Preparation method of 2 '-modified inosine monomer

Abstract

The invention discloses a preparation method of a 2 '-modified inosine monomer, and belongs to the technical field of synthesis of medical intermediates. The method comprises the following steps: by taking a compound I as a starting raw material, preparing a compound II under the action of adenosine deaminase, then reacting the compound II with DMTrCl to generate a compound III, purifying the compound III, and reacting with a phosphorus reagent to prepare a compound IV. According to the method, adenosine deaminase is used as a deamination catalytic enzyme, an adenosine derivative is converted into an inosine derivative, the reaction can be carried out in pure water, the reaction is fast, the condition is mild, almost no by-product is generated, after the reaction is finished, a solvent is used for crystal transformation, a compound II with the purity of 99% + can be obtained, DMTr is added to the compound II, a compound III is formed, the compound III does not need column chromatography, and the yield is high. And then further reacting with a phosphorus reagent to obtain the 2 '-modified inosine monomer derivative with the purity of 99% +.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical intermediates, and particularly relates to a preparation method of 2'-modified inosine monomers. BACKGROUND

[0002] Inosine (I, hypoxanthine nucleoside) is a widely existing RNA modification form in the human transcriptome. By changing the structure and stability of the RNA double strand, the splicing, translation, stability and protein binding capacity of mRNA are affected. Hypoxanthine nucleoside is widely used as a "degenerate base" substitute in the field of molecular biology due to its special structure and flexible base pairing ability. 2'-modified inosine monomers are also widely used in oligonucleotide drugs, such as the soon-to-be-marketed lipid-lowering drug Plozasiran, which uses 2'-methoxy inosine monomers in its sequence.

[0003] The prior art (US20180105551, Journal of Medicinal Chemistry (2003), 46(10), 1878-1885) about the preparation of inosine derivatives adopts a diazotization deamination method, uses acetic acid and nitrite as diazotization reagents, converts adenosine into inosine, and then performs DMT protection, phosphorus loading and other steps to obtain the monomer. The process is as follows: .

[0004] The diazotization reaction needs to be strictly controlled in terms of reaction temperature to prevent the risk of explosion caused by local heat release unevenness, which undoubtedly increases the difficulty of industrial production. Therefore, it is particularly important to find an alternative deamination method. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of 2'-modified inosine monomers. The synthesis method mainly improves the prior art by using an enzyme catalysis process for deamination, simple post-treatment to obtain inosine derivatives, and then DMT protection, phosphorus loading and other steps to obtain the target compound.

[0006] The purpose of the present application can be achieved by the following technical solutions: A preparation method of 2'-modified inosine monomers, which uses compound I as a starting material, and through the action of adenosine deaminase, compound II is prepared, then compound II reacts with DMTrCl (4,4'-dimethoxytrityl chloride) to generate compound III, and after purification, compound III reacts with a phosphorus reagent to obtain compound IV. The synthesis path is as follows: R=OMe (methoxy), OMOE (methoxyethoxy), F (fluorine), etc.

[0007] Specifically, the method comprises the following steps: (1) reacting compound I with adenosine deaminase to obtain compound II; (2) reacting compound II with DMTrCl to obtain compound III; (3) reacting compound III with a phosphorus reagent to obtain compound IV; As a specific embodiment, the adenosine deaminase in step (1) is an enzyme solution, and the amount of the enzyme solution is 0.1-0.5 times the volume of compound I; As a specific embodiment, the reaction solvent in step (1) is selected from one of pure water or a buffer solution with pH=7.5±0.5; As a specific embodiment, the reaction temperature in step (1) is 35-42℃; As a specific embodiment, a pH regulator can be added in step (1) to maintain the pH of the system at 7.5±0.5, and the pH regulator is selected from one or more of hydrochloric acid solution, sulfuric acid solution, and hydrogen cation resin; As a specific embodiment, the pH regulator in step (1) can be added during the reaction or after the reaction; As a specific embodiment, the post-treatment method in step (1) is as follows: adjusting the pH of the system to 7, using a resin to adjust the pH, then performing a filtration operation, then removing water under reduced pressure, then crystallizing with an alcohol solvent, and then filtering and drying to obtain compound II.

