A method for preparing modified nucleoside triphosphate

Abstract

The invention discloses a method for preparing a modified nucleoside triphosphate compound as shown in formula I. The method adopts cheap and readily available organic amine and pyrophosphate reagent to prepare ammonium pyrophosphate, and then prepares a series of modified nucleoside triphosphate compounds in one pot. The method has the characteristics of simple operation, low cost, high yield, safety, stable process, etc., and is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemical synthesis, and specifically relates to a method for preparing modified nucleoside triphosphates. Background Art

[0002] With the development of nucleic acid drugs, mRNA is seen as a new option for drug manufacturing. In 1990, a segment of mRNA was injected into mice and successfully encoded a protein. This segment of mRNA was obtained through a technology called in vitro transcription. Subsequently, a 1992 study found that injection of antidiuretic hormone-encoding mRNA could successfully induce neural activity in the hypothalamus of rats. Although mRNA shows good biological activity, it is far from being used in the treatment of clinical diseases due to its own instability, strong immunogenicity and difficulty in in vivo delivery.

[0003] The synthesis of novel modified nucleoside triphosphates and their incorporation into nucleic acid sequences provide many possibilities for changing the chemical properties of nucleotides, which greatly broadens the practical application areas of nucleotides, can reduce the autoimmunogenicity of mRNA, improve the stability of mRNA itself, and further enhance the expression time and efficiency of mRNA in target cells, so that mRMA can be truly used in the pharmaceutical field.

[0004] It can be seen that modified nucleoside triphosphates are important in mRNA synthesis, so it is of great significance to determine a safe, efficient and stable method for preparing modified nucleoside triphosphates. At present, there are few methods for preparing high-purity modified nucleoside triphosphates. The document "Nucleosides, Nucleotides and Nucleic acids, 2016, 35, 356-362" reports a method for preparing nucleoside triphosphates by a one-pot method using nucleotides as raw materials, but this method requires the use of highly toxic reagent tributylamine, and the purchase price of tributylammonium pyrophosphate is expensive, which is not conducive to industrial production. Self-made tributylammonium pyrophosphate requires tetrasodium pyrophosphate to pass through ion exchange resin and be eluted with highly toxic tributylamine solution, which is cumbersome and highly dangerous.

[0005]

[0006] Therefore, it is of great significance to develop a method for preparing modified nucleoside triphosphates that is lower in cost, safer, more environmentally friendly, and more suitable for industrial large-scale production. Summary of the invention

[0007] In order to solve the technical problems in the prior art of needing to use expensive tributylammonium pyrophosphate and highly toxic reagent tributylamine, high reaction cost, great difficulty in synthesis and low yield of the final product, the present invention provides a preparation method of a modified nucleoside triphosphate structure as shown in formula I, the preparation method adopts cheap and readily available organic amine and pyrophosphate reagent to prepare ammonium pyrophosphate, and then prepares the modified nucleoside triphosphate compound by a one-pot method, greatly reducing the process production cost, while avoiding the use of dangerous reagents and cumbersome operations in the preparation process of tributylammonium pyrophosphate, having the characteristics of simple operation, low cost, high yield, safety, stable process, etc., and being suitable for industrial large-scale production.

[0008] The present invention provides a method for preparing a compound of formula I and a salt thereof, comprising the following steps:

[0009] (1) In a reaction vessel 1, compound III reacts with phosphorus oxychloride in a solvent 1 to prepare an intermediate of formula II;

[0010] (2) In reaction vessel 2, a pyrophosphate reagent and an organic base react in solvent 2 to prepare an ammonium pyrophosphate solution;

[0011] (3) mixing the intermediate II with an ammonium pyrophosphate solution to prepare compound I;

[0012]

[0013] The group B is selected from bases.

[0014] Furthermore, the base is selected from or Preferably or

[0015] Furthermore, the salt of the compound of formula I is selected from triethylamine salt, sodium salt or potassium salt.

[0016] Furthermore, the pyrophosphate reagent is selected from pyrophosphate, sodium pyrophosphate or potassium pyrophosphate.

