A method for synthesizing L-γ-glutamyl-3-carboxyl-4-nitroaniline ammonium salt

Abstract

The invention discloses a method for synthesizing L-γ-glutamyl-3-carboxyl-4-nitroaniline ammonium salt, and relates to the technical field of organic synthesis. According to the invention, Glupa-C is synthesized by using 5-amino-2-nitrobenzoic acid and 3-nitrophthaloyl-L-glutamic anhydride as starting materials through a condensation reaction and a hydrazinolysis reaction under the catalytic action of a Lewis acid. Compared with traditional alkaline preparation conditions, the use of highly toxic reagents and the safety hazard of explosion during the preparation process are avoided, production risks are reduced, and energy consumption is lowered. In addition, the synthesis method provided by the invention can achieve a total yield of 47.8% and a purity of 99.96% for the product Glupa-C, and is suitable for industrial production.

Description

Technical field:

[0001] The present invention relates to the technical field of organic synthesis, and in particular to a method for synthesizing L-γ-glutamyl-3-carboxyl-4-nitroaniline ammonium salt. Background technology:

[0002] L-γ-glutamyl-3-carboxyl-4-nitroaniline ammonium salt (Glupa-C) is a good substrate for the determination of transpeptidase and is widely used in clinical tests.

[0003] U.S. Patent No. 3,979,447A discloses a method for preparing Glupa-C, which involves condensing 5-amino-2-nitrobenzoic acid with phthaloyl-L-glutamic anhydride in the presence of tri-n-butylamine. The resulting condensate is then subjected to a hydrazinolysis reaction to obtain the product Glupa-C. However, the patent does not provide detailed condensation and hydrazinolysis conditions, nor does it describe the purification method or quality standards for the product Glupa-C. Furthermore, tri-n-butylamine is a highly toxic substance, making it unsuitable for industrial scale-up.

[0004] Chinese patent CN1076688A discloses a method for synthesizing Glupa-C using 5-amino-2-nitrobenzoic acid and phthaloyl-L-glutamic anhydride as raw materials. The condensation reaction temperature is 140-142°C, and the hydrazinolysis reaction temperature is 40-50°C. In this method, the nitro compound (5-amino-2-nitrobenzoic acid) needs to undergo condensation at high temperatures, which is prone to explosion. Industrial production is energy-intensive and highly dangerous.

[0005] Chinese patent CN113045446A discloses a method for synthesizing Glupa-C using 5-amino-2-nitrobenzoic acid and phthaloyl-L-glutamic anhydride as raw materials. The condensation reaction solvent is an acidic solvent (formic acid or acetic acid), the condensation reaction temperature is 20-200°C, and the hydrazinolysis reaction temperature is 0-50°C. However, this method uses a large amount of acidic solvent, generates a large amount of wastewater and is highly polluting, while the overall yield is only 36.85%. Summary of the invention:

[0006] The technical problem to be solved by the present invention is to provide a method for synthesizing L-γ-glutamyl-3-carboxyl-4-nitroaniline ammonium salt, with the aim of increasing product yield while reducing energy consumption and improving the environmental protection and safety of the process.

[0007] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

[0008] A method for synthesizing L-γ-glutamyl-3-carboxyl-4-nitroaniline ammonium salt comprises the following steps:

[0009] (1) 5-amino-2-nitrobenzoic acid and 3-nitrophthaloyl-L-glutamic anhydride undergo condensation reaction under the catalysis of Lewis acid to obtain an intermediate;

[0010] (2) The intermediate undergoes hydrazine hydrolysis reaction with hydrazine hydrate and is ammonium radicalized under the action of anion exchange resin to obtain the target compound Glupa-C.

[0011] The Lewis acid is one or more of AlCl3, FeCl3, LiCl, and ZnCl2.

[0012] The molar ratio of the 5-amino-2-nitrobenzoic acid to the 3-nitrophthaloyl-L-glutamic anhydride is (0.9-1):(1-1.2).

[0013] The amount of the Lewis acid used is 0.1 to 200% of the mass of 3-nitrophthaloyl-L-glutamic anhydride.

[0014] The temperature of the condensation reaction is 0-100° C. The reaction progress is monitored by HPLC or LC-MS, and the complete disappearance of 3-nitrophthaloyl-L-glutamic anhydride is regarded as the reaction endpoint.

[0015] The solvent for the condensation reaction is one or more of methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, dichloromethane, chloroform, and toluene.

[0016] The usage ratio of the intermediate to hydrazine hydrate is 1g:(1-20)mL, and the hydrazine hydrate has a volume fraction of 28%.

[0017] The temperature of the hydrazinolysis reaction is 0-30° C. The reaction progress is monitored by HPLC, LC-MS or NMR, and the complete disappearance of the intermediate is regarded as the reaction endpoint.

