CN117126163A

CN117126163A - Benzo heterocyclic compound, and preparation method and application thereof

Info

Publication number: CN117126163A
Application number: CN202210568356.XA
Authority: CN
Inventors: 李瑛颖; 冀斌; 叶兆丰; 杨子翊; 陈明键
Original assignee: China Israel Hyde Artificial Intelligence Drug Research And Development Co ltd
Current assignee: China Israel Hyde Artificial Intelligence Drug Research And Development Co ltd
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2023-11-28

Abstract

The application provides a benzo heterocyclic compound, a pharmaceutical composition, a preparation method and application thereof, and application thereof in treating hepatitis B or tumors, and also provides application of a compound shown in a general formula I or pharmaceutically acceptable salts, solvates, hydrates, crystal forms and stereoisomers thereof in preparing a medicament for treating or preventing hepatitis B. The application also provides a pharmaceutical composition for use in the treatment of hepatitis B or tumors comprising a compound of formula IPharmaceutically acceptable salts, solvates, hydrates, crystal forms, stereoisomers thereof, and optionally one or more additional therapeutic and/or prophylactic agents, and optionally one or more pharmaceutically acceptable carriers.

Description

Benzo heterocyclic compound, and preparation method and application thereof

Technical Field

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a benzo heterocyclic compound, a pharmaceutical composition, a preparation method and application thereof.

Background

Hepatitis b is an infectious disease caused by Hepatitis B Virus (HBV) and is mainly a liver lesion. Clinically, it is mainly manifested by anorexia, nausea, epigastric discomfort, pain in liver region and hypodynamia. Some patients may have jaundice fever and liver large with liver function impairment. Some patients may become chronicized, even develop cirrhosis, and a few may develop liver cancer.

The treatment of hepatitis B is a long-term process, and the treatment aims to inhibit or eliminate HBV to the maximum extent, reduce inflammation necrosis of liver cells and hepatic fibrosis, delay and prevent disease progression, reduce and prevent liver decompensation, cirrhosis, hepatocellular carcinoma and complications thereof, thereby improving life quality and prolonging survival time.

There are many hepatitis b therapeutic drugs on the market at present, and antiviral treatment is mainly performed by using interferon or nucleoside analogues. For interferon, recombinant DNA leukocyte interferon (IFN-. Alpha.) can inhibit HBV replication. However, when interferon is used for treating hepatitis B, serious adverse reactions, including myelosuppression, influence on thyroid function, depression and the like are often accompanied.

Nucleoside analogs inhibit HBV production primarily by inhibiting reverse transcriptase activity during HBV replication, clinically useful drugs include: lamivudine, famciclovir, acyclovir, adefovir, entecavir, tenofovir, sodium phosphonoformate and the like, which have certain HBV inhibiting effect.

Although these reverse transcriptase inhibitors can effectively reduce HBV DNA level and control HBV level, they have no direct effect on HBV cccDNA and HBsAg clearance because the action target is RNA reverse transcription to DNA. Therefore, the single-drug treatment of nucleoside analogues has extremely low probability of seroconversion of HBsAg, and can not truly cure hepatitis B, and patients need to take medicines for a long time or even for a whole life.

Under the condition of taking the medicines for a long time, the problems of drug resistance, huge medical cost, serious side effects of the medicines and the like are heavy burden for hepatitis B patients. The key point is that at present, no medicine can completely remove viruses to cure hepatitis B. Therefore, there is an urgent need in the art to provide a new drug for treating hepatitis b, capable of eliminating HBsAg, and achieving a functional cure.

Disclosure of Invention

The application provides a benzo-heterocycle compound, a pharmaceutical composition, a preparation method and application thereof, and the benzo-heterocycle compound can be used for preventing and/or treating hepatitis B, can reduce the HBsAg level and the HBeAg level of hepatitis B, is expected to become a new hepatitis B treatment drug, and can also be used for treating and/or preventing lung cancer, gastric cancer, breast cancer, cervical cancer, tongue squamous carcinoma, oral squamous carcinoma, head and neck cancer, esophageal carcinoma, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, soft tissue tumor of limbs, melanoma, skin cancer, lymphoma and other tumors.

In one aspect, the present application provides a compound, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, having a structure represented by formula I:

Wherein:

R ₁ and R is ₂ Each independently selected from:

wherein,

R _a and R is _b Each independently selected from the group consisting of halogen and H,

a is selected from-C (=O) NR ₆ -、-NR ₆ C(＝O)-、-S(＝O) ₂ NR ₆ -、-NR ₆ S(＝O) ₂ -、-C(＝O)O-、-OC(＝O)-、-S(＝O) ₂ O-and-OS (=O) ₂ -, wherein R is ₆ Selected from C1-4 alkyl, H and halogenated C1-4 alkyl,

R _c 、R _d and R is _e Each independently selected from C1-4 alkyl, H and halogenated C1-4 alkyl;

x and Y are each independently selected from N, O, S, -C (=O) O-, -OC (=O) -, -NR ₇ -and-CR ₈ -, wherein R is ₇ And R is ₈ Each independently selected from halogen, C1-4 alkyl, H and halogenated C1-4 alkyl; and is also provided with

R ₃ 、R ₄ And R is ₅ Each independently selected from C1-4 alkyl, H and halogenated C1-4 alkyl.

In one embodiment, R ₁ Selected from:

wherein,

R _a and R is _b Each of which is independently selected from the group consisting of halogen,

a is selected from-C (=O) NR ₆ -, wherein R is ₆ Selected from methyl and H, wherein the carbonyl end is attached to the pyridine ring,

R _c 、R _d and R is _e Each independently selected from C1-4 alkyl and H.

In one embodiment, R ₂ Selected from:

wherein,

R _c 、R _d and R is _e Each independently selected from C1-4 alkyl and H.

In one embodiment, X is selected from N, -NR ₇ -, O, S, -C (=o) O-and-OC (=o) -, wherein R ₇ Selected from C1-4 alkyl, H and halogenated C1-4 alkyl.

In one embodiment, Y is selected from N, -NR ₇ -and-CR ₈ -, wherein R is ₇ And R is ₈ Each independently selected from C1-4 alkyl and H.

In one embodiment, R ₃ H.

In one embodiment, R ₄ Selected from C1-4 alkyl.

In one embodiment, R ₅ Selected from C1-4 alkyl.

In one embodiment, the compounds of the present application have a structure represented by formula II, formula III or formula IV:

wherein each group is as defined above.

In another aspect, the application provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the application (e.g., a compound of formula I, formula II, formula III, or formula IV) or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, and optionally one or more additional therapeutic and/or prophylactic agents, and optionally one or more pharmaceutically acceptable carriers.

