CN115177624A - Method for treating HBV by increasing viral empty capsid protein through oral administration - Google Patents

Abstract

The present invention provides a method for treating HBV by increasing viral empty capsid protein through oral administration. In particular, the present invention provides compounds of formula I useful for: (ii) (a) increasing viral empty capsids; (b) increasing IFN- γ production in the liver; (c) Reduction of CD4 in liver ⁺ T _reg The number of cells; (d) activating the cGAS/STING/IRF3 signaling pathway in liver tissue; and/or (e) reducing the number of virally infected hepatocytes. The compounds of the invention are capable of conferring tolerance to the immune rings in the liverThe environment is changed into an immunogenic environment, so that the virus-infected liver cells can be effectively eliminated, and the antiviral effect is achieved.

Description

Method for treating HBV by increasing viral empty capsid protein through oral administration

Technical Field

The invention relates to the field of biological immunity, in particular to a method for treating HBV (hepatitis B virus) by increasing virus empty capsid protein through oral administration.

Background

The establishment of chronic HBV is a result of the virus effectively evading the innate immune system. The HBV virus has stealth, so that the virus can effectively escape from the innate immune system, avoid activating the IFN/ISG reaction in the liver and establish immune tolerance in the liver tissue of a patient. Under conditions of immune tolerance, myeloid cells (Kupffer cells, DCs, M-MDSCs) are capable of secreting TGF- β and promoting differentiation of FoxP3+ regulatory T cells, thereby inducing T cell failure and immune tolerance. During chronic HBV infection, myeloid cells and regulatory T cells can produce IL-10, which on the one hand can inhibit the production of pro-inflammatory cytokines by hepatocytes, and on the other hand can inhibit the response of virus-specific T cells. The immune tolerance of HBV is also due to the suppression of the immune system by precore or HBeAg proteins, HBsAg and HBV virions.

The key of the body for eliminating HBV is that the immune tolerance environment is changed into the immunogenic environment, and the virus-infected liver cells are effectively eliminated. Currently, there is still a lack of effective techniques for inducing the body to eliminate HBV in vivo.

Disclosure of Invention

The present invention aims at providing a method for treating HBV by increasing the empty capsid protein of the virus by oral administration.

In a first aspect of the invention, there is provided the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the preparation of a formulation or composition for use in:

(a) Increasing viral empty capsids;

(b) Increasing IFN- γ production in the liver;

(c) Reduction of CD4 in liver ⁺ T _reg The number of cells;

(d) Activating a cGAS/STING/IRF3 signal pathway in liver tissue; and/or

(e) Reducing the number of virus-infected hepatocytes.

In the formula (I), the compound is shown in the specification,

R ₁ ，R ₂ ，R ₃ and R ₄ Each independently selected from the group consisting of: H. halogen, cyano, substituted or unsubstituted C ₃ -C ₄ Cycloalkyl of (a), substituted or unsubstituted C ₁ -C ₄ Alkyl, substituted or unsubstituted C ₁ -C ₄ Alkoxy group of (a); wherein, the substitution refers to the replacement of hydrogen atoms on the group by one or more substituents selected from the group consisting of: halogen, C ₁ -C ₄ Alkyl (e.g., difluoromethyl, difluoroethyl, monofluoromethyl, trifluoromethyl, trifluoromethoxy);

R ₅ selected from the group consisting of: H. halogen, -CN, hydroxy, amino, carboxy, - (C = O) -substituted or unsubstituted C ₁ -C ₈ Alkyl, substituted or unsubstituted C ₁ -C ₈ Alkyl, substituted or unsubstituted C ₂ -C ₆ Alkenyl, substituted or unsubstituted C ₂ -C ₆ Alkynyl, substituted or unsubstituted C ₁ -C ₈ Alkylamino radical, substituted or unsubstituted C ₁ -C ₈ Alkoxy, substituted or unsubstituted C ₃ -C ₁₀ Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C ₆ -C ₁₀ Aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O;

R _g selected from the group consisting of: H. halogen, -CN, hydroxy, amino, carboxy, - (C = O) -substituted or unsubstituted C ₁ -C ₈ Alkyl, substituted or unsubstituted C ₁ -C ₈ Alkyl, substituted or unsubstituted C ₂ -C ₆ Alkenyl, substituted or unsubstituted C ₂ -C ₆ Alkynyl, substituted or unsubstituted C ₁ -C ₈ Alkylamino radical, substituted or unsubstituted C ₁ -C ₈ Alkoxy, substituted or unsubstituted C ₃ -C ₁₀ Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C ₆ -C ₁₀ Aryl, or substituted or unsubstituted aryl having 1 to 3 substituents selected from the group consisting of N, S and5-10 membered heteroaryl of a heteroatom of O;

unless otherwise specified, "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, a group selected from the group consisting of unsubstituted or substituted with one or more substituents selected from the group consisting of: C6-C10 aryl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, halogenated 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O; the substituents are selected from the following group: halogen, C1-C6 alkoxy.

In another preferred embodiment, said increasing viral empty capsids comprises:

(a1) Increasing the ratio R1 of the number of empty capsids E1 to the number of normal capsids of the nucleic acid virus E0 (i.e., E1/E0);

(a2) Increasing the number or level of capsids;

(a3) Facilitating assembly of empty capsids; and/or

(a4) The ratio R2 of the number of viral nucleic acids C1 within the capsid to the number of viral capsids C0 (i.e. C1/C0) is reduced, wherein the number of viral nucleic acids within the capsid of one complete virus is scored as 1 viral nucleic acid.

In another preferred embodiment, R1 is 10 or more, preferably 50 or more, and more preferably 100 or more.

In another preferred embodiment, R2 is less than or equal to 1/10, preferably less than or equal to 1/50, and more preferably less than or equal to 1/100.

In another preferred embodiment, the viral nucleic acid comprises viral RNA or viral DNA.

