Compound, pharmaceutical composition comprising same, and use thereof

Abstract

A compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof. The present invention further relates to a pharmaceutical composition comprising the compound, a preparation method, and a use.

Description

A compound, a pharmaceutical composition containing the same and its use Technical Field

[0001] The present invention relates to the fields of medicinal chemistry and biopharmaceuticals. Specifically, the present invention relates to a compound having a novel structure of formula (I) or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the same, and use thereof in the preparation of a medicament for preventing or treating hepatitis virus infection. Background Art

[0002] Chronic hepatitis caused by hepatitis B virus (HBV) infection is a global public health problem, affecting nearly 300 million people worldwide. The incidence of liver cancer caused by HBV is approximately 8.5 per 100,000 people. Long-term chronic HBV infection can progress to cirrhosis and hepatocellular carcinoma, with complications resulting in over 800,000 deaths annually. Clinical treatments primarily consist of nucleoside analogs (NAs) and interferons (IFNs), which can ameliorate the progression of HBV-related pathogenesis, reduce viral load, and alleviate hepatitis. However, these drugs are associated with long treatment cycles and drug resistance, making them incurable. The main reasons for the difficulty in treating HBV are, firstly, the ability of HBV DNA to integrate into host cell DNA after infection; and, secondly, the generation of pregenomic RNA (pgRNA) from covalently closed circular DNA (cccDNA), which serves as a template for continuous replication and release of HBV, leading to infection of new hepatocytes, thus maintaining chronic infection and expanding the cccDNA pool. Therefore, a combined drug strategy of "preventing external troubles" and "eliminating internal evils" may be a good choice for the treatment of HBV infection. Developing HBV entry inhibitors to prevent new cells from being infected and combining them with antiviral reverse transcriptase drugs is a powerful means to reduce the cccDNA pool, which is of great significance for the treatment of HBV infection.

[0003] Hepatitis D is an infectious disease caused by hepatitis D virus (HDV) and hepatotropic DNA viruses such as hepatitis B virus. HDV is a defective single-stranded negative-sense RNA virus that requires a capsid from hepatotropic DNA viruses such as HBV for replication. HDV is present in the hepatocyte nuclei and serum of HDV-infected individuals who are positive for hepatitis B surface antigen (HBsAg). Replication primarily occurs within hepatocytes. HDV infection significantly inhibits HBV DNA synthesis, and the appearance of hepatitis D virus antigen (HDAg) coincides with a decrease in serum HBV DNA. With the conversion of HDAg to negative levels and the development of anti-HDV antibodies, HBV DNA levels return to their original levels. Over-infection of HDV with HBV can exacerbate liver damage and predispose to the development of chronic active hepatitis, cirrhosis, and severe hepatitis.

[0004] Sodium-taurocholate cotransporter (NTCP) is a functional receptor for hepatitis B virus (HBV) / hepatitis D virus (HDV) to enter hepatocytes. It is a transmembrane bile acid transporter expressed on the basolateral membrane of hepatocytes, providing a new target for the development of anti-HBV drugs.

[0005] Currently, there are reports of a variety of compounds that target NTCP and inhibit HBV infection, including Myrcludex B, taurocholic acid (TCA), cyclosporin A (CsA) and its analogs, dimeric bile acid derivatives (such as DBA-41), irbesartan and ezetimibe, etc. However, only two large molecules (Myrcludex B and Hepalatide) have entered clinical trials, and no small molecules have entered clinical trials, and most small molecule inhibitors have low activity. The present invention provides a new NTCP inhibitor, which is of great significance and role for clinical research and treatment of HBV. Summary of the Invention

[0006] In a first aspect, the present invention provides a compound having the structure of the following formula (I):

[0007] or a pharmaceutically acceptable salt thereof,

[0008] Among them, X, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 、R 13 , m, n, o and p are as defined herein.

[0009] In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

[0010] In a third aspect, the present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating, ameliorating or preventing a condition responsive to inhibition of the sodium-taurocholate cotransporter. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A shows the inhibitory effect of the compounds of the present application on hepatitis B E antigen (HBeAg); Figure 1B shows the inhibitory effect of the compounds of the present application on HBV 3.5-kb RNA; Figure 1C shows the inhibitory effect of the compounds of the present application on taurocholic acid-d4 uptake.

[0012] FIG2 shows the test results of the toxicity test of the compound JH-B10 of the present application.

[0013] FIG3 shows the test results of the inhibitory effect of the present invention compound Rapavir (JH-B10) on taurocholic acid-d4 uptake, HBV 3.5-kb RNA and hepatitis B E antigen (HBeAg).

[0014] FIG4 shows a schematic diagram of the dosing regimen for the humanized liver mouse model.

[0015] FIG5 shows the test results of the in vivo efficacy verification test of the compound JH-B10 of the present application. DETAILED DESCRIPTION

[0016] The present invention will be described in further detail below. This description is for illustrative purposes only and is not intended to limit the present invention. Those skilled in the art will readily appreciate the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments. Those skilled in the art may make various modifications and alterations without departing from the spirit of the present invention.

[0017] General Terms and Definitions

[0018] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. In the event of a conflict, the definitions provided herein shall prevail. The technology used herein refers to technology generally understood in the art, including variants and equivalent replacements apparent to those skilled in the art. Although it is believed that the following terms are readily understood by those skilled in the art, the following definitions are set forth to better illustrate the present invention. When a trade name appears herein, it refers to the corresponding commodity or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference.

[0019] When a certain amount, concentration or other numerical value or parameter is described in the form of a range, preferred range or preferred upper limit or preferred lower limit, it should be understood to be equivalent to specifically disclosing any range formed by combining any upper limit or preferred value with any lower limit or preferred value, regardless of whether the range is clearly stated. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range. For example, the expression "m is an integer from 0 to 6" means that m is any integer from 0 to 6, for example, m can be 0, 1, 2, 3, 4, 5 or 6. Other similar expressions such as n, o, p and q etc. should also be understood in a similar manner.

[0020] Unless the context clearly dictates otherwise, singular forms such as "a", "an", and "the" include plural forms. The expression "one or more" or "at least one" may mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0021] The terms "about" and "approximately" when used with a numerical variable generally mean that the value of the variable and all values ​​of the variable are within the range of experimental error (e.g., within a 95% confidence interval about the mean) or within ±10% or more of the stated value.

[0022] The expressions "comprising," "including," "containing," and "having" are open ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of excludes any elements, steps, or ingredients not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or ingredients, as well as the optional presence of elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0023] The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. 1-20 Alkyl", such as C 1- 6 alkyl, C 1-4 Alkyl, C 1-2 Alkyl, C3 alkyl, C4 alkyl, C 3-6 Alkyl. "C 1-6 "Alkyl" is used to represent a straight or branched chain saturated hydrocarbon group consisting of 1 to 6 carbon atoms. 1-6 Alkyl groups include C 1-5 Alkyl, C 1-4 Alkyl, C 1-3 Alkyl, C 1-2 Alkyl, C 2-6 Alkyl, C 2-4C6 alkyl, C5 alkyl, etc. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or isomers thereof.

[0024] A divalent group refers to a group obtained by removing a hydrogen atom from a carbon atom with free valence electrons of a corresponding monovalent group. A divalent group has two attachment sites connected to the rest of the molecule. For example, "alkylene" or "alkylene group" refers to a saturated straight or branched divalent hydrocarbon group. Examples of "alkylene" include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H8-), and alkylene (-C5H6-). 10 -), hexamethylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene or ethylpropylene, etc. For example, as used herein, -(CH2) m -、-(CH2) n -、-(CH2) o - and -(CH2) p -All belong to the case of divalent groups.

[0025] The term "alkenyl" is used to refer to a straight or branched hydrocarbon group containing one or more carbon-carbon double bonds, which may be located at any position of the group. Non-limiting examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, piperyl, hexadienyl, and the like.

