TW201609683A - Crystalline form of icaritin, medicaments containing the same and the use thereof - Google Patents

Abstract

The present invention provides a crystalline form of icaritin, medicaments containing the same and the use thereof. The crystalline form is a solvent-less crystalline form and peaks at the 2[theta] ranges of 6.00 ± 0.20, 11.40 ± 0.20, 13.10 ± 0.20 and 18.90 ± 0.20 in X-ray powder diffraction with Cu-Ka radiation. The present invention also provides the use of the crystalline form, which may be used in the preparation of medicaments for treating diseases related to abnormal cell proliferation. The crystalline form of the present invention is highly stable and does not transform into other crystalline forms; and is thus suitable for transportation and storage under extreme weather.

Description

Crystal form of acrolatine compound, drug containing the crystal form, and use thereof

The present invention relates to a crystalline form of a compound in the field of medicine and its use, and in particular to a crystalline form of an acrolatine compound, a medicament containing the crystalline form, and uses thereof.

Akoradine, also known as icariin and icariin, is a new effective monomer obtained by enzymatic conversion of the main active ingredient extract of Epimedium sagittatum extracted from Chinese medicinal material Epimedium. The formula is as shown in the following formula (I):

In the "Chinese Experimental Formulation", Vol. 18, No. 14, 2012, "Effects of Icariin on the Role of Estrogen Dependent Breast Cancer MCF-7 Cells" was disclosed, and epimedium and estradiol were revealed through research. The combined action has the effect of inhibiting the proliferation of human breast cancer MCF-7 cells induced by E2.

In the "Chinese Journal of Comparative Medicine", No. 6, 2011, the article "The effect of icariin on the proliferation of lymphoma cells in vitro" was published, and the article revealed the effect of icariin on tumor cell proliferation.

Epimedium is disclosed in Chinese Patent No. 200710099025.1 The preparation method comprises the enzymatic hydrolysis reaction of icariin by β-glucosidase, and recrystallization by acetone-water after enzymatic hydrolysis to obtain pure product of icariin.

In Chinese patent No. 200910184282.4, "a kind of kinky sheep is disclosed. A method for preparing aglycone, which is obtained by enzymatically lysing a crude extract of Epimedium sinensis by snail enzyme, and then re-crystallizing the obtained icariin with ethanol, methanol, ethyl acetate or acetone to obtain Epimedium Pure product.

However, the inventors of the present application have found that the Acola timing is obtained by the above method. There are many crystal forms coexisting in the pure acrodipine. When a variety of crystal forms coexist, the drug made by cocoadine and excipients has poor stability during storage and poses certain risks to the quality of the drug. Therefore, it is necessary to develop a crystal form having high stability.

It is an object of the present invention to provide a solventless crystalline form of an aclapidine compound. The crystal form has good stability, and the drug prepared by the crystal form has a long shelf life.

Another object of the present invention is to provide a use of the crystalline form of the present invention in a medicament for use in a disease associated with abnormal cell proliferation.

It is still another object of the present invention to provide an acolide drug.

Still another object of the present invention is to provide a use of a medicament containing the acyclodine crystal form of the present invention for the preparation of a medicament for use in a disease associated with abnormal cell proliferation.

In one aspect the present invention provides a crystalline form A can cephradine, a crystalline form of solvent-free crystalline form, the crystalline form X-ray powder diffraction using Cu-Ka radiation is measured in the 2θ 6.0 ⁰ ± 0.2 ^0, 11.4 ⁰ ± 0.2 ⁰ , 13.1 ⁰ ± 0.2 ⁰ and 18.9 ⁰ ± 0.2 ⁰ peak.

In an embodiment of the invention, the X-ray powder diffraction obtained by using the Cu-Ka ray measurement of the crystal form is still 12.1 ⁰ ± 0.2 ⁰ , 20.2 ⁰ ± 0.2 ⁰ , 25.3 ⁰ ± 0.2 ⁰ and 30.8 ⁰ ± 0.2 at 2θ. ^There is a peak at ⁰ .

