Synthesis method for stable-isotope labeled florfenicol and florfenicol amine

Abstract

A synthesis method for stable-isotope labeled florfenicol and florfenicol amine. The method uses florfenicol as a raw material, and comprises hydrolyzing same to obtain florfenicol amine, performing hydrogen-deuterium exchange under an alkaline condition to obtain stable-isotope labeled florfenicol amine, and further performing acylation to obtain stable-isotope labeled florfenicol. The synthesis method involves a short synthesis route, high atom utilization rate, high yield and cheap and easily available raw materials. The chemical purity and deuterium isotope abundance of the prepared stable-isotope labeled florfenicol and florfenicol amine both reach 98.0% or higher, and therefore the stable-isotope labeled florfenicol and florfenicol amine can be used as isotope internal standard substances for analysis and detection.

Description

Synthesis method of stable isotope-labeled florfenicol and florfenicol amide Technical Field

[0001] The present invention relates to a method for synthesizing stable isotope-labeled florfenicol and florfenicol amide. The florfenicol and florfenicol amide prepared by the method of the present invention have a purity and labeled isotope abundance of more than 98%, meeting the requirements for use as analytical internal standards. Background Art

[0002] Florfenicol, also known as fluoroquinolone, is a chemically synthesized broad-spectrum amide antibiotic for animal use. It is highly active against Gram-negative and Gram-positive bacteria, as well as mycoplasmas, and possesses strong antibacterial properties. Due to its low price and effective efficacy, it is widely used in animal disease prevention and treatment. However, it still exhibits certain embryotoxicity, and large-scale use can also cause significant harm to humans. The "National Food Safety Standard Maximum Residue Limits of 41 Veterinary Drugs in Food" (GB 31650.1-2022) stipulates that the maximum residue limit for the sum of florfenicol and florfenicol amide in animal foods is 10 μg / kg.

[0003] "National Food Safety Standard - Determination of Residues of Amide Alcohols and Their Metabolites in Animal Foods by Liquid Chromatography-Tandem Mass Spectrometry" (GB 31658.20-2022) stipulates the liquid chromatography-tandem mass spectrometry method for the determination of residues of amide alcohols and their metabolites in animal foods, and uses chloramphenicol-d5, thiamphenicol-d3, florfenicol-d3, and florfenicol amide-d3 as internal standards for quantification.

[0004] The most common stable isotope labeling method involves isotope exchange, which involves mixing florfenicol with heavy water, allowing the deuterium atoms in the heavy water to exchange with the hydrogen atoms in florfenicol, achieving isotope labeling. However, this method suffers from a low isotope abundance in the product, typically exceeding 80%.

[0005] Chinese invention patent application CN107827688 discloses a method for producing isotope-labeled florfenicol. The method uses p-bromobenzaldehyde and isotope-labeled dimethyl sulfoxide as raw materials to synthesize isotope-labeled p-methylmercaptobenzaldehyde, which is further oxidized to obtain isotope-labeled p-methylsulfonylbenzaldehyde. The product is then condensed with diphenylmethane to form an imine. The imine is then reacted with (R)-2,2'-diphenyl-3,3'-(4-biphenanthrol) and triphenyl borate to form an ethyl diazoacetate amine fragment. Finally, the ethyl imine is ring-opened to produce isotope-labeled florfenicol through ester reduction, hydroxyl fluorination, and dichloroacetic acid. The experimental procedure involves seven complex steps and results in a low overall yield.

[0006] As can be seen from the aforementioned prior art, stable isotope labeling of florfenicol 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 florfenicol, requiring the target compound to have a purity and labeled isotope abundance of more than 98% to meet the requirements for its use as an analytical internal standard.

[0007] Summary of the Invention

[0008] The present invention aims to provide a method for synthesizing stable isotope-labeled florfenicol and florfenicol amide with low cost, simple steps 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.

