CN113185565B - Method for synthesizing and purifying 25-hydroxycholesterol - Google Patents
Method for synthesizing and purifying 25-hydroxycholesterol Download PDFInfo
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Abstract
The invention relates to the technical field of organic chemical synthesis, in particular to a method for synthesizing and purifying 25-hydroxycholesterol, which comprises the following steps: (1) mixing a compound of formula (I) and a compound of formula (II)Contacting the compound with an addition reagent to obtain an addition product; wherein the content of the compound shown in the formula (I) is 10-60wt% based on the total weight of the mixture; the addition reagent has a structure shown in a formula (III); in the formulae (I) and (II), R1Acyl of H or C1-C4; in the formula (III), R2Acyl of C1-C4; and, R1And R2The same or different; (2) saponifying the addition product with an alkali to obtain a saponified product; then the 25-hydroxycholesterol is obtained by crystallization and separation. The method for preparing 25-hydroxycholesterol provided by the invention has the advantages of easily available raw materials, simple process steps and safety.
Description
Technical Field
The invention relates to the technical field of organic chemical synthesis, in particular to a method for synthesizing and purifying 25-hydroxycholesterol.
Background
The 25-hydroxy vitamin D3 has wide application prospect and high economic value, and the 25-hydroxy cholesterol is an important raw material for synthesizing the 25-hydroxy vitamin D3. Existing methods for preparing 25-hydroxycholesterol generally include, for example:
(a) hydroxmhg process
The method (C.R. Chimie,6,79-82,2003) takes 24-dehydrocholesterol (or derivatives) as a raw material, adopts mercury acetate as a hydromercurization reaction, uses a highly toxic mercury reagent, has great pollution, has great potential safety hazard, is difficult to remove mercury residues, and is difficult to realize industrialization;
(b) epoxy oxidation process
The method (chem. pharm. Bull.21(2),457-458,1973) uses 24-dehydrocholesterol as a starting material, and performs epoxidation reaction under the condition that m-chloroperoxybenzoic acid is used as an epoxidation reagent, and after the epoxidation reaction is finished, the ring opening is performed by using lithium aluminum hydride. The method uses a peroxide and strong reducing lithium aluminum hydride, has high operation requirement and larger potential safety hazard, and m-CPBA epoxidizes the double bond of C24-C25 to generate oxide with poor selectivity, epoxidizes the double bond of C5-C6 to obtain a random mixture, causes that 25-hydroxycholesterol and a side reaction product are difficult to separate, and is not suitable for industrialization.
(c) Lewis acid catalyzed hydration process
The process route (CN104130306A) takes 24-dehydrocholesterol (or derivatives) as a raw material, under the catalysis of a Lewis acid catalyst and under the action of 1-100 eq of a hydration reagent, a double bond at the 24-position is opened, and the hydration reagent (R) is2H or acyl of C1-C4) and 24-dehydrocholesterol (or derivatives). The hydrated product is saponified with an alkali to obtain 25-hydroxycholesterol. The raw material of the route is high-content 24-dehydrocholesterol (or derivatives), which is difficult to obtain in industrial production, and a Lewis acid catalyst which is difficult to remove is used, even a Lewis acid catalyst containing heavy metal is used to obtain higher conversion rate, so that the route has potential safety hazard and large environmental protection pressure. It is difficult to realize industrialization.
In summary, in the prior art, pure 24-dehydrocholesterol (or derivative) is required to be used as a starting material for preparing 25-hydroxycholesterol, but the preparation of pure 24-dehydrocholesterol (or derivative) is high in cost and complex in operation, and is not beneficial to industrial production. Meanwhile, the existing method for preparing 25-hydroxycholesterol has the defects of complex process and potential safety hazard, and the raw material 24-dehydrocholesterol (or derivative) is not easy to obtain; therefore, a method for preparing 25-hydroxycholesterol, which is easy to obtain raw materials, simple in process and safe, is required.
Disclosure of Invention
The invention aims to overcome the technical problems of difficult raw material acquisition, complex preparation process and potential safety hazard in the prior art, and provides a method for preparing 25-hydroxycholesterol, which has the advantages of easy raw material acquisition, simple process steps, high safety factor and the like and is beneficial to realizing industrialization.
The inventor of the present invention has found through extensive research that, in the preparation of 25-hydroxycholesterol, a mixed material of 24-dehydrocholesterol (or derivative) + cholesterol (or derivative) can be used as a starting material, 24-dehydrocholesterol (or derivative) with high purity does not need to be obtained, a large amount of purification cost is saved (for example, pure 24-dehydrocholesterol is obtained by high-efficiency industrial preparation column purification, the cost is very high), and the preparation yield can reach a level equivalent to that of the prior art.
