RU2793346C1 - Compound of brd4 inhibitor in solid form, method for its production and application - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to a compound of formula (I) wherein the compound is in crystalline form, wherein the crystalline form of the compound of formula (I) is crystalline form A or crystalline form B. Crystalline form A has an X-ray powder diffraction pattern having characteristic X-ray diffraction peaks at the following angles 2θ: 7.03±0.2°, 11.28±0.2°, 14.00±0.2°, 15.11±0.2°, 17.31±0.2°, 19.48±0.2°, 20.07±0.2°, 22.86±0.2°. Crystal Form B has an X-ray powder diffraction pattern with the characteristic X-ray diffraction peaks at the following angles 2θ: 5.50±0.2°, 8.36±0.2°, 11.87±0.2°, 12.39±0.2°, 12.66±0.2°, 15.11±0.2°, 17.35±0.2°, 18.70±0.2°. A process for preparing a compound of formula (I) in crystalline form A comprises (1) adding a compound of formula (I) to a solvent to form a suspension or solution; where the solvent is selected from the group consisting of methyl tert-butyl ether, ethyl acetate, acetonitrile, ethanol, acetone, methanol, methyl ethyl ketone, a mixed solvent of acetone-water with a volume ratio of 2:1 and a mixed solvent of ethanol-water with a ratio of volumes of 3:1; (2) mixing the suspension or solution in a thermomixer while maintaining a constant temperature at 25–45°C followed by centrifugation and drying to obtain crystalline form A of the compound of formula (I). A process for preparing a compound of formula (I) in crystalline form B comprises (1) adding a compound of formula (I) to tetrahydrofuran to form a suspension or solution; (2) mixing the suspension or solution in a thermomixer while maintaining a constant temperature at 25–45°C followed by centrifugation and drying to obtain crystalline form B of the compound of formula (I).

EFFECT: compound of formula (I) in crystalline form for the treatment of diseases associated with BRD4.

13 cl, 5 dwg, 10 tbl, 9 ex

Description

Technical field

The present invention relates to solid forms and crystalline forms of a compound of formula (I) as a small molecular weight inhibitor of BRD4, and to a method for its preparation, and also relates to its use in the preparation of a medicament for the treatment of diseases associated with BRD4.

State of the art

Histone acetylation regulates gene transcription and chromosome structure, and also plays an important role in epigenetics. Proteins of the BET family (bromodomain and extraterminal domain), as "readers" of the histone acetylation recognition gene, can specifically bind to acetylated lysine residues and attract other transcription factors. Participating in protein-protein interactions, a mediator complex is formed that phosphorylates RNA polymerase, which activates gene transcription and regulates c-Myc and other genes that regulate subsequent parts of signaling cascades. An important role in the proliferation of cancer cells is played by increased expression of specific genes (for example, c-Myc), on which it is highly dependent. Studies have found that tumor cells are highly dependent on the work of specific genes, which makes them very sensitive to BET inhibitors. In the presence of BET inhibitors, BET proteins do not bind to acetylated histone lysine, and thus block the expression of Myc by transcription factors, which leads to suppression of tumor growth.

The BET protein family includes 4 members: BRD2, BRD3, BRD4 and BRDT, each of which consists of two N-terminal tandem regions (BD1 and BD2), an extraterminal domain, several conserved regions (A, B, seed region) and C -terminal motif (SCM). The most studied representative among them is BRD4. It has been found that the occurrence of hematological tumors, including lymphomas (for example, acute myelolymphoma, etc.), leukemias (for example, acute lymphoblastic leukemia, etc.), myelomas (for example, multiple myelomas, etc.) and solid tumors, such as neurocytoma, gliomas, breast cancer (eg, triple-negative breast cancer, etc.), tumors of the gastrointestinal tract (eg, colon and rectal cancer, etc.), prostate cancer, etc., is associated with BRD4 overexpression, but So far, no drugs targeting BRD4 have been approved on the market.

Disclosure of the essence of the invention

According to one aspect of the present invention, the present invention provides a compound of formula (I) in solid form,

In some embodiments of the present invention, the solid form compound is in crystalline form.

In some embodiments of the present invention, the crystalline form is a crystalline Form A of the compound of formula (I) having an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2° , 14.00±0.2°.

In some embodiments of the present invention, crystalline Form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 20.07±0.2°.

In some embodiments of the present invention, crystalline Form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 19.48±0.2°, 20.07±0.2°, 26.05±0.2°.

In some embodiments of the present invention, crystalline Form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 17.31±0.2°, 19.48±0.2°, 20.07±0.2°, 26.05±0.2°.

In some embodiments of the present invention, crystalline Form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 14.00±0.2°, 15.11±0.2°, 17.31±0.2°, 19.48±0.2°, 20.07±0.2°, 22.86±0.2°.

In some embodiments of the present invention, crystalline Form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 14.00±0.2°, 15.11±0.2°, 17.31±0.2°, 19.48±0.2°, 20.07±0.2°, 26.05±0.2°.

In some embodiments of the present invention, crystalline Form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 12.39±0.2°, 14.00±0.2°, 15.11±0.2°, 17.31±0.2°, 19.48±0.2°, 20.07±0.2°, 22.86±0 .2°, 26.05±0.2°.

In some embodiments of the present invention, crystalline Form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 12.39±0.2°, 14.00±0.2°, 14.87±0.2°, 15.11±0.2°, 17.31±0.2°, 19.48±0.2°, 20.07±0 .2°, 22.86±0.2°.

In some embodiments of the present invention, crystalline Form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 12.39±0.2°, 14.00±0.2°, 14.87±0.2°, 15.11±0.2°, 17.31±0.2°, 19.48±0.2°, 20.07±0 .2°, 22.86±0.2°, 26.05±0.2°.

In some embodiments of the present invention, crystalline Form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 12.39±0.2°, 14.00±0.2°, 14.87±0.2°, 15.11±0.2°, 17.31±0.2°, 17.68±0.2°, 19.48±0 .2°, 20.07±0.2°, 22.86±0.2°.

In some embodiments of the present invention, crystalline Form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 12.39±0.2°, 14.00±0.2°, 14.87±0.2°, 15.11±0.2°, 17.31±0.2°, 17.68±0.2°, 19.48±0 .2°, 20.07±0.2°, 22.86±0.2°, 26.05±0.2°.

In some embodiments of the present invention, crystalline Form A has an XRPD pattern substantially as shown in FIG. 1.

In some embodiments of the present invention, XRPD analysis data for crystalline Form A are shown in Table 1:

In some embodiments of the present invention, crystalline form A has a differential scanning calorimetry (DSC) curve with an endothermic peak at 289.22±3°C.

In some embodiments of the present invention, the DSC curve of crystalline form A, essentially as shown in FIG. 2.

In some embodiments of the present invention, the weight loss of crystalline form A according to the thermogravimetric analysis (TGA) thermogram is 1.626% at 300.00±3°C.

