RU2846531C2 - Crystalline form of 1h-pyrrolo[2,3-c]pyridine compound and a method for production thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to crystalline form B of a compound of formula (II), characterized by an X-ray powder diffraction pattern which contains characteristic diffraction peaks at diffraction angle values 2θ 4.579±0.200°, 6.898±0.200°, 13.019±0.200°, 15.918±0.200°, 19.697±0.200° and 25.200±0.200°. Invention relates to use of crystalline form B for preparing a medicinal agent associated with treatment of tenosynovial giant cell tumour.

EFFECT: stable crystalline form of 1H-pyrrolo[2,3-c]pyridine compound of formula (II), with low hygroscopic property, which inhibits BCR-FMS-BaF3 cell proliferation.

10 cl, 9 tbl, 8 dwg, 6 ex

Description

AREA OF TECHNOLOGY

The present invention relates to a crystalline form of a 1H-pyrrolo[2,3-c]pyridine compound and a method for producing the same. It also provides the use of the compound and its crystalline form for producing a medicinal product for treating the corresponding diseases.

LEVEL OF TECHNOLOGY

Colony-stimulating factor 1 (CSF-1, also known as macrophage colony-stimulating factor, M-CSF) is an important growth factor that controls the growth of myeloid progenitor cells, monocytes, macrophages, and osteoclasts and dendritic cells differentiated from macrophages. To exert its biological effect, it must bind to its unique cell surface receptor CSF-1R. CSF-1R is encoded by the c-FMS proto-oncogene and is therefore also called c-FMS, which is a receptor tyrosine kinase. Binding of CSF-1 and CSF-1R at the extracellular domain induces dimerization of CSF-1R, which subsequently leads to self-phosphorylation of the intracellular kinase region of CSF-1R. Once phosphorylated, CSF-1R serves as a docking site for several cytoplasmic signaling molecules and ultimately triggers a series of signaling cascades. For example, phosphorylation of tyrosine residue 697 of CSF-1R can activate the MAPK signaling pathway, whereas phosphorylation of tyrosine residue 721 of CSF-1R can activate the PI3K and PLC γ signaling pathway.

Colony-stimulating factor-1 receptor (CSF-1R) is a key target for the regulation of tumor-associated macrophages in the tumor microenvironment. Many tumor cells can secrete growth factors such as CSF-1 during the growth process, and the latter can attract macrophages (tumor-associated macrophages, TAMs) to infiltrate the tumor site. Macrophages and tumor cells can also secrete CSF-1, and their involvement contributes to the formation of a complex tumor microenvironment, which can help tumor cells develop immune tolerance to autoimmune function and promote proliferation, invasion, and metastasis of tumor cells in vivo. Blockade of the CSF-1/CSF1R pathway has been shown to significantly reduce macrophage infiltration into tumor foci, inhibit the growth of primary tumors, and reduce tumor metastasis. Thus, inhibition of macrophage survival/activation by inhibiting CSF-1/CSF1R signaling has become an important strategy for cancer immunotherapy.

Recent studies have shown that CSF-1R inhibitors have the potential to be used in the treatment of diseases in a variety of ways. They can be used alone or in combination with various types of cancer therapies, such as antiangiogenic therapy, adoptive T-cell transfer, radiotherapy, chemotherapy, and immune checkpoint therapy. Many drugs on the market have CSF-1R inhibitory activity, such as imatinib, dasatinib, and sunitinib, but there are no selective CSF-1R inhibitors on the market. Pexidartinib (PLX-3397), developed by Plexxikon and acquired by Daiichi Sankyo, is a dual inhibitor of CSF-1R and c-Kit. It was approved by the FDA in August 2019 for the treatment of tenosynovial giant cell tumor (TGCT).

ESSENCE OF THE INVENTION

The present application provides a crystalline form A of the compound of formula (I), wherein its powder X-ray diffraction pattern contains characteristic diffraction peaks at diffraction angles 2θ values of 6.12±0.20°, 9.25±0.20° and 14.45±0.20°,

.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form A contains characteristic diffraction peaks at diffraction angles 2θ values of 6.12±0.20°, 7.98±0.20°, 9.25±0.20°, 14.45±0.20°, 16.02±0.20° and 24.52±0.20°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form A contains characteristic diffraction peaks at diffraction angles 2θ values of 6.12±0.20°, 7.98±0.20°, 9.25±0.20°, 12.21±0.20°, 14.45±0.20°, 16.02±0.20°, 20.20±0.20° and 24.52±0.20°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form A comprises characteristic diffraction peaks at diffraction angles 2θ values of 6.12±0.20°, 7.98±0.20°, 9.25±0.20°, 10.08±0.20°, 12.21±0.20°, 14.45±0.20°, 15.13±0.20°, 16.02±0.29°, 29.29±9.29°, 21.35±9.29°, 21.96±9.29° and 24.52±9.29°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form A comprises characteristic diffraction peaks at diffraction angles 2θ values of 6.12°, 7.98°, 9.25°, 10.08°, 12.21°, 14.45°, 15.13°, 16.02°, 18.34°, 20.20°, 21.35°, 21.96°, 23.41°, 24.52°, 25.43°, 27.39°, 28.56°, 29.03°, 29.96°, 31.75° and 37.38°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form A comprises characteristic diffraction peaks at diffraction angles 2θ values of 6.12±0.20°, 9.25±0.20°, and/or 14.45±0.20°, and/or 7.98±0.20°, and/or 16.02±0.20°, and/or 24.52±0.20°, and/or 12.21±0.20°, and/or 20.20±0.20°, and/or 10.08±0.20°, and/or 15.13±0.20°, and/or 18.34±0.20°, and/or 21.35±0.20°, and/or 21.96±0.20°, and/or 23.41±0.20°, and/or 25.43±0.20°, and/or 27.39±0.20°, and/or 28.56±0.20°, and/or 29.03±0.20°, and/or 29.96±0.20°, and/or 31.75±0.20°, and/or 37.38±0.20°.

