Synthesis method of stable isotope-labeled florfenicol and florfenicol amide

Abstract

The invention discloses a method for synthesizing florfenicol and florfenicol amine labeled with a stable isotope. Florfenicol is used as a raw material, hydrolyzed to obtain florfenicol amine, hydrogen-deuterium exchanged under alkaline conditions to obtain florfenicol amine labeled with a stable isotope, and further acylated to obtain florfenicol labeled with a stable isotope. The synthetic route of the invention is short, the atomic utilization rate is high, the yield is excellent, and the raw materials are cheap and easy to obtain. The chemical purity and deuterium isotope abundance of the prepared florfenicol and florfenicol amine both reach more than 98.0%, and can be used as an isotope internal standard for analysis and detection.

Description

Technical Field

[0001] The present invention relates to a method for synthesizing florfenicol and florfenicol amine labeled with stable isotopes. The purity and labeled isotope abundance of florfenicol and florfenicol amine prepared by the method of the present invention are both above 98%, meeting the use requirements of them as analytical internal standards. Background Art

[0002] Florfenicol, also known as fluorometholone, is a chemically synthesized broad-spectrum amide antibiotic for animals. It has strong activity against Gram-negative bacteria, Gram-positive bacteria and mycoplasma, and has strong antibacterial ability. It is widely used in animal disease prevention and treatment due to its low price and good efficacy. However, it still has certain embryotoxicity, and large-scale use can also cause great harm to the human body. The "National Food Safety Standard Maximum Residue Limits of 41 Veterinary Drugs in Food" (GB 31650.1-2022) stipulates that the maximum residue limit of florfenicol and florfenicol amine in animal food is 10μg / kg.

[0003] The National Food Safety Standard for the Determination of Amide Alcohols and Their Metabolites Residues in Animal Foods - Liquid Chromatography-Tandem Mass Spectrometry (GB 31658.20-2022) stipulates the determination of amide alcohols and their metabolites residues in animal foods by liquid chromatography-tandem mass spectrometry, and cites chloramphenicol-d 5 , thiamphenicol-d 3 , Florfenicol-d 3 , Florfenicolamide-d 3 It was used as an internal standard for quantification.

[0004] The most common stable isotope labeling method includes the isotope exchange method, which is to mix florfenicol with heavy water so that the deuterium atoms in the heavy water exchange with the hydrogen atoms in florfenicol to achieve the purpose of isotope labeling. However, the disadvantage of this method is that the product labeling isotope abundance is low, generally difficult to exceed 80%.

[0005] Chinese invention patent application CN107827688 discloses a method for preparing isotope-labeled florfenicol, which 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, which is then condensed with diphenylamine to form an imine, and then the imine structure fragment is constructed with ethyl diazoacetate under the action of (R)-2,2'-diphenyl-3,3'-(4-biphenylphenol) and triphenyl borate, and finally the imine is ring-opened to isotope-labeled florfenicol under the conditions of ester reduction, hydroxyl fluorination, and dichloroacetic acid. The experimental steps require seven steps of reaction, which are cumbersome and have a low overall yield.

[0006] From the above-mentioned prior art, it can be seen that the stable isotope labeling of florfenicol is very difficult and still far from commercialization, and the synthesis method needs to be further optimized and improved. Therefore, the art still needs to develop a low-cost, simple-step, and effective stable isotope-labeled synthesis method of florfenicol, requiring the purity of the target compound and the abundance of the labeled isotope to be above 98%, meeting the requirements for its use as an analytical internal standard. Summary of the invention

[0007] The purpose of the present invention is to provide a method for synthesizing florfenicol and florfenicol amine labeled with stable isotopes 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.

[0008] Therefore, one aspect of the present invention relates to a method for synthesizing stable isotope-labeled florfenicol amide, comprising:

[0009] Florfenicol amide, alkali and heavy water are subjected to hydrogen-deuterium exchange reaction to obtain florfenicol amide-d 3 ; Wherein, the hydrogen-deuterium exchange reaction temperature is 50-70°C and the reaction time is 22-26h.

