Cyclopeptide with stable serum as well as preparation and application thereof

Abstract

The invention discloses a serum stable cyclic peptide which has a structure as shown in a general formula I, the invention also discloses a preparation method and application of the cyclic peptide. Researches show that the cyclopeptide serum is high in stability, and the half-life period is longer than 24 h. The cyclopeptide disclosed by the invention can effectively inhibit proliferation of B16F10 melanoma cells, and the in-vitro anti-tumor cell proliferation inhibition activity is stably maintained along with the prolongation of incubation time. The cyclopeptide can also effectively inhibit tumor proliferation of melanoma cell tumor-bearing mice, and can be applied to preparation of anti-melanoma drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a serum-stable cyclic peptide, synthesis and application thereof. BACKGROUND

[0002] Melanoma is a highly malignant tumor derived from melanocytes, which is metastatic and invasive, and is the most lethal and invasive skin cancer. In the past decade, immune checkpoint inhibitors and targeted therapy drugs such as BRAF and MEK inhibitors have changed the treatment landscape of melanoma, although there are still many limitations in these therapies, such as insufficient response in patients treated with immune checkpoint inhibitors, and drug resistance to small molecule targeted drugs in patients treated with small molecule BRAF / MEK inhibitors. Therefore, it is urgent to develop new anti-melanoma drugs with new mechanisms of action.

[0003] Oncolytic peptide is a cationic amphiphilic short peptide that exerts an anti-tumor effect by directly destroying tumor cell membranes and inducing immunogenic cell death. Oncolytic peptide provides a new direction for traditional melanoma tumor treatment, and its effect is very rapid, killing tumor cells through non-specific destruction, and is not prone to drug resistance. LTX-315 is the most rapidly developed representative drug in oncolytic peptides, which can effectively inhibit tumor growth in in vitro and in vivo experiments, and has entered phase II clinical trials for the treatment of melanoma. However, studies have found that LTX-315, as a linear peptide, has a short half-life (<4 hours) and is easily degraded by proteases, which limits the wide application of LTX-315. SUMMARY

[0004] The purpose of the present application is to provide a cyclic peptide.

[0005] A cyclic peptide having the structure described in general formula (I):

[0006]

[0007] In formula (1):

[0008] A1~A9 are mutually independent amino acid residues; as a preference, A1~A9 are independently from lysine, tryptophan, diphenylalanine, aspartic acid, glutamic acid, ornithine, 2,4-diaminobutyric acid, arginine, histidine, serine, serine, asparagine, glutamine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine;

[0009] and at least one residue in A1~A9 has a carboxyl side chain, and at least one residue has an amino side chain; as a preference, the carboxyl side chain or the amino side chain contains C1~C5 alkylene segments;

[0010] R1 and R2 are independently selected from amino, H, and -NH-C1~C6 alkyl groups; the hydrogen atoms or methylene groups in the alkyl group can be further independently substituted by one or more of hydroxyl, amino, CO, -C(O)-NH2, and carboxyl groups; preferably, the hydrogen atoms in the alkyl group can be further independently substituted by one or more of hydroxyl, amino, -C(O)-NH2, and carboxyl groups; the methylene groups in the alkyl group can be further substituted by CO, etc.

[0011] The dashed arc " The Cy1 ring is formed by the cyclization of the carboxyl and amino groups on the side chains of A1 to A9 via amide bonds; the Cy1 ring can be further substituted with R3, where R3 is selected from H, -(CH2)n1-C(O)-NH-(CH2)n2-R 33 n1 and n2 are each independently selected from 1, 2, 3, 4, 5, R. 33 Selected from amino and C6-C10 aryl groups.

[0012] Furthermore, the -A1-A2-A3-A4-A5-A6-A7-A8-A9- have the structure shown in formula (II):

[0013] -C1-C2-X1-X2-C3-C4-X3-X4-C5-(II)

[0014] The connection position of the dotted arc can be independently selected from C1 and C3, C2 and C4, X1 and X3, X2 and X4, X2 and X3, C3 and C5, and C2 and C3.

[0015] Furthermore, C1-C5 can be independently selected from lysine (Lys), or specified α-amino acids containing carboxyl side chains or α-amino acids containing amino side chains;

[0016] X1-X2 can be independently selected from tryptophan (Trp) or a specified α-amino acid containing a carboxyl side chain;

[0017] X3 can be independently selected from tryptophan (Trp) or a specified α-amino acid containing an amino side chain;

[0018] X4 can be independently selected from diphenylalanine (Dip) or a specified α-amino acid containing an amino side chain.

[0019] The “α-amino acid containing a carboxyl side chain” is selected from: aspartic acid (Asp) and glutamic acid (Glu);

[0020] The “α-amino acid containing an amino side chain” is selected from: lysine (Lys), ornithine (Orn), and 2,4-diaminobutyric acid (Dab).

[0021] Further, R1may be independently selected from hydrogen;

[0022] R2may be independently selected from amino, hydroxyl, cysteinamide, asparaginamide, lysinamide.

[0023] Further, R3is selected from:

[0024] .

[0025] In the peptide chain, there is a linking structure composed of amino acid side chains, which is formed by the amide condensation or ugi reaction of the side chain carboxyl of the specified α-amino acid containing carboxyl side chain and the side chain amino of the specified α-amino acid containing amino side chain, and the connection position can be independently selected from C1 and C3, C2 and C4, X1 and X3, X2 and X4, C3 and C5, C2 and C3, and the structure is -CH2CONR3(CH2)4- or -(CH2)4NR3COCH2- or -CH2CH2CONR3(CH2)4- or -(CH2)4NR3COCH2CH2- or -CH2CONR3(CH2)3- or -(CH2)3NR3COCH2- or -CH2CONR3(CH2)2- or -(CH2)2NR3COCH2- or -CH2CH2CONR3(CH2)3- or -(CH2)3NR3COCH2CH2- or -CH2CH2CONR3(CH2)2- or -(CH2)2NR3COCH2CH2-.

[0026] As preferred, the above cyclic peptide can be selected from, but not limited to, any of the following structures:

[0027] ; the letter combinations in the above formula are all abbreviations corresponding to amino acid residues; in the above formula or The amino acid residues at both ends are the amino acid residues connected at the corresponding end; taking I-A as an example, The amino acid residues at both ends are the amino acid residues connected at the corresponding end; taking I-A as an example,

[0028] Further, the cyclic peptide has a structure shown in one of the following:

[0029] .

[0030] The application further provides a preparation method of the cyclic peptide.

[0031] (1) loading a target polypeptide on a solid phase carrier by a solid phase synthesis method to obtain compound 1, wherein Rc and Rn are carboxyl protecting groups and amino protecting groups respectively, and the amino groups or / and carboxyl groups not involved in the reaction in steps (1)-(3) of A1-A9 still have corresponding amino protecting groups or / and carboxyl protecting groups:

[0032]

[0033] (2) removing the carboxyl protecting group Rc and the amino protecting group Rn on COORc and NHRn to obtain compound 2:

[0034]

[0035] (3) intramolecular cyclization of compound 2, and when R3 is not H, reacting again with a precursor compound of R3 to obtain compound 3:

[0036]

[0037] (4) cleaving the cyclic peptide structure by using a cleavage solution, and simultaneously removing the protecting groups of the amino groups or / and carboxyl groups of A1-A9 to obtain the cyclic peptide (I):

[0038] .

[0039] Further, the amino acid residue structure in which the COORc is located is:

[0040] or

[0041] The amino acid residue structure in which the RnHN is located is:

[0042] .

[0043] Further, another object of the application is to provide a preparation method of the cyclic peptide, which is achieved by the following preparation steps:

[0044] (1) Preparation of polypeptide resin: the target polypeptide is loaded on Rink-Amide resin by standard solid-phase synthesis method to obtain compound 1.

[0045] (2) Preparation of compound 2: compound 1 is subjected to removal of Alloc and Allyl protecting groups under catalysis of tetraphenylphosphonium and with dimethylamine borane complex as a reducing agent, and dichloromethane (DCM) as a solvent to obtain compound 2.

[0046] (3) Preparation of compound 3 or compound 4: intramolecular amide condensation of compound 2 is carried out with condensing agent 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and acid binding agent N,N-diisopropylethylamine (DIEA) in N,N-dimethylformamide (DMF) as a solvent to obtain compound 3; intramolecular macrocyclization of compound 2 is carried out with polyformaldehyde and isonitrile of different structures in dichloromethane (DCM) and trifluoroethanol (TFE) as solvents to obtain compound 4.

[0047] (4) Preparation of compound 5 or compound 6: compound 3 or compound 4 is cut off with trifluoroacetic acid to obtain crude compound 5 or compound 6, and the final product is obtained after purification by preparative liquid chromatography.

[0048]

[0049]

[0050] The application also provides a use for preparing a medicine, i.e., the use of the compound in any of the above technical solutions in the preparation of an anti-melanoma medicine.

