Method for preparing uridine 5'-diphosphate (UDP), its salt or hydrate thereof

Abstract

A method for preparing uridine 5'-diphosphate (UDP), its salt or its hydrate of the present invention, comprising the following steps: (step 1) deionizing the salt of uridine 5'-monophosphate (UMP) represented by chemical formula 2a with an acid, thereby preparing the UMP represented by chemical formula 2a; (step 2) reacting the UMP represented by chemical formula 2a with a tertiary amine, a carbodiimide condensing agent and imidazole, thereby preparing the uridine-5'-phosphoryl imidazole compound represented by chemical formula 2b; and (step 3) reacting the uridine-5'-phosphoryl imidazole compound represented by chemical formula 2b with a monophosphorylating agent and a tertiary amine, thereby preparing the UDP represented by chemical formula 3a, its salt or its hydrate. Therefore, the present invention can simplify the process by not using an ion exchange resin for deionization, and can be effectively used commercially by mass production with a relatively simple process without separate production equipment.

Description

Technical Field

[0001] The present invention relates to a method for preparing uridine 5'-diphosphate (hereinafter referred to as UDP), which is an intermediate compound used in preparing polyphosphate dinucleoside compounds.

[0002] background

[0003] Generally, the compound P represented by the following formula 1 1 ,P 4 -Bis(uridine 5'-)tetraphosphate (hereinafter referred to as "UP4U") or a salt thereof is useful as a therapeutic agent for corneal and conjunctival epithelial diseases associated with dry eye or dry tear syndrome, and is a compound expected to be developed as an expectorant or a therapeutic agent for pneumonia due to its expectorant-inducing effect.

[0004] [Formula 1]

[0005]

[0006] International Unexamined Patent Publication No. 1999 / 05155 reports a method for preparing UP4U, which comprises dissolving uridine, uridine 5'-monophosphate (hereinafter referred to as UMP), UDP or uridine 5'-triphosphate (hereinafter referred to as UTP) and their respective salts and uridine nucleotide compounds in a polar aprotic organic solvent and a hydrophobic amine, phosphorylating the resulting solution using a monophosphorylating agent or a diphosphorylating agent as a phosphorylating agent, and using carbodiimide, an active carbonyl group or an active phosphorus as an activator.

[0007] In addition, Korean Unexamined Patent Application No. 10-2018-0091672 (published on August 16, 2018) reports a method for preparing high-purity UP4U with high yield, which is prepared by reacting a carbodiimide condensation agent and metal ions with nucleoside phosphate compounds represented by the following formulas 2a, 3a and 4a as starting materials in the presence of water (an environmentally friendly solvent).

[0008] [Formula 2a]

[0009]

[0010] [Formula 3a]

[0011]

[0012] [Formula 4a]

[0013]

[0014] J. Org. Chem. 1987, 52, 1794-1801 reported a method for synthesizing UDP by reacting uridine with a diphosphorylating agent. This method has a low yield and requires separate purification using an ion exchange resin, making it difficult to commercialize.

[0015] In addition, Synthetic Communications 36: 3393-3399, 2006 reported a method for synthesizing UDP by converting UMP into uridine-5'-phosphoroimidazolide and reacting it with phosphoric acid and trialkylamine salt. However, this preparation method has a low yield and produces a large amount of by-products, so it needs to be additionally purified by anion exchange resin and fast protein liquid chromatography (FPLC), which makes it difficult to commercialize.

[0016] Therefore, for the above reasons, a preparation method capable of commercially synthesizing UDP by effective purification is required to economically synthesize UDP.

[0017] [Related technical references]

[0018] (Patent Document 1) International Unexamined Patent Publication No. 1999 / 05155

[0019] (Patent Document 2) Korean Unexamined Patent Application No. 10-2018-0091672

[0020] (Non-patent document 1) Synthesis Communications 36, 3393-3399, 2006 (published on November 23, 2006)

[0021] (Non-patent document 2) J. Org. Chem. 1987, 52, 1794-1801 DETAILED DESCRIPTION OF THE INVENTION

[0023] Technical issues

[0024] The object of the present invention is to provide a method for preparing uridine 5'-diphosphate (UDP), its salt or its hydrate, which is economical and suitable for large-scale production while improving purity and yield.

