CN108341810A

CN108341810A - Epithio carbamide compounds and application thereof

Info

Publication number: CN108341810A
Application number: CN201810053503.3A
Authority: CN
Inventors: 王喆; 王晓光; 范国钦; 卢涔宾; 杨赛
Original assignee: Shanghai Longwood Pharmaceutical Co Ltd
Current assignee: Shanghai Longwood Pharmaceutical Co Ltd
Priority date: 2017-01-23
Filing date: 2018-01-19
Publication date: 2018-07-31
Anticipated expiration: 2038-01-19
Also published as: WO2018133846A1; CN108341810B

Abstract

The present invention relates to a kind of epithio carbamide compounds and application thereof.Specifically, the invention discloses a kind of compound with structure shown in chemical formula (A) for making hbv replication inhibitor or its stereoisomers or tautomer or its pharmaceutically acceptable salt, hydrate or solvate.Purposes the invention further relates to the pharmaceutical composition comprising above compound and its in treating hepatitis B.

Description

Episulfide urea compound and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to an episulfide urea compound for treating hepatitis B and application thereof.

Background

Hepatitis B Virus (HBV) is a enveloped, partially double-stranded DNA (dsDNA), virus of the Hepadnaviridae family (Hepadnaviridae). Its genome comprises 4 overlapping reading frames: the pronuclear/nuclear gene, the polymerase gene, the UM and S genes (which encode the three envelope proteins), and the X gene. Before infection, the partially double-stranded DNA genome is converted in the host cell nucleus (open circular DNA, rcDNA) into covalently closed circular DNA (cccDNA) and the viral mRNA is transcribed. Once encapsidated, the pregenomic RNA (pgRNA), which also encodes the core protein and Pol, serves as a template for reverse transcription, which regenerates the portion of the dsDNA genome (rcDNA) in the nucleocapsid.

Transmission of hepatitis b virus results from exposure to infectious blood or body fluids, while viral DNA is detected in saliva, tears, and urine of chronic carriers with high titers of DNA in serum. The choice of direct therapy is currently limited to interferon and the following antiviral drugs; tenofovir, lamivudine, adefovir, entecavir and telbivudine.

In addition, heteroaryl dihydropyrimidine (HAPs) has been identified as a class of HBV inhibitors in tissue culture as well as in animal models (Weber et al, Antiviral Res. 54: 69-78). WO 2013/006394 and WO2013/096744 also disclose sulfamoyl-aryl amide compounds that are related to anti-HBV activity.

However, these direct HBV antiviral agents suffer from various problems such as toxicity, mutagenicity, lack of selectivity, poor therapeutic effect, poor bioavailability, and difficulty in synthesis.

Therefore, there is a need in the art to develop HBV inhibitors with advantages such as high potency, lower toxicity, etc.

Disclosure of Invention

The invention aims to provide a novel episulfide urea compound which can be used as an HBV inhibitor.

The invention provides a compound shown as a formula A, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof in a first aspect,

wherein,

R₁、R₂each independently selected from the group consisting of: hydrogen, substituted or unsubstituted C₁-C₁₀Alkyl, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C₆-C₁₀Aryl, halogen or substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, R₁、R₂Wherein said substitution is substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: -OH, halogen, C₁-C₆Alkyl, halogenated C₁-C₆Alkyl radical, C₁-C₆Alkoxy, ═ O, -O-;

x is CR₁₁R₁₂or-CR₁₁＝CR₁₂-; wherein R is₁₁And R₁₂Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C₁-C₁₀Alkyl, substituted or unsubstituted C₂-C₆Alkenyl, substituted or unsubstituted C₂-C₆Alkynyl, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, substituted or unsubstituted C_1-3alkyl-R₇、-C(＝O)OC_1-4An alkyl group; wherein said R₇Selected from the group consisting of: halogen, C₁-C₃Alkyl, substituted or unsubstituted 5-10 membered heteroaryl with 1-2 heteroatoms selected from the group consisting of N, S and O, 3-7 membered heterocycloalkyl with 1-3 heteroatoms selected from the group consisting of N, S and O, -NR₉R₁₀Wherein, said R₉、R₁₀Each independently selected from: H. c₁-C₃Alkyl, halogenated C₁-C₃An alkyl group;

or,

the R is₁₁And R₁₂Together with the adjacent C atom, form a substituted or unsubstituted 3-7 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O, wherein substitution of said 3-7 membered heterocycloalkyl means substitution with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: -OH, halogen, methoxy, -O-, -C (═ O) OC_1-4Alkyl, benzyl, C_1-4Alkyl, halogenated C_1-4An alkyl group, a carboxyl group,

and, said R₁₁、R₁₂Wherein said substitution is substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: -OH, halogen, C₁-C₆Alkyl, halogenated C₁-C₆Alkyl, -OH substituted C₁-C₆Alkyl radical, C₁-C₆Alkoxy, -C (═ O) OC_1-4An alkyl group;

y is substituted or unsubstituted C₁-C₇Alkylene or C₂-C₇Alkenylene, wherein in Y, the substitution means being substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: c₁-C₄Alkyl, halogen, -OH, preferably C₁-C₄Alkyl or-OH;

z is selected from the group consisting of: NH, O or a bond;

ring C is a substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O, wherein said substitution is by one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: c₁-C₃Alkyl (preferably methyl), C₃-C₄Cycloalkyl, -CN or halogen;

ring B is substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted having 1 to 3 substituents selected from the group consisting of N, S and O5-10 membered heteroaryl of a heteroatom; in the ring B, the substituted means being substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: c₁-C₃Alkyl radical, C₃-C₄Cycloalkyl, -CN or halogen;

R_a、R_b、R_c、R_dis a substituent at any position on the ring B, each of which is independently selected from the group consisting of: H. halogen, -CN, hydroxy, amino, carboxy, - (C ═ O) -substituted or unsubstituted C₁-C₈Alkyl, substituted or unsubstituted C₁-C₈Alkyl, substituted or unsubstituted C₂-C₆Alkenyl, substituted or unsubstituted C₂-C₆Alkynyl, substituted or unsubstituted C₁-C₈Alkylamino radical, substituted or unsubstituted C₁-C₈Alkoxy, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C₆-C₁₀Aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O; the R is_a、R_b、R_c、R_dWherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C₁-C₆Alkyl, halogenated C₁-C₆Alkyl radical, C₁-C₆Alkoxy, halogenated C₁-C₆Alkoxy radical, C₃-C₈Cycloalkyl, halogenated C₃-C₈Cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, C₆-C₁₀Aryl, halogenated C₆-C₁₀Aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O, halogenated 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O.

