CN118026948A - Tri-aromatic ring compound and preparation method, pharmaceutical composition and application thereof - Google Patents

Abstract

The invention discloses a tri-aromatic ring compound, a preparation method, a pharmaceutical composition and application thereof. The structure of the triple aromatic ring compound is shown as a formula I, and the triple aromatic ring compound also comprises stereoisomers, meso, racemates, prodrugs, crystals, pharmaceutically acceptable salts or mixtures thereof, has PD-L1 inhibitory activity, can remarkably inhibit PD-1/PD-L1 protein-protein interaction and block PD-1/PD-L1 signal paths, so that the triple aromatic ring compound can be widely applied to the preparation of immunomodulators for preventing and/or treating tumors, infectious diseases, inflammatory diseases, autoimmune diseases and organ transplant rejection, and meanwhile, the preparation method of the compound is beneficial to the structure expansion.

Description

Tri-aromatic ring compound and preparation method, pharmaceutical composition and application thereof

Technical Field

The invention relates to a tri-aromatic ring compound, a preparation method thereof, a pharmaceutical composition and application thereof, in particular to a tri-aromatic ring 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, 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 can be expressed at low levels in thymus in addition to mature T cells, CD4 ^-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-L1 is mainly through inhibiting PI3K-AKT and RAS signal paths, blocking activation of transcription factors having important roles in T cell activation, proliferation, function and survival, such as activin-1 (AP-1), activated T cell Nuclear Factor (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-L1 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-L1 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-L1 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-L1 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-L1 signaling pathway is overactivated. In addition, blocking the PD-1/PD-L1 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.

Currently, there are allAnd/>More than 10 PD-1/PD-L1 monoclonal antibody medicaments are marketed, are applied to clinically treating malignant melanoma, non-small cell lung cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer and other solid tumors and hematological cancers, greatly improve prognosis of tumor patients and break the treatment bottleneck of various cancers. 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 to the inherent immunogenicity, it is prone to cause adverse events associated with drug-induced immunity in patients (irAEs); 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: the first object of the invention is to provide a tri-aromatic ring compound, the second object is to provide a preparation method of the compound, the third object is to provide a pharmaceutical composition containing the compound, and the fourth object is to provide a pharmaceutical application of the compound and the pharmaceutical composition thereof.

The technical scheme is as follows: the tri-aromatic ring compound has the structure of the formula I, and also comprises a stereoisomer, a meso form, a racemate, a prodrug, a crystal, a pharmaceutically acceptable salt or a mixture thereof,

Wherein:

A. B is C, N or O;

X is N, O or S;

R ¹、R² is independently selected from hydrogen, halogen, nitro, cyano, hydroxy, C ₁-C₄ alkyl, C ₁-C₄ alkoxy, C ₁-C₄ haloalkyl, 5-7 membered heterocycle, 5-7 membered aromatic heterocycle or-O (CH ₂)_n Ar; N is selected from integers from 0-4 and Ar is selected from 5-7 membered aryl or aromatic heterocycle; the aryl, heterocycle or aromatic heterocycle contains one or more heteroatoms selected from O, S or N; the C ₁-C₄ alkyl, aryl, aromatic heterocycle or heterocycle is substituted with one or more W groups);

W is selected from hydrogen, halogen, cyano, hydroxy, mercapto, carboxyl, C ₁-C₆ alkyl, C ₁-C₆ alkoxy, C ₁-C₆ alkylamino or C ₁-C₆ haloalkyl;

R ³、R⁴ is each independently selected from hydrogen, C ₁-C₈ alkyl, C ₁-C₈ alkoxy, C ₁-C₈ alkylamino, C ₃-C₈ cycloalkyl, a 5-7 membered heterocycle, or R ³、R⁴ together with the nitrogen atom to which they are attached form a 5-7 membered heterocycle; the heterocyclic ring comprises one or more heteroatoms selected from O, S or N; the C ₁-C₈ alkyl, C ₁-C₈ alkoxy, C ₁-C₈ alkylamino, C ₃-C₈ cycloalkyl or 5-7 membered heterocycle is substituted with one or more Y groups;

Y is selected from hydrogen, halogen, hydroxy, mercapto, methylthio, carbonyl, carboxyl, amino, guanidino, furyl, tetrahydropyrrolyl, morpholino, N-methylpiperazino, C ₁-C₄ alkyl, -CO ₂R⁵、-NHCOR⁵、-NR⁶R⁷ or-CONR ⁶R⁷; the C ₁-C₄ alkyl is substituted with one or more hydroxy or halogen;

R ⁵ is selected from C ₁-C₈ alkyl;

R ⁶、R⁷ is each independently selected from hydrogen, C ₁-C₈ alkyl, C ₁-C₈ alkoxy, C ₃-C₈ cycloalkyl, or R ⁸、R⁹ together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring; the C ₁-C₈ alkyl, C ₁-C₈ alkoxy, C ₃-C₈ cycloalkyl or 5-7 membered heterocycle is substituted with one or more Z groups;

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

Preferably, in the structure:

A. B, X is selected from any one of the following:

(1) When A is N, B is N or O, X is N, O or S;

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

r ¹ is selected from hydrogen, halogen, methyl, trifluoromethyl, methoxy, nitro, cyclopropyl, thiophene, [2,3] pyrrole, or [2,3] -1, 4-dioxane;

R ² is selected from hydrogen, halogen, nitro or benzyloxy;

R ³、R⁴ is each independently selected from hydrogen, C ₁-C₅ alkyl, or R ³、R⁴ together with the nitrogen atom to which they are attached form a 5-6 membered N-containing heterocycle; said C ₁-C₅ alkyl or 5-6 membered heterocycle being substituted with one or more Y groups;

Y is selected from hydrogen, hydroxy, carbonyl, carboxyl, guanidino, C ₁-C₄ alkyl, -CO ₂CH₃, amino or-CONH ₂; the C ₁-C₄ alkyl group is substituted with one or more hydrogen or hydroxy groups.

Preferably, in the structure:

Selected from: Selected from:

Selected from:

Preferably, the tri-aromatic ring compound is selected from any one of the following compounds:

Preferably, the pharmaceutically acceptable salt is a salt of the compound with an acid or base, the acid being hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid; the alkali is inorganic alkali containing alkali metal cation, alkaline earth metal cation or ammonium cation salt, or choline, piperazine, morpholine, triethylamine, diisopropylamine and trimethylamine.

"Pharmaceutically acceptable salts" refers to salts of compounds prepared from compounds having a particular substituent with a relatively non-toxic acid or base. When compounds contain relatively acidic functionalities, base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When compounds contain relatively basic functional groups, the acid addition salts may be obtained by contacting the free form of such compounds with a sufficient amount of acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogenphosphates, dihydrogenphosphates, sulfuric acid (forming sulfates or bisulphates), hydroiodic acid, phosphorous acid, and the like, and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like, and salts of organic acids including amino acids (such as arginine and the like), glucuronic acid, and the like.

