Novel estriol crystal form and preparation method thereof

Abstract

The invention relates to a novel estriol crystal form and a preparation method thereof. The novel estriol crystal form provided by the invention is radiated by Cu-K alpha, an X-ray powder diffraction pattern expressed by a 2 theta angle has characteristic peaks at 13.121 degrees, 14.720 degrees, 19.899 degrees, 20.220 degrees, 20.519 degrees and 26.418 degrees, the error range of each characteristic peak 2 theta is + / -0.2 degrees, the crystal form has relatively good stability and druggability, and the estriol crystal form can be used for preparing a novel estriol crystal form. Therefore, the generation of impurities in the medicine storage and development process can be well reduced, and the bioavailability and the drug effect can be improved. The invention also provides a preparation method of the novel estriol crystal form, the novel estriol crystal form prepared by the method provided by the invention has the characteristic of high purity, the purity of the novel estriol crystal form is as high as 99.93%, the purity of a single impurity does not exceed 0.05%, and the safety of a medicine can be effectively ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of drug crystal forms, and particularly relates to a new crystal form of estriol and a preparation method thereof. Background Art

[0002] Estriol (estriol, estra-1,3,5(10)-triene-3,16α,17β-triol) is a steroidal compound with the chemical formula C 18 H 24 O3 is a white crystalline powder, and its structure is shown in the following formula I: Estriol is a metabolite of estrone and estradiol (β-Estradiol). As a natural estrogen with physiological activity, it can bind to estrogen receptors in the body and plays an important role in maintaining gender characteristics, regulating the female reproductive system and bone health. It can be used for cervicitis, prostate hypertrophy, prostate cancer, estrogen deficiency, etc., and has very good application prospects in the drug market.

[0003] Because estriol contains three hydroxyls, two chiral carbons, there are four kinds of special chemical structures of stereoisomers, so that the physicochemical properties of its isomeric impurities are very close, especially with less solubility difference, and the effect of general purification method is often not good. In addition, because estriol is on the market earlier, there are more reports about the synthetic method of estriol, and there are fewer reports about its crystal formation, and there may be significant differences in the solubility, stability, mechanical properties, bioavailability and safety and effectiveness of different crystal formations of the same drug. Therefore, systematic polymorphic research is carried out on drugs, and it is found that other advantageous crystal formations are used as the medicinal crystal formations of estriol, which provides a new possibility for further improving its preparation product performance.

[0004] In document 1 ([1] Xu Juan, Ning Lifeng. Study on the cocrystal of estriol [J]. Pharmaceutical Herald, 2022, 41(05): 630-636.), a crystalline form of estriol purchased from China Resources Zizhu Pharmaceutical Co., Ltd., batch number: 20190601, with a content of 99.90% (such as Figure 1 The corresponding X-ray powder diffraction (XRPD or PXRD) has characteristic peaks at 2θ=9.542°, 12.898°, 15.737°, 17.042°, 26.362°, and 30.038°, and the diffraction peak intensities are 55.1%, 57.8%, 65.0%, 88.8%, 100.0%, and 54.8%, respectively. It is disclosed that the solubility of the crystalline estriol in water is about 0.4 μg / mL, but the stability and drugability of estriol are not evaluated.

[0005] Therefore, the art still needs to study a new estriol crystal form with good stability and good drugability and a preparation method thereof to meet the commercial production in the pharmaceutical market. Summary of the invention

[0006] In view of the defects of the prior art, the present invention aims to provide a new crystal form of estriol and a preparation method thereof.

[0007] The new crystal form of estriol provided by the present invention is completely different from the crystal form disclosed in the prior art, and the new crystal form has good stability and drugability, and complies with the pharmacopoeias and drug preparation requirements of EP11.0, USP2022, and JP18. In addition, the preparation method adopted by the present invention not only makes the obtained new crystal form have a higher yield and purity, but also has simple operation and stable process, and is suitable for industrial production.

[0008] In a first aspect, the present invention provides a new crystal form of estriol.

[0009] A new crystal form of estriol has characteristic peaks at 13.121°, 14.720°, 19.899°, 20.220°, 20.519° and 26.418° in an X-ray powder diffraction spectrum expressed in 2θ angles using Cu-Kα radiation, wherein the error range of 2θ of each characteristic peak is ±0.2°.

