阅读说明：本技术 用于治疗乙型肝炎的组合 (Combination for treating hepatitis B ) 是由 胡彦宾 孙飞 丁照中 陈曙辉 吴文强 周义鑫 于 2021-05-14 设计创作，主要内容包括：本发明涉及一类用于治疗乙型肝炎的组合,具体涉及用于治疗乙型肝炎的式(V)所示组合与其他治疗乙型肝炎药物的组合,以及该组合在制备治疗乙型肝炎药物中的用途。(The invention relates to a combination for treating hepatitis B, in particular to a combination of a combination shown in a formula (V) for treating hepatitis B and other medicines for treating hepatitis B, and application of the combination in preparing medicines for treating hepatitis B.)

1. A combination comprising a compound of formula (V), an isomer thereof or a pharmaceutically acceptable salt thereof and another compound,

wherein the content of the first and second substances,

with "-" carbon atoms as chiral carbon atoms, in the form of (R) or (S) single enantiomers or enriched in one enantiomer;

R₁selected from H, OH, CN, NH₂Or is selected from optionally substituted with 1,2 or 3R: c_1-6Alkyl radical, C_1-6Heteroalkyl group, C_2-5Alkenyl radical, C_2-5Heteroalkenyl, C_3-6Cycloalkyl or 3-to 6-membered heterocycloalkyl;

R₂selected from H, OH, CN, NH₂Halogen, or selected from optionally substituted with 1,2 or 3R: c_1-3Alkyl radical, C_1-3Heteroalkyl group, C_3-6Cycloalkyl or 3-to 6-membered heterocycloalkyl;

R₃selected from optionally substituted with 1,2 or 3R: c_1-6Alkyl radical, C_3-6A cycloalkyl group;

m is selected from: 0. 1,2,3, 4 or 5;

when m is 0, R₁Is not selected from: OH, CN, NH₂；

R is selected from H, halogen, OH, CN, NH₂Or is selected from optionally substituted with 1,2 or 3R': c_1-3Alkyl radical, C_1-3A heteroalkyl group;

r' is selected from: F. cl, Br, I, OH, CN, NH₂、CH₃、CH₃CH₂、CH₃O、CF₃、CHF₂、CH₂F；

"hetero" means a heteroatom or a heteroatom group, said C_1-6Heteroalkyl group, C_2-5Heteroalkenyl, 3-to 6-membered heterocycloalkyl, C_1-3Each of the "hetero" of heteroalkyl groups is independently selected from: -C (═ O) N (R) -, -C (═ NR) -, - (R) C ═ N-, -S (═ O)₂N(R)-、-S(＝O)N(R)-、N、-O-、-S-、＝O、＝S、-C(＝O)O-、-C(＝O)-、-C(＝S)-、-S(＝O)-、-S(＝O)₂-、-N(R)C(＝O)N(R)-；

In any of the above cases, the number of heteroatoms or heteroatom groups is independently selected from 1,2 or 3;

characterized in that the further compound is selected from the group consisting of an immunomodulator, a nucleotide reverse transcriptase inhibitor and a nucleoside reverse transcriptase inhibitor.

2. The combination of claim 1, wherein the immunomodulator is selected from pegylated interferon alfa-2 a.

3. A combination according to claim 1 wherein the nucleotide reverse transcriptase inhibitor is selected from tenofovir disoproxil fumarate and tenofovir disoproxil fumarate.

4. The combination of claim 1, wherein the nucleoside reverse transcriptase inhibitor is selected from entecavir.

5. A combination according to any one of claims 1 to 4 wherein R is selected from H, F, Cl, Br, I, OH, CN, NH₂、CH₃、CH₃CH₂、CH₃O、CF₃、CHF₂And CH₂F。

6. A combination according to any one of claims 1 to 4 wherein R₁Selected from H, OH, CN, NH₂Or is selected from optionally substituted with 1,2 or 3R: c_1-3Alkyl, aryl, heteroaryl, and heteroaryl,C_1-3Heteroalkyl group, C_2-3Alkenyl radical, C_2-3Heteroalkenyl, C_3-6Cycloalkyl and 3-6 membered heterocycloalkyl.

7. A combination according to claim 6, wherein R₁Selected from H, OH, CN and NH₂Or is selected from optionally substituted with 1,2 or 3R: CH (CH)₃、

8. A combination according to claim 7, wherein R₁Selected from H, OH, CN, NH₂、

9. A combination according to any one of claims 1 to 4 wherein R₂Selected from H, OH, CN, NH₂And halogen, or selected from optionally substituted with 1,2 or 3R: c_1-3Alkyl radical, C_1-3Heteroalkyl group and C_3-6A cycloalkyl group.

10. A combination according to claim 9, wherein R₂Selected from H, OH, CN, NH₂F, Cl, Br and I, or selected from optionally substituted with 1,2 or 3R: CH (CH)₃、

11. A combination according to claim 10, wherein R₂Selected from Cl,Br、CN、CH₃、

12. A combination according to any one of claims 1 to 4 wherein R₃Selected from optionally substituted with 1,2 or 3R: c_1-4Alkyl and C_3-6A cycloalkyl group.

13. The combination of claim 12, wherein R₃Is selected from

14. A combination according to any one of claims 1 to 4 wherein m is selected from 0, 1,2,3 and 4 and when m is 0, R is₁Is not selected from OH, CN and NH₂。

15. A combination according to any one of claims 1 to 4, wherein the structural elements areIs selected from

16. A combination according to any one of claims 1 to 4 wherein the ` carbon atom is a chiral carbon atom, present in (R) single enantiomer or enriched in one enantiomer.

17. A combination according to any one of claims 1 to 4 wherein the compound, isomer or pharmaceutically acceptable salt thereof is selected from

Wherein the content of the first and second substances,

with "-" carbon atoms as chiral carbon atoms, in the form of (R) or (S) single enantiomers or enriched in one enantiomer;

R₄selected from H, or selected from optionally substituted with 1,2 or 3R: c_1～3Alkyl and C_1～3A heteroalkyl group;

x is selected from C and N;

y is selected from O and C;

L₁and L₂Are each independently selected from the group consisting of single bond, - (CH)₂) n-and-C (═ O) -;

and L is₁And L₂Not being a single bond at the same time;

n is selected from 1 and 2;

m、R、R₂、R₃and C_1～3The "hetero" in the heteroalkyl group is as defined in claims 1-4, and R is R when m is 0₄Is not H.