[0025] Technical Solution

[0026] In order to economically synthesize expensive UDP, the present invention can provide a preparation method capable of commercially synthesizing UDP, wherein the method uses UMP as a starting material to synthesize uridine-5'-phosphoimidazole, reacts with a monophosphorylating agent and a tertiary amine to synthesize crude UDP, and then efficiently purifies it under conditions without purification by ion exchange resin.

[0027] The method for preparing uridine 5'-diphosphate (UDP), a salt thereof or a hydrate thereof represented by formula 3a according to the present invention may include:

[0028] (Step 1) deionizing a salt of uridine 5'-monophosphate (UMP) represented by Formula 2a with an acid to prepare UMP represented by Formula 2a;

[0029] (Step 2) reacting the UMP represented by Formula 2a with a tertiary amine, a carbodiimide condensing agent, and imidazole to prepare a uridine-5'-phosphoimidazole represented by Formula 2b; and

[0030] (Step 3) The uridine-5′-phosphoimidazolide represented by Formula 2b is reacted with a monophosphorylating agent and a tertiary amine to prepare UDP represented by Formula 3a, a salt thereof or a hydrate thereof.

[0031] [Formula 2a]

[0032]

[0033] [Formula 2b]

[0034]

[0035] [Formula 3a]

[0036]

[0037] Step 1

[0038] The salt of uridine 5'-monophosphate (UMP) represented by formula 2a in step 1 may be a UMP metal salt. The UMP metal salt may be selected from the group consisting of lithium, sodium, potassium, magnesium, calcium, barium, cerium, iron, nickel, copper, zinc and boron, and may preferably be an alkali metal salt of lithium, sodium or potassium and an alkaline earth metal salt of calcium or magnesium.

[0039] In one embodiment, the acid used in the deionization process of step 1 above may be nitric acid, sulfuric acid or hydrochloric acid. In this case, the amount of the acid may be 0.8 to 1.2 molar equivalents, preferably 0.9 to 1.1 molar equivalents, based on 1 molar equivalent of UMP.

[0040] In one embodiment, the reaction solvent in step 1 above may be a mixed solvent of alcohol and water, and the mixing ratio of alcohol and water may be in the range of 4:1 to 10:1, preferably in the range of 6:1 to 8:1. The type of alcohol used in the reaction may be methanol, ethanol, isopropanol, n-butanol or isobutanol, which may be used alone or in combination of two or more thereof. The alcohol used as the reaction solvent in step 1 above is preferably methanol or ethanol.

[0041] The reaction temperature of step 1 may be in the range of 10 to 40° C., preferably in the range of 20 to 30° C. In addition, the reaction time of step 1 may be in the range of 10 to 60 minutes, preferably in the range of 20 to 40 minutes, but is not limited thereto.

[0042] In one embodiment, when the UMP metal salt is UMP sodium salt, the reaction in step 1 above may be as shown in the following reaction formula 1.

[0043] [Reaction 1]

[0044]

[0045] According to the above step 1, the UMP metal salt can be deionized with an acid in a mixed solvent of alcohol and water without using an ion exchange resin, and thus can be easily converted into UMP free acid.

[0046] In the above step 1, uridine 5'-monophosphate (UMP) represented by formula 2a may be prepared without using an ion exchange resin, thereby reducing the reaction steps and reaction time, thereby minimizing the generation of impurities.

[0047] Step 2

[0048] The above step 2 may be a step of preparing uridine-5'-phosphoryl imidazolide by using the UMP free acid obtained according to the above step 1.

[0049] The carbodiimide condensing agent in the above step 2 can be a compound represented by R1-N=N-R2 (wherein R1 and R2 are the same or different, and each independently is a straight chain, branched or cyclic alkyl group having 1 to 6 carbon atoms, wherein the alkyl group can be optionally substituted by an alkylamine group), and can be added as a reaction aid in the condensation reaction.

[0050] The carbodiimide condensing agent in the above step 2 can be 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) or its salt, N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), etc., which can be used alone or in combination. In this case, the amount of the carbodiimide condensing agent can be 1 to 3 molar equivalents based on 1 molar equivalent of UMP, preferably 1.5 to 2.5 molar equivalents.

[0051] The tertiary amine in step 2 can be trimethylamine, triethylamine or tributylamine, which can be used alone or in combination of two or more thereof. Preferably, the tertiary amine in step 2 can be tributylamine. In this case, the amount of the tertiary amine can be 1 to 2 molar equivalents, preferably 1.4 to 1.6 molar equivalents, based on 1 molar equivalent of UMP.

