Method for purifying phosphoramidite precursor
By using the combination of inorganic salt and organic base as additive B in the solvent, the salt-forming purification method of the phosphoramidite precursor is solved, and the purification effect of high purity and low cost is achieved.
Patent Information
- Application Number
- CN202311109365.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-30
- Publication Date
- 2025-02-28
AI Technical Summary
The existing purification methods of phosphoramidite precursors are costly, have poor repeatability, and are difficult to effectively control purity.
The compound as shown in formula II is purified by combining an inorganic salt and an organic base as additive B by performing a salt formation reaction in a solvent. The method includes carrying out a salt-forming reaction under stirring conditions until a solid precipitates, and performing post-treatment steps such as filtration, wetting and drying.
Purification of phosphoramidite precursors with low cost, high repeatability, non-stick walls and good yields was achieved, with a purity of 99.5%.
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Figure CN119529010A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for purifying a phosphoramidite precursor. Background Art
[0002] Oligonucleotide drugs have gradually become a hot spot in the research and development of new drugs. Phosphoramidite compounds are synthetic monomers of oligonucleotide molecules. High-purity phosphoramidite monomer raw materials are the guarantee for the preparation of high-quality oligonucleotide drugs. The preparation process of phosphoramidite compounds is mainly to control the purity of its precursors to obtain high-purity phosphoramidite monomers.
[0003] There are three processes that are widely recognized at present. 1. Column purification process. This process requires the use of a large amount of solvent, with a large PMI, which increases production costs and production time, and the purity cannot be effectively controlled. 2. Product solidification process. The compound is dissolved in a benign solvent, and then the solution is added dropwise to a poor solvent. The compound obtained by this method often has no crystal form. The repeatability is poor. 3. Crystallization process. This method can obtain samples with very high purity, but crystal studies have shown that most phosphoramidite precursors are amorphous compounds, and it is difficult to develop an efficient crystallization process. Summary of the invention
[0004] Aiming at the defects of the existing purification method with high production cost and poor repeatability, a method for purifying phosphoramidite precursor is provided. The purification method of the invention is simple, does not require special equipment, has low cost, good repeatability, does not stick to the wall and has good yield.
[0005] The present invention solves the above technical problems through the following technical solutions.
[0006] The present invention also provides a method for purifying a compound as shown in Formula II, which comprises the following steps: in a solvent, in the presence of an additive B, subjecting the compound as shown in Formula II to a salt-forming reaction; the additive B is a combination of an inorganic salt and an organic base; the inorganic salt is a lithium salt, a potassium salt, a cesium salt, a copper salt, a magnesium salt or an iron salt; the organic base is TEA or DBU; the solvent is a nitrile solvent or a ketone solvent, and when the solvent is a ketone solvent, the inorganic salt is a lithium salt, an iron salt or a copper salt;
[0007]
[0008] In formula II, R3 is 4-methoxytrityl, 4,4'-dimethoxytrityl or trityl, and R4 is H, C 1-6 Alkoxy or one or more R 4-1 Substituted C 1-6 Alkoxy; each R 4-1 C 1-6 Alkoxy; B1 is a purine nucleotide or a derivative thereof.
[0009] In one embodiment, R3 is 4,4'-dimethoxytrityl.
[0010] In one embodiment, R4 is H or is replaced by one or more R 4-1 Substituted C 1-6 Alkoxy; for example H or one or more R 4-1 Substituted ethoxy; each R 4-1 It is a methoxy group.
[0011] In one embodiment, R4 is H or
[0012] In one solution, B1 is
[0013] In a certain embodiment, when the solvent is a nitrile solvent, the nitrile solvent is acetonitrile.
[0014] In a certain embodiment, when the solvent is a ketone solvent, the ketone solvent is methyl isobutyl ketone.
[0015] In a certain embodiment, among the inorganic salts, the copper salt is preferably present in the form of a monovalent copper salt, and the iron salt is preferably present in the form of a trivalent iron salt.
