WO2020221280A1 - 二氢嘧啶类化合物及其在药物中的应用 - Google Patents

二氢嘧啶类化合物及其在药物中的应用 Download PDF

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WO2020221280A1
WO2020221280A1 PCT/CN2020/087700 CN2020087700W WO2020221280A1 WO 2020221280 A1 WO2020221280 A1 WO 2020221280A1 CN 2020087700 W CN2020087700 W CN 2020087700W WO 2020221280 A1 WO2020221280 A1 WO 2020221280A1
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Prior art keywords
alkyl
compound
present
alkylene
hbv
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PCT/CN2020/087700
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English (en)
French (fr)
Inventor
任青云
张英俊
刘辛昌
S•戈尔德曼
颜光华
王猛
雷斗兴
李凤
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东莞市东阳光新药研发有限公司
广东东阳光药业有限公司
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Priority to US17/605,456 priority Critical patent/US20220194951A1/en
Publication of WO2020221280A1 publication Critical patent/WO2020221280A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of medicine. Specifically, the present invention relates to a dihydropyrimidine compound and its use as a medicine, especially its use as a medicine for the treatment and/or prevention of hepatitis B.
  • the present invention also relates to a composition composed of these dihydropyrimidine compounds and other antiviral agents, and their application in the treatment and/or prevention of hepatitis B virus (HBV) infection.
  • HBV hepatitis B virus
  • Hepatitis B virus belongs to the family of hepatitis. It can cause acute and/or progressive chronic diseases. Hepatitis B virus can also cause many other clinical manifestations in the pathological form-especially chronic inflammation of the liver, cirrhosis and canceration of liver cells. In addition, co-infection with hepatitis D will have an adverse effect on the development of the disease.
  • interferon has only moderate activity and high toxic and side effects; although lamivudine has good activity, its drug resistance increases rapidly during treatment, and often after stopping treatment The rebound effect appears, and the IC 50 value of lamivudine (3-TC) is 300 nM (Science, 299 (2003), 893-896).
  • HAP heteroaromatic ring substituted dihydropyrimidine
  • heteroaromatic ring-substituted dihydropyrimidine compounds pass through the core
  • the 113-143 amino acid residues of the protein change the angle between the dimers that form the nucleocapsid, resulting in the formation of an unstable expanded nucleocapsid and accelerating the degradation of the core protein (Biochem.Pharmacol.66(2003) , 2273-2279).
  • the invention relates to a novel dihydropyrimidine compound and its use in the preparation of a medicine for treating and preventing HBV infection.
  • the present invention relates to a novel dihydropyrimidine compound and a pharmaceutically acceptable composition thereof.
  • the compound has good solubility, good stability, basically no induction effect on liver drug enzymes, and low toxicity. And other advantages, especially with very good pharmacokinetic properties.
  • the compound of the present invention can effectively inhibit HBV infection, and has good application prospects in anti-HBV.
  • the present invention relates to a compound represented by formula (I) or (Ia) or a stereoisomer, tautomer, and nitrogen oxide of a compound represented by formula (I) or (Ia) , Solvate, metabolite, pharmaceutically acceptable salt or its prodrug,
  • each of R 1 , R 1b and R 1a is independently hydrogen, deuterium, F, Cl, Br, I, cyano, methyl, ethyl, methoxy, ethoxy, methylamino, ethylamino, nitro Group, 4-trifluoromethylphenyl, 3,5-bis(trifluoromethyl)phenyl or trifluoromethyl;
  • R 2 is C 1-6 alkyl or C 1-6 haloalkyl
  • W is CH or N
  • R y is hydrogen, R 4 is methyl, ethyl, n-propyl, methoxy, ethoxy, n-propoxy, isopropoxy, F or Cl; or
  • R y is F or Cl, and R 4 is hydrogen, F or Cl;
  • n 0, 1, 2, 3 or 4;
  • j 1, 2 or 3.
  • R 2 in the present invention is methyl, ethyl, n-propyl, isopropyl, monofluoromethyl, difluoromethyl or trifluoromethyl;
  • the present invention also provides a pharmaceutical composition comprising the compound of the present invention and pharmaceutically acceptable excipients.
  • the pharmaceutical composition of the present invention further comprises other anti-HBV drugs.
  • the pharmaceutical composition of the present invention wherein the other anti-HBV drugs are HBV polymerase inhibitors, immunomodulators or interferons.
  • the pharmaceutical composition of the present invention wherein the other anti-HBV drugs are lamivudine, telbivudine, tenofovir dipivoxil, entecavir, adefovir dipivoxil, Alfaferone, Alloferon , Simo interleukin, clavudine, emtricitabine, faprovir, interferon, baoganling CP, interferon, interferon alpha-1b, interferon alpha, interferon alpha-2a, interferon beta -1a, Interferon Alpha-2, Interleukin-2, Milvotate, Nitrazoxanide, Pegylated Interferon Alpha-2a, Ribavirin, Ruinterferon-A, Cizonan, Euforavac, Ampligen, Phosphazid, Heplisav, Interferon ⁇ -2b, Levamisole, or Propagermanium.
  • the other anti-HBV drugs are lamivudine
  • the present invention also provides the use of the compound or the pharmaceutical composition in the preparation of a medicament for the prevention, treatment or alleviation of viral diseases in patients.
  • the use of the present invention wherein the viral disease refers to hepatitis B infection or a disease caused by hepatitis B infection.
  • the use of the present invention, wherein the disease caused by hepatitis B infection refers to liver cirrhosis or hepatocellular carcinoma.
  • the present invention relates to the use of the compound or pharmaceutical composition to prepare a medicament for the prevention, treatment or alleviation of hepatitis B disease in patients, including administering the compound or pharmaceutical composition to the patient.
  • the effective therapeutic dose of the pharmaceutical composition is administered to the patient.
  • Another aspect of the present invention relates to a method for preventing, treating or alleviating a patient's HBV condition, said method comprising administering to the patient a pharmaceutically acceptable effective dose of the compound of the present invention.
  • Another aspect of the present invention relates to a method for preventing, treating or alleviating HBV disorders in a patient, the method comprising administering to the patient a pharmaceutically acceptable effective dose of a pharmaceutical composition containing the compound of the present invention.
  • Another aspect of the present invention relates to the use of a compound of the present invention to produce a medicament for preventing or treating a patient's HBV disease and reducing its severity.
  • Another aspect of the present invention relates to the use of a pharmaceutical composition containing the compound of the present invention to produce a medicament for preventing or treating a patient's HBV disease and reducing its severity.
  • Another aspect of the present invention relates to a method of inhibiting HBV infection, which method comprises contacting a cell with a compound or pharmaceutical composition of the present invention in a dose effective to inhibit HBV. In other embodiments, the method further comprises contacting the cell with another anti-HBV therapeutic agent.
  • Another aspect of the present invention relates to a method of treating a patient with HBV disease, which method comprises administering an effective therapeutic dose of the compound of the present invention or a pharmaceutical composition thereof to the patient in need of treatment. In other embodiments, the method further comprises administering an effective therapeutic dose of other anti-HBV drugs to the patient in need of treatment.
  • Another aspect of the present invention relates to a method for inhibiting HBV infection in a patient, which method comprises administering an effective therapeutic dose of the compound of the present invention or a pharmaceutical composition thereof to the patient in need of treatment. In other embodiments, the method further comprises administering an effective therapeutic dose of other anti-HBV drugs to the patient in need of treatment.
  • Another aspect of the present invention relates to methods for the preparation, separation and purification of compounds contained in formula (I) or formula (Ia).
  • the present invention will list in detail the documents corresponding to the determined specific content, and the examples are accompanied by diagrams of structural formulas and chemical formulas.
  • the present invention prospectively covers all options, variants and equivalents, which may be included in the current invention field as defined by the claims.
  • Those skilled in the art will recognize many methods and substances similar or equivalent to those described herein, which can be applied in the practice of the present invention.
  • the invention is by no means limited to the description of methods and materials. There are many documents and similar materials that differ or conflict with the application of the present invention, including but not limited to the definition of terms, the usage of terms, the described technology, or the scope controlled by the application of the present invention.
  • the compounds of the present invention can be optionally substituted by one or more substituents, such as the compounds of the general formula above, or the special examples, subclasses, and subclasses contained in the examples.
  • substituents such as the compounds of the general formula above, or the special examples, subclasses, and subclasses contained in the examples.
  • a class of compounds such as the compounds of the general formula above, or the special examples, subclasses, and subclasses contained in the examples.
  • substituted means that one or more hydrogen atoms in a given structure are replaced by a specific substituent.
  • an optional substituent group may have a substituent at each substitutable position of the group.
  • the substituents can be substituted at each position with the same or different substitutions.
  • C 1-6 alkyl refers particularly to the disclosure independently methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, and C 6 alkyl.
  • alkyl as used in the present invention includes saturated linear or branched monovalent hydrocarbon groups of 1-20 carbon atoms, wherein the alkyl groups can be independently optionally substituted with one or more substituents described in the present invention.
  • the alkyl group contains 1-12 carbon atoms, in other embodiments, the alkyl group contains 1-10 carbon atoms, and in other embodiments, the alkyl group contains 1-8 carbon atoms.
  • alkyl groups include, but are not limited to, methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), n-propyl (n-Pr, -CH 2 CH 2 CH 3 ), isopropyl (i-Pr, -CH(CH 3 ) 2 ), n-butyl (n-Bu, -CH 2 CH 2 CH 2 CH 3 ), 2-methylpropyl or isobutyl (i-Bu, -CH 2 CH(CH 3 ) 2 ), 1-methylpropyl or sec-butyl (s-Bu, -CH(CH 3 )CH 2 CH 3 ), tert-butyl (t-Bu , -C(CH 3 ) 3 ), n-pentyl (-CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-
  • alkylene refers to a saturated divalent or multivalent hydrocarbon group obtained by removing two or more hydrogen atoms from a saturated linear or branched hydrocarbon group. Unless otherwise specified, alkylene groups contain 1-12 carbon atoms. In some embodiments, the alkylene group contains 1-6 carbon atoms; in other embodiments, the alkylene group contains 1-4 carbon atoms; in some embodiments, the alkylene group The group contains 1-3 carbon atoms; in still other embodiments, the alkylene group contains 1-2 carbon atoms.
  • alkylene examples include, but are not limited to, methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), n-propylene (-CH 2 CH 2 CH 2 -), isopropylidene Group (-CH(CH 3 )CH 2 -) and so on.
  • hydroxyalkyl and “hydroxyalkoxy” mean alkyl or alkoxy, as the case may be, substituted by one or more hydroxy groups, where "hydroxyalkyl” and “hydroxyalkylene “And “hydroxyalkyl” can be used interchangeably, such examples include, but are not limited to, hydroxymethyl (-CH 2 OH), hydroxyethyl (-CH 2 CH 2 OH, -CHOHCH 3 ), Hydroxypropyl (eg, -CH 2 CH 2 CH 2 OH, -CH 2 CHOHCH 3 , -CHOHCH 2 CH 3 ), hydroxymethoxy (-OCH 2 OH), etc.
  • haloalkyl means an alkyl group, an alkenyl or alkoxy group is substituted with one or more halogen atoms, among which, alkyl, alkenyl and alkoxy
  • the radical has the meaning described in the present invention.
  • alkoxy means that the alkyl group is connected to the rest of the molecule through an oxygen atom, where the alkyl group has the meaning as described in the present invention. Unless otherwise specified, the alkoxy group contains 1-12 carbon atoms. In some embodiments, the alkoxy group contains 1-8 carbon atoms; in other embodiments, the alkoxy group contains 1-6 carbon atoms; in other embodiments, the alkoxy group Groups contain 1-4 carbon atoms; in still other embodiments, alkoxy groups contain 1-3 carbon atoms. The alkoxy group may be optionally substituted with one or more substituents described herein.
  • alkoxy groups include, but are not limited to, methoxy (MeO, -OCH 3 ), ethoxy (EtO, -OCH 2 CH 3 ), 1-propoxy (n-PrO, n- Propoxy, -OCH 2 CH 2 CH 3 ), 2-propoxy (i-PrO, i-propoxy, -OCH(CH 3 ) 2 ), 1-butoxy (n-BuO, n- Butoxy, -OCH 2 CH 2 CH 2 CH 3 ), 2-methyl-l-propoxy (i-BuO, i-butoxy, -OCH 2 CH(CH 3 ) 2 ), 2-but Oxygen (s-BuO, s-butoxy, -OCH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propoxy (t-BuO, t-butoxy, -OC(CH 3 ) 3 ), 1-pentyloxy (n-pentyloxy, -OCH 2 CH 2 CH 2 CH 2 CH 3 ), 2-(
  • halogen or "halogen atom” refers to F, Cl, Br or I.
  • unsaturated means that a part contains one or more degrees of unsaturation.
  • aryl means a monocyclic, bicyclic and tricyclic carbocyclic ring system containing 6-14 ring atoms, or 6-12 ring atoms, or 6-10 ring atoms, wherein at least one ring system is Aromatic, where each ring system contains a ring of 3-7 atoms, and has one or more points of attachment to the rest of the molecule.
  • aryl can be used interchangeably with the term “aromatic ring”. Examples of aryl groups may include phenyl, naphthyl, and anthracenyl. The aryl group may be independently optionally substituted with one or more substituents described in the present invention.
  • heteroaryl refers to monocyclic, bicyclic and tricyclic systems containing 5-12 ring atoms, in which at least one ring is aromatic, and at least one aromatic ring contains one or more heteroatoms, each of which A ring system contains a ring consisting of 5-7 ring atoms, and has one or more points of attachment to the rest of the molecule.
  • heteroaryl can be used interchangeably with the terms “aromatic heterocycle", “heteroaromatic ring” or “heteroaromatic compound”.
  • the heteroaryl group is a monocyclic heteroaryl group consisting of 5-7 ring atoms including 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, sulfur, and oxygen.
  • the heteroaryl group is a monocyclic heteroaryl group consisting of 5-6 ring atoms containing 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, sulfur, and oxygen.
  • the heteroaryl group is a bicyclic heteroaryl group composed of 7-12 ring atoms including 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, sulfur, and oxygen.
  • the heteroaryl group is a bicyclic heteroaryl group consisting of 8-10 ring atoms including 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, sulfur, and oxygen.
  • the heteroaryl group is a bicyclic heteroaryl group consisting of 9-10 ring atoms including 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, sulfur, and oxygen.
  • heteroaromatic rings include, but are not limited to, the following monocyclic rings: 1,2,4-oxadiazole-5(4H)-thioketo group, 1,2,4-thiadiazole-5( 4H)-keto, 1,2,4-oxadiazole-5(4H)-keto, 1,3,4-oxadiazole-2(3H)-thioketo, 1H-1,2,4 -Triazole-5(4H)-keto, 2-furyl, 3-furyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4 -Isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl , 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyr
  • 2-pyrazolyl isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2, 3-triazolyl, 1,2,3-thiodiazolyl, 1,3,4-thiodiazolyl, 1,2,5-thiodiazolyl, pyrazinyl, 1,3, 5-triazinyl, diazolyl, thiadiazolyl, triazinyl, etc.; also include the following bicyclic rings, but not limited to these bicyclic rings: benzothiazolyl, benzimidazolyl, benzofuranyl, benzo Thienyl, indolyl (such as 2-indolyl), purinyl, quinolinyl (such as 2-quinolinyl, 3-quinolinyl, 4-quinolinyl), isoquinolinyl (such as 1- Isoquinolinyl, 3-isoquinolinyl or 4-isoquinolinyl)
  • MM is composed of 1 ring atoms
  • the cyclic group is composed of MM 1 ring atoms, and the ring atoms include carbon atoms and/or O, N, S, P and other heteroatoms.
  • heterocyclic group consisting of 3-6 ring atoms represents a heterocyclic group consisting of 3, 4, 5 or 6 ring atoms.
  • alkoxyalkyl and “alkoxyalkylene” are used interchangeably to indicate that the alkyl group may be substituted by one or more alkoxy groups which may be the same or different, wherein the alkoxy group and the alkyl group
  • the radical group has the meaning as described in the present invention. Examples of such include, but are not limited to, cyclohexylmethyl, cyclopropylethyl, methoxyethyl, ethoxymethyl, and the like.
  • the ring system formed by attaching a substituent to the central ring by drawing a bond means that the substituent can be substituted at any substitutable position on the ring, as shown in formula b, c, d, e, f, g, and h.
  • the structural formula described in the present invention includes all isomeric forms (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers): for example, R containing an asymmetric center , S configuration, double bond (Z), (E) isomers, and (Z), (E) conformational isomers. Therefore, a single stereochemical isomer of the compound of the present invention or its enantiomer Mixtures of isomers, diastereomers, or geometric isomers (or conformational isomers) are all within the scope of the present invention.
  • prodrug represents the conversion of a compound into a compound represented by formula (I) in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or the enzymatic conversion of the prodrug into the maternal structure in the blood or tissue.
  • the prodrug compounds of the present invention can be esters.
  • esters can be used as prodrugs including phenyl esters, aliphatic (C 1-24 ) esters, acyloxymethyl esters, and carbonates. , Carbamates and amino acid esters.
  • a compound in the present invention contains a hydroxyl group, which can be acylated to obtain a compound in the form of a prodrug.
  • prodrug forms include phosphate esters.
  • these phosphate ester compounds are obtained by phosphorylation of the parent hydroxyl group.
  • T. Higuchi and V. Stella Pro-drugs as Novel Delivery Systems, Vol. 14 of the ACSSymposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, J.
  • Metal refers to the product obtained by the metabolism of a specific compound or its salt in the body.
  • the metabolites of a compound can be identified by techniques well known in the art, and its activity can be characterized by experimental methods as described in the present invention. Such products can be obtained by methods such as oxidation, reduction, hydrolysis, amidation, deamidation, esterification, degreasing, and enzymatic cleavage of the administered compound.
  • the present invention includes the metabolites of the compound, including the metabolites produced by fully contacting the compound of the present invention with a mammal for a period of time.
  • stereochemistry and the usage of conventions in this invention usually refer to the following documents: SPParker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S ., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.
  • the compound of the present invention may contain an asymmetric center or a chiral center, so there are different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including but not limited to, diastereomers, enantiomers, atropisomers, and their mixtures, such as racemic mixtures, constitute the present invention Part.
  • optically active compounds that is, they have the ability to rotate the plane of plane-polarized light.
  • the prefixes D, L or R, S are used to indicate the absolute configuration of the molecular chiral center.
  • the prefixes d, l or (+), (-) are used to name the symbols of the plane-polarized light rotation of the compound, (-) or l means that the compound is levorotatory, and the prefix (+) or d means that the compound is dextrorotatory.
  • the chemical structures of these stereoisomers are the same, but their stereo structures are different.
  • a specific stereoisomer may be an enantiomer, and a mixture of isomers is usually called an enantiomeric mixture.
  • racemic mixture A 50:50 mixture of enantiomers is called a racemic mixture or a racemate, which may result in no stereoselectivity or stereospecificity during chemical reactions.
  • racemic mixture and racemate refer to an equimolar mixture of two enantiomers, lacking optical activity.
  • tautomer or "tautomeric form” means that the structural isomers of different energies can be converted into each other through a low energy barrier.
  • proton tautomers ie, proton-transferred tautomers
  • Valence (valence) tautomers include the interconversion of recombined bond electrons. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.
