Pyrrolo-ring compound and application thereof in medicine

Abstract

The invention relates to a pyrrolo-ring compound or a pharmaceutically acceptable salt, a stereoisomer or a deuterated compound thereof, a composition of the pyrrolo-ring compound and application of the pyrrolo-ring compound in medicine.

Description

Technical Field

[0001] The invention relates to a pyrrolocyclic compound and its application in medicine. Background Art

[0002] FGFR, or fibroblast growth factor receptor, is a family of four homologous, highly conserved transmembrane tyrosine kinase receptors (FGFR1-4). FGFR binds to its ligand, fibroblast growth factor (FGF), and FGFR dimerizes and autophosphorylates, activating downstream signaling pathways such as the JAK / STAT pathway and the phospholipase C pathway. These signaling pathways play a vital role in a variety of physiological processes such as cell proliferation, differentiation, migration, and apoptosis. FGFR fusion and dysregulated expression are closely related to the occurrence and development of a variety of cancers. FGFR gene mutations have been reported in more than 10 types of malignant tumors.

[0003] Currently, all drugs marketed for this target are Pan-FGFR inhibitors, which have limited therapeutic effects, with over 50% of patients showing insufficient responses; acquired drug resistance has already emerged; and they have high toxic side effects, mainly manifested as hyperphosphatemia and diarrhea, which are caused by the targeting effect of FGFR1 / 4 inhibition. Therefore, it is necessary to develop a new generation of highly active and highly selective FGFR2 and / or FGFR3 inhibitors, as well as methods for treating cancer and other diseases. Summary of the invention

[0004] The object of the present invention is to provide a pyrrolocyclic compound as a FGFR2 and / or FGFR3 inhibitor and its application in the preparation of anti-tumor drugs.

[0005] One or more embodiments of the present invention provide a compound or a pharmaceutically acceptable salt, stereoisomer or deuterated substance thereof, wherein the compound is selected from, but not limited to, the following structures:

[0006]

[0007]

[0008]

[0009] One or more embodiments of the present invention provide a pharmaceutical composition, comprising:

[0010] (1) a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer or deuterated substance thereof;

[0011] (2) optionally one or more other active ingredients; and

[0012] (3) Pharmaceutically acceptable carriers and / or excipients.

[0013] One or more embodiments of the present invention provide use of the pharmaceutical composition of the present invention or the compound or a pharmaceutically acceptable salt, stereoisomer or deuterated substance thereof in the preparation of an anti-tumor drug.

[0014] Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

[0015] "Pharmaceutically acceptable salt" or "pharmaceutically acceptable salt thereof" refers to a salt of the compound of the present invention that retains the biological effectiveness and properties of the free acid or free base, and the free acid is obtained by reacting with a non-toxic inorganic base or organic base, and the free base is obtained by reacting with a non-toxic inorganic acid or organic acid.

[0016] "Pharmaceutical composition" refers to a mixture of one or more compounds described herein, their pharmaceutically acceptable salts or prodrugs and other chemical components, wherein "other chemical components" refers to pharmaceutically acceptable carriers, excipients and / or one or more other therapeutic agents.

[0017] "Carrier" refers to a material that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0018] "Excipient" refers to an inert substance added to a pharmaceutical composition to facilitate administration of a compound. Non-limiting examples include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders, and disintegrants.

[0019] "Stereoisomers" refer to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and conformational isomers.

[0020] "Optional" or "optionally" or "selective" or "selectively" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "heterocyclyl optionally substituted with alkyl" means that the alkyl may but need not be present, and the description includes instances where the heterocyclyl is substituted with alkyl and instances where the heterocyclyl is not substituted with alkyl. DETAILED DESCRIPTION

[0021] The following embodiments illustrate the technical solutions of the present invention in detail, but the protection scope of the present invention includes but is not limited to them.

[0022] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or (and) mass spectrometry (MS). NMR shifts (δ) are given in units of 10-6 (ppm). NMR measurements were performed using (Bruker Avance III 400 and Bruker Avance 300) NMR spectrometers, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD) as the solvent, and tetramethylsilane (TMS) as the internal standard;

[0023] MS was measured using (Agilent 6120B (ESI) and Agilent 6120B (APCI));

[0024] HPLC determination was performed using an Agilent 1260DAD high pressure liquid chromatograph (Zorbax SB-C18 100×4.6 mm, 3.5 μM);

[0025] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) uses a specification of 0.15mm-0.20mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm-0.5mm;

[0026] Column chromatography generally uses Yantai Huanghai Silica Gel 200-300 mesh silica gel as the carrier;

[0027] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as Titan Technology, Anage Chemical, Shanghai Demo, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, Bailingwei Technology, and Jiangsu Aikon Biomedicine Research and Development.

[0028] Intermediate 1

[0029] 2-Cyclopropyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acrylamide intermediate 1

[0030] 2-cyclopropyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acryla mide

[0031]

[0032]

[0033] 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline compound 1A (1.96 g, 8.9 mmol) and 2-cyclopropylacrylic acid 1B (1 g, 9.0 mmol) were dissolved in acetonitrile (20 mL), and diisopropylethylamine (2.3 g, 17.8 mmol) and HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 4.07 g, 10.7 mmol) were added. The reaction flask was replaced with nitrogen and placed at 50°C for reaction. After reaction for 2 hours, it was concentrated, and water (40 mL) was added after concentration. Ethyl acetate (6×30 mL) was added for extraction. The organic phases were combined and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE:EA=5:1) to obtain intermediate 1 (white solid, 1.2 g, yield 63%).

[0034] LCMS m / s=452.16[M+1].