In another preferred embodiment, R₁、R₂Each independently selected from the group consisting of: hydrogen, substituted or unsubstituted C₁-C₁₀Alkyl, substitutedOr unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C₆-C₁₀Aryl, or substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, and R₁、R₂Wherein said substitution means substitution with one or more substituents selected from the group consisting of: -OH, halogen, C₁-C₆Alkyl, halogenated C₁-C₆Alkyl radical, C₁-C₆Alkoxy, -O-;

x is CR₁₁R₁₂or-CR₁₁＝CR₁₂-; wherein R is₁₁And R₁₂Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C₁-C₁₀Alkyl, substituted or unsubstituted C₂-C₆Alkenyl, substituted or unsubstituted C₂-C₆Alkynyl, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, C_1-3alkyl-R₇or-C (═ O) OC_1-4An alkyl group; wherein said R₇Selected from the group consisting of: halogen, C₁-C₃Alkyl, substituted or unsubstituted 5-10 membered heteroaryl with 1-2 heteroatoms selected from the group consisting of N, S and O, 3-7 membered heterocycloalkyl with 1-3 heteroatoms selected from the group consisting of N, S and O, -NR₉R₁₀Wherein, said R₉、R₁₀Each independently selected from: H. c₁-C₃Alkyl, halogenated C₁-C₃An alkyl group;

or,

the R is₁₁And R₁₂Together with the adjacent C atom, form a substituted or unsubstituted 3-7 membered heterocycloalkyl having 1-3 heteroatoms selected from group N, S and O, wherein said 3-7 membered heterocycloalkylSubstituted means substituted with one or more substituents selected from the group consisting of: -OH, halogen, methoxy, -O-, -C (═ O) OC_1-4Alkyl, benzyl, C_1-4Alkyl, halogenated C_1-4An alkyl group, a carboxyl group,

and, said R₁₁、R₁₂Wherein said substitution means substitution with one or more substituents selected from the group consisting of: -OH, halogen, C₁-C₆Alkyl, halogenated C₁-C₆Alkyl, -OH substituted C₁-C₆Alkyl radical, C₁-C₆Alkoxy, -C (═ O) OC_1-4An alkyl group;

R_a、R_b、R_c、R_dis a substituent at any position on a benzene ring, and is defined as the above.

In another preferred embodiment, R₁Is H, unsubstituted C₁-C₁₀Alkyl radical, C₃-C₁₀Cycloalkyl, C substituted by-OH, ═ O, -O-or halogen₁-C₁₀An alkyl group.

In another preferred embodiment, R₁₁Selected from the group consisting of: H. substituted or unsubstituted C₁-C₁₀Alkyl, -C (═ O) OC_1-4Alkyl radical, C_1-3alkyl-R₇Substituted or unsubstituted C_2-4An alkynyl group, a 3-7 membered heterocycloalkyl group having 1-3 heteroatoms selected from the group consisting of N, S and O, or a substituted or unsubstituted 5-10 membered heteroaryl group having 1-3 heteroatoms selected from the group consisting of N, S and O.

In another preferred embodiment, R₁₂Selected from the group consisting of: H. substituted or unsubstituted C₁-C₁₀Alkyl, preferably H, substituted or unsubstituted C₁-C₆Alkyl, more preferably H or methyl.

In another preferred embodiment, ring C is a substituted or unsubstituted 5-or 6-membered heteroaryl, said substitution being by one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: methyl, -CN or halogen.

In another preferred embodiment, ring B is phenyl or a substituted or unsubstituted 6 membered heteroaryl, preferably phenyl or pyridyl.

In another preferred embodiment, R is_a、R_b、R_c、R_dEach independently selected from the group consisting of: H. halogen, -CHF₂、-CF₂-methyl, -CH₂F、-CF₃、-OCF₃、-CN、-C₃-C₄Cycloalkyl, or-C₁-C₄An alkyl group.

In another preferred embodiment, the ring C isOrWherein,

R₄is H, -C₁-C₃Alkyl (preferably methyl), -C₃-C₄A cycloalkyl group;

R₅is H or halogen (preferably F);

R₆selected from H, methyl, -CN or halogen.

In another preferred embodiment, Z is O or a bond.

In another preferred embodiment, R is₇Is a substituted or unsubstituted 5-10 membered heteroaryl having 1-2 heteroatoms selected from the group consisting of N, S and O.

In another preferred embodiment, the compound of formula a is selected from the group consisting of:

in a second aspect of the invention, there is provided a process for the preparation of a compound according to the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the compound of formula a is a compound of formula VII-1, said process comprising the step (I):

or,

the compound of formula A is a compound of formula VIII-1, the method comprising step (II):

in the step (I) or (II), R₂、R_a、R_b、R_c、R_dThe definition of the ring C and the ring B is the same as that of the ring C and the ring B, and m and n are positive integers of 1-5 respectively;

wherein R is₃Selected from the group consisting of: H. halogen, substituted or unsubstituted C₁-C₁₀Alkyl, substituted or unsubstituted C₂-C₆Alkenyl, substituted or unsubstituted C₂-C₆Alkynyl, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C₆-C₁₀Aryl, or substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, C_1-3alkyl-R₇、-C(＝O)OC_1-4An alkyl group; wherein said R₇Selected from the group consisting of: halogen, C₁-C₃Alkyl, substituted or unsubstituted 5-10 membered heteroaryl with 1-2 heteroatoms selected from the group consisting of N, S and O, 3-7 membered heterocycloalkyl with 1-3 heteroatoms selected from the group consisting of N, S and O, -NR₉R₁₀Wherein, said R₉、R₁₀Each independently selected from: H. c₁-C₃Alkyl, halogenated C₁-C₃An alkyl group.

