Synthesis method for stable-isotope labeled thiamphenicol and thiamphenicol amine

Abstract

Disclosed in the present invention is a synthesis method for stable-isotope labeled thiamphenicol and thiamphenicol amine. The synthesis method uses thiamphenicol as a raw material, and comprises performing hydrolysis to obtain thiamphenicol amine, carrying out hydrogen-deuterium exchange under an alkaline condition to obtain stable-isotope labeled thiamphenicol amine, and further carrying out acylation to obtain stable-isotope labeled thiamphenicol. The synthesis method involves a short synthesis route, high atom utilization rate, high yields and cheap and easily available raw materials. The chemical purity and the deuterium isotope abundance of the prepared stable-isotope labeled thiamphenicol and thiamphenicol amine both reach 98.0% or higher, and therefore the prepared stable-isotope labeled thiamphenicol and thiamphenicol amine can be used as isotope internal standard substances for measurement of thiamphenicol residues in foods.

Description

Synthesis method of stable isotope-labeled thiamphenicol and thiamphenicol amine Technical Field

[0001] The present invention relates to the field of production of stable isotope-labeled compounds, and in particular to a method for synthesizing stable isotope-labeled thiamphenicol and thiamphenicol amine. Background Art

[0002] Thiamphenicol, a derivative of chloramphenicol, is a broad-spectrum amide antimicrobial drug synthesized by replacing the para-nitro group in the chloramphenicol molecular structure with a methylsulfonyl group. It is widely used clinically to prevent and treat infectious diseases in animals. However, due to its toxic side effects on the blood system and other organs, its residues in animal foods may pose a threat to human health. To ensure the safety of animal-derived foods, GB 31650.1-2022 stipulates a maximum residue limit of 10 μg / kg for thiamphenicol in animal foods. In recent years, the number of analytical methods for the detection of chloramphenicol residues in food has continued to increase, with high-performance liquid chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry with isotope internal standard methods being the primary methods for the detection of chloramphenicol.

[0003] Chinese invention patent CN107827791 discloses a method for preparing thiamphenicol-d3. It uses p-bromobenzaldehyde and isotope-labeled dimethyl sulfoxide as raw materials to synthesize isotope-labeled p-methylmercaptobenzaldehyde. This is then oxidized to obtain isotope-labeled p-methylsulfonylbenzaldehyde. Next, the product is condensed with diphenylmethane to form an imine. Then, in the presence of (R)-2,2'-diphenyl-3,3'-(4-biphenanthrol) and triphenyl borate, the product is reacted with ethyl diazoacetate to form an ethylenimine structure. Finally, the ethylenimine ring is opened with dichloroacetic acid and the ester group is reduced to synthesize thiamphenicol-d3. The experimental procedure involves six cumbersome steps and results in a low overall yield of only 38%. Neither thiamphenicol-d4 nor the intermediate thiamphenicolamine-d3 has been reported in the literature.

[0004] As can be seen from the aforementioned prior art, stable isotope labeling of thiamphenicol is very difficult and still far from commercialization, and further optimization and improvement of the synthesis method are urgently needed. Therefore, there is still a need in the art to develop a low-cost, simple, and effective synthesis method for stable isotope-labeled thiamphenicol, requiring the target compound to have a purity and labeled isotope abundance of above 98%, meeting the requirements for its use as an analytical internal standard.

[0005] Summary of the Invention

[0006] The present invention aims to provide a method for synthesizing stable isotope-labeled thiamphenicol and thiamphenicolamine with low cost, simple steps, high atom utilization rate and good effect, and the purity and isotopic abundance of the target compound are both above 98%, meeting the requirements for use as an analytical internal standard.

[0007] Therefore, one aspect of the present invention relates to a method for synthesizing stable isotope-labeled thiamphenicol amine, comprising:

[0008] Thiamphenicol amine, base and heavy water are subjected to a hydrogen-deuterium exchange reaction to prepare thiamphenicol amine-d3; wherein the hydrogen-deuterium exchange reaction temperature is 100-120° C. and the reaction time is 46-50 hours.

