Use of Palovarotene in the Treatment of HBV Virus

Abstract

The present application relates to the field of antiviral treatment of hepatitis B, in particular to the use of compounds of formula I in the treatment against the HBV virus. [Formula 1] TIFF2023528995000020.tif50114 The present invention relates to the use of a compound represented by

Description

[Technical field]

[0001] This application claims the benefit of priority from Chinese Application No. 202010531845.9, filed on June 11, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Technical Field This application relates to the field of antiviral drug therapy for Hepatitis B, and in particular to compounds of formula I

[0003] [ka]

[0004] The present invention relates to the use of a small molecule drug represented by the formula (e.g., palovarotene) in anti-HBV therapy. [Background technology]

[0005] Background technology Viral hepatitis B, abbreviated as hepatitis B, is a disease caused by infection of the body with the hepatitis B virus (HBV). The World Health Organization estimates that 3.5% of the world's population (257 million people) are infected with the hepatitis B virus, with approximately 5 million new infections each year. Worldwide, over 800,000 people die each year from various liver diseases caused by HBV infection, including chronic active hepatitis, cirrhosis, and hepatocellular carcinoma. China is a moderate or high endemic area for hepatitis B virus infection. Although widespread vaccination with hepatitis B vaccine has effectively reduced the number of new HBV infections, it is estimated that there are still more than 70 million chronic HBV infected people and 20 to 30 million chronic hepatitis B (CHB) patients in China, with approximately 500,000 people dying from HBV infection each year and approximately 500,000 to 1 million new cases of hepatitis B diagnosed each year. Hepatitis B virus infection poses serious risks to human health and leads to long-term disease burden, and has currently become one of the most prominent public health problems in China.

[0006] Currently, there are two main types of treatment for chronic hepatitis B patients: nucleoside (nucleotide) analogues (NA) and interferon alpha (IFNα), of which interferon alpha is pegylated interferon alpha (PEGIFNα). NAs approved by the EU for the treatment of HBV include adefovir (ADV), entecavir (ETV), lamivudine (LAM), telbivudine (TBV), tenofovir (TDF) and tenofovir averamide (TAF). These drugs inhibit viral replication, delay or suppress disease progression, and reduce the risk of developing cirrhosis and liver cancer.

[0007] However, the current clinical use of PEGIFNα / NA drugs and their optimized combinations still has difficulty in achieving a high rate of "clinical cure" (elimination of serum hepatitis B surface antigen (HBsAg); elimination of hepatitis B e antigen (HBeAg), which is an important indicator of viral replication and the prognosis of patients' antiviral treatment; regardless of whether hepatitis B surface antibody is positive or not). Existing studies have shown that PEGIFNα / NA can prevent viral replication, but it cannot effectively reduce HBsAg levels or directly affect covalently closed circular DNA (cccDNA; considered to be the transcription and replication template of HBV) stably present in the liver, and therefore it is believed that most patients will experience virological rebound immediately after withdrawal, even if viral replication has been controlled for a long time by treatment. Summary of the Invention [Problem to be solved by the invention]

[0008] The development of antiviral drugs that can effectively reduce the levels of serum HBsAg, HBeAg, HBV DNA and cccDNA in patients with chronic hepatitis B is of urgent practical significance for the antiviral treatment and prognosis of patients with chronic hepatitis B. [Means for solving the problem]

[0009] Contents of the invention The present inventors have surprisingly found in their own research that palovarotene (structural formula shown in Figure 1) treatment can significantly reduce the levels of HBsAg and HBeAg in cell culture supernatants, and the levels of intracellular HBV DNA and cccDNA in in vitro research models (infection models: HepG2-hNTCP 2B1, HepaRG M14a, PHH; replication model: HepAD38). Based on the above findings, the present application has been completed.

[0010] The present application relates to a compound of formula I

[0011] [ka]

[0012] [Wherein, R is hydroxyl, amino, C 1-6 Alkyl-amino-, di(C 1-6 Alkyl)-amino-, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, C 3-6 cycloalkoxy, phenoxy or benzyloxy. or various pharma- ceutically acceptable modifications thereof.

[0013] In particular, the present application relates to the use of a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma-ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma-ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by formula I, a stereoisomer, a solvate, a pharma-ceutically acceptable salt, a hydrate or solvate of a pharma-ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma-ceutically acceptable modifications thereof, in the manufacture of an anti-HBV therapeutic product.

[0014] The present application also relates to the use of a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma-ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma-ceutically acceptable modifications thereof, in the manufacture of a product as an HBV inhibitor.

[0015] The present application also relates to the use of a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma-ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma-ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by formula I, a stereoisomer, a solvate, a pharma-ceutically acceptable salt, a hydrate or solvate of a pharma-ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma-ceutically acceptable modifications thereof, in the manufacture of a product for inhibiting the replication or propagation of HBV in a cell (e.g., a mammalian cell).

[0016] The present application also relates to the use of a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, in the manufacture of a product for the treatment and / or prevention of a disease or infection caused by HBV.

[0017] The present application also relates to the use of a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma- ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma- ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, in the manufacture of a product for the elimination of HBV in a cell (e.g., a mammalian cell).

[0018] The present application also relates to a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma-ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma-ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by formula I, a stereoisomer, a solvate, a pharma-ceutically acceptable salt, a hydrate or solvate of a pharma-ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma-ceutically acceptable modifications thereof, for use in anti-HBV therapy.

[0019] The present application also relates to a compound represented by Formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by Formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma-ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, for use as an HBV inhibitor.

[0020] The present application also relates to a compound represented by Formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma- ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by Formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma- ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, for use in inhibiting the replication or propagation of HBV in a cell (e.g., a mammalian cell).

[0021] The present application also relates to a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma- ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, for use in the treatment and / or prevention of a disease or infection caused by HBV.

[0022] The present application also relates to a compound represented by Formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma- ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by Formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma- ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof, for use in the clearance of HBV in a cell (e.g., a mammalian cell).

[0023] The present application relates to a method for anti-HBV therapy, comprising administering to a subject in need thereof a therapeutically effective amount of a compound represented by Formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma-ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma-ceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by Formula I, a stereoisomer, a solvate, a pharma-ceutically acceptable salt, a hydrate or solvate of a pharma-ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma-ceutically acceptable modifications thereof.

[0024] The present application also relates to a method for inhibiting the replication or propagation of HBV in a cell (e.g., a mammalian cell), comprising administering to a subject in need thereof an effective amount of a compound represented by Formula I, a stereoisomer, a solvate, a pharmaceutically acceptable salt, a hydrate or solvate of a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by Formula I, a stereoisomer, a solvate, a pharmaceutically acceptable salt, a hydrate or solvate of a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or various pharmaceutically acceptable modifications thereof.

