CN108250122B

CN108250122B - Sulfonamide-aryl amide compounds and pharmaceutical use thereof for treating hepatitis B

Info

Publication number: CN108250122B
Application number: CN201711466237.9A
Authority: CN
Inventors: 王喆; 王晓光; 卢涔宾; 范国钦
Original assignee: Shanghai Longwood Biopharmaceuticals Co Ltd
Current assignee: Shanghai Longwood Biopharmaceuticals Co Ltd
Priority date: 2016-12-28
Filing date: 2017-12-28
Publication date: 2022-11-11
Anticipated expiration: 2037-12-28
Also published as: CN108250122A; WO2018121689A1; CN108250121A

Abstract

The invention relates to a sulfonamide-aryl amide compound and a medicinal application thereof in treating hepatitis B. Specifically, the invention discloses a compound which can be used as an HBV replication inhibitor and has a structure shown in a chemical formula (A), or a stereoisomer, a cis-trans isomer or a tautomer thereof, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof, wherein the definition of each group is detailed in the specification. The invention also relates to a pharmaceutical composition containing the compound and application thereof in treating hepatitis B.

Description

Sulfonamide-aryl amide compounds and pharmaceutical use thereof for treating hepatitis B

Technical Field

The invention belongs to the field of chemical medicine, and particularly relates to a sulfonamide-aryl amide compound and a pharmaceutical application thereof in treating hepatitis B.

Background

Hepatitis B Virus (HBV) is a enveloped, partially double-stranded DNA (dsDNA), virus of the Hepadnaviridae family (Hepadnaviridae). Its genome contains 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.

HBV causes epidemics in asia and parts of africa, and it is endemic in china. HBV has infected approximately 20 million people worldwide, of which approximately 3.5 million people develop chronic infectious diseases. The virus causes hepatitis b disease and chronic infectious diseases are associated with a high increased risk of development of cirrhosis and liver cancer.

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.

While there is currently an effective and well-tolerated vaccine, the options for direct treatment are currently limited to interferon and the following antiviral drugs; tenofovir, tenofovir Alafenamide (TAF), lamivudine, adefovir, entecavir, and telbivudine.

In addition, heteroaryl dihydropyrimidines (HAPs) have been identified as a class of HBV inhibitors in tissue culture as well as in animal models (Weber et al, antiviral research 54-69-78).

WO 2013/006394 (disclosed on 10.1.2013) and WO 2013/096744 (disclosed on 27.6.2013) also disclose sulfamoyl-arylamides that are involved in anti-HBV activity.

However, problems of toxicity, mutagenicity, lack of selectivity, poor therapeutic effect, poor bioavailability and difficulty in synthesis are encountered in these direct HBV antiviral agents.

Therefore, in order to overcome the above curves, it is necessary to develop HBV inhibitors having advantages of high potency, lower toxicity, and the like.

Disclosure of Invention

The object of the present invention is to provide a class of compounds of novel structure which are useful as HBV inhibitors.

The invention provides a compound shown as a formula A, or a stereoisomer, a cis-trans isomer or a tautomer thereof, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof,

wherein, the first and the second end of the pipe are connected with each other,

R ₁ 、R ₂ each independently hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O; or R ₁ 、R ₂ Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3-10 membered heterocycloalkyl having 1N and 0-3 heteroatoms selected from the group consisting of N, S, and O;

R ₃ is hydrogen, halogen, -CN, hydroxy, amino, carboxy, - (C = O) -substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkylamino, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 heteroatoms selected from the following groups N, S and O, orSubstituted or unsubstituted C6-C10 aryl, and substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S, and O;

R ₄ 、R ₅ and R ₆ Is a substituent at any position on the phenyl ring, each of which is independently hydrogen, halogen, -CN, hydroxyl, amino, carboxyl, - (C = O) -substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C8 alkylamino, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 heteroatoms selected from the following groups N, S and O, substituted or unsubstituted C6-C10 aryl, and substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the following groups N, S and O;

R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ and R ₆ The "substituted" means being substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, C6-C10 aryl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from the following groups N, S and O, halogenated 5-10 membered heteroaryl having 1-3 heteroatoms selected from the following groups N, S and O;

m is O, S, CR ₇ R _7’ Or NR ₈ (ii) a Wherein R is ₇ 、R _7’ 、R ₈ Each independently is hydrogen, halogen, -CN, hydroxy, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl; wherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, -CN, hydroxy, amino, carboxy;

x is NR ₉ Carbonyl (- (CO) -), haloC 1-C4 alkyl (e.g. CF) ₂ ) Or hydroxyoxime (= N-OH); wherein R is ₉ Is hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C8 cycloalkyl; wherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, -CN, hydroxy, amino, carboxy;

y is carbonyl (- (CO) -) or sulfonyl (-SO) ₂ -)；

And the following conditions are satisfied: x and Y can not be carbonyl at the same time, and Y can not be sulfonyl when X is carbonyl;

z is N or CR ₁₀ (ii) a Wherein R is ₁₀ Each independently is hydrogen, halogen, -CN, hydroxy, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S, and O, substituted or unsubstituted C6-C10 aryl, and substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S, and O; wherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, C6-C10 aryl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from the following groups N, S and O, halogenated 5-10 membered heteroaryl having 1-3 heteroatoms selected from the following groups N, S and O.

In another preferred embodiment, among the compounds,

R ₁ 、R ₂ each independently hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O; or R ₁ 、R ₂ Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3-10 ring having 1N and 0-3 heteroatoms selected from the group consisting of N, S and OA heterocyclic alkyl group;

R ₃ is hydrogen, halogen, -CN, hydroxyl, amino, carboxyl, - (C = O) -substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkylamino, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the following groups N, S and O, substituted or unsubstituted C6-C10 aryl, and substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the following groups N, S and O;

m is NR ₈ (ii) a Wherein R is ₈ Is methyl;

x is NR ₉ (ii) a Wherein R is ₉ Is hydrogen;

y is sulfonyl (-SO) ₂ -)；

Z is N or CR ₁₀ (ii) a Wherein R is ₁₀ Each independently hydrogen.

In another preferred embodiment, among the compounds,

m is NR ₈ (ii) a Wherein R is ₈ Is methyl;

x is NR ₉ (ii) a Wherein R is ₉ Is hydrogen;

y is sulfonyl (-SO) ₂ -)；

Z is CR ₁₀ (ii) a Wherein R is ₁₀ Is hydrogen.