[0016] In a certain embodiment, the inorganic salt is in the form of a halogenated salt, and the organic base is TEA.
[0017] In one embodiment, the additive B is potassium fluoride and TEA, potassium chloride and TEA, potassium bromide and TEA, ferric chloride and TEA, cuprous chloride and TEA, cesium chloride and TEA, cesium iodide and TEA, magnesium chloride and TEA, or lithium chloride and TEA.
[0018] In one embodiment, the compound shown in formula II is The solvent is methyl isobutyl ketone, and the additive B is lithium chloride and TEA.
[0019] In one embodiment, the compound shown in formula II is The solvent is acetonitrile, and the additive B is potassium fluoride and TEA, potassium chloride and TEA, potassium bromide and TEA, ferric chloride and TEA, cuprous chloride and TEA, cesium chloride and TEA, cesium iodide and TEA, or magnesium chloride and TEA.
[0020] In one embodiment, the compound shown in formula II is The solvent is methyl isobutyl ketone, and the additive B is ferric chloride and TEA or cuprous chloride and TEA.
[0021] In one embodiment, the compound shown in formula II is The solvent is acetonitrile, and the additive B is potassium chloride and TEA or potassium bromide and TEA.
[0022] In one embodiment, the molar ratio of the inorganic salt to the compound of formula II is (1-4):1, for example 3:1.
[0023] In one embodiment, the molar ratio of the organic base to the compound of formula II is (1-4):1, for example 3:1.
[0024] In one embodiment, the salt-forming reaction is carried out under stirring conditions until solid is precipitated (preferably stirring for 1-45 hours, such as 1-2 hours, 3 hours, 17 hours, 19 hours or 45 hours).
[0025] In one embodiment, the reaction temperature of the salt-forming reaction is 5-45°C, such as 5-15°C, 20-30°C or 35-45°C.
[0026] In a certain embodiment, the purification method further comprises the following post-treatment steps: filtration and rinsing (preferably rinsing with the solvent) and drying.
[0027] In the present invention, the derivatives of purine nucleotides refer to compounds formed by condensation of the amino group in the purine nucleotides with conventional amino protecting groups in the art.
[0028] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.
[0029] The reagents and raw materials used in the present invention are commercially available.
[0030] The positive improvement effect of the present invention is that the purification method of the present invention is simple, does not require special equipment, has low cost, good repeatability, does not stick to the wall and has a good yield. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is the XRPD spectrum of the lithium salt crystal obtained in Example 1. DETAILED DESCRIPTION
[0032] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.
[0033] Detection method:
[0034] The purity was tested by high performance liquid chromatography under the following conditions:
[0035]
[0036]
[0037] X-ray powder diffraction analysis (XRPD): the test conditions are Cu, the working voltage is 40KV, the working current is 40mA, the scanning angle range is 4-40deg, and the interval is 0.02deg / step.
[0038] Example 1
[0039]
[0040] Under nitrogen protection, 2g of crude dG3 (purity: 98.25%) was placed in a MIBK (5V) solution and stirred at 20-30 degrees. Then, LiCl (3eq.) was added to the dG3 solution. TEA (3eq.) was slowly added dropwise to the reaction solution and stirred at 20-30 degrees for 1-2 hours. Solids precipitated. Filtered, the filter cake was rinsed with MIBK (2V), and dried to obtain 1.28g of white crystals. Purity: 99.5%. Its XRPD data is shown in Figure 1 .
