CN111039942A

CN111039942A - Nitrogen-containing heterocyclic compound, and preparation method, pharmaceutical composition and application thereof

Info

Publication number: CN111039942A
Application number: CN201811191323.8A
Authority: CN
Inventors: 王喆; 张基勇; 曾志宏
Original assignee: Shanghai Longwood Biopharmaceuticals Co Ltd
Current assignee: Shanghai Longwood Biopharmaceuticals Co Ltd
Priority date: 2018-10-12
Filing date: 2018-10-12
Publication date: 2020-04-21
Anticipated expiration: 2038-10-12
Also published as: CN111039942B

Abstract

The invention provides a nitrogen-containing heterocyclic compound, and a preparation method, a pharmaceutical composition and application thereof, and particularly provides a compound shown as a formula I, or an optical isomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof; wherein, the definition of each group is described in the specification. The compounds of formula I are useful for treating diseases associated with the PD-1/PD-L1 signaling pathway.

Description

Nitrogen-containing heterocyclic compound, and preparation method, pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of small molecule medicines, and particularly provides a small molecule compound which can be used for treating diseases related to a PD-1/PD-L1 signal pathway.

Background

The immune system plays a crucial role in controlling and curing many diseases, such as various cancers, diseases caused by viruses, and the like. Cancer cells often multiply rapidly by evading or suppressing the immune system in some way. One such way is to alter the expression of activating and inhibitory molecules on immune cells. Blocking the suppressive immune checkpoint, like PD-1, has proven to be a very effective method of suppressing cancer cells.

PD-1 is programmed cell death protein-1, also known as CD 279. It is expressed mainly in activated T cells and B cells and functions to inhibit the activation of cells, which is a normal homeostatic mechanism of the immune system, and PD-1 is a protective wall of our human body because excessive T/B cell activation causes autoimmune diseases. PD-1 is a type I transmembrane glycoprotein composed of 268 amino acids, and its structure mainly includes an outer immunoglobulin variable region, a hydrophobic transmembrane region, and an intracellular region. The intracellular region contains two phosphorylation sites, located in the immunoreceptor tyrosine inhibitory motif and immunoreceptor tyrosine switch motif, which also demonstrates that PD-1 is capable of back-regulating T cell receptor-mediated signaling. PD-1 has two ligands, PD-L1 and PD-L2, which differ in the manner of expression. PD-L1, which is up-regulated in many tumor cells, binds to PD-1 on T cells, inhibits T cell proliferation and activation, leaves T cells in an inactive state, and ultimately induces immune escape.

PD-1/PD-L1 exerts a reverse immunomodulatory effect. When PD-1 binds to PD-L1, tyrosine in the tyrosine-converting motif domain of the immune receptor of T cells can be phosphorylated, and the phosphorylated tyrosine can bind to phosphatase protein tyrosinase 2 and protein tyrosinase 1. This can block activation of extracellular signal-regulated kinase, and can block activation of phosphoinositide 3-kinase (PI3K) and serine-threonine protein kinase (Akt), thereby inhibiting T lymphocyte proliferation and secretion of related cytokines. While the PD-1/PD-L1 signal inhibits the activation and proliferation of T cells, the secretion of cytokines interleukin 2, interferon gamma and IL-10 can be realized. In addition, PD-1/PD-L1 signal has similar immune function to B cell, and when PD-1 binds to B cell antigen receptor, PD-1 cytoplasm region acts with tyrosinase containing protein tyrosinase 2 binding site, thereby preventing B cell activation.

PD-1/PD-L1-based immunotherapy is a new generation of immunotherapy that is of great interest. In recent years, a series of surprising research results prove that the PD-1/PD-L1 inhibitor has strong antitumor activity on various tumors. PD-1/PD-L1 antibody inhibitors that are currently marketed are Ninolumab from BMS, Lamboluzumab from Merck, and Atezolizumab from Roche. In addition to this, there are many studied PD-1/PD-L1 antibody inhibitors, including Cure Tech's Pidilizumab, GSK's AMP-224, and AstraZeneca's MEDI-4736.

Although tumor immunotherapy is considered as a new generation of revolution for targeted post-treatment cancer therapy. However, currently marketed and researched PD-1 single drug has its own defects, including injection administration, oral administration, instability in vivo, easy protease decomposition, easy immune cross reaction, difficult purification, high production cost, etc. Therefore, small molecule inhibitors of the PD-1/PD-L1 interaction are better candidates for tumor immunotherapy.

In view of the foregoing, there is a pressing need in the art to develop novel small molecule inhibitors of the PD-1/PD-L1 interaction.

Disclosure of Invention

The invention aims to provide a novel small molecule inhibitor of PD-1/PD-L1 interaction.

In a first aspect of the present invention, there is provided a compound represented by the following formula I, or an optical isomer, hydrate, solvate, or a pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

L₁、L₂and L₃Each independently selected from the group consisting of: a bond, substituted or unsubstituted C1-C4 alkylene, substituted or unsubstituted C2-C4 alkenylene, substituted or unsubstituted C2-C4 alkynylene, -S-, -O-, substituted or unsubstituted-NH-, -S (O)₂-, substituted or unsubstituted-NHC (O) NH-, or,

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Is a group having the structure shown below:

wherein the content of the first and second substances,

Z¹selected from the group consisting of: o, S, NRf, N-O-Rf; wherein, said Rf is selected from the group consisting of: H. substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted C₆-C₁₀Heteroaryl, cyano, -C (═ O) -NRdRe, -C (═ O) -substituted or unsubstituted C₁-C₆Alkoxy, -C (═ O) -substituted or unsubstituted C₁-C₆Alkyl, -C (═ O) -substituted or unsubstituted C₃-C₁₀Cycloalkyl, -C (═ O) -substituted or unsubstituted C₂-C₆Alkenyl, -C (═ O) -substituted or unsubstituted C₂-C₆An alkynyl group;

Z²、Z³、Z⁴each independently selected from the group consisting of: NH, N, CH₂N-O, SO or SO₂；

Y¹、Y²、Y³Each independently selected from the group consisting of: CH. CH (CH)₂NH, N-O, CH, O, S, SO or SO₂；

Is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

r is H, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₆-C₁₀Aryl, cyano, -C (═ O) -NRdRe, -C (═ O) -substituted or unsubstituted C₁-C₆Alkoxy, -C (═ O) -substituted or unsubstituted C₁-C₆Alkyl, -C (═ O) -substituted or unsubstituted C₃-C₁₀Cycloalkyl, -C (═ O) -substituted or unsubstituted C₂-C₆Alkenyl, -C (═ O) -substituted or unsubstituted C₂-C₆Alkynyl, or- (L)_1a)_r-(L_2a)_s-(L_3a)_s-, in which,

each L_1aEach independently is a group selected from the group consisting of; chemical bond, substituted or unsubstituted C₁-C₇Alkylene, substituted or unsubstituted C2-C4 alkenylene, substituted or unsubstituted C2-C4 alkynylene, -S-, -O-, substituted or unsubstituted-NH-, -S (O)₂-、

L_2aSelected from the group consisting of: substituted or unsubstituted C6-C12 arylene, substituted or unsubstituted 5-12 membered heteroarylene having 1-3 heteroatoms, substituted or unsubstituted C3-C8 cycloalkylene, substituted or unsubstituted 5-10 membered heterocyclylene having 1-3 heteroatoms;

L_3aselected from the group consisting of: substituted or unsubstituted C1-C10 alkyl, C1-C10 aryl, -CN, hydroxy, amino, carboxy, -CO-NH-SO₂-R_g、-NH-SO₂-R_g、-SO₂-NH-CO-R_g、

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

rd, Re are each independently selected from the group consisting of: H. substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted C₆-C₁₀An aryl group;

selected from the group consisting of: substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 membered heterocyclyl, substituted or unsubstituted 5-12 membered C5-C12 cyclyl, wherein said heterocyclyl has 1-3 heteroatoms; or the said

Is absent;

or the said

Is equal to

Each independently selected from the group consisting of: substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted 5-12 membered (preferably 5-7 membered) heteroarylene having 1-3 heteroatoms, substituted or unsubstituted 5-12 membered heterocyclylene, substituted or unsubstituted 5-12 membered C5-C12 cycloalkylene; or the said

Is absent;

and said

Is not absent at the same time;

R₁、R₂、R₃and R₄Each independently selected from the group consisting of: H. halogen, substituted or unsubstitutedC1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 membered heteroaryl having 1-3 heteroatoms, substituted or unsubstituted 5-12 membered heterocyclyl having 1-4 heteroatoms, substituted or unsubstituted C6-C10 aryl

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

Unless otherwise specified, "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen: including but not limited to-F, Cl, Br, -CH₂Cl、-CHCl₂、-CCl₃、-CH₂F、-CHF₂、-CF₃Oxo, -CN, hydroxy, amino, C1-C6 alkylamino, carboxy, -NHAc, a group selected from the group consisting of unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkoxy, C6-C10 aryl, C3-C8 cycloalkyl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O; the substituents are selected from the following group: halogen, hydroxy, carboxy, cyano, C1-C6 alkoxy, C1-C6 alkylamino;

in the formulae above, any of the heteroatoms is selected from the group consisting of: n, S and O.

In another preferred embodiment, the

A bicyclic structure of 5-7 membered ring and 5-6 membered ring.

In another preferred embodiment, the

Is a bicyclic structure selected from the group consisting of: a 6-membered and 6-membered ring, a 6-membered and 5-membered ring, a 5-membered and 6-membered ring.

In another preferred embodiment, the

Is a structure shown in the following formula (II):

wherein:

X¹、X²、X³、X⁴、X⁵、X⁶each independently selected from the group consisting of: NH, N, CH₂Or CH ═ CH;

is a single bond or a double bond.

In another preferred embodiment, the

Is equal to

In another preferred embodiment, the

Is a ring-forming group selected from the group consisting of: benzene, pyridine, pyrimidine, pyridazine, tetrazine, triazine, pyrrole, thiophene, furan, tetrazole, triazole, imidazole, thiazole, oxazole, pyrazole, isothiazole, isoxazole, oxadiazole, thiadiazole, naphthalene, indole, triazine, pyridine, thiophene, and thiophene,Indazole, quinoline, isoquinoline, morpholine, dihydropiperidine, thiomorpholine, piperidine, piperazine, tetrahydropyran, dihydropyran, pyrroline, tetrahydrothiophene, tetrahydrofuran, oxetane, thietane, azetidine, benzofuran, benzothiophene, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, benzisoxazole, benzotriazole.

In another preferred embodiment, the

Is a ring-forming group selected from the group consisting of:

in another preferred embodiment, the

Each independently selected from the group consisting of: substituted or unsubstituted phenylene, substituted or unsubstituted 5-7 membered heteroarylene having 1-3 heteroatoms, substituted or unsubstituted 5-7 membered heterocyclylene, substituted or unsubstituted C5-C7 cycloalkylene.

