A vinyl indazole deuterated FGFR inhibitor drug and its use

Abstract

The present invention discloses a vinyl indazole deuterated FGFR inhibitor, as shown in the following formula I: The present invention relates to a deuterated FGFR inhibitor, a pharmaceutical composition and use thereof, which has excellent FGFR inhibitory activity and anti-tumor activity, and can improve the pharmacokinetic properties of the FGFR inhibitor, reduce the dosage and possible toxic side effects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to deuterated FGFR inhibitor drugs and uses. Background Art

[0002] Fibroblast growth factor (FGF) is a member of the receptor tyrosine kinase (Rtk) family, including four receptor subtypes (FGFR1, FGFR2, FGFR3 and FGFR4). It is a glycoprotein composed of an extracellular immunoglobulin (Ig)-like domain, a hydrophobic transmembrane region and a cytoplasmic part including a tyrosine kinase region. Under the stimulation of fibroblast growth factor (FGF), FGFR dimerizes, followed by receptor autophosphorylation and activation of downstream signaling pathways. In the FGF signal transduction pathway, after fibroblast growth factor binds to the FGFR receptor, it induces FGFR dimerization and phosphorylates the tyrosine at the end of the FGFR cytoplasm, activating the downstream FRS2-Ras-MAPK, PLCγ and P13K-AKT / PKB signaling pathways. When FGFR dimerizes with specific ligands, it autophosphorylates and generates a cascade reaction, thereby activating downstream channel conduction and participating in functions such as embryonic development, cell differentiation, neurovascular regeneration, and wound healing. Abnormal conduction can cause diseases and is closely related to tumor formation. FGFR gene mutations are commonly found in solid tumors such as lung cancer, liver cancer, intrahepatic bile duct cancer, breast cancer, gastric cancer, uterine cancer, and urothelial carcinoma. A variety of FGFR inhibitors have been used in the treatment of clinical cancers. Therefore, FGFR is recognized as an important target for the development of anti-tumor drugs and is currently one of the hottest targets for drug development.

[0003] At present, the research and development of FGFR inhibitors at home and abroad is in the clinical trial stage. Therefore, providing an effective structural FGFR inhibitor will find new directions and inspirations for the treatment of cancer.

[0004] The present invention is a deuterated FGFR inhibitor drug, which can further improve the pharmacokinetic properties of current FGFR inhibitor drugs and reduce the dosage and possible toxic side effects. Summary of the invention

[0005] The present invention provides vinylindazole deuterated compounds of vinylindazole deuterated FGFR inhibitors and pharmaceutically acceptable salts thereof, which can further improve the pharmacokinetic properties of the vinylindazole deuterated compounds of FGFR inhibitors and pharmaceutically acceptable salts thereof, and reduce the dosage and possible toxic side effects.

[0006] In order to achieve the above object, the present invention provides a vinyl indazole deuterated compound and a pharmaceutically acceptable salt thereof as shown in the following formula I:

[0007]

[0008] The vinyl indazole deuterated compound and its pharmaceutically acceptable salt described in the present invention are selected from methanesulfonate, maleate, hydrochloride or phosphate.

[0009] The vinyl indazole deuterated compounds and pharmaceutically acceptable salts thereof of the present invention include applications thereof in the preparation of anti-tumor drugs.

[0010] The vinyl indazole deuterated compound and the pharmaceutically acceptable salt thereof of the present invention comprise the vinyl indazole deuterated compound and the pharmaceutically acceptable salt thereof as active ingredients and a pharmaceutically acceptable carrier.

[0011] The pharmaceutical composition of the vinyl indazole deuterated compound and the pharmaceutically acceptable salt thereof of the present invention is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0013] The present invention provides a class of vinyl indazole deuterated FGFR inhibitor drugs, which have excellent FGFR inhibitory activity and anti-tumor activity, and can also improve the pharmacokinetic properties of FGFR inhibitors, reduce the dosage and possible toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Represents the compound described in Example 1 1 HNMR (500 MHz, Chloroform-d) spectrum. DETAILED DESCRIPTION

[0015] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0016] Example 1: Synthesis of compounds

[0017] The synthetic route of the compound of the present application is as follows:

[0018]

[0019] Intermediate 1 can be prepared by referring to the method of patent CN 102421769;

[0020] Dissolve the intermediate 1 (2 mmol) in DMF (20 mL), add tributylamine (2.4 mmol) and 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (3 mmol), stir at room temperature for 10 min, add bis(triphenylphosphine)palladium chloride (0.4 mmol), heat to 100°C and stir overnight, cool the reaction solution to 40°C, add 4-iodo-1-(2-(tetrahydro-2H-furan-2-yloxy)ethyl)-1-pyrazole (3 mmol) and an aqueous solution of barium hydroxide (3M, 3 mL), then add bis(triphenylphosphine)palladium chloride (0.4 mmol), continue to heat to 100°C and stir overnight. Filter, and the filtrate is subjected to column chromatography to obtain the intermediate 2.

