isoquinolinyl, quinoxalinyl, quin Lolinyl, 1-naphthyl, 2-naphthyl, 4-biphenylyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, Pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4
  • leaving group refers to a functional group or atom which may be substituted by another functional group or atom by a substitution reaction (for example, an affinity substitution reaction).
  • substituent groups include triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters and the like; acyloxy groups such as acetoxy, trifluoroacetoxy and the like.
  • protecting group includes, but is not limited to, "amino protecting group", “hydroxy protecting group” or “thiol protecting group”.
  • amino protecting group refers to a protecting group suitable for preventing side reactions at the amino nitrogen position.
  • Representative amino protecting groups include, but are not limited to, formyl; acyl, such as alkanoyl (e.g., acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, e.g., tert-butoxycarbonyl (Boc) Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1, 1-di -(4'-methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS) and tert-
  • hydroxy protecting group refers to a protecting group suitable for use in preventing hydroxy side reactions.
  • Representative hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl and t-butyl groups; acyl groups such as alkanoyl groups (e.g., acetyl); arylmethyl groups such as benzyl (Bn), para Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and the like.
  • alkyl groups such as methyl, ethyl and t-butyl groups
  • acyl groups such as alkanoyl groups (e.g., acetyl)
  • arylmethyl groups such as benzyl (Bn), para Oxybenzyl (PMB), 9-fluor
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, combinations thereof with other chemical synthetic methods, and those well known to those skilled in the art. Equivalent alternatives, preferred embodiments include, but are not limited to, embodiments of the invention.
  • the solvent used in the present invention is commercially available.
  • DMF stands for N,N-dimethylformamide
  • Na 2 CO 3 stands for sodium carbonate
  • K 2 CO 3 stands for potassium carbonate
  • Cs 2 CO 3 stands for cesium carbonate
  • CuI stands for iodide Copper
  • MeI stands for methyl iodide
  • EtOAc stands for ethyl acetate
  • EA stands for ethyl acetate
  • THF stands for tetrahydrofuran
  • LiHMDS stands for lithium hexamethyldisilazide
  • MeOH stands for methanol
  • DCM stands for dichloromethane
  • DMSO stands for dimethyl Sulfone
  • PE stands for petroleum ether
  • EtOH stands for ethanol
  • IPA stands for isopropanol
  • CH 3 CN stands for acetonitrile
  • MTBE stands for methyl tert-butyl ether
  • TFA stands for trifluoroacetic acid
  • NH 3 ⁇ H 2 O stands for am
  • Figure 1 is a graph showing the change of Log (HBV DNA copy number) per unit volume of plasma in mice of each test group after receiving the administration of Example 6 for 1 to 7 days.
  • Figure 2 is a log (HBV DNA copy number) level in liver tissue per mouse of each test group on day 7 after administration of the same Example 6
  • Figure 3 is a graph showing the change of Log (HBV DNA copy number) per unit volume of plasma in mice of each test group after administration of Example 23 for 1 to 7 days.
  • Figure 4 is a log (HBV DNA copy number) level in liver tissue per mouse of each test group on day 7 of administration of the mice in Example 23.
  • Phenyl chloroformate (58.54 g, 46.83 ml) was slowly added dropwise to a solution of 3,4,5-trifluoroaniline (50.00 g) and pyridine (29.58 g, 30.18 ml) in dichloromethane (300 ml) at 0 °C. ) in solution. After the reaction mixture was stirred at 25 ° C for 3 hours, it was quenched by adding 250 ml of water to precipitate a white precipitate. The precipitate was filtered and dried under reduced pressure to give Intermediate A-1.
  • the preparation of the intermediate A-5 can be carried out by referring to the preparation method of the intermediate A-1 by replacing 3,4,5-trifluoroaniline with 2-chloro-4-aminopyridine.
  • Step B Synthesis of Compound 1-3
  • N-Boc-(S)-2-methyl-4-piperidone (0.50 g) was dissolved in diethyl ether (8 mL) at -70 ° C under nitrogen, followed by hexamethyldisilylamino Lithium (2.34 ml, 1 mol/l). After the reaction mixture was stirred at -70 °C for 0.5 hr, a solution of Compound 1-2 (374.04 mg) in diethyl ether (2 ml) was added dropwise. The reaction solution was naturally warmed to room temperature and stirring was continued for 3 hours. The reaction solution was poured into 1 mol / liter of hydrochloric acid (40 ml), and ethyl acetate (25 ml. The combined organic layers were washed with EtOAc EtOAc m.
  • Example 2 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with benzoic acid.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALPAK AS-H (specification: 250 mm ⁇ 30 mm, particle size: 5 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, EtOH]; B% Example 2 was obtained from 30% to 30%, 7.8 min.
  • Example 3 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with 2-fluorobenzoic acid.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (specification: 250 mm ⁇ 30 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, MeOH]; B%: 20 Example 3 was obtained in %-20%, 4.0 min).
  • Example 4 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with 3-fluorobenzoic acid.
  • the crude product was separated by SFC preparation (SFC separation method: separation column: DAICEL CHIRALPAK AS (specification: 250 mm ⁇ 50 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, IPA]; B%: 50
  • Example 4 was obtained in %-50%, 1.9 min).
  • Example 5 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with 4-fluorobenzoic acid.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALPAK AS-H (specification: 250 mm ⁇ 30 mm, particle size: 5 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, EtOH]; B% : Example 30 was obtained from 30% to 30%, 4.2 min.
  • SFC separation method separation column: DAICEL CHIRALPAK AS-H (specification: 250 mm ⁇ 30 mm, particle size: 5 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, EtOH]; B% : Example 30 was obtained from 30% to 30%, 4.2 min.
  • Example 6 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with 2,4-difluorobenzoic acid.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALPAK AS (specification: 250 mm ⁇ 30 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, IPA]; B%: 30 Example 6 was obtained in %-30%, 9 min).
  • Example 7 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with 2-methoxybenzoic acid.
  • the crude product was separated by SFC preparation (SFC separation method: separation column: DAICEL CHIRALPAK AD (specification: 250 mm ⁇ 30 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, MeOH]; B%: 30 Example 7 was obtained in %-30%, 2.5 min).
  • Example 8 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with 3-methoxybenzoic acid.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (specification: 250 mm ⁇ 30 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, EtOH]; B%: 30
  • Example 8 was obtained in %-30%, 3.0 min).
  • Example 9 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with 4-methoxybenzoic acid.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALCEL OD (specification: 250 mm ⁇ 50 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, IPA]; B%: 30
  • Example 9 was obtained in %-30%, 4.2 min).
  • Example 10 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with 3-cyanobenzoic acid.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (specification: 250 mm ⁇ 30 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, IPA]; B%: 30
  • Example 10 was obtained in %-30%, 3.1 min).
  • Example 11 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with 3-chlorobenzoic acid.
  • the crude product was separated by SFC preparation (SFC separation method: separation column: DAICEL CHIRALPAK AS-H (specification: 250 mm ⁇ 30 mm, particle size: 5 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, MeOH]; B% Example 11 was obtained from 35% to 35%, 5 min.
  • Example 12 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with 4-chlorobenzoic acid.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALPAK AS-H (specification: 250 mm ⁇ 30 mm, particle size: 5 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, EtOH]; B% : Example 30 was obtained from 30% to 30%, 8.3 min.
  • SFC separation method separation column: DAICEL CHIRALPAK AS-H (specification: 250 mm ⁇ 30 mm, particle size: 5 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, EtOH]; B% : Example 30 was obtained from 30% to 30%, 8.3 min.
  • Example 13 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with pyrazine-2-carboxylic acid.
  • the crude product was separated by SFC preparation (SFC separation method: separation column: DAICEL CHIRALPAK AS-H (specification: 250 mm ⁇ 30 mm, particle size: 5 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, MeOH]; B% : Example 60 was obtained from 60% to 60%, 4.5 min.
  • Example 14 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with thiazole-5-carboxylic acid.
  • the crude product was separated by SFC preparation (SFC separation method: separation column: DAICEL CHIRALCEL OD (specification: 250 mm ⁇ 30 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, MeOH]; B%: 30
  • Example 14 was obtained in %-30%, 2.3 min).
  • Example 15 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with thiazole-4-carboxylic acid.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (specification: 250 mm ⁇ 30 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, IPA]; B%: 30
  • Example 15 was obtained in %-30%, 3.0 min).
  • Example 16 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with thiazole-2-carboxylic acid.
  • the crude product was separated by SFC preparation (SFC separation method: separation column: DAICEL CHIRALPAK AS-H (specification: 250 mm ⁇ 30 mm, particle size: 5 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, MeOH]; B% Example 16 was obtained from 35% to 35%, 6.3 min.
  • Example 17 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with isothiazole-4-carboxylic acid.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (specification: 250 mm ⁇ 30 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, IPA]; B%: 30
  • Example 17 was obtained in %-30%, 30 min).
  • Example 18 Referring to Preparation Schemes A through E of Example 1, 5-fluoropyridine-2-carboxylic acid was replaced with isothiazole-3-carboxylic acid.
  • the crude product was separated by SFC preparation (SFC separation method: separation column: DAICEL CHIRALPAK AD-H (specification: 250 mm ⁇ 30 mm, particle size: 5 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, MeOH]; B% Example 18 was obtained from 35% to 35%, 2.1 min.
  • Example 19 The preparation method of Example 19 can be referred to the preparation procedures A to E of Example 1, replacing 5-fluoropyridine-2-carboxylic acid with 2,4-difluorobenzoic acid; and replacing intermediate A-1 with intermediate A- 2.
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALCEL OJ (specification: 250 mm ⁇ 30 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, EtOH]; B%: 25 Example 19 was obtained in %-25%, 2.4 min).
  • Example 21 For the preparation of Example 21, refer to Preparation Schemes A to E of Example 1, replacing 5-fluoropyridine-2-carboxylic acid with 2,4-difluorobenzoic acid; and replacing intermediate A-1 with intermediate A-4. .
  • the crude product was separated by SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (specification: 250 mm ⁇ 30 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, EtOH]; B%: 20 Example 21 was obtained in %-20%, 5.5 min).
  • Example 22 For the preparation of Example 22, refer to Preparation Schemes A to E of Example 1, replacing 5-fluoropyridine-2-carboxylic acid with 2,4-difluorobenzoic acid; and replacing intermediate A-1 with intermediate A-5. .
  • the crude product was separated by SFC preparation (SFC separation method: separation column: DAICEL CHIRALPAK AD (specification: 250 mm ⁇ 50 mm, particle size: 10 ⁇ m); mobile phase: [0.1% NH 3 ⁇ H 2 O, MeOH]; B%: 30 Example 22 was obtained in %-30%, 1.8 min).
