CN118684674A - Crystal form of compound used as kinase inhibitor, preparation method and application thereof - Google Patents

Abstract

The invention prepares the crystal form A of the compound of the formula I for the first time. The form has very important significance for improving the characteristics of drug production, quality control, solid oral preparation development prospect and the like. The physical and chemical stability evaluation result shows that the crystal form A is a monohydrate, and cannot lose water or have hygroscopicity under the conventional conditions. And the stability under high temperature, high humidity, acceleration experiment and illumination experiment is obviously better than that of amorphous and crystal form E. Further, form a has better flowability than form E and amorphous. Finally, the bioavailability of form a is significantly higher than that of amorphous form. Based on good physicochemical stability, fluidity and bioavailability of the crystal form A, the crystal form A has great potential in the aspect of subsequent development and production.

Description

Crystal form of compound used as kinase inhibitor, preparation method and application thereof

Technical Field

The present invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to a crystal form of a compound used as a kinase inhibitor, a preparation method and an application thereof.

Background Art

The tropomyosin receptor kinase (TRK) family belongs to transmembrane receptor tyrosine kinases (RTKs), which are involved in regulating synaptic growth and functional maintenance of the mammalian nervous system, the development of memory, and protecting neurons from damage. TRK kinase is a type of nerve growth factor receptor. Its family consists of highly homologous tropomyosin-related kinase A (TRKA), tropomyosin-related kinase B (TRKB), and tropomyosin-related kinase C (TRKC), which are encoded by NTRK1, NTRK2, and NTRK3 genes, respectively. The complete TRK kinase consists of three parts: the extracellular region, the transmembrane region, and the intracellular region. Like other RTKs, the extracellular region of TRK kinase forms a dimer after binding to the corresponding ligand, which can cause autophosphorylation of the intracellular region of TRK kinase, thereby activating its own kinase activity and further activating the downstream signal transduction pathway. TRK kinases affect cell proliferation, differentiation, metabolism and apoptosis through downstream pathways such as Ras/MAPK, PI3K/AKT and PLCγ. When NTRKs genes are fused or mutated, the extracellular domain receptors will be changed or eliminated (Greco, A. et al. Mol. Cell. Biol. 1995, 15, 6118; Oncogene 1998, 16, 809). The fused or mutated TRK protein is in a highly activated kinase activity state without the need for ligand binding, thereby continuously activating the downstream signal transduction pathway, which can lead to abnormal regulation of TRK kinase downstream signaling pathways, induce cell proliferation, and promote the occurrence and development of tumors. NTRKs gene fusions appear in a variety of adult and pediatric solid tumors, including breast cancer, colorectal cancer, non-small cell lung cancer, papillary thyroid cancer, Spitz-like melanoma, glioma, and various sarcomas. In common cancers, such as non-small cell lung cancer and colorectal cancer, the incidence of NTRK gene fusion is low, roughly 1%-3%, but in some rare cancers, such as infantile fibrosarcoma and breast secretory carcinoma, the incidence of NTRK gene fusion can reach more than 90%. The earliest TPM3-TRKA fusion protein was discovered in colon cancer cells. Later, more types of NTRK fusion proteins were found in different clinical tumor patient samples such as breast cancer, non-small cell lung cancer, papillary thyroid cancer, Spitz-like melanoma, glioma, etc., such as CD74-NTRKA, MPRIP-NTEKA, QKI-NTRKB, ETV6-NTRKC, BTB1-NTRKC, etc. Therefore, in recent years, NTRK fusion protein has become an effective anti-cancer target and a hot spot in the development of anti-cancer drugs. With the further in-depth understanding of TRK kinases in recent years, more TRK fusion protein types and mutation types have been discovered (Russo, M. et al. Cancer; Discovery, 2016, 6, 36; Drilon, A. et al., Annals of Oncology, 2016, 27, 920). Therefore, there is an urgent need to develop new NTRK inhibitors with better activity and wider effects in clinical practice to solve the treatment problems of tumors caused by these NTRK protein fusions or mutations.

