Octapeptides, cyclic octapeptides and derivatives, methods of preparation and use in the preparation of antitumor drugs

Abstract

The application discloses an octapeptide, a cyclic octapeptide and derivatives, a preparation method and application in preparation of an antitumor drug. The amino acid residue sequence of the octapeptide S3 is as follows: Asp-Ser-Phe-Gly-Leu-Ser-Trp-Leu; the cyclic octapeptide S4 is a cyclization product of S3, and the amino acid residue sequence is as follows: Cyclo(Ser-Phe-Gly-Leu-Ser-Trp-Leu-Asp). Experimental results show that the octapeptide and the derivatives can significantly enhance the expression of two proteins of TIMP-1 and TIMP-2, inhibit the activity of MMP-2 and MMP-9, and thus weaken the ability of non-small cell lung cancer A549 cell metastasis and invasion, thereby providing a path for the treatment of malignant tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to an octapeptide, a cyclized product of the octapeptide, a preparation method of the two peptides, and an application in the preparation of an anti-tumor drug. BACKGROUND

[0002] Cancer and malignant tumor is a kind of disease of cell malignant proliferation, which has the potential of invasion, diffusion and distant metastasis. In recent years, although great progress has been made in cancer chemotherapy, radiotherapy and immunotherapy, cancer is still the most important cause of death in the world. The incidence of non-small cell lung cancer (NSCLC) is increasing year by year in the world, and about 75% of bronchogenic carcinoma is non-small cell lung cancer. Due to early metastasis and lack of effective early metastasis treatment, the five-year survival rate of lung cancer patients is only 8%-14%, so inhibiting lung cancer cell metastasis is one of the keys to lung cancer treatment.

[0003] The metastasis and invasion behavior of tumor cells is the result of the joint regulation of multiple gene changes and mutations in cancer cells, mainly involving MMPs, c-Met, CD44, EGFR family and PAK family, etc. The metastasis and invasion behavior of cancer cells is closely related to the degradation of extracellular matrix (ECM), and the matrix metalloproteinase family (MMPs) plays a crucial role in the degradation of extracellular matrix by degrading extracellular matrix components.

[0004] Type IV collagen is the main component of the basement membrane barrier of ECM, and type IV collagenase (MMP-2 / MMP-9) is the most studied class of matrix metalloproteinases. MMP-2 and MMP-9 are overexpressed in many tumors. Metalloproteinase inhibitors (TIMPs) are a class of small proteins that can regulate MMP activity, usually binding to the active site of MMP with a stoichiometry of 1:1, and the dynamic balance of the expression levels of the two determines the degree of extracellular matrix degradation, and in turn determines the invasion and metastasis of tumor cells. Studies have shown that the interaction between TIMP-1, TIMP-2 and MMP-2, MMP-9 is related to the invasion and metastasis of tumor cells, and the ratio can be used as an index for judging the degree of malignancy and prognosis of some tumors.

[0005] The present application constructs an octapeptide, a cyclic octapeptide and a derivative thereof targeting the transcription level of TIMP-1 / TIMP-2, which can enhance the expression of TIMP-1 and TIMP-2 proteins, and thereby inhibit the activity of MMP-2 and MMP-9, thereby weakening the ability of non-small cell lung cancer A549 cells to metastasize and invade, providing a way for the treatment of malignant tumors. SUMMARY

[0006] The present application is based on the above research, and provides an octapeptide, a cyclic octapeptide and derivatives, a preparation method, and application of the octapeptide and the cyclic octapeptide in preparation of an antitumor drug. The octapeptide and the cyclic octapeptide can effectively inhibit invasion and metastasis of A549 cells, improve mRNA levels and protein expression levels of metalloproteinase 1 (TIMP-1) and metalloproteinase 2 (TIMP-2), and effectively inhibit activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), thus solving the problems of lung cancer cell migration and invasion.

[0007] In order to achieve the above object, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect of the present application, an octapeptide S3 is provided, characterized in that the amino acid sequence of the octapeptide is as shown in Asp-Ser-Phe-Gly-Leu-Ser-Trp-Leu (SEQ ID NO. 1), and the structure is as shown in formula I:

[0009]

[0010] In a second aspect of the present application, a cyclic octapeptide S4 is provided, which is a cyclic substance of the above octapeptide, and the amino acid sequence is as shown in SEQ ID NO. 2: Cyclo (Ser-Phe-Gly-Leu-Ser-Trp-Leu-Asp), and the structure is as shown in formula II:

[0011]

[0012] In a third aspect of the present application, derivatives of the above octapeptide and the cyclic octapeptide are further provided. The derivative of the octapeptide is a product obtained by performing conventional modification on the side chain group of the amino acid of the octapeptide, the amino terminal or the carboxyl terminal, or a product obtained by connecting a tag for polypeptide or protein detection or purification to the octapeptide; and the derivative of the cyclic octapeptide is a product obtained by performing conventional modification on the side chain group of the amino acid of the cyclic octapeptide, or a product obtained by connecting a tag for polypeptide or protein detection or purification to the cyclic octapeptide.