In some embodiments of the present invention, said crystalline form A has an X-ray powder diffraction (XRD) pattern substantially as shown in Figure 1.

In some embodiments of the present invention, the data from the analysis of the XRD pattern of the said crystalline form A are given in Table 1.

In some embodiments of the present invention, said crystalline form A has a differential scanning calorimetry curve containing an endothermic peak beginning at 200.9±3.0°C.

In some embodiments of the present invention, crystalline Form A has a differential scanning calorimetry (DSC) curve shown in Figure 2.

The present application also provides a compound of formula (II),

.

The present application provides crystalline form B of the compound of formula

(II), while its powder X-ray diffraction pattern contains characteristic diffraction peaks at diffraction angles 2θ values of 4.579±0.200°, 6.898±0.200° and 15.918±0.200°,

.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form B contains characteristic diffraction peaks at diffraction angles 2θ values of 4.579±0.200°, 6.898±0.200°, 13.019±0.200°, 15.918±0.200°, 19.697±0.200° and 25.200±0.200°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form B comprises characteristic diffraction peaks at diffraction angles 2θ values of 4.579±0.200°, 6.898±0.200°, 7.941±0.200°, 11.218±0.200°, 13.019±0.200°, 15.918±0.200°, 19.697±9.299° and 25.209±0.290°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form B comprises characteristic diffraction peaks at diffraction angles 2θ values of 4.579±0.200°, 6.898±0.200°, 7.941±0.200°, 11.218±0.200°, 13.019±0.200°, 14.701±0.200°, 15.918±0.200°, 17.338±0.200°, 19.697±0.200°, 22.540±0.200°, 25.200±0.200° and 27.798±0.200°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form B comprises characteristic diffraction peaks at diffraction angles 2θ values of 4.579±0.200°, 6.898±0.200°, and/or 15.918±0.200°, and/or 13.019±0.200°, and/or 19.697±0.200°, and/or 25.200±0.200°, and/or 7.941±0.200°, and/or 11.218±0.200°, and/or 9.070±0.200°, and/or 9.423±0.200°, and/or 13.796±0.200°, and/or 14.701±0.200°, and/or 15.182±0.200°, and/or 17.338±0.200°, and/or 18.939±0.200°, and/or 19.461±0.200°, and/or 20.700±0.200°, and/or 21.602±0.200°, and/or 22.200±0.200°, and/or 22.540±0.200°, and/or 22.818±0.200°, and/or 23.919±0.200°, and/or 24.461±0.200°, and/or 25.639±0.200°, and/or 26.563±0.200°, and/or 27.120±0.200°, and/or 27.798±0.200°, and/or 28.156±0.200°, and/or 28.595±0.200°, and/or 29.000±0.200°, and/or 29.339±0.200°, and/or 30.739±0.200°, and/or 32.064±0.200°, and/or 32.355±0.200°, and/or 33.201±0.200°, and/or 34.156±0.200°, and/or 35.020±0.200°, and/or 36.063±0.200°, and/or 39.176±0.200°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form B comprises characteristic diffraction peaks at diffraction angles 2θ values of 4.579°, 6.898°, 7.941°, 9.070°, 9.423°, 11.218°, 13.019°, 13.796°, 14.701°, 15.182°, 15.918°, 17.338°, 18.939°, 19.461°, 19.697°, 20.700°, 21.602°, 22.200°, 22.540°, 22.818°, 23.919°, 24.461°, 25.200°, 25.639°, 26.563°, 27.120°, 27.798°, 28.156°, 28.595°, 29.000°, 29.339°, 30.739°, 32.064°, 32.355°, 33.201°, 34.156°, 35.020°, 36.063° and 39.176°.

In some embodiments of the present invention, said crystalline form B has a XRD pattern substantially as shown in Figure 3.

In some embodiments of the present invention, the data from the analysis of the XRD pattern of the said crystalline form B are given in Table 2.

In some embodiments of the present invention, said crystalline form B has a differential scanning calorimetry curve with an endothermic peak beginning at 240.16±3.0°C and an exothermic peak with a peak apex at 282.04±3.0°C.

In some embodiments of the present invention, said crystalline form B has a differential scanning calorimetry curve shown in Figure 4.

In some embodiments of the present invention, the mass loss of said crystalline form B is 0.3643% at 200.0±3°C according to its thermogravimetric analysis curve.

In some embodiments of the present invention, said crystalline form B has a thermogravimetric analysis curve shown in Figure 5.