In some embodiments of the present invention, the TGA thermogram of crystalline Form A, substantially as shown in FIG. 3.

In some embodiments of the present invention, the crystalline form is a crystalline Form B of the compound of formula (I) having an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 5.50±0.2°, 8.36±0.2 °, 11.87±0.2°.

In some embodiments of the present invention, crystalline Form B has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 5.50±0.2°, 8.36±0.2°, 12.66±0.2° .

In some embodiments of the present invention, crystalline Form B has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 5.50±0.2°, 8.36±0.2°, 11.87±0.2°, 12.39±0.2°, 12.66±0.2°, 15.11±0.2°, 17.35±0.2°, 18.70±0.2°.

In some embodiments of the present invention, crystalline Form B has an XRPD pattern substantially as shown in FIG. 4.

In some embodiments of the present invention, XRPD analysis data for crystalline form B are shown in Table 2.

In another aspect of the present invention, the present invention also provides a process for preparing a compound of formula (I) in solid form, wherein the solid form is crystalline Form A, comprising:

(1) adding a compound of formula (I) to a solvent to form a suspension or solution;

(2) stirring the suspension or solution in a thermomixer while maintaining a constant temperature, then separating and drying to obtain crystalline form A of the compound of formula (I).

In some embodiments of the present invention, the separation in step (2) in the production method is centrifugation or filtration.

In some embodiments of the present invention, the separation in step (2) in the production method is centrifugation.

In some embodiments of the present invention, the solvent in the preparation process is a simple solvent selected from the group consisting of C _1-4 alkyl-O-C _1-4 alkyl, C _1-4 alkylC(=O)OC _1-4 alkyl, C _1-4 alkyl-CN, C _1-4 alkyl-OH, or C _1-4 alkylC(=O)C _1-4 alkyl.

In some embodiments of the present invention, the simple solvent in the production process is methyl tert-butyl ether, ethyl acetate, acetonitrile, ethanol, acetone, methanol, or methyl ethyl ketone.

In some embodiments of the present invention, the solvent in the preparation process is a mixed solvent of C _1-4 alkylC(=O)C _1-4 alkyl and water, or a mixed solvent of C _1-4 alkyl-OH and water.

In some embodiments of the present invention, for a mixed solvent consisting of C _1-4 alkylC(=O)C _1-4 alkyl and water, in the preparation method, the volume ratio of C _1-4 alkylC(=O)C _1-4 alkyl to the volume of water is 1-5:1, preferably 2:1; or for a mixed solvent consisting of C _1-4 alkyl-OH and water, the volume ratio of C _1-4 alkyl-OH to the volume of water is 1-5:1, preferably 3:1.

In some embodiments of the present invention, the mixed solvent in the production process is a mixed solvent of acetone and water or a mixed solvent of ethanol and water.

In some embodiments of the present invention, the mixed solvent in the production process is a 2:1 v/v acetone-water mixed solvent or a 3:1 v/v ethanol-water mixed solvent.

The term "C _1-4 alkyl" refers to any straight or branched chain group containing from 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, etc. P. And each "C _1-4 alkyl" may be the same or different.

In some embodiments of the present invention, the ratio of the mass of the compound to the volume of the solvent in the method of preparation is 1 g: 5-15 ml or 1 g: 5-12 ml, or 1 g: 5-10 ml.

In some embodiments of the present invention, the mixing temperature in the production method is 25°C-45°C.

In some embodiments of the present invention, the mixing time in the production method is from 12 hours to 50 hours, or from 12 hours to 48 hours, or from 12 hours to 24 hours.

In another aspect of the present invention, the present invention also provides a process for preparing a compound of formula (I) in solid form, wherein the solid form is crystalline Form B, comprising:

(1) adding a compound of formula (I) to a solvent to form a suspension or solution;

(2) stirring the suspension or solution in a thermomixer while maintaining a constant temperature, then separating and drying to obtain crystalline form B of the compound of formula (I).

In some embodiments of the present invention, the separation in step (2) in the production process is centrifugation or filtration.

In some embodiments of the present invention, the separation in step (2) in the production process is centrifugation.

In some embodiments of the present invention, the solvent in the production process is tetrahydrofuran.

In some embodiments of the present invention, the ratio of the mass of the compound to the volume of the solvent in the preparation method is 1 g: 5-10 ml.

In some embodiments of the present invention, the mixing temperature in the production method is 25°C-45°C.

In some embodiments of the present invention, the mixing time in the production method is from 12 hours to 50 hours, or from 12 hours to 48 hours, or from 12 hours to 24 hours.

In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising, as described above, a compound of formula (I) in solid form or a crystalline mixture of any two or more crystalline forms.

In another aspect of the present invention, the present invention also provides the use of a compound of formula (I) in solid form or a pharmaceutical composition as described above for the preparation of a medicament for the treatment of diseases associated with BRD4.

In some aspects of the present invention, the use is characterized in that diseases associated with BRD4 include tumors.

In some embodiments of the present invention, the use is characterized in that tumors include hematological tumors and solid tumors in the late stages, while hematological tumors include leukemia, lymphoma and myeloma, and solid tumors in the late stages include neurocytoma, glioma, breast cancer, tumor of the gastrointestinal -intestinal tract and prostate cancer; preferably, the leukemia is acute lymphoblastic leukemia, or the lymphoma is acute myeloid lymphoma, or the breast cancer is triple-negative breast cancer, or the gastrointestinal tumor is colon and rectal cancer.

In another aspect of the present invention, the present invention also relates to a compound of formula (I) in solid form, or a pharmaceutical composition as described above, for use in the treatment of diseases associated with BRD4.

In some embodiments of the present invention, a solid form of a compound of formula (I) or a pharmaceutical composition as described above, wherein the diseases associated with BRD4 include tumors.

In some embodiments of the present invention, the solid form of the compound of formula (I) or the pharmaceutical composition described above, where the tumors include hematological tumors and advanced solid tumors, while hematological tumors include leukemia, lymphoma and myeloma, and advanced solid tumors include neurocytoma, glioma, breast cancer, tumor of the gastrointestinal tract and prostate cancer; preferably, the leukemia is acute lymphoblastic leukemia, or the lymphoma is acute myeloid lymphoma, or the breast cancer is triple-negative breast cancer, or the gastrointestinal tumor is colon and rectal cancer.

In another aspect of the present invention, the present invention also relates to a method of treating a disease associated with BRD4 in a subject in need thereof, comprising administering to the subject a compound of formula (I) in solid form or a pharmaceutical composition as described above.

In some embodiments of the present invention, a method of treating a disease in a subject, where the disease includes tumors.

In some embodiments of the present invention, a method of treating a disease in a subject, wherein the tumors include hematologic tumors and advanced solid tumors, wherein the hematologic tumors include leukemia, lymphoma, and myeloma, and the advanced solid tumors include neurocytoma, glioma, breast cancer , tumor of the gastrointestinal tract and prostate cancer; preferably, the leukemia is acute lymphoblastic leukemia, or the lymphoma is acute myeloid lymphoma, or the breast cancer is triple-negative breast cancer, or the gastrointestinal tumor is colon and rectal cancer.

