WO2021228222A1 - 用于治疗乙肝的组合 - Google Patents

用于治疗乙肝的组合 Download PDF

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Publication number
WO2021228222A1
WO2021228222A1 PCT/CN2021/093805 CN2021093805W WO2021228222A1 WO 2021228222 A1 WO2021228222 A1 WO 2021228222A1 CN 2021093805 W CN2021093805 W CN 2021093805W WO 2021228222 A1 WO2021228222 A1 WO 2021228222A1
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Prior art keywords
combination according
group
combination
compound
present
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PCT/CN2021/093805
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English (en)
French (fr)
Inventor
蔡哲
孙飞
胡彦宾
丁照中
陈曙辉
吴文强
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福建广生堂药业股份有限公司
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Application filed by 福建广生堂药业股份有限公司 filed Critical 福建广生堂药业股份有限公司
Priority to US17/925,067 priority Critical patent/US20230183263A1/en
Priority to EP21804169.7A priority patent/EP4151220A4/en
Priority to CN202180034220.4A priority patent/CN115515595A/zh
Priority to JP2022569632A priority patent/JP2023526345A/ja
Publication of WO2021228222A1 publication Critical patent/WO2021228222A1/zh

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with heterocyclic ring 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
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the invention belongs to the field of biomedicine, and relates to a combination of a compound represented by formula (I) for the treatment of hepatitis B and other drugs for the treatment of hepatitis B, and the use of the combination in the preparation of drugs for the treatment of hepatitis B.
  • Hepatitis B virus referred to as hepatitis B
  • HBV hepatitis B virus
  • Hepatitis B virus is a hepatotropic virus that mainly exists in liver cells and damages liver cells, causing inflammation, necrosis, and fibrosis of liver cells.
  • Acute hepatitis B can heal itself through its own immune mechanism in most adults.
  • chronic hepatitis B CHB
  • CHB chronic hepatitis B
  • HCC chronic hepatitis B
  • HBsAg hepatitis B virus surface antigen
  • the surface antigen protein of hepatitis B virus plays a very important role in the process of HBV invading liver cells, and is of great significance for the prevention and treatment of HBV infection.
  • Surface antigen proteins include large (L), medium (M) and small (S) surface antigen proteins, which share a common C-terminal S region. They are expressed in an open reading frame, and their different lengths are determined by the three AUG start codons in the reading frame.
  • the three surface antigen proteins include pre-S1/pre-S2/S, pre-S2/S and S domains.
  • the HBV surface antigen protein is integrated into the endoplasmic reticulum (ER) membrane, which is initiated by the N-terminal signal sequence.
  • SVPs spherical and filamentous subviral particles
  • HBsAg spherical and filamentous subviral particles
  • SVP contains most of the S surface antigen protein.
  • the L protein is essential for the interaction between the morphogenesis of the virus and the nucleocapsid, but it is not necessary for the formation of SVP. Due to their lack of nucleocapsid, the SVPs are non-infectious. SVPs are greatly involved in disease progression, especially the immune response to hepatitis B virus.
  • HBsAg can also inhibit human innate immunity, can inhibit the production of cytokines induced by polysaccharides (LPS) and IL-2, inhibit the function of dendritic cells and the interference of LPS on ERK-1/2 and c-Jun N-terminal kinase-1/ 2 Inducible activity in monocytes. It is worth noting that the disease progression of liver cirrhosis and hepatocellular carcinoma is also largely related to the continuous secretion of HBsAg. The results of these studies indicate that HBsAg plays an important role in the development of chronic hepatitis.
  • anti-HBV drugs currently approved for marketing are mainly immunomodulators (interferon- ⁇ and peginterferon- ⁇ -2 ⁇ ) and antiviral drugs (lamivudine, adefovir dipivoxil, entecavir, Bivudine, Tenofovir, Kravudine, etc.).
  • antiviral drugs belong to nucleoside or nucleotide drugs, and their mechanism of action is to inhibit the synthesis of HBV DNA, and cannot directly reduce HBsAg levels.
  • nucleotide drugs show HBsAg clearance speed similar to natural observations.
  • WO2016128335A1 discloses a series of 2-oxo-6,7-dihydrobenzo[a]quinazine 3-carboxylic acid derivatives for the treatment or prevention of hepatitis B virus infection, the series of fused ring compounds still have molecules The problem of strong rigidity, unsatisfactory solubility and easy aromatization. Therefore, in terms of clinical application, there is still a demand for drugs with better pharmaceutical properties.
  • the present invention provides a combination comprising a compound represented by formula (I) and other drugs for the treatment of hepatitis B.
  • the present invention provides a composition comprising the combination of the present invention and a pharmaceutically acceptable carrier and/or excipient.
  • the present invention provides a kit comprising the composition of the present invention.
  • the present invention provides the use of a combination, composition or kit in the preparation of a medicine for treating hepatitis B.
  • the present invention also provides a method for treating hepatitis B, which comprises administering to a subject an effective amount of the combination, composition or kit of the present invention.
  • the present invention also provides the use of the combination, composition or kit of the present invention in the treatment of hepatitis B.
  • the present invention provides the use of the compound represented by formula (I) in combination with other drugs for the treatment of hepatitis B in the preparation of drugs for the treatment of hepatitis B.
  • the present invention also provides a method for treating hepatitis B, which comprises administering to a subject an effective amount of a compound represented by formula (I) and other drugs for treating hepatitis B.
  • the invention also provides the use of the compound represented by formula (I) in combination with other drugs for the treatment of hepatitis B to treat hepatitis B.
  • the present invention provides a combination comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from H, OH, CN, NH 2 , C 1-5 alkyl, C 1-5 heteroalkyl, C 2-5 alkynyl, C 3-6 cycloalkyl and 3-6 membered heterocycloalkane
  • the C 1-5 alkyl group, C 1-5 heteroalkyl group, C 2-5 alkynyl group, C 3-6 cycloalkyl group and 3-6 membered heterocycloalkyl group are optionally selected by 1, 2 or 3 R substitution;
  • R 2 is selected from H, halogen, C 1-3 alkyl and C 1-3 heteroalkyl, the C 1-3 alkyl and C 1-3 heteroalkyl are optionally substituted by 1, 2 or 3 R ;
  • n is selected from 0, 1, 2, 3, 4 and 5;
  • A is selected from a phenyl group and a 5- to 6-membered heteroaryl group, the phenyl group and a 5- to 6-membered heteroaryl group are optionally substituted with 1, 2 or 3 R;
  • the above-mentioned combination comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an immunomodulator, characterized in that the immunomodulator is selected from interferon, preferably polyethylene Alcohol interferon alpha-2a or interferon alpha-2b (IFN alpha-2b).
  • interferon preferably polyethylene Alcohol interferon alpha-2a or interferon alpha-2b (IFN alpha-2b).
  • the above-mentioned combination includes a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an immunomodulator, which is characterized in that it also includes a nucleotide reverse transcriptase inhibitor.
  • the above-mentioned combination includes a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, an immunomodulator and a nucleotide reverse transcriptase inhibitor, characterized in that the nucleoside
  • the acidic reverse transcriptase inhibitor is selected from tenofovir disoproxil fumarate and tenofovir fumarate.
  • the above-mentioned combination includes a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, an immunomodulator and a nucleotide reverse transcriptase inhibitor, characterized in that the immunomodulator
  • the agent is selected from interferons, preferably polyethylene glycol interferon alpha-2a or interferon alpha-2b, and the nucleotide reverse transcriptase inhibitor is selected from tenofovir disoproxil fumarate and fumarate Tenofovir propofol acid.
  • the present invention also provides a combination comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof, which is characterized in that it also comprises a nucleotide reverse transcriptase inhibitor or a nucleoside reverse transcriptase inhibitor .
  • the above-mentioned combination includes a compound of formula (I) or a pharmaceutically acceptable salt thereof and a nucleotide reverse transcriptase inhibitor, characterized in that the nucleotide reverses
  • the transcriptase inhibitor is selected from tenofovir disoproxil fumarate and tenofovir fumarate.
  • the above-mentioned combination includes the compound of formula (I) or a pharmaceutically acceptable salt thereof and a nucleoside reverse transcriptase inhibitor, characterized in that the nucleoside reverse transcriptase The inhibitor is selected from Entecavir.
  • the above combination wherein R is selected from H, F, Cl, Br, OH, CH 3 , CH 3 O, CF 3 , CHF 2 , CH 2 F, and other variables are as defined in the present invention .
  • R 2 is selected from H, F, Cl, Br, CH 3 , CH 3 CH 2 , CH 3 O, CH 3 CH 2 O and The CH 3 , CH 3 CH 2 , CH 3 O, CH 3 CH 2 O and Optionally substituted by 1, 2 or 3 R, other variables are as defined in the present invention.
  • R 1 , R 2 , R and m are as defined in the present invention.
  • the present invention also provides the use of the above-mentioned combination in the preparation of a medicine for treating hepatitis B.
  • the present invention also provides a composition comprising the above-mentioned combination and at least one pharmaceutically acceptable carrier and/or excipient.
  • the present invention also provides a kit, which comprises the above-mentioned combination or the above-mentioned composition.
  • the present invention also provides the use of the above-mentioned composition or the above-mentioned kit in the preparation of a medicine for treating hepatitis B.
  • the above-mentioned combination is a combination for the treatment of chronic hepatitis B.
  • the above-mentioned composition is a pharmaceutical composition for treating chronic hepatitis B.
  • the above-mentioned kit is a kit for treating chronic hepatitis B.
  • the above kit also includes instructions for the combined use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and other drugs for the treatment of hepatitis B to treat hepatitis B.
  • the pharmaceutical composition of the compound represented by formula (I) includes the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and/or excipient .
  • the pharmaceutical composition of the compound represented by formula (I) is in the form of a solid preparation, preferably a capsule or a tablet.
  • the pharmaceutical composition of other drugs for treating hepatitis B includes other drugs for treating hepatitis B, and pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutical composition of the other drugs for the treatment of hepatitis B is in the form of liquid preparations, preferably water-soluble injections.
  • the water-soluble injections include, but are not limited to, water-soluble preparations that have not been lyophilized or Reconstituted water-soluble formulation of lyophilized powder.
  • the components of the combination of the present invention can be separately formulated into a pharmaceutical composition, or part or all of them can be formulated together as a pharmaceutical composition.
  • the combination of the present invention can be formulated as a pharmaceutical composition suitable for single or multiple administration.
  • the components of the combination of the present invention may be administered individually, or some or all of them may be administered together.
  • the components of the combination of the present invention may be administered at substantially different times, or some or all of them may be administered at substantially the same time.
  • the components in the combination of the present invention may have the same or different administration cycles.
  • the components of the combination of the present invention can be independently administered by various suitable routes, including, but not limited to, oral or parenteral (by intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration). Route, such as by injection or infusion). In some schemes, the components of the combination of the present invention may each be independently administered orally or by injection, such as intravenous injection or intraperitoneal injection.
  • the components of the combination of the present invention can each independently be a suitable dosage form, including, but not limited to, tablets, troches, pills, capsules (such as hard capsules, soft capsules, enteric capsules, microcapsules),
  • suitable dosage form including, but not limited to, tablets, troches, pills, capsules (such as hard capsules, soft capsules, enteric capsules, microcapsules),
  • the components of the combination of the present invention may each independently contain a pharmaceutically acceptable carrier and/or excipient.
