A method for synthesizing stable isotope-labeled oxolinic acid

Abstract

A method for synthesizing stable isotope-labeled oxolinic acid belongs to the field of organic synthesis. The invention uses deuterated iodoethane labeled with a stable isotope as a stable isotope labeling source, uses piperidine as a raw material, reacts with EMME, forms a ring, undergoes a deuterated ethylation reaction to obtain a deuterated oxolinic acid ester, and finally hydrolyzes to obtain a stable isotope-labeled oxolinic acid product. The stable isotope raw material used in the invention is cheap and easy to obtain, the synthesis process is simple, the product is easy to separate and purify, and the chemical purity and isotope abundance of the obtained product both reach more than 99%, which meets the technical requirements of a stable isotope internal standard reagent required for liquid chromatography tandem mass spectrometry in food safety testing, and has important market application value.

Description

Technical Field

[0001] The invention belongs to the field of organic synthesis, and particularly relates to a method for synthesizing stable isotope-labeled oxolinic acid. Background Art

[0002] Oxolinic acid, also known as oxocyclooxolinic acid, oxisolinic acid, and olinic acid, is an antimicrobial drug for aquaculture. It is often used as a feed additive for livestock and poultry and aquatic product farming. It is widely used in the prevention and treatment of animal diseases and growth promotion. However, studies have found that long-term use of quinolones can cause adverse reactions to the central nervous system, aggravate the symptoms of myasthenia gravis, weaken respiratory muscles, and endanger life. It has potential carcinogenicity and heritability, and is prone to cause bacteria to develop drug resistance and other hazards, which has attracted widespread attention.

[0003] In 2019, the Ministry of Agriculture and Rural Affairs, the National Health Commission and the State Administration for Market Regulation issued Announcement No. 114 of 2019, GB31650-2019 "National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Foods" (replacing the corresponding part in Announcement No. 235 of the Ministry of Agriculture), stipulating that oxolinic acid is allowed for therapeutic use, but is prohibited in cattle / pigs / chickens during the egg-laying period and must not be detected during this period.

[0004] Stable isotope dilution mass spectrometry is internationally recognized as the "gold standard" for trace detection. It can significantly reduce the matrix effect and improve the accuracy of the test results. Using the stable isotope-labeled compound corresponding to the compound as an internal standard and using the internal standard method for quantitative detection will significantly improve the accuracy of the test results and reduce the detection limit of the compound.

[0005] There is no corresponding stable isotope labeled internal standard compound supply in China, and the imported foreign products are expensive, with a long supply cycle and unstable supply. Therefore, the development of stable isotope labeled oxolinic acid internal standard reagent is of great significance to the relevant fields of food safety supervision and inspection in my country. At present, the synthesis of natural abundant oxolinic acid has been maturely reported. With piperonyl as raw material, N-ethyl-3,4-(methyleneoxy)aniline is synthesized by nitration and catalytic hydrogenation ethylation reaction, and then oxolinic acid ester is synthesized with diethyl ethoxymethylenemalonate (EMME), and hydrolyzed to obtain, but there is no report on the synthesis of stable isotope labeled oxolinic acid. The reported synthesis method of natural abundant oxolinic acid cannot be directly applied to the synthesis of stable isotope labeled oxolinic acid. Based on the above situation, the present invention creatively designs a more reasonable synthesis route, and synthesizes stable isotope labeled oxolinic acid with high chemical purity and isotope abundance through readily available raw materials. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for synthesizing stable isotope-labeled oxolinic acid with high yield and high abundance.

[0007] The technical solution adopted by the present invention to solve the technical problem is: a method for synthesizing stable isotope-labeled oxolinic acid, characterized in that the synthesis route is:

[0008] .

