Preparation method of stable isotope labeled pencycuron internal standard reagent

Abstract

The invention relates to a preparation method of a stable isotope labeled pencycuron internal standard reagent for food safety detection, and belongs to the field of research and development of standard substances for food safety detection. According to the preparation method of the stable isotope labeled pencycuron internal standard reagent, commercially available starting raw materials and stable isotope labeled halogenated benzene are adopted as raw materials, and stable isotope labeled pencycuron is obtained through two-step reaction. The method has the advantages of easily available raw materials, simple operation, no use of highly toxic intermediates, mild reaction conditions, short reaction route, high final product total yield, easy separation and purification, and no dilution of isotope abundance. The chemical purity of the final product stable isotope labeled pencycuron is greater than 98%, and the isotope abundance is greater than 99%. The stable isotope labeled pencycuron internal standard reagent prepared by the preparation method can meet the technical requirements of the stable isotope labeled internal standard reagent required by a liquid chromatography-tandem mass spectrometry method for food safety detection.

Description

Technical Field

[0001] This invention relates to a method for preparing a stable isotope-labeled pendimethalin internal standard reagent for food safety testing, belonging to the field of research and development of standard substances for food safety testing. Background Technology

[0002] Pencycuron is a urea fungicide jointly developed by Nippon Specialty Pesticides Co., Ltd. and Bayer AG of Germany. It is non-systemic and possesses good protective and persistent activity. It strongly inhibits mycelial growth, resulting in morphological disruption during mycelial development. It can be used to control various crop damping-off and seed-borne diseases caused by Rhizoctonia solani, such as Rhizoctonia solani on rice, leaf rot and root rot in beets, Rhizoctonia solani on potatoes, rice sheath blight, Juncus effusus stem rot, and black scurf in potatoes. In recent years, the use of pencycuron in the production of plant-derived foods such as rice and tea has been increasing. Because it is sprayed directly on crops and enters the human body through consumption, excessive residues inevitably pose a health hazard. The United States, Japan, and the European Union have successively set maximum residue limits for pencycuron in rice, and the European Union officially implemented new pesticide residue limits for imported tea in 2014. The relevant standards for detecting this substance in my country include: GB 23200.121-2021 National Food Safety Standard for the Determination of Residues of 331 Pesticides and Their Metabolites in Plant-Derived Foods by Liquid Chromatography-Mass Spectrometry; GB 23200.13-2016 National Food Safety Standard for the Determination of Residues of 448 Pesticides and Related Chemicals in Tea by Liquid Chromatography-Mass Spectrometry; and GB 23200.34-2016 National Food Safety Standard for the Determination of Residues of 65 Pesticides, Including Aldisulfiram, Pyraclostrobin, and Azoxystrobin, in Food by Liquid Chromatography-Mass Spectrometry / Mass Spectrometry.

[0003] The aforementioned detection standards all employ external standard methods for quantitative detection. Stable isotope dilution mass spectrometry is internationally recognized as the "gold standard" for trace detection, significantly reducing matrix effects and improving the accuracy of detection results. Using stable isotope-labeled pendimethalin with internal standard methods for quantitative detection will significantly improve the accuracy of detection results and lower the detection limit of this compound. Therefore, the development of stable isotope-labeled pendimethalin internal standard reagents is of great significance for improving my country's food safety supervision and testing capabilities.

[0004] Currently, the existing synthetic route for compounds with this natural abundance is as follows: Zhang Zhenming, Jia Yonggang, Wang Canming. Synthesis of urea fungicide pendimethalin. Pesticides, 2003, 42(11): 19-20. Pendimethalin is prepared by reacting phenyl isocyanate and N-(4-chlorobenzyl)cyclopentanamine as raw materials.

[0005] The existing synthetic route for stable isotope-labeled pentylene is as follows: Isao Ueyama, Shin Kurogochi, Itsuroh Kobori et al. Use of Ion Cluster Analysis in a Metabolic Study of Pencycuron, a Phenylurea Fungicide, in Rabbits[J]. Journal of Agriculture and Food Chemistry, 1982, 30(6): 1061-1067. The synthetic method of pentylene-D5 reported in the article is also to prepare pentylene-D5 by reacting phenyl isocyanate-D5 and N-(4-chlorobenzyl)cyclopentanamine as raw materials.

[0006] All of the above routes use highly toxic phenyl isocyanates as raw materials, therefore this route is not suitable for synthesizing stable isotope-labeled pendimethalin internal standard reagents.

