A method for the synthesis of a stable isotope labeled ornipressin

Abstract

A method for synthesizing a stable isotope-labeled toltrazuril-D3 belongs to the field of organic synthesis technology. This method uses methylamine hydrochloride-D3 as the stable isotope labeling source, reacting it with p-trifluoromethylthiophenol and 1-chloro-2-methyl-5-nitrobenzene as starting materials, undergoing sequential condensation and reduction to obtain an aminodiphenyl ether intermediate. Simultaneously, methylamine hydrochloride-D3 reacts with sodium cyanate to generate methylurea-D3, which then reacts with carbonyl diimidazole to obtain an activated intermediate. Finally, the aminodiphenyl ether intermediate condenses with the activated intermediate, followed by base-catalyzed cyclization to obtain the target product, toltrazuril-D3. This invention features a rationally designed synthetic route, readily available raw materials, mild reaction conditions, simple operation, and easy product separation and purification. The obtained product achieves a chemical purity and isotope abundance of over 99%.

Description

Technical Field

[0001] A method for synthesizing stable isotope-labeled toltrazuril-D3 belongs to the field of organic synthesis technology. Background Technology

[0002] Toltrazuril is a chemically synthesized triazine ketone anticoccidial drug widely used in poultry farming to prevent and treat coccidiosis. Toltrazuril is almost completely metabolized in animals to its sulfone compound, toltrazuril sulfone, which is the final metabolite and residual marker of toltrazuril and still retains its antigenic biological activity.

[0003] Stable isotope dilution mass spectrometry is currently recognized internationally as the "gold standard" for trace substance detection. This method uses a stable isotope-labeled compound (such as a deuterated label) corresponding to the analyte as an internal standard, effectively correcting for matrix effects and recovery differences during sample pretreatment and instrumental analysis. This significantly improves the precision and accuracy of the detection results and lowers the method's detection limit. Therefore, developing a stable isotope-labeled toltrazuril-D3 internal standard is of vital importance for establishing a highly sensitive method for detecting toltrazuril residues and strengthening my country's food safety regulatory capabilities.

[0004] The toltrazuril synthesis processes reported in the literature currently have significant drawbacks. Existing process routes can be mainly divided into three categories: The first category is relatively long, requiring expensive and high-pressure (e.g., 9 MPa) Renny nickel catalysts for the catalytic hydrogenation step, and highly toxic phosgene in the critical isocyanation step, resulting in harsh process conditions and high safety risks; the second category, although shorter, requires difficult-to-obtain key raw materials (such as methyl isocyanate and chlorocarbonyl isocyanate), and its production also relies on highly toxic phosgene; the third category, while showing some improvement in seeking "green" preparation methods, still requires the preparation of methyl isocyanate or similar highly reactive intermediates in its core steps. This existing process route requires the preparation of isocyanates, which are too reactive and unstable, easily reacting with substrates to produce byproducts, leading to low yields and low abundance. These inherent process defects not only restrict the large-scale and safe production of ordinary toltrazuril, but also make it difficult to directly apply to the synthesis of stable isotope-labeled compounds (such as toltrazuril-D3) that have extremely high requirements for reaction conditions, intermediate stability, and product purity. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a method for synthesizing a stable isotope labeling toltrazuril-D3 with a simple synthetic route, high product purity and isotope abundance, controllable cost, and suitability for industrial production.

[0006] The technical solution adopted by this invention to solve its technical problem is: a method for synthesizing a stable isotope-labeled toltrazuril-D3, the synthetic route of which is as follows: .