In order to achieve the above object, the present invention provides a method for synthesizing and purifying 25-hydroxycholesterol, the method comprising the steps of:
(1) contacting a mixture containing a compound shown in a formula (I) and a compound shown in a formula (II) with an addition reagent to obtain an addition product; wherein the content of the compound shown in the formula (I) is 10-60wt% based on the total weight of the mixture; the addition reagent has a structure shown in a formula (III);
in the formulae (I) and (II), R1Acyl of H or C1-C4; in the formula (III), R2Acyl of C1-C4; and, R1And R2The same or different;
(2) saponifying the addition product with an alkali to obtain a saponified product; then the 25-hydroxycholesterol is obtained by crystallization and separation.
Compared with the prior art, the method for synthesizing and purifying 25-hydroxycholesterol at least comprises the following technical advantages:
1) the invention provides a new route for synthesizing 25-hydroxycholesterol, which is simple to operate, adopts addition reagents which are commonly and easily obtained, can be recycled under the optimal condition, greatly reduces the production cost of 25-hydroxycholesterol, and is environment-friendly; more importantly, the mixture containing the compound shown in the formula (I) and the compound shown in the formula (II) (namely the mixture of cholesterol and derivatives thereof and 24-dehydrocholesterol and derivatives thereof) is used as a starting material, the raw material is easy to obtain, and the pure 24-dehydrocholesterol and derivatives thereof are not required to be separated, so that the cost is greatly reduced;
2) the method adopts simple organic acid as an addition reagent to carry out addition with the compound shown in the formula (I) and the 24-dehydrocholesterol and the derivatives thereof in the mixture shown in the formula (II), does not need a catalyst, avoids using a reagent (such as a Hg-containing reagent) which has great harm to the environment, and has mild addition reaction conditions, simple process and less side reaction;
3) compared with cholesterol, 24-dehydrocholesterol only has one more double bond at C24-C25, and the two compounds have similar properties, so that the separation is difficult by the conventional method; however, the inventors of the present invention have unexpectedly found that 25-hydroxycholesterol can be separated well by crystallization compared to cholesterol, and based on this, the present invention also provides a method for purifying 25-hydroxycholesterol from the reacted mixture (containing unreacted cholesterol), which not only allows separation of cholesterol, which is difficult to separate from the starting materials, in the synthesized product, but also provides the target 25-hydroxycholesterol with high purity.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a method for preparing 25-hydroxycholesterol, which comprises the following steps:
(1) contacting a mixture containing a compound shown in a formula (I) and a compound shown in a formula (II) with an addition reagent to obtain an addition product; wherein the content of the compound shown in the formula (I) is 10-60wt% based on the total weight of the mixture; the addition reagent has a structure shown in a formula (III);
in the formulae (I) and (II), R1Acyl which is H or C1-C4 (which may be, for example
Etc.); preferably, R1Acyl of H or C1-C2; in the formula (III), R2Acyl of C1-C4; and, R1And R2The same or different;
(2) saponifying the addition product with an alkali to obtain a saponified product; then the 25-hydroxycholesterol is obtained by crystallization and separation.
According to some embodiments of the present invention, the compound represented by formula (I) may be contained in an amount of 15 to 50wt% based on the total weight of the mixture.
In the present invention, preferably, the content of the compound represented by formula (I) in the mixture refers to an external standard content. Wherein, the external standard content is a liquid phase method which uses a pure product of the component to be detected as a reference substance and compares response signals of the reference substance and the component to be detected in a sample for quantification, is called an external standard method and can be detected by high performance liquid chromatography.
According to a preferred embodiment of the invention, the total content of the compound of formula (I) and the compound of formula (II) in the mixture is ≥ 97% by weight.
According to some embodiments of the invention, the addition reagent may be selected from at least one of trifluoroacetic acid, dichloroacetic acid, formic acid, acetic acid, propionic acid, butyric acid, oxalic acid and lactic acid, preferably from at least one of trifluoroacetic acid, dichloroacetic acid, formic acid, acetic acid and lactic acid.
In the present invention, it is preferable that when the addition reagent is in a liquid state, the addition reagent can serve as both the addition reagent and a solvent for the addition reaction.
According to some embodiments of the invention, the addition reagent is used in an amount of 1 to 100g, preferably 5 to 80g, with respect to 1g of the mixture.
According to some embodiments of the invention, the conditions of the contacting may comprise: the temperature is 0-115 ℃; the time is 1-10h, preferably 2-7 h.
In the present invention, the conditions of the contact (particularly, the temperature of the contact) may be varied depending on the addition agent.
According to some embodiments of the invention, the contacting is carried out in the presence of a first solvent, preferably the first solvent is selected from at least one of toluene, xylene, chlorobenzene, dichloromethane, chloroform, 1-dichloroethane and 1, 2-dichloroethane, more preferably from at least one of toluene, xylene, chlorobenzene, dichloromethane and 1, 2-dichloroethane.
According to some embodiments of the invention, the amount of the first solvent is 2 to 50mL, preferably 5 to 20mL, relative to 1g of the mixture.
According to some embodiments of the invention, the base may be selected from at least one of sodium hydroxide, potassium hydroxide and lithium hydroxide, preferably from sodium hydroxide and/or potassium hydroxide.