[0026] The term "alkynyl" is used to refer to a straight or branched hydrocarbon group containing one or more carbon-carbon triple bonds, which may be located at any position of the group. Non-limiting examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like.

[0027] The term "cycloalkyl" refers to a cyclic saturated aliphatic group composed of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond, including monocyclic, bicyclic or tricyclic ring systems, wherein bicyclic and tricyclic ring systems include spirocyclic, fused and bridged rings. Cycloalkyl can have 3-10 carbon atoms, i.e. "C 3-10"Cycloalkyl" refers to a divalent cycloalkyl radical, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. "Cycloalkylene" refers to a divalent cycloalkyl radical.

[0028] The term "heterocyclyl" refers to a cycloalkyl group in which one or more carbon atoms are replaced by a heteroatom selected from nitrogen, oxygen, and sulfur, such as azepine, oxa-, or thiirane, azepine, oxa-, or thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, or tetrahydrothiopyranyl. A heteroatom may occupy the position at which the heterocyclyl group is attached to the rest of the molecule. "Heterocyclylene" refers to a divalent cycloalkyl group.

[0029] The term "alkoxy" represents an alkyl group having a specific number of carbon atoms connected through an oxygen bridge. Non-limiting examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy. The term "C 1-6 "Alkoxy" means an alkyl group containing 1 to 6 carbon atoms attached to the rest of the molecule through an oxygen atom. The C 1-6 Alkoxy groups include C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-4 , C6, C5, C4 and C3 alkoxy, etc. 1-6 Non-limiting examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy, s-butoxy and t-butoxy), pentoxy (including n-pentoxy, isopentoxy and neopentoxy), hexyloxy, and the like.

[0030] The terms "aromatic ring" and "aryl" are used interchangeably. The term "aromatic ring" or "aryl" refers to a polyunsaturated carbocyclic ring system, which can be a monocyclic, bicyclic, or polycyclic ring system, wherein at least one ring is aromatic, and the rings in the bicyclic and polycyclic ring systems are fused together. Examples of aryl groups include, but are not limited to, phenyl, naphthyl (including 1-naphthyl and 2-naphthyl, etc.).

[0031] The terms "heteroaromatic ring" and "heteroaryl" are used interchangeably, and the term "heteroaryl" refers to an aromatic group (or aromatic ring) containing 1, 2, 3, or 4 heteroatoms independently selected from B, N, O, and S, which can be a monocyclic, bicyclic, or tricyclic ring system. A heteroaryl group can be attached to the rest of the molecule through a heteroatom. Non-limiting examples of the heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl and 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl and 5-thiazolyl), 1-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), thienyl (including 2-thienyl and 3-thienyl, etc.), pyridyl (including 2-pyridyl, 3-pyridyl and 4-pyridyl, etc.), pyrazinyl, pyrimidinyl (including 2-pyrimidinyl and 4-pyrimidinyl, etc.), benzothiazolyl (including 5-benzothiazolyl, etc.), purinyl, benzimidazolyl (including 2-benzimidazolyl, etc.), indolyl (including 5-indolyl, etc.), isoquinolyl (including 1-isoquinolyl and 5-isoquinolyl, etc.), quinoxalinyl (including 2-quinoxalinyl and 5-quinoxalinyl, etc.), quinolyl (including 3-quinolyl and 6-quinolyl, etc.), pyrazinyl, purinyl, phenyloxazolyl.

[0032] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, fumarates, gluconates, glucuronates, saccharates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1-1-methylene-bis(2-hydroxy-3-naphthoate)). The compounds used in the present invention can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum salts, calcium salts, lithium salts, magnesium salts, potassium salts, sodium salts, zinc salts, bismuth, and diethanolamine salts. Pharmaceutically acceptable salts are reviewed in Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0033] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0034] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or not substituted, and unless otherwise specified, the type and number of substituents can be any on the basis of chemical achievable.

[0035] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0036] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0037] Unless otherwise indicated, the term "cis-trans isomers" or "geometric isomers" arises from the inability to rotate freely about double bonds or single bonds forming ring carbon atoms.

[0038] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0039] Unless otherwise indicated, "(D)" or "(+)" indicates dextrorotatory, "(L)" or "(-)" indicates levorotatory, and "(DL)" or "(±)" indicates racemic.

[0040] Unless otherwise specified, use a solid wedge key. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed key Indicate the relative configuration of stereocenters with a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Indicates a straight solid bond and straight dashed bond

[0041] The term "pharmaceutically acceptable excipient" refers to carrier substances that are non-irritating to organisms and do not impair the biological activity and properties of the active compound. "Pharmaceutically acceptable excipients" include, but are not limited to, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavorings, surfactants, wetting agents, dispersants, disintegrants, stabilizers, solvents, or emulsifiers.

[0042] The term "pharmaceutical composition" or "pharmaceutically active ingredient composition" refers to a composition comprising one or more active ingredients and optionally one or more pharmaceutically acceptable excipients.

[0043] The term "HBcAg" refers to hepatitis B core antigen. HBcAg plays an important role in HBV infection. It can reflect the presence of Dane particles in serum and the replication of HBV in the liver, and can cooperate and complement each other with other HBV serological markers.

[0044] The term "Dane particle" refers to the large spherical particle of hepatitis B virus, which is an infectious, intact HBV particle.

[0045] The term "HBeAg" refers to the hepatitis B E antigen, a soluble protein in the core particle of the hepatitis B virus. Normally, HBeAg is embedded within HBcAg. When HBcAg cleaves, HBeAg dissolves from the viral particle into the serum. It appears later than HBsAg but disappears earlier than HBsAg, making it the second serological antigen marker to appear after HBsAg following HBV infection.

[0046] The term "HBsAg" refers to the surface antigen of hepatitis B virus, which is not the complete hepatitis B virus itself, but the outer shell of hepatitis B virus. It is not contagious but has antigenicity and is only one of the signs of hepatitis B virus infection.

[0047] Compounds of the present invention

[0048] The present invention provides a compound having the structure of the following formula (I):

[0049] or a pharmaceutically acceptable salt thereof,

[0050] in,

[0051] X is selected from O or NR 14 ;

[0052] R 1 Selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl-C 1-4 Alkyl, C 3-10 Heterocyclic group, C 3-10 Heterocyclyl-C 1-4 Alkyl, C 6-10 Aryl, C 6-10 Aryl-C 1-4 Alkyl, C 5-10 Heteroaryl and C 5- 10 Heteroaryl-C 1-4 Alkyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is unsubstituted or substituted with at least one independently selected from R X Substituents substituted;

[0053] R 2 Selected from hydrogen, halogen, amino, cyano, nitro, C 1-6 Alkyl, C 1-6 Alkyl-SC 1-6 Alkyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Alkyl-C6-10 Aryl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl-C 1-4 Alkyl, C 3-10 Heterocyclic group, C 3-10 Heterocyclyl-C 1-4 Alkyl, C 6-10 Aryl, C 6-10 Aryl-C 1-4 Alkyl, C 5-10 Heteroaryl, C 5-10 Heteroaryl-C 1-4 Alkyl, CN and NO2, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is unsubstituted or substituted by at least one independently selected from R X Substituents substituted;

[0054] R 3 Selected from hydrogen and C 1-6 alkyl;

[0055] When R 4 for i) When the formula (I) has the following structure (II-1):

[0056] When R 4 for ii) When the formula (I) has the following structure (II-2):

[0057] R 5 Selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl-C 1-4 Alkyl, C 3-10 Heterocyclic group, C 3-10 Heterocyclyl-C 1-4 Alkyl, C 6-10 Aryl, C 6-10 Aryl-C 1-4 Alkyl, C 5-10 Heteroaryl, C 5- 10 Heteroaryl-C 1-4 Alkyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is unsubstituted or substituted with at least one independently selected from R X Substituents substituted;

