A method for purifying and preparing sitagliptin free base

Abstract

The invention discloses a method for purifying and preparing sitagliptin free base, and belongs to the field of sitagliptin preparation. The method comprises: acidifying the reaction solution of preparing sitagliptin by enzymatic method to remove the enzyme; using Fe3O4 / polystyrene magnetic nanocomposite material to pack the column, dynamically adsorbing the reaction solution after enzyme removal in step S1, washing with water and eluting with eluent in sequence after saturated adsorption, and obtaining sitagliptin eluate; the eluate is vacuum rotary evaporation concentrated to dryness, static filtration at room temperature, and drying to obtain sitagliptin free base powder. The present invention utilizes Fe3O4 / polystyrene magnetic nanocomposite material to adsorb and purify sitagliptin, with good separation effect, high yield, high product quality, and reusability, and the desorption organic solvent is ethanol, with a small amount of use, low requirements on equipment and production environment, and low recovery difficulty; the method is simple to operate, low toxicity, and extremely low damage to the operator's human body.

Description

Technical Field

[0001] The invention relates to the preparation of sitagliptin, and in particular to a method for purifying and preparing sitagliptin free base. Background Art

[0002] Sitagliptin free base is a key intermediate before sitagliptin phosphate is salified. There are both total synthesis and semi-synthesis methods for the preparation of sitagliptin and its intermediates. The semi-synthesis method is mainly prepared by chemical-enzymatic combined preparation. The premise of enzymatic preparation is to obtain an ω-aminotransferase that can convert sitagliptin precursor ketone into sitagliptin. Enzymatic preparation has the advantages of simple operation, low cost, suitability for industrial production, and high economic benefits. The existing sitagliptin purification method mainly comprises first preparing the sitagliptin reaction solution by enzymatic method, adding diatomaceous earth to remove the enzyme after the reaction solution is acidified, and then using a large amount of organic solvents such as ethyl acetate or dichloromethane to continuously extract the sitagliptin in the reaction solution, and then adding saturated sodium chloride and saturated sodium bicarbonate to the organic layer to wash away most of the impurities in the organic layer, obtaining the sitagliptin organic layer, adding liquid alkali to alkalize, and removing part of the water and organic solvent by vacuum distillation at 50°C, and finally obtaining the sitagliptin free base solid powder. This method uses an extraction method to purify sitagliptin, which has high industrial requirements, uses a large amount of organic solvents, has a high risk factor, and has extremely high requirements for equipment and the environment. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a method for purifying and preparing sitagliptin free base.

[0004] Technical solution: A method for purifying and preparing sitagliptin free base, comprising the following steps:

[0005] S1, acidifying the reaction solution of preparing sitagliptin by enzymatic method to remove the enzyme;

[0006] S2, using Fe3O4 / polystyrene magnetic nanocomposite to pack the column, dynamically adsorbing the reaction solution after enzyme removal in step S1, and washing with water and eluting with an eluent in sequence after saturated adsorption to obtain a sitagliptin eluate;

[0007] S3. The eluate was concentrated to 1 / 10 volume by vacuum rotary evaporation at 60° C. in a water bath, cooled to 15° C., filtered to obtain sitagliptin free base solid, and then dried in a forced air drying oven at 60° C. to obtain sitagliptin free base powder.

[0008] Preferably, in step S1, the preparation process of the reaction solution is: 500mM isopropylamine, 1600mM DMSO, 50mM triethanolamine, 1mM PLP, 2L crude enzyme solution, add 20mM PBS buffer to a total system of 4000mL, 300g sitagliptin precursor ketone dissolved in 500mL DMSO as a feeding liquid, react at 45°C and 300r / min for 20h to prepare the sitagliptin reaction solution.

[0009] Preferably, in step S1, HCl is added to the reaction solution for acidification, the pH is adjusted to 2-3, and the enzyme is removed by filtration.

[0010] Preferably, in step S2, the eluent is 10-20% ethanol.

