Paclitaxel-polypeptide conjugate and use thereof

Abstract

The present invention relates to a polypeptide conjugate and the use thereof, and particularly to a paclitaxel- or docetaxel-polypeptide conjugate and the use thereof. The polypeptide-drug conjugate is selectively released in the tumor microenvironment, solves the problem in the prior art of the limited clinical use of a paclitaxel or docetaxel drug due to the lack of a targeting property and strong side effects, and can be clinically administered in higher doses during cancer treatment, thereby improving the overall therapeutic effect.

Description

A paclitaxel-based polypeptide conjugate and its application Technical Field

[0001] This invention relates to a polypeptide conjugate and its application, particularly to a tumor-targeting activated paclitaxel and docetaxel polypeptide conjugate and its application. Background Technology

[0002] Paclitaxel (trade name Taxol) is a tricyclic diterpenoid compound naturally occurring in the bark and needles of the yew tree. Due to its unique anticancer mechanism, it is one of the most successful and widely used natural anticancer drugs. Unlike other tubulin-binding anticancer drugs that prevent tubulin assembly into microtubules, paclitaxel promotes tubulin assembly into microtubules and prevents microtubule dissociation, thus blocking cell cycle progression, preventing mitosis, and inhibiting cancer cell growth. It is also used for coronary heart disease, skin diseases, kidney and liver fibrosis, inflammation, and axonal regeneration, and is currently undergoing clinical trials for degenerative brain diseases.

[0003] Docetaxel is a taxane-based antimitotic chemotherapy drug. These drugs preferentially bind to β-tubulin, inhibiting microtubule dynamics and disrupting cell division, thereby effectively inducing apoptosis. As an FDA-approved drug, docetaxel has become a standard treatment for many types of cancer, including advanced prostate cancer.

[0004] 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 has been paclitaxel and docetaxel, with or without ifosfamide. Several phase II trials have demonstrated the activity of paclitaxel and docetaxel in anthracycline- and alkylating agent-resistant soft tissue sarcomas, suggesting their use as second- and third-line therapy, and showing significant activity in liposarcoma and leiomyosarcoma subtypes. Paclitaxel and docetaxel have shown favorable toxicity profiles and are approved for the treatment of metastatic soft tissue sarcomas in over 70 countries.

[0005] Ovarian cancer can occur at any age, but it is more common in patients over 50. Patients typically present with nonspecific pelvic or abdominal symptoms. Prognosis is usually determined by cancer stage and grade, and is often not ideal, as 70% of cases are diagnosed at stage III or IV, thus associated with poor prognosis. Prophylactic visits provide an opportunity to identify and educate women at increased risk of ovarian cancer, but routine screening is not recommended. More robust solutions are needed to improve patient outcomes while optimizing treatment.

[0006] Breast cancer is the most common cancer and a leading cause of cancer death among women worldwide. In 2008, approximately 1.38 million new cases of breast cancer were diagnosed, with nearly 50% of breast cancer patients and about 60% of deaths occurring in developing countries. There are significant disparities in breast cancer survival rates globally, with an estimated 5-year survival rate of 80% in developed countries and less than 40% in developing countries. Developing countries face resource and infrastructure constraints, which challenge the goal of improving breast cancer outcomes through timely identification, diagnosis, and management. In developed countries like the United States, approximately 232,340 women were diagnosed with breast cancer in 2013, and 39,620 women died. The lifetime risk of breast cancer for women in the United States is 12.38%. From 1975 to 2000, there was a significant decline in the mortality rate from breast cancer in the United States, attributed to increased mammography screening and management. For patients diagnosed with breast cancer, various treatment strategies are used, such as targeted therapy, hormone therapy, radiation therapy, surgery, and chemotherapy. For patients with distant metastases, treatment is generally aimed at improving quality of life and survival. The long-term side effects of breast cancer treatment are one of the most motivating factors in the search for alternative treatments.

[0007] Existing technical document 1: CN113274507A

[0008] Currently, there are existing technologies for preparing other anticancer compounds (drugs) into conjugates to enhance drug targeting and reduce toxicity. For example, CN113274507A discloses an "immunostimulatory conjugate for targeted delivery and activation," comprising an MI group, a selective group S, a tripeptide group C cleaved by asparagine endopeptidase, an auxiliary linker arm A, and the conjugated drug. The tripeptide group C is preferably AAN (alanine-alanine-asparagine), and the auxiliary linker arm A is preferably L (leucine) or PAB (p-aminobenzyl alcohol). Although the conjugates of these existing technologies offer some optimization in reducing toxicity and improving efficacy compared to the anticancer compound (payload) itself, the results are still unsatisfactory and cannot meet the actual needs of clinical applications in cancer treatment. In fact, when treating cancer patients in clinical practice, improving drug targeting, enhancing drug efficacy, and reducing toxic side effects are all very important. Even when drug efficacy is similar, reducing drug toxicity can significantly alleviate patients' side effects (such as diarrhea, loss of appetite, anemia, etc.), and reduced toxicity means that larger doses can be used for treatment, thereby improving the overall treatment effect.

