CN111848580B

CN111848580B - Crystal form of quinoline compound containing 1,2, 4-triazine-3, 5-diketone as well as preparation method and application thereof

Info

Publication number: CN111848580B
Application number: CN202010342453.8A
Authority: CN
Inventors: 徐华
Original assignee: Wuhan Shengyun Biomedical Technology Co Ltd
Current assignee: Wuhan Shengyun Biomedical Technology Co Ltd
Priority date: 2019-04-30
Filing date: 2020-04-27
Publication date: 2021-09-14
Anticipated expiration: 2040-04-27
Also published as: CN111848580A; CN111840299A

Abstract

The invention belongs to the technical field of chemical drugs and crystal form processes, and particularly relates to a crystal form of a quinoline compound, and a preparation method and application thereof. The crystal form of the quinoline compound is characterized by means of X-ray powder diffraction, differential thermal analysis, thermogravimetric analysis, infrared spectrum and the like. It can form a crystalline state with characteristic diffraction peaks at 3.3 DEG and 18.1 DEG with a tolerance of + -0.2 deg. And the purity is high, the stability is good, and the wetting is not easy to cause, so that the preparation method can be used for preparing medicines for preventing and/or treating cancers caused by the abnormal high expression of EGFR or VEGFR-1 tyrosine kinase.

Description

Crystal form of quinoline compound containing 1,2, 4-triazine-3, 5-diketone as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of chemical drugs and crystal form processes, and particularly relates to a crystal form of a quinoline compound containing 1,2, 4-triazine-3, 5-diketone as well as a preparation method and application thereof.

Background

The compound shown in the following structural formula I belongs to quinoline compounds of 1,2, 4-triazine-3, 5-diketone. The chemical name is N- { 3-fluoro-4- { 6-methoxy-7- [3- (4-morpholinyl) propoxy group]Quinoline-4-oxy } phenyl } -2-phenyl-4-methyl-3, 5-dioxo-2, 3,4, 5-tetrahydro-1, 2, 4-triazine-6-carboxamide having the molecular formula C₃₄H₃₃FN₆O₇。

US8993566B2 discloses a process for the preparation of quinolines comprising formula i and pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof. In vitro activity screening shows that the compound has antitumor activity. No crystalline form of this compound is reported in US8993566B 2. The compound prepared according to US8993566B2 was in an amorphous state by XRPD characterization. For drugs, different crystal forms may affect the dissolution and absorption in vivo, and further may affect the clinical efficacy and safety of the drugs to a certain extent; in particular, in some poorly soluble oral solid or semisolid formulations, the effect of the crystalline form is greater. Therefore, for polymorphic drugs, the research on the crystal form is beneficial to selecting a crystal form which is meaningful in clinical treatment and is stably controllable.

The crystal form research comprises two stages of crystal discovery and crystal form optimization. By changing crystallization conditions, solvents, temperature and other external factors, a new crystal form is obtained. The crystal form obtained through optimization is characterized by adopting various characterization means, such as single crystal X-ray diffraction, X-ray powder diffraction (XRPD) and Differential Scanning Calorimetry (DSC), High Performance Liquid Chromatography (HPLC), infrared spectrum (IR) and the like, to study the physicochemical properties of the prepared crystal form, and the hygroscopicity, the chemical stability, the physical state stability and the preparation uniformity of different crystal forms are compared.

The invention aims to provide a crystalline quinoline compound with more improved properties so as to meet the requirements of pharmaceutical preparation purity, stability and the like.

Disclosure of Invention

The invention provides a crystal form A of quinoline compounds with the following structural formula I, and X-ray powder thereof

The diffraction pattern has characteristic diffraction peaks at diffraction angles 2 theta of 3.3 degrees and 18.1 degrees with an error of + -0.2 degrees

Preferably, the 2 θ angle corresponds to peak relative intensities of 100% and 54.7%, respectively.

Preferably, the representation of the single crystal X-ray diffraction diagram shows that the crystal form A belongs to a monoclinic system and has a space group P2₁C, unit cell parameters: 25.9839(14) and 12.7821(9) respectively,

beta-92.144 (5) ° unit cell volume

The number of molecules in the unit cell, Z, is 4.

Preferably, the melting point of the crystal form A is 184.00-186.00 ℃.

Preferably, the form a does not contain water of crystallization or crystallization solvent.