[0010] Another aspect of the present invention relates to a method for synthesizing stable isotope-labeled florfenicol, characterized in that it comprises:

[0011] Take Florfenicol Amine-d 3 , dissolved in a solvent, and then mixed with triethylamine and methyl dichloroacetate, and acylated to obtain florfenicol-d 3 ; wherein the acylation reaction temperature is 30-50°C and the reaction time is 22-26h. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a compound of the invention according to Example 1 of the present invention. 3 H NMR spectrum.

[0013] Figure 2 is a compound of the invention according to Example 1 of the present invention. 3 HPLC spectrum of.

[0014] Figure 3 is a compound of the invention according to Example 1 of the present invention. 3 LC-MS spectrum of

[0015] Figure 4 is the florfenicol-d according to Example 1 of the present invention. 3 H NMR spectrum.

[0016] Figure 5 is the florfenicol-d according to Example 1 of the present invention. 3 HPLC spectrum of.

[0017] Figure 6 is the florfenicol-d according to Example 1 of the present invention. 3 LC-MS spectrum of DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following general description and definition of the terms and uses mentioned in the specification and claims are provided. Unless otherwise specified, the technical and scientific terms used herein are the common meanings understood by those skilled in the art for the present invention. In case of conflict, the definitions in this specification shall prevail.

[0019] In this article, all features defined in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0020] The above-mentioned features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this specification can be used in combination with 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.

[0021] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly described below in conjunction with the specific embodiments of the present invention and the corresponding drawings. Obviously, the described embodiments 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 work are within the scope of protection of the present invention.

[0022] The inventors of the present invention have found through research 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 better than those of florfenicol, and when florfenicol amine is converted into florfenicol through an acylation reaction, its labeled isotope abundance is not affected. The present invention is completed on this basis.

[0023] 1. Stable isotope labeled florfenicol amide

[0024] The synthetic method of the stable isotope-labeled florfenicol amide of the present invention comprises:

[0025] Florfenicol amide, alkali and heavy water are subjected to hydrogen-deuterium exchange reaction to obtain florfenicol amide-d 3 ; Wherein, the hydrogen-deuterium exchange reaction temperature is 50-70°C and the reaction time is 22-26h.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Suitable bases include barium oxide, sodium deuteride, potassium carbonate or cesium carbonate.

[0030] 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 preferably 58-62°C.

[0031] 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.

[0032] As a preferred embodiment of the present invention, the molar ratio of florfenicol 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.

[0033] 2. Stable isotope labeled florfenicol

[0034] The present invention also relates to a method for synthesizing stable isotope-labeled florfenicol, comprising:

[0035] Take Florfenicol Amine-d 3 , dissolved in a solvent, and then mixed with triethylamine and methyl dichloroacetate, and acylated to obtain florfenicol-d 3 ; wherein the acylation reaction temperature is 30-50°C and the reaction time is 22-26h.

[0036] Florfenicol amide-d suitable for the above method of the present invention 3It can be florfenicol amide-d obtained by the above method of the present invention 3 , or it can be florfenicol amide-d 3 .

[0037] 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.

[0038] The reaction temperature suitable 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.

[0039] The reaction time suitable for the above method of the present invention is 22 to 26 hours, preferably 23 to 25 hours.

[0040] In one example of the present invention, the synthesis method of the labeled florfenicol comprises:

[0041] (1) subjecting florfenicol amide, alkali and heavy water to a hydrogen-deuterium exchange reaction to obtain florfenicol amide-d 3 ; Wherein, the reaction temperature of the hydrogen-deuterium exchange reaction is 50-70°C, and the reaction time is 22-26h;

[0042] (2) Take florfenicol amide-d 3 , dissolved in a solvent, and then mixed with triethylamine and methyl dichloroacetate, and acylated to obtain florfenicol-d 3 ; wherein the reaction temperature of the acylation reaction is 30-50°C and the reaction time is 22-26h.