The term "subject" includes all representatives of animals, including, but not limited to, mammals (eg, mice, rats, felines, monkeys, canines, horses, pigs, etc.) and humans.

The term "substantially as shown in FIG." means that at least 50% or at least 60% or at least 70% or at least 80% or at least 90% or at least 95% or at least 99% of the peaks in the powder x-ray diffraction pattern or DSC curve or TGA thermogram are shown in the corresponding drawing.

Definitions and descriptions

The following terms and phrases used in this application have the following meanings, unless otherwise indicated. A particular phrase or term should not be considered vague or obscure in the absence of a clear definition and should be construed according to its generally accepted meaning. Where a trade mark name is used in this application, it is understood to refer to the relevant commercial product or its active ingredient.

The intermediates of the present invention may be prepared by various synthetic methods well known to those skilled in the art, including particular embodiments of the present invention exemplified below, embodiments of the present invention created by combining synthetic methods with other chemical synthesis methods, and their equivalents well known to those skilled in the art, and preferred embodiments, including but not limited to examples of the present invention.

Chemical reactions of private embodiments of the present invention are carried out using suitable solvents that are compatible with the chemical reactions presented in this application, as well as the reagents and materials necessary for this. To prepare the compounds of the present invention, it is sometimes necessary for those skilled in the art to vary or select synthetic steps or reaction schemes based on existing embodiments of the present invention.

The present invention will be described in detail below with examples, which are illustrative only and do not limit the present invention in any way.

All solvents used in the present invention are commercially available and can be used without further purification.

The solvent used in the present invention is commercially available. The following abbreviations are used in the present invention: DCM - dichloromethane; DMF - N,N-dimethylformamide; DMSO - dimethyl sulfoxide; EtOH - ethanol; MeOH - methanol; TFA - trifluoroacetic acid; TsOH - p-toluenesulfonic acid; mp is the melting point; EtSO ₃ H - ethanesulfonic acid; MeSO ₃ H - methanesulfonic acid; ATP - adenosine triphosphate; HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA - ethylenediaminetetraacetic acid; MgCl ₂ - magnesium dichloride; MnCl ₂ - manganese dichloride; DTT - dithiothreitol.

Technical results of the invention

The compound of formula (I) described in this application has good crystal form stability and is easy to use for drug preparation; the crystalline forms of the present invention exhibit excellent efficacy against BRD4, have better pharmacokinetic properties and a high oral absorption rate, have characteristics such as high activity, good metabolic stability, good solubility, suitability for oral administration, and the like. and may provide more effective treatment for diseases caused by abnormal expression of BRD4.

1.1 Powder x-ray diffraction (powder x-ray diffractometer, XRPD)

Instrument model: X-ray diffractometer Bruker D8 ADVANCE

Test Method: Approximately 10-20 mg of sample was taken for XRPD diffraction determination.

A detailed description of the XRPD diffraction parameters is given below:

X-ray generator: Cu, kα,

Tube voltage: 40 kV, tube current: 40 mA

Emission gap: 1 deg

Height Limiting Slot: 10mm

Slit dispersion: 1 deg

Receiving slot: 0.15 mm

Monochromator: stationary monochromator

Scan range: for crystalline form A: 4-33 deg; and for crystalline form B: 4-35 deg

Scanning speed: 10 deg/min

1.2 Differential Scanning Calorimetry (Differential Scanning Calorimeter, DSC)

Instrument model: TA Q2000 differential scanning calorimeter

Test Method: A sample (0.5-1 mg) was placed in an aluminum DSC test cell, and the sample was heated from 30°C to 300°C under 50 ml/min N ₂ at a heating rate of 10°C/min.

1.3 Thermogravimetric analysis (thermogravimetric analyzer, TGA)

Instrument model: thermogravimetric analyzer TA Q5000IR

Test method: a sample (2-5 mg) was placed in a platinum TGA test crucible and the sample was heated from room temperature to a temperature at which the weight loss was 20% under conditions of supply of 25 ml/min N ₂ at a heating rate of 10°C/ min.

1.4 Dynamic Vapor Sorption (VVA) analysis in this application

Instrument Model: SMS DVS Advantage Dynamic Vapor Sorption Analyzer

Test Conditions: A sample (10-20 mg) was placed in chipboard sample trays for testing.

Detailed description of chipboard parameters:

Temperature: 25°C

Balancing: dm/dt=0.01%/min (fast: 10 min, long: 180 min)

Drying: drying for 120 minutes at 0% relative humidity (RH).

RH step (%) in test: 10%

RH step range (%) in test: 0% - 90% - 0%

The hygroscopicity of the samples was evaluated as follows:

Description of drawings

Fig. 1 is an XRPD diffraction pattern of the Cu-Kα radiation of the crystalline form A of the compound of formula (I).

Fig. 2 is a DSC curve of the crystalline form A of the compound of formula (I).

Fig. 3 is a TGA thermogram of crystalline form A of the compound of formula (I).

Fig. 4 is an XRPD diffraction pattern of the Cu-Kα radiation of the crystalline form B of the compound of formula (I).

Fig. 5 is a DSP isotherm of crystalline form A of the compound of formula (I).

Private embodiments of the invention

For a more complete understanding of the present invention, the following are specific examples that are purely illustrative and do not limit the present invention in any way.

Example 1 Preparation of a Compound of Formula (I)

Stage 1:

Compound 1-1 (25.00 g, 139.20 mmol, 1.00 eq.), 2-butanone (11.04 g, 153.12 mmol, 13.63 ml, 1.10 eq.) and morpholine ( 12.13 g, 139.20 mmol, 12.25 ml, 1.00 eq.) was dissolved in ethanol (200.00 ml), then freeze-dried sulfur (4.46 g, 139.20 mmol, 1.00 eq. .). The suspension was heated to 70° C. and stirred for 12 hours under a protective nitrogen atmosphere. The reaction mixture was first evaporated under reduced pressure to give a yellow oil, to which water (500 ml) was added and extracted with ethyl acetate (200 ml×4). The combined organic phases were collected, washed with brine (200 ml), dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The obtained crude substance was purified on a silica gel column (petroleum ether/ethyl acetate=10/1) to obtain compound 1-2. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 7.47 (d, J=8.0 Hz, 2H), 7.38 (d, J=8.0 Hz, 2H), 6.43 (br s , 2H), 2.13 (s, 3H), 1.56 (s, 3H).

