TW202317563A - A crystalline form of a biphenyl derivative inhibitor and its preparation method therefor - Google Patents

Abstract

The present invention relates to a crystalline form of a biphenyl derivative inhibitor and a preparation method therefor. Specifically, the present invention relates to a crystalline form of a compound of formula (I), a preparation method therefor, a pharmaceutical composition containing a therapeutically effective amount of the crystalline form thereof, and a use thereof as a PD-1/PD-L1 inhibitor in the treatment of cancer, infectious diseases and autoimmune diseases.

Description

Crystal form of biphenyl derivative inhibitor and preparation method thereof

The invention belongs to the technical field of medicinal chemistry, and in particular relates to a crystal form of a biphenyl derivative inhibitor, a preparation method and application thereof.

The immune system plays an important role in controlling many diseases, including cancer. However, tumor cells evade immune attack or suppress the activation of the immune system in various ways. Blocking signaling at immune-suppressive checkpoints, such as programmed cell death protein 1 (PD-1), has proven to be a potential therapeutic modality.

PD-1, a member of the CD28 superfamily, is an immunosuppressive receptor on the surface of immune cells, especially cytotoxic T cells. PD-1 has two ligands, PD-L1 and PD-L2, among which PD-L1, programmed cell death receptor-ligand 1, is expressed in a variety of cells, such as macrophages and dendritic cells, and It is generally highly expressed on tumor cells. PD-L1 plays an immunosuppressive role by combining with PD-1 and makes tumor cells escape the killing of T cells, inhibits the activation of T cells and the production of corresponding cytokines, weakens infectious immunity and tumor immunity, and promotes infectious diseases and tumors. Progress. Use of PD-L1 inhibitors such as antibodies Or small molecule inhibitors can relieve the immunosuppressive effect of PD-L1 and promote the immune clearance of tumors, so as to achieve the effect of treating tumors.

PD-1/PD-L1 is a hot spot in the research of tumor immunotherapy in recent years. The breadth, depth and durability of the monoclonal antibody drug response are very rare. At present, there are many PD-1/PD-L1 monoclonal antibody drugs in clinical practice. marketed and achieved great success. PD-L1 inhibitors can be used to treat almost all major cancers, including non-small cell lung cancer, liver cancer, gastric cancer, intestinal cancer, kidney cancer, etc., and have great clinical application value.

PD-L1 inhibitors from macromolecules to small molecules are becoming a new research and development trend and hotspot. Small molecule inhibitors have many natural advantages in terms of administration methods and production costs, and have the potential to replace antibody macromolecules. Currently, they include BMS and Foreign pharmaceutical companies including companies such as Incyte are actively developing PD-L1 small molecule inhibitors.

The oral small molecule inhibitor developed by BMS is currently in the preclinical research stage, and several patents have been published consecutively. The small molecule inhibitor INCB086550 developed by Incyte is in the first phase of clinical research. The small molecule inhibitor developed by Aurigene/Curis CA-170 is in Phase II clinical research.

International applications WO2015034820, WO2015160641, WO2014151634, WO2017066227, WO2017070089, WO2017106634, WO2017112730, WO2017192961, WO2017222976, WO2018013789 , WO2018044783, WO2018119224, WO2018119236, WO2018119263, WO2018119266 and WO2018119286 have reported that PD-1 or PD-L1 small molecule compounds inhibit agent. In addition, international applications WO2014151634, WO2011161699, WO2012168944, WO2013132317, WO2013144704, WO2015033299, WO2015033301, WO2015033303 and WO2015036927 reported macrocyclic and peptide compounds PD- 1 or PD-L1 Inhibitors. However, there is still a great demand for small molecule inhibitors of PD-L1 that are more effective on the PD-1/PD-L1 pathway, have better drug metabolism properties, and are druggable.

PD-L1 small-molecule inhibitors have good application prospects as drugs in the pharmaceutical industry. First, PD-L1 small-molecule inhibitors can be administered orally, which has the advantage of greater compliance than antibody drugs for intravenous administration, and can avoid antibody Serious side effects such as colitis caused by long-term residence in the body. Secondly, small molecule inhibitors of PD-L1 have a unique mechanism of action that binds to PD-L1 and endocytizes it, which may show different efficacy from antibodies in clinical practice. Finally, the production and quality control costs of PD-L1 small-molecule inhibitors are lower, and they have a price advantage that is far lower than the cost of macromolecular drugs. Like PD-L1 antibody inhibitors, they can be applied to a variety of major tumors and have huge market potential .

The patent application of Jiangsu Hansoh Pharmaceutical Group Co., Ltd. (application number: PCT/CN2020/141307) disclosed a series of inhibitor structures containing biphenyl derivatives. In subsequent research and development, in order to make the product easy to handle, filter , drying, easy storage, long-term product stability, high bioavailability, etc., the present invention has carried out comprehensive research on the crystal forms of the above substances, and is committed to obtaining the most suitable crystal forms.

All content involved in the patent application PCT/CN2020/141307 is added to the present invention by reference.

The object of the present invention is to provide a kind of crystal form of the compound shown in general formula (I), and its structure is as follows:

in:

或

； L is selected from the key,

or

;

Ring A is selected from 4-8 membered heterocyclic groups; preferably 4-membered, 7-membered or 8-membered heterocyclic groups;

More preferably the following groups:

_R is selected from hydrogen, deuterium, halogen, hydroxyl, cyano, carboxyl, aldehyde, pendant oxygen, C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl, C _{1 -6} alkoxy, C _1-6 hydroxyalkyl, C _3-8 cycloalkyl, -(CH ₂ ) _n R _a , -(CH ₂ ) _n OR _a , -OC(O)R _a , -C (O)R _a , -C(O)OR _a , -C(O)NR _a R _b , -(CH ₂ ) _n NR _a C(O)R _b or -SO ₂ R _a , wherein the C _{1- 6} alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 hydroxyalkyl and C _3-8 cycloalkyl, if necessary, further deuterated , halogen, hydroxyl, cyano or one or more substituents in C _1-6 alkyl;

R _a and R _b are each independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, carboxyl, C _1-6 alkyl, C _1-6 deuterated alkyl, C _{1-6 6} haloalkyl or C _3-8 cycloalkyl, wherein the C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl and C _3-8 cycloalkyl, if necessary further Substituted by one or more substituents in deuterium, halogen, hydroxyl, cyano or C _1-6 alkyl;

n is 0, 1 or 2; and

x is 0, 1 or 2.

In a preferred embodiment of the present invention, the structure of the compound is shown in general formula (II):

in:

M is O, -NR ₂ or -CR ₃ R ₄ ;

R _is selected from hydrogen, deuterium, aldehyde, C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 haloalkyl, C _1-3 alkoxy, C _1-3 hydroxyalkyl , C _3-6 cycloalkyl, -(CH ₂ ) _n R _a , -(CH ₂ ) _n OR _a , -OC(O)R _a , -C(O)R _a , -C(O)OR _a , -C(O)NR _a R _b , -(CH ₂ ) _n NR _a C(O)R _b or -SO ₂ R _a ;

R ₃ and R ₄ are each independently selected from hydrogen, deuterium, halogen, hydroxyl, cyano, carboxyl, aldehyde, side oxygen, C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 Haloalkyl, C _1-3 alkoxy, C _1-3 hydroxyalkyl, C _3-6 cycloalkyl, -(CH ₂ ) _n R _a , -(CH ₂ ) _n OR _a , -OC(O )R _a , -C(O)R _a , -C(O)OR _a , -C(O)NR _a R _b , -(CH ₂ ) _n NR _a C(O)R _b or -SO ₂ R _a , wherein the C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 haloalkyl, C _1-3 alkoxy, C _1-3 hydroxyalkyl and C _3-6 cycloalkyl , if necessary, further substituted by one or more substituents in deuterium, halogen, hydroxyl, cyano or C _1-3 alkyl;

R _a and R _b are each independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, carboxyl, C _1-3 alkyl, C _1-3 deuterated alkyl, C _{1-3 3} haloalkyl or C _3-6 cycloalkyl, wherein the C _1-3 alkyl, C _1-3 fluoroalkyl, C _1-3 haloalkyl and C _3-6 cycloalkyl, if necessary further Substituted by one or more substituents in deuterium, halogen, hydroxyl, cyano or C _1-3 alkyl;

n is 0, 1 or 2.

In a preferred embodiment of the present invention, the crystal form of the compound is the crystal form of the following compound:

N-(2,2'-dichloro-3'-(6-methoxy-5-((oxetan-3-ylamino)methyl)pyridin-2-yl)-[1,1' -biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(5-((3-fluoroazetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-[1,1 '-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide;

1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-carbazamidylamino<acetylamino>)-[1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)azetine Pyridine-3-yl acetate;

N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptan-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide;

N-(3'-(5-((2-Oxa-6-azaspiro[3.3]heptane-6-yl)methyl)-6-methoxypyridin-2-yl)-2,2 '-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c ]pyridine-2-carboxamide;

N-(3'-(5-((5-oxa-2-azaspiro[3.4]octane-2-yl)methyl)-6-methoxypyridin-2-yl)-2,2 '-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c ]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-carbonyl-2,5-diazaspiro[3.4]octane-2-yl)methyl) Pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c] pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(5-((3-hydroxyazetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-[1,1 '-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(5-((3-hydroxy-3-methylazetidin-1-yl)methyl)-6-methoxypyridin-2-yl) -[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2 - formamide;

1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-formamido)-[1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)azetidine-3-carboxylic acid;

(S)1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5- c] Pyridine-2-carbazimidyl <glycolamide>)-[1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl ) azetidine-2-carboxylic acid;

(S)-N-(2,2'-dichloro-3'-(5-((2-(hydroxymethyl)azetidin-1-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(5-((3-hydroxy-3-(hydroxymethyl)azetidin-1-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide;

N-(3'-(5-((3-Acetamidoazetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-2,2'-dichloro-[ 1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-methanol Amide;

N-(3'-(5-((3-(Acetamidomethyl)azetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-2,2'- Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-Formamide;

N-(2,2'-dichloro-3'-(5-((3-(cyanomethyl)azetidin-1-yl)methyl)-6-methoxypyridin-2-yl) -[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2 - formamide;

N-(2,2'-dichloro-3'-(5-((3-(dimethylaminoformyl)azetidin-1-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(6-methoxy-5-((3-(methylaminoformyl)azetidin-1-yl)methyl)pyridine-2- base)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-Formamide;

N-(2,2'-dichloro-3'-(5-((6-hydroxy-2-azaspiro[3.3]heptane-2-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(5-((6-isobutyryl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(5-((6-(cyclopropylcarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6 -Methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-propionyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl )pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4, 5-c]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-(2,2,2-trifluoroacetyl)-2,6-diazaspiro[ 3.3] Heptane-2-yl)methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7 - Tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(5-((6-(2,2-difluoroacetyl)-2,6-diazaspiro[3.3]heptan-2-yl )methyl)-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetra Hydrogen-1H-imidazo[4,5-c]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(5-((6-(2-cyanoacetyl)-2,6-diazaspiro[3.3]heptane-2-yl)methanol Base)-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro- 1H-imidazo[4,5-c]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-(methylsulfonyl)-2,6-diazaspiro[3.3]heptane-2-yl )methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(5-((6-formyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide;

N-(3'-(5-((7-acetyl-2,7-diazaspiro[3.5]nonan-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide;

N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.4]octane-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide;

N-(2,2'-dichloro-3'-(5-((6-isobutyryl-2,6-diazaspiro[3.4]octane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide; or

N-(3'-(5-((6-Acetamido-2-azaspiro[3.3]heptane-2-yl)methyl)-6-methoxypyridin-2-yl)-2 ,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c] pyridine-2-carboxamide.

In a preferred embodiment of the present invention, the crystal form is the compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl )methyl)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4 , crystalline form of 5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide.

In a preferred embodiment of the present invention, the compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl) -6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6 , Crystal forms A~D of 7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide, wherein:

Form A:

The X-ray powder diffraction pattern of the crystal form A has a diffraction peak at 2θ of 7.8±0.2°; or has a diffraction peak at 14.1±0.2°; or has a diffraction peak at 15.3±0.2°; or has a diffraction peak at 19.5±0.2°; Diffraction peak at 0.2°; or diffraction peak at 12.0±0.2°; or diffraction peak at 12.4±0.2°; or diffraction peak at 13.2±0.2°; or diffraction peak at 14.6±0.2° or have a diffraction peak at 21.2±0.2°; or have a diffraction peak at 22.0±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them;

or, Form B:

The X-ray powder diffraction pattern of the crystal form B has a diffraction peak at 2θ of 4.2±0.2°; or has a diffraction peak at 12.2±0.2°; or has a diffraction peak at 12.6±0.2°; or has a diffraction peak at 16.5±0.2°; Diffraction peak at 0.2°; or diffraction peak at 16.8±0.2°; or diffraction peak at 18.4±0.2°; or diffraction peak at 18.9±0.2°; or diffraction peak at 21.1±0.2° or have a diffraction peak at 22.2±0.2°; or have a diffraction peak at 22.4±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them;

or, Form C:

The X-ray powder diffraction pattern of the crystal form C has a diffraction peak at 2θ of 11.4±0.2°; or has a diffraction peak at 12.5±0.2°; or has a diffraction peak at 21.1±0.2°; or has a diffraction peak at 23.0±0.2°; Diffraction peak at 0.2°; or diffraction peak at 26.1±0.2°; or diffraction peak at 26.6±0.2°; or diffraction peak at 13.0±0.2°; or diffraction peak at 14.0±0.2° or have a diffraction peak at 14.7±0.2°; or have a diffraction peak at 15.8±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them;

or, Form D:

The X-ray powder diffraction pattern of the crystal form D has a diffraction peak at 9.5±0.2° at 2θ ; or has a diffraction peak at 10.4±0.2°; or has a diffraction peak at 14.5±0.2°; or has a diffraction peak at 19.9±0.2°; Diffraction peak at 0.2°; or diffraction peak at 20.8±0.2°; or diffraction peak at 24.8±0.2°; or diffraction peak at 11.7±0.2°; or diffraction peak at 13.3±0.2° or have a diffraction peak at 17.2±0.2°; or have a diffraction peak at 23.8±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them.

In a further preferred solution of the present invention, the X-ray powder diffraction pattern of the crystal form A contains at least one or more diffraction peaks at 2θ of 7.8±0.2°, 14.1±0.2°, and 15.3±0.2°, Preferably two of them are included, more preferably three of them; if necessary, at least one of 19.5±0.2°, 12.0±0.2°, 12.4±0.2°, 13.2±0.2°, 14.6±0.2° can be further included , preferably including 2, 3, 4 or 5 of them;

Or, the X-ray powder diffraction pattern of the crystal form B contains at least one or more diffraction peaks located at 2θ of 4.2±0.2°, 12.2±0.2°, 12.6±0.2°, preferably two of them , more preferably including three; as required, it may further include at least one of 16.5±0.2°, 16.8±0.2°, 18.4±0.2°, 18.9±0.2°, 21.1±0.2°, preferably including 2 of them strips, 3 strips, 4 strips or 5 strips;

Or, the X-ray powder diffraction pattern of the crystal form C contains at least one or more diffraction peaks located at 2θ of 11.4±0.2°, 12.5±0.2°, 21.1±0.2°, preferably two of them , more preferably include three; if necessary, may further include at least one of 23.0±0.2°, 26.1±0.2°, 26.6±0.2°, 13.0±0.2°, 14.0±0.2°, preferably include 2 of them strips, 3 strips, 4 strips or 5 strips;

Or, the X-ray powder diffraction pattern of the crystal form D includes at least one or more diffraction peaks located at 2θ of 9.5±0.2°, 10.4±0.2°, 14.5±0.2°, preferably two of them , more preferably including three; as required, it may further include at least one of 19.9±0.2°, 20.8±0.2°, 24.8±0.2°, 11.7±0.2°, 13.3±0.2°, preferably including 2 of them Strips, 3, 4 or 5.

