Use of Palovarotene in anti-HBV virus treatment

Abstract

This application relates to the field of hepatitis B antiviral therapy, specifically to the use of the compound shown in Formula I in the treatment of anti-HBV virus,

Description

[0001] This application is based on an application with a CN application number of 202010531845.9 and a filing date of June 11, 2020, and claims its priority. The disclosure of this CN application is hereby incorporated herein by reference in its entirety. Technical Field

[0002] This application relates to the field of hepatitis B antiviral therapy, and more particularly to the use of small molecule drugs represented by Formula I (such as Palovarotene) in the treatment of anti-HBV virus.

[0003] Background Art

[0004] Hepatitis B virus (HBV) infection can cause hepatitis B, which is a serious global health problem. According to the World Health Organization, approximately 3.5% (257 million) of the world's population is infected with HBV, with about 5 million new infections each year. More than 800,000 people die each year from liver diseases caused by HBV infection, including chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma. In China, which is a medium to high prevalence area of HBV infection, although the widespread vaccination of 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 in the country, including 20 to 30 million patients with chronic hepatitis B. Approximately 500,000 people die from HBV infection each year, and about 500,000 to 1 million new hepatitis B cases occur each year. HBV infection seriously endangers human health and brings a long-term disease burden, making it one of the most prominent public health problems in China at present.

[0005] Currently, there are mainly two types of therapeutic drugs for chronic hepatitis B patients: including nucleos(t)ide analogues (NAs) or interferon α (IFNα), among which interferon α uses pegylated interferon α (PEGIFNα). The NAs approved for HBV treatment in the European Union include adefovir dipivoxil (ADV), entecavir (ETV), lamivudine (LAM), telbivudine (TBV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF). These drugs have been proven to inhibit virus replication, delay or prevent disease progression, and reduce the risk of cirrhosis and liver cancer.

[0006] However, the currently clinically used PEGIFNα / NAs drugs and their optimized combinations are still difficult to achieve a high proportion of "clinical cure" (serological negative conversion of hepatitis B surface antigen (HBsAg); negative conversion of hepatitis B e antigen (HBeAg), an important indicator of virus replication and the prognosis of antiviral treatment in patients; with or without positive conversion of hepatitis B surface antibody). Existing studies have shown that although the two drugs PEGIFNα / NAs can prevent virus replication, they cannot effectively reduce the level of HBsAg, and even less directly affect the covalently closed circular DNA (cccDNA) that stably exists in the liver (considered as the transcription and replication template of HBV), resulting in virological rebound in most patients soon after drug withdrawal even when virus replication is long-term controlled by treatment.

[0007] Developing an antiviral drug 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 disease prognosis of these patients.

[0008] Application content

[0009] The inventor of the present application surprisingly found in the research that treatment with Palovarotene (structural formula as Figure 1 shown) can significantly reduce the levels of HBsAg and HBeAg in the cell culture supernatant of in vitro HBV research models (infection models: HepG2-hNTCP 2B1, HepaRG M14a, PHH; replication model: HepAD38), as well as the levels of intracellular HBV DNA and cccDNA. Based on the above findings, the present application was completed.

[0010] This application relates to the use of the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof,

[0011]

[0012] wherein R is hydroxyl, amino, C 1-6 alkyl-amino-, di(C 1-6 alkyl)-amino-, C 1 -C 6 alkoxy, C 1-6 haloalkoxy, C 1 -C 6 alkyl, C 1-6 haloalkyl, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkoxy, phenoxy or benzyloxy.

[0013] Specifically, this application relates to the use of the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, hydrates or solvates of its pharmaceutically acceptable salts, or various pharmaceutically acceptable modifications thereof, in the preparation of a product for anti-HBV virus treatment.

[0014] This application also relates to the use of the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, in the preparation of a product as an HBV virus inhibitor.

[0015] The present application also relates to the use of the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, in the preparation of a product for inhibiting the replication or propagation of HBV virus in cells (such as mammalian cells).

[0016] The present application also relates to the use of the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, in the preparation of a product for treating and / or preventing diseases or infections caused by HBV virus.

[0017] The present application also relates to the use of the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, in the preparation of a product for clearing HBV virus from cells (such as mammalian cells).

[0018] The present application also relates to the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, hydrates or solvates of its pharmaceutically acceptable salts, or various pharmaceutically acceptable modifications thereof, for anti-HBV virus treatment.

[0019] This application also relates to a compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, which is used as an HBV virus inhibitor.

[0020] This application also relates to a compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, which is used to inhibit the replication or propagation of HBV virus in cells (such as mammalian cells).

[0021] This application also relates to a compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, which is used for the treatment and / or prevention of diseases or infections caused by HBV virus.

[0022] This application also relates to a compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing the compound represented by formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, which is used to eliminate HBV virus in cells (such as mammalian cells).

[0023] The present application relates to methods for anti-HBV virus treatment, including administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing a compound of formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, hydrates or solvates of its pharmaceutically acceptable salts, or various pharmaceutically acceptable modifications thereof.

[0024] The present application also relates to methods for inhibiting the replication or propagation of HBV virus in cells (such as mammalian cells), including administering to a subject in need thereof an effective amount of a compound of formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing a compound of formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof.

[0025] The present application also relates to methods for treating and / or preventing diseases or infections caused by HBV virus, including administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of a compound of formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing a compound of formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof.

[0026] The present application also relates to methods for clearing HBV virus in cells (such as mammalian cells), including administering to a subject in need thereof an effective amount of a compound of formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof, or a pharmaceutical composition containing a compound of formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof.

[0027] The present application also relates to a composition for anti-HBV virus treatment, which comprises the compound shown in Formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof.

[0028] The present application also relates to a composition for inhibiting the replication or propagation of HBV virus in cells (such as mammalian cells), which comprises the compound shown in Formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof.

[0029] The present application also relates to a composition for treating and / or preventing diseases or infections caused by HBV virus, which comprises the compound shown in Formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof.

