CN111909240A

CN111909240A - PD-1/PD-L1 polypeptide inhibitor and medical application thereof

Info

Publication number: CN111909240A
Application number: CN201910391782.9A
Authority: CN
Inventors: 蒋晟; 周光飚; 郝海平; 苗琪; 吴筱星; 张阔军; 邱亚涛
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-05-10
Filing date: 2019-05-10
Publication date: 2020-11-10
Anticipated expiration: 2039-05-10
Also published as: CN111909240B

Abstract

The invention discloses a cyclopeptide compound or pharmaceutically acceptable salt, ester, solvate, prodrug, racemate or isomer thereof, and a pharmaceutical composition containing the compound as an active ingredient, and tests prove that the cyclopeptide compound or the pharmaceutically acceptable salt, ester, solvate, prodrug, racemate or isomer thereof have activity in inhibiting a PD-1/PD-L1 pathway, and have application in preparing a tumor medicament for preventing and treating a tumor with the PD-1/PD-L1 pathway as a target; the tumor includes blood cancer, nervous system cancer, gastrointestinal cancer, esophageal cancer, urinary system cancer, lung cancer, liver cancer, skin cancer, etc.

Description

PD-1/PD-L1 polypeptide inhibitor and medical application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a PD-1/PD-L1 polypeptide compound or a pharmaceutically acceptable salt, ester or solvent compound thereof, and an application thereof in preparation of PD-1/PD-L1 inhibitor drugs.

Background

PD-1 is also named CD279, is a type I transmembrane protein with a relative molecular weight of 55000-60000, belongs to an immunoglobulin superfamily member, and mainly comprises an extracellular immunoglobulin variable region (IgV) like structural domain, a hydrophobic transmembrane region and an intracellular region. The intracellular domain includes C-terminal and N-terminal amino acid residues, contains 2 independent phosphorylation sites, which are an immunoreceptor tyrosine based inhibition motif (ITIM) and an immunoreceptor tyrosine based switch motif (ITSM), respectively. PD-1 is mainly expressed on activated CD4+ T cells, CD8+ T cells, B cells, NK cells, monocytes, dendritic cells and other immune cells, and promotes the maturation of T cells. The ligands of PD-1 are members of the B7 family, including PD-L1 (aka B7-H1, CD274) and PD-L2 (aka B7-DC), both highly expressed in placental tissue; low expression in spleen, lymph node, thymus; no expression in brain tissue. Wherein PD-L1 is I type transmembrane protein, and is mainly expressed in antigen presenting cells, B cells, T cells, epithelial cells, muscle cells, endothelial cells and the like. PD-1 and PD-L1 jointly form a PD-1/PD-L1 signal channel, inhibit the generation of growth factors and cell proliferation, and play an important role in the activation of T cells and the regulation and control of immune response. The PD-1/PD-L1 pathway, after activation, suppresses the immune system in cancer, pregnancy, tissue transplantation and autoimmune diseases. At present, the development of PD-1/PD-L1 inhibitors is mainly focused on the field of monoclonal antibodies, and monoclonal antibodies such as Nivolumab, Lambolizumab, Atezolizumab, Durvalumab, Avelumab and the like are sold in the market at home and abroad, and can be used for treating diseases with poor effects of conventional treatment methods such as non-small cell lung cancer, melanoma and the like, and have obvious treatment effects. Compared with monoclonal antibody research and development, the peptide inhibitor in the field is slow to progress. Therefore, the research and development of peptide inhibitors for inhibiting the interaction of PD-1/PD-L1 have important clinical significance.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides a cyclic peptide compound or a pharmaceutically acceptable salt, ester or solvent compound thereof, and also provides a pharmaceutical composition containing an effective amount of the cyclic peptide compound or the pharmaceutically acceptable salt, ester or solvent compound thereof, and pharmaceutical application of the cyclic peptide compound or the pharmaceutically acceptable salt, ester or solvent compound thereof.

