Preparation of phosphoramidite monomers of 2 '-OMe modified pseudouridine and N1-methyl pseudouridine

Abstract

The invention provides a preparation method of 2 '-OMe modified pseudouridine and a phosphoramidite monomer of N1-methyl pseudouridine, and belongs to the field of medicine synthesis, and the preparation method comprises the following steps: methylating 2'-OH of pseudouridine / N1-methyl pseudouridine by using trimethylsilyl diazomethane; reacting the methylation product with 4, 4 '-dimethoxytriphenylmethyl chloride to obtain an intermediate with a DMTr protecting group at the 5'position; and reacting the obtained intermediate with bis (diisopropylamino) (2-cyano ethyoxyl) phosphine to obtain a target product. According to the invention, pseudouridine / N1-methyl pseudouridine is taken as a raw material, the 2-OMe modified pseudouridine / N1-methyl pseudouridine phosphoramidite monomer can be prepared by only three steps, the synthesis route is greatly shortened, the total yield is more than 40%, the purity of the target product is more than 99%, and the method is very suitable for large-scale industrial synthesis.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical synthesis, and in particular to a method for preparing phosphoramidite monomers of 2'-OMe modified pseudouridine and N1-methyl pseudouridine. Background Art

[0002] 2'-O modified nucleic acids are one of the most well-studied RNA analogs in ASOs, siRNAs, and aptamers. Depending on the natural structure of the nucleotide, chemical modifications can be made at the phosphate backbone, ribose moiety, or bases to increase their stability and reduce activation of the innate immune system. Modifications at the 2' position have been shown to be highly suitable for applications because the conformational maintenance of modifications at this site enhances chemical stability and prevents 2'-OH-mediated chain breaks. In addition, the 2' position is better protected from nuclease attack due to its proximity to adjacent phosphates. The use of 2'-OMe modification in siRNA can improve the loading efficiency of the RISC complex, enhance the silencing activity of siRNA, and further enhance the anti-tumor activity of siRNA molecules.

[0003] Small nucleic acid drugs, also known as oligonucleotide drugs, are short-chain nucleic acids composed of a dozen to dozens of nucleotides in series. The high-quality preparation of its core material, phosphoramidite monomers (nucleoside monomers), is a key technology in the solid phase synthesis process. Pseudouridine is the most abundant modified nucleoside on RNA and is also known as the "fifth nucleoside" of RNA. Moreover, existing studies have shown that introducing pseudouridine or N1-methylpseudouridine into RNA may effectively reduce the immunogenicity of RNA vaccines, enhance the stability of RNA, and enhance its protein expression ability. Therefore, the phosphoramidite monomers of 2'-OMe-modified pseudouridine / N1-methylpseudouridine have great application potential in siRNA drugs.

[0004] However, in previous syntheses, when methylating agents are used directly to methylate nucleosides, 3'-OMe-modified pseudouridine will be produced as a byproduct, which is difficult to separate and purify from 2'-OMe-modified pseudouridine, and the yield is low; if the 3,5-position OH is first protected and then methylated, and then deprotected, this method is long and the overall yield is not high. With the large-scale launch of siRNA drugs, a simple, cheap, and easy-to-industrial synthetic route for 2'-OMe-modified phosphoramidite monomers is in urgent need. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present invention provides a method for preparing 2'-OMe modified pseudouridine and N1-methyl pseudouridine phosphoramidite monomers, aiming to solve the technical problems of long synthesis route, low yield and / or difficult separation of 2'-OMe modified pseudouridine / N1-methyl pseudouridine phosphoramidite monomers.

[0006] In order to achieve the above object, the present invention specifically adopts the following technical solutions.

[0007] A method for preparing a phosphoramidite monomer of 2'-OMe modified pseudouridine / N1-methyl pseudouridine comprises the following steps: S1, using trimethylsilyldiazomethane (TMSCHN 2 ) Methylate the 2'-OH of pseudouridine / N1-methylpseudouridine; S2, reacting the product obtained in step S1 with 4,4'-dimethoxytrityl chloride (DMTrCl) to obtain an intermediate with a DMTr protecting group at the 5' position; S3, the intermediate obtained in step S2 and bis(diisopropylamino)(2-cyanoethoxy)phosphine (PN 2 ) reaction to obtain the target product.

[0008] Preferably, in the above preparation method, the reaction conditions of step S1 include the following: The molar ratio of pseudouridine / N1-methylpseudouridine to trimethylsilyldiazomethane is 1:(1.5 ~2.6); The reaction is carried out in a first solvent under nitrogen protection and the action of a first catalyst, wherein the first catalyst is SnCl 2 The first solvent is any one of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and 1,4-dioxane.

