TRKA(G667C) and FLT3 targeted inhibitors and their compositions with tucidinostat

Abstract

The present invention discloses N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide, a TRKA(G667C), FLT3, and FLT3(D835Y) target inhibitor represented by formula (I), its preparation method, a pharmaceutical composition containing it and tucidinostat, and its application. Through extensive research, the present inventors have discovered that this compound has excellent TRKA(G667C) and FLT3 kinase inhibitory activity and exhibits unexpected synergistic effects when used in combination with tucidinostat. The present invention also provides for its use as a control for testing related substances in tucidinostat tablets to perform quality control on tucidinostat tablets. [C8] TIFF2025528495000013.tif68166

Description

[Technical Field]

[0001] The present invention belongs to the field of medicine, specifically to a TRKA(G667C) and FLT3 targeted inhibitor and its composition with tucidinostat. [Background technology]

[0002] TRK (Tropomyosin-Related Kinase) is a class of neurotrophic factor receptors belonging to the receptor tyrosine kinase family, encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively, and including three isoforms: TRKA, TRKB, and TRKC. When the NTRK gene is fused with other genes, it induces receptor dimerization and phosphorylation, resulting in high TRK expression or sustained increase in TRK activity, and activates the downstream PI3K / Akt / mTOR, PLCγ, and Ras / Raf / MEK / ERK signaling cascade pathways, thereby regulating tumor cell growth, proliferation, invasion, migration, angiogenesis, and drug resistance.

[0003] Alterations in the TRK signaling pathway primarily involve gene fusions, protein overexpression, single nucleotide changes, and splice mutations. Among these, NTRK gene fusions are the most clearly defined oncogenic drivers found in both adult and pediatric tumors. NTRK gene fusions occur at a rate of over 90% in patients with congenital kidney tumors, infantile sarcomas, salivary gland carcinomas (MASCs), and secretory breast cancer. NTRK gene fusions occur at a rate of 5% to 25% in patients with papillary thyroid carcinoma, gastrointestinal stromal tumors, and spitzoid melanoma. In most other tumors, NTRK gene fusions occur at a rate of less than 5%.

[0004] There are two commercially available first-generation TRK inhibitors: larotrectinib and entrectinib. In 2018, larotrectinib was marketed in the United States for the treatment of adult and pediatric patients with locally advanced or metastatic solid tumors with NTRK gene fusions. In 2019, entrectinib was marketed in the United States for the treatment of adult and pediatric patients with advanced or recurrent solid tumors with NTRK gene fusions and metastatic NSCLC patients with ROS1 rearrangements.

[0005] However, first-generation TRK inhibitors can sometimes exhibit on-target drug resistance after use. TRKA G595R, G667C, F589L, etc. and TRKC G623R, G696A, etc. have been frequently reported in different cancer types.

[0006] FLT3 (FMS-like tyrosine kinase 3) belongs to the type III receptor tyrosine kinase family. Its protein structure contains an extracellular region consisting of five immunoglobulin-like domains, one transmembrane region, one juxtamembrane region, and two intracellular tyrosine kinase domains separated by a kinase insert region. Normally, when FLT3 ligands bind to FLT3, they subsequently activate multiple signaling pathways, including signal transducer and activator of transcription 5 (STAT5), Ras / mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) / AKT pathways. FLT3 plays an important role in the proliferation, differentiation, and apoptosis of hematopoietic cells.

[0007] First-generation FLT3 inhibitors include tandutinib, sunitinib, midostaurin, lestaurtinib, and sorafenib. Because first-generation FLT3 inhibitors were not initially screened for sensitivity and selectivity against activated FLT3 kinase, second-generation FLT3 inhibitors with higher selectivity and stronger inhibitory activity have been developed, among which gilteritinib, quizartinib, and crenolanib (CP-868596) have been approved for clinical use in Japan, Europe, and the United States.

[0008] However, the emergence of drug resistance to FLT3 inhibitors limits further improvement of their clinical therapeutic efficacy.