[0010] In some embodiments, the new crystal form of estriol uses Cu-Kα radiation, and the X-ray powder diffraction spectrum expressed in 2θ angles has characteristic peaks at 12.817°, 13.121°, 14.720°, 15.400°, 18.499°, 19.899°, 20.220°, 20.519°, 21.318°, 26.418°, 37.341°, and 41.200°, wherein the error range of 2θ for each characteristic peak is ±0.2°.

[0011] In some specific embodiments, the new crystal form of estriol uses Cu-Kα radiation, and the X-ray powder diffraction spectrum expressed in 2θ angles has characteristic peaks at 10.938°, 12.817°, 13.121°, 14.720°, 15.400°, 18.499°, 19.899°, 20.220°, 20.519°, 21.318°, 26.418°, 29.922°, 29.958°, 37.341°, and 41.200°, wherein the error range of each characteristic peak 2θ is ±0.2°.

[0012] In a non-limiting manner, in a specific embodiment of the present invention, the X-ray powder diffraction spectrum of the new estriol crystal form is as follows: Figure 1 shown.

[0013] In a second aspect, the present invention also provides a method for preparing a new crystal form of estriol, comprising the following steps: mixing estriol with a benign solvent, heating and refluxing to dissolve until the solution is clear, then cooling, concentrating under reduced pressure to evaporate part of the benign solvent, continuing to cool to -5°C to 15°C, keeping the temperature at this temperature for crystallization for 0.5 to 10 hours, filtering and drying.

[0014] In some embodiments, the benign solvent is one or more of tetrahydrofuran, acetonitrile, methanol or ethanol, preferably methanol.

[0015] Furthermore, the mass volume ratio of estriol to benign solvent is 1:25 to 1:70 g / mL, preferably 1:30 to 1:40 g / mL.

[0016] Furthermore, the reduced pressure concentration to evaporate part of the benign solvent may evaporate 1 / 4 to 3 / 4 volume of the benign solvent, preferably 1 / 2 volume of the benign solvent.

[0017] In some embodiments, the heat preservation crystallization time may be preferably 2 to 5 hours. Further, after the crystallization, filtration and drying are performed according to the conventional selection in the art. The filtration method is preferably suction filtration, pressure filtration or filter cake filtration. Further, after the filtration, the drying method is preferably vacuum drying at 40°C to 50°C.

[0018] In some specific embodiments, the preparation method of the new crystal form of estriol comprises the following steps: mixing estriol with a benign solvent, heating under reflux to dissolve until the solution is clear, then cooling to 40°C to 50°C, concentrating under reduced pressure to evaporate 1 / 4 to 3 / 4 volume of methanol, continuing to cool to -5°C to 15°C, keeping warm at this temperature for crystallization for 0.5 to 10 hours, filtering and drying; the mass volume ratio of estriol to methanol is 1:25 to 1:70 g / mL.

[0019] The beneficial effects of the new estriol crystal form and the preparation method thereof provided by the present invention are:

[0020] (1) The benign solvent used in the preparation method provided by the present invention is cheap and readily available, the crystallization conditions are mild, the operation is simple, the process is stable, the product yield is as high as 86%, the HPLC purity is as high as 99.93%, and the single impurity does not exceed 0.05%, which is conducive to industrial scale-up production and has broad application prospects;

[0021] (2) The new crystal form provided by the present invention has good stability and drugability, can meet the pharmaceutical requirements of production, processing, transportation and storage, is also conducive to storage and quality control at room temperature, and improves the bioavailability and efficacy of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the XRPD spectrum of estriol disclosed in Document 1;

[0023] Figure 2 is the XRPD spectrum of the new crystal form of estriol prepared in Example 2;

[0024] Figure 3 This is the XRPD superposition comparison spectrum of the new crystal form of estriol under accelerated stability conditions. DETAILED DESCRIPTION

[0025] The present invention is further described in detail in conjunction with the following specific examples and drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims are the scope of protection. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention does not specifically limit the content.

[0026] In the present invention, the “HPLC” refers to high performance liquid chromatography, and the “LC-MS / MS” refers to liquid chromatography-tandem mass spectrometry.

[0027] Example 1 Preparation of crude estriol

[0028] It can be prepared by referring to the preparation scheme provided in the experimental part of WO2012032529A1 patent or by the following method:

[0029]

[0030] Add 24.0 g of the solid of formula II and 960 mL of methanol into a 2L three-necked flask, start stirring, cool to 20°C, add 53.5 g of sodium triacetoxyborohydride twice, keep warm at 20°C-25°C for 3.5 h, then transfer the reaction solution into 1L of purified water, stir and crystallize for 1.5 h, filter to obtain a filter cake, and vacuum dry at 45°C to obtain 21.2 g of crude estriol (formula I), with a crude yield of 88% and a purity of 98.93% by HPLC analysis.

