A method for removing impurities from a mixture

Abstract

The present disclosure relates to a method for removing impurities from a mixture, wherein the mixture contains a main component and an impurity, wherein the impurity is a compound containing an imide group, and the method comprises contacting the mixture with an organic strong base under acid-base reaction conditions, wherein the main component does not react with the organic strong base. The method is simple to operate, significantly reduces the impurity content, and avoids hydrolysis of the phosphoramidite group.

Description

Technical Field

[0001] The present invention belongs to the field of chemistry, and particularly relates to a method for removing impurities from a mixture. Background Art

[0002] In the chemical industry and pharmaceutical industry, removing impurities from mixtures is a key step to ensure product quality and purity. Traditional impurity removal methods include purification methods such as washing, extraction, and column chromatography. Although these methods have many advantages, they do not show the expected effect when dealing with compounds containing specific functional groups such as diimide groups. For example, multiple purification steps are required, which not only increases production costs but also may lead to the loss of target products. In addition, the increase in steps requires the use of a large amount of organic solvents, which is contrary to the current trend of green chemistry and sustainable development.

[0003] CN 101580528A discloses a method for purifying cytidine (deoxy) and its derivatives, which comprises the following steps: first, loading an aqueous solution containing cytidine (deoxy) and its derivatives onto a basic resin; then, performing a water elution step to obtain high-purity cytidine (deoxy) and its derivatives, wherein the impurities in the aqueous solution include uridine (deoxy) and its derivatives. Although the method is easy to operate, it is not suitable for compounds whose main components are compounds containing groups that are easily hydrolyzed. Summary of the invention

[0004] The inventors have found in the research of oligonucleotide drugs that it is difficult to remove the impurities containing diimide groups in nucleoside monomers or intermediates of nucleoside monomers by conventional purification methods such as washing, extraction, column chromatography, etc. The impurities containing diimide groups are key impurities that affect the purity of oligonucleotide drugs and need to be removed in nucleoside monomers or intermediates of nucleoside monomers.

[0005] The present disclosure provides a method for removing impurities from a mixture, wherein the mixture contains a main component and impurities, wherein the impurities are compounds containing imide groups, and the method comprises contacting the mixture with a strong organic base under acid-base reaction conditions, wherein the main component does not react with the strong organic base.

[0006] In some embodiments, the method further comprises adding a non-polar solvent after contacting the mixture with the strong organic base.

[0007] In some embodiments, the method further comprises separating a main component from the mixture after the acid-base reaction.

[0008] Beneficial Effects

[0009] The method for removing impurities from a mixture provided by the present disclosure has the following benefits: (1) preventing the main component from being hydrolyzed during the purification process due to the presence of easily hydrolyzed groups, such as phosphoramidite groups; (2) simplifying the purification operation without affecting the product quality, making it suitable for large-scale expansion; (3) effectively removing impurities. Compared with the method of directly performing column chromatography and washing without performing an acid-base reaction, the content of the main component after purification by this method is further increased, and the impurity content is significantly reduced. After purification by the method provided by the present disclosure, the impurity content is reduced by 6-10 times.

[0010] Incorporated by Reference

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. DETAILED DESCRIPTION

[0012] The specific embodiments of the present disclosure are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0013] It will be understood by those skilled in the art that for any group comprising one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and / or inherently unstable.

[0014] The present disclosure provides a method for removing impurities from a mixture, wherein the mixture contains a main component and impurities, wherein the impurities are compounds containing diimide groups, and the method comprises contacting the mixture with a strong organic base under acid-base reaction conditions, wherein the main component does not react with the strong organic base.

