A method for synthesizing a maleic polyethylene glycol drug conjugate

Abstract

The present application relates to a kind of synthesis method of maleic polyethylene glycol drug conjugate.The present application is used to solve the problems of complex synthesis process, high cost, difficult extraction and containing high-risk chemical reagents of maleic polyethylene glycol drug conjugate in prior art. The synthesis method of polyethylene glycol drug modifier disclosed in the present application adopts a new synthesis route, changes the synthesis order of functional groups, protects the stability of maleic functional group during synthesis and improves the purity of product, the synthesis route is short, simple operation, reduces the synthesis period, thereby greatly reduces the synthesis cost. The maleic polyethylene glycol drug conjugate provided by the present application can change the hydrophilicity and hydrophobicity of maleic polyethylene glycol drug conjugate by changing the length of carbon chain and polyethylene glycol, thereby better used for drug and bioactive molecule coupling, and linking of other substances, greatly improve the application range of maleic polyethylene glycol drug conjugate.

Description

Technical Field

[0001] This invention relates to a drug coupling agent preparation technique, and more particularly to a method for synthesizing a maleic polyethylene glycol drug coupling agent. Background Technology

[0002] Polyethylene glycol (PEG) possesses excellent water solubility, compatibility, lubricity, adhesion, and thermal stability, and its products are non-toxic and non-irritating, thus it has long been widely used in the pharmaceutical industry. For example, it is used as a solvent for injectable drugs, an excipient and modifier for tablets, and in clinical applications such as the combination of PEG-based interferon with other drugs to treat diseases. With the development of biotechnology, protein-based biological drugs are being increasingly developed and applied due to their advantages such as high activity, strong specificity, well-defined biological functions, and low toxicity. However, problems such as short half-life, poor stability, poor water solubility, immunogenicity, and rapid renal clearance have limited their further development. To address these issues, researchers have made various attempts, and PEG modification of protein drugs is one of the most important chemical modification techniques.

[0003] With the development of biotechnology, protein-based biopharmaceuticals are being developed and applied more and more widely due to their advantages such as high activity, strong specificity, well-defined biological functions, and low toxicity. The development of polyethylene glycol (PEG) derivatives has progressed through the first generation of random modification, the second generation of specific and functional modification, and the third generation of branched structure applications. PEG modifiers are constantly being improved and innovated, and their specific linkages with different drugs are being explored. Their applications have also expanded from simple drug modification to areas such as biosensing and drug delivery.

[0004] Maleic polyethylene glycol (PEG) drug conjugates possess several advantages: 1. They can be used to conjugate drugs with bioactive molecules (such as antibodies and peptides) to form drug-conjugates. These conjugates enable targeted delivery and release in vivo, improving therapeutic efficacy and reducing side effects. 2. They can modify the surface of biomaterials (such as biosensors and biochips) to improve their biocompatibility and stability. 3. Maleimide groups can react with thiols or amino groups in polymers to achieve crosslinking, which improves the mechanical properties, thermal stability, and chemical stability of the polymers. 4. They can modify the surface properties of polymer materials, such as hydrophilicity, hydrophobicity, and biocompatibility. This modification can alter the application properties of polymer materials and expand their application areas.

[0005] Maleic polyethylene glycol (PEG) drug coupling agents have wide applications in multiple fields, and the diversity of coupling targets requires a variety of PEG drug coupling agent structures. In recent years, there has been much research on such coupling agents, such as Route 1.

[0006]

[0007] In the synthesis process of Route 1, sodium azide is used. Sodium azide is a highly toxic reagent and is also a highly dangerous explosive material, which limits its use in industrial production.

[0008] Route 2,

[0009]

[0010] Route 2 uses easily explosive hydrazine hydrate in the synthesis process, and the synthesis of amino groups involves too many solid impurities that are difficult to purify. In addition, the raw materials used are more expensive.

