CN114644664A - Method for preparing symmetric uridine diphosphate - Google Patents

Method for preparing symmetric uridine diphosphate Download PDF

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CN114644664A
CN114644664A CN202011503324.9A CN202011503324A CN114644664A CN 114644664 A CN114644664 A CN 114644664A CN 202011503324 A CN202011503324 A CN 202011503324A CN 114644664 A CN114644664 A CN 114644664A
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uridine
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tetraphosphate
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李建超
高永宏
刘振亚
周晓东
李殿庆
文东升
龙慎杰
范传文
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Qilu Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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Abstract

The invention relates to the technical field of medicines, in particular to a method for preparing symmetrical bis-uridine tetraphosphate. The invention has the characteristics of simple process, convenient operation, short production period and high yield.

Description

Method for preparing symmetric uridine diphosphate
Technical Field
The invention relates to the technical field of organic chemistry and medicine, in particular to a method for preparing symmetric bisuridine tetraphosphate.
Background
Diquafosol Tetrasodium salt (Diquafosol Tetrasodium) with chemical name P1,P4-tetra sodium bis (uridine 5' -) tetraphosphate, EnglishThe academic name is Tetrasodium P1,P4Bis (5' -uridyl) tetraphosphate tetrahydronate, developed jointly by Shentian pharmacy and Yinsye pharmaceutical company, is the first P2Y2 receptor agonist on the market worldwide and treats dry eye with a new mechanism of action. Clinical studies conducted in japan on diquafosol eye drops have shown that dry eye is ameliorated by promoting the secretion of mucin and water, thereby bringing the tear film closer to a normal state. In addition, no serious eye and systemic adverse reaction is found in clinical research, and the tolerance of long-term administration is good.
The existing methods for preparing the diquafosol tetrasodium mainly comprise four methods. One method is to activate uridine 5 '-monophosphate and then react with uridine 5' -triphosphate to prepare diquafosol tetrasodium, as reported in patent applications CN105026414A, CN1265114A and CN102317300A, according to the following reaction formula:
Figure BDA0002844134130000011
another method is to activate uridine 5 '-diphosphate and then react with uridine 5' -diphosphate to prepare diquafosol tetrasodium, as reported in patent applications CN105026414A and CN1265114A, according to the following reaction formula:
Figure BDA0002844134130000021
the third method is to react uridine 5 ' -monophosphate with pyrophosphate to form uridine 5 ' -triphosphate, and then react with uridine 5 ' -monophosphate to prepare diquafosol tetrasodium, as reported in patent applications CN1502622A, CN104592338A, CN106928269A and WO2008024169A, wherein the reaction formula is as follows:
Figure BDA0002844134130000022
the fourth method is to firstly prepare uridine 5' -triphosphate (UTP) in N, N-dimethylformamide and N, N-dimethylethylThe cyclic triphosphate (UTP-3 Bu) is formed by the action of a carbodiimide condensing agent in a single or mixed solvent of amide, formamide, pyridine, dioxane and dimethyl sulfoxide3NH), and uridine 5' -monophosphate (UMP-2 Bu)3NH), a process for the preparation of diquafosoturdium, as reported in both patent applications CN101495497A and CN1265114A, the reaction formula is as follows:
Figure BDA0002844134130000023
the preparation methods all have certain defects:
(1) virulent tri-n-butylamine (2015 edition of virulent chemical catalogue, serial number: 1923, CAS: 102-82-9) is adopted as an organic base to participate in the reaction, and the material is difficult to purchase, transport and the like, and has potential influence on environment, personnel and product quality when used in the synthetic process of medical products;
(2) the first, second and third preparation methods have the defects of low purity of reaction liquid and low yield;
(3) the four preparation methods are all operated by removing the polar aprotic solvent with high boiling point by adopting a high-temperature reduced-pressure concentration mode after the reaction is finished; high-temperature degradation of part of products is easily caused in the high-temperature concentration process, and the risk of high-temperature degradation of the products is increased along with the enlargement of production scale and the extension of the concentration period;
(4) the four preparation methods have the defects of complicated operation of the post-treatment purification process and long production period.