[0009] Regarding paclitaxel-based drugs, current research lacks information on improving the targeting of paclitaxel and docetaxel as anti-tumor drugs and reducing their side effects. Therefore, there is an urgent need to research a compound that can simultaneously and significantly improve the targeting of paclitaxel and docetaxel, enhance their efficacy, and significantly reduce their side effects, in order to achieve better cancer treatment. Summary of the Invention

[0010] This invention provides a tumor-targeting activated paclitaxel and docetaxel peptide conjugate and its application, which solves the problem that the clinical application of paclitaxel and docetaxel is limited due to their lack of targeting and strong side effects in the prior art. The peptide conjugate of this invention transports paclitaxel and docetaxel to tumor tissue via peptides. In the acidic microenvironment of the tumor, the paclitaxel and docetaxel peptide conjugate can be activated by Legumin, which is highly expressed by tumor cells and tumor-associated macrophages. This allows for the selective release of paclitaxel and docetaxel in the tumor microenvironment, inducing immunogenic death of tumor cells, stimulating the body's anti-tumor immune function, and reducing the toxic side effects of paclitaxel and docetaxel. It exhibits good targeting, achieving multiple effects of inhibiting tumor growth, promoting anti-tumor immunity, and reducing drug toxicity.

[0011] This invention provides a polypeptide conjugate, the structural formula of which is as follows:

[0012] E-nPEG-L1-L2-L3-L4-L5-D

[0013] in,

[0014] E is a maleimide group, a methyl sulfone compound, or a halogen group;

[0015] nPEG stands for n-polyethylene glycol, and its structure is selected from:

[0016] Where n is an integer greater than or equal to 1 and less than or equal to 40;

[0017] L1 is glycine, alanine, or serine;

[0018] L2 is alanine;

[0019] L3 is asparagine;

[0020] L4 is glycine, leucine, or absent;

[0021] L5 is a p-aminobenzyl alcohol-ethylenediamine derivative;

[0022] D represents paclitaxel, docetaxel, or their derivatives.

[0023] Furthermore, when the E group in the polypeptide conjugate provided by the present invention is a maleimide group, its structure is as follows:

[0024] Where n is an integer greater than or equal to 1 and less than or equal to 18;

[0025] When the E group is a methyl sulfone compound, its structural formula is one of the following:

[0026] Where n is an integer greater than or equal to 0 and less than or equal to 10;

[0027] When the E group is a halogen group, its structure is as follows:

[0028] Where X is a halogen atom;

[0029] Furthermore, the L5 group in the polypeptide conjugate provided by the present invention is selected from the following structures: Wherein, R1 and R2 are hydrogen or methyl, and R1 and R2 may be the same or different; n1 and n2 are both integers greater than or equal to 0 and less than or equal to 20, and n1 and n2 may be the same or different.

[0030] Furthermore, the polypeptide conjugate of the present invention may be selected from any of the following structures:

[0031] Among them, G: glycine, A: alanine, N: asparagine, PAB: aminobenzyl alcohol, S: serine, and L: leucine.

[0032]

Example 1

[0033] The synthetic method for the MC-6PEG-AAN-PAB-DAM-4PEG-Paclitaxel peptide conjugate is as follows:

[0034] The specific synthesis steps are as follows:

[0035] 1. Synthesis of Compound 1-III

[0036] Compound 1-I (350 mg, 1.28 mmol) was dissolved in N,N-dimethylformamide (25 mL), and compound 1-II (770 mg, 1.28 mmol) and DIEA (N,N-diisopropylethylamine) (350 mg, 2.72 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 8:1) to give compound 1-III as a pale yellow solid (330 mg, yield 33.9%).

[0037] 2. Synthesis of Compound 1-IV

[0038] Compound 1-III (330 mg, 0.43 mmol) was dissolved in N,N-dimethylformamide (25 mL), and p-aminobenzyl alcohol (60 mg, 0.49 mmol), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (250 mg, 0.66 mmol), and DIEA (N,N-diisopropylethylamine) (200 mg, 1.55 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 4 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 12:1) to give compound 1-IV as a pale yellow solid (320 mg, yield 85.9%).

[0039] 3. Synthesis of compounds 1-V

[0040] Compound 1-IV (320 mg, 0.37 mmol) was dissolved in N,N-dimethylformamide (25 mL), and bis(p-nitrobenzene) carbonate (135 mg, 0.44 mmol) and DIEA (N,N-diisopropylethylamine) (130 mg, 1.01 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was slurried twice with a mixture of ethyl acetate and methyl tert-butyl ether (1:2) to give compound 1-V as a yellow solid (330 mg, yield 86.5%).

[0041] 4. Synthesis of compounds 1-VII

[0042] Compound 1-VI (5.0 g, 25.87 mmol) was dissolved in methanol (250 mL), and N-Boc-(methylamino)acetaldehyde (4.5 g, 25.98 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 1 hour. The reaction mixture was then cooled to 0–5 °C in an ice-water bath, and sodium borohydride (1.2 g, 31.72 mmol) was added. The ice bath was removed, and the reaction mixture was slowly heated to room temperature (25 °C) for 18 hours. The reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 8:1) to give compound 1-VII as a pale yellow oil (7.3 g, yield 80.5%).