[0009] Therefore, one aspect of the present invention relates to a method for synthesizing stable isotope-labeled florfenicol amide, comprising:

[0010] Florfenicol amide, base and heavy water are subjected to a hydrogen-deuterium exchange reaction to prepare florfenicol amide-d3; wherein the hydrogen-deuterium exchange reaction temperature is 50-70° C. and the reaction time is 22-26 hours.

[0011] Another aspect of the present invention relates to a method for synthesizing stable isotope-labeled florfenicol, characterized in that it comprises:

[0012] Florfenicol amide-d3 is dissolved in a solvent, mixed with triethylamine and methyl dichloroacetate, and subjected to an acylation reaction to obtain florfenicol-d3; wherein the acylation reaction temperature is 30-50° C., and the reaction time is 22-26 h. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0014] FIG2 is a HPLC spectrum of florfenicol amide-d3 according to Example 1 of the present invention.

[0015] FIG3 is an LC-MS spectrum of florfenicol amide-d3 according to Example 1 of the present invention.

[0016] FIG4 is a hydrogen nuclear magnetic resonance spectrum of florfenicol-d3 according to Example 1 of the present invention.

[0017] FIG5 is a HPLC spectrum of Florfenicol-d3 according to Example 1 of the present invention.

[0018] FIG6 is an LC-MS spectrum of Florfenicol-d3 in Example 1 according to the present invention. DETAILED DESCRIPTION

[0019] 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.

[0020] 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).

[0021] 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.

[0022] 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.

[0023] The inventors of the present invention have discovered that, in terms of isotope exchange, the exchange efficiency of florfenicol amine with heavy water and the labeled isotope abundance of the final product are significantly superior to those of florfenicol. Furthermore, when florfenicol amine is converted into florfenicol through an acylation reaction, its labeled isotope abundance is not affected. The present invention was completed based on this finding.

[0024] 1. Stable isotope-labeled florfenicol amide

[0025] The synthesis method of stable isotope-labeled florfenicol amide of the present invention comprises:

[0026] Florfenicol amide, base and heavy water are subjected to a hydrogen-deuterium exchange reaction to prepare florfenicol amide-d3; wherein the hydrogen-deuterium exchange reaction temperature is 50-70° C. and the reaction time is 22-26 hours.

[0027] There is no particular limitation on the applicable florfenicol amide, which can be purchased from the market. In one embodiment of the present invention, the florfenicol amide is prepared by heating florfenicol under reflux under acidic conditions.

[0028] 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.

[0029] 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.

[0030] Suitable bases include barium oxide, sodium deuteride, potassium carbonate or cesium carbonate.

[0031] As a preferred embodiment of the present invention, the reaction temperature of the hydrogen-deuterium exchange reaction is 50-70°C, preferably 52-68°C, more preferably 55-65°C, and most preferably 58-62°C.

[0032] As a preferred embodiment of the present invention, the reaction time of the hydrogen-deuterium exchange reaction is 22 to 26 hours, preferably 23 to 25 hours.

[0033] As a preferred embodiment of the present invention, the molar ratio of florfenicol amide, 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.

[0034] 2. Stable isotope-labeled florfenicol

[0035] The present invention also relates to a method for synthesizing stable isotope-labeled florfenicol, comprising:

[0036] Florfenicol amide-d3 is dissolved in a solvent, mixed with triethylamine and methyl dichloroacetate, and subjected to an acylation reaction to obtain florfenicol-d3; wherein the acylation reaction temperature is 30-50° C., and the reaction time is 22-26 h.

[0037] The florfenicol amide-d3 suitable for the above method of the present invention can be the florfenicol amide-d3 prepared by the above method of the present invention, or can be the florfenicol amide-d3 purchased on the market.

[0038] The 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.

[0039] The reaction temperature for the above method of the present invention is 30 to 50 ° C, preferably 32 to 48 ° C, more preferably 35 to 45 ° C, preferably 38 to 42 ° C.

[0040] The reaction time suitable for the above method of the present invention is 22 to 26 hours, preferably 23 to 25 hours.