[0009] Preferably, in the above preparation method, the product obtained in step S1 is not purified, and the crude product is directly used as a raw material to carry out the reaction described in step S2.

[0010] More preferably, in the above preparation method, the reaction conditions of step S2 include: The mass ratio of the crude product to 4,4'-bismethoxytrityl chloride is (1.2-1.8):(1.6-2.8); The reaction is carried out in the second solvent under nitrogen protection, wherein the second solvent is pyridine (Py).

[0011] In some embodiments of the present invention, step S2 specifically includes the following: S21, dissolving the crude product obtained in step S1 in a second solvent, cooling to 0° C. under nitrogen protection, adding 4,4'-bismethoxytrityl chloride, and naturally returning to room temperature for reaction; S22. Treat the reaction solution to purify the intermediate with the DMTr protecting group; wherein the purification operation is specifically as follows: add the reaction solution to ice-saturated sodium bicarbonate, add dichloromethane (DCM) and stir to separate the liquids, wash the obtained organic phase with ice-saturated sodium bicarbonate and ice-saturated brine in turn, and then perform column chromatography on the organic phase obtained by separation, and the eluent used for column chromatography is DCM and MeOH containing triethylamine.

[0012] Preferably, in the above preparation method, the reaction in step S3 further contains a second catalyst, specifically 4,5-dicyanoimidazole (DCI) or tetrazole.

[0013] More preferably, in the above preparation method, the reaction conditions of step S3 include: The molar ratio of the intermediate to bis(diisopropylamino)(2-cyanoethoxy)phosphine and the catalyst is 1:(1.1-1.5):(0.8-1.2); The reaction is carried out in a third solvent under nitrogen protection, and the third solvent is dichloromethane or acetonitrile.

[0014] In some embodiments of the present invention, step S3 includes the following operations: S31, adding the intermediate, bis(diisopropylamino)(2-cyanoethoxy)phosphine and the second catalyst into a third solvent, and reacting under nitrogen protection; S32. Add icy dichloromethane to the reaction solution, wash the separated liquid with icy saturated sodium bicarbonate, concentrate the organic phase to dryness under reduced pressure, and perform gradient elution using acetonitrile and water as mobile phases.

[0015] It is understandable that the phosphoramidite monomer of 2'-OMe modified pseudouridine / N1-methyl pseudouridine prepared according to the method of the present invention also falls within the protection scope of the present invention.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The method of the present invention does not need to protect the 3,5-position OH in advance, and directly performs methylation treatment on pseudouridine / N1-methylpseudouridine. More importantly, the methylation product does not need to be separated and purified, and can be directly used in the next step of reaction; moreover, after the subsequent DMTr protecting group modification and phosphoramidite monomer preparation steps, the total yield of the three steps can still reach more than 40%.

[0017] The present invention controls the methylation conditions to obtain a dimethylated product with relatively high selectivity, avoiding separation at this step, and obtaining a high-purity product by subsequent DMTr and column chromatography separation, thereby avoiding the long route method of removing the protecting group on the modified nucleoside and the difficulty of direct separation after methylation.

[0018] The method of the invention has the advantages of short route, mild reaction conditions, low cost, stable process, simple purification, easy removal of impurities, high purity of target product, etc., and is suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 The synthetic route of the phosphoramidite monomer of 2'-OMe modified pseudouridine in the embodiment; Figure 2 The synthetic route of the phosphoramidite monomer of 2'-OMe modified N1-methyl pseudouridine in the embodiment; Figure 3 is the HNMR spectrum of the phosphoramidite monomer of 2'-OMe modified pseudouridine prepared in the example; Figure 4 The ESI-MS spectrum of the phosphoramidite monomer of 2'-OMe modified pseudouridine prepared in the example Figure 5 This is a graph showing the HPLC detection results of the phosphoramidite monomer of 2'-OMe modified pseudouridine prepared in the example; Figure 6 is the HNMR spectrum of the phosphoramidite monomer of 2'-OMe modified N1-methylpseudouridine prepared in the example; Figure 7 is the ESI-MS spectrum of the phosphoramidite monomer of 2'-OMe modified N1-methyl pseudouridine prepared in the example; Figure 8 This is a graph showing the HPLC detection results of the phosphoramidite monomer of 2'-OMe modified N1-methylpseudouridine prepared in the example. DETAILED DESCRIPTION

[0021] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field of the present invention; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "including" and any variations thereof herein are intended to cover non-exclusive inclusions. The character " / " herein indicates that the objects associated with each other are in an "or" relationship. The terms "first", "second", etc. herein are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the number, specific order or primary and secondary relationship of the indicated technical features. The term "room temperature" herein specifically refers to 20℃~35℃.

