Pharmaceutical composition containing TLR7 / 8 selective agonist and HBV capsid assembly inhibitor and application thereof

Abstract

The invention belongs to the field of pharmaceutical compositions, and particularly relates to a pharmaceutical composition containing a TLR7 / 8 selective agonist and an HBV capsid assembly inhibitor and application of the pharmaceutical composition. The invention provides a pharmaceutical composition containing a TLR7 / 8 selective agonist and an HBV (Hepatitis B Virus) capsid assembly inhibitor. The pharmaceutical composition comprises the TLR7 / 8 selective agonist, the HBV capsid assembly inhibitor and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used for preparing drugs for treating or preventing virus infection, and preferably, the virus is hepatitis B virus (HBV).

Description

[0001] The present invention relates to a composition and method for treating hepatitis B virus infection. Specifically, the present invention relates to a combination therapy comprising administering a TLR7 / 8 selective agonist compound of formula (III) or a compound of formula (IV) or a compound of formula (V) or a compound of formula (VI) disclosed in WO2022063278 or WO2022156678 or WO2023104165 or PCT / CN2023 / 082607 and a HBV capsid assembly regulator compound of formula (I) or a compound of formula (II) disclosed in WO2022121844 or WO2022171072, which is used to treat chronic hepatitis B patients. Field of the Invention

[0002] Hepatitis B (HBV) is an infectious disease caused by the hepatitis B virus (HBV), characterized by inflammation and necrosis of the liver. It is highly contagious, has complex transmission routes, is widely prevalent, and has a high incidence rate. Some patients with hepatitis B may develop chronic hepatitis B (CHB) and may develop into cirrhosis and primary hepatocellular carcinoma.

[0004] HBV is a double-stranded DNA virus belonging to the Hepadnaviridae family. HBV has an icosahedral nucleocapsid, which is composed of 240 capsid (or core) proteins. The main biological function of the capsid protein is to act as a structural protein to encapsidate the pregenomic RNA and form immature capsid particles in the cytoplasm, which is a prerequisite for viral DNA replication.

[0005] A large number of studies have shown that TLR7 and TLR8 play an important role in host innate immunity and adaptive immunity, and can be used as effective targets for antiviral therapy and tumor therapy. Toll-like receptors are a class of structurally conserved pattern recognition receptors (PRRs), which are an important part of the innate immune system. They mainly recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). TLRs can activate downstream signaling pathways and promote the production of inflammatory cytokines by recognizing endogenous molecules released after invasive microorganisms and tissue damage or non-physiological cell death. There are 10 known members of the TLRs family in the human body (TLR1-10). TLR1, 2, 4, 5, 6, and 10 are located on the cell membrane and can bind to ligands on the cell surface; TLR3, 7, 8, and 9 are located in endosomes and mainly bind to nucleic acid receptors. Among them, TLR7 and 8 can recognize single-stranded RNA viruses rich in GU and AU sequences. They are considered to be pattern recognition receptors for many ssRNA viruses, such as influenza virus, HIV-1, vesicular stomatitis virus, Sendai virus, flavivirus, and coronavirus. The role of different TLRs in immune response varies depending on the viruses, cell types, and infection models they recognize. TLR7 is mainly expressed in B cells, monocytes, and plasmacytoid dendritic cells (pDCs), and TLR8 is mainly expressed in monocytes, neutrophils, and myeloid dendritic cells. Both TLR7 and 8 activate downstream inflammatory signaling molecules through Myd-88-dependent signaling pathways: TLR7 activation is more likely to produce type I interferon (IFN), such as IFN-α; TLR8 is more likely to activate NF-κB (nuclear factor-κB), thereby producing a variety of inflammatory cytokines, such as TNF-α, IFN-γ, etc. These cytokines can recruit or activate T cells, B cells, and NK cells in the disease microenvironment, enhance innate and adaptive immune responses, and produce antiviral and anti-tumor activities.

[0006] As for hepatitis B, the medical community still lacks a method to completely cure it. "Functional cure" has become the focus of current hepatitis B treatment, that is, after a limited course of antiviral drug treatment, the hepatitis B virus is limited to a very low level, so low that hepatitis B virus DNA and hepatitis B surface antigen (HBsAg) cannot be detected in the blood. Inducing HBsAg loss and / or serological conversion through a combination of different antiviral therapies is expected to increase the functional cure rate and has clear clinical treatment value. Summary of the invention

[0007] The present invention relates to a pharmaceutical composition comprising a TLR7 / 8 selective agonist and a HBV capsid assembly regulator in a pharmaceutically acceptable carrier.

[0008] In the first aspect of the present invention, a pharmaceutical composition is provided, characterized in that the pharmaceutical composition comprises a TLR7 / 8 selective agonist, a HBV capsid assembly inhibitor and a pharmaceutically acceptable carrier; preferably, in the pharmaceutical composition, the weight ratio of the TLR7 / 8 selective agonist to the HBV capsid assembly inhibitor is 1-20:1-10000.

[0009] In another preferred embodiment, the weight ratio of the TLR7 / 8 selective agonist to the HBV capsid assembly inhibitor is 1-20:1-8000, preferably 1-20:1-5000, and more preferably 1-20:1-4000.

[0010] In another preferred embodiment, the weight ratio of the TLR7 / 8 selective agonist to the HBV capsid assembly inhibitor is 1-20:1-3000, preferably 1-20:1-2000, and more preferably 1-20:1-1000.

[0011] In another preferred embodiment, the weight ratio of the TLR7 / 8 selective agonist to the HBV capsid assembly inhibitor is 1-20:1-800, preferably 1-20:1-600, and more preferably 1-20:1-500.

[0012] In another preferred embodiment, the weight ratio of the TLR7 / 8 selective agonist to the HBV capsid assembly inhibitor is 1-15:1-400, preferably 1-10:1-200, and more preferably 1-5:1-100.

[0013] In another preferred embodiment, the HBV capsid assembly inhibitor is a compound of formula (II), a solvate thereof or a pharmaceutically acceptable salt thereof:

[0014]

[0015] in,

[0016] W is O or S;

[0017] R 2 , R 3 and R 4 are independently H, chloro, fluoro, bromo, methyl, cyano, methoxy or trifluoromethyl;

[0018] R 5 C 1-3 Alkyl; preferably methyl or ethyl;

[0019] L 21 is a single bond or a C1-C3 alkylene group;

[0020] Q is a 5-8 membered bridged cycloalkyl or a 5-8 membered heterocycloalkyl; the 5-8 membered heterocycloalkyl is connected by a bridge ring;

[0021] R 10 COOH, CN, unsubstituted or replaced by one or more R A Substituted C1-C6 alkyl, NR B R C or

[0022] R A is hydroxyl, halogen, C1-C6 alkoxy, COOH, CN, C4-C6 heterocycloalkyl substituted by a carbonyl group,

[0023] R B and R C Independently for H, C1-C6 alkyl, or -C1-C6 alkyleneCOOH;

[0024] R D C4-C6 heterocycloalkyl, NH2-, -NHC1-C6 alkyl,

[0025] Unless otherwise specified, the ring skeleton of the heterocycloalkyl group includes 1, 2 or 3 identical or different heteroatoms selected from N, O and S;

[0026] The carbon atom with “*” indicates that when it is a chiral carbon atom, the carbon atom with “*” is in S configuration, R configuration or a mixture thereof.

[0027] In another preferred embodiment, the compound of formula (II) of the HBV capsid assembly inhibitor is selected from the following group:

[0028]

[0029]

[0030] or a solvate thereof or a pharmaceutically acceptable salt thereof;

[0031] Preferably, the compound of formula (II) of the HBV capsid assembly inhibitor is selected from the following group:

[0032]

[0033] or a solvate thereof or a pharmaceutically acceptable salt thereof;

[0034] Further preferably, the compound of formula (II) of the HBV capsid assembly inhibitor is or a solvate thereof or a pharmaceutically acceptable salt thereof.

