Crystalline form and applications of FGFR4 inhibitors

Abstract

The present invention relates to the crystalline form and application of FGFR4 inhibitor. The present invention provides a crystalline form of compound of formula B, which has excellent FGFR4 kinase inhibitory activity and Huh-7 tumor cell proliferation inhibition, and has better pharmacokinetic effect than its optical isomer or racemate, and its crystalline form, especially its preferred crystalline form, has good solubility, stable quality, thermodynamic stability, and is easy to develop drugs.

Description

Detailed description of the invention

[0001] [Technical Field] This invention belongs to the field of pharmacochemistry and, more specifically, relates to the crystalline form of FGFR4 selective inhibitors and their applications.

[0002] [Background technology] FGFR (fibroblast growth factor receptor) is a receptor protein tyrosine kinase with four receptors: FGFR1, FGFR2, FGFR3, and FGFR4. It plays a crucial role in maintaining cell growth, proliferation, apoptosis, and migration. FGFR-activating mutations can not only promote the proliferation of malignant tumor cells and inhibit apoptosis, but can also play an important role in processes such as tumor neoangiogenesis, tumor invasion, and metastasis. Since high FGFR expression is present in non-small cell lung cancer, liver cancer, breast cancer, bladder cancer, and several other types of cancer, FGFR small molecule kinase inhibitors are a promising treatment method for tumor patients expressing FGFR abnormalities, and research and development of selective FGFR small molecule inhibitors is attracting increasing attention.

[0003] Liver cancer is one of the most common malignancies in terms of incidence and mortality, with 466,000 new cases and 422,000 deaths from liver cancer annually in China. Studies have shown that the FGFR4-FGF19 signaling pathway is closely correlated with hepatocellular carcinoma (HCCs), that FGFR4 is a highly expressed FGFR isoform in human liver cells, and that multiple FGFR4 mutations are found in liver cancer patients. Selectively inhibiting FGFR4 without inhibiting other isoforms such as FGFR1, FGFR2, and FGFR3 could avoid certain toxicity and is likely to be an important target for the treatment of liver cancer. Clinical studies have already shown that FGFR inhibitors are useful in treating multiple types of cancer, but there is an urgent need for the development of selective FGFR4 inhibitors for the treatment of multiple tumors, particularly liver cancer.

[0004] Chinese patent CN108948004A discloses an FGFR4 inhibitor represented by the following formula (I).

[0005] [ka]

[0006] Of these, Example 5 discloses a compound having the following structure.

[0007] [ka]

[0008] [Summary of the Invention] Compound A has two isomers: an S-isomer (shown in formula B below) and an R-isomer (shown in formula C below). The inventors have found that compound B has even better pharmacokinetic properties and is more advantageous for drug discovery compared to compound A or compound C. Based on this, the inventors have systematically studied the crystal form of compound B and found a stable crystal form useful for subsequent development.

[0009] [ka]

[0010] To solve the above problems, in a first embodiment, the present invention provides a crystalline compound of formula B.

[0011] [ka]

[0012] In some embodiments, the above crystalline form of compound B is an anhydrous.

[0013] In a second embodiment, the present invention provides a crystalline form I of compound B of formula B, wherein the X-ray powder diffraction of crystalline form I of compound B of formula B, expressed at a 2θ angle using Cu-Kα radiation, has characteristic peaks at 7.4±0.2° and 25.0±0.2°.

[0014] In some embodiments, the crystalline form I of the above compound B has characteristic peaks at 7.4±0.2°, 15.9±0.2°, 17.4±0.2°, 20.9±0.2°, 25.0±0.2° in the X-ray powder diffraction represented by the 2θ angle using Cu-Kα radiation.

[0015] In some embodiments, the crystalline form I of the above compound B has characteristic peaks at 7.4±0.2°, 9.8±0.2°, 15.9±0.2°, 17.4±0.2°, 20.9±0.2°, 25.0±0.2° in the X-ray powder diffraction represented by the 2θ angle using Cu-Kα radiation.

[0016] In some embodiments, the crystalline form I of the above compound B has characteristic peaks at 7.4±0.2°, 9.8±0.2°, 15.9±0.2°, 17.4±0.2°, 20.9±0.2°, 22.4±0.2°, 25.0±0.2° in the X-ray powder diffraction represented by the 2θ angle using Cu-Kα radiation.

[0017] In some embodiments, the crystalline form I of the above compound B has characteristic peaks at 7.4±0.2°, 9.8±0.2°, 11.7±0.2°, 12.0±0.2°, 14.6±0.2°, 15.9±0.2°, 17.4±0.2°, 20.9±0.2°, 22.4±0.2°, 25.0±0.2° in the X-ray powder diffraction represented by the 2θ angle using Cu-Kα radiation.

[0018] In some embodiments, the crystalline form I of the above compound B has characteristic peaks at 7.4±0.2°, 9.8±0.2°, 11.7±0.2°, 12.0±0.2°, 14.6±0.2°, 15.9±0.2°, 17.4±0.2°, 19.7±0.2°, 20.9±0.2°, 22.4±0.2°, 23.6±0.2°, 25.0±0.2° in the X-ray powder diffraction represented by the 2θ angle using Cu-Kα radiation.