18. A combination according to claim 17, wherein the carbon atom with "x" is a chiral carbon atom, present in (R) single enantiomer or enriched in one enantiomer.

19. A combination according to any one of claims 1 to 4 wherein the compound is selected from

20. The combination according to claim 19, wherein the compound is selected from

21. Use of a combination according to any one of claims 1 to 20 for the preparation of a medicament for the treatment of hepatitis b.

22. A pharmaceutical composition comprising a combination according to any one of claims 1 to 20 and at least one pharmaceutically acceptable carrier and/or excipient.

23. A kit comprising a combination according to any one of claims 1 to 20 or a composition according to claim 22.

24. Use of a pharmaceutical composition according to claim 22 or a kit according to claim 23 in the manufacture of a medicament for the treatment of hepatitis b.

Technical Field

The invention belongs to the field of biological medicine, and relates to a combination of a combination shown as a formula (V) for treating hepatitis B and other medicines for treating hepatitis B, and application of the combination in preparing medicines for treating hepatitis B.

Background

Hepatitis B Virus (HBV, Hepatitis B for short) infection is a serious public health burden worldwide, and chronic infection of more than 2.5 million people is defined as positive for Hepatitis B surface antigen (HBsAg). Approximately one-fourth of the patients may develop severe liver diseases such as cirrhosis and hepatocellular carcinoma (HCC).

HBV belongs to the hepadnaviridae (hepadnaviridae) and assembles a DNA virion with a diameter of 42 nm, which contains a partially double-stranded relaxed circular DNA (relaxed circular DNA) with a length of up to 3200 base pairs. HBV DNA is produced by reverse transcription of pregenomic RNA enclosed in nucleocapsid under the catalysis of reverse transcriptase. Hepatitis b virus is endocytosed into hepatocytes by binding to the sodium taurocholate transporter (NTCP). After entering the hepatocytes, rcDNA is first converted into minichromosomal covalently closed circular dna (cccdna) in the hepatocyte nucleus. Conserved Open Reading Frames (ORFs) translate HBsAg, HBcAg, HBeAg, DNA reverse transcriptase gold, and protein X, respectively. cccDNA transcribes 4 HBV RNAs. pgRNA was used as a template for reverse transcription of HBV DNA. Notably, HBsAg is also produced by the process of transcribing integrated viral DNA.

In clinical diagnosis, high levels of HBsAg are considered to be one of the hallmarks of chronic HBV infection. HBsAg may lead to depletion of antiviral CD8+ T cells. It negatively regulates HBV-specific immune responses. The persistent presence of HBsAg is a major marker for the risk of developing chronic liver disease and advanced HCC. HBV "functional cure", defined as HBsAg clearance, and subsequent seroconversion and detectable anti-HBs antibody levels, is an ideal endpoint for HBV treatment. Thus, drugs that reduce HBsAg levels would provide a potential treatment for chronic HBV infection and reduce the risk of severe liver disease.

The goal of chronic hepatitis b treatment is to suppress HBV replication for a long period of time to the maximum extent, and delay and reduce the occurrence of complications, thereby improving quality of life and prolonging life. At present, the medicines for treating chronic hepatitis B mainly comprise immunomodulators, nucleotide reverse transcriptase inhibitors and nucleoside reverse transcriptase inhibitors. A representative drug for immunomodulators is PEG-IFN alpha-2 a (PegIFN alpha-2 a). Nucleoside reverse transcriptase inhibitors are required to achieve long-lasting viral inhibition for the long-term treatment of chronic hepatitis B. Entecavir (ETV), Tenofovir (TDF) and propionofovir fumarate (TAF) are first-line monotherapy drugs of chronic hepatitis b that are all very effective in inhibiting the virus according to recommendations in the guidelines for anti-hepatitis b drugs revised by the national biomedical research center for liver and digestive disorders in spain.

Currently, when these first-line marketed drugs are used for treating chronic hepatitis B patients, the serological conversion rate of HBsAg is low, long-term administration or lifetime administration is required, and relapse is likely to occur after withdrawal. Therefore, the HBsAg inhibitor with high response rate and stronger effect is urgently needed in clinic, the functional cure rate is improved by single medicine or the combination of the existing first-line medicines, the goal of no relapse after medicine withdrawal is realized, and the prognostic life quality of patients is obviously improved.

Disclosure of Invention

In one aspect, the invention provides a combination comprising a compound of formula (V) and another agent for the treatment of hepatitis B.

In another aspect, the present invention provides a composition comprising a combination of the invention and a pharmaceutically acceptable carrier and/or excipient.

In another aspect, the invention provides a kit comprising a composition of the invention.

In another aspect, the invention provides the use of a combination, composition or kit for the manufacture of a medicament for the treatment of hepatitis b. The present invention also provides a method of treating hepatitis b comprising administering to a subject an effective amount of a combination, composition or kit of the invention. The invention also provides the use of a combination, composition or kit of the invention in the treatment of hepatitis b.

In another aspect, the invention provides the use of a compound of formula (V) in combination with other agents for treating hepatitis B for the manufacture of a medicament for treating hepatitis B. The invention also provides a method for treating hepatitis B, which comprises the step of administering an effective amount of the compound shown as the formula (V) and other medicines for treating the hepatitis B to a subject. The invention also provides the application of the compound shown in the formula (V) in combination with other medicaments for treating hepatitis B to treat the hepatitis B.

The invention provides a combination, which comprises a compound shown as a formula (V) and other medicaments for treating hepatitis B,

the invention provides a combination comprising a compound of formula (V), an isomer thereof or a pharmaceutically acceptable salt thereof and other compounds,

wherein the content of the first and second substances,

with "-" carbon atoms as chiral carbon atoms, in the form of (R) or (S) single enantiomers or enriched in one enantiomer;

R₁selected from H, OH, CN, NH₂Or is selected from optionally substituted with 1,2 or 3R: c_1-6Alkyl radical, C_1-6Heteroalkyl group, C_2-5Alkenyl radical, C_2-5Heteroalkenyl, C_3-6Cycloalkyl or 3-to 6-membered heterocycloalkyl;

R₂selected from H, OH, CN, NH₂Halogen, or selected from optionally substituted with 1,2 or 3R: c_1-3Alkyl radical, C_1-3Heteroalkyl group, C_3-6Cycloalkyl or 3-to 6-membered heterocycloalkyl;