  • Dess-Martin reagent (82.10 g) was added portionwise to a solution of compound 23-3 (40.0 g) in dichloromethane (400 mL). The reaction mixture was naturally warmed to room temperature and stirred for 2 hours, and then washed twice with a saturated sodium hydrogen carbonate solution and a saturated sodium thiosulfate solution in a volume ratio of 1:1, and then washed successively with saturated sodium hydrogen carbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. MS (ESI) m/z: 226[M+H + ].
  • N,N-Diisopropylethylamine (110.31 mg) and Intermediate A-2 were added to a solution of compound 23-8 (61.52 mg) in N,N-dimethylformamide (2 mL) 72.90 mg). The reaction mixture was stirred at 70 ° C for 1 hour. The reaction solution was diluted with water (20 ml) and then evaporated. The organic phase was dried over anhydrous sodium sulfate and filtered.
  • Example 24 can be carried out by referring to Preparation Schemes A to H of Example 23, replacing Intermediate A-2 with Intermediate A-3.
  • Example 25 can be carried out by referring to Preparation Schemes A to H of Example 23, replacing Intermediate A-2 with Intermediate A-1.
  • Example 26 can be carried out by referring to Preparation Schemes A to H of Example 23, replacing Intermediate A-2 with Intermediate A-6.
  • HBV DNA Detection of HBV DNA by real-time quantitative PCR: The total DNA in the supernatant was extracted with QIAamp 96 DNA Blood Kit kit, and the HBV DNA content was quantitatively detected by HBV-specific primers and probes. 20 microliters of the PCR master mix was added to a quantitative PCR plate for reaction with 5 microliters of HBV DNA sample or HBV plasmid standard. The HBV plasmid standard was diluted 7 points per microliter in a 10-fold gradient from 10 7 to 10 copies. The quantitative PCR reaction procedure was as follows: predenaturation at 95 ° C for 10 minutes; denaturation at 95 ° C for 15 seconds, reaction at 60 ° C for 1 minute, and repeating the cycle 40 times. The inhibition rate of each well to HBV DNA was calculated according to the following formula, and the inhibition rate data of the compound was subjected to nonlinear fitting analysis using GraphPad Prism software to obtain the EC 50 value of the compound.
  • HBV DNA inhibition rate % (1 - sample HBV DNA copy number / DMSO control HBV DNA copy number) ⁇ 100%
  • This experiment was designed to evaluate the pharmacokinetic behavior of a compound after single intravenous or intragastric administration in mice.
  • Intravenous administration the compound was formulated into a clear solution of 0.5 mg/mL, vehicle: 5% DMSO / 5% sodium dodecyl citrate / 90% water; administered by intragastric administration, the compound was formulated into 2 mg / mL Suspension, solvent: 0.5% sodium carboxymethylcellulose / 0.2% Tween 80 / 99.3% water.
  • the concentration of the compound in plasma was determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • the retention time, chromatogram acquisition, and chromatogram integration of the compound and the internal standard (diclofenac) were processed using the software Analyst (Applied Biosystems), and the statistics of the data were processed using software Watson LIMS (Thermo Fisher Scientific) or Analyst (Applied Biosystems).
  • the analyte concentration in the sample is in ng/mL, retaining 3 significant figures, and all values expressed as a percentage (eg, % deviation and % coefficient of variation, etc.) are retained to one decimal place.
  • Each calibration curve contains at least 6 concentration levels.
  • the preparation of the calibration standard requires the use of a stock solution from a different source than the quality control sample.
  • the deviation between the concentration calculated by the calibration standard and the indicated value exceeds ⁇ 15.0% (the lower limit of quantitation exceeds ⁇ 20.0%).
  • the standard should be rejected in the regression analysis.
  • the rejected calibration standards should be less than 25%, and each calibration curve contains at least 6 calibration standards that meet the acceptance criteria. If the lower limit of quantitation and the upper limit of quantitation need to be rejected, the upper and lower limits of quantitation of the analysis batch will be correspondingly increased and decreased.
  • noncompartmental pharmacokinetic software processing plasma concentration calculated using the linear trapezoidal method to the method of the number of pharmacokinetic parameters.
  • the pharmacokinetic parameters to be calculated include, but are not limited to, (data permitting) T 1/2 , Vdss, CL, AUC 0-24h in the intravenous group; C max , T max , AUC 0-24h in the oral gavage group, Bioavailability (F%).
  • mice The pharmacokinetic related parameters of the present invention in mice are shown in Table 2 below.
  • HBV plasmid DNA was extracted using pAAV2-HBV 1.3mer using a Qiagen EndoFree Plasmid Giga kit at a concentration of 1000 ng/ ⁇ L. Dilute with normal saline before use and store at 4 ° C until use.
  • the drug formulation is shown in Table 4 below:
  • a qPCR reaction mixture was prepared (see Table 6).
  • the qPCR reaction mix, samples and standards were added to a 96 well reaction plate.
  • the standard product was plasmid DNA containing the full-length sequence of D-type HBV, and the standard was diluted from 107 copies/ ⁇ L, 10-fold serially, and 10 6 to 10 1 copy number/ ⁇ L of DNA standard was sequentially obtained.
  • PCR reaction 95 ° C, 10 minutes; 95 ° C, 15 seconds, 60 ° C, 1 minute, 40 cycles.
  • HBV DNA content DNA content detected by HBV primer - DNA content detected by pAAV2 primer.
  • the liver tissue was taken and homogenized using a tissue grinder. After centrifugation, the supernatant was transferred to a new centrifuge tube and digested with proteinase K digestion for 3 hours. After cooling, RNase A was added for 30 minutes. The RNase A treated mixture was extracted twice with an equal volume of phenol chloroform isoamyl alcohol to remove residual protein. Transfer the supernatant to a new centrifuge tube and add isopropanol to precipitate the DNA. The DNA pellet was washed twice with 70% ethanol. The pellet was air-dried, and DNA was dissolved by adding TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).