[0019] In some embodiments, the crystalline form I of the above formula B compound exhibits characteristic peaks at 7.4±0.2°, 9.8±0.2°, 11.7±0.2°, 12.0±0.2°, 14.6±0.2°, 15.5±0.2°, 15.9±0.2°, 17.4±0.2°, 18.8±0.2°, 19.7±0.2°, 20.9±0.2°, 22.0±0.2°, 22.4±0.2°, 23.6±0.2°, 25.0±0.2°, and 27.8±0.2° when measured using Cu-Kα radiation and expressed at a 2θ angle.

[0020] In some embodiments, the crystalline form I of the above formula B compound exhibits characteristic peaks at 7.4±0.2°, 9.8±0.2°, 11.7±0.2°, 12.0±0.2°, 14.6±0.2°, 15.5±0.2°, 15.9±0.2°, 17.4±0.2°, 18.5±0.2°, 18.8±0.2°, 19.7±0.2°, 20.9±0.2°, 22.0±0.2°, 22.4±0.2°, 23.6±0.2°, 25.0±0.2°, and 27.8±0.2° when X-ray powder diffraction is performed using Cu-Kα radiation and expressed at a 2θ angle.

[0021] In some embodiments, the crystalline form I of the above formula B compound exhibits characteristic peaks at 7.4±0.2°, 9.8±0.2°, 11.7±0.2°, 12.0±0.2°, 14.6±0.2°, 15.5±0.2°, 15.9±0.2°, 17.4±0.2°, 18.8±0.2°, 19.7±0.2°, 20.9±0.2°, 22.0±0.2°, 22.4±0.2°, 23.6±0.2°, 25.0±0.2°, 26.0±0.2°, and 27.8±0.2° when X-ray powder diffraction is performed using Cu-Kα radiation and expressed at a 2θ angle.

[0022] In some embodiments, the crystalline form I of the above formula B compound exhibits characteristic peaks at 7.4°, 9.8°, 11.7°, 12.0°, 14.6°, 15.5°, 15.9°, 17.4°, 18.5°, 18.8°, 19.6°, 20.9°, 22.0°, 22.4°, 23.6°, 24.8°, and 27.7° when measured using Cu-Kα radiation and expressed at a 2θ angle in X-ray powder diffraction.

[0023] In some embodiments, the crystalline form I of the above formula B compound has characteristic peaks at 7.5°, 9.9°, 11.8°, 12.1°, 14.7°, 15.6°, 16.1°, 17.6°, 19.0°, 19.9°, 21.1°, 22.2°, 22.6°, 23.8°, 25.2°, 26.0°, and 28.0° when measured using Cu-Kα radiation and expressed at 2θ angles.

[0024] In some embodiments, the crystalline form I of compound B of the above formula has, using Cu-Kα radiation, basically the X-ray powder diffraction (XRPD) pattern shown in Figure 1 or Figure 3.

[0025] In some embodiments, the crystalline form I of compound B described above has a single crystal that uses CuKα radiation, is monoclinic, belongs to the P21 space group, and has unit cell parameters {a=10.8374(2)Å, b=23.6358(3)Å, c=10.96160(10)Å, α=90°, β=117.360(2)°, γ=90°, V=2493.73(7)Å}. 3}

[0026] In a third embodiment, the present invention provides a crystalline form II of a compound of formula B, of which X-ray powder diffraction using Cu-Kα radiation and expressed at a 2θ angle has characteristic peaks at 7.6±0.2° and 14.0±0.2°.

[0027] In some embodiments, the crystalline form II of the above compound B has characteristic peaks at 7.6±0.2°, 14.0±0.2°, 15.2±0.2°, 20.4±0.2°, and 24.8±0.2° when X-ray powder diffraction is performed using Cu-Kα radiation and expressed at a 2θ angle.

[0028] In some embodiments, the crystalline form II of the above formula B compound exhibits characteristic peaks at 7.6±0.2°, 9.5±0.2°, 14.0±0.2°, 15.2±0.2°, 16.3±0.2°, 20.4±0.2°, 21.6±0.2°, 21.9±0.2°, 23.2±0.2°, 23.5±0.2°, and 24.8±0.2° when measured using Cu-Kα radiation and expressed at a 2θ angle.

[0029] In some embodiments, the crystalline form II of the above formula B compound exhibits characteristic peaks at 7.6±0.2°, 9.5±0.2°, 10.2±0.2°, 12.7±0.2°, 14.0±0.2°, 15.2±0.2°, 16.3±0.2°, 16.8±0.2°, 18.3±0.2°, 20.4±0.2°, 21.6±0.2°, 21.9±0.2°, 23.2±0.2°, 23.5±0.2°, and 24.8±0.2° when measured using Cu-Kα radiation and expressed at a 2θ angle.

[0030] In some embodiments, the crystalline form II of the above formula B compound exhibits characteristic peaks at 7.6±0.2°, 9.5±0.2°, 10.2±0.2°, 12.7±0.2°, 14.0±0.2°, 15.2±0.2°, 16.3±0.2°, 16.8±0.2°, 17.9±0.2°, 18.3±0.2°, 20.4±0.2°, 21.6±0.2°, 21.9±0.2°, 23.2±0.2°, 23.5±0.2°, 24.8±0.2°, 26.8±0.2°, 27.7±0.2°, and 28.2±0.2° when measured using Cu-Kα radiation and expressed at a 2θ angle.

[0031] In some embodiments, the crystalline form II of the above formula B compound has, using Cu-Kα radiation, basically the X-ray powder diffraction (XRPD) pattern shown in Figure 4.