  • the compound of formula (I) of the present invention is a hepatitis B surface antigen inhibitor, can effectively reduce the content of HBsAg, and has a significant inhibitory effect on HBV.
  • the compound of formula (I) is used in combination with the immunomodulator, or the compound of formula (I) is used in combination with nucleoside or nucleotide reverse transcriptase inhibitors, or the combination of drugs using these three types of mechanisms Use, can enhance the effect on HBsAg, play a synergistic inhibitory effect, at the same time multi-channel inhibition of HBV virus, achieve the purpose of improving curative effect, reducing toxic side effects, shortening the treatment cycle and dosage, reducing drug resistance, etc., and can achieve HBV carrier The amount is reduced, HBsAg is reduced or even eliminated.
  • pharmaceutically acceptable salt refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts.
  • the acid addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid (see Berge et al., "
  • the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the neutral form of the compound.
  • the parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.
  • “Pharmaceutically acceptable salt” as used herein is a derivative of the compound of the present invention, wherein the parent compound is modified by forming a salt with an acid or a salt with a base.
  • pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acid radicals such as carboxylic acids, and the like.
  • Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of parent compounds, such as salts formed by non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic acids and organic acids.
  • the inorganic or organic acids are selected from 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, Benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, Hydrobromic acid, hydrochloric acid, hydroiodide, hydroxyl, hydroxylnaphthalene, isethionic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, Pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid,
  • the pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods.
  • such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or an organic solvent or a mixture of both.
  • non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • the compounds provided by the present invention also exist in prodrug forms.
  • the prodrugs of the compounds described herein easily undergo chemical changes under physiological conditions to transform into the compounds of the invention.
  • prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in the in vivo environment.
  • Certain compounds of the present invention may exist in unsolvated or solvated forms, including hydrated forms.
  • the solvated form is equivalent to the unsolvated form, and both are included in the scope of the present invention.
  • Certain compounds of the present invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers and individual isomers are all included in the scope of the present invention.
  • wedge keys and dotted keys Express the absolute configuration of a three-dimensional center, using Represents the relative configuration of a three-dimensional center.
  • the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, they include E and Z geometric isomers. Likewise, all tautomeric forms are included within the scope of the present invention.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Conformers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to Within the scope of the present invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention.
  • optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If you want to obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
  • the molecule when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with an appropriate optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered.
  • the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which uses a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).
  • the compound of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C). All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.
  • pharmaceutically acceptable carrier refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects to the host or patient.
  • Representative carriers include water, oil, Vegetables and minerals, cream base, lotion base, ointment base, etc. These bases include suspending agents, tackifiers, penetration enhancers and the like. Their formulations are well known to those skilled in the field of cosmetics or topical medicine. For other information about the carrier, you can refer to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), and the content of this document is incorporated herein by reference.
  • the term "effective amount” or “therapeutically effective amount” refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect.
  • the "effective amount” of one active substance in the composition refers to the amount required to achieve the desired effect when combined with another active substance in the composition.
  • the determination of the effective amount varies from person to person, and depends on the age and general conditions of the recipient, as well as the specific active substance. The appropriate effective amount in each case can be determined by those skilled in the art according to routine experiments.
  • active ingredient refers to a chemical entity that can effectively treat the target disorder, disease or condition.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by substituents, and may include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable of.
  • Ketone substitution does not occur on aromatic groups.
  • optionally substituted means that it can be substituted or unsubstituted. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis that they can be chemically realized.
  • any variable such as R
  • its definition in each case is independent.
  • the group can optionally be substituted with up to two Rs, and R has independent options in each case.
  • combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • substituents When a substituent is vacant, it means that the substituent is absent. For example, when X in AX is vacant, it means that the structure is actually A. When the bond of a substituent can be cross-connected to two atoms on a ring, the substituent can be bonded to any atom on the ring. When the listed substituents do not indicate through which atom they are connected to the compounds included in the general formula of the chemical structure but are not specifically mentioned, such substituents may be bonded through any of its atoms. Combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds. For example, the structural unit It means that it can be substituted at any position on the cyclohexyl or cyclohexadiene.
  • ring means substituted or unsubstituted cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aryl, or heteroaryl.
  • the so-called ring includes a single ring, a bicyclic ring, a spiro ring, a parallel ring or a bridged ring.
  • the number of atoms in a ring is generally defined as the number of ring members. For example, "5- to 7-membered ring" means that 5 to 7 atoms are arranged around. Unless otherwise specified, the ring optionally contains 1 to 3 heteroatoms.
  • the "5- to 7-membered ring” includes, for example, phenyl, pyridine, and piperidinyl; on the other hand, the term “5- to 7-membered heterocycloalkyl ring” includes pyridyl and piperidinyl, but does not include phenyl.
  • the term “ring” also includes a ring system containing at least one ring, where each "ring" independently meets the above definition.
  • heterocycle or “heterocyclic group” means a stable monocyclic, bicyclic or tricyclic ring containing heteroatoms or heteroatoms, which may be saturated, partially unsaturated or unsaturated ( Aromatic), they contain carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S, wherein any of the above heterocycles can be fused to a benzene ring to form a bicyclic ring. Nitrogen and sulfur heteroatoms can optionally be oxidized (ie, NO and S(O)p, where p is 1 or 2).
  • the nitrogen atom may be substituted or unsubstituted (ie, N or NR, where R is H or other substituents already defined herein).
  • the heterocyclic ring can be attached to any heteroatom or carbon atom pendant group to form a stable structure. If the resulting compound is stable, the heterocyclic ring described herein can be substituted at the carbon or nitrogen position.
  • the nitrogen atom in the heterocyclic ring is optionally quaternized.
  • a preferred solution is that when the total number of S and O atoms in the heterocycle exceeds 1, these heteroatoms are not adjacent to each other. Another preferred solution is that the total number of S and O atoms in the heterocycle does not exceed 1.
  • aromatic heterocyclic group or “heteroaryl” means a stable 5-, 6, 7-membered monocyclic or bicyclic or 7, 8, 9 or 10-membered bicyclic heterocyclic aromatic ring, It contains carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S.
  • the nitrogen atom may be substituted or unsubstituted (ie, N or NR, where R is H or other substituents already defined herein).
  • Nitrogen and sulfur heteroatoms can optionally be oxidized (ie, NO and S(O)p, where p is 1 or 2). It is worth noting that the total number of S and O atoms in the aromatic heterocycle does not exceed 1.
  • Bridged rings are also included in the definition of heterocyclic rings.
  • a bridged ring is formed when one or more atoms (ie, C, O, N, or S) connect two non-adjacent carbon atoms or nitrogen atoms.
  • Preferred bridged rings include, but are not limited to: one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms and one carbon-nitrogen group. It is worth noting that a bridge always converts a single ring to a three ring. In bridged rings, substituents on the ring can also appear on the bridge.
  • heterocyclic compounds include, but are not limited to: acridinyl, azeinyl, benzimidazolyl, benzofuranyl, benzomercaptofuranyl, benzomercaptophenyl, benzoxazolyl, benzoxanyl Azolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, Carboline, chromanyl, chromene, cinnolinyldecahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b] Tetrahydrofuryl, furanyl, furzanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indoleny
  • hydrocarbyl or its subordinate concepts (such as alkyl, alkenyl, alkynyl, aryl, etc.) by itself or as part of another substituent means linear, branched or cyclic Hydrocarbon radicals or combinations thereof, can be fully saturated (such as alkyl), unit or polyunsaturated (such as alkenyl, alkynyl, aryl), can be mono- or multi-substituted, and can be monovalent (such as Methyl), divalent (such as methylene) or multivalent (such as methine), can include divalent or multivalent atom groups, with a specified number of carbon atoms (such as C 1 -C 12 represents 1 to 12 carbon , C 1-12 is selected from C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 and C 12 ; C 3-12 is selected from C 3.
  • Hydrocarbon group includes but is not limited to aliphatic hydrocarbon groups and aromatic hydrocarbon groups.
  • the aliphatic hydrocarbon groups include chain and cyclic, specifically including but not limited to alkyl groups, alkenyl groups, and alkynyl groups.
  • the aromatic hydrocarbon groups include but are not limited to 6-12 members. The aromatic hydrocarbon group, such as benzene, naphthalene, etc.
  • hydrocarbyl refers to linear or branched atomic groups or combinations thereof, which can be fully saturated, unitary or polyunsaturated, and can include divalent and multivalent atomic groups.
  • saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, isobutyl, cyclohexyl, (cyclohexyl) Methyl, cyclopropylmethyl, and homologs or isomers of atomic groups such as n-pentyl, n-hexyl, n-heptyl, and n-octyl.
  • Unsaturated hydrocarbon groups have one or more double bonds or triple bonds, examples of which include but are not limited to vinyl, 2-propenyl, butenyl, crotyl, 2-isopentenyl, 2-(butadienyl) , 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers body.
  • heterohydrocarbyl or its subordinate concepts (such as heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, etc.) by itself or in combination with another term means a stable linear, branched chain Cyclic or cyclic hydrocarbon radicals or combinations thereof, consisting of a certain number of carbon atoms and at least one heteroatom.
  • heteroalkyl by itself or in combination with another term means a stable linear or branched hydrocarbon radical or a combination thereof, consisting of a certain number of carbon atoms and at least one heteroatom.
  • the heteroatoms are selected from B, O, N, and S, wherein nitrogen and sulfur atoms are optionally oxidized, and nitrogen heteroatoms are optionally quaternized.
  • the heteroatom or heteroatom group can be located in any internal position of the heterohydrocarbyl group, including the position where the hydrocarbyl group is attached to the rest of the molecule, but the terms "alkoxy”, “alkylamino” and “alkylthio” (or thioalkoxy) ) Is a customary expression and refers to those alkyl groups that are connected to the rest of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively.
  • Up to two heteroatoms can be continuous, for example -CH 2 -NH-OCH 3 .
  • cycloalkyl "heterocycloalkyl” or its subordinate concepts (such as aryl, heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl , Heterocycloalkynyl, etc.) by itself or in combination with other terms respectively represent cyclized “hydrocarbyl” and “heterohydrocarbyl”.
  • a heteroatom may occupy the position where the heterocycle is attached to the rest of the molecule.
  • cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • heterocyclic groups include 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofurindol-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl and 2-piperazinyl.
  • alkyl is used to denote a linear or branched saturated hydrocarbon group, which may be mono-substituted (such as -CH 2 F) or multi-substituted (such as -CF 3 ), and may be monovalent (such as Methyl), divalent (such as methylene) or multivalent (such as methine).
  • alkyl groups examples include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl) , T-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl) and the like.
  • alkynyl refers to an alkyl group having one or more carbon-carbon triple bonds at any point in the chain, which can be mono- or multi-substituted, and can be monovalent, divalent, or multivalent.
  • alkynyl groups include ethynyl, propynyl, butynyl, pentynyl and the like.
  • cycloalkyl includes any stable cyclic or polycyclic hydrocarbon group, any carbon atom is saturated, can be mono- or poly-substituted, and can be monovalent, divalent or multivalent.