[0030] The present application also relates to a composition for clearing HBV virus in cells (such as mammalian cells), which comprises the compound shown in Formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof.

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

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

[0033] According to certain 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 certain embodiments of the present application, the product described in the present application is a drug.

[0035] According to certain embodiments of the present application, the pharmaceutical composition described in the present application further comprises a pharmaceutically acceptable carrier or excipient.

[0036] According to certain embodiments of the present application, the pharmaceutical composition described in the present application is a solid preparation (such as tablets, capsules, pills, granules, powders), injection, topical preparation, spray, liquid preparation, or compound preparation.

[0037] According to certain embodiments of the present application, the pharmaceutical composition described in the present application can be administered by oral, injection, implantation, topical, spray or inhalation means.

[0038] According to certain embodiments of the present application, the compound of formula I described in the present application, wherein R is hydroxy, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, C 5 linear or branched alkyl, C 6 linear or branched 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 or benzyloxy, chloromethyl, chloroethyl, dichloroethyl, trifluoromethyl, difluoromethyl, monofluoromethyl, chloromethoxy, chloroethoxy, dichloroethoxy, trifluoromethoxy, difluoromethoxy, monofluoromethoxy.

[0039] According to certain embodiments of the present application, the compound of formula I described in the present application, wherein R is hydroxy, and in this case the compound is Palovarotene.

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

[0041] According to certain embodiments of the present application, the compound of formula I described in the present application, wherein R is amino.

[0042] According to certain embodiments of the present application, the compound of formula I described in the present application, wherein R is methylamino or ethylamino.

[0043] According to certain embodiments of the present application, the compound of formula I described in the present application, wherein R is dimethylamino or diethylamino.

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

[0045] According to certain embodiments of the present application, the compound of formula I described in the present application, wherein R is methoxy.

[0046] According to certain embodiments of the present application, the compound of formula I described in the present application, wherein R is ethoxy.

[0047] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is n-propoxy. According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is isopropoxy.

[0048] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is n-butoxy.

[0049] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is isobutoxy.

[0050] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is tert-butoxy.

[0051] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is pentyloxy.

[0052] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is hexyloxy.

[0053] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is cyclopropoxy, cyclobutoxy, cyclopentyloxy or cyclohexyloxy.

[0054] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is cyclopropoxy or cyclobutoxy.

[0055] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is phenoxy or benzyloxy.

[0056] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, C 5 a straight-chain or branched-chain alkyl, or C 6 a straight-chain or branched-chain alkyl.

[0057] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is methyl or ethyl.

[0058] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is n-propyl.

[0059] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0060] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is chloromethyl, chloroethyl, dichloroethyl, trifluoromethyl, difluoromethyl or monofluoromethyl.

[0061] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is chloromethyl or chloroethyl.

[0062] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is trifluoromethyl, difluoromethyl or monofluoromethyl.

[0063] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is chloromethoxy, chloroethoxy, dichloroethoxy, trifluoromethoxy, difluoromethoxy or monofluoromethoxy.

[0064] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is chloromethoxy or chloroethoxy.

[0065] According to certain embodiments of the present application, for the compound of formula I described in the present application, wherein R is trifluoromethoxy, difluoromethoxy or monofluoromethoxy.

[0066] According to certain embodiments of the present application, the pharmaceutically acceptable salts described in the present application include their inorganic or organic acid salts, as well as inorganic or organic base salts. For example, the pharmaceutically acceptable salts include but are not limited to: alkali metal salts including sodium salt, potassium salt, lithium salt of the compound; alkaline earth metal salts including calcium salt, magnesium salt; metal salts including aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt, etc.; meglumine salt, ammonium salt, tert-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, monoethanolamine, diethanolamine, triethanolamine, N-methyl-D-glucosamine, ethylenediamine salt, N-methylglucosamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-N-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris(hydroxymethyl)aminomethane salt and other amine salts.

[0067] According to certain embodiments of the present application, when a carboxyl group is present in the compound of formula I described in the present application, it can form a pharmaceutically acceptable ester with alcohols. The pharmaceutically acceptable esters include C 1 -C 6 alkyl esters such as methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, sec-butyl ester, tert-butyl ester, pentyl ester, hexyl ester, etc.; C 3 -C 6 cycloalkyl esters such as cyclopentyl ester, cyclohexyl ester, etc.; C 6 -C 10 aryl esters such as phenyl ester, naphthyl ester, etc.; C 6 -C 10 aryl C 1 -C 6Alkyl esters; or esters that can be hydrolyzed in vivo, such as (5-methyl-2-oxo-1,3-dioxol-4-yl) methyl ester, (pivaloyloxy) methyl ester, benzofuranone-based ester, [(isopropoxycarbonyl) oxy] methyl ester, [(cyclohexyloxycarbonyl) oxy] methyl ester, 1-[(cyclohexyloxycarbonyl) oxy] ethyl ester, etc.

[0068] The carriers described in this application include but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering 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 substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin.

[0069] The term "excipient" used in this application refers to additives other than the main drug in pharmaceutical preparations. It is stable in nature, has no incompatibility with the main drug, does not produce side effects, does not affect the efficacy, is not easily deformed, cracked, mildewed, moth-eaten at room temperature, is harmless to the human body, has no physiological effects, does not produce chemical or physical effects with the main drug, and does not affect the determination of the content of the main drug, etc. Such as binders, fillers, disintegrants, lubricants in tablets; preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, coloring agents, etc. in oral liquid preparations can all be called excipients, and so on.

[0070] The term "C 1-6 alkyl" used in this application refers to a straight-chain or branched-chain alkyl having 1 to 6 carbon atoms, such as C 1-4 alkyl, C 1-2 alkyl, C 1 alkyl, C 2 alkyl, C 3 straight-chain or branched-chain alkyl, C 4 straight-chain or branched-chain alkyl, C 5 straight-chain or branched-chain alkyl or C 6 straight-chain or branched-chain alkyl. Specific examples include but are not limited to methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0071] The term "C 1-6 alkoxy" used in this application refers to C 1-6 alkyl as defined above that is connected to the parent molecular moiety through an oxygen atom. Representative examples of C 1-6 alkoxy include but are not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy.

