CN111961117A

CN111961117A - Cyclopeptide compound and application thereof

Info

Publication number: CN111961117A
Application number: CN202010840057.8A
Authority: CN
Inventors: 卜宪章; 孙海霞; 伍伟健
Original assignee: National Sun Yat Sen University
Current assignee: Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2020-08-19
Filing date: 2020-08-19
Publication date: 2020-11-20
Anticipated expiration: 2040-08-19
Also published as: CN111961117B

Abstract

The invention provides a cyclopeptide compound and application thereof, wherein the structural formula of the cyclopeptide compound is shown as a formula I. The cyclic peptide compound can obviously inhibit the interaction of programmed death ligand 1(PD-1) and programmed death receptor 1(PD-L1), can obviously promote the killing of PBMC to tumor cells, can obviously reduce the growth rate and the tumor quality of tumors in tumor-bearing animals, and shows that the cyclic peptide compound can promote the killing of tumors by an immune system through blocking the immunosuppressive effect in the tumor environment. In addition, the cyclic peptide compound can reduce the increase of the content of cytokines IFN-gamma and IL-10 caused by the activation of T cells, and shows that the generation of immune related side effects can be reduced.

Description

Cyclopeptide compound and application thereof

Technical Field

The invention relates to the field of medicines, and particularly relates to a cyclopeptide compound and application thereof.

Background

Malignant tumor seriously harms human health, and although traditional treatment methods such as operation, radiotherapy, chemotherapy and the like are widely applied to clinic, the malignant tumor still has serious defects of drug resistance, toxic and side effects, damage to immune system and the like. In recent years, the tumor immunotherapy of killing tumor and inhibiting tumor growth by utilizing the body's own immune system is attracting much attention, and the current clinical tumor therapy mode mainly based on "exogenous therapy" is innovated, is an emerging therapeutic means following traditional operations, chemotherapy, radiotherapy and targeted therapy, and has become a hotspot of the current research on antitumor drugs. Among them, the restoration of the antitumor immune effect by blocking immune checkpoint-related proteins is of general interest.

Programmed death ligand 1(PD-L1) is expressed on most tumor cells and immune cells, and inhibits the function of T cells by binding with programmed death receptor 1(PD-1) expressed on the surface of activated T cells, thereby generating tumor immune escape. Preventing the binding of PD-1/PD-L1 effectively restored the lethality of T cells to tumors. The existing medicines targeting the action axis of PD-1/PD-L1 have a plurality of antibody medicines applied to clinic, and the use of the medicines greatly prolongs the survival period of patients and brings the hope of long-term survival for late-stage patients. However, these drugs still have the disadvantages of low clinical response rate, severe immune-related inflammation and other toxic and side effects.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a cyclopeptide compound and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that: a cyclopeptide compound has a structural formula shown in formula I,

wherein R is₁is-CH₃、-CH(CH₃)₂、-CH(CH₃)OR₇One of (1);

R₂is-CH₂CH(CH₃)₂、-CH₃、-CHCH₃C₂H₅、-CH₂CH₂CH₂CH₃One of (1);

R₄is 4- ((1- (4, 4-dimethylcyclohexadidene) ethyl) amino) butanyl and R₃Is H, or the R3 and R4 are linked to form-CH₂CH₂CH₂-and-CH₂CH₂CH₂-is linked to the C-N bond in which R3, R4 is located to form a five-membered heterocyclic ring containing one nitrogen;

R₅is-CHCH₃C₂H₅or-CH (CH)₃)₂；

R₆is-CH₂C₆H₅Or- (1H-indol-3-yl) methanol;

R₇is H or-CO- (CH)₂)_nCH₃And n is a positive integer from 0 to 18.

The inventor discovers a series of novel cyclic peptide compounds through research, the cyclic peptide compounds have a structural formula shown as a formula I, the inventor discovers that the cyclic peptide compounds can remarkably inhibit the interaction between programmed death ligand 1 and programmed death receptor 1, the cyclic peptide compounds can remarkably promote the killing of Peripheral Blood Mononuclear Cells (PBMC) to tumor cells, and animal in vivo experiments show that the cyclic peptide compounds can remarkably reduce the growth rate and the tumor mass of tumors, and the cyclic peptide compounds can reduce the increase of IFN-gamma and IL-10 contents caused by the activation of a cluster 3(CD3) antibody or the activation of T cells (CD3+ T cells) containing CD3 by the tumor cells.

Preferably, said R is₁is-CH (CH)₃)OR₇Said R is₇Is H or-CO- (CH)₂)_nCH₃And n is a positive integer from 0 to 18.

The inventor finds that the cyclic peptide compound is R₁is-CH (CH)₃)OR₇In this case, the cyclic peptide compound has a higher effect of inhibiting the interaction between programmed death ligand 1 and programmed death receptor 1, and the cyclic peptide compound has a higher effect of promoting the killing of tumor cells by PBMC.

Preferably, said R is₇is-CO- (CH)₂)_nCH₃And n is a positive integer of 10-16.

Preferably, said R is₃Is H and R₄Is 4- ((1- (4,4-dimethyl-2, 6-dioxacyclohexadiene) ethyl) amino) butanyl.

The inventor discovers that R is₃Is H and R₄The cyclic peptide compound of 4- ((1- (4,4-dimethyl-2, 6-dioxacyclohexadiene) ethyl) amino) butanyl has better effect on inhibiting the interaction of programmed death ligand 1 and programmed death receptor 1, and the cyclic peptide compound has better effect on promoting the killing of tumor cells by PBMC.

Preferably, the cyclic peptide compound has a structural formula shown in formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII or formula XIII;

the inventor finds that the cyclic peptide compound can remarkably inhibit the interaction of programmed death ligand 1 and programmed death receptor 1, the cyclic peptide compound can remarkably promote the killing of PBMC to tumor cells, and animal in vivo experiments show that the cyclic peptide compound shown by the formula IX remarkably reduces the growth speed and the quality of tumors. Moreover, the cyclic peptide compound shown in the formula IX can reduce the increase of IFN-gamma and IL-10 contents caused by CD3 antibody activation or tumor cell activation CD3+ T. The cyclic peptide compound of the formula X, XI, XII or XIII is in the form of a prodrug of the formula IX and can be converted into a cyclic peptide compound of the formula IX under a meta-acidic condition, and thus can be converted into a cyclic peptide compound of the formula IX in vivo, and shows the activity of the cyclic peptide compound of the formula IX.

The invention also provides application of any one of the cyclic peptide compounds and pharmaceutically acceptable salts thereof as an active ingredient in preparation of medicines for treating tumor diseases.

