Maleimide derivative with rapid activation capability as well as lipidosome and application thereof

Abstract

The invention provides a maleimide derivative with rapid activation capability and a liposome and application thereof. The derivative provided by the invention has a rapid activation connecting bond, and is adaptive to drugs containing hydroxyl or amino groups, such as R848 (R848), camptothecin (CPT), docetaxel (DTX), paclitaxel (PTX) and the like. The active drug-loading liposome is composed of the derivative, phospholipid, cholesterol, PEGylated phospholipid and a water-soluble sulfydryl substance. The derivative disclosed by the invention is high in activation rate in plasma, in-vitro experiments prove that the corresponding liposome can effectively kill cancer cells, has an excellent in-vitro anti-tumor effect, and can be used for preparing medicines for preventing or treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulations, specifically relating to a prodrug with a rapidly activated linker bond, its liposomes, and its applications. Background Technology

[0002] Liposomes are vesicle-like nanocarriers formed by the self-assembly of lipid materials such as phospholipids and cholesterol. Their core function is to improve drug distribution in the body, reduce the toxicity of chemotherapy drugs to normal tissues, and achieve sustained drug release at the lesion site to enhance therapeutic efficacy. They also address the challenge of poor water solubility for some hydrophobic drugs. Currently, several liposomal drugs have been approved for marketing by the U.S. Food and Drug Administration (FDA), including liposomal formulations of doxorubicin, irinotecan, doxorubicin, vincristine, and paclitaxel for anti-tumor treatment, as well as compound liposomal formulations composed of cytarabine and daunorubicin. In the field of chemotherapy drug delivery, liposomes are one of the most successful examples of clinical translation. Summary of the Invention

[0003] Many drugs cannot be stably encapsulated in liposomes due to their inherent physicochemical properties. Typically, hydrophobic drugs, once encapsulated within the liposome membrane, easily leak from the phospholipid membrane during blood circulation, making sustained release difficult. Research has found that by first encapsulating glutathione (GSH) in the aqueous phase of a liposome, and then designing the drug as a prodrug linked to maleimide (MAL) via an ester or carbonate bond, active drug loading is achieved through the specific reaction between MAL and the thiol group. This method is more robust than ion binding and can significantly reduce drug leakage. After release from the liposome, the prodrug needs rapid activation to exert its pharmacological activity; a slow activation rate may lead to treatment failure. However, during development, it was found that most prodrugs have low activation rates in vivo. Therefore, there is an urgent need to develop a prodrug linker that can achieve rapid prodrug activation.

[0004] Prodrug modification primarily involves covalent bonding with the parent drug molecule. Common functional groups in the parent drug molecule include hydroxyl, amino, and carboxyl groups. These functional groups, when combined with carrier groups, can form structures such as ester bonds, carbonate bonds, amide bonds, and carbamate bonds. Ester prodrugs are the most common, and their ester bonds can be hydrolyzed by esterases widely present in blood, liver, and other organs or tissues, thereby releasing the active drug from the prodrug molecule. Some prodrugs exhibit strong enzymatic stability in vivo, which can hinder the rapid release of the active drug.

[0005] Based on this, the present invention provides a prodrug with a linker bond having rapid activation capability, its liposome formulation, and its application.

[0006] In a first aspect, the present invention provides a linker bond and its derivatives having rapid activation capability, with the following general formula: .

[0007] In the above general formula of the present invention, the drug is a drug containing hydroxyl or amino groups, preferably rethimod (R848), camptothecin (CPT), docetaxel (DTX) and paclitaxel (PTX).

[0008] In this invention, n is 1 or 2, and m is selected from integers from 0 to 5.

[0009] In this invention, the inventors intentionally named the connecting bond in the left diagram of the general formula as 2B; and the connecting bond in the right diagram when n=1 as 2BP. Taking camptothecin as an example, the camptothecin (CPT)-2B-MAL derivative and the CPT-2BP-MAL derivative have the following structures: Furthermore, the derivatives of R848-2B-MAL, DTX-2B-MAL, DTX-2BP-MAL, and PTX-2BP-MAL are: According to a second aspect of the present invention, a drug-rapidly activated linker-maleimide (MAL) derivative active drug-carrying liposome is provided.

[0010] The active drug-loaded liposome of the present invention comprises the above-mentioned drug-rapidly activated linker-maleimide (MAL) derivative, phospholipids, cholesterol, PEGylated phospholipids, and water-soluble thiol substances.

