CN116041277A - Phenyl and biphenyl substituted five-membered heterocyclic compound, preparation method, pharmaceutical composition and application thereof - Google Patents

Abstract

The invention discloses a phenyl and biphenyl substituted five-membered heterocyclic compound, a preparation method, a pharmaceutical composition and application thereof. The structure of the phenyl and biphenyl substituted five-membered heterocyclic compounds is shown as a formula I, and the compounds also comprise stereoisomers, meso forms, racemates, prodrugs, crystals, pharmaceutically acceptable salts or mixtures thereof. The compounds have PD-L1 inhibition activityThe peptide can obviously inhibit PD-1/PD-L1 protein-protein interaction and block PD-1/PD-L1 signal paths, so that the peptide can be used for preparing immunomodulator medicines for preventing and/or treating tumors, infectious diseases, inflammatory diseases, autoimmune diseases and organ transplant rejection.

Description

Phenyl and biphenyl substituted five-membered heterocyclic compound, preparation method, pharmaceutical composition and application thereof

Technical Field

The invention relates to a phenyl and biphenyl substituted five-membered heterocyclic compound, a preparation method thereof, a pharmaceutical composition and application thereof, in particular to a phenyl and biphenyl substituted five-membered heterocyclic compound with inhibitory activity on PD-1/PD-L1 protein-protein interaction, a preparation method thereof, a pharmaceutical composition and application thereof.

Background

Immune escape is a fundamental biological feature of malignancy. Under normal physiological conditions, the immune system of the human body can recognize the isohexide and clear it in time. However, for tumor patients, due to the low immunity of the organism and the special biological characteristics of tumor cells, the tumor cells can escape the recognition and killing of the immune system through various different mechanisms, and finally can occur and develop in vivo. Tumor immune escape is a complex pathological process in which escape mechanisms mediated by immune checkpoints are of great interest.

Immune checkpoints are regulators of the immune system in humans, consisting of a series of co-stimulatory molecules and co-inhibitory molecules, playing an important regulatory role in the immune system of the organism. Co-stimulatory molecules of immune checkpoints include predominantly CD27, CD40, OX40, GITR, CD137, OX40, ICOS, etc., while co-inhibitory molecules are predominantly CTLA-4, PD-1, PD-L2, TIM-3, VISTA, IDO, etc. Wherein, the co-stimulatory molecules can enhance the immune response of the organism, thereby being beneficial to the immune cells to remove the isohexide, and the co-inhibitory molecules play a negative regulation role on the immune response, thereby maintaining the immune homeostasis of the organism and avoiding the damage of normal tissues of the host caused by excessive immunity. However, tumor cells are able to utilize immune checkpoints to achieve immune evasion. Among these, a common evasion mechanism is that tumor cells inhibit activation of T lymphocytes by inducing over-expression of co-suppressor molecules on surfaces of themselves, antigen Presenting Cells (APCs), T lymphocytes, and the like. Among them, the programmed death receptor 1 (PD-1) and its ligand PD-L1/2 are widely focused as important co-inhibitory molecules in immune checkpoints, and the PD-1/PD-L1 is fully confirmed as a target point of tumor immunotherapy at present.

PD-1 expresses CD4 in thymus at low levels in addition to mature T cells ^- CD8 ^- T cells, B cells, dendritic Cells (DCs), and Natural Killer (NK) cells. PD-1 has two ligands, where PD-L1 is expressed primarily in mature T cells, B cells, and some non-hematopoietic cells, but PD-L1 can be expressed on a variety of cells under the induction of inflammatory factors such as IFN-gamma, TNF-alpha, and VEGF. PD-L2 expression ranges are relatively narrow, mainly in macrophages and DC cells. Tyrosine in the ITSM domain of the cytoplasmic domain is phosphorylated when PD-1 binds to its ligand, thereby inhibiting activation of TCR proximal kinase by recruiting SHP-2 phosphatase in the vicinity of the TCR, resulting in reduced levels of TCR-CD3 molecules and Lck-mediated ZAP-70 phosphorylation, which in turn activates its downstream signaling pathway. Negative regulation of immunity by PD-1/PD-L is mainly achieved by inhibiting PI3K-AKT and RAS signal channels to block transcription factors having important effects on T cell activation, proliferation, function and survivalActivation of the subunits, such as activin-1 (AP-1), nuclear factor of activated T cells (NFAT), and NF- κB. In addition, T cell function can also be inhibited by up-regulating expression of the transcription factor bat.

Under normal physiological conditions, the PD-1/PD-L signaling pathway can induce and maintain tolerance of peripheral tissues during immune responses to prevent excessive immune responses in the tissues. Overactivation of the PD-1/PD-L signaling pathway inhibits secretion of immunostimulatory factors such as IFN-gamma, TNF-alpha and IL-2 and expression of survivin when the body is in a pathological state. Numerous studies have shown that abnormalities in the PD-1/PD-L signaling pathway are closely associated with viral infections, diabetes, neurodegenerative diseases, organ transplant rejection, autoimmune diseases, and the like.

In addition, numerous studies have shown that abnormalities in the PD-1/PD-L signaling pathway are closely related to the occurrence, progression and prognosis of various human tumors. In tumor microenvironments, tumor cells can survive by anti-apoptotic signaling and inhibiting the activity of antigen-specific T lymphocytes after the PD-1/PD-L signaling pathway is overactivated. In addition, blocking the PD-1/PD-L signaling pathway with PD-1 or PD-L1 antibodies can inhibit tumor cell growth. The method mainly comprises the steps of reactivating T lymphocytes by reversing the influence on T lymphocyte signal transduction, promoting the generation of effector T lymphocytes and memory T lymphocytes and inhibiting the differentiation of regulatory T lymphocytes, and finally enhancing the immune killing capacity of the T lymphocytes in a tumor microenvironment, so that the aim of treating tumors is fulfilled.

At present, more than 10 PD-1/PD-L1 monoclonal antibody medicines such as Keystuda and OPdivo are marketed globally, and are applied to clinically treating various solid tumors and blood cancers such as malignant melanoma, non-small cell lung cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer and the like, so that prognosis of tumor patients is greatly improved, and treatment bottlenecks of various cancers are broken. However, there are some significant disadvantages to PD-1/PD-L1 mAbs. For example, most tumor patients cannot benefit from it due to their primary and/or acquired resistance; due to its lack of oral bioavailability, it cannot be administered orally, and patient compliance is poor; in addition, the immunogenicity of the medicine is easy to cause the occurrence of drug-induced immune related adverse events (irAEs) of patients; in addition, the preparation and purification of monoclonal antibodies are difficult and inconvenient to transport, resulting in high treatment costs. These problems limit the clinical application of PD-1/PD-L1 monoclonal antibodies. It is worth mentioning that the small molecule drug has low production cost by virtue of the unique pharmacokinetic property and pharmacodynamic property, and is hopeful to solve the defects of the monoclonal antibody drug, so that the research and development of the PD-1/PD-L1 small molecule inhibitor has important application value. However, the development of the small molecule inhibitor is challenging, so that the development of the small molecule inhibitor is still in the early development stage at present and is far behind the monoclonal antibody medicament, and therefore, the development of a novel PD-L1 small molecule inhibitor with high activity and good patentability is urgently required.

Disclosure of Invention

The invention aims to: aiming at the defects of poor patent medicine property and the like of the existing PD-1/PD-L1 small molecule inhibitor, the invention aims to provide a small molecule medicine with remarkable PD-L1 inhibition activity, and a preparation method, a medicine composition and application thereof.

The technical scheme is as follows: as a first aspect of the present invention, the 2-phenyl-5-biphenyl substituted five-membered heterocyclic compounds of the present invention have the structure of formula I, further comprising stereoisomers, meso, racemates, prodrugs, crystals, pharmaceutically acceptable salts or mixtures thereof,

wherein:

(1) When X is O, A and N, B is C; when X is O, A and C, B is N or C;

(2) When X is S, A, B is N or C;

(3) When X is NH, A is N, B is N;

(4) When X is N, A and N, B is O; when X is N, A and C, B is O or NH; when X is N, A is O, B is C or N;

R ¹ selected from methyl, cyano, hydroxy or halogen;

R ² selected from hydrogen, halogen, nitro,Cyano, hydroxy, C ₁ -C ₄ Alkyl, C ₁ -C ₄ Alkoxy, C ₁ -C ₄ Haloalkyl or-O (CH) ₂ ) _n Ar is as follows; wherein n is an integer from 0 to 4; ar is selected from aryl or aromatic heterocycle; the aromatic heterocycle comprises one or more heteroatoms selected from O, S or N; the C is ₁ -C ₄ Alkyl, aryl or aromatic heterocyclic groups are substituted with one or more W groups;

w is selected from hydrogen, halogen, cyano, hydroxy, mercapto, carboxyl, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Alkoxy, C ₁ -C ₆ Alkylamino or C ₁ -C ₆ A haloalkyl group;

R ³ 、R ⁴ each independently selected from hydrogen, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Alkoxy, C ₁ -C ₈ Alkylamino, C ₃ -C ₈ Cycloalkyl, 5-7 membered heterocyclyl or R ³ And R is ⁴ Together with the nitrogen atom to which they are attached, form a 5-7 membered heterocyclyl; the heterocyclic group may optionally contain one or more heteroatoms selected from O, S or N; the C is ₁ -C ₈ Alkyl, C ₁ -C ₈ Alkoxy, C ₁ -C ₈ Alkylamino, C ₃ -C ₈ Cycloalkyl or 5-7 membered heterocyclyl is substituted with one or more Y groups;

y is selected from hydrogen, halogen, hydroxy, mercapto, methylthio, carbonyl, carboxyl, amino, guanidino, furyl, tetrahydropyrrolyl, morpholinyl, N-methylpiperazinyl, C ₁ -C ₄ Alkyl, -CO ₂ R ⁵ 、-NHCOR ⁵ 、-NR ⁶ R ⁷ or-CONR ⁶ R ⁷ The method comprises the steps of carrying out a first treatment on the surface of the The C is ₁ -C ₄ Alkyl groups are substituted with one or more hydroxy groups or halogen;

R ⁵ selected from C ₁ -C ₈ An alkyl group;

R ⁶ 、R ⁷ each independently selected from hydrogen, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Alkoxy, C ₃ -C ₈ Cycloalkyl or R ⁸ And R is ⁹ Connected to themThe nitrogen atoms together form a 5-7 membered heterocyclic group; the C is ₁ -C ₈ Alkyl, C ₁ -C ₈ Alkoxy, C ₃ -C ₈ Cycloalkyl or 5-7 membered heterocyclyl is substituted with one or more Z groups;

z is selected from hydrogen, halogen, hydroxy, mercapto, carboxyl, amino or acetamido.