In an embodiment of the invention, the X-ray powder diffraction obtained by using the Cu-Ka ray measurement of the crystal form is still at 22.0 ⁰ ± 0.2 ⁰ , 22.9 ⁰ ± 0.2 ⁰ , 24.4 ⁰ ± 0.2 ^{0 ,} and 26.5 ⁰ ± 0.2 ⁰ . Peak.

Another aspect of the present invention also provides the use of the crystalline form of the present invention for the preparation of a medicament for use in a disease associated with abnormal cell proliferation.

In an embodiment of the invention, the disease associated with abnormal cell proliferation is a malignant tumor or aplastic anemia.

In an embodiment of the invention, the malignant tumor comprises breast cancer, cervical cancer, ovarian cancer, colon cancer, endometrial cancer, liver cancer, lung cancer, bone marrow cancer, prostate cancer or gastric cancer.

In still another aspect of the present invention, there is provided an acoradine drug comprising the acyclodine crystal form of the present invention and a pharmaceutically acceptable carrier.

In one embodiment of the invention, the pharmaceutically acceptable carrier is: a vegetable oil and a solubilizer.

The use of the acrolatine of the present invention for the preparation of a medicament for use in a disease associated with abnormal cell proliferation.

In an embodiment of the invention, the disease associated with abnormal cell proliferation is a malignant tumor or aplastic anemia.

In an embodiment of the invention, the malignant tumor comprises breast cancer, cervical cancer, ovarian cancer, colon cancer, endometrial cancer, liver cancer, lung cancer, bone marrow cancer, prostate cancer or gastric cancer.

The invention has the beneficial effects that the alcapidine solvent-free crystal form in the invention has high stability and no risk of conversion to other crystal forms, and the pharmaceutical composition prepared by the alacidine solvent-free crystal form of the invention High stability, long shelf life and suitable for transport and storage in extreme weather conditions.

The above and other objects, features and advantages of the present invention will be more It is apparent that the preferred embodiment is described below in detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the thermogravimetric analysis and differential scanning calorimetry curves of the crystals of the acrolatine acetonate prepared by the method of the first embodiment of the present invention.

Fig. 2 is a view showing a nuclear magnetic resonance spectrum of a crystal of a corbinidine acetate prepared by the method of Example 1 of the present invention.

Fig. 3 is a view showing an X-ray powder diffraction pattern of the crystal of the acrolatine acetonate of Example 1 of the present invention.

Figure 4 is a graph showing the thermogravimetric analysis and differential scanning calorimetry curves of the acoradine solvent-free crystal of the present invention.

Figure 5 is a graph showing the nuclear magnetic resonance spectrum of the alcapidine solvent-free crystal form of the present invention.

Figure 6 shows an X-ray powder diffraction pattern of the alcating solventless crystal of the present invention.

Figure 7 is a graph showing the thermogravimetric analysis and the differential scanning calorimetry curve of the crystal form C of the acroradine methanolate of the present invention.

Figure 8 is a graph showing the nuclear magnetic resonance spectrum of the crystal form C of the acrolatine methanolate of the present invention.

Figure 9 shows an X-ray powder diffraction pattern of the crystal form C of the acrolatine methanolate of the present invention.

Figure 10 shows an X-ray powder diffraction pattern of a mixed crystal form of Form B and Form C of the present invention.

Figure 11 shows the thermogravimetric analysis and differential scanning calorimetry curves of the crystal form D of the acrolatine acetate of the present invention.

Figure 12 is a graph showing the nuclear magnetic resonance spectrum of the crystal form D of the acrolatine acetate of the present invention.

Figure 13 shows the X-ray powder derivative of the crystal form D of the acrolatine acetate of the present invention. Shoot the map.

Figure 14 is a graph showing the thermogravimetric analysis and the differential scanning calorimetry curve of the crystal form E of the acrolatine methanolate of the present invention.

Figure 15 shows a nuclear magnetic resonance spectrum of the crystal form E of the acroradine methanolate of the present invention.

Figure 16 shows an X-ray powder diffraction pattern of the crystal form E of the acrolatine methanolate of the present invention.