[0009] Preferably, the synthesis method of the above-mentioned stable isotope-labeled oxolinic acid comprises the following preparation steps:

[0010] 1) reacting 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate, wherein the molar ratio of 3,4-methylenedioxyaniline to diethyl ethoxymethylenemalonate is between 1:0.9 and 1:1.1; reacting at 120°C to 140°C for 90min to 150min; evaporating ethanol; and purifying by column to obtain compound 1;

[0011] 2) Dissolve compound 1 in diphenyl ether to form a solution, heat to 220°C~230°C, and continue to react for 1.5h~2.5h; cool to 40°C~60°C; filter and wash to obtain a crude compound 2;

[0012] 3) Compound 2 and potassium carbonate are mixed, wherein the molar ratio of compound 2 to potassium carbonate is between 1:0.95 and 1:1.15, and DMF is added under nitrogen protection to prepare a solution; deuterated iodoethane is added under stirring at room temperature, wherein the molar ratio of deuterated iodoethane to compound 2 is between 1.2:1 and 1.4:1, and the reaction is stirred for 8h to 10h; filtration and washing are performed to obtain crude deuterated oxolinate D5; this step of the reaction must be reacted at a temperature below 30°C. The higher the reaction temperature, the more obvious the decrease in isotope abundance, and the use requirements cannot be met. The reason for the decrease in abundance in this step of the reaction is that the side reaction of ester exchange is more likely to occur in the reaction system after the temperature increases, and when the temperature is below 30°C, the ester exchange reaction basically does not occur in the reaction system, so that the isotope abundance can meet the use requirements.

[0013] 4) Mix the crude deuterated oxolinic acid ester D5 with 4.5wt%~5.5wt% sodium hydroxide aqueous solution; stir at room temperature for 2h~3h; cool, filter, adjust the pH to 6.5~7.5 with acetic acid, let stand to precipitate solid, filter and wash to obtain the stable isotope labeled compound oxolinic acid D5.

[0014] The invention uses stable isotope-labeled deuterated iodoethane as a stable isotope labeling source, uses 3,4-methylenediethoxyaniline (piperamide) as a raw material, and reacts with diethyl ethoxymethylmalonate (EMME) to form a ring, undergoes a deuterated ethylation reaction to obtain deuterated oxolinic acid ester, and finally hydrolyzes to obtain a stable isotope-labeled oxolinic acid product.

[0015] Preferably, in the preparation step of the stable isotope-labeled oxolinic acid, the reaction temperature in step 1) is 128°C to 132°C, and the reaction time is 110min to 130min. The preferred reaction temperature can ensure the full progress of the reaction while avoiding side reactions or raw material decomposition that may be caused by excessively high temperatures, thereby improving the selectivity and yield of the product. The preferred reaction time can ensure the completion of the reaction without excessively extending the reaction time, thereby improving production efficiency.

[0016] Preferably, in the preparation step of the stable isotope-labeled oxolinic acid, the column material used for column purification in step 1) is silica gel. Silica gel is a common material for column chromatography and has good adsorption properties, which helps to improve the purity of the target compound.

[0017] Preferably, in the preparation step of the stable isotope labeled oxolinic acid, the washing in step 2) is to use ethyl acetate to slurry the filter cake and then filter it again. In this step, washing with ethyl acetate can better wash out the impurities in compound 2, while minimizing the dissolution of compound 2 in the washing liquid, thereby ensuring the yield.

[0018] Preferably, in the preparation step of the stable isotope labeled oxolinic acid, the molar ratio of compound 2 to potassium carbonate in step 3) is 1:1 to 1.1. The preferred ratio helps to achieve complete reaction of the raw materials, reduce the waste of unreacted raw materials, and improve the yield of the target compound. The molar ratio of deuterated iodoethane to compound 2 is 1.3:1, which can ensure the full reaction of deuterated iodoethane while avoiding the subsequent purification difficulties that may be caused by excessive amounts.

[0019] Preferably, in the preparation step of the stable isotope-labeled oxolinic acid, the washing in step 3) is to filter out the DMF and then use dichloromethane to slurry, remove the upper floating matter and filter again. In this step, washing with dichloromethane can better wash out the impurities in the deuterated oxolinic acid ester D5, while minimizing the dissolution of the deuterated oxolinic acid ester D5 in the washing liquid, thereby ensuring the yield.

[0020] Preferably, in the above steps for preparing stable isotope-labeled oxolinic acid, the washing in step 4) is to slurry the filter cake obtained by filtration using a mixture of dichloromethane and methanol in any proportion and then filter again.

[0021] More preferably, in the above-mentioned preparation step of stable isotope-labeled oxolinic acid, the volume ratio of dichloromethane and methanol used for washing and pulping is 1:0.9-1.1. The mixed solution with the preferred ratio as the washing liquid is helpful to fully dissolve the impurities in the compound and improve the effect of washing and purification.