[0009] Although the discovery of TRKA and FLT3 inhibitors has contributed greatly to tumor treatment and saved patients' lives, the emergence of drug resistance is inevitable, ultimately leading to disease progression and limiting their clinical application. Genetic mutations have also been found to be one of the causes of drug resistance. Multitargeted inhibitors represent a new direction in tumor drug development. Research has shown that multitargeted single-entity drugs have superior therapeutic effects compared to single-targeted drugs, have fewer side effects, and can overcome drug resistance. Therefore, the development of novel inhibitors targeting TRKA and FLT3, especially those targeting gene mutation sites, is of great significance for the treatment of related diseases. Summary of the Invention

[0010] Tushidinostat is a histone deacetylase inhibitor whose chemical name is N-(2-amino-4-fluorophenyl)-4-[N-[(E)-3-(3-pyridine)acryloyl]aminomethyl]benzamide and whose chemical structure is as follows:

[0011] [ka] Chinese Patent CN1284772C describes the pharmacological activity of tucidinostat. Tucidinostat is an isoform-selective histone deacetylase inhibitor that mainly inhibits class I HDACs HDAC1, HDAC2, HDAC3 and class IIb HDACs HDAC10, and is used to treat diseases associated with abnormal histone deacetylase activity, such as cancers including lymphomas, solid tumors, and hematological tumors.

[0012] In 2014, 5 mg tucidinostat tablets were approved for marketing in China for the indication of peripheral T-cell lymphoma (PTCL). In 2019, 5 mg tucidinostat tablets were approved in China for the indication of breast cancer.

[0013] The present inventors have confirmed that tucidinostat tablets are 18 After extensive research, we unexpectedly discovered that an unknown substance peak was present at a retention time of 18.2 min in the HPLC chromatogram using a Shimadzu VP-ODS 5 μm 4.6 mm × 150 mm column, mobile phase: methanol-water-glacial acetic acid (30:70:0.4), detection wavelength: 256 nm, flow rate: 1.0 mL / min.

[0014] Through extensive research, the present inventors have found that the unknown substance is N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide, the structural formula of which is shown in formula (I).

[0015] [ka] Tucidinostat tablet related substance test: Column: C 18The HPLC analysis was performed using a Shimadzu VP-ODS 5 μm column (4.6 mm × 150 mm), a mobile phase of methanol-water-glacial acetic acid (30:70:0.4), a detection wavelength of 256 nm, and a flow rate of 1.0 mL / min. A control solution of N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide (compound of formula (I)) was prepared, a chromatogram was recorded, and the retention time of the substance was measured. A sample solution of tushidinostat tablets was prepared, a chromatogram was recorded, and the content of the substance in tushidinostat tablets was calculated using the area normalization method based on the retention time of N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide.

[0016] By studying the activity of this substance, the present inventors have unexpectedly found that it has excellent inhibitory effects on the kinases TRKA(G667C), FLT3, and FLT3(D835Y).

[0017] Based on the above research results, in a first aspect, the present invention provides a TRKA(G667C), FLT3, and FLT3(D835Y) targeted inhibitor compound N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide represented by formula (I) or a pharmaceutically acceptable salt thereof, which has significant inhibitory activity against the kinases TRKA(G667C), FLT3, and FLT3(D835Y).

[0018] [ka] In a second aspect, the present invention provides a method for producing the compound represented by the above formula (I), which is obtained by reacting tucidinostat with an acid.

[0019] In one embodiment, the acid includes an inorganic acid including hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, hydroiodic acid, or hydrobromic acid, and an organic acid including acetic acid, trifluoroacetic acid, lactic acid, succinic acid, fumaric acid, maleic acid, citric acid, benzoic acid, methanesulfonic acid, or p-toluenesulfonic acid.

[0020] In one embodiment, the compound represented by the above formula (I) is obtained by reacting tucidinostat with hydrochloric acid.

[0021] [ka] In one embodiment, tushidinostat is reacted with 0.2 mol / L hydrochloric acid under reflux, then neutralized with sodium hydroxide solution, suction filtered, washed with water, and dried to obtain the compound of formula (I).

[0022] It should be pointed out that the hydrochloric acid and reflux reaction conditions used in the above reaction pathway are merely illustrative and not limiting, and those skilled in the art can appropriately modify and adjust them.

[0023] In a third aspect, the present invention provides a pharmaceutical composition comprising a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0024] In a fourth aspect, the present invention provides use of a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for preventing and / or treating a disease associated with a TRK and / or FLT3 target.

[0025] In one embodiment, the disease is a disease associated with TRKA(G667C) and / or FLT3 and / or FLT3(D835Y) targets.

[0026] In one embodiment, the disease comprises a tumor.