[0015] Any suitable acid-base reaction in the art can be used for the acid-base reaction of the present disclosure. In some embodiments, the acid-base reaction described in the present disclosure is a strong base weak acid acid-base reaction. In some embodiments, the strong base is an organic strong base. In some embodiments, the acid in the acid-base reaction refers to the presence of one or more electron-withdrawing groups in the compound, resulting in the compound being acidic. In some embodiments, the acid-base reaction is the reaction of an organic strong base with a compound containing an imide group. The hydrogen atoms in the compound containing the imide group are weakly acidic and can react with the organic strong base to form a strong base weak acid reaction. In some embodiments, the impurity is one or more compounds containing an imide group. In some embodiments, the impurity is one or more compounds containing an imide group, and reacts with the organic strong base in contact, and the main component does not react with the organic strong base.

[0016] Those skilled in the art are aware of various acid-base reaction conditions, and any condition that allows the compound containing an imide group in the mixture mentioned in the present disclosure to undergo an acid-base reaction after contact with an organic strong base can be used in the present disclosure. In some embodiments, the solvent used in the acid-base reaction conditions is a polar organic solvent. The conditions for the acid-base reaction include the temperature, time, amount ratio between the reactants, and amount ratio of the reactants to the solvent when the reactants are in contact with each other during the acid-base reaction. In some embodiments, the conditions for the acid-base reaction include a contact temperature of 5-35°C, a contact time of 10-60min, a weight ratio of the mixture to the organic strong base of 1:0.02-0.5, and a weight-to-volume ratio of the mixture to the polar organic solvent of 1:0.5-8 g / ml. In some embodiments, the conditions of the acid-base reaction include a contact temperature of 15-30°C, a contact time of 20-40 min, a weight ratio of the mixture to the organic strong base of 1:0.05-0.20, and a weight volume ratio of the mixture to the polar organic solvent of 1:0.8-6 g / ml.

[0017] In some embodiments, the organic strong base is an organic strong base capable of carrying out the acid-base reaction of the present disclosure; or, the organic strong base is an alkoxide, and the alkoxide is selected from a metal salt of an alcohol; or, the alkoxide is an alkali metal alkoxide. Considering the material cost, in some embodiments, the alkali metal alkoxide is at least one of sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, potassium tert-butoxide, or sodium tert-butoxide; for example, at least one of potassium tert-butoxide, sodium tert-butoxide, or sodium methoxide.

[0018] In some embodiments, the polar organic solvent is selected from at least one of esters, halogenated hydrocarbons, nitriles and aromatic hydrocarbons. In some embodiments, the esters are fatty acid esters, or the fatty acid esters are C1-C6 saturated fatty acid esters, or the C1-C6 saturated fatty acid esters are the C1-C6 saturated fatty acid alkyl esters, or the C1-C6 saturated fatty acid alkyl esters are selected from at least one of ethyl acetate, isopropyl acetate or n-butyl acetate; the halogenated hydrocarbons are selected from at least one of chloroform, dichloromethane or 1,2-dichloroethane; the nitriles are selected from acetonitrile or propionitrile; the aromatic hydrocarbons are selected from substituted benzenes, or the substituted benzenes are selected from alkyl-substituted benzenes, or the alkyl-substituted benzenes are selected from C1-C6 alkyl-substituted benzenes or C1-C3 alkyl-substituted benzenes, or the C1-C3 alkyl-substituted benzenes are selected from at least one of toluene, xylene or ethylbenzene. In some embodiments, the polar organic solvent is selected from at least one of ethyl acetate, isopropyl acetate, n-butyl acetate or toluene.

[0019] In some embodiments, the impurity is a compound represented by general formula (I):

[0020] (I)

[0021] Among them, R 1 is hydrogen, a hydroxyl protecting group or a structure represented by formula (R1-1),

[0022] Formula (R1-1)

[0023] in, represents the group attachment site, B1 and B'1 are the same or different and are independently selected from substituted or unsubstituted -C1-C5 alkyl, and B2 is selected from one of -C1-C5 alkylene-CN;

[0024] R 2 is selected from hydrogen, -C1-C5 alkyl, halogen;

[0025] R 3 Selected from hydrogen, hydroxyl, -O-C1-C5 alkyl, halogen, -O-hydroxyl protecting group or the structure represented by formula (R3-1):

[0026] Formula (R3-1),

[0027] in, represents the group attachment site, B3 and B'3 are the same or different and are independently selected from substituted or unsubstituted -C1-C5 alkyl, and B4 is selected from one of -C1-C5 alkylene-CN;

[0028] X is hydrogen, halogen, -O-(C1-C5 alkyl), -O-(C1-C5 alkylene)-O-(C1-C5 alkyl), -O-(C1-C5 alkylene)-CN or -O-tert-butyldimethylsilyl.