[0011] The phospholipids are selected from natural, semi-synthetic or fully synthetic phospholipids, specifically including egg yolk lecithin (EPC), soybean phospholipids, sphingomyelin, hydrogenated soybean phospholipids (HSPC), distearate acylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), etc., with hydrogenated soybean phospholipids (HSPC) being preferred.

[0012] The PEGylated phospholipid is a coupling formed by polyethylene glycol (PEG) and distearate phosphatidylethanolamine (DSPE) linked by an amide bond, preferably DSPE-PEG. 2000 .

[0013] The water-soluble thiol substance is a water-soluble compound containing a thiol group, and specific examples include, but are not limited to, dipeptides, tripeptides and glutathione (GSH) containing cysteine, with glutathione (GSH) being preferred.

[0014] Based on a third aspect of the present invention, the above-mentioned drug-rapidly activated linker-MAL derivative or the above-mentioned active drug-loaded liposome can be used to prepare drugs for the prevention or treatment of tumors. Beneficial effects

[0015] The drug-rapidly activated linker-maleimide (MAL) derivative provided by this invention exhibits an extremely high activation rate in plasma, enabling stable drug loading in actively loaded liposomes. The drug-loaded liposomes of this invention achieve active drug loading by reacting the drug-rapidly activated linker-MAL derivative with thiol substances to generate a hydrophilic prodrug, thus possessing the core advantages of high drug loading stability and rapid activation rate. In vitro toxicity experiments confirm that the actively loaded liposomes of this invention can effectively kill cancer cells and exhibit better in vitro antitumor effects. Attached Figure Description

[0016] Figure 1 The appearance changes and HPLC analysis of R848-2B-LIP (top figure) in Example 10 and R848-2C-LIP (bottom figure) in Example 11 during drug loading process are shown. Figure 2 This is a comparison of drug loading rates during the drug loading process of R848-2B-MAL and R848-2C-MAL liposomes; Figure 3 The appearance changes and HPLC analysis of CPT-2BP-LIP (top figure) in Example 12 and CPT-2C-LIP (bottom figure) in Example 13 during drug loading process; Figure 4 This is a graph showing the drug activation rates of free R848-2B-GSH and free R848-2C-GSH in plasma in Example 14; Figure 5 This is a graph showing the drug activation rates of free CPT-2BP-GSH, free CPT-2B-GSH, and free CPT-2C-GSH in plasma in Example 15; Figure 6 This is a graph showing the drug activation rates of free DTX-2BP-GSH, free DTX-2B-GSH, and free DTX-2C-GSH in plasma in Example 16; Figure 7 This refers to the in vitro cytotoxicity experiments of CPT-2BP-GSH free drug and liposomes, CPT-2C-GSH free drug and liposomes, and free CPT in Example 17. Detailed Implementation

[0017] In the following embodiments, the invention has only been described illustratively, but those skilled in the art can make various modifications to the invention without departing from the spirit and scope of the invention after reading this patent application.

[0018] Example 1: Synthesis of R848-2B-MAL 600 mg of 3-maleimide propionic acid was dissolved in 20 mL of dichloromethane. 1.0 eq of dicyclohexylcarbodiimide was added, and the mixture was reacted at room temperature for 5 minutes. Then, 2 eq of p-hydroxybenzyl alcohol and a catalytic amount of DMAP (approximately 0.05 eq) were added, and the reaction was continued for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na₂SO₄ was added to remove water. The mixture was filtered, and the solution was evaporated to dryness. 10 mL of ethyl acetate was added, and the mixture was cooled at -20 °C for 10 minutes. DCU was removed by filtration. The filtrate was passed through a silica gel plate and eluted with a dichloromethane:methanol (20:1 v / v) mixture to give intermediate 1. Intermediate 1 was dissolved in dry dichloromethane. Under nitrogen protection, 0.35 eq of triphosgene and 0.7 eq of DMAP were added, and the mixture was reacted for 10 minutes. The mixture was then transferred to another volumetric flask under nitrogen protection containing 1.0 eq of R848, and the reaction was continued for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na₂SO₄ was added to remove water. After filtration, the filtrate was passed through a silica gel plate and eluted with a mixed solvent of dichloromethane and methanol (volume ratio 10:1) to obtain the product R848-2B-MAL. The product was identified by nuclear magnetic resonance spectroscopy. 1 H NMR (400 MHz, CHLOROFORM- D ) δ 7.49 (dd, J = 7.6,5.4 Hz, 1H), 7.39 (s, 1H), 7.37 (d, J = 1.8 Hz, 1H), 7.10 – 7.06 (m, 2H), 6.72 (d, J = 1.1 Hz, 2H), 4.68 (s, 2H), 3.96 (t, J = 7.0 Hz, 2H), 2.89 (td, J = 7.0, 1.1 Hz, 2H), 1.29 – 1.21 (m, 3H), confirming successful synthesis.

