A kind of preparation method of estradiol

Abstract

The invention provides a method for preparing estradiol, which comprises: using boldenone prepared by ADD enzyme conversion reduction as a raw material, and preparing estradiol only through a one-step reaction; the existing estradiol preparation process uses estrone prepared by ADD through three steps of ketal protection, aromatization and deprotection as a raw material, and preparing estradiol through reduction; compared with the existing estradiol preparation process, the invention shortens the overall process route steps, reduces the use of materials, reduces energy consumption and pollution, and effectively reduces the content of isomer impurity 17α-estradiol in the estradiol product, thereby improving the product yield and product quality.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical compounds, and in particular to a method for preparing estradiol. Background Art

[0002] As a naturally occurring estrogen, estradiol is rich in content and highly active, and is mainly used to treat female sexual dysfunction, osteoporosis and menopausal syndrome. The most common estradiol synthesis route currently uses estrone as the raw material and obtains estradiol through reduction. Estrone is prepared from 1,4-androstadiene-3,17-dione (ADD) through ethylene glycol acetal protection, aromatization reaction, and deprotection reaction in three steps. The entire estradiol process involves four steps of reaction as follows:

[0003]

[0004] In the process of preparing estrone by ADD, in order to avoid the influence on the 17-position keto group during the aromatization reaction, the keto group needs to be protected and deprotected in the route, resulting in the additional use of protection reagents and deprotection reagents, resulting in an increase in production costs and waste emissions. In addition, due to the increase in reaction steps, the total yield of the product is inevitably reduced. On the other hand, when estrone is reduced to prepare estradiol, the by-product 17α-estradiol isomer is inevitably produced, and the content is generally 0.5-1.0%, which exceeds the control limit of the European Pharmacopoeia (impurity B requires ≤0.3%). It is necessary to focus on removing this impurity during the refining process, which leads to a decrease in yield.

[0005] The patent with publication number CN106866764B discloses a preparation process and a preparation system of estradiol, which uses ADD as a raw material and prepares estradiol through a complex process including ketal protection, aromatization reaction, deprotection, decolorization and refining, reduction reaction and decolorization and refining. The yield after refining is low, and the removal of 17α-estradiol isomers is not mentioned. Summary of the invention

[0006] In view of this, the present invention proposes a method for preparing estradiol, which solves the problems of multiple process steps, extra material consumption, and high content of isomer impurities in the product in the prior art.

[0007] The technical solution of the present invention is achieved in that the present invention provides a method for preparing estradiol, comprising the following steps:

[0008] S1. Dissolve biphenyl and diphenylmethane in tetrahydrofuran, introduce nitrogen protection, add metallic lithium, raise the temperature to initiate the reaction, react for 2 to 4 hours to prepare the lithium reagent, and cool down to 40 to 50°C;

[0009] S2, adding a tetrahydrofuran solution of boldenone to the reaction solution in step S1, reacting at 40-50° C. for 0.5-1 h, until TLC shows that the raw material has reacted completely, cooling to below 10° C., adding methanol dropwise for quenching, adding hydrochloric acid solution to adjust the pH value of the reaction solution to 2-5, and then concentrating under reduced pressure using a rotary evaporator at 40-50° C. until the organic solvent is spin-dried;

[0010] S3, adding an organic solvent immiscible with water to the concentrate in step S2 to dissolve the crystals, and then filtering and drying to obtain estradiol.

[0011] Based on the above technical solution, preferably, in step S1, the ratio of biphenyl, diphenylmethane, metallic lithium and tetrahydrofuran is 1:(0.6-0.8):(0.09-0.18):(5-15) (w / w / w / v).

[0012] On the basis of the above technical solution, preferably, the initiation temperature for preparing the lithium reagent in step S1 is 65-70°C.

[0013] On the basis of the above technical solution, preferably, in the tetrahydrofuran solution of boldenone in step S2, the ratio of boldenone to tetrahydrofuran is 1:(3-6) (w / v).

[0014] On the basis of the above technical solution, preferably, in step S2, the mass ratio of boldenone to biphenyl is (1-3):1.

[0015] On the basis of the above technical solution, preferably, in step S2, the TLC developing agent includes toluene and ethanol, the color developing agent includes vanillin ethanol solution, and the volume ratio of toluene to ethanol is (15-10):1.

