Cyclic peptide targeting integrin alpha v beta 3 and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a cyclic peptide targeting integrin alpha v beta 3 and application thereof. The structural general formula of the cyclic peptide targeting integrin alpha v beta 3 is shown as a formula I, a formula II or a formula III. The novel thioether bridge bond-containing cyclic peptide which has selectivity and affinity and targets alpha v beta 3 is obtained by a solid-phase synthesis method. A cell binding experiment, an endocytosis experiment and an integrin-extracellular matrix interaction experiment based on ELISA (Enzyme-Linked Immuno Sorbent Assay) detection show that the cyclopeptide has relatively good affinity and specificity on integrin alpha v beta 3, and can be used as a targeting peptide for alpha v beta 3 receptor mediated drug delivery. Furthermore, the cyclic peptide is prepared into a corresponding polypeptide conjugate, the polypeptide conjugate has a nanomole-level inhibition effect on tumor cell inhibition, and compared with a single load drug, the toxicity is obviously reduced, the safety is high, and the polypeptide conjugate shows huge potential and application value in tumor treatment of targeting integrin alpha v beta 3.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to cyclic peptides targeting integrin αvβ3 and their applications. Background Technology

[0002] Integrins are heterodimeric transmembrane receptor proteins located on the cell membrane surface, responsible for mediating cell-extracellular matrix (ECM) adhesion and cell-cell interactions. They play crucial roles in cell signaling, migration, proliferation, differentiation, and tissue homeostasis, and are closely related to various diseases such as cancer, inflammation, and thrombosis. To date, 24 integrins have been identified in mammals, each composed of pairs of 18 α subunits and 8 β subunits. Based on the binding ligands, integrins can be classified into four categories: RGD (arginine-glycine-aspartate)-binding integrins, collagen-binding integrins, leukocyte-specific binding integrins, and laminin-binding integrins. In vivo, RGD-binding integrins specifically bind to ECM components such as fibronectin, osteopontin, vitreous connexin, and fibrinogen via the conserved arginine-glycine-aspartate (RGD) sequence, mediating intracellular and extracellular signal transduction. Therefore, they play important roles in fibrosis, cancer, and cardiovascular diseases.

[0003] αvβ3 is an important member of the integrin family. Studies have shown that its expression is upregulated in endothelial cells, contributing to angiogenesis, and it is also expressed in invasive tumor cells, macrophages, activated leukocytes, and osteoclasts. Therefore, αvβ3 has been explored as a therapeutic target for nearly 40 years, but to date, no αvβ3 antagonists have been approved for human treatment.

[0004] Currently reported peptides targeting αvβ3 include monocyclic peptides such as cilengitide, c(RGDfV), c(RGDfK), and c(RGDyK), as well as bicyclic peptides RGD4C and CT3HPQcT3RGDcT3, with the following specific structures:

[0005]

[0006]

[0007] Cilengiline exhibits good affinity for αvβ3, but also shows some affinity for αvβ5 and α5β1. Furthermore, cilengiline lacks an extension site beyond its conserved RGD sequence, limiting its application in drug delivery. Cilengiline has been clinically evaluated for the treatment of approximately 30 cancers, but failed in a phase III clinical trial due to its lack of efficacy in treating glioblastoma, failing to improve overall survival and progression-free survival. Compared to cilengiline, c(RGDfV) has a similar affinity, but also lacks an extension site and shows affinity for αvβ6. In contrast, c(RGDfK) and c(RGDyK), due to the presence of lysine (Lys), are more often used in drug delivery studies, but they also show some affinity for αvβ6. RGD4C has a lower affinity for αvβ3 compared to other cyclic peptides, and its cyclization is accomplished via disulfide bonds, which results in relatively poor stability and is rarely used in research.

[0008] Therefore, existing αvβ3-targeting cyclic peptides have one or more of the following problems: 1) poor stability; 2) poor selectivity; 3) low practicality; 4) low affinity for some cyclic peptides.

[0009] Overall, drug development targeting αvβ3 holds great promise. The conserved RGD sequence offers an advantage for peptide drug development, but existing αvβ3-targeting peptides suffer from poor stability and low affinity. Therefore, designing and developing novel, highly selective, and highly stable αvβ3-targeting peptide drugs is of significant importance and value for the precision diagnosis and treatment of cancer and other diseases. Summary of the Invention

[0010] To address the shortcomings mentioned in the background art, the primary objective of this invention is to provide cyclic peptides targeting integrin αvβ3. This invention utilizes a solid-phase synthesis method to obtain novel sulfide-bridged cyclic peptides targeting αvβ3, including bicyclic and monocyclic peptides obtained by this method.

[0011] A second objective of this invention is to provide a peptide-conjugated drug based on a cyclic peptide targeting integrin αvβ3.

[0012] A third objective of this invention is to provide the application of the above-mentioned cyclic peptides and polypeptide conjugates in drug delivery and in tumor-inhibiting drugs.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] A class of cyclic peptides of integrin αvβ3, the general structural formula of which is shown in Formula I, Formula II or Formula III:

[0015]

[0016] In Equation I:

[0017] R1 group is -H or

[0018] The R2 group can be One of them;

[0019] Preferably, the R2 group is

[0020] The R3 group can be -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2OH, or -CH(CH3)OH;

[0021] Most preferably, the R3 group is -H;

[0022] The R4 group can be -CH2COOH, -(CH2)2COOH, -CH2CONH2 or -(CH2)2CONH2;

[0023] Most preferably, the R4 group is -CH2COOH;

[0024] The R5 group is -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -COOH, or...

[0025] Where X is -CH2-S-indole- or -indole-SC(CH2)2-, and the hydrogen atom on the indole can be replaced by hydroxyl, halogen, halocarbon, alkynyl, alkene, cyano-protected carboxylic acid ester or cyano-protected carboxylamide, and the sulfur atom in X can be oxidized to sulfoxide or sulfone group.

[0026] Preferably, X is -indole-S-CH2-;

[0027] In Formula II:

[0028] R 1’ The radical group can be -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3;

[0029] Most preferably, R 1’ The functional group is -CH3;

[0030] R 2’ Groups can be

[0031] Most preferably, R 2’ Group is

[0032] R 3’ The radical group can be -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2OH or -CH(CH3)OH;

[0033] Most preferably, R 3’ The radical is -H;

[0034] R 4’ The group can be -CH2COOH, -(CH2)2COOH, -CH2CONH2 or -(CH2)2CONH2;

[0035] Most preferably, R 4’ The functional group is -CH2COOH;

[0036] The definition of X in Equation II is the same as that in Equation I;

[0037] Y is hydrogen, a substituted halogen, a hydroxyl group, an amino group, a methyl group, an ethyl group, a mercapto group, or a cyano group;

[0038] Preferably, X is -indole-S-CH2-; Y is hydrogen or hydroxyl.

