Polypeptide conjugate of Sting small molecule agonist and application of polypeptide conjugate

Abstract

The invention belongs to the technical field of polypeptide conjugate drugs, and particularly relates to a polypeptide conjugate of a Sting small molecule agonist and application of the polypeptide conjugate, and the structural formula of the polypeptide conjugate is shown as the following formula: E-P-L1-L2-L3-L4-L5-D. The polypeptide conjugate of the Sting small molecule agonist has the beneficial effects that the polypeptide conjugate of the Sting small molecule agonist can block the activity of the Sting small molecule agonist, and the polypeptide conjugate can be activated by Legumain highly expressed by tumor cells and tumor-related macrophages in a tumor slightly acidic microenvironment, so that a medicine is selectively released in the tumor microenvironment; the compound has the functions of inhibiting tumor growth, inducing immunogenic death of tumor cells and stimulating anti-tumor immunity of a body, reduces the toxic and side effects of the Sting small-molecule agonist, has good targeting property, and achieves multiple effects of inhibiting tumor growth, promoting anti-tumor immunity and reducing the toxic and side effects of drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide-conjugated drugs, and particularly relates to a polypeptide conjugate of a Sting small molecule agonist and its application. Background Art

[0002] In the past few years, the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway has attracted much attention due to its ability to activate innate and adaptive immunity, which contributes to host defense against cancer. Based on the key role of the cGAS-STING signaling pathway in anti-cancer immunity, various STING agonists have been developed and proven to be a promising cancer treatment strategy.

[0003] The development of STING agonists mainly includes cyclic dinucleotides (CDNs) and small molecule compounds. Cyclic dinucleotides are natural ligands of STING, such as c-diGMP, c-diAMP, 3′,3′-cGAMP, and 2′,3′-cGAMP. However, the clinical development of natural CDNs is limited by the rapid hydrolysis by phosphodiesterase, poor cytoplasmic delivery, and the lack of the ability to activate all human STING subtypes, which greatly restricts their application. Therefore, the development of small molecule Sting agonists has received extensive attention. Summary of the Invention

[0004] The present application provides a polypeptide conjugate of a Sting small molecule agonist and its application, aiming to solve the problem that the existing Sting small molecule agonists lack targeting and have strong side effects, resulting in limited clinical application.

[0005] The first aspect of the present application provides a polypeptide conjugate of a Sting small molecule agonist, and the structural formula of the polypeptide conjugate is shown as follows: E-P-L1-L2-L3-L4-L5-D;

[0006] Wherein, E includes the following groups:

[0007]

[0008] The "1" position is connected to P or L1, 1 ≤ n ≤ 18, and X includes a halogen;

[0009] P may or may not exist. When P exists, P includes polyethylene glycol;

[0010] L1 and L2 each independently include one or more of glycine, alanine, and serine;

[0011] L3 includes asparagine;

[0012] L4 may or may not exist. When L4 exists, L4 includes glycine and / or leucine;

[0013] L5 is present or absent. When L5 is present, L5 includes the following structure:

[0014]

[0015] The "4" position is connected to the carboxyl terminus of L3 or L4, the "5" position is connected to D, 0 ≤ n2 ≤ 40 and n2 is an integer, and R1 includes hydrogen and / or methyl;

[0016] D includes a Sting small molecule agonist.

[0017] In some embodiments of the polypeptide conjugate of the Sting small molecule agonist according to the present application, the structural formula of the polyethylene glycol is as shown in the following formula:

[0018]

[0019] Wherein, the "2" position is connected to E, the "3" position is connected to the amino group of L1, 1 ≤ n1 ≤ 40 and n1 is an integer.

[0020] In some embodiments of the polypeptide conjugate of the Sting small molecule agonist according to the present application, the structural formula of D is as shown in the following formula:

[0021]

[0022] Wherein, the "6" position is connected to the carboxyl terminus of L5, L4 or L3, 0 ≤ n3 ≤ 20 and n3 is an integer.

[0023] In some embodiments of the polypeptide conjugate of the Sting small molecule agonist according to the present application, the structural formula of the polypeptide conjugate is as shown in Formula X1-X19:

[0024]

[0025]

[0026]

[0027]

[0028]

[0029] The second aspect of the present application provides a pharmaceutical composition, comprising the polypeptide conjugate described in the first aspect of the present application or a pharmaceutically acceptable carrier thereof.

[0030] The third aspect of the present application provides an application of the polypeptide conjugate described in the first aspect of the present application or the pharmaceutical composition described in the second aspect of the present application in the preparation of an anti-tumor drug for treatment or prevention.

[0031] According to some embodiments of the application described herein, the tumor includes one or more of gastrointestinal cancer, colorectal cancer, colon cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, gastric cancer, genitourinary system cancer, bladder cancer, testicular cancer, cervical cancer, malignant mesothelioma, 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.

[0032] The beneficial effects of the present application include: The polypeptide conjugate of the Sting small molecule agonist described in the present application can block the activity of the Sting small molecule agonist, and it 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 drug is selectively released in the tumor microenvironment, causing immunogenic death of tumor cells, stimulating the anti-tumor immune function of the body, reducing the toxic and side effects of the Sting small molecule agonist, having good targeting, and achieving multiple effects of inhibiting tumor growth while promoting anti-tumor immunity and reducing the toxic and side effects of the drug. Detailed implementation manners

[0033] 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, and should not be construed as limiting the present invention.

[0034] 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.

[0035] Example 1

[0036] Synthesis of MC-6PEG-AANG-CX-ST01 (X1)

[0037] The synthetic route of the compound shown in X1 includes:

[0038]

[0039] The specific synthesis method includes the following steps:

[0040] (1) Synthesis of Compound 1-II

[0041] Compound 1-I (10 g, 43.4 mmol) was dissolved in dichloromethane (150 mL), and the temperature was lowered to 0 °C in an ice-water bath. A dichloromethane solution of boron tribromide (180 mL, 1 M, 180 mmol) was slowly added dropwise to the dichloromethane. The ice bath was removed, and the reaction solution was slowly warmed to room temperature (25 °C) and reacted for 18 h. HPLC detected that the raw material reaction was complete. The reaction solution was poured into 3 L of ice hydrate, stirred for 30 min, filtered, and the solid was dried to obtain Compound 1-II as a yellowish-brown solid (6.5 g, yield 69.2%).

[0042] (2) Synthesis of Compound 1-IV

[0043] Compound 1-II (1.5 g, 6.9 mmol) and Compound 1-III (2.6 g, 7.2 mmol) were added to N,N-dimethylformamide (150 mL), and then potassium carbonate (2 g, 14.5 mmol) was added to form a reaction solution. The above reaction solution was heated to 70 °C and reacted for 8 h. HPLC detected that the reaction was complete. The reaction solution was poured into 800 mL of water, stirred at room temperature (25 °C) for 1 h, filtered, and the solid was air-dried and then purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 25:1) to obtain Compound 1-IV as a yellow solid (2.5 g, yield 73.1%).

