Synthesis method for deuterated tinidazole

Abstract

The present invention relates to a synthesis method for a deuterated tinidazole. The deuterated tinidazole is as represented by formula I, wherein one or more of R1-R5 are deuterium, and the rest is hydrogen. The synthesis method comprises the step of oxidizing an intermediate B with m-chloroperoxybenzoic acid to prepare the deuterated tinidazole, wherein the oxidation is carried out under low-temperature and alkaline conditions; and the intermediate B is as represented by formula II. By using the synthesis method of the present invention, the problems of deuterium shedding and isotope dilution in the reaction process can be avoided, and the stable isotope-labeled deuterated tinidazole with the isotope abundance of > 98% is obtained with a higher yield. The prepared deuterated tinidazole can be used as an isotope internal standard reagent for the quantitative detection of tinidazole, which, for example, can be used to detect residual tinidazole in meat products by means of an isotope internal standard method, and has the advantages of having high recovery efficiency from a matrix, and being environmentally friendly and safe.

Description

Synthesis method of deuterated tinidazole Technical Field

[0001] The present invention relates to a method for synthesizing a deuterated tinidazole. Background Art

[0002] Tinidazole, a derivative of metronidazole (Metronidazole), chemically known as 1-(2-ethylsulfonylethyl)-2-methyl-5-nitro-1H-imidazole, is a highly effective anti-amoebic and anti-anaerobic drug, commonly used clinically to treat anaerobic and protozoal infections. It can also kill Helicobacter pylori. Compared with the traditional anti-anaerobic drug metronidazole, tinidazole offers advantages such as higher efficacy and fewer side effects. Regarding the preparation of tinidazole, most reported synthetic routes involve using hydroxyethyl sulfide and 2-methyl-5-nitroimidazole as raw materials, dissolving them in methyl isobutyl ketone and subjecting them to a Lewis acid-catalyzed condensation reaction to obtain the condensate, 1-(ethylthioethyl)-2-methyl-5-nitroimidazole. The final product is then oxidized with 30% hydrogen peroxide under acidic conditions. This process has the following drawbacks: it requires a large number of chemicals, generates significant site pollution, is complex, has a long production cycle, and produces poor product quality.

[0003] A safe food supply supports a country's economy, trade, and tourism, fostering food and nutrition security and supporting sustainable development. Numerous methods have been reported for detecting drug residues in meat products. Liquid chromatography-mass spectrometry (LC-MS / MS) combines the advantages of ultra-high performance liquid chromatography (UPLC) and mass spectrometry (MS / MS). It offers high specificity, high sensitivity, and suitability for analyzing trace components in complex matrices, making it the primary method for veterinary drug residue detection. According to the standard test method "SN / T 1626-2019: Determination of Metronidazole, Tinidazole, Ornidazole, Ronidazole, Dimetridazole, and Secnidazole Residues in Exported Meat and Meat Products by Liquid Chromatography-Mass Spectrometry / Mass Spectrometry," the detection of tinidazole in meat products uses an external standard method. Stable isotope internal standard detection reduces the effects of matrix interference (the matrix refers to components in the sample other than the analyte. Since the matrix often significantly interferes with the analysis of the analyte and affects the accuracy of the analytical results, these effects and interferences are known as matrix effects), resulting in higher sensitivity and accuracy.

[0004] During the synthesis of stable isotope internal standard reagents, to ensure that their isotopic abundance (>98%) is not diluted and high purity, it is necessary to rationally design the synthesis route of the stable isotope-labeled product, considering the mild feasibility of each reaction process. During the reaction process, the isotope raw material 2-hydroxyethyl ethyl sulfide deuterated is not currently available on the market, so it needs to be synthesized independently. When designing the synthesis route, it is necessary to consider experimental costs and the optimal route. Currently, most literature uses ethanethiol and 2-bromoethanol to react to obtain 2-hydroxyethyl ethyl sulfide. However, the market price of stable isotope-labeled ethanethiol deuterated is high and the market supply is small. Therefore, a better synthetic raw material is needed to synthesize the desired target product.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a method for synthesizing a deuterated tinidazole labeled with a stable isotope and having an isotope abundance of more than 98%.

[0007] In order to achieve the above object, the present invention provides a method for synthesizing a deuterated tinidazole, wherein the deuterated tinidazole is as shown in Formula I:

[0008] wherein one or more of R1 to R5 are deuterium, and the rest are hydrogen,

[0009] The method comprises the following steps: intermediate B is oxidized by m-chloroperbenzoic acid to prepare the deuterated tinidazole, wherein:

[0010] The oxidation is carried out at low temperature and alkaline conditions.

[0011] The intermediate B is shown in Formula II:

[0012] In some embodiments of the present invention, the alkaline condition is to add the intermediate B and m-chloroperbenzoic acid into an alkaline solution to carry out the oxidation.

[0013] In some embodiments of the present invention, the alkaline solution is an aqueous solution of one or more of sodium hydroxide and sodium methoxide.