[0019]

[0020] Example 2: Synthesis of R848-2C-MAL 1 g of 3-maleimide propionic acid was dissolved in 20 mL of dichloromethane. 1.0 eq of dicyclohexylcarbodiimide was added, and the mixture was reacted at room temperature for 5 minutes. Then, excess ethylene glycol and a catalytic amount of DMAP (approximately 0.05 eq) were added, and the reaction was carried out in an ice bath for 2 hours. After the reaction was complete, the reaction mixture was evaporated to dryness, 20 mL of ethyl acetate was added, and the mixture was filtered. The product was separated by silica gel column chromatography (ethyl acetate:petroleum ether, v / v ratio 1:1) to obtain intermediate 2. Intermediate 2 was dissolved in dry dichloromethane, and under nitrogen protection, 0.35 eq of triphosgene and 0.7 eq of DMAP were added, followed by 1.0 eq of R848. The reaction was carried out for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na₂SO₄ was added to remove water. After filtration, the filtrate was passed through a silica gel plate and eluted with a mixed solvent of dichloromethane and methanol (volume ratio 20:1) to obtain the product R848-2C-MAL. The product was identified by nuclear magnetic resonance spectroscopy. 1 H NMR (400 MHz, CHLOROFORM- D ) δ 8.15 (d, J = 8.3 Hz, 2H), 7.61 (t, J = 7.8 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 6.72 (s, 2H), 4.93(s, 2H), 4.80 (s, 2H), 4.57 – 4.28 (m, 4H), 3.87 (td, J = 6.7, 3.2 Hz, 2H), 3.67 (q, J = 7.1 Hz, 2H), 2.74 – 2.66 (m, 2H), 1.26 (t, J = 7.0 Hz, 4H), confirming successful synthesis.

[0021] Example 3: Synthesis of CPT-2BP-MAL 400 mg of 3-maleimide propionic acid was dissolved in 20 mL of dichloromethane. 1.0 eq of dicyclohexylcarbodiimide was added, and the mixture was reacted at room temperature for 5 minutes. Then, 1 eq of tert-butyl 2-(2-hydroxyethoxy)acetate and a catalytic amount of DMAP (approximately 0.05 eq) were added, and the reaction was continued for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na₂SO₄ was added to remove water. The mixture was filtered, and the solution was evaporated to dryness. 10 mL of ethyl acetate was added, and the mixture was cooled at -20 °C for 10 minutes. DCU was removed by filtration. The filtrate was passed through a silica gel plate and eluted with a dichloromethane:methanol (20:1 v / v) mixture to give intermediate 3. Intermediate product 3 was added to 4 mL of dichloromethane, followed by 4 mL of trifluoroacetic acid. The mixture was stirred for 1 h, washed with pure water to remove most of the unreacted trifluoroacetic acid, and then eluted with a dichloromethane:methanol (20:1 v / v) mixture to obtain intermediate product 4. 280 mg of intermediate product 4 was dissolved in 20 mL of dichloromethane, and 1.0 eq of dicyclohexylcarbodiimide was added. After reacting at room temperature for 5 min, 2 eq of p-hydroxybenzyl alcohol and a catalytic amount of DMAP (approximately 0.05 eq) were added, and the reaction was continued for 2 h. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, dehydrated with anhydrous Na₂SO₄, filtered, and evaporated to dryness. 10 mL of ethyl acetate was added, and the mixture was cooled at -20 °C for 10 min. DCU was removed by filtration. The filtrate was passed through a silica gel plate and eluted with a dichloromethane:methanol (20:1 v / v) mixture to obtain intermediate product 5. Intermediate product 5 was dissolved in dry dichloromethane. Under nitrogen protection, 0.35 eq triphosgene and 0.7 eq DMAP were added, and the reaction was allowed to proceed for 10 minutes. The mixture was then transferred to another volumetric flask containing 1.0 eq CPT under nitrogen protection, and the reaction was allowed to proceed for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na₂SO₄ was added to remove water. The solution was filtered, and the filtrate was passed through a silica gel plate and eluted with a dichloromethane:methanol (volume ratio 10:1) mixture to obtain the product CPT-2BP-MAL. The product was identified by nuclear magnetic resonance spectroscopy. 1 H NMR (400 MHz, CHLOROFORM- D ) δ 8.41 (d, J = 1.1 Hz, 1H),8.25 – 8.21 (m, 1H), 7.96 (dd, J = 8.2, 1.4 Hz, 1H), 7.86 (ddd, J = 8.5, 6.9,1.5 Hz, 1H), 7.69 (ddd, J= 8.1, 6.9, 1.2 Hz, 1H), 7.40 – 7.36 (m, 2H), 7.30(s, 1H), 7.08 – 7.03 (m, 2H), 6.69 (s, 2H), 5.70 (d, J = 17.2 Hz, 1H), 5.39(d, J = 17.1 Hz, 1H), 5.17 – 5.08 (m, 2H), 4.30 – 4.27 (m, 2H), 4.25 (d, J =0.5 Hz, 2H), 3.84 (t, J = 7.0 Hz, 2H), 3.81 – 3.78 (m, 2H), 2.68 (t, J = 7.0Hz, 2H), 2.31 – 2.24 (m, 1H), 2.15 (dd, J = 14.0, 7.5 Hz, 1H), 1.00 (t, J =7.5 Hz, 3H). and mass spectrometry identification MS (ES+): calcd for C 39 H 33 N3O 13 (M+H) + : 752.2013, found:752.2089, confirming successful synthesis.