[0025] The present application also relates to a method for treating and / or preventing a disease or infection caused by HBV, comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of a compound represented by Formula I, a stereoisomer, a solvate, a pharmaceutically acceptable salt, a hydrate or solvate of a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by Formula I, a stereoisomer, a solvate, a pharmaceutically acceptable salt, a hydrate or solvate of a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or various pharmaceutically acceptable modifications thereof.

[0026] The present application also relates to a method for eliminating HBV in a cell (e.g., a mammalian cell), comprising administering to a subject in need thereof an effective amount of a compound represented by Formula I, a stereoisomer, a solvate, a pharmaceutically acceptable salt, a hydrate or solvate of a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition comprising a compound represented by Formula I, a stereoisomer, a solvate, a pharmaceutically acceptable salt, a hydrate or solvate of a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or various pharmaceutically acceptable modifications thereof.

[0027] The present application also relates to a composition for anti-HBV therapy, comprising a compound represented by Formula I, a stereoisomer, a solvate, a pharma- ceutically acceptable salt, a hydrate or solvate of a pharma- ceutically acceptable salt, a pharma-ceutically acceptable ester, or various pharma- ceutically acceptable modifications thereof.

[0028] The present application also relates to a composition for inhibiting the replication or propagation of HBV in a cell (e.g., a mammalian cell), comprising a compound represented by Formula I, a stereoisomer, a solvate, a pharmaceutically acceptable salt, a hydrate or solvate of a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or various pharmaceutically acceptable modifications thereof.

[0029] The present application also relates to a composition for the treatment and / or prevention of a disease or infection caused by HBV, comprising a compound represented by formula I, a stereoisomer, a solvate, a pharmaceutically acceptable salt, a hydrate or solvate of a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or various pharmaceutically acceptable modifications thereof.

[0030] The present application also relates to a composition for the clearance of HBV in a cell (e.g., a mammalian cell), comprising a compound represented by Formula I, a stereoisomer, a solvate, a pharmaceutically acceptable salt, a hydrate or solvate of a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or various pharmaceutically acceptable modifications thereof.

[0031] According to some embodiments of the present application, the disease caused by HBV described in the present application is viral hepatitis B.

[0032] According to some embodiments of the present application, the disease caused by HBV described in the present application is hepatitis B-associated cirrhosis or primary liver cancer.

[0033] According to some embodiments of the present application, the viral hepatitis B described in the present application is chronic viral hepatitis B, acute viral hepatitis B or chronic active hepatitis.

[0034] According to some embodiments of the present application, the products described in the present application are pharmaceutical products.

[0035] According to some embodiments of the present application, the pharmaceutical compositions described herein further comprise a pharma- ceutically acceptable carrier or excipient.

[0036] According to some embodiments of the present application, the pharmaceutical compositions described in the present application are solid formulations (e.g., tablets, capsules, pills, granules, powders), injectables, topical formulations, sprays, liquid formulations or combination formulations.

[0037] According to some embodiments of the present application, the pharmaceutical compositions described in the present application can be administered orally, by injection, by implantation, by topical application, by aerosol, or by inhalation.

[0038] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is hydroxy, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, linear or branched C5 alkyl, linear or branched C6 alkyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, methylamino, ethylamino, propylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, phenoxy, benzyloxy, chloromethyl, chloroethyl, dichloroethyl, trifluoromethyl, difluoromethyl, monofluoromethyl, chloromethoxy, chloroethoxy, dichloroethoxy, trifluoromethoxy, difluoromethoxy, or monofluoromethoxy.

[0039] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is hydroxyl, in which case the compound is palovarotene.

[0040] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is amino, methylamino, ethylamino, propylamino, butylamino, dimethylamino, diethylamino, dipropylamino, or dibutylamino.

[0041] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is amino.

[0042] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is methylamino or ethylamino.

[0043] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is dimethylamino or diethylamino.

[0044] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, or hexyloxy.

[0045] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is methoxy.

[0046] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is ethoxy.

[0047] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is n-propoxy.

[0048] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is isopropoxy.

[0049] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is n-butoxy.

[0050] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is isobutoxy.

[0051] According to some embodiments of the present application, in the compounds represented by formula I described herein, R is tert-butoxy.

[0052] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is pentyloxy.

[0053] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is hexyloxy.

[0054] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is cyclopropoxy, cyclobutoxy, cyclopentyloxy, or cyclohexyloxy.

[0055] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is cyclopropoxy or cyclobutoxy.

[0056] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is phenoxy or benzyloxy.

[0057] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, linear or branched C5 alkyl, or linear or branched C6 alkyl.

[0058] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is methyl or ethyl.

[0059] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is n-propyl.

[0060] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0061] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is chloromethyl, chloroethyl, dichloroethyl, trifluoromethyl, difluoromethyl, or monofluoromethyl.

[0062] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is chloromethyl or chloroethyl.

[0063] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is trifluoromethyl, difluoromethyl, or monofluoromethyl.

[0064] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is chloromethoxy, chloroethoxy, dichloroethoxy, trifluoromethoxy, difluoromethoxy, or monofluoromethoxy.

[0065] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is chloromethoxy or chloroethoxy.

[0066] According to some embodiments of the present application, in the compounds represented by formula I described in the present application, R is trifluoromethoxy, difluoromethoxy, or monofluoromethoxy.

[0067] According to some embodiments of the present application, the pharma- ceutically acceptable salts described in the present application include inorganic or organic acid salts as well as inorganic or organic base salts. For example, pharma- ceutically acceptable salts include, but are not limited to, alkali metal salts of the compounds, including sodium, potassium, and lithium salts; alkaline earth metal salts of the compounds, including calcium and magnesium salts; metal salts of the compounds, including aluminum, iron, zinc, copper, nickel, and cobalt salts; amine salts of the compounds, including meglumine, ammonium, tert-octylamine, dibenzylamine, morpholine, glucosamine, phenylglycine alkyl ester salts, monoethanolamine, diethanolamine, triethanolamine, N-methyl-D-glucamine, ethylenediamine, N-methylglucamine, guanidine, diethylamine, triethylamine, bicyclohexylamine, N,N'-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine, N-benzyl-N-phenethylamine, piperazine, tetramethylammonium, and tris(hydroxymethyl)aminomethane salts.