[0032] In a fourth embodiment, the present invention provides a crystalline form III of compound B of formula, of which X-ray powder diffraction using Cu-Kα radiation and expressed at a 2θ angle has a characteristic peak at 4.4 ± 0.2°.

[0033] In some embodiments, the crystalline form III of the above compound B has characteristic peaks at 4.4±0.2°, 10.4±0.2°, 13.4±0.2°, 25.3±0.2°, and 25.8±0.2° when measured using Cu-Kα radiation and expressed at a 2θ angle in X-ray powder diffraction.

[0034] In some embodiments, the crystalline form III of the above formula B compound exhibits characteristic peaks at 4.4±0.2°, 10.4±0.2°, 13.4±0.2°, 14.0±0.2°, 18.1±0.2°, 18.5±0.2°, 20.5±0.2°, 24.3±0.2°, 25.3±0.2°, and 25.8±0.2° when X-ray powder diffraction is performed using Cu-Kα radiation and expressed at a 2θ angle.

[0035] In some embodiments, the crystalline form III of the above formula B compound basically has the X-ray powder diffraction (XRPD) pattern shown in Figure 5 when using Cu-Kα radiation.

[0036] In a fifth embodiment, the present invention further provides a pharmaceutical composition comprising the above-mentioned crystalline form of compound B, or a mixture of one or more crystalline forms I, II, and III of the above-mentioned compound B.

[0037] In some embodiments, the pharmaceutical composition comprises a crystalline form of compound B, or a mixture of one or more crystalline forms I, II, and III of compound B, and one or more pharmaceutically acceptable carriers.

[0038] In a sixth embodiment, the present invention further provides applications of the above-mentioned crystalline form of compound B, or crystalline forms I, II, III of compound B, or the above-mentioned pharmaceutical composition, in the manufacture of a drug for treating a disease related to FGFR4 activity or expression.

[0039] In some embodiments, the present invention further provides the above-mentioned crystalline form of compound B, or crystalline forms I, II, III of compound B, or the above-mentioned pharmaceutical composition for treating diseases related to FGFR4 activity or expression.

[0040] In some embodiments, the present invention further provides a method for treating a disease related to FGFR4 activity or expression, comprising administering an effective amount of the above-mentioned crystalline form of compound B, or crystalline forms I, II, III, or the above-mentioned pharmaceutical composition to a subject in need.

[0041] In some embodiments, the disease associated with FGFR4 activity or expression is selected from cancers such as lung cancer, bladder cancer, breast cancer, gastric cancer, liver cancer, salivary gland sarcoma, ovarian cancer, prostate cancer, cervical cancer, epithelial cell carcinoma, multiple myeloma, pancreatic cancer, lymphoma, chronic myeloid leukemia, lymphocytic leukemia, and cutaneous T-cell lymphoma, and the lung cancer is preferably non-small cell lung cancer.

[0042] In some embodiments, the diseases associated with FGFR4 activity or expression are selected from skeletal disorders such as dysplasia, chondrodysplasia, dwarfism, and Cruzon syndrome.

[0043] In some embodiments, the diseases associated with FGFR4 activity or expression are selected from T-cell regulated inflammation and autoimmune diseases such as rheumatoid arthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, psoriasis, juvenile diabetes mellitus, Sjögren's syndrome, thyroid disease, sarcoidosis, inflammatory bowel disease, and celiac disease.

[0044] The above-mentioned drugs are administered to "subjects" or "patients" in an effective dose. The above-mentioned "subjects" and "patients" include, but are not limited to, all members of the animal kingdom, including mammals (e.g., mice, rats, cats, monkeys, dogs, horses, pigs, etc.) and humans.

[0045] Definition and Description Unless otherwise specified, the following terms and phrases used herein are intended to have the meanings set forth below. Unless otherwise defined, certain words or phrases should not be considered uncertain or ambiguous, but should be understood in their ordinary sense.

[0046] The crystalline forms of compound B of formula as referred to in this application include the anhydrous and solvent-free form, the hydrate form, the solvate form, and the cocrystalline form of compound B of formula B.

[0047] In X-ray powder diffraction patterns, the terms “basically” or “basically shown in the figure” refer to a crystal form that is basically pure and appears in the pattern providing 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 96%, or at least 97%, or at least 98%, or at least 99% of the peaks in that X-ray powder diffraction pattern. Furthermore, as the content of a certain crystal form in the product gradually decreases, some of the diffraction peaks attributed to that crystal form in the X-ray powder diffraction pattern may decrease due to factors related to the detection sensitivity of the instrument. Also, for any given crystal form, there may be slight errors in the position of the peaks, which is well known in the field of crystallography. For example, changes in temperature during sample analysis, sample movement, or instrument calibration can cause the position of the peaks to shift, and the measurement error of the 2θ value is sometimes about ±0.3°, and usually about ±0.2°. Therefore, this error should be taken into account when determining the structure of each crystal form, and the terms “basically” or “basically as shown in the figure” are intended to include such differences in diffraction peak positions, which are ±0.3°, preferably ±0.2°.

[0048] The term "tumor" includes benign tumors, malignant tumors, and borderline tumors, of which malignant tumors are also collectively referred to as cancer.

[0049] As used herein, the term “prevention” means, when used for a disease or condition (e.g., cancer), that the compound or drug (e.g., the combination product claimed herein) can reduce the frequency of symptoms of the medical condition in vivo in a subject or delay their onset compared to a subject who has not been administered the compound or drug.

