CN115677665B - Biphenyl-containing derivative and medical application thereof - Google Patents

Abstract

The invention relates to the field of pharmaceutical chemistry, and discloses biphenyl derivatives with PD-1/PD-L1 inhibitory activity and medical application thereof. The invention also discloses a composition containing the biphenyl derivative with PD-1/PD-L1 inhibitory activity or pharmaceutically acceptable salt and pharmaceutically acceptable carrier thereof, and application of the composition in preparation of PD-1/PD-L1 inhibitor, which can be used for treating various cancers or tumors related to immune checkpoint PD-1/PD-L1 and has wide applicability.

Description

Biphenyl-containing derivative and medical application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to derivatives containing biphenyl structures and application of the compounds in preparation of medicines for treating tumors.

Background

In recent years, tumor immunotherapy has become a focus in the field of tumor therapy, and in contrast to the conventional treatment methods directed against tumor cells, tumor immunotherapy kills tumor cells by using the human autoimmune system. Activation of immune checkpoint pathways inhibits T cell activation, prevents excessive activation of the human immune system, maintains immune tolerance in normal organisms, and avoids autoimmune disease. Tumor cells express an excessive amount of negative immune checkpoint proteins, which, upon binding to lymphocytes, overactivate immune checkpoint pathways leading to the occurrence of tumor immune escape. Among these immune checkpoints, the overactivation of PD-1/PD-L1 plays a crucial role in the development of tumors. When the blocking agent is used for blocking the PD-1/PD-L1 interaction, immune cells can be enabled to be recognized again and kill tumor cells, so that the aim of treating tumors is fulfilled. At present, the PD-1/PD-L1 monoclonal antibody has excellent clinical results and has better curative effects in treating tumors such as melanoma, colon cancer, non-small cell lung cancer and the like (Clinical and Translational Oncology,2019,21:702–712;The Oncologist,2019,24(Suppl 1):S31-S41;Human Vaccines&Immunotherapeutics,2014,10(11):3111-6;Journal of Medicinal Chemistry,2020,63,(22):13825–13850).

In recent years, various PD-1/PD-L1 monoclonal antibodies have been approved for sale, and these monoclonal antibodies have made breakthrough progress in the clinical treatment of various tumors. The survival of many tumor patients is significantly prolonged and some patients are completely relieved. Although the clinical effect of the monoclonal antibody is remarkable, the monoclonal antibody has long half-life and can cause serious immune-related adverse reactions due to long binding time with the target. The production process of the monoclonal antibody medicine is complex, the price is high, the storage and the transportation are inconvenient, and the common patient can only look at the medicine. Compared with monoclonal antibodies, small molecule drugs have the advantages of low price, oral administration, crossing biological barriers, convenient transportation and storage, good membrane permeability, non-immunogenicity and the like. At present, no PD-1/PD-L1 small molecule inhibitor is marketed, so that the development of the PD-1/PD-L1 protein-protein interaction inhibitor has great practical significance and potential application prospect.

Disclosure of Invention

The invention aims to: aiming at the prior art, the invention provides biphenyl derivatives with PD-1/PD-L1 inhibitory activity, a preparation method thereof and pharmaceutical application as PD-1/PD-L1 protein-protein interaction inhibitor.

The technical scheme is as follows: the invention discloses a biphenyl derivative shown in a general formula (I) or pharmaceutically acceptable salt thereof:

Wherein:

x, Y and Z each independently represent: n or CH;

a represents A substituted phenyl or aromatic heterocyclic group, wherein the aromatic heterocyclic group is A five-membered or six-membered aromatic ring containing 1-3O, N or S atoms, and the substituent is H, F, cl, br, CN, NH ₂、OH、CF₃、OCF₃、C₁～C₄ alkyl or C ₁-C₄ alkoxy;

m=0, 1 or 2; n=0, 1 or 2;

R ¹ and R ² each independently represent NR ⁶R⁷、OR⁷ or a substituted quaternary, penta-or hexa-membered heterocycloalkyl containing 1 to 2O or N atoms

R ⁶ represents hydrogen or alkyl of C ₁～C₃;

R ⁷ represents substituted C ₁～C₆ alkyl, wherein the substituent is OH, NH ₂、COOH、CONH₂、COOCH₃、COOCH₂CH₃、C₁～C₄ alkoxy which can be monosubstituted or polysubstituted;

The substituted quaternary, five-membered or six-membered heterocyclic alkyl containing 1-2O or N atoms is substituted tetrahydropyrrole-1-yl, substituted piperidine-1-yl, substituted morpholin-1-yl, substituted piperazin-1-yl or substituted azetidin-1-yl, wherein the substituent is OH、NH₂、COOH、CONH₂、COOCH₃、COOCH₂CH₃、CF₃、OCF₃、C₁～C₄ alkoxy, C ₁～C₄ alkyl, and can be monosubstituted or polysubstituted;

R ³ and R ⁴ each independently represent an alkyl group or a cyclopropyl group of H, F, cl, br, CN, CF ₃、C₁～C₃;

r ⁵ represents an alkyl group or a cyclopropyl group of H, F, cl, br, CN, CF ₃、OCH₃、OCH₂CH₃、OCF₃、C₁～C₄.

When m=1, a is a1, 4-disubstituted 1,2, 3-triazole ring, the compounds of the invention are preferably of the following formula (II):

Wherein: x, Y, Z, R ¹、R²、R³、R⁴、R⁵ and n are as defined above.

N is preferably 0 or 1.

X is preferably N or CH.

Y and Z are preferably CH.

R ¹ and R ² are each preferably OH,Wherein R ⁸ represents CH ₃、CH₂CH₃、CH₂CH₂ OH, formyl, acetyl, cyclopropyl and the like; r ⁹ and R ¹⁰ each independently represent H、OH、COOH、CH₂COOH、CH₂NH₂、CH₂OH、CH₂CH₂OH、F、Cl、Br、CH₃、CH₂CH₃、 cyclopropyl or the like; r ¹¹ represents OH、NH₂、NHCH₃、NHCH₂CH₃、CH₃、OCH₃、OCH₂CH₃;R¹² represents CONH ₂、NHCOCH₃、OH、CH₂OH、CH₂CH₂OH、COOH、CH₂ COOH or the like; r ¹³ represents H, CH ₃、CH₂CH₃、CH₂OH、CH₂CH₂ OH or the like; r ¹⁴ and R ¹⁵ each independently represent H、COOH、NH₂、F、Cl、Br、CH₃、CH₂CH₃、CH₂OH、CH₂CH₂OH、CONH₂、 cyclopropyl or the like; w represents CH ₂、O、NH、N-CH₃、N-CH₂CH₃、N-CH₂CH₂OH、N-COCH₃ or the like; p represents 0 or 1.

R ³ and R ⁴ are each preferably F, cl or CH ₃.

R ⁵ is preferably H, F, CH ₃ or OCH ₃.

Wherein:

X is more preferably N.

R ¹ and R ² are more preferably-OH, -NHCH ₂CONH₂、-NHCH₂CH₂ OH,

R ³ and R ⁴ are more preferably CH ₃.

R ⁵ is more preferably OCH ₃.

Pharmaceutically acceptable salts of the above compounds are acid addition salts of the compounds of general formula (I) or (II), wherein the acid used to form the salt is: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.

The compounds of the general formula (I) according to the invention can be prepared by the following process:

when X represents N or CH; y and Z represent CH; a represents a1, 4-disubstituted 1,2, 3-triazole ring; m=1; when n=0 or 1, the compound (I) can be prepared by the following method:

wherein: x, n, R ¹、R²、R³、R⁴ and R ⁵ are as defined above.

