Polypeptide conjugate and use thereof

Abstract

The present invention provides a polypeptide conjugate and use thereof. In particular, the present invention relates to a belotecan drug and a derivative polypeptide conjugated drug thereof. The polypeptide conjugated drug provided by the present invention is selectively released in a tumor microenvironment, thereby resolving the issue of limited clinical application caused by the lack of targeting capability and strong side effects of the belotecan drug in the prior art. This allows the use of higher doses for cancer treatment in clinical practice, improving overall therapeutic efficacy.

Description

A polypeptide conjugate and its application Technical Field

[0001] The present invention relates to an application of a polypeptide conjugated drug, in particular to a tumor-targeted activated polypeptide conjugate of belotecan and its derivatives and an application thereof. Background Art

[0002] Colorectal cancer, also known as large intestinal cancer, refers to cancers originating from the large intestinal epithelium, including colon cancer and rectal cancer. Adenocarcinoma is the most common pathological type. In my country, colon cancer is a common malignant tumor of the gastrointestinal tract. The early symptoms are not obvious. As the tumor grows, symptoms such as changes in bowel habits, blood in the stool, diarrhea, alternating diarrhea and constipation, and local abdominal pain appear. In the late stage of cancer, systemic symptoms such as anemia and weight loss are manifested. The incidence and mortality of colorectal cancer in my country are on the rise. New therapeutic drugs are needed to improve the treatment effect. Early diagnosis and early surgical resection can improve the postoperative disease-free survival rate and prolong the overall survival.

[0003] Osteosarcoma is one of the rare tumors, with an incidence rate of 4-5 per million people. There are about 5,000 new cases in my country each year. All drug treatments in current clinical diagnosis and treatment plans have poor effects.

[0004] Breast cancer is currently the most common cancer, with over 2.3 million new cases worldwide in 2022. Currently, targeted drugs are used as adjuncts to chemotherapy. These drugs often target only one type of genetic mutation, making them difficult to achieve broad-spectrum efficacy. Chemotherapy, on the other hand, has significant side effects and limited efficacy. Given this situation, there is an urgent need to develop new, effective treatments for breast cancer.

[0005] Belotecan is a synthetic, water-soluble camptothecin analog. It exhibits broad antitumor activity against various human tumor cells, with efficacy comparable to or greater than that of camptothecin and topotecan, and has demonstrated promising therapeutic effects against lung, ovarian, and colorectal cancers. However, belotecan's clinical application is limited by its significant side effects, including neutropenia, thrombocytopenia, anemia, and gastrointestinal toxicity-induced diarrhea and anorexia.

[0006] Prior art document 1: CN113274507A

[0007] At present, there is an existing technology for preparing other anticancer compounds (drugs) into coupled complexes to enhance drug targeting and reduce toxicity. For example, CN113274507A discloses a "targeted delivery and activated immunostimulatory coupled complex", comprising an MI group, a selective group S, a tripeptide group C cleaved by asparagine endopeptidase, an auxiliary connecting arm A and a coupled drug, wherein the tripeptide group C is preferably AAN (alanine-alanine-asparagine), and the auxiliary connecting arm A is preferably L (leucine) or PAB (p-aminobenzyl alcohol). Although the coupled complexes of the above-mentioned prior art have certain optimizations in reducing toxicity and improving drug efficacy compared to the anticancer compound (Payload) itself, the effect is still unsatisfactory and cannot meet the actual needs of clinical applications in cancer treatment. In fact, when treating cancer patients clinically, it is very important to improve drug targeting, enhance drug efficacy, and reduce toxic side effects. Even when the drug efficacy is not much different, reducing drug toxicity can significantly alleviate the patient's side effects (such as diarrhea, anorexia, anemia, etc.), and reduced toxicity means that larger doses can be used for treatment, thereby improving the overall treatment effect.

[0008] Currently, there is no research on improving the targeting and reducing the side effects of belotecan, an anti-tumor drug. Therefore, there is an urgent need to develop a compound that can significantly enhance the targeting, improve the efficacy, and significantly reduce the side effects of belotecan, thereby achieving better cancer treatment results. Summary of the Invention

[0009] The present invention provides a tumor-targeted polypeptide conjugate, its preparation method, and its application, addressing the existing limitations of belotecan's clinical application due to its lack of targeting and significant side effects. The polypeptide conjugate can be selectively released in the tumor microenvironment while exhibiting excellent stability and virtually no release in normal cells, demonstrating excellent targeting. This significantly reduces the toxic side effects of belotecan, allowing for the use of higher doses in clinical cancer treatment, improving overall therapeutic efficacy.

[0010] The present invention provides a polypeptide conjugate, the polypeptide conjugate has the following structural formula:

[0011] E-nPEG-L1-L2-L3-L4-D

[0012] in,

[0013] E is a maleimide-containing group;

[0014] nPEG is n-polyethylene glycol, where n is an integer greater than or equal to 2 and less than or equal to 40;

[0015] L1 and L2 are glycine, alanine, phenylalanine, threonine, serine, or none;

[0016] L3 is glycine, alanine, lysine, guanidine or asparagine;

[0017] L4 is glycine, proline, p -aminobenzyl alcohol, or none;

[0018] D is belotecan or a derivative thereof.

[0019] The present invention also provides a polypeptide conjugate, the polypeptide conjugate has the following structural formula:

[0020] E-nPEG-L1-L2-L3-L4-D

[0021] in,

[0022] E is a maleimide-containing group;

[0023] nPEG is n-polyethylene glycol, where n is an integer greater than or equal to 2 and less than or equal to 40;

[0024] L1 and L2 are glycine, alanine, phenylalanine, threonine, serine, or none;

[0025] L3 is glycine, alanine, lysine, guanidine or asparagine;

[0026] L4 is leucine, isoleucine, or none;

[0027] D is belotecan or a derivative thereof.

[0028] The present invention also provides a polypeptide conjugate, the polypeptide conjugate has the following structural formula:

[0029] E-nPEG-L1-L2-L3-L4-D

[0030] in,

[0031] E is a maleimide-containing group;

[0032] nPEG is n-polyethylene glycol, where n is an integer greater than or equal to 2 and less than or equal to 40;

[0033] L1 and L2 are glycine, alanine, phenylalanine, threonine, serine, or none;

[0034] L3 is glycine, alanine, lysine, guanidine or asparagine;

[0035] L4 is glycine, proline, p-aminobenzyl alcohol, leucine, isoleucine, or none;

[0036] D is belotecan or a derivative thereof. The present invention provides a polypeptide conjugate having the following structural formula:

[0037] E-nPEG-L1-L2-L3-L4-D

[0038] in,

[0039] E is a maleimide-containing group;

[0040] nPEG is n-polyethylene glycol, where n is an integer greater than or equal to 2 and less than or equal to 40;

[0041] L1 and L2 are glycine, alanine, phenylalanine, threonine, and serine;

[0042] L3 is glycine, alanine, lysine, guanidine or asparagine;

[0043] L4 is glycine, proline, and p-aminobenzyl alcohol:

[0044] D is belotecan or a derivative thereof.

[0045] The present invention provides a polypeptide conjugate, the polypeptide conjugate has the following structural formula:

[0046] E-nPEG-L1-L2-L3-L4-D

[0047] in,

[0048] E is a maleimide-containing group;

[0049] nPEG is n-polyethylene glycol, where n is an integer greater than or equal to 2 and less than or equal to 40;

[0050] L1 and L2 are glycine, alanine, phenylalanine, threonine, and serine:

[0051] L3 is glycine, alanine, lysine, guanidine or asparagine;

[0052] L4 is glycine;

[0053] D is belotecan or a derivative thereof.

[0054] The present invention also provides a polypeptide conjugate, the polypeptide conjugate has the following structural formula:

[0055] E-nPEG-L1-L2-L3-L4-D

[0056] in,

[0057] E is a maleimide-containing group;

[0058] nPEG is n-polyethylene glycol, where n is an integer greater than or equal to 2 and less than or equal to 40;

[0059] L1 and L2 are glycine, alanine, phenylalanine, threonine, and serine;

[0060] L3 is glycine, alanine, lysine, guanidine or asparagine;

[0061] L4 is leucine and isoleucine;

[0062] D is belotecan or a derivative thereof.