[0068] According to some embodiments of the present application, when there is a carboxyl group in the compound represented by formula I described in the present application, the compound can form a pharma- ceutically acceptable ester with an alcohol, and the pharma- ceutically acceptable ester includes the following: 1-6 Alkyl esters, such as methyl esters, ethyl esters, propyl esters, isopropyl esters, butyl esters, sec-butyl esters, tert-butyl esters, pentyl esters and hexyl esters; 3-6 Cycloalkyl esters, such as cyclopentyl esters and cyclohexyl esters; 6-10 Aryl esters, such as phenyl esters and naphthyl esters; 6-10 Aryl-C 1-6Alkyl esters, such as benzyl ester, phenylethyl ester, α-methylbenzyl ester, 3-phenylpropyl ester, 4-phenylbutyl ester, 6-phenylhexyl ester, diphenylmethyl ester and triphenylmethyl ester; or esters which are hydrolysable in vivo, such as (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl ester, (pivaloyloxy)methyl ester, benzofuranonyl ester, [(isopropoxycarbonyl)oxy]methyl ester, [(cyclohexyloxycarbonyl)oxy]methyl ester and 1-[(cyclohexyloxycarbonyl)oxy]ethyl ester.

[0069] Carriers described in this application include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, wool fat.

[0070] The term "excipient" as used in this application refers to additives other than the main active ingredient in a pharmaceutical formulation. The excipient is essentially stable, does not have incompatibility with the main active ingredient, does not cause side effects, does not affect efficacy, does not easily deform, crack, grow moldy or be eaten by insects at room temperature, is harmless to the human body, has no physiological effect, does not cause chemical or physical interaction with the main active ingredient, does not affect the content determination of the main active ingredient, etc. Examples include binders, fillers, disintegrants, and lubricants in tablets; preservatives, antioxidants, flavoring agents, flavoring agents, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, coloring agents, etc. in oral liquid formulations, all of which can be referred to as excipients.

[0071] As used in this application, 1-6 The term "alkyl" refers to a straight or branched chain alkyl having 1 to 6 carbon atoms, e.g., C 1-4 Alkyl, C 1-2 It refers to alkyl, C1 alkyl, C2 alkyl, straight or branched C3 alkyl, straight or branched C4 alkyl, straight or branched C5 alkyl, or straight or branched C6 alkyl. Specific examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl.

[0072] As used in this application, 1-6 The term "alkoxy" refers to any C alkyl group, as defined above, attached to the parent molecule through an oxygen atom. 1-6 Refers to alkyl. 1-6 Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and hexyloxy.

[0073] As used in this application, 1-6 The term "haloalkyl" refers to a C alkyl group as defined above which is mono- or polysubstituted with halogen, for example fluorine, chlorine, bromine or iodine.1-6 Refers to alkyl. 1-6 Representative examples of haloalkyl include, but are not limited to, chloromethyl, chloroethyl, dichloroethyl, trifluoromethyl, difluoromethyl, and monofluoromethyl.

[0074] As used in this application, 1-6 The term "alkyl-amino-" refers to the C 1-6 Refers to an amino group that is monosubstituted with alkyl. 1-6 Representative examples of "alkyl-amino" include, but are not limited to, methylamino, ethylamino, propylamino, and butylamino.

[0075] As used in this application, 1-6 The term "alkyl)-amino-" refers to the C 1-6 Refers to an amino group that is disubstituted with alkyl. 1-6 Representative examples of "alkyl)-amino-" include, but are not limited to, dimethylamino, diethylamino, dipropylamino, and dibutylamino.

[0076] As used in this application, 3-6 The term "cycloalkyl" refers to saturated cyclic hydrocarbyl having 3 to 6 carbon atoms and having a monocyclic or bicyclic structure or multiple fused rings, including fused and bridged ring systems. 3-6 Representative examples of "cycloalkyl" include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0077] As used in this application, 3-6 The term "cycloalkoxy" refers to a C alkyl group, as defined above, attached to the parent molecule through an oxygen atom. 3-6 Refers to cycloalkyl. 3-6Representative examples of "cycloalkoxy" include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy.

[0078] As used in this application, 1-6 The term "haloalkoxy" refers to a C, as defined above, bonded to the parent molecule through an oxygen atom. 1-6 Refers to haloalkyl. 1-6 Representative examples of haloalkoxy include, but are not limited to, chloromethoxy, chloroethoxy, dichloroethoxy, trifluoromethoxy, difluoromethoxy, and monofluoromethoxy.

[0079] As used in this application, the term "subject" includes mammals. Mammals referred to in this application include cows, horses, goats, boars, dogs, cats, rodents and primates, with the preferred mammal being humans.

[0080] The term "effective amount" as used in this application refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a therapeutically effective amount refers to an amount sufficient to cure or at least partially suppress a disease and its complications in a patient already suffering from the disease. A prophylactically effective amount refers to an amount that can effectively prevent, suppress or delay the onset of a disease. Determination of such effective amounts is well within the capabilities of a person skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the overall condition of the patient, such as age, weight and sex, the route of administration of the drug, and other therapeutic agents administered at the same time.

[0081] The amount of the compound represented by formula I, stereoisomer, solvate, pharmaceutically acceptable salt, hydrate or solvate of pharmaceutically acceptable salt, pharmaceutically acceptable ester or various pharmaceutically acceptable modifications thereof administered to a subject depends on the type and severity of the disease and the condition and characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs, and also depends on factors such as the type of formulation, the route of administration of the drug, and the duration or interval of administration. Those skilled in the art will be able to determine the appropriate dosage based on these and other factors. In general, the compound represented by formula I, stereoisomer, solvate, pharmaceutically acceptable salt, hydrate or solvate of pharmaceutically acceptable salt, pharmaceutically acceptable ester or various pharmaceutically acceptable modifications thereof may be administered in a daily dose of about 0.0001-1000 mg / kg body weight / day for treatment, and the daily dose can be administered in one or more batches as appropriate.

[0082] The embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are used only to illustrate the present application, rather than to limit the scope of the present application. Various objects and advantages of the present application will become apparent to those skilled in the art from the drawings and the following detailed description of the preferred embodiments. [Brief description of the drawings]

[0083] [Figure 1] FIG. 1 is a diagram showing the chemical structure of palovarotene. [Diagram 2] FIG. 1 shows the inhibitory effect of palovarotene on HBV infection in the infection model HepG2-hNTCP 2B1. [Diagram 3] FIG. 1 shows the inhibitory effect of palovarotene on HBV infection in the infection model HepRG M14a. [Figure 4] FIG. 1 shows the therapeutic effect of palovarotene on the HBV-infected infection model HepG2-hNTCP 2B1. [Diagram 5]FIG. 1 shows the results of evaluation of palovarotene in the high-copy replication model HepAD38. [Figure 6] FIG. 1 shows the inhibitory effect of palovarotene on HBV infection in human primary hepatocytes. [Figure 7] FIG. 1 shows the therapeutic effect of palovarotene on HBV-infected primary cultured human hepatocytes. [Figure 8] FIG. 1 shows the results of a safety evaluation of palovarotene in an in vitro cell model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] A specific model for carrying out the invention If no specific technique or condition is given in the following examples, it shall be followed as described in the literature or product specifications in the art. Unless otherwise stated, the experimental methods are conventional, and the materials, reagents or equipment used without manufacturer's instructions are conventional products available commercially. All experiments were repeated in triplicate wells, and the results were averaged.