[0009] Another aspect of the present invention relates to a method for synthesizing stable isotope-labeled thiamphenicol-d3, comprising:

[0010] Thiamphenicol amine-d3 is dissolved in a non-deuterated solvent, then mixed with triethylamine and methyl dichloroacetate, and subjected to an acylation reaction to obtain thiamphenicol-d3; wherein the acylation reaction temperature is 30-50°C and the reaction time is 22-26 hours.

[0011] Yet another aspect of the present invention relates to a method for synthesizing another stable isotope-labeled thiamphenicol-d4, comprising:

[0012] Thiamphenicol amine-d3 is dissolved in a deuterated solvent, then mixed with triethylamine and methyl dichloroacetate, and subjected to an acylation reaction to obtain thiamphenicol-d4; wherein the acylation reaction temperature is 30-50°C and the reaction time is 2-4 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG1 is a hydrogen nuclear magnetic resonance spectrum of thiamphenicol amine-d3 according to Example 1 of the present invention.

[0014] FIG2 is an LC-MS spectrum of thiamphenicol-d3 in Example 1 according to the present invention.

[0015] FIG3 is a hydrogen nuclear magnetic resonance spectrum of thiamphenicol-d3 according to Example 1 of the present invention.

[0016] FIG4 is a HPLC spectrum of thiamphenicol-d3 in Example 1 according to the present invention.

[0017] FIG5 is an LC-MS spectrum of thiamphenicol-d3 in Example 1 according to the present invention.

[0018] FIG6 is a hydrogen nuclear magnetic resonance spectrum of thiamphenicol-d4 in Example 2 according to the present invention.

[0019] FIG7 is a HPLC spectrum of thiamphenicol-d4 in Example 2 according to the present invention.

[0020] FIG8 is an LC-MS spectrum of thiamphenicol-d4 in Example 2 according to the present invention. DETAILED DESCRIPTION

[0021] To help those skilled in the art understand the features and effects of the present invention, the following general description and definitions of terms and expressions used in the specification and claims are provided. Unless otherwise indicated, technical and scientific terms used herein have the ordinary meanings understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0022] Throughout this document, all features, such as values, amounts, amounts, and concentrations, defined in numerical ranges or percentage ranges are provided for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to encompass and specifically disclose all possible subranges and individual values ​​within those ranges (including integers and fractions).

[0023] The above-mentioned features mentioned in the present invention or the features mentioned in the embodiments can be combined in any combination. All features disclosed in this specification can be used in any combination form. As long as there is no contradiction in the combination of these features, all possible combinations should be considered to be within the scope of this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0024] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly described below in conjunction with specific embodiments of the present invention and corresponding drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] The inventors of the present invention have discovered that the isotope exchange efficiency of thiamphenicol with heavy water and the abundance of the labeled isotope in the final product are significantly superior to those of thiamphenicol. Furthermore, the abundance of the labeled isotope is not affected when thiamphenicol is converted to thiamphenicol through an acylation reaction. The present invention was completed based on this finding.

[0026] 1. Stable isotope-labeled thiamphenicol amine

[0027] The synthesis method of stable isotope-labeled thiamphenicol amine of the present invention comprises:

[0028] Thiamphenicol amine, base and heavy water are subjected to a hydrogen-deuterium exchange reaction to prepare thiamphenicol amine-d3; wherein the hydrogen-deuterium exchange reaction temperature is 100-120° C. and the reaction time is 46-50 hours.

[0029] The thiamphenicol amine is prepared by heating thiamphenicol under reflux under acidic conditions.

[0030] As a preferred embodiment of the present invention, the acidic condition is selected from 0.8 to 1.3 mol / L hydrochloric acid aqueous solution, preferably 1 mol / L hydrochloric acid aqueous solution.

[0031] As a preferred embodiment of the present invention, the reaction conditions of heating under reflux are heating under reflux at 80-100°C, preferably at 85-95°C, more preferably at 88-92°C for 4-8 hours, preferably 5-7 hours, more preferably 6 hours.

[0032] Suitable bases include barium oxide, sodium deuteride, potassium carbonate or cesium carbonate.