[0035] In another preferred embodiment, the HBV capsid assembly inhibitor is a compound of formula (I), a solvate thereof or a pharmaceutically acceptable salt thereof:

[0036]

[0037] in,

[0038] R 2 , R 3 and R 4 each independently is H, chloro, fluoro, bromo, methyl, cyano, methoxy or trifluoromethyl;

[0039] R 5 C 1-3 Alkyl; preferably methyl or ethyl;

[0040] R 6 is H, halogen, hydroxyl or C 1-3 Alkyl; preferably hydrogen, methyl or fluorine;

[0041] R 7 H, halogen, C 1-3 Alkyl, hydroxy or methoxy;

[0042] R 13 is H, unsubstituted or substituted with one or more (e.g. 1, 2, 3 or 4) R 1b Substituted group selected from the group consisting of: phenyl-L 1 -, pyridyl-L 2 -, 3-7 membered monocyclic cycloalkyl-L 3 -、C 1-6 Alkyl-L 4 -; and when the R 1b When there are multiple substituents, each R 1b Same or different;

[0043] -L 1 -、-L 2 -、-L 3 -and-L 4 - are each independently a linking bond, unsubstituted or substituted with one or more R 8 Substituted C 1-3 Alkylene; wherein R 8 Independently for C1-3 Alkyl or 3-7 membered cycloalkyl;

[0044] R 1b are independently -CO2H, OH, halogen, CN, unsubstituted or substituted with one or more R 1c Substituted groups selected from the group consisting of: -CO2-C 1-3 Alkyl, -(C 1-6 Alkylene)-CO2H, -(C 1-6 Alkylene)-CO2-C 1-3 Alkyl, -(C 2-6 Alkenylene)-CO2H, -(C 2-6 Alkenylene)-CO2-C 1-3 Alkyl, C 1-3 Alkyl-O-, C 1-3 Alkyl-O-(C 1-6 Alkylene)-, C 1-6 Alkyl, C 1-3 Alkylsulfonyl, 3-7 membered cycloalkyl; wherein R 1c are independently halogen, C 1-4 Alkyl, 3-7 membered cycloalkyl;

[0045] The carbon atom with "*" indicates that when it is a chiral carbon atom, it is in S configuration, R configuration or a mixture thereof.

[0046] In another preferred embodiment, the compound of formula (I) of the HBV capsid assembly inhibitor is selected from the following group:

[0047]

[0048] or a solvate thereof or a pharmaceutically acceptable salt thereof;

[0049] Preferably, the compound of formula (I) of the HBV capsid assembly inhibitor is selected from the following group:

[0050]

[0051] or a solvate thereof or a pharmaceutically acceptable salt thereof.

[0052] In another preferred embodiment, the TLR7 / 8 selective agonist is a compound of formula (III) or formula (IV), a solvate thereof, or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof:

[0053]

[0054] in,

[0055] A is O, S, (S=O)2;

[0056] Y is N or CR Y ;

[0057] R Y is cyano, or C1-C4 alkyl substituted by halogen; preferably cyano or trifluoromethyl;

[0058] B is unsubstituted or replaced by R 1-1 Substituted C2~C 10 Alkylene, unsubstituted or replaced by R 1-1 Substituted C2~C 10 Unsaturated alkylene, or -(CH2) n -L 25 -(CH2) r -O-;

[0059] L 25 is an unsubstituted or halogen-substituted C3-C6 cycloalkylene group, an unsubstituted or halogen-substituted 3-6-membered heterocycloalkylene group; n and r are independently 1, 2 or 3;

[0060] R 1-1 is halogen, C1-C6 alkyl;

[0061] R 31 , R 32 and R 33 are independently H, halogen, unsubstituted or halogen-substituted C1-C6 alkyl, C1-C6 alkoxy;

[0062] Or, R 31 and R 32 , or R 32 and R 33 Each independently forms with the carbon atom to which they are attached a group selected from the group consisting of: unsubstituted or substituted with R 1-4 substituted 4-7 membered heterocycloalkylene, unsubstituted or replaced by R 1-5 Substituted C4-C7 cycloalkylene; wherein one or more arbitrary methylene groups in the heterocycloalkylene or cycloalkylene are independently replaced by a carbonyl group or S(=O)2;

[0063] R 1-4 and R 1-5 Each independently selected from the group consisting of OH, halogen, CN, unsubstituted or replaced by R 1-6 Substituted C1-C6 alkyl, unsubstituted or R 1-9 Substituted C1-C6 alkoxy, -S(=O)2R 1-7 、-C(=O)R 1-8 NR 1-10 R 1-11 、COOR1-12 , SR 1-13 , unsubstituted or replaced by R 1-19 Substituted C3-C7 cycloalkyl, unsubstituted or R 1-20 substituted 4-7 membered heterocycloalkyl, unsubstituted or replaced by R 1 -21 substituted 5-10 membered heteroaryl, or -G(CR 1-14 R 1-15 ) u -COOR 1-16 ; wherein, G is O, S, S(=O)2 or NH; u is 1, 2 or 3;

[0064] R 1-6 and R 1-9 Each independently selected from the group consisting of halogen, amino, CN, OH, -COOR 1-17 、-S(=O)2R 1-31 , -C(=O)NH2, -S(=O)2NH2, C1-C3 alkoxy, unsubstituted or substituted with COOR 1-18 Substituted C3-C7 cycloalkyl, unsubstituted or R 1 -22 substituted 4-7 membered heterocycloalkyl, unsubstituted or replaced by R 1-23 substituted 5-10 membered heteroaryl;

[0065] R 1-7 and R 1-8 Independently selected from the following groups: C1-C3 alkyl, unsubstituted or replaced by R 1-24 Substituted C3-C7 cycloalkyl, unsubstituted or R 1-25 substituted 4-7 membered heterocycloalkyl, unsubstituted or replaced by R 1-26 substituted 5-10 membered heteroaryl, unsubstituted or R 1-27 Substituted C6~C 10 Aryl or -NR 1-28 R 1-29 ;

[0066] R 1-10 , R 1-11 , R 1-12 , R 1-16 , R 1-17 , R 1-18 , R 1-28 and R 1-29 are independently H or C1-C3 alkyl; R 1-31 is a C1-C3 alkyl group;

[0067] R 1-13 is H, unsubstituted or halogen-substituted C1-C6 alkyl;

[0068] R1-14 and R 1-15 are independently H, unsubstituted or halogen-substituted C1-C6 alkyl;

[0069] R 1-19 , R 1-20 , R 1-21 , R 1-22 , R 1-23 and R 1-24 are independently OH, halogen, amino, CN, C1-C6 alkyl;

[0070] Unless otherwise specified, the ring skeleton of the heterocycloalkylene or heterocycloalkyl group includes 1, 2 or 3 identical or different heteroatoms selected from N, O and S; the ring skeleton of the heteroaryl group includes 1, 2, 3 or 4 identical or different heteroatoms selected from N, O and S; and when R 1-1 , R 1-4 , R 1-5 , R 1-6 , R 1-9 , R 1-19 , R 1-20 , R 1-21 , R 1-22 , R 1-23 , R 1-24 , R 1-25 , R 1-26 and R 1-27 When substituted, each of the above substituents may be one or more.

[0071] In another preferred embodiment, the compound of formula (III) or formula (IV) of the TLR7 / 8 selective agonist is selected from the following group:

[0072]

[0073]

[0074] a solvate thereof or a pharmaceutically acceptable salt thereof; Preferably, the compound of formula (III) or formula (IV) of the TLR7 / 8 selective agonist is selected from the following group:

[0075] or a solvate thereof or a pharmaceutically acceptable salt thereof;

[0076] More preferably, the compound of formula (III) or formula (IV) of the TLR7 / 8 selective agonist is or a solvate thereof or a pharmaceutically acceptable salt thereof.