Any one of the above cyclic peptide compounds the above cyclic peptide compound can significantly inhibit the interaction between programmed death ligand 1 and programmed death receptor 1, and the above cyclic peptide compound can significantly promote the killing of tumor cells by PBMC.

The invention also provides application of any one of the cyclic peptide compounds and pharmaceutically acceptable salts thereof as an active ingredient in preparation of medicines for inhibiting binding of programmed death ligand 1 and programmed death receptor 1.

The invention also provides a pharmaceutical composition comprising any one of the cyclic peptide compounds described above and pharmaceutically acceptable salts thereof.

Preferably, the pharmaceutical composition further comprises a pharmaceutical excipient, for use in the treatment of a neoplastic disease or for inhibiting the binding of programmed death ligand 1 and programmed death receptor 1.

The invention has the beneficial effects that: the invention provides a cyclopeptide compound and application thereof, the cyclopeptide compound can obviously inhibit the interaction of programmed death ligand 1 and programmed death receptor 1, and can obviously promote the killing of PBMC to tumor cells, and animal in vivo experiments show that the cyclopeptide compound obviously reduces the growth rate and the tumor quality of tumors, and shows that the cyclopeptide compound can promote the killing of immune systems to the tumors by blocking the immunosuppressive effect in the tumor environment. In addition, the cyclic peptide compound can reduce the increase of IFN-gamma and IL-10 content caused by CD3 antibody activation or tumor cell activation CD3+ T, and shows that the generation of immune related side effects can be reduced.

Drawings

FIG. 1 is a general chemical structure diagram of a cyclic peptide compound of the present invention.

FIG. 2 is a graph showing the effect of the cyclic peptide compounds of the present invention in inhibiting the interaction of programmed death ligand 1 and programmed death receptor 1.

FIG. 3 is a graph showing the effect of the cyclic peptide compounds of the present invention in promoting the killing of tumor cells by PBMC.

FIG. 4 is a graph showing the effect of the cyclic peptide compound of the present invention on the reduction of tumor growth rate and tumor mass.

FIG. 5 is a graph showing the effect of the cyclic peptide compound of the present invention in inhibiting the release of immune-related factors.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

The invention provides a cyclopeptide compound, the structural formula of which is shown in a formula I,

wherein R is₁is-CH₃、-CH(CH₃)₂、-CH(CH₃)OR₇One of (1);

R₄is 4- ((1- (4, 4-dimethylcyclohexadidene) ethyl) amino) butanyl and R₃Is H, or said R₃And R₄Is linked to form-CH₂CH₂CH₂-and-CH₂CH₂CH₂-and said R₃、R₄The C-N bond is connected with a five-membered heterocyclic ring containing nitrogen;

R₅is-CHCH₃C₂H₅or-CH (CH)₃)₂；

R₆is-CH₂C₆H₅Or- (1H-indol-3-yl) methanol;

R₇is H or-CO- (CH)₂)_nCH₃And n is a positive integer from 0 to 18.

The cyclic peptide compound is prepared by adopting Fmoc/tBu synthesis strategy solid-phase synthesis (Fmoc/tBu solid-phase synthesis) and solution cyclization.

The preparation method of the cyclopeptide compound comprises the following steps:

(1) synthesizing linear peptide with protected side chain, which comprises the following steps: adding 0.5g of 2-chlorotrityl chloride resin into a reactor with a filter element, adding 10mL of dry dichloromethane, swelling for 30min, removing the solvent, adding the first amino acid raw material (3eq.) to be loaded, diisopropylethylamine (3eq.) and dichloromethane as the solvent, and reacting for 3h at room temperature. The amino acid raw material is that the main chain amino group is protected by Fmoc, if the side chain of the amino acid has active groups, the amino acid needs to be protected by a protective group suitable for Fmoc/tBu synthesis strategy. If R is₇is-OCO- (CH)₂)_nCH₃(n is a positive integer of 0 to 18), wherein threonine requires a threonine starting material in which the side chain is protected with fatty acids of different lengths and the amino group is protected with Fmoc.

After the reaction is finished, unreacted sites are blocked by 8mL of dichloromethane, 1.5mL of MeOH and 0.5mL of N, N-Diisopropylethylamine (DIPEA), the reaction is carried out at room temperature for 30min, then the solvent is removed, 5X 5mL of dichloromethane is used for washing, Fmoc removal is carried out by using a Dimethylformamide (DMF) solution of piperidine with the mass concentration of 5% and 2% of 1, 8-diazabicycloundecen-7-ene (DBU), and the reaction is carried out at room temperature for 30 min. The removal of Fmoc was examined by Kaiser's reaction. If the reaction is positive, the next amino acid starting material may be uploaded. The amino acid condensation adopts 3 eq.amino acid raw material, 3eq.HOBT and 3 eq.DIC. After the reaction was completed, the solvent was removed, the resin was washed with DMF, and the loading of the amino acid was examined by Kaiser's reaction. If the reaction is negative, Fmoc removal and the next round of amino acid uploading can be performed.

After the final loading of amino acid was complete, Fmoc was removed and washed 5X 5mL with methanol and 5X 5mL with dichloromethane. 10mL of a 20% trifluoroethanol solution in methylene chloride was added and reacted for 1 hour to obtain a linear peptide containing a side chain protecting group.

(2) Synthesizing cyclic peptide, wherein the synthesized cyclic peptide specifically comprises the following steps: the linear peptide containing side chain protecting groups was dissolved in anhydrous precooled dichloromethane to a final concentration of 1 mM. 3eq. Pybop, 3eq. HOBT, 5eq. DIPEA were added. The reaction was stopped by HPLC detection of complete disappearance of linear peptide. The solvent was removed, 10mL of a deprotection reagent (trifluoroacetic acid: phenol: triisopropylsilane: water: 88:5:5:2) was added, the reaction mixture was concentrated after 2 hours at room temperature, and the resulting product was precipitated in cold ether to obtain a crude product, which was purified by HPLC.