[0033] As a preferred embodiment of the present invention, the reaction temperature of the hydrogen-deuterium exchange reaction is 100-120°C, preferably the 100-120°C includes 105-120°C, 110-120°C, 115-120°C, 100-115°C, 105-115°C, 110-115°C, 100-110°C, 105-110°C, 100-105°C, etc.

[0034] As a preferred embodiment of the present invention, the reaction time of the hydrogen-deuterium exchange reaction is 46 to 50 hours, preferably the 46 to 50 hours includes 47 to 50 hours, 48 ​​to 50 hours, 49 to 50 hours, 46 to 49 hours, 47 to 49 hours, 48 ​​to 49 hours, 47 to 48 hours, etc.

[0035] As a preferred embodiment of the present invention, the molar ratio of thiamphenicol amine, base and heavy water is 1:0.3-0.7:200-400, preferably 1:0.4-0.6:250-350, and more preferably 1:0.45-0.55:280-320.

[0036] 2. Synthesis of Thiamphenicol-d3

[0037] The present invention also relates to a method for synthesizing thiamphenicol-d3, comprising:

[0038] Thiamphenicol amine-d3 is dissolved in a non-deuterated solvent, then mixed with triethylamine and methyl dichloroacetate, and subjected to an acylation reaction to obtain thiamphenicol-d3; wherein the acylation reaction temperature is 30-50°C and the reaction time is 22-26 hours.

[0039] The non-deuterium solvent suitable for the above method of the present invention can be selected from alcohol solvents and / or nitrile solvents. In a preferred embodiment of the present invention, the solvent is selected from methanol and / or acetonitrile.

[0040] The reaction temperature suitable for the above method of the present invention is 30-50°C, preferably 32-48°C, more preferably 35-45°C, and preferably 38-42°C.

[0041] The reaction time suitable for the above method of the present invention is 22 to 26 hours, preferably 23 to 25 hours.

[0042] 3. Synthesis of Thiamphenicol-d4

[0043] The present invention also relates to a method for synthesizing thiamphenicol-d4, comprising:

[0044] Thiamphenicol amine-d3 is dissolved in a deuterated solvent, mixed with triethylamine and methyl dichloroacetate, and subjected to an acylation reaction to obtain thiamphenicol-d3; wherein the acylation reaction temperature is 30-50°C and the reaction time is 22-26 hours.

[0045] The deuterated solvent suitable for the above method of the present invention can be selected from deuterated alcohol solvents. In a preferred embodiment of the present invention, the solvent is selected from methanol-d4 and / or methanol-d1.

[0046] The reaction temperature suitable for the above method of the present invention is 30-50°C, preferably 32-48°C, more preferably 35-45°C, and preferably 38-42°C.

[0047] The reaction time suitable for the above method of the present invention is 22 to 26 hours, preferably 23 to 25 hours.

[0048] In one embodiment of the present invention, the synthesis method of the labeled thiamphenicol comprises:

[0049] (1) subjecting thiamphenicol, base and heavy water to a hydrogen-deuterium exchange reaction to obtain thiamphenicol-d3; wherein the hydrogen-deuterium exchange reaction temperature is 100-120° C. and the reaction time is 46-50 h.

[0050] (2) dissolving thiamphenicol amine-d3 in a non-deuterated solvent, mixing with triethylamine and methyl dichloroacetate, and subjecting the mixture to an acylation reaction to obtain thiamphenicol-d3; wherein the acylation reaction temperature is 30-50° C., and the reaction time is 22-26 h.

[0051] In one embodiment of the present invention, the synthesis method of the labeled thiamphenicol comprises:

[0052] (1) subjecting thiamphenicol, base and heavy water to a hydrogen-deuterium exchange reaction to obtain thiamphenicol-d3; wherein the hydrogen-deuterium exchange reaction temperature is 100-120° C. and the reaction time is 46-50 h.

[0053] (2) Thiamphenicol amine-d3 is dissolved in a deuterated solvent, mixed with triethylamine and methyl dichloroacetate, and subjected to an acylation reaction to obtain thiamphenicol-d4; wherein the acylation reaction temperature is 30-50° C., and the reaction time is 22-26 h.