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising (1) a compound of the first aspect, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; (2) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition further comprises other drugs for preventing and/or treating hepatitis B virus infection.

in another preferred embodiment, the other agent for preventing and/or treating hepatitis B virus infection may be selected from the group consisting of an immunomodulator (e.g., interferon- α (IFN- α), pegylated interferon- α) or a stimulator of the innate immune system (e.g., Toll-like receptor 7 and/or 8 agonists).

In another preferred embodiment, the other agent for preventing and/or treating hepatitis b virus infection may be selected from the group consisting of: tenofovir, lamivudine, adefovir, entecavir, telbivudine, or combinations thereof.

In a fourth aspect of the invention, there is provided a compound of the formula:

in a fifth aspect of the present invention, there is provided a use of a compound according to the first aspect of the present invention, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to the third aspect of the present invention, for the preparation of a medicament for the prophylaxis and/or treatment of hepatitis b virus infection.

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

Detailed Description

The present inventors have conducted extensive and intensive studies and have found a novel group of episulfide urea compounds having an excellent therapeutic effect on hepatitis b. The compound of the present invention has a novel mother nucleus in structure, particularly a structural part with cyclic thiourea, and thus, not only has excellent anti-HBV activity, but also has lower cytotoxicity (particularly for liver cells). On this basis, the inventors have completed the present invention.

Definition of

As used herein, the term "alkyl" includes straight or branched chain alkyl groups. E.g. C₁-C₈Alkyl represents a straight or branched chain alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.

As used herein, the term "alkenyl" includes straight or branched chain alkenyl groups. E.g. C₂-C₆Alkenyl means a straight or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

As used herein, the term "alkynyl" includes straight or branched chain alkynyl groups. E.g. C₂-C₆Alkynyl means straight or branched chain alkynyl having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

As used herein, the term "C₃-C₁₀Cycloalkyl "refers to cycloalkyl groups having 3 to 10 carbon atoms. It may be a single ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may also be in the form of a double ring, for example a bridged or spiro ring.

As used herein, the term "C₁-C₈Alkylamino "is defined as being substituted by C₁-C₈The amino group substituted by the alkyl can be mono-substituted or di-substituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-tert-butylamino and the like.

As used herein, the term "C₁-C₈Alkoxy "means a straight or branched chain alkoxy group having 1 to 8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy and the like.

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

As used herein, the term "C₆-C₁₀Aryl "means an aryl group having 6 to 10 carbon atoms, for example, phenyl or naphthyl and the like.

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

The radicals mentioned in the present invention are "substituted" unless otherwise specifiedOr unsubstituted ", otherwise the groups of the invention may each be substituted by a substituent selected from the group consisting of: halogen, nitrile group, nitro group, hydroxyl group, amino group, C₁-C₆Alkyl-amino, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₁-C₆Alkoxy, halo C₁-C₆Alkyl, halo C₂-C₆Alkenyl, halo C₂-C₆Alkynyl, halo C₁-C₆Alkoxy, allyl, benzyl, C₆-C₁₂Aryl radical, C₁-C₆alkoxy-C₁-C₆Alkyl radical, C₁-C₆Alkoxy-carbonyl, phenoxycarbonyl, C₂-C₆Alkynyl-carbonyl, C₂-C₆Alkenyl-carbonyl, C₃-C₆Cycloalkyl-carbonyl, C₁-C₆Alkyl-sulfonyl, and the like.

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

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

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

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

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

Active ingredient

As used herein, "compound of the present invention" refers to a compound represented by formula (a), and also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compound of formula (a).

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

Pharmaceutical compositions and methods of administration

Since the compound of the present invention has excellent inhibitory activity against Hepatitis B Virus (HBV), the compound of the present invention and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and a pharmaceutical composition containing the compound of the present invention as a main active ingredient can be used for the prevention and/or treatment (stabilization, alleviation or cure) of infection by hepatitis b virus or for the prevention and/or treatment (stabilization, alleviation or cure) of diseases associated with hepatitis b virus (e.g., hepatitis b, progressive hepatic fibrosis, inflammation and necrosis leading to liver cirrhosis, end-stage liver disease, ethyl liver cancer).

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention in combination with a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-200mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

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

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

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

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

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

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

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

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

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

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

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 20 to 500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The compounds of the present invention:

the main advantages of the invention include:

1. the compound of the invention has novel structure and excellent effect of resisting hepatitis B virus infection.

2. The compounds of the present invention have very low toxicity to normal cells.

3. The compound and the pharmaceutical composition containing the compound as the main active ingredient can be used for preventing and/or treating hepatitis B virus infection.

4. The compound of the present invention and the pharmaceutical composition containing the compound of the present invention as a main active ingredient can be used for preventing and/or treating diseases associated with hepatitis b virus (e.g., hepatitis b, progressive liver fibrosis, inflammation and necrosis leading to cirrhosis, end-stage liver disease, ethyl liver cancer).

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The test materials and reagents used in the following examples are commercially available without specific reference.

The synthesis of compounds of type 10 is as follows:

example 1: synthesis of Compound 10a

Step 1:

dissolving the compound 1(1.5g) in acetic acid (20mL), adding iron powder (1.9g) into a reaction system, raising the temperature to 50 ℃ for reaction for 2 hours, adding ethyl acetate (30mL) into the reaction system, adding water (30mL), extracting with ethyl acetate (3X 20mL), drying with anhydrous sodium sulfate, spin-drying an organic phase, and performing crude column chromatography to obtain a compound 2(1 g). ESI-MS (M + H189.04)

Step 2:

dissolving chlorosulfonic acid isocyanate (200mg) in dichloromethane (5mL), reducing the temperature of the system to 0 ℃, adding tert-butyl alcohol (105mg) into the reaction system, stirring for 30min, adding triethylamine (214mg) and a compound 2(266mg) into the reaction system at 0 ℃, heating to room temperature after the addition, stirring for reaction for 2H, adding water (20mL) into the reaction system, extracting with dichloromethane (3X 20mL), drying with anhydrous sodium sulfate, spin-drying an organic phase, and performing crude column chromatography to obtain a compound 3(150mg), ESI-MS (M + H-368.06)