ROS1 (c-ros oncogene 1 receptor kinase) is a tyrosine protein kinase encoded by the ROS1 proto-oncogene in the human body. It is located on chromosome 6q22.1 and belongs to the tyrosine kinase insulin receptor gene. It consists of three parts: the intracellular tyrosine kinase active region, the transmembrane region and the extracellular region, encoding a chimeric protein with tyrosine kinase activity. The basic structure consists of the extracellular N-terminal ligand binding region (amino acids 1-1861), the transmembrane region (amino acids 1862-1882) and the intracellular C-terminal 464 amino acids consisting of the tyrosine kinase active region (amino acids 1883-2347). When the ROS1 gene is rearranged, the extracellular region is lost, and the transmembrane region and the intracellular tyrosine kinase region are retained. The rearrangement site mainly occurs in exons 32 to 36 of the ROS1 gene. ROS1 gene mutations mainly occur in lung cancer patients, with a patient ratio of 1%-2%. In NSCLC, the ROS1 gene mainly fuses with SLC34A2 and CD74, and continuously activates the ROS1 tyrosine kinase region and downstream JAK/STAT, PI3K/AKT, RAS/MAPK and other signaling pathways, thereby causing tumorigenesis. It has been confirmed in a large number of literatures and clinical studies that by inhibiting the activity of mutated ROS1 kinase, diseases caused by excessive activation of ROS1, especially cancer, can be treated. Currently, the therapeutic drugs for ROS1-positive non-small cell lung cancer on the market include crizotinib and entrectinib, both of which are first-generation small molecule ROS1 inhibitors. However, during the treatment of crizotinib or entrectinib, drug resistance will occur in about 15 months, and disease progression will occur. Among patients with drug resistance, the most common drug resistance mutation is solvent front mutation such as G2032R. For patients with drug resistance, there is currently no therapeutic drug on the market. Therefore, there is an urgent need to develop new inhibitors against ROS1, especially new ROS1 inhibitors for clinical treatment of patients who have developed resistance to first-generation ROS1 inhibitors such as crizotinib or entrectinib.

2-5% of NSCLC cases are rearranged with anaplastic lymphoma kinase (ALK), which is a receptor-type protein tyrosine phosphokinase of the insulin receptor superfamily. ALK was first discovered in anaplastic large cell lymphoma as an activated fusion oncogene. Subsequent studies have found fusion forms of ALK in a variety of cancers, including systemic dysplasia, inflammatory myofibroblastic carcinoma, and non-small cell lung cancer. The mutation and abnormal activity of ALK in a variety of cancers have made it a drug target for the treatment of ALK-positive cancers. Currently, multiple ALK kinase inhibitors are on the market. With the clinical application of these drugs, patients will develop drug-resistant mutations. If drug-resistant mutations such as G1202R occur, these drugs will lose their efficacy.

With the further in-depth understanding of kinases such as ROS1, NTRK, and ALK in recent years, as well as the increase in clinical drug-resistant patients, there is an urgent need to develop new tyrosine kinase inhibitors with better activity and broader effects in clinical practice, so as to solve the treatment problems of tumors caused by fusion or mutation of kinase proteins such as ROS1, NTRK, and ALK.

Patent document CN 112867717 A discloses a kinase inhibitor that can simultaneously act on oncogenic proteins such as NTRK, ALK and/or ROS1 - a compound shown in the following formula I:

However, to date, no literature has been found to report on the preparation of the above-mentioned crystal form of the compound of formula I. Those skilled in the art understand that the discovery of compound forms that are beneficial to the purification and quality control of drug compounds is of great significance for improving the production, quality control and development prospects of solid oral preparations of drugs. However, although the phenomenon of homogeneous polymorphism of drug compounds is relatively common, specifically for a certain specific drug, which crystal form is more suitable for purification and quality control, and more suitable for drug formulation, often requires a large amount of synthesis and screening, continuous adjustment, and even their combination with solvents, in order to obtain the desired product. Moreover, developing products with improved efficacy on this basis has become a further demand for a long time. Therefore, one of the important tasks of drug workers includes discovering those compound forms that are closely related to purification, quality control and drug formulation, and is committed to eliminating those compound forms with poor purification, quality control and drug formulation prospects as soon as possible. However, the solution to these problems is rarely as simple as they seem afterwards. An efficient drug development process must focus on the comprehensive consideration of product quality, repeatability, durability and cost-effectiveness. More importantly, since different drug crystal forms affect the drug formulation, bioavailability and efficacy of the drug, the study of drug crystal forms is of great significance. However, how to obtain the superior crystal form is difficult for researchers.