[0043] In one example of the present invention, the synthesis method of the labeled florfenicol comprises:

[0044] (1) heating florfenicol under acidic conditions at 80-110° C. and reflux for 6 h to obtain florfenicol amide;

[0045] (2) subjecting florfenicol amide, alkali and heavy water to a hydrogen-deuterium exchange reaction to obtain florfenicol amide-d 3 ; Wherein, the reaction temperature of the hydrogen-deuterium exchange reaction is 50-70°C, and the reaction time is 22-26h;

[0046] (3) Take florfenicol amide-d 3 , dissolved in a solvent, and then mixed with triethylamine and methyl dichloroacetate, and acylated to obtain florfenicol-d 3 ; wherein the reaction temperature of the acylation reaction is 30-50°C and the reaction time is 22-26h.

[0047] The synthesis method of the present invention has a short synthesis route, high atomic utilization rate, excellent yield, cheap and readily available raw materials, and the target compound florfenicol-d 3 , Florfenicolamide-d 3 The purity and abundance are both above 98%, and it can be used as an internal standard for analysis and detection.

[0048] Accordingly, the present invention provides a low-cost, simple step, and high purity and isotopic abundance florfenicol-d 3 Florfenicolamide-d 3 The synthesis method.

[0049] The main compounds involved in the present invention are shown in the following table:

[0050]

[0051] As used in the present invention, "a compound having a structure as shown in Formula I" and "a compound of Formula I" both refer to the compound numbered I in the above table. The same applies to compounds of 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-d 3 For a compound as shown in formula II, florfenicol amide-d 3 It is a compound with a structure as shown in Formula IV.

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

[0054]

[0055] In one embodiment of the present invention, in the step (1), florfenicol is dissolved in a 1 mol / L hydrochloric acid aqueous solution; the reaction temperature is 100° C. and 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-d 3 In step (3), weigh florfenicol amide-d 3 In a three-necked flask, the solvent was dissolved, triethylamine and methyl dichloroacetate were added at 1-2 drops / second, and the reaction was stirred at 40°C for 24 hours to obtain florfenicol-d 3 .

[0056] Purity and isotope abundance detection methods used in the examples:

[0057] Florfenicolamide-d 3 Purity testing

[0058] (1) Florfenicolamide-d 3 HPLC test conditions

[0059] Chromatographic column Athena C18-WP, (250mmx4.6mm, 5μm)

[0060] Column temperature 30℃

[0061] Mobile phase A: acetonitrile; B: 0.1% formic acid in water

[0062] Gradient 0min 5%A; 3min 5%A; 15min 95%A; 24min 95%A; 26min 5%A; 30min 5%A

[0063] Flow rate 1mL / min

[0064] Detector DAD

[0065] Sample 2 μL, 1.0 mg / mL in methanol

[0066] (2) The chromatographic purity was calculated using the HPLC area normalization method.

[0067] Florfenicolamide-d 3 Isotope abundance detection

[0068] Determination of Florfenicol Amine-d by the “Simplified Mass Cluster” Method 3 The peak areas of the quantitative ion pairs 251-233, 250-232, 249-231, and 248-230 were measured by mass spectrometry and were approximately equal to florfenicol amide-d 3 , Florfenicolamide-d 2 , Florfenicolamide-d 1 , Florfenicolamide-d 0 The peak area is normalized to obtain the isotope abundance percentage of d3-d0 based on the detection basis that "the mass spectrum peak intensity is proportional to the number of molecules". Finally, the formula is used:

[0069]

[0070] Wherein, D%: represents isotope abundance; i: represents the number of D labels, i=0-3; Ri: represents the percentage of the peak area of ​​i D labels, taking 3 D labels 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 labels, n=3.