In another preferred embodiment, the

Each independently a ring-forming group selected from the group consisting of: benzene, pyridine, pyrimidine, pyridazine, tetrazine, triazine, pyrrole, thiophene, furan, tetrazole, triazole, imidazole, thiazole, oxazole, pyrazole, isothiazole, isoxazole, oxadiazole, thiadiazole, naphthalene, indole, indazole, quinoline, isoquinoline, morpholine, dihydropiperidine, thiomorpholine, piperidine, piperazine, tetrahydropyran, dihydropyran, pyrroline, tetrahydrothiophene, tetrahydrofuran, oxetane, thietane, azapineCyclobutane, benzofuran, benzothiophene, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, benzisoxazole, benzotriazole.

In another preferred embodiment, the

Each independently a ring-forming group selected from the group consisting of:

in another preferred embodiment, the

Each independently is a structure represented by the following formula (III):

wherein:

Z^1a、Z^2a、Z^3a、Z^4aeach independently selected from the group consisting of: n, CH are provided.

In another preferred embodiment, the structure of formula II or formula III may be substituted or unsubstituted.

In another preferred embodiment, the structure of formula III is

In another preferred embodiment, R is₂Selected from the group consisting of: C1-C6 alkyl, CN.

In another preferred embodiment, the ring is

Having a substituent represented by formula IV below:

wherein, L is₄Selected from the group consisting of: substituted or unsubstituted C1-C4 alkylene, -S-, -O-, -NRa-, -S (O) -, -S (O)₂-; preferably a substituted or unsubstituted C1-C4 alkylene group;

selected from the group consisting of: a substituted or unsubstituted 5-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O; preferably, said

Is a 5-7 membered nitrogen containing heterocyclic group;

R₅selected from the group consisting of: substituted or unsubstituted C1-C6 alkyl, -CN, hydroxy, amino, carboxy; wherein the substituents are selected from the group consisting of: halogen, hydroxy, carboxy, cyano, C1-C6 alkoxy.

In another preferred embodiment, R is₄Selected from the group consisting of: -O (substituted or unsubstituted C1-C6 alkyl), -O (CH)₂)_n- (substituted or unsubstituted 5-to 7-membered heteroaryl having 1 to 3 heteroatoms selected from N, S and O).

In another preferred embodiment, the compound of formula I is selected from a compound of formula Ia, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

L₁selected from the group consisting of: a bond, substituted or unsubstituted C1-C4 alkylene, substituted or unsubstituted C2-C4 alkenylene, substituted or unsubstituted C2-C4 alkynylene, -S-, -O-, substituted or unsubstituted-NH-, -S (O)₂-, substituted or unsubstituted-NHC(s) ((s))O)NH-、

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Is a group having the structure shown below:

wherein the content of the first and second substances,

Z²、Z³、Z⁴each independently selected from the group consisting of: NH, N, CH₂N-O, SO or SO 2;

Y¹、Y²、Y³each independently selected from the group consisting of: CH. CH (CH)₂NH, N-O, CH, O, S, SO, or SO 2;

is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

Is absent; or the said

Is equal to

R₁、R₂、R₃And R₄Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl having 1 to 3 heteroatoms, substituted or unsubstituted having 1 to 4 heteroatoms5-to 12-membered heterocyclic group of (a), substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

Preferably, the compound is selected from compounds 1-125.

In another preferred embodiment, the compound of formula I is selected from compounds of formula Ib, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

L₁selected from the group consisting of: a bond, substituted or unsubstituted C1-C4 alkylene, substituted or unsubstituted C2-C4 alkenylene, substituted or unsubstituted C2-C4 alkynylene, -S-, -O-, substituted or unsubstituted-NH-, -S (O)₂-, substituted or unsubstituted-NHC (O) NH-, or,

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Is a group having the structure shown below:

wherein the content of the first and second substances,

Z¹selected from the group consisting of: o, S, NRf, N-O-Rf; wherein, said Rf is selected from the group consisting of: H. substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted C₆-C₁₀Heteroaryl, cyano, -C (═ O) -NRdRe, -C (═ O) -substituted or unsubstituted C₁-C₆Alkoxy, -C (═ O) -substituted or unsubstituted C₁-C₆Alkyl, -C (═ O) -substituted or unsubstitutedC of (A)₃-C₁₀Cycloalkyl, -C (═ O) -substituted or unsubstituted C₂-C₆Alkenyl, -C (═ O) -substituted or unsubstituted C₂-C₆An alkynyl group;

is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

L_2aSelected from the group consisting of: substituted or unsubstituted C6-C12 arylene, substituted or unsubstituted 5-12 membered heteroarylene having 1-3 heteroatoms, substituted or unsubstituted C3-C8 cycloalkyleneA substituted or unsubstituted 5-10 membered heterocyclylene group having 1-3 heteroatoms;

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

Is absent; or the said

Is equal to

R₁、R₂、R₃And R₄Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino)) Substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl having 1 to 3 heteroatoms, substituted or unsubstituted 5-to 12-membered heterocyclic group having 1 to 4 heteroatoms, substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

Preferably, the compound is selected from compounds 126-202.

In another preferred embodiment, the compound of formula I is selected from compounds of formula Ic, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Is a group having the structure shown below:

wherein the content of the first and second substances,

is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

each L_1aEach independently is a group selected from the group consisting of; chemical bond, substituted or unsubstituted C₁-C₇Alkylene, substituted or unsubstituted C2-C4 alkenylene, substituted or unsubstituted C2-C4 alkynylene, -S-, -O-, substituted or unsubstitutedunsubstituted-NH-, -S (O)₂-、

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

Is absent; or the said

Is equal to

R₁、R₂、R₃And R₄Each independently selected from the group consisting of:H. halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl having 1 to 3 heteroatoms, substituted or unsubstituted 5-to 12-membered heterocyclyl having 1 to 4 heteroatoms, substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

Preferably, the compound is selected from the group consisting of compounds 203-313.

In another preferred embodiment, the compound of formula I is selected from the group consisting of formulae Id-1 and Id-2, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

L₁and L₃Each independently selected from the group consisting of: a bond, substituted or unsubstituted C1-C4 alkylene, substituted or unsubstituted C2-C4 alkenylene, substituted or unsubstituted C2-C4 alkynylene, -S-, -O-, substituted or unsubstituted-NH-, -S (O)₂-, substituted or unsubstituted-NHC (O) NH-, or,

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Rf is selected from the group consisting of: H. substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted C₆-C₁₀Heteroaryl, cyano, -C (═ O) -NRdRe, -C (═ O) -substituted or unsubstituted C₁-C₆Alkoxy, -C (═ O) -substituted or unsubstituted C₁-C₆Alkyl, -C (═ O) -substituted or unsubstituted C₃-C₁₀Cycloalkyl, -C (═ O) -substituted or unsubstituted C₂-C₆Alkenyl, -C (═ O) -substituted or unsubstituted C₂-C₆An alkynyl group;

Z²and Z³Each independently selected from the group consisting of: NH, N, CH₂N-O, SO or SO 2;

Y¹and Y²Each independently selected from the group consisting of: CH. CH (CH)₂NH, N-O, CH, O, S, SO, or SO 2;

is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

Is absent; or the said

Is equal to

R₁、R₂And R₃Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstitutedC6-C10 aryl, substituted or unsubstituted 5-12 membered heteroaryl having 1-3 heteroatoms, substituted or unsubstituted 5-12 membered heterocyclyl having 1-4 heteroatoms, substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

Preferably, the compound is selected from the group consisting of compounds 314 and 355.

In another preferred embodiment, the compound of formula I is selected from a compound of formula Ie, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Is a group having the structure shown below:

wherein the content of the first and second substances,

is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

L_2aSelected from the group consisting of: substituted or notSubstituted C6-C12 arylene, substituted or unsubstituted 5-12 membered heteroarylene having 1-3 heteroatoms, substituted or unsubstituted C3-C8 cycloalkylene, substituted or unsubstituted 5-10 membered heterocyclylene having 1-3 heteroatoms;

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

Is absent; or the said

Is equal to

R₁、R₂、R₃And R₄Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstitutedUnsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl having 1 to 3 heteroatoms

Substituted or unsubstituted 5-12 membered heterocyclic group having 1-4 hetero atoms, substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

Preferably, the compound is selected from the group consisting of 356-360.

In another preferred embodiment, the pharmaceutically acceptable salts include salts formed in combination with inorganic acids, organic acids, alkali metal ions, alkaline earth metal ions, or organic bases capable of providing physiologically acceptable cations, as well as ammonium salts.

In another preferred embodiment, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; the organic acid is selected from methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, lycic acid, tartaric acid maleate, fumaric acid, citric acid or lactic acid; the alkali metal ions are selected from lithium ions, sodium ions and potassium ions; the alkaline earth metal ions are selected from calcium ions and magnesium ions; the organic base capable of providing a physiologically acceptable cation is selected from methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris (2-hydroxyethyl) amine.

In a second aspect of the invention, there is provided a process for the preparation of a compound of formula I as described in the first aspect of the invention, said process comprising a step selected from the group consisting of those depicted in schemes 1,2 or 3:

synthesis scheme 1

(e) Taking halide 1-1 and appropriate coupling reagent 1-2 (such as boric acid, boric acid ester, tin reagent or Grignard reagent) as basic raw materials, and obtaining an intermediate compound 1-3 through coupling reaction (such as Suzuki, Stille or Kumada coupling) catalyzed by palladium or copper;

(f) taking the intermediate 1-3 as a raw material, and reacting with carboxylic acid 1-4 under the action of a condensing agent (such as HATU, EDCI or HBTU) to obtain an amide intermediate 1-5;

(g) taking the intermediate 1-5 as a raw material, and removing a protecting group (Boc) under an acidic condition to obtain an intermediate 1-6;

(h) taking the intermediate 1-6 as a raw material, and carrying out nucleophilic substitution reaction with a halide under an alkaline condition, or carrying out reductive amination reaction with aldehyde or ketone under the action of a reducing agent to obtain a target compound I;

synthesis scheme 2:

(e) taking carboxylic ester 2-1 as a raw material, and carrying out an aminolysis reaction with amine 2-2 under the catalysis of Lewis acid to obtain an intermediate compound 2-3;

(f) taking the intermediate 2-3 and a proper coupling reagent 2-4 (such as boric acid, boric acid ester, a tin reagent or a Grignard reagent) as basic raw materials, and obtaining an intermediate compound 2-5 through a coupling reaction (such as Suzuki, Stille or Kumada coupling) catalyzed by palladium or copper;

(g) taking the intermediate 2-5 as a raw material, and removing a protecting group (Boc) under an acidic condition to obtain an intermediate 2-6;

(h) taking the intermediate 2-6 as a raw material, and carrying out nucleophilic substitution reaction with a halide under an alkaline condition, or carrying out reductive amination reaction with aldehyde or ketone under the action of a reducing agent to obtain a target compound I;

synthesis scheme 3:

(e) using carboxylic ester 3-1 as a raw material, and carrying out Buchwald-Hartwig coupling or Ullmann coupling with amine 3-2 under the catalysis of palladium or copper to obtain an intermediate compound 3-3;

(f) taking the intermediate 3-3 and a proper coupling reagent 3-4 (such as boric acid, boric acid ester, a tin reagent or a Grignard reagent) as basic raw materials, and obtaining an intermediate compound 3-5 through a coupling reaction (such as Suzuki, Stille or Kumada coupling) catalyzed by palladium or copper;

(g) taking the intermediate 3-5 as a raw material, and removing a protecting group (Boc) under an acidic condition to obtain an intermediate 3-6;

(h) taking the intermediate 3-6 as a raw material, and carrying out nucleophilic substitution reaction with a halide under an alkaline condition, or carrying out reductive amination reaction with aldehyde or ketone under the action of a reducing agent to obtain a target compound I;

said Cy is

Is composed of

Y¹、Y²、Z¹、Z²、Z³And R is as defined above.