[0027] In a fifth aspect, the present invention provides a pharmaceutical composition comprising a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof, and tucidinostat.

[0028] In a sixth aspect, the present invention provides the use of a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof as a standard or control.

[0029] In one embodiment, the present invention provides use of the compound represented by formula (I) above or a pharmaceutically acceptable salt thereof in the quality control of tucidinostat or a formulation containing tucidinostat.

[0030] In one embodiment, the present invention provides use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of tucidinostat or a control product of a formulation containing tucidinostat.

[0031] In one embodiment, the formulation comprising tucidinostat is a tucidinostat tablet.

[0032] In one embodiment, the present invention provides use of the compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof as a control for related substance testing in tucidinostat tablets.

[0033] Terms and Definitions The term "treatment" means the administration or application of an actual treatment to a subject or the administration of a step or mode to a subject to obtain a therapeutic benefit for a disease or health-related condition.

[0034] The term "control" is known to pharmaceutical researchers as a relatively pure compound that can be used as a "control." A control generally refers to a standard substance that identifies, tests, measures, and corrects the performance of a calibration instrument.

[0035] Beneficial effects of the present invention: (1) The present inventors have unexpectedly discovered that the unknown substance appearing at a retention time of 18.2 min in the HPLC chromatogram of tushidinostat tablets is the compound N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide represented by the above formula (I), a compound with a completely novel structure, and have confirmed its preparation and structure.

[0036] (2) The present invention thoroughly studies the activity of the compound represented by formula (I), N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide, and finds that it has excellent TRKA (G667C), FLT3 and FLT3 (D835Y) kinase inhibitory activity and IC 50 were found to be 40.69 nM, 142.6 nM and 103.8 nM, respectively, and based on this, the present invention provides a TRKA (G667C) and FLT3 targeted inhibitor having a completely novel structure and a pharmaceutical composition containing the same.

[0037] (3) Through further intensive research, the present invention has found that the compound represented by formula (I), N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide, and tucidinostat have an unexpected synergistic effect when used in combination. Based on this finding, the present invention provides a pharmaceutical composition comprising the compound represented by formula (I), N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide, and tucidinostat.

[0038] (4) The present invention provides a compound represented by formula (I), N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide, as a control for related substance testing in tucidinostat tablets for quality control of tucidinostat. [Brief explanation of the drawings]

[0039] [Figure 1]1 shows the results of a test of the effect of the compound N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide (Compound A) and tushidinostat on SHP77 cell viability. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will be further described below with reference to examples, but the scope of the present invention is not limited to these examples. All percentages described in the present invention are weight percentages unless otherwise specified. Any units of measurement or numerical ranges, such as percentages, described in the specification are intended to provide clear and unambiguous written references. Those skilled in the art will be able to achieve the desired results when practicing this patent based on the teachings and principles of the present invention, even when using temperatures, concentrations, amounts, etc. outside these ranges or different from the individual numerical values.

[0041] Starting materials and test equipment: Tushidinostat: Manufactured by Shenzhen Weixin Pharmaceutical Co., Ltd., with lot number 20220708 and purity >99%.

[0042] High-performance liquid chromatography: Instrument: UltiMate3000, Column: C 18 Column, Shimadzu VP-ODS 5 μm 4.6 mm × 150 mm.

[0043] Proton nuclear magnetic resonance: Instrument: Avance NEO 400, Solvent: DMSO-d6.

[0044] High-resolution mass spectrometry: Instrument: maXis impact.

[0045] Infrared spectrum: Instrument: PerkinElmer Spectrum.

[0046] Example 1 Preparation of compound N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide [ka] 2.0 g of tushidinostat and 50 mL of 0.2 mol / L hydrochloric acid were added to a reaction flask, dissolved by ultrasound, and heated under reflux for approximately 4 hours. The reaction solution was cooled to room temperature and neutralized with 1 mol / L sodium hydroxide solution until the pH was >12. The solution was then suction filtered and washed with purified water until the filtrate reached a pH of 5-7. The solid was collected and dried to give a weight of 1.6 g with a purity (HPLC) of 99.9%. HRMS (M + +1)(C 22 H 18 FN4O), calculated value (%): 373.1420, observed value (%): 373.1413. 1 H NMR (DMSO-d6) δ 4.53 (brs, 2H),6.86 (d, 1H),7.07 (m, 1H),7.32~7.68 (m, 6H),8.03 (d, 1H),8.15 (d, 2H,),8.58 (d, 1H),8.80 (br, 2H),13.03 (brs, 1H). IR(cm -1 )3432,3267,2917,1658,1625,1537,1495,1453,1423,1229,843,689.