The small molecule compound has good PD-1/PD-L1 protein-protein interaction inhibition activity, and can be used for treating and/or preventing various related diseases caused by PD-1/PD-L1 mediated immunosuppression.

Preferably, in the above structure:

R ¹ selected from methyl or halogen;

R ² selected from hydrogen, nitro or halogen;

R ³ 、R ⁴ each independently selected from hydrogen, C ₁ -C ₅ Alkyl or R ³ And R is ⁴ Together with the nitrogen atom to which they are attached, form a 5-6 membered N-containing heterocyclyl; the C is ₁ -C ₅ Alkyl or 5-6 membered heterocyclyl is substituted with one or more Y groups;

y is selected from hydrogen, hydroxy, carbonyl, carboxyl, guanidino, and C ₁ -C ₄ Alkyl, -CO ₂ R ⁵ 、-NR ⁶ R ⁷ or-CONR ⁶ R ⁷ ；C ₁ -C ₄ Alkyl groups are substituted with one or more hydrogen or hydroxy groups;

R ⁵ selected from C ₁ -C ₄ An alkyl group;

R ⁶ 、R ⁷ each independently selected from hydrogen or C ₁ -C ₄ An alkyl group.

Preferably, in the above structure:

R ¹ selected from methyl or chlorine;

R ² selected from hydrogen, nitro, fluorine, chlorine or bromine;

R ³ 、R ⁴ each independently selected from hydrogen, C ₁ -C ₅ Alkyl or R ³ And R is ⁴ Together with the nitrogen atom to which they are attached, form a 5-to 6-membered heterocyclic group containing one N atom; the saidC of (2) ₁ -C ₅ Alkyl or 5-6 membered heterocyclyl is substituted with one or more Y groups;

y is selected from hydrogen, hydroxy, carbonyl, carboxyl, guanidino, and C ₁ -C ₄ Alkyl, -CO ₂ CH ₃ Amino or-CONH ₂ ；C ₁ -C ₄ Alkyl groups are substituted with one or more hydrogen or hydroxy groups.

In particular, the method comprises the steps of,

selected from the following ring systems: />

Selected from the following groups:

more specifically, the above compound is selected from any one of the following compounds:

as a second aspect of the present invention, the above-mentioned compound is produced by the following method:

the method comprises the following steps: when X is S, A and B are N, the compound a-1 is used as a raw material to prepare a compound with a general formula (I) through Suzuki coupling, esterification, hydrazinolysis, condensation, cyclization and condensation reaction, or the compound with the general formula (I) is prepared through further alkali hydrolysis;

the second method is as follows: when X is NH, A and B are N, the compound d-1 is subjected to cyclization, reduction, oxidation and reductive amination to obtain a compound of the general formula (I), or is subjected to further alkali hydrolysis to obtain the compound of the general formula (I);

and a third method: when X is N, A is O and B is N, the compound a-2 is subjected to addition, cyclization, reduction, oxidation and reductive amination to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

the method four: when X and A are N and B is O, the compound a-3 is subjected to Suzuki coupling, addition, condensation, reduction, oxidation and reductive amination to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

and a fifth method: when X is O, A is C and B is N, the compound a-4 is subjected to coupling, suzuki coupling, bromination, amination, condensation, cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

The method six: when X is O, A is N and B is C, the compound B-1 is subjected to chlorination, condensation, cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

and a seventh method: when X is N, A is O and B is C, the compound B-1 is subjected to amination, cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkaline hydrolysis to prepare the compound of the general formula (I);

method eight: when X is N, A is C and B is O, the compound C-5 is subjected to cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

method nine: when X is N, A is C, and B is NH, the compound is prepared into a compound of the general formula (I) through cyclization, reduction, halogenation and condensation reaction of C-5, or is further prepared into the compound of the general formula (I) through alkaline hydrolysis;

method ten: when X is S, A is C and B is N, the compound e-5-1 is subjected to cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkaline hydrolysis to prepare the compound of the general formula (I);

method eleven: when X is S, A is N, B is C, the compound C-6 is subjected to cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

Method twelve: when X is S or O and A and B are C, the compound a-4 is subjected to coupling, bromination, suzuki coupling and reductive amination reaction to obtain a compound of the general formula (I), or is subjected to further alkali hydrolysis to obtain the compound of the general formula (I);

wherein R is ¹ 、R ² 、R ³ 、R ⁴ Is as defined in any one of claims 1 to 4.

As a third aspect to which the present invention relates, the above-described compounds can be prepared as PD-L1 inhibitor drugs and immunomodulator drugs, in particular, drugs for the prophylaxis and/or treatment of tumors, infectious diseases, inflammatory diseases, organ transplant rejection and autoimmune diseases.

As a fourth aspect of the present invention, the above-mentioned compounds and a pharmaceutically acceptable carrier form a pharmaceutical composition, and the specific formulation forms are tablets, capsules, powders, pills, granules, injections, oral liquids, syrups, inhalants, ointments, patches or suppositories.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:

the compound has high inhibitory activity on PD-1/PD-L1 protein-protein interaction. Pharmacological experiment results show that the phenyl-substituted five-membered heterocyclic compounds have strong binding capacity with PD-L1, can effectively reverse the immunosuppression effect mediated by PD-1/PD-L1 and promote CD8 ⁺ Proliferation of T lymphocytesIncrease secretion of cytokine interferon-gamma and decrease CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Regulatory T cell production reduces PCNA protein expression. The in vivo pharmacodynamics evaluation results show that the compounds of the invention can obviously inhibit the growth of mouse transplanted tumors with various tumor types, but have no influence on the growth of nude mouse transplanted tumors with immune system defects, which indicates that the compounds play an anti-tumor role by activating host immune response.

Drawings

FIG. 1 shows the effect of compounds of the invention on Lewis lung cancer cell viability at various concentrations.

FIG. 2 shows the effect of compounds of the invention on dose-dependent reversal of PD-1/PD-L1 inhibition of INF-gamma secretion by PBMC.

FIG. 3 shows that the compounds of the present invention inhibit growth of Lewis lung carcinoma mouse transplants in a dose-dependent manner.

FIG. 4 is a graph showing the effect of a compound of the invention on T lymphocyte infiltration in a mouse engraftment tumor, wherein: a) CD45 ⁺ A cell; b) CD45 ⁺ CD3 ⁺ A cell; c) CD4 ⁺ CD45 ⁺ CD3 ⁺ A cell; d) CD8 ⁺ CD45 ⁺ CD3 ⁺ And (3) cells.

Detailed Description

The technical scheme of the invention is further described below by referring to examples.

Reagent and material: all reagents required for the experiments were not specifically described as commercially available chemically pure or analytically pure products.

Instrument: ¹ H NMR was measured using Bruker AV-300 and 400MHz nuclear magnetic resonance apparatus, chemical shift values (delta) in ppm, coupling constants (J) in Hz, TMS as internal standard. The Mass Spectrum (MS) analysis instrument is a Shimadzu LCMS-2020 mass spectrometer for measurement; thin Layer Chromatography (TLC) using HG/T2354-92 type GF254 thin layer chromatography silica gel produced by Qingdao ocean chemistry Co., ltd., ZF7 type three-purpose ultraviolet analyzer 254nm color development; column chromatography uses crude pore (ZCX-II) 300-400 mesh column chromatography silica gel of Qingdao ocean chemical plant.

Example 1: (3- (5- (2-methyl- [1,1' -biphenyl)]-3-yl) -1,3, 4-thiadiazol-2-yl) benzyl) glycine (1)Synthesis of hydrochloride (1 s) thereof

Synthesis of 2-methyl- [1,1' -biphenyl ] -3-carboxylic acid (1A)

3-bromo-2-methylbenzoic acid (10.00 g,46.51 mmol), phenylboronic acid (10.21 g,83.72 mmol), potassium carbonate (7.71 g,55.71 mmol) and Pd (PPh) ₃ ) ₄ (0.53 g,0.46 mmol) was added to 100mL of 1,4 dioxane and 10mL of water and reacted at 80℃for 12h under nitrogen. Concentrating under reduced pressure, adjusting pH to 2 with 4M HCl, filtering, and drying to obtain white solid 9.53g with 96% yield. MS (EI) m/z 211[ M-H ]] ^- ； ¹ H NMR(300MHz,Chloroform-d)δ(ppm)7.74-7.67(m,1H),7.50-7.38(m,3H),7.38-7.34(m,2H),7.33-7.28(m,2H),2.29(s,3H).

Synthesis of methyl 2-methyl- [1,1' -biphenyl ] -3-carboxylate (1B)

1A (9.53 g,44.90 mmol) was added to 100mL of methanol, and 5mL of concentrated sulfuric acid was added dropwise with stirring and reacted under reflux for 4h. Cooled, concentrated under reduced pressure, extracted with 300mL of water, washed with saturated brine, and the organic phases were combined to give 9.96g of a yellow oily liquid in 98% yield. MS (EI) m/z 225[ M-H ] ^- ； ¹ H NMR(300MHz,Chloroform-d)δ(ppm)7.45-7.32(m,4H),7.30-7.15(m,3H),7.07(d,J＝7.5Hz,1H),3.98(s,3H)，2.32(s,3H).