[0011] Preferably, in step S2, the Fe3O4 / polystyrene magnetic nanocomposite material is pre-activated with 50% ethanol.

[0012] Preferably, in step S2, the preparation process of the Fe3O4 / polystyrene magnetic nanocomposite material is:

[0013] (1) Dissolve 4 mmol FeCl3·6H2O and 2.8 mmol FeSO4·7H2O in 10 L purified water, add 15 L of a mixed solution of 2 mmol CTAB and 25 mmol NaOH under stirring, react at 80°C for 2 h, filter and wash with purified water for 3 times to obtain Fe3O4 nanoparticles;

[0014] (2) Take 6 L of cyclohexane, add 14 L of purified water, 100 ml of styrene, and 0.015 mol of potassium oleate, stir, add n-butanol dropwise, take the Fe3O4 nanoparticles obtained in (1) and add them to the solution, add APS dropwise, react at 80°C for 8 hours, filter, wash with purified water for 3 times, and dry at 60°C for 12 hours to obtain Fe3O4 / polystyrene magnetic nanocomposite materials.

[0015] Beneficial effects: The present invention utilizes polystyrene groups to adsorb sitagliptin, and has excellent purification effect. Conventional polystyrene-based macroporous resins have poor chemical stability and insufficient mechanical strength, resulting in poor regeneration performance and low reuse rate. Therefore, Fe3O4 / polystyrene magnetic nanocomposite materials are used to adsorb and purify sitagliptin, which has good separation effect, high yield, high product quality, and can be reused. The desorption organic solvent is ethanol, which has a small usage amount, low requirements on equipment and production environment, and low recovery difficulty. The method is simple to operate, has low toxicity, and extremely low harm to the operator's body. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a purity analysis chart of sitagliptin in the upper column liquid in Example 1;

[0017] Figure 2 is a purity analysis chart of the sitagliptin eluate in Example 1;

[0018] Figure 3 is a purity analysis chart of sitagliptin free base dry powder in Example 1;

[0019] Figure 4 is a purity analysis chart of the sitagliptin eluate in Example 2;

[0020] Figure 5 is a purity analysis chart of sitagliptin free base dry powder in Example 2;

[0021] Figure 6 is a purity analysis chart of the sitagliptin eluate in Example 3;

[0022] Figure 7 is a purity analysis chart of sitagliptin free base dry powder in Example 3;

[0023] Figure 8 This is a purity analysis diagram of the sitagliptin eluate in Comparative Example 1;

[0024] Fig. 9 This is a purity analysis chart of sitagliptin free base dry powder in Comparative Example 1;

[0025] Fig.10 This is an analysis chart of the proportion of sitagliptin adsorption effluent in Comparative Example 2;

[0026] Fig.11 It is a purity analysis chart of sitagliptin eluate in comparison column 2;

[0027] Fig.12 This is a purity analysis chart of sitagliptin free base dry powder in comparison column 2. DETAILED DESCRIPTION

[0028] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Liquid chromatography analysis

[0030] Instruments and parameters

[0031] Instrument: Thermo Instrument 3000

[0032] Chromatographic column: Inertsil CN-3 5um, 4.6×150mm

[0033] Instrument parameters:

[0034] Mobile phase A: 1.36 g / L potassium dihydrogen phosphate aqueous solution, pH adjusted to 2.0 with phosphoric acid

[0035] Mobile phase B: acetonitrile

[0036] The degree gradient is shown in Table 1:

[0037] Table 1 Liquid chromatography analysis program gradient

[0038]

[0039] Flow rate: 1ml / min

[0040] Column temperature: 30°C

[0041] Detection wavelength: UV, 205nm

[0042] Injection volume: 10 μL

[0043] Example 1

[0044] (1) Preparation of sitagliptin reaction solution: 300g of sitagliptin precursor ketone, 500mM isopropylamine, 2L DMSO, 50mM triethanolamine, 1mM PLP, 2L transaminase solution, add 20mM PBS buffer to a total system of 4000mL, react at 45°C and 300r / min for 20h to prepare sitagliptin reaction solution. HCl was added to the reaction solution to adjust the pH to 2-3, and the enzyme was removed by filtration to obtain the sitagliptin supernatant (liquid chromatography analysis as shown in Figure 1 As shown in the figure), the concentration is 70g / L and the purity of sitagliptin is 33.75%.

