Tumor-targeted activated mitomycin c complex and use thereof

Abstract

The application belongs to the technical field of cancer treatment, and particularly relates to a tumor-targeting activated mitomycin C compound. The structural formula of the tumor-targeting activated mitomycin C compound is shown in the following formula: E-(PEG)x-L1-L2-L3-L4-D. The application has the following beneficial effects: the tumor-targeting activated mitomycin C compound has good water solubility and strong tumor targeting property; after the tumor-targeting activated mitomycin C compound is used, the tumor-targeting activated mitomycin C compound releases mitomycin C when reaching the tumor site, so as to kill tumor cells. Meanwhile, the compound releases less in normal tissues and blood, and has slight toxic and side effects on normal tissues, so that the purposes of targeting tumor tissues and reducing toxic and side effects can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of cancer treatment technology, specifically relating to a tumor-targeting and activated mitomycin C complex. Background Technology

[0002] Mitomycin C is a broad-spectrum antitumor antibiotic isolated and extracted from Streptomyces capitella culture. It has anticancer effects against various cancers. Its mechanism of action involves depolymerizing cellular DNA and inhibiting DNA replication, thereby suppressing tumor cell division. It has a broad antitumor spectrum and rapid onset of action, but its therapeutic index is low and its toxicity is relatively high. Clinically, it is suitable for gastrointestinal cancers, such as gastric cancer, intestinal cancer, liver cancer, and pancreatic cancer, with good efficacy. It is also effective against lung cancer, breast cancer, cervical cancer, and choriocarcinoma. It can also be used for malignant lymphoma and malignant pleural and peritoneal effusions.

[0003] Soft tissue sarcomas are a group of rare tumors originating from mesenchymal tissue, accounting for approximately 1% of adult cancers. There are over 60 different histological subtypes, each with its own unique biological behavior and response to systemic therapy. Patients with metastatic soft tissue sarcomas have a poor prognosis, and available systemic treatment options are limited. For decades, the primary treatment approach has been mitomycin C, with or without ifosfamide. Several phase II trials have demonstrated the activity of mitomycin C in anthracycline- and alkylating agent-resistant soft tissue sarcomas, suggesting its use as second- and third-line therapy. Recently, mitomycin C has shown similar progression-free survival to doxorubicin in first-line treatment and significant activity in liposarcoma and leiomyosarcoma subtypes. Mitomycin C has shown favorable toxicity profiles and has been approved for the treatment of metastatic soft tissue sarcomas in over 70 countries.

[0004] Colorectal cancer (CRC) is one of the most common cancers in the world, and it is quite harmful. It causes serious damage to the patient's intestinal health and causes trouble in daily life. Patients will experience pain in their intestines, which will affect their daily life and work. However, there are currently no new and effective drugs for the treatment of colorectal cancer. Summary of the Invention

[0005] This application provides a tumor-targeting activated mitomycin C complex and its application, aiming to solve the problems of high toxicity and insufficient efficacy of existing mitomycin C.

[0006] The first aspect of this application provides a tumor-targeting activated mitomycin C complex, the structural formula of which is shown below: E-(PEG). x -L1-L2-L3-L4-D,

[0007] The structural formula for E is shown below:

[0008]

[0009] 1≤n≤18, wavy lines are connecting keys;

[0010] PEG is polyethylene glycol, and the degree of polymerization x is: 2≤x≤20;

[0011] L1 and L2 may or may not exist. When L1 and L2 are present, L1 and L2 are each independently selected from one or more of glycine, alanine, phenylalanine, threonine and serine.

[0012] L3 is selected from one or more of glycine, alanine, lysine, phenylalanine, guanidinine, and asparagine;

[0013] L4 may or may not be present. When L4 is present, L4 is selected from one or more of glycine, leucine, isoleucine, and proline.

[0014] The structural formula for D is shown below:

[0015]

[0016] The tumor-targeting activated mitomycin C complex described in this application comprises a mitomycin derivative (the compound shown in structural formula D), a linker, and a polypeptide group, with the mitomycin derivative and polypeptide coupled via the linker. The polypeptide transports the mitomycin derivative to the tumor tissue, where it is activated by Legumin, a protein highly expressed by tumor cells and tumor-associated macrophages, in the acidic microenvironment of the tumor. This releases the cytotoxic mitomycin derivative, inducing immunogenic death of tumor cells, stimulating the body's anti-tumor immune function, and reducing the toxic side effects and cardiac function of the mitomycin derivative. This achieves the effect of inhibiting tumor growth while promoting anti-tumor immunity and reducing drug toxicity.

