A method for purifying diquafosol

Abstract

The present application relates to a method for purifying deferasirox sodium, which uses a complexing agent to remove metal catalysts and crystallizes under acidic conditions to obtain deferasirox sodium that meets pharmaceutical standards. The purification method of the present application is simple to operate and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of drug purification, and more specifically to diquafosol sodium (P 1 ,P 4 Purification method of bis(5'-uridine)tetraphosphate tetrasodium (U2P4). Background Technology

[0002] The sodium diquafosol (P) shown in Formula I 1 ,P 4 1,5-di(5'-uridine)tetraphosphate tetrasodium (U2P4) is used as a treatment for corneal epithelial disorders associated with dry eye. Additionally, it is a compound expected to be developed as an expectorant with expectorant-inducing effects or as a treatment for pneumonia.

[0003]

[0004] Existing technical documents disclose several methods for preparing diquafosol sodium (P 1 ,P 4 The method of bis(5'-uridine)tetraphosphate tetrasodium (U2P4).

[0005] Patent document 1: CN1147502C (international publication WO199905155) discloses a method for preparing diquaphosphonosodium (U2P4): uridine, UMP, UDP or UTP and their salts, as well as uridine nucleotide compounds, are dissolved in polar, aprotic organic solvents and hydrophobic amines, and diquaphosphonosodium is prepared by condensation using carbodiimide, active carbonyl or active phosphorus as activators. The conversion rate is low, there are many side reactions, the product is purified by resin, and the operation is cumbersome.

[0006] Patent document 2: CN105026414B (international publication WO2014103704) discloses an improved condensation method: using UMP, UDP or pyrophosphate as starting materials, the corresponding active intermediate phosphoryl imidazole is prepared with suitable reagents, and then condensed with the sodium salt of UTP, UDP or UMP in aqueous solution under the catalysis of metal ions (iron II, iron III, aluminum III, lanthanum III, cerium III) to obtain diquaphosphonosodium (U2P4). The conversion rate is high, but since metal ions other than sodium are used, ion exchange chromatography is required to remove other metal ions and purify the product. Diquaphosphonosodium is obtained by recrystallization under neutral conditions. Ion exchange chromatography is cumbersome to operate, and the impurity removal rate is low when recrystallized under neutral conditions.

[0007] Patent document 3, CN110655545A, discloses a method for preparing sodium diquafosol (U2P4): using UMP, UDP, or pyrophosphate as starting materials, the corresponding active intermediate phosphoryl imidazole is prepared with suitable reagents, and then condensed with the sodium salt of UTP, UDP, or UMP in an aqueous solution under the catalysis of metal ions (gadolinium III, samarium III) to obtain sodium diquafosol (U2P4). The conversion rate is high. The post-treatment uses an alkaline catalyst to remove metal ions, and recrystallization under alkaline conditions yields sodium diquafosol. The impurity removal rate is low under alkaline conditions.

[0008] Patent document 4: CN109096346A discloses a method for preparing diquaphosphonophosphate sodium (U2P4): UDP (metal salt or amine salt) undergoes a condensation reaction in a carbodiimide condensing agent, metal ions (calcium, magnesium, cerium, iron, lithium, aluminum, titanium, sodium) salts (halogen, carbonate, acetate, nitrate, trifluoromethanesulfonate, sulfate), and water, an organic solvent, or a mixture of water and an organic solvent to generate diquaphosphonophosphate sodium. After adding an organic solvent, a solid (crude product) precipitates. The crude product is dissolved in water, and ion exchange chromatography is used to remove other metal ions and impurities. The eluent is recrystallized under neutral conditions to obtain diquaphosphonophosphate sodium. Ion exchange chromatography is cumbersome, and recrystallization under neutral conditions results in low impurity removal rates.

[0009] Patent document 5: CN109305991A discloses a method for purifying diquaphos sodium (U2P4): crude diquaphos sodium is dissolved in purified water, added to an anion exchange resin for adsorption, eluted and purified with sodium chloride solution, concentrated by membrane filtration of the eluent, and recrystallized under neutral conditions to obtain diquaphos sodium. The ion exchange chromatography method is cumbersome, and the impurity removal rate is low under neutral conditions. Summary of the Invention

[0010] The purpose of this invention is to provide a method for purifying diquafosol sodium as shown in Formula I.