[0085] In the following examples, the test drug was Palovarotene (purchased from MCE), and the positive control drugs included Entecavir (ETV, purchased from Yuanye Bio-Technology Co., Ltd.) and Interferon IFNα (purchased from Sigma, SRP4595-100UG). During the experiment, the test drug or positive control drug was diluted with culture medium to the desired final concentration, and the culture medium was determined according to the cells used, which was specifically described in each example.

[0086] In the following examples, the formulation of 10 L of PBS used was 80 g NaCl, 2 g KCl, 29 g Na2HPO4·12H2O, and 2.4 g KH2PO4, which was mixed well with water, adjusted to a pH of 7.4 and a weighed volume of 10 L, then filtered through a double-layer 0.22 μm filtration membrane, subpackaged, and sterilized by autoclaving for later use.

[0087] In the following examples, the DMEM medium used was purchased from SIGMA-ALDRICH (Art. No. D6429); FBS was purchased from ThermoFisher (Art. No. 10099141); puromycin was purchased from InvivoGen (Art. No. ant-pr-1); doxycycline was purchased from SIGMA-ALDRICH (Art. No. D9891); penicillin and streptomycin were purchased from Shandong Lukang Pharmaceutical Co., Ltd.; 4% PEG 8000 was purchased from AMRESCO (Art. No. 0159); WME medium was purchased from SIGMA-ALDRICH (Art. No. W1878); glutamine was purchased from SIGMA-ALDRICH (Art. No. G8540); insulin was purchased from Jiangsu Wanbang Pharmaceuticals (Art. No. G8540); Biopharmaceuticals; hydrocortisone was purchased from Cayman (Art. No. 18226); tetracycline was purchased from USB (Art. No. 22105-25g); B-27™ Supplement (50×) was purchased from GIBCO (Art. No. 17504044); forskolin was purchased from MCE (Art. No. HY-15371); SB431542 was purchased from Tocris (Art. No. 1614); IWP2 was purchased from MCE (Art. No. HY-13912); DAPT was purchased from MCE (Art. No. HY-13027); LDN193189 was purchased from MCE (Art. No. HY-12071A).

[0088] In the following examples, medium A used was DMEM medium containing 10% FBS, 1 μg / mL puromycin, 1 μg / mL doxycycline, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0089] In the following examples, medium B used was DMEM medium containing 10% FBS, 1 μg / mL puromycin, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0090] In the following examples, medium C used was DMEM medium (freshly prepared immediately before use) containing 4 w / v% PEG 8000, 1 μg / mL puromycin, 1 μg / mL doxycycline, 100 U / mL penicillin, 100 μg / mL streptomycin and 5% FBS.

[0091] In the following examples, medium D used was WME medium containing 10% FBS, 100 mM glutamine, 5 μg / mL insulin, 50 μM hydrocortisone, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0092] In the following examples, medium E used was WME medium (freshly prepared immediately before use) containing 4 w / v% PEG 8000, 100 mM glutamine, 5 μg / mL insulin, 50 μM hydrocortisone, 100 U / mL penicillin, 100 μg / mL streptomycin and 5% FBS.

[0093] In the following examples, the medium F used was a DMEM medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0094] In the following examples, the medium G used was a DMEM medium containing 10% FBS, 1 μg / mL tetracycline, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0095] In the following examples, the medium H used was a WME medium containing 1 v / v% B-27™ Supplement (diluted at the time of use in a ratio of 1:100, i.e., 1 mL of B-27™ Supplement for every 100 mL of medium H), 20 μmol / L forskolin, 10 μmol / L SB431542, 0.5 μmol / L IWP2, 5 μmol / L DAPT, 0.1 μmol / L LDN193189, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0096] In the following examples, Medium I used was WME medium containing 4 w / v% PEG 8000, 20 μmol / L forskolin, 10 μmol / L SB431542, 0.5 μmol / L IWP2, 5 μmol / L DAPT, 0.1 μmol / L LDN193189, 100 U / mL penicillin, 100 μg / mL streptomycin and 0.5 v / v% B-27™ Supplement (diluted at the time of use in a 1:200 ratio, i.e., for every 100 mL of Medium I, 0.5 mL of B-27™ Supplement is contained).

[0097] Example 1: Evaluation of the inhibitory effect of test drugs on HBV infection in the infection model HepG2-hNTCP 2B1 We engineered human sodium taurocholate cotransporting polypeptide (hNTCP) gene and red fluorescent protein (mCherry) gene in a bicistronic expression cassette controlled by a tetracycline-inducible promoter, stably integrated the expression cassette into HepG2-Tet On cells by using a lentivirus system, and obtained a stable cell line HepG2-hNTCP 2B1 that highly expresses hNTCP by using flow cytometry sorting technology. This cell model confirmed HBV infection and produced HBV-related indicators. (For information on how to generate this cell model, see: Ya-Li Zhang, Ying Gao, Jia-Li Cao, Jing-Hua Zhao, Tian-Ying Zhang, ChuanLai Yang, Hua-Long Xiong, Ying-Bin Wang, Shan-Hai Ou, Tong Cheng, Chang-Rong Chen, Quan Yuan & Ning-Shao Xia (2019). Robust in vitro assay for analyzing the neutralization activity of serum specimens against hepatitis B virus, Emerging Microbes & Infections, Volume 8:1, Pages 724-733, DOI:10.1080 / 22221751.2019.1619485).

[0098] The experiment was carried out in a 24-well culture plate. Each test drug in the experimental group had three final concentrations, 1 μM, 5 μM and 25 μM, and the positive control group had an entecavir (ETV) final concentration of 1 μM. Here, the test drugs and positive control drugs were diluted with medium A to the desired concentration, and the blank group was added with the same volume of medium A without drugs.

[0099] Approximately 1.5×10 5HepG2-hNTCP 2B1 cells were plated in each well and 500 μL of medium B was added. After the cells attached, medium A was used for replacement throughout the entire experiment for induction with doxycycline.