In another preferred embodiment, R ₁ 、R ₂ Each independently hydrogen, substituted or unsubstituted C2-C8 alkyl, substituted or unsubstituted C3-C4 cycloalkyl; wherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: fluorine, chlorine, bromine, iodine.

In another preferred embodiment, R ₁ 、R ₂ Each independently hydrogen, substituted or unsubstituted C2-C4 alkyl, substituted or unsubstituted C3-C4 cycloalkyl; wherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: fluorine, chlorine, bromine, iodine.

In another preferred embodiment, R ₃ Is hydrogen, fluorine, chlorine, bromine, iodine, -CN, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C4 cycloalkyl; wherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C4 alkyl, halogenated C1-C4 alkyl, -CN, hydroxy.

In another preferred embodiment, R ₃ Hydrogen, fluorine, chlorine, bromine and iodine.

In another preferred embodiment, R ₄ 、R ₅ And R ₆ Are independent of each otherIs hydrogen, fluorine, chlorine, bromine, iodine, -CN, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C4 cycloalkyl; wherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: fluorine, chlorine, bromine, iodine, C1-C4 alkyl, halogenated C1-C4 alkyl.

In another preferred embodiment, R ₄ 、R ₅ And R ₆ Each independently is hydrogen, fluoro, chloro, bromo, iodo, -CN, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C4 cycloalkyl; wherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: fluorine, chlorine, bromine, iodine.

In another preferred embodiment, X is NH, CF ₂ Or hydroxamic (= N-OH).

In another preferred embodiment, M is NR ₈ (ii) a Wherein R is ₈ Is hydrogen, halogen, -CN, hydroxy, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C3-C4 cycloalkyl; wherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C4 alkyl, halogenated C1-C4 alkyl, -CN, hydroxyl, amino, carboxyl.

In another preferred embodiment, Z is N or CR ₁₀ (ii) a Wherein R is ₁₀ Each independently is hydrogen, fluoro, chloro, bromo, iodo, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C4 cycloalkyl; wherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: fluorine, chlorine, bromine, iodine, C1-C4 alkyl, halogenated C1-C4 alkyl.

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

in a second aspect, the present invention provides a process for the preparation of a compound of the first aspect, or a stereoisomer, a cis-trans isomer, or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof,

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

(a1) Reacting compound III-1 with compound IV-1 in an inert solvent to form compound V-1;

(a2) Subjecting compound V-1 to a hydrolysis reaction in an inert solvent to form compound VI-1;

(a3) Reacting compound VI-1 with compound VII-1 in an inert solvent to form compound VIII-1;

in the formulae, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ And Z is as defined above;

or the method (a'): the compound of formula A is a compound of formula XIII-3, the method comprising the steps of:

(a' 1) subjecting compound IX-3 to a hydrolysis reaction in an inert solvent, thereby forming compound X-3;

(a' 2) reacting compound X-3 with compound VII-1 in an inert solvent to form compound XI-3;

(a' 3) subjecting compound XI-3 to a reduction reaction in an inert solvent, thereby forming compound XII-3;

(a' 4) reacting compound X-3 with compound IV-1 in an inert solvent to form compound XIII-3;

in the formulae, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ Z and M are as defined above;

or method (b): the compound of formula a is a compound of formula VIII-2, the method comprising the steps of:

(b1) Reacting compound VII-2 with hydroxylamine in an inert solvent to form compound VIII-2; in the formulae, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ And Z is as defined above;

or process (c): a compound of formula a is a compound of formula IX-2, the method comprising the steps of:

(c1) Reacting compound VII-2 with a fluorinating agent in an inert solvent to form compound IX-2; in the formulae, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ And Z is as defined above;

or method (d): a compound of formula a is a compound of formula IX-3, the method comprising the steps of:

(d1) Reacting compound V-3 with a sulfonyl chloride in an inert solvent to form compound VI-3;

(d2) Reacting the compound VI-3 with HNR in an inert solvent ₁ R ₂ Reacting to form compound VII-3;

(d3) Hydrolyzing compound VII-3 in an inert solvent to form compound VIII-3;

(d4) Reacting compound VIII-3 with compound VII-1 in an inert solvent to form compound IX-3;

in the formulae, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ And R ₆ The definition of (1) is as before;

or process (e): the compound of formula a is a compound of formula VI-5, the method comprising the steps of:

(e1) Reacting compound I-5 with a fluorinating agent in an inert solvent to form compound II-5;

(e2) Subjecting compound II-5 to a hydrolysis reaction in an inert solvent to form compound III-5;

(e3) Reacting the compound III-5 with R in an inert solvent ₁ R ₂ NH, thereby forming compound IV-5;

(e4) Subjecting compound IV-5 to a hydrolysis reaction in an inert solvent to form compound V-5;

(e5) Reacting compound V-5 with compound VII-1 in an inert solvent to form compound VI-5;

in the formulae, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ And R ₆ The definition of (1) is as before.

In another preferred embodiment, the compound of formula VII-2 can be prepared by using the compound of formula I-2 as a starting material. The preparation method specifically comprises the following steps:

are of the formulaeIn, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ And Z is as defined above.

In another preferred embodiment, the compound of formula V-3 can be prepared by using the compound of formula I-3 as a starting material.

The preparation method specifically comprises the following steps:

in the formulae, R ₃ The definition of (1) is as before.

In a third aspect of the invention, there is provided an intermediate represented by the formula, or a stereoisomer, cis-trans isomer or tautomer thereof,

in the formulae, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ And Z is as defined above.

In another preferred embodiment, the intermediate represented by the formula V-1 is

In another preferred embodiment, the intermediate represented by the formula VI-1 is

In a fourth aspect, the present invention provides a pharmaceutical composition comprising (1) a compound of the first aspect, or a stereoisomer, cis-trans-isomer, 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: immunomodulators (e.g., interferon-alpha (IFN-alpha), pegylated interferon-alpha) or stimulators 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, tenofovir Alafenamide (TAF), lamivudine, adefovir, entecavir, and telbivudine.

In a fifth aspect, the present invention provides a use of the compound of the first aspect, or a stereoisomer, a cis-trans isomer, or a tautomer thereof, or a pharmaceutically acceptable salt, a hydrate, or a solvate thereof, or the pharmaceutical composition of the fourth aspect, for the preparation of a medicament for the prevention and/or treatment of hepatitis b virus infection.