[0011] In summary, current methods for synthesizing maleic polyethylene glycol (PEG) drug coupling agents are complex, costly, and suffer from drawbacks such as difficult extraction and the presence of hazardous chemicals. Based on these findings, this invention proposes a novel method for synthesizing PEG drug coupling agents that effectively addresses these problems. Summary of the Invention

[0012] This invention provides a method for synthesizing maleic polyethylene glycol drug coupling agents; solving the problems of complex synthesis processes, high costs, difficult extraction, and the presence of high-risk chemical agents in existing technologies for synthesizing maleic polyethylene glycol drug coupling agents.

[0013] The above-mentioned technical problems of the present invention are mainly solved by the following technical solution: a method for synthesizing a maleic polyethylene glycol drug coupling agent, comprising the following steps:

[0014] S1, Cbz-NH(CH2)mCOOH and dichloromethane were added to the reactor, followed by the sequential addition of EDCI and NH2-PEGn-CH2CH2COOtBu. The entire reaction system was then kept at a constant temperature, and the reaction endpoint was determined by TLC. The reaction system was then subjected to an extraction and washing process to obtain Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOtBu.

[0015] S2, Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOtBu was placed in a reactor, and dichloromethane and trifluoroacetic acid were added in a volume ratio of 1 to 2:1. The entire reaction system was then kept at a constant temperature, and the reaction endpoint was determined by TLC. The reaction system was then treated by an acid-base method to obtain Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOH.

[0016] S3, Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOH is added to a hydrogenation reactor and dissolved in methanol. Then, 2-10% palladium on carbon catalyst is added, and hydrogen gas is introduced. The reaction ends when hydrogen gas is no longer consumed. Post-treatment, the reaction solution is filtered to remove palladium on carbon, and the filtrate is concentrated to obtain crude NH2(CH2)mCONH-PEGn-CH2CH2COOH.

[0017] S4, under a nitrogen atmosphere, NH2(CH2)mCONH-PEGn-CH2CH2COOH and maleic anhydride were placed into a reactor, and glacial acetic acid was added for reflux reaction. The reaction solution was dried under reduced pressure to obtain crude product. The crude product was then purified by column chromatography and recrystallized to obtain white solid powder Mal-(CH2)mCONH-PEGn-CH2CH2COOH.

[0018] S5, Mal-(CH2)mCONH-PEGn-CH2CH2COOH and N,N'-disuccinimidyl carbonate were placed in a reactor, and dichloromethane was added for dispersion. Then, DMAP catalyst was added, and the entire reaction system was kept at a constant temperature. The endpoint of the reaction was determined by TLC. The reaction solution was filtered to remove unreacted N,N'-disuccinimidyl carbonate. The filter cake was washed with 50 ml of dichloromethane, and the filtrate was collected. After extraction and crystallization, a white solid powder Mal-(CH2)mCONH-PEGn-CH2CH2COONHS was obtained.

[0019] In the above synthesis method, Cbz represents benzyloxycarbonyl, Mal represents maleimide, and NHS represents N-hydroxysuccinimide. The following is the synthetic route formula for the maleic polyethylene glycol drug coupling agent disclosed in this invention.

[0020] This invention mainly relates to the synthesis of compounds of the Mal-(CH2)mCONH-PEGn-CH2CH2COONHS class, where n is the number of ethylene glycols, m is the number of carbon chains, and m is an integer from 1 to 5.

[0021] The synthetic method for the maleic polyethylene glycol drug coupling agent of this invention employs a novel synthetic route, altering the synthetic sequence of functional groups. This approach helps protect the stability of the maleic functional groups during synthesis and improves product purity. Compared to existing synthetic routes, this method offers advantages such as a shorter synthetic route, simpler operation, reduced synthesis cycle, and significantly lower synthesis costs. The scheme disclosed in this invention avoids the highly toxic and explosive sodium azide used in Route 1, improving both production safety and yield. Furthermore, it avoids the use of easily explosive hydrazine hydrate in Route 2, preventing issues such as excessive solid impurities during amino synthesis that are difficult to purify and the high cost of raw materials.

