An ester chain-containing pd-1 / pd-l1 small molecule inhibitor and a preparation method and application thereof

Abstract

The application discloses a PD-1 / PD-L1 small molecule inhibitor containing an ester chain and a preparation method and application thereof. The application is applied to the preparation of medicines for treating immune regulation related diseases, anti-tumor medicines and anti-infection medicines. The compound has significant inhibitory activity on PD-1 / PD-L1. Meanwhile, a representative compound ZY22 shows excellent anti-tumor effect in an animal tumor model. In general, the compound can solve the defects of long half-life, low oral bioavailability and high price of the existing PD-1 / PD-L1 monoclonal antibody, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of small molecule compound technology, and relates to an ester-chain PD-1 / PD-L1 small molecule inhibitor, its preparation method and application. Background Technology

[0002] Tumor immunotherapy refers to activating the body's immune system to kill cancer cells, and its unique treatment methods have received widespread attention in the field of tumor treatment. Immunotherapy is mainly divided into three categories: chimeric antigen receptor T-cell (CAR-T) therapy, immune checkpoint inhibitors (ICIs) therapy, and tumor vaccines. Among them, immune checkpoint inhibitors, represented by PD-1 / PD-L1, are the most widely used and are the most effective treatment methods in clinical practice. Programmed cell death protein-1 (PD-1) is a member of the CD28 immune receptor family, and its main function is to regulate T cell activation. It is highly expressed in immune cells, such as T cells, B cells, tumor-associated macrophages (TAMs), and natural killer cells (NK cells). Programmed cell death ligand-1 (PD-L1), expressed on the surface of tumor cells, acts as a ligand for PD-1 and has functions such as inducing the survival and development of regulatory T cells. Its overexpression can transmit incorrect regulatory signals to T cells, thereby inhibiting T cell-mediated anti-tumor immune responses and promoting tumor cell escape.

[0003] Therefore, blocking the interaction between PD-1 and PD-L1 is considered a promising therapeutic strategy, as it can not only rapidly restore the body's immune system but also trigger tumor-specific T-cell responses. To date, the US FDA has approved six humanized monoclonal antibodies (Mabs): three PD-1 inhibitors—nivolumab, pembrolizumab, and cemipilimab—and three PD-L1 inhibitors—atezolizumab, durvalumab, and avelumab—which have shown significant therapeutic efficacy in various cancers. However, their equally significant drawbacks, such as long half-lives, poor penetration, and high prices, greatly limit their wider application.

[0004] Therefore, designing small molecule inhibitors of PD-1 / PD-L1 is a work of great significance. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a small molecule inhibitor containing an ester chain PD-1 / PD-L1.

[0006] Another object of the present invention is to provide a method for preparing the ester-chain PD-1 / PD-L1 small molecule inhibitor.

[0007] Another object of the present invention is to provide the application of the ester-chain PD-1 / PD-L1 small molecule inhibitor.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] Compounds, stereoisomers, or pharmaceutically acceptable salts thereof with the structure shown in Formula I:

[0010]

[0011] Wherein, R1 is independently hydrogen, deuterium, halogen, substituted or unsubstituted hydroxyl or amino, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy;

[0012] R2 is selected from hydrogen, deuterium, halogen, substituted alkyl or substituted alkoxy, substituted or unsubstituted hydroxyl, substituted or unsubstituted amino.

[0013] R3 is selected from any one of the three positions on the benzene ring, and the substituent is independently hydrogen, deuterium, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy.

[0014] The substituents in the substituted or unsubstituted alkyl and alkoxy groups described above are one or more of the following groups: halogen, C1-C5 alkyl, hydroxyl, C1-C5 alkoxy, C1-C5 carboxyl, C1-C5 ester, or C1-C5 amide. The substituents in the substituted hydroxyl or substituted amino groups are one or more of the following groups: C1-C5 alkyl, C1-C5 alkoxy, C1-C5 carboxyl, C1-C5 ester, or C1-C5 amide.

[0015] As a preferred embodiment of the present invention, in general formula (I)

[0016] R1 is preferably an independent hydrogen atom or a substituted hydroxyl group, and R2 is preferably... R4 and R5 are preferably independent hydrogen, substituted or unsubstituted alkyl, alkoxy, hydroxyalkyl or aminoalkyl, and R3 is selected from any one of the three positions on the benzene ring, preferably hydrogen, halogen, substituted or unsubstituted alkoxy.