[0018] The solvent for the hydrazinolysis reaction is one or more of methanol, ethanol, isopropanol, acetonitrile and tetrahydrofuran.

[0019] The specific synthetic route is as follows:

[0020]

[0021] The present invention has the beneficial effects of synthesizing Glupa-C using 5-amino-2-nitrobenzoic acid and 3-nitrophthaloyl-L-glutamic anhydride as starting materials through a condensation reaction and a hydrazinolysis reaction under the catalytic action of a Lewis acid. Compared with traditional alkaline preparation conditions, the present invention avoids the use of highly toxic reagents and the safety hazard of explosion during the preparation process, reduces production risks, and lowers energy consumption. In addition, the synthesis method provided by the present invention can achieve a total yield of 47.8% and a purity of 99.96% for the product Glupa-C, and is suitable for industrial production. Description of the drawings:

[0022] Figure 1 Glupa-C 1 H NMR spectra;

[0023] Figure 2 is the LC-MS chart of the product Glupa-C;

[0024] Figure 3 This is the HPLC chart of the product Glupa-C. Specific implementation method:

[0025] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific embodiments and illustrations.

[0026] Example 1

[0027] (1) Dichloromethane (600 mL), 5-amino-2-nitrobenzoic acid (91 g, 0.5 mol), 3-nitrophthaloyl-L-glutamic anhydride (143 g, 0.47 mol), and AlCl3 (31 g, 0.24 mol) were added to a 2 L reaction flask in sequence. The reaction solution was heated to 40°C and stirred for 6 h. HPLC analysis showed that the reaction of 3-nitrophthaloyl-L-glutamic anhydride was complete. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to obtain 220 g of a crude product. The crude product was recrystallized from methanol and water, filtered, and dried in vacuo to obtain 150.5 g of the intermediate with a yield of 65.9%.

[0028] (2) 20 g of the intermediate was dissolved in ethanol (300 mL) at 0°C, and then 28% hydrazine hydrate solution (25 mL) was slowly added dropwise. The mixture was stirred at room temperature overnight. HPLC analysis showed that the intermediate was completely reacted. The reaction solution was filtered, the filter cake was dissolved in water, and the pH value was adjusted to 8-10 with 1N aqueous ammonium carbonate. The resulting solution was ammoniumized on an anion resin column and the crude product was collected. The crude product was recrystallized from a ternary system of ethylene glycol, acetone, and water to obtain 9.8 g of Glupa-C as a white powder with a yield of 72.6% and an HPLC purity of 99.967%.

[0029] LC-MS: [M+H] + =312.1035. 1 HNMR(500MHz,DMSO-d6)δppm 2.02-2.07

[0030] (m,2H),2.51-2.65(m,2H),3.25-3.34(m,1H),7.02-7.70(brs,4H)7.72(m,1H),7.76(m,1H),7.81(m,1H)11.16(brs,1H).

[0031]

[0032] Example 2

[0033] To a 2L reaction flask, tetrahydrofuran (600 mL), 5-amino-2-nitrobenzoic acid (91 g, 0.5 mol), 3-nitrophthaloyl-L-glutamic anhydride (143 g, 0.47 mol), and AlCl₃ (31 g, 0.24 mol) were added sequentially. The reaction mixture was heated to 40°C and stirred for 6 hours. HPLC analysis showed that the 3-nitrophthaloyl-L-glutamic anhydride had reacted completely. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated to obtain 196 g of a crude product. The crude product was recrystallized from methanol and water, filtered, and dried under vacuum to obtain 138.7 g of the intermediate, with a yield of 58.2%.

[0034]

[0035] Example 3

[0036] To a 2L reaction flask, toluene (600 mL), 5-amino-2-nitrobenzoic acid (91 g, 0.5 mol), 3-nitrophthaloyl-L-glutamic anhydride (143 g, 0.47 mol), and AlCl₃ (31 g, 0.24 mol) were added sequentially. The reaction mixture was heated to 40°C and stirred for 6 hours. HPLC analysis showed that the 3-nitrophthaloyl-L-glutamic anhydride had reacted completely. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated to obtain 158 g of crude product. The crude product was recrystallized from methanol and water, filtered, and dried under vacuum to obtain 95.7 g of the intermediate, with a yield of 40.1%.

[0037]

[0038] Comparison of Example 1, Example 2, and Example 3 shows that the type of reaction solvent has a certain impact on the yield of the intermediate. Compared with tetrahydrofuran and toluene, dichloromethane is more suitable as a reaction solvent for the synthesis of intermediates.

