Novel synthesis method of 4-formyl succinimidyl benzoate

Abstract

The invention discloses a novel synthesis method of 4-formyl benzoic acid succinimido ester, which comprises the following steps: by taking p-formylbenzoic acid, NHS and EDAC as raw materials and dichloromethane as a solvent, adding a catalyst for reaction, and removing the solvent in vacuum to obtain a solid reaction product; and then the solid product is further subjected to the steps of dissolving, drying, vacuum solvent removal and the like to obtain a final product. By optimizing the synthesis conditions of the 4-formyl succinimidyl benzoate, the subsequent separation process is simplified, and the operation is simpler and more convenient; by adopting the catalyst, the yield is greatly improved and can reach 96% or above at most. The synthesis method disclosed by the invention is high in yield, mild in reaction condition and simple and convenient to operate, and can meet the requirements of large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of chemical synthesis, and particularly relates to a new method for synthesizing N-succinimidyl-4-formylbenzoate. Background Art

[0002] The structural formula of N-succinimidyl-4-formylbenzoate (S-4FB) is This molecule contains both a succinimidyl ester functional group and an aldehyde group, and both of these functional groups can react with amino groups. Therefore, it is commonly used for the conjugation labeling of macromolecular carriers such as antibodies, antigens, polypeptides, enzymes, oligonucleotides, and microspheres.

[0003] According to literature reports, there are mainly two methods for the synthesis of S-4FB: The first method is represented by the literature US2011306551 and WO2010102886, which uses p-formylbenzoic acid (FBA) and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) as starting materials, DMF as a solvent, and triethylamine as a catalyst to react for 16 h to obtain; The second method uses p-formylbenzoic acid and N-hydroxysuccinimide (NHS) as starting materials, and obtains them under the action of a condensing agent and a catalyst in a suitable solvent and reaction conditions. Representative literatures include: the paper "Single-Step Conjugation of Bioactive Peptides to Proteins via a Self-Contained Succinimidyl Bis-Arylhydrazone" by Joy A. Phillips et al. (Bioconjugate Chem. 2009, 20, 1950-1957), "Galactose conjugated boron dipyrromethene and hydrogen bonding promoted J-aggregates for efficiently targeted NIR-II fluorescenc assistant photothermaltherapy" by Huiping et al. (Journal of Colloid and Interface Science, 2022, 612, 287-297), "Bio-orthogonal Coupling as a Means of Quantifying the Ligand Density on Hydrophilic Quantum Dots" by Nanqian Zhan et al. (Journal of the American Chemical Society, 2016, 138, (9), 3190-3201), "Synthesis of a Styryl-Based Two-Photon Fluorophore Containing Succinimidyl Ester" by Li Min et al. (Shandong Chemical Industry, 2016, 45(3), 3-5), "Synthesis and self-assembly of nonamphiphilic hyperbranched polyoximes" by Yue Jin et al. (Soft Matter, 2012, 8, 10017-10025), CN109609487, CN110475779, EP0354729, etc.

[0004] The synthetic route of the first method is as follows:

[0005]

[0006] As introduced in US2011306551 and WO2010102886, after the full reaction of p-formylbenzoic acid and TSTU, it is diluted with ethyl acetate, washed with 10% sodium bisulfate solution, the obtained ethyl acetate phase is further washed with saturated brine, dried with magnesium sulfate, and recrystallized with ethyl acetate to obtain the corresponding product, with a final yield of 57.4%. Due to the low yield, the subsequent separation and purification process is relatively complex.

[0007] The synthetic route of the second method is as follows:

[0008]

[0009] There are many reports using NHS as the starting material. According to the types of condensing agents (dehydrating agents) used, they are divided into two categories: N,N-dicyclohexylcarbodiimide (DCC) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC). The article "Synthesis of a Bisstyryl-based Two-Photon Fluorophore Containing Succinimidyl Ester" uses DCC as the dehydrating agent, dichloromethane as the solvent, and 4-dimethylaminopyridine (DMAP) as the catalyst. After reacting for 48 h, filtration is carried out, and the filtrate is washed with saturated sodium bicarbonate, sodium chloride, and water for the dichloromethane phase, extracted with dichloromethane, dried with anhydrous sodium sulfate, and then concentrated and purified by column chromatography to obtain the product. The preparation time of this scheme is long, and the product yield is 49.2%.

[0010] EP0354729 also reports using DCC as the condensing agent, 1,4-dioxane as the solvent. After the reaction, the solvent is removed and recrystallized with acetone, and the final yield of the obtained product is 42.5%.

