Preparation method of pyruvic acid-1-13C

Abstract

The invention provides a preparation method of pyruvic acid-1-13C, and relates to the technical field of chemical drugs, and the preparation method comprises the following steps: carrying out reaction and post-treatment on 13C-carbon dioxide and a Grignard reagent to obtain pyruvic acid-1-13C; wherein the Grignard reagent comprises a Grignard reagent obtained by reacting magnesium with acetyl halide. The technical problems that in the prior art, a pyruvic acid-1-13C synthesis method has safety risks, raw materials are not easy to obtain, the number of reaction reagent types is large, the number of process steps is large, and the yield is low are solved, the technical effects that safety and environmental protection are achieved, the raw materials are easy to obtain, the process is simple, and the synthesis route is short are achieved, and the obtained pyruvic acid-1-13C is high in purity and high in yield. And clinical test requirements can be met.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical drugs, and in particular to a preparation method of pyruvic-1-13C. BACKGROUND

[0002] Hyperpolarized (HP) 13C MRI is a cutting-edge molecular imaging technology that can rapidly, non-invasively and specifically study dynamic metabolism and physiological processes that were previously difficult to detect. It can quickly evaluate the distribution of 13C diagnostic drugs and their metabolites in the body. At present, this technology is being widely used in exploratory research of various important diseases such as cancer, cardiovascular disease, liver disease and kidney disease. Pyruvic-1-13C, also known as Pyruvic-1-13C acid, as an important reagent for HP 13C MRI, can be used for imaging solid tumor metabolism flux and evaluating the response of tumors to chemotherapy, radiotherapy and targeted mTOR drugs after being hyperpolarized and injected into the body. It has been used in a number of clinical research trials. As a diagnostic chemical in the body, pyruvic-1-13C has strict requirements for its purity in clinical practice.

[0003] The main synthetic routes of pyruvic-1-13C reported at present are as follows:

[0004] 1. Cambridge Isotope Laboratories, Inc., Andover, MA reported the following synthetic route in patent US7754913B2:

[0005]

[0006] This synthetic route uses 2-propenyl magnesium bromide reagent as raw material, which reacts with CO2 at low temperature to obtain sodium C-2-methyl acrylate, which is then protected by benzyl to obtain C-benzyl pyruvate, and finally hydrogenation is carried out to remove the benzyl protection to obtain pyruvic-1-13C. 13 13 C-2-methyl acrylate, which is then protected by benzyl to obtain C-benzyl pyruvate, and finally hydrogenation is carried out to remove the benzyl protection to obtain pyruvic-1-13C. 13 C-benzyl pyruvate, and finally hydrogenation is carried out to remove the benzyl protection to obtain pyruvic-1-13C.

[0007] The first and third steps of this route need to be carried out at-78℃ ultra-low temperature, which is difficult to control and has certain safety risks; ozone oxidation is used for double bond, ozone needs to be freshly prepared, and the purity is low, the amount of ozone is not easy to measure, and the end point of the reaction is difficult to accurately judge. In addition, the removal of benzyl protection needs to use palladium carbon and hydrogen, which further increases the safety risk of the process.

[0008] ​2. Patents CN116947621A, CN116947622A, and WO2023197522A1 (family patent US2024010595A1) are all patents for pyruvate-1-13C filed by Haidewei Company. The company also published a journal article with essentially the same content as the patents, titled "Research on the Synthesis of Pyruvate-1-13C," which reports the following synthesis method:

[0009] A two-step synthetic route for pyruvate-1-13C:

[0010]

[0011] One-pot synthetic route for pyruvate-1-13C:

[0012]

[0013] The preparation route of Haidewei is a simplification based on US patent US7754913B2, which eliminates the protection and deprotection steps of the carboxyl group, but still requires the use of ultra-low temperature reaction and ozone oxidation.

[0014] 3. Patent US8198484B2 (family patents US2010069675A1 and US2006178534A1) reports two synthetic routes:

[0015] Route 1:

[0016]

[0017] This route uses 1-13C-phenyl sulfoxide as a starting material to prepare alkyl lithium reagents, which are then reacted with methyl acetate to obtain 1-phenyl sulfoxide-[1- 13 C]-acetone, followed by dichloroation, dephenyl sulfoxide group removal, and dechlorination to obtain pyruvate-1-13C.

[0018] The route is relatively long, and the starting materials are not easy to obtain. The route uses lithium reagent LDA, which has poor stability and poses certain risks.

[0019] Route 2:

[0020]

[0021] This route uses anisole as a raw material to prepare alkyllithium reagents, and the alkyllithium reagents are combined with... 13 CO2 reaction yields 1-phenylmercapto-[1- 13 [C]-acetic acid, followed by acylation, amidation, and then chlorination and ethanol substitution reactions, yields [1- 13[C]-N,N-dimethyl oxalate ethyl ester was reacted with an alkyl lithium reagent to obtain a ketone carbonyl compound, which was then subjected to ketone carbonyl reduction, dephenylene sulfoxide group removal, and hydrolysis with hydrochloric acid to obtain the target product.