Preferably, said form a has an infrared absorption spectrum comprising peaks at least at the following wavenumbers: 3074. 2933, 2784, 1733, 1708, 1507-1651, 1351-1430, 1117-1306 cm^-1。

The invention also provides a preparation method of the quinoline compound crystal form A, which comprises the following steps:

s1, dissolving a crude product of the quinoline compound shown in the formula I in a solvent, raising the temperature to 73-100 ℃, and stirring until the crude product is completely dissolved;

s2, slowly cooling, keeping the temperature and stirring for 0.5-10 hours when crystallization is just started to separate out crystals, and then cooling to room temperature;

and S3, carrying out suction filtration, washing and drying on the precipitated crystal to obtain a recrystallized product.

Preferably, the ratio of the mass of the crude quinoline compound to the volume of the solvent in step S1 is: 1g is 2-50 mL.

Preferably, the solvent in step S1 is selected from any one or two of water, Tetrahydrofuran (THF), methanol, acetonitrile, N-Dimethylformamide (DMF), ethanol (EtOH), dioxane or anhydrous methanol.

Preferably, the solvent is a mixed solvent of EtOH and THF, and the volume ratio is 3: 2.

The invention also provides a pharmaceutical composition which comprises the crystal form A of the quinoline compound shown in the formula I as an active ingredient and a pharmaceutically acceptable excipient.

The application of the pharmaceutical composition in preparing medicines for preventing and/or treating cancers, in particular cancers related to protein tyrosine kinase receptors VEGFR-1 and EGFR. In cancers such as pancreatic cancer, renal cancer, liver cancer, and lung cancer, high expression or abnormal expression of VEGFR, EGFR, and the like occurs, and invasion and metastasis of tumor cells are caused. The present invention relates to compounds of formula I having form A which inhibit VEGFR and/or EGFR and related signaling pathways, thereby inhibiting tumor growth.

The amorphous state is unstable and is easily influenced by factors such as storage conditions, storage time and the like, so that the stability and the efficacy of the medicine are influenced. The crystal form A is not easy to deliquesce, has high purity and stable components, and can reduce the influence caused by adverse factors in the processes of pharmacy and storage. Compared with the amorphous state of the prior art, the crystal form a of the present invention has the following beneficial properties:

(1) is a crystalline solid with high crystallinity, and is more suitable to be used as a component of a pharmaceutical preparation;

(2) the decomposition temperature is high, the thermal stability is good, and crystal transformation is not easy to occur;

(3) the purity is up to more than 99%, the chromatographic purity is still maintained at more than 99% after high temperature, high humidity and illumination for 10 days, the appearance character is not changed, and the crystal form is stable;

(4) moisture is not easy to be attracted;

(5) the dissolution rate of the medicine is more than 97 percent, and is higher than that of an amorphous form;

(6) can be used for preparing a medicinal preparation for treating cancers caused by the abnormally high expression of EGFR or VEGFR-1 tyrosine kinase.

In the present invention, experimental error depends on instrument model, sample preparation and sample purity. The spectra will generally vary with the instrument conditions. The intensity of the peak may vary with experimental conditions, so the angle of the peak cannot be the sole or determining factor, usually allowing an error of ± 0.2 °. Thus, it will be appreciated by those skilled in the art that any crystalline form having characteristic peaks that are the same as or similar to the XRPD pattern of the invention is within the scope of the invention.

Drawings

FIG. 1 is an XRPD spectrum of the amorphous compound of comparative example 1;

FIG. 2 is a DSC of the amorphous compound of comparative example 1;

FIG. 3 is a TGA spectrum of the amorphous compound of comparative example 1;

FIG. 4 is a single crystal X-ray diffraction pattern of the compound provided in example 1;

figure 5 is an XRPD spectrum of the compound provided in example 1;

FIG. 6 is an IR spectrum of the compound provided in example 1;

figure 7 is an XRPD spectrum of the compound provided in example 2;

FIG. 8 is an IR spectrum of the compound provided in example 2;

FIG. 9 is a DSC spectrum of the compound provided in example 2;

figure 10 is a TGA profile of the compound provided in example 2;

FIG. 11 shows the results of kinase assays.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the present invention is not limited thereto. Various changes and modifications may be made by those skilled in the art without departing from the spirit of the invention and within the scope of the appended claims.