[0014] In some embodiments of the present invention, the molar concentration of the alkaline solution is 0.40M-1.0M.

[0015] In some embodiments of the present invention, the molar ratio of the intermediate B to meta-chloroperbenzoic acid is 1:(2.0-2.5).

[0016] In some embodiments of the present invention, the step of: performing a Mitsunobu reaction of intermediate A with 2-methyl-5-nitroimidazole at low temperature to prepare intermediate B is further included. The reaction formula is as follows:

[0017] In some embodiments of the present invention, the Mitsunobu reaction comprises the steps of:

[0018] In a first reaction vessel, intermediate A, 2-methyl-5-nitroimidazole and triphenylphosphine are dissolved in tetrahydrofuran.

[0019] Diisopropyl azodicarboxylate dissolved in tetrahydrofuran was added dropwise to the first reaction vessel.

[0020] 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).

[0021] In some embodiments of the present invention, the low temperature is 0-5°C.

[0022] In some embodiments of the present invention, the step of: preparing intermediate A by nucleophilic substitution of 2-bromoethane deuterate with 2-mercaptoethanol is further included, and the reaction formula is as follows:

[0023] In some embodiments of the present invention, the nucleophilic substitution comprises the steps of:

[0024] In a second reaction vessel:

[0025] a). 2-mercaptoethanol, and

[0026] b) one or more of potassium carbonate, sodium bicarbonate, sodium carbonate, cesium carbonate, and

[0027] c) One or both of potassium iodide and cuprous iodide,

[0028] Dissolve in acetone or tetrahydrofuran under nitrogen protection.

[0029] To the second reaction vessel was added 2-bromoethane deuterate.

[0030] In some embodiments of the present invention, the molar ratio of 2-mercaptoethanol:deuterated bromoethane is 1:(1.5-2.2).

[0031] In some embodiments of the present invention, the deuterated tinidazole is:

[0032] The synthesis method of the present invention avoids deuterium shedding and isotope dilution during the reaction process, resulting in a stable isotope-labeled deuterated tinidazole with an isotope abundance of >98% and a high yield. The prepared deuterated tinidazole can be used as an isotope internal standard reagent for the quantitative detection of tinidazole, such as in the isotope internal standard method for detecting tinidazole residues in meat products. It also offers the advantages of high matrix recovery efficiency and environmental safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a synthetic process route for deuterated tinidazole.

[0034] FIG2 is a hydrogen nuclear magnetic resonance spectrum of the stable isotope labeled tinidazole-D5 obtained in Example 1 of the present invention ( 1 H-NMR—) spectrum.

[0035] FIG3 is a liquid chromatography purity (LC) spectrum of the stable isotope-labeled tinidazole-D5 obtained in Example 1 of the present invention.

[0036] FIG4 is a liquid chromatography-mass spectrometry (LC MS) spectrum of the stable isotope-labeled tinidazole-D5 obtained in Example 1 of the present invention.

[0037] Figure 5 is a stable isotope labeled tinidazole-D3 obtained in Example 3 of the present invention 1 H-NMR spectrum.

[0038] FIG6 is an LC spectrum of the stable isotope labeled tinidazole-D3 obtained in Example 3 of the present invention. DETAILED DESCRIPTION

[0039] 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 the event of a conflict, the descriptions and definitions herein shall prevail.

[0040] The term "deuterated tinidazole" refers to a compound having the general formula shown in Formula I:

[0041] Among them, one or more of R1 to R5 is deuterium (-D), and the rest are hydrogen (-H). According to the number of deuterium in the molecular structure of the deuterated compound, the deuterated compound of tinidazole is also referred to as "tinidazole-D" in this application. n ", where n is an integer from 1 to 5, corresponding to the number of deuterium in the molecular structure. For example, tinidazole-D5 means that R1 to R5 are all deuterium, tinidazole-D4 means that 4 of R1 to R5 are deuterium and the remaining one is hydrogen, tinidazole-D3 means that 3 of R1 to R5 are deuterium and the remaining 2 are hydrogen, and so on.

[0042] The term "low temperature" refers to a temperature between 0 and 5°C.

[0043] The reaction principle of the intermediate chloroperbenzoic acid used as an oxidant to oxidize the sulfide group (-S-) to the sulfone group (-SO2-) in the present application is known.

[0044] The reaction principle of preparing thioether by nucleophilic substitution used in the present application is known.

