Method for preparing vitamin D2 with high conversion rate and high selectivity

Abstract

The invention discloses a method for preparing vitamin D2 with high conversion rate and high selectivity, and belongs to the technical field of organic photosynthesis reaction.The method comprises the steps that an ergosterol solution passes through a first-stage illumination reactor, the illumination wavelength is controlled to be 254 nm, the illumination time is 20-25 min, and an ergosterol ring-opening product reaction solution is obtained through a first photoreaction; the ergosterol ring-opening product reaction liquid passes through a second-stage illumination reactor, the illumination wavelength is controlled to be 365-415 nm, the illumination time is 30-35 min, and a VD2 precursor reaction liquid is obtained through a second photoreaction; the VD2 precursor reaction liquid is heated and converted into VD2 converted oil; and carrying out esterification, crystallization, hydrolysis and recrystallization on the VD2 conversion oil to obtain a VD2 finished product. According to the method, the conversion rate of ergosterol and the selectivity of a target product can be remarkably improved, the purity of a VD2 finished product is improved, the impurity content is reduced, and the VD2 impurity limit requirement specified by European Pharmacopoeia is met.

Description

Technical Field

[0001] The invention belongs to the technical field of organic photosynthesis reactions, and specifically relates to a method for preparing vitamin D2 with high conversion rate and high selectivity. Background Art

[0002] Vitamin D2, chemically known as ergocalciferol, is a fat-soluble vitamin that is a colorless needle-shaped crystal or white crystalline powder. It is unstable to light, oxygen, and temperature, and is easily deteriorated. It needs to be stored at low temperature and away from light. Its main function is to help the human body absorb calcium and phosphorus, improve immune function, and promote muscle function.

[0003] Vitamin D2 is synthesized by photochemical reaction using ergosterol as raw material. The main reaction and side reactions involved are as follows: Figure 1 As shown, after ergosterol is irradiated with light, the singlet excited state B ring bond breaks to generate pre-VD2, and then 1,7-hydrogen migration, rearrangement, and pericyclic reaction to obtain VD2. In the preparation of VD2, the VD2 precursor generated after ergosterol is irradiated with light will continue to undergo secondary photochemical reactions to generate by-products luminosterol and tachysterol. To increase the conversion rate of ergosterol and the proportion of vitamin D2 precursors in the irradiated products, it is necessary to control the wavelength of the photoreaction of the ring-opening step of ergosterol and improve the utilization rate of light energy. Therefore, the selection of light source and illumination wavelength is particularly important.

[0004] Most existing technologies use high-pressure mercury lamps as light sources, which have obvious disadvantages: low luminous efficiency; short bulb life, usually 8,000 to 10,000 hours; environmental pollution, the lamp releases harmful substances such as mercury during use; it is not easy to dim, and cannot be dimmed well, making it difficult to meet the lighting requirements of different scenes; the startup time is long, and a special preheating device is required, which increases the cost and complexity of use.

[0005] Chinese patent CN106496088A reports the use of a 10-30kw high-pressure mercury lamp as a light source. Since the radiation spectrum of the high-pressure mercury lamp is difficult to stably exist between 280 and 300nm, by-products are easily generated. The yield of VD2 conversion oil is 10-15%, and the ergosterol conversion rate is extremely low. A large amount of unconverted ergosterol needs to be recovered, and the process is relatively complicated. In addition, VD2 is an ester-soluble vitamin with extremely high solubility in ethyl acetate. This patent uses ethyl acetate to directly crystallize and purify VD2 from the chromatographic oil, which does not conform to the recrystallization principle and results in a great loss of product.