  • TE 10 mM Tris-HCl, pH 8.0, 1 mM EDTA
  • the DNA concentration was measured with Nanodrop, and the DNA concentration of all the samples was adjusted to 10 ng/ ⁇ L. 5 ⁇ L of the sample was added to the quantitative PCR reaction system for quantitative PCR.
  • HBV DNA content DNA content detected by HBV primer - DNA content detected by pAAV2 primer.
  • HBV DNA concentration (Log HBV DNA) was as shown in Table 7 below:
  • HBV plasmid DNA was extracted using pAAV2-HBV 1.3mer using a Qiagen EndoFree Plasmid Giga kit at a concentration of 1000 ng/ ⁇ L. Dilute with normal saline before use and store at 4 ° C until use.
  • the drug formulation is shown in Table 10 below:
  • HBV DNA concentration Log HBV DNA

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Abstract

一类抗HBV的四氢异噁唑并[4,3-c]吡啶类化合物及其药学上可接受的盐或其异构体被公开，所述化合物由式（I）表示。

Description

抗HBV的四氢异噁唑并[4,3-c]吡啶类化合物
相关申请的交叉引用
本申请主张如下2个优先权：中国专利申请CN201711138922.9，申请日2017年11月16日；中国专利申请CN201810339723.2，申请日2018年04月16日，其全文均以引用的方式并入本文中。
技术领域
本发明涉及药物领域，具体涉及一类新的抗HBV的四氢异噁唑并[4,3-c]吡啶类化合物、其制备方法及其在制备治疗乙型肝炎药物中的应用。
背景技术
据WHO统计，目前全世界约有2.4亿人感染HBV(乙型肝炎病毒)，每年直接或间接地导致约68万人死亡。中国是乙肝大国，感染人口七千多万。长期感染HBV可导致肝衰竭、肝硬化和肝癌等恶性疾病。(World Health Organization,Hepatitis B:World Health Organization Fact Sheet(2016).)
目前被许可用于治疗慢性乙肝的常规药物只有核苷(酸)类化合物和干扰素两类。核苷(酸)类药物，如拉米夫定、恩替卡韦、替诺福韦(酯)等，可以抑制HBV DNA复制，但这类药物并不能清除cccDNA，停药后往往出现病情反弹。患者需长期用药，部分患者容易出现耐药现象。干扰素类药物能部分激活病人的免疫系统，通过人体自身免疫作用抑制乙肝病毒，但这类药物副作用较大，患者耐受性不足，更为严重的是不同人群对干扰素治疗的应答率存在显著差异，但总体上看应答率较低(通常低于30％)(Nat.Rev.Gastro.Hepat.8(2011)，275-284)。
HBV感染的病人，在宿主的肝细胞核内形成稳定的共价闭合环状DNA，即cccDNA，作为HBV不断复制的模板。所有次基因组的RNA(sgRNA)和前基因组的RNA(pgRNA)均由cccDNA转录形成。出细胞核后，sgRNA翻译成X蛋白和其他三个包膜蛋白，pgRNA翻译成核心蛋白和病毒聚合酶。pgRNA与核心蛋白在聚合酶的作用下发生自组装，形成包裹了核衣壳的RNA。在核衣壳内，pgRNA逆转录成负链的DNA，并由此进一步合成出DNA正链，形成rcDNA。核衣壳包裹的rcDNA一方面重新脱壳进入细胞核，进一步使cccDNA扩增；另一方面重新与包膜蛋白结合，通过内质网释放出细胞，形成新的HBV。在HBV的复制循环中，核衣壳的的合成是HBV基因组复制过程中的关键一步，病毒DNA的合成只能特异性的发生在核衣壳的内部。核衣壳的组装是限制HBV多样性的一个进化制约过程，即使对细微的分子干扰也非常敏感。对于开发新的针对不同乙肝病毒基因型和耐药菌株的疗法，作用于核衣壳的合成和降解过程的靶标极具前景。一些与核衣壳相关的抗HBV化合物已被报道。NVR 3-778(WO 2015109130A1)、JNJ-56136379、GLS-4JHS等几个相关化合物均处于临床研究阶段。
发明内容
一方面，本发明提供了式(I)所示化合物、其药学上可接受的盐或其异构体，
Figure PCTCN2018115857-appb-000001
其中，环A为6-12元芳基或5-6元杂芳基；
各R独立地为F、Cl、Br、I、CN、-OH、-NR aR b、-S(＝O) 2NR aR b、-S(＝O) 2R c、-C(＝O)OR d、C 1-6烷氧基或任选被1、2或3个独立选自F、Cl、Br、I、-OH、-OCH 3、-NH 2或-CN的取代基所取代C 1-6烷基；
n为0、1、2或3；
T为N或CR 3；
R 1和R 5各自独立地为H、F、Cl、Br、I、CN、-OH、-NR aR b、C 1-3烷氧基或任选被1、2或3个独立选自F、Cl、Br、I、-OH、-NH 2、-CN或-NO 2的取代基所取代C 1-3烷基；
R 2和R 4各自独立地为H、F、Cl、Br、I、CN、-OH、-NR aR b、C 1-6烷氧基或任选被1、2或3个独立选自F、Cl、Br、I、-OH、-NH 2、-CN或-NO 2的取代基所取代C 1-6烷基；
R 3为F、Br或CN；
R a、R b、R c和R d各自独立地为H或C 1-6烷基；
所述5-6元杂芳基包含1、2或3个独立选自-O-、-S-、N或-NH-的杂原子或杂原子团。
在本发明的一些方案中，各R独立地为F、Cl、Br、I、CN、-OH、-NR aR b、-S(＝O) 2NR aR b、-S(＝O) 2R c、-C(＝O)OR d、C 1-6烷氧基或任选被1、2或3个独立选自F、Cl、Br、I、-OH、-NH 2或-CN的取代基所取代C 1-6烷基，其他变量如本发明所定义。
在本发明的一些方案中，上述环A为苯基或5-6元杂芳基，其他变量如本发明所定义。
在本发明的一些方案中，上述环A为苯基、噻唑基、异噻唑基、吡唑基、吡咯基、吡嗪基、嘧啶基或吡啶基，其他变量如本发明所定义。
在本发明的一些方案中，上述R a、R b、R c和R d各自独立地为H、-CH 3、-CH 2CH 3、-CH 2CH 2CH 3或-CH 2(CH 3) 2，其他变量如本发明所定义。
在本发明的一些方案中，上述各R独立地为F、Cl、Br、I、CN、-OH、-OCH 3、-OCH 2CH 3、-NH 2、-N(CH 3) 2、-S(＝O) 2NH 2、-S(＝O) 2CH 3、-C(＝O)OCH 3、-C(＝O)OCH 2CH 3、-CH 3、-CH 2CH 3、CF 3或-CH 2OH，其他变量如本发明所定义。
在本发明的一些方案中，上述各R独立地为F、Cl、Br、I、CN、C 1-3烷氧基或C 1-3烷基，其他变量如本发明所定义。
在本发明的一些方案中，上述各R独立地为F、Cl、CN或甲氧基，其他变量如本发明所定义。
在本发明的一些方案中，上述n为0、1或2，其他变量如本发明所定义。
在本发明的一些方案中，上述R 1和R 5各自独立地为H、F、Cl、Br、I、CN、-OH、-OCH 3、-NH 2、-CH 3、-CHF、-CHF 2或-CF 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 1和R 5各自独立地为H，其他变量如本发明所定义。
在本发明的一些方案中，上述R 2和R 4各自独立地为H、F、Cl、Br、I、CN、-OH、-NH 2、C 1-3烷氧基或任选被1、2或3个独立选自F、Cl、Br、I的取代基所取代C 1-3烷基，其他变量如本发明所定义。
在本发明的一些方案中，上述R 2和R 4各自独立地为H、F、Cl、CN或任选2个F所取代的甲基，其他变量如本发明所定义。
在本发明的一些方案中，上述R 2和R 4各自独立地为H、F、Cl、Br、I、CN、-OH、-OCH 3、-OCH 2CH 3、-NH 2、-CH 3、-CH 2CH 3、-CHF、-CHF 2或-CF 3，其他变量如本发明所定义。
在本发明的一些方案中，上述结构单元
Figure PCTCN2018115857-appb-000002
为
Figure PCTCN2018115857-appb-000003
Figure PCTCN2018115857-appb-000004
R和n及其他变量如本发明所定义；在本发明的一些实施方案中，上述结构单元
Figure PCTCN2018115857-appb-000005
为
Figure PCTCN2018115857-appb-000006
Figure PCTCN2018115857-appb-000007
R和n及其他变量如本发明所定义。
在本发明的一些方案中，上述结构单元
Figure PCTCN2018115857-appb-000008
为
Figure PCTCN2018115857-appb-000009
Figure PCTCN2018115857-appb-000010
Figure PCTCN2018115857-appb-000011
R及其他变量如本发明所定义。
在本发明的一些方案中，上述结构单元
Figure PCTCN2018115857-appb-000012
为
Figure PCTCN2018115857-appb-000013
Figure PCTCN2018115857-appb-000014
Figure PCTCN2018115857-appb-000015
其他变量如本发明所定义。
在本发明的一些方案中，上述结构单元
Figure PCTCN2018115857-appb-000016
为
Figure PCTCN2018115857-appb-000017
R 1、R 2、R 3、R 4和R 5及其他变量如本发明所定义。
在本发明的一些方案中，上述结构单元
Figure PCTCN2018115857-appb-000018
为
Figure PCTCN2018115857-appb-000019
R 2、R 3和R 4及其他变量如本发明所定义。
在本发明的一些方案中，上述结构单元
Figure PCTCN2018115857-appb-000020
为
Figure PCTCN2018115857-appb-000021
R 2和R 4及其他变量如本发明所定义。
在本发明的一些方案中，上述结构单元
Figure PCTCN2018115857-appb-000022
为
Figure PCTCN2018115857-appb-000023
Figure PCTCN2018115857-appb-000024
其他变量如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(I-1)所示结构：
Figure PCTCN2018115857-appb-000025
其中，环A、R 1、R 2、R 4、R 5、R、n、T或结构单元
Figure PCTCN2018115857-appb-000026
如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-a)或式(III-a)所示结构：
Figure PCTCN2018115857-appb-000027
其中，环A、R 2、R 4、R、n或结构单元
Figure PCTCN2018115857-appb-000028
如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-a-1)或式(III-a-1)所示结构：
Figure PCTCN2018115857-appb-000029
其中，环A、R 2、R 4、R、n或结构单元
Figure PCTCN2018115857-appb-000030
如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-b)、(II-c)、(III-b)或(III-c)所示结构：
Figure PCTCN2018115857-appb-000031
其中，T 1和T 2各自独立地为N或CH；R 2、R 4、R和n如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-b-1)、(II-c-1)、(III-b-1)或(III-c-1)所示结构：
Figure PCTCN2018115857-appb-000032
Figure PCTCN2018115857-appb-000033
其中，T 1和T 2各自独立地为N或CH；R 2、R 4、R和n如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-d)、(II-e)、(II-f)、(II-k)、(III-d)、(III-e)、(III-f)或(III-k)所示结构：
Figure PCTCN2018115857-appb-000034
