Covalent cyclic peptide targeting chimera as well as preparation method and application thereof

Abstract

The invention relates to the technical field of polypeptide drug modification, and discloses a covalent cyclic peptide targeting chimera as well as a preparation method and application thereof. In order to solve the problems that a current polypeptide drug is short in binding life with a receptor, poor in drug effect persistence and rapid in-vivo removal, the CCP-TAC is obtained by introducing a SuFEx reaction group into a cyclopeptide tyrosine residue through a hexavalent sulfur-fluorine exchange (SuFEx) reaction, then performing deprotection, alkynyl connection and click chemical reaction, and coupling with an azide-containing BMS molecule. The CCP-TAC provided by the invention has excellent conformational rigidity, covalent labeling capability and biological safety, can selectively degrade PD-L1 and remodel a tumor immune microenvironment, and shows a good application prospect in tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of peptide drug modification technology, and in particular to a covalently cyclic peptide targeted chimera, its preparation method, and its application. Background Technology

[0002] Cancer, also known as malignant tumors, is a leading cause of death worldwide. It is characterized by the uncontrolled growth and spread of abnormal cells within the body. Cancer cells can invade nearby tissues and organs and can spread to other parts of the body via the bloodstream or lymphatic system, forming new tumors in the process. There are many types of cancer, each with different effects on the body, but all ultimately harm human health. Cancer treatment options include surgery, chemotherapy, radiation therapy, targeted therapy, and immunotherapy.

[0003] Tumor cells can suppress the surveillance and clearance functions of the host immune system through various mechanisms during their growth; this process is known as immune escape. Among these mechanisms, the PD-1 / PD-L1 pathway is one of the most representative immunosuppressive pathways. PD-L1 is a co-inhibitory molecule expressed on the surface of various tumor cells. After binding to the PD-1 receptor on the surface of T cells, it can significantly reduce T cell activation and effector function, thereby weakening the anti-tumor immune response. By upregulating PD-L1 expression, tumor cells can form an immunosuppressive microenvironment, promoting their continuous proliferation and survival. Therefore, intervention targeting the PD-1 / PD-L1 pathway has become an important strategy for enhancing the anti-tumor immune response.

[0004] Lysosomal targeted chimeras (LYTACs) and related targeted degradation technologies hijack the cell's inherent lysosomal degradation pathway by linking the extracellular domains of target proteins to lysosomal transport receptors on the cell surface, inducing endocytosis and selective degradation of the target proteins. This strategy has attracted widespread attention in chemical biology and drug discovery due to its ability to effectively regulate cell membrane protein homeostasis. Integrins are transmembrane receptors that are widely expressed in various reproductive system-related tumor cells and cancers such as non-small cell lung cancer. They themselves undergo endocytosis during the uptake of exogenous substances and membrane protein turnover. Therefore, this endocytic property of integrins provides a potential avenue for targeted strategies based on membrane protein degradation.

[0005] Peptides are increasingly recognized as powerful regulators of protein-protein interactions (PPIs), which are fundamental to basic cellular processes and drive many disease pathways. However, peptide therapy is severely hampered by inherent limitations such as low membrane permeability, short half-life, and low bioavailability. Rational chemical modifications to peptides can often enhance their biological activity and pharmacokinetics. Summary of the Invention

[0006] To address the problem of chemical modification of anticancer peptide drugs, and more specifically, to address the issues of short receptor binding lifespan, low pharmacological persistence, and rapid systemic clearance of the peptide drugs, this invention provides a covalently cyclic peptide-targeting chimeric compound (CCP-TAC), its preparation method, and its applications.

[0007] The specific technical solution of this invention is as follows:

[0008] In a first aspect, the present invention provides a covalently cyclic peptide targeting chimera, the chemical structural formula of which is:

[0009] .

[0010] This invention constructs a covalently cyclic peptide-targeting chimeric complex, CCP-TAC. Experiments have demonstrated that CCP-TAC possesses enhanced conformational rigidity and covalent reactivity, as well as long-lasting covalent labeling capability and good biocompatibility. It can selectively degrade PD-L1 and remodel the tumor immune microenvironment.