[0054] In one embodiment of the present invention, the synthesis method of the labeled thiamphenicol comprises:

[0055] (1) thiamphenicol is heated under reflux at 80-110°C for 6 h under acidic conditions to obtain thiamphenicolamine;

[0056] (2) subjecting thiamphenicol, base and heavy water to a hydrogen-deuterium exchange reaction to obtain thiamphenicol-d3; wherein the hydrogen-deuterium exchange reaction temperature is 100-120° C. and the reaction time is 46-50 h.

[0057] (3) dissolving thiamphenicol amine-d3 in a non-deuterated solvent, mixing with triethylamine and methyl dichloroacetate, and subjecting the mixture to an acylation reaction to obtain thiamphenicol-d3; wherein the acylation reaction temperature is 30-50° C., and the reaction time is 22-26 h.

[0058] In one embodiment of the present invention, the synthesis method of the labeled thiamphenicol comprises:

[0059] (1) thiamphenicol is heated under reflux at 80-110°C for 6 h under acidic conditions to obtain thiamphenicolamine;

[0060] (2) subjecting thiamphenicol, base and heavy water to a hydrogen-deuterium exchange reaction to obtain thiamphenicol-d3; wherein the hydrogen-deuterium exchange reaction temperature is 100-120° C. and the reaction time is 46-50 h.

[0061] (3) dissolving thiamphenicol amine-d3 in a deuterated solvent, mixing with triethylamine and methyl dichloroacetate, and subjecting to an acylation reaction to obtain thiamphenicol-d4; wherein the acylation reaction temperature is 30-50° C., and the reaction time is 22-26 h.

[0062] The synthesis method of the present invention utilizes readily available, inexpensive raw materials, low synthesis costs, and requires only three steps, resulting in a short and economical synthesis route. Thiamphenicol-d3 can be synthesized in just three steps, significantly shortening the technical route and achieving excellent yields exceeding 90%. The target compounds, thiamphenicol-d3 and thiamphenicol-d4, exhibit high yields, with purity and isotopic abundance exceeding 98%, and can be used as internal standards for analytical testing.

[0063] Based on this, the present invention provides a method for synthesizing thiamphenicol-d3 and thiamphenicol-d4 with low cost, simple steps, high purity and isotopic abundance.

[0064] The main compounds involved in the present invention are shown in the following table:

[0065] As used herein, "a compound having a structure as shown in Formula I" and "a compound of Formula I" both refer to the compound numbered 1 in the above table. The same applies to compounds with other structures.

[0066] In the present invention, the thiamphenicol is a compound with a structure as shown in Formula I, and the thiamphenicol amine is a compound with a structure as shown in Formula IV; the target compound thiamphenicol-d3 is a compound with a structure as shown in Formula II, and thiamphenicol-d4 is a compound with a structure as shown in Formula III.

[0067] Preferably, the synthetic route of the present invention is as follows:

[0068] In one embodiment of the present invention, thiamphenicol is dissolved in a 1 mol / L aqueous hydrochloric acid solution; the reaction temperature is 100°C and refluxed for 6 hours to obtain thiamphenicol; thiamphenicol, barium oxide, and heavy water are sequentially added to a sealed tube, stirred and dissolved, and heated at a reaction temperature of 110°C for 48 hours to obtain thiamphenicol-d3; thiamphenicol-d3 is weighed and dissolved in 40 mL of acetonitrile, triethylamine and methyl dichloroacetate are added at 1 to 2 drops / second, and the reaction is heated at 40°C for 3 hours to obtain thiamphenicol-d3.

[0069] In one embodiment of the present invention, thiamphenicol is dissolved in a 1 mol / L aqueous hydrochloric acid solution; the reaction temperature is 100°C and refluxed for 6 hours to obtain thiamphenicolamine; thiamphenicolamine, barium oxide, and heavy water are sequentially added to a sealed tube, stirred and dissolved, and heated at a reaction temperature of 110°C for 48 hours to obtain thiamphenicolamine-d3; thiamphenicolamine-d3 is weighed and dissolved in 40 mL of methanol-d1, triethylamine and methyl dichloroacetate are added at 1 to 2 drops / second, and the reaction is heated at 40°C for 3 hours to obtain thiamphenicol-d4.