[0039] In Formula III:

[0040] R 1” The group can be -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CONH2, -(CH2)2CONH2, -CH2COOH or -(CH2)2COOH;

[0041] Preferably, R 1” The radical group is -CH3, -CH2COOH, or -CH2CONH2;

[0042] R 2” The group can be -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CONH2, -(CH2)2CONH2, -CH2COOH or -(CH2)2COOH;

[0043] Preferably, R 2” The radical is -CH3 or -CH2CONH2;

[0044] R 3” Groups can be

[0045] Most preferably, R 3” Group is

[0046] R 4” The radical can be -H, -CH3, -CH2OH or -CH(CH3)OH;

[0047] Most preferably, R 4” The radical is -H;

[0048] R 5” The group can be -CH2COOH, -(CH2)2COOH, -CH2CONH2 or -(CH2)2CONH2;

[0049] Most preferably, R 5” The functional group is -CH2COOH;

[0050] The definition of X in Equation III is the same as that in Equation I;

[0051] The definition of Y in Equation III is the same as that in Equation II.

[0052] This invention also protects the use of the cyclic peptide targeting integrin αvβ3 in the preparation of drug delivery systems and / or in the encapsulation of oncology drugs.

[0053] Furthermore, the tumor includes any one of pancreatic cancer, colon cancer, lung cancer, rectal cancer, uterine cancer, cervical cancer, skin cancer, stomach cancer, liver cancer, brain tumor, bladder cancer, and lymphoma.

[0054] Based on the aforementioned cyclic peptide, this invention protects a polypeptide-conjugated drug, wherein the structural formula of the polypeptide-conjugated drug is A. n -LB; A represents the cyclic peptide targeting integrin αvβ3 as described in claim 1; n represents the number of links of the cyclic peptide, where n is any integer from 0 to 5; L represents the linker; B represents the loaded drug.

[0055] Furthermore, the linker can be cleavable or non-cleavable, and theoretically it can be any linker that can be used in antibody-drug conjugates (ADCs) or peptide-drug conjugates (PDCs).

[0056] Further, B represents an anti-tumor drug, a molecularly targeted drug, a peptide drug, or an antibody drug. The anti-tumor drug is selected from one or more of microtubule inhibitors, topoisomerase inhibitors, radionuclides, and PD-L1 inhibitors. The molecularly targeted drug is selected from one of imatinib, nilotinib, dasatinib, everolimus, erlotinib, sunitinib, sorafenib, ibrutinib, regorafenib, vemurafenib, and olaparib. The peptide drug is selected from one of p53 activating peptide, melitrix venom peptide, and scorpion venom peptide. The antibody drug is selected from one of rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, and nivolumab.

[0057] Furthermore, anti-tumor drugs such as microtubule inhibitors MMAE, MMAF, and PTX; topoisomerase inhibitors Dxd, DOX, and SN38; and radionuclides such as 68 Ga、 177 Lu and its ligands such as DOTA / NOTA; PD-L1 inhibitors such as BMS-8; fluorescent reagents such as Cy5.

[0058] Compared with the prior art, the beneficial effects of the present invention are:

[0059] This invention utilizes a solid-phase synthesis method to obtain a novel thioether-bridged cyclic peptide targeting αvβ3 with both selectivity and affinity. Cell binding assays, endocytosis assays, and ELISA-based integrin-extracellular matrix interaction assays demonstrate that the cyclic peptide exhibits good affinity and specificity for integrin αvβ3, making it suitable as a targeting peptide for αvβ3 receptor-mediated drug delivery. Further preparation of the cyclic peptide into corresponding conjugates revealed nanomolar inhibitory activity against tumor cells, with significantly reduced toxicity and high safety compared to the single loading drug MMAE, indicating its significant potential and application value in tumor therapy targeting integrin αvβ3. Attached Figure Description

[0060] Figure 1 This is a flowchart of the synthesis scheme of the present invention;

[0061] Figure 2 The figure shows the results of the experiment on the binding of 5-K-Cy5 to cells;

[0062] Figure 3 Figure showing the results of the endocytosis experiment of 5-K-Cy5 in MDA-MB-231 cells;

[0063] Figure 4 Comparison of tumor suppression effects of PDC1, MMAE, and blank control in vivo;

[0064] Figure 5 This is a diagram showing the in vivo tumor-suppressing effect of PDC1.

[0065] Figure 6 This is a graph showing the changes in body weight of mice in an in vivo tumor suppression experiment. Detailed Implementation

[0066] The technical solution of the present invention will be clearly and completely described below with reference to experimental examples and comparative examples. Obviously, the described experimental examples are only a part of the experimental examples of the present invention, and not all of the experimental examples. Based on the implementation examples in the present invention, all other experimental examples obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0067] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0068] like Figure 1 The diagram illustrates the preparation process of the cyclic peptide of this invention. The preparation steps are as follows:

[0069] Step 1: First, linear peptide (I) on resin is obtained by solid-phase synthesis;

[0070] Step 2: Cyclization was performed on the resin using a 2 mg / ml solution of elemental iodine (I2) DMF to obtain a monocyclic peptide (II) containing a thioether bridge.

[0071] Step 3: Remove the Fmco protecting group of (II) using a 20% piperidine DMF solution to obtain (III);

[0072] Step 4: The cyclic peptide is cleaved from the resin using TFA solution, precipitated with diethyl ether and purified to obtain a monocyclic peptide (V);

[0073] Alternatively, step 5: Following step 3, the cyclic peptide is cleaved using a 30% hexafluoroisopropanol (HFIP) solution in dichloromethane (DCM), and then cyclized using the condensing agent HATU to obtain a bicyclic peptide with a side-chain protecting group.

[0074] Step 6: Remove the side-chain protecting group of the bicyclic peptide from Step 5 with TFA solution, precipitate with diethyl ether and purify to obtain the bicyclic peptide (IV).