[0050] As used herein, the term “treatment” means the reduction, alleviation, or improvement of the symptoms of a disease or condition; the improvement of symptoms caused by underlying metabolism; the inhibition of a disease or condition, for example, the cessation of the progression of a disease or condition; the alleviation of a disease or condition; the induction of regression of a disease or condition; the alleviation of conditions caused by a disease or condition; or the inhibition of symptoms of a disease or condition.

[0051] The terms "medicinal carrier," "pharmaceutically acceptable carrier," or "pharmaceutically acceptable adjuvant" refer to carriers or adjuvants that do not have apparent irritant effects on organisms and do not impair the biological activity and performance of active compounds.

[0052] All solvents used in this invention are commercially available and can be used without further purification.

[0053] Beneficial effects This invention provides a crystalline form of compound B of formula. The inventors have unexpectedly discovered that compound B of formula has even better pharmacokinetic effects in its optical isomers or racemates, its crystalline form, particularly its preferred crystalline form, is stable, thermodynamically stable, and facilitates drug discovery.

[0054] [Brief explanation of the drawing] [Figure 1] This is the XRPD pattern of crystalline form I of compound B. [Figure 2] This is a single crystal PLM diagram of crystalline form I of compound B. [Figure 3] This is the XRPD pattern calculated from a single crystal of the compound of formula B in crystal form I. [Figure 4] This is the XRPD pattern of crystalline form II of compound B. [Figure 5] This is the XRPD pattern of crystalline form III of compound B.

[0055] [Modes for carrying out the invention] The technical aspects of the present invention will be described in more detail below in accordance with specific embodiments. The following embodiments are merely illustrative and should not be interpreted as limiting the scope of the claims of the present invention. Any technology realized based on the above-described aspects of the present invention is included within the scope of the claims of the present invention.

[0056] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available or can be manufactured by known methods.

[0057] In the following examples, the pattern detection conditions are as follows:

[0058] 1. XRPD detection conditions Equipment: German BRUKER D8 Advance X-ray Powder Diffractometer (BRUKER GER) Conditions: Cu-Kα radiation, tube pressure 40 kV, tube current 40 mA, 2θ scanning range 3-40°, scanning step length 0.02°, scanning speed 0.2 sec / step, sample pan was a zero-background sample pan.

[0059] 2. Dynamic water adsorption and desorption analysis (DVS) Equipment DVS Intrinsic plus (SMS, UK).

[0060] Method: Samples were placed in a tare-filled sample basket, weighed automatically, dried at 40°C / 0% RH until the dm / dt ratio was less than 0.002%, and then cooled to 25°C. Instrument parameters are shown in Table 1.

[0061] [Table 1]

[0062] 3. Single-crystal X-ray diffraction analysis Equipment: Rigaku XtaLAB Synergy-DW diffractometer Method Single-crystal X-ray data was collected at 180 K, using Cu Kα radiation (λ=1.54184 Å) as the light source. Data reduction and empirical absorption correction were performed using the CrysAlisPro program, and the structure was analyzed using the SHELXT program employing a dual-space algorithm. Based on the difference Fouriegram, the coordinates of all non-hydrogen atoms were determined, and the hydrogen atoms were theoretically hydrogenated by being fixed relative to their parent atoms. Using the SHELXL program, F 2 The final structure was refined using the full matrix least squares method.

[0063] 4. Polarized light microscopy (PLM) Equipment: ECLIPSE LV100POL polarizing microscope (Nikon, Japan) Method A small sample was taken and placed on a glass slide. Oil was added and the sample was immersed in it, then the glass slide was covered to allow for some even dispersion. Microscopic observation and photography were performed using a 4-20× objective lens and polarizing light.

[0064] Manufacturing Example 1: Preparation of Compound B

[0065] [ka]

[0066] Compound B-1 (6 g, 34.5 mmol) and N,N-carbonyldi(1,2,4-triazole) (17 g, 103.4 mmol) were dissolved in dry N,N-dimethylformamide (60 mL), and the mixture was reacted with stirring at room temperature for 2 hours. Then, compound B-2 (8.7 g, 24.1 mmol, obtained by referring to Example 10 of WO2017202390A1 and adopting configuration 10a) was added, and the reaction system was stirred overnight at room temperature. After monitoring the completion of the reaction by HPLC, the reaction mixture was quenched with 10% lithium chloride solution, extracted three times with ethyl acetate, and the organic layers were washed together with saturated brine. After drying over anhydrous sodium sulfate and concentrating, the crude product obtained was subjected to column chromatography (dichloromethane / methanol = 25 / 1 to 20 / 1) to obtain white solid compound B-3 (1.5 g, yield 11%).