And step 3:

dissolving compound 3(100mg) and 1, 4-dibromobutane (58.8mg) in acetone (10mL), adding cesium carbonate (266mg) into the reaction system, reacting at 60 ℃ for 24H, adding water (15mL) into the reaction system, extracting with ethyl acetate (3X 20mL), drying over anhydrous sodium sulfate, spin-drying the organic phase, and separating the crude product by column chromatography to obtain yellow solid 80mg, ESI-MS, (M + H-422.11)

And 4, step 4:

dissolving compound 4(80mg) in tetrahydrofuran (2mL), water (0.5mL) and methanol (0.5mL), adding lithium hydroxide monohydrate (80mg) into the reaction system at room temperature, reacting at 40 ℃ for 5H, adjusting the pH value of the system to 3-4 by using 1N hydrochloric acid, extracting by using ethyl acetate (3X 15mL), drying by using anhydrous sodium sulfate, and spin-drying an organic phase to obtain yellow solid (50mg) ESI-MS, (M + H ═ 408.09)

And 5:

dissolving compound 5(50mg), triethylamine (70mg) and 4-fluoro-3-acetonitrile-1-aniline (22mg) in dichloromethane (3mL), cooling to about 5 ℃, adding TBTU (70mg) into the reaction system, reacting at room temperature for 12H, adding water (15mL), extracting with dichloromethane (3X 20mL), drying with anhydrous sodium sulfate, spin-drying the organic phase, and performing column chromatography to obtain compound 6(40mg) ESI-MS (M + H-526.13)

Step 6:

dissolving a compound 6(40mg) in dichloromethane (2mL), adding 4NHCl (1mL) into a reaction system, reacting at 30 ℃ for 2 hours, spin-drying the reaction liquid, adjusting the pH value of a saturated sodium bicarbonate solution to 7-8, extracting with ethyl acetate (3X 10mL), drying with anhydrous sodium sulfate, spin-drying an organic phase, and separating a crude product by column chromatography to obtain a compound 10a (8mg)

Example 2: synthesis of Compound 10b

Compound 6b was prepared according to steps 1 to 5 of example 1, except that 1, 3-dibromopropane was used in place of 1, 4-dibromobutane in step 3.

According to step 6 of example 1, only compound 6b was used in place of compound 6, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 1: 1) on the target product 10b (11 mg).

Example 3: synthesis of Compound 10c

Compound 6c was prepared according to example 1, steps 1 to 5, except that 1, 5-dibromopentane was used in place of 1, 4-dibromobutane in step 3.

According to step 6 of example 1, only compound 6c was used in place of compound 6, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 2: 1) for target product 10c (6 mg).

Example 4: synthesis of Compound 10d

Compound 6d was prepared according to example 1, steps 1-5, except that 2, 5-dibromopentane was used in place of 1, 4-dibromobutane in step 3.

According to step 6 of example 1, only compound 6d was used in place of compound 6, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 2: 1) for target product 10d (7 mg).

Example 5: synthesis of Compound 10e

Compound 6e was prepared according to steps 1-5 of example 1, except that 2-difluoromethyl-5-bromopropane was used in place of 1, 4-dibromobutane in step 3.

According to step 6 of example 1, only compound 6e was used in place of compound 6, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 2: 1) on the target product 10e (11 mg).

Example 6: synthesis of Compound 10f

Compound 6f was prepared by reference to steps 1-5 of example 1, except that 2-methylisoxazobenzyl-2, 5-dibromohexane was used in place of 1, 4-dibromobutane in step 3.

According to step 6 of example 1, only compound 6f was used in place of compound 6, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 1: 1) for target product 10f (8 mg).

Example 7: synthesis of Compound 10g

Compound 6g was prepared according to example 1, steps 1 to 5, except that 1-isopropyl-1, 4-dibromohexane was used in place of 1, 4-dibromobutane in step 3 and 3, 4-difluoro-aniline was used in place of 4-fluoro-3-cyanoaniline in step 5.

According to step 6 of example 1, only compound 6g was used in place of compound 6, and column chromatography was performed (n-heptane: ethyl acetate ═ 1: 1) using 10g (4mg) of the objective product, without changing the conditions.

Example 8: synthesis of Compound 10h

Compound 6h was prepared according to example 1, steps 1 to 5, except that 1-isopropyl-1, 4-dibromohexane was used in place of 1, 4-dibromobutane in step 3 and 4-fluoro-3-difluoromethylaniline was used in place of 4-fluoro-3-cyanoaniline in step 5.

According to step 6 of example 1, compound 6 was replaced with compound 6h, and the desired product was purified by column chromatography (n-heptane: ethyl acetate ═ 1: 1) for 10h (9mg) under otherwise unchanged conditions.

Example 9: synthesis of Compound 20a

Step 11:

adding allyl bromide (1.4g) dropwise into acetonitrile (30mL) solution of compound 2(2g) and potassium carbonate (2.3g), raising the temperature of the system to 85 ℃, reacting for 1H, adding water into the reaction system, extracting with ethyl acetate (3X 20mL), drying with anhydrous sodium sulfate, spin-drying the organic phase, and performing column chromatography to obtain compound 12(2.1g) ESI-MS, (M + H-229.07)

Step 12:

dissolving chlorosulfonic acid isocyanate (2g) in dichloromethane (40mL), reducing the temperature of the system to 0 ℃, then adding tert-butyl alcohol (1.3g) into the reaction system, stirring for 30min, adding triethylamine (3g) and a compound 12(2.0g) into the reaction system at 0 ℃, heating to room temperature after the addition, stirring for reaction for 2H, adding water (50mL) into the reaction system, extracting dichloromethane (3X 50mL), drying with anhydrous sodium sulfate, spin-drying an organic phase, and carrying out crude column chromatography to obtain a compound 13(1.4g), ESI-MS (M + H-408.10)

Step 13:

dissolving compound 13(1.2g) and allyl bromide (1.1g) in acetonitrile, adding cesium carbonate into the reaction system, heating to 80 ℃, reacting for 2H, adding water (20mL) into the reaction system, extracting with ethyl acetate (3X 20mL), drying with anhydrous sodium sulfate, and spin-drying the organic phase to obtain compound 14(1g) ESI-MS, (M + H ═ 448.12)