"Pharmaceutically acceptable salts" can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

Preferably, the stereoisomer is an isomer introduced by chiral C, N in R ³、R⁴.

Preferably, the prodrug is an ester prodrug introduced by the carboxyl group in R ³、R⁴, more preferably a C ₁-C₄ alkyl ester.

The preparation method of the tri-aromatic ring compound is selected from any one of the following methods:

the method comprises the following steps: when A is O, B and X are N, the compound a-1 reacts with hydroxylamine hydrochloride, and then the compound of the formula I is prepared through cyclization, reduction, oxidation, reductive amination and hydrolysis;

the second method is as follows: when X is O and A and B are N, the compound a-2 is subjected to esterification, hydrazinolysis, condensation, ring closure, reduction, oxidation, reductive amination and hydrolysis to prepare a compound of the formula I;

and a third method: when X is S, A and B are N, the compound d-2 is subjected to cyclization, reduction, oxidation, reductive amination and hydrolysis to prepare a compound of the formula I;

the method four: when X is O, A is N and B is C, the compound a-4 is subjected to bromination, ammonolysis, condensation, cyclization, reduction, oxidation, reductive amination and hydrolysis to obtain a compound of the formula I;

And a fifth method: when A is O, B is C and X is N, the compound a-5 is subjected to condensation, cyclization, reduction, oxidation, reductive amination and hydrolysis to prepare the compound of the formula I;

The method six: when B is NH, X is N and A is C, the compound a-6 is subjected to cyclization, reduction, oxidation, reductive amination and hydrolysis to prepare the compound of the formula I;

And a seventh method: when X is S, A is N and B is C, the compound a-7 is subjected to two-step Suzuki coupling, reductive amination and hydrolysis to obtain a compound of the formula I;

Method eight: when X is S and A and B are C, the compound a-8 is subjected to Suzuki coupling, bromination, suzuki coupling, reductive amination and hydrolysis to obtain a compound of the formula I;

wherein R ¹、R²、R³、R⁴ is as defined above;

And salifying the compound of the formula I prepared by the method with corresponding acid or alkali to obtain the pharmaceutically acceptable salt.

The pharmaceutical composition comprises the tri-aromatic ring and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical composition is in the form of tablet, capsule, powder, pill, granule, injection, oral liquid, syrup, inhalant, ointment, patch or suppository.

The pharmaceutically acceptable carrier can be an auxiliary material widely used in the field of medicine production. Adjuvants are primarily used to provide a safe, stable and functional pharmaceutical composition, and may also provide means for allowing the subject to dissolve at a desired rate after administration, or for promoting effective absorption of the active ingredient after administration of the composition. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients can comprise one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, sizing agents, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption retarders, dissolution inhibitors, enhancing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

The pharmaceutical compositions of the present invention may be prepared according to the disclosure using any method known to those of skill in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical compositions of the present invention may be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular). The pharmaceutical compositions of the invention may also be in controlled or sustained release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder formulations which may be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic formulations; aerosols, such as nasal sprays or inhalants; a liquid dosage form suitable for parenteral administration; suppositories and lozenges.

The tri-aromatic ring compound or the pharmaceutical composition thereof disclosed by the invention is applied to the preparation of PD-L1 inhibitor drugs.

The tri-aromatic ring compound or the pharmaceutical composition thereof is applied to the preparation of immunomodulator medicines.

Preferably, the medicament is a medicament for preventing or treating a tumor, an infectious disease, an inflammatory disease, organ transplant rejection, or an autoimmune disease.

Further preferred, the tumor is one or more of malignant melanoma, lung cancer, breast cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, lymphatic cancer, leukemia, prostate cancer, testicular cancer, renal cancer, brain cancer, head and neck cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelial cancer, thyroid tumor, liver cancer, esophageal cancer.

Further preferably, the infectious disease is an infection caused by one or more of human immunodeficiency virus, hepatitis b virus, hepatitis c virus, influenza virus, polio virus, cytomegalovirus, coxsackievirus, human papilloma virus, epstein-barr virus, varicella-zoster virus.

Further preferred, the autoimmune disease is one or more of rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, nodular vasculitis, multiple sclerosis, myasthenia gravis, mixed connective tissue disease, psoriasis, autoimmune response due to infection.

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

the compound has remarkable inhibitory activity on the protein-protein interaction of PD-1/PD-L1, and the inhibitory activity IC ₅₀ reaches the nanomolar concentration level, so that the application is wide. Meanwhile, the preparation method of the compound is beneficial to expanding various structure types.

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 BrukerAV-300 and 400MHz nuclear magnetic resonance apparatus, chemical shift value (delta) in ppm, coupling constant (J) value 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; the column chromatography uses crude pore (ZCX-II) 300-400 mesh column chromatography silica gel of Qingdao ocean chemical plant.

Example 1: synthesis of (3- (5- (3-fluorophenyl) -1,2, 4-oxadiazol-3-yl) benzyl) glycine hydrochloride (1) and hydrochloride (1 s)

Synthesis of methyl 3- (N-hydroxycarbamoyl) benzoate (1A)

Methyl meta-cyanobenzoate (2.61 g,16.21 mmol) was added to 30mL of absolute ethanol, hydroxylamine hydrochloride (3.91 g,56.27 mmol) and sodium hydrogencarbonate (5.30 g,63.10 mmol) were added in this order, and the reaction was refluxed for 12h. Cooled, concentrated under reduced pressure, extracted with ethyl acetate, the organic phases were combined, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and purified by column chromatography to give 2.53g of a white solid in yield 80％.MS(ESI)m/z 195[M+H]⁺;¹H NMR(300MHz,DMSO-d₆)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- (3-fluorophenyl) -1,2, 4-oxadiazol-3-yl) benzoate (1B)

2-Fluorobenzoic acid (0.32 g,2.28 mmol), 1-hydroxybenzotriazole (0.33 g,2.44 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.48 g,2.50 mmol) and potassium carbonate (0.34 g,2.46 mmol) were added to 5mL of DMF and reacted at room temperature for 0.5h, intermediate 1A (0.40 g,2.06 mmol) was added and reacted at 110℃for 12h under N ₂. Cooling, extracting with ethyl acetate, mixing the organic phases, washing with saturated NaCl solution, drying with anhydrous sodium sulfate, and purifying by column chromatography to obtain white solid 0.35g, yield 52％.MS(ESI)m/z 299[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.15(d,J＝8.4Hz,2H),8.12-8.00(m,3H),7.86(s,1H),7.71(d,J＝7.8Hz,2H),3.87(s,3H).