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

In a fourth aspect of the present invention, there is provided a use of the compound according to the first aspect of the present invention or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or the pharmaceutical composition according to the second aspect of the present invention, for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease associated with an activity or an expression amount of PD-1/PD-L1.

In a fifth aspect of the invention, there is provided a PD-1/PD-L1 inhibitor, said inhibitor comprising a compound according to the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

In another preferred embodiment, the pharmaceutical composition is used for treating a disease selected from the group consisting of: cancer, infectious disease, autoimmune disease.

In another preferred embodiment, the cancer is selected from skin cancer, lung cancer, urinary tumor, hematological tumor, breast cancer, glioma, digestive tumor, reproductive tumor, lymphoma, nervous system tumor, brain tumor, head and neck cancer.

In another preferred embodiment, the infectious disease is selected from bacterial infection and viral infection.

In another preferred embodiment, the autoimmune disease is selected from organ-specific autoimmune disease, systemic autoimmune disease.

In another preferred embodiment, the organ-specific autoimmune disease comprises chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, goodpasture's syndrome, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis.

In another preferred embodiment, the systemic autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia.

In a sixth aspect of the present invention, there is provided a method for inhibiting the PD-1/PD-L1 interaction in vitro, comprising the steps of: contacting a compound according to the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, with PD-L1 protein.

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

Detailed Description

The inventor has extensively and intensively studied and found a PD-1/PD-L1 interaction inhibitor with excellent inhibitory effect. On this basis, the inventors have completed the present invention.

Definition of

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

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

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

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

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

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

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

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

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

Unless specifically stated to be "substituted or unsubstituted", the groups of the present invention may be substituted with a substituent selected from the group consisting of: halogen, nitrile group, nitro group, hydroxyl group, amino group, C₁-C₆Alkyl-amino, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₁-C₆Alkoxy, halo C₁-C₆Alkyl, halo C₂-C₆Alkenyl, halo C₂-C₆Alkynyl, halo C₁-C₆Alkoxy, allyl, benzyl, C₆-C₁₂Aryl radical, C₁-C₆alkoxy-C₁-C₆Alkyl radical, C₁-C₆Alkoxy-carbonyl, phenoxycarbonyl, C₂-C₆Alkynyl-carbonyl, C₂-C₆Alkenyl-carbonyl, C₃-C₆Cycloalkyl-carbonyl, C₁-C₆Alkyl-sulfonyl, and the like.

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

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

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

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

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

Active ingredient

As used herein, "compounds of the invention" refers to compounds of formula I, and also includes various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of the compounds of formula I.

Preferred compounds of the present invention include compounds 1-360 (including various classes of R configuration and/or S configuration stereoisomers of each compound, and/or E-/Z-cis-trans isomers).

All such salts within the scope of the present invention may be prepared by conventional methods. During the preparation of the compounds of formula I and solvates and salts thereof, different crystallization conditions may occur as polycrystals or co-crystals.

Preparation of Compounds of formula I

For the preparation of the compounds of the general formula I according to the invention, the preparation of the compounds of the general formula I according to the invention can be obtained by the following synthetic route, depending on the structure of the general formula I.

(a) Taking halide 1-1 and appropriate coupling reagent 1-2 (such as boric acid, boric acid ester, tin reagent or Grignard reagent) as basic raw materials, and obtaining an intermediate compound 1-3 through coupling reaction (such as Suzuki, Stille or Kumada coupling) catalyzed by palladium or copper;

(b) taking the intermediate 1-3 as a raw material, and reacting with carboxylic acid 1-4 under the action of a condensing agent (such as HATU, EDCI or HBTU) to obtain an amide intermediate 1-5;

(c) taking the intermediate 1-5 as a raw material, and removing a protecting group (Boc) under an acidic condition to obtain an intermediate 1-6;

(d) taking the intermediate 1-6 as a raw material, and carrying out nucleophilic substitution reaction with a halide under an alkaline condition, or carrying out reductive amination reaction with aldehyde or ketone under the action of a reducing agent to obtain a target compound I;

the second method comprises the following steps:

(a) taking carboxylic ester 2-1 as a raw material, and carrying out an aminolysis reaction with amine 2-2 under the catalysis of Lewis acid to obtain an intermediate compound 2-3;

(b) taking the intermediate 2-3 and a proper coupling reagent 2-4 (such as boric acid, boric acid ester, a tin reagent or a Grignard reagent) as basic raw materials, and obtaining an intermediate compound 2-5 through a coupling reaction (such as Suzuki, Stille or Kumada coupling) catalyzed by palladium or copper;

(c) taking the intermediate 2-5 as a raw material, and removing a protecting group (Boc) under an acidic condition to obtain an intermediate 2-6;

(d) taking the intermediate 2-6 as a raw material, and carrying out nucleophilic substitution reaction with a halide under an alkaline condition, or carrying out reductive amination reaction with aldehyde or ketone under the action of a reducing agent to obtain a target compound I;

the method 3 comprises the following steps:

(a) using carboxylic ester 3-1 as a raw material, and carrying out Buchwald-Hartwig coupling or Ullman coupling with amine 3-2 under the catalysis of palladium or copper to obtain an intermediate compound 3-3;

(b) taking the intermediate 3-3 and a proper coupling reagent 3-4 (such as boric acid, boric acid ester, a tin reagent or a Grignard reagent) as basic raw materials, and obtaining an intermediate compound 3-5 through a coupling reaction (such as Suzuki, Stille or Kumada coupling) catalyzed by palladium or copper;

(c) taking the intermediate 3-5 as a raw material, and removing a protecting group (Boc) under an acidic condition to obtain an intermediate 3-6;

(d) taking the intermediate 3-6 as a raw material, and carrying out nucleophilic substitution reaction with a halide under an alkaline condition, or carrying out reductive amination reaction with aldehyde or ketone under the action of a reducing agent to obtain a target compound I;

said Cy is

Is composed of

Y¹、Y²、Z¹、Z²、Z³And R is as defined above.

In addition, the starting materials and intermediates in the above reactions are readily available, and the reactions in each step can be readily synthesized according to reported literature or by conventional methods in organic synthesis to those skilled in the art. The compounds of formula I may exist in the form of solvates or non-solvates, and crystallization using different solvents may give different solvates.

Pharmaceutical compositions and methods of administration

The compound of the present invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound of the present invention as a main active ingredient can be used for preventing and/or treating (stabilizing, alleviating or curing) diseases (e.g., cancer, infectious diseases, autoimmune diseases) associated with the PD-1/PD-L1 interaction, because the compound of the present invention has excellent inhibitory activity of the PD-1/PD-L1 interaction.

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

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

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

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

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

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

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

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

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

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

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

When administered in combination, the pharmaceutical composition further comprises one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds. One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds may be used simultaneously, separately or sequentially with the compounds of the invention for the prevention and/or treatment of a disease associated with the PD-1/PD-L1 interaction.

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

The main advantages of the invention include:

(1) the compound has high inhibitory activity on the interaction of PD-1/PD-L1, has strong binding capacity with PD-L1 protein, and has the capacity of relieving the inhibition of IFN gamma by PD-L1.

(2) The compound of the invention has better solubility; the toxicity to normal cells is very low and can therefore be applied to the treated subject over a wide dose range.

(3) Compared with the prior art, the compound of the invention has better solubility and good drug forming property, and the compound of the invention has good bioavailability in vivo experiments, and in addition, compared with the prior art, the compound of the invention can be easily prepared into pharmaceutically acceptable salts, thereby being beneficial to further preparation.

(4) The in vivo efficacy research shows that the compound can obviously inhibit the growth of subcutaneous tumors no matter on the tumor volume or the tumor weight, and can obviously increase the number of lymphocytes in the blood and the spleen of a mouse.

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

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

General materials and test methods:

the instruments and materials involved in the examples are described below:

the NMR spectrum was obtained by analysis with a Bruker AV-400(400MHz) NMR spectrometer.

Chemical shifts are reported in ppm units using tetramethylsilane as an internal standard (CDC 1)₃Delta 7.26 ppm). The data reported are the chemical shifts and their split and coupling constants (s: singlet; d: doublet; t: triplet; q: quartet; br: broad; m: multiplet).

Mass spectrometry data were analyzed using a liquid mass spectrometer from the Finnigen advanced LCQ company (Finnigan LCQ Advantage), all reactions operated under anhydrous and oxygen-free conditions under dry argon protection, except for other requirements. The solid organometallic compound was stored in an argon protected dry box.

The tetrahydrofuran and the diethyl ether are obtained by distillation, and metal sodium and benzophenone are added into the tetrahydrofuran and the diethyl ether during the distillation. Methylene chloride, pentane and hexane were treated with calcium hydride.

The special raw materials and intermediates involved in the present invention are provided by custom-made processing of Tianjin Changsen pharmaceutical Co., Ltd, and all other chemical reagents are purchased from reagent suppliers such as Shanghai chemical reagent company, Aldrich company (Aldrich), Acros company (Acros). If the intermediates or products needed by the reaction in the synthesis process are not enough for the next experiment, the synthesis is repeated for a plurality of times until the intermediates or products are enough.

The raw materials and reagents related to the invention can be obtained by commercial or customized processing and purchase except for special instructions.

The compounds of the invention may contain one or more asymmetric centres and so the series of compounds may be in racemic or single enantiomeric form. The compound prepared by the invention is a heterocyclic compound with the purity higher than 95 percent, and the structural representation of each final product is respectively represented by MS or/and hydrogen spectrum nuclear magnetic resonance (¹H NMR) analysis. The synthesis of the various compounds and intermediates of the invention is illustrated by the examples below.

Example 1: synthesis of (R) -3 ' - ((7- ((3-hydroxypyrrol-1-yl) methyl) quinolin-4-yl) oxy) -2,2 ' -dimethyl- [1,1 ' -diphenyl ] -3-carboxylic acid

Step 1:

diethyl ethoxymethylenemalonate (150mL, 749mmol) and m-toluidine (80mL, 738mmol) were placed in a flask, and the mixture was heated at 110 ℃ for 1 hour. The reaction mixture was cooled to room temperature. The flask was placed in a refrigerator and the precipitated solid was collected and washed with hexane to give the product as a white solid (120g, 58% yield).