[0047] Example 2 Inhibitory activity (IC) of compound N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide (Compound A) against kinases TRKA and FLT3 50 ) The inhibitory effect of the compound of the present invention, N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide (hereinafter referred to as Compound A), on the kinases TRKA(G667C), FLT3, and FLT3(D835Y) was detected using a biochemical-based homogeneous time-resolved fluorescence (HTRF) technique.

[0048] HTRF is a technique for detecting analytes in pure liquid systems based on two techniques, Fluorescence Resonance Energy Transfer (FRET) and Time-Resolved Fluorescence (TRF), where the fluorescence intensity produced is detected by a microplate reader and the ratio of 665 nm / 620 nm reflects the degree of inhibition of Compound A on the activity of the kinases TRKA(G667C), FLT3, and FLT3(D835Y).

[0049] The specific experimental method is as follows.

[0050] Reagents and consumables: [Table 1]

[0051] Tucidinostat was obtained from Shenzhen Weixin Pharmaceutical Co., Ltd.

[0052] Compound A (N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide) was obtained from Shenzhen Weixin Biotechnology Co., Ltd.

[0053] 1. Preparation of Compound A and Kinase Reaction Buffer a) Preparation of Compound A or Tucidinostat: The initial stock solution concentration of Compound A or Tucidinostat was 20 mM, and the solution was diluted to 0.2 mM, which is 200 times the initial detection concentration, using 100% DMSO as a solvent.

[0054] b) Kinase reaction buffer: Assay buffer was prepared according to the following composition. [Table 2]

[0055] 2. Preparation of Detection Compound A, Kinase Solution, and Substrate Mixture a) Compound A or tucidinostat was diluted in kinase reaction buffer to 10 concentrations corresponding to 200-fold final concentrations of 1000 nM, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM, and 0.03 nM.

[0056] b) Using kinase reaction buffer, TRKA(G667C), FLT3, or FLT3(D835Y) kinase solutions were prepared at twice the final concentration, and a mixture of ATP and biotin-labeled substrate (ATP + TK Substrate - biotin polypeptide substrate) was prepared at twice the final concentration using kinase reaction buffer. The final concentrations of each substance during the reaction are shown in the table below. [Table 3]

[0057] 3. HTRF Detection 1) Using a separatory tube Echo655, 25 nL of the diluted compound A or tushidinostat was added to the compound wells of a 384-well reaction plate, and 25 nL of 100% DMSO was added to both the negative control wells and the positive control wells. Next, 2.5 μL of kinase solution at twice the final concentration was added to the compound wells and the positive control wells of the 384-well reaction plate, and mixed evenly. 2.5 μL of kinase reaction buffer was added to the negative control wells, and the plate was incubated at 25°C for 10 minutes.

[0058] 2) 2.5 μL of the substrate mixture at twice the final concentration was added to the 384 reaction plate, and the plate was incubated at 25° C. for 50 minutes.

[0059] 3) A mixture of XL665-labeled avidin and europium (Eu)-labeled phosphorylated TK antibody was prepared at twice the final concentration using detection buffer, and 5 μL of the mixture was added to each reaction well and incubated at 25° C. for 60 minutes.

[0060] 4) The 384-well plate was centrifuged at 1000 rpm for 30 seconds, shaken, and mixed uniformly. The fluorescence intensity at 665 nm and 620 nm was then read using EnVision, and the TR-FRET ratio (665 nm / 620 nm) was calculated.

[0061] 4. Data Analysis 1) The formula for calculating the activity inhibition rate is as follows:

[0062] Inhibition rate (%) = 100% - [(TR-FRET ratio_compound) - (TR-FRET ratio_positive control)] / [(TR-FRET ratio_negative control) - (TR-FRET ratio_positive control)] × 100%

[0063] 2) Dose-effect curve fitting Dose-effect curves were fitted using the analytical software GraphPad 7.0, log (concentration of compound) vs (dose response-variable slope), with the log value of concentration on the X axis and the inhibition rate on the Y axis, to obtain the IC50 of compound A on the activity of kinases TRKA(G667C), FLT3, and FLT3(D835Y). 50 The values ​​are obtained and the results are as follows: [Table 4]

[0064] From the above table, it can be seen that the compound N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide (Compound A) has significant inhibitory activity against the kinases TRKA(G667C), FLT3, and FLT3(D835Y) and is a TRKA(G667C), FLT3, and FLT3(D835Y) target inhibitor, whereas tushidinostat has no inhibitory activity against the above kinase targets.