In a further preferred solution of the present invention, the X-ray powder diffraction pattern of the crystal form A may also include, if necessary, 2 θ at 21.2±0.2°, 22.0±0.2°, 22.6±0.2°, 25.0±0.2°, 28.3± One or more diffraction peaks at 0.2°, 16.7±0.2° or 24.5±0.2°; preferably at least including any 2-3, or 4-5, or 6-7; more preferably , including any 2, 3, 4, 5, 6, or 7 of them;

Or, the X-ray powder diffraction pattern of the crystalline form B may also include 2θ at 22.2±0.2°, 22.4±0.2°, 22.9±0.2°, 23.6±0.2°, 26.6±0.2°, 13.8±0.2° Or one or more diffraction peaks at 14.5±0.2°; preferably at least including any 2-3, or 4-5, or 6-7 of them; more preferably, including any 2 of them , 3 places, 4 places, 5 places, 6 places, 7 places;

Or, the X-ray powder diffraction pattern of the crystal form C may also include 2θ at 14.7±0.2°, 15.8±0.2°, 18.0±0.2°, 19.8±0.2°, 23.6±0.2°, 9.1±0.2° Or one or more diffraction peaks at 16.7±0.2°; preferably at least include any 2-3, or 4-5, or 6-7; more preferably, include any 2, 3 places, 4 places, 5 places, 6 places, 7 places;

Or, the X-ray powder diffraction pattern of the crystal form D may also include 2θ at 17.2±0.2°, 23.8±0.2°, 25.5±0.2°, 27.1±0.2°, 15.3±0.2°, 15.8±0.2° Or one or more diffraction peaks at 21.4±0.2°; preferably at least including any 2-3, or 4-5, or 6-7; more preferably, including any 2, 3 places, 4 places, 5 places, 6 places, 7 places.

In a preferred solution of the present invention, the X-ray powder diffraction pattern of the crystal form A may optionally include 2 θ at 7.8±0.2°, 14.1±0.2°, 15.3±0.2°, 19.5±0.2°, 12.0± 0.2°, 12.4±0.2°, 13.2±0.2°, 14.6±0.2°, 21.2±0.2°, 22.0±0.2°, 22.6±0.2°, 25.0±0.2°, 28.3±0.2°, 16.7±0.2°, or 24.5± One or more diffraction peaks at 0.2°,

Preferably, there are diffraction peaks at 4, 5, 6, 8 or 10 places as required;

Or, the X-ray powder diffraction pattern of the crystalline form B may optionally include positions at 2θ of 4.2±0.2°, 12.2±0.2°, 12.6±0.2°, 16.5±0.2°, 16.8±0.2°, 18.4±0.2°, One of 18.9±0.2°, 21.1±0.2°, 22.2±0.2°, 22.4±0.2°, 22.9±0.2°, 23.6±0.2°, 26.6±0.2°, 13.8±0.2° or 14.5±0.2°, or multiple diffraction peaks,

Preferably, there are diffraction peaks at 4, 5, 6, 8 or 10 positions as required; for example, the X-ray powder diffraction pattern of the crystal form B has diffraction peaks at the following positions at 2θ :

Or, the X-ray powder diffraction pattern of the crystal form C optionally includes positions at 2θ of 11.4±0.2°, 12.5±0.2°, 21.1±0.2°, 23.0±0.2°, 26.1±0.2°, 26.6±0.2°, One of 13.0±0.2°, 14.0±0.2°, 14.7±0.2°, 15.8±0.2°, 18.0±0.2°, 19.8±0.2°, 23.6±0.2°, 9.1±0.2° or 16.7±0.2° or multiple diffraction peaks,

Preferably, there are diffraction peaks at 4, 5, 6, 8 or 10 places as required;

Or, the X-ray powder diffraction pattern of the crystalline form D optionally includes positions at 2θ of 9.5±0.2°, 10.4±0.2°, 14.5±0.2°, 19.9±0.2°, 20.8±0.2°, 24.8±0.2°, One of 11.7±0.2°, 13.3±0.2°, 17.2±0.2°, 23.8±0.2°, 25.5±0.2°, 27.1±0.2°, 15.3±0.2°, 15.8±0.2° or 21.4±0.2° or multiple diffraction peaks,

Preferably, there are diffraction peaks at 4, 5, 6, 8 or 10 places as required.

In a further preferred embodiment of the present invention, the crystal form is N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl Base)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5 , crystalline form B of 6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide;

The X-ray powder diffraction pattern of the crystal form B has a diffraction peak at 4.0±0.2°; or has a diffraction peak at 12.0±0.2°; or has a diffraction peak at 12.4±0.2°; or has a diffraction peak at 16.4±0.2° Have a diffraction peak; or have a diffraction peak at 16.6±0.2°; or have a diffraction peak at 18.2±0.2°; or have a diffraction peak at 18.7±0.2°; or have a diffraction peak at 20.9±0.2°; or Have a diffraction peak at 22.0±0.2°; or have a diffraction peak at 22.3±0.2°; preferably include any 2-5, or 3-5, or 3-6, or 3- 8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them;

Preferably, the X-ray powder diffraction pattern of the crystal form B includes at least one or more diffraction peaks at 2θ of 4.0±0.2°, 12.0±0.2°, and 12.4±0.2°, preferably including Two of them, preferably including three; as required, can further include at least one of 16.4±0.2°, 16.6±0.2°, 18.2±0.2°, 18.7±0.2°, 20.9±0.2°, preferably Contains 2, 3, 4 or 5 of these;

More preferably, the X-ray powder diffraction pattern of the crystal form B may also include 2 θ at 22.0±0.2°, 22.3±0.2°, 22.7±0.2°, 23.5±0.2°, 26.5±0.2°, 13.6 One or more diffraction peaks at ±0.2° or 14.3±0.2°; preferably at least include any 2-3, or 4-5, or 6-7 of them; more preferably, include any of them 2, 3, 4, 5, 6, 7;

Further preferably, the X-ray powder diffraction pattern of the crystalline form B may optionally include 4.0±0.2°, 12.0±0.2°, 12.4±0.2°, 16.4±0.2°, 16.6±0.2°, 18.2±0.2 °, 18.7±0.2°, 20.9±0.2°, 22.0±0.2°, 22.3±0.2°, 22.7±0.2°, 23.5±0.2°, 26.5±0.2°, 13.6±0.2° or 14.3±0.2° One or more diffraction peaks, preferably, including 4, 5, 6, 8 or 10 diffraction peaks as required;

Even more preferably, the X-ray powder diffraction pattern of the crystal form B is shown in FIG. 10 .

In a further preferred embodiment of the present invention, Example 4 compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl Base)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5 , Form A of 6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide, using Cu-Kα radiation, X expressed in terms of 2 θ angles and interplanar spacing d values - Ray characteristic diffraction peaks are shown in Table 1.

Compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methoxy in Example 4 of the present invention Pyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro- The crystal form A of 1H-imidazo[4,5-c]pyridine-2-carboxamide, its X-ray powder diffraction pattern is basically shown in Figure 1; its DSC pattern is basically shown in Figure 2.

In a further preferred embodiment of the present invention, Example 4 compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl Base)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5 , Form B of 6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide, using Cu-Kα radiation, X expressed in terms of 2 θ angles and interplanar spacing d values - Ray characteristic diffraction peaks are shown in Table 2.

Table 2 Example 4 The XRPD ray diffraction data of the free base crystal form B of the compound

Compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methoxy in Example 4 of the present invention Pyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro- The X-ray powder diffraction pattern of 1H-imidazo[4,5-c]pyridine-2-carboxamide Form B is basically shown in Figure 3; its DSC pattern is basically shown in Figure 4.

In a further preferred embodiment of the present invention, Example 4 compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl Base)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5 , Form C of 6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide, using Cu-Kα radiation, X expressed in terms of 2 θ angles and interplanar spacing d values - Ray characteristic diffraction peaks are shown in Table 3.

Table 3 Example 4 The XRPD ray diffraction data of the free base crystal form C of the compound

Compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methoxy in Example 4 of the present invention Pyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro- The X-ray powder diffraction pattern of 1H-imidazo[4,5-c]pyridine-2-carboxamide Form C is basically shown in Figure 5; its DSC pattern is basically shown in Figure 6.

In a further preferred embodiment of the present invention, Example 4 compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl Base)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5 , Form D of 6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide, using Cu-Kα radiation, X expressed in terms of 2 θ angles and interplanar spacing d values - Ray characteristic diffraction peaks are shown in Table 4.

Table 4 Example 4 The XRPD ray diffraction data of the free base crystal form D of the compound

Compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methoxy in Example 4 of the present invention Pyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7- For crystal form D of tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide, its X-ray powder diffraction pattern is basically shown in Figure 7; its DSC pattern is basically shown in Figure 8.

In a further preferred scheme of the present invention, Example 4 compound N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl )-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5, The X-ray powder diffraction patterns of 6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide in the form A, form B, form C and form D are relatively The peak intensity is the 2 θ error between the top ten diffraction peak positions and the corresponding position diffraction peaks in Figure 1, Figure 3, Figure 5 and Figure 7, preferably ±0.2°~±0.5°, preferably ±0.2°~±0.3°, The best is ±0.2°;

The error mentioned in the present invention refers to the X-ray powder diffraction of different batches of samples caused by experimental error factors such as the difference in the sample preparation technology level, the standardization of operation, the thickness of the sample sample preparation, and the use of different sample racks during the experiment. The position of the diffraction peak in the spectrum produces a certain degree of overall displacement. For those skilled in the art, the overall displacement of the diffraction peak position due to experimental errors can be expected. The following tables 5, 6, 7 and 8 are four different The X-ray characteristic diffraction peak data table of the batch crystal form B, and its X-ray powder diffraction patterns are basically shown in Figures 9, 10, 11 and 12:

Table 5 XRPD ray diffraction data of batch 1 of free base crystal form B

Table 6 XRPD ray diffraction data of free base crystal form B batch 2

Table 7 XRPD ray diffraction data of free base crystal form B batch 3

Table 8 XRPD ray diffraction data of free base crystal form B batch 4

By comparing the data in any of Tables 5 to 8 with the data in Table 2, it can be known that the positions of the corresponding diffraction peaks have shifted to varying degrees. , 7, and 8 are 3.901 ° (the degree of displacement is 0.282°), 4.054° (the degree of displacement is 0.129°), 3.973° (the degree of displacement is 0.210°), and 4.039° (the degree of displacement is 0.144°), respectively. or for example in table 2, 2 θ is the peak of 16.789 °, and its corresponding 2 θ in table 5, 6, 7, 8 is respectively 16.512 ° (displacement degree is 0.277 °), 16.634 ° (displacement degree is 0.155 °) , 16.535° (the degree of displacement is 0.254°), 16.612° (the degree of displacement is 0.177°); or for example, the peak of 2 θ in Table 2 is 22.182°, and its corresponding 2 θ in Table 5, 6, 7, and 8 respectively 21.915° (displacement degree is 0.267°), 22.020° (displacement degree is 0.162°), 21.933° (displacement degree is 0.249°), 22.021° (displacement degree is 0.161°);

Although the positions of the corresponding diffraction peaks in Tables 5 to 8 are shifted within 0.3° from those in Table 2, these compounds are all compounds N-(3'-(5-((6-acetyl-2,6-di Azaspiro[3.3]heptane-2-yl)methyl)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3- crystalline form B of -1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide.

In a further preferred solution of the present invention, the crystal form is an anhydrate.

In a further preferred solution of the present invention, the crystal form is a hydrate, and the number of water is 0.2-3, preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3, more preferably 0.5, 1, 2 or 3.

In a further preferred solution of the present invention, the preparation method of the crystal form of the above-mentioned compound specifically includes the following steps:

1) Weigh an appropriate amount of free base, and dissolve it in a benign solvent at a certain temperature, preferably 0-50°C;

2) If necessary, add anti-solvent to the solution obtained above, and stir until solids are precipitated;

3) If necessary, stirring, cooling and crystallization to obtain the target product;

in:

The good solvent is selected from methanol, acetone, ethyl formate, ethyl acetate, acetonitrile, ethanol, 88% acetone, tetrahydrofuran, dichloromethane, 1,4-dioxane, benzene, toluene, isopropanol, n-butanol One or more of , isobutanol, N,N-dimethylformamide, N,N-dimethylacetamide, n-propanol, tertiary butanol, 2-butanone or 3-pentanone ; Preferably one or more of ethyl formate, isopropanol or absolute ethanol;

The anti-solvent is selected from one or more of heptane, cyclohexane, n-hexane, n-pentane, water, methyl tertiary butyl ether, toluene or isopropyl ether; One or more of tributyl ether or cyclohexane.

In a further preferred solution of the present invention, the preparation method of the crystal form of the above-mentioned compound specifically includes the following steps:

1) Weighing an appropriate amount of free alkali or its crystal form, beating with a poor solvent at a certain temperature, preferably 0-50°C;

in:

The poor solvent is selected from one or more of heptane, cyclohexane, n-hexane, n-pentane, water, methyl tertiary butyl ether, toluene or isopropyl ether; One or more of tributyl ether or cyclohexane.

The object of the present invention is also to provide a pharmaceutical composition, which contains a therapeutically effective amount of any crystal form of the above compound, and one or more pharmaceutically acceptable carriers, diluents or excipients.

The object of the present invention is also to provide the application of the crystal form of any of the above-mentioned compounds or the above-mentioned pharmaceutical composition in the preparation of PD-1/PD-L1 inhibitor drugs.

The purpose of the present invention is also to provide the crystal form of any one of the above compounds or the application of the above pharmaceutical composition in the preparation of medicines for treating diseases selected from cancer, infectious diseases, and autoimmune diseases; wherein the cancer is selected from skin cancer, lung cancer , urological tumor, blood tumor, breast cancer, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor, head and neck cancer; the infectious disease is selected from bacterial infection, viral infection; the autoimmune The disease is selected from organ-specific autoimmune diseases, systemic autoimmune diseases, wherein the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colon Inflammation, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritic syndrome, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, systemic autoimmune diseases including rheumatoid arthritis , Systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia.

Terminology Explanation

Unless stated to the contrary, the terms used in the specification and claims for invention have the following meanings.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably 1 to 6 3 carbon atoms, most preferably 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, iso Propyl, n-butyl, isobutyl, tertiary butyl, second butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-di Methylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethyl Propyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methyl Hexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2 -Ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethyl Dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl , 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n- Decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, Dibutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl Base, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2- Dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methyl Amylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably the substituents are one or more of the following groups independently selected from Alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendant oxy, carboxyl or carboxylate, the present invention is preferably methyl, ethyl, isopropyl, third Butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl, and hydroxy-substituted alkyl.

The term "(extended) alkylene" means that one hydrogen atom of the alkyl group is further substituted, for example: "methylene" refers to -CH ₂ -, "(extended) ethylidene" refers to -(CH ₂ ) ₂ - , "(extended) propylidene" refers to -(CH ₂ ) ₃ -, "(extended) butylene" refers to -(CH ₂ ) ₄ -, etc. The term "alkenyl" means an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, for example vinyl, 1-propenyl, 2-propenyl, 1-, 2- or 3- -butenyl etc. Alkenyl may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio radical, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring contains 3 to 20 carbon atoms, preferably contains 3 to 10 carbon atoms, more preferably contains 3 to 8 carbon atoms, more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene group, cyclooctyl group, etc.; polycyclic cycloalkyl group includes spiro ring, fused ring and bridged ring cycloalkyl group, preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

The cycloalkyl ring may be fused to an aryl, heteroaryl, or heterocycloalkyl ring where the ring bonded to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl , Benzocycloheptyl, etc. Cycloalkyl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy , alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, ring Alkylthio, heterocycloalkylthio, pendant oxy, carboxyl or carboxylate.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2), but excluding ring portions of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably contains 3 to 10 ring atoms; further preferably contains 3 to 8 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include oxazinone, pyrazinone, pyridinonyl, pyrrolidinyl, tetrahydropyrrolyl, tetrahydropyrrolidinyl, azetidinyl, oxa Cyclobutanyl, oxanyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuryl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperyl Pyridone group, piperazinyl group, morpholinyl group, thiomorpholinyl group, homopiperazinyl group, tetrahydropyranyl group and pyranyl group, etc.; preferably oxazinone group, pyrazinone group, pyridone group , pyrrolidinyl, tetrahydropyrrolyl, tetrahydropyrrolidinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, pyrazolidinyl, morpholinyl, piperazinyl, piperidinyl, Piperidinonyl, tetrahydropyranyl and pyranyl; more preferably oxazinone, tetrahydrofuranyl, isoxazolidinyl, tetrahydropyrrolyl, tetrahydropyrrolidinyl, azetidine group, oxetanyl group, piperidinyl group, piperidinonyl group and tetrahydropyranyl group. Polycyclic heterocyclic groups include spiro rings, fused rings and bridged ring heterocyclic groups; the spiro rings, condensed rings and bridged ring heterocyclic groups involved are connected with other groups by single bonds as required, or by Any two or more atoms on the ring are further linked with other cycloalkyl, heterocyclyl, aryl and heteroaryl groups.

The heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring where the ring bonded to the parent structure is a heterocyclyl, non-limiting examples of which include:

The heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy , alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, hetero Cycloalkylthio, pendant oxy, carboxyl or carboxylate.

The term "aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) group having a conjugated π-electron system, preferably 6 to 10 members, More preferred are 6 to 8 members such as phenyl and naphthyl. More preferably, it is phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, where the ring bonded to the parent structure is an aryl ring, non-limiting examples of which include:

Aryl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio radical, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 10 members, more preferably 5 to 8 members, most preferably 5 or 6 members, such as imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl , pyrrolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, thiadiazole, pyridazinyl or pyrazinyl, etc.; preferably triazolyl, thienyl, thiazolyl, pyridyl, imidazolyl, Pyrazolyl, pyridazinyl, pyrazinyl or pyrimidinyl; more preferably pyridyl, imidazolyl, pyrazolyl, pyridazinyl, pyrazinyl or pyrimidinyl. The heteroaryl Rings may be fused to aryl, heterocyclyl, or cycloalkyl rings, where the ring bonded to the parent structure is a heteroaryl ring, non-limiting examples of which include:

Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy , alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, ring Alkylthio, heterocycloalkylthio, carboxyl or carboxylate.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above, preferably an alkyl group containing 1 to 8 carbon atoms, more Preferably it is an alkyl group of 1 to 6 carbon atoms, most preferably an alkyl group of 1 to 3 carbon atoms. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy , alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, ring Alkylthio, heterocycloalkylthio, carboxyl or carboxylate.

"Haloalkyl" means an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

"Haloalkoxy" means an alkoxy group substituted with one or more halogens, wherein alkoxy group is as defined above.

"Hydroxyalkyl" means an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

"Haloalkyl" means an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

"Haloalkoxy" means an alkoxy group substituted with one or more halogens, wherein alkoxy group is as defined above.

"Hydroxyalkyl" means an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

"Hydroxy" means an -OH group.

"Halogen" means fluorine, chlorine, bromine or iodine.

"Amino" refers to _-NH2 .

"Cyano" refers to -CN.

"Nitro" refers to _-NO2 .

"Carboxy" refers to -C(O)OH.

"THF" means tetrahydrofuran.

"EtOAc" means ethyl acetate.

"MeOH" means methanol.

"DMF" refers to N,N-dimethylformamide.

"TFA" means trifluoroacetic acid.

"MeCN" refers to acetonitrile.

"DMA" refers to N,N-dimethylacetamide.

" _Et2O " means diethyl ether.

"DCE" means 1,2 dichloroethane.

"DIPEA" refers to N,N-diisopropylethylamine.

"NBS" refers to N-bromosuccinimide.

"NIS" refers to N-iodosuccinimide.

"Cbz-Cl" refers to benzyl chloroformate.

"Pd ₂ (dba) ₃ " refers to tris(dibenzylideneacetone)dipalladium.

"Dppf" refers to 1,1'-bisdiphenylphosphinoferrocene.

"HATU" refers to 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

"KHMDS" refers to potassium hexamethyldisilazide.

"LiHMDS" refers to lithium bistrimethylsilylamide.

"MeLi" means methyllithium.

"n-BuLi" refers to n-butyllithium.

"NaBH(OAc) ₃ " refers to sodium triacetyloxyborohydride.

" t -BuONO" refers to tertiary butyl nitrite.

"EA" means ethyl acetate.

"PE" means petroleum ether.

"DCM" means dichloromethane.

"STAB" refers to sodium triacetoxyborohydride.

"Pd(dcypf)Cl ₂ " refers to dichloro[1,1'-bis(dicyclohexylphosphino)ferrocene]palladium.

Different terms such as "X is selected from A, B, or C", "X is selected from A, B, and C", "X is A, B, or C", "X is A, B, and C" all express the same The meaning means that X can be any one or several of A, B, and C.

The hydrogen atoms described in the present invention can be replaced by its isotope deuterium, and any hydrogen atom in the example compounds involved in the present invention can also be replaced by a deuterium atom.

"Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes cases where the heterocycle group is substituted with an alkyl group and cases where the heterocycle group is not substituted with an alkyl group .

"Substituted" refers to one or more hydrogen atoms in a group, preferably at most 5, more preferably 1 to 3 hydrogen atoms are independently substituted by a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions and that a person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amine or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (eg, ethylenic) bond.

"Pharmaceutical composition" means a mixture containing one or more of the compounds herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipient. The purpose of the pharmaceutical composition is to promote the administration to the living body, facilitate the absorption of the active ingredient and thus exert the biological activity.

Figure 1 is the XRPD pattern of free base crystal form A.

Figure 2 is the DSC chart of free base crystal form A.

Figure 3 is the XRPD pattern of free base crystal form B.

Figure 4 is the DSC chart of free base crystal form B.

Fig. 5 is an XRPD pattern of free base crystal form C.

Figure 6 is the DSC chart of free base crystal form C.

Figure 7 is the XRPD pattern of free base crystal form D.

Figure 8 is the DSC chart of free base crystal form D.

Figure 9 is the XRPD pattern of batch 1 of free base crystal form B.

Fig. 10 is the XRPD pattern of free base crystal form B batch 2.

Fig. 11 is the XRPD pattern of batch 3 of free base crystal form B.

Figure 12 is the XRPD pattern of free base crystal form B batch 4.

The present invention is further described below in conjunction with examples, but these examples do not limit the scope of the present invention.

Example

The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) or/and liquid chromatography-mass chromatography (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). The determination of NMR is carried out with Bruker AVANCE-400 nuclear magnetic instrument, and the determination solvent is deuterated dimethyl sulfide (DMSO-d ₆ ), deuterated methanol (CD ₃ OD) and deuterated chloroform (CDCl ₃ ), and the internal standard is four Methylsilane (TMS).

Agilent 1200 Infinity Series mass spectrometer was used for LC-MS determination. The determination of HPLC uses Agilent 1200DAD high pressure liquid chromatography (Sunfire C18 150×4.6mm chromatographic column) and Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150×4.6mm chromatographic column).

The thin-layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specification used for TLC is 0.15mm~0.20mm, and the specification used for thin-layer chromatography separation and purification products is 0.4mm~0.5mm. Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

The starting materials in the examples of the present invention are known and commercially available, or can be synthesized using or following methods known in the art.

Unless otherwise specified, all the reactions in the present invention are carried out under a dry nitrogen or argon atmosphere under continuous magnetic stirring, the solvent is a dry solvent, and the unit of the reaction temperature is Celsius.

Example 1

N-(2,2'-dichloro-3'-(6-methoxy-5-((oxetan-3-ylamino)methyl)pyridin-2-yl)-[1,1' -biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide

The first step: the preparation of 6-(3-bromo-2-chlorophenyl)-2-methoxynicotinaldehyde

6-chloro-2-methoxynicotinaldehyde (400mg, 2.34mmol), (3-bromo-2-chlorophenyl)boronic acid (600mg, 2.57mmol), Pd(PPh ₃ ) ₄ (266mg, 0.23mmol) and Potassium carbonate (646mg, 4.68mmol) was dissolved in a mixed solvent of dioxane (20mL) and water (2mL), ventilated, and heated to 95°C and stirred overnight under nitrogen protection. After the reaction was complete, the reaction solution was cooled, filtered, and the filtrate was concentrated. The residue was separated by flash silica gel column chromatography (PE:EA=4:1) to obtain the title compound (600mg, 78%).

MS m/z (ESI): 326.7[M+H] ⁺ .

The second step: 6-(2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)phenyl)-2- Preparation of methoxynicotinaldehyde

6-(3-Bromo-2-chlorophenyl)-2-methoxynicotinaldehyde (600mg, 1.83mmol), pinacol bis-boronate (607mg, 2.39mol), Pd(dppf)Cl ₂ DCM The complex (149mg, 0.18mmol) and potassium acetate (538mg, 5.49mmol) were dissolved in dioxane (17mL), gas exchanged, nitrogen protection, heated and stirred at 95°C overnight. After the reaction was complete, the reaction solution was cooled and concentrated. The residue was separated by flash silica gel column chromatography (PE:EA=3:1) to obtain the title compound (600mg, 87%).

MS m/z (ESI): 375.7[M+H] ⁺ .

The third step: N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3- base)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide

N-(3-bromo-2-chlorophenyl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-methyl Amide (170mg, 0.444mmol), 6-(2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl) -2-Methoxynicotinaldehyde (216mg, 0.577mol), Pd(dppf)Cl ₂ DCM complex (73mg, 0.09mmol) and cesium carbonate (360mg, 1.11mmol) were dissolved in dioxane (10mL) and water (2 mL) in a mixed solvent, pumped for gas, nitrogen protection, heated and stirred at 95°C overnight. After the reaction was complete, the reaction solution was cooled and concentrated. The residue was separated by reverse phase to obtain the title compound (200 mg, 82%).

MS m/z (ESI): 550.7[M+H] ⁺ .

The fourth step: N-(2,2'-dichloro-3'-(6-methoxy-5-((oxetan-3-ylamino)methyl)pyridin-2-yl)-[ 1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-methanol Preparation of amides

N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)- 1,5-Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (100mg, 0.181mmol) and oxetane-3- The amine (52 mg, 0.726 mmol) was dissolved in methanol (3 mL), and after adding 4 drops of acetic acid, the mixture was stirred at room temperature for 3 hours. After adding sodium cyanoborohydride (22mg, 0.362mmol), stirring was continued at room temperature overnight. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by reverse phase to obtain the title compound (10.3 mg, 9%).

MS m/z (ESI): 607.2[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.91(s,1H),8.35(d,J=8.3Hz,1H),7.79(d,J=7.5Hz,1H),7.67(d,J=7.6 Hz,1H),7.51(dt,J=25.0,7.7Hz,2H),7.41(d,J=7.6Hz,1H),7.26(d,J=7.5Hz,1H),7.18(d,J=7.6 Hz,1H),4.59(t,J=6.5Hz,2H),4.32(t,J=6.2Hz,2H),3.91(d,J=8.4Hz,7H),3.63(s,2H),3.37( s,2H),2.69(s,4H),2.38(s,3H).

Example 2

N-(2,2'-dichloro-3'-(5-((3-fluoroazetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-[1,1 '-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)- 1,5-Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (55.1 mg, 0.1 mmol) and 3-fluoroazetidine Pyridine hydrochloride (33.3 mg, 0.3 mmol) was dissolved in methanol (3 mL), neutralized by adding DIPEA, then added 3 drops of acetic acid, and stirred at room temperature for 3 hours. After adding sodium cyanoborohydride (18.6 mg, 0.3 mmol), the stirring was continued at room temperature overnight. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by reverse phase to obtain the title compound (14 mg, 23%).

MS m/z (ESI): 609.3[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.91(s,1H),8.35(d,J=8.4Hz,1H),7.69(dd,J=12.5,7.5Hz,2H),7.51(dt,J =23.9,7.9Hz,2H),7.41(d,J=7.6Hz,1H),7.27(d,J=7.5Hz,1H),7.18(d,J=7.6Hz,1H),5.27(d,J =7.3Hz,1H),5.12(d,J=7.1Hz,1H),3.94-3.85(m,5H),3.66(d,J=8.0Hz,4H),3.39(s,2H),3.30-3.22 (m,1H),3.20(s,1H),2.70(s,4H),2.39(s,3H).

Example 3

1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-carbazamidylamino<acetylamino>)-[1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)azetine Pyridine-3-yl acetate

N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)- 1,5-Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (73mg, 0.106mmol) and azetidine-3- Ethyl acetate trifluoroacetate (61 mg, 0.266 mmol) was dissolved in methanol (4 mL), neutralized by adding DIPEA, then added 4 drops of acetic acid, and stirred at room temperature for 3 hours. After adding sodium cyanoborohydride (16mg, 0.266mmol), stirring was continued at room temperature overnight. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by flash silica gel column chromatography (DCM:MeOH=10:1) to obtain the crude product, and then the title compound (9.9 mg, 15%) was obtained by reverse phase purification.

MS m/z (ESI): 649.3[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.91(s,1H),8.38-8.31(m,1H),7.74-7.64(m,2H),7.54(t,J=7.6Hz,1H),7.48 (t,J=7.9Hz,1H),7.41(dd,J=7.5,1.7Hz,1H),7.26(d,J=7.5Hz,1H),7.18(dd,J=7.6,1.6Hz,1H) ,4.96(t,J=5.8Hz,1H),3.91(d,J=3.9Hz,6H),3.70-3.60(m,4H),3.37(s,2H),3.11(dd,J=8.5,5.4 Hz,2H),2.69(s,4H),2.38(s,3H),2.03(s,3H).

Example 4

N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptan-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)- 1,5-Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (115mg, 0.208mmol) and 1-(2,6 -Diazaspiro[3.3]heptan-2-yl)ethan-1-one hydrochloric acid (110mg, 0.625mmol) was dissolved in methanol (4mL), neutralized by adding DIPEA, added 8 drops of acetic acid, at room temperature Stirring was continued for 3 hours. After adding sodium cyanoborohydride (38.8 mg, 0.625 mmol), the stirring was continued at room temperature overnight. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by reverse phase to obtain the title compound (23.4 mg, 17%).

MS m/z (ESI): 674.3[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ ) δ 9.91(s,1H),8.34(dd, J =8.3,1.5Hz,1H),7.75-7.64(m,2H),7.52(dt, J =24.3, 7.8Hz,2H),7.41(dd, J =7.6,1.7Hz,1H),7.28(d, J =7.4Hz,1H),7.18(dd, J =7.6,1.6Hz,1H),4.19(s, 2H), 3.91(d, J =4.1Hz, 7H), 3.53(d, J =65.9Hz, 9H), 2.74(d, J =20.1Hz, 4H), 2.43(s, 3H), 1.72(s, 3H).

Example 5

N-(3'-(5-((2-Oxa-6-azaspiro[3.3]heptane-6-yl)methyl)-6-methoxypyridin-2-yl)-2,2 '-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c ]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)- 1,5-Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (50mg, 0.091mmol) and 2-oxa-6 - Dissolve azaspiro[3.3]heptane (27 mg, 0.272 mmol) in methanol (3 mL), add 2 drops of acetic acid, and stir at room temperature for 2 hours. After adding sodium cyanoborohydride (14 mg, 0.227 mmol), the stirring was continued overnight at room temperature. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by reverse phase to obtain the title compound (10 mg, 18%).

MS m/z (ESI): 633.3[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.91(s,1H),8.35(d,J=8.2Hz,1H),7.67(t,J=5.8Hz,2H),7.51(dt,J=23.1 ,7.8Hz,2H),7.40(d,J=7.5Hz,1H),7.25(d,J=7.5Hz,1H),7.18(d,J=7.7Hz,1H),4.62(s,4H), 3.90(s,6H),3.51(s,2H),3.38(d,J=6.6Hz,6H),2.69(s,4H),2.38(s,3H).