[0058] R 6Selected from hydrogen, amino, cyano, nitro, C 1-6 Alkyl, C 1-6 Alkyl-C 3-10 Cycloalkyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Alkyl-SC 1-6 Alkyl, C 1-6 Alkyl-C 6-10 Aryl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl-C 1-4 Alkyl, C 3-10 Heterocyclic group, C 3-10 Heterocyclyl-C 1-4 Alkyl, C 6-10 Aryl, C 6-10 Aryl-C 1-4 Alkyl, C 5-10 Heteroaryl and C 5-10 Heteroaryl-C 1-4 Alkyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is unsubstituted or substituted with at least one independently selected from R X Substituents substituted;

[0059] or R 5 and R 6 Together with the atoms to which it is attached, it forms a 5-8 membered heterocyclic group containing 1, 2 or 3 heteroatoms, wherein the 5-8 membered heterocyclic group is unsubstituted or substituted by at least one independently selected from R X Substituents substituted;

[0060] R 7 For hydrogen and C 1-6 alkyl;

[0061] R 8 Selected from hydrogen, amino, cyano, nitro, C 1-6 Alkyl, C 1-6 Alkyl-C 3-10 Cycloalkyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Alkyl-SC 1-6 Alkyl, C 1-6 Alkyl-C 6-10 Aryl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl-C 1-4 Alkyl, C 3-10 Heterocyclic group, C 3-10Heterocyclyl-C 1-4 Alkyl, C 6-10 Aryl, C 6-10 Aryl-C 1-4 Alkyl, C 5-10 Heteroaryl and C 5-10 Heteroaryl-C 1-4 Alkyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is unsubstituted or substituted with at least one independently selected from R X Substituents substituted;

[0062] R 9 Selected from hydrogen and C 1-6 alkyl;

[0063] R 10 and R 11 Each independently selected from hydrogen, hydroxy, halogen, C 1-6 Alkyl and C 1-6 Alkoxy, wherein each alkyl and alkoxy are unsubstituted or substituted with at least one independently selected from R X Substituents substituted;

[0064] R 12 and R 13 Each independently selected from hydrogen, hydroxy, halogen, C 1-6 Alkyl and C 1-6 Alkoxy, wherein each alkyl and alkoxy are unsubstituted or substituted with at least one independently selected from R X Substituents substituted;

[0065] R 14 Selected from hydrogen and C 1-6 alkyl;

[0066] R X Selected from hydroxyl, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl-C 1-4 Alkyl, C 3-10 Heterocyclic group, C 3-10 Heterocyclyl-C 1-4 Alkyl, C 6-10 Aryl, C 6-10 Aryl-C 1-4 Alkyl, C 5-10 Heteroaryl and C 5-10 Heteroaryl-C 1-4 Alkyl, wherein each alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is unsubstituted or substituted with at least one independently selected from R YSubstituents substituted;

[0067] R Y Selected from hydroxyl, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, C 3-10 Cycloalkyl-C 1-4 Alkyl, C 3-10 Heterocyclic group, C 3-10 Heterocyclyl-C 1-4 Alkyl, C 6-10 Aryl, C 6-10 Aryl-C 1-4 Alkyl, C 5-10 Heteroaryl and C 5-10 Heteroaryl-C 1-4 alkyl;

[0068] m, n, o and p are each independently selected from integers of 0, 1, 2, 3, 4, 5 and 6;

[0069] *1 C. *2 C and *3 C represents and R 4 、R 6 and R 8 Connected chiral carbon atoms.

[0070] In one embodiment, X is O.

[0071] In one embodiment, R 1 Selected from C 1-6 In one embodiment, R 1 It is a methyl group.

[0072] In one embodiment, R 2 Selected from hydrogen and C 1-6 In one embodiment, R 2 is selected from hydrogen, methyl and isopropyl.

[0073] In one embodiment, R 3 For hydrogen.

[0074] In one embodiment, R 4 for Formula (I) has the following structure:

[0075] Among them, X, R 1 、R 2 、R 3 、R 5 、R 6、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 、R 13 , m, n, o and p are as defined herein.

[0076] In one embodiment, R 4 for Formula (I) has the structure of the following formula (II-1):

[0077] Among them, X, R 1 、R 2 、R 3 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 、R 13 , m, n, o and p are as defined herein.

[0078] In one embodiment, R 5 Selected from hydrogen and C 1-6 In one embodiment, R 5 is selected from hydrogen and methyl.

[0079] In one embodiment, R 6 Selected from C 1-6 Alkyl, C 1-6 Alkyl-C 3-10 Cycloalkyl and C 1-6 Alkyl-C 6-10 In one embodiment, R 6 Selected from methyl,

[0080] In one embodiment, R 5 and R 6 Together with the atoms to which it is attached, it forms a 5-6 membered heterocyclic group containing 1 nitrogen heteroatom, wherein the 5-6 membered heterocyclic group is unsubstituted or substituted by at least one independently selected group consisting of R X In one embodiment, R 5 and R 6 Together with the atoms it is connected to, it forms

[0081] In one embodiment, R 7 For hydrogen.

[0082] In one embodiment, R8 Selected from C 1-6 Alkyl-C 6-10 Aryl, wherein the alkyl, aryl is unsubstituted or substituted by at least one independently selected from R X In one embodiment, R 8 Selected from

[0083] In one embodiment, R 9 Selected from C 1-6 In one embodiment, R 9 It is a methyl group.

[0084] In one embodiment, R 10 Selected from C 1-6 In one embodiment, R 10 It is a methyl group.

[0085] In one embodiment, R 11 Selected from C 1-6 In one embodiment, R 11 It is a methyl group.

[0086] In one embodiment, R 12 Selected from C 1-6 In one embodiment, R 12 It is a methoxy group.

[0087] In one embodiment, R 13 Selected from C 1-6 In one embodiment, R 13 It is a methoxy group.

[0088] In one embodiment, R X Selected from hydroxyl, halogen, C 1-6 Alkyl and C 1-6 In one embodiment, R X Selected from hydroxy, fluoro, methyl, methoxy and

[0089] In one embodiment, m is 1. In one embodiment, n is 1. In one embodiment, o is 1. In one embodiment, p is 1.

[0090] In one embodiment, *1 The C chiral carbon atom is in S configuration. In one embodiment, *2 The chiral carbon atom C is in S or R configuration. In a preferred embodiment, *2 The C chiral carbon atom is in S configuration. In one embodiment, *3 The chiral carbon atom C is in S or R configuration. In a preferred embodiment,*3 The C chiral carbon atom is in S configuration.

[0091] The compound provided by the present invention is selected from the following structures:

[0092] or a pharmaceutically acceptable salt thereof.

[0093] In a preferred embodiment, the compound of the present invention is

[0094] Pharmaceutically acceptable salts of the present invention

[0095] Those skilled in the art will appreciate that the compound according to the present invention can exist in the form of a pharmaceutically acceptable salt. As a pharmaceutically acceptable salt, for example, the following examples can be provided: metal salts, ammonium salts, salts formed with organic bases, inorganic acids, organic acids, alkaline or acidic amino acids, etc. Pharmaceutically acceptable salts according to the present invention can be prepared by conventional chemical methods by compounds containing acidic or basic groups. Usually, the compound can be prepared by reacting the suitable base or acid of stoichiometry in water, organic solvent or its mixture in the form of free acid or base. Usually, preferred non-aqueous media are such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, etc.

[0096] Administration, pharmaceutical compositions and kits

[0097] The compounds according to the present invention will be administered alone or in combination with additional therapeutic agents in an effective amount by any common and acceptable means known in the art. The effective amount may vary depending on the severity of the disease, the age and relative health of the subject, the efficacy of the compound used, and other factors known to those skilled in the art.

[0098] As a general example, a daily dosage of about 0.001 to about 100 mg / kg body weight can be used, or more particularly about 0.03 to 2.5 mg / kg body weight. In larger mammals, such as humans, the daily dosage can be in the range of about 0.5 mg to about 2000 mg.