In a sixth aspect, the present invention provides a method for treating hepatitis b, comprising administering to a subject in need thereof a compound of the first aspect, or a stereoisomer, a cis-trans isomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition of the fourth aspect.

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 class of compounds having an excellent therapeutic effect on hepatitis b. On the basis of this, the inventors have completed the present invention.

Definition of

As used herein, the term "alkyl" includes straight or branched chain alkyl groups. For example, C1-C8 alkyl represents a straight or branched chain alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.

As used herein, the term "alkenyl" includes straight or branched chain alkenyl groups. For example, C2-C6 alkenyl means a straight or branched chain 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. For example, C2-C6 alkynyl means straight or branched chain alkynyl having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

As used herein, the term "C3-C10 cycloalkyl" refers to a cycloalkyl group having 3 to 10 carbon atoms. It may be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may also be in the form of a double ring, such as a bridged or spiro ring.

As used herein, the term "C1-C8 alkylamino" refers to an amino group substituted with a C1-C8 alkyl group, which may be mono-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-C8 alkoxy" refers to a straight or branched chain alkoxy group having 1-8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy and the like.

As used herein, "3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the following groups N, S and O" refers to a saturated or partially saturated cyclic group having 3-10 atoms and in which 1-3 atoms are heteroatoms selected from the following groups N, S and O. It may be monocyclic or may be in the form of a double ring, for example in the form of 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-C10 aryl" refers to an aryl group having 6-10 carbon atoms, for example, phenyl or naphthyl and the like.

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

Unless specifically stated to be "substituted or unsubstituted", the groups of the present invention may be substituted with a substituent selected from the group consisting of: halogen, nitrile group, nitro group, hydroxyl group, amino group, C1-C6 alkyl-amino group, C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C1-C6 alkoxy group, halogenated C1-C6 alkyl group, halogenated C2-C6 alkenyl group, halogenated C2-C6 alkynyl group, halogenated C1-C6 alkoxy group, allyl group, benzyl group, C6-C12 aryl group, C1-C6 alkoxy-C1-C6 alkyl group, C1-C6 alkoxy-carbonyl group, phenoxycarbonyl group, C2-C6 alkynyl-carbonyl group, C2-C6 alkenyl-carbonyl group, C3-C6 cycloalkyl-carbonyl group, C1-C6 alkyl-sulfonyl group and the like.

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

Unless otherwise specified, the structural formulae depicted herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, the R and S configuration containing asymmetric center, the (Z) and (E) isomers of double bond, etc. 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 compound of the present invention coordinated to solvent molecules to form a complex in a specified 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 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.

Preparation method

The compounds of the present invention can be prepared according to methods commonly used in the art. Can also be prepared according to the scheme route given below by using the conventional synthesis conditions (such as reaction temperature or reaction time). The reaction temperature and the reaction time can be determined by those skilled in the art according to the conventional reaction conditions for the reaction. The reaction temperature can be-78 ℃ to reflux; preferably-20 deg.C to reflux or 0-100 deg.C. The reaction time may be shorter than 0.1 hour to 3 days, or 0.1 hour to 24 hours, or 0.1 hour to 5 hours.

The first scheme is as follows:

(1) Carrying out nitration reaction on the compound I-1 in an inert solvent at a certain temperature for a period of time to form a compound II-1;

(2) Carrying out reduction reaction on the compound II-1 in an inert solvent at a certain temperature for a certain period of time to form a compound III-1;

(3) Reacting compound III-1 with compound IV-1 in an inert solvent at a temperature and for a time to form compound V-1;

(4) Hydrolyzing compound V-1 in an inert solvent at a temperature and for a time to form compound VI-1; and

(5) Reacting compound VI-1 with compound VII-1 in an inert solvent at a temperature and for a time to form compound VIII-1;

Scheme two and scheme three:

(i) Hydrolyzing compound I-2 in an inert solvent at a temperature and for a period of time to form compound II-2;

(ii) Reacting compound II-2 with an aniline compound in an inert solvent at a temperature and for a time to form compound III-2;

(iii) Reacting compound III-2 with compound IV-2 in an inert solvent at a temperature and for a time to form compound V-2;

(iv) Hydrolyzing compound V-2 in an inert solvent at a temperature and for a time to form compound VI-2;

(v) In an inert solvent, at a certain temperature, the compound VI-2 is mixed with HNR ₁ R ₂ To carry out the inverseFor a time period to form compound VII-2; and

(vi-1) reacting compound VII-2 with hydroxylamine in an inert solvent at a temperature and for a time to form compound VIII-2; or

(vi-2) reacting compound VII-2 with a fluorinating agent in an inert solvent at a temperature and for a time to form compound IX-2;

In another preferred embodiment, the fluorinating agent is a nucleophilic fluorinating agent capable of converting a carbonyl group into a difluoromethylene group, such as DAST, BAST, ishikawa reagent 1-fluoropyridine tetrafluoroborate and the like.

And the scheme is as follows:

(a) Reacting the compound I-3 with methylhydrazine in an inert solvent at a certain temperature for a period of time to form a compound II-3;

(b) Carrying out bromination reaction on the compound II-3 in an inert solvent at a certain temperature for a certain period of time to form a compound III-3;

(c) Reacting compound III-3 in an inert solvent at a temperature and for a time to form compound IV-3;

(d) Subjecting compound IV-3 to an amination reaction in an inert solvent at a temperature and for a time to form compound V-3;

(e) Reacting compound V-3 with sulfonyl chloride in an inert solvent at a temperature and for a time to form compound VI-3;

(f) In an inert solvent, at a certain temperature, the compound VI-3 is mixed with HNR ₁ R ₂ Conducting the reaction for a time period to form compound VII-3;

(g) Hydrolyzing compound VII-3 in an inert solvent at a temperature and for a time to form compound VIII-3;

(h) Reacting compound VIII-3 with aniline in an inert solvent at a temperature and for a time to form compound IX-3;

And a fifth scheme:

in the formulae, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ Z and M are as defined above.