  • the "pharmaceutically acceptable salt” used in the present invention refers to the organic and inorganic salts of the compound of the present invention.
  • Pharmaceutically acceptable salts are well known to us in the field, as described in the literature: SMBerge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19, 1977.
  • Pharmaceutically acceptable non-toxic acid salts include, but are not limited to, inorganic acid salts formed by reaction with amino groups include hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, And organic acid salts such as acetate, oxalate, maleate, tartrate, citrate, succinate, malonate, or other methods described in books and literature such as ion exchange These salts.
  • salts include adipate, malate, 2-hydroxypropionate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate , Borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate Acid salt, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, caproate, hydroiodide, 2-hydroxy-ethanesulfonate, lacturonate, lactic acid Salt, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate , Pectinate, pers
  • Salts obtained with appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 alkyl) 4 salts.
  • the present invention also contemplates the quaternary ammonium salt formed by any compound containing the N group.
  • Water-soluble or oil-soluble or dispersed products can be obtained by quaternization.
  • Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Pharmaceutically acceptable salts further include appropriate, non-toxic ammonium, quaternary ammonium salts and amine cations that are resistant to counter ion formation, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1 -8 Sulfonates and aromatic sulfonates.
  • solvate of the present invention refers to an association formed by one or more solvent molecules and the compound of the present invention.
  • Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.
  • hydrate refers to the association formed by the solvent molecule being water.
  • protecting group refers to when a substituent reacts with another functional group, it is usually used to block or protect a particular functionality.
  • amino protecting group refers to a substituent connected to an amino group to block or protect the functionality of the amino group in a compound. Suitable amino protecting groups include acetyl, trifluoroacetyl, and tert-butoxycarbonyl. (BOC), benzyloxycarbonyl (CBZ) and 9-fluorenemethyleneoxycarbonyl (Fmoc).
  • hydroxyl protecting group refers to a substituent of a hydroxyl group used to block or protect the functionality of the hydroxyl group.
  • Suitable protecting groups include acetyl and silyl.
  • Carboxy protecting group refers to the substituent of the carboxyl group used to block or protect the functionality of the carboxyl group.
  • General carboxyl protecting groups include -CH 2 CH 2 SO 2 Ph, cyanoethyl, 2-(trimethylsilane Base) ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrobenzenesulfonyl)ethyl, 2-(diphenyl) (Phosphino) ethyl, nitroethyl, etc.
  • protecting groups please refer to the literature: T W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991; and PJ Kocienski, Protecting Groups, Thieme, Stuttgart, 2005.
  • the compound involved in the present invention and the pharmaceutically acceptable composition thereof can effectively inhibit HBV infection.
  • the present invention relates to a compound represented by formula (I) or (Ia) or a stereoisomer, tautomer, and nitrogen oxide of a compound represented by formula (I) or (Ia) , Solvate, metabolite, pharmaceutically acceptable salt or its prodrug,
  • each of R 1 , R 1b and R 1a is independently hydrogen, deuterium, F, Cl, Br, I, cyano, methyl, ethyl, methoxy, ethoxy, methylamino, ethylamino, nitro Group, 4-trifluoromethylphenyl, 3,5-bis(trifluoromethyl)phenyl or trifluoromethyl;
  • R 2 is C 1-6 alkyl or C 1-6 haloalkyl
  • W is CH or N
  • R y is hydrogen, R 4 is methyl, ethyl, n-propyl, methoxy, ethoxy, n-propoxy, isopropoxy, F or Cl; or
  • R y is F or Cl, and R 4 is hydrogen, F or Cl;
  • n 0, 1, 2, 3 or 4;
  • j 1, 2 or 3.
  • R 2 in the present invention is methyl, ethyl, n-propyl, isopropyl, monofluoromethyl, difluoromethyl or trifluoromethyl;
  • the present invention relates to one of the following compounds or their stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, pharmaceutically acceptable salts or their prodrugs, But not limited to these compounds:
  • the present invention also provides a pharmaceutical composition comprising the compound of the present invention and pharmaceutically acceptable excipients.
  • the pharmaceutical composition of the present invention further comprises other anti-HBV drugs.
  • the pharmaceutical composition of the present invention wherein the other anti-HBV drugs are HBV polymerase inhibitors, immunomodulators or interferons.
  • the pharmaceutical composition of the present invention wherein the other anti-HBV drugs are lamivudine, telbivudine, tenofovir dipivoxil, entecavir, adefovir dipivoxil, Alfaferone, Alloferon , Simo interleukin, clavudine, emtricitabine, faprovir, interferon, baoganling CP, interferon, interferon alpha-1b, interferon alpha, interferon alpha-2a, interferon beta -1a, Interferon Alpha-2, Interleukin-2, Milvotate, Nitrazoxanide, Pegylated Interferon Alpha-2a, Ribavirin, Ruinterferon-A, Cizonan, Euforavac, Ampligen, Phosphazid, Heplisav, Interferon ⁇ -2b, Levamisole, or Propagermanium.
  • the other anti-HBV drugs are lamivudine
  • the present invention also provides the use of the compound or the pharmaceutical composition in the preparation of a medicament for the prevention, treatment or alleviation of viral diseases in patients.
  • the use of the present invention wherein the viral disease refers to hepatitis B infection or a disease caused by hepatitis B infection.
  • the use of the present invention, wherein the disease caused by hepatitis B infection refers to liver cirrhosis or hepatocellular carcinoma.
  • the compound or the pharmaceutical composition of the present invention is used to prepare drugs for preventing, treating or alleviating viral diseases in patients.
  • the use of the compound or the pharmaceutical composition of the present invention, wherein the viral disease refers to hepatitis B infection or a disease caused by hepatitis B infection.
  • the use of the compound or the pharmaceutical composition of the present invention, wherein the disease caused by hepatitis B infection refers to liver cirrhosis or hepatocellular carcinoma.
  • the present invention relates to a method for preventing, treating or alleviating a viral disease in a patient, wherein the method comprises administering to the patient a pharmaceutically acceptable effective dose of the compound or pharmaceutical composition of the present invention.
  • the method of the present invention wherein the viral disease refers to hepatitis B infection or a disease caused by hepatitis B infection.
  • the method of the present invention, wherein the disease caused by hepatitis B infection refers to liver cirrhosis or hepatocellular carcinoma.
  • the present invention relates to the use of the compound or pharmaceutical composition in the preparation of a medicine for preventing, treating or alleviating hepatitis B disease in patients.
  • Another aspect of the present invention relates to a method for preventing, treating or alleviating a patient's HBV condition, said method comprising administering to the patient a pharmaceutically acceptable effective dose of the compound of the present invention.
  • Another aspect of the present invention relates to a method for preventing, treating or alleviating HBV disorders in a patient, the method comprising administering to the patient a pharmaceutically acceptable effective dose of a pharmaceutical composition containing the compound of the present invention.
  • Another aspect of the present invention relates to the use of a compound of the present invention to produce a medicine for preventing or treating a patient's HBV disease and reducing its severity.
  • Another aspect of the present invention relates to the use of a pharmaceutical composition containing the compound of the present invention to produce a medicine for preventing or treating a patient's HBV disease and reducing its severity.
  • the patient is a mammal, and in other embodiments, the patient is a human.
  • the use further comprises contacting the cell with an anti-HBV therapeutic agent.
  • Another aspect of the present invention relates to a method of inhibiting HBV infection, which method comprises contacting a cell with a compound or pharmaceutical composition of the present invention in a dose effective to inhibit HBV. In other embodiments, the method further comprises contacting the cell with another anti-HBV therapeutic agent.
  • Another aspect of the present invention relates to a method of treating a patient with HBV disease, which method comprises administering an effective therapeutic dose of the compound of the present invention or a pharmaceutical composition thereof to the patient in need of treatment.
  • the method further comprises administering an effective therapeutic dose of another anti-HBV therapeutic agent to the patient in need of treatment.
  • Another aspect of the present invention relates to a method for inhibiting HBV infection in a patient, which method comprises administering an effective therapeutic dose of the compound of the present invention or a pharmaceutical composition thereof to the patient in need of treatment. In other embodiments, the method further comprises administering an effective therapeutic dose of another anti-HBV therapeutic agent to the patient in need of treatment.
  • Another aspect of the present invention relates to methods for the preparation, separation and purification of compounds contained in formula (I) or formula (Ia).
  • the present invention also relates to the application of the compound of the present invention and the pharmaceutically acceptable salt thereof in the production of medicinal products to effectively inhibit HBV infection, and the application of the compound of the present invention in the production of drugs for effectively inhibiting HBV infection.
  • the compound of the present invention is also used in the production of a medicine for reducing, preventing, controlling or treating patients with hepatitis B.
  • the salt is a pharmaceutically acceptable salt.
  • pharmaceutically acceptable includes that the substance or composition must be chemically or toxicologically suitable, related to the other components of the formulation and the mammal used for treatment.
  • the salts of the compounds of the present invention also include intermediates used in the preparation or purification of compounds represented by formula (I) or (Ia) or salts of enantiomers separated from compounds represented by formula (I) or (Ia), but It is not necessarily a pharmaceutically acceptable salt.
  • the desired salt can be prepared by any suitable method provided in the literature, for example, using inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like.
  • organic acids such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, malic acid, 2-hydroxypropionic acid, citric acid, oxalic acid, glycolic acid and salicylic acid ; Pyranonic acid, such as glucuronic acid and galacturonic acid; ⁇ -hydroxy acids, such as citric acid and tartaric acid; amino acids, such as aspartic acid and glutamic acid; aromatic acids, such as benzoic acid and cinnamic acid; Sulfonic acids, such as p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, trifluoromethanesulfonic acid, etc. or a combination thereof.
  • organic acids such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, malic acid,
  • the desired salt can be prepared by a suitable method, for example, using inorganic or organic bases such as ammonia (primary, secondary, tertiary), alkali metal hydroxide, ammonium , N + (R 14 ) 4 salts and alkaline earth metal hydroxides, etc.
  • inorganic or organic bases such as ammonia (primary, secondary, tertiary), alkali metal hydroxide, ammonium , N + (R 14 ) 4 salts and alkaline earth metal hydroxides, etc.
  • Suitable salts include, but are not limited to, organic salts derived from amino acids, such as glycine and arginine, ammonia, such as primary, secondary and tertiary ammonia, and N + (R 14 ) 4 salts, such as R 14 H, C 1-4 alkyl, C 6-10 aryl, C 6-10 aryl C 1-4 alkyl, etc., and cyclic ammonia, such as piperidine, morpholine and piperazine, etc., and from sodium, Calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium give inorganic salts.
  • amino acids such as glycine and arginine
  • ammonia such as primary, secondary and tertiary ammonia
  • N + (R 14 ) 4 salts such as R 14 H, C 1-4 alkyl, C 6-10 aryl, C 6-10 aryl C 1-4 alkyl, etc.
  • cyclic ammonia such as piperidine, morpho
  • ammonium, quaternary ammonium salts and amine cations that are resistant to counterion formation, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8 sulfonates and Aromatic sulfonate.
  • the characteristics of the pharmaceutical composition of the present invention include the compound of formula (I) or (Ia), the compounds listed in the present invention, or the compounds of the examples, and pharmaceutically acceptable excipients.
  • the compound in the pharmaceutical composition of the present invention can effectively inhibit hepatitis B virus, and is suitable for the treatment of virus-induced diseases, especially acute and chronic persistent HBV infection. Chronic viral diseases caused by HBV may lead to serious disease. Hepatitis virus infection can cause liver cirrhosis and/or hepatocellular carcinoma in many cases.
  • the area of disease treatment that may be mentioned is, for example, the treatment of acute and chronic viral infections that may cause infectious hepatitis, such as hepatitis B virus infection.
  • the compounds of the present invention are particularly suitable for the treatment of chronic hepatitis B infection and acute and chronic hepatitis B virus infections.
  • the present invention includes pharmaceutical preparations, in addition to non-toxic and inert pharmacologically suitable excipients, it also contains one or more of the compounds of formula (I) or (Ia) or pharmaceutical compositions of the present invention or contains one or more
  • the active ingredient is a compound of formula (I) or (Ia) or the pharmaceutical composition of the present invention.
  • compositions may also contain other active pharmaceutical ingredients other than the compound of formula (I) or (Ia).
  • pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable prodrugs, salts, esters, salts of esters, or any other that can be administered directly or indirectly according to the needs of patients Adducts or derivatives, compounds described in other aspects of the invention, their metabolites or their residues.
  • the pharmaceutical composition of the present invention contains any one of the compounds represented by formula (I) or (Ia) of the present invention, and further contains pharmaceutically acceptable excipients, such as those used in the present invention.
  • pharmaceutically acceptable excipients such as those used in the present invention.
  • Including any solvents, solid excipients, diluents, binders, disintegrating agents, or other liquid excipients, dispersing agents, flavoring or suspending agents, surfactants, isotonic agents, thickening agents, Emulsifiers, preservatives, solid binders or lubricants, etc. are suitable for specific target dosage forms.
  • Remington The Science and Practice of Pharmacy, 21st edition, 2005, ed.
  • Substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers; aluminum; aluminum stearate; lecithin; serum proteins, such as human serum proteins; buffer substances such as phosphate; glycine; sorbic acid; Potassium sorbate; a mixture of partial glycerides of saturated plant fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silicon; magnesium trisilicate; poly Vinylpyrrolidone; polyacrylate; wax; polyethylene-polyoxypropylene-blocking polymer; lanolin; sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxylate Sodium methyl cellulose, ethyl cellulose and cellulose acetate; gum powder; malt; gelatin; talc; excipients
  • the pharmaceutical composition of the compound of the present invention can be administered in any of the following ways: oral administration, spray inhalation, topical administration, rectal administration, nasal administration, topical administration, vaginal administration, parenteral administration Drugs such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, or intracranial injection or infusion, or the use of an explanted reservoir.
  • oral administration intramuscular injection, intraperitoneal administration or intravenous injection.
  • the compound of the present invention or its pharmaceutical composition can be administered in a unit dosage form.
  • the dosage form for administration can be a liquid dosage form or a solid dosage form.
  • Liquid dosage forms can be true solutions, colloids, microparticles, and suspensions.
  • Other dosage forms such as tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, freeze-dried powder injections, inclusion compounds, implants, patches, rubs ⁇ etc.
  • Oral tablets and capsules may contain excipients such as binders, such as syrup, gum arabic, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, amino Acetic acid; lubricants, such as magnesium stearate, talc, polyethylene glycol, silica; disintegrating agents, such as potato starch; or acceptable moisturizers such as sodium lauryl sulfate.
  • binders such as syrup, gum arabic, sorbitol, tragacanth, or polyvinylpyrrolidone
  • fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, amino Acetic acid
  • lubricants such as magnesium stearate, talc, polyethylene glycol, silica
  • disintegrating agents such as potato starch
  • acceptable moisturizers such as sodium lauryl sul
  • Oral liquids can be made into hydrated oil suspensions, solutions, emulsions, syrups or elixirs, or they can be made into dry products, supplemented with water or other suitable media before use.
  • This liquid preparation may contain conventional additives such as suspending agent, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated food Oils, emulsifiers, such as lecithin, sorbitan monooleate, gum arabic; or non-aqueous excipients (may contain edible oils), such as almond oil, fats such as glycerin, ethylene glycol, or ethanol; preservatives, Such as methyl or propyl p-hydroxybenzoate, sorbic acid. Flavoring or coloring agents can be added if necessary.
  • Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.
  • the liquid dosage form is usually made of a compound and a sterilizing excipient.
  • Water is the first choice for auxiliary materials.
  • the compound can be dissolved in the excipients or made into a suspension solution.
  • the injection solution the compound is first dissolved in water, filtered and sterilized, and then filled into a sealed bottle or ampoule.
  • the compound of the present invention When applied topically to the skin, the compound of the present invention can be made into an appropriate ointment, lotion, or cream form, in which the active ingredient is suspended or dissolved in one or more excipients, and the excipients that can be used in ointment preparations include but Not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; auxiliary materials that can be used for lotions and creams include but are not limited to: mineral oil, sorbitan mono Stearate, Tween 60, cetyl ester wax, hexadecenyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the total amount of the active compound administered according to the invention is about 0.5-500 mg, preferably 1-100 mg/kg body weight, if appropriate, per 24 hours. Multiple single doses are administered to achieve the desired effect.
  • the amount of active compound contained in a single dose is preferably about 1-80 mg, more preferably 1-50 mg/kg body weight, but the above-mentioned dose may not be followed, that is, it depends on the type and weight of the subject to be treated, the nature and severity of the disease , The type of preparation and the way of drug administration, as well as the administration cycle or time interval.
  • the pharmaceutical composition provided by the present invention also contains anti-HBV drugs.
  • the anti-HBV drugs are HBV polymerase inhibitors, immunomodulators or interferons.
  • the anti-HBV drugs include lamivudine, telbivudine, tenofovir dipivoxil, entecavir, adefovir dipivoxil, Alfaferone, Alloferon, simo interleukin, clavudine, emtricitabine, and fapro Wei, Interferon, Baoganling CP, Interferon, Interferon ⁇ -1b, Interferon ⁇ , Interferon ⁇ -2a, Interferon ⁇ -1a, Interferon ⁇ -2, Interleukin-2, Mivo Ester, Nitrazoxanide, Pegylated Interferon Alpha-2a, Ribavirin, Rointerferon-A, Cizonan, Euforavac, Ampligen, Phosphazid, Heplisav, Interferon Alpha-2b, Levamisole Or propa germanium and so on.
  • Hepatitis B disease refers to liver diseases caused by hepatitis B virus infection or hepatitis B infection, including acute hepatitis, chronic hepatitis, cirrhosis and stem cell cancer.
  • Acute hepatitis B virus infection can be asymptomatic or manifest as symptoms of acute hepatitis.
  • Patients with chronic viral infections have active diseases, which can develop into cirrhosis and liver cancer.
  • the anti-HBV drug can be administered separately from the composition containing the compound of the invention as part of a multiple dosing regimen.
  • those drugs may be part of a single dosage form, mixed with the compounds of the invention to form a single composition. If the administration is part of a multiple dosing regimen, the two active agents can be delivered to each other continuously or over a period of time to achieve the target agent activity.
  • the amount of compound and composition (those containing a composition as described in the present invention) that can be combined with adjuvant materials to produce a single dosage form varies depending on the main treatment and the particular mode of administration. Normally, the amount of the composition of the present invention will not exceed the amount normally administered where the composition contains as the sole active agent. On the other hand, the range of the amount of the presently disclosed composition is about 50%-100% of the normal amount of the existing composition, and the included agent is the only active therapeutic agent. Among those included compositions, the composition will act synergistically with the compound of the present invention.
  • the compound of the present invention shows a strong antiviral effect.
  • Such compounds have unexpected antiviral activity against HBV, so they are suitable for the treatment of various diseases caused by viruses, especially those caused by acute and chronic persistent HBV virus infection.
  • Chronic viral diseases caused by HBV can cause various syndromes of varying severity. It is well known that chronic hepatitis B virus infection can cause liver cirrhosis and/or hepatocellular carcinoma.
  • indications that can be treated with the compounds of the present invention are: acute and chronic viral infections that can lead to infectious hepatitis, such as hepatitis B virus infection. Particularly preferred are chronic hepatitis B infection and acute hepatitis B virus infection.
  • the present invention also relates to the use of the compound and pharmaceutical composition of the present invention for the preparation of drugs for the treatment and prevention of viral diseases, especially hepatitis B.