[0100] After 72 hours of induction with doxycycline, 500 μL of drug-containing medium A was added to each well for drug treatment for 24 hours, and then the cells were infected with HBV. The viral infection dose per well was 100 MOI. Before infection, HBV solution was obtained by diluting HBV with medium C. The infection volume was 250 μL per well. During infection, the cell supernatant was first removed by aspiration, HBV solution was added to the cells, and then the cells were incubated for 16-20 hours, and then the supernatant was collected as the day 0 sample. Then, the cells were washed three times with warmed PBS in a 37°C bath, and fresh, drug-containing medium A (500 μL) was used for replacement, and then the supernatant was collected every 2 days for detection, and fresh, drug-containing medium A was added, and on the 8th day, all the supernatants and cells were collected for relevant detection. The detection items included: HBsAg / HBeAg, HBV DNA, HBV cccDNA.

[0101] Detection of HBsAg / HBeAg: HBsAg (chemiluminescence enzyme immunoassay (CLEIA), product specification number: YZB / Guo 0346-2014) and HBeAg (enzyme-linked immunosorbent assay (ELISA), product specification number: YZB / Guo 0216-2013) were detected using a detection kit from Beijing Wantai Company, and the specific detection procedures were performed according to the detection method described in the instructions in the kit.

[0102] HBV DNA detection: After the collected cells were washed with PBS, HBV DNA was obtained by automatic extraction using a viral DNA & RNA extraction kit (Beijing GenMagBio) on a nucleic acid extraction workstation (purchased from GenMagBio, model: DOF-9696) (please refer to the instruction manual for the extraction procedure). HBV DNA was quantified by probe real-time fluorescent quantitative PCR. Here, the reagent used was Premix Ex Taq™ (Takara), and the instrument used was Roche's LightCycler® 96. The primer sequences used for fluorescent quantification are shown in Table 1, and the fluorescent quantitative reaction system is shown in Table 2.

[0103] Detection of HBV cccDNA: HBV cccDNA was extracted by using a kit using the modified Hirt method. The kit used was Plasmid Mini Kit (TIANGEN BIOTECH (BEIJING) CO., LTD.). The solutions included in this kit were Buffer I (50 mM Tris, 10 mM EDTA, pH 7.5), Buffer II (1.2% SDS), and Buffer III (3 M CsCl, 1 M potassium acetate, 0.67 M acetic acid). For specific methods, please refer to the plasmid extraction method described in the kit instructions. HBV cccDNA was quantitatively detected by probe real-time fluorescent quantitative PCR, and mitochondrial DNA (mtDNA) was simultaneously detected as an internal reference. Here, the instrument used was Roche's LightCycler® 96. The primers used for fluorescent quantification are shown in Table 1, and the fluorescent quantitative reaction system is shown in Table 2.

[0104] The fluorescent reaction procedure for quantitative detection was as follows: (1) HBV DNA quantification: The quantification program was 95°C, 30 sec; (95°C, 5 sec; 60°C, 30 sec) for 45 cycles, and the fluorescence channel was Hex.

[0105] (2) Quantification of cccDNA: The quantitative program was 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) for 45 cycles, and the fluorescence channel was FAM.

[0106] (3) Quantification of mtDNA: The quantitative program was 95°C, 5 min; (95°C, 30 sec; 55°C, 40 sec) for 45 cycles, and the fluorescence channel was Hex.

[0107] Calculation of cccDNA normalization: The specific formula was as follows: 2 (mtDNA-cccDNA) [where the values ​​in parentheses are the Ct values ​​of mtDNA and cccDNA, respectively].

[0108] The experimental results are shown in Figure 2. The results showed that, compared with the blank group, palovarotene treatment could effectively inhibit the levels of HBsAg (p<0.05) and HBeAg (p<0.05) in cell supernatants, and the levels of intracellular HBV DNA (p<0.001) and HBV cccDNA (p<0.0001), and the inhibition degree was dose-dependent to some extent.

[0109] This demonstrated that palovarotene could significantly inhibit HBV infection of HepG2-hNTCP 2B1.

[0110] [Table 1]

[0111] [Table 2]

[0112] Example 2: Evaluation of the inhibitory effect of test drugs on HBV infection in the infection model HepaRG M14A HepaRG was a hepatic progenitor cell line with differentiation potential isolated from tumor tissue of a female hepatitis C patient. Two weeks after cell plating, cell differentiation was induced with 2% DMSO (2 mL of DMSO was added per 100 mL of cell culture medium) for two weeks. Finally, about 50% of the cells differentiated into hepatocyte-like cells, and the other 50% of the cells differentiated into cholangiocyte-like cells. Among them, the hepatocyte-like cells supported HBV infection with an infection efficiency of about 20%. Through genetic modification of HepaRG, HNF4A and FoxM1 genes were incorporated into HepaRG cells, and a HepaRG M14A cell line was obtained by screening, which could complete differentiation and support HBV infection independently of DMSO. (For the method of constructing this cell model, please refer to Zhao Jinghua. Development, Optimization and Preliminary Application of in vitro models supporting HBV Infection [D]. Xiamen University, 2016).

[0113] The experiment was carried out in a 24-well cell culture plate. Each test drug in the experimental group had three final concentrations of 1 μM, 5 μM, and 25 μM, and the positive control group had an entecavir (ETV) final concentration of 1 μM. Here, the test drugs and positive control drugs were diluted with medium D to the desired concentration, and the blank group was added with the same volume of medium D without drugs. Approximately 1.5×10 5HepaRG M14A cells were plated in each well, and 500 μL of medium D was added to each well. After cell plating, medium D was replaced every 2 days. 13 days after cell plating, medium D was replaced with medium D containing drugs for drug treatment. After 24 hours of drug treatment, cells were infected with HBV. The amount of HBV for infection in each well was 1000 MOI. Before infection, HBV solution was obtained by diluting HBV with medium E. The infection volume was 250 μL per well. During infection, the cell supernatant was first removed by aspiration, HBV solution was added to the cells, and then the cells were incubated for 24 hours, and then the supernatant was collected as the day 0 sample. Then, cells were washed three times with PBS warmed in a 37°C bath, and fresh, drug-containing medium D (500 μL per well) was used for replacement, and then, until the 8th day, the supernatant was collected every 2 days for detection, fresh, drug-containing medium D was used for replacement, and all the supernatant and cells were collected for related detection. Detection items included HBsAg, HBeAg, HBV DNA, HBV cccDNA. For detection method, please refer to Example 1.

[0114] The experimental results are shown in Figure 3. The results showed that in the HepaRG M14a-infected cell model, palovarotene treatment before infection could significantly reduce the levels of HBsAg (p<0.05) and HBeAg (p<0.05) in the cell supernatant, as well as the levels of intracellular HBV DNA (p<0.0001) and cccDNA (p<0.0001).

[0115] The above results demonstrated that palovarotene could significantly inhibit HBV infection to HepaRG M14A.