[0052] The amount of imidazole used in the above step 2 can be 1 to 3 molar equivalents, preferably 1.5 to 2.5 molar equivalents, based on 1 molar equivalent of UMP.

[0053] The reaction solvent in step 2 above may be a mixed solvent of alcohol and water. In this case, the mixing ratio of alcohol and water may be in the range of 4:1 to 10:1, preferably 6:1 to 8:1. The type of alcohol used in the reaction may be methanol, ethanol, isopropanol, n-butanol or isobutanol, which may be used alone or in combination of two or more thereof. The alcohol used as the reaction solvent in step 2 above is preferably methanol or ethanol.

[0054] The reaction temperature of step 2 may be in the range of 60 to 80° C., preferably in the range of 65 to 75° C. In addition, the reaction time of step 2 may be in the range of 30 to 120 minutes, preferably in the range of 60 to 90 minutes, but is not limited thereto.

[0055] In one embodiment, step 2 is a step for preparing uridine-5'-phosphoryl imidazolide represented by the above formula 2b, which can be represented by the following reaction formula 2.

[0056] [Reaction 2]

[0057]

[0058] Step 3

[0059] The monophosphorylation reagent used in the above step 3 can be a tertiary amine salt of phosphoric acid (such as phosphoric acid (H3PO4)) Bu3NH·H2PO4, etc. The amount of the monophosphorylation reagent used in the above step 3 can be 1 to 10 molar equivalents, preferably 2 to 4 molar equivalents, based on 1 molar equivalent of uridine 5'-phosphoryl imidazolide.

[0060] The description of the tertiary amine in the above step 3 may be substantially the same as that in the above step 2, and thus will not be repeated. The type of the tertiary amine used in the above step 3 may be the same as or different from that used in the above step 2. In this case, the amount of the tertiary amine used in the above step 3 may be 1 to 10 molar equivalents, preferably 2 to 4 molar equivalents, based on 1 molar equivalent of uridine 5'-phosphoimidazole.

[0061] In one embodiment, the molar equivalent ratio of the monophosphorylation reagent to the tertiary amine in step 3 above may be 1:1.

[0062] The reaction solvent in step 3 can be alcohols such as methanol, ethanol, isopropanol, acetone, acetonitrile, N,N'-dimethylformamide, or the like. They can be used alone or in combination of two or more thereof. In one embodiment, the reaction solvent in step 3 can be methanol or acetone.

[0063] The reaction temperature of the above step 3 may be in the range of 20° C. to 50° C., preferably in the range of 30° C. to 40° C. In addition, the reaction time of the above step 3 may be in the range of 30 to 180 minutes, preferably in the range of 60 to 120 minutes.

[0064] In one embodiment, as shown in the following reaction formula 3, the above step 3 can be performed by reacting uridine-5'-phosphoimidazole with a monophosphorylating agent and a tertiary amine. Through the above step 3, a crude UDP of UDP represented by the above formula 3a can be obtained.

[0065] [Reaction 3]

[0066]

[0067] Purification process: Steps 4-1 to 4-3

[0068] In one embodiment, the method for preparing uridine 5'-diphosphate (UDP), a salt thereof or a hydrate thereof represented by formula 3a according to the present invention may further include, after performing the above step 3, purifying the crude UDP obtained in the above step 3 to obtain the UDP represented by formula 3a.

[0069] Specifically, the method for preparing UDP, its salt or its hydrate represented by formula 3a of the present invention may further include, after performing the above step 3:

[0070] (Step 4-1) preparing UDP represented by Formula 3a by adsorption purification and elution of UDP represented by Formula 3a;

[0071] (Step 4-2) converting the UDP represented by Formula 3a into its metal salt and solidifying it; and

[0072] (Step 4-3) A metal salt of UDP is converted into UDP represented by Formula 3a, a salt thereof, or a hydrate thereof.

[0073] Step 4-1

[0074] In the above step 4-1, uridine 5'-diphosphate (UDP) represented by formula 3a can be prepared from the crude UDP obtained in the above step 3. In this case, the UDP represented by formula 3a can be prepared by absorption purification and elution. When synthesizing crude UDP in the above step 3, an excess of tertiary amine and phosphorylation reagent may be used to complete the reaction, and many phosphate byproducts that cannot be detected by ultraviolet light may remain in the process, so they can be removed in the above step 4-1.