[0023] Phosphoramidite monomers of 2'-OMe-modified pseudouridine / N1-methylpseudouridine play an important role in the development and synthesis of siRNA drugs. However, there are many problems with the existing synthetic route, especially the long synthetic route and low overall yield, which greatly limit its application. In order to solve the problem of synthesizing phosphoramidite monomers of 2'-OMe-modified pseudouridine / N1-methylpseudouridine, the present invention provides a new synthetic route, wherein the purpose of shortening the synthetic route of phosphoramidite monomers is achieved by optimizing the 2'-OMe modification mode of pseudouridine / N1-methylpseudouridine, and the overall yield reaches more than 40%.

[0024] Please refer to Figure 1 and Figure 2 The present invention provides a method for preparing a phosphoramidite monomer of 2'-OMe modified pseudouridine / N1-methyl pseudouridine, comprising the following steps: A method for preparing a phosphoramidite monomer of 2'-OMe modified pseudouridine / N1-methyl pseudouridine comprises the following steps: (1) SnCl 2 As a catalyst, trimethylsilyldiazomethane was used to methylate the 2'-OH of pseudouridine / N1-methylpseudouridine; (2) The methylated product is reacted with 4,4'-dimethoxytrityl chloride in pyridine to obtain intermediate 3a / 3b with a DMTr protecting group at the 5' position; (3) The obtained intermediate is reacted with bis(diisopropylamino)(2-cyanoethoxy)phosphine and 4,5-dicyanoimidazole in dichloromethane to obtain the target product 4a / 4b.

[0025] In the above preparation method, the reaction conditions are mild, that is, the three-step reaction can be carried out at room temperature; in addition, the raw materials in the reaction solution can be detected by TLC to determine the reaction progress.

[0026] Compared with the existing synthetic routes, the route of the scheme of the present invention is short, no purification is required in the first step, and purification is only required in the second and third steps. The purification operations of these two steps are simple, and impurities are easy to remove. The purity of the obtained target product 4a / 4b is as high as more than 99%, and the total yield of the synthesis (three steps) is also much higher than the yield obtained by the existing method (for example, the total yields of Examples 1 and 2 reached 41% and 43%, respectively).

[0027] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0028] Example 1 This example provides a method for synthesizing a phosphoramidite monomer of 2'-OMe-modified pseudouridine, which specifically comprises the following operations: (1) Methylation treatment

[0029] Add 200 g of pseudouridine and 3060 ml of DMF solvent into a three-necked flask, stir to dissolve, and add SnCl 2 •H 2 O 11g, nitrogen protection, stirred at room temperature for 1 hour, then added 1600ml trimethylsilyldiazomethane solution (1M hexane solution), stirred at room temperature for 16 hours; TLC developer (DCM / MeOH=5 / 1, v / v) detection, after the reaction of the raw materials was completed, the reaction solution was spin-dried with DMF at 55℃ using an oil pump to obtain the crude product 2a, which was directly used for the next step. The yield was 62.1%. ESI-MS m / z calcd for C 10 H 14 N 2 O 6 [M+H] + 259.1; found .259.1.

[0030] (2)DMTr protection.

[0031] Take 268g of the crude product obtained in step (1) and 2000ml of pyridine, add them to a three-necked flask, stir and dissolve, protect with nitrogen, cool to 0°C, add 415g of 4,4'-dimethoxytrityl chloride, naturally return to room temperature and stir overnight; detect with TLC developing agent (DCM / MeOH=10 / 1, v / v). After the reaction of the raw materials is completed, pour the reaction solution into 400ml of ice-cold saturated sodium bicarbonate, add 1000ml of dichloromethane (DCM) and stir to separate the liquids, stir and add 1000ml of ice-cold saturated sodium bicarbonate to the organic phase, wash 3 times, wash 3 times with 1000ml of ice-cold saturated brine, separate the liquids, add 800g of silica gel powder to the organic phase, mix with the sample, and concentrate under reduced pressure (30°C) to dryness. The sample was separated and purified by column (glass column 22cm*800cm loaded with 4kg silica gel powder), eluted with eluent, DCM / MeOH (0.1% triethylamine) = 50 / 1→40 / 1→30 / 1→25 / 1→20 / 1, two column volumes for each gradient, the product solution collected at the 20 / 1 product point was concentrated to dryness at 30℃ to obtain 234g of 3a. ESI-MS m / z calcd for C 31 H 32 N 2 O 8 [M+H] + 561.2; found.561.1. The yield is 82.6%.