[0013] The conventional modification is any one of aminoization, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cholesterification, biotinylation, fluorescent group modification, polyethylene glycol modification and immobilization modification.

[0014] In a fourth aspect of the present application, a preparation method of the octapeptide S3 and the cyclic octapeptide S4 is provided, and the preparation method is specifically as follows:

[0015] I. Synthesis of the octapeptide

[0016] (1) The first amino acid coupling: after the swelling of the solid phase carrier resin, the 1-carboxylic acid of the first amino acid is coupled with the solid phase carrier under the action of condensing agent, the condensing agent used is HOBt-DMAP condensing system, the activator is DIC, and the solvent is DMF; the molar ratio among the amino acid, HOBt, DIC, and DAP is 3:3:3:1;

[0017] (2) Blocking: a mixed solution of pyridine and acetic anhydride is added, and after oscillation at 25°C, the resin is washed with DCM, DMF, and anhydrous ether for multiple times, and then the resin is dried by vacuum;

[0018] (3) Fmoc protection removal: a mixed solution of Oxyma, piperidine, and DMF is added to the resin in step (1) until the resin is completely immersed, and after oscillation, the Fmoc group on the resin is removed, and then the resin is washed with DCM and DMF in turn;

[0019] (4) Connection of the second amino acid: according to the conditions in step (1), the next amino acid is connected under the action of condensing agent, and according to the polypeptide sequence, Fmoc amino acid, Oxyma, and DIC are mixed in NMP and added to the resin, and after oscillation at 60°C for a certain time, the resin is washed with DCM and DMF for multiple times;

[0020] (5) Repeat the deprotection-coupling-deprotection operation until all the amino acids are connected;

[0021] (6) Preparation and purification of octapeptide crude product: the peptide chain is cut from the carrier using a cutting reagent, and the octapeptide is obtained after purification, wherein the cutting reagent is a mixed solution of TIPS, H2O, and TFA, and the purification method is reverse phase high performance liquid chromatography.

[0022] The preferred conditions of each step are as follows:

[0023] In step (1), the resin is swelled with DCM, and the sample loading capacity of the resin is 0.57 mmol / g; the temperature of the coupling reaction is 50-60°C (preferably 55°C), and the time of the coupling reaction is 20-30 min, preferably 20 min;

[0024] In step (2), the volume ratio of pyridine and acetic anhydride is 1:1, the oscillation time during the reaction is 20 min, and then the resin is washed with DCM, DMF, and anhydrous ether for 3 times respectively, and then the resin is dried by vacuum;

[0025] In step (3), the mass volume ratio of Oxyma, piperidine, and DMF solution is 0.71:1:4 (mg / ml / ml), and when deprotection is performed, the Fmoc group on the resin is removed after continuous oscillation at 20-30°C for 5 min twice, and then the resin is washed with DCM and DMF for 3 times respectively;

[0026] In step (4), the oscillation time at 60°C is 20 min when the amino acid is coupled, and then the resin is washed with DCM, DMF, respectively, for 3 times;

[0027] In step (6), the volume ratio of TIPS, H2O, PhOH and TFA in the cleavage reagent is 2.5:5:5:87.5, the volume / mass ratio of the cleavage reagent to the linear peptide is 1:10 mL / mg, the cleavage temperature is 20-30°C (preferably 25°C), and the cleavage time is 4 h;

[0028] The purification conditions are as follows: column: CST Daiso C18 column, 10 μm, 30*250 mm; mobile phase: mobile phase A is 0.1% TFA / water, and mobile phase B is 0.1% TFA / acetonitrile; gradient elution program: 25% B elution for 0-5 min, 25% B to 65% B for 5-60 min; flow rate is 15 ml / min, sample amount is 5 ml, and detection wavelength is 214 nm.