[0070] Purity and isotope abundance detection methods used in the examples:

[0071] Thiamphenicol-d3 purity test

[0072] (1) HPLC test conditions for thiamphenicol-d3

[0073] Column: Athena UHPLC C18 (100mm*2.1mm, 1.8μm)

[0074] Column temperature: 30°C

[0075] Mobile phase: A: acetonitrile; B: 0.1% phosphoric acid water

[0076] Gradient: 0min 5%A; 2min 5%A; 8min 100%A; 12min 100%A; 12.1min 5%A; 15min 5%A

[0077] Flow rate: 0.3 mL / min

[0078] Detector: DAD 226nm

[0079] Sample: 0.5 μL, 1.02 mg / mL in methanol

[0080] (2) The chromatographic purity was calculated using the HPLC area normalization method.

[0081] Thiamphenicol-d3 isotope abundance detection

[0082] The "simple mass cluster" method was used to determine the isotopic abundance of thiamphenicol-d3. The peak areas of the quantitative ion pairs 357-188, 356-187, 355-186, and 354-185 were measured by mass spectrometry and approximated to the peak areas of thiamphenicol-D3, thiamphenicol-D2, thiamphenicol-D1, and thiamphenicol-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 D3-D0 were normalized. Finally, the formula:

[0083] Wherein, D% represents isotope abundance; i represents the number of D markers, i=0-3; Ri represents the percentage of the peak area of ​​i D markers. Taking 3 D markers as an example, the calculation formula is: R3=A(D3) / [A(D3)+A(D2)+A(D1)+A(D0)]; n represents the total number of D markers, n=3.

[0084] Mass spectrometry conditions:

[0085] Detection mode: Multiple ion scan

[0086] Ionization mode: negative ion mode

[0087] Ion source voltage: 3500

[0088] Ion transfer tube temperature: 300°C

[0089] Steam temperature: 200℃

[0090] Scan range: 50-400 (m / z)

[0091] Flow rate: 20 μL / min

[0092] Qualifying ion collision energy: 15

[0093] Quantitative ion pairs: 354-185, 355-186, 356-187, 357-188

[0094] Thiamphenicol-d4 purity test

[0095] (1) HPLC test conditions for thiamphenicol-d4

[0096] Column: Athena UHPLC C18 (100mm*2.1mm, 1.8μm)

[0097] Column temperature: 30°C

[0098] Mobile phase: A: acetonitrile; B: 0.1% phosphoric acid water

[0099] Gradient: 0min 5%A; 2min 5%A; 8min 100%A; 12min 100%A; 12.1min 5%A; 15min 5%A

[0100] Flow rate: 0.3 mL / min

[0101] Detector: DAD 226 nm

[0102] Sample: 0.5 μL, 1.02 mg / mL in methanol

[0103] (2) The chromatographic purity was calculated using the HPLC area normalization method.

[0104] Thiamphenicol-d4 isotope abundance detection

[0105] The "simple mass cluster" method was used to determine the isotopic abundance of thiamphenicol-d4. The peak areas of the quantitative ion pairs 358-188, 357-187, 356-186, 355-185, and 354-184 were measured by mass spectrometry and approximated to the peak areas of thiamphenicol-D4, thiamphenicol-D3, thiamphenicol-D2, thiamphenicol-D1, and thiamphenicol-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 D4-D0 were normalized. Finally, the formula:

[0106] Wherein, D% represents isotope abundance; i represents the number of D markers, i=0-4; Ri represents the percentage of the peak area of ​​i D markers. Taking 4 D markers as an example, the calculation formula is: R4=A(D4) / [A(D4)+A(D3)+A(D2)+A(D1)+A(D0)]; n represents the total number of D markers, n=4.