Therefore, since the properties of the above compounds, such as stability, fluidity, drugability, quality control and bioavailability, etc., still need to be improved, it is necessary to develop suitable forms of the above compounds and preparation methods thereof.

Summary of the invention

In order to solve the above technical problems, the present invention first provides a crystalline form A of a monohydrate of a compound of formula I, which has characteristic peaks at one or more of 9.35±0.2°, 11.42±0.2°, 12.06±0.2°, 18.71±0.2° and 21.16±0.2° in an X-ray powder diffraction pattern expressed in 2θ angles, wherein the chemical structure of the compound of formula I is as follows:

Preferably, the crystalline form A has characteristic peaks at one or more of 9.97±0.2°, 13.16±0.2°, 19.15±0.2°, 19.97±0.2° and 21.00±0.2° in the X-ray powder diffraction pattern expressed in 2θ angles.

Also preferably, the X-ray powder diffraction of the crystalline form A expressed in 2θ angle has an absorption peak at the following position,

More preferably, the crystalline form A has an X-ray powder diffraction pattern substantially as shown in FIG1 .

Preferably, the thermogravimetric analysis (TGA) spectrum of the crystalline form A has a weight loss of 4.216% between room temperature and 130°C.

Preferably, the crystalline form A has a thermogravimetric analysis spectrum substantially as shown in FIG. 2 .

Preferably, the differential scanning calorimetry (DSC) spectrum of the crystalline form A has endothermic peaks at about 87.11°C and 142.34°C.

Preferably, the crystalline form A has a differential scanning calorimetry spectrum substantially as shown in FIG. 2 .

Preferably, the crystal form A is a block crystal with a size of 20-100 μm.

Preferably, the crystalline form A has a morphology substantially as shown in FIG. 3 .

Preferably, the crystal form A is monoclinic, with a space group of P2(1) and a unit cell parameter of α=γ=90°β=93.349(8)°, deviation factor R ₁ =0.0562, Z=4.

The present invention also provides a method for preparing the crystal form A, comprising: slurrying the compound of formula I using a mixed solvent of acetonitrile and water.

According to an embodiment of the present invention, the volume ratio of acetonitrile to water is 1:(1-3), for example 1:1.

According to an embodiment of the present invention, beating is performed at room temperature.

According to an embodiment of the present invention, the following method is used to prepare Form A: a compound of Formula I is taken, a mixed solvent of acetonitrile and water is added to perform a slurrying test, and after slurrying for 1 to 4 days, the crystalline powder solid is collected by centrifugation.

The present invention also provides an amorphous form of the compound of Formula I having an X-ray powder diffraction pattern substantially as shown in FIG. 4 .

The present invention also provides Form E of the compound of Formula I, which has an X-ray powder diffraction pattern substantially as shown in FIG. 5 .

The present invention also provides use of the crystalline form A of the compound of formula I in preparing a pharmaceutical composition.

The present invention also provides the use of the crystalline form A of the compound of formula I in the preparation of a drug for preventing and/or treating diseases with pathological characteristics mediated by ROS1, NTRK, ALK, etc.

According to an embodiment of the present invention, the diseases with pathological characteristics mediated by ROS1, NTRK, ALK, etc. include cancer, sarcoma and pain.

According to an embodiment of the present invention, the cancer is any one of breast cancer, cervical cancer, colon cancer, lung cancer, gastric cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, peritoneal tumor, melanoma, glioma, glioblastoma, head and neck cancer, papillary nephroma, leukemia, lymphoma, myeloma, and thyroid tumor.

The crystalline form A of the compound of formula I described in the present invention is used to prevent and/or treat the following diseases: inflammation, cancer, cardiovascular disease, infection, immune disease, and metabolic disease.