[0008] As a specific embodiment, the reaction solvent in step (2) is selected from one or more of pyridine, DCM (dichloromethane), acetonitrile, and ethyl acetate; As a specific embodiment, a basic catalyst is required in step (2), and the basic catalyst is selected from one or more of pyridine, 2,6-dimethylpyridine, DMAP (4-dimethylaminopyridine), 2,4,6-trimethylpyridine, DIPEA (N,N-diisopropylethylamine), and triethylamine; As a specific embodiment, the reaction temperature in step (2) is 0-50℃, and preferably 20-40℃; As a specific embodiment, the post-treatment method in step (2) is as follows: after the reaction is completed, sequentially washing with a citric acid solution, a sodium bicarbonate solution, and salt water, concentrating and drying the organic phase, and then crystallizing with an organic solvent; As a specific embodiment, the organic solvent for crystallization in step (2) is selected from one or more of ethyl acetate, acetonitrile, isopropyl acetate, diethyl ether, isopropyl ether, toluene, methyl tert-butyl ether, and acetone. As a specific embodiment, the reaction solvent in step (3) is selected from one or more of DCM, acetonitrile, THF (tetrahydrofuran), 2-MeTHF (2-methyltetrahydrofuran); As a specific embodiment, the phosphorus reagent in step (3) is selected from one of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite; As a specific embodiment, the catalyst in step (3) is selected from one or more of tetrazole, diisopropylethylamine tetrazole, ethylthio tetrazole, DCI (4,5-dicyanoimidazole), pyridine trifluoroacetate, DIPEA, N-methylmorpholine; As a specific embodiment, the post-processing method in step (3) is: adding sodium bicarbonate solution to quench the reaction, extracting with dichloromethane, washing the organic phase with saturated sodium chloride solution three times, drying with anhydrous sodium sulfate, rotary evaporation and column chromatography.

[0009] Advantages of the present application: The present application uses adenosine deaminase as a deamination catalytic enzyme to convert adenosine derivatives into inosine derivatives. The reaction can be carried out in pure water, the reaction is fast, the conditions are mild, there is almost no by-product, and after the reaction is completed, solvent crystallization is used to obtain compound II with a purity of 99%+. After compound II is subjected to DMTr, compound III is formed, which does not need column chromatography and can be crystallized by solvent, and then further reacts with a phosphorus reagent to obtain a 2'-modified inosine monomer derivative with a purity of 99%+. BRIEF DESCRIPTION OF DRAWINGS

[0010] The present application will be further described below with reference to the accompanying drawings.

[0011] Figure 1 The hydrogen spectrum of compound IVa; Figure 2 The phosphorus spectrum of compound IVa; Figure 3 The hydrogen spectrum of compound IVb; Figure 4 The phosphorus spectrum of compound IVb; Figure 5 The hydrogen spectrum of compound IVc; Figure 6 The phosphorus spectrum of compound IVc. DETAILED DESCRIPTION

[0012] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0013] Example 1 Preparation of compound IVa (R=OMe) Compound Ia 100 g and purified water 1 L were sequentially added to a reaction tank, stirred uniformly (not dissolved), heated to 35-37°C, and then ADA enzyme liquid 20 mL was added. The reaction was stirred until the system was completely dissolved and clear. After the reaction system was clear, HPLC monitoring was started. After the reaction end point was confirmed by HPLC, cationic resin was added to adjust the pH to 7, and then the temperature was continuously increased to 90±5°C, and the stirring was continued for 2 hours. Hot filtration was performed to remove the resin and mechanical impurities. The filtrate was concentrated to dryness, and then ethanol was added to remove water. Ethanol was added to the concentrated solution again, and then the temperature was increased to 70±5°C to reflux for 2 hours. The temperature was decreased to 15-20°C, and then the stirring was continued for 16-18 hours to precipitate crystals. Filtration was performed, and then the filter cake was washed with a small amount of ethanol. The solid was dried to obtain compound IIa 90 g, with a yield of 90% and a purity of 99.5%.

[0014] Compound IIa 90 g, pyridine 90 mL and DCM 1.5 L were added to a reaction bottle. The temperature was decreased to 0±5°C, and then the stirring was continued until the temperature was decreased to the specified temperature. Then, DMTrCl 130 g was added, and the stirring was continued at a temperature of 0-5°C until the reaction was completed. After the reaction was completed, methanol was added to the reaction tank to quench the reaction. Then, sodium bicarbonate was sequentially used twice for washing, saturated laboratory was used once for washing, and sodium sulfate was used for drying. Filtration was performed to obtain an organic phase. The solvent was removed by concentration under reduced pressure. Then, ethyl acetate was added for recrystallization to obtain white solid IIIa 160 g, with a molar yield of 86% and a purity of 99%.