In one embodiment, the compounds of the application are selected from:

in one embodiment, the additional therapeutic and/or prophylactic agent is selected from interferon, pegylated interferon, or a nucleoside analog, and preferably wherein the nucleoside analog is selected from entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

In another aspect, the application provides the use of a compound of the application (e.g., a compound of formula I, formula II, formula III or formula IV) or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, in the manufacture of a medicament for the treatment and/or prophylaxis of hepatitis b or a tumor.

In another aspect, the application provides a compound of the application (e.g., a compound of formula I, formula II, formula III, or formula IV) or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, for use in the treatment and/or prevention of hepatitis b or a tumor.

In another aspect, the application provides a method of treating and/or preventing hepatitis b or a tumor, the method comprising administering to a subject in need thereof an effective amount of a compound of the application (e.g., a compound of formula I, formula II, formula III or formula IV) or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof.

In one embodiment, the tumor is selected from lung cancer, stomach cancer, breast cancer, cervical cancer, tongue squamous carcinoma, oral squamous carcinoma, head and neck cancer, esophageal cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, soft tissue tumors of the extremities, melanoma, skin cancer, lymphoma, a compound of the application is selected from:

In another aspect, the present application provides a process for preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, comprising deprotecting a compound of formula I-1 to give a compound of formula I,

wherein PG is an amino protecting group, and the remaining groups are as defined above.

In some embodiments, pharmaceutically acceptable salts of the compounds of the present application or stereoisomers thereof include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, caproate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinic acid, oxalate, thiocyanate, toluenesulfonate, and undecanoate.

In some embodiments, a compound of the application or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition or a medicament comprising the same is administered by a route selected from the group consisting of: oral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

In some embodiments, the compounds described herein, or stereoisomers or pharmaceutically acceptable salts thereof, pharmaceutical compositions or medicaments comprising the same, are administered orally.

In some embodiments, the compounds of the application or stereoisomers or pharmaceutically acceptable salts thereof, pharmaceutical compositions or medicaments comprising the same are oral formulations. In some embodiments, the compounds described herein, or stereoisomers or pharmaceutically acceptable salts thereof, pharmaceutical compositions or medicaments comprising the same, are in the form of tablets or capsules.

Drawings

FIG. 1 shows the result of the inhibition of IAP by HTCP 81-06.

FIG. 2 is an EC50 result of HTCP81-06 on MDA-MB-231.

FIG. 3 shows the IC50 results of HTCP81-06 on ME-180, hela, UPCI-SCC-152 and UPCI-SCC-090.

Detailed Description

Compounds or pharmaceutically acceptable salts, solvates, hydrates, crystal forms, stereoisomers thereof

The inventor provides a benzoheterocycle compound or pharmaceutically acceptable salt, solvate, hydrate, crystal form and stereoisomer thereof, wherein the compound has a structure shown in a general formula I:

wherein:

R ₁ and R is ₂ Each independently selected from:

wherein,

R _c 、R _d and R is _e Each independently selected from C1-4 alkyl, H andhalogenated C1-4 alkyl;

In one embodiment, R ₁ Selected from:

wherein,

R _c 、R _d and R is _e Each independently selected from C1-4 alkyl and H.

In one embodiment, R ₁ Selected from:

wherein,

a is selected from-C (=O) NR ₆ -, wherein R is ₆ Selected from methyl and H, wherein the carbonyl end is attached to the pyridine ring.

In one embodiment, R ₁ Selected from:

wherein,

R _c 、R _d and R is _e Each independently selected from C1-4 alkyl and H.

In a preferred embodiment, R ₁ Selected from:

in a more preferred embodiment, R ₁ Is that

In one embodiment, R ₂ Selected from:

wherein,

R _c 、R _d and R is _e Each independently selected from C1-4 alkyl and H.

In one embodiment, R ₂ Selected from:

wherein,

In one embodiment, R ₂ Selected from:

wherein,

R _c 、R _d and R is _e Each independently selected from C1-4 alkyl and H.

In a preferred embodiment, R ₂ Selected from:

in a more preferred embodiment, R ₂ Is that

In one embodiment, X is selected from N, -NR ₇ -, O, S, -C (=o) O-and-OC (=o) -, wherein R ₇ Selected from C1-4 alkyl, H and halogenated C1-4 alkyl. In a preferred embodiment, X is selected from N, -NR ₇ -, S, -C (=o) O-and-OC (=o) -, wherein R ₇ Selected from C1-4 alkyl and H. In a more preferred embodiment, X is N or-NH-.

In one embodiment, Y is selected from N, -NR ₇ -and-CR ₈ -, wherein R is ₇ And R is ₈ Each independently selected from C1-4 alkyl and H. In a preferred embodiment, Y is N or-NH-.

In one embodiment, R ₃ H. In one embodiment, R ₄ Selected from C1-4 alkyl; preferably, R ₄ Is methyl. In one embodiment, R ₅ Selected from C1-4 alkyl; preferably, R ₅ Is methyl.

In one embodiment, R ₃ Is H, and R ₄ And R is ₅ Each independently selected from C1-4 alkyl. In a preferred embodiment, R ₃ Is H, and R ₄ And R is ₅ Is methyl.

In one embodiment, the compound of formula I has a structure represented by formula II, formula III, or formula IV:

wherein each group is as defined above.

In one embodiment, the compounds of the application are selected from:

in a preferred embodiment, the compounds of the application are

In another aspect, the application provides the use of a compound, or a pharmaceutically acceptable salt, solvate, hydrate, crystal form, stereoisomer thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of hepatitis b or a tumour, wherein the compound has the structure of formula I:

Wherein:

R ₁ and R is ₂ Each independently selected from:

wherein,

R ₃ 、R ₄ And R is ₅ Each independently selected from C1-4 alkyl,H and halogenated C1-4 alkyl.

In one embodiment, the tumor is selected from lung cancer, stomach cancer, breast cancer, cervical cancer, tongue squamous carcinoma, oral squamous carcinoma, head and neck cancer, esophageal cancer, liver cancer, pancreatic cancer, renal cancer, bladder cancer, soft tissue tumor of extremities, melanoma, skin cancer, lymphoma.