[0017] Further, the organic base is selected from one or a combination of two or more of organic amines or pyridines, preferably organic amines;

[0018] Preferably, the organic amine is selected from triethylamine, tripropylamine, tri-n-pentylamine, tri-n-hexylamine, tri-n-heptylamine, tri-n-octylamine, N,N-diisopropylethylamine, 1,8-diazobispiro[5.4.0]undec-7-ene, triethylenediamine or aniline; the pyridine is selected from pyridine or 4-dimethylaminopyridine;

[0019] More preferably, the organic amine is selected from tri-n-pentylamine, tri-n-hexylamine, tri-n-heptylamine, tri-n-octylamine, triethylenediamine or N,N-diisopropylethylamine;

[0020] More preferably, the organic amine is selected from tri-n-hexylamine, triethylenediamine or N,N-diisopropylethylamine.

[0021] Further, the solvent 1 is selected from one or a combination of two or more of phosphates, ethers, sulfoxides, amides, and nitriles, preferably phosphates; the solvent 2 is selected from one or a combination of two or more of phosphates, ethers, sulfoxides, amides, nitriles, or halogenated hydrocarbons, preferably phosphates, nitriles, or amides;

[0022] Preferably, the phosphate ester is selected from trimethyl phosphate or triethyl phosphate; the ether is selected from diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran or 1,4-dioxane; the sulfoxide is selected from dimethyl sulfoxide; the amide is selected from N,N-dimethylacetamide or N,N-dimethylformamide; the nitrile is selected from acetonitrile or propionitrile; the halogenated hydrocarbon is selected from dichloromethane or chloroform.

[0023] Further, the solvent 1 is selected from one or a combination of two or more of trimethyl phosphate, triethyl phosphate, N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile or tetrahydrofuran, preferably trimethyl phosphate or triethyl phosphate; the solvent 2 is selected from one or a combination of two or more of trimethyl phosphate, triethyl phosphate, N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, dichloromethane or chloroform, preferably trimethyl phosphate, triethyl phosphate, acetonitrile or N,N-dimethylformamide, more preferably acetonitrile.

[0024] Furthermore, the reaction temperature of the compound III and phosphorus oxychloride is -80 to 0°C, preferably -40 to 0°C, and more preferably -20 to 0°C; the reaction temperature of the pyrophosphoric acid reagent and the organic base is -80 to 0°C, preferably -40 to 0°C, and more preferably -20 to 0°C; the reaction temperature of the intermediate II and the ammonium pyrophosphate solution is -20 to 0°C, and preferably -10 to 0°C.

[0025] The beneficial effects brought by the present invention are:

[0026] (1) Compared with the prior art, the present invention avoids the use of expensive tributylammonium pyrophosphate reagent (about 100,000 / kg), adopts cheap and readily available organic amine and pyrophosphate reagent (about 4000 / kg) to prepare ammonium pyrophosphate, and then prepares the modified nucleoside triphosphate compound by a one-pot method, the material cost is reduced to 4% of the original, the yield of the obtained product is higher than that of the prior art, the production cost is significantly reduced, and it is more suitable for industrial large-scale production.

[0027] (2) The preparation conditions of the modified nucleoside triphosphate compound of the present invention are milder, more environmentally friendly, and safer to operate, while avoiding the use of dangerous reagents and cumbersome operations in the preparation process of tributylammonium pyrophosphate. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with the embodiments, but the present invention is not limited thereto. Any equivalent substitutions in the art made according to the disclosure of the present invention shall fall within the protection scope of the present invention.

[0029] The structures of compounds were determined by mass spectrometry (MS) or nuclear magnetic resonance (NMR). 1 HNMR) to determine.

