WO2023006019A1

WO2023006019A1 - Polysubstituted benzene pd-l1 inhibitor, composition and use thereof

Info

Publication number: WO2023006019A1
Application number: PCT/CN2022/108538
Authority: WO
Inventors: 吴一哲; 周星露; 刘兴国; 胡苗; 朱建荣
Original assignee: 杭州和正医药有限公司
Priority date: 2021-07-28
Filing date: 2022-07-28
Publication date: 2023-02-02
Also published as: CN115677689A

Abstract

Disclosed in the present invention are a polysubstituted benzene PD-L1 inhibitor, a composition and the use thereof. The compound has a structure as represented by general formula (I). The inventor of the present invention confirmed by means of an experiment that the compound of the present invention exhibits a strong PD-1/PD-L1 blocking activity and a significant PD-L1 occupancy activity, and can reverse a PD-L1-inhibited T cell function. Moreover, the compound of the present invention can be orally absorbed, has a good pharmacokinetic property, and has a good anti-tumor inhibitory activity in vivo. Thus, the compound of the present invention can be used alone or in combination with other drugs in the treatment of diseases, disorders or conditions which would benefit from the inhibition of PD1 or PD-L1 activity, including infectious diseases, immune diseases, inflammatory diseases and cancers.

Description

Multi-substituted benzene PD-L1 inhibitors, compositions and applications thereof

technical field

The invention belongs to the field of medicine, and in particular relates to a multi-substituted benzene compound, a pharmaceutical composition and applications thereof. The compound can block the interaction between PD1 and PD-L1, and can be used to treat various diseases and disorders, including infectious diseases, immune diseases, and cancer.

Background technique

The human immune system plays an extremely important regulatory role in the process of many diseases including tumors. During the development of malignant tumors, in order to escape the surveillance of the human immune system, a variety of immune escape mechanisms have evolved. Among them, changing the expression of co-stimulatory or co-inhibitory molecules on the surface of tumor cells or immune cells is one of the most critical tumor immune escape mechanisms. At present, blocking the interaction of inhibitory immune checkpoint molecules (such as PD1/PD-L1) has become one of the effective tumor immunotherapy strategies. (Postow et al., J. Clinical Oncology 2015, 1-9).

PD-1 is a surface receptor that can be expressed on a variety of immune cells. It plays an extremely important immune negative feedback regulatory role in the body and can effectively prevent autoimmune diseases caused by excessive activation of T cells (Sharpe et al., Nat. Immunol., 2007, 8, 239-245). The PD-1 receptor has two corresponding endogenous ligands, PD-L1/PD-L2, in the body. PD-L1 and PD-L2 are different in expression, and PD-L1 is mostly expressed in dendritic cells, macrophages, B cells and T cells (Greenwald et al., Annu.Rev.Immunol.2005, 23:515-548 ; Okazaki and Honjo, Trends Immunol 2006, (4): 195-201), and can also be highly expressed on a variety of tumor cells. In contrast, PD-L2 is usually only expressed on dendritic cells. Blocking the interaction of PD-1/PD-L1 with drugs is currently the hottest direction in the field of tumor immunotherapy.

At present, multiple PD-1 and PD-L1 monoclonal antibodies have been clinically used for the treatment of more than 10 tumor indications. However, antibody drugs have the disadvantages that they cannot be administered orally, have poor tissue permeability, and are prone to produce anti-drug antibodies. Therefore, the development of new small molecule inhibitors for blocking PD1/PD-L1 is expected to overcome the defects of antibody drugs and become a new direction for the development of immune checkpoint inhibitors.

Contents of the invention

The object of the present invention is to provide a novel multi-substituted benzene compound or its stereoisomer or its stereoisomer mixture or a pharmaceutically acceptable salt thereof.

The present invention also provides a pharmaceutical composition comprising the above compound or its stereoisomer or its stereoisomer mixture or a pharmaceutically acceptable salt thereof.

The present invention also provides the above-mentioned compound or its stereoisomer or its stereoisomer mixture or a pharmaceutically acceptable salt thereof for the preparation and treatment of diseases and disorders benefiting from the inhibition of PD1 or PD-L1 activity Or the application in the medicine of disease.

The present invention adopts following technical scheme:

A kind of compound, has the structure of general formula (I):

or a stereoisomer thereof or a mixture of stereoisomers thereof or a pharmaceutically acceptable salt thereof;

X is selected from: N or CH;

R ₁ is selected from: C ₁ -C ₄ alkoxy, C ₃ -C ₅ cycloalkoxy, C ₁ -C ₄ haloalkoxy, C ₃ -C ₅ cycloalkyl, C ₁ -C ₄ haloalkyl;

R is selected from _: methyl group, cyano group, chlorine atom;

R ₃ is selected from: methyl group, cyano group, chlorine atom;

R ₄ is selected from: H, C ₁ -C ₄ alkoxy, C ₃ -C ₅ cycloalkoxy, C ₁ -C ₄ haloalkoxy, C ₃ -C ₅ cycloalkyl, C ₁ -C ₄ haloalkane base;

R _W , R _E are independently selected from:

Ra and Ra' are independently selected from: H, methyl, trifluoromethyl, ethyl, -OH, -COOH, -COOMe,

n is selected from: 0, 1, 2;

m is selected from: 1,2.

Furthermore, preferred compounds of the present invention have structures of general formula (IIa), (IIb):

or a stereoisomer thereof or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.

Furthermore, as a preferred solution:

R ₁ is preferably selected from:

Furthermore, as a preferred solution:

R2 is preferably selected from: methyl _;

R _W is preferably selected from:

R _E is preferably selected from:

Preferably, the preferred compound of the present invention has the structure represented by general formula (IIb).

As a further preference, the preferred compound of the present invention has a structure shown in general formula (IIb), and:

R1 is selected from _:

R ₂ is selected from: methyl, Cl; R ₃ is selected from: methyl; R ₄ is selected from difluoromethyl; R _E is selected from

R _w selected from

Preferably, it is selected from the following compounds or their stereoisomers or their stereoisomer mixtures or pharmaceutically acceptable salts thereof:

1-((6-cyclopropyl-2-(3'-((3-((3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-yl)amino)- 2,2'-Dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid 001

1-((6-cyclopropyl-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2 -d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl)methyl) Pyrrolidine-3-carboxylic acid 002

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-)((3-hydroxypyrrolidin-1-yl)methyl)pyridine [3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole-5- Base) methyl) pyrrolidine-3-carboxylic acid 004

4-(2-(((6-cyclopropyl-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido [3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole-5- Base) methyl) amino) ethyl) bicyclo [2.2.1] heptane-1-carboxylic acid 005

1-((2-(2'-chloro-3'-((3-((3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-yl)amino)-2 -Methyl-[1,1'-biphenyl]-3-yl)-6-(difluoromethoxy)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid 006

1-((2-(2'-chloro-3'-((3-((3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-yl)amino)-2 -Methyl-[1,1'-biphenyl]-3-yl)-6-(difluoromethoxy)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid Methyl ester 007

1-((2-(2'-chloro-3'-((3-((3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridin-8-yl)amino)-2 -Methyl-[1,1'-biphenyl]-3-yl)-6-(difluoromethoxy)benzo[d]oxazol-5-yl)methyl)piperidine-2-carboxylic acid 008

(Pivaloyloxy)methyl 1-((2-(2'-chloro-3'-((3-((3-hydroxypyrrolidin-1-yl)methyl)-1,7-naphthyridine -8-yl)amino)-2-methyl-[1,1'-biphenyl]-3-yl)-6-(difluoromethoxy)benzo[d]oxazol-5-yl)methyl base) pyrrolidine-3-carboxylate 009

4-((((6-cyclopropyl-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3 ,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl) Methyl)amino)methyl)bicyclo[2.2.1]heptane-1-carboxylic acid 010

1-((2-(2-chloro-3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2-d ]pyrimidin-4-yl)amino)-2'-methyl-[1,1'-biphenyl]-3-yl)-6-(difluoromethoxy)benzo[d]oxazole-5- Base) methyl) pyrrolidine-3-carboxylic acid 011

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido [3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl )methyl)piperidine-4-carboxylic acid 012

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido [3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl )methyl)piperidine-3-carboxylic acid 013

4-((((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl) Pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole-5 -yl)methyl)amino)methyl)bicyclo[2.2.1]heptane-1-carboxylic acid 014

4-(2-(((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methoxy) Base) pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole -5-yl)methyl)amino)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid 015

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido [3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl )methyl)azetidine-3-carboxylic acid 016

1-((2-(2-cyano-3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2- d]pyrimidin-4-yl)amino)-2'-methyl-[1,1'-biphenyl]-3-yl)-6-(difluoromethoxy)benzo[d]oxazole-5 -yl)methyl)pyrrolidine-3-carboxylic acid 017