Stage 2:

Compound 1-2 (10.00 g, 37.63 mmol, 1.00 eq.) was dissolved in chloroform (100.00 ml) and 2-chloroacetyl chloride (6.37 g, 56.45 mmol, 4. 49 ml, 1.50 eq.), then the reaction mixture was stirred at 70°C for 1 hour. The reaction mixture was washed with saturated sodium bicarbonate solution (100 ml) and brine (50 ml), then dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The resulting crude material was recrystallized from methanol (40 ml) to give compound 1-3. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 11.81 (br s, 1H), 7.58 (dd, J=2.0, 6.4 Hz, 2H), 7.45 (dd, J= 2.2, 8.6 Hz, 2H), 4.25(s, 2H), 2.29(s, 3H), 1.72(s, 3H).

Stage 3:

Compound 1-3 (11.00 g, 32.14 mmol, 1.00 eq.) and sodium iodide (9.63 g, 64.28 mmol, 2.00 eq.) were added to tetrahydrofuran (50.00 ml) and the mixture was stirred at 60° C. for 2 hours. The reaction mixture was evaporated under reduced pressure to give compound 1-4, which was used directly in the next step without purification. Liquid chromatography-mass spectrometry (LCMS) with electric field sputter ionization (ESI) m/z: 433.9 (M+1).

Stage 4:

Compound 1-4 (14.00 g, 32.28 mmol, 1.00 eq.) was dissolved in tetrahydrofuran (100.00 ml), cooled to -60°C and saturated with ammonia gas for 30 minutes. The reaction mixture was slowly heated to 20° C. and stirred for 3 hours. The reaction mixture was evaporated under reduced pressure. The resulting solid was dissolved in ethyl acetate (150 ml), washed with water (50 ml×3) and brine (50 ml), dried over anhydrous sodium sulfate, filtered and then evaporated under reduced pressure to give compound 1-5, which was used in the next step directly without cleaning. LCMS (ESI) m/z: 322.9 (M+1), 344.9 (M+Na).

Stage 5:

Compound 1-5 (10.00 g, 30.98 mmol, 1.00 eq) was dissolved in isopropanol (150.00 ml) and glacial acetic acid (50.00 ml) and stirred at 90°C for 3 h The solvent was removed from the reaction solution under reduced pressure. The remaining mixture was dissolved in chloroform (20 ml), washed with saturated sodium bicarbonate solution (20 ml) and brine (20 ml), dried over anhydrous sodium sulfate, filtered and then evaporated under reduced pressure. The resulting crude product was recrystallized from ethyl acetate (50 ml) to give compound 1-6. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 8.98 (br s, 1H), 7.46 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.4 Hz , 2H), 4.80 (d, J=8.8 Hz, 1H), 3.93 (d, J=8.6 Hz, 1H), 2.28 (s, 3H), 1.59 (s , 3H).

Stage 6:

Phosphorus pentasulfide (17.07 g, 76.79 mmol, 8.17 ml, 3.60 eq.) was added to a continuously stirred suspension of sodium carbonate (4.07 g, 38.39 mmol, 1.80 eq.) in 1 ,2-dichloroethane (200.00 ml), stirred at 20° C. for 1 hour, then compound 1-6 (6.50 g, 21.33 mmol, 1.00 eq.) was added. The resulting suspension was kept at 65°C for 5 hours. The reaction mixture was cooled to 20° C. and filtered, and the filter cake was dissolved in ethyl acetate (2 L), washed with brine (500 ml), dried over sodium sulfate, filtered and then evaporated under reduced pressure. The obtained crude substance was purified on a silica gel column (petroleum ether/ethyl acetate=5/1) to obtain compound 1-7.

Stage 7:

Hydrazine hydrate (1.67 g, 32.72 mmol, 1.62 ml, 98% pure, 3.00 eq.) at 0°C and the reaction mixture was stirred at 0°C for 1 hour. . LCMS (ESI) m/z: 318.9 (M+1).

Stage 8:

To a mixture of compound 1-8 (2.50 g, 7.84 mmol, 1.00 eq.) in toluene (100.00 ml) was added triethyl orthoacetate (3.82 g, 23.52 mmol, 4.29 ml, 3.00 equiv.). The reaction mixture was stirred at 80° C. for 1 hour. The reaction mixture was evaporated under reduced pressure, and the resulting crude material was recrystallized from ethyl acetate (10 ml) to give compound 1-9. LCMS (ESI) m/z: 344.9 (M+1).

Stage 9:

To a solution of compound 1-9 (1.50 g, 4.38 mmol, 1.00 eq.) in tetrahydrofuran (180 ml) was added LiHMDS (1 M, 8.76 ml, 2.00 eq.) dropwise at 70° WITH. The reaction mixture was stirred at this temperature for 1 hour, then a solution of tert-butyl 2-bromoacetate (1.28 g, 6.57 mmol, 970.82 μl, 1.50 eq.) in tetrahydrofuran (20 ml) was added dropwise . After the addition, the reaction mixture was slowly heated to 20° C. and stirred for 5 hours. The reaction mixture was quenched with saturated NH ₄ Cl solution (50 ml), extracted with ethyl acetate (100 ml), washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered and then evaporated under reduced pressure. The resulting crude material was purified by flash column chromatography and the resulting compound was separated by supercritical fluid chromatography (SFC) to give compound 1-10 (basic-EtOH, column: AS (250 mm×30 mm, 5 µm), mobile phase B : 30%, flow rate (ml/min): 55) ([α] ²⁵ _D +54 (C 0.6, CHCl ₃ )). LCMS (ESI) m/z: 457.0 (M+1).

Stage 10:

Compound 1-10 (150.00 mg, 328.23 µmol, 1.00 eq.) was dissolved in dichloromethane (5.00 ml) and trifluoroacetic acid (1.00 ml) and the reaction mixture was stirred for 4 h at 20° WITH. The reaction mixture was evaporated under reduced pressure to give compound 1, which was used directly in the next step. LCMS (ESI) m/z: 401.0 (M+1).

Stage 11:

Compound 2 (0.78 g, 3.66 mmol, 1 eq), tert-butyl carbamate (643.39 mg, 5.49 mmol, 1.5 eq), tris(dibenzylideneacetone)dipalladium (335.29 mg, 366.15 µmol, 0.1 eq), cesium carbonate (2.39 g, 7.32 mmol, 2 eq) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (211.86 mg, 366.15 µmol, 0.1 eq.) was added to 1,4-dioxane (10 ml) and kept at 100°C for 12 hours under a protective nitrogen atmosphere. Water (20 ml) was added to the reaction mixture, ethyl acetate (20 ml) was added, the insoluble matter was separated by filtration, the aqueous phase was extracted with ethyl acetate (10 ml), the combined organic phases were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. Purification was carried out using a flash column to give compound 3. LCMS (ESI) m/z: 250.1 (M+1).

Stage 12:

Trifluoroacetic acid (3.85 g, 33.77 mmol, 2.5 ml, 18.30 eq.) was added to compound 3 (0.46 g, 1.85 mmol, 1 eq.) in anhydrous dichloromethane (20 ml) and kept at 20°C for 12 hours after the addition. The reaction mixture was washed with water (20 ml), the pH of the aqueous phase was adjusted to 7 with saturated sodium bicarbonate solution, the aqueous phase was extracted with dichloromethane (10 ml×2), the combined organic phases were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. Compound 4 was obtained, which was used in the next step without further purification. LCMS (ESI) m/z: 149.8 (M+1).