Method for preparing compound or pharmaceutically acceptable salt, solvate, hydrate, crystal form and stereoisomer thereof

The present application provides a general synthetic route for the synthesis of the compounds of the present application. The application provides a method for preparing a compound shown in a general formula I or pharmaceutically acceptable salt, solvate, hydrate, crystal form and stereoisomer thereof, which comprises the steps of deprotecting a compound shown in a formula I-1 to obtain the compound shown in the formula I,

wherein PG is an amino protecting group, preferably t-butoxycarbonyl (Boc) or p-methoxytrityl (MMT), the remaining groups being as defined above. In an embodiment of the application, if a chiral carbon is present in the compound, the application includes isomers formed based on any of the stereoconfigurations of the chiral carbon, including, for example, racemates or any of the mirror image isomers. Moreover, the present application includes all other stereoisomers that may occur. That is, the compounds of the present application include all enantiomers, diastereomers, cis-trans isomers, racemates and the like.

Here, it is possible to use,Or->Depicting the chemical bond of the compounds of the present application. Use-to depict bonds to asymmetric carbon atoms is intended to include all possible stereoisomers at that carbon atom (e.g., specific enantiomers, racemic mixtures, etc.). Use- > Or->Depicted bonds to asymmetric carbon atoms are intended to indicate the presence of the stereoisomers shown. When present in the racemic mixture, +.>And->To define the relative stereochemistry, not the absolute stereochemistry. Unless otherwise indicated, the compounds of the present invention are intended to exist as stereoisomers (which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotamers, conformational isomers, atropisomers, and mixtures thereof). The compounds of the invention may exhibit more than one type of isomerism and are formed from mixtures thereof (e.g. racemic mixtures and diastereomersIsomer pairs).

Stereoisomers of the compounds of the invention may be obtained by: reacting an optically active starting material under conditions that do not cause racemization or epimerization; or reacting the appropriate starting materials with "chiral auxiliary" which may then be removed at a suitable stage by derivatization; or the racemic or other mixtures obtained may be separated based on their different physicochemical properties by well-known means, for example, fractional crystallization or HPLC techniques. Enantiomers can also be obtained by separation using chiral chromatography columns.

The pharmaceutically acceptable salts of the compounds of the application or stereoisomers thereof may be formed by methods known in the art by reacting the N atom on the structure of the compound or stereoisomer thereof with an inorganic or organic acid. The mineral acids include, but are not limited to: hydrochloric acid, hydrobromic acid, semi-sulfuric acid, hydroiodic acid, and the like. The organic acids include, but are not limited to: acetic acid, adipic acid, alginic acid, aspartic acid, benzoic acid, benzenesulfonic acid, butyric acid, citric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid, digluconic acid, dodecylsulfuric acid, ethanesulfonic acid, fumaric acid, glucoheptanoic acid, glycerophosphate, heptanoic acid, caproic acid, 2-hydroxyethanesulfonic acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, nicotinic acid, oxalic acid, thiocyanic acid, toluenesulfonic acid, and undecanoic acid.

The present application encompasses all possible crystalline forms or polymorphs of the compounds of the present application, which may be single polymorphs or mixtures of any ratio of more than one polymorphs.

Pharmaceutical composition

The application also provides a pharmaceutical composition comprising a compound of the application or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, and optionally one or more additional therapeutic and/or prophylactic agents, and optionally one or more pharmaceutically acceptable carriers. In one embodiment, the compounds of the present application have the structure shown in formula I:

Wherein:

R ₁ and R is ₂ Each independently selected from:

wherein,

In one embodiment, the compounds of the application are selected from:

in one embodiment, the additional therapeutic or prophylactic agent in the pharmaceutical composition is selected from interferon, pegylated interferon, or a nucleoside analog, and preferably wherein the nucleoside analog is selected from entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

In one embodiment, the pharmaceutically acceptable salts of the compounds of the present application or stereoisomers thereof include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, caproate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinic acid, oxalate, thiocyanate, toluenesulfonate, and undecanoate.

In one embodiment, the additional therapeutic or prophylactic agent may be any one or more additional therapeutic or prophylactic agents as described in the "additional therapeutic or prophylactic agent" section below.

In one embodiment, the additional therapeutic or prophylactic agent may be selected from at least one of interferon, pegylated interferon, or a nucleoside analog.

In one embodiment, the nucleoside analog is selected from entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

In one embodiment, the present application provides a pharmaceutical composition comprising a compound of formula I, formula II, formula III or formula IV of the present application (e.g., compound HTCP 81-06), or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer, and nucleoside analog thereof, and a pharmaceutically acceptable carrier.

In one embodiment, the present application provides a pharmaceutical composition comprising a compound of formula I, formula II, formula III or formula IV of the present application (e.g., compound HTCP 81-06), or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer, and nucleoside analog thereof, selected from entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide, in combination with a pharmaceutically acceptable carrier.

In the pharmaceutical composition of the present application, the compound of the present application or a pharmaceutically acceptable salt, solvate, hydrate, crystal form, stereoisomer and another therapeutic or prophylactic agent thereof may be formulated into one dosage form, or may be formulated into separate dosage forms, respectively, for sequential or simultaneous administration as a combined product.

In one embodiment, the pharmaceutical composition may be administered by a route selected from the group consisting of: oral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural. In one embodiment, the pharmaceutical composition is administered orally. In one embodiment, the pharmaceutical composition is administered by intravenous injection.

In one embodiment, the pharmaceutical composition is an oral formulation. In one embodiment, the pharmaceutical composition is in the form of a tablet or capsule.

Use of a compound or a pharmaceutically acceptable salt, solvate, hydrate, crystal form, stereoisomer thereof

The application provides the use of a compound of general formula I, general formula II, general formula III or general formula IV as defined above or a pharmaceutically acceptable salt, solvate, hydrate, crystal form, stereoisomer thereof in the manufacture of a medicament for the treatment and/or prophylaxis of hepatitis B, and for the treatment and/or prophylaxis of tumours.

In some embodiments, the tumor is selected from lung cancer, stomach cancer, breast cancer, cervical cancer, tongue squamous carcinoma, oral squamous carcinoma, head and neck cancer, esophageal cancer, liver cancer, pancreatic cancer, renal cancer, bladder cancer, soft tissue tumors of the extremities, melanoma, skin cancer, lymphoma.

In some embodiments, the compounds of the application are selected from:

in one embodiment, the present application provides the use of a compound of formula I, formula II, formula III or formula IV as defined above, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, in the manufacture of a medicament for reducing the level of HBsAg of hepatitis b.

In one embodiment, the present application provides the use of a compound of formula I, formula II, formula III or formula IV as defined above, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, in the manufacture of a medicament for reducing the level of HBeAg of hepatitis b.

In one embodiment, the present application provides the use of a compound of formula I, formula II, formula III or formula IV as defined above, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, in the manufacture of a medicament for reducing HBV DNA levels.