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxyazetidin-1-yl)methyl )pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole- 5-yl)methyl)pyrrolidine-3-carboxylic acid 018

4-(2-(((2-(2-chloro-3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3 ,2-d]pyrimidin-4-yl)amino)-2'-methyl-[1,1'-biphenyl]-3-yl)-6-(difluoromethoxy)benzo[d]oxa Azol-5-yl)methyl)amino)ethyl)bicyclo[2.2.1]heptane-1-carboxylic acid 019

(R)-1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-(((R)-3-hydroxypyrrolidine-1 -yl)methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[ d] oxazol-5-yl) methyl) pyrrolidine-3-carboxylic acid 020

(S)-1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-(((R)-3-hydroxypyrrolidine-1 -yl)methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[ d] oxazol-5-yl) methyl) pyrrolidine-3-carboxylic acid 021

(S)-1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-(((S)-3-hydroxypyrrolidine-1 -yl)methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[ d] oxazol-5-yl) methyl) pyrrolidine-3-carboxylic acid 022

(R)-1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-(((S)-3-hydroxypyrrolidine-1 -yl)methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[ d] oxazol-5-yl) methyl) pyrrolidine-3-carboxylic acid 023

4-(2-(((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methoxy) Base) pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole -5-yl)methyl)amino)ethyl)bicyclo[2.2.1]heptane-1-carboxylate methyl ester 024

4-((((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl) Pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole-5 -yl)methyl)amino)methyl)bicyclo[2.2.1]heptane-1-carboxylic acid methyl ester 025

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido [3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl )methyl)-3-methylpyrrolidine-3-carboxylic acid 026

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido [3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl )methyl)piperidine-2-carboxylic acid 027

((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3 ,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl)methanol base) proline 028

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-(((2-hydroxypropyl)amino)methyl)pyrido[ 3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl) Methyl)pyrrolidine-3-carboxylic acid 029

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((((S)-2-hydroxypropyl)amino)methyl )pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole- 5-yl)methyl)pyrrolidine-3-carboxylic acid 030

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((((R)-2-hydroxypropyl)amino)methyl )pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole- 5-yl)methyl)pyrrolidine-3-carboxylic acid 031

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxy-3-methylpyrrolidin-1-yl) Methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxa Azol-5-yl)methyl)pyrrolidine-3-carboxylic acid 032

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-(((R)-3-hydroxy-3-methylpyrrolidine- 1-yl)methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo [d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid 033

1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-(((S)-3-hydroxy-3-methylpyrrolidine- 1-yl)methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo [d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid 034

1-((6-cyclopropyl-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2 -d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl)methyl) Piperidine-3-carboxylic acid 035

1-((6-cyclopropyl-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2 -d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl)methyl) Piperidine-4-carboxylic acid 036

(R)-1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl Base) pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole -5-yl)methyl)piperidine-4-carboxylic acid 037

(S)-1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl Base) pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole -5-yl)methyl)piperidine-4-carboxylic acid 038

(R)-1-((2-(2-chloro-3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3 ,2-d]pyrimidin-4-yl)amino)-2'-methyl-[1,1'-biphenyl]-3-yl)-6-(difluoromethoxy)benzo[d]oxa Azol-5-yl)methyl)piperidine-4-carboxylic acid 039

(R)-1-((2-(2'-chloro-3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[ 3,2-d]pyrimidin-4-yl)amino)-2-methyl-[1,1'-biphenyl]-3-yl)-6-(difluoromethoxy)benzo[d]oxa Azol-5-yl)methyl)piperidine-4-carboxylic acid 040

Glossary

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art. Unless otherwise stated, all patent documents, publicly disclosed materials, etc. referred to in the present invention are fully incorporated by reference. If there are multiple definitions of the same term in the present invention, the definition in this section shall prevail.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any claims. It should be noted that in the description and the appended claims, unless otherwise stated in the context, singular referents such as "a", "an", and "the" include plural referents. It should also be noted that unless otherwise stated, "or" means "and/or". Furthermore, terms "comprises", "includes" and the like are not limiting.

"Substituted" means that a hydrogen atom is replaced by a substituent. Note that the substituents on a particular atom are restricted by its valence. In the Definitions section, "C _i -C _j " refers to an inclusive range where i and j are integers representing the number of carbon atoms. For example, C _1- C ₄ , C _1- C ₈ , C _3- C ₈ , etc.

The term "alkyl" used in the present invention refers to a straight-chain saturated monovalent hydrocarbon group with one to six carbon atoms or a branched saturated monovalent hydrocarbon group with three to six carbon atoms, preferably methyl, ethyl, propyl , isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, etc. The alkyl group can be unsubstituted or monosubstituted or multi-substituted, and the substituents can be the same or different when multi-substituted; the substituents of the alkyl group are selected from halogen, nitro, hydroxyl, carboxyl, methyl carboxylate, ethyl carboxylate, Isopropyl ester, carbamoyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₁₀ cycloalkyl, alkoxycarbonyl, alkylthio, alkylsulfonyl, alkyl Amide, hydroxyalkylamide, sulfonamide, 3- to 10-membered heterocyclic group, or amino or monosubstituted or multisubstituted amino, wherein the amino substituents can be the same or different, selected from hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxyalkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₁₀ cycloalkyl, 3 to 10 membered heterocyclyl, C ₆ -C ₁₂ aryl, C ₅ - C ₁₄ heteroaryl.

The term "alkoxy" as used herein refers to -O-alkyl, wherein alkyl is as defined above. Examples of "alkoxy" as used herein include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy and tert-butoxy. "Alkoxy" also includes substituted alkoxy, whose substituents can be halogen, amino, hydroxy, C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxyalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ cycloalkyl, 3- to 10-membered heterocyclic group, C ₆ -C ₁₂ aryl, C ₅ -C ₁₄ heteroaryl.

The term "cycloalkyl" used in the present invention refers to a non-aromatic monovalent monocyclic or polycyclic ring (two monocyclic rings are connected by a chemical bond or bridged ring or spiro ring or fused) with three to ten carbon atoms. Hydrocarbyl, preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., wherein one or two carbon atoms may be replaced by an oxo group. The cycloalkyl group can be unsubstituted or substituted, and its substituent is selected from halogen, nitro, hydroxyl, carboxyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxyalkyl, C ₁ -C ₆ alkoxy radical, halogenated C ₁ -C ₆ alkyl, halogenated C ₁ -C ₆ hydroxyalkyl, halogenated C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, halogenated C ₃ -C ₆ Cycloalkyl, alkoxycarbonyl (methyl carboxylate, ethyl carboxylate), alkylthio, alkylsulfonyl, alkylamide (formamide), hydroxyalkylamide, sulfonamide, 3 to 10-membered heterocyclic group, or amino or monosubstituted or polysubstituted amino, wherein the substituents of amino can be the same or different, selected from hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxyalkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₁₀ cycloalkyl, 3- to 10-membered heterocyclic group, C ₆ -C ₁₂ aryl, C ₅ -C ₁₄ heteroaryl.

The term "haloalkyl" as used herein refers to an alkyl group substituted by halogen, preferably fluorine, chlorine, bromine, iodine, wherein alkyl is as defined above. There may be one or more halogen substituents in "haloalkyl", and the substitution may be on one atom or on different atoms.

The term "cycloalkoxy" as used herein refers to -O-cycloalkyl, wherein cycloalkyl is as defined above. "Cycloalkoxy" also includes substituted cycloalkoxy, whose substituents can be halogen, amino, hydroxy, C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxyalkyl, C ₁ -C ₆ alkoxy , C ₁ -C ₆ cycloalkyl, 3 to 10 membered heterocyclic group, C ₆ -C ₁₂ aryl, C ₅ -C ₁₄ heteroaryl.

The term "haloalkoxy" as used herein refers to the group -O-haloalkyl, wherein haloalkyl is as defined above. "Haloalkoxy" also includes substituted haloalkoxy, whose substituents can be amino, hydroxy, C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxyalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ cycloalkyl, 3- to 10-membered heterocyclic group, C ₆ -C ₁₂ aryl, C ₅ -C ₁₄ heteroaryl.

In the present invention, salts of the compounds described in the present invention can be prepared by methods well known to those skilled in the art. Described salt can be organic acid salt, inorganic acid salt etc., and described organic acid salt comprises citrate, fumarate, oxalate, malate, lactate, camphorsulfonate, p- Tosylate, methanesulfonate, etc.; the inorganic acid salts include hydrohalides, sulfates, phosphates, nitrates, etc. For example, with lower alkylsulfonic acids, such as methanesulfonic acid, trifluoromethanesulfonic acid, etc., can form mesylate, trifluoromethanesulfonate; with arylsulfonic acids, such as benzenesulfonic acid or p-toluenesulfonic acid, etc. It can form p-toluenesulfonate and benzenesulfonate; it can form corresponding salts with organic carboxylic acids, such as acetic acid, fumaric acid, tartaric acid, oxalic acid, maleic acid, malic acid, succinic acid or citric acid; it can form corresponding salts with amino acids , Such as glutamic acid or aspartic acid can form glutamate or aspartate. Corresponding salts can also be formed with inorganic acids, such as hydrohalic acids (such as hydrofluoric acid, hydrobromic acid, hydroiodic acid, hydrochloric acid), nitric acid, carbonic acid, sulfuric acid or phosphoric acid.