[0017] The substituents in the substituted or unsubstituted alkyl and alkoxy groups described above are one or more of the following groups: halogen, C1-C5 alkyl, hydroxyl, C1-C5 alkoxy, C1-C5 carboxyl, C1-C5 ester, or C1-C5 amide; the substituents in the substituted hydroxyl or substituted amino groups are one or more of the following groups: C1-C5 alkyl, C1-C5 alkoxy, C1-C5 carboxyl, C1-C5 ester, or C1-C5 amide.

[0018] As a preferred embodiment of the present invention, in general formula (I)

[0019] R1 is selected from H;R2 is selected from R3 is selected from H,

[0020] As a preferred embodiment of the present invention, the compound represented by general formula (I) is selected from any one of the following:

[0021]

[0022]

[0023] Preparation method of the compound described in this invention:

[0024] (1) When R1 is selected from H, the synthesis route is as follows:

[0025]

[0026]

[0027] (2) When R1 is selected The synthesis route is as follows:

[0028]

[0029] A pharmaceutical composition comprising a pharmaceutically effective amount of an active ingredient and pharmaceutically acceptable excipients; said active ingredient comprising one or more of the compounds, stereoisomers, or pharmaceutically acceptable salts thereof described in this invention.

[0030] In the pharmaceutical composition of the present invention, the excipients include pharmaceutically acceptable carriers, diluents, and / or excipients.

[0031] Depending on the therapeutic purpose, the pharmaceutical composition can be formulated into various types of dosage forms, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, and injections (solutions or suspensions), with tablets, capsules, liquids, suspensions, and injections (solutions or suspensions) being preferred.

[0032] The compounds of the present invention or other pharmaceutical compositions may be administered in unit dose form via enteral or non-enteric routes, such as oral, intramuscular, subcutaneous, or rectal administration, with injections being preferred.

[0033] The use of the compounds, stereoisomers or pharmaceutically acceptable salts thereof or the pharmaceutical compositions described herein in the preparation of PD-1 / PD-L1 inhibitors.

[0034] The application of the compounds, stereoisomers, or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions described herein in the preparation of immune-regulatory diseases, tumors, and infectious diseases caused by PD-1 / PD-L1 overexpression.

[0035] As a preferred embodiment of the present invention, the immune regulation-related diseases are selected from organ-specific autoimmune diseases or systemic autoimmune diseases; the tumors are selected from melanoma, colon cancer, lung cancer, breast cancer, cervical cancer, glioma, or liver cancer.

[0036] Compared to existing technologies, our compounds have the following characteristics and advantages:

[0037] 1. The applicant has designed a novel small molecule inhibitor with a novel scaffold that targets PD-L1 using a novel linker chain. This inhibitor primarily inhibits tumor cell proliferation through immune activation. In vitro and in vivo experiments have demonstrated that this small molecule compound effectively activates the immune microenvironment, thereby inhibiting tumor growth.

[0038] 2. In vivo experiments show that the small molecule inhibitor of the present invention not only has a good therapeutic effect, but also shows no proliferative toxicity in normal organs of mice.

[0039] 3. The compounds of this invention have a broad anticancer spectrum and are applicable to a variety of cancers such as melanoma, glioma, and colon cancer.

[0040] In summary, the compounds disclosed in this invention have great application prospects in the field of tumor immunotherapy and will provide new ideas and methods for the development of PD-1 / PD-L1-based drugs. Attached Figure Description

[0041] Figure 1 In vivo antitumor assay. (A) Anatomical diagram of 4T1 tumor tissue; (B) Photograph of mouse model; (C) Tumor volume changes during treatment; (D) Mouse body weight changes during treatment; (E) Tumor weight on day 21 after treatment. Figure 2 4T1 tumor pathological section. (A)CD8 + Representative immunohistochemical assays of cell-infiltrating tumor tissue. (B) H&E staining of major organs; scale bar, 50 μM. Detailed Implementation

[0042] Synthetic route

[0043] Scheme 1 outlines the synthesis of ZY1-ZY9:

[0044] The commercially available raw material compound 1 was reacted with compound 2 (Suzuki-Miyaura) to obtain intermediate 3. Intermediates 3 and 4, 6-8 were then subjected to Steglich esterification via DMAP catalysis and DCC as a coupling agent to obtain compounds 5, 9-11. Finally, compound 5 was subjected to reductive amination in the presence of sodium cyanoborohydride to obtain the target compounds ZY1-ZY9.

[0045]

[0046] In step c, reactants 1a, 2a, and 3a correspond to products ZY1, ZY2, and ZY3, respectively.