Synthesis of 2-methyl- [1,1' -biphenyl ] -3-carbohydrazide (1C)

1B (9.96 g,44.02 mmol) was added to 100mL of ethanol, 5mL of hydrazine hydrate was added and the reaction was refluxed for 5h. Cooling, concentrating under reduced pressure, adding 100mL of ice water, precipitating solid, filtering, and drying to obtain 9.24g of white solid with a yield of 93%. MS (EI) m/z 225[ M-H] ^- ； ¹ H NMR(300MHz,Chloroform-d)δ8.02(s,1H),7.45(d,J＝7.5Hz,3H),7.37(s,1H),7.33(d,J＝1.8Hz,3H),2.52(s,3H).

Synthesis of N '- (3- (chloromethyl) benzoyl) -2-methyl- [1,1' -biphenyl ] -3-formylhydrazine (1D)

3- (chloromethyl) benzoic acid (0.25 g,1.46 mmol) was added to 5mL of anhydrous dichloromethane, 1mL of oxalyl chloride was added dropwise, reacted for 3 hours, concentrated under reduced pressure, dissolved with 3mL of anhydrous dichloromethane, and cooled dropwise by ice bathIn a solution of 1C (0.32 g,1.33 mmol) and triethylamine (0.21 g,1.99 mmol) in dichloromethane for 3h, suction filtered and dried to give 0.45g of a white solid in 90% yield. MS (EI) m/z 377[ M-H ]] ^- ； ¹ H NMR(300MHz,Chloroform-d)δ10.02(s,1H),8.74(s,1H),8.02(s,4H),7.37(td,J＝15.0,13.5,9.0Hz,8H),4.70(s,2H),2.31(s,3H).

Synthesis of 2- (3- (chloromethyl) phenyl) -5- (2-methyl- [1,1' -biphenyl ] -3-yl) -1,3, 4-thiadiazole (1E)

1D (0.45 g,1.19 mmol) was added to 10mL toluene and reacted at reflux for 5h as L.Lawson reagent (0.58 g,1.43 mmol). Concentrating under reduced pressure, extracting with ethyl acetate, drying with anhydrous sodium sulfate, and purifying by column chromatography [ petroleum ether: ethyl acetate=15:1 (V: V)]0.32g of white solid was obtained in 71% yield. MS (EI) m/z 375[ M-H ] ] ^- ； ¹ H NMR(300MHz,Chloroform-d)δ8.11(s,1H),8.01(dt,J＝6.9,1.8Hz,1H),7.68(dd,J＝6.6,2.4Hz,1H),7.56(s,1H),7.48(s,1H),7.46(s,1H),7.43(s,1H),7.41(d,J＝2.7Hz,2H),7.39(d,J＝1.8Hz,2H),7.36(t,J＝1.5Hz,1H),4.70(s,2H),2.46(s,3H).

Synthesis of methyl (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -1,3, 4-thiadiazol-2-yl) benzyl) glycinate (1 m)

1E (0.32 g,0.85 mmol), K ₂ CO ₃ (0.41 g,2.97 mmol) glycine methyl ester hydrochloride (0.27 g,2.12 mmol) was added to 5mL acetonitrile and reacted for 8h under reflux. Cooling, concentrating under reduced pressure, extracting with ethyl acetate, drying with anhydrous sodium sulfate, and purifying by column chromatography [ petroleum ether: ethyl acetate=3:1 (V: V)]0.15g of white solid was obtained in 41% yield. MS (EI) m/z 428[ M-H ]] ^- ； ¹ H NMR(300MHz,Chloroform-d)δ8.05-7.98(m,2H),7.68-7.63(m,1H),7.51(s,1H),7.48(s,1H),7.45(s,1H),7.43(s,1H),7.36(t,J＝6.9Hz,5H)’4.24(s,2H),3.98(s,3H),3.77(s,2H),2.33(s,3H).

Synthesis of (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -1,3, 4-thiadiazol-2-yl) benzyl) glycine (1)

1M (0.15 g,0.36 mmol), liOH (25 mg,1.05 mmol) was added to 3mL of methanol and reacted at room temperature for 5h, the solvent was removed by swirling, 2mL of water was added, pH was adjusted to 3 with 4M hydrochloric acid, suction filtration and drying to give 135mg of white solid in 92% yield. MS (ESI) m/z 416[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.08(d,J＝7.5Hz,2H),7.79(d,J＝7.8Hz,2H),7.70(d,J＝6.6Hz,1H),7.44(dd,J＝11.4,8.1Hz,7H),4.13(s,2H),3.65(s,2H),2.30(s,3H).

Synthesis of (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -1,3, 4-thiadiazol-2-yl) benzyl) glycine hydrochloride (1 s)

1 (32 mg,0.078 mmol) was added to 1mL of a 4M 1, 4-dioxane hydrochloric acid solution, stirred at room temperature overnight, concentrated under reduced pressure, washed with anhydrous diethyl ether, filtered off with suction, and dried to give 27mg of a white solid in 93% yield. MS (ESI) m/z 416[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ ) Delta 8.36 (s, 1H), 8.15 (d, j=7.2 hz, 1H), 8.05 (d, j=6.9 hz, 1H), 7.80 (s, 1H), 7.70 (s, 1H), 7.47 (q, j=8.1, 7.8hz, 5H), 7.37 (d, j=6.6 hz, 3H), 4.21 (s, 2H), 3.75 (s, 2H), 2.23 (s, 3H) using a procedure similar to example 1, the following compounds were prepared:

Example 2: synthesis of (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -4H-1,2, 4-triazol-3-yl) benzyl) glycine (60) and hydrochloride (60 s) thereof

Synthesis of methyl 3-carbamoylbenzoate hydrochloride (2A)

Methyl 3-cyanobenzoate (7.50 g,46.54 mmol) was dissolved in 80mL of methanol, sodium methoxide (2.77 g,51.19 mmol) was added, and the mixture was reacted at 40℃for 12 hours, followed by ammonium chloride (4.98 g,93.08 mmol) and reacted at 50℃overnight. Cooling, suction filtration, spin drying of the filtrate, recrystallization with PE: ea=5:1, suction filtration, drying, gave 6.90g of white solid in 69% yield. MS (EI) m/z 179[ M+H ]] ⁺ ； ¹ H NMR(300MHz,Chloroform-d)δ7.95(d,J＝6.9Hz,1H),7.52(d,J＝6.9Hz,1H),7.39-7.35(m,2H)3.87(s,3H).

Synthesis of methyl 3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -4H-1,2, 4-triazol-3-yl) benzoate (2B)

2A (3.50 g,16.31 mmol) and sodium methoxide (1.17 g,21.74 mmol) were added to 20mL of absolute ethanol and reacted at room temperature for 1h, suction filtered, and the filtrate was added to 1C (2.46 g,10.87 mmol) and stirred at reflux overnight. Cooling, suction filtering, extracting with ethyl acetate, drying with anhydrous sodium sulfate, and purifying by column chromatography [ petroleum ether: ethyl acetate=20:1 (V: V)]2.61g of white solid was obtained in 65% yield. MS (ESI) m/z 370[ M+H ]] ⁺ ； ¹ H NMR(300MHz,Chloroform-d)δ8.77(s,1H),8.37(d,J＝7.8Hz,1H),8.23(d,J＝7.8Hz,1H),8.02(d,J＝6.6Hz,1H),7.64(t,J＝7.8Hz,1H),7.51-7.30(m,7H),3.99(s,3H),2.61(s,3H).

Synthesis of (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -4H-1,2, 4-triazol-3-yl) phenyl) methanol (2C)

2B (2.62 g,7.04 mmol) was added to 30mL dry THF and LiAlH was added with ice-bath cooling ₄ (0.41 g,10.56 mmol) for 4h. 40mL of ice water was added, suction filtration and drying were carried out to obtain 2.21g of a white solid with a yield of 94%. MS (ESI) m/z 342[ M+H ]] ⁺ ； ¹ H NMR(300MHz,Chloroform-d)δ8.37(s,1H),8.18(d,J＝7.8Hz,1H),8.12(dd,J＝7.5,1.8Hz,1H),7.83(d,J＝7.8Hz,1H),7.70(t,J＝7.8Hz,1H),7.56-7.35(m,7H),4.54(s,2H),2.33(s,3H).

Synthesis of 3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -4H-1,2, 4-triazol-3-yl) benzaldehyde (2D)

2C (2.20 g,6.44 mmol) was dissolved in 35 mM DS MSO and IBX (2.71 g,9.67 mmol) was added and reacted for 2h at room temperature. Ethyl acetate extraction, column chromatography purification [ petroleum ether: ethyl acetate=20:1 (V: V)]1.40g of white solid was obtained in 60% yield. MS (ESI) m/z 340[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ9.85(s,1H),8.37(s,1H),8.15(d,J＝7.8Hz,1H),8.07(dd,J＝7.5,1.8Hz,1H),7.81(d,J＝7.8Hz,1H),7.68(t,J＝7.8Hz,1H),7.62-7.32(m,7H),2.54(s,3H).

Synthesis of methyl (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -4H-1,2, 4-triazol-3-yl) benzyl) glycinate (60 m)

2D (0.15 g,0.44 mmol) was added to 5mL of DMF, methyl glycinate hydrochloride (0.14 g,0.89 mmol), TEA (0.13 g,1.33 mmol), glacial acetic acid (0.13 g,2.21 mmol) and sodium cyanoborohydride (0.14 g,2.21 mmol) were added in sequence, reacted at room temperature for 4h, 10mL of water was added, extracted with ethyl acetate, and purified by column chromatography [ Petroleum ether: ethyl acetate=1:1 (V: V)]0.12g of white solid was obtained in 60% yield. MS (ESI) m/z 413[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.05-7.98(m,2H),7.68-7.63(m,1H),7.51(s,1H),7.48(s,1H),7.45(s,1H),7.43(s,1H),7.36(t,J＝6.9Hz,5H)’4.24(s,2H),3.98(s,3H),3.77(s,2H),2.33(s,3H).