R₃selected from optionally substituted with 1,2 or 3R: c_1-6Alkyl radical, C_3-6A cycloalkyl group;

m is selected from: 0. 1,2,3, 4 or 5;

when m is 0, R₁Is not selected from: OH, CN, NH₂；

R is selected from H, halogen, OH, CN, NH₂Or is selected from optionally substituted with 1,2 or 3R': c_1-3Alkyl radical, C_1-3A heteroalkyl group;

r' is selected from: F. cl, Br, I, OH, CN, NH₂、CH₃、CH₃CH₂、CH₃O、CF₃、CHF₂、CH₂F；

"hetero" means a heteroatom or a heteroatom group, said C_1-6Heteroalkyl group, C_2-5Heteroalkenyl, 3-to 6-membered heterocycloalkyl, C_1-3Each of the "hetero" of heteroalkyl groups is independently selected from: -C (═ O) N (R) -, -C (═ NR) -, - (R) C ═ N-, -S (═ O)₂N(R)-、-S(＝O)N(R)-、N、-O-、-S-、＝O、＝S、-C(＝O)O-、-C(＝O)-、-C(＝S)-、-S(＝O)-、-S(＝O)₂-、-N(R)C(＝O)N(R)-；

In any of the above cases, the number of heteroatoms or heteroatom groups is independently selected from 1,2 or 3;

characterized in that the further compound is selected from the group consisting of an immunomodulator, a nucleotide reverse transcriptase inhibitor and a nucleoside reverse transcriptase inhibitor.

In some embodiments of the present invention, the combination comprises a compound of formula (V) or a pharmaceutically acceptable salt thereof and an immunomodulator, wherein the immunomodulator is selected from the group consisting of pegylated interferon alfa-2 a.

In some embodiments of the present invention, the combination comprises a compound represented by formula (V) or a pharmaceutically acceptable salt thereof and a nucleotide reverse transcriptase inhibitor, wherein the nucleotide reverse transcriptase inhibitor is selected from the group consisting of tenofovir disoproxil fumarate and tenofovir disoproxil fumarate.

In some embodiments of the present invention, the combination comprises a compound represented by formula (V) or a pharmaceutically acceptable salt thereof and a nucleoside reverse transcriptase inhibitor, wherein the nucleoside reverse transcriptase inhibitor is selected from entecavir.

In the inventionIn some embodiments, the above combination, wherein R is selected from H, F, Cl, Br, I, OH, CN, NH₂、CH₃、CH₃CH₂、CH₃O、CF₃、CHF₂And CH₂F, other variables are as defined herein.

In some embodiments of the invention, the above combination, wherein R₁Selected from H, OH, CN, NH₂Or is selected from optionally substituted with 1,2 or 3R: c_1-3Alkyl radical, C_1-3Heteroalkyl group, C_2-3Alkenyl radical, C_2-3Heteroalkenyl, C_3-6Cycloalkyl and 3-6 membered heterocycloalkyl, the other variables being as defined herein.

In some embodiments of the invention, the above combination, wherein R₁Selected from H, OH, CN and NH₂Or is selected from optionally substituted with 1,2 or 3R: CH (CH)₃、 Other variables are as defined herein.

In some embodiments of the invention, the above combination, wherein R₁Selected from H, OH, CN, NH₂、 Other variables are as defined herein.

In some embodiments of the invention, the above combination, wherein R₂Selected from H, OH, CN, NH₂And halogen, or selected from optionally substituted with 1,2 or 3R: c_1-3Alkyl radical, C_1-3Heteroalkyl group and C_3-6Cycloalkyl, the other variables are as defined herein.

In some embodiments of the invention, the above combination, wherein R₂Selected from H, OH, CN, NH₂F, Cl, Br and I, or selected from optionally substituted with 1,2 or 3R: CH (CH)₃、Other variables are as defined herein.

In some embodiments of the invention, the above combination, wherein R₂Selected from Cl, Br, CN, CH₃、Other variables are as defined herein.

In some embodiments of the invention, the above combination, wherein R₃Selected from optionally substituted with 1,2 or 3R: c_1-4Alkyl and C_3-6Cycloalkyl, the other variables are as defined herein.

In some embodiments of the invention, the above combination, wherein R₃Is selected fromOther variables are as defined herein.

In some embodiments of the invention, the above combination, wherein m is selected from 0, 1,2,3 and 4, and when m is 0, R is₁Is not selected from OH, CN and NH₂The other variables are as defined herein.

In some embodiments of the invention, the above combinations wherein the structural elementsIs selected from Other variables are as defined herein.

In some embodiments of the invention, the combination wherein the carbon atom with the "x" is a chiral carbon atom is present in the form of (R) a single enantiomer or in a form enriched in one enantiomer, the other variables being as defined herein.

In some embodiments of the invention, the above combination, wherein the compound, isomer thereof or pharmaceutically acceptable salt thereof is selected from

Wherein the content of the first and second substances,

with "-" carbon atoms as chiral carbon atoms, in the form of (R) or (S) single enantiomers or enriched in one enantiomer;

R₄selected from H, or selected from optionally substituted with 1,2 or 3R: c_1～3Alkyl and C_1～3A heteroalkyl group;

x is selected from C and N;

y is selected from O and C;

L₁and L₂Are each independently selected from the group consisting of single bond, - (CH)₂) n-and-C (═ O) -;

and L is₁And L₂Not being a single bond at the same time;

n is selected from 1 and 2;

m、R、R₂、R₃and C_1～3The "hetero" in heteroalkyl is as described above, and R is when m is 0₄Is not H.

In some embodiments of the invention, the combination wherein the carbon atom with the "x" is a chiral carbon atom is present in the form of (R) a single enantiomer or in one enantiomer.

In some embodiments of the invention, the above combination, wherein the compound is selected from

In some embodiments of the invention, the above combination, wherein the compound is selected from

The invention also provides the application of the composition in preparing a medicament for treating hepatitis B.

The invention also provides a composition, which is characterized by comprising the combination and at least one pharmaceutically acceptable carrier and/or excipient.

The present invention also provides a kit comprising the above combination or the above composition.

The invention also provides application of the composition or the kit in preparing a medicament for treating hepatitis B.

In some embodiments of the present invention, the above combination is a combination for treating chronic hepatitis b.

In some embodiments of the present invention, the composition is a pharmaceutical composition for treating chronic hepatitis b.

In some embodiments of the present invention, the kit is a kit for treating chronic hepatitis b.

In some embodiments of the present invention, the kit further comprises instructions for the use of the compound of formula (V) in combination with other agents for treating hepatitis B.