Stage 13:

POCl ₃ (76.50 mg, 498.90 µmol, 46.36 µl, 2 eq.) was added to a solution of compound 1 (100 mg, 249.45 µmol, 1 eq.) and compound 4 (44.65 mg, 299 .34 µmol, 1.2 eq.) in pyridine (2 ml) at 0°C, after the addition the temperature was raised to 20°C for 1.5 hours. The reaction mixture was quenched by adding water (3 ml) and the pH of the aqueous phase was adjusted to 7 with 2N hydrochloric acid. The aqueous phase was extracted with dichloromethane (5 ml×3), the organic phase was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. Purification was carried out by thin layer chromatography (dichloromethane/methanol=10/1) to obtain the compound of formula (I) as a glass paste or foam adhering to the wall of the vial. LCMS (ESI) m/z: 532.1 (M+1). ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 9.98 (br s, 1H), 7.94 (d, J=6.8 Hz, 1H), 7.67 (d, J=8.0 Hz , 1H), 7.32-7.36 (m, 3H), 7.24-7.27 (m, 2H), 5.08-5.16 (m, 2H), 4.57-4.61 (m, 1H), 3.78-3.84 (m, 1H), 3.45-3.50 (m, 1H), 2.63 (s, 3H), 2.36 (s, 3H), 1.63(s, 3H).

Example 2 Preparation of Crystalline Form A of the Compound of Formula (I)

About 50 mg of a compound of formula (I) was weighed and added to 1.5 ml glass vials, respectively, and the appropriate amount of solvents was added (see Table 3) until a suspension was formed, which was sealed with a sealing film and mixed in a thermomixer while maintaining a constant temperature at 40°C for 48 hours. Then the samples were centrifuged in a centrifuge and the centrifuged solid was placed in a vacuum oven overnight to dry at 30°C to obtain crystalline Form A of the compound of formula (I).

Example 3 Preparation of Crystalline Form B of the Compound of Formula (I)

About 50 mg of the compound of formula (I) was weighed and added to a 1.5 ml glass vial and tetrahydrofuran (0.2 ml) was added until a suspension was formed, which was sealed with a sealing film. The mixture was stirred in a thermomixer while maintaining a constant temperature at 40°C for 48 hours. The samples were then centrifuged and the centrifuged solid was placed in a vacuum oven overnight to dry at 30° C. to give crystalline form B of the compound of formula (I).

Experimental Example 1 Solid State Stability Test of Crystalline Form A of the Compound of Formula (I)

In accordance with the guidelines for testing the stability of active pharmaceutical ingredients (APIs) and formulations (The general guidelines of the Chinese Pharmacopoeia 2015 edition volume IV 9001), the stability of the crystalline form A of the compound of formula (I) was investigated in accelerated mode (40 ° C / 75 % RH, sealed) and long-term (25°C/60% RH, sealed) tests.

1.5 g of crystalline form A of the compound of formula (I) was weighed, placed at the bottom of a glass sample bottle, and spread thinly, respectively, with samples for testing at 25°C/60%RH and 40°C/75%RH packaged in double-layer low-density polyethylene (LDPE) bags. Each layer of LDPE bags was sealed separately, and then the LDPE bag was placed in an aluminum foil bag and sealed. Samples under different conditions were taken and examined on day 90, the test result was compared with the initial test result on day 0, and the test results are presented in the following table 4:

Conclusion: Crystal Form A of the compound of formula (I) has good stability.

Experimental Example 2 Hygroscopicity Study of the Crystalline Form A of the Compound of Formula (I)

Materials for the experiment:

Dynamic Vapor Sorption Analyzer SMS DVS Advantage

Experiment method:

10-15 mg of crystalline form A of the compound of formula (I) was placed on a chipboard sample tray for testing.

Experiment results:

A DSP isotherm of crystalline form A of the compound of formula (I) is shown in FIG. 5, ΔW=1.789%.

Conclusion of the experiment:

The crystalline form A of the compound of formula (I) has a weight gain of 1.789% at 25° C. and 80% RH due to moisture sorption and is thus slightly hygroscopic.

Example 3 BRD4 Biochemical Activity Assay

Experiment preparation:

1) BRD4-BD1 and BRD4-BD2 proteins from BPS were used for experiments; as well as polypeptides from ANASPEC; developing reagents from Perkinelmer;

2) compounds were screened using the experimental TR-FRET mechanism;

3) tested compounds.

The stages of the experiment are presented below:

1) Preparation of plates for compounds:

The preparation of plates for compounds in the experiment was carried out using the Echo method:

The compound was diluted using the Echo method to 10 concentrations with a 3-fold decrease in each subsequent concentration: 20,000, 6666.67, 2222.22, 740.74, 246.91, 82.305, 27.435, 9.145, 3.048, 1.016 nM.

2) Preparation of reagents for the reaction:

The necessary reagents were prepared on the day of the experiment:

a) preparation of 1× assay buffer (test buffer);

b) preparation of 3× solution of components for the experiment:

1. Reagents were placed on ice to spontaneously melt for later use;

2. 1× assay buffer (test buffer solution) was taken to prepare "solution A" (protein solution), "solution B" (polypeptide solution) and "solution C" (reagent solution for analysis) for experiments, so that the components in the reaction 3× solutions were formed in the system, and the amount of solutions A, B, C was sufficient to carry out the required number of experiments.

3) The stages of the experiment are presented below:

Assay plates are plates containing gradient concentrations of compounds and appropriate DMSO solutions prepared before the experiment using the Echo method:

a) take out the assay plate, add 5 µl/well of “solution A” (protein solution) to columns 2-23 of the assay plate, and then add 5 µl/well of 1× assay buffer to columns 1 and 24 of the assay plate as Min control in the experimental system;

b) centrifuged at 1000 rpm for 30 seconds;

c) incubated plate at 23°C for 20 minutes;

d) add 5 µl/well of "solution B" (polypeptide solution) to columns 1-24 of the assay plate after 20 minutes of incubation;

e) centrifuged at 1000 rpm for 30 seconds;

f) incubated plate at 23°C for 20 minutes;

g) add 5 µl/well of "solution C" (assay reagent solution) to columns 1-24 of the assay plate after 20 minutes of incubation;

h) centrifuged at 1000 rpm for 30 seconds;

i) incubated the tablet at 23°C for 40 minutes;

j) read the plates on the EnVision plate analyzer.

4) Data analysis:

a) Calculate the Z' value for each assay plate using the respective Max control (maximum control) and Min control (minimum control) for each assay plate and ensure that the Z' value for each assay plate is greater than 0.5;

b) calculate the IC ₅₀ value from the signal of the test compounds using XLFIT5 and ensure that it remains within the 3-fold average value according to the literature data, the results are presented in table 5.