Synthesis of 3- (5- (3-fluorophenyl) -1,2, 4-oxadiazol-3-yl) benzyl alcohol (1C)

1B (0.30 g,1.00 mmol) was added to 4mL dry THF, stirred in an ice bath for 5min, liAlH ₄ (76 mg,2.00 mmol) was slowly added and reacted in an ice bath for 3h. Quenching by dropwise adding saturated NaCl solution, regulating pH to 5 with dilute HCl solution, extracting with ethyl acetate, mixing organic phases, washing with saturated NaCl solution, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and drying to obtain white solid 0.24g, yield 90％.MS(ESI)m/z 271[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.21(d,J＝8.4Hz,2H),8.18-8.07(m,3H),7.86(s,1H),7.79(d,J＝7.8Hz,2H),4.60(s,2H).

Synthesis of 3- (5- (3-fluorophenyl) -1,2, 4-oxadiazol-3-yl) benzaldehyde (1D)

1C (0.24 g,0.89 mmol) was added to 4mL of anhydrous DCM, and dess-Martin oxidant (0.40 g,0.99 mmol) was slowly added and reacted at room temperature for 4h. Quenching the saturated sodium thiosulfate solution, extracting with ethyl acetate, combining organic phases, washing with saturated NaCl solution, drying with anhydrous sodium sulfate, and purifying by column chromatography to obtain 0.23g of white solid with yield 96％.MS(ESI)m/z 269[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)10.02(s,1H),8.34(s,1H),8.21-8.15(m,2H),8.12(d,J＝8.1Hz,2H),7.98(t,J＝8.1Hz,1H),7.33-7.22(m,2H).

Synthesis of methyl (3- (5- (3-fluorophenyl) -1,2, 4-oxadiazol-3-yl) benzyl) glycinate (1 m)

1D (0.11 g,0.42 mmol), glycine methyl ester hydrochloride (0.16 g,1.25 mmol), triethylamine (0.17 mL,1.25 mmol) were added to 5mL of anhydrous DCM, reacted at room temperature for 0.5h, acetic acid (0.17 mL,2.50 mmol), sodium cyanoborohydride (0.13 g,2.10 mmol) was added and reacted at room temperature for 5h. Concentrating under reduced pressure, adding saturated NaCl solution 5mL, extracting with ethyl acetate, mixing organic phases, washing with saturated NaCl solution, drying with anhydrous sodium sulfate, and purifying by column chromatography to obtain white solid 0.10g, yield 70％.MS(ESI)m/z 342[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.35(s,1H),8.25-8.17(m,2H),8.15(d,J＝8.1Hz,2H),7.97(t,J＝8.1Hz,1H),7.3-7.22(m,2H),4.20(s,2H),3.97(s,3H),3.87(s,2H).

Synthesis of ethyl (3- (5- (3-fluorophenyl) -1,2, 4-oxadiazol-3-yl) benzyl) glycinate (1 e)

Referring to the preparation method of 1m, white solid is obtained from glycine ethyl ester hydrochloride in yield 65％.MS(EI)m/z 356[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.32(s,1H),8.23(t,J＝1.8Hz,1H),8.19-8.12(m,2H),7.84(dd,J＝7.8,1.8Hz,1H),7.76-7.64(m,2H),7.58(t,J＝7.8Hz,1H),4.29(s,2H),4.16(q,J＝6.9Hz,2H),3.90(s,2H),1.27(t,J＝6.9Hz,3H).

Synthesis of isopropyl (3- (5- (3-fluorophenyl) -1,2, 4-oxadiazol-3-yl) benzyl) glycinate (1 i)

Referring to the preparation method of 1m, the white solid is obtained from isopropyl glycinate hydrochloride in yield 61％.MS(EI)m/z 370[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.29-8.18(m,2H),8.00-7.90(m,2H),7.70-7.55(m,4H),5.10(p,J＝6.3Hz,1H),4.21(s,2H),3.88(s,2H),1.25(d,J＝6.3Hz,6H).

Synthesis of (3- (5- (3-fluorophenyl) -1,2, 4-oxadiazol-3-yl) benzyl) glycine (1) and hydrochloride (1 s) thereof

1M (0.1 g,0.29 mmol) was added to 2mL of anhydrous methanol, and lithium hydroxide hydrate (25 mg,0.59 mmol) was added and reacted at room temperature for 3h. Concentrating under reduced pressure, adding saturated NaCl solution, adjusting pH to 6 with HCl solution (4 mol/L), vacuum filtering, and oven drying to obtain white solid 76mg with a yield of 81%. 1 (76 mg,0.23 mmol) was added to a solution of dioxane hydrochloride (4 mol/L) and reacted at room temperature for 4 hours. Concentrating under reduced pressure, washing the solid with anhydrous diethyl ether to obtain 76mg of pale yellow solid in yield 91％.MS(ESI)m/z 328[M+H]⁺;¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.51(s,1H),8.35(s,1H),8.25-8.15(m,2H),8.13(d,J＝8.1Hz,2H),7.97(t,J＝8.1Hz,1H),7.31-7.22(m,2H),4.20(s,2H),3.87(s,2H).

By operating in a similar manner to example 1, the following compounds were prepared:

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example 24: synthesis of (4- (5- (2-fluorophenyl) -1,3, 4-oxadiazol-2-yl) benzyl) glycine (24) and hydrochloride (24 s) thereof

Synthesis of methyl 2-fluorobenzoate (24A)

2-Fluorobenzoic acid (1.40 g,10.00 mmol) was added to 10mL of anhydrous methanol, 2mL of 98% concentrated sulfuric acid was slowly added dropwise under ice-bath conditions, and the reaction was refluxed for 5 hours. Concentrating under reduced pressure, adding saturated NaCl solution 5mL, vacuum filtering, oven drying to obtain white solid 1.44g, and obtaining yield 95％.MS(ESI)m/z 155[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)7.98-7.94(m,1H),7.57-7.52(m,2H),7.25-7.14(m,1H),3.96(s,3H).

Synthesis of 2-fluorobenzoyl hydrazine (24B)

24A (1.44 g,9.50 mmol) was added to 15mL of absolute ethanol, 80% hydrazine hydrate (2.87 mL,47.50 mmol) was added and the reaction was refluxed for 5h. Concentrating under reduced pressure, adding 10mL saturated NaCl solution, vacuum filtering, oven drying to obtain white solid 1.39g, and obtaining yield 96％.MS(ESI)m/z 155[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.08-7.99(m,1H),7.57-7.52(m,2H),7.25-7.14(m,1H).

