Polypeptide coupling medicine and application thereof

Abstract

The invention belongs to the technical field of biomedical polypeptides, and particularly relates to a polypeptide coupling drug and application thereof in preparation of drugs for preventing, treating and treating cancer-related diseases. The polypeptide coupling drug is shown as a formula (I). The polypeptide coupling drug provided by the invention improves the solubility of the drug, and has the characteristics of high activity, strong specific binding with a target spot, obvious endocytosis, capability of improving the ability of the drug to penetrate through the blood brain barrier, and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polypeptide conjugate drug and its use in the preparation of a drug for preventing, treating, and treating cancer-related diseases. Background Art

[0002] Chlorotoxin (CTX) is a peptide segment composed of 36 amino acids extracted from the venom of the Israeli gold scorpion of the genus Leiurus Leiurus quinquestriatus) It contains 36 amino acid residues, including 4 disulfide bonds, and its amino acid sequence is: MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQ -CLCR (Disulfide bridge: Cys 2 -Cys 19 , Cys 5 -Cys 28 , Cys 16 -Cys 33 , Cys 20 -Cys 35 ).

[0003] Chlorotoxin has great application prospects as a new selective targeting drug and a specific marker for diagnosis (including grade judgment) of human gliomas.

[0004] Many previous studies have shown that chlorotoxin is a chloride channel blocker, which can specifically bind to tumor cells. At the same time, chlorotoxin has been proven to cross the blood-brain barrier. Again, chlorotoxin has good biocompatibility and has no obvious toxic effect on normal tissue cells. In addition, chlorotoxin has a slow metabolism in the body, providing sufficient time for researchers to perform imaging and treatment. Therefore, chlorotoxin can be used as a targeting agent to deliver cytotoxic agents and / or imaging agents to various tumors, including metastatic tumors and brain tumors such as malignant gliomas.

[0005] Studies have found that human brain astrocytomas produce a unique voltage-dependent chloride current (referred to as the GCC current, and its chloride channel is called the GCC channel), which is not produced in normal human cells (including normal glial cells) and human tumor cells of non-glial origin. This chloride current can be effectively inhibited by the Cl - channel inhibitor chlorotoxin, and it leads to a change in the proliferation rate of brain tumor cells. It is speculated that Cl -Channels are involved in the regulation of astrocytoma growth. GCC currents sensitive to chlorotoxin persist in implanted glioma cells but not in adjacent normal host glial cells. It is confirmed that this unique chloride channel is specific to gliomas. The selective binding of chlorotoxin to this chloride channel provides an effective new approach for the identification and treatment of gliomas. Chlorotoxin selectively binds to glioma cells, while normal human tissues including the brain, kidney, intestine, etc. show negative immunostaining for chlorotoxin.

[0006] Currently in application, chlorotoxin is mainly developed as a carrier to transport radioactive isotopes, fluorescent molecules, etc. into tumors for tumor imaging, so that tumor tissues can be completely and accurately resected during surgery, and normal brain tissues can be preserved to the greatest extent. As a carrier, chlorotoxin can also transport nanoparticles and drugs into tumors, reducing the damage of drugs to other organs of the body and reducing side effects. The property of chlorotoxin binding to glioma cells was first studied in 125 I-labeled small peptides ( 125 I -CTX) and 131 I-labeled small peptides ( 131 I-CTX). The results showed that 125 I -CTX could accumulate in the tumors of tumor-bearing mice. At the same time, 125 I -CTX could specifically bind to glioma cells but not to normal astrocytes. And 131 I-CTX is the most widely studied chlorotoxin complex at present. The radiation emitted by it can be detected to identify and locate brain tumors. Currently, 131I and indocyanine green (ICG)-labeled chlorotoxin have passed preclinical safety trials in the United States and entered phase II / I clinical trials respectively. In another study, it was found that chlorotoxin can bind to tumors of neuroectodermal origin (tumors with a common embryonic origin with central nervous system cells). It was also found that biotin-conjugated chlorotoxin can bind to biopsy samples of more than 200 malignant gliomas and other tumors at different stages, including melanoma, neuroblastoma, medulloblastoma, and small cell lung cancer, but not to normal tissues of the brain, skin, kidney, and lung. At the same time, some fluorescent dyes, such as Cy5.5, BLZ-100, 800CW, can specifically target tumors in vivo after being conjugated with chlorotoxin. In addition, chlorotoxin can transport nanoprobes, magnetic resonance imaging contrast agents, and therapeutic drugs to tumor tissues. Other chlorotoxin conjugates, including fusion proteins, such as chlorotoxin-GST fusion protein conjugated to saponin, have also been shown to significantly and selectively kill tumor cells. In order to develop new tools for the diagnosis and treatment of gliomas, many CTX-based nanoparticles have been constructed. In addition, chlorotoxin has the potential to be a carrier for specifically delivering anticancer drugs to cancer cells. Chlorotoxin shows binding to glioma cells, but not to normal rat astrocytes and human rhabdomyosarcoma cell lines. It has great application prospects as a new specific marker for selective targeting of human tumors and diagnosis (including grade determination).