[0044] (3) Synthesis of Compound 1-VI

[0045] Compound 1-I (1.5 g, 6.5 mmol) was added to a 100 mL pressure-resistant bottle. Anhydrous ethanol (25 mL), Compound 1-V (1.5 g, 8.1 mmol), and N,N-diisopropylethylamine (DIEA, 2.8 g, 21.7 mmol) were added to the pressure-resistant bottle to form a reaction solution. The reaction solution was heated to 120 °C and reacted for 18 hours. After the reaction was completed, the reaction solution was cooled to room temperature (25 °C), evaporated to dryness under reduced pressure, and 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 15:1) to obtain Compound 1-VI as a light brown solid (1.6 g, yield 64.7%).

[0046] (4) Synthesis of Compound 1-VII

[0047] Compound 1-VI (1.6 g, 4.2 mmol) was dissolved in methanol (20 mL), and then 4M dioxane hydrochloride (10 mL, 40 mmol) was added to the methanol to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 2 hours. The reaction solution was evaporated to dryness under reduced pressure, and the product obtained by evaporation to dryness under reduced pressure was slurried with tert-butyl methyl ether (50 mL * 3) to obtain Compound 1-VII as a yellowish-brown solid (1.1 g, yield 82.7%).

[0048] (5) Synthesis of Compound 1-VIII

[0049] Compound 1-VII (1.1 g, 3.5 mmol) was added to n-butanol (15 mL), and then sodium bicarbonate (600 mg, 7.1 mmol) and N,N-diisopropylethylamine (DIEA, 1.8 g, 14.0 mmol) were added to the n-butanol to form a reaction solution. The reaction solution was stirred at room temperature (25 °C) for 10 min, and then Compound 1-IV (1.6 g, 3.2 mmol) was added. Then the reaction solution was heated to 120 °C and reacted for 18 h. After the reaction was completed, the reaction solution was cooled to room temperature 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 10:1) to obtain a brown solid (1.2 g). The above solid was dissolved in methanol (30 mL), and under ice-bath cooling, an aqueous solution of sodium dithionite (4.0 g, 23.0 mmol) in 15 mL and 6 mL of ammonia water were added to the methanol, and then the temperature was raised to room temperature (25 °C) and reacted for 30 min. After the reaction was completed, extraction was carried out with ethyl acetate (100 mL * 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate 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 8:1) to obtain Compound 1-VIII as a yellow solid (410 mg, yield 17.2%).

[0050] (6) Synthesis of Compound CX-ST01

[0051] Compound 1-VIII (410 mg, 0.60 mmol) was dissolved in methanol (10 mL), and then bromine cyanide (450 mg, 4.25 mmol) was added to the methanol to form a reaction solution. The reaction solution was stirred at room temperature (25 °C) for 30 min, and then the reaction solution was filtered. The solid obtained by filtration was washed with ethyl acetate and dried in air to obtain Compound 1-IX (360 mg, yield: 81.7%);

[0052] Compound 1-IX (360 mg, 0.49 mmol) was dissolved in N,N-dimethylformamide (10 mL). Then, 1-ethyl-3-methyl-1H-pyrazole-5-carboxylic acid (200 mg, 1.30 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 500 mg, 1.32 mmol), 1-hydroxybenzotriazole (HOBt, 100 mg, 0.76), and N,N-diisopropylethylamine (DIEA, 350 mg, 2.71 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 18 h. Then, 1 mL of 5 N sodium hydroxide solution was added to the reaction solution, and the mixture was stirred at room temperature for 30 min. Subsequently, 50 mL of water was added to the reaction solution, and the mixture was filtered. The solid 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 CX-ST01 (180 mg, yield 38.5%).

[0053] (7) Synthesis of compound MC-6PEG-AANG-CX-ST01

[0054] Compound 1-X (200 mg, 0.24 mmol) and compound CX-ST01 (180 mg, 0.23 mmol) were dissolved in N,N-dimethylformamide (10 mL). Then, HATU (120 mg, 0.32 mmol) and DIEA (120 mg, 0.93 mmol) were added to the N,N-dimethylformamide, and the mixture was reacted at room temperature (25 °C) for 5 h. The reaction was monitored by HPLC, and when the raw materials were completely reacted, the reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparative chromatography to obtain the off-white solid of MC-6PEG-AANG-CX-ST01 (110 mg, yield 30.3%).

[0055] Example 2

[0056] Synthesis of MC-6PEG-AANL-CX-ST01 (X2)

[0057] The synthetic route of the compound shown in X2 includes:

[0058]

[0059] The specific synthesis steps include:

[0060] Dissolve compound 2-I (200 mg, 0.23 mmol) and compound CX-ST01 (180 mg, 0.23 mmol) in N,N-dimethylformamide (10 mL). Then add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) to the N,N-dimethylformamide to form a reaction solution. React the reaction solution at room temperature (25 °C) for 5 hours. Detect by HPLC that the raw materials have completely reacted. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-AANL-CX-ST01 (78 mg, yield 20.8%).

[0061] Example 3

[0062] Synthesis of MC-6PEG-AAN-CX-ST01 (X3)

[0063] The synthesis route of the compound shown in X3 includes:

[0064]

[0065] The specific synthesis steps include:

[0066] Dissolve compound 3-I (190 mg, 0.25 mmol) and compound CX-ST01 (180 mg, 0.23 mmol) in N,N-dimethylformamide (10 mL). Then add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) to the N,N-dimethylformamide to form a reaction solution. React the reaction solution at room temperature (25 °C) for 5 hours. Detect by HPLC that the raw materials have completely reacted. Evaporate the reaction solution to dryness under reduced pressure. Purify the product obtained by evaporation to dryness under reduced pressure by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-AAN-CX-ST01 (89 mg, yield 25.4%).

[0067] Example 4

[0068] Synthesis of MC-6PEG-AAN-PAB-CX-ST01 (X4)

[0069] The synthesis route of the compound shown in X4 includes:

[0070]

[0071] The specific synthesis steps include:

[0072] Compound 4-I (260 mg, 0.25 mmol) and compound CX-ST01 (180 mg, 0.23 mmol) were dissolved in N,N-dimethylformamide (10 mL). Then, N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) was added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and HPLC was used to detect that the raw materials had completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain MC-6PEG-AAN-PAB-CX-ST01 as a white solid (106 mg, yield 27.6%).