Internal invention
Aiming at the defects of the prior art, the invention provides a preparation method of diquafosol tetrasodium, which is simple and convenient to operate, environment-friendly and suitable for industrial production, and mainly researches the following aspects.
Firstly, adopting tri-n-pentylamine or tri-n-propylamine to replace tri-n-butylamine as organic base for use, and preparing the obtained P1,P4The chemical purity of the reaction liquid of the ammonium salt of the (uridine 5' -) tetraphosphate is more than 80 percent, and the method is the same as the method disclosed by the prior artThe method is equivalent, and the use of a hypertoxic tri-n-butylamine is avoided.
Second, preparation of the resulting P1,P4The reaction solution of the-di (uridine 5' -) tetraphosphate amine salt is added with an anti-solvent for crystallization, so that solid and liquid can be effectively separated, the operation of concentrating the polar aprotic solvent at high temperature is avoided, the production period is shortened, and the risk of high-temperature degradation of the product is reduced. Meanwhile, different types of anti-solvents are screened, and the anti-solvents are mainly considered from the aspects of solid precipitation, difficulty of solid-liquid separation, purity of products and the like, and researches show that one or more combinations of benzene, toluene, tetrahydrofuran, acetonitrile, C1-C4 fatty acid esters, C1-C4 alcohols, C2-C6 ethers, C2-C6 ketones, dichloromethane and trichloromethane can obtain better solid forms and realize effective separation of solid and liquid.
Thirdly, the dosage of the anti-solvent adopted in the post-treatment process is considered. Research shows that the anti-solvent consumption is small, the solid obtained by crystallization has good shape and is easy to separate solid from liquid, but the yield is low, and the solid obtained by crystallization is difficult to realize subsequent solid-liquid separation operation along with the increase of the anti-solvent consumption. By experiment, it was finally determined that the volume of the antisolvent is preferably 0.5 to 5 times, preferably 1 to 3 times the volume of the polar aprotic solvent.
The main technical scheme of the invention is as follows:
the invention provides a P1,P4A method for preparing sodium-bis (uridine 5' -) tetraphosphate comprising the steps of:
(1) reacting a compound shown in the formula I with a carbodiimide condensing agent in a polar aprotic solvent to obtain a compound shown in the formula II, and then reacting with a compound shown in the formula III in the presence of a magnesium salt to obtain a reaction liquid of a compound shown in the formula IV, wherein X is tri-n-pentylamine or tri-n-propylamine;
(2) adding an anti-solvent after the reaction is finished, and crystallizing to obtain a compound shown in the formula IV;
(3) purifying the compound shown in the formula IV by column chromatography, performing ion exchange and recrystallizing to obtain P1, P4-di (uridine 5' -) sodium tetraphosphate;
Figure BDA0002844134130000041
in particular, P of the invention1,P4A method for preparing sodium-bis (uridine 5' -) tetraphosphate comprising the steps of:
(1) dissolving a compound shown in the formula I in a polar aprotic solvent, adding a carbodiimide condensing agent, and reacting at 0-50 ℃ to obtain a solution of a compound shown in the formula II;
(2) dissolving a compound shown in a formula III in a polar aprotic solvent, adding the polar aprotic solvent into the reaction solution obtained in the step (1), adding a magnesium salt, and reacting at 0-50 ℃ to obtain a reaction solution of a compound shown in a formula IV;
(3) adding an anti-solvent into the reaction liquid of the compound shown in the formula IV, and crystallizing to obtain the compound shown in the formula IV;
(4) purifying the compound shown in the formula IV by anion column chromatography, performing gradient elution by using a saline solution, collecting eluent with the purity of more than 97 percent, and concentrating the eluent to obtain a concentrated solution;
(5) carrying out ion exchange on the concentrated solution obtained in the step (4) through cation resin, and concentrating eluent;
(6) the residue obtained by the concentration in the step (5) was recrystallized using water and an alcohol solvent to obtain P1, P4-bis (uridine 5' -) sodium tetraphosphate.
In the above method, the reaction temperature in step (1) or (2) is 0 to 50 ℃, preferably 20 to 35 ℃.
