6- ((5, 6-Diphenyl-1, 2, 4-triazin-3-yl) (isopropyl) amino) -N- (methylsulfonyl) hexanamide crystal form B and application and preparation method thereofCross Reference to Related Applications
The present application claims priority from chinese patent application CN202410169645.1, whose application date is 2024, 2, 6, and the present application incorporates the entirety of the above-mentioned chinese patent application.
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to a crystal form of a compound 6- ((5, 6-diphenyl-1, 2, 4-triazin-3-yl) (isopropyl) amino) -N- (methylsulfonyl) hexanamide, and a pharmaceutical composition, application and a preparation method thereof.
Background
Pulmonary arterial hypertension (Pulmonary arterial hypertension, PAH) is a rare, difficult to cure pulmonary vascular disorder that can gradually lead to right heart failure and ultimately death. PAH is characterized by pulmonary microvascular remodeling, which leads to progressive increases in pulmonary arterial resistance (PVR) and hence right heart failure, making it a progressive and fatal disease, with 75% of PAH patients dying within 5 years after diagnosis, with an average survival time of 1.9 years after symptoms appear, and is therefore also known as "malignancy in the cardiovascular domain".
Currently, global therapies for PAH include both conventional therapies, which often only improve symptoms and targeted therapies, which are not effective in preventing the progression of the disease. In terms of targeted therapy, there are three pathways for PAH targeted drugs, namely the nitric oxide pathway, the endothelin pathway, and the prostacyclin pathway (prostacyclin, PGI 2). PGI2 is an important vascular endothelial relaxing factor that causes pulmonary vascular smooth muscle to relax and inhibits smooth muscle growth by stimulating the production of cyclic adenosine monophosphate (cAMP). PGI2 deficiency can cause pulmonary hypertension, and thus PGI2 class of drugs are currently the most active method for treating PAH. PGI2 class drugs include PGI2 analogs and PGI2 receptor agonists. PGI2 analogues have a natural PGI2 skeleton in structure, have a fast in vivo metabolism rate, a very short biological half-life, require high frequency administration or intravenous infusion administration, and have poor patient compliance. In addition, the target selectivity is poor, the therapeutic effect is difficult to separate from other effects, and adverse reactions are easy to generate.
The chemical name of the compound I is 6- ((5, 6-diphenyl-1, 2, 4-triazine-3-yl) (isopropyl) amino) -N- (methylsulfonyl) hexanamide, the structural formula is shown as formula I, and the compound I is a PGI2 receptor agonist, and has novel structure and good patentability. The compound I has strong target selectivity, and the agonistic activity of the prostacyclin IP receptor is more than 1000 times of that of the rest 7 prostacyclin IP receptor targets, and the prostacyclin IP receptor has higher efficacy and safety compared with the similar medicines on the market by activating the IP receptor, promoting cAMP generation in pulmonary artery smooth muscle cells, further inhibiting abnormal contraction of pulmonary artery, inhibiting proliferation of pulmonary artery smooth muscle cells and reducing pulmonary artery pressure.
At present, no crystal form condition of the compound I is disclosed, and the invention provides a drug crystal form with good stability, and a drug composition, application and a preparation method thereof based on the crystal form research of the compound I in the drug development process.
Disclosure of Invention
As a result of a great deal of research, it is found that different crystal forms exist in the compound I, and a great deal of research is conducted on the crystal forms of the compound I, so that the crystal forms meeting the medicinal requirements can be determined and prepared. Based on these studies, the present invention provides form B of compound I, which has no hygroscopicity and good storage stability, and is suitable for formulation development. The invention also provides a pharmaceutical composition and application of the compound I crystal form B, and provides a preparation method of the compound I crystal form B, wherein the preparation process condition is mild, and the preparation method is suitable for large-scale production.
In order to achieve the purpose of the invention, the following technical scheme is adopted:
It is an object of the present invention to provide a form B of compound I having an X-ray powder diffraction pattern expressed in terms of 2θ having diffraction peaks at 3.36±0.2°, 10.07±0.2°, 13.90±0.2°, 16.51±0.2°, 20.20±0.2°, 21.10±0.2°.