  • examples of these cycloalkyl groups include, but are not limited to, cyclopropyl, norbornyl, [2.2.2] dicyclooctane, [4.4.0] dicyclodecane, and the like.
  • halogen or “halogen” by itself or as part of another substituent represents a fluorine, chlorine, bromine or iodine atom.
  • haloalkyl is intended to include monohaloalkyl and polyhaloalkyl.
  • halo(C 1 -C 4 )alkyl is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc. Wait.
  • examples of haloalkyl include, but are not limited to: trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.
  • Alkoxy represents the above-mentioned alkyl group having a specified number of carbon atoms connected by an oxygen bridge. Unless otherwise specified, C 1-6 alkoxy includes C 1 , C 2 , C 3 , C 4 , C 5 and C 6 's alkoxy group. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy and S- Pentoxy.
  • aryl means a polyunsaturated aromatic hydrocarbon substituent, which may be mono- or poly-substituted, monovalent, divalent or polyvalent, and it may be monocyclic or polycyclic ( For example, 1 to 3 rings; at least one of which is aromatic), they are fused together or covalently linked.
  • heteroaryl refers to an aryl group (or ring) containing one to four heteroatoms. In an exemplary example, the heteroatoms are selected from B, N, O, and S, wherein nitrogen and sulfur atoms are optionally oxidized, and nitrogen atoms are optionally quaternized. Heteroaryl groups can be attached to the rest of the molecule through heteroatoms.
  • Non-limiting examples of aryl or heteroaryl groups include phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, phenyl-oxazolyl, isooxanyl Azolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidinyl, benzothiazolyl, purinyl, benzimidazolyl, indolyl, isoquinolinyl, quinoxalinyl, quinolinyl, 1 -Naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-ox
  • aryl when used in conjunction with other terms (e.g., aryloxy, arylthio, aralkyl) includes aryl and heteroaryl rings as defined above. Therefore, the term “aralkyl” is meant to include those atomic groups to which the aryl group is attached to the alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including where the carbon atom (e.g., methylene) has been replaced by, for example, oxygen. Those alkyl groups substituted by atoms, for example, phenoxymethyl, 2-pyridyloxymethyl 3-(1-naphthyloxy)propyl and the like.
  • the purpose of this study is to use human primary hepatocytes (PHH) isolated from humanized FRG mouse models to evaluate the relationship between test compounds and TAF (tenofovir fumarate) and Peg- In vitro anti-hepatitis B virus efficacy of IFN- ⁇ 2a (pegylated interferon ⁇ 2a) combined administration.
  • PHL human primary hepatocytes isolated from humanized FRG mouse models
  • HBV was added to infect PHH (2000GE/cell).
  • Set up two compound treatment methods one cell plate was treated with test compound and Peg-IFN ⁇ -2a for four hours on the first day, and then replaced with HBV-containing medium to infect the cells (while adding compound to continue treatment); another plate The test compound and Peg-IFN ⁇ -2a were added for treatment on the 2nd day. Replace the fresh medium with the compound every 1-2 days.
  • the final concentration of DMSO in the culture broth is 2%.
  • the initial concentration of the test compound was 1000 nM, and the initial concentration of the control compound Peg-IFN ⁇ -2a was 500 IU/mL, 5 times dilution, 7 concentration points, three multiple wells (see Table 3 for compound concentration settings).
  • the cell culture supernatants collected on days 8, 14 and 22 will be used for HBV DNA, HBeAg and HBsAg detection.
  • HBV was added to infect PHH (2000GE/cell).
  • PHH 2000GE/cell
  • test compound and Peg-IFN ⁇ -2a are both diluted 3 times at 5 concentration points, tested in a 5 ⁇ 5 matrix, and three-folded plates.
  • the starting concentrations of the test compound and Peg-IFN ⁇ -2a were 400 nM and 100 IU/mL, respectively.
  • the arrangement of the combined action compound is shown in Table 2, and the test concentration of the compound is shown in Table 3.
  • the final concentration of DMSO in the cell culture medium is 2%.
  • the medium containing the compound was changed.
  • the culture supernatant was collected for detection of HBV DNA, HBeAg and HBsAg. After the supernatant was collected, CellTiter-Glo was added to detect cell viability.
  • test compound and Peg-IFN ⁇ -2a are diluted 3 times at 5 concentration points, tested in a 5 ⁇ 5 matrix, and three-folded plates.
  • the initial concentrations of the test compound and Peg-IFN ⁇ -2a were 400 nM and 100 IU/mL, respectively, and a concentration point of 0.3 nM was set for TAF.
  • the arrangement of the combined action compound is shown in Table 4, and the test concentration of the compound is shown in Table 5.
  • the final concentration of DMSO in the culture broth is 2%.
  • the medium was changed with the medium containing the compound.
  • the culture supernatant was collected for HBV DNA, HBeAg and HBsAg detection. After the cell supernatant was collected, CellTiter-Glo was added to detect cell viability.
  • Cell viability test After collecting the cell culture supernatant, dilute CellTiter-Glo with culture medium 1:1, add it to the cell plate, leave it at room temperature for 10 minutes, and detect the luminescence value with a microplate reader.
  • HBV DNA detection Extract the DNA in the supernatant according to the DNA extraction kit (Qiagen-51162), and use the qPCR method to quantify the HBV DNA in the sample.
  • the qPCR reaction system is shown in Table 6.
  • HBV plasmid DNA is used as a standard, 10-fold serial dilution, the standard range is from 1.0 ⁇ 10 7 copies/ ⁇ l to 10 copies/ ⁇ l.
  • the PCR reaction program is: heating at 95°C for 10 minutes, and then entering the cycle mode, heating at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles. Calculate the HBVDNA content in the sample based on the standard curve and the Ct value of each sample.
  • Detection of HBeAg and HBsAg Detect the levels of HBeAg and HBsAg in the supernatant according to the instructions of the ELISA kit. The method is briefly described as follows: Take 50 ⁇ L of standard substance, sample and control substance into the detection plate, then add 50 ⁇ L of enzyme conjugate to each well, incubate at 37°C for 60 minutes, wash the plate with washing solution and blot dry, then add 50 ⁇ L of premix The luminescent substrate was incubated for 10 minutes at room temperature in the dark, and finally the luminescence value was measured by a microplate reader. The content of HBeAg and HBsAg in the sample was calculated according to the standard curve and the chemiluminescence value of each sample.
  • Cell activity% (sample luminescence value-blank control well average value) / (DMSO control luminescence value-blank control well average value) ⁇ 100
  • Inhibition rate% (1- HBV DNA content or HBsAg content or HBeAg content in the sample/HBV DNA content or HBsAg content or HBeAg content in the DMSO control group) ⁇ 100
  • the CC 50 and EC 50 were calculated by GraphPad Prism software, and the inhibition curve fitting method was sigmoidal dose-response (variable slope).
  • the combined efficacy was analyzed by MacSynergy TM II software.
  • the combined efficacy is calculated based on the 95% confidence interval of the synergy/antagonism, and the results are explained in accordance with the MacSynergy guidelines as follows:
  • ⁇ 25 Insignificant synergism/antagonism insignificant synergy or antagonism (addition);
  • ⁇ 1000 Possible errors-check data and repeat experiment may be wrong-check the data and repeat the experiment;
  • the results of the combination of test compounds showed that the combination of WWS0 and Peg-IFN ⁇ -2a had a moderate synergistic effect on the inhibition of HBsAg and an additive effect on the inhibition of HBeAg.
  • the test compound WWS0 combined with TAF and Peg-IFN ⁇ -2a has a moderate synergistic effect on the inhibition of HBsAg and an additive effect on the inhibition of HBeAg.
  • test compound WWS0 showed no obvious cytotoxicity in the test concentration range in the single-drug and combined effect experiments.
  • HepG2-NTCP cell in vitro infection model was used to evaluate the inhibitory activity of compound WWS0 and entecavir (ETV) in vitro on HBV. Detect the content of HBV DNA in the cell supernatant by real-time fluorescent quantitative PCR (qPCR) method, detect the content of HBeAg and HBsAg by ELISA, and determine the anti-HBV activity of the combined action of the compound. At the same time, CellTiter-Glo detects the effect of the test compound on cell viability. Influence.
  • qPCR real-time fluorescent quantitative PCR
  • HepG2-NTCP cell line was constructed and provided by WuXi AppTec.
  • HepG2-NTCP cells are HepG2 cells stably transfected and continuously express the human NTCP gene, which is easy to be infected by HBV.
  • the cell culture medium is DMEM medium supplemented with 10% fetal bovine serum, 1% Penicillin-Streptomycin, 1% glutamine, 1% non-essential amino acids and G418 (500 ⁇ g/mL).
  • Type D HBV is concentrated from HepG2.2.15 cell culture supernatant.
  • the main reagents used in this study include FastStart Universal Probe Master (Roche), DNA extraction kit (Qiagen), 96-well plate (Costar), HBsAg ELISA kit (Autobio) and HBeAg ELISA kit (Autobio).
  • the main instruments used in this study are real-time fluorescent quantitative PCR instrument (Thermo, QuantStudio TM 6 Flex) and multifunctional microplate reader (BioTek, Synergy 2).
  • Test compounds WWS0 and ETV.
  • ETV is a drug that has been marketed to treat HBV.
  • HepG2-NTCP cells were seeded into a 48-well cell culture plate at a density of 75,000 cells/well, and then the cells were cultured overnight under the conditions of 5% CO 2 and 37°C.
  • HBV was added to infect HepG2-NTCP cells.
  • HBV DNA was quantified by qPCR method.
  • HBV plasmid DNA is used as the standard. The concentration of standard HBV plasmid DNA starts from 10 7 copies/ ⁇ L with a 10-fold dilution at 7 points. Fit the standard curve with the HBVDNA copy number and CT value of each standard, and calculate the HBVDNA copy number in each test sample.
  • the index is positive, it is synergistic, and if the index is negative, it is antagonistic; the absolute value of the index is less than 25, that is, the additive effect; the absolute value of the index is in the range of 25-50, that is, mild but clear synergy or antagonism; the absolute value of the index is in The range of 50-100, that is, moderate synergistic or antagonistic effects, may have important implications for in vivo effects.
  • the absolute value of the index is in the range of >100, that is, a highly synergistic or antagonistic effect, which is likely to be of great significance to the effect in vivo.
  • test compound WWS0 inhibits HBV DNA, HBeAg and HBsAg in a dose-dependent manner; the combination of WWS0 and ETV shows that HBeAg and HBsAg have synergistic or superimposed anti-HBV activity; the test compound WWS0 In single-drug and combined action experiments, none of them showed obvious cytotoxicity within the tested concentration range.
  • Example 1 According to the method of Example 1, the test compound WWS. Replace with compound WWS01 WWS02 WWS03
  • test compounds WWS01, WWS02, WWS03 and Peg-IFN ⁇ -2a has a synergistic or additive effect on the inhibition of HBsAg and HBeAg.
  • the synergy index is within the range of 20.00-50.00 in the 95% confidence interval, and the antagonism index is 0. .
  • the ETV Entecavir
  • TDF Teofovir Disoproxil fumarate
  • the combination with TDF has a synergistic effect on the inhibition of HBsAg and HBeAg.
  • the synergy index is 86.55 and 25.49 in the 95% confidence interval, and the antagonism index is 0 and 1.06.