[0041] In one embodiment of the present invention, the synthesis method of the labeled florfenicol comprises:

[0042] (1) subjecting florfenicol amide, base, and heavy water to a hydrogen-deuterium exchange reaction to obtain florfenicol amide-d3; wherein the reaction temperature of the hydrogen-deuterium exchange reaction is 50-70° C., and the reaction time is 22-26 hours;

[0043] (2) Florfenicol amide-d3 is dissolved in a solvent, and then mixed with triethylamine and methyl dichloroacetate, and subjected to an acylation reaction to obtain florfenicol-d3; wherein the reaction temperature of the acylation reaction is 30-50° C., and the reaction time is 22-26 h.

[0044] In one embodiment of the present invention, the synthesis method of the labeled florfenicol comprises:

[0045] (1) Reflux florfenicol at 80-110° C. under acidic conditions for 6 h to obtain florfenicol amide;

[0046] (2) subjecting florfenicol amide, base, and heavy water to a hydrogen-deuterium exchange reaction to obtain florfenicol amide-d3; wherein the reaction temperature of the hydrogen-deuterium exchange reaction is 50-70° C., and the reaction time is 22-26 hours;

[0047] (3) Taking florfenicol amide-d3, dissolving it in a solvent, and then mixing it with triethylamine and methyl dichloroacetate, and performing an acylation reaction to obtain florfenicol-d3; wherein the reaction temperature of the acylation reaction is 30-50° C., and the reaction time is 22-26 h.

[0048] The synthesis method of the present invention has a short synthesis route, high atom utilization rate, excellent yield, and inexpensive and easily available raw materials. The purity and abundance of the target compounds florfenicol-d3 and florfenicol amine-d3 are both above 98%, and the compounds can be used as internal standards for analysis and detection.

[0049] Accordingly, the present invention provides a method for synthesizing florfenicol-d3 and florfenicol amine-d3 with low cost, simple steps, high purity and isotopic abundance.

[0050] The main compounds involved in the present invention are shown in the following table:

[0051] 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.

[0052] In the present invention, the florfenicol is a compound with a structure as shown in Formula I, and the florfenicol amine is a compound with a structure as shown in Formula III; the target product florfenicol-d3 is a compound with a structure as shown in Formula II, and the florfenicol amine-d3 is a compound with a structure as shown in Formula IV.

[0053] Preferably, the synthetic route of the present invention is as follows:

[0054] In one embodiment of the present invention, in the step (1), florfenicol is dissolved in a 1 mol / L aqueous hydrochloric acid solution; the reaction temperature is 100° C. and the mixture is heated under reflux for 6 h to obtain florfenicol amide; in the step (2), florfenicol amide, barium oxide, and heavy water are sequentially added to a sealed tube, stirred and dissolved, and the reaction temperature is 60° C. and heated for 24 h to obtain florfenicol amide-d3; in the step (3), florfenicol amide-d3 is weighed in a three-necked flask, dissolved in a solvent, triethylamine and methyl dichloroacetate are added at 1 to 2 drops / second, and the mixture is stirred and reacted at 40° C. for 24 h to obtain florfenicol-d3.

[0055] Purity and isotope abundance detection methods used in the examples:

[0056] Florfenicol amide-d3 purity test

[0057] (1) HPLC test conditions for florfenicol amide-d3

[0058] Chromatographic column Athena C18-WP, (250mmx4.6mm, 5μm)

[0059] Column temperature 30℃

[0060] Mobile phase A: acetonitrile; B: 0.1% formic acid in water

[0061] Gradient 0min 5%A; 3min 5%A; 15min 95%A; 24min 95%A; 26min 5%A; 30min 5%A

[0062] Flow rate 1mL / min

[0063] Detector DAD

[0064] Sample 2 μL, 1.0 mg / mL in methanol

[0065] (2) The chromatographic purity was calculated using the HPLC area normalization method.