[0016] Based on the above technical solution, preferably, the molar concentration of the hydrochloric acid solution in step S2 is 1.5-2M.

[0017] On the basis of the above technical solution, preferably, the organic solvent immiscible with water in step S3 includes dichloromethane or chloroform.

[0018] On the basis of the above technical solution, preferably, the dissolving process in step S3 includes stirring under ice water cooling, and the stirring time is 0.5-1h.

[0019] On the basis of the above technical solution, preferably, the drying process in step S3 includes forced air drying, the drying temperature is 40-50° C., and the drying time is 4-6 hours.

[0020] The preparation method of estradiol of the present invention has the following beneficial effects compared with the prior art:

[0021] (1) The present invention uses boldenone as a raw material, and only uses an ADD enzyme reduction reaction to prepare estradiol. Compared with the current estrone reduction method, the raw material estrone is protected by ADD ketal, aromatized and prepared, which shortens the overall process route steps and reduces material usage and energy consumption.

[0022] (2) The crude estradiol obtained by the existing estradiol preparation process has an isomer 17α-estradiol content of about 0.5-1%, while the content of 17α-estradiol in the present application is only 0.05%, which effectively reduces the content of the isomer impurity 17α-estradiol in the estradiol product and greatly improves the product quality.

[0023] (3) The optimized post-treatment process of the present application directly dissolves and crystallizes the reaction solution concentrate through a mixed system consisting of a water-immiscible solvent and water, thereby removing inorganic salts and organic impurities in the product at one time, and directly obtaining estradiol with a purity of more than 98%, thereby avoiding multiple extractions and solvent consumption in conventional extraction operations, as well as the concentration and crystallization process, and greatly simplifying the post-treatment operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 HPLC spectrum of estradiol prepared by the present invention (ethyl acetate post-treatment);

[0026] Figure 2 HPLC spectrum of estradiol prepared by the present invention (dichloromethane post-treatment);

[0027] Figure 3 HPLC spectrum of estradiol prepared from estrone by sodium borohydride reduction;

[0028] Figure 4 The figure is the MS spectrum of the impurity isomer 17α-estradiol separated by the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] A method for preparing estradiol comprises the following steps:

[0032] S1. Dissolve biphenyl and diphenylmethane in tetrahydrofuran, introduce nitrogen protection, add metallic lithium, raise the temperature to 65°C to initiate the reaction, react at 65°C for 2h to prepare the lithium reagent, and then cool to 40°C;

[0033] S2, to the reaction solution in step S1, add a tetrahydrofuran solution of boldenone dropwise at a ratio of 1:5 (w / v) of boldenone to tetrahydrofuran, react at 40°C for 0.5h, until TLC shows that the raw material reaction is complete (developing agent: toluene / ethanol volume ratio is 15:1, color developer: vanillin ethanol solution), cool to 5°C, add methanol dropwise for quenching, add 1.5M hydrochloric acid solution to adjust the pH value of the reaction solution to 2, and then use a rotary evaporator to concentrate under reduced pressure at 40°C until the organic solvent is spin-dried;

[0034] S3. Add dichloromethane to the concentrate in step S2 to dissolve the crystals, cool with ice water, stir for 0.5 h, filter, drain, rinse with a small amount of cold dichloromethane, drain, and dry with air at 40° C. for 4 h to obtain estradiol.

[0035] Example 2

[0036] A method for preparing estradiol comprises the following steps:

[0037] S1. Dissolve biphenyl and diphenylmethane in tetrahydrofuran, introduce nitrogen protection, add metallic lithium, heat to 70°C to initiate reaction, react at 70°C for 4h to prepare lithium reagent, and cool to 50°C;

[0038] S2, to the reaction solution in step S1, add a tetrahydrofuran solution of boldenone dropwise at a ratio of 1:5 (w / v) of boldenone to tetrahydrofuran, react at 50°C for 1 h, until TLC shows that the raw material reaction is complete (developing agent: toluene / ethanol volume ratio is 10:1, color developer: vanillin ethanol solution), cool to 0°C, add methanol dropwise for quenching, add 2M hydrochloric acid solution to adjust the pH value of the reaction solution to 5, and then use a rotary evaporator at 50°C to concentrate under reduced pressure until the organic solvent is spin-dried;

[0039] S3. Add dichloromethane to the concentrate in step S2 to dissolve the crystals, cool with ice water, stir for 1 hour, filter, drain, rinse with a small amount of cold dichloromethane, drain, and dry with air at 50° C. for 6 hours to obtain estradiol.