[0047]

[0048] Among them, the reactants and products corresponding to each target compound are as follows:

[0049] reactants in step a Step a product Step b reactants ZY4 6 9 1a ZY5 6 9 2a ZY6 6 9 4a ZY7 7 10 1a ZY8 8 11 2a ZY9 8 11 4a

[0050]

[0051]

[0052] Synthesis of Scheme 2 ZY22, ZY10-ZY13:

[0053] Intermediate 3 undergoes Steglich esterification with different aldehydes 12 and 13 to generate compounds 14 and 15. Then, the resulting compounds 14 and 15 are combined with compound 16 and undergo a substitution reaction under alkaline conditions of Cs2CO3 to generate compounds 17 and 18. Finally, compounds 17 and 18 are subjected to reductive amination in the presence of sodium cyanoborohydride to obtain the target compounds ZY22, ZY10-ZY13.

[0054]

[0055] Among them, the reactants and products corresponding to each target compound are as follows:

[0056]

[0057]

[0058] The structural formulas for 1a and 4a are the same as above;

[0059]

[0060] The following is a detailed implementation plan of the optimal scheme and preferred compound for the above preparation method.

[0061] Example 1

[0062] Preparation of (2-(methoxy-4-((2-methyl-[1,1'-biphenyl]-3-formyl)oxy)benzyl)-L-serine (ZY1):

[0063] 1.00 g of phenylboronic acid and 0.80 g of 3-bromo-2-methylbenzoic acid were weighed and dissolved in 30 mL of a dioxane / H₂O mixture (7:1). 0.15 g of Pd(dppf)Cl₂ and 2.00 g of CS₂CO₃ were added to the above reaction solution. The mixture was replaced with N₂. The reaction mixture was heated at 90 °C for 12 hours, and the reaction was monitored by TLC until completion. The crude product was purified by column chromatography to give a white solid intermediate 3 (1.20 g).

[0064] Compound 3 (1.20 g) was dissolved in 30 mL of dichloromethane, and 4-hydroxy-2-methoxybenzaldehyde (0.90 g), DMAP (0.05 g), and DCC (1.80 g) were added. The reaction mixture was stirred at room temperature for 6 h, and the reaction was monitored by TLC until it was complete. The crude product was purified by column chromatography to give a white solid intermediate 5 (0.90 g).

[0065] Intermediate 5 (0.10 g) and L-serine (compound 1a, 0.05 g) were added to 8 mL of DMF solution, along with 6 drops of acetic acid. The mixture was stirred at room temperature for 2 hours, followed by the addition of sodium cyanoborohydride (0.09 g). Stirring continued at room temperature for 12 hours, and TLC showed the reaction was complete. The reaction solution was concentrated to obtain a residue, which was dissolved in dichloromethane and washed with water. The organic layer was dried and evaporated to obtain a crude product, which was purified by column chromatography to give a white solid in 34% yield. 1 H NMR (600MHz, DMSO-d6) δ8.05(d,J=7.4Hz,1H),7.48(q,J=8.2Hz,4H),7.42(t,J=7.4Hz,1H),7.35(d,J=7.5Hz,2H),7.08(s,1H),6.93(d, J=8.2Hz,1H),4.05(s,2H),3.85(s,3H),3.77(dd,J=11.1,4.3Hz,2H),3.67(dd,J=11.4,6.7Hz,1H),3.23(d,J=5.6Hz,1H),2.42(s,3H). 13CNMR(151MHz,DMSO-d6)δ172.63,166.14,162.77,153.64,143.95,141.09,136.85,134.49,133.79,130.09,129 .65,128.87,127.87,126.38,119.49,116.29,114.51,106.49,63.24,56.60,54.19,42.23,18.70.HRMS:[M+Na] + calcd for C 27 H 30 NO4436.1755 found 436.1755.

[0066] Example 2

[0067] Preparation of 4-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)methyl)-3-methoxyphenyl-2-methyl-[1,1'-biphenyl]-3-carboxylate (ZY2):

[0068] The procedure is the same as for compound ZY1. White solid, yield 45%; 1 H NMR (600MHz, DMSO-d6) δ8.05(dd,J=7.2,2.1Hz,1H),7.49(td,J=7.9,4.1Hz,5H),7.42(t,J=7.3Hz,1H),7.37–7.32(m, 2H),7.07–7.02(m,1H),6.91(dd,J=8.1,2.3Hz,1H),4.92(s,3H),4.05(s,2H),3.84(s,3H),3.57(s,6H),2.42(s,3H). 13 C NMR (151MHz, DMSO-d6) δ158.43,157.13,143.86,143.61,141.16,136.58,130.87,129.66,129.64,128.85,128.77,127. 82,126.34,126.12,115.75,113.65(d,J=6.2Hz),106.82,105.42,99.12,56.05,55.38,46.08,18.68,15.72.HRMS:[M+H] + calcd for C 27 H 30 NO4432.2169 found432.2172.