[0011] When the literature "Synthesis and self-assembly of nonamphiphilic hyperbranched polyoximes" uses DCC as the condensing agent, DMF / dichloromethane is used as the solvent, and the reaction is carried out at 0 °C under a nitrogen atmosphere. After post-treatment such as solvent removal, the product is obtained with a yield of 50%. Relatively speaking, the yield is low and the reaction conditions are relatively harsh. The literature "Bio-orthogonal Coupling as a Means of Quantifying the Ligand Density on Hydrophilic Quantum Dots" has made improvements. Using tetrahydrofuran (THF) as the solvent, the yield is increased to 80%, but still requires nitrogen protection and reaction at 0 °C, and column chromatography is needed for separation, which is relatively complex in operation.

[0012] The literature "Galactose conjugated boron dipyrromethene and hydrogen bonding promoted J-aggregates for efficiently targeted NIR-II fluorescence assistant photothermal therapy" also uses THF as the reaction solvent, DCC as the condensing agent, reacts under a nitrogen reaction atmosphere, filters to remove the filter cake, washes with diethyl ether, rotary evaporates to remove the solvent, and after column chromatography treatment with a PE / EA developing agent, S-4FB is obtained. The reaction conditions of this method are room temperature, and the yield reaches 90%. Although the product yield has been greatly improved, the process of nitrogen protection and column operation cannot be avoided.

[0013] The literature "Single-Step Conjugation of Bioactive Peptides to Proteins via a Self-Contained Succinimidyl Bis-Arylhydrazone" uses acetonitrile as the reaction solvent and EDAC as the condensing agent. After the reaction is completed, the solvent is removed, dissolved in dichloromethane, washed with water, dried, filtered, and the product is obtained with a yield of 91%. It is not only simple to operate but also has a high yield. The disadvantage is that the reaction solvent acetonitrile used is toxic. Summary of the Invention

[0014] Aiming at the technical problems existing in the above prior art, the purpose of the present invention is to overcome the above deficiencies and provide a synthesis method with high yield, mild reaction conditions, and simple operation.

[0015] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0016] The present invention provides a new synthesis method of N-succinimidyl 4-formylbenzoate, which comprises the following steps:

[0017] (1) Dissolve p-formylbenzoic acid, NHS, and EDAC in a reaction solvent, add a catalyst and react, and then remove the solvent under vacuum to obtain a solid reaction product;

[0018] (2) Post-treat and purify the solid reaction product to obtain the final product N-succinimidyl 4-formylbenzoate.

[0019] Further, in the new synthesis method of the present invention, the catalyst is one of N-methylpyrrolidone, N-methylmorpholine, N-methylpiperidine, tributylamine, and triethylamine.

[0020] Further, in the novel synthesis method of the present invention, the catalyst is N-methylpyrrolidone.

[0021] Further, in the novel synthesis method of the present invention, the mass-volume ratio of p-formylbenzoic acid to the catalyst is 100:(2 - 10) g / mL.

[0022] Further, in the novel synthesis method of the present invention, the mass-volume ratio of p-formylbenzoic acid to the catalyst is 100:5 g / mL.

[0023] Further, in the novel synthesis method of the present invention, the post-treatment purification steps are as follows: dissolve the reaction product in dichloromethane, wash the dichloromethane solution with water, then add a drying agent for drying; filter the drying agent and wash it with dichloromethane, combine the dichloromethane phases of the two operations before and after, filter again, and remove the solvent under vacuum to obtain the final product.

[0024] Further, in the novel synthesis method of the present invention, the reaction temperature is 20 - 25 °C.

[0025] Further, in the novel synthesis method of the present invention, the reaction time is 12 - 18 h.

[0026] Further, in the novel synthesis method of the present invention, the reaction time is 16 h.

[0027] Further, in the novel synthesis method of the present invention, the drying agent is anhydrous sodium sulfate.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention optimizes and screens conditions such as reaction solvent, condensing agent, and reaction temperature. It preferentially uses EDAC as the condensing agent, conducts the reaction at room temperature, and introduces a catalyst during the reaction. This not only reduces the generation of impurities but also significantly improves the reaction yield, further simplifies the post-treatment process of refining and purification, and eliminates the cumbersome column chromatography steps. Compared with the prior art, the present invention shows significant advantages in optimizing reaction conditions and applying catalysts, resulting in a substantial increase in yield, up to over 96% at most, providing a more efficient and convenient method for the production of S-4FB, which can meet the requirements of large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of succinimidyl 4-formylbenzoate. DETAILED DESCRIPTION OF THE INVENTION

[0031] To make the above objectives, features, and advantages of the present application more obvious and understandable, the following provides a detailed description of the specific implementation manners of the present application with reference to the accompanying drawings. Many specific details are set forth in the following description to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present application. Therefore, the present application is not limited by the specific embodiments disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0033] The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources without special instructions.