¹H NMR(400MHz,CDCl₃)δ10.99(d,J＝13.8Hz,1H),8.54(d,J＝13.8Hz,1H),7.30–7.25(m,1H),7.02–6.92(m,3H),4.37–4.22(m,4H),2.38(s,3H),1.37(dt,J＝19.5,7.1Hz,6H).ESI m/z 278.2(M+H).

Step 2:

diethyl 2- ((m-tolylamino) methylene) malonate (148g, 534mmol) was suspended in diphenyl ether (200mL) and the mixture was refluxed for 45 minutes. The solution was allowed to cool to room temperature and the solid formed was collected and bubbled with hexane to give the product as a white solid (26.7g, 22% yield).

¹H NMR(400MHz,DMSO-d₆)δ12.19(s,1H),8.49(d,J＝6.5Hz,1H),8.04(d,J＝8.2Hz,1H),7.38(s,1H),7.24(dd,J＝8.3,1.5Hz,1H),4.21(q,J＝7.1Hz,2H),2.80(s,1H),2.44(s,3H),1.28(t,J＝7.1Hz,3H).ESI m/z 232.0(M+H).

And step 3:

ethyl-4-hydroxy-7-methylquinoline-3-carboxylate (43g, 187mmol) was added to 5M sodium hydroxide (200mL) and heated at reflux for 1 h. The mixture was cooled to room temperature and acidified to pH 4 by addition of 2M HCl. The solid formed was collected, washed with water and dried to give 4-hydroxy-7-methylquinoline-3-carboxylic acid as a white solid (37.8g, 90% yield).

ESI m/z 204.0(M+H).

And 4, step 4:

4-hydroxy-7-methylquinoline-3-carboxylic acid (35g, 172mmol) was suspended in diphenyl ether (100 mL). The mixture was refluxed for 1.5 hours and then cooled to room temperature. The precipitated solid was collected and washed with hexane to give 7-methylquinolin-4-ol as a white solid (16.8g, 62% yield).

ESI m/z 160.0(M+H).

And 5:

to a solution of 7-methylquinolin-4-ol (26g, 162mmol) in chloroform (150mL) at 0 deg.C was added phosphorus oxychloride (50 mL). The mixture was then refluxed for 3 hours, then cooled to room temperature and neutralized with 2M NaOH. The resulting solid was collected, washed with water and dried to give 4-chloro-7-methylquinoline as a white solid (17g, 59% yield).

¹H NMR(400MHz,CDCl₃)δ8.76(d,J＝4.8Hz,1H),8.14(d,J＝8.5Hz,1H),7.97–7.91(m,1H),7.50(dd,J＝8.6,1.7Hz,1H),7.45(d,J＝4.8Hz,1H),2.61(d,J＝0.9Hz,3H).ESIm/z 178.0(M+H).

Step 6:

to CCl of 4-chloro-7-methylquinoline (7g, 39mmol)₄To the solution (60mL) was added NBS (8g, 45mmol) and benzoyl peroxide (0.8g, 3.3 mmol). The solution was refluxed for 5 hours. After completion of the reaction, the solvent was evaporated and the resulting residue was purified by flash chromatography to give the product as a white solid (2.48g, 25% yield).

¹H NMR(400MHz,CDCl₃)δ8.82(d,J＝4.7Hz,1H),8.27(d,J＝8.7Hz,1H),8.15–8.11(m,1H),7.72(dd,J＝8.7,1.8Hz,1H),7.53(d,J＝4.7Hz,1H),4.71(s,2H).ESI m/z 258.0(M+H).

And 7:

(R) -pyrrolidin-3-ol (957mg, 11mmol) was added to 7- (bromomethyl) -4-chloroquinoline (2.56g, 10mmol) and Et 3N (2.16ml, 15mmol) in dichloromethane (20 ml). After stirring at room temperature for 4 hours, the solvent was removed and the resulting residue was purified by flash chromatography to give the product (2.35g, 90% yield). ESI M/z 263.0(M + H).

Step 8

To a solution of methyl 3 ' -hydroxy-2, 2 ' -dimethyl- [1,1 ' -biphenyl ] -3-carboxylate (256mg, 1mmol) in DMF (3mL) at 0 deg.C was added NaH (80mg, 2 mmol). The mixture was stirred at this temperature for 30 minutes, then (R) -1- ((4-chloroquinolin-7-yl) methyl) pyrrolidin-3-ol (262mg, 1mmol) was added. The reaction was heated at 120 ℃ overnight and then cooled to room temperature. The mixture was purified by prep-HPLC to give the product (79mg, 17% yield).

ESI m/z 469.2(M+H).

Example 2: 5- (((trans) -4- (methoxycarbonyl) cyclohexyl) methyl) -1-methyl-4, 5,6, 7-tetrahydro-1H-imidazo [4,5-c ] pyridine-2-carboxylic acid

The synthetic route is as follows:

step 1:

dissolving the compound 1(474mg, 2mmol) in 10mL of anhydrous THF under the protection of nitrogen, cooling to-78 ℃, slowly dropwise adding 1mL of 2.5M butyl lithium n-hexane solution, continuing to stir for 30min after the dropwise adding is finished, dropwise adding benzyl chloroformate (511mg, 3mmol), continuing to stir for 1h after the dropwise adding is finished, heating to room temperature, quenching by using saturated sodium bicarbonate solution, performing post-treatment and performing column chromatography to obtain the product.

Step 2:

the compound 2(300mg) was added to 10mL of 4N HCl/dioxane solution, stirred at room temperature for 1h, TLC followed the disappearance of the starting material, and concentrated under reduced pressure to give a solid which was used directly in the next reaction.

And step 3:

under the protection of argon, DMSO (3.12g, 40mmol) is added into DCM (50ml), after stirring for 10min, the mixture is cooled to-78 ℃, oxalyl chloride (2.54g, 20mmol) is added dropwise, after the addition is finished, the mixture is stirred for 10min, then a DCM solution of a compound 4(3.44g, 20mmol) is added dropwise, after stirring for 10min, triethylamine (5.26g, 52mmol) is slowly added dropwise, after stirring for 10min, the mixture is moved to room temperature and is stirred for about 30min, DCM and water are added for quenching, extraction, drying and concentration are carried out to obtain an oily liquid.

And 4, step 4:

under the protection of argon, hydrochloride (31mg, 0.1mmol) of compound 3, compound 5(22.3mg, 0.13mmol) and potassium carbonate (13.9mg, 0.1mmol) were added to a solution of DCM (1.0ml), placed in an ice bath and stirred for 30min to add NaBH (OAc) in portions₃(49.1mg, 0.23mmol) and then allowed to warm to room temperature and stirred overnight. Conversion to product was detected by TLC and LCMS.

And 5:

compound 6(100mg, 0.24mmol) was dissolved in THF (5ml), 10% Pd/C (100mg) was added, hydrogenation was carried out at normal pressure for 5 hours, filtration and concentration of the solvent gave a solid.

LCMS found 336[M+H]⁺

Example 3: synthesis of 5- (2- ((tert-butyldiphenylsilyl) oxy) ethyl) -1-methyl-4-oxo-4, 5-dihydro-1H-pyrrolo [3,2-c ] pyridine-2-carboxylic acid

The synthetic route is as follows:

step 1:

adding raw material aldehyde 1(25g, 1.0eq) into methanol (250ml), protecting with argon, cooling (the internal temperature is-10 ℃ to-15 ℃) and adding azide raw material 2(57g, 2.5eq), wherein the system is clear and does not heat up; dropwise adding 30% sodium methoxide methanol solution (81g, 2.5eq) to control the temperature at minus 12 +/-2 ℃, and maintaining the temperature for 1 h; overnight at 0 ℃; the system was cloudy and the viscosity gradually increased. And (3) treatment: pouring the reaction solution into a mixed system of ice water and ammonium chloride solid, stirring for 30 minutes, filtering, adding water into the solid, rinsing for 3 times, and pumping by an oil pump to obtain 58g of brown yellow solid (quantitative yield), and directly putting the brown yellow solid into the next step.

¹H NMR(400MHz,CDCl₃)δ8.49(dd,J＝7.6,1.9Hz,1H),8.11(dd,J＝5.0,1.9Hz,1H),7.25(d,J＝11.9Hz,1H),6.93(dd,J＝7.7,4.9Hz,1H),3.99(s,3H),3.92(s,3H).

LCMS found 208[M+H-N₂]⁺

Step 2:

adding the raw material 3(26g) into o-dichlorobenzene (500ml), and reacting for 1 hour at 145 ℃ by external bath; and (3) treatment: cooling to-10 deg.C, stirring for 10min, filtering, rinsing the solid with n-heptane, and drying to obtain light yellow solid 7g (yield: 50%)

¹H NMR(400MHz,DMSO-d₆)δ12.36(s,1H),7.83(d,J＝5.9Hz,1H),7.13(d,J＝2.0Hz,1H),7.02(d,J＝5.9Hz,1H),3.97(s,3H),3.87(s,3H).

LCMS:found 207[M+H]⁺

And step 3:

adding the raw material 4(6.8g, 1.0eq) into DMAc (70ml), dissolving to be clear, adding potassium carbonate (9.12g, 2.0eq), dropwise adding methyl iodide (7g, 1.5eq), and reacting at room temperature overnight; and (3) treatment: water (50ml), ammonia (20ml), EA (100ml x 3) was added for extraction, column: elution with ethyl acetate/n-heptane 15/85 gave 6.05g of a yellow solid (yield: 83.4%).

¹H NMR(400MHz,CDCl₃)δ7.94(d,J＝6.1Hz,1H),7.39(d,J＝0.9Hz,1H),7.26(s,1H),6.97–6.83(m,1H),4.11(s,3H),4.04(s,3H),3.91(s,3H).

LCMS:found 221[M+H]⁺

And 4, step 4:

adding 5(5g, 1.0eq) of raw material into acetic acid (82g, 30eq), adding 47% of hydrobromic acid aqueous solution (55g, 30eq), dissolving, and heating to 95 ℃ for reaction for 2 hours; and (3) treatment: concentration to give 9g (yield: quantitative) of pale yellow solid, which was directly fed to the next step.

LCMS:found 193[M+H]⁺

And 5:

adding 6(4.36g, 1.0eq) of raw material into methanol (90ml) for insolubilization, adding concentrated sulfuric acid (5g, 1.0eq), and carrying out reflux reaction at 85 ℃ in an external bath overnight; and (3) treatment: under ice bath, 70ml of saturated sodium bicarbonate is added dropwise, the pH value is 7-8, a solid is precipitated, the solid is filtered, rinsed with water and dried to obtain 4.43g of a white solid (yield: 94.8%).

¹H NMR(400MHz,DMSO-d₆)δ11.02(s,1H),7.24(s,1H),7.20(t,J＝6.5Hz,1H),6.58(d,J＝7.4Hz,1H),3.93(s,3H),3.81(s,3H).

LCMS found:207[M+H]⁺

Step 6:

adding a raw material 7(4.2g, 1.0eq) into DMAc (50ml), protecting with argon, adding cesium carbonate (13.3g, 2.0eq), adding a silanol raw material 8(9.62g, 1.3eq), and reacting at room temperature overnight; and (3) treatment: adding water (100ml) and 1M hydrochloric acid (80ml) to adjust the pH value to 5-6; EA extraction 3 times, column chromatography: 20% EA/n-heptane rinse, concentrate to give 4.5g of white solid, yield: 45.4 percent.