[0067] Compound B-3 (1.5 g, 2.7 mmol) was dissolved in tetrahydrofuran (30 mL), and then HCl (2 M, aqueous solution) (20 mL) was added dropwise to the solution at room temperature. The reaction mixture was allowed to react at room temperature for 3 hours with stirring. The reaction mixture was poured into an appropriate amount of saturated sodium bicarbonate solution, extracted three times with dichloromethane, and the organic layers were combined and washed with saturated brine. After drying over anhydrous sodium sulfate and concentrating, the resulting crude product was separated and purified by column chromatography (dichloromethane / methanol = 20 / 1). The obtained compound B was a white-like solid (0.44 g, yield 32%). 1HNMR(500 MHz, CDCl3) δ 13.56 (s, 1H), 10.17 (s, 1H), 8.11 (s, 1H), 7.85 (s, 1H), 7.57 (s, 1H), 5.21 (s, 1H), 5.00 (dd, J = 46.1, 15.7 Hz, 2H), 4.07 - 3.96 (m, 2H), 3.45 (ddd, J = 12.3, 9.6, 4.8 Hz, 1H), 3.23 (ddd, J = 16.8, 10.7, 6.3 Hz, 2H), 2.98 (dt, J = 12.2, 4.3 Hz, 1H), 2.91 (ddd, J = 9.5, 7.6, 4.8 Hz, 1H), 2.85 (t, J = 6.3 Hz, 2H), 2.79 - 2.69 (m, 1H), 2.61 - 2.51 (m, 2H), 2.23 - 2.14 (m, 1H), 2.00 - 1.94 (m, 2H), 1.94 - 1.69 (m, 3H), 0.93 - 0.85 (m, 2H), 0.63 - 0.55 (m, 2H). MS 515.20 [M+H] + .

[0068] XRPD testing revealed that the solid was in an amorphous state.

[0069] Manufacturing Example 2: Manufacturing of Compound C

[0070] [ka]

[0071] Except for replacing B-2 with the R configuration, the manufacturing method of Production Example 1 was followed to obtain compound C of formula. 1HNMR(500 MHz, CDCl3) δ 13.56 (s, 1H), 10.17 (s, 1H), 8.11 (s, 1H), 7.85 (s, 1H), 7.57 (s, 1H), 5.21 (s, 1H), 5.00 (dd, J = 47.8, 15.7 Hz, 2H), 4.09 - 3.96 (m, 2H), 3.50 - 3.39 (m, 1H), 3.30 - 3.14 (m, 2H), 3.02 - 2.95 (m, 1H), 2.95 - 2.88 (m, 1H), 2.85 (t, J = 6.2 Hz, 2H), 2.79 - 2.71 (m, 1H), MS 515.16 [M+H] + .

[0072] Example 1: Preparation of crystalline form I of compound B The compound B (20 mg) obtained in Preparation Example 1 was added to a reaction flask, 3 mL of dimethyl sulfoxide was added, and the mixture was stirred at 60°C for 1 hour. The solution was filtered while still hot to obtain a saturated solution. The filtered filtrate was stirred overnight at room temperature, the resulting solid was filtered, and the mixture was vacuum-dried at 50°C for 12 hours to obtain crystalline form I of the compound B.

[0073] The obtained crystal form I exhibited good crystallinity, and its XRPD characterization pattern is basically shown in Figure 1, with the characterization data shown in Table 2. The sample hardly absorbed any moisture, and the weight increase after moisture absorption was approximately 0.24% under conditions of 0-90% RH, and the crystal form of the sample did not change after the DVS test.

[0074] [Table 2]

[0075] Example 2: Preparation of crystalline form I of compound B Approximately 5 mg of the compound of formula B obtained in Production Example 1 was dissolved in 1 mL of acetonitrile / dichloromethane (V:V = 1:1) at 50°C. After filtration, a very small amount of hypromellose (HPMC) was added to the filtrate as a template, and the sample bottle was covered with a perforated seal film and placed in a fume hood to gradually volatilize at room temperature. After 1 day, plate-like single crystals were obtained and are shown in Figure 2. The single crystal sample was used for single crystal X-ray diffraction analysis. The single crystal structure belongs to the monoclinic space group P21, and the molecular formula is C 27 H 30 N8O3. There were 2 molecules of the compound of formula B in each asymmetric unit, and there were 2 asymmetric units in each crystal cell. The unit cell parameters were {a = 10.8374(2) Å, b = 23.6358(3) Å, c = 10.96160(10) Å, α = 90°, β = 117.360(2)°, γ = 90°, V = 2493.73(7) Å 3}.

[0076] The XRPD pattern calculated from the single crystal structure is shown in Figure 3, and the results are shown in Table 3, which basically coincides with the diffraction peak positions of the XRPD pattern of Crystal Form I obtained in Example 1.

[0077] [Table 3]

[0078] Examples 3 to 21 Production of Crystal Form I of the Compound of Formula B (1) Cooling Crystallization Method Referring to the production method of Example 1, the following solvent systems in the table were adopted to obtain Crystal Form I of the compound of formula B.

[0079] [Table 4]

[0080] (2) Suspension Percussion Method After adding 20 mg of compound B obtained in Preparation Example 1 to a reaction flask, suspensions were prepared by adding different solvents. All suspensions were stirred for 3 days at room temperature or 50°C, and the resulting solids were filtered and then XRPD characterized, which revealed crystalline form I.

[0081] [Table 5]

[0082] Example 22: Preparation of crystalline form II of compound B The compound B (20 mg) obtained in Preparation Example 1 was added to a reaction flask, dissolved with dichloromethane (4 mL), and acetonitrile (8 mL) was gradually added at room temperature, resulting in the precipitation of a large amount of solid. The obtained solid was characterized by XRPD and was found to be crystal form II. Its XRPD characterization pattern is basically shown in Figure 4, and the characterization data is shown in Table 6.