[0029] II. Preparation of cyclic octapeptide:

[0030] (1) After the solid-phase carrier resin is swelled, the 1-carboxylic acid of the first amino acid is coupled with the solid-phase carrier under the action of a condensing agent, the condensing agent used is HOBt-DMAP condensing system, the activating agent is DIC, and the solvent is DMF; the molar ratio among the amino acid, HOBt, DIC and DMAP is 3:3:3:1;

[0031] (2) Blocking: a mixed solution of pyridine and acetic anhydride is added, and after oscillation at 25°C, the resin is washed with DCM, DMF and anhydrous ether, respectively, for multiple times, and then the resin is dried by vacuum;

[0032] (3) Fmoc protection removal: a mixed solution of Oxyma, piperidine and DMF is added to the resin in step (2) until the resin is completely immersed, the Fmoc on the resin is removed after two oscillations, and then DCM and DMF are used for washing, respectively;

[0033] (4) Coupling of the second amino acid: Fmoc amino acid, Oxyma and DIC are mixed in NMP, and then added to the resin, oscillated at 60°C for a certain time, and then the resin is washed with DCM and DMF for multiple times;

[0034] (5) Repeat the Fmoc protection removal-coupling-Fmoc protection removal operation until all the amino acids are coupled;

[0035] (6) Deprotection and solid phase cyclization: using a mixed solution of tetrakis triphenylphosphine palladium, phenylsilane and DCM as a reaction agent, avoiding light reaction to remove OAllyl protection, then using a mixed solution of piperidine and DMF to remove Fmoc protection, and then adding a mixed solution of PyAOP, HOAt, NMM and anhydrous DMF to perform solid phase cyclization, which is repeated once;

[0036] (7) Preparation and purification of octapeptide crude product: using a cleavage reagent to cut the peptide chain from the carrier, and obtaining the octapeptide after purification, wherein the cleavage reagent is a mixed solution of TIPS, H2O and TFA, and the purification method is reverse phase high performance liquid chromatography.

[0037] In step (1), Wang resin is selected as the resin, DCM is used for swelling, and the loading capacity of the resin is 0.59 mmol / g; the temperature of the coupling reaction is 25-28°C (preferably 28°C), and the time of the coupling reaction is 10-15h (preferably 15h).

[0038] In step (2), the volume ratio of pyridine to acetic anhydride is 1:1, and the oscillation time during the reaction is 20 min, and then the resin is washed with DCM, DMF and anhydrous ether for 3 times respectively, and then the resin is dried by vacuum.

[0039] In step (3), the mass volume ratio of Oxyma, piperidine and DMF solution is 0.71:2:4 (mg / ml / ml), and when deprotection is performed, the Fmoc group on the resin is removed after continuous oscillation for 5 min at 20-30°C for two times, and then the resin is washed with DCM and DMF for 3 times respectively.

[0040] In steps (4) and (5), the molar ratio among amino acid, Oxyma, DIC and NMP is 1:1:1:10; when the amino acid is connected, the oscillation time is 20 min at 60°C, and then the resin is washed with DCM and DMF for 3 times respectively.

[0041] In step (6), the molar ratio of linear peptide, tetrakis triphenylphosphine palladium and phenylsilane is 5:1:100; the molar ratio of linear peptide, PyAOP, HOAt and NMM is 1:5:5:10, and the concentration of the mixed solution is 0.02M.

[0042] In step (7), the volume ratio of TIPS, PhOH, H2O and TFA in the cleavage reagent is 2.5:5:5:87.5, the volume mass ratio of the cleavage reagent to the linear peptide is 1:10 mL / mg, the cleavage temperature is 20-30°C, and the cleavage time is 4h;

[0043] The purification conditions are as follows: chromatographic column: CST Daiso C18 column, 10 μm, 30*250 mm; mobile phase: mobile phase A is 0.1% TFA / water, mobile phase B is 0.1% TFA / acetonitrile; gradient elution procedure: 25% B elution 0-5 min, 25% B-65% B, 5-60 min; flow rate is 15 ml / min, injection amount is 5 ml, detection wavelength is 214 nm.

[0044] In a fifth aspect of the present application, the use of the octapeptide, cyclic octapeptide and derivatives thereof is provided, particularly in the preparation of an anti-tumor drug. Preferably, the anti-tumor drug is a drug that enhances the mRNA expression or protein expression of TIMP-1 and TIMP-2 in tumor cells.

[0045] The results show that the octapeptide and cyclic octapeptide can effectively inhibit the invasion and metastasis of A549 cells, increase the mRNA level and protein expression level of metalloproteinase 1 (TIMP-1) and metalloproteinase 2 (TIMP-2), and effectively inhibit the activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), thereby solving the problem of lung cancer cell migration and invasion. Therefore, the tumor is preferably lung cancer.

[0046] In a sixth aspect of the present application, derivatives of the octapeptide and cyclic octapeptide are provided, such as a recombinant expression vector containing the octapeptide or cyclic octapeptide, a cell containing the recombinant expression vector, and an anti-tumor pharmaceutical composition. The pharmaceutical composition includes an active ingredient, which is the only active ingredient of the octapeptide or cyclic octapeptide and its derivatives, and a pharmaceutically acceptable excipient. The active ingredient can also be used in combination with other anti-tumor drugs.