[0072] The term "C1-6 "Halogenated alkyl" refers to a C alkyl as defined above that is mono- or poly-substituted by a halogen such as fluorine, chlorine, bromine or iodine. 1-6 alkyl. C 1-6 Representative examples of halogenated alkyls include, but are not limited to, chloromethyl, chloroethyl, dichloroethyl, trifluoromethyl, difluoromethyl, monofluoromethyl, etc.

[0073] The term "C 1-6 alkyl-amino-" used in this application refers to an amino group mono-substituted by a C 1-6 alkyl as defined above. "C 1-6 alkyl-amino" typical examples include, but are not limited to, methylamino, ethylamino, propylamino, butylamino, etc.

[0074] The term "di(C 1-6 alkyl)-amino-" used in this application refers to an amino group di-substituted by a C 1-6 alkyl as defined above. "Di(C 1-6 alkyl)-amino-" typical examples include, but are not limited to, dimethylamino, diethylamino, dipropylamino, dibutylamino, etc.

[0075] The term "C 3-6 cycloalkyl" used herein refers to a saturated cyclic hydrocarbon group having 3 to 6 carbon atoms and having a monocyclic or bicyclic or multiple fused rings (including fused and bridged ring systems). "C 3-6 cycloalkyl" typical examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0076] The term "C 3-6 cycloalkoxy" used herein refers to a C 3-6 cycloalkyl as defined above that is connected to the parent molecular moiety through an oxygen atom. "C 3-6 cycloalkoxy" typical examples include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.

[0077] The term "C 1-6 haloalkoxy" used herein refers to a C 1-6 halogenated alkyl as defined above that is connected to the parent molecular moiety through an oxygen atom. C 1-6 Representative examples of haloalkoxys include, but are not limited to, chloromethoxy, chloroethoxy, dichloroethoxy, trifluoromethoxy, difluoromethoxy, monofluoromethoxy, etc.

[0078] The term "subject" used in this application includes mammals.

[0079] The mammals described in this application include bovines, equines, ovines, felines, canines, rodents, primates, and among them, the preferred mammal is human.

[0080] As used herein, the term "effective amount" means an amount sufficient to achieve or at least partially achieve the desired effect. For example, a therapeutically effective amount means an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. A prophylactically effective amount means an amount capable of effectively preventing, arresting or delaying the occurrence of a disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly, etc.

[0081] The amount of the compound of formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, the hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof administered to a subject depends on the type and severity of the disease or condition and the characteristics of the subject, such as general health, age, sex, weight and tolerance to the drug, and also depends on factors such as the type of formulation and the mode of administration of the drug, as well as the dosing cycle or time interval. Those skilled in the art can determine the appropriate dose based on these and other factors. Generally, the daily dose for therapeutic use of the compound of formula I, its stereoisomers, its solvates, its pharmaceutically acceptable salts, the hydrates or solvates of its pharmaceutically acceptable salts, its pharmaceutically acceptable esters, or various pharmaceutically acceptable modifications thereof can be about 0.0001 - 1000 mg / kg body weight / day, and this daily dose can be administered once or in divided doses as appropriate.

[0082] The embodiments of the present application will be described in detail below in conjunction with the drawings and examples. However, those skilled in the art will understand that the following drawings and examples are only for illustrating the present application and not for limiting the scope of the present application. From the following detailed description of the drawings and preferred embodiments, various objects and advantageous aspects of the present application will become apparent to those skilled in the art. Description of the Drawings

[0083] Figure 1 It is the chemical structural formula of Palovarotene.

[0084] Figure 2 It is the inhibitory effect of Palovarotene on HBV infection in the infection model HepG2 - hNTCP 2B1.

[0085] Figure 3 It is the inhibitory effect of Palovarotene on HBV infection in the infection model HepRG M14a.

[0086] Figure 4 The therapeutic effect of Palovarotene after HBV infection in the infection model HepG2-hNTCP 2B1.

[0087] Figure 5 The evaluation results of Palovarotene in the high-copy replication model HepAD38.

[0088] Figure 6 The inhibitory effect of Palovarotene on HBV infection in primary human hepatocytes.

[0089] Figure 7 The therapeutic effect of Palovarotene after HBV infection in primary human hepatocytes.

[0090] Figure 8 The safety evaluation results of Palovarotene in in vitro cell models. Detailed implementation manners

[0091] For those without specific technologies or conditions indicated in the following examples, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. Unless otherwise specified, the experimental methods are all conventional methods. For materials, reagents or equipment without indicating the manufacturer, they are all conventional products that can be obtained by purchase. All experiments are set up with three replicates, and the results are averaged.

[0092] In the following examples, the drug to be tested is Palovarotene (purchased from MCE), and the positive control drugs include entecavir (ETV, purchased from Yuanye Bio-Technology Co., Ltd.) and interferon IFNα (purchased from Sigma, SRP4595-100UG). During the experiment, the drug to be tested or the positive control drug is diluted to the required final concentration with the culture medium, and the culture medium is determined by the cells used, which is specifically described in each example.

[0093] In the following examples, the formula for 10 L of the used PBS is: 80 g of NaCl, 2 g of KCl, Na 2 HPO 4 ·12H 2 O 29 g, KH 2 PO 4 2.4 g, adjust the pH value to 7.4, make up the volume to 10 L, mix well, then filter and dispense with a double-layer 0.22 μm filter membrane, and sterilize by high-pressure steam for later use.