The technical scheme is as follows: the cyclopeptide compound or pharmaceutically acceptable salt, ester, solvate, prodrug, racemate or isomer thereof is shown as a general formula I:

wherein A is selected from:

one kind of (1).

The pharmaceutical composition of the present invention comprises the cyclic peptide compound of claim 1 or a pharmaceutically acceptable salt, ester or solvate thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be tablets, capsules, granules, powder, syrup, oral liquid or injection.

The application discloses application of the cyclic peptide compound or pharmaceutically acceptable salt, ester, solvate, prodrug, racemate or isomer thereof in preparing medicaments for preventing and treating tumors. Furthermore, the medicine is a peptide inhibitor taking a PD-1/PD-L1 pathway as a target point.

The tumors include hematologic cancer, nervous system cancer, gastrointestinal cancer, esophageal cancer, urinary system cancer, lung cancer, liver cancer, and skin cancer. More specifically, the tumors include lymphoma, non-small cell lung cancer, cell cancer of head and neck, glioma, neuroblastoma, squamous carcinoma of lung, adenocarcinoma of lung, bladder cancer, stomach cancer, colon cancer, colorectal cancer, kidney cancer, bile duct cancer, stomach cancer, esophageal squamous carcinoma, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, brain cancer, melanoma, multiple myeloma, skin cancer, epithelial cell cancer, leukemia, cervical cancer and the like, including metastatic lesions of other tissues or organs far away from the primary site of the tumor.

The medicine can be used as cancer immunotherapy medicine, cancer chemotherapy medicine or cancer targeted therapy medicine.

The application discloses application of the cyclic peptide compound or pharmaceutically acceptable salt, ester, solvate, prodrug, racemate or isomer thereof in preparing PD-1/PD-L1 pathway inhibitors.

Has the advantages that: the invention discloses a cyclic peptide compound or pharmaceutically acceptable salt, ester, solvate, prodrug, racemate or isomer thereof, and a pharmaceutical composition containing the compound as an active ingredient, and tests prove that the cyclic peptide compound or pharmaceutically acceptable salt, ester, solvate, prodrug, racemate or isomer thereof have activity in inhibiting a PD-1/PD-L1 pathway, and have application in preparing medicaments for preventing and treating tumors with the PD-1/PD-L1 pathway as a target.

Detailed Description

The invention will be further illustrated with reference to some specific examples of compounds, but the invention is not limited to these examples.

The synthesis of cyclic peptide compounds described herein can be performed by the following methods:

1. general procedure for solid phase Synthesis

Solid phase amino acid synthesis was performed in a solid phase reaction synthesis tube using rink amide-AM resin (Merrifield polymer supported (2, 4-dimethoxyphenyl) (4-alkoxyphenyl) methane, where the 4-alkoxy group is the position and type of bond attached to the resin, with a loading of 0.7 mmol/g). After the reagents used in the reaction were dissolved in DMF and DCM, they were added to the reaction tube along the tube wall and shaken for the desired time after passing nitrogen through the tube. Thereafter, the reaction solution was evacuated from the lower side of the reaction tube by a vacuum pump. The solvents and reagents used in the reaction were: DMF ═ N, N-dimethylformamide; DCM ═ dichloromethane; HATU ═ 1- [ bis (dimethylamino) methylene]-3-oxo-1H-1, 2, 3-triazolo [4,5-b]Pyridine hexafluorophosphate; deptt ═ 3- (diethoxyphosphoryloxy) -1,2, 3-benzotriazin-4-one; TBTU ═ O-benzotriazol-N, N' -tetramethyluronium tetrafluoroborate; DIPEA ═ diisopropylethylamine. The swelling process of the resin is described below in "resin swelling step". The condensation step is described below as the "condensation step". Amino acid derivatives used andthe terminal carboxylic acids are shown below (side chain protecting groups placed in parentheses): Fmoc-L-Gly-OH, Fmoc-L-Cys (Trt) -OH, Fmoc-L-Leu-OH, Fmoc-L- [ N-Me]Nle-OH，Fmoc-L-Trp(CH₂COOtBu)-OH，Fmoc-L-Trp(Boc)-OH，Fmoc-L-Dab(Boc)-OH，Fmoc-L-[O-tBu]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH，ClCH₂COOH。