The present application proposes a crystalline form C of the compound of formula (III), wherein its powder X-ray diffraction pattern contains characteristic diffraction peaks at diffraction angles 2θ values of 5.35±0.20°, 8.66±0.20° and 14.22±0.20°,

.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form C contains characteristic diffraction peaks at diffraction angles 2θ values of 5.35±0.20°, 5.91±0.20°, 8.66±0.20°, 13.12±0.20°, 14.22±0.20° and 25.19±0.20°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form C contains characteristic diffraction peaks at diffraction angles 2θ values of 5.35±0.20°, 5.91±0.20°, 8.66±0.20°, 13.12±0.20°, 14.22±0.20°, 17.61±0.20°, 25.19±0.20° and 26.34±0.20°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form C comprises characteristic diffraction peaks at diffraction angles 2θ values of 5.35±0.20°, 5.91±0.20°, and/or 8.66±0.20°, and/or 13.12±0.20°, and/or 14.22±0.20°, and/or 17.61±0.20°, and/or 25.19±0.20°, and/or 26.34±0.20°, and/or 7.17±0.20°, and/or 10.63±0.20°, and/or 17.34±0.20°, and/or 21.22±0.20°, and/or 22.28±0.20°, and/or 29.39±0.20°, and/or 31.03±0.20°, and/or 32.10±0.20°, and/or 34.91±0.20°.

In some embodiments of the present invention, the powder X-ray diffraction pattern of said crystalline form C contains characteristic diffraction peaks at diffraction angles 2θ values of 5.35°, 5.91°, 7.17°, 8.66°, 10.63°, 13.12°, 14.22°, 17.34°, 17.61°, 21.22°, 22.28°, 25.19°, 26.34°, 29.39°, 31.03°, 32.10° and 34.91°.

In some embodiments of the present invention, said crystalline form C has a XRD pattern substantially as shown in Figure 6.

In some embodiments of the present invention, the data from the analysis of the XRD pattern of the said crystalline form C are given in Table 3.

In some embodiments of the present invention, crystalline form C has a differential scanning calorimetry curve with an endothermic peak beginning at 223.2±3.0°C.

In some embodiments of the present invention, the DSC spectrum of said crystalline form C comprises a differential scanning calorimetry curve shown in Figure 7.

The present application also provides a method for preparing crystalline form B of the compound of formula (II), comprising:

1) adding a compound of formula (I) to a solvent to obtain a solution; and

2) adding 12 N concentrated hydrochloric acid to the said solution, stirring at a certain temperature for a certain time, filtering and drying the filter cake in a vacuum;

at the same time

the solvent is an alcohol solvent;

the mixing temperature is from 30°C to 50°C

and the stirring time is from 12 hours to 24 hours.

The present application also provides a method for preparing crystalline form B of the compound of formula (II), comprising:

1) adding a compound of formula (I) to a solvent to obtain a solution; and

2) adding 12 N concentrated hydrochloric acid to the said solution, stirring at a certain temperature for a certain time, filtering and drying the filter cake in a vacuum;

at the same time

the solvent is an alcohol solvent;

the mixing temperature is from 40°C to 45°C

and the stirring time is from 12 hours to 16 hours.

The present application also provides the use of crystalline form A, crystalline form B or crystalline form C of the above-described compound of formula (II) for the preparation of a medicinal product related to the treatment of tenosynovial giant cell tumor.

Definition and description

Unless otherwise specified, the following terms and phrases as used in this specification are intended to have the following meanings. In the absence of a specific definition, a particular phrase or term is not to be regarded as vague or unclear, but is to be understood in accordance with its generally accepted meanings. When a trade name is mentioned in this application, it is assumed that the name refers to the corresponding product or its active ingredient.

The intermediate compound of the present invention can be prepared by various synthetic methods known to those skilled in the art, including the detailed embodiments listed below, detailed embodiments obtained by combining it with other chemical synthetic methods, and equivalent substitution methods known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples described in this application.

The chemical reaction in the detailed embodiments of the present invention is carried out in a suitable solvent, which must be suitable for the chemical changes described in this application and the necessary reagents and materials. To obtain a compound according to the present invention, those skilled in the art sometimes need to modify or select a synthetic step or reaction process based on existing embodiments.

The structure of the compound according to the present invention can be confirmed by a conventional method known to those skilled in the art. If the present application relates to the absolute configuration of the compound, said absolute configuration can be confirmed by technical means conventional in the art. For example, a single crystal X-ray diffraction (SXRD) method can obtain diffraction intensity data of a cultured single crystal using a Bruker D8 venture diffractometer, the light source is CuKα radiation and the scanning method is ϕ/ω scanning. After obtaining the corresponding data, the crystal structure is further analyzed by a direct method (Shelxs97), and the absolute configuration can be confirmed.

This application will be described in detail using the following examples, which do not imply any limitations with respect to this application.

All solvents used in this application are commercially available and can be used without further purification.

The solvent used in the present application may be commercially available.

Compounds are named according to technically accepted naming conventions or using ChemDraw® software, and names of commercial compounds are given according to the supplier's catalogue.

Technical result

The compound of the present invention has a stable crystalline form and exhibits low hygroscopicity; and the crystalline form of the compound of the present invention can significantly inhibit the proliferation of BCR-FMS-BaF3 cells.

Powder X-ray diffraction method (powder X-ray diffractometer, PXRD) according to the present invention

Crystalline form A of the compound of formula (I) and crystalline form C of the compound of formula (III):

Name of the device: X-ray diffractometer PANalytical X 'pert ³ ,

Test method: Approximately 10 mg of sample was evenly spread on a monocrystalline silicon sample plate.

Crystalline form B of the compound of formula (II):

Name of the device: X-ray diffractometer;

Device model: DX-2700BH;

Manufacturer of the instrument: Dandong Haoyuan Instrument Co., Ltd.