  • the compounds of the present invention can be prepared by the methods described in the present invention, unless there are further instructions, wherein the definition of substituents is as shown in formula (I) or (Ia).
  • the following synthesis schemes and examples are used to further illustrate the content of the present invention.
  • MS mass spectrometry
  • MS data is also measured by Agilent 6120 series LC-MS spectrometer equipped with G1311A quaternary pump and G1316A TCC (column temperature maintained at 30°C). G1329A automatic sampler and G1315D DAD detector are used for analysis , ESI source is used in LC-MS spectrometer.
  • the above two spectrometers are equipped with Agilent Zorbax SB-C18 column, the specification is 2.1 ⁇ 30mm, 5 ⁇ m.
  • the injection volume is determined by the sample concentration; the flow rate is 0.6 mL/min; the HPLC peak is recorded and read by UV-Vis wavelengths at 210 nm and 254 nm.
  • the mobile phases are 0.1% formic acid acetonitrile solution (phase A) and 0.1% formic acid ultrapure aqueous solution (phase B).
  • the gradient elution conditions are shown in Table 1:
  • the purification of the compound was evaluated by Agilent 1100 series high performance liquid chromatography (HPLC), with UV detection at 210nm and 254nm, Zorbax SB-C18 column, specification 2.1 ⁇ 30mm, 4 ⁇ m, 10 minutes, flow rate 0.6mL/min , 5-95% (0.1% formic acid acetonitrile solution) (0.1% formic acid aqueous solution), the column temperature is maintained at 40°C.
  • HPLC high performance liquid chromatography
  • LiOH.H 2 O Lithium hydroxide monohydrate 1atm 101.325kPa
  • each of R 1 , R 2 , R 3 , R 1a , R 1b , R 4 , R 4a , R 4b and R y has the meaning as described in the present invention.
  • Compound (a-8) can be prepared by the method described in Synthesis Scheme 1. First, compound (a-1) reacts with thionyl chloride and methanol to obtain compound (a-2); then, compound (a-2) and compound (a-3) are subjected to alkaline conditions (such as cesium carbonate, etc.) , The catalyst (such as palladium acetate, etc.), ligands (such as X-PHOS, t-BuX-PHOS, etc.) and a suitable solvent (such as 1,4-dioxane, etc.) react to produce compound (a-4); Next, compound (a-4) undergoes an ester hydrolysis reaction under alkaline conditions (such as lithium hydroxide aqueous solution, etc.) to produce compound (a-5), then Boc is removed to obtain compound (a-6); finally, Compound (a-6) or its salt and compound (a-7) (compound (a-7) (compound (a-7) can be prepared by referring to the synthesis scheme 1 in WO20150745
  • F1-0 (3g, 13.7mmol) was dissolved in methanol (30mL), and thionyl chloride (1.2mL, 16mmol) was slowly added dropwise under ice bath. After the addition was completed, the reaction mixture was reacted at room temperature for 12h. After the reaction, the solvent was evaporated under reduced pressure, and petroleum ether (90 mL) was added to the residue for extraction. The organic layer was washed with saturated aqueous sodium bicarbonate solution (30 mL ⁇ 3) and saturated sodium chloride solution (30 mL) successively, and anhydrous sulfuric acid Dry with sodium and concentrate under reduced pressure to obtain F1 as a colorless oil (3.05 g, 95.6%).
  • F1-2 (2.5g, 6.35mmol), methanol (10mL), tetrahydrofuran (20mL) and lithium hydroxide monohydrate (0.8g, 19mmol) were added to the dry reaction flask in sequence.
  • the reaction mixture was reacted at 50°C for 12h and then reduced in pressure Concentrate, add water (100 mL) to dilute the obtained residue, adjust the pH to 4-5 with 1M hydrochloric acid, and filter to obtain F1-3 as a white solid (2.31 g, 95.8%).
  • Test 1 HepAD38 cell evaluation compound inhibits HBV DNA replication activity (qPCR method)
  • HepAD38 Ladner et al. (Ladner, Otto et al. 1997) ligated the tetracycline-sensitive cytomegalovirus CMV promoter to the PBR322 plasmid and ligated it with the ayw subtype HBV DNA into the ptetHBV plasmid, and transfected HepG2 cells to obtain the HepAD38 cell line. Due to the destruction of the pre-C region gene, HBV DNA yield is about 11 times higher than that of HepG2.2.15 cells. Tetracycline can be used to regulate HBV replication, and the time required for culture is only half of that of HepG2.2.15 cells.
  • HepAD38 was cultured in DMEM/F-12K medium containing 10% FBS and 1% double antibody (including Tetracycline at a final concentration of 300 ng/ml and G418 at a final concentration of 400 ⁇ g/ml).
  • the virus particle DNA secreted by HepAD38 cells can be quantified by qPCR method, and thus the influence of the compound on virus replication can be detected.
  • Tetracycline final concentration of 300ng/ml
  • G418 final concentration of 400 ⁇ g/ml
  • the virus does not express in the presence of Tetracycline.
  • 10 5 /mL cell suspension inoculate 100 ⁇ L per well in a 96-well plate (the whole plate is covered), and incubate in a 37°C, 5% CO 2 constant temperature incubator for 24 hours.
  • Compound preparation and cell treatment in antiviral experiments dissolve the compound with DMSO to 20 mM, further dilute the compound with DMSO to 800 ⁇ M, and then perform a 4-fold dilution of 8 dilutions, with the highest concentration of 800 ⁇ M.
  • TDF tenofovir disoproxil fumarate, Selleck, Cat S1400
  • Table 2 EC 50 values of the compounds of the present invention on HBV replication
  • Test 2 HepG2.2.15 cell evaluation compound inhibits HBV DNA replication activity
  • HepG2.2.15 cells The chromosomes of HepG2.2.15 cells (SELLS, PNAS, 1987 and SELLS, JV, 1988) integrate a complete HBV genome, and stably express viral RNA and viral proteins.
  • HepG2.2.15 cells can secrete mature hepatitis B virus particles, HBsAg and HBeAg into the culture medium.
  • HepG2.2.15 was cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin, 100U/mL streptomycin, 1% non-essential amino acids, 1mM sodium pyruvate 300 ⁇ g/mL G418.
  • the virus particle DNA secreted by HepG2.2.15 cells can be quantified by qPCR method, and thus the influence of the compound on virus replication can be detected.
  • HepG 2.2.15 cells were seeded into a 96-well cell culture plate per well, and cultured at 37°C and 5% CO 2 for 3 days until the cells grew to full well. On day 0 of the test, discard the old medium and add 200 ⁇ L of fresh test medium (5% FBS).
  • compound preparation and cell treatment dissolve the compound with DMSO to 30 mM, further dilute the compound with DMSO to 800 ⁇ M, and then perform 8 dilutions of 4-fold dilution, with the highest concentration of 800 ⁇ M.
  • TDF tenofovir disoproxil fumarate, Selleck, Cat S1400
  • Table 3 EC 50 values of the compounds of the present invention on HBV replication
  • Graphpad Prism 5 software was used to process the concentration-cytotoxicity (%) data, and the CC 50 was calculated by a four-parameter nonlinear regression model. A CC 50 greater than 50 indicates that the toxicity is relatively low.
  • Test 4 Pharmacokinetic experiment of the compound of the present invention in beagle dogs, mice, rats and cynomolgus monkeys
  • Beagle dogs were given 2.5 mg/kg or 5 mg/kg or intravenously 1 mg/kg or 2 mg/kg of test compound by gavage.
  • Plasma samples were quantitatively analyzed on a triple quadrupole tandem mass spectrometer using multiple reactive ion monitoring (MRM). WinNonlin 6.3 software was used to calculate the pharmacokinetic parameters using the non-compartmental model method.
  • mice purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., weight 20-25g, male, age 45-60 days, oral administration of 3 mice per group, intravenous injection of 3 mice per group):
  • ICR mice were orally administered 10 mg/kg or 2 mg/kg or 10 mg/kg of test compound via tail vein injection.
  • Plasma samples were quantitatively analyzed on a triple quadrupole tandem mass spectrometer using multiple reactive ion monitoring (MRM). WinNonlin 6.3 software was used to calculate the pharmacokinetic parameters using the non-compartmental model method.
  • Rats were orally administered 2.5 mg/kg or 5 mg/kg or 1 mg/kg intravenously with the test compound.
  • PK determination method of compound in cynomolgus monkey (purchased from Guangdong Chunsheng Biotechnology Development Co., Ltd., weight 3-6kg, male, age 4-6 years old, 3 animals per group by oral administration and 3 animals per group by intravenous injection):
  • Cynomolgus monkeys were given 2.5 mg/kg or 5 mg/kg or intravenously 0.5 mg/kg or 1 mg/kg of the test compound.
  • Test 5 Stability test of the compound of the present invention in liver microsomes of different species
  • the compound of the present invention has better stability in liver microsomes of different species.
  • Dissolution means that 1g (mL) of solute can be dissolved in 10 ⁇ 30mL of solvent;
  • Slightly soluble means that 1g (mL) of the solute can be dissolved in 30 to less than 100 mL of solvent;
  • Slightly soluble means that the solute lg (mL) can be dissolved in 100 to less than 1000 mL of solvent;
  • Very slightly soluble means that 1g (mL) of the solute can be dissolved in 1,000 to less than 10,000 mL of solvent;
  • Test 7 hERG test method
  • Test 8 Liver drug enzyme induction test
  • cryopreserved human liver cells (Baltimore, MD, USA) After resuscitation of cryopreserved human liver cells (Baltimore, MD, USA), trypan blue staining and a cell counter were used to determine the cell number and cell viability. After counting, dilute the hepatocytes with pre-warmed seed plate culture medium to 700,000 living cells per milliliter. Inoculate the diluted hepatocyte suspension on a 48-well plate with pre-coated collagen at 0.2mL/well, and incubate in an incubator for at least 4 hours. When the cells are attached, incubate with 2% base matrigel The culture medium replaces the seed plate culture medium.
  • the incubation medium to prepare the administration working solution every day, including the test product (concentration not less than 0.1 ⁇ M), CYP1A2, CYP2B6, CYP3A4 positive inducer omeprazole, phenobarbital, rifampicin, diluted 1000 times DMSO stock solution.
  • the information of the dosing working fluid is shown in the table below.
  • the culture system After the culture system is established, discard the upper culture medium of the sandwich medium, and add 200 ⁇ L of the administration working solution (including test product, positive control, negative) that has been preheated to 37°C and freshly prepared into each cell culture well Control and matrix control), place the cell culture plate in the incubator and continue to culture for 24 hours. After culturing for 24 hours, replace the freshly prepared administration working fluid and continue culturing for 24 hours. The entire incubation time is 48 hours. Make three parallels for each drug concentration and control concentration.
  • the administration working solution including test product, positive control, negative
  • the potential toxicity of the test product is evaluated by the release of lactate dehydrogenase (LDH) in liver cells. Take out 100 ⁇ L of the dosing working solution after incubating with hepatocytes for 24 hours and 48 hours respectively, and detect the concentration of lactate dehydrogenase with a commercial LDH kit. The cell lysis solution was used as a positive control for the experiment, and the incubation medium was used as a blank control.
  • LDH lactate dehydrogenase
  • RNA is extracted using a fully automatic nucleic acid extraction workstation. Randomly sample samples that exceed 10% of the total amount of the sample at different positions on the sample plate, use the ND2000 micro-spectrophotometer to determine the OD values of 260nM and 280nM, and calculate the ratio of the two to determine the purity of the total RNA. Reverse transcription to obtain cDNA. Use CFX connectTM real-time fluorescent quantitative PCR instrument to quantitatively analyze the selected genes in real time. Set the reaction conditions as follows: 50°C for two minutes; 95°C for ten minutes; 40 cycles of the following two steps: 95°C for fifteen seconds and 60°C for one minute. The endogenous control 18S rRNA was used as an internal standard.
  • This project uses the ⁇ Ct relative quantitative method to compare the differences in gene expression between different treatment groups, and uses 18S rRNA as the internal reference gene to correct the gene expression of each sample.
  • statistical analysis was performed by the method of 2- ⁇ Ct to compare the change of multiples between the treatment group and the blank control group.
  • the experimental data showed the production of CYP1A2, CYP2B6 and CYP3A4 enzyme metabolites.
  • the change in enzyme activity is shown by comparing the fold induction of the corresponding cytochrome enzyme in the presence or absence of the compound.
  • the calculation method of the induction factor and the calculation method of the induction ratio with the control compound are as follows:
  • Induction factor enzyme activity in the sample treated with the test substance/enzyme activity in the sample treated with matrix control
  • the induction ratio with the control compound (the induction factor of the sample treated with the test substance-1)/(the induction factor of the sample treated with the control compound-1) ⁇ 100%.
  • liver drug enzyme induction test show that the compound of the present invention basically has no induction effect on liver drug enzyme.
  • Test 9 Experiment on the effect of human serum on the anti-HBV efficacy of the compound
  • HepG2.2.15 cells integrate a complete HBV genome, and stably express viral RNA and viral proteins.
  • HepG2.2.15 cells can secrete mature hepatitis B virus particles, HBsAg and HBeAg into the culture medium.
  • the viral DNA secreted by HepG2.2.15 cells can be quantified by qPCR method. Different concentrations of human serum are added during the treatment of the test compound, and the effect of the human serum on the antiviral efficacy of the compound is detected.
  • Step 1 Pour 15,000 HepG2.2.15 cells per well in a 96-well cell culture plate, and 200 ⁇ L cell culture medium per well.
  • Step 2 Incubate in a 37°C, 5% CO 2 cell incubator for 3 days until the cells grow to full wells.
  • Step: 3 On day 0 of the test, discard the old medium and add 200 ⁇ L of fresh test medium containing 2% FBS and different human serum (HS) concentrations, including 0% HS, 5% HS, and 10% HS , 20% HS, 40% HS and 50% HS.
  • HS human serum
  • Step 4 Compound preparation and cell treatment in antiviral experiment: Dissolve the compound with DMSO to 30 mM, further dilute the compound with DMSO to 800 ⁇ M, and then perform a 4-fold dilution of 8 dilutions, the highest concentration is 800 ⁇ M. Add 1 ⁇ L of serially diluted compound to each well of the cell plate prepared in step 3. The highest final concentration of the experiment is 4 ⁇ M (200-fold dilution).
  • Step 5 The experiment set TDF (tenofovir disoproxil fumarate, Selleck, Cat S1400) as a positive control compound under 2% FBS conditions, with a maximum concentration of 4 ⁇ M. Add 1 ⁇ L of DMSO to the negative control well, and the final concentration of the experiment is 0.5%.
  • TDF tenofovir disoproxil fumarate, Selleck, Cat S1400
  • Step 6 Incubate the 96-well cell test plate in a 37°C CO 2 incubator for 11 days, change the solution every other day (day 2, 4, 6, 8, and 10), and add 1 ⁇ L of freshly prepared chemical test compound , See steps 3 to 5 for the method.
  • Step 7 Take 150 ⁇ L of supernatant from each well on the 11th day for qPCR detection of viral DNA.
  • Step 8 Compound preparation and cell treatment in cytotoxicity experiment: Prepare a series of diluted compounds with the Bravo liquid handling system, 11 dilutions, 3 times dilution, the highest concentration is 30mM. Use Echo550 to add 0.25 ⁇ L of serially diluted compound to a 384-well cytotoxic cell plate (Greiner 781098). Prepare HepG2.2.15 cells and resuspend them in different concentrations of human serum (50%, 40%, 20%, 10%, 5% and 0%) in the culture medium. Add 50 ⁇ L (4000 cells) of HepG2.2.15 cells prepared above each well to the 384-well cytotoxic cell plate, the highest final concentration of the experiment is 150 ⁇ M (200-fold dilution). The cytotoxicity test was carried out after 4 days incubation in a 37°C CO 2 incubator.
  • Step 1 Dilute the supernatant under 20% HS experimental conditions with DPBS for 2 times, the supernatant under 40% HS experimental conditions with DPBS for 4-fold dilution, and the supernatant under 50% HS experimental conditions with DPBS Make a 5-fold dilution. After mixing, take 1 ⁇ L for qPCR detection.
  • Step 2 Directly take 1 ⁇ L of the supernatant under the experimental conditions of 0% HS, 5% HS and 10% HS for qPCR detection.
  • Step 3 Prepare the qPCR reaction system according to the following ingredients:
  • Step 4 Set up the ABI ViiA7 qPCR instrument according to the following conditions
  • Step 1 Equilibrate PromegaCelltiter-Glo reagent to room temperature.
  • Step 2 Discard the cytotoxicity test plate medium, and add 50 ⁇ L DPBS to each well.
  • Step 3 Add 10 ⁇ L CellTiter-Glo reagent to each well.
  • Step 4 Vibrate on the vibrator for 2 minutes.
  • Step 5 Equilibrate for 10 minutes at room temperature and avoid light.
  • Step 6 Read on Envision plate reader (0.1 seconds/well)
  • plasmid containing the HBV genome (virus copy number: 2 ⁇ 10E6, 2 ⁇ 10E5, 2 ⁇ 10E4, 2 ⁇ 10E3) to make a standard curve, and use the standard curve to calculate the virus copy number.
  • Cytotoxicity% 100-(detected value/average value of DMSO control well ⁇ 100).
  • the cytotoxicity% data was processed with Graphpad Prism 5 software and the curve was drawn, and the CC 50 was calculated by a four-parameter nonlinear regression model.