[0007] Due to the lack of targeting selectivity for tumor cells, traditional treatment methods damage normal tissue cells while killing tumor cells. At the same time, due to the existence of the BBB, finding a new targeted drug delivery system has become an important direction in tumor treatment research. Therefore, there is a great unmet clinical need for the treatment of brain tumors, and the research and development of new brain tumor drugs are also imminent. Chinese Patent CN102844044A discloses a lysine chlorotoxin polypeptide having no more than one available binding site. In some embodiments, the provided lysine-reduced chlorotoxin polypeptide and / or its conjugate can be used in medicine (for example, in various therapeutic and / or diagnostic contexts). Based on the characteristics of chlorotoxin, the present invention provides a novel polypeptide conjugate drug of chlorotoxin analog, which is expected to develop a drug for brain tumors with good development prospects, and can further exert its own advantages through artificial modification, further increasing its stability and efficacy and reducing toxicity. Summary of the Invention

[0008] Only some aspects of the present invention are generally described below and are not limited thereto. These aspects and other parts are more fully described later. All references in this specification are incorporated herein by reference in their entirety. When there is a difference between the disclosure of this specification and the cited literature, the disclosure of this specification shall prevail.

[0009] To solve the deficiencies in the prior art, the present invention provides a polypeptide conjugate drug and its use.

[0010] On the one hand, the present invention provides a polypeptide conjugate drug, the structure of which is shown in formula (I),

[0011] Formula (I) Wherein, R 1 is selected from none, , or ; The Dxd structure is: ; L 1 are each independently selected from the following structures or combinations thereof:

[0012] p is an integer selected from 0 to 6; m are each independently selected from 0 or 1; represents a linking site, and L 1 is linked to the amino group of the side chain of lysine (K).

[0013] Preferably, 0 or 1 or 2 of the L 1 are selected from none.

[0014] Preferably, the L 1 are each independently selected from the following structures or combinations thereof:

[0015] p is an integer selected from 0 to 6.

[0016] Preferably, the L 1 are each independently selected from the following structures:

[0017] On the other hand, the present invention provides a polypeptide conjugate drug, and the structure of the polypeptide conjugate drug is selected from one of the following structures:

[0018]

[0019]

[0020]

[0021] On the other hand, the present invention provides a pharmaceutical composition, which comprises the above-mentioned polypeptide conjugate drug.

[0022] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0023] On the other hand, the present invention provides the use of the above-mentioned polypeptide conjugate drug or pharmaceutical composition in the preparation of a drug for preventing, treating, or alleviating cancer.

[0024] Preferably, the cancer includes breast cancer, lung cancer, prostate cancer, kidney cancer, leukemia, ovarian cancer, gastric cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, brain tumor, skin cancer, lymphoma, or multiple myeloma. Further, the cancer is a brain tumor. Still further, the cancer is glioma.

[0025] The term "naturally occurring amino acid" refers to any one of the 20 L-amino acids commonly found in peptides synthesized in nature, namely alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V) in their L-isomeric forms.

[0026] The polypeptides of the present invention can be modified to form polypeptide derivatives. As is well known to those skilled in the art, various modifications can be made to polypeptides. Typical modifications include, but are not limited to, N-terminal acetylation, C-terminal amidation, D-amino acid substitution, non-natural amino acid substitution, fatty acid modification, or combinations of the above various modifications. The present invention includes any modification of polypeptides that is well known. For example, polypeptide derivatives can include chemical modifications of polypeptides, such as alkylation, acylation, carbamylation, iodination, or any other modification that produces polypeptide derivatives. Modifications of polypeptides can include modified amino acids, such as hydroxyproline or carboxyglutamic acid, and can include amino acids linked by non-peptide bonds.