[0094] In the following examples, the DMEM medium used was purchased from SIGMA-ALDRICH, product number D6429; FBS was purchased from ThermoFisher, product number 10099141; Puromycin was purchased from InvivoGen, product number ant-pr-1; Doxycycline was purchased from SIGMA-ALDRICH, product number D9891; penicillin and streptomycin were purchased from Shandong Lukang Pharmaceutical Co., Ltd.; 4% PEG 8000 was purchased from AMRESCO, product number 0159; WME medium was purchased from SIGMA-ALDRICH, product number W1878; glutamine was purchased from SIGMA-ALDRICH, product number G8540; insulin was purchased from Jiangsu Wanbang Pharmaceutical Co., Ltd.; hydrocortisone was purchased from Cayman, product number 18226; Tetracycline was purchased from USB, product number 22105-25g; B-27 TM Supplement (50×) was purchased from GIBCO, product number 17504044; Forskolin was purchased from MCE, product number HY-15371; SB431542 was purchased from Tocris, product number 1614; IWP2 was purchased from MCE, product number HY-13912; DAPT was purchased from MCE, product number HY-13027; LDN193189 was purchased from MCE, product number HY-12071A.

[0095] In the following examples, the medium A used was DMEM medium, which contained: 10% FBS, 1 μg / mL Puromycin, 1 μg / mL Doxycycline, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0096] In the following examples, the medium B used was DMEM medium, which contained: 10% FBS, 1 μg / mL Puromycin, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0097] In the following examples, the medium C used was DMEM medium (prepared and used immediately), which contained 4 w / v% PEG8000, 1 μg / mL Puromycin, 1 μg / mL Doxycycline, 100 U / mL penicillin, 100 μg / mL streptomycin, and 5% FBS.

[0098] In the following examples, the medium D used was WME medium, which contained: 10% FBS, 100 mM glutamine, 5 μg / mL insulin, 50 μM hydrocortisone, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0099] In the following examples, the medium E used was WME medium (prepared freshly before use), which contained 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.

[0100] In the following examples, the medium F used was DMEM medium, which contained: 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0101] In the following examples, the medium G used was DMEM medium, which contained: 10% FBS, 1 μg / mL Tetracycline, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0102] In the following examples, the medium H used was WME medium, which contained: 1 v / v% B-27 TM Supplement (diluted at a ratio of 1:100 when used, i.e., 1 mL of B-27 Supplement was contained in every 100 mL of medium H) TM Supplement, 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.

[0103] In the following examples, the medium I used was WME medium (prepared freshly before use), which contained 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 TM Supplement (diluted at a ratio of 1:200 when used, i.e., 0.5 mL of B-27 Supplement was contained in every 100 mL of medium I) TM Supplement.

[0104] Example 1 evaluated the inhibitory effect of the drug to be tested on HBV infection in the infection model HepG2-hNTCP 2B1

[0105] The human sodium taurocholate cotransporting polypeptide (hNTCP) gene and the red fluorescent protein (mCherry) gene were constructed in a dicistronic expression cassette regulated by a tetracycline-inducible promoter. The expression cassette was stably integrated into HepG2-TetOn cells using the lentiviral system. Based on flow cytometry sorting technology, a stable cell line HepG2-hNTCP 2B1 with high expression of human NTCP was obtained. This cell model supports HBV infection and generates HBV-related indicators. (For the construction method of this cell model, please refer to: 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, 8:1, 724-733, DOI: 10.1080 / 22221751.2019.1619485).

[0106] This experiment was carried out in a 24-well culture plate. Each test drug in the experimental group was set at three final concentrations of 1 μM, 5 μM, and 25 μM. The positive control group was entecavir (ETV) at a final concentration of 1 μM. Among them, the test drug and the positive control were diluted to the required concentration with medium A, and the blank group was added with the same volume of medium A without adding drugs.

[0107] Approximately 1.5×10 5 HepG2-hNTCP 2B1 cells were seeded in each well and 500 μL of medium B was added. After the cells adhered, medium A was replaced for full-course Doxycycline induction.

[0108] After 72 hours of induction with doxycycline, 500 μL of medium A containing the drug was added to each well for drug treatment. The drug treatment lasted for 24 hours, and then the cells were infected with HBV virus at a virus amount of 100 MOI per well. Before infection, the HBV virus was first diluted in medium C to obtain the HBV virus solution, and the infection volume was 250 μL per well. When infecting, the cell supernatant was first aspirated, and the HBV virus solution was added to the cells. After incubation for 16 - 20 hours, the supernatant was collected as Day0, and then washed 3 times with PBS pre-warmed at 37°C, and fresh medium A containing the drug (500 μL) was replaced. After that, the supernatant was collected every 2 days for testing, and fresh medium A containing the drug was added until all the supernatant and cells were collected on the 8th day for relevant tests. The test items included: HBsAg / HBeAg, HBV DNA, and HBV cccDNA.

[0109] Detection of HBsAg / HBeAg:

[0110] Both HBsAg (chemiluminescent immunoassay CLEIA, product standard number: YZB / Guo 0346 - 2014) and HBeAg (enzyme-linked immunosorbent assay ELISA, product standard number: YZB / Guo 0216 - 2013) were detected using the detection kits from Beijing Wantai Company, and the detection operations were carried out according to the test methods described in the kit instructions.

[0111] Detection of HBV DNA:

[0112] After washing the collected cells once with PBS, HBV DNA was obtained by automated extraction using a virus DNA & RNA extraction kit (Beijing Kingmag) on a nucleic acid extraction workstation (purchased from GenMagBio Company, model DOF - 9696) (the extraction procedure was referred to the instrument instruction manual). The quantification of HBV DNA was performed by probe-based real-time fluorescence quantitative PCR method. The reagent used was Premix Ex TaqTM (Takara), and the instrument used was Roche's 96, and the primer sequences for fluorescence quantification are shown in Table 1, and the fluorescence quantitative reaction system is shown in Table 2.

