Maleimide polyethylene glycol activated lipid and method of making same

Abstract

This invention belongs to the field of chemical pharmaceutical technology, and particularly relates to a method for preparing an anti-puncture flash evaporation material. Addressing the issue that the hydrophilicity of the molecular structure in existing technologies needs improvement, this invention provides a maleic methylamine polyethylene glycol activated ester and its preparation method, comprising a reaction condensation of 3-maleimide propionic acid and tert-butyl methylamine polyethylene glycol propionate, followed by acid hydrolysis of the ester, and then adding the activated ester for a catalytic reaction to obtain the final product Mal-CH2CH2COO-N(CH3)-(PEG). n -CH2CH2COONHS. This invention creatively designs a maleic methylamine polyethylene glycol activated lipid structure. The methylamine structure can increase solubility in aqueous media, making it more hydrophilic than the existing Mal-CH2CH2COO-PEG-COONHS molecular structure, which is more conducive to the binding of drugs to the human body environment.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical technology, and particularly relates to a maleic methylamine polyethylene glycol activated ester and its preparation method. Background Technology

[0002] Mal-CH2CH2COO-PEG-COONHS can be used to prepare antibody-drug conjugates (ADCs) by organically combining monoclonal antibodies and cytotoxins. This combines the advantages of both antibodies and cytotoxic drugs, resulting in strong targeting, high cytotoxicity, low toxicity, and a long degradation half-life. Structurally, an ADC consists of three components: an antibody, a small molecule cytotoxin, and a linker. The antibody's role is to achieve targeting, the cytotoxin's role is to kill target cells, and the linker's role is to achieve the organic binding of the antibody and cytotoxin, forming an integrated whole. Linker design is crucial for ADC drugs. As the hub and bridge connecting monoclonal antibodies and small molecule cytotoxins, the properties of the linker directly affect the efficacy and safety of the ADC. As an ADC linker, it must ensure drug stability in the bloodstream and allow for rapid and effective release of the active toxin upon reaching the target tissue. Linker construction has become a core element and a major challenge restricting the development of ADCs.

[0003] Mal-CH2CH2COO-PEG-COONHS has various applications in scientific research, primarily in the biomedical field. This chemical substance is composed of carboxyl groups (COOH), polyethylene glycol (PEG), and maleimide (Mal). The introduction of PEG not only increases the compound's water solubility but also improves its biocompatibility, giving it potential advantages in biomedical applications. Specifically, the applications of Mal-CH2CH2COO-PEG-COONHS include: Bioconjugation and Modification: The maleimide (Mal) group in Mal-CH2CH2COO-PEG-COONHS is reactive and can undergo Michael addition or covalent coupling reactions with compounds containing thiol or amino groups. This allows it to be used to link, modify, or couple other biomolecules (such as proteins, peptides, nucleic acids, etc.) to construct more complex bioconjugates or for applications such as biolabeling and drug delivery.

[0004] Crosslinking of protein carriers and ligands: Mal-CH2CH2COO-PEG-COONHS is widely used for crosslinking of protein carriers and ligands. This is achieved by linking PEG to the desired material or small molecule drugs and peptides to improve drug solubility, stability and bioavailability.

[0005] Targeted drug research: In targeted drug research, Mal-CH2CH2COO-PEG-COONHS is used to study and develop drug carriers with specific targeting capabilities to improve drug efficacy and reduce side effects.

[0006] Peptide synthesis support: In addition, Mal-CH2CH2COO-PEG-COONHS is also used for peptide synthesis support, facilitating the synthesis and purification of peptides by providing a stable synthesis platform.

[0007] Synthesis of PROTAC; it can also be used as a PEG-based PROTAC linker.

[0008] Although Mal-CH2CH2COO-PEG-COONHS has many applications as mentioned above, its solubility in aqueous media is not ideal, and its hydrophilicity needs to be improved to facilitate the combination of the human body environment and drugs. Summary of the Invention

[0009] The purpose of this invention is to address the above-mentioned problems by providing a maleic methylamine polyethylene glycol activated ester.

[0010] Another objective of this invention is to provide a method for preparing maleic methylamine polyethylene glycol activated ester in response to the above-mentioned problems.

[0011] To achieve the above objectives, the present invention adopts the following technical solutions: A maleic methylamine polyethylene glycol activated ester, the structural formula of which is shown below: ; Where n is an integer from 1 to 24.