[0075] Example 1: Synthesis of Bicyclic Peptide 5

[0076]

[0077] The specific synthesis steps are as follows: First, weigh 1g of 2-CTC resin and place it in a solid-phase synthesis dish. Swell it with 15ml of dichloromethane (DCM) for 30 minutes and then filter out the solution. Dissolve Fmoc-Pro-OH (0.3mmol) in 15ml of N,N-dimethylformamide (DMF) and add it to the treated 2-CTC resin. Then add N,N-diisopropylethylamine (DIEPA) (1.5mmol, 5eq.) and react at room temperature for two hours. Then filter out the solution and wash the resin 5 times with 10ml of DMF. Add 15ml of a mixed solvent of MeOH / DIPEA / DCM (1:1:8) to block the resin for 30 minutes. Then filter out the solvent and wash the resin 5 times with 10ml of DMF. Next, the Fmoc protecting group was removed using a 20% piperidine DMF solution. Fmoc-Ala-OH (1.2 mmol, 4.0 eq.), condensing agent TBTU (1.2 mmol, 4.0 eq.), and DIPEA (3.6 mmol, 12 eq.) were added to the resin, and the reaction was carried out at 40°C for 1 h. The resin was then washed five times with DMF. The above steps were then repeated sequentially to condense amino acids Fmoc-Trp-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-nor-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Asn(Trt)-OH, respectively, to obtain linear peptide 1. The resin containing linear peptide 1 was then transferred to a 250 mL solid-phase synthesis tube, and 150 mL of a 2 mg / mL elemental iodine DMF solution was added. The reaction was carried out at room temperature under nitrogen purging for 2 h to obtain monocyclic peptide 2. The peptide was then washed five times with DMF, followed by removal of the Fmoc protecting group with 20% piperidine DMF solution (2 × 10 ml), washed five times with DMF, and then three times with DCM. Subsequently, 15 ml of HFIP:DCM (3:7) cleavage reagent was added, and cleavage was performed at room temperature for 2 h. The filtrate was collected, and the solvent was evaporated to obtain monocyclic peptide 3 (232 mg, 0.16 mmol). Finally, monocyclic peptide 3 was dissolved in 500 ml of DMF solvent, and condensing agents HATU (0.62 mmol, 2.0 eq.) and DIEPA (0.69 mmol, 2.2 eq.) were added. The reaction was carried out at room temperature for 12 h, and the DMF was evaporated to obtain bicyclic peptide 4. No purification was required. 20 ml of a TFA:TIS:H2O (95:2.5:2.5) mixed solvent was added, and the reaction was carried out at room temperature for 30 min. After all protecting groups were removed, the sample was precipitated with diethyl ether and purified by HPLC to obtain bicyclic peptide 5, which was lyophilized to a white powder of 39 mg, with a yield of 14.7%.

[0078] 1H NMR(400MHz,DMSO-d6)δ12.47(s,1H),11.17(s,1H),9.16(s,1H),8.76–8.65(m ,1H),7.91(d,J=5.9Hz,2H),7.55(t,J=7.3Hz,2H),7.44(s,1H),7.38(d,J=9.3 Hz,1H),7.26(d,J=8.1Hz,1H),7.09(d,J=7.1Hz,2H),7.02–6.95(m,2H),6.77( s,2H),4.96(d,J=11.8Hz,1H),4.52(s,1H),4.46(d,J=10.4Hz,1H),4.29(d,J=5 .1Hz,1H),4.23(dd,J=6.9,4.1Hz,1H),4.16(d,J=8.0Hz,1H),3.92(d,J=3.6Hz ,1H),3.88(d,J=3.9Hz,1H),3.76(d,J=14.0Hz,2H),3.58(dd,J=15.2,6.3Hz,1H ),3.28–3.09(m,8H),2.88–2.76(m,2H),2.73–2.62(m,2H),2.06(dd,J=11.6,6 .7Hz,1H),2.01–1.86(m,3H),1.73(d,J=17.4Hz,2H),1.30(d,J=6.9Hz,3H).(HR ESI-MS) m / z calculated for C 37 H 50 N 13 O 11 S + (M+H) + :884.3468,found 884.3479.

[0079] Example 2: Synthesis of Bicyclic Peptide 6

[0080] The method is the same as in Example 1. The arginine (Arg) used in Example 2 is the natural L-configuration, while the amino acid in Example 1 is the non-natural L-configuration nor-Arg. That is, the amino acid sequence of Example 2 is proline (Pro)-alanine (Ala)-tryptophan (Trp)-arginine (Arg)-glycine (Gly)-aspartic acid (Asp)-cysteine ​​(Cys)-asparagine (Asn).

[0081] 1H NMR (400MHz, DMSO-d6) δ12.34(s,1H),11.22(d,J=24.1Hz,1H),8.75(d,J=7.0Hz,1H),8.5 9–8.31(m,1H),8.23(dd,J=9.3,4.7Hz,1H),7.97(d,J=40.1Hz,2H),7.78(d,J=8.1Hz,1H) ,7.70–7.62(m,1H),7.56(dt,J=27.8,7.1Hz,2H),7.39(d,J=22.0Hz,1H),7.34–7.14(m,3 H),7.12–7.05(m,1H),6.96(dq,J=7.6,4.6,4.2Hz,2H),5.15–4.85(m,1H),4.83–4.59(m, 2H),4.52(d,J=6.4Hz,1H),4.42(td,J=9.1,3.6Hz,1H),4.34(p,J=7.8,7.3Hz,1H),4.23( q,J=10.0,9.1Hz,1H),4.13–3.98(m,1H),3.88–3.72(m,1H),3.67–3.49(m,2H),3.18–2.9 5(m,3H),2.92–2.73(m,2H),2.72–2.53(m,2H),2.40–2.21(m,1H),2.14–1.81(m,3H),1.8 1–1.64(m,2H),1.63–1.40(m,2H),1.39–1.09(m,5H),1.01–0.79(m,1H),0.53(s,1H).(HR ESI-MS)m / z calculated for C 38 H 52 N 13 O 11 S + (M+H) + :898.3624,found898.3623.

[0082]

[0083] Example 3: Synthesis of Bicyclic Peptide 7

[0084] The method is the same as in Example 1. Example 3 is a seven-membered bicyclic peptide. Compared with Example 1, Example 3 lacks asparagine (Asn), and the arginine (Arg) used is the natural L-configuration. Example 1 uses the non-natural L-configuration amino acid nor-Arg. That is, the amino acid sequence of Example 3 is alanine (Ala)-tryptophan (Trp)-arginine (Arg)-glycine (Gly)-aspartic acid (Asp)-cysteine ​​(Cys)-proline (Pro).

[0085] 1 H NMR (400MHz, DMSO-d6) δ12.47(s,1H),11.22(s,1H),8.72(d,J=6.6Hz,1H),8.21(d,J=5.5Hz,1H),7.99(d,J=7.1Hz,1H),7.76(d,J=8.2Hz, 1H),7.44(d,J=8.1Hz,3H),7.21(dd,J=11.0,8.4Hz,2H),7.05(t,J=7.6Hz,1H),6.92(t,J=7.5Hz,1H),4.91–4.82(m,1H),4.76(s,1H),4.35 (q,J=7.8,6.8Hz,1H),4.06(t,J=7.6Hz,1H),3.88(d,J=16.4Hz,3H),3.80–3.56(m,10H),3.06(s,2H),2.70(dd,J=16.6,5.0Hz,1H),2.14(d ,J=6.5Hz,1H),2.00(dd,J=11.9,6.1Hz,1H),1.78(d,J=24.5Hz,4H),1.49–1.32(m,2H),1.23(s,1H),0.72(d,J=7.1Hz,3H).HR(ESI-MS)m / z calculated for C 34 H 46 N 11 O9S + [M+H] + :784.3201,found.784.3205.