Synthesis of Compound 60 and hydrochloride salt thereof (60 s)

According to the method of example 1, 60m was hydrolyzed to give 60 as a white solid in a yield of 60%. Then 60 is salified with hydrochloric acid to prepare white solid with the yield of 94 percent for 60 seconds. MS (ESI) m/z 399[ M+H ] ] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.93(s,1H),8.86(s,1H),8.36(s,1H),8.15(d,J＝7.2Hz,1H),8.05(d,J＝6.9Hz,1H),7.80(s,1H),7.70(s,1H),7.47(q,J＝8.1,7.8Hz,5H),7.37(d,J＝6.6Hz,3H),4.21(s,2H),3.88(d,J＝12.3Hz,2H),3.74(s,1H),2.23(s,3H).

By operating in a similar manner to example 2, the following compounds were prepared:

example 3: synthesis of (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -1,2, 4-oxadiazol-3-yl) benzyl) glycine (72) and hydrochloride (72 s) thereof

Synthesis of methyl 3- (N-hydroxycarbamoyl) benzoate (3A)

Methyl meta-cyanobenzoate (2.61 g,16.01 mmol) was dissolved in 30mL of absolute ethanol, hydroxylamine hydrochloride (3.91 g,56.02 mmol) and sodium hydrogencarbonate (5.30 g,64.02 mmol) were added sequentially with stirring, and the reaction was refluxed for 12 hours. Cooling, suction filtration, extraction with ethyl acetate, drying over anhydrous sodium sulfate, and column chromatography purification (PE: ea=15:1) gave 2.53g of a white solid in 80% yield. MS (ESI) m/z 195[ M+H ]] ⁺ ； ¹ H-NMR(300MHz,DMSO-d ₆ ):δ(ppm)8.76-8.68(m,2H),8.49(s,1H),7.89(s,1H),7.28(s,1H),6.90(s,1H),6.68(s,1H),3.98(s,3H).

Synthesis of methyl 3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -1,2, 4-oxadiazol-3-yl) benzoate (3B)

1A (4.80 g,22.71 mmol), 1-hydroxybenzotriazole (3.31 g,24.82 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.81 g,24.80 mmol) and potassium carbonate (3.41 g,24.80 mmol) were dissolved in 50mL DMF and stirred at room temperature for 0.5h, intermediate 3A (4.01 g,20.62 mmol), N were added ₂ The reaction is carried out for 12 hours at 110 ℃. Cooling, extraction with ethyl acetate, drying over anhydrous sodium sulfate, and column chromatography purification (PE: ea=20:1) gave 3.51g of a white solid in 46% yield. MS (ESI) m/z 371[ M+H ] ] ⁺ ； ¹ H NMR(300MHz,Chloroform-d)δ8.86(s,1H),8.39(d,J＝7.8Hz,1H),8.22(d,J＝8.0Hz,1H),8.12(dd,J＝7.5,1.8Hz,1H),7.62(t,J＝7.8Hz,2H),7.52-7.39(m,5H),7.38-7.33(m,1H),3.99(s,3H),2.63(s,3H).

Synthesis of (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -1,2, 4-oxadiazol-3-yl) phenyl) methanol (3C)

Referring to the procedure of example 2, intermediate 3B was reacted with LiAlH ₄ Reduction reaction was carried out to obtain 3C as a white solid in a yield of 81%. MS (ESI) M/z343[ M+H] ⁺ ； ¹ H NMR(300MHz,Chloroform-d)δ8.16(dd,J＝7.1,4.2Hz,2H),8.04-7.98(m,1H),7.62-7.55(m,2H),7.45(dt,J＝9.8,4.3Hz,5H),7.39-7.34(m,2H),4.85(s,1H),4.84(s,2H),2.62(s,3H).

Synthesis of 3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -1,2, 4-oxadiazol-3-yl) benzaldehyde (3D)

Referring to the procedure of example 2, intermediate 3C was reacted with IBX to give 3D as a white solid in 80% yield. MS (ESI) m/z 341[ M+H ]] ⁺ ； ¹ H NMR(300MHz,Chloroform-d)δ10.12(s,1H),8.20(s,3H),8.02(d,J＝7.0Hz,1H),7.45(t,J＝6.9Hz,5H),7.36(d,J＝6.6Hz,2H),3.99(s,3H),2.63(s,3H).

Synthesis of methyl (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) -1,2, 4-oxadiazol-3-yl) benzyl) glycinate (72 m)

Referring to the procedure of example 2, intermediate 3E and glycine methyl ester hydrochloride were subjected to reductive amination to afford 72m as a white solid in 69% yield. MS (ESI) m/z 414[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.16(d,J＝7.2Hz,2H),8.09(s,1H),7.78(d,J＝7.1Hz,2H),7.54(s,2H),7.52-7.46(m,3H),7.40(d,J＝6.4Hz,2H),4.26(s,2H),3.9(s,3H),3.83(s,2H),2.54(s,3H).

Synthesis of Compound 72 and hydrochloride (72 s) thereof

With reference to the procedure of example 1, 72m was hydrolyzed to give a white solid 72 in 61% yield. Then 72 is salified with hydrochloric acid to prepare white solid with the yield of 93 percent for 72 s. MS (ESI) m/z 400[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ9.54(s,1H),8.16(d,J＝7.2Hz,2H),8.09(s,1H),7.78(d,J＝7.1Hz,2H),7.54(s,2H),7.52-7.46(m,3H),7.40(d,J＝6.4Hz,2H),4.26(s,2H),3.83(s,2H),2.54(s,3H).

By operating in a similar manner to example 3, the following compounds were prepared:

example 4: synthesis of 3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) oxazol-2-yl) benzyl) glycine (146) and hydrochloride (146 s) thereof

1- (3-bromo-2-methylphenyl) ethane-1-one (4A)

1, 3-dibromo-2-methylbenzene (10.01 g,40.01 mmol) was dissolved in 85mL of DMF and tributyl (1-ethoxyethylene) tin (14.41 g,40.01 mmol) and Pd (PPh) were added with stirring ₃ ) ₄ (0.46 g,0.40 mmol) was reacted at 85℃for 12h under nitrogen. Cooled, extracted with ethyl acetate and concentrated under reduced pressure. 20mL of 4M HCl was added and the reaction was carried out at room temperature for 3h. Ethyl acetate extraction, drying over anhydrous sodium sulfate, column chromatography purification [ petroleum ether: ethyl acetate=15:1 (V: V)]8.04g of oil was obtained in 94% yield. MS (EI) m/z 213[ M+H ]] ⁺ ； ¹ H NMR(300MHz,Chloroform-d)δ7.59(d,J＝7.8Hz,1H),6.93(s,2H),2.60(s,3H),2.30(s,3H).

Synthesis of 1- (2-methyl- [1,1' -biphenyl ] -3-yl) ethan-1-one (4B)

With reference to the method of example 1, 4A and phenylboronic acid were subjected to a coupling reaction to give 4B as an oil in 87% yield. MS (EI) m/z 211[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ7.70(dd,J＝7.2,2.4Hz,1H),7.50-7.26(m,7H),2.58(d,J＝2.7Hz,3H),2.22(d,J＝3.0Hz,3H).

Synthesis of 2-bromo-1- (2-methyl-1, 1' -biphenyl) -3-yl) ethan-1-one (4C)

4B (1.50 g,7.13 mmol) and copper bromide (3.51 g ) were added to 20mL ethyl acetate and reacted under reflux for 8h. Cooling, suction filtering, and concentrating under reduced pressure. 10mL of tetrahydrofuran, diethyl phosphite (0.26 g,1.87 mmol) and triethylamine (0.21 g,2.06 mmol) were added and reacted at room temperature for 2 hours. Ethyl acetate extraction, drying over anhydrous sodium sulfate, column chromatography purification [ petroleum ether: ethyl acetate=20:1 (V: V)]1.36g of white solid was obtained in 65% yield. MS (EI) m/z 289[ M+H ] ] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ7.80(dd,J＝6.3,2.7Hz,1H),7.50-7.36(m,5H),7.35-7.26(m,2H),4.91(s,2H),2.21(s,3H).

Synthesis of 2-amino-1- (2-methyl- [1,1' -biphenyl ] -3-yl) ethan-1-one (4D)

4C (1.21 g,4.15 mmol) and sodium diformylamide (0.39 g,4.15 mmol) were added to 15mL of acetonitrile and reacted at 75℃for 12h, filtered off while hot, concentrated under reduced pressure, 5mL of 4M HCl was added and refluxed for 1h. Concentrated under reduced pressure, and ethyl acetate was recrystallized to give 0.65g of a white solid with a yield of 70%. MS (EI) m/z 226[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.39(s,3H),7.90-7.84(m,1H),7.53-7.39(m,6H),4.52(d,J＝6.0Hz,2H),2.28(s,3H).

Synthesis of methyl 3- (2- (2-methyl- [1,1' -biphenyl ] -3-yl) -2-oxoethyl) carbamoyl) benzoate (4E)

With reference to the procedure of example 1, 3- (methoxycarbonyl) benzoic acid and 4D were subjected to acylation reaction to give 4E as a white solid in 61% yield. MS (EI) m/z 388[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ9.21(s,1H),8.16-7.98(m,4H),7.75(d,J＝8.1Hz,1H),7.53-7.31(m,6H),4.61(d,J＝5.7Hz,2H),3.89(s,3H),2.22(s,3H).

Synthesis of methyl 3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) oxazol-2-yl) benzoate (4F)

4E (0.20 g,0.51 mmol) was added to 3mL of acetic anhydride, 4 drops of concentrated sulfuric acid were added, and the reaction was carried out at 80℃for 2h. Cooling, acetic acidEthyl ester extraction and concentration under reduced pressure gave 4F as a white solid in 62% yield. MS (EI) m/z 370[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.35-8.09(m,4H),7.95-7.70(m,2H),7.45(ddt,J＝26.4,19.2,9.6Hz,7H),3.91(d,J＝7.5Hz,3H),2.36(s,3H).

