A method for preparing 2' modified basic nucleoside

Abstract

The invention discloses a method for preparing a 2'-modified alkaline nucleoside, the preparation method comprising: using a nucleoside initiator as a raw material, reacting with a reagent containing a protecting group, and protecting the 2'-hydroxyl group of the nucleoside initiator; after the reaction is complete, performing post-treatment: quenching the reaction solution, and adjusting the system pH to 7-8 after quenching; purifying the mixture obtained by the post-treatment, and the purification is performed using a cation exchange resin. The method of the invention has the advantages of simple operation, good separation and purification effect, low cost, high safety factor and high yield, and can better solve the separation and purification problem of nucleosides in which the hydroxyl group is protected by various protecting groups.

Description

Technical Field

[0001] The invention belongs to the field of nucleoside compound synthesis, and in particular relates to a method for preparing a 2'-modified basic nucleoside. Background Art

[0002] Non-natural nucleosides and nucleotides can be called nucleoside and nucleotide analogs. From the perspective of chemical structure, they have different degrees of similarity with natural nucleosides and nucleotides, so they can be confused with the real ones in the body, thereby interfering with or directly acting on the metabolic process of nucleic acids and blocking the biosynthesis of proteins and nucleic acids. Therefore, these compounds have a very important position in antiviral and anti-tumor chemotherapy drugs and occupy a large proportion. In particular, the research and development of drugs in this area has been very fast in the past decade. It can be said that as long as the structure of natural nucleosides is modified or changed in various aspects, new anti-cancer and anti-viral drugs may be obtained.

[0003] Natural nucleosides include uridine, adenine, guanosine, and cytosine, which are the basic units of RNA / DNA. From the perspective of chemical properties, uridine has a weak acidity, while adenine, guanosine, and cytosine have a weak alkalinity. Currently, these types of nucleoside compounds are fermented and are widely used. Some non-natural nucleosides are also developed on this basis, such as 2'-fluoro-modified nucleosides, 2'-methoxy-modified nucleosides, 2'-methoxyethyl-modified nucleosides, and so on. Chemically modified nucleosides are currently widely used in ASO, siRNA, saRNA, and sgRNA, but there is a common problem, that is, due to the high polarity of the nucleoside compounds themselves, 2'-fluoro-modified nucleosides, 2'-methoxy-modified nucleosides, and 2'-methoxyethyl-modified nucleosides have certain difficulties in separation and purification. The current separation methods include not separating the crude product at present and allowing it to proceed to the next step, which often leads to disadvantages such as difficulty in separating the intermediates and low yields, or using reverse phase preparative separation, which leads to disadvantages such as high costs.

[0004] In addition, the modified basic nucleosides sold on the market, such as 2'-fluoro modified cytidine, adenosine, guanosine, 2'-methoxy modified cytidine, adenosine, guanosine nucleosides, 2'-methoxyethyl modified cytidine, adenosine, guanosine nucleosides, currently have problems such as insufficient purity or too high key single impurities, which makes it difficult to purify intermediates or finished products. The main reason is that these nucleosides are obtained by chemical methods and have disadvantages such as high polarity and poor solubility. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing 2'-modified alkaline nucleosides, which solves the problem that the existing method leads to difficulty in separating intermediates and low yield. The method of the present invention has the advantages of simple operation, good separation and purification effect, low cost, high safety factor and high yield, and can better solve the problem of separation and purification of nucleosides in which hydroxyl groups are protected by various protecting groups.

[0006] In order to achieve the above object, the present invention provides a method for preparing a 2'-modified basic nucleoside, the preparation method comprising:

[0007]

[0008] In formula I, OR is selected from OMe, -OCH2CH2OMe or F; Base is selected from cytosine base, adenine base or guanine base; a nucleoside initiator is used as a raw material, reacted with a reagent containing a protecting group, and the 2'-hydroxyl group of the nucleoside initiator is protected; after the reaction is complete, post-treatment is performed: the reaction solution is quenched, and the pH of the system is adjusted to 7-8 after quenching; the mixture obtained by the post-treatment is purified, and the purification is performed using a cation exchange resin.