[0022] The route is too lengthy, and the reaction uses butyllithium, which is highly dangerous.

[0023] 4. Patent CN109096092A discloses a method for preparing pyruvate-1-13C from acetaldehyde, the process route of which is as follows:

[0024]

[0025] This route consists of four steps:

[0026] (1) Acetaldehyde is used as a raw material to condense with thiol to obtain the intermediate thioacetal;

[0027] (2) Thioacetal reacts with n-butyllithium to give intermediate dithioacetal alkyllithium solution.

[0028] (3) Dithioacetal alkyl lithium solution and 13 The reaction with CO2 yields the intermediate dithioacetal propionic acid.

[0029] (4) The intermediate dithioacetal propionic acid was deprotected to obtain the final product. 13 C-labeled pyruvate.

[0030] This route uses the hazardous reagent n-butyllithium, and the process requires column chromatography purification, making it unsuitable for industrial production.

[0031] 5. Patent CN101031534A (family patent WO2006038811) mentions the use of 13 The method for synthesizing pyruvate-1-13C from CCuCN is as follows:

[0032]

[0033] This route is relatively short and consists entirely of conventional reactions, but the starting material Cu... 13 CN is not easily obtained. If sodium cyanide or potassium cyanide (C13) is used as a substitute, it is also difficult to obtain and is a highly toxic substance, posing a safety risk.

[0034] In summary, the existing methods for synthesizing pyruvate-1-13C have certain safety risks, some raw materials are not easy to obtain, a variety of reaction reagents are used, there are many synthetic steps, and the yield is low.

[0035] In view of this, the present invention is hereby proposed. Summary of the Invention

[0036] The present application aims to provide a preparation method of pyruvic acid-1-13C, which has a short synthesis route, high yield, and meets the quality requirements of clinical use, and solves the technical problems of safety risk, difficulty in obtaining raw materials, multiple reaction reagents, multiple process steps, and low yield in the prior art.

[0037] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted:

[0038] A preparation method of pyruvic acid-1-13C, comprising the following steps:

[0039] reacting 13C-carbon dioxide with a Grignard reagent, and post-treating to obtain pyruvic acid-1-13C;

[0040] The Grignard reagent comprises a Grignard reagent obtained by reacting magnesium with acetyl halide.

[0041] Further, the preparation method of the Grignard reagent comprises the following steps:

[0042] reacting magnesium ribbon with acetyl halide in an organic solvent to obtain the Grignard reagent;

[0043] Preferably, the organic solvent comprises at least one of tetrahydrofuran and diethyl ether.

[0044] Further, the acetyl halide comprises at least one of acetyl chloride, acetyl bromide, and acetyl iodide.

[0045] Preferably, the acetyl halide is acetyl chloride or acetyl bromide.

[0046] Further, the molar ratio of the magnesium ribbon to the acetyl halide is (1.0-1.2):1.

[0047] Further, the temperature for the reaction of the 13C-carbon dioxide with the Grignard reagent is -78℃ to -20℃.

[0048] Further, the molar ratio of the amount of the 13C-carbon dioxide to the acetyl halide is 1:1.

[0049] Further, the post-treatment comprises the following steps:

[0050] After the reaction is completed, the reaction is quenched by an acid first, and then extracted to obtain an organic phase, and the organic phase is concentrated to obtain a concentrate;

[0051] The concentrate is purified to obtain pyruvic acid-1-13C.

[0052] Further, the acid comprises at least one of hydrochloric acid and sulfuric acid.

[0053] Furthermore, the organic solvent used in the extraction includes at least one of ethyl acetate and dichloromethane.

[0054] Furthermore, the purification includes the following steps:

[0055] After dissolving the obtained concentrate, the pH was adjusted to 9-12, and the liquid was separated to obtain the aqueous phase.

[0056] The pH of the resulting aqueous phase was adjusted to 1-5, and then extracted to obtain pyruvate-1-13C.

[0057] Preferably, the alkali used to adjust the pH to 9-12 includes sodium hydroxide;

[0058] Preferably, the acid used to adjust the pH to 1-5 includes hydrochloric acid.

[0059] Compared with the prior art, the present invention has at least the following beneficial effects:

[0060] The method for preparing pyruvate-1-13C provided by this invention uses acetyl halide as a raw material and 13C-carbon dioxide as an isotope source. The raw materials are readily available and the cost is low. At the same time, the synthesis route of this invention is short, the yield is high, and the quality of the obtained pyruvate-1-13C meets clinical requirements. This invention solves the technical problems of existing pyruvate-1-13C synthesis methods, such as safety risks, difficulty in obtaining raw materials, multiple types of reaction reagents, multiple process steps, and low yield. Attached Figure Description

[0061] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0062] Figure 1 A schematic diagram of the synthetic route of pyruvate-1-13C provided in one embodiment of the present invention;

[0063] Figure 2 The pyruvate-1-13C provided in Example 1 of this invention 1 H-NMR spectrum;

[0064] Figure 3 The mass spectrum of pyruvate-1-13C provided in Example 1 of this invention. Detailed Implementation

[0065] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0066] This invention provides a method for preparing pyruvate-1-13C, comprising the following steps:

[0067] 13C-carbon dioxide was reacted with Grignard reagent, followed by post-treatment, to obtain pyruvate-1-13C;

[0068] Grignard reagents include, but are not limited to, Grignard reagents obtained by reacting magnesium with acetyl halide.