[0006] Chinese patent CN114634434A reported that a 500-watt or 1000-watt high-pressure mercury lamp was used as a light source, and the light was irradiated twice. A double light filtering system was used. The filter cold trap material of the first light reaction was quartz glass, which could transmit ultraviolet light below 280nm, so that the B ring of ergosterol was opened to generate pre-VD2. The conversion rate of ergosterol in the first light reaction was 28-35%. The filter cold trap material of the second light reaction was borosilicate glass, which could filter out light below 280nm, and ultraviolet light above 280nm from the high-pressure mercury lamp was used to convert tachysterol into pre-VD2. Although the conversion rate has been improved, 65-70% of the raw materials are still not converted.

[0007] Chinese patent CN116283696A reports that a high-pressure mercury lamp is used for the first illumination to obtain a ring-opened photoreaction liquid, and then a 365-465nm LED lamp is used for the second illumination. The conversion rate of ergosterol can reach nearly 70%, and no column purification is required. The VD2 yield can reach 45%, and nearly 30% of ergosterol is still unconverted. In addition, a large amount of tachysterol, photosterol impurities and unreacted ergosterol will be generated during the photoreaction. They have similar structures and are all isomers of VD2. It is generally difficult to use organic solvents to directly crystallize and purify them to reduce them to the VD2 impurity limit requirements specified in the European Pharmacopoeia (the impurity limits of the European Pharmacopoeia are: impurity A≤0.5%, impurity F≤0.5%, impurity G≤1.5%, impurity B≤0.2%, other unknown impurities≤0.1%, and total impurities≤2%).

[0008] Chinese patent CN117986170A reports a method for preparing VD2 using deep ultraviolet microfluidic technology, using a 265-295nm LED lamp as a light source and quartz glass as a light-transmitting material. The VD2 reaction liquid is obtained by one illumination, and the reaction liquid is concentrated and then pulped with acetone to recover ergosterol. The filtrate is concentrated and dried to obtain the VD2 finished product, with a yield of up to 40%. The illumination reaction of ergosterol is a balanced reaction, which will inevitably generate impurities such as tachysterol and luminosterol. Their solubility is similar to that of VD2. Only filtering cannot effectively remove impurities, so that the product cannot meet the impurity limit requirements of the European Pharmacopoeia; in addition, like other existing technologies, there are still a large number of raw materials that have not been converted.

[0009] Chinese patent CN110724081A reported that ergosterol photo-ring-opening reaction was carried out at a wavelength of 260-300nm. In the photochemical ring-opening reaction, the conversion rate of ergosterol could reach 75%, and then photochemical isomerization reaction was carried out at a wavelength of 310-360nm. After crystallization and recovery, the total yield of the product could exceed 55%, and the purity was above 95%. However, there are still a lot of ergosterol raw materials that are not converted in this method, and the raw materials need to be recovered; more importantly, the product prepared by this method has a high impurity content and cannot meet the impurity limit requirements of the European Pharmacopoeia. Summary of the invention

[0010] The present invention aims to provide a method for preparing vitamin D2 with high conversion rate and high selectivity. The method adopts two photoreactions and dual-wavelength illumination. Under the coordination of specific illumination wavelength and time, the conversion rate of ergosterol and the selectivity of the target product can be significantly improved, the purity of the VD2 finished product can be improved, the impurity content can be reduced, and the VD2 impurity limit requirements specified in the European Pharmacopoeia can be met.

[0011] The present invention is achieved through the following technical solution: a method for preparing vitamin D2 with high conversion rate and high selectivity, comprising the following steps: S1. The ergosterol solution is passed through the first stage photoreactor, the light wavelength is controlled to be 254nm, the illumination time is 20 to 25min, and the ergosterol ring-opening product reaction liquid is obtained by the first photoreaction; S2. The ergosterol ring-opening product reaction solution is passed through a second-stage light reactor, the light wavelength is controlled to be 365 to 415 nm, the illumination time is 30 to 35 min, and the VD2 precursor reaction solution is obtained by the second light reaction; S3. The VD2 precursor reaction solution is heated and converted into VD2 conversion oil; S4. The VD2 conversion oil is subjected to esterification, crystallization, hydrolysis and recrystallization to obtain the VD2 finished product.