¹H NMR(400MHz,DMSO-d₆)δ7.54(d,J＝7.5Hz,1H),7.45(d,J＝7.3Hz,4H),7.39(t,J＝7.5Hz,2H),7.32–7.23(m,5H),6.66(d,J＝7.5Hz,1H),4.13(t,J＝5.0Hz,2H),3.95(s,3H),3.86(t,J＝5.0Hz,2H),3.83(s,3H),0.93(s,9H).

LCMS found:489[M+H]⁺。

Step 7

Taking raw material 9(4.5g,1.0eq) and adding into THF (45ml), adding water (45ml) after dissolving, adding lithium hydroxide hydrate (0.97g,2.5eq) and reacting for 2 hours; and (3) treatment: 1M hydrochloric acid (50ml) and water (50ml) were added to precipitate a white solid, which was stirred for 30 minutes, the solid was filtered and dried to obtain 4.05g of 5- (2- ((tert-butyldiphenylsilyl) oxy) ethyl) -1-methyl-4-oxy-4, 5-dihydro-1H-pyrrolo [3,2-c ] pyridine-2-carboxylic acid as a white solid, yield: 92.6 percent.

¹H NMR(400MHz,DMSO-d₆)δ12.82(s,1H),7.52(d,J＝7.5Hz,1H),7.47–7.42(m,4H),7.43–7.36(m,2H),7.28(t,J＝7.4Hz,4H),7.21(s,1H),6.65(d,J＝7.5Hz,1H),4.13(t,J＝5.0Hz,2H),3.94(s,3H),3.86(t,J＝4.9Hz,2H),0.93(s,9H).

LCMS found:475[M+H]⁺。

Example 4: synthesis of 5- (2- ((tert-butyldiphenylsilyl) oxy) ethyl) -4-oxo-4, 5-dihydrofuran [3,2-c ] pyridine-2-carboxylic acid

The synthetic route is as follows:

step 1:

to a reaction flask were added compound 1(8.7g,56.7mmol,1.0eq), 30% MeONa-MeOH solution (40mL) in sequence; heating the outer bath to 75 ℃, and stirring; TLC monitoring reaction is completed; cooling, adding water, and extracting with ethyl acetate for 3 times; the organic phases were combined, washed 4 times with 15% saline and 1 time with saturated saline. Drying with anhydrous sodium sulfate; and (6) spin-drying. 7.6g of a yellow solid are obtained, yield 89.7%.

LCMS found:150[M+H]⁺

Step 2:

adding 2(7.6g,51.0mmol,1.0eq) into a reaction bottle in sequence, and newly steaming anhydrous THF under the protection of inert gas; cooling the dry ice-acetone to-78 ℃; n-BuLi (30.5mL,76.5mmol,1.5eq,2.5M) was added dropwise; stirring for 30min at low temperature; introducing carbon dioxide dried by concentrated sulfuric acid and anhydrous calcium chloride into the reaction solution; reacting for 3 hours, and slowly heating to 0 ℃ below zero; adding saturated solution of ammonium chloride for quenching; adding water and ethyl acetate; separating out an organic phase; the aqueous phase was washed once with ethyl acetate; adjusting the pH value of the water phase to 2; a large amount of solid was precipitated, filtered and dried to obtain 9.4g of solid with a yield of 95.5%.

¹H NMR(400MHz,DMSO-d₆)δ8.15(d,J＝6.0Hz,1H),7.60(s,1H),7.39(dd,J＝6.0,0.9Hz,1H),4.02(s,3H).

LCMS found:194[M+H]⁺

And step 3:

compound 3(9.0g, 46.6mmol, 1.0eq), ethanol (270mL) and concentrated sulfuric acid (11.5mL) were added to a reaction flask and heated to reflux; stirring overnight, evaporating ethanol, cooling the residue with ice water bath, and adding water dropwise to precipitate solid; filtration and drying gave 7.4g of solid in 76.7% yield.

¹H NMR(400MHz,DMSO-d₆)δ11.68(s,1H),7.61(d,J＝2.3Hz,1H),7.50(d,J＝7.5Hz,1H),6.73(d,J＝7.4Hz,1H),4.33(q,J＝7.1Hz,2H),1.31(td,J＝7.1,2.9Hz,3H).

LCMS found:208[M+H]⁺

And 4, step 4:

4(7.0g, 33.8mmol, 1.0eq), 70mL DMAc, cesium carbonate (22.0g,67.5mmol,2.0eq) and compound 5(14.7g, 40.5mmol, 1.2eq) were added to a reaction flask, stirred at room temperature for 2h, water and ethyl acetate were added, ethyl acetate was separated, the aqueous phase was extracted once with ethyl acetate, the organic phases were combined, washed with saturated sodium chloride, and spin-dried to give compound 6, which was directly fed to the next step without purification.

LCMS found 490[M+H]⁺

And 5:

the crude compound 6 from the previous step, ethanol (70mL) and water (18mL) were added to the reaction flask and LiOH solid (1.22g, 20.9mmol, 1.5eq) was added. Stirring at room temperature for 0.5h, concentrating ethanol, and adding water and ethyl acetate to the residue; separating out ethyl acetate; water phase ice water bath, adjusting pH to 5, and separating out a large amount of solid; filtration and drying gave 10.3g of solid 7, yield 66.1% (two steps).

¹H NMR(400MHz,DMSO-d₆)δ7.82(d,J＝7.5Hz,1H),7.54(d,J＝0.9Hz,1H),7.48–7.34(m,7H),7.29(t,J＝7.3Hz,4H),6.80(dd,J＝7.5,0.9Hz,1H),4.19(t,J＝4.9Hz,2H),3.88(t,J＝4.9Hz,2H),0.92(s,9H).

LCMS found:462[M+H]⁺

Step 6: 2-amino-5- (2- ((tert-butyldiphenylsilyl) oxy) ethyl) furan [3,2-c ] pyrido-4 (5H) -one

Adding the compound 7(462mg, 1mmol) into anhydrous THF (5mL), then adding triethylamine (233mg, 2.3mmol) and azido diphenyl phosphate (316mg,1.15mmol), stirring at room temperature for 2h, then heating to reflux for 2h, then adding water (1.0mL), continuing to reflux for overnight, cooling, then adding EA for extraction, concentrating, and separating by column chromatography to obtain the product. LCMS found:462[ M + H ]]⁺

Example 5: synthesis of 5- (2- ((tert-butyldiphenylsilyl) oxy) ethyl) -7-cyano-1-methyl-4-oxo-4, 5-dihydro-1H-pyrrolo [3,2-c ] pyridine-2-carboxylic acid

The synthetic route is as follows:

step 1:

adding 25g of the compound 1 into 75ml of methanol, cooling to 0 ℃, dropwise adding 62.89g of thionyl chloride, and heating to room temperature for reaction for 2d after dropwise adding; and (3) sampling LCMS, completely reacting the raw materials, evaporating most of methanol, pouring ice water into residues, extracting by EA, combining organic phases, washing with salt, drying and spin-drying to obtain 22.7g of crude products. The crude product is directly put into the next step.

Step 2:

adding 10.7g of compound 2 into 54ml of methanol, adding 14.6g of sodium methoxide/methanol (30% wt), and after the addition is finished, heating and refluxing overnight (the system turns turbid from clear); sample LCMS and starting material was essentially reacted to completion and worked up. The methanol was evaporated, the residual DCM diluted, washed with ice water, brine, dried and spin dried to give 8.9g of crude yellow solid. The crude product is directly put into the next step. Yield: 84.7 percent

Step 3

Cooling 180ml of anhydrous THF to below-10 ℃ under the protection of argon; adding 2.8g of LiAiH4 in portions; dissolving 8.9g of compound 3 in 50ml of THF, and dropwise adding at a temperature below-10 ℃; keeping the temperature for reaction for 30min after dripping, sampling LCMS, basically completely reacting the raw materials, controlling the temperature to be less than-10 ℃, stirring 3ml of water and anhydrous sodium sulfate into sand, and adding the sand in batches; after the addition, filtering, and washing a filter cake EA; the combined organic phases were washed with salt, dried overnight, spun dried, and applied to a column to yield 4.7g of white solids (15% EA in HEP). Yield: 59.5 percent

Step 4

Dissolving 3.5g of compound 4 in 70ml of DCM, adding 10.2g of dess-Martin in batches, and reacting for 2h at room temperature after the addition is finished (the system is white and turbid); sampling LCMS to show that the raw materials are basically reacted completely, pouring into a saturated aqueous solution of ice sodium bicarbonate and sodium thiosulfate (1: 1), and extracting the aqueous phase with DCM for 1 time; the combined organic phases were washed with saturated brine, dried, spun-dried and chromatographed to give 3.1g of a white solid. Yield: 90.6 percent

Step 5

Dissolving 4g of compound 5 in 40ml of methanol, and cooling to the temperature of minus 15 ℃ to minus 10 ℃ under the protection of argon; 4.8g of ethyl azidoacetate are added; controlling the temperature to be-15 to-10 ℃, and dropwise adding 10g of sodium methoxide/methanol (30 percent by weight); after dripping, the reaction is kept for 2h (the system is changed from clear to white and turbid); carrying out external bath at-10 ℃, and keeping the temperature to react overnight; sampling LCMS, pouring the raw materials into ice saturated ammonium chloride aqueous solution after the raw materials are basically completely reacted, filtering the solid, extracting the aqueous phase for 2 times by EA, combining the organic phase soluble solid, washing with salt, drying and spin-drying to obtain 6g of light yellow solid. The crude product is directly put into the next step. Yield: 97.8 percent

Step 6

Dissolving 10.1g of compound 6 in 300ml of THF (tetrahydrofuran), and cooling to-40 to-30 ℃ under the protection of argon; 7.0g of methanesulfonyl chloride was added; controlling the temperature to be between 40 ℃ below zero and 30 ℃ below zero, dripping 21.6g of triethylamine, and keeping the temperature to react for 1.5h (the system turns turbid from clear); sampling LCMS, pouring the residual 7 percent of the raw materials into ice water, EA extracting, combining organic phases, washing with salt, drying, spin-drying, mixing the sample and passing through a column to obtain 4.8g of light yellow solid (10 percent of EA in HEP product). Yield: 50.2 percent

Step 7

Dissolving 4.8g of compound 7 in 200ml of o-dichlorobenzene, heating to 140 ℃ under the protection of argon, and reacting for 1h (the system is clear); LCMS is sampled, the raw materials are reacted completely, and the dried o-dichlorobenzene is concentrated under reduced pressure by an oil pump. The crude product is directly put into the next step.