[0022] The synthesis method of the stable isotope-labeled oxolinic acid of the present invention, the chemical structure of the obtained isotope-labeled oxolinic acid is shown in formula (I)

[0023]

[0024] Compared with the prior art, the synthesis method of a stable isotope-labeled oxolinic acid of the present invention has the beneficial effects that: the present invention provides a synthesis method of a stable isotope-labeled oxolinic acid, which can efficiently synthesize high-purity and high-abundance stable isotope-labeled oxolinic acid through carefully optimized reaction conditions and purification steps. This synthesis process only involves simple operations, greatly simplifies the operation process, and improves the yield and the economy of raw materials. The selected raw materials are not only easy to obtain, but also have high utilization rates in the entire synthesis process, reducing costs and environmental impacts. In addition, the environmental friendliness of the synthesis method is reflected in the easy recovery and treatment of the solvents and reagents used. The synthesized stable isotope-labeled oxolinic acid is widely used in the fields of drug metabolism research, environmental pollutant analysis and veterinary drug residue detection because of its chemical properties that are the same as those of non-labeled compounds, greatly improving the accuracy and reliability of related detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the HNMR spectrum of the stable isotope-labeled oxolinic acid obtained in Example 1.

[0026] Figure 2 This is the LC-MS chromatogram of the stable isotope-labeled oxolinic acid obtained in Example 1.

[0027] Figure 3 This is the LC-MS positive ion spectrum of the stable isotope-labeled oxolinic acid obtained in Example 1.

[0028] Figure 4 This is the HNMR spectrum of unlabeled oxolinic acid.

[0029] Figure 5 is the HNMR spectrum of compound 1 obtained in Example 1.

[0030] Figure 6 This is the LC-MS chromatogram of compound 1 obtained in Example 1.

[0031] Figure 7 This is the LC-MS positive ion spectrum of compound 1 obtained in Example 1.

[0032] Figure 8 This is the LC-MS chromatogram of compound 2 obtained in Example 1.

[0033] Fig. 9 This is the LC-MS positive ion spectrum of compound 2 obtained in Example 1. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific embodiments, wherein Embodiment 1 is the best embodiment. Example 1

[0035] 1) 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate were reacted in a molar ratio of 1:1 at 130° C. for 2 h; ethanol was evaporated; and compound 1 was obtained by column purification.

[0036] 2) Add compound 1 to 80 ml of diphenyl ether; heat to 220°C, continue the reaction for 2 hours, cool to 50°C, a large amount of solid precipitates, filter, and slurry the filter cake with ethyl acetate and filter to obtain crude compound 2.

[0037] 3) The crude compound 2 and potassium carbonate were reacted in a 1:1 molar ratio, and 20 ml of DMF was added under nitrogen protection; deuterated iodoethane was added at room temperature with stirring, wherein the molar ratio of the crude compound 2 and deuterated iodoethane was 1:1.2, and the reaction was stirred at room temperature for 8 hours; filtered, the DMF was spin-dried, slurried with dichloromethane to remove impurities, and filtered again to obtain crude deuterated oxolinate D5.

[0038] 4) Mix the crude deuterated oxolinic acid ester D5 with a 5wt% sodium hydroxide aqueous solution; stir at room temperature for 2.5h; cool, filter, adjust the pH to 7.0 with acetic acid, precipitate a large amount of solid, filter; use a 1:1 volume ratio of dichloromethane and methanol to slurry, filter to obtain a white solid, i.e., the stable isotope labeled compound oxolinic acid D5. The total yield is 84.3% based on the amount of isotope labeled oxolinic acid obtained based on 3,4-methylenedioxyaniline, and the abundance of the obtained stable isotope labeled compound oxolinic acid D5 is 99.77%.

[0039] See attached Figure 1 The isotopic abundance of the obtained stable isotope labeled compound oxolinic acid D5 is shown in Table 1:

[0040] Table 1

[0041]

[0042] The isotopic abundance of oxolinic acid-D5 is calculated according to the atomic percentage of D at the labeling site:

[0043] D(atom%)=∑(n·xn) / n=99.77%. Example 2

[0044] 1) 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate were reacted in a molar ratio of 1:1 at 130° C. for 2 h; ethanol was evaporated; and compound 1 was obtained by column purification.