  • the interferon Peg-IFN ⁇ -2a was replaced with IFN ⁇ -2b, and the test compound WWS0 combined with IFN ⁇ -2b and WWS0 combined with TAF and IFN ⁇ -2b were obtained.
  • the results showed The synergy index of the two groups of combined use on HBsAg and HBeAg is greater than 25, and has a synergistic effect on the inhibition of HBsAg and HBeAg.

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Abstract

本发明涉及一类用于治疗乙型肝炎的式(I)所示化合物与其他治疗乙型肝炎药物的组合，以及该组合在制备治疗乙型肝炎药物中的用途。

Description

用于治疗乙肝的组合
本申请要求申请日为2020年5月15日的中国专利申请CN202010412771.7的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物医药领域，涉及一类用于治疗乙型肝炎的式(I)所示化合物与其他治疗乙型肝炎药物的组合，以及该组合在制备治疗乙型肝炎药物中的用途。
背景技术
乙型病毒性肝炎，简称乙肝，是一种由乙型肝炎病毒(Hepatitis B Virus，简称HBV)感染机体后所引起的疾病。乙型肝炎病毒是一种嗜肝病毒，主要存在于肝细胞内并损害肝细胞，引起肝细胞炎症、坏死、纤维化。乙型病毒性肝炎分急性和慢性两种。急性乙型肝炎在成年人中大多数可通过其自身的免疫机制而自愈。但是慢性乙型肝炎(CHB)已成为全球健康保健所面临的极大挑战，同时也是引起慢性肝病，肝硬化(cirrhosis)和肝癌(HCC)的主要原因。据估计，全球有20亿人感染了慢性乙型肝炎病毒，超过3亿5千万人口已发展成为了乙型肝炎，每年近60万人死于慢性乙型肝炎的并发症。我国是乙肝高发区，乙型肝炎累积病人多，危害严重。据资料显示，我国现有乙型肝炎病毒感染者约9300万，而其中约2000万患者确诊为慢性乙型肝炎，当中10％-20％可演变成肝硬化，1％-5％可发展成肝癌。
乙肝功能性治愈的关键是清除HBsAg(乙型肝炎病毒表面抗原)，产生表面抗体。HBsAg量化是一个非常重要的生物指标。在慢性感染病人中，很少能观察到HBsAg的减少和血清转化，这是目前治疗的终点。
乙型肝炎病毒(HBV)的表面抗原蛋白在HBV侵入肝细胞的过程中起非常重要的作用，对于防治HBV的感染有重要意义。表面抗原蛋白包括大(L)、中(M)和小(S)的表面抗原蛋白，共享共同的C端S区。它们从一个开放读码框中表达，其不同的长度是由读码框三个AUG起始密码子决定的。这三个表面抗原蛋白包括前S1/前S2/S，前S2/S和S域。HBV表面抗原蛋白被整合到内质网(ER)膜，由N端信号序列启动。它们不仅构成了病毒体的基本结构，而且还形成球状和丝状亚病毒颗粒(SVPs，HBsAg)，聚集在ER，宿主ER和前高尔基体器，SVP包含大多S表面抗原蛋白。L蛋白，在病毒的形态发生与核衣壳相互作用方面是至关重要的，但对于SVP的形成是没有必要的。由于它们缺乏核衣壳，所述的SVPs是非感染性的。SVPs大大参与了疾病进展，尤其是对乙肝病毒的免疫应答，在感染者的血液里，SVPs的量至少是病毒数量的10,000倍，诱捕了免疫系统，削弱人体对乙肝病毒的免疫反应。HBsAg也可抑制人的天然免疫，能够抑制多糖(LPS)和IL-2诱导的细胞因子产生，抑制树状细胞DC功能以及LPS对ERK-1/2和c-Jun N端的干扰激酶-1/2在单核细胞的诱导活性。值得注意的是，肝硬化和肝细胞癌的疾病进展很大程度也与持续分泌的HBsAg相关。这些研 究结果表明HBsAg在慢性肝炎的发展中起重要作用。
目前被批准上市的抗HBV药物主要是免疫调节剂(干扰素-α和聚乙二醇干扰素-α-2α)和抗病毒治疗药物(拉米夫定、阿德福韦酯、恩替卡韦、替比夫定、替诺福韦、克拉夫定等)。其中，抗病毒治疗药物属于核苷类或核苷酸类药物，其作用机制是抑制HBV DNA的合成，并不能直接减少HBsAg水平。与延长治疗一样，核苷酸类药物显示HBsAg清除速度类似于自然观察结果。
临床已有疗法降低HBsAg疗效不佳，因此，开发能够有效降低HBsAg的小分子口服抑制剂是目前临床用药所亟需的。
虽然WO2016128335A1公开了一系列用于治疗或者预防乙型肝炎病毒感染的2-氧代-6，7二氢苯并[a]喹嗪3-羧酸衍生物，但是该系列稠环化合物仍然有分子刚性较强、溶解性不够理想以及易于芳构化的问题。因此，就临床应用而言，对成药性更佳的药物的需求依然存在。
发明内容
一方面，本发明提供一种组合，所述的组合包括式(I)所示化合物与其他治疗乙型肝炎药物。
另一方面，本发明提供一种组合物，其包括本发明的组合和药学上可接受的载体和/或赋形剂。
另一方面，本发明提供一种试剂盒，其包括本发明的组合物。
另一方面，本发明提供一种组合、组合物或试剂盒在制备治疗乙型肝炎药物的用途。本发明还提供治疗乙型肝炎的方法，其包括向受试者给予有效量的本发明的组合、组合物或试剂盒。本发明还提供本发明的组合、组合物或试剂盒在治疗乙型肝炎中的用途。
另一方面，本发明提供式(I)所示化合物与其他治疗乙型肝炎药物联用在制备治疗乙型肝炎药物的用途。本发明还提供治疗乙型肝炎的方法，其包括向受试者给予有效量式(I)所示化合物与其他治疗乙型肝炎药物。本发明还提供式(I)所示化合物与其他治疗乙型肝炎药物联用治疗乙型肝炎的用途。
本发明提供一种组合，包括式(I)所示化合物或其药学上可接受的盐，
Figure PCTCN2021093805-appb-000001
其中，
R 1选自H、OH、CN、NH 2、C 1-5烷基、C 1-5杂烷基、C 2-5炔基、C 3-6环烷基和3～6元杂环烷基，所述C 1-5烷基、C 1-5杂烷基、C 2-5炔基、C 3-6环烷基和3～6元杂环烷基任选被1、2或3个R取代；
R 2选自H、卤素、C 1-3烷基和C 1-3杂烷基，所述C 1-3烷基和C 1-3杂烷基任选被1、2或3个R取代；
m选自0、1、2、3、4和5；
A选自苯基和5～6元杂芳基，所述苯基和5～6元杂芳基任选被1、2或3个R取代；
R选自H、卤素、OH、CN、NH2、＝O、CH 3、CH 3CH 2、CH 3O、CF 3、CHF 2、CH 2F；
所述C 1-5杂烷基、3～6元杂环烷基、C 1-3杂烷基和5～6元杂芳基分别独立地包含1、2或3个分别独立地选自N、-O-、＝O、-S-、-NH-、-(C＝O)-、-(S＝O)-和-(S＝O)2-的杂原子或杂原子团，
其特征在于，还包括免疫调节剂。
在本发明的一些方案中，上述组合，包括式(I)所示化合物或其药学上可接受的盐和免疫调节剂，其特征在于，所述免疫调节剂选自干扰素，优选聚乙二醇干扰素α-2a或干扰素α-2b(IFNα-2b)。
在本发明的一些方案中，上述组合，包括式(I)所示化合物或其药学上可接受的盐和免疫调节剂，其特征在于，还包括核苷酸类逆转录酶抑制剂。
在本发明的一些方案中，上述组合，包括式(I)所示化合物或其药学上可接受的盐、免疫调节剂和核苷酸类逆转录酶抑制剂，其特征在于，所述核苷酸类逆转录酶抑制剂选自富马酸替诺福韦二吡呋酯和富马酸丙酚替诺福韦。
在本发明的一些方案中，上述组合，包括式(I)所示化合物或其药学上可接受的盐、免疫调节剂和核苷酸类逆转录酶抑制剂，其特征在于，所述免疫调节剂选自干扰素，优选聚乙二醇干扰素α-2a或干扰素α-2b，所述核苷酸类逆转录酶抑制剂选自富马酸替诺福韦二吡呋酯和富马酸丙酚替诺福韦。
本发明还提供一种组合，包括如式(I)所述化合物或其药学上可接受的盐，其特征在于，还包括核苷酸类逆转录酶抑制剂或核苷类逆转录酶抑制剂。
在本发明的一些方案中，上述组合，包括如式(I)所述化合物或其药学上可接受的盐和核苷酸类逆转录酶抑制剂，其特征在于，所述核苷酸类逆转录酶抑制剂选自富马酸替诺福韦二吡呋酯和富马酸丙酚替诺福韦。
在本发明的一些方案中，上述组合，包括如式(I)所述化合物或其药学上可接受的盐和核苷类逆转录酶抑制剂，其特征在于，所述核苷类逆转录酶抑制剂选自恩替卡韦。
在本发明的一些方案中，上述组合，其中，R选自H、F、Cl、Br、OH、CH 3、CH 3O、CF 3、CHF 2、CH 2F，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，R 1选自H、OH、CN、NH 2、CH 3、CH 3CH 2、CH 3CH 2CH 2、CH 3CH 2CH 2CH 2、CH 3O、CH 3CH 2O、CH 3S、CH 3S(＝O)、CH 3S(＝O) 2、CH 3SCH 2、CH 3CH 2S、CH 3NH、
Figure PCTCN2021093805-appb-000002
吡咯烷基、哌啶基、四氢吡喃基、吗啉基、2-吡咯烷酮基和3-吡咯烷酮基，所述CH 3、CH 3CH 2、CH 3CH 2CH 2、CH 3CH 2CH 2CH 2、CH 3O、CH 3CH 2O、CH 3S、CH 3S(＝O)、CH 3S(＝O) 2、CH 3SCH 2、CH 3CH 2S、CH 3NH、
Figure PCTCN2021093805-appb-000003
吡咯烷基、哌啶基、四氢吡喃基、吗啉基、2-吡咯烷酮基和3-吡咯烷酮基任选被1、2或3个R取代，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，R 1选自H、OH、CN、NH 2、CH 3、CH 3CH 2、CH 3CH 2CH 2、CH 3CH 2CH 2CH 2、CH 3O、CH 3CH 2O、CH 3S、CH 3S(＝O)、CH 3S(＝O) 2、CH 3SCH 2、CH 3CH 2S、CH 3NH、
Figure PCTCN2021093805-appb-000004
Figure PCTCN2021093805-appb-000005
所述CH 3、CH 3CH 2、CH 3CH 2CH 2、CH 3CH 2CH 2CH 2、CH 3O、CH 3CH 2O、CH 3S、CH 3S(＝O)、CH 3S(＝O) 2、CH 3SCH 2、CH 3CH 2S、CH 3NH、
Figure PCTCN2021093805-appb-000006
Figure PCTCN2021093805-appb-000007
任选被1、2或3个R取代，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，R 1选自H、OH、CH 3、CHF 2、CH 3O、
Figure PCTCN2021093805-appb-000008
Figure PCTCN2021093805-appb-000009
其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，R 2选自H、F、Cl、Br、CH 3、CH 3CH 2、CH 3O、CH 3CH 2O和
Figure PCTCN2021093805-appb-000010
所述CH 3、CH 3CH 2、CH 3O、CH 3CH 2O和
Figure PCTCN2021093805-appb-000011