[0035] Intermediate 2

[0036] N-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-cyclopropylacrylamide intermediate 2

[0037] N-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-cycloprop ylacrylamide

[0038]

[0039] 2-Cyclopropylacrylic acid 1B (300 mg, 2.6 mmol) was dissolved in N,N-dimethylformamide (8 ml), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.38 g, 3.6 mmol) and triethylamine (614 mg, 6.07 mmol) were added. After reacting at room temperature for 30 minutes, compound 2A (616 mg, 2.4 mmol) was added. The reaction flask was replaced with nitrogen and reacted at room temperature for 2 hours. Water (8 mL) was added for dilution, and ethyl acetate (3×15 mL) was added for extraction. After the extraction was completed, the organic phase was washed with saturated brine (3×15 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure to obtain intermediate 2 (white solid, 200 mg, yield 23.7%).

[0040] LCMS m / s=348.65[M+1].

[0041] Example 1

[0042] N-(4-(4-amino-7-ethoxy-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-2-yl)phenyl]-2-cyclopropylacrylamide Compound 1

[0043] N-(4-(4-amino-7-ethoxy-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-2-yl)phenyl)-2-cyclopropylacrylamide

[0044]

[0045]

[0046] first step:

[0047] 1-Methyl-1H-pyrrole-2,3-dicarboxylic acid dimethyl ester 1b

[0048] dimethyl 1-methyl-1H-pyrrole-2,3-dicarboxylate

[0049] 1a (5 g, 27.3 mmol) and sodium hydride (2.2 g, 54.6 mmol) were dissolved in dimethyl sulfoxide (100 mL) at room temperature. After stirring for 10 minutes, iodomethane (2.6 mL, 41 mmol) was added to the system and reacted at room temperature for 2 hours. After the reaction was completed, the system was quenched with water, extracted with ethyl acetate (3×100 mL), and dried to obtain compound 1b (white solid, 5.37 g, yield 99%).

[0050] LCMS m / s=198.55[M+1].

[0051] Step 2:

[0052] 1-Methyl-5,6-dihydro-1H-pyrrolo[2,3-d]pyridazine-4,7-dione 1c

[0053] 1-methyl-5,6-dihydro-1H-pyrrolo[2,3-d]pyridazine-4,7-dione

[0054] 1b (5.37 g, 27.3 mmol) and hydrazine hydrate (20 mL, 409.5 mmol) were dissolved in ethanol (100 mL) at room temperature. The system was heated to 75 ° C under nitrogen protection for 4 hours. After the reaction was completed, the temperature was cooled to room temperature. The system was concentrated and washed with a solvent of dichloromethane: methanol = 10:1. The system was filtered to obtain compound 1c (white solid, 4.5 g, yield 99%).

[0055] LCMS m / s=166.09[M+1].

[0056] Step 3:

[0057] 4,7-Dichloro-1-methyl-1H-pyrrolo[2,3-d]pyridazine 1d

[0058] 4,7-dichloro-1-methyl-1H-pyrrolo[2,3-d]pyridazine

[0059] Under nitrogen protection, compound 1c (4.5 g, 27.3 mmol) was added to phosphorus oxychloride (90 mL) and stirred at 80°C for 24 hours. The system was concentrated and extracted with saturated sodium bicarbonate aqueous solution (50 mL), water (50 mL), and saturated brine (50 mL) in sequence, and the organic phases were combined and concentrated to obtain compound 1d (yellow solid, 4.135 g, yield 75%).

[0060] LCMS m / s=203.08[M+1].

[0061] Step 4:

[0062] 4-Chloro-7-ethoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazine 1e

[0063] 4-chloro-7-ethoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazine

[0064] Compound 1d (2 g, 10 mmol) was added to a round-bottom flask and dissolved in tetrahydrofuran (40 mL). Then, a solution of sodium ethoxide (750 mg, 11 mmol) in ethanol (22 mL) was slowly added dropwise to the system and reacted at room temperature for 3 hours. After the reaction was completed, the system was concentrated, water was added, and the system was filtered to obtain compound 1e (white solid, 2.3 g, yield 99%).

[0065] LCMS m / s=212.11[M+1].

[0066] Step 5:

[0067] 7-Ethoxy-N-(4-methoxybenzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine 1f

[0068] 7-ethoxy-N-(4-methoxybenzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine

[0069] Compound 1e (2.3 g, 1.16 mmol), 4-methoxybenzylamine (2.5 g, 23.2 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (2.9 g, 4.64 mmol) and palladium acetate (570 mg, 2.32 mmol) were dissolved in dioxane (50 mL). The reaction bottle was replaced with nitrogen and placed in an 80°C oil bath for reaction. After 4 hours of reaction, the system was filtered and concentrated. The crude product was purified by column chromatography (PE:EA=1:2) to obtain compound 1f (yellow solid, 950 mg, yield 28%).

[0070] LCMS m / s=313.18[M+1].

[0071] Step 6:

[0072] 7-Ethoxy-3-iodo-N-(4-methoxybenzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine 1g

[0073] 7-ethoxy-3-iodo-N-(4-methoxybenzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine

[0074] Compound 1f (800 mg, 2.56 mmol) was dissolved in DCM (15 mL), trifluoroacetic acid (877 mg, 7.69 mmol) was added to the system, and the system was cooled to 0°C, and N-iodosuccinimide (575 mg, 2.56 mmol) was slowly added. After the system was warmed to room temperature and reacted for 2 hours, it was quenched with saturated aqueous sodium sulfite solution (30 mL), and the system was extracted with DCM (3×20 mL). After the extraction was completed, the organic phase was washed with saturated brine (3×20 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA=1:1) to obtain compound 1g (yellow solid, 630 mg, yield 56%).

[0075] LCMS m / s=438.26[M+1].