[0116] Example 3. Evaluation of the therapeutic effect of test drugs on the HBV-infected infection model HepG2-hNTCP 2B1 The experiment was carried out in a 24-well culture plate. Each test drug in the experimental group had three final concentrations of 1 μM, 5 μM, and 25 μM, and the ETV in the positive control group had a final concentration of 1 μM. Here, the test drugs and positive control drugs were diluted with medium A to the desired concentration, and the blank group was added with the same volume of medium A without drugs. Approximately 1.5 × 10 5 HepG2-hNTCP 2B1 cells were plated in each well, and 500 μL of medium B was added to each well. After the cells attached, medium A was used for replacement throughout the entire experiment for induction with doxycycline.

[0117] HBV infection was performed 4 days after induction with doxycycline. The amount of HBV in each well was 100 MOI. Before infection, HBV was diluted with medium C to obtain HBV solution. The infection volume was 250 μL per well. During infection, cell supernatant was first removed by aspiration, HBV solution was added to cells, cells were incubated for 24 hours, and then the supernatant was collected as the sample on day 0. Then, cells were washed with PBS three times, and fresh medium C was used for replacement. After that, the supernatant was collected every 2 days for detection, and fresh medium C was used for replacement. On day 6, fresh drug-containing medium A was used for replacement to start drug treatment, and then the supernatant was collected every 2 days for detection, and fresh drug-containing medium A was used for replacement, and until day 12, all the supernatants and cells were collected for related detection. The detection items included HBsAg, HBeAg, HBV DNA, and HBV cccDNA. For the detection method, please refer to Example 1.

[0118] Southern blot experiments were performed using a 10 cm 2 The experiment was carried out on a culture plate. Approximately 5 × 10 6 HepG2-hNTCP 2B1 cells were placed in 10cm 2The cells were plated in a culture plate and medium B was added. After the cells attached, medium A was used for replacement throughout the entire experiment to perform induction with doxycycline. HBV infection was performed 4 days after induction with doxycycline. The amount of HBV for infection was 1000 MOI. Before infection, HBV was diluted with medium C to obtain HBV solution. The infection volume was 5 mL. During infection, the cell supernatant was first removed by aspiration, HBV solution was added to the cells, the cells were incubated for 24 hours, and then the supernatant was collected as the day 0 sample. The cells were then washed three times with PBS, and fresh medium A was used for replacement. Thereafter, the supernatant was collected every 2 days for detection, and fresh medium A was used for replacement. On the 6th day, fresh drug-containing medium was used for replacement to start drug treatment (drug concentration for treatment was 5 μM, control group used interferon IFNα at a concentration of 500 IU for treatment), then supernatant was collected every 2 days for detection, fresh drug-containing medium A was used for replacement, until the 12th day, all the supernatant and cells were collected for Southern blot detection and immunofluorescence detection.

[0119] Southern Blot Detection HBV cccDNA was extracted by using a kit using a modified Hirt method. The kit used was Qiagen Plasmid Midi Kit. For specific extraction procedures, please refer to the plasmid extraction method of the kit. The specific steps of Southern blot were as follows: A loading buffer (purchased from TaKaRa Company) was added to the extracted HBV cccDNA sample, and then the DNA was separated by electrophoresis through 1.2% agarose gel at 80V for 2 hours in 1×TAE buffer (40mmol / L Tris base, 1mmol / L EDTA, 20mmol / L sodium acetate). The gel was then placed in depurination buffer (0.2mol / L HCl) and soaked for 15 minutes with shaking at room temperature, then rinsed twice with sterile ultrapure water for 1 minute each time. The gel was then placed in denaturation buffer (0.5mol / L NaOH, 1.5mol / L NaCl) and soaked for 30 minutes with shaking at room temperature, then rinsed twice with sterile ultrapure water for 1 minute each time, and finally soaked in neutralization buffer (1mol / L Tris base, 1.5mol / L NaCl) for 10 minutes.

[0120] b. Gel-to-membrane transfer: The gel was immersed in 10×SSC (1.5 mol / L NaCl, 0.15 mol / L trisodium citrate, pH 7.0) for 5 min, during which the filter paper and nylon membrane (purchased from Roche) were first wetted with sterile ultrapure water and then immersed in 10×SSC. The nucleic acids were transferred to the nylon membrane using a vacuum blotting device (the pressure was adjusted to 5 MPa, and the transfer time was 90 min). At the same time, the hybridization solution (DIG Easy Hyb™ Granules, purchased from Roche, Art. No. 38716500) was preheated at 42°C. After transfer, the blotted nylon membrane was cross-linked with 1.5 J / cm2 UV cross-linking instrument SiGmA SH4 B type (purchased from Shanghai Sigma Co., Ltd) at 1.5 J / cm2. 2 The crosslinking was carried out for 3 minutes under a radiation dose of 1000 x g.

[0121] c. Hybridization: The membrane was immersed in pre-warmed hybridization solution at 42°C, placed in a hybridization oven, and incubated at 42°C for 2 hours. At the same time, the probe was heated at 95°C for 10 minutes, then quickly inserted into ice-water mixture for 5 minutes (to prevent denaturation), then diluted with hybridization solution (300ng of probe was added per 10mL of hybridization solution) and hybridized with the membrane at 42°C for 12 hours. After hybridization was completed, the membrane was washed with washing buffer I (2xSSC, 0.1% SDS) at room temperature for 3 times, 5 minutes each time, and then washed with washing buffer II (0.5xSSC, 0.1% SDS) at 65°C for 3 times, 15 minutes each time.

[0122] d. Antibody incubation: First, the membrane was immersed in maleic acid buffer (0.1 mol / L maleic acid, 0.15 mol / L NaCl, pH 7.5) for 2 minutes, then blocked with 1x blocking buffer (purchased from SIGMA-ALDRICH, Art. No. B6429-500ML) (diluted with maleic acid buffer) at room temperature for 1 hour. After blocking, anti-Dig-AP antibody (purchased from SIGMA-ALDRICH, Art. No. Roche-11093274910) was diluted in 1x blocking buffer, and the membrane and antibody were incubated at room temperature for 40 minutes. After incubation, the membrane was washed with rinsing solution (0.1 mol / L maleic acid, 0.15 mol / L NaCl, 0.3% by volume Tween 20, pH 7.5) three times at room temperature for 15 minutes each time.

[0123] e. Detection: First, the membrane was immersed in equilibration solution (0.1 mol / L Tris base, 0.1 mol / L NaCl, pH 9.5) for 3 min, then the membrane was taken out, a chromogenic substrate (CDP-star, purchased from Roche) was added to the membrane, and finally it was exposed on a biomolecular imaging device (ImageQuant LAS 4000 mini, GE).