[0045] (2) Take 1L of sitagliptin sample solution and perform dynamic adsorption on a Fe3O4 / polystyrene magnetic nanocomposite (pre-activated with 50% ethanol). After adsorption, wash 3BV with water until the effluent DMSO is less than 1%, then elute with 3BV of 20% ethanol, and collect the sitagliptin eluate. The yield is 95.2% and the HPLC purity is 99.33% (such as Figure 2 as shown).

[0046] (3) The eluate was concentrated by rotary evaporation to 1 / 10 volume at 60°C in a water bath, cooled to 15°C, and filtered to obtain solid sitagliptin free base. The solid was dried in a forced air drying oven at 60°C to obtain 61.5 g of sitagliptin free base dry powder with a yield of 92.27% and a HPLC purity of 99.91% (e.g. Figure 3 as shown).

[0047] Example 2

[0048] (1) The preparation method of sitagliptin reaction solution is the same as that in Scheme 1.

[0049] (2) The Fe3O4 / polystyrene magnetic nanocomposite was used for the second time, and 50% ethanol 3BV was used for activation, and then the ethanol was washed out with purified water. 1L of sitagliptin sample solution was taken for dynamic adsorption. After the adsorption was completed, 3BV of water was washed until the effluent DMSO was less than 1%, and then 3BV of 20% ethanol was used for elution. The sitagliptin eluate was collected, with a yield of 96.3% and a HPLC purity of 99.85% (such as Figure 4 as shown).

[0050] (3) The eluate was concentrated by rotary evaporation to 1 / 10 volume at 60°C in a water bath, cooled to 15°C, and filtered to obtain solid sitagliptin free base. The solid was dried in a forced air drying oven at 60°C to obtain 62.3 g of sitagliptin free base dry powder, with a yield of 94.41% and a HPLC purity of 99.84% (e.g. Figure 5 as shown).

[0051] Example 3

[0052] (1) The preparation method of sitagliptin reaction solution is the same as that in Scheme 1.

[0053] (2) The Fe3O4 / polystyrene magnetic nanocomposite was used for the 10th time and activated with 3BV of 50% ethanol, and then the ethanol was washed out with purified water. 1L of the sitagliptin sample solution was taken for dynamic adsorption. After the adsorption was completed, 3BV of water was washed until the effluent DMSO was less than 1%, and then eluted with 3BV of 20% ethanol. The sitagliptin eluate was collected, with a yield of 95.8% and a HPLC purity of 99.88% (such as Figure 6 as shown).

[0054] (3) The eluate was concentrated by rotary evaporation to 1 / 10 volume at 60°C in a water bath, cooled to 15°C, and filtered to obtain solid sitagliptin free base. The solid was dried in a forced air drying oven at 60°C to obtain 63.31 g of sitagliptin free base dry powder with a yield of 94.4% and a HPLC purity of 99.79% (e.g. Figure 7 as shown).

[0055] Comparative Example 1

[0056] (1) The preparation method of sitagliptin reaction solution is the same as that in Scheme 1.

[0057] (2) Take 1L of sitagliptin sample solution and put it into a chromatography column filled with polystyrene macroporous adsorption resin for dynamic adsorption (the resin was pre-activated with 50% ethanol). Wash 3BV with water until the effluent DMSO is less than 1%, then elute with 3BV 20% ethanol, collect the sitagliptin eluate, the yield is 90.33%, and the HPLC purity is 99.51% (such as Figure 8 as shown).

[0058] (3) The eluate was concentrated by rotary evaporation to 1 / 10 volume at 60°C in a water bath, cooled to 15°C, and filtered to obtain solid sitagliptin free base. The solid was dried in a forced air drying oven at 60°C to obtain 58.5 g of sitagliptin free base dry powder with a yield of 92.37% and a HPLC purity of 99.29% (e.g. Fig. 9 as shown).