In another preferred embodiment, the viral empty capsid protein is an empty capsid protein free of viral nucleic acid.

In another preferred embodiment, the virus is selected from hepatitis b virus, hepatitis d virus and/or hepatitis c virus.

In another preferred embodiment, the virus is selected from HBV of different genotypes, e.g. A, B, C, D, E, F, G, H, I, J genotypes.

In another preferred embodiment, the formulation or composition is further used for:

(f) Increasing the number of kupffer cells (kupffer cells and monocytes), dendritic cells and M-MDSC subpopulations;

(g) Promoting phagocytic activity of kupffer cells;

(h) Promotion of CD8 ⁺ Maturation of T cells.

In another preferred embodiment, said "inhibiting CD4 ⁺ T _reg Cell "means to reduce CD4 ⁺ T _reg Cells in CD4 ⁺ The proportion in T cells is preferably said "reduced" by an amount of at least 20%, more preferably at least 30%, most preferably at least 40%, e.g.30-50%.

In another preferred embodiment, said "inhibiting CD 4" is ⁺ T _reg By cells is meant cells used to reduce CD4 in immunosuppressed focal sites, such as tumor sites ⁺ T _reg Cells in CD4 ⁺ Proportion in T cells.

In another preferred embodiment, the formulation or composition is for administration to a subject and:

(a) Increasing viral empty capsids;

(b) Increasing IFN- γ production in the liver;

(c) Reduction of CD4 in liver ⁺ T _reg The number of cells;

(d) Activating a cGAS/STING/IRF3 signaling pathway in liver tissue; and/or

(e) Reducing the number of virally infected hepatocytes;

(f) Increasing the number of kupffer cells (kupffer cells and monocytes), dendritic cells and M-MDSC subpopulations;

(g) Promoting the phagocytic activity of kupffer cells.

(h) Promotion of CD8 ⁺ Maturation of T cells.

In another preferred embodiment, said inhibiting CD4 ⁺ T _reg The cells comprise CD 4-inhibiting ⁺ T _reg Cellular function, and/or reduction of CD4 ⁺ T _reg Number or level of cells.

In another preferred embodiment, the subject includes human and non-human mammals.

In another preferred embodiment, the compound of formula I is selected from the compounds shown in table 1 or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the compound of formula I is selected from: 10a1, 10b1, 10u1, 10v1, 20a1, 20b1, 20u1, 20v1, 20u2, 100a01, 100a03, 100a05, 100u01, 100a07, 100b01, 100b05.

In a second aspect of the invention, there is provided a pharmaceutical composition for use in humans or animals, the pharmaceutical composition comprising:

(i) A compound of formula I, or a pharmaceutically acceptable salt thereof, and

(ii) A pharmaceutically acceptable carrier;

the pharmaceutical composition is for one or more applications selected from the group consisting of:

(a) Increasing viral empty capsids;

(b) Reducing the number of virally infected hepatocytes;

(c) Increasing IFN- γ production in the liver;

(d) Reduction of CD4 in liver ⁺ T _reg The number of cells; and/or

(d) Enhancing immune function;

wherein the compound of formula I is as described in the first aspect of the invention.

In another preferred embodiment, the pharmaceutical composition further comprises: (iii) additional antineoplastic and/or antiviral drugs.

In another preferred embodiment, the pharmaceutical composition comprises 0.001-99wt%, preferably 0.1-90wt%, more preferably 1-80wt% of the compound of formula I, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, based on the total weight of the composition.

In another preferred embodiment, the anti-tumor drug is selected from the group consisting of: oxaliplatin, paclitaxel, docetaxel, capecitabine, rituximab, gefitinib, axitinib, regorafenib, cabozantinib, lenvatinib, apatinib, sorafenib, nivolumab, pembrolizumab, astuzumab, or ipilimumab, aviruzumab, doxoruzumab, or a combination thereof.

In another preferred embodiment, the antiviral drug is selected from the group consisting of: acyclovir, telbivudine, zidovudine, entecavir (ETV), tenofovir disoproxil and virgine.

In a third aspect of the present invention, there is provided an antiviral method comprising the steps of: administering to a subject in need thereof a compound of formula I, or an optical isomer or racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein the compound of formula I is as described in the first aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal (e.g., a rodent).

In another preferred embodiment, the virus is selected from hepatitis B virus, hepatitis D virus and/or hepatitis C virus.

In another preferred embodiment, the virus is selected from HBV of different genotypes, e.g. A, B, C, D, E, F, G, H, I, J genotypes.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

FIG. 1 shows the mechanism of action (MOA) of LW231 in clearing HBV infection.

Fig. 2 shows Western blot detection of HBV capsid, wherein a, hepg2.2.15 cells were treated with LW231 and other reference compounds at indicated concentrations for 6 days. The cell lysates were subjected to electrophoresis in a non-denaturing agarose gel. The intact HBV capsids were detected by Western blotting using polyclonal rabbit anti-HBV core antibody. Encapsidated HBV DNA and RNA were detected by Southern and Northern blotting, respectively. The core protein was detected by SDS-PAGE with monoclonal mouse anti-HBV core antibody. Beta-actin served as loading control. B, the effect of LW231 treatment on HBV capsid morphology was evaluated by electron microscopy. DMSO control, LW231, and reference compound AT130 and GLS4 treated groups are shown, respectively. Images are typically acquired at 110000 times magnification.

FIG. 3 shows that LW231 increases IFN-. Gamma.production in liver CTLs. Wherein vehicle is solvent, wk(s) is week number.

FIG. 4 shows LW231 down-regulates the amount of regulatory T cells (T-reg). wk(s) is the number of weeks.

FIG. 5 shows the PK/PD dependence of LW231. AAV-HBV mice were divided into 3 groups (n = 3) and LW231 was orally administered. Blood samples were collected at the indicated time points and analyzed for LW231 plasma concentrations. The unbound concentration was converted from the total concentration to 13.6% of the modulator from the plasma protein binding assay. EC (EC) ₅₀ ，EC ₉₀ And CC ₅₀ The concentration of (b) is estimated according to in vitro experiments.