[0083] [Table 6]

[0084] Examples 23-32: Preparation of crystalline form II of compound B Referring to the manufacturing method in Example 22, 20 mg of compound B obtained in Manufacturing Example 1 was dissolved in different solvents to prepare 4-10 mg / mL solutions. A poor solvent was then gradually added at room temperature or 60°C until a large amount of solid precipitated. The resulting solid was characterized by XRPD and was found to be of crystal form II.

[0085] [Table 7]

[0086] Example 33: Preparation of crystalline form III of compound B The compound B (20 mg) obtained in Preparation Example 1 was added to a reaction flask, dichloromethane / methanol (V:V = 2:1, 3 mL) was added, and the mixture was stirred at room temperature for 30 minutes. After filtration, the filtrate was further allowed to gradually evaporate the solvent at room temperature to obtain crystalline form III. Its XRPD characterization pattern is basically shown in Figure 5, and the characterization data is shown in Table 8.

[0087] [Table 8]

[0088] Comparative Example: Preparation of other crystalline forms of compound B (1) Cooling crystallization method Referring to the manufacturing method of Example 1, the solvent systems shown in the table below were used to produce the crystalline form of compound B, and the results were as follows.

[0089] [Table 9]

[0090] (2) Slow evaporation crystals Referring to the manufacturing method in Example 33, the solvent systems shown in the table below were used to produce the crystalline form of compound B, and the results were as follows.

[0091] [Table 10]

[0092] Test Example 1: Competitive Suspension Experiment Equal amounts (6 mg each) of the crystalline form I of compound B obtained in Example 1, the crystalline form II of compound B obtained in Example 22, and the crystalline form III of compound B obtained in Example 33 were mixed and stirred in the following solvents at room temperature (25°C), high temperature (50°C) for 24 hours, 2 days, and 7 days, respectively. The remaining solids were then subjected to XRPD testing, and the results are shown in the table.

[0093] [Table 11]

[0094] Test Example 2: Solubility Test The crystalline form I sample (5 mg) obtained in Example 1 and the crystalline form II sample (5 mg) obtained in Example 22 were added to a sample bottle, and then 1 mL of SGF (simulated gastric juice), FaSSIF (simulated intestinal fluid under fasting conditions), FeSSIF (simulated intestinal fluid under feeding conditions), and water were added. The resulting suspension was shaken at 200 rpm for 24 hours at 37°C. After filtration, the resulting filtrate was subjected to concentration testing, and the obtained solid was characterized by XRPD. The results are shown in Table 13.

[0095] [Table 12]

[0096] [Table 13]

[0097] Test Example 3: Solid Stability Study 5 mg each of the crystalline form I samples obtained in Example 1 and the crystalline form II samples obtained in Example 22 were weighed and placed in sample bottles. Solid stability studies were conducted under conditions of 60°C (closed) and 40°C / 75%RH (open), with samples taken over 7 days and characterized by XRPD. The results are shown in Table 14.

[0098] [Table 14]

[0099] Test Example 4: Polishing Stability Study Samples of crystal form I (3-5 mg) obtained in Example 1 were polished in a solvent or the solid was polished directly for 5 minutes. The remaining solid was collected and XRPD characterized, and the results are shown in Table 15.

[0100] [Table 15]

[0101] Test Example 5: In vitro drug efficacy experiment Compound B obtained in Test Drug Production Example 1 1. Experiment to inhibit FGFR4 kinase activity FGFR4 protein kinase activity was measured using a caliper mobility shift assay. The compound was dissolved in DMSO, diluted in kinase buffer, and 5 μL of the compound at its 5x final reaction concentration (10% DMSO) was added to a 384-well plate. 10 μL of 2.5x enzyme (FGFR4) solution was added, followed by incubation at room temperature for 10 minutes. Then, 10 μL of 2.5x primer (FAM-labeled peptide and ATP) solution was added. After incubation at 28°C for 30–60 minutes, 25 μL of stop solution was added to halt the reaction. Conversion rate data was read using a Caliper EZ Reader II (Caliper Life Sciences). The conversion rate was converted to inhibition rate data (% inhibition rate = (max - sample conversion rate) / (max - min) × 100). Of these, max referred to the conversion rate of the DMSO control, and min referred to the conversion rate of the control without enzyme activity. The curve was plotted with compound concentration and inhibition rate as the x and y coordinates, and the curve was fitted using the XLFit Excel Add-in version 4.3.1 software to obtain IC. 50 I calculated it.

[0102] The results showed inhibition of FGFR4 kinase activity (IC) of the compound of formula B of the present invention. 50 We showed that (nM) < 5 nM.