[0065] Example 3 Effect of the combination of compound N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide (Compound A) and tucidinostat on SHP77 cell viability 1. Experimental materials and equipment 1.1 Materials and Sources Both SHP77 cell lines were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium (Hyclone) containing 1% double antibody (penicillin-streptomycin, Hyclone) and 10% fetal bovine serum (Gibco).

[0066] Tucidinostat was obtained from Shenzhen Weixin Pharmaceutical Co., Ltd.

[0067] Compound A (N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide) and tushidinostat were both obtained from Shenzhen Weichen Biotechnology Co., Ltd. and dissolved in dimethyl sulfoxide (DMSO, Shanghai Weichen Biotechnology Co., Ltd., DN3039A) to prepare a 20 mM working solution. This solution was then stored in a refrigerator at -20°C for use, and working solutions of the corresponding final concentrations were prepared as needed for each experiment.

[0068] CellTiter 96® AQueous MTS Reagent Powder (Promega, G1111) Phenazine methosulfate, PMS(Sigma, P9625) 1.2 Experimental Reagents and Equipment: Ultra Clean Bench (Su Jing Antai) CO2 cell incubator (RS Biotech, Galaxy S) Inverted fluorescence microscope (Guangzhou Mingmei Science and Technology Co., Ltd.) Cell counting play (Shanghai Sperm Biochemical Reagent Equipment Co., Ltd.) Tecan infinite F50 light absorbance microplate reader

[0069] 2. Experimental Method 2.1 Preparation of MTS and PMS solutions MTS solution: 42 mg of MTS Reagent Powder was placed in a light-shielding container, 21 mL of PBS was added, and the mixture was gently stirred with a magnetic stirrer for 15 minutes until the MTS was completely dissolved. The pH of the solution was measured, and if the reading was higher than pH 6.5, the solution was adjusted to pH 6.5 with 1 M HCl. The solution was sterilized by filtration using a 0.2 μM filter and stored at -20°C, protected from light.

[0070] PMS solution: 21.7 mL of PBS was added to 1 g of PMS to prepare a 150 μM stock solution, which was then sterilized by filtration, dispensed, and stored at -20°C in the dark.

[0071] 2.2 Cell inoculation: SHP77 cells were inoculated into a 96-well cell culture plate at 15,000 cells / 180 μL per well. BLANK wells were not inoculated with cells, but instead received the same volume of culture medium. The wells were cultured at 37°C in 5% CO2.

[0072] 2.3 Administration: After inoculation and overnight culture, Compound A and tucidinostat were first diluted with DMSO to 1000 times the final concentration in each well, and then the DMSO solutions of Compound A and tucidinostat were diluted 1:50 in culture medium. 10 μL of the corresponding solution was added in combination with 0 or 0.5 μM tucidinostat to achieve final concentrations of Compound A of 0, 0.625, or 1.25 μM, respectively.

[0073] 2.4 Detection: After 72 hours of drug exposure, the 96-well plate was removed. SHP77 cells were directly added to 20 μL of a mixture of MTS and PMS (MTS:PMS = 20:1). After incubation for approximately 1 hour, the absorbance of each well was measured at 490 nm using a microplate reader.

[0074] 2.5 Data Processing and Analysis The reading of the blank well is OD 490-BLANK (background value), and the background value was subtracted from the reading of each well to obtain the OD of each treatment well. 490-T and OD of the control well with 0 drug concentration 490-T0 The cell viability of the control well was set to 100%, and the relative cell viability of each treatment well was calculated as follows: Relative cell viability (%) = (OD 490-T ÷OD 490-T0 ) × 100%.