其中，R 2、R 4、R和n如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-d-1)、(II-e-1)、(II-f-1)、(II-k-1)、(III-d-1)、(III-e-1)、(III-f-1)或(III-k-1)所示结构：
Figure PCTCN2018115857-appb-000035
其中，R 2、R 4、R和n如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-m)或(III-m)所示结构：
Figure PCTCN2018115857-appb-000036
其中，R 2和R 4如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，，其具有式(II-m-1)或(III-m-1)所示结构：
Figure PCTCN2018115857-appb-000037
其中，R 2和R 4如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-g)～(II-i)或式(III-g)～(III-i)所示结构：
Figure PCTCN2018115857-appb-000038
其中，R 2、R 4、R和n如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-j)～(II-n)或式(III-j)～(III-n)所示结构：
Figure PCTCN2018115857-appb-000039
Figure PCTCN2018115857-appb-000040
其中，R 2、R 4、R和n如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-g-1)～(II-i-1)或式(III-g-1)～(III-i-1)所示结构：
Figure PCTCN2018115857-appb-000041
其中，R 2、R 4、R和n如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(II-j-1)～(II-n-1)或式(III-j-1)～(III-n-1)所示结构：
Figure PCTCN2018115857-appb-000042
Figure PCTCN2018115857-appb-000043
其中，R 2、R 4、R和n如本发明所定义。
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体选自：
Figure PCTCN2018115857-appb-000044
Figure PCTCN2018115857-appb-000045
在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体选自：
Figure PCTCN2018115857-appb-000046
Figure PCTCN2018115857-appb-000047
Figure PCTCN2018115857-appb-000048
本发明还有一些方案是由上述变量任意组合而来。
另一方面，本发明还提供了一种药物组合物，包括作为活性成分的治疗有效量的上述化合物、其药学上可接受的盐或其异构体以及药学上可接受的载体。
另一方面，本发明还提供了上述化合物、其药学上可接受的盐或其异构体及其上述药物组合物在制备抗乙型肝炎药物中的应用。
另一方面，本发明还提供了上述化合物、其药学上可接受的盐或其异构体及其上述药物组合物在预防或治疗乙型肝炎中的应用。
另一方面，本发明还提供了一种治疗或预防乙型肝炎的方法，包括对需要该治疗或预防的哺乳动物，优选人类，给予治疗有效量的上述化合物、其药学上可接受的盐或其异构体及其上述药物组合物。
另一方面，本发明还提供了用于治疗或预防乙型肝炎的上述化合物、其药学上可接受的盐或其异构体及其上述药物组合物。
本发明涉及的一类新的HBV抑制剂，母核结构区别于现有技术。本发明涉及的化合物展现出抑制HBV DNA复制活性。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂 中用足够量的碱与这类化合物接触的方式获得碱加成盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(+)”表示右旋，“(-)”表示左旋，“(±)”表示外消旋。
除非另有说明，用楔形实线键
Figure PCTCN2018115857-appb-000049
和楔形虚线键
Figure PCTCN2018115857-appb-000050
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2018115857-appb-000051
和直形虚线键
Figure PCTCN2018115857-appb-000052
表示立体中心的相对构型，用波浪线
Figure PCTCN2018115857-appb-000053
表示楔形实线键 或楔形虚线键
Figure PCTCN2018115857-appb-000055
或用波浪线
Figure PCTCN2018115857-appb-000056
表示直形实线键
Figure PCTCN2018115857-appb-000057
和直形虚线键
Figure PCTCN2018115857-appb-000058
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成 氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
术语“药学上可接受的载体”是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂或载体介质代表性的载体包括水、油、蔬菜和矿物质、膏基、洗剂基质、软膏基质等。这些基质包括悬浮剂、增粘剂、透皮促进剂等。它们的制剂为化妆品领域或局部药物领域的技术人员所周知。
术语“赋形剂”通常是指配制有效的药物组合物所需要载体、稀释剂和/或介质。
词语“包括(comprise)”或“包含(comprise)”及其英文变体例如comprises或comprising应理解为开放的、非排他性的意义，即“包括但不限于”。
术语“治疗”意为将本申请所述化合物或制剂进行给药以预防、改善或消除疾病或与所述疾病相关的一个或多个症状，且包括：
(i)预防疾病或疾病状态在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病状态，但尚未被诊断为已患有该疾病状态时；
(ii)抑制疾病或疾病状态，即遏制其发展；
(iii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
针对药物或药理学活性剂而言，术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。对于本发明中的口服剂型，组合物中一种活性物质的“有效量”是指与该组合物中另一种活性物质联用时为了达到预期效果所需要的用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
术语“活性成分”、“治疗剂”，“活性物质”或“活性剂”是指一种化学实体，它可以有效地治疗目标紊乱、疾病或病症。
“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
本文中的C m-n，是该部分具有给定范围中的整数个碳原子。例如“C 1-6”是指该基团可具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子或6个碳原子。例如C 1-3是指该基团可具有1个碳原子、2个碳原子、3个碳原子。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。
当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
Figure PCTCN2018115857-appb-000059
中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
Figure PCTCN2018115857-appb-000060
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
Figure PCTCN2018115857-appb-000061
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，术语“杂”表示杂原子或杂原子团(即含有杂原子的原子团)，包括碳(C)和氢(H)以外的原子以及含有这些杂原子的原子团，例如包括氧(O)、氮(N)、硫(S)、硅(Si)、锗(Ge)、铝(Al)、硼(B)、-O-、-S-、＝O、＝S、-C(＝O)O-、-C(＝O)-、-C(＝S)-、-S(＝O)、-S(＝O) 2-，以及任选被取代的-C(＝O)N(H)-、-N(H)-、-C(＝NH)-、-S(＝O) 2N(H)-或-S(＝O)N(H)-。
除非另有规定，环上原子的数目通常被定义为环的元数，例如，“5-7元环”是指环绕排列5-7个原子的“环”。
除非另有规定，“环”表示被取代或未被取代的环烷基、杂环烷基、环烯基、杂环烯基、环炔基、杂环炔基、芳基或杂芳基。所谓的环包括单环、联环、螺环、并环或桥环。除非另有规定，该环任选地包含1～3个杂原子。因此，“5～7元环”包括例如苯基、吡啶和哌啶基；另一方面，术语“5～7元杂环烷基环”包括吡啶基和哌啶基，但不包括苯基。术语“环”还包括含有至少一个环的环系，其中的每一个“环”均独立地符合上述定义。
除非另有规定，术语“烃基”指只含碳、氢两种原子的官能团，术语“烃基”或者其下位概念(比如烷基、烯基、炔基、芳基等等)本身或者作为另一取代基的一部分表示直链的、支链的或环状的烃原子团或其组合，可以是完全饱和的(如烷基)、单元或多元不饱和的(如烯基、炔基、芳基)，可以是单取代或多取代的，可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)，可以包括二价或多价原子团，具有指定数量的碳原子(如C 1-C 12表示1至12个碳，C 1-12选自C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11和C 12；C 3-12选自C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11和C 12。)。“烃基”包括但不限于脂肪烃基和芳香烃基，所述脂肪烃基包括链状和环状，具体包括但不限于烷基、烯基、炔基，所述芳香烃基包括但不限于6-12元的芳香烃基，例如苯、萘等。在一些实施方案中，术语“烃基”表示直链的或支链的原子团或它们的组合，可以是完全饱和的、单元或多元不饱和的，可以包括二价和多价原子团。饱和烃原子团的实例包括但不限于甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、仲丁基、异丁基、环己基、(环己基)甲基、环丙基甲基，以及正戊基、正己基、正庚基、正辛基等原子团的同系物或异构体。不饱和烃基具有一个或多个双键或三键，其实例包括但不限于乙烯基、2-丙烯基、丁烯基、巴豆基、2-异 戊烯基、2-(丁二烯基)、2,4-戊二烯基、3-(1,4-戊二烯基)、乙炔基、1-和3-丙炔基，3-丁炔基，以及更高级的同系物和异构体。
除非另有规定，术语“烷基”用于表示直链或支链的饱和烃基，可以是单取代(如-CH 2F)或多取代的(如-CF 3)，可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。烷基的例子包括甲基(Me)，乙基(Et)，丙基(如，n-丙基和异丙基)，丁基(如，n-丁基，异丁基，s-丁基，t-丁基)，戊基(如，n-戊基，异戊基，新戊基)等。
除非另有规定，术语“卤代素”或“卤素”本身或作为另一取代基的一部分表示氟、氯、溴或碘原子。此外，术语“卤代烷基”意在包括单卤代烷基和多卤代烷基。例如，术语“卤代(C 1-C 4)烷基”意在包括但不仅限于三氟甲基、2,2,2-三氟乙基、4-氯丁基和3-溴丙基等等。除非另有规定，卤代烷基的实例包括但不仅限于：三氟甲基、三氯甲基、五氟乙基，和五氯乙基。
“烷氧基”代表通过氧桥连接的具有特定数目碳原子的上述烷基，除非另有规定，C 1-6烷氧基包括C 1、C 2、C 3、C 4、C 5和C 6的烷氧基。烷氧基的例子包括但不限于：甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、正戊氧基和S-戊氧基。
除非另有规定，术语“芳基”表示多不饱和的芳族烃取代基，可以是单取代或多取代的，可以是一价、二价或者多价，它可以是单环或多环(比如1至3个环；其中至少一个环是芳族的)，它们稠合在一起或共价连接。例如，术语“杂芳基”是指含有一、二、三、四个杂原子、或含有一、二、三、四形成任意数量区间杂原子的芳基(或环)。在一个示范性实施方案中，杂原子选自B、N、O和S，其中氮和硫原子任选地被氧化，氮原子任选地被季铵化。杂芳基可通过杂原子连接到分子的其余部分。芳基或杂芳基的非限制性实施例包括苯基、萘基、联苯基、吡咯基、吡唑基、咪唑基、吡嗪基、噁唑基、苯基-噁唑基、异噁唑基、噻唑基、异噻唑基、呋喃基、噻吩基、吡啶基、嘧啶基、苯并噻唑基、嘌呤基、苯并咪唑基、吲哚基、异喹啉基、喹喔啉基、喹啉基、1-萘基、2-萘基、4-联苯基、1-吡咯基、2-吡咯基、3-吡咯基、3-吡唑基、2-咪唑基、4-咪唑基、吡嗪基、2-噁唑基、4-噁唑基、2-苯基-4-噁唑基、5-噁唑基、3-异噁唑基、4-异噁唑基、5-异噁唑基、2-噻唑基、4-噻唑基、5-噻唑基、2-呋喃基、3-呋喃基、2-噻吩基、3-噻吩基、2-吡啶基、3-吡啶基、4-吡啶基、2-嘧啶基、4-嘧啶基、5-苯并噻唑基、嘌呤基、2-苯并咪唑基、5-吲哚基、1-异喹啉基、5-异喹啉基、2-喹喔啉基、5-喹喔啉基、3-喹啉基和6-喹啉基。上述任意一个芳基和杂芳基环系的取代基选自下文所述的可接受的取代基。