5) Conclusion:

The compound of formula (I) has a significant inhibitory effect on BRD4-BD1 and BRD4-BD2.

Example 4 In vivo study of the pharmacodynamics of a compound of formula (I) in MDA-MB-231_luc cancer cells of a subcutaneous xenograft tumor model of human breast cancer

1. Experiment planning

2. Materials for the experiment

2.1 Experimental animals

Style: mouse

Breed: hairless mouse BALB/c

Age in weeks and weight: age 6-8 weeks, body weight 18-22 grams

Gender: female

Supplier: Shanghai SIPPR-Bk Laboratory Animal Co., Ltd.

3. Order and stages of the experiment

3.1 Cell culture

Human breast cancer cells MDA-MB-231_luc were cultured in a monolayer in vitro. Culture conditions: culture medium RPMI-1640 (supplier: Gibco; part number: 22400-089; production lot number: 4868546) with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The culture was grown at 37°C in 5% CO ₂ . Traditional treatment of the gastrointestinal tract with a mixture of panreatin-EDTA was carried out twice a week for better absorption of the drug. When the cells reached the exponential growth phase, the cells were harvested, counted and inoculated.

3.2 Inoculation of tumor cells

0.2 ml of 10×10 ⁶ MDA-MB-231_luc cells were subcutaneously inoculated into the right back of each nude mouse (PBS: Matrigel=1:1). Division into groups and administration of the drug started when the average tumor volume reached 100-150 mm ³ .

3.3 Tumor Measurement and Experimental Performance

The experimental parameters made it possible to find out whether the growth of the tumor was suppressed, slowed down, or the tumor was cured. Tumor diameter was measured twice a week with a caliper. The following equation was used to calculate tumor volume: V=0.5 a ×b ² where a and b were the large and small tumor diameters, respectively.

The tumor growth inhibiting effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%) reflects the rate of tumor growth inhibition. TGI (%) was calculated as follows: TGI (%)=[(1-(mean tumor volume at the end of the drug cycle in the treated group-mean tumor volume at the beginning of the drug cycle in this treated group))/ (mean tumor volume at end of treatment in vehicle control group-average tumor volume at start of treatment in vehicle control group)]×100%.

The relative tumor proliferation rate T/C (%) was calculated according to the following equation: T/C %=T _rtv /C _rtv ×100% (T _rtv : RTV of treated group; C _rtv : RTV of negative control group). Relative tumor volume (RTV) was calculated according to the tumor measurement results. The calculation equation was as follows RTV=V _t /V ₀ , where V ₀ is the average tumor volume measured during grouping and drug administration (i.e. d ₀ ) and V _t is the average tumor volume at the time of measurement. T _rtv and C _rtv were obtained from data collected on the same day.

At the end of the experiment, the mass of the tumor was measured and the percentage of T/C _mass was calculated. T _mass and C _mass were the mass of the tumor in the drug group and the vehicle control group, respectively.

3.4 Statistical analysis

Statistical analysis included determination of the mean and standard error of the mean (SEM) of each group's tumor volume at each time point. The drug group showed the best result from the treatment on the 21st day after drug administration at the end of the experiment, so statistical analysis was performed based on these data to assess differences between groups. A T-test was performed to compare two groups, and a comparison of three or more groups was performed using a one-way ANOVA. If the F value was significantly different, the Games-Howell test was used. If the F value did not differ significantly, Dunnett's test (2-tailed) was used for analysis. Complete data analysis was performed using SPSS 17.0 software. p<0.05 was considered a significant difference.

4. Conclusion of the experiment

On the 21st day after drug administration, for the compound of formula (I), the rate of tumor growth inhibition was TGI=54.85%, T/C=52.99%, p<0.05; there was no significant change in body weight of the animals, and they tolerated the drug well.

Example 5 In Vivo Pharmacodynamic Study of a Compound of Formula (I) in PC-3 Cancer Cells of a Subcutaneous Xenotransplant Tumor Model of Human Prostate Cancer

1. Experiment planning

The method of obtaining the test substance was the same as presented in table 6, and the distribution of animals into groups and dosing regimen were the same as presented in table 7.

2. Materials for the experiment

2.1 Experimental animals

Style: mouse

Breed: hairless mouse BALB/c

Age in weeks and weight: age 6-8 weeks, body weight 18-22 grams

gender: male

Supplier: Shanghai SIPPR-Bk Laboratory Animal Co., Ltd.

3. Order and stages of the experiment

3.1 Cell culture

Human prostate cancer cells PC-3 were cultured in a monolayer in vitro. Culture conditions: F-12K culture medium (supplier: Gibco; part number: 21127-022; production lot number: 1868870) with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. The culture was grown at 37°C in 5% CO ₂ . Traditional treatment of the gastrointestinal tract with a mixture of panreatin-EDTA was carried out twice a week for better absorption of the drug. When the cells reached the exponential growth phase, the cells were harvested, counted and inoculated.

3.2 Inoculation of tumor cells

0.1 ml of 10×10 ⁶ PC-3 cells were subcutaneously inoculated into the right back of each hairless mouse. Division into groups and administration of the drug started when the average tumor volume reached 100-150 mm ³ .

3.3 Tumor measurement, experimental performance and statistical analysis were the same as those presented in the MDA-MB-231 model.

4. Conclusion of the experiment

On the 21st day after the administration of the drug, compared with the control group treated with the vehicle, the test compound of formula (I) had a significant inhibitory effect on the tumor (T/C=44.63%, TGI=58.4%, p=0.033 ); the animals tolerated the drug well.

Example 6 Antitumor activity of the compound of formula (I) in vivo in MC38 mouse colon cancer cells in an animal tumor transplantation model.

1. Experiment planning

2. Material for the experiment

2.1 Experimental animals

Style: mouse

Breed: Mouse C57BL6

Age in weeks and weight: age 6-7 weeks, body weight 16-20 grams

Gender: female

Supplier: Shanghai SLAC Laboratory Animal Co., Ltd.

3. Order and stages of the experiment

3.1 Cell culture

MC38 mouse colon cancer cells (OBiO Technology (Shanghai) Corp., Ltd.) were cultured in a monolayer in vitro. Culture conditions: DMEM culture medium (Gibco; part number: 12100) with 10% fetal bovine serum. The culture was grown at 37°C in 5% CO ₂ in an incubator. Traditional treatment of the gastrointestinal tract with a 0.25% panreatin-EDTA mixture was carried out twice a week for better absorption of the drug. When the cells reached the exponential growth phase and the density was 80%-90%, the cells were harvested, counted and inoculated.

3.2 Inoculation of tumor cells

0.1 ml of 2×10 ⁵ MC38 cells were subcutaneously inoculated into the right back of each mouse. Random division into groups and administration of the drug was carried out in accordance with the volume of the tumor, when the average tumor volume reached about 70 mm ³ .