[0027] The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publish- ing Company, 1995). Pharmaceutically acceptable carriers and / or excipients include, but are not limited to: pH regulators, surfactants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, preservatives, stabilizers. For example, pH regulators include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl and its analogues. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc. Description of the Drawings

[0028] Figure 1 : Liquid phase detection results of Compound 1; Figure 2 : LC-MS detection results of Compound 1; Figure 3 : Killing curve of Compound 1 against glioma cell U87-MG; Figure 4 : Mouse plasma stability results of Compound 1. Detailed Description of the Invention

[0029] The polypeptide compound and its derivatives provided by the present disclosure are synthesized by solid-phase synthesis to obtain their linear precursors, and the synthesis carrier is Rink Amide-AM Resin. During the synthesis process, first, the Rink Amide-AM Resin is fully swollen in N,N-dimethylformamide (DMF), and then the solid-phase carrier and the activated amino acid derivatives are repeatedly subjected to the operations of condensation → washing → deprotection of Fmoc → washing → the next round of amino acid condensation to reach the length of the polypeptide chain to be synthesized. Then, N-terminal amidation is completed on the solid phase, AEEA and FITC are coupled, or tetradecanedioic acid is coupled. After that, a mixed solution of trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) is reacted with the resin to cleave the polypeptide from the solid-phase carrier, and then the solid crude product of the linear precursor is obtained by precipitation with cold methyl tert-butyl ether. The crude linear precursor after cleavage is oxidized by disulfide bonds in a weakly alkaline solution, and then purified and separated by a C-18 reversed-phase preparative chromatographic column using an acetonitrile / water system containing 0.1% trifluoroacetic acid to obtain the oxidized polypeptide. The obtained oxidized polypeptide is coupled with a PTX conjugate in the liquid phase, and after the reaction, it is purified and separated by a C-18 reversed-phase preparative chromatographic column using an acetonitrile / water system containing 0.1% trifluoroacetic acid to obtain the pure product of the polypeptide and its derivatives.

[0030] Experimental reagents

[0031]

[0032]

[0033] Step 1: Synthesis of the linear precursor peptide chain The linear precursor peptide chain of Compound 1: M-C-M-P-C-F-T-T-D-H-Q-M-A-R-K-C-D-D-C-C-G-G-K-G-R-G-K-C-Y-G-P-Q-C-L-C-R.

[0034] 294 mg (0.2 mmol) of Rink Amide-AM Resin was fully swollen in DMF for 1 h. Then, the synthesis was carried out in the order from the carboxyl terminus to the amino terminus according to the linear precursor sequence. Each coupling cycle was carried out as follows: 20% piperidine / DMF (20% v / v, 10 mL) was used for Fmoc-deprotection twice, 8 min each time.

[0035] The resin was rinsed with DMF 6 - 8 times until the pH was neutral.

[0036] Dissolve 1.0 mmol of Fmoc-AA, 1.0 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, add the resin and react at room temperature for 1 h.

[0037] Before coupling the next amino acid, rinse the resin with DMF 4 - 6 times.

[0038] After synthesizing the linear polypeptide, rinse the resin with DMF 5 times.

[0039] Step 2: N-terminal acetylation Prepare 10 mL of acetylation reagent: Dissolve 500 μL of acetic anhydride and 500 μL of DIEA in 9 mL of DMF. Add 10 mL of the prepared acetylation reagent to the resin obtained in Step 1, shake well, and oscillate for 10 min. After the reaction, drain the reaction solution, rinse the resin with DMF 6 - 8 times, and rinse the resin with DCM 5 times. Dry the resin in vacuo.

[0040] Step 3: Cleavage of the linear precursor peptide chain Add freshly prepared cleavage cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) to the resin obtained in Step 2, and oscillate the reaction at room temperature for 2 h. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain the crude product. Wash the crude product with MTBE 3 times and dry it in vacuo.

[0041] Step 4: Formation of intramolecular disulfide bonds Add the crude product obtained in Step 3 to DMSO to dissolve it completely (the volume of DMSO is 20% of the total volume of the reaction system). Then add 2 mM of GSH to 50 mM ammonium bicarbonate buffer (pH = 8.0, containing 30% acetonitrile), and slowly add the dissolved polypeptide solution to the above buffer until the final concentration is 1 mg / mL, and oscillate at room temperature for 16 h. Monitor the reaction result by LC-MS, and directly carry out purification and preparation after the reaction ends.