Synthesis of methyl 4- (2- (2-fluorobenzoyl) hydrazine-1-carbonyl) benzoate (24C)

24B (0.15 g,1 mmol) and triethylamine (0.46 mL,3.30 mmol) were added to 5mL of anhydrous DCM and methyl 4-chloroformylbenzoate (0.22 g,1.10 mmol) was slowly added dropwise under ice-bath conditions and reacted at room temperature for 0.5h. 10mL of saturated NaCl solution is added, suction filtration and drying are carried out to obtain 0.32g of white solid with yield 100％.MS(ESI)m/z 317[M+H]⁺;¹HNMR(300MHz,Chloroform-d)δ(ppm)8.32-8.26(m,2H),8.01-7.82(m,2H),7.76-7.70(m,3H),7.55-7.42(m,1H),3.89(s,3H).

Synthesis of methyl 4- (5- (2-fluorophenyl) -1,3, 4-oxadiazol-2-yl) benzoate (24D)

24C (0.32 g,1.00 mmol) and 1mL phosphorus oxychloride were added to the reaction flask and reacted at 80℃for 8h. Quenched by dropwise addition of saturated NaCl solution, extracted with ethyl acetate, the organic phases were combined, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 0.30g of a white solid in yield 99％.MS(ESI)m/z 299[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.35-8.31(m,2H),8.12-7.99(m,2H),7.76-7.70(m,3H),7.55-7.42(m,1H),3.89(s,3H).

Synthesis of 4- (5- (2-fluorophenyl) -1,3, 4-oxadiazol-2-yl) benzyl alcohol (24E)

Referring to the method of example 1, intermediate 24D was reduced with LiAlH ₄ to give a white liquid 24E in yield 90％.MS(ESI)m/z 271[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.39-8.30(m,2H),8.11-8.03(m,2H),7.83-7.76(m,3H),7.51-7.42(m,1H),4.49(s,2H).

Synthesis of 4- (5- (2-fluorophenyl) -1,3, 4-oxadiazol-2-yl) benzaldehyde (24F)

Referring to the procedure of example 1, intermediate 24E was oxidized with dess-martin oxidant to give 24F as a pale yellow solid in yield 95％.MS(ESI)m/z 269[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)9.90(s,1H),8.32-8.26(m,2H),8.01-7.82(m,2H),7.76-7.70(m,3H),7.55-7.42(m,1H).

Synthesis of methyl (4- (5- (2-fluorophenyl) -1,3, 4-oxadiazol-2-yl) benzyl) glycinate (24 m)

Referring to the procedure of example 1, intermediate 24F and glycine methyl ester hydrochloride were subjected to reductive amination to give 24m as a white solid in yield 69％.MS(ESI)m/z 342[M+H]⁺.¹H NMR(300MHz,Chloroform-d)δ(ppm)8.30-8.21(m,2H),8.05-7.92(m,2H),7.72-7.61(m,3H),7.54-7.42(m,1H),4.29(s,2H),3.98(s,3H),3.90(s,2H).

Synthesis of (4- (5- (2-fluorophenyl) -1,3, 4-oxadiazol-2-yl) benzyl) glycine (24) and hydrochloride (24 s) thereof

With reference to the procedure of example 1, 24m was hydrolyzed to give 24 as a white solid in 80% yield. Then salifying 24 with dioxane hydrochloride to obtain white solid 24s, yield 85％.MS(ESI)m/z 328[M+H]⁺;¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.83(s,1H),8.31-8.22(m,2H),8.01-7.82(m,2H),7.78-7.79(m,3H),7.51-7.41(m,1H),4.29(s,2H),3.90(s,2H).

By operating in a similar manner to example 24, the following compounds were prepared:

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example 47: synthesis of (4- (5- (2-fluorophenyl) -1,3, 4-thiadiazol-2-yl) benzyl) glycine (47) and hydrochloride (47 s) thereof

Synthesis of methyl 4- (5- (3-fluorophenyl) -1,3, 4-thiadiazol-2-yl) benzoate (47A)

Methyl 4- (2- (3-fluorobenzoyl) hydrazine-1-carbonyl) benzoate (0.32 g,1.00 mmol) and Lawson's reagent (0.44 g,1.10 mmol) were added to 5mL anhydrous toluene and reacted at reflux for 4h. Concentrating under reduced pressure, purifying by column chromatography to obtain white solid 0.30g, and obtaining yield 95％.MS(ESI)m/z 315[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.10-7.99(m,3H),7.90(d,J＝8.4Hz,2H),7.77(d,J＝8.4Hz,2H),7.42(s,1H),3.83(s,3H).

Synthesis of 4- (5- (3-fluorophenyl) -1,3, 4-thiadiazol-2-yl) benzyl alcohol (47B)

Referring to the method of example 1, intermediate 47A was subjected to reduction with LiAlH ₄ to give a pale yellow liquid 47B in yield 91％.MS(ESI)m/z 287[M+H]⁺;MS(ESI)m/z 358[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.08-7.95(m,3H),7.88(d,J＝8.4Hz,2H),7.71(d,J＝8.4Hz,2H),7.48(s,1H),4.56(s,2H).

Synthesis of 4- (5- (3-fluorophenyl) -1,3, 4-thiadiazol-2-yl) benzaldehyde (47C)

Referring to the procedure of example 1, intermediate 47B was oxidized with dess-martin oxidant to give 47C as a pale yellow solid in yield 88％.MS(ESI)m/z 285[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)9.84(s,1H),8.11-8.01(m,3H),7.85(d,J＝8.4Hz,2H),7.73(d,J＝8.4Hz,2H),7.50(s,1H).

Synthesis of methyl (4- (5- (3-fluorophenyl) -1,3, 4-thiadiazol-2-yl) benzyl) glycinate (47 m)

Referring to the procedure of example 1, intermediate 47C and glycine methyl ester hydrochloride were subjected to reductive amination to give 47m as a pale yellow solid in yield 80％.MS(ESI)m/z 358[M+H]⁺.¹H NMR(300MHz,Chloroform-d)δ(ppm)8.08-7.95(m,3H),7.88(d,J＝8.4Hz,2H),7.71(d,J＝8.4Hz,2H),7.48(s,1H),4.20(s,2H),3.90(s,3H),3.70(s,2H).

Synthesis of (4- (5- (3-fluorophenyl) -1,3, 4-thiadiazol-2-yl) benzyl) glycine (47) and hydrochloride (47 s) thereof

With reference to the procedure of example 1, 47m was hydrolyzed to give 47 as a pale yellow solid in 90% yield. Then the salt is formed by 47 and hydrochloric acid to prepare white solid 47s, the yield 85％.MS(ESI)m/z 344[M+H]⁺;¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.59(s,1H),8.02-7.84(m,3H),7.68(d,J＝8.4Hz,2H),7.61(d,J＝8.4Hz,2H),7.42(s,1H),4.21(s,2H),3.70(s,2H).