[0124] Immunofluorescence detection HBcAg immunofluorescence staining related to this experiment was performed directly on the adherent cells. The following operations were performed on a shaker at 100 rpm / min. The cells in the cell culture plate were washed once with PBS. Fixation: 4% paraformaldehyde (purchased from Beyotime, Art. No. P0099-100ml) was added to cover the cells, and then the cells were incubated at room temperature for 10 minutes in the dark. Washing: The cells were washed three times with PBS, each time for 3 minutes. Permeabilization: 0.2% Triton X-100 (purchased from AMRESCO) was added, and the cells were incubated at room temperature for 10 minutes. Washing: The cells were washed three times with PBS, each time for 3 minutes. Blocking: 3% BSA (BSA powder purchased from SIGMA-ALDRICH was weighed out at 3 g and dissolved in 100 mL of PBS) was added, and the cells were incubated at room temperature for 60 minutes or at 4°C overnight. Primary antibody: Anti-HBcAg (2A7, mouse antibody) was diluted in 3% BSA at a ratio of 1:1000, then added to the wells at 200 μL per well, then the cells were incubated at room temperature for 1 hour or at 4° C. overnight. Wash: Cells were washed with PBS three times for 3 minutes each time. Secondary antibody: Secondary antibody (Alexa Fluor® 488 donkey anti-rabbit IgG (H+L) (Invitrogen)) was diluted in 3% BSA at a ratio of 1:1000, then added to the wells at 200 μL per well, then the cells were incubated at room temperature for 30 minutes. Wash: Cells were washed with PBS three times for 3 minutes each time. Nuclear staining: DAPI (Invitrogen) was diluted in 3% BSA at a ratio of 1:2000, then added to the wells at 200 μL per well, then the cells were incubated at room temperature for 5 minutes. Wash: Cells were washed with PBS three times for 3 minutes each time. Photography: Photographs were taken using a high-content confocal microscope (PerkinElmer, Operetta CLS) and the mean fluorescence intensity of green fluorescence was analyzed.

[0125] The experimental results are shown in Figure 4. The results showed that after drug treatment performed 6 days after HBV infection, high concentrations of palovarotene had a significant inhibitory effect on HBeAg (p<0.05) and could significantly reduce the levels of HBV DNA (p<0.05) and HBV cccDNA (p<0.05), whereas the control group (ETV) and blank group showed no significant difference. Southern blot results showed that the level of HBV cccDNA after drug treatment at a concentration of 5 μM was significantly lower than that of interferon treatment in the control group and the blank group without drug. Immunofluorescence results showed that palovarotene could significantly reduce the positive rate of HBcAg in HepG2-hNTCP 2B1 cells after infection. This indicates that drug treatment could inhibit HBV infection.

[0126] High-concentration palovarotene treatment could significantly inhibit HBV viral replication and e antigen levels after infection.

[0127] Example 4. Evaluation of the therapeutic effect of test drugs on HepAD38 capable of producing high levels of HBV DNA HepAD38 is a hepatocellular carcinoma cell line (D genotype) that stably integrates the HBV genome and expresses HBV under conditions that can be controlled with tetracycline. In the presence of tetracycline, this cell line does not synthesize viral particles due to the suppression of HBV pgRNA synthesis, and in the absence of tetracycline, the HepAD38 cell line transcribes HBV pgRNA, synthesizes subviral particles, and secretes virion-like particles into the cell supernatant. HepAD38 has approximately 10-fold higher DNA levels than HepG2.2.15 and is therefore a high-copy HBV in vitro replicating cell model (for a method for generating this cell model, see Ladner SK, Otto MJ, Barker CS, et al. Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother. 1997;41(8):1715-1720).

[0128] This experiment was carried out in a 24-well culture plate. Each test drug in the experimental group had two final concentrations, 1 μM and 5 μM, and the ETV in the positive control group had a final concentration of 1 μM. Here, the test drugs and positive control drugs were diluted with medium F to the desired concentrations, and the blank group was added with the same volume of medium F without drugs. Approximately 4 × 10 5 HepAD38 cells were plated in each well, 500 μL of medium G containing tetracycline hydrochloride (tetracycline) was added to each well, 48 hours after the cells were plated was set as day 0, medium G containing tetracycline hydrochloride (tetracycline) was removed at the same time, fresh medium F containing drug was used for replacement, and then fresh medium F containing drug was used for replacement every 2 days until day 8, and the level of HBsAg in the cell supernatant and the level of intracellular HBV DNA were detected on day 8. For the method for detection, please refer to Example 1.

[0129] Southern blot experiments were performed using a 6 cm 2 The experiment was carried out on a culture plate. Approximately 4.2 × 10 6 HepAD38 cells, 6 cm 2 Plated in a culture plate, medium G containing tetracycline hydrochloride (tetracycline) was added. During the experiment, medium G containing tetracycline hydrochloride was removed, and medium F containing drug was added at the same time for drug treatment. Here, the treatment concentration of the test drug palovarotene was 5 μM, and the control group used entecavir (ETV) at a concentration of 5 μM for treatment, and the treatment time was 8 days.

[0130] The specific Southern blot detection method was carried out with reference to Example 3. The results are shown in Figure 5. The results showed that palovarotene at a concentration of 5μM had a significant inhibitory effect on the surface antigen production (p<0.01) and HBV DNA (p<0.05) of the replication model HepAD38. Southern blot results showed that the drug could significantly inhibit the level of HBV cccDNA, but the inhibitory effect was inferior to that of the control group (ETV).

[0131] Palovarotene treatment was able to significantly inhibit viral replication levels in the replication model HepAD38.

[0132] Example 5: Evaluation of the inhibitory effect of test drugs on HBV infection in human primary hepatocytes In this experiment, an experimental group, a positive control group, and a blank group were set up. Here, each test drug (palovarotene) in the experimental group had three final concentrations of 1 μM, 5 μM, and 25 μM, and the ETV in the positive control group had a final concentration of 1 μM. Here, the test drugs and the positive control drug were diluted with medium H to the desired concentration, and the blank group was added with the same volume of medium H without drugs.