[0063] The present invention also provides a polypeptide conjugate, the polypeptide conjugate has the following structural formula:

[0064] E-nPEG-L1-L2-L3-L4-D

[0065] in,

[0066] E is a maleimide-containing group;

[0067] nPEG is n-polyethylene glycol, where n is an integer greater than or equal to 2 and less than or equal to 40;

[0068] L1 and L2 are glycine, alanine, phenylalanine, threonine, and serine;

[0069] L3 is glycine, alanine, lysine, guanidine or asparagine;

[0070] L4 is glycine, proline, p-aminobenzyl alcohol, leucine, and isoleucine;

[0071] D is belotecan or its derivatives. In the polypeptide conjugate of the present invention, the structure of the D group is:

[0072] Wherein, R is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, cyclopropyl, cyclobutyl, or cyclopentyl.

[0073] In the polypeptide conjugate of the present invention, the structure of the E group is:

[0074] Where n = 1-18

[0075] The polypeptide conjugate of the present invention can be selected from any of the following structures:

[0076] Among them, G: glycine, A: alanine, T: threonine, S: serine, K: lysine, N: asparagine, F: phenylalanine, PAB: aminobenzyl alcohol, L: leucine, I: isoleucine, C: citrulline.

[0077] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide conjugate of the present invention and a pharmaceutically acceptable carrier.

[0078] The present invention also provides a use of the polypeptide conjugate or the pharmaceutical composition in preparing anti-tumor drugs.

[0079] Compared to the existing belotecan, the polypeptide conjugate provided by the present invention significantly reduces side effects, significantly enhances tumor cell inhibition, and significantly improves safety. It is well released in tumor tissue while not being released in normal tissue, achieving targeted release. This allows for the use of higher doses in clinical cancer treatment, improving overall therapeutic efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figures 1-1, 1-2, and 1-3 are statistical graphs of experimental data on the efficacy of the belotecan polypeptide conjugate provided by the present invention in the treatment of SJSA-1 sarcoma animal model (Example 40);

[0081] Figures 2-1, 2-2, and 2-3 are statistical graphs of experimental data on the efficacy of the belotecan polypeptide conjugate provided by the present invention in the treatment of A2780 ovarian cancer animal model (Example 41);

[0082] FIG3 is a statistical graph of experimental data on the efficacy of the belotecan polypeptide conjugate provided by the present invention in the treatment of colo320 colorectal cancer animal model (Example 42);

[0083] FIG4 is a statistical graph of experimental data on the efficacy of the belotecan polypeptide conjugate provided by the present invention in the treatment of colo320 colorectal cancer animal model (Example 43);

[0084] FIG5 is a statistical diagram of the maximum tolerated dose (MTD) detection experimental results of the belotecan polypeptide conjugate provided by the present invention (Example 44). DETAILED DESCRIPTION

[0085] [Example 1] Synthesis of MI-6PEG-AAN-PAB-Belotecan

[0086] The synthesis method for MI-6PEG-AAN-PAB-Belotecan peptide conjugate is as follows:

[0087] The specific synthesis steps are as follows:

[0088] 1. Synthesis of Compound 1-II

[0089] Compound 1-I (500 mg, 0.83 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM):methanol (MeOH) = 10:1) to afford compound 1-II as a pale yellow solid (280 mg, 88.9% yield).

[0090] 2. Synthesis of Compound 1-IV

[0091] Compound 1-II (280 mg, 0.74 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (450 mg, 0.75 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 10:1) to afford compound 1-IV as a yellow solid (310 mg, 48.4% yield).

[0092] 3. Synthesis of Compound 1-V

[0093] Compound 1-IV (310 mg, 0.36 mmol) was dissolved in N,N-dimethylformamide (15 mL), and di(p-nitrobenzene) carbonate (180 mg, 0.59 mmol) was added. The reaction mixture was allowed to react at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM):methanol (MeOH) = 100:1 to 12:1) to afford compound 1-V as a yellow solid (190 mg, 51.2% yield).

[0094] 4. Synthesis of compound MI-6PEG-AAN-PAB-Belotecan

[0095] Belotecan hydrochloride (80 mg, 0.17 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-V (190 mg, 0.18 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 4 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by high-pressure reverse phase preparative chromatography to obtain MI-6PEG-AAN-PAB-Belotecan as a yellow solid (18 mg, 8.0% yield).

[0096] [Example 2] Synthesis of MI-6PEG-GGFG-Belotecan

[0097] The synthesis method for MI-6PEG-GGFG-Belotecan peptide conjugate is as follows:

[0098] The specific synthesis steps are as follows:

[0099] 1. Synthesis of Compound 2-II

[0100] Belotecan hydrochloride (705 mg, 1.50 mmol) and compound 2-I (850 mg, 1.52 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 2-II as a yellow solid (813 mg, yield 55.6%).

[0101] 2. Synthesis of Compound 2-III

[0102] Compound 2-II (813 mg, 0.83 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 2-III as a yellow solid (560 mg, 89.7% yield).

[0103] 3. Synthesis of compound MI-6PEG-GGFG-Belotecan

[0104] Compound 2-III (560 mg, 0.74 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (580 mg, 0.96 mmol) and diisopropylethylamine (400 mg, 3.10 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase column chromatography to obtain MI-6PEG-GGFG-Belotecan as an off-white solid (284 mg, 31.0% yield).

[0105] [Example 3] Synthesis of MI-6PEG-AANG-Belotecan

[0106] The synthesis method for MI-6PEG-AANG-Belotecan peptide conjugate is as follows:

[0107] The specific synthesis steps are as follows:

[0108] 1. Synthesis of Compound 3-II

[0109] Belotecan hydrochloride (705 mg, 1.50 mmol) and compound 3-I (800 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature (25°C) for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 3-II as a yellow solid (860 mg, yield 59.2%).

[0110] 2. Synthesis of Compound 3-III

[0111] Compound 3-II (860 mg, 0.89 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at 25°C for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 3-III as a yellow solid (580 mg, 87.6% yield).

[0112] 3. Synthesis of compound MI-6PEG-AANG-Belotecan

[0113] Compound 3-III (580 mg, 0.78 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain MI-6PEG-AANG-Belotecan as a yellow solid (220 mg, 22.9% yield).

[0114] [Example 4] Synthesis of MI-6PEG-AANG-D1

[0115] The synthesis method for MI-6PEG-AANG-D1 polypeptide conjugate is as follows:

[0116] The specific synthesis steps are as follows:

[0117] 1. Synthesis of Compound 4-I

[0118] Compound D1 hydrochloride (663 mg, 1.50 mmol) and compound 3-I (800 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature (25°C) for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 4-I as a yellow solid (855 mg, yield 60.6%).

[0119] 2. Synthesis of Compound 4-II

[0120] Compound 4-I (855 mg, 0.91 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 4-II as a yellow solid (615 mg, 94.0% yield).

[0121] 3. Synthesis of compound MI-6PEG-AANG-D1

[0122] Compound 4-II (615 mg, 0.86 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-AANG-D1 as an off-white solid (190 mg, 18.3% yield).

[0123] [Example 5] Synthesis of MI-6PEG-GGFG-D1

[0124] The synthesis method for MI-6PEG-GGFG-D1 polypeptide conjugate is as follows:

[0125] The specific synthesis steps are as follows:

[0126] 1. Synthesis of Compound 8-I

[0127] Compound D1 hydrochloride (663 mg, 1.50 mmol) and compound 4-I (850 mg, 1.52 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 8-I as a yellow solid (694 mg, yield 48.9%).

[0128] 2. Synthesis of Compound 8-II

[0129] Compound 8-I (694 mg, 0.73 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was allowed to react at room temperature at 25°C for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 8-II as a yellow solid (498 mg, 94.3% yield).

[0130] 3. Synthesis of compound MI-6PEG-GGFG-D1

[0131] Compound 8-II (498 mg, 0.69 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (580 mg, 0.96 mmol) and diisopropylethylamine (400 mg, 3.10 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase column chromatography to obtain compound MI-6PEG-GGFG-D1 as an off-white solid (191 mg, 22.9% yield).

[0132] [Example 6] Synthesis of MI-6PEG-GGFL-D1

[0133] The synthesis method for MI-6PEG-GGFL-D1 peptide conjugate is as follows:

[0134] The specific synthesis steps are as follows:

[0135] 1. Synthesis of Compound 9-I

[0136] Compound D1 hydrochloride (663 mg, 1.50 mmol) and compound 5-I (935 mg, 1.52 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 9-I as a yellow solid (742 mg, yield 49.4%).

