Tumor targeted activated mitomycin C compound and application thereof

Abstract

The invention belongs to the technical field of cancer treatment, and particularly relates to a tumor targeted activated mitomycin C compound. The structural formula of the tumor targeted activated mitomycin C compound is shown as the following formula: E-(PEG) x-L1-L2-L3-L4-D. The invention further discloses a preparation method of the tumor targeted activated mitomycin C compound. The tumor targeted activated mitomycin C compound has the beneficial effects that the tumor targeted activated mitomycin C compound has relatively good water solubility and relatively strong tumor targeting property, after the tumor targeted activated mitomycin C compound is used, the tumor targeted activated mitomycin C compound can release mitomycin C when reaching a tumor part, so that the tumor targeted activated mitomycin C compound has a good tumor targeting effect, and the tumor targeted activated mitomycin C compound can be applied to tumor treatment. Therefore, tumor cells are killed. Meanwhile, the compound is less released in normal tissues and blood and has slight toxic and side effects on the normal tissues, so that the purposes of targeting tumor tissues and reducing toxic and side effects can be achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer treatment, and particularly relates to a tumor-targeted activated mitomycin C complex. Background Art

[0002] Mitomycin C is a broad-spectrum antitumor antibiotic isolated from the culture broth of Streptomyces caespitosus. It has antitumor effects on various cancers. Its mechanism of action can depolymerize the DNA of cells and at the same time hinder the replication of DNA, thereby inhibiting the division of tumor cells. It has a relatively broad antitumor spectrum and rapid action, but the therapeutic index is not high and the toxicity is relatively large. Clinically, it is applicable to digestive tract cancers such as gastric cancer, intestinal cancer, liver cancer, and pancreatic cancer, and has good curative effects. It is also effective for lung cancer, breast cancer, cervical cancer, and choriocarcinoma. It can also be used for malignant lymphoma and cancerous pleural and peritoneal effusions.

[0003] Soft tissue sarcomas are a group of rare tumors originating from mesenchymal tissues, accounting for about 1% of adult cancers. There are more than 60 different histological subtypes, each with its own unique biological behavior and response to systemic therapy. The prognosis of patients with metastatic soft tissue sarcomas is poor, and there are few available systemic treatment options. For decades, the main treatment modality has been mitomycin C with or without ifosfamide. Several phase II trials have shown that mitomycin C has activity in anthracycline- and alkylating agent-resistant soft tissue sarcomas and is recommended for second-line and third-line treatment. Recently, mitomycin C has shown a progression-free survival similar to doxorubicin in first-line treatment and has significant activity in the liposarcoma and leiomyosarcoma subtypes. Mitomycin C has shown a favorable toxicity profile and has been approved for the treatment of metastatic soft tissue sarcoma in more than 70 countries / regions.

[0004] Colorectal cancer (CRC) is one of the most common tumors worldwide, with relatively serious harm, severely damaging the intestinal health of patients, and bringing troubles to daily life. The patient's intestine will be accompanied by pain symptoms, affecting daily life and work. However, there is currently a lack of new effective therapeutic drugs for the treatment of colorectal cancer. Summary of the Invention

[0005] The present application provides a tumor-targeted activated mitomycin C complex and its application, aiming to solve the problems of large toxicity and insufficient efficacy of existing mitomycin.

[0006] The first aspect of the present application provides a tumor-targeted activated mitomycin C complex, and the structural formula of the tumor-targeted activated mitomycin C complex is shown as the following formula: E-(PEG) x -L1-L2-L3-L4-D,

[0007] wherein, the structural formula of E is shown as the following formula:

[0008]

[0009] 1 ≤ n ≤ 18, and the wavy line is a connecting bond;

[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 exist, 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, citrulline, and asparagine;

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

[0014] The structural formula of D is shown as follows:

[0015]

[0016] The tumor-targeted activated mitomycin C complex described in the present application includes a mitomycin derivative (the compound shown in structural formula D), a linker, and a polypeptide group. The mitomycin derivative and the polypeptide are conjugated through the linker. The mitomycin derivative is transported to the tumor tissue through the polypeptide, and the mitomycin derivative can be activated by Legumain highly expressed in tumor cells and tumor-associated macrophages in the slightly acidic microenvironment of the tumor, so that the cytotoxic drug mitomycin derivative is released, causing immunogenic death of tumor cells, stimulating the anti-tumor immune function of the body, reducing the toxic side effects of the mitomycin derivative and the impact on cardiac function, and achieving the effect of inhibiting tumor growth while promoting anti-tumor immunity and reducing the drug's toxic side effects.