Scheme six:

Intermediate product

Intermediates useful in the preparation of the compounds of the present invention include the following compounds:

The compound of the formula V-1 can be prepared by taking the compound of the formula I-1 as a starting material. The preparation method specifically comprises the following steps:

in the formulae, R ₁ 、R ₂ 、R ₃ And Z is as defined above.

The compound of the formula VI-1 can be prepared by using the compound of the formula I-1 as a starting material. The preparation method specifically comprises the following steps:

in the formulae, R ₁ 、R ₂ 、R ₃ And Z is as defined above.

The compound of the formula VII-3 can be prepared by taking the compound of the formula I-1 as a starting material. The preparation method specifically comprises the following steps:

in the formulae, R ₁ 、R ₂ And R ₃ The definition of (1) is as before.

The compound of the formula VIII-3 can be prepared by using the compound of the formula I-3 as a starting material. The preparation method specifically comprises the following steps:

in the formulae, R ₁ 、R ₂ And R ₃ The definition of (1) is as before.

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, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.)

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

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

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

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

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used 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 using pharmaceutical compositions, a safe and effective amount of a compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the administration is a pharmaceutically acceptable and effective dose, and the daily dose for a human of 60kg body weight is usually 1 to 2000mg, preferably 20 to 500mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention include:

the compound of the invention has novel structure and excellent anti-hepatitis B virus infection effect.

The compounds of the present invention are effective in inhibiting the assembly of hepatitis B virus nucleocapsid, and thus are effective in inhibiting hepatitis B virus.

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

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.

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).

The compounds of the invention show superior dissolution properties and good pharmacokinetic properties over the reference compound.

The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions, or according to conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The materials and equipment used in the examples of the present invention are commercially available unless otherwise specified.

Example 1: synthesis of Compound 8

Step 1: synthesis of Compound 2

Substrate 1 (1.0 g) was dissolved in acetic anhydride (2 mL), the temperature of the system was lowered to 0 ℃, fuming nitric acid (0.5 mL) was then added to the reaction system, the reaction was carried out at 0 ℃ for 3 hours, and methyl iodide (668 mg) was then added to the reaction system, and the reaction was carried out at room temperature for 3 hours. TLC detected the starting material was completely reacted, water (20 mL) was added to the reaction system, ethyl acetate (3 × 30ml) was extracted, the organic phase was dried, spun dried and crude column chromatographed (n-heptane: ethyl acetate =1. MS (M +1= 219).

Step 2: synthesis of Compound 3

Substrate 2 (0.4 g) was dissolved in acetic acid (5 mL), iron powder (1.8 g) was added to the reaction system at room temperature, reaction was carried out at room temperature for 3h, starting material reaction was detected by tlc, after drying acetic acid, pH of the residue was adjusted to 9-10, ethyl acetate (3 x 25ml) was extracted, organic phase was dried, dried by spinning, crude column chromatography (n-heptane: ethyl acetate = 1) was carried out to obtain product 3 (200 mg. MS (M +1= 189).

And step 3: synthesis of Compound 5

Substrate 3 (0.2 g) was dissolved in acetonitrile (8 mL), triethylamine (0.6 g) was added to the reaction system at room temperature, then compound 4 (0.35 g) was added to the reaction system, heating and refluxing were performed for 8h, tlc detected that the raw materials reacted completely and a new spot was generated, water (20 mL) was added to the reaction system, ethyl acetate (3 × 25ml) was extracted, the organic phase was dried and spun, and crude column chromatography (n-heptane: ethyl acetate = 1) was performed to obtain product 5 (80 mg. ¹ H-NMR(CDCl ₃ ,400MHz)δ:1.33(d,J＝6.8Hz,3H)，3.85(s,3H),3.86(s,3H),3.95-4.02(m,1H),4.80(d,J＝9.2Hz,1H)，6.19(s,1H),6.90(s,1H).

And 4, step 4: synthesis of Compound 6

Substrate 5 (80 mg) was dissolved in tetrahydrofuran (2 mL), methanol (0.5 mL) and water (0.5 mL), and then lithium hydroxide (0.21 g) was added to the reaction system, followed by reaction at room temperature for 8h, and a small amount of starting material remained and a new spot was formed by TLC. Water (20 mL) was added to the reaction system, the pH was adjusted to 3-4 with 1N hydrochloric acid, dichloromethane (3 × 25ml) was extracted, the organic phase was dried over anhydrous sodium sulfate, spun dry, and the crude product was isolated by column chromatography (N-heptane: ethyl acetate = 1) to give compound 6 (50 mg) as a yellow solid. MS (M +1= 350).

And 5: synthesis of Compound 8

Dissolving the substrate 6 (50 mg) in DMF (36 mL), adding the compound 7 (20 mg) and DIPEA (74 mg) into the reaction system, adding HATU (78 mg) into the reaction system at 0 ℃ for reaction for 5h at about 30 ℃, and detecting by TLC that the raw materials are completely reacted to obtainAnd generating a new point. 20mL of saturated brine was added to the reaction system, extracted with ethyl acetate (3X 20mL), the organic phase was dried over anhydrous sodium sulfate, and dried by spinning, and the crude high performance liquid phase (n-heptane: ethyl acetate = 1) was separated to obtain Compound 8 (5 mg) as a pink solid. ¹ H-NMR(CDCl ₃ ,400MHz)δ:1.16(d,J＝6.8Hz,3H)，3.76(s,3H),3.92-3.99(m,1H)，7.52-7.57(m,1H),7.67(s,1H),7.94-7.98(m,1H),8.16-8.18(m,1H),8.49(br,1H)10.68(s,1H).MS(M+1＝468)。

EXAMPLE 2 Synthesis of Compound 8a

According to the step 5, the target product 8a is obtained by only replacing the compound 7 with the compound 7a and performing high performance liquid chromatography on the mixture under the same other conditions. MS (M +1= 461).

EXAMPLE 3 Synthesis of Compound 8b

According to the step 5, the target product 8b is obtained by high performance liquid chromatography separation only by replacing the compound 7 with the compound 7b without changing other conditions. MS (M +1= 457).

EXAMPLE 4 Synthesis of Compound 8c

According to the step 5, the target product 8c is obtained by high performance liquid chromatography separation with the compound 7 replaced by the compound 7c and other conditions unchanged. MS (M +1= 477).

EXAMPLE 5 Synthesis of Compound 8d

According to the step 5, only the compound 7 needs to be replaced by the compound 7d, other conditions are not changed, and the target product 8d is obtained through high performance liquid chromatography separation. MS (M +1= 495).