In the above method, the antisolvent in the step (3) is one or more of benzene, toluene, tetrahydrofuran, acetonitrile, fatty acid esters of C1-C4, alcohols of C1-C4, ethers of C2-C6, ketones of C2-C6, dichloromethane, and chloroform; the volume of the anti-solvent is 0.5-5 times of that of the polar aprotic solvent.
In the above method, the antisolvent is preferably dichloromethane, ethyl acetate, acetonitrile or butanone, and the volume of the antisolvent is preferably 1 to 3 times the volume of the polar aprotic solvent.
In the above method, the polar aprotic solvent is one or a combination of N, N-dimethylformamide, N-dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone.
In the above method, the carbodiimide-based condensing agent is one or a combination of N, N '-Diisopropylcarbodiimide (DIC), N' -Dicyclohexylcarbodiimide (DCC), and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), and the molar ratio thereof to the compound of formula i is 1.0 to 2.0.
In the above method, the magnesium salt is magnesium chloride, and the molar ratio of the magnesium salt to the compound of formula I is 0.5-2.0.
In the above method, the alcoholic solvent used for recrystallization is one or a combination of two or more of C1 to C4 alcohols, preferably one or a combination of two or methanol and ethanol.
More specifically, P of the present invention1,P4A method for preparing sodium-bis (uridine 5' -) tetraphosphate comprising the steps of:
(1) dissolving uridine 5 '-tri-N-pentylamine triphosphate in N-methylpyrrolidone, adding N, N' -diisopropylcarbodiimide, and reacting at the temperature of 0-50 ℃; adding N-methylpyrrolidone solution of uridine 5 '-mono-phosphoric acid tri-N-pentylamine salt into reaction liquid of uridine 5' -tri-N-pentylamine salt, adding magnesium chloride hexahydrate, stirring and reacting at 0-50 ℃, adding dichloromethane, and performing suction filtration to obtain P1,P4-tri-n-pentylamine bis (uridine 5' -) tetraphosphate;
(2) p obtained in the step (1)1,P4Dissolving the-di (uridine 5' -) tripentylamine tetraphosphate in purified water, purifying by DEAE Sephadex A-25 column chromatography, performing gradient elution by using 0.1-0.8 mol/L ammonium bicarbonate water solution, collecting eluent with the purity of more than 97%, removing 1/2-2/3 water by reduced evaporation, performing ion exchange by using Na + type 732 cationic resin, and concentrating the eluent after the exchange to obtain residue. Recrystallizing with purified water and methanol to obtain P1,P4-sodium bis (uridine 5' -) tetraphosphate.
The preparation of uridine 5 '-tri-n-pentylamine triphosphate and uridine 5' -tri-n-pentylamine monophosphate is carried out by using tri-n-pentylamine or tri-n-propylamine instead of tri-n-butylamine, referring to the methods of uridine 5 '-tri-n-butylamine triphosphate and uridine 5' -tri-n-butylamine monophosphate in the prior art. Uridine 5 '-triphosphate trisodium salt and uridine 5' -monophosphate disodium salt are all outsourced raw materials, and the manufacturer is the Wenhu Huaren science and technology Co.
The invention has the following beneficial effects:
(1) the method adopts the anti-solvent for crystallization to obtain the uridine diphosphate amine salt, saves the operation of high-temperature concentration, saves the production time, and avoids the risk of impurities possibly generated by high-temperature operation; the method has the advantages of low cost, simple operation and short process period.
(2) The method adopts the anti-solvent for crystallization to obtain the uridine diphosphate amine salt, the chemical purity reaches more than 85 percent, the qualified product can be obtained without complicated purification steps, and the production efficiency is improved.
(3) The method avoids the use of a highly toxic tri-n-butylamine, is environment-friendly and is suitable for industrial production.
Detailed Description
The foregoing and other aspects of the present invention will become more apparent from the following detailed description, which is provided for illustration and explanation of the present invention and is not to be construed as limiting the scope of the present invention in any way, as the technology embodied in the above-described manner falls within the scope of the present invention. The materials and methods of operation used in the present invention are well known in the art unless otherwise indicated. The starting materials and solvents used in the present invention are commercially available or can be prepared by known methods.