术语“离去基团”是指可以被另一种官能团或原子通过取代反应(例如亲和取代反应)所取代的官能团或原子。例如，代表性的离去基团包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等等。
术语“保护基”包括但不限于“氨基保护基”、“羟基保护基”或“巯基保护基”。术语“氨基保护基”是指适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：甲酰基；酰基，例如链烷酰基(如乙酰基、三氯乙酰基或三氟乙酰基)；烷氧基羰基，如叔丁氧基羰基(Boc)；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；芳基甲基，如苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。术语“羟基保护基”是指适合用于阻止羟基副反应的保护基。代表性羟基保护基包括但不限于：烷基，如甲基、乙基和叔丁基； 酰基，例如链烷酰基(如乙酰基)；芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明所使用的溶剂可经市售获得。
本发明采用下述缩略词：DMF代表N,N-二甲基甲酰胺；Na 2CO 3代表碳酸钠；K 2CO 3代表碳酸钾；Cs 2CO 3代表碳酸铯；CuI代表碘化亚铜；MeI代表碘甲烷；EtOAc代表乙酸乙酯；EA代表乙酸乙酯；THF代表四氢呋喃；LiHMDS代表六甲基二硅基氨基锂；MeOH代表甲醇；DCM代表二氯甲烷；DMSO代表二甲基亚砜；PE代表石油醚；EtOH代表乙醇；IPA代表异丙醇；CH 3CN代表乙腈；MTBE代表甲基叔丁基醚；TFA代表三氟乙酸；NH 3·H 2O代表氨水；TEA代表三乙胺；DIPEA代表N,N-二异丙基乙胺；Boc 2O代表二碳酸二叔丁酯；代表Boc代表叔丁氧羰基，是氨基的一种保护基；NH 2OH·HCl代表羟胺的盐酸盐；Pd(PPh 2)Cl 2代表双三苯基磷二氯化钯；DMP代表戴斯-马丁试剂；PIFA代表二(三氟乙酰氧基)碘代苯；EDTA代表乙二胺四乙酸二钠；LCMS代表液质联用色谱；HPLC代表液相色谱；SFC超临界流体色谱；PO代表灌胃给药；IV代表静脉注射给药；QD代表一天一次给药；BID代表一天两次给药；MPK代表毫克/千克；T 1/2代表半衰期；Vdss代表稳态表观分布容积；CL代表清除率；AUC 0-24h代表给药后0～24小时血药浓度-时间曲线下面积；C max代表最大血药浓度；T max代表达到最大血药浓度的时间。
附图说明
图1为小鼠接受实施例6给药1～7天，各受试组小鼠单位体积血浆中Log(HBV DNA拷贝数)的变化曲线
图2为小鼠接受实施例6给药第7天，各受试组小鼠单位重量肝脏组织中Log(HBV DNA拷贝数)水平
图3为小鼠接受实施例23给药1～7天，各受试组小鼠单位体积血浆中Log(HBV DNA拷贝数)的变化曲线
图4为小鼠接受实施例23给药第7天，各受试组小鼠单位重量肝脏组织中Log(HBV DNA拷贝数)水平
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
中间体的制备
中间体A-1
Figure PCTCN2018115857-appb-000062
中间体A-1由下面的方法制备：
Figure PCTCN2018115857-appb-000063
0摄氏度下将氯甲酸苯酯(58.54克，46.83毫升)缓慢滴加到溶有3,4,5-三氟苯胺(50.00克)和吡啶(29.58克，30.18毫升)的二氯甲烷(300毫升)溶液中。反应混合物在25摄氏度下搅拌3小时后，加入250毫升水淬灭，析出白色沉淀。该沉淀过滤后，减压干燥，得到中间体A-1。
1H NMR(400MHz，CDCl 3)δ：7.46-7.39(m，2H)，7.32-7.27(m，1H),7.21-7.11(m，4H)，4.82(br.s.，1H)；MS(ESI)m/z：268[M+H +]。
中间体A-2
Figure PCTCN2018115857-appb-000064
中间体A-2的制备可参考中间体A-1的制备方法，将3,4,5-三氟苯胺替换成3-氰基-4-氟苯胺。
1H NMR(400MHz，CDCl 3)δ：7.85-7.74(m，1H)，7.67(dd，J＝3.8，8.3Hz，1H)，7.46-7.41(m，2H)，7.31-7.18(m，5H)；MS(ESI)m/z：257[M+H +]。
中间体A-3
Figure PCTCN2018115857-appb-000065
中间体A-3的制备可参考中间体A-1的制备方法，将3,4,5-三氟苯胺替换成3-氯-4-氟苯胺。
1H NMR(400MHz，CDCl 3)δ：7.64(d，J＝4.3Hz，1H)，7.51-7.38(m，3H)，7.32-7.29(m，1H)，7.22-7.10(m，3H)，6.95(br.s.，1H)；MS(ESI)m/z：266[M+H +]。
中间体A-4
Figure PCTCN2018115857-appb-000066
中间体A-4的制备可参考中间体A-1的制备方法，将3,4,5-三氟苯胺替换成3-甲基-4-氟苯胺。
1H NMR(400MHz，CDCl 3)δ：7.49-7.33(m，3H)，7.28-7.15(m，4H)，7.03-6.93(m，1H)，6.96(br.s.，1H)，2.29(s，3H)。MS(ESI)m/z：246[M+H +]。
中间体A-5
Figure PCTCN2018115857-appb-000067
中间体A-5的制备可参考中间体A-1的制备方法，将3,4,5-三氟苯胺替换成2-氯-4-氨基吡啶。
1H NMR(400MHz，CDCl 3)δ：8.19(d，J＝5.6Hz，1H)，7.46(d，J＝1.6Hz，2H)，7.38-7.31(m，2H)，7.25-7.20(m，2H)，7.10(d，J＝7.7Hz，2H)。MS(ESI)m/z：249[M+H +]。
中间体A-6
Figure PCTCN2018115857-appb-000068
中间体A-6的制备可参考中间体A-1的制备方法，将3,4,5-三氟苯胺替换成3,4-二氟苯胺。
1H NMR(400MHz，CDCl 3)δ：7.55-7.47(m，1H)，7.45-7.40(m，2H)，7.32-7.26(m，2H)，7.21-7.20(m，1H)，7.17-7.01(m，3H)。MS(ESI)m/z：250[M+H +]。
实施例1
Figure PCTCN2018115857-appb-000069
实施例1的制备：
Figure PCTCN2018115857-appb-000070
步骤A：化合物1-2的合成
0摄氏度下，向化合物1-1(3.00克)的二氯甲烷(30毫升)溶液中加入草酰氯(5.40克，42.52摩尔)，并随后加入1～2滴N,N-二甲基甲酰胺。该反应混合物自然升至室温，继续搅拌14小时后，减压浓缩得到化合物1-2的粗品。
步骤B：化合物1-3的合成
在-70摄氏度，氮气保护下，将N-Boc-(S)-2-甲基-4-哌啶酮(0.50克)溶解在乙醚(8毫升)中，随后加入六甲基二硅基氨基锂(2.34毫升，1摩尔/升)。反应混合物在-70摄氏度下搅拌0.5小时后，向体系中滴加化合物1-2(374.04毫克)的乙醚(2毫升)溶液。反应液自然升至室温，继续搅拌3小时。将反应液倒入1摩尔/升的盐酸(40毫升)中，用乙酸乙酯(25毫升×2)萃取。合并的有机相经饱和食盐水(40 毫升)洗涤，无水硫酸钠干燥、过滤后，滤液减压浓缩得到化合物1-3的粗品。
步骤C：化合物1-4的合成
搅拌下，向2.5毫升乙醇中依次加入化合物1-3(300.00毫克，粗品)，盐酸羟胺(334.69毫克)和吡啶(2.5毫升)。反应混合物逐渐升温至100摄氏度，并在该温度下搅拌1小时。自然冷却后，减压蒸馏，剩余物用乙酸乙酯(30毫升)稀释，然后依次用1摩尔/升盐酸(20毫升)和饱和食盐水(20毫升)洗涤。有机相经无水硫酸钠干燥、过滤后，滤液减压浓缩。剩余物经制备TLC板分离(展开剂：石油醚：乙酸乙酯＝2：1)得到化合物1-4。
1H NMR(400MHz，CDCl 3)δ：8.63-8.48(m，1H)，7.92(dd，J＝4.2，8.6Hz，1H)，7.60-7.45(m，1H)，5.96-4.79(m，1H)，4.58-4.21(m，1H)，3.29-3.05(m，1H)，3.02-2.72(m，2H)，1.59-1.35(m，12H)。MS(ESI)m/z：334[M+H +]。
步骤D：化合物1-5的合成
室温下，将化合物1-4(93.00毫克)加入到盐酸/1,4-二氧六环溶液(4摩尔/升，5毫升)中。该反应混合物在室温下搅拌0.5小时后，减压蒸馏得到粗产品化合物1-5(盐酸盐)，直接用于下一步反应。MS(ESI)m/z：234[M+H +]。
步骤E：实施例1的合成
室温下，将化合物1-5(盐酸盐，75.00毫克)溶解在2毫升N,N-二甲基甲酰胺中，随后依次加入N,N-二异丙基乙胺(107.82毫克)和中间体A-1(74.31毫克)。反应混合物在70摄氏度下搅拌1小时后，缓慢倒入30毫升水中，然后用乙酸乙酯(20毫升×2)萃取。合并的有机相经饱和食盐水(30毫升)洗涤，无水硫酸钠干燥、过滤后，滤液减压浓缩。剩余物经过制备高效液相色谱纯化(分离柱：Phenomenex Synergi C18，150×30mm×4μm，流动相：[水(0.225％三氟乙酸)-乙腈]；B％：50％-80％，10.5min)得到实施例1。
1HNMR(400MHz，CD 3OD)δ：8.63(d，J＝2.8Hz，1H)，8.00-7.96(m，1H)，7.78-7.75(m，1H)，7.34-7.14(m，2H)，5.29(d，J＝17.5Hz，1H)，4.95(quin，J＝6.4Hz，1H)，4.50(d，J＝17.4Hz，1H)，3.06(dd，J＝5.7，16.4Hz，1H)，2.87(dd，J＝1.2，16.3Hz，1H)，1.22(d，J＝7.0Hz，3H)。MS(ESI)m/z：407[M+H +]。
实施例2
Figure PCTCN2018115857-appb-000071
实施例2可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成苯甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AS-H(规格：250mm×30mm，粒径：5μm)；流动相：[0.1％NH 3·H 2O，EtOH]；B％：30％-30％，7.8min)得到实施例2。
1H NMR(400MHz，CD 3OD)δ：7.86-7.77(m，2H)，7.69-7.48(m，3H)，7.36-7.21(m，2H)，5.21(d，J＝15.94Hz，1H)，4.65-4.47(m，2H)，3.17-3.02(m，1H)，2.90(dd，J＝1.44，16.38Hz，1H)， 1.27(d，J＝6.90Hz，3H)。MS(ESI)m/z：388[M+H +]。
实施例3
Figure PCTCN2018115857-appb-000072
实施例3可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成2-氟苯甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AD(规格：250mm×30mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，MeOH]；B％：20％-20％，4.0min)得到实施例3。
1H NMR(400MHz，CD 3OD)δ：7.81(dt，J＝1.63，7.53Hz，1H)，7.67-7.54(m，1H)，7.43-7.20(m，4H)，5.10(d，J＝16.69Hz，1H)，4.92-4.78(m，1H)，4.38(d，J＝16.81Hz，1H)，3.10(dd，J＝5.90，16.56Hz，1H)，2.91(d，J＝16.31Hz，1H)，1.29(d，J＝6.90Hz，3H)。MS(ESI)m/z：406[M+H +]。
实施例4
Figure PCTCN2018115857-appb-000073
实施例4可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成3-氟苯甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AS(规格：250mm×50mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，IPA]；B％：50％-50％，1.9min)得到实施例4。
1H NMR(400MHz，CD 3OD)δ：7.62-7.56(m，2H)，7.52(dd，J＝1.3，8.7Hz，1H)，7.31-7.21(m，3H)，5.16(d，J＝16.1Hz，1H)，4.99(quin，J＝6.3Hz，1H)，4.49(d，J＝16.1Hz，1H)，3.09-3.00(m，1H)，2.92-2.83(m，1H)，1.23(d，J＝6.8Hz，3H)。MS(ESI)m/z：406[M+H +]。
实施例5
Figure PCTCN2018115857-appb-000074
实施例5可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成4-氟苯甲酸。粗产品经SFC 制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AS-H(规格：250mm×30mm，粒径：5μm)；流动相：[0.1％NH 3·H 2O，EtOH]；B％：30％-30％，4.2min)得到实施例5。