Example 6

N-(3'-(5-((5-oxa-2-azaspiro[3.4]octane-2-yl)methyl)-6-methoxypyridin-2-yl)-2,2 '-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c ]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)- 1,5-Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (76mg, 0.134mmol) and 5-oxa-2 -Azaspiro[3.4]octane hydrochloride (31mg, 0.206mmol) was dissolved in methanol (4mL), neutralized by adding DIPEA, then added 4 drops of acetic acid, stirred at room temperature for 2 hours. After adding sodium cyanoborohydride (17mg, 0.274mmol), stirring was continued at room temperature overnight. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by reverse phase to obtain the title compound (6.9 mg, 8%).

MS m/z (ESI): 647.3[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.91(s,1H),8.35(d,J=8.2Hz,1H),7.69(dd,J=15.2,7.6Hz,2H),7.51(dt,J =23.6,7.8Hz,2H),7.40(d,J=7.5Hz,1H),7.26(d,J=7.5Hz,1H),7.18(d,J=7.6Hz,1H),3.90(s,6H ),3.67(t,J=6.8Hz,2H),3.59(s,2H),3.37(s,4H),3.09(d,J=6.9Hz,2H),2.69(s,4H),2.38(s ,3H),2.04(t,J=7.2Hz,2H),1.82(q,J=6.7Hz,2H).

Example 7

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-carbonyl-2,5-diazaspiro[3.4]octane-2-yl)methyl) Pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-carbonyl-2,5-diazaspiro[3.4]octane-2-yl)methyl) Pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c] The preparation of pyridine-2-carboxamide refers to Example 1 .

MS m/z (ESI): 660.2[M+H] ⁺ .

Example 8

N-(2,2'-dichloro-3'-(5-((3-hydroxyazetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-[1,1 '-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)- 1,5-Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (30mg, 0.055mmol) and azetidine-3- Alcohol hydrochloride (24 mg, 0.217 mmol) was dissolved in methanol (3 mL), neutralized by adding DIPEA, then added 3 drops of acetic acid, and stirred at room temperature for 3 hours. After adding sodium cyanoborohydride (6.7 mg, 0.109 mmol), the stirring was continued at room temperature overnight. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by reverse phase to obtain the title compound (5.3 mg, 16%).

MS m/z (ESI): 607.2[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.91(s,1H),8.35(d,J=8.3Hz,1H),7.68(t,J=7.1Hz,2H),7.51(dt,J=23.4 ,7.8Hz,2H),7.40(d,J=7.8Hz,1H),7.26(d,J=7.5Hz,1H),7.18(d,J=7.7Hz,1H),5.33(s,1H), 4.26-4.18(m,1H),3.90(d,J=3.1Hz,5H),3.58(d,J=11.1Hz,4H),3.37(s,2H),2.84(t,J=6.7Hz,2H ),2.69(s,4H),2.38(s,3H),2.05-1.97(m,1H).

Example 9

N-(2,2'-dichloro-3'-(5-((3-hydroxy-3-methylazetidin-1-yl)methyl)-6-methoxypyridin-2-yl) -[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2 - formamide

N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)- 1,5-Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (30mg, 0.055mmol) and 3-methylazetine Pyridine-3-ol hydrochloric acid (27 mg, 0.218 mmol) was dissolved in methanol (3 mL), neutralized by adding DIPEA, then added 3 drops of acetic acid, and stirred at room temperature for 3 hours. After adding sodium cyanoborohydride (6.8 mg, 0.109 mmol), the stirring was continued at room temperature overnight. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by reverse phase to obtain the title compound (6.4 mg, 19%).

MS m/z (ESI): 621.2[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ 9.91(s,1H),8.35(d,J=8.4Hz,1H),7.69(t,J=9.3Hz,2H),7.51(dt,J=23.5 ,7.8Hz,2H),7.40(d,J=7.6Hz,1H),7.27(d,J=7.4Hz,1H),7.18(d,J=7.7Hz,1H),3.93-3.86(m,6H ),3.59(s,2H),3.37(s,2H),3.29(d,J=6.6Hz,2H),2.97(d,J=6.6Hz,2H),2.69(s,4H),2.39(d ,J=3.4Hz,3H),1.39(s,3H).

Example 10

1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-formamido)-[1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)azetidine-3-carboxylic acid

1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine Reference for the preparation of -2-formamido)-[1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)azetidine-3-carboxylic acid Example 1 .

MS m/z (ESI): 635.2[M+H] ⁺ .

Example 11

(S) -1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c]pyridine-2-carbazoylamino<glycolamide>)-[1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methanol base) azetidine-2-carboxylic acid

Add (2S) -azetidine-2-carboxylic acid (11.02 mg, 109.00 μmol) to N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridine- 2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c ] Pyridine-2-carboxamide (50mg, 90.84μmol) in a mixed solution of AcOH (10.00μL), DMF (0.5mL) and MeOH (0.5mL), stirred at room temperature for 2 hours, then added sodium cyanoborohydride (95.83mg, 454.19μmol), stirred at room temperature for 16 hours. The reaction solution was separated and purified by prep-HPLC to obtain the title compound as a white solid (16.5 mg, 26%).

MS m/z (ESI): 635.2[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.90(s,1H),8.35(dd,J=8.3,1.6Hz,1H),7.79(d,J=7.5Hz,1H),7.68(dd,J =7.7,1.7Hz,1H),7.54(t,J=7.6Hz,1H),7.48(t,J=8.0Hz,1H),7.41(dd,J=7.6,1.7Hz,1H),7.28(d ,J=7.5Hz,1H),7.18(d,J=7.6Hz,1H),3.94-3.87(m,8H),3.76-3.70(m,1H),3.44-3.41(m,1H),3.37( s,2H),3.13-3.08(m,1H),2.71-2.64(m,4H),2.39(s,3H),2.26-2.16(m,2H).

Example 12

(S) -N-(2,2'-dichloro-3'-(5-((2-(hydroxymethyl)azetidin-1-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide

A solution of isobutyl chloroformate (3.01 mg, 22.01 μmol) in THF (0.1 mL) was added dropwise to (S) -1-((6-(2,2'-dichloro-3'-( 1,5-Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carbazamidyl <acetamide>)-[ 1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)azetidine-2-carboxylic acid formate (10 mg, 14.67 μmol) and DIPEA (6.64 mg, 51.35μmol) in THF (2mL), reacted at this temperature for 10 minutes, then added NaBH ₄ (0.83mg, 22.01μmol) in water (0.5mL), slowly raised to room temperature and stirred for 1 hour. The reaction solution was separated and purified by prep-HPLC to obtain the title compound as an off-white solid (2.3 mg, 23%).

MS m/z (ESI): 621.2[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.90(s,1H),8.35(dd,J=8.3,1.6Hz,1H),7.75(d,J=7.5Hz,1H),7.67(dd,J =7.7,1.8Hz,1H),7.54(t,J=7.6Hz,1H),7.48(t,J=7.9Hz,1H),7.40(dd,J=7.5,1.7Hz,1H),7.25(d ,J=7.5Hz,1H),7.18(d,J=7.5Hz,1H),3.91(s,3H),3.90(s,3H),3.79-3.74(m,1H),3.53-3.47(m, 1H),3.41-3.30(s,7H),2.81-2.75(m,1H),2.72-2.63(s,4H),2.38(s,3H),2.00-1.80(m,2H).

Example 13

N-(2,2'-dichloro-3'-(5-((3-hydroxy-3-(hydroxymethyl)azetidin-1-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-((3-hydroxy-3-(hydroxymethyl)azetidin-1-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] The preparation of pyridine-2-formamide refers to Example 11 .

MS m/z (ESI): 637.2[M+H] ⁺ .

Example 14

N-(3'-(5-((3-Acetamidoazetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-2,2'-dichloro-[ 1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-methanol Amide

Add DIPEA (140.88 mg, 1.09 mmol, 189.86 μL ) to N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1 ,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-formyl Amine (400mg, 726.70 μ mol) and N-(azetidin-3-yl) acetamide hydrochloride (164.17mg, 1.09mmol) in dichloroethane (5mL) solution, stirred at room temperature for 1 Hours, sodium acetate borohydride (462.18mg, 2.18mmol) was added and stirred at room temperature for 2 hours. The reaction solution was quenched with saturated aqueous sodium bicarbonate (10 mL), extracted with DCM (20 mL), the organic layer was concentrated under reduced pressure, and the residue was separated and purified by prep-HPLC to obtain the title compound as a white solid (259 mg, 51%).

MS m/z (ESI): 648.2[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.90(s,1H),8.38-8.29(m,2H),7.71(d,J=7.5Hz,1H),7.68(dd,J=7.7,1.8Hz ,1H),7.54(t,J=7.6Hz,1H),7.48(t,J=7.9Hz,1H),7.40(dd,J=7.6,1.7Hz,1H),7.27(d,J=7.5Hz ,1H),7.18(dd,J=7.6,1.6Hz,1H),4.30(q,J=6.9Hz,1H),3.91(s,3H),3.90(s,3H),3.62-3.56(m, 4H),3.39(s,2H),3.00-2.93(m,2H),2.75-2.64(m,4H),2.40(s,3H),1.80(s,3H).

Example 15

N-(3'-(5-((3-(Acetamidomethyl)azetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-2,2'- Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-Formamide

The first step: tertiary-butyl ((1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H -Imidazolo[4,5-c]pyridine-2-carbazamidyl<glycolamide>)-[1,1'-biphenyl]-3-yl)-2-methoxy Preparation of pyridin-3-yl)methyl)azetidin-3-yl)methyl)carbamate

Add DIPEA (35.22 mg, 272.51 μmol , 47.47 μL ) to N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[ 1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-methanol Amide (100mg, 181.67 μ mol) and 3-BOC-aminomethylazetidine hydrochloride (60.69mg, 272.51 μ mol) in dichloroethane (2mL) solution, stirred at room temperature for 0.5 hours, Add sodium acetate borohydride (191.67 mg, 908.37 μ mol) and stir at room temperature for 16 hours. The reaction solution was diluted with DCM (20 mL), washed with saturated aqueous sodium bicarbonate (10 mL), and the organic layer was concentrated under reduced pressure to give the title compound as a brown oil (75 mg, 58%).

MS m/z (ESI): 720.2[M+H] ⁺ .

The second step: N-(3'-(5-((3-(aminomethyl)azetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-2,2 '-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c ] The preparation of pyridine-2-carboxamide

TFA (0.3 mL) was added to tert-butyl ((1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6 ,7-Tetrahydro-1H-imidazo[4,5-c]pyridine-2-carbazamidyl <acetamide>)-[1,1'-biphenyl]-3-yl )-2-methoxypyridin-3-yl)methyl)azetidin-3-yl)methyl)carbamate (75mg, 104.07μmol ) in DCM (0.7mL) solution, room temperature Stir for 2 hours. The reaction solution was concentrated under reduced pressure, the residue was diluted with DCM (10 mL), and then concentrated under reduced pressure, the residue was separated in aqueous sodium bicarbonate (5 mL) and DCM (10 mL), the organic layer was dried over anhydrous sodium sulfate, filtered, and reduced Concentration under reduced pressure gave the title compound as a brown oil (60 mg, 93%).

MS m/z (ESI): 620.2[M+H] ⁺ .

The third step: N-(3'-(5-((3-(acetylaminomethyl)azetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-2 ,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c] Preparation of pyridine-2-carboxamide

A solution of acetyl chloride (11.38 mg, 145.03 μmol , 10.35 μL ) in DCM (0.1 mL) was added dropwise to N-(3'-(5-((3-(aminomethyl)acridine) at -20°C Butidin-1-yl)methyl)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5 -Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (60mg, 96.69 μ mol), DIPEA (37.49mg, 290.06 μ mol , 50.52 μ L) in DCM (1 mL) solution, rise to room temperature and react for 10 minutes. The reaction solution was concentrated under reduced pressure, and the residue was separated and purified by prep-HPLC to obtain the title compound as a white solid (8.1 mg, 11%).

MS m/z (ESI): 662.2[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.90(s,1H),8.35(d,J=8.2Hz,1H),7.91(s,1H),7.72-7.64(m,2H),7.54(t ,J=7.6Hz,1H),7.48(t,J=7.9Hz,1H),7.40(dd,J=7.6,1.7Hz,1H),7.26(d,J=7.5Hz,1H),7.18(d ,J=7.6Hz,1H),3.91(s,3H),3.90(s,3H),3.54(s,2H),3.37(s,2H),3.29(s,2H),3.24(d,J= 6.4Hz,2H),2.94(t,J=6.4Hz,2H),2.73-2.64(q,4H),2.38(s,3H),2.35-2.30(m,1H),1.79(s,3H).

Example 16

N-(2,2'-dichloro-3'-(5-((3-(cyanomethyl)azetidin-1-yl)methyl)-6-methoxypyridin-2-yl) -[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2 - formamide

Add DIPEA (9.39 mg, 72.67 μmol , 12.66 μL ) to N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[ 1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-methanol Amide (20mg, 36.33 μ mol) and 2-(azetidin-3-yl) acetonitrile hydrochloride (9.64mg, 72.67 μ mol) in dichloroethane (0.5mL) solution, stirred at room temperature After 0.5 hour, sodium acetate borohydride (38.33 mg, 181.67 μmol ) was added and stirred at room temperature for 1 hour. The reaction solution was diluted with DCM (10 mL), then washed with saturated aqueous sodium bicarbonate (5 mL), the organic layer was concentrated under reduced pressure, and the residue was separated and purified by prep-HPLC to obtain the title compound as a white solid (18.6 mg, 76%).

MS m/z (ESI): 630.2[M+H] ⁺ .

Example 17

N-(2,2'-dichloro-3'-(5-((3-(dimethylaminoformyl)azetidin-1-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide

Add DIPEA (28.18 mg, 218.01 μmol , 37.97 μL ) to N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[ 1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-methanol Amide (60mg, 109.00 μ mol) and N,N-dimethylazetidine-3-formamide hydrochloride (27.94mg, 218.01 μ mol) in dichloroethane (2mL) solution, Stir at room temperature for 0.5 hour, then add sodium acetate borohydride (34.50 mg, 163.51 μ mol), and stir at room temperature for 0.5 hour. The reaction solution was diluted with DCM (10 mL), then washed with saturated aqueous sodium bicarbonate (5 mL), the organic layer was concentrated under reduced pressure, and the residue was separated and purified by prep-HPLC to obtain the title compound as a white solid (28.3 mg, 36%).

MS m/z (ESI): 662.2[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.83(s,1H),8.28(dd,J=8.2,1.5Hz,1H),7.66-7.57(m,2H),7.47(t,J=7.6Hz ,1H),7.41(t,J=8.0Hz,1H),7.33(dd,J=7.6,1.7Hz,1H),7.19(d,J=7.4Hz,1H),7.11(dd,J=7.6, 1.5Hz,1H),3.84(s,3H),3.83(s,3H),3.47(s,2H),3.47-3.41(m,3H),3.31(s,2H),3.16(t,J=3.6 Hz,2H),2.76(s,3H),2.74(s,3H),2.67-2.58(m,4H),2.32(s,3H).

Example 18

N-(2,2'-dichloro-3'-(6-methoxy-5-((3-(methylaminoformyl)azetidin-1-yl)methyl)pyridine-2- base)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-Formamide

N-(2,2'-dichloro-3'-(6-methoxy-5-((3-(methylaminoformyl)azetidin-1-yl)methyl)pyridine-2- base)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -Refer to Example 11 for the preparation of 2-formamide.

MS m/z (ESI): 648.2[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ 9.90(s,1H),8.35(dd,J=8.3,1.5Hz,1H),7.80-7.74(m,1H),7.73-7.64(m,2H) ,7.54(t,J=7.6Hz,1H),7.48(t,J=7.9Hz,1H),7.40(dd,J=7.6,1.7Hz,1H),7.26(d,J=7.5Hz,1H) ,7.18(dd,J=7.6,1.6Hz,1H),3.91(s,3H),3.90(s,3H),3.54(s,2H),3.46-3.40(m,2H),3.36(s,2H ),3.24-3.17(m,2H),3.18-3.10(m,1H),2.74-2.66(m,4H),2.58(d,J=4.6Hz,3H),2.38(s,3H).

Example 19

N-(2,2'-dichloro-3'-(5-((6-hydroxy-2-azaspiro[3.3]heptane-2-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-((6-hydroxy-2-azaspiro[3.3]heptane-2-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] The preparation of pyridine-2-formamide refers to Example 11 .