[0099] The compounds of the present invention are typically administered in the form of a pharmaceutical composition comprising a pharmaceutically active ingredient and various other pharmaceutically acceptable components, for example, see Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred or desired form depends on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition may also include a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a carrier commonly used to formulate a pharmaceutical composition for administration to animals or humans. The choice of diluent does not affect the biological activity of the combination. Examples of diluents include, but are not limited to, distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0100] The compounds of the present invention can be administered in the form of pharmaceutical compositions by any conventional route; for example, enterally, such as orally, for example in the form of tablets or capsules; parenterally, for example in the form of injectable solutions or suspensions; or topically, for example, ophthalmically or nasally, for example in the form of emulsions, gels, ointments, creams or suppositories.

[0101] Therefore, the present invention also provides a pharmaceutical composition comprising a compound according to the present invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. The compound of the present invention may be present in free form or in the form of a pharmaceutically acceptable salt in combination with at least one pharmaceutically acceptable carrier and may be prepared in a conventional manner, for example, by mixing, granulation, coating, dissolution or lyophilization processes.

[0102] In one embodiment, the pharmaceutical composition is a solution of the active ingredient, including a suspension or dispersion, such as an isotonic aqueous solution. For a lyophilized composition comprising only the active ingredient or comprising the active ingredient and a carrier (such as mannitol), a dispersion or suspension can be prepared before use.

[0103] The limiting examples of carriers include fillers, such as sugars, such as lactose, sucrose, mannitol or sorbitol, cellulose preparations and / or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate, and binders, such as starches, such as corn, wheat, rice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinyl pyrrolidone, and / or if necessary, disintegrants, such as the above-mentioned starches, carboxymethyl starch, cross-linked polyvinyl pyrrolidones, alginic acid or its salts, such as sodium alginate. Other carriers include, but are not limited to, rheology modifiers and lubricants, such as silicic acid, talc, stearic acid or its salts, such as magnesium or calcium stearate, and / or polyethylene glycol or its derivatives.

[0104] The present invention also provides a pharmaceutical combination, such as a kit, comprising a) a first agent, which is a compound according to the present invention or a pharmaceutically acceptable salt thereof, and b) at least one additional agent. The kit may further comprise instructions for administration thereof.

[0105] Methods of treatment and uses of the compounds of the present invention

[0106] The present invention provides a method for treating a condition responsive to inhibition of the sodium-taurocholate cotransporter, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention.

[0107] The present invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition for treating, ameliorating or preventing a condition responsive to inhibition of the sodium ion taurocholate cotransporter.

[0108] The present invention also provides use of a compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the preparation of a drug for treating, ameliorating or preventing conditions responsive to inhibition of the sodium ion taurocholate cotransporter.

[0109] In one embodiment, conditions responsive to inhibition of the sodium-taurocholate cotransporter include, but are not limited to, hepatitis virus infection. In one embodiment, the hepatitis virus infection is hepatitis B virus and / or hepatitis D virus infection. Beneficial effects

[0110] The present invention provides a novel small-molecule inhibitor of NTCP, which exhibits excellent inhibitory effects against NTCP. Compared to macromolecular drugs, the small-molecule inhibitors of the present invention are simple to synthesize and may have better drugability and pharmacokinetic properties, providing more options for clinical research on small-molecule drugs for the treatment of HBV and HDV.

[0111] Example

[0112] The solution of the present invention is further described in detail below with reference to specific embodiments.

[0113] It should be noted that the following examples are merely examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the present invention. For those skilled in the art, other variations or modifications may be made based on the description of the present invention. It is not necessary and is not possible to exhaustively enumerate all embodiments herein, and the obvious variations or modifications derived therefrom are still within the scope of protection of the present invention. Unless otherwise indicated, the instruments, equipment, and reagents used herein are all commercially available.

[0114] The sources of raw materials used in the present invention are as follows:

[0115] Table 1 Sources of conventional reagents

[0116] Table 2 Amino acid information

[0117] Example 1 Synthesis of Compound JH-B10

[0118] 1.1 Synthesis of compound FKBD fragment

[0119] i) KOH, H2O / EtOH, rt; ii) Pd / C (10%), H2, MeOH, rt; iii) tert-butyl 2-bromoacetate, K2CO3, DMF, rt; iv) (+)-DIPCl, THF, -20℃-rt; v) S8, benzoyl chloride, DMAP, Et3N, DCM / THF, rt; vi) TFA (10%), DCM, rt.

[0120] i) 3,4-Dimethoxybenzaldehyde (8.31 g, 50 mmol) and 3-hydroxyacetophenone (6.81 g, 50 mmol) were dissolved in ethanol (EtOH) (50 mL), and NaOH (0.5 g dissolved in 4 mL of water) was added; the reaction mixture was stirred at room temperature until the reaction was complete as determined by TLC (a yellow-brown precipitate slurry appeared after completion of the reaction); the reaction mixture was then diluted with ethyl acetate (EtOAc) and extracted three times with water. The organic phase was concentrated to obtain a crude product which was used directly in the next step.

[0121] ii) The crude product obtained in i) was dissolved in methanol (MeOH) (40 mL). The oxygen in the reaction system was replaced with an inert gas, and then Pd / C (2.3 g, 10%) was added. H2 was then introduced for gas replacement to fill the reaction system with H2. The reaction progress was monitored by TLC. After the reaction was complete, the H2 in the system was replaced with an inert gas, and the Pd / C was filtered out. The filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum ether:ethyl acetate (PE:EtOAc) = 3:1) to obtain 10.3 g of compound S1 as a light yellow oily liquid. The total yield of the two-step reaction was 72%.

[0122] iii) Compound S1 (10.3 g, 36 mmol) was dissolved in 30 mL of N,N-dimethylformamide (DMF), followed by the addition of tert-butyl 2-bromoacetate (6.4 mL, 7.72 g, 39.6 mmol) and K2CO3 (5.97 g, 43.2 mmol). The reaction mixture was stirred at room temperature and monitored by TLC until complete consumption of Compound S1. After completion, the reactant was dissolved in EtOAc and washed with 1N HCl. The organic phase was washed with saturated brine and dried over anhydrous Na2SO4. The organic phase was then concentrated and purified by silica gel column chromatography (eluent: PE:EtOAc = 4:1) to afford 11.68 g of Compound S2, in an 81% yield.

[0123] iv) Compound S2 (11.68 g, 29.16 mmol) was dissolved in dry THF (40 mL) and cooled to -20°C. Subsequently, (+)-diisopinocampheylborane ((+)-DIPCl) (1.6 M in hexane, 27.3 mL, 43.74 mmol) was slowly added dropwise, and the temperature was slowly raised to room temperature. After completion of the reaction, 2,2'-(ethylenedioxy)diethylamine (equal to (+)-DIPCl) was added to quench the reaction to form an insoluble complex. After stirring at room temperature for 30 minutes, the suspension was filtered through a celite pad and concentrated. The suspension was separated and purified by silica gel column chromatography (eluent: PE:EtOAc = 2:1) to obtain 10.56 g of colorless liquid compound S3 in a yield of 90%.

[0124] 1H NMR (600MHz, CDCl3) δ7.26(t,J=7.2Hz,1H),6.96(d,J=7.2Hz,1H),6.93(S,1H),6.82-6.77(m,2H),6.74-6.70(m,2H),4.68-4.64(m,1H ),4.52(s,2H),3.86(s,3H),3.85(s,3H),2.73-2.66(m,1H),2.65-2.58(m,1H),2.12-2.05(m,1H),2.00-1.95(m,1H),1.48(s,9H)ppm.