[0103] 2. Inhibition testing of compounds against Huh-7 tumor cell proliferation Huh7 cell suspensions were prepared in DMEM + 2 mM Glutamine + 10% FBS medium to a concentration of 5 × 10⁴ e⁴ / mL or 2 × 10⁴ e⁴ / mL. 100 μL of the cell suspension was added to each well of a 96-well cell culture plate, with final cell concentrations of 5000 cells / well (72 hours) or 2000 cells / well (168 hours). Compounds awaiting measurement were dissolved in DMSO as a stock solution of 10 mmol. Compounds at 200X final concentrations were prepared using the stock solution and DMSO, and dilutions in a 3X series gradient were also prepared, each then diluted 20-fold in culture medium. Finally, 10 μL of the corresponding 10-fold solution was added to each well of each cell line, and each drug concentration was added to one well. The final treatment concentrations of each compound were 3000 nM, 1000 nM, 333.3 nM, 111.1 nM, 37.04 nM, 12.35 nM, 4.12 nM, and 1.37 nM, respectively, and the final DMSO concentration in each well was 0.5%. Cells were incubated at 37°C in a 5% CO2 incubator for 72 or 168 hours. 72 or 168 hours after drug treatment, 100 μL of pre-thawed and equilibrated CTG solution at room temperature was added to each well according to the CTG instruction manual, mixed uniformly with a microplate shaker for 2 minutes, left at room temperature for 10 minutes, and then the chemiluminescence signal value was measured with an EnSpire plate reader. Cell viability was expressed by formula (V sample -V blank ) / (V vehicle control -V blank It was calculated as ) × 100%. Of which, V sample V represents the reading for the drug treatment group, V vehicle control represents the mean value for the solvent control group, V blank This was the mean value of the blank control well. Using GraphPad Prism 5.0 software, an S-type dose-survival curve was created using a nonlinear regression model, and IC 50 The value was calculated.

[0104] The results showed that Huh7 tumor cell proliferation (IC) was induced by compound B of the present invention. 50 We showed that inhibition of (nM) was <10 nM.

[0105] Study Example 6: Pharmacokinetic study of compound B in rats in vivo The compound of formula B obtained in manufacturing example 1 of the test drug.

[0106] The equipment consisted of an LC-MS / MS-13 (TQ5500, Triple quad), Xcalibur 2.0.7 operating software (Thermo Fisher Scientific, USA), an LC-10ADvp (Shimadzu Corporation) liquid chromatography system, a Shimadzu LC-20AD liquid pump, a Shimadzu CBM-20A system controller, a Shimadzu CTO-20A column oven, and a SIL-20AC auto sample injector (Shimadzu Corporation). The column was an Agilent Zorbax SB-CN 3.5 u 100×2.1 mm, the column temperature was 40°C, mobile phase A was a 0.1% formic acid aqueous solution, and mobile phase B was a 0.1% formic acid acetonitrile solution. The flow rate was 0.60 mL / min. Gradient elution was employed, increasing from 1% mobile phase B to 90% mobile phase B over 0-0.5 minutes, maintaining 90% B for 0.5-1.2 minutes, changing back to 1% mobile phase B at 1.21 minutes, and stopping elution at 1.60 minutes.

[0107] Six male SD rats were provided by Shanghai Xipur-Bikai Laboratory Animals Co., Ltd. Their weight ranged from 180 to 220 grams. After purchase, they were reared in the laboratory at the Laboratory Animal Center for two days before use. They were fasted for 10-14 hours before administration and for 4 hours after administration, and were given free access to water during the study period. The rats were randomly divided into two groups of three rats each; one group received intravenous injection, and the other received intragastric administration.

[0108] Preparation of intragastric administration solution: Compound B (4.85 mg) was precisely weighed, and a mixed aqueous solution containing 0.5% methylcellulose and 0.4% Tween-80 (9.70 mL) was added to each. The solution was then sonicated at room temperature to prepare a 0.5 mg / mL suspension. Preparation of intravenous administration solution: Compound B (2.79 mg) was precisely weighed, and DMSO (21 μL), PEG200 (2.79 mL), and physiological saline (4.164 mL) were added to prepare a 0.4 mg / mL solution.

[0109] For the intragastric administration group, blood samples were collected approximately 0.25 hours before administration and at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 hours after administration. For the intravenous administration group, blood samples were collected approximately 0.25 hours before administration and at 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, and 24 hours after administration. Blood samples were collected at 0.25 mL / time point via jugular vein or other venipuncture, anticoagulated with EDTA-K2 or EDTA-K3, and placed on ice after collection. After blood sample collection, plasma was separated by centrifugation (centrifugation conditions: 6800 g / min, 6 minutes, 2-8°C). The collected plasma samples were stored in a -80°C refrigerator before analysis in preparation for testing.

[0110] Compound B was accurately weighed and prepared to different concentrations, and quantitative analysis was performed by mass spectrometry to establish a standard curve. Then, the concentration of compound B in the plasma was measured to obtain the concentration of compound B at different time points. All measurement data were collected and processed using relevant software, and pharmacokinetic parameters (primarily kinetic parameters T) were calculated using the statistical moment method. max , T 1 / 2 , C max AUC 0-t (including the above)

[0111] Results: Compound B was administered intravenously at a dose of 2.0 mg / kg, and its AUC was... 0-t The AUC after intragastric administration of 5 mg / kg was 3978.65 h × ng / mL, with a half-life of 1.88 hours, a volume distribution of 0.883 L / kg, and a clearance rate of 517.78 mL / h / kg. 0-t The blood drug concentration peaked at 4835.41 h × ng / mL after 2.67 hours, and C max The concentration was 1218.83 ng / mL, the half-life was 1.95 hours, and the absolute bioavailability was 48.61%.

[0112] Study Example 7: Pharmacokinetic study of compound C in rats in vivo The compound of formula C obtained in test drug manufacturing example 2.

[0113] The pharmacokinetics of compound C in rats were studied in vivo using the same method as in Test Example 6.