Step 8

Dissolving 4.37g of the compound 8 in 50ml of DMAc, adding 4.24g of potassium carbonate under the protection of argon, and dropwise adding 2.61g of methyl iodide at room temperature; after dripping, reacting at room temperature for 1 h; sample LCMS, the raw material reaction is complete, pour into ice ammonia water, EA extract, combine organic phase, salt wash, dry, spin dry, mix sample and get 4.7g light yellow solid. Yield: 100 percent

Step 9

Dissolving 2.8g of compound 9 in 33ml of acetic acid, adding 33ml of hydrobromic acid, and after the addition is finished, heating to 93 ℃ (external oil bath) for reaction for 1 h; sampling LCMS, cooling the residual 3 percent of the raw material and 20 percent of saponified acid to room temperature, and drying the raw material at 60 ℃ by an oil pump to obtain yellow solid; dissolving in 30ml methanol, dripping 3ml concentrated sulfuric acid, heating and refluxing, and monitoring by LCMS until the saponified acid content is less than 8 percent, then processing: most of the methanol is removed by rotation, EA is diluted, and the yellow solid crude product is obtained by salt washing, drying and rotary drying. Yield: 89.8 percent

Step 10

Dissolving 1.36g of compound 10 in 70ml of DMAc, adding 3.11g of cesium carbonate, adding 2.08g B, and reacting at room temperature for 3 hours after the addition is finished; sample LCMS and the material was essentially reacted completely, poured into ice water, extracted with 2-MeTHF, combined organic phases, washed with salt, dried, spun dry, and chromatographed on a column to give 2.4g of a pale yellow oil. Yield: 88.6 percent

Step 11

Dissolving 4.3g of compound 11 in 86ml of NMP, adding 2.0g of cuprous cyanide, and after the addition, heating to 170 ℃ (external oil bath) for reaction for 6 h; sampling LCMS, cooling the residual 3 percent of the raw materials to room temperature, pouring into ice water, extracting by EA, combining organic phases, washing by salt, drying, spin-drying, mixing the sample and passing through a column to obtain 1.8g of light yellow solid. Yield: 46.2 percent of

¹H NMR(400MHz,CDCl₃)δ7.77(s,1H),7.52(s,1H),7.51–7.47(m,4H),7.37–7.30(m,2H),7.28(dd,J＝7.2,1.1Hz,4H),4.33(s,3H),4.13(dd,J＝5.4,3.9Hz,2H),3.97(dd,J＝5.4,3.9Hz,2H),3.91(s,3H),1.06(s,9H).

Step 12

Dissolving 1.7g of compound 12 in 17ml of THF, dissolving 0.4g of LiOH in 17ml of H2O, and dropwise adding the reaction solution to react for 1H at room temperature after dropwise adding; TLC was sampled, the starting material was reacted substantially completely, THF was spun off at room temperature, pH was adjusted to 1 with 2M aqueous hydrochloric acid in ice bath, stirring was carried out for 10min, the filter cake was washed with water, and 2g of crude pale yellow solid was obtained with water in toluene. Yield: 100 percent

Example 6: (R) -3- ((3- ((tert-butyldiphenylsilyl) oxy) pyrrol-1-yl) methyl) -8-chloro-1, 7-diazanaphthalene

The synthetic route is as follows:

step 1:

compound 1(21.8g,110.6mmol,1eq) was dissolved in TFA-H₂SO₄(4:1,350mL,16V) in the mixed solution, heating to 40 ℃, stirring overnight, taking a light yellow solution, sampling LCMS to show that the reaction is finished, concentrating the system, dripping the residue into ice water, separating out a large amount of colorless crystals, extracting IPAc once, adjusting the pH of an aqueous phase with 10M NaOH aqueous solution to 8-9, extracting the aqueous phase with IPAc three times again, combining all organic phases, washing with saturated sodium carbonate and saturated sodium chloride aqueous solution in sequence, drying with anhydrous sodium sulfate, filtering, concentrating, and obtaining a compound 2 which is a white crystal and 23.2g in total. The yield thereof was found to be 97.5%.

¹H NMR(400MHz,DMSO-d₆)δ8.54(d,J＝2.1Hz,1H),8.05(d,J＝2.1Hz,1H),7.96(s,1H),7.55(s,1H).，2.50(s,3H)

LCMS:found 215:217＝1:1[M+H]⁺

The other method comprises the following steps:

mixing the compound 1-1(18.4g,85.2mmol,1eq) and thionyl chloride (180mL,10V) to form white turbidity, stirring and heating to reflux, changing the system into a yellow solution after about 1h, stopping heating and concentrating, adding THF (tetrahydrofuran) into the residue to dissolve, dropwise adding ammonia water into a 1, 4-dioxane solution (0.4M,178.9mL, 2.1eq) under ice bath, after dropwise adding, returning the system to room temperature and stirring for 30min, concentrating, adding water into the residue, extracting IOAc for three times, combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, filtering the filtrate and concentrating to obtain 20.3g of a white foamy solid, and quantifying the yield.

Step 2:

compound 2(40.8g,189.7mmol,1eq) and DMF-DMA (33.9g,284.6mmol,1.5eq) were dissolved in toluene (612mL,15V), heated to reflux overnight with stirring, LCMS indicated complete consumption of starting material, concentrated under reduced pressure, and the residue was taken up in the next reaction without isolation.

LCMS:found 270:272＝1:1[M+H]⁺

And step 3:

dissolving the compound 3(49.4g,182.9mmol,1eq) in tetrahydrofuran (1.5L, KF 0.05%, 30V 10mL/g), adding potassium tert-butoxide (30.8g,274.4mmol,1.5eq) after nitrogen replacement, stirring and heating to reflux after nitrogen replacement again, changing the system from reddish brown to brown black, cooling to room temperature, adding water to quench the reaction, adjusting the pH to 7-8 with hydrochloric acid, concentrating, extracting the residue with 2-methyltetrahydrofuran three times, combining organic phases, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate, and carrying out column chromatography on the residue to obtain 12g of the compound 4 which is yellow to reddish brown solid. The total yield of the two steps is 29 percent.

¹H NMR(400MHz,DMSO-d₆)δ11.63(s,1H),8.80(d,J＝2.2Hz,1H),8.48(d,J＝2.2Hz,1H),7.32(d,J＝7.1Hz,1H),6.51(d,J＝7.1Hz,1H).

LCMS:found 225:227＝1:1[M+H]

And 4, step 4:

after compound 4(13.5g,60.0mmol,1eq) and phosphorus oxychloride (135mL,10V) were mixed, heated under reflux for 2h under stirring, LCMS showed the reaction was complete, concentrated, the residue was diluted with water, EA was added and slurried for 1h, filtered, the filter cake was washed with water and dried to give the product as a pale yellow solid, 9.6g, 65.7% yield.

¹H NMR(400MHz,DMSO-d₆)δ9.21(d,J＝2.3Hz,1H),8.93(d,J＝2.3Hz,1H),8.46(d,J＝5.6Hz,1H),7.92(d,J＝5.6Hz,1H).

LCMS:found 243:245:247＝3:3:1[M+H]⁺.

And 5:

the starting material compound 5(9.6g,39.4mmol,1eq), vinyl boronic acid pinacol ester (6.68g,43.4mmol,1.1eq), sodium carbonate (20.9g,197.1mmol,5eq) were added to a THF-water (4:1,480mL,50V) mixed solvent, after nitrogen substitution, tetrakistriphenylphosphine palladium (911.2mg,0.79mmol,0.02eq) was added, after nitrogen substitution again, stirring was carried out to reflux, and after 1.5h, TLC and LCMS analysis of the remaining small amount of starting material. After stopping heating, the system was concentrated, and the residue was extracted three times with EA, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and subjected to column chromatography (EA in Hep 15%) to obtain the product compound 6 as a pale yellow solid 5.9g with a yield of 76.1%.

¹H NMR(400MHz,DMSO-d₆)δ9.31(d,J＝2.2Hz,1H),8.50(d,J＝2.2Hz,1H),8.38(d,J＝5.5Hz,1H),7.89(d,J＝5.5Hz,1H),7.00(dd,J＝17.7,11.1Hz,1H),6.31(d,J＝17.7Hz,1H),5.67(d,J＝11.1Hz,1H).

LCMS:found 191/193＝3:1[M+H]⁺

Step 6:

compound 6(4.0g,21.1mmol,1.0eq), NMO (4.9g,41.8mmol,2.0eq) and K were added in that order₂OsO₄(0.38g,1.03mmol,0.049eq) THF H was added₂Stirring overnight at room temperature in solvent (100ml) of 3:1, monitoring reaction by TLC (PE: EA: 1) until the starting material point disappears, and adding aqueous solution of sodium sulfite to quench the reaction; extracting with ethyl acetate (4 times), mixing the organic phases, washing with saturated brine, spin-drying the solvent, and adding THF: H₂Dissolving in solvent (3: 1) and adding NaIO₄Solid (11.2g,52.4mmol,2.49eq), stirred, TLC monitored reaction was complete, water was added to dissolve the solid, extracted with ethyl acetate (4 times), the organic phases were combined, washed with saturated chloride, dried over anhydrous sodium sulfate, solvent was dried by rotary drying to give 3.45g of product 7 as a flocculent solid in 85.3% yield.

1H NMR(400MHz,DMSO-d6)δ10.32(s,1H),9.48(d,J＝2.0Hz,1H),9.10(d,J＝2.0Hz,1H),8.54(d,J＝5.5Hz,1H),8.18(d,J＝5.5Hz,1H).

LCMS found 193:195＝3:1[M+H]

Step 7

Under nitrogen protection, compound 7(3.45g,17.9mmol,1.0eq), compound 8(6.5g,17.96mmol,1.0eq) and Et were added₃N (7.35g,72.6mmol,4.1eq) was added to DCM (100ml), the temperature was reduced in an ice-water bath, after stirring for about 15min, NaBH (OAc)₃(5.7g,26.9mmol,1.5eq) was added to a reaction flask, stirred in an ice water bath for-2 h and then warmed to room temperature and stirred overnight, TLC monitored reaction (DCM: MeOH ═ 20:1) was complete, quenched with aqueous sodium hydroxide solution, the organic phase separated, the aqueous phase extracted with DCM, combined organic phases and column purified to give the product as a light brown oily liquid, totaling 5.6 g; the yield thereof was found to be 62.3%.

¹H NMR(400MHz,DMSO-d₆)δ9.04(d,J＝2.1Hz,1H),8.39(d,J＝5.5Hz,1H),8.34(d,J＝2.0Hz,1H),7.92(d,J＝5.5Hz,1H),7.57(ddt,J＝11.4,6.3,1.8Hz,4H),7.48–7.32(m,6H),4.37(dq,J＝9.8,3.6Hz,1H),3.89(d,J＝14.0Hz,1H),3.79(d,J＝14.0Hz,1H),2.69(dt,J＝9.8,6.6Hz,2H),1.95(dq,J＝14.3,7.3Hz,1H),1.82–1.62(m,1H),1.23(dd,J＝7.0,4.6Hz,2H),0.98(s,9H).