Method parameters:

Light tube: Cu, k-Alphal (λ=1.54184 Å);

Light tube voltage: 40 kV, light tube current: 30 mA;

Divergence gap: 1 mm;

Detector slit: 0.3 mm;

Anti-scatter gap: 1 mm;

Scanning range: 3-40 degrees;

Step diameter: 0.02 degrees;

Step size: 0.5 seconds.

Test method: A sample was taken and placed on the sample plate in such a way as to ensure that the surface of the sample plate was flat. Finally, the sample plate was placed in the X-ray diffractometer for examination.

The method of differential scanning calorimetry (differential scanning calorimeter, DSC) according to the present invention

Crystalline form A of the compound of formula (I) and crystalline form C of the compound of formula (III):

Device model: differential scanning calorimeter TA2500

Crystalline form B of the compound of formula (II):

Instrument model: Mettler Toledo DSC 1 Differential Scanning Calorimeter;

Test method: A sample (2.44 mg) was taken and placed in a high-pressure DSC crucible, examined after pressing and sealing and heating from 40°C to 350°C at a heating rate of 10°C/min.

A method using a thermogravimetric analyzer (TGA) according to the present invention:

Device model: TGA2SF/1100;

Test method: A sample (2.745 mg) was taken and placed in an aluminum oxide crucible for testing, and then the sample was heated from 40°C to 500°C at a heating rate of 10°C/min.

The method of dynamic vapor sorption (DVS) according to the present invention:

Device model: SMS DVS Intrinsic device for studying dynamic vapor sorption;

Test conditions: A sample (10 mg) was collected and placed on a particle board sample plate for testing.

The detailed parameters of the chipboard were as follows:

Temperature: 25°C;

Equilibrium: dm/dt=0.01%/min;

Drying: 25°C, 0% relative humidity (RH) drying for 2 hours;

Relative humidity step during testing (%): 5% RH;

Relative humidity step range during testing (%): 0%-95%-0%RH.

Note: ΔW% represents the increase in hygroscopic mass of the test sample at 25°C±1°C and 80%±2% relative humidity.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Figure 1 shows the powder X-ray diffraction pattern (PXRD) of compound A of formula (I) obtained using Cu-Kα radiation.

Figure 2 shows the differential scanning calorimetry (DSC) curve of compound A of formula (I).

Figure 3 shows the XRD pattern of compound B of formula (II) obtained using Cu-Kα radiation.

Figure 4 shows the DSC curve of compound B of formula (II).

Figure 5 shows the thermogravimetric analysis (TGA) curve of compound B of formula (II).

Figure 6 shows the XRD pattern of compound C of formula (III) obtained using Cu-Kα radiation.

Figure 7 shows the DSC spectrum of compound C of formula (III).

Figure 8 shows the dynamic vapor sorption (DVA) curve of compound B of formula (II).

DETAILED DESCRIPTION OF THE INVENTION

For a better understanding of the contents of this application, additional explanations are provided below in combination with detailed examples without limiting the contents of this application.

Example 1: Preparation of a compound of formula I

Step 1: Synthesis of compound 1-B

Compound 1-A (125 g, 819.24 mmol) was dissolved in methanol (2.5 L), and toluene (2.5 L), dichloromethane-1,1'-bis(diphenylphosphino)ferrocene palladium(II) dichloride complex (40 g, 48.98 mmol) and triethylamine (331.59 g, 3.28 mol) were added to the resulting solution, followed by reaction with stirring for 40 hours in a high-pressure autoclave under a carbon monoxide atmosphere (2 MPa) and an external temperature of 110°C. After completion of the reaction, the obtained product was filtered using diatomite and concentrated under reduced pressure to remove the solvent to obtain a primary product. The primary product was purified by column chromatography (n-heptane:ethyl acetate=1:1) to give compound 1-B.

MS mass/charge: 177.2 [M+H] ⁺ .

Step 2: Synthesis of compound 1-C

Compound 1-B (249.9 g, 1.42 mol) was dissolved in N,N-dimethylformamide (2.5 L) to obtain a reaction solution. The reaction solution was cooled to 0 °C, N-iodosuccinimide (351.05 g, 1.56 mol) was added thereto, and the reaction solution was stirred at 0 °C to 5 °C for 1 hour. The reaction solution was slowly poured into water (9 L), stirred for 15 minutes, and then filtered under reduced pressure to obtain a filter cake. The filter cake was washed twice with 4 L of water. After the filter cake was dried in a drying oven under reduced pressure, ethanol (800 mL) was added thereto, stirred for 1 hour, and then filtered under reduced pressure to obtain compound 1-C.

MS mass/charge: 302.7 [M+H] ⁺ .

Step 3: Synthesis of compound 1-D

Compound 1-C (200 g, 662.10 mmol) was dissolved in tetrahydrofuran (3 L), the resulting reaction system was cooled to a temperature of 0 °C to 5 °C, sodium hydride (39.73 g, 993.15 mmol, content 60%) was slowly added in portions, followed by stirring at a temperature of 0 °C to 5 °C for 0.5 hour, then 2-chloromethyl-2-(trimethylsilyl)ethyl ether (139.09 g, 834.25 mmol) was added to the above system and the temperature of the resulting reaction system was naturally raised to 20 °C, after which it was stirred for 1 hour. Under a weak nitrogen flow, the reaction solution was slowly poured into 3 L of water, stirred and quenched. _Ethyl acetate (3 L) was added to perform extraction, and the resulting organic phase was washed with water (3 L), and then washed with saturated brine (3 L). The organic phase was dried with anhydrous _Na2SO4 , filtered, and concentrated under reduced pressure to remove the solvent to obtain the primary product. The primary product was purified by column chromatography (n-heptane: tetrahydrofuran=7:1 to 4:1) to obtain the purified primary product, then n-heptane (2 L) was added to the purified primary product, stirred for 1 hour, and then filtered under reduced pressure to obtain compound 1-D.