[0011] Secondly, the present invention provides a method for preparing a covalently cyclic peptide targeting chimera, comprising the following steps:

[0012] Step S1: Take 9h of cyclic peptide and react it with SO2F2 gas to obtain a covalent cyclic peptide precursor containing SuFEx, denoted as compound 10g;

[0013] Step S2: Mix 10g of the cyclic peptide with trifluoroacetic acid, remove the protecting group, and obtain the intermediate, denoted as compound 10g';

[0014] Step S3: React 10 g' of the intermediate obtained in step S2 with 5-hexynyl succinimide ester to obtain the intermediate, denoted as SuFEx-RGD-Alkyne;

[0015] Step S4: The intermediate SuFEx-RGD-Alkyne obtained in step S3 is reacted with N3-BMS via a click reaction to obtain the product, namely the covalent cyclic peptide targeting chimera, denoted as CCP-TAC.

[0016] The chemical structural formula of the cyclic peptide 9h is as follows:

[0017] .

[0018] To address the issues of short receptor binding lifetime, poor sustained efficacy, and rapid in vivo clearance of cyclic peptide 9h, this invention introduces a SuFEx reactive group into the tyrosine residues of the cyclic peptide (cyclic peptide 9h) via a hexavalent sulfonyl fluoride exchange (SuFEx) reaction. Following deprotection, alkyne linkage, and click chemistry, it is coupled with an azide-containing BMS molecule to ultimately prepare a covalently cyclic peptide-targeting chimeric compound, CCP-TAC. Experiments have demonstrated that CCP-TAC possesses enhanced conformational rigidity and covalent reactivity, as well as long-lasting covalent labeling ability and good biocompatibility. It can selectively degrade PD-L1 and remodel the tumor immune microenvironment. CCP-TAC shows promising potential for anti-tumor therapy.

[0019] As a preferred embodiment of the above preparation method, in step S1, a base is added to the reaction. For example, one or more of DIEA (N,N-diisopropylethylamine), Et3N (triethylamine), and NaHCO3 (sodium bicarbonate) are used. The base promotes the modification of the phenol on the cyclic peptide precursor to aryl fluorosulfate after 9 hours, yielding 10g of the cyclic peptide precursor.

[0020] In step S1, the cyclic peptide 9h is reacted with SO2F2 gas, with MeCN as solvent and DIEA as base protectant. The reaction has a good modification effect on the SuFEx group, and the cyclic peptide precursor, i.e., compound 10g, is obtained.

[0021] Optionally, step S1 can be as follows: Add alkali to the stirred solution of the cyclic peptide 9h in MeCN; the color immediately changes from colorless to yellow. Vacuum the reaction flask and backfill with SO2F2 gas three times. Stir the reaction mixture at room temperature; during this period, the solution rapidly changes from yellow to colorless. After 3 minutes, remove volatiles under vacuum. Purify the crude product by PTLC to obtain 10g of colorless cyclic peptide precursor.

[0022] As a preferred embodiment of the above preparation method, in step S2, the intermediate obtained in step S2 is added to a TFA solution, and after the reaction, the protecting groups -Pbf, -Boc, and -tBu are removed.

[0023] Optionally, step S2 can be as follows: Dissolve 10g of the cyclic peptide precursor in an acidic solution and stir the reaction mixture at room temperature. After the reaction is complete, add the reaction mixture to ice-cold diethyl ether, precipitate the solid, filter, wash three times with diethyl ether, and dry to obtain the intermediate, i.e., 10g of compound.

[0024] The role of diethyl ether is to promote the precipitation of 10g of the intermediate compound solid. The addition of trifluoroacetic acid can promote the removal of the acid-sensitive protecting group and the release of the side chain protecting group of the cyclic peptide precursor.

[0025] As a preferred embodiment of the above preparation method, in step S3, an alkali is added to the reaction. For example, one or more of DIEA, Et3N, and NaHCO3.

[0026] In step S3, the purpose of adding the base is to neutralize trifluoroacetic acid and promote the linkage of 5-hexynic acid with the cyclic peptide precursor 10g'.

[0027] Optionally, step S3 can be as follows: Dissolve 10 g of the cyclic peptide precursor in DMF, then add DIEA, and stir the reaction at room temperature. Alternatively, dissolve 5-hexynyl succinimide ester in a certain volume of DMF, then add it to the reaction mixture, and stir at room temperature for 1 hour. After the reaction is complete, purify the crude mixture to obtain a white solid, SuFEx-RGD-Alkyne.

[0028] As a preferred embodiment of the above preparation method, in step S4, the click reaction is carried out under the conditions of a reducing agent and a catalyst.