By operating in a similar manner to example 47, the following compounds were prepared:

example 55: synthesis of (4- (2- (3-chlorophenyl) oxazol-5-yl) benzyl) glycine (55) and its hydrochloride salt (55 s)

Synthesis of methyl 4- (2-bromoacetyl) benzoate (55A)

Methyl 4-acetylbenzoate (1.78 g,10.00 mmol) and copper bromide (4.48 g,20.00 mmol) were added to 20mL ethyl acetate and reacted under reflux for 8h. The sodium thiosulfate/sodium carbonate mixed solution was quenched, extracted with ethyl acetate, the organic phases were combined, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. 10mL of THF, triethylamine (0.76 mL,5.50 mmol) and diethyl phosphite (0.69 g,5.00 mmol) were added slowly with stirring and reacted at room temperature for 2h. Concentrating under reduced pressure, purifying by column chromatography to obtain red liquid 2.30g, and obtaining yield 89％.MS(ESI)m/z 257[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.13(d,J＝8.4Hz,2H),8.02(d,J＝8.4Hz,2H),4.45(s,2H),3.94(s,3H).

Synthesis of methyl 4- (diformylglycoamino) benzoate hydrochloride (55B)

55A (2.30 g,8.90 mmol) was added to 10mL of acetonitrile, sodium diformylamide (1.20 g,10.70 mmol) was added and reacted at 80℃for 8h under an N ₂ atmosphere. Suction filtration, concentration under reduced pressure, addition of 10mLHCl (4 mol/L) solution and reaction at 80℃for 2h. Suction filtering and drying to obtain white solid 1.33g, yield 50％.MS(ESI)m/z 194[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.13(d,J＝8.4Hz,2H),8.02(d,J＝8.4Hz,2H),4.52(s,2H),3.93(s,3H).

Synthesis of methyl 4- ((3-chlorobenzoyl) GANAMINAMINE) benzoate (55C)

55B (1.33 g,4.40 mmol) and triethylamine (2.62 mL,19.00 mmol) were added to 10mL of anhydrous DCM and 3-chlorobenzoyl chloride (0.84 g,4.80 mmol) was slowly added dropwise under ice-bath conditions and reacted at room temperature for 2h. Concentrating under reduced pressure, adding saturated NaCl solution, vacuum filtering, oven drying to obtain white solid 1.44g, and obtaining yield 99％.MS(ESI)m/z 332[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.08(s,1H),7.99(d,J＝2.4Hz,2H),7.96(d,J＝2.4Hz,2H),7.66-7.60(m,3H),5.12(s,2H),3.81(s,3H).

Synthesis of methyl 4- (2- (3-chlorophenyl) oxazol-5-yl) benzoate (55D)

56C (1.44 g,4.40 mmol) was added to 8mL of glacial acetic acid, and 1mL of 98% concentrated sulfuric acid was slowly added dropwise and reacted at 80℃for 4h. Adding saturated sodium carbonate solution for quenching, filtering and drying to obtain white solid 1.36g, yield 99％.MS(ESI)m/z 314[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.15(s,1H),8.08(s,1H),7.89(d,J＝2.4Hz,2H),7.76(d,J＝2.4Hz,2H),7.61-7.52(m,3H),3.81(s,3H).

Synthesis of 4- (2- (3-chlorophenyl) oxazol-5-yl) benzyl alcohol (55E)

Referring to the procedure of example 1, intermediate 55D was reduced with LiAlH ₄ to give a white solid 55E in yield 88％.MS(ESI)m/z 286[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.25(s,1H),8.12(s,1H),8.03(d,J＝2.4Hz,2H),7.96(d,J＝2.4Hz,2H),7.84-7.72(m,3H),4.41(s,2H).

Synthesis of 4- (2- (3-chlorophenyl) oxazol-5-yl) benzaldehyde (55F)

Referring to the procedure of example 1, intermediate 5D was oxidized with dess-martin oxidant to give 55F as a pale yellow solid in yield 90％.MS(ESI)m/z 284[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)10.11(s,1H),8.20(s,1H),8.04(s,1H),7.93(d,J＝2.4Hz,2H),7.86(d,J＝2.4Hz,2H),7.64-7.53(m,3H).

Synthesis of methyl (4- (2- (3-chlorophenyl) oxazol-5-yl) benzyl) glycinate (55 m)

Referring to the procedure of example 1, intermediate 55F and glycine methyl ester hydrochloride were subjected to reductive amination to afford 55m as a white solid in yield 88％.MS(ESI)m/z 357[M+H]⁺.¹H NMR(300MHz,Chloroform-d)δ(ppm)8.15(s,1H),8.08(s,1H),7.99(d,J＝2.5Hz,2H),7.96(m,J＝2.5Hz,2H),7.66-7.60(m,3H),4.22(s,2H),3.94(s,3H),3.88(s,2H).

Synthesis of (4- (2- (3-chlorophenyl) oxazol-5-yl) benzyl) glycine (55) and its hydrochloride salt (55 s)

With reference to the procedure of example 1, 55m was hydrolyzed to give 55 as a white solid in 95% yield. Then 55 is salified with hydrochloric acid to prepare white solid 55s, yield 89％.MS(ESI)m/z 343[M+H]⁺;¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.71(s,1H),8.12(s,1H),8.01(s,1H),7.94(d,J＝2.4Hz,2H),7.80(d,J＝2.4Hz,2H),7.76-7.62(m,3H),4.22(s,2H),3.88(s,2H).

By operating in a similar manner to example 55, the following compounds were prepared:

Example 59: synthesis of (3- (2- (3-chlorophenyl) oxazol-4-yl) benzyl) glycine (59) and its hydrochloride salt (59 s)

Synthesis of methyl 3- (2- (3-chlorophenyl) oxazol-4-yl) benzoate (59A)

3-Chlorobenzamide (0.77 g,5.00 mmol) and methyl 3- (2-bromoacetyl) benzoate (1.40 g,6.00 mmol) were added to 1.5 mLN-methylpyrrolidone, 0.5mL glacial acetic acid was added, and the mixture was reacted at 120℃for 8 hours under N ₂. Adding saturated sodium carbonate solution, extracting with ethyl acetate, mixing organic phases, drying with anhydrous sodium sulfate, and purifying by column chromatography to obtain white solid 0.80g, yield 51％.MS(ESI)m/z 314[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.86(s,1H),8.09-8.00(m,2H),8.00-7.90(m,2H),7.70-7.55(m,4H),3.86(s,3H).

