Preparation method of trisodium uridine triphosphate and product thereof

Abstract

The present application relates to a kind of preparation method of triphosphoryl uridine trisodium and its preparation.The present application uses ionic liquid as reaction solvent, 2-chloro-4H-1,3,2-benzene dioxaphosphorin-4-ketone (compound 1) is reacted with tributylammonium pyrophosphate to generate cyclic intermediate 2, then uridine is reacted, and then it is obtained by oxidation, basic hydrolysis ring-opening step triphosphoryl uridine trisodium.The present application uses ionic liquid as solvent to promote the site selectivity of the hydroxyl group in the ribose structure of uridine in the reaction, which can make the reaction intermediate 2 react with the hydroxyl group of the 5' position of uridine with high selectivity, so as to obtain UTP with high yield, and the present application also prepares UTP into the form of trisodium salt to facilitate purification and preservation.The present application solves the problems of long steps and low yield in the prior art when using chemical synthesis method to synthesize UTP.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis, specifically relating to a method for preparing trisodium uridine triphosphate and its product. Background Technology

[0002] Urate triphosphate, also known as uridine-5'-triphosphate (UTP), is a uracil nucleotide composed of one uracil, one ribose, and three phosphates linked to the 5'-OH carbon of the ribose. UTP is usually stored as a trisodium salt, i.e., trisodium uridine triphosphate. UTP is closely related to carbohydrate metabolism. UTP reacts with glucose-1-phosphate through enzymatic catalysis to produce UDP-glucose and pyrophosphate; in addition, UDP-galactose, UDP-galactosamine, and UDP-glucuronic acid are also produced.

[0003] UTP participates in the synthesis of pyrimidine ribonucleotides and is a raw material for RNA synthesis (transcription). Additionally, UTP can also be used as...

[0004] UTPs are an energy source, functioning similarly to ATP but less abundant. They are involved in many energy metabolism pathways in organisms. UTPs also participate in the activation of certain G protein-coupled receptors (e.g., P2Y, P2Y2, P2Y11), thereby activating chloride channels in epithelial cells, increasing ciliary beating frequency, inducing goblet cell degranulation in respiratory epithelial cells, and influencing inflammatory cell activity and vascular reactivity. Furthermore, UTPs are commonly used in the diagnosis and treatment of certain diseases (e.g., INS316, lung cancer). They also play important roles in sinusitis, chronic bronchitis, otitis media, dry eye syndrome, and gastrointestinal diseases. As a major substrate for RNA synthesis, UTPs are indispensable in the production and development of mRNA vaccines.

[0005] Many methods for preparing UTP have been reported in the prior art, among which enzymatic and fermentation methods are the most commonly used. There are also reports of pure chemical synthesis methods for preparing UTP or its analogues. However, chemical synthesis methods usually require protection of certain reaction sites of the raw materials and intermediates to avoid the formation of byproducts, and finally, the removal of protecting groups. The synthetic steps are usually very lengthy and inefficient. For example, Zhen Huang's research group reported a one-pot synthesis of 2'-deoxynucleoside 5'-triphosphates in their article "Protection-Free One-Pot Synthesis of 2'-Deoxynucleoside 5'-Triphosphates and DNA Polymerization," and the reaction steps, products, and yields are shown below.

[0006]

[0007] However, the one-pot preparation of 2'-deoxynucleoside-5'-triphosphate in the article by Zhen Huang's research group mentioned above is obviously limited by the reaction substrate. They did not attempt to prepare UTP using non-deoxynucleosides such as uridine as reaction substrates, but only obtained some 2'-deoxynucleoside-5'-triphosphate, and the yield of the prepared 2'-deoxynucleoside-5'-triphosphate was also relatively low.

[0008] Chinese patent CN103314001B also reports a similar method for preparing 2'-deoxynucleoside-5'-triphosphate and nucleoside-5'-triphosphate, claiming that the separation yield was greatly improved through optimization of conditions. However, the reaction conditions for preparing nucleoside triphosphate in this patent are basically the same as those in the aforementioned article, and it also does not clearly describe the reaction yield for preparing UTP.