[0137] 2. Synthesis of Compound 9-II

[0138] Compound 9-I (742 mg, 0.74 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 9-II as a yellow solid (454 mg, 78.7% yield).

[0139] 3. Synthesis of compound MI-6PEG-GGFL-D1

[0140] Compound 9-II (454 mg, 0.58 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (520 mg, 0.86 mmol) and diisopropylethylamine (350 mg, 2.71 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase column chromatography to obtain compound MI-6PEG-GGFL-D1 as an off-white solid (159 mg, 21.6% yield).

[0141] [Example 7] Synthesis of MI-6PEG-AANG-D2

[0142] The synthesis method for MI-6PEG-AANG-D2 polypeptide conjugate is as follows:

[0143] The specific synthesis steps are as follows:

[0144] 1. Synthesis of Compound 10-I

[0145] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 3-I (800 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 10-I as a yellow solid (810 mg, yield 56.5%).

[0146] 2. Synthesis of Compound 10-II

[0147] Compound 10-I (810 mg, 0.85 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 10-II as a yellow solid (545 mg, 86.5% yield).

[0148] 3. Synthesis of compound MI-6PEG-AANG-D2

[0149] Compound 10-II (545 mg, 0.74 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain MI-6PEG-AANG-D2 as an off-white solid (138 mg, 15.3% yield).

[0150] [Example 8] Synthesis of MI-6PEG-GGFG-D2

[0151] The synthesis method for MI-6PEG-GGFG-D2 polypeptide conjugate is as follows:

[0152] The specific synthesis steps are as follows:

[0153] 1. Synthesis of Compound 11-I

[0154] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 4-I (850 mg, 1.52 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 11-I as a yellow solid (615 mg, yield 42.7%).

[0155] 2. Synthesis of Compound 11-II

[0156] Compound 11-I (615 mg, 0.64 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was allowed to react at room temperature at 25°C for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 11-II as a yellow solid (400 mg, 84.7% yield).

[0157] 3. Synthesis of compound MI-6PEG-GGFG-D2

[0158] Compound 11-II (400 mg, 0.54 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (580 mg, 0.96 mmol) and diisopropylethylamine (400 mg, 3.10 mmol) were added. The reaction mixture was allowed to react at room temperature at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase column chromatography to obtain compound MI-6PEG-GGFG-D2 as an off-white solid (93 mg, 14.1% yield).

[0159] [Example 9] Synthesis of MI-6PEG-GANG-D2

[0160] The synthesis method for MI-6PEG-GANG-D2 polypeptide conjugate is as follows:

[0161] The specific synthesis steps are as follows:

[0162] 1. Synthesis of Compound 12-II

[0163] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 12-I (810 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature (25°C) for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 12-II as a yellow solid (765 mg, yield 54.2%).

[0164] 2. Synthesis of Compound 12-III

[0165] Compound 12-II (765 mg, 0.81 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 12-III as a yellow solid (498 mg, 85.5% yield).

[0166] 3. Synthesis of compound MI-6PEG-GANG-D2

[0167] Compound 12-III (498 mg, 0.69 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-GANG-D2 as an off-white solid (115 mg, 13.8% yield).

[0168] [Example 10] Synthesis of MI-6PEG-TANG-D2

[0169] The synthesis method for MI-6PEG-TANG-D2 peptide conjugate is as follows:

[0170] The specific synthesis steps are as follows:

[0171] 1. Synthesis of Compound 13-II

[0172] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 13-I (875 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 13-II as a yellow solid (846 mg, yield 57.3%).

[0173] 2. Synthesis of Compound 13-III

[0174] Compound 13-II (846 mg, 0.86 mmol) was dissolved in N,N-dimethylformamide (20 mL) and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 13-III as a yellow solid (552 mg, 84.1% yield).

[0175] 3. Synthesis of compound MI-6PEG-TANG-D2

[0176] Compound 13-III (552 mg, 0.72 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-TANG-D2 as an off-white solid (165 mg, 18.3% yield).

[0177] [Example 11] Synthesis of MI-6PEG-SANG-D2

[0178] The synthesis method for MI-6PEG-SANG-D2 polypeptide conjugate is as follows:

[0179] The specific synthesis steps are as follows:

[0180] 1. Synthesis of Compound 14-II

[0181] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 14-I (855 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature (25°C) for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 14-II as a yellow solid (893 mg, yield 61.3%).

[0182] 2. Synthesis of Compound 14-III

[0183] Compound 14-II (893 mg, 0.92 mmol) was dissolved in N,N-dimethylformamide (20 mL) and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 14-III as a yellow solid (601 mg, 87.2% yield).

[0184] 3. Synthesis of compound MI-6PEG-SANG-D2

[0185] Compound 14-III (601 mg, 0.80 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-SANG-D2 as an off-white solid (225 mg, 22.8% yield).

[0186] [Example 12] Synthesis of MI-6PEG-FANG-D2

[0187] The synthesis method for the MI-6PEG-FANG-D2 polypeptide conjugate is as follows:

[0188] The specific synthesis steps are as follows:

[0189] 1. Synthesis of Compound 15-II

[0190] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 15-I (945 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 15-II as a yellow solid (922 mg, yield 59.6%).

[0191] 2. Synthesis of Compound 15-III

[0192] Compound 15-II (922 mg, 0.89 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 15-III as a yellow solid (624 mg, 86.7% yield).

[0193] 3. Synthesis of compound MI-6PEG-FANG-D2

[0194] Compound 15-III (624 mg, 0.77 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-FANG-D2 as an off-white solid (200 mg, 20.1% yield).

[0195] [Example 13] Synthesis of MI-6PEG-AAAG-D2

[0196] The synthesis method for MI-6PEG-AAAG-D2 polypeptide conjugate is as follows:

[0197] The specific synthesis steps are as follows:

[0198] 1. Synthesis of Compound 16-II

[0199] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 16-I (765 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 16-II as a yellow solid (695 mg, yield 50.8%).

[0200] 2. Synthesis of Compound 16-III

[0201] Compound 16-II (695 mg, 0.76 mmol) was dissolved in N,N-dimethylformamide (20 mL) and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 16-III as a yellow solid (488 mg, 93.1% yield).

[0202] 3. Synthesis of compound MI-6PEG-AAAG-D2

[0203] Compound 16-III (488 mg, 0.71 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-AAAG-D2 as an off-white solid (145 mg, 17.4% yield).

[0204] [Example 14] Synthesis of MI-6PEG-AAKG-D2

[0205] The synthesis method for MI-6PEG-AAKG-D2 peptide conjugate is as follows:

[0206] The specific synthesis steps are as follows:

[0207] 1. Synthesis of Compound 17-II

[0208] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 17-I (1.0 g, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 17-II as a yellow solid (900 mg, yield 56.1%).

[0209] 2. Synthesis of Compound 17-III

[0210] Compound 17-II (900 mg, 0.84 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 17-III as a yellow solid (680 mg, 95.6% yield).

[0211] 3. Synthesis of compound MI-6PEG-AAKG-D2

[0212] Compound 17-III (680 mg, 0.80 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction solution was reacted at room temperature at 25°C for 3 hours. The reaction solution was filtered and the filtrate was evaporated to dryness under reduced pressure. The residue was dissolved in dichloromethane (10 mL), and 3 mL of trifluoroacetic acid was added. The reaction solution was reacted at room temperature at 25°C for 2 hours. LC-MS detection showed that the reaction was complete. The reaction solution was evaporated to dryness under reduced pressure, and the residue was purified by reverse phase column to obtain compound MI-6PEG-AAKG-D2 as an off-white solid (185 mg, yield 18.8%).

[0213] [Example 15] Synthesis of MI-6PEG-AACG-D2

[0214] The synthesis method for MI-6PEG-AACG-D2 peptide conjugate is as follows:

[0215] The specific synthesis steps are as follows:

[0216] 1. Synthesis of Compound 18-II

[0217] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 18-I (915 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 18-II as a yellow solid (860 mg, yield 56.7%).

[0218] 2. Synthesis of Compound 18-III

[0219] Compound 18-II (860 mg, 0.85 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 18-III as a yellow solid (596 mg, 88.8% yield).

[0220] 3. Synthesis of compound MI-6PEG-AACG-D2

[0221] Compound 18-III (596 mg, 0.75 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-AACG-D2 as an off-white solid (119 mg, 12.4% yield).