[0011] The sodium diquafosol shown in Formula I is abbreviated as U2P4.

[0012]

[0013] The purification method of the present invention includes the following steps:

[0014] Sodium diquarophosporine was prepared under the catalysis of a metal ion catalyst. First, a complexing agent was added to remove the metal ion catalyst, and then sodium diquarophosporine was obtained by crystallization under acidic conditions.

[0015] The metal ion catalyst is selected from: calcium ions, magnesium ions, iron ions, ferrous ions, gadolinium ions, samarium ions, zinc ions, copper ions, lanthanum ions, aluminum ions, barium ions, and cerium ions. More preferably, it is selected from: calcium ions, magnesium ions, gadolinium ions, samarium ions, and iron ions.

[0016] The complexing agent is selected from: oxalic acid, ammonium oxalate, alkali metal oxalate salts; phosphoric acid, ammonium phosphate, alkali metal phosphate salts. More preferably, it is selected from oxalic acid and phosphoric acid.

[0017] The acidic condition pH is selected from 1 to 6, preferably 2 to 5.

[0018] The crystallization solvent in the crystallization step is selected from methanol, ethanol, isopropanol and water system, and more preferably from methanol aqueous solution or ethanol aqueous solution.

[0019] According to one embodiment, the purification method of the present invention includes the following steps:

[0020] Add sodium uridine-5′-bisphosphate and purified water to the reaction flask, cool to below 10°C, add 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and gadolinium chloride, stir until the reaction is complete as detected by HPLC, add oxalic acid dihydrate to the reaction flask, adjust the pH of the system to 2-5 with sodium hydroxide, filter, the filter cake is gadolinium oxalate, add methanol to the filtrate to crystallize, filter, and dry the filter cake to obtain P. 1 ,P 4 - Tetrasodium bis(5'-uridylyl)tetraphosphate (U2P4) crude product.

[0021] P 1 ,P 4 Crude di(5'-uridine)tetraphosphate tetrasodium (U2P4) was dissolved in water, and the pH of the system was adjusted to 2-5 with hydrochloric acid. Methanol or ethanol was added to induce crystallization. The crystals were then filtered, and the filter cake was dried to obtain P. 1 ,P 4 -Di(5'-uridine)tetraphosphate sodium (U2P4).

[0022] According to one embodiment, the purification method of the present invention includes the following steps:

[0023] Add uridine-5′-bisphosphate disodium salt and purified water to the reaction flask, cool to below 10°C, add 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and calcium chloride, stir the reaction until the reaction is complete as detected by HPLC, add oxalic acid dihydrate to the reaction flask, adjust the pH of the system to 2-5 with sodium hydroxide, filter, add methanol to the filtrate to crystallize, filter, and dry the filter cake to obtain crude diquaphosphodium sodium.

[0024] After dissolving crude diquafosol sodium in water, the pH of the system was adjusted to 2-5 with hydrochloric acid, and methanol or ethanol was added for crystallization. The mixture was then filtered, and the filter cake was dried to obtain diquafosol sodium.

[0025] According to one embodiment, the purification method of the present invention includes the following steps:

[0026] Under nitrogen protection, N,N′-carbonyldiimidazole (CDI) and acetonitrile were added to a reaction flask and stirred. A solution of uridine-5'-diphosphate tripentamine salt in acetonitrile was added dropwise at room temperature. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in purified water. The mixture was separated, and uridine-5'-diphosphate disodium salt was added to the aqueous phase. The mixture was stirred until the solid dissolved, and the solution was adjusted to acidity with hydrochloric acid. Samarium trichloride was added, and the reaction was stirred until the reaction was complete as monitored by HPLC. Oxalic acid dihydrate was added, and the system was filtered to remove the precipitate samarium oxalate. The solution was then adjusted to alkalinity with sodium hydroxide aqueous solution, and the carbonate was hydrolyzed. The pH of the solution was adjusted to 2-5 with hydrochloric acid, and anhydrous ethanol was added to crystallize the mixture. The mixture was filtered, and the filter cake was washed with anhydrous ethanol and dried under vacuum to obtain crude diquaphosphodium sodium.