In some embodiments, the following amino acids and terminal carboxylic acids are also used: Fmoc-L-Trp (Cbz) -OH, Fmoc-L-dab (Cbz) -OH, Fmoc-L- [ O-Bn ] Hyp-OH, Fmoc-L-dap (Cbz) -OH, Fmoc-L- [ O-Bn ] Tyr-OH and acrylic acid.

In some embodiments, the following amino acid derivatives may also be used: 2-fluorenylmethoxycarbonylamino-4-butenoic acid

2. Resin swelling step

Rink Amide-AM resin (286mg, 0.2mmol) was added to a 10mL solid phase synthesis reaction tube, 8mL DCM was added, and the mixture was allowed to stand for half an hour. After that, the DCM was pumped out by a vacuum pump to complete the swelling of the resin.

3. Step of condensation

6mL of 20% piperidine/DMF solution was added to the reaction tube and shaken for 30 minutes. The reaction solution was drained, and the resin was washed with dry DMF (10mL), dry methanol (10mL) and dry DCM (10mL) for 3 times, after which the sample was colored with chloranil, the resin was blue indicating the end of the removal of the protecting group.

After deprotection was complete, the desired amino acid (0.6mmol), HATU (228mg, 0.6mmol), DMF (6mL) and DIPEA (210. mu.L, 1.2mmol) were added sequentially to a dry round bottom flask and clarified by ultrasonic-assisted dissolution. Adding the mixed solution into a solid-phase synthesis reaction tube, reacting for 3 hours at 25 ℃, sampling, and developing color through chloranil, wherein the resin is colorless and transparent and can indicate that the condensation reaction is finished. The resin was washed 3 times with dry DMF (10mL), dry methanol (10mL) and dry DCM (10mL) for the next condensation.

4. Peptide liberation procedure

After completion of the desired linear peptide synthesis, the resin was washed with anhydrous DMF 1 time and then pumped to dryness. To the dried resin was added a cleavage solution (trifluoroacetic acid: thioanisole: 1, 2-ethanedithiol: anisole ═ 90:5:2.5:2.5, volume ratio). The mixture was shaken at 10 ℃ for 3 hours. After the reaction is finished, the cutting solution is filtered out by suction, the cutting solution is concentrated to 1/2 of the original volume, and the concentrated solution is added into 10 times of anhydrous ether with the temperature of-20 ℃ dropwise. And (4) carrying out suction filtration to form a precipitate to obtain the crude peptide, and directly carrying out the next reaction without treatment.

RP-HPLC separation step

Dissolving the obtained crude peptide in a certain amount of purified water, adjusting the pH to 7 by using triethylamine or 2M HCl, adding acetonitrile to clarify, and freeze-drying to obtain a crude solid. An amount of acetonitrile was added to dissolve it completely, and filtered through a 0.33. mu.M filter. Separation was performed using an agent eclipse XDB-C18 column, mobile phase A: 0.1% TFA/H₂O; mobile phase B: 0.1% TFA/MeCN. The chromatographic condition is 10% B-100% B for 60 min.