Synthesis of 3- (2- (3-chlorophenyl) oxazol-4-yl) benzyl alcohol (59B)

Referring to the method of example 1, intermediate 59A was reduced with LiAlH ₄ to give 59B as a white solid in yield 87％.MS(ESI)m/z 286[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.87(s,1H),8.19-8.12(m,2H),8.10-7.98(m,2H),7.77-7.52(m,4H),4.46(s,2H).

Synthesis of 3- (2- (3-chlorophenyl) oxazol-4-yl) benzaldehyde (59C)

Referring to the procedure of example 1, intermediate 59B was oxidized with dess-martin oxidant to give 59C as a white solid in yield 81％.MS(ESI)m/z 284[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)10.16(s,1H),8.81(s,1H),8.00-7.89(m,2H),7.82-7.63(m,2H),7.60-7.51(m,4H).

Synthesis of methyl (3- (2- (3-chlorophenyl) oxazol-4-yl) benzyl) glycinate (59 m)

Referring to the procedure of example 1, intermediate 59C and glycine methyl ester hydrochloride were subjected to reductive amination to give 59m as a white solid in yield 90％.MS(ESI)m/z 357[M+H]⁺.¹H NMR(300MHz,Chloroform-d)δ(ppm)8.86(s,1H),8.09-8.00(m,2H),8.00-7.90(m,2H),7.70-7.55(m,4H),4.21(s,2H),3.97(s,3H),3.88(s,2H).

Synthesis of (3- (2- (3-chlorophenyl) oxazol-4-yl) benzyl) glycine (59) and its hydrochloride salt (59 s)

With reference to the procedure of example 1, 59m was hydrolyzed to give 59 as a white solid in 90% yield. Then the 59 and the hydrochloric acid form salt to prepare white solid 59s with yield 80％.MS(ESI)m/z 343[M+H]⁺;¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.78(s,1H),8.91(s,1H),8.18-8.10(m,2H),8.05-7.93(m,2H),7.81-7.62(m,4H),4.21(s,2H),3.88(s,2H).

By operating in a similar manner to example 59, the following compounds were prepared:

Example 61: synthesis of (3- (4- (3-chlorophenyl) -1H-imidazol-2-yl) benzyl) glycine (61) and hydrochloride salt (61 s) thereof

Synthesis of methyl 3- (4- (3-chlorophenyl) -1H-imidazol-2-yl) benzoate (61A)

Methyl 3-carbamoyl-benzoate (1.06 g,4.95 mmol) and NaHCO ₃ (0.48 g,5.71 mmol) were added to 5mL of THF, and after 5min of reflux, 2-bromo-1- (3-chlorophenyl) ethan-1-one (1.10 g,3.80 mmol) was added and the reaction was continued under reflux for 5h. Column chromatography purification gave 0.56g of a white solid in yield 47％.MS(ESI)m/z 313[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)δ(ppm)8.15(s,2H),8.12-7.99(m,3H),7.95(s,1H),7.85(s,1H),7.43(s,1H),7.30(s,1H),3.89(s,3H).

Synthesis of 3- (4- (3-chlorophenyl) -1H-imidazol-2-yl) benzyl alcohol (61B)

Referring to example 1, intermediate 61A was reduced with LiAlH ₄ to give a white solid 61B in yield 70％.MS(ESI)m/z 285[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.22(s,1H),8.07(d,J＝8.4Hz,2H),7.96(s,1H),7.90(d,J＝7.8Hz,1H),7.56(d,J＝7.8Hz,2H),7.47(s,1H),7.37(s,1H),5.33(s,1H),4.60(s,2H).

Synthesis of 3- (4- (3-chlorophenyl) -1H-imidazol-2-yl) benzaldehyde (61C)

Referring to the procedure of example 1, intermediate 61B was oxidized with dess-martin oxidant to give 61C as a pale yellow solid in yield 80％.MS(ESI)m/z 283[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)10.21(s,1H),8.33(d,J＝8.1Hz,2H),8.21(s,1H),8.02-7.92(m,2H),7.71(d,J＝8.1Hz,2H),7.61-7.40(m,2H).

Synthesis of methyl (3- (4- (3-chlorophenyl) -1H-imidazol-2-yl) benzyl) glycinate (61 m)

Referring to the procedure of example 1, intermediate 61 and glycine methyl ester hydrochloride were subjected to reductive amination to give 61m as a white solid in yield 85％.MS(ESI)m/z 356[M+H]⁺.¹H NMR(300MHz,Chloroform-d)δ(ppm)8.20-7.93(m,3H),7.79(s,1H),7.64(s,1H),7.39(s,2H),7.24(d,J＝7.4Hz,1H),7.14(s,1H),4.11-3.94(m,2H),3.86(s,3H),3.48(s,2H).

Synthesis of (3- (4- (3-chlorophenyl) -1H-imidazol-2-yl) benzyl) glycine (61) and hydrochloride salt (61 s) thereof

With reference to the procedure of example 1, 61m was hydrolyzed to give 61 as a white solid in 95% yield. Then the 61 is salified with hydrochloric acid to prepare white solid 61s, yield 90％.MS(ESI)m/z 342[M+H]⁺;¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.86(s,1H),9.62(s,1H),8.32(d,J＝8.1Hz,2H),8.22(s,1H),8.01-7.93(m,2H),7.79(d,J＝8.1Hz,2H),7.62-7.48(m,2H),4.27(s,2H),3.90(s,2H).

Example 62: synthesis of (4- (5- (3-chlorophenyl) thiazol-2-yl) benzyl) glycine (62) and hydrochloride salt (62 s) thereof

Synthesis of 4- (5-bromothiazol-2-yl) benzaldehyde (62A)

2, 5-Dibromothiazole (2.41 g,10.00 mmol), 4-formylphenylboronic acid (2.25 g,15.00 mmol), potassium carbonate (2.76 g,20.00 mmol) and Pd (PPh ₃)₄ (0.58 g,0.50 mmol) were added to 25mL of 1, 4-dioxane and 3mL of water, reacted at 80℃under N ₂ atmosphere for 12h, concentrated under reduced pressure, added with 10mL of saturated NaCl solution, extracted with ethyl acetate, combined with the organic phase, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and purified by column chromatography to give 2.05g of pale yellow solid in yield 77％.MS(ESI)m/z 268[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)8.51(s,1H),8.13(d,J＝8.1Hz,2H),8.02(d,J＝8.1Hz,2H),3.94(s,3H).

4- (5- (3-Chlorophenyl) thiazol-2-yl) benzaldehyde (62B)

62A (2.05 g,7.70 mmol), 3-chlorobenzeneboronic acid (1.80 g,11.55 mmol), potassium carbonate (2.12 g,15.40 mmol) and Pd (PPh ₃)₄ (0.44 g,0.38 mmol) were added to 25mL of 1, 4-dioxane and 3mL of water, reacted at 80℃under N ₂ atmosphere for 12h. Concentrated under reduced pressure, 50mL of water was added, extracted with ethyl acetate, the organic phases were combined, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and purified by column chromatography to give a yellow solid 2.07g, yield 90％.MS(ESI)m/z 300[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)10.27(s,1H),8.42(s,1H),7.91-7.86(m,2H),7.82-7.78(m,1H),7.62-7.56(m,1H),7.51-7.46(m,3H),7.40-7.32(m,1H).