[0073] Example 5

[0074] Synthesis of MC-6PEG-GANG-CX-ST01 (X5)

[0075] The synthetic route of the compound shown in X5 includes:

[0076]

[0077] The specific synthesis method includes the following steps:

[0078] Compound 5-I (200 mg, 0.25 mmol) and compound CX-ST01 (180 mg, 0.23 mmol) were dissolved in N,N-dimethylformamide (10 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and HPLC was used to detect that the raw materials had completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain MC-6PEG-GANG-CX-ST01 as a white solid (113 mg, yield 31.4%).

[0079] Example 6

[0080] Synthesis of MC-6PEG-SANG-CX-ST01 (X6)

[0081] The synthetic route of the compound shown in X6 includes:

[0082]

[0083] The specific synthesis method includes the following steps:

[0084] Compound 6-I (210 mg, 0.25 mmol) and compound CX-ST01 (180 mg, 0.23 mmol) were dissolved in N,N-dimethylformamide (10 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and HPLC was used to detect that the raw materials had completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-SANG-CX-ST01 (31 mg, yield 8.4%).

[0085] Example 7

[0086] Synthesis of MSPP-6PEG-AANG-CX-ST01 (X7)

[0087] The synthetic route of the compound shown in X7 includes:

[0088]

[0089] The specific synthesis method includes the following steps:

[0090] Compound 7-I (230 mg, 0.25 mmol) and compound CX-ST01 (180 mg, 0.23 mmol) were dissolved in N,N-dimethylformamide (10 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and HPLC was used to detect that the raw materials had completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a white solid of MSPP-6PEG-AANG-CX-ST01 (64 mg, yield 16.6%).

[0091] Example 8

[0092] Synthesis of BA-6PEG-AANG-CX-ST01 (X8)

[0093] The synthetic route of the compound shown in X8 includes:

[0094]

[0095] The specific synthesis method includes the following steps:

[0096] Compound 8-I (190 mg, 0.24 mmol) and compound CX-ST01 (180 mg, 0.23 mmol) were dissolved in N,N-dimethylformamide (10 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and HPLC was used to detect that the raw materials had completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a pale yellow solid of BA-6PEG-AANG-CX-ST01 (13 mg, yield 3.6%).

[0097] Example 9

[0098] Synthesis of MC-6PEG-AANG-AM-CX-ST02 (X9)

[0099] The synthetic route of the compound shown in X9 includes:

[0100]

[0101] The specific synthesis method includes the following steps:

[0102] (1) Synthesis of compound 9-II

[0103] Compound 1-II (1.5 g, 6.9 mmol) and compound 9-I (1.9 g, 7.5 mmol) were added to N,N-dimethylformamide (150 mL). Then, potassium carbonate (2 g, 14.5 mmol) was added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was heated to 50 °C and reacted for 18 h, and HPLC was used to detect that the reaction was complete. 800 mL of water was poured into the reaction solution, and it was stirred at room temperature (25 °C) for 1 h. After filtration, the solid was dried and purified by silica gel column (eluted with dichloromethane (DCM):methanol (MeOH) with a volume ratio of 100:1 to 25:1) to obtain a yellow solid of compound 9-II (2.6 g, yield 96.9%).

[0104] (2) Synthesis of compound 9-III

[0105] Compound 1-VII (1.1 g, 3.5 mmol) was added to n-butanol (15 mL). Then, sodium bicarbonate (600 mg, 7.1 mmol) and N,N-diisopropylethylamine (DIEA, 1.8 g, 14.0 mmol) were added to the n-butanol to form a reaction solution. The reaction solution was stirred at room temperature (25 °C) for 10 min, then compound 9-II (1.25 g, 3.2 mmol) was added, and the temperature was raised to 120 °C for reaction for 18 h. Then, the reaction solution was cooled to room temperature 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 10:1) to obtain a brown solid (1.4 g). The above solid was dissolved in methanol (30 mL), and an aqueous solution of sodium dithionite (4.0 g, 23.0 mmol) and 6 mL of ammonia water were added under ice bath cooling, and the temperature was raised to room temperature (25 °C) for reaction for 30 min. Then, extraction was carried out with ethyl acetate (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate 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 8:1) to obtain a yellowish-brown solid of compound 9-III (720 mg, yield: 39.3%).

[0106] (3) Synthesis of Compound CX-ST02

[0107] Compound 9-III (720 mg, 1.26 mmol) was dissolved in methanol (16 mL). Then, bromine cyanide (900 mg, 8.50 mmol) was added to the methanol to form a reaction solution. The reaction solution was stirred at room temperature (25 °C) for 30 min, the reaction solution was filtered, and the solid obtained by filtration was washed with ethyl acetate and dried to obtain compound 9-IV (665 mg, yield: 84.9%).

[0108] Compound 9-IV (665 mg, 1.07 mmol) was dissolved in N,N-dimethylformamide (16 mL). Then, 1-ethyl-3-methyl-1H-pyrazole-5-carboxylic acid (400 mg, 2.60 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 1.0 g, 2.64 mmol), 1-hydroxybenzotriazole (HOBt, 200 mg, 1.52) and N,N-diisopropylethylamine (DIEA, 700 mg, 5.42 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 18 h. Then, 2 mL of 5N sodium hydroxide solution was added to the reaction solution, and the mixture was stirred at room temperature for 30 min. Next, 100 mL of water was added to the reaction solution, and the mixture was filtered. The solid 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 CX-ST02 as a yellow solid (402 mg, yield 40.9%).

[0109] (4) Synthesis of compound 9-VI

[0110] Compound CX-ST02 (600 mg, 0.77 mmol) and 9-V (300 mg, 0.81 mmol) were dissolved in dichloromethane (50 mL). Then, pyridinium p-toluenesulfonate (80 mg, 0.32 mmol) was added to the dichloromethane to form a reaction solution. The reaction solution was heated to reflux and reacted for 8 hours. After the reaction was completed, it was cooled to room temperature (25 °C), and the solvent was evaporated under reduced pressure. The product obtained after evaporation under reduced pressure was purified by silica gel column chromatography (eluted with dichloromethane (DCM):methanol (MeOH) with a volume ratio of 100:1 to 10:1) to obtain compound 9-VI as a yellow solid (390 mg, yield 46.6%).

[0111] (5) Synthesis of compound 9-VII

[0112] Compound 9-VI (390 mg, 0.36 mmol) was dissolved in N,N-dimethylformamide (20 mL). Then, piperidine (100 mg, 1.17 mmol) was added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 2 hours. HPLC detection showed that the raw materials had completely reacted. The solvent of the reaction solution was evaporated under reduced pressure, and the product obtained after evaporation under reduced pressure was purified by reverse-phase preparation to obtain compound 9-VII as an off-white solid (165 mg, yield 52.9%).