[0125] v) Compound S3 (10.56 g, 26.24 mmol), compound S8 (9.8 g, 31.4 mmol) and 4-dimethylaminopyridine (DMAP) (3.2 g, 26.24 mmol) were dissolved in anhydrous tetrahydrofuran (THF) and dichloromethane (DCM) (60 mL, THF / DCM = 1:1). Under argon protection, triethylamine (Et3N) (6.2 mL, 4.51 g, 44.6 mmol) and benzoyl chloride (6.0 mL, 7.28 g, 39.36 mmol) were slowly added in sequence. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC. When complete conversion was achieved, the reaction mixture was diluted with 300 mL of EtOAc and 5% acetylcholine. The organic phase was washed with saturated brine and dried over anhydrous Na2SO4; after concentration, the product was purified by silica gel column chromatography (eluent: PE:EtOAc = 10:1-3:1) to obtain 12.78 g of light yellow solid compound S4, with a yield of 70%.

[0126] vi) Compound S4 (12.78 g, 18.37 mmol) was dissolved in DCM (60 mL). Trifluoroacetic acid (TFA) (17.83 mL, 27.37 g, 0.24 mol) was added portionwise under argon and stirred at room temperature until complete conversion. After completion of the reaction, the solvent and TFA were removed and the mixture was purified by silica gel column chromatography (eluent: PE:EtOAc = 3:1-1:1) to obtain 10.8 g of the FKBD fragment (Compound B) as a light yellow foamy solid in a yield of 92%.

[0127] 1H NMR (600MHz, CDCl3) δ7.28 (d, J=7.8Hz, 1H), 6.92 (d, J=7.8Hz, 1H), 6.90 (dd, J1=8. 4,J2=2.4Hz,1H),6.86(s,1H),6.79(d,J=8.4Hz,1H),6.70-6.66(m,2H),6.37(d,J =17.4Hz,1H),6.04(dd,J1=17.4,J2=10.2Hz,1H),5.81(d,J=10.8Hz,1H),5.73(dd ,J1=8.4,J2=5.4Hz,1H),5.28(d,J=5.4Hz,1H),4.71(d,J=16.2,1H),4.66(d,J=16. 2,1H),4.33(d,J=10.8Hz,1H),4.26(d,d,J=10.8,1H),3.87(s,3H),3.86(s,3H),3 .49-3.44(m,1H),3.25-3.20(m,1H),2.67-2.61(m,1H),2.59-2.54(m,1H),2.40(d ,J=14.4Hz,1H),2.29-2.20(m,1H),2.10-2.04(m,1H),1.83-1.73(m,2H),1.63(d, J=12.6Hz,1H),1.54-1.46(m,1H),1.43-1.35(m,1H),1.32(s,3H),1.31(s,3H)ppm.

[0128] 1.2 Synthesis of intermediate compound S8

[0129] i) Allyl bromide, Cs2CO3, DMF, rt; ii) DMAP, TFA, DCM, rt; iii) Toluene, reflux; iv) Acryloyl chloride, DIPEA, DCM, 0°C; v) N-Methylaniline, Pd(PPh3)4, THF, rt.

[0130] i) N-Boc-homoproline (11.47 g, 50 mmol) was dissolved in DMF (70 mL) and CsCO (32.58 g, 100 mmol) was added. The resulting suspension was stirred at room temperature for 5 minutes, followed by the addition of allyl bromide (6.35 g, 52.5 mmol). The reaction mixture was stirred at room temperature until complete by TLC. The suspension was filtered through a pad of Celite, rinsed with EtOAc (60 mL), and washed with HCl (1 M, 50 mL x 3). The organic phase was dried over anhydrous NaSO and co-evaporated with toluene (30 mL x 2). 14.66 g of crude product was obtained as a yellow oil, which was used in the next step without further purification.

[0131] ii) The crude product obtained in i) was dissolved in DCM (30 mL) and TFA (7.5 mL) was added. The mixture was stirred at room temperature until the reaction of the raw material was complete. The mixture was concentrated to obtain 5.6 g of yellow oily crude product S5, which was used directly in the next reaction.

[0132] iii) The crude product S5 (5.6 g, 33.09 mmol) obtained in ii), dihydro-4,4-dimethyl-2,3-furandione (4.24 g, 33.09 mmol) and DMAP (808.5 mg, 6.62 mmol) were dissolved in anhydrous toluene (PhMe) (35 mL) and heated to reflux in an oil bath for 16 hours. The solvent was then removed and the product was separated and purified by silica gel column chromatography (eluent: PE:EtOAc = 3:1) to obtain 9.05 g of yellow oily compound S6. The total yield of the three reactions was 61%.

[0133] 1 H NMR (600MHz, CDCl3) δ5.96-5.88(m,1H),5.36(d,J=17.4Hz,1H),5.30-5.27(m,2H),4.70-4.65(m,2H),3.71-3.60(m,2H),3.50(d,J=15. 6Hz,1H),3.32(s,1H),3.20(td,J1=13.2,J2=3.0Hz,1H),2.36(d,J=13.8Hz,1H),1.81-1.62(m,3H),1.56-1.36(m,2H),1.24(s,6H)ppm.

[0134] iv) Compound S6 (9.05 g, 30.4 mmol) and DIPEA (6.53 mL, 5.11 g, 39.52 mmol, 1.3 equivalents) were dissolved in anhydrous DCM (30 mL), and acryloyl chloride (2.7 mL, 3.026 g, 33.44 mmol) was slowly added dropwise. The reaction mixture was stirred at room temperature until complete and then quenched with saturated NaHCO₃ solution. The organic phase was washed with water, extracted, dried over anhydrous Na₂SO₄, concentrated, and purified by silica gel chromatography (eluent: PE:EtOAc = 5:1) to afford 8.12 g of compound S7 as a colorless oil in a 76% yield.

[0135] 1H NMR (600MHz, CDCl3) δ6.39 (d, J=17.4Hz, 1H), 6.07 (dd, J1=17.4, J2=10.2Hz, 1H), 5.93-5.87 (m, 1H), 5.83 (d, J= 10.8Hz,1H),5.34(d,J=16.8Hz,1H),5.28-5.25(m,2H),4.67-4.64(m,2H),4.37(d,J=10.8Hz,1H),4.26(d,J=10 .8Hz,1H),3.51(d,J=12.0Hz,1H),3.22(td,J1=13.2,J2=3.0Hz,1H),2.34(d,J=13.8Hz,1H),1.81-1.76(m,1H) ,1.73-1.68(m,1H),1.64(d,J=13.2Hz,1H),1.56-1.48(m,1H),1.44-1.38(m,1H),1.35(s,3H),1.34(s,3H)ppm.

[0136] v) Compound S7 (8.12 g, 23.1 mmol), Pd(PPh3)4 (800.8 mg, 0.693 mmol, 3%), and N-methylaniline (7.5 mL, 7.425 g, 69.3 mmol) were dissolved in dry THF (35 mL) and stirred at room temperature for 6 hours. The reaction mixture was then diluted with EtOAc (50 mL) and extracted with HCl (1 M, 40 mL x 3). The organic phase was dried over Na2SO4, filtered, and concentrated. The crude product was separated and purified by silica gel chromatography (eluent: DCM:MeOH = 30:1) to give 5.47 g of white solid compound S8 in a yield of 86%.

[0137] 1.3 Linker Synthesis

[0138] i) m-CPBA, K2HPO4, DCM, rt, 12h; ii) H2SO4, THF:H2O=1:1, reflux; iii) NaIO4, MeOH, 20h; iv) NaBH4, MeOH, 0℃; v) TsCl, Ag2O, KI, DCM, rt; vi) 2-chlorotriphenylmethyl chloride resin, DIPEA, THF, 50℃, 24h; vii) MeNH2, THF, 40℃, 12h.

