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

Abstract

The present invention belongs to the field of pharmaceutical compositions, and in particular relates to a pharmaceutical composition containing a TLR7 / 8 selective agonist and an HBV capsid assembly inhibitor and the use thereof. Provided in the present invention is a pharmaceutical composition containing a TLR7 / 8 selective agonist and an HBV capsid assembly inhibitor. The pharmaceutical composition comprises a TLR7 / 8 selective agonist, an HBV capsid assembly inhibitor and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used for preparing a drug for treating or preventing viral infections, and preferably, the virus is the hepatitis B virus (HBV).

Description

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

[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 compound of formula (IV) or compound of formula (V) or 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 compound of formula (II) disclosed in WO2022121844 or WO2022171072, for treating patients with chronic hepatitis B. Field of the Invention

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

[0003] The World Health Organization (WHO) estimates that 2 billion people worldwide have been or are currently infected with HBV, with as many as 240 million of them chronically infected with HBV surface antigen (HBsAg). Globally, approximately 650,000 people die annually from complications of chronic hepatitis B. China is one of the regions with a high incidence of hepatitis B. According to a report published in The Lancet, there are currently approximately 86 million HBsAg-positive patients in mainland China, of whom 20 million are chronic hepatitis B patients. There are 1 million new cases each year, 5 million cases of cirrhosis caused by hepatitis B, and 300,000 cases of hepatitis B-related liver cancer.

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

[0005] Numerous studies have shown that TLR7 and TLR8 play an important role in host innate and adaptive immunity and can serve as effective targets for antiviral and tumor treatment. Toll-like receptors (TLRs) are a class of structurally conserved pattern recognition receptors (PRRs) that are important components of the innate immune system. They primarily recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). By recognizing invasive microorganisms and endogenous molecules released after tissue damage or non-physiological cell death, TLRs can activate downstream signaling pathways and promote the production of inflammatory cytokines. There are 10 known members of the TLR family in humans (TLRs 1–10). TLRs 1, 2, 4, 5, 6, and 10 are located on the cell membrane and can bind cell surface ligands. TLRs 3, 7, 8, and 9 are located in the endosome and primarily bind nucleic acid receptors. TLRs 7 and 8 recognize single-stranded RNA viruses rich in GU and AU sequences and are considered pattern recognition receptors for many ssRNA viruses, such as influenza virus, HIV-1, vesicular stomatitis virus, Sendai virus, flaviviruses, and coronaviruses. The roles of different TLRs in immune responses vary depending on the viruses they recognize, the cell type, and the infection model. TLR 7 is primarily expressed in B cells, monocytes, and plasmacytoid dendritic cells (pDCs), while TLR 8 is primarily expressed in monocytes, neutrophils, and myeloid dendritic cells. Both TLR7 and 8 activate downstream inflammatory signaling molecules through Myd-88-dependent signaling pathways: Activation of TLR7 favors the production of type I interferons (IFNs), such as IFN-α; while TLR8 favors activation of nuclear factor-κB (NF-κB), which in turn produces a variety of inflammatory cytokines, such as TNF-α and IFN-γ. These cytokines can recruit or activate T cells, B cells, and NK cells in the disease microenvironment, enhancing innate and adaptive immune responses and producing antiviral and anti-tumor activities.

[0006] Currently, the medical community lacks a complete cure for hepatitis B. "Functional cure" has become the current focus of hepatitis B treatment. This involves a limited course of antiviral drug therapy to reduce the hepatitis B virus to very low levels, so low that HBV DNA and hepatitis B surface antigen (HBsAg) are undetectable in the blood. Combining different antiviral therapies to induce HBsAg loss and / or seroconversion is expected to increase the rate of functional cure and has clear clinical therapeutic value.

[0007] Summary of the Invention

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] In another preferred embodiment, the HBV capsid assembly inhibitor is a compound of formula (II), a solvate thereof or a pharmaceutically acceptable salt thereof:

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

[0020] Q is a 5-8 membered bridged cycloalkyl group or a 5-8 membered heterocycloalkyl group; the 5-8 membered heterocycloalkyl group 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 H, C1-C6 alkyl, or -C1-C6 alkyleneCOOH;

[0024] 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;

[0025] The carbon atom marked with “*” indicates that when it is a chiral carbon atom, the carbon atom marked with “*” is in S configuration, R configuration or a mixture thereof.