In some embodiments, form B of compound I, having an X-ray powder diffraction pattern expressed in terms of 2θ, has characteristic peaks at 3.36±0.2°, 6.72±0.2°, 10.07±0.2°, 13.90±0.2°, 16.51±0.2°, 17.67±0.2°, 19.50 ±0.2°, 20.20±0.2°, 21.10±0.2°.
In some embodiments, form B of compound I, which has an X-ray powder diffraction pattern expressed in terms of 2θ, is shown in fig. 1.
In some embodiments, form B of compound I, when characterized by TGA/DSC, has a TGA profile that determines that form B does not contain water of crystallization or a solvate.
In some embodiments, form B of compound I, when characterized by TGA/DSC, has a DSC profile showing a melting point (extrapolated onset temperature) of 140.5±2 ℃.
In some embodiments, form B of compound I has a TGA/DSC profile as shown in figure 2.
The second object of the invention is to provide a preparation method of the compound I crystal form B, which comprises the following steps of heating and dissolving the compound I in a solvent, cooling to 50-60 ℃, preserving heat until solid is separated out, continuing crystallization, separating and drying to obtain the crystal form B.
In some embodiments, the solvent is one or a mixture of alcohols, ethers, esters, alkanes, ketones, acetonitrile, water.
In some embodiments, the solvent is one or a mixture of methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, ethyl acetate, n-hexane, acetone, water.
In certain preferred embodiments, the solvent is one or a mixture of isopropanol, ethanol, water, tetrahydrofuran, and n-hexane.
In certain preferred embodiments, the solvent is isopropanol.
In some embodiments, the mass to volume ratio (g/mL) of the compound I to the solvent is 1:3.5-15.
In some embodiments, the heating may be performed in a single solvent or a mixed solvent, or may be performed by heating in one solvent and then adding one or more solvents.
In some embodiments, the heating conditions are heated reflux.
In some embodiments, the temperature is reduced to 50 ℃ to 60 ℃, the temperature reduction modes are various, and the temperature reduction modes can be natural slow temperature reduction by closing heating, or slow temperature reduction by adding other solvents under heating or non-heating conditions, so long as the temperature range of the temperature reduction can be ensured, and various temperature reduction modes can be selected.
In some embodiments, the temperature is reduced to 50 ℃ to 60 ℃, more preferably the temperature is reduced to 55 ℃ to 60 ℃.
In some embodiments, the heat preservation is performed until solids are precipitated, and crystallization can be performed by standing crystallization or crystallization under stirring, such as paddle stirring, suspension stirring, and the like. Preferably, stirring and crystallization are performed, and stirring can be performed faster than standing.
In some embodiments, the temperature is kept at 1 ℃ to 2 ℃ above and below the previous temperature reduction temperature until solid is precipitated.
In some embodiments, the heat preservation is continued until the solid is separated out, and various crystallization modes are adopted, and conventional crystallization methods can be adopted. For example, the crystallization can be carried out by naturally cooling, then continuously cooling to 0-10 ℃, and preserving heat for crystallization.
Further, the natural cooling crystallization is performed at room temperature.
Further, the natural cooling crystallization mode can be standing crystallization or crystallization under stirring, such as paddle stirring, suspension stirring and the like. Preferably, stirring and crystallization are performed, and stirring can be performed faster than standing.
Further, the natural cooling crystallization time is 1-15 h, preferably 1-2 h.
Further, the temperature is continuously reduced to 0-10 ℃, the temperature reduction condition is to reduce the temperature in a cold water bath, an ice bath or an ice salt bath, and various temperature reduction conditions can be selected as long as the temperature range of the temperature reduction can be ensured.
Further, the temperature is reduced to 0-10 ℃ and then the crystallization is carried out by heat preservation, and the crystallization mode can be standing crystallization or crystallization under stirring, such as paddle stirring, suspension stirring and the like. Preferably, stirring and crystallization are performed, and stirring can be performed faster than standing.