Example 1

According to the aforementioned "resin swelling step", after swelling the resin, according to the "condensation step", the amino acids selected are Fmoc-L-Gly-OH, Fmoc-L-Cys (Trt) -OH, Fmoc-L-Leu-OH, Fmoc-L- [ N-Me, respectively]Nle-OH，Boc-L-Trp-OH，Fmoc-L-Trp(Boc)-OH，Fmoc-L-Dab(Boc)-OH，Fmoc-L-[O-tBu]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-Pro-OH，Fmoc-L-Asn(Trt)-OH，Fmoc-L-[N-Me]Ala-OH，Fmoc-L-[O-tBu]Tyr-OH. After completion of the condensation, the selection is made according to the "peptide liberation step" described aboveThe corresponding amino acid gives crude linear peptide precursor. 60mg of the crude product were dissolved in acetonitrile: 0.1M ammonium carbonate ═ 1:1 (volume ratio) to a total of 600mL of solution, the reaction was stirred at room temperature for 5 hours. And (4) carrying out spin drying on the reaction liquid, and carrying out freeze drying to obtain a powdery mixture. The mixture was dissolved in acetonitrile: the cyclic peptide precursor was purified according to the aforementioned "RP-HPLC separation procedure" in a ratio of 1:1 (volume ratio) of water. Taking 5mg of the pure precursor, dissolving in acetonitrile: water 1:1 (volume ratio) to a total of 0.6mL, 0.07mL of 30% H was added to the solution₂O₂And stirred at room temperature for 3 hours. After the reaction is finished, the excessive hydrogen peroxide is destroyed by ascorbic acid, the solution is dried by spinning, and the crude product can be obtained by freeze drying. The target product was obtained according to the "RP-HPLC separation procedure" described above. t is t_R＝18.760min。HRESIMS:[M+H]⁺＝1916.9560。

Example 2

Obtained according to the method described in "example 1" above, t_R＝27.251min。HRESIMS:[M+H]⁺＝1916.9601。

Example 3

The oxidation step was changed to the following synthesis according to the previous example 1: taking 5mg of the pure precursor, dissolving in methanol: water 1:1 (volume ratio) in 500. mu.L. To the mixture was added 0.5mg of potassium peroxymonosulfonate, and the mixture was stirred at room temperature. The reaction can be judged to be finished by RP-HPLC. Ascorbic acid was added to the reaction solution to destroy peroxides, and the product was obtained according to the "RP-HPLC separation step" described above after lyophilization of the reaction solution. t is t_R＝20.230min。HRESIMS:[M+Na]⁺＝1896.9402。

Example 4

The amino acid derivative Fmoc-L-Cys (Trt) -OH in example 1 was replaced with 2-fluorenylmethoxycarbonylamino-4-butenoic acid and the terminal carboxylic acid was replaced with acrylic acid according to the aforementioned "resin swelling step", "condensation step" and "peptide liberation step", to obtain a crude linear peptide precursor. 10mg of the crude product was taken, dissolved in dichloromethane and Grubb's 2 added^ndCatalyst (0.2equiv), reflux 12 hours. The crude product was dissolved in DMSO and filtered to remove the catalyst. Lyophilization of the solution, a "RP-HPLC separation step" gives a Z/E mixture. t is t_R＝15.245min。HRESIMS:[M+Na]⁺＝1896.9801。

Example 5

10mg of the compound obtained in example 4 was dissolved in 0.5mL of methanol, 1mg of Pd/C was added, and the reaction was carried out at 45 ℃ for 8 hours under a hydrogen atmosphere. And adding dichloromethane into the reaction solution for dilution, and obtaining a pure product according to an RP-HPLC separation step after spin-drying. t is t_R＝15.258min。HRESIMS:[M+2Na]⁺/2＝1869.0002。

Example 6

The specific synthesis method comprises the following steps:

