Icaritin acetone solvate and preparation method thereof

Abstract

The application belongs to the field of medicine crystal form, and particularly relates to a icariogenin acetone solvate and a preparation method thereof. The icariogenin acetone solvate and the preparation method thereof provided by the application have X-ray diffraction spectra using Cu-K alpha radiation, and the X-ray diffraction spectra have diffraction peaks at 6.04+ / -0.2 degrees, 7.03+ / -0.2 degrees, 14.12+ / -0.2 degrees, 19.44+ / -0.2 degrees, 22.39+ / -0.2 degrees and 26.18+ / -0.2 degrees expressed in 2 theta; the icariogenin acetone solvate provided by the application has excellent properties, high solubility and stability, provides a high-quality raw material selection for the preparation of icariogenin, and the preparation method is simple to operate, easy to control and suitable for industrial amplification.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical co-crystals, and particularly relates to icaritin methanol solvate and a preparation method thereof. BACKGROUND

[0002] Icaritin (IT), also known as icariin, is a monomeric component of multi-hydroxyl flavones in Epimedium, and its structural formula is shown in the following formula (I):

[0003]

[0004] Pharmacological studies show that icaritin has stronger anti-osteoporosis activity than other flavone glycosides in Epimedium, and has the effects of promoting the activity of osteoblasts and inhibiting the activity of osteoclasts in vitro. Traditional Chinese medicine Epimedium has the effects of tonifying kidney and yang, strengthening muscles and bones, dispelling wind and dampness, washing sores and killing insects, and relieving fatigue and pain. Icaritin, as one of the main effective components, has attracted the attention of many scholars at home and abroad in recent years, and its pharmacological effects have been extensively studied. So far, it has been found that the main physiological activity of icaritin lies in improving the function of the cardiovascular system, enhancing the body's immunity, and regulating endocrine, and it also has the effects of anti-tumor, anti-liver toxicity, anti-hypoxia reoxygenation, and bone strengthening.

[0005] Although icaritin has good clinical application prospects, its poor water solubility greatly limits its clinical application. At present, the researches on solving the solubility of icaritin mainly focus on two aspects, one is to study the preparation of new crystal forms of icaritin or its derivatives, and the other is to improve the solubility through the means of preparation. For example, Jia Dongsheng et al. significantly improved the solubility of the raw material and physical mixture by preparing icaritin phospholipid complex (Jia Dongsheng, Zhao Jiangli, Shi Feng, et al. Preparation of icaritin phospholipid complex and research on solid dispersion [J]. Chinese Traditional and Herbal Drugs, 2010, 9(41)), but this method has the problems of possible damage to the structure of the drug by the dissolution medium or evaporation of the dissolution medium; Wang Jinyan et al. studied the solubilization of icaritin by microemulsion technology (Jinyan, Chen Yan, Zhang Zhenhai, et al. Preliminary study on the intestinal absorption characteristics of icaritin self-microemulsion in Caco-2 cell model [J]. Chinese Traditional and Herbal Drugs, 2012, 3(43)), which can improve the solubility and absorption of icaritin, but a large amount of emulsifiers and co-emulsifiers are used in the process, which has certain quality and safety risks.

[0006] At present, there are few reports on the research of icariin raw material form. Although patent CN101200743A reports the preparation of crystalline icariin, the solubility characteristics are not improved. Patent CN104860958A reports the crystal form A and crystal form B of dehydrated icariin, and the research results show that the solubility of dehydrated icariin is improved compared with amorphous powder, but it is not significantly improved. Patent CN103936705A discloses four icariin solvates and one anhydrous crystal form B, and reports that the crystal forms of the solvates are unstable and are easily converted into anhydrous crystal form B. Patents CN104230870A and CN104945364A disclose two icariin hydrate crystal forms, and study the light stability of icariin hydrate crystal form and anhydrous crystal form B, and report that the anhydrous crystal form B is not stable under light, and the hydrate crystal form has improved light stability to a certain extent. Patent CN112294765A discloses an icariin amorphous form, which has improved solubility and bioavailability compared with icariin crystal form, but the amorphous form generally has poor stability, which is not conducive to its application in drugs.