[0076] Step 7:

[0077] 7-Ethoxy-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-N-(4-methoxybenzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine 1i

[0078] 7-ethoxy-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-N-(4-methoxyben zyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine

[0079] Compound 1g (630 mg, 1.438 mmol) and 2-(2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-4-methylpyrimidine 1h (498 mg, 1.51 mmol) were dissolved in DMF:H2O=5:1 (11 ml), potassium phosphate (915 mg, 4.315 mmol), 1,1-bis(diphenylphosphino)diferronichloridopalladium (158 mg, 0.216mmol), the reaction bottle was replaced with nitrogen and placed in a 90℃ oil bath for reaction. After 4 hours of reaction, water (15mL) was added for dilution and extracted with ethyl acetate (3×10mL). After the extraction, the organic phase was washed with saturated brine (3×20mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA=1:5) to obtain compound 1i (yellow solid, 256mg, yield 35%).

[0080] LCMS m / s=514.13[M+1].

[0081] Step 8:

[0082] 7-Ethoxy-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine 1j

[0083] 7-ethoxy-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-1-methyl-1H-pyrr olo[2,3-d]pyridazin-4-amine

[0084] Compound 1i (256 mg, 0.5 mmol) was dissolved in trifluoroacetic acid (10 ml), and the system was heated to 75°C and reacted for 1 hour. After the reaction was completed, saturated sodium bicarbonate aqueous solution was added to the system to adjust the pH to alkaline, and extracted with dichloromethane (3×10 mL). The organic phases were combined and concentrated to obtain compound 1j (yellow solid, 190 mg, yield 98%).

[0085] LCMS m / s=551.59[M+1].

[0086] Step 9:

[0087] 7-Ethoxy-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-2-iodo-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine 1k

[0088] 7-ethoxy-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-2-iodo-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine

[0089] Compound 1j (170 mg, 0.43 mmol) was dissolved in DCM (10 mL), trifluoroacetic acid (245 mg, 2.15 mmol) was added to the system, and the system was cooled to 0°C, and N-iodosuccinimide (963 mg, 4.3 mmol) was slowly added. After the system was warmed to room temperature and reacted for 2 hours, it was quenched with saturated aqueous sodium sulfite solution (30 mL), and the system was extracted with DCM (3×20 mL). After the extraction was completed, the organic phase was washed with saturated brine (3×20 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH=100:6) to obtain compound 1k (yellow solid, 146 mg, yield 65%).

[0090] LCMS m / s=521.26[M+1].

[0091] Step 10:

[0092] N-(4-(4-amino-7-ethoxy-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-2-yl)phenyl]-2-cyclopropylacrylamide Compound 1

[0093] N-(4-(4-amino-7-ethoxy-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-2-yl)phenyl)-2-cyclopropylacrylamide

[0094] Compound 1k (146 mg, 0.28 mmol) and intermediate 1 (97 mg, 0.308 mmol) were dissolved in DMF:H2O=20:1 (5 ml), and potassium phosphate (180 mg, 0.84 mmol) and 1,1-bis(diphenylphosphine)dichloroferrocene palladium (31 mg, 0.042 mmol) were added. The reaction flask was replaced with nitrogen and placed in an oil bath at 90°C for reaction. After 2 hours of reaction, water (5 mL) was added to quench the reaction, and ethyl acetate (3×10 mL) was added for extraction. After the extraction was completed, the organic phase was washed with saturated brine (3×10 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH=10:1) and reverse phase column chromatography to obtain compound 1 (white solid, 100 mg, 30%).

[0095] LCMS m / s=580.26[M+1].

[0096] 1 HNMR(400MHz,DMSO-d6)δ10.06(s,1H),8.47(d,J=5.0Hz,1H),7.81(d,J=8.5Hz,2H) ,7.33(m,J=23.4,15.0,10.0Hz,6H),7.19(d,J=5.0Hz,1H),7.11(d,J=7.9Hz,1H),5 .62(s,1H),5.29(s,1H),4.49(q,J=6.9Hz,2H),3.89(s,3H),2.40(s,3H),1.74(td, J=8.4,4.4Hz,1H),1.47(t,J=7.0Hz,3H),0.83–0.69(q,2H),0.56(q,J=5.7Hz,2H).

[0097] Example 2

[0098] N-(4-(4-amino-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-2-yl)phenyl]-2-cyclopropylacrylamide Compound 2

[0099] N-(4-(4-amino-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-2-yl)phenyl)-2-cyclopropylacrylamide

[0100]

[0101]

[0102] first step

[0103] 6-Chloro-5-iodo-3-methoxypyridazin-4-amine 2b

[0104] 6-chloro-5-iodo-3-methoxypyridazin-4-amine

[0105] 6-Chloro-3-methoxypyridazin-4-amine 2a (4.2 g, 26.3 mmol) and N-iodosuccinimide (8.9 g, 39.5 mmol) were added to acetic acid (80 mL), and the mixture was heated in an oil bath at 65°C for 6 hours. The reaction solution was concentrated, and saturated sodium bicarbonate solution (20 mL) and ethyl acetate (2×20 mL) were added and the layers were separated. The organic layer was washed with saturated sodium sulfite solution (20 mL) and brine (20 mL), dried and concentrated, and the crude product was purified by column chromatography (PE:EA=3:1) to obtain compound 2b (yellow solid, 6.5 g, yield 87%).

[0106] LCMS m / s=285.90[M+1].

[0107] Step 2

[0108] (E)-6-Chloro-5-(2-ethoxyvinyl)-3-methoxypyridazin-4-amine 2c

[0109] (E)-6-chloro-5-(2-ethoxyvinyl)-3-methoxypyridazin-4-amine

[0110] Compound 2b (6.5 g, 22.81 mmol) and (E)-1-ethoxyvinyl-2-boronic acid pinacol ester (4.97 g, 25.1 mmol) were dissolved in a mixed solution of 1,4-dioxane:H2O=10:1 (70 ml), and sodium carbonate (7.25 g, 68.44 mmol) and 1,1-bis(diphenylphosphine)diphenylferric palladium chloride (2.5 g, 3.42 mmol) were added. The reaction flask was replaced with nitrogen and placed in an oil bath at 90°C for reaction. After 16 hours of reaction, water (70 mL) was added for dilution and the mixture was extracted with ethyl acetate (3×50 mL). After the extraction, the organic phase was washed with saturated brine (3×50 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA=1:1) to obtain compound 2c (brown solid, 3.3 g, yield 63%).