[0015] Compound IIIa 100 g and 2 L DCM were added to a reaction bottle, and then the stirring was continued. Then, 59 g of phosphorus reagent (2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite) and 18 g of DCI were sequentially added. The stirring was continued under nitrogen protection at a room temperature of 20-30°C overnight until the reaction was completed. After the reaction was completed, saturated NaHCO3 was used for extraction once, and saturated NaCl was used for extraction once. Anhydrous Na2SO4 was used for drying, and then silica gel column purification was performed to obtain compound IVa 110.6 g, with a molar yield of 82.4%, an HPLC purity of 99.1%, and a P-NMR of 99.5%, as shown in FIGS. 1 and 2. Figure 1 and Figure 2 FIGS. 1 and 2 respectively show the hydrogen spectrum and the phosphorus spectrum of the obtained compound IVa.

[0016] Example 2 Preparation of compound IVb (R=OMOE) Into a reaction flask, add compound Ib 100 g, purified water 1 L, stir to dissolve, warm to 35-37 °C, add ADA enzyme liquid 30 mL, stir to react until the system is completely dissolved, after the reaction system is dissolved, start HPLC monitoring. After confirming the end point of the reaction by HPLC, adjust the pH to 7 by adding cation exchange resin, filter to remove the resin and mechanical impurities, concentrate the filtrate to dryness, add methanol to remove water, add ethanol to the concentrated solution, warm to 70 ± 5 °C and reflux for 2 hours, cool to 15-20 °C and stir for 16-18 hours, filter, wash the filter cake with a small amount of methanol, dry the solid, and obtain compound IIb 90.5 g, yield: 90%, purity: 99.7%.

[0017] Into a reaction flask, add IIb 90 g, pyridine 900 mL. Cool to 0 ± 5 °C, stir evenly, and after cooling to the specified temperature, add DMTrCl 98.1 g, stir at a temperature of 0-5 °C, and after the reaction is complete, add methanol to the reaction flask to quench the reaction, then add dichloromethane, then sequentially wash with sodium bicarbonate 2 times, saturated laboratory 1 time, and dry with sodium sulfate, filter to obtain the organic phase, concentrate under reduced pressure to remove the solvent, then recrystallize with ethyl acetate to obtain white solid IIIb 150 g, molar yield: 91.7%, purity: 98.6%.

[0018] Into a reaction flask, add compound IIIb 140 g, 1.4 L DCM, stir to mix, sequentially add 75 g of phosphorus reagent (2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite) and 25 g of DCI, protect with nitrogen, stir at room temperature 20-30 °C overnight, after the reaction is complete, extract with saturated NaHCO3 once and saturated NaCl once, dry with anhydrous Na2SO4, and purify with a silica gel column to obtain compound IVb 155 g, molar yield: 83.9%, HPLC purity: 99.4%, P-NMR: 99.2%, as shown in Figure 3 and Figure 4 , which are the hydrogen spectrum and phosphorus spectrum of the obtained compound IVb, respectively.

[0019] Example 3 Preparation method of compound IVc (R = F) Into a reaction flask, add compound Ib 100 g, purified water 1 L, stir to dissolve, warm to 35-37 °C, add ADA enzyme liquid 30 mL, stir to react until the system is completely dissolved, after the reaction system is dissolved, start HPLC monitoring. After confirming the end point of the reaction by HPLC, adjust the pH to 7 by adding cation exchange resin, filter to remove the resin and mechanical impurities, concentrate the filtrate to dryness, add methanol to remove water, add ethanol to the concentrated solution, warm to 70 ± 5 °C and reflux for 2 hours, cool to 15-20 °C and stir for 16-18 hours, filter, wash the filter cake with a small amount of methanol, dry the solid, and obtain compound IIb 90.5 g, yield: 90%, purity: 99.7%.