[0007] For the currently reported icariin crystal forms, neither the stability nor the solubility problem is well solved, and the improvement of the medicinal effect is not ideal, so it is still necessary to study new crystal forms which significantly improve the solubility and stability of icariin, to fundamentally solve the solubility and stability problems of icariin, and to minimize the addition of auxiliary materials in the preparation process and to minimize the drug hazards. SUMMARY

[0008] In view of the drawbacks of the prior art, the present application successfully prepares an icariin acetone solvate crystal by co-crystal technology, which can significantly improve the solubility characteristics of icariin and has good physicochemical stability.

[0009] The specific technical content of the present application is as follows:

[0010] In the first aspect of the present application, an icariin acetone solvate is provided, characterized in that the molar ratio of icariin to acetone in the crystal unit structure is 1:1.

[0011] Preferably, the icariin acetone solvate has characteristic peaks at 6.04±0.2°, 7.03±0.2°, 14.12±0.2°, 19.44±0.2°, 22.39±0.2°, and 26.18±0.2° in the X-ray diffraction spectrum expressed in 2θ using Cu-Kα radiation.

[0012] Preferably, the icariellin acetone solvate has an X-ray powder diffraction pattern using Cu-Ka radiation, in terms of 2 theta, with characteristic peaks at 6.04±0.2°, 7.03±0.2°, 14.12±0.2°, 15.66±0.2°, 16.32±0.2°, 19.44±0.2°, 19.86±0.2°, 22.39±0.2°, 25.55±0.2°, 26.18±0.2°, 35.89±0.2°.

[0013] Preferably, the icariellin acetone solvate has the following main crystallographic parameters: triclinic crystal system, space group P-1; cell parameters: α = 102.6997(15)°, β = 97.6527(15)°, γ = 92.5379(14)°, cell volume

[0014] Preferably, the icariellin acetone solvate has an X-ray powder diffraction pattern using Cu-Ka radiation, in terms of 2 theta, with characteristic peaks as shown in the X-ray powder diffraction pattern. Figure 1

[0015] In a second aspect, the present application provides a preparation method of icariellin acetone solvate, comprising the following steps: dissolving icariellin in a mixed solvent of acetone and organic solvent D, heating and stirring, cooling and crystallizing, filtering and drying to obtain icariellin acetone solvate.

[0016] Preferably, the organic solvent D is selected from one or two of acetonitrile and tetrahydrofuran.

[0017] Preferably, the mass-volume ratio of icariellin to acetone is 6-12:1, mg / mL; preferably 8-9:1, mg / mL.

[0018] Preferably, the volume ratio of acetone to organic solvent D is 4-18:1, mg / mL; preferably 10-12:1, mg / mL.

[0019] Preferably, the heating and stirring time is 3-7 hours, preferably 4-5 hours.

[0020] Preferably, the temperature of the dissolution and heating is 40-57°C.

[0021] Preferably, the cooling and crystallization temperature is -5-10°C; preferably 0-5°C.

[0022] Preferably, the crystallization time is 2-5 days.

[0023] ​Preferably, the specific steps of the preparation method of the icariellin acetone solvate are as follows: dissolving icariellin in a mixed solvent of acetone and organic solvent D, heating and stirring at 40-57℃ for 3-7 hours, cooling to -5-10℃ for crystallization for 2-5 days, filtering, and drying the filter cake to obtain icariellin acetone solvate.

[0024] The drying is a conventional drying method in the art, such as vacuum drying at 40-70℃.

[0025] In a third aspect of the present application, a pharmaceutical composition is provided, which contains the icariellin acetone solvate of the present application and pharmaceutically acceptable other components.