[0022] Furthermore, in S1, the molar ratio of NH2-PEGn-CH2CH2COOtBu, EDCI, and Cbz-NH(CH2)mCOOH is 1:1.05-1.5:1.05-1.5, the reaction temperature is maintained between 0 and 25°C, and the holding time is 2 to 6 hours.

[0023] Furthermore, in S2, the ratio of Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOtBu, dichloromethane, and trifluoroacetic acid is 1 mmol: 1 ml: 1 ml, the reaction temperature is maintained between 0 and 25°C, and the holding time is 2 to 6 hours.

[0024] Furthermore, in S3, the mass ratio of Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOH to the palladium-carbon catalyst is 1:0.01 to 0.1, and the reaction temperature is maintained between 25 and 55°C.

[0025] Furthermore, in S4, the molar ratio of NH2(CH2)mCONH-PEGn-CH2CH2COOH to maleic anhydride is 1:1.0 to 1.2, the reaction temperature is maintained between 110 and 120°C, and the reaction time is 4 to 6 hours.

[0026] Furthermore, in S5, the molar ratio of Mal-(CH2)mCONH-PEGn-CH2CH2COOH, the N,N'-disuccinimidyl carbonate, and the DMAP is 1:1.05-1.2:0.03-0.05, the reaction temperature is maintained between 5 and 25°C, and the reaction time is 2 to 8 hours.

[0027] Therefore, the present invention has the following characteristics compared with the prior art: 1. The synthesis method of the polyethylene glycol drug modifier of the present invention adopts a new synthesis route, changes the synthesis order of functional groups, which is beneficial to protect the stability of the maleic functional groups in the synthesis process and improve the purity of the product. The synthesis route is short, the operation is simple, and the synthesis cycle is reduced, thereby greatly reducing the cost of synthesis.

[0028] 2. The maleic polyethylene glycol drug coupling agent provided by this invention can change the hydrophilicity and hydrophobicity of the maleic polyethylene glycol drug coupling agent by changing the length of the carbon chain and polyethylene glycol, thereby making it better suited for the coupling of drugs and bioactive molecules, as well as the linking of other substances, and greatly improving the application range of the maleic polyethylene glycol drug coupling agent. Attached Figure Description

[0029] Figure 1The 1H NMR spectrum of the coupling agent Mal-(CH2)2CONH-PEG4-CH2CH2COOH;

[0030] Figure 2 The 1H NMR spectrum of the coupling agent Mal-(CH2)2CONH-PEG4-CH2CH2COONHS;

[0031] Figure 3 The 1H NMR spectrum of the coupling agent Mal-(CH2)2CONH-PEG8-CH2CH2COOH;

[0032] Figure 4 The image shows the 1H NMR spectrum of the coupling agent Mal-(CH2)2CONH-PEG8-CH2CH2COONHS. Detailed Implementation

[0033] The technical solution of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings.

[0034] Example 1: S1, Cbz-NH(CH2)2COOH (24.5 g, 0.11 mol) and 200 mL of dichloromethane were added to a 500 mL reactor, followed by EDCI (23.0 g, 0.12 mol) and NH2-PEG4-CH2CH2COOtBu (32.1 g, 0.1 mol). The reaction system was then kept at 5 °C for 4 hours, and the reaction endpoint was determined by TLC. The reaction solution was then transferred to a separatory funnel and washed twice with 30 mL of water and once with 30 mL of sodium bicarbonate aqueous solution. The organic phase was dried, concentrated, and dried under vacuum with anhydrous magnesium sulfate to obtain 50.6 g of Cbz-NH(CH2)2CONH-PEG4-CH2CH2COOtBu with a purity of 98.3% (yield of 96.2%), which can be directly used for the next synthesis.