[0045] The reaction principle of the Mitsunobu reaction used in the present 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)

[0046] In view of the problem that isotope dilution is prone to occur in the synthesis methods of deuterated tinidazole in the prior art, the present invention provides a different synthesis method of deuterated tinidazole, comprising the steps of: oxidizing intermediate B with m-chloroperbenzoic acid to obtain the deuterated tinidazole, wherein the oxidation is carried out at low temperature and alkaline conditions, and the intermediate B is shown in Formula II:

[0047] The molecular formula of meta-chloroperbenzoic acid is shown in Formula III:

[0048] In the above reaction, meta-chloroperbenzoic acid, as a relatively mild oxidant, can oxidize the sulfide group -S- in Formula II to a sulfone group -SO2- to obtain a deuterated tinidazole. Low temperature can avoid the problem of deuterium shedding that occurs during a vigorous reaction. In addition, an ice bath can effectively control the reaction temperature and absorb the heat generated by the reaction, thereby avoiding deuterium shedding that occurs during a vigorous reaction. Specifically, one of the feasible reaction operation steps of the above reaction includes dissolving meta-chloroperbenzoic acid in an alkaline solution, then adding intermediate B, and reacting at low temperature to prepare the deuterated 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 0.40M-1.0M. Preferably, the molar ratio of intermediate B: meta-chloroperbenzoic acid is 1:(2.0-2.5).

[0049] Furthermore, intermediate B can be prepared by Mitsunobu reaction of intermediate A with 2-methyl-5-nitroimidazole, as shown in the following reaction formula:

[0050] In the above-mentioned step of preparing intermediate A from intermediate B, the Mitsunobu reaction is used. Under the action of triphenylphosphine (PPh3) and diethyl azodicarboxylate (DEAD), the amino group on 2-methyl-5-nitroimidazole acts as a nucleophile in the Mitsunobu reaction, replacing the hydroxyl group on intermediate A to generate intermediate B. The inventors have found that when intermediate A is used to prepare intermediate B using the Mitsunobu reaction principle, there are fewer by-products, which can effectively improve the yield of intermediate B, and the reaction process can also avoid the shedding of deuterium. Specifically, one of the feasible reaction operation steps includes: dissolving intermediate A, 2-methyl-5-nitroimidazole and triphenylphosphine in tetrahydrofuran, slowly adding diisopropyl azodicarboxylate dissolved in tetrahydrofuran, and carrying out the Mitsunobu reaction in an ice-water bath. Preferably, the molar ratio of intermediate A: triphenylphosphine: diisopropyl azodicarboxylate is 1: (1.0-2.0): (1.0-2.0).

[0051] Furthermore, intermediate A can be prepared by nucleophilic substitution of 2-bromoethane deuterate with 2-mercaptoethanol, and the reaction formula is as follows:

[0052] There is currently no public method for preparing intermediate A. This application selects 2-bromoethane deuterated product and 2-mercaptoethanol as reaction raw materials, and realizes the synthesis of intermediate A through nucleophilic substitution reaction. In addition, the two reaction raw materials selected are relatively easy to obtain commercial products, and the raw material price is low, which makes the cost of synthesis lower. Specifically, one of the feasible reaction operation steps includes: adding 2-mercaptoethanol, potassium carbonate and potassium iodide to a reaction vessel in sequence, dissolving them in acetone under nitrogen protection, and then adding 2-bromoethane deuterated product to carry out the nucleophilic reaction. Preferably, the molar ratio of 2-mercaptoethanol: deuterated bromoethane is 1: (1.5-2.2).

[0053] The above preparation method is further described below through specific examples.

[0054] Related reagents

[0055] All raw materials used in the examples are commercially available and have industrial grade purity.

[0056] Deuterated bromoethane (Nanjing Haolu Biotechnology Co., Ltd.); concentrated sulfuric acid and boric acid (Sinopharm Group 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, and methanol (analytical grade, Shanghai Anpu Laboratory Technology Co., Ltd.); 2-methyl-5-nitroimidazole and diisopropyl azodicarboxylate (Shanghai MacLean Biochemical Technology Co., Ltd.); m-chloroperbenzoic acid and triphenylphosphine (Shanghai Aladdin Biochemical Technology Co., Ltd.). Unless otherwise specified, all reagents were used as received.

[0057] Example 1 Synthesis of Tinidazole-D5

[0058] Intermediate A1: Synthesis of 2-hydroxyethyl ethyl sulfide-D5

[0059] 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 in sequence and dissolved in acetone (30 mL) under nitrogen protection. 2-bromoethane-D5 (1.30 mL, 2.00 eq) was then added and refluxed for 12 hours. The reaction progress (V) was monitored by TLC. 二氯甲烷 :V 甲醇 =20:1, potassium permanganate color development). The reaction formula is as follows:

[0060] After the reaction, the reaction flask was cooled to room temperature, the precipitate was removed by filtration, the solvent was dried and 50 mL of ether was added to dissolve it, and the mixture was washed with water, NaHCO3, and NaCl aqueous solution in sequence. The organic phase was finally concentrated and dried in vacuo to obtain a crude product. The crude product was purified by column chromatography (V 正己烷 :V 乙酸乙酯 =2:1~V 二氯甲烷 :V 甲醇 =50:1) to give a transparent oil (730 mg, 6.90 mmol) with a yield of 87.45% [based on 2-mercaptoethanol].