[0077] In another preferred embodiment, the TLR7 / 8 selective agonist is a compound of formula (V) or formula (VI), a solvate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof:

[0078]

[0079] L 51 Selected from the group consisting of -O-, -NH-, -S-, -S(=O)- or -S(=O)2-; preferably -O- or -NH-;

[0080] R 51 Selected from the following group: H, unsubstituted or hydroxy-substituted C 1-12 Alkyl, C 1-12 Alkoxy, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl or 4-12 membered heterocycloalkyl; wherein said R 51 can be further represented by one or more R a Substituents, and the R a Selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, -NR a1 R a2 、-NHC(=O)-R a3 , one or more R a4 Substituted 5-6 membered heteroaryl, -OC(=O)R a5 、-C(=O)R a5 、-OC(=O)OR a5 、-C(=O)OR a5 ;

[0081] The R a1 , R a2 , R a3 or R a4 Select from the following group: C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl;

[0082] The R a5 Select from the following group: C 1-24 Alkyl, halogenated C 1-24 Alkyl, C 1-24 Heteroalkyl, wherein the heteroatom is selected from one or more of NH, N, O and S;

[0083] m is 0, 1 or 2;

[0084] B does not exist, or B is selected from the following group: C 3-12 Cycloalkyl, 4-12 membered heterocyclic group, C 6-12 Aryl, 5-12 membered heteroaryl or wherein each A is selected from C, CH or N, and R6 and R7 together with their adjacent carbon atoms form C 4-7 Cycloalkylene or C 4-7 Heterocycloalkylene, the C 4-7 One or more arbitrary methylene groups in the cycloalkylene or 4-7 membered heterocycloalkylene may be independently replaced by a carbonyl group or S(=O)2; the ring skeleton of the 4-7 membered heterocycloalkylene includes 1, 2 or 3 identical or different heteroatoms selected from N, O and S;

[0085] L 52 Selected from the following group: -(CR b R c ) p -(NR d ) q -、-O-、-S-、-(CR b R c ) p -C(=O)-、-(CR b R c ) p -C(=O)NH-, -(CR b R c ) p -NHC(=O)-, -S(=O)- or -S(=O)2-; wherein R b , R c Selected from the group consisting of hydrogen, halogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl or C 1-6 Haloalkyl, R d Selected from the group consisting of hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, C 1-6 C substituted with halogenated alkyl or hydroxyl 1-6 Alkyl, p is 0, 1, 2, 3, 4, 5 or 6, q is 0 or 1;

[0086] R 54 Selected from the group consisting of hydrogen, halogen, cyano, amino, hydroxyl, C 1-12 Alkyl, C 1-12 Alkoxy, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl, 4-12 membered heterocyclic group, C 6-12 Aryl or 5-12 membered heteroaryl; and said R 54Optionally, one or more R e Substituent substitution, where R e Selected from the group consisting of hydrogen, halogen, hydroxy, carboxylic acid, amino, C 1-6 Alkyl, one or more R e1 Substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-12 Cycloalkyl, 4-12 membered heterocycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, -N(R e2 R e3 R e4 )、-C(=O)OR e2 、-C(=O)NH-R e2 、-S(=O)2-R e2 ;

[0087] R e1 Selected from the group consisting of halogen, hydroxyl, -P(O)(OR e2 )2;

[0088] R e2 , R e3 Selected from the group consisting of hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 1-6 Haloalkyl, C substituted by hydroxy 1-6 alkyl;

[0089] R e4 Not present or selected from the following group: C 1-6 Alkyl, C 3-6 Cycloalkyl or C 1-6 Haloalkyl;

[0090] R 55 Selected from the following group: halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl;

[0091] n is 0, 1, 2, 3 or 4;

[0092] Wherein, each of the heterocyclic groups may be saturated or partially unsaturated (but not having an aromatic structure), and the ring skeleton of the heterocyclic group includes 1, 2, 3 or 4 (preferably 1 or 2) identical or different heteroatoms selected from N, O and S; the ring skeleton of the heteroaryl group includes 1, 2 or 3 identical or different heteroatoms selected from N, O and S.

[0093] In another preferred embodiment, in the formula (V) or formula (VI) of the TLR7 / 8 selective agonist:

[0094] L 51 Selected from the group consisting of: -NH-;

[0095] R 51 Selected from the following group: unsubstituted or hydroxy-substituted C 1-12 alkyl;

[0096] m is 1;

[0097] B is selected from phenyl or pyridyl;

[0098] The remaining groups are as defined above.

[0099] In another preferred embodiment, the compound of formula (V) or formula (VI) of the TLR7 / 8 selective agonist is selected from the following group:

[0100]

[0101]

[0102]

[0103] a solvate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof;

[0104] Preferably, the compound of formula (V) or formula (VI) of the TLR7 / 8 selective agonist is selected from the following group:

[0105]

[0106]

[0107] or a solvate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof;

[0108] Further preferably, the compound of formula (V) or formula (VI) of the TLR7 / 8 selective agonist is A solvate thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof.

[0109] In another preferred embodiment, the pharmaceutical composition consists of a TLR7 / 8 selective agonist, a HBV capsid assembly inhibitor and a pharmaceutically acceptable carrier.

[0110] In another preferred embodiment, the composition further comprises one or more other antiviral drugs;

[0111] Preferably, the other antiviral drugs are selected from lamivudine, adefovir, tenofovir, telbivudine and entecavir.

[0112] In the second aspect of the present invention, a method for preparing the pharmaceutical composition as described in the first aspect of the present invention is provided, the method comprising the steps of: mixing a HBV capsid assembly inhibitor, a TLR7 / 8 selective agonist and a pharmaceutically acceptable carrier to form a pharmaceutical composition.

[0113] In a third aspect of the present invention, a drug combination (kit) is provided, the drug combination comprising a first therapeutic component and a second therapeutic component, and the first therapeutic component comprises: a TLR7 / 8 selective agonist, and a pharmaceutically acceptable carrier;

[0114] The second therapeutic component includes: an HBV capsid assembly inhibitor, and a pharmaceutically acceptable carrier.

[0115] In the fourth aspect of the present invention, there is provided a use of the pharmaceutical composition as described in the first aspect of the present invention, or the pharmaceutical combination as described in the third aspect of the present invention, for preparing a drug for treating or preventing viral infection; preferably, the virus is hepatitis B virus HBV.

[0116] In another preferred embodiment, the dosage form of the drug is a solution, injection, infusion, oral solution, tablet, capsule, powder, granule, ointment, gel, aerosol or spray.

[0117] In a fifth aspect of the present invention, a method for treating or preventing viral infection is provided, characterized in that the method comprises the steps of: administering a TLR7 / 8 selective agonist as a first therapeutic component and a HBV capsid assembly inhibitor as a second therapeutic component to a subject in need thereof;

[0118] In another preferred embodiment, the virus is hepatitis B virus HBV.

[0119] In another preferred embodiment, the TLR7 / 8 selective agonist and the HBV capsid assembly inhibitor are co-administered in the same formulation or in different formulations.

[0120] In another preferred embodiment, the TLR7 / 8 selective agonist and the HBV capsid assembly inhibitor are intended to be administered to an individual via the same route or different routes.

[0121] In another preferred embodiment, the TLR7 / 8 selective agonist and the HBV capsid assembly inhibitor are intended to be administered to an individual by parenteral or oral administration.

[0122] In another preferred embodiment, the administration is simultaneous administration or sequential administration.