[0012] A method for preparing maleic methylamine polyethylene glycol activated ester includes the following steps: Step 1: Add 3-maleimide propionic acid and an organic solvent to the reactor. After cooling, add a condensation reagent. The molar ratio of 3-maleimide propionic acid to the condensation reagent is 1:1.1-1.5. Then, add tert-butyl methylamine polyethylene glycol propionate dropwise. The molar ratio of 3-maleimide propionic acid to tert-butyl methylamine polyethylene glycol propionate is 1:1-1.2. After the reaction is complete, wash the reaction system with water, dry the organic phase, filter, and then evaporate to dryness to obtain intermediate 1, Mal-CH2CH2COO-N(CH3)-(PEG). n-CH2CH2COOtBU; Step 2: Add intermediate 1 to the reactor, dissolve it with an organic solvent, cool it down, and then add acid dropwise to react. The molar ratio of intermediate 1 to acid is 1:2-4. After the reaction is complete, extract and wash to obtain the crude product. Then purify the crude product by column chromatography or recrystallization to obtain intermediate 2, Mal-CH2CH2COO-N(CH3)-(PEG). n -CH2CH2COOH; Step 3: Add intermediate 2 to the reactor, dissolve it in an organic solvent, then add the activated lipid, cool to -5℃~5℃ and maintain the temperature for 5-15 minutes, finally add the catalyst. The molar ratio of intermediate 2, activated lipid, and catalyst is 1:1.2:0.025-0.1. React for 4-6 hours. After the reaction, wash the reaction system with water, dry the organic phase, filter, and then evaporate to dryness to obtain the crude product. Purify the crude product by column chromatography or recrystallization to obtain the final product, Mal-CH2CH2COO-N(CH3)-(PEG). n -CH2CH2COONHS.

[0013] In the above-mentioned method for preparing maleic methylamine polyethylene glycol activated ester, the organic solvent in steps one, two and three is 1,4-dioxane, tetrahydrofuran or dichloromethane.

[0014] In the above-mentioned method for preparing maleic methylamine polyethylene glycol activated ester, the reaction temperature in step one is -10℃ to -20℃.

[0015] In the above-mentioned method for preparing maleic methylamine polyethylene glycol activated ester, the condensation reagent in step one is EDC.HCl, DCC, or HATU.

[0016] In the above-mentioned method for preparing maleic methylamine polyethylene glycol activated ester, the acid in step two is trifluoroacetic acid, formic acid, hydrochloric acid, or acetic acid.

[0017] In the above-mentioned method for preparing maleic methylamine polyethylene glycol activated ester, the reaction temperature in step two is -5℃ to 5℃.

[0018] In the above-mentioned method for preparing a maleic methylamine polyethylene glycol activated ester, the activated ester in step three is DSC or NHS.

[0019] In the above-mentioned method for preparing maleic methylamine polyethylene glycol activated ester, the catalyst in step three is 4-dimethylaminopyridine or 2,6-dimethylpyridine.

[0020] Compared with existing technologies, the advantages of this invention are: 1. This invention creatively designs a maleic methylamine polyethylene glycol activated lipid structure. The methylamine structure can increase solubility in aqueous media, making it more hydrophilic than the existing Mal-CH2CH2COO-PEG-COONHS molecular structure, which is more conducive to the combination of human body environment and drug.

[0021] 2. This invention provides a method for preparing maleic methylamine polyethylene glycol activated ester. The preparation method is simple to operate, has a short process flow, and the overall yield is also quite ideal. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the chemical reaction involved in the present invention; Figure 2 The partial proton NMR spectrum of intermediate 2 from Example 1; Figure 3 The partial proton NMR spectrum of intermediate 2 from Example 1; Figure 4 The partial proton NMR spectrum of intermediate 2 from Example 1; Figure 5 The image shows a partial hydrogen NMR spectrum of the product from Example 1. Figure 6 The image shows a partial hydrogen NMR spectrum of the product from Example 1. Figure 7 This is a partial hydrogen NMR spectrum of the product from Example 1. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0024] Example 1 This embodiment provides a maleic methylamine polyethylene glycol activated ester and its preparation method, including the following steps: Step 1: Weigh 16.9 g (0.1 mol) of 3-maleimide propionic acid into a 1 L reactor, add 200 ml of anhydrous dichloromethane, and maintain the stirring temperature at -20°C for 15 min. Then add the weighed EDC.HCl (22.98 g, 0.12 mol) and stir at this temperature for 15 min. Next, slowly add N(CH3)-PEG2-CH2CH2COOtBU (24.7 g, 0.1 mol) dropwise at a molar ratio of (1:1.2:1). React for 4-6 hours, and determine the endpoint by TLC. For post-processing, wash the reaction system three times with 50 ml of water each time. Dry the dichloromethane phase with anhydrous magnesium sulfate, filter, and evaporate to dryness to obtain intermediate 1, Mal-CH2CH2COO-N(CH3)-(PEG)2-CH2CH2COOtBU, totaling 38.3 g, with a yield of 96.2%.