[0045] 2) Add compound 1 to 80 ml of diphenyl ether; heat to 220°C, continue the reaction for 2 hours, cool to 50°C, a large amount of solid precipitates, filter, and slurry the filter cake with ethyl acetate and filter to obtain crude compound 2.

[0046] 3) The crude compound 2 and potassium carbonate were reacted in a 1:1 molar ratio, and 20 ml of DMF was added under nitrogen protection; deuterated iodoethane was added at room temperature with stirring, wherein the molar ratio of the crude compound 2 and deuterated iodoethane was 1:1.2, and the reaction was stirred at room temperature for 8 hours; filtered, the DMF was spin-dried, slurried with dichloromethane to remove impurities, and filtered again to obtain crude deuterated oxolinate D5.

[0047] 4) Mix the crude deuterated oxolinic acid ester D5 with a 5wt% sodium hydroxide aqueous solution; stir at room temperature for 2.5h; cool, filter, adjust the pH to 7.0 with acetic acid, precipitate a large amount of solid, filter; use dichloromethane to slurry, filter to obtain a white solid, i.e., the stable isotope labeled compound oxolinic acid D5. The total yield is 84.2% based on the amount of isotope labeled oxolinic acid obtained based on 3,4-methylenedioxyaniline, and the abundance of the obtained stable isotope labeled compound oxolinic acid D5 is 99.76%. Example 3

[0048] 1) 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate were reacted in a molar ratio of 1:1 at 128° C. for 130 min; ethanol was evaporated; and compound 1 was obtained by purification by silica gel column.

[0049] 2) Add compound 1 to 80 ml of diphenyl ether; heat to 225°C, continue the reaction for 2 hours, cool to 50°C, a large amount of solid precipitates, filter, and slurry the filter cake with ethyl acetate and filter to obtain crude compound 2.

[0050] 3) The crude compound 2 and potassium carbonate were reacted in a 1:1 molar ratio, and 20 ml of DMF was added under nitrogen protection; deuterated iodoethane was added at room temperature with stirring, wherein the molar ratio of the crude compound 2 and deuterated iodoethane was 1:1.3, and the reaction was stirred at room temperature for 9 hours; filtered, the DMF was spin-dried, slurried with dichloromethane to remove impurities, and filtered again to obtain crude deuterated oxolinate D5.

[0051] 4) Mix the crude deuterated oxolinic acid ester D5 with a 5.0wt% sodium hydroxide aqueous solution; stir at room temperature for 2.5h; cool, filter, adjust the pH to 7 with acetic acid, precipitate a large amount of solid, filter; slurry with a mixture of dichloromethane and methanol in a volume ratio of 1:1, filter to obtain a white solid, i.e., the stable isotope labeled compound oxolinic acid D5. The total yield is 84.1% based on the amount of isotope labeled oxolinic acid obtained based on 3,4-methylenedioxyaniline, and the abundance of the obtained stable isotope labeled compound oxolinic acid D5 is 99.75%. Example 4

[0052] 1) 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate were reacted in a molar ratio of 1:1 at 132° C. for 110 min; ethanol was evaporated; and compound 1 was obtained by purification by silica gel column.

[0053] 2) Add compound 1 to 80 ml of diphenyl ether; heat to 225°C, continue the reaction for 2 hours, cool to 50°C, a large amount of solid precipitates, filter, and slurry the filter cake with ethyl acetate and filter to obtain crude compound 2.

[0054] 3) The crude compound 2 and potassium carbonate were reacted in a molar ratio of 1:1.1, and 20 ml of DMF was added under nitrogen protection; deuterated iodoethane was added at room temperature with stirring, wherein the molar ratio of the crude compound 2 and deuterated iodoethane was 1:1.3, and the reaction was stirred at room temperature for 9 hours; filtered, the DMF was spin-dried, slurried with dichloromethane to remove impurities, and filtered again to obtain crude deuterated oxolinate D5.