[0061] Intermediate B1: Synthesis of 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5

[0062] 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. Diisopropyl azodicarboxylate (1.38 g, 1.00 eq) dissolved in tetrahydrofuran (5 mL) was then slowly added dropwise. The mixture was reacted in an ice-water bath for 3 hours. The reaction progress was monitored by TLC (V 二氯甲烷 :V 甲醇 =5:1). The reaction formula is as follows:

[0063] After the reaction, the solvent was dried, the organic phase was concentrated, and the crude product was obtained by vacuum drying. 二氯甲烷 :V 甲醇 =50:1-20:1) was purified to give a white solid (900 mg, 4.16 mmol) with a yield of 60.29% [based on 2-hydroxyethyl ethyl sulfide-D5].

[0064] Synthesis of Tinidazole-D5

[0065] At 0°C, m-chloroperbenzoic acid (1.58 g, 2.20 eq) was dissolved in 0.50 M aqueous sodium hydroxide solution (100 mL), and then 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5 (900 mg, 4.16 mmol) was added. The reaction was allowed to proceed at low temperature for 2 hours. The reaction progress was monitored by TLC during the reaction (V 二氯甲烷 :V 甲醇 =3:1). The reaction formula is as follows:

[0066] After the reaction, NaCl-saturated aqueous phase was added to the solution, and then extracted with dichloromethane, dried over anhydrous sodium sulfate, and the organic phase was concentrated and dried in vacuo to obtain a crude product, which was purified by column chromatography (V 二氯甲烷 :V 甲醇 =50:1-20:1). Fine purification: The crude tinidazole was dissolved in 70% ethanol water with a mass twice that of the crude tinidazole, and decolorized with activated carbon (1.8% by weight of the crude tinidazole-D5) for 30 minutes. The mixture was filtered while hot, and the filtrate was cooled and crystallized. The solution was filtered and dried to give a white solid (716 mg, 2.84 mmol). The yield was 68.26% [based on 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5].

[0067] The qualitative analysis of H NMR spectrum (see Figure 2) shows that the H NMR spectrum data of tinidazole is 1H NMR (CD3OD, 400MHz, ppm) δ: 7.95 (s, 1H), 4.81 (t, J = 6.9Hz, 2H), 3.63 (t, J = 6.8Hz, 2H), 3.15 (q, J = 7.4Hz, 2H), 2.57 (s, 3H), 1.34 (d, J = 7.4Hz, 3H); The H NMR spectrum data of tinidazole-D5 are 1 H 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). It can be seen that the absorption peaks at δ: 7.95 (s, 1H), 4.80 (d, J = 6.8 Hz, 2H), 3.63 (t, J = 6.8 Hz, 2H), and 2.57 (s, 3H) are consistent, while the absorption peaks at δ: 3.15 (q, J = 7.4 Hz, 2H) and 1.34 (d, J = 7.4 Hz, 3H) have disappeared. This proves that none of the five deuterium groups on the ethyl group in the structure have been detached, and the resulting compound structure is tinidazole-D5.

[0068] The content of tinidazole-D5 was determined by high performance liquid chromatography (mobile phase: 0.1% phosphoric acid water; acetonitrile) to be 99.97%. (See Figure 3)

[0069] Tinidazole-D5 isotope abundance detection

[0070] The MRM mode in tandem mass spectrometry was used to determine the isotope abundance. The parent ion-quantification ion of each substance was selected, and the peak area obtained was approximately equivalent to its respective isotope label. The measured peak area was then normalized to obtain the isotope abundance.

[0071] The "simple mass cluster" method was used for determination, taking the 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 measured by mass spectrometry (see Figure 4), and the peak areas were approximately equal to those of tinidazole-D5, tinidazole-D4, tinidazole-D3, tinidazole-D2, tinidazole-D1, and tinidazole-D0, respectively. 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.

[0072] Table 1 Isotopic abundance of tinidazole-D5 in Example 1

[0073] Finally, through the formula:

[0074] in,

[0075] D% indicates isotope abundance, atom%D;

[0076] i represents the number of D markers, i = 0 to 5;

[0077] R i It represents the percentage of the peak area of ​​i D markers. Taking 5 D markers as an example, the calculation formula is: R6 = A(D) / [A(D5)+A(D4)+A(D3)+A(D2)+A(D1)+A(D0)];

[0078] n represents the total number of D markers, n=5.

[0079] According to formula (i), the isotopic abundance of tinidazole-D5 in this example is 98.81%, which proves that the synthesis method does not cause isotope dilution and can obtain a stable isotope-labeled product.

[0080] Example 2 Synthesis of Tinidazole-D5

[0081] Intermediate A1: Synthesis of 2-hydroxyethyl ethyl sulfide-D5

[0082] In a 100mL three-necked round-bottom flask, 2-mercaptoethanol (0.40mL, 5.63mmol), sodium bicarbonate (1.41g, 2.50eq) and potassium iodide (56mg, 0.06eq) were added in sequence and dissolved in acetone (25mL) under nitrogen protection. Then, 2-bromoethane-D5 (1.00mL, 2.20eq) was added and refluxed for 12 hours. The reaction progress was monitored by TLC (V dichloromethane: V methanol = 20:1, potassium permanganate color development). After the reaction, the reaction flask was cooled to room temperature, the precipitate was removed by filtration, the solvent was dried and 50mL of ether was added to dissolve it, and the mixture was washed and extracted with water, NaHCO3, and NaCl aqueous solution in sequence. Finally, the organic phase was concentrated and dried in vacuo to obtain a crude product, which was then purified by column chromatography (V 正己烷 :V 乙酸乙酯 =2:1~V 二氯甲烷 :V 甲醇 =50:1) to give a transparent oil (508 mg, 4.80 mmol) with a yield of 85.27% [based on 2-mercaptoethanol].