EXAMPLE 6 Synthesis of Compound 18

Step 11: synthesis of Compound 12

Substrate 11 (0.2 g) was dissolved in tetrahydrofuran (2 mL), methanol (0.5 mL) and water (0.5 mL), then lithium hydroxide (0.24 g) was added to the reaction system, and the reaction was carried out at room temperature for 8h, and a small amount of starting material remained and a new spot was formed by TLC. Water (20 mL) was added to the reaction system, pH adjusted to 3-4 with 1N hydrochloric acid, dichloromethane (3 x 25ml) extracted, the organic phase dried over anhydrous sodium sulfate, spun dry and the crude product isolated by column chromatography (N-heptane: ethyl acetate = 1) to yield compound 12 (0.16 g). MS (M +1= 160).

Step 12: synthesis of Compound 13

Substrate 12 (120 mg) was dissolved in DMF (3 mL), then Compound 12a (112 mg), DIPEA (292 mg) was added to the reaction system, HATU (372 mg) was added to the reaction system at 0 ℃ and reacted for 5h at about 30 ℃ and the starting material was reacted completely by TLC to detect the formation of a new spot. Saturated brine (20 mL) was added to the reaction system, extracted with ethyl acetate (3 × 20ml), the organic phase was dried over anhydrous sodium sulfate, spin-dried, and the crude product was separated by column chromatography (n-heptane: ethyl acetate = 1) to give compound 13 (90 mg) as a yellow solid. MS (M +1= 278).

Step 13: synthesis of Compound 15

Dissolving a substrate 13 (90 mg) in DCM (3 mL), reducing the temperature of the system to 0 ℃, then adding a dichloromethane solution of a compound 14 (112 mg) into the reaction system, stirring at 0 ℃ for 30min, then adding aluminum trichloride (144 mg) into the reaction system in batches, reacting at 0 ℃ for 3h, detecting by TLC that the raw materials are completely reacted, and generating new spots. The reaction solution was poured into ice water (10 mL), dichloromethane (3 × 25ml) was extracted, the organic phase was washed with 20mL of saturated brine, dried over anhydrous sodium sulfate, spun-dried, and the crude product was isolated by column chromatography (n-heptane: ethyl acetate = 1) to give compound 15 (55 mg) as a yellow solid. MS (M +1= 378). ¹ H-NMR(CDCl ₃ ,400MHz)δ:1.38-1.42(m,3H)，4.02(s,3H),4.35-4.41(m,1H)，7.19-7.21(m,1H),7.67-7.72(m,1H),7.92(s,1H),8.06-8.08(m,1H),8.66(br,1H).

Step 14: synthesis of Compound 16

The substrate 15 (50 mg) was dissolved in tetrahydrofuran (1 mL) and ethanol (3 mL), and then an aqueous solution of sodium hydroxide (16 mg) was added to the system, followed by reaction at 30 ℃ for 0.5h, and TLC detection to confirm that the starting material had reacted completely and a new spot was formed. Water (20 mL) was added to the reaction, pH adjusted to 3-4 with 1N hydrochloric acid, dichloromethane (3 x 25ml) extracted, organic phase dried over anhydrous sodium sulfate and spin dried to give compound 16 (0.16 g) as a yellow solid.

Step 15: synthesis of Compound 17

The substrate 16 (25 mg) was dissolved in DMF (2 mL), tert-butylamine (6.8 mg) and DIPEA (74 mg) were added to the reaction system, HATU (37 mg) was added to the reaction system at 0 ℃ and the reaction was completed at about 30 ℃ for 5h, and TLC was used to detect the completion of the reaction of the starting materials and the formation of new spots. Adding saturated brine (20 mL) into the reaction system, extracting with ethyl acetate (3X 20mL), drying the organic phase with anhydrous sodium sulfate, and concentratingDry, the crude product was separated by high performance liquid column (n-heptane: ethyl acetate = 1). ¹ H-NMR(CDCl ₃ ,400MHz)δ:1.41(s,9H)，4.00(s,3H),7.19-7.23(m,1H),7.69-7.77(m,1H),8.04-8.06(m,1H),8.54(br,1H)8.68(s,1H).Ms(ESI)m/z＝403(M-1)。

Step 16: synthesis of Compound 18

Substrate 17 (15 mg) was dissolved in ethanol (2 mL), and then hydroxylamine hydrochloride (13 mg) was added to the reaction system, and the reaction temperature was raised to 60 ℃ for 10 hours. After the reaction solution was spin-dried and the residual solid was dissolved in acetonitrile, compound 18-1 (1.8 mg), 18-2 (2.0 mg) was obtained by reverse phase separation with high performance liquid. 18-1 of ¹ H-NMR(CDCl ₃ 400 MHz) delta 1.42 (s, 9H), 3.96 (s, 3H), 6.54-6.57 (m, 1H), 7.16-7.20 (m, 1H), 7.69-7.72 (m, 1H), 8.02 (s, 1H) 8.53 (d, J =8.0Hz, 1H), 18-2 ¹ H-NMR(CDCl ₃ ,400MHz)δ:1.42(s,9H)，3.96(s,3H),6.54-6.57(m,1H),7.16-7.20(m,1H),7.69-7.72(m,1H),8.02(s,1H)8.53(d,J＝8.0Hz,1H).

EXAMPLE 7 Synthesis of Compound 18a

Compound 17a was prepared according to steps 11-15 except isopropylamine was used instead of tert-butylamine.

According to the step 16, the target product 18a is obtained by high performance liquid chromatography separation of the compound 17 only by replacing the compound 17 with the compound 17a without changing other conditions.

EXAMPLE 8 Synthesis of Compound 18b

Compound 17b was prepared according to steps 11-15, except that ethylamine was used instead of tert-butylamine.

According to the step 16, the target product 18 b) can be obtained by only replacing the compound 17 with the compound 17b and performing high performance liquid chromatography without changing other conditions.

EXAMPLE 9 Synthesis of Compound 18c

Preparation of Compound 17c according to steps 11-15, except that trifluoroisopropylamine (CH) is used ₃ (CF ₃ )CHNH ₂ ) Instead of tert-butylamine.

According to the step 16, the target product 18c is obtained by high performance liquid chromatography separation of the compound 17 by only replacing the compound 17 with the compound 17c under the same other conditions.