MS m/z (ESI): 647.2[M+H] ⁺ .

Example 20

N-(2,2'-dichloro-3'-(5-((6-isobutyryl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide

The first step: tertiary-butyl 6-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazole And[4,5-c]pyridine-2-carbazamidyl <glycolamide>)-[1,1'-biphenyl]-3-yl)-2-methoxypyridine- Preparation of 3-yl)methyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate

N-(2,2'-dichloro-3'-(5-formyl-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)- 1,5-Dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (195 mg, 354 μmol ) and 2,6-di A solution of tert-butyl azaspiro[3.3]heptane-2-carboxylate (117mg, 590 μmol ) in dichloroethane (2mL) was stirred at room temperature for 0.5 hours, then sodium acetate borohydride (226mg, 1.06mmol) was added ), stirred at room temperature for 0.5 hours. The reaction solution was diluted with DCM (20 mL), then washed with saturated aqueous sodium bicarbonate (10 mL), the organic layer was concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the title compound as a brown oil (125 mg, 48%).

MS m/z (ESI): 732.2[M+H] ⁺ .

The second step: N-(3'-(5-(2,6-diazaspiro[3.3]heptane-2-ylmethyl)-6-methoxypyridin-2-yl)-2,2 '-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c ] The preparation of pyridine-2-carboxamide

TFA (0.5 mL) was added to tert-butyl 6-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7 -Tetrahydro-1H-imidazo[4,5-c]pyridine-2-carbazamidyl <acetamide>)-[1,1'-biphenyl]-3-yl)- 2-methoxypyridin-3-yl)methyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (157 mg, 214 μmol ) in DCM (2 mL), room Stir warm for 1 hour. The reaction solution was concentrated under reduced pressure, the residue was diluted with DCM (10 mL), and then concentrated under reduced pressure, the residue was separated in aqueous sodium bicarbonate (5 mL) and DCM (10 mL), the organic layer was dried over anhydrous sodium sulfate, filtered, and reduced Concentration under reduced pressure gave the title compound as a brown oil (116 mg, 86%).

MS m/z (ESI): 632.2[M+H] ⁺ .

The third step: N-(2,2'-dichloro-3'-(5-((6-isobutyryl-2,6-diazaspiro[3.3]heptane-2-yl)methyl) -6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H- Preparation of imidazo[4,5-c]pyridine-2-carboxamide

A solution of isobutyryl chloride (8 mg, 75.9 μmol ) in DCM (0.1 mL) was added dropwise to N-(3'-(5-(2,6-diazaspiro[3.3]heptane-2 -ylmethyl)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl- 4,5,6,7-Tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (40 mg, 63.3 μmol ), DIPEA (24 mg, 190 μmol ) in DCM (1 mL) In the solution, react at 0°C for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was separated and purified by prep-HPLC to obtain the title compound as a white solid (7.8 mg, 17%).

MS m/z (ESI): 702.2[M+H] ⁺ .

Example 21

N-(2,2'-dichloro-3'-(5-((6-(cyclopropylcarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6 -Methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-((6-(cyclopropylcarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6 -Methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo The preparation of [4,5-c]pyridine-2-formamide refers to Example 20 .

MS m/z (ESI): 700.2[M+H] ⁺ .

Example 22

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-propionyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl )pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4, 5-c]pyridine-2-carboxamide

A solution of propionyl chloride (5.48mg, 59.28μmol, 5.17μL ) in DCM (0.1mL) was added dropwise to N-(3'-(5-(2,6-diazaspiro[3.3]hept Alkyl-2-ylmethyl)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-di Methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide (25mg, 39.52 μ mol), DIPEA (25.54mg, 197.60 μ mol, 34.42 μL ) in DCM (1 mL), react at room temperature for 0.5 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated and purified by prep-HPLC to obtain the title compound as a white solid (8.9 mg, 31%).

MS m/z (ESI): 688.2[M+H] ⁺ .

Example 23

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-(2,2,2-trifluoroacetyl)-2,6-diazaspiro[ 3.3] Heptane-2-yl)methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7 -Tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-(2,2,2-trifluoroacetyl)-2,6-diazaspiro[ 3.3] Heptane-2-yl)methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7 - The preparation of tetrahydro-1H-imidazo[4,5-c]pyridine-2-formamide refers to Example 20 .

MS m/z (ESI): 728.2[M+H] ⁺ .

Example 24

N-(2,2'-dichloro-3'-(5-((6-(2,2-difluoroacetyl)-2,6-diazaspiro[3.3]heptan-2-yl )methyl)-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetra Hydrogen-1H-imidazo[4,5-c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-((6-(2,2-difluoroacetyl)-2,6-diazaspiro[3.3]heptan-2-yl )methyl)-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetra The preparation of hydrogen-1H-imidazo[4,5-c]pyridine-2-carboxamide refers to Example 20 .

MS m/z (ESI): 710.2[M+H] ⁺ .

Example 25

N-(2,2'-dichloro-3'-(5-((6-(2-cyanoacetyl)-2,6-diazaspiro[3.3]heptane-2-yl)methanol Base)-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro- 1H-imidazo[4,5-c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-((6-(2-cyanoacetyl)-2,6-diazaspiro[3.3]heptane-2-yl)methanol Base)-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro- The preparation of 1H-imidazo[4,5-c]pyridine-2-carboxamide refers to Example 20 .

MS m/z (ESI): 699.2[M+H] ⁺ .

Example 26

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-(methylsulfonyl)-2,6-diazaspiro[3.3]heptane-2-yl )methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(6-methoxy-5-((6-(methylsulfonyl)-2,6-diazaspiro[3.3]heptane-2-yl )methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo The preparation of [4,5-c]pyridine-2-formamide refers to Example 20 .

MS m/z (ESI): 710.2[M+H] ⁺ .

Example 27

N-(2,2'-dichloro-3'-(5-((6-formyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-((6-formyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 , 5-c] The preparation of pyridine-2-formamide refers to Example 20 .

MS m/z (ESI): 660.2[M+H] ⁺ .

Example 28

N-(3'-(5-((7-acetyl-2,7-diazaspiro[3.5]nonan-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide

N-(3'-(5-((7-acetyl-2,7-diazaspiro[3.5]nonan-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 , 5-c] The preparation of pyridine-2-carboxamide refers to Example 1 .

MS m/z(ESI): 702.2[M+H]+.

Example 29

N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.4]octane-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide

N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.4]octane-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 , 5-c] The preparation of pyridine-2-carboxamide refers to Example 1 .

MS m/z (ESI): 688.2[M+H] ⁺ .

Example 30

N-(2,2'-dichloro-3'-(5-((6-isobutyryl-2,6-diazaspiro[3.4]octane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide

N-(2,2'-dichloro-3'-(5-((6-isobutyryl-2,6-diazaspiro[3.4]octane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 , 5-c] The preparation of pyridine-2-carboxamide refers to Example 1 .

MS m/z (ESI): 716.2[M+H] ⁺ .

Example 31

N-(3'-(5-((6-Acetamido-2-azaspiro[3.3]heptane-2-yl)methyl)-6-methoxypyridin-2-yl)-2 ,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c]pyridine-2-carboxamide

N-(3'-(5-((6-Acetamido-2-azaspiro[3.3]heptane-2-yl)methyl)-6-methoxypyridin-2-yl)-2 ,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c] The preparation of pyridine-2-carboxamide refers to Example 1 .

MS m/z (ESI): 688.2[M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ ) δ 9.90(s,1H),8.35(dd, J =8.3,1.6Hz,1H),8.03(d, J =7.5Hz,1H),7.72-7.65(m ,2H),7.54(t, J =7.7Hz,1H),7.48(t, J =8.0Hz,1H),7.40(dd, J =7.6,1.7Hz,1H),7.25(d, J =7.5Hz ,1H),7.18(dd, J =7.6,1.6Hz,1H),4.08-3.97(m,1H),3.93-3.88(m,6H),3.52(s,2H),3.37(s,2H), 3.27(s,2H),3.16(s,2H),2.71-2.64(m,4H),2.43-2.32(m,5H),2.01-1.94(m,2H),1.74(s,3H).

Biology Test Evaluation

The following further describes and explains the present invention in combination with test examples, but these examples are not meant to limit the scope of the present invention.

Test Example 1 Determination of the binding inhibitory effect of the compound of the present invention on human PD-1/PD-L1

Purpose of the experiment: The purpose of this test case is to measure the activity of the compound on human PD-1/PD-L1 binding inhibition.

Experimental equipment: a centrifuge (5810R) was purchased from Eppendorf, a pipette was purchased from Eppendorf or Rainin, a microplate reader was purchased from BioTek, USA, and the model was H1MFD full-function microplate reader.

Experimental reagents: Binding Domain diluent buffer was purchased from Cisbio Company, product number 62DLBDDF; MAb Anti-6HIS Eu cryptate Gold was purchased from Cisbio Company, product number 61HI2KLA; PAb Anti Human IgG-XL665 was purchased from Cisbio Company, product number 61HFCXLB; PD-L1 -His protein was purchased from Abcam Company, the article number is ab167713; PD-1-Fc protein was purchased from R&D Company, the article number was 1086-PD; Detection buffer was purchased from Cisbio Company, the article number was 62SDBRDD; 384-well plates were purchased from PerkinElmer Company, the article number was 6007290.

Experimental method: In order to test the inhibitory activity of the compound on the binding of human PD-1/PD-L1, the time-resolved fluorescence resonance energy transfer (TR-FRET) method was used in this experiment to test the compound's ability to bind to human PD-1/PD-L1. Inhibitory effect, and the half-inhibitory concentration IC ₅₀ of the compound's inhibitory activity on human PD-1/PD-L1 binding was obtained.

The specific experimental operation is as follows:

This experiment was carried out in a 384-well plate, the total reaction system was 20 μL, and the compound was diluted to different concentrations (10 μM or 1 μM starting, 3-fold dilution, 11 doses) using the experimental buffer Binding Domain diluent buffer (Cisbio # 62DLBDDF) , the PD-L1-His protein (19~238 amino acids) (Abcam # ab167713) was diluted to 10nM with the assay buffer, and the PD-1-Fc protein (25~167 amino acids) (R&D # 1086-PD ) was diluted to 20nM with experimental buffer, and the compound, PD-L1 protein and PD-1 protein were added to a 384-well plate, with a total volume of 10 μL, placed in a centrifuge at 1000rpm for 1 minute to mix evenly, and incubated at room temperature for 60 Add 10 μL of the mixed solution of MAb Anti-6HIS Eu cryptate Gold (Cisbio# 61HI2KLA) and PAb Anti Human IgG-XL665 (Cisbio# 61HFCXLB) diluted with HTRF Detection buffer to each well, and put it in a centrifuge at 1000rpm for 1 minute to mix evenly. React for 3 hours at room temperature or overnight at 4°C, put into a microplate reader (BioTek Synergy H1) for reading, the excitation wavelength is 340nm, and the emission wavelength is 620nm and 665nm.

Experimental data processing method:

The ratio of the fluorescence readings at 620nm and 665nm (665nm/620nm) was calculated, and the inhibition rate was calculated. The compound concentration and inhibition rate were used for nonlinear regression fitting using Graphpad Prism software to obtain the IC ₅₀ value, as shown in Table 9 below.

Table 9 IC ₅₀ values of compounds binding to human PD-1/PD-L1

Experimental conclusion: the compounds of the examples of the present invention showed good binding inhibitory activity in the PD-1/PD-L1 inhibition test.

Test example 2 Determination of endocytic activity of PD-L1 protein on tumor cell surface induced by compounds of the present invention

Purpose of the experiment: The purpose of this test case is to test the activity of the compound in inducing endocytosis of PD-L1 on the surface of tumor cells.

Experimental equipment: a centrifuge (5702R) was purchased from Eppendorf, a pipette was purchased from Eppendorf or Rainin, and a flow cytometer was purchased from Beckman Coulter, model DxFlex.

Experimental reagents: PE Mouse Anti-Human CD274 antibody was purchased from BD Pharmingen Company, the product number is 557924; BSA was purchased from Sigma Company, the product number was B2064-100G; PBS was purchased from Gibco Company, the product number was 10010049; 24-well plates were purchased from Corning Company, The item number is 3526.

Experimental method: In order to test the activity of compounds inducing PD-L1 endocytosis, this experiment tested the activity of compounds inducing PD-L1 endocytosis by detecting the degree of change of PD-L1 on the surface of tumor cells with a fluorescently labeled anti-PD-L1 antibody , and the IC ₅₀ of the compound inducing the endocytic activity of PD-L1 protein was obtained.

The specific experimental operation is as follows:

Mouse colon cancer cells MC38-hPDL1 that highly express hPD-L1 were collected, adjusted to a suitable density, spread on 24-well plates, and placed in a 37°C, 5% CO ₂ incubator to adhere to the wall overnight. Compounds were added to 24-well plates at different concentrations using culture medium, and solvent control wells were set up, and incubated in a 37°C, 5% CO ₂ incubator for 16 hours, then the 24-well plates were taken out, and the different treatments in the plates were collected. The MC38-hPDL1 cells were washed once with FACS buffer (PBS containing 0.5% BSA), and the cells were prepared to a suitable density with FACS buffer, and PE Mouse Anti-Human CD274 antibody (BD Pharmingen# 557924) was added, at room temperature Incubate on a shaker in the dark for 30 minutes, wash the cells twice with FACS buffer, resuspend in 100 μL of PBS, use flow cytometry to detect the fluorescent signal on the cell surface, and set the isotype control as a negative control.

Experimental data processing method:

The endocytosis rate of the compound was calculated using the fluorescence signals of different treatment groups, and the compound concentration and endocytosis rate were used for nonlinear regression fitting using GraphPad Prism software to obtain the IC ₅₀ value, as shown in Table 10.

Table 10 The maximum endocytosis rate and IC ₅₀ value of the compounds on the surface of tumor cells PD-L1

Experimental conclusion: the compounds shown in the present invention can well induce the endocytosis of PD-L1 on the surface of tumor cells.

Test example 3 Inhibitory effect of the compound of the present invention on CHO-PDL1/Jurkat-PD1 cell pathway

Experimental purpose: The purpose of this test case is to test the inhibitory effect of the compound on the CHO-PDL1/Jurkat-PD1 cellular pathway.

Experimental equipment: a centrifuge (5810R) was purchased from Eppendorf, a pipette was purchased from Eppendorf or Rainin, a microplate reader was purchased from BioTek, USA, and the model was H1MFD full-function microplate reader.

Experimental reagents: CHO-PDL1 cells and NFAT-luc2/PD1 Jurkat cells are from Promega Company, the product number is J1252; RPMI 1640 is purchased from Gibco Company, the product number is 22400089; FBS was purchased from Gibco Company, product number 10091148; One-Glo reagent was purchased from Promega Company, product number E6120; 96-well plate was purchased from Corning Company, product number 3610.

Experimental method: In this experiment, two stably transfected cell lines were included, CHO-K1 cells stably transfected with PD-L1 and Jurkat cells stably transfected with PD-1 and NFAT luciferase reporter gene. After incubation, due to its inhibitory effect on the PD-1/PD-L1 pathway, the activation degree of Jurkat cells changes, thereby changing the level of the reporter gene NFAT in the middle and downstream of Jurkat cells. By detecting the signal value of luciferase To characterize the level of NFAT, in order to characterize the inhibitory effect of the test compound on the CHO-PDL1/Jurkat-PD1 pathway.

The specific experimental operation is as follows:

Cultivate CHO-PDL1 cells (Promega, #J1252) to a suitable cell density. After digestion, use complete medium to resuspend the cell suspension to a suitable density. Spread 100 μL per well on a 96-well plate (Corning, #3610) and place in 37 ℃, 5% CO ₂ incubator for overnight culture, use the analysis medium (RPMI 1640+2%FBS, RPMI 1640 product number is Gibco, #22400089, FBS product number is Gibco, #10091148) to prepare different concentrations of compound solutions, and collect NFAT - luc2/PD1 Jurkat cells (Promega, #J1252), resuspended to appropriate cell density using assay medium. Aspirate the supernatant of the CHO-PDL1 cell culture plate, add 40 μL of the prepared compound solution and 40 μL of the resuspended NFAT-luc2/PD1 Jurkat cells, put it in a 37°C, 5% CO ₂ incubator for 6 hours, and take out the cell plate , add 40 μL of One-Glo reagent (Promega, #E6120) to each well, shake and mix well, incubate at room temperature in the dark for 10 minutes, put into a microplate reader and use the luminescence program to read the luminescence value of the cell plate.