MS mass/charge: 433.1 [M+H] ⁺ .

Step 4: Synthesis of compound 1-F

Compound 1-E (2.5 kg, 15.77 mol) and cesium carbonate (10.28 kg, 31.54 mol) were dissolved in tetrahydrofuran (10 L). The reaction system was cooled to 0°C to 10°C, and cyclopropanol (1.14 kg, 19.71 mol) was added dropwise thereto. After completion of the dropping, the reaction solution was slowly heated to 25°C to 30°C and stirred for 16 hours. The reaction solution was filtered under reduced pressure to obtain a filter cake, the obtained filter cake was washed twice with tetrahydrofuran (1 L), and the resulting filtrates were combined. The resulting filtrate was slowly added to water (36 L), stirred for 30 minutes, and then filtered under reduced pressure to obtain a filter cake. Water (5 L) was added to the resulting filter cake, stirred for 30 minutes, and then filtered under reduced pressure to obtain compound 1-F.

MS mass/charge: 180.8 [M+H] ⁺ .

Step 5: Synthesis of compound 1-G

Compound 1-F (100 g, 555.06 mmol) was dissolved in ethanol (1 L), and Pd/C (5 g, 555.06 mmol, 10% content) was added thereto to carry out the reaction under hydrogen atmosphere (30 psi (201 kPa) to 40 psi (276 kPa)), and stirred at 25°C to 30°C for 16 h. The reaction solution was filtered using diatomite. Care should be taken not to drain the diatomite. The resulting filtrate was subjected to rotary evaporation under reduced pressure to remove the solvent to obtain compound 1-G.

MS mass/charge: 151.2 [M+H] ⁺ .

Step 6: Synthesis of compound 1-H

Compound 1-G (154 g, 1.03 mol) was dissolved in 1,4-dioxane (2.25 L) and water (45 mL), and 2,5-dibromo-4-methylpyridine (308.77 g, 1.23 mol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (29.67 g, 51.27 mmol), and potassium carbonate (354.31 g, 2.56 mol) were added to the resulting solution. The reaction system was exchanged for nitrogen three times, then tris(dibenzylideneacetone)dipalladium (14.74 g, 25.64 mmol) was added thereto, and then the reaction system was exchanged for nitrogen three times, and the reaction solution was stirred for 16 hours under the protection of nitrogen at a temperature of 55°C to 60°C. The resulting reaction solution was filtered using diatomite to obtain a filter cake, and the filter cake was washed with 1,4-dioxane (500 mL×3). The resulting filtrate (about 6 L) was concentrated under reduced pressure until the residual volume reached about 3.5 L, and then slowly added to n-heptane (17.5 L) and stirred for 0.5 hour, and then filtered under reduced pressure to obtain a filter cake. Water (3 L) was added to the above precipitate, stirred for 20 minutes, and then filtered under reduced pressure to obtain compound 1-H.

MS mass/charge: 319.9 [M+H] ⁺ .

Step 7: Synthesis of compound 1-I

Compound 1-H (2.18 kg, 6.80 mol) was dissolved in dichloromethane (17.5 L), 4-dimethylaminopyridine (41.56 g, 340.21 mmol) was added thereto, and then di-tert-butyl dicarbonate (1.93 kg, 8.85 mol) was added dropwise. The resulting reaction solution was stirred at an external temperature of 30°C to 40°C for 3 hours. Water (10 L) was added to the reaction solution for washing. The organic phase was washed with saturated brine (10 L), and then the organic phase was collected, dried over anhydrous sodium sulfate (1 kg), and concentrated under reduced pressure to obtain a primary product. Then n-heptane (5 L) was added to the primary product and stirred in an ice bath at 0°C, so that after 15 minutes the solid gradually precipitated. Stirring was continued for 1 hour, and then the resulting solution was subjected to suction filtration to give compound 1-I.

MS mass/charge: 420.1 [M+H] ⁺ .

Step 8 Synthesis of compound 1-J

Compound 1-I (500.31 g, 1.19 mol), bis(pinacolato)diboron (392.81 g, 1.55 mol), dioxane (5 L), and potassium acetate (236.73 g, 2.38 mol) were added sequentially into the reactor. Nitrogen gas was replaced three times in the reaction system, and 1,N-bis(diphenylphosphino)ferrocene palladium chloride (43.71 g, 0.08 mol) was added thereto. Nitrogen gas was replaced three times in the reaction system, and protection was turned on using a nitrogen stream. The reaction system was heated to a temperature of 70°C to 80°C and stirred for 12 hours to 16 hours. The resulting reaction solution was subjected to suction filtration using diatomite (200 g) under reduced pressure to obtain a filter cake. The filter cake was washed with ethyl acetate (1 L×2), and the filtrates were combined and concentrated to give the primary product. The primary product was purified by column chromatography (eluent: n-heptane: tetrahydrofuran = 10/1) to give compound 1-J.