Preparing a compound V from a compound III and a compound IV through a suzuki reaction, wherein the solvent is selected from toluene, N-dimethylformamide, N-dimethylacetamide, ethylene glycol dimethyl ether, ethylene glycol monomethyl ether, 1, 4-dioxane, tetrahydrofuran, methanol, ethanol, acetonitrile, acetone, water or a mixed solvent formed by any two solvents, preferably a mixed solvent of 1, 4-dioxane and water; the base is selected from sodium ethoxide, potassium acetate, sodium hydroxide, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium carbonate or tripotassium phosphate, preferably potassium carbonate; the catalyst used is selected from Pd (PPh ₃)₄、Pd(dppf)Cl₂、Pd(PPh₃)₂Cl₂、Pd(OAc)₂ or NiCl ₂ (dppf), preferably Pd (PPh ₃)₄; the reaction temperature is selected from 50 to 120 ℃, preferably from 60 to 100 ℃).

The compound V is firstly salified with sulfuric acid, then reacts with sodium nitrite to obtain diazonium salt, and finally reacts with pinacol ester of biboronate to prepare the compound VI, wherein the solvent is selected from methanol, ethanol, tetrahydrofuran, 1, 4-dioxane, ethylene glycol dimethyl ether, acetonitrile, water or a mixed solvent formed by the solvents, and the mixed solvent of the methanol and the water is preferred.

Preparing a compound VIII from a compound VI and a compound VII through a suzuki reaction, wherein the solvent is selected from toluene, N-dimethylformamide, N-dimethylacetamide, ethylene glycol dimethyl ether, ethylene glycol monomethyl ether, 1, 4-dioxane, tetrahydrofuran, methanol, ethanol, acetonitrile, acetone, water or a mixed solvent formed by any two solvents, and preferably 1, 4-dioxane and water; the base is selected from sodium ethoxide, potassium acetate, sodium hydroxide, potassium carbonate, sodium bicarbonate, potassium bicarbonate, tripotassium phosphate, sodium carbonate or triethylamine, preferably potassium carbonate; the catalyst used is Pd (PPh ₃)₄、Pd(dppf)Cl₂、Pd(PPh₃)₂Cl₂、Pd(OAc)₂ or NiCl ₂ (dppf), preferably Pd (PPh ₃)₄. The reaction temperature is selected from 50 to 120 ℃, preferably 60 to 100 ℃.

The target compound II is prepared by the reductive amination reaction of the compound VIII and the corresponding amine, wherein the solvent is selected from dichloromethane, dichloroethane, chloroform, carbon tetrachloride, tetrahydrofuran, methanol, toluene, ethanol, acetonitrile, N-dimethylformamide or a mixed solvent consisting of any two or three solvents, preferably dichloromethane or a mixed solvent of dichloromethane and methanol; the reducing agent is selected from sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, preferably sodium triacetoxyborohydride; the reaction temperature is selected from 0 to 80 ℃, preferably 25 to 40 ℃.

The application of the compound (including chiral isomer) with the general formula (I) and the hydrate, solvate or crystal thereof in preparing PD-1/PD-L1 inhibitor medicines is also within the protection scope of the invention.

Further, the PD-1/PD-L1 inhibitor can be used for preparing medicines for treating cancers such as non-small cell lung cancer, colon cancer, melanoma and the like.

Pharmacological experiments show that the biphenyl derivatives can generate good inhibition effect on the interaction of PD-1/PD-L1 in a homogeneous phase time resolved fluorescence experiment (HTRF). In a surface plasmon resonance experiment, the biphenyl derivative has good affinity to human PD-L1. The biphenyl derivatives not only can well inhibit the combination of PD-1/PD-L1, but also can promote the recovery of the activity of T cells and promote the secretion of immune factor INF-gamma, so that the biphenyl derivatives can be used for the immunotherapy of tumors. The biphenyl derivative disclosed by the invention has excellent activity, so that the development of the biphenyl inhibitor of PD-1/PD-L1 has great practical significance and potential application prospect.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: (1) The provided novel biphenyl derivatives can obviously inhibit the interaction of PD-1/PD-L1, and particularly importantly, can obviously block the inhibition effect of PD-L1 on CD3 ⁺ T cells, and have the effect of promoting the secretion of immune factor INF-gamma by the T cells. In the experiment of co-incubation of tumor cells and T cells, the biphenyl compound has higher INF-gamma expression promotion effect than positive medicine BMS-202, so that the biphenyl compound has the effect of enhancing the anti-tumor effect of the T cells; therefore, the biphenyl derivatives can be used as an immune checkpoint PD-1/PD-L1 inhibitor for preparing medicines for tumor immunotherapy. (2) The synthesis route of the biphenyl derivatives is ingenious, simple and easy to implement, the raw materials are cheap and easy to obtain, the synthesis process is safe and environment-friendly, and the large-scale production is easy to realize. (3) The medicine taking the compound as an active ingredient can be used for treating various cancers or tumors related to immune checkpoint PD-1/PD-L1, and has wide applicability.

Drawings

FIG. 1 is an INF-gamma expression experiment in co-culture of tumor cells and T cells.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1

N, N '- ((((((2, 2' -dimethyl- [1,1 '-biphenyl ] -3,3' -diyl) bis (2-methoxypyridine-6, 3-diyl)) bis (methylene)) bis (1H-1, 2, 3-triazole-1, 4-diyl)) bis (methylene)) bis (azadiyl)) bis (ethane-2, 1-diyl)) diacetamide (II-1：n＝1,X＝N,R¹＝R²＝-NHCH₂CH₂NHCOCH₃,R³＝R⁴＝CH₃,R⁵＝OCH₃)

Synthesis of 2,2' -dimethyl- [1,1' -biphenyl ] -3,3' -diamine (V)

3-Bromo-2-methylaniline (III) (10.00 g,53.8 mmol) and compound IV (12.50 g,53.8 mmol) were dissolved in dioxane (150 mL), a solution of potassium carbonate (20.83 g,150.6 mmol) in water (15 mL) was added dropwise to the reaction solution, nitrogen was added under protection, pd (PPh ₃)₄ (1.55 g,1.35 mmol) was added, the reaction was allowed to proceed to 80℃for 12 hours, TLC (petroleum ether: ethyl acetate=8:1) was monitored for complete reaction, heating was stopped, cooled to room temperature, insoluble material was removed by suction filtration, the filtrate was diluted with water (100 mL), ethyl acetate (120 mL. Times.3) was extracted, saturated sodium chloride solution was washed (100 mL. Times.3), anhydrous sodium sulfate was dried by suction filtration, the filtrate was distilled off under reduced pressure to obtain crude product, and yellow solid powder 8.58g was purified by column chromatography (petroleum ether: ethyl acetate=15:1) to obtain yield 75.2％.m.p.134～136℃.¹H NMR(400MHz,Chloroform-d)δ7.07(t,J＝7.7Hz,2H,ArH),6.72(d,J＝7.9Hz,2H,ArH),6.63(d,J＝7.6Hz,2H,ArH),3.37(br,4H,NH₂),1.90(s,6H,CH₃).