In some embodiments of the present invention, the pharmaceutical composition of the compound of formula (V) comprises a compound of formula (V) and a pharmaceutically acceptable carrier and/or excipient.

In some embodiments of the present invention, the pharmaceutical composition of the compound of formula (V) is in the form of a solid formulation, preferably a capsule or tablet.

In some embodiments of the present invention, the pharmaceutical composition of the other medicament for treating hepatitis b comprises the other medicament for treating hepatitis b, and a pharmaceutically acceptable carrier and/or excipient.

In some embodiments of the present invention, the pharmaceutical composition of the other medicament for treating hepatitis b is in the form of a liquid preparation, preferably an aqueous injection, including but not limited to an aqueous preparation that is not lyophilized or an aqueous preparation that is reconstituted from a lyophilized powder.

Mode of administration

The following does not limit the mode of administration of the combination of the invention.

The components of the combination of the invention may each be formulated separately, or some or all of them may be co-formulated as a pharmaceutical composition. In some embodiments, the combination of the invention may be formulated as a pharmaceutical composition suitable for single or multiple administration.

The components of the combination of the invention may each be administered separately or some or all of them may be co-administered. The components of the combination of the invention may be administered substantially simultaneously, or some or all of them may be administered substantially simultaneously. The components of the combination of the invention may have the same or different administration periods.

The components of the combination of the invention may each independently be administered by a suitable variety of routes including, but not limited to, oral or parenteral (by intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion). In some embodiments, the components of the combination of the invention may each be administered orally or parenterally, independently, for example intravenously or intraperitoneally.

The components of the combination of the invention may each independently be in a suitable dosage form including, but not limited to, tablets, troches, pills, capsules (e.g., hard capsules, soft capsules, enteric capsules, microcapsules), elixirs, granules, syrups, injections (intramuscular, intravenous, intraperitoneal), granules, emulsions, suspensions, solutions, dispersions and sustained release formulations for oral or non-oral administration.

The components of the combination of the invention may each independently contain a pharmaceutically acceptable carrier and/or excipient.

Technical effects

The compound of the formula (V) is a hepatitis B surface antigen inhibitor and can effectively reduce the content of HBsAg. By combining the compound in the formula (V) with the immunomodulator or combining the compound in the formula (V) with a nucleoside or nucleotide reverse transcriptase inhibitor, the effects on HBsAg, HBeAg and HBV DNA are obviously enhanced, a synergistic inhibition effect is generated, the aims of improving the curative effect, reducing toxic and side effects, shortening the administration treatment period and dosage, reducing drug resistance and the like can be fulfilled, and the effects of reducing the HBV loading and reducing or even eliminating HBsAg are achieved.

Definitions and explanations

As used herein, the following terms and phrases are intended to have the following meanings, unless otherwise indicated. A particular term or phrase, unless specifically defined, should not be considered as indefinite or unclear, but rather construed according to ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient. The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts. Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt.

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two.

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

Unless otherwise indicated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

Unless otherwise indicated, the term "cis-trans isomer" or "geometric isomer" results from the inability of a double bond or a single bond to rotate freely within a ring-forming carbon atom.

Unless otherwise indicated, the term "diastereomer" refers to a stereoisomer in which the molecules have two or more chiral centers and a non-mirror image relationship between the molecules.

Unless otherwise indicated, "(+)" means dextrorotation, "(-) -means levorotation," (±) "means racemization.

By solid wedge-shaped keys (unless otherwise indicated)) And wedge dotted bond: () Representing the absolute configuration of a solid center by means of straight solid-line bonds () And straight dotted bonds: () Showing the relative configuration of the centres of solids by wavy lines (a), (b), (c), (d) and (d)) Represents a wedge-shaped solid-line key () Or wedge dotted bond () Or by wavy lines () Represents a straight solid-line key () And straight dotted bonds: ()。

The compounds of the invention may be present specifically. Unless otherwise indicated, the term "tautomer" or "tautomeric form" means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be rapidly interconverted. If tautomers are possible (e.g., in solution), then the chemical equilibrium of the tautomers can be reached. For example, proton tautomers (prototropic tautomers), also known as proton transfer tautomers (prototropic tautomers), include interconversions by proton transfer, such as keto-enol isomerization and imine-enamine isomerization. Valence isomers (valencetatomer) include interconversion by recombination of some of the bonding electrons. A specific example of where keto-enol tautomerism is the interconversion between two tautomers of pentane-2, 4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise indicated, when a compound of the present invention is an isomer, there is "enriched in one isomer", "enriched in one enantiomer", or "enriched in enantiomer". The term "enriched in one isomer", "isomer enriched", "enantiomer enriched" or "enantiomeric enrichment" refers to a content of one isomer or enantiomer of less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, when a compound of the invention is an isomer, there is an "isomeric excess" or an "enantiomeric excess". The term "enantiomeric excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80%.

Optically active (R) -and (S) -isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one of the enantiomers of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and the pure enantiomers are recovered. Furthermore, separation of enantiomers and diastereomers is typically accomplished by using chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amines). The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be labelled with radioactive isotopes, such as tritium (A), (B), (C) and C)³H) Iodine-125 (¹²⁵I) Or C-14(¹⁴C) In that respect For example, deuterium can be used to replace hydrogen to form a deuterated drug, the bond formed by deuterium and carbon is stronger than the bond formed by common hydrogen and carbon, and compared with an undeuterated drug, the deuterated drug has the advantages of reducing toxic and side effects, increasing the stability of the drug, enhancing the curative effect, prolonging the biological half-life period of the drug and the like. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The term "pharmaceutical composition" refers to a mixture of one or more of the active ingredients of the present application or a pharmaceutical combination thereof with pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate administration of the compounds of the present application, or a pharmaceutical combination thereof, to a subject.

The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium capable of delivering an effective amount of an active agent of the present invention, without interfering with the biological activity of the active agent, and without toxic side effects to the host or patient, and representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. These include suspending agents, viscosity enhancers, skin penetration enhancers, and the like. Their preparation is known to those skilled in the cosmetic or topical pharmaceutical field.

The term "excipient" generally refers to a carrier, diluent, and/or vehicle necessary to formulate an effective pharmaceutical composition.

The words "comprise" or "comprise" and variations thereof such as "comprises" or "comprising," are to be understood in an open, non-exclusive sense, i.e., "including but not limited to.