Synthesis of methyl (4- (5- (3-chlorophenyl) thiazol-2-yl) benzyl) glycinate (62 m)

Referring to example 1, intermediate 62B and glycine methyl ester hydrochloride were subjected to reductive amination to afford 63m as a yellow solid in yield 80％.MS(ESI)m/z 373[M+H]⁺.¹H NMR(300MHz,Chloroform-d)δ(ppm)8.45(s,1H),7.97-7.91(m,2H),7.82-7.73(m,1H),7.70-7.61(m,1H),7.58-7.52(m,3H),7.49-7.41(m,1H),4.19(s,2H),3.85(s,3H),3.78(s,2H).

Synthesis of methyl (4- (5- (3-chlorophenyl) thiazol-2-yl) benzyl) glycinate (62) and hydrochloride (62 s) thereof

With reference to the procedure of example 1, 62m was hydrolyzed to give 62 as a yellow solid in 98% yield. Then the yellow solid 62s is prepared by salifying 62 with hydrochloric acid, the yield is 99％.MS(ESI)m/z 359[M+H]⁺;¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.64(s,1H),8.47(s,1H),7.96-7.90(m,2H),7.89-7.86(m,1H),7.71-7.66(m,1H),7.58-7.51(m,3H),7.50-7.46(m,1H),4.19(s,2H),3.78(s,2H).

By operating in a similar manner to example 62, the following compounds were prepared:

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example 66: synthesis of (4- (5- (3-chlorophenyl) thiophen-2-yl) benzyl) glycine (66) and hydrochloride salt (66 s) thereof

Synthesis of 4- (thiophen-2-yl) benzaldehyde (66A)

4-Bromobenzaldehyde (0.92 g,5.00 mmol), 2-thiopheneboronic acid (0.96 g,7.50 mmol), potassium carbonate (1.38 g,10.00 mmol) and Pd (PPh ₃)₄ (58 mg,0.05 mmol) were added to 10mL of 1, 4-dioxane and 1mL of water, reacted at 80℃under N ₂ atmosphere for 8h, concentrated under reduced pressure, extracted with ethyl acetate, combined organic phases, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and purified by column chromatography to give a pale yellow solid 0.56g, yield 60％.MS(ESI)m/z 189[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)9.99(s,1H),7.88(d,J＝8.4Hz,2H),7.76(d,J＝8.4Hz,2H),7.47-7.45(m,1H),7.40-7.39(m,1H),7.14-7.12(m,1H).

Synthesis of 4- (5-bromothiophen-2-yl) benzaldehyde (66B)

66A (0.56 g,3.00 mmol) and NBS (0.53 g,3.00 mmol) were added to 8mL of DMF and reacted at room temperature for 4h. 50mL of saturated NaCl solution is added, suction filtration and drying are carried out to obtain 0.80g of yellow solid with yield 99％.MS(ESI)m/z 267[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)9.97(s,1H),7.90(d,J＝8.4Hz,2H),7.76(d,J＝8.4Hz,2H),7.57-7.55(m,1H),7.49-7.41(m,1H).

Synthesis of 4- (5- (3-chlorophenyl) thiophen-2-yl) benzaldehyde (66C)

66B (0.80 g,3.00 mmol), 3-chlorobenzeneboronic acid (0.70 g,4.50 mmol), potassium carbonate (0.83 g,6.00 mmol) and Pd (PPh ₃)₄ (35 mg,0.03 mmol) were added to 10mL of 1, 4-dioxane and 1mL of water, reacted at 80℃under N ₂ for 8h, concentrated under reduced pressure, extracted with ethyl acetate, the organic phases were combined, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and purified by column chromatography to give a yellow solid 0.65g, yield 73％.MS(ESI)m/z 299[M+H]⁺;¹H NMR(300MHz,Chloroform-d)δ(ppm)10.08(s,1H),7.88(s,1H),7.78(d,J＝8.4Hz,2H),7.67-7.60(m,3H),7.58(d,J＝7.2Hz,2H),7.40(d,J＝7.2Hz,2H).

Synthesis of methyl (4- (5- (3-chlorophenyl) thiophen-2-yl) benzyl) glycinate (66 m)

Referring to example 1, intermediate 66C and glycine methyl ester hydrochloride were subjected to reductive amination to afford 67m as a yellow solid in yield 91％.MS(ESI)m/z 372[M+H]⁺.¹H NMR(300MHz,Chloroform-d)δ(ppm)7.88(s,1H),7.78(d,J＝8.4Hz,2H),7.65-7.60(m,3H),7.55(d,J＝7.2Hz,2H),7.38(d,J＝7.2Hz,2H),4.18(s,2H),3.85(s,3H),3.77(s,2H).

Synthesis of (4- (5- (3-chlorophenyl) thiophen-2-yl) benzyl) glycine (66) and hydrochloride salt (66 s) thereof

With reference to the procedure of example 1, 66m was hydrolyzed to afford yellow solid 66 in 94% yield. Then the 66 and the hydrochloric acid form salt to prepare yellow solid 67s, the yield 92％.MS(ESI)m/z 358[M+H]⁺;¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.81(s,1H)7.89(s,1H),7.72(d,J＝8.4Hz,2H),7.66-7.60(m,3H),7.58(d,J＝7.2Hz,2H),7.40(d,J＝7.2Hz,2H),4.16(s,2H),3.75(s,2H).

By operating in a similar manner to example 68, the following compounds were prepared:

Example 70: inhibitory Activity of Compounds of the invention on PD-1/PD-L1 protein-protein interactions

1. Purpose of experiment

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

2. Main experiment 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.

3. Instrument for measuring and controlling the intensity of light

Centrifuge (Eppendorf, model: 5430); enzyme label instrument (PERKIN ELMER, model: enVision)

4. Experimental method

(1) 1X Assaybuffer 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 a1 Xassay buffer.

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

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

(6) A PD-1-Eu and Dye labeled acceptor mixture was prepared in 1X Assaybuffer.

(7) Add 15. Mu.LPD-1-Eu and Dye labeled acceptor mix.

(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.

5. Technical formula

Wherein: ratio _sample is the Ratio of sample wells; ratio _min: negative control Kong Bizhi mean; ratio _max: positive control Kong Bizhi mean. Compound IC ₅₀ values were calculated using Graphpad.