[0030] Nuclear magnetic resonance (NMR) 1 HNMR) shifts (δ) are given in parts per million (ppm); nuclear magnetic resonance ( 1 HNMR) was measured using a Bruker AVANCE-400 nuclear magnetic spectrometer, the solvent was deuterated dimethyl sulfoxide (DMSO), and the internal standard was tetramethylsilane (TMS);

[0031] Mass spectrometry (MS) was performed using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Therm, model: Finnigan LCQ advantage MAX);

[0032] Unless otherwise specified in the present invention, the solution mentioned in the reaction of the present invention is an aqueous solution; Example 1I-1 Preparation of triethylammonium salt

[0033]

[0034] Add 10g III-1 to reaction flask ①, add 200mL trimethyl phosphate, add 11.9g phosphorus oxychloride to the dropping funnel, ventilate with nitrogen, place reaction flask ① in a 0℃ low temperature bath with stirring, when the system temperature is 0℃, start to drop phosphorus oxychloride, control the system temperature <5℃ during the dropwise addition, and continue the reaction at 0℃ after the dropwise addition.

[0035] Add 17.3 g of pyrophosphoric acid to reaction bottle ②, followed by 200 mL of acetonitrile, and add 50.2 g of N,N-diisopropylethylamine to the dropping funnel. Ventilate with nitrogen, place reaction bottle ② in a -10°C low-temperature bath with stirring, and when the temperature drops to ≤-5°C, begin to drop N,N-diisopropylethylamine. After the drop is complete, continue the reaction at -10°C.

[0036] Monitor the reaction system in reaction bottle ① by liquid phase. When the raw material III-1 is consumed, quickly transfer the material in reaction bottle ① to reaction bottle ②. Meanwhile, adjust the reaction temperature of reaction bottle ② to 0℃ for reaction. Monitor the reaction of intermediate II-1 by liquid phase. Add 600ml of water to quench the reaction. Extract with dichloromethane three times (150ml*3). Adjust the pH of the obtained aqueous phase to about 6.5. Pass the column through DEAE Sepharose and linearly elute with 0-1M TEAB aqueous solution. Add ethanol to the collected product and azeotropically concentrate in vacuum to obtain 25.6g of triethylammonium salt product of I-1 with a yield of 73%.

[0037] 1 H NMR (D2O, 400MHz) δ7.67 (s, 1H), 5.88 (d, J = 4.0Hz, 1H), 4.35–4.10 (m, 5H), 3.04 (q, J = 4.0Hz, 8H), 1.89 (s, 3H), 1.12 (t, J = 4.0Hz, 12H); 31 P NMR(D2O,162MHz)δ-7.73(d,J=19.4Hz,1P),-11.10(d,J=21.0Hz,1P),-21.76(t,J=16.8Hz,1P); MS(m / z):498[M+H] +

[0038] Example 2 Preparation of I-1 Sodium Salt

[0039]

[0040] Add 10g III-1 to reaction flask ①, add 200mL trimethyl phosphate, add 11.9g phosphorus oxychloride to the dropping funnel, ventilate with nitrogen, place reaction flask ① in a 0℃ low temperature bath with stirring, when the system temperature is 0℃, start to drop phosphorus oxychloride, control the system temperature <5℃ during the dropwise addition, and continue the reaction at 0℃ after the dropwise addition.

[0041] Add 17.3 g of pyrophosphoric acid to reaction bottle ②, followed by 200 mL of acetonitrile, and add 50.2 g of N,N-diisopropylethylamine to the dropping funnel. Ventilate with nitrogen, place reaction bottle ② in a -10°C low-temperature bath with stirring, and when the temperature drops to ≤-5°C, begin to drop N,N-diisopropylethylamine. After the drop is complete, continue the reaction at -10°C.

[0042] Monitor the reaction system in reaction bottle ① by liquid phase. When the raw material III-1 is consumed, quickly transfer the material in reaction bottle ① to reaction bottle ②. Meanwhile, adjust the reaction temperature of reaction bottle ② to 0°C for reaction. Monitor the reaction of intermediate II-1 by liquid phase. Add 600ml of water to quench the reaction. Extract with dichloromethane three times (150ml*3). Adjust the pH of the obtained aqueous phase to about 6.5. Pass the column through DEAE Sepharose and linearly elute with 0-1M TEAB aqueous solution. Add ethanol to the collected product and azeotropically concentrate in vacuo to obtain a white solid of I-1. Add sodium perchlorate acetone solution (50g sodium perchlorate is dissolved in 1000ml acetone) to the white solid. Stir at 0-5°C for 1 hour. Filter. Repeat the treatment of the obtained solid with sodium perchlorate acetone solution again. Dry the obtained white solid to obtain 13.9g sodium salt of I-1 with a yield of 61%.