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

[0018]

[0019]

[0020]

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

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

[0023] The third aspect of the present application provides an application of the tumor-targeted activated mitomycin C complex described in the first aspect of the present application in the preparation of a medicament for treating and / or preventing cancer.

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

[0025] According to some embodiments of the application described in the present application, the cancer includes one or more of gastrointestinal cancer, colorectal cancer, colon cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, gastric cancer, urogenital system cancer, bladder cancer, testicular cancer, cervical cancer, malignant mesothelioma, osteogenic sarcoma, 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, blood cancer, leukemia, acute lymphoblastic 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, kidney cancer, epidermoid carcinoma, skin cancer, melanoma, and oral cancer.

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

[0027] The beneficial effects of the present application include:

[0028] The tumor-targeted activated mitomycin C complex described in the present application has good water solubility and strong tumor targeting. After using the tumor-targeted activated mitomycin C complex described in the present application, when the tumor-targeted activated mitomycin C complex reaches the tumor site, mitomycin C will be released, thereby killing tumor cells. At the same time, less of the complex is released in normal tissues and blood, so its toxic and side effects on normal tissues are relatively mild. Therefore, it can achieve the purpose of targeting tumor tissues and reducing toxic and side effects. Detailed Description of the Embodiments

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

[0030] In the present invention, the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0031] The materials, instruments, reagents, etc. used in the embodiments of this application can be obtained from commercial channels without special instructions. The technical means used in the embodiments are conventional means well-known to those skilled in the art without special instructions.

[0032] Example 1

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

[0034]

[0035] The synthesis equation of the compound shown in Formula X1 is:

[0036]

[0037] The specific synthesis steps include:

[0038] (1) Synthesize compound 1-II

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

[0040] TLC analysis showed that the reaction was complete. Water (80 mL) was added to the reaction solution, and the mixture was separated by liquid-liquid extraction. The aqueous phase was extracted with dichloromethane (50 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residue obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) in a volume ratio of 100:1 to 8:1) to give compound 1-II as a yellowish 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 the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 1 hour.

[0043] TLC analysis showed that the reaction was complete. The reaction solution was evaporated under reduced pressure. The residue obtained by evaporation under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) in 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) Synthesis of 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 compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction was carried out at room temperature (25 °C) for 3 hours.

[0046] TLC analysis showed that the reaction was complete. The reaction solution was evaporated under reduced pressure. The residue 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-targeted activated mitomycin C complex, the structural formula of which is as shown in X9:

[0049]

[0050] The synthesis equation of the compound shown in formula X9 is:

[0051]

[0052] The specific synthesis steps include:

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

[0054] Dissolve compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 2-I (920 mg, 1.51 mmol) in dichloromethane (30 mL). Under ice-bath cooling, add O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) to form a reaction solution, and react at room temperature (25 °C) for 2 hours.

[0055] Detect that the reaction is complete by TLC. Add water (80 mL) to the reaction solution, separate the layers, extract the aqueous phase with dichloromethane (50 mL × 2), combine the organic phases, dry over anhydrous sodium sulfate, and evaporate to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain Compound 2-II as a yellow solid (580 mg, yield 41.8%).

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

[0057] Dissolve compound 2-II (580 mg, 0.63 mmol) in N,N-dimethylformamide (20 mL), and then add piperidine (1 mL) to N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 1 hour.

[0058] Detect that the reaction is complete by TLC. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 5:1) to obtain Compound 2-III as a yellow solid (289 mg, yield 65.2%).

[0059] (3) Synthesis of Compound MI-6PEG-AANL-Mitomycin-C (the compound shown in Formula X9)

[0060] Dissolve compound 2-III (289 mg, 0.41 mmol) in N,N-dimethylformamide (15 mL), and then add compound 1-IV (300 mg, 0.50 mmol) and diisopropylethylamine (250 mg, 1.94 mmol) to N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 3 hours.

[0061] The TLC detection showed that the reaction was complete. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by reverse-phase column to obtain the 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, whose structural formula is shown as X12:

[0064]

[0065] The synthesis equation of the compound shown in formula X12 is:

[0066]

[0067] The specific synthesis steps include:

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

[0069] Dissolve compound Mitomycin-C (the compound shown in formula D, 500 mg, 1.50 mmol) and compound 3-I (935 mg, 1.52 mmol) in dichloromethane (30 mL). Under ice-bath cooling, add O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) to form a reaction solution, and react at room temperature (25 °C) for 2 hours.