[0222] [Example 16] Synthesis of MI-6PEG-GGGP-D2

[0223] The synthesis method for MI-6PEG-GGGP-D2 polypeptide conjugate is as follows:

[0224] The specific synthesis steps are as follows:

[0225] 1. Synthesis of Compound 19-II

[0226] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 19-I (765 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature (25°C) for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 19-II as a yellow solid (585 mg, yield 42.9%).

[0227] 2. Synthesis of Compound 19-III

[0228] Compound 19-II (585 mg, 0.64 mmol) was dissolved in N,N-dimethylformamide (20 mL) and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 19-III as a yellow solid (410 mg, 93.1% yield).

[0229] 3. Synthesis of compound MI-6PEG-GGGP-D2

[0230] Compound 19-III (410 mg, 0.60 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-GGGP-D2 as an off-white solid (90 mg, 12.8% yield).

[0231] [Example 17] Synthesis of MI-6PEG-AAGP-D2

[0232] The synthesis method for MI-6PEG-AAGP-D2 polypeptide conjugate is as follows:

[0233] The specific synthesis steps are as follows:

[0234] 1. Synthesis of Compound 20-II

[0235] Compound D2 hydrochloride (685 mg, 1.50 mmol) and compound 20-I (805 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature (25°C) for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 20-II as a yellow solid (615 mg, yield 43.7%).

[0236] 2. Synthesis of Compound 20-III

[0237] Compound 20-II (615 mg, 0.66 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 20-III as a yellow solid (422 mg, 89.3% yield).

[0238] 3. Synthesis of compound MI-6PEG-AAGP-D2

[0239] Compound 20-III (422 mg, 0.59 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-AAGP-D2 as an off-white solid (115 mg, 16.2% yield).

[0240] [Example 18] Synthesis of MI-6PEG-AANG-D3

[0241] The synthesis method for the MI-6PEG-AANG-D3 polypeptide conjugate is as follows:

[0242] The specific synthesis steps are as follows:

[0243] 1. Synthesis of Compound 21-I

[0244] Compound D3 hydrochloride (705 mg, 1.50 mmol) and compound 3-I (800 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 21-I as a yellow solid (760 mg, yield 52.3%).

[0245] 2. Synthesis of Compound 21-II

[0246] Compound 21-I (760 mg, 0.78 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 21-II as a yellow solid (420 mg, 72.1% yield).

[0247] 3. Synthesis of compound MI-6PEG-AANG-D3

[0248] Compound 21-II (420 mg, 0.56 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain MI-6PEG-AANG-D3 as an off-white solid (86 mg, 12.5% ​​yield).

[0249] [Example 19] Synthesis of MI-6PEG-GGFG-D3

[0250] The synthesis method for MI-6PEG-GGFG-D3 polypeptide conjugate is as follows:

[0251] The specific synthesis steps are as follows:

[0252] 1. Synthesis of Compound 22-I

[0253] Compound D3 hydrochloride (705 mg, 1.50 mmol) and compound 4-I (850 mg, 1.52 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 22-I as a yellow solid (545 mg, yield 37.3%).

[0254] 2. Synthesis of Compound 22-II

[0255] Compound 22-I (545 mg, 0.56 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 22-II as a yellow solid (320 mg, 76.0% yield).

[0256] 3. Synthesis of compound MI-6PEG-GGFG-D3

[0257] Compound 22-II (320 mg, 0.42 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (580 mg, 0.96 mmol) and diisopropylethylamine (400 mg, 3.10 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase column chromatography to obtain compound MI-6PEG-GGFG-D3 as an off-white solid (102 mg, 19.6% yield).

[0258] [Example 20] Synthesis of MI-6PEG-AANG-D4

[0259] The synthesis method for the MI-6PEG-AANG-D4 polypeptide conjugate is as follows:

[0260] The specific synthesis steps are as follows:

[0261] 1. Synthesis of Compound 23-I

[0262] Compound D4 hydrochloride (725 mg, 1.50 mmol) and compound 3-I (800 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 23-I as a yellow solid (820 mg, yield 55.6%).

[0263] 2. Synthesis of Compound 23-II

[0264] Compound 23-I (820 mg, 0.83 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 23-II as a yellow solid (595 mg, 94.2% yield).

[0265] 3. Synthesis of compound MI-6PEG-AANG-D4

[0266] Compound 23-II (595 mg, 0.78 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-AANG-D4 as an off-white solid (135 mg, 13.7% yield).

[0267] [Example 21] Synthesis of MI-6PEG-GGFG-D4

[0268] The synthesis method for MI-6PEG-GGFG-D4 polypeptide conjugate is as follows:

[0269] The specific synthesis steps are as follows:

[0270] 1. Synthesis of Compound 24-I

[0271] Compound D4 hydrochloride (725 mg, 1.50 mmol) and compound 4-I (850 mg, 1.52 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 24-I as a yellow solid (610 mg, yield 41.2%).

[0272] 2. Synthesis of Compound 24-II

[0273] Compound 24-I (610 mg, 0.62 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was allowed to react at room temperature at 25°C for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 24-II as a yellow solid (403 mg, 84.9% yield).

[0274] 3. Synthesis of compound MI-6PEG-GGFG-D4

[0275] Compound 24-II (403 mg, 0.53 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (580 mg, 0.96 mmol) and diisopropylethylamine (400 mg, 3.10 mmol) were added. The reaction mixture was allowed to react at room temperature at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase column chromatography to obtain compound MI-6PEG-GGFG-D4 as an off-white solid (122 mg, 18.4% yield).

[0276] [Example 22] Synthesis of MI-6PEG-AANG-D5

[0277] The synthesis method for the MI-6PEG-AANG-D5 polypeptide conjugate is as follows:

[0278] The specific synthesis steps are as follows:

[0279] 1. Synthesis of Compound 25-I

[0280] Compound D5 hydrochloride (725 mg, 1.50 mmol) and compound 3-I (800 mg, 1.50 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 25-I as a yellow solid (489 mg, yield 33.2%).

[0281] 2. Synthesis of Compound 25-II

[0282] Compound 25-I (489 mg, 0.50 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 25-II as a yellow solid (350 mg, 84.1% yield).

[0283] 3. Synthesis of compound MI-6PEG-AANG-D5

[0284] Compound 25-II (350 mg, 0.42 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain compound MI-6PEG-AANG-D5 as an off-white solid (42 mg, 8.0% yield).

[0285] [Example 23] Synthesis of MI-6PEG-GGFG-D5

[0286] The synthesis method for MI-6PEG-GGFG-D5 polypeptide conjugate is as follows:

[0287] The specific synthesis steps are as follows:

[0288] 1. Synthesis of Compound 26-I

[0289] Compound D5 hydrochloride (725 mg, 1.50 mmol) and compound 4-I (850 mg, 1.52 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 24-I as a yellow solid (410 mg, yield 27.7%).

[0290] 2. Synthesis of Compound 26-II

[0291] Compound 26-I (410 mg, 0.41 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was allowed to react at room temperature at 25°C for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 26-II as a yellow solid (298 mg, 94.9% yield).

[0292] 3. Synthesis of compound MI-6PEG-GGFG-D5

[0293] Compound 26-II (298 mg, 0.39 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (480 mg, 0.79 mmol) and diisopropylethylamine (400 mg, 3.10 mmol) were added. The reaction mixture was allowed to react at room temperature at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase column chromatography to obtain compound MI-6PEG-GGFG-D5 as an off-white solid (33 mg, 6.8% yield).

[0294] Example 24 Comparison of Toxicity of Belotecan Compounds in Sarcoma and Ovarian Cancer Cell Lines (CCK8)

[0295] Different cell types were cultured in complete medium (RPMI1640 (DMEM high-glucose medium) + 10% fetal bovine serum + 1X P / S + 1 mM sodium pyruvate) in a 37°C, 5% CO2 incubator until sufficient cell numbers were reached. Cells were harvested, centrifuged at 1000g for 5 minutes, and resuspended in an appropriate volume of 10% RPMI1640 (DMEM high-glucose medium) and counted. 100 μL of cell culture medium containing various drug concentrations was added to 96-well plates. Control wells containing the corresponding drug solvent (0.1% DMSO) without drug were also set up, as were blank wells containing only culture medium without cells. Three replicate wells were set up for each group. After counting, 100 μL of cell suspension was plated into 96-well plates, with a seeding density of 5000 cells (100 μL) per well. The plates were then incubated at 37°C in a 5% CO2 incubator for 48 hours. After 48 hours, 10 μL of cell proliferation staining reagent (CCK8) was added to each well, and the cells were incubated in a cell culture incubator for about 2 hours, and the absorbance at 450 nm was detected.