3.3 Tumor measurement

Tumor diameter was measured twice a week with a caliper. The following equation was used to calculate tumor volume: V=0.5× a ×b ² where a and b were the large and small tumor diameters, respectively.

The tumor growth inhibiting effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%)=T _rtv /C _rtv ×100% (T _rtv : RTV of treated group; C _rtv : RTV of negative control group). Relative tumor volume (RTV) was calculated according to the tumor measurement results. Calculation equation: RTV=V _t /V ₀ , where V ₀ is the average tumor volume measured during grouping and drug administration (ie D0), and V _t is the average tumor volume at the time of measurement. T _rtv and C _rtv were obtained from data collected on the same day.

TGI (%) reflects the rate of tumor growth inhibition. TGI (%)=[(1-(mean tumor volume at the end of the drug cycle in the treated group-mean tumor volume at the beginning of the drug cycle in this treated group))/(mean tumor volume at the end of treatment in the control group) vehicle-treated group-average tumor volume at the start of treatment in the vehicle-treated control group)]×100%.

At the end of the experiment, the mass of the tumor was measured and the percentage T _mass /C _mass was calculated. T _mass and C _mass were the mass of the tumor in the drug group and the vehicle control group, respectively.

3.4 Statistical analysis

Statistical analysis was performed using SPSS software based on tumor volume and tumor weight at the end of the experiment. A T-test was performed to compare two groups, and a comparison of three or more groups was performed using a one-way ANOVA. If the variance was homogeneous (the F value did not differ significantly), the LSD method was used for analysis. If the variance was not uniform (the F value was significantly different), the Games-Howell method was used for analysis. p<0.05 was considered a significant difference.

4. Conclusion of the experiment

On the 20th day after administration of the test compound of formula (I), for the 15 mg/kg dose group: the relative rate of tumor proliferation was T/C=33.68%, the rate of tumor growth inhibition was TGI=68.81%. , p<0.0001; for the group of administration of the drug at a dosage of 25 mg/kg: the relative rate of tumor proliferation was T/C=27.59%, TGI=75.21%, p<0.0001; and for the 50 mg/kg administration group: T/C=10.04%, TGI=93.46%, p<0.0001. A significant tumor inhibitory effect was observed in each group of well-tolerated animals treated with the drug.

Example 7 In vivo pharmacokinetic study of a compound of formula (I) in mice

Balb/c female mice were used as experimental animals. The compound of formula (I) was administered to mice intravenously and intragastrically, then by liquid chromatography with tandem mass spectrometry (LC-MS/MS) the concentration of the drug substance in blood plasma was determined at different time points. The pharmacokinetic action of the compound of formula (I) in vivo in mice was studied and its pharmacokinetic characteristics were evaluated.

1. Description of the experiment

1.1 Experimental drug substance: Compound of formula (I)

1.2 Experimental Animals: Sixteen healthy adult female Balb/c mice were divided into four body weight groups of four mice per group. The animals were purchased from Shanghai Lingchang BioTech Co., Ltd. from Shanghai SLAC Laboratory Animal Co., Ltd., animal breeding license No. SCXK (Shanghai) 2013-0018.

1.3 Preparation of medicinal substance

An appropriate amount of sample was taken, 5% of the final volume of DMSO was added and then 95% of the final volume of 20% HP-β-CD. The mixture was stirred with ultrasound until a clear solution was obtained at a concentration of 0.5 mg/ml. After filtration, it was used for intravenous administration.

An appropriate amount of sample was taken and dissolved in a 0.5% sodium carboxymethylcellulose solution. The mixture was stirred with ultrasound until a homogeneous suspension of 0.5 mg/ml was obtained, which was used for intragastric administration.

1.4 Administration of the drug

Eight female Balb/c mice were divided into two groups. After an overnight fast, the first group received the drug intravenously in a volume of 2.5 ml/kg and a dosage of 1 mg/kg. The second group was administered the drug intragastrically in a volume of 5 ml/kg and a dosage of 3 mg/kg.

2. Experiment

After intravenous administration of the drug, 30 µl of blood was taken from female Balb/c mice at each time point: 0.0833, 0.25, 0.5, 1, 2, 4, 8 and 24 hours, and placed in tubes containing 2 µl EDTA K2; after intragastric administration of the drug, 30 µl of blood was taken from female Balb/c mice at each time point: 0.0833, 0.25, 0.5, 1, 2, 4, 8 and 24 hours, and placed in tubes containing 2 µl EDTA K2. The tube was centrifuged at 3000 g for 15 minutes to separate the plasma and the separated plasma was stored at -60°C. Animals were allowed to feed 2 hours after drug administration.

The LC-MS/MS method was used to measure the content of the test compound in blood plasma after intravenous and intragastric administration of the drug to mice. The linear range of the method was 2.00-6000 nmol/l; plasma samples were analyzed after protein precipitation by treatment with acetonitrile. The results of the study of pharmacokinetic parameters are presented in table 8.

Conclusion of the experiment: the compound of formula (I) has a strong action when administered orally, a low rate of excretion of the drug substance and high bioavailability when administered orally.

Example 8 Pharmacokinetic study of the compound of formula (I) in vivo in rats

Male SD rats were used as experimental animals. The compound of formula (I) was administered intravenously and intragastrically to rats, and then plasma drug concentration was determined by LC-MS/MS at different time points. The pharmacokinetic action of the compound of formula (I) in vivo in rats was studied and its pharmacokinetic characteristics were evaluated.

1. Description of the experiment

1.1 Experimental drug substance: Compound of formula (I) (crystal form A)

1.2 Experimental animals: 4 healthy adult male SD rats were divided into 2 groups by weight, 2 rats in each group. Animals were purchased from Beijing Weitonglihua Laboratory Animals Ltd., Animal Breeding License No. SCXK (Beijing) 2016-0006.

1.3 Preparation of medicinal substance

An appropriate amount of sample was taken, 5% of the final volume of DMSO was added and then 95% of the final volume of 20% HP-β-CD. The mixture was stirred with ultrasound until a clear solution was obtained at a concentration of 0.5 mg/ml. After filtration, it was used for intravenous administration.

An appropriate amount of sample was taken and dissolved in a 0.5% sodium carboxymethylcellulose solution. The mixture was stirred with ultrasound until a homogeneous suspension with a concentration of 1 mg/ml was obtained, which was used for intragastric administration.

1.4 Administration of the drug

Four male SD rats were divided into two groups. After an overnight fast, the first group was administered the drug intravenously in a volume of 4 ml/kg and a dosage of 2 mg/kg. The second group was administered the drug intragastrically in a volume of 10 ml/kg and a dosage of 10 mg/kg.

2. Experiment

After intravenous administration of the drug, 100 µl of blood was taken from male SD rats at each time point: 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours, and placed in tubes containing 2 µl EDTA K2; after intragastric administration of the drug, 100 μl of blood was taken from SD rats at each time point: 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours, and placed in tubes containing 2 μl of EDTA K2. The tube was centrifuged at 3000 g for 15 minutes to separate the plasma and the separated plasma was stored at -60°C. Animals were allowed to feed 2 hours after drug administration.