[0051] Compared with the prior art, the application has the beneficial effects that a cyclic peptide is designed and synthesized by using a cyclization strategy, which retains the anti-proliferative activity on melanoma and has significantly improved serum stability than LTX-315. The serum stability experiment and the in-vitro anti-proliferative inhibition experiment show that the serum half-life of the cyclic peptide is more than 10 times that of LTX-315, and the cyclic peptide has significantly higher anti-proliferative inhibition activity than LTX-315 as the incubation time with melanoma cells is prolonged. The application provides a more stable cyclic peptide medicine for the treatment of melanoma.

[0052] Research shows that the cyclic peptide has high serum stability and a half-life of more than 24 h. The cyclic peptide effectively inhibits the proliferation of B16F10 melanoma cells, and the in-vitro anti-tumor cell proliferation inhibition activity is stably maintained as the incubation time is prolonged. The cyclic peptide can also effectively inhibit the tumor proliferation of melanoma cell-bearing mice and can be applied to the preparation of an anti-melanoma medicine. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1. Degradation curve in in vitro serum stability experiment, the horizontal axis is the incubation time of polypeptide with serum, and the vertical axis is the percentage of remaining polypeptide measured at the corresponding time of incubation;

[0054] Figure 2 . Time-effect curve of polypeptide inhibiting tumor cell proliferation, the horizontal axis is the incubation time of polypeptide with tumor cells, and the vertical axis is the inhibition rate of tumor cell proliferation when the concentration of polypeptide is 62.5 μM;

[0055] Figure 3 . Time-effect curve of polypeptide inhibiting tumor cell proliferation, the horizontal axis is the incubation time of polypeptide with tumor cells, and the vertical axis is the half maximal inhibitory concentration IC 50 value measured at the corresponding time of incubation;

[0056] Figure 4 . Body weight change of tumor-bearing C57B / 6J mice in each group after administration;

[0057] Figure 5 . Tumor volume change of tumor-bearing C57B / 6J mice in each group after administration;

[0058] Figure 6 . Tumor weight of each group at the end of treatment;

[0059] Figure 7 . Photographs of tumor tissues in each administration group, each row represents tumor tissues of different groups, and each row is arranged from left to right according to the volume from large to small. DETAILED DESCRIPTION

[0060] The present application will be further described in detail below in conjunction with the accompanying drawings and examples, but the embodiments of the present application are not limited thereto, and various substitutions and modifications can be made according to ordinary technical knowledge and conventional means in the art without departing from the technical idea of the present application, which should be included in the scope of the present application.

[0061] Unless otherwise specified in the examples, the techniques or conditions described in the literature in the art or according to the product manual are used. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased from a regular channel.

[0062] The molecular weight and purity of the polypeptides were determined by high performance liquid chromatography and high resolution mass spectrometry. LC-HRMS: Agilent 1290-HPLC-6224 liquid chromatography-mass spectrometer. HPLC: Agilent 1220 Infinity LC. Column chromatography was performed using 200-300 mesh silica gel. The chromatographic column was selected from Aglient ZORBAX SB-C18 5 pm 9.5 x 250 mm chromatographic column and Aglient Eclipse Plus C18 5 pm 9.5 x 150 mm chromatographic column.

[0063] Example 1. Synthesis of 1-6

[0064] In this example, all target polypeptides were prepared using 9-fluorenylmethoxycarbonyl-based solid-phase polypeptide synthesis technology (Fmoc-SPPS). Fmoc-protected amino acids were used for one-by-one condensation (including condensation, deprotection, elution cycle, deprotection of side chain to lactam ring), cleavage, filtration, concentration, precipitation, filtration and drying to obtain crude cyclic peptide, and then purified, freeze-dried to obtain finished product. Unless otherwise specified, the present application selects Rink Amide resin (degree of substitution 0.36 mmol / g) to synthesize polypeptides containing amide terminals, amino acids are L-type amino acids, and the scale of synthesized polypeptides is generally 0.36 mmol, and the reaction temperature is room temperature (25-30°C).

[0065] 1 g of Rink Amide resin was weighed into a solid-phase reaction column and bubbled with nitrogen. 5-6 mL of DMF solvent was added to soak and swell the resin at room temperature for 1 h, and the resin was pre-activated. After 1 h, the solvent was removed by filtration. 6 mL of 20% piperidine-DMF solution was added, bubbled with nitrogen for 1 h, and the solvent was removed by vacuum filtration. 6 mL of DMF was added to wash five times to remove the Fmoc protecting group of the Rink Amide resin, exposing the free amino group for subsequent condensation reaction.

[0066] The synthesis steps of 1-6 are as follows. The sequence of 1-6 is H-Asp-Lys-Trp-Trp-Lys-Lys-Trp-Dip-Lys-NH2, and the amino acids are sequentially condensed from the C-terminal to the N-terminal to obtain the target peptide chain. 1 g of Rink Amide resin (degree of substitution 0.36 mmol / g) was weighed, swelled and activated, and the Fmoc protecting group was removed.

[0067] Synthesis of 1-6-1

[0068]

[0069] Condensation of the first amino acid at the C-terminus: 1 g of Rink Amide resin, swelled and deprotected from Fmoc protection, was added to a solid phase reaction column, followed by pre-activation of Fmoc-Lys(Boc)-OH (797 mg, 1.7 mmol), HBTU (580 mg, 1.53 mmol), DIEA (533 μL, 3.06 mmol) in 6 mL of dry DMF for 3 min at room temperature, and addition of the above-mentioned solid phase reaction column containing the deprotected Rink Amide resin to the reaction column, followed by nitrogen bubbling for 2.5 h at room temperature. The solvent was removed by suction filtration under reduced pressure, and 6 mL of DMF were added for washing four times.

[0070] End-capping of the resin: 6 mL of acetic anhydride and 4 mL of pyridine were added to the solid phase reaction column, and bubbling was performed for 30 min, followed by washing with 6 mL of DMF four times, 6 mL of dry methanol twice, and 6 mL of DCM twice. Fmoc-Lys(Boc)-Rink Amide resin, i.e. 1-6-1, was obtained.

[0071] Monitoring: a small amount (about 10 mg) of the resin was taken for monitoring the reaction, 1 mL of cleavage solution (TFA / TIPS / H2O = 95 / 2.5 / 2.5) was added, and the reaction was shaken for 1 h, the filtrate was collected by filtration, and TFA was removed by evaporation under reduced pressure. Chromatographic methanol was added for dilution, and LC-HRMS was used to detect the amino acid Fmoc-Lys-NH2 cleaved from the resin. MS (ESI + ): m / z found: 368.1975, calculated: 368.1974 [M+H] + .

[0072] Synthesis of 1-6-2

[0073]

[0074] Deprotection: 1-6-1 was added to a solid phase reaction column, 6 mL of a 20% solution of piperidine in DMF was added, and nitrogen bubbling was performed for 7 min, followed by removal of the solvent by suction filtration under reduced pressure; the above-mentioned operation was repeated once, and 6 mL of DMF was added for washing five times.

[0075] Condensation: Fmoc-Dip-OH (204 mg, 0.44 mmol), HBTU (167 mg, 0.44 mmol), DIEA (153 μL, 0.88 mmol) were dissolved in 6 mL dry DMF, pre-activated for 3 min at room temperature, added to a solid phase reaction column containing the Fmoc-protected Rink Amide resin, and reacted under nitrogen bubbling for 2.5 h at room temperature. The solvent was removed by suction filtration under reduced pressure, and the resin was washed four times with 6 mL DMF. A small amount of resin (about 10 mg) was taken, washed twice with methanol, and 0.5 mL ninhydrin color development solution (preparation: 0.03 g hydrindantin and 300 μL glacial acetic acid in 10 mL n-butanol) was added, and heated to 120 °C for 3 min. If the resin did not change color, the reaction was complete, and Fmoc-Dip-Lys(Boc)-Rink Amide resin was obtained. If the resin turned purple, the resin still had unreacted amino groups, and the above coupling procedure was repeated.

[0076] Monitoring: A small amount (about 10 mg) of resin was taken for monitoring the reaction, 1 mL cleavage solution (TFA / TIPS / H2O = 95 / 2.5 / 2.5) was added to the resin, shaken for 1 h, the filtrate was collected by filtration, and the TFA was removed by evaporation under reduced pressure. The residue was diluted with chromatographic methanol, and the dipeptide Fmoc-Dip-Lys-NH2 cleaved from the resin was detected by LC-HRMS. MS (ESI + ): m / z found: 591.2966, calculated: 591.2971 [M+H] + .

[0077] Synthesis of 1-6-3

[0078]

[0079] The synthesis steps of compound 1-6-2 were followed, 1-6-2 was added to the solid phase reaction column, Fmoc-Trp(Boc)-OH (348 mg, 0.66 mmol) was used instead of Fmoc-Dip-OH (204 mg, 0.44 mmol), and the other operations were the same. A small amount (about 10 mg) of resin was taken for monitoring the reaction, 1 mL cleavage solution (TFA / TIPS / H2O = 95 / 2.5 / 2.5) was added to the resin, shaken for 1 h, the filtrate was collected by filtration, and the TFA was removed by evaporation under reduced pressure. The residue was diluted with chromatographic methanol, and the tripeptide Fmoc-Trp-Dip-Lys-NH2 cleaved from the resin was detected by LC-HRMS. MS (ESI + ): m / z found: 777.3764, calculated: 777.3764 [M+H] + .