Example 1

As the cyclopeptide compound of the embodiment of the invention, the structural formula of the cyclopeptide compound is shown as a formula II,

the cyclic peptide compound represented by formula II is named as cyclo (-Leu-DPhe-Pro-Ala-Asp-Leu-DPhe-Pro-Val-Arg-), and R is represented by the general formula of the peptide compound represented by formula I₁＝-CH₃，R₂＝-CH₂CH(CH₃)₂，R₃And R₄Is linked to form-CH₂CH₂CH₂-and-CH₂CH₂CH₂-and said R₃、R₄In which the C-N bond is linked to a five-membered heterocyclic ring containing one nitrogen, R₅＝-CH(CH₃)₂，R₆＝-CH₂C₆H₅。

The preparation method of the cyclic peptide compound of the present example includes the following steps:

(1) synthesizing a linear peptide Leu-DPhe-Pro-Ala-Asp (OtBu) -Leu-DPhe-Pro-Val-Arg (Pbf), and specifically comprising the following steps: adding 0.5g of 2-chlorotrityl chloride resin into a reactor with a filter element, adding 10mL of dry dichloromethane, swelling for 30min, removing the solvent, adding a first carbon-terminal amino acid raw material Fmoc-Arg (Pbf) -OH (3eq.), diisopropylethylamine (3eq.), dichloromethane serving as a solvent, reacting for 3h at room temperature, blocking unreacted sites by using 8mL of dichloromethane, 1.5mL of methanol and 0.5mL of N, N-Diisopropylethylamine (DIPEA) after the reaction is finished, removing the solvent after reacting for 30min at room temperature, washing for 5 x 5mL by using dichloromethane, removing the Fmoc, and reacting for 30min at room temperature by using a Dimethylformamide (DMF) solution of piperidine with the mass concentration of 5% and 2% 1, 8-diazabicycloundecen-7-ene (DBU). The removal of Fmoc was examined by Kaiser's reaction. If the reaction is positive, the next amino acid may be sequentially loaded, and the loading order of the amino acids may be sequentially from the carbon terminus to the nitrogen terminus in the target sequence. The amino acid condensation was carried out by using 3eq. amino acid, 3eq. HOBT, and 3eq. DIC. After the reaction was completed, the solvent was removed, the resin was washed with DMF, and the loading of the amino acid was examined by Kaiser's reaction. If the reaction is negative, Fmoc removal and the next round of amino acid uploading can be performed.

After the final loading of amino acid was complete, Fmoc was removed and washed 5X 5mL with methanol and 5X 5mL with dichloromethane. Adding 10mL of 20% trifluoroethanol dichloromethane solution, and reacting for 1h to obtain linear peptide containing side chain protecting groups;

NMR spectrum of the product purified by HPLC in this example (¹HNMR) High Resolution and Mass Spectrometry (HRMS) detection with the result that¹H NMR(600MHz,MeOD)8.70-8.69(amide),7.37–7.24(m,10H),5.08(t,J＝7.5Hz,1H),5.04–4.97(m,1H),4.69(dt,J＝14.5,6.9Hz,2H),4.61–4.48(m,3H),4.43–4.37(m,2H),4.31(t,J＝8.2Hz,1H),3.80(m,2H),3.33–3.21(m,2H),3.13–2.96(m,5H),2.81(dd,J＝17.1,9.0Hz,1H),2.70(dd,J＝16.1,7.3Hz,1H),2.70(dd,J＝16.1,7.3Hz,1H),2.52(dd,J＝17.1,8.7Hz,1H),2.23–2.16(m,2H),2.07–1.98(m,1H),1.93–1.87(m,1H),1.85–1.78(m,1H),1.77–1.63(m,8H),1.63–1.50(m,4H),1.44(dd,J＝13.4,6.9Hz,1H),1.39(d,J＝7.1Hz,3H),1.36–1.30(m,1H),0.98–0.86(m,18H).HRMS:calcd for C₅₈H₈₅N₁₃O₁₂[M+2H]²⁺m/z 578.8293, found, 578.8306. It can be illustrated that the product obtained in this example is a cyclic peptide compound represented by formula II.

Example 2

The structural formula of the cyclopeptide compound is shown as formula III,

the compound shown in formula III is named as cyclo (-Leu-DPhe-Pro-Ala-Asp-Ala-DPhe-Pro-Val-Arg-), and relative to the general formula of the peptide compound shown in formula I, R₁＝-CH₃，R₂＝-CH₃，R₃And R₄Is linked to form-CH₂CH₂CH₂-and-CH₂CH₂CH₂-and said R₃、R₄In which the C-N bond is linked to a five-membered heterocyclic ring containing one nitrogen, R₅＝-CH(CH₃)₂，R₆＝-CH₂C₆H₅。

The difference between the preparation method of the cyclic peptide compound of this example and example 1 is: 1, synthesizing linear peptide compound Leu-DPhe-Pro-Ala-Asp (OtBu) -Ala-DPhe-Pro-Val-Arg (pbf), 2, and sequentially loading amino acid raw materials according to the difference of linear peptide sequences during synthesis.

NMR spectrum of the product purified by HPLC in this example (¹HNMR) High Resolution and Mass Spectrometry (HRMS) detection with the result that¹H NMR(600MHz,MeOD)8.52-7.73(amide),7.38–7.16(m,10H),5.13(t,J＝7.5Hz,1H),4.95(t,J＝7.5Hz,2H),4.72–4.59(m,3H),4.55(dt,J＝5.3,4.7Hz,2H),4.41–4.36(m,2H),4.31(t,J＝7.9Hz,1H),3.84(dd,J＝12.7,5.4Hz,1H),3.78–3.71(m,1H),3.27(m,2H),3.12–3.03(m,3H),3.02–2.95(m,2H),2.70(dd,J＝15.8,7.7Hz,2H),2.53(dd,J＝17.3,8.5Hz,1H),2.21(m,1H),2.07–1.96(m,2H),1.90(m,1H),1.82–1.63(m,8H),1.62–1.52(m,3H),1.51–1.45(m,1H),1.41(d,J＝7.1Hz,3H),1.27(d,J＝6.8Hz,3H),0.95–0.89(m,12H).HRMS:calcd for C₅₅H₇₉N₁₃O₁₂[M+2H]²⁺m/z 557.8058, found,557.8062, which indicates that the product obtained in this example is a cyclic peptide compound of formula III.

Example 3

As a cyclopeptide compound of the embodiment of the invention, the structural formula of the cyclopeptide compound is shown in a formula IV,

the cyclic peptide compound shown in formula IV is named as cyclo (-Leu-DPhe-Pro-Val-Asp-Leu-DPhe-Pro-Val-Arg-), and relative to the general formula of the peptide compound shown in formula I, R is₁＝-CH(CH₃)₂，R₂＝-CH₂CH(CH₃)₂，R₃And R₄Is linked to form-CH₂CH₂CH₂-and-CH₂CH₂CH₂-and said R₃、R₄In which the C-N bond is linked to a five-membered heterocyclic ring containing one nitrogen, R₅＝-CH(CH₃)₂，R₆＝-CH₂C₆H₅。

The difference between the preparation method of the cyclic peptide compound of this example and example 1 is: 1, synthesizing a linear peptide compound Leu-DPhe-Pro-Val-Asp (OtBu) -Leu-DPhe-Pro-Val-Arg (Pbf); 2, the amino acid starting materials sequentially loaded during synthesis differ depending on the linear peptide sequence.