[0026] In another preferred embodiment, the compound of formula (II) of the HBV capsid assembly inhibitor is selected from the following group:

[0027]

[0028]

[0029] or a solvate or a pharmaceutically acceptable salt thereof;

[0030] Preferably, the compound of formula (II) of the HBV capsid assembly inhibitor is selected from the following group:

[0031] or a solvate or a pharmaceutically acceptable salt thereof;

[0032] Further preferably, the compound of formula (II) of the HBV capsid assembly inhibitor is or a solvate or a pharmaceutically acceptable salt thereof.

[0033] In another preferred embodiment, the HBV capsid assembly inhibitor is a compound of formula (I), a solvate thereof or a pharmaceutically acceptable salt thereof:

[0034] in,

[0035] R 2 、R 3 and R 4 are each independently H, chloro, fluoro, bromo, methyl, cyano, methoxy or trifluoromethyl;

[0036] R 5 C 1-3 Alkyl; preferably methyl or ethyl;

[0037] R 6 is H, halogen, hydroxyl or C 1-3 Alkyl; preferably hydrogen, methyl or fluorine;

[0038] R 7 H, halogen, C 1-3 Alkyl, hydroxy or methoxy;

[0039] R 13 is H, unsubstituted or substituted with one or more (e.g. 1, 2, 3 or 4) R 1b Substituted groups 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;

[0040] -L 1 -、-L 2 -、-L 3 -and-L 4 - are each independently a linking bond, unsubstituted or replaced by one or more R 8 Substituted C 1-3 Alkylene; wherein R 8 Independently C 1-3 Alkyl or 3-7 membered cycloalkyl;

[0041] R 1b is independently -CO2H, OH, halogen, CN, unsubstituted or replaced by one or more R1c 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;

[0042] The carbon atom marked with "*" indicates that when it is a chiral carbon atom, it is in S configuration, R configuration or a mixture thereof.

[0043] In another preferred embodiment, the compound of formula (I) of the HBV capsid assembly inhibitor is selected from the following group:

[0044]

[0045]

[0046] or a solvate or a pharmaceutically acceptable salt thereof;

[0047] Preferably, the compound of formula (I) of the HBV capsid assembly inhibitor is selected from the following group:

[0048]

[0049] or a solvate or a pharmaceutically acceptable salt thereof.

[0050] 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:

[0051] in,

[0052] A is O, S, (S=O)2;

[0053] Y is N or CR Y ;

[0054] RY is cyano, or C1-C4 alkyl substituted by halogen; preferably cyano or trifluoromethyl;

[0055] 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-;

[0056] 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;

[0057] R 1-1 is halogen, C1-C6 alkyl;

[0058] R 31 、R 32 and R 33 are independently H, halogen, unsubstituted or halogen-substituted C1-C6 alkyl, C1-C6 alkoxy;

[0059] Or, R 31 and R 32 , or R 32 and R 33 Each independently forms together with the carbon atom to which they are attached a group selected from the group consisting of: unsubstituted or replaced by 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;

[0060] 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 replaced by R 1-20substituted 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;

[0061] 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 COOR 1-18 Substituted C3-C7 cycloalkyl, unsubstituted or replaced by R 1-22 substituted 4-7 membered heterocycloalkyl, unsubstituted or replaced by R 1-23 substituted 5-10 membered heteroaryl;

[0062] 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 replaced by R 1-25 substituted 4-7 membered heterocycloalkyl, unsubstituted or replaced by R 1-26 substituted 5-10 membered heteroaryl, unsubstituted or replaced by R 1-27 Substituted C6~C 10 Aryl or -NR 1-28 R 1-29 ;

[0063] 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;

[0064] R 1-13 is H, unsubstituted or halogen-substituted C1-C6 alkyl;

[0065] R 1-14 and R 1-15 are independently H, unsubstituted or halogen-substituted C1-C6 alkyl;

[0066] R 1-19 、R 1-20 、R1-21 、R 1-22 、R 1-23 and R 1-24 are independently OH, halogen, amino, CN, or C1-C6 alkyl;

[0067] 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, the above-mentioned substituents may be one or more.