[0027] After dissolving the crude diquafosol sodium in water, the pH of the system was adjusted to 2-5 with hydrochloric acid, and methanol or ethanol was added for crystallization. The mixture was then filtered, and the filter cake was dried to obtain diquafosol sodium.

[0028] According to one embodiment, the purification method of the present invention includes the following steps:

[0029] Under nitrogen protection, N,N′-carbonyldiimidazole (CDI) and propionitrile were added to a reaction flask and stirred. A propionitrile solution of uridine-5'-diphosphate tributylamine salt was added dropwise at room temperature. After the addition was complete, the mixture was stirred at room temperature. The reaction solution was then concentrated under reduced pressure. The residue was dissolved in purified water to obtain a uridine-5'-diphosphate phosphorylimidazole solution. The solution was cooled to 0-10°C with stirring. Sodium uridine-5'-diphosphate salt was added and stirred until the solid dissolved. The pH of the solution was adjusted to 4.9 with hydrochloric acid. Then, ferric chloride aqueous solution was added and the reaction was stirred at 10°C until the reaction was complete as monitored by HPLC. Phosphoric acid was added, and ferric phosphate precipitated. The ferric phosphate was removed by filtration. The filtrate was then adjusted to alkalinity with sodium hydroxide aqueous solution, and the carbonate was hydrolyzed. The pH of the solution was adjusted to 2-5 with hydrochloric acid. Anhydrous ethanol was added to crystallize the solution. The solution was filtered, and the filter cake was washed with anhydrous ethanol and dried under vacuum to obtain crude diquaphosphonophosphate sodium.

[0030] After dissolving the crude diquafosol sodium in water, the pH of the system was adjusted to 2-5 with hydrochloric acid, and methanol or ethanol was added for crystallization. The mixture was then filtered, and the filter cake was dried to obtain diquafosol sodium.

[0031] According to one embodiment, the purification method of the present invention includes the following steps:

[0032] Under nitrogen protection, a solution of N,N-dimethylformamide (DMF) of uridine-5'-triphosphate tripentamine salt was added to a reaction flask and stirred. Then, N,N'-diisopropylcarbodiimide was added to prepare a solution of N,N-dimethylformamide (DMF) of uridine-5'-triphosphate (UTP), a cyclic phosphoric anhydride intermediate of uridine-5'-triphosphate (UTP).

[0033] Add a solution of uridine-5'-monophosphate tripentamine salt in N,N-dimethylformamide (DMF) to the above solution, then add magnesium chloride. Stir the reaction at room temperature, take a sample and monitor the reaction completion by HPLC, concentrate to remove the solvent, add water to dissolve the residue, add phosphoric acid, adjust the pH of the solution to 2-5 with hydrochloric acid, filter the system to remove magnesium phosphate, add anhydrous ethanol to the filtrate to crystallize, filter, wash the filter cake with anhydrous ethanol, and vacuum dry to obtain crude diquaphosphodium sodium.

[0034] After dissolving the crude diquafosol sodium in water, the pH of the system was adjusted to 2-5 with hydrochloric acid, and methanol or ethanol aqueous solution was added for crystallization. The mixture was then filtered, and the filter cake was dried to obtain diquafosol sodium.

[0035] Most preferably, the purification method of the present invention includes the following steps:

[0036] Add uridine-5′-bisphosphate sodium salt (UDP, 150 g) and purified water (900 g) to the reaction flask, cool to below 10°C, add 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl, 64.5 g) and gadolinium chloride (124.5 g), and stir until the reaction is complete as detected by HPLC (HPLC: U2P4, 90.7%, UDP: 0.3%, see [link]). Figure 1 66 g of oxalic acid dihydrate was added to the reaction flask, and the pH of the system was adjusted to 2-5 with sodium hydroxide. The mixture was filtered, and the filter cake was gadolinium oxalate. Methanol (1800 mL) was added to the filtrate to induce crystallization. The mixture was filtered again, the filter cake was washed with methanol, and dried under vacuum to obtain 130 g of P. 1 ,P 4 Crude di(5'-uridine)tetraphosphate tetrasodium (U2P4) (HPLC: U2P4, 97.95%, see...) Figure 2 ).