[0107] Mass spectrometry detection conditions:

[0108] Detection mode: Multiple ion scan

[0109] Ionization mode: negative ion mode

[0110] Ion source voltage: 3500

[0111] Ion transfer tube temperature: 300°C

[0112] Steam temperature: 200℃

[0113] Scan range: 50-400 (m / z)

[0114] Flow rate: 20 μL / min

[0115] Qualifying ion collision energy: 15

[0116] Quantitative ion pairs: 358-188, 357-187, 356-186, 355-185, 354-184

[0117] Example 1 Synthesis of Thiamphenicol-d3 and Thiamphenicolamine-d3

[0118] A method for synthesizing stable isotope-labeled thiamphenicol and thiamphenicol amine comprises the following steps:

[0119] (1) Thiamphenicol hydrolysis

[0120] In a 250 mL three-necked flask, thiamphenicol (3.56 g, 10 mmol) was dissolved in 100 mL of dilute hydrochloric acid (1 mol / L). The mixture was refluxed at 100 ° C overnight. The reaction was completed by TLC monitoring. The reaction solution was cooled to room temperature and extracted twice with ethyl acetate to remove impurities. The aqueous phase was dried to obtain thiamphenicol amine, 2.40 g, with a yield of 97.84%.

[0121] (2) Synthesis of Thiamphenicol-d3

[0122] In a 250 mL sealed tube, stir and dissolve thiamphenicol (2.40 g, 9.78 mmol), barium oxide (750.09 mg, 4.89 mmol), and deuterated water (78.38 g, 3.91 mol). Heat and stir at 110°C for 48 hours, cool to room temperature, filter to remove insoluble impurities, and spin-dry to obtain thiamphenicol-d3. The isotopic abundance of thiamphenicol-d3 is 98.82%, D3: 96.64%, D2: 3.22%, D1: 0.09%, and D0: 0.05%.

[0123] (3) Thiamphenicol-d3 synthesis

[0124] Thiamphenicol amine-d3 (1 g, 4.03 mmol) was dissolved in 40 mL of acetonitrile, and 1 mL of triethylamine and methyl dichloroacetate (1.73 g, 12.08 mmol) were added at a rate of 1 to 2 drops per second. The reaction was heated at 40°C for 3 h. TLC monitored the reaction to be complete. The reaction solution was spin-dried, recrystallized from methanol / dichloromethane, allowed to stand at low temperature, and then filtered. The filter cake was vacuum-dried to obtain the target product, thiamphenicol-d3, 1.43 g, in a yield of 98.62%. Analysis by the purity and isotopic abundance detection method revealed that the target product, thiamphenicol-d3, had a chromatographic purity of 99.63% and an isotopic abundance of 98.83%.

[0125] Thiamphenicol-d3 was detected by ESI-MS, and its mass spectrum in negative ion mode is shown in Figure 5. Measured molecular ion peak [MH] - =357.08, which is consistent with the theoretical calculation result of 357.02 (C 12 H 11 D3Cl2NO5S - The calculated isotopic abundance of thiamphenicol-d3 is 98.83atom%D, with no significant dilution of isotopic abundance.

[0126] Example 2 Synthesis of Thiamphenicol-d4 and Thiamphenicolamine-d3

[0127] A method for synthesizing stable isotope-labeled thiamphenicol and thiamphenicol amine comprises the following steps:

[0128] (1) Thiamphenicol hydrolysis

[0129] In a 250 mL three-necked flask, thiamphenicol (3.56 g, 10 mmol) was dissolved in 100 mL of dilute hydrochloric acid (1 mol / L). The mixture was refluxed at 100 ° C overnight. The reaction was completed by TLC monitoring. The reaction solution was cooled to room temperature and extracted twice with ethyl acetate to remove impurities. The aqueous phase was dried to obtain thiamphenicol amine, 2.40 g, with a yield of 97.84%.

[0130] (2) Synthesis of Thiamphenicol-d3

[0131] Take 250 mL sealed tube, stir and dissolve thiamphenicol (2.40 g, 9.78 mmol), barium oxide (750.09 mg, 4.89 mmol), and heavy water (78.38 g, 3.91 mol), heat and stir at 110 ° C for 48 h, cool to room temperature, filter to remove insoluble impurities, and dry the filtrate to obtain thiamphenicol-d3.