任选被1、2或3个R取代，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，R 2选自Cl和CH 3O，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，A选自任选被1、2或3个R取代的：苯基、噻吩基、噻唑基、异噻唑基、恶唑基和异恶唑基，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，A选自任选被1、2或3个R取代的：
Figure PCTCN2021093805-appb-000012
Figure PCTCN2021093805-appb-000013
其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，A选自
Figure PCTCN2021093805-appb-000014
Figure PCTCN2021093805-appb-000015
其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，A选自
Figure PCTCN2021093805-appb-000016
Figure PCTCN2021093805-appb-000017
Figure PCTCN2021093805-appb-000018
其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，m为3，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，R 2选自Cl和CH 3O，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，R 1为CH 3O，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，m为1，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，R 2为Cl，其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，R 1为
Figure PCTCN2021093805-appb-000019
其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，A选自任选被1、2或3个R取代的：
Figure PCTCN2021093805-appb-000020
Figure PCTCN2021093805-appb-000021
其他变量如本发明所定义。
在本发明的一些方案中，上述组合，其中，化合物选自：
Figure PCTCN2021093805-appb-000022
其中，
R 1、R 2、R、m如本发明所定义。
在本发明的一些方案中，上述组合，其中，化合物选自：
Figure PCTCN2021093805-appb-000023
Figure PCTCN2021093805-appb-000024
Figure PCTCN2021093805-appb-000025
Figure PCTCN2021093805-appb-000026
Figure PCTCN2021093805-appb-000027
在本发明的一些方案中，上述组合，其中，化合物选自：
Figure PCTCN2021093805-appb-000028
Figure PCTCN2021093805-appb-000029
本发明还提供上述组合在制备治疗乙型肝炎药物中的用途。
本发明还提供一种组合物，其包括上述组合和至少一种药学上可接受的载体和/或赋形剂。
本发明还提供一种试剂盒，其包括上述组合或者上述组合物。
本发明还提供上述组合物或上述试剂盒在制备治疗乙型肝炎药物中的用途。
在本发明的一些方案中，上述组合是用于治疗慢性乙型肝炎的组合。
在本发明的一些方案中，上述组合物是用于治疗慢性乙型肝炎的药物组合物。
在本发明的一些方案中，上述试剂盒是用于治疗慢性乙型肝炎的试剂盒。
在本发明的一些方案中，上述试剂盒还包括式(I)所示化合物或其药学上可接受的盐与其他治疗乙型肝炎药物的联合使用治疗乙型肝炎的说明。
在本发明的一些方案中，所述式(I)所示化合物的药物组合物包括式(I)所示化合物或其药学上可接受的盐和药学上可接受的载体和/或赋形剂。
在本发明的一些方案中，所述式(I)所示化合物的药物组合物呈固体制剂形式，优选胶囊或片剂。
在本发明的一些方案中，所述其他治疗乙型肝炎药物的药物组合物包括其他治疗乙型肝炎药物、和药学上可接受的载体和/或赋形剂。
在本发明的一些方案中，所述其他治疗乙型肝炎药物的药物组合物呈液体制剂形式，优选水溶性注射液，所述水溶性注射液包括但不限于未经冻干的水溶性制剂或冻干粉重构的水溶性制剂。
施用方式
下述内容并非限制本发明组合的施用方式。
本发明的组合中的组分可以各自分开配制成药物组合物，或者其中的部分或全部共同配制成药物组合物。在一些方案中，本发明的组合可以配制成适合于单次或多次施用的药物组合物。
本发明的组合中的组分可以各自单独施用，或者其中的部分或全部共同施用。本发明的组合中的组分可以基本上不同时施用，或者其中的部分或全部基本上同时施用。本发明的组合中的组分可以具有相同或不同的施用周期。
本发明的组合中的组分可以各自独立地以适合的各种途径施用，包括，但不限于，口服或肠胃外 (通过静脉内、肌肉内、皮下、腹膜内、脊柱或其他胃肠外施用途径，例如通过注射或输注)。在一些方案中，本发明的组合的组分可以各自独立地口服施用或注射施用，例如静脉注射或腹腔注射。
本发明的组合中的组分可以各自独立地是适合的剂型，包括，但不限于，片剂、含片、丸剂、胶囊剂(例如硬胶囊、软胶囊、肠溶胶囊、微囊剂)、酏剂、颗粒剂、糖浆剂、注射剂(肌肉内、静脉内、腹腔内)、颗粒剂、乳剂、悬浮液、溶液、分散剂和用于口服或非口服给药的缓释制剂的剂型。
本发明的组合中的组分可以各自独立地含有药学上可接受的载体和/或赋形剂。
技术效果
本发明的式(I)化合物为乙肝表面抗原抑制剂，能够有效降低HBsAg含量，对HBV抑制作用显著。通过式(I)化合物与所述免疫调节剂组合使用，或通过式(I)化合物与核苷类或核苷酸类逆转录酶抑制剂的组合使用，或者通过这三类机制的药物的组合使用，能够增强对HBsAg的作用效果，起到协同抑制作用，同时多通道抑制HBV病毒，达到提高疗效、减少毒副作用、缩短给药治疗周期和剂量、减少耐药性等目的，可达到HBV载量降低，HBsAg减少甚至清除的效果。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机氨或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐(参见Berge et al.，″Pharmaceutical Salts″，Journal of Pharmaceutical Science 66：1-19(1977))。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
优选地，以常规方式使盐与碱或酸接触，再分离母体化合物，由此再生化合物的中性形式。化合物的母体形式与其各种盐的形式的不同之处在于某些物理性质，例如在极性溶剂中的溶解度不同。
本文所用的“药学上可接受的盐”属于本发明化合物的衍生物，其中，通过与酸成盐或与碱成盐的 方式修饰所述母体化合物。药学上可接受的盐的实例包括但不限于：碱基比如胺的无机酸或有机酸盐、酸根比如羧酸的碱金属或有机盐等等。药学上可接受的盐包括常规的无毒性的盐或母体化合物的季铵盐，例如无毒的无机酸或有机酸所形成的盐。常规的无毒性的盐包括但不限于那些衍生自无机酸和有机酸的盐，所述的无机酸或有机酸选自2-乙酰氧基苯甲酸、2-羟基乙磺酸、乙酸、抗坏血酸、苯磺酸、苯甲酸、碳酸氢根、碳酸、柠檬酸、依地酸、乙烷二磺酸、乙烷磺酸、富马酸、葡庚糖、葡糖酸、谷氨酸、乙醇酸、氢溴酸、盐酸、氢碘酸盐、羟基、羟萘、羟乙磺酸、乳酸、乳糖、十二烷基磺酸、马来酸、苹果酸、扁桃酸、甲烷磺酸、硝酸、草酸、双羟萘酸、泛酸、苯乙酸、磷酸、多聚半乳糖醛、丙酸、水杨酸、硬脂酸、亚乙酸、琥珀酸、氨基磺酸、对氨基苯磺酸、硫酸、单宁、酒石酸和对甲苯磺酸。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。一般地，优选醚、乙酸乙酯、乙醇、异丙醇或乙腈等非水介质。
除了盐的形式，本发明所提供的化合物还存在前药形式。本文所描述的化合物的前药容易地在生理条件下发生化学变化从而转化成本发明的化合物。此外，前体药物可以在体内环境中通过化学或生化方法被转换到本发明的化合物。
本发明的某些化合物可以以非溶剂化形式或者溶剂化形式存在，包括水合物形式。一般而言，溶剂化形式与非溶剂化的形式相当，都包含在本发明的范围之内。
本发明的某些化合物可以具有不对称碳原子(光学中心)或双键。外消旋体、非对映异构体、几何异构体和单个的异构体都包括在本发明的范围之内。
除非另有说明，用楔形键和虚线键
Figure PCTCN2021093805-appb-000030
表示一个立体中心的绝对构型，用
Figure PCTCN2021093805-appb-000031
表示一个立体中心的相对构型。当本文所述化合物含有烯属双键或其它几何不对称中心，除非另有规定，它们包括E、Z几何异构体。同样地，所有的互变异构形式均包括在本发明的范围之内。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固 定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
术语“药学上可接受的载体”是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂或载体介质代表性的载体包括水、油、蔬菜和矿物质、膏基、洗剂基质、软膏基质等。这些基质包括悬浮剂、增粘剂、透皮促进剂等。它们的制剂为化妆品领域或局部药物领域的技术人员所周知。关于载体的其他信息，可以参考Remington：The Science and Practice of Pharmacy，21 st Ed.，Lippincott，Williams&Wilkins(2005)，该文献的内容通过引用的方式并入本文。
针对药物或药理学活性剂而言，术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。对于本发明中的口服剂型，组合物中一种活性物质的“有效量”是指与该组合物中另一种活性物质联用时为了达到预期效果所需要的用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
术语“活性成分”、“治疗剂”，“活性物质”或“活性剂”是指一种化学实体，它可以有效地治疗目标紊乱、疾病或病症。
“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为酮基(即＝O)时，意味着两个氢原子被取代。酮取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR) 0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。当一个取代基的键可以交叉连接到一个环上的两个原子时，这种取代基可以与这个环上的任意原子相键合。当所列举的取代基中没有指明其通过哪一个原子连接到化学结构通式中包括但未具体提及的化合物时，这种取代基可以通过其任何原子相键合。取代基和/或其变体的组合只有在这样的组 合会产生稳定的化合物的情况下才是被允许的。例如，结构单元