The compounds described in the present invention include compounds in which certain atoms are substituted by isotopes. Isotopes refer to atoms having the same atomic number but different atomic masses. Isotopes such as hydrogen include deuterium and tritium. Among the atoms of the compounds of the present invention, one or more atoms may be replaced by natural or unnatural isotopes. The hydrogen atoms in some embodiments can be replaced by one or more deuterium atoms. Methods for the synthesis of compounds containing isotopic atoms are known in the art.

The second object of the present invention is to provide a pharmaceutical composition, comprising one or more of the compounds described in any one of the above technical schemes. The pharmaceutical composition of the present invention can be composed of one or more of the compounds described in any one of the above technical schemes and other compounds, or one or more of the compounds described in any of the above technical schemes composition.

In another aspect, the present invention provides the use of the compounds described in general formula (I), general formula (IIa), general formula (IIb) disclosed herein or their stereoisomers or their stereoisomer mixtures or their pharmaceutical preparations Use of an acceptable salt above to treat a disease, disorder or condition that would benefit from inhibition of PD1 or PD-L1 activity.

In a further preferred embodiment, the present invention provides a method of blocking the interaction between PD1 and PD-L1 in the subject by administering a composition containing a therapeutically effective amount of at least one compound to the subject in need. The method, wherein the structural formula of the compound is general formula (I), general formula (IIa), general formula (IIb).

In some embodiments, the subject in need thereof suffers from cancer including hematological and solid tumors, such as melanoma, glioblastoma, esophageal tumors, nasopharyngeal carcinoma, uveal melanoma, lymphoma, Lymphocytic lymphoma, primary central nervous system lymphoma, T-cell lymphoma, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, prostate cancer, castration-resistant prostate cancer, chronic myeloid Leukemia, Kaposi's sarcoma, fibrosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, angiosarcoma, lymphangiosarcoma, synovium, meningioma, leiomyosarcoma, rhabdomyosarcoma, soft tissue sarcoma, sarcoma, biliary tract tumor, basal cell Carcinoma, thymic tumor, thyroid cancer, parathyroid cancer, uterine cancer, adrenal cancer, Merkel cell carcinoma, neurotumor, follicular center lymphoma, colon cancer, Hodgkin's disease, non-Hodgkin's lymphoma , leukemia, chronic or acute leukemia, including acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, ovarian neoplasm, myelodysplastic syndrome, skin or intraocular malignant melanoma , renal cell carcinoma, small cell lung cancer, lung cancer, mesothelioma, breast cancer, squamous non-small cell lung cancer (SClC), non-squamous non-small cell lung cancer, colorectal cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic cancer Carcinoma, pancreatic ductal adenocarcinoma, head and neck squamous cell carcinoma, head and neck cancer, gastrointestinal tract, bone cancer, skin cancer, rectal cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer , vaginal cancer, vulvar cancer, esophagus cancer, small intestine cancer, endocrine system cancer, urethra cancer, penile cancer, bladder cancer, kidney cancer, ureter cancer, renal pelvis cancer, central nervous system tumor (CNS), tumor angiogenesis, spinal cord axis tumor , brainstem glioma, pituitary adenoma, epidermoid carcinoma, asbestosis, adenocarcinoma, papillary carcinoma, cystadenocarcinoma, bronchial carcinoma, renal cell carcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, Embryonal carcinoma, Wilm's tumor, pleomorphic adenoma, hepatocellular papilloma, tubular adenoma, cystadenoma, papilloma, adenoma, leiomyoma, rhabdomyoma, hemangioma, lymphangioma, Osteomas, chondromas, lipomas and fibromas.

In a further embodiment, the subject in need thereof suffers from an infectious disease, an immune disease, and an inflammatory disease, such as sepsis, liver infection, HIV, hepatitis A, hepatitis B, hepatitis C, hepatitis D, Herpes virus, papilloma virus, influenza, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, Crohn's disease, colitis, autoimmune hemolytic anemia, ankylosing spondylitis, Pemphigus, urticaria, asthma, optic neuritis, psoriasis, chronic obstructive airway disease, dermatitis, alopecia.

The present invention also provides the application of the compound of the present invention or a pharmaceutically available salt thereof in the preparation of blocking the interaction between PD1 and PD-L1, especially the application of the preparation in the treatment of cell proliferation diseases. Said cell proliferative diseases include cancer. In other words, the present invention also provides the compounds described in general formula (I), general formula (IIa), general formula (IIb) or their pharmaceutically acceptable salts alone or in combination with other drugs in the treatment of proliferative diseases (such as cancer ) in the application.

The drug used in combination with the compound or composition of the present invention can be selected from antimicrobial agents, antiviral agents, cytotoxic agents, gene expression regulators, chemotherapeutic agents, anticancer agents, antiangiogenic agents, immunotherapeutic agents (such as immune checkpoint inhibitors), antihormonal drugs (such as kinase inhibitors), antifibrotic agents, radiation therapy, radiotherapeutic agents, antineoplastic agents, or antiproliferative agents.

A preparation comprising one or more of the compounds described in any of the above schemes.

The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption accelerators, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and fragrances can also be added if necessary agents, sweeteners, etc.

The medicine of the present invention can be made into various forms such as tablets, powders, capsules, injection preparations, granule preparations, sprays, etc., and the medicines in the above dosage forms can be prepared according to conventional methods in the field of pharmacy.

The compounds of the invention may also be used in combination with other methods of treating cancer, for example by chemotherapy, radiation therapy, tumor-targeted therapy, adjuvant therapy, immunotherapy or surgery. Examples of immunotherapy include cytokine therapy (e.g. interferon, GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccines, monoclonal antibodies, recipient T cell transfer, Toll receptor agonism agents, STING agonists, oncolytic virotherapy and immunomodulatory small molecules, including thalidomide or JAK1/2 inhibitors, etc.

The inventors of the present invention have confirmed through experiments that the compound of the present invention exhibits strong PD-1/PD-L1 blocking activity and can reverse the T cell function inhibited by PD-L1. At the same time, the compound of the invention can be orally absorbed, has good pharmacokinetic properties, and has good antitumor effect in vivo. Therefore, the compounds of the present invention can be used in the treatment of diseases, disorders or conditions benefiting from the inhibition of PD1 or PD-L1 activity alone or in combination with other drugs, including infectious diseases, immune diseases, inflammatory diseases disease and cancer.

Detailed ways

The implementability of the present invention is illustrated by the following examples. Those skilled in the art should understand that modifying or replacing the corresponding technical features according to the teaching of the prior art still belongs to the protection scope of the present invention.

Synthetic route of intermediate 1

Synthesis of Intermediate 1-1

Methyl 2-bromo-4-methoxybenzoate (0.1 mmol), cyclopropylboronic acid (0.3 mmol), SPhos-Pd-G2 (chloro(2-dicyclohexylphosphino-2',6'-di Methoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II)) (0.005mmol), potassium phosphate (0.2mmol) dissolved in toluene , N ₂ protection, react overnight at 110°C. The reaction solution was concentrated and subjected to silica gel column chromatography to obtain intermediate 1-1: methyl 2-cyclopropyl-4-methoxybenzoate. LC-MS (ESI-MS): 207 [M+H] ⁺ .

Synthesis of Intermediate 1-2

Intermediate 1-1 (0.1 mmol) was dissolved in anhydrous dichloromethane, boron tribromide (0.7 mmol) was diluted in anhydrous DCM, and added dropwise to the DCM solution of intermediate 1-1 at -10°C, maintaining the temperature React for 3 hours. Water was added at low temperature to quench the reaction. After extraction and concentration, intermediate 1-2: methyl 2-cyclopropyl-4-hydroxybenzoate was obtained by silica gel column chromatography. LC-MS (ESI-MS): 193 [M+H] ⁺ .

Synthesis of Intermediates 1-3

Intermediate 1-2 (10 g) was dissolved in 40 mL of glacial acetic acid, 10 mL of acetic anhydride was added, and cooled in an ice bath. 10 mL (5 mL+5 mL) of a mixture of concentrated nitric acid and glacial acetic acid was added dropwise. After the dropwise addition, the temperature was slowly raised to room temperature and the reaction was continued for 2 hours. After the reaction, the reaction solution was poured into ice water and filtered to obtain intermediate 1-3: 2-cyclopropyl-4-hydroxy-5-nitrobenzoic acid methyl ester, which was directly carried out to the next reaction. LC-MS (ESI-MS): 238 [M+H] ⁺ .