[0133] Primary hepatocytes were isolated from humanized mice transplanted with primary human hepatocytes by perfusion techniques (see Foquet, Lander et al., "Successful engraftment of human hepatocytes in uPA-SCID and FRG®KO mice," Hepatocyte Transplantation. Humana Press, New York, NY, 2017, pp. 117-130; Wan-Chun Li et al., "Isolation and Culture of Adult Mouse Hepatocytes," Mouse Cell Culture. Humana Press, 2010, pp. 185-196) and counted (approximately 5 × 10 per well). 5After 24 h of incubation, the cells were directly cultured in 24-well plates (collagen I pre-coated 24-well plates, purchased from Thermo Scientific) and medium H was added to maintain hepatocyte morphology (see Xiang, Chengang et al., "Long-term functional maintenance of primary human hepatocytes in vitro," Science 364.6438 (2019): pp. 399-402). After 24 h of incubation, medium H containing drugs was used to replace medium H without drugs for 24 h of drug treatment, and then infection with HBV was performed. The amount of HBV for infection in each well was 2000 MOI. Before infection, HBV was first diluted with medium I to obtain HBV solution. The infection volume was 250 μL / well. During infection, the cell supernatant was first removed by aspiration, HBV solution was added to the cells, the cells were incubated for 24 h, and then the supernatant was collected as the day 0 sample. Then, cells were washed three times with PBS warmed in a 37°C bath, and fresh, drug-containing medium H (500 μL per well) was used for replacement, and then the supernatant was collected every two days for detection, and fresh, drug-containing medium H was used for replacement, and until the 8th day, all the supernatant and cells were collected for related detection. Detection items included HBsAg, HBeAg, HBV DNA, HBV cccDNA. For detection method, please refer to Example 1.

[0134] The experimental results are shown in Figure 6. The results showed that in human primary hepatocytes, pre-infection palovarotene treatment could significantly inhibit the levels of HBsAg (p<0.05) and HBeAg (p<0.05) in the cell supernatant, as well as the levels of intracellular HBV DNA (p<0.05) and cccDNA (p<0.05).

[0135] Palovarotene treatment was able to significantly inhibit HBV infection of primary human hepatocytes.

[0136] Example 6: Evaluation of the therapeutic effect of test drugs on HBV-infected human primary hepatocytes In this experiment, an experimental group, a positive control group and a blank group were set up, where each test drug (palovarotene) in the experimental group had three final concentrations of 1 μM, 5 μM and 25 μM, and the ETV in the positive control group had a final concentration of 1 μM. The test drugs and the positive control drugs were diluted with medium H to the required concentration, and the blank group was added with the same volume of medium H without drugs.

[0137] From the humanized mice transplanted with primary human cultured hepatocytes, primary cultured hepatocytes were isolated by perfusion technique and counted (5 x 10 per well). 5 After 1000 cells / well of primary cultured hepatocytes, the cells were directly cultured in 24-well plates and medium H was added to maintain hepatocyte morphology. After 24 hours of culture, infection with HBV was performed. The amount of HBV for infection in each well was 2000 MOI. Before infection, HBV was first dissolved in medium I to obtain HBV solution. The infection volume was 250 μL / well. During infection, the cell supernatant was first removed by aspiration, HBV solution was added to the cells, the cells were incubated for 24 hours, and then the supernatant was collected as the sample on day 0. Then, the supernatant was collected every 2 days for detection, and fresh medium H was used for replacement. On day 8, fresh drug-containing medium H was used for replacement to start drug treatment, and until day 14, all the supernatants and cells were collected for related detection. The detection items included HBsAg, HBeAg, HBV DNA, and HBV cccDNA. For the detection method, please refer to Example 1.

[0138] The experimental results are shown in Figure 7. The results showed that palovarotene significantly inhibited the levels of HBeAg (p<0.05) and HBsAg (p<0.05) in the cell supernatant when the drug was added for treatment 8 days after infection, and the inhibitory effect of palovarotene at concentrations of 5 μM and 25 μM was more obvious. At the concentration of 25 μM, palovarotene had a significant inhibitory effect on HBV cccDNA (p<0.01) and HBV DNA (p<0.05).

[0139] Palovarotene treatment could significantly inhibit HBV viral replication and the e antigen and surface antigen levels of HBV-infected human primary hepatocytes.

[0140] Example 7: Safety evaluation of test drugs in an HBV in vitro cell model The experiment was carried out in a 96-well culture plate. 4 The corresponding cells were plated in each well. The corresponding medium volume was 100 μL. Here, each test drug in the HepG2-hNTCP 2B1, HepaRG M14A and primary cultured hepatocyte experimental group had three final concentrations of 1 μM, 5 μM and 25 μM, and each test drug in the HepAD38 cell experimental group had two final concentrations of 1 μM and 5 μM (the drug was diluted with the corresponding medium to the final concentration; see Example 1, Example 2, Example 4 and Example 5), and the blank group was added with the same volume of medium without drug. After the cells grew to a suitable density, the drug was added and treated for 6 days, during which the medium was replaced with fresh medium containing drug every 2 days. After drug treatment, 110 μL of Beyotime CCK-8 cell activity detection reagent (purchased from Beyotime, Art. No. C0038) (volume ratio of medium to CCK-8 reagent was 100:10) was used for replacement, cells were incubated at 37°C for 30 minutes, and then all the supernatants were collected for absorbance detection. The absorbance value was read on a PHOMO microplate reader purchased from Zhengzhou Autobio.

[0141] The results are shown in Figure 8. The results show that after HepG2 hNTCP 2B1, HepaRG M14a, HepAD38 and human primary hepatocytes were treated with the drug palovarotene at concentrations of 1 μM, 5 μM and 25 μM, the CCK-8 absorbance values ​​of each experimental group were not significantly different from the absorbance value of the blank group, suggesting that the drug has no significant effect on cell activity at these concentrations.

[0142] The examples of the present application show that the compound of formula I (including palovarotene) can significantly reduce the levels of HBsAg and HBeAg in cell culture supernatant and the levels of intracellular HBV DNA and cccDNA in an in vitro research model, and treatment with a drug dose of 5 μM can achieve better effects in the antiviral therapy of cells.This drug may be developed as a therapeutic drug against hepatitis B virus.

[0143] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, not to limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should know that the specific embodiments of the present application can be modified or some technical features can be replaced with equivalents. All of them should be included in the scope of the technical solutions described in the claims of this application without departing from the spirit of the technical solutions of the present application.

Claims

1. 1. A composition for anti-HBV therapy, for inhibiting the replication or proliferation of HBV in cells, for treating and / or preventing a disease or infection caused by HBV, or for anti-HBV therapy or for eliminating HBV in cells, comprising a compound of formula I 【Chemistry 1】 wherein R is hydroxyl. or a hydrate or solvate of said pharma- ceutically acceptable salt.

2. The composition of claim 1 , wherein the cell is a mammalian cell.

3. The composition of claim 1 or 2, wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or excipient.

4. 4. The composition of claim 3, wherein the compound, stereoisomer, solvate, pharma- ceutically acceptable salt, or hydrate or solvate of the pharma- ceutically acceptable salt is present in a therapeutically and / or prophylactically effective amount.

5. The composition according to claim 1 or 2, wherein the pharmaceutical composition is a solid formulation, an injectable formulation, an external preparation, a spray formulation, a liquid formulation or a combination formulation.

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