[0043] 5. Synthesis of Compound 1-VIII

[0044] Paclitaxel (500 mg, 0.59 mmol) was dissolved in N,N-dimethylformamide (50 mL), and p-nitrobenzene chloroformate (140 mg, 0.70 mmol), DMAP (dimethylaminopyridine) (20 mg, 0.16 mmol), and DIEA (N,N-diisopropylethylamine) (200 mg, 1.55 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 4 hours. HPLC analysis showed that 20% of the starting material remained. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 15:1) to give compound 1-VIII as a pale yellow solid (235 mg, yield 39.1%).

[0045] 6. Synthesis of compound 1-IX

[0046] Compound 1-VIII (235 mg, 0.23 mmol) was dissolved in N,N-dimethylformamide (30 mL), and compound 1-VII (100 mg, 0.29 mmol) and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. HPLC (high performance liquid chromatography) was used to confirm complete reaction of the starting materials. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 10:1) to give compound 1-IX as a pale yellow solid (220 mg, yield 77.8%).

[0047] 7. Synthesis of compound 1-X

[0048] Compound 1-IX (220 mg, 0.18 mmol) was added to dichloromethane (15 mL), followed by trifluoroacetic acid (8 mL). The reaction mixture was reacted at room temperature (25 °C) for 2 hours. HPLC (high performance liquid chromatography) confirmed that the reaction proceeds were complete. The reaction mixture was evaporated to dryness under reduced pressure to give compound 1-X as a brownish-yellow solid (195 mg, yield 96.4%).

[0049] 8. Synthesis of compound MC-6PEG-AAN-PAB-DAM-4PEG-Paclitaxel

[0050] Compound 1-X (195 mg, 0.17 mmol) and compound 1-V (210 mg, 0.20 mmol) were dissolved in N,N-dimethylformamide (25 mL), and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 8 hours, and the reaction proceeded to completion as determined by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to obtain MC-6PEG-AAN-PAB-DAM-4PEG-Paclitaxel as a white solid (69 mg, yield 20.1%).

[0051]

Example 2

[0052] The synthetic method for the MC-6PEG-GAN-PAB-DAM-4PEG-Paclitaxel peptide conjugate is as follows:

[0053] The specific synthesis steps are as follows:

[0054] 1. Synthesis of compound 2-II

[0055] Compound 2-I (350 mg, 1.34 mmol) was dissolved in N,N-dimethylformamide (25 mL), and compound 1-II (810 mg, 1.35 mmol) and DIEA (N,N-diisopropylethylamine) (350 mg, 2.72 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 8:1) to give compound 2-II as a pale yellow solid (410 mg, yield 41.0%).

[0056] 2. Synthesis of Compound 2-III

[0057] Compound 2-II (410 mg, 0.55 mmol) was dissolved in N,N-dimethylformamide (25 mL), and p-aminobenzyl alcohol (75 mg, 0.61 mmol), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (300 mg, 0.79 mmol), and DIEA (N,N-diisopropylethylamine) (200 mg, 1.55 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 4 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 12:1) to give compound 2-III as a pale yellow solid (354 mg, yield 75.6%).

[0058] 3. Synthesis of compound 2-IV

[0059] Compound 2-III (354 mg, 0.42 mmol) was dissolved in N,N-dimethylformamide (25 mL), and bis(p-nitrobenzene) carbonate (150 mg, 0.49 mmol) and DIEA (N,N-diisopropylethylamine) (130 mg, 1.01 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was slurried twice with a mixture of ethyl acetate and methyl tert-butyl ether (1:2) to give compound 2-IV as a yellow solid (350 mg, yield 81.9%).

[0060] 4. Synthesis of compound MC-6PEG-GAN-PAB-DAM-4PEG-Paclitaxel

[0061] Compound 1-X (200 mg, 0.18 mmol) and compound 2-IV (200 mg, 0.20 mmol) were dissolved in N,N-dimethylformamide (25 mL), and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 8 hours, and the reaction proceeded to completion by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to obtain MC-6PEG-GAN-PAB-DAM-4PEG-Paclitaxel as a white solid (126 mg, yield 34.9%).

[0062]

Example 3

[0063] The synthetic method for the MC-6PEG-SAN-PAB-DAM-4PEG-Paclitaxel peptide conjugate is as follows:

[0064] The specific synthesis steps are as follows:

[0065] 1. Synthesis of compound 3-II

[0066] Compound 3-I (350 mg, 1.21 mmol) was dissolved in N,N-dimethylformamide (25 mL), and compound 1-II (730 mg, 1.21 mmol) and DIEA (N,N-diisopropylethylamine) (350 mg, 2.72 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 8:1) to give compound 3-II as a pale yellow solid (315 mg, yield 33.5%).

[0067] 2. Synthesis of Compound 3-III

[0068] Compound 3-II (315 mg, 0.41 mmol) was dissolved in N,N-dimethylformamide (25 mL), and p-aminobenzyl alcohol (60 mg, 0.49 mmol), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (300 mg, 0.79 mmol), and DIEA (N,N-diisopropylethylamine) (200 mg, 1.55 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 4 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 12:1) to give compound 2-III as a pale yellow solid (285 mg, yield 78.8%).

[0069] 3. Synthesis of compound 3-IV

[0070] Compound 3-III (285 mg, 0.32 mmol) was dissolved in N,N-dimethylformamide (25 mL), and bis(p-nitrobenzene) carbonate (150 mg, 0.49 mmol) and DIEA (N,N-diisopropylethylamine) (130 mg, 1.01 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was slurried twice with a mixture of ethyl acetate and methyl tert-butyl ether (1:2) to give compound 3-IV as a yellow solid (268 mg, 80.0% yield).