FIG. 6 shows the inhibitory effect of LW231 on serum HBV viral markers. AAV-HBV mice were randomly grouped according to serum HBsAg, HBeAg and HBV DNA levels and treated with LW231 at doses of 50, 100 and 200mg/kg bid. Entecavir ETV (0.1 mg/kg, q.d.) treated mice or vehicle (vehicle) were used as positive and negative controls, respectively. A combination of LW231 and ETV of 100mg/kg was also tested. All three viral indicators were monitored weekly and changes in predose levels plotted on a logarithmic scale. A. Changes in serum HBV DNA. B. Change in serum HBsAg. C. Change in serum HBeAg.

Detailed Description

The inventor of the invention has extensively and deeply studied and screened a lot to obtain a compound (represented by LW231 compound) with a novel structure, which can increase virus empty capsid, promote the assembly of empty capsid without virus nucleic acid, lead HBV virus DNA to accumulate in cytoplasm, further stimulate the innate immune system of cells, activate hepatophagocytic cells and dendritic cells, convert the immune tolerance environment in liver into the immunogenic environment, and finally lead to the elimination of HBV-infected liver cells. The present invention has been completed based on this finding.

Term(s) for

As used herein, the terms "nucleic acid-free capsid," "nucleic acid-free viral capsid," "empty viral particle," "empty virus-like particle," "empty VLP" are used interchangeably to refer to a viral capsid or viral particle that does not contain intact viral nucleic acid inside the viral capsid or viral particle. In the present invention, since the empty viral capsid does not contain a viral nucleic acid in the inside thereof, the viral nucleic acid is lacking even after endocytosis by the cell, and thus viral proteins cannot be replicated in the cell, and the cell cannot be re-infected. In addition, the empty viral capsid has a spatial structure that is nearly identical to that of an intact virus, and thus can stimulate an immune response in a body (e.g., humans and non-human mammals).

As used herein, the term "alkyl" includes straight or branched chain alkyl groups. E.g. C ₁ -C ₈ Alkyl represents a straight or branched chain alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.

As used herein, the term "alkenyl" includes straight or branched chain alkenyl groups. E.g. C ₂ -C ₆ Alkenyl means a straight or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

As used herein, the term "alkynyl" includes straight or branched chain alkynyl groups. Such as C ₂ -C ₆ Alkynyl means straight or branched chain alkynyl having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

As used herein, the term "C ₃ -C ₁₀ Cycloalkyl "refers to cycloalkyl groups having 3 to 10 carbon atoms. It may be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may also be in the form of a double ring, for example a bridged or spiro ring.

As used herein, the term "C ₁ -C ₈ Alkylamino "is defined as being substituted by C ₁ -C ₈ The amino substituted by the alkyl can be mono-substituted or di-substituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylaminoAmino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-tert-butylamino and the like.

As used herein, the term "C ₁ -C ₈ Alkoxy "means a straight or branched chain alkoxy group having 1 to 8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy and the like.

As used herein, the term "3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the following groups N, S and O" refers to a saturated or partially saturated cyclic group having 3-10 atoms and wherein 1-3 atoms are heteroatoms selected from the following groups N, S and O. It may be monocyclic or may be in the form of a double ring, for example a bridged or spiro ring. Specific examples may be oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, pyrrolidinyl, and the like.

As used herein, the term "C ₆ -C ₁₀ Aryl "means an aryl group having 6 to 10 carbon atoms, for example, phenyl or naphthyl and the like.

As used herein, the term "5-10 membered heteroaryl group having 1-3 heteroatoms selected from the following groups N, S and O" refers to a cyclic aromatic group having 5-10 atoms, wherein 1-3 atoms are heteroatoms selected from the following groups N, S and O. It may be a single ring or a condensed ring form. Specific examples thereof may be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1, 2, 3) -triazolyl and (1, 2, 4) -triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl and the like.

Unless specifically stated to be "substituted or unsubstituted", the groups of the present invention may be substituted with a substituent selected from the group consisting of: halogen, nitrile group, nitro group, hydroxyl group, amino group, C ₁ -C ₆ Alkyl-amino, C ₁ -C ₆ Alkyl radical, C ₂ -C ₆ Alkenyl radical, C ₂ -C ₆ Alkynyl, C ₁ -C ₆ Alkoxy, halo C ₁ -C ₆ Alkyl, halo C ₂ -C ₆ Alkenyl, halo C ₂ -C ₆ Alkynyl, halo C ₁ -C ₆ Alkoxy, allyl, benzyl, C ₆ -C ₁₂ Aryl radical, C ₁ -C ₆ alkoxy-C ₁ -C ₆ Alkyl radical, C ₁ -C ₆ Alkoxy-carbonyl, phenoxycarbonyl, C ₂ -C ₆ Alkynyl-carbonyl, C ₂ -C ₆ Alkenyl-carbonyl, C ₃ -C ₆ Cycloalkyl-carbonyl, C ₁ -C ₆ Alkyl-sulfonyl, and the like.

As used herein, "halogen" or "halogen atom" refers to F, cl, br, and I. More preferably, the halogen or halogen atom is selected from F, cl and Br. "halogenated" means substituted with an atom selected from F, cl, br, and I.

Unless otherwise specified, the structural formulae depicted herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, the asymmetric center-containing R and S configuration, and the double bond (Z) and (E) isomers. Thus, individual stereochemical isomers of the compounds of the present invention or mixtures of enantiomers, diastereomers or geometric isomers (or conformers) thereof are within the scope of the present invention.

As used herein, the term "tautomer" means that structural isomers having different energies may exceed the low energy barrier, thereby converting with each other. For example, proton tautomers (i.e., proton transmutations) include interconversion by proton shift, such as 1H-indazoles and 2H-indazoles. Valence tautomers include interconversion by some recombination of bonding electrons.