[0055] 4) Mix the crude deuterated oxolinic acid ester D5 with a 5.0wt% sodium hydroxide aqueous solution; stir at room temperature for 2.3h; cool, filter, adjust the pH to 7.2 with acetic acid, precipitate a large amount of solid, filter; use a 1:1 volume ratio of dichloromethane and methanol to slurry, filter to obtain a white solid, i.e., the stable isotope labeled compound oxolinic acid D5. The total yield is 84.0% based on the amount of isotope labeled oxolinic acid prepared based on 3,4-methylenedioxyaniline, and the abundance of the obtained stable isotope labeled compound oxolinic acid D5 is 99.76%. Example 5

[0056] 1) 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate were reacted in a molar ratio of 1:0.9 at 120° C. for 150 min; ethanol was evaporated; and compound 1 was obtained by purification by silica gel column.

[0057] 2) Add compound 1 to 80 ml of diphenyl ether; heat to 220°C, continue the reaction for 1.5 h, cool to 40°C, a large amount of solid precipitates, filter, and slurry the filter cake with ethyl acetate and filter to obtain crude compound 2.

[0058] 3) The crude compound 2 and potassium carbonate were reacted in a molar ratio of 1:0.95, and 20 ml of DMF was added under nitrogen protection; deuterated iodoethane was added at room temperature with stirring, wherein the molar ratio of the crude compound 2 and deuterated iodoethane was 1:1.2, and the reaction was stirred at room temperature for 8 hours; filtered, the DMF was spin-dried, and the mixture was slurried with dichloromethane to remove impurities, and filtered again to obtain crude deuterated oxolinate D5.

[0059] 4) Mix the crude deuterated oxolinic acid ester D5 with a 4.5wt% sodium hydroxide aqueous solution; stir at room temperature for 3h; cool, filter, adjust the pH to 6.5 with acetic acid, precipitate a large amount of solid, filter; use a mixture of dichloromethane and methanol in a volume ratio of 1:0.9 to slurry, filter to obtain a white solid, i.e., the stable isotope labeled compound oxolinic acid D5. The total yield is 82.7% based on the amount of isotope labeled oxolinic acid obtained based on 3,4-methylenedioxyaniline, and the abundance of the obtained stable isotope labeled compound oxolinic acid D5 is 99.56%. Example 6

[0060] 1) 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate were reacted in a molar ratio of 1:1.1 at 140° C. for 90 min; ethanol was evaporated; and compound 1 was obtained by purification by silica gel column.

[0061] 2) Add compound 1 to 80 ml of diphenyl ether; heat to 230°C, continue the reaction for 2.5 hours, cool to 60°C, a large amount of solid precipitates, filter, and slurry the filter cake with ethyl acetate and filter to obtain crude compound 2.

[0062] 3) The crude compound 2 and potassium carbonate were reacted in a molar ratio of 1:1.15, and 20 ml of DMF was added under nitrogen protection; deuterated iodoethane was added at room temperature with stirring, wherein the molar ratio of the crude compound 2 and deuterated iodoethane was 1:1.4, and the reaction was stirred at room temperature for 10 hours; filtered, the DMF was spin-dried, slurried with dichloromethane to remove impurities, and filtered again to obtain crude deuterated oxolinate D5.

[0063] 4) Mix the crude deuterated oxolinic acid ester D5 with a 5.5wt% sodium hydroxide aqueous solution; stir at room temperature for 2h; cool, filter, adjust the pH to 7.5 with acetic acid, precipitate a large amount of solid, filter; use a mixture of dichloromethane and methanol in a volume ratio of 1:1.1 to slurry, filter to obtain a white solid, i.e., the stable isotope labeled compound oxolinic acid D5. The total yield is 82.1% based on the amount of isotope labeled oxolinic acid obtained based on 3,4-methylenedioxyaniline, and the abundance of the obtained stable isotope labeled compound oxolinic acid D5 is 99.62%.

[0064] Comparative Example 1

[0065] 1) 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate were reacted in a molar ratio of 1:1 at 130° C. for 2 h; ethanol was evaporated; and compound 1 was obtained by column purification.

[0066] 2) Add compound 1 to 80 ml of diphenyl ether; heat to 220°C, continue the reaction for 2 hours, cool to 50°C, a large amount of solid precipitates, filter, and slurry the filter cake with butyl acetate and filter to obtain crude compound 2.