The term "treating" means administering a compound or formulation described herein to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) preventing the occurrence of a disease or condition in a mammal, particularly when such mammal is susceptible to the disease condition, but has not yet been diagnosed as having the disease condition;

(ii) inhibiting the disease or disease state, i.e., arresting its development;

(iii) alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "effective amount" or "therapeutically effective amount" with respect to a drug or pharmacologically active agent refers to a sufficient amount of the drug or agent that is non-toxic but achieves the desired effect. For oral dosage forms of the invention, an "effective amount" of one active agent in a composition is the amount required to achieve the desired effect when combined with another active agent in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient and also on the particular active substance, and an appropriate effective amount in an individual case can be determined by a person skilled in the art according to routine tests.

The term "administering" means physically introducing a composition comprising a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration of the immune checkpoint inhibitor (e.g., anti-PD-1 antibody or anti-PD-L1 antibody) include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In certain embodiments, the immune checkpoint inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) is administered by a non-parenteral route, and in certain embodiments, orally. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, e.g., intranasally, vaginally, rectally, sublingually or topically. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods of time.

The term "subject" is a mammal. In some embodiments, the subject is a mouse. In some embodiments, the subject is a human.

As used herein, "in combination" or "in combination" means that two or more active substances may each be administered to a subject simultaneously as a single formulation, or sequentially in any order each as a single formulation.

The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

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

The term "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent, and may include variations of deuterium and hydrogen, so long as the valency of the particular atom is normal and the substituted compound is stable. When the substituent is a keto group (i.e., ═ O), it means that two hydrogen atoms are substituted. The keto substitution does not occur on the aromatic group. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the kind and number of substituents may be arbitrary on the basis of chemical realizability.

When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2R, the group may optionally be substituted with up to two R, and there are separate options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

When the number of one linking group is 0, e.g. - (CRR)₀-, represents that the linking group is a single bond.

When one of the variables is selected from a single bond, it means that the two groups to which it is attached are directly connected, for example, where L represents a single bond in A-L-Z means that the structure is actually A-Z.

When a substituent is absent, it indicates that the substituent is absent, e.g., when X is absent in A-X, it indicates that the structure is actually A. When a substituent may be attached to more than one atom of a ring, such substituent may be bonded to any atom of the ring, e.g. a building blockIndicates that the substituent R can be substituted at any position on the cyclohexyl or the cyclohexadiene. When a substituent is listed it is not indicated through which atom it passesWhen a group is substituted, such substituent may be bonded through any atom thereof, for example, a pyridyl group as a substituent may be bonded to the substituted group through any carbon atom on the pyridyl ring. When the listed linking groups do not indicate their direction of attachment, the direction of attachment is arbitrary, for example,wherein the linking group L is-M-W-, in which case-M-W-can be formed by connecting the ring A and the ring B in the same direction as the reading sequence from left to rightThe ring A and the ring B may be connected in the reverse direction of the reading sequence from left to rightCombinations of the linking groups, substituents, and/or variants thereof are permissible only if such combinations result in stable compounds.

Unless otherwise specified, the term "hetero" denotes a heteroatom or a heteroatom group (i.e., a heteroatom-containing radical) including atoms other than carbon (C) and hydrogen (H) and radicals containing such heteroatoms, including, for example, oxygen (O), nitrogen (N), sulfur (S), silicon (Si), germanium (Ge), aluminum (Al), boron (B), -O-, -S-, ═ O, ═ S, -C (═ O) O-, -C (═ O) -, -C (═ S) -, -S (═ O)₂-, and optionally substituted-C (═ O) n (h) -, -C (═ NH) -, -S (═ O)₂N (h) -or-S (═ O) n (h) -.

Unless otherwise specified, "cyclic" means substituted or unsubstituted cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aryl, or heteroaryl. The term "ring" includes monocyclic, bicyclic, spiro, fused or bridged rings. The number of atoms in the ring is generally defined as the number of ring members, for example, "5 to 7 membered ring" means 5 to 7 atoms arranged around the ring. Unless otherwise specified, the ring optionally contains 1-3 heteroatoms. Thus, "5 to 7 membered ring" includes, for example, phenyl, pyridine and piperidinyl; in another aspect, the term "5-to 7-membered heterocycloalkyl ring" includes pyridyl and piperidyl, but does not include phenyl. The term "ring" also includes ring systems containing at least one ring, each of which "ring" independently conforms to the above definition.

Unless otherwise specified, the term "heterocycle" or "heterocyclyl" means a stable heteroatom or heteroatom group containing monocyclic, bicyclic, or tricyclic ring which may be saturated, partially unsaturated, or unsaturated (aromatic), which contains carbon atoms and 1,2,3, or 4 ring heteroatoms independently selected from N, O and S, wherein any of the above heterocycles can be fused to a benzene ring to form a bicyclic ring. The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., NO and S (O) p, p being 1 or 2). The nitrogen atom may be substituted or unsubstituted (i.e. N or NR, wherein R is H or other substituents already defined herein). The heterocyclic ring may be attached to any heteroatom or carbon pendant group to form a stable structure. The heterocyclic rings described herein may be substituted at the carbon or nitrogen position if the resulting compound is stable. The nitrogen atoms in the heterocycle are optionally quaternized. In a preferred embodiment, when the total number of S and O atoms in the heterocycle exceeds 1, these heteroatoms are not adjacent to each other. In another preferred embodiment, the total number of S and O atoms in the heterocycle does not exceed 1. As used herein, the term "aromatic heterocyclic group" or "heteroaryl" means a stable 5,6, 7 membered monocyclic or bicyclic or 7, 8, 9 or 10 membered bicyclic heterocyclic group aromatic ring comprising carbon atoms and 1,2,3 or 4 ring heteroatoms independently selected from N, O and S. The nitrogen atom may be substituted or unsubstituted (i.e. N or NR, wherein R is H or other substituents already defined herein). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., NO and S (O) p, p being 1 or 2). It is noted that the total number of S and O atoms on the heteroaromatic ring does not exceed 1. Bridged rings are also included in the definition of heterocyclic. Bridged rings are formed when one or more atoms (i.e., C, O, N or S) connect two non-adjacent carbon or nitrogen atoms. Preferred bridged rings include, but are not limited to: one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms and one carbon-nitrogen group. It is worth noting that a bridge always converts a single ring into a three ring. In bridged rings, ring substituents may also be present on the bridge.