[0007] Specifically, it includes the following steps: (1) Condensation reaction: using p-trifluoromethylthiophenol and 1-chloro-2-methyl-5-nitrobenzene as raw materials, amino diphenyl ether intermediate was prepared by condensation and reduction reaction; Reduction reaction: the obtained nitrobenzene intermediate was dissolved in ethyl acetate, tin chloride dihydrate was added, and the mixture was heated to reflux for reduction reaction. After the reaction was completed, the amino diphenyl ether intermediate was obtained by reduction post-treatment. (2) Preparation of methylurea: Methylamine hydrochloride-D3 and sodium cyanate were used as raw materials and heated under reflux in deuterium water. After the reaction was completed, methylurea-D3 was obtained by post-treatment. (3) Preparation of activated intermediate: Dissolve the methylurea-D3 obtained in step (2) in toluene, add pyridine, and add a toluene solution of carbonyl diimidazole dropwise under heating and stirring. Continue the reaction and obtain the activated intermediate after post-treatment after the reaction is completed. (4) Formation of biuret: The aminodiphenyl ether intermediate obtained in step (1) is dissolved in N,N-dimethylformamide, the activated intermediate obtained in step (3) is added, the reaction is heated, and the deuterated biuret intermediate is obtained after the reaction is completed. (5) Cyclization reaction: The deuterated biuret intermediate obtained in step (4) is dissolved in dimethyl carbonate, dry sodium methoxide is added, and the reaction is heated to generate toltrazuril-D3. After the reaction is completed, the final product is obtained by post-treatment and purification.

[0008] This invention provides a novel synthetic route for stable isotope-labeled toltrazuril-D3. The method uses readily available methylamine hydrochloride-D3 as the stable isotope source, and through multiple steps including condensation, reduction, urea formation, activation, condensation, and cyclization with p-trifluoromethylthiophenol and 1-chloro-2-methyl-5-nitrobenzene as starting materials, the target product is efficiently synthesized. The entire process uses readily available raw materials, mild reaction conditions, simple operation steps, and the product is easy to separate and purify. The obtained toltrazuril-D3 product has a chemical purity and isotope abundance of over 99%, exhibiting excellent quality and fully meeting the high requirements for internal standard reagents in trace residue detection using liquid chromatography-tandem mass spectrometry. This method successfully breaks the foreign technological monopoly, providing a stable, reliable, and cost-effective source of internal standard material for food safety testing, and has significant industrial application value.

[0009] Preferably, in step (1), p-trifluoromethylthiophenol and 1-chloro-2-methyl-5-nitrobenzene are used as reactants. The reaction is carried out in the presence of potassium carbonate in N,N-dimethylformamide solvent under nitrogen protection and heating. After the reaction, a nitrodiphenyl ether intermediate is obtained through post-processing. Specifically, the molar ratio of p-trifluoromethylthiophenol to 1-chloro-2-methyl-5-nitrobenzene is 1.3~1.4:1, the condensation and reduction reaction temperature is 130℃~140℃, and the reaction time is monitored by TLC until the reaction is complete. By precisely controlling the reactant ratio and reaction temperature, the reaction equilibrium is effectively shifted to the right, significantly improving the reaction rate and reactant conversion rate, and suppressing the formation of by-products. Combined with real-time TLC monitoring, the reaction endpoint is accurately determined, thereby simplifying the post-processing procedure and improving the stability and repeatability of the process while ensuring high yield.

[0010] Preferably, step (1) of the condensation reaction also includes a post-condensation treatment step: the reaction solution is poured into water, extracted with petroleum ether, and the organic phase is dried, filtered, and concentrated. The crude product obtained is then purified by silica gel column chromatography, with petroleum ether as the eluent. This post-treatment process efficiently separates and removes residual polar solvent DMF and water-soluble impurities from the reaction through water precipitation and petroleum ether extraction. It is simple to operate and low in cost. Subsequent column chromatography with petroleum ether can selectively separate and obtain high-purity nitrodiphenyl ether intermediates, effectively removing byproducts and raw materials, laying a solid foundation for the smooth progress of subsequent steps, and significantly improving the overall purification efficiency and yield of the product.

[0011] Preferably, in step (1), the molar ratio of the nitrodiphenyl ether intermediate to tin chloride dihydrate is 1:4.4~4.6. This preferred molar ratio ensures a large excess of reducing agent, which can drive the nitro reduction reaction to proceed completely, effectively suppress any possible side reactions in the reaction system, thereby significantly improving the yield and chemical purity of the aminodiphenyl ether intermediate, and providing high-quality raw materials for the subsequent key cyclization steps.