[0007] Therefore, there is an urgent need to develop a method for preparing a stable isotope-labeled pendimethalin internal standard reagent that uses readily available raw materials, is simple to operate, avoids the use of highly toxic raw materials, has mild reaction conditions, a short reaction route, a high total yield of the final product, is easy to separate and purify, and has no dilution of isotope abundance, in order to meet the growing application demand of this compound in domestic food safety supervision and regulation. Summary of the Invention

[0008] The purpose of this invention is to provide a preparation route and method for a stable isotope-labeled pendimethalin internal standard reagent suitable for food safety testing. This preparation route and method uses readily available raw materials, is simple to operate, avoids the use of highly toxic intermediates, employs mild reaction conditions, has a short reaction route, and results in a high overall yield of the final product that is easy to separate and purify, without dilution of isotope abundance.

[0009] The objective of this invention can be achieved through the following technical solutions: A method for preparing a stable isotope-labeled pendimethalin internal standard reagent, the chemical structure of which is shown in the following formula:

[0010] Where * represents 12 C or 13 C and X are H or D, characterized in that its preparation route is as follows:

[0011] The preparation steps are as follows: (1) Add cyanate and catalyst to water, then add N-(4-chlorobenzyl)cyclopentanamine, and react for 1 to 12 hours at 20℃ to 100℃. After the reaction of the raw materials is complete by TLC, the reaction mixture is extracted with organic solvent, the organic phases are combined, and washed successively with water, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The mixture is dried with desiccant, and the solvent is removed by vacuum distillation. The residue is then subjected to column chromatography to obtain 1-(4-chlorobenzyl)-1-cyclohexylurea (II). The reaction in this step is shown below: ;

[0012] In step (1), the catalyst is selected from hydrochloric acid, sulfuric acid, and 4-dodecylbenzenesulfonic acid; the cyanate is selected from sodium cyanate, potassium cyanate, and ammonium cyanate; the molar ratio of compound (I) to catalyst in step (1) is (1:1) to (1:3), the molar ratio of compound (I) to cyanate is (1:1.5) to (1:5), the reaction temperature is 20℃ to 100℃, and the reaction time is 1 to 12 hours.

[0013] (2) In the presence of catalyst, ligand, base and solvent, 1-(4-chlorobenzyl)-1-cyclohexylurea (II) was added, followed by stable isotope-labeled halobenzene (III). The reaction was carried out at 30℃~150℃ for 6~48 hours. After the reaction of the raw materials was completed by TLC, the reaction mixture was quenched with water, filtered, and the filtrate was extracted with organic solvent. The organic phases were combined and washed successively with water, saturated sodium bicarbonate solution and saturated sodium chloride solution. The mixture was dried with desiccant and the solvent was removed by vacuum distillation. The residue was subjected to column chromatography to obtain stable isotope-labeled pendimethalin. The reaction in this step is shown below:

[0014] The catalyst in step (2) is selected from tris(dibenzylacetone)palladium, bis(dibenzylacetone)palladium, palladium acetate, tetra(triphenylphosphine)palladium, bis(diphenylphosphine ferrocene)palladium dichloride or bis(triphenylphosphine)palladium dichloride; the ligand is selected from tris(tert-butyl)phosphine, 1,1'-binaphthyl-2,2'-bisdiphenylphosphine, 4,5-bisdiphenylphosphine-9,9-dimethyloxanthracene, 2-dicyclohexylphosphine-2'-(N,N-dimethylamine)-biphenyl, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl or 1,3-bis(diphenylphosphine)propane; the base is selected from sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium tert-butoxide or potassium tert-butoxide; the organic solvent is selected from N,N-dimethylformamide, dioxane, tetrahydrofuran or toluene.

[0015] In step (2), the molar ratio of compound (III) to catalyst is (1:0.001) to (1:0.05), the molar ratio of compound (III) to ligand is (1:0.005) to (1:0.1), the molar ratio of compound (III) to base is (1:1) to (1:4), and the molar ratio of compound (III) to 1-(4-chlorobenzyl)-1-cyclohexylurea (II) is (1:1 to 1:1.5); the reaction temperature is 80℃ to 120℃, and the reaction time is 12 to 24 hours.