[0029] In some embodiments, the hydroxy protecting group is selected from trityl, 4-methoxytrityl or 4,4'-dimethoxytrityl.

[0030] In some embodiments, B1 and B'1 are the same and are each independently selected from methyl, ethyl, propyl or isopropyl.

[0031] In some embodiments, B2 is selected from cyanoethyl (-CH 2 CH 2 CN).

[0032] In some embodiments, the structure represented by formula (R1-1-1) is the structure represented by formula (R1-1-1):

[0033] Formula (R1-1-1).

[0034] In some embodiments, R1 is selected from hydrogen, trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, or a structure represented by formula (R1-1-1).

[0035] In some embodiments, the -C1-C5 alkyl group is selected from methyl, ethyl, propyl, or isopropyl.

[0036] In some embodiments, the halogen is selected from fluorine, chlorine, bromine, or iodine.

[0037] In some embodiments, R2 is selected from hydrogen, methyl, ethyl, fluoro, or chloro.

[0038] In some embodiments, B3 and B'3 are the same and are each independently selected from methyl, ethyl, propyl or isopropyl.

[0039] In some embodiments, B4 is selected from cyanoethyl (-CH 2 CH 2 CN).

[0040] In some embodiments, the structure represented by formula (R3-1) is the structure represented by formula (R3-1-1):

[0041] Formula (R3-1-1).

[0042] In some embodiments, R3 is selected from hydrogen, hydroxyl, -O-trityl, -O-4-methoxytrityl, -O-4,4'-dimethoxytrityl or the structure represented by formula (R1-1-1).

[0043] In some embodiments, -O-(C1-C5 alkyl) is selected from -O-methyl, -O-ethyl, -O-propyl, or -O-isopropyl.

[0044] In some embodiments, -O-(C1-C5 alkylene) -O-(C1-C5 alkyl) is selected from -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl or -O-propylene-O-ethyl.

[0045] In some embodiments, -O-(C1-C5 alkylene)-CN is selected from -O-CH 2 CH 2 -CN or -O-CH 2 -CN.

[0046] In some embodiments, X is selected from fluorine, chlorine, bromine, -O-methyl, -O-ethyl, -O-propyl, -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl, or -O-propylene-O-ethyl.

[0047] In some embodiments, R 1 is hydrogen or a hydroxyl protecting group, R 2 is hydrogen or -C1-C5 alkyl, R 3 It is hydrogen, hydroxyl, -O-C1-C5 alkyl, -O-hydroxy protecting group or the structure represented by formula (3-1), and X is hydrogen, halogen or -O-(C1-C5 alkylene)-O-(C1-C5 alkyl).

[0048] In some embodiments, R 1 is trityl, 4-methoxytrityl or 4,4'-dimethoxytrityl; R 2 is hydrogen, methyl, ethyl, propyl or isopropyl, R 3 It is the structure shown in formula (3-1-1); X is fluorine, chlorine, -O-methyl, -O-ethyl, -O-propyl, -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl or -O-propylene-O-ethyl.

[0049] In some embodiments, the impurities of formula (I) are selected from: , and At least one of .