[0069] Example 3

[0070] Preparation of 4-(((1,3-dihydroxypropyl-2-yl)amino)methyl)-3-methoxyphenyl-2-methyl-[1,1'-biphenyl]-3-carboxylate (ZY3):

[0071] The procedure is the same as for compound ZY1. White solid, yield 60%; 1 H NMR (600MHz, DMSO-d6) δ8.05 (dd, J=7.3, 2.0Hz, 1H), 7.49 (ddt, J=7.6, 5.2, 3.0 Hz,5H),7.42(t,J=7.4Hz,1H),7.37–7.32(m,2H),7.05(d,J=2.3Hz,1H),6.91( dd,J=8.2,2.2Hz,1H),4.92(s,3H),4.00(s,2H),3.84(s,3H),3.58(dd,J=11.2 ,5.3Hz,2H),3.53(dd,J=11.3,5.5Hz,2H),2.83(t,J=5.4Hz,1H),2.42(s,3H). 13 C NMR(151MHz,DMSO-d6)δ166.45,158.42,157.14,143.87,143.62,130.86,129.66,129.63,128.85,128.77,127.83,127.71 ,126.35,115.74,106.83,105.61,99.12,59.06,56.14,55.49(d,J=34.0Hz),18.68,18.55(d,J=2.6Hz),15.72.HRMS:[M+H] + calcd for C 25 H 28 NO5422.1962 found 422.1963.

[0072] Example 4

[0073] Preparation of ((2-methyl-[1,1'-biphenyl]-3-formyl)oxy)benzyl)-D-serine (ZY4):

[0074] The procedure is the same as for compound ZY1. White solid, yield 45%; 1H NMR (500MHz, DMSO-d6) δ8.01(d,J=7.1Hz,1H),7.53–7.44(m,5H),7.42(t,J=7.3Hz,2H),7.35(d,J=7.4Hz,1H),7.27 (d,J=8.2Hz,2H),3.89(d,J=14.2Hz,1H),3.83(dd,J=10.9,7.0Hz,1H),3.73(dd,J=15.9,10.0Hz,1H),2.40(s,1H). 13 C NMR (151MHz, DMSO-d6) δ166.59,149.91,143.86(d,J=3.6Hz),141.16(d,J=3.7Hz),138.06,136.55,134.55–133.95( m),130.86,129.85,129.66,128.84,127.82,126.37,122.15,63.49,60.62(d,J=86.6Hz),54.59,18.64.HRMS:[M+H] + calcd for C 27 H 30 NO4406.1649 was found.

[0075] Example 5

[0076] Preparation of 2-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)methyl)phenyl-2-methyl-[1,1'-biphenyl]-carboxylate (ZY5):

[0077] The procedure is the same as for compound ZY1. White solid, yield 42%; 1 H NMR(500MHz,DMSO-d6)δ8.03(dd,J=7.0,2.3Hz,1H),7.61(d,J=8.1Hz,2H),7.51–7.45(m,4H),7.41 (t,J=7.4Hz,1H),7.34(dd,J=11.1,7.9Hz,4H),5.19(s,3H),4.15(s,2H),3.62(s,6H),2.41(s,1H). 13C NMR(151MHz,DMSO-d6)δ166.50,162.79,151.10,143.89,141.11,136.61,134.34,132.07,130 .69,129.92,129.66,128.86,127.84,126.41,122.29,58.38,45.26,36.26,18.66.HRMS:[M+H] + calcd for C 27 H 30 NO4422.1962 found 422.1959.

[0078] Example 6

[0079] Preparation of (2-((2-methyl-[1,1'-biphenyl]-3-formyl)oxy)benzyl)serine (ZY6):

[0080] The procedure is the same as for compound ZY1. White solid, yield 39%; 1 H NMR (500MHz, DMSO-d6) δ8.01(dd,J=7.1,2.1Hz,1H),7.48(tt,J=7.6,4.0Hz,5H),7.42(q,J=7.3,6.6Hz,2H),7.35(d,J=7 .4Hz,2H),7.27(d,J=8.3Hz,2H),3.89(d,J=14.3Hz,1H),3.85–3.79(m,1H),3.73(dd,J=16.0,10.9Hz,1H),2.40(s,3H). 13 C NMR(151MHz,DMSO-d6)δ166.59,149.91,143.87,141.15,138.10,136.56,134.24,130.85,129 .89,129.85,129.66,128.84,127.82,126.37,122.15,63.50,60.95,54.60,18.65.HRMS:[M+H] + calcd for C 27 H 30 NO4406.1649 found406.1651.