Figure PCTCN2021093805-appb-000032
表示其可在环己基或者环己二烯上的任意一个位置发生取代。
除非另有规定，术语“杂”表示杂原子或杂原子团(即含有杂原子的原子团)，包括碳(C)和氢(H)以外的原子以及含有这些杂原子的原子团，例如包括氧(O)、氮(N)、硫(S)、硅(Si)、锗(Ge)、铝(Al)、硼(B)、-O-、-S-、＝O、＝S、-C(＝O)O-、-C(＝O)-、-C(＝S)-、-S(＝O)、-S(＝O) 2-，以及任选被取代的-C(＝O)N(H)-、-N(H)-、-C(＝NH)-、-S(＝O) 2N(H)-或-S(＝O)N(H)-。
除非另有规定，“环”表示被取代或未被取代的环烷基、杂环烷基、环烯基、杂环烯基、环炔基、杂环炔基、芳基或杂芳基。所谓的环包括单环、联环、螺环、并环或桥环。环上原子的数目通常被定义为环的元数，例如，“5～7元环”是指环绕排列5～7个原子。除非另有规定，该环任选地包含1～3个杂原子。因此，“5～7元环”包括例如苯基、吡啶和哌啶基；另一方面，术语“5～7元杂环烷基环”包括吡啶基和哌啶基，但不包括苯基。术语“环”还包括含有至少一个环的环系，其中的每一个“环”均独立地符合上述定义。
除非另有规定，术语“杂环”或“杂环基”意指稳定的含杂原子或杂原子团的单环、双环或三环，它们可以是饱和的、部分不饱和的或不饱和的(芳族的)，它们包含碳原子和1、2、3或4个独立地选自N、O和S的环杂原子，其中上述任意杂环可以稠合到一个苯环上形成双环。氮和硫杂原子可任选被氧化(即NO和S(O)p，p是1或2)。氮原子可以是被取代的或未取代的(即N或NR，其中R是H或本文已经定义过的其他取代基)。该杂环可以附着到任何杂原子或碳原子的侧基上从而形成稳定的结构。如果产生的化合物是稳定的，本文所述的杂环可以发生碳位或氮位上的取代。杂环中的氮原子任选地被季铵化。一个优选方案是，当杂环中S及O原子的总数超过1时，这些杂原子彼此不相邻。另一个优选方案是，杂环中S及O原子的总数不超过1。
如本文所用，术语“芳族杂环基团”或“杂芳基”意指稳定的5、6、7元单环或双环或7、8、9或10元双环杂环基的芳香环，它包含碳原子和1、2、3或4个独立地选自N、O和S的环杂原子。氮原子可以是被取代的或未取代的(即N或NR，其中R是H或本文已经定义过的其他取代基)。氮和硫杂原子可任选被氧化(即NO和S(O)p，p是1或2)。值得注意的是，芳香杂环上S和O原子的总数不超过1。桥环也包含在杂环的定义中。当一个或多个原子(即C、O、N或S)连接两个不相邻的碳原子或氮原子时形成桥环。优选的桥环包括但不限于：一个碳原子、两个碳原子、一个氮原子、两个氮原子和一个碳-氮基。值得注意的是，一个桥总是将单环转换成三环。桥环中，环上的取代基也可以出现在桥上。
杂环化合物的实例包括但不限于：吖啶基、吖辛因基、苯并咪唑基、苯并呋喃基、苯并巯基呋喃基、苯并巯基苯基、苯并恶唑基、苯并恶唑啉基、苯并噻唑基、苯并三唑基、苯并四唑基、苯并异恶唑基、苯并异噻唑基、苯并咪唑啉基、咔唑基、4aH-咔唑基、咔啉基、苯并二氢吡喃基、色烯、噌啉基十氢喹啉基、2H，6H-1，5，2-二噻嗪基、二氢呋喃并[2，3-b]四氢呋喃基、呋喃基、呋咱基、咪唑烷基、咪唑啉基、咪唑基、1H-吲唑基、吲哚烯基、二氢吲哚基、中氮茚基、吲哚基、3H-吲哚基、异苯并呋喃基、异吲哚基、异二氢吲哚基、异喹啉基、异噻唑基、异恶唑基、亚甲二氧基苯基、吗啉基、萘啶 基，八氢异喹啉基、恶二唑基、1，2，3-恶二唑基、1，2，4-恶二唑基、1，2，5-恶二唑基、1，3，4-恶二唑基、恶唑烷基、恶唑基、羟吲哚基、嘧啶基、菲啶基、菲咯啉基、吩嗪、吩噻嗪、苯并黄嘌呤基、酚恶嗪基、酞嗪基、哌嗪基、哌啶基、哌啶酮基、4-哌啶酮基、胡椒基、蝶啶基、嘌呤基、吡喃基、吡嗪基、吡唑烷基、吡唑啉基、吡唑基、哒嗪基、吡啶并恶唑、吡啶并咪唑、吡啶并噻唑、吡啶基、吡咯烷基、吡咯啉基、2H-吡咯基、吡咯基、喹唑啉基、喹啉基、4H-喹嗪基、喹喔啉基、奎宁环基、四氢呋喃基、四氢异喹啉基、四氢喹啉基、四唑基，6H-1，2，5-噻二嗪基、1，2，3-噻二唑基、1，2，4-噻二唑基、1，2，5-噻二唑基、1，3，4-噻二唑基、噻蒽基、噻唑基、异噻唑基噻吩基、噻吩并恶唑基、噻吩并噻唑基、噻吩并咪唑基、噻吩基、三嗪基、1，2，3-三唑基、1，2，4-三唑基、1，2，5-三唑基、1，3，4-三唑基和呫吨基。还包括稠环和螺环化合物。
除非另有规定，术语“烃基”或者其下位概念(比如烷基、烯基、炔基、芳基等等)本身或者作为另一取代基的一部分表示直链的、支链的或环状的烃原子团或其组合，可以是完全饱和的(如烷基)、单元或多元不饱和的(如烯基、炔基、芳基)，可以是单取代或多取代的，可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)，可以包括二价或多价原子团，具有指定数量的碳原子(如C 1-C 12表示1至12个碳，C 1-12选自C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11和C 12；C 3-12选自C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11和C 12。)。“烃基”包括但不限于脂肪烃基和芳香烃基，所述脂肪烃基包括链状和环状，具体包括但不限于烷基、烯基、炔基，所述芳香烃基包括但不限于6-12元的芳香烃基，例如苯、萘等。在一些实施例中，术语“烃基”表示直链的或支链的原子团或它们的组合，可以是完全饱和的、单元或多元不饱和的，可以包括二价和多价原子团。饱和烃原子团的实例包括但不限于甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、仲丁基、异丁基、环己基、(环己基)甲基、环丙基甲基，以及正戊基、正己基、正庚基、正辛基等原子团的同系物或异构体。不饱和烃基具有一个或多个双键或三键，其实例包括但不限于乙烯基、2-丙烯基、丁烯基、巴豆基、2-异戊烯基、2-(丁二烯基)、2，4-戊二烯基、3-(1，4-戊二烯基)、乙炔基、1-和3-丙炔基，3-丁炔基，以及更高级的同系物和异构体。
除非另有规定，术语“杂烃基”或者其下位概念(比如杂烷基、杂烯基、杂炔基、杂芳基等等)本身或者与另一术语联合表示稳定的直链的、支链的或环状的烃原子团或其组合，有一定数目的碳原子和至少一个杂原子组成。在一些实施例中，术语“杂烷基”本身或者与另一术语联合表示稳定的直链的、支链的烃原子团或其组合物，有一定数目的碳原子和至少一个杂原子组成。在一个典型实施例中，杂原子选自B、O、N和S，其中氮和硫原子任选地被氧化，氮杂原子任选地被季铵化。杂原子或杂原子团可以位于杂烃基的任何内部位置，包括该烃基附着于分子其余部分的位置，但术语“烷氧基”、“烷氨基”和“烷硫基”(或硫代烷氧基)属于惯用表达，是指分别通过一个氧原子、氨基或硫原子连接到分子的其余部分的那些烷基基团。实例包括但不限于-CH 2-CH 2-O-CH 3、-CH 2-CH 2-NH-CH 3、-CH 2-CH 2-N(CH 3)-CH 3、-CH 2-S-CH 2-CH 3、-CH 2-CH 2、-S(O)-CH 3、-CH 2-CH 2-S(O) 2-CH 3、-CH＝CH-O-CH 3、-CH 2-CH＝N-OCH 3和-CH＝CH-N(CH 3)-CH 3。至多两个杂原子可以是连续的，例如-CH 2-NH-OCH 3。
除非另有规定，术语“环烃基”、“杂环烃基”或者其下位概念(比如芳基、杂芳基、环烷基、杂环 烷基、环烯基、杂环烯基、环炔基、杂环炔基等等)本身或与其他术语联合分别表示环化的“烃基”、“杂烃基”。此外，就杂烃基或杂环烃基(比如杂烷基、杂环烷基)而言，杂原子可以占据该杂环附着于分子其余部分的位置。环烃基的实例包括但不限于环戊基、环己基、1-环己烯基、3-环己烯基、环庚基等。杂环基的非限制性实例包括1-(1，2，5，6-四氢吡啶基)、1-哌啶基、2-哌啶基，3-哌啶基、4-吗啉基、3-吗啉基、四氢呋喃-2-基、四氢呋喃吲哚-3-基、四氢噻吩-2-基、四氢噻吩-3-基，1-哌嗪基和2-哌嗪基。
除非另有规定，术语“烷基”用于表示直链或支链的饱和烃基，可以是单取代(如-CH 2F)或多取代的(如-CF 3)，可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。烷基的例子包括甲基(Me)，乙基(Et)，丙基(如，n-丙基和异丙基)，丁基(如，n-丁基，异丁基，s-丁基，t-丁基)，戊基(如，n-戊基，异戊基，新戊基)等。
除非另有规定，“炔基”指在链的任何位点上具有一个或多个碳碳三键的烷基，可以是单取代或多取代的，可以是一价、二价或者多价。炔基的例子包括乙炔基，丙炔基，丁炔基，戊炔基等。
除非另有规定，环烷基包括任何稳定的环状或多环烃基，任何碳原子都是饱和的，可以是单取代或多取代的，可以是一价、二价或者多价。这些环烷基的实例包括，但不限于，环丙基、降冰片烷基、[2.2.2]二环辛烷、[4.4.0]二环癸烷等。
除非另有规定，术语“卤代素”或“卤素”本身或作为另一取代基的一部分表示氟、氯、溴或碘原子。此外，术语“卤代烷基”意在包括单卤代烷基和多卤代烷基。例如，术语“卤代(C 1-C 4)烷基”意在包括但不仅限于三氟甲基、2，2，2-三氟乙基、4-氯丁基和3-溴丙基等等。除非另有规定，卤代烷基的实例包括但不仅限于：三氟甲基、三氯甲基、五氟乙基，和五氯乙基。
“烷氧基”代表通过氧桥连接的具有特定数目碳原子的上述烷基，除非另有规定，C 1-6烷氧基包括C 1、C 2、C 3、C 4、C 5和C 6的烷氧基。烷氧基的例子包括但不限于：甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、正戊氧基和S-戊氧基。
除非另有规定，术语“芳基”表示多不饱和的芳族烃取代基，可以是单取代或多取代的，可以是一价、二价或者多价，它可以是单环或多环(比如1至3个环；其中至少一个环是芳族的)，它们稠合在一起或共价连接。术语“杂芳基”是指含有一至四个杂原子的芳基(或环)。在一个示范性实例中，杂原子选自B、N、O和S，其中氮和硫原子任选地被氧化，氮原子任选地被季铵化。杂芳基可通过杂原子连接到分子的其余部分。芳基或杂芳基的非限制性实施例包括苯基、萘基、联苯基、吡咯基、吡唑基、咪唑基、吡嗪基、恶唑基、苯基-恶唑基、异恶唑基、噻唑基、呋喃基、噻吩基、吡啶基、嘧啶基、苯并噻唑基、嘌呤基、苯并咪唑基、吲哚基、异喹啉基、喹喔啉基、喹啉基、1-萘基、2-萘基、4-联苯基、1-吡咯基、2-吡咯基、3-吡咯基、3-吡唑基、2-咪唑基、4-咪唑基、吡嗪基、2-恶唑基、4-恶唑基、2-苯基-4-恶唑基、5-恶唑基、3-异恶唑基、4-异恶唑基、5-异恶唑基、2-噻唑基、4-噻唑基、5-噻唑基、2-呋喃基、3-呋喃基、2-噻吩基、3-噻吩基、2-吡啶基、3-吡啶基、4-吡啶基、2-嘧啶基、4-嘧啶基、5-苯并噻唑基、嘌呤基、2-苯并咪唑基、5-吲哚基、1-异喹啉基、5-异喹啉基、2-喹喔啉基、5-喹喔啉基、3-喹啉基和6-喹啉基。上述任意一个芳基和杂芳基环系的取代基选自下文所述的 可接受的取代基。
除非另有规定，芳基在与其他术语联合使用时(例如芳氧基、芳硫基、芳烷基)包括如上定义的芳基和杂芳基环。因此，术语“芳烷基”意在包括芳基附着于烷基的那些原子团(例如苄基、苯乙基、吡啶基甲基等)，包括其中碳原子(如亚甲基)已经被例如氧原子代替的那些烷基，例如苯氧基甲基、2-吡啶氧甲基3-(1-萘氧基)丙基等。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施.方式进行各种变化和改进将是显而易见的。