In one embodiment, the present application provides the use of a compound of formula I, formula II, formula III or formula IV as defined above, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, in the manufacture of a medicament for the treatment and/or prophylaxis of a tumor.

In one embodiment, the medicament further comprises one or more additional therapeutic or prophylactic agents. The one or more additional therapeutic or prophylactic agents may be any one or more additional therapeutic or prophylactic agents as described in the "additional therapeutic or prophylactic agent" section below.

In one embodiment, the additional therapeutic or prophylactic agent is selected from at least one of an interferon, a pegylated interferon, or a nucleoside analog.

In one embodiment, the medicament is administered by a route selected from the group consisting of: oral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural. In one embodiment, the medicament is administered orally. In one embodiment, the medicament is administered by intravenous injection.

In one embodiment, the medicament is an oral formulation. In one embodiment, the medicament is in the form of a tablet or capsule.

In the medicament of the application, the compound of the general formula I, the general formula II, the general formula III or the general formula IV or pharmaceutically acceptable salts, solvates, hydrates, crystal forms, stereoisomers and other therapeutic agents and/or preventive agents thereof can be prepared into one dosage form, can also be prepared into independent dosage forms respectively and can be used as a combined product to be applied sequentially or simultaneously.

Hepatitis B

Hepatitis b is an infectious disease caused by hepatitis b virus and is mainly a liver lesion. Clinically, it is mainly manifested by anorexia, nausea, epigastric discomfort, pain in liver region and hypodynamia. Some patients may have jaundice fever and liver large with liver function impairment. Some patients may become chronicized, even develop cirrhosis, and a few may develop liver cancer.

The pathogen of viral hepatitis B is hepatitis B virus, abbreviated as HBV, and hepatitis B virus is DNA virus. The genome is double-stranded, circular, incompletely closed DNA. The outermost layer of the virus is the outer membrane or coating of the virus, the inner layer of which is the core, the nucleoprotein is the core antigen (HBcAg) and cannot be detected in serum. Serum from HBsAg positive subjects was visualized under electron microscopy for 3 particles: round and filiform particles of diameter 22nm, and also less spherical particles of diameter 42 angstroms, also known as Dane's particles, are intact HBV particles.

The markers for hepatitis b were detected as follows: (1) HBsAg and anti-HBs: HBsAg positive indicates that HBV is currently in the infectious stage, anti-HBs positive for immunoprotective antibodies indicates that immunity to HBV has developed. The diagnosis basis of the chronic HBsAg carrier is that the chronic HBsAg carrier has no clinical symptoms and signs and normal liver function, and the HBsAg is continuously positive for more than 6 months. (2) HBeAg with anti-HBe: HBeAg positive is an index of active replication and strong infectivity of HBV, and the change of the tested serum from HBeAg positive to anti-HBe positive indicates that the disease is relieved and the infectivity is weakened. (3) HBcAg with anti-HBc: HBcAg positive suggests that there is a direct reaction of intact HBV particles and that HBV active replication is rarely used clinically due to the complex detection method. anti-HBc is a marker of HBV infection, and positive IgM for anti-HBc suggests that there is viral replication in the body at the early stage of infection. Three positive HBsAg, HBeAg and anti-HBc in chronic mild hepatitis B and HBsAg carriers have high infectious index that is difficult to turn negative.

Additional therapeutic or prophylactic agents

In one embodiment, a pharmaceutical or pharmaceutical composition comprising a compound of formula I, formula II, formula III or formula IV, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, as described herein, may further comprise one or more additional therapeutic and/or prophylactic agents.

In one embodiment, the additional therapeutic or prophylactic agent is selected from an interferon or a nucleoside analogue.

In one embodiment, the additional therapeutic or prophylactic agent is selected from nucleoside analogs.

In some embodiments, the additional therapeutic or prophylactic agent is selected from one or more of entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide, for example, from one of entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide, or from at least two of entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

Entecavir (ETV) is known under the chemical name 2-amino-1, 9-dihydro-9- [ (1S, 3R, 4S) -4-hydroxy-3- (hydroxymethyl) -2-methylenecyclopentane ] -6H-purin-6-one, and has the following structural formula:

U.S. patent No. 5206244a discloses entecavir and its use for the treatment of hepatitis b virus; a novel entecavir synthesis method is disclosed in WO9809964 A1; WO0164421A1 discloses low dose entecavir solid formulations.

Entecavir is a highly potent antiviral agent, developed by the american schdule company in the 90 th century, with a potent anti-HBV effect. It can be phosphorylated to an active triphosphate, which has a half-life of 15h in the cell. Entecavir triphosphates inhibit all three activities of the viral polymerase (reverse transcriptase) by competing with deoxyguanosine triphosphate, the natural substrate of HBV polymerase: (1) initiation of HBV polymerase; (2) formation of negative strand of pregenomic mRNA reverse transcription; (3) Synthesis of HBV DNA plus strand.

The chemical name of tenofovir disoproxil fumarate (Tenofovir disoproxil fumarate, TDF) is (R) - [ [2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phosphonic acid diisopropyl oxycarbonyl methyl ester fumarate, is an ester precursor of tenofovir, belongs to a novel nucleotide reverse transcriptase inhibitor, and has the effect of inhibiting HBV virus activity.

TDF is another novel ring-opened nucleoside phosphonate that was successfully developed by the us gilid company following adefovir dipivoxil, and was first marketed in the us 10 months in 2001, and has been marketed in countries such as europe, australia and canada.

TDF inhibits viral polymerase in vivo by competitively binding to natural deoxyribose substrates and terminates DNA strand synthesis by insertion into DNA. The main action mechanism is that the tenofovir is hydrolyzed into tenofovir after oral administration, the tenofovir is phosphorylated by cell kinase, and a metabolite tenofovir diphosphate with pharmacological activity is generated, the metabolite tenofovir diphosphate competes with 5 '-deoxyadenosine triphosphate to participate in the synthesis of virus DNA, and after entering the virus DNA, the DNA is prolonged and blocked due to the lack of 3' -OH groups, so that the replication of the virus is blocked. Clinical application shows that TDF has obvious anti-HBV virus curative effect and small toxic side effect, thus having great clinical application prospect.