[0022] Example 4: Synthesis of CPT-2B-MAL The synthesis method of intermediate product 1 is as described in Example 1. 100 mg of intermediate product 1 was weighed and dissolved in dry dichloromethane. Under nitrogen protection, 0.35 eq triphosgene and 0.7 eq DMAP were added, and the reaction was carried out for 10 minutes. The mixture was then added to another volumetric flask containing 1.0 eq CPT under nitrogen protection, and the reaction was carried out for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na₂SO₄ was added to remove water. The mixture was filtered, and the filtrate was passed through a silica gel plate and eluted with a mixed solvent of dichloromethane:methanol (volume ratio 20:1) to obtain the product CPT-2B-MAL. The product was identified by nuclear magnetic resonance spectroscopy. 1 H NMR (400 MHz, CHLOROFORM- D ) δ 8.40 (d, J= 1.0 Hz,1H), 8.25 – 8.21 (m, 1H), 7.97 – 7.93 (m, 1H), 7.85 (ddd, J = 8.5, 6.9, 1.5Hz, 1H), 7.68 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.37 – 7.33 (m, 2H), 7.28 (s,1H), 6.71 (s, 2H), 5.29 – 5.28 (m, 2H), 2.73 (td, J = 7.0, 4.7 Hz, 2H), 2.27(dd, J = 14.1, 7.5 Hz, 1H), 2.14 (dd, J = 14.0, 7.5 Hz, 1H), 0.99 (t, J = 7.5Hz, 3H). and mass spectrometry (ES+): calcd for C 35 H 27 N3O 10 (M+H) + : 650.1696, found: 650.1778 Confirmed successful synthesis.

[0023]

[0024] Example 5: Synthesis of CPT-2C-MAL The synthesis method of intermediate product 2 is as described in Example 2. 100 mg of intermediate product 2 was weighed and dissolved in dry dichloromethane. Under nitrogen protection, 0.35 eq triphosgene and 0.7 eq DMAP were added, followed by 1.0 eq CPT. The reaction was allowed to proceed for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na₂SO₄ was added to remove water. The solution was filtered, and the filtrate was passed through a silica gel plate and eluted with a mixed solvent of dichloromethane and methanol (volume ratio 20:1) to obtain product CPT-2C-MAL. The product was identified by nuclear magnetic resonance spectroscopy. 1 H NMR (400 MHz, CHLOROFORM- D ) δ 8.40 (d, J = 1.1 Hz, 1H), 8.21 – 8.17 (m, 1H), 7.93 (dd, J = 8.2, 1.4Hz, 1H), 7.83 (ddd, J= 8.5, 6.9, 1.5 Hz, 1H), 7.67 (ddd, J = 8.1, 6.9, 1.2Hz, 1H), 7.33 (s, 1H), 6.64 (s, 2H), 5.70 (d, J = 17.1 Hz, 1H), 5.38 (d, J =17.2 Hz, 1H), 5.30 (d, J = 1.2 Hz, 2H), 3.76 (td, J = 6.9, 4.0 Hz, 2H), 2.62(t, J = 6.8 Hz, 2H), 2.28 (dd, J = 14.0, 7.5 Hz, 1H), 2.20 – 2.10 (m, 1H), 0.99 (t, J = 7.5 Hz, 3H). and mass spectrometry MS (ES+): calcd for C 30 H 25 N3O 10 (M+H) + :588.1540, found: 588.1616 Identification confirms successful synthesis.