The detection instrument and the method comprise the following steps:

x-ray powder diffractometer (XRPD): the instrument model DX-2700X-ray diffractometer adopts CuK alpha radiation, tube voltage 50KV, tube current 300mA, 8 degree/min, 0.02 degree step length.

Single crystal X-ray diffractometer: the instrument model Agilent science and technology Xcaliibur, Atlas, Gemini ultra single crystal diffractometer adopts CuK alpha radiation, the diameter phi of a collimator is 0.50mm, the distance d between a crystal and a CCD detector is 53mm, the pipe pressure is 40kV, the pipe flow is 40mA, an omega scanning mode is adopted, the maximum 2 theta angle is 133.236 degrees, the independent diffraction points are 5472, and observable points (| F |²≥2σ|F|²) It was 2982 with data integrity of 98.6%.

Differential thermal analysis (DSC) with an instrument model Shimadzu TA60 differential scanning calorimeter, the test conditions are temperature ranges: 30-250 ℃; the heating rate is as follows: 10 ℃/min; protective gas: n is a radical of₂(purity of>99.99%) using an aluminum sample pan.

Thermogravimetric analysis (TGA): the instrument model Shimadzu TA60 differential scanning calorimeter has the test conditions of temperature range: 30-400 ℃, heating rate: 10 ℃/min; protective gas: n is a radical of₂(purity of>99.99%); reference substance: aluminum oxide.

Infrared Spectrum (IR) instrument model BRUKER TENSOR 27 with test condition of step length of 2nm and scan range of 3996-358 cm^-1。

High Performance Liquid Chromatography (HPLC): octadecylsilane bonded silica gel as a packing (recommended column: WelchromC18 column (250X 4.6mm, 5 μm)); the buffer (1 ml of trifluoroacetic acid in 1000ml of water, with pH adjusted to 2.5 with triethylamine) -methanol (50:50) was used as the mobile phase, the detection wavelength was 240nm, the flow rate was 1.0ml per minute, and the column temperature was 40 ℃.

Comparative example 1

Comparative example 1 is a quinoline compound obtained according to the method of US8993566B2 example 3 (prior art). The melting point can be measured to be 93.1-105.3 ℃. The XRPD pattern is shown in figure 1, and has no sharp diffraction peak and disordered peak shape, which indicates that the compound of comparative example 1 is not crystallized and is in an amorphous state. The DSC chart is shown in figure 2, and an exothermic peak and an endothermic peak appear between 270 ℃ and 320 ℃. The TGA profile is shown in FIG. 3, and the compound of comparative example 1 shows weight loss below 100 deg.C, probably due to the presence of water of crystallization or crystallization solvent in the compound. As can be seen from fig. 1 to 3, the compound of comparative example 1 is in an amorphous state, and is prone to weight loss and moisture absorption at a lower temperature.

Examples 1 to 18

Structural analysis of crystal form A

Adding 20g of quinoline compound shown in the formula I in the comparative example 1 into 460mL of absolute ethyl alcohol/tetrahydrofuran (volume ratio of 3:2) mixed solvent, heating to reflux (80 ℃), preserving heat for 1h, completely dissolving solid, adding 1g of active carbon (5%) for decoloring for 1h, carrying out suction filtration while the solution is hot, and washing a filter cake by using 20mL of absolute ethyl alcohol/tetrahydrofuran (3:2) mixed solvent at 60 ℃. Slowly cooling to 53 deg.C, separating out solid, maintaining at the temperature for 5 hr, naturally cooling to 25 deg.C, maintaining for 1 hr, vacuum filtering, washing filter cake with anhydrous ethanol (2v/m), and vacuum drying to obtain the final product. Air-blast drying at 25 ℃ for 4h gave the compound of example 1.

The single crystal X-ray diffraction pattern of the crystal form A of the embodiment 1 is shown in figure 4. Example 1 is a non-solvated, non-hydrated crystalline form. Is colorless and transparent, and belongs to monoclinic system with space group P2₁C, unit cell parameters: 25.9839(14) and 12.7821(9) respectively,

β is 92.144(5) °. Cell volume

The number of molecules in the unit cell, Z, is 4. The XRPD spectrum of example 1 is figure 5. The crystal form a of example 1 was confirmed to be a crystalline state by having multiple sharp diffraction peaks at 2 θ angles of 3.347 (100%), 6.811 (2.5%), 7.589 (18.9%), 9.611 (15.7%), 12.146 (3.3%), 13.242 (46.8%), 13.640 (11.6%), 15.287 (20.2%), 16.942 (29.4%), 17.640 (8.5%), 18.114 (54.7%), 20.920 (12.2%), 23.015 (28.1%), 23.779 (20.2%), 24.852 (32.5%), 27.907 (30.3%), 29.493 (13.8%).

