Synthesis of a deuterated derivative of tinidazole

Abstract

The present application relates to a method for synthesizing tinidazole deuterium analogues, such as shown in formula I, wherein one or more of R1-R5 are deuterium and the rest are hydrogen, comprising the step of preparing the tinidazole deuterium analogues from intermediate B by oxidation with meta-chloroperoxybenzoic acid, wherein the oxidation is carried out under low temperature and alkaline conditions, and the intermediate B is shown in formula II: The method for synthesizing tinidazole deuterium analogues according to the present application can avoid the problems of deuterium loss and isotopic dilution during the reaction process, obtain stable isotope-labeled tinidazole deuterium analogues with an isotopic abundance of >98%, and have a high yield. The prepared tinidazole deuterium analogues can be used as isotopic internal standard reagents for quantitative detection of tinidazole, for example, in the isotopic internal standard method for detecting the residual amount of tinidazole in meat products, and have the advantages of high recovery efficiency from the matrix and green safety.

Description

Technical Field

[0001] This invention relates to a method for synthesizing tinidazole deuterated compounds. Background Technology

[0002] Tinidazole, a derivative of metronidazole, chemically named 1-(2-ethanesulfonylethyl)-2-methyl-5-nitro-1H-imidazole, is a highly effective anti-amoebic and anti-anaerobic drug, generally used clinically to treat anaerobic bacterial and protozoan infections. It can also kill Helicobacter pylori. Compared with the traditional anti-anaerobic drug metronidazole, tinidazole has advantages such as higher efficacy and fewer side effects. Regarding the preparation methods of tinidazole, most synthetic routes reported in the literature involve using hydroxyethyl sulfide and 2-methyl-5-nitroimidazole as raw materials, dissolving them in methyl isobutyl ketone, and undergoing a Lewis acid-catalyzed condensation reaction to obtain the condensate 1-(ethyl thioethyl)-2-methyl-5-nitroimidazole, followed by oxidation with 30% hydrogen peroxide under acidic conditions to obtain the final product. This process has disadvantages such as requiring a large number of reagents, significant site pollution, complex process, long production cycle, and poor product quality.

[0003] A secure food supply supports a nation's economy, trade, and tourism, promotes food and nutrition security, and supports sustainable development. Numerous methods have been reported for detecting drug residues in meat products. Among them, liquid chromatography-mass spectrometry (LC-MS) effectively combines the advantages of ultra-high performance liquid chromatography (UHPLC) and mass spectrometry (MS), offering high specificity, high sensitivity, and suitability for trace component analysis in complex matrices. It has become the primary method for veterinary drug residue detection. According to the standard detection method in SN / T 1626-2019, "Determination of Residues of Metronidazole, Tinidazole, Ornidazole, Lomonidazole, Dimetronidazole, and Secnidazole in Exported Meat and Meat Products - Liquid Chromatography-Mass Spectrometry / Mass Spectrometry," the detection of tinidazole content in meat products uses the external standard method. However, the stable isotope internal standard method can reduce the influence of matrix interference (matrix refers to components in the sample other than the analyte; since the matrix often significantly interferes with the analysis process of the analyte and affects the accuracy of the analytical results, these effects and interferences are called matrix effects), resulting in higher sensitivity and accuracy.

[0004] In the synthesis of stable isotope internal standards, to ensure that the isotopic abundance (>98%) is not diluted and that the purity is high, it is necessary to rationally design the synthetic route for the stable isotope-labeled product, considering the mildness and feasibility of each reaction process. The isotopic raw material, 2-hydroxyethyl ethyl sulfide deuterated compound, is not currently commercially available and needs to be synthesized in-house. Therefore, experimental costs and optimal routes must be considered when designing the synthetic route. Currently, most literature uses the reaction of ethanethiol and 2-bromoethanol to obtain 2-hydroxyethyl ethyl sulfide; however, the market price of stable isotope-labeled ethanethiol deuterated compound is high and the market supply is limited. Therefore, better synthetic raw materials are needed to synthesize the desired target product. Summary of the Invention

[0005] The main objective of this invention is to provide a method for synthesizing a stable isotope-labeled deuterated tinidazole with an isotopic abundance of >98%.

[0006] To achieve the above objectives, the present invention provides a method for synthesizing a deuterated tinidazole compound, wherein the deuterated tinidazole compound is shown in Formula I. Formula I Of these, one or more of R1 to R5 are deuterium, and the rest are hydrogen. The steps include: intermediate B is prepared by oxidation of m-chloroperoxybenzoic acid to prepare the deuterated tinidazole, wherein, The oxidation is carried out under low temperature and alkaline conditions. The intermediate B is shown in Formula II: Formula II.

[0007] In some embodiments of the present invention, the alkaline conditions are as follows: intermediate B and m-chloroperoxybenzoic acid are added to an alkaline solution to carry out the oxidation.

[0008] In some embodiments of the present invention, the alkaline solution is an aqueous solution of one or more of sodium hydroxide and sodium methoxide.

[0009] In some embodiments of the present invention, the molar concentration of the alkaline solution is 0.40 M-1.0 M.

[0010] In some embodiments of the present invention, the molar ratio of intermediate B to m-chloroperoxybenzoic acid is 1:(2.0-2.5).

[0011] In some embodiments of the present invention, the step of preparing intermediate B by photo-extending reaction of intermediate A with 2-methyl-5-nitroimidazole at low temperature is further included, as shown in the following reaction formula: .

[0012] In some embodiments of the present invention, the photoelongation reaction includes the following steps: In the first reaction vessel, intermediate A, 2-methyl-5-nitroimidazole, and triphenylphosphine were dissolved in tetrahydrofuran. Diisopropyl azodicarbonate dissolved in tetrahydrofuran is added dropwise to the first reaction vessel.