Synthesis of Intermediates 1-4

Intermediate 1-3 (5 g) was dissolved in methanol, 0.5 g of Pd/C was added, and hydrogen was reduced overnight at room temperature. After filtering Pd/C with celite, the reaction solution was concentrated, and the intermediate 1-4 was obtained by silica gel column chromatography: methyl 5-amino-2-cyclopropyl-4-hydroxybenzoate. LC-MS (ESI-MS): 208 [M+H] ⁺ .

Synthesis of Intermediates 1-5

Intermediate 1-4 (0.1 mmol) was dissolved in methanol, 3-bromo-2-methylbenzaldehyde (0.09 mmol) was added, reacted at 80°C for 3 hours, and concentrated under reduced pressure. After concentration, it was re-dissolved in DCM, DDQ (2,3-dichloro-5,6-dicyanobenzoquinone) (0.15 mmol) was added, and reacted at room temperature for 1 hour. The reaction solution was diluted with DCM, and the organic phase was washed with sodium thiosulfate solution. The organic phase was concentrated, and intermediate 1-5 was obtained by silica gel column chromatography: 2-(3-bromo-2-methylphenyl)-6-cyclopropylbenzo[d]oxazole-5-carboxylic acid methyl ester . LC-MS (ESI-MS): 386 [M+H] ⁺ .

Synthesis of Intermediates 1-6

Intermediate 1-5 (0.2 mmol) was dissolved in anhydrous THF, and LiAlH ₄ (0.3 mmol) was added at -20°C. Slowly warm up to room temperature and react for 2 hours, then add 1 mL of 10% sodium hydroxide solution to quench the reaction. The organic phase was washed with hydrochloric acid and saturated brine. Intermediate 1-6: (2-(3-bromo-2-methylphenyl)-6-cyclopropylbenzo[d]oxazol-5-yl)methanol was obtained by silica gel column chromatography. LC-MS (ESI-MS): 358 [M+H] ⁺ .

Synthesis of Intermediates 1-7

Dissolve 1 g of intermediate 1-6 in anhydrous THF, add 1.7 g of Dess-Martin reagent, react at room temperature for 2 hours, filter through diatomaceous earth, and obtain intermediate 1-7 by silica gel column chromatography: 2-(3- Bromo-2-methylphenyl)-6-cyclopropylbenzo[d]oxazole-5-carbaldehyde. LC-MS (ESI-MS): 356 [M+H] ⁺ .

Synthesis of Intermediates 1-8

Intermediate 1-7 (1 g) was dissolved in methanol, 316 mg of acetic acid and 1.8 g of tert-butyl pyrrole-3-carboxylate were added, stirred at room temperature for 30 minutes, sodium cyanoborohydride (520 mg) was added, and reacted at 60°C for 3 hours. Cooled to room temperature, added DCM to dilute the reaction solution, washed with saturated brine, and purified by column to obtain intermediate 1-8: 1-((2-(3-bromo-2-methylphenyl)-6-cyclopropane tert-butyl benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylate. LC-MS (ESI-MS): 511 [M+H] ⁺ .

Synthesis of Intermediate 1

Intermediate 1-8 (700 mg) was dissolved in 1,4-dioxane solution, added pinacol diboronate (1.2 g), potassium acetate (110 mg), Pd(dppf)Cl ₂ (100 mg), N ₂ reaction overnight at 100°C under protection. After the reaction, the reaction solution was concentrated, and the intermediate 1 was obtained by silica gel column chromatography: 1-((6-cyclopropyl-2-(2-methyl-3-(4,4,5,5-tetramethyl- tert-butyl 1,3,2-dioxaborolan-2-yl)phenyl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylate. LC-MS (ESI-MS): 559 [M+H] ⁺ .

Referring to the synthetic route and method of intermediate 1, the following intermediate structure is synthesized:

Synthesis of Intermediate 6

Synthesis of Intermediate 6-1

Take a one-necked bottle, add methyl 2,4-dihydroxybenzoate (10.0g, 59.47mmol, 1.0eq.), glacial acetic acid (44mL), acetic anhydride (11mL), cool the solution to 10°C, add 65% nitric acid (8.3g, 79.20mmol, 1.3eq.) and glacial acetic acid (6mL), the mixture was replaced with nitrogen three times, then stirred at 10°C for 1 hour, then heated to 15°C, and stirred for 1 hour. Sampling and testing showed that the reaction was complete. Add water (40mL) to the reaction solution, stir for 10 minutes, filter and rinse with a small amount of water, collect the solid, add methanol (25mL), stir for 10 minutes, filter and rinse with a small amount of methanol, collect the product and dry it in vacuum to obtain 2,4-dihydroxy - Methyl 5-nitrobenzoate (4.5 g, 20.06 mmol, yield 33.7%). LC-MS: (ESI) [M+H] ⁺ = _214.0tR = 1.77min.

Synthesis of Intermediate 6-2

Take a 250mL single-necked bottle, add 2,4-dihydroxy-5-nitrobenzoic acid methyl ester (4.0g, 18.77mmol, 1.0eq.), and methanol (100mL), add 10% palladium hydroxide carbon catalyst (2.0g , 50% m/m), the mixture was replaced with hydrogen three times, and the hydrogen pressure was maintained at 15psi, stirred at 25°C for 3 hours, TLC spot plate confirmed that the reaction was complete, filtered to remove the catalyst, and washed the filter cake with methanol (100mL), collected the filtrate, and reduced Concentrated by pressure distillation, and the residue was purified by Flash column (petroleum ether: ethyl acetate = 10:1) to obtain methyl 5-amino-2,4-dihydroxybenzoate (2.0 g, 9.83 mmol, yield 52.37%) . LC-MS: (ESI) [M+H] ⁺ = _184.0tR = 1.06min.

Synthesis of Intermediate 6-3

Take a 500mL single-necked bottle, add 5-amino-2,4-dihydroxybenzoic acid methyl ester (5.0g, 27.30mmol, 1.0eq.), 3-bromo-2-methylbenzaldehyde (6.5g, 32.76mmol, 1.2 eq.) and methanol (150 mL). The mixture was replaced with nitrogen three times and stirred at 80°C for 5 hours. Then the reaction solution was concentrated under reduced pressure, dried, added dichloromethane (150mL), and then added DDQ (2,3-dichloro-5,6-dicyanobenzoquinone) (8.7g, 38.22mmol, 1.4eq.) , and stirred at 25°C for 12 hours. Sampling and testing showed that the reaction was complete. The reaction solution was extracted three times with ethyl acetate (100mL×3), the organic phases were combined, and washed three times with water (20mL×3), the organic phase was concentrated to 40mL under reduced pressure, stirred for 1 hour, filtered, and collected to obtain 2-(3- Bromo-2-methylphenyl)-6-hydroxybenzo[d]oxazole-5-carboxylic acid methyl ester (7.5 g, 19.67 mmol, yield 72.0%). LC-MS: (ESI) [M+H] ⁺ = _361.6tR = 2.02min.

Synthesis of Intermediate 6-4

Take a 100mL single-necked bottle, add 2-(3-bromo-2-methylphenyl)-6-hydroxybenzo[d]oxazole-5-carboxylic acid methyl ester (3.8g, 10.49mmol, 1.0eq.), N,N-Dimethylformamide (40 mL), cesium carbonate (13.7 g, 41.97 mmol, 4.0 eq.) and sodium 2-bromo-2,2-difluoroacetate (8.3 g, 41.97 mmol, 4.0 eq.) . The mixture was replaced with nitrogen three times and stirred at 100°C for 14 hours. Sampling and testing showed that the reaction was complete. The reaction solution was extracted with ethyl acetate (30mL×3), the organic phases were combined, concentrated by distillation under reduced pressure, and the residue was purified with a Flash column (petroleum ether:ethyl acetate=20:1) to obtain 2-(3-bromo-2 -Methylphenyl)-6-(difluoromethoxy)benzo[d]oxazole-5-carboxylate (800 mg, 1.75 mmol, yield 16.7%). LCMS: (ESI) [M+H] ⁺ = 411.7, t _R = 1.85 min.