[0009] Traditional purification processes often use silica gel purification or reverse phase purification, in which organic solvents such as dichloromethane, methanol, and acetonitrile are used. The present invention uses cation exchange resin for purification and uses low-toxic deionized water as a solvent. The invention is non-toxic and harmless, has a short construction period, does not require additional treatment of halogenated solvents, reduces material costs, and is suitable for large-scale industrial production.

[0010] In addition, in the traditional purification process, due to the high polarity and poor solubility of the raw materials, the yield is generally low, usually only 20-30%, and it often takes two or even three purifications to get a qualified product. The yield of the method of the present invention can reach 40-50 mol%, which reduces the separation difficulty of the purification step and improves the yield.

[0011] Preferably, it is characterized in that the cation exchange resin is American Dow resin UP6150.

[0012] Preferably, in the purification, water is first used as the mobile phase, and then any one or more of ammonium acetate aqueous solution, ammonium bicarbonate aqueous solution, triethylamine-carbonic acid aqueous solution, triethylamine aqueous solution or ammonia aqueous solution with a concentration gradient is used as the mobile phase.

[0013] Preferably, the amount of the cation exchange resin used is 20 to 30 times the mass of the nucleosides in the mixture obtained by post-treatment.

[0014] Preferably, the quenching agent used in the quenching is a saturated ammonium chloride solution.

[0015] Preferably, the preparation method comprises:

[0016]

[0017] In formula I, OR is selected from OMe, -OCH2CH2OMe; Base is selected from cytosine base, adenine base or guanine base; the nucleoside starting material is used as a raw material, the temperature is lowered to 0°C in a polar organic solvent, an ionic hydride is added, and after reacting at room temperature, a reagent containing a protective group is added, and the reaction is carried out at room temperature for 0.5 to 4 hours.

[0018] More preferably, the reagent containing a protecting group is selected from methoxyethyl bromide, methyl p-toluenesulfonate or dimethyl sulfate.

[0019] More preferably, the polar organic solvent is selected from N,N-dimethylformamide.

[0020] More preferably, the ionic hydride is selected from sodium hydride.

[0021] Preferably, the preparation method further comprises:

[0022]

[0023] The nucleoside starting material is placed in a polar organic solvent, a reagent containing a protective group is added, and the reaction is heated; wherein the reagent containing a protective group is selected from tetrabutylammonium fluoride or pyridine hydrogen fluoride.

[0024] Preferably, the 2'-modified basic nucleoside is selected from any one of the following:

[0025] .

[0026] More preferably, if the 2'-modified basic nucleoside is as shown in Formula II, the mobile phase is water-5% ammonia water-10% ammonia water-15% ammonia water-25% ammonia water; if the 2'-modified basic nucleoside is as shown in Formula III, the mobile phase is water-3% ammonium bicarbonate aqueous solution-7% ammonium bicarbonate aqueous solution-10% ammonium bicarbonate aqueous solution-15% ammonium bicarbonate aqueous solution-20% ammonium bicarbonate aqueous solution-30% ammonium bicarbonate aqueous solution; if the 2'-modified basic nucleoside is as shown in Formula IV, the mobile phase is water-1% triethylamine-carbonic acid aqueous solution-5% triethylamine-carbonic acid aqueous solution-8% triethylamine-carbonic acid aqueous solution-11% triethylamine-carbonic acid aqueous solution-15% triethylamine-carbonic acid aqueous solution.