Synthesis of 3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) oxazol-2-yl) phenylmethanol (4G)

Referring to the method of example 2, 4F and LiAlH ₄ Reduction was performed to obtain 4G as a white solid in 79% yield. MS (EI) m/z 342[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.18-8.02(m,2H),7.83(d,J＝10.5Hz,1H),7.72-7.59(m,1H),7.59-7.34(m,8H),7.29(s,1H),5.36(s,1H),4.59(s,2H),2.35(s,3H).

Synthesis of 2- (3-chloromethyl) phenyl-5- (2-methyl- [1,1' -biphenyl ] -3-yl) oxazole (4H)

4G (0.60G, 1.76 mmol) was added to 5mL of methylene chloride, and 0.3mL of thionyl chloride was added dropwise thereto under ice-bath cooling and reacted at room temperature for 3 hours. Ethyl acetate extraction and concentration under reduced pressure gave 0.53g of a white solid in 83% yield. MS (EI) m/z 360[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.18-8.02(m,2H),7.83(d,J＝10.5Hz,1H),7.72-7.59(m,1H),7.59-7.34(m,8H),7.29(s,1H),4.64(s,2H),2.33(s,3H).

Synthesis of methyl (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) oxazol-2-yl) benzyl) glycinate (146 m)

Referring to the procedure of example 1, 4H and glycine methyl ester hydrochloride were subjected to condensation reaction to obtain 146m as a white solid in 58% yield. MS (EI) m/z 413[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.04(d,J＝7.8Hz,2H),7.83(d,J＝7.8Hz,1H),7.62(s,1H),7.57-7.35(m,8H),7.29(dd,J＝7.5,1.5Hz,1H),3.80(s,2H),3.64(s,3H),3.35(s,2H),2.36(s,3H).

Synthesis of Compound 146 and its hydrochloride (146 m)

With reference to the method of example 1, 146m was hydrolyzed to yield 146 in 84%. And then salifying 146 with hydrochloric acid to prepare 146s with 78% yield. MS (ESI) m/z 399[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.86(s,1H),8.15(d,J＝7.2Hz,1H),8.05(d,J＝6.9Hz,1H),7.80(s,1H),7.70(s,1H),7.47(q,J＝8.1,7.8Hz,5H),7.37(d,J＝6.6Hz,4H),4.21(s,2H),3.75(s,2H),2.23(s,3H).

By operating in a similar manner to example 4, the following compounds were prepared:

example 5:3- (2-methyl- [1,1' -biphenyl)]Synthesis of (3-yl) oxazol-4-yl) benzyl) glycine (199) and its hydrochloride (199 s)

Synthesis of 2-methyl- [1,1' -biphenyl ] -3-carboxamide (5A)

1C (2.00 g,9.42 mmol), ammonia (0.66 g,18.85 mmol), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (5.37 g,14.13 mmol), N, N-diisopropylethylamine (2.44 g,18.85 mmol) were added to 80mL of DMF and reacted overnight at room temperature. 200mL of water was added, suction filtration and drying were carried out to obtain 1.63g of a white solid with a yield of 82%. MS (EI) m/z 212[ M+H ] ] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ7.70(dd,J＝7.2,2.4Hz,1H),7.50-7.26(m,7H),2.22(s,3H).

Synthesis of methyl 3- (2- (2-methyl- [1,1' -biphenyl ] -3-yl) oxazol-4-yl) benzoate (5B)

The cyclization reaction was carried out on 5A to obtain white solid 5B, the yield was 53%. MS (EI) m/z 370[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.90(s,1H),8.44(s,1H),8.14(d,J＝7.8Hz,1H),7.94(t,J＝6.6Hz,2H),7.63(t,J＝7.8Hz,1H),7.52-7.44(m,3H),7.44-7.35(m,4H),3.89(s,3H),2.52(s,3H).

Synthesis of 3- (2-methyl- [1,1' -biphenyl ] -3-yl) oxazol-4-yl) phenylmethanol (5C)

Referring to the method of example 2, 5B and LiAlH ₄ Reduction was carried out to obtain 5C as a white solid in 71% yield. MS (EI) m/z 342[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.91(s,1H),8.42(s,1H),8.14(d,J＝7.8Hz,1H),7.94(t,J＝6.6Hz,2H),7.63(t,J＝7.8Hz,1H),7.52-7.44(m,3H),7.45-7.34(m,4H),5.27(s,2H),2.52(s,3H).

Synthesis of 4- (3-chloromethyl) phenyl-2- (2-methyl- [1,1' -biphenyl ] -3-yl) oxazole (5D)

Referring to the procedure of example 4, 5C was reacted to give 5D as a white solid in 83% yield. MS (EI) m/z 360[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.91(s,1H),8.42(s,1H),8.14(d,J＝7.8Hz,1H),7.94(t,J＝6.6Hz,2H),7.63(t,J＝7.8Hz,1H),7.52-7.44(m,3H),7.45-7.34(m,4H),5.27(s,2H),2.52(s,3H).

Synthesis of methyl 3- (2-methyl- [1,1' -biphenyl ] -3-yl) oxazol-4-yl) benzyl) glycinate (199 m)

Reference to the formulation of example 1The method is to react 5D with glycine methyl ester hydrochloride to prepare white solid 199m with the yield of 80%. MS (EI) m/z 413[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.77(s,1H),8.08(s,1H),7.92(dd,J＝10.9,6.1Hz,2H),7.53(d,J＝4.8Hz,2H),7.51-7.44(m,3H),7.43(s,1H),7.39(d,J＝3.3Hz,2H),7.36(s,1H),3.82(t,J＝9.3Hz,2H),3.62(d,J＝7.2Hz,2H),3.89(s,3H),2.35(s,3H).

Synthesis of Compound 199 and hydrochloride salt (199 s) thereof

199m was hydrolyzed to give 199 as a white solid in 58% yield by the method of example 1. 199 was then salified with hydrochloric acid to give 199s as a white solid in 88% yield. MS (ESI) m/z 399[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ9.02(s,1H),8.36(s,1H),8.15(d,J＝7.2Hz,1H),8.05(d,J＝6.9Hz,2H),7.47(q,J＝8.1,7.8Hz,6H),7.37(d,J＝6.6Hz,3H),4.21(s,2H),3.75(s,2H),2.23(s,3H).

By operating in a similar manner to example 5, the following compounds were prepared:

example 6: synthesis of (3- [4- (2-methyl- [1,1' -biphenyl ] -3-yl) -1H-imidazol-2-yl) benzyl) -L-alanine (227) and its hydrochloride salt (227 s)

Synthesis of methyl 3- (4- (2-methyl- [1,1' -biphenyl ] -3-yl)) -1H-imidazol-2-yl) benzoate (6A)

2A (2.00 g,9.23 mmol) and K ₂ CO ₃ (1.93 g,13.98 mmol) was added to 20mL of tetrahydrofuran and 5mL of water, stirred for 0.5h, 4C (2.42 g,8.39 mmol) was added and reacted at 70℃for 6h. Cooling, ethyl acetate extraction, drying over anhydrous sodium sulfate, column chromatography purification [ petroleum ether: ethyl acetate=4:1 (V: V)]1.06g of yellow oily substance was obtained in 52% yield. MS (EI) m/z 367[ M-H ]] ^- ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.18-8.02(m,2H),7.83(d,J＝10.5Hz,1H),7.72-7.59(m,1H),7.59-7.34(m,8H),7.29(s,1H),3.89(s,3H),2.35(s,3H).

Synthesis of (3- [4- (2-methyl- [1,1' -biphenyl ] -3-yl) -1H-imidazol-2-yl) phenyl) methanol (6B)

Referring to the method of example 2, 6A and LiAlH ₄ The reaction gave 6B as a yellow oil in 94% yield. MS (EI) m/z 339[ M-H ]] ^- ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.18-8.02(m,2H),7.83(d,J＝10.5Hz,1H),7.72-7.59(m,1H),7.59-7.34(m,8H),7.29(s,1H),4.59(s,2H),2.35(s,3H).

Synthesis of 2- (3- (chloromethyl) phenyl) -4- (2-methyl- [1,1' -biphenyl ] -3-yl) -1H-imidazole (6C)

Referring to the procedure of example 4, 6B was reacted to give 6C as a yellow oil in 97% yield. MS (EI) m/z 357[ M-H ]] ^- ； ¹ HNMR(300MHz,DMSO-d ₆ )δ8.18-8.02(m,2H),7.83(d,J＝10.5Hz,1H),7.72-7.59(m,1H),7.59-7.34(m,8H),7.29(s,1H),4.64(s,2H),2.33(s,3H).

Synthesis of methyl (3- [4- (2-methyl- [1,1' -biphenyl ] -3-yl) -1H-imidazol-2-yl) benzyl) -L-alaninate (227 m)

Referring to example 1, 6C and L-alanine methyl ester hydrochloride were reacted to give 227m as a yellow oil in a yield of 63％。MS(EI)m/z 425[M-H] ^- ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.43(s,1H),8.31(d,J＝7.8Hz,1H),7.95(s,1H),7.88(d,J＝7.8Hz,1H),7.73(t,J＝7.8Hz,1H),7.63(dd,J＝7.8,1.5Hz,1H),7.55-7.29(m,7H),3.92(s,3H),3.57(s,2H),3.39(s,1H),2.30(s,3H),1.57(d,J＝7.2Hz,3H).

Synthesis of Compound 227 and its hydrochloride (227 s)

With reference to the procedure of example 1, 227m was hydrolyzed to give 227 as a white solid in 80% yield. And then salifying 227 with hydrochloric acid to obtain a white solid 227s with a yield of 88%. MS (ESI) m/z 413[ M+H ] ] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.36(s,1H),8.15(d,J＝7.2Hz,1H),8.05(d,J＝6.9Hz,2H),7.80(s,1H),7.70(s,1H),7.47(q,J＝8.1,7.8Hz,5H),7.37(d,J＝6.6Hz,3H),4.21(s,2H),3.75-3.65(m,1H),2.23(s,3H),1.53(d,J＝4.3Hz,3H).