[0296] Calculate the cell survival rate and the half-maximal inhibitory concentration of the drug on the cells.

[0297] The specific experimental results of the half-maximal inhibitory concentration (IC50) of the drug on cells are shown in the following chart (unit is ug / mL):

[0298] Table 1 Toxicity of peptide conjugates, irinotecan and belotecan in different cell lines

[0299] As shown in Table 1, the IC50 values ​​of the peptide conjugates in these cells are generally more than 100-fold higher than those of belotecan and its derivatives, with some peptide conjugates even exceeding 1,000-fold. This indicates that the peptide conjugates exhibit significantly reduced toxicity compared to belotecan, significantly reducing the side effects of the drug when used in cancer treatment.

[0300] [Example 25] Study on the efficacy of polypeptide conjugates in the treatment of SJSA-1 sarcoma animal model

[0301] Experimental purpose: To study the anti-tumor efficacy of the above compounds in the SJSA-1 sarcoma animal tumor model.

[0302] Experimental drugs: peptide conjugate, irinotecan, belotecan and normal saline control group.

[0303] Experimental animals: 6-8 week old BALB / c mice, all female.

[0304] Preparation of tumor model:

[0305] Experimental purpose: To study the anti-tumor efficacy of the above-mentioned peptide conjugate, irinotecan and belotecan in tumor models, with dosages of 15 mg / kg and 45 mg / kg (the maximum tolerated dose of the peptide conjugate is about 200-400 mg / kg, 45 mg / kg is about one-fifth to one-eighth of the maximum tolerated dose, the maximum tolerated dose of irinotecan is about 70 mg / kg, 15 mg / kg is one-fifth of the maximum tolerated dose). SJSA-1 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Passage was performed every three days, and cells within 15 generations were used. 2×10 7 The corresponding cells were injected subcutaneously into the right chest of nude mice. 3 Afterwards, the mice were randomly divided into groups of three. Treatment began on day one. The belotecan positive control group received a dose of 5 mg / kg, while the irinotecan positive control group received a dose of 15 mg / kg. The control group received normal saline. Dosing continued once a week for three weeks.

[0306] Control and dosage instructions: 15mg / kg peptide conjugate and 5mg / kg irinotecan are used as controls. When the amount of the substances is equal, the concentration of irinotecan contained in 15mg / kg peptide conjugate is 5mg / kg. 45mg / kg: The maximum tolerated dose of the peptide conjugate is about 200-400mg / kg, and 45mg / kg is about one-fifth to one-eighth of the maximum tolerated dose. The maximum tolerated dose of irinotecan is about 70mg / kg, and 15mg / kg is one-fifth of the maximum tolerated dose.

[0307] Table 2

[0308] According to the data, it can be seen that the therapeutic effect of the polypeptide conjugate of the present invention is far superior to that of irinotecan, and it has a stronger inhibitory efficiency and is safer than belotecan.

[0309] [Example 26] Study on the efficacy of polypeptide conjugates in the treatment of colo320 colorectal cancer animal model

[0310] Experimental purpose: To study the anti-tumor efficacy of the above compounds in the colo320 colorectal cancer animal tumor model.

[0311] Experimental drugs: peptide conjugate, irinotecan, belotecan and normal saline control group.

[0312] Experimental animals: 6-8 week old BALB / c mice, all female.

[0313] Preparation of tumor model:

[0314] Experimental purpose: To study the anti-tumor efficacy of the above-mentioned peptide conjugates, irinotecan and belotecan in tumor models, with dosages of 15 mg / kg and 45 mg / kg. Colo320 cells were purchased from Auris Biotech (Shanghai) Co., Ltd. and cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Passage was performed every three days, and cells within 15 generations were used. 7.5×106 corresponding cells were subcutaneously injected into the right chest of nude mice. The tumor reached at least 120 mm 3 Afterwards, the mice were randomly divided into groups of 3. Treatment began on the first day. The belotecan positive control group received a dose of 5 mg / kg, while the irinotecan positive control group received a dose of 15 mg / kg. The control group received normal saline. Dosing was continued once a week for four weeks.

[0315] Control and dosage instructions: 15mg / kg peptide conjugate and 5mg / kg pelotecan are used as controls. When the amount of the substances is equal, the concentration of pelotecan contained in 15mg / kg peptide conjugate is 5mg / kg. 45mg / kg: The maximum tolerated dose of the peptide conjugate is about 200-400mg / kg, and 45mg / kg is about one-fifth to one-eighth of the maximum tolerated dose. The maximum tolerated dose of irinotecan is about 70mg / kg, and 15mg / kg is one-fifth of the maximum tolerated dose.

[0316] Table 3

[0317] According to the data in the above table, it can be seen that the polypeptide conjugate of the present invention has stronger inhibitory efficiency and is safer than belotecan.

[0318] [Example 27] Maximum tolerated dose (MTD) of polypeptide conjugates, belotecan, irinotecan and other compounds

[0319] Detection experiment

[0320] Experimental Animals: All female mice, 6-8 weeks old, were randomly divided into groups of six. Mice were dosed with a gradient of drug concentrations and monitored for 14 days. Mice were euthanized when they lost 20% of their initial body weight and were considered to have died from toxicity. The maximum tolerated dose (MTD) was defined as the highest dose level at which no mice died from the drug, no individual mouse lost more than 20% of their body weight, or the average group body weight loss did not exceed 15%.

[0321] Table 4

[0322] According to the data in the above table, it can be seen that the maximum tolerated dose of the polypeptide conjugate of the present invention is increased by dozens of times compared with belotecan alone, which is significantly higher than belotecan and is safer.

[0323] [Example 28] Peptide conjugate tumor homogenate activation efficiency experiment

[0324] Buffer configuration: 50 mM pH buffer (MES), 250 mM sodium chloride, adjusted to pH 5.0 with 0.5 M sodium hydroxide.

[0325] The peptide conjugate was prepared in a pH 5.0 buffer solution at a concentration of 1 mg / ml. 100 μg of tumor tissue homogenate (prepared using a Jingxin F6 / 10 handheld homogenizer) was added and the solution was incubated at 37°C for 2 hours. The tumor tissue homogenate effectively activated the peptide conjugate and released belotecan. High-performance liquid chromatography (HPLC) was used to measure the decrease in compound and the increase in belotecan, comparing the drug's activation efficiency in tumor tissue.

[0326] Release percentage = belotecan peak area / total peak area of ​​belotecan and all other belotecan-containing compounds

[0327] Table 5 Release efficiency percentage of polypeptide conjugates in different tissues (%)

[0328] Based on the above examples and the results of Example 28, it can be seen that the maximum tolerated dose of the polypeptide conjugate is increased by several dozen times compared to belotecan alone, and the polypeptide conjugate is well released in tumor tissue while not released in normal tissue, achieving targeted release. Compared to irinotecan, a commonly used first-line chemotherapy drug, the polypeptide conjugate has a much better tumor inhibitory effect at a dose of one-tenth the maximum tolerated dose than irinotecan at a dose of one-fifth the maximum tolerated dose. Furthermore, it has demonstrated excellent therapeutic efficacy in a variety of cancers, including colorectal, sarcoma, lung, and ovarian cancers, effectively extending the safety window and making it a preferred choice for patients with various solid tumors in the chest and abdomen.

[0329] [Example 29] Synthesis of MI-6PEG-AANL-Belotecan

[0330] The synthesis method for MI-6PEG-AANL-Belotecan peptide conjugate is as follows:

[0331] The specific synthesis steps are as follows:

[0332] 1. Synthesis of Compound 2-II

[0333] Belotecan hydrochloride (705 mg, 1.50 mmol) and compound 2-I (920 mg, 1.51 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 2-II as a yellow solid (890 mg, yield 57.9%).

[0334] 2. Synthesis of Compound 2-III

[0335] Compound 2-II (890 mg, 0.87 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at 25°C for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 2-III as a yellow solid (630 mg, 90.2% yield).

[0336] 3. Synthesis of compound MI-6PEG-AANL-Belotecan

[0337] Compound 2-III (630 mg, 0.78 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography to obtain MI-6PEG-AANL-Belotecan as a yellow solid (185 mg, 18.4% yield).