[0113] Detection of HBV cccDNA:

[0114] The improved Hirt method was used to extract HBV cccDNA with a kit. The kit used was a plasmid miniprep kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). The solutions involved 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). The specific method referred to the plasmid extraction method described in the kit instructions. Real-time fluorescence quantitative PCR with a probe was used to quantitatively detect HBV cccDNA, and mitochondrial DNA (mtDNA) was simultaneously detected as an internal reference. The instrument used was Roche's 96. The primers for fluorescence quantification are shown in Table 1, and the reaction system for fluorescence quantification is shown in Table 2.

[0115] The fluorescence reaction program for quantitative detection is as follows:

[0116] (1) HBV DNA quantification: The quantification program was 95°C, 30 s; (95°C, 5 s; 60°C, 30 s) for 45 cycles, and the fluorescence channel was Hex.

[0117] (2) cccDNA quantification: The quantification program was 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) for 45 cycles, and the fluorescence channel was FAM.

[0118] (3) mtDNA quantification: The quantification program was 95°C, 5 min; (95°C, 30 s; 55°C, 40 s) for 45 cycles, and the fluorescence channel was Hex.

[0119] cccDNA normalization calculation: The specific formula is as follows: 2^(mtDNA - cccDNA), where the values in the parentheses are the Ct values of mtDNA and cccDNA respectively.

[0120] The experimental results are as Figure 2 shown. The results showed that compared with the blank group, the treatment with Palovarotene could effectively inhibit the levels of HBsAg (p < 0.05) and HBeAg (p < 0.05) in the cell supernatant, and the levels of intracellular HBV DNA (p < 0.001) and HBV cccDNA (p < 0.0001), and the degree of inhibition showed a certain dose-dependence.

[0121] This indicates that Palovarotene can significantly inhibit the infection of HBV to HepG2-hNTCP 2B1.

[0122] Table 1. PCR primer sequences

[0123]

[0124]

[0125] Table 2. qPCR quantitative reaction system

[0126]

[0127] Example 2. Evaluate the inhibitory effect of the drug to be tested on HBV infection in the HepaRG M14A infection model

[0128] HepaRG is a hepatic progenitor cell line with differentiation potential isolated from the tumor tissue of a female hepatitis C patient. Two weeks after cell seeding, a two-week differentiation induction with 2% DMSO (2 mL DMSO added to every 100 mL cell culture medium) was carried out. Eventually, about 50% of the cells differentiated into hepatocyte-like cells, and the other 50% differentiated into cholangiocyte-like cells. Among them, the hepatocyte-like cells support HBV infection, and the infection efficiency is about 20%. By genetically modifying HepaRG, the HNF4A and FoxM1 genes were integrated into HepaRG cells, and a HepaRG M14A cell line that can complete differentiation and support HBV infection without depending on DMSO was screened. (For the construction method of this cell model, refer to: Zhao Jinghua. Construction, optimization and preliminary application of an in vitro hepatitis B virus infection model [D]. Xiamen University, 2016.)

[0129] This experiment was carried out in a 24-well cell culture plate. For each drug to be tested in the experimental group, three final concentrations of 1 μM, 5 μM, and 25 μM were set. The positive control group was entecavir (ETV) at a final concentration of 1 μM. Among them, the drug to be tested and the positive control were diluted to the required concentration with medium D, and the blank group was added with the same volume of medium D without adding drugs. Approximately 1.5×10 5 HepaRG M14A cells were seeded in each well, and 500 μL of medium D was added to each well. After cell seeding, the medium D was changed every 2 days. On the 13th day after cell seeding, the medium D was changed to the drug-containing medium D for drug treatment. After 24 h of drug treatment, the cells were infected with HBV virus, and the amount of HBV virus infected per well was 1000 MOI. Before infection, the HBV virus was first diluted in medium E to obtain the HBV virus solution, and the infection volume was 250 μL per well. When infecting, first aspirate the cell supernatant, add the HBV virus solution to the cells, incubate for 24 hours, and collect the supernatant as Day0. Then, wash the cells 3 times with PBS pre-warmed at 37 °C, and replace it with fresh drug-containing medium D (500 μL per well). After that, collect the supernatant for testing every 2 days, and replace it with fresh drug-containing medium D until the 8th day when all the supernatant and cells were collected for relevant detection. The detection items included HBsAg, HBeAg, HBV DNA, and HBV cccDNA, and the detection methods were referred to Example 1.

[0130] The experimental results are as Figure 3 shown. The results showed that in the HepaRG M14a-infected cell model, pretreatment with Palovarotene 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).

[0131] The above results indicate that Palovarotene can significantly inhibit the infection of HBV in HepaRG M14A.

[0132] Example 3: Evaluate the therapeutic effect of the drug to be tested on HBV infection in the HepG2-hNTCP 2B1 infection model.

[0133] This experiment was carried out in a 24-well culture plate. Each drug to be tested in the experimental group had three final concentrations of 1 μM, 5 μM, and 25 μM. The positive control group was ETV with a final concentration of 1 μM. The drug to be tested and the positive control were diluted to the required concentration with Medium A. The blank group was added with the same volume of Medium A without adding drugs. Approximately 1.5×10 5 HepG2-hNTCP 2B1 cells were seeded in each well, and 500 μL of Medium B was added to each well. After the cells adhered, Medium A was replaced for the whole process of Doxycycline induction.

[0134] After 4 days of Doxycycline induction, the cells were infected with HBV virus. The amount of HBV virus infected in each well was 100 MOI. Before infection, the HBV virus was first diluted in Medium C to obtain the HBV virus solution, and the infection volume was 250 μL per well. When infecting, the cell supernatant was first aspirated, and the HBV virus solution was added to the cells. After incubating for 24 h, the supernatant was collected as Day0, then washed 3 times with PBS, and fresh Medium C was replaced. After that, the supernatant was collected every two days for testing, and fresh Medium C was replaced. On the 6th day, fresh Medium A containing the drug was replaced to start the drug treatment. After that, the supernatant was collected every two days for testing, and fresh Medium A containing the drug was replaced until the supernatant and cells were collected on the 12th day for relevant detection. The detection items included HBsAg, HBeAg, HBV DNA, and HBV cccDNA. The detection method was referred to Example 1.