Tenofovir alafenamide (Tenofovir Alafenamide), a prodrug of Tenofovir (Tenofovir), a novel Nucleoside Reverse Transcriptase Inhibitor (NRTI) developed by the american gilid science company. Compared with the previous generation of anti-hepatitis B similar drug tenofovir disoproxil TDF, the antiviral activity of tenofovir alafenamide is 10 times of that of tenofovir alafenamide, the stability in blood plasma is 200 times of that of tenofovir alafenamide, and the half life is 225 times higher than that of tenofovir alafenamide. Compared with TDF, tenofovir alafenamide only needs one tenth of TDF administration dose, and can achieve the same antiviral effect as TDF. Therefore, the tenofovir alafenamide is used for preventing or/and treating Hepatitis B Virus (HBV) infection, and has better curative effect, higher safety and lower drug resistance.

Route of administration

The medicament or pharmaceutical composition of the application may be administered by any route suitable for the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal and parenteral, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural. Those skilled in the art will appreciate that the particular route of administration may vary depending upon, for example, the pharmaceutical dosage form, the condition of the recipient.

In one embodiment, the medicament or pharmaceutical composition of the application may be administered by intravenous injection.

One advantage of the medicaments or pharmaceutical compositions of the present application is that they are orally bioavailable and can be administered orally. Thus, in one embodiment, the medicament or pharmaceutical composition of the application may be administered orally. In one embodiment, the medicament or pharmaceutical composition of the present application may be administered orally in the form of a tablet or capsule.

Pharmaceutical composition or formulation and formulation of a drug

In certain embodiments, the compound of formula I, formula II, formula III or formula IV, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, is administered in a pharmaceutical composition. The pharmaceutical compositions of the present application may be formulated with conventional carriers and excipients which will be selected in accordance with common practice. The tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form and are generally isotonic when used for delivery by non-oral administration. The pharmaceutical compositions or medicaments of the application or all formulations thereof will optionally contain excipients, for example as described in "Handbook of Pharmaceutical Excipients" (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkyl celluloses, hydroxyalkyl methylcellulose, stearic acid and the like. The pH of the formulation ranges from about 3 to about 11, but is typically from about 7 to 10. In some embodiments, the pH of the formulation ranges from about 2 to about 5, but is typically from about 3 to 4.

Formulations include those suitable for the aforementioned routes of administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing co., easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which is composed of one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then shaping the product as necessary.

Formulations of the present application suitable for oral administration may exist as: discrete units, such as capsules or tablets, each containing a predetermined amount of the active ingredient; powder or granules; solutions or suspensions in aqueous or non-aqueous liquids; or an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.

Tablets are made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by: the active ingredient in free-flowing form, such as a powder or granules, is pressed in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally formulated so as to provide slow or controlled release of the active ingredient therein.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

The pharmaceutical compositions or medicaments of the present application may also be in the form of a sterile injectable preparation, for example as a sterile injectable aqueous or oleaginous suspension. The suspensions may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol, or as a lyophilized powder. Acceptable carriers and solvents that can be used include water, ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Acceptable carriers and solvents that can be used include water, ringer's solution, isotonic sodium chloride solution, and hypertonic sodium chloride solution.

Definition of the definition

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 the claimed subject matter belongs.

In the present application, the singular is used to include the plural unless specifically stated otherwise. It should also be noted that the use of "or" means "and/or" unless otherwise indicated.

The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, according to the definition below, "optionally substituted alkyl" refers to "unsubstituted alkyl" (alkyl substituted with a substituent) or "substituted alkyl" (alkyl substituted with a substituent).

In the present application, the term "halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

In the present application, the term "alkyl" means a straight or branched chain group consisting of only carbon and hydrogen atoms, free of unsaturated bonds and linked to the rest of the molecule by a single bond. The term "C _1-4 Alkyl "refers to an alkyl group having 1 to 4 carbon atoms. C (C) _1-4 Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl, and the like.

In the present application, the term "halogenated C _1-4 Alkyl "means C substituted by one or more halogen atoms _1-4 Alkyl, wherein C _1-4 Alkyl is as defined above. Halogenated C _1-4 Examples of alkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2-difluoroethyl, chloromethyl, 2-chloroethyl, dichloromethyl, trichloromethyl, 1, 2-dichloroethyl, bromomethyl, 3-bromopropyl, iodomethyl, and the like.

In the present application, the term "5-10 membered heteroaryl" means a 5-, 6-, 7-, 8-, 9-or 10-membered ring system group having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur in the ring, and at least one aromatic ring. The 5-10 membered heteroaryl group may be a monocyclic or bicyclic ring system. The nitrogen, carbon or sulfur atoms in the 5-to 10-membered heteroaryl group may optionally be oxidized; the nitrogen atom may optionally be quaternized. Examples of 5-10 membered heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, benzimidazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, imidazopyridyl, thienopyridinyl, thienopyrimidinyl, thienopyrazinyl, thienopyridazinyl, thiazolopyridinyl, isothiazolopyridinyl, furopyrimidinyl, furopyridazinyl, triazolopyridinyl, imidazopyrimidinyl, imidazopyrazinyl, imidazopyridazinyl, indolinyl, indazolyl, isoindazolyl, purinyl, quinolinyl, isoquinolinyl, quinoxalinyl, pteridinyl, cinnolinyl, quinazolinyl, indolizinyl, and the like.

In the present application, the term "stereoisomer" refers to an isomer produced by the spatial arrangement of atoms in a molecule. The compounds of the application contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomers as well as mixtures, including racemic mixtures, of the compounds of the application are part of the application. Diastereomeric mixtures can be separated into the individual diastereomers, based on their different physicochemical properties, by well known means, for example, resolution of an enantiomer can be converted into the diastereomer by reaction with an appropriate optically active material, which is separated and converted (e.g., hydrolyzed) into the corresponding individual isomer. Enantiomers may also be separated using chiral chromatography columns.

In the present application, the term "pharmaceutically acceptable" refers to a substance that does not affect the biological activity or properties of the compounds of the present application and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological reaction or interacting in an adverse manner with any of the components contained in the composition.

In the present application, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness of the free acid and free base of the compounds of the present application and has no adverse effect on biology or other aspects.

In the present application, the term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered, and which is suitable for contacting the tissues of humans and/or other animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment.

In the present application, the term "hydrate" refers to a substance formed by associating a compound with water molecules.

In the present application, the term "solvate" refers to a substance formed by association of a compound with a solvent molecule.

In the present application, the term "therapeutically effective amount" or "effective amount" refers to an amount that is effective at that dose and for the period of time required to achieve the desired therapeutic result. The therapeutically effective amount will depend on factors such as the nature and severity of the hepatitis b or symptoms thereof, the particular therapeutic agent, the general condition of the recipient (e.g., height, weight, age, and physical condition), and the like, and can be determined by standard clinical techniques known to those skilled in the art.