[0017] According to some embodiments of the tumor-targeting activated mitomycin C complex described in this application, the structural formula of the tumor-targeting activated mitomycin C complex is shown in formulas X1-X13:

[0018]

[0019]

[0020]

[0021] According to some embodiments of the tumor-targeting activated mitomycin C complex described in this application, the degree of polymerization x of the polyethylene glycol is: 5 ≤ x ≤ 10.

[0022] A second aspect of this application provides a pharmaceutical composition comprising the tumor-targeting activated mitomycin C complex described in the first aspect of this application, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[0023] A third aspect of this application provides the use of the tumor-targeting activated mitomycin C complex described in the first aspect of this application in the preparation of a drug for treating and / or preventing cancer.

[0024] The fourth aspect of this application provides the use of the pharmaceutical composition described in the second aspect of this application in the preparation of a medicament for treating and / or preventing cancer.

[0025] According to some embodiments of the application described in this application, the cancers include one or more of the following: gastrointestinal cancer, colorectal cancer, colon cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, gastric cancer, genitourinary system cancer, bladder cancer, testicular cancer, cervical cancer, malignant mesothelioma, osteosarcoma, esophageal cancer, laryngeal cancer, prostate cancer, hormone-resistant prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, triple-negative breast cancer, hematologic cancer, leukemia, acute primitive lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, ovarian cancer, brain cancer, neuroblastoma, Ewing sarcoma, renal cancer, epidermoid carcinoma, skin cancer, melanoma, and oral cancer.

[0026] According to some embodiments of the application described in this application, the cancer includes one or more of non-small cell lung cancer, small cell lung cancer, breast cancer, gastric cancer, esophageal cancer, colorectal cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer, and sarcoma.

[0027] The beneficial effects of this application include:

[0028] The tumor-targeting activated mitomycin C complex described in this application has good water solubility and strong tumor targeting. When this complex reaches the tumor site, it releases mitomycin C, thereby killing tumor cells. Simultaneously, the complex releases relatively little into normal tissues and blood, resulting in milder toxicity to normal tissues. Therefore, it can achieve the goal of targeting tumor tissue while minimizing toxicity. Detailed Implementation

[0029] The embodiments of the present invention are described in detail below. These embodiments are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0030] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0031] Unless otherwise specified, all materials, instruments, and reagents used in the embodiments of this application are commercially available. Unless otherwise specified, the technical means used in the embodiments are conventional methods well-known to those skilled in the art.

[0032] Example 1

[0033] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X1:

[0034]

[0035] The synthetic equation for the compound shown in formula X1 is as follows:

[0036]

[0037] The specific synthesis steps include:

[0038] (1) Synthesis of compound 1-II

[0039] Compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 1-I (850 mg, 1.54 mmol) were dissolved in dichloromethane (30 mL). Under ice bath cooling, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added to dichloromethane to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours.

[0040] The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction mixture, and the mixture was 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 product obtained under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 1-II as a light yellow solid (620 mg, yield 47.5%).

[0041] (2) Synthesis of compound 1-III

[0042] Compound 1-II (620 mg, 0.71 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour.

[0043] The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 1-III as a yellow foamy solid (415 mg, yield 90.0%).

[0044] (3) Synthesize compound MI-6PEG-AANG-Mitomycin-C (the compound shown in formula X1).

[0045] Compound 1-III (415 mg, 0.64 mmol) was dissolved in N,N-dimethylformamide (15 mL), and then compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours.

[0046] The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-AANG-Mitomycin-C as a purple solid (136 mg, yield 18.7%).

[0047] Example 2

[0048] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X9:

[0049]

[0050] The synthetic equation for the compound shown in formula X9 is as follows:

[0051]

[0052] The specific synthesis steps include:

[0053] (1) Synthesis of compound 2-II

[0054] Compound Mitomycin-C (the compound shown in Formula D, 500 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'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours.

[0055] The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction mixture, and the mixture was separated. The aqueous phase was extracted with dichloromethane (50 mL x 2), and the organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 2-II as a yellow solid (580 mg, yield 41.8%).

[0056] (2) Synthesis of compound 2-III

[0057] Compound 2-II (580 mg, 0.63 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour.

[0058] The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 2-III as a yellow solid (289 mg, yield 65.2%).

[0059] (3) Synthesize compound MI-6PEG-AANL-Mitomycin-C (the compound shown in formula X9).