[0035] S2, weigh 42.1 g (0.08 mol) of Cbz-NH(CH2)2CONH-PEG4-CH2CH2COOtBu into a 250 ml reactor, add 80 ml of dichloromethane and 80 ml of trifluoroacetic acid, then maintain the reaction temperature at 15 °C and react for 4 hours. The reaction endpoint was determined by TLC. The reaction system was diluted with 200 ml of dichloromethane and washed 5-6 times with 80 ml of water. The organic phase was evaporated to dryness to obtain the crude product. The crude product was dissolved in 200 ml of alkaline water and extracted 3 times with 50 ml of ethyl acetate to remove impurities. The aqueous phase was adjusted to pH 4 with hydrochloric acid, and the product was extracted three times with 100 ml of dichloromethane. The dichloromethane phase was collected, washed once with 100 ml of water, dried, and concentrated to obtain 34.6 g of Cbz-NH(CH2)2CONH-PEG4-CH2CH2COOH with a purity of 98.5% (yield 92%).

[0036] S3, weigh 32.9 g (0.07 mol) of Cbz-NH(CH2)2CONH-PEG4-CH2CH2COOH and add it to a hydrogenation reactor. Add 150 ml of methanol to dissolve the precipitate, then add palladium-carbon catalyst and purge with hydrogen gas. The reaction system is carried out at 35 °C. The reaction ends when hydrogen gas is no longer consumed. Post-processing, filter the reaction solution to remove palladium-carbon, concentrate the filtrate to obtain 23.5 g of crude NH2(CH2)2CONH-PEG4-CH2CH2COOH (yield approximately 100%). The crude product can be used directly in the next synthesis without purification.

[0037] S4, see Figure 1 Under a nitrogen atmosphere, 23.5 g (0.07 mol) of NH2(CH2)2CONH-PEG4-CH2CH2COOH and 6.9 g (0.07 mol) of maleic anhydride were weighed and placed into a 250 ml reactor. 100 ml of glacial acetic acid was added and the mixture was refluxed for 4 hours. The glacial acetic acid was evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by column chromatography and recrystallized to obtain 13.4 g of white solid powder Mal-(CH2)2CONH-PEG4-CH2CH2COOH with a purity of 98.7% (yield 46%).

[0038] S5, see Figure 2Weigh out 10.4 g (0.025 mol) of Mal-(CH2)2CONH-PEG4-CH2CH2COOH and 7.7 g (0.03 mol) of N,N'-disuccinimidyl carbonate and place them in a 250 ml reactor. Add 100 ml of dichloromethane for dispersion, and then add 0.15 g (1.3 mol) of DMAP catalyst. The reaction system (mmol) was kept at 15℃ for 3 hours. The endpoint of the reaction was determined by TLC. The reaction solution was filtered to remove unreacted N,N'-disuccinimidyl carbonate. The filter cake was washed with 50 ml of dichloromethane. The filtrate was collected and transferred to a separatory funnel. It was washed three times with 50 ml of water and once with 50 ml of sodium chloride aqueous solution. The organic phase was dried with anhydrous magnesium sulfate and concentrated to obtain the crude product. The crude product was purified by recrystallization of ethyl acetate / petroleum ether = 7 / 3 to obtain 11.5 g of white solid powder Mal-(CH2)2CONH-PEG4-CH2CH2COONHS with a purity of 99.1% (yield 90%).

[0039] Example 2: S1, Cbz-NH(CH2)2COOH (49.1 g, 0.22 mol) and 400 mL of dichloromethane were added to a 1000 mL reactor, followed by EDCI (23.0 g, 0.24 mol) and NH2-PEG8-CH2CH2COOtBu (99.4 g, 0.2 mol). The reaction system was then kept at 5 °C for 4 hours, and the reaction endpoint was determined by TLC. The reaction solution was then transferred to a separatory funnel and washed twice with 100 mL of water and once with 100 mL of sodium bicarbonate aqueous solution. The organic phase was dried with anhydrous magnesium sulfate, concentrated, and dried under vacuum to obtain 133.4 g of Cbz-NH(CH2)2CONH-PEG8-CH2CH2COOtBu with a purity of 98.8% (yield of 95.0%), which can be directly used for the next synthesis.