[0066] Florfenicol amide-d3 isotope abundance detection

[0067] The "simple mass cluster" method was used to determine the isotopic abundance of florfenicol amide-d3. The peak areas of the quantitative ion pairs 251-233, 250-232, 249-231, and 248-230 were measured by mass spectrometry and approximated to the peak areas of florfenicol amide-d3, florfenicol amide-d2, florfenicol amide-d1, and florfenicol amide-d0, respectively. Based on the detection principle that "mass spectrometry peak intensity is proportional to the number of molecules", the isotopic abundance percentages of d3-d0 were normalized. Finally, the formula:

[0068] 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.

[0069] Mass spectrometry conditions:

[0070] Detection mode: multiple ion scan

[0071] Ionization mode: positive ion mode

[0072] Ion source voltage: 3500

[0073] Ion transfer tube temperature: 300°C

[0074] Steam temperature: 200℃

[0075] Scan range: 100-350 (m / z)

[0076] Flow rate: 20 μL / min

[0077] Qualifying ion collision energy: 15

[0078] Quantitative ion pairs: 248-230, 249-231, 250-232, 251-233

[0079] Florfenicol-d3 purity test

[0080] (1) HPLC test conditions for Florfenicol-d3

[0081] Chromatographic column: Syncronis aQ (250mm*4.6mm, 5μm)

[0082] Column temperature: 30°C

[0083] Mobile phase: A: acetonitrile; B: 0.1% phosphoric acid water

[0084] Gradient: 0min 5%A; 2min 5%A; 20min 100%A; 21min 100%A; 22min 5%A; 25min 5%A

[0085] Flow rate: 1 mL / min

[0086] Detector: DAD 225nm

[0087] Sample: 5 μL, 0.64 mg / mL in methanol

[0088] (2) The chromatographic purity was calculated using the HPLC area normalization method.

[0089] Florfenicol-d3 isotope abundance detection

[0090] The "simple mass cluster" method was used to determine the isotopic abundance of florfenicol-d3. The peak areas of the quantitative ion pairs 359-339, 358-338, 357-337, and 356-336 were measured by mass spectrometry and approximated to the peak areas of florfenicol-d3, florfenicol-d2, florfenicol-d1, and florfenicol-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:

[0091] 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.

[0092] Mass spectrometry conditions:

[0093] Detection mode: multiple ion scan

[0094] Ionization mode: negative ion mode

[0095] Ion source voltage: 3500

[0096] Ion transfer tube temperature: 300°C

[0097] Steam temperature: 200℃

[0098] Scan range: 60-450 (m / z)

[0099] Flow rate: 20 μL / min

[0100] Qualifying ion collision energy: 15

[0101] Qualifying ion pairs: 356-335, 357-336, 358-338, 359-339

[0102] Example 1 Synthesis of Stable Isotope-Labeled Florfenicol and Florfenicol Amine

[0103] A method for synthesizing stable isotope-labeled florfenicol and florfenicol amide comprises the following steps:

[0104] (1) Florfenicol hydrolysis

[0105] In a 250 mL three-necked flask, florfenicol (8 g, 10 mmol) was dissolved in 100 mL of dilute hydrochloric acid (1 mol / L) and refluxed at 100 ° C for 6 h. 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 spin-dried and recrystallized to obtain florfenicol amine, 4.75 g, with a yield of 86.01%.

[0106] (2) Synthesis of Florfenicol Amine-d3

[0107] Take 250mL sealed tube, florfenicol amine (2.40g, 9.71mmol), barium oxide (744.05mg, 4.85mmol), heavy water (77.75g, 3.88mol) stirring and dissolving, 60 ℃ of heating and stirring for 24h, cool to room temperature, filter to remove insoluble impurities, add silica gel and be spin-dried, column chromatography purification obtains florfenicol amine-d3, 1.71g, yield 70.39%. According to the purity and isotopic abundance method, the chromatographic purity of florfenicol amine-d3 is 98.89%, and the isotopic abundance is 98.73%.