EXAMPLE 10 Synthesis of Compound 18d

Compound 17d was prepared according to steps 11-15 except cyclopropylamine was used instead of tert-butylamine.

According to the step 16, the compound 17 is replaced by the compound 17d, other conditions are not changed, and the target product 18d is obtained by high performance liquid chromatography separation.

EXAMPLE 11 Synthesis of Compound 19

Step 19-A

Compound 19-1 (1g, 5.8mmol, 1equiv) was dissolved in DCM (30 mL) and oxalyl chloride monoethyl ester (2g, 14.5mmol, 2.5euqiv) was added dropwise to the above solution under Ar protection and at 0 ℃. AlCl ₃ (3.9g，29mmol，5euqiv) add to the reaction mixture and stir overnight at room temperature. Water (2.25 mL) and EtOAc (7.5 mL) were added dropwise to the reaction solution while cooling on ice. The reaction was poured into ice water and diluted with dilute hydrochloric acid and extracted with EtOAc. The organic phase was dried and concentrated to give MPLC (heptane: etOAc = 50) as a brown oil LW107-44-56 (125 mg). ¹ H NMR (400 MHz, chloroform-d) δ 7.80 (s, 1H), 4.37 (q, J =7.1hz, 2h), 3.95 (s, 3H), 3.91 (s, 3H), 1.40 (t, J =7.2hz, 3h).

Step 19-B

19-2 (100mg, 0.38mmol, 1equiv) and DAST (1.2g, 7.3mmol, 20equiv) were dissolved in DCM (5 mL) and stirred overnight at 40 ℃ under argon. Pouring the reaction solution into NaHCO ₃ Saturated aqueous solution and extraction with DCM, organic phase washed with saturated brine and dried to give LW107-44-73 (35 mg) as a brown oil-solid by preparative TLC (heptane: etOAc = 3. ¹ H NMR (400 MHz, chloroform-d) δ 7.04 (s, 1H), 4.35 (q, J =7.1hz, 2h), 3.90 (d, J =7.9hz, 6H), 1.34 (t, J =7.1hz, 3h).

Step 19-C

19-3(60mg，0.2mmol，1equiv)，LiOH H ₂ O (9mg, 0.2mmol, 1equiv) at 5 ℃ under Dioxane: h ₂ O (5mL. The reaction was separated by adding water and EtOAc, the aqueous phase was acidified to pH 2-3 with concentrated hydrochloric acid in ice bath and extracted with EtOAc and then Na ₂ SO ₄ Dried and concentrated to give LW107-44-83 (40 mg) as a white solid. ¹ H NMR(400MHz,DMSO-d ₆ )δ7.53(s,1H),3.85(s,3H),3.80(s,3H).MS(ESI ⁺ ):m/z calcd.for[M+H] ⁺ :268,found:268.

Step 19-D

19-4 (1235mg, 4.63mmol, 1equiv), tert-butylamine (1350mg, 18.5mmol, 4equiv), DIPEA (3g, 23.2mmol, 5euqiv) were dissolved in DMF (60 mL). HATU (3581mg, 9.26mmol, 2equiv) was added to the above reaction solution at 0 ℃ and allowed to return to room temperature overnight. The reaction mixture was separated by adding brine and EtOAc, and the organic phase was washed with brine and concentrated. From MPLC (heptane: etOAc =88 21-82) we obtained LW107-44-87 (960 mg) as a white solid. ¹ H NMR (400 MHz, chloroform-d) δ 7.45 (s, 1H), 4.08 (d, J =1.7hz, 6H), 1.61 (s, 9H). MS (ESI) ⁺ ):m/z calcd.for[M+H] ⁺ :323,found:323.

Step 19 to E

19-5 (300mg, 0.93mmol, 1equiv) and LiOH H ₂ O (313mg, 7.45mmol, 8equiv) in MeOH: THF: water (3. The reaction was separated by adding water and EtOAc, the aqueous phase was acidified to pH 2-3 with concentrated hydrochloric acid in ice bath and extracted with EtOAc and then Na ₂ SO ₄ Dried and concentrated to give LW107-68-5 (258 mg) as a white solid. ¹ H NMR(400MHz,DMSO-d ₆ )δ13.05(s,1H),8.00(s,1H),7.36(s,1H),3.84(s,3H),1.32(s,9H).MS(ESI ⁺ ):m/z calcd.for[M+H] ⁺ :309,found:309.

Step 19-F

19-6 (80mg, 0.26mmol, 1equiv), 3-fluoro-4-cyano-aniline (71mg, 0.52mmol, 2equiv) and TEA (131mg, 1.3mmol, 5equiv) were dissolved in DCM (5 mL). TBTU (167mg, 0.52mmol, 2equiv) was added at 0 ℃ and allowed to warm to room temperature and stir overnight. The reaction solution was acidified by 8% hydrochloric acid and separated with DCM, and the organic phase was NaHCO ₃ The solution was washed and MPLC (Heptane: etOAc = 70. Preparation of LC (water: ACN =45, c-18, det =260nm, peak =50min, v = 17ml/min) lyophilized to give 19 (25 mg) as a white solid. ¹ H NMR (400 MHz, chloroform-d) δ 8.11 (d, J =8.5hz, 1h), 7.64-7.40 (m, 3H), 7.36 (s, 1H), 6.47 (s, 1H), 3.95 (s, 3H), 1.46 (s, 9H).

EXAMPLE 13 Synthesis of Compound 19b

Compound 19-6b was prepared according to step 19-A-19-E, except that ethylamine was used instead of tert-butylamine.

According to the synthesis of the step 19-F, only the compound 19-6 is replaced by the compound 19-6b, other conditions are not changed, and the target product 19b is obtained through high performance liquid chromatography separation.

EXAMPLE 14 Synthesis of Compound 19c

Preparation of Compounds 19-6c according to steps 19-A-19-E, except that Trifluoroisopropylamine (CH) ₃ (CF ₃ )CHNH ₂ ) Instead of tert-butylamine.

According to the synthesis of the step 19-F, only the compound 19-6 is replaced by the compound 19-6c, other conditions are not changed, and the target product 19c is obtained by high performance liquid chromatography separation.

EXAMPLE 15 Synthesis of Compound 19d

Compounds 19-6d are prepared according to steps 19-A-19-E except cyclopropylamine is used instead of tert-butylamine.