[0086]

[0087] Example 4: Synthesis of Bicyclic Peptide 8

[0088] The method is the same as in Example 1. Example 4 is a seven-membered bicyclic peptide. Compared with Example 1, Example 4 lacks asparagine (Asn), and the arginine (Arg) used is in the natural L configuration. In Example 1, the amino acid is a non-natural L configuration, nor-Arg. That is, the amino acid sequence of Example 4 is alanine (Ala)-cysteine ​​(Cys)-arginine (Arg)-glycine (Gly)-aspartic acid (Asp)-tryptophan (Trp)-proline (Pro).

[0089] 1 H NMR (400MHz, DMSO-d6) δ12.46(s,1H),11.54(s,1H),9.06(s,1H),8.90(d,J=7.5Hz,1H),8.55(d,J=5.8Hz,1H),7.97(d,J=9.6Hz,1H),7.67(d,J=7. 5Hz,1H),7.61–7.41(m,2H),7.28(d,J=7.8Hz,1H),7.21–7.05(m,3H),6.9 7(s,2H),5.68(ddd,J=12.1,9.4,5.7Hz,1H),4.78(dt,J=8.9,3.3Hz,1H), 4.63–4.43(m,3H),4.19(d,J=7.4Hz,1H),4.14–4.01(m,1H),3.99–3.85(m ,2H),3.84–3.67(m,3H),3.08(ddd,J=16.0,12.1,4.9Hz,3H),2.92–2.77( m,2H),2.70–2.57(m,1H),2.16–2.04(m,1H),2.02–1.87(m,1H),1.82–1.6 2(m,3H),1.59–1.33(m,3H),1.32–1.18(m,1H),0.72(d,J=7.5Hz,3H).(HR ESI-MS)m / zcalculated for C 34 H 46 N 11 O9S + (M+H) + :784.3195,found 784.3206.

[0090]

[0091] Example 5: Synthesis of Bicyclic Peptide 9

[0092] The method is the same as in Example 1. The amino acid sequence of Example 5 is proline (Pro)-asparagine (Asn)-cysteine ​​(Cys)-arginine (Arg)-glycine (Gly)-aspartic acid (Asp)-tryptophan (Trp)-alanine (Ala).

[0093] 1 H NMR (400MHz, DMSO-d6) δ12.50(s,1H),11.18(s,1H),9.15(s,1H),8.77–8.57(m,2H),7.98(s,1H),7.85(s,1H),7.56(d,J=8.1Hz,1H),7.45(d,J =9.1Hz,1H),7.40(s,1H),7.25(t,J=6.6Hz,2H),7.08(t,J=7.6Hz,2H), 6.99(t,J=7.6Hz,2H),6.90(s,1H),6.76(s,1H),4.95(s,1H),4.47(d,J =6.4Hz,2H),4.27(s,2H),4.18(q,J=7.3,6.1Hz,3H),3.93–3.86(m,1H),3.69(d,J=10.9Hz,1H),3.55(dd,J=15.1,5.9Hz,1H),3.25–3.05(m,8H ),2.81(d,J=16.0Hz,2H),2.71–2.62(m,2H),2.04(d,J=7.0Hz,1H),1.96(s,1H),1.72(s,4H),1.54(d,J=7.5Hz,2H),1.27(d,J=6.9Hz,3H).(HR ESI-MS)m / z calculated forC 38 H 52 N 13 O 11 S + (M+H) + :898.3624,found 898.3635.

[0094]

[0095] Example 6: Synthesis of Bicyclic Peptide 10

[0096] The method is the same as in Example 1. The amino acid sequence of Example 6 is asparagine (Asn)-alanine (Ala)-cysteine ​​(Cys)-arginine (Arg)-glycine (Gly)-aspartic acid (Asp)-tryptophan (Trp)-proline (Pro).

[0097] 1H NMR (400MHz, DMSO-d6) δ12.27(s,1H),11.18(s,1H),8.83(d,J=56.0Hz,3H),7.64(d,J=8.5Hz,3H),7.44(s,3H),7.31( s,1H),7.26(d,J=8.1Hz,1H),7.10(d,J=7.9Hz,1H),7.02(t,J=7.4Hz,1H),6.79(s,2H),5.18(s,1H),4.51(s,1H),4.3 4–4.14(m,5H),4.02(s,1H),3.77(s,2H),3.50–3.42(m,2H),3.21–3.05(m,4H),2.90–2.71(m,3H),2.41(dd,J=15.5,7 .8Hz,1H),2.36–2.27(m,1H),2.09(d,J=9.5Hz,3H),1.92(d,J=12.3Hz,4H),1.81–1.70(m,1H),1.56–1.30(m,5H).(HR ESI-MS)m / z calculated forC 38 H 52 N 13 O 11 S + (M+H) + :898.3624,found 898.3633.

[0098]

[0099] Example 7: Synthesis of monocyclic peptide 11

[0100] The method is the same as in Example 1. Example 7 is a monocyclic peptide. Compared with Example 6, the amino acid sequence of Example 7 is asparagine (Asn)-alanine (Ala)-cysteine ​​(Cys)-arginine (Arg)-glycine (Gly)-aspartic acid (Asp)-tryptophan (Trp)-proline (Pro), but Example 7 was not bicyclic.

[0101] 1H NMR (400MHz, DMSO-d6) δ12.37 (s, 2H), 11.41 (s, 1H), 9.07 (d, J = 8.4Hz, 1H), 8. 97–8.88(m,1H),8.64(d,J=7.4Hz,1H),8.54(dd,J=13.4,6.3Hz,1H),8.00(s, 3H),7.70(dd,J=18.8,8.7Hz,2H),7.59(d,J=5.3Hz,1H),7.32(dd,J=18.2,8. 1Hz,2H),7.26–7.05(m,4H),6.87(d,J=8.5Hz,1H),4.93(s,1H),4.74–4.65(m, 1H),4.64–4.56(m,1H),4.54–4.43(m,1H),4.22(q,J=7.1,6.5Hz,1H),4.14–3 .89(m,3H),3.84–3.72(m,1H),3.28–3.10(m,4H),3.07(dd,J=13.1,7.4Hz,2H) ,3.00–2.81(m,3H),2.66–2.57(m,2H),1.74(s,1H),1.69–1.42(m,4H),1.35( ddd,J=18.6,13.3,5.7Hz,2H),1.23(s,3H),1.17–1.06(m,3H).HR(ESI-MS)m / z calculated for C 38 H 54 N 13 O 12 S + [M+H] + :916.3736,found.916.3737.