[0068] In another preferred embodiment, the compound of formula (III) or formula (IV) of the TLR7 / 8 selective agonist is selected from the following group:

[0069]

[0070]

[0071] A solvate 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:

[0072]

[0073] or a solvate or a pharmaceutically acceptable salt thereof;

[0074] More preferably, the compound of formula (III) or formula (IV) of the TLR7 / 8 selective agonist is or a solvate or a pharmaceutically acceptable salt thereof.

[0075] 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:

[0076] L 51Selected from the group consisting of -O-, -NH-, -S-, -S(=O)- or -S(=O)2-; preferably -O- or -NH-;

[0077] R 51 Selected from the group consisting of 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, the R 51 Can be further replaced by one or more R a Substituted by a substituent, and the R a Selected from the group consisting of hydrogen, halogen, hydroxy, 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 ;

[0078] The R a1 、R a2 、R a3 or R a4 Select from the following group: C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 3-6 Cycloalkyl;

[0079] 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;

[0080] m is 0, 1, or 2;

[0081] 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 C4-7 Heterocycloalkylene, the C 4-7 One or more arbitrary methylene groups in a cycloalkylene group or a 4-7 membered heterocycloalkylene group may be independently replaced by a carbonyl group or S(=O)2; the ring skeleton of the 4-7 membered heterocycloalkylene group includes 1, 2 or 3 identical or different heteroatoms selected from N, O and S;

[0082] L 52 Selected from the group consisting of: -(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 haloalkyl or hydroxy 1-6 Alkyl, p is 0, 1, 2, 3, 4, 5 or 6, q is 0 or 1;

[0083] 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 substituted, 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 1-6Alkoxy, 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 ;

[0084] R e1 Selected from the group consisting of halogen, hydroxy, -P(O)(OR e2 )2;

[0085] 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;

[0086] R e4 Not present or selected from the following group: C 1-6 Alkyl, C 3-6 Cycloalkyl or C 1-6 alkyl halide;

[0087] R 55 Selected from the group consisting of halogen, hydroxy, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl;

[0088] n is 0, 1, 2, 3 or 4;

[0089] Wherein, each heterocyclic group 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.

[0090] In another preferred embodiment, in the formula (V) or formula (VI) of the TLR7 / 8 selective agonist:

[0091] L 51 Selected from the group consisting of: -NH-;

[0092] R 51 Selected from the group consisting of: unsubstituted or hydroxy-substituted C 1-12 alkyl;

[0093] m is 1;

[0094] B is selected from phenyl or pyridyl;

[0095] The remaining groups are as defined above.

[0096] In another preferred embodiment, the compound of formula (V) or formula (VI) of the TLR7 / 8 selective agonist is selected from the following group:

[0097] a solvate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof;

[0098] Preferably, the compound of formula (V) or formula (VI) of the TLR7 / 8 selective agonist is selected from the following group:

[0099]

[0100]

[0101] or a solvate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof;

[0102] 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.

[0103] In another preferred embodiment, the pharmaceutical composition consists of a TLR7 / 8 selective agonist, a HBV capsid assembly inhibitor and a pharmaceutically acceptable carrier.

[0104] In another preferred embodiment, the composition further comprises one or more other antiviral drugs;

[0105] Preferably, the other antiviral drugs are selected from lamivudine, adefovir, tenofovir, telbivudine and entecavir.

[0106] 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.

[0107] In a third aspect of the present invention, a drug combination (kit) is provided, comprising a first therapeutic component and a second therapeutic component, wherein the first therapeutic component comprises: a TLR7 / 8 selective agonist, and a pharmaceutically acceptable carrier;

[0108] The second therapeutic component includes: an HBV capsid assembly inhibitor and a pharmaceutically acceptable carrier.

[0109] 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.

[0110] 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.

[0111] 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;

[0112] In another preferred embodiment, the virus is hepatitis B virus HBV.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] In another preferred embodiment, the administration is simultaneous administration or sequential administration.