[0135] The Southern blot experiment was carried out using a 10 cm 2 culture plate. Approximately 5×10 2 cells were seeded in each 10 cm 6One HepG2-hNTCP 2B1 cell was added with Medium B. After the cells adhered to the wall, Medium A was replaced for the whole process of Doxycycline induction. After 4 days of Doxycycline induction, the cells were infected with HBV at an HBV infection dose of 1000 MOI. Before infection, the HBV virus was first dissolved in Medium C to obtain an HBV virus solution, and the infection volume was 5 mL. During infection, the cell supernatant was first aspirated, and the HBV virus solution was added to the cells. After incubation for 24 h, the supernatant was collected as Day 0, then the cells were washed 3 times with PBS, and fresh Medium A was replaced. After that, the supernatant was collected every two days for testing, and fresh Medium A was replaced. On the 6th day, fresh medicated medium was replaced to start drug treatment (the drug treatment concentration was 5 μM, and the control group used interferon IFNα at a concentration of 500 IU). After that, the supernatant was collected every two days for testing, and fresh medicated Medium A was replaced until the supernatant and cells were collected on the 12th day for Southern blot detection and immunofluorescence detection.

[0136] Southern blot detection

[0137] The HBV cccDNA was extracted using a kit with the modified Hirt method. The kit used was the Qiagen Midiprep Kit, and the specific extraction operation was referred to the plasmid midiprep extraction method of the kit. The specific steps of Southern blot detection are as follows:

[0138] a. The extracted HBV cccDNA sample was added with loading buffer (purchased from TaKaRa) and then electrophoresed for 2 h in 1.2% agarose gel at 80 V in 1×TAE buffer (40 mmol / L Tris Base, 1 mmol / L EDTA, 20 mmol / L sodium acetate) to separate the DNA; then the gel was placed in depurination buffer (0.2 mol / L HCl) and soaked with shaking at room temperature for 15 min, and then rinsed twice with sterile ultrapure water for 1 min each time; the gel was placed in denaturing buffer (0.5 mol / L NaOH, 1.5 mol / L NaCl) and soaked with shaking at room temperature for 30 min, and then rinsed twice with sterile ultrapure water for 1 min each time; finally, it was soaked in neutralization buffer (1 mol / L Tris-base, 1.5 mol / L NaCl) for 10 min.

[0139] b. 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 this period, the filter paper and nylon membrane (purchased from Roche) were first wetted with sterile ultrapure water and then immersed in 10×SSC. The nucleic acid was transferred to the nylon membrane using a vacuum transfer apparatus (the pressure was adjusted to 5 MPa and the membrane transfer time was 90 min). At the same time, the hybridization solution (DIG Easy Hyb TM Granules, purchased from Roche, catalog number 38716500) was preheated at 42°C. After the membrane transfer, the transferred nylon membrane was cross-linked using a UV cross-linker SiGmA (purchased from Shanghai Sigma Co., Ltd.) model SH4 B at an irradiation dose of 1.5 J / cm2 for 3 min.

[0140] c. Hybridization: The membrane was immersed in the preheated hybridization solution at 42°C and placed in a hybridization oven at 42°C for incubation for 2 h. At the same time, the probe was heated at 95°C for 10 min and then quickly inserted into an ice-water mixture for 5 min (to prevent renaturation), and then diluted in the hybridization solution (300 ng of the probe was added to every 10 mL of the hybridization solution), and hybridized with the membrane at 42°C for 12 h. After hybridization was completed, it was washed 3 times at room temperature with Wash buffer I (2×SSC, 0.1% SDS), 5 min each time, and then washed 3 times at 65°C with Wash buffer II (0.5×SSC, 0.1% SDS), 15 min each time.

[0141] 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 min, and then blocked at room temperature for 1 h with 1×Blocking buffer (purchased from SIGMA-ALDRICH, catalog number B6429-500ML) (diluted with maleic acid buffer). After blocking, the anti-Dig-AP antibody (purchased from SIGMA-ALDRICH, catalog number Roche-11093274910) was diluted with 1×Blocking buffer, and the membrane was incubated with the antibody at room temperature for 40 min. After incubation was completed, it was washed 3 times at room temperature with the washing solution (0.1 mol / L maleic acid, 0.15 mol / L NaCl, 0.3% (v / v) Tween 20, pH 7.5), 15 min each time.

[0142] e. Detection: First, immerse the membrane in the detection equilibration solution (0.1 mol / L Tris-base, 0.1 mol / L NaCl, pH 9.5) for 3 min, then take out the membrane and add the chromogenic substrate (CDP-star, purchased from Roche) onto the membrane. Finally, expose it on a biomolecular imager (ImageQuant LAS 4000 mini, GE).

[0143] Immunofluorescence detection

[0144] All the HBcAg immunofluorescence stainings involved in this experiment were direct adherent stainings. The following operations were all carried out on a shaker at a rotation speed of 100 rpm / min. Wash the cells in the cell culture plate once with PBS. Fixation: Add 4% paraformaldehyde (purchased from Beyotime, product number: P0099-100ml) to cover the cells and incubate at room temperature in the dark for 10 min. Washing: Wash 3 times with PBS, 3 min for each time. Permeabilization: Add 0.2% Triton X-100 (purchased from AMRESCO) and incubate at room temperature for 10 min. Washing: Wash 3 times with PBS, 3 min for each time. Blocking: Add 3% BSA (weigh 3 g of BSA powder and dissolve it in 100 mL of PBS, the BSA powder is purchased from SIGMA-ALDRICH) and incubate at room temperature for 60 min or overnight at 4°C. Primary antibody: Dilute Anti-HBcAg (2A7, mouse anti-) with 3% BSA at a ratio of 1:1000, 200 μL per well, and incubate at room temperature for 1 hour or overnight at 4°C. Washing: Wash 3 times with PBS, 3 min for each time. Secondary antibody: Dilute the secondary antibody (Alexa 488 Donkey Anti-Rabbit IgG(H+L) (Invitrogen)) with 3% BSA at a ratio of 1:1000, 200 μL per well, and incubate at room temperature for 30 min. Washing: Wash 3 times with PBS, 3 min for each time. Nuclear staining: Dilute DAPI (Invitrogen) with 3% BSA at a ratio of 1:2000, 200 μL per well, and incubate at room temperature for 5 min. Washing: Wash 3 times with PBS, 3 min for each time. Photographing: Take pictures and analyze the average fluorescence intensity of the green fluorescence with a high-content confocal microscope (PerkinElmer, Operetta CLS).