[0047] The pharmaceutical composition of the present application can be prepared into various dosage forms with commonly used pharmaceutical excipients, for example, it can be a decoction, powder, pill, injection, oral ampoule, tablet, capsule, etc. The administration mode is not limited to oral administration, injection, etc. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The synthetic flow chart of the linear octapeptide S3 is shown in the figure;

[0049] Figure 2 The synthetic flow chart of the cyclic octapeptide S4 is shown in the figure;

[0050] Figure 3 The high performance liquid chromatogram of the purified octapeptide S3 and cyclic octapeptide S4 is shown in the figure;

[0051] Figure 4 The mass spectrum of the purified octapeptide S3 and cyclic octapeptide S4 is shown in the figure;

[0052] Figure 5To analyze the inhibitory effects of octapeptide S3 and cyclic octapeptide S4 on the proliferation of A549 cells using CCK8 reagent;

[0053] Figure 6 The results show the inhibitory effect of peptides on A549 cell invasion.

[0054] Figure 7 The results show the effects of the peptide on the protein levels of TIMP-1, TIMP-2, MMP-2, and MMP-9 in A549 cells;

[0055] Figure 8 The results show the changes in the mRNA levels of TIMP-1 and TIMP-2 in tumor cells after the peptide was applied to A549 cells.

[0056] Figure 9 The structure of Sun A, an analogue of Sungsanpin. Detailed Implementation

[0057] The implementation of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0059] Example 1: Preparation of linear peptide S3

[0060] 1. Synthesis of Octapeptide S3

[0061] Synthesis process as follows Figure 1 As shown, the specific synthesis steps are as follows:

[0062] (1) Preparation of compound 1

[0063] ① Swelling: Add 0.1 mmol of Wang resin (sample loading amount is 0.59 mmol / g) to the solid-phase synthesis reaction tube, soak it in DCM for 20 min to allow the resin to fully swell, and then dry it for later use.

[0064] ② Connect the first amino acid: Dissolve 0.3 mmol Fmoc-Leu-OH, 0.3 mmol HOBt, 0.1 mmol DMAP, and 0.3 mmol DIC in 10 ml DMF, add to the resin, shake at room temperature, and let stand overnight. Wash the resin three times each with DCM and DMF.

[0065] (3) Preparation of the target octapeptide S3

[0066] (4) Fmoc deprotection: add 20% piperidine-DMF solution (0.1M Oxyma) to completely submerge the resin, oscillate at 25°C for 5min x 2 to remove Fmoc on the resin, and then wash the resin with DCM, DMF, and anhydrous ether for 3 times each.

[0067] (2) Preparation of linear peptide resin 2

[0068] Repeat steps 2 and 3, and according to the polypeptide sequence, sequentially dissolve 1.0mmol Fmoc amino acid, 1.0mmol Oxyma, and 1.0mmol DIC in 10ml NMP, add to the resin, oscillate at 60°C for 20min, and repeat deprotection → condensation → deprotection until all amino acids are connected.

[0069] Fmoc deprotection: add 20% piperidine-DMF solution (0.1M Oxyma) to completely submerge the resin, oscillate at 25°C for 5min x 2 to remove Fmoc on the resin, and then wash the resin with DCM, DMF, and anhydrous ether for 3 times each.

[0070] (3) Preparation of the target octapeptide S3

[0071] Wash the resin and dry it, then add TIPS:PhOH:H2O:TFA = 2.5:5:5:87.5 (V / V / V / V) 15mL, oscillate at room temperature for 4h, filter, wash the resin with a little TFA, and collect the filtrate. Blow with argon for 30min, pour into ice ethanol to precipitate and centrifuge, discard the supernatant, and continue to wash with ice ethanol repeatedly for 3 times and centrifuge to obtain the crude octapeptide.

[0072] 2. Purification of the target octapeptide S3

[0073] Dissolve the crude peptide with acetonitrile and water, and purify by preparative RP-HPLC. The separation conditions are as follows:

[0074] Instrument: Pre-HPLC, Agilent 1100 high performance liquid chromatograph;

[0075] Chromatographic column: CST Daiso C18 column, 10μm, 30*250mm;

[0076] Mobile phase: mobile phase A is 0.1% TFA in water, mobile phase B is 0.1% TFA in acetonitrile;

[0077] Step and parameter: eluted with 25% B for 0-5 min, 25% B-65% B for 5-60 min; flow rate is 10 ml / min, injection volume is 5 ml, detection wavelength is 214 nm.

[0078] Example 2 Preparation of cyclic peptide S4 of the application

[0079] 1. Synthesis of cyclic octapeptide S4

[0080] The synthesis process is shown as follows: Figure 2 The specific synthesis steps are as follows:

[0081] (1) Preparation of compound 3

[0082] ① Swelling: 0.1 mmol Wang resin (loading capacity is 0.59 mmol / g) was taken into a solid-phase synthesis reaction tube, soaked with DCM for 20 min to make the resin fully swell, and then dried for use.