[0060] Compound 2-III (289 mg, 0.41 mmol) was dissolved in N,N-dimethylformamide (15 mL), and then compound 1-IV (300 mg, 0.50 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) were added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours.

[0061] The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-AANL-Mitomycin-C as a purple solid (77 mg, yield 15.8%).

[0062] Example 3

[0063] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X12:

[0064]

[0065] The synthetic equation for the compound shown in formula X12 is as follows:

[0066]

[0067] The specific synthesis steps include:

[0068] (1) Synthesis of compound 3-II

[0069] Compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 3-I (935 mg, 1.52 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours.

[0070] The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction mixture, and the mixture was 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 product obtained under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 3-II as a yellow solid (760 mg, yield 54.4%).

[0071] (2) Synthesis of compound 3-III

[0072] Compound 3-II (760 mg, 0.82 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour.

[0073] The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 3-III as a yellow solid (495 mg, yield 85.2%).

[0074] (3) Synthesize compound MI-6PEG-GGFL-Mitomycin-C (the compound shown in formula X12).

[0075] Compound 3-III (495 mg, 0.70 mmol) was dissolved in N,N-dimethylformamide (15 mL), and then compound 1-IV (520 mg, 0.86 mmol) and diisopropylethylamine (350 mg, 2.71 mmol) were added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours.

[0076] The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-GGFL-Mitomycin-C as a purple solid (287 mg, yield 34.3%).

[0077] Example 4

[0078] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X13:

[0079]

[0080] The synthetic equation for the compound shown in formula X13 is as follows:

[0081]

[0082] The specific synthesis steps include:

[0083] (1) Synthesis of compound 4-II

[0084] Compound Mitomycin-C (the compound shown in Formula D, 500 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'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours.

[0085] The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction mixture, and the mixture was 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 product obtained under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 4-II as a yellow solid (845 mg, yield 64.4%).

[0086] (2) Synthesis of compound 4-III

[0087] Compound 4-II (845 mg, 0.97 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 4-III as a yellow solid (600 mg, yield 94.8%).

[0088] (3) Synthesize compound MI-6PEG-GGFG-Mitomycin-C (the compound shown in formula X13).

[0089] Compound 4-III (600 mg, 0.92 mmol) was dissolved in N,N-dimethylformamide (15 mL), and then compound 1-IV (580 mg, 0.96 mmol) and diisopropylethylamine (400 mg, 3.10 mmol) were added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-GGFG-Mitomycin-C as a purple solid (345 mg, yield 32.9%).

[0090] Example 5

[0091] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X2:

[0092]

[0093] The synthetic equation for the compound shown in formula X2 is as follows:

[0094]

[0095] The specific synthesis steps include:

[0096] (1) Synthesis of compound 5-II

[0097] Compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 5-I (830 mg, 1.54 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours.

[0098] The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction mixture, and the mixture was 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 product obtained under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 5-II as a yellow solid (608 mg, yield 47.4%).

[0099] (2) Synthesis of compound 5-III

[0100] Compound 5-II (608 mg, 0.71 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 5-III as a yellow foamy solid (398 mg, yield 88.5%).

[0101] (3) Synthesize compound MI-6PEG-GANG-Mitomycin-C (the compound shown in formula X2).

[0102] Compound 5-III (398 mg, 0.63 mmol) was dissolved in N,N-dimethylformamide (15 mL). Compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were then added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours. TLC was used to confirm the completeness of the reaction. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-GANG-Mitomycin-C as a purple solid (149 mg, yield 21.1%).

[0103] Example 6

[0104] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X3:

[0105]

[0106] The synthetic equation for the compound shown in formula X3 is as follows:

[0107]

[0108] The specific synthesis steps include:

[0109] (1) Synthesis of compound 6-II

[0110] Compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 6-I (900 mg, 1.54 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours.

[0111] The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction mixture, and the mixture was separated. The aqueous phase was extracted with dichloromethane (50 mL x 2), and the organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 6-II as a yellow solid (496 mg, yield 36.7%).

[0112] (2) Synthesis of compound 6-III

[0113] Compound 6-II (496 mg, 0.55 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 6-III as a yellow foamy solid (243 mg, yield 65.2%).

[0114] (3) Synthesize compound MI-6PEG-TANG-Mitomycin-C (the compound shown in formula X3).

[0115] Compound 6-III (243 mg, 0.36 mmol) was dissolved in N,N-dimethylformamide (15 mL). Compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were then added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours. TLC was used to confirm the complete reaction. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-TANG-Mitomycin-C as a purple solid (51 mg, yield 12.2%).