[0067] 3) The crude compound 2 and potassium carbonate were reacted in a 1:1 molar ratio, and 20 ml of DMF was added under nitrogen protection; deuterated iodoethane was added at room temperature with stirring, wherein the molar ratio of the crude compound 2 and deuterated iodoethane was 1:1.2, and the reaction was stirred at room temperature for 8 hours; filtered, the DMF was spin-dried, slurried with dichloromethane to remove impurities, and filtered again to obtain crude deuterated oxolinate D5.

[0068] 4) Mix the crude deuterated oxolinic acid ester D5 with a 5wt% sodium hydroxide aqueous solution; stir at room temperature for 2.5h; cool, filter, adjust the pH to 7.0 with acetic acid, precipitate a large amount of solid, filter; use a 1:1 volume ratio of dichloromethane and methanol to slurry, filter to obtain a white solid, i.e., the stable isotope labeled compound oxolinic acid D5. The total yield is 79.5% based on the amount of isotope labeled oxolinic acid obtained based on 3,4-methylenedioxyaniline, and the abundance of the obtained stable isotope labeled compound oxolinic acid D5 is 99.77%.

[0069] Comparative Example 2

[0070] 1) 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate were reacted in a molar ratio of 1:1 at 130° C. for 2 h; ethanol was evaporated; and compound 1 was obtained by column purification.

[0071] 2) Add compound 1 to 80 ml of diphenyl ether; heat to 220°C, continue the reaction for 2 hours, cool to 50°C, a large amount of solid precipitates, filter, and slurry the filter cake with ethyl acetate and filter to obtain crude compound 2.

[0072] 3) The crude compound 2 and potassium carbonate were reacted in a 1:1 molar ratio, and 20 ml of DMF was added under nitrogen protection; deuterated iodoethane was added at room temperature with stirring, wherein the molar ratio of the crude compound 2 and deuterated iodoethane was 1:1.2, and the reaction was stirred at room temperature for 8 hours; filtered, the DMF was spin-dried, and methanol was used to slurry to remove impurities, and filtered again to obtain crude deuterated oxolinate D5.

[0073] 4) Mix the crude deuterated oxolinic acid ester D5 with a 5wt% sodium hydroxide aqueous solution; stir at room temperature for 2.5h; cool, filter, adjust the pH to 7.0 with acetic acid, precipitate a large amount of solid, filter; use a 1:1 volume ratio of methanol to slurry, filter to obtain a white solid, i.e., the stable isotope labeled compound oxolinic acid D5. The total yield is 78.7% based on the amount of isotope labeled oxolinic acid obtained with 3,4-methylenedioxyaniline, and the abundance of the obtained stable isotope labeled compound oxolinic acid D5 is 99.75%.

[0074] Comparative Example 3

[0075] 1) 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate were reacted in a molar ratio of 1:1 at 130° C. for 2 h; ethanol was evaporated; and compound 1 was obtained by column purification.

[0076] 2) Add compound 1 into 80 ml of chlorobenzene; heat to 220°C, continue the reaction for 2 h, cool to 50°C, solid precipitates, filter, slurry the filter cake with ethyl acetate, filter, and obtain crude compound 2.

[0077] 3) The crude compound 2 and potassium carbonate were reacted in a 1:1 molar ratio, and 20 ml of DMF was added under nitrogen protection; deuterated iodoethane was added at room temperature with stirring, wherein the molar ratio of the crude compound 2 and deuterated iodoethane was 1:1.2, and the reaction was stirred at room temperature for 8 hours; filtered, the DMF was spin-dried, slurried with dichloromethane to remove impurities, and filtered again to obtain crude deuterated oxolinate D5.

[0078] 4) Mix the crude deuterated oxolinic acid ester D5 with a 5wt% sodium hydroxide aqueous solution; stir at room temperature for 2.5h; cool, filter, adjust the pH to 7.0 with acetic acid, precipitate a large amount of solid, filter; use a 1:1 volume ratio of dichloromethane and methanol to slurry, filter to obtain a white solid, i.e., the stable isotope labeled compound oxolinic acid D5. The total yield is 76.1% based on the amount of isotope labeled oxolinic acid obtained with 3,4-methylenedioxyaniline, and the abundance of the obtained stable isotope labeled compound oxolinic acid D5 is 99.21%.

[0079] Comparative Example 4

[0080] 1) 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate were reacted in a molar ratio of 1:1 at 130° C. for 2 h; ethanol was evaporated; and compound 1 was obtained by column purification.