[0123] In another preferred embodiment, the TLR7 / 8 selective agonist is a compound as shown in formula (III), formula (IV), formula (V) or formula (VI), or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

[0124] In another preferred embodiment, the HBV capsid assembly inhibitor is a compound represented by formula (I) or formula (II) or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

[0125] The sixth aspect of the present invention provides a method for treating or preventing hepatitis B virus infection, which comprises administering to an individual an effective first amount of a TLR7 / 8 selective agonist or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof; and a second amount of a HBV capsid assembly inhibitor or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof; or administering to an individual an effective first amount of a HBV capsid assembly inhibitor or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof; and a second amount of a TLR7 / 8 selective agonist or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1 HBsAg (A), HBeAg (B), and HBV DNA (C) levels in mouse serum in the AAV-HBV mouse model treated with a combination of a TLR7 / 8 agonist (Compound III-I-60) and a HBV capsid assembly regulator (Compound II-22).

[0127] Figure 2 HBsAg (A), HBeAg (B), and HBV DNA (C) levels in mouse serum in the AAV-HBV mouse model treated with a combination of a TLR7 / 8 agonist (Compound VI-162) and a HBV capsid assembly regulator (Compound II-22). DETAILED DESCRIPTION

[0128] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0129] the term

[0130] In the present invention, the halogen is F, Cl, Br or I.

[0131] In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.

[0132] In the present invention, the term "alkyl" refers to a straight or branched alkyl group, preferably having 1 to 6 carbon atoms. In the present invention, examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl, etc.; preferably ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl. The term "alkylene" refers to a divalent group formed by further losing a hydrogen atom from an alkyl group.

[0133] The term "heteroalkyl" refers to a group formed by replacing one or more carbon atoms (optionally including one or more H atoms carried therein) on an alkane chain with a heteroatom selected from the group consisting of NH, N, O and S. In particular, when NH replaces a carbon atom, and the heteroalkyl group may have a substituent, the hydrogen atom on the NH may also be substituted.

[0134] In the present invention, the term "alkoxy" refers to a linear or branched alkoxy group, preferably having 1 to 6 carbon atoms. In the present invention, examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, and the like.

[0135] In the present invention, the term "cycloalkyl" refers to a cyclic alkyl group, preferably having 3 to 12 (e.g., 3 to 7) carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. The terms "C5-C6 cycloalkyl" and "C3-C6 cycloalkyl" have similar meanings.

[0136] In the present invention, the term "aromatic ring" or "aryl group" has the same meaning, preferably "aryl group" is "C6-C 12 Aryl" or "C6-C 10 The term "C6-C 12 "Aryl" refers to an aromatic ring group having 6 to 12 carbon atoms without heteroatoms in the ring, such as phenyl, naphthyl, etc. The term "C6-C 10 "Aryl" has a similar meaning.

[0137] In the present invention, the term "aromatic heterocycle" or "heteroaryl" has the same meaning and refers to a heteroaromatic group containing one to multiple heteroatoms. The heteroatoms referred to here include oxygen, sulfur and nitrogen. For example, furanyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. The heteroaryl group can be optionally substituted or unsubstituted.

[0138] In the present invention, the term "carbocyclic group" refers to a saturated or unsaturated (non-aromatic ring, including monocyclic, cyclic, spirocyclic, bridged ring, etc.) cyclic group, whose ring skeleton structure only includes carbon atoms, preferably, the carbocyclic group is 3-12 members, such as 4-9 members, 5-6 members, etc. Typical examples include cyclopentyl, cyclohexyl, etc.

[0139] In the present invention, the term "heterocyclic group" refers to a saturated or unsaturated (non-aromatic ring, including monocyclic, cyclic, spirocyclic, bridged, etc.) cyclic group containing 1, 2 or 3 heteroatoms selected from oxygen, sulfur and nitrogen on the ring. Preferably, the heterocyclic group is 3-12 members, such as 4-9 members, 3-7 members, 5-6 members, etc. Typical examples include dioxolanyl, etc.

[0140] In the present invention, the term "substituted" refers to one or more hydrogen atoms on a specific group being replaced by a specific substituent. The specific substituent is a substituent described above or a substituent appearing in the embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable position of the group, and the substituent may be the same or different at each position. A cyclic substituent, such as a heterocycloalkyl, may be connected to another ring, such as a cycloalkyl, to form a spirobicyclic system, for example, the two rings have a common carbon atom. It will be understood by those skilled in the art that the combinations of substituents contemplated by the present invention are those that are stable or chemically feasible. The substituents are, for example (but not limited to): C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, C 3-8 Cycloalkyl, 3- to 12-membered heterocyclic group, aryl, heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C 1-8 Aldehyde, C 2-10 Acyl, C 2-10 Ester group, amino group, alkoxy group, C 1-10 Sulfonyl, etc.

[0141] As used herein, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties such as melting points, boiling points, spectral properties, activities and reactivities.

[0142] As used herein, the term "enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0143] As used herein, the term "pharmaceutically acceptable salt" refers to salts that do not have biologically or otherwise undesirable properties. Pharmaceutically acceptable salts include acid addition salts and base addition salts.

[0144] As used herein, the term "prodrug" refers to a form of a compound or derivative of a compound that is metabolized in vivo, for example by a subject's biological fluids or enzymes after administration, to a pharmacologically active form of the compound to produce the desired pharmacological effect.

[0145] The term "pharmaceutically acceptable acid addition salt" refers to those pharmaceutically acceptable salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid.

[0146] The term "pharmaceutically acceptable base addition salt" refers to those pharmaceutically acceptable salts formed with organic or inorganic bases. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethylamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine and polyamine resins.

[0147] Compounds containing one or more chiral centers may exist as racemates, diastereomeric mixtures or optically active single isomers.

[0148] As used herein, "PO" refers to oral gavage.

[0149] As used herein, "SC" refers to subcutaneous injection.

[0150] As used herein, "BIW" means a dosing frequency of twice a week.

[0151] As used herein, "BID" means dosing frequency is twice a day.

[0152] As used herein, "QD" means that the dosing frequency is once a day.

[0153] As used herein, "QW" means dosing frequency is once a week.

[0154] As used herein, "AAV" refers to adeno-associated virus.

[0155] As used herein, "AAV-HBV" refers to a recombinant virus carrying 1.3 copies of the HBV genome packaged in the AAV8 capsid. A persistent HBV infection mouse model was established by injection of AAV-HBV into the mouse tail vein. In this mouse model, active HBV replication leads to persistent HBV viral markers (e.g., HBV DNA, HBsAg, HBeAg, etc.).

[0156] As used herein, "HBsAg" refers to hepatitis B surface antigen.

[0157] As used herein, "HBeAg" refers to hepatitis B e antigen.

[0158] As used herein, "anti-HBs" refers to antibodies against HBsAg.

[0159] As used herein, "TLR7 / 8" refers to Toll-like receptor 7 and / or Toll-like receptor 8 of any species (eg, human, mouse, woodchuck, etc.).

[0160] As used herein, "TLR7 / 8 agonist" refers to a compound that is a TLR7 agonist, a TLR8 agonist, and a TLR7 / 8 agonist. Unless otherwise indicated, a TLR7 / 8 agonist can include any pharmaceutically acceptable form of the compound, including any isomer (e.g., diastereomer or enantiomer), salt, solvate, polymorph, etc. The TLR agonism of a particular compound can be determined in any suitable manner.

[0161] As used herein, "hepatitis B virus" or "HBV" refers to a member of the Hepadnaviridae family with a small double-stranded DNA genome of approximately 3,200 base pairs and hepatocyte tropism. "HBV" includes hepatitis B viruses that infect any of various mammalian (e.g., human, non-human primate, etc.) and avian (ducks, etc.) hosts. "HBV" includes any known HBV genotype, such as serotypes A, B, C, D, E, F, and G; any HBV serotype or HBV subtype; any HBV isolate; HBV variants, such as HBeAg-negative variants, drug-resistant HBV variants (e.g., lamivudine-resistant mutants; adefovir-resistant mutants; tenofovir-resistant mutants; entecavir-resistant mutants, etc.); and the like.