[0083] ② Connection of the first amino acid: 0.3 mmol Fmoc-Asp(OAllyl)-OH, 0.3 mmol HOBt, 0.1 mmol DMAP, and 0.3 mmol DIC in 10 ml DMF were mixed and added to the resin, which was shaken at room temperature overnight, and then the resin was washed with DCM and DMF for 3 times respectively.

[0084] ③ Capping: 10 ml of pyridine:acetic anhydride (1:1) mixture was added and shaken at 25°C for 20 min, and then the resin was washed with DCM, DMF, and anhydrous ether for 3 times respectively, and then the resin was dried by vacuum.

[0085] (2) Preparation of compound 4

[0086] ① Fmoc removal: 20% piperidine-DMF solution (0.1M Oxyma) was added to completely immerse the resin, which was shaken at 25°C for 5 min x 2 to remove Fmoc on the resin, and then the resin was washed with DCM and DMF for 3 times respectively.

[0087] ② Connection of the second amino acid: 1.0 mmol Fmoc-Leu-OH, 1.0 mmol Oxyma, and 1.0 mmol DIC in 10 ml NMP were mixed and added to the resin, which was shaken at 60°C for 20 min, and then the resin was washed with DCM and DMF for 3 times respectively.

[0088] (3) Preparation of compound 5

[0089] Repeat the steps of ①② in (2), dissolve 1.0 mmol Fmoc amino acid, 1.0 mmol Oxyma and 1.0 mmol DIC in 10 ml NMP according to the polypeptide sequence, add to the resin, oscillate at 60℃ for 20 min, repeat deprotection → condensation → deprotection until all amino acid connection is completed, to obtain linear peptide resin 5.

[0090] (4) Preparation of compound 6

[0091] ① Remove OAllyl protection: take the linear octapeptide-resin complex from the solid-phase peptide tube into a glass tube, add anhydrous DCM to the resin, swell the resin, add 0.02 mmol tetrakis triphenylphosphine palladium, 2 mmol phenylsilane and a mixed solution of DCM (2 ml). Avoid light reaction for 4 h, after the reaction is completed, wash the resin with DCM, DMF 10 times respectively, and dry the resin under vacuum.

[0092] ② Remove Fmoc protection: add 20% piperidine-DMF solution (0.1M Oxyma) to completely submerge the resin, oscillate at 25℃ for 5 min x 2 to remove Fmoc on the resin, wash the resin with DCM, DMF 3 times respectively.

[0093] ③ Solid-phase cyclization: add 2.5 mmol PyAOP, 2.5 mmol HOAt, 2.5 mmol NMM and a mixed solution of anhydrous DMF (15 ml), react for 4 h. Remove the reaction solution, add 2.5 mmol PyAOP, 2.5 mmol HOAt, 2.5 mmol NMM and a mixed solution of anhydrous DMF (15 ml) again, and react overnight, wash the resin with DCM, DMF 3 times respectively.

[0094] (5) Preparation of target cyclic octapeptide S4

[0095] Wash the resin and dry it, add TIPS:PhOH:H2O:TFA=2.5:5:5:87.5(V / V / V / V) 15 mL, oscillate at room temperature for 4 h, filter, wash the resin with a little TFA, collect the filtrate. Blow away the excess TFA with argon, pour into ice ethyl ether to precipitate and centrifuge, discard the supernatant, continue to wash with ice ethyl ether repeatedly and centrifuge for three times, blow dry to obtain the crude cyclic octapeptide.

[0096] 2, Purification of target S4 cyclic octapeptide

[0097] Dissolve the crude cyclic octapeptide in acetonitrile and water, and purify by preparative RP-HPLC. The separation conditions are as follows:

[0098] Instrument: Pre-HPLC Agilent 1100 high performance liquid chromatograph;

[0099] Column: CST Daiso C18 column, 10 μm, 30*250 mm;

[0100] Mobile phase: mobile phase A is 0.1% TFA in water, mobile phase B is 0.1% TFA in acetonitrile;

[0101] Step and parameter: eluted with 25% B for 0-5 min, 25% B-65% B for 5-60 min; flow rate is 15 ml / min, injection volume is 5 ml, detection wavelength is 214 nm.

[0102] The high performance liquid chromatogram of octapeptide S3 and cyclic octapeptide S4 is shown in Figure 3 , and the mass spectrum is shown in Figure 4 . The purity of the target polypeptide is determined to be 90% by analytical high performance liquid chromatography, and the structure is correct by mass spectrometry.