[0075] In one embodiment, an adsorbent may be used in the adsorption purification and elution in step 4-1, and the adsorbent may be activated carbon.

[0076] In one embodiment, the eluent used for elution can be selected from the group consisting of dilute hydrochloric acid, sodium chloride aqueous solution, ammonium carbonate aqueous solution, dilute ammonia water and sodium hydroxide aqueous solution, preferably dilute ammonia water. The concentration of the eluent can be in the range of 0.5 to 2.5 mol / L, preferably 1.0 to 2.0 mol / L.

[0077] In one embodiment, the above step 4-1 can be performed by dissolving crude UDP in water, adsorbing it onto activated carbon, removing impurities by filtering, and eluting the adsorbed activated carbon. For example, after the crude UDP is dissolved in water and a small amount of dilute hydrochloric acid is added until it is completely dissolved at pH 2 to 3, the crude UDP can be adsorbed on activated carbon. At this time, the volume (mL) of water to be diluted can be 40 to 60 times, preferably 45 to 55 times, of the weight (g) of UDP. At this time, the amount (weight) of activated carbon used can be 3 to 7 times, preferably 4 to 6 times, of the weight of UDP.

[0078] Step 4-2

[0079] The metal salt of UDP in the above step 4-2 (hereinafter referred to as UDP metal salt) may be a divalent cation metal salt.

[0080] In one embodiment, the UDP metal salt may be a UDP calcium salt represented by the following formula 3b.

[0081] [Formula 3b]

[0082]

[0083] In one embodiment, the above step 4-2 can be performed by adding a calcifying agent to the UDP represented by formula 3a eluted in the above step 4-1 to convert it into a UDP calcium salt represented by formula 3b, and then crystallizing it to obtain a solid.

[0084] In one embodiment, the reaction in which the UDP metal salt in step 4-2 is a UDP calcium salt can be represented by the following reaction formula 4.

[0085] [Reaction 4]

[0086]

[0087] In one embodiment, when a calcifying agent is used in step 4-2, the calcifying agent can be calcium chloride, calcium acetate, calcium carbonate, calcium sulfate or calcium phosphate, preferably calcium chloride or calcium carbonate. In this case, the amount of the calcifying agent can be 2 to 6 molar equivalents, preferably 3 to 5 molar equivalents, based on 1 molar equivalent of UDP.

[0088] The crystallization of step 4-2 can be carried out using an alcohol solvent, and examples of alcohol solvents can include methanol, ethanol, isopropanol, etc., preferably methanol. During the crystallization process, the amount (volume) of methanol can be 0.2 to 0.4 times, preferably 0.25 to 0.35 times, of the volume of the UDP eluent.

[0089] Step 4-3

[0090] In the above step 4-3, the solid phase UDP metal salt obtained by the above step 4-2 can be converted to prepare UDP represented by formula 3a.

[0091] In one embodiment, the above step 4-3 may include the step of converting the solidified UDP metal salt obtained in the above step 4-2 into the UDP represented by formula 3a by deionization. At this time, the above step 4-3 may also include the step of adding sodium hydroxide to the UDP represented by formula 3a obtained by deionization to obtain the sodium salt of UDP represented by formula 3a.

[0092] Specifically, the above step 4-3 can be performed by converting the solid phase UDP calcium salt represented by formula 3b obtained in the above step 4-2 into UDP free acid with an acid, and then adding sodium hydroxide to convert it into UDP sodium salt again. The above step 4-3 can be represented by the following reaction formula 5.

[0093] [Reaction 5]

[0094]

[0095] The acid used in the deionization of step 4-3 can be a strong acid such as sulfuric acid, hydrochloric acid, nitric acid and phosphoric acid, preferably sulfuric acid or hydrochloric acid. In this case, the amount of the acid can be 0.8 to 1.2 molar equivalents, preferably 0.9 to 1.1 molar equivalents, based on 1 molar equivalent of UDP calcium salt.

[0096] The reaction temperature of the above step 4-3 may be in the range of 10° C. to 40° C., preferably in the range of 20° C. to 30° C. In addition, the reaction time of the above step 4-3 may be in the range of 30 to 90 minutes, preferably in the range of 50 to 70 minutes.