Synthesis of Intermediate 6-5

Take a single-necked bottle, add 2-(3-bromo-2-methylphenyl)-6-(difluoromethoxy)benzo[d]oxazole-5-carboxylic acid methyl ester (100mg, 0.24mmol, 1.0eq.) was dissolved in THF (5mL), under ice bath at 0°C, lithium aluminum tetrahydrogen (11mg, 0.29mmol, 1.2eq.) was slowly added, stirred at 0°C for 30 minutes, and TLC was spotted to confirm that the reaction was complete. A small amount of water (3 mL) was added to quench, the reaction solution was extracted three times with ethyl acetate (10 mL×3), the organic phases were combined, concentrated by distillation under reduced pressure, and purified by preparative TLC (petroleum ether: ethyl acetate = 3:1), Obtained (2-(3-bromo-2-methylphenyl)-6-(difluoromethoxy)benzo[d]oxazol-5-yl)methanol (40 mg, 0.09 mol, yield 38.6%) . LCMS: (ESI) [M+H] ⁺ = 383.8, t _R = 1.73 min.

Synthesis of Intermediate 6-6

Take a one-necked bottle, add (2-(3-bromo-2-methylphenyl)-6-(difluoromethoxy)benzo[d]oxazol-5-yl)methanol (20mg, 0.05mmol, 1.0 eq.) was dissolved in dichloromethane (5 mL), manganese dioxide (226 mg, 2.60 mmol, 50.0 eq.) was added, and stirred at 25°C for 18 hours. Sampling and testing showed that the reaction was complete. The reaction solution was filtered to remove manganese dioxide, extracted three times with ethyl acetate (5mL×3), the organic phases were combined, concentrated under reduced pressure, and the residue was purified by preparative TLC (petroleum ether: ethyl acetate = 5:1) to obtain 2- (3-Bromo-2-methylphenyl)-6-(difluoromethoxy)benzo[d]oxazole-5-carbaldehyde (17 mg, 0.05 mmol, yield 85.5%). LCMS: (ESI) [M+H] ⁺ = 381.6, t _R = 1.92 min. ¹ H NMR (400MHz, DMSO) δ10.35(s, 1H), 8.27(s, 1H), 8.11(d, J=7.6Hz, 1H), 8.03–7.92(m, 2H), 7.67–7.25(m ,2H),2.83(s,3H).

Synthesis of intermediates 6-7

In a 50 mL round bottom flask, 2-(3-bromo-2-methylphenyl)-6-(difluoromethoxy)benzo[d]oxazole-5-carbaldehyde (85 mg, 0.22 mmol, 1.0 eq.), triethylamine (68 mg, 0.67 mmol, 3.0 eq.) and methyl pyrrolidine-3-carboxylate (58 mg, 0.45 mmol, 2.0 eq.) were added to DCM (10 mL). The reaction mixture was stirred at 0°C for 2 hours, sodium cyanoborohydride (17mg, 0.27mmol, 1.2eq.) was added, and stirred at 25°C for 14 hours. Sampling and testing showed that the reaction was complete. The reaction solution was extracted three times with ethyl acetate (10 mL×3), and the organic phases were combined and concentrated to dryness. The residue was purified by preparative silica gel plate (developing solvent: ethyl acetate) to give 1-((2-(3-bromo-2-methylphenyl)-6-(difluoromethoxy)benzo[d] Oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid methyl ester (30 mg, 0.06 mmol, 26.8%). LCMS: (ESI) [M+H] ⁺ = 495.1.

Synthesis of Intermediate 6

1-((2-(3-bromo-2-methylphenyl)-6-(difluoromethoxy)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxy Methyl ester (184mg, 0.37mmol, 1.0eq.), pinacol diboronate (141mg, 0.56mmol, 1.5eq.) and potassium acetate (73mg, 0.74mmol, 2.0eq.) were dissolved in dioxane and Water (3 mL and 1 mL), then [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (27 mg, 0.037 mmol, 0.1 eq.) was added. Nitrogen was replaced 3 times at room temperature, heated to 100°C, and stirred for 18 hours. Sampling and testing showed that the reaction was complete. Cooled to room temperature, filtered with celite, the filtrate was spin-dried, and the residue was purified by Flash column (petroleum ether: ethyl acetate = 3:1) to obtain 1-((6-(difluoromethoxy)-2-( 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)benzo[d]oxazol-5-yl) Methyl)pyrrolidine-3-carboxylate (141 mg, yield: 70%). LC-MS: (ESI) [M+H] ⁺ = 543.6, t _R = 1.575 min.

With reference to the synthetic route and method of intermediate 6, the following intermediate structure is synthesized:

Synthesis of intermediate 22

Synthesis of Intermediate 22-1

Take a one-necked bottle, add 3-bromo-8-chloro-1,7-naphthyridine (800mg), cesium carbonate (2.14g,), potassium vinyl fluoroborate (572mg), [1,1'-bis(diphenyl phosphino)ferrocene]palladium dichloride (268 mg), 1,4-dioxane and water. The mixture was purged three times, then stirred at 90°C for 1.5 hours. The reaction solution was cooled to room temperature, then water (15 mL) was added, extracted three times with ethyl acetate (30 mL*3), the organic phases were combined, backwashed once with saturated brine (30 mL), and purified by silica gel column chromatography to obtain the intermediate 22-1: 8-Chloro-3-vinyl-1,7-naphthyridine. LC-MS (ESI-MS): 191 [M+H] ⁺ .

Synthesis of Intermediate 22-2

Take 100 mL of sealed tube, add 8-chloro-3-vinyl-1,7-naphthyridine (300 mg), 3-bromo-2-methylaniline (325 mg), tert-butanol (5 mL) and hydrochloric acid 1, 4-dioxane solution. The reaction mixture was stirred at 120 °C for 2 h. The reaction solution was cooled to room temperature, then neutralized to pH=7 with saturated sodium carbonate solution, extracted three times with ethyl acetate (30mL*3), combined the organic phases, and washed once with saturated brine (30mL), anhydrous sodium sulfate After drying, it was purified by silica gel column chromatography to obtain intermediate 22-2: N-(3-bromo-2-methylphenyl)-3-vinyl-1,7-naphthyridin-8-amine. LC-MS (ESI-MS): 340 [M+H] ⁺ .

Synthesis of intermediate 22-3

Take a 100mL single-necked bottle, add intermediate 22-2 (750mg), potassium osmate (53.0mg), 1,4-dioxane and water. Sodium periodate (1.33 g) was added to the reaction solution at 0°C. The reaction mixture was stirred at room temperature for 3 hours. Then quench the reaction with saturated sodium sulfite, extract three times with ethyl acetate, combine the organic phases, wash once with saturated brine, and then purify by silica gel column chromatography to obtain intermediate 22-3: 8-((3-bromo-2- Methylphenyl)amino)-1,7-naphthyridine-3-carbaldehyde. LC-MS (ESI-MS): 342 [M+H] ⁺ .

Synthesis of intermediate 22

Take a 100mL single-necked bottle, add intermediate 22-3 (210mg), dichloromethane, triethylamine (231mg) and pyrrolidin-3-ol (63mg). The mixture was stirred for 1 hour, and sodium cyanoborohydride (56.0 mg) was added to the above mixture at 0°C. The mixture was stirred at room temperature for 16 hours. Add water to quench the reaction, extract with dichloromethane, combine the organic phases, and purify by silica gel column chromatography to obtain intermediate 22: 1-((8-((3-bromo-2-methylphenyl)amino)-1 ,7-naphthyridin-3-yl)methyl)pyrrolidin-3-ol. LC-MS (ESI-MS): 413 [M+H] ⁺ .

Synthesis of Intermediate 23

Referring to the synthesis of intermediate 22, in the synthesis of intermediate 22-2, intermediate 23 was obtained by replacing 3-bromo-2-methylaniline with 3-bromo-2-chloroaniline. LC-MS (ESI-MS): 433 [M+H] ⁺ .

Synthesis of intermediate 24

Synthesis of intermediate 24-1

Take a one-necked bottle, dissolve 2,5-dibromo-3-nitropyridine (3.00g, 10.64mmol, 1.00eq.) in acetonitrile (20mL), add cuprous cyanide (1.05g, 11.71mmol, 1.10eq. ), stirred at 85° C. for 16 hours, took a sample and detected, and the reaction was complete. The reaction solution was cooled to room temperature, filtered, the filtrate was collected, concentrated, added water (100mL), extracted three times with dichloromethane (100mL×3), combined the organic phases, concentrated by distillation under reduced pressure to obtain the crude product, and the crude product was used on a Flash column (petroleum Ether: dichloromethane=3:1) Purification gave 5-bromo-3-nitropyridine-2-carbonitrile (1.80 g, 7.11 mmol, yield: 66.8%). ¹ H NMR: [M+H] ⁺ = 227.9. (400MHz, CDCl3) δ9.06(s, 1H), 8.77(s, 1H).

Synthesis of Intermediate 24-2

Take a one-necked bottle, slowly add 5-bromo-3-nitropyridine-2-carbonitrile (1.40g, 6.14mmol, 1.00eq.) into concentrated sulfuric acid (8mL), heat to 100°C, and stir for 2 hours. Sampling and testing showed that the reaction was complete. The reaction solution was cooled to room temperature, poured into ice water, precipitated solid, filtered, washed the filter cake with water and sucked dry to obtain 5-bromo-3-nitropyridine-2-carboxamide (1.25g, 4.57mmol, yield: 74.5% ). LCMS (ESI) [M+H] ⁺ = 245.9, t _R = 1.079 min.