According to some embodiments of the invention, the base may be used in an amount of 0.05 to 0.5g, preferably 0.1 to 0.4g, relative to 1g of the mixture.
According to some embodiments of the invention, the method further comprises: extracting the addition product prior to the saponification reaction; wherein the extraction temperature is 0-110 ℃.
Wherein the extraction is carried out in the presence of a second solvent; preferably, the second solvent is selected from at least one of n-hexane, n-heptane, n-octane, petroleum ether, cyclohexane, iso-heptane and iso-octane; wherein the amount of the second solvent may be 5 to 50mL with respect to 1g of the mixture.
In the present invention, in order to prevent the occurrence of a reversible reaction during extraction, it is preferable that the temperature of the extraction be the same as the temperature of the addition reaction (the temperature of the contact).
According to some embodiments of the invention, the saponification conditions may include: the temperature is-15 ℃ to 80 ℃, preferably-5 ℃ to 50 ℃; the time is 1-10h, preferably 2-5 h.
According to some embodiments of the present invention, the saponification reaction is performed in the presence of a third solvent and a fourth solvent, wherein the third solvent may be a polar solvent selected from at least one of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, ethylene glycol, glycerol and acetone, preferably selected from methanol and/or ethanol; the fourth solvent may be a non-polar solvent selected from at least one of toluene, xylene, n-hexane, n-heptane, n-octane, petroleum ether, cyclohexane, isoheptane and isooctane; preferably at least one selected from the group consisting of toluene, n-hexane, n-heptane and n-octane.
In the present invention, the amount of the third solvent and the fourth solvent is not particularly limited as long as the requirements of the present invention can be satisfied, and preferably, the amount of the third solvent may be 2 to 5mL and the amount of the fourth solvent may be 1 to 4mL with respect to 1g of the mixture.
In the present invention, it is preferable that after the completion of the saponification, the method further comprises adding an acid to the system after the saponification, neutralizing the remaining alkali, and adjusting the pH of the system to 7 to 8. Among them, the acid is preferably a 10 to 36 wt% aqueous hydrochloric acid solution.
According to some embodiments of the invention, the step of crystallization separation may comprise: and sequentially carrying out thermal dissolution, water washing desalting, reflux water diversion and cooling crystallization on the saponified product.
Wherein the temperature of the thermal dissolution is preferably 40 to 100 ℃, more preferably 50 to 90 ℃.
The temperature of the water washing for desalting is 40-80 ℃, and more preferably 50-70 ℃. In the water-washing salt removal, the amount of water used is preferably 1 to 5g relative to 1g of the mixture.
The temperature of the reflux water separation is preferably 60-140 ℃, and more preferably 90-110 ℃. The amount of water removed in the reflux water separation is preferably 0.01 to 0.1g relative to 1g of the mixture.
The temperature of the temperature reduction crystallization is preferably 10-30 ℃, and more preferably 20-25 ℃.
In the present invention, the pressure of the reflux water separation is not particularly limited as long as the requirements of the present invention can be satisfied, and for example, the reflux water separation can be performed under normal pressure or reduced pressure.
According to a preferred embodiment of the present invention, the cooling rate of the temperature-decreasing crystallization is 15-25 ℃/h.
According to some embodiments of the present invention, the thermal dissolution is performed in the presence of a fifth solvent, which may be selected from at least one of toluene, xylene, ethyl acetate, n-hexane, n-heptane, n-octane, petroleum ether, cyclohexane, iso-heptane, and iso-octane, preferably from at least one of toluene, xylene, and n-octane.
According to a preferred embodiment of the present invention, the amount of the fifth solvent is 5 to 100mL, preferably 10 to 50mL, relative to 1g of the mixture.
In the present invention, preferably, the crystallization separation further comprises a first recrystallization (recrystallization) of the crude 25-hydroxycholesterol after cooling crystallization to obtain crude 25-hydroxycholesterol, to obtain 25-hydroxycholesterol (filter cake) and a filtrate; wherein the solvent for the first recrystallization (recrystallization) is selected from at least one of toluene, xylene, and n-octane.
In the present invention, preferably, after the first recrystallization (recrystallization) filtrate is evaporated to dryness, 25-hydroxycholesterol contained therein may be further recovered; wherein the cholesterol remaining after the separation by the crystallization and the separation by the first recrystallization (recrystallization) and the unreacted 24-dehydrocholesterol can be further removed by the second recrystallization to obtain a mixture having a 24-dehydrocholesterol content of 10 to 60wt%, and the mixture is used again for the addition reaction to prepare 25-hydroxycholesterol. Wherein the solvent for the second recrystallization may be selected from at least one of methanol, ethanol, n-propanol, isopropanol, acetic acid, n-hexane, n-heptane, n-octane, toluene, xylene, and ethyl acetate.
The present invention will be described in detail below by way of examples.