[0083] Intermediate B1: Synthesis of 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5

[0084] 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. Diisopropyl azodicarboxylate (1.25 g, 1.30 eq) dissolved in tetrahydrofuran (4 mL) was then slowly added dropwise. The mixture was reacted in an ice-water bath for 3 hours. The reaction progress was monitored by TLC (V 二氯甲烷 :V 甲醇 =5:1). After the reaction, the solvent was dried, the organic phase was concentrated, and vacuum dried to obtain a crude product, which was then purified by column chromatography (V 二氯甲烷 :V 甲醇 =50:1-20:1) was purified to give a white solid (552 mg, 2.83 mmol) with a yield of 59.11% [based on 2-hydroxyethyl ethyl sulfide-D5].

[0085] Synthesis of Tinidazole-D5

[0086] At 0°C, m-chloroperbenzoic 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 allowed to proceed at low temperature for 2-3 hours. The reaction progress was monitored by TLC during the reaction (V 二氯甲烷 :V 甲醇 =3:1). After the reaction, NaCl-saturated aqueous phase was added to the solution, and then extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the organic phase was concentrated and dried in vacuo to obtain a crude product, which was purified by column chromatography (V 二氯甲烷 :V 甲醇 =50:1-20:1). Fine purification: The crude tinidazole was dissolved in 70% ethanol water with a mass twice that of the crude tinidazole, and decolorized with activated carbon (1.5% by weight of the crude tinidazole-D5) for 30 minutes. The mixture was filtered while hot, and the filtrate was cooled and crystallized. The solution was filtered and dried to give a white solid (469 mg, 1.86 mmol). The yield was 65.72% [based on 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5].

[0087] The content of tinidazole-D5 in this comparative example was determined by high performance liquid chromatography (mobile phase: 0.1% phosphoric acid water; acetonitrile) to be 99.95%.

[0088] Tinidazole-D5 isotope abundance detection

[0089] The same detection steps as in Example 1 were used to normalize the isotopic abundance percentages of D5-D0. The data are listed in Table 2.

[0090] Table 2 Isotopic abundance of tinidazole-D5 in Example 2

[0091] The isotopic abundance of tinidazole-D5 in this example is 98.75%, which proves that the synthesis method does not cause isotope dilution and can obtain a stable isotope-labeled product.

[0092] Example 3 Synthesis of Tinidazole-D3

[0093] Intermediate A2: Synthesis of 2-hydroxyethyl ethyl sulfide-D3

[0094] 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 (67 mg, 0.05 eq) were added in sequence and dissolved in tetrahydrofuran (30 mL) under nitrogen protection. 2-bromoethane-D3 (1.21 mL, 2.10 eq) was then added and refluxed for 12 hours. The reaction progress (V) was monitored by TLC. 二氯甲烷 :V 甲醇 =20:1, potassium permanganate color development). The reaction formula is as follows:

[0095] After the reaction, the reaction flask was cooled to room temperature, the precipitate was removed by filtration, the solvent was dried and 50 mL of ether was added to dissolve it, and the mixture was washed with water, NaHCO3, and NaCl aqueous solution in sequence. The organic phase was finally concentrated and dried in vacuo to obtain a crude product. The crude product was purified by column chromatography (V 正己烷 :V 乙酸乙酯 =2:1~V 二氯甲烷 :V 甲醇 =50:1) to give a transparent oil (625 mg, 5.91 mmol) with a yield of 83.96% [based on 2-mercaptoethanol].

[0096] Intermediate B2: Synthesis of 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D3

[0097] 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. Diisopropyl azodicarboxylate (1.42 g, 1.20 eq) dissolved in tetrahydrofuran (5 mL) was then slowly added dropwise. The mixture was reacted in an ice-water bath for 3 hours. The reaction progress was monitored by TLC (V 二氯甲烷 :V 甲醇 =5:1). The reaction formula is as follows:

[0098] After the reaction, the solvent was dried, the organic phase was concentrated, and the crude product was obtained by vacuum drying. 二氯甲烷 :V 甲醇 =50:1-20:1) was purified to give a white solid (750 mg, 3.47 mmol) with a yield of 58.78% [based on 2-hydroxyethyl ethyl sulfide-D3].