[0009] In fact, Zhen Huang's research group already mentioned in their article that during the reaction of intermediate 2 with 2'-deoxynucleosides, the reaction of the hydroxyl group at the 3' carbon position on the ribose with intermediate 2 is the main side reaction. That is, when intermediate 2 reacts with 2'-deoxynucleosides, it lacks specific selectivity for the hydroxyl group at the 5' carbon position, leading to an increase in byproducts. When non-deoxynucleosides, such as uridine, react with intermediate 2, there are three hydroxyl reaction sites on its ribose. Due to the poor selectivity of these reaction sites, the reaction yield for preparing UTP using this method is very low.

[0010] There have been many reports on the application of ionic liquids as solvents or catalysts in organic chemical reactions. Ionic liquids can also promote the site selectivity of organic reactions. Some reports show that using ionic liquids as solvents can improve the reaction selectivity of hydroxyl groups on ribose. However, there are no reports on the application of ionic liquids in the synthesis of UTP. Summary of the Invention

[0011] This invention solves the problems of lengthy steps and low yield in the synthesis of UTP using existing chemical synthesis methods. This invention relates to a method for preparing trisodium uridine triphosphate and the product thereof. This invention uses an ionic liquid as the reaction solvent, reacting 2-chloro-4H-1,3,2-benzodioxophosphoro-4-one (compound 1) with tributylammonium pyrophosphate to generate a cyclic intermediate 2, followed by a uridine reaction, and then oxidation and alkaline hydrolysis ring-opening steps to obtain trisodium uridine triphosphate.

[0012] Specifically, this invention provides a method for preparing uridine triphosphate trisodium, the reaction equation of which is as follows:

[0013]

[0014] In steps 1) and 2), ionic liquids are used as reaction solvents.

[0015] Furthermore, the ionic liquid in steps 1) and 2) is selected from any one of [Bmim]PF6, [Bmim]BF4, and [Bmim]TfO, with [Bmim]PF6 being preferred.

[0016] Furthermore, all reaction steps 1), 2), 3), and 4) are carried out at 25-35°C.

[0017] Furthermore, all reaction steps 1), 2), 3), and 4) are carried out under inert gas protection conditions.

[0018] Furthermore, the preparation method is a one-pot reaction, and no post-treatment of the reaction solution or separation of reaction intermediates is required after reaction steps 1), 2), and 3).

[0019] Furthermore, the reaction time of step 1) is 0.5-1.5h, the reaction time of step 2) is 1-2h, the reaction time of step 3) is 15-30min, and the reaction time of step 4) is 1-3h.

[0020] Further, in step 1), the reagent that reacts with compound 1 is tributylammonium pyrophosphate, and the molar ratio of compound 1 to tributylammonium pyrophosphate is 1:1.5-2.5, preferably 1:2.

[0021] Furthermore, in reaction step 2), the molar amount of uridine is the same as the molar amount of compound 1.

[0022] Furthermore, in step 3), the elemental iodine is dissolved in a mixed solution of pyridine and water, the mass concentration of the iodine solution is 3-8%, preferably 5%; the volume ratio of pyridine to water is 12:1-6:1, preferably 9:1.

[0023] Furthermore, the NaOH in step 4) is in the form of an aqueous solution.

[0024] Furthermore, step 4) after the reaction also includes the steps of refrigeration, filtration, and recrystallization.

[0025] Furthermore, the recrystallization is performed using an ethanol solution as the recrystallization solvent.

[0026] Further, the recrystallization operation is as follows: dissolve the crude uridine triphosphate trisodium obtained by filtration in an appropriate amount of water, adjust the pH to 3-4 with dilute hydrochloric acid, add ethanol to the mass fraction of ethanol to 70-85%, stir for 15-30 minutes, place in a refrigerator at 4-8°C for 8-12 hours, filter, and dry under low temperature vacuum to obtain pure uridine triphosphate trisodium.

[0027] Furthermore, to further improve the purity of uridine triphosphate trisodium, the recrystallization step can be repeated 2-3 times.

[0028] In addition, the present invention also provides a product of uridine triphosphate trisodium obtained by the above preparation method, which is used in the preparation of scientific research reagents and drugs.