Synthesis of 2,2'- (2, 2' -dimethyl- [1,1 '-biphenyl ] -3,3' -diyl) bis (4, 5-tetramethyl-1, 3, 2-dioxaborane) (VI)

Compound V (4.00 g,18.9 mmol) was dissolved in methanol (40 mL), HCl solution (37.7 mL,3 mol/L) and water (20 mL) were added sequentially, and stirred at room temperature for 30 min. Cooling to 0 ℃, slowly dropwise adding an aqueous solution of sodium nitrite (18.86 mL,2.2 mol/L), keeping the temperature and stirring for 30 minutes after the dropwise adding, dissolving the bis-pinacolato borate (19.20 g,75.6 mmol) in methanol (40 mL), slowly dropwise adding the solution into the reaction solution, and returning to room temperature and stirring for 2 hours after the dropwise adding, wherein TLC monitoring (petroleum ether: ethyl acetate=30:1) is carried out to complete the raw material reaction. After dilution with methylene chloride (50 mL), the aqueous phase was separated, extracted with methylene chloride (50 mL. Times.2), the methylene chloride layers were combined, washed with saturated sodium chloride solution (50 mL. Times.3), and dried over anhydrous magnesium sulfate. Suction filtration, vacuum distillation of the filtrate to remove the solvent, column chromatography (petroleum ether: ethyl acetate=250:1) purification to obtain white solid powder 3.54g, yield 42.9％.m.p.167-168℃.¹H NMR(300MHz,Chloroform-d)δ7.77(dd,J＝7.3,1.7Hz,2H,ArH),7.23(t,J＝7.4Hz,2H,ArH),7.15(dd,J＝7.5,1.7Hz,2H,ArH),2.23(s,6H,ArCH₃),1.38(s,24H,CH₃).

Synthesis of 1- ((6-chloro-2-methoxypyridin-3-yl) methyl) -1H-1,2, 3-triazole-4-carbaldehyde (VII-1)

Synthesis of 6-chloro-2-methoxypyridine-3-carbaldehyde (2)

6-Chloro-2-methoxypyridine (2.00 g,13.9 mmol) was dissolved in anhydrous THF (10 mL) under nitrogen. Cooled to-10 ℃, and isopropyl magnesium chloride (6.95 mL,6.95mmol,1.00 mol/L) was slowly injected. After the completion of the dropping, the mixture was reacted at-10℃for 30 minutes. N-butyllithium (6.67 mL,16.7mmol,2.50 mol/L) was continuously injected at-10deg.C. After the addition, stirring was continued at this temperature for 2 hours, anhydrous DMF (2.21 mL,27.8 mmol) was added dropwise. After the dripping, stirring was continued for 1.5 hours. TLC monitored complete reaction of starting material and warmed to room temperature. The reaction mixture was slowly added to a mixture of acetic acid (5.00 g), concentrated hydrochloric acid (2.00 g), isopropyl alcohol (20.00 mL) and water (20.00 mL), and after quenching the reaction, the temperature was raised to 50℃for 2 hours. Cooling to room temperature, evaporating to remove organic solvent, cooling to obtain solid, vacuum filtering to obtain yellow solid, vacuum drying to obtain 1.58g compound 2, yield 66.1％.m.p.62-64℃.¹H NMR(300MHz,DMSO-d₆)δ10.18(s,1H,CHO),8.13(d,J＝7.9Hz,1H,ArH),7.28(d,J＝7.9,1H,ArH),4.02(s,3H,OCH₃).

Synthesis of (6-chloro-2-methoxy) -3-pyridinemethanol (3)

Compound 2 (2.20 g,12.8 mmol) was dissolved in anhydrous tetrahydrofuran (8.00 mL) and anhydrous methanol (3.00 mL). Sodium borohydride (0.59 g,15.4 mmol) was added slowly in portions, cooled to 0deg.C. After the addition, the ice bath was removed, and the mixture was left at room temperature and stirred for 30min. TLC (petroleum ether: ethyl acetate=8:1) monitored complete reaction of starting material, stopped the reaction, quenched with water. The organic solvent was distilled off under reduced pressure, the residue was extracted with ethyl acetate (10 mL. Times.3), and the organic phases were combined, washed with a saturated sodium chloride solution (20 mL. Times.3), and dried over anhydrous sodium sulfate. Removing anhydrous sodium sulfate by suction filtration, evaporating solvent to obtain crude product, separating by column chromatography to obtain white solid powder 1.64g, yield 73.7％.m.p.58-61℃.¹H NMR(300MHz,Chloroform-d)δ7.56(d,J＝7.5,1H,ArH),6.92(d,J＝7.6Hz,1H,ArH),4.63(s,2H,CH₂),4.00(s,3H,OCH₃).

Synthesis of 6-chloro-3- (chloromethyl) -2-methoxypyridine hydrochloride (4)

Compound 3 (11.80 g,68.0 mmol) was dissolved in dichloromethane (70 mL) and thionyl chloride (6.01 mL,81.6 mmol) was slowly added dropwise and the reaction was stirred overnight at room temperature from cloudy to clear. TLC (Petroleum ether: ethyl acetate=8:1) monitored complete reaction of the starting materials, toluene (20 mL) was added to the reaction solution, the solvent was distilled off under reduced pressure, and after cooling, 15.34g of pale green crystals were obtained in 98.8% yield. m.p.62-64 ℃. MS (ESI) M/z [ M+H ] +. Calcd for C ₇H₈Cl₂ NO:191.9; found 192.0.

Synthesis of 3- (azidomethyl) -6-chloro-2-methoxypyridine (5)

Compound 4 (15.34 g,79.9 mmol) and DIPEA (15.49 g,119.9 mmol) were added to DMF (150 mL) and stirred at room temperature for 1 hour. CsF (14.57 g,95.9 mmol) and TMSN ₃ (12.6 mL,95.9 mmol) were added and the reaction was allowed to proceed at 80℃for 5 hours. TLC (petroleum ether: ethyl acetate=30:1) monitored complete reaction of starting material. Stopping heating and cooling to room temperature. The reaction solution was poured into ice water (300 mL), extracted with ethyl acetate (150 mL. Times.3), and the organic layers were combined, washed with saturated sodium chloride (100 mL. Times.3), and dried over anhydrous sodium sulfate. The anhydrous sodium sulfate is removed by suction filtration, the solvent is removed by distillation to obtain crude product, 13.06g of colorless transparent oily liquid is obtained by column chromatography separation, and the yield is 82.3%. MS (ESI) M/z [ M+H ] ⁺Calcd for C₇H₈ClN₄ O199.0; found 199.1.

Synthesis of 6-chloro-3- ((4- (diethoxymethyl) -1H-1,2, 3-triazol-1 yl) methyl) -2-methoxypyridine (6)

Compound 5 (12.50 g,62.9 mmol) and propionaldehyde diethyl acetal (9.67 g,75.5 mmol) were dissolved in acetonitrile (72 mL) and water (24 mL), and CuI (0.60 g,3.16 mmol) was added. The temperature is raised to 30 ℃ and the reaction is carried out for 12 hours. TLC (petroleum ether: ethyl acetate=4:1) monitored complete reaction of starting material, stopped heating, and cooled to room temperature. Poured into ice water (50 mL), extracted with ethyl acetate (100 mL. Times.2), the organic layers were combined, washed with saturated aqueous sodium chloride (100 mL. Times.2), and dried over anhydrous sodium sulfate. Insoluble matter was removed by suction filtration, and the solvent was distilled off to give 15.53g of a colorless transparent oily liquid, which was used in the next reaction without purification.