[0029] Optionally, the reducing agent is NaVc and / or TCEP.

[0030] Optionally, the catalyst is one or more of CuSO4∙5H2O, Cu(OAc)2, and CuI.

[0031] In step S3, the CAS number of 5-hexyneic acid succinimide ester is 906564-59-8, and its structural formula is:

[0032] .

[0033] In step S4, SuFEx-RGD-Alkyne is mixed with N3-BMS, and the click reaction of alkyne and azide is promoted by a reducing agent and a copper catalyst to obtain the final product CCP-TAC.

[0034] Optionally, step S4 can be as follows: SuFEx-RGD-Alkyne and N3-BMS are dissolved in DMF and reacted with stirring at room temperature. CuSO4∙5H2O and NaVc are dissolved in H2O, respectively, and then added to the reaction mixture, followed by stirring at room temperature for 1 hour. After completion, the crude mixture is purified to obtain a white solid CCP-TAC.

[0035] Specifically, the chemical structural formula of the N3-BMS is as follows:

[0036] .

[0037] Optionally, the synthesis process of the N3-BMS is as follows:

[0038] .

[0039] More specifically, the synthesis process of the N3-BMS is as follows:

[0040] BMS-8 was dissolved in DMF, and then DIEA was added. The mixture was stirred at -10°C. Separately, HATU was dissolved in ice-cold DMF and then added to the reaction mixture, followed by stirring at -10°C for 30 minutes. Next, 3-aminopropyl azide, pre-dissolved in DMF, was added to the mixture, and the mixture was stirred at room temperature for 1 hour. After completion, the mixture was diluted with EtOAc, and the organic layer was washed successively with 1M HCl, saturated NaHCO3 aqueous solution, and saturated brine, and then dried over anhydrous Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by PTLC to give compound N3-BMS.

[0041] More specifically, the reaction process for preparing CCP-TAC is as follows:

[0042] , .

[0043] Based on the above-mentioned covalent cyclic peptide targeting chimera or the above-mentioned preparation method, the present invention provides an application of the covalent cyclic peptide targeting chimera in the preparation of tumor immunotherapy drugs.

[0044] Compared with the prior art, the present invention has the following technical effects:

[0045] (1) The present invention uses the antitumor drug cyclic peptide 9h as a precursor to construct a covalent cyclic peptide targeting chimera CCP-TAC. CCP-TAC has a long-lasting covalent labeling ability and can selectively degrade PD-L1 and reshape the tumor immune microenvironment. It shows effective antitumor activity in in vitro and in vivo experiments, can inhibit tumor growth and promote immune cell infiltration. In addition, CCP-TAC shows good biosafety in vivo, with no significant weight loss, organ toxicity or liver and kidney function damage. Furthermore, CCP-TAC can also enhance the systemic antitumor immune response by promoting dendritic cell maturation and T cell differentiation.

[0046] (2) The present invention successfully prepared a covalent cyclic peptide targeting chimera CCP-TAC by converting cyclic peptide 9h into the intermediate SuFEx-RGD-Alkyne and BMS-8 into N3-BMS, and then connecting cyclic peptide 9h and BMS-8 through SuFEx-RGD-Alkyne and N3-BMS. Attached Figure Description

[0047] Figure 1 This is the liquid phase spectrum of compound N3-BMS in this invention.

[0048] Figure 2 This is the liquid phase spectrum of compound 10g' in this invention.

[0049] Figure 3 This is the liquid phase spectrum of the compound SuFEx-RGD-Alkyne used in this invention.

[0050] Figure 4 The liquid phase spectrum of CCP-TAC obtained in Example 1 of this invention is shown.

[0051] Figure 5 This is the mass spectrum of CCP-TAC obtained in Example 1 of the present invention.

[0052] Figure 6 This is an immunofluorescence staining image of CCP-TAC obtained in Example 1 of the present invention (PD-L1 degradation).

[0053] Figure 7 This is a Western blot analysis diagram (PD-L1 expression) of CCP-TAC obtained in Example 1 of the present invention.

[0054] Figure 8 This is a graph showing the changes in tumor volume during the in vivo antitumor experiment of CCP-TAC obtained in Example 1 of the present invention.

[0055] Figure 9 This is a comparison chart of in vivo tumor weight of CCP-TAC obtained in Example 1 of the present invention.