[0117] In another preferred embodiment, the TLR7 / 8 selective agonist is a compound represented by Formula (III), Formula (IV), Formula (V) or Formula (VI), or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

[0118] 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.

[0119] 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 an HBV capsid assembly inhibitor or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof; or administering to an individual an effective first amount of an 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

[0120] 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).

[0121] 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

[0122] 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.

[0123] the term

[0124] In the present invention, the halogen is F, Cl, Br or I.

[0125] In the present invention, unless otherwise specified, the terms used have the general meanings commonly known to those skilled in the art.

[0126] In the present invention, the term "alkyl" refers to a straight or branched chain 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, with ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl being preferred. The term "alkylene" refers to a divalent group formed by further losing a hydrogen atom from an alkyl group.

[0127] The term "heteroalkyl" refers to a group in which one or more carbon atoms (optionally including one or more H atoms therein) on an alkane chain are replaced by 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.

[0128] In the present invention, the term "alkoxy" refers to a straight or branched chain 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.

[0129] 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.

[0130] In the present invention, the term "aromatic ring" or "aryl" has the same meaning, preferably "aryl" is "C6-C 12 Aryl" or "C6-C 10 Aryl. The term "C6-C 12 "Aryl" refers to an aromatic ring group with 6 to 12 carbon atoms and no heteroatoms in the ring, such as phenyl, naphthyl, etc. The term "C6-C 10 "Aryl" has a similar meaning.

[0131] In the present invention, the terms "aromatic heterocycle" or "heteroaryl" have the same meaning and refer to a heteroaromatic group containing one to multiple heteroatoms. The heteroatoms referred to herein include oxygen, sulfur, and nitrogen. Examples include furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0132] In the present invention, the term "carbocyclic group" refers to a saturated or unsaturated (non-aromatic ring, including monocyclic, fused, spirocyclic, bridged, 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 and cyclohexyl.

[0133] In the present invention, the term "heterocyclic group" refers to a saturated or unsaturated (non-aromatic ring, including monocyclic, fused, spirocyclic, bridged, etc.) cyclic group containing 1, 2, or 3 heteroatoms selected from oxygen, sulfur, and nitrogen. Preferably, the heterocyclic group is 3-12 members, such as 4-9 members, 3-7 members, 5-6 members, etc. Typical examples include dioxolane, etc.

[0134] In the present invention, the term "substituted" means that one or more hydrogen atoms on a specific group are replaced by a specific substituent. The specific substituent is the substituent described above, or the substituent appearing in the embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable site 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.

[0135] 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.

[0136] As used herein, the term "enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0137] 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.

[0138] 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 produce the desired pharmacological effect.

[0139] The term "pharmaceutically acceptable acid addition salts" 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 the group consisting of 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.

[0140] 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 salts, potassium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, 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.

[0141] Compounds containing one or more chiral centers may exist as racemates, diastereomeric mixtures or optically active single isomers.

[0142] As used herein, "PO" refers to oral gavage.

[0143] As used herein, "SC" refers to subcutaneous injection.

[0144] As used herein, "BIW" refers to a dosing frequency of twice a week.

[0145] As used herein, "BID" refers to a dosing frequency of twice a day.

[0146] As used herein, "QD" refers to a dosage frequency of once a day.

[0147] As used herein, "QW" refers to a dosing frequency of once a week.

[0148] As used herein, "AAV" refers to adeno-associated virus.

[0149] As used herein, "AAV-HBV" refers to a recombinant virus carrying 1.3 copies of the HBV genome packaged in an AAV8 capsid. A mouse model of persistent HBV infection was established by tail vein injection of AAV-HBV. In this mouse model, active HBV replication resulted in persistent HBV viral markers (e.g., HBV DNA, HBsAg, HBeAg, etc.).

[0150] As used herein, "HBsAg" refers to hepatitis B surface antigen.

[0151] As used herein, "HBeAg" refers to hepatitis B e antigen.

[0152] As used herein, "anti-HBs" refers to antibodies against HBsAg.

[0153] 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.).

[0154] As used herein, "TLR7 / 8 agonist" refers to a compound that acts as 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.

[0155] 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 a variety of 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.