In the following examples, the external standard content was measured by high performance liquid chromatography external standard method; the yield of 25-hydroxycholesterol was calculated based on external standard content.
The yield of 25-hydroxycholesterol is (weight of 25-hydroxycholesterol obtained by crystallization × weight of external standard content of 25-hydroxycholesterol in 25-hydroxycholesterol obtained by crystallization × molecular weight of 24-dehydrocholesterol (or derivative)/(weight of mixture × weight of 24-dehydrocholesterol (or derivative) in mixture × molecular weight of 25-hydroxycholesterol).
Example 1
42.6g of the mixture (external standard content: 24-dehydrocholesterol acetyl ester content: 31.3 wt% (13.3g, 31.4mmol), cholesterol acetyl ester content: 67.6 wt%) was added to a 10L three-necked flask, and the mixture was dissolved in 500mL of xylene, and 4500g (50mol) of lactic acid was added dropwise with stirring, 0.5h after completion of addition reaction, and after addition reaction at 55 ℃ for 5h with stirring, the reaction was followed by HPLC until 24-dehydrocholesterol acetyl ester did not decrease any more; extracting the reaction solution after the addition reaction by using n-octane (1000mL multiplied by 2 times) at the temperature of 55 ℃, layering to obtain an n-octane phase and a residual acid phase, wherein the residual acid phase can be continuously used for the addition reaction, combining the n-octane phases obtained by the two extractions, evaporating to dryness to obtain 45.1g of an adduct, sequentially adding 11.2g (200mmol) of potassium hydroxide, 160mL of methanol and 100mL of n-octane into the adduct, reacting for 3 hours at the temperature of 25 ℃, and then carrying out HPLC tracking reaction until the reaction is complete; and (2) dropwise adding 32 wt% hydrochloric acid solution into a system after saponification reaction to adjust the pH value to 7, evaporating to dryness to obtain a saponification product, cooling to 80 ℃, adding 500mL of n-octane into the saponification product, washing with water at 60 ℃ to remove salt, carrying out reduced pressure reflux water diversion at 110 ℃, reducing the water removal amount to 1.3g, cooling to 20 ℃ for crystallization, filtering to obtain a 25-hydroxycholesterol crude product, and recrystallizing with 500mL of n-octane to obtain 10.8g (26.9mmol) of 25-hydroxycholesterol, wherein the external standard content is more than 99%, and the yield is 85.2%.
Characterization of compound (25-hydroxycholesterol):
melting point: 179.4-180.8 ℃; nuclear magnetism:1H-NMR(CDCl3400M) delta 5.34(M, 1H), 3.51(M, 1H), 2.27(M, 2H), 1.99(M, 2H), 1.83(M, 3H), 1.22-1.69(M, 18H), 1.21(s, 6H,), 1.06-1.16(M, 4H), 1.00(s, 3H), 0.93(d, 3H), 0.68(s, 3H); mass spectrum: MS-EI: 403.7[ M +1 ]]+。
Example 2
Adding 42.6g of the mixture (external standard content: 24-dehydrocholesterol acetyl ester content: 31.3 wt% (13.3g, 31.4mmol), cholesterol acetyl ester content: 67.6 wt%) into a 10L three-necked flask, dissolving the mixture with 500mL of xylene, then dropwise adding 3600g (40mol) of acetic acid under stirring, 0.5h after dropwise addition, and carrying out addition reaction at 100 ℃ for 4h under stirring, and then carrying out HPLC tracking reaction until the 24-dehydrocholesterol acetyl ester is not reduced any more; extracting the reaction solution after the addition reaction by using n-octane (1000mL multiplied by 2 times) at the temperature of 100 ℃, layering to obtain an n-octane phase and a residual acid phase, wherein the residual acid phase can be continuously used for the addition reaction, combining the n-octane phases obtained by the 2 times of extraction, evaporating to dryness to obtain 44.8g of an adduct, sequentially adding 11.2g (200mmol) of potassium hydroxide, 160mL of methanol and 100mL of xylene into the adduct, reacting for 3 hours at the temperature of 25 ℃, and then carrying out HPLC tracking reaction until the reaction is complete; and (2) dropwise adding 32 wt% hydrochloric acid solution into a system after saponification reaction to adjust the pH value to 7, evaporating to dryness to obtain a saponification product, cooling to 80 ℃, adding 500mL of n-octane into the saponification product, washing with water at 60 ℃ to remove salt, using 150mL of water, carrying out reduced pressure reflux water separation at 105 ℃, removing 1.5g of water, cooling to 20 ℃ to crystallize, filtering to obtain a 25-hydroxycholesterol crude product, recrystallizing with 500mL of n-octane to obtain 10.5g (26.1mmol) of 25-hydroxycholesterol, wherein the external standard content is more than 99%, and the yield is 83.1%.
The compound was characterized as 25-hydroxycholesterol.