[0037] P 1 ,P 4 120 g of crude bis(5'-uridine)tetraphosphate tetrasodium (U2P4) was dissolved in water (480 g), and the pH of the system was adjusted to 2-5 with hydrochloric acid. Methanol (960 mL) was added to induce crystallization. The mixture was filtered, the filter cake was washed with methanol, and dried under vacuum to obtain 100 g of P. 1 ,P 4-Di(5'-uridine)tetraphosphate tetrasodium (HPLC: U2P4, 99.85%, U2P4) (see...) Figure 3 ).

[0038] The purification method of this invention was discovered after extensive experimental research. When oxalic acid, ammonium oxalate, alkali metal oxalate salts, phosphoric acid, ammonium phosphate, and alkali metal phosphate salts are added to the diquaphosphophosphate reaction solution containing metal ion catalysts, the metal ion catalysts readily crystallize, while diquaphosphophosphate does not crystallize. The metal ion catalysts can be removed by simple filtration, and excess oxalate and phosphate can be removed by recrystallization. After removing the metal ion catalysts, diquaphosphophosphate exhibits better crystallinity. The inventors also discovered during their research that impurities in diquaphosphophosphate are removed more efficiently under acidic conditions (especially impurities U2P5 and U2P6).

[0039] The method of this invention can not only effectively solve the problem of removing metal ion catalysts in the preparation process of sodium diquaphosphoside, but also more effectively solve the problem of removing impurities in sodium diquaphosphoside, especially for impurities U2P5 and U2P6.

[0040] The purification method of this invention avoids the cumbersome ion exchange chromatography and activated carbon column chromatography, utilizes complexation to efficiently remove metal ion catalysts, and crystallization under acidic conditions to efficiently remove process impurities, thus obtaining pharmaceutical-grade diquafosol sodium (P... 1 ,P 4 -Di(5'-uridine)tetraphosphate tetrasodium, U2P4).

[0041] The English abbreviations appearing in the instruction manual are further explained and clarified here:

[0042] UMP: uridine-5'-monophosphate

[0043] UDP: uridine-5'-bisphosphate

[0044] UTP: uridine-5'-triphosphate

[0045] U2P2:P 1 ,P 2 -Di(5'-uridine)diphosphate disodium salt

[0046] U2P3: P 1 ,P 3 -Di(5'-uridine)triphosphate trisodium salt

[0047] U2P4:P 1 ,P 4 Tetrasodium di(5'-uridine)tetraphosphate

[0048] U2P5:P 1,P 5 5'-uridineyl)pentaphosphate pentasodium salt

[0049] U2P6:P 1 ,P 6 -Di(5'-uridine)hexaphosphate hexasodium salt

[0050] EDC.HCl: 1-Ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride

[0051] CDI: N,N′-carbonyldiimidazole

[0052] DIC: N,N'-Diisopropylcarbodiimide Attached image description:

[0053] Figure 1 The present invention is used to prepare the HPLC analysis chromatogram of the reaction endpoint.

[0054] Figure 2 HPLC analysis chromatogram of the crude product after metal ion removal catalyst prepared using the present invention.

[0055] Figure 3 The HPLC analysis chromatogram of the purified acidic recrystallized product was prepared using this invention. Detailed implementation method:

[0056] Examples are presented below to better understand the invention. The method provided by the present invention has general applicability, and the synthesis of diquaphosphodium can be prepared by existing technologies CN105026414B, CN101495497B, CN109096346A, CN110655545A, but is not limited thereto.

[0057] The following embodiments are merely illustrative of the present invention, but the present invention is not limited to these embodiments in any way.