[0025] Example 6: Synthesis of DTX-2BP-MAL The synthesis of intermediate product 5 was as described in Example 3. 100 mg of intermediate product 5 was weighed and dissolved in dry dichloromethane. Under nitrogen protection, 0.35 eq triphosgene and 0.7 eq DMAP were added, and the reaction was carried out for 10 minutes. The mixture was then added to another volumetric flask containing 1.0 eq DTX under nitrogen protection, and the reaction was carried out for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na₂SO₄ was added to remove water. The mixture was filtered, and the filtrate was passed through a silica gel plate and eluted with a dichloromethane:methanol (volume ratio 20:1) mixture to obtain the product DTX-2BP-MAL. The product was identified by mass spectrometry (MS (ES+): calcd for C). 62 H 70 N2O 23 (M+Na) + : 1233.4369, found: 1233.4260, confirming successful synthesis.

[0026] Example 7: Synthesis of DTX-2B-MAL The synthesis method of intermediate product 1 is as described in Example 1. 100 mg of intermediate product 1 was weighed and dissolved in dry dichloromethane. Under nitrogen protection, 0.35 eq triphosgene and 0.7 eq DMAP were added, and the reaction was carried out for 10 minutes. The mixture was then added to another volumetric flask containing 1.0 eq DTX under nitrogen protection, and the reaction was carried out for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na2SO4 was added to remove water. The mixture was filtered, and the filtrate was passed through a silica gel plate and eluted with a mixed solvent of dichloromethane:methanol (volume ratio 20:1) to obtain product DTX-2B-MAL. The product was identified by mass spectrometry (MS (ES+): calcd for C). 58 H 64 N2O 20 (M+H) + : 1109.4052, found: 1109.4120, confirmed successful synthesis.

[0027] Example 8: Synthesis of DTX-2C-MAL The synthesis method of intermediate product 2 is as described in Example 2. 100 mg of intermediate product 2 was weighed and dissolved in dry dichloromethane. Under nitrogen protection, 0.35 eq triphosgene and 0.7 eq DMAP were added, followed by 1.0 eq DTX. The reaction was carried out for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na2SO4 was added to remove water. The solution was filtered, and the filtrate was passed through a silica gel plate and eluted with a mixed solvent of dichloromethane and methanol (volume ratio 20:1) to obtain product DTX-2C-MAL. The product was identified by mass spectrometry (MS (ES+): calcd for C). 53 H 62 N2O 20 (M+Na) + :1069.3896,found: 1069.3796, confirming successful synthesis.

[0028] Example 9: Synthesis of PTX-2BP-MAL The synthesis of intermediate product 5 was as described in Example 3. 100 mg of intermediate product 5 was weighed and dissolved in dry dichloromethane. Under nitrogen protection, 0.35 eq triphosgene and 0.7 eq DMAP were added, and the reaction was allowed to proceed for 10 minutes. The mixture was then added to another volumetric flask containing 1.0 eq PTX under nitrogen protection, and the reaction was allowed to proceed for 2 hours. After the reaction was complete, the reaction solution was washed once with citric acid and twice with saturated NaCl solution. The organic phase was collected, and anhydrous Na₂SO₄ was added to remove water. The mixture was filtered, and the filtrate was passed through a silica gel plate and eluted with a mixed solvent of dichloromethane and methanol (volume ratio 20:1) to obtain the product PTX-2BP-MAL. The product was identified by mass spectrometry (MS (ES+): calcd for C). 66 H 68 N2O 23 (M+Na) + :1279.4213, found: 1279.4102, confirming successful synthesis.