[0162] As used herein, "HBV capsid assembly regulator" refers to an agent that inhibits and / or destroys and / or accelerates and / or hinders and / or delays and / or reduces and / or modifies normal HBV capsid assembly (e.g., during maturation) and / or normal capsid disassembly (e.g., during infection) and / or perturbs capsid stability, thereby inducing abnormal capsid morphology and function.

[0163] The present invention relates to a pharmaceutical composition comprising a TLR7 / 8 selective agonist and a HBV capsid assembly inhibitor in a pharmaceutically acceptable carrier.

[0164] Pharmaceutical composition containing active ingredients

[0165] Since the compounds of the present invention have excellent selective activation of TLR7 / 8 receptors or inhibition of HBV capsid assembly, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to prevent and / or treat diseases or conditions related to viral infection; preferably, the virus is preferably hepatitis B virus HBV.

[0166] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 0.01-99.99% by weight of the compound of the present invention / dose, and more preferably, contains 0.1-99.9% by weight of the compound of the present invention / dose. Preferably, the "one dose" is a capsule or tablet.

[0167] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0168] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration or subcutaneous administration).

[0169] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0170] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0171] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0172] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0173] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0174] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0175] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds (eg, anti-HBV agents).

[0176] When administered in combination, the pharmaceutical composition also includes one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds. One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds can be used simultaneously, separately or sequentially with the compound of the present invention to prevent and / or treat diseases or conditions caused by viral infection (particularly hepatitis B virus HBV infection).

[0177] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage (e.g., 0.001 g-100 g / kg body weight). Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0178] Example 1

[0179] HEK-Blue TM hTLR7 and HEK-Blue TM hTLR8 assay:

[0180] 1. Weigh the TLR7 / 8 agonist compound to be tested and prepare it to a concentration of 10 mM with DMSO (Sigma). Use DMSO to dilute the compound to be tested by 3.16 times in a gradient, with a total of 10 concentration gradients, and the highest concentration is 1 mM.

[0181] 2. The test compound and DMSO control were diluted 10-fold using DPBS buffer (Gibco) and added to a 96-well cell culture plate (Corning) in a volume of 20 μL.

[0182] 3. Digest HEK-Blue using cell separation buffer (Gibco) TM hTLR7 and / or HEK-Blue TMhTLR8 cells (Invivogen) were cultured and counted (TC-20 cell counter, Bio-rad). The cells were diluted to 4.4E5 / mL using DMEM cell culture medium (Gibco). 180 μL was added to a 96-well cell culture plate. The plates were incubated in a 37°C, 5% CO2 incubator for 16-22 hours.

[0183] 4. Prepare QUANTI-Blue detection reagent (Invivogen), take 180 μL and add it to a new 96-well plate. Take 20 μL of cell culture supernatant from the cell culture plate and add it to the 96-well plate, and incubate at 37°C for 1-3 hours.

[0184] 5. Neo2 multifunctional microplate reader (Bio-tek) was used to measure the light absorption at 630 nm, and GraphPad Prism was used to calculate the half effective concentration (EC) of the drug. 50 .

[0185] Example 2

[0186] The combination of a TLR7 / 8 agonist (Compound III-I-60) and a HBV capsid assembly modulator (Compound II-22) potently reduces HBsAg, HBeAg, and HBV DNA in the AAV-HBV mouse model

[0187] AAV-HBV mouse model

[0188] Male C57BL / 6 mice aged 4-5 weeks were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Mice were housed in single cages in polycarbonate cages with corncob bedding. The environmental control conditions of the animal room included temperature, relative humidity, and light. The temperature was controlled between 21°C and 25°C, the relative humidity was controlled between 40% and 70%, and a 12-hour light / 12-hour dark cycle was used (lighting time was from 7 am to 7 pm). Animals were fed and watered freely during the feeding process.

[0189] Recombinant adeno-associated virus type 8 (rAAV8-1.3HBV) carrying 1.3 times the HBV genome was purchased from Wuhan Shumi Brain Science Technology Co., Ltd. and stored at -70°C for future use. The experimental mice were injected with 200 μL of rAAV8-1.3HBV virus suspension through the tail vein on the first day after adaptation, and blood was collected through the mandibular vein on the 22nd, 29th and 36th days (21st, 28th and 35th days after AAV-HBV injection). 10 μL of serum was prepared from each mouse for quantitative detection of HBsAg, HBeAg and HBV DNA. The mice were grouped according to the levels of HBV biomarkers.

[0190] Experimental groups (see Table 2-1)

[0191] Compound III-I-60 was prepared in solvent 1 (20% HP-β-CD dissolved in water) at a concentration of 1 mg / mL. Compound II-22 was prepared in solvent 2 (2% Klucel, 0.1% Tween 80, 0.09% methylparaben and 0.01% propylparaben dissolved in water) at a concentration of 1 mg / mL.

[0192] The experimental mice were divided into groups and given oral gavage (PO) to the vehicle, compound III-I-60, compound II-22, and the drug combination of compound III-I-60 and compound II-22. The drug was administered for a total of 6 weeks, followed by a 3-week drug withdrawal observation period. The mice in the vehicle group were given vehicle 1 by gavage twice a week (BIW) and vehicle 2 by gavage twice a day (BID). In the single treatment group, mice in the compound III-I-60 group were given 10 mg / kg of the compound by gavage twice a week, and mice in the compound II-22 group were given 20 mg / kg of the compound by gavage twice a day. The combination treatment group was given 10 mg / kg of compound III-I-60 by gavage twice a week and 20 mg / kg of compound II-22 by gavage twice a day. Serum samples were collected and prepared during the experiment, (15+15) μL per mouse, once a week, and the serum samples were transferred to the clinical laboratory for quantitative detection of HBsAg, HBeAg, HBV DNA and ALT levels. An additional 15 μL of serum was collected on Day 36, Day 43, and Day 64 for quantitative determination of anti-HBsAg antibody (HBsAb) levels.

[0193] Table 2-1 AAV-HBV mouse model combination study design for compound III-I-60 and compound II-22

[0194]

[0195] Experimental Results

[0196] like Figure 1 As shown, compared with the vehicle control group, the serum HBsAg and HBV DNA levels of mice in the TLR7 / 8 agonist compound III-I-60 monotherapy group continued to decrease significantly during the six-week dosing period and gradually rebounded after stopping the dosing. Compared with the control group, after six weeks of administration, the serum HBsAg and HBV DNA levels of mice in the compound III-I-60 monotherapy group decreased by 2.28Log10 and 2.76Log10, respectively. Compound III-I-60 monotherapy did not have a significant effect on the serum HBeAg level of mice.

[0197] like Figure 1As shown, compared with the vehicle control group, the capsid assembly regulator compound II-22 monotherapy mice serum HBsAg, HBeAg and HBV DNA levels continued to decrease significantly during the six-week dosing period. After drug withdrawal, the mouse serum HBsAg and HBeAg remained at a relatively low level, while the serum HBV DNA level gradually rebounded. Compared with the control group, after six weeks of administration, the serum HBsAg, HBeAg and HBV DNA levels of mice in the compound II-22 monotherapy group decreased by 1.68Log10, 1.24Log10 and 5.57Log10, respectively.

[0198] like Figure 1 As shown, compared with the vehicle control group, the serum HBsAg, HBeAg and HBV DNA levels of mice in the combination treatment group of compound III-I-60 and compound II-22 decreased significantly during the six-week administration period and remained at a relatively low level after drug withdrawal. Compared with the control group, after six weeks of administration, the serum HBsAg, HBeAg and HBV DNA levels of mice in the combination treatment group decreased by 3.41Log10, 2.17Log10 and 5.83Log10, respectively. And the decline rate of serum HBsAg in mice in the combination treatment group was significantly faster than that in any single treatment group.