[0056] Figure 10 The results of mouse weight change obtained by CCP-TAC in Example 1 of this invention are shown.

[0057] Figure 11 This is a flow cytometry analysis diagram of CCP-TAC obtained in Example 1 of the present invention (DC maturation and T cell infiltration).

[0058] Figure 12 This is a graph showing the levels of different immune-active cytokines in CCP-TAC obtained in Example 1 of the present invention.

[0059] Figure 13 This is a diagram showing the H&E staining results of organ tissues obtained by CCP-TAC in Example 1 of the present invention. Detailed Implementation

[0060] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0061] In one embodiment, a covalently cyclic peptide targeting chimera CCP-TAC is provided, prepared according to the following steps:

[0062] Step S1: Take 9h of cyclic peptide and react it with SO2F2 gas to obtain a covalent cyclic peptide precursor containing SuFEx, denoted as compound 10g;

[0063] Step S2: Mix 10g of the cyclic peptide with trifluoroacetic acid, remove the protecting group, and obtain the intermediate, denoted as compound 10g';

[0064] Step S3: React 10 g' of the intermediate obtained in step S2 with 5-hexynyl succinimide ester to obtain the intermediate, denoted as SuFEx-RGD-Alkyne;

[0065] Step S4: The intermediate SuFEx-RGD-Alkyne obtained in step S3 is reacted with N3-BMS via a click reaction to obtain the product, namely the covalent cyclic peptide targeting chimera, denoted as CCP-TAC.

[0066] The chemical structural formula of the cyclic peptide 9h is as follows:

[0067] .

[0068] In this embodiment, a covalently cyclic peptide-targeting chimeric compound, CCP-TAC, was prepared by converting cyclic peptide 9h into the intermediate SuFEx-RGD-Alkyne and BMS-8 into N3-BMS, followed by the linkage of cyclic peptide 9h and BMS-8 via SuFEx-RGD-Alkyne and N3-BMS. In one embodiment, experiments demonstrated that CCP-TAC possesses enhanced conformational rigidity and covalent reactivity, as well as long-lasting covalent labeling ability and good biocompatibility. It can selectively degrade PD-L1 and remodel the tumor immune microenvironment. CCP-TAC shows promising potential for anti-tumor therapy.

[0069] In one embodiment, in step S1, a base is added to the reaction. For example, one or more of DIEA (N,N-diisopropylethylamine), Et3N (triethylamine), and NaHCO3 (sodium bicarbonate). The base serves to promote the phenolic modification of the cyclic peptide precursor to aryl fluorosulfate at 9h, yielding 10g of the cyclic peptide precursor.

[0070] In step S1, the cyclic peptide 9h is reacted with SO2F2 gas, with MeCN as solvent and DIEA as base protectant. The reaction has a good modification effect on the SuFEx group, and the cyclic peptide precursor, i.e., compound 10g, is obtained.

[0071] In another embodiment, step S1 is as follows: Base is added to the stirred solution of the cyclic peptide 9h in MeCN; the color immediately changes from colorless to yellow. The reaction flask is then evacuated and backfilled three times with SO2F2 gas. The reaction mixture is stirred at 15–30°C, during which time the solution rapidly changes from yellow to colorless. After 3 minutes, volatiles are removed under vacuum. The crude product is purified by PTLC to obtain 10g of a colorless cyclic peptide precursor.

[0072] In one embodiment, in step S2, the intermediate obtained in step S2 is added to a TFA solution, and after the reaction, the protecting groups -Pbf, -Boc, and -tBu are removed.

[0073] Optionally, step S2 can be as follows: Dissolve 10g of the cyclic peptide precursor in an acidic solution and stir the reaction mixture at 15-30°C. After the reaction is complete, add the reaction mixture to ice-cold diethyl ether, precipitate the solid, filter, wash three times with diethyl ether, and dry to obtain the intermediate, i.e., 10g of compound.

[0074] The role of diethyl ether is to promote the precipitation of 10g of the intermediate compound solid. The addition of trifluoroacetic acid can promote the removal of the acid-sensitive protecting group and the release of the side chain protecting group of the cyclic peptide precursor.

[0075] In one embodiment, in step S3, a base is added to the reaction. For example, one or more of DIEA, Et3N, and NaHCO3.

[0076] In step S3, the purpose of adding the base is to neutralize trifluoroacetic acid and promote the linkage of 5-hexynic acid with the cyclic peptide precursor 10g'.