As used herein, the term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio.

As used herein, the term "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

Active ingredient

As used herein, "compound of the invention" refers to a compound of formula I, and also includes and various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of the compound of formula I:

as used herein, "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base, which is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed with acids from the compounds of the present invention. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

In another preferred embodiment, R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R _g Each independently is a group corresponding to each compound in table 1.

Preferred compounds of the invention are shown in table 1:

TABLE 1

In another preferred embodiment, a preferred compound of the invention is compound 10a1, 10b1, 10u1, 10v1, 20a1, 20b1, 20u2, 20v1, 100a01, 100a03, 100a05, 100u01, 100a07, 100b01, 100b05 of table 1 or a pharmaceutically acceptable salt thereof.

Pharmaceutical compositions and methods of administration

Since the compounds of the present invention have excellent activities of enhancing immune functions of a subject, for example, increasing innate immune system functions including activation of intracellular cGAG/STING innate immune system signaling pathway, increase of innate immune system cells, increase and activation of antigen presenting cells, CD8 ⁺ Increase in T cell Activity, T _reg The reduction of the number of cells, and thus the compound of the present invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound of the present invention as a main active ingredient can be used for the prevention and/or treatment (stabilization, alleviation or cure) of tumors, or diseases infected with viruses.

In another preferred embodiment, the tumor is selected from the group consisting of: pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, melanoma, neuroendocrine cancer, central nervous system cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small cell lung cancer or colon cancer, skin cancer, lung cancer, urologic tumors, hematologic tumors, glioma, digestive system tumors, reproductive system tumors, lymphoma, nervous system tumors, brain tumors, head and neck cancer.

In another preferred embodiment, the virus is selected from the group consisting of: hepatitis B virus, measles virus, mumps virus, rabies virus, influenza virus, EB virus, hepatitis C virus, hepatitis D virus and avian influenza virus.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention in combination with 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. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-200mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and between the compounds of the present invention without significantly diminishing the pharmaceutical effectiveness of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).

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, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) Disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary amine compounds; (g) Wetting agents, such as cetyl alcohol and glycerol 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 capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such a composition may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, especially cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

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

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 nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

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

When administered in combination, the pharmaceutical composition further comprises one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds (e.g., anti-HBV agents). One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds (e.g., anti-HBV agents) may be used simultaneously, separately or sequentially with the compounds of the present invention for the prevention and/or treatment of HBV infection or HBV-related disease.

When using pharmaceutical compositions, a safe and effective amount of a compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the administration is a pharmaceutically acceptable and effective dose, and the daily dose for a human of 60kg body weight is usually 1 to 2000mg, preferably 20 to 500mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Mechanism for eliminating HBV by the present invention

LW231 enhances the assembly of empty capsids (fig. 1), which will result in the accumulation of HBV viral DNA in the cytoplasm. Thus, the cGAS/STING signaling channel of the innate immune system is activated by naked HBV viral DNA in the cytoplasm, resulting in increased phosphorylation of the downstream transcription factor IRF3, which enters the nucleus and activates transcriptional expression of type I interferon. The released type I interferon can stimulate a series of immune responses, including autocrine stimulation of JAK/STAT signal channels and induction of interferon stimulation gene expression. Type I interferons released by hepatocytes may also stimulate kupffer cells. Studies have demonstrated that activated kupffer cells are able to clear infected hepatocytes by direct phagocytosis and induce hepatocyte apoptosis through the release of toxic molecules such as granzyme B and perforin (Boltjes et al, 2014).

In the chronic infection stage, liver HBV DNA exists in the nucleus either in the form of cDNA or in the form of chromosomal integration. Transcription of HBV DNA produces 4 mRNAs such as pgRNA, 0.7, 2.1, 2.4, and 3.5kb, respectively. From these mRNAs, viral proteins including core protein (HBcAg), surface protein (HBsAg) and polymerase can be produced. pgRNA is responsible for polymerase ligation, complex formation, and then encapsidation by the core protein, forming the pgRNA capsid, which is further encapsidated by the surface protein to form the virion, which is then released into the circulation. Within the pgRNA capsid, the pgRNA is reverse transcribed as single stranded DNA and then transcribed as rcDNA. rcDNA capsids are also further encapsulated by surface proteins and released into the circulation. In addition, virus-infected cells form a large number of empty capsids that do not contain viral RNA/DNA and are released from the cells after surface protein encapsulation. In the blood of patients infected with HBV, the number of empty capsids is about 100 times that of virions. In addition, infected cells also secrete 1000 to 10,000 times more empty enveloped particles or filaments than virions (Tang et al, 2017).

LW231 enhances the assembly of empty capsids in HBV-infected liver cells (Zhang and Wang 2020), thereby leading to a substantial reduction in virion formation containing hepatitis b virus nucleic acid, resulting in accumulation of viral DNA in the cytoplasm, activation of and innate immunity of the cGAS/STING signaling pathway. Subsequently, phosphorylation of the downstream transcription factor IRF3 increases. Phosphorylated IRF3 enters the nucleus and activates transcription of type I IFN. Type I interferons can stimulate the JAK1/Tyk2/STAT signaling pathway (Gonz a lez-Navajas et al, 2012) in an autocrine manner, phosphorylate STAT1 homodimers and STAT1/STAT2 heterodimers, increasing transcription of interferon-stimulated genes (ISGs). Shortly after treatment, release of type I interferon and other proinflammatory cytokines from infected hepatocytes increases kupffer cells (kupffer)French cells and monocytes), dendritic cells and subsets of M-MDSCs. LW231 treatment promotes phagocytic activity of kupffer cells, resulting in clearance of infected hepatocytes. In addition, activated kupffer cells and dendritic cells may enhance CD8 ⁺ Maturation of T cells, differentiation into Cytotoxic T Lymphocytes (CTLs), enhances IFN- γ production. In addition, activated Kupffer cells and CTLs can secrete cytokines IL-1, IL-18, and IFN- γ that inhibit viral propagation (Boltjes et al, 2014).