[0116] Example 7

[0117] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X4:

[0118]

[0119] The synthetic equation for the compound shown in formula X4 is as follows:

[0120]

[0121] The specific synthesis steps include:

[0122] (1) Synthesis of compound 7-II

[0123] Compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 7-I (877 mg, 1.54 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours.

[0124] The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction mixture, and the mixture was separated. The aqueous phase was extracted with dichloromethane (50 mL x 2), and the organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 7-II as a yellow solid (525 mg, yield 39.5%).

[0125] (2) Synthesis of compound 7-III

[0126] Compound 7-II (525 mg, 0.59 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 7-III as a yellow foamy solid (299 mg, yield 76.4%).

[0127] (3) Synthesize compound MI-6PEG-SANG-Mitomycin-C (the compound shown in formula X4).

[0128] Compound 7-III (299 mg, 0.45 mmol) was dissolved in N,N-dimethylformamide (15 mL). Compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were then added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours. TLC was used to confirm the completeness of the reaction. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-SANG-Mitomycin-C as a purple solid (98 mg, yield 18.9%).

[0129] Example 8

[0130] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X5:

[0131]

[0132] The synthetic equation for the compound shown in formula X5 is as follows:

[0133]

[0134] The specific synthesis steps include:

[0135] (1) Synthesis of compound 8-II

[0136] Compound Mitomycin-C (compound shown in Formula D, 500 mg, 1.50 mmol) and compound 8-I (970 mg, 1.54 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours. The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction solution, and the mixture was 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 product obtained by vacuum evaporation was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 8-II as a yellow solid (696 mg, yield 49.1%).

[0137] (2) Synthesis of compound 8-III

[0138] Compound 8-II (696 mg, 0.74 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 8-III as a yellow foamy solid (415 mg, yield 77.5%).

[0139] (3) Synthesize compound MI-6PEG-FANG-Mitomycin-C (the compound shown in formula X5).

[0140] Compound 8-III (415 mg, 0.57 mmol) was dissolved in N,N-dimethylformamide (15 mL). Compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were then added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours. TLC was used to confirm the completeness of the reaction. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-FANG-Mitomycin-C as a purple solid (185 mg, yield 26.8%).

[0141] Example 9

[0142] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X6:

[0143]

[0144] The synthetic equation for the compound shown in formula X6 is as follows:

[0145]

[0146] The specific synthesis steps include:

[0147] (1) Synthesis of compound 9-II

[0148] Compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 9-I (786 mg, 1.54 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours.

[0149] The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction mixture, and the mixture was 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 product obtained under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 9-II as a yellow solid (668 mg, yield 52.5%).

[0150] (2) Synthesis of compound 9-III

[0151] Compound 9-II (668 mg, 0.80 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 9-III as a yellow foamy solid (460 mg, yield 95.1%).

[0152] (3) Synthesize compound MI-6PEG-AAAG-Mitomycin-C (the compound shown in formula X6).

[0153] Compound 9-III (460 mg, 0.76 mmol) was dissolved in N,N-dimethylformamide (15 mL). Compound 1-IV (550 mg, 0.92 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were then added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours. TLC was used to confirm the completeness of the reaction. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-AAAG-Mitomycin-C as a purple solid (306 mg, yield 36.9%).

[0154] Example 10

[0155] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X7:

[0156]

[0157] The synthetic equation for the compound shown in formula X7 is as follows:

[0158]

[0159] The specific reaction steps include:

[0160] (1) Synthesis of compound 10-II

[0161] Compound Mitomycin-C (compound shown in Formula D, 500 mg, 1.50 mmol) and compound 10-I (1.03 g, 1.54 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours. The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction solution, and the mixture was 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 product obtained by vacuum evaporation was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 10-II as a yellow solid (960 mg, yield 65.0%).

[0162] (2) Synthesis of compound 10-III

[0163] Compound 10-II (960 mg, 0.98 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 10-III as a yellow foamy solid (785 mg, yield 81.4%).

[0164] (3) Synthesize compound MI-6PEG-AAKG-Mitomycin-C (the compound shown in formula X7).