[0070] The TLC detection showed that the reaction was complete. Add water (80 mL) to the reaction solution, separate the layers, extract the aqueous phase with dichloromethane (50 mL × 2), combine the organic phases, dry over anhydrous sodium sulfate and evaporate to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by silica gel column (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain the compound 3-II as a yellow solid (760 mg, yield 54.4%).

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

[0072] Dissolve compound 3-II (760 mg, 0.82 mmol) in N,N-dimethylformamide (20 mL), and then add piperidine (1 mL) to the N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 1 hour.

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

[0074] (3) Synthesis of 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 the N,N-dimethylformamide to form a reaction solution, which was reacted at room temperature (25 °C) for 3 hours.

[0076] TLC analysis indicated that the reaction was complete. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by reverse-phase column chromatography to obtain 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, whose structural formula is as shown in X13:

[0079]

[0080] The synthesis equation of the compound shown in Formula X13 is:

[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), and 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-bath cooling to form a reaction solution, which was reacted at room temperature (25 °C) for 2 hours.

[0085] TLC detection showed that the reaction was complete. Water (80 mL) was added to the reaction solution, and liquid separation was performed. The aqueous phase was extracted with dichloromethane (50 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain the yellow solid of compound 4-II (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 the N,N-dimethylformamide to form a reaction solution, which was reacted at room temperature (25 °C) for 1 hour. TLC detection showed that the reaction was complete. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 5:1) to obtain the yellow solid of compound 4-III (600 mg, yield 94.8%).

[0088] (3) Synthesis of compound MI-6PEG-GGFG-Mitomycin-C (compound shown in formula X13)

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

[0090] Example 5

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

[0092]

[0093] The synthesis equation of the compound shown in formula X2 is:

[0094]

[0095] The specific synthesis steps include:

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

[0097] Dissolve compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 5-I (830 mg, 1.54 mmol) in dichloromethane (30 mL). Under ice-bath cooling, add O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) to form a reaction solution, and react at room temperature (25 °C) for 2 hours.

[0098] Detect that the reaction is complete by TLC. Add water (80 mL) to the reaction solution, separate the layers, extract the aqueous phase with dichloromethane (50 mL × 2), combine the organic phases, dry over anhydrous sodium sulfate and evaporate to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure by silica gel column chromatography (eluting with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain compound 5-II as a yellow solid (608 mg, yield 47.4%).

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

[0100] Dissolve compound 5-II (608 mg, 0.71 mmol) in N,N-dimethylformamide (20 mL), and then add piperidine (1 mL) to the N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 1 hour. Detect that the reaction is complete by TLC. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure by silica gel column chromatography (eluting with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 5:1) to obtain compound 5-III as a yellow foamy solid (398 mg, yield 88.5%).

[0101] (3) Synthesis of compound MI-6PEG-GANG-Mitomycin-C (the compound shown in Formula X2)

[0102] Dissolve compound 5-III (398 mg, 0.63 mmol) in N,N-dimethylformamide (15 mL), and then add compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) to the N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 3 hours. Detect that the reaction is complete by TLC. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure by reverse-phase column chromatography to obtain compound MI-6PEG-GANG-Mitomycin-C as a purple solid (149 mg, yield 21.1%).

[0103] Example 6

[0104] A tumor-targeted activated mitomycin C complex, whose structural formula is as shown in X3:

[0105]

[0106] The synthesis equation of the compound shown in Formula X3 is as follows:

[0107]

[0108] The specific synthesis steps include:

[0109] (1) Synthesize compound 6-II

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

[0111] Detect that the reaction is complete by TLC. Add water (80 mL) to the reaction solution, separate the layers, extract the aqueous phase with dichloromethane (50 mL × 2), combine the organic phases, dry over anhydrous sodium sulfate and evaporate to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain compound 6-II as a yellow solid (496 mg, yield 36.7%).

[0112] (2) Synthesize compound 6-III

[0113] Dissolve compound 6-II (496 mg, 0.55 mmol) in N,N-dimethylformamide (20 mL), and then add piperidine (1 mL) to N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 1 hour. Detect that the reaction is complete by TLC. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 5:1) to obtain 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). Then, compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were added to the N,N-dimethylformamide to form a reaction solution, which was reacted at room temperature (25 °C) for 3 hours. The reaction was monitored by TLC and found to be complete. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by reverse-phase column chromatography to obtain compound MI-6PEG-TANG-Mitomycin-C as a purple solid (51 mg, yield 12.2%).

[0116] Example 7

[0117] A tumor-targeted activated mitomycin C complex, whose structural formula is shown as X4:

[0118]

[0119] The synthesis equation of the compound shown in formula X4 is:

[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). Under ice-bath cooling, 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 to form a reaction solution, which was reacted at room temperature (25 °C) for 2 hours.