Synthesis of Intermediate 24-3

Take a one-necked bottle, dissolve 5-bromo-3-nitropyridine-2-carboxamide (210mg, 0.85mmol, 1.00eq.) in methanol (4mL), add water (2mL), ammonium chloride (365mg, 6.83mmol , 8.00eq.) and iron powder (238mg, 4.27mmol, 5.00eq.), heated to 100°C and stirred for 30min, sampled and tested, the reaction was complete. The reaction solution was filtered, concentrated, added water (10 mL), extracted 3 times with dichloromethane (10 mL×3), and concentrated to obtain a crude product. The crude product was purified by preparative TLC (petroleum ether:ethyl acetate=3:1) to obtain 3-amino-5-bromopyridine-2-carboxamide (140 mg, 0.65 mmol, yield: 75.9%). LCMS: (ESI) [M+H] ⁺ = 216.0, t _R = 1.453 min. ¹ H NMR: (400MHz, DMSO) δ 7.92(s, 1H), 7.82(s, 1H), 7.41(s, 2H), 7.06(s, 2H).

Synthesis of Intermediate 24-4

In a 100 mL round bottom flask, 3-amino-5-bromopyridine-2-carboxamide (6.70 g, 31.01 mmol, 1.0 eq.) was dissolved in DCM (70 mL) and DMF (10 mL). 2,2-Difluoroacetic anhydride (10.80 g, 62.03 mmol, 2.0 eq.) was added dropwise under ice bath. After dropping, the reaction solution was stirred at 25° C. for 18 hours, and a sample was taken for detection, and the reaction was complete. The reaction solution was concentrated and dried to obtain 5-bromo-3-(2,2-difluoroacetamido)pyridine-2-carboxamide (7.20 g, 22.04 mmol, yield: 71.0%). LCMS: (ESI) [M+H] ⁺ = 294.0, t _R = 1.08 min.

Synthesis of intermediate 24-5

In a 250mL round bottom flask, add 5-bromo-3-[(2,2-difluoroacetyl)amino]pyridine-2-carboxamide (9.80g, 33.33mmol, 1.0eq.), ethanol (45mL) and Water (11 mL), sodium hydroxide (1.33 g, 33.33 mmol, 1.0 eq.) was added in portions. The reaction mixture was stirred at 50 °C for 2 hours. Sampling LCMS detection, the reaction conversion is complete. The mixture was cooled to room temperature, and the reaction mixture was poured into water (500 mL). Stir at room temperature for 30 minutes, and filter to obtain 7-bromo-2-(difluoromethyl)pyrido[3,2-d]pyrimidin-4-ol (6.00 g, 20.65 mmol, 62.0%). LC-MS: (ESI) [M+H] ⁺ = 275.9, t _R = 1.401 min.

Synthesis of Intermediate 24-6

In a 250mL round bottom flask, add 7-bromo-2-(difluoromethyl)pyrido[3,2-d]pyrimidin-4-ol (4.50g, 16.30mmol, 1.0eq.), cesium carbonate (10.62 g, 32.60mmol, 2.0eq.), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.19g, 1.63mmol, 0.1eq.), ethylene Potassium trifluoroborate (3.27g, 24.45mmol, 1.5eq.) and dioxane (45mL), water (15mL). Under nitrogen protection, the mixture was warmed to 80°C and stirred for 12 hours. Sampling LCMS detection, the reaction conversion is complete. The reaction solution was filtered, and the filtrate was concentrated in vacuo to obtain 2-(difluoromethyl)-7-vinylpyrido[3,2-d]pyrimidin-4-ol (3.03g, 12.77mmol, yield: 78.3%) The crude product was used directly in the next step without purification. LC-MS: (ESI) [M+H] ⁺ = 224.0, t _R = 1.359 min.

Synthesis of Intermediate 24-7

In a 500 mL round bottom flask, 2-(difluoromethyl)-7-vinylpyrido[3,2-d]pyrimidin-4-ol (15.00 g, 54.34 mmol, 1.0 eq.) was dissolved in toluene (150 mL ), diisopropylethylamine (21.07g, 163.02mmol, 3.0eq.) and phosphorus oxychloride (25.00g, 163.02mmol, 3.0eq.) were added to the mixture. Under nitrogen protection, the mixture was warmed to 80 °C and stirred for 2 hours. Sampling LCMS detection, the reaction conversion is complete. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate=20/1—10/1) to obtain 4-chloro-2-(difluoromethyl)-7-vinylpyrido[3, 2-d]pyrimidine (6.05g, 19.36mmol, yield: 35.6%). LC-MS: (ESI) [M+H] ⁺ = 242.0, t _R = 1.957 min. ¹ H NMR (400MHz, DMSO) δ9.49 (d, J = 2.0Hz, 1H), 8.68 (d, J = 2.0Hz, 1H), 7.32–7.00 (m, 2H), 6.52 (d, J = 17.6 Hz,1H),5.81(d,J=11.2Hz,1H).

Synthesis of intermediate 24-8

4-Chloro-2-(difluoromethyl)-7-vinylpyrido[3,2-d]pyrimidine (1.55 g, 6.42 mmol, 1.0 eq.) was dissolved in tert-butanol (15 mL) and 3- Bromo-2-methylphen-1-amine (1.43g, 7.70mmol, 1.2eq.) was added. The reaction mixture was stirred at 50 °C for 12 hours. Sampling LCMS detection, the reaction conversion is complete. The reaction mixture was cooled to room temperature, and the reaction mixture was poured into water (100 mL). After stirring at room temperature for 30 minutes, it was filtered, and the resulting filter cake was vacuum-dried to obtain N-(3-bromo-2-methylphenyl)-2-(difluoromethyl)-7-vinylpyrido[3, 2-d] Pyrimidin-4-amine (2.02 g, 4.86 mmol, yield: 75.7%). LC-MS: (ESI) [M+H] ⁺ = 391, t _R = 1.799 min.

Synthesis of intermediate 24-9

In a three-necked flask, N-(3-bromo-2-methylphenyl)-2-(difluoromethyl)-7-vinylpyrido[3,2-d]pyrimidin-4-amine (2.01g , 5.11 mmol, 1.0 eq.) was dissolved in dichloromethane (1000 mL). The reaction mixture was cooled to -78°C. Ozone was then introduced into the reaction solution until the reaction solution was slightly blue. Sampling LCMS detection, the reaction conversion is complete. Triethylamine (1.55 g, 15.34 mmol, 3.0 eq.) was then added and the reaction mixture was stirred at -78°C for an additional 1 hour. The reaction mixture was warmed to room temperature, and the residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate=20/1—1/3) to obtain 4-((3-bromo-2-methylphenyl) Amino)-2-(difluoromethyl)pyrido[3,2-d]pyrimidine-7-carbaldehyde (800 mg, 1.87 mmol, yield: 36.6%). LC-MS: (ESI) [M+H] ⁺ = 392.6, t _R = 1.654 min. ¹ H NMR (400MHz, DMSO) δ10.81(s, 1H), 10.35(s, 1H), 9.36(d, J=1.6Hz, 1H), 8.80(d, J=1.6Hz, 1H), 7.60( d,J=8.0Hz,1H),7.53(d,J=7.6Hz,1H),7.25(t,J=8.0Hz,1H),6.74(m,1H),2.29(s,3H).

Synthesis of intermediate 24

In a three-necked flask, 4-((3-bromo-2-methylphenyl)amino)-2-(difluoromethyl)pyrido[3,2-d]pyrimidine-7-carbaldehyde (550mg, 1.40mmol, 1.0 eq.) was dissolved in dichloromethane (15 mL), and pyrrolidin-3-ol (244 mg, 2.80 mmol, 2.0 eq.) and triethylamine (580 mg, 5.74 mmol, 4.0 eq.) were added to the mixture. The mixture was stirred at 25°C for 2 hours. Sodium cyanoborohydride (132mg, 2.10mmol, 1.5eq.) was then added and the reaction mixture was stirred at 25°C for a further 12 hours. Sampling LCMS detection, the reaction conversion is complete. The mixture was added to water (50 mL), extracted with ethyl acetate (100 mL×2). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate=20/1—10/1) to obtain 1-((4-((3-bromo-2-methylphenyl)amino)- 2-(Difluoromethyl)pyrido[3,2-d]pyrimidin-7-yl)methyl)pyrrolidin-3-ol (320 mg, 0.63 mmol, yield: 45.3%). LC-MS: (ESI) [M+H] ⁺ = 464.1, t _R = 1.40, 3 min.