[0029] The beneficial effects of this invention are as follows: This invention innovatively introduces ionic liquids as solvents into the chemical synthesis of UTP to promote site selectivity of the reaction for the hydroxyl groups in the ribonucleotide structure of uridine. This allows reaction intermediate 2 to react with the hydroxyl group at the 5' position of uridine with high selectivity, thereby obtaining UTP in high yield. This invention also prepares UTP in the form of trisodium salt for convenient purification and storage. In existing chemical synthesis methods for UTP preparation, due to low reaction yields, it is difficult to directly purify UTP or its trisodium salt by recrystallization. Purification is usually performed using a separation column. However, due to the high reaction yield and high concentration of the target product in the reaction solution, this invention eliminates the need for complex purification methods and allows for the acquisition of UTP trisodium salt simply by recrystallization. This purification process is simpler, more convenient, and cost-effective. Detailed Implementation

[0030] The present invention will be described in more detail below with reference to specific embodiments.

[0031] Example 1

[0032] Weigh 40 mmol (2 eq) of tributylammonium pyrophosphate and dissolve it in an appropriate amount of ionic liquid [Bmim]PF6, then add 20 mL of anhydrous tributylamine and stir at 25 °C. Weigh 20 mmol (1 eq) of 2-chloro-4H-1,3,2-benzodioxophosphoro-4-one (i.e., compound 1) into a reaction flask and dissolve it in an appropriate amount of ionic liquid [Bmim]PF6. Slowly add the solution of compound 1 dropwise to the solution of tributylammonium pyrophosphate. After the addition is complete, stir at 25 °C for 1 h to generate cyclic compound 2. Then weigh 20 mmol (1 eq) of uridine, dissolve it in an appropriate amount of ionic liquid, and add it to the aforementioned reaction solution containing cyclic compound 2. Stir at 25 °C for 1.5 h to generate compound 3. Then slowly add 5% iodine solution (iodine dissolved in 9:1 Py:H2O) until the reaction solution turns a deep red color that does not fade, indicating that the oxidation reaction is complete. Then, an aqueous solution of NaOH (containing 80 mmol of NaOH) was added, and the mixture was stirred at 25°C for 2 hours. A large amount of solid precipitated from the reaction liquid. All the above reactions were carried out under the protection of an inert gas.

[0033] The reaction system was placed at 8°C for 2 hours, filtered, and the solid was washed with cold ethanol to obtain crude uridine triphosphate trisodium. The crude uridine triphosphate trisodium obtained by filtration was dissolved in an appropriate amount of water, the pH was adjusted to 3-4 with dilute hydrochloric acid, ethanol was added until the ethanol mass fraction was 75%, stirred for 30 minutes, placed in a refrigerator at 8°C for 10 hours, filtered, and dried under low temperature vacuum to obtain 15.3 mmol of pure uridine triphosphate trisodium, with a yield of 76.5% and a purity of 97%.

[0034] Example 2

[0035] Weigh 40 mmol (2 eq) of tributylammonium pyrophosphate and dissolve it in an appropriate amount of ionic liquid [Bmim]BF4, then add 20 mL of anhydrous tributylamine and stir at 35 °C. Weigh 20 mmol (1 eq) of 2-chloro-4H-1,3,2-benzodioxophosphoro-4-one (i.e., compound 1) into a reaction flask and dissolve it in an appropriate amount of ionic liquid [Bmim]BF4. Slowly add the solution of compound 1 dropwise to the solution of tributylammonium pyrophosphate. After the addition is complete, stir at 35 °C for 1 h to generate cyclic compound 2. Then weigh 20 mmol (1 eq) of uridine, dissolve it in an appropriate amount of ionic liquid, and add it to the aforementioned reaction solution containing cyclic compound 2. Stir at 35 °C for 1 h to generate compound 3. Then slowly add 3% iodine solution (iodine dissolved in 6:1 Py:H2O) until the reaction solution turns a deep red color that does not fade, indicating that the oxidation reaction is complete. Then, an aqueous solution of NaOH (containing 80 mmol of NaOH) was added, and the mixture was stirred at 35°C for 1 hour, during which a large amount of solid precipitated from the reaction liquid. All the above reactions were carried out under the protection of an inert gas.