Examples of heterocyclic compounds include, but are not limited to: acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzomercaptofuranyl, benzomercaptophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4 aH-carbazolyl, carbolinyl, chromanyl, chromene, cinnolinyl decahydroquinolinyl, 2H,6H-1,5, 2-dithiazinyl, dihydrofuro [2,3-b ] tetrahydrofuranyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, dihydroindolyl, and the like, Methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2, 3-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazine, phenothiazine, benzoxanthinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, quinazolinyl, quinolyl, 4H-quinolizinyl, quinoxalinyl, Quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2, 5-thiadiazinyl, 1,2, 3-thiadiazolyl, 1,2, 4-thiadiazolyl, 1,2, 5-thiadiazolyl, 1,3, 4-thiadiazolyl, thianthrenyl, thiazolyl, isothiazolylthienyl, thienooxazolyl, thienothiazolyl, thienoimidazolyl, thienyl, triazinyl, 1H-1,2, 3-triazolyl, 2H-1,2, 3-triazolyl, 1H-1,2, 4-triazolyl, 4H-1,2, 4-triazolyl, and xanthenyl. Fused ring and spiro compounds are also included.

Unless otherwise specified, the term "hydrocarbyl" or its progeny (e.g., alkyl, alkenyl, alkynyl)An alkyl group, an aryl group, etc.) by itself or as part of another substituent means a straight, branched or cyclic hydrocarbon radical or combination thereof, which may be fully saturated (e.g., alkyl), mono-or poly-unsaturated (e.g., alkenyl, alkynyl, aryl), which may be mono-or poly-substituted, which may be mono-or poly-valent (e.g., methyl), di-or poly-valent (e.g., methine), which may include di-or poly-valent radicals having the specified number of carbon atoms (e.g., C)₁-C₁₂Represents 1 to 12 carbons, C_1-12Is selected from C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁And C₁₂；C_3-12Is selected from C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁And C₁₂. ). "hydrocarbyl" includes, but is not limited to, aliphatic hydrocarbyl including linear and cyclic, specifically including, but not limited to, alkyl, alkenyl, alkynyl, and aromatic hydrocarbyl including, but not limited to, 6-12 membered aromatic hydrocarbyl such as benzene, naphthalene, and the like. In some embodiments, the term "hydrocarbyl" denotes a straight or branched chain radical or a combination thereof, which may be fully saturated, mono-or polyunsaturated, and may include divalent and polyvalent radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, isobutyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, and homologs or isomers of radicals such as n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated hydrocarbon groups have one or more double or triple bonds, examples of which include, but are not limited to, ethenyl, 2-propenyl, butenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers.

Unless otherwise specified, the term "heterohydrocarbyl" or its subset (such as heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and the like) by itselfOr in combination with another term, means a stable linear, branched, or cyclic hydrocarbon radical, or combinations thereof, having a number of carbon atoms and at least one heteroatom. In some embodiments, the term "heteroalkyl," by itself or in combination with another term, means a stable straight-chain, branched-chain hydrocarbon radical, or combination thereof, having a number of carbon atoms and at least one heteroatom constituent. In one exemplary embodiment, the heteroatoms are selected from B, O, N and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatom or heteroatom group may be located at any internal position of the heterohydrocarbyl group, including the position at which the hydrocarbyl group is attached to the remainder of the molecule, but the terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional expressions to refer to those alkyl groups that are attached to the remainder of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively. Examples include, but are not limited to-CH₂-CH₂-O-CH₃、-CH₂-CH₂-NH-CH₃、-CH₂-CH₂-N(CH₃)-CH₃、-CH₂-S-CH₂-CH₃、-CH₂-CH₂、-S(O)-CH₃、-CH₂-CH₂-S(O)₂-CH₃、-CH＝CH-O-CH₃、-CH₂-CH＝N-OCH₃and-CH ═ CH-N (CH)₃)-CH₃. Up to two heteroatoms may be consecutive, e.g. -CH₂-NH-OCH₃。

Unless otherwise specified, the terms "cycloalkyl", "heterocycloalkyl", or a subset thereof (e.g., aryl, heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, etc.) by themselves or in combination with other terms, mean cyclized "alkyl", "heteroalkyl", respectively. Furthermore, in the case of a heterohydrocarbyl or heterocycloalkyi (e.g., heteroalkyl, heterocycloalkyl), a heteroatom may occupy the position of the heterocycle attached to the rest of the molecule. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Non-limiting examples of heterocyclyl groups include 1- (1,2,5, 6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran indol-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, and 2-piperazinyl.

Unless otherwise specified, the term "alkyl" is intended to mean a straight-chain or branched-chain saturated hydrocarbon radical, which may be monosubstituted (e.g., -CH)₂F) Or polysubstituted (e.g. -CF)₃) And may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like.

Unless otherwise specified, "alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds at any position in the chain, which may be mono-or poly-substituted, and which may be mono-, di-or polyvalent. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, piperylene, hexadienyl, and the like.

Unless otherwise specified, "alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds at any position in the chain, which may be mono-or poly-substituted, and which may be mono-, di-or polyvalent. Examples of alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, and the like.

Unless otherwise specified, cycloalkyl includes any stable cyclic or polycyclic hydrocarbon group, any carbon atom is saturated, may be mono-or poly-substituted, and may be mono-, di-or polyvalent. Examples of such cycloalkyl groups include, but are not limited to, cyclopropyl, norbornyl, [2.2.2] bicyclooctane, [4.4.0] bicyclodecane, and the like.

Unless otherwise specified, cycloalkenyl includes any stable cyclic or polycyclic hydrocarbon radical containing one or more unsaturated carbon-carbon double bonds at any position on the ring, which may be mono-or poly-substituted, and which may be mono-, di-or polyvalent. Examples of such cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexenyl, and the like.

Unless otherwise specified, cycloalkynyl includes any stable cyclic or polycyclic hydrocarbon radical containing one or more carbon-carbon triple bonds at any position in the ring, which may be mono-or poly-substituted, and which may be mono-, di-or polyvalent.

Unless otherwise specified, the term "halogen" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom. Furthermore, the term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C)₁-C₄) Alkyl "is intended to include, but not be limited to, trifluoromethyl, 2, 2, 2-trifluoroethyl, 4-chlorobutyl, and 3-bromopropyl, and the like. Unless otherwise specified, examples of haloalkyl include, but are not limited to: trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.

"alkoxy" represents the above alkyl group having the specified number of carbon atoms attached through an oxygen bridge, unless otherwise specified, C_1-6Alkoxy radicals comprising C₁、C₂、C₃、C₄、C₅And C₆Alkoxy group of (2). Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy and S-pentoxy.