[0059] Comparative Example 2

[0060] (1) The preparation method of sitagliptin reaction solution is the same as that in Scheme 1.

[0061] (2) The polystyrene macroporous adsorption resin was used for the third time and activated with 3 BV of 50% ethanol, and then the ethanol was washed out with purified water. 1 L of sitagliptin sample solution was taken for dynamic adsorption. After 650 ml of the product was adsorbed on the column, the product effluent was tested (such as Fig.10 As shown), wash 3BV with water until the effluent DMSO is less than 1%, then elute with 3BV 20% ethanol, collect the sitagliptin eluate, the yield is 57.3%, and the HPLC purity is 99.75% (as shown Fig.11 as shown).

[0062] (3) The eluate was concentrated by rotary evaporation to 1 / 10 volume at 60°C in a water bath, cooled to 15°C, and filtered to obtain solid sitagliptin free base. The solid was dried in a forced air drying oven at 60°C to obtain 33.24 g of sitagliptin free base dry powder, with a yield of 83.3% and a HPLC purity of 99.88% (e.g. Fig.12 as shown).

[0063] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for purifying and preparing sitagliptin free base, characterized in that: The following steps are involved: S1, acidifying the reaction solution of preparing sitagliptin by enzymatic method to remove the enzyme; S2, using Fe3O4 / polystyrene magnetic nanocomposite to pack the column, dynamically adsorbing the reaction solution after enzyme removal in step S1, and washing with water and eluting with an eluent in sequence after saturated adsorption to obtain a sitagliptin eluate; S3. The eluate was concentrated to 1 / 10 volume by vacuum rotary evaporation at 60° C. in a water bath, cooled to 15° C., filtered to obtain sitagliptin free base solid, and then dried in a forced air drying oven at 60° C. to obtain sitagliptin free base powder.

2. The method for preparing sitagliptin free base by purification according to claim 1, characterized in that: In step S1, the reaction solution is prepared as follows: 500mM isopropylamine, 1600mM DMSO, 50mM triethanolamine, 1mM PLP, 2L crude enzyme solution, 20mM PBS buffer is added to the total system of 4000mL, 300g of sitagliptin precursor ketone is dissolved in 500mL DMSO as a feeding liquid, and the reaction is carried out at 45°C and 300r / min for 20h to prepare the sitagliptin reaction solution.

3. The method for preparing sitagliptin free base by purification according to claim 1, characterized in that: In step S1, HCl is added to the reaction solution for acidification, the pH is adjusted to 2-3, and the enzyme is removed by filtration.

4. The method for preparing sitagliptin free base by purification according to claim 1, characterized in that: In step S2, the eluent is 10-20% ethanol.

5. The method for preparing sitagliptin free base by purification according to claim 1, characterized in that: In step S2, the Fe3O4 / polystyrene magnetic nanocomposite is activated in advance with 50% ethanol.

6. The method for preparing sitagliptin free base by purification according to claim 1, characterized in that: In step S2, the preparation process of Fe3O4 / polystyrene magnetic nanocomposite material is as follows: (1) Dissolve 4 mmol FeCl3·6H2O and 2.8 mmol FeSO4·7H2O in 10 L purified water, add 15 L of a mixed solution of 2 mmol CTAB and 25 mmol NaOH under stirring, react at 80°C for 2 h, filter and wash with purified water for 3 times to obtain Fe3O4 nanoparticles; (2) Take 6 L of cyclohexane, add 14 L of purified water, 100 ml of styrene, and 0.015 mol of potassium oleate, stir, add n-butanol dropwise, take the Fe3O4 nanoparticles obtained in (1) and add them to the solution, add APS dropwise, react at 80°C for 8 hours, filter, wash with purified water for 3 times, and dry at 60°C for 12 hours to obtain Fe3O4 / polystyrene magnetic nanocomposite materials.

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