[0099] Synthesis of Tinidazole-D3

[0100] At 0°C, m-chloroperbenzoic 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 allowed to proceed at low temperature for 2-3 hours. The reaction progress was monitored by TLC during the reaction (V 二氯甲烷 :V 甲醇 =3:1). The reaction formula is as follows:

[0101] After the reaction, NaCl-saturated aqueous phase was added to the solution, and then extracted with dichloromethane, dried over anhydrous sodium sulfate, and the organic phase was concentrated and dried in vacuo to obtain a crude product, which was purified by column chromatography (V 二氯甲烷 :V 甲醇 =50:1-20:1). Fine purification: The crude tinidazole was dissolved in 70% ethanol water with a mass twice that of the crude tinidazole, and decolorized with activated carbon (1.6% by weight of the crude tinidazole-D3) for 30 minutes. The mixture was filtered while hot, and the filtrate was cooled and crystallized. The solution was filtered and dried to give a white solid (584 mg, 2.32 mmol). The yield was 66.96% [based on 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D3].

[0102] The qualitative analysis of H NMR spectrum (see Figure 5) shows that the H NMR spectrum data of tinidazole is 1 H NMR (CD3OD, 400MHz, ppm) δ: 7.95 (s, 1H), 4.81 (t, J = 6.9Hz, 2H), 3.63 (t, J = 6.8Hz, 2H), 3.15 (q, J = 7.4Hz, 2H), 2.57 (s, 3H), 1.34 (d, J = 7.4Hz, 3H); The H NMR spectrum data of tinidazole-D3 are 1H 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). It can be seen that the absorption peaks at 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), and 2.48 (s, 3H) are consistent, while the absorption peak at δ: 1.34 (d, J = 7.4 Hz, 3H) disappears. This proves that the three deuterium groups on the methyl group in the structure have not been detached, and the resulting compound structure is tinidazole-D3.

[0103] The content of tinidazole-D3 was determined by high performance liquid chromatography (mobile phase: 0.1% phosphoric acid water; acetonitrile) to be 99.81%. (See Figure 6)

[0104] Tinidazole-D3 isotope abundance detection

[0105] The same detection steps as in Example 1 were used to normalize the isotopic abundance percentages of D3-D0. The data are listed in Table 3.

[0106] Table 3 Isotopic abundance of tinidazole-D3 in Example 3

[0107] The isotopic abundance of tinidazole-D3 in this example is 98.41%, which proves that the synthesis method does not cause isotope dilution and can obtain a stable isotope-labeled product.

[0108] Comparative Example

[0109] This application selected two existing synthesis methods (Comparative Example 1 and Comparative Example 2) to synthesize tinidazole-D5, and detected 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.

[0110] In short, the main difference between the synthesis scheme of Comparative Example 1 and that of Example 1 is that the Mitsunobu reaction is not used in the preparation of Intermediate B1, and a conventional concentrated sulfuric acid dehydration esterification reaction is used instead. In the step of oxidizing Intermediate B1 to prepare tinidazole D5, hydrogen peroxide is used as the oxidant. The main difference between the synthesis scheme of Comparative Example 2 and that of Example 1 is that the Mitsunobu reaction is not used in the preparation of Intermediate B1, and a conventional concentrated sulfuric acid dehydration esterification reaction is used instead.

[0111] Comparative Example 1

[0112] Synthesis of intermediate A1

[0113] 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 in sequence and dissolved in acetone (30 mL) under nitrogen protection. 2-bromoethane-D5 (0.87 mL, 1.50 eq) was then added and refluxed for 12 hours. The reaction progress (V) was monitored by TLC. 二氯甲烷 :V 甲醇 =20:1, potassium permanganate color development). After the reaction, the reaction flask was cooled to room temperature, the precipitate was filtered off, the solvent was dried, 50 mL of ether was added to dissolve, and the mixture was washed with water, NaHCO3, and NaCl aqueous solution in sequence. The organic phase was finally concentrated and dried in vacuo to obtain a crude product, which was then purified by column chromatography (V 正己烷 :V 乙酸乙酯 =2:1~V 二氯甲烷 :V 甲醇 =50:1) to give a transparent oil (596 mg, 5.64 mmol) with a yield of 80.05% [based on 2-mercaptoethanol].

[0114] Synthesis of intermediate B1

[0115] In a 100 mL three-necked round-bottom flask, 10 mL of toluene was added, followed by 2-hydroxyethyl ethyl sulfide-D5 (596 mg, 5.64 mmol) and 2-methyl-5-nitroimidazole (657 mg, 1.00 eq). The temperature was raised to 50° C. while stirring, and concentrated sulfuric acid (0.30 mL, 1.00 eq) was slowly added dropwise while maintaining the temperature at this temperature. After the addition was complete, the temperature was raised to 88° C. and reacted for 6 hours, then cooled to 35° C., and the pH was adjusted to neutral with aqueous ammonia. An appropriate amount of toluene was added to the reaction solution, filtered, and the filter cake was washed with toluene. The filtrate was collected and the solvent was dried, the organic phase was concentrated, and vacuum dried to obtain a crude product, which was purified by column chromatography (V 二氯甲烷 :V 甲醇 =50:1-20:1) was purified to give a white solid (653 mg, 3.01 mmol) with a yield of 53.36% [based on 2-hydroxyethyl ethyl sulfide-D5].