[0071] 4. Synthesis of compound MC-6PEG-SAN-PAB-DAM-4PEG-Paclitaxel

[0072] Compound 1-X (200 mg, 0.18 mmol) and compound 3-IV (210 mg, 0.20 mmol) were dissolved in N,N-dimethylformamide (25 mL), and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 8 hours, and the reaction proceeded to completion as determined by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to obtain MC-6PEG-SAN-PAB-DAM-4PEG-Paclitaxel as a white solid (77 mg, yield 21.0%).

[0073]

Example 4

[0074] The synthetic method for the MC-6PEG-AANG-PAB-DAM-4PEG-Paclitaxel peptide conjugate is as follows:

[0075] The specific synthesis steps are as follows:

[0076] 1. Synthesis of compound 4-I

[0077] p-Aminobenzyl alcohol (500 mg, 4.06 mmol) and Boc-glycine (710 mg, 4.05 mmol) were dissolved in dichloromethane (50 mL), along with HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (2.2 g, 5.79 mmol) and DIEA (N,N-diisopropylethylamine) (1.5 g, 11.63 mmol). The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by HPLC. 100 mL of purified water 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, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol (MeOH) = 20:1) to give compound 4-I as a white solid (960 mg, yield 84.7%).

[0078] 2. Synthesis of Compound 4-II

[0079] Compound 4-I (960 mg, 3.42 mmol) was dissolved in dichloromethane (50 mL) and trifluoroacetic acid (10 mL). The reaction solution was reacted at room temperature (25 °C) for 3 hours, and the reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction solution was evaporated to dryness under reduced pressure to give compound 4-II as a pale yellow solid (620 mg, 100% yield).

[0080] 3. Synthesis of Compound 4-III

[0081] Compounds 1-III (2.6 g, 3.42 mmol) and 4-II (620 mg, 3.42 mmol) were dissolved in N,N-dimethylformamide (100 mL), and HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (2.0 g, 5.26 mmol) and DIEA (N,N-diisopropylethylamine) (1.5 g, 11.63 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 20:1 to 8:1) to give compound 4-III as a yellow solid (1.82 g, yield 57.7%).

[0082] 4. Synthesis of compound 4-IV

[0083] Compound 4-III (1.82 g, 1.97 mmol) was dissolved in N,N-dimethylformamide (100 mL), and bis(p-nitrobenzene) carbonate (1.0 g, 3.29 mmol) and DIEA (N,N-diisopropylethylamine) (1.5 g, 11.63 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was slurried twice with ethyl acetate:methyl tert-butyl ether (1:5) to give compound 4-IV as a yellow solid (1.58 g, yield 73.7%).

[0084] 5. Synthesis of compound MC-6PEG-AANG-PAB-DAM-4PEG-Paclitaxel

[0085] Compound 1-X (200 mg, 0.18 mmol) and compound 4-IV (220 mg, 0.20 mmol) were dissolved in N,N-dimethylformamide (25 mL), and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 8 hours, and the reaction proceeded to completion as determined by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to obtain MC-6PEG-AANG-PAB-DAM-4PEG-Paclitaxel as a white solid (115 mg, yield 30.7%).

[0086]

Example 5

[0087] The synthetic method for the MC-6PEG-AANL-PAB-DAM-4PEG-Paclitaxel peptide conjugate is as follows:

[0088] The specific synthesis steps are as follows:

[0089] 1. Synthesis of compound 5-I

[0090] p-Aminobenzyl alcohol (500 mg, 4.06 mmol) and Boc-leucine (920 mg, 3.98 mmol) were dissolved in dichloromethane (50 mL), along with HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (2.2 g, 5.79 mmol) and DIEA (N,N-diisopropylethylamine) (1.5 g, 11.63 mmol). The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by HPLC. 100 mL of purified water 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, filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol (MeOH) = 20:1) to give compound 5-I as a white solid (1.22 g, yield 91.1%).

[0091] 2. Synthesis of Compound 5-II

[0092] Compound 5-I (1.22 g, 3.63 mmol) was dissolved in dichloromethane (50 mL) and trifluoroacetic acid (10 mL). The reaction solution was reacted at room temperature (25 °C) for 3 hours, and the reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction solution was evaporated to dryness under reduced pressure to give compound 5-II as a pale yellow solid (770 mg, yield 89.8%).

[0093] 3. Synthesis of Compound 5-III

[0094] Compounds 1-III (500 mg, 0.66 mmol) and 5-II (160 mg, 0.68 mmol) were dissolved in N,N-dimethylformamide (30 mL), and HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (500 mg, 1.32 mmol) and DIEA (N,N-diisopropylethylamine) (500 mg, 3.88 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 20:1 to 8:1) to give compound 5-III as a yellow solid (483 mg, yield 74.7%).

[0095] 4. Synthesis of compound 5-IV

[0096] Compound 5-III (483 mg, 0.49 mmol) was dissolved in N,N-dimethylformamide (30 mL), and bis(p-nitrobenzene) carbonate (220 mg, 0.72 mmol) and DIEA (N,N-diisopropylethylamine) (200 mg, 1.55 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was slurried twice with ethyl acetate:methyl tert-butyl ether (1:3) to give compound 5-IV as a yellow solid (565 mg, 100% yield).