[0113] (6) Synthesis of compound MC-6PEG-AANG-AM-CX-ST02

[0114] Compound 9-VII (165 mg, 0.19 mmol) and compound 3-I (180 mg, 0.24 mmol) were dissolved in N,N-dimethylformamide (25 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and the raw materials were completely reacted as detected by HPLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-AANG-AM-CX-ST02 (83 mg, yield 27.1%).

[0115] Example 10

[0116] Synthesis of MC-6PEG-AANL-AM-CX-ST02 (X10)

[0117] The synthetic route of the compound shown in X10 includes:

[0118]

[0119] The specific synthesis method includes the following steps:

[0120] (1) Synthesis of compound 10-II

[0121] Compound 10-I (10 g, 24.3 mmol) was dissolved in dry tetrahydrofuran (150 mL), and then toluene (50 mL), pyridine (2.31 mL) and lead tetraacetate (13.62 g, 29.3 mmol) were added to form a reaction solution. The reaction solution was purged with nitrogen and heated to 80 °C for 5 hours. After the reaction was completed, the insoluble substances were filtered off, the filtrate was rotary evaporated, and the residue was dissolved in ethyl acetate and washed with water 3 times. The organic phase was dried over anhydrous sodium sulfate, filtered, 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 petroleum ether (PE):ethyl acetate (EA) with a volume ratio of 3:1 to 1:4) to obtain a white solid of compound 10-II (7.2 g, yield 73%).

[0122] (2) Synthesis of compound 10-III

[0123] Compound CX-ST02 (600 mg, 0.77 mmol) and 10-II (350 mg, 0.82 mmol) were dissolved in dichloromethane (50 mL). Pyridinium p-toluenesulfonate (80 mg, 0.32 mmol) was added to the dichloromethane to form a reaction solution. The reaction solution was heated to reflux for 8 hours. After the reaction was completed, the reaction solution was cooled to room temperature (25 °C) 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 10:1) to obtain compound 10-III as a pale yellow solid (396 mg, yield 45.0%).

[0124] (3) Synthesis of compound 10-IV

[0125] Compound 10-III (396 mg, 0.35 mmol) was dissolved in N,N-dimethylformamide (20 mL). Piperidine (100 mg, 1.17 mmol) was added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 2 hours. HPLC detection showed that the raw materials had completely reacted. 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 preparation to obtain compound 10-IV as an off-white solid (159 mg, yield 49.8%).

[0126] (4) Synthesis of compound MC-6PEG-AANL-AM-CX-ST02

[0127] Compound 10-IV (159 mg, 0.17 mmol) and compound 3-I (150 mg, 0.20 mmol) were dissolved in N,N-dimethylformamide (25 mL). 2-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 100 mg, 0.26 mmol) and N,N-diisopropylethylamine (DIEA, 100 mg, 0.78 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours. HPLC detection showed that the raw materials had completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain MC-6PEG-AANL-AM-CX-ST02 as an off-white solid (37 mg, yield 13.1%).

[0128] Example 11

[0129] Synthesis of MC-6PEG-AAN-AM-CX-ST02 (X11)

[0130] The synthetic route of the compound shown in X11 includes:

[0131]

[0132] The specific synthesis method includes the following steps:

[0133] (1) Synthesis of Compound 11-II

[0134] Compound 11-I (2 g, 4.03 mmol) and tert-butyl glycinate (530 mg, 4.04 mmol) were dissolved in N,N-dimethylformamide (50 mL). Then, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 3.2 g, 8.42 mmol) and N,N-diisopropylethylamine (DIEA, 2.5 g, 19.4 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and the raw materials were detected to have completely reacted by HPLC. The reaction solution was evaporated to dryness under reduced pressure. Dichloromethane (50 mL) and trifluoroacetic acid (15 mL) were added to the product obtained by evaporation to dryness under reduced pressure, and the mixture was reacted at room temperature (25 °C) for 2 hours. After detecting that the raw materials had completely reacted by HPLC, it 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 12:1) to obtain 11-II as a yellow solid (850 mg, yield 38.1%).

[0135] (2) Synthesis of Compound 11-III

[0136] Compound 11-II (850 mg, 1.54 mmol) was dissolved in dry tetrahydrofuran (20 mL), and then toluene (6 mL), pyridine (0.3 mL) and lead tetraacetate (1 g, 2.26 mmol) were added to form a reaction solution. The reaction solution was purged with nitrogen and heated to 80 °C for 5 hours. The insoluble substances were filtered off, the filtrate was rotary evaporated, and the residue was purified by silica gel column chromatography (eluted with dichloromethane (DCM):methanol (MeOH) with a volume ratio of 100:1 to 15:1) to obtain 11-III as a yellow solid (460 mg, yield 52.6%).

[0137] (3) Synthesis of Compound 11-IV

[0138] Compound CX-ST02 (600 mg, 0.77 mmol) and 11-III (460 mg, 0.81 mmol) were dissolved in dichloromethane (50 mL). Then, pyridinium p-toluenesulfonate (80 mg, 0.32 mmol) was added to the dichloromethane to form a reaction solution. The reaction solution was heated to reflux for 8 hours. After the reaction was completed, the reaction solution was cooled to room temperature (25 °C) 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 15:1) to obtain 11-IV as a brownish-yellow solid (382 mg, yield 38.6%).

[0139] (4) Synthesis of Compound 11-V

[0140] Compound 11-IV (382 mg, 0.30 mmol) was dissolved in N,N-dimethylformamide (20 mL), and piperidine (100 mg, 1.17 mmol) was added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 2 hours. HPLC detection showed that the raw materials had completely reacted. The reaction solution was evaporated to dryness under reduced pressure, and the product obtained after evaporation to dryness under reduced pressure was purified by reverse-phase preparative chromatography to obtain Compound 11-V as an off-white solid (171 mg, yield 53.5%).

[0141] (5) Synthesis of Compound MC-6PEG-AAN-AM-CX-ST02

[0142] Compound 11-V (171 mg, 0.16 mmol) and Compound 11-VI (120 mg, 0.20 mmol) were dissolved in N,N-dimethylformamide (25 mL), and N,N-diisopropylethylamine (DIEA, 100 mg, 0.78 mmol) was added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours. HPLC detection showed that the raw materials had completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparative chromatography to obtain MC-6PEG-AAN-AM-CX-ST02 as an off-white solid (64 mg, yield 25.8%).

[0143] Example 12

[0144] Synthesis of MC-6PEG-AANG-CX-ST03 (X12)

[0145] The synthetic route of the compound shown in X12 includes:

[0146]

[0147]

[0148] The specific synthesis method includes the following steps:

[0149] (1) Synthesis of Compound 12-II

[0150] Compound 1-II (1.5 g, 6.9 mmol) and Compound 12-I (2.3 g, 7.5 mmol) were added to N,N-dimethylformamide (150 mL). Then, potassium carbonate (2 g, 14.5 mmol) was added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was heated to 50 °C and reacted for 18 h. The reaction was detected to be complete by HPLC. 800 mL of water was poured into the reaction solution, and it was stirred at room temperature (25 °C) for 1 h. After filtration, the solid was dried and purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 15:1) to obtain Compound 12-II as a yellow solid (2.5 g, yield 81.8%).