[0081] 2) Add compound 1 to 80 ml of diphenyl ether; heat to 220°C, continue the reaction for 2 hours, cool to 50°C, a large amount of solid precipitates, filter, and slurry the filter cake with ethyl acetate and filter to obtain crude compound 2.

[0082] 3) The crude compound 2 and potassium carbonate were reacted in a 1:1 molar ratio, and 20 ml of DMF was added under nitrogen protection; deuterated iodoethane was added at room temperature with stirring, wherein the molar ratio of the crude compound 2 and deuterated iodoethane was 1:1.2, and the reaction was stirred at room temperature for 8 hours; filtered, the DMF was spin-dried, slurried with dichloromethane to remove impurities, and filtered again to obtain crude deuterated oxolinate D5.

[0083] 4) Mix the crude deuterated oxolinic acid ester D5 with a 5wt% sodium hydroxide aqueous solution; stir at 40°C for 2.5h; cool, filter, adjust the pH to 7.0 with acetic acid, precipitate a large amount of solid, filter; slurry with a mixture of dichloromethane and methanol in a volume ratio of 1:1, filter to obtain a white solid, i.e., the stable isotope labeled compound oxolinic acid D5. The total yield is 67.9% based on the amount of isotope labeled oxolinic acid obtained based on 3,4-methylenedioxyaniline, and the abundance of the obtained stable isotope labeled compound oxolinic acid D5 is 97.41%.

[0084] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for synthesizing stable isotope-labeled oxolinic acid, characterized in that: The synthetic route is: ； The method comprises the following preparation steps: 1) reacting 3,4-methylenedioxyaniline and diethyl ethoxymethylenemalonate, wherein the molar ratio of 3,4-methylenedioxyaniline to diethyl ethoxymethylenemalonate is between 1:0.9 and 1.1; reacting at 120°C to 140°C for 90min to 150min; evaporating ethanol; and purifying by column to obtain compound 1; 2) Compound 1 was dissolved in diphenyl ether to form a solution, heated to 220°C~230°C, and the reaction was continued for 1.5h~2.5h; Cool to 40°C~60°C; filter and wash to obtain crude compound 2; 3) Compound 2 and potassium carbonate are mixed, wherein the molar ratio of compound 2 to potassium carbonate is between 1:0.95 and 1.15, and DMF is added under nitrogen protection to prepare a solution; deuterated iodoethane is added under stirring at room temperature, wherein the molar ratio of deuterated iodoethane to compound 2 is between 1.2 and 1.4:1, and the mixture is stirred for reaction for 8 h to 10 h; filtering and washing to obtain a crude deuterated oxolinate D5; 4) Mix the crude deuterated oxolinic acid ester D5 with 4.5wt%~5.5wt% sodium hydroxide aqueous solution; stir at room temperature for 2h~3h; cool, filter, adjust the pH to 6.5~7.5 with acetic acid, let stand to precipitate solid, filter and wash to obtain the stable isotope labeled compound oxolinic acid D5; The washing in step 2) is to slurry the filter cake with ethyl acetate and then filter it again; The washing in step 3) is to filter out the DMF and then use dichloromethane to slurry, remove the upper floating matter and then filter again; The washing in step 4) is to slurry the filter cake obtained by filtration with a mixture of dichloromethane and methanol in any proportion and then filter again, and the volume ratio of dichloromethane and methanol used for washing and slurrying is 1:0.9~1.

1.

2. The method for synthesizing stable isotope-labeled oxolinic acid according to claim 1, characterized in that: The reaction temperature in step 1) is 128° C. to 132° C., and the reaction time is 110 min to 130 min.

3. The method for synthesizing stable isotope-labeled oxolinic acid according to claim 1, characterized in that: The column material used for column purification in step 1) is silica gel.

4. The method for synthesizing stable isotope-labeled oxolinic acid according to claim 1, characterized in that: The molar ratio of compound 2 to potassium carbonate in step 3) is 1:1-1.1, and the molar ratio of deuterated iodoethane to compound 2 is 1.3:

1.

5. The method for synthesizing stable isotope-labeled oxolinic acid according to claim 1, characterized in that: The chemical structure of the obtained isotope-labeled oxolinic acid is shown in formula (I): （Ⅰ）。

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