[0097] 5. Synthesis of compound MC-6PEG-AANL-PAB-DAM-4PEG-Paclitaxel

[0098] Compound 1-X (200 mg, 0.18 mmol) and compound 5-IV (230 mg, 0.20 mmol) were dissolved in N,N-dimethylformamide (25 mL), and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 8 hours, and the reaction proceeded to completion as determined by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to obtain MC-6PEG-AANL-PAB-DAM-4PEG-Paclitaxel as a white solid (74 mg, yield 19.3%).

[0099]

Example 6

[0100] The synthetic method for the MSPP-6PEG-AAN-PAB-DAM-4PEG-Paclitaxel peptide conjugate is as follows:

[0101] The specific synthesis steps are as follows:

[0102] 1. Synthesis of Compound 6-III

[0103] Compound 6-I (500 mg, 1.41 mmol) was dissolved in N,N-dimethylformamide (20 mL), and compound 6-II (520 mg, 1.42 mmol) and DIEA (N,N-diisopropylethylamine) (450 mg, 3.49 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 10:1) to give compound 6-III as a pale yellow solid (780 mg, yield 90.5%).

[0104] 2. Synthesis of Compound 6-IV

[0105] Compound 6-III (780 mg, 1.28 mmol) was added to dichloromethane (50 mL), along with N-hydroxysuccinimide (180 mg, 1.56 mmol) and DCC (N,N'-dicyclohexylcarbodiimide) (400 mg, 1.94 mmol). The reaction mixture was reacted at room temperature (25 °C) for 3 hours, and the reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 15:1) to give compound 6-IV as a pale yellow solid (396 mg, yield 44.1%).

[0106] 3. Synthesis of compound 6-V

[0107] Compound 1-I (160 mg, 0.58 mmol) was dissolved in N,N-dimethylformamide (20 mL), and compound 6-IV (396 mg, 0.57 mmol) and DIEA (N,N-diisopropylethylamine) (200 mg, 1.55 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography to give compound 6-V as a pale yellow solid (290 mg, yield 59.7%).

[0108] 4. Synthesis of compound 6-VI

[0109] Compound 6-V (290 mg, 0.34 mmol) was dissolved in N,N-dimethylformamide (20 mL), and p-aminobenzyl alcohol (50 mg, 0.41 mmol), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (200 mg, 0.53 mmol), and DIEA (N,N-diisopropylethylamine) (180 mg, 1.40 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 10:1) to give compound 6-VI as a pale yellow solid (240 mg, yield 73.1%).

[0110] 5. Synthesis of compound 6-VII

[0111] Compound 6-VI (240 mg, 0.25 mmol) was dissolved in N,N-dimethylformamide (20 mL), and bis(p-nitrobenzene) carbonate (160 mg, 0.53 mmol) and DIEA (N,N-diisopropylethylamine) (150 mg, 1.16 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was slurried twice with ethyl acetate:methyl tert-butyl ether (1:3) to give compound 6-VII as a yellow solid (190 mg, yield 67.3%).

[0112] 6. Synthesis of compound MSPP-6PEG-AAN-PAB-DAM-4PEG-Paclitaxel

[0113] Compound 1-X (180 mg, 0.16 mmol) and compound 6-VII (190 mg, 0.17 mmol) were dissolved in N,N-dimethylformamide (25 mL), and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 8 hours, and the reaction proceeded to completion by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to obtain MSPP-6PEG-AAN-PAB-DAM-4PEG-Paclitaxel as a white solid (92 mg, yield 27.3%).

[0114]

Example 7

[0115] The synthetic method for the BA-6PEG-AAN-PAB-DAM-4PEG-Paclitaxel peptide conjugate is as follows:

[0116] The specific synthesis steps are as follows:

[0117] 1. Synthesis of compound 7-II

[0118] Compound 7-I (500 mg, 1.41 mmol) was dissolved in N,N-dimethylformamide (20 mL), and bromoacetic acid (200 mg, 1.44 mmol), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (750 mg, 1.97 mmol), and DIEA (N,N-diisopropylethylamine) (450 mg, 3.49 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 30:1 to 10:1). The resulting solid was dissolved in dichloromethane (50 mL), and trifluoroacetic acid (10 mL) was added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by HPLC. The reaction solution was evaporated under reduced pressure to dryness to give compound 7-II as a pale yellow solid (535 mg, yield 80.0%).

[0119] 2. Synthesis of Compound 7-III

[0120] Compound 7-II (535 mg, 1.13 mmol) was added to dichloromethane (50 mL), along with N-hydroxysuccinimide (180 mg, 1.56 mmol) and DCC (N,N'-dicyclohexylcarbodiimide) (450 mg, 2.18 mmol). The reaction mixture was reacted at room temperature (25 °C) for 3 hours, and the reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 12:1) to give compound 7-III as a pale yellow solid (356 mg, yield 55.1%).

[0121] 3. Synthesis of compound 7-IV

[0122] Compound 1-I (170 mg, 0.62 mmol) was dissolved in N,N-dimethylformamide (20 mL), and compound 7-III (356 mg, 0.62 mmol) and DIEA (N,N-diisopropylethylamine) (200 mg, 1.55 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography to give compound 7-IV as a pale yellow solid (284 mg, yield 62.7%).