In the invention, liver cells are separated from LW231 administration mice, single-cell RNA sequencing analysis is carried out on thousands of liver cells, and transcriptome of liver macrophagocyte is analyzed, so that the biological processes related to cytokine production and phagocytosis can be enhanced after LW231 administration, and the important role of liver macrophages in eliminating HBV-infected liver cells is proved. Hepatic dendritic cells, on the other hand, show negative regulation of the immunogenic process, which may be an important protective mechanism to protect liver tissue from immunogenic damage by kupffer cells. The literature shows that activated macrophages and dendritic cells are able to stimulate liver infiltrating CD8 ⁺ T cells differentiate into CTLs, leading to the formation of IFN- γ (Crispe, 2009, eckert et al, 2015, a. Et al, 2011.

The present invention demonstrates that LW231 treatment can increase IFN- γ production in liver CTLs (fig. 3), suggesting that LW231 can simultaneously activate both innate and adaptive immune systems, leading to clearance of AAV-HBV infection. Activation of CTLs may be due to down-regulation of regulatory T cells by LW231 (fig. 4).

The LW231 treatment demonstrated not only an increase in myeloid subpopulations such as Kupffer cells, but also an enhancement of these effector functions, particularly enhancement of phagocytic activity of Kupffer cells.

Thus, the present invention reveals that activation of macrophage and CTL immune functions by LW231 therapy results in clearance of virus-infected hepatocytes, and that dendritic cells negatively regulate immunogenicity as the virus clears in liver tissue. At the same time, LW231 treated an increased subpopulation of M-MDSC in liver tissue. These cells may play a role in maintaining hepatocyte homeostasis.

The invention discloses an action mechanism of LW231 for causing liver AAV-HBV clearance. LW231 converts the immune tolerance environment into an immunogenic environment in AAV-HBV infected liver by activating the innate cGAS/TING pathway, where the number and effector functions of liver macrophages, dendritic cells and other immune cells are induced, leading to clearance of virus-infected hepatocytes.

In the present invention, there is also provided a mechanism of action (MOA) of LW231 treatment to cause HBV clearance in AAV-HBV model and it was revealed that the induction of innate immunity cGAS/STING signaling channel and the activation of kupffer cells and dendritic cells by LW231 are the main mechanism of action for HBV clearance.

According to the results of the present invention, LW231 cleared the MOA of HBV infection as shown in FIG. 1.

The main advantages of the present invention include:

(1) The LW231 of the invention converts the immune tolerance environment in the AAV-HBV infected liver into an immunogenic environment, so that the virus-infected liver cells can be effectively eliminated;

(2) The compound has good drug forming property;

(3) The compound and the pharmaceutical composition containing the compound as the main component can be used for treating related diseases such as hepatitis B and the like.

(4) The compound of the invention can simultaneously activate the functions of the innate immune system and the acquired immune system, and improve the antiviral and anticancer abilities of the organism.

The invention is further described below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally according to conventional conditions such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

General procedure

Effect of LW231 on hepatitis B Virus capsid Assembly

HepG2.2.15 cells were cultured in DMEM/F12 medium supplemented with 2% fetal bovine serum (FBS, hyClone). Compounds were tested for 6 days. Cells were lysed with NP-40. Lysates were assessed by non-denaturing agarose gel electrophoresis. HBV capsid was detected by Western blot using polyclonal rabbit anti-HBV core antibody. Encapsidated HBV DNA and RNA were detected by Southern blot and Northern blot analysis. Intracellular HBV core protein was assessed by western blot analysis using monoclonal mouse anti-HBV core antibodies. Beta-actin was evaluated by western blot analysis and used as an internal control. The results of western blot analysis were visually observed.

Quantification of liver HBV DNA

Liver tissue was homogenized with Qiagen TissueLyser and then digested with proteinase K. The aqueous phase was collected and digested with RNase A (Sigma, R4642). Digestion of the mixture with phenol: chloroform: isoamyl alcohol was extracted twice. DNA was precipitated with isopropanol and dissolved in H ₂ O HBV DNA was quantified by TaqMan real-time PCR assay.

Quantification of liver 3.5kb HBV RNA

Liver tissue was homogenized using Qiagen tissue Lyser and TRIzol (Life Technologies). The aqueous phase was collected and extracted twice with chloroform. RNA was precipitated with isopropanol and dissolved in DEPC treated H ₂ And (O). cDNA was synthesized by reverse transcription using the FastQuant RT kit (with gDNase) according to the manual. The obtained cDNA was quantified by TaqMan real-time PCR assay, supplemented with FastStart Universal Probe (ROX) (Roche, 04914058001) with 3.5kb HBV RNA specific probe and primers (3.5 kb HBV RNA specific primer F and 3.5kb HBV RNA specific primer (R) as reference genes, and GAPDH expression level was determined using specific primers with ID Mm99999915 from ABI.

The following formula is used:

Δ Ct = Ct average of target gene-Ct average of housekeeping gene.

Δ Δ Ct = Δ Ct for the Δ Ct-carrier group of compound-treated samples.

Serum HBV RNA quantification

According to the handbook, pureLink is used ^TM Serum RNA was extracted from Pro 96 virus RNA/DNA kit. After digestion of the DNA with DNase, HBV RNA was reverse transcribed into cDNA using 3' RACE primer containing HBV specific sequence, anchor sequence and poly T sequence. The resulting cDNA was amplified by real-time PCR using HBV X gene specific primers, fluorophore-labeled TaqMan probe in combination with anchor sequence primers.