[0156] 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.

[0157] 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.

[0158] Pharmaceutical compositions containing active ingredients

[0159] 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.

[0160] The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention and a pharmaceutically acceptable excipient or carrier. "Safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 0.01-99.99% by weight of the compound of the present invention per dose, more preferably 0.1-99.9% by weight of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0161] "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 compound of the present invention and with each other without significantly reducing the efficacy of the compound. 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 (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0162] 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).

[0163] 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 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.

[0164] Solid dosage forms such as tablets, dragees, 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 opacifying agents, and the release of the active compound or compounds in such compositions can be delayed in a certain portion of the digestive tract. Examples of useful encapsulating components are polymeric substances and waxes. If desired, the active compound can also be microencapsulated with one or more of the above-mentioned excipients.

[0165] 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, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0166] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0167] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0168] Compositions for parenteral injection may comprise 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.

[0169] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (eg, anti-HBV agents).

[0170] When administered in combination, the pharmaceutical composition further comprises 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).

[0171] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage (e.g., 0.001 g to 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 condition, all of which are within the skill of a skilled physician.

[0172] Example 1

[0173] HEK-Blue TM hTLR7 and HEK-Blue TM hTLR8 assay:

[0174] 1. Weigh the TLR7 / 8 agonist compound to be tested and prepare it to a concentration of 10 mM with DMSO (Sigma). Dilute the compound to be tested 3.16-fold using DMSO for a total of 10 concentration steps, with the highest concentration being 1 mM.

[0175] 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.

[0176] 3. Digest HEK-Blue using cell separation solution (Gibco) TM hTLR7 and / or HEK-Blue TMhTLR8 cells (Invivogen) were cultured and counted (TC-20 cell counter, Bio-rad). Cells were diluted to 4.4E5 / mL using DMEM cell culture medium (Gibco). 180 μL of each was added to each 96-well cell culture plate. The cells were incubated in a 37°C, 5% CO2 incubator for 16-22 hours.

[0177] 4. Prepare QUANTI-Blue detection reagent (Invivogen) and add 180 μL to a new 96-well plate. Remove 20 μL of cell culture supernatant from the cell culture plate and add it to the 96-well plate. Incubate at 37°C for 1-3 hours.

[0178] 5. The absorbance at 630 nm was measured using a Neo2 multifunctional microplate reader (Bio-tek), and the half effective concentration (EC) of the drug was calculated using GraphPad Prism. 50 .

[0179] Example 2

[0180] 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 an AAV-HBV mouse model

[0181] AAV-HBV mouse model

[0182] Male C57BL / 6 mice, 4–5 weeks old, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Mice were housed individually in polycarbonate cages with corncob bedding. The animal room maintained environmental conditions including temperature, relative humidity, and light intensity. The temperature was maintained between 21°C and 25°C, and the relative humidity was maintained between 40% and 70%. A 12-h light / 12-h dark cycle (lights on from 7:00 AM to 7:00 PM) was used. Animals had free access to food and water.

[0183] Recombinant adeno-associated virus type 8 (rAAV8-1.3HBV) carrying a 1.3-fold increase in the HBV genome was purchased from Wuhan Shumi Brain Science and Technology Co., Ltd. and stored at -70°C until use. On the first day of acclimatization, mice were injected with 200 μL of rAAV8-1.3HBV suspension via the tail vein. Blood was collected via the mandibular vein on days 22, 29, and 36 (21, 28, and 35 after AAV-HBV injection). 10 μL of serum was prepared from each mouse for quantitative HBsAg, HBeAg, and HBV DNA detection. The mice were grouped according to HBV biomarker levels.

[0184] Experimental groups (see Table 2-1)

[0185] Compound III-I-60 was prepared in solvent 1 (20% HP-β-CD 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 in water) at a concentration of 1 mg / mL.