[0058] Example 1: Preparation of sodium diquaphosphochloride (U2P4) using gadolinium chloride as a metal ion catalyst

[0059] Add uridine-5′-bisphosphate sodium salt (UDP, 150 g) and purified water (900 g) to the reaction flask, cool to below 10°C, add 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl, 64.5 g) and gadolinium chloride (124.5 g), and stir until the reaction is complete as detected by HPLC (HPLC: U2P4, 90.7%, UDP: 0.3%, see [link]). Figure 1(A portion of the reaction solution was subjected to different post-treatment methods, and the results are shown in Table 1). Oxalic acid dihydrate (66g) was added to the reaction flask, and the pH of the system was adjusted to 2-5 with sodium hydroxide. The mixture was filtered, and the filter cake was gadolinium oxalate. Methanol (1800mL) was added to the filtrate to induce crystallization. The mixture was then filtered, the filter cake was washed with methanol, and dried under vacuum to obtain 130g of P. 1 ,P 4 Crude di(5'-uridine)tetraphosphate tetrasodium (U2P4) (HPLC: U2P4, 97.95%, see...) Figure 2 ).

[0060] Table 1: Comparison of different treatment methods for reaction solutions

[0061]

[0062]

[0063] According to the results in Table 1, sodium diquafosol exhibits better crystallinity after the addition of a complexing agent to remove gadolinium ions; acidity is beneficial for crystallization and impurity removal, while ethanol and methanol have comparable effects.

[0064] P 1 ,P 4 120 g of crude bis(5'-uridine)tetraphosphate tetrasodium (U2P4) was dissolved in water (480 g), and the pH of the system was adjusted to 2-5 with hydrochloric acid. Methanol (960 mL) was added to induce crystallization. The mixture was filtered, the filter cake was washed with methanol, and dried under vacuum to obtain 100 g of P. 1 ,P 4 -Di(5'-uridine)tetraphosphate tetrasodium (HPLC: U2P4, 99.85%, U2P4) (see...) Figure 3 ).

[0065] P 1 ,P 4 Crude di(5'-uridine)tetraphosphate tetrasodium (U2P4) (9g) was dissolved in water (36g) and divided into three equal portions. The pH of the system was adjusted to (0-1, 6.5-8, 9-11) with hydrochloric acid or sodium hydroxide. Methanol (24mL) was added to each portion for crystallization. The crystals were filtered, the filter cakes were washed with methanol, and dried under vacuum to obtain P. 1 ,P 4 -Di(5'-uridine)tetraphosphate tetrasodium, the specific results are shown in Table 2.

[0066] Table 2: Comparison of Recrystallization Purification Effects of Crude Products at Different pH Levels

[0067]

[0068] According to the results in Table 2, acidity is helpful in removing impurities (U2P5, U2P6 and impurity RRT1.15) after the main peak of diquaphosphodium; however, if the acidity is too strong (pH < 1), diquaphosphodium undergoes significant degradation to generate UMP, UDP and UTP. Although recrystallization can remove UMP, UDP and UTP, the yield decreases significantly.

[0069] To better investigate the degradation of sodium diquafosol under acidic conditions, we conducted a solution stability test under reflux conditions in an acidic (pH: 2–5) methanol-water system. We found that sodium diquafosol degraded, mainly producing UMP, UDP, and UTP, while other impurities showed no significant changes. The specific results are shown in Table 3.

[0070] Table 3. Stability data of sodium diquafosol in acidic (pH: 2–5) methanol-water system under reflux conditions.

[0071]

[0072]

[0073] Example 2: Preparation of sodium diquaphosphoside (U2P4) using calcium chloride as a metal ion catalyst

[0074] Add sodium uridine-5′-bisphosphate (UDP, 5 g) and purified water (30 g) to the reaction flask, cool to below 10 °C, add 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl, 3.2 g) and calcium chloride (1.9 g), and react until HPLC detection is complete (HPLC: U2P4: 91.5%, UDP: 0.9%). Add oxalic acid dihydrate (3.3 g) to the reaction flask, adjust the pH of the system to 2-5 with sodium hydroxide, filter, the filter cake is calcium oxalate, add methanol (50 mL) to the filtrate to crystallize, filter and dry to obtain 4.2 g of P. 1 ,P 4 Crude bis(5'-uridine)tetraphosphate tetrasodium (U2P4), HPLC: 97.5%.