[0080] Synthesis of 1-6-4

[0081]

[0082] For the synthesis of 1-6-2, 1-6-3 was added to a solid-phase reaction column, and Fmoc-Dip-OH (204 mg, 0.44 mmol) was replaced with Fmoc-Lys(Boc)-OH (309 mg, 0.66 mmol), with other procedures remaining the same. A small amount (approximately 10 mg) of resin was used to monitor the reaction. 1 mL of lysis buffer (TFA / TIPS / H2O = 95 / 2.5 / 2.5) was added to the resin, and the mixture was shaken for 1 h. The filtrate was collected by filtration, TFA was removed by evaporation under reduced pressure, and the solution was diluted with chromatographic methanol. The tetrapeptide Fmoc-Lys-Trp-Dip-Lys-NH2 cleaved from the resin was detected using LC-HRMS (ESI). + m / z measured value: 905.4719, calculated value: 905.4714 [M+H] + .

[0083] Synthesis of 1-6-5

[0084]

[0085] For the synthesis of 1-6-2, 1-6-4 was added to a solid-phase reaction column, and Fmoc-Dip-OH (204 mg, 0.44 mmol) was replaced with Fmoc-Lys(Alloc)-OH (199 mg, 0.66 mmol), with other procedures remaining the same. A small amount (approximately 10 mg) of resin was used to monitor the reaction. 1 mL of lysis buffer (TFA / TIPS / H2O = 95 / 2.5 / 2.5) was added to the resin, and the mixture was shaken for 1 h. The filtrate was collected by filtration, TFA was removed by evaporation under reduced pressure, and the solution was diluted with chromatographic methanol. The pentapeptide Fmoc-Lys(Alloc)-Lys-Trp-Dip-Lys-NH2 cleaved from the resin was detected using LC-HRMS (ESI). + m / z measured value: 1117.5879, calculated value: 1117.5875 [M+H] + .

[0086] Synthesis of 1-6-6

[0087]

[0088] Synthesis as for 1-6-2, adding 1-6-5 to the solid phase reaction column, replacing Fmoc-Dip-OH (204 mg, 0.44 mmol) by Fmoc-Trp(Boc)-OH (348 mg, 0.66 mmol), and otherwise identical. Monitor the reaction by taking a small amount (about 10 mg) of resin: add 1 mL of cleavage solution (TFA / TIPS / H20 = 95 / 2.5 / 2.5) to the resin, shake the reaction for 1 h, collect the filtrate by filtration, evaporate the TFA under reduced pressure, dilute with chromatographic methanol, and use LC-HRMS to detect the heptapeptide Fmoc-Trp-Trp-Lys(Alloc)-Lys-Trp-Dip-Lys-NH2 cleaved from the resin. MS (ESI + ): m / z found: 1489.7457, calc. 1489.7461 [M+H] + .

[0089] Synthesis of 1-6-7

[0090]

[0091] Synthesis as for 1-6-2, adding 1-6-5 to the solid phase reaction column, replacing Fmoc-Dip-OH (204 mg, 0.44 mmol) by Fmoc-Trp(Boc)-OH (348 mg, 0.66 mmol), and otherwise identical. Monitor the reaction by taking a small amount (about 10 mg) of resin: add 1 mL of cleavage solution (TFA / TIPS / H20 = 95 / 2.5 / 2.5) to the resin, shake the reaction for 1 h, collect the filtrate by filtration, evaporate the TFA under reduced pressure, dilute with chromatographic methanol, and use LC-HRMS to detect the heptapeptide Fmoc-Trp-Trp-Lys(Alloc)-Lys-Trp-Dip-Lys-NH2 cleaved from the resin. MS (ESI + ): m / z found: 1489.7457, calc. 1489.7461 [M+H] + .

[0092] Synthesis of 1-6-8

[0093]

[0094] For the synthesis of 1-6-2, 1-6-7 was added to a solid-phase reaction column, and Fmoc-Dip-OH (204 mg, 0.44 mmol) was replaced with Fmoc-Lys(Boc)-OH (309 mg, 0.66 mmol), with other procedures remaining the same. A small amount (approximately 10 mg) of resin was used to monitor the reaction: 1 mL of lysis buffer (TFA / TIPS / H2O = 95 / 2.5 / 2.5) was added to the resin, the mixture was shaken for 1 h, the filtrate was collected by filtration, TFA was removed by evaporation under reduced pressure, and the solution was diluted with chromatographic methanol. The octapeptide Fmoc-Lys-Trp-Trp-Lys(Alloc)-Lys-Trp-Dip-Lys-NH2 cleaved from the resin was detected using LC-HRMS (ESI). + m / z measured value: 1617.8415, calculated value: 1617.8411 [M+H] + .

[0095] Synthesis of 1-6-9

[0096]

[0097] For the synthesis of 1-6-2, 1-6-8 was added to a solid-phase reaction column, and Fmoc-Dip-OH (204 mg, 0.44 mmol) was replaced with Fmoc-Asp(OAll)-OH (174 mg, 0.66 mmol), with other procedures remaining the same. A small amount (approximately 10 mg) of resin was used to monitor the reaction: 1 mL of lysis buffer (TFA / TIPS / H2O = 95 / 2.5 / 2.5) was added to the resin, the mixture was shaken for 1 h, the filtrate was collected, TFA was removed by evaporation under reduced pressure, and the solution was diluted with chromatographic methanol. The nonapeptide Fmoc-Asp(OAll)-Lys-Trp-Trp-Lys(Alloc)-Lys-Trp-Dip-Lys-NH2 cleaved from the resin was detected using LC-HRMS (ESI). + m / z measured value: 1772.8996, calculated value: 1772.8993 [M+H] + .

[0098] Synthesis of 1-6-10

[0099]

[0100] Alloc removal and Allyl protection: 1-6-9 was added to a solid-phase reaction column. The 1-6-9 resin was washed three times with 6 ml of DCM, dried under vacuum, and then transferred to a three-necked flask. Under nitrogen protection, 10 mL of anhydrous DCM was added, followed by the addition of DMAB. (85 mg, 0.22 mmol), magnetic stirring, Pd(PPh3)4 (55 mg, 0.022 mmol) was added, the reaction was carried out for 30 min, the solvent was removed by suction filtration, the resin was washed with DCM three times, the above operation was repeated, DMAB and Pd(PPh3)4 were continuously added under nitrogen protection, the solvent was removed by suction filtration, the resin was washed with DCM three times, the resin was washed with 0.2% TFA in DCM twice, DCM three times, 5% DIEA in DCM three times, DCM three times, to obtain Fmoc-Asp-Lys(Boc)-Trp(Boc)-Trp(Boc)-Lys-Lys(Boc)-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin, ninhydrin color developing solution was used for monitoring, the resin turned purple, indicating that the Alloc protecting group had been removed, and the free amino group of the peptide resin was exposed.

[0101] Monitoring: a small amount (about 10 mg) of resin was taken for reaction monitoring: 1 mL of lysis solution (TFA / TIPS / H2O = 95 / 2.5 / 2.5) was added to the resin, the reaction was shaken for 1 h, the filtrate was collected by filtration, TFA was removed by evaporation under reduced pressure, chromatographic methanol was added for dilution, and LC-HRMS was used to detect the polypeptide Fmoc-Asp-Lys-Trp-Trp-Lys-Lys-Trp-Dip-Lys-NH2 cut from the resin. MS (ESI + ): m / z found: 1648.8654, calculated: 1648.8649 [M+H] + .

[0102] Synthesis of 1-6-11

[0103]

[0104] Solid phase cyclization: 1-6-10 resin was added to a solid phase reaction tube, PyBOP (benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, 194 mg, 0.44 mmol), DIEA (N,N-diisopropylethylamine, 153 μL, 0.44 mmol), 6 mL DMF, nitrogen bubbling reaction for 12 h. The solvent was removed by suction filtration under reduced pressure, washed with DMF four times, and DCM three times to obtain Fmoc-cyclo[Asp-Lys(Boc)-Trp(Boc)-Trp(Boc)-Lys]-Lys(Boc)-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin.

[0105] Monitoring: Take a small amount (about 10 mg) of resin to monitor the reaction, add 1 mL of lysis solution (TFA / TIPS / H2O = 95 / 2.5 / 2.5) to the resin, shake the reaction for 1 h, collect the filtrate by filtration, evaporate the TFA under reduced pressure, dilute with chromatography methanol, and use LC-HRMS to detect the polypeptide Fmoc-cyclo[Asp-Lys-Trp-Trp-Lys]-Lys-Trp-Dip-Lys-NH2 cut from the resin. MS (ESI + ): m / z found: 1630.8360, calculated: 1630.8357 [M+H] + .

[0106] Synthesis of 1-6

[0107]

[0108] Deprotection: Add 6 mL of 20% piperidine in DMF, bubble nitrogen into the reaction for 7 min, remove the solvent by filtration under reduced pressure; repeat the above operation once, and wash with 6 mL of DMF for five times. Wash with DCM for three times, and dry the resin in vacuum to obtain cyclo[Asp-Lys(Boc)-Trp(Boc)-Trp(Boc)-Lys]-Lys(Boc)-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin.