[0032] (3) Preparation of phosphoramide monomers.

[0033] 234 g of 3a was added into a three-necked flask, and 2000 ml of dichloromethane, 4,5-dicyanoimidazole (1 molar equivalent) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.2 molar equivalent) were added respectively. The mixture was stirred at room temperature for 6 h under nitrogen protection and the TLC developing solvent was EA / MeOH = 25 / 1, v / v.

[0034] Add 500 ml of ice dichloromethane to dilute the reaction solution, then add 200 ml of ice saturated sodium bicarbonate to the reaction solution for washing three times, separate the liquids, concentrate the organic phase under reduced pressure, and concentrate to dryness at 30°C to obtain a white foam solid. Use high pressure reverse phase preparation (DAC), the mobile phase B is acetonitrile, the phase A is water (containing 0.08% triethylamine), the flow rate is 40 ml / min, and the column packing C 18 , and gradient elution was performed as shown in Table 1. The eluate of 50-65 min was collected and concentrated to dryness under reduced pressure to obtain 257 g of product 4a, i.e., the phosphoramidite monomer of 2'-OMe modified pseudouridine. The yield was 81.3%. The product HNMR was as follows Figure 3 ESI-MS m / z calcd for C 40 H 49 N 4 O 9P[MH] - 759.3; found .759.3, e.g. Figure 4 As shown. HPLC Figure 5 As shown, the purity of the target product 4a was calculated to be 99.1%.

[0035] Table 1

[0036] Example 2 This example provides a method for synthesizing a phosphoramidite monomer of 2'-OMe-modified N1-methylpseudouridine, which specifically comprises the following operations: (1) Methylation treatment

[0037] Add 210 g of N1-methyl pseudouridine and 3060 ml of DMF solvent into a three-necked flask, stir to dissolve, and add SnCl 2 •H 2 O 12g, nitrogen protection, stirred at room temperature for 1 hour, then added 1600ml trimethylsilyldiazomethane solution (1M hexane solution), stirred at room temperature for 16 hours; TLC developing agent (DCM / MeOH=6 / 1, v / v) detection, after the reaction of the raw materials is completed, the reaction liquid is spin-dried with DMF at 55℃ using an oil pump to obtain the crude product 2b, which is directly used for the next step. The yield is 64.1%. ESI-MS m / z calcd for C 11 H 16 N 2 O 6 [M+H] + 273.1; found .273.1.

[0038] (2)DMTr protection.

[0039] Take 282g of the crude product obtained in step (1) and 2000ml of pyridine, add them to a three-necked flask, stir and dissolve, protect with nitrogen, cool to 0°C, add 437g of 4,4'-dimethoxytrityl chloride, naturally return to room temperature and stir overnight; detect with TLC developing agent (DCM / MeOH=10 / 1, v / v). After the reaction of the raw materials is completed, pour the reaction solution into 400ml of ice-cold saturated sodium bicarbonate, add 1000ml of dichloromethane and stir to separate the liquids, stir and add 1000ml of ice-cold saturated sodium bicarbonate to the organic phase, wash 3 times, wash 3 times with 1000ml of ice-cold saturated brine, separate the liquids, add 800g of silica gel powder to the organic phase, mix with the sample, and concentrate under reduced pressure (30°C) to dryness. The sample was separated and purified by column (glass column 22cm*800cm loaded with 4kg silica gel powder), eluted with eluent, DCM / MeOH (0.1% triethylamine) = 60 / 1→50 / 1→40 / 1→30 / 1→25 / 1, two column volumes for each gradient, the product solution collected at the 25 / 1 product point was concentrated to dryness at 30℃ to obtain 246g of 3b. The yield was 82.6%. ESI-MS m / z calcd for C 32 H 34 N 2 O 8 [M+H] + 574.2;found.574.1.

[0040] (3) Preparation of phosphoramidite monomers.

[0041] Add 246 g of 3b into a three-necked flask, and add 2000 ml of dichloromethane, 4,5-dicyanoimidazole (1 molar equivalent) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.2 molar equivalent) respectively. Protect with nitrogen, stir at room temperature for 8 h, and use TLC developing solvent (EA / MeOH=25 / 1, v / v).