[0012] Preferably, the post-reduction treatment in step (1) includes: concentrating to remove the solvent, adding water, adjusting the pH to alkaline with sodium hydroxide aqueous solution, and then extracting with ethyl acetate. The organic phase is then concentrated to obtain the aminodiphenyl ether intermediate. This post-reduction treatment effectively dissolves and removes excess tin salt under alkaline conditions, avoiding impurity contamination. Subsequent extraction with ethyl acetate can efficiently separate the product, while maintaining a clear operating interface, effectively avoiding emulsion formation, simplifying the separation process, and significantly improving the recovery efficiency and purity of the amino intermediate.

[0013] Preferably, the molar ratio of methylurea-D3 to carbonyl diimidazole in step (3) is 1:1.09~1.11, and the reaction temperature is 50℃~60℃. These preferred conditions, by controlling a slight excess of carbonyl diimidazole, ensure the efficient and complete activation reaction of methylurea-D3, maximizing its conversion into the target activation intermediate and effectively reducing interference from unreacted raw materials in subsequent steps. Simultaneously, the suitable reaction temperature guarantees a sufficient reaction rate while avoiding the decomposition of the active intermediate that may occur due to excessively high temperatures, thereby significantly improving the yield and product stability of the activation step.

[0014] Preferably, the post-reaction treatment in step (3) includes: after cooling the reaction solution, adding methanol, filtering and collecting the precipitated solid to obtain the activated intermediate. This post-reaction treatment method uses methanol as the precipitation solvent, which enables the activated intermediate to precipitate from the reaction system efficiently and selectively, and the operation is extremely simple. This method eliminates the need for cumbersome extraction and concentration steps, which not only significantly shortens the processing time and reduces the use and consumption of organic solvents, but also effectively removes residual imidazole and other lipid-soluble impurities, thereby obtaining a high-purity solid product with a high yield, ensuring its stability and reactivity in subsequent condensation reactions.

[0015] Preferably, the molar ratio of the aminodiphenyl ether intermediate to the activated intermediate in step (4) is 1:1.06~1.08, and the reaction temperature is 75℃~85℃. These preferred conditions, by slightly exceeding the activated intermediate, ensure sufficient reaction of the amino group and effectively reduce raw material residue. Simultaneously, the suitable reaction temperature of 75-85℃ guarantees a sufficient reaction rate while avoiding the formation of byproducts, thus efficiently and with high yield, obtaining a high-purity deuterated biuret intermediate, laying a solid foundation for the final cyclization reaction.

[0016] Preferably, the molar ratio of the deuterated biuret intermediate to sodium methoxide in step (5) is 1:2.8~3.1, and the reaction temperature is 80℃~90℃. These preferred conditions ensure a sufficient excess of the strong base sodium methoxide, providing ample basicity for the cyclization reaction, while the reaction temperature effectively promotes the intramolecular cyclization reaction. This combination ensures high reaction efficiency while maximally suppressing side reactions, ultimately yielding a high-quality toltrazuril-D3 product with a chemical purity and isotopic abundance exceeding 99%.

[0017] Preferably, the purification method in step (5) is as follows: after the reaction solution is filtered and concentrated, the crude product is purified by silica gel column chromatography using a mixed solvent of petroleum ether and ethyl acetate as the eluent. Gradient elution is performed, the product fraction is collected, concentrated, and then recrystallized using a mixed solvent of petroleum ether and ethyl acetate. This purification method achieves deep purification of the final product through the combination of column chromatography and recrystallization. Gradient elution can effectively separate impurities with similar structures and polarities, while subsequent recrystallization further refines the product, ultimately ensuring that the obtained toltrazuril-D3 product has extremely high chemical purity and isotopic abundance, fully meeting the quality requirements of trace analysis internal standard reagents.

[0018] Compared with existing technologies, the beneficial effects of the synthetic method for stable isotope-labeled toltrazuril-D3 of this invention are as follows: This invention successfully designed and optimized a novel synthetic route for toltrazuril-D3 using methylamine hydrochloride-D3 as the isotope source. This route abandons traditional strategies that may start from expensive and complex isotope raw materials or have lengthy routes. Through ingenious reaction design, especially the preparation of key activation intermediates and efficient condensation and cyclization strategies, the synthetic route is significantly simplified. The reaction conditions at each step are mild and controllable, and the selected raw materials are inexpensive and readily available. By precisely optimizing the molar ratio of materials, reaction temperature, and post-processing purification, side reactions are effectively suppressed, resulting in higher reaction conversion rates and purer products. The final product not only has a chemical purity exceeding 99%, but more importantly, its isotope abundance also stably reaches over 99%, far exceeding the quality of imported similar products. This method is suitable for large-scale production, providing a stable, reliable, and high-quality internal standard reagent for food safety testing in my country. Attached Figure Description

[0019] Figure 1 The chromatogram of toltrazuril-D3 prepared in Example 1 of this invention at a wavelength of 254 nm is shown.