[0075] Example 3: Preparation of sodium diquaphosphoside (U2P4) using samarium chloride as a metal ion catalyst.

[0076] Under nitrogen protection, N,N′-carbonyldiimidazole (CDI) (5.8 g, 36.0 mmol) and acetonitrile (12 ml) were added to a 100 ml three-necked flask and stirred. A solution of uridine-5′-diphosphate tripentamine salt in acetonitrile (12.0 mmol) was added dropwise at room temperature, maintaining the internal temperature ≤30℃ during the addition. After 1 hour, the reaction solution was concentrated under reduced pressure. The residue was dissolved in purified water (20 ml), separated, and cooled to 0-10℃ with stirring in the aqueous phase. Sodium uridine-5′-diphosphate (3.0 g, 6.0 mmol) was added, and the mixture was stirred until the solid dissolved. The pH of the solution was adjusted to 4 with hydrochloric acid (6N), and then samarium trichloride (SmCl3) (0.31 g, 1.2 mmol) was added. The reaction was stirred while maintaining the internal temperature at 10-15℃ until the reaction was complete as monitored by HPLC (HPLC: U2P4: 87.8%, UDP: 2.0%). Add 0.31 g of oxalic acid dihydrate, filter the system to remove the precipitate samarium oxalate, then adjust the pH of the solution to 10.0 with sodium hydroxide aqueous solution, hydrolyze the carbonate, add hydrochloric acid to adjust the pH of the solution to 2-5, add anhydrous ethanol (90 ml) to crystallize, stir at room temperature for 2.5 hours, filter, wash the filter cake with anhydrous ethanol, vacuum dry to obtain 7.5 g of white solid, take a sample for HPLC analysis, U2P4: 95.6%.

[0077] Example 4: Preparation of sodium diquaphosphoside (U2P4) using ferric chloride as a metal ion catalyst.

[0078] Under nitrogen protection, N,N′-carbonyldiimidazole (CDI) (2.9 g, 18.0 mmol) and propionitrile (6 ml) were added to a 50 ml three-necked flask and stirred. A propionitrile solution of uridine-5′-diphosphate tributylamine salt (6.0 mmol) was added dropwise at room temperature. After the addition was complete, the mixture was stirred at room temperature for 30 minutes. The reaction solution was then concentrated under reduced pressure, and the residue was dissolved in purified water (10 ml) to obtain a uridine-5′-diphosphate phosphorylimidazole solution. The solution was cooled to 0-10 °C with stirring, and uridine-5′-diphosphate sodium salt (1.5 g, 3.0 mmol) was added. The mixture was stirred until the solid dissolved, and the pH of the solution was adjusted to 4.0 with hydrochloric acid. Then, ferric chloride (FeCl3) aqueous solution (1N) (60 μL, 0.06 mmol) was added, and the reaction was stirred at 10 °C until the reaction was complete as monitored by HPLC (HPLC: U2P4: 85.6%, UDP: 4.0%). Phosphoric acid was added, and ferric phosphate precipitated. The ferric phosphate was removed by filtration. The pH of the filtrate was then adjusted to 10.0 with sodium hydroxide aqueous solution. The carbonate was hydrolyzed, and the pH of the solution was adjusted to 2-5 with hydrochloric acid. Anhydrous ethanol (30 ml) was added to crystallize, and a solid precipitated in the system. The solid was filtered, and the filter cake was washed with anhydrous ethanol. The solid was then dried under vacuum (40℃, -0.1 MPa) to constant weight, yielding 3.8 g of yellow solid. The solid was sampled and analyzed by HPLC, and the U2P4 was 92.5%.

[0079] Example 5: Preparation of sodium diquaphosphoside (U2P4) using magnesium chloride as a metal ion catalyst.

[0080] Under nitrogen protection, a DMF solution of uridine-5′-tripentylamine triphosphate salt (0.18 mol / L) (50 ml, 9.0 mmol) was added to a 250 ml three-necked flask and stirred. Then, N,N'-diisopropylcarbodiimide (DIC) (1.5 g, 11.9 mmol) was added and stirred at room temperature for 3 hours to obtain a DMF solution of uridine-5′-triphosphate, the cyclic phosphoric anhydride intermediate uridine-5′-triphosphate (c-UTP).