[0013] In some embodiments of the present invention, the molar ratio of intermediate A: triphenylphosphine: diisopropyl azodicarboxylate is 1:(1.0-2.0):(1.0-2.0).

[0014] In some embodiments of the present invention, the low temperature is 0-5°C.

[0015] In some embodiments of the present invention, the step of preparing intermediate A by nucleophilic substitution of 2-bromoethane deuterated product with 2-mercaptoethanol is further included, as shown in the following reaction formula: .

[0016] In some embodiments of the present invention, the nucleophilic substitution includes the step of: In the second reaction vessel: a) 2-Mercaptoethanol, and b) One or more of potassium carbonate, sodium bicarbonate, sodium carbonate, and cesium carbonate, and c) One or both of potassium iodide or cuprous iodide. Soluble in acetone or tetrahydrofuran under nitrogen protection. Add the 2-bromoethane deuterated product to the second reaction vessel.

[0017] In some embodiments of the present invention, the molar ratio of 2-thioethanol to 2-bromoethane deuterated product is 1:(1.5-2.2).

[0018] In some embodiments of the present invention, the deuterated tinidazole is: ,or .

[0019] The synthesis method of this invention avoids the problems of deuterium loss and isotope dilution during the reaction process, obtaining stable isotopically labeled tinidazole deuterated products with an isotopic abundance >98% and a high yield. The prepared tinidazole deuterated products can be used as isotopic internal standards for the quantitative detection of tinidazole, such as for the detection of tinidazole residues in meat products using isotopic internal standard methods. Furthermore, it has the advantages of high recovery efficiency from the matrix and is environmentally friendly and safe. Attached Figure Description

[0020] Figure 1 This is a process route diagram for the synthesis of deuterated tinidazole.

[0021] Figure 2 The 1H NMR spectrum of tinidazole-D5, a stable isotope label obtained in Example 1 of this invention (…). 1 1H-NMR spectrum.

[0022] Figure 3 The image shows the liquid chromatography purity (LC) spectrum of tinidazole-D5, a stable isotope label obtained in Example 1 of this invention.

[0023] Figure 4 The image shows the liquid chromatography-mass spectra of tinidazole-D5, a stable isotope label obtained in Example 1 of this invention.

[0024] Figure 5 Tinidazole-D3, a stable isotope marker obtained in Example 3 of this invention. 1 H-NMR spectrum.

[0025] Figure 6 The image shows the LC spectrum of tinidazole-D3, a stable isotope label obtained in Example 3 of this invention. Detailed Implementation

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the descriptions and definitions herein shall prevail.

[0027] The term "tinidazole deuterated derivative" represents compounds with the general formula shown in Formula I: Formula I In this compound, one or more of R1 to R5 are deuterium (-D), and the rest are hydrogen (-H). Based on the number of deuterium atoms in the deuterated molecule, this application also refers to the deuterated tinidazole compound as "tinidazole-D". n ", where n is an integer from 1 to 5, corresponding to the number of deuterium atoms in the molecular structure. For example, tinidazole-D5 has R1 to R5 all containing deuterium, tinidazole-D4 has 4 R1 to R5 containing deuterium and the remaining one is hydrogen, tinidazole-D3 has 3 R1 to R5 containing deuterium and the remaining 2 are hydrogen, and so on.

[0028] The term "low temperature" refers to a temperature between 0 and 5°C.

[0029] The reaction principle of using intermediate chloroperoxybenzoic acid as an oxidant to oxidize thioether groups (-S-) to sulfone groups (-SO2-) is known.

[0030] The reaction principle for preparing thioethers using nucleophilic substitution in this application is known.

[0031] The reaction principle of the photoelongation reaction used in this application is known (for example, see Selective mono-and di-N-alkylation of aromatic amines with alcohols and acylation of aromatic amines using Ph3P / DDQ, Nasser Iranpoor et al., Tetrahedron (2009) 65, 3893-3899, DOI: 10.1016 / j.tet.2009.02.078). In view of the problem of isotope dilution in the existing synthesis methods of tinidazole deuterated products, the present invention provides a different synthesis method of tinidazole deuterated products, comprising the steps of: oxidizing intermediate B with m-chloroperoxybenzoic acid to obtain the tinidazole deuterated product, wherein the oxidation is carried out under low temperature and alkaline conditions, and intermediate B is as shown in Formula II: Formula II.

[0032] The molecular formula of m-chloroperoxybenzoic acid is shown in Formula III: Formula III.

[0033] In the above reaction, m-chloroperoxybenzoic acid, as a relatively mild oxidizing agent, can oxidize the thioether group -S- in Formula II to a sulfone group -SO2- to obtain the deuterated product of tinidazole. Low temperature avoids deuteration problems that occur during vigorous reactions. Furthermore, an ice bath effectively controls the reaction temperature and absorbs the heat generated during the reaction, thus preventing deuteration under vigorous conditions. Specifically, one feasible reaction step includes dissolving m-chloroperoxybenzoic acid in an alkaline solution, subsequently adding intermediate B, and reacting at low temperature to prepare the deuterated product of tinidazole. Preferably, the alkaline solution is an aqueous solution of one or more of sodium hydroxide and sodium methoxide. Preferably, the molar concentration of the alkaline solution is 0.40 M-1.0 M. Preferably, the molar ratio of intermediate B to m-chloroperoxybenzoic acid is 1:(2.0-2.5).

[0034] Furthermore, intermediate B can be prepared by photo-tempering reaction of intermediate A with 2-methyl-5-nitroimidazole, as shown in the following reaction formula: .