[0097] In the above step 4-3, sodium hydroxide may be used to convert into UDP sodium salt, wherein the reaction may be carried out in the range of pH 4 to pH 5, preferably pH 4.4 to pH 4.6.

[0098] The reaction of obtaining the sodium salt in step 4-3 above can use a crystallization solvent, wherein the volume (mL) of methanol used as the crystallization solvent can be 5-13 times, preferably 8-10 times, the weight (g) of the UDP calcium salt.

[0099] Beneficial effects

[0100] According to the present invention, UDP as a precursor of diquafosol can be produced in large quantities by simplified procedures, thereby can be used commercially. In the present invention, crude UDP can be purified by using activated carbon instead of ion exchange resin, and the process can be simplified by not using ion exchange resin to carry out deionization process. In addition, high-purity UDP can be obtained in high yield by being converted into a sparingly soluble UDP metal salt and solidifying the metal salt after purification, so UDP can be mass-produced for commercial use by relatively simple processes without separate production equipment.

[0101] BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 The NMR analysis of the uridine-5'-phosphoimidazole synthesized in step 2 of Example 1 ( 1 HNMR) result diagram.

[0103] Figure 2 The UDP sodium salt synthesized in step 4-3 of Example 2 1 H NMR results.

[0104] Figure 3 This is a diagram showing the results of liquid chromatography (HPLC) analysis of uridine-5'-phosphoryl imidazolide synthesized in step 2 of Example 1.

[0105] Figure 4 This is a graph showing the HPLC results of the crude UDP synthesized in step 3 of Example 1.

[0106] Figure 5 This is a graph showing the HPLC results of UDP obtained in step 4-1 of Example 2.

[0107] Figure 6 This is a graph showing the HPLC results of the UDP calcium salt obtained in step 4-2 of Example 2.

[0108] Figure 7 This is a graph showing the HPLC results of the UDP sodium salt obtained in step 4-3 of Example 2.

[0109] Modes for Carrying Out the Invention

[0110] In order to better understand the present invention, the present invention will be described in detail by preferred embodiments. However, the following embodiments are provided only for the purpose of illustrating the present invention, and therefore the present invention is not limited thereto.

[0111] In addition, unless otherwise specified, the reagents and solvents mentioned below were purchased from Sigma-Aldrich and TCI. 1 H NMR data and HLPC data were measured using Agilent Technologies 1200 series.

[0112] The HPLC conditions used in the present invention are as follows, and the purity is measured after the reaction or in the reaction mixture.

[0113] Detector: UV absorption spectrophotometer (wavelength: 260nm)

[0114] Chromatographic column: YMC-Pack Pro C18 (4.6mm X 250mm, 5μm)

[0115] Mobile phase: Dissolve 40g potassium dihydrogen phosphate in 1L water

[0116] Flow rate: 0.800 to 1.200 mL / min

[0117] [Table 1]

[0118] time Flow rate 0.00 0.800 10.00 0.800 10.50 1.200 30.00 1.200

[0119] Sample: 10 mg / mobile phase 10 mL

[0120] Injection volume: 10 μL

[0121] Example 1: Synthesis of crude uridine 5'-diphosphate (UDP)

[0122] (Step 1) Synthesis of uridine 5'-monophosphate (UMP)

[0123]

[0124] 20 g (54.3 mmol) of UMP sodium salt was added to a 250 mL three-necked round-bottom flask, and 80 mL of ethanol (EtOH), 10 mL of purified water and 2.9 mL (54.3 mmol) of sulfuric acid were added. After stirring at room temperature for 30 minutes, the mixture was filtered and washed with a mixture of 5 mL of ethanol and 0.63 mL of purified water. The filtrate was used in the next step of the reaction.

[0125] (Step 2) Synthesis of Uridine-5'-Phosphoryl Imidazolide

[0126]

[0127] 19.4 mL (81.5 mmol) of tributylamine, 16.8 mL (108.6 mmol) of N,N'-diisopropylcarbodiimide (DIC) and 7.4 g (108.6 mmol) of imidazole were added to the filtrate of step 1 containing 17.6 g (54.3 mmol) of UMP in sequence, and the temperature was raised to 70°C and stirred at 70°C for 1 hour. After the reaction was completed, the reaction solution was cooled to 10°C or below, and after stirring at 10°C or below for 30 minutes, the generated solid was filtered out. 400 mL of acetone was added to a separate 1L three-necked round-bottom flask, and the previously filtered filtrate was slowly added dropwise in reverse. 8.38 g (59.7 mmol) of NaClO4·1H2O was added, and it was stirred at room temperature for 30 minutes to crystallize, and then the obtained solid was filtered, washed with 25 mL of acetone, and dried with nitrogen.