[0020] Figure 2 The image shows the electrospray ionization mass spectrum of the toltrazuril-D3 sample prepared in Example 1 of this invention.

[0021] Figure 3 The image shows the hydrogen nuclear magnetic resonance spectrum of toltrazuril-D3 prepared in Example 1 of this invention. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, wherein Embodiment 1 is the preferred embodiment.

[0023] Example 1 Under nitrogen protection, 53.6 g (0.276 mol) of p-trifluoromethylthiophenol, 34.3 g (0.2 mol) of 1-chloro-2-methyl-5-nitrobenzene, 38 g (0.276 mol) of potassium carbonate, and 300 mL of N,N-dimethylformamide (DMF) were added sequentially to a dry 500 mL three-necked flask. The reaction system was heated to 135 °C and stirred for 24 hours. The reaction progress was monitored by TLC. If the reaction was incomplete, 2 g of sodium hydroxide was added and the reaction was continued until complete. After the reaction was completed, the reaction solution was poured into 2.5 L of water and extracted with 1 L of petroleum ether (PE). The organic phase was separated, dried with anhydrous sodium sulfate, filtered, and the solvent was concentrated under reduced pressure to obtain 70 g of crude product. Purification was performed using a 100 g silica gel column with petroleum ether as the eluent. The target fraction was collected, concentrated, and 42 g of high-purity nitrobenzene ether intermediate was obtained.

[0024] (1) Preparation of aminodiphenyl ether intermediate by condensation and reduction reaction: 21 g (63.78 mmol) of the intermediate and 250 mL of ethyl acetate were added to a 500 mL single-necked flask, followed by 64.8 g (287 mmol) of tin chloride dihydrate. The mixture was heated under reflux overnight. After the reaction was completed, the ethyl acetate was removed by concentration under reduced pressure. Water was added to the residue, and the pH was adjusted to approximately 10 with sodium hydroxide aqueous solution. The residue was then extracted with ethyl acetate. The organic phase was dried and concentrated to obtain 21 g of aminodiphenyl ether intermediate.

[0025] (2) Preparation of methylurea-D3: In another 50 mL single-necked flask, 5.7 g (73.7 mmol) of methylamine hydrochloride-D3, 5.76 g (88.5 mmol) of sodium cyanate and 20 mL of deuterium water were added, and the mixture was heated under reflux for 3 hours. After the reaction solution was concentrated, 40 mL of methanol was added to dissolve it, and the mixture was filtered. The filtrate was concentrated, and the residue was recrystallized with 20 mL of methanol and dried under vacuum to obtain 5.4 g of methylurea-D3.

[0026] (3) Preparation of the activated intermediate: In a 100 mL single-necked flask, 3.5 g (45.4 mmol) of methylurea-D3 was dissolved in 25 mL of toluene, and 4.3 g (54.48 mmol) of pyridine was added. The temperature was raised to 55 °C. 8.04 g (49.95 mmol) of carbonyl diimidazole was dissolved in 20 mL of toluene and slowly added dropwise to the reaction system. The reaction was continued at 55 °C for 1 hour. After the reaction was completed, the mixture was cooled, 30 mL of methanol was added, and the precipitated solid was collected by filtration to obtain 6.5 g of the activated intermediate.

[0027] (4) Formation of the deuterated biuret intermediate: In another 100 mL single-necked flask, 7.4 g (24.7 mmol) of aminodiphenyl ether intermediate and 50 mL of DMF were added, followed by 5.2 g (26.5 mmol) of activation intermediate. The reaction was carried out at 80 °C for 3 hours. After the reaction was completed, the reaction solution was poured into a large amount of water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and recrystallized from petroleum ether / ethyl acetate to obtain 6.2 g of deuterated biuret intermediate.