The LC-MS/MS method was used to measure the content of the test compound in blood plasma after intravenous and intragastric administration of the drug to rats. The linear range of the method was 2.00-6000 nmol/l; plasma samples were analyzed after protein precipitation by treatment with acetonitrile. The results of the study of pharmacokinetic parameters are presented in table 9.

Conclusion of the experiment: the compound of formula (I) has a strong action when administered orally, a low rate of excretion of the drug substance and high bioavailability when administered orally.

Example 9: Analysis of the effect of a compound of formula (I) on the hERG potassium channel

The effect of a compound of formula (I) on hERG potassium channel current was investigated in CHO cells (Chinese Hamster Ovary cells) stably expressing the hERG potassium channel using QPatch fully automatic patch clamp technology.

1. Description of the experiment

1.1 Experimental drug substance: compound of formula (I)

1.2 Experimental system: CHO-hERG cell line

1.3 Cell preparation

CHO-hERG cells were cultured in a 175 cm ² culture flask. After increasing the cell density to 60-80%, the culture medium was removed, the cells were washed once with 7 ml of phosphate buffer solution (PBS) and then 3 ml of Detachin was added for digestion.

After digestion was completed, 7 ml of culture medium was added to neutralize, and then the mixture was centrifuged. The supernatant was removed and then 5 ml of culture medium was added for resuspension to bring the cell concentration to 2~5×10 ⁶ /ml.

1.4 Solution preparation

The method of preparing the solution is presented in table 10 below.

2. Experiment

A 20 mM compound stock solution was diluted with extracellular fluid and 5 μl of a 20 mM compound stock solution was added to 2495 μl of extracellular fluid, diluted to 40 μM at a 500-fold dilution, and then subjected to a 3-fold serial dilution serial dilution with extracellular fluid containing 0.2% DMSO , to obtain the required final concentration for testing. The maximum concentration in the test was 40 μM, all concentrations that were investigated, in turn, were: 40, 13.33, 4.44, 1.48, 0.49, 0.16 μM, respectively, for a total of 6 concentrations. The final concentration of DMSO in the test did not exceed 0.2%, which had no effect on the hERG potassium channel.

The formation of a single-cell high-impedance seal and the whole cell was automatically completed using the QPatch instrument. After recording in whole cell mode, the cells were clamped with a voltage of -80 millivolts. Cells were first pre-stressed at -50 millivolts for 50 milliseconds, then depolarized at +40 millivolts for 5 seconds, then repolarized at -50 millivolts for 5 seconds, after which the voltage was restored to -80 millivolts. Such voltage stimulation was performed every 15 seconds. Data was recorded for 2 minutes, then extracellular fluid was injected, after which data was recorded for 5 minutes. Then the drug was started. Administration of test compound was started at the lowest concentration, each concentration was tested for 2.5 minutes. After continuous administration of all concentrations, 3 μM cisapride was administered as a positive control. With the introduction of each concentration examined at least three cells (n≥3). Experimental data were analyzed using XLFit software.

3. Conclusion

The results show that the compound of formula (I) inhibits hERG potassium current at an IC ₅ 0 >40 μM.

Claims (22)

1. Compound of formula (I)

, where the compound is in crystalline form, where the crystalline form of the compound of formula (I) is a crystalline form A or a crystalline form B, where crystalline Form A has an X-ray powder diffraction pattern having characteristic X-ray diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 14.00±0.2°, 15.11± 0.2°, 17.31±0.2°, 19.48±0.2°, 20.07±0.2°, 22.86±0.2°; where crystalline Form B has an X-ray powder diffraction pattern having characteristic X-ray diffraction peaks at the following 2θ angles: 5.50±0.2°, 8.36±0.2°, 11.87±0.2°, 12.39± 0.2°, 12.66±0.2°, 15.11±0.2°, 17.35±0.2°, 18.70±0.2°. 2. The compound of formula (I) according to claim 1, wherein the crystalline form A has an X-ray powder diffraction pattern having characteristic X-ray diffraction peaks at the following 2θ angles: 7.03±0.2°, 11.28±0.2°, 12 .39±0.2°, 14.00±0.2°, 15.11±0.2°, 17.31±0.2°, 19.48±0.2°, 20.07±0, 2°, 22.86±0.2°, 26.05±0.2°. 3. The compound of formula (I) according to claim 1, wherein crystalline form A has a differential scanning calorimetry (DSC) curve with an endothermic peak at 289.22±3°C. 4. The compound of formula (I) according to claim 1, wherein the mass loss of crystalline form A is 1.626% at 300.00±3° C. as measured by a thermogravimetric analysis (TGA) thermogram. 5. The method of obtaining the compounds of formula (I) in crystalline form A according to any one of paragraphs. 1-4, including: (1) adding a compound of formula (I) to a solvent to form a suspension or solution; where the solvent is selected from the group consisting of methyl tert-butyl ether, ethyl acetate, acetonitrile, ethanol, acetone, methanol, methyl ethyl ketone, a mixed solvent of acetone-water with a volume ratio of 2:1 and a mixed solvent of ethanol-water with a ratio of volumes of 3:1 ; (2) stirring the suspension or solution in a thermomixer while maintaining a constant temperature at 25-45°C, then centrifuging and drying to obtain a crystalline Form A of the compound of formula (I). 6. The method according to claim 5, where the ratio of the mass of the compound to the volume of the solvent is 1 g: 5-10 ml. 7. A method for obtaining a compound of formula (I) in crystalline form B according to claim 1, including: (1) adding a compound of formula (I) to tetrahydrofuran to form a suspension or solution; (2) stirring the suspension or solution in a thermomixer while maintaining a constant temperature at 25-45°C, then centrifuging and drying to obtain a crystalline Form B of the compound of formula (I). 8. The method according to p. 7, where the ratio of the mass of the compound to the volume of tetrahydrofuran is 1 g: 5-10 ml. 9. Pharmaceutical composition for the treatment of diseases associated with BRD4, where the composition contains a therapeutically effective amount of a compound of formula (I) according to any one of paragraphs. 1-4. 10. The use of compounds of formula (I) according to any one of paragraphs. 1-4 or a pharmaceutical composition according to claim 9 for the preparation of a medicament for the treatment of diseases associated with BRD4. 11. The use of claim 10 wherein the diseases associated with BRD4 include tumors. 12. The use of claim 11 wherein the tumors include hematologic tumors and advanced solid tumors, wherein the hematologic tumors include acute lymphoblastic leukemia, lymphoma, and myeloma, and the advanced solid tumors include neurocytoma, breast cancer, and prostate cancer. 13. The use of claim 12 wherein the lymphoma is acute myeloid lymphoma or the breast cancer is triple negative breast cancer.

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