[0025] Step 2: 38.3 g (0.096 mol) of Mal-CH2CH2COO-N(CH3)-(PEG)2-CH2CH2COOtBU was placed in a 500 ml single-necked flask. 40 ml of dichloromethane was added, and the temperature was maintained at 0°C. Trifluoroacetic acid (21.9 g, 0.192 mol) was slowly added dropwise at a rate of one drop per second, with a molar ratio of (1:2). After the addition was complete, the reaction was allowed to proceed for 4-8 hours. The reaction endpoint was determined by TLC. For post-treatment, the reaction system was diluted with dichloromethane by half, then washed 4-5 times with 30 ml of water each time. The dichloromethane phase was dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness to obtain the crude product. The product was dissolved in 500 ml of ethyl acetate and refrigerated overnight at -40°C. The solid product was obtained the next day and filtered in a -40°C freezer. The product was then dried by an oil pump to obtain intermediate 2, Mal-CH2CH2COO-N(CH3)-(PEG)2-CH2CH2COOH, totaling 26.8 g, with a yield of 81.6%.

[0026] Step 3: Add Mal-CH2CH2COO-N(CH3)-(PEG)2-CH2CH2COOH (26.8 g, 0.078 mol) to a 500 ml single-necked reaction flask, dilute to 100 ml with dichloromethane, then add the weighed N,N-disuccinimidyl carbonate (24.0 g, 0.094 mol) to the reaction flask, maintain at 0℃ for 10 min, and finally add the catalytically measured amount of 4-dimethylaminopyridine (0.475 g, 0.0039 mol) to the reaction, with a molar ratio of (1:1.2:0.05). React for 4-6 hours, with the reaction endpoint measured by TLC. For post-treatment, wash the reactants 3-4 times with 20 ml of water each time, dry with magnesium sulfate, filter, and evaporate to dryness. The crude product Mal-CH2CH2COO-N(CH3)-(PEG)2-CH2CH2COONHS was obtained. The crude product was mixed with silica gel powder at a ratio of 1:1 (by mass). The silica gel powder was packed into the chromatography column at a ratio of 1.5 times (by mass). The column was flushed with a mixture of 30% ethyl acetate and petroleum ether. When the TLC was single-point, the product was flushed with 60% ethyl acetate and petroleum ether. The product was evaporated to dryness by rotary evaporation. A total of 29.59 g of product Mal-CH2CH2COO-N(CH3)-(PEG)2-CH2CH2COONHS was obtained, with a yield of 86.4%.

[0027] The proton NMR spectra of intermediate 2 and the product are as follows: Figure 2-4 and Figure 5-7 As shown.

[0028] Example 2 This embodiment provides a maleic methylamine polyethylene glycol activated ester and its preparation method, including the following steps: Step 1: Weigh 5g (0.029mol) of 3-maleimide propionic acid into a 250ml reactor, add 50ml of anhydrous dichloromethane, and maintain the stirring temperature at -20℃ for 15min. Then add 6.79g (0.035mol) of EDC.HCl and stir at this temperature for 15min. Next, slowly add 14.82g (0.029mol) of N(CH3)-PEG8-CH2CH2COOtBU, with a molar ratio of (1:1.2:1). React for 4-6 hours, and determine the endpoint of the reaction by TLC. For post-treatment, the reaction system is washed three times with 10ml of water each time. The dichloromethane phase is dried with anhydrous magnesium sulfate, filtered, and evaporated to obtain intermediate 1, Mal-CH2CH2COO-N(CH3)-(PEG)8-CH2CH2COOtBU, totaling 17.93g, with a yield of 93.4%.

[0029] Step 2: Place 17.93 g (0.027 mol) of Mal-CH2CH2COO-N(CH3)-(PEG)8-CH2CH2COOtBU into a 250 ml single-necked flask, add 20 ml of dichloromethane, and maintain the temperature at 0°C. Slowly add 7.72 g (0.067 mol) of trifluoroacetic acid at a rate of one drop per second, with a molar ratio of (1:2.5). After the addition is complete, allow the reaction to proceed for 4-8 hours. The reaction endpoint is determined by TLC. For post-treatment, dilute the reaction system with dichloromethane by half, then wash 4-5 times with 10 ml of water each time. Dry the dichloromethane phase with anhydrous magnesium sulfate, filter, and evaporate to dryness to obtain the crude product. Dissolve the product in 300 ml of ethyl acetate and refrigerate overnight at -40°C. The solid product was obtained the next day and refrigerated overnight in a -40°C refrigerator. The solid product was obtained the next day and filtered in the refrigerator to obtain the product. The product was then dried by an oil pump to obtain intermediate 2, Mal-CH2CH2COO-N(CH3)-(PEG)8-CH2CH2COOH, totaling 12.73 g, with a yield of 78.4%.