[0069] This invention uses acetyl halide as a raw material and 13C-carbon dioxide as an isotope source. The raw materials are readily available and the cost is low. The synthetic route of this invention is short and the yield is high. The quality of the obtained pyruvate-1-13C meets clinical requirements. It solves the technical problems of existing pyruvate-1-13C synthesis methods, such as safety risks, difficulty in obtaining raw materials, multiple types of reaction reagents, multiple process steps, and low yield.

[0070] In a preferred embodiment, the method for preparing the Grignard reagent includes the following steps:

[0071] Magnesium strips were reacted with acetyl halide in an organic solvent to obtain Grignard reagents;

[0072] The organic solvents include, but are not limited to, at least one of tetrahydrofuran and diethyl ether.

[0073] Magnesium strips are first reacted with acetyl halide in an organic solvent to obtain Grignard reagent. The obtained Grignard reagent is then cooled and then 13C-carbon dioxide is introduced to carry out the reaction.

[0074] It should be noted that when tetrahydrofuran is used as an organic solvent, the reaction temperature between magnesium strip and acetyl halide can be 40℃~60℃, and more preferably 50℃~60℃; when diethyl ether is used as an organic solvent, the reaction temperature between magnesium strip and acetyl halide can be 20℃~35℃, and more preferably 30℃~35℃.

[0075] In a preferred embodiment, the acetyl halide includes, but is not limited to, at least one of acetyl chloride, acetyl bromide, and acetyl iodide, and is further preferably acetyl chloride or acetyl bromide.

[0076] In a preferred embodiment, the molar ratio of magnesium strip to acetyl halide can be (1.0 to 1.2):1, with typical but non-limiting molar ratios such as 1.0:1, 1.1:1, and 1.2:1.

[0077] In a preferred embodiment, the reaction temperature of 13C-carbon dioxide with Grignard reagent is -78°C to -20°C. Typical but non-limiting reaction temperatures include, for example, -78°C, -70°C, -60°C, -50°C, -40°C, -30°C, and -20°C. It is further preferred to be -40°C to -20°C, which is more conducive to further improving the product yield and the quality of pyruvate-1-13C.

[0078] In a preferred embodiment, the molar ratio of 13C-carbon dioxide to acetyl halide can be 1:1. The volume of 13C-carbon dioxide can be measured by a flow meter, and the amount introduced is the same as the molar amount of acetyl halide, which is more conducive to further reducing the amount of 13C-carbon dioxide used and reducing costs.

[0079] In a preferred embodiment, post-processing includes the following steps:

[0080] After the reaction is complete, the reaction is first quenched with acid, then extracted to obtain an organic phase. This organic phase is then concentrated to obtain a concentrate.

[0081] The concentrate was purified to obtain pyruvate-1-13C.

[0082] In this invention, 13C-carbon dioxide reacts with Grignard reagent. After the reaction is complete, acid is added to quench the reaction, followed by extraction with an organic solvent to obtain an organic phase. The obtained organic phase is then concentrated until it is essentially solvent-free to obtain a concentrate.

[0083] The acid used for quenching includes, but is not limited to, at least one of hydrochloric acid and sulfuric acid. The hydrochloric acid can be concentrated hydrochloric acid with a concentration of 12 mol / L, and the sulfuric acid can be dilute sulfuric acid with a mass fraction of 50% to 60%.

[0084] In a preferred embodiment, the organic solvent used for extraction includes, but is not limited to, at least one of ethyl acetate and dichloromethane, which is more conducive to fully extracting the product and further improving the product yield.

[0085] In a preferred embodiment, purification includes the following steps:

[0086] After dissolving the obtained concentrate, the pH was adjusted to 9-12, and the liquid was separated to obtain the aqueous phase.

[0087] The pH of the resulting aqueous phase was adjusted to 1-5, and the sample was extracted to obtain pyruvate-1-13C.

[0088] In this invention, an organic solvent is first added to the concentrate for dissolution, then the pH is adjusted for purification, and then an organic solvent is used for extraction. The resulting organic phase is dried and filtered, and then concentrated to dryness to obtain the target compound pyruvate-1-13C, which has high purity and can meet the requirements of clinical trials.