[0040] S2, weigh 70.2 g (0.1 mol) of Cbz-NH(CH2)2CONH-PEG8-CH2CH2COOtBu into a 500 ml reactor, add dichloromethane / trifluoroacetic acid (100 ml / 100 ml), and then incubate the reaction at 15°C for 4 hours. The reaction endpoint is determined by TLC. Dilute the reaction system with 200 ml of dichloromethane, wash 5-6 times with 100 ml of water, and evaporate the organic phase to dryness to obtain the crude product. Dissolve the crude product in 500 ml of alkaline water, and extract 3 times with 100 ml of ethyl acetate to remove impurities. Adjust the pH of the aqueous phase to 4 with hydrochloric acid, extract the product three times with 150 ml of dichloromethane, collect the dichloromethane phase, wash once with 100 ml of water, dry the dichloromethane phase, and concentrate to obtain Cbz-NH(CH2)2CONH-PEG8-CH2CH2COOH 60.1 g, with a purity of 98.7% (yield of 93%);

[0041] S3, weigh 51.7 g (0.08 mol) of Cbz-NH(CH2)2CONH-PEG8-CH2CH2COOH and add it to a hydrogenation reactor. Add 250 ml of methanol to dissolve the precipitate, add palladium-carbon catalyst, and purge with hydrogen gas. The reaction system is carried out at 35 °C. The reaction ends when hydrogen gas is no longer consumed. Post-processing, filter the reaction solution to remove palladium-carbon, concentrate the filtrate to obtain 41.0 g of crude NH2(CH2)2CONH-PEG8-CH2CH2COOH (yield approximately 100%). The crude product can be used directly in the next synthesis without purification.

[0042] S4, see Figure 3 Under nitrogen protection, 41.0 g (0.08 mol) of NH2(CH2)2CONH-PEG8-CH2CH2COOH and 7.8 g (0.08 mol) of maleic anhydride were weighed and placed into a 250 ml reactor. 100 ml of glacial acetic acid was added and the mixture was refluxed for 4 hours. The reaction solution was evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by column chromatography and recrystallized to obtain 21.3 g of white solid powder Mal-(CH2)2CONH-PEG8-CH2CH2COOH with a purity of 98.5% (yield 45%).

[0043] S5, see Figure 4Weigh out 21.3 g (0.036 mol) of Mal-(CH2)2CONH-PEG8-CH2CH2COOH and 11.0 g (0.043 mol) of N,N'-disuccinimidyl carbonate and place them in a 500 ml reactor. Add 150 ml of dichloromethane for dispersion, and then add the catalyst DMAP (0.22 g, 1.8 mol). The reaction system (mmol) was kept at 15℃ for 4 hours. The endpoint of the reaction was determined by TLC. The reaction solution was filtered to remove unreacted N,N'-disuccinimidyl carbonate. The filter cake was washed with 100 ml of dichloromethane. The filtrate was collected and transferred to a separatory funnel. It was washed three times with 50 ml of water and once with 50 ml of sodium chloride aqueous solution. The organic phase was dried with anhydrous magnesium sulfate and concentrated to obtain the crude product. The crude product was purified by recrystallization of ethyl acetate / petroleum ether = 4 / 1 to obtain 22.6 g of white solid powder Mal-(CH2)2CONH-PEG8-CH2CH2COONHS with a purity of 99.0% (yield 91%).

[0044] It will be apparent to those skilled in the art that the present invention can be modified in various ways, and such modifications are not considered to depart from the scope of the invention. All such modifications that are obvious to those skilled in the art are included within the scope of the claims.