[0075] 3. Experimental Results The results of the test on the effect of compound A and tucidinostat on SHP77 cell viability are shown in Figure 1. After 72 hours of treatment with compound A alone at final concentrations of 0.625 and 1.25 μM, the relative cell viabilities of SHP77 were 77.43 ± 1.18% and 44.09 ± 6.59%, respectively. After 72 hours of treatment with tucidinostat alone at a final concentration of 0.5 μM, the relative cell viabilities were 95.45 ± 0.81%. Meanwhile, after two doses of compound A and tucidinostat were combined to treat SHP77, the relative cell viabilities were 38.22 ± 1.92% and 20.01 ± 2.87%, respectively. These results demonstrate that compound A can inhibit tumor cell proliferation in a dose-dependent manner. Surprisingly, the combination of compound A and tucidinostat on tumors produced a significant synergistic tumor inhibitory effect.

[0076] Example 4 Use of the Compound N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide as a Control for Related Substance Testing in Tucidinostat Tablets 1. Test conditions Instrument: UltiMate3000, Column: C 18 Column: Shimadzu VP-ODS 5 μm 4.6 mm × 150 mm, mobile phase: methanol-water-glacial acetic acid (30:70:0.4), detection wavelength: 256 nm, flow rate: 1.0 mL / min.

[0077] 2. Test method (1) Approximately 6 mg of N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide (Lot No.: 000043001-01) was precisely weighed and placed in a 100 mL measuring flask. 30 mL of methanol was added and dissolved by ultrasound. The solution was diluted to the mark with water and shaken uniformly. 5 mL was precisely dispensed into a 100 mL measuring flask, diluted to the mark with the solvent methanol:water (30:70), and shaken uniformly. 1 mL was precisely dispensed into a 50 mL measuring flask, diluted to the mark with the solvent methanol:water (30:70), and shaken uniformly to prepare a control solution of N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide. 60 L was precisely dispensed and injected into a liquid chromatograph, the chromatogram was recorded, and the time at which the peak of N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide appeared was measured.

[0078] (2) An appropriate amount of tushidinostat tablets (lot number: 20220708) was taken, precisely weighed, finely pulverized, and mixed uniformly. An appropriate amount (equivalent to approximately 20 mg of tushidinostat) was precisely weighed and placed in a 100 mL measuring flask. An appropriate amount of methanol was added, and the mixture was dissolved by ultrasound. The mixture was diluted to the mark with methanol, shaken uniformly, and filtered. 15 mL of the filtrate was placed in a 50 mL measuring flask and diluted to the mark with water to obtain a sample solution of tushidinostat tablets. 60 μL was precisely weighed and injected into a liquid chromatograph. The chromatogram was recorded, and the content of N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide was calculated based on the retention time of this substance using the area normalization method.

[0079] 3. Measurement results Based on the chromatogram of the N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide control solution, the time for the appearance of the N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide peak was measured at 18.2 min.

[0080] Based on the chromatogram of the tucidinostat tablet sample solution, the content of N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide (peak appearance time: 18.2 min) in the tucidinostat tablet was determined to be 0.07%.

[0081] The present invention has been described by way of example with reference to specific embodiments. However, the present invention is not limited to these specific embodiments. Those skilled in the art can make various modifications and variations within the scope of the present invention, and can combine the technical features described in each part of this specification without departing from the spirit and scope of the present invention. All such modifications and variations are within the scope of the present invention.

Claims

1. The compound represented by formula (I) N-[4-(6-fluoro-1H-2-benzimidazolyl)benzyl]-(E)-3-(3-pyridine)acrylamide or a pharmaceutically acceptable salt thereof: 【Chemistry 6】

2. A method for producing the compound of formula (I) according to claim 1, which is obtained by reacting tucidinostat with an acid, A manufacturing method characterized by:

3. The acid includes inorganic acids and organic acids, and is preferably obtained by reacting tucidinostat with hydrochloric acid. The method according to claim 2 . 【Chemistry 7】

4. A compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

5. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 in the manufacture of a medicament for preventing and / or treating a disease associated with a TRK and / or FLT3 target. use.

6. Preferably, the disease is a disease associated with TRKA(G667C) and / or FLT3 and / or FLT3(D835Y) targets, more preferably, the disease comprises a tumor.

6. The use according to claim 5.

7. The compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, and tucidinostat. Pharmaceutical compositions.

8. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 as a standard or control. use.

9. In the quality control of a formulation containing tucidinostat, the compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, use.

10. The formulation containing tucidinostat is a tucidinostat tablet.

10. The use according to claim 9.

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