[0021] Intermediate 2 (1 mmol) and cesium carbonate (1.2 mmol) were dissolved in acetonitrile (20 mL), stirred at 60 °C for 10 min, and then (S)-ethyl 1-(3,5-dichloropyridin-4-yl)methanesulfonate was added and stirred overnight. Filtered, the filtrate was concentrated and subjected to column chromatography to obtain intermediate 3.

[0022] To a solution of intermediate 3 (0.5 mmol) in N,N-dimethylformamide (10 mL) were added potassium hydroxide (2 mmol, 4 eq) and iodine (1 mmol, 2 eq), and the mixture was reacted at room temperature for 3 hours. After the reaction was completed as monitored by TLC, a saturated sodium sulfite solution was added to quench the reaction. The aqueous phase was extracted with ethyl acetate (10 mL*2), washed with water (20 mL*2), washed with saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, and concentrated by column chromatography to obtain intermediate 4.

[0023] To a deuterated acetic acid solution (8 mL) of intermediate 4 (0.5 mmol) was added sodium acetate (1 mmol, 2 eq), and the mixture was added dropwise over 2 hours. The mixture was reacted at room temperature for 24 hours. The reaction was complete when detected by TLC. The mixture was concentrated under reduced pressure and subjected to column chromatography to obtain intermediate 5.

[0024] Acetyl chloride (3 mL) was slowly added to a methanol (5 mL) solution at 0°C. Under stirring, a methanol solution (2 mL) of intermediate 5 (1 mmol) was added dropwise. The mixture was moved to room temperature and the reaction was continued for 4 hours. A saturated sodium bicarbonate solution was added to quench the mixture, the mixture was filtered, the solid was washed with water, and dried to obtain Example 1. 1H NMR (500MHz, Chloroform-d) δ8.42 (s, 2H), 7.72 (d, J=3.5Hz, 2H), 7.70-7.64 (m, 2H), 7.49 (d, J=2.8Hz, 1H), 6.97 (dd, J=8.4, 2 .7Hz, 1H), 6.18 (q, J=6.0Hz, 1H), 4.05 (td, J=6.6, 0.9Hz, 2H), 4.00-3.87 (m, 2H), 3.69 (t, J=7.3Hz, 1H), 1.62 (d, J=6.0Hz, 3H).

[0025] Test Example 1: FGFR kinase inhibitory activity test

[0026] The cells were fixed with 10 mM 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) pH 7.5, 8 mM Tris-HCl pH 7.5, 5.0 mM dithiothreitol (DTT), 10.0 μM adenosine triphosphate (ATP), 10 mM MnCl2, 150 mM NaCl, 0.01% TMTONX-100, 0.5 μCi 33 FGFR1 or FGFR3 kinase (0.15 ng / μL human FGFR1 or 0.32 ng / μL human FGFR3) was incubated in 50 μL buffer containing 10 μP-ATP and 0.05 μg / μL Poly(Glu-Tyr). The reaction was carried out at room temperature in a volume of 50 μL for 30 minutes, followed by quenching the reaction by adding 130 μL 10% H3PO4. The reaction (120 μL) was transferred to a 96-well 1.0 μm glass fiber filter plate, incubated at room temperature for 20-30 minutes, and then Wash 3 times with 0.5% H3PO4 on Zoom. Air dry each well and add 40uL MicroScint TM 20 (Packard) followed by counting on a Wallac Micobeta counter. For compound inhibition, compounds were provided as 10 mM stocks in dimethyl sulfoxide (DMSO).

[0027] Compounds were serially diluted 1:3 in 20% DMSO to generate a 10-point concentration response curve and diluted 1:5 (diluted to 20 μM to 0.001 μM final concentration in 4% final DMSO concentration) to the reaction plate, and the reaction mixture was then added to the filter plate to determine compound activity. Control wells contained only 4% DMSO, and a baseline was established based on control wells containing 0.1 M ethylenediaminetetraacetate (EDTA). The percent inhibition value for each of the 10 concentrations was calculated relative to the control wells on each plate, and then the absolute IC of the compound of Example 1 was calculated using the Activity Base software (IDBS) to fit the curve. 50 value.

[0028] Table 1: FGFR kinase activity inhibition (IC 50 , nM)

[0029] Compound FGFR1 FGFR3 Example 1 Compound 1.3 1.9 LY-2874455 7.4 8.6

[0030] As can be seen from Table 1, the compound of Example 1 has excellent FGFR1 and FGFR3 inhibitory activity. Compared with the positive drug LY-2874455, the inhibitory activity of the compound of Example 1 is significantly better than that of the positive drug.