The present invention also provides a treatment method, which comprises the steps of administering the crystalline form A of the compound of formula I described in the present invention to a subject in need of treatment, for selectively inhibiting fusion mutations and drug-resistant mutations of ROS1, NTRK, ALK, etc.

The present invention prepares the crystal form A of the compound of formula I for the first time. The above-mentioned form is of great significance for improving the production, quality control and development prospects of solid oral preparations of drugs. The results of the physical and chemical stability evaluation show that the crystal form A is a monohydrate, which does not lose water or have hygroscopicity under normal conditions. And the stability is significantly better than that of the amorphous form and the crystal form E under high temperature, high humidity, accelerated test and light test. Furthermore, the fluidity of the crystal form A is better than that of the crystal form E and the amorphous form. Finally, the bioavailability of the crystal form A is significantly higher than that of the amorphous form.

In summary, based on the good physicochemical stability, fluidity and bioavailability of Form A, it has great potential in subsequent development and production.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the XRD spectrum of Form A.

FIG. 2 is a thermogravimetric analysis and differential scanning calorimetry (TGA-DSC) diagram of Form A.

FIG. 3 is a polarized light microscope (PLM) spectrum of Form A.

Figure 4 is the XRD spectrum of amorphous.

FIG5 is an XRD spectrum of Form E.

FIG6 is the DVS test result of Form A.

FIG. 7 is the DVS test result of Form E.

FIG8 shows the DVS test results of the amorphous sample.

FIG. 9 shows the flowability results of Form A, Form E and amorphous samples.

Figure 10 is a blood concentration-time curve of rats administered with 10 mg/kg of the compound of formula I (crystalline form A).

Figure 11 is a blood concentration-time curve of rats administered with 10 mg/kg of the compound of formula I (amorphous form).

Figure 12 is a single crystal structure diagram of Form A of the compound of Formula I (Figure 12 is a modified unit, containing 2 molecules of compound and 1 molecule of crystal water, wherein C7 and C7' are both R configurations, and the FLACK parameter is 0.26 (12)).

DETAILED DESCRIPTION

The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

Example 1 Preparation of Form A

15.3 mg of the compound of formula I was placed in a 1.5 mL HPLC bottle, and 0.2 mL ACN/H ₂ O (1:1, v:v) was added to perform a room temperature slurry test. After slurrying for about 4 days, the crystalline powder solid was collected by centrifugation, which was Form A. Its XRD spectrum is shown in Figure 1 (test conditions are shown in Table 1). Thermogravimetric analysis and differential scanning calorimetry (TGA-DSC) are shown in Figure 2. The PLM spectrum is shown in Figure 3.

Table 1 XRPD test parameters

The single crystal structure of Form A is shown in Figure 12. The single crystal data are: monoclinic system, P2(1) space group, unit cell parameters are α=γ=90°β=93.349(8)°, deviation factor R ₁ =0.0562, Z=4.

Example 2 Preparation of amorphous

Weigh 400 mg of the Form A sample prepared in Example 1, dissolve it in 20 mL of dichloromethane at room temperature, filter and quickly remove the solvent from the filtrate by rotary evaporation, and perform corresponding characterization tests on the obtained solid sample, and its XRD spectrum is shown in Figure 4 (test conditions are shown in Table 1). After the amorphous sample was placed under ambient conditions (room temperature, >20% RH) for one night, the PLM was observed again, and it was found that part of the sample was transformed into crystals. After the sample was placed under the same conditions for two days, the XRD results showed that the crystal form of the sample after crystallization was basically the same as Form A. It can be seen from the above results that the amorphous sample is unstable under ambient conditions and recrystallizes into Form A after hygroscopic absorption.

Example 3 Preparation of Form E

The Form E sample was obtained by suspending and stirring the amorphous sample prepared in Example 2 in a 1,4-dioxane/n-heptane solvent system at room temperature. The XRD results are shown in FIG5 (the test conditions are shown in Table 1). The results show that the Form E sample is transformed into Form A after being exposed to air at room temperature for about 1 day, and it is inferred that Form E is a metastable form.