NMR spectrum of the product purified by HPLC in this example (¹HNMR) High Resolution and Mass Spectrometry (HRMS) detection with the result that¹H NMR(600MHz,MeOD)8.69-7.32(amide),7.31(dt,J＝9.6,4.5Hz,5H),7.24(m,5H),5.06(t,J＝7.6Hz,1H),5.01(dd,J＝13.9,7.0Hz,1H),4.69(dd,J＝9.7,6.3Hz,1H),4.67–4.60(m,1H),4.60–4.53(m,1H),4.51(dd,J＝11.1,5.2Hz,1H),4.44–4.34(m,2H),4.26(t,J＝8.4Hz,1H),4.13(t,J＝8.9Hz,1H),3.87–3.79(m,1H),3.78–3.71(m,1H),3.61(q,J＝7.0Hz,1H),3.25(td,J＝6.9,3.2Hz,2H),3.09–3.04(m,2H),3.03–2.99(m,1H),2.99–2.93(m,1H),2.82(dd,J＝17.1,9.3Hz,1H),2.66(dd,J＝16.2,7.2Hz,1H),2.53(dd,J＝17.1,9.4Hz,1H),2.26(td,J＝13.6,6.8Hz,1H),2.19–2.12(m,1H),2.05–1.99(m,2H),1.90–1.79(m,2H),1.74(dd,J＝14.6,6.3Hz,1H),1.72–1.68(m,2H),1.68–1.62(m,3H),1.62–1.57(m,2H),1.57–1.51(m,2H),1.49–1.39(m,1H),1.29(d,J＝5.1Hz,2H),1.18(t,J＝7.0Hz,1H),0.97–0.84(m,24H).HRMS:calcd for C₆₀H₈₉N₁₃O₁₂[M+2H]²⁺m/z 592.8450, found,592.8462, which indicates that the product obtained in this example is a cyclic peptide compound of formula IV.

Example 4

As the cyclopeptide compound of the embodiment of the invention, the structural formula of the cyclopeptide compound is shown in a formula V,

the cyclic peptide compound shown in formula V is named as cyclo (-Leu-DPhe-Pro-Thr-Asp-Leu-DPhe-Pro-Val-Arg-), and relative to the general formula of the peptide compound shown in formula I, R is₁＝-CH(CH₃)OH,R₂＝-CH₂CH(CH₃)₂，R₃And R₄Is linked to form-CH₂CH₂CH₂-and-CH₂CH₂CH₂-and said R₃、R₄The C-N bond is connected with a five-membered heterocyclic ring containing nitrogen,

R₅＝-CH(CH₃)₂，R₆＝-CH₂C₆H₅。HRMS：calcd for C₅₉H₈₇N₁₃O₁₃。

the difference between the preparation method of the cyclic peptide compound of this example and example 1 is: 1, synthesizing a linear peptide compound Leu-DPhe-Pro-Thr (tBu) -Asp (OtBu) -Leu-DPhe-Pro-Val-Arg (Pbf); 2, the amino acid starting materials sequentially loaded during synthesis differ depending on the linear peptide sequence.

NMR spectrum of the product purified by HPLC in this example (¹HNMR) High Resolution and Mass Spectrometry (HRMS) detection, the resultIs composed of¹H NMR(600MHz,MeOD)8.77-7.76(amide),7.39–7.20(m,10H),5.10(t,J＝7.4Hz,1H),4.97(t,J＝7.2Hz,1H),4.68(dd,J＝16.0,7.7Hz,2H),4.65–4.51(m,2H),4.45(d,J＝7.4Hz,1H),4.41–4.35(m,2H),4.32(t,J＝7.7Hz,1H),4.12(m,1H),3.88–3.75(m,2H),3.32–3.27(m,1H),3.24(dt,J＝13.7,6.9Hz,1H),3.13–2.94(m,5H),2.80(dd,J＝16.9,8.7Hz,1H),2.71(dd,J＝16.0,7.3Hz,1H),2.52(dd,J＝17.1,9.5Hz,1H),2.23–2.15(m,1H),2.09–2.01(m,2H),1.91–1.48(m,15H),1.48–1.42(m,1H),1.16(d,J＝6.3Hz,3H),0.92(m,18H).HRMS:calcd for C₅₉H₈₇N₁₃O₁₃[M+2H]²⁺m/z 593.8346, found,593.8317, which indicates that the product obtained in this example is a cyclic peptide compound of formula V.

Example 5

As the cyclopeptide compound of the embodiment of the invention, the structural formula of the cyclopeptide compound is shown in a formula VI,

the cyclic peptide compound shown in formula VI is named as cyclo (-Leu-DTrp-Pro-Val-Asp-Leu-DPhe-Pro-Val-Arg-), and relative to the general formula of the peptide compound shown in formula I, R is₁＝-CH(CH₃)₂，R₂＝-CH₂CH(CH₃)₂，R₃And R₄Is linked to form-CH₂CH₂CH₂-and-CH₂CH₂CH₂-and said R₃、R₄In which the C-N bond is linked to a five-membered heterocyclic ring containing one nitrogen, R₅＝-CH(CH₃)₂，R₆＝(1H-indol-3-yl)methanyl。HRMS：calcd for C₆₂H₉₀N₁₄O₁₂。

The difference between the preparation method of the cyclic peptide compound of this example and example 1 is: 1, synthesizing a linear peptide compound Leu-DTrp (Boc) -Pro-Val-Asp (OtBu) -Leu-DPhe-Pro-Val-Arg (Pbf); 2, the amino acid starting materials sequentially loaded during synthesis differ depending on the linear peptide sequence.