[0012] The ergosterol solution is prepared by dissolving ergosterol and an antioxidant in a reaction solvent, and the concentration of ergosterol in the ergosterol solution is controlled to be 1-1.5%.

[0013] The antioxidant is 2,6-di-tert-butyl-p-cresol, and the weight ratio of the antioxidant to ergosterol is controlled to be 1:200-500.

[0014] The reaction solvent is methanol, ethanol, methyl tert-butyl ether, tetrahydrofuran, a mixed solvent of methanol and n-heptane, a mixed solvent of ethanol and n-heptane, a mixed solvent of methanol and n-hexane, or a mixed solvent of ethanol and n-hexane.

[0015] The first-stage light irradiation reactor and the second-stage light irradiation reactor are both microfluidic reactors, and the light-transmitting material is both quartz glass.

[0016] The light sources for the first light reaction and the second light reaction are both LED lamps.

[0017] During the heating conversion, the heating temperature is controlled to be 50-55° C. and the heating time is 5-7 hours.

[0018] During the esterification, the VD2 conversion oil is subjected to an esterification reaction with triethylamine and 3,5-dinitrobenzoyl chloride to obtain VD2 ester.

[0019] The hydrolysis is carried out by adding sodium hydroxide or potassium hydroxide into a solvent, and the solvent is anhydrous ethanol.

[0020] The solvent used in the crystallization is a mixed solvent of ethanol and ethyl acetate; the solvent used in the recrystallization is anhydrous methanol.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention adopts the above-mentioned VD2 synthesis process, and through dual-wavelength irradiation, the ergosterol conversion rate is greater than 90%, which is significantly better than the existing technology.

[0022] (2) The present invention selects a specific photoreaction wavelength during the photoreaction process, so that the target product generated is highly selective. The total amount of VD2 and VD2 precursor in the photoreaction accounts for more than 75%. At the same time, there are few by-products and no column chromatography is required. Therefore, the VD2 production process is simplified and the generation of a large amount of column chromatography filler solid waste is avoided. The crude reaction product can be directly subjected to subsequent esterification, hydrolysis, and recrystallization steps, with high production efficiency and suitable for industrial production.

[0023] (3) The method of the present invention also provides a new refining condition for crude VD2 ester. Instead of using acetone, a solvent that is easy to make drugs, as a crystallization solvent, a mixed solvent of ethanol and ethyl acetate is used for crystallization, which can reduce the single impurity content of VD2 ester to less than 0.1%. That is, among all the impurities of VD2 ester, the content of a single impurity is reduced to less than 0.1%.

[0024] (4) The present invention adopts a microfluidic reactor to prepare VD2. Compared with the traditional kettle-type photoreaction equipment, when the reaction is carried out in the flow channel, the mass transfer and heat transfer are very fast, which can effectively avoid the "temperature runaway" phenomenon and has high safety; the residence time of the reactants is short, the reaction is more efficient, and the productivity is higher; the microchannel reactor can be scaled up by "number increase", which relies on increasing the number of parallel flow channels. It is easier to scale up than the kettle process by increasing the volume of the kettle body; the light-transmitting material is quartz glass, which has good light transmittance and high light energy utilization.

[0025] (5) The light source of the present invention is an LED lamp. Compared with a high-pressure mercury lamp, the high-pressure mercury lamp has a wide spectrum range and produces more impurities, while the LED lamp has a narrow wavelength, a relatively simple illumination product, and fewer by-products. The LED lamp is a cold light source and releases less heat than a high-pressure mercury lamp, thus saving costs and having high efficiency.

[0026] In summary, the present invention can greatly improve the conversion rate of ergosterol and the selectivity of the target product through the above-mentioned VD2 synthesis process, so that the prepared VD2 meets the requirements of relevant substances of European Pharmacopoeia VD2, and the quality level is significantly better than the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the photochemical reaction synthesis of VD2.