[0338] [Example 30] Synthesis of MI-6PEG-GGFL-Belotecan

[0339] The synthesis method for MI-6PEG-GGFL-Belotecan peptide conjugate is as follows:

[0340] The specific synthesis steps are as follows:

[0341] 1. Synthesis of Compound 5-II

[0342] Belotecan hydrochloride (705 mg, 1.50 mmol) and compound 5-I (935 mg, 1.52 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 5-II as a yellow solid (720 mg, yield 46.6%).

[0343] 2. Synthesis of Compound 5-III

[0344] Compound 5-II (720 mg, 0.70 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 5-III as a yellow solid (465 mg, 82.2% yield).

[0345] 3. Synthesis of compound MI-6PEG-GGFL-Belotecan

[0346] Compound 5-III (465 mg, 0.58 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (520 mg, 0.86 mmol) and diisopropylethylamine (350 mg, 2.71 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase column chromatography to obtain MI-6PEG-GGFL-Belotecan as an off-white solid (265 mg, 35.3% yield).

[0347] [Example 31] Synthesis of MI-6PEG-AANL-D1

[0348] The synthesis method for MI-6PEG-AANL-D1 peptide conjugate is as follows:

[0349] The specific synthesis steps are as follows:

[0350] 2. Synthesis of Compound 6-I

[0351] Compound D1 hydrochloride (663 mg, 1.50 mmol) and compound 2-I (920 mg, 1.51 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 6-I as a yellow solid (950 mg, yield 63.5%).

[0352] 3. Synthesis of Compound 6-II

[0353] Compound 6-I (950 mg, 0.95 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1.5 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 6-II as a yellow solid (580 mg, 78.8% yield).

[0354] 4. Synthesis of compound MI-6PEG-AANL-D1

[0355] Compound 6-II (580 mg, 0.75 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (500 mg, 0.83 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse phase column chromatography to obtain compound MI-6PEG-AANL-D1 as an off-white solid (153 mg, 16.2% yield).

[0356] [Example 32] Synthesis of MI-6PEG-GGFL-D1

[0357] The synthesis method for MI-6PEG-GGFL-D1 peptide conjugate is as follows:

[0358] The specific synthesis steps are as follows:

[0359] 1. Synthesis of Compound 9-I

[0360] Compound D1 hydrochloride (663 mg, 1.50 mmol) and compound 5-I (935 mg, 1.52 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice-cooling. The reaction mixture was allowed to react at room temperature at 25°C for 2 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. Water (80 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM): methanol (MeOH) = 100:1 to 8:1) to obtain compound 9-I as a yellow solid (742 mg, yield 49.4%).

[0361] 2. Synthesis of Compound 9-II

[0362] Compound 9-I (742 mg, 0.74 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added. The reaction mixture was allowed to react at room temperature (25°C) for 1 hour. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified on a silica gel column (dichloromethane (DCM) : methanol (MeOH) = 100:1 to 5:1) to afford compound 9-II as a yellow solid (454 mg, 78.7% yield).

[0363] 3. Synthesis of compound MI-6PEG-GGFL-D1

[0364] Compound 9-II (454 mg, 0.58 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-III (520 mg, 0.86 mmol) and diisopropylethylamine (350 mg, 2.71 mmol) were added. The reaction mixture was allowed to react at 25°C for 3 hours. Thin-layer chromatography (TLC) confirmed the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase column chromatography to obtain compound MI-6PEG-GGFL-D1 as an off-white solid (159 mg, 21.6% yield).

[0365] Example 33 Comparison of toxicity of peptide conjugates in sarcoma and ovarian cancer cell lines (CCK8)

[0366] Different cell types were cultured in complete culture medium (RPMI1640 (DMEM high-glucose medium) + 10% fetal bovine serum + 1X P / S + 1 mM sodium pyruvate) in a 37°C, 5% CO2 incubator until sufficient cell mass was reached. Cells were harvested, centrifuged at 1000g for 5 minutes, and resuspended in an appropriate volume of 10% RPMI1640 (DMEM high-glucose medium) to the appropriate density. 100 μL of cell culture medium containing various drug concentrations was added to 96-well plates. Control wells containing the corresponding drug solvent (0.1% DMSO) without drug were also set up, as were blank wells containing only culture medium without cells. Three replicate wells were set up for each group. After counting, cells were seeded onto 96-well plates, with 100 μL of cell suspension per well, at a seeding density of 5000 cells (100 μL) per well. The plates were then incubated at 37°C in a 5% CO2 incubator for 48 hours. After 48 hours, 10 μL of cell proliferation staining reagent (CCK8) was added to each well, and the cells were incubated in a cell culture incubator for about 2 hours, and the absorbance at 450 nm was detected.

[0367] The cell survival rate and the half-maximal inhibitory concentration (IC50) of the drug on the cells were calculated.

[0368] The specific experimental results of the half-maximal inhibitory concentration (IC50) of the drug on cells are shown in the following chart (unit: ug / mL):

[0369] Table 6 Toxicity of peptide conjugates, belotecan, irinotecan, etc. in different cell lines

[0370] As shown in Table 6, the IC50 values ​​of the polypeptide conjugates of the present invention in the above cells are generally more than 100-fold higher than those of irinotecan and belotecan. The IC50 values ​​of some polypeptide conjugates are even more than 1,000-fold higher than that of belotecan. This indicates that the polypeptide conjugates exhibit significantly reduced toxicity compared to the belotecan compound itself, significantly reducing the side effects of medication when used to treat cancer in patients.

[0371] [Example 34] Study on the efficacy of polypeptide conjugates in the treatment of SJSA-1 sarcoma animal model

[0372] Experimental purpose: To study the anti-tumor efficacy of the above compounds in the SJSA-1 sarcoma animal tumor model.

[0373] Experimental drugs: peptide conjugate, irinotecan, belotecan and normal saline control group.

[0374] Experimental animals: 6-8 week old BALB / c mice, all female.

[0375] Preparation of tumor model:

[0376] Experimental purpose: To investigate the anti-tumor efficacy of the above-mentioned peptide conjugate, irinotecan, and belotecan in a tumor model at doses of 15 mg / kg and 45 mg / kg. SJSA-1 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were passaged every three days, and cells within passage 15 were used. 2×10 7 The corresponding cells were injected subcutaneously into the right chest of nude mice. 3 Afterwards, the mice were randomly divided into groups of three. Treatment began on day one. The belotecan positive control group received a dose of 5 mg / kg, while the irinotecan positive control group received a dose of 15 mg / kg. The control group received normal saline. Dosing continued once a week for three weeks.

[0377] Control and dosage instructions: 15mg / kg peptide conjugate and 5mg / kg pelotecan are used as controls. When the amount of the substances is equal, the concentration of pelotecan contained in 15mg / kg peptide conjugate is 5mg / kg. 45mg / kg: The maximum tolerated dose of the peptide conjugate is about 150-200mg / kg, and 45mg / kg is about one-third to one-fifth of the maximum tolerated dose. The maximum tolerated dose of irinotecan is about 70mg / kg, and 15mg / kg is one-fifth of the maximum tolerated dose.

[0378] Table 7

[0379] The data indicate that the therapeutic effects of the polypeptide conjugates of the present invention are comparable to those of irinotecan and belotecan, and some conjugates have better efficacy than irinotecan and belotecan. Furthermore, compared with irinotecan and belotecan, the polypeptide conjugates of the present invention have significantly improved targeting and significantly reduced toxicity. Therefore, overall, they have a good effect in treating cancer.

[0380] [Example 35] Study on the efficacy of polypeptide conjugates in the treatment of colo320 colorectal cancer animal model

[0381] Experimental purpose: To study the anti-tumor efficacy of the above compounds in the colo320 colorectal cancer animal tumor model.

[0382] Experimental drugs: peptide conjugate, irinotecan, belotecan and normal saline control group.

[0383] Experimental animals: 6-8 week old BALB / c mice, all female.

[0384] Preparation of tumor model:

[0385] Experimental purpose: To study the anti-tumor efficacy of the above-mentioned peptide conjugates, irinotecan and belotecan in tumor models, with dosages of 15 mg / kg and 45 mg / kg. Colo320 cells were purchased from Auris Biotech (Shanghai) Co., Ltd. and cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Passage was performed every three days, and cells within 15 generations were used. 7.5×106 corresponding cells were subcutaneously injected into the right chest of nude mice. The tumor reached at least 120 mm 3 Afterwards, the mice were randomly divided into groups of 3. Treatment began on the first day. The belotecan positive control group received a dose of 5 mg / kg, while the irinotecan positive control group received a dose of 15 mg / kg. The control group received normal saline. Dosing was continued once a week for four weeks.