Synthesis of 1- ((6-chloro-2-methoxypyridin-3-yl) methyl) -1H-1,2, 3-triazole-4-carbaldehyde (VI I-1)

Compound 6 (15.00 g,45.9 mmol) was dissolved in dichloromethane (50 mL) and trifluoroacetic acid (6.82 mL,91.8 mmol) was slowly added dropwise. After the completion of the dropping, the reaction was carried out at room temperature for 1 hour. TLC (petroleum ether: ethyl acetate=2:1) monitored reaction completion. The pH was adjusted to alkaline with 2N NaOH solution, extracted with dichloromethane (50 mL. Times.3), the organic layers combined, washed with saturated NaCl solution (50 mL. Times.2) and dried over anhydrous sodium sulfate. Removing anhydrous sodium sulfate by suction filtration, evaporating solvent to obtain crude product, purifying by column chromatography to obtain 11.14g white solid powder, and obtaining yield 96.1％.m.p.128-130℃.¹H NMR(300MHz,Chloroform-d)δ10.20(s,1H,CHO),8.22(s,1H,ArH),7.61(d,J＝7.7Hz,1H,ArH),7.03(d,J＝7.7Hz,1H,ArH),5.60(s,2H,CH₂),4.09(s,3H,OCH₃).

Synthesis of 1,1'- ((2, 2' -dimethyl- [1,1 '-biphenyl ] -3,3' -diyl) bis (2-methoxypyridine-6, 3-diyl)) bis (methylene)) bis (1H-1, 2, 3-triazole-4-carbaldehyde) (VIII-1)

Sequentially adding a water (2 mL) solution of a compound VI I-1 (1.42 g,3.27 mmol), a compound VI I (2.06 g,8.17 mmol), pd (PPh ₃)₄(0.38g,0.33mmol)、K₂CO₃ (1.27 g,9.17 mmol) and dioxane (20 mL) into a eggplant-shaped bottle, protecting nitrogen, heating to 80 ℃ and stirring for 12 hours, monitoring the raw materials to react completely by TLC (petroleum ether: ethyl acetate=2:1), stopping heating, cooling to room temperature, filtering to remove insoluble matters by suction, extracting by ethyl acetate (50 mL×3), merging organic layers, washing by saturated sodium chloride solution (50 mL×2), drying by suction, removing insoluble matters by suction, evaporating solvent under reduced pressure to obtain crude products, purifying by column chromatography (petroleum ether: ethyl acetate=4:1-1.5:1) to obtain white solid powder of 1.56g, and obtaining the yield 77.6％.m.p.179-181℃.¹H NMR(400MHz,DMSO-d₆)δ10.05(s,2H,CHO),8.96(s,2H,ArH),7.70(d,J＝7.6Hz,2H,ArH),7.45(dd,J＝7.7,1.5Hz,2H,ArH),7.38(t,J＝7.6Hz,2H,ArH),7.24–7.21(m,4H,ArH),5.72(s,4H,CH₂),3.92(s,6H,OCH₃),2.05(s,6H,CH₃).

Synthesis of N, N '- (((((((2, 2' -dimethyl- [1,1 '-biphenyl ] -3,3' -diyl) bis (2-methoxypyridine-6, 3-diyl)) bis (methylene)) bis (1H-1, 2, 3-triazole-1, 4-diyl)) bis (methylene)) bis (azadiyl)) bis (ethane-2, 1-diyl)) diacetamide (II-1)

Compound VIII-1 (0.20 g,0.33 mmol), N-acetyl ethylenediamine (0.10 g,0.98 mmol) and acetic acid (2 drops) were dissolved in dichloromethane (5 mL) and methanol (2 mL), and the reaction was stirred at room temperature for 1 hour. NaBH (OAc) ₃ (0.28 g,1.32 mmol) was added in portions to the reaction solution and stirred at room temperature for 4 hours. TLC (dichloromethane: methanol=10:1) monitored complete reaction of starting materials, pH was adjusted to 7 with saturated aqueous sodium bicarbonate, extracted with dichloromethane (10 ml×3), combined organic layers were washed with saturated aqueous sodium chloride (10 ml×2) and dried over anhydrous sodium sulfate. Removing insoluble substances by suction filtration, evaporating solvent under reduced pressure to obtain crude product, purifying by silica gel column chromatography to obtain yellow solid powder 0.09g, and obtaining yield 34.2％.m.p.86-88℃.¹H NMR(300MHz,DMSO-d₆)δ8.02(s,2H,ArH),7.83(s,2H,2CONH),7.54(d,J＝7.6Hz,2H,ArH),7.46(dd,J＝7.7,1.7Hz,2H,ArH),7.39(t,J＝7.5Hz,2H,ArH),7.22(td,J＝7.4,2.6Hz,4H,ArH),5.60(s,4H,2ArCH₂),3.95(s,6H,2OCH₃),3.78(s,4H,ArCH₂NH),3.14(q,J＝6.4Hz,4H,2CH₂NHCH₂),2.59(t,J＝6.5Hz,4H,2CONHCH₂),2.07(s,6H,2ArCH₃),1.80(s,6H,2COCH₃).HRMS(ESI):m/z[M+H]⁺Calcd for C₄₂H₅₁N₁₂O₄:787.4156;Found:787.4149.

Example 2

Synthesis of 2,2'- (((((2, 2' -dimethyl- [1,1 '-biphenyl ] -3,3' -diyl) bis (2-methoxypyridine-6, 3-diyl)) bis (methylene)) bis (1H-1, 2, 3-triazole-1, 4-diyl)) bis (methylene)) bis (azadiyl)) bis (ethane-1-ol )(II-2：n＝1,X＝N,R¹＝R²＝-NHCH₂CH₂OH,R³＝R⁴＝CH₃,R⁵＝OCH₃)

Using compound VII I-1 (0.20 g,0.33 mmol) and ethanolamine (0.06 g,0.98 mmol) as raw materials, the same procedure as for compound II-1 gave 0.08g of yellow solid powder, yield 34.9％.m.p.112-114℃.¹H NMR(300MHz,DMSO-d₆)δ8.10(d,J＝7.9Hz,2H,ArH),7.55(t,J＝7.2Hz,2H,ArH),7.45(d,J＝7.6Hz,2H,ArH),7.38-7.28(m,2H,ArH),7.25–7.09(m,4H,ArH),5.65–5.48(m,4H,ArCH₂),4.05(s,6H,OCH₃),3.90(s,4H,2NHCH₂Ar),2.54–2.49(m,4H,CH₂CH₂OH),2.76–2.66(m,4H,CH₂CH₂OH),2.03(s,6H,2ArCH₃).HRMS(ESI):m/z[M+H]⁺Calcd for C₃₈H₄₅N₁₀O₄:705.3625;Found:705.3615.

Example 3

2,2'- ((((((2, 2' -Dimethyl- [1,1 '-biphenyl ] -3,3' -diyl) bis (2-methoxypyridine-6, 3-diyl)) bis (methylene)) bis (1H-1, 2, 3-triazole-1, 4-diyl)) bis (methylene)) bis (azadiyl)) diacetamide (II-3：n＝1,X＝N,R¹＝R²＝-NHCH₂CONH₂,R³＝R⁴＝CH₃,R⁵＝OCH₃)

Using Compound VII I-1 (0.20 g,0.33 mmol) and glycinamide hydrochloride (0.15 g,1.32 mmol) as raw materials, the synthesis of Compound II-1 was operated to obtain yellow solid powder 0.11g, yield 46.3％.m.p.86-88℃.¹H NMR(300MHz,DMSO-d₆)δ8.08(s,2H,ArH),7.56(d,J＝7.5Hz,2H,ArH),7.47(dd,J＝7.7,1.7Hz,2H,ArH),7.40(t,J＝7.6Hz,2H,ArH),7.34(br,2H,CONH₂),7.30–7.18(m,4H,ArH),7.09(s,2H,CONH₂),5.61(s,4H,2ArCH₂),3.97(s,6H,2OCH₃),3.77(s,4H,2ArCH₂NH),3.10(s,4H,2COCH₂),2.65(s,2H,2NH),2.08(s,6H,2ArCH₃).HRMS(ESI):m/z[M+H]⁺Calcd for C₃₈H₄₃N₁₂O₄:731.3530;Found:731.3528.