Immunohistochemistry

Immunohistochemical (IHC) staining was used to detect HBsAg and HBcAg expression in liver tissue sections. Mouse liver was fixed with 10% nbf, processed by conventional methods, dehydrated, paraffin embedded and sectioned. Section samples with a thickness of 4 μm were prepared from formalin-fixed, paraffin-embedded (FFPE) livers and subjected to deparaffinization, antigen recovery, serum blocking, primary and secondary antibody incubation and DAB staining as a routine IHC analysis.

With citric acid antigen recovery solution (MXB Biotechnologies, MVS-0100) and 3% ₂ O ₂ Liver tissue was treated and blocked with goat serum. Polyclonal horse anti-HBsAg antibody (Abcam, ab 9193) and rabbit anti-horse IgG H were used&L (HRP) (Abcam, ab 6921) detects intrahepatic HBsAg. Polyclonal rabbit anti-HBsAg antibodies (Abcam, ab 115992) and donkey anti-rabbit IgG H were used&L (HRP) (Abcam, ab 6802) detects intrahepatic HBcAg. Positive hepatocytes were stained with DAB (Vector Laboratories, SK-4105) and stained brown; the nuclei were stained with hematoxylin (Harris, BASO, BA 4041) and stained blue. HBcAg ⁺ Quantification of hepatocytes was performed with an automated slide scanner (Aperio versia 200 Brightfield)&Fluorescence).

Antiviral assay of HepG2.2.15 cells

HepG2.2.15 cells were cultured in DMEM/F12 medium supplemented with 2% fetal bovine serum (FBS; hyClone). Mixing the raw materials in a ratio of 1: serial dilutions of 3 were assayed at 8 concentrations in triplicateTest compounds were prepared. Cells were treated with compound for 6 days. HBV DNA was extracted from the cell culture supernatant using QIAamp 96 DNA Blood Kit (Qiagen). HBV DNA was quantified by TaqMan real-time PCR analysis. HBV DNA copy number was determined from Ct values and standard curve for each sample. Percent inhibition was calculated by the following formula: % Inh. = (1-number of HBV in sample/number of HBV in DMSO control) × 100.EC (EC) ₅₀ And EC ₉₀ Values were plotted using GraphPad Prism using the "four parameter logistic equation". Cytotoxicity of the compounds was measured using CellTiter-Glo (Promega) and CC was calculated ₅₀ And CC ₉₀ The value is obtained. SI was calculated as the mean CC of LW231 measured in HepG2.2.15 cells ₅₀ Or CC ₉₀ Values and mean EC of LW231 measured in HepG2.2.15 cells ₅₀ Or EC ₉₀ The ratio of the values.

Efficacy study of AAV-HBV mice

Male C57BL/6 mice, 4-5 weeks old, were purchased from Shanghai Lingchang Biotechnology Ltd, shanghai, china. After an adaptation period of 7 days, mice were injected intravenously with 1X 10 injections via the tail vein ¹¹ rAAV8-1.3HBV (batch number: 2019032703, genotype D, serotype ayw) was purchased from the molecular medicine institute of FivePlus, beijing, china, and model-induced in 200. Mu.L Phosphate Buffered Saline (PBS). Five weeks after infection, eligible AAV-HBV-infected mice were selected for treatment. The HBeAb and HBV DNA levels were randomized according to serum HBsAb. LW231 bid, 50, 100 and 150mg/kg, was then administered orally to mice, and all three viral indicators were monitored weekly. ETV was used as a positive control treatment at 0.1mg/s q. A combination of LW231 and ETV at 100mg/kg was also administered. Six weeks after treatment, only selected groups continued with an extended dosing period until day 112, half of the animals were sacrificed 4 hours after the last dose to collect serum and liver tissue for further analysis.

Preparation of compounds

The preparation method of the compound in the invention is referred to the preparation method in the embodiment of Chinese application CN 2019100275736.

Test compound LW231 is one of the preferred compounds of the invention 10a1, 10b1, 10u1, 10v1, 20a1, 20b1, 20u2, 20v1, 100a01, 100a03, 100a05, 100u01, 100a07, 100b01, 100b05.

Example 1 LW231 enhances the nucleic acid capsid free content in HBV-infected HepG2.2.15 hepatocytes

Experiment HBV-infected HepG2.2.15 hepatocytes were cultured for 6 days with LW231 added at final concentrations of 10, 100, 1000nM, and the cell lysates were electrophoresed in non-denaturing agarose gels and the intact HBV capsids were detected by Western blotting.

As shown in fig. 2A, LW231 increased the level of nucleocapsid and exhibited a concentration dependence. LW231 did not promote the expression of nucleocapsid monomers in cells at final concentrations of 100nM and 1000 nM. The increase in nucleocapsid (iv in FIG. 2A) is due to the promotion of nucleocapsid protein polymerization. LW231 at 100nM and 1000nM final concentrations completely inhibited complete HBV capsid loading of HBV DNA detected by Southern blotting (ii of fig. 2A) and HBV RNA detected by Northern blotting (iii of fig. 2A) compared to DMSO negative controls.

LW231, starting at 100nM, enhances the formation of empty capsids without HBV DNA or RNA. The reference compound AT130 also enhanced the formation of nucleic acid-free capsids only AT 1000 nM. In contrast, the CpAM-type reference compound GLS4 resulted in abnormal assembly of the capsid. Thus, intact capsids could not be detected in the treated cells. As expected, treatment with ETV inhibited DNA formation.

HBV capsid examined by electron microscopy showed normal capsid morphology in LW231 treated cells, but was also observed in AT130 treated cells, but was different from the abnormal capsid morphology of GLS4 treated cells (fig. 2B). Thus, LW231 can enhance the formation of morphologically intact nucleic acid-free capsids. As can be seen from the figure, at 100nM of the compound of the invention, there is almost no viral nucleic acid inside the nucleocapsid, which suggests that the ratio R2 of the number of viral nucleic acids C1 inside the capsid to the number of viral capsids C0 (i.e., C1/C0) is close to 0, i.e., much less than 1/100 (where the number of viral nucleic acids inside the capsid of one complete virus is denoted as 1 viral nucleic acid). In other words, the ratio R1 of the number of empty capsids E1 to the number of normal capsids E0 (i.e., E1/E0) is much greater than 100.