Test Example 1 Evaluation of Crystal Hygroscopicity

Dynamic Vapor Sorption (DVS) was used to study the hygroscopicity of different crystalline APIs.

Determination method:

Instrument: dynamic water vapor adsorption instrument; temperature: 25°C; protective gas and flow rate: N ₂ , 200mL/min;

dm/dt: 0.002%/min; RH range: 0%RH-100%RH; cycle: 1 complete cycle.

Experimental results:

The DVS test results of Form A are shown in Figure 6. As can be seen from Figure 6, when the humidity is 0%, the Form A sample loses about 3-4% of its weight, and the weight loss result is equivalent to the mass of crystal water. The weight gain is obvious from 0% to 20% humidity, and there is almost no water absorption and weight gain from 20% to 80% humidity. When the humidity is above 90%, the moisture absorption is obvious. The weight loss continues when the humidity drops from 100% to 0%, and the weight change trajectories of adsorption and desorption can completely overlap.

The DVS test results of Form E are shown in Figure 7. As can be seen from Figure 7, the Form E sample loses weight continuously when the humidity increases from 0% to 90%: the weight loss is about 2% when the humidity increases from 0% to 30%, and the weight loss suddenly increases from 40% to 50%. When the humidity increases from 90% to 100%, the weight gain is about 4%; when the humidity increases from 100% to 90%, the weight loss is about 2%, and the weight gain due to moisture absorption is greater than the weight loss. When the humidity is below 20%, the weight loss is significantly up to 3%. The weight change trajectories of adsorption and desorption are basically not overlapped.

The DVS experimental results of amorphous are shown in Figure 8. As can be seen from Figure 8, the amorphous sample continues to gain weight when the humidity increases from 0% to 100%; the weight gain rate is uniform from 0% to 80% and the weight gain increases suddenly from 80% to 100%. The weight gain is about 3.5% from 90% to 100%; the amorphous sample continues to lose weight when the humidity decreases from 100% to 0%, and the weight loss rate is fast from 100% to 90%, about 1.5%, which is less than the weight gain; the weight loss rate is uniform from 90% to 0%. The weight change trajectories of adsorption and desorption do not overlap at all.

In summary, Form A is a monohydrate, which does not lose water or have hygroscopic properties under normal conditions; Form E is a solvate, which will cause the loss of the solvent when the humidity in the normal environment changes, and has hygroscopic properties; the amorphous form has hygroscopic properties. In summary, Form A is significantly more stable than Form E and amorphous form under normal storage and production conditions.

Test Example 2: Physical and Chemical Stability Evaluation

In order to verify the stability of Form A, Form E and amorphous, high temperature, high humidity, accelerated tests and light experiments were also carried out. The specific test methods and results are shown below.

2.1 High temperature test

The high temperature test investigation method is shown in Table 2 below.

Table 2

2.2 The high humidity test inspection method is shown in Table 3 below.

Table 3

2.3 Lighting test

The illumination test investigation method is shown in Table 4 below.

Table 4

2.4 Accelerated testing

The accelerated test investigation method is shown in Table 5 below.

Table 5

The results of related substances in each crystal form under high temperature conditions (50℃±2℃) are shown in Table 6.

HPLC test results of related substances under high temperature conditions:

Table 6

After being placed under high temperature conditions (50℃±2℃) for 32 days, the content of specific impurity RRT0.66 in crystal form A and amorphous did not change significantly with the extension of high temperature time (increase ≤0.02%), and the content in crystal form E increased by 0.05%. The content of specific impurity RRT0.71 did not change significantly in the three crystal forms with the extension of high temperature time. The specific impurity RRT1.02 did not change significantly in crystal form A, and increased by 0.19% and 0.03% in crystal form E and amorphous, respectively. The specific impurity RRT1.08 increased by 0.05%, 0.23% and 0.05% in crystal form A, crystal form E and amorphous, respectively. The content of other non-specific impurities was not more than 0.10%. The total impurities increased by 0.16%, 0.51% and 0.09% in crystal form A, crystal form E and amorphous, respectively, and the total impurity content was not more than 2.0%. In summary, under high temperature conditions, the stability of Form A and the amorphous form is comparable, but both are better than the stability of Form E.