In the present application, the term "treatment" may refer to, for example, alleviation of symptoms, prolongation of survival, improvement of quality of life, and the like. The treatment need not be a "cure". Treatment may also refer to functional cure and clearance of hepatitis b virus.

In the present application, the term "reducing Hepatitis B Virus (HBV) load" means reducing the amount of hepatitis b virus DNA in the blood of a patient that is detectable.

In the present application, the term "reducing the level of HBsAg and/or HBeAg" refers to reducing the amount of HBsAg and/or HBeAg of hepatitis B virus in the blood of a patient that is detectable. The amount of HBsAg and/or HBeAg is generally closely related to the functional cure of hepatitis B.

Examples

Example 1: preparation of Compound HTCP81-06

Step 1:

6-bromo-4-nitro-3H-1, 3-benzodiazepine-2-amine (1.4 g, 5.4476 mmol,1 equiv) was dissolved in N, N-dimethylformamide (20 mL) and stirred well, then 2- [ (tert-butoxycarbonyl) (methyl) amino ] propionic acid (1.107 g, 5.4476 mmol,1 equiv), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (3.106 g,8.169mmol,1.5 equiv), N, N-diisopropylethylamine (2.112 g, 16.336 mmol,3 equiv) were added sequentially at 20deg.C. The reaction was stirred at room temperature for 20h. LCMS monitoring shows that the reaction is completed, pouring the reaction solution into water, extracting with ethyl acetate three times, combining the organic layers, washing the organic layers with saturated saline, drying the organic layers with anhydrous sodium sulfate, concentrating, and performing silica gel column chromatography (eluent: ethyl acetate and petroleum ether in a volume ratio of 1:1) to obtain brick red solid tert-butyl N- {1- [ (6-bromo-4-nitro-3H-1, 3-benzodiazol-2-yl) carbamoyl ] ethyl } -N-methylcarbamate (1 g, yield: 40.59%, purity: 97.7%).

LCMS conditions, kinetex EVO; the length is 30mm, and the inner diameter is 3.0mm; mobile phase A water/5 mM ammonium bicarbonate; mobile phase B, acetonitrile; the time is 2.00min; rt is 1.318min.

ESI-MS m/z:442，444[M+H]+.

Step 2:

tert-butyl N- {1- [ (6-bromo-4-nitro-3H-1, 3-benzodiazepine-2-yl) carbamoyl ] ethyl } -N-methylcarbamate (1 g,2.260mmol,1 equiv) was dissolved in 1, 4-dioxane (10 mL) at room temperature, and after stirring well, pinacol biborate (1.148 g,4.520mmol,2 equiv), potassium acetate (665.73 g,6.780mmol,3 equiv), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex (184.20 mg,0.226mmol,0.1 equiv) was added in sequence and stirred under nitrogen at 80℃overnight. After the completion of the reaction, the reaction mixture was poured into water, extracted three times with ethyl acetate, the organic layers were combined, the organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a crude product as a black oil, which was used as it is in the next step.

LCMS conditions, kinetex EVO; the length is 30mm, and the inner diameter is 3.0mm; mobile phase A water/5 mM ammonium bicarbonate; mobile phase B, acetonitrile; the time is 2.00min; rt is 1.078min.

ESI-MS m/z:408[M+H]+.

Step 3:

2- {2- [ (tert-Butoxycarbonyl) (methyl) amino ] propanamido } -7-nitro-1H-1, 3-benzodiazol-5-ylboronic acid (330 mg,0.810mmol,1 equiv) was dissolved in dioxane (5 mL) and stirred well at room temperature. 7-chloro-1, 3, 5-trimethylpyrazolo [4,3-d ] pyrimidine (175.30 mg,0.891mmol,1.1 equiv), sodium carbonate (257.69 mg,2.430mmol,3 equiv), water (0.5 mL), and [1,1' -bis (diphenylphosphine) ferrocene ] palladium dichloride dichloromethane complex (59.30 mg,0.081mmol,0.1 equiv) were added sequentially, and the mixture was stirred under nitrogen at 80℃overnight. After the completion of the reaction, the reaction mixture was poured into water, extracted three times with ethyl acetate, the organic layers were combined, the organic layers were washed with saturated brine and dried over anhydrous sodium sulfate, and after concentration, silica gel column chromatography (eluent: ethyl acetate and petroleum ether at a volume ratio of 1:1) was performed to obtain tert-butylmethyl (1- ((7-nitro-5- (1, 3, 5-trimethyl-1H-pyrazolo [4,3-d ] pyrimidin-7-yl) -1H-benzo [ d ] imidazol-2-yl) amino) -1-oxopropan-2-yl) carbamate (109 mg, yield: 23.82%, purity: 92.7%) as a pale yellow solid.

LCMS conditions, kinetex EVO; the length is 30mm, and the inner diameter is 3.0mm; mobile phase A water/5 mM ammonium bicarbonate; mobile phase B, acetonitrile; the time is 2.00min; rt is 1.194min.

ESI-MS m/z:524[M+H]+.

Step 4:

tert-butylmethyl (1- ((7-nitro-5- (1, 3, 5-trimethyl-1H-pyrazolo [4,3-d ] pyrimidin-7-yl) -1H-benzo [ d ] imidazol-2-yl) amino) -1-oxopropan-2-yl) carbamate (107 mg,0.204mmol,1 equiv) was dissolved in absolute ethanol (1 mL) and water (0.1 mL) at room temperature and stirred well. Ammonium chloride (109.32 mg,2.040mmol,10 equiv) and iron powder (91.31 mg,1.632mmol,8 equiv) were then added in this order, followed by stirring for 1h at 80 ℃. After the reaction was completed, the reaction solution was filtered through celite, the cake was washed three times with absolute ethanol, and the filtrate was concentrated under reduced pressure. Silica gel column chromatography (eluent: methanol and dichloromethane at a volume ratio of 10:1) gave t-butyl N- {1- [ (4-amino-6- {1,3, 5-trimethylpyrazolo [4,3-d ] pyrimidin-7-yl } -3H-1, 3-benzodiazol-2-yl) carbamoyl ] ethyl } -N-methylcarbamate (80 mg, yield: 71.38%, purity: 50.3%) as a pale green solid.

LCMS conditions, kinetex EVO; the length is 30mm, and the inner diameter is 3.0mm; mobile phase A water/5 mM ammonium bicarbonate; mobile phase B, acetonitrile; the time is 2.00min; rt is 1.012min.

ESI-MS m/z:494[M+H]+.