NMR spectrum of the product purified by HPLC in this example (¹HNMR) high scoreThe result of the discrimination and mass spectrometry (HRMS) detection is¹H NMR(600MHz,MeOD)8.70-7.71(amide),7.50(d,J＝8.0Hz,1H),7.38(d,J＝8.2Hz,1H),7.34–7.18(m,6H),7.14(t,J＝7.6Hz,1H),7.03(t,J＝7.5Hz,1H),5.07(dd,J＝14.9,7.4Hz,2H),4.72(m,2H),4.62–4.55(m,1H),4.52(dd,J＝9.9,6.2Hz,1H),4.39(dd,J＝8.4,2.1Hz,1H),4.32(dd,J＝14.0,7.2Hz,1H),4.26(d,J＝7.0Hz,1H),4.09(t,J＝9.0Hz,1H),3.89–3.83(m,1H),3.58(dd,J＝13.9,6.7Hz,1H),3.30(dd,J＝13.8,6.9Hz,2H),3.25–3.19(m,2H),3.13–3.01(m,3H),2.87(dd,J＝17.1,9.0Hz,1H),2.73–2.65(m,1H),2.33–2.24(m,2H),2.18(m,1H),2.09–1.99(m,1H),1.92–1.81(m,3H),1.81–1.69(m,3H),1.69–1.59(m,4H),1.59–1.51(m,3H),1.48(dt,J＝13.2,5.5Hz,1H),1.41–1.30(m,4H),1.29–1.18(m,2H),0.98–0.86(m,21H).HRMS:calcd for C₆₂H₉₀N₁₄O₁₂[M+2H]²⁺m/z 612.3504, found,612.3525, which indicates that the product obtained in this example is a cyclic peptide compound of formula VI.

Example 6

As a cyclopeptide compound of the embodiment of the invention, the structural formula of the cyclopeptide compound is shown in a formula VII,

the cyclic peptide compound represented by the formula VII is named as cyclo (-Leu-DTrp-Pro-Thr-Asp-Ile-DPhe-Pro-Ile-Arg-), and relative to the general formula of the peptide compound represented by the formula I, R is₁＝-CH(CH₃)OH，R₂＝-CHCH₃C₂H₅，R₃And R₄Is linked to form-CH₂CH₂CH₂-and-CH₂CH₂CH₂-and said R₃、R₄In which the C-N bond is linked to a five-membered heterocyclic ring containing one nitrogen, R₅＝-CHCH₃C₂H₅，R₆＝(1H-indol-3-yl)methanyl。

The difference between the preparation method of the cyclic peptide compound of this example and example 1 is: 1, synthesizing a linear peptide compound Leu-DTrp (Boc) -Pro-Thr (tBu) -Asp (OtBu) -Ile-DPhe-Pro-Ile-Arg (Pbf); 2, the amino acid starting materials sequentially loaded during synthesis differ depending on the linear peptide sequence.

NMR spectrum of the product purified by HPLC in this example (¹HNMR) High Resolution and Mass Spectrometry (HRMS) detection with the result that¹H NMR(600MHz,MeOD)8.85-8.39(amide),7.72–7.64(m,1H),7.50(d,J＝8.0Hz,1H),7.37(d,J＝8.2Hz,1H),7.33–7.28(m,2H),7.26(dd,J＝14.5,7.0Hz,2H),7.17(s,1H),7.12(t,J＝7.5Hz,1H),7.02(t,J＝7.4Hz,1H),5.40–5.34(m,1H),4.97–4.91(m,1H),4.83(s,1H),4.66(dd,J＝16.3,7.5Hz,1H),4.55–4.48(m,2H),4.40(dd,J＝8.8,6.6Hz,2H),4.28(t,J＝9.4Hz,2H),4.20(t,J＝9.2Hz,1H),4.06–3.98(m,1H),3.73–3.66(m,1H),3.61(dd,J＝14.0,7.0Hz,1H),3.30–3.23(m,1H),3.22(d,J＝8.0Hz,1H),3.06(dd,J＝12.6,5.2Hz,1H),3.01–2.94(m,1H),2.90(dd,J＝15.3,6.4Hz,1H),2.65(dd,J＝15.3,8.3Hz,1H),2.53(dd,J＝17.0,9.4Hz,1H),2.24(dd,J＝16.6,9.2Hz,1H),2.10(dd,J＝15.0,6.6Hz,1H),2.02(dd,J＝14.7,8.5Hz,1H),1.85(dd,J＝15.6,10.2Hz,3H),1.78–1.71(m,1H),1.72–1.63(m,3H),1.59(dt,J＝19.8,7.0Hz,3H),1.55–1.46(m,3H),1.44–1.38(m,1H),1.36–1.26(m,3H),1.22–1.14(m,3H),1.12(d,J＝6.3Hz,3H),0.95–0.82(m,18H).HRMS:calcd for C₆₂H₉₀N₁₄O₁₃[M+2H]²⁺m/z 620.3479, found,620.3486, which indicates that the product obtained in this example is a cyclic peptide compound represented by formula VII.

Example 7

As a cyclopeptide compound of the embodiment of the invention, the structural formula of the cyclopeptide compound is shown in a formula VIII,

the cyclic peptide compound represented by the formula VIII is named as cyclo (-Leu-DTrp-Pro-Thr-Asp-Nle-DPhe-Pro-Val-Arg-), and R is relative to the general formula of the peptide compound represented by the formula I₁＝-CH(CH₃)OH，R₂＝-CH₂CH₂CH₂CH₃，R₃And R₄Is linked to form-CH₂CH₂CH₂-and-CH₂CH₂CH₂-and said R₃、R₄The C-N bond is connected with a five-membered heterocyclic ring containing nitrogen,R₅＝-CH(CH₃)₂，R₆＝(1H-indol-3-yl)methanyl。

the difference between the preparation method of the cyclic peptide compound of this example and example 1 is: 1, synthesizing a linear peptide compound Leu-DTrp (Boc) -Pro-Thr (tBu) -Asp (OtBu) -Nle-DPhe-Pro-Val-Arg (Pbf); 2, the amino acid starting materials sequentially loaded during synthesis differ depending on the linear peptide sequence.