Step 14:

dissolving the compound 14(400mg) in dichloromethane (400mL), adding a Zanza catalyst CatB (42mg) into a reaction system, reacting at 30 ℃ for 12H, adding silica gel into the reaction system, spin-drying to obtain white powder, and performing column chromatography to obtain a compound 15(130mg) ESI-MS, (M + H ═ 420.09)

Step 15:

dissolving compound 15(80mg) in tetrahydrofuran (2mL), water (0.5mL) and methanol (0.5mL), adding lithium hydroxide monohydrate (80mg) into the reaction system at room temperature, reacting at 40 ℃ for 5H, adjusting the pH value of the system to 3-4 by using 1N hydrochloric acid, extracting by using ethyl acetate (3X 15mL), drying by using anhydrous sodium sulfate, and rotatably drying an organic phase to obtain yellow solid 16(40mg) ESI-MS (M + H-406.07)

Step 16:

dissolving compound 16(40mg), triethylamine (60mg) and 4-fluoro-3-acetonitrile-1-aniline (22mg) in dichloromethane (3mL), cooling to about 5 ℃, adding TBTU (70mg) into the reaction system, reacting at room temperature for 12H, adding water (15mL), extracting with dichloromethane (3X 20mL), drying with anhydrous sodium sulfate, spin-drying the organic phase, and performing column chromatography to obtain compound 17(40mg) ESI-MS (M + H-524.11)

And step 17:

dissolving a compound 17(40mg) in dichloromethane (2mL), adding 4NHCl (1mL) into a reaction system, reacting at 30 ℃ for 2 hours, spin-drying the reaction liquid, adjusting the pH value of a saturated sodium bicarbonate solution to 7-8, extracting with ethyl acetate (3X 10mL), drying with anhydrous sodium sulfate, spin-drying an organic phase, and separating a crude product by column chromatography to obtain a compound 20a (12mg)

Example 10: synthesis of Compound 20b

Compound 17b was prepared by reference to steps 11-16 of example 9, except that 4-bromo-1-butene was used in place of the propylene bromide in step 13.

According to step 17 of example 9, only compound 17b was used instead of compound 17, and the other conditions were unchanged, the target product 20b (6mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).

Example 11: synthesis of Compound 20c

Compound 17c was prepared by reference to steps 11-16 of example 9, except that 3-bromo-1-butene was used in place of the propylene bromide in step 13.

According to step 17 of example 9, only compound 17c was used instead of compound 17, and the other conditions were unchanged, the objective product 20c (13mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).

Example 12: synthesis of Compound 20d

Compound 17d was prepared by reference to steps 11-16 of example 9, except that 4-methylisoxazobenzyl-1-pentene was used in place of propylenebromide in step 13.

According to step 17 of example 9, only compound 17d was used in place of compound 17, and the other conditions were unchanged, the objective product 20d (22mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).

Example 13: synthesis of Compound 20e

Compound 17e was prepared according to example 9, steps 11-16, except that in step 13, 1-isopropyl-1-propene was used instead of propene bromide, and in step 16, 3, 4-difluoro-aniline was used instead of 4-fluoro-3-cyanoaniline.

According to step 17 of example 9, only compound 17e was used instead of compound 17, and the other conditions were unchanged, the objective product 20e (14mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 1: 1).

Example 14: synthesis of Compound 20f

Compound 17f was prepared according to example 9, Steps 11-16, except that 1-isopropyl-1-propene was used in place of propene bromide in step 13 and 4-fluoro-3-difluoromethylaniline was used in place of 4-fluoro-3-cyanoaniline in step 16.

According to step 17 of example 9, only compound 17f was used in place of compound 17, and the other conditions were unchanged, the target product 20f (8mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 1: 1).

Example 15: synthesis of Compound 30a

Step 21:

dissolving the compound 21(1.5g) in acetic acid (20mL), adding iron powder (1.9g) into a reaction system, raising the temperature to 50 ℃ for reaction for 2 hours, adding ethyl acetate (30mL) into the reaction system, adding water (30mL), extracting with ethyl acetate (3X 20mL), drying with anhydrous sodium sulfate, spin-drying an organic phase, and performing crude column chromatography to obtain a compound 22(1 g). ESI-MS (M + H190.03)

Step 22:

dissolving chlorosulfonic acid isocyanate (200mg) in dichloromethane (5mL), reducing the temperature of the system to 0 ℃, adding tert-butyl alcohol (105mg) into the reaction system, stirring for 30min, adding triethylamine (214mg) and a compound (22) (266mg) into the reaction system at 0 ℃, heating to room temperature after the addition, stirring for reaction for 2H, adding water (20mL) into the reaction system, extracting with dichloromethane (3X 20mL), drying with anhydrous sodium sulfate, spin-drying an organic phase, and performing crude column chromatography to obtain a compound (23 (150mg), ESI-MS (M + H-369.06)

Step 23:

dissolving compound 23(100mg) and 1, 4-dibromobutane (58.8mg) in acetone (10mL), adding cesium carbonate (266mg) to the reaction system, reacting at 60 ℃ for 24H, adding water (15mL) to the reaction system, extracting with ethyl acetate (3 × 20mL), drying over anhydrous sodium sulfate, spin-drying the organic phase, and separating the crude product by column chromatography to obtain yellow solid compound 24(80mg), ESI-MS, (M + H ═ 422.11)

Step 24:

dissolving compound 24(80mg) in tetrahydrofuran (2mL), water (0.5mL) and methanol (0.5mL), adding lithium hydroxide monohydrate (80mg) into the reaction system at room temperature, reacting at 40 ℃ for 5H, adjusting the pH value of the system to 3-4 by using 1N hydrochloric acid, extracting by using ethyl acetate (3X 15mL), drying by using anhydrous sodium sulfate, and rotatably drying an organic phase to obtain yellow solid 25(50mg) ESI-MS, (M + H ═ 408.09)

Step 25:

dissolving compound 25(50mg), triethylamine (70mg) and 4-fluoro-3-acetonitrile-1-aniline (22mg) in dichloromethane (3mL), cooling to about 5 ℃, adding TBTU (70mg) into the reaction system, reacting at room temperature for 12H, adding water (15mL), extracting with dichloromethane (3X 20mL), drying with anhydrous sodium sulfate, spin-drying the organic phase, and performing column chromatography to obtain compound 26(40mg) ESI-MS (M + H-526.13)

Step 26:

dissolving compound 26(40mg) in dichloromethane (2mL), adding 4N HCl (1mL) into the reaction system, reacting at 30 ℃ for 2h, spin-drying the reaction solution, adjusting pH to 7-8 with saturated sodium bicarbonate solution, extracting with ethyl acetate (3X 10mL), drying with anhydrous sodium sulfate, spin-drying the organic phase, and separating the crude product by column chromatography to obtain compound 30a (8mg)

Example 16: synthesis of Compound 30b

Compound 26b was prepared according to example 15, steps 21 to 25, except that 2, 4-dibromopentane was used in place of 1, 4-dibromobutane in step 23.