[0081] Add a DMF solution of uridine-5'-monophosphate tripentamine salt (0.22 mol / L) (50 mL, 11.0 mmol) to the above solution, then add magnesium chloride (1.1 g, 11.6 mmol), and stir at room temperature for 20 hours. After the reaction is complete, HPLC analysis is performed (U2P4: 63.5%, UMP: 0.7%, UTP: 3.3%). The solvent is removed by concentration, and water is added to the residue to dissolve it. Phosphoric acid (0.4 g) is added, and the pH of the solution is adjusted to 2–5 with hydrochloric acid (6N). The system is filtered to remove magnesium phosphate. Anhydrous ethanol (90 mL) is added to the filtrate for crystallization, and the mixture is stirred at room temperature for 2.5 hours. After filtration, the filter cake is washed with anhydrous ethanol and dried under vacuum to obtain 3.0 g of white solid. HPLC analysis shows U2P4: 95.6%.

[0082] Based on the above embodiments, in order to better evaluate the post-processing schemes of various processes, a list is provided below.

[0083] Table 4. Comparison of Post-processing for Various Processes

[0084]

[0085] According to the results in Table 4, the addition of a complexing agent to remove metal ion catalysts resulted in better crystallization and a high impurity removal rate.

[0086] To better evaluate the removal rates of oxalate and phosphate by metal ion catalysts, the water solubility of various salts was statistically analyzed. The solubility in the reaction system was lower than that in the pure water system.

[0087] Table 5. Solubility of oxalate and phosphate in water (100g water)

[0088]

[0089]

[0090] Comparative Example 1: Preparation of sodium diquaphosphoside using calcium chloride as a metal ion catalyst (CN109096346A)

[0091] Add sodium uridine-5′-bisphosphate (UDP, 10 g) and purified water (30 g) to the reaction flask, cool to below 10 °C, add 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl, 4.3 g) and calcium chloride (2.5 g), and react until HPLC detection is complete (HPLC: U2P4: 91.3%, UDP: 1.0%). Add purified water (20 g) to the reaction flask, add ethanol (100 mL), and crystallize (some solids agglomerate into spheres). After filtration and drying, 9.0 g of P is obtained. 1 ,P 4 Crude di(5'-uridine)tetraphosphate tetrasodium (U2P4), HPLC: 97.4%. Ion-liquid chromatography analysis revealed that the crude product was a calcium and sodium mixed salt of diquaphosphoric acid, and calcium ions could not be removed by recrystallization purification.

[0092] Based on existing literature, and to better illustrate the advantages of this patent, post-processing comparisons are shown in Table 6.

[0093] Table 6. Comparison of Dequaphos sodium metal catalyst removal methods and crude product purification

[0094]

[0095] The above comparison shows that using complexing agents to remove metal catalysts is more efficient and easier to operate than ion exchange chromatography; and using crystallization under acidic conditions to remove process impurities is more efficient, simpler to operate, and has comparable yields compared to adsorption column chromatography and activated carbon column chromatography.

Claims

1. A method for purifying diquafosol sodium, characterized in that, Includes the following steps: Add sodium uridine-5'-diphosphate and purified water to the reaction flask, cool to below 10°C, add 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and gadolinium chloride, stir until the reaction is complete as detected by HPLC, add oxalic acid dihydrate to the reaction flask, adjust the pH of the system to 2-5 with sodium hydroxide, filter, the filter cake is gadolinium oxalate, add methanol to the filtrate to crystallize, filter, and dry the filter cake to obtain crude P1,P4-di(5'-uridine)tetraphosphate tetrasodium U2P4; dissolve the crude P1,P4-di(5'-uridine)tetraphosphate tetrasodium U2P4 in water, adjust the pH of the system to 2-5 with hydrochloric acid, add methanol or ethanol to crystallize, filter, and dry the filter cake to obtain P1,P4-di(5'-uridine)tetraphosphate sodium U2P4; or Add uridine-5'-bisphosphate disodium salt and purified water to the reaction flask, cool to below 10°C, add 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride EDC.HCl and calcium chloride, stir the reaction until HPLC detection shows the reaction is complete, add oxalic acid dihydrate to the reaction flask, adjust the pH of the system to 2-5 with sodium hydroxide, filter, add methanol to the filtrate to crystallize, filter, dry the filter cake to obtain crude diquafosol sodium, dissolve the crude diquafosol sodium in water, adjust the pH of the system to 2-5 with hydrochloric acid, add methanol or ethanol to crystallize, filter, dry the filter cake to obtain diquafosol sodium.