Further, after the temperature is reduced to 0-10 ℃, the temperature is kept for crystallization, and the crystallization time is 1-15 h, preferably 1-2 h.
In some embodiments, the crystallization is continued until the solid is separated out, and the crystallization mode can be that heating is started, the temperature is raised to 55-60 ℃ for heat preservation and stirring, natural cooling crystallization is performed, then the temperature is continuously lowered to 0-10 ℃, and the heat preservation crystallization is performed.
Further, the stirring is performed at a temperature of 20min to 3h, preferably 30min to 1h.
Further, the natural cooling crystallization is to turn off heating and naturally and slowly cool in a hot oil bath.
Further, the natural cooling crystallization mode can be standing crystallization or crystallization under stirring, such as paddle stirring, suspension stirring and the like. Preferably, stirring and crystallization are performed, and stirring can be performed faster than standing.
Further, the natural cooling crystallization time is 5-24 hours, preferably 5-8 hours.
Further, the temperature is continuously reduced to 0-10 ℃, the temperature reduction condition is to reduce the temperature in a cold water bath, an ice bath or an ice salt bath, and various temperature reduction conditions can be selected as long as the temperature range of the temperature reduction can be ensured.
Further, the heat preservation crystallization mode can be standing crystallization mode or crystallization mode can be performed under stirring, such as paddle stirring, suspension stirring and the like. Preferably, stirring and crystallization are performed, and stirring can be performed faster than standing.
Further, the heat preservation crystallization is carried out, and the crystallization time is 1-20 hours, preferably 1-2 hours.
In some embodiments, the heat preservation is carried out until the solid is separated out, then crystallization is continued, and the crystallization mode can be natural cooling crystallization.
Further, the natural cooling crystallization can be carried out by removing an oil bath, and naturally cooling at room temperature or in a hot oil bath.
Further, the natural cooling crystallization mode can be standing crystallization or crystallization under stirring, such as paddle stirring, suspension stirring and the like. Preferably, stirring and crystallization are performed, and stirring can be performed faster than standing.
Further, the natural cooling crystallization time is 5-24 hours, preferably 10-20 hours.
In some embodiments, compound I may be further seeded.
In some embodiments, the seed crystal is added in an amount of 0.1% -5% by mass of the compound I.
In some embodiments, after the heating and dissolving, the seed crystal of the compound I may be added during the cooling to 50 ℃ to 60 ℃ or during the heat preservation after the cooling to 50 ℃ to 60 ℃ to further accelerate the precipitation of the crystal form B.
Further, the seed crystal may be various kinds of seed crystals of the compound I, such as seed crystal of form a, seed crystal of form B, and the like. The crystal form A of the compound I is described in patent '6- ((5, 6-diphenyl-1, 2, 4-triazin-3-yl) (isopropyl) amino) -N- (methylsulfonyl) hexanamide crystal form A, the pharmaceutical composition, the application and the preparation method' of the inventor on the same application date.
In some embodiments, the separation may be a conventional process, such as centrifugation or filtration.
In some embodiments, the drying is a conventional drying process, such as vacuum drying.
In some embodiments, the drying requires drying to a constant weight.
A third object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective dose of compound I form B and a pharmaceutically acceptable excipient.
Further, the pharmaceutical compositions may be formulated into a variety of dosage forms for convenient administration, e.g., oral formulations (e.g., tablets, capsules, solutions or suspensions), injectable formulations (e.g., injectable solutions or suspensions, or injectable dry powders, for immediate use after addition to a pharmaceutical vehicle prior to injection).
A fourth object of the present invention is to provide the use of a therapeutically effective dose of form B of compound I or of said pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of a disease or condition.
In certain preferred embodiments, the disease or condition is associated with PGI2 receptor agonism.
In certain preferred embodiments, the disease or condition is selected from pulmonary hypertension, platelet aggregation-associated cardiovascular and cerebrovascular diseases, diabetic nephropathy, and the like.