[0124] The reaction was monitored by TLC and found to be complete. Water (80 mL) was added to the reaction solution, and liquid separation was carried out. The aqueous phase was extracted with dichloromethane (50 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain 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 the N,N-dimethylformamide to form a reaction solution, which was reacted at room temperature (25 °C) for 1 hour. The reaction was detected to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 5:1) to obtain compound 7-III as a yellow foamy solid (299 mg, yield 76.4%).

[0127] (3) Synthesis of compound MI-6PEG-SANG-Mitomycin-C (compound shown in formula X4)

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

[0129] Example 8

[0130] A tumor-targeted activated mitomycin C complex, whose structural formula is as shown in X5:

[0131]

[0132] The synthesis equation of the compound shown in formula X5 is:

[0133]

[0134] The specific synthesis steps include:

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

[0136] The compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and the compound 8-I (970 mg, 1.54 mmol) were dissolved in dichloromethane (30 mL). Under ice-bath cooling, 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 to form a reaction solution, which was reacted at room temperature (25 °C) for 2 hours. The reaction was detected to be complete by TLC. Water (80 mL) was added to the reaction solution, and liquid separation was performed. The aqueous phase was extracted with dichloromethane (50 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain the compound 8-II as a yellow solid (696 mg, yield 49.1%).

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

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

[0139] (3) Synthesis of compound MI-6PEG-FANG-Mitomycin-C (the compound shown in Formula X5)

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

[0141] Example 9

[0142] A tumor-targeted activated mitomycin C complex, whose structural formula is as shown in X6:

[0143]

[0144] The synthesis equation of the compound shown in Formula X6 is as follows:

[0145]

[0146] The specific synthesis steps are as follows:

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

[0148] Dissolve compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 9-I (786 mg, 1.54 mmol) in dichloromethane (30 mL). Under ice-bath cooling, add O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) to form a reaction solution, and react at room temperature (25 °C) for 2 hours.

[0149] Detect that the reaction is complete by TLC. Add water (80 mL) to the reaction solution, separate the layers, extract the aqueous phase with dichloromethane (50 mL × 2), combine the organic phases, dry over anhydrous sodium sulfate and evaporate to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain compound 9-II as a yellow solid (668 mg, yield 52.5%).

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

[0151] Dissolve compound 9-II (668 mg, 0.80 mmol) in N,N-dimethylformamide (20 mL), and then add piperidine (1 mL) to N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 1 hour. Detect that the reaction is complete by TLC. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 5:1) to obtain compound 9-III as a yellow foamy solid (460 mg, yield 95.1%).

[0152] (3) Synthesis of compound MI-6PEG-AAAG-Mitomycin-C (the compound shown in Formula X6)

[0153] Dissolve compound 9-III (460 mg, 0.76 mmol) in N,N-dimethylformamide (15 mL), then add compound 1-IV (550 mg, 0.92 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) to the N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 3 hours. TLC detection shows that the reaction is complete. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a reverse-phase column to obtain compound MI-6PEG-AAAG-Mitomycin-C as a purple solid (306 mg, yield 36.9%).

[0154] Example 10

[0155] A tumor-targeted activated mitomycin C complex, whose structural formula is shown as X7:

[0156]

[0157] The synthesis equation of the compound shown in formula X7 is:

[0158]

[0159] The specific reaction steps include:

[0160] (1) Synthesize compound 10-II

[0161] Dissolve compound Mitomycin-C (the compound shown in formula D, 500 mg, 1.50 mmol) and compound 10-I (1.03 g, 1.54 mmol) in dichloromethane (30 mL), add O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) under ice-bath cooling to form a reaction solution, and react at room temperature (25 °C) for 2 hours. TLC detection shows that the reaction is complete. Add water (80 mL) to the reaction solution, separate the layers, extract the aqueous phase with dichloromethane (50 mL * 2), combine the organic phases, dry over anhydrous sodium sulfate and evaporate to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain compound 10-II as a yellow solid (960 mg, yield 65.0%).

[0162] (2) Synthesize 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 the N,N-dimethylformamide to form a reaction solution, which was reacted at room temperature (25 °C) for 1 hour. The reaction was detected to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 5:1) to obtain Compound 10-III as a yellow foamy solid (785 mg, yield 81.4%).