[0028] (5) Cyclization reaction to prepare toltrazuril-D3: Finally, in a 250 mL single-necked flask, 6.17 g (15.35 mmol) of deuterated biuret intermediate, 100 mL of dimethyl carbonate, and 2.5 g (46 mmol) of sodium methoxide dried at 70 °C for 1 hour were added, and the mixture was reacted at 85 °C for 3 hours. The reaction solution was filtered and concentrated, and the crude product was purified by silica gel column chromatography (eluting with petroleum ether:ethyl acetate = 5:1 to 1:1, gradient elution). The product fraction was collected, concentrated, and further recrystallized with a petroleum ether / ethyl acetate mixed solvent. After vacuum drying, 3.9 g of the final product toltrazuril-D3 was obtained, with a total yield of 68% based on the initial feed amount of 1-chloro-2-methyl-5-nitrobenzene. The chemical purity was greater than 99.5% by high performance liquid chromatography (HPLC).

[0029] The isotopic abundance was greater than 99.5% as determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS); the chromatogram, electrospray ionization mass spectrum, and proton nuclear magnetic resonance spectrum are shown below. Figure 1 , Figure 2 and Figure 3 .

[0030] Example 2 The main difference between this embodiment and Example 1 lies in the material ratio and temperature in step (1). Specifically, under nitrogen protection, 53.6 g (0.276 mol) of p-trifluoromethylthiophenol, 34.3 g (0.2 mol) of 1-chloro-2-methyl-5-nitrobenzene (molar ratio 1.38:1), 38 g (0.276 mol) of potassium carbonate, and 300 mL of DMF were added sequentially to a dry 500 mL three-necked flask. The reaction system was heated to 130 °C and stirred until complete by TLC. The post-processing method was the same as in Example 1 to obtain the nitrodiphenyl ether intermediate.

[0031] Subsequently, referring to the reduction reaction method of step (1) of Example 1 (taking raw materials in the same molar ratio as the obtained nitrodiphenyl ether intermediate for reduction), steps (2), (3), (4), and (5), the amount of reagents was scaled up proportionally, and the reaction and post-processing methods were the same as in Example 1.

[0032] The final yield of toltrazuril-D3 was approximately 58%, with a chemical purity of over 99.2% and an isotopic abundance of over 99.5%.

[0033] Example 3 The main difference between this embodiment and Example 1 lies in the material ratio and temperature in step (1). Specifically, under nitrogen protection, 57.7 g (0.296 mol) of p-trifluoromethylthiophenol, 34.3 g (0.2 mol) of 1-chloro-2-methyl-5-nitrobenzene (molar ratio 1.48:1), 38 g (0.276 mol) of potassium carbonate and 300 mL of LDM were added sequentially to a dry 500 mL three-necked flask. The reaction system was heated to 140 °C and stirred until complete by TLC. The post-processing method was the same as in Example 1 to obtain the nitrodiphenyl ether intermediate.

[0034] Subsequently, referring to the reduction reaction in step (1), steps (2), (3), (4), and (5) of Example 1, the amount of reagents was scaled up proportionally, and the reaction and post-processing methods were the same as in Example 1.

[0035] The final yield of toltrazuril-D3 was 66%. Testing showed that its chemical purity was greater than 99.5% and its isotopic abundance was greater than 99.5%.

[0036] Example 4 The difference between this embodiment and Example 1 lies in the amount of reducing agent used in step (1). Specifically, the condensation reaction is the same as in Example 1, yielding 21g (63.78mmol) of nitrodiphenyl ether intermediate. Subsequently, the 21g intermediate and 250mL of ethyl acetate are added to a 500mL single-necked flask, followed by 63.0g (279.6mmol) of tin chloride dihydrate (molar ratio 1:4.4). The reaction is heated to reflux, and the reaction rate is slightly slower than in Example 1, requiring an appropriate extension of the reaction time to ensure complete reaction. The post-treatment method is the same as in Example 1, yielding aminodiphenyl ether intermediate.

[0037] Subsequently, referring to steps (2), (3), (4), and (5) of Example 1, the amount of reagents was scaled up proportionally, and the reaction and post-processing methods were the same as in Example 1.