[0109] Peptide chain cleavage: 1 g of dry cyclo[Asp-Lys(Boc)-Trp(Boc)-Trp(Boc)-Lys]-Lys(Boc)-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin is added into a three-necked flask, 8 mL of lysis solution (TFA / TIPS / H2O = 95 / 2.5 / 2.5) is added, and the reaction is stirred at room temperature for 2.5 h. The filtrate is collected by filtration, the resin is washed with a small amount of TFA and DCM, the filtrate is collected, the TFA is removed by rotary evaporation under reduced pressure, 20 mL of ice ethyl ether is added at 0°C to precipitate the polypeptide, the supernatant is removed by filtration or centrifugation, and the polypeptide is washed with ice ethyl ether for three times. The crude product 1-6 is obtained by drying under reduced pressure, and the polypeptide sequence is cyclo[Asp-Lys-Trp-Trp-Lys]-Lys-Trp-Dip-Lys-NH2. Polypeptide purification: use Aglient ZORBAX SB-C18 5 µm 9.5 × 250mm chromatographic column, 0.1% trifluoroacetic acid aqueous solution and methanol as the mobile phase, gradient elution, and the elution ratio is as follows:

[0110]

[0111] The target peak fraction was collected. (Retention time 9.704 min) Concentration and lyophilization gave 27 mg of pure product with a purity of 98.3% by HPLC, a yield of 7.7%, MS (ESI + ): m / z found: 1408.7680, calcd: 1408.7677.

[0112] Example 2

[0113] Synthesis of 1-7: (In the following examples, Fmoc-AA-OH corresponds to the structure of the product of a plurality of amino acids, not a specific amino acid structure, such as for Example 2, Fmoc-AA-OH corresponds to Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OAll)-OH, Fmoc-Lys(Boc)-OH, etc.)

[0114]

[0115] Reference Example 1, sequentially coupling amino acids on a solid phase resin, removing the side chain protecting group, cyclizing after cutting the polypeptide, to synthesize 1-7.

[0116] Synthesis of 1-7-9:

[0117] 1 g of Rink Amide resin was weighed and placed in a solid phase reaction column. After swelling and Fmoc deprotection according to Reference Example 1, Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OAll)-OH, Fmoc-Lys(Boc)-OH were sequentially coupled by condensation / deprotection cycles. During the sequential coupling process, ninhydrin was used to monitor whether the reaction was completely coupled. Fmoc-Lys(Boc)-Asp(OAll)-Trp(Boc)-Trp(Boc)-Lys(Boc)-Lys(Alloc)-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin was obtained. LC-HRMS was used to detect the nonapeptide Fmoc-Lys-Asp(OAll)-Trp-Trp-Lys-Lys(Alloc)-Trp-Dip-Lys-NH2 cut from the resin. MS (ESI +LC-MS (Method 1): m / z found: 1772.8988, calculated: 1772.8993 [M+H] + .

[0118] Synthesis of 1-7:

[0119] Reference Example 1, resin 1-7-9 was treated with DMAB and Pd(PPh3)4to remove Alloc and Allyl groups, exposing the side chain amino group of Lys and the side chain carboxyl group of Asp, and then subjected to solid phase cyclization on the resin using PyBOP as the condensing agent. The N-terminal Fmoc protecting group was removed using 20% piperidine in DMF, and the cleavage solution was cut to give crude 1-7, which was purified by preparative HPLC using the same conditions as in Reference Example 1. The fractions containing the desired product were collected (retention time 9.785 minutes). The product was concentrated and lyophilized to give 80 mg of pure product in 26.0% yield with a purity of 95.7% and MS (ESI + ): m / z found: 1408.7682, calculated: 1408.7677 [M+H] + .

[0120] Example 3

[0121] Synthesis of 1-8:

[0122]

[0123] Reference Example 1, resin 1-7-9 was treated with DMAB and Pd(PPh3)4to remove Alloc and Allyl groups, exposing the side chain amino group of Lys and the side chain carboxyl group of Asp, and then subjected to solid phase cyclization on the resin using PyBOP as the condensing agent. The N-terminal Fmoc protecting group was removed using 20% piperidine in DMF, and the cleavage solution was cut to give crude 1-7, which was purified by preparative HPLC using the same conditions as in Reference Example 1. The fractions containing the desired product were collected (retention time 9.785 minutes). The product was concentrated and lyophilized to give 80 mg of pure product in 26.0% yield with a purity of 95.7% and MS (ESI

[0124] Synthesis of 1-8-9:

[0125] Take 1 g Rink Amide resin, placed in a solid phase reaction column, after swelling, deprotection of Fmoc protection according to Example 1, through the cycle of condensation / deprotection, Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OAll)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH are sequentially coupled, and the reaction is monitored by ninhydrin to determine whether the coupling is complete; Fmoc-Lys(Boc)-Lys(Boc)-Asp(OAll)-Trp(Boc)-Lys(Boc) Lys(Boc)-Lys(Alloc)-Dip-Lys(Boc)-Rink Amide resin is obtained. The nonapeptide Fmoc-Lys-Lys-Asp(OAll)-Trp-Lys-Lys-Lys(Alloc)-Dip-Lys-NH2 cut from the resin is detected by LC-HRMS. MS (ESI + ): m / z found: 1656.9307, calculated: 1656.9306 [M+H] + .

[0126] Synthesis of 1-8:

[0127] According to Example 1, the resin of 1-8-9 is removed with DMAB and Pd(PPh3)4 to expose the side chain amino group of Lys and the side chain carboxyl group of Asp. PyBOP is used as a condensing agent for solid phase cyclization on the resin. A 20% piperidine solution in DMF is used to remove the N-terminal Fmoc protecting group. The cleavage solution is cut to obtain crude 1-8, which is purified by preparative HPLC. The stationary phase, mobile phase and elution ratio are as described in Example 1. The target peak fraction is collected. (Retention time 7.464 minutes) Concentrate and lyophilize to obtain 50.2 mg of pure product, with a yield of 17.6%, a purity of 95.8%, and MS (ESI + ): m / z found: 1292.7994, calculated: 1292.7990 [M+H] + .

[0128] Example 4

[0129] Synthesis of 1-9:

[0130]

[0131] According to Example 1, amino acids are sequentially coupled on the solid phase resin, the side chain protecting groups are removed, and the polypeptide is cleaved after cyclization to synthesize 1-9.

[0132] Synthesis of 1-9-9:

[0133] Take 1 g Rink Amide resin, place it in a solid phase reaction column, swell, remove Fmoc protection according to Example 1, then sequentially couple Fmoc-Lys(Alloc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OAll)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH by condensation / deprotection cycles, and monitor the reaction by ninhydrin to ensure complete coupling. Fmoc-Lys(Boc)-Lys(Boc)-Trp(Boc)-Trp(Boc)-Asp(OAll)-Lys(Boc)-Trp(Boc)-Dip-Lys(Alloc)-Rink Amide resin is obtained. The nonapeptide Fmoc-Lys-Lys-Trp-Trp-Asp(OAll)-Lys-Trp-Dip-Lys(Alloc)-NH2 cleaved from the resin is detected by LC-HRMS. MS (ESI + ): m / z found: 1772.8984, calculated: 1772.8988 [M+H] + .

[0134] Synthesis of 1-9:

[0135] According to Example 1, the resin of 1-9-9 is removed from the Alloc and Allyl groups using DMAB and Pd(PPh3)4, exposing the side chain amino group of Lys and the side chain carboxyl group of Asp. PyBOP is used as a condensing agent for solid phase cyclization on the resin. A 20% piperidine solution in DMF is used to remove the N-terminal Fmoc protecting group. The cleavage solution is cut to obtain crude 1-9, which is purified by preparative HPLC. The stationary phase, mobile phase, and elution ratio are as described in Example 1. The target peak fraction is collected. (Retention time 9.910 minutes) Concentrate and lyophilize to obtain 70.3 mg of pure product, with a yield of 22.4%, a purity of 95.3%, and MS (ESI + ): m / z found: 1408.7674, calculated: 1408.7677 [M+H] + .

[0136] Example 5

[0137] Synthesis of 1-10:

[0138]

[0139] Referring to Example 1, amino acids were sequentially coupled onto a solid resin, side-chain protecting groups were removed, and the polypeptide was cyclized and cleaved to synthesize 1-10.

[0140] Synthesis of 1-10-9:

[0141] Weigh 1 g of Rink Amide resin was placed in a solid-phase reaction column and swollen and deprotected according to Example 1. Then, Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OAll)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Alloc)-OH, and Fmoc-Lys(Boc)-OH were coupled sequentially through condensation / deprotection cycles. During the sequential coupling process, the completeness of the coupling reaction was monitored by ninhydrin. The resulting resin was Fmoc-Lys(Boc)-Lys(Alloc)-Trp(Boc)-Trp(Boc)-Lys(Boc)-Asp(OAll)-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin. The nonapeptide Fmoc-Lys-Lys(Alloc)-Trp-Trp-Lys-Asp(OAll)-Trp-Dip-Lys-NH2 cleaved from the resin was detected by LC-HRMS (ESI). + m / z measured value: 1772.8993, calculated value: 1772.8988 [M+H] + Synthesis of 1-10:

[0142] Referring to Example 1, the 1-10-9 resin was treated with DMAB and Pd(PPh3)4 to remove the Alloc and Allyl groups, exposing the side-chain amino groups of Lys and the side-chain carboxyl groups of Asp. PyBOP was used as a condensing agent for solid-phase cyclization on the resin. The N-terminal Fmoc protecting group was removed with a 20% piperidine DMF solution. The lysis buffer was used to obtain crude 1-10 product, which was then purified by HPLC. The stationary phase, mobile phase, and elution ratio were the same as in Example 1. The target peak fraction was collected. (Retention time 10.371 min) The product was concentrated and lyophilized to obtain 11.8 mg of pure product, yield 3.8%, purity 95.5%. MS (ESI) was then analyzed. + m / z measured value: 1408.7682, calculated value: 1408.7677 [M+H] + .