[0035] In the above steps for preparing intermediate A from intermediate B, a photoelongation reaction is employed. Under the action of triphenylphosphine (PPh3) and diethyl azodicarbonate (DEAD), the amino group on 2-methyl-5-nitroimidazole acts as a nucleophile in the photoelongation reaction, replacing the hydroxyl group on intermediate A to generate intermediate B. The inventors have discovered that when intermediate A is prepared into intermediate B using the photoelongation reaction principle, fewer byproducts are produced, effectively increasing the yield of intermediate B, and the reaction process also avoids deuterium loss. Specifically, one feasible reaction operation step includes: dissolving intermediate A, 2-methyl-5-nitroimidazole, and triphenylphosphine in tetrahydrofuran, slowly adding diisopropyl azodicarbonate dissolved in tetrahydrofuran, and carrying out the photoelongation reaction in an ice-water bath. Preferably, the molar ratio of intermediate A:triphenylphosphine:diisopropyl azodicarbonate is 1:(1.0-2.0):(1.0-2.0).

[0036] Furthermore, intermediate A can be prepared by nucleophilic substitution of the 2-bromoethane deuterated product with 2-mercaptoethanol, as shown in the following reaction formula: .

[0037] Currently, there is no publicly disclosed method for preparing intermediate A. This application selects 2-bromoethane deuterated product and 2-mercaptoethanol as reaction raw materials to synthesize intermediate A through a nucleophilic substitution reaction. Furthermore, the two selected reaction raw materials are readily available commercial products with low prices, resulting in lower synthesis costs. Specifically, one feasible reaction operation step includes: sequentially adding 2-mercaptoethanol, potassium carbonate, and potassium iodide to a reaction vessel, dissolving them in acetone under nitrogen protection, and then adding the 2-bromoethane deuterated product to carry out the nucleophilic reaction. Preferably, the molar ratio of 2-thioethanol to 2-bromoethane deuterated product is 1:(1.5-2.2).

[0038] The preparation method described above will be further explained below through specific embodiments.

[0039] Related reagents All raw materials used in the examples are commercially available and of industrial grade purity.

[0040] Deuterated bromoethane (Nanjing Haolv Biotechnology Co., Ltd.); concentrated sulfuric acid, boric acid (Sinopharm Group Pharmaceutical Co., Ltd.); hydrogen peroxide (Shanghai Aladdin Biochemical Technology Co., Ltd.); 2-mercaptoethanol, potassium iodide, cuprous iodide, potassium carbonate, sodium carbonate, cesium carbonate, sodium bicarbonate, sodium hydroxide, anhydrous sodium sulfate, toluene, xylene, acetone, tetrahydrofuran, ethyl acetate, n-hexane, dichloromethane, ethanol, methanol (analytical grade, Shanghai Anpu Experimental Technology Co., Ltd.); 2-methyl-5-nitroimidazole, diisopropyl azodicarbonate (Shanghai Maclean Biochemical Technology Co., Ltd.); m-chloroperoxybenzoic acid, triphenylphosphine (Shanghai Aladdin Biochemical Technology Co., Ltd.). Unless otherwise specified, all reagents are for direct use.

[0041] Example 1 Synthesis of Tinidazole-D5 Synthesis of intermediate A1: 2-hydroxyethyl ethyl sulfide-D5 In a 100 mL three-necked round-bottom flask, 2-mercaptoethanol (0.56 mL, 7.89 mmol), potassium carbonate (2.03 g, 2.00 eq), and potassium iodide (78 mg, 0.05 eq) were added sequentially and dissolved in acetone (30 mL) under nitrogen protection. Then, 2-bromoethane-D5 (1.30 mL, 2.00 eq) was added, and the mixture was refluxed for 12 hours. The reaction progress was monitored by TLC (V0.05). 二氯甲烷 V 甲醇 =20:1, potassium permanganate for color development). The reaction formula is as follows: After the reaction was complete, the reaction flask was cooled to room temperature, the precipitate was removed by filtration, the solvent was evaporated, and 50 mL of diethyl ether was added to dissolve the precipitate. The mixture was then washed and extracted successively with water, NaHCO3, and NaCl aqueous solution. Finally, the organic phase was concentrated, dried under vacuum to obtain the crude product, and subjected to column chromatography (V... 正己烷 V 乙酸乙酯 =2:1~V 二氯甲烷 V 甲醇 The solution was purified by a ratio of 50:1 to obtain a transparent oil (730 mg, 6.90 mmol), with a yield of 87.45% [based on 2-mercaptoethanol].

[0042] Synthesis of Intermediate B1: 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazolium-D5 At 0 °C, 2-hydroxyethyl ethyl sulfide-D5 (730 mg, 6.90 mmol), 2-methyl-5-nitroimidazole (877 mg, 1.00 eq), and triphenylphosphine (1.81 g, 1.00 eq) were dissolved in tetrahydrofuran (10 mL) and placed in a 50 mL three-necked round-bottom flask. Then, diisopropyl azodicarbonate (1.38 g, 1.00 eq) dissolved in tetrahydrofuran (5 mL) was slowly added dropwise. The reaction was carried out in an ice-water bath for 3 hours, and the reaction progress was monitored by TLC (V0.00).二氯甲烷 V 甲醇 =5:1). The reaction formula is as follows: After the reaction was complete, the solvent was evaporated, the organic phase was concentrated, and the crude product was obtained by vacuum drying. The crude product was then subjected to column chromatography (V... 二氯甲烷 V 甲醇 The product was purified from 50:1 to 20:1 to give a white solid (900 mg, 4.16 mmol) in a yield of 60.29% [based on 2-hydroxyethyl ethyl sulfide-D5].