[0128] Purity 97.0%(HPLC)

[0129] 1 H NMR (D2O, 400MHz): δ3.95(m,1H),4.04(m,1H),4.09(m,2H),4.19(m,1H),5.80(m,2H),7.03(m,1H),7.21(t,1H),7.58(d,1H),7.84(s,1H)

[0130] (Step 3) Synthesis of crude uridine 5'-diphosphate (UDP)

[0131]

[0132] 16 g (42.8 mmol) of uridine-5'-phosphoryl imidazole and 160 mL of acetone were added to a 250 mL three-necked round-bottom flask. 80 mL of acetone, 5.87 mL (85.5 mmol) of 85% phosphoric acid and 20.4 mL (85.5 mmol) of tributylamine were added to a separate 250 mL three-necked round-bottom flask, stirred for 10 minutes, and then added dropwise in reverse. 6.29 g (42.8 mmol) of calcium chloride dihydrate was added, the temperature was raised to 40°C and stirred at 40°C for 1 to 2 hours. After the reaction was completed, 12.57 g (85.5 mmol) of NaClO4·1H2O was added, stirred at room temperature for 1 hour, and then the obtained solid was filtered, washed with 40 mL of acetone, and dried. 100 mL of methanol and 50 mL of purified water were added to the dried material, and the slurry was stirred at room temperature for 1 hour. The obtained solid was filtered, washed with 40 mL of acetone, and dried with nitrogen.

[0133] Yield: 21.92 g, UDP content: 62.6%, UDP purity: 88.8% (HPLC)

[0134] Embodiment 2:

[0135] (Step 4-1) Purification of uridine 5'-diphosphate (UDP)

[0136] For the crude uridine 5'-diphosphate (UDP) obtained in step 3 of Example 1 above, 16g of crude UDP (UDP 10g, 49.5mmol) and 1L of purified water were added to a 2L conical flask, and 6N hydrochloric acid was slowly added while stirring until the pH reached 2.5. After clarification and dissolution, 100g of activated carbon was added and stirred at room temperature for 30 minutes for adsorption. After filtration, impurities were removed with the filtrate, washed with 1L of purified water to remove impurities, and 1.4N ammonia water was used to elute and purify UDP.

[0137] Eluent: 0.6L, purity: 95.3% (HPLC)

[0138] (Step 4-2) Synthesis of uridine 5'-diphosphate (UDP) calcium salt

[0139]

[0140] 29.1 g (198 mmol) of calcium chloride dihydrate was added to 0.6 L of the UDP (49.5 mmol) solution eluted in step 4-1, and 240 mL of methanol was added thereto. After stirring at room temperature for 1 hour, the resulting solid was filtered to obtain UDP calcium salt, and dried with nitrogen.

[0141] Yield 96%, content 80%, purity 94.9% (HPLC)

[0142] (Step 4-3) Synthesis of uridine 5'-diphosphate (UDP) sodium salt

[0143]

[0144] 12.3g (27.9mmol) UDP calcium salt and 30mL purified water were added to a 250mL three-necked round-bottom flask and stirred at room temperature. Dilute sulfuric acid was slowly added dropwise until the pH reached 2.5, and stirred at room temperature for 1 hour. The resulting solid was removed by filtration, and a 5N aqueous sodium hydroxide solution was slowly added dropwise to the filtrate until the pH reached 4.5. The resulting solution was reversely added dropwise to 100mL methanol and stirred at room temperature for 1 hour, and then the resulting solid was filtered to obtain the UDP sodium salt, and dried with nitrogen.

[0145] Yield 86.6%, content 99.1%, purity 99.0% (HPLC)

[0146] 1 H NMR (D2O, 400MHz): δ4.14(m,2H),4.19(m,1H),4.30(m,2H),5.89(m,2H),7.91(d,1H)

[0147] The reaction results of the above steps 1 to 4-3 are shown in Table 2 below.