[0111] LCMS m / s=230.10[M+1].

[0112] Step 3

[0113] 4-Chloro-7-methoxy-1H-pyrrolo[2,3-d]pyridazine 2d

[0114] 4-chloro-7-methoxy-1H-pyrrolo[2,3-d]pyridazine

[0115] Compound 2c (3.0 g, 13.09 mmol) was added to 3N hydrochloric acid, and the mixture was reacted at room temperature for 12 hours. 3N sodium hydroxide was slowly added dropwise to the mixture under ice bath for neutralization. Solid precipitated, and the solid was collected by filtration and dried to obtain compound 2d (yellow solid, 2.2 g, yield 91%).

[0116] LCMS m / s=184.10[M+1].

[0117] Step 4

[0118] 4-Chloro-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazine 2e

[0119] 4-chloro-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazine

[0120] Compound 2d (2.4 g, 13.07 mmol) and cesium carbonate (8.5 g, 26.14 mmol) were added to N,N-dimethylformamide (10 mL), and then iodomethane (2.3 g, 15.68 mmol) was added. After reacting at room temperature for 4 hours, water (20 mL) was added for dilution, and the mixture was extracted with ethyl acetate (3×10 mL). After the extraction, the organic phase was washed with saturated brine (3×10 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA=1:1) to obtain compound 2e (yellow solid, 2.0 g, yield 77%).

[0121] LCMS m / s=198.10[M+1].

[0122] Step 5

[0123] 7-Methoxy-N-(4-methoxybenzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine 2f

[0124] 7-methoxy-N-(4-methoxybenzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine

[0125] Compound 2e (2.0 g, 10.15 mmol) and 4-methoxybenzylamine (2.1 g, 15.22 mmol) were dissolved in 1,4-dioxane (30 ml), and cesium carbonate (10.0 g, 30.45 mmol), palladium acetate (456 mg, 2 mmol), and 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (1.9 g, 3 mmol) were added. The reaction flask was replaced with nitrogen and placed in an oil bath at 100°C for reaction. After reacting for 5 hours, water (40 mL) was added for dilution and the mixture was extracted with ethyl acetate (3×30 mL). After the extraction, the organic phase was washed with saturated brine (3×30 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH=30:1) to obtain compound 2f (brown solid, 1.5 g, yield 49%).

[0126] LCMS m / s=299.10[M+1].

[0127] Step 6

[0128] 3-iodo-7-methoxy-N-(4-methoxybenzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine 2g

[0129] 3-iodo-7-methoxy-N-(4-methoxybenzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine

[0130] Compound 2f (880 mg, 2.95 mmol) was dissolved in DCM (5 ml), trifluoroacetic acid (1.0 g, 8.85 mmol) was added, and the temperature was cooled to 0°C, and N-iodosuccinimide (665 mg, 2.95 mmol) was added. After the reaction flask was replaced with nitrogen and reacted for 3 hours, the reaction system was quenched with Na2SO3, and DCM (3×5 mL) was added for extraction. After the extraction was completed, the organic phase was washed with saturated brine (3×5 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH=50:1 to obtain compound 2g (yellow solid, 840 mg, yield 67%).

[0131] LCMS m / s=425.10[M+1].

[0132] Step 7

[0133] 3-(3-Fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-N-(4-methoxybenzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine 2h

[0134] 3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-N-(4-methoxyb enzyl)-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine

[0135] Compound 2g (1.1 g, 2.59 mmol) and compound 1h (1.1 g, 3.37 mmol) were dissolved in DMF:H2O=5:1 (15 ml), and potassium phosphate (1.65 g, 7.78 mmol) and 1,1-bis(diphenylphosphine)dichloropalladium iron (190 mg, 0.26 mmol) were added. The reaction flask was replaced with nitrogen and placed in a 90°C oil bath for reaction. After 4 hours of reaction, water (20 mL) was added for dilution and the mixture was extracted with ethyl acetate (3×10 mL). After the extraction, the organic phase was washed with saturated brine (3×10 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH=40:1) to obtain compound 2h (yellow solid, 700 mg, yield 53%).

[0136] LCMS m / s=501.20[M+1].

[0137] Step 8

[0138] 3-(3-Fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine 2i

[0139] 3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-py rrolo[2,3-d]pyridazin-4-amine

[0140] Compound 2h (600 mg, 1.19 mmol) was dissolved in trifluoroacetic acid (5 ml), and the mixture was heated in an oil bath at 75°C for 2 hours. The reaction solution was concentrated, and saturated sodium bicarbonate solution (5 mL) and ethyl acetate (2×5 mL) were added and the layers were separated. The organic layer was dried and concentrated, and the crude product was purified by column chromatography (DCM:MeOH=20:1) to obtain compound 2i (brown solid, 310 mg, yield 67%).

[0141] LCMS m / s=381.10[M+1].

[0142] Step 9

[0143] 2,2,2-Trifluoro-N-(3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-yl)acetamide 2j

[0144] 2,2,2-trifluoro-N-(3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methox y-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-yl)acetamide

[0145] Compound 2i (400 mg, 1.05 mmol), 4-dimethylaminopyridine (12 mg, 0.1 mmol), triethylamine (214 mg, 2.1 mmol) were dissolved in dichloromethane (5 ml), and the mixture was reacted at room temperature for 6 hours. Water (5 ml) was added to the mixture and the layers were separated, the organic layer was dried and concentrated, and the crude product was purified by column chromatography (DCM: MeOH = 50: 1) to obtain compound 2j (yellow solid, 290 mg, yield 58%).