[0041] Example 2-11
[0042]
[0043] Under the following conditions, the experiment was carried out in the same manner as in Example 1, and the results are as follows:
[0044]
[0045]
[0046] Examples 12-13
[0047]
[0048] Under the following conditions, the experiment was carried out in the same manner as in Example 1, and the results are as follows:
[0049]
[0050] Comparative Examples 1-6
[0051]
[0052] Under the following conditions, the experiment was carried out in the same manner as in Example 1, and the results are as follows:
[0053]
Claims
1. A method for purifying a compound as shown in formula II, comprising the following steps: in a solvent, in the presence of an additive B, subjecting the compound as shown in formula II to a salt-forming reaction; the additive B is a combination of an inorganic salt and an organic base; the inorganic salt is a lithium salt, a potassium salt, a cesium salt, a copper salt, a magnesium salt or an iron salt; the organic base is TEA or DBU; the solvent is a nitrile solvent or a ketone solvent, and when the solvent is a ketone solvent, the inorganic salt is a lithium salt, an iron salt or a copper salt; In formula II, R3 is 4-methoxytrityl, 4,4'-dimethoxytrityl or trityl, and R4 is H, C 1-6 Alkoxy or one or more R 4-1 Substituted C 1-6 Alkoxy; each R 4-1 C 1-6 Alkoxy; B1 is a purine nucleotide or a derivative thereof.
2. The purification method according to claim 1, characterized in that It meets one or both of the following conditions: (1) R3 is 4,4'-dimethoxytrityl; and (2) R4 is H or is replaced by one or more R 4-1 Substituted C 1-6 Alkoxy; for example H or one or more R 4-1 Substituted ethoxy; each R 4-1 It is a methoxy group.
3. The purification method according to claim 2, characterized in that It meets one or both of the following conditions: (1) R4 is H or and (2)B1 is 4. The purification method according to claim 1, characterized in that It meets one or more of the following conditions: (1) When the solvent is a nitrile solvent, the nitrile solvent is acetonitrile; (2) When the solvent is a ketone solvent, the ketone solvent is methyl isobutyl ketone; (3) In the inorganic salt, the copper salt exists in the form of monovalent copper salt; (4) In the inorganic salt, the iron salt exists in the form of trivalent iron salt; and (5) The inorganic salt is in the form of a halogenated salt, and the organic base is TEA.
5. The purification method according to claim 1, characterized in that The additive B is potassium fluoride and TEA, potassium chloride and TEA, potassium bromide and TEA, ferric chloride and TEA, cuprous chloride and TEA, cesium chloride and TEA, cesium iodide and TEA, magnesium chloride and TEA or lithium chloride and TEA.
6. The purification method according to claim 1, characterized in that It meets any of the following conditions: (1) The compound shown in formula II is The solvent is methyl isobutyl ketone, and the additive B is lithium chloride and TEA; (2) The compound shown in formula II is The solvent is acetonitrile, and the additive B is potassium fluoride and TEA, potassium chloride and TEA, potassium bromide and TEA, ferric chloride and TEA, cuprous chloride and TEA, cesium chloride and TEA, cesium iodide and TEA, or magnesium chloride and TEA; (3) The compound shown in formula II is The solvent is methyl isobutyl ketone, and the additive B is ferric chloride and TEA or cuprous chloride and TEA; (4) The compound shown in formula II is The solvent is acetonitrile, and the additive B is potassium chloride and TEA or potassium bromide and TEA.
7. The purification method according to claim 1, characterized in that It meets one or both of the following conditions: (1) the molar ratio of the inorganic salt to the compound represented by formula II is (1-4):1; and (2) The molar ratio of the organic base to the compound shown in Formula II is (1-4):
1.
8. The purification method according to claim 7, characterized in that It meets one or both of the following conditions: (1) the molar ratio of the inorganic salt to the compound of formula II is 3:1; and (2) The molar ratio of the organic base to the compound represented by formula II is 3:
1.
9. The purification method according to claim 1, characterized in that It meets one or more of the following conditions: (1) the salt-forming reaction is carried out under stirring conditions until solid is precipitated; the stirring time is 1-45 hours; and (2) The reaction temperature of the salt-forming reaction is 5-45°C.
10. The purification method according to claim 9, characterized in that It meets one or more of the following conditions: (1) the stirring time is 1-2 hours, 3 hours, 17 hours, 19 hours or 45 hours; and (2) the reaction temperature of the salt-forming reaction is 5-15°C, 20-30°C or 35-45°C.