[0089] In a preferred embodiment, the base used to adjust the pH to 9-12 includes, but is not limited to, sodium hydroxide, which can be prepared as a solution with a mass fraction of 10%-30%; the acid used to adjust the pH to 1-5 includes, but is not limited to, hydrochloric acid, which can be concentrated hydrochloric acid with a concentration of 12 mol / L.

[0090] A typical preparation method for pyruvate-1-13C is shown in the schematic diagram of the synthetic route. Figure 1 This includes the following steps:

[0091] (1) Magnesium strips were reacted with acetyl halide in an organic solvent to obtain Grignard reagent;

[0092] Among them, the acetyl halide can be acetyl chloride or acetyl bromide;

[0093] The molar ratio of magnesium strip to acetyl halide can be (1.0–1.2):1;

[0094] Meanwhile, at least one of tetrahydrofuran and diethyl ether can be selected as the organic solvent;

[0095] It should be noted that when tetrahydrofuran is used as the organic solvent, the reaction temperature can be 50℃~60℃; when diethyl ether is used as the organic solvent, the reaction temperature can be 30℃~35℃.

[0096] (2) Cool the obtained Grignard reagent, pass 13C-carbon dioxide into it to react, after the reaction is complete, add acid to quench it, and then add an organic solvent to extract it to obtain an organic phase.

[0097] Among them, 13C-carbon dioxide can be measured by volume using a flow meter, and the amount introduced is the same as the molar amount of acetyl halide;

[0098] The reaction temperature can be -40℃ to -20℃;

[0099] The acid used for quenching can be at least one of hydrochloric acid and sulfuric acid. The hydrochloric acid can be concentrated hydrochloric acid with a concentration of 12 mol / L, and the sulfuric acid can be dilute sulfuric acid with a mass fraction of 50% to 60%.

[0100] The organic solvent used for extraction can be at least one of ethyl acetate and dichloromethane;

[0101] The resulting organic phase was concentrated until it was essentially solvent-free to obtain a concentrate.

[0102] (3) Add an organic solvent to the obtained concentrate to dissolve it. First, adjust the pH to 9-12 with sodium hydroxide solution, then separate the liquid to obtain the aqueous phase.

[0103] The resulting aqueous phase was then adjusted to pH 1-5 with hydrochloric acid, followed by extraction with an organic solvent. The resulting organic phase was dried, filtered, and concentrated to dryness to obtain the target compound, pyruvate-1-13C.

[0104] In summary, this invention, through a two-step chemical reaction and a one-step purification process, with the synergistic coordination of each step and its parameters, can synthesize pyruvate-1-13C of clinically required purity in a high yield. At the same time, the synthesis process of this invention consists of conventional reactions, mild conditions, safe and simple operation, no special equipment requirements, and is suitable for large-scale industrial production.

[0105] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0106] Example 1

[0107] A method for preparing pyruvate-1-13C includes the following steps:

[0108] (a) Add 26.74 g (1.1 mol) of magnesium strip to a dry reaction flask equipped with a thermometer and a reflux condenser, then add 400 ml of tetrahydrofuran, evacuate the flask, replace with nitrogen three times, and start stirring and heating.

[0109] When the temperature reaches 50℃, heating is stopped, and 78.50g (1.0mol) of acetyl chloride is slowly added dropwise. The dropping rate is controlled to maintain the system temperature at 50℃~60℃. After the addition is completed, the temperature is lowered to room temperature and reacted for 1h to obtain Grignard reagent.

[0110] (b) Cool the Grignard reagent to -30℃ to -20℃, evacuate, and introduce 22.4L (about 1mol) of 13C-carbon dioxide, controlling the introduction rate so that the system temperature does not exceed -10℃;

[0111] After the carbon dioxide 13C was completely introduced, the reaction was carried out at a controlled temperature for 1 hour. Then, 92 ml of concentrated hydrochloric acid was slowly added to quench the reaction, while keeping the temperature below 20°C. After the addition was complete, the mixture was stirred for 5 minutes. The remaining magnesium strip was removed by filtration. 500 ml of water was added to the system, and 500 ml of ethyl acetate was added each time for extraction three times. The organic phases were combined and concentrated under reduced pressure at 45°C to 50°C until there was almost no solvent, thus obtaining the concentrate.

[0112] (c) Add 800 ml of ethyl acetate to the concentrate, stir to dissolve, adjust the pH of the system to 10-11 with 20% sodium hydroxide aqueous solution, separate the liquids, wash the organic phase with 400 ml of water, and combine the aqueous phases;

[0113] Add concentrated hydrochloric acid to the aqueous phase to adjust the pH to 2-3. Extract three times with 300 ml of ethyl acetate each time. Combine the organic phases, add 40 g of anhydrous sodium sulfate to the organic phase and dry for 1 h. Filter and concentrate under reduced pressure at 45℃-50℃ to dryness to obtain colorless liquid pyruvate-1-13C. Actual yield: 72.31 g (theoretical yield: 89.05 g).