Fig. 17 shows the case where the Hec1A cells of the present invention are exposed to a gradually increasing concentration of a coradinate solvent-free crystal form B.

Figure 18 is a graph showing the growth curve of the alcatidine solvent-free crystal form B of the present invention inhibiting Huh-7 tumor cells.

Figure 19 is a graph showing the effect of the alcating solventless crystalline form B of the present invention on the proliferation of acute myeloid leukemia cells overexpressing FLT-3, MV-4-11.

Unless otherwise stated, the term "solvent free form" as used herein refers to a particular crystalline form formed by the arrangement of a particular molecular lattice of a compound molecule or atom without the inclusion of water or other solvent in the acrolatine compound.

The term "abnormal cell proliferation" as used herein, unless otherwise indicated, refers to the growth, differentiation, and apoptosis of cells that deviate from the normal growth cycle of the cell.

The term "anti-cell abnormal proliferation-related disease" as used herein, unless otherwise indicated, refers to the treatment or prevention of a disease associated with abnormal cell proliferation.

The term "pharmaceutically acceptable carrier" as used herein, unless otherwise indicated, refers to a related pharmaceutical excipient that is required to form a pharmaceutical formulation with the crystalline form of the present invention.

Unless otherwise stated, the term "thermogravimetric analysis" as used herein refers to a thermal analysis technique that measures the relationship between the quality of a sample to be tested and temperature changes at a programmed temperature.

Unless otherwise stated, the term "differential scanning thermal analysis" as used herein refers to An analytical technique for measuring the difference in energy of a measured object with respect to temperature over a unit of time per unit of temperature under varying temperature conditions.

Example 1

Preparation of acrolatine by enzymatic hydrolysis of epimedium extract

The extract of Epimedium in this example was purchased from Shaanxi Jiahe Plant Chemical Co., Ltd., and the trade name was "Icaria extract", which contained icariin with a mass fraction of 90%.

Step 1: Preparation of acorridin by enzymatic hydrolysis

A commercially available 80g Epimedium extract containing 90% of icariin in a concentration of 1 mol/L of pH 5.2 in disodium hydrogen phosphate-potassium dihydrogen phosphate buffer 2.0 L Add 0.6L of ethanol and 1400g of RAPIDASE pectinase, a total of 1.4L in a 5L reactor, and carry out enzymatic hydrolysis under the condition of 50 °C. The specific conditions are as shown in Table 1:

The reaction system was transformed by HPLC for 70 hours, and the conversion rate of icariin to acaradine was 90%.

Step 2: Purification of acorridine

After completion of the reaction for 70 hours, the reaction solution was centrifuged, the supernatant was removed, the precipitate was dissolved in 2 L of acetone, filtered, and about 1 L of distilled water was added to the filtrate, and the solution was refluxed at 75 ° C, and the mixture was allowed to stand at 20 ° C for crystallization. After crystallization for 24 hours, filtration gave a pale yellow crystal. The obtained crystals were continuously dried at 20 ° C until the weight of the crystals no longer changed, and crystals were obtained. Thermogravimetric analysis and differential scanning calorimetry were performed to obtain a map as shown in FIG.

In Fig. 1, differential scanning calorimetry: that is, the DSC curve shows that the sample exhibits a desolvation endothermic peak at an initial temperature of 56.1 ° C, and a melting endothermic peak at 253.6 ° C, and the melting enthalpy is 117.79 J/g. Thermogravimetric analysis curve, ie weight loss when the sample is heated to 100 ° C in TGA It is 14.3%.

It can be seen from the magnetic resonance spectrum of Fig. 2 that the displacement is 2.09 ppm. A peak having an integral value of 5.7, which represents acetone, is estimated, and thus a single crystal form of the acetone solvate of acrolatine in the experimental sample of nuclear magnetic resonance is estimated, that is, the crystal form A.