[0031] Example 2 Preparation of a new crystal form of estriol

[0032] Weigh 20.0 g of estriol from Example 1 and add it to 800 mL of methanol. Stirring is started. After heating and reflux at 60 ° C., stirring and dissolving, the temperature is then cooled to 45 ° C., concentrated under reduced pressure to evaporate three quarters of the methanol, and the temperature is further cooled to -2 ° C. At this temperature, the crystallization is stirred and kept for 2 hours, and filtered to obtain 20.5 g of a white solid wet product. The product is dried in vacuo at 45 ° C. to obtain 17.2 g of a new crystal form of estriol, with a yield of 86%. The purity of the HPLC analysis is 99.93%; the XRPD spectrum of the new crystal form of estriol is shown in Figure 1 The main data of its XRPD diffraction peaks are shown in Table 1.

[0033] Table 1 XPRD diffraction peak data of estriol crystal form

[0034]

[0035] Example 3 Preparation of a new crystal form of estriol

[0036] Weigh 20.0 g of estriol from Example 1 and add it to 1200 mL of methanol. Stirring is started. After heating and reflux at 60°C and stirring to dissolve, the temperature is then lowered to 40°C, and concentrated under reduced pressure to evaporate half of the methanol. The temperature is further lowered to 4°C, and crystallization is carried out by stirring at this temperature for 2.5 hours. Filter by suction to obtain 15.0 g of a white solid wet product. Dry under vacuum at 40°C to obtain 14.2 g of a new crystal form of estriol, with a yield of 71% and a purity of 99.54% by HPLC. XRPD determination shows that the obtained crystal form is the same as that of Example 2.

[0037] Example 4 Preparation of a new crystal form of estriol

[0038] Weigh 20.0 g of estriol from Example 1 and add it to 550 mL of methanol. Stirring is started. After heating and reflux at 60°C and stirring to dissolve, the temperature is then lowered to 40°C, concentrated under reduced pressure to evaporate a quarter of the methanol, and the temperature is further lowered to -4°C. At this temperature, the mixture is stirred and crystallized for 5 hours, and filtered to obtain 16.7 g of a white solid wet product. The product is dried under vacuum at 40°C to obtain 15.8 g of a new crystal form of estriol, with a yield of 79% and a purity of 99.12% by HPLC. The obtained crystal form is the same as that of Example 2 as determined by XRPD.

[0039] Example 5 Preparation of a new crystal form of estriol

[0040] Weigh 20.0 g of estriol from Example 1 and add it to 1000 mL of acetonitrile, start stirring, heat and reflux at 80°C to dissolve, then cool to 50°C, concentrate under reduced pressure to evaporate half of the acetonitrile, continue to cool to 4°C, keep stirring at this temperature for 2.5 hours, filter and obtain 17.5 g of a white solid wet product, and dry in vacuo at 40°C to obtain 15.4 g of a new crystal form of estriol, with a yield of 77% and a purity of 99.65% by HPLC. XRPD determination shows that the obtained crystal form is the same as that of Example 2.

[0041] Example 6 Preparation of a new crystal form of estriol

[0042] Weigh 20.0 g of estriol from Example 1 and add it to 1200 mL of tetrahydrofuran, start stirring, heat and reflux at 65°C to dissolve, then cool to 40°C, concentrate under reduced pressure to evaporate half of the tetrahydrofuran, continue to cool to 4°C, keep stirring at this temperature to crystallize for 2.5 hours, filter and obtain 16.8 g of a white solid wet product, and dry in vacuo at 40°C to obtain 15.6 g of a new crystal form of estriol, with a yield of 78% and a purity of 99.27% ​​by HPLC. XRPD determination shows that the obtained crystal form is the same as that of Example 2.