[0165] Compound 10-III (785 mg, 0.80 mmol) was dissolved in N,N-dimethylformamide (15 mL). Compound 1-IV (550 mg, 0.92 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were then added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours. TLC analysis showed the reaction was complete. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation under reduced pressure was dissolved in dichloromethane (50 mL), and 10 mL of trifluoroacetic acid was added. The reaction was carried out at room temperature (25 °C) for 3 hours. TLC analysis showed the reaction was complete. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-AAKG-Mitomycin-C as a purple solid (226 mg, yield 24.6%).

[0166] Example 11

[0167] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X8:

[0168]

[0169] The synthetic equation for the compound shown in formula X8 is as follows:

[0170]

[0171] The specific synthesis steps include:

[0172] (1) Synthesis of compound 11-II

[0173] Compound Mitomycin-C (compound shown in Formula D, 500 mg, 1.50 mmol) and compound 11-I (940 mg, 1.54 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours. The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction solution, and the mixture was 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 product obtained by vacuum evaporation was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 11-II as a yellow solid (800 mg, yield 57.5%).

[0174] (2) Synthesis of compound 11-III

[0175] Compound 11-II (800 mg, 0.86 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 11-III as a yellow foamy solid (590 mg, yield 97.3%).

[0176] (3) Synthesize compound MI-6PEG-AACG-Mitomycin-C (the compound shown in formula X8).

[0177] Compound 11-III (590 mg, 0.84 mmol) was dissolved in N,N-dimethylformamide (15 mL). Compound 1-IV (550 mg, 0.92 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were then added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours. TLC was used to confirm the completeness of the reaction. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-AACG-Mitomycin-C as a purple solid (255 mg, yield 25.5%).

[0178] Example 12

[0179] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X10:

[0180]

[0181] The synthetic equation for the compound shown in formula X10 is as follows:

[0182]

[0183] The specific reaction steps include:

[0184] (1) Synthesis of compound 12-II

[0185] Compound Mitomycin-C (compound shown in Formula D, 500 mg, 1.50 mmol) and compound 12-I (783 mg, 1.54 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours. The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction solution, and the mixture was 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 product obtained by vacuum evaporation was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 12-II as a yellow solid (545 mg, yield 44.0%).

[0186] (2) Synthesis of compound 12-III

[0187] Compound 12-II (545 mg, 0.66 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (1 mL) was added to N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 12-III as a yellow foamy solid (369 mg, yield 92.8%).

[0188] (3) Synthesize compound MI-6PEG-GGGP-Mitomycin-C (the compound shown in formula X10).

[0189] Compound 12-III (369 mg, 0.61 mmol) was dissolved in N,N-dimethylformamide (15 mL). Compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were then added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at 25 °C for 3 hours. TLC was used to confirm the completeness of the reaction. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-GGGP-Mitomycin-C as a purple solid (168 mg, yield 25.3%).

[0190] Example 13

[0191] A tumor-targeting activated mitomycin C complex, the structural formula of which is shown in X11:

[0192]

[0193] The synthetic equation for the compound shown in formula X11 is as follows:

[0194]

[0195] The specific synthesis steps include:

[0196] (1) Synthesis of compound 13-II

[0197] Compound Mitomycin-C (compound shown in Formula D, 500 mg, 1.50 mmol) and compound 13-I (826 mg, 1.54 mmol) were dissolved in dichloromethane (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) were added under ice bath cooling to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 2 hours. The reaction was confirmed to be complete by TLC. Water (80 mL) was added to the reaction solution, and the mixture was 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 product obtained by vacuum evaporation was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 8:1) to give compound 13-II as a yellow solid (558 mg, yield 43.6%).

[0198] (2) Synthesis of compound 13-III

[0199] Compound 13-II (558 mg, 0.65 mmol) was dissolved in N,N-dimethylformamide (20 mL). Piperidine (1 mL) was added to the N,N-dimethylformamide to form a reaction solution, and the reaction was carried out at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluting with dichloromethane (DCM):methanol (MeOH) at a volume ratio of 100:1 to 5:1) to give compound 13-III as a yellow foamy solid (363 mg, yield 88.6%).

[0200] (3) Synthesize compound MI-6PEG-AAGP-Mitomycin-C (the compound shown in formula X11).

[0201] Compound 13-III (363 mg, 0.58 mmol) was dissolved in N,N-dimethylformamide (15 mL). Compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were then added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours. TLC was used to confirm the completeness of the reaction. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation under reduced pressure was purified by reverse-phase column chromatography to give compound MI-6PEG-AAGP-Mitomycin-C as a purple solid (134 mg, yield 20.7%).