Figure 3 shows an X-ray powder diffraction pattern of Form A, from which it can be seen that Form A is about 5.4 ⁰ , about 6.0 ⁰ , about 9.4 ⁰ , about 9.8 ⁰ , about 12.5 ⁰ , about 14.3 ⁰ at 2θ angles. , about 16.5 ⁰ , about 18.9 ⁰ , about 19.1 ⁰ , about 19.7 ⁰ , about 21.9 ⁰ , about 25.0 ⁰ , about 25.4 ⁰ , about 28.7 ⁰ , about 29.1 ⁰ , about 30.6 ⁰ , about 34.5 ^{0 ,} and about 38.5 ⁰ peak.

Example 2

The crystal obtained in Example 1 was continuously dried at 80 ° C until the crystal was The weight no longer changes. The weight of the crystal after drying at 80 ° C was weighed, which was reduced relative to the weight of the crystal in Example 1.

Thermogravimetric analysis and differential scanning calorimetry for crystals obtained according to the above method Analysis, the map shown in Figure 4 is obtained.

In Figure 4, differential scanning thermal analysis: that is, the DSC curve is stored at 253.0 ° C At the melting endothermic peak, the melting enthalpy was 137.29 J/g. Moreover, in the thermogravimetric analysis curve, that is, the weight loss of the sample when heated to 150 ° C in the TGA is 0.35%, the crystal obtained in the present embodiment can be considered to be a single crystal form, that is, the crystal form B, by the above curve.

Figure 5 shows a nuclear magnetic resonance spectrum of Form B of the present invention, which is shown in Figure 2 and Figure 2 When the NMR spectrum was compared, it was found that there was no peak at a displacement of 2.09 ppm, and other peaks were the same as in Fig. 2, so that it was confirmed that the crystal form B was a solventless crystal form of acrolatine.

Figure 6 shows an X-ray powder diffraction pattern of the solventless Form B of a coradine. As it can be seen by the powder diffraction pattern Form B Angle 2θ at about 6.0 ^0, 11.4 ^0, 12.1 ^0, 13.1 ^0, 18.9 ^0, 20.2 ^{0, 0} to about 22.0, ⁰ 22.9, 24.4 ⁰ , about 25.3 ⁰ and about 26.5 ⁰ and 30.8 ⁰ peak.

Example 3

Weigh 15 mg of the acrolatine solvent-free crystal form sample obtained in Example 2 to In a 1.5 mL vial, 1.2 mL of methanol solvent was added to obtain a suspension. The suspension was stirred at 20 ° C for 4 days, and the obtained crystals were collected by centrifugation, subjected to thermogravimetric analysis and differential scanning calorimetry to obtain a map as shown in FIG. It can be seen from the spectrum that the sample has a weight loss of 5.5% when heated to 100 ° C, a desolvation endothermic peak at 89.0 ° C and 107.3 ° C, and a melting endotherm at 253.8 ° C, and the melting enthalpy is 117.51 J/g.

It can be seen from the nuclear magnetic resonance image of FIG. 8 that the displacement is compared with FIG. A peak having an integral value of 1.04 at 3.17 ppm is inferred from the displacement to indicate a characteristic peak of methanol, since the methoxy group in the structural formula of the acrolatine is known to have a peak at 3.85 ppm, and the integral value is 3.00. It is inferred that the crystal in this example is a crystal form of 1/3 of the methanol solvate of acrolatine, that is, crystal form C.

FIG. 9 shows an X-ray powder diffraction pattern of Form C by the pattern, it can be seen at about 4.8 ^0, 7.2 ^{0, 0} to about 8.6, from about 9.7 ^{0, 0} to about 12.3, about 14.5 ^0, 15.2 ^0, about Diffraction peaks appear at 16.5 ⁰ , about 19.6 ⁰ , about 21.1 ^{0 ,} and about 22.5 ⁰ .

Example 4

1.0 g of the alacidine solvent-free crystal form prepared in Example 2 and 100 mL of methanol was placed in a 250 mL single-necked flask, heated to reflux for 1 hour, slowly cooled to 20 ° C, allowed to stand for 24 hours, and filtered. The filter cake was dried in a 25 ° C blast oven for 24 hours to obtain a product of 0.82 g, and the obtained product was subjected to an X-ray powder diffraction pattern, as shown in FIG.