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Abstract

一种二氢嘧啶类化合物及其在药物中的应用，尤其是作为治疗和预防乙型肝炎的药物的应用。具体地说，涉及通式(I)或式(Ia)所示的化合物或其立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐或它的前药，其中各变量如说明书所定义。还涉及通式(I)或式(Ia)所示的化合物或或其立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物或药学上可接受的盐作为药物的用途，尤其是作为治疗和预防乙型肝炎的药物的用途。

Description

二氢嘧啶类化合物及其在药物中的应用 技术领域
本发明属于医药领域。具体地，本发明涉及一种二氢嘧啶类化合物及其作为药物的用途，尤其是作为用于治疗和/或预防乙型肝炎的药物的用途。本发明还涉及这些二氢嘧啶类化合物同其他抗病毒剂组成的组合物，及其在用于治疗和/或预防乙型肝炎病毒(HBV)感染的应用。
背景技术
乙型肝炎病毒属于肝病毒科。它可引起急性的和/或持续渐进的慢性病。乙型肝炎病毒还可引起病理形态中的许多其他的临床表征——尤其是肝脏的慢性炎症、肝硬化和肝细胞的癌变。另外，与丁型肝炎的共同感染在疾病的发展过程中会产生不利影响。
被许可用于治疗慢性肝炎的常规药物是干扰素和拉米夫定(lamivudine)。然而，干扰素只具有中等的活性，并具有较高的毒副反应；虽然拉米夫定(lamivudine)具有良好的活性，但其耐药性在治疗过程中增幅迅速，并在停止治疗之后常常出现反弹效应，拉米夫定(3-TC)的IC 50值为300nM(Science,299(2003),893-896)。
Deres等报道了以Bay41-4109、Bay39-5493为代表的杂芳环取代的二氢嘧啶类(HAP)化合物，该类化合物能够通过阻止正常核衣壳的形成起到抑制HBV复制的作用。Bay41-4109在临床研究中表现出较好的药物代谢性质(Science,299(2003),893-896)，通过对其作用机制的研究发现，杂芳环取代的二氢嘧啶类化合物通过与核心蛋白的113-143氨基酸残基作用，改变了形成核衣壳的二聚体之间的夹角，导致形成不稳定的膨胀核衣壳，加速核心蛋白的降解(Biochem.Pharmacol.66(2003),2273-2279)。
目前仍然需要有新的能够有效地用作抗病毒药物的化合物，尤其是用作治疗和/或预防乙型肝炎的药物。
发明内容
本发明涉及新型的二氢嘧啶类化合物和其在制备用于治疗与预防HBV感染的药物中的用途。特别地，本发明涉及一种新型的二氢嘧啶类化合物，及其药学上可接受的组合物，该化合物具有溶解性好、稳定性好、对肝药酶基本无诱导作用和较小的毒性等优点，尤其具有非常好的药代动力学性质。本发明化合物可以有效抑制HBV感染，其在抗HBV方面有很好的应用前景。
一方面，本发明涉及一种如式(I)或(Ia)所示的化合物或如式(I)或(Ia)所示的化合物的立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐或它的前药，
Figure PCTCN2020087700-appb-000001
其中，各R 1、R 1b和R 1a独立地为氢、氘、F、Cl、Br、I、氰基、甲基、乙基、甲氧基、乙氧基、甲氨基、乙氨基、硝基、4-三氟甲基苯基、3,5-二(三氟甲基)苯基或三氟甲基；
R 2为C 1-6烷基或C 1-6卤代烷基；
R 3为苯基、咪唑基、呋喃基、噻吩基或噻唑基，其中所述苯基、咪唑基、呋喃基、噻吩基和噻唑基各自独立地未被取代或被1、2、3、4或5个选自氘、F、Cl、Br、OH、CN、C 1-6烷基、羟基C 1-6烷基、C 1-6烷基-OC(＝O)-、C 1-6烷基-OC(＝O)-C 1-6亚烷基、HOOC-C 1-6亚烷基、C 1-6烷氧基-C 1-6亚烷基和C 1-6烷基-S(＝O) 2-的取代基所取代；
W为CH或N；
X 1为-C(＝O)-、-S(＝O) 2-或-(CR 5R 6) j-；
各R 4a、R 4b、R 5和R 6独立地为氢、氘、F、Cl、Br、氨基、C 1-6烷基、NH 2C(＝O)-、C 1-6烷基-OC(＝O)-、羟基C 1-6烷基、C 1-4烷氧基C 1-4亚烷基或C 1-6卤代烷基；
各R 7独立地为氢、氘、F、Cl、Br、氨基、C 1-6烷基、NH 2C(＝O)-、C 1-6烷基-OC(＝O)-、羧基、羧基C 1-6亚烷基、羟基C 1-6烷基、C 1-4烷氧基C 1-4亚烷基或C 1-6卤代烷基；
R y为氢，R 4为甲基、乙基、正丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、F或Cl；或
R y为F或Cl，R 4为氢、F或Cl；
m为0、1、2、3或4；
j为1、2或3。
在一些实施例中，本发明所述的R 2为甲基、乙基、正丙基、异丙基、一氟甲基、二氟甲基或三氟甲基；
R 3为苯基、咪唑基、呋喃基、噻吩基或噻唑基，其中所述苯基和噻唑基各自独立地未被取代或被1、2、3、4或5个选自氘、F、Cl、Br、OH、CN、甲基、乙基、正丙基、异丙基、叔丁基、羟基C 1-4烷基、C 1-4烷基-OC(＝O)-、C 1-4烷基-OC(＝O)-C 1-3亚烷基-、HOOC-C 1-3亚烷基-、C 1-4烷氧基-C 1-3亚烷基-和C 1-4烷基-S(＝O) 2-的取代基所取代。
在一些实施例中，本发明所述的各R 4a、R 4b、R 5和R 6独立地为氢、氘、F、Cl、Br、氨基、甲基、乙基、正丙基、异丙基、正丁基、叔丁基、NH 2C(＝O)-、C 1-4烷基-OC(＝O)-、羟基C 1-4烷基、C 1-4烷氧基C 1-2 亚烷基或C 1-4卤代烷基；
各R 7独立地为氘、F、Cl、Br、氨基、甲基、乙基、正丙基、异丙基、正丁基、叔丁基、NH 2C(＝O)-、C 1-4烷基-OC(＝O)-、羧基、羧基C 1-4亚烷基、羟基C 1-4烷基、C 1-4烷氧基C 1-2亚烷基或C 1-4卤代烷基。
另一方面，本发明还提供了一种药物组合物，包含本发明所述的化合物，及药学上可接受的辅料。
在一些实施方案中，本发明所述的药物组合物，其进一步地包含其它抗HBV药物。
在一些实施方案中，本发明所述的药物组合物，其中所述其它抗HBV药物为HBV聚合酶抑制剂、免疫调节剂或干扰素。
在一些实施方案中，本发明所述的药物组合物，其中所述其它抗HBV药物为拉米夫定、替比夫定、替诺福韦酯，恩替卡韦、阿德福韦酯、Alfaferone、Alloferon、西莫白介素、克拉夫定、恩曲他滨、法普洛韦、干扰素、宝甘灵CP、因特芬、干扰素α-1b、干扰素α、干扰素α-2a、干扰素β-1a、干扰素α-2、白细胞介素-2、米伏替酯、硝唑尼特、聚乙二醇干扰素α-2a、病毒唑、罗扰素-A、西佐喃、Euforavac、安普利近、Phosphazid、Heplisav、干扰素α-2b、左旋咪唑或丙帕锗。
另一方面，本发明还提供了所述化合物或所述药物组合物在制备用于预防、治疗或减轻患者病毒性疾病的药物中的用途。
在一些实施方案中，本发明所述的用途，其中所述病毒性疾病是指乙型肝炎感染或乙型肝炎感染引起的疾病。
在另外一些实施方案中，本发明所述的用途，其中所述乙型肝炎感染引起的疾病是指肝硬化或肝细胞癌变。
另一方面，本发明涉及所述的化合物或药物组合物来制备用于预防、治疗或减轻患者乙型肝炎疾病的药物中的用途，包括给予患者如本发明所述的化合物或本发明所述的药物组合物的有效治疗剂量。
本发明另一方面涉及预防、治疗或减轻患者HBV病症的方法，所述方法包含使用本发明的化合物药学上可接受的有效剂量对患者进行给药。
本发明另一方面涉及预防、治疗或减轻患者HBV病症的方法，所述方法包含使用含有本发明的化合物的药物组合物的药学上可接受的有效剂量对患者进行给药。
本发明另一方面涉及使用一种本发明的化合物来生产用于预防或治疗患者HBV病症，并减轻其严重程度的药物的用途。
本发明另一方面涉及使用一种包含本发明的化合物的药物组合物来生产用于预防或治疗患者HBV病症，并减轻其严重程度的药物的用途。
本发明另一方面涉及一种抑制HBV感染的方法，该方法包含将细胞与能有效抑制HBV的剂量的本发明的化合物或药物组合物接触。另外一些实施例是，所述方法更进一步地包含将细胞与其它抗HBV治疗剂接触。
本发明另一方面涉及对患者HBV疾病的治疗方法，该方法包含向需要治疗的患者施用有效治疗剂量的本发明的化合物或其药物组合物。另外一些实施例是，所述方法更进一步地包含向需要治疗的患者施用有效治疗剂量的其它抗HBV药物。
本发明另一方面涉及一种抑制患者HBV感染的方法，该方法包含向需要治疗的患者施用有效治疗剂量的本发明的化合物或其药物组合物。另外一些实施例是，所述方法更进一步地包含向需要治疗的患者施用有效治疗剂量的其它抗HBV药物。
本发明另一方面涉及式(I)或式(Ia)所包含的化合物的制备、分离和纯化的方法。
前面所述内容只概述了本发明的某些方面，但并不限于这些方面。这些方面及其他的方面的内容将在下面作更加具体完整的描述。
本发明的详细说明书
定义和一般术语
本发明将会把确定的具体化的内容所对应的文献详细列出，实施例都伴随有结构式和化学式的图解。本发明有预期地涵盖所有的选择余地、变体和同等物，这些可能像权利要求所定义的那样包含在现有发明领域。所属领域的技术人员将识别许多类似或等同于在此所描述的方法和物质，这些可以应用于本发明的实践中去。本发明绝非限于方法和物质的描述。有很多文献和相似的物质与本发明申请相区别或抵触，其中包括但绝不限于术语的定义，术语的用法，描述的技术，或像本发明申请所控制的范围。
本发明将应用以下定义除非其他方面表明。根据本发明的目的，化学元素根据元素周期表，CAS版本和化学药品手册，75， thEd，1994来定义。另外，有机化学一般原理见"Organic Chemistry,"Thomas Sorrell,University Science Books,Sausalito:1999,and"March's Advanced Organic Chemistry,"by Michael B.Smith and Jerry March,John Wiley&Sons,New York:2007，因此所有的内容都融合了参考文献。
像本发明所描述的，本发明的化合物可以任选地被一个或多个取代基所取代，如上面的通式化合物，或者像实施例里面特殊的例子，子类，和本发明所包含的一类化合物。
一般而言，术语“取代的”，表示所给结构中的一个或多个氢原子被具体取代基所取代。除非其他方面表明，一个任选的取代基团可以有一个取代基在基团各个可取代的位置进行取代。当所给出的结构式中不止一个位置能被选自具体基团的一个或多个取代基所取代，那么取代基可以相同或不同地在各个位置取代。
在本说明书的各部分，本发明化合物的取代基按照基团种类或范围公开。特别指出，本发明包括这些基团种类和范围的各个成员的每一个独立的次级组合。例如，术语“C 1-6烷基”特别指独立公开的甲基、乙基、C 3烷基、C 4烷基、C 5烷基和C 6烷基。
本发明使用的术语“烷基”包括1-20个碳原子饱和直链或支链的单价烃基，其中烷基可以独立任选地被一个或多个本发明所描述的取代基所取代。其中一些实施例是，烷基基团含有1-12个碳原子，另外一些实施例是，烷基基团含有1-10个碳原子，另外一些实施例是，烷基基团含有1-8个碳原子，另外一些实施例是，烷基基团含有1-6个碳原子，另外一些实施例是，烷基基团含有1-4个碳原子，另外一些实施例是，烷基基团含有1-3个碳原子。烷基基团更进一步的实例包括，但并不限于，甲基(Me，-CH 3)、乙基(Et，-CH 2CH 3)、正丙基(n-Pr，-CH 2CH 2CH 3)、异丙基(i-Pr，-CH(CH 3) 2)、正丁基(n-Bu，-CH 2CH 2CH 2CH 3)、2-甲基丙基或异丁基(i-Bu，-CH 2CH(CH 3) 2)、1-甲基丙基或仲丁基(s-Bu，-CH(CH 3)CH 2CH 3)、叔丁基(t-Bu，-C(CH 3) 3)、正戊基(-CH 2CH 2CH 2CH 2CH 3)、2-戊基(-CH(CH 3)CH 2CH 2CH 3)、3-戊基(-CH(CH 2CH 3) 2)、2-甲基-2-丁基(-C(CH 3) 2CH 2CH 3)、3-甲基-2-丁基(-CH(CH 3)CH(CH 3) 2)、3-甲基-1-丁基(-CH 2CH 2CH(CH 3) 2)、2-甲基-1-丁基(-CH 2CH(CH 3)CH 2CH 3)、正己基(-CH 2CH 2CH 2CH 2CH 2CH 3)、2-己基(-CH(CH 3)CH 2CH 2CH 2CH 3)、3-己基(-CH(CH 2CH 3)(CH 2CH 2CH 3))、2-甲基-2-戊基(-C(CH 3) 2CH 2CH 2CH 3)、3-甲基-2-戊基(-CH(CH 3)CH(CH 3)CH 2CH 3)、4-甲基-2-戊基(-CH(CH 3)CH 2CH(CH 3) 2)、3-甲基-3-戊基(-C(CH 3)(CH 2CH 3) 2)、2-甲基-3-戊基(-CH(CH 2CH 3)CH(CH 3) 2)、2,3-二甲基-2-丁基(-C(CH 3) 2CH(CH 3) 2)、3,3-二甲基-2-丁基(-CH(CH 3)C(CH 3) 3)、正庚基、正辛基，等等。
术语“亚烷基”表示从饱和的直链或支链烃基中去掉两个或多个氢原子所得到的饱和的二价或多价烃基基团。除非另外详细说明，亚烷基基团含有1-12个碳原子。在一些实施方案中，亚烷基基团含有1-6个碳原子；在另一些实施方案中，亚烷基基团含有1-4个碳原子；还在一些实施方案中，亚烷基基团含有1-3个碳原子；还在另一些实施方案中，亚烷基基团含有1-2个碳原子。亚烷基的实例包括，但不限于亚甲基(-CH 2-)，亚乙基(-CH 2CH 2-)，亚正丙基(-CH 2CH 2CH 2-),亚异丙基(-CH(CH 3)CH 2-)等等。
术语“羟基烷基”和“羟基烷氧基”表示烷基或烷氧基，视情况而定，被一个或多个羟基基团所取代，其 中，“羟基烷基”、“羟基亚烷基”与“羟烷基”相互之间可以交换使用，这样的实例包含，但并不限于，羟基甲基(-CH 2OH)、羟基乙基(-CH 2CH 2OH,-CHOHCH 3)、羟基丙基(如，-CH 2CH 2CH 2OH,-CH 2CHOHCH 3,-CHOHCH 2CH 3)、羟基甲氧基(-OCH 2OH)等。
术语“卤代烷基”、“卤代烯基”或“卤代烷氧基”表示烷基，烯基或烷氧基基团被一个或多个卤素原子所取代，其中，烷基、烯基和烷氧基具有本发明所述的含义。这样的实例包含，但并不限于，二氟乙基(-CH 2CHF 2,-CF 2CH 3,-CHFCH 2F)、三氟乙基(-CH 2CF 3,-CF 2CH 2F,-CFHCHF 2)、三氟甲基(-CF 3)、三氟甲氧基(-OCF 3)、氟乙烯基(-CH＝CHF,-CF＝CH 2)等。
术语“烷氧基”表示烷基基团通过氧原子与分子其余部分相连，其中烷基基团具有如本发明所述的含义。除非另外详细说明，所述烷氧基基团含有1-12个碳原子。在一些实施方案中，烷氧基基团含有1-8个碳原子；在另一些实施方案中，烷氧基基团含有1-6个碳原子；在另一些实施方案中，烷氧基基团含有1-4个碳原子；在又一些实施方案中，烷氧基基团含有1-3个碳原子。所述烷氧基基团可以任选地被一个或多个本发明描述的取代基所取代。
烷氧基基团的实例包括，但并不限于，甲氧基(MeO、-OCH 3)，乙氧基(EtO、-OCH 2CH 3)，1-丙氧基(n-PrO、n-丙氧基、-OCH 2CH 2CH 3)，2-丙氧基(i-PrO、i-丙氧基、-OCH(CH 3) 2)，1-丁氧基(n-BuO、n-丁氧基、-OCH 2CH 2CH 2CH 3)，2-甲基-l-丙氧基(i-BuO、i-丁氧基、-OCH 2CH(CH 3) 2)，2-丁氧基(s-BuO、s-丁氧基、-OCH(CH 3)CH 2CH 3)，2-甲基-2-丙氧基(t-BuO、t-丁氧基、-OC(CH 3) 3)，1-戊氧基(n-戊氧基、-OCH 2CH 2CH 2CH 2CH 3)，2-戊氧基(-OCH(CH 3)CH 2CH 2CH 3)，3-戊氧基(-OCH(CH 2CH 3) 2)，2-甲基-2-丁氧基(-OC(CH 3) 2CH 2CH 3)，3-甲基-2-丁氧基(-OCH(CH 3)CH(CH 3) 2)，3-甲基-l-丁氧基(-OCH 2CH 2CH(CH 3) 2)，2-甲基-l-丁氧基(-OCH 2CH(CH 3)CH 2CH 3)，等等。
术语“卤素”或“卤原子”是指F、Cl、Br或I。
本发明所使用的术语“不饱和的”表示部分含有一个或多个不饱和度。
术语“芳基”表示含有6-14个环原子，或6-12个环原子，或6-10个环原子的单环、双环和三环的碳环体系，其中，至少有一个环体系是芳香族的，其中每一个环体系包含3-7个原子组成的环，且有一个或多个连接点与分子的其余部分相连。术语“芳基”可以和术语“芳香环”交换使用。芳基基团的实例可以包括苯基、萘基和蒽基。所述芳基基团可以独立任选地被一个或多个本发明所描述的取代基所取代。
术语“杂芳基”表示含有5-12个环原子的单环、双环和三环体系，其中至少有一个环是芳香环的，且至少有一个芳香环包含一个或多个杂原子，其中每一个环体系包含5-7环个原子组成的环，且有一个或多个连接点与分子其余部分相连。术语“杂芳基”可以与术语“芳杂环”、“杂芳环”或“杂芳族化合物”交换使用。在一些实施方案中，杂芳基为包含1，2，3或4个独立选自氮、硫和氧的杂原子的5-7个环原子组成的单环杂芳基。一些实施方案中，杂芳基为包含1，2，3或4个独立选自氮、硫和氧的杂原子的5-6个环原子组成的单环杂芳基。在一些实施案中，杂芳基为包含1，2，3或4个独立选自氮、硫和氧的杂原子的7-12个环原子组成的双环杂芳基。在一些实施案中，杂芳基为包含1，2，3或4个独立选自氮、硫和氧的杂原子的8-10个环原子组成的双环杂芳基。在一些实施案中，杂芳基为包含1，2，3或4个独立选自氮、硫和氧的杂原子的9-10个环原子组成的双环杂芳基。
杂芳环的实例包括以下的单环，但并不限于这些单环：1,2,4-噁二唑-5(4H)-硫酮基、1,2,4-噻二唑-5(4H)-酮基、1,2,4-噁二唑-5(4H)-酮基、1,3,4-噁二唑-2(3H)-硫酮基、1H-1,2,4-三唑-5(4H)-酮基、2-呋喃基、3-呋喃基、N-咪唑基、2-咪唑基、4-咪唑基、5-咪唑基、3-异噁唑基、4-异噁唑基、5-异噁唑基、2-噁唑基、4-噁唑基、5-噁唑基、N-吡咯基、2-吡咯基、3-吡咯基、2-吡啶基、3-吡啶基、4-吡啶基、2-嘧啶基、4-嘧啶基、5-嘧啶基、哒嗪基(如3-哒嗪基)、2-噻唑基、4-噻唑基、5-噻唑基、四唑基(如5-四唑基)、三唑基(如2-三唑基和5-三唑基)、2-噻吩基、3-噻吩基、吡喃基、吡唑基(如2-吡唑基)、异噻唑基、1,2,3-噁二唑基、1,2,5-噁二唑基、1,2,4-噁二唑基、1,2,3-三唑基、1,2,3-硫代二唑基、1,3,4-硫代二唑基、1,2,5-硫代二唑基、 吡嗪基、1,3,5-三嗪基、二唑基、噻二唑基、三嗪基等；也包括以下的双环，但绝不限于这些双环：苯并噻唑基、苯并咪唑基、苯并呋喃基、苯并噻吩基、吲哚基(如2-吲哚基)、嘌呤基、喹啉基(如2-喹啉基、3-喹啉基、4-喹啉基)、异喹啉基(如1-异喹啉基、3-异喹啉基或4-异喹啉基)等。
术语“M-M 1个环原子组成的”表示所述环状基团由M-M 1个环原子所组成，所述的环原子包括碳原子和/或O、N、S、P等杂原子。例如，“3-6个环原子组成的杂环基”代表其包括3、4、5或6个环原子组成的杂环基。
术语“烷氧基烷基”“烷氧基亚烷基”可相互交换使用，表示烷基基团可以被一个或多个相同或不同的烷氧基基团所取代，其中烷氧基和烷基基团具有如本发明所述的含义。这样的实例包括，但并不限于环己基甲基，环丙基乙基，甲氧基乙基，乙氧基甲基等。
像本发明所描述的，取代基画一个键连接到中心的环上形成的环体系(如式a所示)代表取代基在环上任何可取代的位置都可以取代，如式b、c、d、e、f、g和h所示。
Figure PCTCN2020087700-appb-000002
另外，需要说明的是，除非以其他方式明确指出，在本文中通篇采用的描述方式“各…和…独立地为”、“…和…各自独立地为”和“…和…分别独立地为”可以互换，应做广义理解，其既可以是指在不同基团中，相同符号之间所表达的具体选项之间互相不影响，也可以表示在相同的基团中，相同符号之间所表达的具体选项之间互相不影响。例如，如式p所示，多个R 7的具体选项互相之间不受影响。
Figure PCTCN2020087700-appb-000003
除非其他方面表明，本发明所描述的结构式包括所有的同分异构形式(如对映异构，非对映异构，和几何异构(或构象异构)：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体，和(Z)、(E)的构象异构体。因此，本发明的化合物的单个立体化学异构体或其对映异构体，非对映异构体，或几何异构体(或构象异构体)的混合物都属于本发明的范围。