[0071] Mass spectrometry conditions:

[0072] Detection mode: multiple ion scan

[0073] Ionization mode: positive ion mode

[0074] Ion source voltage: 3500

[0075] Ion transfer tube temperature: 300°C

[0076] Steam temperature: 200℃

[0077] Scan range: 100-350 (m / z)

[0078] Flow rate: 20 μL / min

[0079] Qualifying ion collision energy: 15

[0080] Quantitative ion pairs: 248-230, 249-231, 250-232, 251-233

[0081] Florfenicol-d 3 Purity testing

[0082] (1) Florfenicol-d 3 HPLC test conditions

[0083] Chromatographic column: Syncronis aQ (250mm*4.6mm, 5μm)

[0084] Column temperature: 30°C

[0085] Mobile phase: A: acetonitrile; B: 0.1% phosphoric acid water

[0086] Gradient: 0min 5%A; 2min 5%A; 20min 100%A; 21min 100%A; 22min 5%A; 25min 5%A

[0087] Flow rate: 1mL / min

[0088] Detector: DAD 225nm

[0089] Sample: 5 μL, 0.64 mg / mL in methanol

[0090] (2) The chromatographic purity was calculated using the HPLC area normalization method.

[0091] Florfenicol-d 3 Isotope abundance detection

[0092] Determination of Florfenicol-d by the “Simplified Mass Cluster” Method 3 The peak areas of the quantitative ion pairs 359-339, 358-338, 357-337, and 356-336 were measured by mass spectrometry and were approximately equal to florfenicol-d 3 , Florfenicol-d 2 , Florfenicol-d 1 , Florfenicol-d 0The peak area is normalized to obtain the isotope abundance percentage of d3-d0 based on the detection basis that "the mass spectrum peak intensity is proportional to the number of molecules". Finally, the formula is used:

[0093]

[0094] Wherein, D%: represents isotope abundance; i: represents the number of D labels, i=0-3; Ri: represents the percentage of the peak area of ​​i D labels, taking 3 D labels 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 labels, n=3.

[0095] Mass spectrometry conditions:

[0096] Detection mode: multiple ion scan

[0097] Ionization mode: Negative ion mode

[0098] Ion source voltage: 3500

[0099] Ion transfer tube temperature: 300°C

[0100] Steam temperature: 200℃

[0101] Scan range: 60-450 (m / z)

[0102] Flow rate: 20 μL / min

[0103] Qualifying ion collision energy: 15

[0104] Qualifying ion pairs: 356-335, 357-336, 358-338, 359-339

[0105] Example 1 Synthesis of Stable Isotope Labeled Florfenicol and Florfenicol Amine

[0106] A method for synthesizing stable isotope-labeled florfenicol and florfenicol amine comprises the following steps:

[0107] (1) Florfenicol hydrolysis

[0108] Take a 250mL three-necked flask, dissolve florfenicol (8g, 10mmol) in 100mL dilute hydrochloric acid (1mol / L), reflux at 100°C for 6h, monitor the reaction completion by TLC, cool the reaction solution to room temperature, extract twice with ethyl acetate to remove impurities, spin dry the aqueous phase, and recrystallize and purify to obtain florfenicol amine, 4.75g, yield 86.01%.

[0109] (2) Florfenicol amide-d 3 synthesis

[0110] Take 250mL sealed tube, stir and dissolve florfenicol amine (2.40g, 9.71mmol), barium oxide (744.05mg, 4.85mmol), and heavy water (77.75g, 3.88mol), heat and stir at 60℃ for 24h, cool to room temperature, filter to remove insoluble impurities, add silica gel and spin dry, and purify by column chromatography to obtain florfenicol amine-d 3 , 1.71 g, yield 70.39%. According to the purity and isotope abundance method, florfenicol amide-d 3 The chromatographic purity is 98.89% and the isotopic abundance is 98.73%.

[0111] Florfenicolamide-d 3 After ESI-MS detection, the mass spectrum of the positive ion mode is as follows Figure 3 As shown. Measured molecular ion peak [M+H] + =251.12, which is consistent with the theoretical calculation result of 251.09 (C 10 H 12 D 3 FNO 3 S + ) is consistent. Calculated florfenicol amide-d 3 The isotopic abundance value is 98.73atom%D, and there is no obvious dilution of isotopic abundance.