[0164] (3) Synthesis of compound MI-6PEG-AAKG-Mitomycin-C (compound shown in formula X7)

[0165] Compound 10-III (785 mg, 0.80 mmol) was dissolved in N,N-dimethylformamide (15 mL), and compound 1-IV (550 mg, 0.92 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were added to the N,N-dimethylformamide to form a reaction solution, which was reacted at room temperature (25 °C) for 3 hours. The reaction was detected to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was dissolved in dichloromethane (50 mL), and 10 mL of trifluoroacetic acid was added and reacted at room temperature (25 °C) for 3 hours. The reaction was detected to be complete by TLC. The reaction solution was evaporated to dryness under reduced pressure, and the product obtained by evaporation to dryness under reduced pressure was purified by reverse phase column chromatography to obtain 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, whose structural formula is as shown in X8:

[0168]

[0169] The synthesis equation of the compound shown in formula X8 is:

[0170]

[0171] The specific synthesis steps include:

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

[0173] Dissolve compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 11-I (940 mg, 1.54 mmol) in dichloromethane (30 mL). Under ice-bath cooling, add O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) to form a reaction solution, and react at room temperature (25 °C) for 2 hours. The reaction is detected to be complete by TLC. Add water (80 mL) to the reaction solution, separate the layers, extract the aqueous phase with dichloromethane (50 mL × 2), combine the organic phases, dry over anhydrous sodium sulfate and evaporate to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain compound 11-II as a yellow solid (800 mg, yield 57.5%).

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

[0175] Dissolve compound 11-II (800 mg, 0.86 mmol) in N,N-dimethylformamide (20 mL), and then add piperidine (1 mL) to the N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 1 hour. The reaction is detected to be complete by TLC. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 5:1) to obtain compound 11-III as a yellow foamy solid (590 mg, yield 97.3%).

[0176] (3) Synthesis of compound MI-6PEG-AACG-Mitomycin-C (the compound shown in Formula X8)

[0177] Dissolve compound 11-III (590 mg, 0.84 mmol) in N,N-dimethylformamide (15 mL), and then add compound 1-IV (550 mg, 0.92 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) to the N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 3 hours. The reaction is detected to be complete by TLC. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a reverse-phase column to obtain compound MI-6PEG-AACG-Mitomycin-C as a purple solid (255 mg, yield 25.5%).

[0178] Example 12

[0179] A tumor-targeted activated mitomycin C complex, whose structural formula is as shown in X10:

[0180]

[0181] The synthesis equation of the compound shown in Formula X10 is as follows:

[0182]

[0183] The specific reaction steps are as follows:

[0184] (1) Synthesize compound 12-II

[0185] Dissolve compound Mitomycin-C (the compound shown in Formula D, 500 mg, 1.50 mmol) and compound 12-I (783 mg, 1.54 mmol) in dichloromethane (30 mL). Under ice-bath cooling, add O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (800 mg, 2.11 mmol) and diisopropylethylamine (550 mg, 4.26 mmol) to form a reaction solution, and react at room temperature (25 °C) for 2 hours. The reaction is detected to be complete by TLC. Add water (80 mL) to the reaction solution, separate the layers, extract the aqueous phase with dichloromethane (50 mL × 2), combine the organic phases, dry over anhydrous sodium sulfate and evaporate to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain compound 12-II as a yellow solid (545 mg, yield 44.0%).

[0186] (2) Synthesize compound 12-III

[0187] Dissolve compound 12-II (545 mg, 0.66 mmol) in N,N-dimethylformamide (20 mL), and then add piperidine (1 mL) to the N,N-dimethylformamide to form a reaction solution, and react at room temperature (25 °C) for 1 hour. The reaction is detected to be complete by TLC. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure through a silica gel column (elute with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 5:1) to obtain 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). Then, compound 1-IV (450 mg, 0.75 mmol) and diisopropylethylamine (300 mg, 2.33 mmol) were added to the N,N-dimethylformamide to form a reaction solution, which was reacted at room temperature (25 °C) for 3 hours. The reaction was monitored by TLC and found to be complete. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation to dryness under reduced pressure was purified by reverse-phase column chromatography to obtain 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 synthesis equation of the compound shown in formula X11 is:

[0194]

[0195] The specific synthesis steps include:

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

[0197] Compound Mitomycin-C (the 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). Under ice-bath cooling, 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 to form a reaction solution, which was reacted at room temperature (25 °C) for 2 hours. The reaction was monitored by TLC and found to be complete. Water (80 mL) was added to the reaction solution, and the layers were separated. The aqueous phase was extracted with dichloromethane (50 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The product obtained after evaporation to dryness under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 8:1) to obtain 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 monitored by TLC and was found to be complete. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) in a volume ratio of 100:1 to 5:1) to obtain Compound 13-III as a yellow foamy solid (363 mg, yield 88.6%).