[0043] Example 7 Preparation of a new crystal form of estriol

[0044] Weigh 20.0 g of estriol from Example 1 and add it to 1200 mL of ethanol. Stirring is started. After heating and reflux at 75°C and stirring to dissolve, the mixture is cooled to 40°C, concentrated under reduced pressure to evaporate half of the ethanol, and then cooled to 4°C. The mixture is stirred and crystallized for 2.5 hours. The mixture is filtered to obtain 16.1 g of a white solid wet product. The mixture is dried under vacuum at 40°C to obtain 15.0 g of a new crystal form of estriol, with a yield of 75% and a purity of 99.35% by HPLC. The obtained crystal form is the same as that of Example 2 as determined by XRPD.

[0045] Comparative Example 1 Refining of estriol

[0046] 9 g of estriol from Example 1, 400 mL of methanol, and 4 g of activated carbon were mixed, heated and refluxed, filtered and decolorized, concentrated to 50% under reduced pressure, discharged, frozen, filtered and dried to obtain 8.1 g of estriol concentrate, with a yield of 90% and a purity of 98.07% according to HPLC. The obtained crystal form was the same as that in Reference 1 as determined by XRPD.

[0047] Comparative Example 2 Refining of estriol

[0048] 10 g of estriol, 200 mL of dioxane, 200 mL of acetone and 5.0 g of p-toluenesulfonic acid monohydrate from Example 1 were heated to reflux and reacted for 1.0 h; then half of the solvent was removed by vacuum concentration, cooled with ice water, crystals were precipitated, filtered, drained, washed with purified water until neutral, and dried to obtain 8.3 g of estriol, with a yield of 83% and a purity of 99.75% according to HPLC analysis. The obtained crystal form was the same as that in Reference 1 as determined by XRPD.

[0049] Comparative Example 3 Preparation of Estriol Crystalline Form

[0050] Weigh 20.0 g of estriol from Example 1 and add it to 800 mL of ethyl acetate. Stirring is started. After heating under reflux and stirring to dissolve, the temperature is lowered to 40°C. Half of the ethyl acetate is evaporated by vacuum concentration. The temperature is further lowered to -4°C. The mixture is stirred and crystallized for 5 hours. The mixture is filtered to obtain 16.4 g of a white solid wet product. The solid estriol is dried under vacuum at 40°C to obtain 14.0 g of solid estriol. The yield is 70%. The purity of the estriol is 96.58% according to HPLC. The obtained crystal form is the same as that of Reference 1 as determined by XRPD.

[0051] Test Example 1 Stability Test of Estriol Crystalline Form

[0052] Referring to the guidelines for stability investigation in Part 4 of the 2020 edition of the Chinese Pharmacopoeia, an accelerated stability test was carried out at a temperature of 40°C ± 2°C and a relative humidity of 75% ± 5% for 3 months. The sample of Example 2 was measured at 0 days, 1 month, and 3 months, and its purity and impurity content were detected by HPLC. The crystal form was characterized by XRPD. The specific test results are shown in Table 2.

[0053] Table 2 Stability test results of estriol crystal form

[0054]

[0055] From the stability test results of estriol crystal form in Table 2, it can be seen that the new estriol crystal form provided by the present invention has low impurity content and good stability, which is conducive to reducing toxic side effects; and under accelerated conditions, the purity of the sample of the new estriol crystal form for 3 months still reaches more than 99.77%, and the appearance and properties have not changed. In addition, the XPRD superposition comparison spectrum of the sample under accelerated stability conditions is as follows Figure 2 As shown, it is proved that they are all target crystal forms, no crystal transformation phenomenon occurs, and the pharmaceutical safety is greatly improved.

[0056] Test Example 2 Solubility Test of Estriol Crystalline Form

[0057] The solubility of estriol samples of Example 2 and Comparative Examples 1-3 in pure water at 37° C. was tested by the internal standard method. The results are shown in Table 3 below.

[0058] Table 3 Solubility test results of estriol crystal form

[0059]

[0060] The results show that the solubility of estriol in comparative examples 1-3 is inferior to the new crystal form of estriol provided by the present invention.

[0061] Test Example 3 Preparation of estriol cream and its in vitro transdermal test

[0062] Example 2 Estriol cream: 0.8 g of the estriol crystal form prepared in Example 2, 35 g of octyldodecanol, 30.92 g of polysorbate 60, 10.00 g of hexadecyl palmitate, 31.36 g of hexadecanol, 70.72 g of octadecanol, and 606.08 g of Span were placed in a 60-80° C. water bath to dissolve and mix uniformly to obtain an oil phase; 509.34 g of purified water, 3.20 g of lactic acid, 6.401 g of sodium hydroxide solution, 0.080 g of chlorhexidine hydrochloride, and 96 g of glycerol were weighed and placed in a 60-80° C. water bath to dissolve and mix uniformly, and then added to the above-mentioned oil phase, mixed, and cooled to 40° C. to obtain an estriol cream.