[0043] 1 H NMR (D2O, 400MHz) δ7.67 (s, 1H), 5.88 (d, J = 4.0Hz, 1H), 4.35–4.10 (m, 5H), 1.89 (s, 3H); 31 P NMR(D2O,162MHz)δ-7.73(d,J=19.4Hz,1P),-11.10(d,J=21.0Hz,1P),-21.76(t,J=16.8Hz,1P); MS(m / z):498[M+H] +

[0044] Example 3 Preparation of I-1 Sodium Salt

[0045]

[0046] Add 5g III-1 to reaction flask ①, add 100mL trimethyl phosphate, add 6g phosphorus oxychloride to the dropping funnel, ventilate with nitrogen, place reaction flask ① in a 0℃ low temperature bath with stirring, when the system temperature is 0℃, start to drop phosphorus oxychloride, control the system temperature <5℃ during the dropwise addition, and continue the reaction at 0℃ after the dropwise addition.

[0047] Add 8.7 g of pyrophosphoric acid to reaction bottle ②, followed by 100 mL of trimethyl phosphate, add 25 g of N,N-diisopropylethylamine to the dropping funnel, ventilate with nitrogen, place reaction bottle ② in a -10℃ low-temperature bath with stirring, wait until the temperature drops to ≤-5℃, start adding N,N-diisopropylethylamine dropwise, and continue the reaction at -10℃ after the addition is complete.

[0048] The reaction system of reaction bottle ① was monitored by liquid phase. After the raw material III-1 was consumed, the material of reaction bottle ① was quickly transferred to reaction bottle ②. At the same time, the reaction temperature of reaction bottle ② was adjusted to 0°C for reaction. After the reaction of intermediate II-1 was monitored by liquid phase, 300 ml of water was added dropwise to quench the reaction. The reaction was extracted with dichloromethane three times (100 ml*3). The pH of the obtained aqueous phase was adjusted to about 6.5, passed through a DEAE Sepharose column, and linearly eluted with 0-1M TEAB aqueous solution. The collected product was azeotropically concentrated in ethanol under vacuum to obtain a white solid of I-1. An acetone solution of sodium perchlorate (25 g of sodium perchlorate was dissolved in 500 ml of acetone) was added to the white solid, stirred at 0-5°C for 1 hour, filtered, and the obtained solid was repeatedly treated with an acetone solution of sodium perchlorate. The obtained white solid was dried to obtain 3.6 g of sodium salt of I-1, with a yield of 32%. The NMR and mass spectra were consistent with those in Example 2.

[0049] Example 4 Preparation of I-2 Sodium Salt

[0050]

[0051] Add 25g III-2 to reaction bottle ①, add 1000mL trimethyl phosphate, add 31.4g phosphorus oxychloride to the dropping funnel, ventilate with nitrogen, place reaction bottle ① in a 0℃ low temperature bath with stirring, when the system temperature is 0℃, start to drop phosphorus oxychloride, control the system temperature <5℃ during the dropwise addition, and continue the reaction at 0℃ after the dropwise addition.

[0052] Add 45.5 g of pyrophosphoric acid to reaction bottle ②, followed by 500 mL of acetonitrile, and add 132 g of N,N-diisopropylethylamine to the dropping funnel. Ventilate with nitrogen, place reaction bottle ② in a -10°C low-temperature bath with stirring, and when the temperature drops to ≤-5°C, begin to drop N,N-diisopropylethylamine. After the dropwise addition is complete, continue the reaction at -10°C.