[0081] Example 7

[0082] Preparation of 2-bromo-4-((((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)methyl)phenyl-2-methyl-[1,1'-biphenyl]-3-carboxylate (ZY7):

[0083] The procedure is the same as for compound ZY1. White solid, yield 53%; 1 H NMR (500MHz, DMSO-d6) δ8.05(dd,J=7.4,1.9Hz,1H),7.72(d,J=8.4Hz,1H),7.63(d,J=2.3Hz,1H),7.48(dt,J=7 .5,5.3Hz,4H),7.45–7.39(m,1H),7.37–7.33(m,3H),4.44–4.23(m,3H),3.91(s,2H),3.47(s,6H),2.41(s,3H). 13 C NMR(151MHz,DMSO-d6)δ167.99,157.38,143.59,141.33,135.84,133.43,132.11,131.36,129.63,129.43 ,128.79,127.71,126.09,123.52,119.10,115.18,64.81,61.76,60.89,59.90,45.52,18.61.HRMS:[M+H] + calcd for C 27 H 30 NO4500.1067found 500.1070

[0084] Example 8

[0085] Preparation of 2-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)methyl)-3-methylphenyl-2-methyl-[1,1'-biphenyl]-3-carboxylate (ZY8):

[0086] The procedure is the same as for compound ZY1. White solid, yield 29%; 1 H NMR(500MHz,DMSO-d6)δ8.01(dd,J=7.0,2.3Hz,1H),7.55(d,J=8.0Hz,1H),7.51–7.45(m,4H),7.45–7.39(m,1H),7 .37–7.32(m,2H),7.16(d,J=7.6Hz,2H),5.09(s,3H),4.12(s,2H),3.62(s,6H),3.18(s,1H),2.42(d,J=8.7Hz,6H). 13C NMR(151MHz,DMSO-d6)δ166.68,149.89,143.86,141.14,138.79,136.43,134.19,130.97,129.78 ,129.66,128.84,127.82,126.38,123.52,119.39,61.77,61.00,43.10,19.06,18.64.HRMS:[M+H] + calcd for C 27 H 30 NO4436.2118 found436.2117.

[0087] Example 9

[0088] Preparation of (2-methyl-4-((2-methyl-[1,1'-biphenyl]-3-formyl)oxy)benzyl)-D-serine (ZY9):

[0089] The procedure is the same as for compound ZY1. White solid, yield 32%; 1 H NMR (500MHz, DMSO-d6) δ8.00(dd,J=7.1,2.1Hz,1H),7.48(tdd,J=7.5,6.1,4.4Hz,5H),7.45–7.39(m,1H),7.37–7.33(m,2H),7.15(t,J=3 .4Hz,1H),7.12(d,J=2.5Hz,1H),3.72(dd,J=10.9,5.0Hz,2H),3.66(dd,J=10.9,5.8Hz,2H),3.25–3.19(m,1H),2.40(s,3H),2.38(s,3H). 13 C NMR(151MHz,DMSO-d6)δ168.81,166.06,159.44,153.36,143.91,141.13,136.89,134.44,130.46 ,130.16,129.65,128.87,127.85,126.34,106.75,99.36,63.48,60.46,56.84,18.73.HRMS:[M+H] + calcd for C 27 H 30 NO4420.1805 found420.1806.

[0090] Example 10

[0091] Preparation of ((3-cyanobenzyl)oxy)-4-((((2-hydroxyethyl)amino)methyl)phenyl-2-methyl-[1,1'-biphenyl]-3-carboxylate (ZY10):

[0092] Compounds 14 and 15 were synthesized according to compound 5 in general formula I. Compound 14 (1.35 g) was dissolved in 20 ml of DMF. Then, compound 16 (0.80 g) and CS2CO3 (1.20 g) were added to the above reactants and stirred at room temperature for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography to obtain a white solid, which was intermediate 17 (0.90 g).