LCMS found 502:504＝3:1[M+H]

Example 7: synthesis of (R) -3 ' - ((3- ((3- ((tert-butyldiphenylsilyl) oxy) pyrrol-1-yl) methyl) -1, 7-diazanaphthalen-8-yl) amino) -2,2 ' -dimethyl- [1,1 ' -diphenyl ] -3-carboxylic acid

Step 1

Block 1(0.3g,0.6mmol,1.0eq), isopropanol, Compound 1(0.145g,0.6mmol,1.0eq) and TosOH-H were added to the reaction flask in this order₂O (0.117g,0.61mmol,1.0eq), heated under reflux for 8h, monitored by TLC (HEP: EA: 3:1) until the starting material point disappeared, EA was added, the organic phase was washed with aqueous sodium bicarbonate, dried, and column chromatographed to give 0.287g of product in 67.8% yield.

LCMS found 721[M+H]

Step 2:

compound 2(287mg, 0.4mmol) was dissolved in THF/H₂O (3:1, 3ml), adding LiOH (28.6mg, 1.2mmol), stirring at room temperature overnight, TLC monitoring reaction until the raw material point disappears, dropwise adding 1N hydrochloric acid to adjust pH to neutral, extracting with ethyl acetate, drying, separating by column chromatography to obtain 280mg of product, and quantifying yield.

LCMS found 707[M+H]

(R) -N- (3-bromo-2-methylphenyl) -3- ((3- ((tert-butyldiphenylsilyl) oxy) pyrrol-1-yl) methyl) -1, 7-diazanaphthalen-8-amine

Synthesized according to the method of step 1 of reference example 7.

(R) -N3- (3- ((3- ((tert-butyldiphenylsilyl) oxy) pyrrol-1-yl) methyl) -1, 7-diazanaphthalen-8-yl) -2,2 ' -dimethyl- [1,1 ' -diphenyl ] -3,3 ' -diamine

Synthesized according to the method of step 1 of reference example 7.

Example 8: synthesis of N- (5- (((3S) -cyclopentyl) methyl) -1-methyl-4-oxo-4, 5-dihydro-1H-imidazo [4,5-c ] pyridin-2-yl) -3- ((7(((R) -3-hydroxypyrrolidin-1-yl) methyl) quinolin-4-yl) oxy) -2,2 ' -dimethyl- [1,1 ' -biphenyl ] -3,3 ' -dicarboxamide.

The synthetic route is as follows:

to a solution of (R) -3 ' - ((7- ((3-hydroxypyrrol-1-yl) methyl) quinolin-4-yl) oxy) -2,2 ' -dimethyl- [1,1 ' -diphenyl ] -3-carboxylic acid (468mg, 1mmol) and 2-amino-5- (((3S) -3-hydroxycyclopentyl) methyl) -1-methyl-1, 5-dihydro-4H-imidazo [4,5-c ] pyridin-4-one (262mg, 1mmol) in DMF (3mL) at room temperature were added HATU (456mg) and DIEA (258 mg). The mixture was then heated to 50 ℃ and stirred overnight, then cooled to room temperature. The mixture was purified by prep-HPLC to give the product.

LCMS found 713.3[M+H]⁺

Example 9: synthesis of N, N '- (2, 2' -dimethyl- [1,1 '-diphenyl ] -3, 3' -diyl) bis (5- (2-hydroxyethyl) -1-methyl-4-oxo-4, 5-dihydro-1H-pyrrolo [3,2-c ] pyridine-2-carboxamide).

Step 1:

200mg of Compound 1 are added to 5.6ml of toluene and 4.5ml of methanol, and a solution of trimethylsilylated diazomethane cyclohexane (2M) is added dropwise until the yellow colour does not disappear anymore (0.25 ml); after stirring for 1min, the reaction was quenched with acetic acid (0.2ml) and adjusted to pH 8 with aqueous sodium bicarbonate. Spin-drying the solvent; EA extraction, organic phase combination, salt washing, drying, spin-dry mixing and column chromatography to obtain 214mg of white solid. Yield: 100 percent

Step 2

Dissolving 300mg of compound 2 and 149mg of compound 3 in 15ml of THF, performing nitrogen protection, performing ice salt bath, dropwise adding 2.5ml (2M) of NaHMDS THF solution, performing heat preservation reaction for 20min, performing LCMS detection, adding water to quench the raw materials to perform reaction, performing EA extraction, combining organic phases, performing salt washing, drying, performing spin-drying mixing, and performing column chromatography to obtain 160mg of light yellow solid. Yield: 37.2 percent

Step 3

Dissolving 160mg of compound 4 in 2ml of methanol, adding 100mg of 10% Pd/C, introducing hydrogen, reacting at room temperature for 30min, sampling, detecting by TLC, completely reacting the raw materials, filtering to remove 10% Pd/C, and spin-drying to obtain 140mg of off-white solid crude product; the crude product is directly put into the next step. Yield: 91.5 percent

Step 4

Dissolving 55mg of compound 2 and 75mg of compound 5 in 3.8ml of THF, performing nitrogen protection, performing ice salt bath, dropwise adding 0.45ml of (2M) NaHMDS THF solution, and reacting for 1h under the condition of keeping temperature after dropwise adding; adding water to quench and react, extracting with EA, combining organic phases, washing with salt, drying, mixing with a rotary drying sample, and passing through a column to obtain 53mg yellow solid. Yield: 41.9 percent

Step 5

53mg of Compound 6 was dissolved in 2.5ml of THF, 2.5ml of triethylamine trihydrofluoride was added thereto, the mixture was reacted at room temperature for 2 hours, TLC was sampled, the reaction of the starting material was substantially completed (. about.30% of the product), THF was distilled off under reduced pressure, and preparative TLC separation was carried out to obtain 6.1mg of THF. Yield: 20.3 percent of

¹H NMR(400MHz,DMSO-d₆)δ9.85(s,1H),7.55(s,1H),7.43(d,J＝7.5Hz,1H),7.39(d,J＝7.9Hz,1H),7.30(t,J＝7.8Hz,1H),7.02(d,J＝7.4Hz,1H),6.62(d,J＝7.5Hz,1H),4.00(t,J＝5.7Hz,2H),3.95(s,3H),3.62(t,J＝5.6Hz,2H),1.96(s,4H),1.24(s,1H).

Example 10: synthesis of (1r,4r) -4- ((2- ((2,2 ' -dimethyl-3 ' - (4-oxo-4, 5,6, 7-tetrahydrothiazolo [4,5-c ] pyridopyridin-2-yl) - [1,1 ' -diphenyl ] -3-yl) carbamoyl) -1-methyl-1, 4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridopyridin-5-yl) methyl) cyclohexane-1-carboxylic acid.

The synthetic route is as follows:

step 1:

1(318mg) was dissolved in dioxane (15mL) at room temperature, and 2(323mg) and 1.5mL Na were added₂CO₃(2N) aqueous solution, vacuum degassing, then addition of Pd (dppf) Cl₂(73.2mg), degassed again and reacted at 90 degrees overnight. After cooling to room temperature, the reaction was extracted with water and EtOAc, the organic phase was washed with saturated NaCl, dried, spun-dried and passed through a column (HEP: EtOAc: 5: 1-2: 1) to yield 150mg of a pale yellow solid.

Step 2:

compound 3(150mg) was dissolved in DMF (2ml) at room temperature, HATU (196.7mg) and DIEA (113mg) were added, and the mixture was stirred for 10 minutes, followed by addition of compound 4(138.7mg), and the reaction was continued at room temperature overnight. The reaction was extracted with water and EtOAc, the organic phase washed with saturated NaCl, dried, spun dried and column filtered (HEP: EtOAc ═ 5: 1-1: 1) to give 100mg of a pale yellow solid.

And step 3:

compound 5(100mg) was dissolved in 4N HCl/dioxane (2ml) at room temperature, stirred for 30 minutes, TLC detected disappearance of starting material and reaction was concentrated and used directly for the next reaction.

And 4, step 4:

compound 6(100mg, N) was dissolved in MeOH/H at room temperature₂O (1ml,1:1), LiOH₂O (12.2mg), and stirred for 2 hours. TLC detection is carried out, the pH value is adjusted to 6-7 by 1N HCl, and the organic phase is prepared into (1r,4r) -4- ((2- ((2,2 '-dimethyl-3' - (4-oxo-4, 5,6, 7-tetrahydrothiazole [4, 5-c) by high performance liquid chromatography]And pyridin-2-yl) - [1, 1' -Diphenyl]-3-yl) carbamoyl) -1-methyl-1, 4,6, 7-tetrahydro-5H-imidazole [4,5-c ]]And pyridin-5-yl) methyl) cyclohexane-1-carboxylic acid. LC-MS found 653.3(M + H)

Example 11: synthesis of (R) -5- (2-hydroxyethyl) -N- (3 ' - ((3- ((3-hydroxypyrrolin-1-yl) methyl) -1, 7-dinaphthalen-8-yl) amino) -2,2 ' -dimethyl- [1,1 ' -diphenyl ] -3-yl) -1-methyl-4-oxo-4, 5-dihydro-1H-pyrrolo [3,2-c ] pyridine-2-carboxamide

The synthetic route is as follows:

step 1:

compound 1(120mg) and compound 2(86.5mg) were dissolved in THF (2ml) under ice-bath, and N was added₂A2M solution of NaHMDS (0.7mL THF) was added with protection and the ice bath was continued for 1 hour. TLC showed complete reaction of starting material, saturated NH was added₄And quenching the reaction by using a Cl aqueous solution, extracting by using EA, and carrying out column chromatography separation to obtain 80mg of yellow solid.

Step 2:

compound 3(90mg) was added to a mixture of THF (1mL) and TEA/3HF (1mL) and reacted for 2 hours. TLC detection for disappearance of raw material, reaction liquid is added with saturated NaHCO₃The aqueous solution was adjusted to pH 9, extracted with EtOAc, and lyophilized to afford a yellow solid, 12.4 mg.

¹H NMR(400MHz,DMSO-d₆)δ10.98(d,J＝94.5Hz,1H),9.88(s,1H),9.09(s,1H),8.52(s,1H),8.18(d,J＝8.3Hz,1H),8.05(d,J＝5.9Hz,1H),7.56(s,1H),7.38(ddd,J＝30.9,13.6,7.3Hz,4H),7.26–7.17(m,1H),7.08(t,J＝6.5Hz,1H),6.97(d,J＝7.6Hz,1H),6.62(d,J＝7.4Hz,1H),4.67(s,2H),4.47(s,1H),4.01(s,2H),3.95(s,3H),3.71–3.60(m,4H),2.93(m,1H),2.34-2.32(s,1H),2.07(s,3H),2.00(s,3H),1.95-1.85(m,1H),1.21-1.15(m,3H).