Experimental data processing method:

The EC ₅₀ value was fitted according to the compound concentration and the luminescent signal value, and the maximum induction factor of each compound relative to the signal value of the untreated group was calculated, as shown in Table 11.

Table 11 Compounds on CHO-PDL1/Jurkat-PD1 cells EC ₅₀ and the maximum induction fold of the compound relative to the signal value of the untreated group

Experimental conclusion: the compounds of the examples of the present invention showed good inhibitory activity in the inhibition test of CHO-PDL1/Jurkat-PD1 cell pathway.

Test Example 4 Determination of the Combination of Invention Compounds to Improve PD-L1 Protein Stability (Melting Temperature)

The purpose of the experiment: To test the ability of the compound to improve the stability of PD-L1 protein and increase the melting temperature of the protein.

Experimental equipment: Quantitative PCR instrument (Quantstudio6 Flex) was purchased from Life Company, and the pipette was purchased from Eppendorf or Rainin Company.

Experimental reagents: Protein Thermal Shift ^TM Dye Kit was purchased from Thermofisher Company, product number 4461146; PD-L1 protein was purchased from Acro Biosystems Company, product number was PD1-H5229; HEPES, 1M Buffer Solution was purchased from Thermofisher Company, product number 15630080; NaCl was purchased from From Sinopharm Chemical Reagent Co., Ltd., the product number is 10019318.

Experimental method: In this experiment, the thermal shift method was used to test the change degree of the melting temperature (Tm) of PD-L1 protein before and after compound binding to characterize the ability of the compound to enhance the stability of PD-L1 protein, thereby showing the binding ability of the compound to PD-L1 protein.

The specific experimental operation is as follows:

Prepare a solution containing 10 μM HEPES, SYPRO Orange and 150 mM NaCl as the experimental buffer, and add human PD-L1 protein at a final concentration of 2 μM. Dispense the above reaction mixture into 8 PCR tubes, 19.5 μL per tube, add 0.5 μL of the test compound or DMSO respectively, the total reaction system is 20 μL, the final concentration of the compound is 10 μM, and set 2.5% DMSO as the solvent control . Put the PCR tube into the PCR machine, and select the melt curve function to detect the melting temperature of PD-L1 protein in different treatment groups (heating from 25°C to 95°C, 0.03°C/s).

Experimental data processing method:

Import the experimental data files of the PCR instrument into the thermal shift software to obtain the melting temperature (Tm) of each treatment group, and subtract the Tm of the DMSO solvent control group to obtain the change value of the melting temperature (△Tm), as shown in the table below :

The variation value of the melting temperature of the compound of table 12

Experimental results:

From the above scheme, it can be concluded that the compounds shown in the present invention have the ability to improve the stability of PD-L1 protein in the test of increasing the melting temperature of PD-L1 protein.

Test Example 5 In vitro cytotoxicity of compounds of the present invention on NFAT-luc2/PD1 Jurkat cells and MC38-hPDL1 cells

Experimental purpose: To test the in vitro cytotoxicity of compounds on NFAT-luc2/PD1 Jurkat cells and MC38-hPDL1 cells.

Experimental equipment: a centrifuge (5810R) was purchased from Eppendorf, a pipette was purchased from Eppendorf or Rainin, a microplate reader was purchased from BioTek, USA, and the model was H1MFD full-function microplate reader.

Experimental reagents: RPMI 1640 was purchased from Gibco Company, product number 22400089; FBS was purchased from Gibco Company, product number 10091148; PBS was purchased from Gibco Company, product number 10010049; Cell Titer-Glo was purchased from Promega Company, the article number is G7573; the cell culture plate was purchased from Corning Company, the article number was 3610.

experimental method:

When the cells are cultured to a suitable degree of confluence, collect the cells, adjust the cells to an appropriate cell concentration using complete medium, spread the cell suspension on a 96-well plate, 90 μL per well, and culture at 37°C, 5% CO ₂ The box was adhered to the wall overnight, and compound solutions of different concentrations were prepared using DMSO and culture medium, and the vehicle control was set, and the compound solution was added to a 96-well plate, 10 μL per well, and placed in a 37°C, 5% CO ₂ incubator to continue culturing After 72 hours, CellTiter-Glo solution was added, shaken and mixed evenly, incubated in the dark for 10 minutes, and read with a BioTek Synergy H1 microplate reader.

Experimental data processing method:

Use the luminescent signal value to calculate the inhibition rate, and use the Graphpad Prism software to perform nonlinear regression curve fitting on the concentration and inhibition rate to obtain the IC ₅₀ value, as shown in the table below:

In Vitro Cytotoxicity of Table 13 Compounds

Experimental results:

From the above results, it can be seen that the compounds shown in the present invention have weaker toxic effects on NFAT-luc2/PD1 Jurkat and MC38-hPDL1 cells.

Test example 6 Balb/C mouse pharmacokinetic determination

1. Research purposes:

Taking Balb/C mice as the experimental animals, the pharmacokinetics of the following compound examples were orally administered in mice at a dose of 5 mg/kg.

2. Test plan

2.1 Test drugs:

Embodiment of the present invention, self-made.

2.2 Experimental animals:

Balb/C Mouse 6/embodiment, male, Shanghai Jiesijie Experimental Animal Co., Ltd., animal production license number (SCXK (Shanghai) 2013-0006 N0.311620400001794).

2.3 Drug preparation:

0.5% HPMC (1% Tween 80), ultrasonically dissolved, prepared as a clear solution or a homogeneous suspension.

2.3 Administration:

Balb/C mice, male; p.o. after fasting overnight, the dose is 5mg/kg, and the administration volume is 10mL/kg.

2.4 Sample collection:

Before and after administration, at 0, 0.5, 1, 2, 4, 6, 8 and 24 hours, 0.1 mL of orbital blood was collected from mice, placed in EDTA-K ₂ test tubes, and centrifuged at 6000 rpm at 4°C for 6 minutes to separate plasma , stored at -80°C.

2.5 Sample handling:

1) Add 40 μL of plasma sample to 160 μL of acetonitrile for precipitation, mix and centrifuge at 3500×g for 5-20 minutes.

2) Take 100 μL of the treated supernatant solution and analyze the concentration of the compound to be tested by LC/MS/MS.

2.6 Liquid phase analysis

●Liquid phase conditions: Shimadzu LC-20AD pump

●Mass spectrometer conditions: AB Sciex API 4000 mass spectrometer

●Chromatographic column: phenomenonex Gemiu 5 μm C18 50×4.6mm

●Mobile phase: liquid A is 0.1% formic acid aqueous solution, liquid B is acetonitrile

●Flow rate: 0.8mL/min

●Eluting time: 0-4.0 minutes, the eluting solution is as follows:

3. Test results and analysis

The main parameters of pharmacokinetics are calculated by WinNonlin 8.2, and the results of drug metabolism experiments in mice are shown in the table below:

4. Experimental conclusion:

It can be seen from the results of drug metabolism experiments in mice in the table that the compounds of the examples of the present invention exhibit good metabolic properties, and both the exposure AUC and the maximum blood drug concentration C _max are good.

Pharmacokinetic determination of test example 7 tumor-bearing nude mouse plasma and tumor

1. Research purposes:

Taking tumor-bearing nude mice as experimental animals, the pharmacokinetic behavior of plasma and tumor in tumor-bearing nude mice was studied by oral administration of the following compound examples at a dose of 30 mg/kg.

2. Test plan

2.1 Test drugs:

Embodiment of the present invention, self-made.

2.2 Experimental animals:

21 tumor-bearing nude mice/example, female, Shanghai Bikai Experimental Animal Co., Ltd., animal production license number (SCXK (Shanghai) 2013-0006 No. 311620400001794).

2.3 Drug preparation:

0.5% HPMC (1% Tween 80), ultrasonically dissolved, prepared as a clear solution or a homogeneous suspension.

2.4 Administration:

Tumor-bearing nude mice, female; p.o. after fasting overnight, the dose is 30mg/kg, and the administration volume is 10mL/kg.

2.5 Sample collection:

Before and after administration of tumor-bearing nude mice, at 0, 1, 2, 4, 6, 8, 16 and 24 hours, 0.1 mL of orbital blood was collected, placed in EDTA-K ₂ test tubes, and centrifuged at 6000 rpm at 4 °C for 6 min Plasma was separated, tumor tissue was weighed after dissection, and stored at -80°C.

2.6 Sample handling:

1) Tumor tissue was homogenized by adding methanol and water at a ratio of 1:3 and then centrifuged to obtain the supernatant. Take 40 μL of the supernatant of the tumor homogenate and plasma samples and add 160 μL of acetonitrile for precipitation. After mixing, centrifuge at 3500×g for 5-20 minutes.

2) Take 100 μL of the treated supernatant solution and analyze the concentration of the compound to be tested by LC/MS/MS.

2.7 Liquid phase analysis

●Liquid phase conditions: Shimadzu LC-20AD pump

●Mass spectrometer conditions: AB Sciex API 4000 mass spectrometer

●Chromatographic column: phenomenonex Gemiu 5 μm C18 50×4.6mm

●Mobile phase: liquid A is 0.1% formic acid aqueous solution, liquid B is acetonitrile

●Flow rate: 0.8mL/min

●Eluting time: 0-4.0 minutes, the eluting solution is as follows:

3. Test results and analysis

The main parameters of pharmacokinetics were calculated by WinNonlin 8.2, and the results of drug metabolism experiments in tumor-bearing nude mice are shown in the table below:

4. Experimental conclusion:

The above data show that the drug concentration of the compound of the embodiment of the present invention in the mouse tumor is higher than the blood drug concentration.

Test example 8 plasma protein binding rate experiment

1. Purpose of the experiment:

The purpose of this experimental method is to detect the plasma protein binding of the compounds of the examples in plasma.

2. Experimental instruments and materials:

Ultra-high performance liquid phase tandem mass spectrometry coupled instrument, refrigerated centrifuge, vortex instrument, electric heating constant temperature oscillating water tank, pipette, continuous liquid adder, 96-well plate, tissue homogenizer (used for tissue sample analysis), Add internal standard acetonitrile solution, blank matrix (plasma, urine or tissue homogenate, etc.)

3. Experimental steps:

3.1 Preparation of plasma

Thaw the frozen plasma at room temperature or in a water bath at 37°C, centrifuge at 3500rpm for 5min, and take the supernatant.

3.2 Preparation of reaction termination solution

Use acetonitrile containing an internal standard as a stop solution and store in a refrigerator at 2-8°C.

3.3 Preparation of compound working solutions

Compound working solution preparation: dilute the stock solution with DMSO to a final concentration of 1 mM.

3.4 Preparation of plasma solution

Take 2.5 μL compound working solution and add it to 497.5 μL blank plasma, the final concentration is 5 μM , shake and mix.

3.5 Equilibrium dialysis

1) Prepare the equilibrium dialysis device, and put the detection membrane device into the equilibrium dialysis 96-well plate;

2) Add 200 μL of prepared plasma solution into the membrane, n=2;

3) Take another 5 μL of plasma solution, dilute it 10 times with 45 μL of the same species of blank plasma, add 200 μL of acetonitrile stop solution containing internal standard, and store it in a -20°C refrigerator;

4) Add 350 μ L of dialysate (100 mM phosphate buffer) outside the membrane;

5) Seal the dialysis plate and place it in a water bath at 37°C for 5 hours;

6) After the dialysis, 5 μL was taken out from the sample holes inside the membrane, and diluted 10 times with 45 μL of blank plasma of the same species; 50 μL of the dialysate was taken out from the sample holes outside the membrane, and 200 μL of Standard acetonitrile stop solution;

7) Take the supernatant after the sample is centrifuged;

8) LC-MS analysis.

4. Chromatographic conditions:

Instrument: Shimadzu LC-30 AD;

Chromatographic column: XBridge® C18 (50*4.6mm, 5 μm particle size);

Mobile phase: A: 0.1% formic acid solution, B: methanol;

Washing gradient: 0.2~1.6min 5%A to 95%A, 3.0~3.1min 95%A to 5%A;

Execution time: 4.0min.

5. Mass spectrometry conditions:

Instrument: API5500 liquid chromatography mass spectrometry, AB Sciex, USA;

Ion source: electrospray ionization source (ESI);

Drying gas: N ₂ , temperature 500°C;

Electrospray voltage: 5000V;

Detection method: positive ion detection;

Scanning method: select the reaction monitoring (MRM) method.

6. Experimental results: as shown in Table 16:

7. Experimental conclusion:

The above data show that the compounds of the examples of the present invention exhibit a high plasma protein binding rate.

Test Example 9 Tumor inhibition experiment on MC38-hPDL1 xenograft tumor model

1. Purpose of the experiment:

To evaluate the antitumor activity of the test compound on MC38-hPDL1 cell subcutaneous transplanted tumor of C57 mice.

2. Experimental instruments and reagents:

2.1 Instruments:

Ultra-clean workbench (BSC-1300II A2, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory);

_CO incubator (311, Thermo);

Centrifuge (Centrifuge 5720R, Eppendorf);

Automatic cell counter (Countess II, Life);

Pipette (10-20 μL , Eppendorf);

Microscope (TS100, Nikon);

Vernier caliper (500-196, Mitutoyo, Japan);

Cell culture flasks (T25/T75/T225, Corning).

2.2 Reagents:

DMEM (11995-065, Gibco);

Fetal bovine serum (FBS) (10099-141, Gibco);

Phosphate buffered saline (PBS) (10010-023, Gibco).

2.3 Test compounds:

The compound of the embodiment of the present invention is self-made.

3. Experimental operation:

Take out MC38-hPDL1 cells from the cell bank, add them to DMEM medium (DMEM+10%FBS) after recovery, and culture them in a CO ₂ incubator (the temperature of the incubator is 37°C, and the concentration of CO ₂ is 5%). MC38-hPDL1 cells were harvested when expanded to the desired number for inoculation in vivo. Count with an automatic cell counter, and resuspend the cells with PBS according to the counting results to make a cell suspension (density 1×10 ⁶ /mL), and place in an ice box until use.

Use 6-8 week old female C57 mice weighing approximately 18-22 g. Mice were raised in SPF grade animal room, and raised in single cages, with 4-5 mice per cage. All cages, litter and water were sterilized before use, and all animals had free access to food and water. Before the experiment started, the C57 mice were marked with a disposable ear tag for rats and rats, and the skin of the inoculation site was disinfected with 75% alcohol before inoculation, and each mouse was subcutaneously inoculated with 0.1 mL (containing 1*10 ⁵ cells) of MC38-hPDL1 on the right back. cell. When the tumor volume reached 40-180mm ³ , group administration began, with 8 rats in each group. Each test compound was orally administered twice a day for 14 days. The tumor volume was measured and the body weight of the mice was weighed twice a week, and the tumor TGI (%) was calculated.

4. Data processing:

Tumor volume (mm ³ ), the calculation formula is: V=0.5*D*d*d, where D and d are the long and short diameters of the tumor, respectively.

Calculation of TGI(%):

When the tumor does not regress, TGI (%)=[(1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group))/(average tumor volume at the end of treatment of the solvent control group - average tumor volume at the beginning of treatment in the solvent control group)] × 100%;

When the tumor has regressed, TGI (%)=[1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group)/average tumor volume at the beginning of administration of this treatment group]× 100%.

5. Experimental results:

The test results are shown in Table 17 below:

6. Test conclusion

The above data show that the compounds of the examples of the present invention have a strong inhibitory effect on MC38-hPDL1 cell subcutaneous transplanted tumors in C57 mice.