Example 3
42.6g of the mixture (external standard content: 24-dehydrocholesterol acetyl ester content: 31.3% by weight (13.3g, 31.4mmol), cholesterol acetyl ester content: 67.6% by weight) was added to a 5L three-necked flask, and the mixture was dissolved in 500mL of 1, 2-dichloroethane, and 1380g (30mol) of formic acid was added dropwise with stirring, 0.5h was added after completion of the addition reaction, and after 4h of addition reaction with stirring at 60 ℃, the reaction was followed by HPLC until 24-dehydrocholesterol acetyl ester did not decrease any more; extracting the reaction liquid of the addition reaction with petroleum ether (1000mL multiplied by 2 times) at 60 ℃, layering to obtain a petroleum ether phase and a residual acid phase, wherein the residual acid phase can be continuously used for the addition reaction, combining the petroleum ether phases obtained by 2 times of extraction, evaporating to dryness to obtain 44.0g of an adduct, sequentially adding 11.2g (200mmol) of potassium hydroxide, 160mL of methanol and 100mL of toluene into the adduct, carrying out saponification reaction for 3 hours under stirring at 25 ℃, tracking the reaction by HPLC to be complete, dripping 32 wt% hydrochloric acid solution into the system after the saponification reaction to adjust the pH to 7, evaporating to dryness to obtain a salt-containing saponification product, cooling to 80 ℃, adding 500mL of xylene into the saponification product, washing with water at 60 ℃, washing with 100mL of water, carrying out water separation under reduced pressure and reflux at 105 ℃, removing 1.1g of water, cooling to 20 ℃ for crystallization, filtering to obtain a crude 25-hydroxyl cholesterol product, then recrystallizing with 500mL xylene to obtain 9.58g (23.84mmol) of 25-hydroxycholesterol, with external standard content of more than 99% and yield of 75.9%.
The compound was characterized as 25-hydroxycholesterol.
Example 4
42.6g of the mixture (external standard content: 24-dehydrocholesterol acetyl ester content: 31.3 wt% (13.3g, 31.4mmol), cholesterol acetyl ester content: 67.6 wt%) was added to a 5L three-necked flask, and 500mL of toluene was used to dissolve the mixture, and then 2220g (30mol) of propionic acid was added dropwise with stirring for 0.5h after completion, and after addition reaction at 60 ℃ for 5h with stirring, HPLC tracing the reaction until 24-dehydrocholesterol acetyl ester did not decrease any more; extracting the reaction solution after the addition reaction by using n-octane (1000mL multiplied by 2 times) at the temperature of 60 ℃, layering to obtain an n-octane phase and a residual acid phase, wherein the residual acid phase can be continuously used for the addition reaction, combining the n-octane phases obtained by the 2 times of extraction, evaporating to dryness to obtain 44.7g of an adduct, sequentially adding 11.2g (200mmol) of potassium hydroxide, 160mL of methanol and 100mL of toluene into the adduct, reacting for 3 hours at the temperature of 25 ℃, and then carrying out HPLC tracking reaction until the reaction is complete; and (2) dropwise adding 32 wt% hydrochloric acid solution into a system after saponification reaction to adjust the pH value to 7, evaporating to dryness to obtain a saponification product, cooling to 80 ℃, adding 500mL of toluene into the saponification product, washing with water at 60 ℃ to remove salt, using 150mL of water, carrying out reduced pressure reflux water separation at 110 ℃, removing 0.9g of water, cooling to 20 ℃ to crystallize, filtering to obtain a 25-hydroxycholesterol crude product, recrystallizing with 500mL of toluene to obtain 9.31g (23.2mmol) of 25-hydroxycholesterol, wherein the external standard content is more than 99%, and the yield is 73.9%.
The compound was characterized as 25-hydroxycholesterol.
Example 5
38.67g of the mixture (external standard content: 24-dehydrocholesterol content: 17.6 wt% (6.81g, 17.7mmol), cholesterol content: 81.6 wt%) was charged into a 5L three-necked flask, and 386mL of xylene was used to dissolve the mixture at 25 ℃, and then 3225g (25mol) of dichloroacetic acid was added dropwise with stirring, 0.5h was taken out after dropwise addition, and after addition reaction was carried out for 3h with stirring at 25 ℃, HPLC followed until 24-dehydrocholesterol was not reduced any more; extracting the reaction solution after the addition reaction by using cyclohexane (1000mL multiplied by 2) at 25 ℃, layering to obtain a cyclohexane phase and a residual acid phase, wherein the residual acid phase can be continuously used for the addition reaction, merging the cyclohexane phases obtained by 2 times of extraction, evaporating to dryness to obtain 43.7g of an adduct, sequentially adding 9.6g (400mmol) of lithium hydroxide, 160mL of methanol and 100mL of cyclohexane into the adduct, performing saponification reaction for 3h under stirring at 25 ℃, performing HPLC tracking reaction until the reaction is complete, dropwise adding 32 wt% hydrochloric acid solution into the system after the saponification reaction to adjust the pH to 8, evaporating to dryness to obtain a salt-containing saponification product, cooling to 80 ℃, adding 500mL of xylene into the saponification product, washing with water at 60 ℃ to remove salt, using 100mL of water, performing reflux and water separation under reduced pressure at 105 ℃, removing 0.8g of water, cooling to 20 ℃ for crystallization, filtering to obtain a crude 25-hydroxyl cholesterol product, then recrystallizing with 500mL xylene to obtain 25-hydroxycholesterol 5.17g (12.86mmol), the external standard content is more than 99%, and the yield is 72.6%.