[0143] Example 6

[0144] Synthesis of 1-11:

[0145]

[0146] Referring to Example 1, amino acids were sequentially coupled onto a solid resin, side-chain protecting groups were removed, and the polypeptide was cyclized and cleaved to synthesize 1-11.

[0147] Synthesis of 1-11-9:

[0148] Weigh 1 g of Rink Amide resin and place it in a solid-phase reaction column. After swelling and deprotection with Fmoc as described in Example 1, sequentially couple Fmoc-Lys(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OAll)-OH, and Fmoc-Trp through condensation / deprotection cycles. During the sequential coupling of p(Boc)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Lys(Boc)-OH, the completeness of the coupling reaction was monitored using ninhydrin. This yielded Fmoc-Lys(Boc)-Lys(Boc)-Trp(Boc)-Asp(OAll)-Lys(Boc)-Lys(Boc)-Trp(Boc)-Lys(Alloc)-Lys(Boc)-Rink Amide resin. The nonapeptide Fmoc-Lys-Lys-Trp-Asp(OAll)-Lys-Lys-Trp-Lys(Alloc)-Lys-NH2 cleaved from the resin was detected using LC-HRMS. MS (ESI) + m / z measured value: 1619.9105, calculated value: 1619.9102 [M+H] + .

[0149] Synthesis of 1-11:

[0150] Referring to Example 1, the 1-11-9 resin was treated with DMAB and Pd(PPh3)4 to remove the Alloc and Allyl groups, exposing the side-chain amino groups of Lys and the side-chain carboxyl groups of Asp. PyBOP was used as a condensing agent for solid-phase cyclization on the resin. The N-terminal Fmoc protecting group was removed with a 20% piperidine DMF solution. The lysis buffer was used to obtain crude 1-11, which was then purified by HPLC. The stationary phase, mobile phase, and elution ratio were the same as in Example 1. The target peak fraction was collected. (Retention time 7.098 min) The fraction was concentrated and lyophilized to obtain 43.9 mg of pure product, yield 15.9%, purity 97.6%. MS (ESI) was then analyzed. + m / z measured value: 1255.7787, calculated value: 1255.7792 [M+H] + .

[0151] Example 7

[0152] Synthesis of 1-12:

[0153]

[0154] Reference Example 1, sequentially coupling amino acids on solid phase resin, removing side chain protecting groups, cyclization after cleavage of the polypeptide, synthesis of 1-12.

[0155] Synthesis of 1-12-9:

[0156] 1 g Rink Amide resin was weighed into a solid phase reaction column, and after swelling and Fmoc deprotection according to Reference Example 1, Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OAll)-OH were sequentially coupled by condensation / deprotection cycles, and during the sequential coupling, the reaction was monitored for complete coupling by ninhydrin. Fmoc-Asp(OAll)-Lys(Boc)-Trp(Boc)-Trp(Boc)-Lys(Alloc)-Lys(Boc)-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin was obtained. The nonapeptide Fmoc-Asp(OAll)-Lys-Trp-Trp-Lys(Alloc)-Lys-Trp-Dip-Lys-NH2 cleaved from the resin was detected by LC-HRMS. MS (ESI + ): m / z found: 1772.8984, calculated: 1772.8988 [M+H] + .

[0157] Synthesis of 1-12:

[0158] Reference Example 1, the resin was added to a solid phase reaction tube, and DMAB and Pd(PPh3)4 were used to remove the Alloc and OAll groups, exposing the side chain amino group of Lys and the side chain carboxyl group of Asp, to obtain Fmoc-Asp-Lys(Boc)-Trp(Boc)-Trp(Boc)-Lys-Lys(Boc)-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin, followed by solid phase ugi multicomponent cyclization:

[0159] The resin was added to a solid phase reaction tube, and paraformaldehyde (26.5 mg, 0.88 mmol), tetrahydropryrrole (75 μL, 0.88 mmol), 6 mL THF / MeOH (1:1) mixed solvent, nitrogen was bubbled for 30 min. The solvent was removed by suction filtration under reduced pressure, washed with DCM four times, washed with DCM / TFE (1:1) mixed solvent twice, added benzyl isonitrile (107 μL, 0.88 mmol), 6 ml DCM / TFE (1:1) mixed solvent, reacted for 12 h, the solvent was removed by suction filtration under reduced pressure, washed with DCM four times.

[0160] Added 6 mL 20% piperidine in DMF solution, nitrogen was bubbled for 7 min, the solvent was removed by suction filtration under reduced pressure; added 6 ml 20% piperidine in DMF solution, continued to bubble for 7 min, suction filtration to remove the solvent, added 6 mL DMF washed five times. Washed with DCM three times, vacuum dried the resin.

[0161] The dried resin was added to a three-necked flask, added 8 mL cleavage solution (TFA / TIPS / H2O = 95 / 2.5 / 2.5), stirred at room temperature for 2.5 h, collected the filtrate by filtration, washed the resin with a small amount of TFA and DCM, collected the filtrate, removed TFA by rotary evaporation under reduced pressure, added 20 mL ice ethyl ether at 0°C to precipitate the polypeptide, removed the supernatant by suction filtration or centrifugation, washed with ice ethyl ether three times, dried under reduced pressure to obtain 1-12 crude product 340 mg, the polypeptide sequence was cyclo[Asp-Lys-Trp-Trp-Lys]-Lys-Trp-Dip-Lys-NH2.

[0162] Purified by preparative HPLC, the stationary phase, mobile phase and elution ratio refer to Example 1, collected the target peak fraction. (Retention time 10.218 min) concentrated and lyophilized to obtain 10.9 mg of pure product, HPLC purity 98.1%, yield 3.2%, MS (ESI + ): m / z found: 1555.8352, calculated: 1555.8361.

[0163] Example 8

[0164] Synthesis of 1-13:

[0165]

[0166] Example 1, amino acids were sequentially coupled on a solid phase resin, the side chain protecting group was removed, the polypeptide was cleaved after cyclization, and 1-13 was synthesized.

[0167] Synthesis of 1-13-9:

[0168] Take 1 g Rink Amide resin, place in a solid phase reaction column, after swelling, deprotection of Fmoc according to Example 1, Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OAll)-OH, Fmoc-Lys(Boc)-OH are sequentially coupled by condensation / deprotection cycles, and the reaction is monitored by ninhydrin to determine whether the coupling is complete; Fmoc-Lys(Boc)-Glu(OAll)-Trp(Boc)-Trp(Boc)-Lys(Boc)-Lys(Alloc)-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin is obtained. The nonapeptide Fmoc-Lys-Glu(OAll)-Trp-Trp-Lys-Lys(Alloc)-Trp-Dip-Lys-NH2 cleaved from the resin is detected by LC-HRMS. MS (ESI + ): m / z found: 1786.9149, calculated: 1786.9150 [M+H] + .

[0169] Synthesis of 1-13:

[0170] According to Example 1, the resin of 1-13-9 is treated with DMAB and Pd(PPh3)4 to remove the Alloc and Allyl groups, exposing the side chain amino group of Lys and the side chain carboxyl group of Glu, and then solid phase cyclization is carried out on the resin using PyBOP as the condensing agent, and the N-terminal Fmoc protecting group is removed with 20% piperidine in DMF, and the cleavage solution is cut to obtain crude 1-13, which is purified by preparative HPLC, using the same mobile phase, stationary phase and elution ratio as in Example 1, and the target peak fraction is collected. (Retention time 10.451 minutes) Concentrate and lyophilize to obtain 44.1 mg of pure product, with a yield of 14.1% and a purity of 96.4%, MS (ESI + ): m / z found: 1422.7834, calculated: 1422.7834 [M+H] + .

[0171] Example 9

[0172] Synthesis of 1-14:

[0173]

[0174] Referring to Example 1, amino acids were sequentially coupled onto a solid resin, side-chain protecting groups were removed, and the polypeptide was cyclized and cleaved to synthesize 1-14.