[0053] Monitor the reaction system in reaction bottle ① by liquid phase. When the raw material III-1 is consumed, quickly transfer the material in reaction bottle ① to reaction bottle ②. Meanwhile, adjust the reaction temperature of reaction bottle ② to 0°C for reaction. Monitor the reaction of intermediate II-1 by liquid phase. Add 3000ml of water to quench the reaction. Extract with dichloromethane three times (1000ml*3). Adjust the pH of the obtained aqueous phase to about 6.5. Pass the column through DEAE Sepharose and linearly elute with 0-1M TEAB aqueous solution. Add ethanol to the collected product and azeotropically concentrate in vacuo to obtain a white solid of I-2. Add sodium perchlorate acetone solution (125g sodium perchlorate is dissolved in 2500ml acetone) to the white solid. Stir at 0-5°C for 1 hour. Filter. Repeat the treatment of the obtained solid with sodium perchlorate acetone solution again. Dry the obtained white solid to obtain 39.8g of sodium salt of I-2 with a yield of 68%.

[0054] 1 H NMR (D2O, 400MHz) δ7.74 (s, 1H), 4.76 (d, J = 4.0Hz, 1H), 4.24–4.03 (m, 5H); 31 PNMR(D2O,162MHz)δ-10.93(d,J=19.4Hz,1P),-11.24(d,J=19.4Hz,1P),-22.34(t,J=19.4Hz,1P); MS(m / z):485[M+H] +

[0055] Example 5 Preparation of I-2 Sodium Salt

[0056]

[0057] Add 25g III-2 to reaction bottle ①, add 1000mL trimethyl phosphate, add 31.4g phosphorus oxychloride to the dropping funnel, ventilate with nitrogen, place reaction bottle ① in a 0℃ low temperature bath with stirring, when the system temperature is 0℃, start to drop phosphorus oxychloride, control the system temperature <5℃ during the dropwise addition, and continue the reaction at 0℃ after the dropwise addition.

[0058] Add 45.5 g of pyrophosphoric acid to reaction bottle ②, followed by 500 mL of acetonitrile, add 276 g of tri-n-hexylamine to the dropping funnel, ventilate with nitrogen, place reaction bottle ② in a -10°C low-temperature bath with stirring, wait until the temperature drops to ≤-5°C, start to drop N,N-diisopropylethylamine, and after the dropwise addition is complete, continue the reaction at -10°C.

[0059] The reaction system of reaction flask ① was monitored by liquid phase. After the raw material III-1 was consumed, the material of reaction flask ① was quickly transferred to reaction flask ②. At the same time, the reaction temperature of reaction flask ② was adjusted to 0°C for reaction. After the reaction of intermediate II-1 was monitored by liquid phase, 3000ml of water was added dropwise to quench the reaction. The reaction was extracted with dichloromethane three times (1000ml*3). The pH of the obtained aqueous phase was adjusted to about 6.5, passed through a DEAE Sepharose column, and linearly eluted with 0-1M TEAB aqueous solution. The collected product was azeotropically concentrated in ethanol under vacuum to obtain a white solid of I-2. An acetone solution of sodium perchlorate (125g of sodium perchlorate was dissolved in 2500ml of acetone) was added to the white solid, stirred at 0-5°C for 1 hour, filtered, and the obtained solid was repeatedly treated with an acetone solution of sodium perchlorate. The obtained white solid was dried to obtain 36.2g of the sodium salt of I-2 with a yield of 62%. The NMR and mass spectra were consistent with those in Example 4.

[0060] Example 6 Preparation of I-3 Sodium Salt

[0061]

[0062] Add 25g III-3 to reaction flask ①, add 500mL trimethyl phosphate, add 29.7g phosphorus oxychloride to the dropping funnel, ventilate with nitrogen, place reaction flask ① in a 0℃ low temperature bath with stirring, when the system temperature is 0℃, start to drop phosphorus oxychloride, control the system temperature <5℃ during the dropwise addition, and continue the reaction at 0℃ after the dropwise addition.

[0063] Add 43.1 g of pyrophosphoric acid to reaction bottle ②, followed by 250 mL of acetonitrile, and add 125 g of N,N-diisopropylethylamine to the dropping funnel. Ventilate with nitrogen, place reaction bottle ② in a -10°C low-temperature bath with stirring, and when the temperature drops to ≤-5°C, begin to drop N,N-diisopropylethylamine. After the drop is complete, continue the reaction at -10°C.