5g of previously activated 4A molecular sieve, LiOH (384mg, 16mmol) was added to anhydrous DMF (50mL) and stirred at room temperature for 20 minutes. Then 6-1(2.01g,8mmol) was added to the mixture and stirred at room temperature for 40 minutes. Allyl bromide (830. mu.L, 9.6mmol) was added to the reaction solution, and the reaction was stirred at room temperature for 12 hours. After the reaction is finished, the reaction solution is diluted by ethyl acetate, filtered and concentrated to be dry to obtain the productAnd (5) crude product. Separation by silica gel column chromatography (n-hexane: ethyl acetate ═ 20:1) gave the objective compound 6-2(1.82g) in 78% yield.¹H-NMR：(DMSO-d6,ppm)7.13(d,J＝7.3Hz,2H),6.86(d,J＝7.3Hz,2H),5.87–5.84(m,1H),5.20-5.17(m,2H),3.86–3.82(m,1H),3.66,(s,3H),3,39-3.10(m,3H),1.42(s,9H).HRESIMS:[M+H]⁺＝292.1830

Compound 6-2(1.37g, 4.7mmol) was dissolved in 30mL of methanol, the reaction mixture was transferred to 0 ℃ and lithium hydroxide (124mg, 5.17mmol) was added to the reaction mixture, and the reaction was allowed to react at 0 ℃ for 1 hour. After the reaction is finished, adding ethyl acetate into the reaction liquid for dilution, adding 2M hydrochloric acid solution to adjust the pH value to 3, separating two phases, and washing an organic layer with saturated solution of sodium chloride for 1 time. Concentrating to dry to obtain compound 6-2, which can be directly used for the next reaction without treatment. HRESIMS: [ M + H ]]⁺＝277.3644

1.4g of the crude compound 6-3 was dissolved in 25mL of 10% sodium carbonate solution (25 mL) and 50mL of acetone, the reaction mixture was transferred to an ice bath, Fmoc-OSu (2.05g, 6.06mmol) was added, and the reaction was stirred at room temperature for 18 hours. After the reaction was completed, the reaction solution was adjusted to pH 3 with 2M hydrochloric acid solution, diluted with ethyl acetate, separated into two phases, the organic phase was washed with water 3 times, washed with saturated sodium chloride solution 1 time, concentrated to dryness, and separated by silica gel column chromatography (DCM: MeOH 300:1) to obtain compound 6-4(1.22 g). The yield in two steps was 61%. .¹H-NMR：(DMSO-d6,ppm)7.90(d,J＝7.1Hz,2H),7.55(d.J＝7.0Hz,2H),7.41-7.29(m,4H),7.11(d,J＝7.3Hz,2H),6.87(d,J＝7.3Hz,2H),5.87–5.84(m,1H),5.20-5.17(m,2H),4.71-4.59(m,3H),3.86–3.82(m,1H),3,39-3.10(m,3H),1.42(s,9H)。

Example 7

The crude linear peptide precursor was obtained by replacing Fmoc-L-Cys (strt) -OH in example 1 with 2-fluorenylmethoxycarbonylamino-4-butenoic acid and Fmoc-L-Tyr (OtBu) -OH in example 6 according to the aforementioned "resin swelling step", "condensation step" and "peptide liberation step". 10mg of the crude product was taken, dissolved in dichloromethane and Grubb's 2 added^ndCatalyst (0.2equiv), reflux 12 hours. The crude product was dissolved in DMSO and filtered to remove the catalyst. Lyophilizing the solution to obtain target product t according to RP-HPLC separation procedure_R＝22.254min。HRESIMS:[M+Na]⁺＝1874.9908。

Example 8

10mg of the compound obtained in example 7 was dissolved in 0.5mL of methanol, 1mg of Pd/C was added, and the reaction was carried out at 45 ℃ for 8 hours under a hydrogen atmosphere. The reaction solution is diluted by adding dichloromethane, and after spin-drying, a pure product can be obtained according to the RP-HPLC separation step. t is t_R＝22.243min。HRESIMS:[M+Na]⁺＝1877.0246。