According to the synthesis of the step 19-F, only the compound 19-6 is replaced by the compound 19-6d, other conditions are not changed, and the target product 19d is obtained through high performance liquid chromatography separation.

EXAMPLE 16 Synthesis of Compound 20

Step 18: synthesis of Compound 22

Dissolving a compound methylhydrazine (10 g) in methanol (20 mL) and water (10 mL), cooling to 0 ℃, stirring for 15min, then dropwise adding a compound 21 (12 g) into a reaction system, heating to 70 ℃, reacting for 10h, then returning the system temperature to room temperature, reacting for 12h, carrying out suction filtration, and washing a filter cake with water (5 mL) to obtain 22 g of light yellow solid, wherein MS (M +1= 157).

Step 19: synthesis of Compound 23

Substrate 22 (5 g) was dissolved in DMF (10 mL), phosphorus oxychloride (10 mL) was added to the reaction system at room temperature, warmed to 100 ℃, reacted for 5h, the reaction system was poured into ice water, extracted with ethyl acetate (3 × 30ml), the organic phase was dried, spun dried, and crude column chromatography (n-heptane: ethyl acetate = 1) gave product 23 (2 g. MS (M +1= 219).

Step 20: synthesis of Compound 24

Substrate 23 (2 g) was dissolved in acetonitrile (10 mL), NBS (4 g) was added to the reaction system at room temperature, then the system temperature was raised to 60 degrees, water (20 mL) was added to the reaction system, ethyl acetate (3 × 25ml) was extracted, the organic phase was dried, spun dry, crude column chromatography (n-heptane: ethyl acetate = 1) gave product 5 (1 g. MS (M +1= 299).

Step 21: synthesis of Compound 25

The substrate 24 (1 g) was dissolved in dichloromethane (10 mL), then aqueous ammonia (5 mL) was added to the reaction system, reacted at room temperature for 2h, water (20 mL) was added to the reaction system, pH was adjusted to 3-4 with 1N hydrochloric acid, dichloromethane (3 x 25ml) was extracted, the organic phase was dried over anhydrous sodium sulfate, spun dry, and the crude product was isolated by column chromatography (N-heptane: ethyl acetate = 1) to give a yellow solid compound 25 (300 mg. MS (M +1= 234).

Step 22: synthesis of Compound 26

After dissolving the substrate 25 (300 mg) in dichloromethane (3 mL), sulfonyl chloride (1 mL) was added to the reaction system, and reacted at 40 ℃ for 5 hours, saturated brine (20 mL) was added to the reaction system, extracted with ethyl acetate (3 × 20ml), dried over anhydrous sodium sulfate as an organic phase, dried by spinning, and subjected to column chromatography (n-heptane: ethyl acetate = 1) to obtain a yellow solid compound 26 (150 mg). MS (M +1= 332).

Step 23: synthesis of Compound 27

Substrate 26 (150 mg) was dissolved in acetonitrile (5 mL), triethylamine (0.1 mL) was added to the reaction system, and then trifluoromethylethylamine hydrochloride (0.5 g) was added to the reaction system, and the reaction was carried out at 80 ℃ for 5 hours, and saturated brine (20 mL) was added to the reaction system, followed by extraction with ethyl acetate (3 × 20ml), drying over anhydrous sodium sulfate for the organic phase, spin-drying, and column chromatography (n-heptane: ethyl acetate = 1) to isolate compound 27 (120 mg). MS (M +1= 409).

And step 24: synthesis of Compound 28

The substrate 27 (120 mg) was dissolved in tetrahydrofuran (2 mL), water (0.5 mL) and methanol (0.5 mL), then lithium hydroxide monohydrate (100 mg) was added to the reaction system, followed by reaction at 40 ℃ for 8h, pH was adjusted to 3-4 with 1N hydrochloric acid, ethyl acetate (3 × 20ml) was extracted, the organic phase was dried over anhydrous sodium sulfate, spin-dried, and column chromatography (N-heptane: ethyl acetate = 1) was performed to isolate the compound 27 (80 mg) as a yellow solid. MS (M +1= 395).

Step 25: synthesis of Compound 20

Substrate 28 (80 mg), compound 29 (50 mg) and DIPEA (70 mg) were dissolved in DMF (5 mL), then HATU (130 mg) was added to the reaction system, followed by reaction at 40 ℃ for 8h, water (20 mL) was added, ethyl acetate (3 × 20ml) was extracted, the organic phase was dried over anhydrous sodium sulfate, spin-dried, and column-chromatographed (n-heptane: ethyl acetate = 1) to isolate compound 20 (10 mg) as a yellow solid.

EXAMPLE 17 Synthesis of Compound 20b

Compound 29 was substituted with 29b according to step 25, and otherwise unchanged, column chromatography (n-heptane: ethyl acetate = 1) was performed to isolate compound 20b (8 mg) as a yellow solid.

EXAMPLE 18 Synthesis of Compound 20c

Compound 29 was substituted with 29c according to step 25, and otherwise unchanged, column chromatography (n-heptane: ethyl acetate = 1) was performed to isolate compound 20c (11 mg) as a yellow solid.

EXAMPLE 19 Synthesis of Compound 30a

Step 26 Synthesis of Compound 32

Substrate 31 (0.8 g) was dissolved in tetrahydrofuran (12 mL), methanol (3 mL) and water (3 mL), then sodium hydroxide (0.44 g) was added to the system, reaction was carried out at 55 ℃ for 1h, TLC detected that the starting material had reacted completely and was newly formed. Adjusting pH to 3-4 with 1N hydrochloric acid, adding 20mL of water into the reaction system, extracting with ethyl acetate (3 × 20mL), drying with anhydrous sodium sulfate, spin-drying, and separating the crude product by column chromatography to obtain a yellow solid 32 (0.5 g).

Step 27 Synthesis of Compound 33

Dissolving a substrate 32 (100 mg) in DMF (3 mL), adding 4-F-3-cyano-aniline (40 mg) and DIPEA (84 mg) into the reaction system, adding TBTU (78 mg) into the reaction system at 0 ℃, reacting for 5h at room temperature (30 ℃), and detecting that the raw materials are completely reacted and new spots are generated by TLC. 20mL of saturated brine was added to the reaction system, and extraction was performed with ethyl acetate (3X 20mL), followed by drying over anhydrous sodium sulfate, spin-drying, and crude product column chromatography to give 33 (100 mg) as a yellow solid.