[0026] Preferably, the preparation method of the pharmaceutical composition is as follows: using standard and conventional techniques, combining icariellin acetone solvate with pharmaceutically acceptable solid or liquid carriers, and optionally combining with pharmaceutically acceptable adjuvants and excipients to prepare a pharmaceutical dosage form.

[0027] Preferably, the pharmaceutically acceptable other components include, but are not limited to, pharmaceutically active ingredients that can be used in combination, excipients, fillers, etc.

[0028] Preferably, the dosage form of the pharmaceutical composition includes, but is not limited to, sprays, tablets, capsules, powder injections, injections, etc.

[0029] In a fourth aspect of the present application, the use of icariellin acetone solvate as an active ingredient in the preparation of drugs for treating liver damage and liver fibrosis, tumors, etc. is provided.

[0030] Confirmation of crystal structure

[0031] X-ray crystal data were collected on a Rigaku XtaLAB Synergy model instrument at a test temperature of 293(2) K, with CuKa radiation, in an omega scan mode, and Lp correction. The crystal structure was calculated using the ShelXT program in the 01ex2 software, and the structure parameters were corrected by least squares method and the atomic species were identified using the ShelXL program, and the positions of all hydrogen atoms were obtained using geometric calculation method and difference Fourier method, with a goodness of fit (goof value) of 1.019, close to 1.0, indicating that the weight scheme is appropriate and the structure is accurate.

[0032] The crystallographic data obtained by testing and analyzing the icariellin acetone compound crystal prepared by the present application are shown in Table 1, and the ORTEP diagram of the icariellin acetone solvate of the present application (see attached Figure 2 ) shows that icariellin and acetone are connected through intramolecular hydrogen bonds; and the crystal packing diagram of icariellin acetone solvate is shown in the attached Figure 3 .

[0033] Table 1 Main crystallographic data of icariellin propionone solvate

[0034]

[0035] The X-ray powder diffraction testing instrument and testing conditions in the present application are as follows: X-ray powder diffraction instrument: PANalytical EMPYREAN; Cu-Ka; sample table: flat plate; incident light path: BBHD; diffraction light path: PLXCEL; voltage: 45 kv, current: 40 mA; divergence slit: 1 / 4; anti-scattering slit: 1; soller slit: 0.04 rad; step length: 0.5 s; scanning range: 3-50°. According to the crystallographic data, the characteristic peaks in the corresponding X-ray powder diffraction pattern (Cu-Ka) are shown in the following Table 2 and the following Figure 1. Figure 1 and Table 2.

[0036] Table 2. Main PXRD peaks of icariellin propionone solvate

[0037]

[0038]

[0039] The samples prepared in Examples 1-4 all meet the X-ray powder diffraction spectrum.

[0040] The present application has the following beneficial effects:

[0041] The icariellin propionone solvate prepared by the present application has good stability, and significantly improves the solubility of icariellin, provides a high-quality raw material selection for the preparation of icariellin, and the preparation method is simple and easy to control, the prepared crystal has high purity, and is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 : X-ray powder diffraction pattern of icariellin propionone solvate eutectic crystal.

[0043] Figure 2 : ORTEP diagram of icariellin propionone solvate eutectic crystal

[0044] Figure 3 : packing diagram of icariellin propionone solvate eutectic crystal. DETAILED DESCRIPTION

[0045] The present application will be further described by the following examples, it should be understood that: the examples of the present application are only used to illustrate the present application, but not limit the present application, so, the simple improvement of the present application under the premise of the method of the present application is within the scope of the present application.

[0046] The raw material icariogenin can be prepared according to any method in the prior art or purchased from a commercially available product (purity ≥ 98%), and the remaining reagent materials are commercially available.