The compound was characterized as 25-hydroxycholesterol.
Example 6
38.67g of the mixture (external standard content: 24-dehydrocholesterol content: 17.6 wt% (6.81g, 17.7mmol), cholesterol content: 81.6 wt%) was added to a 5L three-necked flask, and the mixture was dissolved in 386mL of methylene chloride at 25 ℃, and 2280g (20mol) of trifluoroacetic acid was added dropwise with stirring for 0.5h after completion, and after addition reaction was carried out at 0 ℃ for 2.5h with stirring, HPLC trace reaction was carried out until 24-dehydrocholesterol was not reduced any more; extracting the reaction solution after the addition reaction by using n-hexane (1000mL multiplied by 2) at 0 ℃, layering to obtain an n-hexane phase and a residual acid phase, wherein the residual acid phase can be continuously used for the addition reaction, combining the n-hexane phases obtained by 2 times of extraction, evaporating to dryness to obtain 42.5g of an adduct, sequentially adding 100mL of ethanol, 80mL of n-hexane and 10g (250mmol) of sodium hydroxide into the adduct, performing saponification reaction for 3 hours under stirring at 25 ℃, and performing HPLC tracking reaction to be complete; and (2) dropwise adding 32 wt% hydrochloric acid solution into a system after saponification reaction to adjust the pH value to 7, evaporating to dryness to obtain a salt-containing saponified product, cooling to 80 ℃, adding 600mL of toluene into the saponified product, washing with water at 60 ℃ to remove salt, using 100mL of water, refluxing and dividing water at 110 ℃ under normal pressure, removing 1.1g of water, cooling to 20 ℃ for crystallization, filtering to obtain a 25-hydroxycholesterol crude product, recrystallizing with 600mL of toluene to obtain 4.9g (12.14mmol) of 25-hydroxycholesterol, wherein the external standard content is more than 99%, and the yield is 68.7%.
The compound was characterized as 25-hydroxycholesterol.
Example 7
42.6g of the mixture (external standard content: 24-dehydrocholesterol acetyl ester content: 31.3 wt% (13.3g, 31.4mmol), cholesterol acetyl ester content: 67.6 wt%) was added to a 5L three-necked flask, and the mixture was dissolved in 500mL of chlorobenzene, and 1800g (30mol) of acetic acid was added dropwise with stirring for 0.5 hour after completion, and after addition reaction was carried out at 60 ℃ for 5 hours with stirring, the reaction was followed by HPLC until 24-dehydrocholesterol acetyl ester did not decrease any more; extracting the reaction solution after the addition reaction by using n-octane (1000mL multiplied by 2 times) at 60 ℃, layering to obtain an n-octane phase and a residual acid phase, wherein the residual acid phase can be continuously used for the addition reaction, combining the n-octane phases obtained by the 2 times of extraction, evaporating to dryness to obtain 44.2g of an adduct, sequentially adding 11.2g (200mmol) of potassium hydroxide, 160mL of methanol and 100mL of toluene into the adduct, reacting for 3 hours at 25 ℃, and then carrying out HPLC tracking reaction until the reaction is complete; and (2) dropwise adding 32 wt% hydrochloric acid solution into a system after saponification reaction to adjust the pH value to 7, evaporating to dryness to obtain a saponified product, cooling to 80 ℃, adding 500mL of n-octane into the saponified product, washing with water at 60 ℃ to remove salt, using 150mL of water, carrying out reduced pressure reflux water separation at 110 ℃, removing 1.5g of water, cooling to 20 ℃ to crystallize, filtering to obtain a 25-hydroxycholesterol crude product, recrystallizing with 500mL of n-octane to obtain 8.65g (21.4mmol) of 25-hydroxycholesterol, wherein the external standard content is more than 99%, and the yield is 68.2%.
The compound was characterized as 25-hydroxycholesterol.