Example 4

5,5'- ((((((2, 2' -Dimethyl- [1,1 '-biphenyl ] -3,3' -diyl) bis (2-methoxypyridine-6, 3-diyl)) bis (methylene)) bis (1H-1, 2, 3-triazole-1, 4-diyl)) bis (methylene)) bis (azadiyl)) bis (methylene)) bis (pyrrolidin-2-one) (II-4:n =1, x=n, R ¹＝R² is (pyrrolidin-2-one-5-yl) methylamino, R ³＝R⁴＝CH₃,R⁵＝-OCH₃)

The synthesis of compound VII I-1 (0.20 g,0.33 mmol) and 5-aminomethyl-2-pyrrolidone (0.15 g,1.32 mmol) was carried out using the same compound II-1 as the starting material to obtain 0.12g of yellow solid powder in yield 45.5％.m.p.111-114℃.1H NMR(300MHz,Chloroform-d)δ7.60(s,2H,ArH),7.51(d,J＝7.5Hz,2H,ArH),7.44–7.38(m,2H,ArH),7.32(t,J＝7.6Hz,2H,ArH),7.24–7.19(m,2H,ArH),7.05(d,J＝7.5Hz,2H,ArH),6.32(br,2H,NHCO),5.54(s,4H,ArCH₂N),4.03(s,6H,OCH₃),3.94(s,4H,ArCH₂NH),3.80-3.69(m,2H,2CH),2.86(dd,J＝12.0,4.0Hz,2H,NHCH₂),2.61(dd,J＝12.0,8.5Hz,2H,NHCH₂),2.33(t,J＝8.0Hz,4H,NHCH₂CH),2.25-2.17(m,2H,CH₂),2.17-2.12(m,2H,CH₂),2.11(s,6H,ArCH₃),2.08-2.00(m,2H,CH₂),1.80-1.69(m,2H,CH₂).HRMS(ESI):m/z[M+Na]⁺Calcd for C₄₄H₅₀N₁₂O₄Na:833.3976;Found:833.3993.

Example 5

Synthesis of 2,2',2",2 '" - ((((((2, 2' -dimethyl- [1,1' -biphenyl ] -3,3' -diyl) bis (2-methoxypyridine-6, 3-diyl)) bis (methylene)) bis (1H-1, 2, 3-triazole-1, 4-diyl)) bis (methylene)) bis (azatriyl)) tetrakis (ethane-1-ol )(II-5：n＝1,X＝N,R¹＝R²＝-N(CH₂CH₂OH)₂,R³＝R⁴＝CH₃,R⁵＝OCH₃))

Using Compound VII I-1 (0.20 g,0.33 mmol) and diethanolamine (0.13 mL,0.98 mmol) as raw materials, the synthesis of Compound II-1 was operated to obtain yellow solid powder 0.11g, yield 42.6％.m.p.90-92℃.¹H NMR(300MHz,DMSO-d₆)δ8.09(s,2H,ArH),7.51(d,J＝7.5Hz,2H,ArH),7.46(dd,J＝7.7,1.6Hz,2H,ArH),7.38(t,J＝7.5Hz,2H,ArH),7.22(td,J＝7.5,2.4Hz,4H,ArH),5.60(s,4H,ArCH₂),4.43(s,4H,4OH),3.94(s,6H,2OCH₃),3.79(s,4H,2NHCH₂),3.39(s,4H,2CH₂CH₂OH),3.19(d,J＝4.3Hz,4H,2CH₂CH₂OH),2.06(s,6H,2ArCH₃).HRMS(ESI):m/z[M+H]⁺Calcd for C₄₂H₅₃N₁₀O₆:793.4150;Found:793.4152.

Example 6

Synthesis of(((2, 2' -dimethyl- [1,1' -biphenyl ] -3,3' -diyl) bis (2-methoxypyridine-6, 3-diyl)) bis (methylene)) bis (1H-1, 2, 3-triazole-1, 4-diyl)) dimethanol (II-6:n =1, X=N, R ¹＝R²＝-OH,R³＝R⁴＝CH₃,R⁵＝OCH₃)

Compound VII I-1 (0.20 g,0.33 mmol) was dissolved in methanol (2 mL) and cooled to 0deg.C. NaBH ₄ (19 mg,0.51 mmol) was added to the reaction and stirred for 30 min. TLC monitored complete reaction of starting material, and water was added to quench excess sodium borohydride. Methanol was distilled off under reduced pressure, and the residue was diluted with dichloromethane (20 mL), separated, and the dichloromethane layer was washed with saturated sodium chloride solution (5 ml×3) and water (5 ml×3), dried over anhydrous sodium sulfate. Filtering to remove insoluble substances, evaporating solvent under reduced pressure to obtain crude product, and performing column chromatography to obtain yellow solid powder 0.17g with yield 84.5％.m.p.144-146℃.¹H NMR(400MHz,DMSO-d₆)δ8.03(s,2H,ArH),7.53(d,J＝7.6Hz,2H,ArH),7.43(d,J＝7.6Hz,2H,ArH),7.36(t,J＝7.5Hz,2H,ArH),7.20(dd,J＝11.1,6.9Hz,4H,ArH),5.58(s,4H,ArCH₂N),5.21(t,J＝5.7Hz,2H,OH),4.53(d,J＝5.6Hz,4H,CH₂),3.93(s,6H,OCH₃),2.04(s,6H,ArCH₃).HRMS(ESI):m/z[M+H]⁺Calcd for C₃₄H₃₅N₈O₄:619.2781;Found:619.2777.

Example 7

N, N '- ((((3, 3' "-dimethoxy-2 ',2" -dimethyl- [1,1':3',1": synthesis of 3',1 '-tetraphenyl ] -4, 4' -diyl) bis (1H-1, 2, 3-triazole-1, 4-diyl) bis (methylene) bis (azadiyl)) bis (ethane-2, 1-diyl)) diacetamide (II-7：n＝0,X＝CH,R¹＝R²＝-NHCH₂CH₂NHCOCH₃,R³＝R⁴＝CH₃,R⁵＝OCH₃)

Synthesis of 1- (4-bromo-2-methoxyphenyl) -1H-1,2, 3-triazole-4-carbaldehyde (VII-2)

Synthesis of 4-bromo-2-methoxyaniline (8)

O-methoxyaniline 7 (20.00 g,162.4 mmol) was dissolved in acetonitrile (100 mL). N-bromosuccinimide (33.8 g,195.0 mmol) was added slowly in portions to the reaction solution, cooled to 0deg.C. After the addition, the reaction was carried out at room temperature overnight. TLC (petroleum ether: ethyl acetate=15:1) monitored complete reaction of starting materials, dilution with water, extraction with ethyl acetate (100 ml×3), combined organic layers, washing with saturated sodium chloride (100 ml×2), drying over anhydrous sodium sulfate. The anhydrous sodium sulfate is removed by suction filtration, the solvent is removed by reduced pressure distillation to obtain crude products, and the crude products are purified by column chromatography (petroleum ether: ethyl acetate=100:1-60:1) to obtain 19.82g of reddish brown solid. Yield is good 60.4％.m.p.60-61℃.¹H NMR(300MHz,Chloroform-d)δ6.95-6.88(m,2H,ArH),6.63(dt,J＝8.4,1.1Hz,1H,ArH),3.89(s,3H,OCH₃),3.81(s,2H,NH₂).