By operating in a similar manner to example 6, the following compounds were prepared:

example 7: synthesis of 3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) thiazol-2-yl) benzyl) glycine (235) and hydrochloride (235 s) thereof

Synthesis of methyl 3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) thiazol-2-yl) benzoate (7A)

Referring to the procedure of example 1, the cyclization reaction of 4E and Lawsen reagent produced 7A as a white solid in 83% yield. MS (EI) m/z 384[ M-H] ^- ； ¹ H NMR(300MHz,Chloroform-d)δ8.11(s,1H),8.01(dt,J＝6.9,1.8Hz,1H),7.68(dd,J＝6.6,2.4Hz,1H),7.56(s,1H),7.48(s,1H),7.46(s,1H),7.43(s,1H),7.41(d,J＝2.7Hz,2H),7.39(d,J＝1.8Hz,2H),7.36(t,J＝1.5Hz,2H),3.90(s,3H),2.46(s,3H).

Synthesis of 3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) thiazol-2-yl) phenylmethanol (7B)

Referring to the method of example 2, 7A and LiAlH ₄ The reaction gave 7B as a white solid in 94% yield. MS (EI) m/z 356[ M-H] ^- ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.18-8.02(m,2H),7.83(d,J＝10.5Hz,1H),7.72-7.59(m,1H),7.59-7.34(m,8H),7.29(s,1H),4.59(s,2H),2.35(s,3H).

Synthesis of 2- (3-chloromethyl) phenyl-5- (2-methyl- [1,1' -biphenyl ] -3-yl) thiazole (7C)

Referring to the procedure of example 4, 7B was reacted to give 7C as a white solid in 95% yield. MS (EI) m/z 374[ M-H] ^- ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.18-8.02(m,2H),7.83(d,J＝10.5Hz,1H),7.72-7.59(m,1H),7.59-7.34(m,8H),7.29(s,1H),4.64(s,2H),2.33(s,3H).

Synthesis of methyl (3- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) thiazol-2-yl) benzyl) glycinate (235 m)

Referring to the procedure of example 1, 7C was reacted with glycine methyl ester hydrochloride to give 235m as a white solid in 76% yield. MS (EI) m/z 427[ M-H ]] ^- ； ¹ H NMR(300MHz,DMSO-d ₆ )δ8.04(d,J＝7.8Hz,2H),7.83(d,J＝7.8Hz,1H),7.62(s,1H),7.57-7.35(m,8H),7.29(dd,J＝7.5,1.5Hz,1H),3.80(s,2H),3.64(s,3H),3.35(s,2H),2.36(s,3H).

Synthesis of Compound 235 and its hydrochloride (235 s)

Referring to the procedure of example 1, 235m was hydrolyzed to give 235 as a white solid in 87% yield. Then 235 and hydrochloric acid are salified to prepare white solid 235s with the yield of 90 percent. MS (ESI) m/z 415[ M+H ] ] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ9.02(s,1H),8.36(s,1H),8.15(d,J＝7.2Hz,1H),8.05(d,J＝6.9Hz,2H),7.80(s,1H),7.70(s,1H),7.47(q,J＝8.1,7.8Hz,5H),7.37(d,J＝6.6Hz,2H),4.21(s,2H),3.75(s,2H),2.23(s,3H).

By operating in a similar manner to example 7, the following compounds were prepared:

example 8: synthesis of (4- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) thiophen-2-yl) benzyl) glycine (245) and hydrochloride (245 s) thereof

Synthesis of 2- (3-bromo-2-methylphenyl) thiophene (8A)

With reference to the synthesis of example 1, 2, 5-dibromotoluene and 2-boric acid thiophene were used as raw materials to obtain 8A as a pale yellow oily liquid, the yield was 89%, and the obtained product was directly fed to the next reaction.

Synthesis of 2- (2-methyl- [1,1' -biphenyl ] -3-yl) thiophene (8B)

With reference to the synthesis of example 1, the coupling reaction of 8A with phenylboronic acid was carried out to give 8B as a yellow oily liquid in 82% yield, which was directly put into the next reaction.

Synthesis of 2-bromo-5- (2-methyl- [1,1' -biphenyl ] -3-yl) thiophene (8C)

8B (2.20 g,8.79 mmol) was dissolved in 5mL of LDMSO, then N-bromosuccinimide (1.56 g,8.79 mmol) was added, the reaction was carried out at room temperature for 3h, after the reaction was completed, 50mL of water was added, suction filtration and drying were carried out to obtain 2.62g of pale yellow solid, the yield was 90%, which was directly put into the next reaction.

Synthesis of 4- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) thiophen-2-yl) benzaldehyde (8D)

8C (2.62 g,7.91 mol) was dissolved in 20mL dioxane, followed by the sequential addition of 2mL of water, 4-formylphenylboronic acid pinacol ester (2.38 g,10.3 mol), potassium carbonate (3.27 g,23.7 mmol), pd (dppf) Cl ₂ (32 mg,0.04 mmol). The reaction was carried out for 8h at 80℃under nitrogen. Cooling, spin-removing solvent, acetic acidEthyl ester extraction, drying over anhydrous sodium sulfate, column chromatography purification (petroleum ether: ethyl acetate=5:1 (V: V)) gave 1.61g of pale yellow solid, yield 57%, which was directly put into the next reaction.

Synthesis of methyl (4- (5- (2-methyl- [1,1' -biphenyl ] -3-yl) thiophen-2-yl) benzyl) glycinate (245 m)

Referring to the procedure of example 2, the reductive amination of 8D and glycine methyl ester hydrochloride produced 245m as a pale yellow solid in 59% yield. MS (ESI) m/z 428[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ7.76(d,J＝8.1Hz,2H),7.62(d,J＝3.6Hz,1H),7.57(d,J＝8.1Hz,3H),7.52-7.40(m,3H),7.40-7.33(m,3H),7.28-7.21(m,2H),4.18(s,2H),3.91(s,3H),3.83(s,2H),2.27(s,3H).

Synthesis of Compound 245 and hydrochloride salt thereof (245 s)

With reference to the procedure of example 1, 245m was hydrolyzed to afford 245 as a yellow solid in 85% yield. And then salifying 245 with hydrochloric acid to obtain pale yellow solid 245s with 96% yield. MS (ESI) m/z 414[ M+H ]] ⁺ ； ¹ H NMR(300MHz,DMSO-d ₆ )δ7.76(d,J＝8.1Hz,2H),7.62(d,J＝3.6Hz,1H),7.57(d,J＝8.1Hz,2H),7.52-7.40(m,4H),7.40-7.33(m,3H),7.28-7.21(m,2H),4.18(s,2H),3.83(s,2H),2.27(s,3H).

By operating in a similar manner to example 8, the following compounds were prepared:

example 9: evaluation of pharmacological Activity

1. Inhibitory Activity of the Compounds of the invention against PD-1/PD-L1 protein-protein interactions

1.1 purpose of experiment

The inhibitory activity of the compounds of the invention on PD-1/PD-L1 protein-protein interactions was tested using the PD-1/PD-L1 binding assay kit kit (BPS Bioscience).

1.2 Main Experimental materials

The PD-1/PD-L1 binding assay kit kit is purchased from BPS Bioscience and contains reagents required by experiments such as PD-1, PD-L1, anti-tag1-Eu, anti-tag2-XL665, volume Buffer, detection Buffer and the like; 384 well microplates were purchased from Perkin Elmer company; positive drugs (BMS-202) were purchased from Selleck.

1.3 instruments

Centrifuge (Eppendorf, model: 5430); enzyme label instrument (Perkin Elmer, model: enVision)

1.4 Experimental methods

(1) 1 XAssay buffer was prepared.

(2) Compound addition: 200nL was transferred to 384 reaction plates with different concentration gradients of the compounds using an Echo550 instrument.

(3) PD-L1-Biotin working solution was prepared in a 1 Xassay buffer.

(4) Adding 5 mu L of PD-L1-Biotin working solution into the compound hole and the positive control hole respectively; mu.L of Assaybuffer was added to the negative control wells.

(5) Centrifugation at 1000rpm for 30 seconds and incubation at room temperature for 15 minutes.

(6) The PD-1-Eu and Dye labeled acceptor mixed solution was prepared in a 1 Xassay buffer.

(7) Add 15. Mu.L of PD-1-Eu and Dye labeled acceptor mixture.

(8) Centrifugation at 1000rpm for 30 seconds and incubation at room temperature for 90 minutes.

(9) EnVision reads 665nm/615nm ratio. The inhibition of protein binding by the compound was calculated from the fluorescence ratio.

1.5 data equation

Wherein: ratio _sample Is the ratio of the sample wells; ratio _min : negative control Kong Bizhi mean; ratio _max : positive control Kong Bizhi mean, compound IC was calculated with Graphpad ₅₀ Values.

1.6 experimental results

The inhibitory activity of the compounds of the present invention on PD-1/PD-L protein-protein interactions is shown in Table 1. Experimental results show that the compound has remarkable inhibitory activity on PD-1/PD-L1 protein-protein interaction. Wherein A represents IC ₅₀ =1 nM-100nM; b represents IC ₅₀ =100.01 nM-500nM; c represents IC ₅₀ ＝500.01nM-20μM。

TABLE 1 inhibitory Activity of the Compounds of the invention on PD-1/PD-L1 interaction

* Control group: BMS-1018 is compound 1018 in WO 2015105641 A2.

2. Toxicity test of the Compounds of the invention on cells

To verify whether the compounds of the present invention have significant cytotoxicity, the effect of the compounds of the present invention on Lewis lung cancer cell viability was examined using the MTT method.