[0199] The body's immune system produces an immune response to HBV viral components, such as the production of anti-HBsAg antibodies HBsAb, which exerts humoral immune responses, which is an important way to inhibit HBV reinfection and promote HBV clearance. As shown in Table X1-2, no HBsAb was produced in the vehicle group (Group 01) and the mice in the capsid assembly regulator compound II-22 monotherapy group (Group 03) after treatment. In the TLR7 / 8 agonist compound III-I-60 monotherapy group (Group 02), 1 and 2 mice produced HBsAb after 5 and 6 weeks of treatment, respectively, and the HBsAb level was low; no mice produced HBsAb after 3 weeks of drug withdrawal. In the combination treatment group (Group 04), 5, 5, and 4 mice produced HBsAb after 5 weeks of treatment, 6 weeks of treatment, and 3 weeks of drug withdrawal, respectively, and the HBsAb level was high, and the HBsAb level showed an increasing trend.

[0200] Table 2-2 Serum anti-HBsAg antibody (HBsAb)

[0201]

[0202] Example 3

[0203] The combination of a TLR7 / 8 agonist (Compound VI-162) and a HBV capsid assembly modulator (Compound II-22) potently reduces HBsAg, HBeAg, and HBV DNA in the AAV-HBV mouse model

[0204] AAV-HBV mouse model

[0205] Male C57BL / 6 mice aged 4-5 weeks were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Mice were housed in single cages in polycarbonate cages with corncob bedding. The environmental control conditions of the animal room included temperature, relative humidity, and light. The temperature was controlled between 21°C and 25°C, the relative humidity was controlled between 40% and 70%, and a 12-hour light / 12-hour dark cycle was used (lighting time was from 7 am to 7 pm). Animals were fed and watered freely during the feeding process.

[0206] Recombinant adeno-associated virus type 8 (rAAV8-1.3HBV) carrying 1.3 times the HBV genome was purchased from Wuhan Shumi Brain Science and Technology Co., Ltd. and stored at -70°C for future use. On the first day after adaptation, the experimental mice were injected with 200 μL of rAAV8-1.3HBV virus suspension through the tail vein, and blood was collected through the mandibular vein on the 22nd and 29th days (21st and 28th days after AAV-HBV injection). 10 μL of serum was prepared from each mouse for quantitative detection of HBsAg, HBeAg and HBV DNA. The mice were grouped according to the levels of HBV biomarkers.

[0207] Experimental groups (see Table 3-1)

[0208] Compound VI-162 was prepared in solvent 1 (0.9% saline) at a concentration of 1 mg / mL and 0.2 mg / mL. Compound II-22 was prepared in solvent 2 (an aqueous solution containing 0.5% hydroxypropylcellulose, 0.75% sodium dodecyl sulfate and 15 mmol / L disodium hydrogen phosphate) at a concentration of 2 mg / mL.

[0209] The experimental mice were divided into 6 groups, 5 in each group. Group 1 was a vehicle control group, which was given vehicle 1 by subcutaneous injection once a week (QW), and vehicle 2 was given by oral gavage once a day (QD). Group 2 was given 20 mg / kg compound II-22 by oral gavage once a day. Group 3 was given 5 mg / kg compound VI-162 by oral gavage once a week. Group 4 was a combination treatment group, which was given 20 mg / kg compound II-22 by oral gavage once a day, and 5 mg / kg compound VI-162 by oral gavage once a week. Group 5 was given 1 mg / kg compound VI-162 by subcutaneous injection once a week. Group 6 was a combination treatment group, which was given 20 mg / kg compound II-22 by oral gavage once a day, and 1 mg / kg compound VI-162 by subcutaneous injection once a week. The drug was administered for a total of 5 weeks, followed by a 3-week withdrawal observation period. During the experiment, serum samples were collected and prepared, (15+15) μL per mouse, once a week, and the serum samples were transferred to the clinical laboratory for quantitative detection of HBsAg, HBeAg, HBV DNA and ALT levels. An additional 15 μL of serum was collected on Day 35, Day 42 and Day 56 for quantitative determination of anti-HBsAg antibody (HBsAb) levels.

[0210] Table 3-1. AAV-HBV mouse model combination study design for compound VI-162 and compound II-22

[0211]

[0212] Experimental Results

[0213] like Figure 2 As shown, compared with the vehicle control group, the capsid assembly regulator compound II-22 monotherapy group (Group 2) mice serum HBsAg, HBeAg and HBV DNA levels continued to decrease significantly during the five-week dosing period. After drug withdrawal, the mouse serum HBsAg and HBeAg remained at a relatively low level, while the serum HBV DNA level gradually rebounded. Compared with the control group, after five weeks of administration, the serum HBsAg, HBeAg and HBV DNA levels of mice in the compound II-22 monotherapy group were reduced by 1.62Log10, 1.15Log10 and 4.89Log10, respectively.

[0214] like Figure 2As shown, compared with the vehicle control group, the serum HBsAg and HBV DNA levels of mice in the TLR7 / 8 agonist compound VI-162 monotherapy group (groups 3 and 5) continued to decrease significantly during the five-week dosing period, and gradually rebounded after stopping the administration. Compared with the control group, after five weeks of administration, the serum HBsAg and HBV DNA levels of mice in the oral gavage monotherapy group of compound VI-162 were reduced by 4.74Log10 and 4.00Log10, respectively, and the serum HBsAg and HBV DNA levels of mice in the subcutaneous injection monotherapy group of compound VI-162 were reduced by 4.54Log10 and 4.03Log10, respectively. Compound VI-162 monotherapy did not have a significant effect on the serum HBeAg level of mice.

[0215] like Figure 2 As shown, compared with the vehicle control group, the serum HBsAg, HBeAg and HBV DNA levels of mice in the combination treatment group (group 4 and group 6) of compound VI-162 and compound II-22 decreased significantly during the five-week administration period and remained at a relatively low level after drug withdrawal. Compared with the control group, after five weeks of administration, the serum HBsAg, HBeAg and HBV DNA levels of mice in the combination treatment group 4 were reduced by 5.11Log10, 1.68Log10 and 6.45Log10, respectively, and the serum HBsAg, HBeAg and HBV DNA levels of mice in the combination treatment group 6 were reduced by 5.11Log10, 1.84Log10 and 6.08Log10, respectively. And the rate of decline of serum HBsAg in mice in the combination treatment group was significantly faster than that in any single treatment group.

[0216] The body's immune system produces an immune response to HBV viral components, such as the production of anti-HBsAg antibodies HBsAb. The humoral immune response is an important way to inhibit HBV reinfection and promote HBV clearance. As shown in Table X2-2, mice in the vehicle group (Group 01) and the capsid assembly regulator compound II-22 monotherapy group (Group 2) did not produce HBsAb after treatment. Mice in the TLR7 / 8 agonist compound VI-162 monotherapy group (Groups 3 and 5) and the combination treatment group (Groups 4 and 6) produced HBsAb after 5 weeks of treatment, and the HbsAb level in the combination treatment group was higher than that in the monotherapy group; after discontinuation of the drug, the HbsAb level in the compound VI-162 monotherapy group showed a downward trend, while the HbsAb level in the combination treatment group showed an upward trend. Table 3-2 Serum anti-HBsAg antibodies (HBsAb)

[0217]

[0218] Conclusions

[0219] In summary, the combination of TLR7 / 8 agonists and HBV capsid regulators is effective in treating HBV, and can reduce or even eliminate HBsAg, HBeAg and HBV DNA faster than monotherapy. Moreover, after combination therapy, viral suppression can last up to 3 weeks without retreatment. Existing treatments for chronic hepatitis B seek functional cure, i.e., HBsAg loss and / or seroconversion (production of HBsAb). However, existing therapies rarely achieve HBsAg seroconversion. In our combination therapy, HBsAb production can be detected during treatment, while the single treatment group cannot produce HBsAg, or the proportion of mice producing HBsAb or HBsAb levels are very low. Moreover, after combination therapy, HBsAb levels can be maintained or tend to increase without retreatment. Therefore, the combination therapy of TLR7 / 8 agonists and HBV capsid regulators provides another key advantage to promote the emergence of HBsAb. This combination therapy is expected to achieve functional cure of chronic hepatitis B and has great clinical development value.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a TLR7 / 8 selective agonist, a HBV capsid assembly inhibitor and a pharmaceutically acceptable carrier; preferably, in the pharmaceutical composition, the weight ratio of the TLR7 / 8 selective agonist to the HBV capsid assembly inhibitor is 1-20:1-10000.