Synthesis of 1-azido-4-bromo-2-methoxybenzene (9)

Compound 8 (6.47 g,32.0 mmol) was dissolved in acetonitrile and tert-butyl nitrite (3.96 g,38.4 mmol) was slowly added dropwise. After the completion of the dropping, the temperature was lowered to 0℃and azido trimethylsilane (4.42 g,38.4 mmol) was slowly added dropwise, and after the completion of the dropping, the reaction was carried out at room temperature for 2 hours. TLC (petroleum ether: ethyl acetate=30:1) monitored reaction completion. The solvent was evaporated, the residue was dissolved in dichloromethane and purified by column chromatography to give 3.66g of a pale brown transparent oily liquid. Yield is good 50.1％.¹H NMR(300MHz,Chloroform-d)δ7.13(dd,J＝8.4,1.6Hz,1H,ArH),7.08(d,J＝1.5Hz,1H,ArH),6.93(d,J＝8.3,1H,ArH),3.94(s,3H,OCH₃).

Synthesis of 1- (4-bromo-2-methoxyphenyl) -4- (diethoxymethyl) -1H-1,2, 3-triazole (10)

Compound 9 (3.88 g,17.0 mmol) was dissolved in acetonitrile (21 mL) and water (7 mL), and propargyl propanal diethyl acetal (2.61 g,20.4 mmol) was slowly added dropwise. CuI (0.16 g,0.85 mmol) was added and the reaction was allowed to proceed at 30℃for 24 hours. TLC (petroleum ether: ethyl acetate=4:1) monitored the reaction was complete, stopped heating, and cooled to room temperature. The reaction solution was poured into ice water (30 mL), extracted with ethyl acetate (20 mL. Times.2), washed with saturated aqueous sodium chloride solution (20 mL. Times.2), and the organic layers were combined and dried over anhydrous magnesium sulfate. Insoluble matter was removed by suction filtration, and the solvent was distilled off to obtain 5.57g of a yellow oily liquid, and the reaction product was directly used for the next reaction without purification.

Synthesis of 1- (4-bromo-2-methoxyphenyl) -1H-1,2, 3-triazole-4-carbaldehyde (VII-2)

Compound 10 (5.57 g,15.6 mmol) was dissolved in dichloromethane (20 mL) and trifluoroacetic acid (2.32 mL,31.2 mmol) was slowly added dropwise at room temperature. After the completion of the dropping, the reaction was carried out at room temperature for 1 hour. TLC monitoring (petroleum ether: ethyl acetate=4:1) reaction was complete, pH was adjusted to 8 with 2N NaOH, dichloromethane (30 ml×2) was extracted to no fluorescence, the organic layers were combined, washed with saturated NaCl solution (20 ml×2), and dried over anhydrous magnesium sulfate. Removing anhydrous magnesium sulfate by suction filtration, evaporating solvent to obtain crude product, and purifying by column chromatography (petroleum ether: ethyl acetate=8:1) to obtain white solid powder 4.18g, yield 94.8％.m.p.137-138℃.¹H NMR(300MHz,DMSO-d₆)δ10.13(s,1H,CHO),9.26(s,1H,ArH),7.69(d,J＝8.4Hz,1H,ArH),7.63(d,J＝2.1Hz,1H,ArH),7.42(dd,J＝8.4,2.0Hz,1H,ArH),3.92(s,3H,OCH₃).

1,1' - (3, 3' "-Dimethoxy-2 ',2" -dimethyl- [1,1': synthesis of 3', 1':3', 1' -tetraphenyl ] -4, 4' -diyl) bis (1H-1, 2, 3-triazole-4-carbaldehyde) (VIII-2)

A solution of compound VI (0.20 g,0.46 mmol), compound VII-2 (0.32 g,1.15 mmol) and K ₃PO₄ (0.10 g,0.46 mm) in water (0.5 mL) was dissolved in tetrahydrofuran, pd (dppf) Cl ₂ (34 mg,0.046 mmol) was added under nitrogen and the temperature was raised to 65℃for reaction for 12 hours. TLC (petroleum ether: ethyl acetate=2:1) starting material reacted completely, stopping heating, and cooling to room temperature. Insoluble substances are removed by suction filtration, the solvent is removed by distillation under reduced pressure to obtain crude products, and the crude products are purified by column chromatography (petroleum ether: ethyl acetate=5:1-3:1) to obtain yellow solid powder of 0.23g, yield 85.4％.m.p.108-110℃.¹H NMR(400MHz,Chloroform-d)δ10.28(s,2H,CHO),8.80(s,2H,ArH),7.96(d,J＝8.1Hz,2H,ArH),7.37(d,J＝7.4Hz,2H,ArH),7.33(d,J＝1.6Hz,2H,ArH),7.18(dd,J＝8.1,1.7Hz,2H,ArH),7.14(d,J＝1.7Hz,2H,ArH),3.98(s,6H,OCH₃),2.06(s,6H,ArCH₃).

N, N '- ((((3, 3' "-dimethoxy-2 ',2" -dimethyl- [1,1':3',1": synthesis of 3',1 '-tetraphenyl ] -4, 4' -diyl) bis (1H-1, 2, 3-triazole-1, 4-diyl) bis (methylene) bis (azadiyl)) bis (ethane-2, 1-diyl)) diacetamide (II-7)

Using compound VIII-2 (0.20 g,0.34 mmol) and N-acetyl ethylenediamine (0.14 g,1.37 mmol) as raw materials, the same procedure as for compound II-1 gave 0.08g of yellow solid powder, yield 30.9％.m.p.82-84℃.¹H NMR(300MHz,DMSO-d₆)δ8.39(s,2H,ArH),7.89(s,2H,CONH),7.71(d,J＝8.1Hz,2H,ArH),7.46-7.32(m,6H,ArH),7.26(dd,J＝7.2,1.9Hz,2H,ArH),7.18(dd,J＝8.1,1.7Hz,2H,ArH),3.94(s,6H,OCH₃),3.92(s,4H,ArCH₂),3.26-3.19(m,6H,ArHCH₂NHCH₂),2.70(t,J＝6.6Hz,4H,CONHCH₂),2.06(s,6H,ArCH₃),1.83(s,6H,COCH₃).HRMS(ESI):m/z[M+H]⁺Calcd for C₄₂H₄₉N₁₀O₄:757.3938;Found:757.3940.

Example 8

2,2' - ((((3, 3' "-Dimethoxy-2 ',2" -dimethyl- [1,1':3',1":3", 1' "-tetraphenyl ] -4,4 '" -diyl) bis (1H-1, 2, 3-triazol-1, 4-diyl)) bis (methylene)) bis (azadiyl)) bis (ethan-1-ol) )(II-8：n＝0,X＝CH,R¹＝R²＝-NHCH₂CH₂OH,R³＝R⁴＝CH₃,R⁵＝OCH₃)

Using compound VIII-2 (0.20 g,0.34 mmol) and ethanolamine (81. Mu.L, 1.37 mmol) as raw materials, the same procedure as for compound II-1 gave 0.10g of yellow solid powder, yield 43.3％.m.p.96-98℃.¹H NMR(300MHz,DMSO-d₆)δ8.38(s,2H,ArH),7.69(d,J＝8.1Hz,2H,ArH),7.43–7.30(m,6H,ArH),7.23(dd,J＝7.1,2.1Hz,2H,ArH),7.16(dd,J＝8.1,1.8Hz,2H,ArH),3.92(s,10H,OCH₃/ArCH₂NH),3.52(t,J＝5.6Hz,4H,2CH₂OH),2.71(t,J＝5.7Hz,4H,2NHCH₂CH₂),2.03(s,6H,2ArCH₃).HRMS(ESI):m/z[M+H]⁺Calcd for C₃₈H₄₃N₈O₄:675.3407;Found:675.3414.