In addition, without nucleocapsid loading, nuclear-produced HBV DNA and HBV DNA will be naked in the cytoplasm, thereby stimulating the innate immune system inherent to the cell.

Example 2 treatment of LW231 increases IFN-. Gamma.in liver CTLs

The percentage of CTLs in total hepatocytes identified by single cell RNA sequencing was not affected by LW231 treatment. Analysis of IFN-. Gamma.expression in CTL showed that the percentage of CTLs expressing IFN-. Gamma.increased significantly at week 1 after LW231 treatment, and this increase did not occur anymore at week 26 (FIG. 3).

The results indicate that LW231 treatment can increase IFN- γ production in liver CTLs, indicating that LW231 can activate both innate and adaptive immune systems, leading to clearance of AAV-HBV infection.

Example 3 treatment of LW231 Down-Regulation of T _reg Cells (regulatory T cells)

Activation of CTLs may also be due to down-regulation of regulatory T cells by LW231 (fig. 4).

To explore how LW231 treatment affected the hepatic immune system to result in clearance of AAV-HBV infection, single cell suspensions were prepared from mouse liver tissue that was continuously dosed LW231 for 1 and 26 weeks and subjected to FACS analysis.

CD4 in liver tissue analyzed by FACS method ⁺ The number of cells was significantly reduced after continuous administration of LW231. For CD4 ⁺ Cell subsets expressing FoxP3 were further analyzed and the results indicated that FoxP3 positive T cells (i.e., T cells) _reg Cells) in CD45 ⁺ The proportion in the cells (myeloid leukocytes) decreased significantly, from 1.0% to 0.6% (p.ltoreq.0.05) and 0.6% (p.ltoreq.0.05) of the baseline level at week 1 and week 26, respectively, with a 40% decrease. This indicates that LW231 administration significantly reduced CD4 ⁺ T _reg Number of cells (FIG. 4).

Example 4 anti-HBV Activity of LW231 against HBV of different genotypes

LW231 was tested for antiviral activity against different HBV genotypes in hepg.2 cells transiently transfected with DNA of HBV genotypes a to J. HBV DNA was extracted from cell culture supernatants and DNA levels were assessed by qPCR. In each experiment, EC50 values were determined from the mean inhibition value of 2 wells per compound concentration.

qPCR analysis of HBV DNA in cell culture supernatants showed that LW231 was effective in inhibiting LW231 with EC50 values of 8-60nM or lower from all genotypes measured (table 2). The EC50 value was measured to be about 10nM for the global dominant genotypes A, B and C.

TABLE 2 EC50 of LW231 against different genotypes of HBV

Ge-based n-gene ot type ype	GeneBank ID	LW231(nM)	ETV(nM)
				A	HE974371	9.86	4.68
B	JN406371	10.13	3.12
				C	AB246346	9.02	3.07
D	U95551	29.23	6.69
				E	HE974380	58.31	2.92
F	HE974369	22.50	3.74
				G	AB064315	17.67	3.78
H	AB179747	22.31	4.35
				I	FJ023673	8.27	5.08
J	AB486012	7.52	5.90

Example 5 pharmacokinetic characterization of LW231

Pharmacokinetic (PK) studies in AAV-HBV mice were performed to evaluate the pharmacological activity of in vivo efficacy studiesAnd (4) dosage. Serum levels of compounds were measured after oral administration of 50, 100 and 200mg/kg of LW231. The concentration of unbound LW231 in plasma was compared to the EC derived from cell-based assays ₅₀ And EC ₉₀ Values are plotted (fig. 5).

The results show that plasma concentrations of unbound LW231 remained EC for more than 12 hours at doses of 100 and 200mg/kg ₉₀ Values above 20.12ng/ml, supporting the b.i.d. (twice daily) dosing regimen in the efficacy study of LW231.

Example 6 treatment of LW231 in mice stably infected with AAV-HBV substantially completely eliminates HBV

Antiviral activity of LW231 was evaluated in mice stably infected with AAV-HBV. The study was divided into two phases. First phase, lasting 6 weeks, with emphasis on assessing the optimal antiviral dose of LW231. As shown in fig. 6, LW231 treatment resulted in a rapid and dose-dependent decrease in serum HBV DNA levels (fig. 6A). By day 42, HBV DNA levels were reduced by 0.24, 2.15 and 2.25 logs compared to pretreatment levels of mice dosed with 50, 100 and 200mg/kg LW231, respectively. Notably, LW231 treatment reduced serum levels of HBsAg by 0.82log and 1.11log, respectively, in mice in the 100mg/kg treatment group (fig. 6b, c). Unexpectedly, the reduction of HBsAg (-0.48 log) and HBeAg (-0.73 log) was smaller in the 200mg/kg treatment group than before treatment, with 100mg/kg being the optimal dose of LW231 for antiviral activity.

In contrast, although ETV treatment effectively inhibited HBV DNA (-3.82 log), there was no effect on HBsAg and HBeAg levels. Combination treatment of ETV with LW231 (100 mg/kg) reduced HBV DNA levels synergistically by 5.08log, but also reduced the inhibitory effects of LW231 on HBsAg (p < 0.05) and HBeAg (p < 0.001).

Example 7 Effect of preferred Compounds of formula I on nucleocapsid-free content in hepatocytes after treatment

In this example, the effect of another of the preferred compounds of formula I on nucleocapsid free content in hepatocytes was tested using the method of example 1.