[0114] The pyruvate-1-13C obtained in this embodiment 1 The H-NMR spectrum results are as follows: Figure 2 As shown, 1 ¹H-NMR (CDCl₃, 600MHz) δ (ppm): 8.28 (s, COOH, 1H), 2.55 (s, CH₃, 3H). Mass spectrometry results for pyruvate-1-13C are as follows: Figure 3 As shown, the molecular weight of pyruvate-1-13C is 89.05, and a peak of 88.13 (M-1) appears in negative ion mode.

[0115] 1 The H-NMR spectrum and mass spectrometry results are consistent with the structure of pyruvate-1-13C, proving that the product prepared in this example is pyruvate-1-13C.

[0116] Example 2

[0117] A method for preparing pyruvate-1-13C includes the following steps:

[0118] (a) Add 26.74 g (1.1 mol) of magnesium strip to a dry reaction flask equipped with a thermometer and a reflux condenser, then add 400 ml of tetrahydrofuran, evacuate the flask, replace with nitrogen three times, and start stirring and heating.

[0119] When the temperature reaches 50℃, heating is stopped, and 122.95g (1.0mol) of acetyl bromide is slowly added dropwise. The dropping rate is controlled to maintain the system temperature at 50℃~60℃. After the addition is completed, the temperature is lowered to room temperature and reacted for 1h to obtain Grignard reagent.

[0120] (b) Cool the Grignard reagent to -30℃ to -20℃, evacuate, and introduce 22.4L (about 1mol) of 13C-carbon dioxide, controlling the introduction rate to keep the system temperature below -15℃;

[0121] After the carbon dioxide 13C was completely introduced, the reaction was carried out at a controlled temperature for 0.5 h. Then, 92 ml of concentrated hydrochloric acid was slowly added for quenching, and the temperature was controlled not to exceed 25 °C. After the addition was completed, the mixture was stirred for 5 min. The remaining magnesium strip was filtered out, and 500 ml of water was added to the system. 500 ml of ethyl acetate was added each time for extraction three times. The organic phases were combined and concentrated under reduced pressure at 45 °C to 50 °C until there was almost no solvent, to obtain the concentrate.

[0122] (c) Add 800 ml of ethyl acetate to the concentrate, stir to dissolve, adjust the pH of the system to 10-11 with 20% sodium hydroxide aqueous solution, separate the liquids, wash the organic phase with 400 ml of water, and combine the aqueous phases;

[0123] Add concentrated hydrochloric acid to the aqueous phase to adjust the pH to 2-3. Extract three times with 300 ml of ethyl acetate each time. Combine the organic phases, add 40 g of anhydrous sodium sulfate to the organic phase and dry for 1 h. Filter and concentrate under reduced pressure at 45℃-50℃ to dryness to obtain colorless liquid pyruvate-1-13C. The actual yield was 73.52 g.

[0124] Example 3

[0125] A method for preparing pyruvate-1-13C includes the following steps:

[0126] (a) Add 24.31 g (1.0 mol) of magnesium strip to a dry reaction flask equipped with a thermometer and a reflux condenser, then add 350 ml of tetrahydrofuran, evacuate the flask, replace with nitrogen three times, and start stirring and heating.

[0127] When the temperature reaches 45℃, heating is stopped, and 78.50g (1.0mol) of acetyl chloride is slowly added dropwise. The dropping rate is controlled to maintain the system temperature between 45℃ and 55℃. After the addition is complete, the temperature is lowered to room temperature and reacted for 1 hour to obtain Grignard reagent.

[0128] (b) Cool the Grignard reagent to -40℃ to -30℃, evacuate, and introduce 22.4L (about 1mol) of 13C-carbon dioxide, controlling the introduction rate so that the system temperature does not exceed -15℃;

[0129] After the carbon dioxide 13C was completely introduced, the reaction was carried out at a controlled temperature for 0.5 h. Then, 92 ml of concentrated hydrochloric acid was slowly added for quenching, and the temperature was controlled not to exceed 25 °C. After the addition was completed, the mixture was stirred for 5 min. The remaining magnesium strip was filtered out, and 500 ml of water was added to the system. 500 ml of ethyl acetate was added each time for extraction three times. The organic phases were combined and concentrated under reduced pressure at 45 °C to 50 °C until there was almost no solvent, to obtain the concentrate.

[0130] (c) Add 800 ml of ethyl acetate to the concentrate, stir to dissolve, adjust the pH of the system to 11-12 with 30% sodium hydroxide aqueous solution, separate the liquids, wash the organic phase with 400 ml of water, and combine the aqueous phases;

[0131] Add concentrated hydrochloric acid to the aqueous phase to adjust the pH to 1-2. Extract three times with 300 ml of ethyl acetate each time. Combine the organic phases, add 40 g of anhydrous sodium sulfate to the organic phase and dry for 1 h. Filter and concentrate under reduced pressure at 45℃-50℃ to dryness to obtain colorless liquid pyruvate-1-13C. The actual yield is 71.43 g.