[0200] (3) Synthesis of 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 added to the N,N-dimethylformamide to form a reaction solution, and the reaction was carried out at room temperature (25 °C) for 3 hours. The reaction was monitored by TLC and was found to be complete. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by reverse phase column chromatography to obtain compound MI-6PEG-AAGP-Mitomycin-C as a purple solid (134 mg, yield 20.7%).

[0202] Performance study of the tumor-targeted activated mitomycin C complex described in Examples 1-13 of this application:

[0203] Cells such as A2780 (the cells listed in Table 1) were cultured in complete medium (RPMI1640 (DMEM high glucose medium) + 10% fetal bovine serum + 1X P / S + 1 mM sodium pyruvate solution), cultured in an incubator at 37 °C and 5% CO2 until the cells grew to a sufficient number, the cells were collected, centrifuged at 1000 g for 5 min, and resuspended in an appropriate volume of 10% RPMI1640 (DMEM high glucose medium) medium, and the cell density was adjusted to 4,000,000 cells per milliliter.

[0204] Add 100 μL of cell culture medium containing different concentrations of the drug into a 96-well culture plate. Set up control wells (0.1% DMSO) with only the corresponding drug solvent without the drug, and zero-adjustment wells (Blank) with only the culture medium without cells. Each group has 3 parallel wells. Then, inoculate the cells after cell counting onto the 96-well culture plate, with 100 μL of cell suspension per well, and the inoculation concentration of the cells is 5000 cells (100 μL) / well. Then place the plate in an incubator at 37°C with 5% carbon dioxide for 48 hours. After 48 hours, add 10 μL of CCK8 (concentration 5 mg / ml) to each well, incubate in the cell incubator for about 2 hours, and measure the absorbance at 450 nm.

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

[0206] Cell survival rate = [(absorbance of the experimental group - absorbance of the blank control) / (absorbance of the control group - absorbance of the blank control)] × 100%

[0207] Based on the cell survival rate, use GraphPad Prism 8 software to calculate the half-maximal inhibitory concentration (IC50) of the drug on the cells. The results are shown in Table 1 and Table 2.

[0208] Note: The different concentrations of the drug in the cell culture medium containing different concentrations of the drug refer to setting the maximum starting concentration of the drug to be tested as 1000 μM, the starting concentrations of the drug with and without Legumain activation as 100 μM, and performing gradient dilution at a ratio of 1:4 into 9 dose groups (3 replicates per group). The concentration of the drug solvent (DMSO) in all wells with added drug is controlled at 0.1%. Only adding the drug solvent (0.1% DMSO) serves as the experimental control group (Control), and the blank group (Blank) with only the culture medium without cells.

[0209] Legumain activation operation: Buffer preparation: distilled water, 50 mM MES, 250 mM sodium chloride, adjust the pH to 5.0 with 0.5 M sodium hydroxide. The concentration of Legumain is selected as 1 mg / mL. Prepare a solution with a concentration of 0.5 μmol / mL of the tumor-targeted activated mitomycin C complex with the buffer. Accurately pipette 50 μL of the above 0.5 μmol / mL solution and 50 μL of the buffer into a centrifuge tube, add 100 μL of Legumain, and place at 37°C for 2 hours.

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

[0211]

[0212]

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

[0214] Compound Name A549 (Non-small cell lung cancer) SW872 (Sarcoma) A2780 (Ovarian cancer) MKN45 (Gastric cancer) Compound shown in Formula D 0.224 umol / L 0.015 umol / L 0.077 umol / L 0.192 umol / L Example 1 0.54 umol / L 0.12 umol / L 0.32 umol / L 0.46 umol / L Example 2 0.47 umol / L 0.18 umol / L 0.45 umol / L 0.39 umol / L Example 5 0.65 umol / L 0.17 umol / L 0.18 umol / L 0.60 umol / L Example 6 0.51 umol / L 0.20 umol / L 0.36 umol / L 0.43 umol / L Example 7 0.42 umol / L 0.092 umol / L 0.43 umol / L 0.40 umol / L Example 8 0.33 umol / L 0.11 umol / L 0.22 umol / L 0.31 umol / L

[0215] As can be seen from Table 1, compared with mitomycin C alone, the cytotoxicity of the unactivated tumor-targeted activated mitomycin C complex has been greatly reduced, indicating that the complex has a very good shielding effect on the toxicity of mitomycin C.