According to step 26 of example 15, only compound 26b was used in place of compound 26, and the other conditions were unchanged, the objective product 30b (11mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 1: 1).

Example 17: synthesis of Compound 30c

Compound 26c was prepared by reference to example 15, Steps 21-25, except that 2-methylisoxazobenzyl-2, 5-dibromohexane was used in place of 1, 4-dibromobutane in step 23.

According to step 26 of example 15, only compound 26c was used instead of compound 26, and the other conditions were unchanged, the objective product 30c (8mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).

Example 18: synthesis of 30d

Compound 26d was prepared by reference to steps 21 to 25 of example 15, except that 1-isopropyl-1, 4-dibromohexane was used in place of 1, 4-dibromobutane in step 23. In step 25 3, 4-difluoro-aniline is used instead of 4-fluoro-3-cyanoaniline. According to step 26 of example 15, only compound 26d was used in place of compound 26, and the other conditions were unchanged, the objective product 30d (9mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).

Example 19: synthesis of Compound 30e

Compound 26e was prepared by reference to steps 21 to 25 of example 15, except that 1-isopropyl-1, 4-dibromohexane was used in place of 1, 4-dibromobutane in step 23. In step 25 3-difluoromethyl-4-fluoro-aniline is used instead of 4-fluoro-3-cyanoaniline. According to step 26 of example 15, only compound 26e was used in place of compound 26, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 2: 1) on the target product 30e (11 mg).

Example 20: synthesis of Compound 40a

Step 31:

adding allyl bromide (1.4g) dropwise into acetonitrile (30mL) solution of compound 31(2g) and potassium carbonate (2.3g), raising the temperature of the system to 85 ℃, reacting for 1H, adding water into the reaction system, extracting with ethyl acetate (3X 20mL), drying with anhydrous sodium sulfate, spin-drying the organic phase, and performing column chromatography to obtain compound 32(2.1g) ESI-MS, (M + H-230.07)

Step 32:

dissolving chlorosulfonic acid isocyanate (2g) in dichloromethane (40mL), reducing the temperature of the system to 0 ℃, then adding tert-butyl alcohol (1.3g) into the reaction system, stirring for 30min, adding triethylamine (3g) and a compound 32(2.0g) into the reaction system at 0 ℃, heating to room temperature after the addition, stirring for reaction for 2H, adding water (50mL) into the reaction system, extracting dichloromethane (3X 50mL), drying with anhydrous sodium sulfate, spin-drying an organic phase, and carrying out crude column chromatography to obtain a compound 33(1.4g), ESI-MS (M + H-409.11)

Step 33:

dissolving compound 33(1.2g) and allyl bromide (1.1g) in acetonitrile, adding cesium carbonate into the reaction system, heating to 80 ℃, reacting for 2H, adding water (20mL) into the reaction system, extracting with ethyl acetate (3X 20mL), drying with anhydrous sodium sulfate, and spin-drying the organic phase to obtain compound 34(1g) ESI-MS, (M + H ═ 449.13)

Step 34:

dissolving the compound 34(400mg) in dichloromethane (400mL), adding a Zanza catalyst CatB (42mg) into a reaction system, reacting at 30 ℃ for 12H, adding silica gel into the reaction system, spin-drying to obtain white powder, and performing column chromatography to obtain a compound 35(130mg) ESI-MS, (M + H ═ 421.10)

Step 35:

dissolving compound 35(80mg) in tetrahydrofuran (2mL), water (0.5mL) and methanol (0.5mL), adding lithium hydroxide monohydrate (80mg) into the reaction system at room temperature, reacting at 40 ℃ for 5H, adjusting the pH value of the system to 3-4 by using 1N hydrochloric acid, extracting by using ethyl acetate (3X 15mL), drying by using anhydrous sodium sulfate, and rotatably drying an organic phase to obtain yellow solid 36(40mg) ESI-MS, (M + H ═ 407.08)

Step 36:

dissolving compound 36(40mg), triethylamine (60mg) and 4-fluoro-3-acetonitrile-1-aniline (22mg) in dichloromethane (3mL), cooling to about 5 ℃, adding TBTU (70mg) into the reaction system, reacting at room temperature for 12H, adding water (15mL), extracting with dichloromethane (3X 20mL), drying with anhydrous sodium sulfate, spin-drying the organic phase, and performing column chromatography to obtain compound 37(40mg) ESI-MS (M + H-525.11)

Step 37:

dissolving the compound 37(40mg) in dichloromethane (2mL), adding 4N Cl (1mL) into the reaction system, reacting at 30 ℃ for 2h, spin-drying the reaction solution, adjusting the pH value of a saturated sodium bicarbonate solution to 7-8, extracting with ethyl acetate (3X 10mL), drying with anhydrous sodium sulfate, spin-drying the organic phase, and separating the crude product by column chromatography to obtain a compound 40a (12mg)

Example 21: synthesis of Compound 40b

Compound 37b was prepared by reference to steps 31-36 of example 20, except that 3-bromo-1-butene was used in place of the propylene bromide in step 32.

According to step 37 of example 20, only compound 37b was used instead of compound 37, and the other conditions were unchanged, the target product 40b (6mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).

Example 22: synthesis of Compound 40c

Compound 37c was prepared according to example 20, steps 31 to 36, except that 4-methylisoxazobenzyl-1-pentene was used instead of propylenebromide in step 32 according to example 20, step 37, except that compound 37c was used instead of compound 37 and the conditions were otherwise unchanged, column chromatography (n-heptane: ethyl acetate: 2: 1) was carried out on the title product 40c (13 mg).