Figure 10 shows at about ⁰ 4.9, from about 6.0 ^0, 11.3 ^0, the diffraction peak appears at about 13.1 and about 18.9 ^{0 0} position.

Synthesis Example 2 Diffraction Results of Form B and Example 3 Form C It can be seen that the present embodiment obtains a mixed crystal of Form B and Form C.

Example 5

Weigh 15 mg of the acrolatine solvent-free crystal form sample obtained in Example 2 to In a 3 mL vial, 1.0 mL of acetic acid solvent was added, and the mixture was stirred at 50 ° C for 2 hours to filter the supernatant. The resulting supernatant was cooled from 50 ° C to 5 ° C at a rate of 0.1 ° C / min and thermostated at 5 ° C for one week. The obtained crystals were collected, subjected to thermogravimetric analysis and differential scanning calorimetry to obtain a map as shown in FIG. It can be seen from the spectrum that the sample has a weight loss of 13.7% when heated to 150 ° C, a desolvation peak at 87.9 ° C, and a melting endotherm at 253.1 ° C, and the melting enthalpy is 112.40 J/g.

It can be seen from the nuclear magnetic resonance spectrum of Fig. 12 that it is in place compared with Fig. 6. When the shift was 1.91 ppm, a peak having an integral value of 3.05 appeared, and it was inferred from the displacement that the peak was a characteristic peak of acetic acid. Therefore, it was estimated that the crystal in this example was a crystal form of the acetic anhydride solvate of acrolatine, that is, a crystal form. D.

13 shows an X-ray powder diffraction pattern Form D by the map, it can be seen at about 6.0 ^{0, 0} to about 8.2, about 10.6 ^0, 11.9 ^0, 14.4 ^0, 16.0 ^0, 16.4 ^0, about Diffraction peaks appear at 17.2 ⁰ , about 17.9 ⁰ , about 18.8 ⁰ , about 24.0 ⁰ , about 24.8 ⁰ , about 25.4 ⁰ , 26.3 ⁰ , 29.1 ⁰ , 30.9 , 31.9 , and about 33.7 ⁰ .

Example 6

15 mg of the crystalline compound obtained in Example 2 was weighed into a 3 mL vial. A 20 mL vial was taken and 4 mL of methanol solvent was added thereto, and a 3 mL vial was placed in a 20 mL vial, sealed, and allowed to stand at 20 ° C for 5 days, and the obtained crystals were collected and tested. Thermogravimetric analysis and differential scanning calorimetry were performed to obtain a map as shown in FIG. It can be seen from the spectrum that the sample has a weight loss of 6.0% when heated to 150 ° C, a desolvation endothermic peak at 69.1 ° C, and a melting endotherm at 253.5 ° C, and the melting enthalpy is 123.37 J/g.

It can be seen from the nuclear magnetic resonance image of Fig. 15 that the displacement is compared with that of Fig. 6. A peak with an integral value of 3.3 at 3.17 ppm is obtained, and it is inferred from the displacement that the peak is a characteristic peak of methanol, since the methoxy group of acrolatine is known to have a peak at 3.85 ppm, and the integral value is 3.00, so the calculation is calculated. The crystal in the examples is a crystal form of a methanol solvate of acrolatine, ie, Form E.

Figure 16 shows an X-ray powder diffraction pattern of Form E, by which it can be seen that at about 5.7 ⁰ , about 10.4 ⁰ , about 11.2 ⁰ , about 11.6 ⁰ , about 12.5 ⁰ , about 14.5 ⁰ , about 15.9 ⁰ , about Diffraction peaks appear at 16.4 ⁰ , about 17.3 ⁰ , about 18.5 ⁰ , about 25.5 ⁰ , about 26.1 ^{0 ,} and about 27.4 ⁰ .