[0116] Synthesis of Tinidazole-D5

[0117] The intermediate of 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 about 35°C for about 100-135 minutes, and the reaction progress (V 二氯甲烷 :V 甲醇=3:1). After the reaction is completed, the mixture is extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the organic phase is concentrated and dried under vacuum to obtain a crude product, which is then purified by column chromatography (V 二氯甲烷 :V 甲醇 =50:1-20:1). Fine purification: The crude tinidazole was dissolved in 70% ethanol water with a mass twice that of the crude tinidazole, and decolorized with activated carbon at a concentration of 1.5% by weight of the crude tinidazole for 30 min. The mixture was filtered while hot, and the filtrate was cooled and crystallized. The solution was filtered and dried to obtain a white solid (404 mg, 1.60 mmol). The yield was 53.35% [based on 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5].

[0118] The content of tinidazole-D5 in this comparative example was determined by high performance liquid chromatography (mobile phase: 0.1% phosphoric acid water; acetonitrile) to be 99.91%.

[0119] Tinidazole-D5 isotope abundance detection

[0120] The same detection steps as in Example 1 were used to normalize the isotopic abundance percentages of D5-D0. The data are listed in Table 4.

[0121] Table 4 Isotopic abundance of tinidazole-D5 in comparative example 1

[0122] After calculation, the isotopic abundance of tinidazole-D5 in this comparative example is 88.41%, which proves that the synthesis method will cause isotope dilution and cannot obtain a stable isotope-labeled product.

[0123] Comparative Example 2

[0124] Synthesis of intermediate A1

[0125] 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 in sequence and dissolved in acetone (30 mL) under nitrogen protection. 2-bromoethane-D5 (1.39 mL, 2.00 eq) was then added and refluxed for 12 hours. The reaction progress (V) was monitored by TLC. 二氯甲烷 :V 甲醇 =20:1, potassium permanganate color development). After the reaction, the reaction flask was cooled to room temperature, the precipitate was filtered off, the solvent was dried, 50 mL of ether was added to dissolve, and the mixture was washed with water, NaHCO3, and NaCl aqueous solution in sequence. The organic phase was finally concentrated and dried in vacuo to obtain a crude product, which was then purified by column chromatography (V 正己烷 :V 乙酸乙酯 =2:1~V 二氯甲烷 :V 甲醇=50:1) to give a transparent oil (705 mg, 6.67 mmol) with a yield of 78.96% [based on 2-mercaptoethanol].

[0126] Synthesis of intermediate B1

[0127] In a 100 mL three-necked round-bottom flask, 10 mL of xylene was added, followed by 2-hydroxyethyl ethyl sulfide-D5 (705 mg, 6.67 mmol) and 2-methyl-5-nitroimidazole (777 mg, 1.00 eq). The temperature was raised to 60° C. while stirring, and concentrated sulfuric acid (0.42 mL, 1.20 eq) was slowly added dropwise while maintaining the temperature at this temperature. After the addition was complete, the temperature was raised to 140° C. and the reaction was continued for 6 hours, then cooled to 30° C. The pH was adjusted to neutral with aqueous ammonia, an appropriate amount of xylene was added to the reaction solution, filtered, and then an appropriate amount of n-pentane was added. The solution was frozen for crystallization, and the solid was subjected to column chromatography (V 二氯甲烷 :V 甲醇 =50:1-20:1) was purified to give a white solid (724 mg, 3.34 mmol) with a yield of 50.21% [based on 2-hydroxyethyl ethyl sulfide-D5].

[0128] Synthesis of Tinidazole-D5

[0129] At 0°C, m-chloroperbenzoic acid (1.43 g, 2.5 eq) was dissolved in 0.75 M sodium bicarbonate (100 mL), and then the intermediate of the second step reaction (724 mg, 3.34 mmol) was added. The reaction was allowed to proceed at low temperature for 2-3 hours. During the reaction, the reaction progress was monitored by TLC (V 二氯甲烷 :V 甲醇 =3:1). NaCl saturated aqueous phase was added to the solution, and then extracted with dichloromethane, dried over anhydrous sodium sulfate, and the organic phase was concentrated and dried under vacuum to obtain a crude product, which was purified by column chromatography (V 二氯甲烷 :V 甲醇 =50:1-20:1), fine purification: the crude tinidazole was dissolved in 70% ethanol water with a mass twice that of the crude tinidazole, and decolorized with activated carbon at a concentration of 1.2% by weight of the crude tinidazole for 30 min. The mixture was filtered while hot, and the filtrate was cooled and crystallized, filtered, and dried to give a white solid (529 mg, 2.10 mmol). The yield was 62.77% [based on 1-(β-ethylthioethyl)-2-methyl-5-nitroimidazole-D5].

[0130] The content of tinidazole-D5 in this comparative example was determined by high performance liquid chromatography (mobile phase: 0.1% phosphoric acid water; acetonitrile) to be 99.95%.