[0145] The experimental results are as Figure 4As shown. The results showed that after adding the drug 6 days after HBV infection, high-concentration 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), while there was no significant difference between the control group (ETV) and the blank group. Southern blot results showed that the level of HBV cccDNA was significantly lower in the group treated with the drug at a concentration of 5 μM than in the control group treated with interferon and the blank group without drug addition. Immunofluorescence results showed that Palovarotene could significantly reduce the positive rate of intracellular HBcAg in HepG2-hNTCP 2B1 after infection, suggesting that the drug treatment could inhibit HBV infection.

[0146] Treatment with high-concentration Palovarotene can significantly inhibit HBV virus replication and e antigen levels after infection.

[0147] Example 4. Evaluation of the therapeutic effect of the drug to be tested on the high-copy replication model HepAD38. HepAD38 is a hepatoma cell line (genotype D) with stable integration of the HBV genome that regulates the expression of HBV virus by tetracycline. In the presence of tetracycline, the synthesis of HBV pgRNA is inhibited and the cells cannot synthesize virus particles; when tetracycline is absent, the HepAD38 cell line transcribes HBV pgRNA, synthesizes subviral particles and secretes virus-like particles into the cell supernatant. The level of HepAD38 DNA is about 10 times that of HepG2.2.15 and is a high-copy HBV in vitro replication cell model (the construction method of this cell model can be referred to: 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.).

[0148] This experiment was carried out in a 24-well culture plate. Each drug to be tested in the experimental group had two final concentrations of 1 μM and 5 μM. The positive control group was ETV with a final concentration of 1 μM. Among them, the drug to be tested and the positive control were diluted to the required concentration with medium F, and the blank group was added with the same volume of medium F without drug addition. Approximately 4×10 5For each well containing HepAD38 cells, add 500 μL of medium G containing tetracycline hydrochloride. After the cells are plated for 48 h, it is designated as Day 0. At the same time, remove medium G containing tetracycline hydrochloride and replace it with fresh medium F containing the drug. Thereafter, replace the fresh medium F containing the drug every two days until Day 8. Detect the levels of HBsAg in the cell supernatant and HBV DNA in the cells on Day 8. The detection method refers to Example 1.

[0149] The Southern blot experiment was carried out on a 6 cm 2 culture plate. For each 6 cm 2 culture plate, approximately 4.2×10 6 HepAD38 cells were plated and medium G containing tetracycline hydrochloride was added. During the experiment, remove medium G containing tetracycline hydrochloride and simultaneously add medium F containing the drug for drug treatment. The treatment concentration of the test drug palovarotene was 5 μM, and the control group used entecavir (ETV) at a concentration of 5 μM. The treatment time was 8 days.

[0150] The specific Southern blot detection method refers to Example 3.

[0151] The results are as Figure 5 shown. The results showed that palovarotene at a concentration of 5 μM had a significant inhibitory effect on the production of surface antigen of the replication model HepAD38 (p<0.01) and HBV DNA (p<0.05). The Southern blot results showed that the drug could significantly inhibit the level of HBV cccDNA, and the inhibitory effect was worse than that of the control group (ETV).

[0152] Palovarotene treatment can significantly inhibit the virus replication level of the replication model HepAD38.

[0153] Example 5: Evaluate the inhibitory effect of the test drug on HBV infection in primary human hepatocytes

[0154] In this experiment, there were an experimental group, a positive control group, and a blank group. Among them, each test drug (palovarotene) in the experimental group had three final concentrations of 1 μM, 5 μM, and 25 μM. The positive control group was ETV at a final concentration of 1 μM. Among them, the test drug and the positive control were diluted to the required concentration with medium H, and the blank group was added with the same volume of medium H without drug treatment.

[0155] Perfuse primary hepatocytes from humanized mice transplanted with human primary hepatocytes (Reference: Foquet, Lander, et al. "Successful engraftment of human hepatocytes in uPA - SCID and KO mice." Hepatocyte Transplantation. Humana Press, New York, NY, 2017. 117 - 130., Wan - Chun Li, et al. "Isolation and Culture of Adult Mouse Hepatocytes." Mouse Cell Culture. Humana Press, 2010. 185 - 196.), and after counting (plating approximately 5×10 5 primary hepatocytes per well), directly culture them in a 24 - well plate (Collagen I pre - coated 24 - well plate, purchased from Thermo Scientific), and add medium H to maintain the hepatocyte morphology (Reference: Xiang, Chengang, et al. "Long - term functional maintenance of primary human hepatocytes in vitro." Science 364.6438 (2019): 399 - 402.). After culturing for 24 h, replace the drug - free medium H with medium H containing the drug for drug treatment for 24 hours, and then perform HBV virus infection. The amount of HBV virus infected per well is 2000 MOI. Before infection, first dilute the HBV virus in medium I to obtain the HBV virus solution. The infection volume is 250 μL / well. When infecting, first aspirate the cell supernatant, add the HBV virus solution to the cells, incubate for 24 hours, and collect the supernatant as Day 0. Then wash the cells 3 times with PBS warmed at 37 °C, and replace it with fresh medium H containing the drug (500 μL per well). After that, collect the supernatant for testing every 2 days, and replace it with fresh medium H containing the drug until the 8th day when all the supernatant and cells are collected for relevant tests. The detection items include HBsAg, HBeAg, HBV DNA, and HBV cccDNA, and the detection method refers to Example 1.