Example 9

The amino acid derivatives Fmoc-L-Trp (Boc) -OH, Fmoc-L-dab (Boc) -OH, Fmoc-L- [ O-tBu ] used in example 1 were subjected to the aforementioned "resin swelling step", "condensation step" and "peptide liberation step", respectively]Hyp-OH，Fmoc-L-Dap(Boc)-OH，Fmoc-L-[O-tBu]Tyr-OH is replaced by Fmoc-L-Trp (Cbz) -OH, Fmoc-L-dab (Cbz) -OH and Fmoc-L- [ O-Bn]Hyp-OH，Fmoc-L-Dap(Cbz)-OH，Fmoc-L-[O-Bn]Tyr-OH can give a direct-linked peptide precursor. 10mg of the crude product was taken and dissolved in 150mL of anhydrous DMF, and the reaction solution was transferred to-20 ℃. HATU (41mg, 109. mu. mol), DEPBT (33mg, 109. mu. mol) and TBTU (35mg, 109. mu. mol) were mixed in advance and added to the reaction mixture. The reaction solution was stirred at-20 ℃ for 1 hour, then at 0 ℃ for 1 hour, and then at room temperature for 3 days. After the reaction was completed by HPLC identification, the reaction mixture was diluted with ethyl acetate and washed with water for 1 time. The organic layer was concentrated to dryness and the desired product was obtained according to the "RP-HPLC separation procedure" described above. t is t_R＝24.236min。HRESIMS:[M+Na]⁺＝1919.0022

Example 10

Determination of in vitro inhibitory Activity

1. Evaluation of homogeneous time fluorometric assay example inhibition of the PD-1/PD-L1 interaction:

the interaction of PD-1 and PD-L1 can be determined using recombinant proteins of the ectodomain portions of the two proteins. The extracellular domains of the PD-1 and PD-L1 proteins are expressed as fusion proteins with a detection tag, the Fc portion of an immunoglobulin (PD-1-Ig) for PD-1 and a 6 histidine motif (PD-L1-His) for PD-L1. Human PD-1(25-167), C-terminal human Fc domain with Immunoglobulin G (IG) epitope tag [ hPD-1(25-167) -3S-IG ], and human PD-L1(18-239) with C-terminal His epitope tag [ hPD-L1(18-239) -TVMV-His1] were expressed in HEK293T cells and purified by protein a affinity chromatography and size exclusion chromatography in sequence.

The interaction studies were all performed in HTRF assay buffer consisting of an additional addition of 0.1% (containing) bovine serum albumin and 0.05% (v/v) Tween-20 in dPBS. For the hPD-L1-His binding assay, the inhibitor was preincubated with PD-L1-His (final concentration 10nM) in 4ul assay buffer for 15 minutes, followed by the addition of PD-1-Ig (final concentration 20 nM). In 1uL assay buffer and further incubation for 15 min. HTRF detection was achieved using europium calcium phosphate-labeled anti-Ig (final concentration 1nM) and isonicotina phthalocyanine (APC) -labeled anti-His (final 20 nM). The antibody was diluted in HTRF detection buffer and 5ul was taken. The reaction mixture was allowed to equilibrate for 30 minutes and the resulting signal (665nm/620nm) was obtained using an EnVision fluorometer.

The results of the determination that each cyclic peptide compound inhibited the PD-1/PD-L1 interaction are shown in Table 1.

TABLE 1

A：IC₅₀<100nM；B：100nM<IC₅₀<10μM；C：10μM<IC₅₀<100μM

Examples	IC₅₀
		1	0.33μM
2	0.13μM
		3	A
4	A
		5	A
7(Z/E)	A
		8	A
9	A

As shown in the table above, the cyclopeptide compounds in the examples all have certain capacity of inhibiting the interaction of PD-1/PD-L1.