[0131] Tinidazole-D5 isotope abundance detection

[0132] The same detection steps as in Example 1 were used to normalize the isotopic abundance percentages of D5-D0, and the data are listed in Table 5.

[0133] Table 5 Isotopic abundance of tinidazole-D5 in comparative example 2

[0134] After calculation, the isotopic abundance of tinidazole-D5 in this comparative example is 93.30%, which proves that the synthesis method will cause isotope dilution and cannot obtain a stable isotope-labeled product.

[0135] In summary, isotope dilution occurred in both Comparative Examples 1 and 2, and it is necessary to consider the mildness and feasibility of the reaction when designing the synthesis route. The use of concentrated sulfuric acid during the condensation reaction will introduce a large amount of protons in the reaction, thereby causing hydrogen-deuterium exchange to lead to isotope dilution, and the scheme is more stringent and troublesome for temperature control. The oxidation reaction is also carried out under more intense reaction conditions, and deuterium shedding is also prone to occur, so that the isotopic abundance of the final product cannot be guaranteed.

[0136] Application of Deuterated Tinidazole (Recovery Data)

[0137] Taking tinidazole-D5 in Example 1 as an example (standard purity >98%), the national standard SN / T 1626-2019 uses an external standard method for the detection of tinidazole content in meat products, while the present invention uses the tinidazole-D5 isotope internal standard method for quantitative testing. The concentration and recovery data of nitroimidazole drugs in the sample matrix in the national standard SN / T 1626-2019 are shown below:

[0138] Table 6 Nitrazol concentration and recovery data in four types of sample matrices

[0139] The specific detection method of the present invention refers to this standard and uses the tinidazole-D5 isotope internal standard method for quantitative testing. Chicken, pork, beef and sausage are selected as samples. The added concentration of tinidazole-D5 in the matrix and the recovery rate data are shown in Table 7:

[0140] Table 7 Concentration and recovery data of tinidazole-D5 in four types of sample matrices

[0141] From the data comparison results, it can be seen that the recovery rate of tinidazole-D5 isotope internal standard method from the matrix is ​​higher than that of tinidazole external standard method, and its method can effectively reduce the influence of matrix interference.

[0142] The embodiments of the present invention are only used to illustrate the present invention and do not limit the scope of the claims. Other substantially equivalent alternatives that can be thought of by those skilled in the art are all within the scope of protection of the present invention.

Claims

1. A method for synthesizing a deuterated tinidazole, wherein the deuterated tinidazole is as shown in Formula I: in, One or more of R1 to R5 is deuterium, and the rest are hydrogen, The method is characterized in that it comprises the steps of: preparing the deuterated tinidazole by oxidizing the intermediate B with m-chloroperbenzoic acid, wherein: The oxidation is carried out under low temperature and alkaline conditions. The intermediate B is shown in Formula II:

2. The method for synthesizing the deuterated tinidazole according to claim 1, characterized in that The alkaline condition is that the intermediate B and m-chloroperbenzoic acid are added into an alkaline solution to carry out the oxidation.

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

5. The method for synthesizing the deuterated tinidazole according to claim 1, characterized in that The molar ratio of the intermediate B to meta-chloroperbenzoic acid is 1:(2.0-2.5).

6. The method for synthesizing the deuterated tinidazole according to claim 1, characterized in that The step further includes: intermediate A and 2-methyl-5-nitroimidazole are subjected to a Mitsunobu reaction at low temperature to prepare intermediate B, and the reaction formula is as follows:

7. The method for synthesizing the deuterated tinidazole according to claim 6, characterized in that The Mitsunobu reaction comprises the steps of: In a first reaction vessel, intermediate A, 2-methyl-5-nitroimidazole and triphenylphosphine are dissolved in tetrahydrofuran, Diisopropyl azodicarboxylate dissolved in tetrahydrofuran was added dropwise to the first reaction vessel.

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

9. The method for synthesizing a deuterated tinidazole according to claim 1 or 6, characterized in that The low temperature is 0-5°C.

10. The method for synthesizing the deuterated tinidazole according to claim 6, characterized in that The step also includes: 2-bromoethane deuteride and 2-mercaptoethanol undergo nucleophilic substitution to prepare intermediate A, and the reaction formula is as follows:

11. The method for synthesizing the deuterated tinidazole according to claim 10, characterized in that The nucleophilic substitution comprises the steps of: In a second reaction vessel place: a). 2-mercaptoethanol, and b) one or more of potassium carbonate, sodium bicarbonate, sodium carbonate, cesium carbonate, and c) One or both of potassium iodide and cuprous iodide, Dissolve in acetone or tetrahydrofuran under nitrogen protection. To the second reaction vessel was added 2-bromoethane deuterate.

12. The method for synthesizing the deuterated tinidazole according to claim 10, characterized in that The molar ratio of 2-mercaptoethanol:deuterated bromoethane is 1:(1.5-2.2).

13. The method for synthesizing the deuterated tinidazole according to claim 1, characterized in that The deuterated tinidazole is:

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