实施例1
实验目的：本研究的目的是应用从人源化FRG小鼠模型中分离得到的人原代肝细胞(PHH)来评价受试化合物与TAF(富马酸丙酚替诺福韦)、Peg-IFN-α2a(聚乙二醇干扰素α2a)联合给药的体外抗乙肝病毒药效。
实验材料：
(1)主要仪器：荧光qPCR仪(Applied Biosystems，型号7500)。酶标仪(Bio Tek，型号Synergy 2)。化学发光成像系统(GE，型号LAS4010)
(2)主要试剂和耗材：FastStart Universal Probe Master(Roche)，DNA提取试剂盒(Qiagen)，96孔板(Costar 3599)和96 well V型板(Axygen WIPP02280)，HBsAg ELISA试剂盒(Autobio)和HBeAg ELISA试剂盒(Autobio)，Cell Counting Kit-8(CCK-8)(Biolite)。
(3)其他：FRG小鼠、D型HBV、受试化合物WWS0
Figure PCTCN2021093805-appb-000033
实验方法：
1.应用PHH模型评价受试化合物单药的体外抗病毒活性
第0天，复苏人原代肝细胞，将细胞铺到2块48孔板中(1.32×10 5细胞/孔)。
第1天，加入HBV感染PHH(2000GE/细胞)。设置2种化合物处理方式：一块细胞板在第1天加入受试化合物和Peg-IFNα-2a处理四小时后，再更换含有HBV的培养基感染细胞(同时加入化合物继续处理)；另一块板第2天开始加入受试化合物和Peg-IFNα-2a进行处理。每1-2天更换带有化合物的新鲜培养基。
培养液中DMSO终浓度为2％。受试化合物起始浓度为1000nM，对照化合物Peg-IFNα-2a起始浓度为500IU/mL，5倍稀释，7个浓度点，三复孔(化合物浓度设置见表3)。
第4、6、8、10、12、14、16、18、20天更换含有化合物的新鲜培养液。第22天，收集培养上 清，并使用CellTiter-Glo检测细胞活力。
收集第8、14和22天的细胞培养上清将用于HBV DNA、HBeAg和HBsAg检测。
表1受试化合物单独用药浓度设置
Figure PCTCN2021093805-appb-000034
2.应用PHH模型评价受试化合物与TAF、Peg-IFNα-2a联合用药的体外抗HBV活性
第0天复苏人原代肝细胞，将细胞铺到6块48孔板中(1.32×10 5细胞/孔)。
第1天，加入HBV感染PHH(2000GE/细胞)。第2天加入不同组的联合药物进行处理：
(1)受试化合物与Peg-IFNα-2a联合用药：受试化合物和Peg-IFN-α2a均3倍稀释5个浓度点，以5×5的矩阵进行测试，三复板。受试化合物和Peg-IFNα-2a起始浓度分别为400nM和100IU/mL。联合作用化合物排布见表2，化合物测试浓度见表3。
细胞培养液中DMSO终浓度为2％。第4、6天更换含有化合物的培养基。第8天，收集培养上清，用于检测HBV DNA、HBeAg和HBsAg，待收集上清后加入CellTiter-Glo检测细胞活力。
表2受试化合物和Peg-IFNα-2a联合用药排布
Figure PCTCN2021093805-appb-000035
a＝受试化合物，b＝Peg-IFNα-2a；0表示无化合物，1-5表示5个浓度
表3受试化合物和Peg-IFNα-2a联合用药浓度设置
Figure PCTCN2021093805-appb-000036
(2)受试化合物与TAF和Peg-IFNα-2a联合用药：受试化合物和Peg-IFNα-2a均3倍稀释5个浓度点，以5×5的矩阵进行测试，三复板。受试化合物和Peg-IFNα-2a起始浓度分别为400nM和100IU/mL，TAF设置1个浓度点0.3nM。联合作用化合物排布见表4，化合物测试浓度见表5。
培养液中DMSO终浓度为2％。第4、6天用含有化合物的培养基换液。第8天，收集培养上清，用于HBV DNA、HBeAg和HBsAg检测，待收集细胞上清后加入CellTiter-Glo检测细胞活力。
表4受试化合物与TAF和Peg-IFNα-2a联合用药排布
Figure PCTCN2021093805-appb-000037
a＝受试化合物，b＝Peg-IFNα-2a，c＝TAF；0表示无化合物，1-5表示5个浓度，Uninf表示未感染对照组
表5受试化合物与TAF和Peg-IFNα-2a联合用药浓度设置
Figure PCTCN2021093805-appb-000038
3.样品检测
细胞活力检测：收集细胞培养上清后，将CellTiter-Glo用培养基1∶1稀释，加入细胞板中，室温放置10分钟，酶标仪检测发光值。
HBV DNA检测：根据DNA提取试剂盒(Qiagen-51162)提取上清中的DNA，使用qPCR法定量样品中的HBV DNA，qPCR反应体系见表6。HBV质粒DNA作为标准品，10倍系列稀释，标准品范围从1.0×10 7copies/μl至10copies/μl。PCR反应程序为：95℃加热10分钟，然后进入循环模式，95℃加热15秒，随后60℃，1分钟，共40个循环。依据标准曲线和各样品的Ct值来计算样品中的HBVDNA含量。
表6 qPCR反应体系
试剂 配置1个孔所需体积(μl)
定量快速启动通用探针试剂 5
正向引物(10μM) 0.4
反向引物(10μM) 0.4
探针(10μM) 0.2
AE溶剂 2
样品的DNA 2
HBeAg和HBsAg检测：根据ELISA试剂盒说明检测上清中的HBeAg和HBsAg水平。方法简述如下：取50μL的标准品，样品和对照品加入到检测板中，然后每孔加入50μL酶结合物，37℃孵育60分钟，用洗液洗板后吸干，然后加入50μL预混发光底物，室温避光孵育10分钟，最后酶标仪测定发光值。依据标准曲线和各样品的化学发光值来计算样品中的HBeAg和HBsAg含量。
4.数据处理及统计分析
细胞活性％＝(样品发光值-空白对照孔平均值)/(DMSO对照组发光值-空白对照孔平均值)×100
抑制率％＝(1-样品中的HBV DNA含量或HBsAg含量或HBeAg含量/DMSO对照组中的HBV DNA含量或HBsAg含量或HBeAg含量)×100
CC 50和EC 50通过GraphPad Prism软件计算，抑制曲线拟合方法为sigmoidal dose-response(variable slope)。
联合药效通过MacSynergy TM II软件分析。联合药效根据协同/拮抗作用到95％的置信区间计算，结果按照MacSynergy准则解释如下：
＜25＝Insignificant synergism/antagonism不显著的协同或拮抗作用(相加)；
25-50＝Minor but significant synergism/antagonism弱但显著的协同或拮抗作用；
50-100＝Moderate synergism/antagonism-may be important in vivo中等的协同或拮抗作用-在体内实验中也许很重要；
＞100＝Strong synergism/antagonism-probably important in vivo强的协同或拮抗作用-在体内实验中可能很重要；
～1000＝Possible errors-check data and repeat experiment可能错误-检查数据并重复实验；
5.实验结果：见表7和8。
表7单药在PHH中的抗HBV活性和细胞毒性(48孔培养板)
Figure PCTCN2021093805-appb-000039
表8受试化合物在PHH中的联合给药结果(95％的置信区间)
Figure PCTCN2021093805-appb-000040
6.实验结论：在PHH体外感染HBV模型中，受试化合物WWS0剂量依赖性抑制HBV DNA、HBeAg和HBsAg，且感染前和感染HBV后2种给药方式，WWS0抑制HBV复制活性基本一致。
受试化合物联合用药结果显示，WWS0与Peg-IFNα-2a联合用药对HBsAg的抑制具有中度协同作用，对HBeAg的抑制有相加作用。受试化合物WWS0与TAF、Peg-IFNα-2a三药联合对HBsAg的抑制具有中度协同作用，对HBeAg的抑制有相加作用。
受试化合物WWS0单药和联合作用实验中，在测试浓度范围内均未显示出明显的细胞毒性。
实施例2
本研究应用HepG2-NTCP细胞体外感染模型评价化合物WWS0与恩替卡韦(ETV)体外联合用药对HBV的抑制活性。通过实时荧光定量PCR(qPCR)方法检测细胞上清中的HBV DNA的含量，ELISA检测HBeAg和HBsAg的含量，测定化合物联合作用的抗HBV活性，同时，CellTiter-Glo检测受试化合物对细胞活力的影响。
1.实验材料：
细胞株：HepG2-NTCP细胞株由药明康德构建并提供。HepG2-NTCP细胞是HepG2细胞稳定转染并持续高表达人NTCP基因，易于被HBV感染。细胞培养基为DMEM培养基添加10％胎牛血清，1％Penicillin-Streptomycin，1％谷氨酰胺，1％非必须氨基酸和G418(500μg/mL)。
病毒：D型HBV从HepG2.2.15细胞培养上清液中浓缩。
试剂：本研究使用的主要试剂包括FastStart Universal Probe Master(Roche)，DNA提取试剂盒(Qiagen)，96孔板(Costar)，HBsAg ELISA试剂盒(Autobio)和HBeAg ELISA试剂盒(Autobio)。
仪器：本研究使用的主要仪器为实时荧光定量PCR仪(Thermo，QuantStudio TM 6 Flex)和多功能酶标仪(BioTek，Synergy2)。
受试化合物：WWS0和ETV。
Figure PCTCN2021093805-appb-000041
ETV为已经上市的治疗HBV的药物。
2.实验方法：
2.1 第0天，将HepG2-NTCP细胞以75，000个细胞/孔的密度种入48孔细胞培养板，然后将细胞在5％CO 2、37℃条件下培养过夜。
2.2 第2天，加入HBV感染HepG2-NTCP细胞。
2.3 第3天，将WWS0、ETV单药分别以7个不同浓度给药，或联合分别以5个不同浓度进行正交配比，加入48孔板中，每个组合为3复板。
表9受试化合物单独用药浓度设置
Figure PCTCN2021093805-appb-000042
表10 WWS0和ETV联合用药排布
Figure PCTCN2021093805-appb-000043
a＝WWS0，b＝ETV；0表示无化合物，1-5表示5个浓度
表11 WWS0和ETV联合用药浓度设置
Figure PCTCN2021093805-appb-000044
2.4 第6天，弃掉细胞上清液，加入含有化合物的新鲜培养基，细胞在5％CO2、37℃条件下培养3天。
2.5 第9天，收集化合物处理后的细胞板上清液，按照QIAamp 96 DNA Blood Kit(12)说明书抽提DNA。按照ELISA检测试剂盒说明检测HBeAg和HBsAg，同时，用CellTiter-Glo试剂盒检测化合物对的细胞毒性，按试剂盒说明书方法，用多功能酶标仪检测各细胞孔的化学发光强度(RLU)。用qPCR方法定量HBV DNA。以HBV质粒DNA为标准品，标准品HBV质粒DNA浓度从10 7copies/μL起始10倍梯度稀释7个点。利用各标准品的HBVDNA拷贝数和CT值拟合标准曲线，计算各检测样本中的HBVDNA拷贝数。