[0077] Optionally, step S3 can be as follows: dissolve 10 g of the cyclic peptide precursor in DMF, then add DIEA, and stir the reaction at 15–30 °C. Alternatively, dissolve 5-hexynyl succinimide ester in a certain volume of DMF, then add it to the reaction mixture, and stir at 15–30 °C for 1 hour. After the reaction is complete, purify the crude mixture to obtain a white solid, SuFEx-RGD-Alkyne.

[0078] In one embodiment, in step S4, the click reaction is carried out under the conditions of a reducing agent and a catalyst.

[0079] Optionally, the reducing agent is NaVc and / or TCEP.

[0080] Optionally, the catalyst is one or more of CuSO4∙5H2O, Cu(OAc)2, and CuI.

[0081] In one embodiment, SuFEx-RGD-Alkyne was mixed with N3-BMS, and the click reaction of alkyne and azide was promoted by a reducing agent and a copper catalyst to obtain the final product CCP-TAC.

[0082] Optionally, step S4 can be as follows: SuFEx-RGD-Alkyne and N3-BMS are dissolved in DMF and reacted with stirring at 15-30°C. CuSO4∙5H2O and NaVc are dissolved in H2O, respectively, and then added to the reaction mixture, followed by stirring at 15-30°C for 1 hour. After completion, the crude mixture is purified to obtain a white solid CCP-TAC.

[0083] In the above embodiments, the chemical structural formula of the N3-BMS is:

[0084] .

[0085] Optionally, the synthesis process of the N3-BMS is as follows:

[0086] .

[0087] More specifically, in one embodiment, the synthesis process of the N3-BMS is as follows:

[0088] BMS-8 was dissolved in DMF, and then DIEA was added. The mixture was stirred at -10°C. HATU was dissolved in ice-cold DMF and then added to the reaction mixture, followed by stirring at -10°C for 30 minutes. Next, 3-aminopropyl azide, pre-dissolved in DMF, was added to the mixture, and the mixture was stirred at 25°C for 1 hour. After completion, the mixture was diluted with EtOAc, and the organic layer was washed successively with 1M HCl, saturated NaHCO3 aqueous solution, and saturated brine, and then dried over anhydrous Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by PTLC to obtain compound N3-BMS. The HPLC spectrum of N3-BMS prepared by this method is shown below. Figure 1 As shown. The N3-BMS mass spectrometry data are: LCMS (ESI) m / z (relative intensity) 576.00 (100) [M+H+ In this invention, the preparation of CCP-TAC is described using the preparation of N3-BMS in this embodiment as an example.

[0089] In this invention and its embodiments, the cyclic peptide 9h, i.e., the starting material, is based on existing technology. The preparation of cyclic peptide 9h can be carried out according to the method described in the specification of Chinese patent application CN2024106559643, as detailed in Example 3 of the specification.

[0090] In this invention and its embodiments, the procedure for purification using semi-preparative reversed-phase high-performance liquid chromatography gradient elution is as follows: a C18 column is used, with 0.1% trifluoroacetic acid aqueous solution and acetonitrile as the mobile phase. The acetonitrile ratio is linearly reduced from 75% to 55% within 60 minutes, the flow rate is 3-5 mL / min, and the detection is performed at a wavelength of 220 nm.

[0091] More specifically, in this embodiment, the preparation reaction process of the above-mentioned CCP-TAC is as follows:

[0092] , .

[0093] To illustrate the invention in more detail, the following embodiments are provided.

[0094] Example 1

[0095] This embodiment provides the preparation of compound CCP-TAC, and the specific reaction process is as follows:

[0096] , .

[0097] S1: Under stirring at 400 rpm, DIEA (9.7 mg, 13 μL, 0.075 mmol) was added to a MeCN (1 mL) solution containing 9 h of cyclic peptide (0.025 mmol). The solution color immediately changed from colorless to yellow. The reaction flask was evacuated and purged three times with SO2F2 gas. The reaction mixture was stirred at room temperature, during which the solution rapidly changed from yellow back to colorless. After 3 minutes, volatiles were removed under reduced pressure. The crude product was purified by PTLC to obtain 10 g of colorless SuFEx-modified cyclic peptide.

[0098] S2: 10 g (0.01 mmol) of the cyclic peptide was treated with a 0.5 mL TFA / H2O mixture (95 / 5) for 1.5 hours at room temperature. After dilution with ice-cold ether, a solid was formed, filtered, and further purified by RP-HPLC to give 10 g' (7.1 mg, 95% yield) of a white solid.