[0386] Control and dosage instructions: 15mg / kg peptide conjugate and 5mg / kg pelotecan are used as controls. When the amount of substances is equal, the concentration of pelotecan contained in 15mg / kg peptide conjugate is 5mg / kg. 45mg / kg: The maximum tolerated dose of the peptide conjugate is about 150-200mg / kg, and 45mg / kg is about one-third to one-fifth of the maximum tolerated dose. The maximum tolerated dose of irinotecan is about 70mg / kg, and 15mg / kg is one-fifth of the maximum tolerated dose.

[0387] Table 8

[0388] The data indicate that the therapeutic effects of the polypeptide conjugates of the present invention are comparable to those of irinotecan and belotecan, and some conjugates have better efficacy than irinotecan and belotecan. Furthermore, compared with irinotecan and belotecan, the polypeptide conjugates of the present invention have significantly improved targeting and significantly reduced toxicity. Therefore, overall, they have a good effect in treating cancer.

[0389] [Example 36] Maximum Tolerated Dose (MTD) Detection Experiment of Peptide Conjugates

[0390] Experimental animals: 6 8-week-old mice, all female, were randomly divided into groups of six. Mice were dosed with different concentration gradients and monitored for 14 days. Mice were euthanized when they lost 20% of their initial body weight and were considered to have died from poisoning. The maximum tolerated dose (MTD) was defined as the highest dose level at which none of the six mice died from the drug, the body weight loss of a single mouse did not exceed 20%, or the average body weight loss within the group did not exceed 15%. The final statistical results, the maximum tolerated dose (MTD) of the peptide conjugate are shown in the table below:

[0391] Table 9

[0392] According to the data in the above table, it can be seen that the maximum tolerated dose of the polypeptide conjugate of the present invention is increased by dozens of times compared with belotecan alone, which is significantly higher than belotecan and is safer.

[0393] [Example 37] Peptide conjugate tumor homogenate activation efficiency experiment

[0394] Buffer configuration: 50 mM pH buffer (MES), 250 mM sodium chloride, adjusted to pH 5.0 with 0.5 M sodium hydroxide.

[0395] The peptide conjugate was prepared in a pH 5.0 buffer solution at a concentration of 1 mg / ml. 100 μg of tumor tissue homogenate (prepared using a Jingxin F6 / 10 handheld homogenizer) was added and the solution was incubated at 37°C for 2 hours. The tumor tissue homogenate effectively activated the peptide conjugate and released belotecan. High-performance liquid chromatography (HPLC) was used to measure the decrease in compound and the increase in belotecan, comparing the drug's activation efficiency in tumor tissue.

[0396] Release percentage = belotecan peak area / total peak area of ​​belotecan and all other belotecan-containing compounds

[0397] The results are as follows:

[0398] Table 10 Release efficiency percentage of polypeptide conjugates in different tissues (%)

[0399] According to the data in the above table, it can be seen that the polypeptide conjugate of the present invention is well released in homogenates of different cancer tissues, but is only slightly activated in the heart.

[0400] [Example 38] Enzyme cleavage activation experiment of berotelecan polypeptide conjugate

[0401] Buffer preparation: 50 mM MES, 250 mM sodium chloride, adjusted to pH 5.0 with 0.5 M sodium hydroxide. Legumain and cathepsin B are used at a concentration of 1 mg / mL. Compounds are prepared in buffer to a concentration of 0.5 μmol / mL. Accurately pipette 50 μL of the 0.5 μmol / mL compound and 50 μL of buffer into a centrifuge tube. Add 100 μL of legumain or cathepsin B and incubate at 37°C for 2 h. Analyze the reaction solution by LC-MS.

[0402] Legumain enzyme is highly expressed in the tumor microenvironment. Belotecan peptide conjugates can be cleaved by Legumain enzyme, which is highly expressed in tumor cells and tumor-associated macrophages, releasing belotecan.

[0403] [Example 39] Enzyme cleavage activation experiment of berotelecan polypeptide conjugate

[0404] Buffer preparation: 50 mM MES, 250 mM sodium chloride, adjusted to pH 5.0 with 0.5 M sodium hydroxide. Legumain and cathepsin B should be used at a concentration of 1 mg / mL. Prepare the compound to a concentration of 0.5 μmol / mL using the buffer. Accurately pipette 50 μL of the 0.5 μmol / mL compound and 50 μL of buffer into a centrifuge tube. Add 100 μL of legumain or cathepsin B and incubate at 37°C for 2 h. Analyze the reaction solution by LC-MS.

[0405] Legumain enzyme is highly expressed in the tumor microenvironment. The belotecan peptide conjugate can be cleaved by legumainase, which is highly expressed in tumor cells and tumor-associated macrophages, releasing belotecan.

[0406] [Example 40] Study on the efficacy of polypeptide conjugates in the treatment of SJSA-1 sarcoma animal model

[0407] Purpose of the experiment: To further compare the anti-tumor efficacy of different peptide linkers in the SJSA-1 sarcoma animal tumor model.

[0408] Test drugs: peptide conjugate and normal saline control group.

[0409] Experimental animals: 6-8 week old BALB / c mice, all female.

[0410] Preparation of tumor model:

[0411] Experimental purpose: To study the anti-tumor efficacy of the above-mentioned peptide conjugate in a tumor model, with a dosage of 30 mg / kg.

[0412] Experimental procedures: SJSA-1 cells were purchased from ATCC and cultured in DMEM containing 10% fetal bovine serum at 37°C in 5% CO2. Cells were passaged every three days and cells within the 15th passage were used. 4 × 10 6 The corresponding cells were injected subcutaneously into the right chest of nude mice. 3 Afterwards, the mice were randomly divided into groups of 5. Treatment began on the first day of treatment. The control group received normal saline. The drug was administered once a week for two weeks.

[0413] The experimental results are as follows:

[0414] Table 11

[0415] The data show that both MI-6PEG-AAGP-D2 and MI-6PEG-AAN-PAB-Belotecan have less than ideal tumor inhibition effects. A comparison of the tetrapeptide linker AANG and AANL clearly demonstrates that AANG exhibits superior tumor inhibition compared to AANL. Furthermore, as shown in Examples 27 and 36, the mouse MTD of AANG is approximately 2-fold higher than that of AANL. Clearly, AANG significantly reduces molecular toxicity compared to AANL. AANG is a more effective peptide linker than AANL and AAN-PAB.

[0416] For schematic diagrams prepared based on the data in Table 11, please refer to Figures 1-1, 1-2, and 1-3 of the specification.

[0417] It can also be seen from Figures 1-1, 1-2 and 1-3 that the comparison of the tumor volume growth curves of the tetrapeptide linkers AANG, AANL and AAN-PAB shows that the tumor volume growth curve of AANG is significantly slower, while the tumor volume growth curves of AANL and AAN-PAB are significantly steeper, indicating that the release rate of the peptide conjugate with AANG as the linker in tumor cells is faster than that of the peptide conjugate with AANL and AAN-PAB as the linker, and can produce better drug efficacy.

[0418] [Example 41] Study on the efficacy of polypeptide conjugates in the treatment of A2780 ovarian cancer animal model

[0419] Purpose of the experiment: To further compare the anti-tumor efficacy of different peptide linkers in the A2780 ovarian cancer animal tumor model.

[0420] Test drugs: peptide conjugate and normal saline control group.

[0421] Experimental animals: 6-8 week old BALB / c mice, all female.

[0422] Experimental purpose: To study the anti-tumor efficacy of the above-mentioned peptide conjugate in a tumor model, with a dosage of 30 mg / kg.

[0423] Experimental procedures: A2780 cells were purchased from ATCC and cultured in DMEM (1640) medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were passaged every three days and cells within the 15th passage were used. 8×10 6The corresponding cells were injected subcutaneously into the right axilla of nude mice. 3 Afterwards, the mice were randomly divided into groups of 5. Treatment began on the first day. The negative control group received normal saline. Dosing continued once a week for two weeks.

[0424] The experimental results are as follows:

[0425] Table 12

[0426] This mouse tumor model experiment further confirmed that the peptide linker AANG has a greater anti-tumor effect and lower toxicity than AANL and AAN-PAB, which is a significant advantage.