Comparative example

Take the crystal forms A, C, D and E as described above for heating, when When the temperature is added to 80-100 ° C, the above crystals have obvious desolvation phenomenon, and the weight of the crystal itself is also reduced. The crystals after solvent removal are again subjected to X-ray powder diffraction, which is confirmed to be converted into a corridin. Solvent-free Form B.

Solvent-free crystal form B of acrolatine by dynamic vapor absorption The (DVS) test showed that Form B had almost no hygroscopicity. No conversion or degradation of Form B was observed for 1 week at 25 ° C and 60% humidity at 80 ° C for 24 hours and 40 ° C for 75% humidity. In addition, it can be seen from the DSC chart of Forms A, B, C, D, and E that the melting enthalpy of Form B is the highest, and thus Form B is the most stable and suitable for medicinal crystal forms.

Example 7

Preparation of a formulation containing araconine crystal form B

1. Grinding acrolatine crystal form B

First, the acrolatine crystal form B was ground to a fineness of less than 90 μm, and the samples before and after the grinding were examined for substances, contents, powder X-ray diffraction, etc., and no significant changes were observed in the substances and contents, and the micronization process was performed. The crystal form B did not undergo a crystal transformation.

2. Screening of a cortiline prescription

According to the experimental results of high bioavailability of acrolatine in oily solvents, the prescriptions related to acrolatidine were studied. The micronized acrolatine was allowed to settle for a period of 5 hours in vegetable oil, and a suspending agent was added to the formulation to increase the suspending. The content of the beeswax was based on the total weight of the acrolimin and corn oil mixture.

The screening prescription for a coradine corn oil suspension is as follows:

The acrolatine is prepared as a suspension according to the prescriptions 1-4 and placed separately In a 10 ml vial, place at 25 ° C ± 1 ° C, 4 ° C ± 1 ° C, and sample at 3, 5, 10, and 15 days. The prescriptions were screened by appearance and sedimentation. The evaluation results are shown in the table below.

Evaluation: Prescription 1-3: The three prescriptions showed rapid sedimentation after 3-15 days of placement, and the appearance did not change.

Prescription 4: Settlement, the appearance is very good.

On the basis of the above test, the prescriptions 3 and 4 were filled with soft capsules to see the sedimentation, appearance, disintegration, and molding in soft capsules, and placed at 25±1°C and 4±1°C for 15 days (3) , 5, 10, 15) sampling for appearance, settling, forming, The time limit for disintegration is the indicator screening prescription, and the results are shown in the table below.

Evaluation: Prescription 3 was placed at 4 ° C for 10 days and sedimentation occurred. There was no sedimentation for 15 days. Prescription 3 had subsided at 4 ° C, and prescription 4 still performed well. According to the above results, prescription 4 was selected as the preferred prescription.

Example 8 Study on the stability of crystal form

The contents of the formulation 4 formulation of Example 7 were taken out, using petroleum ether The vegetable oil and beeswax were removed by washing, and the obtained solid particles were naturally air-dried at 20 ° C, and subjected to X-ray powder diffraction, and the diffraction pattern was the same as that of the crystal form B of FIG. Therefore, it is indicated that its solvent-free crystal form is very stable in the formulation or in the formulation.

Example 9

Effect of aclidine crystal form B on human endometrial carcinoma Hec1A cells (ATCC HTB-112 ^TM )

The cells were cultured in 96-well culture plates at a concentration of 5 × 10 ³ cells/well in DMEM containing high glucose medium. Cells were starved overnight for serum and exposed to acrolatine for 24 hours. The concentrations of acolat were 0 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 3 μM and 5 μM, respectively, and then the cells were treated with 0.5 mg/mL of 3-(4,5-dimethylthiazole-2). -Based -2,5-diphenyltetrazolium bromide (MTT) was incubated together at 37 ° C for 4 hours, wherein MTT was reduced to insoluble purple formazan by active mitochondrial reductase in living cells. Excess liquid MTT was then removed from the reaction mixture and 150 [mu]L of DMSO was added to dissolve the purple formazan into a colored solution. The optical density of the colored solution is linear with the number of cells in the solution, thereby enabling accurate quantification of cells.