NMR spectrum of the product purified by HPLC in this example (¹HNMR) High Resolution and Mass Spectrometry (HRMS) detection with the result that¹H NMR(600MHz,MeOD)8.76-7.73(amide),7.49(d,J＝8.0Hz,1H),7.37(d,J＝8.2Hz,1H),7.33–7.26(m,2H),7.24(t,J＝6.9Hz,3H),7.18(s,1H),7.12(t,J＝7.6Hz,1H),7.02(t,J＝7.5Hz,1H),5.09(t,J＝7.1Hz,1H),5.03–4.94(m,1H),4.68(dt,J＝16.2,7.5Hz,2H),4.57–4.50(m,1H),4.46(dd,J＝15.8,8.1Hz,1H),4.37(d,J＝7.9Hz,1H),4.35–4.30(m,1H),4.26(d,J＝7.4Hz,1H),4.13–4.05(m,1H),3.86–3.78(m,1H),3.61(dt,J＝7.0,6.3Hz,1H),3.37–3.33(m,1H),3.28–3.23(m,1H),3.22(d,J＝8.2Hz,2H),3.03(dd,J＝11.4,5.6Hz,2H),2.98(dd,J＝15.8,7.6Hz,1H),2.78–2.73(m,1H),2.70(dd,J＝15.8,7.3Hz,1H),2.24(dd,J＝16.2,9.0Hz,1H),2.21–2.14(m,1H),2.02(dd,J＝11.4,5.2Hz,1H),1.90–1.80(m,3H),1.81–1.70(m,4H),1.70–1.62(m,3H),1.62–1.50(m,4H),1.49–1.41(m,1H),1.26(dd,J＝17.6,9.8Hz,3H),1.23–1.15(m,3H),1.13(d,J＝6.3Hz,3H),0.96–0.86(m,15H).HRMS:calcd for C₆₁H₈₈N₁₄O₁₃[M+2H]²⁺m/z 613.3400, found,613.3382, which indicates that the product obtained in this example is a cyclic peptide compound of formula VIII.

Example 8

The cyclic peptide compound of the embodiment of the invention has a structural formula shown in formula IX,

the cyclic peptide compound of formula IX is named cyclo (-Leu-DTrp-Pro-Thr-Asp-Leu-DPhe-Lys (Dde) -Val-Arg-), R is relative to the general formula of the peptide compound of formula I₁＝-CH(CH₃)OH，R₂＝-CH₂CH(CH₃)₂，R₃＝H，R₄＝4-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)amino)butanyl，R₅＝-CH(CH₃)₂，R₆＝(1H-indol-3-yl)methanyl，HRMS：calcd for C₇₂H₁₀₅N₁₅O₁₅。

The difference between the preparation method of the cyclic peptide compound of this example and example 1 is: 1, synthesizing a linear peptide compound Leu-DTrp (Boc) -Pro-Thr (tBu) -Asp (OtBu) -Leu-DPhe-Lys (Dde) -Val-Arg (Pbf); 2, the amino acid starting materials sequentially loaded during synthesis differ depending on the linear peptide sequence.

NMR spectrum of the product purified by HPLC in this example (¹HNMR) High Resolution and Mass Spectrometry (HRMS) detection with the result that¹H NMR(600MHz,MeOD),8.98–7.64(amide),7.48(d,J＝8.0Hz,1H),7.41(d,J＝8.2Hz,1H),7.28–7.23(m,2H),7.23–7.20(m,3H),7.19–7.13(m,2H),7.05(t,J＝7.5Hz,1H),5.06(t,J＝7.5Hz,1H),4.89(m,1H),4.71–4.64(dd,J＝14.4,7.3Hz,1H),4.59(dd,J＝9.5,6.9Hz,1H),4.54(m,2H),4.32–4.26(m,1H),4.26–4.20(m,2H),4.13–4.04(m,2H),3.56(d,J＝9.4Hz,2H),3.42–3.32(m,2H),3.24(m,4H),2.98(m,3H),2.71(dd,J＝16.4,7.3Hz,1H),2.52(s,3H),2.36(s,4H),2.18(m,2H),1.93–1.84(m,2H),1.80(m,1H),1.72(m,1H),1.68–1.54(m,6H),1.55–1.39(m,8H),1.19(m,2H),1.11(d,J＝6.4Hz,3H),1.00(s,6H),0.93(d,J＝6.7Hz,2H),0.90(d,J＝6.5Hz,2H),0.89(m,2H),0.88(d,J＝6.5Hz,2H),0.86(d,J＝6.2Hz,2H),0.84(d,J＝6.3Hz,2H).HRMS:calcd for C₇₂H₁₀₅N₁₅O₁₅[M+2H]²⁺m/z 710.9030, found,710.9027, which indicates that the product obtained in this example is a cyclic peptide compound of formula IX.

Example 9

As a cyclopeptide compound of the embodiment of the invention, the structural formula of the cyclopeptide compound is shown in the formula X,

the cyclic peptide compound represented by the formula IX is named as cyclo (-Leu-DTrp-Pro-Thr (laurate) -Asp-Leu-DPhe-Lys (Dde) -Val-Arg-, R being relative to the general formula of the peptide compound of formula I₁＝-CH(CH₃)OR₇，R₂＝-CH₂CH(CH₃)₂，R₃＝H，R₄＝4-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)amino)butanyl，R₅＝-CH(CH₃)₂，R₆＝(1H-indol-3-yl)methanyl，R₇＝-CO-(CH₂)₁₀CH₃。

The difference between the preparation method of the cyclic peptide compound of this example and that of example 8 is: the linear peptide compound Leu-DTrp (Boc) -Pro-Thr (Laurate) -Asp (OtBu) -Leu-DPhe-Lys (Dde) -Val-Arg (Pbf) was synthesized.

The HPLC purified product of this example was subjected to high resolution mass spectrometry detection, HRMS: calcd for C₈₄H₁₂₇N₁₅O₁₆，[M+2H]²⁺The product obtained in this example is a cyclic peptide compound of formula X, as shown in m/z 801.9849, found, 801.9830.

Example 10

The cyclopeptide compound of the embodiment of the invention has the structural formula XI,

the cyclic peptide compound shown as the formula XI is named as cyclo (-Leu-DTrp-Pro-Thr (Myristate) -Asp-Leu-DPhe-Lys (Dde) -Val-Arg-), and relative to the general formula of the peptide compound shown as the formula I, R is₁＝-CH(CH₃)OR₇，R₂＝-CH₂CH(CH₃)₂，R₃＝H，R₄＝4-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)amino)butanyl，R₅＝-CH(CH₃)₂，R₆＝(1H-indol-3-yl)methanyl，R₇＝-CO-(CH₂)₁₂CH₃。

The only difference between the preparation method of the cyclic peptide compound of this example and that of example 9 is: the linear peptide compound Leu-DTrp (Boc) -Pro-Thr (Myristate) -Asp-Leu-DPhe-Lys (Dde) -Val-Arg (Pbf) was synthesized.

The product purified by HPLC in this example was subjected to high resolution mass spectrometry detection, HRMS: calcd for C₈₆H₁₃₁N₁₅O₁₆，[M+2H]²⁺The formula xi, wherein m/z is 815.9952, found, 815.9943, indicates that the product obtained in the embodiment is the cyclic peptide compound shown as the formula xi.