The results of related substances in each crystal form under high humidity conditions (90% RH ± 5% RH) are shown in Table 7.

The HPLC test results of related substances under high humidity conditions are as follows:

Table 7

After being placed under high humidity conditions (90% RH ± 5% RH) for 32 days, the contents of specific impurities RRT0.66 and RRT0.71 in the three crystal forms were basically unchanged (increase ≤ 0.02%). The specific impurity RRT1.02 was basically unchanged in both crystal form A and crystal form E, and increased by 0.03% in the amorphous form. The specific impurity RRT1.08 was basically unchanged in crystal form A, and increased by 0.04% and 0.05% in crystal form E and amorphous form, respectively. The contents of other non-specific impurities were not more than 0.10%, and the total impurities increased by 0.03%, 0.05%, and 0.10% in crystal form A, crystal form E, and amorphous form, respectively, and the total impurity content was not more than 2.0%. In summary, the stability of crystal form A under high humidity conditions is better than that of crystal form E and amorphous form.

The results of related substances in each crystal form under lighting conditions (4500lx±500lx) are shown in Table 8.

The HPLC test results of the relevant substances under illumination conditions are as follows:

Table 8

Note: N.D means not detected.

After being placed under illumination conditions (4500lx±500lx) for 32 days, the content of specific impurity RRT0.66 in crystalline form A, crystalline form E and amorphous form increased by 0.09%, 0.04% and 0.03% respectively. Specific impurities RRT0.71 and RRT1.02 did not increase in crystalline form A and crystalline form E, but increased by 0.05% and 0.06% in amorphous form respectively. Specific impurity RRT1.08 increased by 0.09%, 0.08% and 0.19% in crystalline form A, crystalline form E and amorphous form respectively. The content of other non-specific impurities was not more than 0.10%, and the total impurities increased by 0.13%, 0.13% and 0.37% in crystalline form A, crystalline form E and amorphous form respectively, and the total impurity content was not more than 2.0%. In summary, the stability of crystalline form A and crystalline form E under illumination conditions is better than that of amorphous form.

The results of the changes of related substances in each crystal form under accelerated conditions (40°C±2°C, 75%RH±5%RH) are shown in Table 8.

The HPLC test results of related substances under accelerated conditions are as follows:

Table 9

After being placed under accelerated conditions (40℃±2℃, 75%RH±5%RH) for 32 days, the specific impurities RRT0.66, RRT0.71 and RRT1.02 showed no significant changes in the three crystal forms (increase ≤0.02%), and the specific impurity RRT1.08 increased by 0.04%, 0.03% and 0.03% in crystal form A, crystal form E and amorphous form, and the increase was equivalent. The content of other non-specific impurities was no more than 0.10%, and the total impurities were no more than 2.0%. This shows that the stability of the three crystal forms is equivalent under accelerated conditions.

Under high temperature (50℃±2℃) and high humidity (90%RH±5%RH) conditions, the stability of Form A is better than Form E and amorphous, respectively. Under illumination conditions (4500lx±500lx), the stability of Form A and Form E is better than amorphous. Under accelerated conditions (40℃±2℃, 75%RH±5%RH), the stability of the three forms is comparable. Compared with other conditions, the stability of the three forms is poor under illumination conditions (4500lx±500lx), suggesting that they should be kept away from light.

In summary, compared with form E and amorphous form, form A is the most stable form and is most suitable for subsequent processing and development.

Test Example 3: Fluidity Test

The method for measuring fluidity is as follows: the angle of repose is measured using a device in which 2 to 3 funnels are staggered and connected in series. The drug powder flows slowly and evenly through the funnel to a stationary base with a diameter of d, forming a symmetrical powder pile with a single layer of powder at the bottom. During the formation of the powder pile, the funnel height must be maintained within a range of 2 to 4 cm from the top of the powder pile. The cone height h is measured, and the angle of repose tanθ = h/(d/2) is calculated.

Experimental results: The powder flowability test results of the three crystalline APIs are shown in Figure 9.