[0103] Example 3 CCK8 experiment for determining the survival rate of tumor cells A549

[0104] This example systematically studies the proliferation inhibition effect of octapeptide S3 and cyclic octapeptide S4 on A549 tumor cells. A549 is seeded in a 96-well plate at a density of 5000 cells per well, and cultured overnight in a humidified sterile incubator at 37°C and 5% CO2, then the cells are treated with polypeptide diluent at a specified concentration (12.5 μM, 25 μM, 50 μM, 100 μM) for 72 h, and then the culture solution is gently aspirated. CCK-8 reagent solution is dissolved in serum-free medium at a volume ratio of 1:10, 100 μL is added to each well, and incubated at 37°C for 1 h, and the absorbance value is recorded at 450 nm by an enzyme-labeled instrument.

[0105] The CCK-8 experiment results are shown in Figure 5 , and the survival rates of cells at different concentrations of octapeptide S3 and cyclic octapeptide S4 are basically the same, indicating that octapeptide S3 and cyclic octapeptide S4 cannot effectively kill A549 cells, and have no obvious inhibition effect on the proliferation of A549 cells.

[0106] Example 4 Inhibition of tumor cell invasion experiment

[0107] This example studies the inhibition effect of octapeptide S3 and cyclic octapeptide S4 on the migration and invasion ability of A549 tumor cells: DMSO is used as a blank control. After 100 μM S3 and S4 diluent respectively treat the cells for 24 hours, the culture medium is aspirated and washed with PBS for 2 times, then the cells are starved with DMEM serum-free medium for 6 h, and then the cells are routinely digested and centrifuged, and the cell concentration is adjusted to 3×10 5, standby. In the 24-well transfer plate, 600 μL of medium containing 10% FBS (containing 1% double antibody) was pre-added and placed in the Transwell chamber. Using the Biozellen 3D organoid culture matrigel kit, matrigel was seeded according to the use steps at the bottom of the upper chamber, and after the matrigel was gelled, 200 μL of each group of cell suspension was respectively introduced into the upper chamber of the Transwell, and cultured in a 37°C, 5% CO2 incubator for 48 h. The Transwell was taken out, washed with PBS once, and the cells on one side of the upper chamber that did not migrate were gently wiped off with a sterile cotton swab, and the cells were fixed with 10% formaldehyde solution for 30 min. The chamber was soaked and washed with PBS for 3 times, 5 min / time, and stained with 5% crystal violet staining solution at room temperature for 20 min. After washing with PBS, the number of cells that migrated through the matrigel and transferred to the lower chamber was counted, and the cell invasion inhibition rate was calculated: invasion inhibition rate (%) = 1- (drug group invasion cell number / control group invasion cell number) x 100%.

[0108] The results are shown in Figure 6 Compared with the blank control group, S3 and S4 had different degrees of inhibition on the invasion and migration ability of A549 lung cancer cells.

[0109] Example 5: Detection of the influence of migration and invasion related protein expression at the protein level by western blot method

[0110] The logarithmic growth period of A549 cells was routinely digested and centrifuged, resuspended and counted, and then seeded in a 6-well plate at a density of 7x10 5 / well, and cultured in a 37°C, 5% CO2 incubator overnight. The cells were treated with SunA (structure see Figure 9 ), S3, S4 diluent at the specified concentration for 24 h, and then the original culture medium was replaced with serum-free medium for 6 h of starvation treatment. Add 120 μL of cell lysis solution, and determine the protein concentration by BCA method. Take 40 μg of protein sample, separate by electrophoresis, and transfer to NC transfer filter membrane, block with 50 mg / mL skim milk powder / PBS for 2 h. Add primary antibody (1:1000 rabbit anti-human monoclonal antibody), incubate at 4°C overnight, the next day wash the membrane 5 times with PBST, add secondary antibody (1:5000 HRP-labeled anti-rabbit antibody), incubate at room temperature for 1 h, wash the membrane 5 times with PBST, add ECL chemiluminescence substrate, scan under the Odyssey infrared laser imaging system, and take pictures with white stripes.

[0111] The results are shown in Figure 7 Compared with the blank control group, the protein expression of matrix metalloproteinase tissue inhibitors TIMP-1 and TIMP-2 in the drug group was increased, and there was no obvious effect on the protein expression of MMP-2 and MMP-9.

[0112] Example 6 The effect of migration and invasion related mRNA expression was detected at the RNA level by qPCR.

[0113] A549 lung cancer cells were treated with 100 μM S3, S4 and positive drug Sungsanpin dilutions for 24 h, and total RNA was extracted according to the instructions of the Beyotime animal RNA extraction kit, and reverse transcribed into cDNA using the RevertAid™ cDNA synthesis reverse transcription kit. Subsequently, PCR analysis was performed on equal amounts of cDNA preparations using Applied Biosystems. The amplification conditions were: 94°C pre-denaturation for 5 min, 40 cycles of 94°C denaturation for 30 s, 60°C denaturation for 30 s.