[0093] The obtained intermediate 17 (0.10 g) and ethanolamine (0.04 g) were dissolved again in 4 ml of DMF. Six drops of acetic acid were added to the reaction system, and the mixture was stirred at room temperature for 2 h. Then, NaBH4CN (0.04 g) was added, and stirring continued for 12 h. The reaction solution was dissolved in water, and the aqueous phase was extracted with ethyl acetate (20 ml × 3). The organic phases were collected and combined. The crude product was purified by column chromatography to obtain a white solid, with a yield of 37%. 1 H NMR (500MHz, DMSO-d6) δ8.05 (dd, J=7.2, 2.1Hz, 1H), 8.00 (d, J=1.7Hz, 1H), 7.88 (dt, J=7. 9,1.5Hz,1H),7.84(dt,J=7.8,1.4Hz,1H),7.64(t,J=7.7Hz,1H),7.54–7.45(m,5H),7.45– 7.40(m,1H),7.37–7.34(m,2H),7.18(d,J=2.1Hz,1H),6.98(dd,J=8.2,2.1Hz,1H),5.25(s ,2H),4.95(s,1H),4.04(s,2H),3.61(t,J=5.5Hz,2H),2.84(t,J=5.5Hz,2H),2.43(s,3H). 13C NMR(151MHz,DMSO-d6)δ166.30,157.40,143.94,141.10,138.76,136.73,13 4.42,132.95,132.28,131.58,130.56,130.24,129.98,129.66,129.63,128 .88,128.76,127.87,126.40,125.98,119.18,114.68,111.96,107.36,69.1 5,55.38,31.67(dd,J=18.9,5.4Hz),29.46(d,J=6.1Hz),18.69.HRMS:[M+H] + calcd forC 27 H 30 NO4493.2122 found 493.2124.

[0094] Example 11

[0095] Preparation of (2R,4S)-1-(2-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-carboxylic acid)benzyl)-4-hydroxypyrrolidine-2-carboxylic acid (ZY22):

[0096] The procedure is the same as for compound ZY10. White solid, yield 36%; 1 H NMR (500MHz, DMSO-d6) δ8.04(dd,J=7.2,2.1Hz,1H),8.00(d,J=1.7Hz,1H),7.89(dt,J=7.9,1.5Hz,1H),7.82(dt,J=7.7,1.5 Hz,1H),7.63(t,J=7.7Hz,1H),7.52–7.45(m,5H),7.45–7.40(m,1H),7.37–7.34(m,2H),7.15(d,J=2.2Hz,1H),6.95(dd,J=8 .2,2.2Hz,1H),5.31–5.22(m,2H),5.12(s,1H),4.27(t,J=5.1Hz,1H),4.12(d,J=13.6Hz,1H),3.98(d,J=13.6Hz,1H),3.62( t,J=7.9Hz,1H),3.28(dd,J=10.5,5.4Hz,1H),2.58(dd,J=10.5,4.1Hz,1H),2.42(s,3H),2.03(ddd,J=12.1,8.3,5.3Hz,2H). 13C NMR(151MHz,DMSO-d6)δ172.41,166.28,157.22,151.82,143.91,141.11,138.81,136.72,134.37,132.89,132.20,131.55,130.61,130.24,12 9.96,129.65,128.87,127.85,126.40,121.77,119.19,114.69,111.91 ,107.38,69.25,69.11,66.06,60.90,53.07,38.92,18.68.HRMS:[M+Na] + calcd for C 27 H 30 NO4585.1996found 585.2000.

[0097] Example 12

[0098] Preparation of (3-((3-cyanobenzyl)oxy)-4-((2-methyl-[1,1'-biphenyl]-3-formyl)oxy)benzyl)serine (ZY11):

[0099] The procedure is the same as for compound ZY10. White solid, yield 38%; 1 H NMR(500MHz,DMSO-d6)δ8.03(dd,J=7.3,2.0Hz,1H),8.00(s,1H),7.91(d,J=8.1Hz,1H),7.81(d,J =7.7Hz,1H),7.62(t,J=7.7Hz,1H),7.49(tt,J=9.2,3.7Hz,5H),7.45–7.39(m,1H),7.35(dd,J=7.3 ,1.6Hz,2H),7.15(d,J=2.2Hz,1H),6.94(dd,J=8.1,2.2Hz,1H),5.24(s,2H),04.03(d,J=5.9Hz,2H ),3.70(t,J=5.6Hz,1H),3.66–3.58(m,1H),3.52–3.31(m,2H),3.16(t,J=6.3Hz,1H),2.41(s,3H). 13C NMR(151MHz,DMSO-d6)δ166.29,157.28,151.78,143.90,141.12,138.76,136.73,134.37,133.01,132.23,131.58,131.52,131.48,130.60, 130.25,130.24,129.97,129.65,128.86,127.84,126.37,119.18,114.56,111.91,107.27,69.15,63.23,61.52,45.65,18.68.HRMS:[M+Na] + calcd for C 27 H 30 NO4559.1840found 559.1839.