Referring to the synthetic route and method of intermediate 24, the following intermediate structure is synthesized:

Example 001. 1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-((3-hydroxypyrrolidin-1-yl)methyl Base) pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl)benzo[d]oxazole -5-yl)methyl)pyrrolidine-3-carboxylic acid (004)

first step:

In a three-necked flask, Intermediate 24 (193 mg, 0.42 mmol, 1.0 eq.) was added to dioxane (3 mL) and H ₂ O (0.4 mL), then Intermediate 6 (271 mg, 0.50 mmol, 1.2 eq. .), K ₃ PO ₄ (265mg, 1.25mmol, 3.0eq.) and XPhos Pd G2 (chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II)) (65 mg, 0.083 mmol, 0.2 eq.). The mixture was degassed 3 times with _N2 , and the reaction was stirred at 100 °C for 3 h. Sampling LCMS detection, the reaction conversion is complete. The mixture was cooled to 25 °C, filtered, and the filtrate was concentrated in vacuo to obtain intermediate 020-1: 1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)- 7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'- Biphenyl]-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylate (330 mg, 0.21 mmol, yield: 49.6%) crude product. LC-MS: [M+H] ⁺ = 800.1, _tR = 2.337min.

Step two:

In a round bottom flask, Intermediate 020-1 (330 mg, 0.21 mmol, 1.0 eq.) was added MeOH (3 mL) and H ₂ O (2 mL), followed by lithium hydroxide monohydrate (20 mg, 0.84 mmol, 4.0 eq.). The mixture was stirred at 25°C for 3 hours. Sampling LCMS detection, the reaction conversion is complete. The pH of the reaction solution was adjusted to 3-4 with 1N hydrochloric acid solution, and the residue was purified by preparative HPLC to obtain 1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl) )-7-((3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1 '-biphenyl]-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid (144 mg, 0.17 mmol, yield: 83.6%). LC-MS: (ESI) [M+H] ⁺ = 786.2. ¹ H NMR: (400MHz, DMSO) δ10.46(s, 1H), 8.99–8.96(m, 1H), 8.16(d, J=1.5Hz, 1H), 8.15–8.09(m, 1H), 7.91( s,1H),7.76–7.70(m,2H),7.56–7.52(m,1H),7.46–7.39(m,2H),7.40–7.03(m,2H),6.88–6.58(m,1H), 4.74(s,1H),4.28–4.18(m,1H),3.95–3.82(m,2H),3.80–3.65(m,2H),3.33(s,2H),2.99–2.90(m,1H), 2.80–2.66(m,4H),2.66–2.55(m,2H),2.47(s,3H),2.45–2.40(m,1H),2.08–1.96(m,6H),1.64–1.55(m,1H ).

Example 002. (R)-1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)-7-(((R)-3-hydroxy Pyrrolidin-1-yl)methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1,1'-biphenyl]-3-yl ) benzo [d] oxazol-5-yl) methyl) pyrrolidine-3-carboxylic acid (020)

Referring to the synthetic route and method of Example 001, compound 020 was synthesized: (R)-1-((6-(difluoromethoxy)-2-(3'-((2-(difluoromethyl)- 7-(((R)-3-hydroxypyrrolidin-1-yl)methyl)pyrido[3,2-d]pyrimidin-4-yl)amino)-2,2'-dimethyl-[1 ,1'-biphenyl]-3-yl)benzo[d]oxazol-5-yl)methyl)pyrrolidine-3-carboxylic acid. LC-MS: (ESI) [M+H] ⁺ = 786.2.t _R = 1.288 min ¹ H NMR: (400MHz, DMSO) δ 10.45 (s, 1H), 8.97 - 8.94 (m, 1H), 8.17( d,J＝1.6Hz,1H),8.14–8.09(m,1H),7.90(s,1H),7.74–7.69(m,2H),7.57–7.50(m,1H),7.43–7.38(m, 2H),7.38–7.03(m,2H),6.87–6.57(m,1H),4.73(s,1H),4.27–4.18(m,1H),3.93–3.81(m,2H),3.79–3.66( m,2H),3.32(s,2H),2.98–2.89(m,1H),2.78–2.65(m,4H),2.64–2.54(m,2H),2.47(s,3H),2.44–2.39( m,1H),2.08–1.98(m,2H),1.96(s,4H),1.63–1.54(m,1H).

With reference to the synthetic route and method of Example 001, the following specific compounds are synthesized:

Example 040: PD-1/PD-L1 blocking activity (Alphalisa method)

Experimental steps:

1. Prepare the compound:

The compound was formulated into a 50 mM (mol/L) stock solution for use.

2. Prepare 1×Immunoassy Buffer:

Dilute 10×Immunoassy Buffer into 1×Immunoassy Buffer with deionized water.

3. Dilute compound:

The compound was diluted in a series of concentration gradients according to the experimental protocol: first diluted with DMSO to a 1000-fold final concentration of the compound, then diluted with PBS to a 100-fold final concentration of the compound, and finally diluted to a 4-fold final concentration with 1× Immunoassy Buffer compound for subsequent injection.

4. Loading: add the diluted compound to HTRF 96 well low volume plate at 5 μL/well.

5. Prepare 4×His tagged PD-L1 (20nM): Add 5 μL 500nM PD-L1 into 120uL 1×Immunoassy Buffer, mix by pipetting, and then add 5 μL/well into HTRF 96 well low volume plate.

6. Prepare 4×His tagged PD-1 (20nM): Add 5 μL 500nM PD-1 to 120 μL 1×Immunoassy Buffer, mix by pipetting, and then add 5 μL/well to HTRF 96 well low volume plate.

7. Prepare 4×Anti-6×His AlphaLISA Acceptor beads (40μg/mL) and 4×Streptavidin (SA) Donor beads (80μg/mL) mixture: Take 1μL 5mg/mL Anti-6×His AlphaLISA Acceptor beads and 2μL Add 5mg/mL SA-Donor beads into 122uL 1×Immunoassy Buffer, pipette to mix, and then add 5μL/well to HTRF 96 well low volume plate, pipette to mix.

8. Incubate at 23°C for 90 minutes in the dark.

9. Use the Alpha parameter of the EnVision multi-function microplate reader to read the absorbance at 615nm.

Table 1: Results of PD-1/PD-L1 blocking activity experiments:

化合物编号Compound number	IC ₅₀ _IC50	化合物编号Compound number	IC ₅₀ _IC50
001001	++	002002	++
004004	++	005005	++
007007	++	008008	++
009009	++	010010	++
011011	++	012012	++
013013	++	014014	++
015015	++	016016	++
017017	++	018018	++
019019	++	020020	++
021021	++	022022	++
023023	++	024024	++
025025	++	026026	++
027027	++	028028	++
030030	++	030030	++
031031	++	032032	++
033033	++	034034	++

035035	++	036036	++
037037	++	038038	++
039039	++	040040	++

Note: +: IC ₅₀ <10nM; ++: 10nM<IC ₅₀ <100nM; +++: 100nM<IC ₅₀ <1000nM; ++++: 1000nM<IC ₅₀

It can be seen from Table 1 that the compounds 001-040 prepared by the present invention have potent PD-1/PD-L1 blocking activity.

Example 041. PD-L1 occupancy (PBMC) test

Healthy human PBMC were placed in a round bottom 96-well plate, 100uL/well. Test compounds were added at different concentrations, followed by 1 ng/ml of IFN-g (R&D System). Incubate at 37°C for 18 hours. Then add PD-L1 flow antibody (PE, M1H1, eBioscience), CD14 flow antibody (BV421,) and stain for 1 hour in the dark. After centrifugation, resuspending cells in PBS, and sieving, the average fluorescence intensity of PD-L1 in CD14+ cell subsets was detected by flow cytometry. Concentration and mean fluorescence intensity were plotted and _IC50 was calculated.

Table 2 PD-L1 occupancy (PBMC)

化合物编号Compound number	PBMC(IC ₅₀) PBMC (IC ₅₀ )	化合物编号Compound number	PBMC(IC ₅₀) PBMC (IC ₅₀ )
004004	<1nM<1nM	029029	<1nM<1nM
011011	<1nM<1nM	034034	<1nM<1nM
012012	<1nM<1nM	037037	<1nM<1nM
014014	<1nM<1nM	039039	<1nM<1nM
015015	<1nM<1nM	化合物acompound a	45nM45nM
020020	<1nM<1nM	the	the

It can be seen from Table 2 that the compound of the present invention exhibits significant PD-L1 occupation activity in PBMC occupation activity.