EXAMPLE 1 preparation of Tri-n-pentylamine uridine 5' -triphosphate
Uridine 5' -triphosphate trisodium salt 70.0g (0.127mol) was dissolved in 210ml of purified water, applied to a pretreated H + 732 cationic resin chromatography column, eluted with purified water, and the eluate with pH < 4 was collected. After the collection, tri-n-pentylamine was added to adjust the pH to neutral. The mixture was concentrated to a residue, and 350ml of N-methylpyrrolidone was added thereto and dissolved by stirring. 70ml of toluene were added, the reduced pressure distillation was continued, and the operation was repeated twice. 76.0g of 4A molecular sieve was added thereto, and the mixture was sealed and stored to obtain an N-methylpyrrolidone solution of uridine 5' -triphosphate, tri-N-pentylamine salt.
EXAMPLE 2 preparation of uridine 5' -monophosphate tri-n-pentylamine salt
Uridine 5' -monophosphate disodium salt 38.0g (0.103mol) was dissolved in 110ml of purified water and loaded to pretreated H+Separating with 732 type cation resin chromatography column, eluting with purified water, and collecting eluate with pH value less than 4. After the collection, tri-n-pentylamine was added to adjust the pH to neutral. Concentrate to a residue, add 200ml of N-methylpyrrolidone, and dissolve with stirring. 50ml of toluene were added and the reduced pressure distillation was continued and the operation was repeated twice. Adding 50.0g of 4A molecular sieve, and sealing and storing to obtain the N-methylpyrrolidone solution of uridine 5' -monophosphate tri-N-pentylamine salt.
Example 3P1,P4Preparation of tri-n-pentylamine-bis (uridine 5' -) tetraphosphate salt
An N-methylpyrrolidone solution of uridine 5 '-tri-N-pentylamine triphosphate salt according to example 1 (equivalent to 50.0g of uridine 5' -triphosphate (0.103mol)) was added with 16.3g N, N '-diisopropylcarbodiimide (0.129mol), and the mixture was reacted at 20 to 35 ℃ for 1.5 hours to activate the uridine 5' -triphosphate salt. Adding the N-methylpyrrolidone solution of uridine 5' -monophosphate tri-N-pentylamine salt obtained in example 2 into the activated reaction solution, adding 42.0g (0.206mol) of magnesium chloride hexahydrate, stirring at 20-35 ℃ for reaction for 8-10 hours, adding 550ml of dichloromethane, and performing suction filtration to obtain a white solid P1,P4-tri-n-pentylamine bis (uridine 5' -) tetraphosphate. Chemical purity 87.8% (HPLC).
Example 4 high purity P1,P4Preparation of sodium-bis (uridine 5' -) tetraphosphate
The product P obtained in example 31,P450.0g (0.029mol) of a solid of-bis (uridine 5' -) tri-n-pentylamine tetraphosphate salt is dissolved in 150ml of purified water, purified by DEAE Sephadex A-25 column chromatography, gradient-eluted with 0.8mol/L aqueous ammonium bicarbonate solution, the eluate of which the purity is more than 97% is collected, most of the water is removed by reduced evaporation, ion-exchanged by Na + -type 732 cationic resin, and the eluate after the exchange is evaporated to dryness, thereby obtaining 18.0g of a foamy solid. Recrystallization from 70ml of purified water and 140ml of methanol gave 17.1g of a white solid with a chemical purity of 99.8% (HPLC) and a yield of 67.1%.
Example 5P1,P4-di (urine)Preparation of glycoside 5' -) tri-n-pentylamine tetraphosphate
A solution of uridine 5 ' -tri-N-pentylamine triphosphate salt of example 1 in N-methylpyrrolidone (equivalent to 50.0g of uridine 5 ' -triphosphate (0.103mol)) was added with 13.0g N, N ' -diisopropylcarbodiimide (0.103mol), and the mixture was reacted at 20 to 35 ℃ for 2.0 hours to activate the enzyme. Adding the N-methylpyrrolidone solution of uridine 5' -monophosphate tri-N-pentylamine salt obtained in example 2 to the activated reaction solution, adding 10.5g (0.052mol) of magnesium chloride hexahydrate, stirring at 20-35 ℃ for reaction for 8-10 hours, adding 1100ml of ethyl acetate, and performing suction filtration to obtain a white solid P1,P4-tri-n-pentylamine bis (uridine 5' -) tetraphosphate. Chemical purity 86.8% (HPLC).