[0151] (2) Synthesis of Compound 12-IV

[0152] Compound 1-ethyl-3-methyl-1H-pyrazole-5-carboxylic acid (15 g, 97.3 mmol) was dissolved in dichloromethane (300 mL). Under ice-bath cooling, N,N-dimethylformamide (0.1 mL) and oxalyl chloride (15 g, 118.2 mmol) were added to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 2 h. The reaction solution was evaporated to dryness under reduced pressure to obtain Compound 12-III (17.5 g).

[0153] Potassium thiocyanate (18.5 g, 190.4 mmol) was dissolved in acetone (450 mL). A 30 mL acetone solution of Compound 12-III (17.5 g) was added dropwise to the acetone under ice-bath. After the addition was complete, it was stirred under ice-bath for 30 min. After filtration, the filtrate 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 petroleum ether (PE): ethyl acetate (EA) with a volume ratio of 100:1 to 10:1) to obtain Compound 12-IV as a pale yellow oil (11.2 g, yield 59.0%).

[0154] (3) Synthesis of Compound 12-V

[0155] Compound 1-VII (1.1 g, 3.5 mmol) was added to n-butanol (15 mL). Then, sodium bicarbonate (600 mg, 7.1 mmol) and N,N-diisopropylethylamine (DIEA, 1.8 g, 14.0 mmol) were added to the n-butanol to form a reaction solution. The reaction solution was stirred at room temperature (25 °C) for 10 min, then compound 12-II (1.5 g, 3.4 mmol) was added and the temperature was raised to 120 °C for reaction for 18 h. After the reaction was completed, the reaction solution was cooled to room temperature 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 10:1) to obtain a brown solid (1.5 g). The above solid was dissolved in methanol (30 mL). An aqueous solution of sodium dithionite (4.0 g, 23.0 mmol) in 15 mL and 6 mL of ammonia water were added under ice bath cooling, and the temperature was raised to room temperature (25 °C) for reaction for 30 min. After the reaction was completed, extraction was carried out with ethyl acetate (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate 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 8:1) to obtain a light brown solid of compound 12-V (684 mg, yield 32.2%).

[0156] (4) Synthesis of Compound CX-ST03

[0157] Compound 12-V (684 mg, 1.09 mmol) was dissolved in N,N-dimethylformamide (25 mL). Then, compound 12-IV (500 mg, 2.56 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (600 mg, 3.13 mmol) and N,N-diisopropylethylamine (DIEA, 500 mg, 3.88 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was stirred at room temperature (25 °C) for 20 h, then the reaction solution was poured into 200 mL of saturated sodium bicarbonate solution and stirred at room temperature for 30 min. It was filtered, and the solid 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 a yellow solid of compound 12-VI (420 mg, yield 40.6%).

[0158] Compound 12-VI (420 mg, 0.44 mmol) was dissolved in a mixed solvent of dichloromethane (8 mL) and methanol (8 mL). Then, 4M hydrochloric acid dioxane (2 mL, 8 mmol) was added to the mixed solvent to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 h, then the reaction solution was evaporated to dryness under reduced pressure. The product after evaporation to dryness was triturated with tert-butyl methyl ether (20 mL × 2) to obtain a yellow solid of compound CX-ST03 (265 mg, yield 68.0%).

[0159] (5) Synthesis of Compound MC-6PEG-AANG-CX-ST03

[0160] Compound CX-ST03 (150 mg, 0.17 mmol) and Compound 1-X (170 mg, 0.21 mmol) were dissolved in N,N-dimethylformamide (25 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and the raw materials were detected by HPLC to have completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-AANG-CX-ST03 (46 mg, yield 16.5%).

[0161] Example 13

[0162] Synthesis of MC-6PEG-AANL-CX-ST03 (X13)

[0163] The synthetic route of the compound shown in X13 includes:

[0164]

[0165] The specific synthesis method includes the following steps:

[0166] Compound CX-ST03 (150 mg, 0.17 mmol) and Compound 2-I (180 mg, 0.21 mmol) were dissolved in N,N-dimethylformamide (25 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and the raw materials were detected by HPLC to have completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-AANL-CX-ST03 (55 mg, yield 19.0%).

[0167] Example 14

[0168] Synthesis of MC-6PEG-AAN-CX-ST03 (X14)

[0169] The synthetic route of the compound shown in X14 includes:

[0170]

[0171] The specific synthesis method includes the following steps:

[0172] Compound CX-ST03 (150 mg, 0.17 mmol) and compound 3-I (160 mg, 0.21 mmol) were dissolved in N,N-dimethylformamide (25 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and the raw materials were detected to have completely reacted by HPLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-AAN-CX-ST03 (41 mg, yield 15.2%).

[0173] Example 15

[0174] Synthesis of MC-6PEG-AAN-PAB-CX-ST03 (X15)

[0175] The synthesis route of the compound shown in X15 includes:

[0176]

[0177] The specific synthesis method includes the following steps:

[0178] Compound CX-ST03 (150 mg, 0.17 mmol) and compound 4-I (230 mg, 0.22 mmol) were dissolved in N,N-dimethylformamide (25 mL). Then, N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) was added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and the raw materials were detected to have completely reacted by HPLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-AAN-PAB-CX-ST03 (72 mg, yield 24.3%).

[0179] Example 16

[0180] Synthesis of MC-6PEG-AANG-CX-ST04 (X16)

[0181] The synthesis route of the compound shown in X16 includes:

[0182]

[0183]

[0184] The specific synthesis method includes the following steps:

[0185] (1) Synthesis of Compound 16-II

[0186] Compound 1-II (1.5 g, 6.9 mmol) and Compound 16-I (2.3 g, 7.5 mmol) were added to N,N-dimethylformamide (150 mL), and then potassium carbonate (2 g, 14.5 mmol) was added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was heated to 50 °C and reacted for 18 h, and the reaction was detected to be complete by HPLC. The reaction solution was poured into 800 mL of water, stirred at room temperature (25 °C) for 1 h, filtered, and the solid was dried and then purified by silica gel column chromatography (eluted with dichloromethane (DCM): methanol (MeOH) with a volume ratio of 100:1 to 15:1) to obtain Compound 16-II as a yellow solid (2.7 g, yield 88.5%).