Test Example 10 Example 4 Compound SD rats repeated gavage toxicity test for 14 days

1. Purpose of the experiment

The purpose of this study is to investigate the possible toxic reaction of the compound of Example 4 given to SD rats after repeated gavage for 14 days.

2. Experimental Materials and Instruments

2.1 Test article

Test article: N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methoxypyridine- 2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H- Imidazo[4,5-c]pyridine-2-carboxamide (compound of Example 4)

2.2 Solvent

Name: 0.5% (w/v) HPMC K4M aqueous solution containing 1% (v/v) Tween 80

2.3 Animal Information

Species & strain: SD rats

Animal grade: SPF grade

Number and sex of animals: 8 males and 8 females/group

2.4 Instruments

ADVIA®2120 series blood analyzer, Sysmex CS-2000i automatic coagulation analyzer, TBA-120FR automatic biochemical analyzer.

3. Experimental method

Repeated intragastric administration of the compound of Example 4 to SD rats was administered once a day for 14 consecutive days, and the rats were regularly observed for clinical observation, body weight, food consumption, clinical pathology, toxicology, metabolic kinetics, general and histopathology. and other indicators were detected to investigate the possible toxic reaction and metabolism of rats after continuous administration of the compound of Example 4 for 14 days.

4. Test data

4.1 Dying/Death

There was no death/moribundity of animals in the 15, 50, 100 mg/kg dose groups.

4.2 Toxicokinetics

Under 15,50,100mg/kg dosage, the average exposure amount (AUC _last ) of the compound of Example 4 in the animal body all increases with dosage; There was no significant gender difference in the exposure of the compound of Example 4 in animals. After 14 days of repeated administration, compared with the first administration, there was no significant accumulation of the compound of Example 4 in the animals of each dose group.

4.3 Clinical observation

1/5 male animals in the 100mg/kg dose group occasionally had soft stools, and no abnormalities were found in the 15 and 50mg/kg dose groups.

4.4 Body weight and food intake

100mg/kg dose group W1 and W2 body weight growth slowed down, food intake decreased, 50mg/kg dose group female animals W2 body weight growth slowed down; 15mg/kg dose group no abnormalities.

4.5 Blood cell count and coagulation function

100mg/kg dose group Neut, FIB increased; 15 and 50mg/kg dose group female animals FIB increased°

4.6 Blood biochemistry

100mg/kg dose group Alb decreased; 15, 50mg/kg dose groups no abnormalities.

4.7 Pathology

Animals in the 15, 50, and 100 mg/kg dose groups had no abnormalities in general observation, and no histopathological examination was performed.

5. Experimental conclusion

The compound of Example 4 was administered to SD rats repeatedly by intragastric administration at doses of 15, 50, and 100 mg/kg, once a day for 14 consecutive days, and the maximum tolerated dose (MTD) was 100 mg/kg.

Crystal Form Study of Compounds

Those of ordinary skill in the art are well aware that the compounds of the above examples have been proven to have good inhibitory activity on the CHO-PDL1/Jurkat-PD1 cell pathway, and their crystal forms often have the same pharmacological activity. On this basis, the inventors further studied the physical and chemical properties of the corresponding compound crystal forms, but the preparation and characterization of the following specific crystal forms do not represent a limitation to the scope of protection of the present invention, and those of ordinary skill in the art can use the present invention as a basis , Obtain more crystals of the compound of the present invention by means of conventional crystallization, and these crystals are the schemes protected by the present invention. details as follows:

1. Experimental equipment

1.1 Some parameters of physical and chemical detection instruments

1.2 Instruments and liquid phase analysis conditions

2. The preparation of embodiment 4 compound free alkali crystal form A～D

2.1 Preparation of free base crystal form A

Add the crude product DCM concentrate and isopropanol (5V), control the temperature at 40-45°C and continue to concentrate to 1-2V. Repeat this operation once. Add isopropanol to 5-6V, keep stirring at 40-45°C for 10-20min. Keep the temperature at 35-45°C for hot filtration and rinse with isopropanol (1-2V). Control the temperature at 40-45°C, concentrate the filtrate to 5V, stir for 10-30min, and the system is clarified. Slowly drop to 15-20°C, and the cooling time is about 1-2 hours (natural cooling is also acceptable). Control the temperature and stir for 16-20 hours, and it can be observed that a solid gradually precipitates out. Filter and rinse the filter cake with isopropanol (1-2V) to obtain a light brown solid. The oil pump is drained to obtain the product solid. The obtained solid detection XRPD is Form A. After detection and analysis, it has an XRPD pattern as shown in FIG. 1 and a DSC pattern as shown in FIG. 2 .

2.2 Preparation of free base crystal form B

Add the crude product DCM concentrate (obtained by reacting 28g of starting material) and absolute ethanol (5V), control the temperature at 40-45°C and continue to concentrate to 1-2V. Repeat this operation once. Add absolute ethanol to 5-6V, keep stirring at 40-45°C for 10-20min. Keep the temperature at 35-45°C for hot filtration and rinse with absolute ethanol (1-2V). Control the temperature at 40-45°C, concentrate the filtrate to 3V, stir for 10-30min, the system is basically clear; control the temperature at 40-45°C, add methyl tertiary butyl ether (112ml, 4V) dropwise (note: the rate of addition should not be too fast , otherwise it is easy to form an oily substance) about half an hour after dropping. Keep warm for 20-30 minutes; slowly drop to 15-20°C, and the cooling time is about 1-2 hours (natural cooling is also acceptable). Control the temperature and stir for 16-20 hours, and it can be observed that a solid gradually precipitates out. Filter and rinse the filter cake with MTBE:EtOH=2:1 (1-2V) to obtain a light brown solid. The obtained solid detection XRPD is crystal form B. After detection and analysis, it has an XRPD pattern as shown in FIG. 3 and a DSC pattern as shown in FIG. 4 .

2.3 Preparation of free base crystal form C

Weigh about 50 mg of free base B, add 1 mL of ethyl formate to dissolve at room temperature, add 1 mL of cyclohexane at room temperature to become cloudy and oily, stir at room temperature until solid precipitates. Finally, the solid was centrifuged quickly, the supernatant was removed, and vacuum-dried at 40° C., the obtained solid was found to be crystal form C by XRPD. After detection and analysis, it has an XRPD pattern as shown in FIG. 5 and a DSC pattern as shown in FIG. 6 .

2.4 Preparation of free base crystal form D

Weigh about 10mg of free base crystal form C, add 0.1mL ethyl acetate and beat at 40°C for 2 hours, then beat at room temperature for 12 hours, and the final solid obtained is crystal form D by XRPD. After detection and analysis, it has an XRPD pattern as shown in FIG. 7 and a DSC pattern as shown in FIG. 8 .

3. Stable crystal form confirmation experiment

3.1 Purpose of the experiment:

The relatively stable crystal form of the compound of Example 4 was found through the crystal form beating experiment and the stability investigation experiment.

3.2 Experimental scheme:

Choose an organic solvent and water with a certain solubility, suspend different crystal forms in the solvent system, stir and beat at a certain temperature, and finally treat the solid, measure the XRPD of the solid and compare them.

3.3 Experimental results:

Weigh about 20mg sample of free base crystal form A, add 0.1mL solvent, and beat at room temperature. The specific results are shown in Table 18 below.

Table 18 Confirmation experiment results of free base stable crystal form

Experimental conclusion: From the DSC data, it can be seen that the crystal form B is the most stable crystal form.

4. Solid stability experiment

4.1 Purpose of the experiment:

Investigate the physical and chemical stability of the compound free base crystal form B of Example 4 under the conditions of high temperature (60°C), room temperature and high humidity (RH=92.5%) and high temperature and high humidity (50°C, RH=75%) It provides a basis for crystal form screening and compound storage.

4.2 Experimental scheme:

In the experiment, about 5mg of free base crystal form B was weighed into a 20mL vial, and placed in high temperature (60°C), room temperature and high humidity (RH=92.5%) and high temperature and high humidity (50°C, RH=75%) three times. Under these conditions, samples were taken at 5 days, 10 days, 20 days, and 30 days, and the content was determined by the HPLC external standard method, and the changes of related substances were calculated by the chromatographic peak area normalization method.

4.3 Experimental results:

The solid stability test results of the free base crystal form B of the compound of Example 4 are shown in Table 19 below:

Table 19 Solid stability test results of free base crystal form B

Experimental conclusion: Free base crystal form B has good stability under high temperature and high humidity conditions, and the maximum simplex increase is less than 0.1%.

5. Moisture test

5.1 Purpose of the experiment

The hygroscopicity of the free base crystal form B of the compound of Example 4 was investigated under different relative humidity conditions to provide a basis for the screening and storage of the compound crystal form.

5.2 Experimental scheme:

The free base crystal form B of the compound is placed in saturated water vapor with different relative humidity, so that the compound and the water vapor reach a dynamic equilibrium, and the percentage of the weight gain of the compound after the equilibrium is calculated.

5.3 Experimental results:

The free base crystal form B has a hygroscopic weight gain of about 0.815% under the condition of RH80%, which is slightly hygroscopic. After one cycle of moisture absorption and desorption under the condition of 0-95% relative humidity, the XRPD spectrum of the free base crystal form B has not changed, that is, the crystal form B has not been transformed.

6. Solubility experiments in different media

6.1 Purpose of experiment

Comparing the solubility of the free base crystal form B of the compound of Example 4 in pH 1-pH 8 buffer solution, water, artificial analogous gastric fluid (SGF), fasting artificial analogous intestinal fluid (FaSSIF) and non-fasting artificial analogous intestinal fluid (FeSSIF) and other media The size provides a basis for the assessment of druggability.

6.2 Experimental scheme:

About 2 mg of free base crystal form B was suspended in different media for 2 hours, and the thermodynamic solubility of the compound at 37°C was determined by HPLC and external standard method.

6.3 Experimental results:

The solubility test results in different media are shown in Table 20 below:

Table 20 Solubility test results in different media of free base crystal form B

Experimental conclusion: Free base crystal form B has good solubility in the pH range and environment of the entire gastrointestinal tract.

Claims (16)

A crystal form of the compound shown in general formula (I), the compound structure is as follows:

in: L is selected from the key,

or

; Ring A is selected from 4-8 membered heterocyclic groups; preferably 4-membered, 7-membered or 8-membered heterocyclic groups; More preferably the following groups:

_{R is} independently selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, carboxyl, aldehyde, pendant oxy, C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl , C _1-6 alkoxy, C _1-6 hydroxyalkyl, C _3-8 cycloalkyl, -(CH ₂ ) _n R _a , -(CH ₂ ) _n OR _a , -OC(O)R _a , -C(O)R _a , -C(O)OR _a , -C(O)NR _a R _b , -(CH ₂ ) _n NR _a C(O)R _b or -SO ₂ R _a , wherein the C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 hydroxyalkyl and C _3-8 cycloalkyl, as required Further substituted by one or more substituents in deuterium, halogen, hydroxyl, cyano or C _1-6 alkyl; R _a and R _b are each independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, carboxyl, C _1-6 alkyl, C _1-6 deuterated alkyl, C _{1-6 6} haloalkyl or C _{3-8 cycloalkyl} , wherein the C _1-6 alkyl, C _1-6 deuterated alkyl, C _1-6 haloalkyl and C _3-8 cycloalkyl, if necessary further Substituted by one or more substituents in deuterium, halogen, hydroxyl, cyano or C _1-6 alkyl; n is 0, 1 or 2; and x is 0, 1 or 2. The crystal form of the compound as claimed in item 1, wherein the structure of the compound is shown in general formula (II):