[0099] S3: 10 g' (20.0 mg, 26.8 μmol) of the cyclic peptide was dissolved in DMF (400 μL), followed by the addition of DIEA (14.0 μL, 80.6 μmol, 3.0 equivalents), and the reaction was stirred at room temperature. Separately, 5-hexynyl succinimide ester (5.6 mg, 26.8 μmol, 1.0 equivalent) was dissolved in DMF (100 μL) and added to the above reaction mixture, and the mixture was stirred for another 1 hour at room temperature. After the reaction was complete, the crude product was purified by RP-HPLC to give 19 mg of SuFEx-RGD-Alkyne white solid, with a yield of 85%.

[0100] S4: SuFEx-RGD-Alkyne (10 mg, 11.9 μmol, 1.0 equivalent) and N3-BMS (8.2 mg, 14.3 μmol, 1.2 equivalent) were dissolved in DMF (1.5 mL), and the reaction mixture was stirred at room temperature. Separately, CuSO4·5H2O (1.8 mg, 7.1 μmol, 0.6 equivalent) and NaVc (9.4 mg, 47.7 μmol, 4.0 equivalent) were dissolved in H2O (750 μL), and then added to the reaction mixture. The mixture was stirred for another 1 hour at room temperature. After the reaction was complete, the crude product was purified by RP-HPLC to obtain 13.8 mg of CCP-TAC as a white solid, with a yield of 82% for this step. The overall yield was calculated as the ratio of the actual molar amount of CCP-TAC obtained to the theoretical molar amount of CCP-TAC corresponding to the molar amount of the cyclic peptide starting material at 9 h, resulting in an overall yield of 64%.

[0101] The product CCP-TAC was analyzed by HPLC and mass spectrometry. Results are shown below. Figure 4 , Figure 5 .like Figure 4 The image shown is an HPLC chromatogram. Figure 5 The mass spectrum is shown. The mass spectrometry data for compound CCP-TAC is: HRMS (ESI) m / z calcd for C 64 H 79 BrFN 13 O 16 S / 2708.7312,found708.7300.

[0102] In step S2, 10g of the cyclic peptide was taken for NMR analysis, and the data are as follows:

[0103] The 1H NMR spectrum data of 10g of cyclic peptide are as follows:

[0104] 1HNMR(500MHz,DMSO-d6)δ8.43(d,J=8.0Hz,1H),8.13(d,J=5.9Hz,1H),8.01(d,J=7.9Hz,1H),7.76(d,J=2.1Hz,2H),7.62-7.44(m,3H),7.19(d,J=7 .9Hz,1H),6.75(d,J=16.0Hz,1H),6.38(s,2H),4.82-4.76(m,1H),4.69( dd,J=11.3,3.1Hz,1H),4.62(q,J=7.4Hz,1H),4.29(td,J=11.0,5.5Hz,2H ),4.20-4.14(m,1H),4.09(dd,J=16.8,6.7Hz,1H),3.73(s,3H),3.52(dd ,J=16.9,3.4Hz,1H),3.04(dd,J=13.4,5.4Hz,1H),3.01-2.90(m,5H),2.7 3(dd,J=16.3,7.0Hz,1H),2.49-2.44(m,4H),2.42(s,3H),2.00(s,3H),1 .64-1.54(m,1H),1.41(s,8H),1.37(d,J=6.6Hz,18H),1.34-1.30(m,2H).

[0105] The carbon NMR spectrum data of 10g of cyclic peptide are as follows: 13 CNMR(126MHz,DMSO)δ172.10,170.75,170.38,169.70,169.62,169.24,165.52,157.89,15 6.45,155.29,146.82,139.54,137.71,135.41,134.68,134.24,131.87,129.83,126.35,1 24.74,122.27,122.12,116.69,86.73,80.74,78.96,63.09,57.34,55.34,52.91,52.11,51.81,49.44,42.95,42.31,37.78,36.53,30.38,28.74,28.53,28.13,19.38,18.03,12.71.

[0106] The NMR fluorine spectrum data of 10g of cyclic peptide are as follows: 19 FNMR (377MHz, DMSO) δ 39.83.