The IR spectrum of form a of example 1 is shown in figure 6. At 3273, 3074, 2949, 2933, 2868, 1733, 1708, 1595, 1507, 1477, 1430, 1391, 1306, 1209, 1071, 1029, 1016, 853 and 816cm^-1Has characteristic peaks.

Crystal forms of quinoline compounds under different recrystallization conditions

Dissolving the compound of comparative example 1 in different solvents, raising the temperature to 73-100 ℃ to completely dissolve the compound, slowly cooling, keeping the temperature and stirring for 0.5-10 h at the beginning of crystallization, then cooling to room temperature, and carrying out suction filtration, washing and drying on the precipitated crystals to obtain examples 2-14. The kind of solvent, the ratio of the solvent, the amount of the solvent used, the crystallization temperature, the crystallization time and the drying method for each batch are shown in table 1 below.

TABLE 1 different recrystallization conditions

The DSC and TGA weight loss temperature results of the products obtained in examples 1-14 are shown in Table 2 after XRPD, DSC, TGA and IR measurement, and other related spectra are shown in figures 7-11.

TABLE 2 DSC and TGA results for samples of examples 1-14

Examples	DSC(℃)	TGA weight loss temperature (. degree.C.)
			Example 1	186.00	>200
Example 2	186.00	>200
			Example 3	184.90	>200
Example 4	184.88	>200
			Example 5	184.00	>200
Example 6	185.68	>200
			Example 7	185.99	>200
Example 8	185.68	>200
			Example 9	185.39	>200
Example 10	185.76	>200
			Example 11	185.71	>200
Example 12	185.60	>200
			Example 13	185.57	>200
Example 14	185.79	>200

According to XRPD spectrograms, under different solvent systems and recrystallization modes, peak shapes of crystallization samples precipitated in examples 1-14 are basically consistent, strong characteristic diffraction peaks exist at positions with 2 theta of 3.3 degrees and 18.1 degrees and errors of +/-0.2 degrees, and the crystallization samples are of the same crystal form A. The XRPD spectrum of example 2 is shown in figure 7.

For example, the IR spectrum of example 2 is shown in FIG. 8, 3074cm^-1The absorption peak is the stretching vibration peak of phenyl C-H; 2935cm^-1The absorption peak is a methylene C-H asymmetric stretching vibration peak, 2785cm^-1The absorption peak is a symmetric stretching vibration peak of methylene; 1733cm^-1And 1708cm^-1The absorption peak is a stretching vibration peak of-CONH carbonyl C ═ O; 1507 to 1651cm^-1The absorption peak is a stretching vibration peak of C ═ C and C ═ N; 1351 to 1430cm^-1The absorption peak is an in-plane bending vibration peak of C-H; 1117-1306 cm^-1The absorption peaks are the stretching vibration peaks of C-N and C-O. Frequency and intensity of IR spectrogram of samples of examples 3-14 under other recrystallization conditionsThe degree is similar to the frequency and the intensity of the main peak in example 2, and no obvious difference exists, so that the crystal forms obtained under different recrystallization conditions can be further proved to be the same.

The DSC curve and TGA profile of each of the samples of examples 1-14 were the same. The map of example 2 is used as an example for space limitation. The DSC spectrum of example 2 is shown in figure 9, and the TGA spectrum is shown in figure 10. As shown in Table 2, the DSC curves of the samples of examples 1-14 all showed a sharp endothermic peak at 184.00-187.25 ℃, and the TG curves were significantly decreased, indicating that the melting points of the samples were 184.00-186.00 ℃. Meanwhile, the decomposition temperature of the sample is high, the thermal stability is good, and the crystal transformation is not easy to occur. In the TGA profile, the weight loss of each sample in examples 1 to 14 starts after the temperature is higher than 260 ℃, and although the weight loss ratio is different when the temperature reaches 400 ℃, the weight loss of each sample starts after melting by combining with the DSC profile. Thus, it is demonstrated that the samples of examples 1-14, which do not contain crystal water or a crystallization solvent, begin to lose weight after the temperature exceeds the melting point.