Example 9

2,2' - (((3, 3' "-Dimethoxy-2 ',2" -dimethyl- [1,1':3',1":3", 1' "-tetraphenyl ] -4,4 '" -diyl) bis (1H-1, 2, 3-triazole-1, 4-diyl) bis (methylene)) bis (azadiyl)) bis (3-hydroxypropionic acid )(II-9：：n＝0,X＝CH,R¹＝R²＝-NHCH(CH₂OH)COOH,R³＝R⁴＝CH₃,R⁵＝OCH₃)

Using compound VIII-2 (0.20 g,0.34 mmol) and serine methyl ester hydrochloride (0.21 g,1.37 mmol) as raw materials, the same procedure as for compound II-1 was followed to give a pale green viscous liquid of 0.10g. The resulting pale green liquid (0.10 g,0.13 mmol) was dissolved in methanol (1 mL) and tetrahydrofuran (1 mL), and a solution of lithium hydroxide (30 mg,1.27 mmol) in water (0.5 mL) was added dropwise and stirred overnight at room temperature. TLC (dichloromethane: methanol=15:1) monitored complete reaction of the starting materials, the organic solvent was distilled off under reduced pressure, pH was adjusted to around 6 with 0.1M HCl, a large amount of solids precipitated, and a grey solid, 50mg, was obtained by suction filtration in yield 19.2％.m.p.＞250℃.¹H NMR(300MHz,DMSO-d₆)δ8.43(s,2H,ArH),7.69(d,J＝8.1Hz,2H,ArH),7.37(q,J＝7.3,6.4Hz,4H,ArH),7.32(s,2H,ArH),7.23(d,J＝7.4Hz,2H,ArH),7.15(d,J＝8.3Hz,2H,ArH),4.09(d,J＝13.8Hz,2H,CH₂OH),3.99(d,J＝14.2Hz,2H,CH₂OH),3.92(s,6H,OCH₃),3.62(d,J＝5.5Hz,4H,NHCH₂),3.19(d,J＝5.5Hz,2H,CH),2.01(s,6H,ArCH₃).

Example 10

((3, 3' "-Dimethoxy-2 ',2" -dimethyl- [1,1':3',1": 3', 1' -tetraphenyl ] -4,4 ' -diyl) bis (1H-1, 2, 3-triazole-1, 4-diyl)) dimethanol (II-10：n＝0,X＝CH,R¹＝R²＝-OH,R³＝R⁴＝CH₃,R⁵＝OCH₃)

Compound VIII-2 (0.20 g,0.34 mmol) was dissolved in methanol (5 mL) and cooled to 0deg.C. NaBH ₄ (19 mg,0.51 mmol) was added to the reaction and stirred for 30 min. TLC monitored complete reaction of starting material, and water was added to quench excess sodium borohydride. The methanol was distilled off under reduced pressure, and the residue was diluted with dichloromethane (10 mL), washed with a saturated sodium chloride solution (5 mL. Times.3) and water (5 mL. Times.3), and dried over anhydrous sodium sulfate. Filtering to remove insoluble substances, evaporating solvent under reduced pressure to obtain crude product, purifying by column chromatography to obtain yellow solid powder 0.13g, and obtaining yield 64.6％.m.p.130-132℃.¹H NMR(300MHz,DMSO-d₆)δ8.38(s,2H,ArH),7.71(d,J＝8.1Hz,2H,ArH),7.42–7.36(m,4H,ArH),7.34(d,J＝1.7Hz,2H,ArH),7.27(dd,J＝7.1,2.0Hz,2H,ArH),7.19(dd,J＝8.1,1.7Hz,2H,ArH),5.34(t,J＝5.6Hz,2H,2OH),4.65(d,J＝5.4Hz,4H,2ArCH₂),3.94(s,6H,2OCH₃),2.07(s,6H,2ArCH₃).HRMS(ESI):m/z[M+H]⁺Calcd for C₃₄H₃₃N₆O₄:589.2563;Found:589.2543.

Example 11

Synthesis of 1,1' - ((3, 3' "-dimethoxy-2 ',2" -dimethyl- [1,1':3',1":3", 1' "-tetraphenyl ] -4,4 '" -diyl) bis (1H-1, 2, 3-triazole-1, 4-diyl)) bis (methylene)) bis (piperidine-2-carboxamide) (II-11:n=0, x=ch, R ¹＝R² is 2-carbamoylpiperidin-1-yl, R ³＝R⁴＝CH₃,R⁵＝OCH₃)

Using compound VIII-2 (0.20 g,0.34 mmol) and 2-piperidinecarboxamide (0.12 g,1.03 mmol) as raw materials, the same procedure as for compound II-1 gave 0.05g of a white solid powder, yield 18.1％.m.p.146-148℃.¹H NMR(300MHz,DMSO-d₆)δ8.41(s,2H,ArH),7.73(d,J＝8.1Hz,2H,ArH),7.42(d,J＝7.2Hz,2H,ArH),7.38(dd,J＝7.7,1.9Hz,2H,ArH),7.34(d,J＝1.7Hz,2H,ArH),7.31(s,2H,CONH₂),7.26(dd,J＝7.1,1.9Hz,2H,ArH),7.19(dd,J＝8.0,1.7Hz,2H,ArH),7.14(s,2H,CONH₂),3.94(s,6H,2OCH₃),3.87(d,J＝14.1Hz,2H,ArCH₂),3.57(d,J＝14.2Hz,2H,ArCH₂),3.35(s,2H,2CH),2.95(d,J＝11.2Hz,2H,1/2Piperidine-CH₂),2.78–2.70(m,2H,Piperidine-CH₂),2.13(d,J＝11.4Hz,2H,1/2Piperidine-CH₂),2.06(s,6H,ArCH₃),1.78(d,J＝12.3Hz,2H,1/2Piperidine-CH₂),1.68(d,J＝12.7Hz,2H,1/2Piperidine-CH₂),1.57(s,2H,1/2Piperidine-CH₂),1.51–1.39(m,2H,1/2Piperidine-CH₂),1.23(d,J＝14.3Hz,2H,1/2Piperidine-CH₂).HRMS(ESI):m/z[M+H]⁺Calcd for C₄₆H₅₃N₁₀O₄:809.4251;Found:809.4263.

Example 12

2,2',2",2 '" - (((3, 3' "-Dimethoxy-2 ',2" -dimethyl- [1,1':3',1": synthesis of 3', 1' -tetraphenyl ] -4,4 ' -diyl) bis (1H-1, 2, 3-triazole-1, 4-diyl) bis (methylene)) bis (azatriyl)) tetrakis (ethane-1-ol )(II-12：n＝0,X＝CH,R¹＝R²＝-N(CH₂CH₂OH),R³＝R⁴＝CH₃,R⁵＝OCH₃)

Using compound VIII-2 (0.20 g,0.34 mmol) and diethanolamine (0.13 mL,1.37 mmol) as raw materials, the same procedure as for compound II-1 gave 0.08g of yellow-green solid, yield 30.7％.m.p.92-94℃.¹H NMR(400MHz,DMSO-d₆)δ8.38(s,2H,ArH),7.70(d,J＝8.0Hz,2H,ArH),7.40(t,J＝7.4Hz,2H,ArH),7.36(dd,J＝7.7,1.9Hz,2H,ArH),7.31(d,J＝1.9Hz,2H,ArH),7.24(dd,J＝7.3,1.8Hz,2H,ArH),7.16(dd,J＝8.1,1.8Hz,2H,ArH),4.40(s,4H,OH),3.92(s,6H,OCH₃),3.87(s,4H,2ArCH₂),3.50(t,J＝6.1Hz,8H,4CH₂OH),2.59(t,J＝6.3Hz,8H,4NCH₂CH₂),2.04(s,6H,2ArCH₃).HRMS(ESI):m/z[M+H]⁺Calcd for C₄₂H₅₁N₈O₆:763.3932;Found:763.3936.