[0043] Synthesis of Tinidazole-D5 At 0 °C, m-chloroperoxybenzoic acid (1.58 g, 2.20 eq) was dissolved in 0.50 M sodium hydroxide aqueous solution (100 mL), followed by the addition of 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5 (900 mg, 4.16 mmol). The reaction was carried out at low temperature for 2 hours, and the reaction progress was monitored by TLC during the reaction (V0.05). 二氯甲烷 V 甲醇 =3:1). The reaction formula is as follows: After the reaction was complete, NaCl saturated aqueous phase was added to the solution, followed by extraction with dichloromethane, drying with anhydrous sodium sulfate, concentrating the organic phase, drying under vacuum to obtain the crude product, and purifying by column chromatography (V). 二氯甲烷 V 甲醇 =50:1-20:1). Fine purification: Crude tinidazole was dissolved in 70% ethanol-water at twice the weight of crude tinidazole, and 1.8% of the weight of crude tinidazole-D5 was added with activated carbon for decolorization for 30 min. The mixture was filtered while hot, and the filtrate was cooled to crystallize. After filtration and drying, a white solid (716 mg, 2.84 mmol) was obtained, with a yield of 68.26% [based on 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5].

[0044] Qualitative analysis by 1H NMR (reference) Figure 2 The 1H NMR data for tinidazole are as follows: 1 H NMR (CD3OD, 400MHz, ppm) δ: 7.95 (s, 1H), 4.81 (t, J = 6.9 Hz, 2H), 3.63 (t, J = 6.8 Hz, 2H), 3.15(q, J = 7.4 Hz, 2H), 2.57 (s, 3H), 1.34 (d, J = 7.4 Hz, 3H); the 1H NMR data of tinidazole-D5 are 1H NMR (CD3OD, 400 MHz, ppm) δ: 7.95 (s, 1H), 4.80 (d, J = 6.8 Hz, 2H), 3.63 (t, J =6.8 Hz, 2H), 2.57 (s, 3H). From this, we can see that δ: 7.95 (s, 1H), 4.80 (d, J = 6.8 Hz, 2H), 3.63(t, J The absorption peaks at 6.8 Hz (2H) and 2.57 s (3H) are consistent, while δ: 3.15 (q, J = 7.4 Hz, 2H), 1.34 (d, J The absorption peaks at 7.4 Hz and 3H all disappeared. This proves that none of the five deuterium groups on the ethyl group in the structure were lost, and the resulting compound structure is tinidazole-D5.

[0045] The content of tinidazole-D5 was determined to be 99.97% by high-performance liquid chromatography (mobile phase: 0.1% phosphoric acid water; acetonitrile). (Reference) Figure 3 ) Tinidazole-D5 Isotope Abundance Detection The MRM mode in tandem mass spectrometry was used to determine the isotope abundance. The parent ion and quantitative ion of each substance were selected, and the peak area obtained was approximately equal to that of their respective isotope labels. The measured peak area was then normalized to obtain the isotope abundance.

[0046] The "simplified mass cluster" method was used for determination, taking tinidazole-D5 prepared in Example 1 as an example. The peak areas of the quantitative ion pairs 126.01-121.00, 125.01-121.00, 124.01-121.00, 123.01-121.00, 122.01-121.00, and 121.01-121.00 were determined by mass spectrometry (see [reference]). Figure 4 The peak areas of tinidazole-D5, tinidazole-D4, tinidazole-D3, tinidazole-D2, tinidazole-D1, and tinidazole-D0 were approximated to these values. Based on the detection principle that "mass spectrometry peak intensity is directly proportional to the number of molecules", the isotopic abundance percentages of D5-D0 were normalized, and the data are listed in Table 1.

[0047] Table 1. Isotopic abundance of tinidazole-D5 in Example 1 Finally, through the formula: D%= Formula i in, D% represents isotopic abundance, atom%D; i represents the number of D tags, i = 0~5; R i This represents the percentage of the peak area occupied by i D labels. Taking 5 D labels as an example, the calculation formula is: R6=A(D) / [A(D5)+A(D4)+A(D3)+A(D2)+A(D1)+A(D0)]; n represents the total number of D tags, n=5.

[0048] According to Equation i, the isotopic abundance in tinidazole-D5 in this embodiment is 98.81%, which proves that the synthesis method does not involve isotopic dilution and can obtain a product with stable isotopic labeling.

[0049] Example 2 Synthesis of Tinidazole-D5 Synthesis of intermediate A1: 2-hydroxyethyl ethyl sulfide-D5 In a 100 mL three-necked round-bottom flask, 2-mercaptoethanol (0.40 mL, 5.63 mmol), sodium bicarbonate (1.41 g, 2.50 eq), and potassium iodide (56 mg, 0.06 eq) were added sequentially and dissolved in acetone (25 mL) under nitrogen protection. Then, 2-bromoethane-D5 (1.00 mL, 2.20 eq) was added and the mixture was refluxed for 12 hours. The reaction progress was monitored by TLC (Vdichloromethane:Vmethanol = 20:1, with potassium permanganate for color development). After the reaction was complete, the flask was cooled to room temperature, the precipitate was removed by filtration, the solvent was evaporated, and 50 mL of diethyl ether was added to dissolve the precipitate. The mixture was then washed and extracted successively with water, NaHCO3, and NaCl aqueous solutions. Finally, the organic phase was concentrated, dried under vacuum to obtain the crude product, and subjected to column chromatography (Vdichloromethane:Vmethanol = 20:1, with potassium permanganate for color development). 正己烷 V 乙酸乙酯 =2:1~V 二氯甲烷 V 甲醇 The product was purified by a 50:1 ratio to obtain a transparent oil (508 mg, 4.80 mmol), with a yield of 85.27% [based on 2-mercaptoethanol].