[0031] Experimental Example 3: SNU-16 tumor cell proliferation inhibition experiment

[0032] Adjust the SNU-16 cell suspension to 5.55x10e in RPMI 1640 medium. 4 / mL. Add 90μL of cell suspension to each well of a 96-well cell culture plate, and the final cell concentration is 5000 cells / well. Dissolve the test compound in DMSO to make a 10mM stock solution. Prepare a 3X series of gradient dilutions with the stock solution and DMSO, and then dilute each 100 times with the culture medium. Finally, add 10μL of the corresponding 10-fold solution to each well of each cell line, and duplicate wells for each drug concentration. The final treatment concentrations of each compound were 1000nM, 333.3nM, 111.1nM, 37.0nM, 12.4nM, 4.1nM, 1.37nM, 0.46nM, and 0.15nM, respectively, and the final DMSO concentration in each well was 0.1%. Culture in a 37°C, 5% CO2 incubator for 72 hours. After 72 hours of drug treatment, according to the CTG operating instructions, 50 μL (1 / 2 culture volume) of pre-melted and room temperature CTG solution was added to each well, mixed with a microplate shaker for 2 minutes, and placed at room temperature for 10 minutes. The fluorescence signal value was measured using the Envision2104 plate reader. The cell viability formula is: V sample / V vehicle control x100% calculation. Where V sample is the reading of the drug-treated group, V vehicle controlThe S-shaped dose-survival rate curve was drawn using the nonlinear regression model and the IC 50 value.

[0033] Table 2: Inhibition of SNU-16 tumor cell proliferation (IC 50 , nM)

[0034] Compound SNU-16 Example 1 Compound 0.23 LY-2874455 1.88

[0035] As can be seen from Table 2, the compound of Example 1 has excellent activity in inhibiting the proliferation of SNU-16 tumor cells. Compared with the positive drug LY-2874455, the inhibitory activity of the compound of Example 1 is significantly better than that of the positive drug.

[0036] Experimental Example 3: Pharmacokinetics of Compounds

[0037] 1. Experimental instruments and materials

[0038] High-speed refrigerated centrifuge, vortex oscillator (Vortex Genius3), high-speed centrifuge (Eppendorf5415D), disposable syringes, pipettes (Eppendorf), SD male rats used in the experiment were purchased from Yangzhou University, EDTA-K2 vacuum blood collection tubes, and normal saline. All rats in the oral group fasted for 12 hours before administration, had free access to water, and had free access to water and food during administration.

[0039] (II) Experimental steps

[0040] Example 1 or bozitinib was dissolved in DMSO / solutol / water (10 / 10 / 80) to prepare a clear solution, and the compound dose for oral administration was 25 mg / kg, and the compound dose for tail vein administration was 5 mg / kg. At 2min, 10min, 30min, 1h, 2h, 3h, 5h, 8h, 12h, 16h, 24h after tail vein administration, 0.5mL of blood was continuously taken from the fundus venous plexus into a heparin tube, and 5min, 15min, 30min, 1h, 2h, 3h, 5h, 8h, 12h, 16h, 24h after oral administration, 0.5mL of blood was continuously taken from the fundus venous plexus into a heparin tube. After the sample was centrifuged at 8000r and 4°C for 10min, 0.15mL of upper plasma was taken and stored at -20°C, and then LC-MS / MS analysis was performed. The data was analyzed by WinNolin non-compartmental model to obtain key pharmacokinetic parameters.

[0041] (III) Experimental results

[0042] Table 1 Pharmacokinetic parameters

[0043]

[0044] Compared with LY-2874455, the half-life and peak concentration of Example 1 after oral administration are significantly improved, which can effectively improve the dosage, thereby reducing the toxic and side effects of high-dose administration.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A vinyl indazole deuterated FGFR inhibitor compound of formula I and a pharmaceutically acceptable salt thereof, 2. The deuterated vinyl indazole compound and its pharmaceutically acceptable salt according to claim 1, characterized in that The pharmaceutically acceptable salt is selected from methanesulfonate, maleate, hydrochloride or phosphate.

3. Use of the deuterated vinyl indazole compound and its pharmaceutically acceptable salt according to claim 1 in the preparation of an anti-tumor drug, wherein the anti-tumor target is FGFR.

4. The pharmaceutical composition of the deuterated vinyl indazole compound and its pharmaceutically acceptable salt according to claim 1, characterized in that: The pharmaceutical composition consists of the vinyl indazole deuterated compound and a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of the vinylindazole deuterated FGFR inhibitor according to claim 4, characterized in that: The pharmaceutical composition is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral solutions, inhalants, ointments, suppositories or patches.

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