[0123] 4. Synthesis of compound 7-V

[0124] Compound 7-IV (284 mg, 0.39 mmol) was dissolved in N,N-dimethylformamide (20 mL), and p-aminobenzyl alcohol (50 mg, 0.41 mmol), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (200 mg, 0.53 mmol), and DIEA (N,N-diisopropylethylamine) (180 mg, 1.40 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 3 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 10:1) to give compound 7-V as a pale yellow solid (255 mg, yield 78.2%).

[0125] 5. Synthesis of compound 7-VI

[0126] Compound 7-V (255 mg, 0.31 mmol) was dissolved in N,N-dimethylformamide (20 mL), and bis(p-nitrobenzene) carbonate (160 mg, 0.53 mmol) and DIEA (N,N-diisopropylethylamine) (150 mg, 1.16 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. The reaction was confirmed to be complete by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was slurried twice with ethyl acetate:methyl tert-butyl ether (1:3) to give compound 7-VI as a yellow solid (230 mg, yield 74.1%).

[0127] 6. Synthesis of compound BA-6PEG-AAN-PAB-DAM-4PEG-Paclitaxel

[0128] Compound 1-X (220 mg, 0.19 mmol) and compound 7-VI (230 mg, 0.23 mmol) were dissolved in N,N-dimethylformamide (25 mL), and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 8 hours, and the reaction proceeded to completion as determined by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to obtain BA-6PEG-AAN-PAB-DAM-4PEG-Paclitaxel as a white solid (123 mg, yield 32.5%).

[0129]

Example 8

[0130] The synthetic method for the MC-6PEG-AANG-PAB-DAM-4PEG-Docetaxel peptide conjugate is as follows:

[0131] The specific synthesis steps are as follows:

[0132] 1. Synthesis of compound 8-I

[0133] Docetaxel (480 mg, 0.59 mmol) was dissolved in N,N-dimethylformamide (50 mL), and p-nitrobenzene chloroformate (140 mg, 0.70 mmol), DMAP (dimethylaminopyridine) (20 mg, 0.16 mmol), and DIEA (N,N-diisopropylethylamine) (200 mg, 1.55 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 4 hours. HPLC analysis showed that 12% of the starting material remained. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 15:1) to give compound 8-I as a pale yellow solid (169 mg, yield 29.4%).

[0134] 2. Synthesis of compound 8-II

[0135] Compound 8-I (169 mg, 0.17 mmol) was dissolved in N,N-dimethylformamide (30 mL), and compound 1-VII (100 mg, 0.29 mmol) and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) were added. The reaction mixture was reacted at room temperature (25 °C) for 6 hours. HPLC (high performance liquid chromatography) was used to confirm the complete reaction of the starting materials. The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 50:1 to 10:1) to give compound 8-II as a yellow solid (165 mg, yield 80.1%).

[0136] 3. Synthesis of Compound 8-III

[0137] Compound 8-II (165 mg, 0.14 mmol) was added to dichloromethane (15 mL), followed by trifluoroacetic acid (8 mL). The reaction mixture was reacted at room temperature (25 °C) for 2 hours. HPLC (high performance liquid chromatography) confirmed that the reaction proceeds were complete. The reaction mixture was evaporated to dryness under reduced pressure to give compound 8-III as a brown solid (139 mg, yield 87.9%).

[0138] 4. Synthesis of compound MC-6PEG-AANG-PAB-DAM-4PEG-Docetaxel

[0139] Compound 8-III (139 mg, 0.12 mmol) and compound 4-IV (210 mg, 0.19 mmol) were dissolved in N,N-dimethylformamide (25 mL), and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 8 hours, and the reaction proceeded to completion by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to obtain MC-6PEG-AANG-PAB-DAM-4PEG-Docetaxel as a white solid (44 mg, yield 18.0%).

[0140]

Example 9

[0141] The synthetic method for the MC-6PEG-AANL-PAB-DAM-4PEG-Docetaxel peptide conjugate is as follows:

[0142] The specific synthesis steps are as follows:

[0143] 1. Synthesis of compound MC-6PEG-AANL-PAB-DAM-4PEG-Docetaxel

[0144] Compound 8-III (120 mg, 0.10 mmol) and compound 5-IV (200 mg, 0.17 mmol) were dissolved in N,N-dimethylformamide (25 mL), and DIEA (N,N-diisopropylethylamine) (120 mg, 0.93 mmol) was added. The reaction mixture was reacted at room temperature (25 °C) for 8 hours, and the reaction proceeded to completion as determined by HPLC (high performance liquid chromatography). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by reverse-phase chromatography under high pressure to obtain MC-6PEG-AANL-PAB-DAM-4PEG-Docetaxel as a white solid (16 mg, yield 7.7%).

[0145] [Example 10] Legumain enzyme and tumor homogenate activation assay of paclitaxel and docetaxel peptide conjugate

[0146] Buffer preparation: 50 mM MES, 250 mM sodium chloride, pH adjusted to 5.0 with 0.5 M sodium hydroxide. Legumain was selected at a concentration of 1 mg / mL. The compound was prepared to a concentration of 0.5 μmol / mL using buffer. 50 μL of the 0.5 μmol / mL compound and 50 μL of buffer were accurately transferred to a centrifuge tube. 100 μL of Legumain or 100 μg of ovarian cancer (A2780) tissue homogenate (tumor homogenate prepared from mouse tumor tissue using a Jingxin F6 / 10 handheld homogenizer) was added, and the mixture was incubated at 37°C for 2 h. The reaction solution was analyzed by LC-MS.