本发明所使用的术语“前药”，代表一个化合物在体内转化为式(I)所示的化合物。这样的转化受前体药物在血液中水解或在血液或组织中经酶转化为母体结构的影响。本发明前体药物类化合物可以是酯，在现有的发明中酯可以作为前体药物的有苯酯类，脂肪族(C 1-24)酯类，酰氧基甲基酯类，碳酸酯，氨基甲酸酯类和氨基酸酯类。例如本发明里的一个化合物包含羟基，即可以将其酰化得到前体药物形式的化合物。其他的前体药物形式包括磷酸酯，如这些磷酸酯类化合物是经母体上的羟基磷酸化得到的。关于前体药物 完整的讨论可以参考以下文献：T.Higuchi and V.Stella,Pro-drugs as Novel Delivery Systems,Vol.14 of the A.C.S.Symposium Series,Edward B.Roche,ed.,Bioreversible Carriers in Drug Design,American Pharmaceutical Association and Pergamon Press,1987,J.Rautio et al,Prodrugs:Design and Clinical Applications,Nature Review Drug Discovery,2008,7,255-270,and S.J.Hecker et al,Prodrugs of Phosphates and Phosphonates,Journal of Medicinal Chemistry,2008,51，2328-2345。
除非其他方面表明，本发明的化合物的所有互变异构形式都包含在本发明的范围之内。另外，除非其他方面表明，本发明所描述的化合物的结构式包括一个或多个不同的原子的富集同位素。
“代谢产物”是指具体的化合物或其盐在体内通过代谢作用所得到的产物。一个化合物的代谢产物可以通过所属领域公知的技术来进行鉴定，其活性可以通过如本发明所描述的那样采用试验的方法进行表征。这样的产物可以是通过给药化合物经过氧化、还原、水解、酰氨化，脱酰氨作用、酯化、脱脂作用，酶裂解等等方法得到。相应地，本发明包括化合物的代谢产物，包括将本发明的化合物与哺乳动物充分接触一段时间所产生的代谢产物。
本发明中立体化学的定义和惯例的使用通常参考以下文献：S.P.Parker,Ed.,McGraw-Hill Dictionary of Chemical Terms(1984)McGraw-Hill Book Company,New York；and Eliel,E.and Wilen,S.,"Stereochemistry of Organic Compounds",John Wiley&Sons,Inc.,New York,1994.本发明的化合物可以包含不对称中心或手性中心，因此存在不同的立体异构体。本发明的化合物所有的立体异构形式，包括但绝不限于，非对映体，对映异构体，阻转异构体，和它们的混合物，如外消旋混合物，组成了本发明的一部分。很多有机化合物都以光学活性形式存在，即它们有能力旋转平面偏振光的平面。在描述光学活性化合物时，前缀D、L或R、S用来表示分子手性中心的绝对构型。前缀d、l或(+)、(-)用来命名化合物平面偏振光旋转的符号，(-)或l是指化合物是左旋的，前缀(+)或d是指化合物是右旋的。这些立体异构体的化学结构是相同的，但是它们的立体结构不一样。特定的立体异构体可以是对映体，异构体的混合物通常称为对映异构体混合物。50：50的对映体混合物被称为外消旋混合物或外消旋体，这可能导致化学反应过程中没有立体选择性或立体定向性。术语“外消旋混合物”和“外消旋体”是指等摩尔的两个对映异构体的混合物，缺乏光学活性。
术语“互变异构体”或“互变异构的形式”是指不同能量的结构的同分异构体可以通过低能垒互相转化。例如质子互变异构体(即质子转移的互变异构体)包括通过质子迁移的互变，如酮式-烯醇式和亚胺-烯胺的同分异构化作用。原子价(化合价)互变异构体包括重组成键电子的互变。除非另外指出，本发明化合物的所有互变异构体形式都在本发明的范围之内。
本发明所使用的“药学上可接受的盐”是指本发明的化合物的有机盐和无机盐。药学上可接受的盐在所属领域是为我们所熟知的，如文献：S.M.Berge et al.,describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences,66:1-19,1977.所记载的。药学上可接受的无毒的酸形成的盐包括，但并不限于，与氨基基团反应形成的无机酸盐有盐酸盐、氢溴酸盐、磷酸盐、硫酸盐、高氯酸盐、和有机酸盐如乙酸盐、草酸盐、马来酸盐、酒石酸盐、柠檬酸盐、琥珀酸盐、丙二酸盐、或通过书籍文献上所记载的其他方法如离子交换法来得到这些盐。其他药学上可接受的盐包括己二酸盐、苹果酸盐、2-羟基丙酸盐、藻酸盐、抗坏血酸盐、天冬氨酸盐、苯磺酸盐、苯甲酸盐、重硫酸盐、硼酸盐、丁酸盐、樟脑酸盐、樟脑磺酸盐、环戊基丙酸盐、二葡萄糖酸盐、十二烷基硫酸盐、乙磺酸盐、甲酸盐、反丁烯二酸盐、葡庚糖酸盐、甘油磷酸盐、葡萄糖酸盐、半硫酸盐、庚酸盐、己酸盐、氢碘酸盐、2-羟基-乙磺酸盐、乳糖醛酸盐、乳酸盐、月桂酸盐、月桂基硫酸盐、苹果酸盐、丙二酸盐、甲磺酸盐、2-萘磺酸盐、烟酸盐、硝酸盐、油酸盐、棕榈酸盐、扑酸盐、果胶酸盐、过硫酸盐、3-苯基丙酸盐、苦味酸盐、特戊酸盐、丙酸盐、硬脂酸盐、硫氰酸盐、对甲苯磺酸盐、十一酸盐、戊酸盐，等等。通过适当的碱得到的盐包括碱金属，碱土金属，铵和N +(C 1-4烷基) 4的盐。本发明也拟构思了任何所包含N的基团的化合物所形成的季铵盐。水溶性或油溶性或分散产 物可以通过季铵化作用得到。碱金属或碱土金属盐包括钠、锂、钾、钙、镁，等等。药学上可接受的盐进一步包括适当的、无毒的铵，季铵盐和抗平衡离子形成的胺阳离子，如卤化物、氢氧化物、羧化物、硫酸化物、磷酸化物、硝酸化物、C 1-8磺酸化物和芳香磺酸化物。
本发明的“溶剂化物”是指一个或多个溶剂分子与本发明的化合物所形成的缔合物。形成溶剂化物的溶剂包括，但并不限于，水、异丙醇、乙醇、甲醇、二甲亚砜、乙酸乙酯、乙酸、氨基乙醇。术语“水合物”是指溶剂分子是水所形成的缔合物。
术语“保护基团”或“Pg”是指一个取代基与别的官能团起反应的时候，通常用来阻断或保护特殊的功能性。例如，“氨基的保护基团”是指一个取代基与氨基基团相连来阻断或保护化合物中氨基的功能性，合适的氨基保护基团包括乙酰基、三氟乙酰基、叔丁氧羰基(BOC)、苄氧羰基(CBZ)和9-芴亚甲氧羰基(Fmoc)。相似地，“羟基保护基团”是指羟基的取代基用来阻断或保护羟基的功能性，合适的保护基团包括乙酰基和甲硅烷基。“羧基保护基团”是指羧基的取代基用来阻断或保护羧基的功能性，一般的羧基保护基包括-CH 2CH 2SO 2Ph、氰基乙基、2-(三甲基硅烷基)乙基、2-(三甲基硅烷基)乙氧基甲基、2-(对甲苯磺酰基)乙基、2-(对硝基苯磺酰基)乙基、2-(二苯基膦基)乙基、硝基乙基，等等。对于保护基团一般的描述可参考文献：T W.Greene,Protective Groups in Organic Synthesis,John Wiley&Sons,New York,1991；and P.J.Kocienski,Protecting Groups,Thieme,Stuttgart,2005。
本发明化合物的描述
本发明所涉及的化合物，及其药学上可接受的组合物，都可以有效抑制HBV感染。
一方面，本发明涉及一种如式(I)或(Ia)所示的化合物或如式(I)或(Ia)所示的化合物的立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐或它的前药，
Figure PCTCN2020087700-appb-000004
其中，各R 1、R 1b和R 1a独立地为氢、氘、F、Cl、Br、I、氰基、甲基、乙基、甲氧基、乙氧基、甲氨基、乙氨基、硝基、4-三氟甲基苯基、3,5-二(三氟甲基)苯基或三氟甲基；
R 2为C 1-6烷基或C 1-6卤代烷基；
R 3为苯基、咪唑基、呋喃基、噻吩基或噻唑基，其中所述苯基、咪唑基、呋喃基、噻吩基和噻唑基各自独立地未被取代或被1、2、3、4或5个选自氘、F、Cl、Br、OH、CN、C 1-6烷基、羟基C 1-6烷基、C 1-6烷基-OC(＝O)-、C 1-6烷基-OC(＝O)-C 1-6亚烷基、HOOC-C 1-6亚烷基、C 1-6烷氧基-C 1-6亚烷基和C 1-6烷基-S(＝O) 2- 的取代基所取代；
W为CH或N；
X 1为-C(＝O)-、-S(＝O) 2-或-(CR 5R 6) j-；
各R 4a、R 4b、R 5和R 6独立地为氢、氘、F、Cl、Br、氨基、C 1-6烷基、NH 2C(＝O)-、C 1-6烷基-OC(＝O)-、羟基C 1-6烷基、C 1-4烷氧基C 1-4亚烷基或C 1-6卤代烷基；
各R 7独立地为氢、氘、F、Cl、Br、氨基、C 1-6烷基、NH 2C(＝O)-、C 1-6烷基-OC(＝O)-、羧基、羧基C 1-6亚烷基、羟基C 1-6烷基、C 1-4烷氧基C 1-4亚烷基或C 1-6卤代烷基；
R y为氢，R 4为甲基、乙基、正丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、F或Cl；或
R y为F或Cl，R 4为氢、F或Cl；
m为0、1、2、3或4；
j为1、2或3。
在一些实施例中，本发明所述的R 2为甲基、乙基、正丙基、异丙基、一氟甲基、二氟甲基或三氟甲基；
R 3为苯基、咪唑基、呋喃基、噻吩基或噻唑基，其中所述苯基和噻唑基各自独立地未被取代或被1、2、3、4或5个选自氘、F、Cl、Br、OH、CN、甲基、乙基、正丙基、异丙基、叔丁基、羟基C 1-4烷基、C 1-4烷基-OC(＝O)-、C 1-4烷基-OC(＝O)-C 1-3亚烷基-、HOOC-C 1-3亚烷基-、C 1-4烷氧基-C 1-3亚烷基-和C 1-4烷基-S(＝O) 2-的取代基所取代。
在一些实施例中，本发明所述的各R 4a、R 4b、R 5和R 6独立地为氢、氘、F、Cl、Br、氨基、甲基、乙基、正丙基、异丙基、正丁基、叔丁基、NH 2C(＝O)-、C 1-4烷基-OC(＝O)-、羟基C 1-4烷基、C 1-4烷氧基C 1-2亚烷基或C 1-4卤代烷基；
各R 7独立地为氘、F、Cl、Br、氨基、甲基、乙基、正丙基、异丙基、正丁基、叔丁基、NH 2C(＝O)-、C 1-4烷基-OC(＝O)-、羧基、羧基C 1-4亚烷基、羟基C 1-4烷基、C 1-4烷氧基C 1-2亚烷基或C 1-4卤代烷基。
另一方面，本发明涉及到以下其中之一的化合物或它们的立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐或它的前药，但绝不限于这些化合物：
Figure PCTCN2020087700-appb-000005
Figure PCTCN2020087700-appb-000006
Figure PCTCN2020087700-appb-000007
Figure PCTCN2020087700-appb-000008
Figure PCTCN2020087700-appb-000009
另一方面，本发明还提供了一种药物组合物，包含本发明所述的化合物，及药学上可接受的辅料。
在一些实施方案中，本发明所述的药物组合物，其进一步地包含其它抗HBV药物。
在一些实施方案中，本发明所述的药物组合物，其中所述其它抗HBV药物为HBV聚合酶抑制剂、免疫调节剂或干扰素。
在一些实施方案中，本发明所述的药物组合物，其中所述其它抗HBV药物为拉米夫定、替比夫定、替诺福韦酯，恩替卡韦、阿德福韦酯、Alfaferone、Alloferon、西莫白介素、克拉夫定、恩曲他滨、法普洛韦、干扰素、宝甘灵CP、因特芬、干扰素α-1b、干扰素α、干扰素α-2a、干扰素β-1a、干扰素α-2、白细胞介素-2、米伏替酯、硝唑尼特、聚乙二醇干扰素α-2a、病毒唑、罗扰素-A、西佐喃、Euforavac、安普利近、Phosphazid、Heplisav、干扰素α-2b、左旋咪唑或丙帕锗。
另一方面，本发明还提供了所述化合物或所述药物组合物在制备用于预防、治疗或减轻患者病毒性疾病的药物中的用途。
在一些实施方案中，本发明所述的用途，其中所述病毒性疾病是指乙型肝炎感染或乙型肝炎感染引起的疾病。
在另外一些实施方案中，本发明所述的用途，其中所述乙型肝炎感染引起的疾病是指肝硬化或肝细胞癌变。
另一方面，使用本发明所述化合物或所述药物组合物用于制备用于预防、治疗或减轻患者病毒性疾病的药物。
在一些实施方案中，本发明所述化合物或所述药物组合物的使用，其中所述病毒性疾病是指乙型肝炎感染或乙型肝炎感染引起的疾病。
在另外一些实施方案中，本发明所述化合物或所述药物组合物的使用，其中所述乙型肝炎感染引起疾病是指肝硬化或肝细胞癌变。
另一方面，本发明涉及预防、治疗或减轻患者病毒性疾病的方法，其中，所述方法包含使用本发明的化合物或药物组合物药学上可接受的有效剂量对患者进行给药。
在一些实施方案中，本发明所述方法，其中所述病毒性疾病是指乙型肝炎感染或乙型肝炎感染引起的疾病。
在另外一些实施方案中，本发明所述方法，其中所述乙型肝炎感染引起的疾病是指肝硬化或肝细胞癌变。
另一方面，本发明涉及所述的化合物或药物组合物在制备用于预防、治疗或减轻患者乙型肝炎疾病的药品的用途。
本发明另一方面涉及预防、治疗或减轻患者HBV病症的方法，所述方法包含使用本发明的化合物药学上可接受的有效剂量对患者进行给药。
本发明另一方面涉及预防、治疗或减轻患者HBV病症的方法，所述方法包含使用含有本发明的化合物的药物组合物的药学上可接受的有效剂量对患者进行给药。
本发明另一方面涉及使用一种本发明的化合物来生产用于预防或治疗患者HBV病症，并减轻其严重程度的药品的用途。
本发明另一方面涉及使用一种包含本发明的化合物的药物组合物来生产用于预防或治疗患者HBV病症，并减轻其严重程度的药品的用途。
其中一些实施方案是，所述患者是哺乳动物，另外一些实施例是，所述患者是人类。另外一些实施例是，所述用途更进一步地包含细胞与抗HBV治疗剂的接触。
本发明另一方面涉及一种抑制HBV感染的方法，该方法包含将细胞与能有效抑制HBV的剂量的本发明的化合物或药物组合物接触。另外一些实施例是，所述方法更进一步地包含将细胞与其它抗HBV治疗剂接触。
本发明另一方面涉及对患者HBV疾病的治疗方法，该方法包含向需要治疗的患者施用有效治疗剂量的本发明的化合物或其药物组合物。
另外一些实施方案是，所述方法更进一步地包含向需要治疗的患者施用有效治疗剂量的其它抗HBV治疗剂。
本发明另一方面涉及一种抑制患者HBV感染的方法，该方法包含向需要治疗的患者施用有效治疗剂量的本发明的化合物或其药物组合物。另外一些实施例是，所述方法更进一步地包含向需要治疗的患者施用有效治疗剂量的其它抗HBV治疗剂。
本发明另一方面涉及式(I)或式(Ia)所包含的化合物的制备、分离和纯化的方法。
本发明还涉及本发明的化合物及其药学上可接受的盐用于生产医药产品来有效抑制HBV感染的应用,本发明的化合物在生产有效抑制HBV感染的药物中的应用。本发明的化合物还用于生产一种医药品用来减轻、阻止、控制或治疗患者乙型肝炎的病症。
除非其他方面表明，本发明的化合物所有的立体异构体，几何异构体，互变异构体，氮氧化物，水合物，溶剂化物，代谢产物，药学上可接受的盐和前药都属于本发明的范围。
具体地说，盐是药学上可接受的盐。术语“药学上可接受的”包括物质或组合物必须是适合化学或毒理学地，与组成制剂的其他组分和用于治疗的哺乳动物有关。
本发明的化合物的盐还包括用于制备或纯化式(I)或(Ia)所示化合物的中间体或式(I)或(Ia)所示化合物分离的对映异构体的盐，但不一定是药学上可接受的盐。
如果本发明的化合物是碱性的，则想得到的盐可以通过文献上提供的任何合适的方法制备得到，例如，使用无机酸，如盐酸、氢溴酸、硫酸、硝酸和磷酸等等。或者使用有机酸、如乙酸、马来酸、琥珀酸、扁桃酸、富马酸、丙二酸、丙酮酸、苹果酸、2-羟基丙酸、枸橼酸、草酸、羟乙酸和水杨酸；吡喃糖酸，如葡萄糖醛酸和半乳糖醛酸；α-羟酸，如柠檬酸和酒石酸；氨基酸，如天门冬氨酸和谷氨酸；芳香族酸，如苯甲酸和肉桂酸；磺酸，如对甲苯磺酸、苯磺酸、甲磺酸、乙磺酸、三氟甲磺酸等等或它们的组合。
如果本发明的化合物是酸性的，则想得到的盐可以通过合适的方法制备得到，如，使用无机碱或有机碱，如氨(伯氨，仲氨，叔氨)，碱金属氢氧化物，铵，N +(R 14) 4的盐和碱土金属氢氧化物，等等。合适的盐包括，但并不限于，从氨基酸得到的有机盐，如甘氨酸和精氨酸，氨，如伯氨、仲氨和叔氨，N +(R 14) 4 的盐，如R 14是H、C 1-4烷基、C 6-10芳基、C 6-10芳基C 1-4烷基等，和环状氨，如哌啶、吗啉和哌嗪等，和从钠、钙、钾、镁、锰、铁、铜、锌、铝和锂得到无机盐。也包括适当的，无毒的铵，季铵盐和抗平衡离子形成的胺阳离子，如卤化物、氢氧化物、羧化物、硫酸化物、磷酸化物、硝酸化物、C 1-8磺酸化物和芳香磺酸化物。
本发明的化合物的药物组合物，制剂，给药和化合物及药物组合物的用途
根据另一方面，本发明的药物组合物的特点包括式(I)或(Ia)的化合物，本发明所列出的化合物，或实施例化合物，和药学上可接受的辅料。本发明的药物组合物中化合物能有效的抑制乙型肝炎病毒，适用于病毒引起的疾病尤其是急性和慢性持续的HBV感染的治疗，HBV引发的慢性病毒病可能导致病态变严重，慢性乙型肝炎病毒感染在许多情况下可导致肝硬化和/或肝细胞癌变。
对本发明的化合物来说，可能被提及的疾病治疗的区域是，例如：可能导致传染性肝炎的急性和慢性病毒感染的治疗，例如，乙肝病毒感染。本发明的化合物尤其适合治疗慢性乙肝感染和急性和慢性乙肝病毒感染。
本发明包括药物制剂，除了无毒，惰性的制药学上合适的辅料外，还含有一种或多种本发明的式(I)或(Ia)化合物或其药物组合物或含有一种或多种活性成分式(I)或(Ia)化合物或本发明的药物组合物。
上述药物制剂也可以包含式(I)或(Ia)化合物以外的其他活性药物成分。
本发明的化合物存在自由形态，或合适的、作为药学上可接受的衍生物。根据本发明，药学上可接受的衍生物包括，但并不限于，药学上可接受的前药，盐，酯，酯类的盐，或能直接或间接地根据患者的需要给药的其他任何加合物或衍生物，本发明其他方面所描述的化合物，其代谢产物或它的残留物。
像本发明所描述的，本发明药物组合物包含任何一种本发明的式(I)或(Ia)所示化合物，进一步包含药学上可接受的辅料，这些辅料，例如像本发明所应用的，包括任何溶剂、固体赋形剂、稀释剂、粘合剂、崩解剂、或其他液体赋形剂、分散剂、矫味剂或悬浮剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂，固体粘合剂或润滑剂，等等，适合于特有的目标剂型。如以下文献所描述的：In Remington:The Science and Practice of Pharmacy,21st edition,2005,ed.D.B.Troy,Lippincott Williams&Wilkins,Philadelphia,and Encyclopedia of Pharmaceutical Technology,eds.J.Swarbrick and J.C.Boylan,1988-1999,Marcel Dekker,New York,综合此处文献的内容，表明不同的辅料可应用于药学上可接受的组合物的制剂和它们公知的制备方法。除了任何常规的辅料与本发明的化合物不相容的范围，例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用，它们的用途也是本发明所考虑的范围。
可作为药学上可接受的辅料的物质包括，但并不限于，离子交换剂；铝；硬脂酸铝；卵磷脂；血清蛋白，如人血清蛋白；缓冲物质如磷酸盐；甘氨酸；山梨酸；山梨酸钾；饱和植物脂肪酸的部分甘油酯混合物；水；盐或电解质，如硫酸鱼精蛋白，磷酸氢二钠，磷酸氢钾，氯化钠，锌盐；胶体硅；三硅酸镁；聚乙烯吡咯烷酮；聚丙烯酸脂；蜡；聚乙烯-聚氧丙烯-阻断聚合体；羊毛脂；糖，如乳糖，葡萄糖和蔗糖；淀粉如玉米淀粉和土豆淀粉；纤维素和它的衍生物如羧甲基纤维素钠，乙基纤维素和乙酸纤维素；树胶粉；麦芽；明胶；滑石粉；辅料如可可豆脂和栓剂蜡状物；油如花生油，棉子油，红花油，麻油，橄榄油，玉米油和豆油；二醇类化合物，如丙二醇和聚乙二醇；酯类如乙基油酸酯和乙基月桂酸酯；琼脂；缓冲剂如氢氧化镁和氢氧化铝；海藻酸；无热原的水；等渗盐；林格(氏)溶液；乙醇；磷酸缓冲溶液；和其他无毒的合适的润滑剂如月桂硫酸钠和硬脂酸镁；着色剂；释放剂；包衣衣料；甜味剂；调味剂；香料；防腐剂和抗氧化剂。
本发明化合物的药物组合物，可以以下方面的任意方式施用：口服给药，喷雾吸入法，局部给药，经直肠给药，经鼻给药，局部给药，阴道给药，非肠道给药如皮下，静脉，肌内，腹腔内，鞘内，心室内， 胸骨内，或颅内注射或输液，或借助一种外植的储器用药。优选的方式为口服给药，肌注，向腹膜内给药或静脉注射。