[0040] Example 3

[0041] A method for preparing estradiol comprises the following steps:

[0042] S1. Dissolve biphenyl and diphenylmethane in tetrahydrofuran, introduce nitrogen protection, add metallic lithium, raise the temperature to 68°C to initiate the reaction, react at 68°C for 3h to prepare the lithium reagent, and then cool to 45°C;

[0043] S2, to the reaction solution in step S1, add a tetrahydrofuran solution of boldenone dropwise at a ratio of 1:4 (w / v) of boldenone to tetrahydrofuran, react at 45°C for 0.8h, until TLC shows that the raw material reaction is complete (developing agent: toluene / ethanol volume ratio is 12:1, color developer: vanillin ethanol solution), cool to 4°C, add methanol dropwise for quenching, add 1.8M hydrochloric acid solution to adjust the pH value of the reaction solution to 3, and then use a rotary evaporator to concentrate under reduced pressure at 45°C until the organic solvent is spin-dried;

[0044] S3. Add chloroform to the concentrate in step S2 to dissolve the crystals, cool with ice water, stir for 0.8 h, filter, drain, rinse with a small amount of cold chloroform, drain, and dry with air at 45° C. for 5 h to obtain estradiol.

[0045] Embodiment 4-6

[0046] A method for preparing estradiol, which differs from Example 1 in that the ratio of biphenyl, diphenylmethane, metallic lithium and tetrahydrofuran and the initiation temperature of the lithium reagent are different.

[0047] Comparative Examples 1-4

[0048] A method for preparing estradiol is different from Example 1 in that the reaction conditions for preparing the lithium reagent, the initiation temperature, the protective gas, or the post-treatment method after the aromatization reaction is different.

[0049] Comparative Example 5

[0050] A method for preparing estradiol, which differs from Example 1 in that estrone is used for preparation, comprises the following steps:

[0051] Step 1: ketal. Add 15g ADD, 18g ethylene glycol, 15g triethyl orthoformate and 0.3g PTS decolorizer into a reactor, react at 18°C ​​for 3h, then add 0.3g triethylamine to adjust the pH to 4, then add water dropwise for hydrolysis, centrifuge and wash with water until the pH is adjusted to 7, dry to obtain ketal and waste water;

[0052] Step 2 aromatization, add the acetal obtained in step 1 into a reactor, then add 162g tetrahydrofuran, 5g biphenyl, 6.5g diphenylmethane, introduce nitrogen, maintain the temperature at 45°C, add 0.81g lithium particles and stir for 0.5h, cool to 24°C, drop the tetrahydrofuran acetal solution, stir at 38°C for 2h, then cool to 5°C, drop methanol, add 16.2g hydrochloric acid solution, adjust the pH to 3.5, heat to 38°C and reflux for 2h, concentrate under reduced pressure, centrifuge, wash with water, and dry to obtain a hydrolyzate;

[0053] In step 3, 106 g of methanol was drawn into the refining kettle, and the hydrolyzate in step 2 was added. 0.96 g of potassium borohydride was added in batches, and the reaction was carried out for 4 h. Then 1.45 g of glacial acetic acid was added to neutralize the pH to 7, and the temperature was raised to 55 ° C. The methanol was concentrated under reduced pressure, water was added for hydrolysis, centrifuged, and dried to obtain crude estradiol.

[0054] Table 1 Screening of reaction conditions for lithium reagent preparation

[0055]

[0056] As shown in Table 1, the possibility that the reaction cannot be initiated at room temperature is very high, and the initiation time fluctuates greatly each time, indicating that the initiation process at room temperature is uncontrollable. Under reflux conditions, the reaction can be successfully initiated each time, so the initiation temperature of the lithium reagent is determined to be 65-70°C;

[0057] Table 2 Reaction concentration screening status of lithium reagent preparation

[0058]

[0059] It can be seen from Table 2 that as the feed concentration decreases, the possibility that the reaction cannot be initiated increases, so the ratio of biphenyl, diphenylmethane, metallic lithium and tetrahydrofuran is 1:(0.6-0.8):(0.09-0.18):(5-15) (w / w / w / v).