[0147] Table 1. Paclitaxel and docetaxel peptide conjugate Legumain and tumor homogenate activation products

[0148] As can be seen from Table 1 above, the paclitaxel and docetaxel polypeptide conjugate of the present invention can be specifically recognized and cleaved by legumain, and can also be activated by tumor homogenate to release paclitaxel or docetaxel.

[0149]

Example 11

[0150] Tumor cells were cultured in complete medium (RPMI 1640 (DMEM high-glucose medium) + 10% fetal bovine serum + 1XP / S + 1mM sodium pyruvate solution) at 37°C in a 5% CO2 incubator until sufficient cell numbers were reached. Cells were collected, centrifuged at 1000g for 5 min, and resuspended in an appropriate volume of 10% RPMI 1640 (DMEM high-glucose medium) to adjust the density to 4,000,000 cells / mL. 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 blank wells (medium-only medium) were also included, with three parallel wells for each group. After cell counting, the cells were seeded onto 96-well plates at a concentration of 5000 cells (100 μL) per well. The plate was 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 plate was incubated in a cell culture incubator for about 2 hours. The absorbance at 450 nm was then measured. The Legumain activation procedure was the same as in Example 10.

[0151] Calculate cell viability and the half-maximal inhibitory concentration (IC50) of the drug on cells. Cell viability % = (OD assay - OD blank control) / (OD assay control - OD blank control) * 100%. Viability (%) was calculated using Excel software, and the dose-response curve of the drug on cells was plotted using Prism 5 software. All indicators are expressed as means, and the coefficient of variation (CV) was used to assess the consistency of the data.

[0152] Based on the above experimental method, the maximum initial concentration of the test drug was set to 10 μM, and the initial concentration of the Legumin-activated complex was set to 50 μM. Nine dose groups were serially diluted at a ratio of 1:3 (three replicates per group). The concentration of the drug solvent (DMSO) in all wells was controlled at 0.1%. A control group (Control) was formed by adding only the drug solvent (0.1% DMSO), and a blank group (Blank) was formed by adding only culture medium and no cells. The tumor cell survival rate (%) of each dose group relative to the control group (Control) was then calculated using the following method:

[0153] Cell viability = [(Experimental group absorbance - Blank control absorbance) / (Control group absorbance - Blank control absorbance)] × 100%

[0154] The specific experimental results are shown in the following table:

[0155] Table 2. Toxicity (IC50) of paclitaxel and docetaxel peptide conjugate (without Legumain activation) in different cell lines.

[0156] Table 3. Toxicity (IC50) of paclitaxel and docetaxel peptide conjugates (after Legumin activation) versus paclitaxel and docetaxel monomers in different cell lines.

[0157] As shown in Tables 2 and 3, the IC50 values ​​of paclitaxel and docetaxel peptide conjugates in the above cells were significantly higher than those of paclitaxel or docetaxel alone. Therefore, the cytotoxicity of paclitaxel and docetaxel drugs provided in Table 2 was greatly reduced without Legumain activation. That is, the paclitaxel and docetaxel peptide conjugates provided in Table 2 have very low toxicity compared to the compounds themselves, but their toxicity increased significantly after Legumain activation. This indicates that paclitaxel and docetaxel peptide conjugates can be activated by Legumain and released in the tumor microenvironment where Legumain is highly expressed, achieving targeted release into the tumor.

[0158] [Example 12] Pharmacodynamic study of paclitaxel and docetaxel peptide conjugate in the treatment of mouse A2780 tumor model

[0159] Objective: To investigate the antitumor efficacy of the above compounds in the A2780 tumor model.

[0160] Test drugs: paclitaxel and docetaxel peptide conjugate, paclitaxel and docetaxel, and saline control group.

[0161] Experimental animals: 6-8 week old BALB / c mice, all of which were female.

[0162] Tumor model preparation: A2780 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were passaged every three days, and cells up to passage 15 were used. 4 × 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 six. Treatment began on day one. Paclitaxel and docetaxel at 15 mg / kg served as the positive control, while paclitaxel and docetaxel peptide conjugate at 75 mg / kg served as the experimental group. The negative control group received saline. Administered once weekly for three weeks. Data are shown in the table below:

[0163] Table 4. Statistical analysis of the efficacy of paclitaxel and docetaxel peptide conjugates in the treatment of A2780 tumor model.

[0164] As can be seen from Example 13, the MTD of paclitaxel is 30 mg / kg, and the dosage of 15 mg / kg in this example is half of the MTD. The MTD of docetaxel is 20 mg / kg, and the dosage of 15 mg / kg in this example is three-quarters of the MTD. The average MTD of paclitaxel peptide conjugates X1-X9 is around 200 mg / kg, and the dosage of 75 mg / kg in this example is approximately one-third of the MTD.