[0146] LCMS m / s=477.10[M+1].

[0147] Step 10

[0148] N-(2-Bromo-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-yl)-2,2,2-trifluoroacetamide 2k

[0149] N-(2-bromo-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-yl)-2,2,2-trifluoroacetamide

[0150] Compound 2j (250 mg, 0.52 mmol) was dissolved in DCM (5 ml), trifluoroacetic acid (180 mg, 1.57 mmol) was added, and the temperature was cooled to 0°C, and N-bromosuccinimide (140 mg, 0.79 mmol) was added. After the reaction flask was replaced with nitrogen and reacted for 3 hours, the reaction system was quenched with Na2SO3, and DCM (3×5 mL) was added for extraction. After the extraction was completed, the organic phase was washed with saturated brine (3×5 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH=50:1) to give compound 2k (white solid, 231 mg, yield 80%).

[0151] LCMS m / s=555.10[M+1].

[0152] Step 11

[0153] 2-Bromo-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine 2l

[0154] 2-bromo-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-4-amine

[0155] Compound 2k (332 mg, 0.6 mmol) was added to 3N hydrochloric acid, and the mixture was reacted at room temperature for 4 hours. The solid was collected by filtration and dried to obtain compound 2l (white solid, 220 mg, yield 80%).

[0156] LCMS m / s=459.10[M+1].

[0157] Step 12

[0158] N-(4-(4-amino-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-2-yl)phenyl]-2-cyclopropylacrylamide Compound 2

[0159] N-(4-(4-amino-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-2-yl)phenyl)-2-cyclopropylacrylamide

[0160] Compound 2l (110 mg, 0.24 mmol) and intermediate 1 (98 mg, 0.31 mmol) were dissolved in DMF:H2O=20:1 (4 ml), and potassium phosphate (153 mg, 0.72 mmol) and 1,1-bis(diphenylphosphine)diferronipalladium dichloride (18 mg, 0.024 mmol) were added. The reaction flask was replaced with nitrogen and placed in an oil bath at 90°C for reaction. After 2 hours of reaction, water (5 mL) was added to quench the reaction. Ethyl acetate (3×10 mL) was added to extract. After the extraction was completed, the organic phase was washed with saturated brine (3×10 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH=10:1) to obtain compound 2 (white solid, 25 mg, 19%).

[0161] LCMS m / s=566.20[M+1].

[0162] 1H NMR(400MHz, DMSO-d6)δ10.05(s,1H),8.47(d,J=5.0Hz,1H),7.82–7.78(m,2H) ,7.39(t,J=8.3Hz,1H),7.36–7.32(m,2H),7.30(dd,J=11.1,2.0Hz,1H),7.19( d,J=5.0Hz,1H),7.15–7.09(m,3H),5.62(s,1H),5.29(s,1H),4.09(s,3H),3.8 8(s,3H),2.40(s,3H),1.78–1.71(m,1H),0.81–0.75(m,2H),0.58–0.53(m,2H).

[0163] Example 3

[0164] N-(4-(4-amino-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-2-yl)-3-chlorophenyl)-2-cyclopropylacrylamide Compound 3

[0165] N-(4-(4-amino-3-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-7-methoxy-1-methyl-1H-pyrrolo[2,3-d]pyridazin-2-yl)-3-chlorophenyl)-2-cyclopropylacrylamide

[0166]

[0167] Compound 21 (100 mg, 0.21 mmol) and intermediate 2 (98 mg, 0.28 mmol) were dissolved in DMF:H2O=20:1 (4 ml), and potassium phosphate (153 mg, 0.72 mmol) and 1,1-bis(diphenylphosphine)diferronipalladium dichloride (18 mg, 0.024 mmol) were added. The reaction flask was replaced with nitrogen and placed in an oil bath at 90°C for reaction. After 2 hours of reaction, water (5 mL) was added to quench the reaction. Ethyl acetate (3×10 mL) was added for extraction. After the extraction was completed, the organic phase was washed with saturated brine (3×10 mL), dried over anhydrous Na2SO4, filtered, and the organic phases were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH=10:1) to obtain compound 3 (white solid, 12 mg, 10%).

[0168] LCMS m / s=600.20[M+1].

[0169] 1 H NMR (400MHz, DMSO-d6) δ10.22(s,1H),8.46(d,J=5.0Hz,1H),8.08(d,J=2.1Hz,1H),7.76( dd,J=8.5,2.1Hz,1H),7.44(d,J=8.5Hz,1H),7.39(t,J=8.4Hz,1H),7.29–7.25(m,1H),7. 22(s,2H),7.19(d,J=5.0Hz,1H),7.08(d,J=8.4Hz,1H),5.66(s,1H),5.34(s,1H),4.09(s ,3H),3.78(s,3H),2.39(s,3H),1.78–1.71(m,1H),0.81–0.76(m,2H),0.58–0.54(m,2H).

[0170] Example 4

[0171] N-(4-(3-amino-2-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azepin-1-yl)phenyl]-2-cyclopropylacrylamide Compound 4

[0172] N-(4-(3-amino-2-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azulen-1-yl)phenyl)-2-cyclopropylacrylamide

[0173]

[0174]

[0175] first step:

[0176] 7-Bromo-1-(3-(((tert-butyldimethylsilyl)oxy)propyl)-4-chloro-1H-pyrrolo[3,2-c]pyridine 4c

[0177] 7-bromo-1-(3-((tert-butyldimethylsilyl)oxy)propyl)-4-chloro-1H-pyrrolo[3,2-c]pyridine

[0178] At 0°C, NaH (520 mg, 13 mmol) was slowly added to DMF (50 mL) of compound 4a (5 g, 21.6 mmol), and the mixture was reacted at 0°C for 30 min. Then 4b (8.2 g, 32.47 mmol) was added, and the mixture was slowly warmed to room temperature and the reaction was continued for 3 h. The reaction was quenched by pouring into water, and ethyl acetate (6×30 mL) was added for extraction. The organic phases were combined and concentrated under reduced pressure to obtain crude product 4c (8 g, yield 92%).