[0187] (2) Synthesis of Compound 16-III

[0188] Compound 1-VII (1.1 g, 3.5 mmol) was added to n-butanol (15 mL), and then sodium bicarbonate (600 mg, 7.1 mmol) and N,N-diisopropylethylamine (DIEA, 1.8 g, 14.0 mmol) were added to the n-butanol to form a reaction solution. The reaction solution was stirred at room temperature (25 °C) for 10 min, then Compound 16-II (1.5 g, 3.4 mmol) was added and the temperature was raised to 120 °C and reacted for 18 h. After the reaction was completed, the reaction solution was cooled to room temperature 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 10:1) to obtain a brown solid (1.4 g). The above solid was dissolved in methanol (30 mL), and an aqueous solution of sodium dithionite (4.0 g, 23.0 mmol) in 15 mL and 6 mL of ammonia water were added under ice bath cooling and the temperature was raised to room temperature (25 °C) and reacted for 30 min. Then, extraction was carried out with ethyl acetate (100 mL * 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate 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 8:1) to obtain Compound 16-III as a light brown solid (755 mg, yield 34.5%).

[0189] (3) Synthesis of Compound CX-ST04

[0190] Compound 16-III (755 mg, 1.21 mmol) was dissolved in N,N-dimethylformamide (25 mL). Then, compound 12-IV (550 mg, 2.82 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (600 mg, 3.13 mmol), and N,N-diisopropylethylamine (DIEA, 500 mg, 3.88 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was stirred at room temperature (25 °C) for 20 h, and then poured into 200 mL of saturated sodium bicarbonate solution and stirred at room temperature for 30 min. After filtration, the solid 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 16-IV as a yellow solid (453 mg, yield 39.5%).

[0191] Compound 16-IV (453 mg, 0.48 mmol) was dissolved in a mixed solvent of dichloromethane (8 mL) and methanol (8 mL). Then, 4M hydrochloric acid dioxane (2 mL, 8 mmol) was added to the mixed solvent to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 h. The reaction solution was evaporated to dryness under reduced pressure, and the residue was triturated with tert-butyl methyl ether (20 mL * 2) to obtain compound CX-ST04 as a yellow solid (236 mg, yield 55.6%).

[0192] (4) Synthesis of compound MC-6PEG-AANG-CX-ST04

[0193] Compound CX-ST04 (150 mg, 0.17 mmol) and compound 1-X (170 mg, 0.21 mmol) were dissolved in N,N-dimethylformamide (25 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 h, and the raw materials were detected by HPLC to have completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained after evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparative chromatography to obtain MC-6PEG-AANG-CX-ST04 as an off-white solid (62 mg, yield 22.1%).

[0194] Example 17

[0195] Synthesis of MC-6PEG-AANL-CX-ST04 (X17)

[0196] The synthetic route of the compound shown in X17 includes:

[0197]

[0198] The specific synthesis method includes the following steps:

[0199] Compound CX-ST04 (150 mg, 0.17 mmol) and compound 2-I (180 mg, 0.21 mmol) were dissolved in N,N-dimethylformamide (25 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and the raw materials were detected to have completely reacted by HPLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-AANL-CX-ST04 (67 mg, yield 23.1%).

[0200] Example 18

[0201] Synthesis of MC-6PEG-AAN-CX-ST04 (X18)

[0202] The synthesis route of the compound shown in X18 includes:

[0203]

[0204] The specific synthesis method includes the following steps:

[0205] Compound CX-ST04 (150 mg, 0.17 mmol) and compound 3-I (160 mg, 0.21 mmol) were dissolved in N,N-dimethylformamide (25 mL). Then, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 120 mg, 0.32 mmol) and N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) were added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and the raw materials were detected to have completely reacted by HPLC. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-AAN-CX-ST04 (75 mg, yield 27.7%).

[0206] Example 19

[0207] Synthesis of MC-6PEG-AAN-PAB-CX-ST04 (X19)

[0208] The synthesis route of the compound shown in X19 includes:

[0209]

[0210] The specific synthesis method includes the following steps:

[0211] Compound CX-ST04 (150 mg, 0.17 mmol) and compound 4-I (230 mg, 0.22 mmol) were dissolved in N,N-dimethylformamide (25 mL), and then N,N-diisopropylethylamine (DIEA, 120 mg, 0.93 mmol) was added to the N,N-dimethylformamide to form a reaction solution. The reaction solution was reacted at room temperature (25 °C) for 5 hours, and HPLC was used to detect that the raw materials had completely reacted. The reaction solution was evaporated to dryness under reduced pressure. The product obtained by evaporation to dryness under reduced pressure was purified by high-pressure reverse-phase preparation to obtain a white solid of MC-6PEG-AAN-PAB-CX-ST04 (48 mg, yield 16.2%).

[0212] 1. Experimental study on the activation of legumain enzyme by the polypeptide conjugate of the Sting small molecule agonist described in Examples 1-19 of the present application

[0213] Buffer preparation: 50 mM MES aqueous solution (2-morpholinoethanesulfonic acid, water is distilled water), 250 mM sodium chloride aqueous solution (water is distilled water), and the pH was adjusted to 5.0 with 0.5 M sodium hydroxide. The concentration of legumain was selected as 1 mg / mL. The polypeptide conjugates described in Examples 1-19 were respectively prepared into a 0.5 μmol / mL solution with the buffer. Accurately pipette 50 μL of the above 0.5 μmol / mL solution and 50 μL of the buffer into centrifuge tubes, and add 100 μL of legumain to each tube, and react at 37 °C for 2 h. The reaction solution was detected by LCMS, and the detection results are shown in Table 1.

[0214] Table 1

[0215] Compound Name Digestion Product MC-6PEG-AANG-CX-ST01(X1) G-CX-ST01 MC-6PEG-AANL-CX-ST01(X2) L-CX-ST01 MC-6PEG-AAN-CX-ST01(X3) CX-ST01 MC-6PEG-AAN-PAB-CX-ST01(X4) CX-ST01 MC-6PEG-GANG-CX-ST01(X5) G-CX-ST01 MC-6PEG-SANG-CX-ST01(X6) G-CX-ST01 MSPP-6PEG-AANG-CX-ST01(X7) G-CX-ST01 BA-6PEG-AANG-CX-ST01(X8) G-CX-ST01 MC-6PEG-AANG-AM-CX-ST02(X9) G-AM-CX-ST02 MC-6PEG-AANL-AM-CX-ST02(X10) L-AM-CX-ST02 MC-6PEG-AAN-AM-CX-ST02(X11) CX-ST02 MC-6PEG-AANG-CX-ST03(X12) G-CX-ST03 MC-6PEG-AANL-CX-ST03(X13) L-CX-ST03 MC-6PEG-AAN-CX-ST03(X14) CX-ST03 MC-6PEG-AAN-PAB-CX-ST03(X15) CX-ST03 MC-6PEG-AANG-CX-ST04(X16) G-CX-ST04 MC-6PEG-AANL-CX-ST04(X17) L-CX-ST04 MC-6PEG-AAN-CX-ST04(X18) CX-ST04 MC-6PEG-AAN-PAB-CX-ST04(X19) CX-ST04

[0216] It can be seen from Table 1 that the polypeptide conjugate of the Sting small molecule agonist described in the present application can be activated by the legumain enzyme highly expressed in tumors.