[0139] i) 1,4-cyclohexadiene (6.41 g, 80.0 mmol) was dissolved in DCM (80 mL), followed by the addition of K2HPO4 (14.63 g, 84.0 mmol, 1.05), and m-chloroperbenzoic acid (m-CPBA) (17.05 g, 84.0 mmol, 85%) was added in 10 portions at 0°C, and the mixture was stirred overnight. After completion of the reaction, the mixture was filtered using a fritted funnel covered with diatomaceous earth, and the filter cake was washed with DCM. The organic phase was washed with Na2S2O3 (150 mL) and saturated NaHCO3 (150 mL). The organic phase was dried over Na2SO4, filtered, and concentrated to obtain the crude product, compound S9, as a colorless, transparent oil, which was used directly in the next step.

[0140] ii) Dissolve the crude product, Compound S9, in a mixed solvent (THF:H2O = 1:1, 40 mL). Slowly add concentrated H2SO4 (3 mL) dropwise with stirring and reflux for 3-4 hours. Monitor the reaction by TLC. After completion, terminate the reaction by adding an appropriate amount of K2CO3 (approximately 4 g). Extract with EtOAc three times and n-butanol three times. Combine the organic phases, dry over anhydrous NaSO4, and concentrate to obtain the crude product, Compound S10, as a light yellow, transparent oil.

[0141] iii) The crude product, Compound S10, was dissolved in MeOH (40 mL), and NaHCO₃ (2.52 g, 29.95 mmol) was added, followed by NaIO₄ (17.1 g, 80.0 mmol) added portionwise. The mixture was allowed to react at room temperature for 3-4 hours, monitored by TLC. Upon completion, the reaction was filtered through a fritted funnel covered with celite, the filter cake was washed with DCM, and the mixture was concentrated to afford the crude product, Compound S11, as a brown, transparent oil, which was used directly in the next reaction.

[0142] iv) The crude product, Compound S11, was dissolved in a solvent mixture (DCM:MeOH = 1:2, 45 mL). NaBH4 (3.02 g, 80.0 mmol) was added portionwise under an ice-water bath. The mixture was stirred for approximately 0.5 hours and monitored by TLC. Upon completion, the reaction was quenched by the addition of a small amount of saturated NH4Cl solution. The product was extracted with EtOAc (30 mL x 2) and n-butanol (20 mL x 3). The product was concentrated and purified by silica gel chromatography (eluent: PE:EtOAc = 2:1) to obtain 3.88 g of Compound S12 as a light yellow oily liquid. The total yield over the four steps was 42%.

[0143] 1 H NMR (600MHz, CDCl3) δ5.59 (t, J = 6.0 Hz, 2H), 3.69 (t, J = 6.0 Hz, 4H), 2.38 (q, J = 6.0 Hz, 4H) ppm.

[0144] v) Compound S12 (3.88 g, 33.4 mmol) was dissolved in anhydrous DCM solution, and p-toluenesulfonyl chloride (TsCl) (5.09 g, 26.72 mmol), KI (831.7 mg, 5.01 mmol) and Ag2O (8.5 g, 36.74 mmol) were added sequentially. The mixture was reacted at room temperature for 2 days and then filtered. The filter cake was washed with DCM, concentrated, and purified by silica gel chromatography (eluent: PE:EtOAc = 2:1) to obtain 4.4 g of colorless oily liquid compound S13. At the same time, the starting compound S12 (1.68 g, recovery rate 43%) was recovered, with a yield of 49% (conversion rate 86%).

[0145] 1 H NMR (600MHz, CDCl3) δ7.79(d,J=7.8Hz,2H),7.34(d,J=7.8Hz,2H),5.56-5.50(m,1H),5.46-5.40(m,1H),4 .04(t,J=6.6Hz,2H),3.63(t,J=6.6Hz,2H),2.45(s,3H),2.44(q,J=6.6Hz,2H),2.28(q,J=6.6Hz,2H)ppm.

[0146] vi) 2-Chlorotriphenylmethyl chloride resin (8.47 g, 10.08 mmol, 1.19 mmol / g) was placed in a 100 mL round-bottom flask and allowed to swell in THF (25 mL) for 15-20 minutes. Compound S13 (3.0 g, 11.09 mmol) and DIPEA (13.3 mL, 10.4 g, 80.64 mmol) were added sequentially. The mixture was stirred slowly at 50°C for 24 hours. After completion of the reaction, the mixture was filtered using a fritted funnel and the resin was washed alternately with DCM and MeOH until clean. The resin was collected and dried under vacuum to yield compound S14, which was used directly in the next reaction.

[0147] vii) Compound S14 was placed in a 100 mL round-bottom flask and allowed to swell in THF (15 mL) for 15-20 minutes. MeNH2·MeOH solution (15 mL) was added and the mixture was slowly stirred at 40°C for 12 hours. After the reaction was complete, the mixture was filtered using a fritted funnel and the resin was washed alternately with DCM and MeOH until clean. The resin was collected and dried under vacuum to yield compound 15 (Linker).

[0148] viii) Determination of the loading of compound S15

[0149] Compound S15 (100 mg) was placed in a threaded standard sample vial and DMF (1-2 mL) was added to swell for 5 minutes. Subsequently, the condensing agent HATU (68.4 mg, 0.18 mmol), N-Fmoc-Tyr(O-tBu) (82.7 mg, 0.18 mmol) and DIPEA (60 μL, 46.5 mg, 0.36 mmol) were added and dissolved. The vial was placed on a decolorization shaker to react for 2-3 hours.

[0150] After the reaction is complete, filter with a sand core funnel, wash the resin alternately with DCM and MeOH until clean, then transfer the resin to a new reaction bottle, add 2 mL of a mixed solvent (TFA:MeOH:DCM=1:1:8), and place on a shaker to react for 1 hour to cut off the Linker-N-Fmoc-Tyr(O-tBu) coupled with one amino acid.

[0151] After the reaction was complete, the resin was filtered using a fritted funnel and washed with DCM and MeOH alternately until clean. The filtrate was collected, concentrated, and weighed to yield 44.8 mg of a light yellow oily substance. The resin loading was calculated based on this mass and found to be 0.78 mmol / g.

[0152] 1.4 Solid-phase synthesis and ring-close metathesis

[0153] Compound S15 (100 mg, 0.078 mmol) was placed in a solid-phase synthesis tube and allowed to swell in DMF (3 mL) for 5 minutes. Fmoc-protected p-fluorophenylalanine (3.0 equiv), HATU (3.0 equiv), and DIPEA (3.5 equiv) were then added sequentially to the reaction tube and allowed to completely dissolve. The reaction tube was placed on a shaker and monitored for completion using Kaiser reagent (typically 1-3 hours). After completion, the tube was drained and rinsed sequentially with DMF, MeOH, and DCM to remove any remaining reactants. The tube was then vacuum dried (approximately 15-30 minutes).

[0154] 2) Add 3 mL of a 20% piperidine-DMF solution (piperidine:DMF = 1:4, volume ratio) to the reaction tube and incubate on a shaker for 5-10 minutes. Repeat this procedure twice to completely remove the Fmoc group from the amino acid. Drain the solvent and rinse with DMF and then DCM to remove the piperidine. Vacuum dry to obtain a resin coupled with one amino acid.

[0155] 3) DMF (3 mL) was added to the system from the previous step and the mixture was swollen for 3-5 min. Fmoc-protected N-Me-phenylalanine (3.0 equiv.), HATU (3.0 equiv.), and DIPEA (3.5 equiv.) were then added sequentially. After the reactants were completely dissolved, the steps 1) and 2) were repeated to obtain a resin coupled with two amino acids.

[0156] 4) Repeat the above steps to sequentially couple tyrosine and methylglycine to obtain a system with four amino acids coupled. Compounds FKBD (1.5 equivalents), HATU (1.6 equivalents), and DIPEA (2.0 equivalents) are then added to the reaction system and completely dissolved in DMF. Shake on a shaker for 3-4 hours to allow for sufficient reaction. After the reaction is complete, drain the solvent and rinse with DMF, MeOH, and DCM, sequentially. Rinse any remaining reactants thoroughly and dry under vacuum.