[0036] The reaction system was placed at 8°C for 2 hours, filtered, and the solid was washed with cold ethanol to obtain crude uridine triphosphate trisodium. The crude uridine triphosphate trisodium obtained by filtration was dissolved in an appropriate amount of water, the pH was adjusted to 3-4 with dilute hydrochloric acid, ethanol was added until the ethanol mass fraction was 75%, stirred for 30 minutes, placed in a refrigerator at 8°C for 10 hours, filtered, and dried under low temperature vacuum to obtain 13.6 mmol of pure uridine triphosphate trisodium, with a yield of 68% and a purity of 97%.

[0037] Example 3

[0038] The difference between this embodiment and Example 1 is that, following existing techniques, the ionic liquid solution was replaced with DMF. The final product obtained from this reaction system is more complex, with more byproducts, making purification by recrystallization difficult. A crude product sample was taken and quantified by high-performance liquid chromatography (HPLC), revealing that the UTP content in the product was <30%.

[0039] While some specific forms of the present invention have been described above, various obvious modifications and combinations made to the present invention without departing from its principles should also fall within the scope of the present invention.

Claims

1. A method for preparing trisodium uridine triphosphate, characterized in that, The reaction equations and reaction steps 1), 2), 3), and 4) are shown below: ； In steps 1) and 2), ionic liquids are used as reaction solvents, and the ionic liquids are selected from any one of [Bmim]PF6, [Bmim]BF4, and [Bmim]TfO. The reaction steps 1), 2), 3), and 4) are all carried out at 25-35℃ and under inert gas protection.

2. The method for preparing uridine triphosphate trisodium as described in claim 1, characterized in that, The ionic liquid is selected from [Bmim]PF6.

3. The method for preparing uridine triphosphate trisodium as described in claim 2, characterized in that, The preparation method is a one-pot reaction, and no post-treatment of the reaction solution or separation of reaction intermediates is required after reaction steps 1), 2), and 3).

4. The method for preparing uridine triphosphate trisodium as described in claim 3, characterized in that, The reaction time for step 1) is 0.5-1.5 h, the reaction time for step 2) is 1-2 h, the reaction time for step 3) is 15-30 min, and the reaction time for step 4) is 1-3 h.

5. The method for preparing uridine triphosphate trisodium as described in claim 3, characterized in that, In step 1), the reagent reacting with compound 1 is tributylammonium pyrophosphate, and the molar ratio of compound 1 to tributylammonium pyrophosphate is 1:1.5-2.5; and / or in step 2), the molar amount of uridine is the same as the molar amount of compound 1; and / or in step 3), the elemental iodine is dissolved in a mixed solution of pyridine and water, the mass concentration of the iodine solution is 3-8%, and the volume ratio of pyridine to water is 12:1-6:1; and / or in step 4), the NaOH is in the form of an aqueous solution.

6. The method for preparing uridine triphosphate trisodium as described in claim 5, characterized in that, In step 1), the molar ratio of compound 1 to tributylammonium pyrophosphate is 1:2; and / or in step 2), the molar amount of uridine is the same as the molar amount of compound 1; and / or in step 3), elemental iodine is dissolved in a mixed solution of pyridine and water, the mass concentration of the iodine solution is 5%, and the volume ratio of pyridine to water is 9:1; and / or in step 4), NaOH is in the form of an aqueous solution.

7. The method for preparing uridine triphosphate trisodium as described in claim 3, characterized in that, Step 4) after the reaction also includes refrigeration, filtration, and recrystallization.

8. The method for preparing uridine triphosphate trisodium as described in claim 7, characterized in that, The recrystallization is performed using an ethanol solution as the recrystallization solvent.

9. The method for preparing uridine triphosphate trisodium as described in claim 8, characterized in that, The recrystallization process is as follows: dissolve the crude uridine triphosphate trisodium obtained by filtration in an appropriate amount of water, adjust the pH to 3-4 with dilute hydrochloric acid, add ethanol to a mass fraction of 70-85%, stir for 15-30 minutes, place in a refrigerator at 4-8°C for 8-12 hours, filter, and dry under low temperature vacuum to obtain pure uridine triphosphate trisodium.

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