Thirdly, study on stability, hygroscopicity and dissolution rate

Stability of

Example 15: 47.2 the quinoline compound of the formula I of comparative example 1 was added to 1085.6mL of a mixed solvent of absolute ethanol/tetrahydrofuran (volume ratio 3:2), after the addition, the temperature was raised (80 ℃) to reflux, the solid was completely dissolved after 1 hour of heat preservation, 2.36g of activated carbon (5%) was added to decolorize for 1 hour, the mixture was filtered while hot, and the filter cake was washed with 50mL of a mixed solvent of absolute ethanol/tetrahydrofuran (3:2) at 60 ℃. Slowly cooling to 53 deg.C, separating out solid, maintaining at the temperature for 5 hr, naturally cooling to 25 deg.C, maintaining for 1 hr, vacuum filtering, washing filter cake with anhydrous ethanol (2v/m), and vacuum drying to obtain the final product. Air-blast drying at 25 ℃ for 4h gave the compound of example 15.

Example 16 and example 17 were prepared by the same recrystallization method. The XRPD patterns of three samples in examples 15-17 are consistent, and stronger diffraction peaks exist at 2 theta angles of 3.3 degrees and 18.1 degrees and errors of +/-0.2 degrees, and the three samples are of the same crystal form A. The compound is placed for a long time under the conditions of high temperature of 60 ℃, high humidity of 92.5% and illumination of 4500Lux, and the stability of the compound of the crystal form A and the compound of the comparative example 1 is researched through appearance properties, HPLC chromatographic purity and XRPD tests. The results are shown in tables 3 to 5.

TABLE 3 chromatographic purity stability results

Table 4 appearance property results

TABLE 5XRPD stability results

From the results in tables 3 to 5, it is clear that the crystal form a of the samples of examples 15 to 17 has a purity of up to 99.00% under the conditions of standing at a high temperature of 60 ℃, a high humidity of 92.5% and illumination of 4500Lux for 10 days, and has no significant change in appearance, remains as white powder, and has no significant changes in crystal diffraction pattern and 2 theta angle. The chromatographic purity of the comparative example 1 is 86.2532%, which is lower than the chromatographic purity of 99.9034-99.9666% of the examples 15-17, the purity is reduced to 75.2985-78.3252% after the high temperature is 60 ℃, the high humidity is 92.5% and the illumination is 4500Lux for 10 days, the purity is still amorphous, white powder is changed into yellow powder after the high temperature and the illumination, and the powder is agglomerated after the high humidity. Therefore, the compound of the crystal form A in a crystalline state is stable and has high purity, and is suitable for being used as a medicinal form.

Moisture-wicking property

The hygroscopicity of a drug refers to the property of the substance to absorb water at a certain temperature and humidity. The samples of examples 15 to 17 and comparative example 1 were analyzed for hygroscopicity by the following specific test methods:

(1) taking a dry glass weighing bottle with a plug, and weighing the bottle one day before the testPlacing in a climatic chamber (set temperature 25 + -1 deg.C, relative humidity 80% + -2%), precisely weighing (m)₁)；

(2) Appropriate amounts of the samples of examples 15 to 17 and comparative example 1 were taken, respectively, and the samples were spread in the above weighing bottles to a thickness of about 1mm and precisely weighed (m)₂)；

(3) Opening the weighing bottle, and placing the weighing bottle and the bottle cap under the constant temperature and humidity condition for 24 hours;

(4) the weighing bottle cap is covered well, and the weight (m) is precisely weighed₃)，

Percent weight gain (m)₃-m₂)/(m₂-m₁)×100％；

(5) Hygroscopicity characterization and definition of hygroscopicity increase:

deliquescence: absorb enough water to form liquid,

has the characteristics of moisture absorption: the weight is increased by not less than 15 percent when the moisture is introduced,

moisture absorption: the moisture-inducing weight gain is less than 15 percent but not less than 2 percent,

slightly hygroscopic: the moisture-inducing weight gain is less than 2 percent but not less than 0.2 percent,

no or almost no hygroscopicity: the moisture-drawing weight gain is less than 0.2 percent.

The hygroscopicity test data for the samples are shown in Table 6, the weight gain percentage of the tablets prepared from the samples of examples 15-17 is 0.40% -0.88%, the hygroscopicity is slight, and the weight gain percentage of the tablets prepared from the sample of comparative example 1 is the highest and is 1.51%. It can be seen that when the compound of the crystal form A prepared by the invention is used as a medicament, the hygroscopicity is lower than that of the compound of the comparative example 1, and the compound of the crystal form A is more suitable for being used as a medicinal preparation.