[0016] Compared with existing technologies, the advantages of this invention are as follows: The preparation method of the stable isotope-labeled pendimethalin internal standard reagent for food safety testing uses commercially available intermediates and stable isotope-labeled halobenzenes as raw materials, directly obtaining stable isotope-labeled pendimethalin through a two-step reaction. The raw materials used in this method are simple, readily available, and inexpensive; it avoids the use of highly toxic phenyl isocyanates as intermediates; the reaction conditions are mild; and the reaction yield is high. It also has advantages such as fewer byproducts in the final product, ease of separation and purification, high chemical purity, and no dilution of isotope abundance. The preparation route and method of this stable isotope-labeled pendimethalin internal standard reagent have not been reported in the literature. The stable isotope-labeled pendimethalin prepared by this method has a chemical purity greater than 98% and an isotope abundance greater than 99%. The stable isotope-labeled pendimethalin internal standard reagent for food safety testing prepared by this method is of great significance to the development of related food safety supervision and testing fields in my country. Attached Figure Description Figure 1 : 1-(4-chlorobenzyl)-1-cyclohexylurea 1 H NMR image Figure 2 LC-MS chromatogram of 1-(4-chlorobenzyl)-1-cyclohexylurea Figure 3 Pendimethalin (phenyl-D5) 1 H NMR image Figure 4 Pendimethalin (phenyl-D5) 13 C NMR spectrum Figure 5 LC-MS chromatogram of pendimethalin (phenyl-D5) Figure 6 HPLC chromatogram of pendimethalin (phenyl-D5) Detailed Implementation

[0017] The present invention will be further described below with reference to the embodiments and accompanying drawings, but this is not intended to limit the scope of the invention.

[0018] Example 1: Preparation of 1-(4-chlorobenzyl)-1-cyclohexylurea

[0019] In a 25 mL three-necked flask, N-(4-chlorobenzyl)cyclopentanamine (419 mg, 2 mmol), 1 M HCl (3 mL, 3 mmol), and potassium cyanate (356 mg, 4.4 mmol) were added. The reaction was carried out at 25 °C for 12 hours. After the reaction was completed by TLC, the reaction solution was cooled to room temperature, 20 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined and washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution. The mixture was dried with anhydrous sodium sulfate, the drying agent was removed by filtration, and the solvent was removed by vacuum distillation. The residue was subjected to column chromatography to obtain 1-(4-chlorobenzyl)-1-cyclohexylurea (II) (450 mg, yield 89%). 1 ¹H NMR (DMSO-D6, 600MHz): δ 7.35 (d, 2H), 7.21 (d, 2H), 7.25 (d, 2H), 5.90 (s, 2H), 4.35 (s, 2H), 4.31–4.28 (m, 1H), 1.68–1.29 (m, 8H), see appendix. Figure 1 LC-MS (ESI+) 253 [M+H] + See appendix Figure 2 .

[0020] Example 2: Synthesis of stable isotope-labeled pendimethalin (phenyl-D5)

[0021] In a 100 mL three-necked flask under nitrogen protection, deuterated bromobenzene (810 mg, 5 mmol), bis(dibenzylacetone)palladium (86 mg, 0.15 mmol), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (187 mg, 0.3 mmol), sodium tert-butoxide (671 mg, 7 mmol), 1-(4-chlorobenzyl)-1-cyclohexylurea (1.38 g, 5.5 mmol), and toluene (20 mL) were added. The reaction was carried out at 100 °C for 12 hours. After the reaction proceeds were completely controlled by TLC, the reaction solution was cooled to room temperature, 20 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined and washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution. The mixture was dried with anhydrous sodium sulfate, the drying agent was removed by filtration, and the solvent was removed by vacuum distillation. The residue was subjected to column chromatography to obtain stable isotope-labeled pendimethalin (phenyl-D5) (1.47 g, yield 88%).

[0022] Example 3: Synthesis of stable isotope-labeled pendimethalin (phenyl-D5) In a 100 mL three-necked flask under nitrogen protection, deuterated bromobenzene (810 mg, 5 mmol), tris(dibenzylacetone)palladium (92 mg, 0.1 mmol), 2-dicyclohexylphosphine-2'-(N,N-dimethylamine)-biphenyl (59 mg, 0.15 mmol), cesium carbonate (3.25 g, 10 mmol), 1-(4-chlorobenzyl)-1-cyclohexylurea (1.52 g, 6 mmol), and dioxane (20 mL) were added. L); The reaction was carried out at 80℃ for 20 hours. After the reaction was completed by TLC, the reaction solution was cooled to room temperature, 20 mL of water was added, and the mixture was extracted with ethyl acetate (3×20 mL). The organic phases were combined and washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution. The mixture was dried with anhydrous sodium sulfate, the desiccant was removed by filtration, the solvent was removed by vacuum distillation, and the residue was purified by column chromatography to obtain stable isotope-labeled pendimethalin (phenyl-D5) (1.52 g, yield 91%). 1 ¹H NMR (DMSO-D6, 600MHz): δ 8.34 (s, 1H), 7.37 (d, 2H), 7.25 (d, 2H), 4.60–4.54 (m, 1H), 4.52 (s, 2H), 1.76–1.39 (m, 8H), see appendix. Figure 3 ; 13 C NMR (150MHz): δ 155.92, 140.74, 140.09, 131.27, 128.56, 128.14, 120.15, 119.83, 57.17, 45.01, 29.64, 23.53, see appendix Figure 4 LC-MS (ESI+) 334 [M+H] + See appendix Figure 3 HPLC analysis showed a chemical purity of 98.86% (see attached document). Figure 5 Isotope abundance = 99.5%, see Table 1.