[0179] LCMS m / s=403.16[M+1].

[0180] Step 2:

[0181] 3-(7-Bromo-4-chloro-1H-pyrrolo[3,2-c]pyridin-1-yl)propan-1-ol 4d

[0182] 3-(7-bromo-4-chloro-1H-pyrrolo[3,2-c]pyridin-1-yl)propan-1-ol

[0183] At room temperature, TBAF (60 mL, 60 mmol) was added to a solution of compound 4c (8 g, 19.85 mol) in THF (100 mL), and the mixture was reacted at room temperature for 30 min. Ethyl acetate (100 mL) was added for dilution, and the mixture was washed with water (3×50 mL). The organic phase was concentrated under reduced pressure to obtain a crude product, which was purified by reverse phase column chromatography to obtain compound 4d (white solid, 4.3 g, yield 75%).

[0184] LCMS m / s=290.34[M+1].

[0185] Step 3:

[0186] 3-Chloro-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azurine 4e

[0187] 3-chloro-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azulene

[0188] Compound 4d (500 mg, 1.73 mmol), Pd2(dba)3 (475 mg, 0.519 mmol), tert-butyl Xphos (441 mg, 1.03 mmol), potassium tert-butoxide (387 mg, 3.46 mmol), and toluene (20 mL) were added to a round-bottom flask, and after nitrogen was replaced, the mixture was placed in an oil bath at 100°C for 48 h. The reaction mixture was cooled to room temperature, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 4e (200 mg, yield 55%).

[0189] LCMS m / s=209.04[M+1].

[0190] Step 4:

[0191] N-(8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azur-3-yl)-1,1-diphenylmethanimine 4g

[0192] N-(8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azulen-3-yl)-1,1-diphenylmethani mine

[0193] Compound 4e (400 mg, 1.92 mmol), benzophenone imine 4f (521 mg, 2.88 mmol), Pd2(dba)3 (176 mg, 0.19 mmol), BINAP (179 mg, 0.288 mmol), sodium tert-butoxide (369 mg, 3.84 mmol), toluene (20 mL) were added to a round-bottom flask, and after nitrogen was replaced, the mixture was placed in an oil bath at 110°C for 12 h. The reaction mixture was cooled to room temperature, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 4g (335 mg, yield 49%).

[0194] LCMS m / s=354.15[M+1].

[0195] Step 5:

[0196] 8,9-Dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azur-3-amine 4h

[0197] 8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azulen-3-amine

[0198] To a solution of 4 g (200 mg, 0.57 mmol) of MeOH (10 mL) was added a solution of hydrochloric acid in ethyl acetate (10 mL), and the mixture was reacted at room temperature for 30 min. The solvent was removed under reduced pressure, ethyl acetate was added, and the solid was collected by filtration for 4 h (78 mg, yield 72%).

[0199] LCMS m / s=190.09[M+1].

[0200] Step 6:

[0201] 2-Bromo-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azur-3-amine 4i

[0202] 2-bromo-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azulen-3-amine

[0203] At room temperature, NBS (74 mg, 0.41 mmol) was slowly added to a solution of compound 4h (78 mg, 0.35 mmol) in dichloromethane (35 mL), and the reaction was continued at room temperature for 30 min. After the reaction was completed, the solvent was removed under reduced pressure, and column chromatography (MeOH / DCM=10%, v / v) was performed to obtain compound 4i (65 mg, yield 70%).

[0204] LCMS m / s=268.01[M+1].

[0205] Step 7:

[0206] 3-(2-Bromo-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azur-3-yl)isoindole-1,3-dione 4j

[0207] 2-(2-bromo-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azulen-3-yl)isoindoline-1,3-dione

[0208] Phthalic anhydride (340 mg, 2.30 mmol) was added to the acetic acid solution of compound 4i (308 mg, 1.15 mmol), and the reaction was placed in an oil bath at 120°C for 6 h. After the reaction was completed, the solvent was removed under reduced pressure, neutralized with saturated sodium bicarbonate, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain a crude compound 4j (200 mg, yield 43%).

[0209] LCMS m / s=398.01[M+1].

[0210] Step 8:

[0211] 2-(2-(3-Fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azuryl-3-carbonyl)benzoic acid 4k

[0212] 2-(2-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azulene-3-carbonyl)benzoic acid

[0213] Compound 4j (200 mg, 0.50 mmol), compound 1h (183 mg, 0.55 mmol), Xphos-Pd-G4 (86 mg, 0.10 mmol), potassium phosphate (320 mg, 1.51 mmol), dioxane (20 mL), and water (1 mL) were added to a round-bottom flask, and after nitrogen was replaced, the mixture was placed in an oil bath at 100°C for 6 h. The reaction mixture was cooled to room temperature, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 4k (170 mg, yield 65%).

[0214] LCMS m / s=522.10[M+1].

[0215] Step 9:

[0216] 2-(2-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-1-iodo-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azepine-3-carbonyl)benzoic acid 4l

[0217] 2-(2-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-1-iodo-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azulene-3-carbonyl)benzoic acid

[0218] To a solution of compound 4k (170 mg, 0.32 mmol) in DCM (10 mL) were added trifluoroacetic acid (111 mg, 0.98 mmol) and NIS (109 mg, 0.49 mmol), and the mixture was reacted at room temperature for 3 h. After the reaction was completed, the solvent was removed under reduced pressure, and compound 4l (145 mg, yield 69%) was obtained by column chromatography (EA / DCM = 20%).