[0148] [Table 2]

[0149] substance Yield purity content Step 1 Uridine 5'-monophosphate Equal - - Step 2 Uridine-5'-phospholipid imidazole 91％ 97.0％ - Step 3 Crude Uridine 5'-diphosphate <![CDATA[65% 1 ]]> 88.8％ 62.6％ Step 4-1 Uridine 5'-diphosphate <![CDATA[82% 1 ]]> 95.3％ - Step 4-2 Uridine 5'-diphosphate calcium salt <![CDATA[96% 1 ]]> 94.9％ 80.0％ Step 4-3 Uridine 5'-diphosphate sodium salt <![CDATA[87% 1 ]]> 99.0％ 99.1％

[0150] 1 Actual output × UDP content / 100

[0151] Comparative Experimental Example 1: Synthesis of Crude Uridine 5'-Diphosphate (UDP)

[0152] (Step A) Synthesis of Uridine-5'-Phosphoryl Imidazolide: Test of Existing Preparation Methods

[0153] N,N'-dimethylformamide (20 mL / g) and TEA (6.6 mol equivalents) were added to UMP free acid (1 mol equivalent) and stirred for 10 minutes. Then, imidazole (4 mol equivalents), 2,2'-dithiodipyridyl (1.6 mol equivalents) and triphenylphosphine (1.6 mol equivalents) were added in sequence. After reacting at 25°C for 3 hours, acetone (140 mL / g) and NaClO4 (4 mol equivalents) were added, cooled to 4°C, stirred for 30 minutes, filtered and dried.

[0154] Yield 84.2%, purity 94.1% (HPLC)

[0155] (Step B) Synthesis of crude UDP: Experimental study of existing preparation methods

[0156] N, N'-dimethylformamide (34mL / g), phosphoric acid (4 molar equivalents) and tributylamine (4 molar equivalents) were added to the uridine-5'-phosphoryl imidazole compound (1 molar equivalent) synthesized in step A above and stirred for 10 minutes. Then, ZnCl2 (0.7 molar equivalents) was added and stirred at 25°C for 15 hours. The reaction solution was analyzed by HPLC to determine the purity of UDP, UMP, UP3U, UP2U and UMP-IMD in the reaction solution. The results are shown in Table 3 below.

[0157] [Table 3]

[0158]

[0159]

[0160] The results of the synthesis process of steps 1 to 3 of Example 1 of the present invention compared with the existing literature process of Comparative Experimental Example 1 are shown in Table 4. The results confirm that the synthesis process of the present invention greatly improves the yield and purity of the product.

[0161] [Table 4]

[0162]

[0163] 1 Actual output × UDP content / 100

[0164] Experimental Example 1: Synthesis of Uridine-5'-Phosphoryl Imidazolide: Effect of Condensation Agent

[0165] 88% ethanol (4.5 mL / g), tributylamine (TBA, 1.5 molar equivalents), a condensing agent (2 molar equivalents) and imidazole (2 molar equivalents) were added to UMP free acid (1 molar equivalent) in sequence, and then the temperature was raised to 70°C and reacted at 70°C for 1 hour.

[0166] The reaction solution was analyzed by HPLC to determine the purity of UMP, uridine-5'-phosphoimidazolide and UP2U in the reaction solution. The results are shown in Table 5 below.

[0167] [Table 5]

[0168]

[0169]

[0170] According to the results of [Table 5] above, in the synthesis of uridine-5'-phosphoimidazole, the reactivity and dimer formation are different depending on the type of condensing agent. The results confirmed that when DIC (N,N'-diisopropylcarbodiimide) is used, the conversion rate of uridine-5'-phosphoimidazole is the best and the formation of dimers is the least.

[0171] Experimental Example 2: Synthesis of Uridine-5'-Phosphoryl Imidazolide: Effect of Tertiary Amine

[0172] 88% ethanol (4.5 mL / g), tertiary amine (1.5 mol equivalent), N,N'-diisopropylcarbodiimide (DIC, 2 mol equivalent) and imidazole (2 mol equivalent) were added to UMP free acid (1 mol equivalent) in sequence, and then the temperature was raised to 70°C and reacted at 70°C for 1 hour. Triethylamine (TEA), tributylamine (TBA) and diisopropylethylamine (DIPEA) were used as tertiary amines, respectively.

[0173] The reaction solution was analyzed by HPLC to determine the purity of UMP, uridine-5'-phosphoimidazolide and UP2U in the reaction solution. The results are shown in Table 6 below.