[0102]

[0103] Example 8: Synthesis of Bicyclic Peptide 12

[0104] The method is the same as in Example 1. Compared with Example 6, in Example 8, proline (Pro) in Example 6 is replaced with sarcosine (Sar), while the types and order of other amino acids remain unchanged. That is, the amino acid sequence of Example 10 is asparagine (Asn)-alanine (Ala)-cysteine ​​(Cys)-arginine (Arg)-glycine (Gly)-aspartic acid (Asp)-tryptophan (Trp)-sarcosine (Sar).

[0105] 1H NMR(400MHz,DMSO-d6)δ12.23(s,1H),11.12(s,1H),9.50(t,J=6.7Hz,1H),9 .15(d,J=5.4Hz,1H),8.76(d,J=8.1Hz,1H),7.79(d,J=8.6Hz,1H),7.72(d,J= 8.1Hz,1H),7.57–7.40(m,1H),7.36–7.18(m,4H),7.10–6.84(m,6H),5.48(dd d,J=13.5,9.8,3.7Hz,1H),4.67(p,J=7.1Hz,1H),4.54(td,J=8.6,4.6Hz,1H) ,4.40–4.20(m,2H),4.17–3.96(m,3H),3.51–3.49(m,4H),3.32–3.17(m,3H), 3.08(t,J=6.6Hz,2H),2.96(s,3H),2.79(dd,J=14.9,4.1Hz,1H),2.63(dd,J= 15.6,4.6Hz,1H),2.42–2.30(m,2H),2.05–1.91(m,1H),1.72(d,J=7.4Hz,4H),1.64(dd,J=17.7,2.7Hz,1H),1.55–1.36(m,2H).HR(ESI-MS)m / zcalculated for C 36 H 50 N 13 O 11 S + [M+H] + 872.3473, found. 872.3477.

[0106]

[0107] Example 9: Synthesis of monocyclic peptide 13

[0108] The method is the same as in Example 1. Example 9 is a five-membered monocyclic peptide with the following amino acid sequence: cysteine ​​(Cys)-arginine (Arg)-glycine (Gly)-aspartic acid (Asp)-tryptophan (Trp).

[0109] 1H NMR (400MHz, DMSO-d6) δ12.39(s,2H),11.32(s,1H),8.96(d,J=9.0Hz,1H),8.83(t,J=6.1Hz,1H),8.59(d,J=8.8Hz,1 H),8.36(s,3H),7.69(d,J=8.0Hz,1H),7.57(d,J=7.8Hz,1H),7.31(d,J=8.1Hz,1H),7.15(t,J=7.0Hz,2H),7.05(t,J =7.6Hz,2H),4.77–4.67(m,1H),4.60(q,J=7.3Hz,1H),4.48–4.37(m,1H),4.04–3.89(m,1H),3.76–3.63(m,1H),3.47 (dd,J=14.7,4.6Hz,2H),3.06(dd,J=9.7,6.2Hz,3H),2.98–2.82(m,3H),2.53(d,J=7.0Hz,1H),1.72–1.31(m,5H).(HR ESI-MS)m / z calculated for C 26 H 36 N9O8S + (M+H) + :634.2402,found 634.2401.

[0110]

[0111] Example 10: Synthesis of Bicyclic Peptide 14

[0112] The method is the same as in Example 1. Compared with Example 1, Example 10 replaces the nor-Arg amino acid in Example 1 with homo-Arg, while the other amino acid types remain unchanged. Its amino acid sequence is proline (Pro)-asparagine (Asn)-cysteine ​​(Cys)-homo-Arg-glycine (Gly)-aspartic acid (Asp)-tryptophan (Trp)-alanine (Ala).

[0113] 1H NMR (400MHz, DMSO-d6) δ12.49(s,1H),11.17(s,1H),9.14(s,1H),8.71(s,1H),7.96(d,J=6.9Hz,1H),7.86(s,1H),7.56(d,J=8.1Hz ,1H),7.46–7.35(m,3H),7.25(t,J=6.8Hz,2H),7.08(t,J=7.3Hz,2H),6.98(t,J=7.6Hz,2H),6.83(s,1H),6.76(s,1H),5.00–4.88(m ,1H),4.53–4.42(m,3H),4.29–4.18(m,3H),3.92–3.85(m,1H),3.76–3.65(m,2H),3.55–3.50(m,1H),3.25–3.15(m,4H),3.09(q,J=6 .8Hz,4H),2.85–2.77(m,2H),2.72–2.62(m,2H),1.77–1.68(m,4H),1.51–1.43(m,3H),1.42–1.32(m,4H),1.28(d,J=6.3Hz,3H).(HR ESI-MS)m / z calculated for C 39 H 54 N 13 O 11 S + (M+H) + :912.3781,found 912.3787.

[0114]

[0115] Example 11: Synthesis of Bicyclic Peptide 15

[0116] The method is the same as in Example 1. Compared with Example 5, Example 11 replaces the natural L-cysteine ​​(Cys) in Example 5 with non-natural homocysteine ​​(homo-Cys), whose amino acid sequence is proline (Pro)-asparagine (Asn)-homocysteine ​​(homo-Cys)-arginine (Arg)-glycine (Gly)-aspartic acid (Asp)-tryptophan (Trp)-alanine (Ala).

[0117] 1H NMR (400MHz, DMSO-d6) δ12.16(s,1H),11.20(s,1H),9.17(s,1H),8.35(d,J=4.8H z,2H),8.24(s,1H),7.83(d,J=6.6Hz,1H),7.61(d,J=8.4Hz,1H),7.37(t,J=8.0H z,2H),7.30(s,1H),7.20(d,J=8.0Hz,1H),7.06–6.95(m,2H),6.91(t,J=8.0Hz,1 H),6.86(d,J=6.9Hz,2H),4.57–4.46(m,1H),4.45–4.39(m,1H),4.32(t,J=7.7Hz, 1H),4.20–4.10(m,2H),4.06(q,J=7.6,7.1Hz,2H),3.94–3.81(m,1H),3.54(dd,J =18.0,4.1Hz,7H),3.15(q,J=7.4,6.9Hz,3H),3.01(dd,J=16.5,6.8Hz,1H),2.95 –2.80(m,2H),2.72(dd,J=15.7,7.1Hz,1H),2.58(dd,J=15.8,4.6Hz,1H),2.02(d ,J=12.5Hz,2H),1.88–1.66(m,5H),1.65–1.46(m,3H),-0.06(d,J=6.5Hz,3H).(HR ESI-MS)m / z calculated forC 39 H 54 N 13 O 11 S + (M+H) + :912.3781,found 912.3797.