[0216] As can be seen from Table 1 and Table 2, as shown in Table 1, the IC50 of the tumor-targeted activated mitomycin C complex in the above cells is higher than that of mitomycin C. Therefore, the cytotoxicity of the tumor-targeted activated mitomycin C complex provided in Table 1 is greatly reduced without legumain activation, that is, the tumor-targeted activated mitomycin C complex provided in Table 1 has lower toxicity than mitomycin C itself.

[0217] As shown in Table 2, after the tumor-targeted activated mitomycin C complex is activated by Legumain, its toxicity increases significantly compared with that before activation, indicating that in the environment with high expression of Legumain in the tumor microenvironment, the tumor-targeted activated mitomycin C complex can be released after activation, realizing directional activation and targeted release.

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

[0219] Test drugs: The tumor-targeted activated mitomycin C complex described in Examples 1-13 of this application, mitomycin C, and a normal saline control group.

[0220] Test animals: 6-8-week-old BALB / c mice, all female.

[0221] Preparation of tumor model:

[0222] A2780 cells were purchased from ATCC and cultured in DMEM (1640) medium containing 10% fetal bovine serum at 37 °C and 5% CO2. Passage was performed every three days, and cells within 15 passages were used. 5×10 6 corresponding cells were subcutaneously injected into the right axilla of nude mice. The tumor reached at least 100 mm 3After that, the mice were randomly divided into groups of 3. Then the treatment began, and the first day of treatment was the first day. Mitomycin C was administered at 2.5 mg / kg as a positive control, while the tumor-targeted activated mitomycin C complex described in Examples 1-13 was administered at 10 mg / kg as the experimental group, and the negative control group was given normal saline. The drug was administered once a week for three weeks. The results are shown in Table 3.

[0223] Table 3

[0224] Compound Name <![CDATA[Tumor final volume (mm 3 )]]> Tumor growth rate Change rate of mouse body weight Normal saline 2435.19 1932.74％ +25.6％ 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 inhibitory effects of other drugs on A2780 tumors were better than those of mitomycin C, indicating that the tumor-targeted activated mitomycin C complex described in this application has a greater improvement in drug 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 MKN45 tumor model

[0227] Test drugs: The tumor-targeted activated mitomycin C complex described in Examples 1-13 of this application, mitomycin C, and a normal saline control group.

[0228] Test animals: 6-8-week-old BALB / c mice, all female.

[0229] Preparation of tumor model:

[0230] MKN45 cells were purchased from ATCC. The cells were cultured in DMEM (1640) medium containing 10% fetal bovine serum at 37 °C and 5% CO2. Passage was performed every three days, and cells within 15 passages were used. 5×10 6 corresponding cells were subcutaneously injected into the right axilla of nude mice. After the tumor reached at least 100 mm 3 , the mice were randomly divided into groups of 3. Then the treatment began, and the first day of treatment was the first day. Mitomycin C was administered at 2.5 mg / kg as a positive control, while the tumor-targeted activated mitomycin C complex conjugate described in Examples 1-13 was administered at 10 mg / kg as the experimental group, and the negative control group was given normal saline. The drug was administered once a week for four weeks. The results are shown in Table 4.

[0231] Table 4

[0232] Compound Name <![CDATA[Tumor final volume (mm 3 )]]> Tumor inhibition rate Change rate of mouse body weight Normal saline 813.94±190.21 0 +15.2％ 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, for the tumor-targeted activated mitomycin C complexes X1-X13 described in this application, their inhibitory effects on MKN45 tumors are better than those of mitomycin C. That is, compared with mitomycin C, the tumor-targeted activated mitomycin C complexes described in this application have a significant improvement in drug efficacy, and the body weights of the mice have not significantly decreased, while the body weights of the mice in the mitomycin C group have significantly decreased, indicating that the toxicity of the tumor-targeted activated mitomycin C complexes in this application has been greatly reduced compared with mitomycin C.

[0234] 4. MTD detection test of the tumor-targeted activated mitomycin C complex described in this application

[0235] Experimental animals: Female mice, 6-8 weeks old. The mice were randomly grouped, with six mice in each group. The mice were administered at 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. The mice were euthanized when they lost 20% of their initial body weight and were considered to have died of poisoning. The results are shown in Table 5.

[0236] MTD is defined as the highest dose level at which none of the six mice died due to the drug, the weight loss of an individual mouse did not exceed 20%, or the average weight loss within the group did not exceed 15%.