After testing, the repose angle of the crystal form A sample is about 32.7°, the repose angle of the crystal form E sample is 37.9°, and the repose angle of the amorphous sample is about 37.5°.

Since the smaller the angle of repose of the powder, the better the fluidity, the fluidity of the crystal form A is better than that of the crystal form E and the amorphous form.

Test Example 4

This test example compares the pharmacokinetic process of crystalline form A and amorphous form in SD rats, and compares their pharmacokinetic characteristics in SD rats.

Experimental methods and materials

Compound Information

Test substance

Name: Compound of formula I (crystal form A)

Name: Compound of formula I (amorphous form)

Internal Standard

Name: verapamil

Experimental animals: 6 healthy adult SD rats, male, divided into two groups, 3 rats in each group, 6-8 weeks old; body weight about 200-300 grams. The animals were kept in rat cages and fasted (not less than 10 hours) but not water from the day before the experiment; they were weighed and marked on the tail on the day of the experiment. Blank blood was collected before administration. Blood was collected from the tail vein. Dosage method: Oral gavage (p.o.): drug suspension was gavaged; preparation of dosing suspension: accurately weigh about 10 mg of the sample to be tested, add 5% DMSO to dissolve, 10% solutol HS-15 and 85% saline, vortex and mix to obtain a suspension with a concentration of 1.0 mg/mL; freshly prepared before use.

Sample collection: Rats were given 10 mg/kg of the drug orally by gavage. After the drug was administered, the timing was started. Blood was collected at 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after administration. 0.1 ml of whole blood was collected in an EDTA-Na ₂ anticoagulant tube, inverted 3-4 times to mix, centrifuged at 10000g for 5 minutes at 4°C to separate plasma, and stored at -80°C for testing. Blood was collected from the tail vein.

Sample preparation: 1) Thaw the sample, take 15 μL of unknown plasma sample before or after administration, standard series solution, single blank and double blank samples in a 1.5 ml centrifuge tube. 2) Add 15 μL protein precipitant (methanol) and 400 μL internal standard solution (verapamil prepared in methanol, about 10 ng/ml) to each sample, vortex mix for 2 minutes, and then centrifuge at 4°C, 12000 rpm for 10 minutes. 3) Take the supernatant for LC/MS/MS detection.

Analysis conditions: Liquid phase conditions: Liquid phase: Shimadzu Nexera X2; Chromatographic column: Agilent ZORBAX XDB-C18 3.5 (2.1×50 mm); Column temperature: 35°C; Mobile phase: A-5% acetonitrile (0.1% formic acid water), B-95% acetonitrile (0.1% formic acid water); Injection volume: 3 μL;

Flow rate: 0.5 mL/min. Use gradient elution. See the table below for the elution program.

Table 10

Mass spectrometry conditions: Electrospray ionization source ESI, positive ion, multiple reaction monitoring (MRM) mode was used for mass spectrometry analysis. The mass spectrometry ion source parameters and compound detection parameters are shown in the table below.

Mass spectrometer ion source parameters

Table 11

instrument Triple QuadTM 6500+AB Mass Spectrometer Origin: Canada Ion source mode ESI Scan Mode MRM Curtain Air 20L/min Atomizing gas 50L/min Auxiliary gas 50L/min Ion source temperature 300℃ Ion spray voltage +5500v(positive MRM)

The main scanning parameters of analytes and internal standards are as follows

Table 12

Compound Name Q1(m/z) Q3(m/z) DP(v) EP(v) CE(v) CXP(v) Compounds of formula I 391.1 159.1 135 10 50 15 IS (verapamil) 455.3 165.2 135 10 35 15

PK parameter processing: Based on each individual's blood drug concentration and the corresponding sampling time data, the software PhoenixWinNonlin was used to calculate the pharmacokinetic (PK) parameters based on the blood drug concentration using a non-compartmental model. (1) The PK parameters used to evaluate pharmacokinetics are: Cmax, AUC0-t, AUC0-∞, Tmax, t1/2.

(2) For bioequivalence evaluation: Cmax, AUC0-t, and AUC0-∞ are used for bioequivalence evaluation.

Cmax: The maximum blood drug concentration measured within a specified period of time, which is the actual measured value.