[0064] Monitor the reaction system in reaction bottle ① by liquid phase. When the raw material III-1 is consumed, quickly transfer the material in reaction bottle ① to reaction bottle ②. Meanwhile, adjust the reaction temperature of reaction bottle ② to 0°C for reaction. Monitor the reaction of intermediate II-1 by liquid phase. Add 1500ml of water to quench the reaction. Extract with dichloromethane three times (750ml*3). Adjust the pH of the obtained aqueous phase to about 6.5. Pass the column through DEAE Sepharose and linearly elute with 0-1M TEAB aqueous solution. Add ethanol to the collected product and azeotropically concentrate in vacuo to obtain a white solid of I-3. Add sodium perchlorate acetone solution (125g sodium perchlorate is dissolved in 2500ml acetone) to the white solid. Stir at 0-5°C for 1 hour. Filter. Repeat the treatment of the obtained solid with sodium perchlorate acetone solution again. Dry the obtained white solid to obtain 39.7g of sodium salt of I-3 with a yield of 70%.

[0065] 1 H NMR (D2O, 400MHz) δ7.70 (s, 1H), 4.68 (d, J = 4.4Hz, 1H), 4.18–3.96 (m, 5H), 3.26 (s, 3H); 31 P NMR(D2O,162MHz)δ-11.00(d,J=21.0Hz,1P),-11.40(d,J=19.4Hz,1P),-23.43(t,J=21.0Hz,1P); MS(m / z):499[M+H] +

[0066] Example 7 Preparation of I-3 Sodium Salt

[0067]

[0068] Add 25g III-3 to reaction flask ①, add 500mL trimethyl phosphate, add 29.7g phosphorus oxychloride to the dropping funnel, ventilate with nitrogen, place reaction flask ① in a 0℃ low temperature bath with stirring, when the system temperature is 0℃, start to drop phosphorus oxychloride, control the system temperature <5℃ during the dropwise addition, and continue the reaction at 0℃ after the dropwise addition.

[0069] Add 43.1 g of pyrophosphoric acid to reaction bottle ②, followed by 250 mL of N,N-dimethylformamide, and 125 g of N,N-diisopropylethylamine to the dropping funnel. Ventilate with nitrogen, place reaction bottle ② in a -10°C low-temperature bath with stirring, and when the temperature drops to ≤-5°C, begin to drop N,N-diisopropylethylamine. After the dropwise addition is complete, continue the reaction at -10°C.

[0070] The reaction system of reaction bottle ① was monitored by liquid phase. After the raw material III-1 was consumed, the material of reaction bottle ① was quickly transferred to reaction bottle ②. At the same time, the reaction temperature of reaction bottle ② was adjusted to 0°C for reaction. After the reaction of intermediate II-1 was monitored by liquid phase, 1500ml of water was added dropwise to quench the reaction. The mixture was extracted with dichloromethane three times (750ml*3). The pH of the obtained aqueous phase was adjusted to about 6.5, passed through a DEAE Sepharose column, and linearly eluted with 0-1M TEAB aqueous solution. The collected product was azeotropically concentrated in ethanol under vacuum to obtain a white solid of I-3. An acetone solution of sodium perchlorate (125g of sodium perchlorate was dissolved in 2500ml of acetone) was added to the white solid, stirred at 0-5°C for 1 hour, filtered, and the obtained solid was repeatedly treated with an acetone solution of sodium perchlorate. The obtained white solid was dried to obtain 30.1g of sodium salt of I-3 with a yield of 53%. The NMR and mass spectra were consistent with those in Example 6.

[0071] Example 8 Preparation of I-3 Sodium Salt

[0072]

[0073] Add 25g III-3 to reaction flask ①, add 500mL trimethyl phosphate, add 29.7g phosphorus oxychloride to the dropping funnel, ventilate with nitrogen, place reaction flask ① in a -80℃ low temperature bath with stirring, when the system temperature is 0℃, start to drop phosphorus oxychloride, control the system temperature <5℃ during the dropwise addition, and continue the reaction at 0℃ after the dropwise addition.

[0074] Add 43.1 g of pyrophosphoric acid to reaction bottle ②, followed by 250 mL of N,N-dimethylformamide, and 125 g of N,N-diisopropylethylamine to the dropping funnel. Ventilate with nitrogen, place reaction bottle ② in a -80°C low-temperature bath with stirring, and when the temperature drops to ≤-5°C, begin to drop N,N-diisopropylethylamine. After the dropwise addition is complete, continue the reaction at -10°C.