[0165] As can be seen from the data in Table 4, the paclitaxel-docetaxel peptide conjugate of the present invention achieves a tumor-suppressive effect comparable to that of monotherapy with a relatively lower dosage compared to monotherapy. Furthermore, the peptide conjugate of the present invention exhibits good targeting, selectively releasing into the tumor microenvironment while maintaining good stability in normal cells with almost no release. Therefore, larger doses can be used clinically to treat cancer, and due to its lower toxicity, patient adverse reactions are significantly reduced, thus improving the overall therapeutic effect in various aspects.

[0166] [Example 13] Detection experiment of MTD of paclitaxel and docetaxel peptide conjugate

[0167] Experimental animals: 6-8 week old Balb / c Nude mice, all female, were randomly divided into groups of six. Mice were administered drugs at different concentration gradients and monitored for 14 days. Mice were euthanized when they lost 20% of their initial body weight, which was considered death due to toxicity. The maximum dose level (MTD) was defined as the highest dose level in which no six mice died from the drug, no individual mouse lost more than 20% of its body weight, or the average body weight loss within the group did not exceed 15%. Final results:

[0168] Table 5. MTD in mice with paclitaxel and docetaxel peptide conjugates

[0169] As shown in Example 13, the maximum tolerated dose of the paclitaxel and docetaxel peptide conjugate in mice is significantly higher than that of paclitaxel and docetaxel alone. This indicates that the toxicity of the paclitaxel and docetaxel peptide conjugate of the present invention is significantly reduced compared to the single drug, and therefore it is expected to be used in clinical practice at higher doses to improve the overall therapeutic effect.

[0170] [Example 14] Experiment on the activation efficiency of paclitaxel and docetaxel peptide conjugate in tumor homogenates and their release in normal tissues

[0171] PBS buffer: Beijing Lanjieke Technology Co., Ltd., Cat. NO: BL302A, Lot. No: 24149299.

[0172] Paclitaxel and docetaxel peptide conjugates were prepared into a 1 mg / mL solution using PBS buffer. 200 μL of this solution was added to 100 μg of mouse tumor or normal tissue homogenate (the tissue homogenate was prepared from mouse tumor or normal tissue using a Jingxin F6 / 10 handheld homogenizer). The solution was incubated at 37°C for 2 h. HPLC (High Performance Liquid Chromatography) was used to detect the decrease in the concentration of the compounds and the increase in paclitaxel and docetaxel, thus comparing the activation efficiency of the drugs in tumor tissue.

[0173] Release percentage = Peak area of ​​paclitaxel or docetaxel / Total peak area of ​​paclitaxel or docetaxel and all other compounds containing paclitaxel or docetaxel

[0174] The results are shown in the table below:

[0175] Table 6. Percentage of release efficiency (%) of paclitaxel and docetaxel peptide conjugates in tumor tissue.

[0176] Table 7. Percentage of release efficiency (%) of paclitaxel and docetaxel peptide conjugate in normal mouse tissues.

[0177] As can be seen from the data in Table 7, paclitaxel and docetaxel peptide conjugates can release large amounts of toxin molecules in tumor homogenates, while the release amount in normal tissues is very small. Therefore, the drug has good tumor selectivity and the damage to normal tissues and organs is significantly reduced compared with paclitaxel and docetaxel themselves, thus effectively improving the safety window and making it possible to use higher doses in clinical practice.

[0178] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide conjugate of paclitaxel or docetaxel, characterized in that, The structural formula of the polypeptide conjugate is as follows: E-nPEG-L1-L2-L3-L4-L5-D in, E is a maleimide group, a methyl sulfone compound, or a halogen group; nPEG stands for n-polyethylene glycol, where n is an integer greater than or equal to 1 and less than or equal to 40. L1 is glycine, alanine, or serine; L2 is alanine; L3 is asparagine; L4 is glycine, leucine, or absent; L5 is a p-aminobenzyl alcohol-ethylenediamine derivative; D represents paclitaxel, docetaxel, or their derivatives.

2. The polypeptide conjugate according to claim 1, characterized in that, The D group is selected from one of the following structures:

3. The polypeptide conjugate according to claim 1, characterized in that, When the E group is a maleimide-containing group, the E group is selected from the following structures: Where n is an integer greater than or equal to 1 and less than or equal to 18.

4. The polypeptide conjugate according to claim 1, characterized in that, When the E group is a methyl sulfone compound, the E group is selected from one of the following structures: Where n is an integer greater than or equal to 0 and less than or equal to 10.

5. The polypeptide conjugate according to claim 1, characterized in that, When the E group is a halogen group, the E group is selected from the following structures: Where X is a halogen atom.

6. The polypeptide conjugate according to claim 1, characterized in that, The nPEG group is selected from one of the following structures: Where n is an integer greater than or equal to 1 and less than or equal to 40.

7. The polypeptide conjugate according to claim 1, characterized in that, The structure of the L5 group is as follows: Wherein, R1 and R2 are hydrogen or methyl, and R1 and R2 may be the same or different; n1 and n2 are both integers greater than or equal to 0 and less than or equal to 20, and n1 and n2 may be the same or different.

8. The polypeptide conjugate according to claim 1, characterized in that, The polypeptide conjugate has any of the following structures:

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

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

11. Use of the polypeptide conjugate of any one of claims 1-8 and the pharmaceutical composition of claim 9 in the preparation of a medicament for treating cancer, wherein the cancer includes 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, 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 malignancies, 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.

Images