Example 8
42.6g of the mixture (external standard content: 24-dehydrocholesterol acetyl ester content: 31.3 wt% (13.3g, 31.4mmol), cholesterol acetyl ester content: 67.6 wt%) was added to a 10L three-necked flask, and the mixture was dissolved in 1000mL of xylene, and 4500g (50mol) of lactic acid was added dropwise with stirring, 0.5h after completion of addition reaction, and after addition reaction at 55 ℃ for 5h with stirring, the reaction was followed by HPLC until 24-dehydrocholesterol acetyl ester did not decrease any more; extracting the reaction solution after the addition reaction by using n-octane (1000mL multiplied by 2 times) at the temperature of 55 ℃, layering to obtain an n-octane phase and a residual acid phase, wherein the residual acid phase can be continuously used for the addition reaction, combining the n-octane phases obtained by the two extractions, evaporating to dryness to obtain 39.1g of an adduct, sequentially adding 11.2g (200mmol) of potassium hydroxide, 160mL of methanol and 100mL of n-octane into the adduct, reacting for 3 hours at the temperature of 25 ℃, and then carrying out HPLC tracking reaction until the reaction is complete; and (2) dropwise adding 32 wt% hydrochloric acid solution into a system after saponification reaction to adjust the pH value to 7, evaporating to dryness to obtain a saponification product, cooling to 80 ℃, adding 500mL of n-octane into the saponification product, washing with water at 60 ℃ to remove salt, carrying out reduced pressure reflux water diversion at 110 ℃, reducing the water removal amount to 1.1g, cooling to 20 ℃ for crystallization, filtering to obtain a 25-hydroxycholesterol crude product, and recrystallizing with 500mL of n-octane to obtain 3.7g (9.20mmol) of 25-hydroxycholesterol, wherein the external standard content is more than 99%, and the yield is 29.3%.
The compound was characterized as 25-hydroxycholesterol.
Example 9
42.6g of the mixture (external standard content: 24-dehydrocholesterol acetyl ester content: 31.3 wt% (13.3g, 31.4mmol), cholesterol acetyl ester content: 67.6 wt%) was added to a 10L three-necked flask, and the mixture was dissolved in 500mL of xylene, and 3600g (40mol) of acetic acid was added dropwise with stirring, 0.5h was taken out after the addition reaction was completed, and after 4h was added with stirring at 50 ℃, the reaction was followed by HPLC until 24-dehydrocholesterol acetyl ester did not decrease any more; extracting the reaction solution after the addition reaction by using n-octane (1000mL multiplied by 2 times) at the temperature of 50 ℃, layering to obtain an n-octane phase and a residual acid phase, wherein the residual acid phase can be continuously used for the addition reaction, combining the n-octane phases obtained by the 2 times of extraction, evaporating to dryness to obtain 41.8g of an adduct, sequentially adding 11.2g (200mmol) of potassium hydroxide, 160mL of methanol and 100mL of xylene into the adduct, reacting for 3 hours at the temperature of 25 ℃, and then carrying out HPLC tracking reaction until the reaction is complete; and (2) dropwise adding 32 wt% hydrochloric acid solution into a system after saponification reaction to adjust the pH value to 7, evaporating to dryness to obtain a saponified product, cooling to 80 ℃, adding 500mL of n-octane into the saponified product, washing with water at 60 ℃ to remove salt, using 150mL of water, carrying out reduced pressure reflux water separation at 105 ℃, removing 1.5g of water, cooling to 20 ℃ to crystallize, filtering to obtain a 25-hydroxycholesterol crude product, recrystallizing with 500mL of n-octane to obtain 4.1g (10.19mmol) of 25-hydroxycholesterol, wherein the external standard content is more than 99%, and the yield is 32.4%.
The compound was characterized as 25-hydroxycholesterol.
Comparative example 1
Preparation of 25-hydroxycholesterol starting from pure 24-dehydrocholesterol acetyl ester:
42.6g of the mixture (external standard content: 98.7 wt% of 24-dehydrocholesterol acetyl ester (98.7mmol) and 1.3 wt% of cholesterol acetyl ester) was added to a 10L three-necked flask, and 500mL of xylene was used to dissolve the mixture, and 4500g (50mol) of lactic acid was added dropwise with stirring, 0.5 hour was taken after the addition reaction was completed, and after 5 hours of addition reaction with stirring at 55 ℃, the HPLC tracing reaction was carried out until 24-dehydrocholesterol acetyl ester was no longer reduced; extracting the reaction solution after the addition reaction by using n-octane (1000mL multiplied by 2) at 55 ℃, layering to obtain an n-octane phase and a residual acid phase, wherein the residual acid phase can be continuously used for the addition reaction, combining the n-octane phases obtained by two times of extraction, evaporating to dryness to obtain 47.5g of an addition product, sequentially adding 11.2g (200mmol) of potassium hydroxide, 160mL of methanol and 100mL of n-octane into the addition product, reacting for 3 hours at 25 ℃, and then carrying out HPLC tracking reaction until the reaction is complete; and (2) dropwise adding 32 wt% hydrochloric acid solution into a system after saponification reaction to adjust the pH value to 7, evaporating to dryness to obtain a saponification product, cooling to 80 ℃, adding 500mL of n-octane into the saponification product, washing with water at 60 ℃ to remove salt, carrying out reduced pressure reflux water diversion at 110 ℃, reducing the water removal amount to 1.3g, cooling to 20 ℃ for crystallization, filtering to obtain a 25-hydroxycholesterol crude product, and recrystallizing with 500mL of n-octane to obtain 34.2g (85.1mmol) of 25-hydroxycholesterol, wherein the external standard content is greater than 99%, and the yield is 86.2%.