[0112] (3) Florfenicol-d 3 synthesis

[0113] Florfenicolamide-d 3 (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 1-2 drops / second. The mixture was heated at 40 °C for 24 h. The reaction was complete after TLC monitoring. The reaction solution was dried and purified by column chromatography to obtain the target product florfenicol-d 3 , 1.34 g, yield 92.85%. According to the purity and isotope abundance method, florfenicol-d 3 The chromatographic purity is 99.55% and the isotopic abundance is 98.98%.

[0114] Florfenicol-d 3 After ESI-MS detection, the mass spectrum of its negative ion mode is as follows Figure 6 As shown. Measured molecular ion peak [MH] - =359.07, which is consistent with the theoretical calculation result of 359.02 (C 12 H 10 D 3 Cl 2 FNO 4 S - ) is consistent. Florfenicol-d3 The isotopic abundance value is 98.98atom%D, and there is no obvious dilution of isotopic abundance.

[0115] Comparative Example 1 Florfenicol Amine-d 3 Synthesis

[0116] Take 250mL sealed tube, stir and dissolve florfenicol amine (2.40g, 9.71mmol), barium oxide (744.05mg, 4.85mmol), and heavy water (77.75g, 3.88mol), heat and stir at 80℃ for 12h, cool to room temperature, filter to remove insoluble impurities, add silica gel and spin dry, and purify by column chromatography to obtain florfenicol amine-d 3 The isotope abundance was detected to be 40.90%, D3: 6.04%, D2: 10.65%, D1: 83.31%, D0: 0.00%.

[0117] Comparative Example 2 Florfenicol-d 3 Synthesis

[0118] 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-d 3 The isotope abundance was 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, It is characterized in that Includes steps: Florfenicol amide, alkali and heavy water are subjected to hydrogen-deuterium exchange reaction to obtain florfenicol amide-d 3 ; Wherein, the hydrogen-deuterium exchange reaction temperature is 55-65°C, and the reaction time is 23-25h; The base is selected from barium oxide or potassium carbonate.

2. A method for synthesizing stable isotope-labeled florfenicol, It is characterized in that Includes steps: Florfenicol amide, alkali and heavy water are subjected to hydrogen-deuterium exchange reaction to obtain florfenicol amide-d 3 ; Wherein, the hydrogen-deuterium exchange reaction temperature is 55-65°C, and the reaction time is 23-25h; The base is selected from barium oxide or potassium carbonate; Take Florfenicol Amine-d 3 , dissolved in a solvent, and then mixed with triethylamine and methyl dichloroacetate, and acylated to obtain florfenicol-d 3 ; wherein the acylation reaction temperature is 30-50°C and the reaction time is 22-26h.

3. The synthesis method according to claim 2, It is characterized in that The solvent is selected from an alcohol solvent, a nitrile solvent or a mixture thereof.

4. The synthesis method according to claim 1 or 2, It is characterized in that The molar ratio of florfenicol amine, alkali and heavy water is 1:0.3-0.7:200-400.

5. The synthesis method according to claim 3, It is characterized in that The solvent is selected from methanol and / or acetonitrile.

6. The synthesis method according to claim 1 or 2, It is characterized in that The florfenicol amine is prepared by heating florfenicol under reflux at 80-110° C. for 4-8 hours under acidic conditions.

7. The synthesis method according to claim 6, It is characterized in that The florfenicol amine is prepared by heating florfenicol under reflux at 80-110° C. for 6 hours under acidic conditions.

8. The synthesis method according to claim 6, It is characterized in that The acidic condition is 0.8-1.3 mol / L hydrochloric acid aqueous solution.

9. The synthesis method according to claim 8, It is characterized in that The acidic condition is 1 mol / L hydrochloric acid aqueous solution.

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