[0042] Step 5: Purification and preparation of the oxidized polypeptide After filtering through a 0.45 μm membrane, separate it with a reverse-phase high-performance liquid chromatography system. The buffers are A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). Among them, the chromatographic column is a BR-C18 (Sepax) reverse-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph is set at 230 nm, the flow rate is 15 mL / min, and the gradient is 20 - 50% acetonitrile in 40 min. Collect the relevant fractions of the product, combine the fractions with a purity >75% after HPLC identification, lyophilize to obtain the oxidized polypeptide.

[0043] Step 6: Synthesize the Dxd conjugate

[0044] Step 6.1 Synthesize Compound 1-2

[0045] Step 6.1.1 Dissolve irinotecan (1 mmol) in an appropriate amount of DMF, add DIPEA (3.5 mmol), and stir the reaction at room temperature for 1 hour under N2 protection. After 1 hour of reaction, add glycolic acid (1.15 mmol) and HATU (1.05 mmol), and stir the reaction at room temperature for 6 hours under N2 protection. Monitor the reaction results by LC-MS, and prepare for purification after the reaction is completed.

[0046] Purification is carried out by HPLC. Dilute the crude product obtained in Step 6.1.1 according to the ratio of stock solution: acetonitrile: pure water = 1:1:1. The A / B phase solvents are pure water and pure acetonitrile respectively. Among them, the chromatographic column is a BR-C18 (Sepax) reverse-phase chromatographic column. The gradient is set to 5%-90% acetonitrile in 40 min, and the flow rate is set to 15 mL / min. After purification, collect the target fraction and lyophilize it to obtain the light yellow solid Compound 1-2.

[0047] Step 6.2 Synthesize Compound 1-3

[0048] Step 6.2.1 Dissolve Compound 1-2 (1 eq) obtained in Step 6.1.2 in an appropriate amount of DMF, add succinic anhydride (2.0 eq) and DIPEA (3.5 eq), and stir the reaction at room temperature for 8 hours under N2 protection. Monitor the reaction results by LC-MS, and prepare for purification after the reaction is completed.

[0049] Purification is carried out by HPLC. Dilute the crude product obtained in Step 6.2.2 according to the ratio of stock solution: acetonitrile: pure water = 1:1:1. The A / B phase solvents are pure water and pure acetonitrile respectively. Among them, the chromatographic column is a BR-C18 (Sepax) reverse-phase chromatographic column. The gradient is set to 30%-50% acetonitrile in 40 min, and the flow rate is set to 15 mL / min. After purification, collect the target fraction and lyophilize it to obtain the light yellow solid Compound 1-3.

[0050] Step 6.3 Synthesize Compound 1-4

[0051] Step 6.3.1 Dissolve the compound 1-3 (1 eq) obtained in Step 6.2.2 in an appropriate amount of DMF and DCM (DMF:DCM = 2:1), add NHS (5.0 eq), EDCI (5.0 eq), and DMAP (0.1 eq), and stir the reaction at room temperature for 16 hours under N 2 protection. Monitor the reaction result by LC-MS. After the reaction is completed, prepare for purification.

[0052] Step 6.3.2 The purification is carried out by HPLC method. Dilute the crude product obtained in Step 6.3.1 according to the ratio of stock solution:acetonitrile:pure water = 1:1:1. The A / B phase solvents are pure water and pure acetonitrile respectively. The gradient is set at 30%-50% in 40 min, and the flow rate is set at 15 mL / min. After purification, lyophilize to obtain the light yellow solid compound 1-4, namely the Dxd conjugate.

[0053] Step 7: Conjugation of polypeptide with Dxd conjugate Weigh 30 mg of the polypeptide obtained in Step 5 and dissolve the polypeptide in 4 mL of DMF. Then add the compound Dxd conjugate (6eq) obtained in Step 6 and DIEA (25eq), and stir the reaction in an oil bath at 30 °C for 16 hours under N2 protection. Monitor the reaction result by LC-MS. After the reaction is completed, directly carry out purification preparation.

[0054] Step 8: Purification and preparation of the target polypeptide After filtration through a 0.45 μm membrane, separate it with a reverse-phase high-performance liquid chromatography system. The buffer solutions are A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). Among them, the chromatographic column is a BR-C18 (Sepax) reverse-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph is set at 230 nm, the flow rate is 15 mL / min, and the gradient is 25-45% acetonitrile in 40 min. Collect the relevant fractions of the product. After identifying the purity by HPLC, combine the fractions with a purity >95%, and lyophilize to obtain the pure polypeptide.