[0050] In some embodiments, the main component is a nucleoside monomer, and the nucleoside monomer is a nucleoside monomer of general formula (II):

[0051] Formula (II),

[0052] B is a non-imide nucleotide base, a non-imide nucleotide base analog or a protected non-imide nucleotide base. In some embodiments, the "non-imide nucleotide base" refers to a natural nucleotide base, which is adenine (A), guanine (G) and cytosine (C). In some embodiments, the "non-imide nucleotide base analog" refers to an analog of a natural nucleotide base, a chemical substance that has the function of replacing the nucleobase in a nucleotide. In some embodiments, the nucleotide base analog is selected from a nucleotide base with a substituent group, and the "substituent group" refers to a "substituted" group at any atom on the nucleotide base analog group, but does not contain a diimide group. In some embodiments, the non-imide nucleotide base analog is selected from a substituted purine, a substituted pyrimidine or a denitrogenated purine. In some embodiments, a substituted purine or a substituted pyrimidine refers to a hydrogen atom on the aromatic ring replaced by a halogen, an amino group, a methoxy group or a C 1 -C 3 Alkyl substitution. In some embodiments, substituted purine or substituted pyrimidine refers to that the oxygen on the aromatic ring is replaced by a sulfur atom. In some embodiments, the nucleotide base analog is selected from one of the non-natural nucleotide base residues of 2-aminoadenine, 7-deazaguanine, 2-amino-7-deazaadenine, 8-azaadenine, 5-azacytosine, 6-methyladenine 2-amino-6-chloropurine, 2-amino-6-iodopurine, 2-chloroadenine, 2-fluoroadenine, isoguanine, 7-methylguanine, 7-deazaadenine, 7-deazaadenine, 6-thioguanine, 5-fluorocytosine or 5-methylcytosine. The selection range and definition of R1, R3 and X are as described above for R1, R3 and X.

[0053] In some embodiments, the non-imide nucleotide base may be: or .

[0054] In some embodiments, the protected non-imide nucleotide base refers to a non-imide nucleotide base protected by a protecting group, wherein the protecting group is a protecting group capable of protecting an amino group, and the protected non-imide nucleotide base may be:

[0055] (C(ac)) (A(bz)) or (5-Me-C(bz)),

[0056] Among them, ac represents an acetyl group, bz represents a benzoyl group, and Me represents a methyl group.

[0057] In some embodiments, the nucleoside monomer of the general formula (II) is selected from: , and At least one of .

[0058] In some embodiments, the impurities are selected from , and At least one of;

[0059] The main component is selected from , and At least one of .

[0060] Those skilled in the art are aware of the preparation method of the mixture mentioned in the present disclosure, for example, it can be prepared with reference to WO2009 / 143369, non-patent document 1 (Andrew McPherson, Daniel Capaldi, Lijian Chen, An Improved Process for the Manufacture of 5'-O-(4,4'-Dimethoxytrityl)-N2-isobutyryl-2'-O-(2-methoxyethyl)guanosine, Organic Process Research&Development, 2020, 11, 2583-2590 and non-patent document 2 (Chaoyu Xie, Michael A. Staszak, John T, etc., Nucleosidic Phosphoramidite Synthesis via Phosphitylation: Activator Selection and Process Development, 2005, 9, 730-737).

[0061] In some embodiments, the method further comprises adding a non-polar solvent after the mixture is in contact with a strong organic base. In some embodiments, the method further comprises adding a non-polar solvent after the mixture is in contact with a strong organic base for an acid-base reaction. In some embodiments, the method further comprises adding a non-polar solvent after the mixture is in contact with a strong organic base for 10-60 minutes. In some embodiments, stirring is performed for 2-30 minutes, or 5-20 minutes after the non-polar solvent is added. In some embodiments, the non-polar solvent is selected from at least one of alkanes or cycloalkanes. In some embodiments, the alkane is at least one of n-pentane, isopentane, hexane or heptane; the cycloalkane is selected from cyclohexane and / or cyclopentane. In some embodiments, the non-polar solvent is selected from at least one of n-hexane, n-heptane, cyclohexane or cyclopentane.

[0062] In some embodiments, the method further comprises separating a main component and impurities from the mixture after the acid-base reaction.