Step 5:

5-chloro-3-fluoropyridine-2-carbonyl chloride (82.53 mg,0.4305mmol,3.00 equiv) was dissolved in dichloromethane (2 mL) under nitrogen and stirred well. N, N-diisopropylethylamine (91.23 mg,0.71 mmol,5 equiv) and tert-butylN- {1- [ (4-amino-6- {1,3, 5-trimethylpyrazolo [4,3-d ] pyrimidin-7-yl } -3H-1, 3-benzodiazol-2-yl) carbamoyl ] ethyl } -N-methylcarbamate (70 mg,0.1435mmol,1 equiv) were then added sequentially at 0deg.C and stirred overnight. After the reaction, the reaction solution is concentrated, the residue is purified by a climbing plate (eluent: methanol and dichloromethane with volume ratio of 10:1), the filter cake is washed three times by absolute ethanol, and the filtrate is concentrated under reduced pressure. Silica gel column chromatography (eluent: ethyl acetate and petroleum ether at a volume ratio of 1:1) gave tert-butyl N- (1- { [4- (5-chloro-3-fluoropyridine-2-amino) -6- {1,3, 5-trimethylpyrazolo [4,3-d ] pyrimidin-7-yl } -3H-1, 3-benzodiazol-2-yl ] carbamoyl } ethyl) -N-methylcarbamate (40 mg, yield: 43.34%, purity: 98%) as a pale green solid.

LCMS conditions: HALO; the length is 30mm, and the inner diameter is 3.0mm; mobile phase A is water/0.05% trifluoroacetic acid; mobile phase B acetonitrile/0.05% trifluoroacetic acid; the time is 2.00min; rt is 1.311min.

ESI-MS m/z:651[M+H]+.

Step 6:

the compound tert-butyl N- (1- { [4- (5-chloro-3-fluoropyridine-2-amino) -6- {1,3, 5-trimethylpyrazolo [4,3-d ] pyrimidin-7-yl } -3H-1, 3-benzodiazol-2-yl ] carbamoyl } ethyl) -N-methylcarbamate (35 mg,0.054mmol,1 equiv) was placed in a 5mL sample bottle and dichloromethane (1 mL) and trifluoroacetic acid (0.2 mL) and reacted at room temperature for 1 hour, LCMS monitoring showed the reaction to be complete. The reaction system was then cooled to room temperature, extracted three times with ethyl acetate, the organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure, and the obtained crude product was purified by column chromatography on silica gel to give the compound 5-chloro-3-fluoro-N- (2- (2- (methylamino) propanamido) -5- (1, 3, 5-trimethyl-1H-pyrazolo [4,3-d ] pyrimidin-7-yl) -1H-benzo [ d ] imidazo-7-yl) pyridine amide (weight: 3.2mg, yield: 10.8%, purity: 98.396%) as a yellow solid. LCMS conditions, column model: kinetex EVO C18-100A; the length is 30mm, and the inner diameter is 3.0mm; mobile phase A water/6.5 mM ammonium bicarbonate; mobile phase B, acetonitrile; the total time is 2.00min; the peak time was 1.003min.

1H NMR(400MHz,DMSO-d6)δ1.57(d,J＝7.1Hz,3H),2.56(s,3H),2.67(d,J＝2.5Hz,3H),2.78(s,3H),3.83(s,3H),4.10(s,1H),7.74(d,J＝1.6Hz,1H),8.38(m,J＝11.1,2.4Hz,1H),8.49–8.58(m,1H),8.72(d,J＝1.9Hz,1H).

Example 2: evaluation of in vitro anti-HBV Activity of Compound HTCP81-06 Using HepG2.2.15 cells

Material

Compound HTCP81-06 was prepared by the method described in example 1;

entecavir is purchased from Shanghai Taitan technologies Co., ltd (lot number: P1214012;99.0% purity);

cell hepg2.2.15 was purchased from Shanghai Minkangde New drug development Co., ltd;

the cell subculture medium was DMEM medium (Gibco, cat No. 11960051) containing 10% fetal bovine serum (FBS, ex cell cat No. FSP 500), 500 μg/ml G418, 1% glutamine, 1% neaa (non-essential amino acids), 1mM sodium pyruvate, 1% penicillin-streptomycin, mainly for subculturing cells. Cell plating medium is DMEM medium (Gibco, cat No. 11960051) containing 2% fetal bovine serum (FBS, ex cell cat No. FSP 500), 500 μg/ml G418, 1% glutamine, 1% neaa (non-essential amino acids), 1mM sodium pyruvate, 1% penicillin-streptomycin, mainly for cell plating and drug addition and fluid change;

QIAamp 96DNA Blood Kit is available from Qiagen (accession number 51162);

HBsAg ELISA kits were purchased from amblygraphic organisms (cat No. CL 0310);

HBeAg ELISA kit was purchased from amblygraphic organism (cat No. CL 0312);

CellTiter-Glo kit was purchased from Roche;

HBV virus was purchased from Shanghai Mingkang New drug development Co.

Cell spreading and compound treatment

On day 0, cells will be platedHepG2.2.15 in the medium was plated into 96-well plates (6X 10) ⁴ Individual cells/well).

On day 1, medium containing the compound was added. Test compound HTCP81-06 was diluted 2 single drug concentrations with DMSO, 9 total concentrations of control compound ETV, 3-fold dilution, and triplicate wells (table 1). DMSO controls containing DMSO alone without compound were also set. Cells at 5% CO ₂ And cultured at 37℃for 3 days.

On day 4, the fresh cell plating medium containing the compound was changed once.

On day 7, the supernatant was collected, and the collected cell supernatant was assayed for HBeAg and HBsAg by ELISA, and HBV DNA levels by qPCR. Meanwhile, cellTiter-Glo detects cell viability and collects cells for cryopreservation (spare).

TABLE 1 test concentration of Compounds

Sample detection

qPCR method for detecting HBV DNA content in cell culture supernatant

DNA was extracted from the cell culture supernatant by reference to QIAamp 96DNA Blood Kit instructions. The HBV DNA content was detected by qPCR method. And (3) PCR reaction: 95 ℃ for 10min;95 ℃,15sec,60 ℃,1min,40 cycles.ELISA method for detection Measurement of HBeAg and HBsAg content in cell culture supernatant

The method is briefly described below with reference to the kit instructions: respectively adding 50 μl of standard substance, sample and reference substance into the detection plate, adding 50 μl of enzyme conjugate into each well, incubating at 37deg.C for 60 min, washing the plate with washing solution, blotting, adding 50 μl of premixed luminescent substrate, incubating at room temperature in dark for 10min, and measuring the luminescence value by enzyme-labeled instrument.