[0029] Example 10: Preparation of R848-2B-MAL drug-loaded liposomes Preparation of blank liposomes: Weigh 42 mg HSPC, 20 mg Chol, and 13 mg DSPE-PEG. 2000 Dissolve 600 mg of glutathione (GSH) in 1 mL of ethanol. Dissolve 600 mg of glutathione (GSH) in 5 mL of pure water to prepare a 400 mM glutathione (GSH) solution. Under vigorous stirring, add the above lipid ethanol solution dropwise to the glutathione (GSH) aqueous solution to obtain a crude liposome solution. Extrude this liposome solution through 0.4 μm and 0.1 μm polycarbonate core-porous membranes at 60 °C. Transfer the obtained liposomes to a dialysis bag with a molecular weight cutoff of 3000 and dialyze at 38 °C in 150 mM NaCl to remove glutathione (GSH) from the liposomes. Change the dialysate every 2 hours for a total of 8 times to obtain blank liposomes loaded with glutathione (GSH).

[0030] 1 mg of R848-2B-MAL was dissolved in 0.1 mL of acetonitrile (10% DMSO) and added dropwise to 0.3 mL of 150 mM NaCl solution at 60 °C with vigorous stirring. Then, 1 mL of the above-mentioned blank liposomes was added, and the mixture was incubated at 60 °C. Samples were taken at specified time points for HPLC analysis. Changes in the clarity of the liposomes were recorded by photography to observe the drug loading process of R848-2B-MAL. Results are as follows: Figure 1 and Figure 2As shown, during the incubation process, R848-2B-MAL gradually reacts with glutathione (GSH) in the liposomes (the reaction is completed in about 10 minutes) to generate the corresponding hydrophilic R848-2B-GSH. At the same time, the liposomes gradually change from turbid to clear, indicating that R848-2B-MAL is successfully encapsulated by the liposomes.

[0031] Example 11: Preparation of R848-2C-MAL drug-loaded liposomes 1 mg of R848-2C-MAL was dissolved in 0.1 mL of acetonitrile (10% DMSO) and added dropwise to 0.3 mL of 150 mM NaCl solution at 60 °C with vigorous stirring. Then, 1 mL of the above-mentioned blank liposomes was added, and the mixture was incubated at 60 °C. Samples were taken at specified time points for HPLC analysis. Changes in the clarity of the liposomes were recorded by photography to observe the drug loading process of R848-2C-MAL. Results are as follows: Figure 1 and Figure 2 As shown, during the incubation process, R848-2C-MAL gradually reacts with glutathione (GSH) in the liposomes (the reaction is complete in about 10 minutes) to generate the corresponding hydrophilic R848-2C-GSH. At the same time, the liposomes quickly change from turbid to clear, indicating that R848-2C-MAL is successfully encapsulated by the liposomes.

[0032] Example 12: Preparation of CPT-2BP-MAL drug-loaded liposomes 1 mg of CPT-2BP-MAL was dissolved in 0.1 mL of acetonitrile (10% DMSO) and added dropwise to 0.3 mL of 150 mM NaCl solution at 60 °C with vigorous stirring. Then, 1 mL of the above-mentioned blank liposomes was added, and the mixture was incubated at 60 °C. Samples were taken at specified time points for HPLC analysis. Changes in the clarity of the liposomes were recorded by photography to observe the drug loading process of CPT-2BP-MAL. Results are as follows: Figure 3 As shown, during incubation, CPT-2BP-MAL gradually reacts with glutathione (GSH) in the liposomes (the reaction is complete in about 10 minutes) to generate the corresponding hydrophilic CPT-2BP-GSH. At the same time, the liposomes quickly change from turbid to clear, indicating that CPT-2BP-MAL is successfully encapsulated by the liposomes.

[0033] Example 13: Preparation of CPT-2C-MAL drug-loaded liposomes 1 mg of CPT-2C-MAL was dissolved in 0.1 mL of acetonitrile (10% DMSO) and added dropwise to 0.3 mL of 150 mM NaCl solution at 60 °C with vigorous stirring. Then, 1 mL of the above-mentioned blank liposomes was added, and the mixture was incubated at 60 °C. Samples were taken at specified time points for HPLC analysis. Changes in the clarity of the liposomes were recorded by photography to observe the drug loading process of CPT-2C-MAL. Results are as follows: Figure 3 As shown, during incubation, CPT-2C-MAL gradually reacts with glutathione (GSH) in the liposomes (the reaction is complete in about 10 minutes) to generate the corresponding hydrophilic CPT-2C-GSH. At the same time, the liposomes quickly change from turbid to clear, indicating that CPT-2C-MAL is successfully encapsulated by the liposomes.