2.6 应用MacSynegy软件(Prichard et al.，1990)对试验数据进行处理，分析化合物联合用药的效果参数。药物联合指数说明：
指数为正值则为协同作用，负值为拮抗作用；指数绝对值＜25，即相加作用；指数绝对值在25-50范围，即轻度但明确的协同或拮抗作用；指数绝对值在50-100范围，即中度协同或拮抗作用，可能对体内作用有重要意义。指数绝对值在＞100范围，即高度协同或拮抗作用，很可能对体内作用有重要意义。
3.实验结果：见表12和表13。
表12单药在抗HBV活性和细胞毒性
Figure PCTCN2021093805-appb-000045
表13 WWS0和ETV的联合给药结果(95％的置信区间)
指标 联合 协同指数 拮抗指数 细胞毒性 结果
HBeAg WWS0+ETV 33.82 -6.03 无 轻度协同
HBsAg WWS0+ETV 136.04 0 无 强协同
4.实验结论：在HepG2-NTCP体外感染HBV模型中，受试化合物WWS0剂量依赖性抑制HBV DNA、HBeAg和HBsAg；WWS0与ETV联合显示HBeAg和HBsAg有协同或叠加抗HBV活性；受试化合物WWS0单药和联合作用实验中，在测试浓度范围内均未显示出明显的细胞毒性。
实施例3
根据实施例1的方法，将受试化合物WWS0
Figure PCTCN2021093805-appb-000046
替换为化 合物WWS01
Figure PCTCN2021093805-appb-000047
WWS02
Figure PCTCN2021093805-appb-000048
WWS03
Figure PCTCN2021093805-appb-000049
得到受试化合物WWS01、WWS02、WWS03与Peg-IFNα-2a两药联用对HBsAg和HBeAg的抑制具有协同或相加作用，在95％置信区间协同指数在20.00～50.00范围内，拮抗指数为0。
实施例4
根据实施例2的方法，将ETV(恩替卡韦)替换为TDF(富马酸替诺福韦二吡呋酯)，得到受试化合物WWS0在HepG2-NTCP体外感染HBV模型中的联合给药结果：WWS0与TDF联用对HBsAg和HBeAg的抑制具有协同作用，在95％置信区间协同指数分别为86.55、25.49，拮抗指数为0、1.06。
实施例5
根据实施例1的方法，将干扰素Peg-IFNα-2a替换为IFNα-2b，得到受试化合物WWS0与IFNα-2b两药联用以及WWS0与TAF、IFNα-2b三药联用结果，结果显示两组联用对HBsAg和HBeAg的协同指数均大于25，对HBsAg和HBeAg的抑制具协同作用。

Claims (32)

  1. 一种组合，包括式(I)所示化合物或其药学上可接受的盐，
    Figure PCTCN2021093805-appb-100001
    其中，
    R 1选自H、OH、CN、NH 2、C 1-5烷基、C 1-5杂烷基、C 2-5炔基、C 3-6环烷基和3～6元杂环烷基，所述C 1-5烷基、C 1-5杂烷基、C 2-5炔基、C 3-6环烷基和3～6元杂环烷基任选被1、2或3个R取代；
    R 2选自H、卤素、C 1-3烷基和C 1-3杂烷基，所述C 1-3烷基和C 1-3杂烷基任选被1、2或3个R取代；
    m选自0、1、2、3、4和5；
    A选自苯基和5～6元杂芳基，所述苯基和5～6元杂芳基任选被1、2或3个R取代；
    R选自H、卤素、OH、CN、NH 2、＝O、CH 3、CH 3CH 2、CH 3O、CF 3、CHF 2、CH 2F；
    所述C 1-5杂烷基、3～6元杂环烷基、C 1-3杂烷基和5～6元杂芳基分别独立地包含1、2或3个分别独立地选自N、-O-、＝O、-S-、-NH-、-(C＝O)-、-(S＝O)-和-(S＝O)2-的杂原子或杂原子团，
    其特征在于，还包括免疫调节剂。
  2. 根据权利要求1所述的组合，其特征在于，所述免疫调节剂选自干扰素，优选聚乙二醇干扰素α-2a或干扰素α-2b。
  3. 根据权利要求1所述的组合，其特征在于，还包括核苷酸类逆转录酶抑制剂。
  4. 根据权利要求3所述的组合，其特征在于，所述核苷酸类逆转录酶抑制剂选自富马酸替诺福韦二吡呋酯和富马酸丙酚替诺福韦。
  5. 根据权利要求4所述的组合，其特征在于，所述免疫调节剂选自干扰素，优选聚乙二醇干扰素α-2a或干扰素α-2b。
  6. 一种组合，包括如式(I)所述化合物或其药学上可接受的盐，其特征在于，还包括核苷酸类逆转录酶抑制剂或核苷类逆转录酶抑制剂。
  7. 根据权利要求6所述的组合，其特征在于，所述核苷酸类逆转录酶抑制剂选自富马酸替诺福韦二吡呋酯和富马酸丙酚替诺福韦。
  8. 根据权利要求6所述的组合，其特征在于，所述核苷类逆转录酶抑制剂选自恩替卡韦。
  9. 根据权利要求1～8任意一项所述的组合，其中，R选自H、F、Cl、Br、OH、CH 3、CH 3O、CF 3、CHF 2、CH 2F。
  10. 根据权利要求1～8任意一项所述的组合，其中，R 1选自H、OH、CN、NH 2、CH 3、CH 3CH 2、CH 3CH 2CH 2、CH 3CH 2CH 2CH 2、CH 3O、CH 3CH 2O、CH 3S、CH 3S(＝O)、CH 3S(＝O) 2、CH 3SCH 2、CH 3CH 2S、 CH 3NH、
    Figure PCTCN2021093805-appb-100002
    吡咯烷基、哌啶基、四氢吡喃基、吗啉基、2-吡咯烷酮基和3-吡咯烷酮基，所述CH 3、CH 3CH 2、CH 3CH 2CH 2、CH 3CH 2CH 2CH 2、CH 3O、CH 3CH 2O、CH 3S、CH 3S(＝O)、CH 3S(＝O) 2、CH 3SCH 2、CH 3CH 2S、CH 3NH、
    Figure PCTCN2021093805-appb-100003
    吡咯烷基、哌啶基、四氢吡喃基、吗啉基、2-吡咯烷酮基和3-吡咯烷酮基任选被1、2或3个R取代。
  11. 根据权利要求10所述的组合，其中，R 1选自H、OH、CN、NH 2、CH 3、CH 3CH 2、CH 3CH 2CH 2、CH 3CH 2CH 2CH 2、CH 3O、CH 3CH 2O、CH 3S、CH 3S(＝O)、CH 3S(＝O) 2、CH 3SCH 2、CH 3CH 2S、CH 3NH、
    Figure PCTCN2021093805-appb-100004
    Figure PCTCN2021093805-appb-100005
    所述CH 3、CH 3CH 2、CH 3CH 2CH 2、CH 3CH 2CH 2CH 2、CH 3O、CH 3CH 2O、CH 3S、CH 3S(＝O)、CH 3S(＝O) 2、CH 3SCH 2、CH 3CH 2S、CH 3NH、
    Figure PCTCN2021093805-appb-100006
    Figure PCTCN2021093805-appb-100007
    任选被1、2或3个R取代。
  12. 根据权利要求11所述的组合，其中，R 1选自H、OH、CH 3、CHF 2、CH 3O、
    Figure PCTCN2021093805-appb-100008
    Figure PCTCN2021093805-appb-100009
  13. 根据权利要求1～8任意一项所述的组合，其中，R 2选自H、F、Cl、Br、CH 3、CH 3CH 2、CH 3O、CH 3CH 2O和
    Figure PCTCN2021093805-appb-100010
    所述CH 3、CH 3CH 2、CH 3O、CH 3CH 2O和
    Figure PCTCN2021093805-appb-100011
    任选被1、2或3个R取代。
  14. 根据权利要求13所述的组合，其中，R 2选自Cl和CH 3O。
  15. 根据权利要求1～8任意一项所述的组合，其中，A选自任选被1、2或3个R取代的：苯基、噻吩基、噻唑基、异噻唑基、恶唑基和异恶唑基。
  16. 根据权利要求15所述的组合，其中，A选自任选被1、2或3个R取代的：
    Figure PCTCN2021093805-appb-100012
    Figure PCTCN2021093805-appb-100013
  17. 根据权利要求16所述的组合，其中，A选自
    Figure PCTCN2021093805-appb-100014
    Figure PCTCN2021093805-appb-100015
  18. 根据权利要求17所述的组合，其中，A选自
    Figure PCTCN2021093805-appb-100016
    Figure PCTCN2021093805-appb-100017
  19. 根据权利要求1～8任意一项所述的组合，其中，m为3。
  20. 根据权利要求19所述的组合，其中，R 2选自Cl和CH 3O。
  21. 根据权利要求20所述的组合，其中，R 1为CH 3O。
  22. 根据权利要求1～8任意一项所述的组合，其中，m为1。
  23. 根据权利要求22所述的组合，其中，R 2为Cl。
  24. 根据权利要求23所述的组合，其中，R 1为
    Figure PCTCN2021093805-appb-100018
  25. 根据权利要求21或24所述的组合，其中，A选自任选被1、2或3个R取代的：
    Figure PCTCN2021093805-appb-100019
    Figure PCTCN2021093805-appb-100020
  26. 根据权利要求1～8任意一项所述的组合，其中，化合物选自：
    Figure PCTCN2021093805-appb-100021
    其中，
    R 1、R 2、R、m如权利要求1～8任意一项所定义。
  27. 根据权利要求1～8任意一项所述的组合，其中，化合物选自：
    Figure PCTCN2021093805-appb-100022
    Figure PCTCN2021093805-appb-100023
    Figure PCTCN2021093805-appb-100024
    Figure PCTCN2021093805-appb-100025
    Figure PCTCN2021093805-appb-100026
  28. 根据权利要求27所述的组合，其中，化合物选自：
    Figure PCTCN2021093805-appb-100027
    Figure PCTCN2021093805-appb-100028
  29. 根据权利要求1～28任意一项所述的组合在制备治疗乙型肝炎药物中的用途。
  30. 一种组合物，其包括如权利要求1～28任意一项所述的组合和至少一种药学上可接受的载体和/或赋形剂。
  31. 一种试剂盒，其包括如权利要求1～28任意一项所述的组合或者如权利要求29所述的组合物。
  32. 根据权利要求30所述的组合物或根据权利要求31所述的试剂盒在制备治疗乙型肝炎药物中的用途。
PCT/CN2021/093805 2020-05-15 2021-05-14 用于治疗乙肝的组合 WO2021228222A1 (zh)

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