[0038] Experimental results show that the reduction reaction can still be completed at this ratio, but the reaction rate is slightly slower than in Example 1, and the reaction time needs to be appropriately extended to ensure complete reaction. The total yield of the final aminodiphenyl ether intermediate and subsequent product toltrazuril-D3 was 55%. The final product had a chemical purity of approximately 99.0% and an isotopic abundance greater than 99.5%.

[0039] Example 5 The difference between this embodiment and Example 1 lies in the amount of alkali used in step (5). Specifically: (5) Cyclization reaction to prepare toltrazuril-D3: In a 250 mL single-necked flask, add 6.17 g (15.35 mmol) of deuterated biuret intermediate, 100 mL of dimethyl carbonate, and 2.85 g (52.8 mmol) of sodium methoxide dried at 70 °C for 1 h (molar ratio 1:3.1), and react at 85 °C for 3 h. The post-treatment and purification methods are the same as in Example 1.

[0040] Experimental results show that the cyclization reaction proceeds smoothly and rapidly under these conditions, with a total yield of 67% for the final product, toltrazuril-D3. Testing revealed that its chemical purity is greater than 99.5% and its isotopic abundance is greater than 99.5%.

[0041] Comparative Example 1 This example aims to verify that a high abundance product cannot be obtained using ordinary methylamine hydrochloride. Except for replacing methylamine hydrochloride-D3 with an equimolar amount of ordinary methylamine hydrochloride (CH3NH2·HCl) and replacing deuterium water with an equal volume of deionized water, the raw materials, steps, and reaction conditions were exactly the same as in Example 1. The final product yield was 65%. Mass spectrometry analysis showed that its isotopic abundance was approximately 0.5% (natural background abundance), indicating no deuteration labeling; its chemical purity was approximately 98.5%.

[0042] Comparative Example 2 The difference between this embodiment and Example 1 lies in the post-processing method of step (4): after the reaction solution is cooled, instead of using methanol precipitation, it is first diluted with water, then extracted with ethyl acetate, the organic phases are combined, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude activated intermediate. The remaining conditions are the same as in Example 1. Experiments have shown that this alternative post-processing scheme has complicated operation steps, consumes a large amount of solvent, and the obtained intermediate is an oily substance or solid, and its purity is lower than that of the product obtained in Example 1. When this intermediate is used in step (5), the yield and purity of the deuterated biuret intermediate are both lower than those in Example 1. The final yield of the toltrazuril-D3 product is 50%. The chemical purity is about 98.8%, and the isotopic abundance is greater than 99.5%.

[0043] Comparative Example 3 The difference between this embodiment and Example 1 lies in the reduction conditions of step (2): the reaction temperature is controlled at 80°C (lower than the reflux temperature of Example 1), and the molar ratio of nitrodiphenyl ether intermediate to tin chloride dihydrate remains 1:4.5. The remaining conditions are the same as in Example 1. Experiments revealed that at this lower temperature, the reduction reaction rate significantly slowed down. Even with extended reaction time, TLC monitoring still showed a significant amount of residual raw materials, indicating incomplete reaction. This ultimately resulted in a low yield of the aminodiphenyl ether intermediate, affecting subsequent steps, with the final product, toltrazuril-D3, having a total yield of only 42%. Its chemical purity was approximately 97.5%, and its isotopic abundance was greater than 99.5%.

[0044] Comparative Example 4 The difference between this embodiment and Example 1 lies in the purification method involved in step (5): after the reaction solution is filtered and concentrated, the crude product is recrystallized directly using a mixed solvent of petroleum ether and ethyl acetate without silica gel column chromatography. The remaining conditions are the same as in Example 1. Experiments showed that recrystallization alone could not effectively remove impurities with structures similar in polarity to the product. The total yield of the obtained toltrazuril-D3 product was 60% (this yield is the crude product yield, as the product contained impurities due to the lack of column chromatography). HPLC analysis showed a chemical purity of 97.0% and an isotopic abundance greater than 99.5%.