1H NMR(400MHz，CD 3OD)δ：7.90-7.80(m，2H)，7.42-7.23(m，4H)，5.24-5.14(m，1H)，5.01(quin，J＝6.3Hz，1H)，4.50(d，J＝16.1Hz，1H)，3.08(dd，J＝5.8，16.3Hz，1H)，2.89(dd，J＝1.2，16.4Hz，1H)，1.26(d，J＝6.9Hz，3H)。MS(ESI)m/z：406[M+H +]。
实施例6
Figure PCTCN2018115857-appb-000075
实施例6可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成2，4-二氟苯甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AS(规格：250mm×30mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，IPA]；B％：30％-30％，9min)得到实施例6。
1H NMR(400MHz，CD 3OD)δ：7.86-7.75(m，1H)，7.26-7.14(m，4H)，5.05(d，J＝16.4Hz，1H)，4.93(quin，J＝6.4Hz，1H)，4.38-4.27(m，1H)，3.06(dd，J＝5.9，16.4Hz，1H)，2.91-2.82(m，1H)，1.25(d，J＝6.9Hz，3H)。MS(ESI)m/z：424[M+H +]。
实施例7
Figure PCTCN2018115857-appb-000076
实施例7可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成2-甲氧基苯甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AD(规格：250mm×30mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，MeOH]；B％：30％-30％，2.5min)得到实施例7。
1H NMR(400MHz，CD 3OD)δ：7.60(dd，J＝1.6，7.6Hz，1H)，7.53-7.45(m，1H)，7.25-7.12(m，3H)，7.07(t，J＝7.5Hz，1H)，4.99(d，J＝16.6Hz，1H)，4.94-4.88(m，1H)，4.24(d，J＝16.6Hz，1H)，3.94(s，3H)，3.04(dd，J＝5.9，16.4Hz，1H)，2.83(dd，J＝1.0，16.3Hz，1H)，1.26(d，J＝6.8Hz，3H)。MS(ESI)m/z：418[M+H +]。
实施例8
Figure PCTCN2018115857-appb-000077
实施例8可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成3-甲氧基苯甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AD(规格：250mm×30mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，EtOH]；B％：30％-30％，3.0min)得到实施例8。
1H NMR(400MHz，CD 3OD)：δ7.51-7.43(m，1H)，7.34-7.23(m，4H)，7.07(dd，J＝2.1，8.2Hz，1H)，5.15(d，J＝16.1Hz，1H)，4.97(quin，J＝6.4Hz，1H)，4.47(d，J＝16.0Hz，1H)，3.88(s，3H)，3.05(dd，J＝5.7，16.3Hz，1H)，2.86(dd，J＝1.1，16.3Hz，1H)，1.24(d，J＝6.8Hz，3H)。MS(ESI)m/z：418[M+H +]。
实施例9
Figure PCTCN2018115857-appb-000078
实施例9可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成4-甲氧基苯甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALCEL OD(规格：250mm×50mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，IPA]；B％：30％-30％，4.2min)得到实施例9。
1H NMR(400MHz，CD 3OD)δ：7.75(d，J＝8.9Hz，2H)，7.33-7.25(m，2H)，7.14(d，J＝9.0Hz，2H)，5.16(d，J＝15.8Hz，1H)，5.05-4.97(m，1H)，4.48(d，J＝15.9Hz，1H)，3.90(s，3H)，3.06(dd，J＝5.6，16.4Hz，1H)，2.87(d，J＝16.3Hz，1H)，1.26(d，J＝6.9Hz，3H)。MS(ESI)m/z：418[M+H +]。
实施例10
Figure PCTCN2018115857-appb-000079
实施例10可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成3-氰基苯甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AD(规格：250mm×30mm，粒径：10μm)； 流动相：[0.1％NH 3·H 2O，IPA]；B％：30％-30％，3.1min)得到实施例10。
1H NMR(400MHz，CD 3OD)δ：8.17-8.06(m，2H)，7.88(d，J＝7.8Hz，1H)，7.81-7.72(m，1H)，7.33-7.22(m，2H)，5.23(d，J＝16.3Hz，1H)，5.08-4.96(m，1H)，4.62-4.58(m，1H)，3.09(dd，J＝5.7，16.4Hz，1H)，2.91(dd，J＝1.3，16.3Hz，1H)，1.26(d，J＝6.9Hz，3H)。MS(ESI)m/z：413[M+H +]。
实施例11
Figure PCTCN2018115857-appb-000080
实施例11可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成3-氯苯甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AS-H(规格：250mm×30mm，粒径：5μm)；流动相：[0.1％NH 3·H 2O，MeOH]；B％：35％-35％，5min)得到实施例11。
1H NMR(400MHz，CD 3OD)δ：7.71-7.55(m，2H)，7.51-7.38(m，2H)，7.16(dd，J＝6.3，10.3Hz，2H)，5.08(d，J＝16.0Hz，1H)，4.94-4.82(m，1H)，4.50-4.37(m，1H)，3.04-2.91(m，1H)，2.78(br.d，J＝16.4Hz，1H)，1.14(d，J＝6.8Hz，3H)。MS(ESI)m/z：422[M+H +]。
实施例12
Figure PCTCN2018115857-appb-000081
实施例12可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成4-氯苯甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AS-H(规格：250mm×30mm，粒径：5μm)；流动相：[0.1％NH 3·H 2O，EtOH]；B％：30％-30％，8.3min)得到实施例12。
1H NMR(400MHz，CD 3OD)δ：7.73-7.63(m，2H)，7.52-7.45(m，2H)，7.17(dd，J＝6.36，10.27Hz，2H)，5.08(d，J＝16.14Hz，1H)，4.94-4.84(m，1H)，4.39(d，J＝16.02Hz，1H)，2.96(dd，J＝5.75，16.38Hz，1H)，2.78(dd，J＝1.28，16.32Hz，1H)，1.14(d，J＝6.97Hz，3H)。MS(ESI)m/z：422[M+H +]。
实施例13
Figure PCTCN2018115857-appb-000082
实施例13可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成吡嗪-2-甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AS-H(规格：250mm×30mm，粒径：5μm)；流动相：[0.1％NH 3·H 2O，MeOH]；B％：60％-60％，4.5min)得到实施例13。
1H NMR(400MHz，CD 3OD)δ：9.17(d，J＝1.5Hz，1H)，8.81(dd，J＝1.6，2.4Hz，1H)，8.67(d，J＝2.5Hz，1H)，7.32-7.22(m，2H)，5.37(d，J＝17.6Hz，1H)，5.03-4.96(m，1H)，4.57(d，J＝17.7Hz，1H)，3.12(dd，J＝5.8，16.4Hz，1H)，2.94(dd，J＝1.4，16.4Hz，1H)，1.26(d，J＝6.9Hz，3H)。MS(ESI)m/z：390[M+H +]。
实施例14
Figure PCTCN2018115857-appb-000083
实施例14可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成噻唑-5-甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALCEL OD(规格：250mm×30mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，MeOH]；B％：30％-30％，2.3min)得到实施例14。
1H NMR(400MHz，CD 3OD)δ：9.12(s，1H)，8.26(s，1H)，7.24-7.11(m，2H)，5.03(d，J＝16.38Hz，1H)，4.90(t，J＝6.72Hz，1H)，4.33(d，J＝16.38Hz，1H)，3.02-2.92(m，1H)，2.79(dd，J＝1.41，16.44Hz，1H)，1.14(d，J＝6.85Hz，3H)。MS(ESI)m/z：395[M+H +]。
实施例15
Figure PCTCN2018115857-appb-000084
实施例15可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成噻唑-4-甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AD(规格：250mm×30mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，IPA]；B％：30％-30％，3.0min)得到实施例15。
1H NMR(400MHz，CD 3OD)δ：9.20(d，J＝2.0Hz，1H)，8.18(d，J＝1.9Hz，1H)，7.37-7.21(m， 2H)，5.30(d，J＝16.8Hz，1H)，5.05-4.95(m，1H)，4.52(d，J＝16.8Hz，1H)，3.09(dd，J＝5.7，16.4Hz，1H)，2.90(d，J＝16.3Hz，1H)，1.26(d，J＝6.9Hz，3H)。MS(ESI)m/z：395[M+H +]。
实施例16
Figure PCTCN2018115857-appb-000085
实施例16可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成噻唑-2-甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AS-H(规格：250mm×30mm，粒径：5μm)；流动相：[0.1％NH 3·H 2O，MeOH]；B％：35％-35％，6.3min)得到实施例16。
1H NMR(400MHz，CD 3OD)δ：9.01(s，1H)，7.75(d，J＝4.8Hz，1H)，7.16(dd，J＝6.4，10.3Hz，2H)，5.21(d，J＝17.2Hz，1H)，4.93-4.84(m，1H)，4.50-4.41(m，1H)，3.05-2.95(m，1H)，2.80(d，J＝16.5Hz，1H)，1.14(d，J＝6.8Hz，3H)。MS(ESI)m/z：395[M+H +]。
实施例17
Figure PCTCN2018115857-appb-000086
实施例17可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成异噻唑-4-甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AD(规格：250mm×30mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，IPA]；B％：30％-30％，30min)得到实施例17。
1H NMR(400MHz，CD 3OD)δ：9.40(s，1H)，9.00(s，1H)，7.33-7.27(m，2H)，5.18(d，J＝16.0Hz，1H)，5.08-5.00(m，1H)，4.50(d，J＝16.0Hz，1H)，3.08(dd，J＝5.7，16.4Hz，1H)，2.94-2.88(m，1H)，1.25(d，J＝7.0Hz，3H)。MS(ESI)m/z：395[M+H +]。
实施例18
Figure PCTCN2018115857-appb-000087