[0132] Example 4

[0133] A method for preparing pyruvate-1-13C includes the following steps:

[0134] (a) Add 24.31 g (1.0 mol) of magnesium strip to a dry reaction flask equipped with a thermometer and a reflux condenser, then add 350 ml of tetrahydrofuran, evacuate the flask, replace with nitrogen three times, and start stirring and heating.

[0135] When the temperature reaches 45℃, heating is stopped, and 122.95g (1.0mol) of acetyl bromide is slowly added dropwise. The dropping rate is controlled to maintain the system temperature between 45℃ and 55℃. After the addition is complete, the temperature is lowered to room temperature and reacted for 1 hour to obtain the Grignard reagent.

[0136] (b) Cool the Grignard reagent to -40℃ to -30℃, evacuate, and introduce 22.4L (about 1mol) of 13C-carbon dioxide, controlling the introduction rate to keep the system temperature below -10℃;

[0137] After the carbon dioxide 13C was completely introduced, the reaction was carried out at a controlled temperature for 0.5 h. Then, 92 ml of concentrated hydrochloric acid was slowly added for quenching, and the temperature was controlled not to exceed 20 °C. After the addition was completed, the mixture was stirred for 5 min. The remaining magnesium strip was filtered out, and 500 ml of water was added to the system. 500 ml of ethyl acetate was added each time for extraction three times. The organic phases were combined and concentrated under reduced pressure at 45 °C to 50 °C until there was almost no solvent, to obtain the concentrate.

[0138] (c) Add 800 ml of ethyl acetate to the concentrate, stir to dissolve, adjust the pH of the system to 11-12 with 30% sodium hydroxide aqueous solution, separate the liquids, wash the organic phase with 400 ml of water, and combine the aqueous phases;

[0139] Add concentrated hydrochloric acid to the aqueous phase to adjust the pH to 1-2. Extract three times with 300 ml of ethyl acetate each time. Combine the organic phases, add 40 g of anhydrous sodium sulfate to the organic phase and dry for 1 h. Filter and concentrate under reduced pressure at 45℃-50℃ to dryness to obtain colorless liquid pyruvate-1-13C. The actual yield is 73.02 g.

[0140] Example 5

[0141] A method for preparing pyruvate-1-13C includes the following steps:

[0142] (a) Add 29.17 g (1.2 mol) of magnesium strip to a dry reaction flask equipped with a thermometer and a reflux condenser, then add 400 ml of diethyl ether, evacuate the flask, purge with nitrogen three times, and start stirring and heating.

[0143] When the temperature reaches 30℃, heating is stopped, and 78.50g (1.0mol) of acetyl chloride is slowly added dropwise. The dropping rate is controlled to maintain the system temperature at 30℃~35℃. After the addition is completed, the temperature is lowered to room temperature and reacted for 1h to obtain Grignard reagent.

[0144] (b) Cool the Grignard reagent to -30℃ to -20℃, evacuate, and introduce 22.4L (about 1mol) of 13C-carbon dioxide, controlling the introduction rate so that the system temperature does not exceed -10℃;

[0145] After the carbon dioxide 13C was completely introduced, the reaction was carried out at a controlled temperature for 1 hour. Then, 92 ml of concentrated hydrochloric acid was slowly added to quench the reaction, while keeping the temperature below 20°C. After the addition was complete, the mixture was stirred for 5 minutes. The remaining magnesium strip was removed by filtration. 500 ml of water was added to the system, and 500 ml of ethyl acetate was added each time for extraction three times. The organic phases were combined and concentrated under reduced pressure at 45°C to 50°C until there was almost no solvent, thus obtaining the concentrate.

[0146] (c) Add 800 ml of ethyl acetate to the concentrate, stir to dissolve, adjust the pH of the system to 10-11 with 20% sodium hydroxide aqueous solution, separate the liquids, wash the organic phase with 400 ml of water, and combine the aqueous phases;

[0147] Add concentrated hydrochloric acid to the aqueous phase to adjust the pH to 3-4. Extract three times with 300 ml of ethyl acetate each time. Combine the organic phases, add 40 g of anhydrous sodium sulfate to the organic phase and dry for 1 h. Filter and concentrate under reduced pressure at 45℃-50℃ to dryness to obtain colorless liquid pyruvate-1-13C. The actual yield is 72.10 g.

[0148] Example 6

[0149] A method for preparing pyruvate-1-13C includes the following steps:

[0150] (a) Add 29.17 g (1.2 mol) of magnesium strip to a dry reaction flask equipped with a thermometer and a reflux condenser, then add 400 ml of diethyl ether, evacuate the flask, purge with nitrogen three times, and start stirring and heating.

[0151] When the temperature reaches 30℃, heating is stopped, and 122.95g (1.0mol) of acetyl bromide is slowly added dropwise. The dropping rate is controlled to maintain the system temperature at 30℃~35℃. After the addition is completed, the temperature is lowered to room temperature and reacted for 1h to obtain Grignard reagent.