[0157] 5) The resin coupled with the four amino acids and compound FKBD in the previous step was weighed into a microwave reaction vial, using DCE (2.0 mL) as the solvent and Hoveyda-Grubbs II (0.3 equivalents) as the catalyst. The vial was sealed and placed in a microwave reactor for reaction at 140°C for 0.5 hours. After the microwave reaction, the vial was cooled to room temperature and opened. The resin was filtered through a fritted funnel and washed with DCM and MeOH. The filtrate was concentrated and purified by HPLC to obtain compound JH-B10 (Rapavir). MS [M+H] + :1239.5631.

[0158] Referring to the synthesis method of compound JH-B10, the amino acids used were replaced with appropriate compounds in Table 2 to prepare the following compounds:

[0159] Example 2 Anti-HBV screening test

[0160] HepG2-NTCP cell culture: HepG2-NTCP cell line was cultured in modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU / mL penicillin, and 100 μg / mL streptomycin, and further cultured in a humidified incubator at 37°C and 5% CO2 until use.

[0161] HepG2-NTCP cells were cultured at 2×10 5Cells were plated in 12-well plates at a density of 100 μg / well and incubated for 48 hours at 37°C in a 5% CO2 environment. The cells were then incubated with a 3 μM concentration of the test compound for 1 hour, with a DMSO control group. Following compound pre-incubation, the HBV infection system (500 vge / cell HBV viral particles + 4% w / v PEG 8000) was added and incubated for 16 hours for HBV infection. After incubation, the cells were washed three times with PBS solution to remove uninfected virus and residual drug, and fresh culture medium was added for continued culture. Cell supernatant was collected and the culture medium was replaced daily. Cells were collected for testing after 5 days.

[0162] Enzyme-linked immunosorbent assay (ELISA): The culture supernatant was centrifuged at 2000g for 5 min, and HBeAg in the supernatant was detected using an enzyme-linked immunosorbent assay kit (Shanghai Kehua) according to the manufacturer's instructions.

[0163] Cellular RNA was extracted using TRNzol Total RNA extraction reagent (Beijing Tiangen, DP405), and the HBV 3.5-kb RNA level in cells was detected by quantitative PCR (qPCR) (QuantStudio6 FLEX Q6, Thermofisher).

[0164] Taurocholic acid-d4 (TCA-d4) uptake assay: HepG2-NTCP cells were cultured at 2×10 5 Cells were plated at a density of 100 μg / well in a 12-well plate and incubated for 48 hours at 37°C in a 5% CO2 environment. The culture medium was removed and the cells were washed twice with 0.5 mL of buffer (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, pH 7.4) per well, followed by pre-incubation in 0.5 mL of buffer at 37°C in a 5% CO2 environment for 15 minutes. The buffer was removed and the cells were washed again with buffer, followed by addition of 0.5 mL of buffer followed by addition of the compound (JH-B01 to JH-B20) to a final concentration of 2 μM. An equal volume of DMSO was added to the control group, with 3 replicates per group, and the cells were incubated at 37°C in a 5% CO2 environment for 15 minutes. TCA-d4 was then added to the cells to a final concentration of 5 μM and incubated for 15 minutes at 37°C in a 5% CO2 environment. Cells were washed three times with 0.5 mL of buffer added to each well and lysed with 200 μl of anhydrous ethanol. The cell lysate was transferred to an EP tube and centrifuged at 12,000 rpm for 10 minutes at 4°C to collect the supernatant. TCA uptake was quantified using LC-MS / MS.

[0165] The test results are shown in Figure 1.

[0166] According to the test results, compared with the control group, the compounds JH-B10 to JH-B20 of the present application have inhibitory effects on taurocholic acid-d4 uptake, HBV 3.5-kb RNA and hepatitis B E antigen (HBeAg).

[0167] Example 3 Toxicity test of the compounds of the present invention

[0168] CCK8 assay was performed using a microplate reader (Synergy H1, Biotek) to detect drug toxicity. HepG2-NTCP cell suspension (2×10 3 After 24 hours of pre-incubation in an incubator (Mod3111, Thermofisher) at 37°C, 5% CO2, various concentrations of compound JH-B10 were added to the culture plates and incubated for a further 48 hours. 10 μL of CCK8 detection solution (HY-K0301, MCE) was added to each well. After 1 hour, absorbance at 450 nm was measured using a microplate reader to analyze cytotoxic activity.

[0169] The test results are shown in Figure 2.

[0170] According to the test results, the half toxic concentration (CC 50 ) exceeds 10μM, has no cytotoxicity, and has good safety.

[0171] Example 4 Inhibitory Effect Test of the Compounds of the Invention

[0172] Compound JH-B10 was prepared into test solutions with different concentration gradients for pre-incubation and IC 50 value.

[0173] HepG2-NTCP cells were cultured at 2×10 5The density of cells / well was plated in a 12-well plate and incubated for 48 hours at 37°C in a 5% CO2 environment. The culture medium was removed and the cells were washed twice with 0.5 mL of buffer (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPEs, pH 7.4) per well, and then pre-incubated for 15 minutes in 0.5 mL of buffer at 37°C in a 5% CO2 environment. The buffer was removed and the cells were washed again with buffer, and then after adding 0.5 mL of buffer, different concentration gradients of the compound Rapavir (final concentrations were 80, 16, 3.2, 0.64, 0.128, 0.0256, 0.00512 nM) were added, and the group to which an equal volume of DMSO was added was set as the control group. Three replicates were set for each group and incubated for 15 minutes at 37°C in a 5% CO2 environment. TCA-d4 was then added to the cells to a final concentration of 5 μM and incubated for 15 minutes at 37°C in a 5% CO2 environment. The cells were washed three times with 0.5 mL of buffer per well and lysed with 200 μL of absolute ethanol. The cell lysate was transferred to an EP tube and the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes at 4°C.

[0174] The HBeAg level was detected by enzyme-linked immunosorbent assay, the HBV 3.5-kb RNA level was detected by quantitative PCR, and the TCA uptake was detected by LC-MS / MS in Example 2.

[0175] The test results are shown in Figure 3.

[0176] According to the test results, the inhibitory activity of the compound Rapavir on taurocholic acid-d4 uptake, relative HBeAg level and relative HBV 3.5-kb RNA level was measured as IC 50 The inhibitory activities of Rapavir on taurocholic acid-d4 uptake, relative HBeAg levels and relative HBV 3.5-kb RNA levels were 11.1 times, 3.2 times and 10.8 times the corresponding inhibitory activities of JH-A32, respectively.

[0177] Example 5 In vivo efficacy verification test of the compounds of the present invention

[0178] By constructing a humanized liver mouse model, the compound JH-B10 (Rapavir) was selected for in vivo efficacy verification.

[0179] Human liver chimeric mice (10 mice) were randomly divided into a treatment group and a control group, with 5 mice in each group, and were administered with compound JH-B10 and DMSO at a dose of 2 mg / kg. The dosing regimen is shown in Figure 4. The two groups of mice were pretreated by administering the drug once 3 days and 1 hour before HBV infection. The time of HBV infection was recorded as day 0, and the drug was administered once on days 1, 2, 3, and 5, and then once a week (i.e., days 15, 22, 29, 36, 43, and 50) for a total of 8 weeks. Blood was collected for biochemical analysis every week (i.e., days 7, 14, 21, 28, 35, 42, 49, and 56). After 8 weeks, the mice were killed and the livers were collected for various index tests. The test results are shown in Figure 5.

[0180] Test results show that during administration, human serum albumin (HSA) levels remained stable, and serum HBV DNA and HBeAg levels were significantly suppressed. Following administration, relative HBV 3.5-kb RNA levels, HBV DNA, and HBV cccDNA copy numbers in the mouse livers decreased significantly compared to the control group, demonstrating that JH-B10 significantly inhibits HBV infection of hepatocytes. JH-B10 exhibits robust anti-HBV activity in animals.