Example 23: synthesis of Compound 40d

Compound 37d was prepared according to example 20, Steps 31-36, except that 1-isopropyl-1-propene was used in place of propene bromide in step 32 and 3, 4-difluoro-aniline was used in place of 4-fluoro-3-cyanoaniline in step 35.

According to step 37 of example 20, only compound 37d was used in place of compound 37, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 2: 1) for target product 40d (12 mg).

Example 24: synthesis of Compound 40e

Compound 37e was prepared according to example 20, Steps 31-36, except that 1-isopropyl-1-propene was used in place of propene bromide in step 32 and 3-difluoromethyl-4-fluoro-aniline was used in place of 4-fluoro-3-cyanoaniline in step 35.

According to step 37 of example 20, only compound 37e was used in place of compound 37, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 2: 1) on the target product 40e (13 mg).

Biological examples- -anti-HBV Activity assay

Experiment one: in vitro anti-hepatitis B virus nucleocapsid assembly activity test method

Main reagents and raw materials:

c150 protein is expressed and purified by the pharmaceutical Mingkuda company;

purchased from semer feishell technologies.

Protein fluorescence labeling:

to each well of the 96-well plate, 150. mu.L of 2% w/v skim milk was added and incubated at room temperature for 2 hours. Sucking off the skimmed milk, washing with deionized water, drying, and storing at room temperature. The C150 protein (3 mg per tube) was purified using a 5ml Hitrap desalting columnAnd (4) desalting. 50mM was added to the desalted C150 protein per tube20 μ l of the fluorescent dye was mixed well and incubated overnight at 4 ℃ in the dark. The fluorescent dye not bound to C150 was removed by Sephadex G-25 gel filtration. The fluorescence labeling efficiency of C150 was calculated as follows:

wherein,

indicates the concentration of the fluorescent label;

[ C150Bo ] indicates the concentration of the fluorescent-labeled protein;

a504 represents the absorbance at wavelength 504 nM;

a280 represents the absorbance at a wavelength of 280 nM;

M^-1represents the reciprocal of the molar concentration.

Compound dilution:

compound stock was diluted to 6mM in DMSO and then to 600. mu.M in 50mM HEPES, followed by a further 3-fold serial dilution of 8 concentrations in 10% DMSO/50mM HEPES.

C150Bo was diluted to 2. mu.M with 50mM HEPES. Compounds were added to 96-well plates at 37.5 μ L C150Bo and 2.5 μ L of each concentration and mixed well and incubated for 15 minutes at room temperature. Mu.l of 750mM NaCl/50mM HEPES was added to the reaction wells at a final concentration of 150mM NaCl.

Control wells were assembled with 0% protein, and 10. mu.L of 50mM HEPES, NaCl at a final concentration of 0mM, was added.

100% protein assembly control wells, 10. mu.L of 5M NaCl/50mM HEPES, 1M NaCl final concentration.

The final DMSO concentration was 0.5%, the maximum final concentration of the compound was 30. mu.M, and the final concentration of C150Bo was 1.5. mu.M. Incubate at room temperature for 1 hour. The fluorescence signal was measured (excitation 485 nm; emission 535 nm).

Data analysis

% protein assembly ═ 1- (sample fluorescence value-1M NaCl fluorescence value)/(0M NaCl fluorescence value-1M NaCl fluorescence value) ] × 100.

IC₅₀The values were calculated by prism software, the equation is as follows:

Y＝Bottom+(Top-Bottom)/(1+10^{((LogIC50-X)*HillSlope)})；

wherein X represents the log of the concentration, Y represents the effect value, and Y is fitted to the top in sigmoid form starting from the bottom;

bottom represents the Bottom of the curve; top represents the Top of the curve;

HillSlope denotes: absolute value of the maximum slope of the curve.

Experiment two: determination of anti-hepatitis B Virus Activity in HepG2.2.15 cells

The main reagents are as follows:

QIAamp 96DNA blood kit (12) (Qiagen, cat # 51162);

FastStart Universal Probe Master (Roche, cat # 04914058001);

cell-titer Glo detection reagent (Promega, cat # G7573).

Compound dilution: in vitro anti-HBV activity experiments and cytotoxicity experiments all compounds were serially diluted 3-fold at 8 concentrations. The final starting concentration of test compound was 30 μ M, the final starting concentration of reference compound GLS4 was 1 μ M, and the final concentration of DMSO was 0.5%.

Inoculation of HepG2.2.15 cells (4X 10)⁴Cells/well) to 96-well plates at 37 ℃, 5% CO₂The culture was carried out overnight. The following day, fresh medium containing different concentrations of the compounds was added to the culture wells. On the fifth day, old culture medium was aspirated from the culture wells, and fresh culture medium containing different concentrations of compounds was added.

And eighthly, collecting the supernatant in the culture hole for extracting HBV DNA in the supernatant, and detecting the HBV DNA content in the HepG2.2.15 supernatant by qPCR. And after collecting the supernatant, adding a culture medium and a Cell-titer Glo reagent into the culture wells, and detecting chemiluminescence values of the wells by using an enzyme-labeling instrument.

The activity calculation formula is as follows:

Y＝Bottom+(Top-Bottom)/(1+10^{((LogIC50-X)*HillSlope)})；

bottom represents the Bottom of the curve; top represents the Top of the curve;

HillSlope denotes: absolute value of the maximum slope of the curve.

Experiment three: cytotoxicity assays

The cytotoxicity of test compounds was tested using HepG2 cells, which were incubated for 4 days in the presence of test compounds. Cell viability was assessed using the resazurin assay.

In the table:

a1 indicates IC50(μ M) at < 1;

a2 represents IC50 (mu M) between 1 and 100;

a3 denotes IC50 (. mu.M) at > 100;

b1 denotes EC50(μ M) between < 1;

b2 represents EC50(μ M) between 1 and 100;

b3 denotes EC50 (. mu.M) at > 100;

wherein the control compound is:

(see WO2014184350A1)

The results show that: the compound of the invention has excellent in vitro anti-hepatitis B virus nucleocapsid assembly activity and anti-hepatitis B virus activity and lower cytotoxicity.