实施例18可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成异噻唑-3-甲酸。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AD-H(规格：250mm×30mm，粒径：5μm)； 流动相：[0.1％NH 3·H 2O，MeOH]；B％：35％-35％，2.1min)得到实施例18。
1H NMR(400MHz，CD 3OD)δ：8.00(d，J＝3.2Hz，1H)，7.75(d，J＝3.2Hz，1H)，7.20-7.13(m，2H)，5.18(d，J＝17.2Hz，1H)，4.92-4.85(m，1H)，4.51-4.37(m，1H)，3.04-2.96(m，1H)，2.85-2.78(m，1H)，1.14(d，J＝7.0Hz，3H)。MS(ESI)m/z：395[M+H +]。
实施例19
Figure PCTCN2018115857-appb-000088
实施例19的制备方法可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成2,4-二氟苯甲酸；将中间体A-1替换成中间体A-2。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALCEL OJ(规格：250mm×30mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，EtOH]；B％：25％-25％，2.4min)得到实施例19。
1H NMR(400MHz，CD 3OD)δ：7.89-7.76(m，2H)，7.72-7.64(m，1H)，7.29-7.16(m，3H)，5.08(d，J＝16.4Hz，1H)，4.96(quin，J＝6.4Hz，1H)，4.36(d，J＝16.6Hz，1H)，3.08(dd，J＝5.8，16.4Hz，1H)，2.93-2.85(m，1H)，1.27(d，J＝6.8Hz，3H)。MS(ESI)m/z：413[M+H +]。
实施例20
Figure PCTCN2018115857-appb-000089
实施例20的制备方法可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成2,4-二氟苯甲酸；将中间体A-1替换成中间体A-3。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AS(规格：250mm×50mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，MeOH]；B％：40％-40％，1.9min)得到实施例20。
1H NMR(400MHz，CD 3OD)δ：7.85-7.79(m，1H)，7.59(dd，J＝2.6，6.8Hz，1H)，7.36-7.08(m，4H)，5.09(dd，J＝1.1，16.6Hz，1H)，4.97-4.94(m，1H)，4.36(dd，J＝1.5，16.6Hz，1H)，3.10(dd，J＝5.9，16.4Hz，1H)，2.90(dd，J＝1.3，16.4Hz，1H)，1.28(d，J＝6.9Hz，3H)。MS(ESI)m/z：422[M+H +]。
实施例21
Figure PCTCN2018115857-appb-000090
实施例21的制备可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成2,4-二氟苯甲酸；将中间体A-1替换成中间体A-4。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AD(规格：250mm×30mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，EtOH]；B％：20％-20％，5.5min)得到实施例21。
1H NMR(400MHz，CD 3OD)δ：7.87-7.83(m，1H)，7.30-7.10(m，4H)，6.96-6.94(m，1H)，5.09(d，J＝16.6Hz，1H)，5.02-4.94(m，1H)，4.35(d，J＝16.4Hz，1H)，3.16-3.06(m，1H)，2.89(d，J＝16.4Hz，1H)，2.24(s，3H)，1.28(d，J＝6.9Hz，3H)。MS(ESI)m/z：402[M+H +]。
实施例22
Figure PCTCN2018115857-appb-000091
实施例22的制备可参考实施例1的制备流程A至E，将5-氟吡啶-2-甲酸替换成2,4-二氟苯甲酸；将中间体A-1替换成中间体A-5。粗产品经SFC制备分离(SFC分离方法：分离柱：DAICEL CHIRALPAK AD(规格：250mm×50mm，粒径：10μm)；流动相：[0.1％NH 3·H 2O，MeOH]；B％：30％-30％，1.8min)得到实施例22。
1H NMR(400MHz，CD 3OD)δ：8.00(d，J＝5.9Hz，1H)，7.77-7.69(m，1H)，7.53(d，J＝1.7Hz，1H)，7.31(dd，J＝1.8，5.7Hz，1H)，7.16-7.06(m，2H)，4.99(d，J＝16.5Hz，1H)，4.90-4.83(m，1H)，4.27(d，J＝16.6Hz，1H)，2.99(dd，J＝5.9，16.5Hz，1H)，2.79(d，J＝16.5Hz，1H)，1.17(d，J＝6.8Hz，3H)。MS(ESI)m/z：405[M+H +]。
实施例23
Figure PCTCN2018115857-appb-000092
实施例23的制备：
Figure PCTCN2018115857-appb-000093
步骤A：化合物23-2的合成
0摄氏度下，化合物向23-1(50.0克)和碳酸钾(77.53克)的乙腈(500毫升)溶液中滴加3-溴丙炔(66.73克)。该反应混合物自然升至室温，继续搅拌12小时后，减压浓缩得到残余物。该残余物用水稀释，并用乙酸乙酯萃取，合并的有机相经饱和食盐水洗涤，无水硫酸钠干燥、过滤后，滤液减压浓缩得到化合物23-2的粗品。MS(ESI)m/z：128[M+H +]。
步骤B：化合物23-3的合成
室温下，向化合物23-2(52.0克)的四氢呋喃(500毫升)溶液中加入碳酸钾(26.56克)和Boc 2O(41.94克)。该反应液在18摄氏度下搅拌12小时后，过滤除去固体，收集滤液并减压浓缩。将得到的残余物溶解在乙酸乙酯(800毫升)中，该有机相依次用水、饱和柠檬酸溶液和饱和碳酸氢钠溶液洗涤，经无水硫酸钠干燥、过滤后，滤液减压浓缩得到化合物23-3的粗品。MS(ESI)m/z：228[M+H +]。
步骤C：化合物23-4的合成
0摄氏度下，向化合物23-3(40.0克)的二氯甲烷(400毫升)溶液中分批加入戴斯-马丁试剂(82.10克)。该反应液混合物自然升至室温并搅拌2小时后，用体积比1:1的饱和碳酸氢钠溶液和饱和硫代硫酸钠溶液洗涤两次，再依次用饱和碳酸氢钠溶液和饱和食盐水洗涤有机相，无水硫酸钠干燥、过滤后，滤液减压浓缩得到化合物23-4的粗品。MS(ESI)m/z：226[M+H +]。
步骤D：化合物23-5的合成
室温下，向化合物23-4(42.0克)的乙醇(400毫升)和水(40毫升)的混合溶液中加入乙酸钠(22.94克)和盐酸羟胺(16.84克)。该反应混合物在室温下搅拌12小后，减压浓缩除去乙醇得到残余物。将该残余物用乙酸乙酯溶解后，依次用水和饱和食盐水洗涤，再经无水硫酸钠干燥、过滤后，滤液减压浓缩得到化合物23-5的粗品。MS(ESI)m/z：241[M+H +]。
步骤E：化合物23-6的合成
室温下，向化合物23-5(300毫克)和2-碘嘧啶(257.16毫克)的N,N-二甲基甲酰胺(8毫升)溶液 中加入碘化亚铜(11.89毫克)，三乙胺(252.66毫克)和双三苯基膦二氯化钯(43.81毫克)。氮气保护下，该反应混合物在12摄氏度下搅拌12小时。将反应液用乙酸乙酯稀释后过滤，滤液减压浓缩，剩余物经制备TLC板分离得到化合物23-6。MS(ESI)m/z：319[M+H +]。
步骤F：化合物23-7的合成
室温下，向化合物23-6(180毫克)的甲醇(4毫升)和水(0.8毫升)的混合溶液中，分批加入二(三氟乙酰氧基)碘代苯(291.76毫克)。该反应混合物在10～20摄氏度下搅拌0.5小时后，用20毫升水稀释，然后用乙酸乙酯萃取。有机相经无水硫酸钠干燥，过滤，滤液减压浓缩，剩余物经制备TLC板分离得到化合物23-7。MS(ESI)m/z：317[M+H +]。
步骤G：化合物23-8的合成
将化合物23-7(90毫克)溶解于盐酸的二氧六环溶液(4摩尔/升，4毫升)中，该反应混合物在20摄氏度下搅拌0.5小时后，减压浓缩得到化合物23-8的粗品。MS(ESI)m/z：217[M+H +]。
步骤H：实施例23的合成
室温下，向化合物23-8(61.52毫克)的N,N-二甲基甲酰胺(2毫升)溶液中加入N,N-二异丙基乙胺(110.31毫克)和中间体A-2(72.90毫克)。该反应混合物在70摄氏度下搅拌1小时。将反应液用20毫升水稀释后，用乙酸乙酯萃取。有机相经无水硫酸钠干燥、过滤。滤液减压浓缩后，剩余物经制备高效液相色谱纯化(分离柱：Phenomenex Synergi C18，150×30mm×4μm，流动相：[水(0.225％三氟乙酸)-乙腈]；B％：35％-65％，10min)得到实施例23。
1H NMR(400MHz，CD 3OD)δ：8.97(d，J＝5.0Hz，2H)，7.83(dd，J＝2.8，5.6Hz，1H)，7.75-7.71(m，1H)，7.52(t，J＝5.0Hz，1H)，7.30(t，J＝9.0Hz，1H)，5.42(d，J＝17.9Hz，1H)，5.01(quin，J＝6.4Hz，1H)，4.59(d，J＝17.7Hz，1H)，3.14(dd，J＝5.7，16.4Hz，1H)，2.95(dd，J＝1.2，16.4Hz，1H)，1.27(d，J＝6.8Hz，3H)。MS(ESI)m/z：379[M+H +]。
实施例24
Figure PCTCN2018115857-appb-000094
实施例24的制备可参考实施例23的制备流程A至H，将中间体A-2替换成中间体A-3。
1H NMR(400MHz，DMSO-d 6)δ：9.04(d，J＝4.9Hz，2H)，9.01(s，1H)，7.74(dd，J＝2.4，6.8Hz，1H)，7.60(t，J＝4.9Hz，1H)，7.44-7.40(m，1H)，7.35-7.27(m，1H)，5.36(d，J＝18.0Hz，1H)，4.98-4.83(m，1H)，4.36(d，J＝18.0Hz，1H)，3.12-2.99(m，1H)，2.94-2.83(m，1H)，1.16(d，J＝6.8Hz，3H)。MS(ESI)m/z：388[M+H +]。
实施例25
Figure PCTCN2018115857-appb-000095
实施例25的制备可参考实施例23的制备流程A至H，将中间体A-2替换成中间体A-1。
1H NMR(400MHz，CDCl 3)δ：8.86(d，J＝4.89Hz，2H)，7.29(t，J＝4.89Hz，1H)，7.08(dd，J＝6.05，9.48Hz，2H)，6.45(s，1H)，5.07(d，J＝16.75Hz，1H)，5.05-4.94(m，1H)，4.55(d，J＝16.87Hz，1H)，3.10-3.00(m，1H)，2.94-2.85(m，1H)，1.17(d，J＝6.97Hz，3H)。MS(ESI)m/z：390[M+H +]。
实施例26
Figure PCTCN2018115857-appb-000096
实施例26的制备可参考实施例23的制备流程A至H，将中间体A-2替换成中间体A-6。
1H NMR(400MHz，DMSO-d 6)δ：9.03(s，3H)，7.60(s，2H)，7.43-7.09(m，2H)，5.36(d，J＝17.9Hz，1H)，4.90(s，1H)，4.36(d，J＝18.1Hz，1H)，3.06(d，J＝12.1Hz，1H)，2.89(d，J＝16.4Hz，1H)，1.15(d，J＝5.4Hz，3H)。MS(ESI)m/z：372[M+H +]。
活性测试
1.体外抗HBV活性测试
1)将100微升的HepG2.2.15细胞以每孔1.2×10 5细胞数种在96孔板细胞培养板里，细胞在37℃含5％二氧化碳(CO 2)的培养箱里培养过夜。第二天，用DMSO三倍梯度稀释待测化合物，共稀释8个浓度，然后用培养基再将化合物稀释100倍，并取100微升稀释的化合物加入含细胞的培养板，终体积是200微升，DMSO的终浓度是0.5％，双复孔。细胞在37℃含5％CO 2的培养箱里培养3天。第五天，用新鲜的含有相同浓度化合物的培养基给细胞培养板换液。当培养到第八天时，收集细胞培养板上清，用于提取HBV DNA。
2)实时定量PCR检测HBV DNA：用QIAamp 96 DNA Blood Kit试剂盒提取上清中总的DNA，通过HBV特异性引物及探针对HBV DNA含量进行定量PCR检测。把20微升的PCR预混液与5微升的HBV DNA样品或HBV质粒标准品加入到定量PCR板进行反应。HBV质粒标准品以10倍梯度从10 7到10拷贝数每微升稀释7个点。定量PCR反应程序如下：95℃预变性10分钟；95℃变性15秒，60℃反应1分钟，重复该循环40次。按下列公式计算各孔对HBV DNA的抑制率，并使用GraphPad Prism软件对化合物的抑制率数据进行非线性拟合分析，得到化合物的EC 50值。
HBV DNA抑制率％＝(1-样品HBV DNA拷贝数/DMSO对照HBV DNA拷贝数)×100％
试验结果如下表1所示。
表1
Figure PCTCN2018115857-appb-000097
2.小鼠药代动力学研究
本实验旨在评价化合物在小鼠体内单次静脉注射或灌胃给药后的药代动力学行为。静脉注射给药，化合物配制成0.5mg/mL的澄清溶液，溶媒：5％DMSO/5％十二羟基硬脂酸脂(solutol)/90％水；灌胃给药，化合物配制成2mg/mL的混悬液，溶媒：0.5％羧甲基纤维素钠/0.2％吐温80/99.3％水。