MS mass/charge: 490.3 [M+H+Na] ⁺ .

Step 9 Synthesis of compound 1-K

Compound 1-D (385.26 g, 0.89 mol), compound 1-J (466.95 g, 0.91 mol), and dioxane (3.85 L) were sequentially added into the reactor and stirred. Potassium phosphate (385.81 g, 1.82 mol) was dissolved in water (385 mL) and added into the reactor at room temperature (20°C to 30°C). The reaction system was exchanged for nitrogen gas three times and then cooled to 0°C to 5°C. Dichloromethane-1,1'-bis(diphenylphosphine)ferrocene palladium chloride complex (65.25 g, 0.09 mol) was added into the above system, and the nitrogen gas was exchanged for three times in the reaction system. The reaction system was heated to 35°C to 40°C and stirred for 12 hours to 16 hours. Ethyl acetate (8 L) and water (8 L) were added to the reaction solution, followed by stirring for 10 minutes and then maintained for liquid/liquid separation to obtain an aqueous phase and an organic phase. The aqueous phase was separated. The organic phase was washed with water (8 L) and saturated sodium chloride solution (8 L). The organic phase was collected, dried over anhydrous sodium sulfate (500 g), filtered under reduced pressure, and concentrated under reduced pressure to obtain the primary product. Ethyl acetate (7.7 L) and powdered activated carbon (193.38 g) were added to the primary product. The resulting mixture was stirred at reflux (75°C to 80°C) for 1 hour, then cooled to 55°C to 60°C and filtered through diatomite (500 g) under reduced pressure to obtain a filter cake. The filter cake was washed with ethyl acetate (15 L) to obtain filtrates. The filtrates were combined and concentrated under reduced pressure (at a temperature not exceeding 45°C) to a volume of about 2 L, to which ethyl acetate (0.3 L) and n-heptane (3 L) were then added and the resulting solution was stirred at 20°C to 30°C for 12 to 16 hours, then filtered to obtain compound 1-K.

MS mass/charge: 646.4 [M+H] ⁺ .

Step 10 Synthesis of compound 1-L

Ethanol (705 mL) was added to a 3 L reaction vessel, followed by adding compound 1-K (352 g, 0.55 mol) and stirring. Then, 260 mL of methylamine ethanol solution was added to the reaction vessel, and stirring was continued to obtain a cloudy reaction solution. The reaction solution was slowly heated to a temperature of 35 °C to 40 °C and stirred for 2 hours to 3 hours. Another 260 mL of methylamine ethanol solution was added to the reaction vessel, and stirring was continued to obtain a cloudy reaction solution. The reaction solution was stirred at a temperature of 35 °C to 40 °C for 20 hours to 24 hours, during which time the reaction solution gradually became transparent. The reaction solution was concentrated under reduced pressure to obtain the primary product 1-L, which was directly used for the next reaction without further purification.

MS mass/charge: 645.2 [M+H] ⁺ .

Step 11 Synthesis of compound 1-M

To the primary product 1-L was added 704 ml of tetrahydrofuran, and 1100 ml of tetrabutylammonium fluoride tetrahydrofuran solution was added to the reaction flask with stirring to obtain a clear reaction solution. Then, 66 ml of anhydrous ethylenediamine was added to the reaction flask. The reaction solution was stirred at 65°C to 70°C for 18 hours. The reaction solution was concentrated under reduced pressure (50°C) until the solvent evaporated, then 2 l of ethyl acetate and 2 l of water were added to perform liquid-liquid extraction, and the organic phase was washed with water (2 l × 4). The organic phase was collected and concentrated under reduced pressure (50°C) until the solvent evaporated. 700 mL of water was added to the rotary evaporation flask to obtain a cloudy solution, which was then transferred to a round-bottomed flask and stirred at 20°C to 30°C for 16 hours. The resulting solution was filtered under reduced pressure using a Buchner funnel to obtain a filter cake. The filter cake was washed with water (50 mL × 3) to obtain a solid. The solid was again stirred with acetonitrile (700 mL) at 20°C to 30°C for 30 minutes. The resulting solution was filtered under reduced pressure using a Buchner funnel to obtain a filter cake. The filter cake was washed with acetonitrile (30 mL × 3) to obtain compound 1-M.

MS mass/charge: 515.3 [M+H] ⁺ .

Step 12 Synthesis of compound of formula I

Ethyl acetate (3.4 L) was placed in a 10 L three-neck bottle. Compound 1-M (168 g) was added to the reaction vessel, and stirring was continued to obtain a clear reaction solution. Hydrochloric acid/ethyl acetate (4 mol/L) 800 mL was slowly added to the reaction vessel, and a solid was precipitated from the reaction solution. The reaction system was gradually heated to a temperature of 20-30 °C to 35-40 °C and stirred for 19 hours under these conditions. The reaction solution was filtered under reduced pressure to obtain a yellow solid, which was washed with ethyl acetate (50 mL×3). The yellow solid was added to 1700 mL of water in the vessel with stirring to obtain a clear reaction system. The pH of this reaction system was adjusted to 9 to 10 using 2.0 L of the prepared _Na2CO3 solution (1 mol/L), a light green solid _was precipitated, and stirring was continued for 0.5 hour to 1 hour. The light green solid was added to a round-bottomed flask containing acetonitrile (1.7 L), and stirred at 20°C to 30°C for 0.5 hour to 1 hour to obtain a reaction mixture. The reaction mixture was filtered under reduced pressure to obtain a compound of formula (I).