[0034] The following describes this application in detail with specific embodiments. These embodiments and comparative examples are for understanding rather than limiting this application.

[0035] Example 1

[0036] Dissolve 100 g of FBA, 84.25 g of NHS, and 140.5 g of EDAC in 2.5 L of dichloromethane solution, add 5 mL of N-methylpyrrolidone, and react overnight. After removing the solvent under vacuum, dissolve the obtained white solid in 1.2 L of dichloromethane, wash the dichloromethane solution with an appropriate amount of distilled water, and dry it with 500 g of anhydrous sodium sulfate. Filter, wash the solid with 3 × 400 mL of dichloromethane, combine the obtained dichloromethane phases, filter again, and remove the solvent under vacuum to obtain 158.4 g of white solid, and the product yield is 96.2%.

[0037] The test results of the nuclear magnetic resonance hydrogen spectrum are as Figure 1 shown, H-NMR(CDCl3-d6): δ2.94(s,4H), δ8.04(d,2H), δ8.31(d,2H), δ10.14(s,1H).

[0038] Example 2

[0039] According to the procedure of Example 1, use N-methylmorpholine as the catalyst, and finally obtain 148.0 g of white solid, with a yield of 89.9%. The H-NMR spectrum is the same as that of Example 1.

[0040] Example 3

[0041] According to the procedure of Example 1, use N-methylpiperidine as the catalyst, and finally obtain 151.0 g of white solid, with a yield of 91.7%. The H-NMR spectrum is the same as that of Example 1.

[0042] Example 4

[0043] According to the procedure of Example 1, tributylamine was used as the catalyst instead, and finally 154.0 g of white solid was obtained with a yield of 93.5%. The H-NMR spectrum was the same as that of Example 1.

[0044] Example 5

[0045] According to the procedure of Example 1, triethylamine was used as the catalyst instead, and finally 142.3 g of white solid was obtained with a yield of 86.4%. The H-NMR spectrum was the same as that of Example 1.

[0046] The above examples only represent several implementation manners of this application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those skilled in the art, without departing from the concept of this application, several modifications and improvements can still be made, and these all belong to the protection scope of this application. Therefore, the protection scope of this application patent shall be subject to the appended claims, and the specification and drawings can be used to explain the content of the claims.

Claims

1. A new method for synthesizing N-succinimidyl 4-formylbenzoate, characterized in that, It includes the following steps: (1) Dissolve p-formylbenzoic acid, NHS, and EDAC in a reaction solvent, add a catalyst and react, and then remove the solvent under vacuum to obtain a solid reaction product; (2) Post-treat and purify the solid reaction product to obtain the final product succinimidyl 4-formylbenzoate.

2. The novel synthesis method according to claim 1, characterized in that, The catalyst is one of N-methylpyrrolidone, N-methylmorpholine, N-methylpiperidine, tributylamine, and triethylamine.

3. The novel synthesis method according to claim 2, wherein, The catalyst is N-methylpyrrolidone.

4. The novel synthesis method according to claim 1, characterized in that, The mass-volume ratio of the p-formylbenzoic acid to the catalyst is 100:(2 - 10) g / mL.

5. The novel synthesis method according to claim 4, characterized in that, The mass-volume ratio of the p-formylbenzoic acid to the catalyst is 100:5 g / mL.

6. The novel synthesis method according to claim 1, wherein, The steps of the post-treatment purification are as follows: dissolve the reaction product in dichloromethane, wash the dichloromethane solution with water, and then add a drying agent for drying; filter the drying agent and wash it with dichloromethane, combine the dichloromethane phases of the two operations before and after, filter again, and remove the solvent under vacuum to obtain the final product.

7. The novel synthesis method according to claim 1, characterized in that, The reaction temperature is 20 - 25 °C.

8. The novel synthesis method according to claim 1, characterized in that, The reaction time is 12 - 18 h.

9. The novel synthesis method according to claim 8, wherein, The reaction time is 16 h.

10. The new synthesis method according to claim 6, characterized in that, The drying agent is anhydrous sodium sulfate.

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