[0186] The experimental mice were divided into groups and orally gavaged (PO) to give the solvent, compound III-I-60, compound II-22, and the drug combination of compound III-I-60 and compound II-22. The drugs were administered for a total of 6 weeks, followed by a 3-week drug withdrawal observation period. The mice in the vehicle group were gavaged with solvent 1 twice a week (BIW) and vehicle 2 twice a day (BID). In the single treatment group, mice in the compound III-I-60 group were gavaged with 10 mg / kg of the compound twice a week, and mice in the compound II-22 group were gavaged with 20 mg / kg of the compound twice a day. The combination treatment group was gavaged with 10 mg / kg of compound III-I-60 twice a week and 20 mg / kg of compound II-22 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.

[0187] Table 2-1 AAV-HBV mouse model combination study design for compound III-I-60 and compound II-22

[0188] Experimental results

[0189] As shown in Figure 1, compared to the vehicle control group, serum HBsAg and HBV DNA levels in mice treated with the TLR7 / 8 agonist compound III-I-60 monotherapy continued to decline significantly over the six-week dosing period, with a gradual rebound after dosing. After six weeks of dosing, serum HBsAg and HBV DNA levels in the compound III-I-60 monotherapy group were reduced by 2.28 Log10 and 2.76 Log10, respectively, compared to the control group. Compound III-I-60 monotherapy did not significantly affect serum HBeAg levels in mice.

[0190] As shown in Figure 1, compared to the vehicle control group, monotherapy with the capsid assembly modulator Compound II-22 resulted in a sustained and significant decrease in serum HBsAg, HBeAg, and HBV DNA levels in mice during the six-week dosing period. Following drug discontinuation, serum HBsAg and HBeAg levels remained relatively low, while serum HBV DNA levels gradually rebounded. After six weeks of dosing, serum HBsAg, HBeAg, and HBV DNA levels in the Compound II-22 monotherapy group decreased by 1.68 Log10, 1.24 Log10, and 5.57 Log10, respectively, compared to the control group.

[0191] As shown in Figure 1, compared to the vehicle control group, serum HBsAg, HBeAg, and HBV DNA levels in mice treated with the combination of Compound III-I-60 and Compound II-22 decreased significantly during the six-week dosing period and remained at relatively low levels after drug discontinuation. After six weeks of dosing, serum HBsAg, HBeAg, and HBV DNA levels in the combination-treated mice decreased by 3.41 Log10, 2.17 Log10, and 5.83 Log10, respectively, compared to the control group. Furthermore, the rate of decline in serum HBsAg in the combination-treated mice was significantly faster than in either of the monotherapy groups.

[0192] The body's immune system produces an immune response to HBV viral components, such as the production of anti-HBsAg antibodies (HBsAbs). This humoral immune response is an important pathway for inhibiting HBV reinfection and promoting HBV clearance. As shown in Table X1-2, no HBsAbs were produced in the vehicle group (Group 01) or in the capsid assembly regulator Compound II-22 monotherapy group (Group 03). In the TLR7 / 8 agonist Compound III-I-60 monotherapy group (Group 02), one and two mice produced HBsAbs after 5 and 6 weeks of treatment, respectively, with low HBsAb levels. No mice produced HBsAbs after 3 weeks of drug withdrawal. In the combination therapy group (Group 04), 5, 5, and 4 mice produced HBsAbs after 5, 6, and 3 weeks of treatment, respectively, with high HBsAb levels, and an increasing trend.

[0193] Table 2-2 Serum anti-HBsAg antibodies (HBsAb)

[0194] Example 3

[0195] 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 an AAV-HBV mouse model

[0196] AAV-HBV mouse model

[0197] Male C57BL / 6 mice, 4–5 weeks old, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Mice were housed individually in polycarbonate cages with corncob bedding. The animal room maintained environmental conditions including temperature, relative humidity, and light intensity. The temperature was maintained between 21°C and 25°C, and the relative humidity was maintained between 40% and 70%. A 12-h light / 12-h dark cycle (lights on from 7:00 AM to 7:00 PM) was used. Animals had free access to food and water.

[0198] Recombinant adeno-associated virus type 8 (rAAV8-1.3HBV) carrying a 1.3-fold increase in the HBV genome was purchased from Wuhan Shumi Brain Science and Technology Co., Ltd. and stored at -70°C until use. On the first day of acclimatization, mice were injected with 200 μL of rAAV8-1.3HBV suspension via the tail vein. Blood was collected via the mandibular vein on days 22 and 29 (21 and 28 after AAV-HBV injection). 10 μL of serum was prepared from each mouse for quantitative HBsAg, HBeAg, and HBV DNA detection. HBV biomarker levels were used to stratify the mice.