[0156] The experimental results are as Figure 6 shown. The results show that in human primary hepatocytes, pretreatment with Palovarotene before infection can significantly inhibit the levels of HBsAg (p < 0.05), HBeAg (p < 0.05) in the cell supernatant, and the levels of intracellular HBV DNA (p < 0.05) and cccDNA (p < 0.05).

[0157] Palovarotene treatment can significantly inhibit the infection of HBV in human primary hepatocytes.

[0158] Example 6. Evaluation of the therapeutic effect of the drug to be tested on HBV-infected human primary hepatocytes

[0159] In this experiment, there were an experimental group, a positive control group and a blank group. Among them, each drug to be tested (Palovarotene) in the experimental group had three final concentrations of 1 μM, 5 μM and 25 μM. The positive control group was ETV with a final concentration of 1 μM. The drug to be tested and the positive control were diluted to the required concentration with medium H. The blank group was added with the same volume of medium H without drug treatment.

[0160] Primary hepatocytes were perfused from humanized mice transplanted with human primary hepatocytes. After counting (5×10 5 primary hepatocytes per well), they were directly cultured in 24-well plates. Medium H was used to maintain the hepatocyte morphology. After culturing for 24 hours, they were infected with HBV virus, and the amount of HBV virus infected per well was 2000 MOI. Before infection, the HBV virus was first dissolved in medium I to obtain an HBV virus solution. The infection volume was 250 μL / well. When infecting, first aspirate the cell supernatant, add the HBV virus solution to the cells, incubate for 24 h, and collect the supernatant as Day 0. Then, the supernatant was collected every two days for testing, and fresh medium H was replaced. On the 8th day, fresh medium H containing the drug was replaced, and drug treatment was started until all the supernatant and cells were collected on the 14th day for relevant detection. The detection items included: HBsAg, HBeAg, HBV DNA, HBV cccDNA. The detection method referred to Example 1.

[0161] The experimental results are as Figure 7 shown. The results showed that when the drug treatment was added 8 days after infection, Palovarotene had a significant inhibitory effect on the levels of HBeAg (p<0.05) and HBsAg (p<0.05) in the cell supernatant, and the inhibitory effects of the 5 μM and 25 μM concentrations were more obvious. The 25 μM concentration had a significant inhibitory effect on both HBV cccDNA (p<0.01) and HBV DNA (p<0.05).

[0162] Palovarotene treatment can significantly inhibit the replication of HBV virus and the levels of e antigen and surface antigen after infection.

[0163] Example 7. Safety evaluation of the drug to be tested on the HBV in vitro cell model

[0164] This experiment was carried out in a 96-well culture plate, with 8×10 4Corresponding cells, with a corresponding culture medium volume of 100 μL each. For each test drug in the experimental groups of HepG2-hNTCP 2B1, HepaRG M14A, and primary hepatocytes, three final concentrations of 1 μM, 5 μM, and 25 μM were set. For each test drug in the experimental group of HepAD38 cells, two final concentrations of 1 μM and 5 μM were set (the drugs were diluted to the final concentration with the corresponding culture medium, specifically referring to Example 1, Example 2, Example 4, and Example 5). The blank group was added with the same volume of culture medium without adding drugs. After the cells grew to an appropriate density, they were treated with drugs for 6 days. During this period, the culture medium was changed every two days, and fresh drug-containing culture medium was replaced. After the drug treatment ended, 110 μL of culture medium containing Beyotime CCK-8 cell viability detection reagent (purchased from Beyotime, catalog number C0038) (the volume ratio of the culture medium to the CCK-8 reagent was 100:10) was replaced and incubated at 37 °C for 30 min, and then all the supernatants were collected to detect the absorbance. The absorbance was read using a Zhengzhou Antu PHOMO microplate reader.

[0165] The results are as Figure 8 shown. The results showed that there was no significant difference in the CCK-8 absorbance between the groups treated with the drug Palovarotene at concentrations of 1 μM, 5 μM, and 25 μM on HepG2hNTCP 2B1, HepaRG M14a, HepAD38, and human primary hepatocytes and the blank group, indicating that the drug had no significant effect on cell viability within this concentration range.

[0166] Examples of this application show that the compound shown in Formula I (including Palovarotene) can significantly reduce the levels of HBsAg and HBeAg in the cell culture supernatant of the HBV in vitro research model, as well as the levels of intracellular HBV DNA and cccDNA. A good antiviral treatment effect on cells can be achieved after treatment with a drug dose of 5 μM. This drug has the potential to be developed into an anti-hepatitis B virus treatment drug.

[0167] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of this application and not to limit them; although this application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: it is still possible to modify the specific implementation manners of this application or perform equivalent replacements for some technical features; without departing from the spirit of the technical solutions of this application, they should all be covered within the scope of the technical solutions claimed in this application.

Claims

1. Use of a compound represented by formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound represented by formula I or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating and / or preventing diseases or infections caused by HBV virus, wherein R is a hydroxyl group.

2. The use according to claim 1, wherein the disease caused by HBV virus is viral hepatitis B, hepatitis B-related liver cirrhosis or primary liver cancer.

3. The use according to claim 2, wherein the viral hepatitis B is chronic viral hepatitis B or acute viral hepatitis B.

4. The use according to claim 3, wherein the chronic viral hepatitis B is chronic active hepatitis.

5. The use according to any one of claims 1-4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

6. The use according to claim 5, wherein the compound represented by formula I or a pharmaceutically acceptable salt thereof is present in a therapeutically and / or prophylactically effective amount.

7. The use according to any one of claims 1-4, wherein the pharmaceutical composition is a solid preparation or a liquid preparation.

8. The use according to any one of claims 1-4, wherein the pharmaceutical composition is a compound preparation.

9. The use according to any one of claims 1-4, wherein the pharmaceutical composition is an injection or a spray.

10. The use according to any one of claims 1-4, wherein the pharmaceutical composition is a topical preparation.

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