2. The pharmaceutical composition according to claim 1, characterized in that The HBV capsid assembly inhibitor is a compound of formula (II), a solvate thereof or a pharmaceutically acceptable salt thereof: in, W is O or S; R 2 , R 3 and R 4 are independently H, chloro, fluoro, bromo, methyl, cyano, methoxy or trifluoromethyl; R 5 C 1-3 Alkyl; preferably methyl or ethyl; L 21 is a single bond or a C1-C3 alkylene group; Q is a 5-8 membered bridged cycloalkyl or a 5-8 membered heterocycloalkyl; the 5-8 membered heterocycloalkyl is connected by a bridge ring; R 10 COOH, CN, unsubstituted or replaced by one or more R A Substituted C1-C6 alkyl, NR B R C or R A is hydroxyl, halogen, C1-C6 alkoxy, COOH, CN, C4-C6 heterocycloalkyl substituted by a carbonyl group, R B and R C Independently for H, C1-C6 alkyl, or -C1-C6 alkyleneCOOH; R D C4-C6 heterocycloalkyl, NH2-, -NHC1-C6 alkyl, Unless otherwise specified, the ring skeleton of the heterocycloalkyl group includes 1, 2 or 3 identical or different heteroatoms selected from N, O and S; The carbon atom with "*" indicates that when it is a chiral carbon atom, the carbon atom with "*" is in S configuration, R configuration or a mixture thereof.

3. The pharmaceutical composition according to claim 2, characterized in that The compound of formula (II) of the HBV capsid assembly inhibitor is selected from the following group: or a solvate thereof or a pharmaceutically acceptable salt thereof; Preferably, the compound of formula (II) of the HBV capsid assembly inhibitor is selected from the following group: or a solvate thereof or a pharmaceutically acceptable salt thereof; Further preferably, the compound of formula (II) of the HBV capsid assembly inhibitor is or a solvate thereof or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 1, characterized in that The HBV capsid assembly inhibitor is a compound of formula (I), a solvate thereof or a pharmaceutically acceptable salt thereof: in, R 2 , R 3 and R 4 each independently is H, chloro, fluoro, bromo, methyl, cyano, methoxy or trifluoromethyl; R 5 C 1-3 Alkyl; preferably methyl or ethyl; R 6 is H, halogen, hydroxyl or C 1-3 Alkyl; preferably hydrogen, methyl or fluorine; R 7 H, halogen, C 1-3 Alkyl, hydroxy or methoxy; R 13 is H, unsubstituted or substituted with one or more (e.g. 1, 2, 3 or 4) R 1b Substituted group selected from the group consisting of: phenyl-L 1 -, pyridyl-L 2 -, 3-7 membered monocyclic cycloalkyl-L 3 -、C 1-6 Alkyl-L 4 -; and when the R 1b When there are multiple substituents, each R 1b Same or different; -L 1 -、-L 2 -、-L 3 -and-L 4 - are each independently a linking bond, unsubstituted or substituted with one or more R 8 Substituted C 1-3 Alkylene; wherein R 8 Independently for C 1-3 Alkyl or 3-7 membered cycloalkyl; R 1b are independently -CO2H, OH, halogen, CN, unsubstituted or substituted with one or more R 1c Substituted groups selected from the group consisting of: -CO2-C 1-3 Alkyl, -(C 1-6 Alkylene)-CO2H, -(C 1-6 Alkylene)-CO2-C 1-3 Alkyl, -(C 2-6 Alkenylene)-CO2H, -(C 2-6 Alkenylene)-CO2-C 1-3 Alkyl, C 1-3 Alkyl-O-, C 1-3 Alkyl-O-(C 1-6 Alkylene)-, C 1-6 Alkyl, C 1-3 Alkylsulfonyl, 3-7 membered cycloalkyl; wherein R 1c are independently halogen, C 1-4 Alkyl, 3-7 membered cycloalkyl; The carbon atom with "*" indicates that when it is a chiral carbon atom, it is in S configuration, R configuration or a mixture thereof.

5. The pharmaceutical composition according to claim 4, characterized in that The HBV capsid assembly inhibitor compound of formula (I) is selected from the following group: or a solvate thereof or a pharmaceutically acceptable salt thereof; Preferably, the compound of formula (I) of the HBV capsid assembly inhibitor is selected from the following group: or a solvate thereof or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 1, characterized in that The TLR7 / 8 selective agonist is a compound of formula (III) or formula (IV), a solvate thereof, or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof: in, A is O, S, (S=O)2; Y is N or CR Y ; R Y is cyano, or C1-C4 alkyl substituted by halogen; preferably cyano or trifluoromethyl; B is unsubstituted or replaced by R 1-1 Substituted C2~C 10 Alkylene, unsubstituted or replaced by R 1-1 Substituted C2~C 10 Unsaturated alkylene, or -(CH2) n -L 25 -(CH2) r -O-; L 25 is an unsubstituted or halogen-substituted C3-C6 cycloalkylene group, an unsubstituted or halogen-substituted 3-6-membered heterocycloalkylene group; n and r are independently 1, 2 or 3; R 1-1 is halogen, C1-C6 alkyl; R 31 , R 32 and R 33 are independently H, halogen, unsubstituted or halogen-substituted C1-C6 alkyl, C1-C6 alkoxy; Or, R 31 and R 32 , or R 32 and R 33 Each independently forms with the carbon atom to which they are attached a group selected from the group consisting of: unsubstituted or substituted with R 1-4 substituted 4-7 membered heterocycloalkylene, unsubstituted or replaced by R 1-5 Substituted C4-C7 cycloalkylene; wherein one or more arbitrary methylene groups in the heterocycloalkylene or cycloalkylene are independently replaced by a carbonyl group or S(=O)2; R 1-4 and R 1-5 Each independently selected from the group consisting of OH, halogen, CN, unsubstituted or replaced by R 1-6 Substituted C1-C6 alkyl, unsubstituted or R 1-9 Substituted C1-C6 alkoxy, -S(=O)2R 1-7 、-C(=O)R 1-8 NR 1-10 R 1-11 、COOR 1-12 , SR 1-13 , unsubstituted or replaced by R 1-19 Substituted C3-C7 cycloalkyl, unsubstituted or R 1-20 substituted 4-7 membered heterocycloalkyl, unsubstituted or replaced by R 1-21 substituted 5-10 membered heteroaryl, or -G(CR 1-14 R 1-15 ) u -COOR 1-16 ; wherein, G is O, S, S(=O)2 or NH; u is 1, 2 or 3; R 1-6 and R 1-9 Each independently selected from the group consisting of halogen, amino, CN, OH, -COOR 1-17 、-S(=O)2R 1-31 , -C(=O)NH2, -S(=O)2NH2, C1-C3 alkoxy, unsubstituted or substituted with COOR 1-18 Substituted C3-C7 cycloalkyl, unsubstituted or R 1-22 substituted 4-7 membered heterocycloalkyl, unsubstituted or replaced by R 1-23 substituted 5-10 membered heteroaryl; R 1-7 and R 1-8 Independently selected from the following groups: C1-C3 alkyl, unsubstituted or replaced by R 1-24 Substituted C3-C7 cycloalkyl, unsubstituted or R 1-25 substituted 4-7 membered heterocycloalkyl, unsubstituted or replaced by R 1-26 substituted 5-10 membered heteroaryl, unsubstituted or R 1-27 Substituted C6~C 10 Aryl or -NR 1-28 R 1-29 ; R 1-10 , R 1-11 , R 1-12 , R 1-16 , R 1-17 , R 1-18 , R 1-28 and R 1-29 are independently H or C1-C3 alkyl; R 1-31 is a C1-C3 alkyl group; R 1-13 is H, unsubstituted or halogen-substituted C1-C6 alkyl; R 1-14 and R 1-15 are independently H, unsubstituted or halogen-substituted C1-C6 alkyl; R 1-19 , R 1-20 , R 1-21 , R 1-22 , R 1-23 and R 1-24 are independently OH, halogen, amino, CN, C1-C6 alkyl; Unless otherwise specified, the ring skeleton of the heterocycloalkylene or heterocycloalkyl group includes 1, 2 or 3 identical or different heteroatoms selected from N, O and S; the ring skeleton of the heteroaryl group includes 1, 2, 3 or 4 identical or different heteroatoms selected from N, O and S; and when R 1-1 , R 1-4 , R 1-5 , R 1-6 , R 1-9 , R 1-19 , R 1-20 , R 1-21 , R 1-22 , R 1-23 , R 1-24 , R 1-25 , R 1-26 and R 1-27 When substituted, each of the above substituents may be one or more.