[0050] Synthesis of Intermediate B1: 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazolium-D5 At 0 °C, 2-hydroxyethyl ethyl sulfide-D5 (508 mg, 4.80 mmol), 2-methyl-5-nitroimidazole (671 mg, 1.10 eq), and triphenylphosphine (1.64 g, 1.30 eq) were dissolved in tetrahydrofuran (8 mL) and placed in a 50 mL three-necked round-bottom flask. Then, diisopropyl azodicarbonate (1.25 g, 1.30 eq) dissolved in tetrahydrofuran (4 mL) was slowly added dropwise. The reaction was carried out in an ice-water bath for 3 hours, and the reaction progress was monitored by TLC (V0.05). 二氯甲烷 V 甲醇=5:1). After the reaction, the solvent was evaporated, the organic phase was concentrated, and the crude product was obtained by vacuum drying. The product was then subjected to column chromatography (V... 二氯甲烷 V 甲醇 The product was purified from 50:1 to 20:1 to give a white solid (552 mg, 2.83 mmol) in a yield of 59.11% [based on 2-hydroxyethyl ethyl sulfide-D5].

[0051] Synthesis of Tinidazole-D5 At 0 °C, m-chloroperoxybenzoic acid (1.18 g, 2.00 eq) was dissolved in 0.40 M sodium methoxide solution (80 mL), followed by the addition of 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5 (552 mg, 2.83 mmol) (900 mg, 4.16 mmol). The reaction was carried out at low temperature for 2–3 hours, with the reaction progress monitored by TLC during the reaction (V0). 二氯甲烷 V 甲醇 =3:1). After the reaction was complete, NaCl saturated aqueous phase was added to the solution, followed by extraction with ethyl acetate, drying with anhydrous sodium sulfate, concentrating the organic phase, drying under vacuum to obtain the crude product, and purifying by column chromatography (V 二氯甲烷 V 甲醇 =50:1-20:1). Fine purification: Tinidazole crude product was dissolved in 70% ethanol-water at twice the weight of crude tinidazole, and then tinidazole was added. -D5 The crude product was decolorized with 1.5% (by weight) activated carbon for 30 min, filtered while hot, the filtrate was cooled to crystallize, filtered, and dried to give a white solid (469 mg, 1.86 mmol), with a yield of 65.72% [based on 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5].

[0052] The content of tinidazole-D5 in this comparative example was determined by high performance liquid chromatography (mobile phase: 0.1% phosphoric acid water; acetonitrile). It is 99.95%. Tinidazole-D5 Isotope Abundance Detection Using the same detection steps as in Example 1, the isotopic abundance percentages of D5-D0 were normalized and the data are listed in Table 2.

[0053] Table 2. Isotopic abundance of tinidazole-D5 in Example 2 The isotopic abundance of tinidazole-D5 in this embodiment is 98.75%, which proves that the synthesis method does not involve isotopic dilution and can obtain a product with stable isotopic labeling.

[0054] Example 3 Synthesis of Tinidazole-D3 Synthesis of Intermediate A2: 2-Hydroxyethyl ethyl sulfide-D3 In a 100 mL three-necked round-bottom flask, 2-mercaptoethanol (0.50 mL, 7.04 mmol), potassium carbonate (2.71 g, 3.00 eq), and iodide ketone (67 mg, 0.05 eq) were added sequentially and dissolved in tetrahydrofuran (30 mL) under nitrogen protection. Then, 2-bromoethane-D3 (1.21 mL, 2.10 eq) was added and refluxed for 12 hours. The reaction progress was monitored by TLC (V0.05). 二氯甲烷 V 甲醇 =20:1, potassium permanganate for color development). The reaction formula is as follows: After the reaction was complete, the reaction flask was cooled to room temperature, the precipitate was removed by filtration, the solvent was evaporated, and 50 mL of diethyl ether was added to dissolve the precipitate. The mixture was then washed and extracted successively with water, NaHCO3, and NaCl aqueous solution. Finally, the organic phase was concentrated, dried under vacuum to obtain the crude product, and subjected to column chromatography (V... 正己烷 V 乙酸乙酯 =2:1~V 二氯甲烷 V 甲醇 The product was purified by a 50:1 ratio to obtain a transparent oil (625 mg, 5.91 mmol), with a yield of 83.96% [based on 2-mercaptoethanol].

[0055] Synthesis of Intermediate B2: 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazolium-D3 At 0 °C, 2-hydroxyethyl ethyl sulfide-D3 (625 mg, 5.91 mmol), 2-methyl-5-nitroimidazole (901 mg, 1.20 eq), and triphenylphosphine (1.86 g, 1.20 eq) were dissolved in tetrahydrofuran (10 mL) and placed in a 50 mL three-necked round-bottom flask. Then, diisopropyl azodicarbonate (1.42 g, 1.20 eq) dissolved in tetrahydrofuran (5 mL) was slowly added dropwise. The reaction was carried out in an ice-water bath for 3 hours, and the reaction progress was monitored by TLC (V0.05). 二氯甲烷 V 甲醇 =5:1). The reaction formula is as follows: After the reaction was complete, the solvent was evaporated, the organic phase was concentrated, and the crude product was obtained by vacuum drying. The crude product was then subjected to column chromatography (V... 二氯甲烷 V 甲醇 The mixture was purified from 50:1 to 20:1 to give a white solid (750 mg, 3.47 mmol) in a yield of 58.78% [based on 2-hydroxyethyl ethyl sulfide-D3].