[0075] The reaction system of reaction bottle ① was monitored by liquid phase. After the raw material III-1 was consumed, the material of reaction bottle ① was quickly transferred to reaction bottle ②. At the same time, the reaction temperature of reaction bottle ② was adjusted to 0°C for reaction. After the reaction of intermediate II-1 was monitored by liquid phase, 1500ml of water was added dropwise to quench the reaction. The mixture was extracted with dichloromethane three times (750ml*3). The pH of the obtained aqueous phase was adjusted to about 6.5, passed through a DEAE Sepharose column, and linearly eluted with 0-1M TEAB aqueous solution. The collected product was azeotropically concentrated in ethanol under vacuum to obtain a white solid of I-3. An acetone solution of sodium perchlorate (125g of sodium perchlorate was dissolved in 2500ml of acetone) was added to the white solid, stirred at 0-5°C for 1 hour, filtered, and the obtained solid was repeatedly treated with an acetone solution of sodium perchlorate. The obtained white solid was dried to obtain 17g of sodium salt of I-3 with a yield of 30%. The NMR and mass spectra were consistent with those in Example 6.

[0076] Example 9 Preparation of I-3 Sodium Salt

[0077]

[0078] Add 25g III-3 to reaction flask ①, add 500mL trimethyl phosphate, add 29.7g phosphorus oxychloride to the dropping funnel, ventilate with nitrogen, place reaction flask ① in a 0℃ low temperature bath with stirring, when the system temperature is 0℃, start to drop phosphorus oxychloride, control the system temperature <5℃ during the dropwise addition, and continue the reaction at 0℃ after the dropwise addition.

[0079] Add 64.4 g of sodium pyrophosphate to reaction bottle ②, followed by 250 mL of N,N-dimethylformamide, and 125 g of N,N-diisopropylethylamine to the dropping funnel. Ventilate with nitrogen, place reaction bottle ② in a -20°C low-temperature bath with stirring, and when the temperature drops to ≤-5°C, begin to drop N,N-diisopropylethylamine. After the dropwise addition is complete, continue the reaction at -10°C.

[0080] The reaction system of reaction bottle ① was monitored by liquid phase. After the raw material III-1 was consumed, the material of reaction bottle ① was quickly transferred to reaction bottle ②. At the same time, the reaction temperature of reaction bottle ② was adjusted to 0°C for reaction. After the reaction of intermediate II-1 was monitored by liquid phase, 1500ml of water was added dropwise to quench the reaction. The mixture was extracted with dichloromethane three times (750ml*3). The pH of the obtained aqueous phase was adjusted to about 6.5, passed through a DEAE Sepharose column, and linearly eluted with 0-1M TEAB aqueous solution. The collected product was azeotropically concentrated in ethanol under vacuum to obtain a white solid of I-3. An acetone solution of sodium perchlorate (125g of sodium perchlorate was dissolved in 2500ml of acetone) was added to the white solid, stirred at 0-5°C for 1 hour, filtered, and the obtained solid was repeatedly treated with an acetone solution of sodium perchlorate. The obtained white solid was dried to obtain 23.8g of the sodium salt of I-3 with a yield of 42%. The NMR and mass spectra were consistent with those in Example 6.

[0081] Example 10 Preparation of I-3 Sodium Salt

[0082]

[0083] Add 25g III-3 to reaction bottle ①, add 500mL triethyl phosphate, add 29.7g phosphorus oxychloride to the dropping funnel, ventilate with nitrogen, place reaction bottle ① in a 0℃ low temperature bath with stirring, when the system temperature is 0℃, start to drop phosphorus oxychloride, control the system temperature <5℃ during the dropwise addition, and continue the reaction at 0℃ after the dropwise addition.