[0175] Synthesis of 1-14-9:

[0176] 1 g of Rink Amide resin was weighed and placed in a solid-phase reaction column. Following swelling and deprotection with Fmoc as described in Example 1, Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OAll)-OH, and Fmoc-Lys(Boc)-OH were sequentially coupled through condensation / deprotection cycles. During this sequential coupling process, the completeness of the coupling was monitored using ninhydrin. The resulting product was Fmoc-Lys(Boc)-Glu(OAll)-Trp(Boc)-Trp(Boc)-Lys(Alloc)-Lys(Boc)-Trp(Boc). -Dip-Lys(Boc)-Rink Amide resin. The nonapeptide Fmoc-Lys-Glu(OAll)-Trp-Trp-Lys(Alloc)-Lys-Trp-Dip-Lys-NH2 cleaved from the resin was detected using LC-HRMS (ESI). + m / z measured value: 1786.9155, calculated value: 1786.9150 [M+H] + .

[0177] Synthesis of 1-14:

[0178] Referring to Example 1, the 1-13-9 resin was treated with DMAB and Pd(PPh3)4 to remove the Alloc and Allyl groups, exposing the side-chain amino groups of Lys and the side-chain carboxyl groups of Glu. PyBOP was used as a condensing agent for solid-phase cyclization on the resin. The N-terminal Fmoc protecting group was removed with a 20% piperidine DMF solution. The lysis buffer was used to cleave the crude 1-14 product, which was then purified by preparative HPLC. The stationary phase, mobile phase, and elution ratio were the same as in Example 1. The target peak fraction was collected. (Retention time 9.630 min) The product was concentrated and lyophilized to obtain 44.1 mg of pure product, yield 14.1%, purity 96.4%. MS (ESI) + m / z measured value: 1422.7830, calculated value: 1422.7834 [M+H] + .

[0179] Example 10

[0180] Synthesis of 1-15:

[0181]

[0182] Referring to Example 1, amino acids were sequentially coupled onto a solid resin, side-chain protecting groups were removed, and the polypeptide was cyclized and cleaved to synthesize 1-15.

[0183] Synthesis of 1-15-9:

[0184] Weigh 1 g of Rink Amide resin was placed in a solid-phase reaction column and swollen and deprotected according to Example 1. Then, Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OAll)-OH, and Fmoc-Lys(Boc)-OH were coupled sequentially through condensation / deprotection cycles. During the sequential coupling process, the completeness of the coupling reaction was monitored by ninhydrin. The resulting resin was Fmoc-Lys(Boc)-Asp(OAll)-Trp(Boc)-Trp(Boc)-Lys(Alloc)-Lys(Boc)-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin. The nonapeptide Fmoc-Lys-Asp(OAll)-Trp-Trp-Lys-Lys(Alloc)-Trp-Dip-Lys-NH2 cleaved from the resin was detected using LC-HRMS. Measured m / z: 1772.8993, Calculated: 1772.8988 [M+H] + .

[0185] Synthesis of 1-15:

[0186] Referring to Example 1, the 1-15-9 resin was treated with DMAB and Pd(PPh3)4 to remove the Alloc and Allyl groups, exposing the side-chain amino groups of Lys and the side-chain carboxyl groups of Asp. PyBOP was used as a condensing agent for solid-phase cyclization on the resin. The N-terminal Fmoc protecting group was removed with a 20% piperidine DMF solution. The lysis buffer was used to cleave the crude 1-15 product, which was then purified by preparative HPLC. The stationary phase, mobile phase, and elution ratio were the same as in Example 1. The target peak fraction was collected. (Retention time 10.169 min) The product was concentrated and lyophilized to obtain 9.9 mg of pure product, yield 3.2%, purity 96.4%. MS (ESI) was then analyzed. + m / z measured value: 1408.7691, calculated value: 1408.7683 [M+H]+ .

[0187] Example 11

[0188] Synthesis of 1-16:

[0189]

[0190] Reference Example 1, sequentially coupling amino acids on solid phase resin, removing side chain protecting groups, cyclization after cleavage of the polypeptide, synthesis of 1-16.

[0191] Synthesis of 1-16-9:

[0192] Take 1 g Rink Amide resin, placed in a solid phase reaction column, swelling, removing Fmoc protection according to reference example 1, through the cycle of condensation / deprotection, sequentially coupling Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OAll)-OH, Fmoc-Lys(Boc)-OH, sequentially coupling process, through the indanone monitoring reaction whether complete coupling; Fmoc-Lys(Boc)-Asp(OAll)-Trp(Boc)-Trp(Boc)-Lys(Boc)-Lys(Alloc)-Trp(Boc)-Dip-Lys(Boc)-Ser(tBu)-Rink Amide resin is obtained. The decapeptide Fmoc-Lys-Asp(OAll)-Trp-Trp-Lys-Lys(Alloc)-Trp-Dip-Lys-Ser-NH2 is cut from the resin using LC-HRMS detection. MS (ESI + ): m / z found: 1859.9307, calculated: 1859.9308 [M+H] + .

[0193] Synthesis of 1-16:

[0194] Reference Example 1, 1-16-9 resin was treated with DMAB and Pd(PPh3)4 to remove Alloc and Allyl groups, exposing the side chain amino group of Lys and the side chain carboxyl group of Asp, and then subjected to solid phase cyclization on the resin using PyBOP as the condensing agent, and the N-terminal Fmoc protecting group was removed using 20% piperidine in DMF. The cleavage solution was cut to obtain 1-16 crude product, which was purified by preparative HPLC. The stationary phase, mobile phase and elution ratio were the same as in Reference Example 1, and the target peak fraction was collected (retention time 9.654 minutes). After concentration and lyophilization, 75.6 mg of pure product was obtained, with a yield of 23.0%, a purity of 99.8%, and MS (ESI + ): m / z found: 1495.7996, calculated: 1495.7997 [M+H] + .

[0195] Example 12

[0196] Synthesis of 1-17:

[0197]

[0198] Reference Example 1, 1-16-9 resin was treated with DMAB and Pd(PPh3)4 to remove Alloc and Allyl groups, exposing the side chain amino group of Lys and the side chain carboxyl group of Asp, and then subjected to solid phase cyclization on the resin using PyBOP as the condensing agent, and the N-terminal Fmoc protecting group was removed using 20% piperidine in DMF. The cleavage solution was cut to obtain 1-16 crude product, which was purified by preparative HPLC. The stationary phase, mobile phase and elution ratio were the same as in Reference Example 1, and the target peak fraction was collected (retention time 9.654 minutes). After concentration and lyophilization, 75.6 mg of pure product was obtained, with a yield of 23.0%, a purity of 99.8%, and MS (ESI

[0199] Synthesis of 1-17-9:

[0200] 1 g of Rink Amide resin was weighed into a solid phase reaction column, and after swelling and Fmoc deprotection according to Reference Example 1, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OAll)-OH, Fmoc-Lys(Boc)-OH were sequentially coupled by cycles of condensation / deprotection, and during the sequential coupling, ninhydrin was used to monitor whether the reaction was completely coupled; Fmoc-Lys(Boc)-Asp(OAll)-Trp(Boc)-Trp(Boc)-Lys(Boc)-Lys(Alloc)-Trp(Boc)-Dip-Lys(Boc)-Lys(Boc)-Rink Amide resin was obtained. LC-HRMS was used to detect the decapeptide Fmoc-Lys-Asp(OAll)-Trp-Trp-Lys-Lys(Alloc)-Trp-Dip-Lys-Lys-NH2 cut from the resin. MS (ESI LC-MS (ESI+): m / z found: 1900.9937, calculated: 1900.9943 [M+H] + .

[0201] Synthesis of 1-17:

[0202] Reference Example 1, 1-17-9 resin was removed with DMAB and Pd(PPh3)4Alloc and Allyl groups, exposing the side chain amino of Lys and the side chain carboxyl of Asp, using PyBOP as condensing agent for solid phase cyclization, 20% piperidine in DMF to remove the N-terminal Fmoc protecting group, cleavage solution was cut to get 1-17 crude product, preparation HPLC purification, using preparation HPLC purification, stationary phase, mobile phase and elution ratio reference Example 1, the target peak fraction was collected. (Retention time 9.077 minutes) concentrated and lyophilized to obtain pure product 17.9 mg, yield 5.3%, purity 98.2%, MS (ESI + ): m / z found: 1536.8636, calculated: 1536.8627 [M+H] + .

[0203] Example 13

[0204] Synthesis of 1-18:

[0205]

[0206] Reference Example 1, 1-17-9 resin was removed with DMAB and Pd(PPh3)4Alloc and Allyl groups, exposing the side chain amino of Lys and the side chain carboxyl of Asp, using PyBOP as condensing agent for solid phase cyclization, 20% piperidine in DMF to remove the N-terminal Fmoc protecting group, cleavage solution was cut to get 1-17 crude product, preparation HPLC purification, using preparation HPLC purification, stationary phase, mobile phase and elution ratio reference Example 1, the target peak fraction was collected. (Retention time 9.077 minutes) concentrated and lyophilized to obtain pure product 17.9 mg, yield 5.3%, purity 98.2%, MS (ESI

[0207] Synthesis of 1-18-9:

[0208] Weigh 1 g of Rink Amide resin and place it in a solid-phase reaction column. After swelling and deprotection with Fmoc as described in Example 1, sequentially couple Fmoc-Asp(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Dip-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, and Fmoc through a condensation / deprotection cycle. During the sequential coupling of Fmoc-Lys(Boc)-OH, Fmoc-Asp(OAll)-OH, and Fmoc-Lys(Boc)-OH, the completeness of the coupling was monitored using ninhydrin. This yielded Fmoc-Lys(Boc)-Asp(OAll)-Trp(Boc)-Trp(Boc)-Trp(Boc)-Lys(Boc)-Lys(Alloc)-Trp(Boc)-Dip-Lys(Boc)-Asp(tBu)-Rink Amide resin. The decapeptide Fmoc-Lys-Asp(OAll)-Trp-Trp-Trp-Lys-Lys(Alloc)-Trp-Dip-Lys-Asp-NH2 cleaved from the resin was detected using LC-HRMS (ESI). + m / z measured value: 1887.9257, calculated value: 1887.9257 [M+H] + .