[0132] (3) Synthesis of thiamphenicol-d4

[0133] Thiamphenicol amine-d3 (1 g, 4.03 mmol) was dissolved in 40 mL of methanol-d1, and 1 mL of triethylamine and methyl dichloroacetate (1.73 g, 12.08 mmol) were added at 1-2 drops / second. The reaction was heated at 40°C for 3 h. The reaction was completed after monitoring by TLC. The reaction solution was spin-dried and recrystallized from methanol / dichloromethane. After standing at low temperature, it was filtered and the filter cake was dried under vacuum to obtain the target product thiamphenicol-d4, 1.19 g, with a yield of 82.26%. Analysis by the purity and isotope abundance detection method showed that the chromatographic purity of the target product thiamphenicol-d4 was 99.64% and the isotope abundance was 99.12%.

[0134] Thiamphenicol-d4 was detected by ESI-MS, and its mass spectrum in negative ion mode is shown in Figure 8. Measured molecular ion peak [MH] - =358.04, which is consistent with the theoretical calculation result of 358.02 (C 10 H 10 D4NO5S - The calculated isotopic abundance of thiamphenicol-d4 is 99.12 atom% D, with no significant dilution of isotopic abundance.

[0135] Comparative Example 1 Synthesis of intermediate product thiamphenicol amine-d3

[0136] Take a 50 mL sealed tube, stir and dissolve thiamphenicol (0.3 g, 1.22 mmol), barium oxide (93.76 mg, 0.61 mmol), and heavy water (9.80 g, 0.49 mol), heat and stir at 80 ° C for 12 h, cool to room temperature, filter to remove insoluble impurities, and dry the filtrate to obtain thiamphenicol-d3 with a stable isotope abundance of 51.86%.

[0137] Comparative Example 2: Synthesis of Thiamphenicol-d3

[0138] Take 250mL sealed tube, stir and dissolve thiamphenicol (3.56g, 10.00mmol), barium oxide (1.07g, 7.00mmol), and heavy water (80.11g, 4.00mol), heat and stir at 110℃ for 48h, cool to room temperature, filter to remove insoluble impurities, and dry the filtrate to obtain thiamphenicol-d3. The isotopic abundance is detected to be 94.02%, D3: 83.26%, D2: 15.59%, D1: 1.09%, and D0: 0.05%.

Claims

1. A method for synthesizing stable isotope-labeled thiamphenicol amine, characterized in that: Includes steps: Thiamphenicol amine, alkali and heavy water are subjected to a hydrogen-deuterium exchange reaction to obtain thiamphenicol amine-d3; wherein the reaction temperature of the hydrogen-deuterium exchange reaction is 100-120° C. and the reaction time is 46-50 hours.

2. A method for synthesizing stable isotope-labeled thiamphenicol, characterized in that: Includes steps: Take thiamphenicol amine-d3, dissolve it in a solvent, mix it with triethylamine and methyl dichloroacetate, and carry out acylation reaction to obtain thiamphenicol labeled with a stable isotope; wherein the reaction temperature of the acylation reaction is 30-50°C, and the reaction time is 2-4h.

3. The synthesis method according to claim 2, characterized in that The solvent is selected from an alcohol solvent, a nitrile solvent or a mixture thereof, preferably selected from methanol and / or acetonitrile, and the stable isotope-labeled thiamphenicol is thiamphenicol-d3.

4. The synthesis method according to claim 2, characterized in that The solvent is selected from deuterated alcohol solvents, preferably methanol-d4 and / or methanol-d1, and the stable isotope-labeled thiamphenicol is thiamphenicol-d4.

5. The synthesis method according to claim 1, characterized in that The molar ratio of thiamphenicol amine, alkali and heavy water is 1:0.3-0.7:200-400.

6. The synthesis method according to claim 1 or 5, characterized in that: The base is selected from barium oxide, sodium deuteride, potassium carbonate or cesium carbonate.

7. The synthesis method according to claim 1 or 5, characterized in that: The thiamphenicol amine is prepared by heating thiamphenicol under acidic conditions at 80-110° C. for 4-8 hours, preferably 6 hours.

8. The synthesis method according to claim 7, characterized in that The acidic condition is 0.8-1.3 mol / L hydrochloric acid aqueous solution, preferably 1 mol / L hydrochloric acid aqueous solution.

9. The synthesis method according to claim 1 or 5, characterized in that: The hydrogen-deuterium exchange reaction temperature is 105-115° C., and the reaction time is 47-49 hours.

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