[0039] Comparative Example 1

[0040] The raw material 3-nitrophthaloyl-L-glutamic anhydride used to synthesize the intermediate in Example 1 was replaced with the same molar amount of phthaloyl-L-glutamic anhydride to obtain Control Example 1.

[0041] To a 2L reaction flask, dichloromethane (600 mL), 5-amino-2-nitrobenzoic acid (91 g, 0.5 mol), phthaloyl-L-glutamic anhydride (122 g, 0.47 mol), and AlCl₃ (31 g, 0.24 mol) were added sequentially. The reaction mixture was heated to 40°C and stirred overnight. HPLC analysis showed that the phthaloyl-L-glutamic anhydride was not completely reacted. The reaction mixture was concentrated and purified by column chromatography to obtain 35 g of (S)-5-(4-carboxy-4-(1,3-dioxoisoindolin-2-yl)butyramido)-2-nitrobenzoic acid in a yield of 14.7%.

[0042]

[0043] Comparison of Example 1 with Control Example 1 shows that, compared with phthaloyl-L-glutamic anhydride, the use of 3-nitrophthaloyl-L-glutamic anhydride as the reaction raw material of the intermediate is beneficial to increasing the yield of the intermediate, and the increase in the yield of the intermediate can indirectly increase the yield of the product Glupa-C.

[0044] Comparative Example 2

[0045] The catalyst AlCl3 used in the synthesis of the intermediate in Example 1 was deleted to obtain Control Example 2.

[0046] To a 2L reaction flask, dichloromethane (600 mL), 5-amino-2-nitrobenzoic acid (91 g, 0.5 mol), and 3-nitrophthaloyl-L-glutamic anhydride (143 g, 0.47 mol) were added sequentially. The reaction mixture was heated to 40°C and stirred overnight. HPLC analysis showed that the 3-nitrophthaloyl-L-glutamic anhydride was not completely reacted. The reaction mixture was concentrated and purified by column chromatography to obtain 30 g of the intermediate in a yield of 12.6%.

[0047]

[0048] Comparing Example 1 with Control Example 2, it can be seen that using AlCl3 as a catalyst for intermediate synthesis is beneficial to improving the yield of the intermediate.

[0049] Comparative Example 3

[0050] The catalyst AlCl3 used to synthesize the intermediate in Example 1 was replaced by the same molar amount of boron trifluoride to obtain Control Example 3.

[0051] To a 2L reaction flask, dichloromethane (600 mL), 5-amino-2-nitrobenzoic acid (91 g, 0.5 mol), 3-nitrophthaloyl-L-glutamic anhydride (143 g, 0.47 mol), and 80 mL of boron trifluoride etherate were added sequentially. The reaction mixture was heated to 40°C and stirred overnight. HPLC analysis showed that the 3-nitrophthaloyl-L-glutamic anhydride was not completely reacted. The reaction mixture was concentrated and purified by column chromatography to obtain 78 g of the intermediate in a yield of 32.8%.

[0052]

[0053] Comparing Example 1 with Control Example 3, it can be seen that, compared with boron trifluoride, the use of AlCl3 as a catalyst for the synthesis of the intermediate is beneficial to improving the yield of the intermediate.

[0054] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for synthesizing L-γ-glutamyl-3-carboxyl-4-nitroaniline ammonium salt, characterized in that: The following steps are involved: (1) 5-amino-2-nitrobenzoic acid and 3-nitrophthaloyl-L-glutamic anhydride undergo condensation reaction under the catalysis of Lewis acid to obtain an intermediate; The Lewis acid is AlCl3; the solvent for the condensation reaction is dichloromethane; (2) The intermediate undergoes hydrazine hydrolysis reaction with hydrazine hydrate and is ammonium radicalized under the action of anion exchange resin to obtain the target compound Glupa-C; The specific synthetic route is as follows:

2. The synthesis method according to claim 1, wherein: The molar ratio of the 5-amino-2-nitrobenzoic acid to the 3-nitrophthaloyl-L-glutamic anhydride is (0.9-1):(1-1.2).

3. The synthesis method according to claim 1, wherein: The amount of the Lewis acid used is 0.1 to 200% of the mass of 3-nitrophthaloyl-L-glutamic anhydride.

4. The synthesis method according to claim 1, wherein: The temperature of the condensation reaction is 0-100°C.

5. The synthesis method according to claim 1, wherein: The usage ratio of the intermediate to hydrazine hydrate is 1g:(1-20)mL, and the hydrazine hydrate has a volume fraction of 28%.

6. The synthesis method according to claim 1, wherein: The temperature of the hydrazinolysis reaction is 0-30°C.

7. The synthesis method according to claim 1, wherein: The solvent for the hydrazinolysis reaction is one or more of methanol, ethanol, isopropanol, acetonitrile and tetrahydrofuran.

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