[0209] Synthesis of 1-18:

[0210] Referring to Example 1, the 1-18-9 resin was treated with DMAB and Pd(PPh3)4 to remove the Alloc and OAll groups, exposing the side-chain amino groups of Lys and the side-chain carboxyl groups of Asp. PyBOP was used as a condensing agent for solid-phase cyclization on the resin. The N-terminal Fmoc protecting group was removed with a 20% piperidine DMF solution. The lysis buffer was used to cleave the crude 1-18 product, which was then purified by preparative HPLC. The stationary phase, mobile phase, and elution ratio were the same as in Example 1. The target peak fraction was collected. (Retention time 9.747 min)

[0211] After concentration and lyophilization, 43.5 mg of pure product was obtained, with a yield of 13% and a purity of 95.7%. MS (ESI) + m / z measured value: 1523.7957, calculated value: 1523.7952 [M+H] + .

[0212] Example 14

[0213] Synthesis of 1-19:

[0214]

[0215] Referring to Example 1, 1-19 was synthesized by sequentially coupling amino acids on a solid phase resin, removing side chain protecting groups, cyclizing, and cleaving the polypeptide.

[0216] Synthesis of 1-19-9:

[0217] Referring to Example 1, 1-19 was synthesized by sequentially coupling amino acids on a solid phase resin, removing side chain protecting groups, cyclizing, and cleaving the polypeptide. + ): m / z found: 1772.8983, calculated: 1772.8988 [M+H] + .

[0218] Synthesis of tert-butyl (4-isocyanatobutyl)carbamate:

[0219]

[0220] Boc-Butanediamine (500 mg, 2.22 mmol), chloroform (178 μL, 2.22 mmol), tetrabutylammonium bromide (6.7 mg, 0.033 mmol) were added to a three-necked flask and dissolved in 2 mL DCM, 2.2 g sodium hydroxide solid was dissolved in 4 mL water, the sodium hydroxide aqueous solution was added dropwise while stirring, and the reaction was allowed to proceed overnight. TLC was used to monitor the progress of the reaction. After the reaction was completed, 6 mL DCM was added and extracted three times. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. Column chromatography with DCM / MeOH 50 / 1 gave 80 mg of yellow solid with a yield of 19.2%. MS (ESI +): m / z found: 199.1448, calc: 199.1447. 1 H NMR (500 MHz, CDCl3) δ 4.58 (s, 1H), 3.42 (m, 2H), 3.16 (m, 2H),1.71 (m, 2H), 1.63 (m, 2H), 1.43 (s, 9H).

[0221] Synthesis of 1-19:

[0222] The method of removing Alloc and Allyl protecting groups was referenced to Example 1, removing Alloc and OAll groups with DMAB and Pd(PPh3)4, exposing the side chain amino group of Lys and the side chain carboxyl group of Asp, to obtain Fmoc-Lys(Boc)-Asp-Trp(Boc)-Trp(Boc)-Lys(Boc)-Lys-Trp(Boc)-Dip-Lys(Boc)-Rink Amide resin, followed by solid-phase ugi multicomponent cyclization, ugi multicomponent cyclization reaction referenced to Example 7:

[0223] The resin was added to the solid-phase reaction tube, and polyformaldehyde (26.5 mg, 0.88 mmol), tetrahydro-pyrrole (75 μL, 0.88 mmol), 6 mL THF / MeOH (1:1) mixed solvent, and nitrogen was bubbled for 30 min. The solvent was removed by vacuum filtration, washed with DCM four times, and washed with DCM / TFE (1:1) mixed solvent twice, and then tert-butyl (4-isocyanobutyl) carbamate (165 mg, 0.88 mmol) was added, 6 mL DCM / TFE (1:1) mixed solvent was reacted for 12 h, the solvent was removed by vacuum filtration, and washed with DCM four times.

[0224] 6 mL 20% piperidine in DMF was added, and nitrogen was bubbled for 7 min, and the solvent was removed by vacuum filtration; 6 mL 20% piperidine in DMF was added again, and the bubbling was continued for 7 min, and the solvent was removed by filtration, and 6 mL DMF was added and washed five times. DCM was washed three times, and the resin was dried under vacuum.

[0225] The dried resin was added into a three-necked flask, 8 ml cleavage solution (TFA / TIPS / H2O = 95 / 2.5 / 2.5) was added, the reaction was stirred at room temperature for 2.5 h, the filtrate was collected by filtration, the resin was washed with a small amount of TFA and DCM, the filtrate was collected, TFA was removed by rotary evaporation under reduced pressure, 20 mL ice-ethanol was added at 0°C to precipitate the polypeptide, the supernatant was removed by suction filtration or centrifugation, washed with ice-ethanol for 3 times, and dried under reduced pressure to obtain 1-12 crude product 340 mg, and the polypeptide sequence was Lys-cyclo[Asp-Trp-Trp-Lys-Lys]-Trp-Dip-Lys-NH2.

[0226] Purified by preparative HPLC, the stationary phase, mobile phase and elution ratio refer to Example 1, and the peak fraction of interest was collected. (Retention time 8.549 minutes) Concentration and lyophilization gave pure product 10.9 mg, HPLC purity 98.1%, yield 3.2%, MS (ESI + ): m / z found: 1536.8622, calculated: 1536.8627.

[0227] Example 15. Serum stability results of the cyclic peptide of the present application

[0228] The polypeptide of the present application was incubated with human serum at 37°C to simulate the in vivo environment, and the polypeptide content was quantitatively tested at different time points to draw a degradation curve and calculate the half-life. The specific operation steps are as follows: 1. The human serum frozen at -20°C was thawed overnight in a 4°C refrigerator, and then activated in a 37°C water bath for 2 hours; 2. Preparation of polypeptide solution: 1 mg of polypeptide was added to 30 μL of acetonitrile / water solution, vortexed for 30 seconds, and 210 μL of PBS was added, vortexed for 30 seconds. 240 μL of polypeptide stock solution was obtained; 3. Incubation: 20 μL of polypeptide stock solution was added to 20 μL of human serum, vortexed for 30 seconds, and then placed in a 37°C water bath for incubation; 4. Sample treatment and detection: the corresponding sample was taken out from the water bath at a specific time point (0 min, 2 h, 4 h, 12 h, 24 h), 200 uL of methanol was added to quench the reaction, and the protein was precipitated and separated, and then placed in a 4°C refrigerator. Centrifugation at 12000 rpm for 10 min, and filtration of 100 μL of supernatant. HPLC analysis was used to draw a degradation curve according to the retention time and peak area, and the half-life was calculated. Half-life calculation: the half-life calculation formula is t 1 / 2 =0.693 / k, k is the elimination rate constant, k = (polypeptide content at 0 min - polypeptide content at 24 h) / 24.

[0229] The results are shown in the following table: Figure 1As shown, the linear peptide LTX-315 is rapidly degraded within 4 h, while more than 50% of the polypeptides of most of the cyclic peptides remain intact at 24 h, especially 1-6, 1-7, 1-8, 1-12, and 1-13, which have significantly improved stability compared to the linear peptide LTX-315. As shown in Table 1, the half-life of the cyclic peptides described in the present application is more than 24 hours, while the half-life of LTX-315 is only 2.16 hours. The above results show that the cyclic peptides described in the present application have good serum stability.

[0230] Table 1. Serum half-life of the cyclic peptides described in the present application

[0231]

[0232] Example 16. Anti-tumor cell proliferation effect of the cyclic peptides described in the present application in B16F10 melanoma cells

[0233] The B16F10 cell line in the logarithmic growth phase was taken, and a cell suspension with a concentration of 20,000 cells / mL was prepared. The cells were seeded in a 96-well plate at 100 μL per well. After 24 h of culture, the cells were treated with different concentrations of polypeptide compounds, and after 24 h of incubation, 20 μL of 5 mg / mL MTT solution was added to each well, and the incubator was incubated for 4 h. The supernatant was discarded, 150 μL of DMSO was added to each well, and the cells were shaken for 10 min to completely dissolve. The zero setting hole was set, and the absorbance was read at 570 nm using a microplate reader. The IC 50 The experiment was repeated three times independently, and the results were expressed as the average value ± standard deviation of three experiments.

[0234] Table 2. Inhibition rate of the cyclic peptides described in the present application on the proliferation of B16F10

[0235]

[0236] As shown in Table 2, the cyclic peptides described in the present application showed obvious killing effect on B16F10 melanoma cells.