[0114] The primer sequences used during amplification are as follows:

[0115] (1) TIMP-1: forward primer 5'-ATCCTGTTGTTGCTGTGGCTGATAG-3' (SEQ ID NO. 3); reverse primer 5'-TGCTGGGTGGTAACTCTTTATTTCA-3' (SEQ ID NO. 4).

[0116] (2) TIMP-2: forward primer 5'-AAACGACATTTATGGCAACCCTATC-3' (SEQ ID NO. 5); reverse primer 5'-ACAGGAGCCGTCACTTCTCTTGATG-3' (SEQ ID NO. 6);

[0117] (3) GAPDH: forward primer 5'-CGGAGTCAACGGATTTGGTCGTAT-3' (SEQ ID NO. 7); reverse primer 5'-AGCCTTCTCCATGGTGGTGAAGAC-3' (SEQ ID NO. 8).

[0118] The results, as shown in Figure 8 S3 can significantly increase the mRNA level of TIMP-2 in tumor cells, and S4 can simultaneously enhance the mRNA expression of TIMP-1 and TIMP-2.

[0119] The above has specifically described the preferred embodiments of the present application, but the present application is not limited to the described embodiments, and those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the present application, and these equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims

1. An octapeptide, characterized in that, The amino acid sequence of the octapeptide is shown as SEQ ID NO.

1.

2. A cyclic octapeptide, characterized in that, The cyclic matter of the octapeptide of claim 1, the amino acid sequence of which is shown as SEQ ID NO. 2: Cyclo (Ser-Phe-Gly-Leu-Ser-Trp-Leu-Asp), the structure of which is shown as follows:

3. A process for the preparation of the octapeptide according to claim 1, characterized in that, The method comprises the following steps: (1) After swelling, the 1-carboxylic acid of the first amino acid is coupled with the solid-phase carrier under the action of a condensing agent, the condensing agent used is an HOBt-DMAP condensing system, the activating agent is DIC, and the solvent is DMF; the molar ratio among the amino acid, HOBt, DIC, and DAP is 3:3:3:1; (2) Blocking: a mixed solution of pyridine and acetic anhydride is added, the resin is washed with DCM, DMF, and anhydrous ether for multiple times after oscillation at 25 DEG C, and the resin is dried by vacuumizing; (3) Fmoc protection removal: a mixed solution of Oxyma, piperidine, and DMF is added to the resin in step (1) until the resin is completely immersed, the Fmoc on the resin is removed after oscillation, and then the resin is washed with DCM and DMF in sequence; (4) Connection of the second amino acid: according to the conditions in step (1), the next amino acid is connected under the action of a condensing agent, and the Fmoc amino acid, Oxyma, and DIC are mixed in NMP according to the polypeptide sequence, and then added to the resin, oscillated at 60 DEG C for a certain time, and then the resin is washed with DCM and DMF for multiple times in sequence; (5) The deprotection-coupling-deprotection operation is repeated until all the amino acids are connected; (6) Preparation and purification of the octapeptide crude product: the peptide chain is cut from the carrier by using a cutting reagent, and the octapeptide is obtained after purification, wherein the cutting reagent is a mixed solution of TIPS, H2O, phenol, and TFA, and the purification method used is reverse-phase high performance liquid chromatography.

4. The preparation method of the octapeptide according to claim 3, wherein: in step (1), the resin is Wang resin, the sample loading amount is 0.57 mmol / g, and DCM is used for swelling; the temperature of the coupling reaction is 50-60 DEG C, and the time of the coupling reaction is 20-30 min, wherein, in step (2), the volume ratio of pyridine and acetic anhydride is 1:1, the oscillation time during the reaction is 20 min, and then the resin is washed with DCM, DMF, and anhydrous ether for 3 times in sequence, and then the resin is dried by vacuumizing, in step (3), the mass-volume ratio of Oxyma, piperidine, and DMF solution is 0.71:1:4 (mg / ml / ml), the Fmoc group on the resin is removed after continuous oscillation for 5 min twice at 20-30 DEG C, and then the resin is washed with DCM and DMF for 3 times in sequence, in step (4), when the amino acid connection is performed, the oscillation time at 60 DEG C is 20 min, and then the resin is washed with DCM and DMF for 3 times in sequence, ​ In step (6), the volume ratio of TIPS, H2O, PhOH and TFA in the cleavage reagent is 2.5:5:5:87.5, the volume / mass ratio of the cleavage reagent to the linear peptide is 1:10 mL / mg, the cleavage temperature is 20-30℃, and the cleavage time is 4 h; The purification conditions are as follows: chromatographic column: CST Daiso C18 column, 10 μm, 30*250 mm; mobile phase: mobile phase A is 0.1% TFA / water, and mobile phase B is 0.1% TFA / acetonitrile; gradient elution program: 25% B elution for 0-5 min, 25% B to 45% B for 5-60 min; flow rate is 15 ml / min, sample injection amount is 5 ml, and detection wavelength is 214 nm.