7. The pharmaceutical composition according to claim 6, wherein the compound of formula (III) or formula (IV) of the TLR7 / 8 selective agonist is selected from the following group: a solvate thereof or a pharmaceutically acceptable salt thereof; Preferably, the compound of formula (III) or formula (IV) of the TLR7 / 8 selective agonist is selected from the following group: or a solvate thereof or a pharmaceutically acceptable salt thereof; More preferably, the compound of formula (III) or formula (IV) of the TLR7 / 8 selective agonist is or a solvate thereof or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 1, characterized in that The TLR7 / 8 selective agonist is a compound of formula (V) or formula (VI), a solvate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof: L 51 Selected from the group consisting of -O-, -NH-, -S-, -S(=O)- or -S(=O)2-; preferably -O- or -NH-; R 51 Selected from the following group: H, unsubstituted or hydroxy-substituted C 1-12 Alkyl, C 1-12 Alkoxy, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl or 4-12 membered heterocycloalkyl; wherein said R 51 can be further represented by one or more R a Substituents, and the R a Selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, -NR a1 R a2 、-NHC(=O)-R a3 , one or more R a4 Substituted 5-6 membered heteroaryl, -OC(=O)R a5 、-C(=O)R a5 、-OC(=O)OR a5 、-C(=O)OR a5 ; The R a1 , R a2 , R a3 or R a4 Select from the following group: C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl; The R a5 Select from the following group: C 1-24 Alkyl, halogenated C 1-24 Alkyl, C 1-24 Heteroalkyl, wherein the heteroatom is selected from one or more of NH, N, O and S; m is 0, 1 or 2; B does not exist, or B is selected from the following group: C 3-12 Cycloalkyl, 4-12 membered heterocyclic group, C 6-12 Aryl, 5-12 membered heteroaryl or wherein each A is selected from C, CH or N, and R6 and R7 together with their adjacent carbon atoms form C 4-7 Cycloalkylene or C 4-7 Heterocycloalkylene, the C 4-7 One or more arbitrary methylene groups in the cycloalkylene or 4-7 membered heterocycloalkylene may be independently replaced by a carbonyl group or S(=O)2; the ring skeleton of the 4-7 membered heterocycloalkylene includes 1, 2 or 3 identical or different heteroatoms selected from N, O and S; L 52 Selected from the following group: -(CR b R c ) p -(NR d ) q -、-O-、-S-、-(CR b R c ) p -C(=O)-、-(CR b R c ) p -C(=O)NH-, -(CR b R c ) p -NHC(=O)-, -S(=O)- or -S(=O)2-; wherein R b , R c Selected from the group consisting of hydrogen, halogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl or C 1-6 Haloalkyl, R d Selected from the group consisting of hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, C 1-6 C substituted with halogenated alkyl or hydroxyl 1-6 Alkyl, p is 0, 1, 2, 3, 4, 5 or 6, q is 0 or 1; R 54 Selected from the group consisting of hydrogen, halogen, cyano, amino, hydroxyl, C 1-12 Alkyl, C 1-12 Alkoxy, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl, 4-12 membered heterocyclic group, C 6-12 Aryl or 5-12 membered heteroaryl; and said R 54 Optionally, one or more R e Substituent substitution, where R e Selected from the group consisting of hydrogen, halogen, hydroxy, carboxylic acid, amino, C 1-6 Alkyl, one or more R e1 Substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 3-12 Cycloalkyl, 4-12 membered heterocycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, -N(R e2 R e3 R e4 )、-C(=O)OR e2 、-C(=O)NH-R e2 、-S(=O)2-R e2 ; R e1 Selected from the group consisting of halogen, hydroxyl, -P(O)(OR e2 )2; R e2 , R e3 Selected from the group consisting of hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 1-6 Haloalkyl, C substituted by hydroxy 1-6 alkyl; R e4 Not present or selected from the following group: C 1-6 Alkyl, C 3-6 Cycloalkyl or C 1-6 Haloalkyl; R 55 Selected from the following group: halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl; n is 0, 1, 2, 3 or 4; Wherein, each of the heterocyclic groups may be saturated or partially unsaturated (but not having an aromatic structure), and the ring skeleton of the heterocyclic group includes 1, 2, 3 or 4 (preferably 1 or 2) identical or different heteroatoms selected from N, O and S; the ring skeleton of the heteroaryl group includes 1, 2 or 3 identical or different heteroatoms selected from N, O and S.

9. The pharmaceutical composition according to claim 8, characterized in that In the formula (V) or formula (VI) of the TLR7 / 8 selective agonist: L 51 Selected from the group consisting of: -NH-; R 51 Selected from the following group: unsubstituted or hydroxy-substituted C 1-12 alkyl; m is 1; B is selected from phenyl or pyridyl; The remaining groups are as defined in claim 8.

10. The pharmaceutical composition according to claim 8, characterized in that The compound of formula (V) or formula (VI) of the TLR7 / 8 selective agonist is selected from the following group: a solvate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof; Preferably, the compound of formula (V) or formula (VI) of the TLR7 / 8 selective agonist is selected from the following group: or a solvate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof; Further preferably, the compound of formula (V) or formula (VI) of the TLR7 / 8 selective agonist is A solvate thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof.

11. The pharmaceutical composition according to any one of claims 1 to 10, characterized in that The composition may also include one or more other antiviral drugs; Preferably, the other antiviral drugs are selected from lamivudine, adefovir, tenofovir, telbivudine and entecavir.

12. A drug combination (kit), characterized in that: The drug combination comprises a first therapeutic component and a second therapeutic component, and the first therapeutic component comprises: a TLR7 / 8 selective agonist, and a pharmaceutically acceptable carrier; The second therapeutic component includes: an HBV capsid assembly inhibitor, and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition according to claims 1 to 11, or use of a pharmaceutical combination according to claim 12, characterized in that: Used for preparing a drug for treating or preventing viral infection; preferably, the virus is hepatitis B virus HBV.

Images