[0237] Example 17. Change of the anti-tumor cell proliferation effect of the cyclic peptides described in the present application in B16F10 melanoma cells with incubation time

[0238] B16F10 cell lines in logarithmic growth phase were resuspended in DMEM medium to prepare a cell suspension concentration of 20,000 cells / mL. 100 μL of each cell was seeded into a 96-well plate. After 24 h of culture, cells were treated with different concentration gradients of cyclic peptide compounds 1-7, with LTX-315 as a positive control. After incubation for 12 h, 24 h, 36 h, 48 h, and 72 h, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well. The cells were shaken for 10 min to dissolve completely. A zeroing well was set, and the absorbance was read at 570 nm using a microplate reader. The IC50 was calculated. 50 The experiment was repeated three times independently, and the results are expressed as the mean ± standard deviation of the three experiments.

[0239] The results are attached. Figures 2-3 As shown, the IC of the positive LTX-315 50 The inhibition rate gradually increased with prolonged incubation time, while at a concentration of 62.5 μM, it gradually decreased with time, indicating that its activity in inhibiting tumor cell proliferation gradually decreased over time. Conversely, cyclic peptides 1-7 exhibited more sustained inhibitory activity against tumor cell proliferation, with an IC50 value of [missing value]. 50 The numerical value and the inhibition rate of 62.5 μM did not change significantly over time. The above results indicate that the cyclic peptide described in this invention not only improves serum stability but also exerts a more sustained effect in inhibiting tumor cell proliferation, demonstrating significant advantages in anti-tumor activity.

[0240] Example 18. Antitumor activity of cyclic peptides 1-7 of the present invention in a B16F10 tumor-bearing mouse model. SPF grade C57B / 6J mice (6-8 weeks old), female, weighing 20 ± 1 g.

[0241] Mouse melanoma B16F10 cells in logarithmic growth phase (density approximately 80%–90%) were selected, digested with trypsin, and then suspended in serum-containing medium. Cells were collected by centrifugation at 1000 rpm for 5 min. The collected cells were washed twice with PBS by centrifugation, and then resuspended in PBS to adjust the cell concentration to 2 × 10⁻⁶ cells / mL. 6 B16F10 cells were inoculated subcutaneously into the right forelimb of mice using a 1 mL syringe, with an inoculation volume of 0.1 mL per mouse (approximately 20,000 cells). Three days after inoculation, small, firm protrusions appeared at the inoculation site, gradually increasing in size. Body weight and tumor volume were measured every two days, and the tumor was allowed to grow until it reached 30-50 mm². 3, and the model was successfully made. The inoculated mice were randomly divided into 4 groups, 5 mice in each group, and were respectively given (I) normal saline, (II) LTX-315 (1 mg, i.t. (intratumoral injection)), (III) cyclopeptide 1-7 (1 mg, i.t. (intratumoral injection)), and (IV) cyclopeptide 1-7 (2 mg, i.t. (intratumoral injection)). The drugs were dissolved in 50 μL of normal saline, and were administered by intratumoral injection, once every two days, for a total of three times of administration, and a total of 7 days of administration. The effects of the drugs on the fur, mental state, and diet and water intake of the mice were observed every day. Meanwhile, the body weight of the mice was measured and recorded every two days.

[0242] When the tumor nodules were formed, the longest diameter (a) and the maximum transverse diameter (b) in the perpendicular direction of the tumor were measured 2-3 times per week using a vernier caliper, the volume of the tumor was calculated, and a tumor growth curve was plotted. The tumor volume V (mm 3 ) = a×b 2 / 2.

[0243] Tumor inhibition rate calculation: After the treatment ended, the mice were sacrificed by cervical dislocation, the tumors were completely dissected, and the tumor weight was measured using an analytical balance. The tumor inhibition rate (IR) was IR (%) = (1- average tumor weight of the administration group / average tumor weight of the control group) x 100%.

[0244] As shown in the accompanying Figure 4 , there was no significant difference in the body weight of the mice in each group. As shown in the accompanying Figure 5 , the tumor volume of the normal saline solvent group significantly increased over time, although the tumor volume of the treatment groups and the positive control group also increased over time, the tumor growth was significantly inhibited, and the tumor weight of the two administration groups of cyclopeptide 1-7 and the positive control group at the treatment endpoint on the sixth day after the administration ended was significantly less than that of the model group, with a significant difference (p < 0.05). As shown in the accompanying Figure 6 , the tumor weight of the positive group LTX-315 (1 mg, i.t. (intratumoral injection)), the equimolar dose intratumoral injection group cyclopeptide 1-7 (1 mg, i.t. (intratumoral injection)), and the high-dose intratumoral injection group cyclopeptide 1-7 (2 mg, i.t. (intratumoral injection)) was 0.146 ± 0.093 g, 0.096 ± 0.118 g, and 0.068 ± 0.060 g, respectively, which was significantly less than that of the model group 1.016 ± 1.048 g, with a significant difference (p < 0.05).

[0245] Table 3

[0246]

[0247] As shown in Table 3, compared with the solvent group, cyclic peptide 1-7 can significantly inhibit tumor growth in tumor-bearing mice, and is superior to LTX-315.

[0248] like Figure 7 As shown, all mice were sacrificed on the sixth day after the drug administration ended, and the final tumor volume of the cyclic peptide 1-7 groups was significantly smaller than that of the model group.

Claims

1. A cyclic peptide, characterized in that, having the structure of general formula (I): ； in formula (1): A1~A9 are independently amino acid residues from lysine, tryptophan, diphenylalanine, aspartic acid, glutamic acid, ornithine, 2,4-diaminobutyric acid, arginine, histidine, serine, serine, asparagine, glutamine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine; and at least one of A1~A9 has a carboxyl side chain and at least one has an amino side chain; R1, R2 are independently selected from amino, H, -NH-C1~C6 alkyl; the hydrogen atoms or methylene groups in the alkyl groups can be further independently substituted by one or more of hydroxyl, amino, CO, -C(O)-NH2, carboxyl; Cy1 ring is formed by the carboxyl and amino groups on the side chains of A1-A9 through amide bonds; the Cy1 ring can be further substituted by R3, R3 is selected from H, -(CH2)n1-C(O)-NH-(CH2)n2-R 33 ; n1, n2 are each independently selected from 1, 2, 3, 4, 5, R 33 is selected from amino, C6-C10 aryl.

2. The cyclic peptide of claim 1, wherein, said -A1-A2- A3-A4- A5-A6- A7-A8- A9- has the structure of formula (II): ； the virtual arc line connection position can be independently selected from between C1 and C3, C2 and C4, X1 and X3, X2 and X4, X2 and X3, C3 and C5, C2 and C3.

3. The cyclic peptide of claim 2, wherein: C1-C5 can be independently selected from the amino acid residues from lysine, or from the specified alpha amino acid containing a carboxyl side chain or alpha amino acid containing an amino side chain; X1, X2 can be independently selected from tryptophan, or from the specified alpha amino acid containing a carboxyl side chain; X3 is selected from tryptophan, or the specified alpha amino acid containing an amino side chain; X4 can be independently selected from diphenylalanine, or from the specified alpha amino acid containing an amino side chain.

4. The cyclic peptide of claim 1, wherein: R1 can be independently selected from hydrogen; R2 can be independently selected from amino, threonine amide, asparagine, lysine amide; R3 is selected from: 。 5. The cyclic peptide of claim 1, wherein the structure of the virtual arc line is -CH2CONR3(CH2)4- or -(CH2)4NR3COCH2- or -CH2CH2CONR3(CH2)4- or -(CH2)4NR3COCH2CH2- or -CH2CONR3(CH2)3- or -(CH2)3NR3COCH2- or -CH2CONR3(CH2)2- or -(CH2)2NR3COCH2- or -CH2CH2CONR3(CH2)3- or -(CH2)3NR3COCH2CH2- or -CH2CH2CONR3(CH2)2- or -(CH2)2NR3COCH2CH2-.

6. The cyclic peptide of claim 1, wherein said cyclic peptide has one of the following structures: ； the letter combinations in the formula are all abbreviations corresponding to amino acid residues.

7. The cyclic peptide of claim 1, wherein has one of the following structures: 。 8. A method of preparing a cyclic peptide of claim 1, characterized by, including: (1) using solid phase synthesis method to load the target polypeptide on the solid phase carrier to obtain compound 1, wherein Rc and Rn are carboxyl protecting group and amino protecting group respectively, and in steps (1)-(3) of the process, the amino groups or / and carboxyl groups not involved in the reaction in A1-A9 retain the corresponding amino group or / and carboxyl protecting group: ； (2) removing the carboxyl protecting group Rc and the amino protecting group Rn on COORc and NHRn to obtain compound 2: ； (3) intramolecular cyclization of compound 2, and when R3 is not H, further reacting with a precursor compound of R3 to obtain compound 3: ； (4) using a cleavage solution to cleave the cyclic peptide structure, and simultaneously removing the protecting groups of the amino groups or / and the carboxyl groups of A1-A9 to obtain the cyclic peptide (I): 。 9. The method for preparing the cyclic peptide according to claim 8, characterized in that, The amino acid residue structure in which the COORc is located is: or ; The amino acid residue structure in which the RnHN is located is: 。 10. Use of the compound of any one of claims 1-7 in the preparation of an anti-melanoma drug.

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