As can be seen from Figure 17, the optical density is exposed with Hec1A cells. It is smoothly reduced in the crystal form B which is gradually increased in concentration. Therefore, it can be seen that the crystal form B of acrolatine has a significant inhibitory effect on the growth of Hec1A cells.

Example 10

Inhibition of proliferative capacity of hepatocellular carcinoma Huh-7 cells by acrolatine crystal form B

The liver cancer cell line Huh-7 in the present example was purchased from Japanese Cancer Research Bank (JCRB), Tokyo, Japan, under the trade name Huh-7, and the commercial number was JCRB0403. The CCK-8 kit was purchased from Dojindo, Inc. under the product name Cell Counting Kit-8 (CCK-8), product number CK04.

Experimental steps:

1. Huh-7 cell culture was cultured in DMEM medium containing 10% fetal bovine serum to a cell density of 80%.

2. The cells were divided into phenol red-free medium containing activated carbon-treated 2.5% fetal bovine serum and cultured for 24 hours.

3. Alcoradin-free solvent-free Form B at concentrations of 2.5, 5.0, 7.5, and 10.0 μM was added to the culture system, respectively, and the final concentration of DMSO in each reaction system was 0.1%. DMSO containing no aclidine was added to the culture system as a blank control group.

4. Use the Cell Counting Kit (CCK-8) to detect cell proliferation

4.1 After culturing the culture system of step 3 for 48 hours, 10 μM of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- Benzene disulfonate-2H-tetrazole monosodium salt (CCK-8 solution);

4.2 Incubate at 37 ° C for 2 hours, and measure the absorbance at 450 and 630 nm by microplate reader;

4.3 Taking the concentration of the solvent-free crystal form B of acrolatine as the abscissa, and plotting the ratio of the cells relative to the blank control group as the ordinate, the growth curve of the drug inhibiting Huh-7 tumor cells was plotted, as shown in Fig. 18.

5. As can be seen from Figure 18, Huk-7 cells were treated with a solvent-free crystal form B for 48 hours. As the drug concentration increased, the degree of inhibition of living cells was deepened, showing a concentration-dependent relationship. Therefore, Akola The solventless Form B had an inhibitory effect on Huh-7 cells and had an IC ₅₀ value of 4.5 μM.

Example 11

Inhibition of acylating form B on MV-4-11 (ATCC CRL-9591 ^TM ) cells in acute myeloid leukemia cells by MTT pharmacodynamic experiment (colorimetric detection)

1. Experimental material fetal bovine serum RPMI IMDM medium (purchased from Gibco Life Technologies, USA), CO ₂ cell culture medium was purchased from Heraeus, Germany, cell culture medium (purchased from PAA, USA), tetrazolium blue (MTT, Purchased from Sigma, USA, DMSO (purchased from Sigma, USA).

2. Cell culture method: human acute myeloid leukemia MV-4-11 cells were cultured in RPMI IMDM medium with 10% fetal bovine serum at 37 ° C, 5% CO ₂ cell incubator, passaged and replaced every other day. .

3. MTT pharmacodynamics experiment (colorimetric detection of efficacy) method

After culturing the cultured MV-4-11 cells, a medium suspension of 8.0 × 10 ⁴ cells/ml was prepared with a medium, and 100 μl per well was added to a 96-well plate. After 24 hours of culture, 100 μl of different concentrations of acomidine were added to each well, with 4 parallel wells per concentration. After 72 hours of culture, 20 μl of 50 mg/ml of MTT serum-free medium was added to each well. After culturing for 4 hours, the medium was discarded, 200 μl of DMSO was added to each well, and the mixture was gently shaken for 10 minutes. After the formazan was sufficiently dissolved, the cell light absorption value (OD value) was measured by a microplate reader at a wavelength of 570 nm. The final concentration of acrolatine was set to 169.66 μM, 84.83 μM, 42.41 μM, 21.21 μM, 10.6 μM, 5.3 μM, 2.65 μM, 1.33 μM, 0.66 μM, and the growth inhibition of MV-4-11 cells in each exposure concentration was calculated. Rate, the cell up inhibition rate curve was fitted by Origin 5.0 software, and the half-effect inhibitory concentration IC _{50 of} MV-4-11 cells was obtained.