Claims

1. A method for synthesizing a maleic polyethylene glycol drug coupling agent, characterized in that, Includes the following steps: S1, Cbz-NH(CH2)mCOOH and dichloromethane were added to the reactor, followed by the sequential addition of EDCI and NH2-PEGn-CH2CH2COOtBu. The entire reaction system was then kept at a constant temperature, and the reaction endpoint was determined by TLC. The reaction system was then subjected to an extraction and washing process to obtain Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOtBu. S2, Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOtBu was placed in a reactor, and dichloromethane and trifluoroacetic acid were added in a volume ratio of 1 to 2:

1. The entire reaction system was then kept at a constant temperature, and the reaction endpoint was determined by TLC. The reaction system was then treated by an acid-base method to obtain Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOH. S3, Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOH is added to a hydrogenation reactor and dissolved in methanol. Then, palladium-carbon catalyst is added, and hydrogen gas is introduced. The reaction ends when hydrogen gas is no longer consumed. Post-treatment, the reaction solution is filtered to remove palladium-carbon, and the filtrate is concentrated to obtain crude NH2(CH2)mCONH-PEGn-CH2CH2COOH. S4, under a nitrogen atmosphere, NH2(CH2)mCONH-PEGn-CH2CH2COOH and maleic anhydride were placed into a reactor, and glacial acetic acid was added for reflux reaction. The reaction solution was dried under reduced pressure to obtain crude product. The crude product was then purified by column chromatography and recrystallized to obtain white solid powder Mal-(CH2)mCONH-PEGn-CH2CH2COOH. S5, Mal-(CH2)mCONH-PEGn-CH2CH2COOH and N,N'-disuccinimidyl carbonate were placed in a reactor, and dichloromethane was added for dispersion. Then, DMAP catalyst was added, and the entire reaction system was kept at a certain temperature. The endpoint of the reaction was determined by TLC. The reaction solution was filtered to remove unreacted N,N'-disuccinimidyl carbonate. The filter cake was washed with 50 ml of dichloromethane, and the filtrate was collected. After extraction and crystallization, a white solid powder Mal-(CH2)mCONH-PEGn-CH2CH2COONHS was obtained. The molar ratio of Mal-(CH2)mCONH-PEGn-CH2CH2COOH, N,N'-disuccinimidyl carbonate and DMAP was 1:1.05-1.2:0.03-0.

05. The reaction temperature was maintained between 5 and 25 °C, and the reaction time was 2 to 8 hours.

2. The method for synthesizing the maleic polyethylene glycol drug coupling agent according to claim 1, characterized in that: In S1, the molar ratio of NH2-PEGn-CH2CH2COOtBu, EDCI, and Cbz-NH(CH2)mCOOH is 1:1.05-1.5:1.05-1.5, the reaction temperature is maintained between 0 and 25°C, and the holding time is 2 to 6 hours.

3. The method for synthesizing the maleic polyethylene glycol drug coupling agent according to claim 1, characterized in that: In S2, the ratio of Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOtBu, dichloromethane, and trifluoroacetic acid is 1 mmol: 1 ml: 1 ml, the reaction temperature is maintained between 0 and 25°C, and the holding time is 2 to 6 hours.

4. The method for synthesizing the maleic polyethylene glycol drug coupling agent according to claim 1, characterized in that: In S3, the mass ratio of Cbz-NH(CH2)mCONH-PEGn-CH2CH2COOH to the palladium-carbon catalyst is 1:0.01 to 0.1, and the reaction temperature is maintained between 25 and 55°C.

5. The method for synthesizing the maleic polyethylene glycol drug coupling agent according to claim 1, characterized in that: In S4, the molar ratio of NH2(CH2)mCONH-PEGn-CH2CH2COOH to maleic anhydride is 1:1.0 to 1.2, the reaction temperature is maintained between 110 and 120°C, and the reaction time is 4 to 6 hours.

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