MS mass/charge: 415.2 [M+H] ⁺ .

Example 2: Preparation of crystalline form B of the compound of formula (II)

Step 1: Synthesis of crystalline form B of the compound of formula (II)

EtOH (2.5 L) was added to a 5 L reaction vessel, then the compound of formula (I) (121.2 g) was added and stirred. Concentrated hydrochloric acid (12 mol/L) 26 mL was slowly added to the reaction vessel, and the reaction solution first dissolved and then became cloudy. The reaction system was gradually heated to a temperature of 40 °C to 45 °C, and stirring was continued for 12 hours to 16 hours. Then, the reaction system was naturally cooled to a temperature of 10 °C to 20 °C, filtered under reduced pressure using a Buchner funnel to obtain a filter cake. The filter cake was washed with ethanol (50 mL×3), and the filter cake was collected. The obtained filter cake was dried in vacuo (45 °C, -0.1 MPa, 12 hours) to obtain Form B crystal of the compound of formula (II). By measuring IC-Cl (measurement of Cl using ion chromatography), it was determined that the compound contained one hydrochloride (Cl ^- % was 7.6%).

MS mass/charge: 415.2 [M+H] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.22 (s, 1H), 10.63 (s, 1H), 9.07 (d, J=4.8 Hz, 1H), 8.37 (d, J=2.8 Hz, 1H), 8.24 (d, J=5.6 Hz, 1H), 7.99 (s, 1H), 7.93-7.90 (m, 2H), 7.71 (d, J=5.6 Hz, 1H) 7.15 (s, 1H) 7.00 (d, J=8.8 Hz, 1H), 4.23-4.19 (m, 1H) 2.91 (d, J=4.8 Hz, 3H) 2.27 (s, 3H) 0.79-0.67 (m, 4H).

Example 3: Preparation of crystalline form A of the compound of formula (I)

1.6 g of crystalline form B of the compound of formula (II) were weighed and dissolved in 25 ml of water and stirred at a temperature of 10°C to 20°C to obtain a solution. 1.88 g of sodium carbonate was dissolved in 5 ml of water and slowly added to the above solution, after which stirring was continued for 0.5 h. The reaction solution turned from clear to cloudy and a solid precipitated. The solid was filtered. The filter cake was dried for 12 h under vacuum to obtain crystalline form A of the compound of formula (I). MS w/z: 415.2 [M+H] ⁺ .

Example 4: Preparation of crystalline form C of compound of formula (III)

Crystalline form A of the compound of formula (I) (1.2 g, 2.90 mmol) was weighed and mixed with ethanol (18 mL), then concentrated HCl (12 M, 603.20 μL, 2.5 equiv) was added thereto and the solid was observed to dissolve. The resulting product was stirred in an oil bath at 50 °C overnight, filtered and dried to obtain crystalline form C of the compound of formula (III), which according to IC-CL measurement contains two hydrochlorides (Cl ^- % was 18.03%).

Example 5: Study of the hygroscopicity of crystalline form B of the compound of formula (II)

Experimental materials:

SMS DVS Internal Dynamic Vapor Adsorption Tester

Method of conducting the experiment:

A sample (10 mg) was collected and placed in a particle board sample plate for testing.

Experiment results:

The DSP curve of crystalline form B of the compound of formula (II) is shown in Figure 8, ΔW=0.28%.

Conclusion based on experimental data:

The increase in hygroscopic mass of crystalline form B of the compound of formula (II) was 0.28% at 80% relative humidity/25°C, indicating that the sample had low hygroscopicity.

Example 6: Solid state stability test of crystalline form B of the compound of formula (II)

About 900 mg of the crystalline form B of the compound of formula (II) was weighed accordingly, placed on the bottom of a glass sample bottle, spread into a thin layer so that the samples were placed to ensure complete exposure. The samples placed at high temperature and humidity were collected for XRD analysis at days 10 and 30; the samples exposed to light were placed in a dish with a clean surface, spread into a thin layer, covered with a quartz glass lid, and collected for XRD analysis at days 5 and 10; for the long-term test and the accelerated test (shadowing), each sample was placed in a double-layer low-density polyethylene (LDPE) bag, each layer of the LDPE bag was folded accordingly and sealed, and then the LDPE bag was placed in an aluminum foil bag together with a desiccant bag and sealed. The samples were placed at 25°C/60%RH, 30°C/65%RH and 40°C/75%RH respectively for testing, the test results were compared with the initial test results on day 0, and the test results are shown below in Table 7.

Conclusion: Crystalline form B of the compound of formula (II) had good stability under the influence of influencing factors, light, under conditions of accelerated and prolonged experiment.

Biological research data:

A luminescent cell viability assay (CellTiter-Glo® method) from Promega was used to detect cell proliferation. The CellTiter-Glo® Luminescent Cell Viability Assay (CellTiter-Glo® Luminescent Cell Viability Kit) was a homogeneous, rapid detection method for determining the viability of living cells in culture by quantifying ATP. ATP was used as an indicator of the metabolism of living cells. Cell lysis and the resulting light signal were proportional to the amount of ATP present, with the amount of ATP being directly proportional to the amount of living cells in the culture. The higher the measured fluorescence value, the higher the ATP content in the cell and thus the higher the cell viability. Thus, the degree of inhibition of cell proliferation by the compound can be monitored by the fluorescence value.