[0028] Figure 2 This is the system suitability spectrum of VD2 impurities A, F, and G.

[0029] Figure 3 This is the HPLC localization map of VD2 impurity B.

[0030] Figure 4 This is the HPLC spectrum of VD2 obtained in Example 1.

[0031] Figure 5 This is the HPLC spectrum of VD2 obtained in Example 2.

[0032] Figure 6 This is the HPLC spectrum of VD2 obtained in Example 3.

[0033] Figure 7 This is the HPLC spectrum of VD2 obtained in Example 4.

[0034] Figure 8 This is the HPLC spectrum of VD2 obtained in Example 5.

[0035] Fig. 9 It is the HPLC spectrum of the reaction solution of Comparative Example 1.

[0036] Fig.10 This is the HPLC spectrum of VD2 obtained in Comparative Example 1. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Figure 2 and Figure 3 Used to locate different impurities in the HPLC spectrum of VD2 in this example.

[0038] Embodiment 1: 10 g of ergosterol and 30 mg of 2,6-di-tert-butyl-p-cresol were dissolved in 100 ml of ethyl acetate under stirring at room temperature, filtered, and insoluble matter was removed. The filtrate was introduced into the first-stage microfluidic photoreactor (the light-transmitting material was quartz glass) through a liquid inlet pump at an appropriate flow rate, and the circulating water was turned on to control the temperature at 25-35°C. The ring-opening reaction was carried out at a wavelength of 254 nm (the light source was an LED lamp), and the illumination time was 20 minutes to obtain a ring-opening product reaction liquid, and the ergosterol conversion rate was 93.2%.

[0039] The ring-opening reaction liquid passes through the second-stage microfluidic photoreactor (translucent material is quartz glass), and the temperature is controlled at 25-35°C by circulating water. Under the illumination of LED lamp at 365-370nm, the ring-opening product is converted into the second-stage illumination product whose main product is VD2 precursor. The illumination time is 30min. The second-stage illumination product is heated at 50-55°C for about 5h, and concentrated under reduced pressure at 40-50°C to obtain 11.33g of VD2 conversion oil. The HPLC area percentage of each product in the VD2 conversion oil is: VD2 (63.21%), VD2 precursor (15.41), ergosterol: 6.09 (%), tachysterol (2.92%), and luminosterol (6.64%).

[0040] Dissolve 11.00 g of VD2 conversion oil and 5.60 g of triethylamine in 100 ml of dichloromethane, cool to 0-10°C, add 6.40 g of 3,5-dinitrobenzoyl chloride, and complete the reaction. Wash with water until neutral, concentrate the organic phase to dryness, add 50 ml of ethyl acetate: ethanol = 1:1 (v / v), and crystallize at 0±5°C for about 2-3 hours. Filter to obtain 10.33 g of VD2 refined ester with a wet weight of 98.55% and a single impurity of less than 0.1%.

[0041] Add 10.00 g of VD2 refined ester to 50 ml of anhydrous ethanol, add 1 g of sodium hydroxide, stir at 25-35°C until hydrolysis is complete, add 200 ml of water for crystallization, and filter to obtain 6.56 g of crude VD2. HPLC purity: 99.25%.

[0042] 6.10 g of crude VD2 was dissolved in 50 ml of anhydrous methanol at room temperature, cooled to 0±5°C, filtered to obtain VD2 product, and dried to obtain 4.85 g, with a yield of 48.5% and a HPLC purity of 99.71%. The HPLC spectrum of the product is shown in Figure 4 .