[0042] Add 500 ml of ice dichloromethane to the reaction solution to dilute the reaction solution, then add 200 ml of ice saturated sodium bicarbonate to the reaction solution and wash three times, separate the liquids, concentrate the organic phase under reduced pressure, and concentrate to dryness at 30°C to obtain a white foam solid. Gradient elution was performed in the same manner as in Example 1. The eluate of 50-65 min was collected and concentrated to dryness under reduced pressure to obtain 269 g of product 4b, i.e., the phosphoramidite monomer of 2'-OMe modified N1-methyl pseudouridine. The yield was 81.2%. The product HNMR is as follows Figure 6 ESI-MS m / zcalcd for C 41 H 51 N 4 O 9 P [MH] - 773.3; found .773.4, e.g. Figure 7 As shown. HPLC Figure 8 As shown, the purity of the target product 4b was calculated to be 99.4%.

[0043] In summary, the present invention uses pseudouridine / N1-methylpseudouridine as a raw material, and only needs three steps to prepare the phosphoramidite monomer of 2-OMe modified pseudouridine / N1-methylpseudouridine, which greatly shortens the synthesis route. Moreover, on the basis of not performing separation and purification in the first step, the total yield of the three steps can still reach more than 40%, and the purity of the target product reaches more than 99%. It can be seen that the present invention is suitable for large-scale industrial synthesis of relevant phosphoramidite monomers, and has important significance in the field of siRNA drug development and preparation.

[0044] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A method for preparing a phosphoramidite monomer of 2'-OMe modified pseudouridine / N1-methyl pseudouridine, characterized in that: The following steps are involved: S1, using trimethylsilyldiazomethane to methylate the 2'-OH of pseudouridine / N1-methylpseudouridine; S2, reacting the product obtained in step S1 with 4,4'-dimethoxytrityl chloride to obtain an intermediate with a DMTr protecting group at the 5' position; S3, reacting the intermediate obtained in step S2 with bis(diisopropylamino)(2-cyanoethoxy)phosphine to obtain the target product.

2. The preparation method according to claim 1, characterized in that: The reaction conditions of step S1 include: The molar ratio of pseudouridine / N1-methylpseudouridine to trimethylsilyldiazomethane is 1:(1.5-2.6), and / or; The reaction is carried out in a first solvent under nitrogen protection and the action of a first catalyst, wherein the first catalyst is SnCl2, and the first solvent is any one of DMF, DMSO, THF, and 1,4-dioxane.

3. The preparation method according to claim 1, characterized in that: The product obtained in step S1 is not purified, and the reaction in step S2 is directly carried out using the crude product as a raw material.

4. The preparation method according to claim 3, characterized in that: The reaction conditions of step S2 include: The mass ratio of the crude product to 4,4'-bismethoxytrityl chloride is (1.2-1.8):(1.6-2.8), and / or; The reaction is carried out in the second solvent under nitrogen protection, and the second solvent is pyridine.

5. The preparation method according to claim 1 or 4, characterized in that: The step S2 comprises the following: S21, dissolving the crude product obtained in step S1 in a second solvent, cooling to 0° C. under nitrogen protection, adding 4,4'-bismethoxytrityl chloride, and naturally returning to room temperature for reaction; S22, treating the reaction solution to purify the intermediate with a DMTr protecting group.

6. The preparation method according to claim 5, characterized in that: The step S22 includes the following operations: The reaction solution was added into icy saturated sodium bicarbonate, and dichloromethane was added followed by stirring and separation. The obtained organic phase was washed successively with icy saturated sodium bicarbonate and icy saturated brine, and then the organic phase obtained by separation was subjected to column chromatography, and the eluents used for column chromatography were dichloromethane and MeOH containing triethylamine.

7. The preparation method according to claim 1, characterized in that: The reaction in step S3 further contains a second catalyst, and the second catalyst is 4,5-dicyanoimidazole or tetrazole.

8. The preparation method according to claim 7, characterized in that: The reaction conditions of step S3 include: The molar ratio of the intermediate to bis(diisopropylamino)(2-cyanoethoxy)phosphine and the catalyst is 1:(1.1-1.5):(0.8-1.2), and / or; The reaction is carried out in a third solvent under nitrogen protection, and the third solvent is dichloromethane or acetonitrile.

9. The preparation method according to claim 8, characterized in that: The step S3 comprises the following: S31, adding the intermediate, bis(diisopropylamino)(2-cyanoethoxy)phosphine and the second catalyst into a third solvent, and reacting under nitrogen protection; S32. Add icy dichloromethane to the reaction solution, wash the separated liquid with icy saturated sodium bicarbonate, concentrate the organic phase to dryness under reduced pressure, and perform gradient elution using acetonitrile and water as mobile phases.

10. The phosphoramidite monomer of 2'-OMe modified pseudouridine / N1-methylpseudouridine prepared according to any one of claims 1 to 9.

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