[0056] Synthesis of Tinidazole-D3 At 0 °C, m-chloroperoxybenzoic acid (1.37 g, 2.30 eq) was dissolved in 0.60 M sodium carbonate aqueous solution (80 mL), followed by the addition of 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D3 (750 mg, 3.47 mmol). The reaction was carried out at low temperature for 2–3 hours, with the reaction progress monitored by TLC during the reaction (VL). 二氯甲烷 V 甲醇 =3:1). The reaction formula is as follows: After the reaction was complete, NaCl saturated aqueous phase was added to the solution, followed by extraction with dichloromethane, drying with anhydrous sodium sulfate, concentrating the organic phase, drying under vacuum to obtain the crude product, and purifying by column chromatography (V). 二氯甲烷 V 甲醇 =50:1-20:1). Fine purification: Tinidazole crude product was dissolved in 70% ethanol-water at twice the weight of crude tinidazole, and then tinidazole was added. -D3 The crude product was decolorized with 1.6% (by weight) activated carbon for 30 min, filtered while hot, and the filtrate was cooled to crystallize. After filtration and drying, a white solid (584 mg, 2.32 mmol) was obtained, with a yield of 66.96% [based on 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D3].

[0057] Qualitative analysis by 1H NMR (reference) Figure 5 The 1H NMR data for tinidazole are as follows: 1 H NMR (CD3OD, 400MHz, ppm) δ: 7.95 (s, 1H), 4.81 (t, J = 6.9 Hz, 2H), 3.63 (t, J = 6.8 Hz, 2H), 3.15(q, J = 7.4 Hz, 2H), 2.57 (s, 3H), 1.34 (d, J = 7.4 Hz, 3H); the 1H NMR data for tinidazole-D3 are: 1 H NMR (CD3OD, 400 MHz, ppm) δ: 7.86 (s, 1H), 4.72 (t, J = 6.9 Hz, 2H), 3.54 (t, J =6.8 Hz, 2H), 3.06 (q, J = 7.4 Hz, 2H), 2.48 (s, 3H). From this, we can see that δ: 7.86 (s, 1H), 4.72 (t, J = 6.9 Hz, 2H), 3.54 (t, J = 6.8 Hz, 2H), 3.06 (q,J The absorption peaks at 7.4 Hz (2H) and 2.48 s (3H) are consistent, while δ: 1.34 d, J The absorption peaks at 7.4 Hz and 3H all disappeared. This proves that the three deuterium groups on the methyl group in the structure did not detach, and the resulting compound structure is tinidazole-D3.

[0058] The content of tinidazole-D3 was determined to be 99.81% by high-performance liquid chromatography (mobile phase: 0.1% phosphoric acid water; acetonitrile). (Reference) Figure 6 ) Tinidazole-D3 Isotope Abundance Detection Using the same detection steps as in Example 1, the isotopic abundance percentages of D3-D0 were normalized and the data are listed in Table 3.

[0059] Table 3. Isotopic abundance of tinidazole-D3 in Example 3 The isotopic abundance of tinidazole-D3 in this embodiment is 98.41%, which proves that the synthesis method does not involve isotopic dilution and can obtain a product with stable isotopic labeling.

[0060] Comparative Example This application selected two existing synthetic methods (Comparative Example 1 and Comparative Example 2) to synthesize tinidazole-D5, and tested the abundance of the stable isotope deuterium contained therein and the yield of tinidazole-D5 and related intermediates to compare and illustrate the advantages of the present invention.

[0061] In summary, the main difference between Comparative Example 1 and Example 1 is that the photoelectrophoresis reaction was not used in the preparation of intermediate B1; instead, a conventional concentrated sulfuric acid dehydration esterification reaction was employed. In the step of oxidizing intermediate B1 to prepare tinidazole D5, hydrogen peroxide was used as the oxidant. The main difference between Comparative Example 2 and Example 1 is that the photoelectrophoresis reaction was not used in the preparation of intermediate B1; instead, a conventional concentrated sulfuric acid dehydration esterification reaction was employed.

[0062] Comparative Example 1 Synthesis of intermediate A1 In a 100 mL three-necked round-bottom flask, 2-mercaptoethanol (0.56 mL, 7.04 mmol), sodium carbonate (1.39 g, 2.00 eq), and potassium iodide (110 mg, 0.08 eq) were added sequentially and dissolved in acetone (30 mL) under nitrogen protection. Then, 2-bromoethane-D5 (0.87 mL, 1.50 eq) was added, and the mixture was refluxed for 12 hours. The reaction progress was monitored by TLC (V0.05). 二氯甲烷V 甲醇 =20:1, potassium permanganate for color development). After the reaction, the reaction flask was cooled to room temperature, the precipitate was removed by filtration, the solvent was evaporated, and 50 mL of diethyl ether was added to dissolve the precipitate. The mixture was then washed and extracted successively with water, NaHCO3, and NaCl aqueous solution. Finally, the organic phase was concentrated, dried under vacuum to obtain the crude product, and subjected to column chromatography (V... 正己烷 V 乙酸乙酯 =2:1~V 二氯甲烷 V 甲醇 The solution was purified by a ratio of 50:1 to obtain a transparent oil (596 mg, 5.64 mmol), with a yield of 80.05% [based on 2-mercaptoethanol].