本发明化合物或其药物组合物可以是以单位剂量形式给药。给药剂型可以是液体剂型、固体剂型。液体剂型可以是真溶液类、胶体类、微粒剂型、混悬剂型。其他剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、栓剂、冻干粉针剂、包合物、埋植剂、贴剂、擦剂等。
口服片剂和胶囊可以含有赋形剂如粘合剂，如糖浆，阿拉伯胶，山梨醇，黄芪胶，或聚乙烯吡咯烷酮；填充剂，如乳糖，蔗糖，玉米淀粉，磷酸钙，山梨醇，氨基乙酸；润滑剂，如硬脂酸镁，滑石，聚乙二醇，硅土；崩解剂，如马铃薯淀粉；或可接受的增润剂如月桂醇钠硫酸盐。片剂可以用制药学上公知的方法包衣。
口服液可以制成水合油的悬浮液，溶液，乳浊液，糖浆或酏剂，也可以制成干品，用前补充水或其它合适的媒质。这种液体制剂可以包含常规的添加剂，如悬浮剂，山梨醇，纤维素甲醚，葡萄糖糖浆，凝胶，羟乙基纤维素，羧甲基纤维素，硬脂酸铝凝胶，氢化的食用油脂，乳化剂，如卵磷脂，山梨聚醣单油酸盐，阿拉伯胶；或非水辅料(可能包含可食用油)，如杏仁油，油脂如甘油，乙二醇，或乙醇；防腐剂，如对羟基苯甲酸甲酯或丙酯，山梨酸。如需要可添加调味剂或着色剂。
栓剂可包含常规的栓剂基质，如可可黄油或其他甘油酯。
对胃外投药，液态剂型通常由化合物和一种消毒的辅料制成。辅料首选水。依照所选辅料和药物浓度的不同，化合物既可溶于辅料中也可制成悬浮溶液，在制成注射用溶液时先将化合物溶于水中，过滤消毒后装入封口瓶或安瓿中。
当皮肤局部施用时，本发明化合物可以制成适当的软膏，洗剂，或霜剂的形式，其中活性成分悬浮或溶解于一种或多种的辅料中，其中软膏制剂可以使用的辅料包括但不局限于：矿物油，液体凡士林，白凡士林，丙二醇，聚氧化乙烯，聚氧化丙烯，乳化蜡和水；洗剂和霜剂可使用的辅料包括但不限于：矿物油，脱水山梨糖醇单硬脂酸酯，吐温60，十六烷酯蜡，十六碳烯芳醇，2-辛基十二烷醇，苄醇和水。
一般而言，已经证明有利的是无论在人体医药还是在兽医药中，本发明活性化合物的给药总量每24小时为约0.5-500mg，优选1-100mg/kg体重，如果合适的话，分多次单剂量给药，以达到所要求的效果。单剂量中含活性化合物的量优选为约1-80mg，更优选为1-50mg/kg体重，但也可以不按照上述的剂量，即取决于治疗对象的种类和体重、疾病的性质和严重程度、制剂的类型和药物的给药方式，以及给药周期或时间间隔。
本发明提供的药物组合物中还包含抗HBV药物。其中抗HBV药物为HBV聚合酶抑制剂、免疫调节剂或干扰素。
所述抗HBV药物有拉米夫定、替比夫定、替诺福韦酯，恩替卡韦、阿德福韦酯、Alfaferone、Alloferon、西莫白介素、克拉夫定、恩曲他滨、法普洛韦、干扰素、宝甘灵CP、因特芬、干扰素α-1b、干扰素α、干扰素α-2a、干扰素β-1a、干扰素α-2、白细胞介素-2、米伏替酯、硝唑尼特、聚乙二醇干扰素α-2a、病毒唑、罗扰素-A、西佐喃、Euforavac、安普利近、Phosphazid，Heplisav、干扰素α-2b、左旋咪唑或丙帕锗等。
本发明另一方面涉及一种本发明的化合物或药物组合物来制备用于预防、治疗或减轻患者乙型肝炎疾病的药品的用途，包括给予患者药学上可接受的有效剂量对患者进行给药。乙型肝炎疾病是指由乙肝病毒感染或乙型肝炎感染导致引起的肝脏疾病，包括急性肝炎、慢性肝炎，肝硬化和干细胞癌。急性乙型肝炎病毒感染可以是无症状或表现为急性肝炎症状。慢性病毒感染患者患有活动性疾病，可发展为肝硬化和肝癌。
抗HBV药物可以与包含本发明的化合物的组合物分开给药，作为多次给药方案的一部分。或者，那 些药物可以是单剂型的一部分，与本发明的化合物混合在一起形成单个组合物。如果给药作为多给药方案的一部分，两个活性剂可以同时连续地或在一段时间内互相传递，从而得到目标试剂活性。
可以结合辅料物质产生单剂型的化合物和组合物的用量(那些包含一个组合物像本发明所描述的)的改变取决于主治和特殊给药模式。正常地，本发明的组合物的量将不超过组合物包含作为唯一的活性剂的正常给药的量。另一方面，现公开的组合物的量的范围大约是现有组合物正常量的50％-100％，包含的试剂作为唯一活性治疗剂。在那些包含的组合物中，组合物将与本发明的化合物起协同作用。
本发明的化合物显示出较强的抗病毒作用。这类化合物对HBV具有出乎预料的抗病毒活性，因此适于用来治疗病毒引起的各种疾病，尤其是急性和慢性持久性HBV病毒感染引起的疾病。由HBV引起的慢性病毒性疾病可以导致各种不同严重程度的综合症状，众所周知，慢性乙肝病毒感染可导致肝硬化和/或肝细胞癌。
可用本发明化合物治疗的适应症的实例有：可导致感染性肝炎的急性和慢性病毒感染，例如乙型肝炎病毒感染。特别优选的是慢性乙型肝炎感染和急性乙型肝炎病毒感染。
本发明还涉及，本发明的化合物和药物组合物用于制备治疗和预防病毒性疾病特别是乙型肝炎的药物的用途。
一般合成方法
一般地，本发明的化合物可以通过本发明所描述的方法制备得到，除非有进一步的说明，其中取代基的定义如式(I)或(Ia)所示。下面的合成方案和实施例用于进一步举例说明本发明的内容。
所属领域的技术人员将认识到：本发明所描述的化学反应可以用来合适地制备许多本发明的其他化合物，且用于制备本发明的化合物的其它方法都被认为是在本发明的范围之内。例如，根据本发明那些非例证的化合物的合成可以成功地被所属领域的技术人员通过修饰方法完成，如适当的保护干扰基团，通过利用其他已知的试剂除了本发明所描述的，或将反应条件做一些常规的修改。另外，本发明所公开的反应或已知的反应条件也公认地适用于本发明其他化合物的制备。
下面所描述的实施例，除非其他方面表明所有的温度定为摄氏度(℃)。试剂购买于商品供应商如Aldrich Chemical Company,Arco Chemical Company and Alfa Chemical Company，使用时都没有经过进一步纯化，除非其他方面表明。一般的试剂从汕头西陇化工厂，广东光华化学试剂厂，广州化学试剂厂，天津好寓宇化学品有限公司，青岛腾龙化学试剂有限公司，和青岛海洋化工厂购买得到。
色谱柱使用硅胶柱，硅胶(200-300目)购于青岛海洋化工厂。核磁共振光谱以CDC1 3,DMSO-d 6,CD 3OD或丙酮-d 6为溶剂(报导以ppm为单位)，用TMS(0ppm)或氯仿(7.25ppm)作为参照标准。当出现多重峰的时候，将使用下面的缩写：s(singlet，单峰),d(doublet，双峰),t(triplet，三重峰),m(multiplet，多重峰),br(broadened，宽峰),dd(doublet of doublets，双二重峰),dt(doublet of triplets，双三重峰),br.s(broadened singlet，宽单峰)。偶合常数J，单位用赫兹(Hz)表示。
低分辨率质谱(MS)数据通过配备G1312A二元泵和a G1316A TCC(柱温保持在30℃)的Agilent 6320系列LC-MS的光谱仪来测定，G1329A自动采样器和G1315B DAD检测器应用于分析，ESI源应用于LC-MS光谱仪。
低分辨率质谱(MS)数据还通过配备G1311A四元泵和G1316A TCC(柱温保持在30℃)的Agilent 6120系列LC-MS的光谱仪来测定，G1329A自动采样器和G1315D DAD检测器应用于分析，ESI源应用于LC-MS光谱仪。
以上两种光谱仪都配备了Agilent Zorbax SB-C18柱，规格为2.1×30mm，5μm。注射体积是通过样品浓度来确定；流速为0.6mL/min；HPLC的峰值是通过在210nm和254nm处的UV-Vis波长来记录读取的。流动相为0.1％的甲酸乙腈溶液(相A)和0.1％的甲酸超纯水溶液(相B)。梯度洗脱条件如表1所示：
表1：梯度洗脱条件
时间(min) A(CH 3CN，0.1％HCOOH) B(H 2O，0.1％HCOOH)
0-3 5-100 95-0
3-6 100 0
6-6.1 100-5 0-95
6.1-8 5 95
化合物纯化是通过Agilent 1100系列高效液相色谱(HPLC)来评价的，其中UV检测在210nm和254nm处，Zorbax SB-C18柱，规格为2.1×30mm，4μm，10分钟，流速为0.6mL/min，5-95％的(0.1％甲酸乙腈溶液)的(0.1％甲酸水溶液)，柱温保持在40℃。
下面简写词的使用贯穿本发明：
MeOH 甲醇                                             Na 2SO 4 硫酸钠
MeOH-d 4 氘代甲醇                                      Et 3N,TEA 三乙胺
DCM,CH 2Cl 2 二氯甲烷                                   H 2O 水
CHCl 3 氯仿，三氯甲烷                                  mL,ml 毫升
CDC1 3 氘代氯仿                                        RT,rt 室温
Ph 3P 三苯基膦                                         Rt 保留时间
LiOH.H 2O 一水氢氧化锂                                 1atm 101.325kPa
tBu XPhos 2-二-叔丁膦基-2',4',6'-三异丙基联苯         h 小时
TFA 三氟乙酸                                          H 2 氢气
(dppf)PdCl 2 [1,1'-双(二苯基膦基)二茂铁]二氯化钯       HCl/EA,HCl/EtOAc 氯化氢的乙酸乙酯
(dppf)PdCl 2·CH 2Cl 2 [1,1'-双(二苯基膦基)二茂铁]二氯化 溶液
钯二氯甲烷络合物                                      HOAt 1-羟基-7-偶氮苯并三氮唑
X-PHOS，X-Phos 2-二环己基磷-2,4,6-三异丙基联          HATU O-(7-氮杂苯并三唑-1-
苯                                                    基)-N,N,N′,N′-四甲基脲六氟磷酸酯
Xantphos 4,5-双二苯基膦-9,9-二甲基氧杂蒽              DIPEA N,N-二异丙基乙胺
Pd/C 钯/碳                                            DMF N,N-二甲基甲酰胺
Boc 叔丁氧羰基                                        THF 四氢呋喃
(Boc) 2O 二碳酸二叔丁酯                                DMSO 二甲基亚砜
Pd 2(dba) 3 三(二亚苄基丙酮)二钯                        CH 3OH 甲醇
CH 3I 碘甲烷                                           N 2 氮气
SOCl 2 二氯亚砜                                        NH 4Cl 氯化铵
PE 石油醚                                             Ac 2O 乙酸酐
EtOAc,EA 乙酸乙酯                                     t 1/2 半衰期
EtOH 乙醇                                             AUC 药时曲线下面积
HCl 盐酸                                              Vss 稳态表观分布容积
K 2CO 3 碳酸钾                                          CL,clearance 清除率
NaHCO 3 碳酸氢钠                                       F,absolute bioavailability 生物利用度
NaOH 氢氧化钠                                         Dose 剂量
NaCl 氯化钠                                           T max 达峰时间
                                                      C max 最大浓度
                                                      hr *ng/mL 血药浓度*时间
合成方法
以下合成方案列出了制备本发明中公开化合物的实验步骤。其中，各R 1、R 2、R 3、R 1a、R 1b、R 4、R 4a、R 4b和R y具有如本发明所述的含义。
合成方案1
Figure PCTCN2020087700-appb-000010
化合物(a-8)可以通过合成方案1描述的方法制备得到。首先，化合物(a-1)与二氯亚砜和甲醇反应得到化合物(a-2)；然后，化合物(a-2)与化合物(a-3)在碱性条件下(如碳酸铯等)，催化剂(如醋酸钯等)、配体(如X-PHOS，t-BuX-PHOS等)和合适的溶剂中(如1,4-二氧六等)反应，生成化合物(a-4)；接下来，化合物(a-4)在碱性条件下(如氢氧化锂水溶液等)发生酯水解反应，生成化合物(a-5)，再脱去Boc，得到化合物(a-6)；最后，化合物(a-6)或其盐与化合物(a-7)(化合物(a-7)可参考WO2015074546中的合成方案1和其中的具体实施例方法制备得到)在碱性条件下(如碳酸钾等)和合适的溶剂(如乙醇等)中反应得到化合物(a-8)。具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
制备实施例
在以下制备实施例中，发明人以本发明的部分化合物为例，详细描述了本发明化合物的制备过程。
片段F1的合成：
Figure PCTCN2020087700-appb-000011
F1-1的合成：
将F1-0(3g,13.7mmol)溶于甲醇(30mL)中，冰浴下缓慢滴加二氯亚砜(1.2mL,16mmol)，滴加完毕后，反应混合物常温反应12h。反应完后，减压蒸除溶剂，向残余物中加入石油醚(90mL)萃取，有机层依次用饱和碳酸氢钠水溶液(30mL×3)和饱和氯化钠溶液(30mL)洗涤，无水硫酸钠干燥，减压浓缩，得F1为无色油状物(3.05g,95.6％)。
F1-2的合成：
于干燥反应瓶中依次加入F1-1(3.05g,13.1mmol)、(R)-3-氧代六氢咪唑并[1,5-a]吡嗪-7(1H)-甲酸叔丁酯(2.9g,12mmol)、Pd 2(dba) 3(0.55g,0.60mmol)、Xantphos(0.70g,1.2mmol)、碳酸铯(5.9g,18mmol)和1,4-二氧六环(30mL)，反应混合物于氮气保护及100℃下搅拌反应24h，然后用硅藻土抽滤，并用二氯甲烷(200mL)洗涤滤饼，旋干滤液，所得残留物经硅胶柱层析分离(PE/EA(V/V)＝2/1)纯化，得F1-2为白色固体(2.5g,53％)。MS(ESI,pos.ion)m/z：416.2[M+Na] +。
F1-3的合成：
于干燥反应瓶中依次加入F1-2(2.5g,6.35mmol)、甲醇(10mL)、四氢呋喃(20mL)和一水合氢氧化锂(0.8g,19mmol)，反应混合物于50℃反应12h然后减压浓缩，所得残留物加入水(100mL)稀释，再用1M盐酸调pH至4～5，过滤得F1-3为白色固体(2.31g,95.8％)。MS(ESI,pos.ion)m/z：402.2[M+Na] +。F1的合成：
将F1-3(300mg,0.79mmol)溶于二氯甲烷(2mL)中，然后加入三氟乙酸(2mL)，反应混合物于室温搅拌反应0.5h，然后减压浓缩，得F1为褐色油状物(311mg,100％)。MS(ESI,pos.ion)m/z：280.0[M+H] +。
片段F2的合成：
Figure PCTCN2020087700-appb-000012
将F2-0替换F1-0，其余实验操作参考片段F1的合成方法，得F2为褐色油状物。 实施例1的合成：
Figure PCTCN2020087700-appb-000013
于干燥反应瓶中依次加入F1(311mg,0.79mmol)、(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸甲酯(350mg,0.79mmol)、乙醇(10mL)和碳酸钾(0.276g,2mmol)，反应混合物于室温搅拌反应12h。反应完后，加入水(50mL)稀释反应混合物，再用1M盐酸调pH＝5～6，然后用二氯甲烷萃取(50mL×3)，合并的有机相减压浓缩。所得残留物经硅胶柱层析(DCM/CH 3OH(V/V)＝50/1)分离纯化，得标题化合物为黄色固体(155mg,30.5％)。MS(ESI,pos.ion)m/z：643.3[M+H] +； 1H NMR(400MHz,DMSO-d 6)δ(ppm):9.70(s,1H),8.03(d,J＝3.0Hz,1H),7.94(d,J＝3.0Hz,1H),7.80–7.73(m,2H),7.70(d,J＝12.0Hz,1H),7.46–7.37(m,2H),7.18(td,J＝8.5,2.3Hz,1H),6.05(s,1H),4.08–4.00(m,1H),4.00–3.91(m,2H),3.90–3.78(m,2H),3.58(dd,J＝8.6,4.1Hz,1H),3.52(s,3H),3.13–3.05(m,1H),2.99–2.92 (m,2H),2.38–2.27(m,1H),2.22(t,J＝10.8Hz,1H)。
实施例2的合成：
Figure PCTCN2020087700-appb-000014
于干燥反应瓶中依次加入F2(311mg,0.79mmol)、(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸甲酯(350mg,0.79mmol)、乙醇(10mL)和碳酸钾(0.276g,2mmol)，室温搅拌反应12h。反应完后，反应混合物加入水(50mL)稀释，再用1M盐酸调pH＝5～6，然后用二氯甲烷萃取(50mL×3)，合并的有机相减压浓缩。所得残留物经硅胶柱层析(DCM/CH 3OH(V/V)＝50/1)分离纯化，得标题化合物为黄色固体(260mg,51.13％)。MS(ESI,pos.ion)m/z：643.2[M+H] +； 1H NMR(400MHz,CDCl 3)δ(ppm):9.60(s,1H),7.98(t,J＝8.6Hz,1H),7.87(d,J＝3.1Hz,1H),7.56(d,J＝13.7Hz,1H),7.49(d,J＝3.1Hz,1H),7.36–7.29(m,2H),7.15(dd,J＝8.5,2.5Hz,1H),6.94(td,J＝8.3,2.5Hz,1H),6.22(s,1H),4.16(d,J＝17.0Hz,1H),4.12–4.01(m,2H),3.97–3.89(m,2H),3.62(s,3H),3.45(dd,J＝9.2,4.9Hz,1H),3.30(td,J＝13.0,3.1Hz,1H),2.96(t,J＝10.5Hz,2H),2.53(td,J＝11.3,2.9Hz,1H),2.28(t,J＝10.6Hz,1H)。
实施例3的合成：
Figure PCTCN2020087700-appb-000015
将(R)-4-(2-溴-4-氟苯基)-6-(溴甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(0.3g,0.6mmol)替换实施例1的(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸甲酯，其余按实施例1方法操作，得黄色固体(0.24g,56.0％)。MS(ESI,pos.ion)m/z：701.1[M+H] +； 1H NMR(400MHz,CDCl 3)δ(ppm):9.60(s,1H),7.94–7.75(m,4H),7.48(d,J＝3.1Hz,1H),7.36–7.29(m,2H),6.99(td,J＝8.3,2.5Hz,1H), 6.22(s,1H),4.22–3.92(m,7H),3.65(d,J＝3.2Hz,1H),3.35–3.25(m,1H),2.95–2.89(m,2H),2.58–2.53(m,1H),2.35(d,J＝9.2Hz,1H),1.15(t,J＝7.1Hz,3H).