[0060] The estradiol samples obtained in all the above embodiments were sent for HPLC detection. The HPLC detection method was as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase was acetonitrile-methanol-water (40:5:55); the detection wavelength was 280 nm; the flow rate was 1.0 ml / min; the injection volume was 20 ul; in addition, the separated isomer 17α-estradiol was collected and sent for GC-MS mass spectrometry detection.

[0061] Table 3 Experimental results of Examples 1-6 and Comparative Examples 1-5

[0062]

[0063]

[0064] As shown in Table 3, after the aromatization reaction is completed, the present application screens the method for isolating the target product estradiol. It can be seen that when the solution system of the aromatization reaction is directly treated with water, estradiol and reactant impurities such as biphenyl and diphenylmethane are insoluble in water and are wrapped together in the form of oil, and estradiol cannot be precipitated and separated; when treated with ethyl acetate, according to the principle of like dissolves like, estradiol, biphenyl and diphenylmethane are all dissolved in the organic layer, and inorganic salt impurities are removed through the water layer by layered extraction, but reactant impurities such as biphenyl and diphenylmethane cannot be removed, and can only be removed by crystallization of ethyl acetate. When treated with dichloromethane, although the solution system is a layered two-phase system, impurities such as biphenyl and diphenylmethane are dissolved in the dichloromethane layer and cannot be precipitated, estradiol has been precipitated in the organic phase, and the inorganic salts in the system are dissolved in the water layer, so by filtering, the target product can be obtained very easily, and all impurities are dissolved in the dichloromethane layer or the water layer.

[0065] The estradiol prepared in this application has an isomer 17α-estradiol content of 0.05% (attached Figure 2 ), compared with the 17α-estradiol content of 0.70% (attached Figure 3 ) was significantly reduced; the molecular weight of the isomer was 272.0 ([M]) (Attached Figure 4 ), which is consistent with the theoretical molecular mass of 272 ([M]).

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing estradiol, characterized in that: The steps include: S1. Dissolve biphenyl and diphenylmethane in tetrahydrofuran, introduce nitrogen protection, add metallic lithium, raise the temperature to initiate the reaction, react for 2-4 hours to prepare the lithium reagent, and cool down to 40-50°C; S2, adding a tetrahydrofuran solution of boldenone to the reaction solution in step S1, reacting at 40-50° C. for 0.5-1 h, until TLC shows that the raw material has reacted completely, cooling to below 10° C., adding methanol dropwise for quenching, adding hydrochloric acid solution to adjust the pH value of the reaction solution to 2-5, and then concentrating under reduced pressure using a rotary evaporator at 40-50° C. until the organic solvent is spin-dried; S3, adding an organic solvent immiscible with water to the concentrate in step S2, dissolving the crystals, filtering, and drying to obtain estradiol; In step S1, the ratio of biphenyl, diphenylmethane, metallic lithium and tetrahydrofuran is 1: (0.6-0.8): (0.09-0.18): (5-15) w / w / w / v; The initiation temperature for preparing the lithium reagent in step S1 is 65-70°C; In step S3, the organic solvent immiscible with water is dichloromethane or chloroform.

2. The method for preparing estradiol according to claim 1, characterized in that: In the tetrahydrofuran solution of boldenone in step S2, the ratio of boldenone to tetrahydrofuran is 1:(3-6) w / v.

3. The method for preparing estradiol according to claim 1, characterized in that: The mass ratio of boldenone to biphenyl in step S2 is (1-3):

1.

4. The method for preparing estradiol according to claim 1, characterized in that: In step S2, the TLC developing agent includes toluene and ethanol, the color developing agent includes vanillin ethanol solution, and the volume ratio of toluene to ethanol is (15-10):

1.

5. The method for preparing estradiol according to claim 1, characterized in that: The molar concentration of the hydrochloric acid solution in step S2 is 1.5-2M.

6. The method for preparing estradiol according to claim 1, characterized in that: The dissolving process in step S3 includes stirring under ice water cooling, and the stirring time is 0.5-1h.

7. The method for preparing estradiol according to claim 1, characterized in that: The drying process in step S3 includes forced air drying, the drying temperature is 40-50° C., and the drying time is 4-6 hours.

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