4. Experimental results

As can be seen from Figure 19, the MTT assay showed that MV-4-11 cells were at 169.66 μM, 84.83 μM, 42.41 μM, 21.21 μM, 10.6 μM, 5.3 μM, 2.65 μM, 1.33 μM, and 0.66 μM of acrolatidine. Form B exposed 72 hours, cell growth inhibition rates were 88.12%, 84.17%, 87.14% , 76.22%, 55.44%, 32.72%, 30.20%, 13.27%, 5.56%, IC 50 of 7.48μM.

Example 12

The alcating solvent-free crystal form B of the present invention was tested by the MTT assay for human breast cancer cell line MDA-MB-231 (ATCC HTB-26 ^TM ), gastric cancer cell line MGC-803 (Shanghai Xinran Industrial Co., Ltd.), Lung cancer cell line H460 (ATCC HTB177 ^TM ), colon cancer cell line LS174T (ATCC CL-188T ^M ), pancreatic cancer cell line PANC-1 (ATCC CRL1469 ^TM ), prostate cancer cell line PC-3 (ATCC CRL1435 ^TM ), cervix , inhibition of cancer cell line Hela (ATCC CCL2 ^TM) ovarian cancer cell line SKOV3 (ATCC HTB-77 ^TM) and the myeloma cell line in RPMI8226 (Shanghai Industrial Co., Ltd. However, letter) of. The IC ₅₀ values of the above various cells are shown in Table 4 below.

Conclusion: The alcating solvent-free crystal form B of the present invention has an inhibitory effect on abnormal cell proliferation.

While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

Claims (10)

Form A may be one kind of pulling given compound, the solvent free crystalline form of polymorph, the polymorph X-ray powder diffraction using Cu-Ka radiation is measured in the 2θ ^{6.0 0 ± 0.2 0, 11.4 0} ± 0.2 0 , 13.1 ⁰ ± 0.2 ⁰ and 18.9 ⁰ ± 0.2 ⁰ peak. The crystal form according to claim 1, wherein the crystal form is obtained by Cu-Ka ray measurement, and the X-ray powder diffraction is still 12.1 ⁰ ± 0.2 ⁰ , 20.2 ⁰ ± 0.2 ⁰ , 25.3 ⁰ ± 2θ. 0.2 ⁰ and 30.8 ⁰ ± 0.2 ⁰ peaks. The crystal form according to claim 1 or 2, wherein the X-ray powder obtained by the Cu-Ka ray measurement of the crystal form is still 22.0 ⁰ ± 0.2 ⁰ , 22.9 ⁰ ± 0.2 ⁰ , 24.4 in 2θ. ⁰ ± 0.2 ⁰ and 26.5 ⁰ ± 0.2 ⁰ peak. The use of the crystalline form of the acortadine compound according to any one of claims 1 to 3 for the preparation of a medicament for use in a disease associated with abnormal cell proliferation. The use of the invention of claim 4, wherein the disease associated with abnormal cell proliferation is a malignant tumor or aplastic anemia. The use of claim 5, wherein the malignant tumor comprises breast cancer, cervical cancer, ovarian cancer, colon cancer, endometrial cancer, liver cancer, lung cancer, bone marrow cancer, prostate cancer or gastric cancer. An acrolatine drug comprising the crystalline form of the avocaine compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. The acrolatine drug of claim 7, wherein the pharmaceutically acceptable carrier is a vegetable oil and a solubilizer. The use of the acrolatine drug of claim 7 or 8 for the preparation of a medicament for use in a disease associated with abnormal cell proliferation. The use according to claim 9, wherein the disease associated with abnormal cell proliferation is a malignant tumor or aplastic anemia, preferably, the malignant tumor includes breast cancer, cervical cancer, ovarian cancer, colon Cancer, endometrial cancer, liver cancer, lung cancer, bone marrow cancer, prostate cancer or stomach cancer.