LCMS found 658[M+H]⁺

Example 12: synthesis of (R) -7-cyano-5- (2-hydroxyethyl) -N- (3 ' - ((3- ((3-hydroxypyrrolin-1-yl) methyl) -1, 7-diazanaphthalin-8-yl) amino) -2,2 ' -dimethyl- [1,1 ' -diphenyl ] -3-yl) -1-methyl-4-oxo-4, 5-dihydro-1H-pyrrolo [3,2-c ] pyridine-2-carboxamide

The synthetic route is as follows:

step 1:

dissolving compound 1(300mg) in toluene (8.4 mL)/methanol (6.6mL) mixed solution at room temperature, adding 2M cyclohexane solution of trimethylsilyl diazomethane (1.08mL) dropwise until yellow color does not disappear, detecting by TLC that raw materials react completely, adding 0.3mL AcOH to quench reaction, adding saturated NaHCO₃The pH was adjusted to 9, the solvent was evaporated under reduced pressure, extracted with EtOAc and passed through a column to give 240mg of a white solid

Step 2:

compound 3(136mg) and compound 4(150mg) were dissolved in THF (3mL) in an ice bath, and NaHMDS (2M in THF, 0.88mL) was added under the protection of N2 and the reaction was continued for 1 hour in an ice bath. TLC shows the reaction is complete, saturated NH4Cl is added to quench the reaction, EA extraction, column chromatography separation gives 80mg of a yellow solid.

And step 3:

compound 5(40mg) was dissolved in THF (0.5mL) at room temperature, and TBAF/THF (0.1mL) was added to react for 3 hours. TLC showed the starting material was reacted completely, the solvent was distilled off under reduced pressure and preparative isolation gave 6.3mg of a yellow solid.

1H NMR(400MHz,DMSO-d6)δ10.02(s,1H),9.31(s,1H),8.87(s,1H),8.43(d,J＝8.3Hz,1H),8.34(s,1H),8.19(s,1H),8.06(d,J＝5.7Hz,1H),7.61(s,1H),7.40(d,J＝8.1Hz,1H),7.38–7.26(m,2H),7.18(d,J＝5.8Hz,1H),7.09(d,J＝7.5Hz,1H),6.88(d,J＝7.6Hz,1H),6.64(s,1H),5.33(t,J＝4.8Hz,1H),4.92(t,J＝5.6Hz,1H),4.72(d,J＝4.6Hz,1H),4.24(s,3H),4.07(d,J＝5.7Hz,1H),3.83(d,J＝10.4Hz,2H),3.65(d,J＝5.7Hz,2H),2.74(d,J＝6.1Hz,1H),2.68(s,1H),2.41(s,1H),2.10(s,3H),2.01-1.90(m,5H),1.46-1.40(m,2H).

LCMS found 683[M+H]⁺

Biological assay

Example A: PD-1/PD-L1 homogeneous time-resolved fluorescence (HTRF) binding assay

The assay was performed in standard black 384 well polystyrene plates with a final volume of 20. mu.L. Firstly, the methodInhibitors were serially diluted in DMSO and added to the plate wells, followed by the addition of the other reaction components. The final concentration of DMSO in the assay was 1%. The assay was performed in PBS buffer (pH7.4) containing 0.05% Tween-20 and 0.1% BSA at 25 ℃. Recombinant human PD-L1 protein (19-238) with a His tag at the C-terminus was purchased from Acro biosystems (PD 1-H5229). Recombinant human PD-1 protein (25-167) with an Fc marker at the C-terminus was also purchased from Acrobiosystems (PD 1-H5257). PD-L1 and PD-1 protein were diluted in assay buffer and 0.1. mu.l of the solution was extracted and added to the plate well. The plates were centrifuged and the proteins were preincubated with inhibitors for 40 minutes. After incubation, 0.1. mu.l of HTRF detection buffer containing europium-blocked labeled anti-human IgG (Perkinelmer-AD0212) Fc specific and anti-His

Allophycocyanin (APC, Perkinelmer-AD0059H) conjugated antibody. After centrifugation, the plates were incubated at 25 ℃ for 60 minutes. The data were read (665nm/620nm ratio) in a PHERAStar FS plate reader. The final concentrations in the assay were-3 nM PD1, 10nM PD-L1, 1nM europium anti-human IgG and 20nM anti-His-allophycocyanin. The activity data were fitted using GraphPad Prism 5.0 software to give IC50 values for the inhibitors.

The compound IC50 values illustrated in the examples are represented in the following manner: IC50 ≤ 100 nM; 100nM < IC50 ≦ 1000 nM; 1000nM < IC50 +++, and

data for the example compounds obtained using the PD-1/PD-L1 Homogeneous Time Resolved Fluorescence (HTRF) binding assay described in example a are provided in table 1.

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

Claims

1. A compound of formula I, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Is a group having the structure shown below:

wherein the content of the first and second substances,

Is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

r is H, substituted or unsubstituted C₁-C₆Alkyl, substitutedOr unsubstituted C₆-C₁₀Aryl, cyano, -C (═ O) -NRdRe, -C (═ O) -substituted or unsubstituted C₁-C₆Alkoxy, -C (═ O) -substituted or unsubstituted C₁-C₆Alkyl, -C (═ O) -substituted or unsubstituted C₃-C₁₀Cycloalkyl, -C (═ O) -substituted or unsubstituted C₂-C₆Alkenyl, -C (═ O) -substituted or unsubstituted C₂-C₆Alkynyl, or- (L)_1a)_r-(L_2a)_s-(L_3a)_s-, in which,

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

Is absent;

or the said

Is equal to

Is absent;

and said

Is not absent at the same time;

R₁、R₂、R₃and R₄Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstitutedSubstituted C6-C10 aryl, substituted or unsubstituted 5-12 membered heteroaryl having 1-3 heteroatoms, substituted or unsubstituted 5-12 membered heterocyclyl having 1-4 heteroatoms, substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

2. The compound of claim 1, or an isomer, optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof

Is a 5-7 membered ring and 5-6Bicyclic structure of the membered ring.

3. The compound of claim 1, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein said compound is

4. The compound of claim 1, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof, wherein said ring is

Having a substituent represented by formula IV below:

Is a 5-7 membered nitrogen containing heterocyclic group;

R₅selected from the group consisting of: substituted or unsubstituted C1-C6 alkyl, -CN, hydroxy, amino, carboxy; wherein, theThe substituents are selected from the group consisting of: halogen, hydroxy, carboxy, cyano, C1-C6 alkoxy.

5. The compound of claim 1, wherein the compound of formula I is selected from a compound of formula Ia, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Is a group having the structure shown below:

wherein the content of the first and second substances,

is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

Is absent; or the said

Is equal to

R₁、R₂、R₃And R₄Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl having 1 to 3 heteroatoms, substituted or unsubstituted 5-to 12-membered heterocyclyl having 1 to 4 heteroatoms, substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

Unless otherwise specified, "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen: including but not limited to-F, Cl, Br, -CH₂Cl、-CHCl₂、-CCl₃、-CH₂F、-CHF₂、-CF₃Oxo, -CN, hydroxy, ammoniaA group, C1-C6 alkylamino, carboxyl, -NHAc, a group selected from the group consisting of unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkoxy, C6-C10 aryl, C3-C8 cycloalkyl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O; the substituents are selected from the following group: halogen, hydroxy, carboxy, cyano, C1-C6 alkoxy, C1-C6 alkylamino;

in the formulae above, any of the heteroatoms is selected from the group consisting of: n, S and O;

preferably, the compound is selected from the following compounds 1-125:

6. the compound of claim 1, wherein the compound of formula I is selected from compounds of formula Ib, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Is a group having the structure shown below:

wherein the content of the first and second substances,

Z²、Z³、Z⁴each independentlySelected from the group consisting of: NH, N, CH₂N-O, SO or SO 2;

is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

Is absent; or the said

Is equal to

R₁、R₂、R₃And R₄Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 membered heteroaryl having 1-3 heteroatomsA substituted or unsubstituted 5-12 membered heterocyclic group having 1-4 heteroatoms, a substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

preferably, the compound is selected from the group consisting of compounds 126-202:

7. the compound of claim 1, wherein the compound of formula I is selected from compounds of formula Ic, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Is a group having the structure shown below:

wherein the content of the first and second substances,

is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

Is absent; or the said

Is equal to

R₁、R₂、R₃And R₄Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 membered heteroaryl having 1-3 heteroatoms, substituted or unsubstituted 5-12 membered heterocyclic ring having 1-4 heteroatomsRadicals, substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

preferably, the compound is selected from the group consisting of compounds 203-313;

8. the compound of claim 1, wherein the compound of formula I is selected from the group consisting of formulae Id-1 and Id-2, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

Is absent; or the said

Is equal to

R₁、R₂And R₃Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl having 1 to 3 heteroatoms, substituted or unsubstituted 5-to 12-membered heterocyclyl having 1 to 4 heteroatoms, substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

preferably, the compound is selected from the group consisting of compounds 314-355:

9. the compound of claim 1, wherein the compound of formula I is selected from a compound of formula Ie, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

wherein n, m, p and q are each independently selected from 0, 1,2,3 or 4;

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Or substituted or unsubstituted

Is a group having the structure shown below:

wherein the content of the first and second substances,

is a single bond or a double bond;

and is

Is an aromatic or non-aromatic moiety;

r is 1,2,3, 4,5, 6;

s is 0, 1,2 respectively;

selected from the group consisting of: substituted or unsubstituted 5-12 membered heteroaryl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstitutedUnsubstituted 5-12 membered heterocyclyl, substituted or unsubstituted 5-12 membered C5-C12 cyclyl, wherein said heterocyclyl has 1-3 heteroatoms; or the said

Is absent; or the said

Is equal to

Substituted or unsubstituted

Or substituted or unsubstituted

Or (L)_1a)_r-(L_2a)_s-(L_3a)_s-；

Unless otherwise specified, "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen: including but not limited to-F, Cl,Br、-CH₂Cl、-CHCl₂、-CCl₃、-CH₂F、-CHF₂、-CF₃Oxo, -CN, hydroxy, amino, C1-C6 alkylamino, carboxy, -NHAc, a group selected from the group consisting of unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkoxy, C6-C10 aryl, C3-C8 cycloalkyl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O; the substituents are selected from the following group: halogen, hydroxy, carboxy, cyano, C1-C6 alkoxy, C1-C6 alkylamino;

preferably, the compound is selected from the group consisting of compounds 356-360:

10. a process for the preparation of a compound of formula I according to claim 1, comprising a step selected from the group consisting of schemes 1,2 and 3:

synthesis scheme 1

synthesis scheme 2:

(a) carboxylic ester 22-1 is taken as a raw material, and is subjected to aminolysis reaction with amine 2-2 under the catalysis of Lewis acid to obtain an intermediate compound 2-3;

synthesis scheme 3:

(a) using carboxylic ester 3-1 as a raw material, and carrying out Buchwald-Hartwig coupling or Ullmann coupling with amine 3-2 under the catalysis of palladium or copper to obtain an intermediate compound 3-3;

said Cy is

Is composed of

Y¹、Y²、Z¹、Z²、Z³And R is as defined above.

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

12. Use of a compound according to claim 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 7, for the preparation of a pharmaceutical composition for the prophylaxis and/or treatment of diseases associated with the activity or expression of PD-1/PD-L1.

13. A PD-1/PD-L1 inhibitor, wherein said inhibitor comprises a compound of claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

14. A method of inhibiting the PD-1/PD-L1 interaction in vitro comprising the steps of: contacting a compound of claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, with PD-L1 protein.