[0217] 2. Tumor homogenate activation experiment of the polypeptide conjugate of the Sting small molecule agonist described in the present application

[0218] The PBS buffer was purchased from Beijing Lanjieke Technology Co., Ltd., Cat. NO: BL302A, Lot. No: 24149299.

[0219] CT26 tumor tissue homogenate: CT26 tumor tissue taken from C57BL / 6 mice with CT26 tumorigenesis model, and the homogenate was prepared with a Jingxin F6 / 10 handheld homogenizer.

[0220] The polypeptide conjugates described in Examples 1-19 of the present application were respectively formulated into a 0.5 mg / ml solution with PBS buffer. 0.5 mL of the above solution was taken and 300 micrograms of CT26 tumor tissue homogenate was added respectively. The above solution was placed at 37 °C for 2 h. The generated compounds were detected by liquid chromatography-mass spectrometry (LC-MS), and the results are shown in Table 2.

[0221] Table 2

[0222]

[0223]

[0224] It can be seen from Table 2 that the polypeptide conjugate of the Sting small molecule agonist described in the present application can be activated by tumor homogenate and release the Sting small molecule agonist.

[0225] 3. Tumor homogenate activation efficiency experiment of the polypeptide conjugate of the Sting small molecule agonist described in the present application

[0226] The PBS buffer was purchased from Beijing Lanjieke Technology Co., Ltd., Cat. NO: BL302A, Lot. No: 24149299;

[0227] Tumor tissue homogenate: CT26 was the CT26 tumor tissue taken from C57BL / 6 mice in the CT26 tumorigenesis model and prepared into homogenate with a Jingxin F6 / 10 handheld homogenizer;

[0228] A549 and HT-1080 were the tumor tissues taken from Balb / c nude mice in the corresponding tumorigenesis models and prepared into homogenate with a Jingxin F6 / 10 handheld homogenizer.

[0229] The polypeptide conjugates described in the examples of the present application were respectively formulated into a 0.5 mg / ml solution with PBS buffer. 0.5 mL of the above solution (three portions were taken for each solution) was added to 300 micrograms of tumor tissue homogenate (the three kinds of tumor tissue homogenates were respectively added to three portions of the solution of the same polypeptide conjugate), and the above solution was placed at 37 °C for 2 h. The activation efficiency of the drug in tumor tissue was compared by detecting the reduction of the compound and the increase of toxic molecules by high performance liquid chromatography (HPLC):

[0230] Release percentage = molar amount calculated from the peak area of toxic molecules (see Table 1) / molar amount before activation of the polypeptide drug conjugate; the results are shown in Table 3:

[0231] Table 3

[0232]

[0233]

[0234] As can be seen from Table 3, the polypeptide conjugate of the Sting small molecule agonist described in the present application can release toxin molecules in different tumor tissue homogenates.

[0235] 4. Stability of the polypeptide conjugate of the Sting small molecule agonist described in the present application in mouse normal tissue homogenates

[0236] PBS buffer: Beijing LanJieKe Technology Co., Ltd., Cat. NO: BL302A, Lot. No: 24149299.

[0237] Mouse normal tissue homogenate: Organs were taken from 8-week-old C57BL / 6 mice after sacrifice and prepared into tissue homogenates using a Jingxin F6 / 10 handheld homogenizer.

[0238] The polypeptide conjugates described in the examples of the present application were respectively prepared into 0.5 mg / ml solutions with PBS buffer. 0.5 mL of each of the above solutions was taken and added to 300 micrograms of normal tissue homogenate, and the above solutions were placed at 37 °C for 2 h. The release percentage of the drug in normal tissues was compared by detecting the reduction of the compound and the increase of the toxic molecule by high performance liquid chromatography (HPLC):

[0239] Release percentage = molar amount calculated from the peak area of the toxic molecule (see Table 1) / molar amount before activation of the polypeptide drug conjugate. The results are shown in Table 4, with the unit of %.

[0240] Table 4

[0241] Compound Heart Liver Kidney Lung Spleen Muscle Plasma MC-6PEG-AANG-CX-ST01(X1) 0.6 5.8 2.6 3.0 1.0 <0.1 <0.1 MC-6PEG-AANL-CX-ST01(X2) 1.1 12.9 5.9 4.6 1.8 <0.1 <0.1 MC-6PEG-AAN-CX-ST01(X3) 1.9 16.6 6.6 7.2 2.2 <0.1 0.2 MC-6PEG-AAN-PAB-CX-ST01(X4) 3.6 22.7 14.5 9.9 6.4 <0.1 2.3 MC-6PEG-GANG-CX-ST01(X5) 2.0 10.6 8.2 9.3 3.1 <0.1 1.2 MC-6PEG-AANG-AM-CX-ST02(X9) 0.2 3.1 2.4 2.6 1.2 <0.1 <0.1 MC-6PEG-AANL-AM-CX-ST02(X10) 0.8 9.5 4.8 7.2 2.0 <0.1 0.9 MC-6PEG-AAN-AM-CX-ST02(X11) 2.2 10.8 4.4 6.5 0.8 <0.1 <0.1 MC-6PEG-AANG-CX-ST03(X12) <0.1 1.8 1.9 1.3 0.4 <0.1 <0.1 MC-6PEG-AANL-CX-ST03(X13) 0.9 4.5 3.6 3.0 0.9 <0.1 1.1 MC-6PEG-AAN-CX-ST03(X14) 1.2 3.0 4.0 2.4 1.1 <0.1 <0.1 MC-6PEG-AANG-CX-ST04(X16) <0.1 2.2 1.7 1.8 0.7 <0.1 <0.1 MC-6PEG-AANL-CX-ST04(X17) 1.5 4.1 4.8 3.3 1.2 <0.1 0.6 MC-6PEG-AAN-CX-ST04(X18) 1.1 2.7 4.0 3.5 0.9 <0.1 <0.1

[0242] As can be seen from Table 4, the polypeptide conjugate of the Sting small molecule agonist described in the present application is partially released in the liver, kidney and lung, very little released in the heart and spleen, and hardly released in the muscle and plasma.