[0114] Results: Compound C underwent intravenous injection at a dose of 2.0 mg / kg, and the AUC was as follows: 0-t The AUC after intragastric administration of 5 mg / kg was 2553.74 h × ng / mL, with a half-life of 1.23 hours, a volume distribution of 0.973 L / kg, and a clearance rate of 803.23 mL / h / kg. 0-t The concentration was 2254.69 h × ng / mL, and the peak blood drug concentration was reached after 2.5 hours. max The concentration was 596.53 ng / mL, the half-life was 1.24 hours, and the absolute bioavailability was 35.32%.

[0115] In summary, the compound of formula B of the present invention possesses excellent FGFR4 kinase inhibitory activity and Huh-7 tumor cell proliferation inhibitory activity, exhibits even better pharmacokinetic effects compared to its optical isomers or racemates, has good solubility in its crystalline form, particularly its preferred crystalline form, is stable in quality, thermodynamically stable, and facilitates drug discovery. [Brief explanation of the drawing]

[0116] [Figure 1] This is the XRPD pattern of crystalline form I of compound B. [Figure 2] This is a single crystal PLM diagram of crystalline form I of compound B. [Figure 3] This is the XRPD pattern calculated from a single crystal of crystalline form I of compound B. [Figure 4] This is the XRPD pattern of crystalline form II of compound B. [Figure 5] This is the XRPD pattern of crystalline form III of compound B.

Claims

1. A crystal of an anhydrous compound shown in formula B, 【Chemistry 1】 X-ray powder diffraction using Cu-Kα radiation, expressed at a 2θ angle, shows characteristic peaks at 7.4±0.2°, 15.9±0.2°, 17.4±0.2°, 20.9±0.2°, and 25.0±0.2°, indicating a crystal of the anhydrous compound shown in formula B.

2. A crystal of the anhydrous compound shown in formula B according to claim 1, wherein the X-ray powder diffraction, expressed as a 2θ angle using Cu-Kα radiation, has characteristic peaks at 7.4±0.2°, 9.8±0.2°, 15.9±0.2°, 17.4±0.2°, 20.9±0.2°, and 25.0±0.2°.

3. A crystal of the anhydrous compound shown in formula B according to claim 1, wherein X-ray powder diffraction, expressed as a 2θ angle using Cu-Kα radiation, has characteristic peaks at 7.4±0.2°, 9.8±0.2°, 15.9±0.2°, 17.4±0.2°, 20.9±0.2°, 22.4±0.2°, and 25.0±0.2°.

4. A crystal of the anhydrous compound shown in formula B according to claim 1, wherein the X-ray powder diffraction, expressed as a 2θ angle using Cu-Kα radiation, has characteristic peaks at 7.4±0.2°, 9.8±0.2°, 11.7±0.2°, 12.0±0.2°, 14.6±0.2°, 15.9±0.2°, 17.4±0.2°, 19.7±0.2°, 20.9±0.2°, 22.4±0.2°, 23.6±0.2°, and 25.0±0.2°.

5. A crystal of the anhydrous compound shown in formula B according to claim 1, wherein the X-ray powder diffraction, expressed as a 2θ angle using Cu-Kα radiation, has characteristic peaks at 7.4±0.2°, 9.8±0.2°, 11.7±0.2°, 12.0±0.2°, 14.6±0.2°, 15.5±0.2°, 15.9±0.2°, 17.4±0.2°, 18.8±0.2°, 19.7±0.2°, 20.9±0.2°, 22.0±0.2°, 22.4±0.2°, 23.6±0.2°, 25.0±0.2°, and 27.8±0.2°.

6. A crystal of the anhydrous compound shown in formula B according to claim 1, having an X-ray powder diffraction pattern shown in Figure 1 or Figure 3, obtained using Cu-Kα radiation. Table 1 Table 2

7. The single crystal of the crystal is obtained using CuKα radiation, and is monoclinic, P2 1 It belongs to the space group, and its unit cell parameters are a=10.8374(2) Å, b=23.6358(3) Å, c=10.96160(10) Å, α=90°, β=117.360(2)°, γ=90°, V=2493.73(7) Å 3 A crystal of the anhydrous compound shown in formula B according to claim 1.

8. A pharmaceutical composition, A pharmaceutical composition comprising crystals of an anhydrous compound represented by formula B as described in any one of claims 1 to 7, and one or more pharmaceutically acceptable carriers optionally.

9. Use of crystals of the anhydrous compound shown in formula B according to any one of claims 1 to 7 in the manufacture of a drug for treating a disease related to FGFR4 activity or expression.

10. The use according to claim 9, wherein the disease associated with FGFR4 activity or expression is selected from cancer, skeletal diseases, T-cell regulated inflammation, and autoimmune diseases.

11. The cancer is lung cancer, bladder cancer, breast cancer, stomach cancer, liver cancer, salivary gland sarcoma, ovarian cancer, prostate cancer, cervical cancer, epithelial cell carcinoma, multiple myeloma, pancreatic cancer, lymphoma, chronic myeloid leukemia, lymphocytic leukemia, or cutaneous T-cell lymphoma; The aforementioned skeletal disorders include dysplasia, chondrodysplasia, dwarfism, or Cruzon syndrome; The aforementioned T-cell regulated inflammation and autoimmune diseases include rheumatoid arthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, psoriasis, juvenile diabetes mellitus, Sjögren's syndrome, thyroid disease, sarcoidosis, inflammatory bowel disease, or celiac disease. The use described in claim 10.

12. The use according to claim 11, wherein the lung cancer is non-small cell lung cancer.

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