化合物在血浆中的浓度由高效液相色谱-串联质谱(LC-MS/MS)进行测定。化合物和内标(双氯芬酸)的保留时间、色谱图采集和色谱图的积分采用软件Analyst(Applied Biosystems)进行处理，数据的统计采用软件Watson LIMS(Thermo Fisher Scientific)或Analyst(Applied Biosystems)进行处理。样品中分析物浓度单位为ng/mL，保留3位有效数字，所有以百分数表示的数值(如：％偏差和％变异系数等)均保留到小数点后一位。每条校正曲线至少包含6个浓度水平。校正标样的配制需采用和质控样品不同来源的储备液。校正标样算得的浓度与标示值的偏差超出±15.0％(定量下限超出±20.0％)回归分析中应拒绝该标样。被拒绝的校正标样应小于25％，且每条校正曲线至少包含6个符合接受标准的校正标样。如定量下限和定量上限校正标样需要拒绝时，该分析批的定量上限和下限将相应的提高和降低。
采用WinNonlin TM Version 6.3(Pharsight,Mountain View,CA)药动学软件的非房室模型处理血浆浓度，使用线性对数梯形法方法计算药动学参数。需计算的药动学参数包含但不局限于(数据允许)静脉注射组的T 1/2、Vdss、CL、AUC 0-24h；口服灌胃组的C max、T max、AUC 0-24h、生物利用度(F％)。
本发明实施例在小鼠体内的药代动力学相关参数如下表2所示。
表2
Figure PCTCN2018115857-appb-000098
3.小鼠尾静脉高压水注射HBV DNA质粒模型体内活性测试
此研究的目的是通过高压尾静脉注射的小鼠模型来检测化合物(实施例6)在小鼠体内对HBV的抑制 效果。本实验采用雌性BABL/c小鼠，鼠龄6-7周。HBV质粒DNA采用pAAV2-HBV 1.3mer，使用Qiagen EndoFree Plasmid Giga试剂盒提取，浓度为1000ng/μL。使用前用正常生理盐水稀释，保存于4℃直至使用。
3.1动物分组
实验动物分组情况如下表3所示：
表3
Figure PCTCN2018115857-appb-000099
*：第7天只给药一次。
3.2药物配制
药物配制情况如下表4所示：
表4
Figure PCTCN2018115857-appb-000100
3.3给药安排
表5
Figure PCTCN2018115857-appb-000101
*：第7天只给一次药。
3.4非终点取血的血样收集及转运
第1、3、5天，当天第一次给药后约4小时每只小鼠取100μL全血，将全血收集到加有肝素钠的管子中，4℃离心，7000转，离心10分钟，取上清，即得到血浆。血浆保存在-80℃冰箱，干冰冷冻条件下运送至分析实验室进行检测。
3.5定量PCR检测小鼠血浆中HBV DNA的含量
1)提取血浆中DNA，实验步骤参照QIAamp 96 DNA Blood Kit说明书。
2)定量PCR检测小鼠血浆中HBV DNA的含量
配制qPCR反应混合液(见表6)。将qPCR反应混合液、样品和标准品加入到96孔反应板中。其中 标准品为含有D型HBV全长序列的质粒DNA，将标准品从107拷贝数/μL，10倍系列稀释，依次得到10 6～10 1拷贝数/μL的DNA标准品。PCR反应：95℃，10分钟；95℃，15秒，60℃，1分钟，40个循环。
表6 qPCR反应成份表
Figure PCTCN2018115857-appb-000102
HBV DNA含量＝HBV引物检测的DNA含量-pAAV2引物检测的DNA含量。
3.6定量PCR检测小鼠肝脏中HBV DNA的含量
1)肝脏总DNA的提取
取肝脏组织，用组织研磨机匀浆。离心后取上清转移到新的离心管中，加入蛋白酶K消化液消化3小时。待冷却后，加入RNA酶A孵育30分钟。将RNA酶A处理后的混合液用等体积的酚氯仿异戊醇抽提两次以去除残余的蛋白。将上清转移到新离心管中，加入异丙醇沉淀DNA。将DNA沉淀用70％乙醇洗两遍。将沉淀风干，加入TE(10mM Tris-HCl，pH 8.0，1mM EDTA)溶解DNA。
2)定量PCR检测小鼠肝脏中HBV DNA的含量
用Nanodrop测定DNA浓度，将所有样品的DNA浓度调整到10ng/μL。加5μL样品到定量PCR反应体系中，进行定量PCR。
HBV DNA含量＝HBV引物检测的DNA含量-pAAV2引物检测的DNA含量。
3.7实验结果
1)各受试组小鼠分别在给药后第1、3、5、7天采集血样中，HBV DNA浓度的检测值(Log HBV DNA)如下表7所示：
表7
Figure PCTCN2018115857-appb-000103
LLOQ＝40拷贝数/μL，Log(LLOQ)＝1.60(LLOQ表示最低检测下限)
这一结果如图1所示。
2)给药7天后，各受试组动物肝脏组织中HBV DNA浓度的检测值(Log HBV DNA)如下表8所示：
表8
Figure PCTCN2018115857-appb-000104
这一结果如图2所示。
4.小鼠尾静脉高压水注射HBV DNA质粒模型体内活性测试
此研究的目的是通过高压尾静脉注射的小鼠模型来检测化合物(实施例23)在小鼠体内对HBV的抑制效果。本实验采用雌性BABL/c小鼠，鼠龄6-7周。HBV质粒DNA采用pAAV2-HBV 1.3mer，使用Qiagen EndoFree Plasmid Giga试剂盒提取，浓度为1000ng/μL。使用前用正常生理盐水稀释，保存于4℃直至使用。
4.1动物分组
实验动物分组情况如下表9所示：
表9
Figure PCTCN2018115857-appb-000105
*：第7天只给药一次。
4.2药物配制
药物配制情况如下表10所示：
表10
Figure PCTCN2018115857-appb-000106
4.3给药安排
表11
Figure PCTCN2018115857-appb-000107
*：第7天只给一次药。
4.4实验结果
1)各受试组小鼠分别在给药后第1、3、5、7天采集血样中，HBV DNA浓度的检测值(Log HBV DNA)如下表12所示：
表12
Figure PCTCN2018115857-appb-000108
这一结果如图3所示。
2)给药7天后，各受试组动物肝脏组织中HBV DNA浓度的检测值(Log HBV DNA)如下表13所示：
表13
Figure PCTCN2018115857-appb-000109
这一结果如图4所示。
Figure PCTCN2018115857-appb-000110

Claims (20)

  1. 根据权利要求1所述的化合物、其药学上可接受的盐或其异构体，其中，所述结构单元
    Figure PCTCN2018115857-appb-100001
    为
    Figure PCTCN2018115857-appb-100002
  2. 根据权利要求8所述的化合物、其药学上可接受的盐或其异构体，其中，所述结构单元
    Figure PCTCN2018115857-appb-100003
    为
    Figure PCTCN2018115857-appb-100004
    Figure PCTCN2018115857-appb-100005
  3. 根据权利要求9所述的化合物、其药学上可接受的盐或其异构体，其中，所述结构单元
    Figure PCTCN2018115857-appb-100006
    为
    Figure PCTCN2018115857-appb-100007
    Figure PCTCN2018115857-appb-100008
    Figure PCTCN2018115857-appb-100009
  4. 根据权利要求1所述的化合物、其药学上可接受的盐或其异构体，其中，所述结构单元
    Figure PCTCN2018115857-appb-100010
    为
    Figure PCTCN2018115857-appb-100011
  5. 根据权利要求1或11所述的化合物、其药学上可接受的盐或其异构体，其中，所述结构单元
    Figure PCTCN2018115857-appb-100012
    为
    Figure PCTCN2018115857-appb-100013
  6. 根据权利要求12所述的化合物、其药学上可接受的盐或其异构体，其中，所述结构单元
    Figure PCTCN2018115857-appb-100014
    为
    Figure PCTCN2018115857-appb-100015
    Figure PCTCN2018115857-appb-100016
  7. 根据权利要求1～4或6～10任一项所述的化合物、其药学上可接受的盐或其异构体，其具有式(II-a)或式(III-a)所示结构：
    Figure PCTCN2018115857-appb-100017
    其中，环A如权利要求1～3或8～10所定义；
    R 2和R 4如权利要求1或7所定义；
    R如权利要求1或5所定义；
    n如权利要求1所定义。
  8. 根据权利要求14所述的化合物、其药学上可接受的盐或其异构体，其具有式(II-a-1)或式(III-a-1)所示结构：
    Figure PCTCN2018115857-appb-100018
    其中，环A如权利要求1～3或8～10所定义；
    R 2和R 4如权利要求1或7所定义；
    R如权利要求1或5所定义；
    n如权利要求1所定义。
  9. 根据权利要求1、5或7所述的化合物、其药学上可接受的盐或其异构体，其具有式(II-b)、(II-c)、(III-b)或(III-c)所示结构：
    Figure PCTCN2018115857-appb-100019
    其中，T 1和T 2各自独立地为N或CH；
    R 2和R 4如权利要求1或7所定义；
    R如权利要求1或5所定义；
    n如权利要求1所定义。
  10. 根据权利要求16所述的化合物、其药学上可接受的盐或其异构体，其具有式(II-b-1)、(II-c-1)、(III-b-1)或(III-c-1)所示结构：
    Figure PCTCN2018115857-appb-100020
    其中，T 1和T 2各自独立地为N或CH；
    R 2和R 4如权利要求1或7所定义；
    R如权利要求1或5所定义；
    n如权利要求1所定义。
  11. 根据权利要求16所述的化合物、其药学上可接受的盐或其异构体，其具有式(II-d)、(II-e)、(II-f)、(II-k)、(III-d)、(III-e)、(III-f)或(III-k)所示结构：
    Figure PCTCN2018115857-appb-100021
    Figure PCTCN2018115857-appb-100022
    其中，R 2和R 4如权利要求1或7所定义；
    R如权利要求1或5所定义；
    n如权利要求1所定义。
  12. 根据权利要求18所述的化合物、其药学上可接受的盐或其异构体，其具有式(II-d-1)、(II-e-1)、(II-f-1)、(II-k-1)、(III-d-1)、(III-e-1)、(III-f-1)或(III-k-1)所示结构：
    Figure PCTCN2018115857-appb-100023
    其中，R 2和R 4如权利要求1或7所定义；
    R如权利要求1或5所定义；
    n如权利要求1所定义。
  13. 根据权利要求18所述的化合物、其药学上可接受的盐或其异构体，其具有式(II-m)或(III-m)所示结构：
    Figure PCTCN2018115857-appb-100024
    其中，R 2和R 4如权利要求1或7所定义。
  14. 根据权利要求20所述的化合物、其药学上可接受的盐或其异构体，其具有式(II-m-1)或(III-m-1)所示结构：
    Figure PCTCN2018115857-appb-100025
    其中，R 2和R 4如权利要求1或7所定义。
  15. 根据权利要求14所述的化合物、其药学上可接受的盐或其异构体，其具有式(II-g)～(II-i)或式(III-g)～(III-i)所示结构：
    Figure PCTCN2018115857-appb-100026
    其中，R 2和R 4如权利要求1或7所定义；
    R如权利要求1或5所定义；
    n如权利要求1所定义。
  16. 根据权利要求22所述的化合物、其药学上可接受的盐或其异构体，其具有式(II-g-1)～(II-i-1)或式(III-g-1)～(III-i-1)所示结构：
    Figure PCTCN2018115857-appb-100027
    其中，R 2和R 4如权利要求1或7所定义；
    R如权利要求1或5所定义；
    n如权利要求1所定义。
  17. 下式化合物、其药学上可接受的盐或其异构体：
    Figure PCTCN2018115857-appb-100028
    Figure PCTCN2018115857-appb-100029
  18. 下式化合物、其药学上可接受的盐或其异构体：
    Figure PCTCN2018115857-appb-100030
    Figure PCTCN2018115857-appb-100031
  19. 一种药物组合物，包括作为活性成分的治疗有效量的根据权利要求1～25任一项所述的化合物、其药学上可接受的盐或其异构体以及药学上可接受的载体。
  20. 根据权利要求1～25任一项所述的化合物、其药学上可接受的盐或其异构体或权利要求26所述的药物组合物在制备抗乙型肝炎药物中的应用。
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