[0027] The method for preparing 2'-modified basic nucleosides of the present invention solves the problems of difficult intermediate separation and low yield in the prior art, and has the following advantages:

[0028] (1) The method of the present invention adopts cationic resin for adsorption separation for the first time, which greatly simplifies the previous process and has the advantages of simple operation, good separation and purification effect, low cost, high safety factor and high yield. It solves the problems of great separation difficulty, low yield, high cost and high toxicity in the prior art, and can better solve the problem of separation and purification of nucleosides whose hydroxyl groups are protected by various protecting groups;

[0029] (2) The method of the present invention can be applied to the preparation process of various nucleosides, and can effectively solve the problems of difficulty in subsequent purification and separation due to the polarity and solubility of modified nucleosides, and low yield of target products. The method of the present invention has wide applicability and can be directly transferred to the synthesis of similar alkaline compounds;

[0030] (3) According to the method of the present invention, the recovery rate of the product in the purification step is as high as 60-70%, and the two-step yield is 40-50 mol%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a purity chart of 2'-methoxyethyl cytidine prepared in Example 1 of the present invention.

[0032] Figure 2 This is a purity chart of 2'-methoxyadenosine prepared in Example 2 of the present invention.

[0033] Figure 3 This is a purity chart of 2'-F cytidine prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] It should be noted that if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased from the market.

[0036] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be deemed to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0037] The features mentioned in the present invention can be combined arbitrarily, as long as there is no contradiction in the combination of these features, all possible combinations should be considered as the scope of this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0038] Example 1

[0039] A method for preparing 2'-methoxyethyl cytidine, the structural formula of the 2'-methoxyethyl cytidine is as follows:

[0040]

[0041] The preparation of 2'-methoxyethyl cytidine comprises the following steps:

[0042] (1) Synthesis of modified nucleosides

[0043]

[0044] Dissolve cytosine nucleoside (20.0 g, 1.0 eq.) in N, N-dimethylformamide (140 mL, 7V), cool to 0°C, add sodium hydride (1.5 eq.) in portions, react at 25°C for 2 h, then add methoxyethyl bromide (1.2 eq.), and react at 25°C for 4 hours.

[0045] After the reaction was completed as monitored by HPLC, the reaction solution was quenched by adding 1V saturated ammonium chloride solution. After quenching, the pH of the system was adjusted to 7-8 with 1N hydrochloric acid before the next purification step.

[0046] (2) Purification of modified nucleosides

[0047] The aqueous solution was directly purified by cation exchange resin (U.S. Dow resin UP6150). The amount of cation exchange resin used as the stationary phase was 30 times the mass of nucleosides in the mixture obtained after post-treatment. The mobile phase was water-5% ammonia water-10% ammonia water-15% ammonia water-25% ammonia water. Pay attention to collecting the infusion liquid. The purified product was concentrated under reduced pressure to obtain 11.2 g of 2'-methoxyethyl cytidine, with a yield of 45.21 mol% and a purity of 99.3%. See Figure 1 .

[0048] The NMR characterization results of 2'-methoxyethyl cytidine are as follows:

[0049] 1H NMR (400 MHz, DMSO-d6 ) δ 8.95-8.92 (d, J=10.3 Hz, 1H), 6.67 (s,2H), 6.21-6.18 (d, J=10.3 Hz, 1H), 6.05-6.03 (d, J = 9.3 Hz, 1H), 4.71-4.65(m, 1H), 4.51 (s, 1H), 4.40-4.35 (m, J=4.5Hz, 1H), 4.27-4.23(m, 1H), 3.94 (s,1H), 3.58-3.55 (m, 2H), 3.54-3.51 (t, J=6.0 Hz, 4H), 3.40 (s,3H).

[0050] Example 2

[0051] A method for preparing 2'-methoxyadenosine, the structural formula of the 2'-methoxyadenosine is as follows:

[0052]

[0053] The preparation method of 2'-methoxyadenosine comprises the following steps:

[0054] (1) Synthesis of modified nucleosides

[0055]

[0056] Adenine nucleoside (20.0 g, 1.0 eq.) was dissolved in N,N-dimethylformamide (140 mL, 7 V), cooled to 0°C, sodium hydride (1.5 eq.) was added in portions, reacted at 25°C for 2 h, then methyl p-toluenesulfonate TsOMe (1.5 eq.) was added, and reacted at 25°C for 4 h.