2.1 Experimental methods

Adding 20 mu L of 4mg/mL MTT solution into each well of a 96-well plate, placing the mixture into a cell culture box for incubation for 4 hours, centrifuging the 96-well plate, carefully sucking out liquid in the well, adding 200 mu L of dimethyl sulfoxide into each well, and placing the mixture on a shaking table for 300r oscillation for 10 minutes to enable the purple crystalline substance to be fully dissolved. Finally, the absorbance at 570nm is detected by an enzyme label instrument. The inhibition was calculated by the Bliss method based on the absorbance.

2.2 experimental results

The experimental results are shown in FIG. 1. The results show that compound 77 of the invention has no significant effect on the viability of Lewis lung cancer cells at various concentrations tested, compared to the model group, indicating that the compound of the invention has no significant cytotoxicity.

3. Effect of the inventive Compounds on cytokine INF-gamma Release

Cytokines are a unique class of molecules with both effector and regulatory effects, with important immunomodulatory effects in lymphocyte responses. Activated human Peripheral Blood Mononuclear Cells (PBMC) release cytokines such as IFN-gamma, IL-2 and TNF-alpha, and when PD-1 expressed on the PBMC membrane binds to its ligand PD-L1, the release of cytokines is inhibited. The purpose of this experiment was to examine whether the compounds of the invention reverse the ability of PD-1/PD-L1 to inhibit the secretion of INF-gamma by PBMC.

3.1 Experimental methods

Human Peripheral Blood Mononuclear Cells (PBMC) are extracted by using human lymphocyte separation liquid, inoculated into a 24-well plate, anti-CD3/anti-CD28 with the final concentration of 1g/mL and compound with different doses are added, ligand protein with the final concentration of 2g/mL, 100L of supernatant is obtained by centrifugation after 48 hours, and the expression quantity of INF-gamma in the supernatant is detected by using INF-gamma enzyme-linked immunosorbent assay kit of Daidae company.

3.2 experimental results

The experimental results are shown in FIG. 2. The results show that compared with the model group, the release of INF-gamma can be obviously promoted when anti-CD3/anti-CD28 is added, and the level of INF-gamma is obviously reduced when PD-L1 is added, which shows that PD-1/PD-L1 obviously inhibits the release of INF-gamma. The ability to significantly increase the levels of INF-gamma and present a dose-dependent profile when added at various concentrations of compound 77 of the invention demonstrates that the compound of the invention can block the inhibition of PBMC by PD-1/PD-L1, thereby restoring T cell activity and thus promoting secretion of INF-gamma.

4. Pharmacokinetic experiments of the Compounds of the invention

Good pharmacokinetic properties are an essential parameter for evaluating the drug formation of a candidate drug. In order to test whether the compound of the invention has good drug formation, the compound 77 of the invention is selected to carry out in vivo pharmacokinetics experiments of rats, and BMS-1018 is a positive drug.

4.1 Experimental methods

The animal selected in the experiment is SD rat, and the weight is 200-250 g. The doses of compound 77 and BMS-1018 were 10mg/kg orally and 2mg/kg intravenously, respectively, 3 rats in the oral group and 5 rats in the intravenous group. Plasma samples were collected at 9 time points post-dose and pharmacokinetic parameters were calculated.

ICR male mice were randomly divided into groups of 3 animals by body weight on the day of the experiment. The water is not forbidden for 12-14 h after 1 day of feeding, and the feed is fed for 4h after the feeding. Each animal was anticoagulated with 0.030mL of blood per orbital, edoak 2, at the time point of collection: (1) group i.g.: 0.0833,0.25,0.5,1,2,4,6,8,24h. (2) group i.v.: 0.25,0.5,1,2,4,6,8,24 hours after administration of the test agent. Blood samples were collected, placed on ice, and plasma was centrifuged over 30 minutes and stored at-80 ℃ prior to analysis.

Taking 10 mu L of a sample (taking the sample out of a refrigerator at-80 ℃, and swirling the sample for 30 seconds after naturally dissolving at room temperature) into a 1.5mL centrifuge tube, adding 100 mu L of an internal standard solution (5.0 ng/mL verapamil, 30.0ng/mL buspirone and 60.0ng/mL dexamethasone acetonitrile solution), and centrifuging the mixture for 3 minutes (12000 rpm) after swirling the mixture for 60 seconds; 75 mu L of supernatant is taken and transferred to a 96-hole sample feeding plate with equal volume of water, and LC-MS/MS sample feeding analysis is carried out after shaking and mixing, wherein the sample feeding amount is 10 mu L.

The data acquisition and control system software is Analyst1.5.1 software. The peak integration mode of the map sample is automatic integration; using samplesThe ratio of the peak area to the internal standard peak area was used as an index, and the concentration of the sample was regressed. Regression mode: linear regression, weight coefficient 1/X2. Pharmacokinetic parameters were analyzed using a non-compartmental model using WinNonlin Professional v 6.3.6.3 (Pharsight, USA). C (C) _max The area under the blood concentration-time curve AUC is the measured maximum blood concentration _(0→t) Calculated by a trapezoid method, T _max Peak time is reached for the blood concentration after administration.

4.2 experimental results

The experimental results are shown in table 2. As can be seen from the table, compound 77 has good pharmacokinetic parameters, in particular it has a long half-life, and an oral bioavailability of 23.94%. It is worth mentioning that the oral administration of BMS-1018 failed to detect blood concentration, and the half-life of intravenous injection was only 0.53h. These experimental results show that the compounds of the present invention have good pharmacokinetic properties.

TABLE 2 results of in vivo pharmacokinetic experiments in rats

5. In vivo pharmacodynamic evaluation of the Compounds of the invention

The Proliferation Cell Nuclear Antigen (PCNA) is a nuclear protein necessary for DNA synthesis in eukaryotic cells, and detection of PCNA can objectively evaluate the proliferation state of tumor cells. For this purpose, in the development of in vivo pharmacodynamic evaluation, T lymphocyte infiltration and IFN- γ and PCNA levels in tumor tissues were detected using immunohistochemistry and TUNEL analysis. BMS-1018 was used as a positive control group.

5.1 Experimental methods

Culture of mice: female mice were selected for 7-8 weeks and kept in SPF-grade animal feeding chambers for one week, each mouse weighing approximately 18-20 g.

Treatment of tumor cells: collecting tumor cells in logarithmic growth phase, centrifuging at 180g for 5min (4deg.C), washing with pre-cooled PBS for 2 times, blowing uniformly, and final cell concentration of 1×10 ⁷ /mL, ice bath for use.

Transplantation of tumor cells: inoculating Lewis lung cancer cell suspension into BALB/c female mouse right armpit subcutaneous, and inoculating tumor cell number of 1×10 ⁶ /only. The tumor size of the mice was measured once every two days using vernier calipers and the body weight of the mice was weighed once. When the average value of the tumor volume reaches 40mm ³ When the dose is left or right, administration is started.

Experimental grouping and dosing methods: BALB/c females transplanted with Lewis lung cancer cells were divided into 4 groups of 6 females each. Model group (solvent: PBS+2% Tween 20+2% DMSO, administered by gavage, once daily), positive control group (BMS-1018, administered by gavage, once daily, dose: 15 mg/kg), drug-treated group 1 (Compound 77, administered by gavage, once daily, dose: 5 mg/kg), drug-treated group 2 (Compound 77, administered by gavage, once daily, dose: 15 mg/kg).

And after the tumor volume reaches a certain size, ending the animal experiment. The mice were weighed, blood was taken from the eyeballs, euthanized, tumor tissue was removed, weighed and photographed. Meanwhile, part of the tissue is placed in 10% neutral fixing solution, paraffin embedded tissue is carried out by sample feeding, paraffin tissue sections are manufactured, and H & E staining, TUNEL and immunohistochemical analysis are carried out. Experimental procedures refer to the test kit instructions.

5.2 experimental results

The experimental results are shown in FIG. 3. The results show that compared with the model group, the compound 77 can obviously inhibit the growth of the Lewis lung cancer mice transplanted tumor at the administration dose of 5mg/kg and 15mg/kg, shows dose dependency and does not influence the weight of the mice. Furthermore, the inhibition activity of compound 77 on transplanted tumor was significantly better than that of control BMS-1018 at the same dose, indicating that the compound of the present invention

The results of immunohistochemistry and TUNEL experiments show that compared with a model group, the compound 77 can obviously promote infiltration of T lymphocytes in tumor tissues at doses of 5mg/kg and 15mg/kg, improve IFN-gamma level, reduce PCNA protein expression, and reverse PD-1/PD-L1 mediated immunosuppression effect of the compound 77 at doses of 5mg/kg is stronger than that of a BMS-1018 control group.

6. Effect of the inventive Compounds on tumor microenvironment T lymphocyte infiltration

T lymphocytes are the core executors of the human immune system and play an important role in tumor immune responses. Tumor Infiltrating Lymphocytes (TILs) refer to those leukocytes which leave the blood stream and enter the tumor. When a large number of tumor infiltrating lymphocytes are present in the tumor microenvironment, it is shown that the body initiates an immune response against the tumor. Activation of the PD-1/PD-L1 signaling pathway inhibits the anti-tumor immune microenvironment, resulting in reduced infiltration of lymphocytes. The purpose of this experiment was to analyze the effect of the compounds of the invention on T lymphocyte infiltration in the tumor microenvironment.

6.1 Experimental methods

Taking part of tumor tissues stripped in experiment 4, cutting the tumor tissues into a 15mL centrifuge tube, adding collagenase IV (0.5 mg/mL) and DNase I (0.5 mg/mL), digesting for 30min at 37 ℃, filtering out residual tissue fragments, centrifuging, re-suspending cells, and then using CD45, CD3, CD4 and CD8 streaming antibodies of different channels to dye for 30min in a dark state, and detecting by a streaming cytometer.