A fifth object of the present invention is to provide a use of a therapeutically effective dose of compound I form B or of said pharmaceutical composition for the preparation of a PGI2 receptor agonist drug.
Definition and description of terms
The term "room temperature" or "RT" as used herein refers to an ambient temperature of 20 to 25 ℃ (68 to 77°f).
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The compound I has the positive progress effects that the compound I has low hygroscopicity, the crystal form B has higher melting point, more outstanding thermal stability and high purity, is not easy to generate impurities, has extremely strong drug stability and crystal form stability in forced degradation test, and is suitable for preparation development. Meanwhile, the preparation process condition is mild, and the preparation method is suitable for large-scale production. Therefore, the crystal form B has obvious advantages in aspects of medicament formation, industrial production and the like, and has very important significance for medicament development.
Drawings
FIG. 1 shows the XRPD pattern for form B of compound I prepared in example 1;
FIG. 2 shows the TGA/DSC profile of form B of compound I prepared in example 1;
FIG. 3 shows1 H-NMR spectra of form B of compound I prepared in example 1;
FIG. 4 shows the IR spectrum of form B of compound I prepared in example 1;
FIG. 5 shows the XRPD patterns for form B of compound I prepared in example 1 at high temperature, high humidity, light for 30 days and day 0;
FIG. 6 shows a graph of the average drug time of Compound I in plasma after intravenous administration of animals of group 1 in the bioavailability test of Experimental example 7;
figure 7 shows a graph of the average drug time of compound I in plasma after oral administration of group 2 animals in the bioavailability test of experimental example 7.
Detailed Description
The following detailed description of the invention is provided to facilitate an understanding of the invention and is not intended to limit the scope of the invention to those skilled in the art to make or use the invention.
Example 1
Heating, refluxing and dissolving 10g of the crude drug of the compound I in 120mL of isopropanol, closing an oil bath kettle for heating, naturally and slowly cooling to 58 ℃, preserving heat, stirring at 58+/-1 ℃ until solid is separated out, then naturally cooling, stirring and crystallizing at room temperature for 2 hours, continuously cooling to 10 ℃ in a cold water bath, preserving heat, stirring and crystallizing for 1.5 hours, filtering, and vacuum drying to constant weight to obtain a compound I crystal form B sample with the yield of 93.5%.
Example 2
Heating, refluxing and dissolving 10g of the crude drug of the compound I in 80mL of absolute ethyl alcohol, closing an oil bath kettle, heating, naturally and slowly cooling to 52 ℃, preserving heat, stirring at 52+/-1 ℃ until solid is separated out, then naturally cooling at room temperature, stirring and crystallizing for 1.5h, cooling to 5 ℃ in an ice bath, preserving heat, stirring and crystallizing for 2.0h, filtering, and vacuum drying to constant weight to obtain a compound I crystal form B sample, wherein the yield is 85.3%. The X-ray powder diffraction pattern of the sample obtained by this method is consistent with the pattern of the sample of compound I form B obtained in example 1.
Example 3
Heating, refluxing and dissolving 10g of the crude drug of the compound I in 50mL of methanol, closing an oil bath for heating, naturally and slowly cooling to 50 ℃, adding 0.1g of crystal form B seed crystal, keeping the temperature at 50+/-1 ℃ and stirring until solid is separated out, then starting heating, heating to 58+/-2 ℃ and keeping the temperature for 30min, closing the oil bath for heating, naturally and slowly cooling in a hot oil bath, stirring and crystallizing for 5h, continuously cooling to 0 ℃ in an ice salt bath, keeping the temperature and stirring for crystallizing for 1h, filtering, and vacuum drying to constant weight to obtain a crystal form B sample of the compound I, wherein the yield is 77.4%. The X-ray powder diffraction pattern of the sample obtained by this method is consistent with the pattern of the sample of compound I form B obtained in example 1.