Unless otherwise specified, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent, which may be mono-or poly-substituted, and which may be mono-, di-or polyvalent, and which may be mono-or polycyclic (e.g., 1 to 3 rings; wherein at least one ring is aromatic), fused together or covalently linked. The term "heteroaryl" refers to an aryl (or ring) containing one to four heteroatoms. In one illustrative example, the heteroatom is selected from B, N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl or heteroaryl include phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, phenyl-oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidinyl, benzothiazolyl, purinyl, benzimidazolyl, indolyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, oxazolyl, 2-oxazolyl, and the like, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl. The substituents for any of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

Unless otherwise specified, aryl when used in combination with other terms (e.g., aryloxy, arylthio, aralkyl) includes aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" is intended to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like), including those alkyl groups in which a carbon atom (e.g., methylene) has been replaced by, for example, an oxygen atom, such as phenoxymethyl, 2-pyridyloxymethyl 3- (1-naphthyloxy) propyl and the like.

The term "leaving group" refers to a functional group or atom that can be substituted by another functional group or atom through a substitution reaction (e.g., an affinity substitution reaction). For example, representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-toluenesulfonate and the like; acyloxy groups such as acetoxy, trifluoroacetyloxy, and the like.

The term "protecting group" includes, but is not limited to, "amino protecting group," hydroxyl protecting group, "or" thiol protecting group. The term "amino protecting group" refers to a protecting group suitable for use in preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: a formyl group; acyl, for example alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups such as benzyl (Bn), trityl (Tr), 1-bis- (4' -methoxyphenyl) methyl; silyl groups, such as Trimethylsilyl (TMS) and t-butyldimethylsilyl (TBS), and the like. The term "hydroxy protecting group" refers to a protecting group suitable for use in preventing side reactions of a hydroxy group. Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups, such as alkanoyl (e.g., acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (benzhydryl, DPM); silyl groups, such as Trimethylsilyl (TMS) and t-butyldimethylsilyl (TBS), and the like.

The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, examples of the present invention.

The solvent used in the present invention can be commercially available.

The invention employs the following abbreviations: DMSO represents dimethyl sulfoxide; HBsAg represents hepatitis B surface antigen; HBeAg stands for hepatitis B e antigen; cccDNA represents a covalent, closed, circular DNA molecule.

The compounds are used according to the conventional naming principle in the fieldThe software names, and the commercial compounds are under the supplier catalog name.

Drawings

FIG. 1 shows the results of HBV inhibition and cell viability in well plates with single or double drug combinations.

FIG. 2 shows the results of cell viability (viatility) of well plates with single or double drug combinations.

Detailed Description

The present invention is described in detail below by way of examples, but is not meant to be limited to any of the disadvantages of the present invention. Having described the invention in detail and having disclosed specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Experimental example 1

Purpose of the experiment: the objective of this study was to use human primary hepatocytes (PHH) isolated from a humanized FRG mouse model to evaluate the in vitro anti-HBV efficacy of test compounds administered in combination with ETV (entecavir), TAF (Propofovir fumarate), and Peg-IFN-. alpha.2a (Peg-IFN-. alpha.2a).

Experimental materials:

(1) the main apparatus is as follows: fluorescent qPCR instruments (Applied Biosystems, model 7500). Microplate reader (Bio Tek, model Synergy 2). Chemiluminescence imaging system (GE, model LAS4010)

(2) Main reagents and consumables: FastStart Universal Probe Master (Roche), DNA extraction Kit (Qiagen), 96-well plate (Costar 3599) and 96well V-plate (Axygen WIPP02280), HBsAg ELISA Kit (Autobio) and HBeAg ELISA Kit (Autobio), Cell Counting Kit-8(CCK-8) (Biolite).

(3) And others: FRG mouse, type D HBV, test Compound WWS1

The experimental method comprises the following steps:

on day 0, PHH (primary humanized hepatocytes) was isolated using collagenase perfusion, and the separated human primary hepatocytes were adjusted to a density of 600000 cells per ml and seeded into 8 48-well cell plates (1.2X 10) in volumes of 200. mu.L and 420. mu.L, respectively⁵One cell/well) and 2 24-well cell plates (2.52X 10)⁵Individual cells/well).

Day 1, human primary hepatocytes (400HBV GE/cell) were infected.

On day 2, different groups of combinations were added for treatment. The 48-well cell plate is provided with a single compound treatment group, a double-drug combined treatment group, a DMSO control group and an uninfected group, the initial concentration of a test compound is 100nM, the initial concentrations of control compounds ETV, TAF and Peg-IFN-alpha 2a are respectively 50pM, 50pM and 50IU/mL, 3-fold dilution is carried out, the total concentration is 5, the test is carried out by a matrix of 5 multiplied by 5, three-fold plate is carried out, the final concentration of DMSO is 2%, and the arrangement and combined drug concentration setting are shown in tables 1,2,3 and 4. A24-well culture plate is provided with a single compound treatment group, a double-drug combined treatment group and a DMSO control group, tested compounds with different concentrations and 10IU/mL and 25IU/mL Peg-IFN-alpha 2a are respectively subjected to combined treatment, the initial concentration of the tested compounds is 100nM, 5-time dilution is carried out, 3 concentration points are formed, three wells are formed, the final concentration of DMSO is 2%, and the arrangement and combined drug concentration are set as shown in tables 1 to 5.

TABLE 1 Placement of test Compounds with Peg-IFN-. alpha.2a

Note: the test compound (a ═ WWS1, b ═ Peg-IFN- α 2a, c ═ ETV, d ═ TAF); 0 represents no compound, 1-5 represents 5 concentrations; DMSO represents DMSO control; uninf represents the uninfected group.

Table 2 alignment of test compounds with ETV

1

2

3

4

5

6

7

8

A

a0；c5

a5；c5

a4；c5

a3；c5

a2；c5

a1；c5

DMSO

Uninf

B

a0；c4

a5；c4

a4；c4

a3；c4

a2；c4

a1；c4

DMSO

Uninf

C

a0；c3

a5；c3

a4；c3

a3；c3

a2；c3

a1；c3

DMSO

Uninf

D

a0；c2

a5；c2

a4；c2

a3；c2

a2；c2

a1；c2

DMSO

Uninf

E

a0；c1

a5；c1

a4；c1

a3；c1

a2；c1

a1；c1

DMSO

Uninf

F

DMSO

a5；c0

a4；c0

a3；c0

a2；c0

a1；c0

DMSO

Uninf

Note: the test compound (a ═ WWS1, b ═ Peg-IFN- α 2a, c ═ ETV, d ═ TAF); 0 represents no compound, 1-5 represents 5 concentrations; DMSO represents DMSO control; uninf represents the uninfected group.