[0243] 5. Maximum tolerated dose (MTD) of the polypeptide conjugate of the Sting small molecule agonist described in the present application in mice

[0244] Test animals: C57BL / 6 mice, 6 - 8 weeks old, all female. The mice were randomly grouped, with six mice in each group. The mice were administered drugs at different concentration gradients (the gradient dosing method is as follows: for CX - ST01, CX - ST02, CX - ST03, and CX - ST04, the initial dose is 10 mg / kg. If there is no toxicity at all, the dose is increased by 5 mg / kg. If it is close to the MTD, the dose is increased or decreased by 5 mg / kg. If the dose is basically within the MTD range, the dose is further increased or decreased by 2 mg / kg for verification. The polypeptide conjugate described in this application uses 100 mg / kg as the starting dose and then increases by 20 mg / kg. If the dose is basically within the MTD range, the dose is further increased or decreased by 20 mg / kg for verification), and monitored for 14 days. The mice were euthanized when they lost 20% of their initial body weight and were considered to have died from poisoning. The maximum tolerated dose (MTD) is defined as the highest dose level at which none of the six mice died due to the drug, and the weight loss of an individual mouse does not exceed 20%, or the average weight loss within the group does not exceed 15%. The experimental results are shown in Table 5.

[0245] Table 5

[0246] Compound Name Maximum Tolerated Dose (MTD) CX-ST01 10mg / kg CX-ST02 10mg / kg CX-ST03 15mg / kg CX-ST04 15mg / kg MC-6PEG-AANG-CX-ST01(X1) 150mg / kg MC-6PEG-AANL-CX-ST01(X2) 80mg / kg MC-6PEG-AAN-CX-ST01(X3) 100mg / kg MC-6PEG-AAN-PAB-CX-ST01(X4) 80mg / kg MC-6PEG-GANG-CX-ST01(X5) 100mg / kg MC-6PEG-SANG-CX-ST01(X6) 120mg / kg MSPP-6PEG-AANG-CX-ST01(X7) 120mg / kg BA-6PEG-AANG-CX-ST01(X8) 120mg / kg MC-6PEG-AANG-AM-CX-ST02(X9) 160mg / kg MC-6PEG-AANL-AM-CX-ST02(X10) 100mg / kg MC-6PEG-AAN-AM-CX-ST02(X11) 120mg / kg MC-6PEG-AANG-CX-ST03(X12) 160mg / kg MC-6PEG-AANL-CX-ST03(X13) 100mg / kg MC-6PEG-AAN-CX-ST03(X14) 100mg / kg MC-6PEG-AAN-PAB-CX-ST03(X15) 100mg / kg MC-6PEG-AANG-CX-ST04(X16) 160mg / kg MC-6PEG-AANL-CX-ST04(X17) 100mg / kg MC-6PEG-AAN-CX-ST04(X18) 120mg / kg MC-6PEG-AAN-PAB-CX-ST04(X19) 100mg / kg

[0247] Note: The structural formula of CX - ST01 is The structural formula of CX - ST02 is: The structural formula of CX - ST03 is: The structural formula of CX - ST04 is:

[0248] It can be seen from Table 5 that for the polypeptide conjugate of the Sting small molecule agonist described in this application, compared with the separate Sting small molecule agonist, the MTD has been significantly improved, indicating that the effect of reducing toxicity is very obvious.

[0249] 6. Pharmacodynamic study of the polypeptide conjugate of the Sting small molecule agonist described in this application in the treatment of CT26 murine colon cancer animal model

[0250] Test objective: To study the anti - tumor pharmacodynamic differences of the polypeptide conjugates of the Sting small molecule agonists shown as X1, X2, X9, X10, X12, and X16 in this application compared with the separate Sting small molecule agonist and the existing chemotherapy drug cisplatin in the CT26 murine colon cancer animal tumor model.

[0251] Test drugs: The polypeptide conjugates of the Sting small molecule agonists shown as X1, X2, X9, X10, X12, and X16, cisplatin, CX - ST01, CX - ST02, CX - ST03, CX - ST04, and the normal saline control group.

[0252] Experimental animals: 6-8-week-old BALB / c mice, all female mice.

[0253] CT26 cells were purchased from ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 °C and 5% CO2. Passage was performed every three days, and cells within 15 passages were used. 5×10 6 corresponding cells were subcutaneously injected into the back of nude mice. When the tumor reached at least 100 - 150 mm 3 (average about 120 mm 3 ), the mice were randomly divided into groups of 5. Then the treatment started, and the day when the treatment started was the first day. The drug was administered once a week for three weeks, and the experimental results are shown in Table 6.

[0254] Table 6

[0255]

[0256] It can be seen from Table 6 that the polypeptide conjugates of the Sting small molecule agonists shown by X1 and X9 in this application have better drug efficacy than the Sting small molecule agonists alone. The polypeptide conjugates of the Sting small molecule agonists shown by X2, X10, X12, and X16 have equivalent drug efficacy to the Sting small molecule agonists, and are much better than the traditional chemotherapeutic drug cisplatin.

[0257] Although the above embodiments have been shown and described, it can 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 protection scope of the present invention.

Claims

1. A polypeptide conjugate of a Sting small molecule agonist, characterized in that: The structural formula of the polypeptide conjugate is shown as follows: EP-L1-L2-L3-L4-L5-D; Wherein, E includes the following groups: The "1" position is connected to P or L1, 1≤n≤18, and X includes halogen; P is present or absent, and when P is present, P comprises polyethylene glycol; L1 and L2 each independently include one or more of glycine, alanine and serine; L3 includes asparagine; L4 is present or absent, and when L4 is present, L4 comprises glycine and / or leucine; L5 may or may not exist. When L5 exists, L5 includes the following structure: The "4" position is connected to the carboxyl end of L3 or L4, the "5" position is connected to D, 0≤n2≤40, and n2 is an integer, and R1 includes hydrogen and / or methyl; D includes Sting small molecule agonists.

2. The polypeptide conjugate of the Sting small molecule agonist according to claim 1, characterized in that: The structural formula of the polyethylene glycol is shown below: Among them, the "2" position is connected to E, the "3" position is connected to the amino group of L1, 1≤n1≤40, and n1 is an integer.

3. The polypeptide conjugate of the Sting small molecule agonist according to claim 1, characterized in that: The structural formula of D is shown below: Wherein, the "6" position is connected to the carboxyl end of L5, L4 or the carboxyl end of L3, 0≤n3≤20, and n3 is an integer.

4. The polypeptide conjugate of the Sting small molecule agonist according to claim 1, characterized in that: The structural formula of the polypeptide conjugate is shown in Formula X1-X19:

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide conjugate according to any one of claims 1 to 4 or a pharmaceutically acceptable carrier thereof.

6. Use of the polypeptide conjugate according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a drug for treating or preventing anti-tumor.

7. The use according to claim 6, characterized in that: The tumors include one or more of gastrointestinal cancer, colorectal cancer, colon cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, gastric cancer, genitourinary system cancer, bladder cancer, testicular cancer, cervical cancer, malignant mesothelioma, 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.

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