[0043] Embodiment 2: Some reaction conditions and parameters in this embodiment are slightly different from those in embodiment 1, as follows: The reaction solvent during the photoreaction was replaced by methyl tert-butyl ether from ethyl acetate, and the concentration of ergosterol in the ergosterol solution was adjusted to 1.2%; The first level of illumination time is 22 minutes, and the second level of illumination time is 32 minutes; After the illumination is completed, the second-stage illumination product is heated at 50-55°C for about 5.5h; The remaining steps such as esterification, hydrolysis, and refining conditions remained unchanged. The final VD2 product yield was 46.6%, HPLC purity was 99.70%, and the product HPLC spectrum was shown in Figure 5 .

[0044] Embodiment 3: Some reaction conditions and parameters in this embodiment are slightly different from those in embodiment 1, including: Adjust the concentration of ergosterol in the ergosterol solution to 1.25%; In the second-stage microfluidic photoreactor, the illumination wavelength was set to 385-390 nm; The first level of illumination time is 24 minutes, and the second level of illumination time is 32 minutes; After the illumination is completed, the second-stage illumination product is heated at 50-55°C for about 6.5h; The remaining steps such as esterification, hydrolysis, and refining conditions remained unchanged. The final VD2 product yield was 48.8%, HPLC purity was 99.74%, and the product HPLC spectrum was shown in Figure 6 .

[0045] Embodiment 4: Some reaction conditions and parameters in this embodiment are slightly different from those in embodiment 1, including: The reaction solvent during the photoreaction was replaced from ethyl acetate to anhydrous ethanol, and the concentration of ergosterol in the ergosterol solution was adjusted to 1.25%; In the second-stage microfluidic photoreactor, the illumination wavelength was set to 385-390 nm; The first level of illumination time is 24 minutes, and the second level of illumination time is 32 minutes; After the illumination is completed, the second-stage illumination product is heated at 50-55°C for about 6.5h; The remaining steps such as esterification, hydrolysis, and refining conditions remained unchanged. The final VD2 product yield was 46.8%, HPLC purity was 99.72%, and the product HPLC spectrum was shown in Figure 7 .

[0046] Embodiment 5: Some reaction conditions and parameters in this embodiment are slightly different from those in embodiment 1, including: Adjust the concentration of ergosterol in the ergosterol solution to 1.3%; In the second-stage microfluidic photoreactor, the wavelength of illumination was set to 395-400 nm; The first level of illumination time is 23 minutes, and the second level of illumination time is 32 minutes; After the illumination is completed, the second-stage illumination product is heated at 50-55°C for about 6h; The remaining steps such as esterification, hydrolysis, and refining conditions remained unchanged. The final VD2 product yield was 47.3%, HPLC purity was 99.74%, and the product HPLC spectrum was shown in Figure 8 .

[0047] Comparative Example 1: This comparative example refers to the method of patent CN110724081B.

[0048] 20g of ergosterol was dissolved in 250ml of cyclohexane, 20mg of 2,6-di-tert-butyl-p-methoxybenzylphenol was added, and the mixture was cooled to 0°C. Nitrogen was introduced into the reaction system, and the solution was irradiated with LED light of wavelength 275nm to undergo photochemical ring-opening reaction. The reaction temperature was controlled at 0±5°C. The reaction solution was taken for HPLC detection, and the remaining ergosterol was 25.4%, and the conversion rate was 74.6% (see Fig. 9 ), a photochemical ring-closing reaction occurred under the irradiation of LED light with a wavelength of 335nm, the reaction temperature was controlled at 0±5℃, the total reaction time was about 4 hours, and after the reaction was completed, the reaction solvent was recovered under reduced pressure, 30ml of ethyl acetate was added and heated under reflux for 5 minutes, then cooled to 0℃, and 3.5g of unconverted ergosterol was recovered by suction filtration.