Tmax: The time to peak plasma concentration is measured, which is the actual value. If the maximum value occurs at more than one time point, Tmax is defined as the first time point with this value.

AUC0-t: The area under the blood drug concentration-time curve from point 0 to the last time point t.

AUC0-∞: The area under the plasma concentration-time curve from zero to infinity. Ct is the last measured concentration, and λz is the terminal phase elimination rate constant.

t1/2: Elimination or terminal half-life, estimated by ln2/λz.

Results: The linear range of the compound of formula I is 2-2000 ng/ml. The blood drug concentrations of the compound of formula I (crystalline form A) and the compound of formula I (amorphous form) administered to rats at 10 mg/kg over time are shown in Tables 13 and 14, Figures 10 and 11, respectively. The main pharmacokinetic parameters in rats are shown in Tables 15 and 16.

Table 13: Blood drug concentration-time data of rats given 10 mg/kg of the compound of formula I (crystalline form A)

Table 14: Blood concentration-time data of rats given 10 mg/kg of the compound of formula I (amorphous form)

Table 15 Some pharmacokinetic parameters of rats after oral administration of 10 mg/kg of the compound of formula I (form A)

Table 16 Partial pharmacokinetic parameters of rats after oral administration of 10 mg/kg of the compound of formula I (amorphous form)

After oral administration of the same dose of the compound of Formula I (form A) or the compound of Formula I (amorphous form) to rats, the Cmax obtained were 578 ng/ml and 151 ng/ml, respectively; the AUClast were 4734 h*ng/ml and 1077 h*ng/ml, respectively, and there was a significant difference between the two (p<0.05), indicating that the bioavailability of form A is significantly higher than that of the amorphous form.

The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A crystalline form A of a monohydrate of a compound of formula I, characterized in that it has characteristic peaks at one or more of 9.35±0.2°, 11.42±0.2°, 12.06±0.2°, 18.71±0.2° and 21.16±0.2° in an X-ray powder diffraction pattern expressed in 2θ angles, wherein the chemical structure of the compound of formula I is as follows: 2. The crystalline form A according to claim 1 is characterized in that the crystalline form A has characteristic peaks at one or more of 9.97±0.2°, 13.16±0.2°, 19.15±0.2°, 19.97±0.2° and 21.00±0.2° in the X-ray powder diffraction pattern expressed in 2θ angles. 3. The crystalline form A according to claim 1, characterized in that the crystalline form A has an X-ray powder diffraction pattern substantially as shown in Figure 1. 4. The crystalline form A according to any one of claims 1 to 3, characterized in that the crystalline form A has a thermogravimetric analysis spectrum and a differential scanning calorimetry spectrum substantially as shown in FIG. 2; More preferably, the crystal form A is monoclinic, with a space group of P2(1) and a unit cell parameter of α=γ=90°β=93.349(8)°, deviation factor R ₁ =0.0562, Z=4. 5. The method for preparing the crystalline form A according to any one of claims 1 to 4, characterized in that it comprises: slurrying the compound of formula I using a mixed solvent of acetonitrile and water. 6. The preparation method according to claim 5, characterized in that the crystalline form A is prepared by the following method: taking the compound of formula I, adding a mixed solvent of acetonitrile and water to perform a slurrying test, and after slurrying for 1 to 4 days, centrifuging and collecting the crystalline powder solid. 7. Use of the crystalline form A according to any one of claims 1 to 4 in the preparation of a pharmaceutical composition. 8. Use of the crystalline form A according to any one of claims 1 to 4 in the preparation of a medicament for preventing and/or treating diseases with pathological characteristics mediated by ROS1, NTRK, or ALK. 9. The use according to claim 8, characterized in that the diseases with pathological characteristics mediated by ROS1, NTRK, and ALK include cancer, sarcoma, and pain. 10. The use according to claim 9 is characterized in that the cancer is any one of breast cancer, cervical cancer, colon cancer, lung cancer, stomach cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, peritoneal tumor, melanoma, glioma, glioblastoma, head and neck cancer, papillary nephroma, leukemia, lymphoma, myeloma, and thyroid tumor.