[0047] I. Preparation of the crystal form

[0048] Example 1

[0049] 1 g of icariogenin was dissolved in a mixed solvent of 125 mL of acetone and 12.5 mL of tetrahydrofuran, stirred at 50-55°C for 4-5 hours, slowly cooled to 0-5°C, and left to stand for 3 days to crystallize, filtered, and the filter cake was dried under vacuum at 50°C to prepare icariogenin acetone solvate, with a yield of 98.0% and a purity of 99.97%.

[0050] Example 2

[0051] 1 g of icariogenin was dissolved in a mixed solvent of 111 mL of acetone and 9 mL of acetonitrile, stirred at 55-57°C for 3-4 hours, slowly cooled to 5-10°C, and left to stand for 5 days to crystallize, filtered, and the filter cake was dried under vacuum at 50°C to prepare icariogenin acetone solvate, with a yield of 97.4% and a purity of 99.97%.

[0052] Example 3

[0053] 1 g of icariogenin was dissolved in a mixed solvent of 83 mL of acetone, 11 mL of tetrahydrofuran, and 10 mL of acetonitrile, stirred at 40-45°C until completely dissolved, slowly cooled to -5-0°C, and left to stand for 2 days to crystallize, filtered, and the filter cake was dried under vacuum at 60°C to prepare icariogenin acetone solvate, with a yield of 96.2% and a purity of 99.94%.

[0054] Example 4

[0055] 1 g of icariogenin was dissolved in a mixed solvent of 167 mL of acetone and 9 mL of acetonitrile, stirred at 45-50°C until completely dissolved, slowly cooled to 0-5°C, and left to stand for 4 days to crystallize, filtered, and the filter cake was dried under vacuum at 60°C to prepare icariogenin methanol solvate, with a yield of 95.4% and a purity of 99.95%.

[0056] Comparative Example 1

[0057] 368.4 mg of icariogenin was dissolved in 30 mL of acetone, filtered, about 15 mL of distilled water was added to the filtrate, dissolved by refluxing at 75°C, and left to stand at 20°C to crystallize. After 24 hours of crystallization, the light yellow crystals were filtered. The obtained crystals were continuously dried at 20°C until the weight of the crystals no longer changed, to obtain icariogenin crystals, with a yield of 90.2% and a purity of 99.89%.

[0058] Comparative Example 2

[0059] The 30 mg icariellin crystals prepared in Comparative Example 1 were continuously dried at 80℃ until the weight of the crystals no longer changed, and the obtained crystals were collected, with a yield of 90.8% and a purity of 99.85%.

[0060] II. Verification Example

[0061] 1. Stability Experiment

[0062] The stability experiment method of the present application was performed according to the guidance method for stability investigation in the fourth part of the Chinese Pharmacopoeia (2020 edition). The high-temperature test condition was 60℃, the strong light irradiation test condition was 4500lx±500lx, and the high-humidity test condition was 92.5%. The purity was detected by HPLC method, and three parallel experiments were performed, and the average value was taken. The specific detection results are shown in Table 3.

[0063] Table 3 Stability test results of icariellin acetone solvent compound under light, high temperature and high humidity conditions

[0064]

[0065] At the same time, it was found that Examples 1-4 of the present application had similar stability test results.

[0066] 2. Solubility Experiment

[0067] Method: 10ml of medium (water, 0.01mol / L HCl solution) was respectively taken in a vial, and an excess of the sample to be tested was added. The vial was sealed and placed in a 25℃ constant temperature water bath for stirring for 1 hour, filtered through a filter membrane, and the filtrate was taken. The absorbance was measured at a wavelength of 270nm, and the solubility was calculated by testing the absorbance of the standard control.

[0068] Table 4 Solubility of icariellin acetone solvent compound in different media (μg / mL)

[0069]

[0070] At the same time, it was found that Examples 1-4 of the present application had similar solubility test results.

[0071] III. Biological Activity Experiment

[0072] 1. Materials

[0073] 1.1 Drug

[0074] Example 1 and Comparative Examples 1-2 were self-made crystal forms.