TABLE 3 Placement of test Compounds with TAF

1

2

3

4

5

6

7

8

A

a0；d5

a5；d5

a4；d5

a3；d5

a2；d5

a1；d5

DMSO

Uninf

B

a0；d4

a5；d4

a4；d4

a3；d4

a2；d4

a1；d4

DMSO

Uninf

C

a0；d3

a5；d3

a4；d3

a3；d3

a2；d3

a1；d3

DMSO

Uninf

D

a0；d2

a5；d2

a4；d2

a3；d2

a2；d2

a1；d2

DMSO

Uninf

E

a0；d1

a5；d1

a4；d1

a3；d1

a2；d1

a1；d1

DMSO

Uninf

F

DMSO

a5；d0

a4；d0

a3；d0

a2；d0

a1；d0

DMSO

Uninf

Note: the test compound (a ═ WWS1, b ═ Peg-IFN- α 2a, c ═ ETV, d ═ TAF); 0 represents no compound, 1-5 represents 5 concentrations; DMSO represents DMSO control; uninf represents the uninfected group.

Table 448 well cell plate combination drug delivery arrangement and concentration settings

Note: medium stands for the Medium in which Peg-IFN-. alpha.2a is present

Table 524 well cell plate combination drug delivery configuration and concentration settings

The culture broth and compound were changed on days 4, 6, 8, 10. On day 12, cell supernatants were collected, DNA in the supernatants was extracted using DNA extraction kit (Qiagen-51162), and HBV DNA in the samples was quantified using qPCR; the levels of HBeAg and HBsAg in the supernatant were measured using ELISA kits. The cells after collecting the supernatant were examined for cell viability using CCK-8. After the cells in the 24-well culture plate are subjected to CCK-8 detection, the cells are washed twice by PBS, Hirt DNA is collected and extracted, and the expression level of cccDNA in the cells is detected by using a Southern blot method (blotting hybridization).

The qPCR reaction system is shown in table 5. HBV plasmid DNA as standard substance, 10 times serial dilution, standard substance range from 1.0X 10⁷copies/. mu.l to 10 copies/. mu.l.

TABLE 5 qPCR reaction System

Reagent	Configuration 1 required volume of well (μ l)
		Quantitative quick-start universal probe reagent	12.5
Forward primer (10. mu.M)	1
		Reverse primer (10. mu.M)	1
Probe (10 μ M)	0.5

The PCR reaction program is: heating at 95 deg.C for 10min, then entering into circulation mode, heating at 95 deg.C for 15sec, then 60 deg.C for 1min, for 40 cycles. Calculating the HBV DNA content of the sample according to the standard curve and the Ct value of each sample.

The HBeAg and HBsAg levels are detected by an ELISA kit, and the HBV HBeAg and HBsAg contents in the samples are calculated according to the standard curve and the chemiluminescence values of the samples.

Inhibition [ [ 1 ] -HBV DNA content or HBsAg content or HBeAg content in sample/HBV DNA content or HBsAg content or HBeAg content in DMSO control group ]. times.100

EC₅₀Values were calculated by Prism software and the inhibition curve fitting method was a sigmoidal dose-response (variable slope). The effect of the 48-well plate combination was analyzed by MacSynergy software.

Cell viability was measured by the CCK-8 method, based on OD of each sample₄₅₀/OD₆₃₀The cell viability was calculated.

Cell viability% (% sample OD value-medium control OD value)/[ DMSO control luminescence value-medium control OD value ] x 100

CC₅₀Values were calculated by Prism software and the inhibition curve fitting method was a sigmoidal dose-response (variable slope).

Intracellular cccDNA levels in 24-well culture plates were detected by Southern blot method.

In 48-well culture plates, the combined efficacy of WWS1 and ETV, TAF, Peg-IFN-. alpha.2a was analyzed by MacSynergy software. The combined efficacy was calculated based on the 95% confidence interval of synergy/antagonism, and the results were interpreted according to the MacSynergy criterion as follows:

<25 > the insigniac syntergism/antagonism did not show significant synergy or antagonism;

25-50. Minor but significant synergy or antagonism of significant positive or negative effects;

moderate synergistic or antagonistic effects of 50-100 (Moderate synergy/antagonism-my be important in vivo-may be important in vivo experiments;

strong synergy or antagonism of >100 ═ Strong synergy/antagon-basic important in vivo-may be important in vivo experiments;

1000. Possible errors-check data and repeat experiments Possible errors-check data and repeat experiments;

the experimental results are as follows: see tables 6, 7, fig. 1 and 2.

TABLE 6 anti-HBV activity and cytotoxicity of single drugs in PHH (48 well plates)

TABLE 7 Combined results of test compounds in PHH (95% confidence interval)

And (4) experimental conclusion: as shown in figure 1, the combined efficacy results of the compound of the invention and ETV or Peg-IFN-alpha 2a show that the combined double drugs have stronger synergistic effect on the inhibition of HBV DNA, HBsAg and HBeAg, and compared with the single drug, the combined double drugs have better inhibition effect on the inhibition of HBV DNA, HBsAg and HBeAg. As shown in FIG. 2, neither the compound of the present invention nor Peg-IFN-. alpha.2a showed significant cytotoxicity in the tested concentration range, and the cell viability was greater than 92%. The combined drug effect result of the compound and ETV or TAF shows that the compound has stronger synergistic effect on the inhibition of HBV DNA, HBsAg and HBeAg after the combination of the two drugs, and has better inhibition effect on the inhibition of the HBV DNA, the HBsAg and the HBeAg after the combination of the two drugs compared with the single drug; none of the tested compounds showed significant cytotoxicity over the tested concentration range.

Experimental example 2

The test compound WWS1 was prepared according to the method of Experimental example 1Respectively replaced by WWS11WWS12WWS13Results of combined administration of test compounds in PHH were obtained: WWS11, WWS12, WWS13 and Peg-IFN alpha-2 a, ETV and TAF have strong synergistic effect on inhibition of HBV DNA, HBsAg and HBeAg, the 95% confidence interval synergy index is 125.24-236.83, and the antagonism index is-10-0.