[0199] Experimental groups (see Table 3-1)

[0200] Compound VI-162 was prepared in solvent 1 (0.9% saline) at concentrations 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 lauryl sulfate, and 15 mmol / L disodium hydrogen phosphate) at a concentration of 2 mg / mL.

[0201] 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 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 treatment was administered for a total of 5 weeks, followed by a 3-week drug withdrawal observation period. Serum samples (15 + 15 μL per mouse) were collected and prepared weekly during the experiment and transferred to the clinical laboratory for quantitative determination 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.

[0202] Table 3-1. AAV-HBV mouse model combination study design for compound VI-162 and compound II-22

[0203] Experimental results

[0204] As shown in Figure 2, compared with the vehicle control group, the serum HBsAg, HBeAg and HBV DNA levels of mice in the capsid assembly regulator compound II-22 monotherapy group (Group 2) continued to decrease significantly during the five-week dosing period. After drug withdrawal, the serum HBsAg and HBeAg of the mice remained at relatively low levels, while the serum HBV DNA level gradually rebounded. Compared with the control group, after five weeks of dosing, the serum HBsAg, HBeAg and HBV DNA levels of mice in the compound II-22 monotherapy group decreased by 1.62Log10, 1.15Log10 and 4.89Log10, respectively.

[0205] As shown in Figure 2, compared with the vehicle control group, the serum HBsAg and HBV DNA levels of mice in the TLR7 / 8 agonist compound VI-162 monotherapy groups (Groups 3 and 5) continued to decline significantly during the five-week dosing period, and gradually rebounded after dosing was stopped. Compared with the control group, after five weeks of dosing, the serum HBsAg and HBV DNA levels of mice in the oral gavage monotherapy group of compound VI-162 decreased 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 decreased by 4.54Log10 and 4.03Log10, respectively. Compound VI-162 monotherapy did not significantly affect the serum HBeAg levels of mice.

[0206] As shown in Figure 2, compared with the vehicle control group, the serum HBsAg, HBeAg, and HBV DNA levels of mice in the combination treatment groups (Groups 4 and 6) of Compound VI-162 and Compound II-22 decreased significantly during the five-week dosing period and remained at a relatively low level after drug withdrawal. Compared with the control group, after five weeks of dosing, the serum HBsAg, HBeAg, and HBV DNA levels of mice in the combination treatment group 4 decreased 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 decreased by 5.11Log10, 1.84Log10, and 6.08Log10, respectively. The rate of decline in serum HBsAg in mice in the combination treatment group was significantly faster than in any of the single treatment groups.

[0207] 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, no HBsAb was produced in the vehicle group (Group 01) and the capsid assembly regulator compound II-22 monotherapy group (Group 2) after treatment. After 5 weeks of 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, 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)

[0208] Research Conclusions

[0209] Taken together, these results suggest that the combination of a TLR7 / 8 agonist and a HBV capsid modulator is effective in treating HBV, achieving faster reduction or even elimination of HBsAg, HBeAg, and HBV DNA compared to monotherapy. Furthermore, viral suppression persisted for up to three weeks after combination therapy, even without retreatment. Current treatments for chronic hepatitis B seek a functional cure, defined as HBsAg loss and / or seroconversion (production of HBsAbs). However, existing therapies rarely achieve HBsAg seroconversion. In our combination therapy, HBsAb production was detectable during treatment, whereas monotherapy groups failed to produce HBsAg or had very low HBsAb production or HBsAb levels. Furthermore, after combination therapy, HBsAb levels were maintained or showed a trend toward improvement, even without retreatment. Therefore, the combination of a TLR7 / 8 agonist and a HBV capsid modulator offers another key advantage in promoting HBsAb production, potentially leading to a functional cure for chronic hepatitis B and possessing significant clinical development potential.

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 its solvent 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 its solvent compound 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 (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 medicine for treating or preventing viral infection; preferably, the virus is hepatitis B virus HBV.

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