[0057] After the reaction was complete as monitored by HPLC, the reaction solution was quenched by adding 1V saturated ammonium chloride solution. After quenching, the pH of the system was adjusted to 7-8 with 1N hydrochloric acid before the next purification step.

[0058] (2) Purification of modified nucleosides

[0059] The above aqueous solution was directly purified by cation exchange resin (U.S. Dow resin UP6150), the amount of cation exchange resin used as the stationary phase was 30 times the mass of nucleosides in the mixture, and the mobile phase was water-3% ammonium bicarbonate aqueous solution-7% ammonium bicarbonate aqueous solution-10% ammonium bicarbonate aqueous solution-15% ammonium bicarbonate aqueous solution-20% ammonium bicarbonate aqueous solution-30% ammonium bicarbonate aqueous solution, and attention was paid to collecting the infusion liquid. The purified product was concentrated under reduced pressure to obtain 11.5 g of 2'-methoxyadenosine, with a yield of 54.63 mol% and a purity of 98.3%, see Figure 2 .

[0060] The NMR characterization results of 2'-methoxyadenosine are:

[0061] 1 H NMR (400 MHz, DMSO-d6 ) δ 8.58(s, 1H), 8.35(s, 1H), 7.09(s, 2H), 6.36-6.34(d, J = 6.9 Hz, 1H), 4.71-4.66(m, 1H), 4.51(s, 1H), 4.46-4.44(m,1H), 4.40-3.96(m, 1H), 3.94(s, 1H), 3.57-3.55(m, 2H), 3.41(s, 3H).

[0062] Example 3

[0063] A method for preparing 2'-F cytidine, wherein the structural formula of the 2'-F cytidine is as shown below:

[0064]

[0065] The preparation method of 2'-F cytidine comprises the following steps:

[0066] (1) Synthesis of modified nucleosides

[0067]

[0068] Dissolve cyclocytosine hydrochloride (20.0 g, 1.0 eq.) in N,N-dimethylformamide (140 mL, 7 V), add tetrabutylammonium fluoride (10 eq.) in portions, and react at 110°C for 4 hours. After the reaction is complete, cool to room temperature to quench and proceed to the next step of purification.

[0069] (2) Purification of modified nucleosides

[0070] The above solution was directly purified by cation exchange resin (U.S. Dow Resin UP6150), the amount of cation exchange resin used as the stationary phase was 30 times the mass of nucleosides in the mixture, and the mobile phase was water-1% triethylamine-carbonic acid aqueous solution-5% triethylamine-carbonic acid aqueous solution-8% triethylamine-carbonic acid aqueous solution-11% triethylamine-carbonic acid aqueous solution-15% triethylamine-carbonic acid aqueous solution, and attention was paid to collecting the infusion liquid. After purification, the product was concentrated under reduced pressure to obtain 10.5 g of 2'-F cytidine, with a yield of 56 mol% and a purity of 99.7%, see Figure 3 .

[0071] The results of NMR characterization are:

[0072] 1 H NMR (400 MHz, DMSO-d6 ) δ 8.95-8.92 (d, J=10.3 Hz, 1H), 6.67 (s,2H), 6.55-6.52(m, 1H), 6.21-6.18 (d, J=10.3 Hz, 1H), 4.55-4.40(m, 2H), 4.37(s, 1H), 4.30-4.28(m, 1H), 3.94(s, 1H), 3.57-3.53(m, 2H).

[0073] Comparative Example 1

[0074] The method is basically the same as Example 1, except that other conditions remain unchanged and only the purification conditions are changed to prepare 2'-methoxyethyl cytidine, and 2'-methoxyethyl cytidine is purified using a reverse phase C18 chromatographic column.