[0107] Among them, cyclic peptide 10g' is the substance obtained by removing the acid-sensitive protecting group from compound 10g. The structural formula of compound 10g' obtained in step S2 is as follows:

[0108] .

[0109] Compound 10g' was analyzed by HPLC and mass spectrometry. The HPLC chromatogram results are shown below. Figure 2 As shown. The mass spectrometry results are as follows:

[0110] The mass spectrometry data of compound 10g' are: HRMS (ESI) m / z calcd for C 28 H 38 FN8O 13 S(M+H) + 745.2258, found745.2234.

[0111] The white solid SuFEx-RGD-Alkyne obtained in step S3 was analyzed by HPLC and mass spectrometry. The HPLC chromatogram results are shown below. Figure 3 The mass spectrometry results are as follows:

[0112] The mass spectrometry data for compound SuFEx-RGD-Alkyne are as follows: HRMS (ESI) m / z calcd for C 34 H 44 FN8O 14 S (M + H + ) 839.2676, found 839.2656.

[0113] Example 2

[0114] This embodiment provides the preparation of compound CCP-TAC. The main difference between this embodiment and Example 1 is that in step S1, 0.075 mmol DIEA is replaced with 0.075 mmol Et3N. Everything else is the same as in Example 1.

[0115] The total yield of CCP-TAC prepared in this embodiment was 51%.

[0116] Example 3

[0117] This embodiment provides the preparation of compound CCP-TAC. The main difference between this embodiment and Example 1 is that in step S3, 0.075 mmol DIEA is replaced with 0.075 mmol Et3N. Everything else is the same as in Example 1.

[0118] The total yield of CCP-TAC prepared in this embodiment was 53%.

[0119] Example 4

[0120] This embodiment provides the preparation of compound CCP-TAC. The main difference between this embodiment and Example 1 is that in step S4, the addition of 7.1 μmol CuSO4·5H2O is replaced with the addition of 7.1 μmol Cu(OAc)2. Everything else is the same as in Example 1.

[0121] The total yield of CCP-TAC prepared in this embodiment was 42%.

[0122] Example 5

[0123] This embodiment provides the preparation of compound CCP-TAC. The main difference between this embodiment and Example 1 is that in step S4, 47.7 μmol NaVc is replaced with 47.7 μmol TCEP. Everything else is the same as in Example 1.

[0124] The total yield of CCP-TAC prepared in this embodiment was 45%.

[0125] Example 6

[0126] 1. In this embodiment, the compound CCP-TAC prepared in Example 1 was used to evaluate its in vitro PD-L1 degradation ability, as follows:

[0127] To assess the degradation ability of CCP-TAC on PD-L1, MDA-MB-231 cells (highly expressing PD-L1) were seeded in culture plates and treated with 0, 0.1, 0.5, and 1 μC of CCP-TAC for 12 hours, respectively. Immunofluorescence staining and Western blot analysis were then performed.

[0128] Immunofluorescence staining results as follows Figure 6 As shown, the fluorescence intensity of PD-L1 on the cell membrane of the CCP-TAC-treated group was significantly reduced in a concentration-dependent manner. Figure 7 The Western blot results shown further confirm that PD-L1 protein expression decreases with increasing CCP-TAC concentration, indicating that CCP-TAC can effectively degrade PD-L1.

[0129] 2. Evaluation of the in vivo antitumor activity of CCP-TAC, as detailed below:

[0130] Using the MDA-MB-231 tumor-bearing BALB / c mouse model, mice were randomly divided into three groups: (I) PBS control group; (II) FOLFOXIRI chemotherapy group (leucovorin 50 mg / kg, 5-fluorouracil 50 mg / kg, oxaliplatin 5 mg / kg, irinotecan 20 mg / kg); (III) FOLFOXIRI combined with CCP-TAC group (in addition to the drugs in the FOLFOXIRI chemotherapy group, CCP-TAC 10 mg / kg was also administered).

[0131] (1) The treatment cycle is 18 days, and the tumor volume and weight are recorded every 3 days.

[0132] The results are as follows Figure 8 As shown, the CCP-TAC combined treatment group exhibited the slowest tumor growth, with a significantly smaller average tumor volume compared to other groups. Tumors were weighed after treatment, and the results are as follows: Figure 9 As shown, the average tumor weight in the CCP-TAC group was only about 0.23g, significantly lower than that in the control group and the chemotherapy group. Meanwhile, there were no significant changes in the body weight of mice in any group. Figure 10 This indicates that CCP-TAC has good biosafety.