[0034] Example 14: Drug activation of free R848-2B-GSH and free R848-2C-GSH in plasma R848-2B-GSH and R848-2C-GSH were diluted in 200 μL of rat plasma, with an R848 equivalent concentration of 10 μg / mL. (Temperature 37 ℃, times 0, 5, 15, 30 and 60 min, 4 h, 16 h). 180 μL of methanol (containing 1% acetic acid) was added to 20 μL of plasma, and the supernatant was collected by centrifugation at 10000 rpm for 5 min. The content of R848 and the two R848-GSHs in the supernatant was measured by HPLC using a UV detector, with the detection wavelength set at 325 nm. The percentage of R848 activation (%) was calculated based on the R848 / (R848-GSH+R848) ratio. Results are as follows. Figure 4 As shown, after incubation in plasma for 0.5 h, R848-2B-GSH was completely activated into the original R848 drug, while only 44.5% of R848-2C-GSH was activated into the original drug at this time. This indicates that the activation rate of the R848 prodrug is greatly increased due to the presence of the fast linker 2B.

[0035] Example 15: Drug activation of free CPT-2BP-GSH, free CPT-2B-GSH and free CPT-2C-GSH in plasma CPT-2BP-GSH, CPT-2B-GSH, and CPT-2C-GSH were diluted in 200 μL of rat plasma, with a CPT equivalent concentration of 10 μg / mL. (Temperature: 37 ℃; Time: 0, 5, 15, 30, and 60 min, 4 h). 180 μL of methanol (containing 1% acetic acid) was added to 20 μL of plasma, and the supernatant was collected by centrifugation at 10000 rpm for 5 min. The concentrations of CPT and the three CPT-GSH compounds in the supernatant were measured by HPLC using a UV detector at a wavelength of 325 nm. The percentage of CPT activation (%) was calculated based on the CPT / (CPT-GSH+CPT) ratio. Results are as follows: Figure 5 As shown, CPT-2C-GSH requires 4 hours of incubation in plasma to achieve complete activation; CPT-2B-GSH exhibits significantly improved activation efficiency, converting completely to free CPT in just 15 minutes; while CPT-2BP-GSH shows the fastest activation rate, completing full activation within 5 minutes. Notably, at the 5-minute time point of complete activation of CPT-2BP-GSH, the activation rate of CPT-2C-GSH is only 12.14%. These data indicate that, compared to traditional linkages (corresponding to CPT-2C-GSH), the rapid activation linkages 2B and 2BP designed in this invention can significantly accelerate the activation process of CPT prodrugs in plasma, with the 2BP linkage showing a superior promoting effect compared to 2B.

[0036] Example 16: Drug activation of free DTX-2BP-GSH, free DTX-2B-GSH and free DTX-2C-GSH in plasma DTX-2BP-GSH, DTX-2B-GSH, and DTX-2C-GSH were diluted in 200 μL of rat plasma, with a DTX equivalent concentration of 10 μg / mL. (Temperature 37 ℃, time: 0, 5, 15, 30 and 60 min, 4 h). 180 μL of methanol (containing 1% acetic acid) was added to 20 μL of plasma, and the supernatant was collected by centrifugation at 10000 rpm for 5 min. The content of DTX and the three DTX-GSHs in the supernatant was measured by HPLC using a UV detector, with the detection wavelength set at 325 nm. The percentage of DTX activation (%) was calculated based on the DTX / (DTX-GSH+DTX) ratio. Results are shown below. Figure 6As shown, only 87.1% of DTX-2C-GSH was activated after 4 hours of incubation in plasma. DTX-2B-GSH showed significantly improved activation efficiency, achieving complete activation within 4 hours, and reaching an activation rate of 84.26% after only 1 hour of incubation, 1.5 times that of DTX-2C-GSH at the same time. DTX-2BP-GSH exhibited the fastest activation rate, reacting completely within 5 minutes. Notably, at the 5-minute time point when DTX-2BP-GSH was fully activated, the activation rates of DTX-2B-GSH and DTX-2C-GSH were 25.27% and 15.4%, respectively. This indicates that rapid activation of the 2B and 2BP linkages can significantly enhance the activation rate of the prodrug, with the 2BP linkage showing a superior promoting effect compared to 2B.