[0063] In some embodiments, the method for separating the main component from the mixture after the acid-base reaction is chromatography, filtration or adsorption; preferably chromatography.

[0064] In some embodiments, a non-polar solvent is added after the acid-base reaction and before separation; or, a non-polar solvent is added after the mixture is contacted with the strong organic base for 10-60 min and before column chromatography.

[0065] Chromatography, filtration and adsorption are commonly used in the art. In some embodiments, the chromatography includes eluting the mixture after the acid-base reaction with an eluent. In some embodiments, the chromatography is eluted in a chromatography column. In some embodiments, the chromatography conditions may include: a chromatography temperature of 5-35°C, a weight ratio of the mixture to the adsorbent of 1:0.5-10, a chromatography pressure of 0.05MPa-0.8MPa, and a ratio of the adsorbent to the eluent of the chromatography of 1:2-6 (g / ml). In some embodiments, the chromatography temperature is 10-30°C, the weight ratio of the mixture to the adsorbent is 1:0.8-5, and the ratio of the adsorbent to the eluent of the chromatography is 1:2.5-5.5 (g / ml) at normal pressure. The chromatography column is filled with an adsorbent, and the adsorbent is silica gel and / or alumina. The eluent is an organic solvent capable of eluting the above mixture. In some embodiments, the eluent comprises a non-polar solvent. In some embodiments, in addition to the non-polar solvent, the eluent further comprises a polar solvent. In some embodiments, polar solvents and non-polar solvents are mutually soluble and are used to separate the main component and impurities. In some embodiments, the eluent comprising a polar solvent and a non-polar solvent has a difference in solubility for the anti-main component and impurities. In some embodiments, compared with the main component and impurities before the acid-base reaction, the main component and impurities are easier to separate after the acid-base reaction. Compared with the acid-base reaction and then column chromatography, the acid-base reaction is followed by column chromatography, and the content of the main component obtained is further improved, and the impurities are reduced by 6-10 times. In the present disclosure, the solvent used for the acid-base reaction and column chromatography is an organic solvent that does not contain water to prevent the compound containing the phosphoramidite group from undergoing a hydrolysis reaction. In some embodiments, the volume ratio of the polar solvent to the non-polar solvent is 1:5-10:1, or 1:2-8:1, or 1:1-5:1. In some embodiments, the polar solvent has the same selection range and definition as the polar organic solvent. In some embodiments, the non-polar solvent has the same selection range and definition as the non-polar solvent. In some embodiments, the eluent is selected from: ethyl acetate and n-hexane, toluene and n-hexane, or ethyl acetate and n-heptane.

[0066] Example

[0067] The present invention and its beneficial effects are described in detail below in conjunction with specific embodiments.

[0068] The sources of the reagents and instruments used in the following examples are shown in Table 1 below:

[0069] Table 1 Sources of reagents and instruments

[0070]

[0071] Example 1

[0072] 200g of DMT-2'-F-dC(ac) phosphoramidite monomer (prepared according to Preparation Example 10 of WO2009 / 143369, the content of the main component DMT-2'-F-dC(ac) phosphoramidite monomer was 99.2% by HPLC, and the content of the impurity DMT-2'-F-dU phosphoramidite monomer was 0.3%) was added with 150ml of ethyl acetate at room temperature, stirred to dissolve, and then 16g of potassium tert-butoxide was added at room temperature, stirred for 30 minutes, and then 100ml of n-hexane was added and stirred for 10 minutes.

[0073] At room temperature, 600 ml of a mixed solvent of ethyl acetate and n-hexane (the volume ratio of ethyl acetate to n-hexane is 3:2) was added to 200 g of silica gel, stirred evenly, poured into a chromatography column, and allowed to settle naturally for 2 hours. The solution after the above reaction was poured into a prepared chromatography column for column chromatography, the pressure of the chromatography column was normal pressure, and the chromatography column was eluted with 800 ml of the mixed solvent of ethyl acetate and n-hexane, the chromatography solution was collected, the solvent was evaporated under reduced pressure, and vacuum dried to obtain 184.6 g of a foamy solid (the DMT-2'-F-dC (ac) phosphoramidite monomer content was 99.45% and the DMT-2'-F-dU phosphoramidite monomer content was 0.03% by HPLC).