Example 11

As a cyclopeptide compound of the embodiment of the invention, the structural formula of the cyclopeptide compound is shown in a formula XII,

the cyclic peptide compound represented by the formula XII is named as cyclo (-Leu-DTrp-Pro-Thr (palmitate) -Asp-Leu-DPhe-Lys (Dde) -Val-Arg-), and R is represented by the general formula of the peptide compound represented by the formula I₁＝-CH(CH₃)OR₇，R₂＝-CH₂CH(CH₃)₂，R₃＝H，R₄＝4-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)amino)butanyl，R₅＝-CH(CH₃)₂，R₆＝(1H-indol-3-yl)methanyl，R₇＝-CO-(CH₂)₁₄CH₃。

The only difference between the preparation method of the cyclic peptide compound of this example and that of example 9 is: the linear peptide compound Leu-DTrp (Boc) -Pro-Thr (palmate) -Asp-Leu-DPhe-Lys (Dde) -Val-Arg (Pbf) was synthesized.

The HPLC purified product of this example was subjected to high resolution mass spectrometry detection, HRMS: calcd for C₈₈H₁₃₅N₁₅O₁₆，[M+2H]²⁺When m/z is 830.0125, found, 829.9980, it can be said that the product obtained in this example is a cyclic peptide compound represented by formula XII.

Example 12

As a cyclopeptide compound of the embodiment of the invention, the structural formula of the cyclopeptide compound is shown in the formula XIII,

the cyclic peptide compound of formula XIII is named Cyclo (-Leu-DTrp-Pro-Thr (Stearate) -Asp-Leu-DPhe-Lys (Dde) -Val-Arg-, R being relative to the general formula of the peptide compound represented by formula I₁＝-CH(CH₃)OR₇，R₂＝-CH₂CH(CH₃)₂，R₃＝H，R₄＝4-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)amino)butanyl，R₅＝-CH(CH₃)₂，R₆＝(1H-indol-3-yl)methanyl，R₇＝-CO-(CH₂)₁₆CH₃。

The only difference between the preparation method of the cyclic peptide compound of this example and that of example 9 is: the linear peptide compound Leu-DTrp (Boc) -Pro-Thr (Stearate) -Asp-Leu-DPhe-Lys (Dde) -Val-Arg (Pbf) was synthesized.

The product purified by HPLC in this example was subjected to high resolution mass spectrometry detection, HRMS: calcd for C₉₀H₁₃₉N₁₅O₁₆，[M+2H]²⁺m/z 884.2567, found, 884.2530, which indicates that the product obtained in this example is a cyclic peptide compound of formula XIII.

Effect example 1

1. The cyclic peptide compounds of examples 1-8 were tested for their ability to inhibit the interaction between PD-1 and PD-L1.

The test is carried out by adopting a PD-1/PD-L1 binding assay kit, and the specific operation of the experiment is carried out by referring to a reagent instruction, which is briefly operated as follows. Cyclopeptide molecules with different concentrations and PD-L1-Tag1 protein (final concentration 5nM) were added to a 384-well plate, 50nM PD-1-Tag2 protein was added, and incubation was carried out at room temperature for 15 min. Adding anti-Tag1-Eu³⁺And anti-Tag2-XL665, and incubating for 3h at room temperature. Detection of fluorescence reading F under 665nm by means of microplate reader FlexStation 3₆₆₅And fluorescence reading F at 620nm₆₂₀. Relative fluorescence value FR as 10000 XF₆₆₅/F₆₂₀。

Inhibition rate calculation Inhibition (%) 100 × (FR-_cyclopeptide–FR_negative)/(FR_positive–FR_negative)。

Wherein, FR_cyclopeptideAnd FR_positiveRepresents the relative fluorescence values obtained by adding a specific concentration of the cyclopeptide molecule or DMSO group respectively; FR_negativeneRepresents the relative fluorescence values obtained for the negative group comprising all components of the positive group except the PD-1 protein.

The results are shown in FIG. 2. The line numbers in fig. 2 correspond to the example numbers. As can be seen from fig. 2, the above cyclic peptide compound can significantly inhibit the interaction between programmed death ligand 1 and programmed death receptor 1. Further, it was found that the inhibitory effects of examples 4 and 6 to 8 are superior to those of examples 1 to 3 and 5. The common feature of example 4, example 6, example 7 and example 8 is that R₁is-CH (CH)₃) OH, examples 1 to 3, example 5 have in common that R₁Is alkyl, i.e. R₁is-CH₃or-CH (CH)₃)₂. And IC of the cyclic peptide compound of example 8 was found₅₀Much lower than the other embodiments.

2. The cyclic peptide compounds of examples 1-8 promote the killing of tumor cells by immune cells.

MDA-MB-231-luc cells were seeded in 96-well plates at 2000/well density 12h in advance, 10. mu.M of cyclic peptide molecules 1-8 and PBMC cells were added, respectively, and culture supernatants were collected after 72h of culture, and tested for LDH according to the LDH test kit (Beyotime Biotechnology, C0016) instructions. At 1h before the predetermined detection time point, the cell culture plate is taken out, the LDH release reagent (the addition volume is 10% of the culture solution volume) is added into the maximum enzyme activity hole, the blowing and beating are repeated for a plurality of times, and then the cell culture plate is put into an incubator for continuous culture. After the predetermined detection time point was reached, 400g was centrifuged for 5min, and 120. mu.L of the supernatant was added to a new 96-well plate. And preparing a detection reagent comprising a 1X INT solution, an LDH detection working solution and the like. And adding 60 mu L of LDH detection working solution into each hole, mixing uniformly, and incubating for 30min at room temperature in a dark place. Absorbance at 490nm was measured using FLUStar Omega-ACU.

And (3) data calculation:

Promotion(％)＝(LD_{cancer+PBMC+peptide}-LD_{PBMC+peptides}-LD_{cancer+peptide})/LD_cancer；

wherein, the% Promotion represents the Promotion percentage of the cyclopeptide compound on the tumor cell inhibition of PBMC; LD_{cancer+PBMC+peptide}Test values representing LDH in the supernatant of wells containing PBMC, tumor cells, cyclic peptides simultaneously; LD_PBMC+peptideTest values representing LDH in the supernatant of wells containing both PBMC and cyclic peptide; LD_{cancer+peptide}Represents the test value of LDH in the supernatant of the well containing both tumor cells and cyclic peptide; LD_cancerRepresenting the maximum amount released by total lysis of the tumor cells.