The results indicate that the compound enhances the formation of morphologically intact nucleic acid-free capsids. At 100nM of the compound, there is almost no viral nucleic acid inside the nucleocapsid.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. Use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the preparation of a formulation or composition for:

(a) Increasing viral empty capsids;

(b) Increasing IFN- γ production in the liver;

(c) Reduction of CD4 in liver ⁺ T _reg The number of cells;

(d) Activating a cGAS/STING/IRF3 signaling pathway in liver tissue; and/or

(e) Reducing the number of virus-infected hepatocytes;

in the formula (I), the compound is shown in the specification,

R ₁ ，R ₂ ，R ₃ and R ₄ Each independently selected from the group consisting of: H. halogen, cyano, substituted or unsubstituted C ₃ -C ₄ Cycloalkyl of (a), substituted or unsubstituted C ₁ -C ₄ Alkyl, substituted or unsubstituted C ₁ -C ₄ Alkoxy of (2); wherein said substitution means that the hydrogen atoms on the group are substituted by one or more substituents selected from the group consisting of: halogen, C ₁ -C ₄ Alkyl (e.g., difluoromethyl, difluoroethyl, monofluoromethyl, trifluoromethyl, trifluoromethoxy);

R ₅ selected from the group consisting of: H. halogen, -CN, hydroxy, amino, carboxyl- (C = O) -substituted or unsubstituted C ₁ -C ₈ Alkyl, substituted or unsubstituted C ₁ -C ₈ Alkyl, substituted or unsubstituted C ₂ -C ₆ Alkenyl, substituted or unsubstituted C ₂ -C ₆ Alkynyl, substituted or unsubstituted C ₁ -C ₈ Alkylamino radical, substituted or unsubstituted C ₁ -C ₈ Alkoxy, substituted or unsubstituted C ₃ -C ₁₀ Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, substituted or unsubstituted C ₆ -C ₁₀ Aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O;

R _g selected from the group consisting of: H. halogen, -CN, hydroxy, amino, carboxy, - (C = O) -substituted or unsubstituted C ₁ -C ₈ Alkyl, substituted or unsubstituted C ₁ -C ₈ Alkyl, substituted or unsubstituted C ₂ -C ₆ Alkenyl, substituted or unsubstituted C ₂ -C ₆ Alkynyl, substituted or unsubstituted C ₁ -C ₈ Alkylamino radical, substituted or unsubstituted C ₁ -C ₈ Alkoxy, substituted or unsubstituted C ₃ -C ₁₀ Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C ₆ -C ₁₀ Aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O;

unless otherwise specified, "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, a group selected from the group consisting of unsubstituted or substituted with one or more substituents selected from the group consisting of: C6-C10 aryl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, halogenated 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O; the substituents are selected from the following group: halogen, C1-C6 alkoxy.

2. The use of claim 1, wherein said increasing the empty capsid of the virus comprises:

(a1) Increasing the ratio R1 of the number of empty capsids E1 to the number of normal capsids of the nucleic acid virus E0 (i.e., E1/E0);

(a2) Increasing the number or level of capsids;

(a3) Facilitating assembly of empty capsids; and/or

(a4) The ratio R2 of the number of viral nucleic acids C1 within the capsid to the number of viral capsids C0 (i.e. C1/C0) is reduced, wherein the number of viral nucleic acids within a capsid of one complete virus is scored as 1 viral nucleic acid.

3. The use of claim 1, wherein said formulation or composition is for administration to a subject and:

(a) Increasing viral empty capsids;

(b) Increasing IFN- γ production in the liver;

(c) Reduction of CD4 in liver ⁺ T _reg The number of cells;

(d) Activating a cGAS/STING/IRF3 signaling pathway in liver tissue; and/or

(e) Reducing the number of virally infected hepatocytes;

(f) Increasing the number of kupffer cells (kupffer cells and monocytes), dendritic cells and M-MDSC subpopulations;

(g) Promoting phagocytic activity of kupffer cells;

(h) Promotion of CD8 ⁺ Maturation of T cells.

4. The use according to claim 1, wherein the compound of formula I is selected from the group consisting of the compounds shown in table 1, or a pharmaceutically acceptable salt thereof.

5. The use according to claim 4, wherein the compound of formula I is selected from: 10a1, 10b1, 10u1, 10v1, 20a1, 20b1, 20u1, 20v1, 20u2, 100a01, 100a03, 100a05, 100u01, 100a07, 100b01, 100b05.

6. A pharmaceutical composition for use in humans or animals, said pharmaceutical composition comprising:

(i) A compound of formula I, or a pharmaceutically acceptable salt thereof, and

(ii) A pharmaceutically acceptable carrier;

the pharmaceutical composition is for one or more applications selected from the group consisting of:

(a) Increasing viral empty capsids;

(b) Reducing the number of virally infected hepatocytes;

(c) Increasing IFN- γ production in the liver;

(d) Reduction of CD4 in liver ⁺ T _reg The number of cells; and/or

(d) Enhancing immune function;

wherein the compound of formula I is as described in claim 1.

7. The pharmaceutical composition of claim 6, wherein said pharmaceutical composition further comprises: (iii) additional antineoplastic and/or antiviral drugs.

8. The pharmaceutical composition of claim 7, wherein the anti-neoplastic drug is selected from the group consisting of: oxaliplatin, paclitaxel, docetaxel, capecitabine, rituximab, gefitinib, axitinib, regorafenib, cabozantinib, lenvatinib, apatinib, sorafenib, nivolumab, pembrolizumab, astuzumab, or ipilimumab, aviruzumab, doxoruzumab, or a combination thereof.

9. The pharmaceutical composition of claim 7, wherein the antiviral drug is selected from the group consisting of: acyclovir, telbivudine, zidovudine, entecavir (ETV), tenofovir disoproxil, virgine.

10. An antiviral method comprising the steps of: administering to a subject in need thereof a compound of formula I, or an optical isomer or racemate thereof, or a solvate or pharmaceutically acceptable salt thereof, wherein the compound of formula I is as set forth in claim 1.

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