[0084] Add 43.1 g of pyrophosphoric acid to reaction bottle ②, followed by 250 mL of N,N-dimethylformamide, and add 108.5 g of triethylenediamine to the dropping funnel. Ventilate with nitrogen, place reaction bottle ② in a 0℃ low-temperature bath with stirring, and wait until the temperature drops to ≤-20℃, then start to drop N,N-diisopropylethylamine. After the dropwise addition is complete, react at 0℃.

[0085] The reaction system of reaction flask ① was monitored by liquid phase. After the raw material III-1 was consumed, the material of reaction flask ① was quickly transferred to reaction flask ②. At the same time, the reaction temperature of reaction flask ② was adjusted to 0°C for reaction. After the reaction of intermediate II-1 was monitored by liquid phase, 1500ml of water was added dropwise to quench the reaction. The mixture was extracted with dichloromethane three times (750ml*3). The pH of the obtained aqueous phase was adjusted to about 6.5, passed through a DEAE Sepharose column, and linearly eluted with 0-1M TEAB aqueous solution. The collected product was concentrated in azeotropic vacuum with ethanol to obtain a white solid of I-3. An acetone solution of sodium perchlorate (125g of sodium perchlorate was dissolved in 2500ml of acetone) was added to the white solid, stirred at 0-5°C for 1 hour, filtered, and the obtained solid was repeatedly treated with an acetone solution of sodium perchlorate. The obtained white solid was dried to obtain 27.8g of the sodium salt of I-3 with a yield of 49%. The NMR and mass spectra were consistent with those in Example 6.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a compound of formula I and a salt thereof, characterized in that: The steps include: (1) In a reaction vessel 1, compound III reacts with phosphorus oxychloride in a solvent 1 to prepare an intermediate of formula II; (2) In reaction vessel 2, a pyrophosphate reagent and an organic amine react in acetonitrile to prepare an ammonium pyrophosphate solution; (3) mixing the intermediate II with an ammonium pyrophosphate solution to prepare compound I; The group B is selected from The organic amine is selected from triethylamine, tripropylamine, tri-n-pentylamine, tri-n-hexylamine, tri-n-heptylamine, tri-n-octylamine, N,N-diisopropylethylamine, 1,8-diazobispiro[5.4.0]undec-7-ene, triethylenediamine or aniline.

2. The method according to claim 1, characterized in that: The pyrophosphate reagent is selected from pyrophosphate, sodium pyrophosphate or potassium pyrophosphate.

3. The method according to claim 1, characterized in that The solvent 1 is selected from one or a combination of two or more of phosphates, ethers, sulfoxides, amides, and nitriles.

4. The method according to claim 3, characterized in that The solvent 1 is a phosphate ester.

5. The method according to claim 3, characterized in that: The phosphates are selected from trimethyl phosphate or triethyl phosphate; the ethers are selected from diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran or 1,4-dioxane; the sulfoxides are selected from dimethyl sulfoxide; the amides are selected from N,N-dimethylacetamide or N,N-dimethylformamide; and the nitriles are selected from acetonitrile or propionitrile.

6. The method according to claim 3, characterized in that The solvent 1 is selected from one or a combination of two or more of trimethyl phosphate, triethyl phosphate, N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile or tetrahydrofuran.

7. The method according to claim 6, characterized in that The solvent 1 is trimethyl phosphate or triethyl phosphate.

8. The method according to claim 1, characterized in that The reaction temperature of the compound III and phosphorus oxychloride is -80 to 0°C; the reaction temperature of the pyrophosphoric acid reagent and the organic base is -80 to 0°C; the reaction temperature of the intermediate II and the ammonium pyrophosphate solution is -20 to 0°C.

9. The method according to claim 8, characterized in that The reaction temperature of the compound III and phosphorus oxychloride is -40 to 0°C.

10. The method according to claim 9, characterized in that The reaction temperature of the compound III and phosphorus oxychloride is -20 to 0°C.

11. The method according to claim 9, characterized in that The reaction temperature of the pyrophosphate reagent and the organic amine is -40 to 0°C.

12. The method according to claim 11, characterized in that The reaction temperature of the pyrophosphate reagent and the organic amine is -20 to 0°C.

13. The method according to claim 1, characterized in that The reaction temperature of the intermediate II and the ammonium pyrophosphate solution is -10 to 0°C.

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