The results are shown in FIG. 3. The abscissa numerals in fig. 3 correspond to the numerals of the embodiments. As is clear from fig. 3, the cyclic peptide compounds of examples 1 to 8 significantly promoted the killing of tumor cells by PBMC, and it was found that the effects of examples 4 and 6 to 8 were superior to those of examples 1 to 3 and 5. And it was found that the cyclic peptide compound of example 8 was significantly superior in effect to the other examples.

3. In vivo antitumor Effect test of Cyclic peptide Compound of example 8

5×10⁵B16F10 was inoculated in the upper abdomen of 5-8 week female C57/BL mice, and 6 days later, administration was started. The administration mode comprises the following steps: 40mg/kg of each was administered by intraperitoneal injection every other day, and the solvent group was injected with 5% DMSO, 20% Kolliphor HS15, 75% physiological saline. Tumor volume, mouse body weight were measured once in two days. Tumor volume calculation formula: v_mm ³＝tumor length×(tumor width)²/2. Two weeks post-treatment, each group of tumor volumes and masses were calculated. The results are shown in FIG. 4. As can be seen from fig. 4, the cyclic peptide compound of example 8 significantly reduced the growth rate (fig. 4A) and the tumor mass (fig. 4B) of the tumor.

4. Reduction of the cyclic peptide compound of example 8 increases representative inflammatory factors in activated T cells.

And (4) setting an antibody activating group and a tumor cell activating group, and detecting the content change of each cytokine by Elisa. CD3+ T cells in the antibody activation group were added to a 96-well plate at a density of 40 ten thousand per well, and a normal group, a 1. mu.g/mL anti-CD3+ 1. mu.g/mL anti-CD28anti group, and a 1. mu.g/mL anti-CD3+ 1. mu.g/mL anti-CD28anti +8 group were set. Incubating for 48h at 37 ℃, taking the culture supernatant, and detecting the content of IFN-gamma and IL-10 in the supernatant according to the requirement of an Elisa kit. In the tumor cell activation group, tumor cells were plated at a density of 2000 cells/well in advance, CD3+ T cells were added to the well plate at a density of 40 ten thousand cells/well, and a group containing only tumor cells, tumor cells + PBMC cells, and tumor cells + PBMC cells +8 cells were set. Incubating for 48h at 37 ℃, taking the culture supernatant, and detecting the content of IFN-gamma and IL-10 in the supernatant according to the requirement of an Elisa kit.

The Elisa detection process is as follows: the culture supernatant was transferred to an EP tube and centrifuged at 1000g at 4 ℃ for 15min to obtain the supernatant. Add 100. mu.L of test supernatant to each well, incubate at 37 ℃ for 2h, and set the standard curve. After the incubation was completed, the solution in the wells was removed, 100. mu.L of Biotin-antibody was added, and the mixture was incubated at 37 ℃ for 1 hour. After incubation, the wells were cleared of solution, and 200. mu.L/well of wash solution was added and washed 3 times for 2min each. 100 μ L of HRP-avidin solution was added and incubated at 37 ℃ for 1 h. After incubation, the solution in the wells was removed, and 200. mu.L/well of washing solution was added and washed 3 times for 2min each. Adding 90 μ L of TMB solution, incubating at 37 deg.C in dark for 15-30min, adding 50 μ L of stop solution when standard curve has obvious variation trend, and reading OD on enzyme-linked immunosorbent assay_450nmThe numerical value of (c). The standard curve is used to calculate the IL-10 or IFN-gamma content in each well to be tested.

The final calculated content for each group was compared to the normal group and the final results are shown in FIG. 5. As shown in FIG. 5, the cyclic peptide compound of example 8 can reduce the increase of IFN-. gamma.and IL-10 levels caused by the activation of CD3 antibody (FIG. 5A) or tumor cell activation (FIG. 5B) with CD3+ T, respectively.

Effect example 2

Hydrolysis of the cyclic peptide compounds of examples 9-12.

The acidic microenvironment in the tumor tissue was simulated with PBS buffer at

pH

5 or 6, 10mg of the cyclic peptide compounds of examples 9-12 were weighed and dissolved in PBS buffer at

pH

5 or 6 at a concentration of 1mM, respectively, and after 24 hours, samples were taken and analyzed by HPLC to calculate the percent release of the cyclic peptide compound of example 8. The results are shown in Table 1.

TABLE 1 hydrolysis efficiency of Cyclic peptide Compounds of examples 9-12

As is clear from Table 1, the cyclic peptide compounds of examples 9 to 12 released the cyclic peptide compound of example 8 under acidic conditions, and the release rate was 45% or more at pH 6 for 24 hours and 75% or more at pH 5 for 24 hours.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A cyclopeptide compound is characterized in that the structural formula of the cyclopeptide compound is shown as a formula I,

wherein R is₁is-CH₃、-CH(CH₃)₂、-CH(CH₃)OR₇One of (1);

R₅is-CHCH₃C₂H₅or-CH (CH)₃)₂；

R₆is-CH₂C₆H₅Or- (1H-indol-3-yl) methanol;

R₇is H or-CO- (CH)₂)_nCH₃And n is a positive integer from 0 to 18.

2. A cyclic peptide compound of claim 1, wherein R is₁is-CH (CH)₃)OR₇Said R is₇Is H or-CO- (CH)₂)_nCH₃And n is a positive integer from 0 to 18.

3. A cyclic peptide compound of claim 1, wherein R is₇is-CO- (CH)₂)_nCH₃And n is a positive integer of 10-16.

4. A cyclic peptide compound according to claim 1 or 2, wherein R is₃Is H and R₄Is 4- ((1- (4,4-dimethyl-2, 6-dioxacyclohexadiene) ethyl) amino) butanyl.

5. A cyclic peptide compound according to claim 1, wherein the structural formula of the cyclic peptide compound is as shown in formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII, or formula XIII;

6. use of a cyclic peptide compound according to any one of claims 1 to 5 and pharmaceutically acceptable salts thereof as an active ingredient in the manufacture of a medicament for the treatment of neoplastic diseases.

7. Use of a cyclic peptide compound according to any one of claims 1 to 5 and pharmaceutically acceptable salts thereof as active ingredient in the manufacture of a medicament for inhibiting the binding of programmed death ligand 1 and programmed death receptor 1.

8. A pharmaceutical composition comprising a cyclic peptide compound of any one of claims 1 to 5, and pharmaceutically acceptable salts thereof.

9. The pharmaceutical composition of claim 8, further comprising a pharmaceutical excipient, for use in treating a neoplastic disease or for inhibiting the binding of programmed death ligand 1 and programmed death receptor 1.