[0063] Estriol cream of Example 1 and Comparative Examples 1-3: Estriol cream samples were prepared by the same method as above, except that the estriol prepared in Example 1 and Comparative Examples 1-3 were used instead of the estriol crystal prepared in Example 2.

[0064] The prepared estriol cream and a commercially available estriol cream product of the same drug specification were subjected to in vitro permeation tests. A Franz diffusion cell with an inner diameter of 1.5 cm and a receiving cell volume of 12 mL was used. Rabbit intestinal mucosa was used as the permeation barrier. The drug amount was 270 mg. The receiving solution was normal saline for in vitro permeation tests. The temperature was set to 32°C, the rotation speed was 600 rpm, the sampling time points were 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h, and the cumulative permeation amount was calculated by using the LC-MS / MS method to determine the estriol concentration. The measurement results are shown in Table 4 below.

[0065] Table 4 Cumulative permeation of estriol cream

[0066]

[0067] The results show that the new crystal form of estriol provided by the present invention has excellent physical properties and is more conducive to the preparation of creams than existing crystals. The estriol in the cream prepared by uniformly dispersing it in the matrix has strong mucosal permeability, which is conducive to improving the bioavailability and the efficacy of the drug in treating diseases.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A new crystal form of estriol, characterized in that: Using Cu-Kα radiation, the X-ray powder diffraction spectrum expressed in 2θ angles has characteristic peaks at 13.121°, 14.720°, 19.899°, 20.220°, 20.519°, and 26.418°, wherein the error range of 2θ for each characteristic peak is ±0.2°.

2. The new crystal form of estriol according to claim 1, characterized in that: The new estriol crystal form uses Cu-Kα radiation, and an X-ray powder diffraction spectrum represented by 2θ angles has characteristic peaks at 12.817°, 13.121°, 14.720°, 15.400°, 18.499°, 19.899°, 20.220°, 20.519°, 21.318°, 26.418°, 37.341°, and 41.200°, wherein the error range of each characteristic peak 2θ is ±0.2°.

3. The new crystal form of estriol according to claim 1, characterized in that: The new estriol crystal form uses Cu-Kα radiation, and an X-ray powder diffraction spectrum represented by 2θ angles has characteristic peaks at 10.938°, 12.817°, 13.121°, 14.720°, 15.400°, 18.499°, 19.899°, 20.220°, 20.519°, 21.318°, 26.418°, 29.922°, 29.958°, 37.341°, and 41.200°, wherein the error range of 2θ for each characteristic peak is ±0.2°.

4. The new crystal form of estriol according to claim 1, characterized in that: The X-ray powder diffraction spectrum of the new estriol crystal form is shown in FIG1 .

5. A method for preparing the new crystal form of estriol according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: mixing estriol with a benign solvent, heating and refluxing to dissolve until the solution is clear, cooling, concentrating under reduced pressure to evaporate part of the benign solvent, further cooling to -5°C to 15°C, keeping the temperature at this temperature for crystallization for 0.5 to 10 hours, filtering and drying.

6. The preparation method according to claim 5, characterized in that: The benign solvent is one or more of tetrahydrofuran, acetonitrile, methanol or ethanol.

7. The preparation method according to claim 5, characterized in that: The mass volume ratio of estriol to the benign solvent is 1:25 to 1:70 g / mL.

8. The preparation method according to claim 5, characterized in that: The reduced pressure concentration evaporates 1 / 4 to 3 / 4 volume of the benign solvent.

9. The preparation method according to claim 5, characterized in that: The heat preservation and crystallization time is 2 to 5 hours, and the drying method is vacuum drying at 40° C. to 50° C.

10. The preparation method according to claim 5, characterized in that: The preparation method of the new crystal form of estriol comprises the following steps: mixing estriol with methanol, heating under reflux to dissolve until the solution is clear, then cooling to 40°C to 50°C, concentrating under reduced pressure to evaporate 1 / 4 to 3 / 4 volume of methanol, further cooling to -5°C to 15°C, keeping the temperature at this temperature for crystallization for 0.5 to 10 hours, filtering and drying; the mass volume ratio of estriol to methanol is 1:25 to 1:70 g / mL.

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