[0174] [Table 6]

[0175]

[0176] From the results in [Table 6] above, it was confirmed that when TEA, TBA, or DIPEA was used as the tertiary amine in the synthesis of uridine-5'-phosphoimidazolide, the purity of the target UMP-IMD was 95% or more.

[0177] Experimental Example 3: Synthesis of crude UDP: Effect of molar equivalents of monophosphorylating agent and tertiary amine

[0178] Add 85% phosphoric acid and tributylamine in molar equivalents to acetone (15 mL / g) to a three-necked round-bottom flask and stir for 10 minutes. Then, add uridine-5'-phosphoryl imidazole (1 molar equivalent) and calcium chloride dihydrate (1 molar equivalent) in sequence, and heat to 30°C for reaction for 1 hour.

[0179] The reaction solution was analyzed by HPLC to determine the purity of UDP, UMP and UP3U in the reaction solution. The results are shown in Table 7 below.

[0180] [Table 7]

[0181]

[0182] The results of the above [Table 7] confirmed the reactivity and the generation of dimer-related substances (UP3U) when using 1 to 10 molar equivalents of monophosphorylating agent and TBA in UDP synthesis. The results confirmed that when 2 molar equivalents or more of monophosphorylating agent and TBA were used, 85% or more of UDP could be synthesized, and as the molar equivalents increased, the purity of UDP increased, and the unreacted and side reactions decreased. However, after the molar equivalent of phosphoric acid was greater than the molar equivalent of TBA, it was confirmed that the purity of the product decreased when the reaction was carried out in an acidic environment.

Claims

1. A method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a, characterized in that: The method comprises: (Step 1) deionizing a salt of uridine 5'-monophosphate (UMP) represented by Formula 2a with an acid to prepare UMP represented by Formula 2a; (Step 2) reacting the UMP represented by Formula 2a with a tertiary amine, a carbodiimide condensing agent, and imidazole to prepare a uridine-5'-phosphoimidazole represented by Formula 2b; and (Step 3) reacting the uridine-5′-phosphoimidazole represented by Formula 2b with a monophosphorylating agent and a tertiary amine to prepare UDP represented by Formula 3a or a salt thereof, Wherein, the carbodiimide condensing agent is at least one selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) or its salt and N,N'-diisopropylcarbodiimide (DIC); [Formula 2a] [Formula 2b] [Formula 3a] 2. The method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a according to claim 1, characterized in that: The acid in step 1 is sulfuric acid or hydrochloric acid.

3. The method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a according to claim 1, characterized in that: In the above step 1, a mixed solvent of water and alcohol is used as the reaction solvent.

4. The method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a according to claim 1, characterized in that: The tertiary amine in step 2 is at least any one selected from triethylamine, tributylamine and diisopropylethylamine.

5. The method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a according to claim 1, characterized in that: In the above step 3, at least any one selected from acetone, alcohol, acetonitrile and N,N'-dimethylformamide is used as a reaction solvent.

6. The method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a according to claim 1, characterized in that: The amounts of the monophosphorylating agent and the tertiary amine used in the above step 3 are 1.0-10.0 molar equivalents based on 1 molar equivalent of the uridine-5'-phosphoryl imidazolide represented by formula 2b.

7. The method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a according to claim 1, characterized in that: The molar equivalent ratio of the monophosphorylation reagent to the tertiary amine in step 3 is 1:

1.

8. The method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a according to claim 1, characterized in that: The method further comprises: after the above step 3, (Step 4-1) preparing UDP represented by Formula 3a by adsorption purification and elution of UDP represented by Formula 3a; (Step 4-2) converting the UDP represented by Formula 3a into its metal salt and solidifying it; and (Step 4-3) A metal salt of uridine 5'-diphosphate is converted into UDP represented by Formula 3a or a salt thereof.

9. The method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a according to claim 8, characterized in that: The adsorbent used in the adsorption purification in step 4-1 above is activated carbon.

10. The method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a according to claim 8, characterized in that: The eluent used for elution in the above step 4-1 is at least any one selected from dilute hydrochloric acid, dilute ammonia water and sodium chloride aqueous solution.

11. The method for preparing uridine 5'-diphosphate (UDP) or a salt thereof represented by formula 3a according to claim 8, characterized in that: The UDP metal salt prepared in the above step 4-2 is a divalent cation metal salt.

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