[0181] The above description is only a specific embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent transformation made by using the present invention, or directly or indirectly applied in other related technical fields, is also included in the protection scope of the present invention.

Claims

1. A compound having the structure of the following formula (I): or a pharmaceutically acceptable salt thereof, wherein, X is selected from O or NR 14 ; R 1 selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, C 3-10 cycloalkyl-C 1-4 alkyl, C 3-10 heterocyclic group, C 3-10 heterocyclic group-C 1-4 alkyl, C 6-10 aryl, C 6-10 aryl-C 1-4 alkyl, C 5-10 heteroaryl and C 5- 10 heteroaryl-C 1-4 alkyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is unsubstituted or substituted with at least one substituent independently selected from R X ; R 2 Selected from hydrogen, halogen, amino, cyano, nitro, C 1-6 alkyl, C 1-6 alkyl-S-C 1-6 alkyl, C 1-6 alkyl-O-C 1-6 alkyl, C 1-6 alkyl-C 6-10 aryl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, C 3-10 cycloalkyl-C 1-4 alkyl, C 3-10 heterocyclic group, C 3-10 heterocyclic group-C 1-4 alkyl, C 6-10 aryl, C 6-10 aryl-C 1-4 alkyl, C 5-10 heteroaryl, C 5-10 heteroaryl-C 1-4 alkyl, CN and NO2, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is unsubstituted or substituted by at least one substituent independently selected from R X ; R 3 selected from hydrogen and C 1-6 alkyl group; When R 4 is i) When, formula (I) has the following structure of formula (II-1): When R 4 is ii) When, the formula (I) has the structure of the following formula (II-2): R 5 Selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, C 3-10 cycloalkyl-C 1-4 alkyl, C 3-10 heterocyclic group, C 3-10 heterocyclic group-C 1-4 alkyl, C 6-10 aryl, C 6-10 aryl-C 1-4 alkyl, C 5-10 heteroaryl, C 5- 10 heteroaryl-C 1-4 alkyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is unsubstituted or substituted with at least one substituent independently selected from R X ; R 6 selected from hydrogen, amino, cyano, nitro, C 1-6 alkyl, C 1-6 alkyl-C 3-10 cycloalkyl, C 1-6 alkyl-O-C 1-6 alkyl, C 1-6 alkyl-S-C 1-6 alkyl, C 1-6 alkyl-C 6-10 aryl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, C 3-10 cycloalkyl-C 1-4 alkyl, C 3-10 heterocyclic group, C 3-10 heterocyclic group-C 1-4 alkyl, C 6-10 aryl, C 6-10 aryl-C 1-4 alkyl, C 5-10 heteroaryl and C 5-10 heteroaryl-C 1-4 alkyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is unsubstituted or substituted by at least one substituent independently selected from R X ; or R 5 and R 6 together with the atom(s) to which it is attached form a 5- to 8-membered heterocyclic group containing 1, 2 or 3 heteroatoms, said 5- to 8-membered heterocyclic group being unsubstituted or substituted by at least one substituent independently selected from R X ; R 7 is hydrogen and C 1-6 alkyl; R 8 selected from hydrogen, amino, cyano, nitro, C 1-6 alkyl, C 1-6 alkyl-C 3-10 cycloalkyl, C 1-6 alkyl-O-C 1-6 alkyl, C 1-6 alkyl-S-C 1-6 alkyl, C 1-6 alkyl-C 6-10 aryl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, C 3-10 cycloalkyl-C 1-4 alkyl, C 3-10 heterocyclic group, C 3-10 heterocyclic group-C 1-4 alkyl, C 6-10 aryl, C 6-10 aryl-C 1-4 alkyl, C 5-10 heteroaryl and C 5-10 heteroaryl-C 1-4 alkyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is unsubstituted or substituted by at least one substituent independently selected from R X ; R 9 selected from hydrogen and C 1-6 alkyl; R 10 and R 11 each independently selected from hydrogen, hydroxy, halogen, C 1-6 alkyl and C 1-6 alkoxy, wherein each alkyl and alkoxy is unsubstituted or substituted with at least one substituent independently selected from R X ; R 12 and R 13 each independently selected from hydrogen, hydroxy, halogen, C 1-6 alkyl and C 1-6 alkoxy, wherein each alkyl and alkoxy is unsubstituted or substituted by at least one substituent independently selected from R X ; R 14 selected from hydrogen and C 1-6 alkyl; R X Selected from hydroxy, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, C 3-10 cycloalkyl-C 1-4 alkyl, C 3-10 heterocyclic group, C 3-10 heterocyclic group-C 1-4 alkyl, C 6-10 aryl, C 6-10 aryl-C 1-4 alkyl, C 5-10 heteroaryl and C 5-10 heteroaryl-C 1-4 alkyl, wherein each alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is unsubstituted or substituted with at least one substituent independently selected from R Y ; R Y selected from hydroxy, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, C 3-10 cycloalkyl-C 1-4 alkyl, C 3-10 heterocyclic group, C 3-10 heterocyclic group-C 1-4 alkyl, C 6-10 aryl, C 6-10 aryl-C 1-4 alkyl, C 5-10 heteroaryl and C 5-10 heteroaryl-C 1-4 alkyl; m, n, o and p are each independently selected from the integers 0, 1, 2, 3, 4, 5 and 6; *1 C, *2 C and *3 C respectively represent chiral carbon atoms connected to R 4 , R 6 and R 8 connected thereto.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, X is O; and / or R 1 selected from C 1-6 alkyl, preferably methyl; and / or R 2 selected from hydrogen and C 1-6 alkyl groups, preferably hydrogen, methyl and isopropyl; and / or R 3 is hydrogen; and / or R 5 selected from hydrogen and C 1-6 alkyl groups, preferably hydrogen and methyl; and / or R 6 selected from C 1-6 alkyl, C 1-6 alkyl-C 3-10 cycloalkyl and C 1-6 alkyl-C 6-10 aryl, preferably methyl, and / or R 5 and R 6 together with the atom to which it is attached form a 5- or 6-membered heterocyclic group containing 1 nitrogen heteroatom, said 5- or 6-membered heterocyclic group being unsubstituted or substituted by at least one substituent independently selected from R X and is preferably and / or R 7 is hydrogen; and / or R 8 selected from C 1-6 alkyl-C 6-10 aryl, wherein the alkyl and aryl are unsubstituted or substituted with at least one substituent independently selected from R X and preferably and / or R 9 selected from C 1-6 alkyl, preferably methyl; and / or R 10 selected from C 1-6 alkyl, preferably methyl; and / or R 11 selected from C 1-6 alkyl, preferably methyl; and / or R 12 selected from C 1-6 alkoxy, preferably methoxy; and / or R 13 Selected from C 1-6 alkoxy groups, preferably methoxy groups.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, R X selected from hydroxy, halogen, C 1-6 alkyl and C 1-6 alkoxy, preferably hydroxy, fluorine, methyl, methoxy and 4. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein, m is 1; and / or n is 1; and / or o is 1; and / or p is 1.

5. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein, *1 The C chiral carbon atom has an S configuration; and / or *2 The C chiral carbon atom is in the S or R configuration, preferably the S configuration; and / or *3 The C chiral carbon atom is in the S or R configuration, preferably the S configuration.

6. The compound according to claim 1, which is selected from the following structures: or a pharmaceutically acceptable salt thereof.

7. A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-6 and at least one pharmaceutically acceptable carrier.

8. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating, ameliorating or preventing a condition responsive to inhibition of the sodium taurocholate cotransporting polypeptide.

9. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing or treating hepatitis virus infection, preferably, the hepatitis virus infection is hepatitis B virus and / or hepatitis D virus infection.

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