[0152] (b) Cool the Grignard reagent to -30℃ to -20℃, evacuate, and introduce 22.4L (about 1mol) of 13C-carbon dioxide, controlling the introduction rate to keep the system temperature below -15℃;

[0153] After the carbon dioxide 13C was completely introduced, the reaction was carried out at a controlled temperature for 0.5 h. Then, 92 ml of concentrated hydrochloric acid was slowly added for quenching, and the temperature was controlled not to exceed 25 °C. After the addition was completed, the mixture was stirred for 5 min. The remaining magnesium strip was filtered out, and 500 ml of water was added to the system. 500 ml of ethyl acetate was added each time for extraction three times. The organic phases were combined and concentrated under reduced pressure at 45 °C to 50 °C until there was almost no solvent, to obtain the concentrate.

[0154] (c) Add 800 ml of ethyl acetate to the concentrate, stir to dissolve, adjust the pH of the system to 10-11 with 20% sodium hydroxide aqueous solution, separate the liquids, wash the organic phase with 400 ml of water, and combine the aqueous phases;

[0155] Add concentrated hydrochloric acid to the aqueous phase to adjust the pH to 3-4. Extract three times with 300 ml of ethyl acetate each time. Combine the organic phases, add 40 g of anhydrous sodium sulfate to the organic phase and dry for 1 h. Filter and concentrate under reduced pressure at 45℃-50℃ to dryness to obtain colorless liquid pyruvate-1-13C. The actual yield was 73.16 g.

[0156] Example 7

[0157] A method for preparing pyruvate-1-13C includes the following steps:

[0158] (a) Add 26.74 g (1.1 mol) of magnesium strip to a dry reaction flask equipped with a thermometer and a reflux condenser, then add 400 ml of tetrahydrofuran, evacuate the flask, replace with nitrogen three times, and start stirring and heating.

[0159] When the temperature reaches 50℃, heating is stopped, and 78.50g (1.0mol) of acetyl chloride is slowly added dropwise. The dropping rate is controlled to maintain the system temperature at 50℃~60℃. After the addition is completed, the temperature is lowered to room temperature and reacted for 1h to obtain Grignard reagent.

[0160] (b) Cool the Grignard reagent to -30℃ to -20℃, evacuate, and introduce 22.4L (about 1mol) of 13C-carbon dioxide, controlling the introduction rate to keep the system temperature below -15℃;

[0161] After the carbon dioxide 13C was completely introduced, the reaction was carried out at a controlled temperature for 1 hour. Then, 215.6 g of 50% dilute sulfuric acid was slowly added to quench the reaction, while controlling the temperature to not exceed 25°C. After the addition was completed, the mixture was stirred for 5 minutes. The remaining magnesium strip was removed by filtration. 500 ml of water was added to the system, and 600 ml of dichloromethane was added each time for extraction three times. The organic phases were combined and concentrated under reduced pressure at 45°C to 50°C until there was almost no solvent, thus obtaining the concentrate.

[0162] (c) Add 900 ml of dichloromethane to the concentrate, stir to dissolve, adjust the pH of the system to 10-11 with 20% sodium hydroxide aqueous solution, separate the liquids, wash the organic phase with 400 ml of water, and combine the aqueous phases;

[0163] Add concentrated hydrochloric acid to the aqueous phase to adjust the pH to 2-3. Extract three times with 300 ml of dichloromethane each time. Combine the organic phases, add 40 g of anhydrous sodium sulfate to the organic phase and dry for 1 h. Filter and concentrate under reduced pressure at 35℃-40℃ to dryness to obtain colorless liquid pyruvate-1-13C. The actual yield is 71.96 g.

[0164] Example 8

[0165] A method for preparing pyruvate-1-13C includes the following steps:

[0166] (a) Add 26.74 g (1.1 mol) of magnesium strip to a dry reaction flask equipped with a thermometer and a reflux condenser, then add 400 ml of tetrahydrofuran, evacuate the flask, replace with nitrogen three times, and start stirring and heating.

[0167] When the temperature reaches 50℃, heating is stopped, and 122.95g (1.0mol) of acetyl bromide is slowly added dropwise. The dropping rate is controlled to maintain the system temperature at 50℃~60℃. After the addition is completed, the temperature is lowered to room temperature and reacted for 1h to obtain Grignard reagent.

[0168] (b) Cool the Grignard reagent to -30℃ to -20℃, evacuate, and introduce 22.4L (about 1mol) of 13C-carbon dioxide, controlling the introduction rate so that the system temperature does not exceed -10℃;

[0169] After the 13C-carbon dioxide was completely introduced, the reaction was carried out at a controlled temperature for 0.5 hours. Then, 179.7 g of 60% dilute sulfuric acid was slowly added to quench the reaction, while keeping the temperature below 20°C. After the addition was complete, the mixture was stirred for 5 minutes. The remaining magnesium strip was removed by filtration. 500 ml of water was added to the system, and 600 ml of dichloromethane was added each time for extraction three times. The organic phases were combined and concentrated under reduced pressure at 45°C to 50°C until there was almost no solvent, thus obtaining the concentrate.