[0074] Example 2

[0075] 100g of DMT-2'-O-Me-A(bz) phosphoramidite monomer (prepared according to Preparation Example 10 of WO2009 / 143369, the content of the main component DMT-2'-O-Me-A(bz) phosphoramidite monomer was 99.5% by HPLC, and the content of the impurity DMT-2'-O-MeU phosphoramidite monomer was 0.3%) was added to 300ml of toluene at room temperature, stirred to dissolve, and then 10g of sodium tert-butoxide was added at room temperature, stirred for 30 minutes, and then 150ml of n-hexane was added and stirred for 10 minutes.

[0076] At room temperature, 600mL of a mixed solvent of toluene and normal hexane (the volume ratio of toluene and normal hexane is 2:1) was added to 200g of silica gel, stirred evenly, poured into a chromatography column, and allowed to settle naturally for 2 hours. The solution after the above reaction was poured into a prepared chromatography column for column chromatography, the pressure of the chromatography column was normal pressure, and the chromatography column was eluted with 500ml of the mixed solvent of the above toluene and normal hexane, the chromatography solution was collected, the solvent was evaporated under reduced pressure, and vacuum dried to obtain 91.6g of a foamy solid (the content of DMT-2'-O-Me-A (bz) phosphoramidite monomer was 99.74% by HPLC, and the content of DMT-2'-O-MeU phosphoramidite monomer was 0.05%).

[0077]

[0078] Example 3

[0079] Add 100g of DMT-2'-O-MOE-5-meC(bz) phosphoramidite monomer (refer to Andrew McPherson, Daniel Capaldi, Lijian Chen, An Improved Process for the Manufacture of 5'-O-(4,4'-Dimethoxytrityl)-N2-isobutyryl-2'-O-(2-methoxyethyl) guanosine, Organic Process Research&Development, 2020, 11, 2583-2590; Chaoyu Xie, MichaelA. Staszak, John T, etc, Nucleosidic Phosphoramidite Synthesis via Phosphitylation: Activator Selection and Process Development, 2005, 9, 730-737), the main component DMT-2'-O-MOE-5-meC (bz) phosphoramidite monomer content was 99.4% by HPLC, and the impurity DMT-2'-O-MOE-T phosphoramidite monomer content was 0.2%), 100 ml of ethyl acetate was added at room temperature, stirred to dissolve, 12 g of sodium methoxide was added at room temperature, stirred for 30 minutes, and then 50 ml of n-heptane was added and stirred for 10 minutes.

[0080] At room temperature, 900ml of a mixed solvent of ethyl acetate and normal heptane (the volume ratio of ethyl acetate to normal heptane is 2:1) is added to 300g of silica gel, stirred evenly, poured into a chromatography column, and allowed to settle naturally for more than 2 hours. The above-mentioned reacted solution is poured into a prepared chromatography column for column chromatography, the pressure of the chromatography column is normal pressure, the chromatography column is eluted with a mixed solvent of 400ml of the above-mentioned ethyl acetate and normal heptane, the chromatography solution is collected, the solvent is evaporated under reduced pressure, and vacuum dried to obtain 90.3g of a foamy solid. (The monomer content of DMT-2'-O-MOE-5-meC (bz) phosphoramidite is 99.57% by HPLC, and the monomer content of DMT-2'-O-MOE-T phosphoramidite is 0.02%).