[0202] Performance studies of the tumor-targeting activated mitomycin C complexes described in Examples 1-13 of this application:

[0203] A2780 cells (cells listed in Table 1) were cultured in complete medium (RPMI 1640 (DMEM high glucose medium) + 10% fetal bovine serum + 1XP / S + 1mM sodium pyruvate solution) and cultured in an incubator at 37°C and 5% CO2 until the cells reached a sufficient number. The cells were then collected, centrifuged at 1000g for 5 min, and resuspended in an appropriate volume of 10% RMPI 1640 (DMEM high glucose medium) medium. The cell density was adjusted to 4,000,000 cells per milliliter.

[0204] 100 μL of cell culture medium containing different concentrations of the drug was added to each 96-well culture plate. Control wells (0.1% DMSO) containing only the corresponding drug solvent and no drug were included, as well as blank wells containing only culture medium and no cells. Three parallel wells were set up for each group. Cells after counting were seeded into the 96-well plates at a concentration of 5000 cells per well (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 CCK8 (5 mg / ml) was added to each well, and the plates were incubated for approximately 2 hours. The absorbance at 450 nm was then measured.

[0205] Calculate cell viability and the half-maximal inhibitory concentration (IC50) of the drug on cells.

[0206] Cell viability = [(Experimental group absorbance - Blank control absorbance) / (Control group absorbance - Blank control absorbance)] × 100%

[0207] The half-maximal inhibitory concentration (IC50) of the drug on cells was calculated using Graphpad Prism 8 software based on cell viability, and the results are shown in Tables 1 and 2.

[0208] Note: Different drug concentrations in cell culture medium containing different drug concentrations refer to setting the maximum initial concentration of the test drug to 1000 μM, and the initial concentrations of the drug after activation and before activation to 100 μM. The drugs were serially diluted at a ratio of 1:4 to form 9 dose groups (3 replicates per group). The concentration of the drug solvent (DMSO) in all wells was controlled at 0.1%. The control group was the one with only drug solvent (0.1% DMSO) and the blank group was the one with only culture medium and no cells.

[0209] Legumain activation procedure: Buffer preparation: distilled water, 50 mM MES, 250 mM sodium chloride, pH adjusted to 5.0 with 0.5 M sodium hydroxide. Use a Legumain concentration of 1 mg / mL. Prepare a 0.5 μmol / mL solution of the tumor-targeting activating mitomycin C complex using buffer. Accurately transfer 50 μL of the 0.5 μmol / mL solution and 50 μL of buffer into a centrifuge tube, add 100 μL of Legumain, and incubate at 37°C for 2 hours.

[0210] Table 1 shows the inactive tumor-targeted activated mitomycin C complex and the toxicity of mitomycin C in different cell lines.

[0211]

[0212]

[0213] Table 2 shows the activated tumor-targeting mitomycin C complex and the toxicity of mitomycin C in different cell lines.

[0214] The compound shown in formula D 0.224umol / L 0.015umol / L 0.077umol / L 0.192umol / L Example 1 0.54umol / L 0.12umol / L 0.32umol / L 0.46umol / L Example 2 0.47umol / L 0.18umol / L 0.45umol / L 0.39umol / L Example 5 0.65umol / L 0.17umol / L 0.18umol / L 0.60umol / L Example 6 0.51umol / L 0.20umol / L 0.36umol / L 0.43umol / L Example 7 0.42umol / L 0.092umol / L 0.43umol / L 0.40umol / L Example 8 0.33umol / L 0.11umol / L 0.22umol / L 0.31umol / L

[0215] As can be seen from Table 1, the cytotoxicity of the unactivated tumor-targeted mitomycin C complex was significantly reduced compared to mitomycin C alone, indicating that the complex effectively shielded the toxicity of mitomycin C.

[0216] As shown in Tables 1 and 2, the IC50 of the tumor-targeted mitomycin C complex in the above cells is higher than that of mitomycin C. Therefore, the tumor-targeted mitomycin C complex provided in Table 1 exhibits significantly reduced cytotoxicity without Legumain activation, meaning that the tumor-targeted mitomycin C complex provided in Table 1 is less toxic than mitomycin C itself.

[0217] As shown in Table 2, the toxicity of the tumor-targeted mitomycin C complex increased significantly after activation by Legumain compared to before activation. This indicates that in the tumor microenvironment with high Legumain expression, the tumor-targeted mitomycin C complex can be activated and released, achieving directed activation and targeted release.

[0218] 2. Pharmacodynamic study of the tumor-targeted activated mitomycin C complex described in this application in the treatment of the A2780 tumor model.