2. Examples evaluation of cell-level inhibition of the PD-1/PD-L1 interaction:

recombinant murine PD-L1 was used in this experiment as the source of PD-L1 in cellular experiments. Mouse splenocytes were obtained by triturating mouse spleen in a 40um cell strainer and further treating with 1mL ACK lysis buffer for 5 minutes at room temperature. After washing with 9mL RPMI complete medium, cells were resuspended in 3mL 1xPBS in a 15mL tube. Carefully add 3mL Histopaque to the bottom of the tube without disturbing the overlaid spleen cell suspension. After centrifugation at 800 Xg for 20 min at room temperature, an opaque layer of splenocytes was collected. The obtained spleen cellsThe cells were washed twice more with cold PBS solution and the total number of cells was counted using trypan blue staining for later cell-level testing. Spleen cells were cultured in RPMI complete medium (RPMI + 10% fetal bovine serum +1mM sodium pyruvate +10,000u/mL penicillin and 10,000ug/mL streptomycin) and maintained at 37 ℃ with 5% CO₂CO of₂An incubator.

CFSE is a dye that passively diffuses into cells and binds to intracellular proteins. 1X 10⁶cells/mL of splenocytes were treated with 5uM CFSE in pre-warmed 1 XPBS/0.1% BSA solution for 10 min at 37 ℃. Excess CFSE quenched 5 volumes of 0 ℃ media into the cells and incubated on ice for 5 minutes. CFSE-labeled splenocytes were further washed three times with 0 ℃ complete RPMI medium. Marking CFSE 1 × 10⁵Splenocytes were added to wells containing either MDA-MB231 cells (1X 10 cells cultured in high glucose DMEM medium) or recombinant human PD-L1(100ng/mL) and test compound. Splenocytes were stimulated with anti-mouse CD3 and anti-mouse CD28 antibodies (1 ug/mL each) and cultures were incubated at 37 ℃ with 5% CO₂The cells were further cultured for 72 hours. Cells were harvested and washed three times with ice cold FACS buffer and analyzed for percent proliferation by flow cytometry with 488nm excitation and 521nm emission filter.

The percentage of splenocyte proliferation was analyzed using FACS program and the percentage of compound reversion to splenocyte proliferation was calculated after subtracting background proliferation value (%) and normalizing stimulated splenocyte proliferation (%, positive control) to 100%.

Stimulated splenocytes: splenocytes + anti-CD3/CD28

Stimulating background proliferation: splenocytes + anti-CD3/CD28+ PD-L1

Proliferation of the compound: splenocyte + anti-CD3/CD28+ PD-L1+ compound

Compound effects were tested by adding the required concentrations. Expression of anti-CD3/CD 28-stimulated splenocytes by compounds in the presence of ligand (PD-L1).

The results of the assay for each cyclic peptide compound at 5nM concentration to inhibit the PD-1/PD-L1 interaction are shown in Table 2.

TABLE 2

A：>90％；B：70％<IC₅₀<90％；C：50％<IC₅₀<70％；D：<50％

Examples	Inhibition rate
		1	92％
2	96％
		3	A
4	A
		5	A
7(Z/E)	A
		8	A
9	A

Claims

1. A cyclic peptide compound shown in the general formula I, or pharmaceutically acceptable salt, ester, solvate, prodrug, racemate or isomer thereof:

wherein A is selected from:

one kind of (1).

2. A pharmaceutical composition comprising the cyclic peptide compound of claim 1, or a pharmaceutically acceptable salt, ester, or solvate thereof, and a pharmaceutically acceptable carrier.

3. Use of the cyclic peptide compound of claim 1, or a pharmaceutically acceptable salt, ester, solvate, prodrug, racemate or isomer thereof for the preparation of a medicament for the prevention and treatment of tumors.

4. The use of claim 3, wherein the tumor comprises a hematologic cancer, a nervous system cancer, a gastrointestinal cancer, an esophageal cancer, a urinary system cancer, a lung cancer, a liver cancer, and a skin cancer.

5. The use of claim 3, wherein the medicament is a peptide inhibitor targeting the PD-1/PD-L1 pathway.

6. Use of the cyclic peptide compound of claim 1, or a pharmaceutically acceptable salt, ester, solvate, prodrug, racemate or isomer thereof, for the preparation of a PD-1/PD-L1 pathway inhibitor.