[0023] Table 1. Isotopic abundance of pendimethalin (phenyl-D5).

[0024] The quality standards meet the quality requirements of stable isotope-labeled internal standard reagents required for liquid chromatography-tandem mass spectrometry (LC-MS / MS) for food safety testing.

[0025] 13 The synthetic route and process for C-labeled stable isotope-labeled pendimethalin are the same as those in the above embodiments.

[0026] The above description is merely a preferred embodiment of the present invention, but the present invention should not be limited to the content disclosed in this embodiment. Therefore, any equivalent or modified versions made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

Claims

1. A method for preparing a stable isotope-labeled pendimethalin internal standard reagent, the chemical structure of which is shown in the following formula: ； Where * represents 12 C or 13 C, X are H or D, characterized in that... Its preparation route is as follows: ； The preparation steps are as follows: (1) Add cyanate and catalyst to water, then add N-(4-chlorobenzyl)cyclopentane (I), and react for 1 to 12 hours at 20℃ to 100℃. After the reaction of the raw materials is complete, the reaction mixture is extracted with organic solvent by TLC, the organic phases are combined, washed with water, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, dried with desiccant, and the solvent is removed by vacuum distillation. The residue is then subjected to column chromatography to obtain 1-(4-chlorobenzyl)-1-cyclohexylurea (II). The reaction in this step is shown below: ; (2) In the presence of catalyst, ligand, base and solvent, 1-(4-chlorobenzyl)-1-cyclohexylurea (II) was added, followed by stable isotope-labeled halobenzene (III). The reaction was carried out at 30℃~150℃ for 6~48 hours. After the reaction of the raw materials was completed by TLC, the reaction mixture was quenched with water, filtered, and the filtrate was extracted with organic solvent. The organic phases were combined and washed successively with water, saturated sodium bicarbonate solution and saturated sodium chloride solution. The mixture was dried with desiccant and the solvent was removed by vacuum distillation. The residue was subjected to column chromatography to obtain stable isotope-labeled pendimethalin. The reaction in this step is shown below: ； In step (1), the catalyst is selected from hydrochloric acid, sulfuric acid, and 4-dodecylbenzenesulfonic acid; the cyanate is selected from sodium cyanate, potassium cyanate, and ammonium cyanate. The catalyst in step (2) is selected from tris(dibenzylacetone)palladium, bis(dibenzylacetone)palladium, palladium acetate, tetra(triphenylphosphine)palladium, bis(diphenylphosphine ferrocene)palladium dichloride or bis(triphenylphosphine)palladium dichloride; the ligand is selected from tris(tert-butyl)phosphine, 1,1'-binaphthyl-2,2'-bisdiphenylphosphine, 4,5-bisdiphenylphosphine-9,9-dimethyloxanthracene, 2-dicyclohexylphosphine-2'-(N,N-dimethylamine)-biphenyl, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl or 1,3-bis(diphenylphosphine)propane; the base is selected from sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium tert-butoxide or potassium tert-butoxide; the organic solvent is selected from N,N-dimethylformamide, dioxane, tetrahydrofuran or toluene.

2. The method for preparing a stable isotope-labeled pendimethalin internal standard reagent according to claim 1, characterized in that: In step (1), the molar ratio of compound (I) to catalyst is (1:1) to (1:3), the molar ratio of compound (I) to cyanate is (1:1.5) to (1:5), the reaction temperature is 20℃ to 100℃, and the reaction time is 1 to 12 hours.

3. The method for preparing a stable isotope-labeled pendimethalin internal standard reagent according to claim 1, characterized in that: In step (2), the molar ratio of compound (III) to catalyst is (1:0.001) ~ (1: 0.05), the molar ratio of compound (III) to ligand is (1:0.005) ~ (1: The molar ratio of compound (III) to base is (1:1) to (1:4), and the molar ratio of compound (III) to 1-(4-chlorobenzyl)-1-cyclohexylurea (II) is (1:1 to 1:1.5); the reaction temperature is 80℃ to 120℃, and the reaction time is 12 to 24 hours.

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