Example 6 high purity P1,P4Preparation of sodium-bis (uridine 5' -) tetraphosphate
Example 5 gave P1,P4Preparation of starting material (Tri-n-pentylamine-bis (uridine 5' -) tetraphosphate), in accordance with example 4, gave 16.3g of white solid with a chemical purity of 99.7% (HPLC) and a yield of 64.0%.
Example 7P1,P4Preparation of tri-n-pentylamine-bis (uridine 5' -) tetraphosphate salt
A solution of uridine 5 ' -tri-N-pentylamine triphosphate salt of example 1 in N-methylpyrrolidone (equivalent to 50.0g of uridine 5 ' -triphosphate (0.103mol)) was added with 26.0g N, N ' -diisopropylcarbodiimide (0.206mol), and the mixture was reacted at 20 to 35 ℃ for 1.0 hour to activate the uridine. Adding the N-methylpyrrolidone solution of uridine 5' -monophosphate tri-N-pentylamine salt obtained in example 2 into the activated reaction solution, adding 21.0g (0.103mol) of magnesium chloride hexahydrate, stirring at 20-35 ℃ for reaction for 10-12 hours, adding 1650ml of acetonitrile, and performing suction filtration to obtain a white solid P1,P4-tri-n-pentylamine bis (uridine 5' -) tetraphosphate. Chemical purity 89.0% (HPLC).
Example 8 high purity P1,P4Preparation of sodium-bis (uridine 5' -) tetraphosphate
P is obtained as in example 71,P4Preparation of starting material of-di (uridine 5' -) tri-n-pentylamine tetraphosphate, embodimentsReferring to example 4, 17.3g of a white solid was obtained with a chemical purity of 99.6% (HPLC) and a yield of 68.0%.
Example 9P1,P4Preparation of tri-n-pentylamine-bis (uridine 5' -) tetraphosphate salt
A solution of uridine 5 '-tri-N-pentylamine triphosphate (reduced to 50.0g of uridine 5' -triphosphate (0.103mol)) obtained in example 1 in N-methylpyrrolidone was added with 19.5g N, N '-diisopropylcarbodiimide (0.155mol), and the mixture was reacted at 20 to 35 ℃ for 1.5 hours to activate the uridine 5' -triphosphate. Adding the N-methylpyrrolidone solution of uridine 5' -monophosphate tri-N-pentylamine salt obtained in example 2 into the activated reaction solution, adding 31.5g (0.155mol) of magnesium chloride hexahydrate, stirring at 20-35 ℃ for reaction for 8-10 hours, adding 1350ml of butanone, and performing suction filtration to obtain a white solid P1,P4-tri-n-pentylamine bis (uridine 5' -) tetraphosphate. Chemical purity 88.1% (HPLC).
Example 10 high purity P1,P4Preparation of sodium-bis (uridine 5' -) tetraphosphate
P is obtained as in example 91,P4Preparation of starting material, tri-n-pentylamine-bis (uridine 5' -) tetraphosphate, specific embodiment referring to example 4, gave 16.3g of white solid with a chemical purity of 99.7% (HPLC), yield of 64.0%.
EXAMPLE 11 preparation of Tri-n-propylamine uridine 5' -triphosphate salt
Tri-n-propylamine was used in place of tri-n-pentylamine and prepared according to example 1 to give uridine 5' -tri-n-propylamine triphosphate salt.
EXAMPLE 12 preparation of Tri-n-propylamine uridine 5' -monophosphate salt
Preparation of uridine 5 '-monophosphate using tri-n-propylamine instead of tri-n-pentylamine according to example 2 gave a tri-n-propylamine salt of uridine 5' -monophosphate.