[0030] Step 3: Add 12.73 g (0.021 mol) of Mal-CH2CH2COO-N(CH3)-(PEG)8-CH2CH2COOH to a 250 ml single-necked reaction flask, dilute to 50 ml with dichloromethane, then add the weighed N,N-disuccinimidyl carbonate (6.45 g, 0.025 mol) to the reaction flask, maintain at 0 °C for 10 min, and finally add a catalytically measured amount of 4-dimethylaminopyridine (0.13 g, 0.001 mol) to the reaction. The molar ratio of reactants to reactants is (1:1.2:0.05). React for 4-6 hours, with the endpoint measured by TLC. For post-treatment, wash the reactants 3-4 times with 10 ml of water each time, dry with magnesium sulfate, filter, and evaporate to dryness. The crude product Mal-CH2CH2COO-N(CH3)-(PEG)8-CH2CH2COONHS was obtained. The crude product was diluted and dissolved in 250 ml of ethyl acetate and then refrigerated at -40°C. After the product precipitated crystals, it was filtered at low temperature to obtain a total of 12.09 g of product Mal-CH2CH2COO-N(CH3)-(PEG)8-CH2CH2COONHS, with a yield of 81.89%.

[0031] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A maleic methylamine polyethylene glycol activated ester, characterized in that, The structural formula is as follows: ； Where n is an integer from 1 to 24.

2. A method for preparing maleic methylamine polyethylene glycol activated ester, characterized in that, Includes the following steps: Step 1: Add 3-maleimide propionic acid and an organic solvent to the reactor. After cooling, add a condensation reagent. The molar ratio of 3-maleimide propionic acid to the condensation reagent is 1:1.1-1.

5. Then, add tert-butyl methylamine polyethylene glycol propionate dropwise. The molar ratio of 3-maleimide propionic acid to tert-butyl methylamine polyethylene glycol propionate is 1:1-1.

2. After the reaction is complete, wash the reaction system with water, dry the organic phase, filter, and then evaporate to dryness to obtain intermediate 1, Mal-CH2CH2COO-N(CH3)-(PEG). n -CH2CH2COOtBU; Step 2: Add intermediate 1 to the reactor, dissolve it with an organic solvent, cool it down, and then add acid dropwise to react. The molar ratio of intermediate 1 to acid is 1:2-4. After the reaction is complete, extract and wash to obtain the crude product. Then purify the crude product by column chromatography or recrystallization to obtain intermediate 2, Mal-CH2CH2COO-N(CH3)-(PEG). n -CH2CH2COOH; Step 3: Add intermediate 2 to the reactor, dissolve it in an organic solvent, then add the activated lipid, cool to -5℃~5℃ and maintain the temperature for 5-15 minutes, finally add the catalyst. The molar ratio of intermediate 2, activated lipid, and catalyst is 1:1.2:0.025-0.

1. React for 4-6 hours. After the reaction, wash the reaction system with water, dry the organic phase, filter, and then evaporate to dryness to obtain the crude product. Purify the crude product by column chromatography or recrystallization to obtain the final product, Mal-CH2CH2COO-N(CH3)-(PEG). n -CH2CH2COONHS.

3. The method for preparing a maleic methylamine polyethylene glycol activated ester as described in claim 2, characterized in that: The organic solvents used in steps one, two, and three are 1,4-dioxane, tetrahydrofuran, or dichloromethane.

4. The method for preparing a maleic methylamine polyethylene glycol activated ester as described in claim 2, characterized in that: The reaction temperature in step one is -10℃ to -20℃.

5. The method for preparing a maleic methylamine polyethylene glycol activated ester as described in claim 2, characterized in that: The condensation reagent in step one is EDC.HCl, DCC, or HATU.

6. The method for preparing a maleic methylamine polyethylene glycol activated ester as described in claim 2, characterized in that: The acid in step two is trifluoroacetic acid, formic acid, hydrochloric acid, or acetic acid.

7. The method for preparing a maleic methylamine polyethylene glycol activated ester as described in claim 2, characterized in that: The reaction temperature in step two is -5℃ to 5℃.

8. The method for preparing a maleic methylamine polyethylene glycol activated ester as described in claim 2, characterized in that: The activated lipid in step three is DSC or NHS.

9. The method for preparing a maleic methylamine polyethylene glycol activated ester as described in claim 2, characterized in that: The catalyst in step three is 4-dimethylaminopyridine or 2,6-dimethylpyridine.

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