in: M is O, -NR ₂ or -CR ₃ R ₄ ; R _is selected from hydrogen, deuterium, aldehyde, C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 haloalkyl, C _1-3 alkoxy, C _1-3 hydroxyalkyl , C _3-6 cycloalkyl, -(CH ₂ ) _n R _a , -(CH ₂ ) _n OR _a , -OC(O)R _a , -C(O)R _a , -C(O)OR _a , -C(O)NR _a R _b , -(CH ₂ ) _n NR _a C(O)R _b or -SO ₂ R _a ; R ₃ and R ₄ are each independently selected from hydrogen, deuterium, halogen, hydroxyl, cyano, carboxyl, aldehyde, side oxygen, C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 Haloalkyl, C _1-3 alkoxy, C _1-3 hydroxyalkyl, C _3-6 cycloalkyl, -(CH ₂ ) _n R _a , -(CH ₂ ) _n OR _a , -OC(O )R _a , -C(O)R _a , -C(O)OR _a , -C(O)NR _a R _b , -(CH ₂ ) _n NR _a C(O)R _b or -SO ₂ R _a , wherein the C _1-3 alkyl, C _1-3 deuterated alkyl, C _1-3 haloalkyl, C _1-3 alkoxy, C _1-3 hydroxyalkyl and C _3-6 cycloalkyl , if necessary, further substituted by one or more substituents in deuterium, halogen, hydroxyl, cyano or C _1-3 alkyl; R _a and R _b are each independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, carboxyl, C _1-3 alkyl, C _1-3 deuterated alkyl, C _{1-3 3} haloalkyl or C _3-6 cycloalkyl, wherein the C _1-3 alkyl, C _1-3 deuterated alkyl _, C _1-3 haloalkyl and C _3-6 cycloalkyl, if necessary further Substituted by one or more substituents in deuterium, halogen, hydroxyl, cyano or C _1-3 alkyl; n is 0, 1 or 2. The crystal form of the compound as claimed in item 1 or 2, wherein the compound is: N-(2,2'-dichloro-3'-(6-methoxy-5-((oxetan-3-ylamino)methyl)pyridin-2-yl)-[1,1' -biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(5-((3-fluoroazetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-[1,1 '-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide; 1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-carbazamidylamino<acetylamino>)-[1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)azetine Pyridine-3-yl acetate; N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.3]heptan-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide; N-(3'-(5-((2-Oxa-6-azaspiro[3.3]heptane-6-yl)methyl)-6-methoxypyridin-2-yl)-2,2 '-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c ]pyridine-2-carboxamide; N-(3'-(5-((5-oxa-2-azaspiro[3.4]octane-2-yl)methyl)-6-methoxypyridin-2-yl)-2,2 '-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c ]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(6-methoxy-5-((6-carbonyl-2,5-diazaspiro[3.4]octane-2-yl)methyl) Pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c] pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(5-((3-hydroxyazetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-[1,1 '-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(5-((3-hydroxy-3-methylazetidin-1-yl)methyl)-6-methoxypyridin-2-yl) -[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2 - formamide; 1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-formamido)-[1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)azetidine-3-carboxylic acid; (S)-1-((6-(2,2'-dichloro-3'-(1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c]pyridine-2-carbazoylamino<glycolamide>)-[1,1'-biphenyl]-3-yl)-2-methoxypyridin-3-yl)methanol Base) azetidine-2-carboxylic acid; (S)-N-(2,2'-dichloro-3'-(5-((2-(hydroxymethyl)azetidin-1-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(5-((3-hydroxy-3-(hydroxymethyl)azetidin-1-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide; N-(3'-(5-((3-Acetamidoazetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-2,2'-dichloro-[ 1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-methanol Amide; N-(3'-(5-((3-(Acetamidomethyl)azetidin-1-yl)methyl)-6-methoxypyridin-2-yl)-2,2'- Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-Formamide; N-(2,2'-dichloro-3'-(5-((3-(cyanomethyl)azetidin-1-yl)methyl)-6-methoxypyridin-2-yl) -[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2 - formamide; N-(2,2'-dichloro-3'-(5-((3-(dimethylaminoformyl)azetidin-1-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(6-methoxy-5-((3-(methylaminoformyl)azetidin-1-yl)methyl)pyridine-2- base)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine -2-Formamide; N-(2,2'-dichloro-3'-(5-((6-hydroxy-2-azaspiro[3.3]heptane-2-yl)methyl)-6-methoxypyridine-2 -yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] Pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(5-((6-isobutyryl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(5-((6-(cyclopropylcarbonyl)-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6 -Methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(6-methoxy-5-((6-propionyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl )pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4, 5-c]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(6-methoxy-5-((6-(2,2,2-trifluoroacetyl)-2,6-diazaspiro[ 3.3] Heptane-2-yl)methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7 - Tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(5-((6-(2,2-difluoroacetyl)-2,6-diazaspiro[3.3]heptan-2-yl )methyl)-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetra Hydrogen-1H-imidazo[4,5-c]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(5-((6-(2-cyanoacetyl)-2,6-diazaspiro[3.3]heptane-2-yl)methanol Base)-6-methoxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro- 1H-imidazo[4,5-c]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(6-methoxy-5-((6-(methylsulfonyl)-2,6-diazaspiro[3.3]heptane-2-yl )methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(5-((6-formyl-2,6-diazaspiro[3.3]heptane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide; N-(3'-(5-((7-acetyl-2,7-diazaspiro[3.5]nonan-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide N-(3'-(5-((6-acetyl-2,6-diazaspiro[3.4]octane-2-yl)methyl)-6-methoxypyridin-2-yl) -2,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide; N-(2,2'-dichloro-3'-(5-((6-isobutyryl-2,6-diazaspiro[3.4]octane-2-yl)methyl)-6-methanol Oxypyridin-2-yl)-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4 ,5-c]pyridine-2-carboxamide; or N-(3'-(5-((6-Acetamido-2-azaspiro[3.3]heptane-2-yl)methyl)-6-methoxypyridin-2-yl)-2 ,2'-Dichloro-[1,1'-biphenyl]-3-yl)-1,5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5 -c] pyridine-2-carboxamide. The crystal form of the compound as described in any one of claims 1 to 3, wherein the crystal form is N-(3'-(5-((6-acetyl-2,6-diazaspiro[ 3.3] Heptane-2-yl)methyl)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1 , Crystal forms A~D of 5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide; The X-ray powder diffraction pattern of the crystal form A has a diffraction peak at 2θ of 7.8±0.2°; or has a diffraction peak at 14.1±0.2°; or has a diffraction peak at 15.3±0.2°; or has a diffraction peak at 19.5±0.2°; Diffraction peak at 0.2°; or diffraction peak at 12.0±0.2°; or diffraction peak at 12.4±0.2°; or diffraction peak at 13.2±0.2°; or diffraction peak at 14.6±0.2° or have a diffraction peak at 21.2±0.2°; or have a diffraction peak at 22.0±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them; The X-ray powder diffraction pattern of the crystal form B has a diffraction peak at 2θ of 4.2±0.2°; or has a diffraction peak at 12.2±0.2°; or has a diffraction peak at 12.6±0.2°; or has a diffraction peak at 16.5±0.2°; Diffraction peak at 0.2°; or diffraction peak at 16.8±0.2°; or diffraction peak at 18.4±0.2°; or diffraction peak at 18.9±0.2°; or diffraction peak at 21.1±0.2° or have a diffraction peak at 22.2±0.2°; or have a diffraction peak at 22.4±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them; The X-ray powder diffraction pattern of the crystal form C has a diffraction peak at 2θ of 11.4±0.2°; or has a diffraction peak at 12.5±0.2°; or has a diffraction peak at 21.1±0.2°; or has a diffraction peak at 23.0±0.2°; Diffraction peak at 0.2°; or diffraction peak at 26.1±0.2°; or diffraction peak at 26.6±0.2°; or diffraction peak at 13.0±0.2°; or diffraction peak at 14.0±0.2° or have a diffraction peak at 14.7±0.2°; or have a diffraction peak at 15.8±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them; The X-ray powder diffraction pattern of the crystal form D has a diffraction peak at 9.5±0.2° at 2θ ; or has a diffraction peak at 10.4±0.2°; or has a diffraction peak at 14.5±0.2°; or has a diffraction peak at 19.9±0.2°; Diffraction peak at 0.2°; or diffraction peak at 20.8±0.2°; or diffraction peak at 24.8±0.2°; or diffraction peak at 11.7±0.2°; or diffraction peak at 13.3±0.2° or have a diffraction peak at 17.2±0.2°; or have a diffraction peak at 23.8±0.2°; preferably include any 2-5, or 3-5, or 3-6 of the above-mentioned diffraction peaks, Or 3-8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them. The crystal form of the compound as claimed in item 4, wherein, The X-ray powder diffraction pattern of crystal form A contains at least one or more diffraction peaks at 2θ of 7.8±0.2°, 14.1±0.2°, and 15.3±0.2°, preferably two of them, more preferably In order to include three; if necessary, it can further include at least one of 19.5±0.2°, 12.0±0.2°, 12.4±0.2°, 13.2±0.2°, 14.6±0.2°, preferably including 2, 3 strips, 4 strips or 5 strips; The X-ray powder diffraction pattern of crystal form B contains at least one or more diffraction peaks at 2θ of 4.2±0.2°, 12.2±0.2°, and 12.6±0.2°, preferably two of them, more preferably In order to include three; as required, it can further include at least one of 16.5±0.2°, 16.8±0.2°, 18.4±0.2°, 18.9±0.2°, 21.1±0.2°, preferably including 2, 3 strips, 4 strips or 5 strips; The X-ray powder diffraction pattern of Form C contains at least one or more diffraction peaks located at 2θ of 11.4±0.2°, 12.5±0.2°, and 21.1±0.2°, preferably including two of them, more preferably In order to include three; as required, it can further include at least one of 23.0±0.2°, 26.1±0.2°, 26.6±0.2°, 13.0±0.2°, 14.0±0.2°, preferably including 2, 3 strips, 4 strips or 5 strips; The X-ray powder diffraction pattern of crystal form D contains at least one or more diffraction peaks at 2θ of 9.5±0.2°, 10.4±0.2°, 14.5±0.2°, preferably two of them, more preferably In order to include three; if necessary, it can further include at least one of 19.9±0.2°, 20.8±0.2°, 24.8±0.2°, 11.7±0.2°, 13.3±0.2°, preferably including 2, 3 strips, 4 strips or 5 strips. The crystal form of the compound as claimed in item 5, wherein, The X-ray powder diffraction pattern of the crystal form A may also include, if necessary, the 2θ at 21.2±0.2°, 22.0±0.2°, 22.6±0.2°, 25.0±0.2°, 28.3±0.2°, 16.7±0.2° or 24.5± One or more diffraction peaks at 0.2°; preferably at least including any 2-3, or 4-5, or 6-7; more preferably, including any 2, 3, 4, 5, 6, 7; The X-ray powder diffraction pattern of the crystal form B may optionally also include 2θ at 22.2±0.2°, 22.4±0.2°, 22.9±0.2°, 23.6±0.2°, 26.6±0.2°, 13.8±0.2° or 14.5± One or more diffraction peaks at 0.2°; preferably at least including any 2-3, or 4-5, or 6-7; more preferably, including any 2, 3, 4, 5, 6, 7; The X-ray powder diffraction pattern of the crystal form C may also include 14.7±0.2°, 15.8±0.2°, 18.0±0.2°, 19.8±0.2°, 23.6±0.2°, 9.1±0.2° or 16.7±0.2° in 2θ . One or more diffraction peaks at 0.2°; preferably at least including any 2-3, or 4-5, or 6-7; more preferably, including any 2, 3, 4, 5, 6, 7; The X-ray powder diffraction pattern of the crystal form D may also include, if necessary , 17.2±0.2°, 23.8±0.2°, 25.5±0.2°, 27.1±0.2°, 15.3±0.2°, 15.8±0.2° or 21.4± One or more diffraction peaks at 0.2°; preferably at least including any 2-3, or 4-5, or 6-7; more preferably, including any 2, 3, 4, 5, 6, 7. The crystal form of the compound as claimed in item 4, wherein, The X-ray powder diffraction pattern of the crystal form A may optionally include 7.8±0.2°, 14.1±0.2°, 15.3±0.2°, 19.5±0.2°, 12.0±0.2°, 12.4±0.2°, 13.2±0.2 °, 14.6±0.2°, 21.2±0.2°, 22.0±0.2°, 22.6±0.2°, 25.0±0.2°, 28.3±0.2°, 16.7±0.2° or 24.5±0.2° at one or more diffractions peak, Preferably, there are diffraction peaks at 4, 5, 6, 8 or 10 places as required; The X-ray powder diffraction pattern of the crystal form B optionally includes 4.2±0.2°, 12.2±0.2°, 12.6±0.2°, 16.5±0.2°, 16.8±0.2°, 18.4±0.2°, 18.9±0.2 °, 21.1±0.2°, 22.2±0.2°, 22.4±0.2°, 22.9±0.2°, 23.6±0.2°, 26.6±0.2°, 13.8±0.2° or 14.5±0.2° at one or more diffractions peak, Preferably, there are diffraction peaks at 4, 5, 6, 8 or 10 places as required; The X-ray powder diffraction pattern of the crystal form C may optionally include 11.4±0.2°, 12.5±0.2°, 21.1±0.2°, 23.0±0.2°, 26.1±0.2°, 26.6±0.2°, 13.0±0.2 One or more diffractions at °, 14.0±0.2°, 14.7±0.2°, 15.8±0.2°, 18.0±0.2°, 19.8±0.2°, 23.6±0.2°, 9.1±0.2° or 16.7±0.2° peak, Preferably, there are diffraction peaks at 4, 5, 6, 8 or 10 places as required; The X-ray powder diffraction pattern of the crystal form D optionally includes 9.5±0.2°, 10.4±0.2°, 14.5±0.2°, 19.9±0.2°, 20.8±0.2°, 24.8±0.2°, 11.7±0.2 °, 13.3±0.2°, 17.2±0.2°, 23.8±0.2°, 25.5±0.2°, 27.1±0.2°, 15.3±0.2°, 15.8±0.2° or 21.4±0.2° at one or more diffractions peak, Preferably, there are diffraction peaks at 4, 5, 6, 8 or 10 places as required. The crystal form of the compound as described in any one of claims 4 to 7, wherein, Form A has an X-ray powder diffraction pattern as shown in Figure 1, or has a DSC pattern as shown in Figure 2; Form B has an X-ray powder diffraction pattern as shown in Figure 3, or has a DSC pattern as shown in Figure 4; Form C has an X-ray powder diffraction spectrum as shown in Figure 5, or has a DSC spectrum as shown in Figure 6; Form D has an X-ray powder diffraction pattern as shown in FIG. 7 , or a DSC pattern as shown in FIG. 8 . The crystal form of the compound as described in any one of claims 1 to 3, wherein the crystal form is N-(3'-(5-((6-acetyl-2,6-diazaspiro[ 3.3] Heptane-2-yl)methyl)-6-methoxypyridin-2-yl)-2,2'-dichloro-[1,1'-biphenyl]-3-yl)-1 , crystalline form B of 5-dimethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-2-carboxamide; The X-ray powder diffraction pattern of the crystal form B has a diffraction peak at 4.0±0.2°; or has a diffraction peak at 12.0±0.2°; or has a diffraction peak at 12.4±0.2°; or has a diffraction peak at 16.4±0.2° Have a diffraction peak; or have a diffraction peak at 16.6±0.2°; or have a diffraction peak at 18.2±0.2°; or have a diffraction peak at 18.7±0.2°; or have a diffraction peak at 20.9±0.2°; or Have a diffraction peak at 22.0±0.2°; or have a diffraction peak at 22.3±0.2°; preferably include any 2-5, or 3-5, or 3-6, or 3- 8, or 5-8, or 6-8, more preferably including any 6, 7 or 8 of them; Preferably, the X-ray powder diffraction pattern of the crystal form B includes at least one or more diffraction peaks at 2θ of 4.0±0.2°, 12.0±0.2°, and 12.4±0.2°, preferably including Two, preferably three; if necessary, at least one of 16.4±0.2°, 16.6±0.2°, 18.2±0.2°, 18.7±0.2°, 20.9±0.2° can be included, preferably including 2, 3, 4 or 5 of them; More preferably, the X-ray powder diffraction pattern of the crystalline form B may also include 2θ at 22.0±0.2°, 22.3±0.2°, 22.7±0.2°, 23.5±0.2°, 26.5±0.2°, 13.6± One or more diffraction peaks at 0.2° or 14.3±0.2°; preferably at least including any 2-3, or 4-5, or 6-7; more preferably, including any 2 place, 3 places, 4 places, 5 places, 6 places, 7 places; Further preferably, the X-ray powder diffraction pattern of the crystalline form B may optionally include 4.0±0.2°, 12.0±0.2°, 12.4±0.2°, 16.4±0.2°, 16.6±0.2, 18.2±0.2 °, 18.7±0.2°, 20.9±0.2°, 22.0±0.2°, 22.3±0.2°, 22.7±0.2°, 23.5±0.2°, 26.5±0.2°, 13.6±0.2° or 14.3±0.2° One or more diffraction peaks, preferably, including 4, 5, 6, 8 or 10 diffraction peaks as required; Even more preferably, the X-ray powder diffraction pattern of the crystal form B is shown in FIG. 10 . The crystal form of the compound as described in any one of claims 4 to 9, wherein, In the X-ray powder diffraction pattern of crystal form A, crystal form B, crystal form C and crystal form D, the relative peak intensity is the top ten diffraction peak positions and the corresponding positions of the diffraction peaks in Figure 1, Figure 3, Figure 5 and Figure 7 The 2θ error is ±0.2°~±0.5°; preferably ±0.2°~±0.3°; optimally ±0.2°. The crystal form of the compound according to any one of claims 1 to 10, wherein the crystal form is an anhydrate. The crystal form of the compound as described in any one of claims 1 to 11, wherein the crystal form is a hydrate, and the number of water is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3 ; more preferably 0.5, 1, 2 or 3. A method for preparing the crystal form of the compound as described in any one of claims 1 to 12, specifically comprising the following steps: 1) Weigh an appropriate amount of free base, and dissolve it in a benign solvent at a certain temperature, preferably 0-50°C; 2) If necessary, add anti-solvent to the solution obtained above, and stir until solids are precipitated; 3) If necessary, stirring, cooling and crystallization to obtain the target product; or 1) Weighing an appropriate amount of free alkali or its crystal form, beating with a poor solvent at a certain temperature, preferably 0-50°C; in: The good solvent is selected from methanol, acetone, ethyl formate, ethyl acetate, acetonitrile, ethanol, 88% acetone, tetrahydrofuran, dichloromethane, 1,4-dioxane, benzene, toluene, isopropanol, n-butanol , isobutanol, N,N-dimethylformamide, N,N-dimethylacetamide, n-propanol, tertiary butanol, 2-butanone or 3-pentanone or one or more ; Preferably one or more of ethyl formate, isopropanol or absolute ethanol; The anti-solvent is selected from one or more of heptane, cyclohexane, n-hexane, n-pentane, water, ethyl acetate, methyl tertiary butyl ether, toluene or isopropyl ether; preferably ethyl acetate One or more of , methyl tertiary butyl ether or cyclohexane; The poor solvent is selected from one or more of heptane, cyclohexane, n-hexane, n-pentane, water, ethyl acetate, methyl tertiary butyl ether, toluene or isopropyl ether; preferably ethyl acetate , one or more of methyl tertiary butyl ether or cyclohexane. A pharmaceutical composition, which contains a therapeutically effective amount of the crystal form of the compound described in any one of claims 1 to 12, and one or more pharmaceutically acceptable carriers, diluents or excipients. Application of a crystal form of the compound as described in any one of claims 1 to 12 or a pharmaceutical composition as described in claim 14 in the preparation of PD-1/PD-L1 inhibitor drugs. Application of a crystal form of the compound as described in any one of claims 1 to 12 or a pharmaceutical composition as described in claim 14 in the preparation of drugs for treating diseases selected from cancer, infectious diseases, and autoimmune diseases; Wherein the cancer is selected from skin cancer, lung cancer, urological tumor, blood tumor, breast cancer Carcinoma, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor, head and neck cancer; the infectious disease is selected from bacterial infection or viral infection; the autoimmune disease is selected from organ-specific autoimmune disease or systemic autoimmune disease, wherein the organ-specific autoimmune disease includes chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis , pulmonary hemorrhagic nephritic syndrome, primary biliary cirrhosis, multiple sclerosis or acute idiopathic polyneuritis, systemic autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, systemic vascular scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, or autoimmune hemolytic anemia.

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