[0100] Example 13

[0101] Preparation of (2-((3-cyanobenzyl)oxy)-6-methyl-4-((2-methyl-[1,1'-biphenyl]-3-formyl)oxy)benzyl)serine (ZY12):

[0102] The procedure is the same as for compound ZY10. White solid, yield 36%; 1 H NMR (500MHz, DMSO-d6) δ8.05–8.01(m,2H),7.95(d,J=7.8Hz,1H),7.82(d,J=7.6Hz,1H),7. 62(t,J=7.7Hz,1H),7.49(dt,J=7.3,4.8Hz,4H),7.42(t,J=7.3Hz,1H),7.37–7.34(m,2H), 7.03(d,J=2.3Hz,1H),6.85(d,J=2.2Hz,1H),5.24(d,J=4.6Hz,2H),4.21–4.04(m,2H),3.7 8(dd,J=11.1,4.3Hz,1H),3.63(dd,J=11.3,6.9Hz,2H),3.23(t,J=5.8Hz,1H),2.42(s,6H). 13C NMR(151MHz,DMSO-d6)δ166.25,157.96,151.71,143.91,141.11,140.82, 138.66,136.70,134.37,133.10,132.83,132.23,132.15,131.66,131.41 ,130.60,130.21,129.94,129.65,128.85,127.84,126.37,119.19,116.7 5,111.91,105.05,69.34,63.75,60.98,42.71,19.65,18.68.HRMS:[M+Na] + calcd for C 27 H 30 NO4573.1996 found 573.2000.

[0103] Example 14

[0104] Preparation of (2-((3-cyanobenzyl)oxy)-6-methyl-4-((2-methyl-[1,1'-biphenyl]-3-formyl)oxy)benzyl)-D-serine (ZY13):

[0105] The procedure is the same as for compound ZY10. White solid, yield 31%; 1 H NMR (500MHz, DMSO-d6) δ8.04(d,J=6.5Hz,2H),7.97(d,J=7.0Hz,1H),7.84(d,J=8.3Hz,1 H),7.63(dh,J=8.1,4.1Hz,2H),7.50(tq,J=7.2,3.6,3.2Hz,4H),7.40–7.35(m,2H),7.0 5(d,J=5.2Hz,1H),6.87(q,J=4.5Hz,1H),5.28–5.24(m,2H),4.23–4.06(m,2H),3.86–3. 77(m,1H),3.66(ddt,J=11.3,7.4,4.2Hz,2H),3.31–3.20(m,2H),2.43(q,J=4.2Hz,6H). 13C NMR(151MHz,DMSO-d6)δ166.27,157.93,151.64,143.91,141.11,140.76,138.69,136.69,134.37,133.09,132.23,132.16,131.65,131.42,130 .62,130.21,129.93,129.66,128.86,127.85,126.38,119.19,116.75, 111.92,105.06,69.34,63.68,61.12,42.71,19.63,18.68.HRMS:[M+Na] + calcdfor C 27 H 30 NO4573.1996 found 573.1993.

[0106] Implementation effect

[0107] The pharmacological experiments and results of some compounds in this invention are as follows:

[0108] The compound's activity in inhibiting PD-1 / PD-L1 interaction:

[0109] Experimental Methods: The PD-1 / PD-L1 binding assay kit was purchased from CISBIO (Cat#64PD1PEG). Compounds were serially diluted according to their concentration gradient. In a 384-well plate, 2 μL of the target compound dilution was mixed with 4 μL of Tag1-PD-L1 protein and 4 μL of Tag2-PD-1 protein. The mixture was incubated at room temperature (RT) for 15 min. Then, 10 μL of a mixture of anti-Tag1-Eu Cryptate reagent and anti-Tag2-XL665 antibody was added, the plate was sealed, and incubated in the dark at RT for 2 h. Fluorescence signals were detected at 665 nm and 620 nm. IC50 was calculated using Graphpad 8.0.2 software. 50 Values. Three subwells were set up for each compound, and each experiment was repeated three times. The experimental results are expressed as mean ± SD.

[0110] Table 1. Inhibition rate of the preferred compounds of the present invention on the inhibition of PD-1 / PD-L1 interaction;

[0111]

[0112] a The data is the mean ± SD of three repeated measurements.

[0113] As shown in Table 1, the inhibitory effects of the compounds of the present invention on PD-1 / PD-L1 are all better than those of the positive control, especially compounds ZY7, ZY8, ZY9 and ZY22. Among them, compound ZY22 has the best effect on inhibiting the interaction of PD-1 / PD-L1. Compared with the positive control BMS-1, the inhibitory activity is increased by 130 times, which shows the potential to be an excellent anti-tumor drug.