Example 13P1,P4Preparation of tri-n-propylamine-bis (uridine 5' -) tetraphosphate salt
The uridine 5 ' -tri-N-propylamine triphosphate obtained in example 11 was dissolved in N, N-dimethylformamide (50.0 g of uridine 5 ' -triphosphate (0.103mol)), and 26.5g N, N ' -dicyclohexylcarbodiimide (0.129mol) was added thereto, and the mixture was reacted at 20 to 35 ℃ for 1.5 hoursThe activation is carried out. Adding the N, N-dimethylformamide solution of uridine 5' -monophosphate tri-N-propylamine salt obtained in example 12 to the activated reaction solution, adding 42.0g (0.206mol) of magnesium chloride hexahydrate, stirring at 20-35 ℃ for reaction for 8-10 hours, adding 550ml of dichloromethane, and performing suction filtration to obtain a white solid P1,P4-tri-n-propylamine bis (uridine 5' -) tetraphosphate. Chemical purity 87.4% (HPLC).
Example 14P1,P4Preparation of tri-n-propylamine-bis (uridine 5' -) tetraphosphate salt
Tri-n-propylamine uridine 5 '-triphosphate salt obtained in example 11 was dissolved in dimethyl sulfoxide (50.0 g of uridine 5' -triphosphate (0.103mol)), 19.7g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.103mol) was added, and the mixture was reacted at 20 to 35 ℃ for 2.0 hours to activate the compound. Adding the dimethyl sulfoxide solution of uridine 5' -monophosphate tri-n-propylamine salt obtained in example 12 into the activated reaction solution, adding 10.5g (0.052mol) of magnesium chloride hexahydrate, stirring and reacting at 20-35 ℃ for 8-10 hours, adding 1100ml of ethyl acetate, and performing suction filtration to obtain a white solid P1,P4-tri-n-pentylamine bis (uridine 5' -) tetraphosphate. Chemical purity 86.5% (HPLC).
Example 15P1,P4Preparation of tri-n-propylamine-bis (uridine 5' -) tetraphosphate salt
The uridine 5 ' -triphosphate tri-N-propylamine salt of example 11 was dissolved in N, N-dimethylacetamide (equivalent to 50.0g of uridine 5 ' -triphosphate (0.103mol)), and 26.0g N, N ' -diisopropylcarbodiimide (0.206mol) was added thereto, followed by reaction at 20 to 35 ℃ for 1.0 hour for activation. Adding the N, N-dimethylacetamide solution of uridine 5' -monophosphate tri-N-propylamine salt obtained in example 2 into the activated reaction solution, adding 21.0g (0.103mol) of magnesium chloride hexahydrate, stirring at 20-35 ℃ for reaction for 10-12 hours, adding 1650ml of acetonitrile, and performing suction filtration to obtain a white solid P1,P4-tri-n-propylamine bis (uridine 5' -) tetraphosphate. Chemical purity 89.0% (HPLC).
Example 16 high purity P1,P4Preparation of sodium-bis (uridine 5' -) tetraphosphate
Example 15 gave P1,P4Preparation of starting material, -di (uridine 5' -) tri-n-propylamine tetraphosphate salt, detailed description referring to example 4, 16.7g of white solid was obtained with a chemical purity of 99.6% (HPLC), yield of 65.7%.

Claims (10)

1. A process for the preparation of sodium P1, P4-bis (uridine 5' -) tetraphosphate comprising the steps of:
(1) reacting a compound shown in the formula I with a carbodiimide condensing agent in a polar aprotic solvent to obtain a compound shown in the formula II, and then reacting with a compound shown in the formula III in the presence of a magnesium salt to obtain a reaction liquid of a compound shown in the formula IV, wherein X is tri-n-pentylamine or tri-n-propylamine;
(2) adding an anti-solvent after the reaction is finished, and crystallizing to obtain a compound shown in the formula IV;
(3) purifying the compound shown in the formula IV by column chromatography, performing ion exchange and recrystallizing to obtain P1, P4-di (uridine 5' -) sodium tetraphosphate;
Figure FDA0002844134120000011
2. the method according to claim 1, wherein the anti-solvent is one or more of benzene, toluene, tetrahydrofuran, acetonitrile, fatty acid esters of C1-C4, alcohols of C1-C4, ethers of C2-C6, ketones of C2-C6, dichloromethane, and chloroform, preferably dichloromethane, ethyl acetate, acetonitrile, butanone; the volume of the antisolvent is 0.5 to 5 times, preferably 1 to 3 times that of the polar aprotic solvent.