[0219] Test drugs: the tumor-targeting and activating mitomycin C complex described in Examples 1-13 of this application, mitomycin C, and a saline control group.

[0220] Experimental animals: 6-8 week old BALB / c mice, all of which were female.

[0221] Preparation of tumor models:

[0222] 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 up to passage 15 were used. 5 × 10⁶ cells were then cultured. 6 One corresponding cell was subcutaneously injected into the right axilla of a nude mouse. The tumor reached at least 100 mm. 3Mice were then randomly divided into groups of three. Treatment began on day one. Mitomycin C was administered at 2.5 mg / kg as a positive control, while the tumor-targeting mitomycin C complex described in Examples 1-13 was administered at 10 mg / kg as the experimental group. The negative control group received saline. Administration was once weekly for three weeks. Results are shown in Table 3.

[0223] Table 3

[0224] physiological saline 2435.19 1932.74％ +25.6％ The compound shown in formula D 1215.86 996.61％ -12.5％ X1 798.24 617.07％ +5.9％ X2 995.18 786.52％ +6.5％ X3 1353.26 1081.40％ +3.2％ X4 1092.54 857.23％ +5.8％ X5 1589.39 1266.65％ +9.5％ X6 1430.28 1131.91％ +9.3％ X7 790.34 614.86％ +0.8％ X8 744.18 606.55％ +1.6％ X9 1254.86 1019.13％ -4.9％ X10 830.95 662.85％ -6.9％ X11 721.46 578.70％ -5.4％ X12 1141.63 902.55％ -8.2％ X13 836.26 672.34％ -7.9％

[0225] As can be seen from Table 3, among the tumor-targeted activated mitomycin C complexes X1-X13, except for X3, X5, X6 and X9, the other drugs have better inhibitory effects on A2780 tumors than mitomycin C, indicating that the tumor-targeted activated mitomycin C complexes described in this application have a significant improvement in efficacy compared to mitomycin C.

[0226] 3. Pharmacodynamic study of the tumor-targeted activated mitomycin C complex described in this application in the treatment of the MKN45 tumor model.

[0227] Test drugs: the tumor-targeting and activating mitomycin C complex described in Examples 1-13 of this application, mitomycin C, and a saline control group.

[0228] Experimental animals: 6-8 week old BALB / c mice, all of which were female.

[0229] Preparation of tumor models:

[0230] MKN45 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 up to passage 15 were used. 5 × 10⁶ cells were then cultured. 6 One corresponding cell was subcutaneously injected into the right axilla of a nude mouse. The tumor reached at least 100 mm. 3 Mice were then randomly divided into groups of three. Treatment began on day one. Mitomycin C was administered at 2.5 mg / kg as a positive control, while the tumor-targeting mitomycin C complex conjugate described in Examples 1-13 was administered at 10 mg / kg as the experimental group. The negative control group received saline. Administration was once weekly for four weeks. Results are shown in Table 4.

[0231] Table 4

[0232] physiological saline 813.94±190.21 0 +15.2％ The compound shown in formula D 770.32±465.69 5.36％ -9.6％ X1 380.47±206.51 53.26％ +9.8％ X2 463.34±84.55 43.07％ +8.4％ X3 589.62±163.42 27.56％ +10.1％ X4 551.08±131.70 32.29％ +10.5％ X5 621.24±221.73 23.67％ +9.6％ X6 703.54±146.33 13.56％ +9.0％ X7 568.55±158.31 30.15％ +8.1％ X8 524.16±136.31 35.60％ +6.5％ X9 665.40±278.06 18.25％ +6.8％ X10 615.35±176.53 24.40％ +11.2％ X11 602.18±149.83 26.02％ +7.8％ X12 768.63±290.38 5.57％ +10.2％ X13 553.37±126.90 32.01％ +6.4％

[0233] As can be seen from Table 4, the tumor-targeted mitomycin C complexes X1-X13 described in this application have better inhibitory effects on MKN45 tumors than mitomycin C. That is, the tumor-targeted mitomycin C complexes described in this application have a significantly improved efficacy compared to mitomycin C, and the weight of mice did not decrease significantly, while the weight of mice in the mitomycin C group decreased significantly. This indicates that the toxicity of the tumor-targeted mitomycin C complexes described in this application is significantly reduced compared to mitomycin C.

[0234] 4. MTD assay for the tumor-targeted activated mitomycin C complex described in this application.