6.2 experimental results

The experimental results are shown in FIG. 4. The results show that compound 77 is able to significantly promote CD45 at doses of 5mg/kg and 15mg/kg compared to the model group ⁺ White blood cells, CD45 ⁺ CD3 ⁺ T lymphocytes, CD8 ⁺ CD45 ⁺ CD3 ⁺ Infiltration of cytotoxic T cells and dose-dependency is exhibited, but for CD4 ⁺ CD45 ⁺ CD3 ⁺ Regulatory T lymphocytes have a weak effect. Furthermore, at the same dose, compound 77 has a greater capacity to promote lymphocyte infiltration than BMS-1018, particularly for CD45 ⁺ CD3 ⁺ CD8 ⁺ The increased infiltration of cytotoxic T lymphocytes is more pronounced. These experiments demonstrate that the compounds of the present invention are capable of effectively reversing the PD-1/PD-L1 mediated immunosuppression and remodelling the anti-tumor immune microenvironment.

It should be pointed out that other compounds in the invention have remarkable anti-tumor effect in mouse transplantation tumor models of various tumor types such as CT26, EMT6, B16F1, PAN02, LLC and the like, and the compounds can promote infiltration of lymphocytes to tumor microenvironment, improve IFN-gamma secretion in tumor tissues and reduce PCNA protein expression. These experiments demonstrate that the compounds of the present invention are capable of blocking PD-1/PD-L1 mediated immunosuppression, activating an anti-tumor immune response.

Claims (10)

1. A phenyl and biphenyl substituted five-membered heterocyclic compound is characterized by having a structure shown in a formula I, and further comprises a stereoisomer, a meso form, a racemate, a prodrug, a crystal, a pharmaceutically acceptable salt or a mixture thereof,

wherein:

(1) When X is O, A and N, B is C; when X is O, A and C, B is N or C;

(2) When X is S, A, B is N or C;

(3) When X is NH, A is N, B is N;

(4) When X is N, A and N, B is O; when X is N, A and C, B is O or NH; when X is N, A is O, B is C or N;

R ¹ selected from methyl, cyano, hydroxy or halogen;

R ² selected from hydrogen, halogen, nitro, cyano, hydroxy, C ₁ -C ₄ Alkyl, C ₁ -C ₄ Alkoxy, C ₁ -C ₄ Haloalkyl or-O (CH) ₂ ) _n Ar is as follows; wherein n is an integer from 0 to 4; ar is selected from aryl or aromatic heterocycle; the aromatic heterocycle comprises one or more heteroatoms selected from O, S or N; the C is ₁ -C ₄ Alkyl, aryl or aromatic heterocyclic groups are substituted with one or more W groups;

w is selected from hydrogen, halogen, cyano, hydroxy, mercapto, carboxyl, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Alkoxy, C ₁ -C ₆ Alkylamino or C ₁ -C ₆ A haloalkyl group;

R ³ 、R ⁴ each independently selected from hydrogen, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Alkoxy, C ₁ -C ₈ Alkylamino, C ₃ -C ₈ Cycloalkyl, 5-7 membered heterocyclyl or R ³ And R is ⁴ Together with the nitrogen atom to which they are attached, form a 5-7 membered heterocyclyl; the heterocyclic group may optionally contain one or more heteroatoms selected from O, S or N; the C is ₁ -C ₈ Alkyl, C ₁ -C ₈ Alkoxy, C ₁ -C ₈ Alkylamino, C ₃ -C ₈ Cycloalkyl or 5-7 membered heterocyclyl is substituted with one or more Y groups;

y is selected from hydrogen, halogen, hydroxy, mercapto, methylthio, carbonyl, carboxyl, amino, guanidino, furyl, tetrahydropyrrolyl, morpholinyl, N-methylpiperazinyl, C ₁ -C ₄ Alkyl, -CO ₂ R ⁵ 、-NHCOR ⁵ 、-NR ⁶ R ⁷ or-CONR ⁶ R ⁷ The method comprises the steps of carrying out a first treatment on the surface of the The C is ₁ -C ₄ Alkyl groups are substituted with one or more hydroxy groups or halogen;

R ⁵ selected from C ₁ -C ₈ An alkyl group;

R ⁶ 、R ⁷ each independently selected from hydrogen, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Alkoxy, C ₃ -C ₈ Cycloalkyl or R ⁸ And R is ⁹ Together with the nitrogen atom to which they are attached, form a 5-7 membered heterocyclyl; the C is ₁ -C ₈ Alkyl, C ₁ -C ₈ Alkoxy, C ₃ -C ₈ Cycloalkyl or 5-7 membered heterocyclyl is substituted with one or more Z groups;

z is selected from hydrogen, halogen, hydroxy, mercapto, carboxyl, amino or acetamido.

2. The phenyl and biphenyl substituted five-membered heterocyclic compound according to claim 1, wherein in the structure:

R ¹ selected from methyl or halogen;

R ² selected from hydrogen, nitro or halogen;

R ³ 、R ⁴ each independently selected from hydrogen, C ₁ -C ₅ Alkyl or R ³ And R is ⁴ Together with the nitrogen atom to which they are attached, form a 5-6 membered N-containing heterocyclyl; the C is ₁ -C ₅ Alkyl or 5-6 membered heterocyclyl is substituted with one or more Y groups;

y is selected from hydrogen, hydroxy, carbonyl, carboxyl, guanidino, and C ₁ -C ₄ Alkyl, -CO ₂ R ⁵ 、-NR ⁶ R ⁷ or-CONR ⁶ R ⁷ ；C ₁ -C ₄ Alkyl groups are substituted with one or more hydrogen or hydroxy groups;

R ⁵ selected from C ₁ -C ₄ An alkyl group;

R ⁶ 、R ⁷ each independently selected from hydrogen or C ₁ -C ₄ An alkyl group.

3. The phenyl and biphenyl substituted five-membered heterocyclic compound according to claim 1, wherein in the structure:

R ¹ selected from methyl or chlorine;

R ² selected from hydrogen, nitro, fluorine, chlorine or bromine;

R ³ 、R ⁴ each independently selected from hydrogen, C ₁ -C ₅ Alkyl or R ³ And R is ⁴ Together with the nitrogen atom to which they are attached, form a 5-to 6-membered heterocyclic group containing one N atom; the C is ₁ -C ₅ Alkyl or 5-6 membered heterocyclyl is substituted with one or more Y groups;

y is selected from hydrogen, hydroxy, carbonyl, carboxyl, guanidino, and C ₁ -C ₄ Alkyl, -CO ₂ CH ₃ Amino or-CONH ₂ ；C ₁ -C ₄ Alkyl groups are substituted with one or more hydrogen or hydroxy groups.

4. The phenyl-and biphenyl-substituted five-membered heterocyclic compound according to claim 1, selected from any one of the following compounds:

5. a process for the preparation of phenyl-and biphenyl-substituted five-membered heterocyclic compounds as described in any one of claims 1-4, characterized by a process selected from any one of the following:

The method comprises the following steps: when X is S, A and B are N, the compound a-1 is used as a raw material to prepare a compound with a general formula (I) through Suzuki coupling, esterification, hydrazinolysis, condensation, cyclization and condensation reaction, or the compound with the general formula (I) is prepared through further alkali hydrolysis;

the second method is as follows: when X is NH, A and B are N, the compound d-1 is subjected to cyclization, reduction, oxidation and reductive amination to obtain a compound of the general formula (I), or is subjected to further alkali hydrolysis to obtain the compound of the general formula (I);

and a third method: when X is N, A is O and B is N, the compound a-2 is subjected to addition, cyclization, reduction, oxidation and reductive amination to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

the method four: when X and A are N and B is O, the compound a-3 is subjected to Suzuki coupling, addition, condensation, reduction, oxidation and reductive amination to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

and a fifth method: when X is O, A is C and B is N, the compound a-4 is subjected to coupling, suzuki coupling, bromination, amination, condensation, cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

the method six: when X is O, A is N and B is C, the compound B-1 is subjected to chlorination, condensation, cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

And a seventh method: when X is N, A is O and B is C, the compound B-1 is subjected to amination, cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkaline hydrolysis to prepare the compound of the general formula (I);

method eight: when X is N, A is C and B is O, the compound C-5 is subjected to cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

method nine: when X is N, A is C, and B is NH, the compound is prepared into a compound of the general formula (I) through cyclization, reduction, halogenation and condensation reaction of C-5, or is further prepared into the compound of the general formula (I) through alkaline hydrolysis;

method ten: when X is S, A is C and B is N, the compound e-5-1 is subjected to cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkaline hydrolysis to prepare the compound of the general formula (I);

method eleven: when X is S, A is N, B is C, the compound C-6 is subjected to cyclization, reduction, halogenation and condensation reaction to prepare a compound of the general formula (I), or is subjected to further alkali hydrolysis to prepare the compound of the general formula (I);

method twelve: when X is S or O and A and B are C, the compound a-4 is subjected to coupling, bromination, suzuki coupling and reductive amination reaction to obtain a compound of the general formula (I), or is subjected to further alkali hydrolysis to obtain the compound of the general formula (I);

Wherein R is ¹ 、R ² 、R ³ 、R ⁴ Is as defined in any one of claims 1 to 4.

6. Use of a phenyl-and biphenyl-substituted five-membered heterocyclic compound as described in any one of claims 1-4 in the preparation of a PD-L1 inhibitor medicament.

7. Use of a phenyl-and biphenyl-substituted five-membered heterocyclic compound as described in any one of claims 1-4 in the preparation of an immunomodulator medicament.

8. The use according to claim 7, wherein the immunomodulator drug is a drug for preventing and/or treating tumors, infectious diseases, inflammatory diseases, organ transplant rejection and autoimmune diseases.

9. A pharmaceutical composition comprising a phenyl-and biphenyl-substituted five-membered heterocyclic compound as described in any one of claims 1-4 and a pharmaceutically acceptable carrier.

10. The pharmaceutical combination according to claim 9, wherein the pharmaceutical formulation is in the form of a tablet, capsule, powder, pill, granule, injection, oral liquid, syrup, inhalant, ointment, patch or suppository.

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