[0075] 1.2 Animals

[0076] Beagle dogs, 18, half male and half female, weighing (12-15 kg), were provided by Guangzhou Medical University.

[0077] 2. Pharmacokinetics experiment in Beagle dogs

[0078] After 2 weeks of adaptive feeding and quarantine, 12 healthy Beagles were selected and divided into 3 groups, and were orally administered with the crystal form of Example 1 and Comparative Example 1-2 at a dose of 15 mg / kg. Blood samples were collected from peripheral veins at 0, 0.5, 1, 2, 4, 6, 8 and 12 hours, and centrifuged at 2-8℃ for 10 minutes to obtain plasma. The concentration of icariin in the plasma of Beagle dogs was determined by LC-MS / MS method (reference: Huang Y, Liu YJ, Hu L, et al. LC-MS / MS method for determination of the concentration of acoradin in Beagle plasma[C]. The 7th National Toxicology Conference and the 8th Hubei Science and Technology Forum. 2015.) by hydrolysis of β-glucuronidase. The pharmacokinetic parameters measured are shown in Table 5, wherein the area under the blood concentration-time curve (AUC) was calculated by trapezoidal method, the elimination half-life (t 1 / 2 ) was calculated by Best Fit method, and the peak concentration (C max ) and the time to peak (T max ) were measured values.

[0079] Table 5 Pharmacokinetic parameters of Beagle dogs after oral administration

[0080]

[0081] Note: 1 group was administered with Example 1; 2 group was administered with Comparative Example 1; 3 group was administered with Comparative Example 2.

Claims

1. An icariin acetone solvate, characterized in that, Using Cu-Kα radiation, the X-ray diffraction pattern, expressed as 2θ, has characteristic peaks at 6.04±0.2°, 7.03±0.2°, 14.12±0.2°, 19.44±0.2°, 22.39±0.2°, and 26.18±0.2°. The molar ratio of icariin to acetone in its crystal unit structure is 1:

1.

2. The icariin acetone solvate according to claim 1, characterized in that, Using Cu-Kα radiation, the X-ray diffraction pattern, expressed as 2θ, shows characteristic peaks at 6.04±0.2°, 7.03±0.2°, 14.12±0.2°, 15.66±0.2°, 16.32±0.2°, 19.44±0.2°, 19.86±0.2°, 22.39±0.2°, 25.55±0.2°, 26.18±0.2°, and 35.89±0.2°.

3. The icariin acetone solvate according to claim 1, characterized in that, Using Cu-Kα radiation, its main crystallographic parameters are: triclinic crystal system, space group P-1; unit cell parameters are: a =7.20903(13)Å, b =8.94926(14)Å, c =17.5802(3)Å, α=102.6997(15)°, β=97.6527(15)°, γ=92.5379(14)°, cell volume V =1093.42(3)Å 3 .

4. The icariin acetone solvate according to claim 1, characterized in that, Its characteristic peaks have the X-ray powder diffraction pattern shown in Figure 1.

5. A method for preparing the icariin acetone solvate according to any one of claims 1-4, characterized in that, Includes the following steps: Icariin is dissolved in a mixed solvent of acetone and organic solvent D, heated and stirred, cooled to crystallize, filtered and dried to obtain icariin acetone solvate, wherein organic solvent D is selected from one or two of acetonitrile and tetrahydrofuran.

6. The preparation method according to claim 5, characterized in that, The mass-to-volume ratio of icariin to acetone is 6–12:1, mg / mL; the volume ratio of acetone to organic solvent D is 4–18:

1.

7. The preparation method according to claim 5, characterized in that, The heating and stirring time is 3 to 7 hours.

8. The preparation method according to claim 5, characterized in that, The specific steps are as follows: Icariin is dissolved in a mixed solvent of acetone and organic solvent D, heated and stirred at 40-57°C for 3-7 hours, cooled to -5-10°C for 2-5 days to crystallize, filtered, and the filter cake is dried to obtain icariin acetone solvate.

Images