2. The purification method according to claim 1, characterized in that it includes the following steps: Add 150 g of uridine-5′-bisphosphate sodium salt (UDP) and 900 g of purified water to a reaction flask, cool to below 10 °C, then add 64.5 g of 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and 124.5 g of gadolinium chloride. Stir the reaction until HPLC analysis shows the reaction is complete: HPLC: U2P4, 90.7%, UDP: 0.3%. Add 66 g of oxalic acid dihydrate to the reaction flask, adjust the pH of the system to 2–5 with sodium hydroxide, filter, and the filter cake is gadolinium oxalate. Add 1800 mL of methanol to the filtrate for crystallization, filter, wash the filter cake with methanol, and vacuum dry to obtain 130 g of P. 1 ,P 4 Crude bis(5'-uridine)tetraphosphate tetrasodium U2P4 (HPLC): U2P4, 97.95%.

3. The purification method according to claim 2, characterized in that it further includes the following step: P 1 ,P 4 120g of crude di(5'-uridine)tetraphosphate tetrasodium U2P4 was dissolved in 480g of water, and the pH of the system was adjusted to 2-5 with hydrochloric acid. 960mL of methanol was added for crystallization. The mixture was filtered, the filter cake was washed with methanol, and dried under vacuum to obtain 100g of P. 1 ,P 4 -Di(5'-uridine)tetraphosphate tetrasodium HPLC: U2P4, 99.85%, U2P4.

4. The purification method according to claim 1, characterized in that it includes the following steps: Add 150 g of uridine-5′-bisphosphate sodium salt (UDP) and 900 g of purified water to a reaction flask, cool to below 10 °C, then add 64.5 g of 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and 124.5 g of gadolinium chloride. Stir the reaction until HPLC analysis shows the reaction is complete: HPLC: U2P4, 90.7%, UDP: 0.3%. Add 66 g of oxalic acid dihydrate to the reaction flask, adjust the pH of the system to 2–5 with sodium hydroxide, filter, and the filter cake is gadolinium oxalate. Add 1800 mL of methanol to the filtrate for crystallization, filter, wash the filter cake with methanol, and vacuum dry to obtain 130 g of P. 1 ,P 4 Crude bis(5'-uridine)tetraphosphate tetrasodium U2P4 HPLC: U2P4, 97.95%; P 1 ,P 4 120g of crude di(5'-uridine)tetraphosphate tetrasodium U2P4 was dissolved in 480g of water, and the pH of the system was adjusted to 2-5 with hydrochloric acid. 960mL of methanol was added for crystallization. The mixture was filtered, the filter cake was washed with methanol, and dried under vacuum to obtain 100g of P. 1 ,P 4 -Di(5'-uridine)tetraphosphate tetrasodium HPLC: U2P4, 99.85%, U2P4.

5. The purification method according to claim 1, characterized in that it includes the following steps: Add 5g of uridine-5′-bisphosphate sodium salt (UDP) and 30g of purified water to the reaction flask, cool to below 10℃, add 3.2g of 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 1.9g of calcium chloride, and react until HPLC detection is complete. HPLC: U2P4: 91.5%, UDP: 0.9%. Add 3.3g of oxalic acid dihydrate to the reaction flask, adjust the pH of the system to 2-5 with sodium hydroxide, filter, the filter cake is calcium oxalate, add 50mL of methanol to the filtrate for crystallization, filter and dry to obtain 4.2g of crude P1,P4-bis(5′-uridine)tetraphosphate tetrasodium salt (U2P4), HPLC: 97.5%.

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