3. The method of claim 1, comprising the steps of:
(1) dissolving a compound shown in the formula I in a polar aprotic solvent, adding a carbodiimide condensing agent, and reacting at 0-50 ℃ to obtain a solution of a compound shown in the formula II;
(2) dissolving a compound shown in a formula III in a polar aprotic solvent, adding the dissolved compound into the reaction solution obtained in the step (1), adding a magnesium salt, and reacting at 0-50 ℃ to obtain a reaction solution of a compound shown in a formula IV;
(3) adding an anti-solvent into the reaction liquid of the compound shown in the formula IV, and crystallizing to obtain the compound shown in the formula IV;
(4) purifying the compound shown in the formula IV by anion column chromatography, performing gradient elution by using a saline solution, collecting eluent with the purity of more than 97 percent, and concentrating the eluent to obtain a concentrated solution;
(5) carrying out ion exchange on the concentrated solution obtained in the step (4) through cation resin, and concentrating the eluent;
(6) the residue obtained after the concentration in the step (5) was recrystallized using water and an alcoholic solvent to obtain P1, P4-bis (uridine 5' -) tetrasodium phosphate.
4. The process according to claim 3, wherein the reaction temperature in step (1) or (2) is 0 to 50 ℃, preferably 20 to 35 ℃.
5. The method according to claim 3, wherein the antisolvent in step (3) is one or more of benzene, toluene, tetrahydrofuran, acetonitrile, fatty acid esters of C1-C4, alcohols of C1-C4, ethers of C2-C6, ketones of C2-C6, dichloromethane, and trichloromethane, preferably dichloromethane, ethyl acetate, acetonitrile, butanone; the volume of the antisolvent is 0.5 to 5 times, preferably 1 to 3 times that of the polar aprotic solvent.
6. The method according to claim 1 or 3, wherein the polar aprotic solvent is one or more of N, N-dimethylformamide, N-dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone, or a combination thereof.
7. The method according to claim 1 or 3, wherein the carbodiimide-based condensing agent is one or more of N, N '-Diisopropylcarbodiimide (DIC), N' -Dicyclohexylcarbodiimide (DCC), and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) in a molar ratio of 1.0 to 2.0.
8. The process according to claim 1 or 3, wherein the magnesium salt is magnesium chloride in a molar ratio of 0.5 to 2.0 with respect to the compound of formula I.
9. The method according to claim 3, wherein the alcoholic solvent used for recrystallization is one or more of C1-C4 alcohols; one or a combination of two of methanol and ethanol is preferable.
10. A method according to claim 3, characterized by the steps of:
(1) dissolving uridine 5 '-tri-N-pentylamine triphosphate in N-methylpyrrolidone, adding N, N' -diisopropylcarbodiimide, and reacting at the temperature of 0-50 ℃; adding an N-methylpyrrolidone solution of uridine 5 '-monophosphate tri-N-pentylamine salt into a reaction solution of uridine 5' -triphosphate tri-N-pentylamine salt, adding magnesium chloride hexahydrate, stirring and reacting at 0-50 ℃, adding dichloromethane, and performing suction filtration to obtain P1,P4-tri-n-pentylamine bis (uridine 5' -) tetraphosphate;
(2) p obtained in the step (1)1,P4Dissolving the-di (uridine 5' -) tripentylamine tetraphosphate in purified water, purifying by DEAE Sephadex A-25 column chromatography, performing gradient elution by using 0.1-0.8 mol/L ammonium bicarbonate water solution, collecting eluent with the purity of more than 97%, removing 1/2-2/3 water by reduced evaporation, performing ion exchange by using Na < + > type 732 cationic resin, concentrating the eluent after the exchange to obtain residue, and performing recrystallization by using purified water and methanol to obtain P1,P4Sodium di (uridine 5' -) tetraphosphate.
CN202011503324.9A 2020-12-18 2020-12-18 Method for preparing symmetric uridine diphosphate Pending CN114644664A (en)

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