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

Claims

1. A compound shown as formula A, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof,

wherein,

R₁、R₂each independently selected from the group consisting of: hydrogen, substituted or unsubstituted C₁-C₁₀Alkyl, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C₆-C₁₀Aryl, halogen or substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, R₁、R₂Wherein said substitution means substitution with one or more substituents selected from the group consisting of: -OH, halogen, C₁-C₆Alkyl, halogenated C₁-C₆Alkyl radical, C₁-C₆Alkoxy, ═ O, -O-;

x is CR₁₁R₁₂or-CR₁₁＝CR₁₂-; wherein R is₁₁And R₁₂Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C₁-C₁₀Alkyl, substituted or unsubstituted C₂-C₆Alkenyl, substituted or unsubstituted C₂-C₆Alkynyl, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, substituted or unsubstituted C_1-3alkyl-R₇or-C (═ O) OC_1-4An alkyl group; wherein said R₇Selected from the group consisting of: halogen, C₁-C₃Alkyl, substituted or unsubstituted 5-10 membered heteroaryl with 1-2 heteroatoms selected from the group consisting of N, S and O, 3-7 membered heterocycloalkyl with 1-3 heteroatoms selected from the group consisting of N, S and O, -NR₉R₁₀Wherein, said R₉、R₁₀Each independently selected from: H. c₁-C₃Alkyl, halogenated C₁-C₃An alkyl group;

or,

the R is₁₁And R₁₂Together with the adjacent C atom, a substituted or unsubstituted 3-7 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O, wherein the substitution of said 3-7 membered heterocycloalkyl means with one or more substituents selected from the group consisting ofAnd (3) substitution: -OH, halogen, methoxy, -O-, -C (═ O) OC_1-4Alkyl, benzyl, C_1-4Alkyl, halogenated C_1-4An alkyl group, a carboxyl group,

y is substituted or unsubstituted C₁-C₇Alkylene or C₂-C₇Alkenylene, wherein in the Y, the substitution means substitution with one or more substituents selected from the group consisting of: c₁-C₄Alkyl, halogen, -OH, preferably C₁-C₄Alkyl or-OH;

z is selected from the group consisting of: NH, O or a bond;

ring C is a substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O, wherein said substitution means being substituted with one or more substituents selected from the group consisting of: c₁-C₃Alkyl radical, C₃-C₄Cycloalkyl, -CN or halogen;

ring B is substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O; in the ring B, the substitution means being substituted with one or more substituents selected from the group consisting of: c₁-C₃Alkyl radical, C₃-C₄Cycloalkyl, -CN or halogen;

R_a、R_b、R_c、R_dis a substituent at any position on the ring B, each of which is independently selected from the group consisting of: H. halogen, -CN, hydroxy, amino, carboxy, - (C ═ O) -substituted or unsubstituted C₁-C₈Alkyl, substituted or unsubstituted C₁-C₈Alkyl, substituted or unsubstituted C₂-C₆Alkenyl, substituted or unsubstituted C₂-C₆Alkynyl, substituted or unsubstituted C₁-C₈Alkylamino radical, substituted or unsubstituted C₁-C₈Alkoxy, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C₆-C₁₀Aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O; the R is_a、R_b、R_c、R_dWherein said "substituted" means substituted with one or more substituents selected from the group consisting of: halogen, C₁-C₆Alkyl, halogenated C₁-C₆Alkyl radical, C₁-C₆Alkoxy, halogenated C₁-C₆Alkoxy radical, C₃-C₈Cycloalkyl, halogenated C₃-C₈Cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, C₆-C₁₀Aryl, halogenated C₆-C₁₀Aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O, halogenated 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O.

2. The compound of claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein R is₁、R₂Each independently selected from the group consisting of: hydrogen, substituted or unsubstituted C₁-C₁₀Alkyl, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C₆-C₁₀Aryl, or substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, and R₁、R₂Wherein said substitution means substitution with one or more substituents selected from the group consisting of: -OH, halogen, C₁-C₆Alkyl, halogenated C₁-C₆Alkyl radical, C₁-C₆Alkoxy, -O-;

or,

the R is₁₁And R₁₂And an adjacent C atom together form a substituted or unsubstituted 3-7 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O, wherein the substitution of said 3-7 membered heterocycloalkyl means substitution with one or more substituents selected from the group consisting of: -OH, halogen, methoxy, -O-, -C (═ O) OC_1-4Alkyl, benzyl, C_1-4Alkyl, halogenated C_1-4An alkyl group, a carboxyl group,

R_a、R_b、R_c、R_dis benzeneThe substituents at any position on the ring are as defined in claim 1.

3. The compound of claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein R is₁Is H, unsubstituted C₁-C₁₀Alkyl radical, C₃-C₁₀Cycloalkyl, or C substituted by-OH, ═ O, -O-or halogen₁-C₁₀An alkyl group.

4. The compound of claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein ring C is a substituted or unsubstituted 5-or 6-membered heteroaryl, said substitution being by one or more substituents selected from the group consisting of: methyl, -CN or halogen.

5. The compound of claim 1, or a stereoisomer or tautomer thereof, or pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein R is_a、R_b、R_c、R_dEach independently selected from the group consisting of: H. halogen, -CHF₂、-CF₂-methyl, -CH₂F、-CF₃、-OCF₃、-CN、-C₃-C₄Cycloalkyl, or-C₁-C₄An alkyl group.

6. The compound of claim 1, or a stereoisomer or tautomer thereof, or pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the compound of formula a is selected from the group consisting of:

7. a process for preparing a compound of formula a, a compound of formula VII-1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, according to claim 1, comprising the step (I):

or,

in the step (I) or (II), R₂、R_a、R_b、R_c、R_dThe definition of the ring C and the ring B is the same as that of claim 1, and m and n are positive integers of 1-5 respectively;

8. A pharmaceutical composition comprising (1) a compound of claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; (2) a pharmaceutically acceptable carrier.

9. The compounds are shown below:

10. use of a compound according to claim 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, or a pharmaceutical composition according to claim 8, for the preparation of a medicament for the prophylaxis and/or treatment of hepatitis b virus infection.