[0133] (2) The remodeling effect of CCP-TAC on the immune microenvironment is as follows:

[0134] The expression of maturation markers CD80 and CD86 of dendritic cells (DCs) in tumor-draining lymph nodes of mice in each group was analyzed by flow cytometry, as well as the expression of CD3 in spleen and tumor tissue. + CD4 + and CD3 + CD8 + T cell infiltration status.

[0135] The results are as follows Figure 11 As shown, the CCP-TAC treatment group exhibited high expression of CD80 and CD86 in DCs, indicating promoted DC maturation. Simultaneously, CD4+ expression was also observed in the tumor tissue of the CCP-TAC group. + and CD8 + The proportion of T cell infiltration was significantly increased (CD4). + T cells accounted for 13.0%, CD8 + T cells accounted for 8.06%, indicating that CCP-TAC can enhance T cell immune responses.

[0136] (3) Evaluate the regulatory effect of CCP-TAC on cytokines, specifically:

[0137] The levels of cytokines IL-6, IFN-γ, and TNF-α in the serum and tumor tissues of mice in each group were detected by ELISA. Results are shown below. Figure 12 .

[0138] Figure 12 The results showed that the levels of the above-mentioned cytokines were significantly upregulated in the CCP-TAC treatment group, and the duration of this upregulation was prolonged, indicating that CCP-TAC can effectively activate the systemic anti-tumor immune response.

[0139] (4) Histopathological analysis of major organs (H&E staining) of mice in each group. After treatment, important organs such as the heart, liver, spleen, lungs, and kidneys of the mice were paraffin-embedded, sectioned, and H&E-stained, and their morphology and structure were observed under a light microscope. The results are shown in […]. Figure 13 .

[0140] The results showed that the major organ structures of mice in the CCP-TAC treatment group were clear, cell morphology was normal, and no obvious pathological changes were observed, such as inflammatory cell infiltration, tissue necrosis, or fibrosis. This result directly proves that CCP-TAC did not cause observable damage to major organs during treatment.

[0141] In summary, the results of this embodiment demonstrate that CCP-TAC can effectively degrade PD-L1, inhibit tumor growth, reshape the tumor immune microenvironment, and has good biosafety, showing broad application prospects in tumor immunotherapy.

[0142] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0143] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A covalently cyclic peptide targeting chimera, characterized in that: The chemical structural formula is: 。 2. The method for preparing the covalently cyclic peptide targeting chimera as described in claim 1, characterized in that: Includes the following steps: Step S1: Take 9h of cyclic peptide and react it with SO2F2 gas to obtain a covalent cyclic peptide precursor containing SuFEx, denoted as compound 10g; Step S2: Mix 10g of compound with trifluoroacetic acid, remove the protecting group to obtain the first intermediate, denoted as compound 10g'; Step S3: React 10 g' of compound with 5-hexynyl succinimide ester to obtain the second intermediate, denoted as compound SuFEx-RGD-Alkyne; Step S4: The compound SuFEx-RGD-Alkyne is subjected to a click reaction with N3-BMS to obtain the product, namely the covalent cyclic peptide targeting chimera, denoted as CCP-TAC; The chemical structural formula of the cyclic peptide 9h is as follows: 。 3. The preparation method according to claim 2, characterized in that: In step S1, an alkali is added to the reaction, and the reaction is carried out in a solvent.

4. The preparation method according to claim 2, characterized in that: In step S2, the protecting groups are -Pbf, -Boc, and -tBu.

5. The preparation method according to claim 2, characterized in that: In step S3, an alkali is added to the reaction.

6. The preparation method according to claim 5, characterized in that: The alkali is selected from one or more of DIEA, Et3N, and NaHCO3.

7. The preparation method according to claim 2, characterized in that: In step S4, the click reaction is carried out under the conditions of a reducing agent and a catalyst.

8. The preparation method according to claim 7, characterized in that: The reducing agent is NaVc and / or TCEP.

9. The preparation method according to claim 7, characterized in that: The catalyst is one or more of CuSO4∙5H2O, Cu(OAc)2, and CuI.

10. The use of the covalent cyclic peptide targeting chimera as described in claim 1, or the covalent cyclic peptide targeting chimera prepared by the preparation method as described in any one of claims 2 to 9, in the preparation of tumor immunotherapy drugs.

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