1.2 Stages of the experiment

1.2.1 Preparation of the working solution concentration of the compound

1) The compound was diluted to 2 mM with 100% DMSO, that is, 5 μL of 10 mM compound stock solution was taken and placed in the first column of a 96-well compound plate, then 20 μL of 100% DMSO was added and mixed well;

2) 20 µl of 100% DMSO was added to columns two through ten of a 96-well plate for coupling;

3) 5 μl of the compound was taken from the first column to the second column and mixed well, and then 5 μl of the compound was taken from the second column to the third column and mixed well. To perform a gradient dilution of the compound, the above steps were repeated up to the ninth column;

4) Preparation of working solution of compound: 2 μl of compound per well from the second column of the compound plate was transferred to a new 96-well compound plate, and 78 μl of cell culture medium was added per well; 2 μl of 100% DMSO was taken from the negative control well, and 78 μl of cell culture medium was added; and

5) The compound concentration was the final concentration, with the blank control well being the well with culture medium without cells, and the negative control being 0.5% DMSO.

1.2.2 Cell inoculation and drug treatment

1) The BCR-FMS-BaF3 cell suspension was collected and centrifuged at 1000 rpm (rotations per minute) for 5 minutes. The supernatant was removed, and the cells were resuspended with pre-warmed medium and counted. After counting, the cell suspension was diluted with cell culture medium to the desired density. The density of each cell type is shown in Table 4-1. Each well was inoculated with 80 μl of the cell suspension in a 96-well cell culture plate.

2) On the day of the experiment, 20 μl of the working solution of the compound was added to the cell plate and incubated for 3 days at 37°C in an incubator with 5% CO ₂ . The incubation time of different cells varied, and the specific cell density and the number of incubation days are shown in Table 8.

3) After completion of incubation, 20 μl of CTG detection reagent (CellTiter-Glo) was added to each well of the cell plate and incubated at 25°C for 10 minutes. After completion of incubation, En Vision was used to detect the luminescence signal.

1.3 Data Analysis

The cell survival rate after compound treatment was calculated by the following formula: % inhibition rate = (RFU _compound -RFU _{negative control} )/(RFU _{blank well} -RFU _{negative control} )×100% (RFU is relative fluorescence unit). Blank well value: the average of the blank control well value; Negative control: the cells were treated with 0.5% DMSO and Prism was used to plot and calculate the _IC50 value of the test compound.

1.4 Experimental results: see table 9.

Conclusion based on experimental data: Crystalline form B of the compound of formula (II) can significantly inhibit the proliferation of BCR-FMS-BaF3 cells.

Claims (11)

1. Crystalline form B of the compound of formula (II), characterized in that its powder X-ray diffraction pattern contains characteristic diffraction peaks at diffraction angles 2θ values of 4.579±0.200°, 6.898±0.200°, 13.019±0.200°, 15.918±0.200°, 19.697±0.200° and 25.200±0.200°, . 2. Crystalline form B according to claim 1, characterized in that its powder X-ray diffraction pattern contains characteristic diffraction peaks at diffraction angles 2θ values of 4.579±0.200°, 6.898±0.200°, 7.941±0.200°, 11.218±0.200°, 13.019±0.200°, 15.918±0.200°, 19.697±0.200 and 25.200±0.200°. 3. Crystalline form B according to claim 2, characterized in that its powder X-ray diffraction pattern contains characteristic diffraction peaks at diffraction angles 2θ values of 4.579±0.200°, 6.898±0.200°, 7.941±0.200°, 11.218±0.200°, 13.019±0.200°, 14.701±0.200°, 15.918±0.200°, 17.338±0.200°, 19.697±0.200°, 22.540±0.200°, 25.200±0.200° and 27.798±0.200°. 4. Crystalline form B according to claim 3, characterized in that its powder X-ray diffraction pattern contains characteristic diffraction peaks at diffraction angles 2θ values of 4.579°, 6.898°, 7.941°, 9.070°, 9.423°, 11.218°, 13.019°, 13.796°, 14.701°, 15.182°, 15.918°, 17.338°, 18.939°, 19.461°, 19.697°, 20.700°, 21.602°, 22.200°, 22.540°, 22.818°, 23.919°, 24.461°, 25.200°, 25.639°, 26.563°, 27.120°, 27.798°, 28.156°, 28.595°, 29.000°, 29.339°, 30.739°, 32.064°, 32.355°, 33.201°, 34.156°, 35.020°, 36.063° and 39.176°. 5. Crystalline form B according to claim 4, characterized in that said crystalline form B has a powder X-ray diffraction pattern shown in Figure 3. 6. Crystalline form B according to any one of claims 1-5, characterized in that said crystalline form B has a differential scanning calorimetry curve with an endothermic peak starting at 240.16±3.0 °C and an exothermic peak with a peak apex at 282.04±3.0 °C. 7. Crystalline form B according to claim 4, characterized in that said crystalline form B has a differential scanning calorimetry curve shown in Figure 4. 8. Crystalline form B according to any one of claims 1-5, characterized in that the loss in mass of said crystalline form B is 0.3643% at 200.0±3 °C according to its thermogravimetric analysis curve. 9. Crystalline form B according to claim 8, characterized in that said crystalline form B has a thermogravimetric analysis curve shown in Figure 5. 10. Use of crystalline form B according to any one of claims 1-9 for the preparation of a medicinal product associated with the treatment of tenosynovial giant cell tumor.