TABLE 6 hygroscopicity

Dissolution rate

The samples of examples 15-17 and comparative example 1 were prepared into tablets having the following formulation:

respectively crushing the samples of examples 15-17 and comparative example 1, uniformly mixing with fine powder of auxiliary materials, sieving, adding into a high-efficiency wet mixing granulator, granulating with starch slurry, drying with a fluidized bed dryer, adding magnesium stearate, uniformly mixing, tabletting and packaging.

Taking the tablets prepared by the samples of the examples 15-17 and the comparative example 1, taking hydrochloric acid solution with pH1.0, acetate buffer solution with pH3.6, acetate buffer solution with pH4.0, acetate buffer solution with pH4.5 and 1000ml as dissolution media according to a dissolution method (0931 second method of the general rule of the four ministry of pharmacopoeia 2015), taking 10ml of solution from a dissolution cup at 5 min, 10 min, 15 min, 20 min, 30 min, 45 min and 60min respectively by operating according to the method, simultaneously supplementing the dissolution media with the same volume, filtering, taking 5ml of subsequent filtrate, placing the subsequent filtrate in a 10ml volumetric flask, adding the dissolution media to dilute to scale, shaking up to be used as a sample solution; measuring absorbance at 230nm according to ultraviolet spectrophotometry (0401 in the four parts of pharmacopoeia 2015 edition); an appropriate amount of the control of comparative example 1 was added to prepare a control solution containing about 110. mu.g of the control per 1mL, and the measurement was carried out in the same manner. And calculating the dissolution amount of each tablet according to an external standard method. The results are shown in Table 7.

TABLE 7

	Traits	Related substance (%)	Dissolution (%)	Content (%)
					Example 15	White colour	Compliance with regulations	98	Compliance with regulations
Example 16	White colour	Compliance with regulations	98	Compliance with regulations
					Example 17	White colour	Compliance with regulations	97	Compliance with regulations
Comparative example 1	White colour	Compliance with regulations	85	Compliance with regulations

The results in table 7 show that the properties, related substances and contents of the drug preparation prepared from the compound of the crystal form a of the present invention are not much different from those of the amorphous compound of comparative example 1, but the dissolution rate of the drug is significantly increased, which indicates that the compound of the crystal form a of the present invention has stable and controllable quality.

Preparation containing quinoline compound crystal form A

The medicament containing the quinoline compound crystal form A generally has various medicament dosage forms, such as tablets, capsules, injections, pastes and films.

Example 18: capsule preparation

Grinding the compound of example 1 and other adjuvants into fine powder, mixing well, and encapsulating.

The experimental steps of the effect of the compound on the tumor cell receptor tyrosine kinase EGFR or VEGFR-1 are as follows:

1. ADP-Glo^TMThe Reagent Kinase kit is dissolved at room temperature, and a Kinase Detection Buffer (Kinase Detection Buffer) and a Kinase Detection Substrate (Kinase Detection Substrate) are mixed to obtain a Kinase Detection solvent (Kinase Detection Reagent).

2. The EGFR Kinase Enzyme System and ATP were dissolved on ice.

3. 400. mu.l of 5 × Reaction Buffer A, 1. mu.l of DTT (0.1M), 1.6. mu.l of MnCl₂(2.5M) and 597.4. mu.l dH₂O mix to make 1ml of 2 XBuffer.

4. Mu.l ATP solution (10mM), 500. mu.l 2 XBuffer and 475. mu.l dH₂O mixing to prepare 250. mu.M ATP Assay Solution.

EGFR (0.1. mu.g/. mu.l) was diluted to 1 ng/. mu.l with 1 Xbuffer, and the compound of example 1 was dissolved in DMSO to prepare a 5mM stock solution, which was stored at 4 ℃ for two weeks; the Cabozantinib is evenly mixed and dissolved in DMSO solution to prepare 5mM drug stock solution, and the drug stock solution is stored at 4 ℃ and used within two weeks.