[0108] Florfenicol amide-d3 was detected by ESI-MS, and its mass spectrum in positive ion mode is shown in Figure 3. The measured molecular ion peak [M+H] + =251.12, which is consistent with the theoretical calculation result of 251.09 (C 10 H 12 D3FNO3S + The calculated isotopic abundance of florfenicol amide-d3 is 98.73atom%D, and there is no obvious dilution of isotopic abundance.

[0109] (3) Synthesis of Florfenicol-d3

[0110] Florfenicol 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 1 to 2 drops per second. The reaction was heated at 40 ° C for 24 hours. The reaction was complete after TLC monitoring. The reaction solution was spin-dried and purified by column chromatography to obtain the target product, florfenicol-d3, 1.34 g, in a yield of 92.85%. The purity and isotopic abundance method was used for detection. The chromatographic purity of florfenicol-d3 was 99.55% and the isotopic abundance was 98.98%.

[0111] Florfenicol-d3 was detected by ESI-MS, and its mass spectrum in negative ion mode is shown in Figure 6. Measured molecular ion peak [MH] - =359.07, which is consistent with the theoretical calculation result of 359.02 (C 12 H 10 D3Cl2FNO4S - The calculated isotopic abundance of florfenicol-d3 is 98.98 atom% D, with no significant dilution of isotopic abundance.

[0112] Comparative Example 1 Synthesis of Florfenicol Amine-d3

[0113] Take 250 mL sealed tube, florfenicol amide (2.40 g, 9.71 mmol), barium oxide (744.05 mg, 4.85 mmol), and heavy water (77.75 g, 3.88 mol) and stir to dissolve, heat and stir at 80 ° C for 12 h, cool to room temperature, filter to remove insoluble impurities, add silica gel and spin dry, and purify by column chromatography to obtain florfenicol amide-d3, the isotopic abundance of which is 40.90%, D3: 6.04%, D2: 10.65%, D1: 83.31%, D0: 0.00%.

[0114] Comparative Example 2 Synthesis of Florfenicol-d3

[0115] Take 250 mL sealed tube, stir and dissolve florfenicol (500 mg, 1.40 mmol), barium oxide (107.01 mg, 0.70 mmol), and heavy water (11.18 g, 0.56 mol), heat and stir at 110 ° C for 16 h, cool to room temperature, filter to remove insoluble impurities, add silica gel and spin dry, and purify by column chromatography to obtain florfenicol-d3. The isotopic abundance is detected to be 79.16%, D3: 68.17%, D2: 13.75%, D1: 5.45%, and D0: 12.62%.

Claims

1. A method for synthesizing stable isotope-labeled florfenicol amide, characterized in that: Includes steps: Florfenicol amide, alkali and heavy water are subjected to a hydrogen-deuterium exchange reaction to obtain florfenicol amide-d3; wherein the hydrogen-deuterium exchange reaction temperature is 50-70° C. and the reaction time is 22-26 hours.

2. A method for synthesizing stable isotope-labeled florfenicol, characterized in that: Includes steps: Take florfenicol amide-d3, dissolve it in a solvent, mix it with triethylamine and methyl dichloroacetate, and prepare florfenicol-d3 through acylation reaction; wherein the acylation reaction temperature is 30-50° C., and the reaction time is 22-26 h.

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

4. The synthesis method according to claim 1 or 3, characterized in that The base is selected from barium oxide, sodium deuteride, potassium carbonate or cesium carbonate.

5. The synthesis method according to claim 2, characterized in that The solvent is selected from alcohol solvents, nitrile solvents or mixtures thereof, preferably selected from methanol and / or acetonitrile.

6. The synthesis method according to claim 1 or 3, characterized in that: The florfenicol amine is prepared by heating florfenicol under acidic conditions at 80-110° C. for 4-8 hours, preferably 6 hours.

7. The synthesis method according to claim 6, 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.

8. The synthesis method according to claim 1 or 3, characterized in that: The hydrogen-deuterium exchange reaction temperature is 55-65° C., and the reaction time is 23-25 ​​hours.

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