[0055] Step 10: Detection and characterization methods Determine the purity of the pure polypeptide in Step 9 and complete the N-terminal acetylation of the compound, the conjugation of Dxd conjugate to the side chains of K15, K23, and K27, and the formation of intramolecular disulfide bonds by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are shown in Figure 1 、 Figure 2 .

[0056] Other compounds of the present invention can be synthesized with reference to the experimental method of Example 1.

[0057] The present invention determines the IC of the polypeptide-conjugated drug molecule against two glioma cell lines U87-MG through a killing experiment 50Evaluate its killing effect on glioma cells in vitro.

[0058] Materials: U87-MG cells (Procell); FBS (EXCELL); MEM (SIGMA); P / S solution 100x (self-made); Trypsin (Gibco); EDTA (SIGMA); DPBS (self-made); 96-well black clear-bottom cell plate (Aligent); Celltiter-blue (Promega); DMSO (aladdin).

[0059] Experimental procedures: U87-MG cells are cultured and grown in a medium (MEM, 10% FBS, 1% P / S). When the cell growth density reaches 80 - 90% of the culture flask, first rinse the cells with DPBS, and then digest the cells with 0.25% trypsin (containing 0.5 mM EDTA); collect the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 3 min, and remove the supernatant medium; add 6 - 8 mL of fresh growth medium to resuspend the cells, and passage the cells at a ratio of 1:3 to 1:8, and place them in an incubator at 37°C and 5% CO 2 Incubate in the incubator. Change the medium or passage the cells every 2 - 3 days after passage.

[0060] Sixteen to twenty-four hours before the experiment, passage and amplify U87-MG cells to the required number of cells, digest and centrifuge the cells to collect the cell pellet, resuspend the cells with an appropriate amount of complete medium, detect the cell viability and count, and then adjust the cell concentration to 2×104 cells / mL with complete medium. Inoculate 100 μL / well into the middle wells of a 96-well plate, and fill the edge wells with 100 μL / well of DPBS of the same volume, and place it in an incubator at 37°C and 5% CO 2 Incubate overnight in the incubator.

[0061] The polypeptide-conjugated drug is dissolved in sterile water or DMSO to 1 mM, and the polypeptide-conjugated drug is diluted with the growth medium of U87-MG cells used in the experiment to a concentration of 1 μM (2× concentration), and then serially diluted 5-fold with the corresponding cell growth medium, for a total of 9 concentrations (500 nM - 0.00128 nM).

[0062] Take out the cells inoculated overnight, aspirate 50 μL of the medium from each well, add 50 μL / well of the polypeptide-conjugated drug working solution, and continue to place it in an incubator at 37°C and 5% CO 2 After incubating for 48 h in the incubator, add 20 μL of Celltiter-blue dye solution equilibrated to room temperature to each well, and incubate at 37°C and 5% CO 2 Detect 560EX / 590EM after incubating for 1 h in the incubator.

[0063] IC of the killing experiment of some polypeptide conjugate drugs of the present invention on tumor cells 50 As shown in Table 1, the killing curve of glioma cell U87-MG is as Figure 3 shown.

[0064] ANG-1005 is a brain-penetrating peptide drug conjugate. ANG-1005 is a taxane derivative composed of three paclitaxel molecules covalently linked to Angiopep-2, designed to cross the blood-brain and blood-brain-spinal cord barriers through the low-density lipoprotein receptor-related protein (LRP1) transport system and penetrate malignant cells, and has entered clinical phase III.

[0065]

[0066] Table 1 IC of the killing experiment of some polypeptide conjugate drug molecules of the present invention on tumor cell U87-MG 50 Results

[0067] It can be seen from the results that the polypeptide conjugate drugs of the present invention have good killing effects on tumor cell U87-MG, and the killing effects are better than those of paclitaxel (PTX) and ANG-1005.

[0068] Experimental materials: Polypeptide conjugate drugs (self-made synthetic polypeptide-conjugated drugs): ANG-1005, polypeptide conjugate drugs of the present invention.

[0069] Experimental related reagents: Na 2 HPO 4 (disodium hydrogen phosphate) (purchased from Aladdin); NaH 2 PO 4 (sodium dihydrogen phosphate) (purchased from Aladdin); ACN (acetonitrile) (purchased from Sigma-Aldrich®); ultrapure water (self-made by ultrapure water machine).

[0070] Experimental instrument: Agilent high performance liquid chromatograph 1260.