Example 13

Pharmacological experiments and results of some of the compounds of the present invention are as follows:

1. Evaluation of inhibitory Activity on PD-1/PD-L1

The purpose of the experiment is as follows: the activity of the compound of formula (I) inhibitor PD-1/PD-L1 interaction was tested using a PD-1/PD-L1 binding assas kit test kit (CISBIO).

Experimental principle: HTRF (homogeneous time resolved fluorescence), homogeneous Time-Resolved Fluorescence, is a technique used to detect analytes in pure liquid systems. It mainly uses energy transfer of two fluorophores, which are divided into energy donor europium (eu+) and energy acceptor. When the donor is externally excited (e.g., a flash lamp or laser), if within a sufficiently close distance from the acceptor, energy resonance can be transferred to the acceptor, which is excited to a specific wavelength. The assay enables simple, rapid characterization of compounds and antibody blockers in a high throughput format using HTRF technology. The interaction between PD-L1 and PD-1 can be detected by using anti-Tag 1 labeled with Eu ⁺ (europium) (HTRF energy donor) and anti-Tag 2 labeled with XL665 (HTRF energy acceptor). The PD-L1 protein and the PD-1 protein were labeled with Tag1 and Tag2, respectively, and Eu ⁺ and XL665 bound to PD-L1 and PD-1, respectively, by antibodies to form complexes. When PD-L1 and PD-1 are combined close to each other, eu ⁺, when excited by an external laser, triggers fluorescence resonance energy transfer towards XL665, which in turn emits specifically at 665 nm. This particular signal is proportional to the extent of PD1/PD-L1 interaction. Thus, compounds or antibodies that prevent PD-1/PD-L1 interactions will result in a decrease in HTRF signal.

Experimental materials: the kit is purchased from CISBIO company as PD-1/PD-L1 binding ASSAY KITS; 96-well plates: and purchasing CISBIO company.

Test instrument: PERKIN ELMER, model: enVision.

Test compounds: a compound of formula (II). Dissolving with DMSO, and diluting with diluent buffer; DMSO concentration does not exceed 0.5%.

The experimental process comprises the following steps: PD1/PD-L1 binding ASSAY KITS was used. A negative group, a positive group and a dosing group were set, with 2 duplicate wells per group. Positive control group, 2 μ L diluent was added to 96-well plate; 4uL PD-L1 and 4uL PD-1 diluted according to the specification; negative control, 6 μ L diluent and 4 μl PD-L1 were added to 96-well plates; the dosing group was dosed with 2. Mu.L of the compound of formula (I) tested (or positive compound BMS-202), 4. Mu.L of LPD-L1 and 4. Mu.L of PD-1 sequentially into 96-well plates. The plates were sealed and centrifuged at 1000rpm for 1 min and incubated at room temperature for 15 min. And uniformly mixing the Anti-Tag-Eu3 ⁺ and the Anti-Tag-XL665 which are diluted by the Buffer in equal volume, adding 10 mu L of mixed solution into each hole, centrifuging at 1000rpm for 1 minute on a sealing plate, and incubating for 2 hours at room temperature. The plate film was removed, the fluorescence intensities of 665nm and 615nm were read using EnVision, and ratio = Signal 665nm/Signal 620nm x 10 ⁴ was calculated. IC ₅₀ for the compounds was calculated using Graphpad. BMS-202 in WO2015034820 patent of BMS company was selected as positive drug for this experiment, and the activity data is shown in Table 1.

A represents 0.50-10nM; b represents 10.01-100nM; c represents 100.01nM to 1. Mu.M.

TABLE 1 blocking of hPD-1/hPD-L1 by Compounds at the protein level

Experimental results show that the compound has remarkable PD-1/PD-L1 protein-protein interaction inhibition activity.

2. Experiments to block the effect of PD-L1 on inhibiting the secretion of INF-gamma by T cells

Experimental principle: hep3B-OS8-hPDL1 cells (Shanghai Ming's research on intelligent chemistry Co., ltd.) stably expressed hPD-L1 protein on the surface; CD3 ⁺ T cells (Shanghai Chemicals research Co., ltd.) express PD-1 on their surface; when two cell lines are co-cultured, hPD-L1 on the cell surface of the Hep3B-OS8-hPDL1 can interact with PD-1 protein on the cell surface of the protein CD3+T cell, so that activation and proliferation of the CD3+T cell and expression of an immune factor INF-gamma are inhibited. When the compound blocks the PD-1/PD-L1 interaction, inhibition of CD3+ T cells will be released, thereby promoting expression of INF-gamma.

The experimental process comprises the following steps: PBMCs were isolated by density gradient centrifugation using EDTA anticoagulant tubes containing whole blood. CD3+ T cells were further isolated from PBMC using EASYSEPTM HUMAN T CELL ISOLATION KIT and resuspended in RPMI-1640 complete medium to a concentration of 5X 10 ⁵/mL. Hep3B-OS8-hPDL1 cells were incubated with 10 μg/mL mitomycin for 1.5h at 37℃and washed 4 times with PBS, and the RPMI-1640 complete medium was resuspended to a cell concentration of 5X 10 ⁵/mL. Hep3B-OS8-hPDL1 (50. Mu.L/well) and T cells (100. Mu.L/well) were added to 96-well round-bottomed microplates. 4 XKeystuda (50. Mu.L/well) and 4 Xtest compound (50. Mu.L/well) were prepared in RPMI-1640 complete medium, and the prepared compound and Keystuda were added to the corresponding wells (final concentration of Keystuda: 5. Mu.g/mL) in a total volume of 200. Mu.L. 3 concentration gradients were set for each drug, double wells, keytruda and BMS-202 as positive control groups. Incubate at 37℃in a 5% CO ₂ incubator for 72 hours. After centrifugation at 350 Xg for 5 min, 150. Mu.L of supernatant was collected and ELISA was used to detect IFN-. Gamma.secretion. GRAPDPAD PRISM6 for data processing.

As shown in the results of FIG. 1, it is evident from the experimental results that the compound II-3 can promote the expression of INF-gamma by blocking the PD-1/PD-L1 interaction, thereby relieving the inhibition of CD3 ⁺ T cells. The compounds II-1, II-3 and II-7 have the effects of promoting the expression of INF-gamma in a dose-dependent manner, are remarkably higher than BMS-202 and slightly lower than Keystuda (5 mug/mL), so that the compounds have the effect of enhancing the anti-tumor effect of T cells; therefore, the biphenyl compound can be used as an immune check point PD-1/PD-L1 inhibitor to prepare medicines for tumor immunotherapy.

Claims (5)

1. A compound of formula II:

；

II

Wherein N represents 0 or 1, X represents N or CH, Y and Z represent CH;

R ³ and R ⁴ each represent F, cl or CH ₃, respectively;

R ⁵ represents H, F, CH ₃ or OCH ₃;

r ¹ and R ² each represent 、/>、/>、/>、、/>And/>。

2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ each represent CH ₃,R⁵ for OCH ₃.

3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an acid addition salt of a compound of formula I with: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.

4. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

5. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 and a pharmaceutical composition according to claim 4 in the preparation of anti-tumor drugs of PD-1/PD-L1 inhibitors.