[0049] Add 60 ml of isooctane to the mother liquor after filtration, heat and reflux for about 0.5 hours, then cool to 0°C to precipitate VD2, and filter to obtain the first batch of VD2 products. Dry in a vacuum dryer to obtain 5.3 g of VD2 with an HPLC purity of 96.41%. The remaining mother liquor is further heated for about 0.5 hours, then partially removed by reduced pressure and cooled to 0°C, and VD2 is precipitated again. The second batch of VD2 products is obtained by filtration, and 1.5 g of VD2 is obtained by drying in a vacuum dryer with an HPLC purity of 97.83%. The remaining mother liquor is further heated for 20 minutes, then partially removed by reduced pressure and cooled to 0°C, and the third batch of VD2 is precipitated. Dry in a vacuum dryer to obtain 0.5 g of VD2 with an HPLC purity of 98.11% and a yield of 36.5%. There are many types of impurities and they do not meet the limit requirements of the European Pharmacopoeia. See the HPLC spectrum of the product. Fig.10 .

[0050] The ergosterol conversion rate of the above examples and comparative examples and the related substances of the VD2 finished product were tested according to the European Pharmacopoeia method, as shown in Table 1 below.

[0051] Table 1 It can be seen that the synthesis method of VD2 provided by the present invention can not only significantly improve the conversion rate of ergosterol, but also improve the selectivity of the target product, effectively control the type and content of impurities in the product, meet the European Pharmacopoeia's limit requirements for impurities, and have a high prospect for industrial conversion due to the reasonable control of the concentration of ergosterol, the light source wavelength and the illumination time during the two-stage illumination reaction.

[0052] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing vitamin D2 with high conversion rate and high selectivity, characterized in that: The following steps are involved: S1. The ergosterol solution is passed through the first stage photoreactor, the light wavelength is controlled to be 254nm, the illumination time is 20 to 25min, and the ergosterol ring-opening product reaction liquid is obtained by the first photoreaction; S2. The ergosterol ring-opening product reaction solution is passed through a second-stage light reactor, the light wavelength is controlled to be 365 to 415 nm, the illumination time is 30 to 35 min, and the VD2 precursor reaction solution is obtained by the second light reaction; S3. The VD2 precursor reaction solution is heated and converted into VD2 conversion oil; S4. The VD2 conversion oil is subjected to esterification, crystallization, hydrolysis and recrystallization to obtain the VD2 finished product.

2. The method according to claim 1, characterized in that: The ergosterol solution is prepared by dissolving ergosterol and an antioxidant in a reaction solvent, and the concentration of ergosterol in the ergosterol solution is controlled to be 1-1.5%.

3. The method according to claim 2, characterized in that: The antioxidant is 2,6-di-tert-butyl-p-cresol, and the weight ratio of the antioxidant to ergosterol is controlled to be 1:200-500.

4. The method according to claim 2, characterized in that: The reaction solvent is methanol, ethanol, methyl tert-butyl ether, tetrahydrofuran, a mixed solvent of methanol and n-heptane, a mixed solvent of ethanol and n-heptane, a mixed solvent of methanol and n-hexane, or a mixed solvent of ethanol and n-hexane.

5. The method according to claim 1, characterized in that: The first-stage light irradiation reactor and the second-stage light irradiation reactor are both microfluidic reactors, and the light-transmitting material is both quartz glass.

6. The method according to claim 1, characterized in that: The light sources for the first light reaction and the second light reaction are both LED lamps.

7. The method according to claim 1, characterized in that: During the heating conversion, the heating temperature is controlled to be 50-55° C. and the heating time is 5-7 hours.

8. The method according to claim 1, characterized in that: During the esterification, the VD2 conversion oil is subjected to an esterification reaction with triethylamine and 3,5-dinitrobenzoyl chloride to obtain VD2 ester.

9. The method according to claim 1, characterized in that: The hydrolysis is carried out by adding sodium hydroxide or potassium hydroxide into a solvent, and the solvent is anhydrous ethanol.

10. The method according to claim 1, characterized in that: The solvent used in the crystallization is a mixed solvent of ethanol and ethyl acetate; the solvent used in the recrystallization is anhydrous methanol.

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