[0020] Reaction bottle is added with IIc 80g, pyridine 800ml. Lower temperature to 0±5℃, stirring uniform, after the temperature is reduced to the specified temperature, add DMTrCl 104g, temperature 20-30℃ stirring reaction, after the reaction is completed, to the reaction tank is added with methanol to quench the reaction, then add dichloromethane, then use sodium bicarbonate wash 2 times, saturated laboratory wash 1 times, sodium sulfate drying, filtration to get organic phase, reduce pressure concentration to remove the solvent, then add acetone to recrystallize, get white solid IIIc 150g, molar yield: 88.8%, purity: 99.3%.

[0021] Reaction bottle is added with compound IIIc 100g, 2LDCM stirring mixed, added with 61g phosphorus reagent (2-cyanoethyl N, N, N', N'-tetraisopropyl phosphorodiamidite), 19.5g DCI, nitrogen protection, room temperature 20-30℃ stirring overnight, after the reaction is completed, add saturated NaHCO3 extraction once, saturated NaCl extraction once, anhydrous Na2SO4 drying, silica gel column purification, get compound IVc 110.6g, molar yield: 82.4%, HPLC purity: 99.6%, P-NMR: 99.1%, as shown in Figure 5 and Figure 6 The hydrogen spectrum and phosphorus spectrum of the obtained compound IVc are shown in the above specific embodiment part.

[0022] The above specific embodiment part specifically introduces the analysis method involved in the present application. It should be noted that the above introduction is only to help the skilled in the art better understand the method and idea of the present application, and is not a limitation on the related content. The skilled in the art can also make appropriate adjustments or modifications to the present application without departing from the principles of the present application, and the above adjustments and modifications should also belong to the protection scope of the present application.

Claims

1. A method for preparing a 2'-modified inosine monomer, characterized in that, Starting with compound I, compound II was prepared by the action of adenosine deaminase. Then, compound II reacted with DMTrCl to generate compound III. After purification, compound III reacted with a phosphorus reagent to obtain compound IV. The synthetic route is as follows: 。 2. The method for preparing a 2'-modified inosine monomer according to claim 1, characterized in that, In the formula, R = OMe, OMOE, or F.

3. The method for preparing a 2'-modified inosine monomer according to claim 1, characterized in that, Includes the following steps: (1) Compound I reacts with adenosine deaminase to form compound II; (2) Compound II was reacted with DMTrCl to prepare compound III; (3) Compound III reacts with phosphorus reagent to form compound IV.

4. The method for preparing a 2'-modified inosine monomer according to claim 3, characterized in that, In step (1), adenosine deaminase is an enzyme solution, and its volume is 0.1 to 0.5 times that of compound I.

5. The method for preparing a 2'-modified inosine monomer according to claim 3, characterized in that, Step (1) The reaction solvent is selected from pure water or a buffer solution with pH=7.5±0.5; the reaction temperature is 35-42℃.

6. The method for preparing a 2'-modified inosine monomer according to claim 3, characterized in that, In step (1) of the reaction, a pH adjuster is added to maintain the pH of the system at 7.5 ± 0.

5. The pH adjuster is selected from one or more of hydrochloric acid solution, sulfuric acid solution, and hydrogen cation exchange resin.

7. The method for preparing a 2'-modified inosine monomer according to claim 3, characterized in that, In step (2), the reaction solvent is selected from one or more of pyridine, DCM, acetonitrile, and ethyl acetate; in step (2), an alkaline catalyst is added to catalyze the reaction, wherein the alkaline catalyst is selected from one or more of pyridine, 2,6-dimethylpyridine, DMAP, 2,4,6-trimethylpyridine, DIPEA, and triethylamine.

8. The method for preparing a 2'-modified inosine monomer according to claim 3, characterized in that, The reaction temperature in step (2) is 0-50℃.

9. The method for preparing a 2'-modified inosine monomer according to claim 3, characterized in that, In step (3), the reaction solvent is selected from one or more of DCM, acetonitrile, THF, and 2-MeTHF; the phosphorus reagent is selected from one of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonium diamine and 2-cyanoethyl N,N-diisopropylchlorophosphonium diamine.

10. The method for preparing a 2'-modified inosine monomer according to claim 3, characterized in that, The reaction in step (3) is carried out under the action of a catalyst, which is selected from one or more of tetrazolium, diisopropylethylaminetetrazolium, ethylthiotetrazolium, DCI, pyridine trifluoroacetate, DIPEA, and N-methylmorpholine.

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