[0219] LCMS m / s=648.10[M+1].

[0220] Step 10:

[0221] 2-(3-Fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-1-iodo-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azur-3-amine 4m

[0222] (3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-1-iodo-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azulen-3-amine

[0223] To a solution of compound 4l (104 mg, 0.16 mmol) in methanol (10 mL) was added hydrazine hydrate (24 mg, 0.48 mmol) and the mixture was reacted at room temperature for 1 h. After completion of the reaction, the mixture was poured into water, extracted with dichloromethane, concentrated under reduced pressure, and subjected to reverse phase column chromatography to obtain compound 4m (77 mg, yield 92%).

[0224] LCMS m / s=518.10[M+1].

[0225] Step 11:

[0226] N-(4-(3-amino-2-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azepin-1-yl)phenyl]-2-cyclopropylacrylamide Compound 4

[0227] N-(4-(3-amino-2-(3-fluoro-4-((4-methylpyrimidin-2-yl)oxy)phenyl)-8,9-dihydro-7H-6-oxa-4,9a-diazabenzo[cd]azulen-1-yl)phenyl)-2-cyclopropylacrylamide

[0228] Compound 4m (57 mg, 0.11 mmol), intermediate 1 (41 mg, 0.13 mmol), Pd(dppf)Cl2 (16 mg, 0.02 mmol), potassium phosphate (70 mg, 0.33 mmol), DMF (4 mL), and water (0.2 mL) were added to a round-bottom flask, and after nitrogen was replaced, the mixture was placed in a 90°C oil bath for 6 h. The reaction mixture was cooled to room temperature, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 4 (5 mg, yield 7.9%).

[0229] LCMS m / s=577.30[M+1]

[0230] 1H NMR (400MHz, DMSO-d6) δ9.95 (s, 1H), 8.49 (d, J = 4.0Hz, 1H), 7.84-7.79 (m, 4H), 7.42 (d, J = 8.0Hz, 2H), 7.36-7.32 (m, 2H), 7.19 (d, J = 4.0Hz, 1H), 5. 65(s,1H),5.28(s,1H),5.04(s,2H),4.35-4.28(m,4H),2.43(s,3H),2.3 6-2.30(m,2H),1.82-1.75(m,1H),0.83-0.78(m,2H),0.60-0.56(m,2H).

[0231] Biological tests

[0232] 1. FGFR1-4 enzymology study

[0233] The HTRF method (PerkinElmer, 62TK0PEJ) was used to detect the inhibitory effects of the compounds on the enzyme activities of FGFR1, 2, 3, and 4. 2× enzyme and 2× substrate / ATP mixed solutions were prepared with buffer solution. Among them, the final concentrations of FGFR1 enzyme (Carna, 08-133), substrate (TK), and ATP were 0.10nM, 1μM, and 40μM, respectively; the final concentrations of FGFR2 (Carna, 08-134), substrate (TK), and ATP were 0.08nM, 1μM, and 20μM, respectively; the final concentrations of FGFR3 (Carna, 08-135), substrate (TK), and ATP were 0.30nM, 1μM, and 50μM, respectively; the final concentrations of FGFR4 (Carna, 08-136), substrate (TK), and ATP were 10nM, 1μM, and 50μM, respectively. The compound stock solution was diluted to 100× working concentration using DMSO. Use Echo to transfer 50nL / well of compound to a 384-well plate with 2 replicates for each concentration. Add 2.5μL / well of 2× kinase solution to the reaction well and mix, incubate at 25°C for 10min. Add 2.5μL / well of 2× substrate / ATP solution to the reaction well and incubate at 25°C for 50min. Prepare a 2× streptavidin-XL665 / Cryptate-antibody mixed solution with detection buffer (containing EDTA). Add 5μL / well of kinase detection reagent to the reaction well and incubate at 25°C for 60min. Read the fluorescence signals at 620nm (Cryptate) and 665nm (XL665) using a multifunctional microplate reader. Calculate the compound IC using GraphPad nonlinear fitting formula 50(half inhibitory concentration), Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X) × HillSlope)), X: logarithmic value of compound concentration; Y: compound inhibition rate (%inhibition).

[0234] The results show that, relative to FGFR1 and FGFR4, the compounds of the present invention have significant inhibitory activity and high selectivity against FGFR2 and / or FGFR3.

[0235] 2. Enzyme studies of FGFR2 and FGFR3 mutations

[0236] The HTRF method (PerkinElmer, 62TKOPEJ) was used to detect the inhibitory effects of the compounds on the FGFR2 and FGFR3 mutant enzyme activities. A 2× enzyme and 2× substrate / ATP mixed solution was prepared using a buffer solution, and the specific reaction system is shown in the following table.

[0237]

[0238] Use DMSO to dilute the compound stock solution to 100× working concentration. Use Echo to transfer 50nL / well compound to a 384-well plate, with 2 replicates for each concentration. Add 2.5μL / well of 2× kinase solution to the reaction well and mix, incubate at 25°C for 10min. Add 2.5μL / well of 2× substrate / ATP solution to the reaction well and incubate at 25°C for 50min. Prepare 2× streptavidin-XL665 / Cryptate-antibody mixed solution with detection buffer (containing EDTA). Add 5μL / well of kinase detection reagent to the reaction well and incubate at 25°C for 60min. Read the fluorescence signals at 620nm (Cryptate) and 665nm (XL665) with a multifunctional microplate reader. Calculate the inhibition rate based on the test results, inhibition rate (%Inhibition) = (signal negative control-signal compound) / (signal negative control-signal positive control)*100%. The IC50 (half maximal inhibitory concentration) of the compound was calculated using the GraphPad nonlinear fitting formula: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X) × HillSlope)), where X: logarithmic value of compound concentration; Y: compound inhibition rate (%inhibition).