[0081]

[0082] Comparative Example 1

[0083] 50 g of DMT-2'-F-dC(ac) phosphoramidite monomer (prepared according to Preparation Example 10 of WO2009 / 143369, the content of the main component DMT-2'-F-dC(ac) phosphoramidite monomer was 99.2% by HPLC, and the content of the impurity DMT-2'-F-dU phosphoramidite monomer was 0.3%) was added to 30 ml of ethyl acetate at room temperature, stirred to dissolve, and then 20 ml of n-hexane was added and stirred.

[0084] At room temperature, 150 ml of a mixed solvent of ethyl acetate and n-hexane (the volume ratio of ethyl acetate to n-hexane is 3:2) was added to 50 g of silica gel, stirred evenly, poured into a chromatography column, and naturally settled for 2 hours. The solution was poured into a prepared chromatography column for column chromatography, the pressure of the chromatography column was normal pressure, and the chromatography column was eluted with 200 ml of the mixed solvent of ethyl acetate and n-hexane, the chromatography solution was collected, the solvent was evaporated under reduced pressure, and vacuum dried to obtain 45.1 g of a foamy solid (detected by HPLC, the DMT-2'-F-dC (ac) phosphoramidite monomer content was 99.2%, and the DMT-2'-F-dU phosphoramidite monomer content was 0.3%).

[0085] Comparative Example 2

[0086] 30g of DMT-2'-F-dC(ac) phosphoramidite monomer (prepared according to Preparation Example 10 of WO2009 / 143369, the content of the main component DMT-2'-F-dC(ac) phosphoramidite monomer was 99.2% by HPLC, and the content of the impurity DMT-2'-F-dU phosphoramidite monomer was 0.3%) was added to 200ml of toluene at room temperature, stirred and dissolved, and then washed with 100ml of DMF: water (the volume ratio of DMF: water was 1:1), washed 3 times in total, and the upper organic phase was detected. (The content of DMT-2'-F-dC(ac) phosphoramidite monomer was 99.2% by HPLC, and the content of DMT-2'-F-dU phosphoramidite monomer was 0.3%).

[0087] It can be seen from the above examples and comparative examples that the washing method and the column chromatography method without acid-base reaction did not change the content of the main components and impurities before and after purification (Comparative Examples 1-2). After the method for removing impurities disclosed in the present invention, the content of the main components was further increased, for example, the content of the main components of Examples 1-3 was increased from 99.2%, 99.5%, and 99.4% to 99.45%, 99.74%, and 99.57%, respectively; the impurity content was reduced from 0.3%, 0.3%, and 0.2% to 0.03%, 0.05%, and 0.02%, and the impurity content was reduced by 6-10 times.

Claims

1. A method for removing impurities from a mixture, the mixture containing a main component and impurities, the method comprising contacting the mixture with an organic strong base under acid-base reaction conditions, wherein: The main component does not react with the organic strong base; the organic strong base is an alkali metal alkoxide; The impurity is a compound represented by the general formula (I): (I)， The main component is a nucleoside monomer of general formula (II): Formula (II), R1 is trityl, 4-methoxytrityl or 4,4'-dimethoxytrityl; R2 is selected from hydrogen, -(C1-C5 alkyl), halogen; R3 is a structure shown in formula (R3-1): Formula (R3-1), in, represents the group attachment site, B3 and B'3 are the same or different and are independently selected from -(C1-C5 alkyl), and B4 is selected from one of -(C1-C5 alkylene)-CN; X is hydrogen, halogen, -O-(C1-C5 alkyl), -O-(C1-C5 alkylene)-O-(C1-C5 alkyl) or -O-(C1-C5 alkylene)-CN; B is: , or ; The solvent used in the acid-base reaction conditions is a polar organic solvent that does not contain water; The method further comprises separating a main component from the mixture after the acid-base reaction; The method for separating the main component from the mixture after the acid-base reaction is chromatography, wherein the chromatography is performed in a chromatography column, and the adsorbent filled in the chromatography column is silica gel and / or alumina. The eluent used for the chromatography comprises a polar organic solvent and a non-polar solvent, and the volume ratio of the polar organic solvent to the non-polar solvent is 1:5-10:

1.