Compound a is selected from Example 24 of CN110582493A:

Example 042. PD-L1 occupancy (MC-38) test

MC-38 cells were placed in a flat-bottomed 96-well plate, 100uL/well. Various concentrations of test compounds were added. Incubate at 37°C for 18 hours. Then add PD-L1 flow antibody (PE, M1H1, eBioscience), and dark staining for 1 hour. After centrifugation, resuspending cells in PBS, and sieving, the average fluorescence intensity of PD-L1 was detected by flow cytometry. Concentration and mean fluorescence intensity were plotted and _IC50 was calculated.

Table 3 PD-L1 occupancy (MC-38)

化合物编号Compound number	MC-38(IC ₅₀) MC-38 (IC ₅₀ )	化合物编号Compound number	MC-38(IC ₅₀) MC-38 (IC ₅₀ )
004004	++	029029	++
011011	++	034034	++
012012	++	037037	++
014014	++	039039	++
015015	++	化合物acompound a	++++++

020

+

the

Note: +: _IC50 <0.5nM; ++: 0.5nM< _IC50 <3nM; +++: 3nM<_IC50;

It can be seen from Table 3 that the compound of the present invention exhibits significant PD-L1 occupation activity in MC-38 cell occupation activity.

Example 043: In vitro co-incubation experiment

Experimental procedure

1. Pretreatment of 293T-OS8-hPDL1 cells:

(1) Take a small culture bottle full of 293T OS8-hPDL1 cells, discard the culture medium, add 4 mL of PBS to wash, discard the PBS, add 1 mL of trypsin to mix, digest the cells, let stand for 2 minutes, and gently The cells were patted off to make them completely detached (you can observe whether they are completely detached with a microscope), and then 4 mL of DMEM-hPDL1 complete culture medium (DMEM culture medium + 10% FBS + 50 μg/mL Hypomycin + 0.5 ng/mL puromycin) was added to stop the digestion.

(2) Subsequently, the liquid was transferred to a 15mL centrifuge tube and centrifuged at 1000r/min for 5min.

(3) Discard the supernatant, add 2 mL DMEM-hPDL1 complete culture solution, add 2 μL Mitomycin C (Mitomycin C), and incubate at room temperature for 1.5 h.

(4) Centrifuge at 1000r/min for 5min, discard the supernatant.

(5) Add 5mL PBS to wash three times (centrifuge, remove supernatant).

(6) Add 2 mL of 1640-hPDL1 complete culture medium (1640 culture medium + 10% serum + 50 μg/mL Hypomycin + 0.5ng/mL puromycin) to resuspend the cells and count them.

2. 293T OS8-hPDL1 cell seeding plate: Seed 293T OS8-hPDL1 cells at 5×10 ⁴ cells/100 μL/well in a 96-well plate, and mix well. Place in a 5% CO ₂ incubator at 37°C and incubate for 2 h.

3. Administration of 293T OS8-hPDL1 cells. Experimental group settings:

Blank control: PBS (Con), DMSO (0.1%)

Positive control: Atezolizumab (10nM)

Test group: each concentration of the compound to be tested

Drug delivery system: 100 μL 293T OS8-hPDL1 cells (50,000/well) + 1 μL compound (final concentration)

After administration, they were placed in a 5% CO ₂ incubator at 37°C and incubated for 3 hours.

4. Preparation of CD3 ⁺ T cells: Resuscitate purchased CD3 ⁺ T cells, add 1 mL of CD3 ⁺ T cell cryopreservation solution to 5 mL of 1640 complete culture medium, centrifuge at 1000 r/min for 5 min, and discard the supernatant. Add 5mL1640 complete culture medium, resuspend the cells, and count.

5. CD3 ⁺ T cell seeding plate: CD3 ⁺ T cells were seeded in the original 96-well plate at 10×10 ⁴ cells/100 μL/well, and mixed well. Total system: 200 μL 293T OS8-hPDL1 cells and CD3 ⁺ T cell mixture + 2 μL compound (final concentration)

6. Co-incubation culture: the cells were placed in a 5% CO ₂ incubator at 37°C and incubated for 48 hours.

7. Sample collection: After 48 hours, the supernatant was collected for ELISA detection of IFN-g.

8. Data processing: (value of compound detection-blank control (PBS))/(DMSO-blank control (PBS))

Table 4 in vitro co-incubation test results

化合物编号Compound number	Folds comparing with DMSO(IFN-g)Folds comparing with DMSO(IFN-g)
012(5nM)012(5nM)	3.93.9
020(5nM)020(5nM)	3.23.2
mAb(10nM)mAb (10nM)	2.72.7

The results of in vitro co-incubation experiments show that the compound prepared by the present invention has a higher expression level of IFN-g than the monoclonal antibody, indicating that the compound of the present invention can better reverse the T cell function inhibited by PD-L1.

Example 044: Mouse Absorption Test

ICR mice were used as experimental animals, and 30 mg/kg (milk white suspension formulated with sodium carboxymethyl cellulose solution) was administered by intragastric administration. The time points of blood collection for intragastric administration were 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours. Take 0.3ml of whole blood, and take 0.1ml of plasma after centrifugation for analysis by LC-MS/MS

Table 5: Experimental results of mouse absorption test

化合物编号Compound number	剂量dose	C _max _Cmax
012012	30mg/kg30mg/kg	++++++
015015	30mg/kg30mg/kg	++++++
020020	30mg/kg30mg/kg	++++++
029029	30mg/kg30mg/kg	++++
034034	30mg/kg30mg/kg	++++

Note: +: C _max <10ng/ml; ++: 10ng/ml<C _max <200ng/ml; +++: 200ng/ml<C _max

It can be seen from Table 5 that the compounds obtained in the present invention have better oral absorption.

Example 045: In vivo anti-tumor efficacy experiment

The right forelimb of female C57BL/6 mice was inoculated with 1 million MC38-hPDL1 cells. After the tumors grew to an average of 90 mm ³ , random average grouping was carried out according to the tumor volume. Different doses of the compounds were administered orally twice a day. At the same time, an equal volume of blank vehicle was orally administered twice a day as a blank control. Durvalumab monoclonal antibody (5 mg/kg) was administered twice a week as a positive control. Tumor volume was measured with a vernier caliper three times a week, and body weight was recorded. When the average tumor volume of the blank control group reached 2000 mm ³ , the experiment was stopped. According to the average tumor volume of each group at different times, the tumor growth curve was drawn.

The test results show that the compound of the present invention has better anti-tumor effect in vivo.

Claims

A kind of compound is characterized in that, has the structure shown in following general formula (I):

or a stereoisomer thereof or a mixture of stereoisomers thereof or a pharmaceutically acceptable salt thereof;

X is selected from: N or CH;

R 1 is selected from: C 1 -C 4 alkoxy, C 3 -C 5 cycloalkoxy, C 1 -C 4 haloalkoxy, C 3 -C 5 cycloalkyl, C 1 -C 4 haloalkyl;

R is selected from : methyl group, cyano group, chlorine atom;

R 3 is selected from: methyl group, cyano group, chlorine atom;

R 4 is selected from: H, C 1 -C 4 alkoxy, C 3 -C 5 cycloalkoxy, C 1 -C 4 haloalkoxy, C 3 -C 5 cycloalkyl, C 1 -C 4 haloalkane base;

R W , R E are independently selected from:

Ra and Ra' are independently selected from: H, methyl, trifluoromethyl, ethyl, -OH, -COOH, -COOMe,

n is selected from: 0, 1, 2;

m is selected from: 1,2.
The compound according to claim 1, characterized in that it has a structure shown in general formula (IIa):

or a stereoisomer thereof or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.
The compound according to claim 1, characterized in that, has a structure shown in general formula (IIb):
The compound according to claim 2 or 3, characterized in that,

R 1 is preferably selected from:
The compound according to claim 4, characterized in that,

R2 is preferably selected from: methyl ;

R W is preferably selected from:

R E is preferably selected from:
The compound according to claim 1, characterized in that it is one or more of the following compounds:

Or a stereoisomer of the above compound or a mixture of stereoisomers of the above compound or a pharmaceutically acceptable salt of the above compound.
A pharmaceutical composition, characterized by comprising one or more of the compounds described in any one of claims 1-6.
A pharmaceutical preparation, characterized by comprising one or more of the compounds described in any one of claims 1-6.
The pharmaceutical preparation according to claim 8, is characterized in that, comprises tablet, powder, capsule, injection preparation, granule preparation, spray.
A compound according to any one of claims 1 to 6 is used in the preparation alone or in combination with other drugs to treat diseases benefiting from the inhibition of PD1 or PD-L1 activity.
The application according to claim 10, wherein the other drugs are selected from the group consisting of antimicrobial agents, antiviral agents, cytotoxic agents, gene expression regulators, chemotherapeutic agents, anticancer agents, antiangiogenic agents, immunotherapy antihormonal, antifibrotic, radiotherapeutic, antineoplastic, or antiproliferative
The use according to claim 10, wherein the disease is selected from one or more of infectious diseases, immune diseases, inflammatory diseases and cancer.