[0075] 2'-methoxyethylcytidine was purified by reverse phase C18 column, and the amount of stationary phase C18 was 20 times the total mass of the mixture; mobile phase: 10 mM ammonium acetate (pH=7.0) was used for aqueous phase, and acetonitrile was used for organic phase, with a gradient of 20% acetonitrile-30% acetonitrile-50% acetonitrile-20% acetonitrile. The purification process was monitored by LC-MS, and attention was paid to collecting the infusion liquid. After purification, the product was concentrated under reduced pressure to obtain 8.2 g of 2'-methoxyethylcytidine, with a yield of 32.10 mol % and a purity of 90.5%.

[0076] Comparative Example 2

[0077] The method is basically the same as Example 1, except that other conditions remain unchanged and only the purification conditions are changed to prepare 2'-methoxyethyl cytidine, and 2'-methoxyethyl cytidine is purified by silica gel flash chromatography.

[0078] 2'-methoxyethylcytidine was purified by silica gel flash chromatography, and the amount of silica gel used as the stationary phase was 20 times the total mass of the mixture; the mobile phase was dichloromethane and 0.05% ammonia methanol solution, and the gradient was: 100% dichloromethane-70% dichloromethane-50% dichloromethane-30% dichloromethane. The purification process was monitored by thin layer chromatography (TLC), and the infusion solution was collected. After purification, the product was concentrated under reduced pressure to obtain 6.3 g of 2'-methoxyethylcytidine, with a yield of 25.4 mol% and a purity of 91.2%.

[0079] In summary, the preparation method of the present invention has the advantages of simple operation, good separation and purification effect, low cost, high safety factor and high yield, etc., which solves the problems of great separation difficulty, low yield, high cost and high toxicity in the prior art, and can better improve or even solve the separation and purification problem of nucleosides in which hydroxyl groups are protected by various protecting groups.

[0080] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for preparing a 2'-modified basic nucleoside, characterized in that: The preparation method comprises: In formula I, OR is selected from OMe, -OCH2CH2OMe; Base is selected from cytosine base, adenine base or guanine base; Using a nucleoside initiator as a raw material, reacting with a reagent containing a protecting group to protect the 2'-hydroxyl group of the nucleoside initiator; After the reaction is complete, post-treatment is performed: the reaction solution is quenched, and the pH of the system is adjusted to 7-8 after quenching; Purifying the mixture obtained by the post-treatment, wherein the purification is performed using a cation exchange resin; The cation exchange resin is American Dow resin UP6150; in the purification, water is first used as the mobile phase, and then the concentration gradient of ammonium bicarbonate aqueous solution or ammonia water is used as the mobile phase.

2. The preparation method according to claim 1, characterized in that: The amount of the cation exchange resin used is 20 to 30 times the mass of the nucleosides in the mixture obtained by post-treatment.

3. The preparation method according to claim 1, characterized in that: The quenching agent used in the quenching is a saturated ammonium chloride solution.

4. The preparation method according to claim 1, characterized in that: The preparation method comprises: In formula I, OR is selected from OMe, -OCH2CH2OMe; Base is selected from cytosine base, adenine base or guanine base; Using nucleoside as starting material, in a polar organic solvent, cool to 0°C, add ionic hydride, react at room temperature, then add reagent containing protective group, and react at room temperature for 0.5-4 h.

5. The preparation method according to claim 4, characterized in that: The reagent containing a protecting group is selected from methoxyethyl bromide, methyl p-toluenesulfonate or dimethyl sulfate.

6. The preparation method according to claim 4, characterized in that: The polar organic solvent is selected from N,N-dimethylformamide.

7. The preparation method according to claim 4, characterized in that: The ionic hydride is selected from sodium hydride.

8. A method for preparing a 2'-modified basic nucleoside, characterized in that: The preparation method comprises: The nucleoside starting material is placed in a polar organic solvent, a reagent containing a protective group is added, and the reaction is heated; a cation exchange resin is used for purification, and the cation exchange resin is selected from American Dow resin UP6150; the purification first uses water as a mobile phase, and then uses a concentration gradient of triethylamine-carbonic acid aqueous solution as a mobile phase, wherein the reagent containing a protective group is selected from tetrabutylammonium fluoride or hydrogen fluoride pyridine.

Images