[0063] Synthesis of intermediate B1 Add 10 mL of toluene to a 100 mL three-necked round-bottom flask, followed by 2-hydroxyethyl ethyl sulfide-D5 (596 mg, 5.64 mmol) and 2-methyl-5-nitroimidazole (657 mg, 1.00 eq). While stirring, raise the temperature to 50 °C, and while maintaining this temperature, slowly add concentrated sulfuric acid (0.30 mL, 1.00 eq). After the addition is complete, raise the temperature to 88 °C and react for 6 hours, then lower the temperature to 35 °C. Adjust the pH to neutral with ammonia. Add an appropriate amount of toluene to the reaction mixture, filter, wash the filter cake with toluene, collect the filtrate, evaporate the solvent, concentrate the organic phase, and dry under vacuum to obtain the crude product. Perform column chromatography (V... 二氯甲烷 V 甲醇 The mixture was purified from 50:1 to 20:1 to give a white solid (653 mg, 3.01 mmol) in a yield of 53.36% [based on 2-hydroxyethyl ethyl thioether-D5].

[0064] Synthesis of Tinidazole-D5 The intermediate from the second step reaction (653 mg, 3.01 mmol) was dissolved in a mixed solution of 30% hydrogen peroxide (183 mg, 1.8 eq) and boric acid (55 mg, 0.3 eq). The mixture was stirred at approximately 35°C for about 100–135 minutes, and the reaction progress was monitored by TLC (Vo). 二氯甲烷 V 甲醇 =3:1). After the reaction was complete, the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, the organic phase was concentrated, dried under vacuum to obtain the crude product, and purified by column chromatography (V). 二氯甲烷 V 甲醇=50:1-20:1). Fine purification: Crude tinidazole was dissolved in 70% ethanol-water at twice the weight of crude tinidazole, and 1.5% of the weight of crude tinidazole was added with activated carbon for decolorization for 30 min. The solution was filtered while hot, and the filtrate was cooled to crystallize. After filtration and drying, a white solid (404 mg, 1.60 mmol) was obtained, with a yield of 53.35% [based on 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5].

[0065] The content of tinidazole-D5 in this comparative example was determined to be 99.91% by high performance liquid chromatography (mobile phase: 0.1% phosphoric acid water; acetonitrile).

[0066] Tinidazole-D5 Isotope Abundance Detection Using the same detection steps as in Example 1, the isotopic abundance percentages of D5-D0 were normalized and the data are listed in Table 4.

[0067] Table 4. Isotopic abundance of tinidazole-D5 in Comparative Example 1 Calculations showed that the isotope abundance in tinidazole-D5 in this comparative example was 88.41%, proving that the synthetic method would result in isotope dilution and could not obtain a product with stable isotope labeling.

[0068] Comparative Example 2 Synthesis of intermediate A1 In a 100 mL three-necked round-bottom flask, 2-mercaptoethanol (0.60 mL, 8.45 mmol), cesium carbonate (3.59 g, 1.50 eq), and potassium iodide (83 mg, 0.05 eq) were added sequentially and dissolved in acetone (30 mL) under nitrogen protection. Then, 2-bromoethane-D5 (1.39 mL, 2.00 eq) was added and the mixture was refluxed for 12 hours. The reaction progress was monitored by TLC (V0.05). 二氯甲烷 V 甲醇 =20:1, potassium permanganate for color development). After the reaction, the reaction flask was cooled to room temperature, the precipitate was removed by filtration, the solvent was evaporated, and 50 mL of diethyl ether was added to dissolve the precipitate. The mixture was then washed and extracted successively with water, NaHCO3, and NaCl aqueous solution. Finally, the organic phase was concentrated, dried under vacuum to obtain the crude product, and subjected to column chromatography (V... 正己烷 V 乙酸乙酯 =2:1~V 二氯甲烷 V 甲醇 The product was purified by a 50:1 ratio to obtain a transparent oil (705 mg, 6.67 mmol), with a yield of 78.96% [based on 2-mercaptoethanol].

[0069] Synthesis of intermediate B1 Add 10 mL of xylene to a 100 mL three-necked round-bottom flask, followed by 2-hydroxyethyl ethyl sulfide-D5 (705 mg, 6.67 mmol) and 2-methyl-5-nitroimidazole (777 mg, 1.00 eq). While stirring, raise the temperature to 60 °C, and while maintaining this temperature, slowly add concentrated sulfuric acid (0.42 mL, 1.20 eq). After the addition is complete, raise the temperature to 140 °C and react for 6 hours, then lower the temperature to 30 °C. Adjust the pH to neutral with ammonia. Add an appropriate amount of xylene to the reaction solution, filter, add an appropriate amount of n-pentane, and freeze to crystallize. Perform column chromatography (V... 二氯甲烷 V 甲醇 The mixture was purified from 50:1 to 20:1 to give a white solid (724 mg, 3.34 mmol) in a yield of 50.21% [based on 2-hydroxyethyl ethyl sulfide-D5].

[0070] Synthesis of Tinidazole-D5 At 0 °C, m-chloroperoxybenzoic acid (1.43 g, 2.5 eq) was dissolved in 0.75 M sodium bicarbonate (100 mL), followed by the addition of the intermediate from the second step reaction (724 mg, 3.34 mmol). The reaction was carried out at low temperature for 2–3 hours, with the reaction progress monitored by TLC (V0.05). 二氯甲烷 V 甲醇 =3:1). NaCl was added to the saturated aqueous phase of the solution, followed by extraction with dichloromethane, drying with anhydrous sodium sulfate, concentrating the organic phase, and vacuum drying to obtain the crude product. The product was then purified by column chromatography (V...). 二氯甲烷 V 甲醇 =50:1-20:1), fine purification: Crude tinidazole was dissolved in 70% ethanol-water at twice the weight of crude tinidazole, and 1.2% of the weight of crude tinidazole was added to activated carbon for decolorization for 30 min. The solution was filtered while hot, and the filtrate was cooled to crystallize. After filtration and drying, a white solid (529 mg, 2.10 mmol) was obtained, with a yield of 62.77% [calculated as 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5].