[0071] Experimental method: After dissolving the polypeptide with an acetonitrile / water solution, it was injected and analyzed by an Agilent high performance liquid chromatograph 1260. The buffer solutions were A (90% H 2 O (20 mM Na 2 HPO 4 + 20 mM NaH 2 PO 4 ) / 10% ACN) and B (70% ACN + 30% H 2O). The chromatographic column was Yuexu C18 reverse phase column (4.6*150mm, 5um, 120Å), the chromatograph detection wavelength was set at 214 nm, the flow rate was 1mL / min, and the gradient was 10% B-95% B in 26 min.

[0072] Experimental results: After HPLC injection analysis, the retention time of the sample (t R ), and substituted into the formula to calculate the CHI of the test compound 7.4 The calculation results are shown in Table 2.

[0073] CHI 7.4 = 14.817 t R - 63.317 r = 0.9999 CHILogD=0.054CHI-1.467 Higher CHILogD values ​​indicate a more lipophilic compound. The difference in lipophilicity at neutral pH reveals whether the overall charge of the molecule at neutral pH is positive or negative.

[0074] Table 2 CHILogD values ​​of compounds

[0075] 1. Experimental materials: methanol (purchased from Sigma); formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin); acetonitrile (purchased from Sigma); dichloromethane (purchased from Sigma). Mouse plasma (heparin sodium) was obtained by Slack.

[0076] 2. Preparation of test compounds Samples were diluted to 0.1 mM using DMSO.

[0077] 3. Pretreatment solution and preparation method

[0078] 4. Experimental steps: (1) Prepare the mixed solution: Take (50 μL × (6 time points) + 1) 350 μL plasma (heparin sodium) and add it to a 1.5 mL EP tube. Prepare at least 3 parallel samples for each time point, prepare 1 tube of mixed solution, and place on ice for 5 min. Add 3.5 μL of the sample to be tested to each tube to make the final concentration 1 μM. Vortex and mix well, and dispense 50 μL into EP tubes according to the time gradient and incubate.

[0079] (2) Incubation: Incubate in a 37°C water bath at six time points: 0 min, 15 min, 30 min, 60 min, 120 min, and 240 min.

[0080] (3)Termination reaction: After incubation, different pretreatment methods are carried out according to the pretreatment methods required by bioanalysis.

[0081] (4)Mixing: Mix well on a vortex oscillator.

[0082] (5)Centrifugation: Centrifuge at 4°C and 13,000 rpm for 10 min in a low-temperature high-speed centrifuge. Take 70 μL of the supernatant, transfer it to an injection vial, and analyze it by LC-MS / MS.

[0083] (6)Data analysis Result evaluation: Detect the peak area of the polypeptide at different time points by LC-MS / MS method, and the results are expressed as the percentage of the remaining rate of the original drug. The results are shown in Table 3 and Table 4, Figure 4 as shown. Table 4 shows the fitting results of the prism software. The half-life of Compound 1 is obtained as 73.84 min using the prism software.

[0084] Table 3 Percentage of polypeptide remaining rate at different time points

[0085] Table 4 Fitting results of the prism software

Claims

1. A polypeptide-conjugated drug, characterized in that: Its structure is shown in formula (I), in, R1 is selected from none, , or ; The Dxd structure is: ； L1 is independently selected from the following structures or their combinations: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ； p is an integer selected from 0 to 6; m is each independently selected from 0 or 1; Indicates the attachment site, L1 is attached to the amino group of the lysine (K) side chain.

2. The polypeptide-conjugated drug according to claim 1, characterized in that: The L1 are each independently selected from the following structures or their combinations: 、 、 、 、 ； p is selected from an integer of 0 to 6.

3. The polypeptide-conjugated drug according to claim 1 or 2, characterized in that: The L1 are each independently selected from the following structures: 、 。 4. The polypeptide-conjugated drug according to claim 1, characterized in that: The peptide-coupled drug structure is selected from one of the following structures: 、 、 、 。 5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide-conjugated drug according to any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

7. Use of the polypeptide-coupled drug according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 or 6 in the preparation of a drug for preventing, treating, curing or alleviating cancer.

8. The use according to claim 7, characterized in that The cancer includes breast cancer, lung cancer, prostate cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, brain tumor, skin cancer, lymphoma, or multiple myeloma.

9. The use according to claim 8, characterized in that The cancer is a brain tumor.

10. The use according to claim 9, characterized in that The cancer is glioma.

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