6. Mu.l of diluted EGFR (1 ng/. mu.l), 5. mu.l of Poly (Glu4, Tyr1) (1mg/ml) and 5. mu.l of 2 XBuffer were added to each well of a 96-well plate, 5. mu.l of Cabozantinib (diluted to 5, 12.5, 25, 50, 100, 200. mu.M with 1 XBuffer) at different concentrations was added to each positive control, and 5. mu.l of the compound of example 1 (diluted to 5, 12.5, 25, 50, 100, 200. mu.M with 1 XBuffer) at different concentrations was added to each experimental. Another 2 blank control groups were set, one of which was not added with EGFR but added with 5 μ l of 1 × Buffer; the other well was dosed with 5. mu.l DMSO without drug.

7. Mu.l of 250. mu.M ATP Assay Solution was added to each well to make the total reaction system 25. mu.l. And (4) uniformly mixing, and incubating for 30-60 min at room temperature.

8. Add 25. mu.l ADP-Glo to each well^TMReagent, mix well and incubate at room temperature for 40 min.

9. 50 mul of Kinase Detection Reagent is added into each well, mixed evenly and incubated at room temperature for 30-60 min.

10. The microporous plate multifunctional microplate reader measures and records the fluorescence intensity of each hole.

11. The inhibition ratio was calculated, which is (1- (fluorescence intensity of added inhibitor-blank fluorescence intensity without enzyme)/(fluorescence intensity without inhibitor-blank fluorescence intensity without enzyme)) × 100.

12. Inputting the logarithm value and the inhibition rate of the concentration of the inhibitor in Graphpadprism 6, drawing a relation curve between the two values, and calculating IC₅₀。IC₅₀Defined as the concentration of compound that inhibits 50% of the enzyme activity under the experimental conditions.

The results of the EGFR kinase assay are shown in fig. 11. Compound of example 1 IC₅₀IC of 4.537 μ M Cabozantinib₅₀3.764 μ M. The same procedure can be used for the VEGFR-1 kinase inhibition assay, Compound IC of example 1₅₀2.833. mu.M IC from Cabozantinib₅₀2.349 μ M. The results of the kinase assay demonstrate that the compound of example 1 significantly inhibits the kinase activity of EGFR or VEGFR-1.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made in the embodiments without departing from the spirit and scope of the invention.

Claims

1. A crystalline form a of a quinoline compound having the following structural formula i:

the X-ray powder diffraction pattern is shown in FIG. 5.

2. Form a of the quinolinic compound according to claim 1, characterized in that: the peak relative intensities for 3.3 ° and 18.1 ° 2 θ angles are 100% and 54.7%, respectively.

3. Form a of the quinolinic compound according to claim 1, characterized in that: the single crystal X-ray diffraction diagram shows that the crystal form belongs to a monoclinic system and has a space group of P2₁C, unit cell parameters: a =25.9839(14), b =12.7821(9), c =9.4654(5) a, β =92.144(5) ° and unit cell volume V =3141.5(3) a³The number of molecules Z =4 in the unit cell.

4. Form a of the quinolinic compound according to claim 1, characterized in that: the melting point of the crystal form A is 184.00-186.00 ℃.

5. Form a of the quinolinic compound according to claim 1, characterized in that: the crystal form A does not contain crystal water or a crystal solvent.

6. Form a of the quinolinic compound according to claim 1, characterized in that: the infrared absorption spectrum of form a comprises peaks at least at the following wavenumbers: 3074. 2933, 2784, 1733, 1708, 1507-1651, 1351-1430, 1117-1306 cm^-1。

7. A method for preparing the crystal form A of the quinoline compound as claimed in any one of claims 1 to 6, comprising the steps of:

s1, dissolving a crude product of the quinoline compound shown in the formula I in a solvent, raising the temperature to 73-100 ℃, and stirring until the crude product is completely dissolved; the solvent is selected from any one or more of water, Tetrahydrofuran (THF), methanol, acetonitrile, N-Dimethylformamide (DMF), ethanol (EtOH), dioxane or anhydrous methanol;

8. The method for preparing the crystal form A of the quinoline compound according to claim 7, wherein the ratio of the mass of the crude quinoline compound to the volume of the solvent in the step S1 is as follows: 1g is 2-50 mL.

9. The method for preparing the quinoline compound in the form A according to claim 7, wherein the solvent in the step S1 is a mixed solvent of EtOH and THF.

10. A pharmaceutical composition comprising the crystalline form A of the quinoline compound of any one of claims 1 to 6 as an active ingredient and a pharmaceutically acceptable excipient.