[0071] The content of tinidazole-D5 in this comparative example was determined to be 99.95% by high performance liquid chromatography (mobile phase: 0.1% phosphoric acid water; acetonitrile).

[0072] Tinidazole-D5 Isotope Abundance Detection Using the same detection steps as in Example 1, the isotopic abundance percentages of D5-D0 were normalized and the data are listed in Table 5.

[0073] Table 5. Isotopic abundance of tinidazole-D5 in Comparative Example 2 Calculations showed that the isotope abundance of tinidazole-D5 in this comparative example was 93.30%, proving that the synthetic method would result in isotope dilution and could not obtain a product with stable isotope labeling.

[0074] In summary, both Comparative Examples 1 and 2 exhibited isotope dilution, highlighting the need to consider the mildness and feasibility of the reaction when designing synthetic routes. Using concentrated sulfuric acid in the condensation reaction introduces a large number of protons, leading to hydrogen-deuterium exchange and isotope dilution. Furthermore, this approach requires strict and complex temperature control. The oxidation reaction, similarly conducted under relatively vigorous conditions, is also prone to deuterium loss, making it impossible to guarantee the isotope abundance of the final product.

[0075] Applications of deuterated tinidazole (recovery data) Taking tinidazole-D5 in Example 1 as an example (standard purity >98%), the national standard SN / T 1626-2019 uses the external standard method to detect the tinidazole content in meat products, while this invention uses the tinidazole-D5 isotope internal standard method for quantitative testing. The national standard SN / T 1626-2019 specifies the concentration and recovery rate data of nitroimidazole drugs added to the sample matrix: Table 6. Concentration and recovery data of nitroazole in four types of sample matrices. The specific detection method of this invention refers to this standard, and quantitative testing is performed using the tinidazole-D5 isotope internal standard method. Chicken, pork, beef, and sausage were selected as samples, and the concentration and recovery rate of tinidazole-D5 in the matrix are shown in Table 7. Table 7. Concentration and recovery data of tinidazole-D5 in four types of sample matrices. The data comparison results show that the tinidazole-D5 isotope internal standard method has a higher recovery rate from the matrix than the tinidazole external standard method, and the method can effectively reduce the influence of matrix interference.

[0076] The embodiments described in this invention are for illustrative purposes only and do not constitute a limitation on the scope of the claims. Other substantially equivalent substitutions that can be conceived by those skilled in the art are all within the scope of protection of this invention.

Claims

1. A method for synthesizing tinidazole deuterated compounds, wherein the tinidazole deuterated compounds are shown in Formula I. Formula I in, One or more of R1 to R5 are deuterium, and the rest are hydrogen. Its characteristic is that it includes the following steps: Intermediate A was reacted with 2-methyl-5-nitroimidazole at 0-5°C to prepare intermediate B, as shown in the following reaction formula: ， Intermediate B is prepared from the deuterated tinidazole by oxidation of m-chloroperoxybenzoic acid, wherein the oxidation is carried out at 0-5°C under alkaline conditions.

2. The method for synthesizing the deuterated tinidazole compound as described in claim 1, characterized in that... The alkaline conditions are as follows: intermediate B and m-chloroperoxybenzoic acid are added to an alkaline solution to carry out the oxidation.

3. The method for synthesizing the deuterated tinidazole compound as described in claim 2, characterized in that... The alkaline solution is an aqueous solution of one or more of sodium hydroxide and sodium methoxide.

4. The method for synthesizing the deuterated tinidazole compound as described in claim 3, characterized in that... The molar concentration of the alkaline solution is 0.40 M-1.0 M.

5. The method for synthesizing the deuterated tinidazole compound as described in claim 1, characterized in that... The molar ratio of intermediate B to m-chloroperoxybenzoic acid is 1:(2.0-2.5).

6. The method for synthesizing the deuterated tinidazole compound as described in claim 1, characterized in that... The aforementioned photoelongation reaction includes the following steps: In the first reaction vessel, intermediate A, 2-methyl-5-nitroimidazole, and triphenylphosphine were dissolved in tetrahydrofuran. Diisopropyl azodicarbonate dissolved in tetrahydrofuran is added dropwise to the first reaction vessel.

7. The method for synthesizing the deuterated tinidazole compound as described in claim 6, characterized in that... The molar ratio of intermediate A: triphenylphosphine: diisopropyl azodicarboxylate is 1:(1.0-2.0):(1.0-2.0).

8. The method for synthesizing the deuterated tinidazole compound as described in claim 1, characterized in that... The process also includes the step of nucleophilic substitution of 2-bromoethane deuterated product with 2-mercaptoethanol to prepare intermediate A, as shown in the following reaction formula: 。 9. The method for synthesizing the deuterated tinidazole compound as described in claim 8, characterized in that... The nucleophilic substitution includes the following steps: In the second reaction vessel: a) 2-Mercaptoethanol, and b) One or more of potassium carbonate, sodium bicarbonate, sodium carbonate, and cesium carbonate, and c) One or both of potassium iodide or cuprous iodide. Soluble in acetone or tetrahydrofuran under nitrogen protection. Add the 2-bromoethane deuterated product to the second reaction vessel.

10. The method for synthesizing the deuterated tinidazole compound as described in claim 8, characterized in that... The molar ratio of 2-thioethanol to 2-bromoethane deuterated product is 1:(1.5-2.2).

11. The method for synthesizing the deuterated tinidazole compound as described in claim 1, characterized in that... The aforementioned deuterated tinidazole is: ,or .

Images