[0170] (c) Add 900 ml of dichloromethane to the concentrate, stir to dissolve, adjust the pH of the system to 10-11 with 20% sodium hydroxide aqueous solution, separate the liquids, wash the organic phase with 400 ml of water, and combine the aqueous phases;

[0171] Add concentrated hydrochloric acid to the aqueous phase to adjust the pH to 2-3. Extract three times with 300 ml of dichloromethane each time. Combine the organic phases, add 40 g of anhydrous sodium sulfate to the organic phase and dry for 1 h. Filter and concentrate under reduced pressure at 35℃-40℃ to dryness to obtain colorless liquid pyruvate-1-13C. The actual yield is 73.45 g.

[0172] Comparative Example 1

[0173] The only difference between this comparative example and Example 1 is that fluoroacetyl is used instead of acetyl chloride;

[0174] The remaining steps and their process parameters are the same as in Example 1.

[0175] Compared to Example 1, the drawback of this comparative example is that it has an adverse effect on the yield and quality of the pyruvate-1-13C product.

[0176] Comparative Example 2

[0177] The only difference between this comparative example and Example 1 is that lithium is used instead of magnesium;

[0178] The remaining steps and their process parameters are the same as in Example 1.

[0179] Compared with Example 1, the drawback of this comparative example is that the Grignard reagent is not easy to generate, and the pyruvate-1-13C product cannot be obtained.

[0180] Comparative Example 3

[0181] The only difference between this comparative example and Example 1 is that an organolithium reagent is used instead of the Grignard reagent.

[0182] The remaining steps and their process parameters are the same as in Example 1.

[0183] Compared with Example 1, the drawback of this comparative example is that the reaction is highly dangerous and the yield and purity of the resulting pyruvate-1-13C product are both low.

[0184] The yield of pyruvate-1-13C obtained from the examples and comparative examples was calculated, and the purity was tested. The data results are shown in Table 1. It can be seen that the two-step reaction and one-step purification preparation method in the examples can obtain pyruvate-1-13C products with high yield and purity that meet clinical requirements.

[0185] Table 1

[0186] Yield (%) Purity (%) Example 1 81.2 99.055 Example 2 82.6 99.106 Example 3 80.2 99.120 Example 4 82.0 99.048 Example 5 81.0 99.086 Example 6 82.2 99.132 Example 7 80.8 99.058 Example 8 82.5 99.060 Comparative Example 1 41.3 53.210 Comparative Example 2 / / Comparative Example 3 63.2 84.056

[0187] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing pyruvate-1-13C, characterized in that, Includes the following steps: 13C-carbon dioxide was reacted with Grignard reagent, followed by post-treatment, to obtain pyruvate-1-13C; The Grignard reagent includes Grignard reagents obtained by reacting magnesium with acetyl halide.

2. The preparation method according to claim 1, characterized in that, The preparation method of the stated reagent includes the following steps: Magnesium strips were reacted with acetyl halide in an organic solvent to obtain the Grignard reagent; Preferably, the organic solvent includes at least one of tetrahydrofuran and diethyl ether.

3. The preparation method according to claim 2, characterized in that, The acetyl halide includes at least one of acetyl chloride, acetyl bromide, and acetyl iodide; Preferably, the acetyl halide is acetyl chloride or acetyl bromide.

4. The preparation method according to claim 2, characterized in that, The molar ratio of the magnesium strip to the acetyl halide is (1.0-1.2):

1.

5. The preparation method according to any one of claims 1-4, characterized in that, The reaction temperature between 13C-carbon dioxide and Grignard reagent is -78℃ to -20℃.

6. The preparation method according to claim 5, characterized in that, The molar ratio of 13C-carbon dioxide to acetyl halide is 1:

1.

7. The preparation method according to claim 5, characterized in that, The post-processing includes the following steps: After the reaction is complete, the reaction is first quenched with acid, then extracted to obtain an organic phase. The organic phase is then concentrated to obtain a concentrate. The concentrate was purified to obtain pyruvate-1-13C.

8. The preparation method according to claim 7, characterized in that, The acid includes at least one of hydrochloric acid and sulfuric acid.

9. The preparation method according to claim 7, characterized in that, The organic solvent used in the extraction includes at least one of ethyl acetate and dichloromethane.

10. The preparation method according to claim 7, characterized in that, The purification process includes the following steps: After dissolving the obtained concentrate, the pH was adjusted to 9-12, and the liquid was separated to obtain the aqueous phase. The pH of the resulting aqueous phase was adjusted to 1-5, and the mixture was extracted to obtain pyruvate-1-13C. Preferably, the alkali used to adjust the pH to 9-12 includes sodium hydroxide; Preferably, the acid used to adjust the pH to 1-5 includes hydrochloric acid.

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