2. The method according to claim 1, wherein: The conditions of the acid-base reaction include a contact temperature of 5-35° C., a contact time of 10-60 min, a weight ratio of the mixture to the organic strong base of 1: 0.02-0.5, and a weight-volume ratio of the mixture to the polar organic solvent of 1: 0.5-8 g / ml.

3. The method according to claim 2, wherein: The conditions of the acid-base reaction include a contact temperature of 15-30°C, a contact time of 20-40 min, a weight ratio of the mixture to the organic strong base of 1: 0.05-0.20, and a weight-volume ratio of the mixture to the polar organic solvent of 1: 0.8-6 g / ml.

4. The method according to claim 1, wherein: The organic strong base is at least one of sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, potassium tert-butoxide or sodium tert-butoxide.

5. The method according to claim 1, wherein: The polar organic solvent is at least one selected from esters, halogenated hydrocarbons, nitriles and alkyl-substituted benzenes.

6. The method according to claim 1, wherein: The polar organic solvent is selected from at least one of fatty acid esters, chloroform, dichloromethane, 1,2-dichloroethane, acetonitrile, propionitrile or C1-C6 alkyl-substituted benzene.

7. The method according to claim 1, wherein: The polar organic solvent is C1-C6 saturated fatty acid ester and / or C1-C3 alkyl substituted benzene.

8. The method according to claim 1, wherein: The polar organic solvent is at least one of C1-C6 saturated fatty acid alkyl ester, toluene, xylene or ethylbenzene.

9. The method according to claim 1, wherein: The polar organic solvent is at least one of ethyl acetate, isopropyl acetate or n-butyl acetate.

10. The method according to claim 1, wherein: R2 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine or iodine; Alternatively, B3 is the same as B'3 and is selected from methyl, ethyl, n-propyl or isopropyl; B4 is selected from cyanoethyl (-CH2CH2CN); Alternatively, X is selected from fluorine, chlorine, -O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-n-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-n-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl, -O-propylene-O-ethyl, -O-CH2CH2-CN or -O-CH2-CN.

11. The method according to claim 1, wherein: R2 is selected from hydrogen or -(C1-C5 alkyl), and X is selected from hydrogen, halogen or -O-(C1-C5 alkylene)-O-(C1-C5 alkyl).

12. The method according to claim 11, wherein: X is selected from -O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-n-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-n-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl or -O-propylene-O-ethyl.

13. The method according to claim 11, wherein the impurity of general formula (I) is selected from: , and At least one of .

14. The method according to claim 1, wherein: R2 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl; X is selected from fluorine, chlorine, -O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -O-methylene-O-methyl, -O-methylene-O-ethyl, -O-methylene-O-n-propyl, -O-methylene-O-isopropyl, -O-ethylene-O-methyl, -O-ethylene-O-ethyl, -O-ethylene-O-n-propyl, -O-ethylene-O-isopropyl, -O-propylene-methyl or -O-propylene-O-ethyl.

15. The method according to claim 14, wherein the nucleoside monomer of general formula (II) is selected from: , and At least one of .

16. The method according to claim 15, wherein: The impurities are selected from: , and At least one of; The main component is selected from , and At least one of .

17. The method according to claim 1, wherein: The method further comprises adding a non-polar solvent after contacting the mixture with the strong organic base.

18. The method according to claim 17, wherein: The non-polar solvent is selected from alkanes and / or cycloalkanes.

19. The method according to claim 17, wherein: The non-polar solvent is selected from at least one of n-pentane, isopentane, hexane, heptane, cyclohexane and cyclopentane.

20. The method according to claim 1, wherein: The temperature of the chromatography is 5-35°C, the weight ratio of the mixture after the acid-base reaction to the adsorbent is 1:0.5-10, the pressure of the chromatography is 0.05MPa-0.8MPa, and the dosage ratio of the adsorbent to the eluent in the chromatography is 1:2-6 g / ml.

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