CellTiter-Glo cell viability assay

Cell viability was determined with reference to CellTiter-Glo kit instructions, briefly as follows: after collecting the cell culture supernatant, cellTiter-Glo (medium 1:1 dilution) was added to each well, incubated at room temperature for 10 minutes, and the luminescence value was measured by an enzyme-labeled instrument.

Data computation

HBV DNA inhibition (%) = (HBV copy number of 1-compound group sample/HBV copy number of DMSO control group) ×100%

HBsAg inhibition (%) = (HBsAg value of 1-sample/HBsAg value of DMSO control) ×100%

HBeAg inhibition (%) = (HBeAg value of 1-sample/HBeAg value of DMSO control group) ×100%

Cell viability (%) = ((sample value-medium control group mean)/(DMSO group mean-medium control group mean) ×100%)

Calculation of EC using GraphPad Prism software (four parameter logistic equations) ₅₀ Values.

Data analysis

The results of the control compound ETV are shown in table 2 below:

TABLE 2

The results of the control compound ETV indicate that the test method is effective. Furthermore, it can be seen from table 2 that ETV has a significant inhibitory effect on HBV DNA, but not on HBeAg and HBsAg.

The results of the test compounds are shown in table 3 below:

TABLE 3 Table 3

As shown in Table 3, the compound HTCP81-06 has better inhibition effect on HBsAg and HBeAg at the concentration of 20 mu M, and can effectively reduce the levels of HBsAg and HBeAg, thereby being expected to remove hepatitis B virus, achieve functional cure and avoid taking medicine for a long time or for a whole life.

Example 3: the inhibition of IAP (apoptosis inhibitor) by compound HTCP81-06 was evaluated by fluorescence luminescence method, and the test results are shown in Table 4 below:

TABLE 4 Table 4

Compounds of formula (I)	IC50(nM)
		HTCP81-06	49.94

As shown in Table 4 and FIG. 1, the IC50 for the compound HTCP81-06 for IAP was 49.94nM, indicating that this compound is a good IAP inhibitor.

Example 4: the anti-tumor activity of compound HTCP81-06 in vitro was evaluated using MDA-MB-231 cells (human breast cancer cells) and the results are shown in Table 5 below:

TABLE 5

As shown in Table 5 and FIG. 2, the compound HTCP81-06 showed good in vitro antitumor activity with an EC50 of 1335nM for MDA-MB-231 cells.

Example 5: compound HTCP81-06 in vitro antitumor activity was evaluated using ME-180 (human cervical epidermoid carcinoma cells), hela (cervical carcinoma cells), UPCI-SCC-152 (human tongue squamous carcinoma cells), UPCI-SCC-090 (human mouth squamous carcinoma cells), 4 cell lines.

In the experimental method, cells of each cell line were plated in 384-well plates, HTCP81-06 was added at the corresponding concentration on the next day, the concentrations were shown in Table 6, and CellTiter-Glo was added after 3 days of incubation to examine the cell viability.

TABLE 6

The test results are shown in table 7 below:

TABLE 7

As shown in table 7 and fig. 3, compound HTCP81-06 showed good in vitro anti-tumor activity.

Although the present application has been described with reference to particular embodiments, those skilled in the art will recognize that changes and modifications may be made to the embodiments without departing from the spirit and scope of the application, which is defined by the appended claims.

Claims

1. A compound having a structure according to formula I:

wherein:

R ₁ and R is ₂ Each independently selected from:

wherein,

R _c 、R _d and R is _e Each independently ofThe site is selected from C1-4 alkyl, H and halogenated C1-4 alkyl;

2. The compound according to claim 1, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, wherein R ₁ Selected from:

wherein,

R _c 、R _d and R is _e Each independently selected from C1-4 alkyl and H;

preferably, R ₁ Selected from:

and is also provided with

More preferably, R ₁ Is that

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, solvate, hydrate, crystal form, stereoisomer thereof, wherein R ₂ Selected from:

wherein,

R _c 、R _d and R is _e Each independently selected from C1-4 alkyl and H;

preferably, R ₂ Selected from:

and is also provided with

More preferably, R ₂ Is that

4. A compound according to any one of claims 1-3, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, wherein X is selected from N, -NR ₇ -, O, S, -C (=o) O-and-OC (=o) -, wherein R ₇ Selected from C1-4 alkyl, H and halogenated C1-4 alkyl;

preferably, X is selected from N, -NR ₇ -, S, -C (=o) O-and-OC (=o) -, wherein R ₇ Selected from C1-4 alkyl and H; and, in addition, the processing unit,

more preferably, X is N or-NH-.

5. The compound according to any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, wherein Y is selected from N, -NR ₇ -and-CR ₈ -, wherein R is ₇ And R is ₈ Each independently selected from C1-4 alkyl and H; and is also provided with

Preferably, Y is N or-NH-.

6. The compound according to any one of claims 1-5A compound or a pharmaceutically acceptable salt, solvate, hydrate, crystal form, stereoisomer thereof, wherein R ₃ Is H, R ₄ And R is ₅ Each independently selected from C1-4 alkyl; and, in addition, the processing unit,

preferably, R ₃ Is H, and R ₄ And R is ₅ Is methyl.

7. The compound of any one of claims 1-6, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, wherein the compound has a structure represented by formula II, formula III, or formula IV:

wherein each group is as defined in any one of claims 1 to 6; and, in addition, the processing unit,

preferably, the compound is selected from:

8. a pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1-7, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, and optionally one or more additional therapeutic and/or prophylactic agents, and optionally one or more pharmaceutically acceptable carriers, and,

preferably, wherein the additional therapeutic or prophylactic agent is selected from interferon, pegylated interferon, or a nucleoside analogue, and preferably wherein the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

9. The use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, hydrate, crystalline form, stereoisomer thereof, for the manufacture of a medicament for the treatment or prophylaxis of hepatitis B or tumor, and,

preferably, the tumor is selected from lung cancer, stomach cancer, breast cancer, cervical cancer, tongue squamous carcinoma, oral squamous carcinoma, head and neck cancer, esophageal cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, soft tissue tumor of extremities, melanoma, skin cancer, lymphoma, wherein the compound is selected from:

10. a method for preparing a compound shown in a general formula I or pharmaceutically acceptable salt, solvate, hydrate, crystal form and stereoisomer thereof, which comprises the steps of deprotecting a compound shown in a formula I-1 to obtain the compound shown in the formula I,

wherein PG is an amino protecting group, preferably t-butoxycarbonyl (Boc) or p-methoxytrityl (MMT), the remaining groups being as defined in any of claims 1 to 7.