[0114] In vivo antitumor experiments of compound ZY22:

[0115] BALB / c female mice (6 weeks old) were purchased from Jiangsu Wukong Biotechnology Co., Ltd. All experiments were conducted according to the guidelines and protocols approved by the Jiangsu Provincial Animal Experimentation and Protection Association. The mice were housed in a sterile environment with sterile food and water under standard laboratory conditions (292±2℃, 55±10% humidity, 12-12h light-dark cycle). 9.0×10 5 4T1 cells were resuspended in PBS and subcutaneously injected into the area near the spine of mice. When the tumor volume reached 80 mm², the cells were inoculated. 3 ~100mm 3 Mice were randomly divided into four groups (n=6 per group): control group, BMS-1 ​​(20 mg / kg), ZY22 (10 mg / kg), and ZY22 (20 mg / kg). The drug dissolution formulation was 5% DMSO, 10% castor oil, and 85% saline. Intraperitoneal administration was performed for 21 consecutive days. The control group received only an equal volume of drug-loaded solution. Tumor volume was measured every two days using calipers and calculated using the following formula: Volume (mm²) 3 ) = Length (mm) 3 )×width 2 (mm 3 × 0.5. After administration, the mice were euthanized, and the tumors and other necessary tissues and organs were dissected and weighed. Figure 1 A portion of the tumor tissue was used for further immune function analysis. Figure 2 In section A, the final analysis of mouse heart, liver, spleen, lung, and kidney sections using HE staining is shown in the figure. Figure 2 B in the middle.

[0116] In vivo experiments showed that ZY22 could better inhibit tumor growth than BMS-1. Immunological analysis of tissues indicated that ZY22 inhibits tumor growth by activating CD8+ in vivo. + T cells thus inhibit tumor growth. HE staining results of some tissues and organs in mice in the drug-treated group showed that the representative compound ZY22 had no obvious toxicity in vivo.

Claims

1. A compound, stereoisomer or pharmaceutically acceptable salt thereof represented by the following formula (I): ###0001### Formula (I) wherein the compound represented by the formula (I) is selected from any one of the following: ###0002### ###0003### ###0004### ###0005### ###0006### ###0007### ###0008### ###0009### ###0010### ###0011### ###0012### ###0013### ###0014### ###0015### ###0016### ###0017### ###0018### ###0019### ###0020### ###0021### ###0022### ###0023### ###0024### ###0025### ###0026### ###0027### ###0028### ###0029### ###0030### ###0031### ###0032### ###0033### ###0034### ###0035### ###0036### ###0037### ###0038### ###0039### ###0040### ###0041### ###0042### ###0043### ###0044### ###0045### ###0046### ###0047### ###0048### ###0049### ###0050### ###0051### ###0052### ###0053### ###0054### ###0055### ###0056### ###0057### ###0058### ###0059### ###0060### ###0061### ###0062### ###0063### ###0064### ###0065### ###0066### ###0067### ###0068### ###0069### ###0070### ###0071### ###0072### ###0073### ###0074### ###0075### ###0076### ###0077### ###0078### ###0079### ###0080### ###0081### ###0082### ###0083### ###0084### ###0085### ###0086### ###0087### ###0088### ###0089### ###0090### ###0091### ###0092### ###0093### ###0094### ###0095### ###0096### ###0097### ###0098### ###0099### ###0100### ###0101### ###0102### ###0103### ###0104### ###0105### ###0106### ###0107### ###0108### ###0109### ###0110### ###0111### ###0112### ###0113### ###0114### ###0115### ###0116### ###0117### ###0118### ###0119### ###0120### ###0121### ###0122### ###0123### ###0124### ###0125### ###0126### ###0127### ###0128### ###0129### ###0130### ###0131### ###0132### ###0133### ###0134### ###0135### ###0136### ###0137### ###0138### ###0139### ###0140### ###0141### ###0142### ###0143### ###0144### ###0145### ###0146### ###0147### ###0148### ###0149### ###0150### ###0151### ###0152### ###0153### ###0154### ###0155### ###0156### ###0157### ###0158### ###0159### ###0160### R1is independently selected from H; R2is selected from R3is selected from H, 2. The compound of formula (I) according to claim 1, stereoisomers or pharmaceutically acceptable salts thereof, wherein, ​ 3. A pharmaceutical composition, characterized by ​ ​ ​ 6. Use according to claim 5, characterized in that, ​

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