The compound was characterized as 25-hydroxycholesterol.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (14)
1. A method for synthesizing and purifying 25-hydroxycholesterol, which is characterized by comprising the following steps:
(1) contacting a mixture containing a compound shown in a formula (I) and a compound shown in a formula (II) with an addition reagent to obtain an addition product; wherein the content of the compound shown in the formula (I) is 10-60wt% based on the total weight of the mixture; the addition reagent has a structure shown in a formula (III);
the addition reagent is selected from at least one of trifluoroacetic acid, dichloroacetic acid, formic acid, acetic acid, propionic acid, butyric acid and lactic acid;
the conditions of the contacting include: the temperature is 0-115 ℃; the time is 2-7 h;
the contacting is carried out in the presence of a first solvent selected from at least one of toluene, xylene, chlorobenzene, dichloromethane, and 1, 2-dichloroethane; the amount of the first solvent is 5-20mL relative to 1g of the mixture;
in the formulae (I) and (II), R1Acyl of H or C1-C4; in the formula (III), R2Acyl of C1-C4; and, R1And R2The same or different;
(2) saponifying the addition product with an alkali to obtain a saponified product; then 25-hydroxycholesterol is obtained through crystallization separation;
the step of crystallization separation comprises: sequentially carrying out thermal dissolution, water washing desalting, reflux water diversion and cooling crystallization on the saponified product; the thermal dissolution is carried out in the presence of a fifth solvent selected from at least one of toluene, xylene, n-heptane and n-octane;
the method further comprises the following steps: extracting the addition product prior to the saponification reaction;
wherein the extraction is carried out in the presence of a second solvent; the second solvent is selected from at least one of n-hexane, n-heptane, n-octane, petroleum ether, cyclohexane, iso-heptane and iso-octane; the amount of the second solvent is 5 to 50mL with respect to 1g of the mixture.
2. The method according to claim 1, wherein the compound of formula (I) is present in an amount of 15-50wt%, based on the total weight of the mixture;
and/or the total content of the compound shown in the formula (I) and the compound shown in the formula (II) in the mixture is more than or equal to 97 wt%.
3. The process according to claim 1 or 2, wherein the addition reagent is selected from at least one of trifluoroacetic acid, dichloroacetic acid, formic acid, acetic acid and lactic acid.
4. The process according to claim 1 or 2, wherein the addition reagent is used in an amount of 1-100g with respect to 1g of the mixture.
5. The process according to claim 1 or 2, wherein the addition reagent is used in an amount of 5-80g with respect to 1g of the mixture.
6. The process according to claim 1 or 2, wherein the base is selected from at least one of sodium hydroxide, potassium hydroxide and lithium hydroxide;
and/or the amount of the base is 0.05 to 0.5g relative to 1g of the mixture.
7. The process according to claim 1 or 2, wherein the base is selected from sodium hydroxide and/or potassium hydroxide;
and/or the amount of the base is 0.1 to 0.4g relative to 1g of the mixture.
8. The process of claim 1 or 2, wherein the saponification conditions include: the temperature is-15 ℃ to 80 ℃; the time is 1-10 h;
and/or, the saponification reaction is carried out in the presence of a third solvent and a fourth solvent, wherein the third solvent is selected from at least one of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, ethylene glycol, glycerol and acetone; the fourth solvent is at least one selected from toluene, xylene, n-hexane, n-heptane, n-octane, petroleum ether, cyclohexane, isoheptane and isooctane.
9. The process of claim 1 or 2, wherein the saponification conditions include: the temperature is-5 ℃ to 50 ℃; the time is 2-5 h;
and/or, the third solvent is selected from methanol and/or ethanol; the fourth solvent is at least one selected from toluene, n-hexane, n-heptane and n-octane.
10. The method of claim 1 or 2, wherein the temperature of the thermal dissolution is 40-100 ℃;
the temperature of the water washing desalination is 40-80 ℃; in the water washing desalting, the amount of water is 1-5g relative to 1g of the mixture;
the temperature of the reflux water diversion is 60-140 ℃; the amount of water removed from the reflux water cut is 0.01 to 0.1g relative to 1g of the mixture;
the temperature of the cooling crystallization is 10-30 ℃.
11. The method of claim 1 or 2, wherein the temperature of the thermal dissolution is 50-90 ℃;
the temperature of the water washing desalination is 50-70 ℃;
the temperature of the reflux water diversion is 90-110 ℃;
the temperature of the cooling crystallization is 20-25 ℃.
12. The method of claim 10, wherein the temperature-decreasing crystallization is performed at a temperature-decreasing rate of 15-25 ℃/h.
13. The method according to claim 1 or 2, wherein the amount of the fifth solvent is 5-100mL with respect to 1g of the mixture.
14. The method according to claim 1 or 2, wherein the amount of the fifth solvent is 10-50mL with respect to 1g of the mixture.
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