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 to 100nM; b represents IC ₅₀ =100.01 to 500nM; c represents IC ₅₀ > 500nM.

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

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Claims (10)

1. A tri-aromatic ring 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:

A. B is C, N or O;

X is N, O or S;

R ¹、R² is independently selected from hydrogen, halogen, nitro, cyano, hydroxy, C ₁-C₄ alkyl, C ₁-C₄ alkoxy, C ₁-C₄ haloalkyl, 5-7 membered heterocycle, 5-7 membered aromatic heterocycle or-O (CH ₂)_n Ar; N is selected from integers from 0-4 and Ar is selected from 5-7 membered aryl or aromatic heterocycle; the aryl, heterocycle or aromatic heterocycle contains one or more heteroatoms selected from O, S or N; the C ₁-C₄ alkyl, aryl, aromatic heterocycle or heterocycle is substituted with one or more W groups);

W is selected from hydrogen, halogen, cyano, hydroxy, mercapto, carboxyl, C ₁-C₆ alkyl, C ₁-C₆ alkoxy, C ₁-C₆ alkylamino or C ₁-C₆ haloalkyl;

R ³、R⁴ is each independently selected from hydrogen, C ₁-C₈ alkyl, C ₁-C₈ alkoxy, C ₁-C₈ alkylamino, C ₃-C₈ cycloalkyl, a 5-7 membered heterocycle, or R ³、R⁴ together with the nitrogen atom to which they are attached form a 5-7 membered heterocycle; the heterocyclic ring comprises one or more heteroatoms selected from O, S or N; the C ₁-C₈ alkyl, C ₁-C₈ alkoxy, C ₁-C₈ alkylamino, C ₃-C₈ cycloalkyl or 5-7 membered heterocycle is substituted with one or more Y groups;

Y is selected from hydrogen, halogen, hydroxy, mercapto, methylthio, carbonyl, carboxyl, amino, guanidino, furyl, tetrahydropyrrolyl, morpholino, N-methylpiperazino, C ₁-C₄ alkyl, -CO ₂R⁵、-NHCOR⁵、-NR⁶R⁷ or-CONR ⁶R⁷; the C ₁-C₄ alkyl is substituted with one or more hydroxy or halogen;

R ⁵ is selected from C ₁-C₈ alkyl;

R ⁶、R⁷ is each independently selected from hydrogen, C ₁-C₈ alkyl, C ₁-C₈ alkoxy, C ₃-C₈ cycloalkyl, or R ⁸、R⁹ together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring; the C ₁-C₈ alkyl, C ₁-C₈ alkoxy, C ₃-C₈ cycloalkyl or 5-7 membered heterocycle is substituted with one or more Z groups;

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

2. The tri-aromatic ring compound according to claim 1, wherein in the structure:

A. B, X is selected from any one of the following:

(1) When A is N, B is N or O, X is N, O or S;

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

r ¹ is selected from hydrogen, halogen, methyl, trifluoromethyl, methoxy, nitro, cyclopropyl, thiophene, [2,3] pyrrole, or [2,3] -1, 4-dioxane;

R ² is selected from hydrogen, halogen, nitro or benzyloxy;

R ³、R⁴ is each independently selected from hydrogen, C ₁-C₅ alkyl, or R ³、R⁴ together with the nitrogen atom to which they are attached form a 5-6 membered N-containing heterocycle; said C ₁-C₅ alkyl or 5-6 membered heterocycle being substituted with one or more Y groups;

Y is selected from hydrogen, hydroxy, carbonyl, carboxyl, guanidino, C ₁-C₄ alkyl, -CO ₂CH₃, amino or-CONH ₂; the C ₁-C₄ alkyl group is substituted with one or more hydrogen or hydroxy groups.

3. The tri-aromatic ring compound according to claim 1, wherein in the structure:

selected from: /(I)

Selected from:

Selected from:

4. the tri-aromatic ring compound according to claim 1, wherein the compound is selected from any one of the following:

5. The tri-aromatic ring compound according to claim 1, wherein the pharmaceutically acceptable salt is a salt of the compound with an acid or base, the acid being hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid; the alkali is inorganic alkali containing alkali metal cation, alkaline earth metal cation or ammonium cation salt, or choline, piperazine, morpholine, triethylamine, diisopropylamine and trimethylamine.

6. A process for the preparation of a tri-aromatic ring compound according to claim 1, selected from any one of the following:

the method comprises the following steps: when A is O, B and X are N, the compound a-1 reacts with hydroxylamine hydrochloride, and then the compound of the formula I is prepared through cyclization, reduction, oxidation, reductive amination and hydrolysis;

the second method is as follows: when X is O and A and B are N, the compound a-2 is subjected to esterification, hydrazinolysis, condensation, ring closure, reduction, oxidation, reductive amination and hydrolysis to prepare a compound of the formula I;

and a third method: when X is S, A and B are N, the compound d-2 is subjected to cyclization, reduction, oxidation, reductive amination and hydrolysis to prepare a compound of the formula I;

the method four: when X is O, A is N and B is C, the compound a-4 is subjected to bromination, ammonolysis, condensation, cyclization, reduction, oxidation, reductive amination and hydrolysis to obtain a compound of the formula I;

And a fifth method: when A is O, B is C and X is N, the compound a-5 is subjected to condensation, cyclization, reduction, oxidation, reductive amination and hydrolysis to prepare the compound of the formula I;

The method six: when B is NH, X is N and A is C, the compound a-6 is subjected to cyclization, reduction, oxidation, reductive amination and hydrolysis to prepare the compound of the formula I;

And a seventh method: when X is S, A is N and B is C, the compound a-7 is subjected to two-step Suzuki coupling, reductive amination and hydrolysis to obtain a compound of the formula I;

Method eight: when X is S and A and B are C, the compound a-8 is subjected to Suzuki coupling, bromination, suzuki coupling, reductive amination and hydrolysis to obtain a compound of the formula I;

wherein R ¹、R²、R³、R⁴ is as defined in claim 1;

and salifying the compound of the formula I prepared by the method with corresponding acid or alkali to obtain pharmaceutically acceptable salt of the compound.

7. A pharmaceutical composition comprising the terphenyl ring of claim 1 and a pharmaceutically acceptable carrier.

8. Use of the tri-aromatic ring compound of claim 1 or the pharmaceutical composition of claim 7 in the preparation of a PD-L1 inhibitor medicament.

9. Use of the tri-aromatic ring compound of claim 1 or the pharmaceutical composition of claim 7 in the preparation of an immunomodulatory agent.

10. The use according to claim 8 or 9, wherein the medicament is a medicament for the prophylaxis or treatment of a tumor, an infectious disease, an inflammatory disease, organ transplant rejection or an autoimmune disease.