实施例4的合成：
Figure PCTCN2020087700-appb-000016
将(R)-4-(2-溴-4-氟苯基)-6-(溴甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(0.3g,0.6mmol)替换实施例2的(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸甲酯，其余按实施例2方法操作，得黄色固体(84mg,20.0％)。MS(ESI,pos.ion)m/z：701.1[M+H] +； 1H NMR(400MHz,CDCl 3)δ(ppm):9.55(s,1H),7.99(t,J＝8.5Hz,1H),7.86(d,J＝3.0Hz,1H),7.56(d,J＝13.8Hz,1H),7.48(d,J＝3.0Hz,1H),7.39–7.29(m,3H),6.99(td,J＝8.4,2.3Hz,1H),6.22(s,1H),4.21–3.92(m,7H),3.45(dd,J＝9.0,4.9Hz,1H),3.35–3.25(m,1H),3.02–2.92(m,2H),2.57–2.48(m,1H),2.27(t,J＝10.6Hz,1H),1.15(t,J＝7.1Hz,3H).
实施例5的合成：
Figure PCTCN2020087700-appb-000017
将(R)-4-(2-溴-4-氟苯基)-6-(溴甲基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸甲酯(0.3g,0.6mmol)替换实施例1的(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸甲酯，其余按实施例1方法操作，得黄色固体(0.3g,73％)。MS(ESI,pos.ion)m/z：687.0[M+H] +； 1H NMR(400MHz,CDCl 3)δ(ppm):9.63(s,1H),7.94–7.72(m,4H),7.48(d,J＝3.1Hz,1H),7.36–7.29(m,2H),6.98(td,J＝8.4,2.5Hz,1H),6.21(s,1H),4.22–3.92(m,5H),3.68–3.58(m,4H),3.30(t,J＝11.1Hz,1H),2.98–2.92(m,2H),2.58–2.51(m,1H),2.37(t,J＝10.0Hz,1H).
实施例6的合成：
Figure PCTCN2020087700-appb-000018
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(0.23g,0.5mmol)替换实施例2的(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸甲酯，其余按实施例2方法操作，得黄色固体(0.13g,39.0％).MS(ESI,pos.ion)m/z：657.1[M+H] +； 1H NMR(400MHz,CDCl 3)δ(ppm):9.57(s,1H),7.98(t,J＝8.5Hz,1H),7.87(d,J＝3.1Hz,1H),7.56(d,J＝13.8Hz,1H),7.48(d,J＝3.0Hz,1H),7.37–7.29(m,2H),7.15(dd,J＝8.5,2.4Hz,1H),6.94(td,J＝8.4,2.4Hz,1H),6.24(s,1H),4.17–3.91(m,7H),3.45(dd,J＝9.2,4.9Hz,1H),3.35–3.25(m,1H),2.98–2.92(m,2H),2.57–2.47(m,1H),2.27(t,J＝10.6Hz,1H),1.14(t,J＝7.1Hz,3H).
实施例7的合成：
Figure PCTCN2020087700-appb-000019
将(R)-6-(溴甲基)-4-(2,4-二氯苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸乙酯(0.38g,0.79mmol)替换实施例1的(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸甲酯，其余按实施例1方法操作，得黄色固体(0.23g,43％).MS(ESI,pos.ion)m/z：673.1[M+H] +； 1H NMR(400MHz,CDCl 3)δ(ppm):7.90–7.75(m,4H),7.47(d,J＝3.1Hz,1H),7.40(d,J＝2.0Hz,1H),7.26(d,J＝1.2Hz,1H),7.19(dd,J＝8.4,2.0Hz,1H),6.22(s,1H),4.21(d,J＝16.5Hz,1H),4.11–3.92(m,6H),3.64(dd,J＝7.7,4.4Hz,1H),3.38–3.28(m,1H),3.02-2.94(m,2H),2.58–2.52(m,1H),2.45–2.36(m,1H),1.13(t,J＝7.1Hz,3H).
实施例8的合成：
Figure PCTCN2020087700-appb-000020
将(R)-6-(溴甲基)-4-(2,4-二氯苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-羧酸乙酯(0.38g,0.79mmol)替换实施例2的(R)-6-(溴甲基)-4-(2-氯-4-氟苯基)-2-(噻唑-2-基)-1,4-二氢嘧啶-5-甲酸甲酯，其余按实施例2方法操作，得黄色固体(0.26g,48％)。MS(ESI,pos.ion)m/z：673.1[M+H] +； 1H NMR(400MHz,CD 3OD-d 4)δ(ppm):7.94(d,J＝3.1Hz,1H),7.89(t,J＝8.7Hz,1H),7.73(d,J＝3.1Hz,1H),7.61(dd,J＝14.1,1.8Hz,1H),7.46(d,J＝2.0Hz,1H),7.40(d,J＝8.4Hz,1H),7.33–7.26(m,2H),6.17(s,1H),4.15(d,J＝16.9Hz,1H),4.07–3.92(m,6H),3.54(dd,J＝9.4,4.5Hz,1H),3.30–3.21(m,1H),3.02(t,J＝8.7Hz,2H),2.52–2.42(m,1H),2.24(t,J＝10.8Hz,1H),1.11(t,J＝7.1Hz,3H).生物学测试
测试1：HepAD38细胞评价化合物抑制HBV DNA复制活性(qPCR法)
HBV细胞株及培养条件
HepAD38：Ladner等(Ladner,Otto et al.1997)将对四环素敏感的巨细胞病毒CMV启动子连接到PBR322质粒上并于ayw亚型HBV DNA连接成ptetHBV质粒，转染HepG2细胞获得HepAD38细胞株，由于前C区基因收到破坏，HBV DNA产量比HepG2.2.15细胞高约11倍。可利用四环素对HBV复制进行调控，培养所需的时间只有HepG2.2.15细胞的一半，适用于研究HBV复制过程和复制中间型以及抗HBV药物筛选。HepAD38培养于含10％FBS及1％双抗的DMEM/F-12K培养基(另包含终浓度为300ng/ml的Tetracycline以及终浓度为400μg/ml的G418)。
HepAD38细胞分泌的病毒粒子DNA可以通过qPCR的方法来定量，并由此检测化合物对病毒复制的影响。
体外抗HBV活性测定
将传代次数少的HepAD38复苏后，待细胞状态良好后，于培养基中加入Tetracycline(终浓度为300ng/ml)以及G418(终浓度为400μg/ml)，Tetracycline存在下病毒不表达，待细胞长满后消化、计数，用含10％FBS的DMEM/F-12K培养基(包含终浓度为300ng/ml的Tetracycline以及终浓度为400μg/ml的G418，1％双抗)稀释成浓度为2×10 5/mL细胞悬液，以每孔100μL的量接种于96孔板(整块板铺满)，置于37℃、5％CO 2恒温培养箱中孵育24h。
抗病毒实验中化合物配制和细胞处理：用DMSO溶解化合物至20mM，进一步用DMSO稀释化合物到800μM，然后进行8个稀释度的4倍稀释，最高浓度为800μM。加系列稀释的化合物1μL每孔到上述细胞板中，实验最高终浓度为4μM(200倍稀释)。TDF(富马酸替诺福韦二吡呋酯,Selleck,Cat S1400)作为阳性对照化合物，最高浓度为4μM。阴性对照孔加入1μL DMSO，终浓度为0.5％。
HBV DNA Q-PCR
使用圣湘生物48人份(PCR-荧光探针法)一步法的乙型肝炎病毒核酸定量测定试剂盒进行QPCR， 吸取2.5μL上清液进行Q-PCR,使用前待试剂盒试剂融化后涡旋混匀，离心后将酶混合液放置冰上待用，并保证后续步骤在冰上完成。于Q-PCR板中每孔加入2.5μL样本释放剂，2.5μL测试样本上清液(实验组，对照组，标准曲线组)。QPCR反应后得到各孔病毒DNA拷贝数。用Graphpad Prism 5软件处理浓度-病毒拷贝数，通过四参数非线性回归模型计算化合物对病毒复制的EC50。实验结果见表2。
表2：本发明化合物对HBV复制的EC 50值
专利实施例 EC 50(nM)
实施例1 20
实施例2 10
实施例3 31
实施例4 16
实施例6 15
实施例7 16
实施例8 13
结论：实验数据表明，本发明化合物对HBV具有较好的抑制活性，在抗HBV病毒方面有很好的应用前景。
测试2：HepG2.2.15细胞评价化合物抑制HBV DNA复制活性
HBV细胞系和培养条件
HepG2.2.15细胞(SELLS,PNAS,1987和SELLS,JV,1988)的染色体整合有完整的HBV基因组，并稳定表达病毒RNA和病毒蛋白质。HepG2.2.15细胞能够向培养基中分泌成熟的乙肝病毒颗粒，HBsAg和HBeAg。HepG2.2.15培养于含有10％胎牛血清、100U/mL青霉素、100U/mL链霉素、1％非必需氨基酸、1mM丙酮酸钠300μg/mL G418的DMEM培养基。
HepG2.2.15细胞分泌的病毒粒子DNA可以通过qPCR的方法来定量，并由此检测化合物对病毒复制的影响。
体外抗HBV活性测定
HepG 2.2.15细胞8,000个每孔接种到96孔细胞培养板，37℃，5％CO 2培养3天至细胞长至满孔。在测试第0天，弃掉旧的培养基并加入200μL新鲜的检测培养基(5％FBS)。
抗病毒实验中化合物配制和细胞处理：用DMSO溶解化合物至30mM，进一步用DMSO稀释化合物到800μM，然后进行8个稀释度的4倍稀释，最高浓度为800μM。加系列稀释的化合物1μL每孔到上述细胞板中，实验最高终浓度为4μM(200倍稀释)。TDF(富马酸替诺福韦二吡呋酯,Selleck,Cat S1400)作为阳性对照化合物，最高浓度为4μM。阴性对照孔加入1μL DMSO，终浓度为0.5％,阳性对照孔加入TDF，终浓度为1μM。
qPCR方法检测病毒基因组DNA
引物：HBV-For-202，CAGGCGGGGTTTTTCTTGTTGA；HBV-Rev-315，GTGATTGGAGGTTGGGGACTGC。使用SYBR Premix Ex Taq II–Takara DRR081S试剂盒，以1μL细胞培养上清作为模板，用包含HBV基因组的质粒做标准曲线，并以标准曲线来计算病毒拷贝数。用Graphpad Prism 5软件处理浓度-病毒拷贝数，通过四参数非线性回归模型计算化合物对病毒复制的EC 50。实验结果见表3。
表3：本发明化合物对HBV复制的EC 50值
专利实施例 EC 50(nM)
实施例2 4
结论：实验数据表明，本发明化合物对HBV具有较好的抑制活性，在抗HBV病毒方面有很好的应用 前景。
测试3：细胞毒和选择性指数
测试化合物细胞毒和选择指数实验方法：
加系列稀释的化合物到384孔细胞毒细胞板中，每孔加入50μL HepG2.2.15细胞(3000个细胞/孔)，实验最高终浓度为150μM(200倍稀释)。37℃，CO 2培养箱中孵育4天后用CellTiter Glo试剂检测化合物的细胞毒作用。
化合物细胞毒性用以下公式计算：细胞毒性(％)＝100-(检测值/DMSO对照孔平均值×100)。用Graphpad Prism 5软件处理浓度-细胞毒性(％)数据，通过四参数非线性回归模型计算CC 50。CC 50大于50说明毒性比较低。
结论：细胞毒实验数据表明，本发明化合物对细胞的毒性小。
测试4：本发明化合物在比格犬、小鼠、大鼠、食蟹猴中的药代动力学实验
(1)比格犬PK测试实验
化合物在比格犬(购自湖南斯莱克景达实验动物有限公司，体重10-12kg，雄性，年龄10-12个月，口服每组3只，静脉注射每组3只)体内的PK测定实验方法：
比格犬经口灌胃给予2.5mg/kg或5mg/kg或经静脉注射1mg/kg或2mg/kg的测试化合物。
给药后按时间点(0.083、0.25、0.5、1、2、4、6、8和24小时)静脉采血，收集于加EDTA-K 2的抗凝管内。血浆样品经液液萃取后，在三重四极杆串联质谱仪上，以多重反应离子监测(MRM)方式进行定量分析。采用WinNonlin 6.3软件用非房室模型法计算药动学参数。
结论：药代实验数据表明，本发明化合物在比格犬体内具有较好的药代动力学性质，在抗HBV病毒方面有很好的应用前景。
(2)小鼠PK测试实验：
化合物在小鼠(购自湖南斯莱克景达实验动物有限公司，体重20-25g，雄性，年龄45-60天，口服每组3只，静脉注射每组3只)体内的PK测定实验方法：
ICR小鼠经口灌胃给予10mg/kg或经尾静脉注射2mg/kg或10mg/kg的测试化合物。
给药后按时间点(0.083，0.25，0.5，1，2，4，6，8和24小时)眼眶静脉采血，收集于加EDTA-K 2的抗凝管内。血浆样品经液液萃取后，在三重四极杆串联质谱仪上，以多重反应离子监测(MRM)方式进行定量分析。采用WinNonlin 6.3软件用非房室模型法计算药动学参数。
结论：药代实验数据表明，本发明化合物在小鼠体内具有较好的药代动力学性质，在抗HBV病毒方面有很好的应用前景。
(3)SD大鼠PK测试实验：
化合物在SD大鼠(购自湖南斯莱克景达实验动物有限公司，体重200-250g，雄性，年龄2-3个月，口服每组3只，静脉注射每组3只)体内的PK测定实验方法：
大鼠经口灌胃给予2.5mg/kg或5mg/kg或经静脉注射1mg/kg的测试化合物。
给药后按时间点(0.083、0.25、0.5、1、2、5、7和24小时)静脉采血，收集于加EDTA-K 2的抗凝管内。血浆样品经液液萃取后，在三重四极杆串联质谱仪上，以多重反应离子监测(MRM)方式进行定量分析。采用WinNonlin 6.3软件用非房室模型法计算药动学参数。
结论：药代实验数据表明，本发明化合物在SD大鼠体内具有较好的药代动力学性质，在抗HBV病毒方面有很好的应用前景。
(4)食蟹猴PK测试实验：
化合物在食蟹猴(购自广东春盛生物科技发展有限公司，体重3-6kg，雄性，年龄4-6岁，口服每组3只，静脉注射每组3只)体内的PK测定实验方法：
食蟹猴经口灌胃给予2.5mg/kg或5mg/kg或经静脉注射0.5mg/kg或1mg/kg的测试化合物。
给药后按时间点(0.083、0.25、0.5、1、2、4、6、8和24小时)静脉采血，收集于加EDTA-K2的抗凝管内。血浆样品经液液萃取后，在三重四极杆串联质谱仪上，以多重反应离子监测(MRM)方式进行定量分析。采用WinNonlin 6.3软件用非房室模型法计算药动学参数。实验结果见表4。
表4：化合物在食蟹猴体内的PK数据
Figure PCTCN2020087700-appb-000021
备注：对照化合物的结构及合成方法参见专利申请WO2015132276说明书第90页实施例25(即，化合物4-((S)-7-(((R)-6-(2-氯-4-氟苯基)-5-(甲氧基羰基)-2-(噻唑-2-基)-3,6-二氢嘧啶-4-基)甲基)-3-氧代六氢咪唑并[1,5-a]吡嗪-2(3H)-基)苯甲酸)。
结论：药代实验数据表明，本发明化合物药时曲线下面积AUClast较大，暴露量较好，说明本发明化合物在食蟹猴体内的吸收好，体内稳定，并且生物利用度高，其药代性质明显犹于对照化合物。因此，本发明化合物在食蟹猴体内具有较好的药代动力学性质，在抗HBV病毒方面有很好的应用前景。
测试5：本发明化合物在不同种属的肝微粒体中的稳定性测试
化合物在不同种属中的肝微粒体稳定性方法：