Step 28 Synthesis of Compound 34

Dissolving a substrate 33 (0.3 g) in acetic acid (5 mL), adding iron powder (1.0 g) into the reaction system at room temperature, reacting for 3h at room temperature, detecting the reaction of raw materials by TLC, drying acetic acid to adjust the pH value to 9-10, extracting ethyl acetate (3 x 45mL), drying and drying, and performing column chromatography to obtain a product 0.15g.

Step 29 Synthesis of Compound 30a

Compound 34 (80 mg) and compound 34a (63 mg) were dissolved in acetonitrile (3 mL), triethylamine (83 mg) was added, the reaction was raised to 85 ℃ and reacted for 8h, water (10 mL) was added to the reaction, ethyl acetate (3 × 20ml) was extracted, dried over anhydrous sodium sulfate, the organic phase was dried by spinning, and the high performance liquid phase was prepared as a white solid (8 mg). Ms/ESI =411.6 (M-1). ¹ H-NMR(CDCl ₃ ,400MHz)δ:1.11(d,J＝6.4Hz，6H)，3.41-3.49(m,1H),3.74(s,3H)，6.93(d,J＝7.2Hz，1H),7.06(s,1H),7.52-7.56(m,1H),7.96-8.00(m,1H),8.19-8.21(m,1H),8.76(s,1H)，10.31(s,1H)Ms/ESI＝411.6(M-H).

EXAMPLE 20 Synthesis of Compound 30b

Compound 34a was substituted with 34b according to step 29, and the other was left unchanged and column chromatographed (n-heptane: ethyl acetate = 1) to isolate compound 30b (11 mg) as a yellow solid. ¹ H-NMR(CDCl ₃ ,400MHz)δ:10.33(s,1H),8.94(s,1H),8.20(dd,J＝5.8,2.6Hz,1H),7.99(ddd,J＝9.1,4.8,2.7Hz,1H),7.60(s,1H),7.54(t,J＝9.1Hz,1H),7.12(s,1H),4.72(d,J＝6.0Hz,2H),4.19(d,J＝6.1Hz,2H),3.75(s,3H),1.65(s,3H).

EXAMPLE 21 Synthesis of Compound 30c

Compound 34a was substituted with 34c according to step 29, and otherwise unchanged, column chromatography (n-heptane: ethyl acetate = 1) was performed to isolate compound 30c (11 mg) as a yellow solid. ¹ H-NMR(CDCl ₃ ,400MHz)δ:10.33(s,1H),8.94(s,1H),8.20(dd,J＝5.8,2.6Hz,1H),7.99(ddd,J＝9.1,4.8,2.7Hz,1H),7.60(s,1H),7.54(t,J＝9.1Hz,1H),7.12(s,1H),4.72(d,J＝6.0Hz,2H),4.19(d,J＝6.1Hz,2H),3.75(s,3H),1.65(s,3H).

Other compounds in table 1 were synthesized according to the synthesis of compounds 8, 18, 19, 20 and 30a of the present invention.

TABLE 1

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:

add 150. Mu.L 2% w/v skim milk to each well of 96-well plate and incubate for 2 hours at room temperature. Sucking off the skimmed milk, washing with deionized water, drying, and storing at room temperature. The C150 protein (3 mg per tube) was desalted using 5ml hitrap desalting column. 50mM was added to the desalted C150 protein per tube

20 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 the content of the first and second substances,

indicating the concentration of the fluorescent label;

[ C150Bo ] represents 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 ^-1 means of moleReciprocal of molar concentration.

Compound dilution:

the compound stock solution was diluted to 6mM with DMSO, then to 600. Mu.M with 50mM HEPES, and then further serially diluted 3-fold with 10% DMSO/50mM HEPES for 8 concentrations.

C150Bo was diluted to 2. Mu.M with 50mM HEPES. 37.5. Mu.L of C150Bo and 2.5. Mu.L of each concentration of compound were added to a 96-well reaction plate 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 was added to a final concentration of 0mM.

Control wells were assembled with 100% protein, 10. Mu.L of 5M NaCl/50mM HEPES, 1M NaCl final concentration was added.

The final concentration of DMSO was 0.5%, the maximum final concentration of 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 485nm; emission 535 nm).

Data analysis

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

IC ₅₀ The values were calculated by prism software, the equation is as follows:

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

wherein the content of the first and second substances,

x represents the log of the concentration, Y represents the effect value, and Y fits to the top in sigmoid form starting from the bottom;

bottom represents the Bottom of the curve;

top indicates Top of the curve;

HillSlope represents: 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 and cytotoxicity experiments all compounds were serially diluted 3-fold at 8 concentrations. The final starting concentration of test compound was 30. Mu.M, the final starting concentration of reference compound GLS4 was 1. Mu.M, and the final concentration of DMSO was 0.5%.

HepG2.2.15 cell (4X 10) ⁴ Cells/well) to 96-well plates, at 37 ℃ 5% ₂ The 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)} )；

wherein the content of the first and second substances,

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

The results of the above experiments are shown in table 2.

Table 2 activity data of compounds

In the table: a1 represents an IC50 (mu M) of 0.001 to 10;

a2 represents an IC50 (mu M) of 10 to 100;

a3 represents an IC50 (μ M) of between 100 and 10000;

b1 represents an EC50 (. Mu.M) of 0.001 to 1;

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

wherein the control compound is:

(see WO2014184350 A1).

In addition, the solubility and the pharmacokinetic performance of the compound of the invention are better than those of the control compound through conventional solubility experiments and pharmacokinetic experiments.

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 a formula A or a pharmaceutically acceptable salt thereof,

A

wherein the content of the first and second substances,

the compound has a structure selected from the group consisting of:

。

2. a process for preparing a compound of claim 1, or a pharmaceutically acceptable salt thereof,

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

or method (a'): the compound of formula A is a compound of formula XIII-3, the method comprising the steps of:

3. A pharmaceutical composition comprising (1) a compound of claim 1, or a pharmaceutically acceptable salt thereof; (2) a pharmaceutically acceptable carrier.

4. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of claim 3 for the manufacture of a medicament for the prevention and/or treatment of hepatitis b virus infection.