Claims

1. A method for synthesizing a stable isotope-labeled toltrazuril-D3, characterized in that, The synthetic route is as follows: 。 2. The method for synthesizing a stable isotope-labeled toltrazuril-D3 according to claim 1, characterized in that, Specifically, the following steps are included: (1) An amino diphenyl ether intermediate was prepared by condensation reaction and reduction reaction of p-trifluoromethylthiophenol and 1-chloro-2-methyl-5-nitrobenzene as reactants; (2) Preparation of methylurea: Methylamine hydrochloride-D3 and sodium cyanate were used as raw materials and heated under reflux in deuterium water. After the reaction was completed, methylurea-D3 was obtained by post-treatment. (3) Preparation of activated intermediate: Dissolve the methylurea-D3 obtained in step (2) in toluene, add pyridine, and add a toluene solution of carbonyl diimidazole dropwise under heating and stirring. Continue the reaction and obtain the activated intermediate after post-treatment after the reaction is completed. (4) Formation of biuret: The aminodiphenyl ether intermediate was dissolved in N,N-dimethylformamide, the activated intermediate obtained in step (3) was added, the reaction was heated, and the deuterated biuret intermediate was obtained after the reaction was completed. (5) Cyclization reaction: The deuterated biuret intermediate obtained in step (4) is dissolved in dimethyl carbonate, dry sodium methoxide is added, and the reaction is heated to generate toltrazuril-D3. After the reaction is completed, the final product is obtained by post-treatment and purification.

3. The method for synthesizing a stable isotope-labeled toltrazuril-D3 according to claim 2, characterized in that: The condensation reaction in step (1) uses p-trifluoromethylthiophenol and 1-chloro-2-methyl-5-nitrobenzene as reactants, and the molar ratio of p-trifluoromethylthiophenol to 1-chloro-2-methyl-5-nitrobenzene is 1.3~1.4:

1.

4. The method for synthesizing a stable isotope-labeled toltrazuril-D3 according to claim 2, characterized in that: The reduction reaction in step (1) involves dissolving the nitrodiphenyl ether intermediate obtained from the condensation reaction in ethyl acetate, adding tin chloride dihydrate, and heating under reflux to carry out the reduction reaction. The molar ratio of the nitrodiphenyl ether intermediate to tin chloride dihydrate is 1:4.4~4.

6. After the reaction is completed, the aminodiphenyl ether intermediate is obtained by post-reduction treatment. The temperature of the condensation reaction and the reduction reaction is 130℃~140℃, and the reaction time is monitored by TLC until the reaction is complete.

5. The method for synthesizing a stable isotope-labeled toltrazuril-D3 according to claim 4, characterized in that: The post-reduction treatment includes: concentrating to remove the solvent, adding water, adjusting the pH to alkaline with sodium hydroxide aqueous solution, extracting with ethyl acetate, and concentrating the organic phase to obtain an aminodiphenyl ether intermediate.

6. The method for synthesizing a stable isotope-labeled toltrazuril-D3 according to claim 2, characterized in that: In step (3), the molar ratio of methylurea-D3 to carbonyl diimidazole is 1:1.09~1.11, and the reaction temperature is 50℃~60℃.

7. The method for synthesizing a stable isotope-labeled toltrazuril-D3 according to claim 2, characterized in that: The post-reaction processing in step (3) includes: after the reaction solution is cooled, methanol is added, the precipitated solid is collected by filtration, and an activated intermediate is obtained.

8. The method for synthesizing a stable isotope-labeled toltrazuril-D3 according to claim 2, characterized in that: In step (4), the molar ratio of the aminodiphenyl ether intermediate to the activated intermediate is 1:1.06~1.08, and the reaction temperature is 75℃~85℃.

9. The method for synthesizing a stable isotope-labeled toltrazuril-D3 according to claim 2, characterized in that: In step (5), the molar ratio of the deuterated biuret intermediate to sodium methoxide is 1:2.8~3.1, and the reaction temperature is 80℃~90℃.

10. The method for synthesizing a stable isotope-labeled toltrazuril-D3 according to claim 2, characterized in that: The purification method in step (5) is as follows: after the reaction solution is filtered and concentrated, the crude product is purified by silica gel column chromatography. The eluent is a mixed solvent of petroleum ether and ethyl acetate. Gradient elution is performed, the product fraction is collected, concentrated, and then recrystallized further using a mixed solvent of petroleum ether and ethyl acetate.

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