[0118]

[0119] Example 12: Synthesis of Bicyclic Peptide 16

[0120] The method is the same as in Example 1. Compared with Example 5, Example 12 replaces the natural glycine (Gly) in Example 5 with non-natural β-alanine (β-Ala), whose amino acid sequence is proline (Pro)-asparagine (Asn)-cysteine ​​(Cys)-arginine (Arg)-β-alanine (β-Ala)-aspartic acid (Asp)-tryptophan (Trp)-alanine (Ala).

[0121] 1H NMR (400MHz, DMSO-d6) δ12.25(s,1H),11.13(d,J=17.8Hz,1H),8.57(t,J=9.4Hz,1H) ,8.38–8.25(m,1H),8.16(d,J=7.6Hz,1H),7.62(d,J=8.1Hz,1H),7.55–7.41(m,2H),7 .37(d,J=8.1Hz,1H),7.31(s,1H),7.25(d,J=8.1Hz,1H),7.20–7.05(m,3H),7.02(t, J=7.7Hz,1H),6.92–6.63(m,2H),4.99–4.82(m,1H),4.61–4.49(m,1H),4.47–4.34(m, 2H),4.31(s,1H),4.26–4.16(m,1H),4.15–4.05(m,1H),3.84–3.56(m,8H),3.21–3.0 7(m,4H),3.01(dd,J=26.1,11.3Hz,1H),2.88(d,J=8.9Hz,1H),2.83–2.72(m,1H),2.7 2–2.52(m,3H),2.48–2.42(m,1H),2.04(dd,J=9.3,4.6Hz,1H),1.92–1.81(m,1H),1. 81–1.69(m,1H),1.68–1.51(m,3H),1.24(t,J=6.7Hz,1H),-0.02(d,J=7.1Hz,3H).(HR ESI-MS)m / z calculated forC 39 H 54 N 13 O 11 S + (M+H) + :912.3781,found 912.3789.

[0122]

[0123] Example 13: Synthesis of Bicyclic Peptide 17

[0124] The method is the same as in Example 1. Compared with Example 5, Example 13 replaces the natural glycine (Gly) in Example 5 with non-natural γ-aminobutyric acid (GABA), whose amino acid sequence is proline (Pro)-asparagine (Asn)-cysteine ​​(Cys)-arginine (Arg)-γ-aminobutyric acid (GABA)-aspartic acid (Asp)-tryptophan (Trp)-alanine (Ala).

[0125] 1H NMR (400MHz, DMSO-d6) δ12.30(s,1H),10.98(s,1H),8.39(d,J=7.0Hz,1H),8.26(d,J=7.1Hz,1H),8.19–8.09(m,1H),7.90(t,J=5.5Hz,1H ),7.57(d,J=7.3Hz,1H),7.43(d,J=5.5Hz,1H),7.29–7.22(m,3H),7.10(q,J=8.9Hz,2H),7.01(q,J=7.6Hz,2H),6.83(s,1H),6.56(s,1H) ,4.74–4.54(m,3H),4.49(d,J=6.8Hz,1H),4.41–4.33(m,2H),4.17(dd,J=18.3,8.2Hz,2H),3.24–3.20(m,1H),3.17–3.05(m,5H),3.03–2 .93(m,3H),2.44–2.31(m,2H),2.18–1.88(m,6H),1.76(dt,J=11.4,3.6Hz,1H),1.62(s,5H),1.54–1.35(m,4H),1.25(d,J=6.9Hz,3H).(HR ESI-MS)m / z calculated for C 40 H 56 N 13 O 11 S + (M+H) + :926.3937,found 926.3947.

[0126]

[0127] Example 14: Synthesis of Bicyclic Peptide 18

[0128] The method is the same as in Example 1. Compared with Example 5, Example 14 replaces the natural L-configuration cysteine ​​(Cys) in Example 5 with non-natural D-configuration cysteine ​​(D-Cys), whose amino acid sequence is proline (Pro)-asparagine (Asn)-D-cysteine ​​(D-Cys)-arginine (Arg)-glycine (Gly)-aspartic acid (Asp)-tryptophan (Trp)-alanine (Ala).

[0129] 1H NMR (400MHz, DMSO-d6) δ12.31(s,1H),11.39(s,1H),9.20(d,J=99.3Hz,2H),8.33(s,1H),8.16(d,J=14.0Hz,1H),7.99(s,1H),7.66(s,1H),7.59(d ,J=8.1Hz,1H),7.52(dd,J=8.8,4.1Hz,1H),7.31(t,J=7.6Hz,2H),7.26– 7.21(m,1H),7.10(d,J=8.0Hz,2H),7.03–6.97(m,2H),6.77(s,1H),5.10( s,1H),4.61(dd,J=18.1,9.4Hz,2H),4.43(s,2H),4.30(s,1H),4.16(d,J =19.5Hz,2H),3.87(d,J=10.0Hz,2H),3.21–2.96(m,6H),2.88(d,J=11.9H z,2H),2.69–2.57(m,2H),2.20–2.06(m,2H),1.74(dd,J=15.6,6.8Hz,3H ),1.64(s,2H),1.51(s,2H),1.30–1.22(m,1H),1.10(d,J=6.8Hz,3H).(HR ESI-MS)m / zcalculated for C 38 H 52 N 13 O 11 S + (M+H) + :898.3624,found 898.3636.

[0130]

[0131] Example 15: Synthesis of Cy5 fluorescently labeled bicyclic peptide and its binding and endocytosis in αvβ3-positive human breast cancer cells (MDA-MB-231) and αvβ3-negative human liver cancer cells (Huh-7).

[0132]

[0133] Synthesis conditions: a) HATU (2.0 eq), DIPEA (2.2 eq), DMF, rt 2 h; b) TFA / TIS / H2O (95:2.5:2.5), reaction at room temperature for 0.5 h.

[0134] Taking bicyclic peptide 5 as the targeting peptide as an example, following the synthesis method in Example 1, alanine in bicyclic peptide 5 was replaced with lysine, which can serve as a linker, during the synthesis process. The lysine side chain amino protecting group Dde was preferentially removed using an orthogonal protection strategy, yielding bicyclic peptide 5-K protected by asparagine (Asn), arginine (Arg), and aspartic acid (Asp) side chains. An appropriate amount of 5-K was condensed with the fluorescent reagent Cy5 under the synthesis conditions (a, b) to obtain the fluorescently labeled compound 5-K-Cy5. Subsequently, 5-K-Cy5 was prepared into a 5 μM test solution using DMEM medium. After pre-cooling at 4°C, it was added to 96-well plates cultured with MDA-MB-231 and Huh-7 cells, respectively. The cells were then incubated at 4°C in the dark for 1 hour, washed, fixed, and the nuclei were stained with DAPI. The results were observed under a confocal microscope. The cell binding experiment results are as follows: Figure 2 As shown, red fluorescence represents the Cy5 signal, and blue fluorescence represents the signal from the cell nucleus after DAPI staining. 5-K-Cy5 showed significant binding with positive MDA-MB-231 cells, but no binding signal with negative Huh-7 cells. The endocytosis experiment results are as follows... Figure 3 As shown, the fluorescence signal indicates that 5-K-Cy5 can be effectively internalized by αvβ3-expressing positive cells MDA-MB-231 and has good lysosomal co-localization.