Example 4
Heating, refluxing and dissolving 10g of the crude drug of the compound I in 50mL of absolute ethyl alcohol and 50mL of isopropanol, closing an oil bath kettle for heating, naturally and slowly cooling to 53 ℃, preserving heat, stirring at 53+/-1 ℃ until solid is separated out, then naturally cooling, standing and crystallizing for 13h at room temperature, cooling to 3 ℃ in an ice bath, preserving heat, stirring and crystallizing for 1h, filtering, and vacuum drying to constant weight to obtain a sample of the compound I in a crystal form B, wherein the yield is 88.7%. The X-ray powder diffraction pattern of the sample obtained by this method is consistent with the pattern of the sample of compound I form B obtained in example 1.
Example 5
Heating, refluxing and dissolving 10g of the crude drug of the compound I in 40mL of ethyl acetate, closing an oil bath kettle for heating, naturally and slowly cooling to 50 ℃, preserving heat for 50+/-1 ℃ and stirring until solid is separated out, then starting heating, heating to 55+/-2 ℃ and preserving heat and stirring for 1h, closing heating, naturally and slowly cooling in a hot oil kettle, standing and crystallizing for 20h, cooling to 2 ℃ in an ice bath, preserving heat, standing and crystallizing for 18h, filtering, and drying in vacuum to constant weight to obtain a sample of the compound I in a crystal form B, wherein the yield is 68.8%. The X-ray powder diffraction pattern of the sample obtained by this method is consistent with the pattern of the sample of compound I form B obtained in example 1.
Example 6
Heating, refluxing and dissolving 10g of the crude drug of the compound I in 90mL of 95% ethanol, closing an oil bath kettle, heating, naturally and slowly cooling to 55 ℃, preserving heat and stirring at 55+/-1 ℃ until solid is separated out, then naturally cooling and crystallizing at room temperature and stirring for 1h, cooling to 5 ℃ in an ice bath, preserving heat and stirring and crystallizing for 2.0h, filtering, and vacuum drying to constant weight to obtain a compound I crystal form B sample with the yield of 86.2%. The X-ray powder diffraction pattern of the sample obtained by this method is consistent with the pattern of the sample of compound I form B obtained in example 1.
Example 7
Heating, refluxing and dissolving 10g of the crude drug of the compound I in 60mL of acetonitrile and 40mL of water, closing an oil bath kettle, heating, naturally and slowly cooling to 51 ℃, preserving heat, stirring at 51+/-1 ℃ until solid is separated out, naturally cooling, crystallizing and stirring at room temperature for 1.5 hours, cooling to 3 ℃ in an ice bath, preserving heat, stirring, crystallizing for 1 hour, filtering, and vacuum drying to constant weight to obtain a compound I crystal form B sample, wherein the yield is 76.1%. The X-ray powder diffraction pattern of the sample obtained by this method is consistent with the pattern of the sample of compound I form B obtained in example 1.
Example 8
Heating, refluxing and dissolving 10g of the crude drug of the compound I in a mixed solvent of 20mL of acetone and 15mL of water, closing an oil bath, heating, naturally and slowly cooling to 55 ℃, preserving heat, stirring at 55+/-1 ℃ until solid is separated out, then naturally cooling, stirring and crystallizing for 1h at room temperature, cooling to 3 ℃ in an ice bath, preserving heat, standing and crystallizing for 15h, filtering, and vacuum drying to constant weight to obtain a compound I crystal form B sample, wherein the yield is 80.3%. The X-ray powder diffraction pattern of the sample obtained by this method is consistent with the pattern of the sample of compound I form B obtained in example 1.
Example 9
Heating, refluxing and dissolving 10g of the crude drug of the compound I in 30mL of tetrahydrofuran, slowly adding 120mL of n-hexane, cooling to 60 ℃, then preserving heat and stirring at 60+/-2 ℃ until solid is separated out, removing an oil bath, naturally cooling at room temperature, stirring and crystallizing for 16h, filtering, and drying in vacuum to constant weight to obtain a compound I crystal form B sample with the yield of 89.3%. The X-ray powder diffraction pattern of the sample obtained by this method is consistent with the pattern of the sample of compound I form B obtained in example 1.