[0237] Table 5

[0238] Compound structure Maximum tolerated dose (MTD) Compound shown in Formula D 4 mg / kg X1 120 mg / kg X2 100 mg / kg X3 120 mg / kg X4 120 mg / kg X5 150 mg / kg X6 120 mg / kg X7 120 mg / kg X8 150 mg / kg X9 70 mg / kg X10 100 mg / kg X11 100 mg / kg X12 80 mg / kg X13 120 mg / kg

[0239] As can be seen from Table 5, the maximum lethal concentration of the tumor-targeted activated mitomycin C complex prepared in the examples of this application in mice has been significantly increased compared with mitomycin C itself. Because it is basically not released in normal tissues, the toxic and side effects are greatly reduced, so it is expected to be used at higher doses clinically.

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

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

[0242] The tumor-targeted activated mitomycin C complexes described in Examples 1-13 of the present application were respectively prepared into 1 mg / ml solutions with PBS buffer. 200 μL of the above tumor-targeted activated mitomycin C complexes were respectively taken and added to 100 micrograms of tumor tissue homogenate or normal tissue homogenate (the tissue homogenate was prepared from the tissues taken after the mice were euthanized using a Jingxin F6 / 10 handheld homogenizer). The solutions were placed at 37 °C for 2 h. The activation efficiency of the drug in the tumor tissue was compared by detecting the reduction of the compound and the increase of mitomycin C through HPLC.

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

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

[0245] Table 6 Activation efficiency of the tumor-targeted activated mitomycin C complexes in different tumor homogenates

[0246] Compound Name A549 (Small cell lung cancer) (%) SW872 (Sarcoma) (%) A2780 (Ovary) (%) 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] It can be seen from Table 6 that the tumor-targeted activated mitomycin C complexes described in Examples 1-13 of the present application have good release efficiency in different cancer tissue homogenates.

[0248] Table 7 Percentage of release efficiency of the tumor-targeted activated mitomycin C complexes in normal tissues of mice

[0249]

[0250]

[0251] It can be seen from Table 7 that the release ratio of the tumor-targeted activated mitomycin C complexes described in the present application in normal tissues is very low. Except for a small amount of release in the liver and blood, there is little release in other tissues.

[0252] It can be seen from Tables 6 and 7 that the tumor-targeted activated mitomycin C complexes described in the present application are released in large amounts in tumor homogenates. This is because mitomycin C can be activated by enzymes highly expressed in the tumor microenvironment, while the release in normal tissues is very little or almost non-existent. Therefore, the damage to normal tissue organs is greatly reduced compared to mitomycin C itself, effectively improving the safety window and potentially allowing for higher doses to be used clinically.

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

Claims

1. A tumor-targeted activated mitomycin C complex, characterized in that: The structural formula of the tumor-targeted activated mitomycin C complex is shown below: E-(PEG) x -L1-L2-L3-L4-D, Among them, the structural formula of E is shown as follows: 1≤n≤18, the wavy line is a connecting key; PEG is polyethylene glycol, and the degree of polymerization x is: 2≤x≤20; L1 and L2 are present or absent. When L1 and L2 are present, L1 and L2 are each independently selected from one or more of glycine, alanine, phenylalanine, threonine and serine; L3 is selected from one or more of glycine, alanine, lysine, phenylalanine, guanidine and asparagine; L4 is present or absent, and when L4 is present, L4 is selected from one or more of glycine, leucine, isoleucine and proline; The structural formula of D is shown below:

2. The tumor-targeted activated mitomycin C complex according to claim 1, characterized in that: The structural formula of the tumor-targeted activated mitomycin C complex is shown in Formula X1-X13:

3. The tumor-targeted activated mitomycin C complex according to claim 1, characterized in that: The polymerization degree x of the polyethylene glycol is: 5≤x≤10.

4. A pharmaceutical composition, characterized in that The invention comprises the tumor-targeted activated mitomycin C complex according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof.

5. Use of the tumor-targeted activated mitomycin C complex according to any one of claims 1 to 3 in the preparation of a drug for treating and / or preventing cancer, or a drug for treating and / or preventing angiogenesis.

6. Use of the pharmaceutical composition according to claim 4 in the preparation of drugs for treating and / or preventing cancer.

7. The use according to claim 6, characterized in that: The cancers include one or more of gastrointestinal cancer, colorectal cancer, colon cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, gastric cancer, genitourinary cancer, bladder cancer, testicular cancer, cervical cancer, malignant mesothelioma, osteogenic sarcoma, 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, blood cancer, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, ovarian cancer, brain cancer, neuroblastoma, Ewing's sarcoma, kidney cancer, epidermoid carcinoma, skin cancer, melanoma and oral cancer.

8. The use according to claim 6, characterized in that: The cancer includes one or more of non-small cell lung cancer, small cell lung cancer, breast cancer, gastric cancer, esophageal cancer, intestinal cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer and sarcoma.

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