[0135] The above experimental results indicate that bicyclic peptide 5 has targeting properties for integrin αvβ3. Substances based on bicyclic peptide 5 and shown in formulas I, II, or III all possess targeting properties and can be used as targeting peptides for αvβ3 receptor-mediated drug delivery.

[0136] Example 16: Affinity activity and selectivity test of cyclic peptide compounds

[0137] The activity and selectivity of cyclic peptide integrin ligands were detected by an ELISA-based integrin-extracellular matrix interaction assay.

[0138] Using c(RGDyK) as the reference reagent, other reagents included ECM protein (extracellular matrix protein), human integrin protein, primary antibody (mouse anti-human antibody), and secondary antibody (peroxidase-labeled anti-mouse IgG), etc. The specific processing is as follows:

[0139] First, the ECM protein was dissolved in carbonate buffer (15mM Na₂CO₃, 35mM NaHCO₃, pH 9.6), and then added to a 96-well plate (100 μl per well) and incubated overnight at 4°C. Then, the plate was washed with PBS-T buffer (phosphate / Tween 20 buffer, 137mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄, 2mM KH₂PO₄, 0.01% Tween 20, pH 7.4; 3 × 200 μl), and blocked at room temperature for 1 hour with TSB buffer (Tris / BSA buffer, 150 μl / well, 20mM Tris-HCl, 150mM NaCl, 1mM CaCl₂, 1mM MgCl₂, 1mM MnCl₂, pH 7.5, 1% BSA). Simultaneously, a series of dilutions of the reference reagent and the test compound were prepared in another 96-well plate, starting from 1 μM and serially diluted 3-fold, for 10 concentration points. After washing the plate three times with PBS-T (200 μl), 50 μl of the corresponding dilution was transferred to each of the BG wells of the plate. 100 μl of TSB solution was added to well A as a blank, and 50 μl of TSB buffer was added to well H. Then, 50 μl of human integrin TSB buffer was added to well HB and incubated at room temperature for 1 h. After washing the plate three times with PBS-T buffer, primary antibody (100 μl per well) was added to the plate. After incubating at room temperature for 1 h, the plate was washed three times with PBS-T buffer. Then, secondary antibody (100 μl per well) was added and incubated at room temperature for 1 h, followed by washing three times with PBS-T buffer. After washing, the substrate SeramunBlau was quickly added to the plate (50 μl per well) and incubated in the dark for 5 minutes. Then add 50 μl of 3M H2SO4 stop solution to each well, and finally use an ELISA reader to detect the absorbance at a wavelength of 450 nm.

[0140] The test results (Table 1) show that the cyclic peptides obtained in most examples have nanomolar affinity for integrin αvβ3 and exhibit high selectivity for integrin αvβ3.

[0141] Table 1: Results of ELISA-based integrin-extracellular matrix interaction assay

[0142]

[0143] (In Table 1, a: 0-10 nM, b: 10-100 nM, c: 100-300 nM, d: 300-500 nM, e: 500-700 nM, nd: not detected)

[0144] Among them, the specific IC values ​​of compounds 5, 6, 9, 10, 11, and 13 are... 50 The test values ​​are shown in Table 2 below:

[0145] Table 2: Data from partial peptide-based ELISA-based integrin-extracellular matrix interaction experiments

[0146]

[0147] The test results (Table 2) show that cyclic peptides 5, 6, 9, 10, 11, and 13 have a sub-nanomolar affinity for integrin αvβ3, but their affinity for integrin αvβ5, αvβ6, αvβ8, and α5β1 is weaker than that for integrin αvβ3. Like other peptides in Table 1, they have both high affinity and selectivity for integrin αvβ3.

[0148] Example 17: Synthesis of peptide-conjugated drugs PDC1 and PDC2

[0149]

[0150] Synthetic conditions:

[0151] a) HATU (2.0eq), DIPEA (2.2eq), DMF, 2h at room temperature;

[0152] b) TFA / TIS / H2O (95:2.5:2.5), 0.5h at room temperature.

[0153] c) 6-azidohexanamido-VA-PABC-MMAE (1.5 eq), CuSO4·5H2O (4.0 eq), sodium ascorbate (8.0 eq). Wherein, 6-azidohexanamido-VA-PABC-MMAE is a cleavable linker-drug complex for antibody-drug conjugates (ADCs), MMAE represents monomethyl auréstatin E, PABC represents p-aminobenzyloxycarbonyl, VA represents valine-alanine, and 6-Azidohexanamido represents 6-azidohexylamide.

[0154] First, taking bicyclic peptide 5 as the targeting peptide as an example, according to the synthesis method of Example 1, alanine in bicyclic peptide 5 was replaced with lysine, which can serve as a linker, during the synthesis process. The lysine side chain amino protecting group Dde was preferentially removed using an orthogonal protection strategy to obtain bicyclic peptide 5-K protected by asparagine (Asn), arginine (Arg), and aspartic acid (Asp) side chains. 30 mg of 5-K was used for the synthesis of PDC1 and PDC2, respectively. Under synthesis conditions (a, b), direct condensation yielded bicyclic peptide 21 containing an alkynyl group. Bicyclic peptide 21 was directly precipitated with diethyl ether, centrifuged, and lyophilized. Under synthesis condition c, it was reacted with 6-azidohexanamido-VA-PABC-MMAE to obtain peptide conjugates PDC1 and PDC2. After purification, 40.2 mg of PDC1 was obtained, with a yield of 79.3%. HR ESI-MS: m / z for C 121 H 188 N 25 O 32 S + (M+H) + :2535.3567, found 2535.3541. PDC2 yielded 35.2 mg, 77.1% yield, HR ESI-MS: m / z for C 112 H 170 N 25 O 24 S + (M+H) + :2281.2566,found 2281.2526.