Experimental example 1:X determination of diffraction angle (2. Theta.) of powder diffraction peak
Regarding the compound I form B sample prepared in example 1, its X-ray powder diffraction (XRPD) spectrum is shown in fig. 1, and the values of diffraction angles (2θ) of its X-ray powder diffraction peaks are shown in table 1.
Physical property data of each crystal described in experimental example 1 were measured under the following conditions.
The detector is a full-automatic multifunctional X-ray diffractometer of Japanese science SmartLab SE.
The operation conditions are that the X-ray tube ball is a counter-cathode, copper, tube pressure is 40kV, tube flow is 30mA, the scanning mode is one-dimensional scanning, the scanning speed is 10 DEG/min, the scanning axis is theta/2 theta, the scanning range is 3-35 DEG, and the stepping interval is 0.01 deg.
As a result of the detection, the characteristic peaks of the crystal form B occur at diffraction angles 2 theta of 3.36+/-0.2 degrees, 6.72+/-0.2 degrees, 10.07+/-0.2 degrees, 13.90+/-0.2 degrees, 16.51+/-0.2 degrees, 17.67+/-0.2 degrees, 19.50 +/-0.2 degrees, 20.20+/-0.2 degrees and 21.10+/-0.2 degrees through X-ray powder diffraction.
TABLE 1 values of diffraction angles (2 theta) of X-ray powder diffraction peaks of sample form B of Compound I prepared in example 1
Experimental example 2 thermogravimetry-differential scanning calorimetric analysis
With respect to the compound I form B sample prepared in example 1, a thermogravimetric-differential scanning calorimetry (TGA/DSC) profile is shown in figure 2.
Detection instrument model synchronous thermal analyzer STA449F3
Test conditions were 25℃temperature, 35% RH humidity
Crucible DSC/TG pan Al2O3
Atmosphere of AIR (80/20) - -/NITROGEN/50/NITROGEN/20
As a result of the detection, the TGA chart revealed that form B contained no crystal water or solvate, and the DSC chart revealed that the melting point (extrapolated onset temperature) of form B was 140.5.+ -. 2 ℃.
Experimental example 3 Nuclear magnetic resonance Spectrometry (1 H-NMR)
The nuclear magnetic resonance (1 H-NMR) spectrum of the sample of form B of compound I prepared in example 1 is shown in FIG. 3.
The measurement conditions were collected on a Bruker 500MHz NMR apparatus with CDCl3 as solvent.
Test results ：1H-NMR：δ：10.181(br,1H),7.493～7.439(m,4H),7.392～7.378(m,1H),7.317～7.261(m,5H),5.072(m,1H),3.608(m,2H),3.230(s,3H),2.376(m,2H),1.729(m,4H),1.460(m,2H),1.303～1.289(m,6H),ppm.
Experimental example 4 Infrared Spectrometry (IR)
With respect to the compound I form B sample prepared in example 1, an Infrared (IR) spectrum thereof is shown in fig. 4.
Instrument model: nicolet.
The detection method comprises the steps of taking a proper amount (about 1-2 mg) of the product, taking a proper amount of potassium bromide which is dried after grinding, putting the potassium bromide into an agate mortar, mixing and grinding the potassium bromide, taking a proper amount of the potassium bromide, tabletting the potassium bromide, and measuring an infrared spectrogram.
The infrared spectrum of the sample has a characteristic peak at 3105±5cm-1、2952±5cm-1、1684±5cm-1、1532±5cm-1、1473±5cm-1、1457±5cm-1、1441±5cm-1、1370±5cm-1、1150±5cm-1、1137±5cm-1、697±5cm-1、688±5cm-1.
Experimental example 5 hygroscopicity
Regarding the sample of the compound I form B prepared in example 1, the hygroscopicity was measured with reference to the fourth general rule 9103 of the chinese pharmacopoeia of 2020 edition. The specific test method is as follows:
the dried glass weighing bottle with plug (outer diameter 50mm, height 15 mm) was placed in a suitable 25.+ -. 1 ℃ constant temperature and humidity dryer one day before the test, and the weight (m1) was precisely weighed.