[0427] For schematic diagrams prepared based on the data in Table 12, please refer to Figures 2-1, 2-2, and 2-3 of the specification.

[0428] It can also be seen from Figures 2-1, 2-2, and 2-3 that the comparison of the tumor volume growth curves of the tetrapeptide linkers AANG, AANL, and AAN-PAB shows that the tumor volume growth curve of AANG is significantly slower, while the tumor volume growth curves of AANL and AAN-PAB are significantly steeper, indicating that the release rate of the peptide conjugate with AANG as the linker in tumor cells is faster than that of the peptide conjugate with AANL and AAN-PAB as the linker, and can produce better drug efficacy.

[0429] [Example 42] Study on the efficacy of polypeptide conjugates in the treatment of colo320 colorectal cancer animal model

[0430] Experimental purpose: To study the anti-tumor efficacy of the above compounds in the colo320 colorectal cancer animal tumor model.

[0431] Experimental drugs: peptide conjugate, irinotecan, belotecan and normal saline control group.

[0432] Experimental animals: 6-8 week old BALB / c mice, all female.

[0433] Preparation of tumor model:

[0434] Experimental purpose: To study the anti-tumor efficacy of the above-mentioned peptide conjugates, irinotecan and belotecan in tumor models, with dosages of 15 mg / kg and 45 mg / kg. Colo320 cells were purchased from Auris Biotech (Shanghai) Co., Ltd. and cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Passage was performed every three days, and cells within 15 generations were used. 7.5×106 corresponding cells were subcutaneously injected into the right chest of nude mice. The tumor reached at least 120 mm 3 Afterwards, the mice were randomly divided into groups of 3. Treatment began on the first day. The belotecan positive control group received a dose of 5 mg / kg, while the irinotecan positive control group received a dose of 15 mg / kg. The control group received normal saline. Dosing was continued once a week for four weeks.

[0435] Table 13

[0436] The schematic diagram prepared according to the data in Table 13 is shown in Figure 3 of the specification.

[0437] Comparison of the tetrapeptide linkers GGFG and GGFL clearly demonstrates that GGFG exhibits superior tumor suppression compared to GGFL. Furthermore, as shown in Examples 27 and 36, the mouse MTD of GGFG is approximately 2-fold higher than that of GGFL. Clearly, GGFG significantly reduces molecular toxicity compared to GGFL. GGFG is a more effective peptide linker than GGFL.

[0438] [Example 43] Study on the efficacy of polypeptide conjugates in the treatment of colo320 colorectal cancer animal model

[0439] Experimental purpose: To study the anti-tumor efficacy of the above compounds in the colo320 colorectal cancer animal tumor model.

[0440] Experimental drugs: peptide conjugate, irinotecan, belotecan and normal saline control group.

[0441] Experimental animals: 6-8 week old BALB / c mice, all female.

[0442] Preparation of tumor model:

[0443] Experimental purpose: To study the anti-tumor efficacy of the above-mentioned peptide conjugates, irinotecan and belotecan in tumor models, with dosages of 15 mg / kg and 45 mg / kg. Colo320 cells were purchased from Auris Biotech (Shanghai) Co., Ltd. and cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Passage was performed every three days, and cells within 15 generations were used. 7.5×106 corresponding cells were subcutaneously injected into the right chest of nude mice. The tumor reached at least 120 mm 3 Afterwards, the mice were randomly divided into groups of 3. Treatment began on the first day. The belotecan positive control group received a dose of 5 mg / kg, while the irinotecan positive control group received a dose of 15 mg / kg. The control group received normal saline. Dosing was continued once a week for four weeks.

[0444] Table 14

[0445] The schematic diagram prepared according to the data in Table 14 is shown in Figure 4 of the specification.

[0446] According to Table 14, Figure 4, and Examples 40, 41, and 42, for the polypeptide conjugates of the present invention having the general formula E-nPEG-L1-L2-L3-L4-D, when the L4 group is G (glycine), the polypeptide conjugates exhibit better efficacy than when the L4 group is L (leucine) or PAB (aminobenzyl alcohol). For specific examples, MI-6PEG-AANG-belotecan and MI-6PEG-AANL-belotecan undergo a first activation step in the tumor microenvironment to generate G-belotecan and L-belotecan, respectively. These steps then enter tumor cells for a second activation step to generate belotecan, thereby killing the tumor cells. G-belotecan is released faster in tumor cells than L-belotecan, resulting in better efficacy. Therefore, MI-6PEG-AANG-belotecan exhibits superior efficacy compared to MI-6PEG-AANL-belotecan.

[0447] [Example 44] Maximum Tolerated Dose (MTD) Detection Experiment of Peptide Conjugates

[0448] Experimental animals: 6-8 week old mice, all female, were randomly divided into groups of six. Mice were dosed with different concentration gradients and monitored for 14 days. Mice were euthanized when they lost 20% of their initial body weight and were considered to have died from poisoning. The maximum tolerated dose (MTD) was defined as the highest dose level at which all six mice did not die from the drug, the body weight loss of a single mouse did not exceed 20%, or the average body weight loss within the group did not exceed 15%. The final statistical results, the maximum tolerated dose (MTD) of the peptide conjugate are shown in the table below:

[0449] Table 15

[0450] For a schematic diagram prepared based on the data in Table 15, please refer to Figure 5 of the specification.

[0451] As shown in Table 15 and Figure 5 , for the polypeptide conjugates of the general formula E-nPEG-L1-L2-L3-L4-D of the present invention, when the L4 group is G (glycine), the polypeptide conjugates exhibit lower toxicity compared to when the L4 group is L (leucine) or PAB (aminobenzyl alcohol). For example, MI-6PEG-AANG-belotecan is more stable than MI-6PEG-AANL-belotecan because AANG is more stable than AANL in blood and normal tissues. The N and L ends of AANL are easily cleaved by proteases in normal tissues, releasing more L-belotecan in normal tissues and blood, causing additional toxicity. AANG, on the other hand, is more stable, releasing less L-belotecan in normal tissues and blood, resulting in lower toxicity.

[0452] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polypeptide conjugate, characterized in that, The structural formula of the polypeptide conjugate is as follows: E-nPEG-L1-L2-L3-L4-D Wherein, E is a group with maleimide; nPEG is n-polyethylene glycol, and n is an integer greater than or equal to 2 and less than or equal to 40: L1 and L2 are glycine, alanine, phenylalanine, threonine, serine; L3 is glycine, alanine, lysine, citrulline or asparagine; L4 is glycine, proline, p-aminobenzyl alcohol; D is belotecan or its derivative.

2. A polypeptide conjugate, characterized in that, The structural formula of the polypeptide conjugate is as follows: E-nPEG-L1-L2-L3-L4-D Wherein, E is a group with maleimide; nPEG is n-polyethylene glycol, and n is an integer greater than or equal to 2 and less than or equal to 40; L1 and L2 are glycine, alanine, phenylalanine, threonine, serine; L3 is glycine, alanine, lysine, citrulline or asparagine; L4 is leucine, isoleucine; D is belotecan or its derivative.

3. The polypeptide conjugate according to claim 1, wherein The structural formula of the polypeptide conjugate is as follows: E-nPEG-L1-L2-L3-L4-D Wherein, E is a group with maleimide; nPEG is n-polyethylene glycol, and n is an integer greater than or equal to 2 and less than or equal to 40; L1 and L2 are glycine, alanine, phenylalanine, threonine, serine; L3 is glycine, alanine, lysine, citrulline or asparagine; L4 is glycine; D is belotecan or its derivative.

4. The polypeptide conjugate according to any one of claims 1-3, characterized in that, The structure of the D group is as follows: Wherein, R is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl.

5. The polypeptide conjugate according to any one of claims 1-3, characterized in that, The E group structure is as follows: Wherein, n = 1-18.

6. The polypeptide conjugate according to claim 1, wherein, The polypeptide conjugate is any of the following structures:

7. The polypeptide conjugate according to claim 3, wherein The polypeptide conjugate is any of the following structures:

8. The polypeptide conjugate according to claim 2, wherein The polypeptide conjugate is any of the following structures:

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition described above comprises the polypeptide conjugate according to any one of claims 1-8 and a pharmaceutically acceptable carrier.

10. Use of the polypeptide conjugate according to any one of claims 1-8 and the pharmaceutical composition according to claim 9 in the preparation of an anti-tumor drug.

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