[0155] Example 18: In vitro cell proliferation inhibition assay of peptide-conjugated drugs (CCK8 assay)

[0156] Human breast cancer cells (MDA-MB-231, 3.0 × 10⁶ m²) were incubated in 96-well cell culture plates. 3 / well) and lung cancer cells (A549 cells, 3.0 × 10⁶) 3After incubating for 24 hours in each well, the culture medium was discarded, and different concentrations of the test drug (5.0 μM, 2.5 μM, 1.0 μM, 333.33 nM, 111.13 nM, 37.04 nM, 12.35 nM, 4.12 nM, 1.37 nM) diluted in the culture medium were added. The cells were then incubated in a 5% CO2 incubator for 72 hours. Following this, the detection reagent was added according to the traditional CCK8 assay protocol, and the cells were incubated in the dark for 3 hours. The OD value of each well was then measured using a microplate reader and compared with the control group. The cell proliferation inhibition results are shown in Table 3. The experimental results show that PDC1 and PDC2 have nanomolar inhibitory effects on both αvβ3-positive cell lines, MDA-MB-231 and A549, and their toxicity is significantly reduced compared to the single loading drug MMAE. Bicyclic peptide 5 alone has no toxic effect on either cell type. These data indicate that bicyclic peptide 5 can be used as a targeting peptide for drug delivery with high safety.

[0157] Table 3: Results of cell proliferation inhibition experiment

[0158]

[0159] Example 19: In vivo tumor inhibition experiment of peptide-conjugated drugs (tumor-bearing mouse sample size n=7, ***p<0.001)

[0160] The experimental mice were 6-week-old nude mice weighing 20-25g. Each mouse received an injection of 1×10⁻⁶ A549 cells into the axilla of its right upper limb. 7 Inject 200 μl. Wait until the tumor grows to 100-150 mm in size. 3 Mice were randomly divided into groups of seven for each group and administered the drug twice daily. Body weight and tumor volume were measured before each administration. The experiment lasted 21 days. A PBS-only injection group served as a negative control, while 1 mg / kg PDC1 and an equivalent dose of MMAE (0.29 mg / kg) were used as control drugs. Results showed that compared to the PBS control group, 1 mg / kg PDC1 significantly inhibited tumor growth (***p<0.001), and there was no significant change in mouse body weight. Figure 4 , Figure 5 and Figure 6 In the MMAE-treated group, mice showed significant weight loss and mortality after four administrations. These results indicate that the PDC drug synthesized based on bicyclic peptide 5 has a significant antitumor effect in vivo and can significantly reduce the toxicity of the single loading drug MMAE. It demonstrates great potential and application value in tumor therapy targeting integrin αvβ3.

[0161] The physicochemical indicators in the above experimental examples are only a portion of the examples, and should not be construed as limiting the specific experimental examples of this invention to these instances and applications. Clearly, the above experimental examples are merely illustrative of the technical solutions of this invention and are not intended to limit the specific experimental methods of this invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of this invention should be included within the scope of protection of the claims of this invention.

Claims

1. A class of cyclic peptides targeting integrin αvβ3, characterized in that, The general structural formula of the cyclic peptide targeting integrin αvβ3 is shown in Formula I, Formula II, or Formula III: In Equation I: R1 group is -H or The R2 group is The R3 group is -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2OH, or -CH(CH3)OH; The R4 group is -CH2COOH, -(CH2)2COOH, -CH2CONH2 or -(CH2)2CONH2; The R5 group is -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -COOH, or... Where X is -CH2-S-indole- or -indole-SC(CH2)2-, and the hydrogen atom on the indole can be replaced by hydroxyl, halogen, halocarbon, alkynyl, alkene, cyano-protected carboxylic acid ester or cyano-protected carboxylamide, and the sulfur atom in X can be oxidized to sulfoxide or sulfone group. In Formula II: R 1’ The radical group is -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3; R 2’ Group is R 3’ The radical group is -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2OH or -CH(CH3)OH; R 4’ The radical group is -CH2COOH, -(CH2)2COOH, -CH2CONH2 or -(CH2)2CONH2; The definition of X in Equation II is the same as the definition of X in Equation I; Y is hydrogen, a substituted halogen, a hydroxyl group, an amino group, a methyl group, an ethyl group, a mercapto group, or a cyano group; In Formula III: R 1” The radicals are -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CONH2, -(CH2)2CONH2, -CH2COOH or -(CH2)2COOH; R 2” The radicals are -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CONH2, -(CH2)2CONH2, -CH2COOH or -(CH2)2COOH; R 3” Group is R 4” The radical group is -H, -CH3, -CH2OH or -CH(CH3)OH; R 5” The radical group is -CH2COOH, -(CH2)2COOH, -CH2CONH2 or -(CH2)2CONH2; The definition of X in Equation III is the same as the definition of X in Equation I; The definition of Y in Equation III is the same as the definition of Y in Equation II.

2. The cyclic peptide targeting integrin αvβ3 according to claim 1, characterized in that, In formula I, X is -indole-S-CH2-; In Formula II, X is -indole-S-CH2-; Y is hydrogen or hydroxyl. In Formula III, X is -indole-S-CH2-; Y is hydrogen or hydroxyl.

3. A cyclic peptide targeting integrin αvβ3 according to claim 1 or 2, characterized in that, In formula I, the R2 group is The R3 group is -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3; The R4 group is -CH2COOH or -(CH2)2COOH; In Formula II: R 2’ Group is R 3’ The radical group is -H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3; R 4’ The functional group is -CH2COOH or -(CH2)2COOH; In Formula III: R 3” Group is R 4” The radical is -H or -CH3; R 5” The group is -CH2COOH or -(CH2)2COOH.

4. The cyclic peptide targeting integrin αvβ3 according to claim 1, characterized in that, The structural formula of the cyclic peptide is shown in any of the following:

5. The use of the cyclic peptide targeting integrin αvβ3 as described in claim 1 in the preparation of drug delivery systems and / or encapsulation of oncology drugs.

6. The application according to claim 5, characterized in that, The tumors include any one of pancreatic cancer, colon cancer, lung cancer, rectal cancer, uterine cancer, cervical cancer, skin cancer, stomach cancer, liver cancer, brain tumor, bladder cancer, and lymphoma.

7. A polypeptide-conjugated drug, characterized in that, The structural formula of the polypeptide-conjugated drug is A. n -LB; A represents the cyclic peptide targeting integrin αvβ3 as described in claim 1; n represents the number of links of the cyclic peptide, where n is any integer from 0 to 5; L represents the linker; B represents the loaded drug.

8. The polypeptide-conjugated drug according to claim 7, characterized in that, B represents an antitumor drug, a molecularly targeted drug, a peptide drug, or an antibody drug. The antitumor drug is selected from one or more of microtubule inhibitors, topoisomerase inhibitors, radionuclides and their ligands, and PD-L1 inhibitors. The molecularly targeted drug is selected from one of imatinib, nilotinib, dasatinib, everolimus, erlotinib, sunitinib, sorafenib, ibrutinib, regorafenib, vemurafenib, and olaparib. The peptide drug is selected from one of p53 activating peptide, melitrix venom peptide, and scorpion venom peptide. The antibody drug is selected from one of rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, and nivolumab.

9. A polypeptide-conjugated drug, characterized in that, The structural formula of the polypeptide-conjugated drug is any one of the following:

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