向96孔板中加入空白溶液与肝微粒体的混合溶液30μL，在各孔中加入15μL含待测化合物的缓冲液，平行做两份样品。37℃预孵育10min后按时间点加入15μL NADPH溶液(8mM)，待测化合物的终浓度为1μM，肝微粒体的浓度为0.5mg/mL，NADPH终浓度为2mM。分别孵育0、15、30、60min，孵育结束后将150μL乙腈(含内标)加入混合体系中。乙腈稀释后的样品在4000rpm下离心5min，取150μL上清液至LC-MS/MS进行分析。
结论：本发明化合物在不同种属的肝微粒体中的稳定性较好。
测试6：溶解度测试方法
化合物溶解度的实验测试方法
除另有规定外，称取研成细粉的供试品或量取液体供试品至25℃±2℃、一定容量的溶剂中，每隔5min强力振摇30s，观察30min内的溶解情况，如无目视可见的溶质颗粒或液滴时，即视为完全溶解。根据中国药典2015版的标准：
极易溶解系指溶质1g(mL)能在溶剂不到1mL中溶解；
易溶系指溶质1g(mL)能在溶剂1～不到10mL中溶解；
溶解系指溶质1g(mL)能在溶剂10～不到30mL中溶解；
略溶系指溶质1g(mL)能在溶剂30～不到100mL中溶解；
微溶系指溶质lg(mL)能在溶剂100～不到1000mL中溶解；
极微溶解系指溶质1g(mL)能在溶剂1000～不到10000mL中溶解；
几乎不溶或不溶系指溶质1g(mL)在溶剂10000mL中不能完全溶解。
结论：溶解度实验数据表明，本发明化合物溶解性较好。
测试7：hERG测试方法
化合物对心脏的实验测试方法
在384孔板中依次加入化合物/阳性对照品/阴性对照品、含有hERG通道的膜片段、与hERG通道具 有高度亲和力的示踪物，25℃，250rpm孵育4小时。用多功能酶标仪测量各孔的荧光偏振值，计算化合物对hERG通道的相对抑制率和50％抑制浓度(IC 50)。
结论：hERG测试实验数据表明，本发明化合物对心脏的毒性小。
测试8：肝药酶诱导测试
细胞培养
所有孵育均在37℃、5％CO2和95％湿度条件的培养箱中进行。
冻存人肝细胞(Baltimore,MD,USA)复苏后，采用台盼蓝染色法和细胞计数器确定细胞数量和细胞活率。计数完毕后用预热的种板培养液将肝细胞稀释到每毫升中含70万个活细胞。将稀释好的肝细胞悬液按0.2mL/孔接种到预铺胶原的48孔板上，于培养箱中孵育培养至少4小时，待细胞呈贴壁状态，用含有2％基底基质胶的孵育培养液替换种板培养液。
每天用孵育培养液新鲜配制给药工作液，包括供试品(浓度不低于0.1μM)，CYP1A2，CYP2B6，CYP3A4的阳性诱导剂omeprazole，phenobarbital，rifampicin，稀释1000倍DMSO储备液得到。给药工作液的信息见下表。
Figure PCTCN2020087700-appb-000022
待培养体系建立好后，弃去三明治培养基的上层培养液，于每个细胞培养孔中加入200μL已预热至37℃并新鲜配制的给药工作液(包含供试品、阳性对照，阴性对照和基质对照)，将细胞培养板放置于培养箱中继续培养24小时。培养24小时后，更换新鲜配制的给药工作液并继续培养24小时。整个孵育时间为48小时。每个药物浓度及对照浓度分别做三个平行。
待细胞与给药工作液孵育48小时后，弃去板中剩余的药物溶液，用0.5mL预热至37℃的HBSS溶液清洗细胞孔2次，于每孔加入100μL已预热至37℃的酶活标记底物工作液孵育30分钟。孵育30分钟后，每孔取出75μL上清样品加入到含有150μL终止液的96孔深孔板中。摇板10分钟，于4℃、3220g离心20分钟，然后取上清溶液按1:4的比例用含有0.1％甲酸的水溶液稀释。稀释后的样品摇板10分钟后，用液相色谱串联质谱(LC/MS/MS)方法对代谢产物的生成量进行检测。
酶活性检测反应结束之后，弃去上清剩余溶液，并用0.5mL预热的HBSS清洗细胞。于每孔加入280μL含有1％β-巯基乙醇的裂解液RLT，封板，摇板10分钟后，转移至-80℃冰箱内保存。
细胞毒性实验
供试品的潜在毒性由肝细胞中乳酸脱氢酶(LDH)的释放量来评估。将与肝细胞孵育24小时和48小时后的给药工作液各自取出100μL，用商品化LDH试剂盒对其乳酸脱氢酶的浓度进行检测。细胞裂解溶液作为实验阳性对照，孵育培养液作为空白对照。
RNA分析检测
室温化冻样品板，将所有样品转移至新的48孔细胞培养板中。应用全自动核酸抽提工作站抽提RNA。在样品板的不同位置随机抽取超过样品总量10％的样品，运用ND2000微量分光光度计测定260nM与280nM的OD值，通过计算两者的比值来判断总RNA的纯度。反转录以得到cDNA。用CFX connectTM实时荧光定量PCR仪实时定量分析选择的基因。按如下程序设置反应条件：50℃两分钟；95℃十分钟；以下两步做40个循环：95℃十五秒，60℃一分钟。内源性对照18S rRNA作为内标。
样品分析检测
利用液相色谱串联质谱(LC/MS/MS)方法测定经蛋白沉淀后肝细胞中三个CYP酶底物的代谢产物(对 乙酰氨基酚(Acetaminophen)、羟基安非他酮(Hydroxybupropion)以及1-羟基咪达唑仑(1’-Hydroxymidazolam))的浓度。分析方法见表4。
表4：诱导试验LCMS分析方法
Figure PCTCN2020087700-appb-000023
基因表达数据计算
该项目采用ΔCt相对定量的方法来比较不同处理组间基因表达的差异，以18S rRNA为内参基因来校正每个样品的基因表达量。目的基因的Ct值减去内参基因的Ct值则为ΔCt，即Ct 目的基因–Ct 18S＝ΔCt。用处理组的ΔCt值减去空白对照组的ΔCt值则为ΔΔCt，即ΔCt处理组–ΔCt空白对照组＝ΔΔCt。最后以2 -ΔΔCt的方法进行统计分析，比较处理组与空白对照组间的倍数的变化。
酶活性数据计算
实验数据显示了CYP1A2，CYP2B6和CYP3A4的酶代谢产物的生成量。酶活性的改变是通过比较在化合物存在或不存在的条件下相应细胞色素酶的诱导倍数来表现的。诱导倍数的计算方法及与对照化合物的诱导比率的计算方法如下：
诱导倍数＝供试品处理过的样品中的酶活性/基质对照处理过的样品中的酶活性
与对照化合物的诱导比率＝(供试品处理过的样品的诱导倍数-1)/(对照化合物处理过的样品的诱导倍数-1)×100％。
结论：肝药酶诱导测试实验数据表明，本发明化合物对肝药酶基本无诱导作用。
测试9：人血清对化合物抗HBV药效影响实验
实验原理
HepG2.2.15细胞的染色体整合有完整的HBV基因组，并稳定表达病毒RNA和病毒蛋白质。HepG2.2.15细胞能够向培养基中分泌成熟的乙肝病毒颗粒、HBsAg和HBeAg。HepG2.2.15细胞分泌的病毒DNA可以通过qPCR的方法来定量，在测试化合物处理的同时加入不同浓度的人血清，并由此检测人血清对化合物抗病毒药效的影响。
实验方法
HepG2.2.15细胞的化合物处理
步骤1：96孔细胞培养板中铺HepG2.2.15细胞15000个每孔，每孔200μL细胞培养基。
步骤2：在37℃，5％CO 2细胞培养箱中培养3天至细胞长至满孔。
步骤:3：在测试第0天，弃掉旧的培养基并加入200μL新鲜的含有2％FBS和不同人血清(HS)浓度的检测培养基，包括0％HS、5％HS、10％HS、20％HS、40％HS和50％HS。
步骤4：抗病毒实验中化合物配制和细胞处理：用DMSO溶解化合物至30mM，进一步用DMSO稀释化合物到800μM，然后进行8个稀释度的4倍稀释，最高浓度为800μM。加系列稀释的化合物1μL每孔到步骤3准备的细胞板中，实验最高终浓度为4μM(200倍稀释)。
步骤5：实验设定在2％FBS条件下TDF(富马酸替诺福韦二吡呋酯,Selleck,Cat S1400)作为阳性对照化合物，最高浓度为4μM。阴性对照孔加入1μLDMSO，其实验终浓度为0.5％。
步骤6：96孔细胞测试板在37℃ CO 2培养箱中孵育11天，每隔一天(第2，4，6，8，10天)换一次液，并加入1μL新鲜配置的化测试合物，方法见步骤3到5。
步骤7：在第11天每孔取150μL上清作病毒DNA的qPCR检测。
步骤8：细胞毒实验中化合物配制和细胞处理：用Bravo liquid handling system配制系列稀释的化合物，11个稀释度，3倍稀释，最高浓度为30mM。用Echo550加系列稀释的化合物0.25μL每孔到384孔细胞毒细胞板中(Greiner 781098)。准备HepG2.2.15细胞，并重悬于不同浓度人血清浓度(50％，40％，20％，10％，5％和0％)的培养基中。加入50μL(4000个细胞)每孔以上制备的HepG2.2.15细胞到384孔细胞毒细胞板中，实验最高终浓度为150μM(200倍稀释)。37℃ CO 2培养箱中孵育4天后进行细胞毒测试。
qPCR方法检测病毒基因组DNA
步骤1：将20％HS实验条件下的上清液用DPBS作2倍稀释，40％HS实验条件下的上清液用DPBS作4倍稀释，50％HS实验条件下的上清液用DPBS作5倍稀释。混匀后取1μL进行qPCR检测。
步骤2：0％HS，5％HS和10％HS实验条件下的上清液直接取1μL进行qPCR检测。
步骤3：按以下成分配制qPCR反应体系：
SYBR Premix Ex TaqTM II(2×) 10μL
HBV-For-202(10μM) 0.8μL
HBV-Rev-315(10μM) 0.8μL
ROX Reference Dye(50×) 0.4μL
病毒上清 1μL
加水至 20μL
步骤4：按以下条件设置ABI ViiA7 qPCR仪
阶段1：
Reps：95℃，30s，1个循环
阶段2：
Reps：95℃，5s和60℃，34s，40个循环
添加溶解曲线
化合物的细胞毒作用检测
步骤1：平衡PromegaCelltiter-Glo试剂至室温。
步骤2：弃去细胞毒实验板培养基，每孔加入50μL DPBS。
步骤3：每孔加入10μL CellTiter-Glo试剂。
步骤4：振板仪上振2分钟。
步骤5：室温避光平衡10分钟。
步骤6：在Envision读板仪上读数(0.1秒/孔)
结果分析
用包含HBV基因组的质粒(病毒拷贝数:2×10E6,2×10E5,2×10E4,2×10E3)做标准曲线，并以标准曲线来计算病毒拷贝数。用Graphpad Prism 5软件处理数据并绘制浓度-病毒拷贝数曲线，通过四参数非线性回归模型计算EC 50。细胞毒性％＝100-(检测值/DMSO对照孔平均值×100)。细胞毒性％数据用Graphpad Prism 5软件处理并绘制曲线，通过四参数非线性回归模型计算CC 50。
结论：实验数据表明，人血清对本发明化合物抗病毒药效的影响较小，在人血清存在的情况下，本发明化合物仍具有良好的HBV抑制效果，说明本发明化合物在人体可起到良好的抗病毒效果。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (11)

  1. 一种化合物，其为如式(I)或(Ia)所示的化合物或如式(I)或(Ia)所示的化合物的立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐或它的前药，
    Figure PCTCN2020087700-appb-100001
    其中，各R 1、R 1b和R 1a独立地为氢、氘、F、Cl、Br、I、氰基、甲基、乙基、甲氧基、乙氧基、甲氨基、乙氨基、硝基、4-三氟甲基苯基、3,5-二(三氟甲基)苯基或三氟甲基；
    R 2为C 1-6烷基或C 1-6卤代烷基；
    R 3为苯基、咪唑基、呋喃基、噻吩基或噻唑基，其中所述苯基、咪唑基、呋喃基、噻吩基和噻唑基各自独立地未被取代或被1、2、3、4或5个选自氘、F、Cl、Br、OH、CN、C 1-6烷基、羟基C 1-6烷基、C 1-6烷基-OC(＝O)-、C 1-6烷基-OC(＝O)-C 1-6亚烷基、HOOC-C 1-6亚烷基、C 1-6烷氧基-C 1-6亚烷基和C 1-6烷基-S(＝O) 2-的取代基所取代；
    W为CH或N；
    X 1为-C(＝O)-、-S(＝O) 2-或-(CR 5R 6) j-；
    各R 4a、R 4b、R 5和R 6独立地为氢、氘、F、Cl、Br、氨基、C 1-6烷基、NH 2C(＝O)-、C 1-6烷基-OC(＝O)-、羟基C 1-6烷基、C 1-4烷氧基C 1-4亚烷基或C 1-6卤代烷基；
    各R 7独立地为氢、氘、F、Cl、Br、氨基、C 1-6烷基、NH 2C(＝O)-、C 1-6烷基-OC(＝O)-、羧基、羧基C 1-6亚烷基、羟基C 1-6烷基、C 1-4烷氧基C 1-4亚烷基或C 1-6卤代烷基；
    R y为氢，R 4为甲基、乙基、正丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、F或Cl；或
    R y为F或Cl，R 4为氢、F或Cl；
    m为0、1、2、3或4；
    j为1、2或3。
  2. 根据权利要求1所述的化合物，其中，R 2为甲基、乙基、正丙基、异丙基、一氟甲基、二氟甲基或三氟甲基；
    R 3为苯基、咪唑基、呋喃基、噻吩基或噻唑基，其中所述苯基、咪唑基、呋喃基、噻吩基和噻唑基各自独立地未被取代或被1、2、3、4或5个选自氘、F、Cl、Br、OH、CN、甲基、乙基、正丙基、异丙基、叔丁基、羟基C 1-4烷基、C 1-4烷基-OC(＝O)-、C 1-4烷基-OC(＝O)-C 1-3亚烷基-、HOOC-C 1-3亚烷基-、C 1-4烷氧基-C 1-3亚烷基-和C 1-4烷基-S(＝O) 2-的取代基所取代。
  3. 根据权利要求1或2所述的化合物，其中，各R 4a、R 4b、R 5和R 6独立地为氢、氘、F、Cl、Br、氨基、甲基、乙基、正丙基、异丙基、正丁基、叔丁基、NH 2C(＝O)-、C 1-4烷基-OC(＝O)-、羟基C 1-4烷基、C 1-4烷氧基C 1-2亚烷基或C 1-4卤代烷基；
    各R 7独立地为氘、F、Cl、Br、氨基、甲基、乙基、正丙基、异丙基、正丁基、叔丁基、NH 2C(＝O)-、C 1-4烷基-OC(＝O)-、羧基、羧基C 1-4亚烷基、羟基C 1-4烷基、C 1-4烷氧基C 1-2亚烷基或C 1-4卤代烷基。
  4. 一种化合物，其包含以下其中之一的结构：
    Figure PCTCN2020087700-appb-100002
    Figure PCTCN2020087700-appb-100003
    Figure PCTCN2020087700-appb-100004
    Figure PCTCN2020087700-appb-100005
    Figure PCTCN2020087700-appb-100006
    或其立体异构体、互变异构体、氮氧化物、溶剂化物、代谢产物、药学上可接受的盐或它的前药。
  5. 一种药物组合物，包含权利要求1-4任意一项所述的化合物，及其药学上可接受的辅料。
  6. 根据权利要求5所述的药物组合物，其更进一步地包含其它抗HBV药物。
  7. 根据权利要求6所述的药物组合物，其中所述其它抗HBV药物为HBV聚合酶抑制剂、免疫调节剂或干扰素。
  8. 根据权利要求7所述的药物组合物，其中所述其它抗HBV药物为拉米夫定、替比夫定、替诺福韦酯，恩替卡韦、阿德福韦酯、Alfaferone、Alloferon、西莫白介素、克拉夫定、恩曲他滨、法普洛韦、干扰素、宝甘灵CP、因特芬、干扰素α-1b、干扰素α、干扰素α-2a、干扰素β-1a、干扰素α-2、白细胞介素-2、米伏替酯、硝唑尼特、聚乙二醇干扰素α-2a、病毒唑、罗扰素-A、西佐喃、Euforavac、安普利近、Phosphazid，Heplisav、干扰素α-2b、左旋咪唑或丙帕锗。
  9. 权利要求1-4任意一项所述的化合物或权利要求5-8任意一项所述的药物组合物在制备预防、处理、治疗或减轻患者病毒性疾病的药物中的用途。
  10. 根据权利要求9所述的用途，其中所述病毒性疾病是指乙型肝炎感染或乙型肝炎感染引起的疾病。
  11. 根据权利要求10所述的用途，其中所述乙型肝炎感染引起的疾病是指肝硬化或肝细胞癌变。
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CN109678859A (zh) * 2017-10-18 2019-04-26 广东东阳光药业有限公司 二氢嘧啶类化合物及其在药物中的应用

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