And (3) a proper amount of a test sample is taken and paved in the weighing bottle, the thickness of the test sample is generally about 1mm, and the weight (m2) is precisely weighed.
The weighing bottle is opened, and the weighing bottle and the bottle cap are placed under the constant temperature and humidity condition for 24 hours, the weighing bottle cap is covered, and the weighing bottle cap is precisely weighed (m3).
As shown in Table 2, the wet weight gain of the 3 batches of samples is less than 0.2%, which indicates that the crystal form B obtained by the method has no wet permeability.
TABLE 2 summary of moisture wicking results
| Lot number | Batch 1 | Batch 2 | Batch 3 |
| Percentage of weight gain | 0.02% | 0.02% | 0.03% |
Experimental example 6 stability test
For stability study of the compound I form B sample prepared in example 1, the sample was left under high temperature (60 ℃) and high humidity (RH 90±5%) and light (4500 lx±500 Lx) conditions for 30 days, respectively, and then XRPD patterns of the sample were measured, respectively, and the change of the crystal form was determined by comparing with the XRPD patterns measured on day 0, while the purity was measured by high performance liquid chromatography to examine the change of the related substances.
The stability test results of the crystal form B sample at 0 day and high temperature, high humidity and light irradiation for 30 days are shown in table 3, and the XRPD superposition graph of the crystal form B sample at high temperature, high humidity and light irradiation for 30 days and at 0 day is shown in fig. 5.
The results in fig. 5 show that form B was a stable form as compared to the XRPD pattern detected on day 0 when the XRPD pattern detected on day 30 was placed under high temperature (60 ℃), high humidity (RH 90 + 5%), and light (4500 Lx + 500 Lx).
According to the data in table 3, form B samples showed no significant change in related substances after 30 days of high temperature, high humidity and light irradiation, and form B had very high and stable purity. The stability research results show that the crystal form is suitable for formulation development.
TABLE 3 results of high temperature, high humidity, 30 day light stability experiments for form B samples
| Experimental conditions and time | Crystal form | Purity of |
| Day 0 | Crystal form B | 99.94% |
| High temperature (60 ℃) for 30 days | Crystal form B | 99.95% |
| High humidity (RH 90+ -5%) for 30 days | Crystal form B | 99.94% |
| Light (4500 Lx.+ -. 500 Lx) for 30 days | Crystal form B | 99.93% |
Experimental example 7 bioavailability test
For the bioavailability test of the compound I form B sample prepared in example 1, a clear solution and suspension were prepared by conventional methods, respectively.
The study used 12 Beagle dogs (male and female halves) randomly divided into 2 groups of 3 male and female animals each. Animals in group 1 were not fasted, given 0.5mg/kg of Compound I (clear solution) by single intravenous injection, blood samples were collected until 24h post-administration, specific collection time points were pre-administration, 0.083, 0.25, 0.5, 1,2,3, 4, 5, 6, 8,12 and 24h post-administration, animals in group 2 were given 0.5mg/kg of Compound I (suspension) by single oral gavage after fasted, blood samples were collected until 24h post-administration, specific collection time points were pre-administration, 0.25, 0.5, 1,2,3, 4, 5, 6, 8,12 and 24h post-administration, and drug formulation homogeneity and concentration analysis was performed using the validated HPLC-UV analysis method. The concentration of compound I in the plasma samples was detected using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis method. By using the blood concentration data, phoenix is applied7.0 Software the bioavailability was calculated using a non-compartmental model.
The mean time profile of compound I in plasma after intravenous administration and the mean time profile of compound I in plasma after oral administration of animals of group 2 in the bioavailability test are shown in fig. 6 and 7, respectively.
The results show that the bioavailability of the crystal form B is 67.48%, the bioavailability is general, but the bioavailability reaches 60% -90%, and the requirements of basic in vivo absorption and drug effect exertion can be met.
The compound I form B samples prepared in examples 2-9 have comparable properties to the compound I form B samples prepared in example 1, including hygroscopicity, stability, bioavailability, and the like.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.