[0239] The results show that the compounds of the present invention have more significant inhibitory activity against different mutations of FGFR2 and FGFR3 than the control compounds, indicating that the compounds of the present invention can overcome acquired drug resistance.

[0240] 3. Pharmacokinetic study in mouse brain tissue

[0241] Weigh an appropriate amount of the test substance, use 10% DMSO + 5% HS-15 + 85% Saline to prepare the test substance into a 1 mg / mL transparent clear solution for oral administration. Take 24 healthy male ICR mice (Viton Liva, SPF grade), 3 animals at each time point. The test substance (10 mg / kg) was administered by oral gavage and blood was collected at 15min, 0.5, 1, 2, 4, 6, 8, and 24h after administration. After the blood sample was anticoagulated with EDTA-K2, it was centrifuged at 4°C, 6000g for 5min to separate the plasma and stored at -70°C for testing. After blood collection, the animal was killed and the cerebrospinal fluid was collected and stored at -70°C for testing. The brain tissue was rinsed with ice-cold saline for residual blood, and the brain tissue was dried with absorbent paper and stored at -70°C for testing. The concentrations of designated compounds in plasma / cerebrospinal fluid / brain tissue were determined by LC-MS / MS (internal standard was gliclazide). The main pharmacokinetic parameters were calculated by Winnolin 8.3 non-compartmental model.

[0242] The results show that the compounds of the present invention have better pharmacokinetic characteristics and better blood-brain barrier penetration ability than the control compounds.

[0243] 4. Pharmacokinetic study in rats

[0244] Weigh an appropriate amount of the test substance, use 10% DMSO + 5% HS-15 + 85% Saline to prepare the test substance into a 0.5 mg / mL transparent clear solution for intravenous administration; use 10% DMSO + 5% HS-15 + 85% Saline to prepare the test substance into a 1 mg / mL uniform suspension for oral administration. Take 6 healthy male SD rats (Viton River, SPF grade), 3 animals in the intravenous and oral administration groups, 3 animals at each time point, and collect blood through the jugular vein. Blood was collected at 5min, 15min, 0.5, 1, 2, 4, 8, and 24h after intravenous administration of the test substance (1 mg / kg), and blood was collected at 15min, 0.5, 1, 2, 4, 6, 8, and 24h after oral administration of the test substance (5 mg / kg). After anticoagulation with EDTA-K2, the blood samples were centrifuged at 4°C, 6000g for 5 min to separate the plasma, and all plasma samples were stored at -70°C for testing. The plasma concentration of the test substance (internal standard: gliclazide) was determined by LC-MS / MS, and the main pharmacokinetic parameters were calculated by Winnolin 8.3 non-compartmental model.

[0245] The results showed that the compounds of the present invention had better pharmacokinetic characteristics than the control compounds.

[0246] 5. Pharmacokinetic study in dogs

[0247] Weigh an appropriate amount of the test substance, use 10% DMSO + 5% HS-15 + 85% Saline to prepare the test substance into a 0.5 mg / mL transparent clear solution for intravenous administration. Use 10% DMSO + 5% HS-15 + 85% Saline to prepare the test substance into a 0.6 mg / mL uniform suspension solution for oral administration. Take 6 healthy male beagle dogs (Viton River, SPF grade), 3 animals in the intravenous and oral administration groups, 3 animals at each time point, and collect blood through the forelimb vein. Blood was collected at 5min, 15min, 0.5, 1, 2, 4, 8, and 24h after intravenous administration of the test substance (1 mg / kg), and blood was collected at 15min, 0.5, 1, 2, 4, 6, 8, and 24h after oral administration of the test substance (3 mg / kg). After anticoagulation with EDTA-K2, the blood samples were centrifuged at 4°C, 3200g for 10 min to separate the plasma, and all plasma samples were stored at -70°C for testing. The plasma concentration of the test substance (internal standard: gliclazide) was determined by LC-MS / MS, and the main pharmacokinetic parameters were calculated by Winnolin 8.3 non-compartmental model.

[0248] The results showed that the compounds of the present invention had better pharmacokinetic characteristics than the control compounds.

[0249] 6. Study on serum phosphorus concentration in tumor-bearing mice

[0250] Human endometrial adenocarcinoma cells AN3CA (Punosai, CL-0505) were cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. When the cells were in the exponential growth phase, they were digested with trypsin, collected, counted, and inoculated in the axilla of female BALB / c nude mice. The inoculated cell amount was 2.5–5×10 6 / mouse, the inoculation volume is 100μL. When the tumor grows to 150mm 3 At about 1:10 pm, the mice were randomly divided into groups according to the tumor size, with 6 mice in each group. The test compound was prepared with 0.5% MC / 2% TPGS and administered by gavage at a dosage of 10 mL / kg. After the last administration, blood samples were collected from the orbital venous plexus of the mice, and serum was collected after centrifugation for phosphorus concentration detection.

[0251] The results showed that the serum phosphorus level in the group administered with the compound of the present invention was significantly lower than that in the group administered with the first-generation FGFR2 inhibitor Futibatinib, indicating that the compound of the present invention has significant safety.

[0252] The specification of the present invention describes the specific implementation scheme in detail. Those skilled in the art should recognize that the above implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt, stereoisomer or deuterated substance thereof, wherein the compound is selected from the following structures:

2. A pharmaceutical composition, comprising: (1) The compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer or deuterated substance thereof; (2) one or more other active ingredients; as well as (3) Pharmaceutically acceptable carriers and / or excipients.

3. Use of the compound according to claim 1 or its pharmaceutically acceptable salt, stereoisomer or deuterated substance or the pharmaceutical composition according to claim 2 in the preparation of anti-tumor drugs.

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