[0235] Experimental animals: 6-8 week old mice, all female, were randomly divided into groups of six. Mice were administered the drug at concentrations of 0.1 μmol / kg, 0.2 μmol / kg, 0.4 μmol / kg, 0.8 μmol / kg, and 1.0 μmol / kg, respectively, and monitored for 14 days. Mice were euthanized when they lost 20% of their initial body weight, which was considered death due to poisoning. The results are shown in Table 5.

[0236] MTD was defined as the highest dose level in which none of the six mice died from the drug, and the weight loss of a single mouse did not exceed 20%, or the average weight loss within the group did not exceed 15%.

[0237] Table 5

[0238] The compound shown in formula D 4mg / kg X1 120mg / kg X2 100mg / kg X3 120mg / kg X4 120mg / kg X5 150mg / kg X6 120mg / kg X7 120mg / kg X8 150mg / kg X9 70mg / kg X10 100mg / kg X11 100mg / kg X12 80mg / kg X13 120mg / kg

[0239] As can be seen from Table 5, the maximum lethal concentration of the tumor-targeting activated mitomycin C complex prepared in the embodiments of this application is significantly higher than that of mitomycin C itself in mice. Because it is basically not released in normal tissues, the toxic side effects are greatly reduced, and therefore it is expected to be used in clinical practice at higher doses.

[0240] 5. Activation efficiency experiment of the tumor-targeting mitomycin C complex described in this application in mouse tumor homogenates and normal tissues.

[0241] PBS buffer: Beijing Lanjieke Technology Co., Ltd., Cat. NO: BL302A, Lot. No: 24149299.

[0242] The tumor-targeting mitomycin C complexes described in Examples 1-13 of this application were each prepared into 1 mg / ml solutions using PBS buffer. 200 μL of each of these complexes was added to 100 μg of tumor tissue homogenate or normal tissue homogenate (the tissue homogenate was prepared from euthanized mice using a Jingxin F6 / 10 handheld homogenizer). The solutions were incubated at 37°C for 2 hours. HPLC was used to detect the decrease in the compound concentration and the increase in mitomycin C concentration, allowing for comparison of the drug's activation efficiency in tumor tissue.

[0243] Activation efficiency = (Total peak area of ​​mitomycin C + Total peak area of ​​mitomycin C and compounds containing one amino acid) / Total peak area of ​​mitomycin C and all other compounds containing mitomycin C

[0244] The results are shown in Tables 6 and 7:

[0245] Table 6 shows the activation efficiency of the tumor-targeting mitomycin C complex in different tumor homogenates.

[0246] X1 88.2 76.5 92.3 X2 87.1 88.3 87.6 X3 79.6 93.1 88.8 X4 87.0 78.6 91.2 X5 74.6 82.1 87.6 X6 89.0 79.6 86.7 X7 76.8 86.7 88.2 X8 77.5 90.4 93.1 X9 74.3 78.4 77.6 X10 88.9 86.6 85.4 X11 87.8 83.4 81.5 X12 79.7 82.1 85.5 X13 93.1 90.0 86.0

[0247] As can be seen from Table 6, the tumor-targeted mitomycin C complex described in Examples 1-13 of this application has good release efficiency in different cancer tissue homogenates.

[0248] Table 7 shows the percentage of release efficiency of the tumor-targeted mitomycin C complex in normal mouse tissues.

[0249]

[0250]

[0251] As can be seen from Table 7, the tumor-targeted mitomycin C complex described in this application is released in a very low proportion in normal tissues. Except for a small amount released in the liver and blood, it is released very little in other tissues.

[0252] As can be seen from Tables 6 and 7, the tumor-targeting activated mitomycin C complex described in this application is released in large quantities in tumor homogenate. This is because mitomycin C can be activated by enzymes highly expressed in the tumor microenvironment, while it is released very little or almost not at all in normal tissues. Therefore, the damage to normal tissues and organs is significantly reduced compared to mitomycin C itself, thereby effectively improving the safety window and making it possible to use higher doses in clinical practice.

[0253] Although the above embodiments have been shown and described, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made to the above embodiments by those skilled in the art are within the protection scope of the present invention.

Claims

1. A tumor-targeting activated mitomycin C complex, characterized in that, The structural formula of the tumor-targeting activated mitomycin C complex is shown below: ， The application of the tumor-targeting activated mitomycin C complex in the preparation of a treatment for A2780 and MKN45 tumors.

2. A pharmaceutical composition, characterized in that, Includes the tumor-targeting activated mitomycin C complex of claim 1, and its pharmaceutically acceptable salt.

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