[0037] Example 17: In vitro cytotoxicity assays of CPT-2BP-GSH, CPT-2BP-LIP, CPT-2C-GSH, CPT-2C-GSH and free CPT Mouse breast cancer cells (4T1 cells) in the logarithmic growth phase were digested with trypsin, centrifuged, and then... 3 Cells were seeded in 96-well plates and incubated overnight. Then, different concentration gradients of CPT-2BP-GSH, CPT-2BP-LIP, CPT-2C-GSH, CPT-2C-LIP, and free CPT were added (each experiment was repeated three times), and the cells were incubated for 48 hours. Afterward, 10 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated for another 4 hours. Finally, 100 μL of DMSO was used to replace the culture medium in each well, and absorbance was measured at 570 nm to plot cell viability curves. Results are as follows: Figure 7 As shown, the cytotoxicity of each drug group exhibited a significant concentration-dependent effect. At a concentration of 40 μg / ml, CPT-2C-GSH showed a cell survival rate of 53.02%, while CPT-2BP-GSH at the same concentration only showed a survival rate of 26.51%, demonstrating significantly superior cytotoxicity compared to CPT-2C-GSH and achieving a toxicity effect very close to that of free CPT. At a concentration of 40 μg / ml, CPT-2C-LIP showed a cell survival rate of 50.8%, while CPT-2BP-LIP showed even better cell-killing ability at the same concentration, with a survival rate decreasing to 29.41%. Encouragingly, at 10 μg / ml, the cytotoxicity of CPT-2BP-LIP (31.29%) even exceeded that of free CPT (45%). This indicates that due to rapid activation of the linkage bond, CPT-2BP-LIP exhibits strong cytotoxicity against the growth of 4T1 cells, suggesting that CPT-2BP-LIP has an enhanced tumor-killing effect.

Claims

1. A drug-rapidly activated linker-maleimide (MAL) derivative having the following general formula: 。 2. The derivative as claimed in claim 1, characterized in that: The drug is selected from resimote (R848), camptothecin (CPT), docetaxel (DTX), or paclitaxel (PTX).

3. The derivative as described in claim 1 or 2, characterized in that: n is 1 or 2, and m is an integer selected from 0 to 5.

4. The derivative as described in claim 1, characterized in that: The derivatives are selected from the following compounds: 。 5. The derivative as described in claim 4, characterized in that: The derivative is resimot-2B-maleimide (R848-2B-MAL).

6. The preparation method of R848-2B-MAL as described in claim 5, wherein intermediate product 1 is first prepared from 3-maleimide propionic acid and p-hydroxybenzyl alcohol as raw materials, and then intermediate product 1 is reacted with ressimolide (R848) under triphosgene and DMAP catalysis to obtain the final product; the reaction route is as follows: 。 7. An active drug-carrying liposome, characterized in that: Includes the drug-rapidly activated linker-maleimide (MAL) derivative as described in any one of claims 1-5, phospholipids, cholesterol, PEGylated phospholipids, and water-soluble thiol substances.

8. The active drug-loaded liposome as described in claim 7, characterized in that: The phospholipids are natural, semi-synthetic, or fully synthetic phospholipids selected from egg yolk lecithin (EPC), soybean phospholipids, sphingomyelin, hydrogenated soybean phospholipids (HSPC), distearate phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), and myristoyl phosphatidylcholine (DMPC); the PEGylated phospholipids are coupling compounds of PEG and DSPE linked by amide bonds; the water-soluble thiol substances are water-soluble substances containing thiol groups, selected from dipeptides, tripeptides, or glutathione (GSH) containing cysteine.

9. The active drug-loaded liposome as described in claim 8, characterized in that: The phospholipid is hydrogenated soybean phospholipid (HSPC); the PEGylated phospholipid is DSPE-PEG. 2000 The water-soluble thiol substance is glutathione (GSH).

10. The use of the drug-rapidly activated linker-maleimide (MAL) derivative as described in any one of claims 1-5 or the active drug-loaded liposome as described in any one of claims 7-9 in the preparation of drugs for the prevention or treatment of tumors.

Images