5. The process for the preparation of a cyclic octapeptide according to claim 2, characterized in that, The method comprises the following steps: (1) After the solid-phase carrier resin is swelled, the 1-carboxylic acid of the first amino acid is coupled to the solid-phase carrier under the action of a condensing agent, the condensing agent used is an HOBt-DMAP condensing system, the activating agent is DIC, and the solvent is DMF; the molar ratio among the amino acid, HOBt, DIC and DMAP is 3:3:3:1; (2) Blocking: a mixed solution of pyridine and acetic anhydride is added, the resin is washed with DCM, DMF and anhydrous ether in sequence after oscillation at 25℃, and then the resin is dried by vacuumizing; (3) Fmoc protection removal: a mixed solution of Oxyma, piperidine and DMF is added to the resin in step (2) until the resin is completely immersed, the Fmoc on the resin is removed after oscillation twice, and then the resin is washed with DCM and DMF in sequence; (4) Coupling of the second amino acid: Fmoc amino acid, Oxyma and DIC are mixed in NMP and added to the resin, the resin is washed with DCM and DMF in sequence after oscillation at 60℃ for a certain time; (5) The operation of Fmoc protection removal-coupling-Fmoc protection removal is repeated until all the amino acids are coupled; (6) Protection removal and solid-phase cyclization: a mixed solution of tetrakis triphenyl phosphine palladium, phenylsilane and DCM is used as a reaction agent to remove the OAllyl protection under light shielding, a mixed solution of piperidine and DMF is used to remove the Fmoc protection, and then a mixed solution of PyAOP, HOAt, NMM and anhydrous DMF is used for solid-phase cyclization, and the process is repeated once; (7) Preparation and purification of the octapeptide crude product: the peptide chain is cut from the carrier by using a cleavage reagent, and the octapeptide is obtained after purification, wherein the cleavage reagent is a mixed solution of TIPS, H2O and TFA, and the purification method used is reverse phase high performance liquid chromatography.

6. The preparation method according to claim 5, wherein: wherein, In step (1), the resin is Wang resin, the loading amount is 0.59 mmol / g, DCM is used for swelling, the coupling reaction temperature is 25-28℃, and the coupling reaction time is 10-15 h; In step (2), the volume ratio of pyridine to acetic anhydride is 1:1, the oscillation time during the reaction is 20 min, the resin is washed with DCM, DMF and anhydrous ether in sequence for 3 times, and then the resin is dried by vacuumizing. In step (3), the mass-volume ratio of Oxyma, piperidine and DMF solution was 0.71:1:4 (mg / ml / ml), and the Fmoc group on the resin was removed after continuous oscillation for 5 min twice at 20-30℃, and then the resin was washed with DCM and DMF for 3 times respectively, In steps (4) and (5), the molar ratio of amino acid, Oxyma, DIC and NMP was 1:1:1:10, and the oscillation time was 20 min at 60℃, and then the resin was washed with DCM and DMF for 3 times respectively, In step (6), the molar ratio of linear peptide, tetrakis triphenyl phosphine palladium and phenylsilane was 5:1:100, and the molar ratio of linear peptide, PyAOP, HOAt and NMM was 1:5:5:10, and the concentration of the mixed solution was 0.02M, In step (7), the volume ratio of TIPS, H2O, PhOH and TFA in the cleavage reagent was 2.5:5:5:87.5, the volume-mass ratio of the cleavage reagent and linear peptide was 1:10 mL / mg, the cleavage temperature was 20-30℃, and the cleavage time was 4h; The purification conditions are as follows: chromatographic column: CST Daiso C18 column, 10μm, 30*250mm; mobile phase: mobile phase A is 0.1% TFA / water, and mobile phase B is 0.1% TFA / acetonitrile; gradient elution program: 25%B elution for 0-5min, 25%B-65%B for 5-60min; flow rate is 15ml / min, sample amount is 5ml, and detection wavelength is 214nm.

7. Use of the octapeptide according to claim 1 or the cyclic octapeptide according to claim 2 for the manufacture of an antitumor medicament, characterized in that, The tumor is lung cancer.

8. Use according to claim 7, characterized in that, The antitumor drug is a drug that enhances the mRNA expression or protein expression of TIMP-1 and TIMP-2 in tumor cells.

9. An antitumor pharmaceutical composition, characterized by comprising the compound or a pharmaceutically acceptable salt thereof according to claim 1. The active ingredient is the octapeptide of claim 1 or the cyclic octapeptide of claim 2, or the octapeptide or the cyclic octapeptide is contained.

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